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Biochemistry / Richard A. Harvey, Denise R. Ferrier ; computer graphics, Michael Cooper. --5th ed.

Rev. ed. of: Biochemistry / Pamela C. Champe, Richard A. Harvey, Denise R. Ferrier. 4th ed. c2008.
Includes bibliographical references and index.
ISBN 978-1-60831-412-6 (alk. paper)
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Boston University School of Medicine
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Computer Graphics:
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This book is dedicated to the memory of our dear friend and
colleague Pamela Champe, whose commitment to her
students and love of biochemistry made her the consummate
teacher and mentor.

Chapter 1: Amino Acids 1
Chapter 2: Structure of Proteins 13
Chapter 3: Globular Proteins 25
Chapter 4:
Chapter 5: Enzymes 53
Fibrous
Proteins
43

Chapter 6: Bioenergetics and Oxidative Phosphorylation 69
Chapter 7: Introduction to Carbohydrates 83
Chapter 8: Glycolysis 91
Chapter 9: Tricarboxylic Acid Cycle 109
Chapter 10: Gluconeogenesis 117
Chapter 11: Glycogen Metabolism 125
Chapter 12: Metabolism of Monosaccharides and Disaccharides 137
Chapter 13: Pentose Phosphate Pathway and NADPH 145
Chapter 14: Glycosaminoglycans, Proteoglycans, and Glycoproteins 157

Chapter 15 : Metabolism of Dietary Lipids 173
Chapter 16 : Fatty Acid and Triacylglycerol Metabolism 181
Chapter 17 : Complex Lipid Metabolism 201
Chapter 18 : Cholesterol and Steroid Metabolism 219

Chapter 19 : Amino Acids: Disposal of Nitrogen 245
Chapter 20 : Amino Acid Degradation and Synthesis 261
Chapter 21 : Conversion of Amino Acids to Specialized Products 277
Chapter 22 : Nucleotide Metabolism 291

Chapter 23: Metabolic Effects of Insulin and Glucagon 307
Chapter 24: The Feed/Fast Cycle 321
Chapter 25: Diabetes Mellitus 337
Chapter 26: Obesity 349
Chapter 27: Nutrition 357
Chapter 28: Vitamins 373

Chapter 29: DNA Structure, Replication and Repair 395
Chapter 30: RNA Structure, Synthesis and Processing 417
Chapter 31: Protein Synthesis 431
Chapter 32: Regulation of Gene Expression 449
Chapter 33: Biotechnology and Human Disease 465
Index
489

Proteins are the most abundant and functionally diverse molecules in living systems. Virtually every life process depends on this class of molecules. For example, enzymes and polypeptide hormones direct and regulate metabolism in the body, whereas contractile proteins in muscle permit movement. In bone, the protein collagen forms a framework for the deposition of calcium phosphate crystals, acting like the steel cables in reinforced concrete. In the bloodstream, proteins, such as hemoglobin and plasma albumin, shuttle molecules essential to life, whereas immunoglobulins fight infectious bacteria and viruses. In short, proteins display an incredible diversity of functions, yet all share the common structural feature of being linear polymers of amino acids. This chapter describes the properties of amino acids. Chapter 2 explores how these simple building blocks are joined to form proteins that have unique three-dimensional structures, making them capable of performing specific biologic functions.

Although more than 300 different amino acids have been described in nature, only 20 are commonly found as constituents of mammalian proteins. [Note: These are the only amino acids that are coded for by DNA, the genetic material in the cell (see p. 395).] Each amino acid (except for proline, which has a secondary amino group) has a carboxyl group, a primary amino group, and a distinctive side chain ("R-group") bonded to the a-carbon atom (Figure 1.1A). At physiologic pH (approximately pH 7.4), the carboxyl group is dissociated, forming the negatively charged carboxylate ion (-COO'), and the amino group is protonated (—NH3*). In proteins, almost all of these carboxyl and amino groups are combined through peptide linkage and, in general, are not available for chemical reaction except for hydrogen bond formation (Figure 1.1B). Thus, it is the nature of the side chains that ultimately dictates the role

Structural features of amino acids (shown in their fully protonated form).
an amino acid plays in a protein. It is, therefore, useful to classify the amino acids according to the properties of their side chains, that is, whether they are nonpolar (have an even distribution of electrons) or polar (have an uneven distribution of electrons, such as acids and bases; Figures 1.2 and 1.3).

Each of these amino acids has a nonpolar side chain that does not gain or lose protons or participate in hydrogen or ionic bonds (Figure 1.2). The side chains of these amino acids can be thought of as "oily" or lipid-like, a property that promotes hydrophobic interactions (see Figure 2.10, p. 19).
- 1. Location of nonpolar amino acids in proteins: In proteins found in aqueous solutions—a polar environment—the side chains of the nonpolar amino acids tend to cluster together in the interior of the protein (Figure 1.4). This phenomenon, known as the hydrophobic

Classification of the 20 amino acids commonly found in proteins, according to the charge and polarity of their side chains at acidic pH is shown here and continues in Figure 1.3. Each amino acid is shown in its fully protonated form, with dissociable hydrogen ions represented in red print. The pK values for the a-carboxyl and o-amino groups of the nonpolar amino acids are similar to those shown for glycine. (Continued in Figure 1.3.)

Classification of the 20 amino acids commonly found in proteins, according to the charge and polarity of their side chains at acidic pH (continued from Figure 1.2).

Location of nonpolar amino acids in soluble and membrane proteins.

Comparison of the secondary amino group found in proline with the primary amino group found in other amino acids, such as alanine.

Hydrogen bond between the phenolic hydroxyl group of tyrosine and another molecule containing a carbonyl group.
effect, is the result of the hydrophobicity of the nonpolar R-groups, which act much like droplets of oil that coalesce in an aqueous environment. The nonpolar R-groups thus fill up the interior of the folded protein and help give it its three-dimensional shape. However, for proteins that are located in a hydrophobic environment, such as a membrane, the nonpolar R-groups are found on the outside surface of the protein, interacting with the lipid environment (see Figure 1.4). The importance of these hydrophobic interactions in stabilizing protein structure is discussed on p. 19.
Sickle cell anemia, a sickling disease of red blood cells, results from the substitution of polar glutamate by nonpolar valine at the sixth position in the B subunit of hemoglobin (see p. 36).
2. Proline: Proline differs from other amino acids in that proline's side chain and a-amino N form a rigid, five-membered ring structure (Figure 1.5). Proline, then, has a secondary (rather than a primary) amino group. It is frequently referred to as an imino acid. The unique geometry of proline contributes to the formation of the fibrous structure of collagen (see p. 45), and often interrupts the a-helices found in globular proteins (see p. 26).

These amino acids have zero net charge at neutral pH, although the side chains of cysteine and tyrosine can lose a proton at an alkaline pH (see Figure 1.3). Serine, threonine, and tyrosine each contain a polar hydroxyl group that can participate in hydrogen bond formation (Figure 1.6). The side chains of asparagine and glutamine each contain a carbonyl group and an amide group, both of which can also participate in hydrogen bonds.
- 1. Disulfide bond: The side chain of cysteine contains a sulfhydryl group (—SH), which is an important component of the active site of many enzymes. In proteins, the —SH groups of two cysteines can become oxidized to form a dimer, cystine, which contains a covalent cross-link called a disulfide bond (-S—S-). (See p. 19 for a further discussion of disulfide bond formation.)
Many extracellular proteins are stabilized by disulfide bonds. Albumin, a blood protein that functions as a transporter for a variety of molecules, is an example.

- Side chains as sites of attachment for other compounds: The polar hydroxyl group of serine, threonine, and, rarely, tyrosine, can serve as a site of attachment for structures such as a phosphate group. In addition, the amide group of asparagine, as well as the hydroxyl group of serine or threonine, can serve as a site of attachment for oligosaccharide chains in glycoproteins (see p. 165).

The amino acids aspartic and glutamic acid are proton donors. At physiologic pH, the side chains of these amino acids are fully ionized, containing a negatively charged carboxylate group (-COO ). They are, therefore, called aspartate or glutamate to emphasize that these amino acids are negatively charged at physiologic pH (see Figure 1.3).

The side chains of the basic amino acids accept protons (see Figure 1.3). At physiologic pH the side chains of lysine and arginine are fully ionized and positively charged. In contrast, histidine is weakly basic, and the free amino acid is largely uncharged at physiologic pH. However, when histidine is incorporated into a protein, its side chain can be either positively charged or neutral, depending on the ionic environment provided by the polypeptide chains of the protein. This is an important property of histidine that contributes to the role it plays in the functioning of proteins such as hemoglobin (see p. 31).

Each amino acid name has an associated three-letter abbreviation and a one-letter symbol (Figure 1.7). The one-letter codes are determined by the following rules:
1. Unique first letter: If only one amino acid begins with a particular letter, then that letter is used as its symbol. For example, | = isoleucine.
. . . _ 2. Most commonly occurring amino acids have priority: If more than one amino acid begins with a particular letter, the most common of these amino acids receives this letter as its symbol. For example, glycine is more common than glutamate, so G = glycine.
3. Similar sounding names: Some one-letter symbols sound like the . i . amino acid they represent. For example, F = phenylalanine, or W = tryptophan ("twyptophan" as Elmer Fudd would say).
4. Letter close to initial letter: For the remaining amino acids, a oneletter symbol is assigned that is as close in the alphabet as possible to the initial letter of the amino acid, for example, K = lysine. Furthermore, B is assigned to Asx, signifying either aspartic acid or asparagine, Z is assigned to Glx, signifying either glutamic acid or glutamine, and X is assigned to an unidentified amino acid.

The a-carbon of an amino acid is attached to four different chemical groups and is, therefore, a chiral or optically active carbon atom. Glycine is the exception because its a-carbon has two hydrogen substituents and, therefore, is optically inactive. Amino acids that have an asymmetric center at the a-carbon can exist in two forms, designated D and L, that are mirror images of each other (Figure 1.8). The two forms in each pair are termed stereoisomers, optical isomers, or enantiomers. All amino acids found in proteins are of the L-configuration. However, D-amino acids are found in some antibiotics and in plant and bacterial cell walls. (See p. 253 for a discussion of D-amino acid metabolism.)
B

A la nine, 1 = =. A la nine, 2 = Ala = A. G ly cine, 1 = =. G ly cine, 2 = Gly = G. L eu cine, 1 = = Leu =. L eu cine, 2 = L. P ro line, 1 = = Pro =. P ro line, 2 = P. T hr eonine, 1 = = Thr. T hr eonine, 2 = = T

Abbreviations and symbols for the commonly occurring amino acids.

Amino acids in aqueous solution contain weakly acidic a-carboxyl groups and weakly basic «-amino groups. In addition, each of the acidic and basic amino acids contains an ionizable group in its side chain. Thus, both free amino acids and some amino acids combined in peptide linkages can act as buffers. Recall that acids may be defined as proton donors and bases as proton acceptors. Acids (or bases) described as "weak" ionize to only a limited extent. The concentration of protons in aqueous solution is expressed as pH, where pH = log 1/[H*] or —log [H*]. The quantitative relationship between the pH of the solution and concentration of a weak acid (HA) and its conjugate base (A) is described by the Henderson-Hasselbalch equation.

Consider the release of a proton by a weak acid represented by HA:
The "salt" or conjugate base, A', is the ionized form of a weak acid. By definition, the dissociation constant of the acid, Ka, is
[Note: The larger the Ka, the stronger the acid, because most of the HA has dissociated into H* and A'. Conversely, the smaller the Ka, the less acid has dissociated and, therefore, the weaker the acid.] By solving for the [H*] in the above equation, taking the logarithm of both sides of the equation, multiplying both sides of the equation by —1, and substituting pH = —log [H* ] and pKa = —log Ka, we obtain the Henderson-Hasselbalch equation:

A buffer is a solution that resists change in pH following the addition of an acid or base. A buffer can be created by mixing a weak acid (HA) with its conjugate base (A'). If an acid such as HCl is then added to such a solution, A" can neutralize it, in the process being converted to HA. If a base is added, HA can neutralize it, in the process being converted to A'. Maximum buffering capacity occurs at a pH equal to the pKa, but a conjugate acid/base pair can still serve as an effective buffer when the pH of a solution is within approximately +1 pH unit of the pKa. If the

lonic forms of alanine in acidic, neutral, and basic solutions.
amounts of HA and A" are equal, the pH is equal to the pKa. As shown in Figure 1.9, a solution containing acetic acid (HA = CH3s—-COOH) and acetate (A" = CH3s—COO) with a pK, of 4.8 resists a change in pH from PH 3.8 to 5.8, with maximum buffering at pH 4.8. At pH values less than the pKa, the protonated acid form (CH3—COOH) is the predominant species. At pH values greater than the pKa, the deprotonated base form (CHs3—COO) is the predominant species in solution.

1. Dissociation of the carboxyl group: The titration curve of an amino acid can be analyzed in the same way as described for acetic acid. Consider alanine, for example, which contains both an a-carboxyl and an a-amino group. At a low (acidic) pH, both of these groups are protonated (shown in Figure 1.10). As the pH of the solution is raised, the -COOH group of Form | can dissociate by donating a proton to the medium. The release of a proton results in the formation of the carboxylate group, -COO . This structure is shown as Form Il, which is the dipolar form of the molecule (see Figure 1.10). This form, also called a zwitterion, is the isoelectric form of alanine, that is, it has an overall (net) charge of zero.

Application of the Henderson-Hasselbalch equation: The dissociation constant of the carboxyl group of an amino acid is called K;, rather than Ka, because the molecule contains a second titratable group. The Henderson-Hasselbalch equation can be used to analyze the dissociation of the carboxyl group of alanine in the same way as described for acetic acid:
where | is the fully protonated form of alanine, and II is the isoelectric form of alanine (see Figure 1.10). This equation can be rearranged and converted to its logarithmic form to yield:

The titration curve of alanine.
3. Dissociation of the amino group: The second titratable group of alanine is the amino (-NH3*) group shown in Figure 1.10. This is a much weaker acid than the -COOH group and, therefore, has a much smaller dissociation constant, Ko. [Note: Its pK, is therefore larger.] Release of a proton from the protonated amino group of Form II results in the fully deprotonated form of alanine, Form Ill (see Figure 1.10).
. PKs of alanine: The sequential dissociation of protons from the carboxyl and amino groups of alanine is summarized in Figure 1.10. Each titratable group has a pK, that is numerically equal to the pH at which exactly one half of the protons have been removed from that group. The pKa for the most acidic group (—COOh) is pK;, whereas the pK, for the next most acidic group (—NHs3*) is pKo.
. Titration curve of alanine: By applying the HendersonHasselbalch equation to each dissociable acidic group, it is possible to calculate the complete titration curve of a weak acid. Figure 1.11 shows the change in pH that occurs during the addition of base to the fully protonated form of alanine (I) to produce the completely deprotonated form (Ill). Note the following:
a. Buffer pairs: The -COOH/—COO™ pair can serve as a buffer in the pH region around pK;, and the —NH3*/—NHp pair can buffer in the region around pKo.
b. When pH = pK: When the pH is equal to pK, (2.3), equal amounts of Forms | and II of alanine exist in solution. When the pH is equal to pKz (9.1), equal amounts of Forms II and III are present in solution.

c. Isoelectric point: At neutral pH, alanine exists predominantly as the dipolar Form II in which the amino and carboxyl groups are ionized, but the net charge is zero. The isoelectric point (pl) is the pH at which an amino acid is electrically neutral, that is, in which the sum of the positive charges equals the sum of the negative charges. For an amino acid, such as alanine, that has only two dissociable hydrogens (one from the a-carboxyl and one from the a-amino group), the pl is the average of pK; and pKo (pl = [2.3 + 9.1]/2 = 5.7, see Figure 1.11). The pl is thus midway between pk; (2.3) and pKo (9.1). pl corresponds to the pH at which the Form II (with a net charge of zero) predominates, and at which there are also equal amounts of Forms | (net charge of +1) and Ill (net charge of —1).
is done at a pH above the pl of the major proteins, thus, the charge on the proteins is negative. In an electric field, the proteins will move toward the positive electrode at a rate determined by their net negative charge. Variations in the mobilSeparation of plasma proteins by charge typically ity pattern are suggestive of certain diseases.
6. Net charge of amino acids at neutral pH: At physiologic pH, amino acids have a negatively charged group (-COO ) and a positively charged group (—NH3*), both attached to the a-carbon. [Note: Glutamate, aspartate, histidine, arginine, and lysine have additional potentially charged groups in their side chains.] Substances, such as amino acids, that can act either as an acid or a base are defined as amphoteric, and are referred to as ampholytes (amphoteric electrolytes).

The Henderson-Hasselbalch equation can be used to calculate how the pH of a physiologic solution responds to changes in the concentration of a weak acid and/or its corresponding "salt" form. For example, in the bicarbonate buffer system, the Henderson-Hasselbalch equation predicts how shifts in the bicarbonate ion concentration, [HCO], and COz influence pH (Figure 1.12A). The equation is also useful for calculating the abundance of ionic forms of acidic and basic drugs. For example, most drugs are either weak acids or weak bases (Figure 1.12B). Acidic drugs (HA) release a proton (H*), causing a charged anion (A') to form.
Weak bases (BH*) can also release a H*. However, the protonated form of basic drugs is usually charged, and the loss of a proton produces the uncharged base (B).
A drug passes through membranes more readily if it is uncharged. Thus, for a weak acid such as aspirin, the uncharged HA can permeate through membranes and A" cannot. For a weak base, such as morphine, the uncharged form, B, penetrates through the cell membrane and BH* does not. Therefore, the effective concentration of the permeable form of each drug at its absorption site is determined by the relative concentrations of the charged and uncharged forms. The ratio between the two forms is determined by the pH at the site of absorption, and by the strength of the weak acid or base, which is represented by the pKa of the ionizable group. The Henderson-Hasselbalch equation is useful in determining how much drug is found on either side of a membrane that separates two compartments that differ in pH, for example, the stomach (pH 1.0—1.5) and blood plasma (pH 7.4).

The Henderson-Hasselbalch equation is used to predict: A, changes in pH as the concentrations of HCO3° or COz are altered; or B, the ionic forms of drugs.

Students sometimes view biochemistry as a blur of facts or equations to be memorized, rather than a body of concepts to be understood. Details provided to enrich understanding of these concepts inadvertently turn into distractions. What seems to be missing is a road map—a guide that provides the student with an intuitive understanding of how various topics fit together to make sense. The authors have, therefore, created a series of biochemical concept maps to graphically illustrate relationships between ideas presented in a chapter, and to show how the information can be grouped or organized. A concept map is, thus, a tool for visualizing the connections between concepts. Material is represented in a hierarchic fashion, with the most inclusive, most general concepts at the top of the map, and the more specific, less general concepts arranged beneath. The concept maps ideally function as templates or guides for organizing information, so the student can readily find the best ways to integrate new information into knowledge they already possess.

1. Concept boxes and links: Educators define concepts as "perceived regularities in events or objects." In our biochemical maps, concepts include abstractions (for example, free energy), processes (for example, oxidative phosphorylation), and compounds (for example, glucose 6-phosphate). These broadly defined concepts are prioritized with the central idea positioned at the top of the page. The concepts that follow from this central idea are then drawn in boxes (Figure 1.13A). The size of the type indicates the relative importance of each idea. Lines are drawn between concept boxes to show which are related. The label on the line defines the relationship between two concepts, so that it reads as a valid statement, that is, the connection creates meaning. The lines with arrowheads indicate in which direction the connection should be read (Figure 1.14).
2. Cross-links: Unlike linear flow charts or outlines, concept maps may contain cross-links that allow the reader to visualize complex relationships between ideas represented in different parts of the map (Figure 1.13B), or between the map and other chapters in this book or companion books in the series (Figure 1.13C). Crosslinks can thus identify concepts that are central to more than one discipline, empowering students to be effective in clinical situations, and on the United States Medical Licensure Examination (USMLE) or other examinations, that bridge disciplinary boundaries. Students learn to visually perceive nonlinear relationships between facts, in contrast to cross-referencing within linear text.

Each amino acid has an a-carboxyl group and a primary a-amino group (except for proline, which has a secondary amino group). At physiologic pH, the a-carboxyl group is dissociated, forming the negatively charged carboxylate ion (-COO_), and the a-amino group is protonated (—NH3*). Each amino acid also contains one of 20 distinctive

Key concept map for amino acids.
side chains attached to the a-carbon atom. The chemical nature of this side chain determines the function of an amino acid in a protein, and provides the basis for classification of the amino acids as nonpolar, uncharged polar, acidic, or basic. All free amino acids, plus charged amino acids in peptide chains, can serve as buffers. The quantitative relationship between the pH of a solution and the concentration of a weak acid (HA) and its conjugate base (A') is described by the Henderson-Hasselbalch equation. Buffering occurs within +1pH unit of the pKa, and is maximal when pH = pKa, at which [A] = [HA]. The a-carbon of each amino acid (except glycine) is attached to four different chemical groups and is, therefore, a chiral or optically active carbon atom. Only the L-form of amino acids is found in proteins synthesized by the human body.

1.1
The letters
A
through
E
designate the
one titration
curve for
of the
is following
certain glycine
(shown statements
regions below).
concerning on
Which this
correct?
- A. Point A represents the region where glycine is
deprotonated.
B. Point B represents a region of minimal buffering.
C. Point C represents the region where the net charge
on glycine is zero.
D. Point D represents the pK of glycine's carboxyl group.
E. Point E represents the pl for glycine.
1.2 Which one of the following statements concerning the
peptide shown below is correct?

A. The peptide contains glutamine.
B. The peptide contains a side chain with a secondary
amino group.
- C. The peptide contains a majority of amino acids with
side chains that would be at 7.
positively charged pH
The peptide is able form internal disulfide
D. to an bond.
1.3 Given that the pl for glycine is 6.1, to which electrode, positive or negative, will glycine move in an electric field at pH 2? Explain.
curve
Correct answer = C. C represents the isoelectric point or pl, and as such is midway between pk; and pKo2 for this monoamino monocarboxylic acid. Glycine is fully protonated at Point A. Point B represents a region of maximum buffering, as does Point D. Point E represents the region where glycine is fully deprotonated.
Correct answer = D. The two cysteine residues can, under oxidizing conditions, form a disulfide bond. Glutamine's 3-letter abbreviation is Gin. Proline (Pro) contains a secondary amino group. Only one (Arg) of the seven would have a positively charged side chain at pH 7.
Correct answer = negative electrode. When the PH is less than the pl, the charge on glycine is positive because the o-amino group is fully protonated. (Recall that glycine has H as its R group).

The 20 amino acids commonly found in proteins are joined together by peptide bonds. The linear sequence of the linked amino acids contains the information necessary to generate a protein molecule with a unique three-dimensional shape. The complexity of protein structure is best analyzed by considering the molecule in terms of four organizational levels, namely, primary, secondary, tertiary, and quaternary (Figure 2.1). An examination of these hierarchies of increasing complexity has revealed that certain structural elements are repeated in a wide variety of proteins, suggesting that there are general "rules" regarding the ways in which proteins achieve their native, functional form. These repeated structural elements range from simple combinations of a-helices and B-sheets forming small motifs, to the complex folding of polypeptide domains of multifunctional proteins (see p. 18).

The sequence of amino acids in a protein is called the primary structure of the protein. Understanding the primary structure of proteins is important because many genetic diseases result in proteins with abnormal amino acid sequences, which cause improper folding and loss or impairment of normal function. If the primary structures of the normal and the mutated proteins are known, this information may be used to diagnose or study the disease.

In proteins, amino acids are joined covalently by peptide bonds, which are amide linkages between the a-carboxyl group of one amino acid and the a-amino group of another. For example, valine and alanine can form the dipeptide valylalanine through the formation of a peptide bond (Figure 2.2). Peptide bonds are not broken by conditions that denature proteins, such as heating or high concentrations of urea (see p. 20). Prolonged exposure to a strong acid or base at elevated temperatures is required to hydrolyze these bonds nonenzymically.

A. Formation of a peptide bond, showing the structure of the dipeptide valylalanine. B. Characteristics of the peptide bond.
1.
Naming the peptide: By convention, the free amino end (N-terminal) of the peptide chain is written to the left and the free carboxyl end (C-terminal) to the right. Therefore, all amino acid sequences are read from the Nto the C-terminal end of the peptide. For example, in Figure 2.2A, the order of the amino acids is "valine, alanine." Linkage of many amino acids through peptide bonds results in an unbranched chain called a polypeptide. Each component amino acid in a polypeptide is called a "residue" because it is the portion of the amino acid remaining after the atoms of water are lost in the formation of the peptide bond. When a polypeptide is named, all amino acid residues have their suffixes (-ine, -an, -ic, or -ate) changed to -yl, with the exception of the C-terminal amino acid. For example, a tripeptide composed of an N-terminal valine, a glycine, and a C-terminal leucine is called valylglycylleucine.
2.
Characteristics of the peptide bond: The peptide bond has a partial double-bond character, that is, it is shorter than a single bond, and is rigid and planar (Figure 2.2B). This prevents free rotation around the bond between the carbonyl carbon and the nitrogen of the peptide bond. However, the bonds between the a-carbons and the a-amino or o-carboxyl groups can be freely rotated (although they are limited by the size and character of the R-groups). This allows the polypeptide chain to assume a variety of possible configurations. The peptide bond is generally a trans bond (instead of cis, see Figure 2.2B), in large part because of steric interference of the R-groups when in the cis position.

3. Polarity of the peptide bond: Like all amide linkages, the -C=O and —NH groups of the peptide bond are uncharged, and neither accept nor release protons over the pH range of 2-12. Thus, the charged groups present in polypeptides consist solely of the N-terminal (a-amino) group, the C-terminal (a-carboxyl) group, and any ionized groups present in the side chains of the constituent amino acids. The -C=O and —NH groups of the peptide bond are polar, and are involved in hydrogen bonds, for example, in a-helices and B-sheet structures, described on pp. 16-17.

The first step in determining the primary structure of a polypeptide is to identify and quantitate its constituent amino acids. A purified sample of the polypeptide to be analyzed is first hydrolyzed by strong acid at 110°C for 24 hours. This treatment cleaves the peptide bonds and releases the individual amino acids, which can be separated by cation-exchange chromatography. In this technique, a mixture of amino acids is applied to a column that contains a resin to which a negatively charged group is tightly attached. [Note: If the attached group is positively charged, the column becomes an anionexchange column.] The amino acids bind to the column with different affinities, depending on their charges, hydrophobicity, and other characteristics. Each amino acid is sequentially released from the chromatography column by eluting with solutions of increasing ionic strength and pH (Figure 2.3). The separated amino acids contained in the eluate from the column are quantitated by heating them with ninhydrin—a reagent that forms a purple compound with most
amino acids, ammonia, and amines. The amount of each amino acid is determined spectrophotometrically by measuring the amount of light absorbed by the ninhydrin derivative. The analysis described above is performed using an amino acid analyzer—an automated machine whose components are depicted in Figure 2.3.

Sequencing is a stepwise process of identifying the specific amino acid at each position in the peptide chain, beginning at the N-terminal end. Phenylisothiocyanate, known as Edman reagent, is used to label the amino-terminal residue under mildly alkaline conditions (Figure 2.4). The resulting phenylthiohydantoin (PTH) derivative introduces an instability in the N-terminal peptide bond that can be selectively hydrolyzed without cleaving the other peptide bonds. The identity of the amino acid derivative can then be determined. Edman reagent can be applied repeatedly to the shortened peptide obtained in each previous cycle.

Many polypeptides have a primary structure composed of more than 100 amino acids. Such molecules cannot be sequenced directly from end to end. However, these large molecules can be cleaved at specific sites, and the resulting fragments sequenced. By using more than one cleaving agent (enzymes and/or chemicals) on separate samples of the purified polypeptide, overlapping fragments can be generated that permit the proper ordering of the sequenced fragments, thus providing a complete amino acid sequence of the large polypeptide (Figure 2.5). Enzymes that hydrolyze peptide bonds are termed peptidases (proteases). [Note: Exopeptidases cut at the ends of proteins, and are divided into aminopeptidases and carboxypeptidases. Carboxypeptidases are used in determining the C-terminal amino acid. Endopeptidases cleave within a protein.]

The sequence of nucleotides in a protein-coding region of the DNA specifies the amino acid sequence of a polypeptide. Therefore, if the nucleotide sequence can be determined, it is possible, from knowledge of the genetic code (see p. 431), to translate the sequence of nucleotides into the corresponding amino acid sequence of that

Determination of the amino-terminal residue of a polypeptide by Edman degradation.

Determination of the amino acid composition of a polypeptide using an amino acid analyzer.

Overlapping of peptides produced by the action of trypsin and cyanogen bromide.

a-Helix showing peptide backbone.
polypeptide. This indirect process, although routinely used to obtain the amino acid sequences of proteins, has the limitations of not being able to predict the positions of disulfide bonds in the folded chain, and of not identifying any amino acids that are modified after their incorporation into the polypeptide (posttranslational modification, see p. 443). Therefore, direct protein sequencing is an extremely important tool for determining the true character of the primary sequence of many polypeptides.

The polypeptide backbone does not assume a random three-dimensional structure, but instead generally forms regular arrangements of amino acids that are located near to each other in the linear sequence. These arrangements are termed the secondary structure of the polypeptide. The a-helix, B-sheet, and B-bend (B-turn) are examples of secondary structures frequently encountered in proteins. [Note: The collagen a-chain helix, another example of secondary structure, is discussed on p. 45.]

Several different polypeptide helices are found in nature, but the a-helix is the most common. It is a spiral structure, consisting of a tightly packed, coiled polypeptide backbone core, with the side chains of the component amino acids extending outward from the central axis to avoid interfering sterically with each other (Figure 2.6). A very diverse group of proteins contains a-helices. For example, the keratins are a family of closely related, fibrous proteins whose structure is nearly entirely o-helical. They are a major component of tissues such as hair and skin, and their rigidity is determined by the number of disulfide bonds between the constituent polypeptide chains. In contrast to keratin, myoglobin, whose structure is also highly o-helical, is a globular, flexible molecule (see p. 26).
1. Hydrogen bonds: An o-helix is stabilized by extensive hydrogen bonding between the peptide-bond carbonyl oxygens and amide hydrogens that are part of the polypeptide backbone (see Figure 2.6). The hydrogen bonds extend up and are parallel to the spiral from the carbonyl oxygen of one peptide bond to the -NH— group of a peptide linkage four residues ahead in the polypeptide. This ensures that all but the first and last peptide bond components are linked to each other through intrachain hydrogen bonds. Hydrogen bonds are individually weak, but they collectively serve to stabilize the helix.
2. Amino acids per turn: Each turn of an a-helix contains 3.6 amino acids. Thus, amino acid residues spaced three or four residues apart in the primary sequence are spatially close together when folded in the o-helix.
3. Amino acids that disrupt an a-helix: Proline disrupts an o-helix because its secondary amino group is not geometrically compatible with the right-handed spiral of the a-helix. Instead, it inserts a kink in the chain, which interferes with the smooth, helical structure. Large numbers of charged amino acids (for example, gluta-

mate, aspartate, histidine, lysine, or arginine) also disrupt the helix by forming ionic bonds, or by electrostatically repelling each other. Finally, amino acids with bulky side chains, such as tryptophan, or amino acids, such as valine or isoleucine, that branch at the B-carbon (the first carbon in the R-group, next to the a-carbon) can interfere with formation of the a-helix if they are present in large numbers.

The B-sheet is another form of secondary structure in which all of the peptide bond components are involved in hydrogen bonding (Figure 2.7A). The surfaces of B-sheets appear "pleated," and these structures are, therefore, often called "B-pleated sheets." When illustrations are made of protein structure, B-strands are often visualized as broad arrows (Figure 2.7B).
1. Comparison of a B-sheet and an a-helix: Unlike the a-helix, B-sheets are composed of two or more peptide chains (B-strands), or segments of polypeptide chains, which are almost fully extended. Note also that in B-sheets the hydrogen bonds are perpendicular to the polypeptide backbone (see Figure 2.7A).
2.
Parallel and antiparallel sheets: A B-sheet can be formed from two or more separate polypeptide chains or segments of polypeptide chains that are arranged either antiparallel to each other (with the N-terminal and C-terminal ends of the B-strands alternating as shown in Figure 2.7B), or parallel to each other (with all the N-termini of the B-strands together as shown in Figure 2.7C). When the hydrogen bonds are formed between the polypeptide backbones of separate polypeptide chains, they are termed interchain bonds. A B-sheet can also be formed by a single polypeptide chain folding back on itself (see Figure 2.7C). In this case, the hydrogen bonds are intrachain bonds. In globular proteins, B-sheets always have a right-handed curl, or twist, when viewed along the polypeptide backbone. [Note: Twisted Bsheets often form the core of globular proteins.]
The a-helix and B-sheet structures provide maximal hydrogen bonding for peptide bond components within the interior of polypeptides.

B-Bends reverse the direction of a polypeptide chain, helping it form a compact, globular shape. They are usually found on the surface of protein molecules, and often include charged residues. [Note: B-Bends were given this name because they often connect successive strands of antiparallel B-sheets.] B-Bends are generally composed of four amino acids, one of which may be proline—the amino acid that causes a "kink" in the polypeptide chain. Glycine, the amino acid with the smallest R-group, is also frequently found in B-bends. B-Bends are stabilized by the formation of hydrogen and ionic bonds.

A. Structure of a B-sheet. B. An antiparallel 6-sheet with the B-strands represented as broad arrows. C. A parallel -sheet formed from a single polypeptide chain folding back on itself.

Some common structural motifs combining «-helices and/or B-sheets. The names describe their schematic appearance.

Formation of a disulfide bond by the oxidation of two cysteine residues, producing one cystine residue.
Approximately one half of an average globular protein is organized into repetitive structures, such as the a-helix and/or B-sheet. The remainder of the polypeptide chain is described as having a loop or coil conformation. These nonrepetitive secondary structures are not "random," but rather simply have a less regular structure than those described above. [Note: The term "random coil" refers to the disordered structure obtained when proteins are denatured (see p. 20).]

Globular proteins are constructed by combining secondary structural elements (a-helices, B-sheets, nonrepetitive sequences). These form primarily the core region—that is, the interior of the molecule. They are connected by loop regions (for example, B-bends) at the surface of the protein. Supersecondary structures are usually produced by packing side chains from adjacent secondary structural elements close to each other. Thus, for example, a-helices and B-sheets that are adjacent in the amino acid sequence are also usually (but not always) adjacent in the final, folded protein. Some of the more common motifs are illustrated in Figure 2.8.
Proteins that bind to DNA contain a limited number of motifs. The helix-loop-helix motif is an example found in a number of proteins that function as transcription factors (see p. 455).

. . . . . . The primary structure of a polypeptide chain determines its tertiary structure. "Tertiary" refers both to the folding of domains (the basic units of structure and function, see discussion below), and to the final arrangement of domains in the polypeptide. The structure of globular proteins in aqueous solution is compact, with a high-density (close packing) of the atoms in the core of the molecule. Hydrophobic side chains are buried in the interior, whereas hydrophilic groups are generally found on the surface of the molecule.

Domains are the fundamental functional and three-dimensional structural units of polypeptides. Polypeptide chains that are greater than 200 amino acids in length generally consist of two or more domains. The core of a domain is built from combinations of supersecondary structural elements (motifs). Folding of the peptide chain within a domain usually occurs independently of folding in other domains. Therefore, each domain has the characteristics of a small, compact globular protein that is structurally independent of the other domains in the polypeptide chain.

The unique three-dimensional structure of each polypeptide is determined by its amino acid sequence. Interactions between the amino acid side chains guide the folding of the polypeptide to form a compact structure. The following four types of interactions cooperate in stabilizing the tertiary structures of globular proteins.

1. Disulfide bonds: A disulfide bond is a covalent linkage formed from the sulfhydryl group (-SH) of each of two cysteine residues, to produce a cystine residue (Figure 2.9). The two cysteines may be separated from each other by many amino acids in the primary sequence of a polypeptide, or may even be located on two different polypeptide chains; the folding of the polypeptide chain(s) brings the cysteine residues into proximity, and permits covalent bonding of their side chains. A disulfide bond contributes to the stability of the three-dimensional shape of the protein molecule, and prevents it from becoming denatured in the extracellular environment. For example, many disulfide bonds are found in proteins such as immunoglobulins that are secreted by cells.
2. Hydrophobic interactions: Amino acids with nonpolar side chains tend to be located in the interior of the polypeptide molecule, where they associate with other hydrophobic amino acids (Figure 2.10). In contrast, amino acids with polar or charged side chains tend to be located on the surface of the molecule in contact with the polar solvent. [Note: Recall that proteins located in nonpolar (lipid) environments, such as a membrane, exhibit the reverse arrangement (see Figure 1.4, p. 4).] In each case, a segregation of R-groups occurs that is energetically most favorable.
3. Hydrogen bonds: Amino acid side chains containing oxygenor nitrogen-bound hydrogen, such as in the alcohol groups of serine and threonine, can form hydrogen bonds with electron-rich atoms, such as the oxygen of a carboxyl group or carbonyl group of a peptide bond (Figure 2.11; see also Figure 1.6, p. 4). Formation of hydrogen bonds between polar groups on the surface of proteins and the aqueous solvent enhances the solubility of the protein.
4. lonic interactions: Negatively charged groups, such as the carboxylate group (-COO ) in the side chain of aspartate or glutamate, can interact with positively charged groups, such as the amino group (—NHs*) in the side chain of lysine (see Figure 2.11).

Hydrophobic interactions between amino acids with nonpolar side chains.

Interactions of side chains of amino acids through hydrogen bonds and ionic bonds (salt bridges).

Interactions between the side chains of amino acids determine how a long polypeptide chain folds into the intricate three-dimensional shape of the functional protein. Protein folding, which occurs within the cell in seconds to minutes, employs a shortcut through the maze of all folding possibilities. As a peptide folds, its amino acid side chains are attracted and repulsed according to their chemical properties. For example, positively and negatively charged side chains attract each other. Conversely, similarly charged side chains repel each other. In addition, interactions involving hydrogen bonds, hydrophobic interactions, and disulfide bonds all exert an influence on the folding process. This process of trial and error tests many, but not all, possible configurations, seeking a compromise in which attractions outweigh repulsions. This results in a correctly folded protein with a low-energy state (Figure 2.12).

Protein denaturation results in the unfolding and disorganization of the protein's secondary and tertiary structures, which are not accompanied by hydrolysis of peptide bonds. Denaturing agents include heat, organic solvents, mechanical mixing, strong acids or bases, detergents, and ions of heavy metals such as lead and mercury. Denaturation may, under ideal conditions, be reversible, in which case the protein refolds into its original native structure when the denaturing agent is removed. However, most proteins, once denatured, remain permanently disordered. Denatured proteins are often insoluble and, therefore, precipitate from solution.

It is generally accepted that the information needed for correct protein folding is contained in the primary structure of the polypeptide. Given that premise, it is difficult to explain why most proteins when denatured do not resume their native conformations under favorable environmental conditions. One answer to this problem is that a protein begins to fold in stages during its synthesis, rather than waiting for synthesis of the entire chain to be totally completed. This limits competing folding configurations made available by longer stretches of nascent peptide. In addition, a specialized group of proteins, named "chaperones," are required for the proper folding of many species of proteins. The chaperones—also known as "heat shock" proteins—interact with the polypeptide at various stages during the folding process. Some chaperones are important in keeping the protein unfolded until its synthesis is finished, or act as catalysts by increasing the rates of the final stages in the folding process. Others protect proteins as they fold so that their vulnerable, exposed regions do not become tangled in unproductive interactions.

Many proteins consist of a single polypeptide chain, and are defined as monomeric proteins. However, others may consist of two or more polypeptide chains that may be structurally identical or totally unrelated. The arrangement of these polypeptide subunits is called the quaternary structure of the protein. Subunits are held together by noncovalent interactions (for example, hydrogen bonds, ionic bonds, and hydrophobic
interactions). Subunits may either function independently of each other, or may work cooperatively, as in hemoglobin, in which the binding of oxygen to one subunit of the tetramer increases the affinity of the other subunits for oxygen (see p. 29).
Isoforms are proteins that perform the same function but have different primary structures. They can arise from different genes or from tissue-specific processing of the product of a single gene. If the proteins function as enzymes, they are referred to as isozymes (see p. 65).

Protein folding is a complex, trial-and-error process that can sometimes result in improperly folded molecules. These misfolded proteins are usually tagged and degraded within the cell (see p. 444). However, this quality control system is not perfect, and intracellular or extracellular aggregates of misfolded proteins can accumulate, particularly as individuals age. Deposits of these misfolded proteins are associated with a number of diseases.

Misfolding of proteins may occur spontaneously, or be caused by a mutation in a particular gene, which then produces an altered protein. In addition, some apparently normal proteins can, after abnormal proteolytic cleavage, take on a unique conformational state that leads to the formation of long, fibrillar protein assemblies consisting of B-pleated sheets. Accumulation of these insoluble, spontaneously aggregating proteins, called amyloids, has been implicated in many degenerative diseases—particularly in the age-related neurodegenerative disorder, Alzheimer disease. The dominant component of the amyloid plaque that accumulates in Alzheimer disease is amyloid B (AB), a peptide containing 40-42 amino acid residues. X-ray crystallography and infrared spectroscopy demonstrate a characteristic Bpleated sheet conformation in nonbranching fibrils. This peptide, when aggregated in a B-pleated sheet configuration, is neurotoxic, and is the central pathogenic event leading to the cognitive impairment characteristic of the disease. The AB that is deposited in the brain in Alzheimer disease is derived by proteolytic cleavages from the larger amyloid precursor protein—a single transmembrane protein expressed on the cell surface in the brain and other tissues (Figure 2.13). The AB peptides aggregate, generating the amyloid that is found in the brain parenchyma and around blood vessels. Most cases of Alzheimer disease are not genetically based, although at least 5-10% of cases are familial. A second biologic factor involved in the development of Alzheimer disease is the accumulation of neurofibrillary tangles inside neurons. A key component of these tangled fibers is an abnormal form of the tau (t) protein, which in its healthy version helps in the assembly of the microtubular structure. The defective t, however, appears to block the actions of its normal counterpart.

One proposed mechanism for multiplication of infectious prion agents.

The prion protein (PrP) has been strongly implicated as the causative agent of transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle (popularly called "mad cow disease").' After an extensive series of purification procedures, scientists were astonished to find that the infectivity of the agent causing scrapie in sheep was associated with a single protein species that was not complexed with detectable nucleic acid. This infectious protein is designated PrPS° (Sc = scrapie). It is highly resistant to proteolytic degradation, and tends to form insoluble aggregates of fibrils, similar to the amyloid found in some other diseases of the brain. A noninfectious form of PrP° (C = cellular), encoded by the same gene as the infectious agent, is present in normal mammalian brains on the surface of neurons and glial cells. Thus, PrP° is a host protein. No primary structure differences or alternate posttranslational modifications have been found between the normal and the infectious forms of the protein. The key to becoming infectious apparently lies in changes in the three-dimensional conformation of PrP°. It has been observed that a number of a-helices present in noninfectious PrP® are replaced by B-sheets in the infectious form (Figure 2.14). It is presumably this conformational difference that confers relative resistance to proteolytic degradation of infectious prions, and permits them to be distinguished from the normal PrP in infected tissue. The infective agent is thus an altered version of a normal protein, which acts as a "template" for converting the normal protein to the pathogenic conformation. The TSEs are invariably fatal, and no treatment is currently available that can alter this outcome.

Central to understanding protein structure is the concept of the native conformation (Figure 2.15), which is the functional, fully-folded protein structure (for example, an active enzyme or structural protein). The unique three-dimensional structure of the native conformation is determined by its primary structure, that is, its amino acid sequence. Interactions between the amino acid side chains guide the folding of the polypeptide chain to form secondary, tertiary, and (sometimes) quaternary structures, which cooperate in stabilizing the native conformation of the protein. In addition, a specialized group of proteins named "chaperones" is required for the proper folding of many species of proteins. Protein denaturation results in the unfolding and disorganization of the protein's structure, which are not accompanied by hydrolysis of peptide bonds. Denaturation may be reversible or, more commonly, irreversible. Disease can occur when an apparently normal protein assumes a conformation that is cytotoxic, as in the case of Alzheimer disease and the transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease. In Alzheimer disease, normal proteins, after abnormal chemical processing, take on a unique conformational state that leads to the formation of neurotoxic amyloid protein assemblies consisting of B-pleated sheets. In TSEs, the infective agent is an altered version of a normal prion protein that acts as a "template" for converting normal protein to the pathogenic conformation.
sec Chapter 31 in Lippincott's Illustrated Reviews: Microbiology for a more detailed discussion of prions.

A. has a partial double-bond character.
B. is ionized at physiologic pH.
C. is cleaved by agents that denature proteins, such as organic solvents and high concentrations of urea.
D. is stable to heating in strong acids.
E. occurs most commonly in the cis configuration.
2.2 Which one of the following statements is correct?
A. The a-helix can be composed of more than one polypeptide chain.
B. B-Sheets exist only in the antiparallel form.
C. B-Bends often contain proline.
D. Domains are a type of secondary structure.
interac-
E. The a-helix is stabilized primarily by ionic tions between the side chains of amino acids.
2.3 Which one of the following statements about protein structure is correct?
A. Proteins consisting of one polypeptide can have quaternary structure.
B. The formation of a disulfide bond in a protein requires that the two participating cysteine residues be adjacent to each other in the primary sequence of the protein.
C. The stability of quaternary structure in proteins is mainly a result of covalent bonds among the subunits.
D. The denaturation of proteins always leads to irreversible loss of secondary and tertiary structure.
E. The information required for the correct folding of a protein is contained in the specific sequence of amino acids along the polypeptide chain.
2.4 An 80-year-old man presented with impairment of
higher intellectual function and alterations in mood and behavior. His family reported progressive disorientation and memory loss over the last 6 months. There is no family history of dementia. The patient was tentatively diagnosed with Alzheimer disease. Which one of the following best describes the disease?
A. It is associated with B-amyloid—an abnormal protein with an altered amino acid sequence.
B. It results from accumulation of denatured proteins
that have
random conformations.

C. It is associated with the accumulation of amyloid precursor protein.
D. It is associated with the deposition of neurotoxic amyloid peptide aggregates.
E. It is an environmentally produced disease not influenced by the genetics of the individual.
F. It is caused by the infectious form of a host-cell protein.
Correct answer = A. The peptide bond has a partial double-bond character. Unlike its components—the o-amino and o-carboxyl groups—the —NH and -C=O of the peptide bond do not accept or give off protons. The peptide bond is not cleaved by organic solvents or urea, but is labile to strong acids. It is usually in the trans configuration.
Correct answer = C. B-Bends often contain proline, which provides a kink. The o-helix differs from the B-sheet in that it always involves the coiling of a single polypeptide chain. The B-sheet occurs in both parallel and antiparallel forms. Domains are elements of tertiary structure. The a-helix is stabilized primarily by hydrogen bonds between the -C=O and —NHgroups of peptide bonds.
Correct answer = E. The correct folding of a protein is guided by specific interactions between the side chains of the amino acid residues of a polypeptide chain. The two cysteine residues that react to form the disulfide bond may be a great distance apart in the primary structure (or on separate polypeptides), but are brought into close proximity by the three-dimensional folding of the polypeptide chain. Denaturation may either be reversible or irreversible. Quaternary structure requires more than one polypeptide chain. These chains associate through noncovalent interactions.

Correct answer = D. Alzheimer disease is associated with long, fibrillar protein assemblies consisting of B-pleated sheets found in the brain and elsewhere. The disease is associated with abnormal processing of a normal protein. The accumulated altered protein occurs ina B-pleated sheet configuration that is neurotoxic. The AB amyloid that is deposited in the brain in Alzheimer disease is derived by proteolytic cleavages from the larger amyloid precursor protein—a single transmembrane protein expressed on the cell surface in the brain and other tissues. Most cases of Alzheimer disease are sporadic, although at least 5-10% of cases are familial. Prion diseases, such as CreutzfeldtJakob, are caused by the infectious form (PrP) of a host-cell protein (PrP°).
I.

The previous chapter described the types of secondary and tertiary structures that are the bricks-and-mortar of protein architecture. By arranging these fundamental structural elements in different combinations, widely diverse proteins can be constructed that are capable of various specialized functions. This chapter examines the relationship between structure and function for the clinically important globular hemeproteins. Fibrous structural proteins are discussed in Chapter 4.

Hemeproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic group. (See p. 54 for a discussion of prosthetic groups.) The role of the heme group is dictated by the environment created by the three-dimensional structure of the protein. For example, the heme group of a cytochrome functions as an electron carrier that is alternately oxidized and reduced (see p. 76). In contrast, the heme group of the enzyme catalase is part of the active site of the enzyme that catalyzes the breakdown of hydrogen peroxide (see p. 148). In hemoglobin and myoglobin, the two most abundant hemeproteins in humans, the heme group serves to reversibly bind oxygen.

Heme is a complex of protoporphyrin IX and ferrous iron (Fe**) (Figure 3.1). The iron is held in the center of the heme molecule by bonds to the four nitrogens of the porphyrin ring. The heme Fe?* can form two additional bonds, one on each side of the planar porphyrin ring. In myoglobin and hemoglobin, one of these positions is coordinated to the side chain of a histidine residue of the globin molecule, whereas the other position is available to bind oxygen (Figure 3.2). (See p. 278 for a discussion of the synthesis and degradation of heme.)

A. Model of myoglobin showing helices A to H. B. Schematic diagram of the oxygen-binding site of myoglobin.

Myoglobin, a hemeprotein present in heart and skeletal muscle, functions both as a reservoir for oxygen, and as an oxygen carrier that increases the rate of transport of oxygen within the muscle cell. Myoglobin consists of a single polypeptide chain that is structurally similar to the individual subunit polypeptide chains of the hemoglobin molecule. This homology makes myoglobin a useful model for interpreting some of the more complex properties of hemoglobin.
= . o-Helical content: Myoglobin is a compact molecule, with approximately 80% of its polypeptide chain folded into eight stretches of a-helix. These a-helical regions, labeled A to H in Figure 3.2A, are terminated either by the presence of proline, whose five-membered ring cannot be accommodated in an o-helix (see p. 16), or by B-bends and loops stabilized by hydrogen bonds and ionic bonds (see p. 17).
2. Location of polar and nonpolar amino acid residues: The interior of the myoglobin molecule is composed almost entirely of nonpolar amino acids. They are packed closely together, forming a structure stabilized by hydrophobic interactions between these clustered residues (see p. 19). In contrast, charged amino acids are located almost exclusively on the surface of the molecule, where they can form hydrogen bonds, both with each other and with water.
3. Binding of the heme group: The heme group of myoglobin sits in a crevice in the molecule, which is lined with nonpolar amino acids. Notable exceptions are two histidine residues (Figure 3.2B). One, the proximal histidine (F8), binds directly to the iron of heme. The second, or distal histidine (E7), does not directly interact with the heme group, but helps stabilize the binding of oxygen to the ferrous iron. The protein, or globin, portion of myoglobin thus creates a special microenvironment for the heme that permits the reversible binding of one oxygen molecule (oxygenation). The simultaneous loss of electrons by the ferrous iron (oxidation) occurs only rarely.

A. Structure of hemoglobin showing the polypeptide backbone. B. Simplified drawing showing the helices.

Hemoglobin is found exclusively in red blood cells (RBCs), where its main function is to transport oxygen (Oz) from the lungs to the capillaries of the tissues. Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide chains—two « chains and two B chains—held together by noncovalent interactions (Figure 3.3). Each subunit has stretches of a-helical structure, and a heme-binding pocket similar to that described for myoglobin. However, the tetrameric hemoglobin molecule is structurally and functionally more complex than myoglobin. For example, hemoglobin can transport H* and COs from the tissues to the lungs, and can carry four molecules of Ox. from the lungs to the cells of the body. Furthermore, the oxygen-binding properties of hemoglobin are regulated by interaction with allosteric effectors (see p. 29).
Obtaining Oz from the atmosphere solely by diffusion greatly limits the size of organisms. Circulatory systems overcome this, but transport molecules such as hemoglobin are also required because Oz is only slightly soluble in aqueous solutions such as blood.
1. Quaternary structure of hemoglobin: The hemoglobin tetramer can be envisioned as being composed of two identical dimers, (a); and (a), in which the numbers refer to dimers one and two. The two polypeptide chains within each dimer are held tightly together, primarily by hydrophobic interactions (Figure 3.4). [Note: In this instance, hydrophobic amino acid residues are localized not only in the interior of the molecule, but also in a region on the surface of each subunit. Interchain hydrophobic interactions form strong associations between a-subunits and B-subunits in the

Schematic diagram showing structural changes resulting from oxygenation and deoxygenation of hemoglobin.
dimers.] lonic and hydrogen bonds also occur between the members of the dimer. In contrast, the two dimers are able to move with respect to each other, being held together primarily by polar bonds. The weaker interactions between these mobile dimers result in the two dimers occupying different relative positions in deoxyhemoglobin as compared with oxyhemoglobin (see Figure 3.4). [Note: The binding of O2 to the heme iron pulls the iron into the plane of the heme. Because the iron is also linked to the proximal histidine (F8), there is movement of the globin chains that alters the interface between the af dimers.]
- a. T form: The deoxy form of hemoglobin is called the "T,' or taut (tense) form. In the T form, the two a8 dimers interact through a network of ionic bonds and hydrogen bonds that constrain the movement of the polypeptide chains. The T form is the lowoxygen-affinity form of hemoglobin.
b. R form: The binding of oxygen to hemoglobin causes the rupture of some of the ionic bonds and hydrogen bonds between the aB dimers. This leads to a structure called the "R," or relaxed form, in which the polypeptide chains have more freedom of movement (see Figure 3.4). The R form is the highoxygen-affinity form of hemoglobin.

Myoglobin can bind only one molecule of oxygen, because it contains only one heme group. In contrast, hemoglobin can bind four oxygen molecules—one at each of its four heme groups. The degree of saturation (Y) of these oxygen-binding sites on all myoglobin or hemoglobin molecules can vary between zero (all sites are empty) and 100% (all sites are full, Figure 3.5).
1. Oxygen dissociation curve: A plot of Y measured at different partial pressures of oxygen (pOz) is called the oxygen dissociation curve. The curves for myoglobin and hemoglobin show important differences (see Figure 3.5). This graph illustrates that myoglobin has a higher oxygen affinity at all pO2 values than does hemoglobin. The partial pressure of oxygen needed to achieve half-saturation of the binding sites (Pso) is approximately 1 mm Hg for myoglobin and 26 mm Hg for hemoglobin. The higher the oxygen affinity (that is, the more tightly oxygen binds), the lower the Pso. [Note: pO2 may also be represented as PO2.]
a. Myoglobin (Mb): The oxygen dissociation curve for myoglobin has a hyperbolic shape (see Figure 3.5). This reflects the fact that myoglobin reversibly binds a single molecule of oxygen. Thus, oxygenated (MbOz) and deoxygenated (Mb) myoglobin exist in a simple equilibrium:
Mb
+
O2
MbOs
The equilibrium is shifted to the right or to the left as oxygen is added to or removed from the system. [Note: Myoglobin is designed to bind oxygen released by hemoglobin at the low pOz found in muscle. Myoglobin, in turn, releases oxygen within the muscle cell in response to oxygen demand.]

- b. Hemoglobin (Hb): The oxygen dissociation curve for hemoglobin is sigmoidal in shape (see Figure 3.5), indicating that the subunits cooperate in binding oxygen. Cooperative binding of oxygen by the four subunits of hemoglobin means that the binding of an oxygen molecule at one heme group increases the oxygen affinity of the remaining heme groups in the same hemoglobin molecule (Figure 3.6). This effect is referred to as heme-heme interaction (see below). Although it is more difficult for the first oxygen molecule to bind to hemoglobin, the subsequent binding of oxygen occurs with high affinity, as shown by the steep upward curve in the region near 20-30 mm Hg (see Figure 3.5).

The ability of hemoglobin to reversibly bind oxygen is affected by the pOxz (through heme-heme interactions as described above), the pH of the environment, the partial pressure of carbon dioxide, pCOs, and the availability of 2,3-bisphosphoglycerate. These are collectively called allosteric ("other site") effectors, because their interaction at one site on the hemoglobin molecule affects the binding of oxygen to heme groups at other locations on the molecule. [Note: The binding of oxygen to myoglobin is not influenced by allosteric effectors. ]
- 1. Heme-heme interactions: The sigmoidal oxygen dissociation curve reflects specific structural changes that are initiated at one heme group and transmitted to other heme groups in the hemoglobin tetramer. The net effect is that the affinity of hemoglobin for the last oxygen bound is approximately 300 times greater than its affinity for the first oxygen bound.

Effect of pH on the oxygen affinity of hemoglobin. Protons are allosteric effectors of hemoglobin.
a. Loading and unloading oxygen: The cooperative binding of oxygen allows hemoglobin to deliver more oxygen to the tissues in response to relatively small changes in the partial pressure of oxygen. This can be seen in Figure 3.5, which indicates pOz in the alveoli of the lung and the capillaries of the tissues. For example, in the lung, the concentration of oxygen is high and hemoglobin becomes virtually saturated (or "loaded") with oxygen. In contrast, in the peripheral tissues, oxyhemoglobin releases (or "unloads") much of its oxygen for use in the oxidative metabolism of the tissues (Figure 3.7).
- b. Significance of the sigmoidal oxygen dissociation curve: The steep slope of the oxygen dissociation curve over the range of oxygen concentrations that occur between the lungs and the tissues permits hemoglobin to carry and deliver oxygen efficiently from sites of high to sites of low pOz. A molecule with a hyperbolic oxygen dissociation curve, such as myoglobin, could not achieve the same degree of oxygen release within this range of partial pressures of oxygen. Instead, it would have maximum affinity for oxygen throughout this oxygen pressure range and, therefore, would deliver no oxygen to the tissues.
2. Bohr effect: The release of oxygen from hemoglobin is enhanced when the pH is lowered or when the hemoglobin is in the presence of an increased pCOz. Both result in a decreased oxygen affinity of hemoglobin and, therefore, a shift to the right in the oxygen dissociation curve (Figure 3.8), and both, then, stabilize the T state. This change in oxygen binding is called the Bohr effect. Conversely, raising the pH or lowering the concentration of CO2 results in a greater affinity for oxygen, a shift to the left in the oxygen dissociation curve, and stabilization of the R state.

- a. Source of the protons that lower the pH: The concentration of both COs and H* in the capillaries of metabolically active tissues is higher than that observed in alveolar capillaries of the lungs, where COz is released into the expired air. [Note: Organic acids, such as lactic acid, are produced during anaerobic metabolism in rapidly contracting muscle (see p. 103).] In the tissues, CO2 is converted by carbonic anhydrase to carbonic acid:
<!-- formula-not-decoded -->
H2CO3
which spontaneously loses a proton, becoming bicarbonate (the major blood buffer):
H2CO;
HCO; + Ht
The H* produced by this pair of reactions contributes to the lowering of pH. This differential pH gradient (lungs having a higher pH, tissues a lower pH) favors the unloading of oxygen in the peripheral tissues, and the loading of oxygen in the lung. Thus, the oxygen affinity of the hemoglobin molecule responds to small shifts in pH between the lungs and oxygen-consuming tissues, making hemoglobin a more efficient transporter of oxygen.
b. Mechanism of the Bohr effect: The Bohr effect reflects the fact that the deoxy form of hemoglobin has a greater affinity for protons than does oxyhemoglobin. This effect is caused by ionizable groups, such as specific histidine side chains that have higher pKas in deoxyhemoglobin than in oxyhemoglobin. Therefore, an increase in the concentration of protons (resulting in a decrease in pH) causes these groups to become protonated (charged) and able to form ionic bonds (also called salt bridges). These bonds preferentially stabilize the deoxy form of hemoglobin, producing a decrease in oxygen affinity.
The Bohr effect can be represented schematically as:
HbO2z
+
H*
2
HbH
+
Oo oxyhemoglobin
deoxyhemoglobin where an increase in protons (or a lower pOz) shifts the equilibrium to the right (favoring deoxyhemoglobin), whereas an increase in pOs (or a decrease in protons) shifts the equilibrium to the left.

3. Effect of 2,3-bisphosphoglycerate on oxygen affinity: 2,3-Bisphosphoglycerate (2,3-BPG) is an important regulator of the binding of oxygen to hemoglobin. It is the most abundant organic phosphate in the RBC, where its concentration is approximately that of hemoglobin. 2,3-BPG is synthesized from an intermediate of the glycolytic pathway (Figure 3.9; see p. 101 for a discussion of 2,3-BPG synthesis in glycolysis).
a. Binding of 2,3-BPG to deoxyhemoglobin: 2,3-BPG decreases the oxygen affinity of hemoglobin by binding to deoxyhemoglobin but not to oxyhemoglobin. This preferential binding stabilizes the taut conformation of deoxyhemoglobin. The effect of binding 2,3-BPG can be represented schematically as:
HbOz
+
2,3BPG
@
Hb-2,3-BPG
+
deoxyhemoglobin
. Binding site of 2,3-BPG: One molecule of 2,3-BPG binds to a pocket, formed by the two B-globin chains, in the center of the deoxyhemoglobin tetramer (Figure 3.10). This pocket contains several positively charged amino acids that form ionic bonds with the negatively charged phosphate groups of 2,3-BPG. [Note: A mutation of one of these residues can result in hemoglobin variants with abnormally high oxygen affinity.] 2,3-BPG is expelled on oxygenation of the hemoglobin.
. Shift of the oxygen dissociation curve: Hemoglobin from which 2,3-BPG has been removed has a high affinity for oxygen. However, as seen in the RBC, the presence of 2,3-BPG significantly reduces the affinity of hemoglobin for oxygen, shifting the oxygen dissociation curve to the right (Figure 3.11). This reduced affinity enables hemoglobin to release oxygen efficiently at the partial pressures found in the tissues.

Synthesis of 2,3-bisphosphoglycerate. [Note: ®is a phosphoryl group.] In older literature 2,3-bisphosphoglycerate (2,3-BPG) may be referred to as 2,3-diphosphoglycerate (2,3-DPG).

Allosteric effect of 2,3-BPG on the oxygen affinity of hemoglobin.

Effect of carbon monoxide on the oxygen affinity of hemoglobin. CO-Hb = carbon monoxyhemoglobin.
d. Response of 2,3-BPG levels to chronic hypoxia or anemia: The concentration of 2,3-BPG in the RBC increases in response to chronic hypoxia, such as that observed in chronic obstructive pulmonary disease (COPD) like emphysema, or at high altitudes, where circulating hemoglobin may have difficulty receiving sufficient oxygen. Intracellular levels of 2,3-BPG are also elevated in chronic anemia, in which fewer than normal RBCs are available to supply the body's oxygen needs. Elevated 2,3-BPG levels lower the oxygen affinity of hemoglobin, permitting greater unloading of oxygen in the capillaries of the tissues (see Figure 3.11).
e.
Role of 2,3-BPG in transfused blood: 2,3-BPG is essential for the normal oxygen transport function of hemoglobin. However, storing blood in the currently available media results in a decrease in 2,3-PBG. Stored blood displays an abnormally high oxygen affinity, and fails to unload its bound oxygen properly in the tissues. Hemoglobin deficient in 2,3-BPG thus acts as an oxygen "trap" rather than as an oxygen transport system. Transfused RBCs are able to restore their depleted supplies of 2,3-BPG in 6-24 hours. However, severely ill patients may be compromised if transfused with large quantities of such 2,3-BPG—'stripped" blood. [Note: The maximum storage time for red cells has been doubled (21 to 42 days, with median time of 15 days) by changes in H*, phosphate and hexose sugar concentration, and by the addition of adenine (see p. 291). Although the content of 2,3-BPG was not greatly affected by these changes, ATP production was increased and improved RBC survival.]
- . Binding of CO2: Most of the COs produced in metabolism is hydrated and transported as bicarbonate ion (see p. 9). However, some COs is carried as carbamate bound to the N-terminal amino groups of hemoglobin (forming carbaminohemoglobin, see Figure 3.7), which can be represented schematically as follows:
Hb—NHp
+

COz
Hb-NH-COO™ + H*
The binding of COs stabilizes the T (taut) or deoxy form of hemoglobin, resulting in a decrease in its affinity for oxygen (see p. 28) and a right shift in the oxygen dissociation. In the lungs, COs dissociates from the hemoglobin, and is released in the breath.
. Binding of CO: Carbon monoxide (CO) binds tightly (but reversibly) to the hemoglobin iron, forming carbon monoxyhemoglobin (or carboxyhemoglobin). When CO binds to one or more of the four heme sites, hemoglobin shifts to the relaxed conformation, causing the remaining heme sites to bind oxygen with high affinity. This shifts the oxygen dissociation curve to the left, and changes the normal sigmoidal shape toward a hyperbola. As a result, the affected hemoglobin is unable to release oxygen to the tissues (Figure 3.12). [Note: The affinity of hemoglobin for CO is 220 times greater than for oxygen. Consequently, even minute concentrations of CO in the environment can produce toxic concentrations of carbon monoxyhemoglobin in the blood. For exam-
ple, increased levels of CO are found in the blood of tobacco smokers. Carbon monoxide toxicity appears to result from a combination of tissue hypoxia and direct CO-mediated damage at the cellular level.] Carbon monoxide poisoning is treated with 100% oxygen at high pressure (hyperbaric oxygen therapy), which facilitates the dissociation of CO from the hemoglobin. [Note: CO inhibits Complex IV of the electron transport chain (see p. 76).] In addition to O2, COz, and CO, nitric oxide gas (NO) also is carried by hemoglobin. NO is a potent vasodilator (see p. 151). It can be taken up (salvaged) or released from RBCs, thus modulating NO availability and influencing vessel diameter.

It is important to remember that human hemoglobin A (Hb A) is just one member of a functionally and structurally related family of proteins, the hemoglobins (Figure 3.13). Each of these oxygen-carrying proteins is a tetramer, composed of two a-globin polypeptides and two B-globin (or B-globin-like) polypeptides. Certain hemoglobins, such as Hb F, are normally synthesized only during fetal development, whereas others, such as Hb Ag, are synthesized in the adult, although at low levels compared with Hb A. Hb A can also become modified by the covalent addition of a hexose. For example, addition of glucose forms the glucosylated hemoglobin derivative, Hb Aic.
1. Fetal hemoglobin (Hb F): Hb F is a tetramer consisting of two a chains identical to those found in Hb A, plus two y chains (a2y2, see Figure 3.13). The y chains are members of the B-globin gene family (see p. 35).
a. Hb F synthesis during development: In the first month after conception, embryonic hemoglobins such as Hb Gower 1, composed of two a-like zeta (¢) chains and two f-like epsilon (e) chains (Cze2), are synthesized by the embryonic yolk sac. In the fifth week of gestation, the site of globin synthesis shifts, first to the liver and then to the marrow, and the primary product is Hb F. Hb F is the major hemoglobin found in the fetus and newborn, accounting for about 60% of the total hemoglobin in the erythrocytes during the last months of fetal life (Figure 3.14). Hb A synthesis starts in the bone marrow at about the eighth month of pregnancy and gradually replaces Hb F. (Figure 3.14 shows the relative production of each type of hemoglobin chain during fetal and postnatal life.) [Note: Hb F represents less than 1% of the Hb in most adults, and is concentrated in RBCs known as F-cells.]

b. Binding of 2,3-BPG to Hb F: Under physiologic conditions, Hb F has a higher affinity for oxygen than does Hb A, as a result of Hb F binding only weakly to 2,3-BPG. [Note: The y-globin chains of Hb F lack some of the positively charged amino acids that are responsible for binding 2,3-BPG in the B-globin chains.] Because 2,3-BPG serves to reduce the affinity of hemoglobin for oxygen, the weaker interaction between 2,3-BPG and Hb F results in a higher oxygen affinity for Hb F relative to Hb A. In contrast, if both Hb A and Hb F are stripped of their 2,3-BPG, they then have a similar affinity for oxygen.

Normal adult human hemoglobins. [Note: The a-chains in these hemoglobins are identical.]

Nonenzymic addition of glucose to hemoglobin.
The higher oxygen affinity of Hb F facilitates the transfer of oxygen from the maternal circulation across the placenta to the RBCs of the fetus.
2. Hemoglobin Az (Hb Az): Hb Az is a minor component of normal adult hemoglobin, first appearing shortly before birth and, ultimately, constituting about 2% of the total hemoglobin. It is composed of two a-globin chains and two 6-globin chains (252, see Figure 3.13).
3. Hemoglobin Aji(HbAj,): Under physiologic conditions, Hb A is slowly and nonenzymically glycosylated, the extent of glycosylation being dependent on the plasma concentration of a particular hexose. The most abundant form of glycosylated hemoglobin is Hb Aic. It has glucose residues attached predominantly to the NH2 groups of the N-terminal valines of the B-globin chains (Figure 3.15). Increased amounts of Hb Aj, are found in RBCs of patients with diabetes mellitus, because their Hb A has contact with higher glucose concentrations during the 120-day lifetime of these cells. (See p. 340 for a discussion of the use of this phenomenon in assessing average blood glucose levels in persons with diabetes.)

To understand diseases resulting from genetic alterations in the structure or synthesis of hemoglobins, it is necessary to grasp how the hemoglobin genes, which direct the synthesis of the different globin chains, are structurally organized into gene families and also how they are expressed.

The genes coding for the o-globin-like and B-globin-like subunits of the hemoglobin chains occur in two separate gene clusters (or families) located on two different chromosomes (Figure 3.16). The a-gene cluster on chromosome 16 contains two genes for the a-globin chains. It also contains the ¢ gene that is expressed early in development as a component of embryonic hemoblobin. [Note: Globin gene famillies also contain globin-like genes that are not expressed (that is, their genetic information is not used to produce globin chains). These are called pseudogenes. ]

A single gene for the B-globin chain is located on chromosome 11 (see Figure 3.16). There are an additional four B-globin-like genes: the ¢ gene (which, like the ¢ gene, is expressed early in embryonic development), two y genes (Gy, and A, that are expressed in Hb F), and the 5 gene that codes for the globin chain found in the minor adult hemoglobin Hb Az.

Expression of a globin gene begins in the nucleus of red cell precursors, where the DNA sequence encoding the gene is transcribed. The RNA produced by transcription is actually a precursor of the messenger RNA (mRNA) that is used as a template for the synthesis of a globin chain. Before it can serve this function, two noncoding stretches of RNA (introns) must be removed from the mRNA precursor sequence, and the remaining three fragments (exons) joined in a linear manner. The resulting mature mRNA enters the cytosol, where its genetic information is translated, producing a globin chain. (A summary of this process is shown in Figure 3.17. A more detailed description of protein synthesis is presented in Chapter 31, p. 431.)

Hemoglobinopathies have traditionally been defined as a family of genetic disorders caused by production of a structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin, or, rarely, both. Sickle cell anemia (Hb S), hemoglobin C disease (Hb C), hemoglobin SC disease (Hb S + Hb C), and the thalassemia syndromes are representative hemoglobinopathies that can have severe clinical consequences. The first three conditions result from production of hemoglobin with an altered amino acid sequence (qualitative hemoglobinopathy), whereas the thalassemias are caused by decreased production of normal hemoglobin (quantitative hemoglobinopathy).

Sickle cell anemia, the most common of the red cell sickling diseases, is a genetic disorder of the blood caused by a single nucleotide alteration (a point mutation) in the gene for B-globin. It is the most common inherited blood disorder in the United States, affecting 80,000 Americans. It occurs primarily in the AfricanAmerican population, affecting one of 500 newborn African-American infants in the United States. Sickle cell anemia is a homozygous, recessive disorder. It occurs in individuals who have inherited two mutant genes (one from each parent) that code for synthesis of the B chains of the globin molecules. [Note: The mutant B-globin chain is designated BS, and the resulting hemoglobin, 285s, is referred to as Hb S.] An infant does not begin showing symptoms of the disease until sufficient Hb F has been replaced by Hb S so that sickling can occur (see below). Sickle cell anemia is characterized by lifelong episodes of pain ("crises"), chronic hemolytic anemia with associated hyperbilirubinemia (see p. 284), and increased susceptibility to infections, usually beginning in early childhood. [Note: The lifetime of an

Diagram of hemoglobins A, S, and C after electrophoresis.
erythrocyte in sickle cell anemia is less than 20 days, compared with 120 days for normal RBCs; hence, the anemia.] Other symptoms include acute chest syndrome, stroke, splenic and renal dysfunction, and bone changes due to marrow hyperplasia. Heterozygotes, representing 1 in 12 African-Americans, have one normal and one sickle cell gene. The blood cells of such heterozygotes contain both Hb S and Hb A. These individuals have sickle cell trait. They usually do not show clinical symptoms and can have a normal life span.
1.
Amino acid substitution in Hb S B chains: A molecule of Hb S contains two normal a-globin chains and two mutant B-globin chains (BS), in which glutamate at position six has been replaced with valine (Figure 3.18). Therefore, during electrophoresis at alkaline pH, Hb S migrates more slowly toward the anode (positive electrode) than does Hb A (Figure 3.19). This altered mobility of Hb S is a result of the absence of the negatively charged glutamate residues in the two B chains, thus rendering Hb S less negative than Hb A. [Note: Electrophoresis of hemoglobin obtained from lysed RBCs is routinely used in the diagnosis of sickle cell trait and sickle cell disease.]
2.

Sickling and tissue anoxia: The replacement of the charged glutamate with the nonpolar valine forms a protrusion on the B-globin that fits into a complementary site on the B chain of another hemoglobin molecule in the cell (Figure 3.20). At low oxygen tension, deoxyhemoglobin S polymerizes inside the RBC, forming a network of fibrous polymers that stiffen and distort the cell, producing rigid, misshapen erythrocytes. Such sickled cells frequently block the flow of blood in the narrow capillaries. This interruption in the supply of oxygen leads to localized anoxia (oxygen deprivation) in the tissue, causing pain and eventually death (infarction) of cells in the vicinity of the blockage. The anoxia also leads to an increase in deoxygenated Hb S. [Note: The mean diameter of RBCs is 7.5 um, whereas that of the microvasculature is 3-4 um. Instead of squeezing through the microvasculature like Hb A—containing RBCs, sickled cells have a decreased ability to deform and an increased tendency to adhere to vessel walls, and so have difficulty moving through small vessels.]
- 3. Variables that increase sickling: The extent of sickling and, therefore, the severity of disease is enhanced by any variable that increases the proportion of Hb S in the deoxy state (that is, reduces the affinity of Hb S for oxygen). These variables include decreased oxygen tension as a result of high altitudes or flying ina nonpressurized plane, increased pCOz, decreased pH, dehydration, and an increased concentration of 2,3-BPG in erythrocytes.
4. Treatment: Therapy involves adequate hydration, analgesics, aggressive antibiotic therapy if infection is present, and transfusions in patients at high risk for fatal occlusion of blood vessels. Intermittent transfusions with packed red cells reduce the risk of stroke, but the benefits must be weighed against the complications of transfusion, which include iron overload (hemosiderosis), bloodborne infections, and immunologic complications. Hydroxyurea, an antitumor drug, is therapeutically useful because it increases circulating levels of Hb F, which decreases RBC sickling. This leads to decreased frequency of painful crises and reduces mortality.
5.

Possible selective advantage of the heterozygous state: The high frequency of the BS mutation among black Africans, despite its damaging effects in the homozygous state, suggests that a selective advantage exists for heterozygous individuals. For example, heterozygotes for the sickle cell gene are less susceptible to malaria, caused by the parasite Plasmodium falciparum. This organism spends an obligatory part of its life cycle in the RBC. One theory is that because these cells in individuals heterozygous for Hb S, like those in homozygotes, have a shorter life span than normal, the parasite cannot complete the intracellular stage of its development. This fact may provide a selective advantage to heterozygotes living in regions where malaria is a major cause of death. Figure 3.21 illustrates that in Africa, the geographic distribution of sickle cell anemia is similar to that of malaria. The morbidity and mortality associated with sickle cell anemia has led to its inclusion in newborn screening panels to allow prophylactic antibiotic therapy to begin soon after the birth of an affected child.

Like Hb S, Hb C is a hemoglobin variant that has a single amino acid substitution in the sixth position of the B-globin chain (see Figure 3.18). In this case, however, a lysine is substituted for the glutamate (as compared with a valine substitution in Hb S). [Note: This substitution causes Hb C to move more slowly toward the anode than Hb A or Hb S does (see Figure 3.19).] Patients homozygous for hemoglobin C generally have a relatively mild, chronic hemolytic anemia. These patients do not suffer from infarctive crises, and no specific therapy is required.

. ; ; a . Hemoglobin SC disease is another of the red cell sickling diseases. In this disease, some B-globin chains have the sickle cell mutation, whereas other B-globin chains carry the mutation found in Hb C disease. [Note: Patients with Hb SC disease are doubly hetero-

A. Distribution of sickle cell in
Africa expressed as a percentage
of the population with disease.
B. Distribution of malaria
in Africa.

A. B-Globin gene mutations in the B-thalassemias. B. Hemoglobin tetramers formed in B-thalassemias.
zygous (compound heterozygote) because both of their B-globin genes are abnormal, although different from each other.] Hemoglobin levels tend to be higher in Hb SC disease than in sickle cell anemia, and may even be at the low end of the normal range. The clinical course of adults with Hb SC anemia differs from that of sickle cell anemia in that symptoms such as painful crises are less frequent and less severe; however, there is significant clinical variability.

Oxidation of the heme component of hemoglobin to the ferric (Fe**) state forms methemoglobin, which cannot bind oxygen. This oxidation may be caused by the action of certain drugs, such as nitrates, or endogenous products, such as reactive oxygen intermediates (see p. 148). The oxidation may also result from inherited defects, for example, certain mutations in the aor B-globin chain promote the formation of methemoglobin (Hb M). Furthermore, a deficiency of NADH-cytochrome bs reductase (also called NADH-methemoglobin reductase), the enzyme responsible for the conversion of methemoglobin (Fe**) to hemoglobin (Fe**), leads to the accumulation of methemoglobin. [Note: The erythrocytes of newborns have approximately half the capacity of those of adults to reduce methemoglobin. They are therefore particularly susceptible to the effects of methemoglobin-producing compounds.] The methemoglobinemias are characterized by "chocolate cyanosis" (a brownish-blue coloration of the skin and mucous membranes) and chocolate-colored blood, as a result of the dark-colored methemoglobin. Symptoms are related to the degree of tissue hypoxia, and include anxiety, headache, and dyspnea. In rare cases, coma and death can occur. Treatment is with methylene blue, which is oxidized as Fet? is reduced.

The thalassemias are hereditary hemolytic diseases in which an imbalance occurs in the synthesis of globin chains. As a group, they are the most common single gene disorders in humans. Normally, synthesis of the aand B-globin chains is coordinated, so that each a-globin chain has a B-globin chain partner. This leads to the formation of O22 (Hb A). In the thalassemias, the synthesis of either the oor the B-globin chain is defective. A thalassemia can be caused by a variety of mutations, including entire gene deletions, or substitutions or deletions of one to many nucleotides in the DNA. [Note: Each thalassemia can be classified as either a disorder in which no globin chains are produced («°-or B°-thalassemia), or one in which some chains are synthesized, but at a reduced level (c*or B*-thalassemia).]
1.
B-Thalassemias: In these disorders, synthesis of B-globin chains is decreased or absent, typically as a result of point mutations that affect the production of functional mRNA; however, a-globin chain synthesis is normal. «-Globin chains cannot form stable tetramers and, therefore, precipitate, causing the premature death of cells initially destined to become mature RBCs. Increase in azy2 (Hb F) and ode (Hb Az) also occurs. There are only two copies of the B-globin gene in each cell (one on each chromosome 11). Therefore, individuals with B-globin gene defects have either B-thalassemia trait (6-thalassemia minor) if they have only one defective B-globin gene, or B-thalassemia major (Cooley anemia)

if both genes are defective (Figure 3.22). Because the B-globin gene is not expressed until late in fetal gestation, the physical manifestations of B-thalassemias appear only several months after birth. Those individuals with B-thalassemia minor make some £ chains, and usually do not require specific treatment. However, those infants born with B-thalassemia major are seemingly healthy at birth, but become severely anemic, usually during the first or second year of life due to ineffective erythropoiesis. Skeletal changes as a result of extramedullary hematopoiesis also are seen.These patients require regular transfusions of blood. [Note: Although this treatment is lifesaving, the cumulative effect of the transfusions is iron overload (a syndrome known as hemosiderosis). Use of iron chelation therapy has improved morbidity and mortality.] The increasing use of bone marrow replacement therapy has been a boon to these patients.
2. a-Thalassemias: These are defects in which the synthesis of a-globin chains is decreased or absent, typically as a result of deletional mutations. Because each individual's genome contains four copies of the a-globin gene (two on each chromosome 16), there are several levels of a-globin chain deficiencies (Figure 3.23). If one of the four genes is defective, the individual is termed a silent carrier of a-thalassemia, because no physical manifestations of the disease occur. If two a-globin genes are defective, the individual is designated as having a-thalassemia trait. If three a-globin genes are defective, the individual has Hb H (B4) disease—a mildly to moderately severe hemolytic anemia. If all four a-globin genes are defective, Hb Bart (v4) disease with hydrops fetalis and fetal death results, because a-globin chains are required for the synthesis of Hb F. [Note: Hemoglobinopathies that result in increased Oo affinity typically are characterized by increased production of RBCs, whereas those that result in decreased Oz affinity are characterized by anemia.]

Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide chains (two o chains and two B chains, ae) held together by noncovalent interactions (Figure 3.24). The subunits occupy different relative positions in deoxyhemoglobin compared with oxyhemoglobin. The deoxy form of hemoglobin is called the "T," or taut (tense) form. It has a constrained structure that limits the movement of the polypeptide chains. The T form is the low-oxygen-affinity form of hemoglobin. The binding of oxygen to hemoglobin causes rupture of some of the ionic and hydrogen bonds. This leads to a structure called the "R," or relaxed form, in which the polypeptide chains have more freedom of movement. The R form is the high-oxygen-affinity form of hemoglobin. The oxygen dissociation curve for hemoglobin is sigmoidal in shape (in contrast to that of myoglobin, which is hyperbolic), indicating that the subunits cooperate in binding oxygen. Cooperative binding of oxygen by the four subunits of hemoglobin means that the binding of an oxygen molecule at one heme group increases the oxygen affinity of the remaining heme groups in the same hemoglobin molecule. Hemoglobin's ability to bind

A.
a-Globin gene deletions in the a-thalassemias. B. Hemoglobin
tetramers formed in a-thalassemias.
oxygen reversibly is affected by the pO (through heme-heme interactions), the pH of the environment, the pCOz, and the availability of 2,3-bisphosphoglycerate (2,3-BPG). For example, the release of O2 from Hb is enhanced when the pH is lowered or the pCOz is increased (the Bohr effect), such as in exercising muscle, and the oxygen dissociation curve of Hb is shifted to the right. To cope long-term with the effects of chronic hypoxia or anemia, the concentration of 2,3-BPG in RBCs increases. 2,3-BPG binds to the Hb and decreases its oxygen affinity, and it, therefore, also shifts the oxygen-dissociation curve to the right. Carbon monoxide (CO) binds tightly (but reversibly) to the hemoglobin iron, forming carbon monoxyhemoglobin (Hb CO). Hemoglobinopathies are disorders caused either by production of a structurally abnormal hemoglobin molecule, synthesis of insufficient quantities of normal hemoglobin subunits, or, rarely, both (Figure 3.25). The sickling diseases sickle cell anemia (Hb S disease) and hemoglobin SC disease, as well as hemoglobin C disease and the thalassemia syndromes are representative hemoglobinopathies that can have severe clinical consequences.

Choose the ONE correct answer.
3.1
Which one of the following statements concerning the hemoglobins is correct?
A.
Fetal blood has a higher affinity for oxygen than does adult blood because Hb F has a decreased affinity for 2,3-BPG.
B. Purified Hb F (stripped of 2,3-BPG) has a higher affinity for oxygen than does purified Hb A.
C. The globin chain composition of Hb F is o282.
D. Hb Ai¢ differs from Hb A by a single, genetically determined amino acid substitution.
E. Hb Ao appears early in fetal life.
3.2 Which one of the following statements concerning the ability of acidosis to precipitate a crisis in sickle cell anemia is correct?
A. Acidosis decreases the solubility of Hb S.
B. Acidosis increases the affinity of hemoglobin for oxygen.
C. Acidosis favors the conversion of hemoglobin from the taut to the relaxed conformation.
D. Acidosis shifts the oxygen dissociation curve to the left.
E. Acidosis decreases the ability of 2,3-BPG to bind to hemoglobin.
3.3 Which one of the following statements concerning the of is correct?
binding oxygen by hemoglobin
- A. The Bohr effect results in a lower affinity for oxygen at
higher pH values.
- B. Carbon dioxide increases the oxygen affinity of hemoglobin by binding to the C-terminal groups of the chains.
polypeptide
C. The oxygen affinity of hemoglobin increases as the percentage saturation increases.
D. The hemoglobin tetramer binds four molecules of 2,3BPG.
E. Oxyhemoglobin and deoxyhemoglobin have the same affinity for protons (H*).

Correct answer = A. Because 2,3-BPG reduces the affinity of hemoglobin for oxygen, the weaker interaction between 2,3-BPG and Hb F results in a higher oxygen affinity for Hb F relative to Hb A. In contrast, if both Hb A and Hb F are stripped of 2,3-BPG, they have a similar affinity for oxygen. Hb F consists of ogy2. Hb Aic is a glycosylated form of Hb A, formed nonenzymically in red cells. Hb Az is a minor component of normal adult hemoglobin, first appearing shortly before birth and rising to adult levels (about 2% of the total hemoglobin) by 6 months of age.
Correct answer = A. Hb S is significantly less soluble in the deoxygenated form, compared with oxyhemoglobin S. A decrease in pH (acidosis) causes the oxygen dissociation curve to shift to the right, indicating a decreased affinity for oxygen. This favors the formation of the deoxy, or taut, form of hemoglobin, and can precipitate a sickle cell crisis. The binding of 2,3BPG is increased, because it binds only to the deoxy form of hemoglobins.
Correct answer = C. The binding of oxygen at one heme group increases the oxygen affinity of the remaining heme groups in the same molecule. Carbon dioxide decreases oxygen affinity because it lowers the pH; moreover, binding of carbon dioxide to the N-termini stabilizes the taut, deoxy form. Hemoglobin binds one molecule of 2,3-BPG. Deoxyhemoglobin has a greater affinity for protons and, therefore, is a weaker acid.

3.4 B-Lysine 82 in hemoglobin A is important for the bind-
ing of 2,3-BPG. In Hb Helsinki, this amino acid has been replaced by methionine. Which of the following should be true Hb Helsinki?
concerning
A. It should be stabilized in the T, rather than the R, form.
Op conse-
B. It should have increased affinity and, quently, decreased delivery of Oz to tissues.
C. Its Oz dissociation curve should be shifted to the right relative to Hb A.
D. It results in anemia.
Correct answer is B. Substitution of lysine by methionine decreases the ability of negatively charged phosphate groups in 2,3-BPG to bind the B subunits of hemoglobin. Because 2,3BPG decreases the Op affinity of hemoglobin, a reduction in 2,3-BPG should result in increased Oz affinity and decreased delivery of Oz to tissues. The R form is the high-oxygen-affinity form of hemoglobin. Increased Ob» affinity (decreased delivery) results in a left shift in the Oz dissociation curve. Decreased Oz delivery is compensated for by increased RBC production.

3.5 A 67-year-old man presented to the emergency depart- ment
with
a
1
week history
of angina
and shortness
of breath.
He
a
“blue angina
complained color.”
that
His medical
treated with
his face
and history
isosorbide extremities
included dinitrate
had chronic
stable and
nitroglycerin.
Blood obtained
for analysis
was chocolate-colored.
Which one of the following is the most likely diagnosis?
A. Sickle cell anemia
B. Carboxyhemoglobinemia
C. Methemoglobinemia.
D. B-Thalassemia
E. Hemoglobin SC disease
Correct answer = C. Oxidation of the heme component of hemoglobin to the ferric (Fe**) state forms methemoglobin. This may be caused by the action of certain drugs, such as nitrates. The methemoglobinemias are characterized by chocolate cyanosis (a brownish-blue coloration of the skin and mucous membranes), and chocolate-colored blood as a result of the dark-colored methemoglobin. Symptoms are related to tissue hypoxia, and include anxiety, headache, dyspnea. In rare cases, coma and death can occur.

Collagen and elastin are examples of common, well-characterized fibrous proteins of the extracellular matrix that serve structural functions in the body. For example, collagen and elastin are found as components of skin, connective tissue, blood vessel walls, and sclera and cornea of the eye. Each fibrous protein exhibits special mechanical properties, resulting from its unique structure, which are obtained by combining specific amino acids into regular, secondary structural elements. This is in contrast to globular proteins, whose shapes are the result of complex interactions between secondary, tertiary, and, sometimes, quaternary structural elements.

Collagen is the most abundant protein in the human body. A typical collagen molecule is a long, rigid structure in which three polypeptides (referred to as "a chains") are wound around one another in a rope-like triple helix (Figure 4.1). Although these molecules are found throughout the body, their types and organization are dictated by the structural role collagen plays in a particular organ. In some tissues, collagen may be dispersed as a gel that gives support to the structure, as in the extracellular matrix or the vitreous humor of the eye. In other tissues, collagen may be bundled in tight, parallel fibers that provide great strength, as in tendons. In the cornea of the eye, collagen is stacked so as to transmit light with a minimum of scattering. Collagen of bone occurs as fibers arranged at an angle to each other so as to resist mechanical shear from any direction.

The collagen superfamily of proteins includes more than 25 collagen types, as well as additional proteins that have collagen-like domains. The three polypeptide a chains are held together by hydrogen bonds between the chains. Variations in the amino acid sequence of the a. chains result in structural components that are about the same size (approximately 1,000 amino acids long), but with slightly different properties. These « chains are combined to form the various types of collagen found in the tissues. For example, the most common collagen, type I, contains two chains called «1 and one chain called a2 (1202), whereas type II collagen contains three «1 chains (013). The

Triple-stranded helix of collagen.
TYPE |, 1 = TISSUE DISTRIBUTION. , 1 = Skin, bone, tendon, blood vessels, cornea. , 1 = Cartilage, intervertebral disk, vitreous body. , 1 = Blood vessels, fetal skin. , 1 = Network-formin'. IV, 1 = Basement membrane. Vil, 1 = Beneath stratified squamous epithelia. , 1 = Fibril-associated. Ix, 1 = Cartilage. Xxil, 1 = Tendon, ligaments, some other tissues

The most abundant types of collagen.
collagens can be organized into three groups, based on their location and functions in the body (Figure 4.2).
1. Fibril-forming collagens: Types |, II, and Ill are the fibrillar collagens, and have the rope-like structure described above for a typical collagen molecule. In the electron microscope, these linear polymers of fibrils have characteristic banding patterns, reflecting the regular staggered packing of the individual collagen molecules in the fibril (Figure 4.3). Type | collagen fibers are found in supporting elements of high tensile strength (for example, tendon and cornea), whereas fibers formed from type II collagen molecules are restricted to cartilaginous structures. The fibers derived from type III collagen are prevalent in more distensible tissues, such as blood vessels.
2. Network-forming collagens: Types IV and VII form a three-dimensional mesh, rather than distinct fibrils (Figure 4.4). For example, type IV molecules assemble into a sheet or meshwork that constitutes a major part of basement membranes.
Basement membranes are thin, sheet-like structures that provide mechanical support for adjacent cells, and function as a semipermeable filtration barrier to macromolecules in organs such as the kidney and the lung.
3. Fibril-associated collagens: Types IX and XII bind to the surface of collagen fibrils, linking these fibrils to one another and to other components in the extracellular matrix (see Figure 4.2).

Collagen fibrils at right have a characteristic banding pattern, reflecting the regularly staggered packing of the individual collagen molecules in the fibril.

1. Amino acid sequence: Collagen is rich in proline and glycine, both of which are important in the formation of the triple-stranded helix. Proline facilitates the formation of the helical conformation of each a chain because its ring structure causes "kinks" in the peptide chain. [Note: The presence of proline dictates that the helical conformation of the a chain cannot be an @ helix.] Glycine, the smallest amino acid, is found in every third position of the polypeptide chain. It fits into the restricted spaces where the three chains of the helix come together. The glycine residues are part of a repeating sequence, —Gly—-X—Y-, where X is frequently proline and Y is often hydroxyproline (but can be hydroxylysine, Figure 4.5). Thus, most of the a chain can be regarded as a polytripeptide whose sequence can be represented as (—Gly—Pro—Hyp-—)333.
2. Triple-helical structure: Unlike most globular proteins that are folded into compact structures, collagen, a fibrous protein, has an elongated, triple-helical structure that places many of its amino acid side chains on the surface of the triple-helical molecule. [Note: This allows bond formation between the exposed R-groups of neighboring collagen monomers, resulting in their aggregation into long fibers.]
3. Hydroxyproline and hydroxylysine: Collagen contains hydroxyproline (hyp) and hydroxylysine (hyl), which are not present in most other proteins. These residues result from the hydroxylation of some of the proline and lysine residues after their incorporation into polypeptide chains (Figure 4.6). The hydroxylation is, thus, an example of posttranslational modification (see p. 443). Hydroxyproline is important in stabilizing the triple-helical structure of collagen because it maximizes interchain hydrogen bond formation.
4. Glycosylation: The hydroxyl group of the hydroxylysine residues of collagen may be enzymatically glycosylated. Most commonly, glucose and galactose are sequentially attached to the polypeptide chain prior to triple-helix formation (Figure 4.7).

The polypeptide precursors of the collagen molecule are formed in fibroblasts (or in the related osteoblasts of bone and chondroblasts of cartilage), and are secreted into the extracellular matrix. After enzymic modification, the mature collagen monomers aggregate and become cross-linked to form collagen fibers.
1.
- Formation of pro-a chains: Collagen is one of many proteins that normally function outside of cells. Like most proteins produced for export, the newly synthesized polypeptide precursors of « chains (prepro-a chains) contain a special amino acid sequence at their N-terminal ends. This sequence acts as a signal that, in the absence of additional signals, targets the polypeptide being synthesized for secretion from the cell. The signal sequence facilitates the binding of ribosomes to the rough endoplasmic reticulum (RER), and directs the passage of the prepro-a chain into the lumen of the RER. The signal sequence is rapidly cleaved in the RER to yield a precursor of collagen called a pro-a chain (see Figure 4.7).

Electron micrograph of a polygonal network formed by association of collagen type IV monomers.

Amino acid sequence of a portion of the a1 chain of collagen. [Note: Hyp is hydroxyproline and Hyl is hydroxylysine.]

Hydroxylation of prolyl residues of pro-a chains of collagen by prolyl hydroxylase.

Synthesis of collagen. RER = rough endoplasmic reticulum. (Continued on the next page)
Synthesis of collagen. (Continued from the previous page)

2. Hydroxylation: The pro-« chains are processed by a number of enzymic steps within the lumen of the RER while the polypeptides are still being synthesized (see Figure 4.7). Proline and lysine residues found in the Y-position of the —Gly-X—-Y— sequence can be hydroxylated to form hydroxyproline and hydroxylysine residues. These hydroxylation reactions require molecular oxygen, Fe?*, and the reducing agent vitamin C (ascorbic acid, see p. 377), without which the hydroxylating enzymes, prolyl hydroxylase and lysyl hydroxylase, are unable to function (see Figure 4.6). In the case of ascorbic acid deficiency (and, therefore, a lack of prolyl and lysyl hydroxylation), interchain H-bond formation is impaired, as is formation of a stable triple helix. Additionally, collagen fibrils cannot be cross-linked (see below), greatly decreasing the tensile strength of the assembled fiber. The resulting deficiency disease is known as scurvy. Patients with ascorbic acid deficiency also often show bruises on the limbs as a result of subcutaneous extravasation of blood due to capillary fragility (Figure 4.8).
3. Glycosylation: Some hydroxylysine residues are modified by glycosylation with glucose or glucosyl-galactose (see Figure 4.7).
4. Assembly and secretion: After hydroxylation and glycosylation, pro-a chains form procollagen, a precursor of collagen that has a central region of triple helix flanked by the nonhelical aminoand carboxyl-terminal extensions called propeptides (see Figure 4.7). The formation of procollagen begins with formation of interchain disulfide bonds between the C-terminal extensions of the pro-a chains. This brings the three « chains into an alignment favorable for helix formation. The procollagen molecules move through the Golgi apparatus, where they are packaged in secretory vesicles. The vesicles fuse with the cell membrane, causing the release of procollagen molecules into the extracellular space.

5. Extracellular cleavage of procollagen molecules: After their release, the procollagen molecules are cleaved by Nand C-procollagen peptidases, which remove the terminal propeptides, releasing triple-helical tropocollagen molecules.

Formation of cross-links in collagen.
Figure 4.10 Stretchy skin of Ehlers-Danlos syndrome.
6. Formation of collagen fibrils: Individual tropocollagen molecules spontaneously associate to form collagen fibrils. They form an ordered, overlapping, parallel array, with adjacent collagen molecules arranged in a staggered pattern, each overlapping its neighbor by a length approximately three-quarters of a molecule (see Figure 4.7).
7.
Cross-link formation: The fibrillar array of collagen molecules serves as a substrate for /ysy! oxidase. This Cu**-containing extracellular enzyme oxidatively deaminates some of the lysyl and hydroxylysyl residues in collagen. The reactive aldehydes that result (allysine and hydroxyallysine) can condense with lysyl or hydroxylysyl residues in neighboring collagen molecules to form covalent cross-links and, thus, mature collagen fibers (Figure 4.9).
|
enzymes. Others include cytochrome oxidase (see p. 76), dopamine hydroxylase (see p. 286), superoxide dismutase (see p.148) and tyrosinase (see p. 273). Disruption in copper homeostasis causes copper deficiency (X-linked Menkes disLysyl oxidase is one of several copper-containing ease) or overload (Wilson disease).

Normal collagens are highly stable molecules, having half-lives as long as several years. However, connective tissue is dynamic and is constantly being remodeled, often in response to growth or injury of the tissue. Breakdown of collagen fibers is dependent on the proteolytic action of collagenases, which are part of a large family of matrix metalloproteinases. For type | collagen, the cleavage site is specific, generating three-quarter and one-quarter length fragments. These fragments are further degraded by other matrix proteinases to their constituent amino acids.

Defects in any one of the many steps in collagen fiber synthesis can result in a genetic disease involving an inability of collagen to form fibers properly and, thus, provide tissues with the needed tensile strength normally provided by collagen. More than 1,000 mutations have been identified in 22 genes coding for 12 of the collagen types. The following are examples of diseases that are the result of defective collagen synthesis.
1.
Ehlers-Danlos syndrome (EDS): This disorder is a heterogeneous group of generalized connective tissue disorders that result from inheritable defects in the metabolism of fibrillar collagen molecules. EDS can result from a deficiency of collagen-processing enzymes (for example, /ysyl hydroxylase or procollagen peptidase), or from mutations in the amino acid sequences of collagen types |, Ill, or V. The most clinically important mutations are found in the gene for type III collagen. Collagen containing mutant chains is not secreted, and is either degraded or accumulated to high levels in intracellular compartments. Because collagen type III is an important component of the arteries, potentially lethal vascular problems
occur. [Note: Although collagen type III is only a minor component of the collagen fibrils in the skin, patients with EDS also show, for unknown reasons, defects in collagen type | fibrils. This results in fragile, stretchy skin and loose joints (Figure 4.10).]
2.

Osteogenesis imperfecta (Ol): This disease, known as brittle bone syndrome, is also a heterogeneous group of inherited disorders distinguished by bones that easily bend and fracture (Figure 4.11). Retarded wound healing and a rotated and twisted spine leading to a "humped-back" (kyphotic) appearance are common features of the disease. Type | Ol is called osteogenesis imperfecta tarda. The disease is the consequence of decreased production of «1 and a2 chains. It presents in early infancy with fractures secondary to minor trauma, and may be suspected if prenatal ultrasound detects bowing or fractures of long bones. Type II Ol is called osteogenesis imperfecta congenita, and is the most severe. Patients die of pulmonary hypoplasia in utero or during the neonatal period. Most patients with severe Ol have mutations in the gene for either the pro-a1 or pro-c2 chains of type | collagen. The most common mutations cause the replacement of glycine residues (in —Gly—-X—Y-) by amino acids with bulky side chains. The resultant structurally abnormal pro-a chains prevent the formation of the required triple-helical conformation.

In contrast to collagen, which forms fibers that are tough and have high tensile strength, elastin is a connective tissue protein with rubber-like properties. Elastic fibers composed of elastin and glycoprotein microfibrils are found in the lungs, the walls of large arteries, and elastic ligaments. They can be stretched to several times their normal length, but recoil to their original shape when the stretching force is relaxed.

Elastin is an insoluble protein polymer synthesized from a precursor, tropoelastin, which is a linear polypeptide composed of about 700 amino acids that are primarily small and nonpolar (for example, glycine, alanine, and valine). Elastin is also rich in proline and lysine, but contains only a little hydroxyproline and hydroxylysine. Tropoelastin is secreted by the cell into the extracellular space. There it interacts with specific glycoprotein microfibrils, such as fibrillin, which function as a scaffold onto which tropoelastin is deposited. Some of the lysyl side chains of the tropoelastin polypeptides are oxidatively deaminated by lysy/ oxidase, forming allysine residues. Three of the allysyl side chains plus one unaltered lysyl side chain from the same or neighboring polypeptides form a desmosine cross-link (Figure 4.12). This produces elastin—an extensively interconnected, rubbery network that can stretch and bend in any direction when stressed, giving connective tissue elasticity (Figure 4.13). Mutations in the fibrillin-1 protein are responsible for Marfan syndrome—a connective tissue disorder characterized by impaired structural integrity in the skeleton, the eye, and the cardiovascular system. With this disease, abnormal fibrillin protein is incorporated into microfibrils along with normal fibrillin, inhibiting the formation of functional microfibrils. [Note: Patients with Ol, EDS, or Marfan syndrome may have blue sclera due to tissue thinning that allows underlying pigment to show through.]

Lethal form (type II) of osteogenesis imperfecta in which the fractures appear in utero, as revealed by this radiograph of a stillborn fetus.

Destruction of alveolar tissue by elastase released from neutrophils activated as part of the immune response to airborne pathogens.

1. o1-Antitrypsin: Blood and other body fluids contain a protein, a4-antitrypsin (o4-AT, A1AT, currently also called o-antiproteinase), that inhibits a number of proteolytic enzymes (also called proteases or proteinases) that hydrolyze and destroy proteins. [Note: The inhibitor was originally named a-antitrypsin because it inhibits the activity of trypsin (a proteolytic enzyme synthesized as trypsinogen by the pancreas, see p. 248.] o1-AT comprises more than 90% of the o4-globulin fraction of normal plasma. o1-AT has the important physiologic role of inhibiting neutrophil elastase—a powerful protease that is released into the extracellular space, and degrades elastin of alveolar walls, as well as other structural proteins in a variety of tissues (Figure 4.14). Most of the a4-AT found in plasma is synthesized and secreted by the liver. The remainder is synthesized by several tissues, including monocytes and alveolar macrophages, which may be important in the prevention of local tissue injury by elastase.
. Role of a-AT in the lungs: In the normal lung, the alveoli are chronically exposed to low levels of neutrophil elastase released from activated and degenerating neutrophils. This proteolytic activity can destroy the elastin in alveolar walls if unopposed by the action of a-AT, the most important inhibitor of neutrophil e/astase (see Figure 4.14). Because lung tissue cannot regenerate, emphysema results from the destruction of the connective tissue of alveolar walls.
.

- Emphysema resulting from o4-AT deficiency: In the United States, approximately 2—5% of patients with emphysema are predisposed to the disease by inherited defects in o4-AT. A number of different mutations in the gene for o1-AT are known to cause a deficiency of this protein, but one single purine base mutation (GAG — AAG, resulting in the substitution of lysine for glutamic acid at position 342 of the protein) is clinically the most widespread. The polymerization of the mutated protein in the endoplasmic reticulum of hepatocytes causes decreased secretion of a1-AT by the liver. The accumulated polymer may result in cirrhosis (scarring of the liver). In the United States, the o-AT mutation is most common in Caucasians of Northern European ancestry. An individual must inherit two abnormal o4-AT alleles to be at risk for the development of emphysema. In a heterozygote, with one normal and one defective gene, the levels of «1-AT are sufficient to protect the alveoli from damage. [Note: A specific o.1-AT methionine is required for the binding of the inhibitor to its target proteases. Smoking causes the oxidation and subsequent inactivation of that methionine residue, thereby rendering the inhibitor powerless to neutralize elastase. Smokers with o.4-AT deficiency, therefore, have a considerably elevated rate of lung destruction and a poorer survival rate than nonsmokers with the deficiency.] The deficiency of elastase inhibitor can be reversed by augmentation therapy—weekly intravenous administration of a4-AT. The o4-AT diffuses from the blood into the lung, where it reaches therapeutic levels in the fluid surrounding the lung epithelial cells.

Collagen and elastin are fibrous proteins (Figure 4.15). Collagen molecules contain an abundance of proline, lysine, and glycine, the latter occurring at every third position in the primary structure. Collagen also contains hydroxyproline, hydroxylysine, and glycosylated hydroxylysine, each formed by posttranslational modification. Collagen molecules typically form fibrils containing a long, stiff, triple-stranded helical structure, in which three collagen polypeptide chains are wound around one another in a rope-like superhelix (triple helix). Other types of collagen form mesh-like networks. Elastin is a connective tissue protein with rubber-like properties in tissues such as the lung. a1-Antitrypsin (01-AT), produced primarily by the liver but also by tissues such as monocytes and alveolar macrophages, prevents elastin degradation in the alveolar walls. A deficiency of «1-AT can cause emphysema and, in some cases, cirrhosis of the liver.

4.1 A 30-year-old woman presented with progressive shortness of breath. She denied the use of cigarettes. A family history revealed that her sister had suffered from unexplained lung disease. Which one of the following etiologies most likely explains this patient's pulmonary symptoms?
A. Deficiency of proline hydroxylase
B. Deficiency of o4-antitrypsin
C. Deficiency in dietary vitamin C
D Decreased elastase activity
E. Increased collagenase activity
4.2 A seven-month-old child "fell over" while crawling, and now presents with a swollen leg. At age 1 month, the infant had multiple fractures in various states of healing (right clavicle, right humerus, right radius). At age 7 months, the infant has a fracture of a bowed femur, secondary to minor trauma (see x-ray at right). The bones are thin, have few trabecula, and have thin cortices. A careful family history ruled out nonaccidental trauma (child abuse) as a cause of the bone fractures. The child is most likely to have a defect in:
A. type | collagen.
B. type Ill collagen.
C. type IV collagen.
D. elastin.
E. fibrillin.
4.3 What is the differential basis of the liver and lung
pathology seen in o-AT deficiency?
Correct answer = B. o-Antitrypsin deficiency is a genetic disorder that can cause pulmonary emphysema even in the absence of cigarette use. A deficiency of a1-antitrypsin permits increased elastase activity to destroy elastin in the alveolar walls, even in nonsmokers. «-Antitrypsin deficiency should be suspected when chronic obstructive pulmonary disease (COPD) develops in a patient younger than 45 years who does not have a history of chronic bronchitis or tobacco use, or when multiple family members develop obstructive lung disease at an early age. Choices A, C, and E refer to collagen, not elastin.

Correct answer = A. The child most likely has osteogenesis imperfecta. Most cases arise from a defect in the genes encoding type | collagen. Bones in affected patients are thin, osteoporotic, often bowed with a thin cortex and deficient trabeculae, and extremely prone to fracture. This patient is affected with type |, osteogenesis imperfecta tarda. The disease presents in early infancy with fractures secondary to minor trauma. The disease may be suspected on prenatal ultrasound through detection of bowing or fractures of long bones. Type II, osteogenesis imperfecta congenita, is more severe, and patients die of pulmonary hypoplasia in utero or during the neonatal period. Defects in type Ill collagen are the most common cause of EhlersDanlos syndrome, characterized by lethal vascular problems and stretchy skin. Type IV collagen forms networks, not fibrils.
With o4-AT deficiency, the liver cirrhosis that can result is due to polymerization and retention of a-AT in the liver, its site of synthesis. The lung pathology is due to this retention-based deficiency in o4-AT (a serin protease inhibitor or serpin) such that elastase (a serine protease) is unopposed.

Virtually all reactions in the body are mediated by enzymes, which are protein catalysts that increase the rate of reactions without being changed in the overall process. Among the many biologic reactions that are energetically possible, enzymes selectively channel reactants (called substrates) into useful pathways. Enzymes thus direct all metabolic events. This chapter examines the nature of these catalytic molecules and their mechanism of action.

Each enzyme is assigned two names. The first is its short, recommended name, convenient for everyday use. The second is the more complete systematic name, which is used when an enzyme must be identified without ambiguity.

Most commonly used enzyme names have the suffix "-ase" attached to the substrate of the reaction (for example, glucosidase and urease), or to a description of the action performed (for example, lactate dehydrogenase and adenylyl cyclase). [Note: Some enzymes retain their original trivial names, which give no hint of the associated enzymic reaction, for example, trypsin and pepsin.]

In the systematic naming system, enzymes are divided into six major classes (Figure 5.1), each with numerous subgroups. For a given enzyme, the suffix -ase is attached to a fairly complete description of the chemical reaction catalyzed, including the names of all the substrates; for example, lactate: NAD* oxidoreductase. [Note: Each enzyme is also assigned a classification number.] The systematic names are unambiguous and informative, but are frequently too cumbersome to be of general use.

The six major classes of enzymes with examples. THF = tetrahydrofolate.

Schematic representation of an enzyme with one active site binding a substrate molecule.

The intracellular location of some important biochemical pathways.
Potentially confusing enzyme nomenclature: synthetase (requires ATP), synthase (no ATP required); phosphatase (uses water to remove phosphoryl group), phosphorylase (uses P; to break a bond and generate a phosphorylated product); dehydrogenase (NAD*/FAD is electron acceptor in redox reaction), oxidase (Oz is acceptor but oxygen atoms are not incorporated into substrate), oxygenase (one or both oxygens atoms are incorporated).

Enzymes are protein catalysts that increase the velocity of a chemical reaction, and are not consumed during the reaction. [Note: Some RNAs can act like enzymes, usually catalyzing the cleavage and synthesis of phosphodiester bonds. RNAs with catalytic activity are called ribozymes (see p. 439), and are much less commonly encountered than protein catalysts.]

Enzyme molecules contain a special pocket or cleft called the active site. The active site contains amino acid side chains that participate in substrate binding and catalysis (Figure 5.2). The substrate binds the enzyme, forming an enzyme-—substrate (ES) complex. Binding is thought to cause a conformational change in the enzyme (induced fit) that allows catalysis. ES is converted to an enzyme—product (EP) complex that subsequently dissociates to enzyme and product.

Enzyme-catalyzed reactions are highly efficient, proceeding from 10°—108 times faster than uncatalyzed reactions. The number of molecules of substrate converted to product per enzyme molecule per second is called the turnover number, or Kea: and typically is 107-104s",

Enzymes are highly specific, interacting with one or a few substrates and catalyzing only one type of chemical reaction. [Note: The set of enzymes made in a cell determines which metabolic pathways occur in that cell.]

Some enzymes require molecules other than proteins for enzymic activity. The term holoenzyme refers to the active enzyme with its nonprotein component, whereas the enzyme without its nonprotein moiety is termed an apoenzyme and is inactive. If the nonprotein moiety is a metal ion such as Zn** or Fe**, it is called a cofactor. If it is a small organic molecule, it is termed a coenzyme. Coenzymes that only transiently associate with the enzyme are called cosubstrates. Cosubstrates dissociate from the enzyme in an altered state (NAD* is an example, see p. 101). If the coenzyme is permanently associated with the enzyme and returned to its original form, it is called a prosthetic group (FAD is an example, see p. 110). Coenzymes frequently are derived from vitamins. For example, NAD* contains niacin and FAD contains riboflavin (see Chapter 28).

Enzyme activity can be regulated, that is, increased or decreased, so that the rate of product formation responds to cellular need.

Many enzymes are localized in specific organelles within the cell (Figure 5.3). Such compartmentalization serves to isolate the reaction substrate or product from other competing reactions. This provides a favorable environment for the reaction, and organizes the thousands of enzymes present in the cell into purposeful pathways.

The mechanism of enzyme action can be viewed from two different perspectives. The first treats catalysis in terms of energy changes that occur during the reaction, that is, enzymes provide an alternate, energetically favorable reaction pathway different from the uncatalyzed reaction. The second perspective describes how the active site chemically facilitates catalysis.

Virtually all chemical reactions have an energy barrier separating the reactants and the products. This barrier, called the free energy of activation, is the energy difference between that of the reactants and a high-energy intermediate that occurs during the formation of product. For example, Figure 5.4 shows the changes in energy during the conversion of a molecule of reactant A to product B as it proceeds through the transition state (high-energy intermediate), T*:
1. Free energy of activation: The peak of energy in Figure 5.4 is the difference in free energy between the reactant and T*, where the high-energy intermediate is formed during the conversion of reactant to product. Because of the high free energy of activation, the rates of uncatalyzed chemical reactions are often slow.
2. Rate of reaction: For molecules to react, they must contain sufficient energy to overcome the energy barrier of the transition state. In the absence of an enzyme, only a small proportion of a population of molecules may possess enough energy to achieve the transition state between reactant and product. The rate of reaction is determined by the number of such energized molecules. In general, the lower the free energy of activation, the more molecules have sufficient energy to pass through the transition state, and, thus, the faster the rate of the reaction.
3. Alternate reaction pathway: An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower free energy of activation (Figure 5.4). The enzyme does not change the free energies of the reactants or products and, therefore, does not change the equilibrium of the reaction (see p. 71). It does, however, accelerate the rate with which equilibrium is reached.

Schematic representation of energy changes accompanying formation of an enzyme-—substrate complex and subsequent formation of a transition state.

The active site is not a passive receptacle for binding the substrate, but rather is a complex molecular machine employing a diversity of chemical mechanisms to facilitate the conversion of substrate to product. A number of factors are responsible for the catalytic efficiency of enzymes, including the following:
- 1. Transition-state stabilization: The active site often acts as a flexible molecular template that binds the substrate and initiates its conversion to the transition state, a structure in which the bonds are not like those in the substrate or the product (see T* at the top of the curve in Figure 5.4). By stabilizing the transition state, the enzyme greatly increases the concentration of the reactive intermediate that can be converted to product and, thus, accelerates the reaction.
2.
Other mechanisms: The active site can provide catalytic groups that enhance the probability that the transition state is formed. In some enzymes, these groups can participate in general acidbase catalysis in which amino acid residues provide or accept protons. In other enzymes, catalysis may involve the transient formation of a covalent ES complex. [Note: The mechanism of action of chymotrypsin, an enzyme of protein digestion in the intestine, includes general base, general acid, and covalent catalysis. A histidine at the active site of the enzyme gains (general base) and loses (general acid) protons, mediated by the pK of histidine in proteins being close to physiologic pH. Serine at the active site forms a covalent link with the substrate.]
3.

Visualization of the transition state: The enzyme-catalyzed conversion of substrate to product can be visualized as being similar to removing a sweater from an uncooperative infant (Figure 5.5). The process has a high energy of activation because the only reasonable strategy for removing the garment (short of ripping it off) requires that the random flailing of the baby results in both arms being fully extended over the head—an unlikely posture. However, we can envision a parent acting as an enzyme, first coming in contact with the baby (forming ES), then guiding the baby's arms into an extended, vertical position, analogous to the ES transition state. This posture (conformation) of the baby facilitates the removal of the sweater, forming the disrobed baby, which here represents product. [Note: The substrate bound to the enzyme (ES) is at a slightly lower energy than unbound substrate (S) and explains the small "dip" in the curve at ES.]

Enzymes can be isolated from cells, and their properties studied in a test tube (that is, in vitro). Different enzymes show different responses to changes in substrate concentration, temperature, and pH. This section describes factors that influence the reaction velocity of enzymes. Enzymic responses to these factors give us valuable clues as to how enzymes function in living cells (that is, in vivo).

1. Maximal velocity: The rate or velocity of a reaction (v) is the number of substrate molecules converted to product per unit time; velocity is usually expressed as umol of product formed per minute. The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached (Figure 5.6). The leveling off of the reaction rate at high substrate concentrations reflects the saturation with substrate of all available binding sites on the enzyme molecules present.
2. Hyperbolic shape of the enzyme kinetics curve: Most enzymes show Michaelis-Menten kinetics (see p. 58), in which the plot of initial reaction velocity (Vo) against substrate concentration ([S]), is hyperbolic (similar in shape to that of the oxygen-dissociation curve of myoglobin, see p. 29). In contrast, allosteric enzymes do not follow Michaelis-Menton kinetics and show a sigmoidal curve (see p. 62) that is similar in shape to the oxygen dissociation curve of hemoglobin (see p. 29).

1. Increase of velocity with temperature: The reaction velocity increases with temperature until a peak velocity is reached (Figure 5.7). This increase is the result of the increased number of . . . molecules having sufficient energy to pass over the energy barrier and form the products of the reaction.
2. Decrease of velocity with higher temperature: Further elevation of the temperature results in a decrease in reaction velocity as a result of temperature-induced denaturation of the enzyme (see Figure 5.7).
|
enzymes is between 35 and 40°C. Human enzymes start to denature at temperatures above 40°C, but thermophilic bacteria found in the hot springs have optimum temperatures of 70°C. The optimum temperature for most human

1. Effect of pH on the ionization of the active site: The concentration of H* affects reaction velocity in several ways. First, the catalytic process usually requires that the enzyme and substrate have specific chemical groups in either an ionized or un-ionized state in order to interact. For example, catalytic activity may require that an amino group of the enzyme be in the protonated form (—NH3"*). At alkaline pH, this group is deprotonated, and the rate of the reaction, therefore, declines.
2. Effect of pH on enzyme denaturation: Extremes of pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic character of the amino acid side chains.

Effect of pH on enzyme-catalyzed reactions.
Figure 5.9 Fftset of substrate concentration on reaction velocities for two enzymes: enzyme 1 with a small Km, and enzyme 2 with a large Km.
3. The pH optimum varies for different enzymes: The pH at which maximal enzyme activity is achieved is different for different enzymes, and often reflects the [H*] at which the enzyme functions in the body. For example, pepsin, a digestive enzyme in the stomach, is maximally active at pH 2, whereas other enzymes, designed to work at neutral pH, are denatured by such an acidic environment (Figure 5.8).

Leonor Michaelis and Maude Menten proposed a simple model that accounts for most of the features of enzyme-catalyzed reactions. In this model, the enzyme reversibly combines with its substrate to form an ES complex that subsequently yields product, regenerating the free enzyme. The model, involving one substrate molecule, is represented below:
where
S is the substrate
E is the enzyme
ES is the enzyme-—substrate complex
P is the product ky, k-4, and kp are rate constants

The Michaelis-Menten equation describes how reaction velocity varies with substrate concentration:
where
Vo = initial reaction velocity
Vmax = Maximal velocity
Km = Michaelis constant = (k-1 + ko)/k;
[S] = substrate concentration
The following assumptions are made in deriving the MichaelisMenten rate equation:
- 1. Relative concentrations of E and S: The concentration of substrate ([S]) is much greater than the concentration of enzyme ([E]), so that the percentage of total substrate bound by the enzyme at any one time is small.
2. Steady-state assumption: [ES] does not change with time (the steady-state assumption), that is, the rate of formation of ES is equal to that of the breakdown of ES (to E + S and to E + P). In general, an intermediate in a series of reactions is said to be in steadystate when its rate of synthesis is equal to its rate of degradation.
- 3. Initial velocity: Initial reaction velocities (Vo) are used in the analysis of enzyme reactions. This means that the rate of the reaction is measured as soon as enzyme and substrate are mixed. At that time, the concentration of product is very small and, therefore, the i i rate of the back reaction from P to S can be ignored.

1. Characteristics of Km: K,—the Michaelis constant—is characteristic of an enzyme and its particular substrate, and reflects the affinity of the enzyme for that substrate. Km is numerically equal to the substrate concentration at which the reaction velocity is equal to %VmaxKm does not vary with the concentration of enzyme.
a. Small Km: A numerically small (low) Km reflects a high affinity of the enzyme for substrate, because a low concentration of substrate is needed to half-saturate the enzyme—that is, to reach a velocity that is /%Vmax (Figure 5.9).
b. Large K,: A numerically large (high) Km reflects a low affinity of enzyme for substrate because a high concentration of substrate is needed to half-saturate the enzyme.
2. Relationship of velocity to enzyme concentration: The rate of the reaction is directly proportional to the enzyme concentration at all substrate concentrations. For example, if the enzyme concentration is halved, the initial rate of the reaction (v.), as well as that of Vmax, are reduced to half that of the original.
3. Order of reaction: When [S] is much less than Km, the velocity of the reaction is approximately proportional to the substrate concentration (Figure 5.10). The rate of reaction is then said to be first order with respect to substrate. When [S] is much greater than Kn, the velocity is constant and equal to Vmax. The rate of reaction is then independent of substrate concentration, and is said to be zero order with respect to substrate concentration (see Figure 5.10).

When \, is plotted against [S], it is not always possible to determine when Vmax has been achieved, because of the gradual upward slope of the hyperbolic curve at high substrate concentrations. However, if 1/Vo is plotted versus 1/[S], a straight line is obtained (Figure 5.11). This plot, the Lineweaver-Burk plot (also called a double-reciprocal plot) can be used to calculate Km and Vmax, aS well as to determine the mechanism of action of enzyme inhibitors.
- 1. The equation describing the Lineweaver-Burk plot is:
where the intercept on the x-axis is equal to —1/K,, and the intercept on the y-axis is equal to 1/Vmax.
Figure 5.10 Efroct of substrate concentration on reaction velocity for an enzymecatalyzed reaction.

Any substance that can diminish the velocity of an enzyme-catalyzed reaction is called an inhibitor. In general, irreversible inhibitors bind to enzymes through covalent bonds. Reversible inhibitors typically bind to enzymes through noncovalent bonds, thus dilution of the enzyme-inhibitor complex results in dissociation of the reversibly bound inhibitor, and recovery of enzyme activity. The two most commonly encountered types of reversible inhibition are competitive and noncompetitive.

This type of inhibition occurs when the inhibitor binds reversibly to the same site that the substrate would normally occupy and, therefore, competes with the substrate for that site.
1. Effect on Vax: The effect of a competitive inhibitor is reversed by increasing [S]. At a sufficiently high substrate concentration, the reaction velocity reaches the Vmax observed in the absence of inhibitor (Figure 5.12).
2. Effect on Km: A competitive inhibitor increases the apparent K,, for a given substrate. This means that, in the presence of a competitive inhibitor, more substrate is needed to achieve /2Vmax.
- 3. Effect on the Lineweaver-Burk plot: Competitive inhibition shows a characteristic Lineweaver-Burk plot in which the plots of the inhibited and uninhibited reactions intersect on the y-axis at 1/Vmax (Vmax is unchanged). The inhibited and uninhibited reactions show different x-axis intercepts, indicating that the apparent Km is increased in the presence of the competitive inhibitor because -1/Km moves closer to zero from a negative value (see Figure 5.12).

A. Effect of a competitive inhibitor on the reaction velocity (vo) versus substrate ([S]) plot. B. Lineweaver-Burk plot of competitive inhibition of an enzyme.
- 4. Statin drugs as examples of competitive inhibitors: This group of antihyperlipidemic agents competitively inhibits the first committed step in cholesterol synthesis. This reaction is catalyzed by hydroxymethylglutaryI-CoA reductase (HMG-CoA reductase), see p. 220). Statin drugs, such as atorvastatin (Lipitor) and pravastatin (Pravachol),' are structural analogs of the natural substrate for this enzyme, and compete effectively to inhibit HMG-CoA reductase. By doing so, they inhibit de novo cholesterol synthesis, thereby lowering plasma cholesterol levels (Figure 5.13).

This type of inhibition is recognized by its characteristic effect on Vmax (Figure 5.14). Noncompetitive inhibition occurs when the inhibitor and substrate bind at different sites on the enzyme. The noncompetitive inhibitor can bind either free enzyme or the ES complex, thereby preventing the reaction from occurring (Figure 5.15).
1. Effect on Vmax: Noncompetitive inhibition cannot be overcome by increasing the concentration of substrate. Thus, noncompetitive inhibitors decrease the apparent Vmax of the reaction.
2. Effect on Km: Noncompetitive inhibitors do not interfere with the binding of substrate to enzyme. Thus, the enzyme shows the same Km in the presence or absence of the noncompetitive inhibitor.
3. Effect on Lineweaver-Burk plot: Noncompetitive inhibition is readily differentiated from competitive inhibition by plotting 1/vo versus 1/[S] and noting that the apparent Vmax decreases in the presence of a noncompetitive inhibitor, whereas Km is unchanged (see Figure 5.14).

Pravastatin competes with HMGCoA for the active site of HMGCoA reductase.
- 4. Examples of noncompetitive inhibitors: Some inhibitors act by forming covalent bonds with specific groups of enzymes. For

A. Effect of a noncompetitive inhibitor on the reaction velocity (Vo) versus substrate ([S]) plot. B. Lineweaver-Burk plot of noncompetitive inhibition of an enzyme.
@'se
Chapter 21 in Lippincott's Illustrated Reviews: Pharmacolog for a more

A noncompetitive inhibitor binding to both free enzyme and enzyme— substrate (ES) complex.

Effects of negative e or positive 6 effectors on an allosteric enzyme. A. Vmax is altered. B. The substrate concentration that gives half-maximal velocity (Kgs) is altered.
example, lead forms covalent bonds with the sulfhydryl side chains of cysteine in proteins. The binding of the heavy metal shows noncompetitive inhibition. Ferrochelatase, an enzyme that catalyzes the insertion of Fe** into protoporphyrin (a precursor of heme, see p. 279), is an example of an enzyme sensitive to inhibition by lead. Other examples of noncompetitive inhibition are certain insecticides, whose neurotoxic effects are a result of their covalent binding at the catalytic site of the enzyme acetylcholinesterase (an enzyme that cleaves the neurotransmitter, acetylcholine). [Note: Even though covalent bonds are formed, if active enzyme can be recovered, the inhibition is reversible.]

At least half of the ten most commonly dispensed drugs in the United States act as enzyme inhibitors. For example, the widely prescribed B-lactam antibiotics, such as penicillin and amoxicillin,? act by inhibiting enzymes involved in bacterial cell wall synthesis. Drugs may also act by inhibiting extracellular reactions. This is illustrated by angiotensin-converting enzyme (ACE) inhibitors. They lower blood pressure by blocking the enzyme that cleaves angiotensin | to form the potent vasoconstrictor, angiotensin 1.3 These drugs, which include captopril, enalapril, and lisinopril, cause vasodilation and a resultant reduction in blood pressure.

The regulation of the reaction velocity of enzymes is essential if an organism is to coordinate its numerous metabolic processes. The rates of most enzymes are responsive to changes in substrate concentration, because the intracellular level of many substrates is in the range of the Km. Thus, an increase in substrate concentration prompts an increase in reaction rate, which tends to return the concentration of substrate toward normal. In addition, some enzymes with specialized regulatory functions respond to allosteric effectors or covalent modification, or they show altered rates of enzyme synthesis (or degradation) when physiologic conditions are changed.

Allosteric enzymes are regulated by molecules called effectors (also called modifiers) that bind noncovalently at a site other than the active site. These enzymes are usually composed of multiple subunits, and the regulatory (allosteric) site that binds the effector may be located on a subunit that is not itself catalytic. The presence of an allosteric effector can alter the affinity of the enzyme for its substrate, or modify the maximal catalytic activity of the enzyme, or both. Effectors that inhibit enzyme activity are termed negative effectors, whereas those that increase enzyme activity are called positive effectors. Allosteric enzymes frequently catalyze the committed step early in a pathway.
1. Homotropic effectors: When the substrate itself serves as an effector, the effect is said to be homotropic. Most often, an allosteric sub-
?See Chapter 32 in Lippincott's Illustrated Reviews: Pharmacology for a qciscussion of inhibitors of bacterial cell wall synthesis. 3See Chapter 19 in Lippincott's Illustrated Reviews: Pharmacology for a discussion of angiotensin-converting enzyme inhibitors.
strate functions as a positive effector. In such a case, the presence of a substrate molecule at one site on the enzyme enhances the catalytic properties of the other substrate-binding sites—that is, their binding sites exhibit cooperativity. These enzymes show a sigmoidal curve when reaction velocity (Vo) is plotted against substrate concentration ([S]), Figure 5.16). This contrasts with the hyperbolic curve characteristic of enzymes following Michaelis-Menten kinetics, as previously discussed. [Note: The concept of cooperativity of substrate binding is analogous to the binding of oxygen to hemoglobin.] Positive and negative effectors of allosteric enzymes can affect either the Vmax, the Ko.s, or both (see Figure 5.16).
2.

Heterotropic effectors: The effector may be different from the substrate, in which case the effect is said to be heterotropic. For example, consider the feedback inhibition shown in Figure 5.17. The enzyme that converts D to E has an allosteric site that binds the endproduct, G. If the concentration of G increases (for example, because it is not used as rapidly as it is synthesized), the first irreversible step unique to the pathway is typically inhibited. Feedback inhibition provides the cell with appropriate amounts of a product it needs by regulating the flow of substrate molecules through the pathway that synthesizes that product. Heterotropic effectors are commonly encountered, for example, the glycolytic enzyme phosphofructokinase-1 is allosterically inhibited by citrate, which is not a substrate for the enzyme (see p. 99).

Many enzymes may be regulated by covalent modification, most frequently by the addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme. Protein phosphorylation is recognized as one of the primary ways in which cellular processes are regulated.
1. Phosphorylation and dephosphorylation: Phosphorylation reactions are catalyzed by a family of enzymes called protein kinases that use adenosine triphosphate (ATP) as a phosphate donor. Phosphate groups are cleaved from phosphorylated enzymes by the action of phosphoprotein phosphatases (Figure 5.18).
. Response of enzyme to phosphorylation: Depending on the specific enzyme, the phosphorylated form may be more or less active than the unphosphorylated enzyme. For example, phosphorylation of glycogen phosphorylase (an enzyme that degrades glycogen) increases activity, whereas the addition of phosphate to glycogen synthase (an enzyme that synthesizes glycogen) decreases activity (see p. 132).

The regulatory mechanisms described above modify the activity of existing enzyme molecules. However, cells can also regulate the amount of enzyme present by altering the rate of enzyme degradation or, more typically, the rate of enzyme synthesis. The increase (induction) or decrease (repression) of enzyme synthesis leads to an alteration in the total population of active sites. Enzymes subject to regulation of synthesis are often those that are needed at only one

Covalent modification by the addition and removal of phosphate groups.
Substrate inhibition, TYPICAL EFFECTOR = Substrate. Substrate inhibition, RESULTS = Change in velocity (v o ). Substrate inhibition, TIME REQUIRED FOR CHANGE = Immediate. Product inhibition, TYPICAL EFFECTOR = Reaction product. Product inhibition, RESULTS = Change in V max and/or K m. Product inhibition, TIME REQUIRED FOR CHANGE = Immediate. Allosteric control, TYPICAL EFFECTOR = Pathway end product. Allosteric control, RESULTS = Change in V max and/or K 0.5. Allosteric control, TIME REQUIRED FOR CHANGE = Immediate. Covalent modification, TYPICAL EFFECTOR = Another enzyme. Covalent modification, RESULTS = Change in V max and/or K m. Covalent modification, TIME REQUIRED FOR CHANGE = Immediate to minutes. Synthesis or degradation of enzyme, TYPICAL EFFECTOR = Hormone or metabolite. Synthesis or degradation of enzyme, RESULTS = Change in the amount of enzyme. Synthesis or degradation of enzyme, TIME REQUIRED FOR CHANGE = Hours to days

Mechanisms for regulating enzyme activity. [Note: Inhibition by pathway end product is also referred to as feedback inhibition.]
stage of development or under selected physiologic conditions. For example, elevated levels of insulin as a result of high blood glucose levels cause an increase in the synthesis of key enzymes involved in glucose metabolism (see p. 105). In contrast, enzymes that are in constant use are usually not regulated by altering the rate of enzyme synthesis. Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days), compared with allosterically or covalently regulated changes in enzyme activity, which occur in seconds to minutes. Figure 5.19 summarizes the common ways that enzyme activity is regulated.

Plasma enzymes can be classified into two major groups. First, a relatively small group of enzymes are actively secreted into the blood by certain cell types. For example, the liver secretes zymogens (inactive precursors) of the enzymes involved in blood coagulation. Second, a large number of enzyme species are released from cells during normal cell turnover. These enzymes almost always function intracellularly, and have no physiologic use in the plasma. In healthy individuals, the levels of these enzymes are fairly constant, and represent a steady state in which the rate of release from damaged cells into the plasma is balanced by an equal rate of removal of the enzyme protein from the plasma. Increased plasma levels of these enzyme may indicate tissue damage (Figure 5.20).

Release of enzymes from normal and diseased or traumatized cells.
Plasma is the fluid, noncellular part of blood. Laboratory assays of enzyme activity most often use serum, which is obtained by centrifugation of whole blood after it has been allowed to coagulate. Plasma is a physiologic fluid, whereas serum is prepared in the laboratory.

Many diseases that cause tissue damage result in an increased release of intracellular enzymes into the plasma. The activities of many of these enzymes are routinely determined for diagnostic purposes in diseases of the heart, liver, skeletal muscle, and other tissues. The level of specific enzyme activity in the plasma frequently correlates with the extent of tissue damage. Thus, determining the degree of elevation of a particular enzyme activity in the plasma is often useful in evaluating the prognosis for the patient.

Some enzymes show relatively high activity in only one or a few tissues. The presence of increased levels of these enzymes in plasma thus reflects damage to the corresponding tissue. For example, the enzyme alanine aminotransferase (ALT, see p. 251) is abundant in the liver. The appearance of elevated levels of ALT in plasma signals possible damage to hepatic tissue. [Note: Measurement of ALT is part of the liver function test panel.] Increases in plasma levels of enzymes with a wide tissue distribution provide a less specific indication of the site of cellular injury and limits their diagnostic value.

Most isoenzymes (also called isozymes) are enzymes that catalyze the same reaction. However, they do not necessarily have the same physical properties because of genetically determined differences in amino acid sequence. For this reason, isoenzymes may contain different numbers of charged amino acids and may, therefore, be separated from each other by electrophoresis (Figure 5.21). Different organs frequently contain characteristic proportions of different isoenzymes. The pattern of isoenzymes found in the plasma may, therefore, serve as a means of identifying the site of tissue damage. For example, the plasma levels of creatine kinase (CK) are commonly determined in the diagnosis of myocardial infarction. They are particularly useful when the electrocardiogram is difficult to interpret, such as when there have been previous episodes of heart disease.
1.
Quaternary structure of isoenzymes: Many isoenzymes contain different subunits in various combinations. For example, creatine kinase (CK) occurs as three isoenzymes. Each isoenzyme is a dimer composed of two polypeptides (called B and M subunits) associated in one of three combinations: CK1 = BB, CK2 = MB, and CK3 = MM. Each CK isoenzyme shows a characteristic electrophoretic mobility (see Figure 5.21). [Note: Virtually all CK in the brain is the BB isoform, whereas in skeletal muscle it is MM. In cardiac muscle, about one-third is MB with the rest as MM.]

Subunit structure, electrophoretic mobility, and enzyme activity of creatine kinase (CK) isoenzymes.

Appearance of creatine kinase (Ck) and cardiac troponin in plasma after a myocardial infarction.
(
2. Diagnosis of myocardial infarction: Measurement of blood levels of proteins with cardiac specificity is used in the diagnosis of myocardial infarction (MI) because myocardial muscle is the only tissue that contains more than 5% of the total CK activity as the CK2 (MB) isoenzyme. Appearance of this hybrid isoenzyme in plasma is virtually specific for infarction of the myocardium. Following an acute MI, this isoenzyme appears approximately 4—8 hours following onset of chest pain, reaches a peak of activity at approximately 24 hours, and returns to baseline after 48-72 hours (Figure 5.22). Troponin T and troponin | are regulatory proteins involved in myocardial contractility. They are released into the plasma in response to cardiac damage. Cardiac troponin | (cTnl) is highly sensitive and specific for damage to cardiac tissue. cTnl appears in plasma within 4—6 hours after an MI, peaks in 8-28 hours, and remains elevated for 3-10 days. Elevated serum troponins then, are more predictive of adverse outcomes in unstable angina or myocardial infarction than the conventional assay of CK2.

Enzymes are protein catalysts that increase the velocity of a chemical reaction by lowering the energy of the transition state (Figure 5.23). Enzymes are not consumed during the reaction they catalyze. Enzyme molecules contain a special pocket or cleft called the active site. The active site contains amino acid side chains that participate in substrate binding and catalysis. The active site binds the substrate, forming an enzyme-substrate (ES) complex. Binding is thought to cause a conformational change in the enzyme (induced fit) that allows catalysis. ES is converted to enzyme-product (EP), which subsequently dissociates to enzyme and product. An enzyme allows a reaction to proceed rapidly under conditions prevailing in the cell by providing an alternate reaction pathway with a lower free energy of activation. The enzyme does not change the free energies of the reactants or products and, therefore, does not change the equilibrium of the reaction. Most enzymes show Michaelis-Menten kinetics, and a plot of the initial reaction velocity (vo) against substrate concentration ([S]) has a hyperbolic shape similar to the oxygen dissociation curve of myoglobin. Any substance that can diminish the velocity of such enzyme-catalyzed reactions is called an inhibitor. The two most commonly encountered types of reversible inhibition are competitive (which increases the apparent Km) and noncompetitive (which decreases the apparent Vmax). In contrast, the multisubunit allosteric enzymes frequently show a sigmoidal curve similar in shape to the oxygen dissociation curve of hemoglobin. They typically catalyze the committed step (often the rate-limiting or slowest step) of a pathway. Allosteric enzymes are regulated by molecules called effectors (also modifiers) that bind noncovalently at a site other than the active site. Effectors can be either positive (accelerate the enzyme-catalyzed reaction) or negative (slow down the reaction). An allosteric effector can alter the affinity of the enzyme for its substrate, or modify the maximal catalytic activity of the enzyme, or both. Enzymes can also be regulated by covalent modification, and by changes in the rate of synthesis or degradation. Enzymes have diagnostic and therapeutic value in medicine.

Key concept map for the enzymes. S = substrate, [S] = substrate concentration, P = product, E = enzyme, Vo = initial velocity, Vmax = maximal velocity, Km, = Michaelis constant, Ko.5 = substrate concentration which gives half maximal velocity.

5.1
In cases of ethylene glycol poisoning and its characteristic metabolic acidosis, treatment involves correction of the acidosis, removal of any remaining ethylene glycol, and administration of an inhibitor of alcohol dehydrogenase (ADH, alcohol:NAD* oxidoreductase), the enzyme that oxidizes ethylene glycol to the organic acids that cause the acidosis. Ethanol (grain alcohol) frequently is the inhibitor given to treat ethylene glycol poisoning; it works by competitively inhibiting ADH. As a competitive inhibitor, ethanol:
A. increases apparent K,, without affecting Vmax.
B. decreases apparent K,, without affecting Vmax.
C. increases apparent Vmax without affecting Km.
D. decreases apparent Vmax without affecting Kn.
E. decreases both apparent Vmax and apparent Km.
5.2 ADH requires NAD* for catalytic activity. In the reaction catalyzed by ADH, an alcohol is oxidized to an aldehyde as NAD* is reduced to NADH and dissociates from the enzyme. The NAD* is functioning as a (an):
. apoenzyme. p>
. coenzyme-cosubstrate.
. coenzyme-prosthetic group.
. cofactor.
. heterotropic effector.
moQgW
5.3
A 70-year-old man was admitted to the emergency room with a 12-hour history of chest pain. Serum creatine kinase (CK) activity was measured at admission (day 1) and once daily (Figure 5.24). On day 2 after admission, he experienced cardiac arrhythmia, which was terminated by three cycles of electric cardioconversion, the latter two at maximum energy. [Note: Cardioconversion is performed by placing two paddles, 12 cm in diameter, in firm contact with the chest wall and applying a short electric voltage.] Normal cardiac rhythm was reestablished. He had no recurrence of arrhythmia over the next several days. His chest pain subsided and he was released on day 10. Which one of the following is most consistent with the data presented?

A. The patient had a myocardial infarction 48 to 64 hours prior to admission.
B. The patient had a myocardial infarction on day 2.
. The patient had angina prior to admission. 9090
. The patient had damage to his skeletal muscle on

- E. The data do not permit any conclusion concerning myocardial infarction prior to, or after, admission to the hospital.

Serum creatine kinase levels.
Correct answer = A. In the presence of a competitive inhibitor, an enzyme appears to have a lower affinity for substrate, but as the substrate level is increased, the observed velocity approaches Vmax. (See panel B of Figures 5.12 and 5.14 to compare effects of competitive and noncompetitive inhibitors.)
Correct answer = B. Coenzymes-cosubstrates are small organic molecules that associate transiently with an enzyme and leave the enzyme ina changed form. Coenzyme-prosthetic groups are small organic molecules that associate permanently with an enzyme and are returned to their original form on the enzyme. Cofactors are metal ions. Heterotropic effectors are not substrates.
Correct answer = D. The CK isoenzyme pattern at admission showed elevated MB isozyme, indicating that the patient had experienced a myocardial infarction in the previous 12-24 hours. [Note: 48-64 hours after an infarction, the MB isozyme would have returned to normal values.] On day 2, 12 hours after the cardioconversions, the MB isozyme had decreased, indicating no further damage to the heart. However, the patient showed an increased MM isozyme after cardioconversion. This suggests damage to muscle, probably a result of the convulsive muscle contractions caused by repeated cardioconversion. Angina is typically the result of transient spasms in the vasculature of the heart, and would not be expected to lead to tissue death that results in elevation in serum creatine kinase.
X
S
UNIT
IL:

Bioenergetics describes the transfer and utilization of energy in biologic systems. It makes use of a few basic ideas from the field of thermodynamics, particularly the concept of free energy. Changes in free energy (AG) provide a measure of the energetic feasibility of a chemical reaction and can, therefore, allow prediction of whether a reaction or process can take place. Bioenergetics concerns only the initial and final energy states of reaction components, not the mechanism or how much time is needed for the chemical change to take place. In short, bioenergetics predicts if a process is possible, whereas kinetics measures how fast the reaction occurs
(see p. 54).

The direction and extent to which a chemical reaction proceeds is determined by the degree to which two factors change during the reaction. These are enthalpy (AH, a measure of the change in heat content of the reactants and products) and entropy (AS, a measure of the change in randomness or disorder of reactants and products, Figure 6.1). Neither of these thermodynamic quantities by itself is sufficient to determine whether a chemical reaction will proceed spontaneously in the direction it is written. However, when combined mathematically (see Figure 6.1), enthalpy and entropy can be used to define a third quantity, free energy (G), which predicts the direction in which a reaction will spontaneously proceed.

Relationship between changes in free energy (G), enthalpy (H), and entropy (S). T is the absolute temperature in degrees Kelvin (°K): °K = °C + 278.
GB
B—A
Transition state
ee
*
Final
AG
is reaction
c
>
=)
2
o
oO
2
ue
Progress of
state positive
——~

Change in free energy (AG) during a reaction. A. The product has a lower free energy (G) than the reactant. B. The product has a higher free energy than the reactant.
oe

The change in free energy is represented in two ways, AG and AG°. The first, AG (without the superscript "o"), represents the change in free energy and, thus, the direction of a reaction at any specified concentration of products and reactants. AG, then, is a variable. This contrasts with the standard free energy change, AG° (with the superscript "o"), which is the energy change when reactants and products are at a concentration of 1 mol/L. [Note: The concentration of protons is assumed to be 107" mol/L, that is, pH = 7.] Although AG®° represents energy changes at these nonphysiologic concentrations of reactants and products, it is nonetheless useful in comparing the energy changes of different reactions. Furthermore, AG®° can readily be determined from measurement of the equilibrium constant (see p. 72). This section outlines the uses of AG; AG° is described on p. 71.

The change in free energy, AG, can be used to predict the direction of a reaction at constant temperature and pressure. Consider the reaction:
A
2
B
_ . Negative AG: If AG is a negative number, there is a net loss of energy, and the reaction goes spontaneously as written—that is, A is converted into B (Figure 6.2A). The reaction is said to be exergonic.
2. Positive AG: If AG is a positive number, there is a net gain of energy, and the reaction does not go spontaneously from B to A (see Figure 6.2B). Energy must be added to the system to make the reaction go from B to A, and the reaction is said to be endergonic.
3. AG is zero: If AG = 0, the reactants are in equilibrium. [Note: When a reaction is proceeding spontaneously—that is, free energy is being lost—then the reaction continues until AG reaches zero and equilibrium is established.]

The free energy of the forward reaction (A > B) is equal in magnitude but opposite in sign to that of the back reaction (B — A). For example, if AG of the forward reaction is —5 kcal/mol, then that of the back reaction is +5 kcal/mol. [Note: AG can also be expressed in kilojoules per mole or kJ/mol (1 kcal = 4.2 kJ).]

The AG of the reaction A — B depends on the concentration of the reactant and product. At constant temperature and pressure, the following relationship can be derived:
where
AG° is the standard free energy change (see below) R is the gas constant (1.987 cal/moldegree) T is the absolute temperature (°K) [A] and [B] are the actual concentrations of the reactant and product
In represents the natural logarithm
A reaction with a positive AG° can proceed in the forward direction (have a negative overall AG) if the ratio of products to reactants ([BJ/[A]) is sufficiently small (that is, the ratio of reactants to products is large). For example, consider the reaction:
Glucose
6-phosphate
2
__
fructose 6-phosphate
Figure 6.3A shows reaction conditions in which the concentration of reactant, glucose 6-phosphate, is high compared with the concentration of product, fructose 6-phosphate. This means that the ratio of the product to reactant is small, and RT In([fructose 6-phosphate]/ [glucose 6-phosphate]) is large and negative, causing AG to be negative despite AG° being positive. Thus, the reaction can proceed in the forward direction.

The standard free energy change, AG®, is so called because it is equal to the free energy change, AG, under standard conditions— that is, when reactants and products are at 1 mol/L concentrations (see Figure 6.3B). Under these conditions, the natural logarithm of the ratio of products to reactants is zero (In1 = 0) and, therefore, the equation shown at the top of this page becomes:

- = . AG° is predictive only under standard conditions: Under standard conditions, AG° can be used to predict the direction a reaction proceeds because, under these conditions, AG° is equal to AG. However, AG° cannot predict the direction of a reaction under physiologic conditions, because it is composed solely of constants (R, T, and Keg) and is, therefore, not altered by changes in product or substrate concentrations.
2. Relationship between AG° and Keg: In a reaction AB, a point of equilibrium is reached at which no further net chemical change takes place—that is, when A is being converted to B as fast as B is being converted to A. In this state, the ratio of [B] to [A] is constant, regardless of the actual concentrations of the two compounds:

AG of a reaction depends on the concentration of reactant (A) and product (B). For the conversion of glucose 6-P to fructose 6-P, AG is negative when the ratio of reactant (A) to product (B) is large (top, panel A); is positive under standard conditions (middle, panel B); and is zero at equilibrium (bottom, panel C).

model of coupling of and unfavorable
Mechanical favorable processes.
where Keg is the equilibrium constant, and [A]eg and [B]eg are the concentrations of A and B at equilibrium. If the reaction A @ Bis allowed to go to equilibrium at constant temperature and pressure, then at equilibrium the overall free energy change (AG) is zero. Therefore,
where the actual concentrations of A and B are equal to the equilibrium concentrations of reactant and product [A]eq and [Bleg, and their ratio as shown above is equal to the Keg. Thus,
- 3. AG° of two consecutive reactions are additive: The standard free energy changes (AG°) are additive in any sequence of consecutive reactions, as are the free energy changes (AG). For example:
Glucose
+
ATP
—
glucose
6-P
+ADP
AG°
=-4,000
cal/mol
Glucose
6-P
-
fructose
6-P
AG°
=
+400
cal/mol
Glucose
+
ATP
>
fructose
6-P
+
ADP
AG°
=-3,600
cal/mol
- 4. AGs of a pathway are additive: This additive property of free energy changes is very important in biochemical pathways through which substrates must pass in a particular direction (for example, A B > C > D -....). As long as the sum of the AGs of the individual reactions is negative, the pathway can potentially proceed as written, even if some of the individual reactions of the pathway have a positive AG. The actual rate of the reactions does, of course, depend on the lowering of activation energies by the enzymes that catalyze the reactions (see p. 55).

Reactions or processes that have a large positive AG, such as moving ions against a concentration gradient across a cell membrane, are made possible by coupling the endergonic movement of ions with a second, spontaneous process with a large negative AG, such as the exergonic hydrolysis of adenosine triphosphate (ATP). [Note: In the absence of enzymes, ATP is a stable molecule because its hydrolysis has a high activation energy.] Figure 6.4 shows a mechanical model of
energy coupling. A gear with an attached weight spontaneously turns in the direction that achieves the lowest energy state, in this case the weight seeks its lowest position (see Figure 6.4A). The reverse motion (see Figure 6.4B) is energetically unfavored and does not occur spontaneously. Figure 6.4C shows that the energetically favored movement of one gear can be used to turn a second gear in a direction that it would not move spontaneously. The simplest example of energy coupling in biologic reactions occurs when the energy-requiring and the energyyielding reactions share a common intermediate.

Two chemical reactions have a common intermediate when they occur sequentially so that the product of the first reaction is a substrate for the second. For example, given the reactions
D is the common intermediate and can serve as a carrier of chemical energy between the two reactions. Many coupled reactions use ATP to generate a common intermediate. These reactions may involve the transfer of a phosphate group from ATP to another molecule. Other reactions involve the transfer of phosphate from an energy-rich intermediate to adenosine diphosphate (ADP), forming ATP.

ATP consists of a molecule of adenosine (adenine + ribose) to which three phosphate groups are attached (Figure 6.5). If one phosphate is removed, ADP is produced; if two phosphates are removed, adenosine monophosphate (AMP) results. The standard free energy of hydrolysis of ATP, AG®, is approximately —7.3 kcal/mol for each of the two terminal phosphate groups. Because of this large negative AG®, ATP is called a high-energy phosphate compound.

Energy-rich molecules, such as glucose, are metabolized by a series of oxidation reactions ultimately yielding CO2 and water (Figure 6.6). The metabolic intermediates of these reactions donate electrons to specific coenzymes—nicotinamide adenine dinucleotide (NAD*) and flavin adenine dinucleotide (FAD)—to form the energy-rich reduced coenzymes, NADH and FADH». These reduced coenzymes can, in turn, each donate a pair of electrons to a specialized set of electron carriers, collectively called the electron transport chain, described in this section. As electrons are passed down the electron transport chain, they lose much of their free energy. Part of this energy can be captured and stored by the production of ATP from ADP and inorganic phosphate (Pj). This process is called oxidative phosphorylation and is described on p. 77. The remainder of the free energy not trapped as ATP is used to drive ancillary reactions such as Ca** transport into mitochondria, and to generate heat.

Structure of a mitochondrion showing schematic representation of the electron transport chain and ATP synthesizing structures on the inner membrane. mtDNA = mitochondrial DNA; mtRNA = mitochondrial RNA. [Note: In contrast to the inner membrane, the outer membrane is highly permeable and the milieu of the intermembrane space is like that of the cytosol.]

The electron transport chain is present in the inner mitochondrial membrane and is the final common pathway by which electrons derived from different fuels of the body flow to oxygen. Electron transport and ATP synthesis by oxidative phosphorylation proceed continuously in all tissues that contain mitochondria.
1. Membranes of the mitochondrion: The components of the electron transport chain are located in the inner membrane. Although the outer membrane contains special pores, making it freely permeable to most ions and small molecules, the inner mitochondrial membrane is a specialized structure that is impermeable to most small ions, including H*, Na*, and K*, and small molecules such as ATP, ADP, pyruvate, and other metabolites important to mitochondrial function (Figure 6.7). Specialized carriers or transport systems are required to move ions or molecules across this membrane. The inner mitochondrial membrane is unusually rich in protein, half of which is directly involved in electron transport and oxidative phosphorylation. The inner mitochondrial membrane is highly convoluted. The convolutions, called cristae, serve to greatly increase the surface area of the membrane.
2. Matrix of the mitochondrion: This gel-like solution in the interior of mitochondria is 50% protein. These molecules include the enzymes responsible for the oxidation of pyruvate, amino acids, fatty acids (by B-oxidation), and those of the tricarboxylic acid (TCA) cycle. The synthesis of glucose, urea, and heme occur partially in the matrix of mitochondria. In addition, the matrix contains NAD* and FAD (the oxidized forms of the two coenzymes that are required as hydrogen acceptors) and ADP and P;, which are used to produce ATP. [Note: The matrix also contains mitochondrial RNA and DNA (mtRNA and mtDNA) and mitochondrial ribosomes.]

The inner mitochondrial membrane can be disrupted into five separate protein complexes, called Complexes |, Il, Ill, IV, and V. Complexes I-IV each contain part of the electron transport chain (Figure 6.8). Each complex accepts or donates electrons to relatively mobile electron carriers, such as coenzyme Q and cytochrome c. Each carrier in the electron transport chain can receive electrons from an electron donor, and can subsequently donate electrons to the next carrier in the chain. The electrons ultimately combine with oxygen and protons to form water. This requirement for oxygen makes the electron transport process the respiratory chain, which accounts for the greatest portion of the body's use of oxygen. Complex V is a protein complex that contains a domain (F,) that spans the inner mitochondrial membrane, and a domain (F;) that appears as a sphere that protrudes into the mitochondrial matrix (see p. 78). Complex V catalyzes ATP synthesis and so is referred to as ATP synthase.

With the exception of coenzyme Q, all members of this chain are proteins. These may function as enzymes as is the case with the

Electron transport chain. [Note: NADH, produced from a variety of oxidative (catabolic) processes, is the substrate for Complex |. Succinate, an intermediate of the TCA cycle, is the substrate for Complex II.]
dehydrogenases, may contain iron as part of an iron-sulfur center, may be coordinated with a porphyrin ring as in the cytochromes, or may contain copper as does the cytochrome a + ag complex.

1. Formation of NADH: NAD* is reduced to NADH by dehydrogenases that remove two hydrogen atoms from their substrate. (For examples of these reactions, see the discussion of the dehydrogenases found in the TCA cycle, p. 112.) Both electrons but only one proton (that is, a hydride ion, :H-) are transferred to the NAD*, forming NADH plus a free proton, H*.
2. NADH dehydrogenase: The free proton plus the hydride ion carried by NADH are next transferred to NADH dehydrogenase, a protein complex (Complex |) embedded in the inner mitochondrial membrane. Complex | has a tightly bound molecule of flavin mononucleotide (FMN, a coenzyme structurally related to FAD, see Figure 28.15, p. 380) that accepts the two hydrogen atoms (2e7 +2H*), becoming FMNH2. NADH dehydrogenase also contains iron atoms paired with sulfur atoms to make iron-sulfur centers (Figure 6.9). These are necessary for the transfer of the hydrogen atoms to the next member of the chain, coenzyme Q (ubiquinone).
3. Coenzyme Q: Coenzyme Q (CoQ) is a quinone derivative with a long, hydrophobic isoprenoid tail. It is also called ubiquinone because it is ubiquitous in biologic systems. CoQ is a mobile carrier and can accept hydrogen atoms both from FMNHs, produced on NADH dehydrogenase (Complex |), and from FADHs, produced on succinate dehydrogenase (Complex II), glycerophosphate dehydrogenase (see p. 79), and acyl CoA dehydrogenase (see p. 192). CoQ transfers electrons to Complex III. CoQ, then, links the flavoproteins to the cytochromes.

lron-sulfur center of Complex I. Note: Complexes II and III also Note: ( ror eeuifur centars.

Site-specific inhibitors of electron transport shown using a mechanical model for the coupling of oxidationreduction reactions. [Note: Figure illustrates normal direction of electron flow.]
4. Cytochromes: The remaining members of the electron transport chain are cytochromes. Each contains a heme group (a porphyrin ring plus iron). Unlike the heme groups of hemoglobin, the cytochrome iron is reversibly converted from its ferric (Fe**) to its ferrous (Fe**) form as a normal part of its function as a reversible carrier of electrons. Electrons are passed along the chain from CoQ to cytochromes bce; (Complex Ill), c, and a + a3 (Complex IV, see Figure 6.8). [Note: Cytochrome c is associated with the outer face of the inner membrane and, like CoQ, is a mobile carrier of electrons.]
5. Cytochrome a + as: This cytochrome complex is the only electron carrier in which the heme iron has an available coordination site that can react directly with Oz, and so also is called cytochrome oxidase. At this site, the transported electrons, Oz, and free protons are brought together, and Os is reduced to water (see Figure 6.8). Cytochrome oxidase contains copper atoms that are required for this complex reaction to occur.
6. Site-specific inhibitors: Site-specific inhibitors of electron transport have been identified and are illustrated in Figure 6.10. These compounds prevent the passage of electrons by binding to a component of the chain, blocking the oxidation/reduction reaction. Therefore, all electron carriers before the block are fully reduced, whereas those located after the block are oxidized. [Note: Inhibition of electron transport inhibits ATP synthesis because these processes are tightly coupled.]
Incomplete reduction of oxygen to water produces reactive oxygen species (ROS), such as superoxide (Ozs), hydrogen peroxide (H2O2) and hydroxyl radicals (OHs). ROS damage DNA and proteins, and cause lipid peroxidation. Enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase are cellular defenses against ROS.

Free energy is released as electrons are transferred along the electron transport chain from an electron donor (reducing agent or reductant) to an electron acceptor (oxidizing agent or oxidant). The electrons can be transferred as hydride ions (:H~) to NAD*, as hydrogen atoms (eH) to FMN, coenzyme Q, and FAD, or as electrons (e7) to cytochromes.
1. Redox pairs: Oxidation (loss of electrons) of one compound is always accompanied by reduction (gain of electrons) of a second substance. For example, Figure 6.11 shows the oxidation of NADH to NAD* accompanied by the reduction of FMN to FMNHp». Such oxidation-reduction reactions can be written as the sum of two separate half-reactions, one an oxidation reaction and the other a reduction reaction (see Figure 6.11). NAD* and NADH form a redox pair, as do FMN and FMNb». Redox pairs differ in their tendency to lose electrons. This tendency is a characteristic of a particular redox pair, and can be quantitatively specified by a constant, E, (the standard reduction potential), with units in volts.
2. Standard reduction potential (E,): The E> of various redox pairs can be ordered from the most negative E, to the most positive. The more negative the E, of a redox pair, the greater the tendency of the reductant member of that pair to lose electrons. The more positive the E,, the greater the tendency of the oxidant member of that pair to accept electrons. Therefore, electrons flow from the pair with the more negative E, to that with the more positive E>. The E> values for some members of the electron transport chain are shown in Figure 6.12. [Note: The components of the electron transport chain are arranged in order of increasingly positive Eo values.]
3. AG° is related to AE,: The change in free energy is related directly to the magnitude of the change in E,:

n = number of electrons transferred (1 for a cytochrome, 2 for NADH, FADH», and coenzyme Q)
F = Faraday constant (23.1 kcal/volt -mol)
AE, = Eo of the electron-accepting pair minus the E, of the electron-donating pair
AG®° = change in the standard free energy
- 4. AG° of ATP: The standard free energy for the phosphorylation of ADP to ATP is +7.3 kcal/mol. The transport of a pair of electrons from NADH to oxygen via the electron transport chain produces 52.58 kcal. Therefore, more than sufficient energy is available to produce three ATP from three ADP and three P; (3 x 7.3 = 21.9 kcal/mol), sometimes expressed as a P:O ratio (ATP made per O atom reduced) of 3:1. The remaining calories are used for ancillary reactions or released as heat. [Note: P:O for FADHz2 is 2:1 because Complex | is bypassed.]

The transfer of electrons down the electron transport chain is energetically favored because NADH is a strong electron donor and molecular oxygen is an avid electron acceptor. However, the flow of electrons from NADH to oxygen does not directly result in ATP synthesis.

The chemiosmotic hypothesis (also known as the Mitchell hypothesis) explains how the free energy generated by the transport of electrons : by the electron transport chain is used to produce ATP from ADP + Pi.
- 1. Proton pump: Electron transport is coupled to the phosphorylation of ADP by the transport ("pumping") of protons (H*) across the inner mitochondrial membrane from the matrix to the intermembrane space at Complexes |, Ill, and IV. This process creates an electrical gradient (with more positive charges on the outside of the membrane than on the inside) and a pH gradient (the outside of the membrane is at a lower pH than the inside, Figure 6.13). The energy generated by this proton gradient is sufficient to drive ATP

Oxidation of NADH by FMN, ted into t t redox pairs. omwe componen

Electron transport chain shown coupled to the transport of protons. [Note: Protons are not pumped at Complex II.]
synthesis. Thus, the proton gradient serves as the common intermediate that couples oxidation to phosphorylation.
2.
ATP synthase: The enzyme complex ATP synthase (Complex V, see Figure 6.13) synthesizes ATP using the energy of the proton gradient generated by the electron transport chain. [Note: It is also called F;/F, ATPase because the isolated enzyme can catalyze the hydrolysis of ATP to ADP and P;.] The chemiosmotic hypothesis proposes that after protons have been pumped to the cytosolic side of the inner mitochondrial membrane, they reenter the matrix by passing through a channel in the membrane-spanning domain (F,) of Complex V, driving rotation of F, and, at the same time, dissipating the pH and electrical gradients. F, rotation causes conformational changes in the extra-membranous F,; domain that allow it to bind ADP + Pi, phosphorylate ADP to ATP, and release ATP.
a. Oligomycin: This drug binds to the F, (hence the letter 0) domain of ATP synthase, closing the H* channel, preventing reentry of protons into the mitochondrial matrix, and thus preventing phosphorylation of ADP to ATP. Because the pH and electrical gradients cannot be dissipated in the presence of this drug, electron transport stops because of the difficulty of pumping any more protons against the steep gradients. This dependency of cellular respiration on the ability to phosphorylate ADP to ATP is known as respiratory control, and is the consequence of the tight coupling of these processes. Electron transport and phosphorylation are, therefore, again shown to be tightly coupled processes. Inhibition of one process inhibits the other. [Note: Respiratory control also results from decreased availability of ADP or Pi.]

Transport of H* across the mitochondrial membrane by 2,4-dinitrophenol.
b. Uncoupling proteins (UCP): UCPs occur in the inner mitochondrial membrane of mammals, including humans. These carrier proteins create a "proton leak," that is, they allow protons to re-enter the mitochondrial matrix without energy being captured as ATP (Figure 6.14). The energy is released as heat, and the process is called nonshivering thermogenesis. UCP1, also called thermogenin, is responsible for the heat production in the brown adipocytes of mammals. UCP1 is activated by fatty acids. Brown fat, unlike the more abundant white fat, uses almost 90% of its respiratory energy for thermogenesis in response to cold in the neonate, and during arousal in hibernating animals. However, humans appear to have little brown fat (except in the newborn), and UCP1 does not appear to play a major role in energy balance. [Note: Other uncoupling proteins (UCP2, UCP3) have been found in humans, but their significance remains unclear.]
c.
Synthetic uncouplers: Electron transport and phosphorylation can also be uncoupled by compounds that increase the permeability of the inner mitochondrial membrane to protons. The classic example is 2,4-dinitrophenol, a lipophilic proton carrier that readily diffuses through the mitochondrial membrane. This uncoupler causes electron transport to proceed at a rapid rate without establishing a proton gradient, much as do the UCPs (see Figure 6.14). Again, energy is released as heat rather than being used to synthesize ATP. In high doses, aspirin and other salicylates uncouple oxidative phosphorylation. This explains the fever that accompanies toxic overdoses of these drugs.

The inner mitochondrial membrane is impermeable to most charged or hydrophilic substances. However, it contains numerous transport proteins that permit passage of specific molecules from the cytosol (or more correctly, the intermembrane space) to the mitochondrial matrix.
1. ATP-ADP transport: The inner mitochondrial membrane requires specialized carriers to transport ADP and Pj from the cytosol (where ATP is used and converted to ADP in many energyrequiring reactions) into mitochondria, where ATP can be resynthesized. An adenine nucleotide carrier imports one molecule of ADP from the cytosol into mitochondria, while exporting one ATP from the matrix back into the cytosol (see Figure 6.13). [Note: A phosphate carrier is responsible for transporting Pj from the cytosol into mitochondria.]
2. Transport of reducing equivalents: The inner mitochondrial membrane lacks an NADH transporter, and NADH produced in the cytosol cannot directly enter the mitochondrial matrix. However, two electrons of NADH (also called reducing equivalents) are transported from the cytosol into the matrix using substrate shuttles. In the glycerophosphate shuttle (Figure 6.15A), two electrons are transferred from NADH to dihydroxyacetone phosphate by cytosolic glycerophosphate dehydrogenase. The glycerol 3-phosphate produced is oxidized by the mitochondrial isozyme as FAD is

Substrate shuttles for the transport of electrons across the inner mitochondrial membrane. A. Glycerol 3-phosphate shuttle. B. Malateaspartate shuttle. DHAP = dihydroxyacetone phosphate.

Muscle fibers from a patient with a mitochondrial myopathy show abnormal mitochondrial proliferation when stained for succinate dehydrogenase, an of II.
enzyme Complex reduced to FADH2. CoQ of the electron transport chain oxidizes FADH2. The glycerophosphate shuttle, therefore, results in the synthesis of two ATPs for each cytosolic NADH oxidized. This contrasts with the malate-aspartate shuttle (Figure 6.15B), which produces NADH (rather than FADHz2) in the mitochondrial matrix and, therefore, yields three ATPs for each cytosolic NADH oxidized by malate dehydrogenase as oxaloacetate is reduced to malate. A transport protein carries malate into the matrix.

Thirteen of the approximately 120 polypeptides required for oxidative phosphorylation are coded for by mtDNA and synthesized in mitochondria, whereas the remaining mitochondrial proteins are synthesized in the cytosol and transported into mitochondria. Defects in oxidative phosphorylation are more likely a result of alterations in mtDNA, which has a mutation rate about ten times greater than that of nuclear DNA. Tissues with the greatest ATP requirement (for example, central nervous system, skeletal and heart muscle, kidney, and liver) are most affected by defects in oxidative phosphorylation. Mutations in mtDNA are responsible for several diseases, including some cases of mitochondrial myopathies (Figure 6.16), and Leber hereditary optic neuropathy, a disease in which bilateral loss of central vision occurs as a result of neuroretinal degeneration, including damage to the optic nerve. The mtDNA is maternally inherited because mitochondria from the sperm cell do not enter the fertilized egg.

The process of apoptosis or programmed cell death may be initiated through the intrinsic (mitochondrial-mediated) pathway by the formation of pores in the outer mitochondrial membrane. These pores allow cytochrome c to leave the intermembrane space and enter the cytosol. Once in the cytosol, cytochrome c, in association with proapoptotic factors, activates a family of proteolytic enzymes (the caspases), causing cleavage of key proteins and resulting in the morphologic and biochemical changes characteristic of apoptosis.

The change in free energy (AG) occurring during a reaction predicts the direction in which that reaction will spontaneously proceed. If AG is negative (that is, the product has a lower free energy than the substrate), the reaction goes spontaneously. If AG is positive, the reaction does not go spontaneously. If AG = 0, the reactions are in equilibrium. The AG of the forward reaction (A — B) is equal in magnitude but opposite in sign to that of the back reaction (B > A). The AGs are additive in any sequence of consecutive reactions, as are the standard free energy changes (AG°s). Therefore, reactions or processes that have a large, positive AG are made possible by coupling with cleavage of adenosine triphosphate (ATP), which has a large, negative AG. The reduced coenzymes NADH and FADHp each donate a pair of electrons to a specialized set of electron carriers, consisting of FMN,

Summary of key concepts for oxidative phosphorylation. [Note: Electron flow and ATP synthesis are envisioned as sets of interlocking gears to emphasize the idea of coupling.]
coenzyme Q, and a series of cytochromes, collectively called the electron transport chain. This pathway is present in the inner mitochondrial membrane, and is the final common pathway by which electrons derived from different fuels of the body flow to oxygen, reducing it to water. The terminal cytochrome, cytochrome oxidase, is the only cytochrome able to bind oxygen. Electron transport is coupled to the transport of protons (H*) across the inner mitochondrial membrane from the matrix to the intermembrane space. This process creates electrical and pH gradients across the inner mitochondrial membrane. After protons have been transferred to the cytosolic side of the inner mitochondrial membrane, they reenter the mitochondrial matrix by passing through the F, channel in ATP synthase (Complex V), dissipating the pH and electrical gradients and causing conformational changes in F, that result in the synthesis of ATP from ADP + Pj. Electron transport and phosphorylation are thus said to be tightly coupled (Figure 6.17). Inhibition of one process inhibits the other. These processes can be uncoupled by uncoupling proteins found in the inner mitochondrial membrane, and by synthetic compounds such as 2,4-dinitrophenol and aspirin, all of which increase the permeability of the inner mitochondrial membrane to protons. The energy produced by the transport of electrons is released as heat rather than being used to synthesize ATP. Mutations in mitochondrial DNA (mtDNA) are responsible for some cases of mitochondrial diseases, such as Leber hereditary optic neuropathy. The release of cytochrome c into the cytoplasm and subsequent activation of proteolytic caspases results in apoptotic cell death.

6.1 A muscle biopsy specimen from a patient with a rare disorder, Luft disease, showed abnormally large mitochondria that contained packed cristae when examined in the electron microscope. Basal ATPase activity of the mitochondria was seven times greater than normal. From these and other data it was concluded that oxidation and phosphorylation were partially uncoupled. Which of the following statements about this patient is correct?
A. The rate of electron transport is abnormally low.
B. The proton gradient across the inner mitochondrial membrane is greater than normal.
C. ATP levels in the mitochondria are greater than nor-
mal.
D. Cyanide would not inhibit electron flow.
E. The patient shows hypermetabolism and elevated core temperature.
6.2 Explain why and how the malate-aspartate shuttle moves NADH reducing equivalents from the cytosol to the mitochondrial matrix.
6.3 CO binds to and inhibits Complex IV of the electron transport chain. What effect, if any, should this respiratory inhibitor have on oxidative phosphorylation?
Correct answer = E. When phosphorylation is partially uncoupled from electron flow, one would expect a decrease in the proton gradient across the inner mitochondrial membrane and, hence, impaired ATP synthesis. In an attempt to compensate for this defect in energy capture, metabolism and electron flow to oxygen is increased. This hypermetabolism will be accompanied by elevated body temperature because the energy in fuels is largely wasted, appearing as heat. The electron transport chain will still be inhibited by cyanide.
There is no transporter for NADH in the inner mitochondrial membrane. However, NADH can be oxidized to NAD* by the cytoplasmic isozyme of malate dehydrogenase as oxaloacetate is reduced to malate. The malate is transported across the inner membrane, and the mitochondrial isozyme of malate dehydrogenase oxidizes it to oxaloacetate as mitochondrial NAD* is reduced to NADH. This NADH can be oxidized by Complex | of the electron transport chain, generating three ATP through the coupled processes of respiration and oxidative phosphorylation.
Inhibition of the electron transport chain by respiratory inhibitors such as CO results in an inability to maintain the proton gradient. Oxidative phosphorylation is therefore inhibited, as are ancillary reactions, because they also require the proton gradient.

Carbohydrates are the most abundant organic molecules in nature. They have a wide range of functions, including providing a significant fraction of the dietary calories for most organisms, acting as a storage form of energy in the body, and serving as cell membrane components that mediate some forms of intercellular communication. Carbohydrates also serve as a structural component of many organisms, including the cell walls of bacteria, the exoskeleton of many insects, and the fibrous cellulose of plants. The empiric formula for many of the simpler carbohydrates is (CH20)n, hence the name "hydrate of carbon."

Monosaccharides (simple sugars) can be classified according to the number of carbon atoms they contain. Examples of some monosaccharides commonly found in humans are listed in Figure 7.1. Carbohydrates with an aldehyde as their most oxidized functional group are called aldoses, whereas those with a keto as their most oxidized functional group are called ketoses (Figure 7.2). For example, glyceraldehyde is an aldose, whereas dihydroxyacetone is a ketose. Carbohydrates that have a free carbonyl group have the suffix —ose. [Note: Ketoses (with some exceptions, for example, fructose) have an additional two letters in their suffix: -ulose, for example, xylulose.] Monosaccharides can be linked by glycosidic bonds to create larger structures (Figure 7.3). Disaccharides contain two monosaccharide units, oligosaccharides contain from three to about ten monosaccharide units, whereas polysaccharides contain more than ten monosaccharide units, and can be hundreds of sugar units in length.

Compounds that have the same chemical formula but have different structures are called isomers. For example, fructose, glucose, mannose, and galactose are all isomers of each other, having the same chemical formula, CgH;20¢. Carbohydrate isomers that differ in configuration around only one specific carbon atom (with the exception of the carbonyl carbon, see "anomers" below) are defined as epimers of each other. For example, glucose and galactose are C-4

Examples of monosaccharides found in humans, classified according to the number of carbons they contain.

C-2 and C-4 epimers and an isomer of glucose.
epimers—their structures differ only in the position of the -OH group at carbon 4. [Note: The carbons in sugars are numbered beginning at the end that contains the carbonyl carbon—that is, the aldehyde or keto group (Figure 7.4).] Glucose and mannose are C-2 epimers. However, galactose and mannose are NOT epimers—they differ in the position of -OH groups at two carbons (2 and 4) and are, therefore, defined only as isomers (see Figure 7.4).

A special type of isomerism is found in the pairs of structures that are mirror images of each other. These mirror images are called enantiomers, and the two members of the pair are designated as a Dand an L-sugar (Figure 7.5). The vast majority of the sugars in humans are D-sugars. In the D isomeric form, the -OH group on the asymmetric carbon (a carbon linked to four different atoms or groups) farthest from the carbonyl carbon is on the right, whereas in the L-isomer it is on the left. Enzymes known as racemases are able to interconvert Dand L-isomers.

Less than 1% of each of the monosaccharides with five or more carbons exists in the open-chain (acyclic) form. Rather, they are predominantly found in a ring (cyclic) form, in which the aldehyde (or keto) group has reacted with an alcohol group on the same sugar, making the carbonyl carbon (carbon 1 for an aldose or carbon 2 for a ketose) asymmetric. [Note: Pyranose refers to a six-membered ring consisting of five carbons and one oxygen, for example, glucopyranose (Figure 7.6), whereas furanose denotes a five-membered ring with four carbons and one oxygen.]
1.
Anomeric carbon: Cyclization creates an anomeric carbon (the former carbonyl carbon), generating the « and B configurations of the sugar, for example, o-D-glucopyranose and B-D-glucopryanose (see Figure 7.6). These two sugars are both glucose but are anomers of each other. [Note: In the a configuration, the OH on the anomeric C projects to the same side as the ring in a modified Fischer projection formula (Figure 7.6A), and is trans to the CH2OH group in a Haworth projection formula (Figure 7.6B). Because the ao and B forms are not mirror images, they are referred to as diastereomers.] Enzymes are able to distinguish between these two structures and use one or the other preferentially. For example, glycogen is synthesized from a-D-glucopyranose, whereas cellulose is synthesized from B-D-glucopyranose. The cyclic « and B anomers of a sugar in solution are in equilibrium with each other, and can be spontaneously interconverted (a process called mutarotation, see Figure 7.6).

2. Reducing sugars: If the hydroxyl group on the anomeric carbon of a cyclized sugar is not linked to another compound by a glycosidic bond, the ring can open. The sugar can act as a reducing agent, and is termed a reducing sugar. Such sugars can react with chromogenic agents (for example, Benedict's reagent or Fehling's solution) causing the reagent to be reduced and colored, with the aldehyde group of the acyclic sugar becoming oxidized. [Note:
Only the state of the oxygen in the aldehyde group determines the sugar is reducing or nonreducing.]
|
urine. A positive result is indicative of an underlying pathology because sugars are not normally present in urine, and can be followed up by more A colorimetric test can detect a reducing sugar in specific tests to identify the reducing sugar.

Monosaccharides can be joined to form disaccharides, oligosaccharides, and polysaccharides. Important disaccharides include lactose (galactose + glucose), sucrose (glucose + fructose), and maltose (glucose + glucose). Important polysaccharides include branched glycogen (from animal sources) and starch (plant sources) and unbranched cellulose (plant sources); each is a polymer of glucose. The bonds that link sugars are called glycosidic bonds. These are formed by enzymes known as glycosyltransferases that use nucleotide sugars such as UDP-glucose as substrates.
- 1. Naming glycosidic bonds: Glycosidic bonds between sugars are named according to the numbers of the connected carbons, and with regard to the position of the anomeric hydroxyl group of the sugar involved in the bond. If this anomeric hydroxyl is in the a configuration, the linkage is an a-bond. If it is in the B configuration, the linkage is a B-bond. Lactose, for example, is synthesized by forming a glycosidic bond between carbon 1 of B-galactose and carbon 4 of glucose. The linkage is, therefore, a B(1—4) glycosidic bond (see Figure 7.3). [Note: Because the anomeric end of the glucose residue is not involved in the glycosidic linkage it (and, therefore, lactose) remains a reducing sugar.]

Carbohydrates can be attached by glycosidic bonds to non-carbohydrate structures, including purine and pyrimidine bases (found in

A The interconversion (mutarotation) of the « and B anomeric forms of glucose shown as modified Fischer projection formulas. B. The interconversion shown as Haworth projection formulas. Carbon 1 is the anomeric carbon. [Note: Glucose is a reducing sugar.]
if

Hydrolysis of a glycosidic bond.
nucleic acids), aromatic rings (such as those found in steroids and ape . . + + bilirubin), proteins (found in glycoproteins and proteoglycans), and lipids (found in glycolipids).
1. Nand O-glycosides: If the group on the non-carbohydrate molecule to which the sugar is attached is an —NHe group, the structure is an N-glycoside and the bond is called an N-glycosidic link. If the group is an —OH, the structure is an O-glycoside, and the bond is an O-glycosidic link (Figure 7.7). [Note: All sugar—sugar glycosidic bonds are O-type linkages.]

The principal sites of dietary carbohydrate digestion are the mouth and intestinal lumen. This digestion is rapid and is catalyzed by enzymes known as glycoside hydrolases (glycosidases) that hydrolyze glycosidic bonds. Because there is little monosaccharide present in diets of mixed animal and plant origin, the enzymes are primarily endoglycosidases that hydrolyze polysaccharides and oliosaccharides, and disaccharidases that hydrolyse triand disaccharides into their reducing sugar components (Figure 7.8). Glycosidases are usually specific for the structure and configuration of the glycosyl residue to be removed, as well as for the type of bond to be broken. The final products of carbohydrate digestion are the monosaccharides, glucose, galactose and fructose, which are absorbed by cells of the small intestine.

The major dietary polysaccharides are of plant (starch, composed of amylose and amylopectin) and animal (glycogen) origin. During mastication, salivary a-amylase acts briefly on dietary starch and glycogen, hydrolyzing random a(1—4) bonds. [Note: There are both a(1—4)and B(1->4)-endoglucosidases in nature, but humans do not produce the latter. Therefore, we are unable to digest cellulose— a carbohydrate of plant origin containing B(1—4) glycosidic bonds between glucose residues.] Because branched amylopectin and glycogen also contain (1-6) bonds, which a@-amylase cannot hydrolyze, the digest resulting from its action contains a mixture of short, branched and unbranched oligosaccharides kown as dextrins (Figure 7.9) [Note: Disaccharides are also present as they, too, are resistant to amylase.] Carbohydrate digestion halts temporarily in the stomach, because the high acidity inactivates salivary a-amylase.

When the acidic stomach contents reach the small intestine, they are neutralized by bicarbonate secreted by the pancreas, and pancreatic a-amylase continues the process of starch digestion.

The final digestive processes occur primarily at the mucosal lining of the upper jejunum, and include the action of several disacchari-
dases (Figure 7.10). For example, isomaltase cleaves the a(1-6) bond in isomaltose and maltase cleaves maltose and maltotriose, each producing glucose, sucrase cleaves sucrose producing glucose and fructose, and lactase ($-galactosidase) cleaves lactose producing galactose and glucose. Trehalose, an a(1-1) disaccharide of glucose found in mushrooms and other fungi, is cleaved by trehalase. These enzymes are secreted through, and remain associated with, the luminal side of the brush border membranes of the intestinal mucosal cells. [Note: The substrates for isomaltase are broader than its name suggests, as it hydrolyzes the majority of maltose.]
Sucrase and isomaltase are enzymic activities of a single protein which is cleaved into two func2 Single Pro! : : . tional subunits that remain associated in the cell membrane, forming the sucrase-isomaltase complex. Maltase forms a similar complex with an exoglucosidase (glucoamylase) that cleaves a(1—4) glycosidic bonds in dextrins.

The duodenum and upper jejunum absorb the bulk of the dietary sugars. However, different sugars have different mechanisms of absorption. For example, galactose and glucose are transported into the mucosal cells by an active, energy-requiring process that requires a concurrent uptake of sodium ions; the transport protein is the sodium-dependent glucose cotransporter 1 (SGLT-1). Fructose uptake requires a sodium-independent monosaccharide transporter (GLUT-5) for its absorption. All three monosaccharides are transported from the intestinal mucosal cell into the portal circulation by yet another transporter, GLUT-2. (See p. 97 for a discussion of these transporters.)

The overall process of carbohydrate digestion and absorption is so efficient in healthy individuals that ordinarily all digestible dietary carbohydrate is absorbed by the time the ingested material reaches the lower jejunum. However, because it is monosaccharides that are -? on . . a absorbed, any defect in a specific disaccharidase activity of the . . . intestinal mucosa causes the passage of undigested carbohydrate into the large intestine. As a consequence of the presence of this osmotically active material, water is drawn from the mucosa into the large intestine, causing osmotic diarrhea. This is reinforced by the bacterial fermentation of the remaining carbohydrate to twoand three-carbon compounds (which are also osmotically active) plus large volumes of COz and Hea gas, causing abdominal cramps, diarrhea, and flatulence.

Degradation of dietary glycogen by salivary or pancreatic a-amylase.

Digestion of carbohydrates. [Note: Indigestible cellulose enters the colon and is excreted.]

Abnormal lactose metabolism.
- 1. Digestive enzyme deficiencies: Genetic deficiencies of the individual disaccharidases result in disaccharide intolerance. Alterations in disaccharide degradation can also be caused by a variety of intestinal diseases, malnutrition, or drugs that injure the mucosa of the small intestine. For example, brush border enzymes are rapidly lost in normal individuals with severe diarrhea, causing a temporary, acquired enzyme deficiency. Thus, patients suffering or recovering from such a disorder cannot drink or eat significant amounts of dairy products or sucrose without exacerbating the diarrhea.
2.
Lactose intolerance: More than three quarters of the world's adults are lactose intolerant (Figure 7.11). This is particularly manifested in certain populations. For example, up to 90% of adults of African or Asian descent are /actase-deficient and, therefore, are less able to metabolize lactose than individuals of Northern European origin. The age-dependent loss of lactase activity represents a reduction in the amount of enzyme rather than a modified inactive enzyme. It is thought to be caused by small variations in the DNA sequence of a region on chromosome 2 that controls expression of the gene for lactase, also on ar : chromosome 2. Treatment for this disorder is to reduce consumption of milk while eating yogurts and cheeses, as well as green vegetables such as broccoli, to ensure adequate calcium intake; to use /actase-treated products; or to take /actase in pill form prior to eating. [Note: Because the loss of /actase is the norm for most of the world's adults, use of the term "adult hypolactasia" for lactose intolerance is becoming more common.]
3. Sucrase-isomaltase complex deficiency: This deficiency results in an intolerance of ingested sucrose. The disorder is found in about 10% of the Inuit people of Greenland and Canada, whereas 2% of North Americans are heterozygous for the deficiency. Treatment includes the dietary restriction of sucrose, and enzyme replacement therapy.
4. Diagnosis: Identification of a specific enzyme deficiency can be obtained by performing oral tolerance tests with the individual disaccharides. Measurement of hydrogen gas in the breath is a reliable test for determining the amount of ingested carbohydrate not absorbed by the body, but which is metabolized instead by the intestinal flora (see Figure 7.11).

Monosaccharides (simple sugars, Figure 7.12) containing an aldehyde group are called aldoses and those with a keto group are called ketoses. Disaccharides, oligosaccharides, and polysaccharides consist of monosaccharides linked by glycosidic bonds. Compounds with the same chemical formula are called isomers. If two monosaccharide isomers differ in configuration around one specific carbon atom (with the exception of the carbonyl carbon), they are defined as epimers of each other. If a pair of sugars are mirror images (enantiomers), the two members of the pair are designated as Dand L-sugars. When a

Key concept map for structure of monosaccharides.
sugar cyclizes, an anomeric carbon is created from the aldehyde group of an aldose or keto group of a ketose. This carbon can have two configurations, « or B. If the aldehyde group on an acyclic sugar gets oxidized as a chromogenic agent gets reduced, that sugar is a reducing sugar. A sugar with its anomeric carbon linked to another structure is called a glycosyl residue. Sugars can be attached either to an -NH2 or an —OH group, producing Nand O-glycosides. Salivary a-amylase acts on dietary polysaccharides (glycogen, amylose, amylopectin), producing oligosaccharides. Pancreatic a-amylase continues the process of polysaccharide digestion. The final digestive processes occur at the mucosal lining of the small intestine. Several disaccharidases [for example, lactase (B-galactosidase), sucrase, maltase, and isomaltase] produce monosaccharides (glucose, galactose, and fructose). These enzymes are secreted by and remain associated with the luminal side of the brush border membranes of intestinal mucosal cells. Absorption of the monosaccharides requires specific transporters. If carbohydrate degradation is deficient (as a result of heredity, intestinal disease, malnutrition, or drugs that injure the mucosa of the small intestine), undigested carbohydrate will pass into the large intestine, where it can cause osmotic diarrhea. Bacterial fermentation of the compounds produces large volumes of COz and Hp gas, causing abdominal cramps, diarrhea, and flatulence. Lactose intolerance, caused by a lack of lactase, is by far the most common of these deficiencies.

. . . 7.1 Which of the following statements best describes glucose?
A. It is a ketose and usually exists as a furanose in solution.
ring
B. It is a C-4 epimer of galactose.
C. It is utilized in biological systems only in the L-isomeric form.
D. It is produced from dietary starch by the action of aamylase.
E. Homopolysaccharides of glucose, formed by the action of glycosyltransferases, are always branched molecules that contain only B-glycosidic linkages.
7.2 A young black man entered his physician's office complaining of bloating and diarrhea. His eyes were sunken and the physician noted additional signs of dehydration. The patient's temperature was normal. He explained that the episode had occurred following a birthday party at which he had participated in an ice cream eating contest. The patient reported prior episodes of a similar nature following ingestion of a significant amount of dairy products. This clinical picture is most probably due to a deficiency in:
A. salivary a-amylase.
B. isomaltase.
C. pancreatic o-amylase.
D.sucrase.
E. lactase.
- 7.3 Routine examination of the urine of an asymptomatic pediatric patient showed a positive reaction with Clinitest (a copper reduction method of detecting reducing sugars), but a negative reaction with the glucose oxidase test. Which one of the following sugars is least likely to be present (assuming a single elevated saccharide)?
A. Lactose
B. Fructose
C. Sucrose
D. Xylulose
E. Galactose
- 7.4 a-Glucosidase inhibitors such as acarbose and miglitol taken with meals are used in the treatment of diabetes. Explain. What effect should these drugs have on the digestion of lactose?

Correct answer = B. Glucose and galactose differ only in configuration around carbon 4, and so are C-4 epimers that are interconvertible by the action of an epimerase. Glucose is an aldose sugar that typically exists as a pyranose ring in solution; fructose, however, is a ketose with a furanose ring. The D-isomeric form of carbohydrates is most typically the form found in biologic systems, in contrast to amino acids. Salivary amylase does not produce monosaccharides. Homopolysaccharides of glucose include branched glycogen in which the glycosidic linkages are the a form, as well as unbranched cellulose that has B linkages.
Correct answer = E. The physical symptoms suggest a deficiency in an enzyme responsible for carbohydrate degradation. The symptoms observed following the ingestion of dairy products suggest that the patient is deficient in lactase.
Correct answer = C. Clinitest is a nonspecific test that produces a change in color if urine is positive for reducing substances, including reducing sugars (glucose, fructose, galactose, xylulose, lactose), amino acids, ascorbic acid, and certain drugs and drug metabolites. Because sucrose is not a reducing sugar, it is not detected by Clinitest. Glucose oxidase method will not detect increased levels of galactose or other sugars in urine. It is therefore important that a copper reduction method be used as a screening test. In those instances when the copper method is positive and the glucose oxidase method is negative, glucosuria is ruled out.
a-Glucosidase inhibitors slow the production of glucose from dietary carbohydrates, thereby reducing the post-prandial rise in blood glucose and facilitating better blood glucose control in diabetics. These drugs have no effect on lactose digestion because the disaccharide lactose contains a B-glycosidic bond, not an a.

In Chapter 5, individual enzymic reactions were analyzed in an effort to explain the mechanisms of catalysis. However, in cells, these reactions rarely occur in isolation, but rather are organized into multistep sequences called pathways, such as that of glycolysis (Figure 8.1). Ina pathway, the product of one reaction serves as the substrate of the subsequent reaction. Different pathways can also intersect, forming an integrated and purposeful network of chemical reactions. These are collectively called metabolism, which is the sum of all the chemical changes occurring in a cell, a tissue, or the body. Most pathways can be classified as either catabolic (degradative) or anabolic (synthetic). Catabolic reactions break down complex molecules, such as proteins, polysaccharides, and lipids, to a few simple molecules, for example, COz, NH3 (ammonia), and water. Anabolic pathways form complex end products from simple precursors, for example, the synthesis of the polysaccharide, glycogen, from glucose. [Note: Pathways that regenerate a component are called cycles.] In the following chapters, this text focuses on the central metabolic pathways that are involved in synthesizing and degrading carbohydrates, lipids, and amino acids.

It is convenient to investigate metabolism by examining its component pathways. Each pathway is composed of multienzyme sequences, and each enzyme, in turn, may exhibit important catalytic or regulatory features. To provide the reader with the "big picture,' a metabolic map containing the important central pathways of energy metabolism is presented in Figure 8.2. This map is useful in tracing connections between pathways, visualizing the purposeful "movement" of metabolic intermediates, and picturing the effect on the flow of intermediates if a pathway is blocked, for example, by a drug or an inherited deficiency of an enzyme. Throughout the next three units of this book, each pathway under discussion will be repeatedly featured as part of the major metabolic map shown in Figure 8.2.

Catabolic reactions serve to capture chemical energy in the form of adenosine triphosphate (ATP) from the degradation of energy-rich fuel molecules. Catabolism also allows molecules in the diet (or nutrient molecules stored in cells) to be converted into building blocks needed for the synthesis of complex molecules. Energy generation by degradation of complex molecules occurs in three stages as shown in Figure 8.3. [Note: Catabolic pathways are typically oxidative, and require coenzymes such as NAD*.]

Important reactions of intermediary metabolism. Several important pathways to be discussed in later chapters are highlighted. Curved reaction arrows ( £7 ) indicate forward and reverse reactions that are catalyzed by different enzymes. The straight arrows ( <> ~ indicate forward and reverse reactions that are catalyzed by the same enzyme. Blue text = intermediates of carbohydrate metabolism; brown text = intermediates of lipid metabolism; green text = intermediates of protein metabolism.

Three stages of catabolism.
1. Hydrolysis of complex molecules: In the first stage, complex molecules are broken down into their component building blocks. For example, proteins are degraded to amino acids, polysaccharides to monosaccharides, and fats (triacylglycerols) to free fatty acids and glycerol.
2. Conversion of building blocks to simple intermediates: In the second stage, these diverse building blocks are further degraded to acetyl coenzyme A (CoA) and a few other, simple molecules. Some energy is captured as ATP, but the amount is small compared with the energy produced during the third stage of catabolism.
3. Oxidation of acetyl CoA: The tricarboxylic acid (TCA) cycle (see p. 109) is the final common pathway in the oxidation of fuel molecules that produce acetyl CoA. Oxidation of acetyl CoA generates large amounts of ATP via oxidative phosphorylation as electrons flow from NADH and FADH+z to oxygen (see p. 73).

Anabolic reactions combine small molecules, such as amino acids, to form complex molecules, such as proteins (Figure 8.4). Anabolic reactions require energy (are endergonic), which is generally provided by the breakdown of ATP to adenosine diphosphate (ADP) and inorganic phosphate (Pj). Anabolic reactions often involve chemical reductions in which the reducing power is most frequently provided by the electron donor NADPH (see p. 147). Note that catabolism is a convergent process—that is, a wide variety of molecules are transformed into a few common end products. By contrast, anabolism is a divergent process in which a few biosynthetic precursors form a wide variety of polymeric or complex products.

The pathways of metabolism must be coordinated so that the production of energy or the synthesis of end products meets the needs of the cell. Furthermore, individual cells do not function in isolation but, rather, are part of a community of interacting tissues. Thus, a sophisticated communication system has evolved to coordinate the functions of the

Some commonly used mechanisms for transmission of regulatory signals between cells.

Structure of a typical G proteincoupled receptor (GPCR) of the plasma membrane.
body. Regulatory signals that inform an individual cell of the metabolic state of the body as a whole include hormones, neurotransmitters, and the availability of nutrients. These, in turn, influence signals generated within the cell (Figure 8.5).

The rate of a metabolic pathway can respond to regulatory signals that arise from within the cell. For example, the rate of a pathway may be influenced by the availability of substrates, product inhibition, or alterations in the levels of allosteric activators or inhibitors. These intracellular signals typically elicit rapid responses, and are important for the moment-to-moment regulation of metabolism.

The ability to respond to extracellular signals is essential for the survival and development of all organisms. Signaling between cells provides for long-range integration of metabolism, and usually results in a response that is slower than is seen with signals that originate within the cell. Communication between cells can be mediated, for example, by surface-to-surface contact and, in some tissues, by formation of gap junctions, allowing direct communication between the cytoplasms of adjacent cells. However, for energy metabolism, the most important route of communication is chemical signaling between cells by bloodborne hormones or by neurotransmitters.

Hormones or neurotransmitters can be thought of as signals, and their receptors as signal detectors. Each component serves as a link in the communication between extracellular events and chemical changes within the cell. Many receptors signal their recognition of a bound ligand by initiating a series of reactions that ultimately result in a specific intracellular response. "Second messenger" molecules—so named because they intervene between the original messenger (the neurotransmitter or hormone) and the ultimate effect on the cell—are part of the cascade of events that translates hormone or neurotransmitter binding into a cellular response. Two of the most widely recognized second messenger systems are the calcium/phosphatidylinositol system (see p. 205), and the adenylyl cyclase system, which is particularly important in regulating the pathways of intermediary metabolism.

The recognition of a chemical signal by some membrane receptors, such as the Band a2-adrenergic receptors, triggers either an increase or a decrease in the activity of adenylyl cyclase (adenylate cyclase). This is a membrane-bound enzyme that converts ATP to 3',5'-adenosine monophosphate (also called cyclic AMP or cAMP). The chemical signals are most often hormones or neurotransmitters, each of which binds to a unique type of membrane receptor. Therefore, tissues that respond to more than one chemical signal must have several different receptors, each of which can be linked to adenylyl cyclase. These receptors, known as G protein-coupled receptors (GPCR), are characterized by an extracellular ligand-binding region, seven transmembrane helices, and an intracellular domain that interacts with G proteins (Figure 8.6).

- 1. GTP-dependent regulatory proteins: The effect of the activated, + + + . occupied GPCR on second messenger formation is not direct but, rather, is mediated by specialized trimeric proteins (a, B, y subunits) of the cell membrane. These proteins, referred to as G proteins because they bind guanosine nucleotides (GTP and GDP), form a link in the chain of communication between the receptor and adenylyl cyclase. In the inactive form of a G protein, the a-subunit is bound to GDP (Figure 8.7). Binding of ligand causes a conformational change in the receptor, triggering replacement of this GDP with GTP. The GTP-bound form of the subunit dissociates from the By subunits and moves to adenylyl cyclase, which is thereby activated. Many molecules of active Go protein are formed by one activated receptor. [Note: The ability of a hormone or neurotransmitter to stimulate or inhibit adenylyl cyclase depends on the type of Ga protein that is linked to the receptor. One family of G proteins, designated Gs, stimulates adenyly! cyclase; another family, designated Gj, inhibits the enzyme (not shown in Figure 8.7).] The actions of the Ga—GTP complex are short-lived because Ga has an inherent G7Pase activity, resulting in the rapid hydrolysis of GTP to GDP. This causes inactivation of the Ga, its dissociation from adenylyl cyclase and reassociation with the By dimer.
Toxins from Vibrio cholerae (cholera) and Bordetella pertussis (whooping cough) cause inappropriate activation of adenylyl cyclase through covalent modification (ADP-ribosylation) of different G proteins. With cholera, the GTPase activity of Gas is inhibited. With whooping cough, Go; is inactivated.

. Protein kinases: The next key link in the cAMP second messenger system is the activation by cAMP of a family of enzymes called cAMP-independent protein kinases, for example, protein kinase A (Figure 8.8). Cyclic AMP activates protein kinase A by binding to its two regulatory subunits, causing the release of active catalytic subunits. The active subunits catalyze the transfer of phosphate from ATP to specific serine or threonine residues of protein substrates. The phosphorylated proteins may act directly on the cell's ion channels, or, if enzymes, may become activated or inhibited. Protein kinase A can also phosphorylate proteins that bind to DNA, causing changes in gene expression. [Note: Several types of protein kinases are not cAMP-dependent, for example, protein kinase C described on p. 205.]
. Dephosphorylation of proteins: The phosphate groups added to proteins by protein kinases are removed by protein phosphatases—enzymes that hydrolytically cleave phosphate esters (see Figure 8.8). This ensures that changes in protein activity induced by phosphorylation are not permanent.
4. Hydrolysis of cAMP: cAMP is rapidly hydrolyzed to 5'-AMP by cAMP. phosphodiesterase, one of a family of enzymes that cleave the cyclic 3',5'-phosphodiester bond. 5'-AMP is not an intracellular signaling molecule. Thus, the effects of neurotransmitteror hormone-mediated

The recognition of chemical signals by certain membrane receptors triggers an increase (or, less often, a decrease) in the activity of adenylyl cyclase.

Actions of cAMP.
increases of cAMP are rapidly terminated if the extracellular signal is removed. [Note: Phosphodiesterase is inhibited by methylxanthine derivatives, such as theophylline and caffeine."

The glycolytic pathway is employed by all tissues for the breakdown of glucose to provide energy (in the form of ATP) and intermediates for other metabolic pathways. Glycolysis is at the hub of carbohydrate metabolism because virtually all sugars—whether arising from the diet or from catabolic reactions in the body—can ultimately be converted to glucose (Figure 8.9A). Pyruvate is the end product of glycolysis in cells with mitochondria and an adequate supply of oxygen. This series of ten reactions is called aerobic glycolysis because oxygen is required to reoxidize the NADH formed during the oxidation of glyceraldehyde 3-phosphate (Figure 8.9B). Aerobic glycolysis sets the stage for the oxidative decarboxylation of pyruvate to acetyl CoA, a major fuel of the TCA (or citric acid) cycle. Alternatively, pyruvate is reduced to lactate as NADH is oxidized to NAD* (Figure 8.9C). This conversion of glucose to lactate is called anaerobic glycolysis because it can occur without the participation of oxygen. Anaerobic glycolysis allows the production of ATP in tissues that lack mitochondria (for example, red blood cells) or in cells deprived of sufficient oxygen.

Glucose cannot diffuse directly into cells, but enters by one of two transport mechanisms: a Na*-independent, facilitated diffusion transport system or a Na*-monosaccharide cotransporter system.

A. Glycolysis shown as one of the essential pathways of energy metabolism. B. Reactions of aerobic glycolysis. C. Reactions of anaerobic glycolysis.

This system is mediated by a family of 14 glucose transporters in cell membranes. They are designated GLUT-1 to GLUT-14 (glucose transporter isoforms 1-14). These transporters exist in the membrane in two conformational states (Figure 8.10). Extracellular glucose binds to the transporter, which then alters its conformation, transporting glucose across the cell membrane.
1. Tissue specificity of GLUT gene expression: The glucose transporters display a tissue-specific pattern of expression. For example, GLUT-3 is the primary glucose transporter in neurons. GLUT-1 is abundant in erythrocytes and blood brain barrier, but is low in adult muscle, whereas GLUT-4 is abundant in adipose tissue and skeletal muscle. [Note: The number of GLUT-4 transporters active in these tissues is increased by insulin. (See p. 311 for a discussion of insulin and glucose transport.)] The other GLUT isoforms also have tissue-specific distributions.
2.
Specialized functions of GLUT isoforms: In facilitated diffusion, glucose movement follows a concentration gradient, that is, from a high glucose concentration to a lower one. For example, GLUT-1, GLUT-3, and GLUT-4 are primarily involved in glucose uptake from the blood. In contrast, GLUT-2, which is found in the liver and kidney, can either transport glucose into these cells when blood glucose levels are high, or transport glucose from these cells when blood glucose levels are low (for example, during fasting). [Note: GLUT-2 is also found in pancreatic B cells.] GLUT-5 is unusual in that it is the primary transporter for fructose (instead of glucose) in the small intestine and the testes.

This is an energy-requiring process that transports glucose "against" a concentration gradient—that is, from low glucose concentrations outside the cell to higher concentrations within the cell. This system is a carrier-mediated process in which the movement of glucose is coupled to the concentration gradient of Na*, which is transported into the cell at the same time. The carrier is a sodium-dependent-glucose transporter or SGLT. This type of transport occurs in the epithelial cells of the intestine (see p. 87), renal tubules, and choroid plexus. [Note: The choroid plexus, part of the blood brain barrier, also contains GLUT-1.]

The conversion of glucose to pyruvate occurs in two stages (Figure 8.11). The first five reactions of glycolysis correspond to an energy investment phase in which the phosphorylated forms of intermediates are synthesized at the expense of ATP. The subsequent reactions of glycolysis constitute an energy generation phase in which a net of two molecules of ATP are formed by substrate-level phosphorylation (see p. 102) per glucose molecule metabolized.

Schematic representation of the facilitated transport of glucose through a cell membrane. [Note: GLUT proteins contain 12 transmembrane helices.]

Effect of glucose concentration on the rate of phosphorylation catalyzed by hexokinase and glucokinase.

Phosphorylated sugar molecules do not readily penetrate cell membranes, because there are no specific transmembrane carriers for these compounds, and because they are too polar to diffuse through the lipid core of membranes. The irreversible phosphorylation of glucose (Figure 8.12), therefore, effectively traps the sugar as cytosolic glucose 6-phosphate, thus committing it to further metabolism in the cell. Mammals have several isozymes of the enzyme hexokinase that catalyze the phosphorylation of glucose to glucose 6-phosphate.
1.
Hexokinase: In most tissues, the phosphorylation of glucose is catalyzed by hexokinase, one of three regulatory enzymes of glycolysis (see also phosphofructokinase and pyruvate kinase). Hexokinase has broad substrate specificity and is able to phosphorylate several hexoses in addition to glucose. Hexokinase is inhibited by the reaction product, glucose 6-phosphate, which accumulates when further metabolism of this hexose phosphate is reduced. Hexokinase has a low Km (and, therefore, a high affinity, see p. 59) for glucose. This permits the efficient phosphorylation and subsequent metabolism of glucose even when tissue concentrations of glucose are low (Figure 8.13). Hexokinase, however, has a low Vmax for glucose and, therefore, cannot sequester (trap) cellular phosphate in the form of phosphorylated hexoses, or phosphorylate more sugars than the cell can use.
2.
Glucokinase: In liver parenchymal cells and B cells of the pancreas, glucokinase (also called hexokinase D, or type IV) is the predominant enzyme responsible for the phosphorylation of glucose. In B cells, glucokinase functions as the glucose sensor, determining the threshold for insulin secretion (see p. 310). In the liver, the enzyme facilitates glucose phosphorylation during hyperglycemia. [Note: Hexokinase also serves as a glucose sensor in neurons of the hypothalamus, playing a key role in the adrenergic response to hypoglycemia (see p. 315.] Despite the popular but misleading name glucokinase, the sugar specificity of the enzyme is similar to that of other hexokinase isozymes.

a. Kinetics: Glucokinase differs from hexokinase in several important properties. For example, it has a much higher Km, requiring a higher glucose concentration for half-saturation (see Figure 8.13). Thus, glucokinase functions only when the intracellular concentration of glucose in the hepatocyte is elevated, such as during the brief period following consumption of a carbohydrate-rich meal, when high levels of glucose are delivered to the liver via the portal vein. Glucokinase has a high Vmax, allowing the liver to effectively remove the flood of glucose delivered by the portal blood. This prevents large amounts of glucose from entering the systemic circulation following a carbohydraterich meal, and thus minimizes hyperglycemia during the absorptive period. [Note: GLUT-2 insures that blood glucose equilibrates rapidly across the membrane of the hepatocyte.]

Glucokinase activity is not directly inhibited by glucose 6-phosphate as are the other hexokinases, but rather is indirectly inhibited by fructose 6-phosphate (which is in equilibrium with
glucose 6-phosphate, a product of glucokinase), and is indirectly stimulated by glucose (a substrate of glucokinase) via the following mechanism. Glucokinase regulatory protein (GKRP) in the liver regulates the activity of glucokinase through reversible binding. In the presence of fructose 6phosphate, glucokinase is translocated into the nucleus and binds tightly to the regulatory protein, thus rendering the enzyme inactive (Figure 8.14). When glucose levels in the blood (and also in the hepatocyte, as a result of GLUT-2) increase, glucokinase is released from the regulatory protein, and the enzyme re-enters the cytosol where it phosphorylates glucose to glucose 6-phosphate. [Note: Fructose 1-phosphate inhibits formation of the glucokinase-GKRP complex.]
Glucokinase functions as a glucose sensor in the maintenance of blood glucose homeostasis. Mutations that decrease the activity of glucokinase are the cause of a rare form of diabetes, maturity onset diabetes of the young type 2 (MODY 2).

The isomerization of glucose 6-phosphate to fructose 6-phosphate is catalyzed by phosphoglucose isomerase (Figure 8.15). The reaction is readily reversible and is not a rate-limiting or regulated step.

The irreversible phosphorylation reaction catalyzed by phosphofructokinase-1 (PFK-1) is the most important control point and the rate-limiting and committed step of glycolysis (Figure 8.16). PFK-7 is controlled by the available concentrations of the substrates ATP and fructose 6-phosphate, and by regulatory substances described below.

Regulation by energy levels within the cell: PFK-7 is inhibited allosterically by elevated levels of ATP, which act as an "energyrich" signal indicating an abundance of high-energy compounds. Elevated levels of citrate, an intermediate in the TCA cycle (see p. 109), also inhibit PFK-1. Conversely, PFK-1 is activated allosterically by high concentrations of AMP, which signal that the cell's energy stores are depleted. [Note: Citrate inhibition favors the use of glucose for glycogen synthesis, see p.125.]
- 2. Regulation by fructose 2,6-bisphosphate: Fructose 2,6-bisphosphate is the most potent activator of PFK-1 (see Figure 8.16), and is able to activate the enzyme even when ATP levels are high. Fructose 2,6-bisphosphate is formed by phosphofructokinase-2 (PFK-2), an enzyme different than PFK-1. PFK-2 is a bifunctional protein that has both the kinase activity that produces fructose 2,6-bisphosphate and a phosphatase activity that dephosphorylates fructose 2,6-bisphosphate back to fructose 6-phosphate. In liver, the kinase domain is active if dephosphorylated and is inactive if phosphorylated (Figure 8.17). [Note: Fructose 2,6-bisphos-

Regulation of glucokinase activity by glucokinase regulatory protein.

Aldose-ketose isomerization of glucose 6-phosphate to fructose 6-phosphate.
phate is an inhibitor of fructose 1,6-bisphosphatase, an enzyme of . . . . gluconeogenesis (see p. 120 for a discussion of the regulation of gluconeogenesis). The reciprocal actions of fructose 2,6-bisphosphate on glycolysis (activation) and gluconeogenesis (inhibition) ensure that both pathways are not fully active at the same time, preventing a futile cycle in which glucose would be converted to pyruvate followed by resynthesis of glucose from pyruvate.]
a. During the well-fed state: Decreased levels of glucagon and elevated levels of insulin, such as occur following a carbohydrate-rich meal, cause an increase in fructose 2,6-bisphosphate and, thus, in the rate of glycolysis in the liver (see Figure 8.17). Fructose 2,6-bisphosphate, therefore, acts as an intracellular signal, indicating that glucose is abundant.
b. During starvation: Elevated levels of glucagon and low levels of insulin, such as occur during fasting (see p. 327), decrease the intracellular concentration of hepatic fructose 2,6-bisphosphate. This results in a decrease in the overall rate of glycolysis and an increase in gluconeogenesis.

/ Energy investment phase (continued): Conversion of fructose 6-phosphate to triose phosphates.
Aldolase cleaves fructose 1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (see Figure 8.16). The reaction is reversible and not regulated. [Note: Aldolase B, the isoform in the liver and kidney, also cleaves fructose 1-phosphate, and functions in the metabolism of dietary fructose (see p. 138).]

Effect of elevated insulin concentration on the intracellular concentration of fructose 2,6-bisphosphate in liver. PFK-2 = phosphofructokinase-2, FBP-2 = fructose bisphosphatase-2.

Triose phosphate isomerase interconverts dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (see Figure 8.16). Dihydroxyacetone phosphate must be isomerized to glyceraldehyde 3-phosphate for further metabolism by the glycolytic pathway. This isomerization results in the net production of two molecules of glyceraldehyde 3-phosphate from the cleavage products of fructose 1,6bisphosphate.

The conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate by glyceraldehyde 3-phosphate dehydrogenase is the first oxidation-reduction reaction of glycolysis (Figure 8.18). [Note: Because there is only a limited amount of NAD* in the cell, the NADH formed by this reaction must be reoxidized to NAD* for glycolysis to continue. Two major mechanisms for oxidizing NADH are: 1) the NADH-linked conversion of pyruvate to lactate (anaerobic, see p. 96), and 2) oxidation of NADH via the respiratory chain (aerobic, see p. 75). The latter requires substrate shuttles (see p. 79.]

Synthesis of 1,3-bisphosphoglycerate (1,3-BPG): The oxidation of the aldehyde group of glyceraldehyde 3-phosphate to a carboxyl group is coupled to the attachment of P; to the carboxyl group. The high-energy phosphate group at carbon 1 of 1,3-BPG conserves much of the free energy produced by the oxidation of glyceraldehyde 3-phosphate. The energy of this high-energy phosphate drives the synthesis of ATP in the next reaction of glycolysis.
2. Mechanism of arsenic poisoning: The toxicity of arsenic is explained primarily by the inhibition of enzymes such as pyruvate dehydrogenase, which require lipoic acid as a coenzyme (see p. 110). However, pentavalent arsenic (arsenate) also can prevent net ATP and NADH production by glycolysis, without inhibiting the pathway itself. The poison does so by competing with inorganic phosphate as a substrate for glyceraldehyde 3-phosphate dehydrogenase, forming a complex that spontaneously hydrolyzes to form 3-phosphoglycerate (see Figure 8.18). By bypassing the synthesis of and phosphate transfer from 1,3BPG, the cell is deprived of energy usually obtained from the glycolytic pathway. [Note: Arsenic also replaces Pj on the F; domain of ATP synthase (see p. 78), resulting in formation of ADP-arsenate that is rapidly hydrolyzed.]

cells: Some of the 1,3-BPG is converted to 2,3-BPG by the action of bisphosphoglycerate mutase (see Figure 8.18). 2,3-BPG, which is found in only trace amounts in most cells, is present at high concentration in red blood cells (increases Oz delivery, see p. 31). 2,3-BPG is hydrolyzed by a phosphatase to 3-phosphoglycerate, which is also an intermediate in glycolysis (see Figure 8.18). In the red blood cell, glycolysis is modified by inclusion of these "shunt" reactions.

Energy generating phase: conversion of glyceraldehyde 3-phosphate to pyruvate.

Covalent modification of hepatic pyruvate kinase results in inactivation of enzyme.

When 1,3-BPG is converted to 3-phosphoglycerate, the high-energy phosphate group of 1,3-BPG is used to synthesize ATP from ADP (see Figure 8.18). This reaction is catalyzed by phosphoglycerate kinase, which, unlike most other kinases, is physiologically reversible. Because two molecules of 1,3-BPG are formed from each glucose molecule, this kinase reaction replaces the two ATP molecules consumed by the earlier formation of glucose 6-phosphate and fructose 1,6-bisphosphate. [Note: This is an example of substrate-level phosphorylation, in which the energy needed for the production of a highenergy phosphate comes from a substrate rather than from the electron transport chain (see J. below and p. 113 for other examples).]

The shift of the phosphate group from carbon 3 to carbon 2 of phosphoglycerate by phosphoglycerate mutase is freely reversible (see Figure 8.18).

The dehydration of 2-phosphoglycerate by enolase redistributes the energy within the 2-phosphoglycerate molecule, resulting in the formation of phosphoenolpyruvate (PEP), which contains a highenergy enol phosphate (see Figure 8.18). The reaction is reversible despite the high-energy nature of the product.

The conversion of PEP to pyruvate is catalyzed by pyruvate kinase, the third irreversible reaction of glycolysis. The equilibrium of the pyruvate kinase reaction favors the formation of ATP (see Figure 8.18). [Note: This is another example of substrate-level phosphorylation.]
1. Feed-forward regulation: In liver, pyruvate kinase is activated by fructose 1,6-bisphosphate, the product of the phosphofructokinase reaction. This feed-forward (instead of the more usual feedback) regulation has the effect of linking the two kinase activities: increased phosphofructokinase activity results in elevated levels of fructose 1,6-bisphosphate, which activates pyruvate kinase.
2. Covalent modulation of pyruvate kinase: Phosphorylation by a cAMP-dependent protein kinase leads to inactivation of pyruvate kinase in the liver (Figure 8.19). When blood glucose levels are low, elevated glucagon increases the intracellular level of cAMP, which causes the phosphorylation and inactivation of pyruvate kinase. Therefore, PEP is unable to continue in glycolysis, but instead enters the gluconeogenesis pathway. This, in part, explains the observed inhibition of hepatic glycolysis and stimulation of gluconeogenesis by glucagon. Dephosphorylation of pyruvate kinase by a phosphoprotein phosphatase results in reactivation of the enzyme.
3. Pyruvate kinase deficiency: The normal, mature erythrocyte lacks mitochondria and is, therefore, completely dependent on glycolysis for production of ATP. This high-energy compound is required to meet the metabolic needs of the red blood cell, and also to fuel the pumps necessary for the maintenance of the biconcave, flexi-

ble shape of the cell, which allows it to squeeze through narrow capillaries. The anemia observed in glycolytic enzyme deficiencies is a consequence of the reduced rate of glycolysis, leading to decreased ATP production. The resulting alterations in the red blood cell membrane lead to changes in the shape of the cell and, ultimately, to phagocytosis by the cells of the reticuloendothelial system, particularly macrophages of the spleen. The premature death and lysis of red blood cells results in hemolytic anemia. Among patients exhibiting the rare genetic defects of glycolytic enzymes, about 95% show a deficiency in pyruvate kinase, and 4% exhibit phosphoglucose isomerase deficiency. PK deficiency is restricted to the erythrocytes, and produces mild to severe chronic hemolytic anemia (erythrocyte destruction), with the severe form requiring regular cell transfusions. The severity of the disease depends both on the degree of enzyme deficiency (generally 5-25% of normal levels), and on the extent to which the individual's red blood cells compensate by synthesizing increased levels of 2,3-BPG (see p. 31). Almost all individuals with PK deficiency have a mutant enzyme that shows abnormal properties— most often altered kinetics (Figure 8.20).
Pyruvate kinase deficiency is the second most common cause (after glucose 6-phosphate dehydrogenase deficiency) of enzyme deficiencyrelated nonspherocytic hemolytic anemia.

Lactate, formed by the action of lactate dehydrogenase, is the final product of anaerobic glycolysis in eukaryotic cells (Figure 8.21). The formation of lactate is the major fate for pyruvate in lens and cornea of the eye, kidney medulla, testes, leukocytes and red blood cells, because these are all poorly vascularized and/or lack mitochondria.
1. Lactate formation in muscle: In exercising skeletal muscle, NADH production (by glyceraldehyde 3-phosphate dehydrogenase and by the three NAD*-linked dehydrogenases of the citric acid cycle, see p. 112) exceeds the oxidative capacity of the respiratory chain. This results in an elevated NADH/NAD* ratio, favoring reduction of pyruvate to lactate. Therefore, during intense exercise, lactate accumulates in muscle, causing a drop in the intracellular pH, potentially resulting in cramps. Much of this lactate eventually diffuses into the bloodstream, and can be used by the liver to make glucose (see p. 118).
2. Lactate consumption: The direction of the lactate dehydrogenase reaction depends on the relative intracellular concentrations of pyruvate and lactate, and on the ratio of NADH/NAD* in the cell. For example, in liver and heart, the ratio of NADH/NAD* is lower than in exercising muscle. These tissues oxidize lactate (obtained from the blood) to pyruvate. In the liver, pyruvate is either converted to glucose by gluconeogenesis or oxidized in the TCA cycle. Heart muscle exclusively oxidizes lactate to CO2 and H2O via the citric acid cycle.

Alterations observed with various mutant forms of pyruvate kinase.

Summary of anaerobic glycolysis. Reactions involving the production or consumption of ATP or NADH are indicated. The three irreversible reactions of glycolysis are shown with thick arrows. DHAP = dihydroxyacetone phosphate.
3. Lactic acidosis: Elevated concentrations of lactate in the plasma, termed lactic acidosis, occur when there is a collapse of the circulatory system, such as in myocardial infarction, pulmonary embolism, and uncontrolled hemorrhage, or when an individual is in shock. The failure to bring adequate amounts of oxygen to the tissues results in impaired oxidative phosphorylation and decreased ATP synthesis. To survive, the cells use anaerobic glycolysis as a backup system for generating ATP, producing lactic acid as the endproduct. [Note: Production of even meager amounts of ATP may be life-saving during the period required to reestablish adequate blood flow to the tissues.] The excess oxygen required to recover from a period when the availability of oxygen has been inadequate is termed the oxygen debt.
The oxygen debt is often related to patient morbidity or mortality. In many clinical situations, measuring the blood levels of lactic acid allows the rapid, early detection of oxygen debt in patients and the monitoring of their recovery.

Despite the production of some ATP during glycolysis, the end products, pyruvate or lactate, still contain most of the energy originally contained in glucose. The TCA cycle is required to release that energy completely (see p. 109).
- 1. Anaerobic glycolysis: Two molecules of ATP are generated for each molecule of glucose converted to two molecules of lactate (Figure 8.22). There is no net production or consumption of NADH.
2.
Aerobic glycolysis: The direct consumption and formation of ATP is the same as in anaerobic glycolysis—that is, a net gain of two ATP per molecule of glucose. Two molecules of NADH are also produced per molecule of glucose. Ongoing aerobic glycolysis requires the oxidation of most of this NADH by the electron transport chain, producing approximately three ATP for each NADH molecule entering the chain (see p. 77). [Note: NADH cannot cross the inner mitochondrial membrane, and substrate shuttles are required (see p. 79).]

The regulation of glycolysis by allosteric activation or inhibition, or the phosphorylation/dephosphorylation of rate-limiting enzymes, is shortterm—that is, they influence glucose consumption over periods of minutes or hours. Superimposed on these moment-to-moment effects are slower, and often more profound, hormonal influences on the amount of enzyme protein synthesized. These effects can result in 10-fold to 20fold increases in enzyme activity that typically occur over hours to days. Although the current focus is on glycolysis, reciprocal changes occur in the rate-limiting enzymes of gluconeogenesis, which are described in
Chapter 10 (see p. 117). Regular consumption of meals rich in carbohydrate or administration of insulin initiates an increase in the amount of glucokinase, phosphofructokinase, and pyruvate kinase in liver (Figure 8.23). These changes reflect an increase in gene transcription, resulting in increased enzyme synthesis. High activity of these three enzymes favors the conversion of glucose to pyruvate, a characteristic of the wellfed state (see p. 321). Conversely, gene transcription and synthesis of glucokinase, phosphofructokinase, and pyruvate kinase are decreased when plasma glucagon is high and insulin is low, for example, as seen in fasting or diabetes.

Oxidative decarboxylation of pyruvate by pyruvate dehydrogenase complex is an important pathway in tissues with a high oxidative capacity, such as cardiac muscle (Figure 8.24). Pyruvate dehydrogenase irreversibly converts pyruvate, the end product of glycolysis, into acetyl CoA, a major fuel for the TCA cycle (see p. 109) and the building block for fatty acid synthesis (see p. 183).

Carboxylation of pyruvate to oxaloacetate (OAA) by pyruvate carboxylase is a biotin-dependent reaction (see Figure 8.24). This reaction is important because it replenishes the citric acid cycle intermediates, and provides substrate for gluconeogenesis (see p. 118).

The conversion of pyruvate to ethanol occurs by the two reactions summarized in Figure 8.24. The decarboxylation of pyruvate by pyruvate decarboxylase occurs in yeast and certain other microorganisms, but not in humans. The enzyme requires thiamine pyrophosphate as a coenzyme, and catalyzes a reaction similar to that described for pyruvate dehydrogenase (see p. 110).

Most pathways can be classified as either catabolic (degrade complex molecules to a few simple products) or anabolic (synthesize complex end products from simple precursors). Catabolic reactions also capture chemical energy in the form of ATP from the degradation of energy-rich molecules. Anabolic reactions require energy, which is generally provided by the breakdown of ATP. The rate of a metabolic pathway can respond to regulatory signals, for example, allosteric activators or inhibitors, that arise from within the cell. Signaling between cells provides for the integration of metabolism. The most important route of this communication is chemical signaling between cells, for example, by hormones or neurotransmitters. Second messenger molecules convey the intent of a chemical signal (hormone or neurotransmitter) to appropriate intracellular responders. Adenylyl cyclase is a membrane-bound enzyme that synthesizes cAMP in response to chemical signals, such

Effect of insulin and glucagon on the synthesis of key enzymes of glycolysis in liver.

as the hormones glucagon and epinephrine. Following binding of a hormone to its cell-surface receptor, a GTP-dependent regulatory protein (G protein) is activated that, in turn, activates adenylyl cyclase. The cAMP produced activates a protein kinase, which phosphorylates a cadre of enzymes, causing their activation or deactivation. Phosphorylation is reversed by protein phosphatases. Aerobic glycolysis, in which pyruvate is the end product, occurs in cells with mitochondria and an adequate supply of oxygen. Anaerobic glycolysis, in which lactic acid is the end product, occurs in cells that lack mitochondria, or in cells deprived of sufficient oxygen. Glucose is transported across membranes by one of 14 glucose transporter isoforms (GLUTs). GLUT-1 is abundant in erythrocytes and brain, GLUT-4 (which is insulin-dependent) is found in muscle and adipose tissue, and GLUT-2 is found in liver and the B cells of the pancreas. The conversion of glucose to pyruvate (glycolysis, Figure 8.25) occurs in two stages: an energy investment phase in which phosphorylated intermediates are synthesized at the expense of ATP, and an energy generation phase, in which ATP is produced. In the energy investment phase, glucose is phosphorylated by hexokinase (found in most tissues) or glucokinase (a hexokinase found in liver cells and the B cells of the pancreas). Hexokinase has a high affinity (low Km) and a small Vmax for glucose, and is inhibited by glucose 6-phosphate. Glucokinase has a large Km and a large Vmax for glucose. It is indirectly inhibited by fructose 6phosphate and activated by glucose, and the transcription of the glucokinase gene is enhanced by insulin. Glucose 6-phosphate is isomerized to fructose 6-phosphate, which is phosphorylated to fructose 1,6-bisphosphate by phosphofructokinase. This enzyme is allosterically inhibited by ATP and citrate, and activated by AMP. Fructose 2,6-bisphosphate, whose synthesis is activated by insulin, is the most potent allosteric activator of this enzyme. A total of two ATP are used during this phase

of glycolysis. Fructose 1,6-bisphosphate is cleaved to form two trioses that are further metabolized by the glycolytic pathway, forming pyruvate. During these reactions, four ATP and two NADH are produced from ADP and NAD*. The final step in pyruvate synthesis from phosphoenolpyruvate is catalyzed by pyruvate kinase. This enzyme is allosterically activated by fructose 1,6-bisphosphate, and hormonally activated by insulin and inhibited by glucagon via the cAMP pathway. Pyruvate kinase deficiency accounts for 95% of all inherited defects in glycolytic enzymes. It is restricted to erythrocytes, and causes mild to severe chronic hemolytic anemia. In anaerobic glycolysis, NADH is reoxidized to NAD* by the conversion of pyruvate to lactic acid. This occurs in cells, such as erythrocytes, that have few or no mitochondria, and in tissues, such as exercising muscle, where production of NADH exceeds the oxidative capacity of the respiratory chain. Elevated concentrations of lactate in the plasma (lactic acidosis) occur when there is a collapse of the circulatory system, or when an individual is in shock. Pyruvate can be: 1) oxidatively decarboxylated by pyruvate dehydrogenase, producing acetyl CoA; 2) carboxylated to oxaloacetate (a TCA cycle intermediate) by pyruvate carboxylase; or 3) reduced by microorganisms to ethanol by pyruvate decarboxylase.

- 8.1 Which one of the following statements concerning
glycolysis is correct?
- A. The conversion of glucose to lactate requires the
presence of oxygen.
B. Hexokinase is important in hepatic glucose metabolism only in the absorptive period following consumption of a carbohydrate-containing meal.
C. Fructose 2,6-bisphosphate is a potent inhibitor of phosphofructokinase.
D. The regulated reactions are also the irreversible reactions.
E. The conversion of glucose to lactate yields two ATP and two NADH.
8.2 The reaction catalyzed by phosphofructokinase-1:
A. is activated by high concentrations of ATP and citrate.
B. uses fructose 1-phosphate as substrate.
C. is the rate-limiting reaction of the glycolytic path-

. is near equilibrium in most tissues. 0
E. is inhibited by fructose 2,6-bisphosphate.
8.3 Compared with the resting state, vigorously contracting skeletal muscle shows:
A. an increased conversion of pyruvate to lactate.
B. decreased oxidation of pyruvate to COz and water.
C. a decreased NADH/NAD* ratio.
D. a decreased concentration of AMP.
E. decreased levels of fructose 2,6-bisphosphate.
8.4 A 43-year-old man presented with symptoms of weak-
ness, fatigue, shortness of breath, and dizziness. His hemoglobin level was less than 7 g/dl (normal for a male being greater than 13.5 g/dl). Red blood cells isolated from the patient showed abnormally low level of lactate production. A deficiency of which one of the following enzymes would be the most likely cause of this patient's anemia?
A. Phosphoglucose isomerase
B. Phosphofructokinase
C. Pyruvate kinase
D. Hexokinase
E. Lactate dehydrogenase
Correct answer = D. Hexokinase, phosphofructokinase, and pyruvate kinase are all irreversible and are the regulated steps in glycolysis. The conversion of glucose to lactate (anaerobic glycolysis) is a process that does not involve a net oxidation or reduction and, thus, oxygen is not required. Glucokinase (not hexokinase) is important in hepatic glucose metabolism only in the absorptive period following consumption of a carbohydrate-containing meal. Fructose 2,6-bisphosphate is a potent activator (not inhibitor) of phosphofructokinase. The conversion of glucose to lactate yields two ATP but no net production of NADH.
Correct answer = C. Phosphofructokinase-1 is the pace-setting enzyme of glycolysis. It is inhibited by ATP and citrate, uses fructose 6-phosphate as substrate, and catalyzes a reaction that is far from equilibrium. The reaction is activated by fructose 2,6-bisphosphate.

Correct answer = A. Vigorously contracting muscle shows an increased formation of lactate and an increased rate of pyruvate oxidation compared with resting skeletal muscle. The levels of AMP and NADH increase, whereas change in the concentration of fructose 2,6-bisphosphate is not a key regulatory factor in skeletal muscle.
Correct answer = C. Decreased lactate production in the erythrocyte indicates a defect in glycolysis. Among patients exhibiting genetic defects of glycolytic enzymes, about 95% show a deficiency in pyruvate kinase. Pyruvate kinase deficiency is the second most common cause (after glucose 6phosphate dehydrogenase deficiency) of enzyme deficiency-related hemolytic anemia.

The tricarboxylic acid cycle (TCA cycle, also called the Krebs cycle or the citric acid cycle) plays several roles in metabolism. It is the final pathway where the oxidative metabolism of carbohydrates, amino acids, and fatty acids converge, their carbon skeletons being converted to COz. This oxidation provides energy for the production of the majority of ATP in most animals, including humans. The cycle occurs totally in the mitochondria and is, therefore, in close proximity to the reactions of electron transport (see p. 73), which oxidize the reduced coenzymes produced by the cycle. The TCA cycle is an aerobic pathway, because Oz is required as the final electron acceptor. Most of the body's catabolic pathways converge on the TCA cycle (Figure 9.1). Reactions such as the catabolism of some amino acids generate intermediates of the cycle and are called anaplerotic reactions. The citric acid cycle also supplies intermediates for a number of important synthetic reactions. For example, the cycle functions in the formation of glucose from the carbon skeletons of some amino acids, and it provides building blocks for the synthesis of some amino acids (see p. 267) and heme (see p. 278). Therefore, this cycle should not be viewed as a closed circle, but instead as a traffic circle with compounds entering and leaving as required.

In the TCA cycle, oxaloacetate is first condensed with an acetyl group from acetyl coenzyme A (CoA), and then is regenerated as the cycle is completed (Figure 9.1). Thus, the entry of one acetyl CoA into one round of the TCA cycle does not lead to the net production or consumption of intermediates. [Note: Two carbons entering the cycle as acetyl CoA are balanced by two COz exiting.]

Pyruvate, the endproduct of aerobic glycolysis, must be transported into the mitochondrion before it can enter the TCA cycle. This is accomplished by a specific pyruvate transporter that helps pyruvate cross the inner mitochondrial membrane. Once in the matrix, pyruvate is converted to acetyl CoA by the pyruvate dehydrogenase complex, which is a multienzyme complex. Strictly speaking, the pyruvate dehydrogenase complex is not part of the TCA cycle proper, but is a major source of acetyl CoA—the two-carbon substrate for the cycle.

The tricarboxylic acid cycle shown as a part of the central pathways of energy metabolism. (See Figure 8.2, p. 92 for a more detailed view of the metabolic map.)
1. Component enzymes: The pyruvate dehydrogenase complex (PDH complex) is a multimolecular aggregate of three enzymes, pyruvate dehydrogenase (PDH or E;, also called a decarboxylase), dihydrolipoyl transacetylase (Ez), and dihydrolipoyl dehydrogenase (E3). Each catalyzes a part of the overall reaction (Figure 9.2). Their physical association links the reactions in proper sequence without the release of intermediates. In addition to the enzymes participating in the conversion of pyruvate to acetyl CoA, the complex also contains two tightly bound regulatory enzymes, pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.
- 2. Coenzymes: The PDH complex contains five coenzymes that act as carriers or oxidants for the intermediates of the reactions shown in Figure 9.2. E; requires thiamine pyrophosphate (TPP), E> requires lipoic acid and CoA, and E3 requires FAD and NAD*.
Deficiencies of thiamine or niacin can cause serious central nervous system problems. This is because brain cells are unable to produce sufficient ATP (via the TCA cycle) if the PDH complex is inactive. Wernicke-Korsakoff, an encephalopathy-psychosis syndrome due to thiamine deficiency, may be seen with alcohol abuse.
- 3. Regulation of the pyruvate dehydrogenase complex: Covalent modification by the two regulatory enzymes that are part of the complex alternately activate and inactivate E; (PDH). The cyclic AMP-independent PDH kinase phosphorylates and, thereby, inhibits E;, whereas PDH phosphatase dephosphorylates and

Mechanism of action of the pyruvate dehydrogenase complex. TPP = thiamine pyrophosphate; L = lipoic acid.
.
activates E; (Figure 9.3). The kinase itself is allosterically activated by ATP, acetyl CoA, and NADH. Therefore, in the presence of these high-energy signals, the PDH complex is turned off. Pyruvate is a potent inhibitor of PDH kinase. Therefore, if pyruvate concentrations are elevated, E; will be maximally active. Calcium is a strong activator of PDH phosphatase, stimulating E; activity. This is particularly important in skeletal muscle, where release of Ca** during contraction stimulates the PDH complex, and thereby energy production. [Note: Although covalent regulation by the kinase and phosphatase is key, the complex is also subject to product (NADH, acetyl CoA) inhibition.]
Pyruvate dehydrogenase deficiency: A deficiency in the E; component of the PDH complex, although rare, is the most common biochemical cause of congenital lactic acidosis. This enzyme deficiency results in an inability to convert pyruvate to acetyl CoA, causing pyruvate to be shunted to lactic acid via lactate dehydrogenase (see p. 103). This causes particular problems for the brain, which relies on the TCA cycle for most of its energy, and is particularly sensitive to acidosis. Symptoms are variable and include neurodegeneration, muscle spasticity and, in the neonatal onset form, early death. The E; defect is X-linked, but because of the importance of the enzyme in the brain, it affects both males and females. Therefore, the defect is classified as X-linked dominant. There is no proven treatment for pyruvate dehydrogenase deficiency: however, dietary restriction of carbohydrate and supplementation with TPP may reduce symptoms in select patients.
Leigh syndrome (subacute necrotizing encephalomyelopathy) is a rare, progressive neurological disorder that is the result of defects in mitochondrial ATP production, primarily as a result of mutations in the PDH complex, the electron transport chain, or ATP synthase. Both nuclear and mtDNA can be affected.

. Mechanism of arsenic poisoning: As previously described (see p.101), arsenic can interfere with glycolysis at the glyceraldehyde 3-phosphate step, thereby decreasing ATP production. "Arsenic poisoning" is, however, due primarily to inhibition of enzymes that require lipoic acid as a coenzyme, including Ez of the PDH complex, a-ketoglutarate dehydrogenase (see below), and branchedchain a-keto acid dehydrogenase (see p. 266). Arsenite (the trivalent form of arsenic) forms a stable complex with the thiol (-SH) groups of lipoic acid, making that compound unavailable to serve as a coenzyme. When it binds to lipoic acid in the PDH complex, pyruvate (and consequently lactate) accumulates. Like pyruvate dehydrogenase deficiency, this particularly affects the brain, causing neurologic disturbances and death.

Regulation of
pyruvate dehydrogenase
complex.
[> denotes product inhibition.]

The condensation of acetyl CoA and oxaloacetate to form citrate (a tricarboxylic acid) is catalyzed by citrate synthase (Figure 9.4). This aldol condensation has an equilibrium far in the direction of citrate synthesis. In humans, citrate synthase is not an allosteric enzyme. It is inhibited by its product, citrate. Substrate availability is another means of regulation for citrate synthase. The binding of oxaloacetate causes a conformational change in the enzyme that generates a binding site for acetyl CoA. [Note: Citrate, in addition to being an intermediate in the TCA cycle, provides a source of acetyl CoA for the cytosolic synthesis of fatty acids (see p. 183). Citrate also inhibits phosphofructokinase, the rate-limiting enzyme of glycolysis (see p. 99), and activates acetyl CoA carboxylase (the rate-limiting enzyme of fatty acid synthesis; see p. 183).]

Citrate is isomerized to isocitrate by aconitase, an Fe-S protein (see Figure 9.4). [Note: Aconitase is inhibited by fluoroacetate, a compound that is used as a rat poison. Fluoroacetate is converted to fluoroacetyl CoA, which condenses with oxaloacetate to form fluorocitrate—a potent inhibitor of aconitase—resulting in citrate accumulation. ]

Isocitrate dehydrogenase catalyzes the irreversible oxidative decarboxylation of isocitrate, yielding the first of three NADH molecules produced by the cycle, and the first release of COs (see Figure 9.4). This is one of the rate-limiting steps of the TCA cycle. The enzyme is allosterically activated by ADP (a low-energy signal) and Ca?*, and is inhibited by ATP and NADH, whose levels are elevated when the cell has abundant energy stores.

The conversion of o-ketoglutarate to succinyl CoA is catalyzed by the a-ketoglutarate dehydrogenase complex, a multimolecular aggregate of three enzymes (Figure 9.5). The mechanism of this oxidative decarboxylation is very similar to that used for the conversion of pyruvate to acetyl CoA by the PDH complex. The reaction releases the second COz and produces the second NADH of the cycle. The coenzymes required are thiamine pyrophosphate, lipoic acid, FAD, NAD*, and CoA. Each functions as part of the catalytic mechanism in a way analogous to that described for the PDH complex (see p. 110). The equilibrium of the reaction is far in the direction of succinyl! CoA—a high-energy thioester similar to acetyl CoA. a-Ketoglutarate dehydrogenase complex is inhibited by its products, NADH and succinyl CoA, and activated by Ca?*. However, it is not regulated by phosphorylation/dephosphorylation reactions as described for PDH complex. [Note: o-Ketoglutarate is also produced by the oxidative deamination (see p. 252) or transamination of the amino acid, glutamate (see p. 250).]

Succinate thiokinase (also called succinyl CoA synthetase—named for the reverse reaction) cleaves the high-energy thioester bond of succinyl CoA (see Figure 9.5). This reaction is coupled to phosphorylation of guanosine diphosphate (GDP) to guanosine triphosphate (GTP). GTP and ATP are energetically interconvertible by the nucleoside diphosphate kinase reaction:
GTP+ADP
@
GDP+ATP
The generation of GTP by succinate thiokinase is another example of substrate-level phosphorylation (see p. 102). [Note: Succinyl CoA is also produced from propionyl CoA derived from the metabolism of fatty acids with an odd number of carbon atoms (see p. 193), and from the metabolism of several amino acids (see pp. 265—266).]

Succinate is oxidized to fumarate by succinate dehydrogenase, as FAD (its coenzyme) is reduced to FADHp (see Figure 9.5). Succinate dehydrogenase is the only enzyme of the TCA cycle that is embedded in the inner mitochondrial membrane. As such, it functions as Complex II of the electron transport chain (see p. 75). [Note: FAD, rather than NAD*, is the electron acceptor because the reducing power of succinate is not sufficient to reduce NAD*.]

Fumarate is hydrated to malate in a freely reversible reaction catalyzed by fumarase (also called fumarate hydratase, see Figure 9.5). [Note: Fumarate is also produced by the urea cycle (see p. 254), in purine synthesis (see p. 294), and during catabolism of the amino acids, phenylalanine and tyrosine (see p. 263).]

Malate is oxidized to oxaloacetate by malate dehydrogenase (Figure 9.6). This reaction produces the third and final NADH of the cycle. The AG? of the reaction is positive, but the reaction is driven in the direction of oxaloacetate by the highly exergonic citrate synthase reaction. [Note: Oxaloacetate is also produced by the transamination of the amino acid, aspartic acid (see p. 250).]

Two carbon atoms enter the cycle as acetyl CoA and leave as COs. The cycle does not involve net consumption or production of oxaloacetate or of any other intermediate. Four pairs of electrons are transferred during one turn of the cycle: three pairs of electrons reducing three NAD* to NADH and one pair reducing FAD to FADH». Oxidation of one NADH by the electron transport chain leads to formation of approximately three ATP, whereas oxidation of FADH2 yields approximately two ATP (see p. 77). The total yield of ATP from the oxidation of one acetyl CoA is shown in Figure 9.7. Figure 9.8 summarizes the reactions of the TCA cycle.

Number of ATP molecules produced from the oxidation of one molecule of acetyl CoA (using both substrate-level and oxidative phosphorylation).

A. Production of reduced coenzymes, ATP, and COs in the citric acid cycle. B. Inhibitors and activators of the cycle.
|

In contrast to glycolysis, which is regulated primarily by phosphofructokinase, the TCA cycle is controlled by the regulation of several enzyme activities (see Figure 9.8). The most important of these regulated enzymes are those that catalyze reactions with highly negative AG°: citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase complex. Reducing equivalents needed for oxidative phosphorylation are generated by the pyruvate dehydrogenase complex and the TCA cycle, and both processes are upregulated in response to a rise in ADP.

Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase (PDH) complex, producing acetyl CoA, which is the major fuel for the tricarboxylic acid cycle (TCA cycle, Figure 9.9). This multienzyme complex requires five coenzymes: thiamine pyrophosphate, lipoic acid, FAD, NAD*, and coenzyme A. PDH complex is regulated by covalent modification of E; (pyruvate dehydrogenase, PDH) by PDH kinase and PDH phosphatase: phosphorylation inhibits PDH. PDH kinase is allosterically activated by ATP, acetyl CoA, and NADH and inhibited by pyruvate; the phosphatase is activated by Ca?*. Pyruvate dehydrogenase deficiency is the most common biochemical cause of congenital lactic acidosis. The central nervous system is particularly affected in this is X-linked dominant disorder. Arsenic poisoning causes inactivation of PDH complex by binding to lipoic acid. Citrate is synthesized from oxaloacetate and acetyl CoA by citrate synthase. This enzyme is subject to product inhibition by citrate. Citrate is isomerized to isocitrate by aconitase. Isocitrate is oxidized and decarboxylated by isocitrate dehydrogenase to a-ketoglutarate, producing CO2 and NADH. The enzyme is inhibited by ATP and NADH, and activated by ADP and Ca**. a-Ketoglutarate is oxidatively decarboxylated to succinyl CoA by the a-ketoglutarate dehydrogenase complex, producing CO2 and NADH. The enzyme is very similar to pyruvate dehydrogenase and uses the same coenzymes. o-Ketoglutarate dehydrogenase complex is activated by calcium and inhibited by NADH and succinyl CoA, but is not covalently regulated. Succinyl CoA is cleaved by succinate thiokinase (also called succinyl CoA synthetase), producing succinate and GTP. This is an example of substrate-level phosphorylation. Succinate is oxidized to fumarate by succinate dehydrogenase, producing FADH2. Fumarate is hydrated

to malate by fumarase (fumarate hydratase), and malate is oxidized to oxaloacetate by malate dehydrogenase, producing NADH. Three NADH, one FADHbp, and one GTP (whose terminal phosphate can be transferred to ADP by nucleoside diphosphate kinase, producing ATP) are produced by one round of the TCA cycle. The generation of acetyl CoA by the oxidation of pyruvate via the PDH complex also produces an NADH. Oxidation of these NADHs and FADH2 by the electron transport chain yields 14 ATP. An additional ATP (GTP) comes from substrate level phosphorylation in the TCA cycle. Therefore, a total of 15 ATPs are produced from the complete mitochondrial oxidation of pyruvate to COp.

9.1 The conversion of pyruvate to acetyl CoA and COs:
A. is reversible.
B. involves the participation of lipoic acid.
C. is activated when pyruvate dehydrogenase (PDH,
E;) of the pyruvate dehydrogenase complex is phos-
phorylated by PDH kinase in the presence of ATP.
D. occurs in the cytosol.
E. depends on the coenzyme biotin.
9.2 Which one of the following conditions decreases the
oxidation of acetyl CoA by the citric acid cycle?
A. A low ATP/ADP ratio
B. A low NADH concentration due to rapid oxidation to NAD* through the respiratory chain
C. A low NAD*/NADH ratio
D. A high concentration of AMP
E. A low GTP/GDP ratio

A + B + FAD + H20 > C + FADHs + NADH
Choose the lettered answer that corresponds to the
missing "A", "B", and "C" in the equation.
A. Succinyl CoA, Reactant B = GDP. A. Succinyl CoA, Reactant C = Succinate. B. Succinate, Reactant B = NAD +. B. Succinate, Reactant C = Oxaloacetate. C. Fumarate, Reactant B = NAD +. C. Fumarate, Reactant C = Oxaloacetate. D. Succinate, Reactant B = NAD +. D. Succinate, Reactant C = Malate. E. Fumarate, Reactant B = GTP. E. Fumarate, Reactant C = Malate

nervous system and lactic acidosis. Enzyme assay for pyruvate dehydrogenase (PDH) activity on extracts of cultured skin fibroblasts showed 5% of normal activity, with a low concentration (1 x 10° mM) of thiamine pyrophosphate (TPP), but 80% of normal activity when the assay contained a high (0.4 mM) concentration of TPP. Which one of the following statements concerning this patient is most correct?
- A. Elevated levels of lactate and pyruvate in the blood
reliably predict the presence of PDH deficiency.
B. The patient is expected to show disturbances in fatty acid degradation.
C. A diet consisting of high carbohydrate intake would be expected to be beneficial in this patient.
D. Alanine concentration in the blood is expected to be less than normal.
E.
- Administration of thiamine is expected to reduce his
serum lactate concentration and improve his clinical symptoms.
Correct answer = B. Lipoic acid is an intermediate acceptor of the acetyl group formed in the reaction. Pyruvate dehydrogenase complex catalyzes an irreversible reaction that is inhibited when the PDH (E1) component is phosphorylated. The enzyme complex is located in the mitochondrial matrix. Biotin is utilized by carboxylases.
Correct answer = C. A low NAD*/NADH ratio limits the rates of the NAD*-requiring dehydrogenases. A low ATP/ADP or GTP/GDP ratio stimulates the cycle. AMP does not directly affect the cycle.
- Correct answer = B. Succinate + NAD* + FAD — oxaloacetate + NADH + FADH2

Correct answer = E. The patient appears to have a thiamine-responsive PDH deficiency. The enzyme fails to bind thiamine pyrophosphate at low concentration, but shows significant activity at a high concentration of the coenzyme. This mutation, which affects the Km of the enzyme for the coenzyme, is present in some, but not all, cases of PDH deficiency. All inborn errors of PDH are associated with elevated levels of lactate, pyruvate, and alanine (the transamination product of pyruvate). Patients routinely show neuroanatomic defects, developmental delay, and often early death. Elevated lactate and pyruvate are also observed in pyruvate carboxylase deficiency, another rare defect in pyruvate metabolism. Because PDH is an integral part of carbohydrate metabolism, a diet low in carbohydrates would be expected to blunt the effects of the enzyme deficiency. By contrast, fatty acid degradation occurs via conversion to acetyl CoA by B-oxidation, a process that does not involve pyruvate as an intermediate. Thus, fatty acid metabolism is not disturbed in this enzyme deficiency.

Some tissues, such as the brain, red blood cells, kidney medulla, lens and cornea of the eye, testes, and exercising muscle, require a continuous supply of glucose as a metabolic fuel. Liver glycogen, an essential postprandial source of glucose, can meet these needs for only 10-18 hours in the absence of dietary intake of carbohydrate (see p. 329). During a prolonged fast, however, hepatic glycogen stores are depleted, and glucose is formed from precursors such as lactate, pyruvate, glycerol (derived from the backbone of triacylglycerols, see p. 190), and a-ketoacids (derived from the catabolism of glucogenic amino acids, see p. 261). The formation of glucose does not occur by a simple reversal of glycolysis, because the overall equilibrium of glycolysis strongly favors pyruvate formation. Instead, glucose is synthesized by a special pathway, gluconeogenesis, that requires both mitochondrial and cytosolic enzymes. During an overnight fast, approximately 90% of gluconeogenesis occurs in the liver, with the kidneys providing 10% of the newly synthesized glucose molecules. However, during prolonged fasting, the kidneys become major glucose-producing organs, contributing an estimated 40% of the total glucose production. Figure 10.1 shows the relationship of gluconeogenesis to other important reactions of intermediary metabolism.

Gluconeogenic precursors are molecules that can be used to produce a net synthesis of glucose. They include intermediates of glycolysis and the tricarboxylic acid (TCA) cycle. Glycerol, lactate, and the a-keto acids obtained from the transamination of glucogenic amino acids are the most important gluconeogenic precursors.

Glycerol is released during the hydrolysis of triacylglycerols in adipose tissue (see p. 190), and is delivered by the blood to the liver. Glycerol is phosphorylated by glycerol kinase to glycerol phosphate, which is oxidized by glycerol phosphate dehydrogenase to dihydroxyacetone phosphate—an intermediate of glycolysis. [Note: Adipocytes cannot phosphorylate glycerol because they essentially lack glycerol kinase.]

The gluconeogenesis pathway shown as part of the essential pathways of energy metabolism. The numbered reactions are unique to gluconeogenesis. (See Figure 8.2, p. 92, for a more detailed map of metabolism.)

Lactate is released into the blood by exercising skeletal muscle, and by cells that lack mitochondria, such as red blood cells. In the Cori cycle, bloodborne glucose is converted by exercising muscle to lactate, which diffuses into the blood. This lactate is taken up by the liver and reconverted to glucose, which is released back into the circulation (Figure 10.2).

Amino acids derived from hydrolysis of tissue proteins are the major sources of glucose during a fast. a-Ketoacids, such as a-ketoglutarate, are derived from the metabolism of glucogenic amino acids (see p. 261). These a-ketoacids can enter the TCA cycle and form oxaloacetate (OAA)—a direct precursor of phosphoenolpyruvate (PEP). [Note: Acetyl coenzyme A (CoA) and compounds that give rise only to acetyl CoA (for example, acetoacetate and amino acids such as lysine and leucine) cannot give rise to a net synthesis of glucose. This is due to the irreversible nature of the pyruvate dehydrogenase reaction, which converts pyruvate to acetyl CoA (see p. 109). These compounds give rise instead to ketone bodies (see p. 195) and are therefore termed ketogenic.]

Seven glycolytic reactions are reversible and are used in the synthesis of glucose from lactate or pyruvate. However, three of the reactions are irreversible and must be circumvented by four alternate reactions that energetically favor the synthesis of glucose. These reactions, unique to gluconeogenesis, are described below.

The first "roadblock" to overcome in the synthesis of glucose from pyruvate is the irreversible conversion in glycolysis of PEP to pyruvate by pyruvate kinase. In gluconeogenesis, pyruvate is first carboxylated by pyruvate carboxylase to OAA, which is then converted to PEP by the action of PEP-carboxykinase (Figure 10.3).
1.
Biotin is a coenzyme: Pyruvate carboxylase requires biotin (see p. 381) covalently bound to the e-amino group of a lysine residue in the enzyme (see Figure 10.3). Hydrolysis of ATP drives the formation of an enzyme-biotin—COz intermediate. This high-energy complex subsequently carboxylates pyruvate to form OAA. [Note: This reaction occurs in the mitochondria of liver and kidney cells, and has two purposes: to provide an important substrate for gluconeogenesis, and to provide OAA that can replenish the TCA cycle intermediates that may become depleted, depending on the synthetic needs of the cell. Muscle cells also contain pyruvate carboxylase, but use the OAA produced only for the latter purpose—they do not synthesize glucose.]
Pyruvate carboxylase is one of several carboxylases that require biotin. Others include acetyl CoA carboxylase (p. 183), propionyl CoA carboxylase (p. 194), and methylcrotonyl CoA carboxylase (p. 266).

Activation and transfer of CO» to pyruvate, followed by transport of oxaloacetate to the cytosol and subsequent decarboxylation. Alternatively, OAA can be converted to PEP that is transported out of the mitochondria.
2. Allosteric regulation: Pyruvate carboxylase is allosterically activated by acetyl CoA. Elevated levels of acetyl CoA in mitochondria signal a metabolic state in which the increased synthesis of OAA is required. For example, this occurs during fasting, when OAA is used for the synthesis of glucose by gluconeogenesis in the liver and kidney. Conversely, at low levels of acetyl CoA, pyruvate carboxylase is largely inactive, and pyruvate is primarily oxidized by the pyruvate dehydrogenase complex to produce acetyl CoA that can be further oxidized by the TCA cycle (see p. 109).

OAA must be converted to PEP for gluconeogenesis to continue. The enzyme that catalyzes this conversion is found in both the mitochondria and the cytosol in humans. The PEP that is generated in the mitochondria is transported to the cytosol by a specific transporter, whereas that generated in the cytosol requires the transport of OAA from the mitochondria to the cytosol. However, OAA is unable to directly cross the inner mitochondrial membrane; it must first be reduced to malate by mitochondrial malate dehydrogenase. Malate can be transported from the mitochondria to the cytosol, where it is reoxidized to oxaloacetate by cytosolic malate dehydrogenase as NAD* is reduced (see Figure 10.3). The NADH produced is used in the reduction of 1,3-BPG to glyceraldehyde 3-phosphate (see p. 101), a step common to both glycolysis and gluconeogenesis.

Oxaloacetate is decarboxylated and phosphorylated to PEP in the cytosol by PEP-carboxykinase (also referred to as PEPCK). The reaction is driven by hydrolysis of guanosine triphosphate (GTP, see Figure 10.3). The combined actions of pyruvate carboxylase and PEPcarboxykinase provide an energetically favorable pathway from pyruvate to PEP. Then, PEP is acted on by the reactions of glycolysis running in the reverse direction until it becomes fructose 1,6-bisphosphate.
The pairing of carboxylation with decarboxylation, as seen in gluconeogenesis, drives reactions that would otherwise be energetically unfavorable. A similar strategy is used in fatty acid synthesis (see pp. 183-184).

Hydrolysis of fructose 1,6-bisphosphate by fructose 1,6-bisphosphatase bypasses the irreversible phosphofructokinase-1 reaction, and provides an energetically favorable pathway for the formation of fructose 6-phosphate (Figure 10.4). This reaction is an important regulatory site of gluconeogenesis.
1. Regulation by energy levels within the cell: Fructose 1,6-bisphosphatase is inhibited by elevated levels of adenosine monophosphate (AMP), which signal an "energy-poor" state in the cell.

Effect of elevated glucagon on the intracellular concentration of fructose 2,6-bisphosphate in the liver. PFK-2 = phosphofructokinase-2, FBP-2 = fructose bisphosphatase-2.
Conversely, high levels of ATP and low concentrations of AMP stimulate gluconeogenesis, an energy-requiring pathway.
2. Regulation by fructose 2,6-bisphosphate: Fructose 1,6-bisphosphatase, found in liver and kidney, is inhibited by fructose 2,6bisphosphate, an allosteric effector whose concentration is influenced by the level of circulating glucagon (Figure 10.5). [Note: The signals that inhibit (low energy, high fructose 2,6-bisphosphate) or favor (high energy, low fructose 2,6-bisphosphate) gluconeogenesis have the opposite effect on glycolysis, providing reciprocal control of the pathways that synthesize and oxidize glucose (see p. 100).]

Hydrolysis of glucose 6-phosphate by glucose 6-phosphatase bypasses the irreversible hexokinase reaction, and provides an energetically favorable pathway for the formation of free glucose (Figure 10.6). Liver and kidney are the only organs that release free glucose from glucose 6-phosphate. This process actually requires two proteins: glucose 6-phosphate translocase, which transports glucose 6-phosphate across the endoplasmic reticulum (ER) membrane, and the ER enzyme, glucose 6-phosphatase (found only in gluconeogenic cells), which removes the phosphate, producing free glucose (see Figure 10.6). [Note: These proteins are also required for the final step of glycogen degradation (see p. 130). Type la glycogen storage disease (see p. 130), due to an inherited deficiency of glucose 6-phosphatase, is characterized by severe fasting hypoglycemia, because free glucose is unable to be produced from either gluconeogenesis or glycogenolysis.] Specific transporters are responsible for releasing free glucose and phosphate back into the cytosol and, for glucose, into blood. [Note: Muscle lacks glucose 6phosphatase, and therefore muscle glycogen can not be used to maintain blood glucose levels.]

Of the 11 reactions required to convert pyruvate to free glucose, seven are catalyzed by reversible glycolytic enzymes (Figure 10.7). The irreversible reactions of glycolysis catalyzed by hexokinase, phosphofructokinase-1, and pyruvate kinase are circumvented by glucose 6-phosphatase, fructose 1,6-bisphosphatase, and pyruvate carboxylase/PEP-carboxykinase. In gluconeogenesis, the equilibria of the seven reversible reactions of glycolysis are pushed in favor of glucose synthesis as a result of the essentially irreversible formation of PEP, fructose 6-phosphate, and glucose catalyzed by the gluconeogenic enzymes. [Note: The stoichiometry of gluconeogenesis from pyruvate couples the cleavage of six high-energy phosphate bonds and the oxidation of two NADH with the formation of each molecule of glucose (see Figure 10.7).]

The moment-to-moment regulation of gluconeogenesis is determined primarily by the circulating level of glucagon, and by the availability of gluconeogenic substrates. In addition, slow adaptive changes in

Dephosphorylation of glucose 6phosphate allows release of free glucose from liver and kidney into blood.

Summary of the reactions of glycolysis and gluconeogenesis,
showing the energy requirements of gluconeogenesis.

Covalent modification of pyruvate kinase results in inactivation of the enzyme. OAA = oxaloacetate. [Note: Only the hepatic isozyme is subject to covalent regulation.]
enzyme activity result from an alteration in the rate of enzyme synthesis or degradation, or both. [Note: Hormonal control of the glucoregulatory system is presented in Chapter 23.]

This hormone from the o cells of pancreatic islets (see p. 313) stimulates gluconeogenesis by three mechanisms.
1. Changes in allosteric effectors: Glucagon lowers the level of fructose 2,6-bisphosphate, resulting in activation of fructose 1,6-bisphosphatase and inhibition of phosphofructokinase-1, thus favoring gluconeogenesis over glycolysis (see Figure 10.5). [Note: See p. 100 for the role of fructose 2,6-bisphosphate in the regulation of glycolysis.]
2. Covalent modification of enzyme activity: Glucagon binds its G protein-coupled receptor (see p. 95) and, via an elevation in cyclic AMP (cAMP) level and cAMP-dependent protein kinase activity, stimulates the conversion of hepatic pyruvate kinase to its inactive (phosphorylated) form. This decreases the conversion of PEP to pyruvate, which has the effect of diverting PEP to the synthesis of glucose (Figure 10.8).
3. Induction of enzyme synthesis: Glucagon increases the transcription of the gene for PEP-carboxykinase, thereby increasing the availability of this enzyme as levels of its substrate rise during fasting. [Note: Insulin causes decreased transcription of the mRNA for this enzyme.]

The availability of gluconeogenic precursors, particularly glucogenic amino acids, significantly influences the rate of hepatic glucose synthesis. Decreased levels of insulin favor mobilization of amino acids from muscle protein, and provide the carbon skeletons for gluconeogenesis. In addition, ATP and NADH, coenzymes-cosubstrates required for gluconeogenesis, are primarily provided by the catabolism of fatty acids.

Allosteric activation of hepatic pyruvate carboxylase by acetyl CoA occurs during fasting. As a result of increased lipolysis in adipose tissue, the liver is flooded with fatty acids (see p. 330). The rate of formation of acetyl CoA by B-oxidation of these fatty acids exceeds the capacity of the liver to oxidize it to CO2 and H20. As a result, acetyl CoA accumulates and leads to activation of pyruvate carboxylase. [Note: Acetyl CoA inhibits pyruvate dehydrogenase (by activating PDH kinase, see p. 111). Thus, this single compound can divert pyruvate toward gluconeogenesis and away from the TCA cycle.]

Fructose 1,6-bisphosphatase is inhibited by AMP—a compound that activates phosphofructokinase-1. This results in a reciprocal regulation of glycolysis and gluconeogenesis seen previously with fructose 2,6-bisphosphate (see p. 121). [Note: Elevated AMP thus stimulates pathways that oxidize nutrients to provide energy for the cell.]

Gluconeogenic precursors include the intermediates of glycolysis and the citric acid cycle, glycerol released during the hydrolysis of triacylglycerols in adipose tissue, lactate released into the blood by cells that lack mitochondria and by exercising skeletal muscle, and a-ketoacids derived from the metabolism of glucogenic amino acids (Figure 10.9). Seven of the reactions of glycolysis are reversible and are used for gluconeogenesis in the liver and kidneys. Three reactions are physiologically irreversible and must be circumvented. These reactions are catalyzed by the glycolytic enzymes pyruvate kinase, phosphofructokinase, and hexokinase. Pyruvate is converted to oxaloacetate (OAA) and then to phosphoenolpyruvate (PEP) by pyruvate carboxylase and PEP-carboxykinase. The carboxylase requires biotin and ATP, and is allosterically activated by acetyl CoA. PEP-carboxykinase requires GTP. The transcription of its mRNA is increased by glucagon and decreased by insulin. Fructose 1,6-bisphosphate is converted to fructose 6-phosphate by fructose 1,6-bisphosphatase. This enzyme is inhibited by elevated levels of AMP and activated when ATP levels are elevated. The enzyme is also inhibited by fructose 2,6-bisphosphate, the primary allosteric activator of glycolysis. Glucose 6-phosphate is converted to glucose by glucose 6-phosphatase. This enzyme of the ER is required for the final step in gluconeogenesis, as well as hepatic and renal glycogen degradation. A deficiency of this enzyme results in severe, fasting hypoglycemia.

- 10.1 The synthesis of glucose from pyruvate by gluconeo-

mogQgwp
. occurs exclusively in the cytosol.
. is inhibited by an elevated level of glucagon.
. requires the participation of biotin.
. involves lactate as an intermediate.
. requires the oxidation/reduction of FAD.
10.2 Which one of the following statements concerning gluconeogenesis is correct?
A. It occurs in muscle.
B. It is stimulated by fructose 2,6-bisphosphate.
C. It is inhibited by elevated levels of acetyl CoA.
D. It is important in maintaining blood glucose during
the normal overnight fast.
- E. It uses carbon skeletons provided by degradation of fatty acids.
10.3 Which one of the following reactions is unique to glu- coneogenesis?
A. Lactate > pyruvate
B. Phosphoenolpyruvate —> pyruvate
C. Oxaloacetate — phosphoenolpyruvate
D. Glucose 6-phosphate — fructose 6-phosphate
E. 1,3-Bis-phosphoglycerate — 3-phosphoglycerate
10.4 The metabolism of ethanol by alcohol dehydrogenase (ADH) produces NADH. What effect is the change in the NAD*/NADH expected to have on gluconeogenesis? Explain.
10.5 Given that acetyl CoA cannot be a substrate for gluconeogenesis, why is its production in fatty acid oxidation essential for gluconeogenesis?
10.6 What effect does AMP have on gluconeogenesis and glycolysis? What enzymes are affected?
Correct answer = C. Biotin is the coenzymeprosthetic group of pyruvate carboxylase. The carboxylation of pyruvate occurs in the mitochondria. Glucagon stimulates gluconeogenesis. Lactate is not an intermediate in the conversion of pyruvate to glucose; however, pyruvate can be produced from lactate. FAD is not involved in gluconeogenesis.

Correct answer = D. During the overnight fast, glycogen is partially depleted and gluconeogenesis provides blood glucose. Gluconeogenesis is inhibited by fructose 2,6-bisphosphate and stimulated by elevated levels of acetyl CoA. Degradation of fatty acids yields acetyl CoA, which cannot be converted to glucose. This is because there is no net gain of carbons from acetyl CoA in the TCA cycle, and the PDH reaction is physiologically irreversible. Carbon skeletons of most amino acids are, however, gluconeogenic.
Correct answer = C. The other reactions are common to both gluconeogenesis and glycolysis.
The increase in NADH as ethanol is oxidized, will decrease the availability of OAA because the reversible oxidation of malate to OAA by malate dehydrogenase of the TCA cycle is driven in the reverse direction by the high availability of NADH. Additionally, the reversible reduction of pyruvate to lactate by lactate dehydrogenase of glycolysis is driven in the forward direction by NADH. Thus, two important gluconeogenic substrates, OAA and pyruvate, are decreased as a result of the increase in NADH during ethanol metabolism. This will result in a decrease in gluconeogenesis.
Acetyl CoA inhibits pyruvate dehydrogenase and activates pyruvate carboxylase, pushing pyruvate to gluconeogenesis.
AMP inhibits gluconeogenesis through inhibition of fructose 1,6-bisphosphatase and favors glycolysis through activation of phosphofructokinase-1. (Fructose 2,6-bisphosphate has a similar effect on these enzymes.)

A constant source of blood glucose is an absolute requirement for human life. Glucose is the greatly preferred energy source for the brain, and the required energy source for cells with few or no mitochondria, such as mature erythrocytes. Glucose is also essential as an energy source for exercising muscle, where it is the substrate for anaerobic glycolysis. Blood glucose can be obtained from three primary sources: the diet, degradation of glycogen, and gluconeogenesis. Dietary intake of glucose and glucose precursors, such as starch, monosaccharides, and disaccharides, is sporadic and, depending on the diet, is not always a reliable source of blood glucose. In contrast, gluconeogenesis (see p. 117) can provide sustained synthesis of glucose, but it is somewhat slow in responding to a falling blood glucose level. Therefore, the body has developed mechanisms for storing a supply of glucose in a rapidly mobilizable form, namely, glycogen. In the absence of a dietary source of glucose, this sugar is rapidly released from liver and kidney glycogen. Similarly, muscle glycogen is extensively degraded in exercising muscle to provide that tissue with an important energy source. When glycogen stores are depleted, specific tissues synthesize glucose de novo, using amino acids from the body's proteins as a primary source of carbons for the gluconeogenic pathway. Figure 11.1 shows the reactions of glycogen synthesis and degradation as part of the essential pathways of energy metabolism.

The main stores of glycogen in the body are found in skeletal muscle and liver, although most other cells store small amounts of glycogen for their own use. The function of muscle glycogen is to serve as a fuel reserve for the synthesis of adenosine triphosphate (ATP) during muscle contraction. That of liver glycogen is to maintain the blood glucose concentration, particularly during the early stages of a fast (Figure 11.2, and see p. 329).

Approximately 400 g of glycogen make up 1—2% of the fresh weight of resting muscle, and approximately 100 g of glycogen make up to 10% of the fresh weight of a well-fed adult liver. What limits the production of glycogen at these levels is not clear. However, in some

Glycogen synthesis and degradation shown as a part of the essential reactions of energy metabolism (see Figure 8.2, p. 92, for a more detailed view of the overall reactions of metabolism).

Branched structure of glycogen, showing o(1— 4) and «(1 6) glycosidic bonds.
glycogen storage diseases (see Figure 11.8), the amount of glycogen in the liver and/or muscle can be significantly higher.

Glycogen is a branched-chain polysaccharide made exclusively from a-D-glucose. The primary glycosidic bond is an o(1—4) linkage. After an average of eight to ten glucosyl residues, there is a branch containing an o(1-6) linkage (Figure 11.3). A single molecule of glycogen can have a molecular mass of up to 10° daltons. These molecules exist in discrete cytoplasmic granules that also contain most of the enzymes necessary for glycogen synthesis and degradation.

Liver glycogen stores increase during the well-fed state (see p. 323), and are depleted during a fast (see p. 329). Muscle glycogen is not affected by short periods of fasting (a few days) and is only moderately decreased in prolonged fasting (weeks). Muscle glycogen is synthesized to replenish muscle stores after they have been depleted following strenuous exercise. [Note: Glycogen synthesis and degradation are cytosolic processes that go on continuously. The differences between the rates of these two processes determine the levels of stored glycogen during specific physiologic states.]

Glycogen is synthesized from molecules of «-D-glucose. The process occurs in the cytosol, and requires energy supplied by ATP (for the phosphorylation of glucose) and uridine triphosphate (UTP).

a-D-Glucose attached to uridine diphosphate (UDP) is the source of all the glucosyl residues that are added to the growing glycogen molecule. UDP-glucose (Figure 11.4) is synthesized from glucose 1-phosphate and UTP by UDP-glucose pyrophosphorylase (Figure 11.5). The high-energy bond in pyrophosphate (PPj), the second product of the reaction, is hydrolyzed to two inorganic phosphates (Pj) by pyrophosphatase, which ensures that the UDP-glucose pyrophosphorylase reaction proceeds in the direction of UDP-glucose production. [Note: Glucose 6-phosphate is converted to glucose 1phosphate by phosphoglucomutase. Glucose 1,6-bisphosphate is an obligatory intermediate in this reaction (Figure 11.6).]

Glycogen synthase is responsible for making the o.(1—>4) linkages in glycogen. This enzyme cannot initiate chain synthesis using free glucose as an acceptor of a molecule of glucose from UDP-glucose. Instead, it can only elongate already existing chains of glucose. Therefore, a fragment of glycogen can serve as a primer in cells whose glycogen stores are not totally depleted. In the absence of a
glycogen fragment, a protein, called glycogenin, can serve as an acceptor of glucose residues from UDP-glucose (see Figure 11.5). The side chain hydroxyl group of a specific tyrosine serves as the site at which the initial glucosyl unit is attached. The reaction is catalyzed by glycogenin itself via autoglucosylation; thus, glycogenin is an . enzyme. Glycogenin then catalyzes the transfer of the next few molecules of glucose from UDP-glucose, producing a short, o:(1—>4)linked glucosyl chain. This short chain serves as a primer that is able to be elongated by glycogen synthase as described below [Note: Glycogenin stays associated with and forms the core of a glycogen granule.]

. . Elongation of a glycogen chain involves the transfer of glucose from UDP-glucose to the nonreducing end of the growing chain, forming a new glycosidic bond between the anomeric hydroxyl of carbon 1 of the activated glucose and carbon 4 of the accepting glucosyl! residue (see Figure 11.5). [Note: The nonreducing end of a carbohydrate chain is one in which the anomeric carbon of the terminal sugar is linked by a glycosidic bond to another compound, making the terminal sugar nonreducing (see p. 84).] The enzyme responsible for making the o«(1—4) linkages in glycogen is glycogen synthase. [Note: The UDP released when the new «(1->4) glycosidic bond is made can be phosphorylated to UTP by nucleoside diphosphate kinase (UDP + ATP @ UTP +ADP, see p. 296).]

Interconversion of glucose 6phosphate and glucose 1-phosphate by phosphoglucomutase.

Cleavage of an «(1 4)-glycosidic bond. PLP= pyridoxal phosphate.

If no other synthetic enzyme acted on the chain, the resulting structure would be a linear (unbranched) molecule of glucosyl residues attached by o(1-4) linkages. Such a compound is found in plant tissues, and is called amylose. In contrast, glycogen has branches located, on average, eight glucosyl residues apart, resulting in a highly branched, tree-like structure (see Figure 11.3) that is far more soluble than the unbranched amylose. Branching also increases the number of nonreducing ends to which new glucosyl residues can be added (and also, as described later, from which these residues can be removed), thereby greatly accelerating the rate at which glycogen synthesis can occur, and dramatically increasing the size of the molecule.
1.
Synthesis of branches: Branches are made by the action of the branching enzyme, amylo-a(1—>4) — a(1->6)-transglucosidase. This enzyme removes a chain of six to eight glucosyl residues from the nonreducing end of the glycogen chain, breaking an a(1—>4) bond to another residue on the chain, and attaches it to a non-terminal glucosyl residue by an o(1-6) linkage, thus functioning as a 4:6 transferase. The resulting new, nonreducing end (see 'j' in Figure 11.5), as well as the old nonreducing end from which the six to eight residues were removed (see "o" in Figure 11.5), can now be further elongated by glycogen synthase.
2. Synthesis of additional branches: After elongation of these two ends has been accomplished by glycogen synthase, their terminal six to eight glucosyl residues can be removed and used to make additional branches.

The degradative pathway that mobilizes stored glycogen in liver and skeletal muscle is not a reversal of the synthetic reactions. Instead, a separate set of cytosolic enzymes is required. When glycogen is degraded, the primary product is glucose 1-phosphate, obtained by breaking «(1—>4) glycosidic bonds. In addition, free glucose is released from each o(1-6)-linked glucosyl residue.

Glycogen phosphorylase sequentially cleaves the a(1—>4) glycosidic bonds between the glucosyl residues at the nonreducing ends of the glycogen chains by simple phosphorolysis (producing glucose 1phosphate) until four glucosyl units remain on each chain before a branch point (Figure 11.7). [Note: This enzyme contains a molecule of covalently bound pyridoxal phosphate (PLP) that is required as a coenzyme.] The resulting structure is called a limit dextrin, and phosphorylase cannot degrade it any further (Figure 11.8).

Branches are removed by the two enzymic activities of a single bifunctional protein, the debranching enzyme (see Figure 11.8). First, oligo-a(1>4)—a(1->4)-glucan transferase activity removes the

Glycogen degradation, showing some of the glycogen storage diseases (GSD). [Note: A GSD can also be caused by defects in branching enzyme, an enzyme of synthesis, resulting in Type IV: Andersen disease and causing death in early childhood.](Continued on next page.)
Figure 11.8 (Continued )
outer three of the four glucosyl residues attached at a branch. It next transfers them to the nonreducing end of another chain, lengthening it accordingly. Thus, an o(1—>4) bond is broken and an (1-4) bond is made, and the enzyme functions as a 4:4 transferase. Next, the remaining single glucose residue attached in an (1-6) linkage is removed hydrolytically by amylo-a(1—6)-glucosidase activity, releasing free glucose. The glucosyl chain is now available again for degradation by glycogen phosphorylase until four glucosyl units from the next branch are reached.

Glucose 1-phosphate, produced by glycogen phosphorylase, is converted in the cytosol to glucose 6-phosphate by phosphoglucomutase—a reaction that produces glucose 1,6-bisphosphate as a temporary but essential intermediate (see Figure 11.6). In the liver, glucose 6-phosphate is transported into the endoplasmic reticulum (ER) by glucose 6-phosphate translocase. There it is converted to glucose by glucose 6-phosphatase—the same enzyme used in the last step of gluconeogenesis (see p. 121). The glucose then moves from the ER to the cytosol. Hepatocytes release glycogen-derived glucose into the blood to help maintain blood glucose levels until the gluconeogenic pathway is actively producing glucose. [Note: In the muscle, glucose 6-phosphate cannot be dephosphorylated because of a lack of glucose 6-phosphatase. Instead, it enters glycolysis, providing energy needed for muscle contraction.]

A small amount (1-3%) of glycogen is continuously degraded by the lysosomal enzyme, o( 1—>4)-glucosidase (acid maltase). The purpose of this pathway is unknown. However, a deficiency of this enzyme causes accumulation of glycogen in vacuoles in the lysosomes, resulting in the serious glycogen storage disease Type II: Pompe disease (see Figure 11.8). [Note: Type Il: Pompe disease is the only glycogen storage disease that is a lysosomal storage disease.]
Lysosomal storage diseases are genetic disorders characterized by the accumulation of abnormal amounts of carbohydrates or lipids primarily due to their decreased lysosomal degradation.

Because of the importance of maintaining blood glucose levels, the synthesis and degradation of its glycogen storage form are tightly regulated. In the liver, glycogenesis accelerates during periods when the body has been well fed, whereas glycogenolysis accelerates during periods of fasting. In skeletal muscle, glycogenolysis occurs during active exercise, and glycogenesis begins as soon as the muscle is again at rest. Regulation of glycogen synthesis and degradation is accomplished on two levels. First, glycogen synthase and glycogen phosphorylase are hormonally regulated to meet the needs of the body as a whole. Second, the pathways of glycogen synthesis and degradation are allosterically controlled to meet the needs of a particular tissue.

Hormonal regulation of glycogen synthesis. [Note: In contrast to glycogen phosphorylase, glycogen synthase is inactivated by phosphorylation.]

The binding of hormones, such as glucagon or epinephrine, to plasma membrane G protein-coupled receptors (GPCR) signals the need for glycogen to be degraded—either to elevate blood glucose levels or to provide energy for exercising muscle.
1. Activation of protein kinase A: Binding of glucagon or epinephrine to their specific hepatocyte GPCR, or of epinephrine to specific myocyte GPCR, results in the G protein-mediated activation of adenylyl cyclase. This enzyme catalyzes the synthesis of cAMP, which activates cAMP-dependent protein kinase A (PKA), as described on page 95. PKA is a tetramer, having two regulatory subunits (R) and two catalytic subunits (C). cAMP binds to the regulatory subunit dimer, releasing individual catalytic subunits that are active (Figure 11.9). PKA then phosphorylates several enzymes of glycogen metabolism, affecting their activity. [Note: When cAMP is removed, the inactive tetramer, R2Co, is again formed.]

2. Activation of phosphorylase kinase: Phosphorylase kinase exists in two forms: an inactive "b" form and an active "a" form. Active PKA phosphorylates the inactive "b" form of phosphorylase kinase, producing the active "a" form (see Figure 11.9). [Note: The phosphorylated enzyme can be inactivated by the hydrolytic removal of its phosphate by protein phosphatase-1. This enzyme is activated by a signal cascade initiated by insulin (see p. 311). Insulin also activates the phosphodiesterase that degrades cAMP, thus opposing the effects of glucagon and epinephrine.]
3. Activation of glycogen phosphorylase: Glycogen phosphorylase also exists in two forms: the dephosphorylated, inactive "b" form and the phosphorylated, active "a" form. Active phosphorylase kinase phosphorylates glycogen phosphorylase b to its active "a" form, which then begins glycogenolysis (see Figure 11.9). Phosphorylase a is reconverted to phosphorylase b by the hydrolysis of its phosphate by protein phosphatase-1. [Note: Protein phosphatase-1 is inactivated by inhibitor proteins that bind in response to their phosphorylation and activation by PKA.]
4.
Summary of the regulation of glycogen degradation: The cascade of reactions listed above results in glycogenolysis. The large number of sequential steps serves to amplify the effect of the hormonal signal, that is, a few hormone molecules binding to their receptors result in a number of protein kinase A molecules being activated that can each activate many phosphorylase kinase molecules. This causes the production of many active glycogen phosphorylase a molecules that can degrade glycogen.

The regulated enzyme in glycogenesis is glycogen synthase. It also exists in two forms, the active "a" form and the inactive "b" form. However, for glycogen synthase, in contrast to phosphorylase kinase and glycogen phosphorylase, the active form is dephosphorylated
whereas the inactive form is phosphorylated (Figure 11.10). Glycogen synthase a is converted to the inactive "b" form by phosphorylation at several sites on the enzyme, with the level of inactivation proportional to its degree of phosphorylation. This conversion process is catalyzed by several different protein kinases that are regulated by cAMP or other signaling mechanisms (see C below). Glycogen synthase b can be reconverted to the "a" form by protein phosphatase-1, which removes the phosphate groups hydrolytically.

In addition to hormonal signals, glycogen synthase and glycogen phosphorylase respond to the levels of metabolites and energy needs of the cell. Glycogenesis is stimulated when substrate availability and energy levels are high, whereas glycogenolysis is increased when glucose and energy levels are low. This allosteric regulation allows a rapid response to the needs of a cell, and can override the effects of hormone-mediated covalent regulation.

In the well-fed state, glycogen synthase b in both liver and muscle is allosterically activated by glucose 6-phosphate which is present in elevated concentrations (Figure 11.11). In contrast, glycogen phosphorylase a is allosterically inhibited by glucose 6phosphate, as well as by ATP, a high-energy signal in the cell. [Note: In liver, but not muscle, non-phosphorylated glucose is also an allosteric inhibitor of glycogen phosphorylase a, making it a better substrate for protein phosphatase-1.]
2.
Activation of glycogen degradation by calcium: Ca?* is released into the cytoplasm in muscle in response to neural stimulation and in liver in response to epinephrine binding to a-adrenergic receptors. The Ca** binds to calmodulin, the most widely distributed member of a family of small, calcium-binding proteins. The binding of four molecules of Ca®* to calmodulin triggers a conformational change such that the activated Ca?*-calmodulin complex binds to and activates protein molecules—often enzymes—that are inactive in the absence of this complex (see Figure 11.12). Thus, calmodulin functions as an essential subunit of many complex proteins. One such protein is phosphorylase kinase b, which is activated by the Ca?*-calmodulin complex without the need for the kinase to be phosphorylated by PKA. [Note: Epinephrine at B-adrenergic receptors signals through a rise in cAMP, not calcium (see p. 131).]

muscle contraction, there is a rapid and urgent need for ATP. This energy is supplied by the degradation of muscle glycogen to glucose, which can then enter glycolysis. Nerve impulses cause membrane depolarization, which promotes Ca** release from the sarcoplasmic reticulum into the sarcoplasm of myocytes. The Ca?* binds calmodulin and the complex activates muscle phosphorylase kinase b (see Figure 11.9).

Allosteric regulation of glycogen synthesis and degradation. A. Liver. B. Muscle. [Note: Ca2+ indirectly activates phosphorylase in both muscle and liver by directly activating phosphorylase kinase.]
(EI

Calmodulin mediates many effects of intracellular calcium.
b. Calcium activation of liver phosphorylase kinase: During "fight or flight" situations, epinephrine is released from the adrenal medulla and signals the need for blood glucose. This glucose initially comes from hepatic glycogenolysis. Binding of epinephrine to hepatocyte a-adrenergic G protein-coupled receptors activates a phospholipid-dependent cascade (see p. 205) that results in movement of Ca** from the ER into the cytoplasm. A Ca?*-calmodulin complex forms and activates hepatic phosphorylase kinase b. [Note: The released Ca?* also helps to activate protein kinase C that can phosphorylate (thus inactivate) glycogen synthase a.]
3. Activation of glycogen degradation in muscle by AMP: Muscle glycogen phosphorylase is active in the presence of the high AMP concentrations that occur in the muscle under extreme conditions of anoxia and ATP depletion. AMP binds to glycogen phosphorylase b, causing its activation without phosphorylation (see Figure 11.9). [Note: Recall that AMP also activates PFK-1 of glycolysis (see p. 99).]

These are a group of genetic diseases that result from a defect in an enzyme required for glycogen synthesis or degradation. They result either in formation of glycogen that has an abnormal structure, or in the accumulation of excessive amounts of normal glycogen in specific tissues as a result of impaired degradation. A particular enzyme may be defective in a single tissue, such as liver (resulting in hypoglycemia) or muscle (muscle weakness), or the defect may be more generalized, affecting liver, muscle, kidney, intestine, and myocardium. The severity of the glycogen storage diseases (GSDs) ranges from fatal in infancy to mild disorders that are not life-threatening. Some of the more prevalent GSDs are illustrated in Figure 11.8.

The main stores of glycogen in the body are found in skeletal muscle, where they serve as a fuel reserve for the synthesis of ATP during muscle contraction, and in the liver, where they are used to maintain the blood glucose concentration, particularly during the early stages of a fast. Glycogen is a highly branched polymer of o-D-glucose. The primary glycosidic bond is an o(1—>4) linkage. After about eight to ten glucosyl residues, there is a branch containing an a(1-6) linkage. UDP-glucose, the building block of glycogen, is synthesized from glucose 1-phosphate and UTP by UDP-glucose pyrophosphorylase (Figure 11.13). Glucose from UDP-glucose is transferred to the nonreducing ends of glycogen chains by primer-requiring glycogen synthase, which makes o(1—4) linkages. The primer is made by glycogenin. Branches are formed by amylo-o(1—4)—0(1-6)-transglucosidase, which transfers a chain of six to eight glucosyl residues from the nonreducing end of the glycogen chain (breaking an o(1—>4) linkage), and attaches it with an a(1-36) linkage to another residue in the chain.

PLP-requiring glycogen phosphorylase cleaves the a(1-4) bonds between glucosyl residues at the nonreducing ends of the glycogen chains, producing glucose 1-phosphate. This sequential degradation continues until four glucosyl units remain on each chain before a branch point. The resulting structure is called a limit dextrin that is degraded by the bifunctional debranching enzyme. Oligo-o(1—4)—a(1-4)-glucan transferase (common name, glucosyl 4:4 transferase) removes the outer three of the four glucosyl residues attached at a branch, and transfers them to the nonreducing end of another chain where they can be converted to glucose 1-phosphate by glycogen phosphorylase. Next, the remaining single glucose residue attached in an a(1-4) linkage is removed hydrolytically by the amylo-(1-6) glucosidase activity of debranching enzyme, releasing free glucose. Glucose 1-phosphate is converted to glucose 6-phosphate by phosphoglucomutase. In the muscle, glucose 6-phosphate enters glycolysis. In the liver, the phosphate is removed by glucose 6-phosphatase, releasing free glucose that can be used to maintain blood glucose levels at the beginning of a fast. A deficiency of the phosphatase causes glycogen storage disease Type 1a (Von Gierke disease). This disease results in an inability of the liver to provide free glucose to the body during a fast. It affects both glycogen degradation and gluconeogenesis. Glycogen synthesis and degradation are reciprocally regulated to meet whole-body needs by the same hormonal signals, namely, an elevated insulin level results in overall increased glycogenesis and decreased glycogenolysis, whereas an elevated glucagon (or epinephrine) level causes increased glycogenolysis and decreased glycogenesis. Key enzymes are phosphorylated by a family of protein kinases, some of which are cAMP-dependent (a compound increased by glucagon and epinephrine). Phosphate groups are removed by protein phosphatase-1 (activated when insulin levels are elevated). Glycogen synthase, phosphorylase kinase and phosphorylase are also allosterically regulated to meet tissues needs. In the well-fed

state, glycogen synthase is activated by glucose 6-phosphate, but glycogen phosphorylase is inhibited by glucose 6-phosphate, as well as by ATP. In the liver, glucose also serves an an allosteric inhibitor of glycogen phosphorylase. The Ca?* released from the endoplasmic reticulum in muscle during exercise and in liver in response to epinephrine activates phosphorylase kinase by binding to the enzyme's calmodulin subunit. This allows the enzyme to activate glycogen phosphorylase, thereby causing glycogen degradation.

Key concept map for glycogen metabolism in liver. [Note: Glycogen fhosphovyiase | is phosphorylated by phosphorylase kinase, the "b" form of which can be activated by Ca*+

- 11.1 A 2-year-old boy was brought into the emergency room, suffering from severe fasting hypoglycemia. On physical examination, he was found to have hepatomegaly. Laboratory tests indicated that he also had hyperlacticacidemia and hyperuricemia. A liver biopsy indicated that hepatocytes contained greater than normal amounts of glycogen that was of normal structure. Enzyme assay likely confirmed a deficiency in which of the following enzymes?
A. Glycogen synthase
B. Glycogen phosphorylase
C. Glucose 6-phosphatase
- D. Amylo-a(1—6)-glucosidase
E. Amylo-o.(14)—a.(1-6)-transglucosidase
- 11.2 The hormones, epinephrine and glucagon have
which one of the following effects on glycogen metabolism in the liver?
A. The net synthesis of glycogen is increased.
B. Glycogen phosphorylase is phosphorylated and active, whereas glycogen synthase is phosphorylated and inactive.
C. Both glycogen phosphorylase and glycogen synthase are activated by phosphorylation but at significantly different rates.
D. Glycogen phosphorylase is inactivated by a rise in
Ca?*, whereas glycogen synthase is activated.
E.
cAMP-dependent protein kinase A is activated, whereas phosphorylase kinase is inactivated.
11.3 In contracting skeletal muscle, a sudden elevation of the cytosolic Ca?* concentration will result in:
A. activation of cAMP-dependent protein kinase A.
B. dissociation of cAMP-dependent protein kinase A into and subunits.
catalytic regulatory
- C. inactivation of phosphorylase kinase caused by
the action of protein phosphatase-1.
D. direct activation of phosphorylase kinase b.
E. direct activation of glycogen phosphorylase b.
F. conversion of cAMP to AMP by phosphodiesterase.
11.4 Explain why the hypoglycemia seen with Type la glycogen storage disease (glucose 6-phosphatase deficiency) is severe, whereas that seen with Type VI (liver phosphorylase deficiency) is mild.

Correct answer = C. A deficiency of glucose 6phosphatase (Von Gierke disease) prevents the liver from releasing free glucose into the blood, causing severe fasting hypoglycemia, hyperlacticacidemia, and hyperuricemia. A deficiency of glycogen phosphorylase would result in a decrease in glycogen degradation, causing fasting hypoglycemia, but not the other symptoms. A deficiency of glycogen synthase would result in lower amounts of stored glycogen. Amylo«(1-6)-glucosidase removes single glucosyl residues attached to the glycogen chain through an a(1-6)-glycosidic bond. A deficiency in this enzyme would result in a decreased ability of the cell to completely degrade glycogen branches. Amylo-a(1—>4)—>a(1—6)-transglucosidase deficiency would decrease the ability of the cell to make branches.
Correct answer = B. Epinephrine and glucagon both cause increased glycogen degradation in the liver through covalent modification (phosphorylation) of key enzymes of glycogen metabolism. Glycogen phosphorylase is phosphorylated and active ("a" form), whereas glycogen synthase is phosphorylated and inactive ("b" form). cAMP-dependent protein kinase A is active and phosphorylates (and activates) its substrate, phosphorylase kinase. It is phosphorylase kinase a that directly phosphorylates and activates phosphorylase.
Correct answer = D. Ca** released from the sarcoplasmic reticulum during exercise binds to the calmodulin subunit of phosphorylase kinase, thereby allosterically activating the "b" form of this enzyme. The other choices are not caused by an elevation of cytosolic calcium.
With Type la, the liver is unable to generate free glucose either from glycogenolysis or gluconeogenesis because both processes produce glucose 6-phosphate. With Type VI, liver is still able to produce free glucose from gluconeogenesis; glycogenolysis, however, is inhibited.

Glucose is the most common monosaccharide consumed by humans, and its metabolism has been discussed extensively. However, two other monosaccharides—fructose and galactose—occur in significant amounts in the diet (primarily in disaccharides), and make important contributions to energy metabolism. In addition, galactose is an important component of cell structural carbohydrates. Figure 12.1 shows the metabolism of fructose and galactose as part of the essential pathways of energy metabolism in the body.

About 10% of the calories contained in the Western diet are supplied by fructose (approximately 55 g/day). The major source of fructose is the disaccharide sucrose, which, when cleaved in the intestine, releases equimolar amounts of fructose and glucose. Fructose is also found as a free monosaccharide in many fruits, in honey, and in high-fructose corn syrup (55% fructose/45% glucose typically), which is used to sweeten soft drinks and many foods. Entry of fructose into cells is not insulindependent (unlike that of glucose into certain tissues, see p. 97), and, in contrast to glucose, fructose does not promote the secretion of insulin.

For fructose to enter the pathways of intermediary metabolism, it must first be phosphorylated (Figure 12.2). This can be accomplished by either hexokinase or fructokinase (also called ketohexokinase). Hexokinase phosphorylates glucose in most cells of the body (see p. 98), and several additional hexoses can serve as substrates for this enzyme. However, it has a low affinity (that is, a high Km, see p. 59) for fructose. Therefore, unless the intracellular concentration of fructose becomes unusually high, the normal presence of saturating concentrations of glucose means that little fructose is converted to fructose 6-phosphate by hexokinase. Fructokinase provides the primary mechanism for fructose phosphorylation (see Figure 12.2). It is found in the liver (which processes most of the dietary fructose), kidney, and the small intestinal mucosa, and converts fructose to fructose 1-phosphate, using ATP as the phosphate donor. [Note: These three tissues also contain aldolase B, discussed below.]

Galactose and fructose metabolism as part of the essential pathways of energy metabolism (see Figure 8.2, p. 92, for a more detailed view of the overall reactions of metabolism).

Fructose 1-phosphate is not phosphorylated to fructose 1,6-bisphosphate as is fructose 6-phosphate (see p. 99), but is cleaved by aldolase B (also called fructose 1-phosphate aldolase) to dihydroxyacetone phosphate (DHAP) and glyceraldehyde. [Note: Humans express three a/dolases, A, B and C, the products of three different genes. Aldolase A (found in most tissues), a/dolase B (in liver), and aldolase C (in brain) all cleave fructose 1,6-bisphosphate produced during glycolysis to DHAP and glyceraldehyde 3-phosphate (see p. 100), but only aldolase B cleaves fructose 1-phosphate.] DHAP can directly enter glycolysis or gluconeogenesis, whereas glyceraldehyde can be metabolized by a number of pathways, as illustrated in Figure 12.3.

The rate of fructose metabolism is more rapid than that of glucose because the trioses formed from fructose 1-phosphate bypass phosphofructokinase-1—the major rate-limiting step in glycolysis (see p. 99).

A deficiency of one of the key enzymes required for the entry of fructose into intermediary metabolic pathways can result in either a benign condition as a result of fructokinase deficiency (essential fructosuria), or a severe disturbance of liver and kidney metabolism as a result of aldolase B deficiency (hereditary fructose intolerance, HFI), which is estimated to occur in 1:20,000 live births (see Figure 12.3). The first symptoms of HFl appear when a baby is weaned from milk (see p. 142) and begins to be fed food containing sucrose or fructose. Fructose 1-phosphate accumulates, resulting in a drop in the level of inorganic phosphate (Pi) and, therefore, of ATP. As ATP falls, AMP rises. In the absence of Pi, AMP is degraded, causing hyperuricemia (and lactic acidosis, see p. 299). The decreased availability of hepatic ATP affects gluconeogenesis (causing hypoglycemia with vomiting), and protein synthesis (causing a decrease in blood clotting factors and other essential proteins). Kidney function may also be affected. Diagnosis of HFl can be made on the basis of fructose in the urine, enzyme assay or by DNA-based testing (see Chapter 33). In some states HFI is part of the newborn screening panel. With HFI, sucrose and sorbitol (a sugar alcohol), as well as fructose, must be removed from the diet to prevent liver failure and possible death. Individuals with HFI display an aversion to sweets and, consequently, have an absence of dental caries.

Mannose, the C-2 epimer of glucose (see p. 84), is an important component of glycoproteins (see p. 166). Hexokinase phosphorylates mannose, producing mannose 6-phosphate, which, in turn, is (reversibly) isomerized to fructose 6-phosphate by phosphomannose isomerase. [Note: There is little mannose in dietary carbohydrates. Most intracellular mannose is synthesized from fructose, or is preexisting mannose produced by the degradation of structural carbohydrates and salvaged by hexokinase.]

Most sugars are rapidly phosphorylated following their entry into cells. They are thereby trapped within the cells, because organic phosphates cannot freely cross membranes without specific transporters. An alternate mechanism for metabolizing a monosaccharide is to convert it to a polyol (sugar alcohol) by the reduction of an aldehyde group, thereby producing an additional hydroxy! group.
- 1. Synthesis of sorbitol: Aldose reductase reduces glucose, producing sorbitol (glucitol, Figure 12.4). This enzyme is found in many tissues, including the lens, retina, Schwann cells of peripheral nerves, liver, kidney, placenta, red blood cells, and in cells of the ovaries and seminal vesicles. In cells of the liver, ovaries, and seminal vesicles, there is a second enzyme, sorbitol dehydrogenase, which can oxidize the sorbitol to produce fructose (see Figure 12.4). The two-reaction pathway from glucose to fructose in the seminal vesicles is for the benefit of sperm cells, which use fructose as a major carbohydrate energy source. The pathway

Sorbitol metabolism.
from sorbitol to fructose in the liver provides a mechanism by which any available sorbitol is converted into a substrate that can enter glycolysis or gluconeogenesis.

insulin is not required for the entry of glucose into the cells listed in the previous paragraph, large amounts of glucose may enter these cells during times of hyperglycemia, for example, in uncontrolled diabetes. Elevated intracellular glucose concentrations and an adequate supply of NADPH cause aldose reductase to produce a significant increase in the amount of sorbitol, which cannot pass efficiently through cell membranes and, therefore, remains trapped inside the cell (see Figure 12.4). This is exacerbated when sorbitol dehydrogenase is low or absent, for example, in retina, lens, kidney, and nerve cells. As a result, sorbitol accumulates in these cells, causing strong osmotic effects and, therefore, cell swelling as a result of water retention. Some of the pathologic alterations associated with diabetes can be attributed, in part, to this phenomenon, including cataract formation, peripheral neuropathy, and microvascular problems leading to nephropathy and retinopathy. (See p. 344 for a discussion of the complications of diabetes.)

The major dietary source of galactose is lactose (galactosyl B-1,4-glucose) obtained from milk and milk products. [Note: The digestion of lactose by B-galactosidase (lactase) of the intestinal mucosal cell membrane was discussed on p. 87.] Some galactose can also be obtained by lysosomal degradation of complex carbohydrates, such as glycoproteins and glycolipids, which are important membrane components. Like fructose, the entry of galactose into cells is not insulin-dependent.

Like fructose, galactose must be phosphorylated before it can be further metabolized. Most tissues have a specific enzyme for this purpose, galactokinase, which produces galactose 1-phosphate (Figure 12.5). As with other kinases, ATP is the phosphate donor.

Galactose 1-phosphate cannot enter the glycolytic pathway unless it is first converted to UDP-galactose (see Figure 12.5). This occurs in an exchange reaction, in which UDP-glucose reacts with galactose 1-phosphate, producing UDP-galactose and glucose 1-phosphate (Figure 12.6). The enzyme that catalyzes this reaction is galactose 1phosphate uridyltransferase (GALT).

For UDP-galactose to enter the mainstream of glucose metabolism, it must first be converted to its C-4 epimer, UDP-glucose, by UDPhexose 4-epimerase. This "new" UDP-glucose (produced from the original UDP-galactose) can then participate in many biosynthetic

Metabolism of galactose.
reactions, as well as being used in the GALT reaction described above. (See Figure 12.5 for a summary of this interconversion.) [Note: Rare deficiencies of the epimerase are known. ]

UDP-galactose can serve as the donor of galactose units in a number of synthetic pathways, including synthesis of lactose (see below), glycoproteins (see p. 166), glycolipids (see p. 210), and glycosaminoglycans (see p. 160). [Note: If galactose is not provided by the diet (for example, when it cannot be released from lactose as a result of a lack of B-galactosidase in people who are lactose-intolerant), all tissue requirements for UDP-galactose can be met by the action of UDP-hexose 4-epimerase on UDP-glucose, which is efficiently produced from glucose 1-phosphate (see Figure 12.5).]

GALT is deficient in individuals with classic galactosemia (see Figure 12.5). In this disorder, galactose 1-phosphate and, therefore, galactose accumulate in cells. Physiologic consequences are similar to those found in hereditary fructose intolerance (see p. 138), but a broader spectrum of tissues is affected. The accumulated galactose is shunted into side pathways such as that of galactitol production.

Lactose synthesis.
This reaction is catalyzed by aldose reductase, the same enzyme that converts glucose to sorbitol (see p. 139). [Note: A deficiency in galactokinase results in a less severe disorder of galactosemia metabolism, although cataracts are common (see Figure 12.5).]

Lactose is a disaccharide that consists of a molecule of B-galactose attached by a B(1-4) linkage to glucose. Therefore, lactose is galactosy! B(1—4)-glucose. Lactose, known as the "milk sugar," is produced by the mammary glands of most mammals. Therefore, milk and other dairy products are the dietary sources of lactose. Lactose is synthesized in the Golgi by lactose synthase (UDP-galactose:glucose galactosyltransferase), which transfers galactose from UDP-galactose to glucose, releasing UDP (Figure 12.7). This enzyme is composed of two proteins, A and B. Protein A is a B-D-galactosyltransferase, and is found in a number of body tissues. In tissues other than the lactating mammary gland, this enzyme transfers galactose from UDP-galactose to N-acetyl-D-glucosamine, forming the same B(1—4) linkage found in lactose, and producing N-acetyllactosamine—a component of the structurally important N-linked glycoproteins (see p. 167). In contrast, protein B is found only in lactating mammary glands. It is a-lactalbumin, and its synthesis is stimulated by the peptide hormone, prolactin. Protein B forms a complex with the enzyme, protein A, changing the specificity of that transferase so that lactose, rather than N-acetyllactosamine, is produced (see Figure 12.7).

The major source of fructose is sucrose, which when cleaved releases equimolar amounts of fructose and glucose (Figure 12.8). Entry of fructose into cells is insulin-independent. Fructose is first phosphorylated to fructose 1-phosphate by fructokinase, and then cleaved by aldolase B to dihydroxyacetone phosphate and glyceraldehyde. These enzymes are found in the liver, kidney, and small intestinal mucosa. A deficiency of fructokinase causes a benign condition (essential fructosuria), but a deficiency of aldolase B causes hereditary fructose intolerance (HFI), in which severe hypoglycemia and liver failure lead to death if fructose (and sucrose) in the diet is not severely limited. Mannose, an important component of glycoproteins, is phosphorylated by hexokinase to mannose 6-phosphate, which is reversibly isomerized to fructose 6-phosphate by phosphomannose isomerase. Glucose can be reduced to sorbitol (glucitol) by aldose reductase in many tissues, including the lens, retina, Schwann cells, liver, kidney, ovaries, and seminal vesicles. In cells of the liver, ovaries, and seminal vesicles, a second enzyme, sorbitol dehydrogenase, can oxidize sorbitol to produce fructose. Hyperglycemia results in the accumulation of sorbitol in those cells lacking sorbitol dehydrogenase. The resulting osmotic events cause cell swelling, and may contribute to the cataract formation, peripheral

neuropathy, nephropathy, and retinopathy seen in diabetes. The major dietary source of galactose is lactose. The entry of galactose into cells is not insulin-dependent. Galactose is first phosphorylated by galactokinase (deficiency results in cataracts) to galactose 1-phosphate. This compound is converted to UDP-galactose by galactose 1-phosphate uridyltransferase (GALT), with the nucleotide supplied by UDP-glucose. A deficiency of this enzyme causes classic galactosemia. Galactose 1-phosphate accumulates, and excess galactose is converted to galactitol by aldose reductase. This causes liver damage, severe mental retardation, and cataracts. Treatment requires removal of galactose (and lactose) from the diet. For UDP-galactose to enter the mainstream of glucose metabolism, it must first be converted to UDP-glucose by UDP-hexose 4-epimerase. This enzyme can also be used to produce UDP-galactose from UDP-glucose when the former is required for the synthesis of structural carbohydrates. Lactose is a disaccharide that consists of galactose and glucose. Milk and other dairy products are the dietary sources of lactose. Lactose is synthesized by lactose synthase from UDP-galactose and glucose in the lactating mammary gland. The enzyme has two subunits, protein A (which is a galactosyl transferase found in most cells where it synthesizes N-acetyllactosamine) and protein B (a-lactalbumin, which is found only in the lactating mammary glands, and whose synthesis is stimulated by the peptide hormone, prolactin). When both subunits are present, the transferase produces lactose.

Key concept map for metabolism of fructose and galactose. [Note: GALT is galactose 1-phosphate uridyltransferase.]

- 12.1 Following the intravenous injection of lactose into a
rat, none
of the
lactose is
metabolized.
However, ingestion
of lactose
disaccharide.
leads to
The rapid
metabolism difference
in these
of this
observations
a
result of:
A. the presence of lactase in the serum.
B. the absence of hepatic galactokinase.
C.the absence of maltase in the serum.
D.the presence of lactase in the intestine.

A. free (nonphosphorylated) galactose is the acceptor of glucose transferred by lactose synthase in the synthesis of lactose.
B. galactose can be produced from a glucose
metabolite by epimerization.
- C. hexokinase can efficiently phosphorylate dietary
galactose to galactose 1-phosphate.
- D. the enzyme deficient in galactosemia is activated
by a hormone produced in the mammary gland.
- E. galactose can be produced from fructose by isomerization.
12.3 A 5-month-old boy is brought to his physician because of vomiting, night sweats, and tremors. History revealed that these symptoms began after fruit juices were introduced to his diet as he was being weaned off breast milk. The physical examination was remarkable for hepatomegaly. Tests on the baby's urine were positive for reducing sugar but negative for glucose. The infant most likely suffers from:
A. aldolase B deficiency.
B. fructokinase deficiency.
C. galactokinase deficiency.
D. B-galactosidase deficiency.
E. glucose 6-phosphatase deficiency.

milk by mammary glands. In lactose synthesis:
- A. galactose from galactose 1-P is transferred to glugener-
cose by galactosyltransferase (protein A), ating lactose.
- B. protein A is used exclusively in the synthesis of
lactose.
C. a-lactalbumin (protein B) regulates the sugar
specificity of protein A by increasing its Kn for glucose.
D. protein B expression is stimulated by prolactin.
is
Correct answer = D. Lactase and maltase are intestinal enzymes not found in the serum. Therefore, ingested lactose is degraded, but injected lactose is not. If hepatic galactokinase is absent, the galactose segment of the lactose is not metabolized, but the glucose segment of the lactose can still be metabolized.
Correct answer = B. UDP-hexose 4-epimerase converts UDP-glucose to UDP-galactose, thus providing the appropriate form of galactose for lactose synthesis. UDP-galactose, not free galactose, is the source of the galactose portion of lactose. Galactose is not converted to galactose 1-phosphate by hexokinase. Galactosemia is the result of a deficiency in GALT. Isomerization of fructose to galactose does not occur in the human body.
Correct answer = A. The symptoms suggest fructose intolerance, a deficiency in aldolase B. Deficiencies in fructokinase or galactokinase result in relatively benign conditions characterized by elevated levels of fructose or galactose in the blood and urine. Deficiency in B-galactosidase (lactase) results in a decreased ability to degrade lactose (milk sugar). Congenital lactase deficiency is quite rare and would have presented much earlier in this baby, and with different symptoms. Typical lactase deficiency (adult hypolactasia) presents at a later age. The symptoms of glucose 6-phosphatase deficiency would result from fasting, and would not be related to ingestion of fruit juice.

Correct answer = D. The expression of o-lactalbumin (protein B) is increased by the hormone, prolactin. UDP-galactose is the form used by the galactosyltransferase (protein A). Protein A is also involved in the synthesis of the amino sugar, N-acetyllactosamine. Protein B increases the affinity of protein A for glucose, and so decreases the Kn.

The pentose phosphate pathway (also called the hexose monophosphate pathway, or 6-phosphogluconate pathway) occurs in the cytosol of the cell. It includes two, irreversible oxidative reactions, followed by a series of reversible sugar—phosphate interconversions (Figure 13.1). No ATP is directly consumed or produced in the cycle. Carbon 1 of glucose 6-phosphate is released as COs, and two NADPH are produced for each glucose 6-phosphate molecule entering the oxidative part of the pathway. The rate and direction of the reversible reactions of the pentose phosphate pathway are determined by the supply of and demand for intermediates of the cycle. The pathway provides a major portion of the body's NADPH, which functions as a biochemical reductant. It also produces ribose 5-phosphate, required for the biosynthesis of nucleotides (see p. 293), and provides a mechanism for the metabolic use of five-carbon sugars obtained from the diet or the degradation of structural carbohydrates in the body.

The oxidative portion of the pentose phosphate pathway consists of three reactions that lead to the formation of ribulose 5-phosphate, COs, and two molecules of NADPH for each molecule of glucose 6-phosphate oxidized (Figure 13.2). This portion of the pathway is particularly important in the liver, lactating mammary glands, and adipose, which are active in the NADPH-dependent biosynthesis of fatty acids (see p. 186), in the testes, ovaries, placenta and adrenal cortex, which are active in the NADPH-dependent biosynthesis of steroid hormones (see p. 237), and in erythrocytes, which require NADPH to keep glutathione reduced (see p. 152).

Glucose 6-phosphate dehydrogenase (G6PD) catalyzes an irreversible oxidation of glucose 6-phosphate to 6-phosphogluconolactone in a reaction that is specific for NADP* as its coenzyme. The pentose phosphate pathway is regulated primarily at the G6PD reaction. NADPH is a potent competitive inhibitor of the enzyme,

Hexose monophosphate pathway shown as a component of the metabolic map (see Figure 8.2, p. 92 for a more detailed view of the metabolic pathway).
and, under most metabolic conditions, the ratio of NADPH/NADP* is sufficiently high to substantially inhibit enzyme activity. However, with increased demand for NADPH, the ratio of NADPH/NADP* decreases and flux through the cycle increases in response to the enhanced activity of G6PD. Insulin upregulates expression of the gene for G6PD, and flux through the pathway increases in the wellfed state.

6-Phosphogluconolactone is hydrolyzed by 6-phosphogluconolactone hydrolase. The reaction is irreversible and not rate-limiting. The oxidative decarboxylation of the product, 6-phosphogluconate is catalyzed by 6-phosphogluconate dehydrogenase. This irreversible reaction produces a pentose sugar—phosphate (ribulose 5-phosphate), CO, (from carbon 1 of glucose), and a second molecule of NADPH (see Figure 13.2).

Reactions of the hexose monophosphate pathway. Enzymes numbered above are: 1,2) glucose 6-phosphate dehydrogenase and 6-phosphogluconolactone hydrolase, 3) 6-phosphogluconate dehydrogenase, 4) ribose 5-phosphate isomerase, 5) phosphopentose epimerase, 6) and 8) transketolase (coenzyme: thiamine pyro reactions; ea hosphate), and 7) transaldolase. = two carbons are transferred in transketolase = three carbons are transferred in the transaldolase reaction.

The nonoxidative reactions of the pentose phosphate pathway occur in all cell types synthesizing nucleotides and nucleic acids. These reactions catalyze the interconversion of sugars containing three to seven carbons (see Figure 13.2). These reversible reactions permit ribulose 5-phosphate (produced by the oxidative portion of the pathway) to be converted either to ribose 5-phosphate (needed for nucleotide synthesis, see p. 293) or to intermediates of glycolysis—fructose 6-phosphate and glyceraldehyde 3-phosphate. For example, many cells that carry out reductive biosynthetic reactions have a greater need for NADPH than for ribose 5-phosphate. In this case, transketolase (which transfers two-carbon units in a thiamine pyrophosphate (TPP)-requiring reaction) and transaldolase (which transfers three-carbon units) convert the ribulose 5-phosphate produced as an endproduct of the oxidative reactions to glyceraldehyde 3-phosphate and fructose 6-phosphate, which are intermediates of glycolysis. In contrast, under conditions in which the demand for ribose for incorporation into nucleotides and nucleic acids is greater than the need for NADPH, the nonoxidative reactions can provide the biosynthesis of ribose 5-phosphate from glyceraldehyde 3-phosphate and fructose 6-phosphate in the absence of the oxidative steps (Figure 13.3).
In addition to transketolases, thiamine pyrophosphate is required by the enzyme complexes pyruvate dehydrogenase, a-ketoglutarate dehydrogenase of the TCA cycle, and branched-chain a-keto acid dehydrogenase of branched-chain amino acid metabolism (see p. 266).

The coenzyme NADP* differs from NAD* only by the presence of a phosphate group on one of the ribose units (Figure 13.4). This seemingly small change in structure allows NADP* to interact with NADP*specific enzymes that have unique roles in the cell. For example, in the cytosol of hepatocytes the steady-state ratio of NADP*/NADPH is approximately 0.1, which favors the use of NADPH in reductive biosynthetic reactions. This contrasts with the high ratio of NAD*/NADH (approximately 1000), which favors an oxidative role for NAD*. This section summarizes some important NADP* or NADPH-specific functions.

NADPH can be thought of as a high-energy molecule, much in the same way as NADH. However, the electrons of NADPH are destined for use in reductive biosynthesis, rather than for transfer to oxygen as is the case with NADH (see p. 75). Thus, in the metabolic transformations of the pentose phosphate pathway, part of the energy of glucose 6-phosphate is conserved in NADPH—a molecule with a negative reduction potential (see p. 77) that, therefore, can be used in reactions requiring an electron donor.

Formation of ribose 5-phosphate from intermediates of glycolysis.

A. Formation of reactive intermediates from molecular oxygen. B. Actions of antioxidant enzymes. G-SH = reduced glutathione; G-S-S-G = oxidized glutathione.

A. Structure of glutathione (G-SH). [Note: Glutamate is linked to cysteine through a y-carboxyl, rather than an a-carboxyl.] B. Glutathione-mediated reduction of hydrogen peroxide by NADPH.
Hydrogen peroxide is one of a family of reactive oxygen species (ROS) that are formed from the partial reduction of molecular oxygen (Figure 13.5A). These compounds are formed continuously as by-products of aerobic metabolism, through reactions with drugs and environmental toxins, or when the level of antioxidants is diminished, all creating the condition of oxidative stress. The highly reactive oxygen intermediates can cause serious chemical damage to DNA, proteins, and unsaturated lipids, and can lead to cell death. These ROS have been implicated in a number of pathologic processes, including reperfusion injury, cancer, inflammatory disease, and aging. The cell has several protective mechanisms that minimize the toxic potential of these compounds.
1.
Enzymes that catalyze antioxidant reactions: Reduced glutathione, a tripeptide-thiol (y-glutamylcysteinylglycine) present in most cells, can chemically detoxify hydrogen peroxide (Figure 13.5B). This reaction, catalyzed by the selenium-requiring glutathione peroxidase, forms oxidized glutathione, which no longer has protective properties. The cell regenerates reduced glutathione in a reaction catalyzed by glutathione reductase, using NADPH as a source of reducing equivalents. Thus, NADPH indirectly provides electrons for the reduction of hydrogen peroxide (Figure 13.6). [Note: Erythrocytes are totally dependent on the pentose phosphate pathway for their supply of NADPH because, unlike other cell types, erythrocytes do not have an alternate source for this essential coenzyme.] Additional enzymes, such as superoxide dismutase and catalase, catalyze the conversion of other toxic oxygen intermediates to harmless products (see Figure 13.5B). As a group, these enzymes serve as a defense system to guard against the toxic effects of reactive oxygen species.

2. Antioxidant chemicals: A number of intracellular reducing agents, such as ascorbate (see p. 377), vitamin E (see p. 391), and B-carotene (see p. 382), are able to reduce and, thus, detoxify oxygen intermediates in the laboratory. Consumption of foods rich in these antioxidant compounds has been correlated with a
reduced risk for certain types of cancers, as well as decreased frequency of certain other chronic health problems. Thus, it is tempting to speculate that the effects of these compounds are, in part, an expression of their ability to quench the toxic effect of oxygen intermediates. However, clinical trials with antioxidants as dietary supplements have failed to show clear beneficial effects. In the case of dietary supplementation with B-carotene, the rate of lung cancer in smokers increased rather than decreased. Thus, the health-promoting effects of dietary fruits and vegetables probably reflects a complex interaction among many naturally occurring compounds, which has not been duplicated by consumption of isolated antioxidant compounds.

Monooxygenases (mixed function oxidases) incorporate one atom from molecular oxygen into a substrate (creating a hydroxyl group), with the other atom being reduced to water. In the cytochrome P450 monooxygenase system, NADPH provides the reducing equivalents required by this series of reactions (Figure 13.7). This system performs different functions in two separate locations in cells. The overall reaction catalyzed by a cytochrome P450 enzyme is:
R-H
+
Oo
+
NADPH
+
H*t
>
R-OH
+
H20
+
NADP*
where R may be a Steroid, drug, or other chemical. [Note: Cytochrome P450s (CYPs) are actually a superfamily of related, heme-containing monooxygenase enzymes that participate in a broad variety of reactions. The name P450 reflects the absorbance at 450 nm by the protein.]

1. Mitochondrial system: The function of the mitochondrial cytochrome P450 monooxygenase system found associated with the inner mitochondrial membrane is the biosynthesis of steroid hormones. In steroidogenic tissues, such as the placenta, ovaries, testes, and adrenal cortex, it is used to hydroxylate intermediates in the conversion of cholesterol to steroid hormones, a process that makes these hydrophobic compounds more water-soluble (see p. 237). The liver uses this same system in bile acid synthesis (see p. 224) and in the hydroxylation of cholecalciferol to 25-hydroxycholecalciferol (vitamin D3, see p. 386), and the kidney uses it to hydroxylate vitamin Dg to its biologically active 1,25-dihydroxylated form.
2. Microsomal system: An extremely important function of the microsomal cytochrome P450 monooxygenase system found associated with the membranes of the smooth endoplasmic reticulum (particularly in the liver) is the detoxification of foreign compounds (xenobiotics). These include numerous drugs and such varied pollutants as petroleum products and pesticides. The microsomal system can be used to hydroxylate these toxins, again using NADPH as the source of reducing equivalents. The purpose of these modifications is two-fold. First, it may itself activate or inactivate a drug or second, make a toxic compound more soluble, thus facilitating its excretion in the urine or feces. Frequently, however,

Cytochrome P450 monooxygenase cycle. Electrons move from NADPH to FAD to FMN and then to the heme iron.

Phagocytosis and the oxygendependent pathway of microbial killing. IgG = the antibody immunoglobulin G.
the new hydroxyl group will serve as a site for conjugation with a polar molecule, such as glucuronic acid (see p. 161), which will significantly increase the compound's solubility.

Phagocytosis is the ingestion by receptor-mediated endocytosis of microorganisms, foreign particles, and cellular debris by cells such as neutrophils and macrophages (monocytes). It is an important body defense mechanism, particularly in bacterial infections. Neutrophils and monocytes are armed with both oxygen-independent and oxygen-dependent mechanisms for killing bacteria.
- 1. Oxygen-independent mechanism: Oxygen-independent mechanisms use pH changes in phagolysosomes and lysosomal enzymes to destroy pathogens.
2.

Oxygen-dependent system: Oxygen-dependent mechanisms include the enzymes, NADPH oxidase and myeloperoxidase (MPO) that work together in killing bacteria (Figure 13.8). Overall, the MPO system is the most potent of the bactericidal mechanisms. An invading bacterium is recognized by the immune system and attacked by antibodies that bind it to a receptor on a phagocytic cell. After internalization of the microorganism has occurred, NADPH oxidase, located in the leukocyte cell membrane, is activated and reduces molecular oxygen from the surrounding tissue into superoxide (Oz*), a free radical. The rapid consumption of molecular oxygen that accompanies formation of superoxide is referred to as the respiratory burst. [Note: Active NADPH oxidase is a membrane-associated complex containing a flavocytochrome plus additional peptides that translocate from the cytoplasm upon activation of the leukocyte. Electrons move from NADPH to Os, via FAD and heme, generating O2*. Genetic deficiencies in NADPH oxidase cause chronic granulomatous disease (CGD) characterized by severe, persistent infections. and the formation of granulomas (nodular areas of inflammation) that sequester the bacteria that were not destroyed.] Next, superoxide is converted to hydrogen peroxide (a ROS), either spontaneously or catalyzed by superoxide dismutase (SOD). In the presence of MPO, a heme-containing lysosomal enzyme present within the phagolysosome, peroxide plus chloride ions are converted to hypochlorous acid (HOCI, the major component of household bleach), which kills the bacteria. The peroxide can also be partially reduced to the hydroxyl radical (OHe), a ROS, or be fully reduced to water by catalase or glutathione peroxidase. [Note: Deficiencies in MPO do not confer increased susceptibility to infection because peroxide from NADPH oxidase is bactericidal.]

Nitric oxide (NO) is recognized as a mediator in a broad array of biologic systems. NO is the endothelium-derived relaxing factor, which causes vasodilation by relaxing vascular smooth muscle. NO also acts as a neurotransmitter, prevents platelet aggregation, and plays an essential role in macrophage function. [Note: NO is a free radical
gas that is often confused with nitrous oxide (N20), the "laughing gas" that is used as an anesthetic and is chemically stable.] NO has a very short half-life in tissues (8-10 seconds) because it reacts with oxygen and superoxide, and then is converted into nitrates and nitrites including peroxynitrite (O=NOO)), a reactive nitrogen species (RNS).
1. Synthesis of NO: Arginine, O2, and NADPH are substrates for cytosolic NO synthase (Figure 13.9). Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), heme, and tetrahydrobiopterin (see p. 268) are coenzymes for the enzyme, and NO and citrulline are products of the reaction. Three NO synthases have been identified. Two are constitutive (synthesized at a constant rate regardless of physiologic demand), Ca?*-calmodulindependent enzymes. They are found primarily in endothelium (eNOS), and neural tissue (nNOS), and constantly produce low levels of NO. An inducible, Ca?*-independent enzyme (iNOS) can be expressed in many cells, including hepatocytes, macrophages, monocytes, and neutrophils. The specific inducers for iNOS vary with cell type, and include tumor necrosis factor-a, bacterial endotoxins, and inflammatory cytokines. These compounds have been shown to promote synthesis of iNOS, which can result in large amounts of NO being produced over hours or even days.

. Actions of NO on vascular endothelium: NO is an important mediator in the control of vascular smooth muscle tone. NO is synthesized by eNOS in endothelial cells, and diffuses to vascular smooth muscle, where it activates the cytosolic form of guanylate cyclase (also known as guanylyl cyclase) to form cGMP. [Note: This reaction is analogous to the formation of cAMP by adenylate cyclase (see p. 94), except that this guanylate cyclase is not membrane-associated.] The resultant rise in cGMP causes activation of protein kinase G, which phosphorylates Ca** channels, causing decreased entry of Ca* into smooth muscle cells. This decreases the calcium-calmodulin activation of myosin light-chain kinase, thereby decreasing smooth muscle contraction and favoring relaxation. Vasodilator nitrates, such as nitroglycerin and nitroprusside, are metabolized to nitric oxide, which causes relaxation of vascular smooth muscle and, therefore, lowers blood pressure. Thus, NO can be envisioned as an endogenous nitrovasodilator. [Note: Sildenafil citrate, used in the treatment of erectile dysfunction, inhibits the phosphodiesterase that inactivates cGMP]
. Role of NO in mediating macrophage bactericidal activity: In macrophages, iNOS activity is normally low, but synthesis of the enzyme is significantly stimulated by bacterial lipopolysaccharide and y-interferon release in response to infection. Activated macrophages form superoxide radicals (see p. 150) that combine with NO to form intermediates that decompose, forming the highly bactericidal OHe radical.
4. Other functions of NO: NO is a potent inhibitor of platelet aggregation (by activating the cGMP pathway). It is also characterized as a neurotransmitter in the brain.

Synthesis and some of the actions of nitric oxide. [Note: FMN, FAD, heme, and tetrahydrobiopterin are additional coenzymes required by NOS.]

Pathways of glucose 6-phosphate metabolism in the erythrocyte. HMP = hexose monophosphate pathway.

Heinz bodies in erythrocytes of a patient with G6PD deficiency.
Glucose 6-phosphate dehydrogenase (G6PD) deficiency is an inherited disease characterized by hemolytic anemia caused by the inability to detoxify oxidizing agents. G6PD deficiency is the most common disease-producing enzyme abnormality in humans, affecting more than 400 million individuals worldwide. This deficiency has the highest prevalence in the Middle East, tropical Africa and Asia, and parts of the Mediterranean. G6PD deficiency is X-linked, and is, in fact, a family of deficiencies caused by more than 400 different mutations in the gene coding for G6PD. Only some of these mutations cause clinical symptoms. [Note: In addition to hemolytic anemia, a clinical manifestation of G6PD deficiency is neonatal jaundice appearing 1—4 days after birth. The jaundice, which may be severe, typically results from increased production of unconjugated bilirubin (see p. 285).] The life span of individuals with a severe form of G6PD deficiency may be somewhat shortened as a result of complications arising from chronic hemolysis. This negative effect of G6PD deficiency has been balanced in evolution by an advantage in survival—an increased resistance to falciparum malaria shown by female carriers of the mutation. [Note: Sickle cell trait and B-thalassemia minor also confer resistance. ]

Diminished G6PD activity impairs the ability of the cell to form the NADPH that is essential for the maintenance of the reduced glutathione pool. This results in a decrease in the cellular detoxification of free radicals and peroxides formed within the cell (Figure 13.10). Glutathione also helps maintain the reduced states of sulfhydryl groups in proteins, including hemoglobin. Oxidation of those sulfhydryl groups leads to the formation of denatured proteins that form insoluble masses (called Heinz bodies) that attach to the red cell membranes (Figure 13.11). Additional oxidation of membrane proteins causes the red cells to be rigid (less deformable), and they are removed from the circulation by macrophages in the spleen and liver. Although G6PD deficiency occurs in all cells of the affected individual, it is most severe in erythrocytes, where the pen-
tose phosphate pathway provides the only means of generating NADPH. Other tissues have alternative sources for NADPH production (such as NADP*-dependent malate dehydrogenases, see Figure 16.11, p. 187) that can keep glutathione reduced. The erythrocyte has no nucleus or ribosomes and cannot renew its supply of the enzyme. Thus, red blood cells are particularly vulnerable to enzyme variants with diminished stability.

Most individuals who have inherited one of the many G6PD mutations do not show clinical manifestations, that is, they are asymptomatic. However, some patients with G6PD deficiency develop hemolytic anemia if they are treated with an oxidant drug, ingest fava beans, or contract a severe infection.
1. Oxidant drugs: Commonly used drugs that produce hemolytic anemia in patients with G6PD deficiency are best remembered from the mnemonic AAA—Antibiotics (for example, sulfamethoxazole and chloramphenicol), Antimalarials (for example, primaquine but not quinine), and Antipyretics (for example, acetanilid but not acetaminophen).
2. Favism: Some forms of G6PD deficiency, for example the Mediterranean variant, are particularly susceptible to the hemolytic effect of the fava (broad) bean, a dietary staple in the Mediterranean region. Favism, the hemolytic effect of ingesting fava beans, is not observed in all individuals with G6PD deficiency, but all patients with favism have G6PD deficiency.
3. Infection: Infection is the most common precipitating factor of hemolysis in G6PD deficiency. The inflammatory response to infection results in the generation of free radicals in macrophages, which can diffuse into the red blood cells and cause oxidative damage.

Almost all G6PD variants are caused by point mutations in the gene for G6PD. Some mutations do not disrupt the structure of the enzyme's active site and, hence, do not affect enzymic activity. However, many mutant enzymes show altered kinetic properties. For example, variant enzymes may show decreased catalytic activity, decreased stability, or an alteration of binding affinity for NADP*, NADPH, or glucose 6-phosphate. The severity of the disease usually correlates with the amount of residual enzyme activity in the patient's red blood cells. For example, variants can be classified as shown in Figure 13.12. G6PD A' is the prototype of the moderate (Class III) form of the disease. The red cells contain an unstable but kinetically normal G6PD, with most of the enzyme activity present in the reticulocytes and younger erythrocytes (Figure 13.13). The oldest cells, therefore, have the lowest level of enzyme activity, and are preferentially removed in a hemolytic episode. G6PD Mediterranean is the prototype of a more severe (Class Il) deficiency in which the enzyme has decreased stability resulting in decreased enzymic activity. Class | mutations (rare) are the most severe and are associated with chronic nonspherocytic anemia, which occurs even in the absence of oxidative stress.

Classification of G6PD deficiency variants. Note: Class V mutations (not shown in table) result in overproduction of G6PD.
Although the activity of the normal enzyme declines as red cells age, even the oldest cells have a sufficient level of activity to provide protection against oxidative damage and hemolysis.

Decline of erythrocyte G6PD activity with cell age for the three most commonly encountered forms of the enzyme.

The cloning of the gene for G6PD and the sequencing of its DNA (see p. 467) have permitted the identification of mutations that cause G6PD deficiency. More than 400 different mutations or mutation combinations have been identified in this gene, a finding that explains the numerous biochemical variants that have been described. Most mutations that result in enzymic deficiency are missense mutations (see p. 433) in the coding region. Both G6PD A" and G6PD Mediterranean represent mutant enzymes that differ from the respective normal variants by a single amino acid. Large deletions or frameshift mutations have not been identified, suggesting that complete absence of G6PD activity is probably lethal.

The pentose phosphate pathway includes two irreversible oxidative reactions followed by a series of reversible sugar—phosphate interconversions (Figure 13.14). No ATP is directly consumed or produced in the cycle. The NADPH-producing oxidative portion of the pentose phosphate pathway is particularly important in liver, mammary glands, and adipose, which are active in the biosynthesis of fatty acids; in the placenta, ovaries, testes, and adrenal cortex, which are active in the synthesis of steroid hormones; in erythrocytes, which require reduced glutathione. Glucose 6-phosphate is irreversibly converted to ribulose 5-phosphate, and two NADPH are produced. The regulated step is glucose 6-phosphate dehydrogenase (G6PD), which is strongly inhibited by NADPH. Reversible nonoxidative reactions interconvert sugars. This part of the pathway is the source of ribose 5-phosphate, required for nucleotide and nucleic acid synthesis. Because the reactions are reversible, they can be entered from fructose 6-phosphate and glyceraldehyde 3-phosphate (glycolytic intermediates) if ribose is needed and glucose 6-phosphate dehydrogenase is inhibited. NADPH is a source of reducing equivalents in reductive biosynthesis, such as the production of fatty acids and steroids. It is also required for the reduction of hydrogen peroxide, providing the reducing equivalents required by glutathione (GSH). GSH is used by glutathione peroxidase to reduce peroxide to water. The oxidized glutathione is reduced by glutathione reductase, using NADPH as the source of electrons. NADPH provides reducing equivalents for the mitochondrial cytochrome P450 monooxygenase system, which is used in steroid hormone synthesis in steroidogenic tissue, bile acid synthesis in liver, and vitamin D activation in liver and kidney. The microsomal system uses NADPH to detoxify foreign compounds (xenobiotics), such as drugs and a variety of pollutants. NADPH provides the reducing equivalents for phagocytes in the process of eliminating invading microorganisms. NADPH oxidase uses molecular oxygen and NADPH electrons to produce superoxide radicals, which, in turn, can be converted to peroxide by superoxide dismutase. Myeloperoxidase catalyzes the formation of bactericidal

hypochlorous acid from peroxide and chloride ion. A genetic defect in NADPH oxidase causes chronic granulomatous disease characterized by severe, persistent, infections and formation of granulomas. NADPH is required for the synthesis of nitric oxide (NO), an important molecule that causes vasodilation by relaxing vascular smooth muscle, acts as a neurotransmitter, prevents platelet aggregation, and helps mediate macrophage bactericidal activity. G6PD deficiency impairs the ability of the cell to form the NADPH that is essential for the maintenance of the reduced glutathione pool. The cells most affected are the red blood cells because they do not have additional sources of NADPH. G6PD deficiency is an X-linked genetic disease characterized by hemolytic anemia. Free radicals and peroxides formed within the cells cannot be neutralized, causing denaturation of cytosolic protein (for example, hemoglobin, forming Heinz bodies) and membrane proteins. The cells become rigid, and they are removed by the reticuloendothelial system of the spleen and liver. Babies with G6PD deficiency may experience neonatal jaundice. Hemolytic anemia can be caused by the production of free radicals and peroxides following the taking of oxidant drugs, ingestion of fava beans, or severe infections. The degree of severity of the anemia depends on the amount of residual enzyme. Class | mutations, the most severe, are associated with chronic nonspherocytic anemia. Class II mutations (for example, G6PD Mediterranean) are also severe. Class III mutations (Such as G6PD A_) cause a more moderate form of the disease.

- 13.1 In preparation for a trip to an area of India
where malaria
is endemic,
a
young man
is given
primaquine prophylactically.
develops
a
Soon hemolytic
thereafter, condition.
he
The cause
of the
hemolysis most
likely is
a
less-than-normal level of which of the following? .
A. Glucose 6-phosphate
ay B. Oxidized form of NAD
. C. Reduced form of glutathione
D. Ribose 5-phosphate
E. Ribulose 5-phosphate
Correct answer = C. Glutathione is essential for red cell integrity and is maintained in its functional (reduced) form by NADPH-dependent glutathione reductase. The NADPH is generated by the oxidative portion of pentose phosphate pathway. Individuals with a deficiency of the initiating and regulated enzyme (G6PD) of this pathway have a decreased ability to generate NADPH, See espe ego eed functional. When treated with an oxidant drug such as primaquine, some patients with G6PD deficiency develop a hemolytic anemia. Primaquine does not affect glucose 6-phosphate levels. NAD* is neither produced by the pentose phosphate pathway nor used as a coenzyme by glutathione reductase. Ribulose 5-phosphate, another product of the oxidative portion of the pentose phosphate pathway, can be isomerized to ribose 5-phosphate, but a deficiency in either does not cause hemolysis.
13.2 In male patients who are hemizygous for glucose 6phosphate dehydrogenase (G6PD) deficiency, pathophysiologic consequences are more apparent in erythrocytes (RBC) than in other cells, such as in the liver. Which one of the following provides the most reasonable explanation for this different response by these individual tissue types?

| . | A. Excess glucose 6-phosphate in the liver, but not in RBCs, can be channeled to glycogen, thus averting cellular damage.
B. Liver cells, in contrast to RBCs, have alternative mechanisms for supplying the NADPH required for keeping metabolic and cellular integrity.
C. Glucose 6-phosphatase activity in RBCs decreases the level of glucose 6-phosphate, thus oe : : resulting in cell damage. This does not happen in the hepatocyte.
. . D. Because RBCs do not have mitochondria, production of ATP required to keep cell integrity depends exclusively on the routing of glucose 6-phosphate to the pentose phosphate pathway.
E.
- The catalytic properties of the liver enzyme are significantly different than those of the RBC enzyme.
13.3 A G6PD A' individual was treated with primaquine from day 0 to day 120 (Figure 13.15). Hemolysis occurred immediately after initiation of drug therapy, as indicated by progressive anemia, hemoglobinuria, and reticulocytosis. However, despite continued administration of the drug, the hemolysis spontaneously decreased and red cell survival improved with time. Red cell G6PD activity measured 2 months after termination of therapy was 10% of normal. Which one of the following statements about this patient is correct?
A. The patient will continue to be resistant to druginduced hemolysis after 6 months or longer.
a . we we ae B. Erythrocytes in this patient exhibit a longer lifetime than in normal individuals.
C. During the period of peak hemolysis, erythrocytes
from the
patient will
show no
G6PD
activity.
D.
E.
- The intracellular concentration of NADPH in the
patient's erythrocytes
is greater
than normal
.
- The patient shows increased bone marrow erythropoiesis.

Correct answer = B. Cellular damage is directly related to decreased ability of the cell to regenerate reduced glutathione, for which large amounts of NADPH are needed, and RBC have no other means of generating NADPH. Catalytic properties of G6PD in liver and RBCs are very similar. The pentose phosphate pathway does not generate ATP. RBCs do not have glucose 6phosphatase.

The course of primaquine-induced hemolysis in a patient with G6PD deficiency.
Correct answer = E. As red cells age, the activity of G6PD declines (see Figure 13.13). Despite this [SCID e ny Ly ee eee Nabe SON eeaee ae face of oxidant stress. In contrast, the G6PD variants with hemolysis have much shorter half-lives. The clinical correlate of this age-related enzyme instability is that hemolysis in patients with G6PD A generally is mild and limited to the older deficient erythrocytes. The anemia is self-limited because the older, vulnerable population of erythrocytes is replaced by younger RBCs with sufficient G6PD activity to withstand an oxidative assault. Although red cell survival remains shortened as long as use of the drug continues, compensation by the erythroid marrow effectively abolishes the anemia in subjects with G6PD A-. The individual's continuing sensitivity to the effects of the drug is revealed by discontinuing the drug for several months to allow the rate of red cell production by the bone marrow to normalize; during this phase, the older red cells are able to survive, and the red cell population is rendered sensitive to drug-induced hemolysis.

: . Glycosaminoglycans are large complexes of negatively charged heteropolysaccharide chains. They are generally associated with a small amount of protein, forming proteoglycans, which typically consist of over 95% carbohydrate. [Note: This is in comparison to the glycoproteins, which consist primarily of protein with a small amount of carbohydrate (see p. 165).] Glycosaminoglycans have the special ability to bind large amounts of water, thereby producing the gel-like matrix that forms the basis of the body's ground substance, which, along with fibrous structural proteins such as collagen and elastin, and adhesive proteins such as fibronectin, make up the extracellular matrix (ECM). The hydrated glycosaminoglycans serve as a flexible support for the ECM, interacting with the structural and adhesive proteins, and as a molecular sieve, influencing movement of materials through the ECM. The viscous, lubricating properties of mucous secretions also result from the presence of glycosaminoglycans, which led to the original naming of these compounds as mucopolysaccharides.

Glycosaminoglycans (GAGs) are long, unbranched, heteropolysaccharide chains generally composed of a repeating disaccharide unit [acidic sugar—amino sugar], (Figure 14.1). The amino sugar is either D-glucosamine or D-galactosamine, in which the amino group is usually acetylated, thus eliminating its positive charge. The amino sugar may also be sulfated on carbon 4 or 6 or on a nonacetylated nitrogen. The acidic sugar is either D-glucuronic acid or its C-5 epimer, L-iduronic acid (Figure 14.2). [Note: A single exception is keratan sulfate, in which galactose rather than an acidic sugar is present.] These acidic sugars contain carboxyl groups that are negatively charged at physiologic pH and, together with the sulfate groups, give GAGs their strongly negative nature.

Because of their large number of negative charges, these heteropolysaccharide chains tend to be extended in solution. They repel each other, and are surrounded by a shell of water molecules. When brought together, they "slip" past each other, much as two magnets

Resilience of glycosaminoglycans.
with the same polarity seem to slip past each other. This produces the "slippery" consistency of mucous secretions and synovial fluid. When a solution of glycosaminoglycans is compressed, the water is "squeezed out" and the glycosaminoglycans are forced to occupy a smaller volume. When the compression is released, the glycosaminoglycans spring back to their original, hydrated volume because of the repulsion of their negative charges. This property contributes to the resilience of synovial fluid and the vitreous humor of the eye (Figure 14.3).

The six major classes of glycosaminoglycans are divided according to monomeric composition, type of glycosidic linkages, and degree and location of sulfate units. The structure of the glycosaminoglycans and their distribution in the body is illustrated in Figure 14.4.

All of the glycosaminoglycans, except hyaluronic acid, are found covalently attached to protein, forming proteoglycan monomers.
1. Structure of proteoglycan monomers: A proteoglycan monomer found in cartilage consists of a core protein to which the linear glycosaminoglycan chains are covalently attached. These chains, which may each be composed of more than 100 monosaccharides, extend out from the core protein, and remain separated from each other because of charge repulsion. The resulting structure resembles a "bottle brush" (Figure 14.5). In cartilage proteoglycan, the species of glycosaminoglycans include chondroitin sulfate and keratan sulfate. [Note: Proteoglycans are now grouped into gene families that code for core proteins with common structural features. The aggrecan family (aggrecan, versecan, neurocan, and brevican), abundant in cartilage, is an example.]
2. Linkage between the carbohydrate chain and the protein: This linkage is most commonly through a trihexoside (galactose-galactose-xylose) and a serine residue, respectively. An O-glycosidic bond is formed between the xylose and the hydroxyl group of the serine (Figure 14.6).
3. Proteoglycan aggregates: The proteoglycan monomers associate with a molecule of hyaluronic acid to form proteoglycan aggregates. The association is not covalent, but occurs primarily through ionic interactions between the core protein and the hyaluronic acid. The association is stabilized by additional small proteins called link proteins (Figure 14.7).

The polysaccharide chains are elongated by the sequential addition of alternating acidic and amino sugars donated by their UDP-derivatives. The reactions are catalyzed by a family of specific glycosyltransferases. The synthesis of the glycosaminoglycans is analogous to that of glycogen (see p. 126) except that the glycosaminoglycans are produced for export from the cell. Their synthesis occurs, therefore, primarily in the Golgi, rather than in the cytosol.

Structure and distribution of glycosaminoglycans (GAGs). Sulfate groups (©) are shown in all possible positions. GIcUA = glucuronic acid; IdUA = iduronic acid; GalNAC = N-acetylgalactosamine; GICNAC = N-acetylglucosamine; GIcN = glucosamine; Gal = galactose.

Amino sugars are essential components of glycosaminoglycans, glycoproteins, glycolipids, and certain oligosaccharides, and are also found in some antibiotics. The synthetic pathway of amino sugars is very active in connective tissues, where as much as 20% of glucose flows through this pathway.

(GalNAc): The monosaccharide fructose 6-phosphate is the precursor of GIcNAc, GalNAc, and the sialic acids, including Nacetylneuraminic acid (NANA, a nine-carbon, acidic monosaccharide). In each of these sugars, a hydroxyl group of the precursor is replaced by an amino group donated by glutamine (Figure 14.8). [Note: The amino groups are then almost always acetylated.] The UDP-derivatives of GIcNAc and GalNAc are synthesized by reactions analogous to those described for UDP-glucose synthesis (see p. 126). These nucleotide sugars are the activated forms of the monosaccharides that can be used to elongate the carbohydrate chains.

a member of the family of sialic acids, each of which is acylated at a different site. These compounds are usually found as terminal carbohydrate residues of oligosaccharide side chains of glycoproteins, glycolipids, or, less frequently, of glycosaminoglycans. The carbons and nitrogens in NANA come from N-acetylmannosamine and phosphoenolpyruvate (an intermediate in the glycolytic pathway, see p. 102). [Note: Before NANA can be added to a growing oligosaccharide, it must be converted into its active form by reacting with cytidine triphosphate (CTP). The enzyme CMP-NANA synthetase catalyzes the reaction. This is the only nucleotide sugar in human metabolism in which the carrier nucleotide is a monophosphate.]

D-Glucuronic acid, whose structure is that of glucose with an oxidized carbon 6 (-CH20H ——COOH), and its C-5 epimer, L-iduronic acid, are essential components of glycosaminoglycans. Glucuronic acid is also required in detoxification reactions of a number of insoluble compounds, such as bilirubin (see p. 282), steroids, and several drugs, including morphine. In plants and mammals (other than guinea pigs and primates, including humans), glucuronic acid serves as a precursor of ascorbic acid (vitamin C). The uronic acid pathway also provides a mechanism by which dietary D-xylulose can enter the central metabolic pathways.
1. Glucuronic acid: Glucuronic acid can be obtained in small amounts from the diet. It can also be obtained from the intracellular lysosomal degradation of glycosaminoglycans, or via the uronic acid pathway. The endproduct of glucuronic acid metabolism in humans is D-xylulose 5-phosphate, which can enter the hexose monophosphate pathway and produce the glycolytic intermediates glyceraldehyde 3-phosphate and fructose 6-phosphate (Figure 14.9; see also Figure 13.2, p. 146). The active form of glucuronic acid that donates the sugar in glycosaminoglycan synthesis and other glucuronylating reactions is UDP-glucuronic acid, which is produced by oxidation of UDP-glucose (Figure 14.10).
2. L-lduronic acid synthesis: Synthesis of L-iduronic acid residues occurs after D-glucuronic acid has been incorporated into the carbohydrate chain. Uronosyl 5-epimerase causes epimerization of the Dto the L-sugar.

The core protein is synthesized on and enters the rough endoplasmic reticulum (RER). The protein is then glycosylated by bound glycosyltransferases located in the Golgi.

Carbohydrate chain formation begins by synthesis of a short linkage region on the core protein on which carbohydrate chain synthesis will be initiated. The most common linkage region is formed by the transfer of a xylose from UDP-xylose to the hydroxyl group of a serine (or threonine) catalyzed by xylosyltransferase. Two galactose molecules are then added, completing the trinexoside. This is followed by sequential addition of alternating acidic and amino sugars (Figure 14.11), and epimerization of some D-glucuronyl to L-iduronyl residues.

Sulfation of the carbohydrate chain occurs after the monosaccharide to be sulfated has been incorporated into the growing carbohydrate chain. The source of the sulfate is 3'-phosphoadenosyl-5'-phosphosulfate (PAPS, a molecule of AMP with a sulfate group attached to the 5'-phosphate). Sulfotransferases cause the sulfation of the carbohydrate chain at specific sites. [Note: An example of the synthesis of a sulfated glycosaminoglycan, chondroitin sulfate, is shown in Figure 14.11.] PAPS is also the sulfur donor in glycosphingolipid synthesis.
A defect in the sulfation of the growing glycosaminoglycan chains results in one of several autosomal recessive disorders (chondrodystrophies) that affect the proper development and maintenance of the skeletal system.

Glycosaminoglycans are degraded in lysosomes, which contain hydrolytic enzymes that are most active at a pH of approximately 5. [Note: Therefore, as a group, these enzymes are called acid hydrolases.] The low pH optimum is a protective mechanism that prevents the enzymes from destroying the cell should leakage occur into the cytosol where the pH is neutral. With the exception of keratan sulfate, which has a half-life of greater than 120 days, the glycosaminoglycans have a relatively short half-life, ranging from about 3 days for hyaluronic acid to 10 days for chondroitin and dermatan sulfate.

Because glycosaminoglycans are extracellular or cell-surface compounds, they must first be engulfed by an invagination of the cell membrane (phagocytosis), forming a vesicle inside of which the glycosaminoglycans are to be degraded. This vesicle then fuses with a lysosome, forming a single digestive vesicle in which the glycosaminoglycans are efficiently degraded (see p. 150 for a discussion of phagocytosis).

The lysosomal degradation of glycosaminoglycans requires a large number of acid hydrolases for complete digestion. First, the polysaccharide chains are cleaved by endoglycosidases, producing oligosaccharides. Further degradation of the oligosaccharides occurs sequentially from the nonreducing end of each chain (see p. 127), the last group (sulfate or sugar) added during synthesis being the first group removed. Examples of some of these enzymes and the bonds they hydrolyze are shown in Figure 14.12.

The mucopolysaccharidoses are hereditary diseases (1:25,000 births) caused by a deficiency of any one of the lysosomal hydrolases normally involved in the degradation of heparan sulfate and/or dermatan sulfate (see Figure 14.12). They are progressive disorders characterized by accumulation of glycosaminoglycans in various tissues, causing a range of symptoms, such as skeletal and extracellular matrix deformities, and mental retardation. Children who are homozygous for any one of these diseases are apparently normal at birth, then gradually deteriorate. In severe cases, death occurs in childhood. All are autosomal recessive diseases except Hunter syndrome, which is X-linked. Incomplete lysosomal degradation of glycosaminoglycans results in the presence of oligosaccharides in the urine. These fragments can be used to diagnose the specific mucopolysaccharidosis by identifying the structure present on the nonreducing end of the oligosaccharide, as that residue would have been the substrate for the missing enzyme. Diagnosis is confirmed by measuring the patient's cellular level of the lysosomal hydrolases. Bone marrow and cord blood transplants have been used to treat Hurler and Hunter syndromes. Here the transplanted macrophages produce the enzymes needed to degrade glycosaminoglycans in the extracellular space. Enzyme replacement therapy (ERT) is currently available for both syndromes.
In addition to the degradation of glycosaminoglycans, lysosomal endoand exoglycosidases are also involved in the degradation of glycoproteins (see p. 170) and glycolipids (see p. 210). Deficiencies in these enzymes result in the accumulation of partially degraded carbohydrates in the lysosomes, leading to cell and tissue damage.

Degradation of the glycosaminoglycan heparan sulfate by lysosomal enzymes, indicating sites of enzyme deficiencies in some representative mucopolysaccharidoses. [Note: Deficiencies in the degradation of keratan sulfate result in Morquio syndrome, A and B.]

Glycoproteins are proteins to which oligosaccharides are covalently attached. They differ from the proteoglycans in that the length of the glycoprotein's carbohydrate chain is relatively short (usually two to ten sugar residues in length, although they can be longer), whereas it can be very long in the glycosaminoglycans (see p. 157). In addition, whereas glycosaminoglycans have diglucosyl repeat units, the carbohydrates of glycoproteins do not have serial repeats. The glycoprotein carbohydrate chains are often branched instead of linear, and may or may not be negatively charged. Glycoproteins contain highly variable amounts of carbohydrate. For example, immunoglobulin IgG, contains less than 4% of its mass as carbohydrate, whereas human gastric glycoprotein (mucin) contains more than 80% carbohydrate. Membranebound glycoproteins participate in a broad range of cellular phenomena, including cell surface recognition (by other cells, hormones, and viruses), cell surface antigenicity (such as the blood group antigens), and as components of the extracellular matrix and of the mucins of the gastrointestinal and urogenital tracts, where they act as protective biologic lubricants. In addition, almost all of the globular proteins present in human plasma are glycoproteins. (See Figure 14.13 for a summary of some of the functions of glycoproteins.)

The oligosaccharide components of glycoproteins are generally branched heteropolymers composed primarily of D-hexoses, with the addition in some cases of neuraminic acid, and of L-fucose—a 6-deoxyhexose.

The oligosaccharide may be attached to the protein through an Nor an O-glycosidic link (see p. 86). In the former case, the sugar chain is attached to the amide group of an asparagine side chain, and in the latter case, to the hydroxyl group of either a serine or threonine R-group. [Note: In the case of collagen, there is an O-glycosidic linkage between galactose or glucose and the hydroxyl group of hydroxylysine (see p. 47).]

A glycoprotein may contain only one type of glycosidic linkage (Nor O-linked), or may have both Oand N-linked oligosaccharides within the same molecule.
1. O-Linked oligosaccharides: The O-linked oligosaccharides may have one or more of a wide variety of sugars arranged in either a linear or a branched pattern. Many O-linked oligosaccharides are found in extracellular glycoproteins or as membrane glycoprotein components. For example, O-linked oligosaccharides on the surface of RBCs help provide the ABO blood group determinants.

Complex (top) and high-mannose (bottom) oligosaccharides. [Note: Members of each class contain the same pentasaccharide core.]
2. N-linked oligosaccharides: The N-linked oligosaccharides fall into two broad classes: complex oligosaccharides and high-mannose oligosaccharides. Both contain the same core pentasaccharide shown in Figure 14.14, but the complex oligosaccharides contain a diverse group of additional sugars, for example, N-acetylglucosamine (GIcNAc), L-fucose (Fuc), and N-acetylneuraminic acid (NANA), whereas the high-mannose oligosaccharides contain primarily mannose (Man).

Proteins destined to function in the cytoplasm are synthesized on free cytosolic ribosomes. However, proteins, including many glycoproteins, that are destined for cellular membranes, lysosomes, or to be exported from the cell, are synthesized on ribosomes attached to the RER. These proteins contain specific signal sequences that act as molecular "address labels," targeting the proteins to their proper destinations. An N-terminal hydrophobic sequence initially directs these proteins to the RER, allowing the growing polypeptide to be extruded into the lumen. The proteins are then transported via secretory vesicles to the Golgi complex, which acts as a sorting center (Figure 14.15). In the Golgi those glycoproteins that are to be secreted from the cell (or are targeted for lysosomes) are packaged into vesicles that fuse with the cell (or lysosomal) membrane and release their contents. Those that are destined to become components of the cell membrane are integrated into the Golgi membrane, which buds off, forming vesicles that add their membrane-bound glycoproteins to the cell membrane. [Note: The membrane glycoproteins are thus oriented with the carbohydrate portion on the outside of the cell (see Figure 14.15).]

The precursors of the carbohydrate components of glycoproteins are nucleotide sugars, which include UDP-glucose, UDP-galactose, UDPN-acetylglucosamine, and UDP-N-acetylgalactosamine. In addition, GDP-mannose, GDP-L-fucose (which is synthesized from GDP-mannose), and CMP-N-acetylneuraminic acid may donate sugars to the growing chain. [Note: When NANA is present, the oligosaccharide has a negative charge at physiologic pH.] The oligosaccharides are covalently attached to specific amino acid R-groups of the protein, where the three-dimensional structure of the protein determines whether or not a specific amino acid R-group is glycosylated.

The synthesis of the O-linked glycosides is very similar to that of the glycosaminoglycans (see p. 158). First, the protein to which the oligosaccharides are to be attached is synthesized on the RER, and extruded into its lumen. Glycosylation begins with the transfer of an N-acetylgalactosamine (from UDP-N-acetylgalactosamine) onto the R-group of a specific serine or threonine.
1. Role of glycosyltransferases: The glycosyltransferases responsible for the stepwise synthesis of the oligosaccharides are bound to the membranes of the Golgi apparatus. They act in a specific order,

Transport of glycoproteins through the Golgi apparatus and their subsequent release or incorporation into a lysosome or the cell membrane.

Synthesis of N-linked glycoproteins. o = N-acetylglucosamine; o= mannose; e = glucose; a = N-acetylgalactosamine; © or <« for example, fucose or N-acetylneuraminic acid.
2.
Final processing of N-linked oligosaccharides: After incorporation into the protein, the N-linked oligosaccharide is processed by the removal of specific mannosyl and glucosyl residues as the glycoprotein moves through the RER. Finally, the oligosaccharide chains are completed in the Golgi by addition of a variety of sugars (for example, N-acetylglucosamine, N-acetylgalactosamine, and additional mannoses, and then fucose or NANA as terminal groups) to produce a complex glycoprotein, or they are not processed further, leaving branched, mannose-containing chains in a high-mannose glycoprotein (see Figure 14.16). The ultimate fate of N-linked glycoproteins is the same as that of the O-linked, for example, they can be released by the cell, or become part of a cell membrane. In addition N-linked glycoproteins can be translocated to the lysosomes. [Note: Nonenzymatic glycosylation of proteins is known as glycation (see p. 345).]

Mechanism for transport of N-linked glycoproteins to the lysosomes.
3.
Enzymes destined for lysosomes: N-linked glycoproteins being processed through the Golgi can be phosphorylated at one or more specific mannosyl residues. Mannose 6-P receptors, located in the Golgi apparatus, bind the mannose 6-P residues of these targeted enzymes, which are then packaged into vesicles and sent to the lysosomes. I-cell disease is a rare syndrome in which the acid hydrolase enzymes normally found in lysosomes are absent, resulting in an accumulation of substrates normally degraded by lysosomal enzymes within these vesicles. [Note: |-cell disease is so-named because of the large inclusion bodies seen in cells of patients with this disease.] In addition, high amounts of lysosomal enzymes are found in the patient's plasma, suggesting that the targeting process to lysosomes (rather than the synthetic pathway of these enzymes) is deficient. It has been determined that individuals with I-cell disease are lacking the enzymic ability to phosphorylate the mannose residues of potential lysosomal enzymes, causing an incorrect targeting of these proteins to extracellular sites, rather than lysosomal vesicles (Figure 14.17). I-cell disease, a lysosomal storage disease, is characterized by skeletal abnormalities, restricted joint movement, coarse (dysmorphic) facial features, and severe psychomotor impairment. Death usually occurs by 8 years of age.

Degradation of glycoproteins is similar to that of the glycosaminoglycans (see p. 162). The lysosomal acid hydrolases are each generally specific for the removal of one component of the glycoprotein. They are primarily exoenzymes that remove their respective groups in sequence in the reverse order of their incorporation ("last on, first off"). If any one degradative enzyme is missing, degradation by the other exoenzymes cannot continue. A group of very rare, autosomal recessive genetic diseases called the glycoprotein storage diseases (oligosaccharidoses), caused by a deficiency of any one of the degradative enzymes, results in accumulation of partially degraded structures in the lysosomes. For example, a-mannosidosis type 1 is a progressive, fatal deficiency of the enzyme, o-mannosidase. Presentation is similar to Hurler syndrome, and immune deficiency is also seen. Mannose-rich oligosaccharide fragments appear in the urine. Diagnosis is by enzyme assay.

Glycosaminoglycans are long, negatively charged, unbranched, heteropolysaccharide chains generally composed of a repeating disaccharide unit [acidic sugar—amino sugar], (Figure 14.18). The amino sugar is either Dglucosamine or D-galactosamine in which the amino group is usually acetylated, thus eliminating its positive charge. The amino sugar may also be sulfated on carbon 4 or 6 or on a nonacetylated nitrogen. The acidic sugar is either D-glucuronic acid or its C-5 epimer, L-iduronic acid. These compounds bind large amounts of water, thereby producing the gel-like matrix that forms the basis of the body's ground substance. The viscous, lubricating properties of mucous secretions are also caused by the presence of glycosaminoglycans, which led to the original naming of these compounds as mucopolysaccharides. As essential components of cell surfaces, glycosaminoglycans play an important role in mediating cell-cell signaling and adhesion. There are six major classes of glycosaminoglycans, including chondroitin 4and 6-sulfates, keratan sulfate, dermatan sulfate, heparin, heparan sulfate, and hyaluronic acid. All of the glycosaminoglycans, except hyaluronic acid, are found covalently attached to protein, forming proteoglycan monomers, which consist of a core protein to which the linear glycosaminoglycan chains are covalently attached. The proteoglycan monomers associate with a molecule of hyaluronic acid to form proteoglycan aggregates. Glycosaminoglycans are synthesized in the Golgi. The polysaccharide chains are elongated by the sequential addition of alternating acidic and amino sugars, donated by their UDP-derivatives. D-glucuronate maybe epimerized to L-iduronate. The last step in synthesis is sulfation of some of the amino sugars. The source of the sulfate is 3'-phosphoadenosyl-5'-phosphosulfate. Glycosaminoglycans are degraded by lysosomal hydrolases. They are first broken down to oligosaccharides, which are degraded sequentially from the nonreducing end of each chain. A deficiency

of any one of the hydrolases results in a mucopolysaccharidosis. These are hereditary disorders in which glycosaminoglycans accumulate in tissues, causing symptoms such as skeletal and extracellular matrix deformities, and mental retardation. Examples of these genetic diseases include Hunter and Hurler syndromes. Glycoproteins are proteins to which oligosaccharides are covalently attached. They differ from the proteoglycans in that the length of the glycoprotein's carbohydrate chain is relatively short (usually two to ten sugar residues long, although they can be longer), may be branched, and does not contain serial disaccharide units. Membrane-bound glycoproteins participate in a broad range of cellular phenomena, including cell surface recognition (by other cells, hormones, and viruses), cell surface antigenicity (such as the blood group antigens), and as components of the extracellular matrix and of the mucins of the gastrointestinal and urogenital tracts, where they act as protective biologic lubricants. In addition, almost all of the globular proteins present in human plasma are glycoproteins. Glycoproteins are synthesized in the endoplasmic reticulum and the Golgi. The precursors of the carbohydrate components of glycoproteins are nucleotide sugars. O-linked glycoproteins are synthesized in the Golgi by the sequential transfer of sugars from their nucleotide carriers to the protein. N-linked glycoproteins contain varying amounts of mannose.
X
They are synthesized by the transfer of a preformed oligosaccharide from its ER membrane lipid carrier, dolichol, to the protein. A deficiency in the phosphorylation of mannose residues in N-linked glycoprotein pre-enzymes destined for the lysosomes results in I-cell disease. Glycoproteins are degraded in lysosomes by acid hydrolases. A deficiency of any one of these enzymes results in a lysosomal glycoprotein storage disease (oligosaccharidosis), resulting in accumulation of partially degraded structures in the lysosome.

- 14.1 Mucopolysaccharidoses are inherited lysosomal stor-
age diseases. They are caused by:
A. increased of synthesis of the carbohydrate
an rate component of proteoglycans.
B. the synthesis of polysaccharides with an altered
structure.
- C. defects in the degradation of the GAGs in proteo-
glycans.
- D. the synthesis of abnormally small amounts of pro-
tein cores.
E.
- an insufficient amount of proteolytic enzymes.

of a patient suggests a deficiency in which one of the enzymes listed below?
Sulfate
Sulfate
|
|
GalNac—GIcUA—GalNAc—
A. Galactosidase
B. Glucosidase
- C. Glucuronidase
D. Mannosidase
E. Sulfatase
14.3 An 8-month-old boy with coarse facial features,
skeletal abnormalities, and delays in both growth and development is diagnosed with I-cell disease based on his presentation, and on histologic and biochemical testing. I-cell disease is the consequence of:
A. decreased production of cell-surface glycoproteins.
B. an inability to ubiquitinate proteins.
C. an inability to glycosylate proteins.
D. incorrect targeting of lysosomal proteins.
E. increased synthesis of proteoglycans.
14. 4 Distinguish between glycoproteins and proteogly-
cans.
Correct answer = C. In mucopolysaccharidoses, synthesis of proteoglycans is unaffected, both in terms of the structure and the amount of material synthesized. The diseases are caused by a deficiency of one of the lysosomal, hydrolytic enzymes responsible for the degradation of glycosaminoglycans (not the core protein).
Correct answer = E. Degradation of glycoproteins follows the rule "last on, first off." Because sulfation is the last step in the synthesis of this sequence, a sulfatase is required for the next step in the degradation of the compound shown.
Correct answer = D. I-cell disease is a lysosomal storage disease caused by a defect in the gene that initiaties the synthesis of the mannose 6phosphate signal that targets acid hydrolases to the lysosome, resulting in accumulation of materials within the lysosome due to decreased degradation. of the other choices relate in any way to |-cell disease or lysosomal function.

Glycoproteins are proteins to which short, branched, oligosaccharide chains are attached. Proteoglycans consist of a core protein to which long, unbranched, glycosaminoglycan (GAG) chains are attached. GAGs are large complexes of negatively charged heteropolysaccharides composed of repeating [acidic sugar-amino sugar]n disaccharide units.

Lipids are a heterogeneous group of water-insoluble (hydrophobic) organic molecules that can be extracted from tissues by nonpolar solvents (Figure 15.1). Because of their insolubility in aqueous solutions, body lipids are generally found compartmentalized, as in the case of membrane-associated lipids or droplets of triacylglycerol in white adipocytes, or transported in plasma in association with protein, as in lipoprotein particles (see p. 227), or on albumin. Lipids are a major source of energy for the body, and they also provide the hydrophobic barrier that permits partitioning of the aqueous contents of cells and subcellular structures. Lipids serve additional functions in the body, for example, some fat-soluble vitamins have regulatory or coenzyme functions, and the prostaglandins and steroid hormones play major roles in the control of the body's homeostasis. Not surprisingly, deficiencies or imbalances of lipid metabolism can lead to some of the major clinical problems encountered by physicians, such as atherosclerosis and obesity.

The average daily intake of lipids by U.S. adults is about 81 g, of which more than 90% is normally triacylglycerol (TAG, formerly called triglyceride). The remainder of the dietary lipids consists primarily of cholesterol, cholesteryl esters, phospholipids, and unesterified ("free") fatty acids. The digestion of dietary lipids is summarized in Figure 15.2.

The digestion of lipids begins in the stomach, catalyzed by an acidstable lipase (lingual lipase) that originates from glands at the back of the tongue. TAG molecules, particularly those containing fatty acids of shortor medium-chain length (fewer than 12 carbons, such as are found in milk fat), are the primary target of this enzyme. These same TAGs are also degraded by a separate gastric lipase, secreted by the gastric mucosa. Both enzymes are relatively acid-

Structures of some common classes of lipids. Hydrophobic portions of the molecules are shown in orange.

Overview of lipid digestion.
stable, with pH optimums of pH 4 to pH 6. These "acid lipases" play a particularly important role in lipid digestion in neonates, for whom milk fat is the primary source of calories. They also become important digestive enzymes in individuals with pancreatic insufficiency, such as those with cystic fibrosis (see below). Lingual and gastric lipases aid these patients in degrading TAG molecules (especially those with shortto medium-chain fatty acids) despite a near or complete absence of pancreatic lipase (see below).
1.
Cystic fibrosis (CF): This is the most common lethal genetic disease in Caucasians of Northern European ancestry, and has a prevalence of about 1:3,000 births. This autosomal recessive disorder is caused by mutations to the gene for the CF transmembrane conductance regulator (CFTR) protein that functions as a chloride channel on epithelium. Defective CFTR results in decreased secretion of chloride and increased reabsorption of sodium and water. In the pancreas, the decreased hydration results in thickened secretions such that pancreatic enzymes are not able to reach the intestine, leading to pancreatic insufficiency. Treatment includes replacement of these enzymes and supplementation with fat-soluble vitamins. [Note: CF also causes chronic lung infections with progressive pulmonary disease.]

The critical process of emulsification of dietary lipids occurs in the duodenum. Emulsification increases the surface area of the hydrophobic lipid droplets so that the digestive enzymes, which work at the interface of the droplet and the surrounding aqueous solution, can act effectively. Emulsification is accomplished by two complementary mechanisms, namely, use of the detergent properties of the bile salts, and mechanical mixing due to peristalsis. Bile salts, made in the liver and stored in the gallbladder, are derivatives of cholesterol (see p. 224). They consist of a sterol ring structure with a side chain to which a molecule of glycine or taurine is covalently attached by an amide linkage (Figure 15.3). These emulsifying agents interact with the dietary lipid particles and the aqueous duodenal contents, thereby stabilizing the particles as they become smaller, and preventing them from coalescing. A more complete discussion of bile salt metabolism is given on p. 225.

The dietary TAG, cholesteryl esters, and phospholipids are enzymically degraded ("digested") by pancreatic enzymes, whose secretion is hormonally controlled.

TAG degradation: TAG molecules are too large to be taken up efficiently by the mucosal cells of the intestinal villi. They are, therefore, acted upon by an esterase, pancreatic lipase, which preferentially removes the fatty acids at carbons 1 and 3. The primary products of hydrolysis are thus a mixture of 2-monoacylglycerol and free fatty acids (see Figure 15.2). [Note: This enzyme is found in high concentrations in pancreatic secretions (2-3% of the total protein present), and it is highly efficient catalytically, thus insuring that only severe pancreatic deficiency, such as that seen in cystic fibrosis, results in significant malabsorption of fat.] A second protein, colipase, also secreted by the pancreas, binds the /ipase at a ratio of 1:1, and anchors it at the lipid-aqueous interface. Colipase restores activity to lipase in the presence of inhibitory substances like bile acids that bind the micelles. [Note: Colipase is secreted as the zymogen, procolipase, which is activated in the intestine by trypsin.] Orlistat, an antiobesity drug, inhibits gastric and pancreatic lipases, thereby decreasing fat absorption, resulting in loss of weight."

Cholesteryl ester degradation: Most dietary cholesterol is present in the free (nonesterified) form, with 10-15% present in the esterified form. Cholesteryl esters are hydrolyzed by pancreatic cholesteryl ester hydrolase (cholesterol esterase), which produces cholesterol plus free fatty acids (see Figure 15.2). Cholestery! ester hydrolase activity is greatly increased in the presence of bile salts.
- Phospholipid degradation: Pancreatic juice is rich in the proenzyme of phospholipase Ap that, like procolipase, is activated by trypsin and, like cholesteryl ester hydrolase, requires bile salts for optimum activity. Phospholipase Az removes one fatty acid from carbon 2 of a phospholipid, leaving a lysophospholipid. For example, phosphatidylcholine (the predominant phospholipid during digestion) becomes lysophosphatidylcholine. The remaining fatty
1See Chapter 29 in Lippincott's Illustrated Reviews: Pharmacology for a discussion of orlistat.

Hormonal control of lipid digestion in the small intestine.
acid at carbon 1 can be removed by /ysophospholipase, leaving a glycerylphosphoryl base (for example, glycerylphosphorylcholine, see Figure 15.2) that may be excreted in the feces, further degraded, or absorbed.
4. Control of lipid digestion: Pancreatic secretion of the hydrolytic enzymes that degrade dietary lipids in the small intestine is hormonally controlled (Figure 15.4). Cells in the mucosa of the lower duodenum and jejunum produce a small peptide hormone, cholecystokinin (CCK), in response to the presence of lipids and partially digested proteins entering these regions of the upper small intestine. CCK acts on the gallbladder (causing it to contract and release bile—a mixture of bile salts, phospholipids, and free cholesterol), and on the exocrine cells of the pancreas (causing them to release digestive enzymes). It also decreases gastric motility, resulting in a slower release of gastric contents into the small intestine (see p. 353). Other intestinal cells produce another small peptide hormone, secretin, in response to the low pH of the chyme entering the intestine. Secretin causes the pancreas and the liver to release a solution rich in bicarbonate that helps neutralize the pH of the intestinal contents, bringing them to the appropriate pH for digestive activity by pancreatic enzymes.

Free fatty acids, free cholesterol, and 2-monoacylglycerol are the primary products of lipid digestion in the jejunum. These, plus bile salts and fat-soluble vitamins (A, D, E, and K), form mixed micelles—diskshaped clusters of amphipathic lipids that coalesce with their hydrophobic groups on the inside and their hydrophilic groups on the outside. Mixed micelles are, therefore, soluble in the aqueous environment of the intestinal lumen (Figure 15.5). These particles approach the primary site of lipid absorption, the brush border membrane of the enterocytes (mucosal cell). This membrane is separated from the liquid contents of the intestinal lumen by an unstirred water layer that mixes poorly with the bulk fluid. The hydrophilic surface of the micelles facilitates the transport of the hydrophobic lipids through the unstirred water layer to the brush border membrane where they are absorbed. Bile salts are absorbed in the ileum. [Note: Relative to other dietary lipids, cholesterol is only poorly absorbed by the enterocytes. Drug therapy (for example, with ezetimibe*) can further reduce cholesterol absorption in the small intestine.] Shortand mediumchain length fatty acids do not require the assistance of mixed micelles for absorption by the intestinal mucosa.

The mixture of lipids absorbed by the enterocytes migrates to the endoplasmic reticulum where biosynthesis of complex lipids takes place. Fatty acids are first converted into their activated form by fatty acyl-CoA synthetase (thiokinase) (Figure 15.6). Using the fatty acyl CoA derivatives, the 2-monoacylglycerols absorbed by the enterocytes are converted to TAGs by the enzyme complex, TAG synthase. This complex synthesizes TAG by the consecutive actions of two enzyme activities—acyl CoA:monoacylglycerol acyltrans-
2See Chapter 21 in Lippincott's Illustrated Reviews: Pharmacology for a discussion of ezetimibe (Zetia).
ferase and acyl CoA:diacylglycerol acyltransferase. Lysophospholipids are reacylated to form phospholipids by a family of acyltransferases, and cholesterol is esterified to a fatty acid primarily by acy/ CoA:cholesterol acyltransferase (see p. 232). [Note: Virtually all long-chain fatty acids entering the enterocytes are used in this fashion to form TAGs, phospholipids, and cholesteryl esters. Shortand medium-chain length fatty acids are not converted to their CoA derivatives, and are not reesterified to 2-monoacylglycerol. Instead, they are released into the portal circulation, where they are carried by serum albumin to the liver.]

Lipid malabsorption, resulting in increased lipid (including the fatsoluble vitamins and essential fatty acids, see p. 182) in the feces (that is, steatorrhea), can be caused by disturbances in lipid digestion and/or absorption (Figure 15.7). Such disturbances can result from several conditions, including CF (causing poor digestion) and shortened bowel (causing decreased absorption).
The ability of shortand medium-chain length fatty acids to be taken up by enterocytes without the aid of mixed micelles has made them important in dietary therapy for individuals with malabsorption disorders.

Absorption of lipids contained in a mixed micelle by an intestinal mucosal cell. [Note: The micelle itself is not taken up.]
The newly resynthesized TAGs and cholesteryl esters are very hydrophobic, and aggregate in an aqueous environment. It is, therefore, necessary that they be packaged as particles of lipid droplets surrounded by a thin layer composed of phospholipids, unesterified cholesterol, and a molecule of the characteristic protein, apolipoprotein B-48 (see p. 228). This layer stabilizes the particle and increases its solubility, thereby preventing multiple particles from

Assembly and secretion of chylomicrons by intestinal mucosal cells. [Note: Shortand medium-chain length fatty acids do not require incorporation into micelles and directly enter into the blood.]

Possible causes of steatorrhea.
coalescing. [Note: Microsomal TAG transfer protein is essential for the assembly of these TAG-rich apolipoprotein B—containing lipoprotein particles in the endoplasmic reticulum.] The particles are released by exocytosis from enterocytes into the lacteals (lymphatic vessels originating in the villi of the small intestine). The presence of these particles in the lymph after a lipid-rich meal gives it a milky appearance. This lymph is called chyle (as opposed to chyme—the name given to the semifluid mass of partially digested food that passes from the stomach to the duodenum), and the particles are named chylomicrons. Chylomicrons follow the lymphatic system to the thoracic duct, and are then conveyed to the left subclavian vein, where they enter the blood. The steps in the production of chylomicrons are summarized in Figure 15.6. (For a more detailed description of chylomicron structure and metabolism, see p. 228.)

Triacylglycerol contained in chylomicrons is broken down primarily in the capillaries of skeletal muscle and adipose tissues, but also those of the heart, lung, kidney, and liver. Triacylglycerol in chylomicrons is degraded to free fatty acids and glycerol by lipoprotein lipase. This enzyme is synthesized primarily by adipocytes and muscle cells. It is secreted and becomes associated with the luminal surface of endothelial cells of the capillary beds of the peripheral tissues. [Note: Familial lipoprotein lipase deficiency (Type | hyperlipoproteinemia) is a rare, autosomal recessive disorder caused by a deficiency of lipoprotein lipase or its coenzyme, apolipoprotein C-Il (see p. 228). The result is fasting chylomicronemia and hypertriacylglycerolemia.]
1. Fate of free fatty acids: The free fatty acids derived from the hydrolysis of TAG may either directly enter adjacent muscle cells or adipocytes, or they may be transported in the blood in association with serum albumin until they are taken up by cells. [Note: Serum albumin is a large protein secreted by the liver. It transports a number of primarily hydrophobic compounds in the circulation, including free fatty acids and some drugs.] Most cells can oxidize fatty acids to produce energy (see p. 190). Adipocytes can also reesterify free fatty acids to produce TAG molecules, which are stored until the fatty acids are needed by the body (see p. 188).
- 2. Fate of glycerol: Glycerol that is released from TAG is used almost exclusively by the liver to produce glycerol 3-phosphate, which can enter either glycolysis or gluconeogenesis by oxidation to dihydroxyacetone phosphate (see p. 190).
3.

Fate of the remaining chylomicron components: After most of the TAG has been removed, the chylomicron remnants (which contain cholesteryl esters, phospholipids, apolipoproteins, fat-soluble vitamins, and some TAG) bind to receptors on the liver (see p. 230) and are then endocytosed. The remnants are then hydrolyzed to their component parts. Cholesterol and the nitrogenous bases of phospholipids (for example, choline) can be recycled by the body. [Note: If removal of remnants by the liver is decreased due to impaired binding to their receptor, they accumulate in the plasma. This is seen in Type Ill hyperlipoproteinemia (rare, also called familial dysbetalipoproteinemia, see p. 231).]

Key concept map for metabolism of dietary lipids. apo = apolipoprotein; TAGs = triacylglycerols.

The digestion of dietary lipids begins in the stomach and continues in the small intestine (Figure 15.8). The hydrophobic nature of lipids requires that the dietary lipids—particularly those that contain long-chain length fatty acids (LCFA)—be emulsified for efficient degradation. Triacylglycerols (TAG) obtained from milk contain shortto medium-chain length fatty acids that can be degraded in the stomach by the acid lipases (lingual lipase and gastric lipase). Cholesteryl esters (CE), phospholipids (PL), and TAG containing LCFAs are degraded in the small intestine by enzymes secreted by the pancreas. The most important of these enzymes are pancreatic lipase, phospholipase Ap, and cholesteryl esterase. The dietary lipids are emulsified in the small intestine using peristaltic action, and bile salts, which serve as a detergent. The primary products resulting from enzymatic degradation of dietary lipid are 2-monoacylglycerol, unesterified cholesterol, and free fatty acids. These compounds, plus the fat-soluble vitamins, form mixed micelles that facilitate the absorption of dietary lipids by intestinal mucosal cells (enterocytes). These cells resynthesize TAG, CE, and PL, and also synthesize protein (apolipoprotein B-48), all of which are then assembled with the fat-soluble vitamins into chylomicrons. These serum lipoprotein particles are released into the lymph, which carries them to the blood. Shortand medium chain fatty acids enter blood directly. Thus, dietary lipids are transported to the peripheral tissues. Problems with fat absorption cause steatorrhea. A deficiency in the ability to degrade chylomicron components, or remove their remnants after TAG has been removed, results in accumulation of these particles in blood.

15.1Which one of the following statements about the digestion of lipids is correct?
oo. . . . . . A. Lipid digestion begins with acid-stable lipases that primarily use TAG with long to very long-chain fatty acids as substrates.
B. Large lipid droplets are emulsified (have their surface area increased) in the mouth through the act of chewing (mastication).
. -— . . C. Colipase facilitates the binding of bile salts to mixed micelles, maximizing the activity of pancreatic lipase.
D. The peptide hormone secretin causes the gallbladder to contract and release bile.
| . on . vege . . E. Patients with cystic fibrosis have difficulties with digestion because their thickened pancreatic secretions are less able to reach the small intestine, the primary site of lipid digestion.
F. Formation of chylomicrons is independent of protein synthesis in the intestinal mucosa.
15.2 Which one of the following statements about the absorption of lipids from the intestine is correct?
A. Dietary triacylglycerol must be completely hydrolyzed to free fatty acids and glycerol before absorption.
. . . B. The TAG carried by chylomicrons is degraded to free fatty acids and glycerol by lipoprotein lipase on the endothelial surface of capillaries in muscle and adipose primarily.
C. Fatty acids that contain ten carbons or less are absorbed and enter the circulation primarily via the lymphatic system.
on . D. Deficiencies in the ability to absorb fat result in excessive amounts of chylomicrons in the blood.

Correct answer = E. Patients with CF, a genetic disease due to a deficiency of functional CFTR, have thickened secretions that impede the flow of pancreatic enzymes into the duodenum. The acid-stable lipases, lingual and gastric lipase, use as substrates TAG with short to mediumchain fatty acids that are abundant in milk. Emulsification occurs) through peristalsis that provides mechanical mixing, and bile salts that function as detergents. Colipase restores activity to pancreatic lipase in the presence of inhibitory bile acids that bind the micelles. CCK (cholecystokinin) is the hormone that causes contraction of the gallbladder and release of stored bile; secretin causes release of bicarbonate. Chylomicron formation requires synthesis of the protein apolipoprotein B-48.
Correct answer = B. The TAG in chylomicrons are degraded to fatty acids and glycerol by lipoprotein lipase on the endothelial surface of capillaries in muscle and adipose, thus providing a source of fatty acids to these tissues for degradation or storage. In the duodenum, TAG are degraded to 1 monoacylglycerol + 2 free fatty acids that get absorbed. Medium and short chain fatty acids enter directly into blood; they do not get packaged into chylomicrons. Chylomicrons contain dietary lipids that were digested and absorbed, thus a defect in fat absorption would result in decreased production of chylomicrons.

Fatty acids exist "free" in the body (that is, they are unesterified), and are also found as fatty acyl esters in more complex molecules, such as triacylglycerols. Low levels of free fatty acids occur in all tissues, but substantial amounts can sometimes be found in the plasma, particularly during fasting. Plasma free fatty acids (transported on serum albumin) are in route from their point of origin (triacylglycerol of adipose tissue or circulating lipoproteins) to their site of consumption (most tissues). Free fatty acids can be oxidized by many tissues— particularly liver and muscle—to provide energy. Fatty acids are also structural components of membrane lipids, such as phospholipids and glycolipids (see p. 201). Fatty acids are attached to certain intracellular proteins to enhance the ability of those proteins to associate with membranes (see p. 206). Fatty acids are also precursors of the hormone-like prostaglandins (see p. 213). Esterified fatty acids, in the form of triacylglycerols stored in adipose cells, serve as the major energy reserve of the body. Figure 16.1 illustrates the metabolic pathways of fatty acid synthesis and degradation, and their relationship to carbohydrate metabolism.

A fatty acid consists of a hydrophobic hydrocarbon chain with a terminal carboxyl group that has a pK, of about 4.8 (Figure 16.2). At physiologic pH, the terminal carboxyl group (-COOH) ionizes, becoming —COO'. This anionic group has an affinity for water, giving the fatty acid its amphipathic nature (having both a hydrophilic and a hydrophobic region). However, for long-chain fatty acids (LCFAs), the hydrophobic portion is predominant. These molecules are highly waterinsoluble, and must be transported in the circulation in association with protein. More than 90% of the fatty acids found in plasma are in the form of fatty acid esters (primarily triacylglycerol, cholesteryl esters, and phospholipids) contained in circulating lipoprotein particles (see p. 227). Unesterified (free) fatty acids are transported in the circulation in association with albumin.

A saturated (A) and an unsaturated (B) fatty acid. Orange denotes hydrophobic portions of the molecules. [Note: Cis double bonds cause a fatty acid to "kink."]

Fatty acid chains may contain no double bonds—that is, be saturated—or contain one or more double bonds—that is, be monoor polyunsaturated. When double bonds are present, they are nearly always in the cis rather than in the trans configuration. (See p. 363 for a discussion of the dietary occurrence of cis and trans unsaturated fatty acids.) The introduction of a cis double bond causes the fatty acid to bend or "kink" at that position (Figure 16.3). If the fatty acid has two or more double bonds, they are always spaced at three-carbon intervals. [Note: In general, addition of double bonds decreases the melting temperature (T) of a fatty acid, whereas increasing the chain length increases the Tm. Because membrane lipids typically contain LCFA, the presence of double bonds in some fatty acids helps maintain the fluid nature of those lipids.]

The common names and structures of some fatty acids of physiologic importance are listed in Figure 16.4. The carbon atoms are numbered, beginning with the carboxyl carbon as carbon 1. The number before the colon indicates the number of carbons in the chain, and those after the colon indicate the numbers and positions (relative to the carboxyl end) of double bonds. For example, as shown in Figure 16.5A, arachidonic acid, 20:4(5,8,11,14), is 20 carbons long and has 4 double bonds (between carbons 5-6, 8-9, 11-12, and 14-15). [Note: Carbon 2, the carbon to which the carboxyl group is attached, is also called the a-carbon, carbon 3 is the B-carbon, and carbon 4 is the y-carbon. The carbon of the terminal methyl group is called the -carbon regardless of the chain length.] The double bonds in a fatty acid can also be denoted relative to the @ (or methyl-terminal) end of the chain. Arachidonic acid is referred to as an w-6 fatty acid acid (also an n-6, Figure 16.5A) because the terminal double bond is six bonds in from the @ end (Figure 16.5B). Another w-6 fatty acid is the essential linoleic acid, 18:2(9,12). In contrast, a-linolenic acid, 18:3(9,12,15), is an essential w-3 fatty acid. (See p. 363 for a discussion of the nutritional significance of w-3 and o-6 fatty acids.)

Two fatty acids are dietary essentials in humans because of our inability to synthesize them: linoleic acid, which is the precursor of @-6 arachidonic acid, the substrate for prostaglandin synthesis (see p. 213), and c-linolenic acid, the precursor of other w-3 fatty acids important for growth and development. Plants provide us with the essential fatty acids. [Note: Arachidonic acid becomes essential if linoleic acid is deficient in the diet.]
Essential fatty acid deficiency can result in a scaly dermatitis (ichthyosis), as well as visual and neurologic abnormalities. Essential fatty acid deficiency, however, is rare.

A large proportion of the fatty acids used by the body is supplied by the diet. Carbohydrates and protein obtained from the diet in excess of the body's needs for these compounds can be converted to fatty acids, which are stored as triacylglycerols. (See p. 321 for a discussion of the metabolism of dietary nutrients in the well-fed state.) In adult humans, fatty acid synthesis occurs primarily in the liver and lactating mammary glands and, to a lesser extent, in adipose tissue. This cytosolic process incorporates carbons from acetyl coenzyme A (CoA) into the growing fatty acid chain, using adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH).

The first step in de novo fatty acid synthesis is the transfer of acetate units from mitochondrial acetyl CoA to the cytosol. Mitochondrial acetyl CoA is produced by the oxidation of pyruvate (see p. 109), and by the catabolism of fatty acids (see p. 190), ketone bodies (see p. 196), and certain amino acids (see p. 266). The CoA portion of acetyl CoA, however, cannot cross the inner mitochondrial membrane; only the acetyl portion enters the cytosol. It does so as part of citrate produced by the condensation of oxaloacetate (OAA) and acetyl CoA (Figure 16.6). [Note: This process of translocation of citrate from the mitochondrion to the cytosol, where it is cleaved by ATP-citrate lyase to produce cytosolic acetyl CoA and OAA, occurs when the mitochondrial citrate concentration is high. This is observed when isocitrate dehydrogenase is inhibited by the presence of large amounts of ATP, causing citrate and isocitrate to accumulate (see p. 112). Therefore, cytosolic citrate may be viewed as a high-energy signal.] Because a large amount of ATP is needed for fatty acid synthesis, the increase in both ATP and citrate enhances this pathway.

The energy for the carbon-to-carbon condensations in fatty acid synthesis is supplied by the process of carboxylation and then decarboxylation of acetyl groups in the cytosol. The carboxylation of acetyl CoA to form malonyl CoA is catalyzed by acetyl CoA carboxylase (Figure 16.7), and requires COz and ATP. The coenzyme is the vitamin, biotin, which is covalently bound to a lysyl residue of the carboxylase (see Figure 28.16).
1. Short-term regulation of acetyl CoA carboxylase (ACC): This carboxylation is both the rate-limiting and the regulated step in fatty acid synthesis (see Figure 16.7). The inactive form of ACC is a protomer (dimer). The enzyme undergoes allosteric activation by citrate, which causes dimers to polymerize, and allosteric inactivation by long-chain fatty acyl CoA (the end product of the pathway), which causes its depolymerization. A second mechanism of shortterm regulation is by reversible phosphorylation. AMP-activated protein kinase (AMPK) phosphorylates and inactivates ACC. AMPK itself is allosterically activated by AMP and covalently activated by phosphorylation via several kinases. At least one of these

Arachidonic acid, illustrating the position of the double bonds. Arachidonic acid, 20:4(5,8,11,14) is an n-6 fatty acid because the double bond furthest from the carboxy end (carbon 1) is 14 carbons from that end: 20-14 = 6. It is also referred to as an w-6 fatty acid because the terminal double bond is six bonds in from the end. Thus, the "o" and "n" designations are equivalent (see * ).

Allosteric regulation of malonyl CoA synthesis by acetyl CoA carboxylase (ACC).The carboxyl group contributed by dissolved COs is shown in blue.

Covalent regulation (phosphorylation) of acetyl CoA carboxylase (ACC) by AMP-dependent kinase (AMPK), which itself is regulated both covalently and allosterically.
AMPK kinases is activated by cAMP-dependent protein kinase A (PKA). Thus, in the presence of counterregulatory hormones, such as epinephrine and glucagon, ACC is phosphorylated and, thereby, inactivated (Figure 16.8). In the presence of insulin, ACC is dephosphorylated and, thereby, activated. [Note: This is analogous to the regulation of glycogen synthase, see p. 131.]
- 2. Long-term regulation of acetyl CoA carboxylase: Prolonged consumption of a diet containing excess calories (particularly highcalorie, high-carbohydrate diets) causes an increase in ACC synthesis, thus increasing fatty acid synthesis. Conversely, a lowcalorie or a high-fat diet causes a reduction in fatty acid synthesis by decreasing the synthesis of ACC. [Note: Synthesis of the carboxylase is upregulated by insulin via a sterol response element binding protein, SREBP-1. The function and regulation of SREBPs are described on p. 222. Fatty acid synthase (see below) is similarly regulated by diet and SREBP-1.] Metformin, used in the treatment of type 2 diabetes, lowers serum TAG through activation of AMPK resulting in inhibition of ACC activity (by phosphorylation) and inhibition of ACC and fatty acid synthase expression (by decreasing SREBP-1). Metformin also lowers blood glucose by increasing AMPK-mediated uptake of glucose by muscle.

The remaining series of reactions of fatty acid synthesis in eukaryotes is catalyzed by the multifunctional, dimeric enzyme, fatty acid synthase (FAS). Each FAS monomer is a multicatalytic polypeptide with seven different enzymic activities plus a domain that covalently binds a molecule of 4'-phosphopantetheine. [Note: 4'-Phosphopantetheine, a derivative of the vitamin pantothenic acid (see p. 381), carries acyl units on its terminal thiol (—-SH) group during fatty acid synthesis. It also is a component of CoA.] In prokaryotes, FAS is a multienzyme complex, and the 4'-phosphopantetheine domain is a separate protein, referred to as the acyl carrier protein (ACP). ACP is used below to refer to the phosphopantetheine-containing domain of eukaryotic FAS. The reaction numbers in brackets below refer to Figure 16.9. [Note: The enzyme activities listed are separate catalytic domains present in each multicatalytic FAS monomer.]
[1] A molecule of acetate is transferred from acetyl CoA to the -SH group of the ACP. Domain: Acetyl! CoA-ACP acetyltransacylase.
[2] Next, this two-carbon fragment is transferred to a temporary holding site, the thiol group of a cysteine residue on the enzyme.
[3] The now-vacant ACP accepts a three-carbon malonate unit from malonyl CoA. Domain: Malonyl CoA-ACP transacylase.
[4] The acetyl group on the cysteine residue condenses with the malonyl group on ACP as the COz originally added by acetyl CoA carboxylase is released. The result is a four-carbon unit attached to the ACP domain. The loss of free energy from the decarboxylation drives the reaction. Domain: 3-Ketoacyl-ACP synthase.

Synthesis of palmitate (16:0) by multifunctional fatty acid synthase (FAS). [Note: Numbers in brackets correspond to bracketed numbers in the text. A second repetition of the steps is indicated by numbers with an asterisk (*). Carbons provided directly by acetyl CoA are shown in red.]

Cytosolic conversion of oxaloacetate to pyruvate with the generation of NADPH. [Note: The pentose phosphate pathway is the primary source of the NADPH.]
The next three reactions convert the 3-ketoacyl group to the corresponding saturated acyl group by a pair of reductions requiring NADPH and a dehydration step.
[5] The keto group is reduced to an alcohol. Domain: 3-KetoacylACP reductase.
[6] A molecule of water is removed to introduce a double bond between carbons 2 and 3 (the aand B-carbons). Domain: 3-Hydroxyacyl-ACP dehydratase.
[7 The double bond is reduced. Domain: Enoyl-ACP reductase.
The result of these seven steps is production of a four-carbon compound (butyryl) whose three terminal carbons are fully saturated, and which remains attached to the ACP. These seven steps are repeated, beginning with the transfer of the butyryl chain from the ACP to the Cys residue [2*], the attachment of a molecule of malonate to the ACP [3*], and the condensation of the two molecules liberating CO» [4*]. The carbonyl group at the B-carbon (carbon 3—the third carbon from the sulfur) is then reduced [5*], dehydrated [6*], and reduced [7*], generating hexanoyl-ACP. This cycle of reactions is repeated five more times, each time incorporating a two-carbon unit (derived from malonyl CoA) into the growing fatty acid chain at the carboxyl end. When the fatty acid reaches a length of 16 carbons, the synthetic process is terminated with palmitoyl-S-ACP. [Note: Shorter-length fatty acids are important endproducts in the lactating mammary gland.] Pa/mitoy! thioesterase cleaves the thioester bond, releasing a fully saturated molecule of palmitate (16:0). [Note: All the carbons in palmitic acid have passed through malonyl CoA except the two donated by the original acetyl CoA, which are found at the methyl-group (w) end of the fatty acid. This underscores the rate-limiting nature of the acetyl CoA carboxylase reaction.]

The hexose monophosphate pathway (see p. 145) is the major supplier of NADPH for fatty acid synthesis. Two NADPH are produced for each molecule of glucose that enters this pathway. The cytosolic conversion of malate to pyruvate, in which malate is oxidized and decarboxylated by cytosolic malic enzyme (NADP*t-dependent malate dehydrogenase), also produces cytosolic NADPH (and COs, Figure 16.10). [Note: Malate can arise from the reduction of OAA by cytosolic NADH-dependent malate dehydrogenase (see Figure 16.10). One source of the cytosolic NADH required for this reaction is that produced during glycolysis (see p. 101). OAA, in turn, can arise from citrate. Recall from Figure 16.6 that citrate was shown to move from the mitochondria into the cytosol, where it is cleaved into acetyl CoA and OAA by ATP-citrate lyase.] A summary of the interrelationship between glucose metabolism and palmitate synthesis is shown in Figure 16.11.

Interrelationship between glucose metabolism and palmitate synthesis.

Although palmitate, a 16-carbon, fully saturated long-chain length fatty acid (16:0), is the primary endproduct of fatty acid synthase activity, it can be further elongated by the addition of two-carbon units in the smooth endoplasmic reticulum (SER). Elongation requires a system of separate enzymes rather than a multifunctional enzyme. Malonyl CoA is the two-carbon donor and NADPH supplies the electrons. The brain has additional elongation capabilities, allowing it to produce the very-long-chain fatty acids (over 22 carbons) that are required for synthesis of brain lipids.

Enzymes (desaturases) also present in the SER are responsible for desaturating long-chain fatty acids (that is, adding cis double bonds). The desaturation reactions require NADH, cytochrome bs and its FAD-linked reductase. The first double bond is typically inserted between carbons 9 and 10, producing primarily 18:1(9) and small amounts of 16:1(9). A variety of polyunsaturated fatty acids can be made through additional desaturation combined with elongation.
Humans have carbon 9, 6, 5 and 4 desaturases, but lack the ability to introduce double bonds from carbon 10 to the w end of the chain. This is the basis for the nutritional essentiality of the polyunsaturated linoleic and linolenic acids.

Mono-, di-, and triacylglycerols consist of one, two, or three molecules of fatty acid esterified to a molecule of glycerol. Fatty acids are esterified through their carboxyl groups, resulting in a loss of negative charge and formation of "neutral fat." [Note: If a species of acylglycerol is solid at room temperature, it is called a "fat"; if liquid, it is called an "oil."]
1. Structure of triacylglycerol (TAG): The three fatty acids esterified to a glycerol molecule are usually not of the same type. The fatty acid on carbon 1 is typically saturated, that on carbon 2 is typically unsaturated, and that on carbon 3 can be either. Recall that the presence of the unsaturated fatty acid(s) decrease(s) the melting temperature (T,) of the lipid. An example of a TAG molecule is shown in Figure 16.12.
2. Storage of TAG: Because TAGs are only slightly soluble in water and cannot form stable micelles by themselves, they coalesce within adipocytes to form oily droplets that are nearly anhydrous. These cytosolic lipid droplets are the major energy reserve of the body.

Pathways for production of glycerol phosphate in liver and adipose tissue.
3. Synthesis of glycerol phosphate: Glycerol phosphate is the initial acceptor of fatty acids during TAG synthesis. There are two pathways for glycerol phosphate production (Figure 16.13). In both liver (the primary site of TAG synthesis) and adipose tissue, glycerol phosphate can be produced from glucose, using first the reactions of the glycolytic pathway to produce dihydroxyacetone phosphate (DHAP, see p. 101). Next, DHAP is reduced by glycerol phosphate dehydrogenase to glycerol phosphate. A second pathway found in the liver, but not in adipose tissue, uses glycerol kinase to convert free glycerol to glycerol phosphate (see Figure 16.13). [Note: The glucose transporter in adipocytes (GLUT-4) is insulin-dependent (see p. 312). Thus, when plasma glucose—and, therefore, plasma insulin—levels are low, adipocytes have only a limited ability to synthesize glycerol phosphate, and cannot produce TAG.]
4. Conversion of a free fatty acid to its activated form: A fatty acid must be converted to its activated form (attached to CoA) before it can participate in metabolic processes such as TAG synthesis. This reaction, illustrated in Figure 15.6, is catalyzed by a family of fatty acyl CoA synthetases (thiokinases).
5. Synthesis of a molecule of TAG from glycerol phosphate and fatty acyl CoA: This pathway involves four reactions, shown in Figure 16.14. These include the sequential addition of two fatty acids from fatty acyl CoA, the removal of phosphate, and the addition of the third fatty acid.

In white adipose tissue, TAG is stored in a nearly anhydrous form as fat droplets in the cytosol of the cells. It serves as "depot fat," ready for mobilization when the body requires it for fuel. Little TAG is stored in the liver. Instead, most is exported, packaged with other lipids and apoproteins to form lipoprotein particles called very-lowdensity lipoproteins (VLDL). Nascent VLDL are secreted directly into the blood where they mature and function to deliver the endogenously derived lipids to the peripheral tissues. [Note: Recall that chylomicrons deliver primarily dietary (exogenously derived) lipids.] Plasma lipoproteins are discussed in Chapter 18.

Fatty acids stored in adipose tissue, in the form of neutral TAG, serve as the body's major fuel storage reserve. TAGs provide concentrated stores of metabolic energy because they are highly reduced and largely anhydrous. The yield from the complete oxidation of fatty acids to CO2 and H20 is 9 kcal/g fat (as compared to 4 kcal/g protein or carbohydrate, see Figure 27.5). [Note: Fatty acids are also supplied to tissues by lipoproteins (see p. 228).]

Hormonal regulation of triacylglycero degradation in the adipocyte.

The mobilization of stored fat requires the hydrolytic release of fatty acids and glycerol from their TAG form. This process is initiated by hormone-sensitive lipase, which removes a fatty acid from carbon 1 and/or carbon 3 of the TAG. Additional lipases specific for diacylglycerol or monoacylglycerol remove the remaining fatty acid(s).
1.
Activation of hormone-sensitive lipase (HSL): This enzyme is activated when phosphorylated by a 3'5'-cyclic AMP(cAMP)dependent protein kinase. 3',5'-Cyclic AMP is produced in the adipocyte when one of several hormones (such as epinephrine or glucagon) binds to receptors on the cell membrane, and activates adenylyl cyclase (Figure 16.15). The process is similar to that of the activation of glycogen phosphorylase (see Figure 11.10). [Note: Because acetyl CoA carboxylase is inhibited by . . : hormone-directed phosphorylation when the cAMP-mediated cascade is activated (see Figure 16.8), fatty acid synthesis is turned off when TAG degradation is turned on.] In the presence of high plasma levels of insulin and glucose, HSL is dephosphorylated, and becomes inactive.
2. Fate of glycerol: The glycerol released during TAG degradation cannot be metabolized by adipocytes because they apparently lack glycerol kinase. Rather, glycerol is transported through the blood to the liver, where it can be phosphorylated. The resulting glycerol phosphate can be used to form TAG in the liver, or can be converted to DHAP by reversal of the glycerol phosphate dehydrogenase reaction illustrated in Figure 16.13. DHAP can participate in glycolysis or gluconeogenesis.

3. Fate of fatty acids: The free (unesterified) fatty acids move through the cell membrane of the adipocyte, and bind to plasma albumin. They are transported to the tissues, enter cells, get activated to their CoA derivatives, and are oxidized for energy. Regardless of their levels, plasma free fatty acids (FFA) cannot be used for fuel by erythrocytes, which have no mitochondria. Brain, too, does not use fatty acids for energy, but the reasons are less clear. [Note: Over 50% of the fatty acids released from adipose TAG are reesterified to glycerol 3-phosphate. White adipose does not express glycerol kinase, and the phosphorylated glycerol is produced by glyceroneogenesis, an incomplete version of gluconeogenesis: pyruvate to PEP to OAA to DHAP to glycerol 3-phosphate. The process reduces plasma FFA, molecules associated with insulin resistance in type 2 diabetes and obesity (see p. 343).]

The major pathway for catabolism of fatty acids is a mitochondrial pathway called B-oxidation, in which two-carbon fragments are successively removed from the carboxyl end of the fatty acyl CoA, producing acetyl CoA, NADH, and FADH».
- 1. Transport of long-chain fatty acids (LCFA) into the mitochondria: After a LCFA enters a cell, it is converted in the cytosol to its CoA derivative by long-chain fatty acyl CoA synthetase (thiokinase), an enzyme of the outer mitochondrial membrane. Because f-oxida-

Carnitine shuttle. [Note: Long-chain fatty acyl CoA synthetase is in the outer mitochondrial membrane; active site faces the cytosol.
tion occurs in the mitochondrial matrix, the fatty acid must be transported across the inner mitochondrial membrane that is impermeable to CoA. Therefore, a specialized carrier transports the long-chain acyl group from the cytosol into the mitochondrial matrix. This carrier is carnitine, and this rate-limiting transport process is called the carnitine shuttle (Figure 16.16).
a. Steps in LCFA translocation: First, the acyl group is transferred from CoA to carnitine by carnitine palmitoyltransferase | (CPT-l—an enzyme of the outer mitochondrial membrane. [Note: CPT-/ is also known as CAT-/ for carnitine acyltransferase |.] This reaction forms acylcarnitine, and regenerates free CoA. Second, the acylcarnitine is transported into the mitochondrial matrix in exchange for free carnitine by carnitine-acylcarnitine translocase. Carnitine palmitoyltransferase II (CPT-II, or CAT-I)—an enzyme of the inner mitochondrial membrane—catalyzes the transfer of the acyl group from carnitine to CoA in the mitochondrial matrix, thus regenerating free carnitine.
b. Inhibitor of the carnitine shuttle: Malonyl CoA inhibits CPT-/, thus preventing the entry of long-chain acyl groups into the mitochondrial matrix. Therefore, when fatty acid synthesis is occurring in the cytosol (as indicated by the presence of malonyl CoA), the newly made palmitate cannot be transferred into the mitochondria and degraded. [Note: Muscle, though it does not synthesize fatty acids, contains the mitochondrial isoform of acetyl CoA carboxylase (ACC2), allowing muscle to regulate B-oxidation.] Fatty acid oxidation is also regulated by the acetyl CoA to CoA ratio: as the ratio increases, the CoArequiring thiolase reaction decreases (Figure 16.17).

c. Sources of carnitine: Carnitine can be obtained from the diet, where it is found primarily in meat products. Carnitine can also be synthesized from the amino acids lysine and methionine by an enzymatic pathway found in the liver and kidney but not in skeletal or heart muscle. Therefore, these latter tissues are totally dependent on uptake of carnitine provided by endogenous synthesis or the diet, and distributed by the blood. [Note: Skeletal muscle contains about 97% of all carnitine in the body.]

Enzymes involved in the B-oxidation of fatty acyl CoA. [Note: Enoyl CoA hydratase requires a trans double bond between carbon 2 and carbon 3.]
d. Carnitine deficiencies: Such deficiencies result in a decreased ability of tissues to use LCFA as a metabolic fuel. Secondary carnitine deficiency occurs in many situations, including: 1) in patients with liver disease causing decreased synthesis of carnitine; 2) in individuals suffering from malnutrition or those on strictly vegetarian diets; 3) in those with an increased requirement for carnitine as a result of, for example, pregnancy, severe infections, burns, or trauma; or 4) in those undergoing hemodialysis, which removes carnitine from the blood. Congenital deficiencies in one of the components of the carnitine palmitoyltransferase system, in renal tubular reabsorption of carnitine, or in carnitine uptake by cells cause primary carnitine deficiency. Genetic CPT-/ deficiency affects the liver, where an inability to use LCFA for fuel greatly impairs that tissue's ability to synthesize glucose during a fast. This can lead to severe hypoglycemia, coma, and death. CPT-I// deficiency occurs primarily in cardiac and skeletal muscle, where symptoms of carnitine deficiency range from cardiomyopathy to muscle weakness with myoglobinemia following prolonged exercise. [Note: This is an example of how the impaired flow of a metabolite from one cell compartment to another results in pathology.] Treatment includes avoidance of prolonged fasts, adopting a diet high in carbohydrate and low in LCFA, but supplemented with medium-chain fatty acids and carnitine.
2. Entry of shortand medium-chain fatty acids into the mitochondria: Fatty acids shorter than 12 carbons can cross the inner mitochondrial membrane without the aid of carnitine or the CPT system. Once inside the mitochondria, they are activated to their CoA derivatives by matrix enzymes, and are oxidized. [Note: Medium-chain fatty acids are plentiful in human milk. Because their oxidation is not dependent on CPT-!I, it is not subject to inhibition by malonyl CoA.]

3. Reactions of B-oxidation: The first cycle of B-oxidation is shown in Figure 16.17. It consists of a sequence of four reactions involving the B-carbon (carbon 3) that results in shortening the fatty acid chain by two carbons. The steps include an oxidation that produces FADHs», a hydration step, a second oxidation that produces NADH, and a thiolytic cleavage that releases a molecule of acetyl CoA. Each step is catalyzed by enzymes with chain-length specificity. These four steps are repeated for saturated fatty acids of even-numbered carbon chains (n/2) — 1 times (where n is the number of carbons), each cycle producing an acetyl group plus one NADH and one FADb:. The final thiolytic cleavage produces two acetyl groups. [Note: Acetyl CoA is a positive allosteric effector of pyruvate carboxylase (see p. 119), thus linking fatty acid oxidation and gluconeogenesis.]
4. Energy yield from fatty acid oxidation: The energy yield from the B-oxidation pathway is high. For example, the oxidation of a molecule of palmitoyl CoA to COz and H20 produces 8 acetyl CoA, 7 NADH, and 7 FADHs, from which 131 ATP can be generated; however, activation of the fatty acid requires 2 ATP. Thus, the net yield from palmitate is 129 ATP (Figure 16.18). A comparison of the processes of synthesis and degradation of long-chain saturated fatty acids with an even number of carbon atoms is provided in Figure 16.19.

Summary of the energy yield from the oxidation of palmitoyl CoA (16 carbons). CC = acetyl CoA. *Activation of palmitate to palmitoyl CoA requires the equivalent of 2 ATP.

In mitochondria, there are four fatty acyl CoA dehydrogenase species, each with a specificity for either short-, medium-, long-, or very-long-chain fatty acids. MCAD deficiency, an autosomal recessive disorder, is one of the most common inborn errors of metabolism, and the most common inborn error of fatty acid oxidation, being found in 1:14,000 births worldwide, with a higher incidence in Northern Europeans. It results in decreased ability to oxidize fatty acids with six to ten carbons (these accumulate and can be measured in urine), and severe hypoglycemia (because the tissues must increase their reliance on glucose). Treatment includes avoidance of fasting. MCAD deficiency has been identified as the cause of some cases originally reported as sudden infant death syndrome (SIDS) or Reye syndrome.

Oxidation of fatty acids with an odd number of carbons: The f-oxidation of a saturated fatty acid with an odd number of carbon atoms proceeds by the same reaction steps as that of fatty acids with an even number, until the final three carbons are reached. This compound, propionyl CoA, is metabolized by a three-step pathway (Figure 16.20). [Note: Propionyl CoA is also produced during the metabolism of certain amino acids (see Figure 20.10).]

Greatest flux through pathway, SYNTHESIS = After carbohydrate-rich meal. Greatest flux through pathway, DEGRADATION = In starvation. Hormonal state favoring pathway, SYNTHESIS = High insulin/glucagon ratio. Hormonal state favoring pathway, DEGRADATION = Low insulin/glucagon ratio. Major tissue site, SYNTHESIS = Primarily liver. Major tissue site, DEGRADATION = Muscle, liver. Subcellular location, SYNTHESIS = Primarily cytosol. Subcellular location, DEGRADATION = Primarily mitochondria. Carriers of acyl/acetyl groups between mitochondria and cytosol, SYNTHESIS = Citrate (mitochondria to cytosol). Carriers of acyl/acetyl groups between mitochondria and cytosol, DEGRADATION = Carnitine (cytosol to mitochondria). Phosphopantetheine-containing active carriers, SYNTHESIS = Acyl carrier protein domain, coenzyme A. Phosphopantetheine-containing active carriers, DEGRADATION = Coenzyme A. Oxidation/reduction coenzymers, SYNTHESIS = NADPH (reduction). Oxidation/reduction coenzymers, DEGRADATION = NAD + , FAD (oxidation). Two-carbon donor/product, SYNTHESIS = Malonyl CoA: donor of one acetyl group. Two-carbon donor/product, DEGRADATION = Acetyl CoA: product of β -oxidation. Activator, SYNTHESIS = Citrate. Activator, DEGRADATION = . Inhibitor, SYNTHESIS = Long-chain fatty acyl CoA (inhibits acetyl CoA carboxylase ). Inhibitor, DEGRADATION = Malonyl CoA (inhibits carnitine palmitoyltransferase-I ). Product of pathway, SYNTHESIS = Palmitate. Product of pathway, DEGRADATION = Acetyl CoA. Repetitive four-step process, SYNTHESIS = Condensation, reduction dehydration, reduction. Repetitive four-step process, DEGRADATION = Dehydrogenation, hydration dehydrogenation, thiolysis

Comparison of the synthesis and degradation of long-chain, even-numbered, saturated fatty acids.
a. Synthesis of D-methylmalonyl CoA: First, propionyl CoA is carboxylated, forming D-methylmalonyl CoA. The enzyme propionyl CoA carboxylase has an absolute requirement for the coenzyme biotin, as do most other carboxylases (see p. 381).
. Formation of L-methylmalonyl CoA: Next, the D-isomer is converted to the L-form by the enzyme, methylmalonyl CoA racemase.
. Synthesis of succinyl CoA: Finally, the carbons of L-methylmalonyl CoA are rearranged, forming succinyl CoA, which can enter the tricarboxylic acid (TCA) cycle (see p. 109). [Note: This is the only example of a glucogenic precursor generated from fatty acid oxidation.] The enzyme, methylmalony! CoA mutase, requires a coenzyme form of vitamin By2 (deoxyadenosylcobalamin) for its action. The mutase reaction is one of only two reactions in the body that require vitamin By2 (see p. 375). [Note: In patients with vitamin B12 deficiency, both propionate and methylmalonate are excreted in the urine. Two types of heritable methylmalonic acidemia and aciduria have been described: one in which the mutase is missing or deficient (or has reduced affinity for the coenzyme), and one in which the patient is unable to convert vitamin By2 into its coenzyme form. Either type results in metabolic acidosis, with developmental retardation seen in some patients.]

7. Oxidation of unsaturated fatty acids: The oxidation of unsaturated fatty acids provides less energy than that of saturated fatty acids because unsaturated fatty acids are less highly reduced and, therefore, fewer reducing equivalents can be produced from these structures. Oxidation of monounsaturated fatty acids, such as 18:1(9) (oleic acid) requires one additional enzyme, 3,2-enoyl CoA isomerase, which converts the 3-trans derivative obtained after three rounds of B-oxidation to the 2-trans derivative required as a substrate by the enoyl CoA hydratase. Oxidation of polyunsaturated fatty acids, such as 18:2(9,12) (linoleic acid), requires an NADPH-dependent 2,4-dienoyl CoA reductase in addition to the isomerase.
8. B-Oxidation in the peroxisome: Very-long-chain fatty acids (VLCFA), or those 22 carbons long or longer, undergo a preliminary B-oxidation in peroxisomes. The shortened fatty acid (linked to carnitine) diffuses to a mitochondrion for further oxidation. In contrast to mitochondrial B-oxidation, the initial dehydrogenation in peroxisomes is catalyzed by an FAD-containing acyl CoA oxidase. The FADH2 produced is oxidized by molecular oxygen, which is reduced to H2Oz; thus, no ATP is generated by this step. The H2Oz is reduced to H2O by catalase (see p. 148). [Note: Genetic defects either in the ability to target matrix proteins to peroxisomes (resulting in Zellweger syndrome—a peroxisomal biogenesis disorder) or in the ability to transport VLCFA across the peroxisomal membrane (resulting in X-linked adrenoleukodystrophy), lead to accumulation of VLCFA in the blood and tissues.]

Branched-chain, 20 carbon fatty acid, phytanic acid: This is not a substrate for acyl CoA dehydrogenase because of the methyl group on its B carbon (Figure 16.21). Instead, it is hydroxylated at the a-carbon by phytanoyl CoA a-hydroxylase (PhyH), carbon 1 is released as COs, and the product, 19 carbon pristanic acid, is activated to its CoA derivative and undergoes f-oxidation. Refsum disease is a rare, autosomal recessive disorder caused by a deficiency of peroxisomal PhyH. This results in the accumulation of phytanic acid in the plasma and tissues. The symptoms are primarily neurologic, and the treatment involves dietary restriction to halt disease progression. [Note: @-Oxidation (at the methyl terminus) also is known, and generates dicarboxylic acids. Normally a minor pathway of the ER, its up-regulation is seen with conditions such as MCAD deficiency that limit fatty acid B-oxidation.]

Liver mitochondria have the capacity to convert acetyl CoA derived from fatty acid oxidation into ketone bodies. The compounds categorized as ketone bodies are acetoacetate, 3-hydroxybutyrate (also called B-hydroxybutyrate), and acetone (a nonmetabolized side product, Figure 16.22). [Note: The two functional ketone bodies are actually organic acids.] Acetoacetate and 3-hydroxybutyrate are transported in the blood to the peripheral tissues. There they can be reconverted to acetyl CoA, which can be oxidized by the TCA cycle. Ketone bodies

Synthesis of ketone bodies. HMG = hydroxymethylglutaryl CoA.
are important sources of energy for the peripheral tissues because 1) they are soluble in aqueous solution and, therefore, do not need to be incorporated into lipoproteins or carried by albumin as do the other lipids; 2) they are produced in the liver during periods when the amount of acetyl CoA present exceeds the oxidative capacity of the liver; and 3) they are used in proportion to their concentration in the blood by extrahepatic tissues, such as the skeletal and cardiac muscle and renal cortex. Even the brain can use ketone bodies to help meet its energy needs if the blood levels rise sufficiently; thus, ketone bodies spare glucose.This is particularly important during prolonged periods of fasting (see p. 332). [Note: Disorders of fatty acid oxidation present with the general picture of hypoketosis (due to decreased availability of acetyl CoA) and hypoglycemia (due to increased reliance on glucose for energy.]

During a fast, the liver is flooded with fatty acids mobilized from adipose tissue. The resulting elevated hepatic acetyl CoA produced primarily by fatty acid degradation inhibits pyruvate dehydrogenase (see p. 111), and activates pyruvate carboxylase (see p. 119). The OAA thus produced is used by the liver for gluconeogenesis rather than for the TCA cycle. Therefore, acetyl CoA is channeled into ketone body synthesis. [Note: Fatty acid oxidation decreases the NAD* to NADH ratio, and the rise in NADH shifts OAA to malate (see p. 113). This pushes acetyl CoA away from gluconeogenesis and into ketogenesis (Figure 16.24).]
1. Synthesis of 3-hydroxy-3-methylglutaryl (HMG) CoA: The first synthetic step, formation of acetoacetyl CoA, occurs by reversal of the thiolase reaction of fatty acid oxidation (see Figure 16.17). Mitochondrial HUG CoA synthase combines a third molecule of acetyl CoA with acetoacetyl CoA to produce HMG CoA. [Note: HMG CoA is also a precursor of cholesterol (see p. 220). These pathways are separated by location in, and conditions of, the cell.] HMG CoA synthase is the rate-limiting step in the synthesis of ketone bodies, and is present in significant quantities only in the liver.
2. Synthesis of the ketone bodies: HMG CoA is cleaved to produce acetoacetate and acetyl CoA, as shown in Figure 16.22. Acetoacetate can be reduced to form 3-hydroxybutyrate with NADH as the hydrogen donor. Acetoacetate can also spontaneously decarboxylate in the blood to form acetone—a volatile, biologically nonmetabolized compound that can be released in the breath. The equilibrium between acetoacetate and 3-hydroxybutyrate is determined by the NAD*/NADH ratio. Because this ratio is low during fatty acid oxidation, 3-hydroxybutyrate synthesis is favored. [Note: The generation of free CoA during ketogenesis allows fatty acid oxidation to continue.]

Although the liver constantly synthesizes low levels of ketone bodies, their production becomes much more significant during fasting

Ketone body synthesis in the liver and use in peripheral tissues. [Note: Thiophorase is also known as succinyl CoA: acetoacetate CoA transferase.]
when ketone bodies are needed to provide energy to the peripheral tissues. 3-Hydroxybutyrate is oxidized to acetoacetate by 3-hydroxybutyrate dehydrogenase, producing NADH (Figure 16.23). Acetoacetate is then provided with a CoA molecule taken from succinyl CoA by succinyl CoA:acetoacetate CoA transferase (thiophorase). This reaction is reversible, but the product, acetoacetyl CoA, is actively removed by its conversion to two acetyl CoA. Extrahepatic tissues, including the brain but excluding cells lacking mitochondria (for example, red blood cells), efficiently oxidize acetoacetate and 3-hydroxybutyrate in this manner. In contrast, although the liver actively produces ketone bodies, it lacks thiophorase and, therefore, is unable to use ketone bodies as fuel.

When the rate of formation of ketone bodies is greater than the rate of their use, their levels begin to rise in the blood (ketonemia) and, eventually, in the urine (ketonuria). This is seen most often in cases of uncontrolled, type 1 diabetes mellitus. In diabetic individuals with severe ketosis, urinary excretion of the ketone bodies may be as high as 5,000 mg/24 hr, and the blood concentration may reach 90 mg/dl (versus less than 3 mg/dl in normal individuals). A frequent symptom of diabetic ketoacidosis is a fruity odor on the breath, which results from increased production of acetone. An elevation of the ketone body concentration in the blood results in acidemia. [Note: The carboxyl group of a ketone body has a pKa of about 4. Therefore, each ketone body loses a proton (H*) as it circulates in the blood, which lowers the pH of the body. Also, excretion of glucose and ketone bodies in the urine results in dehydration of the body. Therefore, the increased number of H*, circulating in a decreased volume of plasma, can cause severe acidosis (ketoacidosis).] Ketoacidosis may also be seen in cases of fasting (see p. 330).

Generally a linear hydrocarbon chain with a terminal carboxyl group, a fatty acid can be saturated or unsaturated. Two fatty acids are essential (must be obtained from the diet): linoleic and a-linolenic acids. Fatty acids are synthesized in the cytosol of liver following a meal containing excess carbohydrate and protein. Carbons used to synthesize fatty acids are provided by acetyl CoA, energy by ATP, and reducing equivalents by NADPH (Figure 16.25). Citrate carries two-carbon acetyl units from the mitochondrial matrix to the cytosol. The regulated step in fatty acid synthesis is catalyzed by acetyl CoA carboxylase, which requires biotin. Citrate is the allosteric activator and long-chain fatty acyl CoA is the inhibitor. The enzyme can also be activated in the presence of insulin and inactivated by AMPK in response to epinephrine, glucagon, or a rise in AMP. The rest of the steps in fatty acid synthesis are catalyzed by the multifunctional enzyme, fatty acid synthase, which produces palmitoyl CoA from acetyl CoA and malonyl CoA, with NADPH (from the pentose phosphate pathway) as the source of reducing equivalents. Fatty acids can be elongated and desaturated in the ER. When fatty acids are required by the body for energy, adipose cell hormone-sensitive lipase (activated by epinephrine or glucagon, and inhibited by insulin) initiates degradation of stored triacylglycerol. Fatty acids are carried by serum albumin to the liver and peripheral tissues, where oxidation of the fatty acids provides energy. The glycerol backbone of the degraded triacylglycerol is carried by the blood to the liver, where it serves as an important gluconeogenic precursor. Fatty acid degradation (B-oxidation) occurs in mitochondria. The carnitine shuttle is required to transport LCFA from the cytosol to the mitochondrial matrix. A translocase and the enzymes carnitine palmitoyltransferases | and Il are required. Carnitine palmitoyltransferase | is inhibited by malonyl CoA. This prevents fatty acids being synthesized in the cytosol from malonyl CoA from being transported into the mitochondria where they would be degraded. Once in the mitochondria, fatty acids are oxidized, producing

acetyl CoA, NADH, and FADH2. The first step in the B-oxidation pathway is catalyzed by one of a family of four acyl CoA dehydrogenases, each of which has a specificity for either short-, medium-, long-, or very-long-chain fatty acids. Medium-chain fatty acyl CoA dehydrogenase (MCAD) deficiency is one of the most common inborn errors of metabolism. It causes a decrease in fatty acid oxidation (process stops once a medium chain fatty acid is produced), resulting in hypoketonemia and severe hypoglycemia. Oxidation of fatty acids with an odd number of carbons proceeds two carbons at a time (producing acetyl CoA) until three carbons remain (propionyl CoA). This compound is converted to methylmalonyl CoA (a reaction requiring biotin), which is then converted to succinyl CoA (a gluconeogenic precursor) by methylmalonyl CoA mutase (requiring vitamin B12). A genetic error in the mutase or vitamin By2 deficiency causes methylmalonic acidemia and aciduria. 3-Oxidation of VLCFA and a-oxidation of branched-chain fatty acids occur in the peroxisome. w-Oxidation occurs in the ER. Liver mitochondria can convert acetyl CoA derived from fatty acid oxidation into the ketone bodies, acetoacetate and 3-hydroxybutyrate. Peripheral tissues possessing mitochondria can oxidize 3-hydroxybutyrate to acetoacetate, which can be reconverted to acetyl CoA, thus producing energy for the cell. Unlike fatty acids, ketone bodies are utilized by the brain and, therefore, are important fuels during a fast. The liver lacks the ability to degrade ketone bodies, and so synthesizes them specifically for the peripheral tissues. Ketoacidosis occurs when the rate of formation of ketone bodies is greater than their rate of use, as is seen in cases of uncontrolled, type 1 diabetes mellitus.

16.1 Triacylglycerol molecules stored in adipose tissue represent the major reserve of substrate providing energy during a prolonged fast. During such a fast:
A. the stored fatty acids are released from adipose
tissue into the plasma as components of the serum lipoprotein particle, VLDL.
B. free fatty acids are produced at a high rate in the plasma by the action of lipoprotein lipase on chylomicrons.
C. glycerol produced by the degradation of triacylglycerol is an important direct source of energy for adipocytes and fibroblasts.
D. hormone-sensitive lipase is phosphorylated and activated by a cCAMP-activated protein kinase.
Correct answer = D. Hormone-sensitive lipase is phosphorylated by cAMP-activated protein kinase, which is itself activated by epinephrine or glucagon. Fatty acids released from adipose tissue are carried in the plasma by serum albumin, not VLDL. During a fast, the amount of circulating triacylglycerol (found in chylomicrons and VLDL) will be low. Therefore, there is little substrate for lipoprotein lipase. The glycerol produced during triacylglycerol degradation cannot be metabolized by adipocytes or fibroblasts, but rather must go to the liver where it can be phosphorylated (by glycerol kinase).

Key concept map for fatty acid and triacylglycerol metabolism.
16.2 A low level of carbon dioxide labeled with '4C is accidentally released into the atmosphere surrounding industrial workers as they resume work following the lunch hour. Unknowingly, they breathe the contaminated air for 1 hour. Which of the following compounds will be radioactively labeled?
A. All of the carbon atoms of newly synthesized fatty acid.
B. About one half of the carbon atoms of newly synthesized fatty acids.
C. The carboxyl atom of newly synthesized fatty acids.
D. About one third of the carbons of newly synthesized malonyl CoA.
E. One half of the carbon atoms of newly synthesized acetyl CoA.
16.3 A teenager, concerned about his weight, attempts to maintain a fat-free diet for a period of several weeks. If his ability to synthesize various lipids were examined, he would be found to be most deficient in his ability to synthesize:
A. triacylglycerol.
B. phospholipids.
C. cholesterol.
D. sphingolipids.
E. prostaglandins.
16.4 A 6-month-old boy was hospitalized following a seizure. History revealed that for several days prior, his appetite was decreased due to a "stomach virus". At admission, his blood glucose was 24 mg/dl (agereferenced normal is 60-100). His urine was negative for ketone bodies, but positive for a variety of dicarboxylic acids. A tentative diagnosis of medium-chain fatty acyl CoA dehydrogenase (MCAD) deficiency is made. In patients with MCAD deficiency, the fasting hypoglycemia is a consequence of:
A. decreased acetyl CoA production.
B. decreased ability to convert acetyl CoA to glucose. acetoac-
C. increased conversion of acetyl CoA to etate.
D. increased production of ATP and NADH.
16.5 Explain why with Zellweger syndrome both very long chain fatty acids (VLCFA) and phytanic acid accumulate, whereas with X-linked adrenoleukodystrophy (XALD) only VLCFA accumulate.

Correct answer = D. Malonyl CoA (three carbons) is synthesized from acetyl CoA (two carbons) by the addition of COs, using the enzyme acetyl CoA carboxylase. Because COz is subsequently removed during fatty acid synthesis, the radioactive label will not appear at any position in newly synthesized fatty acids.
Correct answer = E. Prostaglandins are synthesized from arachidonic acid. Arachidonic acid is synthesized from linoleic acid, an essential fatty acid obtained by humans from dietary lipids. The teenager would be able to synthesize all other compounds, but presumably in somewhat depressed amounts.
Correct answer = A. Impaired oxidation of fatty acids less than 12 carbons in length results in decreased production of acetyl CoA, the allosteric activator of pyruvate carboxylase, a gluconeogenic enzyme; thus, glucose levels fall. Acetyl CoA cannot be used for the net synthesis of glucose. Acetoacetate is a ketone body, and with MCAD deficiency ketogenesis is decreased. Impaired fatty acid oxidation means that less ATP and NADH are made, and both are needed for gluconeogenesis.
Zellweger syndrome is caused by an inability to target matrix proteins to the peroxisome; thus, all peroxisomal activities are affected because functional peroxisomes are not able to be formed. In X-ALD, the defect is an inability to transport VLCFA into the peroxisome—other peroxisomal functions such as « oxidation are normal.

Phospholipids are polar, ionic compounds composed of an alcohol that is attached by a phosphodiester bridge to either diacylglycerol or sphingosine. Like fatty acids, phospholipids are amphipathic in nature, that is, each has a hydrophilic head (the phosphate group plus whatever alcohol is attached to it, for example, serine, ethanolamine, and choline, highlighted in blue in Figure 17.1A), and a long, hydrophobic tail (containing fatty acids or fatty acid—derived hydrocarbons, shown in orange in Figure 17.1A). Phospholipids are the predominant lipids of cell membranes. In membranes, the hydrophobic portion of a phospholipid molecule is associated with the nonpolar portions of other membrane constituents, such as glycolipids, proteins, and cholesterol. The hydrophilic (polar) head of the phospholipid extends outward, interacting with the intracellular or extracellular aqueous environment (see Figure 17.1A). Membrane phospholipids also function as a reservoir for intracellular messengers, and, for some proteins, phospholipids serve as anchors to cell membranes. Nonmembrane-bound phospholipids serve additional functions in the body, for example, as components of lung surfactant and essential components of bile, where their detergent properties aid in the solubilization of cholesterol.

There are two classes of phospholipids: those that have glycerol as a backbone and those that contain sphingosine. Both classes are found as structural components of membranes, and both play a role in the generation of lipid-signaling molecules.

Phospholipids that contain glycerol are called glycerophospholipids (or phosphoglycerides). Glycerophospholipids constitute the major class of phospholipids. All contain (or are derivatives of) phosphatidic acid (diacylglycerol with a phosphate group on the third carbon, Figure 17.1B). Phosphatidic acid is the simplest phosphoglyceride, and is the precursor of the other members of this group.

A. Structures of some glycero- phospholipids. B. Phosphatidic acid. ® = phosphate, PO,1-.

A. The plasmalogen phosphatidalethanolamine. B. Platelet-activating factor.
1. Glycerophospholipids are formed from phosphatidic acid (PA) and an alcohol: The phosphate group on PA can be esterified to another compound containing an alcohol group (see Figure 17.1). For example:
Serine
+
PA
—
phosphatidylserine
Ethanolamine
+
PA
—
phosphatidylethanolamine (cephalin)
Choline
+
PA
—
phosphatidylcholine (lecithin)
Inositol
+
PA
—
phosphatidylinositol
Glycerol
+
PA
—
phosphatidylglycerol

2. Cardiolipin: Two molecules of PA esterified through their phosphate groups to an additional molecule of glycerol is called cardiolipin (diphosphatidylglycerol, Figure 17.2). Cardiolipin is found in bacteria and eukaryotes. In eukaryotes, cardiolipin is virtually exclusive to the inner mitochondrial membrane, where it appears to be required for the maintenance of certain respiratory complexes of the electron transport chain. [Note: Cardiolipin is antigenic, and is recognized by antibodies raised against Treponema pallidum, the bacterium that causes syphilis.]
3. Plasmalogens: When the fatty acid at carbon 1 of a glycerophospholipid is replaced by an unsaturated alkyl group attached by an ether (rather than by an ester) linkage to the core glycerol molecule, a plasmalogen is produced. For example, phosphatidalethanolamine (abundant in nerve tissue, Figure 17.3A) is the plasmalogen that is similar in structure to phosphatidylethanolamine. Phosphatidalcholine (abundant in heart muscle) is the other quantitatively significant ether lipid in mammals.
4. Platelet-activating factor (PAF): This is an unusual ether glycerophospholipid, with a saturated alkyl group in an ether link to carbon 1 and an acetyl residue (rather than a fatty acid) at carbon 2 of the glycerol backbone (Figure 17.3B). PAF is synthesized and released by a variety of cell types. It binds to surface receptors, triggering potent thrombotic and acute inflammatory events. For example, PAF activates inflammatory cells and mediates hypersensitivity, acute inflammatory, and anaphylactic reactions. It causes platelets to aggregate and degranulate, and neutrophils and alveolar macrophages to generate superoxide radicals (see p. 148 for a discussion of the role of superoxides in killing bacteria). [Note: PAF is one of the most potent bioactive molecules known, causing effects at concentrations as low as 10 '? mol/L.]

The backbone of sphingomyelin is the amino alcohol sphingosine, rather than glycerol (Figure 17.4). A long-chain fatty acid is attached to the amino group of sphingosine through an amide linkage, producing a ceramide, which can also serve as a precursor of glycolipids (see p. 209). The alcohol group at carbon 1 of sphingosine is esterified to phosphorylcholine, producing sphingomyelin, the only
significant sphingophospholipid in humans. Sphingomyelin is an important constituent of the myelin of nerve fibers. [Note: The myelin sheath is a layered, membranous structure that insulates and protects neuronal fibers of the central nervous system.]

Glycerophospholipid synthesis involves either the donation of phosphatidic acid from CDP-diacylglycerol to an alcohol, or the donation of the phosphomonoester of the alcohol from CDP-alcohol to 1,2-diacylglycerol (Figure 17.5). [Note: CDP is the nucleotide cytidine diphosphate (see p. 292).] In both cases, the CDP-bound structure is considered an "activated intermediate,' and cytidine monophosphate (CMP) is released as a side product of glycerophospholipid synthesis. A key concept in phosphoglyceride synthesis, therefore, is activation—either of diacylglycerol or the alcohol to be added—by linkage with CDP. [Note: This is similar in principle to the activation of sugars by their attachment to uridine diphosphate (UDP) (see p. 126).] The fatty acids esterified to the glycerol alcohol groups can vary widely, contributing to the heterogeneity of this group of compounds. Most phospholipids are synthesized in the smooth endoplasmic reticulum. From there, they are transported to the Golgi apparatus and then to membranes of organelles or the plasma membrane, or are secreted from the cell by exocytosis. [Note: Ether lipid synthesis occurs in peroxisomes. ]

PA is the precursor of many other phosphoglycerides. The steps in its synthesis from glycerol phosphate and two fatty acyl coenzyme A (CoA) molecules were illustrated in Figure 16.14, p. 189, in which PA is shown as a precursor of triacylglycerol.
Essentially all cells except mature erythrocytes can synthesize phospholipids, whereas triacylglycerol synthesis occurs essentially only in liver, adipose tissue, lactating mammary glands, and intestinal mucosal cells.

PC and PE are the most abundant phospholipids in most eukaryotic cells. The primary route of their synthesis uses choline and ethanolamine obtained either from the diet or from the turnover of the body's phospholipids. [Note: In the liver, PC also can be synthesized from phosphatidylserine (PS) and PE (see below).]
1. Synthesis of PE and PC from preexisting choline and ethanolamine: These synthetic pathways involve the phosphorylation of choline or ethanolamine by kinases, followed by conversion to the activated form, CDP-choline or CDP-ethanolamine. Finally, choline-phosphate or ethanolamine-phosphate is transferred from the nucleotide (leaving CMP) to a molecule of diacylglycerol (see Figure 17.5).

Structure of sphingomyelin, showing sphingosine (in green box) and ceramide components (in dashed box).

Activation of either diacylglycerol or an alcohol by linkage to a nucleoside diphosphate (CDP) promotes phospholipid synthesis.

Synthesis of phosphatidylcholine from phosphatidylserine in the liver.
2.
a. Significance of choline reutilization: The reutilization of choline is important because, whereas humans can synthesize choline de novo, the amount made is insufficient for our needs. Thus, choline is an essential dietary nutrient with an Adequate Intake (see p. 358) of 550 mg for men and 425 mg for women. [Note: Choline is also used for the synthesis of acetylcholine— a neurotransmitter.]
b. Role of PC in lung surfactant: The pathway described above is the principal pathway for the synthesis of dipalmitoylphosphatidylcholine (DPPC, or dipalmitoyl lecithin). In DPPC, positions 1 and 2 on the glycerol are occupied by palmitate. DPPC, made and secreted by Type II pneumocytes, is the major lipid component of lung surfactant—the extracellular fluid layer lining the alveoli. Surfactant serves to decrease the surface tension of this fluid layer, reducing the pressure needed to reinflate alveoli, thereby preventing alveolar collapse (atelectasis). [Note: Surfactant is a complex mixture of lipids (90%) and proteins (10%), with DPPC being the major component for reducing surface tension.] Respiratory distress syndrome (RDS) in preterm infants is associated with insufficient surfactant production and/or secretion, and is a significant cause of all neonatal deaths in Western countries.
Lung maturity of the fetus can be gauged by determining the ratio of DPPC to sphingomyelin, usually written as the L (for lecithin)/S ratio, in amniotic fluid. A ratio of two or above is evidence of maturity, because it reflects the major shift from sphingomyelin to DPPC synthesis that occurs in the pneumocytes at about 32 weeks of gestation.

Lung maturation can be accelerated by giving the mother glucocorticoids shortly before delivery. Administration of natural or synthetic surfactant (by intratracheal instillation) is also used in the prevention and treatment of infant RDS. RDS due to an insufficient amount of surfactant can also occur in adults whose surfactant-producing pneumocytes have been damaged or destroyed, for example, by infection or trauma.
Synthesis of PC from phosphatidylserine (PS) in the liver: The liver requires a mechanism for producing PC, even when free choline levels are low, because it exports significant amounts of PC in the bile and as a component of serum lipoproteins. To provide the needed PC, PS is decarboxylated to PE by PS decarboxylase, an enzyme requiring pyridoxal phosphate as a coenzyme. PE then undergoes three methylation steps to produce PC, as illustrated in Figure 17.6. S-adenosylmethionine is the methyl group donor (see p. 264).

The primary pathway for synthesis of PS in mammalian tissues is provided by the base exchange reaction, in which the ethanolamine of PE is exchanged for free serine (see Figure 17.6). This reaction, although reversible, is used primarily to produce the PS required for membrane synthesis.

PI is synthesized from free inositol and CDP-diacylglycerol as shown in Figure 17.5. PI is an unusual phospholipid in that it often contains stearic acid on carbon 1 and arachidonic acid on carbon 2 of the glycerol. Pl, therefore, serves as a reservoir of arachidonic acid in membranes and, thus, provides the substrate for prostaglandin synthesis when required (see p. 213 for a discussion of these compounds).
EEE

Figure 17.7 Stoncture of phosphatidylinositol 4,5-bisphosphate (PIP2). Cleavage by phospholipase C produces IP3 and DAG.
across phorylation of membrane-bound phosphatidylinositol produces polyphosphoinositides, for example, phosphatidylinositol 4,5bisphosphate (PIP2, Figure 17.7). The degradation of PIP2 by phospholipase C occurs in response to the binding of a variety of neurotransmitters, hormones, and growth factors to receptors on the cell membrane (Figure 17.8). The products of this degradation, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), mediate the mobilization of intracellular calcium and the activation

Role of inositol trisphosphate and diacylglycerol in intracellular signaling.

Example of a glycosyl phosphatidylinositol (GP!) membrane protein anchor. GIcN = glucosamine.
of protein kinase C, respectively, which act synergistically to evoke specific cellular responses. Signal transmission across the membrane is thus accomplished.
2.
Role of PI in membrane protein anchoring: Specific proteins can be covalently attached via a carbohydrate bridge to membranebound PI (Figure 17.9). [Note: Examples of such proteins include alkaline phosphatase (a digestive enzyme found on the surface of the small intestine that attacks organic phosphates), and acetylcholine esterase (an enzyme of the postsynaptic membrane that degrades the neurotransmitter acetylcholine). Cell surface proteins bound to glycosyl phosphatidylinositol (GPI) are also found in a variety of parasitic protozoans (for example, trypanosomes and leishmania).] Being attached to a membrane lipid (rather than being an integral part of the membrane) allows GPl-anchored proteins increased lateral mobility on the surface of the plasma membrane. The protein can be cleaved from its anchor by the action of phospholipase C (see Figure 17.8), releasing diacylglycerol. [Note: A deficiency in the synthesis of GPI in hematopoietic cells results in a hemolytic disease, paroxysmal nocturnal hemoglobinuria]

Phosphatidylglycerol occurs in relatively large amounts in mitochondrial membranes and is a precursor of cardiolipin. It is synthesized by a two-step reaction from CDP-diacylglycerol and glycerol 3-phosphate. Cardiolipin (diphosphatidylglycerol, see Figure 17.2) is composed of two molecules of phosphatidic acid connected by a molecule of glycerol. It is synthesized by the transfer of diacylglycerophosphate from CDP-diacylglycerol to a preexisting molecule of phosphatidylglycerol.

Sphingomyelin, a sphingosine-based phospholipid, is a major structural lipid in the membranes of nerve tissue. The synthesis of sphingomyelin is shown in Figure 17.10. Briefly, palmitoy! CoA condenses with serine, as CoA and the carboxyl group (as COz) of serine are lost. [Note: This reaction, like the decarboxylation reactions involved in the synthesis of PE from PS, and of regulators from amino acids—for example, the catecholamines from tyrosine (see p. 286)— requires pyridoxal phosphate (a derivative of vitamin Bg) as a coenzyme (see p. 378).] The product is reduced in an NADPH-requiring reaction to sphinganine, which is acylated at the amino group with one of a variety of long-chain fatty acids, and then desaturated to produce a ceramide—the immediate precursor of sphingomyelin.
A ceramide containing a fatty acid 30 carbons long is a major component of skin, and regulates skin's water permeability.
Phosphorylcholine from phosphatidylcholine is transferred to the ceramide, producing sphingomyelin and diacylglycerol. [Note: Sphingomyelin of the myelin sheath contains predominantly longer-
9
CHa(CHa);,-C-CoA
:
900
HgN-C-H
CH,OH
CH,(CH,);2°
CH
CHa-C—C-CHOH
NADP*
:
RA
Bile
NADPH
+H*
Sphinganine
NH
Palmitoyl
CoA
OH
Serine chain fatty acids such as lignoceric acid and nervonic acid, whereas gray matter of the brain has sphingomyelin that contains primarily stearic acid.]

The degradation of phosphoglycerides is performed by phospholipases found in all tissues and pancreatic juice (for a discussion of phospholipid digestion, see p. 175). A number of toxins and venoms have phospholipase activity, and several pathogenic bacteria produce phospholipases that dissolve cell membranes and allow the spread of infection. Sphingomyelin is degraded by the lysosomal phospholipase, sphingomyelinase.

+
+
FAD
O
CH,(CH2)n-G-CoA
Fatty acyl
CoA
FADH
CoA
H
OH
roworrg-€-oH0n|
H
NH
c-0
(CH2),
CH3
Ceramide
Phosphatidylcholine
Diacylglycerol
HH
R-CH+CH-G-C-CH,O-P-O
Oo
OH
NH
=0
c
(CHa)n
CH,
Sphingomyelin

Synthesis of sphingomyelin. PLP = pyridoxal phosphate.
m,
Phospholipases hydrolyze the phosphodiester bonds of phosphoglycerides, with each enzyme cleaving the phospholipid at a specific site. The major enzymes responsible for degrading phosphoglycerides are shown in Figure 17.11. [Note: Removal of the fatty acid from carbon 1 or 2 of a phosphoglyceride produces a lysophosphoglyceride, which is the substrate for /ysophospholipases.] Phospholipases release molecules that can serve as messengers (for example, DAG and IP3), or that are the substrates for synthesis of messengers (for example, arachidonic acid). [Note: Phospholipases are responsible not only for degrading phospholipids, but also for "remodeling" them. For example, phospholipases A; and Az

Degradation of glycerophospholipids by phospholipases.
‘On
Sphingosine

Accumulation of lipids in spleen cells from a patient with NiemannPick disease.
remove specific fatty acids from membrane-bound phospholipids; these can be replaced with alternative fatty acids using fatty acyl CoA transferase. This mechanism is used as one way to create the unique lung surfactant, DPCC (see p. 204), and to insure that carbon 2 of PI (and sometimes of PC) is bound to arachidonic acid.]

Sphingomyelin is degraded by sphingomyelinase, a lysosomal enzyme that hydrolytically removes phosphorylcholine, leaving a ceramide. The ceramide is, in turn, cleaved by ceramidase into sphingosine and a free fatty acid (Figure 17.12). [Note: The ceramide and sphingosine released regulate signal transduction pathways, in part by influencing the activity of protein kinase C and, thus, the phosphorylation of its protein substrates. They also promote apoptosis.] Niemann-Pick disease (Types A and B) is an autosomal recessive disease caused by the inability to degrade sphingomyelin. The deficient enzyme is sphingomyelinase—a type of phospholipase C. In the severe infantile form (Type A—less than 1% normal activity), the liver and spleen are the primary sites of lipid deposits and are, therefore, tremendously enlarged. The lipid consists primarily of the sphingomyelin that cannot be degraded (Figure 17.13). Infants with this lysosomal storage disease experience rapid and progressive neurodegeneration as a result of deposition of sphingomyelin in the central nervous system, and they die in early childhood. A less severe variant (Type B—5% or more) causes little to no damage to neural tissue, but lungs, spleen, liver, and bone marrow are affected, resulting in a chronic form of the disease, with a life expectancy into adulthood. Although NiemannPick disease occurs in all ethnic groups, Type A occurs with greater frequency in the Ashkenazi Jewish population than in the general population.

Glycolipids are molecules that contain both carbohydrate and lipid components. Like the phospholipid sphingomyelin, glycolipids are derivatives of ceramides in which a long-chain fatty acid is attached to the amino alcohol sphingosine. They are, therefore, more precisely called glycosphingolipids. [Note: Ceramides, then, are the precursors of both phosphorylated and glycosylated sphingolipids.] Like the phospholipids, glycosphingolipids are essential components of all membranes in the body, but they are found in greatest amounts in nerve tissue. They are located in the outer leaflet of the plasma membrane, where they interact with the extracellular environment. As such, they play a role in the regulation of cellular interactions, growth, and development.
When cells are transformed (that is, when their cell division and growth are dysregulated), there is a dramatic change in the glycosphingolipid composition of the plasma membrane.
Glycosphingolipids are antigenic, and they have been identified as a source of blood group antigens, various embryonic antigens specific for particular stages of fetal development, and some tumor antigens. [Note: The carbohydrate portion of a glycolipid is the antigenic determinant.] They also serve as cell surface receptors for cholera and tetanus toxins, as well as for certain viruses and microbes. Genetic disorders associated with an inability to properly degrade the glycosphingolipids result in lysosomal accumulation of these compounds.

The glycosphingolipids differ from sphingomyelin in that they do not contain phosphate, and the polar head function is provided by a monosaccharide or oligosaccharide attached directly to the ceramide by an O-glycosidic bond (Figure 17.14). The number and type of carbohydrate moieties present help determine the type of glycosphingolipid.

The simplest neutral (uncharged) glycosphingolipids are the cerebrosides. These are ceramide monosaccharides that contain either a molecule of galactose (galactocerebroside—the most common cerebroside found in membranes, see Figure 17.14) or glucose (glucocerebroside, which serves primarily as an intermediate in the synthesis and degradation of the more complex glycosphingolipids). [Note: Members of a group of galactocerebrosides (or glucocerebrosides) may also differ from each other in the type of fatty acid attached to the sphingosine.] As their name implies, cerebrosides are found predominantly in the brain and peripheral nervous tissue, with high concentrations in the myelin sheath. Ceramide oligosaccharides (or globosides) are produced by attaching additional monosaccharides (including GalNAc) to a glucocerebroside. Examples of these compounds include:
Cerebroside (glucocerebroside): Cer-Glc
Cer-Glc-Gal
Cer-Glc-Gal-Gal-GalNac-GalNac
Globoside (lactosylceramide):
Globoside (Forssman antigen):
(Cer = ceramide, Glc = glucose, Gal = galactose, GalNac = N-acetylgalactosamine)

Acidic glycosphingolipids are negatively charged at physiologic pH. The negative charge is provided by N-acetylneuraminic acid (NANA, a sialic acid, see Figure 17.15) in gangliosides, or by sulfate groups in sulfatides.
- 1. Gangliosides: These are the most complex glycosphingolipids, and are found primarily in the ganglion cells of the central nervous system, particularly at the nerve endings. They are derivatives of ceramide oligosaccharides, and contain one or more molecules of NANA. The notation for these compounds is G (for ganglioside),

Structure of a neutral glyco- sphingolipid, galactocerebroside
( is a fatty acid hydrocarbon chain).

Structure of galactocerebroside 3sulfate.
plus a subscript M, D, T, or Q to indicate whether there is one (mono), two (di), three (tri), or four (quatro) molecules of NANA in the ganglioside, respectively. Additional numbers and letters in the subscript designate the monomeric sequence of the carbohydrate attached to the ceramide. (See Figure 17.15 for the structure of Gwe.) Gangliosides are of medical interest because several lipid storage disorders involve the accumulation of NANA-containing glycosphingolipids in cells (see Figure 17.20, p. 212).
2. Sulfatides: Sulfoglycosphingolipids (sulfatides) are cerebrosides that contain sulfated galactosyl residues, and are therefore negatively charged at physiologic pH. Sulfatides are found predominantly in nerve tissue and kidney.

Synthesis of glycosphingolipids occurs primarily in the Golgi by sequential addition of glycosyl monomers transferred from UDP-—sugar donors to the acceptor molecule. The mechanism is similar to that used in glycoprotein synthesis (see p. 166).

The enzymes involved in the synthesis of glycosphingolipids are glycosyl transferases, each specific for a particular sugar nucleotide and acceptor. [Note: These enzymes may recognize both glycosphingolipids and glycoproteins as substrates.]

A sulfate group from the sulfate carrier, 3'-phosphoadenosine-5'phosphosulfate (PAPS, Figure 17.16), is added by a sulfotransferase to the 3'-hydroxyl group of the galactose in a galactocerebroside. Galactocerebroside 3-sulfate is the major sulfatide in the brain (Figure 17.17). [Note: PAPS is also the sulfur donor in glycosaminoglycan synthesis (see p. 162), and steroid hormone catabolism (see p. 240).] An overview of the synthesis of sphingolipids is shown in Figure 17.18.

Glycosphingolipids are internalized by endocytosis as described for the glycosaminoglycans. All of the enzymes required for the degradative process are present in lysosomes, which fuse with the endocytotic vesicles. The lysosomal enzymes hydrolytically and irreversibly cleave specific bonds in the glycosphingolipid. As seen with the glycosaminoglycans (see p. 163) and glycoproteins (see p. 170), degradation is a sequential process following the rule "last on, first off," in which the last group added during synthesis is the first group removed in degradation. [Note: Defects in the degradation of the polysaccharide chains in these three glycoconjugates also result in lysosomal storage diseases. ]

a . . . In a normal individual, synthesis and degradation of glycosphingolipids are balanced, so that the amount of these compounds pre-

Overview of sphingolipid synthesis.
sent in membranes is constant. If a specific lysosomal hydrolase required for degradation is partially or totally missing, a sphingolipid accumulates. Lysosomal lipid storage diseases caused by these deficiencies are called sphingolipidoses. The result of a specific hydrolase deficiency may be seen dramatically in nerve tissue, where neurologic deterioration can lead to early death. [Note: Ganglioside turnover in the central nervous system is extensive during neonatal development.] Figure 17.20 provides an outline of the pathway of sphingolipid degradation and descriptions of some sphingolipidoses.
1.
Common properties: A specific lysosomal hydrolytic enzyme is deficient in each disorder. Therefore, usually only a single sphingolipid (the substrate for the deficient enzyme) accumulates in the involved organs in each disease. [Note: The rate of biosynthesis of the accumulating lipid is normal.] The disorders are progressive and, although many are fatal in childhood, extensive phenotypic variability is seen leading to the designation of different clinical types, such as Types A and B in Niemann-Pick disease. Genetic variability is also seen because a given disorder can be caused by any one of a variety of mutations within a single gene. The sphingolipidoses are autosomal recessive diseases, except for Fabry disease, which is X-linked. The incidence of the sphingolipidoses is low in most populations, except for Gaucher and Tay-Sachs diseases, which, like Niemann-Pick disease, show a high frequency in the Ashkenazi Jewish population.

2. Diagnosis and treatment: A specific sphingolipidosis can be diagnosed by measuring enzyme activity in cultured fibroblasts or peripheral leukocytes, or by analysis of DNA (see p. 473). Histologic examination of the affected tissue is also useful. [Note: Shell-like inclusion bodies are seen in Tay-Sachs, and a wrinkled tissue paper appearance of the cytosol is seen in Gaucher disease (Figure 17.19).] Prenatal diagnosis, using cultured amniocytes or chorionic villi, is available. Gaucher disease, in which macrophages become engorged with glucocerebroside, and Fabry disease, in which globosides accumulate in the vascular endothelial lysosomes of the brain, heart, kidneys, and skin, are treated by recombinant human enzyme replacement therapy, but the mone-

Degradation of sphingolipids showing the lysosomal enzymes affected in related genetic diseases, the sphingolipidoses. All of the diseases are autosomal recessive except Fabry disease, which is X-linked, and all can be fatal in early life. (Cer = ceramide).
tary cost is extremely high. Gaucher has also been treated by bone marrow transplantation (because macrophages are derived from hematopoietic stem cells), and by substrate reduction therapy through pharmacological reduction of glucosylceramide, the substrate for the deficient enzyme.

Prostaglandins, and the related compounds thromboxanes and leukotrienes, are collectively known as eicosanoids to reflect their origin from polyunsaturated fatty acids with 20 carbons. They are extremely potent compounds that elicit a wide range of responses, both physiologic (inflammatory response) and pathologic (hypersensitivity). They ensure gastric integrity and renal function, regulate smooth muscle contraction (intestine and uterus are key sites) and blood vessel diameter, and maintain platelet homeostasis. Although they have been compared to hormones in terms of their actions, eicosanoids differ from the true hormones in that they are produced in very small amounts in almost all tissues rather than in specialized glands. They also act locally rather than after transport in the blood to distant sites, as occurs with true hormones such as insulin. Eicosanoids are not stored, and they have an extremely short half-life, being rapidly metabolized to inactive products. Their biologic actions are mediated by plasma membrane G protein-coupled receptors (see p. 94), which are different in different organ systems. Examples of prostaglandins and related structures are shown in Figure 17.21.

The dietary precursor of the prostaglandins is the essential fatty acid, linoleic acid, an w-6 fatty acid. It is desaturated and elongated to arachidonic acid (also an w-6), the immediate precursor of the predominant class of prostaglandins (those with two double bonds) in humans (Figure 17.22). [Note: Arachidonic acid is incorporated into membrane-bound phospholipids. It is released by phospholipase Az in response to a variety of signals (Figure 17.23).]
1. Synthesis of PGHz2: The first step in prostaglandin synthesis is the oxidative cyclization of free arachidonic acid to yield PGH»2 by prostaglandin endoperoxide synthase (PGH synthase). This enzyme is an endoplasmic reticulum membrane-bound protein that has two catalytic activities: fatty acid cyclooxygenase (COX), which requires two molecules of Os, and peroxidase, which is dependent on reduced glutathione (see p. 148). PGHz is converted to a variety of prostaglandins and thromboxanes, as shown in Figure 17.23, by cell-specific synthases.
a. Ilsozymes of PGH synthase: Two isozymes, usually denoted as COX-1 and COX-2, of PGH synthase are known. COX-1 is made constitutively in most tissues, and is required for maintenance of healthy gastric tissue, renal homeostasis, and platelet aggregation. COX-2 is inducible in a limited number of tissues in response to products of activated immune and inflammatory cells. [Note: The increase in prostaglandin synthesis subsequent to the induction of COX-2 mediates the pain, heat, redness, and swelling of inflammation, and the fever of infection.]

Examples of prostaglandin structures. Prostaglandins are named as follows: PG plus a third letter (for example, A, D, E, F), which designates the type and arrangement of functional groups in the molecule. The subscript number indicates the number of double bonds in the molecule. PGla is known as prostacyclin. Thromboxanes are designated by TX and leukotrienes by LT.

Oxidation and cyclization of arachidonic acid by the two catalytic activities of prostaglandin endoperoxide synthase. G-SH = reduced glutathione; GS-SG = oxidized glutathione.
2.
Inhibition of prostaglandin synthesis: The synthesis of prostaglandins can be inhibited by a number of unrelated compounds. For example, cortisol (a steroidal anti-inflammatory agent) inhibits phospholipase Az activity (see Figure 17.23) and, therefore, the precursor of the prostaglandins, arachidonic acid, is not made available from membrane phospholipids. Aspirin, indomethacin, and phenylbutazone (all nonsteroidal anti-inflammatory agents [NSAIDS]) inhibit both COX-1 and COX-2 and, thus, prevent the synthesis of the parent prostaglandin, PGHo. [Note: Systemic inhibition of COX-1, with subsequent damage to the stomach and the kidneys, and impaired clotting of blood, is the basis of aspirin's toxicity.] Inhibitors specific for COX-2 (for example, celecoxib') were designed to reduce pathologic inflammatory processes while maintaining the physiologic functions of COX-1; however, their use has been associated with increased risk of heart attacks.

Arachidonic acid is converted to a variety of linear hydroperoxy acids by a separate pathway involving a family of lipoxygenases (LOXs). For example, 5-lipoxygenase converts arachidonic acid to 5hydroxy-6,8,11,14 eicosatetraenoic acid (5-HPETE, see Figure 17.23). 5-HPETE is converted to a series of leukotrienes, the nature of the final products varying according to the tissue. Leukotrienes are mediators of allergic response and inflammation. Their synthesis is not affected by NSAIDs. [Note: Aspirin-induced asthma is a response to overproduction of leukotrienes with NSAID use. NSAIDs, however, also favor synthesis of lipoxins (LXs), lipid mediators with anti-inflammatory effects.] Inhibitors of 5-lipoxygenase and leukotriene receptor antagonists are used in the treatment of asthma."

Thromboxane Az (TXAz) is produced by COX-7 in activated platelets. It promotes adherence and aggregation of circulating platelets, and contraction of vascular smooth muscle, thus promoting formation of blood clots (thrombi). Prostacyclin (PGl2), produced by COX-2 in vascular endothelial cells, inhibits platelet aggregation and stimulates vasodilation, and so impedes thrombogenesis. The opposing effects of TXAz and PGlbz limit thrombi formation to sites of vascular injury. [Note: Aspirin has an antithrombogenic effect. It inhibits COX-1 (and TXAz synthesis) in platelets, and COX-2 (and PGle synthesis) in endothelial cells by irreversible acetylation of these isozymes (Figure 17.24). The inhibition of COX-7 cannot be overcome in platelets, which lack nuclei. However, the inhibition of COX-2 can be overcome in endothelial cells, because they have a nucleus and, therefore, can generate more of the enzyme. This difference is the basis of low-dose aspirin therapy used to lower the risk of stroke and heart attacks by decreasing formation of thrombi.]
QOS 1See Chapter 41 in Lippincott's Illustrated Reviews: Pharmacology for a (SG) discussion of anti-inflammatory drugs. SaeS 2See Chapter 27 in Lippincott's Illustrated Reviews: Pharmacology for a \O®) discussion of the treatment of asthma.

Overview of the biosynthesis and function of some important prostaglandins, leukotrienes, and a thromboxane from arachidonic acid.

Phospholipids are polar, ionic compounds composed of an alcohol (for example, choline or ethanolamine) attached by a phosphodiester bridge to either diacylglycerol (producing phosphatidylcholine or phosphatidylethanolamine) or to sphingosine (Figure 17.25). The alcohol sphingosine attached to a long-chain fatty acid produces a ceramide. Addition of a phosphorylcholine produces the phospholipid sphingomyelin, which is the only significant sphingophospholipid in humans. Phospholipids are the predominant lipids of cell membranes. Nonmembrane-bound phospholipids serve as components of lung surfactant and bile. Dipalmitoylphosphatidylcholine (DPPC, also called dipalmitoyl lecithin, DPPL) is the major lipid component of lung surfactant. Insufficient surfactant production causes respiratory distress syndrome. Phosphatidylinositol (Pl) serves as a reservoir for arachidonic acid in membranes. The phosphorylation of membrane-bound PI produces phosphatidylinositol 4,5-bisphosphate (PIP). This compound is degraded by phospholipase C in response to the binding of a variety of neurotransmitters, hormones, and growth factors to membrane receptors. The products of this degradation, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol mediate the mobilization of intracellular calcium and the activation of protein kinase C, which act synergistically to evoke cellular responses. Specific proteins can be covalently attached via a carbohydrate bridge to membrane-bound phosphatidylinositol (glycosyl phosphatidylinositol, or GPI). A deficiency in the synthesis of GPI in hematopoietic cells results in a hemolytic disease, paroxysmal nocturnal hemoglobinuria. The degradation of phosphoglycerides is performed by phospholipases found in all tissues and pancreatic juice. Sphingomyelin is degraded to a ceramide plus phosphorylcholine by the lysosomal enzyme sphingomyelinase. A deficiency in sphingomyelinase causes Niemann-Pick (A + B) disease.

Glycolipids (glycosphingolipids) are derivatives of ceramides to which carbohydrates have been attached. When one sugar molecule is added to the ceramide, a cerebroside is produced. If an oligosaccharide is added, a globoside is produced. If an acidic N-acetylneuraminic acid (NANA) molecule is added, a ganglioside is produced. Glycolipids are found predominantly in cell membranes of the brain and peripheral nervous tissue, with high concentrations in the myelin sheath. They are very antigenic. Glycolipids are degraded in the lysosomes by hydrolytic enzymes. A deficiency of one of these enzymes produces a sphingolipidosis, in each of which a characteristic sphingolipid accumulates. Prostaglandins (PG), thromboxanes (TX), and leukotrienes (LT) are produced in very small amounts in almost all tissues, act locally and have an extremely short half-life. They serve as mediators of the inflammatory response. The dietary precursor of the eicosanoids is the essential fatty acid, linoleic acid. It is desaturated and elongated to arachidonic acid—the immediate precursor of prostaglandins—which is stored in the membrane as a component of a phospholipid, generally phosphatidylinositol. Arachidonic acid is released from the phospholipid by phospholipase Az. Synthesis of the PG and TX begins with the oxidative cyclization of free arachidonic acid to yield PGHs2 by prostaglandin endoperoxide synthase—an ER membrane protein that has two catalytic activities: fatty acid cyclooxygenase (COX) and peroxidase. Opposing effects of PGl2 and TXAz limit clot formation. There are two isozymes of the synthase: COX-1 (constitutive) and COX-2. NSAIDs inhibit both. LT are linear molecules produced by the 5-lipoxygenase pathway. They mediate allergic response and are unaffected by NSAIDs.

17.1 Aspirin-induced asthma (AIA) is a severe reaction to nonsteroidal anti-inflammatory drugs (NSAIDs) characterized by bronchoconstriction 30 minutes to several hours after ingestion. It is seen in as many as 20% of adults. Which of the following statements best explains the symptoms seen in patients with AIA?
A.
NSAIDs inhibit
the activity
of the
CFTR
protein, resulting
in thickened
secretions that
block air-
ways.
B. NSAIDs inhibit COX but not lipoxygenase, resulting in the flow of arachidonic acid to leukotriene synthesis.
C. NSAIDs activate the COX activity of PGH syn-
thase, resulting
in increased
synthesis of
prostaglandins that
promote vasodilation.
- D. NSAIDs activate phospholipases, resulting in decreased amounts of dipalmytoylphosphatidylcholine and alveolar collapse (atelectasis).
17.2 An infant, born at 28 weeks of gestation, rapidly gave evidence of respiratory distress. Lab and x-ray results supported the diagnosis of infant respiratory distress syndrome (RDS). Which of the following statements about this syndrome is true?
A. It is unrelated to the baby's premature birth.
B. It is a consequence of too few Type II pneumocytes.
C. The lecithin/sphingomyelin ratio in the amniotic
fluid is
likely to
be greater
than two.
- D. The concentration of dipalmitoylphosphatidyl-
choline in
the amniotic
fluid would
be expected
to be
lower than
that of
a
full-term baby.
E.
- RDS is an easily treated disorder with low mortality.
17.3 A 25-year-old woman with a history that included hepatosplenomegaly with eventual removal of the spleen, bone and joint pain with several fractures of the femur, and a liver biopsy that showed wrinkledlooking cells with accumulations of glucosylceramides was presented at Grand Rounds. The likely diagnosis for this patient is:

A. Fabry disease.
B. Farber disease.
C. Gaucher disease.
D. Krabbe disease.
E. Niemann-Pick disease.
Correct answer = B. NSAIDs inhibit COX but not lipoxygenase, so any arachidonic acid available is used for the synthesis of bronchoconstricting-leukotrienes. NSAIDs have no effect on the CFTR protein, defects in which are the cause of cystic fibrosis. Steroids, not NSAIDs, inhibit phospholipase Az. COX is inhibited by NSAIDs, not activated. NSAIDs have no effect on phospholipases.
Correct answer = D. Dipalmitoylphosphatidylcholine (DPPC, or dipalmitoyl lecithin) is the lung surfactant found in mature, healthy lungs. RDS can occur in lungs that make too little of this compound. If the lecithin/sphingomyelin ratio in amniotic is greater than two, a newborn's lungs are considered to be sufficiently mature—premature lungs would be expected to have a ratio lower than two. The RDS would not be due to too few Type II pneumocytes—these cells would simply be secreting sphingomyelin rather than DPPC at 28 weeks of gestation.
Correct answer = C. The adult form of Gaucher disease causes hepatosplenomegaly, osteoporosis of the long bones, and the characteristic wrinkled appearance of the cytosol of cells. This is also the sphingolipidosis in which glucosylceramides accumulate. The deficient enzyme is Bglucosidase (a glucocerebrosidase).

Cholesterol, the characteristic steroid alcohol of animal tissues, performs a number of essential functions in the body. For example, cholesterol is a structural component of all cell membranes, modulating their fluidity, and, in specialized tissues, cholesterol is a precursor of bile acids, steroid hormones, and vitamin D. It is therefore of critical importance that the cells of the body be assured an appropriate supply of cholesterol. To meet this need, a complex series of transport, biosynthetic, and regulatory mechanisms has evolved. The liver plays a central role in the regulation of the body's cholesterol homeostasis. For example, cholesterol enters the livers cholesterol pool from a number of sources including dietary cholesterol, as well as cholesterol synthesized de novo by extrahepatic tissues and by the liver itself. Cholesterol is eliminated from the liver as unmodified cholesterol in the bile, or it can be converted to bile salts that are secreted into the intestinal lumen. It can also serve as a component of plasma lipoproteins sent to the peripheral tissues. In humans, the balance between cholesterol influx and efflux is not precise, resulting in a gradual deposition of cholesterol in the tissues, particularly in the endothelial linings of blood vessels. This is a potentially life-threatening occurrence when the lipid deposition leads to plaque formation, causing the narrowing of blood vessels (atherosclerosis) and increased risk of cardio-, cerebroand peripheral vascular disease. Figure 18.1 summarizes the major sources of liver cholesterol and the routes by which cholesterol leaves the liver.

Cholesterol is a very hydrophobic compound. It consists of four fused hydrocarbon rings (A-D) called the "steroid nucleus"), and it has an eight-carbon, branched hydrocarbon chain attached to carbon 17 of the D ring. Ring A has a hydroxyl group at carbon 3, and ring B has a double bond between carbon 5 and carbon 6 (Figure 18.2).

Steroids with eight to ten carbon atoms in the side chain at carbon 17 and a hydroxyl group at carbon 3 are classified as sterols.

Sources of liver cholesterol (influx) and routes by which cholesterol leaves the liver (efflux).

Synthesis of HMG CoA.
Cholesterol is the major sterol in animal tissues. [Note: Plant sterols, such as B-sitosterol are poorly absorbed by humans. After entering the enterocytes, they are actively transported back into the intestinal lumen. Because some cholesterol is transported as well, plant sterols appear to reduce the absorption of dietary cholesterol. This has led to clinically useful dietary treatment of hypercholesteremia. Daily ingestion of plant steroid esters (in the form of commercially available trans fatty acid-free margarine) is one of a number of dietary strategies leading to the reduction of plasma cholesterol levels (see p. 363).]

Most plasma cholesterol is in an esterified form (with a fatty acid attached at carbon 3, see Figure 18.2), which makes the structure even more hydrophobic than free (unesterified) cholesterol. Cholesteryl esters are not found in membranes, and are normally present only in low levels in most cells. Because of their hydrophobicity, cholesterol and its esters must be transported in association with protein as a component of a lipoprotein particle (see p. 227) or be solubilized by phospholipids and bile salts in the bile (see p. 226).

Cholesterol is synthesized by virtually all tissues in humans, although liver, intestine, adrenal cortex, and reproductive tissues, including ovaries, testes, and placenta, make the largest contributions to the body's cholesterol pool. As with fatty acids, all the carbon atoms in cholesterol are provided by acetate, and NADPH provides the reducing equivalents. The pathway is endergonic, being driven by hydrolysis of the high-energy thioester bond of acetyl coenzyme A (CoA) and the terminal phosphate bond of adenosine triphosphate (ATP). Synthesis requires enzymes in both the cytosol and the membrane of the smooth endoplasmic reticulum (ER). The pathway is responsive to changes in cholesterol concentration, and regulatory mechanisms exist to balance the rate of cholesterol synthesis within the body against the rate of cholesterol excretion. An imbalance in this regulation can lead to an elevation in circulating levels of plasma cholesterol, with the potential for vascular disease.

The first two reactions in the cholesterol synthetic pathway are similar to those in the pathway that produces ketone bodies (see Figure 16.22, p. 196). They result in the production of HMG CoA (Figure 18.3). First, two acetyl CoA molecules condense to form acetoacetyl CoA. Next, a third molecule of acetyl CoA is added, producing HMG CoA, a six-carbon compound. [Note: Liver parenchymal cells contain two isoenzymes of HMG CoA synthase. The cytosolic enzyme participates in cholesterol synthesis, whereas the mitochondrial enzyme functions in the pathway for ketone body synthesis.]

The next step, the reduction of HMG CoA to mevalonate, is catalyzed by HMG CoA reductase, and is the rate-limiting and key regulated step in cholesterol synthesis. It occurs in the cytosol, uses two molecules of NADPH as the reducing agent, and releases CoA, making the reaction irreversible (Figure 18.4). [Note: HMG CoA reductase is an intrinsic membrane protein of the ER, with the enzyme's catalytic domain projecting into the cytosol. Regulation of HMG CoA reductase activity is discussed below.]

The reactions and enzymes involved in the synthesis of cholesterol from mevalonate are illustrated in Figure 18.5. [Note: The numbers shown in brackets below correspond to numbered reactions shown in this figure.]
[1] Mevalonate is converted to 5-pyrophosphomevalonate in two steps, each of which transfers a phosphate group from ATP.
[2] A five-carbon isoprene unit—isopentenyl pyrophosphate (IPP)— is formed by the decarboxylation of 5-pyrophosphomevalonate. The reaction requires ATP. [Note: IPP is the precursor of a family of molecules with diverse functions, the isoprenoids. Cholesterol is a sterol isoprenoid. Nonsterol isoprenoids include dolichol (see p. 167) and ubiquinone (Coenzyme Q) (see p. 75).]
[3] IPP is isomerized to 3,3-dimethylallyl pyrophosphate (DPP).

[4] IPP and DPP condense to form ten-carbon geranyl pyrophosphate (GPP).
[5] A second molecule of IPP then condenses with GPP to form 15carbon farnesyl pyrophosphate (FPP). [Note: Covalent attachment of farnesyl to proteins, a process known as "prenylation," is one mechanism for anchoring proteins to plasma membranes.]
[6] Two molecules of FPP combine, releasing pyrophosphate, and are reduced, forming the 30-carbon compound squalene. [Note: Squalene is formed from six isoprenoid units. Because three ATP are hydrolyzed per mevalonate residue converted to IPP, a total of 18 ATP are required to make the polyisoprenoid squalene.]
[7] Squalene is converted to the sterol lanosterol by a sequence of reactions catalyzed by ER-associated enzymes that use molecular oxygen and NADPH. The hydroxylation of squalene triggers the cyclization of the structure to lanosterol.
[8] The conversion of lanosterol to cholesterol is a multistep process, resulting in the shortening of the carbon chain from 30 to 27 carbons, removal of the two methyl groups at carbon 4, migration of the double bond from carbon 8 to carbon 5, and reduction of the double bond between carbon 24 and carbon 25. [Note: This ER-associated pathway includes several different enzymatic reactions. Smith-Lemli-Opitz syndrome (SLOS), a relatively common autosomal recessive disorder of cholesterol biosynthesis, is caused by a partial deficiency in 7-dehydrocholesterol-7-reductase—an enzyme involved in the migration of the double bond. SLOS is one of several multisystem, embryonic malformation syndromes associated with impaired cholesterol synthesis. ]

HMG CoA reductase, the rate-limiting enzyme, is the major control point for cholesterol biosynthesis, and is subject to different kinds of metabolic control.
1. Sterol-dependent regulation of gene expression: Expression of the gene for HMG CoA reductase is controlled by the transcription factor, SREBP-2 (sterol regulatory element-binding protein-2) that binds DNA at the cis-acting sterol regulatory element (SRE) of the reductase gene. SREBP is an integral protein of the ER membrane, and associates with a second ER membrane protein, SCAP (SREBP cleavage—activating protein). When sterol levels in
the cell are low, the SREBP-SCAP complex is sent out of the ER to the Golgi. In the Golgi, SREBP is sequentially acted upon by two proteases, which generate a soluble fragment that enters the nucleus, binds the SRE, and functions as a transcription factor. This results in increased synthesis of HMG CoA reductase and, therefore, increased cholesterol synthesis (Figure 18.6). If sterols are abundant, however, they bind SCAP at its sterol-sensing domain and induce the binding of SCAP to yet other ER membrane proteins (insigs). This results in the retention of the SCAPSREBP complex in the ER, thus preventing the activation of SREBP, and leading to down-regulation of cholesterol synthesis.

2. Sterol-accelerated enzyme degradation: The reductase itself is a sterol-sensing integral protein of the ER membrane. When sterol levels in the cell are high, the reductase binds to insig proteins. Binding leads to ubiquitination and proteasomal degradation of the reductase (see p. 247).
3. Sterol-independent phosphorylation/dephosphorylation: HMG CoA reductase activity is controlled covalently through the actions of adenosine monophosphate (AMP)—activated protein kinase (AMPK, see p. 183) and a phosphoprotein phosphatase (see Figure 18.6). The phosphorylated form of the enzyme is inactive, whereas the dephosphorylated form is active. [Note: AMPK is activated by AMP, so cholesterol synthesis, like fatty acid synthesis, is decreased when ATP availability is decreased.]
4. Hormonal regulation: The amount (and, therefore, the activity) of HMG CoA reductase is controlled hormonally. An increase in insulin and thyroxine favors up-regulation of the expression of the gene for HMG CoA reductase. Glucagon and the glucocorticoids have the opposite effect.

Regulation of HUG CoA reductase. SRE = sterol regulatory element; SREBP = sterol regulatory elementbinding protein; SCAP = SREBP cleavage—activating protein.

Structural similarity of HMG and pravastatin, a clinically useful cholesterol-lowering drug of the "statin" family.
5. Inhibition by drugs: The statin drugs (atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, and simvastatin) are structural analogs of HMG CoA, and are (or are metabolized to) reversible, competitive inhibitors of HMG CoA reductase (Figure 18.7). They are used to decrease plasma cholesterol levels in patients with hypercholesterolemia.'

The ring structure of cholesterol cannot be metabolized to COz and H20 in humans. Rather, the intact sterol nucleus is eliminated from the body by conversion to bile acids and bile salts, which are excreted in the feces, and by secretion of cholesterol into the bile, which transports it to the intestine for elimination. Some of the cholesterol in the intestine is modified by bacteria before excretion. The primary compounds made are the isomers coprostanol and cholestanol, which are reduced derivatives of cholesterol. Together with cholesterol, these compounds make up the bulk of neutral fecal sterols.

Bile consists of a watery mixture of organic and inorganic compounds. Phosphatidylcholine (lecithin, see p. 202) and bile salts (conjugated bile acids) are quantitatively the most important organic components of bile. Bile can either pass directly from the liver where it is synthesized into the duodenum through the common bile duct, or be stored in the gallbladder when not immediately needed for digestion.

The bile acids contain 24 carbons, with two or three hydroxyl groups and a side chain that terminates in a carboxyl group. The carboxyl group has a pKa of about six and, therefore, is not fully ionized at physiologic pH—hence, the term "bile acid." The bile acids are amphipathic in that the hydroxyl groups are a in orientation (they lie "pelow' the plane of the rings) and the methyl groups are f (they lie "above" the plane of the rings). Therefore, the molecules have both a polar and a nonpolar face, and can act as emulsifying agents in the intestine, helping prepare dietary triacylglycerol and other complex lipids for degradation by pancreatic digestive enzymes.

Bile acids are synthesized in the liver by a multistep, multiorganelle pathway in which hydroxyl groups are inserted at specific positions on the steroid structure, the double bond of the cholesterol B ring is reduced, and the hydrocarbon chain is shortened by three carbons, introducing a carboxyl group at the end of the chain. The most common resulting compounds, cholic acid (a triol) and chenodeoxycholic acid (a diol, Figure 18.8), are called "primary" bile acids. [Note: The rate-limiting step in bile acid synthesis is the introduction of a hydroxyl group at carbon 7 of the steroid nucleus by cholesterol-7-a-hydroxylase, an ER-associated cytochrome P450 (CYP) enzyme found only in liver. The enzyme is down-regulated by cholic acid (Figure 18.9)]

Before the bile acids leave the liver, they are conjugated to a molecule of either glycine or taurine (an endproduct of cysteine metabolism) by an amide bond between the carboxyl group of the bile acid and the amino group of the added compound. These new structures include glycocholic and glycochenodeoxycholic acids, and taurocholic and taurochenodeoxycholic acids (Figure 18.10). The ratio of glycine to taurine forms in the bile is approximately 3:1. Addition of glycine or taurine results in the presence of a carboxyl group with a lower pKa (from glycine) or a sulfonate group (from taurine), both of which are fully ionized (negatively charged) at physiologic pH; thus, the conjugated forms are called bile salts. Bile salts are more effective detergents than bile acids because of their enhanced amphipathic nature. Therefore, only the conjugated forms—that is, the bile salts—are found in the bile. Individuals with genetic deficiencies in the conversion of cholesterol to bile acids are treated with exogenously supplied chenodeoxycholic acid.
Bile salts provide the only significant mechanism for cholesterol excretion, both as a metabolic product of cholesterol and as a solubilizer of cholesterol in bile.

Bacteria in the intestine can remove glycine and taurine from bile salts, regenerating bile acids. They can also convert some of the primary bile acids into "secondary" bile acids by removing a hydroxyl group, producing deoxycholic acid from cholic acid and lithocholic acid from chenodeoxycholic acid (Figure 18.11).

Bile salts secreted into the intestine are efficiently reabsorbed (greater than 95%) and reused. The liver converts both primary and secondary bile acids into bile salts by conjugation with glycine or taurine, and secretes them into the bile. The mixture of bile acids and bile salts is absorbed primarily in the ileum via a Na*-bile salt cotransporter. They are actively transported out of the ileal mucosal cells into the portal blood, and are efficiently taken up by the hepatocytes via an isoform of the cotransporter. [Note: Bile acids are hydrophobic and require a carrier in the portal blood. Albumin carries them in a noncovalent complex, just as it transports fatty acids in blood (see p. 181).] The continuous process of secretion of bile salts into the bile, their passage through the duodenum where some are converted to bile acids, their uptake in the ileum, and subsequent return to the liver as a mixture of bile acids and salts is termed the enterohepatic circulation (see Figure 18.11). Between 15 and 30 g of bile salts are secreted from the liver into the duodenum each day, yet only about 0.5 g (less than 3%) is lost daily in the feces. Approximately 0.5 g/day is synthesized from cholesterol in the liver to replace the lost bile acids. Bile acid sequestrants, such as cholestyramine, bind bile acids in the gut, prevent their reabsorption,

Enterohepatic circulation of bile salts and bile acids.
and so promote their excretion. They are used in the treatment of hypercholesterolemia because the removal of bile acids relieves the inhibition on bile acid synthesis in the liver, thereby diverting additional cholesterol into that pathway. [Note: Dietary fiber also binds bile acids and increases their excretion (see p. 366).]
——

The movement of cholesterol from the liver into the bile must be accompanied by the simultaneous secretion of phospholipid and bile salts. If this dual process is disrupted and more cholesterol enters the bile than can be solubilized by the bile salts and phosphatidyl choline present, the cholesterol may precipitate in the gallbladder, leading to cholesterol gallstone disease—cholelithiasis (Figure 18.12). This disorder is typically caused by a decrease of bile acids in the bile, which may result from: 1) gross malabsorption of bile acids from the intestine, as seen in patients with severe ileal disease; 2) obstruction of the biliary tract, interrupting the enterohepatic circulation; 3) severe hepatic dysfunction, leading to decreased synthesis of bile salts, or other abnormalities in bile production; or 4) excessive feedback suppression of bile acid synthesis as a result of an accelerated rate of recycling of bile acids. Cholelithiasis also may result from increased biliary cholesterol excretion, as seen with the use of fibrates. [Note: Fibrates, such as gemfibrozil, are derivatives of fibric acid. They are used to reduce triacylglycerol levels in blood through up-regulation of fatty acid B-oxidation.] Laparoscopic cholecystectomy (surgical removal of the gallbladder through a small incision) is currently the treatment of choice. However, for patients who are unable to undergo surgery, oral administration of chenodeoxycholic acid to supplement the body's supply of bile acids results in a gradual (months to years) dissolution of the gallstones.

The plasma lipoproteins are spherical macromolecular complexes of lipids and specific proteins (apolipoproteins or apoproteins). The lipoprotein particles include chylomicrons (CM), very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). They differ in lipid and protein composition, size, density (Figure 18.13), and site of origin. Lipoproteins function both to keep their component lipids soluble as they transport them in the plasma and to provide an efficient mechanism for transporting their lipid contents to (and from) the tissues. In humans, the transport system is less perfect than in other animals and, as a result, humans experience a gradual deposition of lipid—especially cholesterol—in tissues. This is a potentially life-threatening occurrence when the lipid deposition contributes to plaque formation, causing the narrowing of blood vessels (atherosclerosis).

Lipoproteins are composed of a neutral lipid core (containing triacylglycerol and cholesteryl esters) surrounded by a shell of amphipathic apolipoproteins, phospholipid, and nonesterified (free) cholesterol (Figure 18.14). These amphipathic compounds are oriented so that their polar portions are exposed on the surface of the lipoprotein, thus making the particle soluble in aqueous solution. The triacylglycerol and cholesterol carried by the lipoproteins are obtained either from the diet (exogenous source) or from de novo synthesis (endogenous source). [Note: Lipoprotein particles constantly interchange lipids and apolipoproteins with each other; therefore, the actual apolipoprotein and lipid content of each class of particles can be somewhat variable.]
- 1. Size and density of lipoprotein particles: Chylomicrons are the lipoprotein particles lowest in density and largest in size, and contain the highest percentage of lipid and the lowest percentage of protein. VLDLs and LDLs are successively denser, having higher ratios of protein to lipid. HDL particles are the densest. Plasma lipoproteins can be separated on the basis of their electrophoretic mobility, as shown in Figure 18.15, or on the basis of their density by ultracentrifugation.
2. Apolipoproteins: The apolipoproteins associated with lipoprotein particles have a number of diverse functions, such as providing recognition sites for cell-surface receptors, and serving as activators or coenzymes for enzymes involved in lipoprotein metabolism. Some of the apolipoproteins are required as essential structural components of the particles and cannot be removed (in fact, the particles cannot be produced without them), whereas others are transferred freely between lipoproteins. Apolipoproteins are divided by structure and function into five major classes, A through E, with most classes having subclasses, for example, apolipoprotein (or apo) A-I| and apo C-Il. [Note: Functions of all of the apolipoproteins are not yet known.]

Approximate size and density of serum lipoproteins. Each family of lipoproteins exhibits a range of sizes and densities; this figure shows typical values. The width of the rings approximates the amount of each component. [Note: Although cholesterol and its esters are shown as one component in the center of each particle, physically cholesterol is a surface component whereas cholesteryl esters are located in the interior of the lipoproteins.]

Electrophoretic mobility of plasma lipoproteins. The order of LDL and VLDL is reversed if ultracentrifugation is used as the separation technique.

Chylomicrons are assembled in intestinal mucosal cells and carry dietary triacylglycerol, cholesterol, fat-soluble vitamins, and cholesteryl esters (plus additional lipids made in these cells) to the peripheral tissues (Figure 18.16). [Note: TAGs account for close to 90% of the lipids in a chylomicron.]
1. Synthesis of apolipoproteins: Apolipoprotein B-48 is unique to chylomicrons. Its synthesis begins on the rough ER; it is glycosylated as it moves through the RER and Golgi. [Note: Apo B-48 is so named because it constitutes the N-terminal, 48% of the protein coded for by the gene for apo B. Apo B-100, which is synthesized by the liver and found in VLDL and LDL, represents the entire protein coded for by the apo B gene. Posttranscriptional editing (see p. 457) of a cytosine to a uracil in intestinal apo B-100 mRNA creates a nonsense codon (see p. 433), allowing translation of only 48% of the mRNA.]
2. Assembly of chylomicrons: The enzymes involved in triacylglycerol, cholesterol, and phospholipid synthesis are located in the smooth ER. Assembly of the apolipoproteins and lipid into chylomicrons requires microsomal triacylglycerol transfer protein (MTP, see p. 178), which loads apo B-48 with lipid. This occurs before transition from the ER to the Golgi, where the particles are packaged in secretory vesicles. These fuse with the plasma membrane releasing the lipoproteins, which then enter the lymphatic system and, ultimately, the blood.

Modification of nascent chylomicron particles: The particle released by the intestinal mucosal cell is called a "nascent" chylomicron because it is functionally incomplete. When it reaches the plasma, the particle is rapidly modified, receiving apolipoprotein E (which is recognized by hepatic receptors) and C. The latter includes apo C-II, which is necessary for the activation of lipoprotein lipase, the enzyme that degrades the triacylglycerol contained in the chylomicron (see below). The source of these apolipoproteins is circulating HDL (see Figure 18.16).

Degradation of triacylglycerol by lipoprotein lipase: Lipoprotein lipase is an extracellular enzyme that is anchored by heparan sulfate to the capillary walls of most tissues, but predominantly those of adipose tissue and cardiac and skeletal muscle. Adult liver does not have this enzyme. [Note: A hepatic lipase is found on the surface of endothelial cells of the liver. It plays some role in triacylglycerol degradation in CM and VLDL, and is particularly important in HDL metabolism (see p. 236).] Lipoprotein lipase, activated by apo C-Il on circulating lipoprotein particles, hydrolyzes the triacylglycerol contained in these particles to yield fatty acids and glycerol. The fatty acids are stored (by the adipose) or used for energy (by the muscle). If they are not immediately taken up by a cell, the long-chain fatty acids are transported by serum albumin until their uptake does occur. Glycerol is used by the liver, for example, in lipid synthesis, glycolysis, or gluconeogenesis. [Note: Patients with a deficiency of lipoprotein lipase or

Metabolism of chylomicrons. CM = chylomicron; TAG = triacylglycerol; C = cholesterol; CE = cholesteryl esters. Apo B-48, apo C-II, and apo E are apolipoproteins found as specific components of plasma lipoproteins. The lipoproteins are not drawn to scale (see Figure 18.13 for details of the size and density of lipoproteins).
apo C-Il (Type 1 hyperlipoproteinemia, or familial /ipoprotein lipase deficiency) show a dramatic accumulation—1000 mg/dl or greater—of chylomicron-triacylglycerol in the plasma (hypertriacylglycerolemia) even in the fasted state.]

synthesis and transfer to the luminal surface of the capillary is stimulated by insulin (signifying a fed state, see p. 321). Furthermore, isomers of lipoprotein lipase have different Km values for triacylglycerol (reminiscent of the hexokinase/glucokinase story, see p. 98). For example, the adipose enzyme has a large Kp (see p. 59), allowing the removal of fatty acids from circulating lipoprotein particles and their storage as triacylglycerols only when plasma lipoprotein concentrations are elevated. Conversely, heart muscle lipoprotein lipase has a small Km, allowing the heart continuing access to the circulating fuel, even when plasma lipoprotein con-
6.
centrations are low. [Note: The highest concentration of /ipoprotein lipase is in cardiac muscle, reflecting the use of fatty acids to provide much of the energy needed for cardiac function.]
Formation of chylomicron remnants: As the chylomicron circulates and more than 90% of the triacylglycerol in its core is degraded by lipoprotein lipase, the particle decreases in size and increases in density. In addition, the C apoproteins (but not apo E) are returned to HDL. The remaining particle, called a "remnant," is rapidly removed from the circulation by the liver, whose cell membranes contain lipoprotein receptors that recognize apo E (see Figure 18.16). Chylomicron remnants bind to these receptors and are taken into the hepatocytes by endocytosis. The endocytosed vesicle then fuses with a lysosome, and the apolipoproteins, cholesteryl esters, and other components of the remnant are hydrolytically degraded, releasing amino acids, free cholesterol, and fatty acids. The receptor is recycled. (A more detailed discussion of the mechanism of receptor-mediated endocytosis is illus- trated for LDL in Figure 18.20.)

Metabolism of VLDL and LDL. TAG = triacylglycerol; VLDL = very-low-density lipoprotein; = low-density-lipoprotein; IDL = intermediate-density lipoprotein; C = cholesterol; CE = cholesteryl esters. Apo B-100, apo C-Il, and apo E are apolipoproteins found as specific components of plasma lipoproteins. Lipoproteins are not drawn to scale (see Figure 18.13 for details of the size and density of lipoproteins).

VLDLs are produced in the liver (Figure 18.17). They are composed predominantly of endogenous triacylglycerol (approximately 60%), and their function is to carry this lipid from the liver (site of synthesis) to the peripheral tissues. There, the triacylglycerol is degraded by /ipoprotein lipase, as discussed for chylomicrons (see p. 228). [Note: "Fatty liver' (hepatic steatosis) occurs in conditions in which there is an imbalance between hepatic triacylglycerol synthesis and the secretion of VLDL. Such conditions include obesity, uncontrolled diabetes mellitus, and chronic ethanol ingestion.]
1. Release of VLDL: VLDL are secreted directly into the blood by the liver as nascent VLDL particles containing apo B-100. They must obtain apo C-Il and apo E from circulating HDL (see Figure 18.17). As with chylomicrons, apo C-Il is required for activation of lipoprotein lipase. [Note: Abetalipoproteinemia is a rare hypolipoproteinemia caused by a defect in microsomal triacylglycerol transfer protein (MTP), leading to an inability to load apo B with lipid. AS a consequence, no VLDL or chylomicrons are formed, and triacylglycerols accumulate in the liver and intestine.]
2. Modification of circulating VLDL: As VLDL pass through the circulation, triacylglycerol is degraded by lipoprotein lipase, causing the VLDL to decrease in size and become denser. Surface components, including the C and E apoproteins, are returned to HDL, but the particles retain apo B-100. Finally, some triacylglycerols are transferred from VLDL to HDL in an exchange reaction that concomitantly transfers some cholesteryl esters from HDL to VLDL. This exchange is accomplished by cholesteryl ester transfer protein (CETP, Figure 18.18).

3. Production of LDL from VLDL in the plasma: With these modifications, the VLDL is converted in the plasma to LDL. Intermediatesized particles, the intermediate-density lipoproteins (IDL) or VLDL remnants, are observed during this transition. IDLs can also be taken up by cells through receptor-mediated endocytosis that uses apo E as the ligand. [Note: Apo E is normally present in three isoforms, E-2, E-3, and E-4. Apo E-2 binds poorly to receptors, and patients who are homozygotic for apo E-2 are deficient in the clearance of chylomicron remnants and IDL. These individuals have familial Type III hyperlipoproteinemia (familial dysbetalipoproteinemia, or broad beta disease), with hypercholesterolemia and premature atherosclerosis. Not yet understood is the fact that the E-4 isoform confers increased susceptibility to and decreased age of onset of late-onset Alzheimer disease, doubling the lifetime risk.]

LDL particles contain much less triacylglycerol than their VLDL predecessors, and have a high concentration of cholesterol and cholesteryl esters (Figure 18.19).
- 1. Receptor-mediated endocytosis: The primary function of LDL particles is to provide cholesterol to the peripheral tissues (or return it to the liver). They do so by binding to cell surface mem-

Transfer of cholesteryl esters (CE) from HDL to VLDL in exchange for triacylglycerol (TAG).

Composition of the plasma lipoproteins. Note the high concentration of cholesterol and cholesteryl esters in LDL.
brane LDL receptors that recognize apo B-100 (but not apo B-48). Because these LDL receptors can also bind apo E, they are known as apo B-100/apo E receptors. A summary of the uptake and degradation of LDL particles is presented in Figure 18.20. [Note: The numbers in brackets below refer to corresponding numbers on that figure.] A similar mechanism of receptor-mediated endocytosis is used for the cellular uptake and degradation of chylomicron remnants and IDLs by the liver.

[1] LDL receptors are negatively charged glycoproteins that are clustered in pits on cell membranes. The cytosolic side of the pit is coated with the protein clathrin, which stabilizes the shape of the pit.
[2] After binding, the LDL-receptor complex is internalized by endocytosis. [Note: A deficiency of functional LDL receptors causes a significant elevation in plasma LDL and, therefore, of plasma cholesterol. Patients with such deficiencies have Type II hyperlipidemia (familial hypercholesterolemia, FH) and premature atherosclerosis. FH can also be caused by increased activity of a protease that degrades the receptor and by defects in apo B-100 that reduce its binding to the receptor.]
[3] The vesicle containing LDL loses its clathrin coat and fuses with other similar vesicles, forming larger vesicles called endosomes.
[4] The pH of the endosome falls (due to the proton-pumping activity of endosomal ATPase), which allows separation of the LDL from its receptor. The receptors then migrate to one side of the endosome, whereas the LDLs stay free within the lumen of the vesicle. [Note: This structure is called CURL—the Compartment for Uncoupling of Receptor and Ligand.]
[5] The receptors can be recycled, whereas the lipoprotein remnants in the vesicle are transferred to lysosomes and degraded by lysosomal acid hydrolases, releasing free cholesterol, amino acids, fatty acids, and phospholipids. These compounds can be reutilized by the cell. [Note: Storage diseases caused by rare autosomal recessive deficiencies in the ability to hydrolyze lysosomal cholesteryl esters (Wolman disease), or to transport unesterified cholesterol out of the lysosome (Niemann-Pick disease, Type C) have been identified.]
2.

Effect of endocytosed cholesterol on cellular cholesterol homeostasis: The chylomicron remnant-, IDL-, and LDL-derived cholesterol affects cellular cholesterol content in several ways (see Figure 18.20). First, HMG CoA reductase is inhibited by high cholesterol, as a result of which, de novo cholesterol synthesis decreases. Second, synthesis of new LDL receptor protein is reduced by decreasing the expression of the LDL receptor gene, thus limiting further entry of LDL cholesterol into cells. [Note: Regulation of the LDL receptor gene involves a SRE and a SREBP (SREBP-2), as was seen in the regulation of the gene for HMG CoA reductase (see p. 222).] Third, if the cholesterol is not required immediately for some structural or synthetic purpose, it is esterified by acyl CoA:cholesterol acyltransferase (ACAT). ACAT

Cellular uptake and degradation of LDL. ACAT = acy! CoA:cholesterol acyltransferase.

Synthesis of intracellular cholesteryl ester by ACAT.
transfers a fatty acid from a fatty acyl CoA derivative to cholesterol, producing a cholesteryl ester that can be stored in the cell (Figure 18.21). The activity of ACAT is enhanced in the presence of increased intracellular cholesterol.
. Uptake of chemically modified LDL by macrophage scavenger receptors: In addition to the highly specific and regulated receptor-mediated pathway for LDL uptake described above, macrophages possess high levels of scavenger receptor activity. These receptors, known as scavenger receptor class A (SR-A), can bind a broad range of ligands, and mediate the endocytosis of chemically modified LDL in which the lipid components or apo B have been oxidized. Unlike the LDL receptor, the scavenger receptor is not down-regulated in response to increased intracellular cholesterol. Cholesteryl esters accumulate in macrophages and cause their transformation into "foam" cells, which participate in the formation of atherosclerotic plaque (Figure 18.22).

HDL comprise a heterogeneous family of lipoproteins with a complex metabolism that is not yet completely understood. HDL particles are formed in blood by the addition of lipid to apo A-1, an apolipoprotein made by the liver and intestine and secreted into blood. Apo A-1 accounts for about 70% of the apoproteins in HDL. HDL perform a number of important functions, including the following:
1. HDL is a reservoir of apolipoproteins: HDL particles serve as a circulating reservoir of apo C-Il (the apolipoprotein that is transferred to VLDL and chylomicrons, and is an activator of lipoprotein lipase), and apo E (the apolipoprotein required for the receptormediated endocytosis of IDLs and chylomicron remnants).
. HDL uptake of unesterified cholesterol: Nascent HDL are diskshaped particles containing primarily phospholipid (largely phosphatidylcholine) and apolipoproteins A, C, and E. They take up cholesterol from non-hepatic (peripheral) tissues and return it to the liver as cholesteryl esters (Figure 18.23). [Note: HDL particles are excellent acceptors of unesterified cholesterol as a result of their high concentration of phospholipids, which are important solubilizers of cholesterol.]
. Esterification of cholesterol: When cholesterol is taken up by HDL, it is immediately esterified by the plasma enzyme lecithin:cholesterol acyltransferase (LCAT, also known as PCAT, in which "P" stands for phosphatidylcholine). This enzyme is synthesized by the liver. LCAT binds to nascent HDL, and is activated by apo A-l. LCAT transfers the fatty acid from carbon 2 of phosphatidylcholine to cholesterol. This produces a hydrophobic cholesteryl ester, which is sequestered in the core of the HDL, and lysophosphatidylcholine, which binds to albumin. [Note: Esterification maintains the cholesterol concentration gradient, allowing continued efflux of cholesterol to HDL.] As the discoidal nascent HDL accumulates cholesteryl esters, it first becomes a

Role of oxidized lipoproteins in plaque formation in arterial wall.
spherical, relatively cholesteryl ester—-poor HDL3 and, eventually, a cholesteryl ester—rich HDL2 particle that carries these esters to the liver. Cholesterol ester transfer protein (CETP, see p. 231) moves some of the cholesteryl esters from HDL to VLDL in exchange for triacylglycerol, relieving product inhibition of LCAT. Because VLDL are catabolized to LDL, the cholesteryl esters are ultimately taken up by the liver.
4. Reverse cholesterol transport: The selective transfer of cholesterol from peripheral cells to HDL, and from HDL to the liver for bile acid synthesis or disposal via the bile, and to steroidogenic cells for hormone synthesis, is a key component of cholesterol homeostasis. This is, in part, the basis for the inverse relationship seen

Metabolism of HDL. PC = phosphatidylcholine; lyso-PC = lysophosphatidylcholine. LCAT = Lecithin cholesterol transferase. CETP = cholesteryl ester transfer protein. ABCA1 = transport protein. [Note: For convenience the size of VLDLs are shown smaller than HDL, whereas VLDLs are larger than HDL.]
between plasma HDL concentration and atherosclerosis, and for HDL designation as the "good" cholesterol carrier. Reverse cholesterol transport involves efflux of cholesterol from peripheral cells to HDL, esterification of cholesterol by LCAT, binding of the cholesteryl ester—rich HDL (HDL2) to liver and steroidogenic cells, the selective transfer of the cholesteryl esters into these cells, and the release of lipid-depleted HDL (HDL3). The efflux of cholesterol from peripheral cells is mediated, at least in part, by the transport protein, ABCA1. [Note: Tangier disease is a very rare deficiency of ABCA\1, and is characterized by the virtual absence of HDL particles due to degradation of lipid-poor apo A-1.] The uptake of cholesteryl esters by the liver is mediated by a cell-surface receptor, SR-B1 (scavenger receptor class B type 1) that binds HDL (see p, 234 for SR-A). It is not yet clear as to whether the HDL particle itself is taken up, the cholesteryl esters extracted, and the lipid-poor HDL released back into the blood, or if there is selective uptake of the cholesteryl ester alone. [Note: Hepatic lipase, with its ability to degrade both TAG and phospholipids, also participates in the conversion of HDL2 to HDL3.]
ABCA1 is an ATP-binding cassette (ABC) protein. ABC proteins use energy from ATP hydrolysis to transport materials, including lipids, in and out of cells and across intracellular compartments. In addition to Tangier disease, defects in specific ABC proteins result in X-linked adrenoleukodystrophy, respiratory distress syndrome due to decreased surfactant secretion, and cystic fibrosis.
|

Lipoprotein (a), or Lp(a), is a particle that, when present in large quantities in the plasma, is associated with an increased risk of coronary heart disease. Lp(a) is nearly identical in structure to an LDL particle. Its distinguishing feature is the presence of an additional apolipoprotein molecule, apo(a), that is covalently linked at a single site to apo B-100. Circulating levels of Lp(a) are determined primarily by genetics. However, factors such as diet may play some role, as trans fatty acids have been shown to increase Lp(a), and estrogen decreases both LDL and Lp(a). [Note: Apo(a) is structurally homologous to plasminogen—the precursor of a blood protease whose target is fibrin, the main protein component of blood clots. It is hypothesized that elevated Lp(a) slows the breakdown of blood clots that trigger heart attacks because it competes with plasminogen for binding to fibrin. Niacin reduces Lp(a) and raises HDL.]

Cholesterol is the precursor of all classes of steroid hormones: glucocorticoids (for example, cortisol), mineralocorticoids (for example, aldosterone), and sex hormones—androgens, estrogens, and progestins (Figure 18.24). [Note: Glucocorticoids and mineralocorticoids are collectively called corticosteroids.] Synthesis and secretion occur in the adrenal cortex (cortisol, aldosterone, and androgens), ovaries and placenta (estrogens and progestins), and testes (testosterone). Steroid hormones are transported by the blood from their sites of synthesis to their target organs. Because of their hydrophobicity, they must be complexed with a plasma protein. Plasma albumin can act as a nonspecific carrier, and does carry aldosterone. However, specific steroid-carrier plasma proteins bind the steroid hormones more tightly than does albumin, for example, corticosteroid-binding globulin (transcortin) is responsible for transporting cortisol. A number of genetic diseases are caused by deficiencies in specific steps in the biosynthesis of steroid hormones. Some representative diseases are described in Figure 18.25.

Synthesis involves shortening the hydrocarbon chain of cholesterol, and hydroxylation of the steroid nucleus. The initial and rate-limiting reaction converts cholesterol to the 21-carbon pregnenolone. It is catalyzed by the cholesterol side-chain cleavage enzyme complex (desmolase, P450sec)— a cytochrome P450 (CYP) mixed function oxidase of the inner mitochondrial membrane (see p. 149). NADPH and molecular oxygen are required for the reaction. The cholesterol substrate can be newly synthesized, taken up from lipoproteins, or released from cholesteryl esters stored in the cytosol of steroidogenic tissues. An important control point is the movement of cholesterol into mito-
Figure 18.24
Key steroid hormones.

Steroid hormone synthesis and associated diseases. [Note: 3-B-hydroxysteroid dehydrogenase, CYP17, and CYP11B2 are bifunctional enzymes. Synthesis of testosterone and the estrogens from cholesterol occurs outside of the adrenal primarily gland.]
chondria. This process is mediated by StAR (steroidogenic acute regulatory protein). Pregnenolone is the parent compound for all steroid hormones (see Figure 18.25). Pregnenolone is oxidized and then isomerized to progesterone which is further modified to the other steroid hormones by hydroxylation reactions that occur in the ER and mitochondria. Like desmolase, the enzymes primarily are CYP proteins. A defect in the activity or amount of an enzyme in this pathway can lead to a deficiency in the synthesis of hormones beyond the affected step, and to an excess in the hormones or metabolites before that step. Because all members of the pathway have potent biologic activity, serious metabolic imbalances occur if enzyme deficiencies are present (see Figure 18.25). Collectively these disorders are known as the congenital adrenal hyperplasias. [Note: Addison disease, due to autoimmune destruction of the adrenal cortex, is characterized by adrenocortical insufficiency. ]

Steroid hormones are secreted on demand from their tissues of origin in response to hormonal signals. The corticosteroids and androgens are made in different regions of the adrenal cortex, and are secreted into blood in response to different signals.
1.
Cortisol: Its production in the middle layer (zona fasciculata) of the adrenal cortex is controlled by the hypothalamus, to which the pituitary gland is attached (Figure 18.26). In response to severe stress (for example, infection), corticotropin-releasing hormone (CRH), produced by the hypothalamus, travels through capillaries to the anterior lobe of the pituitary, where it induces the production and secretion of adrenocorticotropic hormone (ACTH). The polypeptide ACTH, the "stress hormone," stimulates the adrenal cortex to synthesize and secrete the glucocorticoid cortisol. Cortisol allows the body to respond to stress through its effects on intermediary metabolism (for example, increased gluconeogenesis) and the inflammatory and immune responses. As cortisol levels rise, the release of CRH and ACTH is inhibited. [Note: ACTH binds to a membrane G-protein coupled receptor, resulting in cAMP production and activation of protein kinase A (see p. 94). PKA phosphorylates the esterase that converts cholesteryl ester to cholesterol and stimulates synthesis of StAR protein.]
2.

Aldosterone: Its production in the outer layer (zona glomerulosa) of the adrenal cortex is induced by a decrease in the plasma Na*/K* ratio, and by the hormone, angiotensin II. Angiotensin II (an octapeptide) is produced from angiotensin | (a decapeptide) by angiotensin-converting enzyme (ACE), an enzyme found predominantly in the lungs, but which is also distributed widely in the body. [Note: Angiotensin | is produced in the blood by cleavage of an inactive precursor, angiotensinogen, secreted by the liver. Cleavage is accomplished by the enzyme renin, made and secreted by the kidney.] Angiotensin II binds to cell-surface receptors. However, in contrast to ACTH, its effects are mediated through the phosphatidylinositol 4,5-bisphosphate (PIP2) pathway (see p. 205) and not by cAMP. Aldosterone's primary effect is on the kidney tubules, where it stimulates sodium uptake and potassium excretion (Figure 18.27). [Note: An effect of aldosterone is an increase in blood pressure. Competitive inhibitors of ACE are used to treat renin-dependent hypertension.]
3. Androgens: Both the inner (zona reticularis) and middle layers of the adrenal cortex produce androgens, primarily dehydroepiandrosterone and androstenedione. Although adrenal androgens themselves are weak, they are converted in peripheral tissues to testosterone—a stronger androgen—and to estrogens.
Estrogens are derived from androstenedione and testosterone by aromatase (CYP19). Aromatase inhibitors are used in the treatment of estrogen-responsive breast cancer in postmenopausal women.

©
Control menstrual cycle.
@ Promote development of female secondary sex characteristics.

© Secretory phase of uterus and mammary glands.
@ Implantation and maturation of fertilized ovum.
TESTES

Stimulates spermatogenesis.
Promotes
©
@
of male
secondary development
sex characteristics.
@ Promotes anabolism.
@ Masculinization of the fetus

The testes and ovaries synthesize hormones necessary for sexual differentiation and reproduction. A single hypothalamic-releasing factor, gonadotropin-releasing hormone, stimulates the anterior pituitary to release the glycoproteins, luteinizing hormone (LH) and folliclestimulating hormone (FSH). Like ACTH, LH and FSH bind to surface receptors and cause an increase in cAMP. LH stimulates the testes to produce testosterone and the ovaries to produce estrogens and progesterone (see Figure 18.27). FSH regulates the growth of ovarian follicles and stimulates testicular spermatogenesis.

Each steroid hormone diffuses across the plasma membrane of its target cell and binds to a specific cytosolic or nuclear receptor. These receptor—ligand complexes accumulate in the nucleus, dimerize, and bind to specific regulatory DNA sequences (hormoneresponse elements, HRE) in association with coactivator proteins, thereby causing promoter activation and increased transcription of targeted genes (Figure 18.28). An HRE is found in the promoter (or an enhancer element, see p. 424) for genes that respond to a specific steroid hormone, thus ensuring coordinated regulation of these genes. Hormone-receptor complexes can also inhibit transcription in association with corepressors. [Note: The binding of a hormone to its receptor causes a conformational change in the receptor that uncovers its DNA-binding domain, allowing the complex to interact through a zinc-finger motif with the appropriate sequence on the DNA. Receptors for the steroid hormones, plus those for thyroid hormone, retinoic acid (see p. 382), and 1,25-dihydroxycholecalciferol (Vitamin D, see p. 386), are members of a "superfamily" of structurally related gene regulators that function in a similar way.]

Steroid hormones are generally converted into inactive metabolic excretion products in the liver. Reactions include reduction of unsaturated bonds and the introduction of additional hydroxyl groups. The resulting structures are made more soluble by conjugation with glucuronic acid or sulfate (from 3'-phosphoadenosyl-5'-phosphosulfate, see p. 162). Approximately 20-30% of these metabolites are secreted into the bile and then excreted in the feces, whereas the remainder are released into the blood and filtered from the plasma in the kidney, passing into the urine. These conjugated metabolites are fairly water-soluble and do not need protein carriers.

Cholesterol is a hydrophobic compound, with a single hydroxyl group— located at carbon 3 of the A ring—to which a fatty acid can be attached, producing an even more hydrophobic cholesteryl ester. Cholesterol is synthesized by virtually all human tissues, although primarily by liver, intestine, adrenal cortex, and reproductive tissues (Figure 18.29). All the carbon atoms in cholesterol are provided by acetate, and NADPH provides the reducing equivalents. The pathway is driven by hydrolysis of the high-energy thioester bond of acetyl CoA and the terminal phos-
phate bond of ATP. Cholesterol is synthesized in the cytoplasm.The rate-limiting and regulated step in cholesterol synthesis is catalyzed by the SER—membrane protein, hydroxymethylglutaryl (HMG) CoA reductase, which produces mevalonate from HMG CoA. The enzyme is regulated by a number of mechanisms: 1) Expression of the gene for HMG CoA reductase is activated when cholesterol levels are low, via the transcription factor, SREBP-2, bound to a sterol response element (SRE), resulting in increased enzyme and, therefore, more cholesterol synthesis; 2) HMG CoA reductase activity is controlled by adenosine monophosphate (AMP)-activated protein kinase (AMPK, which phosphorylates and inactivates HMG CoA reductase) and an insulin-activated protein phosphatase (which activates HMG CoA reductase); 3) Expression of the gene for the reductase is upregulated by insulin and downregulated by glucagon. Statins are competitive inhibitors of HMG CoA reductase. These drugs are used to decrease plasma cholesterol in patients with hypercholesterolemia. The ring structure of cholesterol can not be degraded in humans.

Cholesterol can be eliminated from the body either by conversion to bile salts or by secretion into the bile. Bile salts and phosphatidylcholine are quantitatively the most important organic components of bile. Bile salts are conjugated bile acids produced by the liver and stored in the gallbladder. The primary bile acids, cholic or chenodeoxycholic acids, are amphipathic, and can serve as emulsifying agents. The rate-limiting step in bile acid synthesis is catalyzed by cholesterol-7-a-hydroxylase, which is inhibited by bile acids. Before the bile acids leave the liver, they are conjugated to a molecule of either glycine or taurine, producing the primary bile salts: glycocholic or taurocholic acid, and glycochenodeoxycholic or taurochenodeoxycholic acid. Bile salts are more amphipathic than bile acids and, therefore, are more effective emulsifiers. In the intestine, bacteria can remove the glycine and taurine, and can remove a hydroxyl group from the steroid nucleus, producing the secondary bile acids—deoxycholic and lithocholic acids. More than 95% of the bile acids and salts are efficiently reabsorbed from the intestine by a sodium-bile salt cotransporter. They are then actively transported out of the ileal mucosal cells into the portal blood, where they are carried by albumin back to the liver (enterohepatic circulation; bile acid sequestrants reduce this) and are taken up by the hepatic form of the cotransporter. In the liver, the primary and secondary bile acids are reconverted to bile salts, and secreted into the bile. If more cholesterol enters the bile than can be solubilized by the available bile salts and phosphatidylcholine, cholesterol gallstone disease (cholelithiasis) can occur.

The plasma lipoproteins include chylomicrons (CM), very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). They function to keep lipids (primarily triacylglycerol (TAG) and cholesteryl esters) soluble as they transport them between tissues. Lipoproteins are composed of a neutral lipid (TAG, cholesteryl esters, or both) surrounded by a shell of amphipathic apolipoproteins, phospholipid, and nonesterified cholesterol. CM are assembled in intestinal mucosal cells from dietary lipids (primarily, TAG). Each nascent CM particle has one molecule of apolipoprotein (apo) B-48. They are released from the cells into the lymphatic system

Activation of transcription by interaction of steroid hormone-receptor complex with hormone response element (HRE).

and travel to the blood, where they receive apo C-Il and apo E from HDLs. Apo C-II activates endothelial lipoprotein lipase, which degrades the TAG in CM to fatty acids and glycerol. The fatty acids that are released are stored (in the adipose) or used for energy (by the muscle). The glycerol is metabolized by the liver. Patients with a deficiency of lipoprotein lipase or apo C-Il show a dramatic accumulation of CM in the plasma (Type | hyperlipoproteinemia or familial lipoprotein lipase deficiency). After most of the TAG is removed, apo C-II is returned to the HDL, and the CM remnant—carrying most of the dietary cholesterol—binds to a receptor on the liver that recognizes apo E. The particle is endocytosed and its contents degraded by lysosomal enzymes. Defective uptake of CM remnants causes Type III hyperlipoproteinemia. Nascent VLDL are produced in the liver, and are composed predominantly of TAG. They contain a single molecule of apo B-100. Like nascent CM, VLDL receive apo C-Il and apo E from HDL in the plasma. The function of VLDL is to carry hepatic TAG to the peripheral tissues where lipoprotein lipase degrades the lipid. As TAG is removed from the VLDL, the particle receives cholesteryl esters from HDL. This process is accomplished by cholesteryl ester transfer protein (CETP). Eventually, VLDL in the plasma is converted to LDL—a much smaller, denser particle. Apo C-Il and apo E are returned to HDL, but the LDL retains apo B-100, which is recognized by receptors on peripheral tissues and the liver. LDL undergo receptor-mediated endocytosis, and their contents are degraded in the lysosomes. A deficiency of functional LDL receptors causes Type II hyperlipoproteinemia (familial hypercholesterolemia). The endocytosed cholesterol inhibits HMG CoA reductase and decreases synthesis of LDL receptors. Some of it can also be esterified by acyl CoA:cholesterol acyltransferase (ACAT) and stored. HDL are created by lipidation of apo A-1 synthesized in the liver and intestine. They have a number of functions, including:

1) serving as a circulating reservoir of apo C-Il and apo E for chylomicrons and VLDL; 2) removing unesterified cholesterol from from peripheral tissues via ABCA1and esterifying it using lecithin:cholesterol acyl transferase (LCAT), a liversynthesized plasma enzyme that is activated by apo A-1; and 3) delivering these cholesteryl esters to the liver (reverse cholesterol transport) for uptake via SR-B1.
Cholesterol is the precursor of all classes of steroid hormones (glucocorticoids, mineralocorticoids, and sex hormones—androgens, estrogens, and progestins). Synthesis, using primarily cytochrome P450 (CYP) mixedfunction oxidases, occurs in the adrenal cortex (cortisol, aldosterone, and androgens), ovaries and placenta (estrogens and progestins), and testes (testosterone). Each steroid hormone diffuses across the plasma membrane of its target cell and binds to a specific cytosolic or nuclear receptor. These receptor—ligand complexes accumulate in the nucleus, dimerize, and bind to specific regulatory DNA sequences (hormone-response elements) in association with coactivator proteins, thereby causing promoter activation and increased transcription of targeted genes. In association with corepressors, transcription is decreased.

18.1 A 35-year-old woman was seen in the emergency room because of recurrent abdominal pain. The history revealed a 2-year pattern of pain in the upper right quadrant, beginning several hours after the ingestion of a meal rich in fried/fatty food. Ultrasonographic examination demonstrated the presence of numerous stones in the gallbladder. The patient initially elected treatment consisting of exogenously supplied chenodeoxycholic acid, but eventually underwent surgery for the removal of the gallbladder, and had a full recovery. The rationale for the initial treatment of this patient with chenodeoxy- cholic acid is that this compound:
A. interferes with the enterohepatic circulation.
B. inhibits cholesterol synthesis.
C. increases de novo bile acid production.
D. increases cholesterol solubility in bile.
Correct answer = D. Chenodeoxycholic acid is a bile acid used in the treatment of gallstones. It is an amphipathic molecule that can act like an emulsifying agent and help solubilize cholesterol. The compound will not affect the enterohepatic circulation, interfere with cholesterol synthesis, increase bile acid production, or stimulate VLDL production.

A young girl with a history of severe abdominal pain was taken to her local hospital at 5 a.m. in severe distress. Blood was drawn, and the plasma appeared milky, with the triacylglycerol level in excess of 2,000 mg/dl (normal = 4-150 mg/dl). The patient was placed on a diet severely limited in fat, but supplemented with medium-chain fatty acids.
- 18.2 Which of the following lipoprotein particles are most
likely responsible for the appearance of the patient's plasma?
A. Chylomicrons.
B. Very-low-density lipoproteins.
C. Intermediate-density lipoproteins.
D. Low-density-lipoproteins.
E. High-density-lipoproteins.
18.3 Medium-chain fatty acids are given because they:
A. are more calorically dense than long-chain fatty acids.
B. enter directly into the portal blood, and can be metabolized by the liver.
. are activators of lipoprotein lipase. oO
D. are more efficiently packed into serum lipoproteins.
E. can be converted into a variety of gluconeogenic precursors.
E. stimulates VLDL production by the liver.
18.4 Complete the following table for an individual with classic 21-o-hydroxylase deficiency relative to a normal individual.
How might the results be changed if the individual was deficient in 17-0-hydroxylase, rather than 21-chydroxylase?
Correct answer = A. The milky appearance of her blood was a result of triacylglycerol-rich chylomicrons. Because 5 a.m. is presumably several hours after her evening meal, she must have difficulty clearing these lipoprotein particles. IDL, LDL, or HDL contain primarily cholesteryl esters and, if one or more of these particles was elevated, it would cause hypercholesterolemia. VLDL do not cause the described "milky appearance" in plasma.

Correct answer = B. Medium-chain fatty acids are not packaged in chylomicrons, but rather are carried by albumin to the liver where they can be metabolized. They have the same caloric density as long-chain fatty acids, and are generally much more ketogenic than glycogenic. Lipoprotein lipase does not play a role in their metabolism.
Aldosterone, Increased = . Aldosterone, Decreased = X. Cortisol, Increased = . Cortisol, Decreased = X. Androstenedione, Increased = X. Androstenedione, Decreased = . ACTH, Increased = X. ACTH, Decreased = . Blood glucose, Increased = . Blood glucose, Decreased = X. Blood pressure, Increased = . Blood pressure, Decreased = X
21-0-Hydroxylase deficiency causes mineralocorticoids (aldosterone) and glucocorticoids (cortisol) to be virtually absent. Because aldosterone increases blood pressure, and cortisol increases blood glucose, their deficiencies result in a decrease in blood pressure and blood glucose, respectively. Cortisol normally feeds back to inhibit ACTH release by the pituitary, and so its absence results in an elevation in ACTH. The loss of 21-a-hydroxylase pushes progesterone and pregnenolone to androgen synthesis, thus, androstenedione levels rise. With 17-a-hydroxylase deficiency, sex hormone synthesis would be inhibited. Mineralocorticoid production would be increased, leading to hypertension.

Unlike fats and carbohydrates, amino acids are not stored by the body, that is, no protein exists whose sole function is to maintain a supply of amino acids for future use. Therefore, amino acids must be obtained from the diet, synthesized de novo, or produced from normal protein degradation. Any amino acids in excess of the biosynthetic needs of the cell are rapidly degraded. The first phase of catabolism involves the removal of the a-amino groups (usually by transamination and subsequent oxidative deamination), forming ammonia and the corresponding a-keto acid—the "carbon skeletons" of amino acids. A portion of the free ammonia is excreted in the urine, but most is used in the synthesis of urea (Figure 19.1), which is quantitatively the most important route for disposing of nitrogen from the body. In the second phase of amino acid catabolism, described in Chapter 20, the carbon skeletons of the a-ketoacids are converted to common intermediates of energy producing, metabolic pathways. These compounds can be metabolized to CO2 and water, glucose, fatty acids, or ketone bodies by the central pathways of metabolism described in Chapters 8-13, and 16.

Amino acid catabolism is part of the larger process of the metabolism of nitrogen-containing molecules. Nitrogen enters the body in a variety of compounds present in food, the most important being amino acids contained in dietary protein. Nitrogen leaves the body as urea, ammonia, and other products derived from amino acid metabolism. The role of body proteins in these transformations involves two important concepts: the amino acid pool and protein turnover.

Urea cycle shown as part of the essential reactions of energy metabolism. (See Figure 8.2, p. 92, for a more detailed view of the metabolic pathway.)

Free amino acids are present throughout the body, for example, in cells, blood, and the extracellular fluids. For the purpose of this discussion, envision all these amino acids as if they belonged to a single entity, called the amino acid pool. This pool is supplied by three sources: 1) amino acids provided by the degradation of body proteins, 2) amino acids derived from dietary protein, and 3) synthesis of nonessential amino acids from simple intermediates of metabolism (Figure 19.2). Conversely, the amino pool is depleted by three routes: 1) synthesis of body protein, 2) amino acids consumed as precursors of essential nitrogen-containing small molecules, and 3) conversion of amino acids to glucose, glycogen, fatty acids, ketone bodies, or COz2 + H2O (Figure 19.2). Although the amino acid pool is small (comprised of about 90-100 g of amino acids) in comparison with the amount of protein in the body (about 12 kg in a 70-kg man), it is conceptually at the center of whole-body nitrogen metabolism.
In healthy, well-fed individuals, the input to the amino acid pool is balanced by the output, that is, the amount of amino acids contained in the pool is constant. The amino acid pool is said to be in a steady state, and the individual is said to be in nitrogen balance.

Most proteins in the body are constantly being synthesized and then degraded, permitting the removal of abnormal or unneeded proteins. For many proteins, regulation of synthesis determines the concentration of protein in the cell, with protein degradation assuming a minor role. For other proteins, the rate of synthesis is constitutive, that is, relatively constant, and cellular levels of the protein are controlled by selective degradation.
1.
Rate of turnover: In healthy adults, the total amount of protein in the body remains constant, because the rate of protein synthesis is just sufficient to replace the protein that is degraded. This process, called protein turnover, leads to the hydrolysis and resynthesis of 300-400 g of body protein each day. The rate of protein turnover varies widely for individual proteins. Short-lived proteins (for example, many regulatory proteins and misfolded proteins) are rapidly degraded, having half-lives measured in minutes or hours. Long-lived proteins, with half-lives of days to weeks, constitute the majority of proteins in the cell. Structural proteins, such as collagen, are metabolically stable, and have half-lives measured in months or years.
- 2. Protein degradation: There are two major enzyme systems responsible for degrading damaged or unneeded proteins: the ATP-dependent ubiquitin-proteasome system of the cytosol, and the ATP-independent degradative enzyme system of the lyso-
somes. Proteasomes degrade mainly endogenous proteins, that is, proteins that were synthesized within the cell. Lysosomal enzymes (acid hydrolases, see p. 162) degrade primarily extracellular proteins, such as plasma proteins that are taken into the cell by endocytosis, and cell-surface membrane proteins that are used in receptor-mediated endocytosis.
a.

Ubiquitin-proteasome proteolytic pathway: Proteins selected for degradation by the ubiquitin-proteasome system are first covalently attached to ubiquitin, a small, globular, non-enzymic protein. Ubiquitination of the target substrate occurs through linkage of the a-carboxyl group of the C-terminal glycine of ubiquitin to the e-amino group of a lysine on the protein substrate by a three-step, enzyme-catalyzed, ATP-dependent process. The consecutive addition of ubiquitin moieties generates a polyubiquitin chain. Proteins tagged with ubiquitin are then recognized by a large, barrel-shaped, macromolecular, proteolytic complex called a proteasome, which functions like a garbage disposal (Figure 19.3). The proteasome unfolds, deubiquitinates, and cuts the target protein into fragments that are then further degraded to amino acids, which enter the amino acid pool. [Note: The ubiquitins are recycled.] It is noteworthy that the selective degradation of proteins by the ubiquitin-proteosome complex (unlike simple hydrolysis by proteolytic enzymes) requires energy in the form of ATP.
b.
Chemical signals for protein degradation: Because proteins have different half-lives, it is clear that protein degradation cannot be random, but rather is influenced by some structural aspect of the protein. For example, some proteins that have been chemically altered by oxidation or tagged with ubiquitin are preferentially degraded. The half-life of a protein is influenced by the nature of the N-terminal residue. For example, proteins that have serine as the N-terminal amino acid are long-lived, with a half-life of more than 20 hours. In contrast, proteins with aspartate as the N-terminal amino acid have a half-life of only 3 minutes. Additionally, proteins rich in sequences containing proline, glutamate, serine, and threonine (called PEST sequences after the one-letter designations for these amino acids) are rapidly degraded and, therefore, exhibit short intracellular half-lives.

Most of the nitrogen in the diet is consumed in the form of protein, typically amounting to 70-100 g/day in the American diet (see Figure 19.2). Proteins are generally too large to be absorbed by the intestine. [Note: An example of an exception to this rule is that newborns can take up maternal antibodies in breast milk.] They must, therefore, be hydrolyzed to yield diand tripeptides as well as individual amino acids, which can be absorbed. Proteolytic enzymes responsible for degrading proteins are produced by three different organs: the stomach, the pancreas, and the small intestine (Figure 19.4).

Digestion of dietary proteins by the proteolytic enzymes of the gastrointestinal tract.

The digestion of proteins begins in the stomach, which secretes gastric juice—a unique solution containing hydrochloric acid and the proenzyme, pepsinogen.
1. Hydrochloric acid: Stomach acid is too dilute (pH 2-3) to hydrolyze proteins. The acid, secreted by the parietal cells, functions instead to kill some bacteria and to denature proteins, thus making them more susceptible to subsequent hydrolysis by proteases.
2. Pepsin: This acid-stable endopeptidase is secreted by the chief cells of the stomach as an inactive zymogen (or proenzyme), pepsinogen. In general, zymogens contain extra amino acids in their sequences that prevent them from being catalytically active. [Note: Removal of these amino acids permits the proper folding required for an active enzyme.] Pepsinogen is activated to pepsin, either by HCl, or autocatalytically by other pepsin molecules that have already been activated. Pepsin releases peptides and a few free amino acids from dietary proteins.

On entering the small intestine, large polypeptides produced in the stomach by the action of pepsin are further cleaved to oligopeptides and amino acids by a group of pancreatic proteases.
1. Specificity: Each of these enzymes has a different specificity for the amino acid R-groups adjacent to the susceptible peptide bond (Figure 19.5). For example, trypsin cleaves only when the carbonyl group of the peptide bond is contributed by arginine or lysine. These enzymes, like pepsin described above, are synthesized and secreted as inactive zymogens.
2. Release of zymogens: The release and activation of the pancreatic zymogens is mediated by the secretion of cholecystokinin and secretin, two polypeptide hormones of the digestive tract (see p. 176).
3. Activation of zymogens: Enteropeptidase (formerly called enterokinase)—an enzyme synthesized by and present on the luminal surface of intestinal mucosal cells of the brush border membrane—converts the pancreatic zymogen trypsinogen to trypsin by removal of a hexapeptide from the N-terminus of trypsinogen. Trypsin subsequently converts other trypsinogen molecules to trypsin by cleaving a limited number of specific peptide bonds in the zymogen. Enteropeptidase thus unleashes a cascade of proteolytic activity, because trypsin is the common activator of all the pancreatic zymogens (see Figure 19.5).
4. Abnormalities in protein digestion: In individuals with a deficiency in pancreatic secretion (for example, due to chronic pancreatitis, cystic fibrosis, or surgical removal of the pancreas), the digestion and absorption of fat and protein are incomplete. This results in the abnormal appearance of lipids (called steatorrhea, see p. 177) and undigested protein in the feces.

Cleavage of dietary protein by proteases from the pancreas. The peptide bonds susceptible to hydrolysis are shown for each of the five major pancreatic proteases. [Note:The first three are serine endopeptidases, whereas the last two are exopeptidases.]
Celiac disease (celiac sprue) is a disease of malabsorption resulting from immune-mediated damage to the small intestine in response to ingestion of gluten (or gliadin produced from gluten), a protein found in wheat, barley and rye.

The luminal surface of the intestine contains aminopeptidase—an exopeptidase that repeatedly cleaves the N-terminal residue from oligopeptides to produce even smaller peptides and free amino acids.

Free amino acids are taken into the enterocytes by a Na'*-linked secondary transport system of the apical membrane. Diand tripeptides, however, are taken up by a H*-linked transport system. The peptides are hydrolyzed in the cytosol to amino acids that are released into the portal system by facilitated diffusion. Thus, only free amino acids are found in the portal vein after a meal containing protein. These amino acids are either metabolized by the liver or released into the general circulation. [Note: Branched-chain amino acids are important examples of amino acids that are not metabolized by the liver, but instead are sent from the liver primarily to muscle via the blood.]

The concentration of free amino acids in the extracellular fluids is significantly lower than that within the cells of the body. This concentration gradient is maintained because active transport systems, driven by the hydrolysis of ATP, are required for movement of amino acids from

Genetic defect seen in cystinuria. [Note: Cystinura is distinct from cystinosis, a rare defect in the transport of cystine out of lyso-. of eyatin crystals with © hone lysosome, and tissue damage.]

Aminotransferase reaction using a-ketoglutarate as the aminogroup acceptor.

Reactions catalyzed during amino acid catabolism. A. Alanine aminotransferase (ALT). B. Aspartate aminotransferase (AST).
the extracellular space into cells. At least seven different transport systems are known that have overlapping specificities for different amino acids. The small intestine and the proximal tubule of the kidney have common transport systems for amino acid uptake; therefore, a defect in any one of these systems results in an inability to absorb particular amino acids into the gut and into the kidney tubules. For example, one system is responsible for the uptake of cystine and the dibasic amino acids, ornithine, arginine, and lysine (represented as "COAL'). In the inherited disorder cystinuria, this carrier system is defective, and all four amino acids appear in the urine (Figure 19.6). Cystinuria occurs at a frequency of 1 in 7,000 individuals, making it one of the most common inherited diseases, and the most common genetic error of amino acid transport. The disease expresses itself clinically by the precipitation of cystine to form kidney stones (calculi), which can block the urinary tract. Oral hydration is an important part of treatment for this disorder. [Note: Defects in the transport of tryptophan (and other neutral amino acids) can result in Hartnup disorder and pellagra-like (see p. 380) dermatologic and neurologic symptoms.]

The presence of the o-amino group keeps amino acids safely locked away from oxidative breakdown. Removing the a-amino group is essential for producing energy from any amino acid, and is an obligatory step in the catabolism of all amino acids. Once removed, this nitrogen can be incorporated into other compounds or excreted, with the carbon skeletons being metabolized. This section describes transamination and oxidative deamination—reactions that ultimately provide ammonia and aspartate, the two sources of urea nitrogen (see p. 253).

The first step in the catabolism of most amino acids is the transfer of their a-amino group to a-ketoglutarate (Figure 19.7). The products are an a-keto acid (derived from the original amino acid) and glutamate. o-Ketoglutarate plays a pivotal role in amino acid metabolism by accepting the amino groups from most amino acids, thus becoming glutamate. Glutamate produced by transamination can be oxidatively deaminated (see below), or used as an amino group donor in the synthesis of nonessential amino acids. This transfer of amino groups from one carbon skeleton to another is catalyzed by a family of enzymes called aminotransferases (formerly called transaminases). These enzymes are found in the cytosol and mitochondria of cells throughout the body—especially those of the liver, kidney, intestine, and muscle. All amino acids, with the exception of lysine and threonine, participate in transamination at some point in their catabolism. [Note: These two amino acids lose their o-amino groups by deamination (see pp. 265—266).]
1. Substrate specificity of aminotransferases: Each aminotransferase is specific for one or, at most, a few amino group donors. Aminotransferases are named after the specific amino group donor, because the acceptor of the amino group is almost always o-ketoglutarate. The two most important aminotransferase reactions are catalyzed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST), Figure 19.8).

a. Alanine aminotransferase (ALT): ALT is present in many tissues. The enzyme catalyzes the transfer of the amino group of alanine to a-ketoglutarate, resulting in the formation of pyruvate and glutamate. The reaction is readily reversible. However, during amino acid catabolism, this enzyme (like most aminotransferases) functions in the direction of glutamate synthesis. Thus, glutamate, in effect, acts as a "collector" of nitrogen from alanine.
b. Aspartate aminotransferase (AST): AST is an exception to the rule that aminotransferases funnel amino groups to form glutamate. During amino acid catabolism, AST transfers amino groups from glutamate to oxaloacetate, forming aspartate, which is used as a source of nitrogen in the urea cycle (see p. 253). [Note: The AST reaction is also reversible.]
. Mechanism of action of aminotransferases: All aminotransferases require the coenzyme pyridoxal phosphate (a derivative of vitamin Bg, see p. 378), which is covalently linked to the €-amino group of a specific lysine residue at the active site of the enzyme. Aminotransferases act by transferring the amino group of an amino acid to the pyridoxal part of the coenzyme to generate pyridoxamine phosphate. The pyridoxamine form of the coenzyme then reacts with an a-keto acid to form an amino acid, at the same time regenerating the original aldehyde form of the coenzyme. Figure 19.9 shows these two component reactions for the reaction catalyzed by AST.
3. Equilibrium of transamination reactions: For most transamination reactions, the equilibrium constant is near one. This allows the reaction to function in both amino acid degradation through removal of «-amino groups (for example, after consumption of a protein-rich meal) and biosynthesis through addition of amino groups to the carbon skeletons of a-keto acids (for example, when the supply of amino acids from the diet is not adequate to meet the synthetic needs of cells).

. Diagnostic value of plasma aminotransferases: Aminotransferases are normally intracellular enzymes, with the low levels found in the plasma representing the release of cellular contents during normal cell turnover. The presence of elevated plasma levels of aminotransferases indicates damage to cells rich in these enzymes. For example, physical trauma or a disease process can cause cell lysis, resulting in release of intracellular enzymes into the blood. Two aminotransferases—AST and ALT—are of particular diagnostic value when they are found in the plasma.
a. Liver disease: Plasma AST and ALT are elevated in nearly all liver diseases, but are particularly high in conditions that cause extensive cell necrosis, such as severe viral hepatitis, toxic injury, and prolonged circulatory collapse. ALT is more specific than AST for liver disease, but the latter is more sensitive because the liver contains larger amounts of AST. Serial enzyme measurements are often useful in determining the course of liver damage. Figure 19.10 shows the early release of ALT into the serum, following ingestion of a liver

Cyclic interconversion of pyridoxal phosphate and pyridoxamine phosphate during the aspartate aminotransferase reaction. [Note: ®= phosphate group.]

Pattern of serum alanine aminotransferase (ALT) and bilirubin in the plasma, following poisoning with the toxic mushroom Amanita phalloides.

Combined actions of aminotransferase and glutamate dehydrogenase reactions.
toxin. [Note: Elevated serum bilirubin results from hepatocellular damage that decreases the hepatic conjugation and excretion of bilirubin (see p. 284).]
- b. Nonhepatic disease: Aminotransferases may be elevated in nonhepatic disease, such as myocardial infarction and muscle disorders. However, these disorders can usually be distinguished clinically from liver disease.

In contrast to transamination reactions that transfer amino groups, oxidative deamination by glutamate dehydrogenase results in the liberation of the amino group as free ammonia (NHs) (Figure 19.11). These reactions occur primarily in the liver and kidney. They provide a-keto acids that can enter the central pathway of energy metabolism, and ammonia, which is a source of nitrogen in urea synthesis.

1. Glutamate dehydrogenase: As described above, the amino groups of most amino acids are ultimately funneled to glutamate by means of transamination with o-ketoglutarate. Glutamate is unique in that it is the only amino acid that undergoes rapid oxidative deamination—a reaction catalyzed by glutamate dehydrogenase (see Figure 19.11). Therefore, the sequential action of transamination (resulting in the collection of amino groups from most amino acids onto a-ketoglutarate to produce glutamate) and the oxidative deamination of that glutamate (regenerating a-ketoglutarate) provide a pathway whereby the amino groups of most amino acids can be released as ammonia.
a. Coenzymes: Glutamate dehydrogenase is unusual in that it can use either NAD* or NADP* as a coenzyme (see Figure 19.11). NAD* is used primarily in oxidative deamination (the simultaneous loss of ammonia coupled with the oxidation of the carbon skeleton (Figure 19.12A), and NADPH is used in reductive amination (the simultaneous gain of ammonia coupled with the reduction of the carbon skeleton, Figure 19.12B).
b. Direction of reactions: The direction of the reaction depends on the relative concentrations of glutamate, o-ketoglutarate, and ammonia, and the ratio of oxidized to reduced coenzymes. For example, after ingestion of a meal containing protein, glutamate levels in the liver are elevated, and the reaction proceeds in the direction of amino acid degradation and the formation of ammonia (see Figure 19.12A). [Note: the reaction can also be used to synthesize amino acids from the corresponding a-keto acids (see Figure 19.12B).]
c. Allosteric regulators: Guanosine triphosphate (GTP) is an allosteric inhibitor of glutamate dehydrogenase, whereas adenosine diphosphate (ADP) is an activator. Thus, when energy levels are low in the cell, amino acid degradation by glutamate dehydrogenase is high, facilitating energy production from the carbon skeletons derived from amino acids.

2. D-Amino acid oxidase: D-Amino acids (see p. 5) are found in plants and in the cell walls of microorganisms, but are not used in the synthesis of mammalian proteins. D-Amino acids are, however, present in the diet, and are efficiently metabolized by the kidney and liver. D-Amino acid oxidase (DAO) is an FAD-dependent peroxisomal enzyme that catalyzes the oxidative deamination of these amino acid isomers, producing a-keto acids, ammonia, and hydrogen peroxide. The a-keto acids can enter the general pathways of amino acid metabolism, and be reaminated to L-isomers, or catabolized for energy. [Note: DAO degrades D-serine, the isomeric form of serine that modulates NMDA-type glutamate receptors. Increased DAO activity has been linked to increased susceptibility to schizophrenia.] L-amino acid oxidases are components of several snake venoms.

Two mechanisms are available in humans for the transport of ammonia from the peripheral tissues to the liver for its ultimate conversion to urea. The first, found in most tissues, uses glutamine synthetase to combine ammonia (NH3) with glutamate to form glutamine—a nontoxic transport form of ammonia (Figure 19.13). The glutamine is transported in the blood to the liver where it is cleaved by glutaminase to produce glutamate and free ammonia (see p. 256). The second transport mechanism, used primarily by muscle, involves transamination of pyruvate (the end product of aerobic glycolysis) to form alanine (see Figure 19.8). Alanine is transported by the blood to the liver, where it is converted to pyruvate, again by transamination. In the liver, the pathway of gluconeogenesis can use the pyruvate to synthesize glucose, which can enter the blood and be used by muscle—a pathway called the glucose-alanine cycle.

Urea is the major disposal form of amino groups derived from amino acids, and accounts for about 90% of the nitrogen-containing components of urine. One nitrogen of the urea molecule is supplied by free ammonia, and the other nitrogen by aspartate. [Note: Glutamate is the immediate precursor of both ammonia (through oxidative deamination by glutamate dehydrogenase) and aspartate nitrogen (through transamination of oxaloacetate by AST).] The carbon and oxygen of urea are derived from COs. Urea is produced by the liver, and then is transported in the blood to the kidneys for excretion in the urine.

The first two reactions leading to the synthesis of urea occur in the mitochondria, whereas the remaining cycle enzymes are located in the cytosol (Figure 19.14). [Note: Gluconeogenesis (see p. 117) and heme synthesis (see p. 278) also involve both the mitochondrial matrix and the cytosol.]
1. Formation of carbamoyl phosphate: Formation of carbamoyl phosphate by carbamoyl! phosphate synthetase | is driven by cleavage of two molecules of ATP. Ammonia incorporated into carbamoyl phosphate is provided primarily by the oxidative deamina-

Reactions of the urea cycle.
tion of glutamate by mitochondrial glutamate dehydrogenase (see Figure 19.11). Ultimately, the nitrogen atom derived from this ammonia becomes one of the nitrogens of urea. Carbamoyl phosphate synthetase | requires N-acetylglutamate as a positive allosteric activator (see Figure 19.14). [Note: Carbamoyl phosphate synthetase II participates in the biosynthesis of pyrimidines (see p. 302). It does not require N-acetylglutamate, uses glutamine as the nitrogen source, and occurs in the cytosol.]

. Formation of citrulline: The carbamoyl portion of carbamoyl phosphate is transferred to ornithine by ornithine transcarbamoylase (OTC) as the high-energy phosphate is released as P;. The reaction product, citrulline, is transported to the cytosol. [Note: Ornithine and citrulline are basic amino acids that participate in the urea cycle, moving across the inner mitochondrial membrane via a cotransporter. They are not incorporated into cellular proteins because there are no codons for these amino acids (see p. 432).] Ornithine is regenerated with each turn of the urea cycle, much in the same way that oxaloacetate is regenerated by the reactions of the citric acid cycle (see p. 109).
. Synthesis of argininosuccinate: Argininosuccinate synthetase combines citrulline with aspartate to form argininosuccinate. The a-amino group of aspartate provides the second nitrogen that is ultimately incorporated into urea. The formation of argininosuccinate is driven by the cleavage of ATP to adenosine monophosphate (AMP) and pyrophosphate. This is the third and final molecule of ATP consumed in the formation of urea.
4. Cleavage of argininosuccinate: Argininosuccinate is cleaved by argininosuccinate lyase to yield arginine and fumarate. The arginine formed by this reaction serves as the immediate precursor of urea. Fumarate produced in the urea cycle is hydrated to malate, providing a link with several metabolic pathways. For example, the malate can be transported into the mitochondria via the malate shuttle, reenter the tricarboxylic acid cycle, and get oxidized to oxaloacetate (OAA), which can be used for gluconeogenesis (see p. 120). Alternatively, the OAA can be converted to aspartate via transamination (see Figure 19.8), and can enter the urea cycle (see Figure 19.14).

5. Cleavage of arginine to ornithine and urea: Arginase cleaves arginine to ornithine and urea, and occurs almost exclusively in the liver. Thus, whereas other tissues, such as the kidney, can synthesize arginine by these reactions, only the liver can cleave arginine and, thereby, synthesize urea.
6. Fate of urea: Urea diffuses from the liver, and is transported in the blood to the kidneys, where it is filtered and excreted in the urine. A portion of the urea diffuses from the blood into the intestine, and is cleaved to COz and NHsg by bacterial urease. This ammonia is partly lost in the feces, and is partly reabsorbed into the blood. In patients with kidney failure, plasma urea levels are elevated, promoting a greater transfer of urea from blood into the gut. The intestinal action of urease on this urea becomes a clinically important source of ammonia, contributing to the hyperammonemia often seen in these patients. Oral administration of neomycin reduces the number of intestinal bacteria responsible for this NH3 production.

Flow of nitrogen from amino acids to urea. Amino groups for urea synthesis are collected in the form of ammonia and aspartate.

Formation and degradation of Nacetylglutamate, an allosteric activator of carbamoyl phosphate synthetase |.

Four high-energy phosphate bonds are consumed in the synthesis of each molecule of urea; therefore, the synthesis of urea is irreversible, with a large, negative AG (see p. 70). [Note: The ATP is replenished by oxidative phosphorylation.] One nitrogen of the urea molecule is supplied by free NHs, and the other nitrogen by aspartate. Glutamate : : : ; ae is the immediate precursor of both ammonia (through oxidative deamination by glutamate dehydrogenase) and aspartate nitrogen (through transamination of oxaloacetate by AS7). In effect, both nitrogen atoms of urea arise from glutamate, which, in turn, gathers nitro-+ 1 = gen from other amino acids (Figure 19.15).

N-Acetylglutamate is an essential activator for carbamoyl phosphate synthetase |—the rate-limiting step in the urea cycle (see Figure 19.14). N-Acetylglutamate is synthesized from acetyl coenzyme A and glutamate by N-acetylglutamate synthase (Figure 19.16), ina reaction for which arginine is an activator. Therefore, the intrahepatic concentration of N-acetylglutamate increases after ingestion of a protein-rich meal, which provides both a substrate (glutamate) and the regulator of N-acetylglutamate synthesis. This leads to an increased rate of urea synthesis.

Ammonia is produced by all tissues during the metabolism of a variety of compounds, and it is disposed of primarily by formation of urea in the liver. However, the level of ammonia in the blood must be kept very low, because even slightly elevated concentrations (hyperammonemia) are toxic to the central nervous system (CNS). There must, therefore, be a metabolic mechanism by which nitrogen is moved from peripheral tissues to the liver for ultimate disposal as urea, while at the same time maintaining low levels of circulating ammonia.

Amino acids are quantitatively the most important source of ammonia, because most Western diets are high in protein and provide excess amino acids, which travel to the liver and undergo transdeamination— the linking of aminotransferase and glutamate dehydrogenase reactions—producing ammonia. However, substantial amounts of ammonia can be obtained from other sources.
1. From glutamine: The kidneys generate ammonia from glutamine by the actions of renal g/utaminase (Figure 19.17) and glutamate dehydrogenase. Most of this ammonia is excreted into the urine as NH,*, which provides an important mechanism for maintaining the body's acid-base balance through the excretion of protons. Ammonia is also obtained from the hydrolysis of glutamine by intestinal glutaminase. The intestinal mucosal cells obtain
glutamine either from the blood or from digestion of dietary protein. [Note: Intestinal glutamine metabolism produces citrulline, which travels to the kidney and is used to synthesize arginine.]
2. From bacterial action in the intestine: Ammonia is formed from urea by the action of bacterial urease in the lumen of the intestine. This ammonia is absorbed from the intestine by way of the portal vein and is almost quantitatively removed by the liver via conversion to urea.
3. From amines: Amines obtained from the diet, and monoamines that serve as hormones or neurotransmitters, give rise to ammonia by the action of amine oxidase (see p. 286).
4. From purines and pyrimidines: In the catabolism of purines and pyrimidines, amino groups attached to the rings are released as ammonia (see Figure 22.15 and p. 304).

Although ammonia is constantly produced in the tissues, it is present at very low levels in blood. This is due both to the rapid removal of blood ammonia by the liver, and the fact that many tissues, particularly muscle, release amino acid nitrogen in the form of glutamine or alanine, rather than as free ammonia (see Figure 19.13).
1. Urea: Formation of urea in the liver is quantitatively the most important disposal route for ammonia. Urea travels in the blood from the liver to the kidneys, where it passes into the glomerular filtrate.
2. Glutamine: This amide of glutamic acid provides a nontoxic storage and transport form of ammonia (Figure 19.18). The ATPrequiring formation of glutamine from glutamate and ammonia by glutamine synthetase occurs primarily in the muscle and liver, but is also important in the CNS where it is the major mechanism for the removal of ammonia in the brain. Glutamine is found in plasma at concentrations higher than other amino acids—a finding consistent with its transport function. Circulating glutamine is removed by the liver and the kidneys and deaminated by glutaminase. |n the liver, the NH3 produced is detoxified through conversion to urea, and in the kidney it can be used in the excretion of protons. The metabolism of ammonia is summarized in Figure 19.19.

The capacity of the hepatic urea cycle exceeds the normal rates of ammonia generation, and the levels of serum ammonia are normally low (5-35 mol/L). However, when liver function is compromised, due either to genetic defects of the urea cycle or liver disease, blood levels can rise above 1,000 umol/L. Such hyperammonemia is a medical emergency, because ammonia has a direct neurotoxic effect on the CNS. For example, elevated concentrations of ammonia in the blood cause the symptoms of ammonia intoxication, which include tremors, slurring of speech, somnolence, vomiting, cerebral edema, and blurring of vision. At high concentrations, ammonia can cause coma and death. The two major types of hyperammonemia are:

Metabolism of ammonia. Urea content in the urine is reported as urinary urea nitrogen or UUN. Urea in blood is reported as BUN (blood urea nitrogen).The enzymes glutamate dehydrogenase, glutamine synthetase, and carbamoyl phosphate synthetase | fix ammonia (NHs3) into organic molecules.
7
\
-

Treatment of patients with urea cycle defects by administration of phenylbutyrate to aid in excretion of ammonia.
Acquired hyperammonemia: Liver disease is a common cause of hyperammonemia in adults, and may be due, for example, to viral hepatitis or to hepatotoxins such as alcohol. Cirrhosis of the . . . . . liver may result in formation of collateral circulation around the liver. AS a result, portal blood is shunted directly into the systemic circulation and does not have access to the liver. The con-> . . 7 . version of ammonia to urea is, therefore, severely impaired, leading to elevated levels of ammonia.
- Congenital hyperammonemia: Genetic deficiencies of each of the five enzymes of the urea cycle have been described, with an overall prevalence estimated to be 1:25,000 live births. Ornithine transcarbamoylase deficiency, which is X-linked, is the most com-. . mon of these disorders, predominantly affecting males, although female carriers may become symptomatic. All of the other urea cycle disorders follow an autosomal recessive inheritance pattern. In each case, the failure to synthesize urea leads to hyperammonemia during the first weeks following birth. [Note: The hyperammonemia seen with arginase deficiency is less severe because arginine contains two waste nitrogens and can be excreted in the urine.] Historically, urea cycle defects had high morbidity (neurological manifestations) and mortality. Treatment included restriction of dietary protein in the presence of sufficient . . : calories to prevent catabolism. Administration of compounds that bind covalently to amino acids, producing nitrogen-containing molecules that are excreted in the urine, has improved survival. For example, phenylbutyrate given orally is converted to phenylacetate. This condenses with glutamine to form phenylacetylglutamine, which is excreted (Figure 19.20).

Nitrogen enters the body in a variety of compounds present in food, the most important being amino acids contained in dietary protein. Nitrogen leaves the body as urea, ammonia, and other products derived from amino acid metabolism (Figure 19.21). Free amino acids in the body are produced by hydrolysis of dietary protein by proteases in the stomach and intestine, degradation of tissue proteins, and de novo synthesis. This amino acid pool is consumed in the synthesis of body protein, metabolized for energy, or its members serve as precursors for other nitrogen-containing compounds. Note that body protein is simultaneously degraded and resynthesized— a process known as protein turnover. For many proteins, regulation of synthesis determines the concentration of the protein in the cell, whereas the amounts of other proteins are controlled by selective degradation. The ATP-dependent ubiquitin/proteasome and ATP-independent lysosomal acid hydrolases are the two major enzyme systems that are responsible for degrading damaged or unneeded proteins. Nitrogen cannot be stored, and amino acids in excess of the biosynthetic needs of the cell are immediately degraded. The first phase of catabolism involves the transfer of the o-amino groups by PLP-dependent transamination, followed by oxidative deamination of glutamate, forming ammonia and the corresponding a-keto acids. A portion of the free ammonia is excreted in the urine, some is used in converting glutamate to glutamine, but most is used in the synthesis of urea, which is quantitatively the most important route for disposing of nitrogen from the body. The two major causes of hyperammonemia (with its CNS effects) are liver disease and inherited deficiencies of enzymes (such as ornithine transcarbamolyase) in the urea cycle.
1.

A. Alanine, o-ketoglutarate.
B. Glutamate,
a-ketoglutarate.
C. Asparate, o-ketoglutarate.
D. Pyruvate, aspartate.
E. Pyruvate, alanine.

urea cycle is correct?
A. The two nitrogen atoms that are incorporated into urea enter the cycle as ammonia and alanine.
- B. Urea is produced directly by the hydrolysis of ornithine.
C. ATP is required for the reaction in which argini- nosuccinate is cleaved to form arginine.
D. Urinary urea is increased by a diet rich in protein.
E. The urea cycle occurs exclusively in the cytosol.

A female neonate did well until approximately 24 hours of age when she became lethargic. A sepsis workup proved negative. At 56 hours, she started showing focal seizure activity. The plasma ammonia level was found to be 1,100 mol/L (normal 5-35 umol/L). Quantitative plasma amino acid levels revealed a marked elevation of argininosuccinate.

- B. Argininosuccinate lyase.
C. Argininosuccinate synthase.
D. Carbamoyl phosphate synthetase I.
E. Ornithine transcarbamoylase.

the blood of this patient?
A. Asparagine.
B. Glutamine.
C. Lysine.
D. Urea.
E. Uric acid.

Correct answer = C. Transaminase reactions always have an amino acid and an a-keto acid as substrates. The products of the reaction are also an amino acid (corresponding to the a-keto substrate) and an a-keto acid (corresponding to the amino acid substrate). Three amino acid aketo acid pairs commonly encountered in metabolism are:
alanine/pyruvate aspartate/oxaloacetate
glutamate/o.-ketoglutarate
In this question, glutamate is deaminated to form a-ketoglutarate, and oxaloacetate is aminated to form aspartate.
Correct answer = D. The amino nitrogen of dietary protein is excreted as urea. The two nitrogens enter the urea cycle as ammonia and aspartate. Urea is produced by the hydrolysis of arginine. The cleavage of argininosuccinate does not require ATP. The urea cycle occurs partly in the mitochondria.
Correct answer = B. Genetic deficiencies of each of the five enzymes of the urea cycle, as well as deficiencies in N-acetyglutamate synthase, have been described. The accumulation of argininosuccinate in the plasma of this patient means that the enzymes required for its synthesis are functional, but the enzyme (argininosuccinate lyase or argininosuccinase) required for its cleavage to arginine plus fumarate is not.
Correct answer = B. With the exception of arginase, deficiencies of the enzymes of the urea cycle result in the failure to synthesize urea and lead to hyperammonemia in the first few weeks after birth. Glutamine will also be elevated because it acts as a nontoxic storage and transport form of ammonia. Thus, elevated glutamine always accompanies hyperammonemia. Asparagine does not serve this sequestering role. Urea would be decreased due to impaired activity of the urea cycle. Lysine and uric acid would not be elevated. Treatment of this patient includes limiting protein in the diet and administering compounds that bind covalently to amino acids, producing nitrogen-containing molecules that are excreted in the urine. For example, phenylbutyrate given orally is converted to phenylacetate. This compound condenses with glutamine to form phenylacetylglutamine, which is excreted.

The catabolism of the amino acids involves the removal of a-amino groups, followed by the breakdown of the resulting carbon skeletons. These pathways converge to form seven intermediate products: oxaloacetate, pyruvate, o-ketoglutarate, fumarate, succinyl coenzyme A (CoA), acetyl CoA, and acetoacetate. These products directly enter the pathways of intermediary metabolism, resulting either in the synthesis of glucose or lipid or in the production of energy through their oxidation to COs by the citric acid cycle. Figure 20.1 provides an overview of these pathways, with a more detailed summary presented later in Figure 20.14 (see p. 269). Nonessential amino acids (Figure 20.2) can be synthesized in sufficient amounts from the intermediates of metabolism or, as in the case of cysteine and tyrosine, from essential amino acids. In contrast, the essential amino acids cannot be synthesized (or produced in sufficient amounts) by the body and, therefore, must be obtained from the diet in order for normal protein synthesis to occur. Genetic defects in the pathways of amino acid metabolism can cause serious disease.

Amino acids can be classified as glucogenic, ketogenic, or both based on which of the seven intermediates are produced during their catabolism (see Figure 20.2).

Amino acids whose catabolism yields pyruvate or one of the intermediates of the citric acid cycle are termed glucogenic. These intermediates are substrates for gluconeogenesis (see p. 117) and, therefore, can give rise to the net formation of glucose in the liver and kidney.
@ BLUE CAPS TEXT = names of seven products of amino acid
Color-coding UEYTo RT Uy chapter:
metabolism
@ Red text = names of glucogenic amino acids
e@ Brown text = names of glucogenic and ketogenic amino acids
@ Green text = names of ketogenic amino acids
@
Cyan text = one-carbon compounds

Amino acid metabolism shown as a part of the central pathways of energy metabolism. (See figure 8.2, p. 92, for a more detailed view of these processes.

Classification of amino acids. [Note: Some amino acids can become conditionally essential. For example, supplementation with glutamine and arginine has been shown to improve outcomes in patients with trauma, post-operative infections, and immunosuppression.]

Metabolism of asparagine and aspartate. [Note: Recall that carbons from aspartate can form fumarate in the urea cycle (see p. 254).]

Amino acids whose catabolism yields either acetoacetate or one of its precursors (acetyl CoA or acetoacetyl CoA) are termed ketogenic (see Figure 20.2). Acetoacetate is one of the ketone bodies, which also include 3-hydroxybutyrate and acetone (see p. 195). Leucine and lysine are the only exclusively ketogenic amino acids found in proteins. Their carbon skeletons are not substrates for gluconeogenesis and, therefore, cannot give rise to the net formation of glucose.

The pathways by which amino acids are catabolized are conveniently organized according to which one (or more) of the seven intermediates listed above is produced from a particular amino acid.

Asparagine is hydrolyzed by asparaginase, liberating ammonia and aspartate (Figure 20.3). Aspartate loses its amino group by transamination to form oxaloacetate (see Figure 20.3). [Note: Some rapidly dividing leukemic cells are unable to synthesize sufficient asparagine to support their growth. This makes asparagine an essential amino acid for these cells, which therefore require asparagine from the blood. Asparaginase, which hydrolyzes asparagine to aspartate, can be administered systemically to treat leukemic patients.' Asparaginase lowers the level of asparagine in the plasma and, therefore, deprives cancer cells of a required nutrient.]

1. Glutamine: This amino acid is converted to glutamate and ammonia by the enzyme glutaminase (see p. 256). Glutamate is converted to a-ketoglutarate by transamination, or through oxidative deamination by glutamate dehydrogenase (see p. 252).
2. Proline: This amino acid is oxidized to glutamate. Glutamate is transaminated or oxidatively deaminated to form a-ketoglutarate.
3. Arginine: This amino acid is cleaved by arginase to produce ornithine. [Note: This reaction occurs primarily in the liver as part of the urea cycle (see p. 255).] Ornithine is subsequently converted to a-ketoglutarate; glutamate semi-aldehyde is an intermediate.
4. Histidine: This amino acid is oxidatively deaminated by histidase to urocanic acid, which subsequently forms N-formiminoglutamate (FlGlu, Figure 20.4). FlGlu donates its formimino group to tetrahydrofolate (THF), leaving glutamate, which is degraded as described above. [Note: Individuals deficient in folic acid excrete increased amounts of FlGlu in the urine, particularly after ingestion of a large dose of histidine. The FlGlu excretion test has been used in diagnosing a deficiency of folic acid.] (See p. 267 for a discussion of folic acid, THF, and one-carbon metabolism.)
1See Chapter 39 in Lippincott's Illustrated Reviews: Pharmacology for a discussion of the use of asparaginase as an antileukemic drug.

1. Alanine: This amino acid loses its amino group by reversible transamination to form pyruvate (Figure 20.5). [Note: Alanine is the major gluconeogenic amino acid.]
2. Serine: This amino acid can be converted to glycine and N°,N'°methylenetetrahydrofolate (Figure 20.6A). Serine can also be converted to pyruvate by serine dehydratase (Figure 20.6B).
3. Glycine: This amino acid can be converted to serine by the reversible addition of a methylene group from N®°,N'°-methylenetetrahydrofolic acid (see Figure 20.6A) or oxidized to COz and NH3. [Note: Glycine can be converted to glyoxylate. Glyoxylate can be oxidized to oxalate, or transaminated to glycine. Deficiency of the transaminase causes overproduction of oxalate and kidney damage (primary oxaluria Type 1).]
4. Cystine: This amino acid is reduced to cysteine, using NADH + H* as a reductant. Cysteine undergoes desulfuration to yield pyruvate. [Note: The sulfate released can be used to synthesize 3'-phosphoadenosine-5'-phosphosulfate (PAPS), an activated sulfur donor to acceptors such as glycosaminoglycans (see p. 162).]
5. Threonine: This amino acid is converted to pyruvate or to a-ketobutyrate, which forms succinyl CoA.

1. Phenylalanine and tyrosine: Hydroxylation of phenylalanine produces tyrosine (Figure 20.7). This reaction, catalyzed by tetrahydrobiopterin-requiring phenylalanine hydroxylase, initiates the catabolism of phenylalanine. Thus, the metabolism of phenylalanine and tyrosine merge, leading ultimately to the formation of fumarate and acetoacetate. Phenylalanine and tyrosine are, therefore, both glucogenic and ketogenic.
2. Inherited deficiencies: Inherited deficiencies in the enzymes of phenylalanine and tyrosine metabolism lead to the diseases phenylketonuria (see p. 270) and alkaptonuria (see p. 274), and the condition of albinism (see p. 273).

Methionine is one of four amino acids that form succinyl CoA. This sulfur-containing amino acid deserves special attention because it is converted to S-adenosylmethionine (SAM), the major methyl-group donor in one-carbon metabolism (Figure 20.8). Methionine is also the source of homocysteine—a metabolite associated with atherosclerotic vascular disease (see p. 265).

A. Interconversion of serine and glycine, and oxidation of glycine. B. Dehydration of serine to form pyruvate.

Degradation and resynthesis of methionine. [Note: The resynthesis of methionine from homocysteine is the only reaction in which THF both carries and donates a methyl group. In all other reactions, SAM is the methyl group carrier and donor.]
4.
b. Synthesis of cysteine: Homocysteine condenses with serine, forming cystathionine, which is hydrolyzed to «-ketobutyrate and cysteine (see Figure 20.8). This vitamin Bg—requiring sequence has the net effect of converting serine to cysteine, and homocysteine to a-ketobutyrate, which is oxidatively decarboxylated to form propiony! CoA. Propionyl CoA is converted to succinyl CoA (see p. 194). Because homocysteine is synthesized from the essential amino acid methionine, cysteine is not an essential amino acid as long as sufficient methionine is available.
Relationship of homocysteine to vascular disease: Elevations in plasma homocysteine levels promote oxidative damage, inflammation, and endothelial dysfunction, and are an independent risk factor for occlusive vascular disease (Figure 20.9). Mild elevations are seen in about 7% of the population. Epidemiologic studies have shown that plasma homocysteine levels are inversely related to plasma levels of folate, Bi2, and Be—the three vitamins involved in the conversion of homocysteine to methionine or cysteine. Supplementation with these vitamins has been shown to reduce circulating levels of homocysteine. However, in patients with established cardiovascular disease, vitamin therapy does not decrease cardiovascular events or death. This raises the question as to whether homocysteine is a cause of the vascular damage or merely a marker of such damage. [Note: Large elevations in plasma homocysteine as a result of rare deficiencies in cystathionine B-synthase are seen in patients with classic homocystinuria. These individuals experience premature vascular disease, with about 25% dying from thrombotic complications before 30 years of age.]

Elevated homocysteine or decreased folic acid levels in pregnant women are associated with increased incidence of neural tube defects (improper closure, as in spina bifida) in the fetus. Periconceptual supplementation with folate reduces the risk of such defects.

Degradation of valine, isoleucine, and threonine also results in the production of succinyl CoA—a tricarboxylic acid (TCA) cycle intermediate and glucogenic compound.
= . Valine and isoleucine: These amino acids are branched-chain amino acids that generate propionyl CoA, which is converted to succinyl CoA by biotinand vitamin B;2—requiring reactions (Figure 20.10).
- Threonine: This amino acid is dehydrated to a-ketobutyrate, which is converted to propionyl CoA and then to succinyl CoA. Threonine can also be converted to pyruvate. [Note: Propionyl CoA, then, is generated by the catabolism of certain amino acids and odd-numbered fatty acids (see p. 193).]

Association between cardiovascular disease mortality and total plasma homocysteine.

Degradation of leucine, valine, and isoleucine. TPP = thiamine pyrophosphate. [Note: 3-Methylcrotonyl CoA carboxylase is one of four biotin-requiring carboxylases we have encountered. The other three are pyruvate carboxylase, acetyl CoA carboxylase, and propionyl CoA carboxylase.]

Leucine, isoleucine, lysine, and tryptophan form acetyl CoA or acetoacetyl CoA directly, without pyruvate serving as an intermediate (through the pyruvate dehydrogenase reaction, see p. 109). As mentioned previously, phenylalanine and tyrosine also give rise to acetoacetate during their catabolism (see Figure 20.7). Therefore, there are a total of six ketogenic amino acids.
1. Leucine: This amino acid is exclusively ketogenic in its catabolism, forming acetyl CoA and acetoacetate (see Figure 20.10). The initial steps in the catabolism of leucine are similar to those of the other branched-chain amino acids, isoleucine and valine (see below).
2. Isoleucine: This amino acid is both ketogenic and glucogenic, because its metabolism yields acetyl CoA and propionyl CoA. The first three steps in the metabolism of isoleucine are similar to the initial steps in the degradation of the other branched-chain amino acids, valine and leucine (see Figure 20.10).
3. Lysine: An exclusively ketogenic amino acid, this amino acid is unusual in that neither of its amino groups undergoes transamination as the first step in catabolism. Lysine is ultimately converted to acetoacetyl CoA.
4. Tryptophan: This amino acid is both glucogenic and ketogenic because its metabolism yields alanine and acetoacetyl CoA.

The branched-chain amino acids, isoleucine, leucine, and valine, are essential amino acids. In contrast to other amino acids, they are metabolized primarily by the peripheral tissues (particularly muscle), rather than by the liver. Because these three amino acids have a similar route of catabolism, it is convenient to describe them as a group (see Figure 20.10).
1. Transamination: Removal of the amino groups of all three amino acids is catalyzed by a single, vitamin Bg—requiring enzyme, branched-chain amino acid aminotransferase.
2. Oxidative decarboxylation: Removal of the carboxyl group of the a-keto acids derived from leucine, valine, and isoleucine is catalyzed by a single multienzyme complex, branched-chain a-keto acid dehydrogenase complex. This complex uses thiamine pyrophosphate, lipoic acid, FAD, NAD*, and CoA as its coenzymes. [Note: This reaction is similar to the conversion of pyruvate to acetyl CoA by pyruvate dehydrogenase (see p. 110) and the oxidation of a-ketoglutarate to succinyl CoA by a-ketoglutarate dehydrogenase (see p. 112).] An inherited deficiency of branched-chain a-keto acid dehydrogenase results in accumulation of the branched-chain o-keto acid substrates in the urine. Their sweet odor prompted the name maple syrup urine disease (see p. 272).
3. Dehydrogenation: Oxidation of the products formed in the above reaction yields o-B-unsaturated acyl CoA derivatives. This reaction is analogous to the FAD-linked dehydrogenation described in the B-oxidation scheme of fatty acid degradation (see p. 192). [Note: Deficiency in the dehydrogenase specific for isovaleryl CoA
causes neurological problems, and is associated with a "sweaty feet" odor in body fluids.]

4. End products: The catabolism of isoleucine ultimately yields acetyl CoA and succinyl CoA, rendering it both ketogenic and glucogenic. Valine yields succinyl CoA and is glucogenic. Leucine is ketogenic, being metabolized to acetoacetate and acetyl CoA. [Note: Branched-chain amino acid catabolism also results in glutamine and alanine being sent out into the blood from muscle.]

Some synthetic pathways require the addition of single carbon groups that exist in a variety of oxidation states, including formyl, methenyl, methylene, and methyl. These single carbon groups can be transferred from carrier compounds such as THF and SAM to specific structures that are being synthesized or modified. The "one-carbon pool" refers to single carbon units attached to this group of carriers. [Note: COz, the dehydrated form of carbonic acid, is carried by the vitamin biotin, which is a prosthetic group for most carboxylation reactions, but is not considered a member of the one-carbon pool. Defects in the ability to add or remove biotin from carboxylases result in multiple carboxylase deficiency; treatment is supplementation with biotin.]

The active form of folic acid, tetrahydrofolic acid (THF), is produced from folate by dihydrofolate reductase in a two-step reaction requiring two NADPH. The carbon unit carried by THF is bound to nitrogen N° or N'°, or to both N° and N'°. THF allows one-carbon compounds to be recognized and manipulated by biosynthetic enzymes. Figure 20.11 shows the structures of the various members of the THF family and their interconversions, and indicates the sources of the one-carbon units and the synthetic reactions in which the specific members participate. [Note: Folate deficiency presents as a megaloblastic anemia due to decreased availability of the purines and of the TMP needed for DNA synthesis (see p. 303).]

Nonessential amino acids are synthesized from intermediates of metabolism or, as in the case of tyrosine and cysteine, from the essential amino acids phenylalanine and methionine, respectively. The synthetic reactions for the nonessential amino acids are described below, and are summarized later in Figure 20.14. [Note: Some amino acids found in proteins, such as hydroxyproline and hydroxylysine (see p. 45), are modified after their incorporation into the protein (posttranslational modification, see p. 443).]

Alanine, aspartate, and glutamate are synthesized by transfer of an amino group to the o-keto acids pyruvate, oxaloacetate, and a-ketoglutarate, respectively. These transamination reactions (Figure 20.12, and see p. 250) are the most direct of the biosynthetic pathways.

Summary of the interconversions and uses of the carrier, tetra- hydrofolate. [Note: N5, N19-Methenyl- THF arises from 5-formimino-THF (see Figure 20.4).]

Formation of alanine, aspartate, and glutamate from the corresponding a-keto acids.

Incidence of inherited diseases of amino acid metabolism. [Note: Cystinuria is the most common genetic error of amino acid transport.]
Glutamate is unusual in that it can also be synthesized by the reverse of oxidative deamination, catalyzed by glutamate dehydrogenase (see p. 252).

1. Glutamine: This amino acid, which contains an amide linkage with ammonia at the y-carboxyl, is formed from glutamate by glutamine synthetase (see Figure 19.18, p. 256). The reaction is driven by the hydrolysis of ATP. In addition to producing glutamine for protein synthesis, the reaction also serves as a major mechanism for the transport of ammonia in a nontoxic form (see p. 256 for a discussion of ammonia metabolism).
- 2. Asparagine: This amino acid, which contains an amide linkage with ammonia at the B-carboxyl, is formed from aspartate by asparagine synthetase, using glutamine as the amide donor. The reaction requires ATP, and, like the synthesis of glutamine, has an equilibrium far in the direction of asparagine synthesis.

Glutamate is converted to proline by cyclization and reduction reactions.

1. Serine: This amino acid arises from 3-phosphoglycerate, an intermediate in glycolysis (see Figure 8.18, p. 101), which is first oxidized to 3-phosphopyruvate, and then transaminated to 3-phosphoserine. Serine is formed by hydrolysis of the phosphate ester. Serine can also be formed from glycine through transfer of a hydroxymethyl group by serine hydroxymethyl transferase using N°,N'°-methylene-THF as the one carbon donor (see Figure 20.6A).
2. Glycine: This amino acid is synthesized from serine by removal of a hydroxymethyl group, also by serine hydroxymethyl transferase (see Figure 20.6A). THF is the one carbon acceptor.
3. Cysteine: This amino acid is synthesized by two consecutive reactions in which homocysteine combines with serine, forming cystathionine that, in turn, is hydrolyzed to a-ketobutyrate and cysteine (see Figure 20.8). Homocysteine is derived from methionine as described on p. 264. Because methionine is an essential amino acid, cysteine synthesis can be sustained only if the dietary intake of methionine is adequate.

Tyrosine is formed from phenylalanine by phenylalanine hydroxylase. The reaction requires molecular oxygen and the coenzyme tetrahydrobiopterin (BH4), which can be synthesized from guanosine triphosphate (GTP) by the body. One atom of molecular oxygen becomes the hydroxyl group of tyrosine, and the other atom is reduced to water. During the reaction, BH, is oxidized to dihydrobiopterin (BH2). BH4 is regenerated from BH2 by NADH-requiring dihydropteridine reductase. Tyrosine, like cysteine, is formed from an essential amino acid and is, therefore, nonessential only in the presence of adequate dietary phenylalanine.

Summary of the metabolism of amino acids in humans. Genetically determined enzyme deficiencies are summarized in white boxes. Nitrogen-containing compounds derived from amino acids are shown in small, yellow boxes. Classification of amino acids is color coded: Red = glucogenic; brown = glucogenic and ketogenic; green = ketogenic. Compounds in BLUE ALL CAPS are the seven metabolites to which all amino acid metabolism converges.

A deficiency in phenylalanine hydroxylase results in the disease phenylketonuria (PKU).

Inborn errors of metabolism are commonly caused by mutant genes that generally result in abnormal proteins, most often enzymes. The inherited defects may be expressed as a total loss of enzyme activity or, more frequently, as a partial deficiency in catalytic activity. Without treatment, the inherited defects of amino acid metabolism almost invariably result in mental retardation or other developmental abnormalities as a consequence of harmful accumulation of metabolites. Although more than 50 of these disorders have been described, many are rare, occurring in less than 1 per 250,000 in most populations (Figure 20.13). Collectively, however, they constitute a very significant portion of pediatric genetic diseases (Figure 20.14). Phenylketonuria is the most important disease of amino acid metabolism because it is relatively common and responds to dietary treatment.
Screening of newborns for a number of the amino acid disorders by tandem mass spectrometry of blood obtained from a heel prick is possible; however, exactly which disorders are screened for currently varies from state to state, and only phenylketonuria screening is mandated by all states.

Phenylketonuria (PKU), caused by a deficiency of phenylalanine hydroxylase (Figure 20.15), is the most common clinically encountered inborn error of amino acid metabolism (prevalence 1:15,000). Biochemically, it is characterized by accumulation of phenylalanine (and a deficiency of tyrosine). Hyperphenylalaninemia may also be caused by deficiencies in any of the several enzymes required to synthesize BH,, or in dihydropteridine reductase, which regenerates BH, from BHo2 (Figure 20.16). Such deficiencies indirectly raise phenylalanine concentrations, because phenylalanine hydroxylase

Biosynthetic reactions involving amino acids and tetrahydrobiopterin.
requires BH, as a coenzyme. BHsg is also required for tyrosine hydroxylase and tryptophan hydroxylase, which catalyze reactions leading to the synthesis of neurotransmitters, such as serotonin and the catecholamines. Simply restricting dietary phenylalanine does not reverse the central nervous system (CNS) effects due to deficiencies in neurotransmitters. Replacement therapy with BH4 or L-DOPA and 5-hydroxytryptophan (products of the affected tyrosine hydroxylase— and tryptophan hydroxylase-catalyzed reactions) improves the clinical outcome in these variant forms of hyperphenylalaninemia, although the response is unpredictable.

a. Elevated phenylalanine: Phenylalanine is present in elevated concentrations in tissues, plasma, and urine. Phenyllactate, phenylacetate, and phenylpyruvate, which are not normally produced in significant amounts in the presence of functional phenylalanine hydroxylase, are also elevated in PKU (Figure 20.17). These metabolites give urine a characteristic musty ("mousey") odor. [Note: The disease acquired its name from the presence of a phenylketone (now known to be phenylpyruvate) in the urine.]
b. CNS symptoms: Mental retardation, failure to walk or talk, seizures, hyperactivity, tremor, microcephaly, and failure to grow are characteristic findings in PKU. The patient with untreated PKU typically shows symptoms of mental retardation by the age of 1 year, and rarely achieves an IQ greater than 50 (Figure 20.18). [Note: These clinical manifestations are now rarely seen as a result of neonatal screening programs.]
c. Hypopigmentation: Patients with phenylketonuria often show a deficiency of pigmentation (fair hair, light skin color, and blue eyes). The hydroxylation of tyrosine by tyrosinase, which is the first step in the formation of the pigment melanin, is competitively inhibited by the high levels of phenylalanine present in PKU.
2. Neonatal screening and diagnosis of PKU: Early diagnosis of phenylketonuria is important because the disease is treatable by dietary means. Because of the lack of neonatal symptoms, laboratory testing for elevated blood levels of phenylalanine is mandatory for detection. However, the infant with PKU frequently has normal blood levels of phenylalanine at birth because the mother clears increased blood phenylalanine in her affected fetus through the placenta. Normal levels of phenylalanine may persist until the newborn is exposed to 24-48 hours of protein feeding. Thus, screening tests are typically done after this time to avoid false negatives. For newborns with a positive screening test, diagnosis is confirmed through quantitative determination of phenylalanine levels.

3. Prenatal diagnosis of PKU: Classic PKU is a family of diseases caused by any of 100 or more different mutations in the gene that codes for phenylalanine hydroxylase (PAH). The frequency of any given mutation varies among populations, and the disease is often doubly heterozygous, that is, the PAH gene has a different mutation in each allele. Despite this complexity, prenatal diagnosis is possible (see p. 477).

Pathways of phenylalanine metabolism in normal individuals and in patients with phenylketonuria.

Typical intellectual ability in untreated PKU patients of different ages.

Changes in IQ scores after discon- tinuation of low-phenylalanine diet in patients with phenylketonuria.
4.
Treatment of PKU: Most natural protein contains phenylalanine, and it is impossible to satisfy the body's protein requirement when ingesting a normal diet without exceeding the phenylalanine limit. Therefore, in PKU, blood phenylalanine is maintained close to the normal range by feeding synthetic amino acid preparations low in phenylalanine, supplemented with some natural foods (such as fruits, vegetables, and certain cereals) selected for their low phenylalanine content. The amount is adjusted according to the tolerance of the individual as measured by blood phenylalanine levels. The earlier treatment is started, the more completely neurologic damage can be prevented. [Note: Treatment must begin during the first 7-10 days of life to prevent mental retardation.] Because phenylalanine is an essential amino acid, overzealous treatment that results in blood phenylalanine levels below normal is avoided because this can lead to poor growth and neurologic symptoms. In patients with PKU, tyrosine cannot be synthesized from phenylalanine and, therefore, it becomes an essential amino acid and so must be supplied in the diet. Discontinuance of the phenyalanine-restricted diet before 8 years of age is associated with poor performance on IQ tests. Adult PKU patients show deterioration of IQ scores after discontinuation of the diet (Figure 20.19). Lifelong restriction of dietary phenylalanine is, therefore, recommended. [Note: Individuals with PKU are advised to avoid aspartame, an artificial sweetener that contains phenylalanine.]
5. Maternal PKU: If women with PKU who are not on a low-phenylalanine diet become pregnant, the offspring are affected with "maternal PKU syndrome." High blood phenylalanine levels in the mother cause microcephaly, mental retardation, and congenital heart abnormalities in the fetus—phenlyalanine is a teratogen. Some of these developmental responses to high phenylalanine occur during the first months of pregnancy. Thus, dietary control of blood phenylalanine must begin prior to conception, and must be maintained throughout the pregnancy.

Maple syrup urine disease (MSUD) is a rare (1:185,000), autosomal recessive disorder in which there is a partial or complete deficiency in branched-chain a-keto acid dehydrogenase, an enzyme complex that decarboxylates leucine, isoleucine, and valine (see Figure 20.10). These amino acids and their corresponding a-keto acids accumulate in the blood, causing a toxic effect that interferes with brain functions. The disease is characterized by feeding problems, vomiting, dehydration, severe metabolic acidosis, and a characteristic maple syrup odor to the urine. If untreated, the disease leads to mental retardation, physical disabilities, and even death.
1. Classification: The term "maple syrup urine disease" includes a classic type and several variant forms of the disorder. The classic form is the most common type of MSUD. Leukocytes or cultured skin fibroblasts from these patients show little or no branchedchain a-keto acid dehydrogenase activity. Infants with classic MSUD show symptoms within the first several days of life. If not diagnosed and treated, classic MSUD is lethal in the first weeks
of life. Patients with intermediate forms have a higher level of enzyme activity (approximately 3-15% of normal). The symptoms are milder and show an onset from infancy to adulthood. Patients with the rare thiamine-dependent variant of MSUD achieve increased activity of branched-chain a-keto acid dehydrogenase if given large doses of this vitamin.
2. Screening and diagnosis: As with PKU, prenatal diagnosis and neonatal screening are available, and most affected individuals are compound heterozygotes.
3. Treatment: The disease is treated with a synthetic formula that contains limited amounts of leucine, isoleucine, and valine—sufficient to provide the branched-chain amino acids necessary for normal growth and development without producing toxic levels. Early diagnosis and lifelong dietary treatment is essential if the child with MSUD is to develop normally. [Note: Branched-chain amino acids are an important energy source in times of metabolic need, and individuals with MSUD are at risk of decompensation during periods of increased protein catabolism.]

Albinism refers to a group of conditions in which a defect in tyrosine metabolism results in a deficiency in the production of melanin. These defects result in the partial or full absence of pigment from the skin, hair, and eyes. Albinism appears in different forms, and it may be inherited by one of several modes: autosomal recessive (primary mode), autosomal dominant, or X-linked. Complete albinism (also called tyrosinase-negative oculocutaneous albinism) results from a deficiency of copper-requiring tyrosinase, causing a total absence of pigment from the hair, eyes, and skin (Figure 20.20). It is the most severe form of the condition. In addition to hypopigmentation, affected individuals have vision defects and photophobia (sunlight hurts their eyes). They are at increased risk for skin cancer.

The homocystinurias are a group of disorders involving defects in the metabolism of homocysteine. The diseases are inherited as autosomal recessive illnesses, characterized by high plasma and urinary levels of homocysteine and methionine and low levels of cysteine. The most common cause of homocystinuria is a defect in the enzyme cystathionine B-synthase, which converts homocysteine to cystathionine (Figure 20.21). Individuals who are homozygous for cystathionine B-synthase deficiency exhibit ectopia lentis (displacement of the lens of the eye), skeletal abnormalities, a tendency to form thrombi (blood clots), osteoporosis, and neurological deficits. Patients can be responsive or nonresponsive to oral administration of pyridoxine (vitamin Bg)—a coenzyme of cystathionine B-synthase. Vitamin Be—-responsive patients usually have a milder and later onset of clinical symptoms compared with Bg-nonresponsive patients. Treatment includes restriction of methionine intake and supplementation with vitamins Bg, B12, and folate.

Patient with oculocutaneous albinism, showing white eyebrows and lashes.

Alkaptonuria is a rare metabolic condition involving a deficiency in homogentisic acid oxidase, resulting in the accumulation of homogentisic acid. [Note: This reaction occurs in the degradative pathway of tyrosine, p. 269.] The condition has three characteristic symptoms: homogentisic aciduria (the patient's urine contains elevated levels of homogentisic acid, which is oxidized to a dark pigment on standing, Figure 20.22A), large joint arthritis, and black ochronotic pigmentation of cartilage and collagenous tissue (Figure 20.22B). Patients with alkaptonuria are usually asymptomatic until about age 40. Dark staining of the diapers sometimes can indicate the disease in infants, but usually no symptoms are present until later in life. Diets low in protein—especially in phenylalanine and tyrosine—help reduce the levels of homogentisic acid, and decrease the amount of pigment deposited in body tissues. Although alkaptonuria is not life-threatening, the associated arthritis may be severely crippling.

Amino acids whose catabolism yields pyruvate or one of the intermediates of the tricarboxylic acid cycle are termed glucogenic. (Figure 20.23).They can give rise to the net formation of glucose in the liver and the kidney. The solely glucogenic amino acids are glutamine, glutamate, proline, arginine, histidine, alanine, serine, glycine, cysteine, methionine, valine, threonine, aspartate, and asparagine. Amino acids whose catabolism yields either acetoacetate or one of its precursors, acetyl coenzyme A (CoA) or acetoacetyl CoA, are termed ketogenic. Leucine and lysine are solely ketogenic. Tyrosine, phenylalanine, tryptophan, and isoleucine are both ketogenic and glucogenic. Nonessential amino acids can be synthesized from metabolic intermediates, or from the carbon skeletons of essential amino acids. Essential amino acids need to be obtained from the diet. They include histidine, methionine, threonine, valine, isoleucine, phenylalanine, tryptophan, leucine and lysine. Phenylketonuria (PKU) is caused by a deficiency of phenylalanine hydroxylase—the enzyme that converts phenylalanine to tyrosine. Hyperphenylalaninemia may also be caused by deficiencies in the enzymes that synthesize or reduce the hydroxylase's coenzyme, tetrahydrobiopterin. Untreated patients with PKU suffer from mental retardation, failure to walk or talk, seizures, hyperactivity, tremor, microcephaly, failure to grow and a characteristic smell of the urine. Treatment involves controlling dietary phenylalanine. Note that tyrosine becomes an essential dietary component for people with PKU. Maple syrup urine disease (MSUD) is a recessive disorder in which there is a partial or complete deficiency in branched-chain o-keto acid dehydrogenase—an enzyme that decarboxylates leucine, isoleucine, and valine. Symptoms include feeding problems, vomiting, dehydration, severe metabolic acidosis, and a characteristic smell of the urine. If untreated, the disease leads to mental retardation, physical

disabilities, and death. Treatment of MSUD involves a synthetic formula that contains limited amounts of leucine, isoleucine, and valine. Other important genetic diseases associated with amino acid metabolism include albinism, homocystinuria, methylmalonyl CoA mutase deficiency, alkaptonuria, histidinemia, tyrosinemia, and cystathioninuria.

- 20.1 Which one of the following statements concerning
amino acids is correct?
- A. An increase in gluconeogenesis from amino acids
results in a decrease in urea formation.
B. All essential amino acids are glycogenic.
C. Ornithine and citrulline are found in tissue proteins.
D. Cysteine is an essential amino acid in individuals
consuming a diet severely limited in methionine.
E.
In the presence of adequate dietary sources of tyrosine, phenylalanine is not an essential amino acid.
20.2 Which one of the following statements concerning a
1-week-old male infant with undetected classic phenylketonuria is correct?
A. Tyrosine is a nonessential amino acid for the infant.
B. High levels of phenylpyruvate appear in his urine.
C. Therapy must begin within the first year of life.
D. A diet devoid of phenylalanine should be initiated immediately.
E. When the infant reaches adulthood, it is recommended that diet therapy be discontinued.
A 4-year-old boy of a first-degree consanguineous couple was noted by the parents to have darkening of the urine to an almost black color when it was left standing. He has a normal sibling, and there are no other medical problems. Growth and development to date are normal. Which of the following is most likely to be elevated in this patient?
A. Methylmalonate.
B. Homogentisate.
C. Phenylpyruvate.
D. a-Ketoisovalerate.
E. Homocysteine.
20.4 What enzyme is deficient in: homocystinuria, methylmalonic acidemia, MSUD, oculocutaneous albinism, PKU?
20.3

Correct answer = D. Methionine is the precursor of cysteine. An increase in the availability of gluconeogenic amino acids from the catabolism of body protein is associated with increased ammonia and results in increased urea production. The essential amino acids leucine and lysine are ketogenic. Ornithine and citrulline are amino acids that are intermediates in the urea cycle, but are not found in tissue proteins because there are no codons for them. Phenylalanine is essential regardless of the level of tyrosine.
Correct answer = B. Phenyllactate, phenylacetate, and phenylpyruvate, which are not normally produced in significant amounts in the presence of functional phenylalanine hydroxylase, are elevated in PKU, and appear in the urine. In patients with PKU, tyrosine cannot be synthesized from phenylalanine and, hence, becomes essential and must be supplied in the diet. Treatment must begin during the first 7-10 days of life to prevent mental retardation. Discontinuance of the phenylalanine-restricted diet before 8 years of age is associated with poor performance on IQ tests. Adult PKU patients show deterioration of attention and speed of mental processing after discontinuation of the diet. Elevated levels of phenylalanine are teratogenic. Lifelong restriction of dietary phenylalanine is, therefore, recommended.
Correct answer = B. Alkaptonuria is a rare metabolic disease involving a deficiency in homogentisic acid oxidase, and the subsequent accumulation of homogentisic acid in the urine, which turns dark upon standing. The elevation of methylmalonate (due to methylmalony! CoA mutase deficiency), phenylpyruvate (due to phenylalanine hydroxylase deficiency), aketoisovalerate (due to branched-chain a-keto acid dehydrogenase deficiency), and homocysteine (due to cystathionine B-synthase deficiency) are inconsistent with a healthy child with darkening of the urine.

Answer: Cystathionine B-synthase, methylmalonyl CoA mutase, branched-chain a-keto acid dehydrogenase, tyrosinase, phenylanine hydroxylase. [Note:Deficiency of dihydropteridine reductase or any of the enzymes needed for BH4 synthesis can also result in hyperphenylalaninemia.]

In addition to serving as building blocks for proteins, amino acids are precursors of many nitrogen-containing compounds that have important physiologic functions (Figure 21.1). These molecules include porphyrins, neurotransmitters, hormones, purines, and pyrimidines.

Porphyrins are cyclic compounds that readily bind metal ions—usually Fe** or Fe°+. The most prevalent metalloporphyrin in humans is heme, which consists of one ferrous (Fe**) iron ion coordinated in the center of the tetrapyrrole ring of protoporphyrin IX (see p. 280). Heme is the prosthetic group for hemoglobin, myoglobin, the cytochromes, catalase, nitric oxide synthase, and peroxidase. These hemeproteins are rapidly synthesized and degraded. For example, 6-7 g of hemoglobin are synthesized each day to replace heme lost through the normal turnover of erythrocytes. Coordinated with the turnover of hemeproteins is the simultaneous synthesis and degradation of the associated porphyrins, and recycling of the bound iron ions.

Porphyrins are cyclic molecules formed by the linkage of four pyrrole rings through methenyl bridges (Figure 21.2). Three structural features of these molecules are relevant to understanding their medical significance.
1. Side chains: Different porphyrins vary in the nature of the side chains that are attached to each of the four pyrrole rings. Uroporphyrin contains acetate (-CH2-COO ) and propionate (—CHz—CH2—COO ) side chains, coproporphyrin contains methyl (—CHs) and propionate groups, and protoporphyrin IX (and heme) contains vinyl (-CH=CH2), methyl, and propionate groups. [Note: The methyl and vinyl groups are produced by decarboxylation of acetate and propionate side chains, respectively.]

Structures of uroporphyrin | and uroporphyrin Ill. A = acetate and P = propionate.
2.

Pathway of porphyrin synthesis: Formation of porphobilinogen. ALAS = 6-aminolevulinic acid synthase. (Continued in Figures 21.4 and 21.5.)
Distribution of side chains: The side chains of porphyrins can be ordered around the tetrapyrrole nucleus in four different ways, designated by Roman numerals | to IV. Only Type III porphyrins, which contain an asymmetric substitution on ring D (see Figure 21.2), are physiologically important in humans. [Note: Protoporphyrin IX is a member of the Type III series.]
- 3. Porphyrinogens: These porphyrin precursors (for example, uroporphyrinogen) exist in a chemically reduced, colorless form, and serve as intermediates between porphobilinogen and the oxidized, colored protoporphyrins in heme biosynthesis.

The major sites of heme biosynthesis are the liver, which synthesizes a number of heme proteins (particularly cytochrome P450 proteins), and the erythrocyte-producing cells of the bone marrow, which are active in hemoglobin synthesis. [Note: Over 85% of all heme synthesis occurs in erythroid tissue.] In the liver, the rate of heme synthesis is highly variable, responding to alterations in the cellular heme pool caused by fluctuating demands for heme proteins. In contrast, heme synthesis in erythroid cells is relatively constant, and is matched to the rate of globin synthesis. The initial reaction and the last three steps in the formation of porphyrins occur in mitochondria, whereas the intermediate steps of the biosynthetic pathway occur in the cytosol (see Figure 21.8). [Note: Mature red blood cells lack mitochondria and are unable to synthesize heme.]
1.
Formation of 5-aminolevulinic acid (ALA): All the carbon and nitrogen atoms of the porphyrin molecule are provided by glycine (a nonessential amino acid) and succinyl coenzyme A (an intermediate in the citric acid cycle) that condense to form ALA ina reaction catalyzed by ALA synthase (ALAS) (Figure 21.3) This reaction requires pyridoxal phosphate (PLP) as a coenzyme, and is the committed and rate-limiting step in porphyrin biosynthesis. [Note: There are two isoforms of ALAS, 1 and 2, each controlled by different mechanisms. Erythroid tissue produces only ALAS2, the gene for which is located on the X-chromosome. Loss of function mutations in ALAS2 result in X-linked sideroblastic anemia.]
a. End-product inhibition of ALAS1 by hemin: When porphyrin production exceeds the availability of the apoproteins that

require it, heme accumulates and is converted to hemin by the oxidation of Fe** to Fe°+. Hemin decreases the activity of hepatic ALAS7 by causing decreased synthesis of the enzyme, through inhibition of MRNA synthesis and use (heme decreases stability of the mRNA), and by inhibiting mitochondrial import of the enzyme. [Note: In erythroid cells, ALAS2 is controlled by the availability of intracellular iron.]

b. Effect of drugs on ALA synthase activity: Administration of any of a large number of drugs results in a significant increase in hepatic ALAS7 activity. These drugs are metabolized by the microsomal cytochrome P450 monooxygenase system—a hemeprotein oxidase system found in the liver (see p. 149). In response to these drugs, the synthesis of cytochrome P450 proteins increases, leading to an enhanced consumption of heme—a component of cytochrome P450 proteins. This, in turn, causes a decrease in the concentration of heme in liver cells. The lower intracellular heme concentration leads to an increase in the synthesis of ALAS7 (derepression), and prompts a corresponding increase in ALA synthesis.
2. Formation of porphobilinogen: The condensation of two molecules of ALA to form porphobilinogen by Zn-containing ALA dehyadratase (porphobilinogen synthase) is extremely sensitive to inhibition by heavy metal ions, for example, lead that replace the zinc (see Figure 21.3). This inhibition is, in part, responsible for the elevation in ALA and the anemia seen in lead poisoning.
3. Formation of uroporphyrinogen: The condensation of four porphobilinogens produces the linear tetrapyrrole, hydroxymethylbilane, which is isomerized and cyclized by uroporphyrinogen III synthase to produce the asymmetric uroporphyrinogen Ill. This cyclic tetrapyrrole undergoes decarboxylation of its acetate groups, generating coproporphyrinogen III (Figure 21.4). These reactions occur in the cytosol.
4. Formation of heme: Coproporphyrinogen III enters the mitochondrion, and two propionate side chains are decarboxylated to vinyl groups generating protoporphyrinogen IX, which is oxidized to protoporphyrin IX. The introduction of iron (as Fe*) into protoporphyrin IX occurs spontaneously, but the rate is enhanced by ferrochelatase, an enzyme that, like ALA dehydratase, is inhibited by lead (Figure 21.5).

Porphyrias are rare, inherited (or occasionally acquired) defects in heme synthesis, resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors (see Figure 21.8). [Note: With few exceptions, porphyrias are inherited as autosomal dominant disorders.] The mutations that cause the porphyrias are heterogenous (not all are at the same DNA locus), and nearly every affected family has its own mutation. Each porphyria results in the accumulation of a unique pattern of intermediates caused by the deficiency of an enzyme in the heme synthetic pathway. [Note: "Porphyria" refers to the purple color caused by pigment-like porphyrins in the urine of some patients with defects in heme synthesis. ]

Pathway of porphyrin synthesis: Formation of protoporphyrin IX. (Continued from Figure 21.3.)

Pathway of porphyrin synthesis: Formation of heme. (Continued from Figures 21.3 and 21.4)

Urine from a patient with porphyria cutanea tarda (right) and from a patient with normal porphyrin excretion (left).
1.
Clinical manifestations: The porphyrias are classified as erythropoietic or hepatic, depending on whether the enzyme deficiency occurs in the erythropoietic cells of the bone marrow or in the liver. Hepatic porphyrias can be further classified as chronic or acute. In general, individuals with an enzyme defect prior to the synthesis of the tetrapyrroles manifest abdominal and neuropsychiatric signs, whereas those with enzyme defects leading to the accumulation of tetrapyrrole intermediates show photosensitivity—that is, their skin itches and burns (pruritus) when exposed to visible light. [Note: Photosenstivity is a result of the oxidation of colorless porphyrinogens to colored porphyrins, which are photosensitizing molecules that are thought to participate in the formation of superoxide radicals from oxygen. These reactive oxygen species can oxidatively damage membranes, and cause the release of destructive enzymes from lysosomes.]
a. Chronic hepatic porphyria: Porphyria cutanea tarda, the most common porphyria, is a chronic disease of the liver. The disease is associated with a deficiency in uroporphyrinogen decarboxylase, but clinical expression of the enzyme deficiency is influenced by various factors, such as hepatic iron overload, exposure to sunlight, alcohol ingestion, and the presence of hepatitis B or C, or HIV infections. Clinical onset is typically during the fourth or fifth decade of life. Porphyrin accumulation leads to cutaneous symptoms (Figure 21.6), and urine that is red to brown in natural light (Figure 21.7), and pink to red in fluorescent light.
b.

Acute hepatic porphyrias: Acute hepatic porphyrias (ALA dehydratase deficiency, acute intermittent porphyria, hereditary coproporphyria, and variegate porphyria) are characterized by acute attacks of gastrointestinal, neuropsychiatric, and motor symptoms that may be accompanied by photosensitivity. Porphyrias leading to accumulation of ALA and porphobilinogen, such as acute intermittent porphyria, cause abdominal pain and neuropsychiatric disturbances, ranging from anxiety to delirium. Symptoms of the acute hepatic porphyrias are often precipitated by administration of drugs such as barbiturates and ethanol, which induce the synthesis of the hemecontaining cytochrome P450 microsomal drug oxidation system. This further decreases the amount of available heme, which, in turn, promotes the increased synthesis of ALAS7.
c. Erythropoietic porphyrias: The erythropoietic porphyrias (congenital erythropoietic porphyria and erythropoietic protoporphyria) are characterized by skin rashes and blisters that appear in early childhood. The diseases are complicated by cholestatic liver cirrhosis and progressive hepatic failure.
. Increased ALA synthase activity: One common feature of the porphyrias is a decreased synthesis of heme. In the liver, heme normally functions as a repressor of the gene for ALAS7. Therefore, the absence of this end product results in an increase in the synthesis of ALA synthase? (derepression). This causes an increased synthesis of intermediates that occur prior to the genetic block. The accumulation of these toxic intermediates is the major pathophysiology of the porphyrias.

Summary of heme synthesis. 'Also referred to as porphobilinogen deaminase.

Formation of bilirubin from heme. UDP = uridine diphosphate.
3. Treatment: During acute porphyria attacks, patients require medical support, particularly treatment for pain and vomiting. The severity of symptoms of the porphyrias can be diminished by intravenous injection of hemin and glucose, which decreases the synthesis of ALAS1. Avoidance of sunlight and ingestion of B-carotene (a free-radical scavenger) are helpful in porphyrias with photosensitivity.

After approximately 120 days in the circulation, red blood cells are taken up and degraded by the reticuloendothelial system, particularly in the liver and spleen (Figure 21.9). Approximately 85% of heme destined for degradation comes from senescent red blood cells, and 15% is from turnover of immature red blood cells and cytochromes from nonerythroid tissues.
- 1. Formation of bilirubin: The first step in the degradation of heme is catalyzed by the microsomal heme oxygenase system of the reticuloendothelial cells. In the presence of NADPH and Oz, the enzyme adds a hydroxyl group to the methenyl bridge between two pyrrole rings, with a concomitant oxidation of ferrous iron to Fe. A second oxidation by the same enzyme system results in cleavage of the porphyrin ring. The green pigment biliverdin is produced as ferric iron and CO are released (see Figure 21.9). [Note: The CO has biologic function, acting as a signaling molecule and vasodilator.] Biliverdin is reduced, forming the redorange bilirubin. Bilirubin and its derivatives are collectively termed bile pigments. [Note: The changing colors of a bruise reflect the varying pattern of intermediates that occurs during heme degradation.]
Bilirubin, unique to mammals, appears to function as an antioxidant. In this role, it is oxidized to biliverdin, which is then reduced by biliverdin reductase, regenerating bilirubin.

2. Uptake of bilirubin by the liver: Bilirubin is only slightly soluble in plasma and, therefore, is transported to the liver by binding noncovalently to albumin. [Note: Certain anionic drugs, such as salicylates and sulfonamides, can displace bilirubin from albumin, permitting bilirubin to enter the central nervous system. This causes the potential for neural damage in infants.] Bilirubin dissociates from the carrier albumin molecule, enters a hepatocyte via facilitated diffusion, and binds to intracellular proteins, particularly the protein ligandin.
3. Formation of bilirubin diglucuronide: In the hepatocyte, the solubility of bilirubin is increased by the addition of two molecules of glucuronic acid. [Note: This process is referred to as conjugation.] The reaction is catalyzed by microsomal bilirubin glucuronyltransferase using uridine diphosphate-glucuronic acid as the glucuronate donor. [Note: Varying degrees of deficiency of this enzyme result in Crigler-Najjar | and II and Gilbert syndrome, with Crigler-Najjar | being the most severe deficiency.]

Catabolism of heme @ = bilirubin; Ha = bilirubin diglucuronide; © = urobilinogen; @ = urobilin; A = stercobilin.
4. Secretion of bilirubin into bile: Bilirubin diglucuronide (conjugated bilirubin) is actively transported against a concentration gradient into the bile canaliculi and then into the bile. This energy-dependent, rate-limiting step is susceptible to impairment in liver disease. [Note: A deficiency in the protein required for transport of conjugated bilirubin out of the liver results in Dubin-Johnson syndrome.] Unconjugated bilirubin is normally not secreted.
- 5. Formation of urobilins in the intestine: Bilirubin diglucuronide is hydrolyzed and reduced by bacteria in the gut to yield urobilinogen, a colorless compound. Most of the urobilinogen is oxidized by intestinal bacteria to stercobilin, which gives feces the characteristic brown color. However, some of the urobilinogen is reabsorbed from the gut and enters the portal blood. A portion of

Jaundiced patient, with the sclerae of his eyes appearing yellow.
BUTIBIA

oT : A ramolstic jaundice D. Neonatal leu ndice. BG = bilirubin glucuronide; B = bilirubin;
U = urobilinogen; S = stercobilin.
this urobilinogen participates in the enterohepatic urobilinogen cycle in which it is taken up by the liver, and then resecreted into the bile. The remainder of the urobilinogen is transported by the blood to the kidney, where it is converted to yellow urobilin and excreted, giving urine its characteristic color. The metabolism of bilirubin is summarized in Figure 21.10.

Jaundice (also called icterus) refers to the yellow color of skin, nail beds, and sclerae (whites of the eyes) caused by deposition of bilirubin, secondary to increased bilirubin levels in the blood (hyperbilirubinemia, Figure 21.11). Although not a disease, jaundice is usually a symptom of an underlying disorder.
- 1. Types of jaundice: Jaundice can be classified into three major forms described below. However, in clinical practice, jaundice is often more complex than indicated in this simple classification. For example, the accumulation of bilirubin may be a result of defects at more than one step in its metabolism.
a. Hemolytic jaundice: The liver has the capacity to conjugate and excrete over 3,000 mg of bilirubin per day, whereas the normal production of bilirubin is only 300 mg/day. This excess capacity allows the liver to respond to increased heme degradation with a corresponding increase in conjugation and secretion of bilirubin diglucuronide. However, massive lysis of red blood cells (for example, in patients with sickle cell anemia, pyruvate kinase or glucose 6-phosphate dehydrogenase deficiency) may produce bilirubin faster than it can be conjugated. Unconjugated bilirubin levels in the blood become elevated, causing jaundice (Figure 21.12A). [Note: More conjugated bilirubin is excreted into the bile, the amount of urobilinogen entering the enterohepatic circulation is increased, and urinary urobilinogen is increased.]

- b. Hepatocellular jaundice: Damage to liver cells (for example, in patients with cirrhosis or hepatitis) can cause unconjugated bilirubin levels in the blood to increase as a result of decreased conjugation. Urobilinogen is increased in the urine because hepatic damage decreases the enterohepatic circulation of this compound, allowing more to enter the blood, from which it is filtered into the urine. The urine thus darkens, whereas stools may be a pale, clay color. Plasma levels of AST and ALT (see p. 251) are elevated. [Note: If conjugated bilirubin is not efficiently secreted from the liver into bile (intrahepatic cholestasis), it can diffuse ("leak") into the blood, causing a conjugated hyperbilirubinemia.]
c. Obstructive jaundice: |n this instance, jaundice is not caused by overproduction of bilirubin or decreased conjugation, but instead results from obstruction of the bile duct (extrahepatic cholestasis). For example, the presence of a tumor or bile stones may block the bile ducts, preventing passage of bilirubin into the intestine. Patients with obstructive jaundice experience gastrointestinal pain and nausea, and produce stools that are a pale, clay color, and urine that darkens upon standing. The liver "regurgitates" conjugated bilirubin into the blood
(hyperbilirubinemia). The compound is eventually excreted in the urine. Urinary urobiloinogen is absent. [Note: Prolonged obstruction of the bile duct can lead to liver damage and a subsequent rise in unconjugated bilirubin.]

2. Jaundice in newborns: Newborn infants, particularly if premature, often accumulate bilirubin, because the activity of hepatic bilirubin glucuronyltransferase is low at birth—it reaches adult levels in about 4 weeks (Figures 21.12B and 21.13). Elevated bilirubin, in excess of the binding capacity of albumin, can diffuse into the basal ganglia and cause toxic encephalopathy (kernicterus). Thus, newborns with significantly elevated bilirubin levels are treated with blue fluorescent light (Figure 21.14), which converts bilirubin to more polar and, hence, water-soluble isomers. These photoisomers can be excreted into the bile without conjugation to glucuronic acid.
3. Determination of bilirubin concentration: Bilirubin is most commonly determined by the van den Bergh reaction, in which diazotized sulfanilic acid reacts with bilirubin to form red azodipyrroles that are measured colorimetrically. In aqueous solution, the watersoluble, conjugated bilirubin reacts rapidly with the reagent (within one minute), and is said to be "direct-reacting." The unconjugated bilirubin, which is much less soluble in aqueous solution, reacts more slowly. However, when the reaction is carried out in methanol, both conjugated and unconjugated bilirubin are soluble and react with the reagent, providing the total bilirubin value. The "indirect-reacting" bilirubin, which corresponds to the unconjugated bilirubin, is obtained by subtracting the direct-reacting bilirubin from the total bilirubin. [Note: In normal plasma, only about 4% of the total bilirubin is conjugated or direct-reacting, because most is secreted into bile.]

Dopamine, norepinephrine, and epinephrine are biologically active (biogenic) amines that are collectively termed catecholamines. Dopamine and norepinephrine are synthesized in the brain and function as neurotransmitters. Norepinephrine is also synthesized in the adrenal as is medulla, epinephrine.
1. Function: Outside the nervous system, norepinephrine and its methylated derivative, epinephrine, are hormone regulators of carbohydrate and lipid metabolism. Norepinephrine and epinephrine are released from storage vesicles in the adrenal medulla in response to fright, exercise, cold, and low levels of blood glucose. They increase the degradation of glycogen and triacylglycerol, as well as increase blood pressure and the output of the heart. These effects are part of a coordinated response to prepare the individual for stress, and are often called the "fight-or-flight" reactions.
2. Synthesis of catecholamines: The catecholamines are synthesized from tyrosine, as shown in Figure 21.15. Tyrosine is first hydroxylated by tyrosine hydroxylase to form 3,4-dihydroxyphenyl-

Metabolism of the catecholamines by catechol-O-methyltranferase (COMT) and monoamine oxidase (MAO).
alanine (DOPA) in a reaction analogous to that described for the hydroxylation of phenylalanine (see p. 268). The tetrahydrobiopterin (BH4)-requiring enzyme is abundant in the central nervous system, the sympathetic ganglia, and the adrenal medulla, and is the rate-limiting step of the pathway. DOPA is decarboxylated in a reaction requiring pyridoxal phosphate (PLP, see p. 378) to form dopamine, which is hydroxylated by dopamine B-hydroxylase to yield norepinephrine in a reaction that requires ascorbate (vitamin C) and copper. Epinephrine is formed from norepinephrine by an N-methylation reaction using S-adenosylmethionine (SAM) as the methyl donor (see p. 264).
Parkinson disease, a neurodegenerative movement disorder, is due to insufficient dopamine production as a result of the idiopathic loss of dopamine-producing cells in the brain. Administration of L-DOPA (levodopa) is the most common treatment.
3. Degradation of catecholamines: The catecholamines are inactivated by oxidative deamination catalyzed by monoamine oxidase (MAO), and by O-methylation carried out by catechol-O-methyltransferase using SAM as the methyl donor (Figure 21.16). The two reactions can occur in either order. The aldehyde products of the MAO reaction are oxidized to the corresponding acids. The metabolic products of these reactions are excreted in the urine as vanillylmandelic acid (VMA) from epinephrine and norepinephrine, and homovanillic acid from dopamine. [Note: VMA is increased with pheochromocytomas, tumors of the adrenal characterized by excessive production of catecholamines.]
4. MAO inhibitors: MAO is found in neural and other tissues, such as the intestine and liver. In the neuron, this enzyme oxidatively deaminates and inactivates any excess neurotransmitter molecules (norepinephrine, dopamine, or serotonin) that may

leak out of synaptic vesicles when the neuron is at rest. MAO inhibitors may irreversibly or reversibly inactivate the enzyme, permitting neurotransmitter molecules to escape degradation and, therefore, to both accumulate within the presynaptic neuron and to leak into the synaptic space. This causes activation of norepinephrine and serotonin receptors, and may be responsible for the antidepressant action of these drugs.

Histamine is a chemical messenger that mediates a wide range of cellular responses, including allergic and inflammatory reactions, gastric acid secretion, and possibly neurotransmission in parts of the brain. A powerful vasodilator, histamine is formed by decarboxylation of histidine in a reaction requiring PLP (Figure 21.17). It is secreted by mast cells as a result of allergic reactions or trauma. Histamine has no clinical applications, but agents that interfere with the action of histamine have important therapeutic applications.

Serotonin, also called 5-hydroxytryptamine (S5HT), is synthesized and stored at several sites in the body (Figure 21.18). By far the largest amount of serotonin is found in cells of the intestinal mucosa. Smaller amounts occur in the central nervous system, where it functions as a neurotransmitter, and in platelets. Serotonin is synthesized from tryptophan, which is hydroxylated in a BH,requiring reaction analogous to that catalyzed by phenylalanine hydroxylase. The product, 5-hydroxytryptophan, is decarboxylated to serotonin, which is also degraded by MAO. Serotonin has multiple physiologic roles, including pain perception, regulation of sleep, appetite, temperature, blood pressure, cognitive functions, and mood (causes a feeling of well-being). [Note: Serotonin is converted to melatonin in the pineal gland via acetylation and methylation.]

Creatine phosphate (also called phosphocreatine), the phosphorylated derivative of creatine found in muscle, is a high-energy compound that provides a small but rapidly mobilized reserve of high-energy phosphates that can be reversibly transferred to ADP (Figure 21.9) to maintain the intracellular level of ATP during the first few minutes of intense muscular contraction. [Note: The amount of creatine phosphate in the body is proportional to the muscle mass.]
1. Synthesis: Creatine is synthesized from glycine and the guanidino group of arginine, plus a methyl group from SAM (see Figure 21.19). Creatine is reversibly phosphorylated to creatine phosphate by creatine kinase, using ATP as the phosphate donor. [Note: The presence of creatine kinase (MB isozyme) in the plasma is indicative of heart damage, and is used in the diagnosis of myocardial infarction (see p. 65).]
- 2. Degradation: Creatine and creatine phosphate spontaneously cyclize at a slow but constant rate to form creatinine, which is excreted in the urine. The amount of creatinine excreted is propor-

Synthesis of creatine.
tional to the total creatine phosphate content of the body, and thus can be used to estimate muscle mass. When muscle mass decreases for any reason (for example, from paralysis or muscular dystrophy), the creatinine content of the urine falls. In addition, any rise in blood creatinine is a sensitive indicator of kidney malfunction, because creatinine normally is rapidly removed from the blood and excreted. A typical adult male excretes about 15 mmol of creatinine per day.

Melanin is a pigment that occurs in several tissues, particularly the eye, hair, and skin. It is synthesized from tyrosine in the epidermis by pigment-forming cells called melanocytes. Its function is to protect underlying cells from the harmful effects of sunlight. [Note: A defect in melanin production results in albinism, the most common form being due to defects in copper-containing tyrosinase (see p. 273).]

Amino acids are precursors of many nitrogen-containing compounds including porphyrins, which, in combination with ferrous (Fe'') iron, form heme (Figure 21.20). The major sites of heme biosynthesis are the liver, which synthesizes a number of heme proteins (particularly cytochrome P450), and the erythrocyte-producing cells of the bone marrow, which are active in hemoglobin synthesis. In the liver, the rate of heme synthesis is highly variable, responding to alterations in the cellular heme pool caused by fluctuating demands for hemeproteins. In contrast, heme synthesis in erythroid cells is relatively constant, and is matched to the rate of globin synthesis. Porphyrin synthesis start with glycine and succinyl CoA. The committed step in heme synthesis is the formation of 5-aminolevulinic acid (ALA). This reaction is catalyzed by ALA synthase-1 in liver (inhibited by hemin, the oxidized form of heme that accumulates in the cell when heme is being underutilized) and ALA synthase-2 in erythroid tissues (regulated by iron). Porphyrias are caused by inherited or acquired defects in heme synthesis, resulting in the accumulation and increased excretion of porphyrins or porphyrin precursors. With few exceptions, porphyrias are inherited as autosomal dominant disorders. Degradation of hemeproteins occurs in the reticuloendothelial system, particularly in the liver and spleen. The first step in the degradation of heme is the production of the green pigment biliverdin, which is subsequently reduced to bilirubin. Bilirubin is transported to the liver, where its solubility is increased by the addition of two molecules of glucuronic acid. Bilirubin diglucuronide is transported into the bile canaliculi, where it is first hydrolyzed and reduced by bacteria in the gut to yield urobilinogen, then oxidized by intestinal bacteria to stercobilin. Jaundice refers to the yellow color of the skin and sclera that is caused by deposition of bilirubin, secondary to increased bilirubin levels in the blood. Three commonly encountered type of jaundice are hemolytic jaundice, obstructive

jaundice, and hepatocellular jaundice. Other important Ncontaining compounds derived from amino acids include the catecholamines (dopamine, norepinephrine, and epinephrine), creatine, histamine, serotonin, and melanin.

21.1 6-Aminolevulinic acid synthase activity:
A. in liver is frequently decreased in individuals
treated with drugs, such as the barbiturate phenobarbital.
- B. catalyzes a rate-limiting reaction in porphyrin
biosynthesis.
C. requires the coenzyme biotin.
D. is strongly inhibited by heavy metal ions such as lead.
E. occurs in the cytosol.

A. occurs in red blood cells.
B. involves the oxidative cleavage of the porphyrin ring.
C. results in the liberation of carbon dioxide.
D. results in the formation of protoporphyrinogen.
E. is the sole source of bilirubin.
A 50-year-old man presented with painful blisters on the backs of his hands. He was a golf instructor, and indicated that the blisters had erupted shortly after the golfing season began. He did not have recent exposure to poison ivy or sumac, new soaps or detergents, or new medications. He denied having previous episodes of blistering. He had partial complex seizure disorder that had begun about three years earlier after a head injury. The patient had been taking phenytoin—his only medication—since the onset of the seizure disorder. He admitted to an average weekly ethanol intake of about eighteen 12oz cans of beer. The patient's urine was reddish orange. Cultures obtained from skin lesions failed to grow organisms. A 24-hour urine collection showed elevated uroporphyrin (1,000 mg; normal, <27mg). The most likely presumptive diagnosis is:

B. acute intermittent porphyria.
C. hereditary coproporphyria.
D. congenital erythropoietic porphyria.
E. erythropoietic protoporphyria.
21.4 A 10-year-old boy is referred to a specialist because of skin that blisters easily, urine that darkens on standing, and stained teeth. Lab studies are remarkable for high levels of uroporphyrin | and coproporphyrin | in plasma, with uroporphyrin | being present in the urine. The most likely biochemical pathology in this case is:
A. deficiency of ALA synthase.
B. deficiency of bilirubin glucuronyltransferase.
C. deficiency of uroporphyrinogen III synthase.
D. down-regulation of tyrosinase.
E. inhibition of ALA dehydratase by lead.
21.3
Correct answer = B. The activity of 5-aminolevulinic acid synthase controls the rate of porphyrin synthesis. The hepatic form of the enzyme is increased in patients treated with certain drugs, and requires pyridoxal phosphate as a coenzyme. Another enzyme in the pathway (5aminolevulinic acid dehydrase) is extremely sensitive to the presence of heavy metals.
Correct answer = B. The cyclic heme molecule is oxidatively cleaved to form biliverdin. The catabolism occurs in the cells of the reticuloendothelial system, particularly the spleen, and results in the liberation of carbon monoxide. Protoporphyrinogen is an intermediate in the synthesis, not degradation, of heme. Cytochromes and other non-hemoglobin hemeproteins are also precursors of bilirubin.

Correct answer = A. The disease is associated with a deficiency in uroporphyrinogen decarboxylase, but clinical expression of the enzyme deficiency is influenced by hepatic injury caused by iron overload, chronic ethanol consumption, and the presence of hepatitis B or C and HIV infections. Exposure to sunlight can also be a precipitating factor. Clinical onset is typically during the fourth or fifth decade of life. Porphyrin accumulation leads to cutaneous symptoms and urine that is red to brown. Treatment of the patient's seizure disorder with phenytoin caused increased synthesis of ALA synthase, and, therefore, of uroporphyrinogen, the substrate of the deficient enzyme. The laboratory and clinical findings are inconsistent with other porphyrias.
Correct answer = C. A deficiency of uroporphyrinogen Ill synthase results in accumulation of hydroxymethylbilane and the spontaneous conversion of this substrate to porphyrins of the Type | series. A deficiency of ALA synthase or inhibition of ALA dehydratase by lead would not allow the synthesis of porphobilinogen, the first pyrrole product in the heme biosynthetic pathway, and thus would not result in uroor coproporphyrin synthesis. Deficiency of the glucuronyltransferase would not present with the systems described, and lab studies would be remarkable for an elevation of unconjugated bilirubin. Down-regulation of tyrosinase would result in decreased pigmentation.

Ribonucleoside and deoxyribonucleoside phosphates (nucleotides) are essential for all cells. Without them, neither RNA nor DNA can be produced and, therefore, proteins cannot be synthesized or cells proliferate. Nucleotides also serve as carriers of activated intermediates in the synthesis of some carbohydrates, lipids, and conjugated proteins, for example, UDP-glucose and CDP-choline, and are structural components of several essential coenzymes, such as coenzyme A, FAD, NAD*, and NADP*. Nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), serve as second messengers in signal transduction pathways. In addition, nucleotides play an important role as "energy currency" in the cell. Finally, nucleotides are important regulatory compounds for many of the pathways of intermediary metabolism, inhibiting or activating key enzymes. The purine and pyrimidine bases found in nucleotides can be synthesized de novo, or can be obtained through salvage pathways that allow the reuse of the preformed bases resulting from normal cell turnover. [Note: Little of the purines and pyrimidines supplied by diet are utilized, and are degraded instead.]

Nucleotides are composed of a nitrogenous base, a pentose monosaccharide, and one, two, or three phosphate groups. The nitrogencontaining bases belong to two families of compounds: the purines and the pyrimidines.

Both DNA and RNA contain the same purine bases: adenine (A) and guanine (G). Both DNA and RNA contain the pyrimidine cytosine (C), but they differ in their second pyrimidine base: DNA contains thymine (T), whereas RNA contains uracil (U). T and U differ in that only T has a methyl group (Figure 22.1). [Note: Unusual (modified) bases are
Figure 22.1
Purines and pyrimidines commonly found in DNA and RNA.

A. Pentoses found in nucleic acids. B. Examples of the numbering systems for purineand pyrimidinecontaining nucleosides.
occasionally found in some species of DNA and RNA, for example, in some viral DNA, and in transfer RNA. Base modifications include methylation, glycosylation, acetylation, or reduction. Some examples of unusual bases are shown in Figure 22.2. The presence of an unusual base in a nucleotide sequence may aid in its recognition by specific enzymes, or protect it from being degraded by nucleases.]

The addition of a pentose sugar to a base produces a nucleoside. If the sugar is ribose, a ribonucleoside is produced; if the sugar is 2-deoxyribose, a deoxyribonucleoside is produced (Figure 22.3A). The ribonucleosides of A, G, C, and U are named adenosine, guanosine, cytidine, and uridine, respectively. The deoxyribonucleosides of A, G, C, and T have the added prefix, "deoxy-," for example, deoxyadenosine. [Note: The compound deoxythymidine is often simply called thymidine, with the "deoxy-" prefix being understood.] The carbon and nitrogen atoms in the rings of the base and the sugar are numbered separately (Figure 22.3B). Note that the carbons in the pentose are numbered 1' to 5'. Thus, when the 5'-carbon of a nucleoside (or nucleotide) is referred to, a carbon atom in the pentose, rather than an atom in the base, is being specified.

The addition of one or more phosphate groups to a nucleoside produces a nucleotide. The first phosphate group is attached by an ester linkage to the 5'-OH of the pentose. Such a compound is called a nucleoside 5'-phosphate or a 5'-nucleotide. The type of pentose is denoted by the prefix in the names "5'-ribonucleotide" and "5'-deoxyribonucleotide." If one phosphate group is attached to the 5'-carbon of the pentose, the structure is a nucleoside monophosphate, like adenosine monophosphate (AMP) (also called adenylate). If a second or third phosphate is added to the nucleoside, a nucleoside diphosphate (for example, adenosine diphosphate or ADP) or triphosphate (for example, adenosine triphosphate or ATP) results (Figure 22.4). The second and third phosphates are each connected to the nucleotide by a "high-energy" bond. [Note: The phosphate groups are responsible for the negative charges associated with nucleotides, and cause DNA and RNA to be referred to as "nucleic acids."]

The atoms of the purine ring are contributed by a number of compounds, including amino acids (aspartic acid, glycine, and glutamine), COs, and N'°-formyltetrahydrofolate (Figure 22.5). The purine ring is constructed primarily in the liver by a series of reactions that add the donated carbons and nitrogens to a preformed ribose 5-phosphate. (See p. 147 for the synthesis of ribose 5-phosphate by the pentose phosphate pathway).

PRPP is an "activated pentose" that participates in the synthesis and salvage of purines and pyrimidines. Synthesis of PRPP from ATP and
ribose 5-phosphate is catalyzed by PRPP synthetase (ribose phosphate pyrophosphokinase, Figure 22.6). This X-linked enzyme is activated by inorganic phosphate and inhibited by purine nucleotides (end-product inhibition). [Note: The sugar moiety of PRPP is ribose, and, therefore, ribonucleotides are the end products of de novo purine synthesis. When deoxyribonucleotides are required for DNA synthesis, the ribose sugar moiety is reduced (see p. 297).]

Synthesis of 5'-phosphoribosylamine from PRPP and glutamine is shown in Figure 22.7. The amide group of glutamine replaces the pyrophosphate group attached to carbon 1 of PRPP. The enzyme, glutamine:phosphoribosylpyrophosphate amidotransferase, is inhibited by the purine 5'-nucleotides AMP and GMP—the end products of the pathway. This is the committed step in purine nucleotide biosynthesis. The rate of the reaction is also controlled by the intracellular concentration of PRPP. [Note: The intracellular concentration of PRPP is normally far below the Km for the amidotransferase. Therefore, any small change in the PRPP concentration causes a proportional change in the rate of the reaction (see p. 59).]

The next nine steps in purine nucleotide biosynthesis leading to the synthesis of inosine monophosphate (IMP, whose base is hypoxanthine) are illustrated in Figure 22.7. This pathway requires ATP as an energy source. Two steps in the pathway require N'°-formyltetrahydrofolate (see p. 267).

Some synthetic inhibitors of purine synthesis (for example, the sulfonamides'), are designed to inhibit the growth of rapidly dividing microorganisms without interfering with human cell functions (see Figure 22.7). Other purine synthesis inhibitors, such as structural analogs of folic acid (for example, methotrexate'), are used pharmacologically to control the spread of cancer by interfering with the synthesis of nucleotides and, therefore, of DNA and RNA (see Figure 22.7).

Sources of the individual atoms in the purine ring. The order in which the atoms are added is shown by the numbers in the black boxes (see Figure 22.7).

Synthesis of 5-phosphoribosyl-1-pyrophosphate (PRPP), showing the activator and inhibitors of the reaction.
1.2.See Chapter 33 in Lippincott's Illustrated Reviews: Pharmacology for a discussion of sulfonamides and methotrexate.

Synthesis of purine nucleotides, showing the inhibitory effect of some structural analogs.
Inhibitors of human purine synthesis are extremely toxic to tissues, especially to developing structures such as in a fetus, or to cell types that normally replicate rapidly, including those of bone marrow, skin, gastrointestinal (Gl) tract, immune system, or hair follicles. As a result, individuals taking such anticancer drugs can experience adverse effects, including anemia, scaly skin, GI tract disturbance, immunodeficiencies, and hair loss.

The conversion of IMP to either AMP or GMP uses a two-step, energy-requiring pathway (Figure 22.8). Note that the synthesis of AMP requires guanosine triphosphate (GTP) as an energy source, whereas the synthesis of GMP requires ATP. Also, the first reaction in each pathway is inhibited by the end product of that pathway. This provides a mechanism for diverting IMP to the synthesis of the species of purine present in lesser amounts. If both AMP and GMP are present in adequate amounts, the de novo pathway of purine synthesis is turned off at the amidotransferase step.

Conversion of nucleoside monophosphates to nucleoside diphosphates and triphosphates.

Nucleoside diphosphates are synthesized from the corresponding nucleoside monophosphates by base-specific nucleoside monophosphate kinases (Figure 22.9). [Note: These kinases do not discriminate between ribose or deoxyribose in the substrate.] ATP is generally the source of the transferred phosphate, because it is present in higher concentrations than the other nucleoside triphosphates. Adenylate kinase is particularly active in liver and muscle, where the turnover of energy from ATP is high. Its function is to maintain an equilibrium among AMP, ADP, and ATP. Nucleoside diphosphates and triphosphates are interconverted by nucleoside diphosphate kinase—an enzyme that, unlike the monophosphate kinases, has broad specificity.

Purines that result from the normal turnover of cellular nucleic acids, or the small amount that is obtained from the diet and not degraded, can be converted to nucleoside triphosphates and used by the body. This is referred to as the "salvage pathway" for purines.
1. Conversion of purine bases to nucleotides: Two enzymes are involved: adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Both enzymes use PRPP as the source of the ribose 5-phosphate group. The release of pyrophosphate and its subsequent hydrolysis by pyrophosphatase makes these reactions irreversible (Figure 22.10). [Note: Adenosine is the only purine nucleoside to be salvaged. It is phosphorylated to AMP by adenosine kinase.]
2.
Lesch-Nyhan syndrome: This syndrome is a rare, X-linked, recessive disorder associated with a virtually complete deficiency of HGPRT. This deficiency results in an inability to salvage hypoxanthine or guanine, from which excessive amounts of uric acid, the end product of purine degradation, are produced (see p. 298). In addition, the lack of this salvage pathway causes increased PRPP levels and decreased IMP and GMP levels. As a result, glutamine:phosphoribosylpyrophosphate amidotransferase (the committed step in purine synthesis) has excess substrate and decreased inhibitors available, and de novo purine synthesis is increased. The combination of decreased purine reutilization and increased purine synthesis results in increased degradation of purines and the production of large amounts of uric acid, making Lesch-Nyhan a heritable cause of hyperuricemia. In patients with Lesch-Nyhan syndrome, the hyperuricemia frequently results in the formation of uric acid stones in the kidneys (urolithiasis) and the deposition of urate crystals in the joints (gouty arthritis) and soft tissues. In addition, the syndrome is characterized by motor dysfunction, cognitive deficits, and behavioral disturbances that include self-mutilation (biting of lips and fingers, Figure 22.11).

The nucleotides described thus far all contain ribose (ribonucleotides). The nucleotides required for DNA synthesis, however, are 2'-deoxyribonucleotides, which are produced from ribonucleoside diphosphates by the enzyme ribonucleotide reductase during the S-phase of the cell cycle (see p. 407). [Note: The same enzyme acts on pyrimidine ribonucleotides.]

Ribonucleotide reductase (ribonucleoside diphosphate reductase) is composed of two nonidentical dimeric subunits, R1 and R2, and is specific for the reduction of purine nucleoside diphosphates (ADP and GDP), and pyrimidine nucleoside diphosphates, cytidine diphosphate (CDP) and uridine diphosphate (UDP) to their deoxyforms (dADP, dGDP, dCDP, and dUDP). The immediate donors of the hydrogen atoms needed for the reduction of the 2'-hydroxyl group are two sulfhydryl groups on the enzyme itself, which, during the reaction, form a disulfide bond (Figure 22.12).

Regeneration of reduced enzyme: In order for ribonucleotide reductase to continue to produce deoxyribonucleotides, the disulfide bond created during the production of the 2'-deoxy carbon must be reduced. The source of the reducing equivalents for this purpose is thioredoxin—a peptide coenzyme of ribonucleotide reductase. Thioredoxin contains two cysteine residues separated by two amino acids in the peptide chain. The two sulfhydryl groups of thioredoxin donate their hydrogen atoms to ribonucleotide reductase, in the process forming a disulfide bond (see p. 19).
2. Regeneration of reduced thioredoxin: Thioredoxin must be converted back to its reduced form in order to continue to perform its function. The necessary reducing equivalents are provided by NADPH + H*, and the reaction is catalyzed by thioredoxin reductase (see Figure 22.12).

Ribonucleotide reductase is responsible for maintaining a balanced supply of the deoxyribonucleotides required for DNA synthesis. To achieve this, the regulation of the enzyme is complex. In addition to the catalytic (active) site, there are allosteric sites on the enzyme involved in regulating its activity (Figure 22.13).
- 1. Activity sites: The binding of dATP to allosteric sites (known as the activity sites) on the enzyme inhibits the overall catalytic activity of the enzyme and, therefore, prevents the reduction of any of the four nucleoside diphosphates. This effectively prevents DNA synthesis, and explains the toxicity of increased levels of dATP seen in conditions such as adenosine deaminase deficiency (see p. 301). In contrast, ATP bound to these sites activates the enzyme.

Lesions on the lips of Lesch-Nyhan patients caused by self-mutilation.
—_—

Regulation of ribonucleotide reductase.
2. Substrate specificity sites: The binding of nucleoside triphosphates to additional allosteric sites (known as the substrate specificity sites) on the enzyme regulates substrate specificity, causing an increase in the conversion of different species of ribonucleotides to deoxyribonucleotides as they are required for DNA synthesis. For example, deoxythymidine triphosphate (dTTP) binding at the specificity sites causes a conformational change that allows reduction of GDP to dGDP at the catalytic site.
The drug hydroxyurea destroys the free radical required for enzymic activity of ribonucleotide reductase, and thus inhibits the generation of substrates for DNA synthesis. Hydroxyurea has been used in the treatment of cancers such as chronic myelogenous leukemia. Hydroxyurea is also used in the treatment of sickle cell disease (see p. 36); however, the increase in fetal hemoglobin seen with hydroxyurea has not been linked to its effect on ribonucleotide reductase.

Degradation of dietary nucleic acids occurs in the small intestine, where a family of pancreatic enzymes hydrolyzes the nucleic acids to nucleotides. Inside the intestinal mucosal cells, purine nucleotides are sequentially degraded by specific enzymes to nucleosides and free bases, with uric acid as the end product of this pathway. [Note: Purine nucleotides from de novo synthesis are degraded in the liver primarily. The free bases are sent out from liver and salvaged by peripheral tissues.]

Ribonucleases and deoxyribonucleases, secreted by the pancreas, hydrolyze dietary RNA and DNA primarily to oligonucleotides. Oligonucleotides are further hydrolyzed by pancreatic phosphodiesterases, producing a mixture of 3'and 5'-mononucleotides. In the intestinal mucosal cells, a family of nucleotidases removes the phosphate groups hydrolytically, releasing nucleosides that are further degraded to free bases. Dietary purine bases are not used to any appreciable extent for the synthesis of tissue nucleic acids. Instead, they are generally converted to uric acid in intestinal mucosal cells. Most of the uric acid enters the blood, and is eventually excreted in the urine. A summary of this pathway is shown in Figure 22.14. [Note: Mammals other than primates express urate oxidase which cleaves the purine ring, generating allantoin. Use of recombinant urate oxidase is a potential therapeutic strategy to lower urate levels.]

A summary of the steps in the production of uric acid and genetic diseases associated with deficiencies of specific degradative enzymes are shown in Figure 22.15. [Note: The bracketed numbers refer to specific reactions in the figure.]
[1] An amino group is removed from AMP to produce IMP by AMP deaminase, or from adenosine to produce inosine (hypoxanthineribose) by adenosine deaminase.
[2] IMP and GMP are converted into their nucleoside forms—inosine and guanosine—by the action of 5'-nucleotidase.
[3] Purine nucleoside phosphorylase converts inosine and guanosine into their respective purine bases, hypoxanthine and guanine. [Note: A mutase interconverts ribose 1and ribose 5-phosphate.]
[4] Guanine is deaminated to form xanthine.
[5] Hypoxanthine is oxidized by xanthine oxidase to xanthine, which is further oxidized by xanthine oxidase to uric acid, the final product of human purine degradation. Uric acid is excreted primarily in the urine.

1. Gout: Gout is a disorder characterized by high levels of uric acid— the end product of purine catabolism—in blood (hyperuricemia), as a result of either the overproduction or underexcretion of uric acid. The hyperuricemia can lead to the deposition of monosodium urate crystals in the joints, and an inflammatory response to the crystals, causing first acute and then progressing to chronic gouty arthritis. Nodular masses of monosodium urate crystals (tophi) may be deposited in the soft tissues, resulting in chronic tophaceous gout (Figure 22.16). Formation of uric acid stones in the kidney (urolithiasis) may also be seen. [Note: Hyperuricemia is typically asymptomatic and does not lead to gout, but gout is preceded by hyperuricemia.] The definitive diagnosis of gout requires aspiration and examination of synovial fluid (Figure 22.17) from an affected joint (or material from a tophus) using polarized light microscopy to confirm the presence of needle-shaped monosodium urate crystals (Figure 22.18).
a. Underexcretion of uric acid: In the vast majority of patients, the hyperuricemia leading to gout is caused by underexcretion of uric acid. Underexcretion can be primary, due to as-yetunidentified inherent excretory defects, or secondary to known disease processes that affect how the kidney handles urate, for example lactic acidosis (lactate and urate compete for the same renal transporter), and to environmental factors such as the use of drugs, for example, thiazide diuretics, or exposure to lead (saturnine gout).

b. Overproduction of uric acid: A less common cause of gout is hyperuricemia from the overproduction of uric acid. Primary hyperuricemia is, for the most part, idiopathic (having no known cause). However, several identified mutations in the gene for X-linked PRPP synthetase result in the enzyme having an increased Vmax (See p. 58) for the production of PRPP, a lower Km (see p. 59) for ribose 5-phosphate, or a decreased sensitivity to purine nucleotides—its allosteric inhibitors (see p. 62). In each case, increased availability of PRPP increases purine production, resulting in elevated levels of plasma uric acid. LeschNyhan syndrome (see p. 296) also causes hyperuricemia as a

Digestion of dietary nucleic acids. [Note: Much of the metabolism of the mononucleotides occurs within the intestinal mucosal cells.]

The degradation of purine nucleotides to uric acid, illustrating some of the genetic diseases associated with this pathway. [Note: The numbers in brackets refer to the corresponding numbered citations in the text.]
result of the decreased salvage of hypoxanthine and guanine, and the subsequent increased availability of PRPP. Secondary hyperuricemia is typically the consequence of increased availability of purines, for example, in patients with myeloproliferative disorders or who are undergoing chemotherapy and so have a high rate of cell turnover. Hyperuricemia leading to gout can also be the result of seemingly unrelated metabolic diseases, such as von Gierke disease (see Figure 11.8 on p. 129) or fructose intolerance (see p. 138).
2.
A diet rich in meat and seafood (particularly shellfish) is associated with increased risk of gout. In addition, a diet rich in low-fat dairy products was shown to be associated with a decreased risk.

c. Treatment of gout: Acute attacks of gout are treated with antiinflammatory agents. Colchicine, steroidal drugs such as prednisone, and nonsteroidal drugs such as indomethacin are used. [Note: Colchicine prevents formation of microtubules, thus decreasing the movement of neutrophils into the affected area. Like the other anti-inflammatory drugs, it has no effect on uric acid levels.] Long-term therapeutic strategies for gout involve lowering the uric acid level below its saturation point, thereby preventing the deposition of urate crystals. Uricosuric agents, such as probenecid or sulfinpyrazone, that increase renal excretion of uric acid, are used in patients who are "underexcretors" of uric acid. Allopurinol, a structural analog of hypoxanthine, inhibits uric acid synthesis and is used in patients who are "overproducers" of uric acid. Allopurinol is converted in the body to oxypurinol, which inhibits xanthine oxidase (XO), resulting in an accumulation of hypoxanthine and xanthine (see Figure 22.15)—compounds more soluble than uric acid and, therefore, less likely to initiate an inflammatory response. In patients with normal levels of HGPRT, the hypoxanthine can be salvaged, thus reducing the levels of PRPP and, therefore, de novo purine synthesis. Febuxostat, a non-purine inhibitor of XO, is now available. [Note: Uric acid levels in the blood normally are close to the saturation point. One reason for this may be the strong antioxidant effects of uric acid.]

Adenosine deaminase (ADA) deficiency: ADA is expressed in a variety of tissues, but, in humans, lymphocytes have the highest activity of this cytoplasmic enzyme. A deficiency of ADA results in an accumulation of adenosine, which is converted to its ribonucleotide or deoxyribonucleotide forms by cellular kinases. As dATP levels rise, ribonucleotide reductase is inhibited, thus preventing the production of all deoxyribose-containing nucleotides (see p. 297). Consequently, cells cannot make DNA and divide. [Note: The dATP and adenosine that accumulate in ADA deficiency lead to developmental arrest and apoptosis of lymphocytes.] In its most severe form, this autosomal recessive disorder causes a type of severe combined immunodeficiency disease (SCID), involving a decrease in T cells, B cells, and natural killer (NK) cells. It is estimated that in the United States, ADA deficiency accounts for approximately 14% of all cases of SCID. Treatment requires either bone marrow transplantation (BMT) or enzyme replacement therapy (ERT). Without appropriate treatment, children with this disorder usually die by the age of two. [Note: Purine nucleoside phosphorylase (PNP) deficiency results in a less severe immunodeficiency involving T-cells primarily.]

Analysis of joint fluid can help to define causes of joint swelling or arthritis, such as infection, gout, and rheumatoid disease.

Gout can be diagnosed by the presence of negatively birefringent monosodium urate crystals in aspirated synovial fluid examined by polarized-light microscopy. Here, crystals are within polymorphonuclear leukocytes.

pources of the individual atoms in © pyrimidine ring.
Cellular location, CPS I = Mitochondria. Cellular location, CPS II = Cytosol. Pathway involved, CPS I = Urea cycle. Pathway involved, CPS II = Pyrimidine synthesis. Source of nitrogen, CPS I = Ammonia. Source of nitrogen, CPS II = γ -Amide group of glutamine. Regulators, CPS I = Activator: N-acetyl- glutamate. Regulators, CPS II = Inhibitor: UTP Activator: PRPP

Summary of the differences between carbamoyl! phosphate synthetase (CPS) | and I.

Unlike the synthesis of the purine ring, which is constructed on a preexisting ribose 5-phosphate, the pyrimidine ring is synthesized before being attached to ribose 5-phosphate, which is donated by PRPP. The sources of the atoms in the pyrimidine ring are glutamine, CO2, and aspartic acid (Figure 22.19). [Note: Glutamine and aspartic acid are thus required for both purine and pyrimidine synthesis.]

The regulated step of this pathway in mammalian cells is the synthesis of carbamoyl phosphate from glutamine and COs, catalyzed by carbamoyl phosphate synthetase (CPS) II. CPS Il is inhibited by UTP (the end product of this pathway, which can be converted into the other pyrimidine nucleotides), and is activated by PRPP. [Note: Carbamoyl phosphate, synthesized by CPS /, is also a precursor of urea (see p. 253). Defects in ornithine transcarbamylase of the urea cycle promote pyrimidine synthesis due to increased availability of carbamoyl phosphate. A comparison of the two enzymes is presented in Figure 22.20.]

The second step in pyrimidine synthesis is the formation of carbamoylaspartate, catalyzed by aspartate transcarbamoylase. The pyrimidine ring is then closed hydrolytically by dihydroorotase. The resulting dihydroorotate is oxidized to produce orotic acid (orotate, Figure 22.21). The enzyme that produces orotate, dihydroorotate dehydrogenase, is associated with the inner mitochondrial membrane. All other enzymes in pyrimidine biosynthesis are cytosolic. [Note: The first three enzymic activities in this pathway (CPS Il, aspartate transcarbamoylase, and dihydroorotase) are actually three different catalytic domains of a single polypeptide chain known as CAD from the first letter in the name of each domain. (See p. 19 for a discussion of domains). This is an example of a multfunctional or multicatalytic polypeptide that facilitates the ordered synthesis of an important compound. Synthesis of the purine nucleotide IMP also involves multifunctional proteins.]

The completed pyrimidine ring is converted to the nucleotide orotidine 5'-monophosphate (OMP) in the second stage of pyrimidine nucleotide synthesis (see Figure 22.21). PRPP is again the ribose 5-phosphate donor. The enzyme orotate phosphoribosyltransferase produces OMP and releases pyrophosphate, thereby making the reaction biologically irreversible. [Note: Both purine and pyrimidine synthesis thus require glutamine, aspartic acid, and PRPP as essential precursors.] OMP, the parent pyrimidine mononucleotide, is converted to uridine monophosphate (UMP) by orotidylate decarboxylase, which removes the acidic carboxyl group. Orotate phosphoribosyltransferase and orotidylate decarboxylase are also catalytic domains of a single polypeptide chain called UMP synthase. Orotic aciduria—a rare genetic defect—may be caused by a deficiency of one or both activities of this bifunctional enzyme, resulting in orotic acid in the urine (see Figure 22.21). UMP is

De novo pyrimidine synthesis.
sequentially phosphorylated to UDP and UTP. [Note: The UDP is a substrate for ribonucleotide reductase, which generates dUDP. The dUDP is phosphorylated to dUTP, which is rapidly hydrolyzed to dUMP by UTP diphosphatase (dUTPase). dUTPase, then, plays an important role in reducing availability of (UTP as a substrate for DNA synthesis, thus preventing erroneous incorporation of uracil into DNA.]

CTP is produced by amination of UTP by CTP synthetase (Figure 22.22), with glutamine providing the nitrogen. [Note: Some CTP is dephosphorylated to CDP, which is a substrate for ribonucleotide reductase. The dCDP generated can be phosphorylated to dCTP for DNA synthesis.]

dUMP is converted to dTMP by thymidylate synthase, which uses N®,N'°-methylene tetrahydrofolate as the source of the methyl group (see p. 267 for a discussion of this coenzyme). This is an unusual reaction in that tetrahydrofolate (THF) contributes not only a onecarbon unit but also two hydrogen atoms from the pteridine ring, resulting in the oxidation of THF to dihydrofolate (DHF) (Figure 22.23). Inhibitors of thymidylate synthase include thymine analogs such as 5-fluorouracil, which serve as successful antitumor agents.

Synthesis of CTP from UTP. [Note: CTP, required for RNA synthesis, is converted to dCTP for DNA synthesis.]

Synthesis of dTMP from dUMP, illustrating sites of action of antineoplastic drugs.
5-Fluorouracil is metabolically converted to 5-FdUMP, which becomes permanently bound to the inactivated thymidylate synthase; for this reason, the drug is called a "suicide" inhibitor. DHF can be reduced to THF by dihydrofolate reductase (see Figure 28.3, p. 374), an enzyme that is inhibited by drugs such as methotrexate. By decreasing the supply of THF, these folate analogs not only inhibit purine synthesis (see Figure 22.7), but, by preventing methylation of (UMP to dTMP, they also lower the cellular concentration of this essential component of DNA. DNA synthesis is inhibited and cell growth slowed. Drugs such as those described above, therefore, are used to decrease the growth rate of cancer cells. [Note: Trimethoprim, a folate analog, has potent antibacterial activity because of its selective inhibition of bacterial dihydrofolate reduct ase. ]

Few pyrimidine bases are salvaged in human cells. However, the pyrimidine nucleosides can be salvaged by nucleoside kinases that utilize ATP in the phosphorylation of the nucleosides to nucleotides. [Note: The salvage of pyrimidine nucleosides is the basis for using uridine in the treatment of hereditary orotic aciduria.]

Unlike the purine ring, which is not cleaved human cells, the pyrimidine ring is opened and degraded to highly soluble products, B-alanine and B-aminoisobutyrate, with the production of NH3 and COs.

Nucleotides are composed of a nitrogenous base (adenine = A, guanine = G, cytosine = C, uracil = U, and thymine = T), a pentose, and one, two, or three phosphate groups (Figure 22.24). A and G are purines; C, U, and T are pyrimidines. If the sugar is ribose, the nucleotide is a ribonucleoside phosphate (for example, AMP), and it can have several functions in the cell, including being a component of RNA. If the sugar is deoxyribose, the nucleotide is a deoxyribonucleoside phosphate (for example, dAMP), and will be found almost exclusively as a component of DNA. The committed step in purine synthesis uses 5-phosphoribosyl-1-pyrophosphate (PRPP, an "activated pentose" that provides the ribose-phosphate group for de novo purine and pyrimidine synthesis and purine salvage) and nitrogen from glutamine to produce phosphoribosyl amine. The enzyme is glutamine:PRPP amidotransferase, and is inhibited by AMP and GMP (the end products of the pathway) and activated by PRPP. Purine nucleotides can also be produced from preformed purine bases by using salvage reactions catalyzed by adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT). A near total deficiency of HGPRT causes Lesch-Nyhan syndrome—a severe, heritable form of hyperuricemia accompanied by compulsive self-mutilation. All deoxyribonucleotides are synthesized from ribonucleotides by the enzyme ribonucleotide reductase. This enzyme is highly regulated, for example, it is strongly inhibited by dATP—a compound that is overproduced in bone marrow cells in individuals with adenosine deaminase deficiency. This syndrome causes severe combined immunodeficiency disease. The end product of purine degradation is uric acid—a compound whose overproduction or undersecretion causes hyperuricemia that, if accompanied by the deposition of urate crystals in joints and soft tissues, and an inflammatory response to those crystals, results in gout. The

first step in pyrimidine synthesis—the production of carbamoyl phosphate by carbamoyl phosphate synthetase Il—is the regulated step in this pathway (it is inhibited by UTP and activated by PRPP). The UTP produced by this pathway can be converted to CTP. JUMP can be converted to dTMP using thymidylate synthase—an enzyme targeted by anticancer drugs such as 5-fluorouracil. The regeneration of THF from DHF produced in the thymidylate synthase reaction requires dihydrofolate reductase—an enzyme targeted by the drug, methotrexate.

Key concept map for nucleotide metabolism. ADA= adenosine deaminase, XO = xanthine oxidase, TS = thymidylate synthase. RNR = ribonucleotide reductase, CPS II = carbamyl phosphate synthetase II.

22.1 A 42-year-old male patient undergoing radiation therapy for prostate cancer develops severe pain in the metatarsal phalangeal joint of his right big toe. Monosodium urate crystals are detected by polarized light microscopy in fluid obtained from this joint by arthrocentesis. Uric acid crystals are present in his urine. This patient's pain is directly caused by the overproduction of the end product of which of the following metabolic pathways?
- A. De novo pyrimidine biosynthesis.
mogagw
. Pyrimidine degradation.
. De novo purine biosynthesis.
. Purine salvage.
- . Purine degradation.
A 1-year-old female patient is lethargic, weak, and anemic. Her height and weight are both low for her age. Her urine contains an elevated level of orotic acid. The administration of which of the following compounds is most likely to alleviate her symptoms?
A. Adenine.
B. Guanine.
C. Hypoxanthine.
- D. Thymidine.
E. Uridine.
22.3 The rate of DNA synthesis in a culture of cells could be most accurately determined by measuring the incorporation of which of the following radiolabeled compounds?
C. Phosphate.
D. Thymidine.
22.4 Which one of the following enzymes of nucleotide metabolism is correctly paired with its pharmacological inhibitor?
A. Dihydrofolate reductase—methotrexate
B. IMP dehydrogenase—hydroxyurea
C. Ribonucleotide reductase—5-fluorouracil
D. Thymidylate synthase—allopurinol
E. Xanthine oxidase—probenecid
22.5 What laboratory test would help in distinguishing an orotic aciduria caused by ornithine transcarbamylase deficiency from that caused by UMP synthase deficiency?
22.2

Correct answer = E. The patient's pain is caused by gout, resulting from an inflammatory response to the crystallization of excess uric acid in his joints. Radiation therapy caused cell death, with degradation of nucleic acids and their constituent purines. Uric acid, the end product of purine degradation, is a relatively insoluble compound that can cause gout and kidney stones. Pyrimidine metabolism is not associated with uric acid production. Overproduction of purines can indirectly result in hyperuricemia. Purine salvage decreases uric acid production.
Correct answer = E. The elevated excretion of orotic acid indicates that the patient has orotic aciduria, a rare genetic disorder affecting the de novo pyrimidine biosynthetic pathway. Deficiencies in the enzyme activities OMP decarboxylase and/or orotate phosphoribosyltransferase (both of which are domains of the enzyme UMP synthase) leave the patient unable to synthesize pyrimidines. Uridine, a pyrimidine nucleoside, is useful in treating this disorder because it bypasses the missing enzymes and can be salvaged to UMP, which can be converted to all the other pyrimidines. Although thymidine is a pyrimidine nucleoside, it cannot be converted to other pyrimidines. Hypoxanthine, guanine, and adenine are all purine bases that have no value in helping to replace the missing pyrimidines.
Correct answer = D. Because thymidine is essentially found only in DNA, its incorporation would most accurately reflect the rate of DNA synthesis. Uridine is found only in RNA and could be used to measure the rate of RNA synthesis. Phosphate, adenine, and guanine are present in both DNA and RNA, and could not be used to specifically measure synthesis of either one.

Correct answer = A. Methotrexate interferes with folate metabolism by acting as a competitive inhibitor of the enzyme dihydrofolate reductase. This starves cells for tetrahydrofolate, and makes them unable to synthesize purines and dTMP. IMP dehydrogenase is inhibited by mycophenolic acid. Ribonucleotide reductase is inhibited by hydroxyurea. Thymidylate synthase is inhibited by 5-fluorouracil. Xanthine oxidase is inhibited by allopurinol; probenecid increases renal excretion of urate, but does not inhibit its production.
Blood ammonia level would expected to be elevated in ornithine transcarbamylase deficiency but not in UMP synthase deficiency.

Four major organs play a dominant role in fuel metabolism: liver, adipose, muscle, and brain. These tissues contain unique sets of enzymes, such that each organ is specialized for the storage, use, or generation of specific fuels. These tissues do not function in isolation, but rather form part of a network in which one tissue may provide substrates to another, or process compounds produced by other organs. Communication between tissues is mediated by the nervous system, by the availability of circulating substrates, and by variation in the levels of plasma hormones (Figure 23.1). The integration of energy metabolism is controlled primarily by the actions of two peptide hormones: insulin and glucagon, with the catecholamines epinephrine and norepinephrine playing a supporting role. Changes in the circulating levels of these hormones allow the body to store energy when food is available in abundance, or to make stored energy available, for example, during "survival crises," such as famine, severe injury, and "fight-orflight" situations. This chapter describes the structure, secretion, and metabolic effects of the two hormones that most profoundly affect energy metabolism.

Insulin is a polypeptide hormone produced by the B cells of the islets of Langerhans—clusters of cells that are embedded in the exocrine portion of the pancreas (Figure 23.2). The islets of Langerhans make up only about 1-2% of the total cells of the pancreas. Insulin is the most

Mechanisms of communication between four major tissues.
Figure 23.2
Islets of Langerhans.
important hormone coordinating the use of fuels by tissues. Its metabolic effects are anabolic, favoring, for example, synthesis of glycogen, triacylglycerols, and protein.

Insulin is composed of 51 amino acids arranged in two polypeptide chains, designated A and B, which are linked together by two disulfide bridges (Figure 23.3A). The insulin molecule also contains an intramolecular disulfide bridge between amino acid residues of the A chain. [Note: Pig (porcine) and beef (bovine) insulin differ from human insulin at one and three amino acid positions, respectively. When used in humans for the treatment of diabetes, antibodies to these foreign proteins develop. Use of human recombinant insulin (see p. 470) has eliminated this problem.]

The processing and transport of intermediates that occur during the synthesis of insulin are shown in Figures 23.3B and 23.4. Note that the biosynthesis involves two inactive precursors, preproinsulin and proinsulin, which are sequentially cleaved to form the active hormone plus the connecting or C-peptide (see Figure 23.4). [Note: The C-peptide is essential for proper insulin folding. Also, because of its longer half-life in the plasma, the C-peptide is a good indicator of insulin production and secretion.] Insulin is stored in the cytosol in granules that, given the proper stimulus (see below), are released by exocytosis. (See p. 166 for a discussion of the synthesis of proteins destined for secretion.) Insulin is degraded by the enzyme insulinase which is present in the liver and, to a lesser extent, in the kidneys. Insulin has a plasma half-life of approximately 6 minutes. This short duration of action permits rapid changes in circulating levels of the hormone.

A. Structure of insulin. B. Formation of human insulin from preproinsulin.

Intracellular movements of insulin and its precursors. RER = rough endoplasmic reticulum.

- 1.
Stimulation of insulin secretion: Insulin secretion by the cells of the islets of Langerhans of the pancreas is closely coordinated with the release of glucagon by pancreatic a cells. The relative amounts of insulin and glucagon released by the pancreas are regulated so that the rate of hepatic glucose production is kept equal to the use of glucose by peripheral tissues. In view of its coordinating role, it is not surprising that the B cell responds to a variety of stimuli. In particular, insulin secretion is increased by:

Changes in blood levels of glucose, insulin, and glucagon after ingestion of a carbohydrate-rich meal.

Regulation of insulin release from pancreatic B cells.
a. Glucose: The f cells are the most important glucose-sensing cells in the body. Like the liver, B cells contain GLUT-2 transporters (see p. 97) and have glucokinase activity (see p. 98), and thus can phosphorylate glucose in amounts proportional to its actual concentration in the blood. Ingestion of glucose or a carbohydrate-rich meal leads to a rise in blood glucose, which is a signal for increased insulin secretion (as well as decreased glucagon synthesis and release, Figure 23.5). Glucose is the most important stimulus for insulin secretion. [Note: Glucose also increases expression of the gene for insulin.]
b. Amino acids: Ingestion of protein causes a transient rise in plasma amino acid levels, which, in turn, induces the immediate secretion of insulin. Elevated plasma arginine, for example, stimulates insulin secretion.
c. Gastrointestinal hormones: Most gastrointestinal hormones favor insulin release. The intestinal peptides cholecystokinin and gastric-inhibitory polypeptide (glucose-dependent insulinotropic peptide) increase insulin secretion in response to oral glucose, and so are referred to as "incretins." They are released from the small intestine after the ingestion of food and cause an anticipatory rise in insulin levels. This may account for the fact that the same amount of glucose given orally induces a much greater secretion of insulin than if given intravenously.
Glucose-dependent release of insulin into blood is mediated through a rise in calcium concentration in the B cell. Glucose taken into B cells is metabolized, with subsequent production of ATP. ATP-sensitive potassium channels close, causing depolarization of the plasma membrane, activation of voltage-gated calcium channels, and influx of calcium into the cell. Calcium causes vesicles containing insulin to be released from the B cell. Sulfonylureas, oral agents used to treat type 2 diabetes, increase insulin secretion by closing ATP-sensitive potassium channels.

- 2. Inhibition of insulin secretion: The synthesis and release of insulin are decreased when there is a scarcity of dietary fuels, and also during periods of stress (for example, fever or infection). These effects are mediated primarily by epinephrine, which is secreted by the adrenal medulla in response to stress, trauma, or extreme exercise. Under these conditions, the release of epinephrine is controlled largely by the nervous system. Epinephrine has a direct effect on energy metabolism, causing a rapid mobilization of energy-yielding fuels, including glucose from the liver (produced by glycogenolysis or gluconeogenesis, see p. 121) and fatty acids from adipose tissue (see p. 189). In addition, epinephrine can override the normal glucose-stimulated release of insulin. Thus, in emergency situations, the sympathetic nervous

Insulin binds to specific, high-affinity receptors in the cell membrane of most tissues, including liver, muscle, and adipose. This is the first step in a cascade of reactions ultimately leading to a diverse array of actions

Insulin receptor. IRS = Insulin receptor substrate.
biologic (Figure 23.7).

Insulin causes the recruitment of glucose transporters (GLUTs) from intracellular stores in skeletal and cardiac muscle and adipose tissue.
1. Insulin receptor: The insulin receptor is synthesized as a single polypeptide that is glycosylated and cleaved into « and B subunits, which are then assembled into a tetramer linked by disulfide bonds (see Figure 23.7). A hydrophobic domain in each B subunit spans the plasma membrane. The extracellular « subunit contains the insulin-binding site. The cytosolic domain of the B subunit is a tyrosine kinase, which is activated by insulin.
SSS

Characteristics of glucose transport in various tissues.
2. Signal transduction: The binding of insulin to the « subunits of the insulin receptor induces conformational changes that are transduced to the B subunits. This promotes a rapid autophosphorylation of specific tyrosine residues on each B subunit (see Figure 23.7). Autophosphorylation initiates a cascade of cellsignaling responses, including phosphorylation of a family of proteins called insulin receptor substrates (IRS). At least four IRS have been identified that show similar structures but different tissue distributions. Phosphorylated IRS proteins interact with other signaling molecules through specific domains, activating a number of pathways that affect gene expression, cell metabolism and growth. The actions of insulin are terminated by dephosphorylation of the receptor.
3. Membrane effects of insulin: Glucose transport in some tissues, such as skeletal muscle and adipocytes, increases in the presence of insulin (Figure 23.8). Insulin promotes the recruitment of insulin-sensitive glucose transporters (GLUT-4) from a pool located in intracellular vesicles. [Note: Many tissues have insulininsensitive systems for glucose transport (Figure 23.9). For example, hepatocytes, erythrocytes, and cells of the nervous system, intestinal mucosa, renal tubules, and cornea do not require insulin for glucose uptake.]

4. Receptor regulation: Binding of insulin is followed by internalization of the hormone-receptor complex. Once inside the cell, insulin is degraded in the lysosomes. The receptors may be degraded but most are recycled to the cell surface. [Note: Elevated levels of insulin promote the degradation of receptors, thus decreasing the number of surface receptors. This is one type of "down-regulation."]
5. Time course of insulin actions: The binding of insulin provokes a wide range of actions. The most immediate response is an increase in glucose transport into adipocytes and skeletal muscle cells that occurs within seconds of insulin binding to its membrane receptor. Insulin-induced changes in enzymic activity in many cell types occur over minutes to hours, and reflect changes in the phosphorylation states of existing proteins. Insulin also initiates an increase in the amount of many enzymes, such as glucokinase, liver pyruvate kinase, acetyl CoA carboxylase, and fatty acid synthase, which requires hours to days. These changes reflect an increase in gene expression through increased transcription (mediated by SREBP-1, see p. 184) and translation.

Glucagon is a polypeptide hormone secreted by the o cells of the pancreatic islets of Langerhans. Glucagon, along with epinephrine, cortisol, and growth hormone (the "counter-regulatory hormones"), opposes many of the actions of insulin (Figure 23.10). Most importantly, glucagon acts to maintain blood glucose levels by activation of hepatic glycogenolysis and gluconeogenesis. Glucagon is composed of 29 amino acids arranged in a single polypeptide chain. [Note: Unlike insulin, the amino acid sequence of glucagon is the same in all mammalian species examined to date.] Glucagon is synthesized as a large precursor molecule (preproglucagon) that is converted to glucagon through a series of selective proteolytic cleavages, similar to those described for insulin biosynthesis (see Figure 23.3). In contrast to insulin, preproglucagon is processed to different products in different tissues.

The « cell is responsive to a variety of stimuli that signal actual or potential hypoglycemia (Figure 23.11). Specifically, glucagon secretion is increased by:
1. Low blood glucose: A decrease in plasma glucose concentration is the primary stimulus for glucagon release. During an overnight or prolonged fast, elevated glucagon levels prevent hypoglycemia (see below for a discussion of hypoglycemia).
2. Amino acids: Amino acids derived from a meal containing protein stimulate the release of both glucagon and insulin. The glucagon effectively prevents hypoglycemia that would otherwise occur as a result of increased insulin secretion that occurs after a protein meal.
3. Epinephrine: Elevated levels of circulating epinephrine produced by the adrenal medulla, or norepinephrine produced by sympathetic innervation of the pancreas, or both, stimulate the release

Regulation of glucagon release from pancreatic « cells. [Note: Amino acids increase release of insulin and glucagon, whereas glucose increases release of insulin only.]

Mechanism of action of glucagon. [Note: For clarity, G-protein activation of adenylyl cyclase has been omitted.] R = regulatory subunit; C = catalytic subunit.
of glucagon. Thus, during periods of stress, trauma, or severe exercise, the elevated epinephrine levels can override the effect on the a cell of circulating substrates. In these situations—regardless of the concentration of blood glucose—glucagon levels are elevated in anticipation of increased glucose use. In contrast, insulin levels are depressed.

Glucagon secretion is significantly decreased by elevated blood glucose and by insulin. Both substances are increased following ingestion of glucose or a carbohydrate-rich meal (see Figure 23.5). The regulation of glucagon secretion is summarized in Figure 23.11.

- 1. Effects on carbohydrate metabolism: The intravenous administration of glucagon leads to an immediate rise in blood glucose. This results from an increase in the breakdown of liver (not muscle) glycogen and an increase in gluconeogenesis.
2.
Effects on lipid metabolism: Glucagon activates lipolysis in adipose. The free fatty acids released are taken up by liver and oxidized to acetyl coenzyme A, which is used in ketone body synthesis. [Note: The catecholamines also activate lipolysis. ]
- 3. Effects on protein metabolism: Glucagon increases uptake of amino acids by the liver, resulting in increased availability of carbon skeletons for gluconeogenesis. As a consequence, plasma levels of amino acids are decreased.

Glucagon binds to high-affinity G protein-coupled receptors on the cell membrane of hepatocytes. The receptors for glucagon are distinct from those that bind insulin or epinephrine. [Note: Glucagon receptors are not found on skeletal muscle.] Glucagon binding results in activation of adenylyl cyclase in the plasma membrane (Figure 23.12, and see p. 94). This causes a rise in cAMP (the "second messenger'), which, in turn, activates cAMP-dependent protein kinase and increases the phosphorylation of specific enzymes or other proteins. This cascade of increasing enzymic activities results in the phosphorylation-mediated activation or inhibition of key regulatory enzymes involved in carbohydrate and lipid metabolism. An example of such a cascade in glycogen degradation is shown on Figure 11.9, p. 131, and p. 132. [Note: Glucagon also affects gene transcription.]

Hypoglycemia is characterized by: 1) central nervous system (CNS) symptoms, including confusion, aberrant behavior, or coma; 2) a simultaneous blood glucose level equal to or less than 40 mg/dl; and 3) symptoms being resolved within minutes following the administration of glucose (Figure 23.13). Hypoglycemia is a medical emergency because the CNS has an absolute requirement for a continuous supply of bloodborne glucose to serve as fuel for energy metabolism. Transient
hypoglycemia can cause cerebral dysfunction, whereas severe, prolonged hypoglycemia causes brain death. It is, therefore, not surprising that the body has multiple overlapping mechanisms to prevent or correct hypoglycemia. The most important hormone changes in combating hypoglycemia are elevated glucagon and epinephrine, combined with the diminished release of insulin.

The symptoms of hypoglycemia can be divided into two categories. Adrenergic symptoms—anxiety, palpitation, tremor, and sweating— are mediated by epinephrine release regulated by the hypothalamus in response to hypoglycemia. Usually adrenergic symptoms (that is, symptoms mediated by elevated epinephrine) occur when blood glucose levels fall abruptly. The second category of hypoglycemic symptoms is neuroglycopenic. Neuroglycopenia—the impaired delivery of glucose to the brain—results in impairment of brain function, causing headache, confusion, slurred speech, seizures, coma, and death. Neuroglycopenic symptoms often result from a gradual decline in blood glucose, often to levels below 40 mg/dl. The slow decline in glucose deprives the CNS of fuel, but fails to trigger an adequate epinephrine response.

A. Actions of some of the glucoregulatory hormones in response to low blood glucose. B. Glycemic thresholds for the various responses to hypoglycemia. + = weak stimulation; ++ = moderate stimulation; +++ = strong stimulation; 0 = no effect. [Note: Normal fasted blood glucose is 70-99 mg/100 ml.]

Reversal of insulin-induced hypoglycemia by administration of subcutaneous glucagon.

Humans have two overlapping glucose-regulating systems that are activated by hypoglycemia: 1) the islets of Langerhans, which release glucagon; and 2) receptors in the hypothalamus, which respond to abnormally low concentrations of blood glucose. The hypothalamic glucoreceptors can trigger both the secretion of epinephrine (mediated by the autonomic nervous system) and release of adrenocorticotropic hormone (ACTH) and growth hormone by the anterior pituitary (see Figure 23.13). [Note: ACTH increases cortisol synthesis and secretion in the adrenal cortex (see p. 239.] Glucagon, epinephrine, cortisol, and growth hormones are sometimes called the "counter-regulatory" hormones because each opposes the action of insulin on glucose use.
1.
Glucagon and epinephrine: Hypoglycemia is combatted by decreased release of insulin and increased secretion of glucagon, epinephrine, cortisol, and growth hormone (see Figure 23.13). Glucagon and epinephrine are most important in the acute, shortterm regulation of blood glucose levels. Glucagon stimulates hepatic glycogenolysis and gluconeogenesis. Epinephrine promotes glycogenolysis and lipolysis, inhibits insulin secretion, and inhibits the insulin-mediated uptake of glucose by peripheral tissues. Epinephrine is not normally essential in combating hypoglycemia, but it can assume a critical role when glucagon secretion is deficient, for example, in the late stages of type 1 (formerly called insulin-dependent) diabetes mellitus (see p. 340). The prevention or correction of hypoglycemia fails when the secretion of both glucagon and epinephrine is deficient.
- 2. Cortisol and growth hormone: These hormones are less important in the short-term maintenance of blood glucose concentrations. They do, however, play a role in the long-term management of glucose metabolism.

Hypoglycemia may be divided into three types: 1) insulin-induced; 2) postprandial (sometimes called reactive hypoglycemia); and 3) fasting hypoglycemia. [Note: Alcohol intoxication in fasting individuals can also be associated with hypoglycemia.]
1. Insulin-induced hypoglycemia: Hypoglycemia occurs frequently in patients with diabetes who are receiving insulin treatment, particularly those striving to achieve tight control of blood glucose levels. Mild hypoglycemia in fully conscious patients is treated by oral administration of carbohydrate. More commonly, patients with hypoglycemia are unconscious or have lost the ability to coordinate swallowing. In these cases, glucagon, administered subcutaneously or intramuscularly, is the treatment of choice (Figure 23.14).
2. Postprandial hypoglycemia: This is the second most common form of hypoglycemia. It is caused by an exaggerated insulin release following a meal, prompting transient hypoglycemia with mild adrenergic symptoms. The plasma glucose level returns to normal even if the patient is not fed. The only treatment usually required is that the patient eat frequent small meals rather than the usual three large meals.
3. Fasting hypoglycemia: Low blood glucose during fasting is rare, but is more likely to present as a serious medical problem. Fasting hypoglycemia, which tends to produce neuroglycopenia symptoms, may result from a reduction in the rate of glucose production by hepatic glycogenolysis or gluconeogenesis. Thus, low blood glucose levels are often seen in patients with hepatocellular damage or adrenal insufficiency, or in fasting individuals who have consumed large quantities of ethanol (see below). Alternately, fasting hypoglycemia may be the result of an increased rate of glucose use by the peripheral tissues due to overproduction of insulin by rare pancreatic tumors. If left untreated, a patient with fasting hypoglycemia may lose consciousness and experience convulsions and coma. [Note: Defects in fatty acid oxidation also result in hypoglycemia.]

4. Hypoglycemia and alcohol intoxication: Alcohol is metabolized in the liver by two oxidation reactions (Figure 23.15). Ethanol is first converted to acetaldehyde by alcohol dehydrogenase. Acetaldehyde is subsequently oxidized to acetate by aldehyde dehydrogenase. [Note: This enzyme is inhibited by disulfiram, a drug that has found some use in patients desiring to stop alcohol ingestion. It causes the accumulation of acetaldehyde in the blood, which results in flushing, tachycardia, hyperventilation, and nausea.] In each reaction, electrons are transferred to NAD*, resulting in a massive increase in the concentration of cytosolic NADH. The abundance of NADH favors the reduction of pyruvate to lactate, and of oxaloacetate (OAA) to malate. [Note: The rise in lactate can result in lactic acidosis and, because lactate competes with urate for excretion by the kidney, can also result in hyperuricemia.] Recall from p. 118 that pyruvate and OAA are both intermediates in the synthesis of glucose by gluconeogenesis. Thus, the ethanol-mediated increase in NADH causes the intermediates of gluconeogenesis to be diverted into alternate reaction pathways, resulting in the decreased synthesis of glucose.

A. Normal gluconeogenesis in the absence of ethanol consumption. B. Inhibition of gluconeogenesis resulting from hepatic metabolism of ethanol.

Effects of chronic alcohol consump- tion on liver morphology.
This can precipitate hypoglycemia, particularly in individuals who have depleted their stores of liver glycogen. [Note: Decreased availability of OAA allows acetyl CoA to be diverted to ketone body synthesis in liver (see p. 195), and can result in alcoholic ketoacidosis.] Hypoglycemia can produce many of the behaviors associated with alcohol intoxication—agitation, impaired judgement, and combativeness. Thus, alcohol consumption in vulnerable individuals—those who are fasted or have engaged in prolonged, strenuous exercise—can produce hypoglycemia that may contribute to the behavioral effects of alcohol. Alcohol consumption can also increase the risk for hypoglycemia in patients using insulin; therefore, patients in an intensive insulin treatment protocol (see p. 340) are counseled about the increased risk of hypoglycemia that generally occurs many hours after the alcohol has been consumed. [Note: Chronic alcohol consumption can also result in alcoholic fatty liver due to increased synthesis of triacylglycerols. This occurs as a result of decreased fatty acid oxidation due to a fall in the NAD*/NADH ratio, and increased lipogenesis due to the increased availability of fatty acids (decreased catabolism) and of glyceraldehyde 3-phosphate (the dehydrogenase is inhibited by the low NAD*/NADH ratio). With continued alcohol consumption, alcoholic fatty liver can progress first to alcoholic hepatitis, and then to alcoholic cirrhosis (Figure 23.16).]

The integration of energy metabolism is controlled primarily by insulin and the opposing actions of glucagon and epinephrine (Figure 23.17). Changes in the circulating levels of these hormones allow the body to store energy when food is available in abundance, or to make stored energy available, for example, during "survival crises," such as famine, severe injury, and "fight-or-flight" situations. Insulin is a polypeptide hormone produced by the B-cells of the islets of Langerhans of the pancreas. The biosynthesis involves two inactive precursors, preproinsulin and proinsulin, which are sequentially cleaved to form the active hormone. A rise in blood glucose is the most important signal for increased insulin secretion. The synthesis and release of insulin are decreased by epinephrine, which is secreted by the adrenal cortex in response to stress, trauma, or extreme exercise. Insulin increases glucose uptake (by muscle and adipose) and the synthesis of glycogen, protein, and triacylglycerol. These actions are mediated by the binding of insulin to the « subunit of the insulin receptor, which initiates a cascade of cell-signaling responses, including phosphorylation by the B subunit of a family of proteins called insulin receptor substrate (IRS) proteins. Glucagon is a polypeptide hormone secreted by the a cells of the pancreatic islets. Glucagon, along with epinephrine, cortisol, and growth hormone (the "counter-regulatory hormones'"), opposes many of the actions of insulin. Glucagon acts to maintain blood glucose during periods of potential hypoglycemia. Glucagon increases glycogenolysis, gluconeogenesis, lipolysis, ketogenesis, and uptake of amino acids. Glucagon secretion is stimulated by low blood glucose, amino acids, and epinephrine. Its secretion is inhibited by elevated blood glucose and by insulin. Glucagon binds to G protein-coupled receptors of hepatocytes. This binding results in the activation of adenylate cyclase, which produces the second messenger, cyclic AMP (cAMP). Subsequent activation of cAMP-dependent protein kinase results in the phosphorylation-mediated activation or inhibition of key regulatory enzymes involved in carbohydrate and lipid metabolism. Both insulin and glucagon affect gene transcription. Hypoglycemia is characterized by: 1) central nervous system

symptoms, including confusion, aberrant behavior, or coma; 2) a simultaneous blood glucose level equal to or less than 40 mg/dl; and 3) resolution of these symptoms within minutes following the administration of glucose. Hypoglycemia most commonly occurs in patients receiving insulin treatment with tight control. The consumption and subsequent metabolism of ethanol inhibits gluconeogenesis, leading to hypoglycemia in individuals with depleted stores of liver glycogen. Alcohol consumption can also increase the risk for hypoglycemia in patients using insulin. Chronic alcohol consumption can cause liver disease.

- 23.1 In which one of the following tissues is glucose trans-
port into the cell insulin-sensitive?
A. Brain.
B. Lens of the eye.
C. Red blood cells.
D. Adipose tissue.
E. Liver.

insulin levels?
A. Increased glycogen synthesis.
B. Decreased gluconeogenesis from lactate.
C. Decreased glycogenolysis.
D. Increased formation of 3-hydroxybutyrate.
E. Decreased action of hormone-sensitive lipase.

glucagon is correct?
A. High levels of blood glucose increase the release of glucagon from the « cells of the pancreas.
B. Glucagon levels decrease following ingestion of a protein-rich meal.
C. Glucagon increases the intracellular levels of CAMP in liver cells, causing an increase in glycogenolysis.
D. Glucagon is the only hormone important in combating hypoglycemia.
E. Glucagon depresses ies by the liver.
the formation of ketone bod-

room complaining of dizziness. She recalls getting up early that morning to do as much shopping as possible and had skipped breakfast. She drank a cup of coffee for lunch and had nothing to eat during the day. She met with friends at 8 p.m. and had a drink at the bar. She soon became weak and dizzy and was transported to the hospital. Following examination, the patient was given orange juice and immediately felt better. Which one of the following best completes this sentence? "The has":
patient
- A. blood glucose greater than 70 mg/dl.
moon
. elevated insulin.
. elevated glucagon.
. elevated liver glycogen.
. presence of an insulinoma.
Correct answer = D. The major tissues in which glucose transport requires insulin are muscle and adipose tissue. The metabolism of the liver responds to insulin, but hepatic glucose transport is determined by blood glucose concentration and does not require insulin. Brain, RBC, and lens of the eye have insulin-insensitive uptake of glucose.
Correct answer = D. 3-Hydroxybutyrate (or Bhydroxybutyrate)—a ketone body—synthesis is enhanced in the liver by low insulin levels, which favor activation of hormone-sensitive lipase and release of fatty acids from adipose tissue. Glycogen synthesis is decreased, whereas gluconeogenesis and glycogenolysis are increased.
Correct answer = C. The cAMP cascade initiated by glucagon causes the liver to degrade glycogen, releasing glucose to the blood. High levels of blood glucose decrease the release of glucagon from the a cells of the pancreas. Glucagon levels increase following ingestion of a protein-rich meal. In addition to glucagon, epinephrine and cortisol are also important in increasing glucose production in hypoglycemia. Glucagon increases the formation of ketone bodies by the liver.
Correct answer = C. The patient's glucagon level will be elevated in response to the hypoglycemia. She is most likely experiencing alcohol-induced fasting hypoglycemia. Blood glucose is expected to be 40 mg/dl or less, insulin secretion depressed because of the low blood glucose, and liver glycogen levels low because of the fast. An insulinoma, an insulin-producing tumor of the pancreas, is unlikely.

The absorptive (fed) state is the twoto four-hour period after ingestion of anormal meal. During this interval, transient increases in plasma glucose, amino acids, and triacylglycerols (TAG) occur, the latter primarily as components of chylomicrons synthesized by the intestinal mucosal cells (See p. 228). Islet tissue of the pancreas responds to the elevated levels of glucose and amino acids with an increased secretion of insulin and a decreased release of glucagon. The elevated insulin to glucagon ratio and the ready availability of circulating substrates make the absorptive state an anabolic period characterized by increased synthesis of TAG and glycogen to replenish fuel stores, and enhanced synthesis of protein. During this absorptive period, virtually all tissues use glucose as a fuel, and the metabolic response of the body is dominated by alterations in the metabolism of liver, adipose tissue, muscle, and brain. In this chapter, an "organ map" is introduced that traces the movement of metabolites between tissues. The goal is to create an expanded and clinically useful vision of whole-body metabolism.

The flow of intermediates through metabolic pathways is controlled by four mechanisms: 1) the availability of substrates; 2) allosteric regulation of enzymes; 3) covalent modification of enzymes; and 4) inductionrepression of enzyme synthesis, primarily through regulation of transcription. This scheme may at first seem unnecessarily redundant; however, each mechanism operates on a different timescale (Figure 24.1), and allows the body to adapt to a wide variety of physiologic situations. In the fed state, these regulatory mechanisms ensure that available nutrients are captured as glycogen, TAG, and protein.

Allosteric changes usually involve rate-determining reactions. For example, glycolysis in the liver is stimulated following a meal by an increase in fructose 2,6-bisphosphate—an allosteric activator of phosphofructokinase-1 (see p. 99). In contrast, gluconeogenesis is inhibited by fructose 2,6-bisphosphate, an allosteric inhibitor of fructose 1,6-bisphosphatase (see p. 121).

Control mechanisms of metabolism and some typical response times. [Note: Response times may vary according to the nature of the stimulus and from tissue to tissue.]

Many enzymes are regulated by the addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the protein. In the absorptive state, most of the enzymes regulated by these covalent modifications are in the dephosphorylated form and are active (Figure 24.2). Three exceptions are glycogen : phosphorylase kinase (see p. 132), glycogen phosphorylase (see p. 132), and hormone-sensitive lipase of adipose tissue (see p. 190), which are inactive in their dephosphorylated form.

Increased (induction of) or decreased (repression of) enzyme synthesis leads to changes in the total population of active sites, rather than influencing the efficiency of existing enzyme molecules. to of are often those that are needed at only one stage of development or under selected physiologic conditions. For example, in the fed state, elevated insulin levels result in an increase in the synthesis of key enzymes, such as acetyl coenzyme A (CoA) carboxylase (see p. 184) and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (see p. 223) involved in anabolic metabolism.
Enzymes subject regulation synthesis (hepatic)

The liver is uniquely situated to process and distribute dietary nutrients because the venous drainage of the gut and pancreas passes through the hepatic portal vein before entry into the general circulation. Thus, after a meal, the liver is bathed in blood containing absorbed nutrients and elevated levels of insulin secreted by the pancreas. During the absorptive period, the liver takes up carbohydrates, lipids, and most amino acids. These nutrients are then metabolized, stored, or routed to other tissues. Thus, the liver smooths out potentially broad fluctuations in the availability of nutrients for the peripheral tissues.

Liver is normally a glucose-producing rather than a glucose-using . a . tissue. However, after a meal containing carbohydrate, the liver becomes a net consumer of glucose, retaining roughly 60 of every ye 100 g of glucose presented by the portal system. This increased use is not a result of stimulated glucose transport into the hepatocyte, because this process is normally rapid and the glucose transporter, GLUT-2 (see p. 97), is insulin insensitive. Rather, hepatic glucose metabolism is increased by the following mechanisms. [Note: The numbers in colored circles in the text refer to Figure 24.3.]

1
Important reactions of intermediary metabolism regulated by enzyme phosphorylation. Blue text = intermediates of carbohydrate metabolism; Brown text = intermediates of lipid metabolism.
- . Increased phosphorylation of glucose: Elevated levels of glucose within the hepatocyte (as a result of elevated extracellular levels) allow glucokinase to phosphorylate glucose to glucose 6-phosphate. (Recall that glucokinase is not subject to product inhibition.) This contrasts with the postabsorptive (fasted) state in which hepatic glucose levels are lower and glucokinase is largely dormant because of its low affinity (high Km) for glucose (Figure 24.3, @).

2. Increased glycogen synthesis: The conversion of glucose 6-phosphate to glycogen is favored by the activation of glycogen synthase—both by dephosphorylation and by increased availability of glucose 6-phosphate, its allosteric effector (see Figure 24.3, ®).
3. Increased activity of the hexose monophosphate pathway (HMP): The increased availability of glucose 6-phosphate in the absorptive state, combined with the active use of NADPH in hepatic lipogenesis, stimulate the HMP (see Chapter 12, p. 145). This pathway typically accounts for 5-10% of the glucose metabolized by the liver (see Figure 24.3, ©).
4. Increased glycolysis: In liver, glycolytic metabolism of glucose is significant only during the absorptive period following a carbohydrate-rich meal. The conversion of glucose to acetyl CoA is stimulated by the elevated insulin to glucagon ratio that results in increased activity (and amount) of the regulated enzymes of glycolysis, for example, pyruvate kinase (see p. 102). Pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl CoA, is active (dephosphorylated) because pyruvate inhibits PDH kinase (see Figure 24.3, @). Acetyl CoA is used as either a building block for fatty acid synthesis, or it provides energy by oxidation in the tricarboxylic acid (TCA) cycle.
5. Decreased gluconeogenesis: Whereas glycolysis is stimulated in the absorptive state, gluconeogenesis is decreased. Pyruvate carboxylase, which catalyzes the first step in gluconeogenesis, is largely inactive due to low levels of acetyl CoA—an allosteric effector essential for enzyme activity (see p. 119). [Note: The acetyl CoA is being used for fatty acid synthesis.] The high insulin to glucagon ratio also favors inactivation of other gluconeogenic

Major metabolic pathways in liver in the absorptive state. [Note: The acetyl CoA is also used for cholesterol synthesis.] The numbers in circles, which appear both in the figure and in the text, indicate important pathways for carbohydrate. fat, or protein metabolism. Blue text = intermediates of carbohydrate metabolism; Brown text = intermediates of lipid metabolism; Green text = intermediates of protein metabolism.

Colorized transmission electron micrograph of adipocytes.
enzymes, such as fructose 1,6-bisphosphatase (see Figure 8.17, p. 100). [Note: Glycogenolysis is also decreased in this period.]

1. Increased fatty acid synthesis: Liver is the primary tissue for de novo synthesis of fatty acids (see Figure 24.3, ®). This pathway occurs in the absorptive period, when dietary caloric intake exceeds energy expenditure by the body. Fatty acid synthesis is favored by the availability of substrates (acetyl CoA and NADPH derived from the metabolism of glucose) and by the activation of acetyl CoA carboxylase, both by dephosphorylation and by the presence of its allosteric activator, citrate. This enzyme catalyzes the formation of malonyl CoA from acetyl CoA—a reaction that is rate-limiting for fatty acid synthesis (see p. 183). [Note: Fatty acid oxidation is inhibited in this period by malonyl CoA.]
- 2. Increased TAG synthesis: TAG synthesis is favored because fatty acyl CoA is available both from de novo synthesis from acetyl CoA and from hydrolysis of the TAG component of chylomicron remnants removed from the blood by hepatocytes (see p. 178). Glycerol 3-phosphate, the backbone for TAG synthesis, is provided by the glycolytic metabolism of glucose (see p. 189). The liver packages TAG into very-low-density lipoprotein (VLDL) particles that are secreted into the blood for use by extrahepatic tissues, particularly adipose and muscle tissue (see Figure 24.3, O).

1. Increased amino acid degradation: In the absorptive period, more amino acids are present than the liver can use in the synthesis of proteins and other nitrogen-containing molecules. The surplus amino acids are not stored, but are either released into the blood for all tissues to use in protein synthesis or are deaminated, with the resulting carbon skeletons being degraded by the liver to pyruvate, acetyl CoA, or TCA cycle intermediates. These metabolites can be oxidized for energy or used in fatty acid synthesis (see Figure 24.3, @). The liver has limited capacity to degrade the branched-chain amino acids leucine, isoleucine, and valine. They pass through the liver essentially unchanged and are preferentially metabolized in muscle (see p. 266).
- 2. Increased protein synthesis: The body cannot store protein in the same way that it maintains glycogen or TAG reserves (see p. 327). However, a transient increase in the synthesis of hepatic proteins does occur in the absorptive state, resulting in replacement of any proteins that may have been degraded during the previous postabsorptive period (see Figure 24.3, ®).

Adipose tissue is second only to the liver in its ability to distribute fuel molecules. In a 70-kg man, adipose tissue weighs approximately 14 kg, or about half as much as the total muscle mass. In obese individuals adipose tissue can constitute up to 70% of body weight. Nearly the entire volume of each adipocyte can be occupied by a droplet of TAG (Figure 24.4).

1. Increased glucose transport: Glucose transport by GLUT-4 into adipocytes is sensitive to the insulin concentration in the blood. Circulating insulin levels are elevated in the absorptive state, resulting in an influx of glucose into adipocytes (Figure 24.5, @).
2. Increased glycolysis: The increased intracellular availability of glucose results in an enhanced rate of glycolysis (see Figure 24.5, ®). In adipose tissue, glycolysis serves a synthetic function by supplying glycerol phosphate for TAG synthesis (see p. 189).
3. Increased activity in the HMP: Adipose tissue can metabolize glucose by means of the HMP, thereby producing NADPH, which is essential for fat synthesis (see p. 186 and Figure 24.5, ®). However in humans, de novo synthesis is not a major source of fatty acids in adipose tissue.

1. Increased synthesis of fatty acids: De novo synthesis of fatty acids from acetyl CoA in adipose tissue is low in humans, except when refeeding a previously fasted individual (see Figure 24.5, @). Instead, most of the fatty acids added to the lipid stores of adipocytes are provided by dietary fat (in the form of chylomicrons), with a lesser amount supplied by VLDL from the liver (see pp. 228 and 231).
2. Increased TAG synthesis: After consumption of a lipid-containing meal, hydrolysis of the TAG of chylomicrons (from the intestine) and VLDL (from the liver) provides adipose tissue with fatty acids (see Figure 24.5, @). The fatty acids are released from lipoproteins by the action of /ipoprotein lipase, an extracellular enzyme attached to the capillary walls in many tissues—particularly adipose and muscle. Because adipocytes lack glycerol kinase, glycerol 3-phosphate used in TAG synthesis comes from the metabolism of glucose (see p. 189). Thus, in the fed state, elevated levels of glucose and insulin favor storage of TAG (see Figure 24.5, @), all the carbons of which are supplied by glucose.
3. Decreased TAG degradation: Elevated insulin favors the dephosphorylated (inactive) form of hormone-sensitive lipase (see p. 190). TAG degradation is thus inhibited in the fed state.

The energy metabolism of skeletal muscle is unique in being able to respond to substantial changes in the demand for ATP that accompanies muscle contraction. At rest, muscle accounts for approximately 30% of the oxygen consumption of the body, whereas during vigorous exercise, it is responsible for up to 90% of the total oxygen consumption. This graphically illustrates the fact that skeletal muscle, despite its potential for transient periods of anaerobic glycolysis, is an oxidative tissue. [Note: Heart muscle differs from skeletal muscle in three important ways: 1) the heart is continuously active, whereas skeletal muscle contracts intermittently on demand; 2) the heart has a completely aerobic metabolism;

Major metabolic pathways in adipose tissue in the absorptive state. [Note: The numbers in the circles, which appear both in the figure and in the corresponding text, indicate important pathways for adipose tissue metabolism.]
SSS

Major metabolic pathways in skeletal muscle in the absorptive state. [Note: The numbers in circles, which appear both in the figure and in the text, indicate important pathways for carbohydrate or protein metabolism.]
and 3) the heart contains negligible energy stores, such as glycogen or lipid. Thus, any interruption of the vascular supply, for example, as occurs during a myocardial infarction, results in rapid death of the myocardial cells.] Heart muscle uses fatty acids, glucose, and ketone bodies as fuels.

1. Increased glucose transport: The transient increase in plasma glucose and insulin after a carbohydrate-rich meal leads to an increase in glucose transport into muscle cells by GLUT-4 (see p. 97 and Figure 24.6, ®). Glucose is phosphorylated to glucose 6phosphate by hexokinase, and metabolized to provide the energy needs of the cells. [Note: In the fasted state fatty acids and ketone bodies are the major fuels of resting muscle.]
2. Increased glycogen synthesis: The increased insulin to glucagon ratio and the availability of glucose 6-phosphate favor glycogen synthesis, particularly if glycogen stores have been depleted as a result of exercise (see p. 126, and Figure 24.6, ®).

Fatty acids are released from chylomicrons and VLDL by the action of lipoprotein lipase (see pp. 228 and 231). However, fatty acids are of secondary importance as a fuel for muscle during the fed state, in which glucose is the primary source of energy.

1.
Increased protein synthesis: A spurt in amino acid uptake and protein synthesis occurs in the absorptive period after ingestion of a meal containing protein (see Figure 24.6, ® and @). This synthesis replaces protein degraded since the previous meal.
- 2. Increased uptake of branched-chain amino acids: Muscle is the principal site for degradation of branched-chain amino acids because it contains the required transaminase (see p. 266). The branched-chain amino acids, leucine, isoleucine, and valine, escape metabolism by the liver, and are taken up by muscle, where they are used for protein synthesis (see Figure 24.6, ®) and as sources of energy.

Although contributing only 2% of the adult weight, the brain accounts for a consistent 20% of the basal oxygen consumption of the body at rest. Because the brain is vital to the proper functioning of all organs of the body, special priority is given to its fuel needs. To provide energy, substrates must be able to cross the endothelial cells that line the blood vessels in the brain (the "blood-brain barrier'). Normally, glucose serves as the primary fuel for the brain. [Note: If blood glucose levels fall below approximately 40 mg/100 ml (normal fasted blood glucose is 70-99 mg/100 ml), cerebral function is impaired. If the hypoglycemia occurs for even a short time, severe and potentially irreversible brain damage may occur.] During a fast, however, ketone bodies play a significant role as a fuel for brain, reducing its dependence on glucose (see p. 196).

In the fed state, the brain uses glucose exclusively as a fuel, completely oxidizing approximately 140 g/day to COz2 and H20. The brain contains no significant stores of glycogen and is, therefore, completely dependent on the availability of blood glucose (Figure 24.7, @).

The brain has no significant stores of TAG, and the fatty acids circulating in the blood make little contribution to energy production because fatty acids bound to albumin do not efficiently cross the blood-brain barrier. The intertissue exchanges characteristic of the absorptive period are summarized in Figure 24.8.

Fasting begins if no food is ingested after the absorptive period. It may result from an inability to obtain food, the desire to lose weight rapidly, or clinical situations in which an individual cannot eat, for example, because of trauma, surgery, cancer, or burns. In the absence of food, plasma levels of glucose, amino acids, and TAG fall, triggering a decline in insulin secretion and an increase in glucagon release. The decreased insulin to glucagon ratio, and the decreased availability of circulating substrates, make the period of nutrient deprivation a catabolic period characterized by degradation of TAG, glycogen, and protein. This sets into motion an exchange of substrates among liver, adipose tissue, muscle, and brain that is guided by two priorities: 1) the need to maintain adequate plasma levels of glucose to sustain energy metabolism of the brain, red blood cells, and other glucose-requiring tissues; and 2) the need to mobilize fatty acids from adipose tissue, and the synthesis and release of ketone bodies from the liver, to supply energy to all other tissues.

The metabolic fuels available in a normal 70—kg man at the beginning of a fast are shown in Figure 24.9. Note the enormous caloric stores available in the form of TAG compared with those contained in glycogen. [Note: Although protein is listed as an energy source, each protein also has a function, for example, as a structural component of the body, an enzyme, and so forth. Therefore, only about one third of the body's protein can be used for energy production without fatally compromising vital functions.]

In fasting (as in the fed state), the flow of intermediates through the pathways of energy metabolism is controlled by four mechanisms: 1) the availability of substrates; 2) allosteric regulation of enzymes; 3) covalent modification of enzymes; and 4) induction-repression of enzyme synthesis. The metabolic changes observed in fasting are generally opposite to those described for the absorptive state (see Figure 24.8). For example, most of the enzymes regulated by covalent

Major metabolic pathways in brain in the absorptive state. [Note: The numbers in circles, which appear both in the figure and in the text, indicate important pathways for carbohydrate metabolism.]

Intertissue relationships in the absorptive state. [Note: Small circles on the perimeter of tissues indicate insulin-dependent transport systems.]
modification are dephosphorylated and active in the fed state, whereas in the fasted state, they are phosphorylated and active. Three exceptions are glycogen phosphorylase (see p. 132), glycogen phosphorylase kinase (see p. 132), and hormone-sensitive lipase of adipose tissue (see p. 190), which are inactive in their dephosphorylated states. In fasting, substrates are not provided by the diet, but are available from the breakdown of stores and/or tissues, for example, lipolysis with release of fatty acids and glycerol from TAG in adipose tissue, and proteolysis with release of amino acids from muscle.
Recognition that the changes in fasting are the reciprocal of those in the fed state is helpful in understanding the ebb and flow of metabolism,

The primary role of liver in energy metabolism during fasting is maintenance of blood glucose through the synthesis and distribution of fuel molecules for use by other organs. Thus, one speaks of "hepatic metabolism" and "extrahepatic" or "peripheral" metabolism.

The liver first uses glycogen degradation and then gluconeogenesis to maintain blood glucose levels to sustain energy metabolism of the brain and other glucose-requiring tissues in the fasted (postabsorptive) state. [Note: Recall the presence of glucose 6-phosphatase in the liver allows the production of free glucose both from glycogenoland from ysis gluconeogenesis (see p. 130).]
1.
Increased glycogen degradation: Figure 24.10 shows the sources of blood glucose after ingestion of 100 g of glucose. During the brief absorptive period, ingested glucose is the major source of blood glucose. Several hours later, blood glucose levels have declined sufficiently to cause increased secretion of glucagon and decreased release of insulin. The increased glucagon to insulin ratio causes a rapid mobilization of liver glycogen stores (which contain about 80 g of glycogen in the fed state) due to phosphorylation (activation) of glycogen phosphorylase (see p. 130). Note that liver glycogen is nearly exhausted after 10-18 hours of fasting; therefore, hepatic glycogenolysis is a transient response to early fasting. Figure 24.11, @, shows glycogen degradation as part of the overall metabolic response of the liver during fasting.

Increased gluconeogenesis: The synthesis of glucose and its release into the circulation are vital hepatic functions during fasting (see Figure 24.11, ®). The carbon skeletons for gluconeogenesis are derived primarily from glucogenic amino acids and lactate from muscle, and glycerol from adipose. Gluconeogenesis, favored by activation of fructose 1,6-bisphosphatase (due to a drop in its inhibitor, fructose 2,6-bisphosphate, see p. 121) and by induction of phosphoenolpyruvate (PEP) carboxykinase by glucagon (see p. 122), begins 4-6 hours after the last meal and becomes fully active as stores of liver glycogen are depleted (see Figure 24.10). Gluconeogenesis plays an essential role in maintaining blood glucose during both overnight and prolonged fasting. [Note: Whereas acetyl CoA cannot be used as a substrate for gluconeogenesis, the acetyl CoA produced by hepatic oxidation of fatty acids supplied from lipolysis in adipose is an allosteric activator of pyruvate carboxylase (and an allosteric inhibitor of pyruvate dehydrogenase), and thus pushes pyruvate to gluconeogenesis (see Figure 8.24).]

Metabolic fuels present in a 70-kg man at the beginning of a fast. Fat stores are sufficient to meet energy needs for about 3 months

Major metabolic pathways in liver during starvation. [Note: The numbers in circles, which appear both in the figure and in the corresponding citation in the text, indicate important metabolic pathways for carbohydrate or fat.]

Concentrations of fatty acids and 3-hydroxybutyrate in the blood during fasting.
1. Increased fatty acid oxidation: The oxidation of fatty acids obtained from TAG hydrolysis in adipose tissue is the major source of energy in hepatic tissue in the postabsorptive state (see Figure 24.11, ®). The fall in malonyl CoA due to phosphorylation (inactivation) of acetyl CoA carboxylase by AMP-activated protein kinase (AMPK) removes the brake on carnitine palmitoyl transferase-1 (CPT-1), allowing B-oxidation to occur (see p. 191). [Note: Fatty acid oxidation provides the NADH and ATP required for gluconeogenesis.]
- 2. Increased synthesis of ketone bodies: The liver is unique in being able to synthesize and release ketone bodies, primarily 3-hydroxybutyrate (formerly called B-hydroxybutyrate), for use as fuel by peripheral tissues (see p. 195), but not by the liver itself. Ketogenesis is favored when the concentration of acetyl CoA, produced from fatty acid metabolism, exceeds the oxidative capacity of the TCA cycle. [Note: Ketogenesis releases CoA, ensuring its availability for continued fatty acid oxidation.] Ketogenesis starts during the first days of fasting (Figure 24.12). The availability of circulating water-soluble ketone bodies is important in fasting because they can be used for fuel by most tissues, including brain tissue, once their level in the blood is sufficiently high. This reduces the need for gluconeogenesis from amino acid carbon skeletons, thus preserving essential protein. Ketogenesis as part of the overall hepatic response to fasting is shown in Figure 24.11, @. [Note: Ketone bodies are organic acids and, when present at high concentrations, can cause ketoacidosis. ]

Glucose transport by insulin-sensitive GLUT-4 into the adipocyte (see p. 97) and its subsequent metabolism are depressed due to low levels of circulating insulin. This leads to a decrease in fatty acid and TAG synthesis.

1. Increased degradation of TAG: The activation of hormonesensitive lipase (see p. 190) and subsequent hydrolysis of stored TAG are enhanced by the elevated catecholamines epinephrine and, particularly, norepinephrine. These compounds, which are released from the sympathetic nerve endings in adipose tissue, are physiologically important activators of hormone-sensitive lipase (Figure 24.13, @). [Note: Glucagon also activates the lipase (see p. 190).]
2. Increased release of fatty acids: Fatty acids obtained from hydrolysis of stored TAG are primarily released into the blood (see Figure 24.13, ®). Bound to albumin, they are transported to a variety of tissues for use as fuel. The glycerol produced from TAG degradation is used as a gluconeogenic precursor by the liver. [Note: Fatty acids can also be oxidized to acetyl CoA, which can enter the TCA cycle, thus producing energy for the adipocyte. They also can be re-esterified to glycerol 3-phosphate (from glyceroneogenesis, see p. 190), generating TAG.]
3. Decreased uptake of fatty acids: In fasting, /ipoprotein lipase activity of adipose tissue is low. Consequently, circulating TAG of lipoproteins is not available to adipose tissue.

Resting muscle uses fatty acids as its major fuel source. By contrast, exercising muscle initially uses its glycogen stores as a source of energy. During intense exercise, glucose 6-phosphate derived from glycogen is converted to lactate by anaerobic glycolysis (see p. 103). As these glycogen reserves are depleted, free fatty acids provided by the mobilization of TAG from adipose tissue become the dominant energy source.

Glucose transport into skeletal muscle cells via insulin-sensitive GLUT-4 proteins in the plasma membrane (see p. 97) and subsequent glucose metabolism are depressed because of low levels of circulating insulin.

During the first 2 weeks of fasting, muscle uses fatty acids from adipose tissue and ketone bodies from the liver as fuels (Figure 24.14, @ and ®). After about 3 weeks of fasting, muscle decreases its use of ketone bodies and oxidizes fatty acids almost exclusively. This

Major metabolic pathways in adipose tissue during starvation. [Note: The numbers in the circles, which appear both in the figure and in the corresponding citation in the text, indicate important pathways for fat metabolism.]

Major metabolic pathways in skeletal muscle during starvation. [Note:The numbers in the circles, which appear both in the figure and in the corresponding citation in the text, indicate important pathways for fat or protein metabolism.]

Major metabolic pathways in the brain during starvation. [Note:The numbers in the circles, which appear both in the figure and in the corresponding citation in the text, indicate important pathways for metabolism of fat or carbohydrates. ]
leads to a further increase in the already elevated level of circulating ketone bodies. [Note: The increased use of ketone bodies by the brain as a result of their increased concentration in the blood is correlated with the decreased use of these compounds by the muscle.]

During the first few days of fasting, there is a rapid breakdown of muscle protein, providing amino acids that are used by the liver for gluconeogenesis (see Figure 24.14, ®). Because muscle does not have glucagon receptors, muscle proteolysis likely is initiated by the fall in insulin and sustained by the rise in glucocorticoids. [Note: Alanine and glutamine are quantitatively the most important gluconeogenic amino acids released from muscle. They are produced by the catabolism of branched-chain amino acids (see p. 267).] By several weeks of fasting, the rate of muscle proteolysis decreases paralleling a decline in the need for glucose as a fuel for the brain, which has begun using ketone bodies as a source of energy.

During the first days of fasting, the brain continues to use glucose exclusively as a fuel (Figure 24.15, @). [Note: Blood glucose is maintained by hepatic gluconeogenesis from glucogenic precursors, such as amino acids from proteolysis and glycerol from lipolysis.] In prolonged fasting (greater than 2-3 weeks), plasma ketone bodies (see Figure 24.12) reach significantly elevated levels, and replace glucose as the primary fuel for the brain (see Figure 24.15, ®, and Figure 24.16). This reduces the need for protein catabolism for gluconeogenesis: ketone bodies spare glucose and, thus, muscle protein. The metabolic changes that occur during fasting ensure that all tissues have an adequate supply of fuel molecules. The response of the major tissues involved in energy metabolism during fasting is summarized in Figure 24.17 (see below).

As fasting continues into early starvation and beyond, the kidney plays important roles. Kidney expresses the enzymes of gluconeogenesis, including glucose 6-phosphatase, and in late fasting about 50% of gluconeogenesis occurs here. [Note: A portion of this glucose is used by kidney itself.] Kidney also provides compensation for the acidosis that accompanies the increased production of ketone bodies (organic acids). The glutamine released from the muscle's metabolism of branched-chain amino acids is taken up by the kidney and acted upon by renal glutaminase and glutamate dehydrogenase (see p. 256), producing a-ketoglutarate that can be used as a substrate for gluconeogenesis, plus ammonia (NH3). The NH3 picks up H* from ketone body dissociation, and is excreted in the urine as NH4*, decreasing the acid load in the body. In long-term fasting, then, there is a switch from nitrogen disposal in the form of urea to disposal in the form of ammonia. [Note: As ketone body concentration rises, enterocytes, typically consumers of glutamine, become consumers of ketone bodies. This allows more glutamine to be available to the kidney.]

The flow of intermediates through metabolic pathways is controlled by four mechanisms: 1) the availability of substrates; 2) allosteric activation and inhibition of enzymes; 3) covalent modification of enzymes; and 4) induction-repression of enzyme synthesis. In the absorptive state, these regulatory mechanisms ensure that available nutrients are captured as glycogen, triacylglycerol, and protein (Figure 24.18). The absorptive state is the twoto four-hour period after ingestion of a normal meal. During this interval, transient increases in plasma glucose, amino acids, and triacylglycerols occur, the last primarily as components of chylomicrons synthesized by the intestinal mucosal cells. The pancreas responds to the elevated levels of glucose and amino acids with an increased secretion of insulin and a decrease in the release of glucagon by the islets of Langerhans. The elevated insulin to glucagon ratio and the ready availability of circulating substrates make the 2-4 hours after ingestion of a meal into an anabolic period. During this absorptive period, virtually all tissues use glucose as a fuel. In addition, the liver replenishes its glycogen stores, replaces any needed hepatic proteins, and increases triacylglycerol synthesis. The latter are packaged in very-low-density lipoproteins, which are exported to the peripheral tissues. The adipose increases triacylglycerol synthesis and storage, whereas the muscle increases protein synthesis to replace protein degraded since the previous meal. In the fed state, the brain uses glucose exclusively as a fuel. In the absence of food, plasma levels of glucose, amino acids, and triacylglycerols fall, triggering a decline in insulin secretion and an increase in glucagon and epinephrine release. The decreased insulin to glucagon ratio, and the decreased availability of circulating substrates, makes the period of fasting a catabolic period. This sets into motion an exchange of substrates among liver, adipose tissue, muscle, and brain that is guided by two priorities: 1) the need to maintain adequate plasma levels of glucose to sustain energy metabolism of the brain and other glucose-requiring tissues; and 2) the need to mobilize fatty acids from adipose tissue and ketone bodies from liver to supply energy to all other tissues. To accomplish these goals, the liver degrades glycogen and initiates gluconeogenesis, using increased fatty acid oxidation as a source of

the energy needed for gluconeogenesis, and to supply the acetyl coenzyme A building blocks for ketone body synthesis. The adipose degrades stored triacylglycerols, thus providing fatty acids and glycerol to the liver. The muscle can also use fatty acids as fuel, as well as ketone bodies supplied by the liver. Muscle protein is degraded to supply amino acids for the liver to use in gluconeogenesis. The brain can use both glucose and ketone bodies as fuels. From late fasting into starvation, the kidneys play important roles by synthesizing glucose and excreting the protons from ketone body dissociation as NH,*.

24.1
Which one of the following is elevated in plasma during the absorptive (fed) period as compared with the postabsorptive (fasted) state?
A. Glucagon.
B. Acetoacetate.
C. Chylomicrons.
D. Free fatty acids.
E. Lactate.

the fed state is correct?
- A. Most enzymes that are regulated by covalent
modification are in the phosphorylated state.
B. Hepatic fructose 2,6-bisphosphate is elevated.
C. The oxidation of acetyl CoA is increased.
D. Insulin stimulates the transport of glucose into hepatocytes.
E. The synthesis of glucokinase is repressed.

is a result of:
A. decreased levels of circulating glucagon.
B. decreased formation of acetyl CoA in the liver.
C. increased levels of free fatty acids in blood.
D. inhibition of B-oxidation of fatty acids in the liver.
E. decreased activity of hormone-sensitive lipase in adipose tissue.

source of blood glucose during the last hours of
a
48-hour fast?
A. Muscle glycogen.
C. Liver glycogen.
D. Amino acids.
Correct answer = C. TAG-rich chylomicrons are synthesized in (and released from) the intestine following ingestion of a meal. Glucagon is depressed in the absorptive period. Acetoacetate, free fatty acids, and lactate are not elevated.
Correct answer = B. The increased insulin and decreased glucagon levels characteristic of the fed state promote the synthesis of fructose 2,6bisphosphate. Most covalently modified enzymes are in the dephosphorylated state and are active. Acetyl CoA is not elevated in the fed state. The transport of glucose in the liver is not insulin sensitive. Synthesis of glucokinase is enhanced in the fed state.
Correct answer = C. Free fatty acids bound to albumin are increased as a result of an increased activity of hormone-sensitive lipase in adipose tissue. Hepatic ketogenesis is stimulated by elevated levels of glucagon. The formation of acetyl CoA is increased. B-Oxidation of fatty acids in liver provides the acetyl CoA for ketogenesis.
Correct answer = D. The carbon skeletons of glucogenic amino acids are used by the liver for gluconeogenesis. Liver glycogen is nearly depleted by 12 hours after a meal, and muscle glycogen cannot give rise to free glucose because muscle lacks glucose 6-phosphatase. Acetoacetate is metabolized to acetyl CoA, which is not glucogenic. Lactate can arise from anaerobic glycolysis in muscle and red blood cells, but is less important than amino acids as a source of glucose.

Diabetes mellitus is not one disease, but rather is a heterogeneous group of multifactorial, polygenic syndromes characterized by an elevation of fasting blood glucose caused by a relative or absolute deficiency in insulin. Diabetes is the leading cause of adult blindness and amputation, and a major cause of renal failure, nerve damage, heart attacks, and strokes. Most cases of diabetes mellitus can be separated into two groups (Figure 25.1), type 1 (formerly called insulin-dependent diabetes mellitus) and type 2 (formerly called noninsulin-dependent diabetes). Approximately 150,000 newly diagnosed cases of type 1 and 1.3 million cases of type 2 diabetes are estimated to occur yearly in the United States. The incidence and prevalence of type 2 disease is increasing because of the aging of the United States population, and the increasing prevalence of obesity and sedentary lifestyles (see p. 349). The increase in children with type 2 diabetes is particularly disturbing.

AGE OF ONSET, Type 1 Diabetes = Usually during childhood or puberty; symptoms develop rapidly. AGE OF ONSET, Type 2 Diabetes = Frequently after age 35; symptoms develop gradually. NUTRITIONAL STATUS AT TIME OF DISEASE ONSET, Type 1 Diabetes = Frequently undernourished. NUTRITIONAL STATUS AT TIME OF DISEASE ONSET, Type 2 Diabetes = Obesity usually present. PREVALENCE, Type 1 Diabetes = 10% of diagnosed diabetics. PREVALENCE, Type 2 Diabetes = 90% of diagnosed diabetics. GENETIC PREDISPOSITION, Type 1 Diabetes = Moderate. GENETIC PREDISPOSITION, Type 2 Diabetes = Very strong. DEFECT OR DEFICIENCY, Type 1 Diabetes = β Cells are destroyed, eliminating production of insulin. DEFECT OR DEFICIENCY, Type 2 Diabetes = Insulin resistance combined with inability of β cells to produce appropriate quantities of insulin. FREQUENCY OF KETOSIS, Type 1 Diabetes = Common. FREQUENCY OF KETOSIS, Type 2 Diabetes = Rare. PLASMA INSULIN, Type 1 Diabetes = Low to absent. PLASMA INSULIN, Type 2 Diabetes = High early in disease; low in disease of long duration. ACUTE COMPLICATIONS, Type 1 Diabetes = Ketoacidosis. ACUTE COMPLICATIONS, Type 2 Diabetes = Hyperosmolar state. RESPONSE TO ORAL HYPOGLYCEMIC DRUGS, Type 1 Diabetes = Unresponsive. RESPONSE TO ORAL HYPOGLYCEMIC DRUGS, Type 2 Diabetes = Responsive. TREATMENT, Type 1 Diabetes = Insulin is always necessary. TREATMENT, Type 2 Diabetes = Diet, exercise, oral hypoglycemic drugs; insulin may or may not be necessary. Reduction of risk factors (smoking cessation, blood pressure control, treatment of dyslipidemia) is essential to therapy.

Insulin secretory capacity during onset of type 1 diabetes. [Note: Rate of autoimmune destruction of B cells may be faster or slower than shown.]

Persons with type 1 diabetes constitute approximately 10% of the nearly 20 million known diabetics in the United States. The disease is characterized by an absolute deficiency of insulin caused by an autoimmune attack on the B cells of the pancreas. In type 1 diabetes, the islets of Langerhans become infiltrated with activated T lymphocytes, leading to a condition called insulitis. Over a period of years, this autoimmune attack on the B cells leads to gradual depletion of the B-cell population (Figure 25.2). However, symptoms appear abruptly when 80-90% of the B cells have been destroyed. At this point, the pancreas fails to respond adequately to ingestion of glucose, and insulin therapy is required to restore metabolic control and prevent life-threatening ketoacidosis. 8 Cell destruction requires both a stimulus from the environment (such as a viral infection) and a genetic determinant that allows the B cells to be recognized as "nonself." [Note: Among monozygotic (identical) twins, if one sibling develops type 1 diabetes mellitus, the other twin has only a 30-50% chance of developing the disease. This contrasts with type 2 disease (see p. 341), in which the genetic influence is stronger, and in virtually all monozygotic twinships, the disease eventually develops in both individuals.]
Patients with type 1 diabetes have virtually no functional B cells, and can neither respond to variations in circulating fuels nor maintain a basal secretion of insulin.

The onset of type 1 diabetes is typically during childhood or puberty, and symptoms develop suddenly. Patients with type 1 diabetes can usually be recognized by the abrupt appearance of polyuria (frequent urination), polydipsia (excessive thirst), and polyphagia (excessive hunger), often triggered by stress or an illness. These symptoms are usually accompanied by fatigue, weight loss, and weakness. The diagnosis is confirmed by a fasting blood glucose (FBG) greater than or equal to 126 mg/dl, commonly accompanied by ketoacidosis. [Note: A FBG of 100-125 mg/dl is categorized as an impaired FBG.] Fasting is defined as no caloric intake for at least 8 hours. When the diagnosis of type 1 diabetes is uncertain by clinical presentation, testing for circulating islet-cell antibodies is recommended. [Note: The oral glucose tolerance test is not routinely used as a diagnostic tool for diabetes because it is difficult to perform in practice and the results are highly variable; however, it is used to screen pregnant women for gestational diabetes (see p. 342).]

The metabolic abnormalities of type 1 diabetes mellitus result from a deficiency of insulin which profoundly affects metabolism in three tissues: liver, muscle, and adipose tissue (Figure 25.3).
- 1. Hyperglycemia and ketoacidosis: Elevated levels of blood glucose and ketones are the hallmarks of untreated type 1 diabetes melli-

Intertissue relationships in type 1 diabetes.
tus (see Figure 25.3). Hyperglycemia is caused by increased hepatic production of glucose, combined with diminished peripheral utilization (muscle and adipose have the insulin-sensitive GLUT-4, see p. 97). Ketosis results from increased mobilization of fatty acids from adipose tissue, combined with accelerated hepatic fatty acid B-oxidation and synthesis of 3-hydroxybutyrate and acetoacetate. [Note: Acetyl coenzyme A from B-oxidation is the substrate for ketogenesis and the allosteric effector of pyruvate carboxylase, a gluconeogenic enzyme.] Diabetic ketoacidosis (DKA, a type of metabolic acidosis) occurs in 25-40% of those newly diagnosed with type 1 diabetes, and may recur if the patient becomes ill (most commonly with an infection) or does not comply with therapy. DKA is treated by replacing fluid and electrolytes, and administering short-acting insulin to gradually correct hyperglycemia without precipitating hypoglycemia.
- 2. Hypertriacylglycerolemia: Not all the fatty acids flooding the liver can be disposed of through oxidation or ketone body synthesis. These excess fatty acids are converted to triacylglycerol, which is packaged and secreted in very-low-density lipoproteins (VLDL, see p. 231). Chylomicrons are synthesized from dietary lipids by the intestinal mucosal cells following a meal (see p. 178). Because

Correlation between mean blood glucose and HbAjc in patients with type 1 diabetes.

Effect of tight glucose control on hypoglycemic episodes in a population of patients on intensive therapy or conventional therapy.
lipoprotein degradation catalyzed by lipoprotein lipase in the capillary beds of muscle and adipose tissue (see. p. 228) is low in diabetics (synthesis of the enzyme is decreased when insulin levels are low), the plasma chylomicron and VLDL levels are elevated, resulting in hypertriacylglycerolemia (see Figure 25.3).

Individuals with type 1 diabetes must rely on exogenous insulin injected subcutaneously to control the hyperglycemia and ketoacidosis. Two therapeutic regimens are currently in use—standard and intensive insulin treatment.
Insulin may also be delivered by a pump, which allows continuous subcutaneous infusion of insulin 24 hours a day at preset levels and the ability to program doses (a bolus) of insulin as needed at meal times.
1.
Standard treatment versus intensive treatment: Standard treatment typically consists of one or two daily injections of recombinant human insulin. Mean blood glucose levels obtained are typically in the 225-275 mg/dl range, with a hemoglobin Aic (HbAjc) level (see p. 34) of 8-9% of the total hemoglobin (blue arrow in Figure 25.4). [Note: The rate of formation of HbAjc is proportional to the average blood glucose concentration over the previous 3 months. Thus, HbAic provides a measure of how well treatment has normalized blood glucose in the diabetic over that time.] In contrast to standard therapy, intensive treatment seeks to more closely normalize blood glucose through more frequent monitoring, and subsequent injections of insulin—typically three or more times a day. Mean blood glucose levels of 150 mg/dl can be achieved, with HbAic approximately 7% of the total hemoglobin (red arrow: Figure 25.4). [Note: Normal mean blood glucose is approximately 100 mg/dl and HbAic is 6% or less (black arrow, see Figure 25.4).] Thus, normalization of glucose values (euglycemia) is not achieved even in intensively treated patients. Nonetheless, patients on intensive therapy show a 50% or more reduction in the long-term microvascular complications of diabetes—retinopathy, nephropathy, and neuropathy—compared with patients receiving standard care. This confirms that the complications of diabetes are related to an elevation of plasma glucose.

2. Hypoglycemia in type 1 diabetes: One of the therapeutic goals in cases of diabetes is to decrease blood glucose levels in an effort to minimize the development of long-term complications of the disease (see p. 344 for a discussion of the chronic complications of diabetes). However, appropriate dosage of insulin is difficult to achieve. Hypoglycemia caused by excess insulin is the most common complication of insulin therapy, occurring in over 90% of patients. The frequency of hypoglycemic episodes, coma, and seizures is particularly high with intensive treatment regimens
designed to achieve tight control of blood glucose (Figure 25.5). Recall that in normal individuals hypoglycemia triggers a compensatory secretion of counter-regulatory hormones, most notably glucagon and epinephrine, which promote hepatic production of glucose. However, patients with type 1 diabetes also develop a deficiency of glucagon secretion. This defect occurs early in the disease and is almost universally present 4 years after diagnosis. These patients thus rely on epinephrine secretion to prevent severe hypoglycemia. However, as the disease progresses, type 1 diabetes patients show diabetic autonomic neuropathy and impaired ability to secrete epinephrine in response to hypoglycemia. The combined deficiency of glucagon and epinephrine secretion creates a condition sometimes called "hypoglycemia unawareness." Thus, patients with long-standing diabetes are par-: ° ; ticularly vulnerable to hypoglycemia. Hypoglycemia can also be caused by strenuous exercise. Exercise promotes glucose uptake into muscle and decreases the need for exogenous insulin. Patients are advised, therefore, to check blood glucose levels before or after intensive exercise to prevent or abort hypoglycemia.
- 3. Contraindications for tight control: Children are not put on a program of tight control of blood glucose because of the risk that episodes of hypoglycemia may adversely affect brain development. Elderly people typically do not go on tight control because hypoglycemia can cause strokes and heart attacks in this population. Also, the major goal of tight control is to prevent complications many years later. Tight control, then, is most worthwhile for otherwise healthy people who can expect to live at least ten more years.

Type 2 diabetes is the most common form of the disease, afflicting approximately 90% of the diabetic population in the United States. [Note: Native Americans, Latinos, African Americans, and Asian Americans have the highest prevalence.] Typically, type 2 diabetes develops gradually without obvious symptoms. The disease is often detected by routine screening tests. However, many individuals with type 2 diabetes have symptoms of polyuria and polydipsia of several weeks duration. Polyphagia may be present, but is less common. Patients with type 2 diabetes have a combination of insulin resistance and dysfunctional B cells (Figure 25.6), but do not require insulin to sustain life, although insulin eventually will be required to control hyperglycemia and keep HbA;, below 7% in 90% of patients. The metabolic alterations observed in type 2 diabetes are milder than those described for type 1, in part, because insulin secretion in type 2 diabetes— although not adequate—does restrain ketogenesis and blunts the development of DKA. Diagnosis is based most commonly on the presence of hyperglycemia—that is, a fasting blood glucose concentration of equal to or greater than 126 mg/dl. Pathogenesis does not involve viruses or autoimmune antibodies. [Note: An acute complication of type 2 in the elderly is a hyperglycemic hyperosmolar state characterized by severe hyperglycemia and dehydration, and altered mental status.]

Major factors contributing to hyperglycemia observed in type 2 diabetes.
Type 2 diabetes mellitus is characterized by hyperglycemia, insulin resistance, and relative impairment in insulin secretion.

Insulin resistance is the decreased ability of target tissues, such as liver, adipose, and muscle, to respond properly to normal (or elevated) circulating concentrations of insulin. For example, insulin resistance is characterized by uncontrolled hepatic glucose production, and decreased glucose uptake by muscle and adipose tissue.
1. Insulin resistance and obesity: Obesity is the most common cause of insulin resistance; however, most people with obesity and insulin resistance do not become diabetic. In the absence of a defect in B-cell function, nondiabetic, obese individuals can compensate for insulin resistance with elevated levels of insulin. For example, Figure 25.7A shows that insulin secretion is two to three times higher in obese subjects than it is in lean individuals. This higher insulin concentration compensates for the diminished effect of the hormone (as a result of insulin resistance), and produces blood glucose levels similar to those observed in lean individuals (Figure 25.7B).
2. Insulin resistance and type 2 diabetes: Insulin resistance alone will not lead to type 2 diabetes. Rather, type 2 diabetes develops in insulin-resistant individuals who also show impaired B-cell function. Insulin resistance and subsequent risk for the development of type 2 diabetes is commonly observed in the elderly, and in individuals who are obese, physically inactive, or in the 3-5% of pregnant women who develop gestational diabetes. These patients are unable to sufficiently compensate for insulin resistance with increased insulin release. Figure 25.8 shows the time course for the development of hyperglycemia and the loss of B-cell function.

Blood insulin and glucose levels in normal weight and obese subjects.

Progression of blood glucose and insulin levels in patients with type 2 diabetes.
3. Causes of insulin resistance: Insulin resistance increases with weight gain and, conversely, diminishes with weight loss. This suggests that fat accumulation is important in the development of insulin resistance. Adipose tissue is not simply an energy storage organ, but also a secretory organ. Regulatory substances produced by adipocytes include leptin (see p. 353), and adiponectin (see p. 353), all of which may contribute to the development of insulin resistance. In addition, the elevated levels of free fatty acids (FFA) that occur in obesity have also been implicated in the development of insulin resistance. [Note: FFA also arise from lipolysis in insulin-resistant adipose.]

In type 2 diabetes, the pancreas initially retains B-cell capacity, resulting in insulin levels that vary from above normal to below normal. However, with time, the B cell becomes increasingly dysfunctional and fails to secrete enough insulin to correct the prevailing hyperglycemia. For example, insulin levels are high in typical, obese, type 2 diabetic patients, but not as high as in similarly obese individuals who are nondiabetic. Thus, the natural progression of the disease results in a declining ability to control hyperglycemia with endogenous secretion of insulin (Figure 25.9). Deterioration of B-cell function may be accelerated by the toxic effects of sustained hyperglycemia and elevated FFA.

The metabolic abnormalities of type 2 diabetes mellitus are the result of insulin resistance expressed primarily in liver, muscle, and adipose tissue (Figure 25.10).
1. Hyperglycemia: Hyperglycemia is caused by increased hepatic production of glucose, combined with diminished peripheral use. Ketosis is usually minimal or absent in type 2 patients because the presence of insulin—even in the presence of insulin resistance—diminishes hepatic ketogenesis. [Note: Metformin, an oral agent for the treatment of type 2 diabetes, inhibits hepatic gluconeogenesis".]
2. Dyslipidemia: In the liver, fatty acids are converted to triacylglycerols, which are packaged and secreted in VLDL. Chylomicrons are synthesized from dietary lipids by the intestinal mucosal cells following a meal (see p. 177). Because lipoprotein degradation catalyzed by lipoprotein lipase in adipose tissue (and muscle, see p. 228) is low in diabetics, the plasma chylomicron and VLDL levels are elevated, resulting in hypertriacylglycerolemia (see Figure 25.10). Low HDL levels are also associated with type 2 diabetes.

The goal in treating type 2 diabetes is to maintain blood glucose concentrations within normal limits, and to prevent the development of long-term complications. Weight reduction, exercise, and medical nutrition therapy (dietary modifications) often correct the hyperglycemia of newly diagnosed type 2 diabetes. Hypoglycemic agents or insulin therapy may be required to achieve satisfactory plasma glucose levels.

As noted previously, available therapies moderate the hyperglycemia of diabetes, but fail to completely normalize metabolism. The long-standing elevation of blood glucose is associated with the chronic complica-
1See
Chapter
24
in
Lippincott’s
Illustrated
Reviews:
Pharmacology for
a
discussion diabetes.
of the
use of
hypoglycemic agents
in the
treatment of

Intertissue relationships in type 2 diabetes.
tions of diabetes—premature atherosclerosis (including cardiovascular disease and stroke), retinopathy, nephropathy, and neuropathy. Intensive treatment with insulin (see p. 340) delays the onset and slows the progression of these long-term complications. For example, the incidence of retinopathy decreases as control of blood glucose improves and HbAic levels decrease (Figure 25.11). The benefits of tight control of blood glucose outweigh the increased risk of severe hypoglycemia in most patients. How hyperglycemia causes the chronic complications of diabetes is unclear. In cells where entry of glucose is not dependent on insulin, elevated blood glucose leads to increased intracellular glucose and its metabolites. For example, increased intracellular sorbitol contributes to the formation of cataracts (see p. 140) in diabetics. Furthermore, hyperglycemia promotes the non-enzymic condensation of glucose with cellular proteins in a reaction analogous to the formation of HbAic (see p. 34). These glycated proteins mediate some of the early microvascular changes of diabetes. There is currently no preventative treatment for type 1 diabetes. The risk for type 2 diabetes can be significantly decreased by a combined regimen of medical nutrition therapy, weight loss, exercise and aggressive control

Effect of body weight and exercise on the development of type 2 diabetes.
of hypertension and dyslipidemias. For example, Figure 25.12 shows the incidence of disease in normal and overweight individuals with varying degrees of exercise. The beneficial effect of intensive therapy on cardiovascular disease has not been shown in individuals with longstanding type 2 diabetes. In contrast, initial intensive control in individuals with newly diagnosed diabetes has long-term benefit in decreasing the risk of myocardial infarction, diabetes-related death, and overall death. The clinical evidence thus supports initiating intensive therapy with the goal of lowering HbAic levels to below 7.0% as early as possible in the course of diabetes.

Diabetes mellitus is a heterogeneous group of syndromes characterized by an elevation of fasting blood glucose that is caused by a relative or absolute deficiency in insulin (Figure 25.13). Diabetes is the leading cause of adult blindness and amputation, and a major cause of renal failure, nerve damage, heart attacks, and stroke. The disease can be classified into two groups, type 1 and type 2. Type 1 diabetics constitute approximately 10% of diabetics in the United States. The disease is characterized by an absolute deficiency of insulin caused by an autoimmune attack on the 8 cells of the pancreas. This destruction requires a stimulus from the environment (such as a viral infection) and a genetic determinant that allows the f cell to be recognized as "nonself"" The metabolic abnormalities of type 1 diabetes mellitus include hyperglycemia, ketoacidosis, and hypertriacylglycerolemia. They result from a deficiency of insulin and a relative excess of glucagon. Type 1 diabetics must rely on exogenous insulin injected subcutaneously to control hyperglycemia and ketoacidosis. Type 2 diabetes has a strong genetic component. It results from a combination of insulin resistance and dysfunctional B cells. Insulin resistance is the decreased ability of target tissues, such as liver, adipose tissue, and muscle, to respond properly to normal (or elevated) circulating concentrations of insulin. Obesity is the most common cause of insulin resistance. However, most people with obesity and insulin resistance do not become diabetic. In the absence of a defect in B-cell function, nondiabetic, obese individuals can compensate for insulin resistance with elevated levels of insulin. Insulin resistance alone will not lead to type 2 diabetes. Rather, type 2 diabetes develops in insulin-resistant individuals who also show impaired B-cell function. The metabolic alterations observed in type 2 diabetes are milder than those described for the insulin-dependent form of the disease, in part, because insulin secretion in type 2 diabetes—although not adequate—does restrain ketogenesis and blunts the development of diabetic ketoacidosis. Available treatments for diabetes moderate the hyperglycemia, but fail to completely normalize metabolism. The long-standing elevation of blood glucose is associated with the chronic complications of diabetes— premature atherosclerosis, retinopathy, nephropathy, and neuropathy.

25.1 Relative or absolute lack of insulin in humans would
result in which one of the following reactions in the liver?
Cc. Decreased glycogenolysis.
25.2 Which one of the following is most often found in
untreated patients
with type
1
and type
2
diabetes?
A. Hyperglycemia.
B. Extremely low levels of insulin synthesis and secretion.
C.
- Synthesis of an insulin with an abnormal amino
acid sequence.
D. A simple pattern of genetic inheritance.
E. Ketoacidosis.

A. usually shows a sudden onset of symptoms.
B. usually has a lower plasma level of insulin than a normal individual.
Cc. usually shows significant improvement in glucose
tolerance if body weight is reduced to normal.
. usually benefits from receiving insulin about 6 hours after a meal.
. usually has lower plasma levels of glucagon than a normal individual.

cell function:
A. usually shows elevated fasting glucose levels.
B. usually shows elevated fasting insulin levels.
Cc. will eventually become diabetic.
D. is rarely obese.
E. is treated by injection of insulin.
25.5 Explain why drugs that inhibit the o-glucosidase activity of the intestinal saccharidases aid in glycemic control in patients with diabetes.
Correct answer = D. Low insulin levels favor the liver producing ketone bodies, using acetyl coenzyme A it generated by B-oxidation of fatty acids provided by the adipose. Low insulin also causes activation of hormone-sensitive lipase, decreased glycogen synthesis, and increased gluconeogenesis and glycogenolysis.
Correct answer = A. Elevated blood glucose occurs in type 1 diabetes as a result of a lack of insulin. In type 2 diabetes, hyperglycemia is due to a defect in B-cell function and insulin resistance. The amino acid sequence of insulin is not changed in diabetes. Both forms of the disease show complex genetics. Ketoacidosis is more common in type 1 disease.
Correct answer = C. Eighty percent of type 2 diabetics are obese, and almost all show some improvement in blood glucose with weight reduction. Symptoms usually develop gradually. These patients have elevated insulin levels, and usually do not require insulin (certainly not 6 hours after a meal). Glucagon levels are typically normal.
Correct answer = B. Insulin resistance is the decreased ability of target tissues, such as liver, adipose, and muscle, to respond properly to normal circulating concentrations of insulin. Obesity is the most common cause of insulin resistance. Most of the people with obesity and insulin resistance do not become diabetic. In the absence of a defect in B-cell function, nondiabetic, obese individuals can compensate for insulin resistance with elevated levels of insulin. The elevated insulin levels normalize fasting blood glucose levels. Insulin resistance without overt diabetes requires no pharmacologic treatment.
a-Glucosidase inhibitors prevent glucose production from those products of carbohydrate digestion in which glucose is attached through an a-glycosidic linkage, thus reducing the postprandial rise in blood glucose. Note that the digestion of lactose is unaffected because it has a -linkage.

Obesity is a disorder of body weight regulatory systems characterized by an accumulation of excess body fat. In primitive societies, in which daily life required a high level of physical activity and food was only available intermittently, a genetic tendency favoring storage of excess calories as fat may have had a survival value. Today, however, the sedentary lifestyle and abundance and wide variety of palatable, inexpensive foods in industrialized societies has undoubtedly contributed to an obesity epidemic. As adiposity has increased so has the risk of developing associated diseases, such as arthritis, diabetes, hypertension, cardiovascular disease, and cancer. Particularly alarming is the explosion of obesity in children and adolescents, which has shown a three-fold increase in prevalence over the last two decades. In the United States, the lifetime risk of becoming overweight or obese is approximately 50% and 25%, respectively. Obesity has increased globally. In fact, by some estimates, there are more obese than undernour- ished individuals worldwide.

The amount of body fat is difficult to measure directly, and is usually determined from an indirect measure—the body mass index (BMI)— which has been shown to correlate with the amount of body fat in most individuals. [Note: Exceptions are athletes who have large amounts of lean muscle mass.] Measuring the waist size with a tape measure is also used to screen for obesity, as this measurement reflects the amount of fat in the central abdominal area of the body. The presence of excess central fat is associated with an increased risk for morbidity and mortality.

The BMI (weight in kg)/(height in meters)? provides a measure of relative weight, adjusted for height. This allows comparisons both within and between populations. The healthy range for the BMI is between 18.5 and 24.9. Individuals with a BMI between 25 and 29.9 are considered overweight, those with a BMI equal to or greater than 30 are defined as obese, and a BMI over 40 is considered extremely obese. Anyone more than 100 pounds overweight is considered severely obese (Figure 26.1). These cutoffs are based on the studies examining the relationship of BMI to premature death, and are similar in men and women. Nearly two thirds of American adults are overweight, and more than one third are obese.

To use the BMI Chart, find height in the left-hand column. Move across the row to weight. Height and weight intersect at the individual's BMI.

A. Individuals with upper body obesity (left) have greater health risks than individuals with lower body obesity (right). B. Visceral fat is located inside the abdominal cavity, packed in between the internal organs; subcutaneaous fat is found underneath the skin.

The anatomic distribution of body fat has a major influence on associated health risks. A waist to hip ratio of more than 0.8 for women and more than 1.0 for men is defined as android, "apple-shaped," or upper body obesity, and is associated with more fat deposition in the trunk (Figure 26.2A). In contrast, a lower waist to hip ratio reflects a preponderance of fat distributed in the hips and thighs and is called gynoid, "pear-shaped," or lower body obesity. It is defined as a waist to hip ratio of less than 0.8 for women and less than 1.0 for men. The pear shape, more commonly found in women, presents a much lower risk of metabolic disease, and some studies indicate it may actually be protective. Thus, the clinician can use simple indices of body shape to identify those who may be at higher risk for metabolic diseases associated with obesity.
1.
Subcutaneous and visceral depots: About 80-90% of the fat stored in the human body is in subcutaneous depots, just under the skin, in the abdominal (upper body) and the gluteal-femoral (lower body) regions. In addition, 10-20% of body fat is stored in so-called visceral depots (omental and mesenteric), which are located within the abdominal cavity in close association with the digestive tract (Figure 26.2B). Excess fat in visceral stores (and also in abdominal subcutaneous fat) increases health risks associated with obesity.

The regional types of fat described above are biochemically different. Subcutaneous adipocytes from the lower body (gluteal-femoral), particularly in women, are larger, very efficient at fat deposition, and tend to mobilize fatty acids more slowly than those from the abdominal subcutaneous depots. Visceral adipocytes are the most metabolically active. Both abdominal subcutaneous and visceral depots of obese subjects have high rates of lipolysis, and contribute to increased availability of free fatty acids. These metabolic differences may contribute to the higher risk found in individuals with upper body obesity.
1. Endocrine function: Adipose tissue, once thought to be a passive bystander in metabolism, is now known to play an active role in body weight regulatory systems. For example, the adipocyte is an endocrine cell that secretes a number of hormones, such as leptin, which regulates appetite as well as metabolism (see p. 353). Adiponectin, an adipocyte-derived cytokine, reduces levels of blood free fatty acids and has been associated with improved lipid profiles, better glycemic control, and reduced inflammation in diabetic patients.
2. Importance of portal circulation: One reason that visceral adipose depots may have such a large influence on metabolic dysfunction in obesity is that cytokines secreted by adipose tissue, as well as free fatty acids released from abdominal fat, enter the portal vein and, therefore, have direct access to the liver. Fatty acids
and inflammatory cytokines released from visceral adipose tissue are taken up by the liver. They may lead to insulin resistance (see p. 342) and increased synthesis of triacylglycerols, which are released as very-low-density lipoprotein (VLDL) particles and contribute to hypertriglyceridemia (see p. 353). By contrast, free fatty acids from subcutaneous body adipose depots enter the general circulation where they can be oxidized in muscle and, therefore, reach the liver in lower concentration.

As triacylglycerols are stored, adipocytes can expand to an average of two to three times their normal volume. (Figure 26.3). However, the ability of a fat cell to expand is limited. With prolonged overnutrition, preadipocytes within adipose tissue are stimulated to proliferate and differentiate into mature fat cells, increasing the number of adipocytes. Thus, most obesity is due to a combination of increased fat cell size (hypertrophy) and number (hyperplasia). Like other tissues, the adipose tissue undergoes continuous remodeling. Contrary to early dogma, we now know that adipocytes can die, but it is uncertain how fast this process occurs; some studies estimate that the average age of an adipocyte is 10 years.
Obese individuals can have up to five times the normal number of fat cells. If excess calories cannot be accommodated within adipose tissue, the excess fatty acids 'spillover' into other tissues, such as muscle and liver. The amount of this so-called 'ectopic fat' is strongly associated with insulin resistance. With weight loss in an obese individual, the size of the fat cells is reduced, but the number of fat cells is not usually affected. Thus, a normal body fat is achieved by decreasing the size of the fat cell below normal. Small fat cells are very efficient at reaccumulating fat, and this may drive appetite and weight regain.

The body weight of most individuals tends to be relatively stable over time. This observation prompted the hypothesis that each individual has a biologically predetermined "set point" for body weight. The body attempts to add to adipose stores when the body weight falls below the set point, and to lose adipose stores when the body weight is higher than the set point. For example, with weight loss, appetite increases and energy expenditure falls, whereas with overfeeding, appetite falls and energy expenditure may slightly increase (Figure 26.4). However, a strict set point model does not explain why some individuals fail to revert to their starting weight after a period of overeating, or the current epidemic of obesity. It appears that factors in the environment (availability of food and exercise) influence a 'settling point' that is defended. Body weight, rather than being irrevocably set, seems to drift around a "settling point," reflecting a balance between environmental factors that influence food intake and energy expenditure, and biologic factors that control body weight.

Hypertrophic and hyperplastic changes are thought to occur in severe obesity.

Weight changes following episodes of overfeeding or underfeeding followed by feeding with no restrictions.

Identical twins with combined weight of 1,300 pounds. Note similarity in body shape.

A. Patient with leptin deficiency before initiation of therapy at age 5 years. B. Patient at age 9 years after 48 months of therapy with subcutaneous injection of recombinant leptin.

It is now evident that genetic mechanisms play a major role in determining body weight. The importance of genetics as a determinant of obesity is indicated by the observation that children who are adopted usually show a body weight that correlates with their biologic rather than adoptive parents. Furthermore, identical twins have very similar BMI (Figure 26.5), whether reared together or apart, and their BMI are more similar than those of nonidentical, dizygotic twins.
1. Mutations: Rare, single gene mutations can cause human obesity. Mutations in the gene for the adipocyte hormone leptin or its receptor produce hyperphagia (increased appetite for and consumption of food) and massive obesity (Figure 26.6), underscoring the importance of the leptin system in regulation of human body weight (section IV). Most obese humans have elevated leptin levels but appear to be resistant to the appetite-regulating effects of this hormone.

The epidemic of obesity occurring over the last decade cannot be simply explained by changes in genetic factors, which are stable on this short time scale. Clearly, environmental factors, such as the ready availability of palatable, energy-dense foods, play a role in the increased prevalence of obesity. Furthermore, sedentary lifestyles encouraged by TV watching, automobiles, computer usage, and energy-sparing devices in the workplace and at home, decrease physical activity and enhance the tendency to gain weight. Eating behaviors, such as snacking, portion size, variety of foods consumed, an individual's unique food preferences, and the number of people with whom one eats also influence food consumption. It is important to note, however, that in this same environment, many individuals do not become obese. The susceptibility to obesity appears to be explained, at least in part, by an interaction of an individual's genes and his or her environment, and can be influenced by additional factors such as maternal underor overnutrition that may 'set' the body regulatory systems to defend a higher or lower level of body fat.

The cause of obesity can be summarized in a deceptively simple statement of the first law of thermodynamics: Obesity results when energy intake exceeds energy expenditure. However, the mechanism underlying this imbalance involves a complex interaction of biochemical, neurologic, environmental, and psychologic factors. The basic neural and humoral pathways that regulate appetite, energy expenditure, and body weight involve systems that regulate short-term food intake (meal to meal), and signals for the long-term (day to day, week to week, year to year) regulation of body weight (Figure 26.7).

1. Leptin: Leptin is an adipocyte hormone that is secreted in proportion to the size of fat stores. When we consume fewer calories than we need, body fat declines and leptin production from the fat cell decreases. The body adapts by minimizing energy utilization (decreasing activity) and increasing appetite, closing the feedback loop that regulates body weight. Unfortunately, in many individuals, the leptin system may be better at preventing weight loss than preventing weight gain. Although a meal or overeating increases leptin and this should, in theory, also dampen appetite and prevent overconsumption of calories, other cues that stimulate appetite can apparently overcome the leptin system in many individuals.
2. Insulin: Obese individuals are also hyperinsulinemic. Like leptin, insulin acts on hypothalamic neurons to dampen appetite.

Short-term signals from the gastrointestinal tract control hunger and satiety, which affect the size and number of meals over a time course of minutes to hours. In the absence of food intake (between meals), the stomach produces ghrelin, an orexigenic (appetite-stimulating) hormone that drives hunger. During a meal, as food is consumed, gut hormones, including cholecystokinin (CCK) and peptide YY (PYY), among others, through actions on the gastric empyting and neural signals to the hypothalamus, cause satiety and meals are terminated. Within the hypothalamus, neuropeptides such as NPY and a-melanocyte stimulating hormone (a-MSH), and neurotransmitters such as serotonin and dopamine are important in regulating hunger and satiety. Long-term and short-term signals interact, as leptin can affect the sensitivity of hypothalamic neurons to shortterm signals such as CCK. Thus, there are many and complex regulatory loops that control the size and number of meals in relationship to the status of body fat stores.

The primary metabolic effects of obesity include dyslipidemias, glucose intolerance (hyperglycemia below that classified as diabetes, see p. 338), and insulin resistance, expressed primarily in the liver, muscle, and adipose tissue. These metabolic abnormalities reflect molecular signals originating from the increased mass of adipocytes.

Abdominal obesity is associated with a cluster of metabolic abnormalities that is referred to as the metabolic syndrome and includes glucose intolerance, insulin resistance, hyperinsulinemia, dyslipidemia (low high-density lipoprotein (HDL) and elevated triacylglycerols), and hypertension (Figure 26.8). The metabolic syndrome is also associated with a state of chronic systemic inflammation that contributes to the pathogenesis of insulin resistance and atherosclerosis. In obesity, low levels of the adipocyte hormone adiponectin

Some signals that influence appetite and satiety. CCK = cholecystokinin, PYY = peptide YY.

Body mass index and the relative risk of death.
that normally dampens inflammation and sensitizes tissues, especially the liver, to insulin, may contribute to the metabolic syndrome and therefore the risk of type 2 diabetes and heart disease.

Obesity is correlated with an increased risk of death (Figure 26.9), and is a risk factor for a number of chronic conditions, including type 2 diabetes, dyslipidemias, hypertension, heart disease, some cancers, gallstones, arthritis, gout, and sleep apnea. The relationship between obesity and associated morbidities is stronger among individuals younger than 55 years. After age 74, there is no longer an association between increased BMI and mortality. Weight loss in obese individuals leads to decreased blood pressure, serum triacylglycerols, and blood glucose levels. HDL levels increase.

Weight reduction can help reduce the complications of obesity, including diabetes and hypertension. To achieve weight reduction, the obese patient must decrease energy intake or increase energy expenditure, though decreasing energy intake is thought to contribute more to inducing weight loss. Typically, a prescription for weight reduction combines dietary change, increased physical activity, and behavioral modification, which can include nutritional education and meal planning, recording and monitoring food intake through food diaries, modifying factors that lead to overeating, and relearning cues to satiety. Once weight loss is achieved, weight maintenance is a separate process that requires vigilance as the majority of patients regain weight after they stop their weight loss efforts.

An increase in physical activity can create an energy deficit. Although adding exercise to a hypocaloric regimen may not produce a greater weight loss initially, exercise is a key component of programs directed at maintaining a weight loss. In addition, physical activity increases cardiopulmonary fitness and reduces the risk of cardiovascular disease, independent of weight loss. Persons who combine caloric restriction and exercise with behavioral treatment may expect to lose about 5—10% of initial body weight over a period of 4-6 months. Studies show that individuals who maintain their exercise program regain less weight after their initial weight loss.

Dieting is the most commonly practiced approach to weight control. Because 1 pound of adipose tissue corresponds to approximately 3,500 kcal, one can estimate the effect of caloric restriction on the reduction in adipose tissue. Weight loss on calorie-restricted diets is determined primarily by energy intake and not nutrient composition. Caloric restriction is ineffective over the long term for many individuals. More than 90% of people who attempt to lose weight regain the lost weight when dietary intervention is suspended. Nonetheless, it is important to recognize that, although few individuals will reach their ideal weight with treatment, weight losses of
10% of body weight over a 6-month period often reduce blood pressure and lipid levels, and enhance control of type 2 diabetes. The health benefits of relatively small be emphasized to the patient. weight losses should, therefore,

Several weight-loss medications are currently approved by the U.S. Food and Drug Administration for higher'. use in adults with a BMI of 30 or Their effects on weight reduction tend to be modest, and weight regain upon termination of drug therapy is common.

Gastric bypass and restriction surgeries are effective in causing weight loss in severely obese individuals. Through mechanisms that remain poorly understood, these operations improve poor blood sugar control in diabetic individuals.
QO:
Chapter 29 in Lippincott's Illustrated Reviews: Pharmacology for a more detailed discussion of drugs used to treat obesity.
|

Obesity—the accumulation of excess body fat—results when energy intake exceeds energy expenditure. Obesity is increasing in industrialized countries because of a reduction in daily energy expenditure, and an increase in energy intake resulting from the increasing availability of palatable, inexpensive foods. The body mass index (BMI) is easy to determine and highly correlated to body fat. Nearly two thirds of American adults are overweight (BMI = 25 kg/m?) and more than 30% are obese (BMI > 30 kg/m'). The anatomic distribution of body fat has a major influence on associated health risks. Excess fat located in the central abdominal area is associated with greater risk for hypertension, insulin resistance, diabetes, dyslipidemia, and coronary heart disease. A person's weight is determined by genetic and environmental factors. Appetite is influenced by afferent, or incoming, signals—neural signals, circulating hormones, and metabolites—that are integrated by the hypothalamus. These diverse signals prompt release of hypothalamic peptides and activate outgoing, efferent neural signals. Obesity is correlated with an increased risk of death, and is a risk factor for a number of chronic conditions. Weight reduction is achieved best with negative energy balance, that is, by decreasing caloric intake. Virtually all diets that limit particular groups of foods or macronutrients lead to short-term weight loss. Long-term maintenance of weight loss is difficult to achieve. Modest reduction in food intake occurs with pharmacologic treatment. Surgical procedures designed to limit food intake are an option for the severely obese patient who has not responded to other treatments.

26.1 A 40-year-old woman, 5 feet, 1 inch (155 cm) tall and weighing 188 pounds (85.5 kg), seeks your advice on how to lose weight. Her waist measured 41 inches and her hips 39 inches. A physical examination and blood laboratory data were all within the normal range. Her only child, who is 14 years old, her sister, and both of her parents are overweight. The patient recalls being obese throughout her childhood and adolescence. Over the past 15 years she had been on seven different diets for periods of 2 weeks to 3 months, losing from 5-25 pounds. On discontinuation of each diet, she regained weight, returning to 185-190 pounds. Which one of the following best describes this patient?
A. She is classified as overweight.
B. She shows an "apple" pattern of fat distribution.
C. She has approximately the same number of fat cells as a normal-weight individual, but each adipocyte is larger.
D. She would be expected to show lower than normal levels of circulating leptin.
E. She would be expected to show lower than normal levels of circulating triacylglycerols.
26.2 Calculate the BMI for the patient presented in question 26.1
Correct answer = B. Her waist to hip ratio is 41/39 = 1.05. Apple shape is defined as a waist to hip ratio of more than 0.8 for women, and more than 1.0 for men. She has, therefore, an apple pattern of fat distribution, more commonly seen in males. Compared with other women of the same body weight who have a gynoid fat pattern, the presence of increased visceral or intra-abdominal adipose tissue places her at greater risk for diabetes, hypertension, dyslipidemia, and coronary heart disease. The calculated BMI indicates that the patient is classified as obese. Individuals with marked obesity and a history dating to early childhood have an adipose depot made up of too many adipocytes, each fully loaded with triacylglycerols. Plasma leptin in obese humans is usually normal for their fat mass, suggesting that resistance to leptin, rather than its deficiency, occurs in human obesity. The elevated circulating fatty acids characteristic of obesity are carried to the liver and converted to triacylglycerol and cholesterol. Excess triacylglycerol and cholesterol are released as VLDL, resulting in elevated serum triacylglycerols.

Nutrients are the constituents of food necessary to sustain the normal functions of the body. All energy is provided by three classes of nutrients: fats, carbohydrates, protein—and in some diets, ethanol (Figure 27.1). The intake of these energy-rich molecules is larger than that of the other dietary nutrients. Therefore, they are called macronutrients. This chapter focuses on the kinds and amounts of macronutrients that are needed to maintain optimal health and prevent chronic disease in adults. Those nutrients needed in lesser amounts, vitamins and minerals, are called micronutrients, and are considered in Chapter 28.

Committees of U.S. and Canadian experts organized by the Food and Nutrition Board of the National Academy of Sciences have compiled Dietary Reference Intakes (DRI)—estimates of the amounts of nutrients required to prevent deficiencies and maintain optimal health and growth. The DRis replace and expand on the Recommended Dietary Allowances (RDA), which have been published with periodic revisions since 1941. Unlike the RDA, the DRI establish upper limits on the consumption of some nutrients, and incorporate the role of nutrients in lifelong health, going beyond deficiency diseases. Both the DRI and the RDA refer to long-term average daily nutrient intakes, because it is not necessary to consume the full RDA every day.

The DRI consist of four dietary reference standards for the intake of nutrients designated for specific age groups, physiologic states, and gender (Figure 27.2).
1. Estimated Average Requirement (EAR): The EAR is the average daily nutrient intake level estimated to meet the requirement of one half of the healthy individuals in a particular life stage and gender group. It is useful in estimating the actual requirements in groups and individuals.
2. Recommended Dietary Allowance (RDA): The RDA is the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97-98%) the individuals in a life stage and gender group. The RDA is not the minimal requirement for healthy individuals; rather, it is intentionally set to provide a margin of safety for most individuals. The EAR serves as the foundation for setting the RDA. If the standard deviation (SD) of the EAR

Essential nutrients obtained from the diet. [Note: Ethanol is not considered an essential component of the diet but may provide a significant contribution to the daily caloric intake of some individuals.]

Dietary Reference Intakes for vitamins and minerals in individuals one year and older. EAR = Estimated Average Requirement; RDA = Recommended Dietary Allowance; Al = Adequate Intake; UL = Tolerable Upper Intake Level; — = no value established.
is available and the requirement for the nutrient is normally distributed, the RDA is set at 2 SDs above the EAR, that is, RDA = EAR + 2SDear.
3. Adequate Intake (Al): The Al is set instead of an RDA if sufficient scientific evidence is not available to calculate an EAR or RDA. The Al is based on estimates of nutrient intake by a group (or groups) of apparently healthy people that are assumed to be adequate. For example, the Al for young infants, for whom human milk is the recommended sole source of food for the first 4-6 months, is based on the estimated daily mean nutrient intake supplied by human milk for healthy, full-term infants who are exclusively breast-fed.
4.
Tolerable Upper Intake Level (UL): The UL is the highest average daily nutrient intake level that is likely to pose no risk of adverse health effects to almost all individuals in the general population. As intake increases above the UL, the potential risk of adverse effects may increase. The UL is not intended to be a recommended level of intake. The UL are useful because of the increased availability of fortified foods and the increased use of dietary supplements. The UL applies to chronic daily use. For some nutrients, there may be insufficient data on which to develop a UL.

Most nutrients have a set of DRI (Figure 27.3). Usually a nutrient has an EAR and a corresponding RDA. Most are set by age and gender, and may be influenced by special factors, such as pregnancy and lactation in women. When the data are not sufficient to estimate an EAR (or an RDA), then an Al is designated. The Al is judged by experts to meet the needs of all healthy individuals in a group, but is based on less data than an EAR and RDA. Intakes below the EAR need to be improved because the probability of adequacy is 50% or less (Figure 27.4). Intakes between the EAR and RDA likely need to be improved because the probability of adequacy is less than 98%, and intakes at or above the RDA can be considered adequate. Intake above the Al can be considered adequate. Intakes between the UL and the RDA can be considered to have no risk for adverse effects.

The Estimated Energy Requirement is the average dietary energy intake predicted to maintain an energy balance (that is, when the calories consumed are equal to the energy expended) in a healthy adult of a defined age, gender, and height whose weight and level of physical activity are consistent with good health. Differences in the genetics, body composition, metabolism, and behavior of individuals make it difficult to accurately predict a person's caloric requirements. However, some simple approximations can provide useful estimates. For example, sedentary adults require about 30 kcal/kg/day to maintain body weight; moderately active adults require 35 kcal/kg/day; and very active adults require 40 kcal/kg/day. [Note: The daily average requirement for energy that is listed on food labels is either 2,000 or 2,500 kcal/day.]

Comparison of the components of the Dietary Reference Intakes. EAR = Estimated Average Requirement; RDA = Recommended Dietary Allowance; Al = Adequate Intake; UL = Tolerable Upper Intake Level.

The energy content of food is calculated from the heat released by the total combustion of food in a calorimeter. It is expressed in kilocalories (kcal, or Cal). The standard conversion factors for determining the metabolic caloric value of fat, protein, and carbohydrate are shown in Figure 27.5. Note that the energy content of fat is more than twice that of carbohydrate or protein, whereas the energy content of ethanol is intermediate between those of fat and carbohydrate. [Note: The joule is a unit of energy widely used in countries other than the United States. For uniformity, many scientists are promoting the use of joules (J), rather than calories. One cal = 4.2 J; 1 Cal (1 kcal, 1 food calorie) = 4.2 kJ. However, kcal still predominates and is used throughout this text.]

The energy generated by metabolism of the macronutrients is used for three energy-requiring processes that occur in the body: resting metabolic rate, thermic effect of food (formerly termed specific dynamic action), and physical activity.
1. Resting metabolic rate: The energy expended by an individual in a resting, postabsorptive state is called the resting (formerly, basal) metabolic rate (RMR). It represents the energy required to carry out the normal body functions, such as respiration, blood flow, ion transport, and maintenance of cellular integrity. In an adult, the RMR is about 1,800 kcal for men (70 kg) and 1,300 kcal for women (50 kg). From 50-70% of the daily energy expenditure in sedentary individuals is attributable to the RMR (Figure 27.6).
2. Thermic effect of food: The production of heat by the body increases as much as 30% above the resting level during the digestion and absorption of food. This effect is called the thermic effect of food or diet-induced thermogenesis. Over a 24-hour period, the thermic response to food intake may amount to 5-10% of the total energy expenditure.

Estimated total energy expenditure 65 One feet 4 inches) tall weighing 50 kg (110 Ib), and engaged in light activity.

Acceptable macronutrient distribution ranges in adults. *A growing body of evidence suggests that higher levels of @-3 polyunsaturated fatty acids provide protection against heart disease.
coronary
3. Physical activity: Muscular activity provides the greatest variation in energy expenditure. The amount of energy consumed depends on the duration and intensity of the exercise. The daily expenditure of energy can be estimated by carefully recording the type and duration of all activities. In general, a sedentary person requires about 30-50% more than the resting caloric requirement for energy balance (see Figure 27.6), whereas a highly active individual may require 100% or more calories above the RMR.

Acceptable Macronutrient Distribution Ranges (AMDR) are defined as a range of intakes for a particular macronutrient that is associated with reduced risk of chronic disease while providing adequate amounts of essential nutrients. The AMDR for adults is 45-65% of their total calories from carbohydrates, 20-35% from fat, and 10-35% from protein (Figure 27.7). Note that there is a range of acceptable intakes for the macronutrients. The biologic properties of dietary fat, carbohydrate, and protein are described below.

The incidence of a number of chronic diseases is significantly influenced by the kinds and amounts of nutrients consumed (Figure 27.8). Dietary fats most strongly influence the incidence of coronary heart disease (CHD); evidence linking dietary fat and the risk for cancer or obesity is much weaker.
In the past, dietary recommendations emphasized decreasing the total amount of fat and cholesterol in the diet. Research now indicates that the type of fat is more important than the total amount of fat consumed.

Plasma cholesterol may arise from the diet or from endogenous biosynthesis. In either case, cholesterol is transported between the tissues in combination with protein and phospholipids as lipoproteins.
1. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL): The level of plasma cholesterol is not precisely regulated, but rather varies in response to the diet. Elevated levels result in an increased risk for CHD (Figure 27.9). The risk increases progressively with higher values for serum total cholesterol. A much stronger correlation exists between the levels of blood LDL cholesterol and heart disease (see p. 232) In contrast, high levels of HDL cholesterol have been associated with a decreased risk for heart disease (see p. 235). Abnormal levels of plasma lipids (dyslipidemias) act in combination with smoking, obesity, seden-
|
tary lifestyle, insulin resistance, and other risk factors to increase the risk of CHD. Elevated plasma triacylglycerols are also a risk factor for CHD, but the association is weaker than that of LDL cholesterol with CHD.
2. Beneficial effect of lowering plasma cholesterol: Clinical trials have demonstrated that dietary or drug treatment of hypercholesterolemia is effective in decreasing LDL, increasing HDL, and reducing the risk for cardiovascular events. The diet-induced changes of plasma lipoprotein concentrations are modest, typically 10-20%, whereas treatment with "statin" drugs' decreases plasma cholesterol by 30-60% (see p. 224).

Triacylglycerols are quantitatively the most important class of dietary fats. The influence of triacylglycerols on blood lipids is determined by the chemical nature of their constituent fatty acids. The absence or presence and number of double bonds (saturated vs. monoand polyunsaturated), the location of the double bonds (w-6 vs w-3), and the cis vs. trans configuration of the unsaturated fatty acids are the most important structural features that influence blood lipids.
1. Saturated fat: Triacylglycerols composed primarily of fatty acids whose hydrocarbon chains do not contain any double bonds are referred to as saturated fats. Consumption of saturated fats is positively associated with high levels of total plasma cholesterol and LDL cholesterol, and an increased risk of CHD. The main sources of saturated fatty acids are dairy and meat products and some vegetable oils, such as coconut and palm oils (a major source of fat in Latin America and Asia, although not in the United States, Figure 27.10). Most experts strongly advise limiting intake of saturated fats.
Saturated fatty acids with carbon chain lengths of 14 (myristic) and 16 (palmitic) are most potent in increasing the serum cholesterol. Stearic acid (18 carbons—found in many foods including chocolate) has little effect on blood cholesterol.
2. Monounsaturated fats: Triacylglycerols containing primarily fatty acids with one double bond are referred to as monounsaturated fat. Unsaturated fatty acids are generally derived from vegetables and fish. When substituted for saturated fatty acids in the diet, monounsaturated fats lower both total plasma cholesterol and LDL cholesterol, but maintain or increase HDL cholesterol. This ability of monounsaturated fats to favorably modify lipoprotein levels may explain, in part, the observation that Mediterranean cultures, with diets rich in olive oil (high in monounsaturated oleic acid), show a low incidence of CHD.
‘See
Chapter
21
in
Reviews:
a
more detailed
discussion of
antihyperlipidemic drugs.

Influence of nutrition on some common causes of death in the United States in the year 2000. Red indicates causes of death in which the diet plays a significant role. Blue indicates causes of death in which excessive alcohol consumption plays a part. (*Diet plays a role in only some forms of cancer.)

Correlation of the death rate from coronary heart disease with the concentration of plasma cholesterol. [Note: The data were obtained from a 6 year study of men with the death rate adjusted for age.]

Compositions of commonly encountered dietary lipids.
a.
The Mediterranean diet: The Mediterranean diet is an example of a diet rich in monounsaturated fatty acids or MUFAs (from olive oil) and w-3 fatty acids (from fish oils and some nuts), but low in saturated fat. For example, Figure 27.11 shows the composition of the Mediterranean diet in comparison with both a Western diet similar to that consumed in the United States and a typical low-fat diet. The Mediterranean diet contains seasonally fresh food, with an abundance of plant material, low amounts of red meat, and olive oil as the principal source of fat. The Mediterranean diet is associated with decreased serum total cholesterol and LDL cholesterol—but little change in HDL cholesterol—when compared with a typical Western diet higher in saturated fats. Plasma triacylglycerols are unchanged.

Composition of typical Mediterranean, Western, and low-fat diets.
- 3. Polyunsaturated fats: Triacylglycerols containing primarily fatty acids with more than one double bond are referred to as polyunsaturated fats. The effects of polyunsaturated fatty acids (PUFAs) on cardiovascular disease is influenced by the location of the double bonds within the molecule.
- a. @-6 Fatty acids: These are long-chain, PUFAs, with the first double bond beginning at the sixth bond position when starting from the methyl end of the fatty acid molecule.
[Note: They are also called n-6 fatty acids (see p. 183).] Consumption of fats containing m-6 PUFAs, principally linoleic acid, 18:2(9,12), obtained from vegetable oils, lowers plasma cholesterol when substituted for saturated fats. Plasma LDL are lowered, but HDL, which protect against CHD, are also lowered. The powerful benefits of lowering LDL are only partially offset because of the decreased HDL. Nuts, avocados, olives, soybeans, and various oils, including sesame, cottonseed, and corn oil, are common sources of these fatty acids (see Figure 27.10). Linoleic acid, along with a-linolenic acid, 18:3(9,12,15), an w-3 fatty acid (see below), are essential fatty acids required for fluidity of membrane structure and synthesis of eicosanoids (see p. 213). [Note: A deficiency of essential fatty acids is characterized by scaly dermatitis, hair loss, and poor wound healing.] A lower boundary level of 5% of calories meets the Al set for linoleic acid. An upper boundary for linoleic acid is set at 10% of total calories because of concern that oxidation of these polyunsaturated fatty acids may lead to deleterious products.

b. @-3 Fatty acids: These are long-chain, PUFAs, with the first double bond beginning at the third bond position from the methyl end. Dietary w-3 polyunsaturated fats suppress cardiac arrhythmias, reduce serum triacylglycerols, decrease the tendency for thrombosis, lower blood pressure, and substantially reduce risk of cardiovascular mortality (Figure 27.12), but they have little effect on LDL or HDL cholesterol levels. The w-3 polyunsatyrated fats, principally a-linolenic acid, 18:3(9,12,15), are found in plants. The acceptable range for o-linolenic acid is 0.6-1.2% of total calories. Fish oil contains the long-chain, w-3 docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Two fatty fish (for example, salmon) meals per week are recommended. [Note: The w-3 long-chain PUFAs are included in infant formulas to promote brain development.]
4. Trans fatty acids: Trans fatty acids (Figure 27.13) are chemically classified as unsaturated fatty acids, but behave more like saturated fatty acids in the body, that is, they elevate serum LDL (but not HDL), and they increase the risk of CHD. Trans fatty acids do not occur naturally in plants, but occur in small amounts in ani-

Response of plasma LDL concentrations to an increase in dietary cholesterol intake.
mals. However, trans fatty acids are formed during the hydrogenation of liquid vegetable oils, for example, in the manufacture of margarine and partially hydrogenated vegetable oil. Trans fatty acids are a major component of many commercial baked goods, such as cookies and cakes, and most deep-fried foods. Many manufacturers have reformulated their products to be free of trans fats. Starting in 2006, the U.S. Food and Drug Administration requires that Nutrition Facts labels portray trans fat content. Some municipalities, for example, New York City, have banned the use of trans fats in restaurants.
5. Dietary cholesterol: Cholesterol is found only in animal products. The effect of dietary cholesterol on plasma cholesterol (Figure 27.14) is less important than the amount and types of fatty acids consumed.

Moderate consumption of alcohol (for example, two drinks a day) decreases the risk of CHD, because there is a positive correlation between moderate alcohol consumption and the plasma concentration of HDL. However, because of the potential dangers of alcohol abuse, health professionals are reluctant to recommend increased alcohol consumption to their patients. Red wine may provide cardioprotective benefits in addition to those resulting from its alcohol content, for example, red wine contains phenolic compounds that inhibit lipoprotein oxidation (see p. 235). [Note: These antioxidants are also present in raisins and grape juice.] Figure 27.15 summarizes the effects of dietary fats.
Trans fatty acid, METABOLIC EFFECTS = LDL HDL. Trans fatty acid, EFFECTS ON DISEASE PREVENTION = Incidence of coronary heart disease. Saturated fatty acid, METABOLIC EFFECTS = LDL Little effect on HDL. Saturated fatty acid, EFFECTS ON DISEASE PREVENTION = Incidence of coronary heart disease; may increase risk of prostate, colon cancer. Monounsaturated fatty acid, METABOLIC EFFECTS = LDL HDL. Monounsaturated fatty acid, EFFECTS ON DISEASE PREVENTION = Incidence of coronary heart disease. Polyunsaturated fatty acids ω -6, METABOLIC EFFECTS = Provide arachidonic acid which is an important precursor of prostaglandins and leukotrienes LDL HDL. Polyunsaturated fatty acids ω -6, EFFECTS ON DISEASE PREVENTION = Incidence of coronary heart disease. Polyunsaturated fatty acids ω -3, METABOLIC EFFECTS = Suppress cardiac arrhythmias, reduce serum triacylglycerols, decrease the tendency for thrombosis, lower blood pressure Little effect on LDL Little effect on HDL. Polyunsaturated fatty acids ω -3, EFFECTS ON DISEASE PREVENTION = Incidence of coronary heart disease Risk of sudden cardiac death

The primary role of dietary carbohydrate is to provide energy. Although caloric intake in the United States has shown a modest increase since 1971, the incidence of obesity has dramatically increased (see p. 349). During this same period, carbohydrate consumption has significantly increased, leading some observers to link obesity with carbohydrate consumption. However, obesity has also been related to increasingly inactive lifestyles, and to calorie-dense foods served in expanded portion size. Carbohydrates are not inherently fattening.

Carbohydrates in the diet are classified as either monosaccharides and disaccharides (simple sugars), polysaccharides (complex sugars), or fiber.

1. Monosaccharides: Glucose and fructose are the principal monosaccharides found in food. Glucose is abundant in fruits, sweet corn, corn syrup, and honey. Free fructose is found together with free glucose and sucrose in honey and fruits.
a. High fructose corn syrup: High-fructose corn syrups (HFCS) are corn syrups that have undergone enzymatic processing to convert their glucose into fructose and have then been mixed with pure corn syrup (100% glucose) to produce a desired sweetness. In the United States, HFCS 55 (containing 55% fructose and 42% glucose) is commonly used as a substitute for sucrose in beverages, including soft drinks, with HFCS 42 used in processed foods. The composition and metabolism of HFCS and sucrose are similar, the major difference being that HFCS is ingested as a mixture of monosaccharides (Figure 27.16). Most studies have shown no significant difference between sucrose and HFCS syrup meals in either postprandial glucose or insulin responses.
2. Disaccharides: The most abundant disaccharides are sucrose (glucose + fructose), lactose (glucose + galactose), and maltose (glucose + glucose). Sucrose is ordinary "table sugar," and is abundant in molasses and maple syrup. Lactose is the principal sugar found in milk. Maltose is a product of enzymic digestion of polysaccharides. It is also found in significant quantities in beer and malt liquors. The term "sugar" refers to monosaccharides and disaccharides. "Added sugars" are those sugars and syrups added to foods during processing or preparation.
3. Polysaccharides: Complex carbohydrates are polysaccharides (most often polymers of glucose), which do not have a sweet taste. Starch is an example of a complex carbohydrate that is found in abundance in plants. Common sources include wheat and other grains, potatoes, dried peas and beans, and vegetables.
4. Fiber: Dietary fiber is defined as the nondigestible carbohydrates and lignin (a complex polymer of phenylpropanoid subunits) present intact in plants. Several different terms are used to

Digestion of HFCS 55 or sucrose leads to absorption of glucose plus fructose.

Blood glucose concentrations following ingestion of food with
low or high glycemic index.
describe this complex group of compounds. For example, functional fiber is the isolated, extracted, or synthetic fiber that has proven health benefits. Total fiber is the sum of dietary fiber and functional fiber. Soluble fiber is the edible parts of plants that is resistant to digestion and absorption in the human small intestine, but is completely or partially fermented to short-chain fatty acids in the large intestine. Insoluble fiber passes through the digestive track largely intact. Dietary fiber provides little energy but has several beneficial effects. First, it adds bulk to the diet (Figure 27.17). Fiber can absorb 10-15 times its own weight in water, drawing fluid into the lumen of the intestine and increasing bowel motility. Soluble fiber delays gastric emptying and can result in a sensation of fullness. This delayed emptying also results in reduced peaks of blood glucose following a meal. Second, consumption of soluble fiber has now been shown to lower LDL cholesterol levels by increasing fecal bile acid excretion and interfering with bile acid absorption. For example, diets rich (25-50 g/day) in the soluble fiber oat bran are associated with a modest, but significant, reduction in risk for cardiovascular disease by lowering total and LDL cholesterol levels. Also, fiberrich diets decrease the risk for constipation, hemorrhoids, and diverticulosis. The recommended daily fiber intake (Al) is 25 g/day for women and 38 g/day for men. However, most American diets are far lower in fiber—approximately 15 g/day.

Some carbohydrate-containing foods produce a rapid rise followed by a steep fall in blood glucose concentration, whereas others result in a gradual rise followed by a slow decline—they differ in their glycemic response. The glycemic index (GI) quantitates these differences in the time course of postprandial glucose concentrations (Figure 27.18). Glycemic index is defined as the area under the blood glucose curves seen after ingestion of a meal with carbohydrate-rich food, compared with the area under the blood glucose curve observed after a meal consisting of the same amount (50g) of carbohydrate either as glucose or white bread. The clinical importance of glycemic index is controversial. Food with a low glycemic index tends to create a sense of satiety over a longer period of time, and may be helpful in limiting caloric intake. [Note: How much a typical serving size of a food raises blood glucose is referred to as the glycemic load (GL). A food, for example, carrots, can have a high Gl and a low GL.]
Many experts assert that high nutrient and fiber content, such as occurs in whole grains, fruits, and vegetables, is a better guide than glycemic index for selecting dietary carbohydrates.

Carbohydrates are not essential nutrients, because the carbon skeletons of most amino acids can be converted into glucose (see p. 261). However, the absence of dietary carbohydrate leads to ketone body production (see p. 262), and degradation of body protein whose constituent amino acids provide carbon skeletons for gluconeogenesis (see p. 118). The RDA for carbohydrate is set at 130 g/day for adults and children, based on the amount of glucose used by carbohydrate-dependent tissues, such as the brain and erythrocytes. However, this level of intake is usually exceeded to meet energy needs. Adults should consume 45-65% of their total calories from carbohydrates. It is recommended that added sugar represent no more than 25% of total energy because of concerns that sugar may displace nutrient-rich foods from the diet, potentially leading to deficiencies of certain micronutrients.

There is no direct evidence that the consumption of simple sugars is harmful. Contrary to folklore, diets high in sucrose do not lead to diabetes or hypoglycemia. Also contrary to popular belief, carbohydrates are not inherently fattening. They yield 4 kcal/g (the same as protein and less than one half that of fat, see Figure 27.5), and result in fat synthesis only when consumed in excess of the body's energy needs. However, there is an association between sucrose consumption and dental caries, particularly in the absence of fluoride treatment.

Humans have no dietary requirement for protein, per se, but the protein in food does provide essential amino acids (see Figure 20.2, p. 262). Nine of the 20 amino acids needed for the synthesis of body proteins are essential—that is, they cannot be synthesized in humans.

The quality of a dietary protein is a measure of its ability to provide the essential amino acids required for tissue maintenance. Most government agencies have adopted the Protein DigestibilityCorrected Amino Acid Score (PDCAAS) as the standard by which to evaluate protein quality. PDCAAS is based on the profile of essential amino acids and the digestibility of the protein. The highest possible score under these guidelines is 1.00. This amino acid score provides a method to balance intakes of poorer-quality proteins with high-quality dietary proteins.

sources: sources (meat, poultry, milk, and fish) have a high quality because they contain all the essential amino acids in proportions similar to those required for synthesis of human tissue proteins (Figure 27.19). [Note: Gelatin prepared from animal collagen is an exception; it has a low biologic value as a result of deficiencies in several essential amino acids.]

Combining two incomplete proteins that have complementary amino acid deficiencies results in a mixture with a higher biologic value.
2. Proteins from plant sources: Proteins from wheat, corn, rice, and beans have a lower quality than do animal proteins. However, proteins from different plant sources may be combined in such a way that the result is equivalent in nutritional value to animal protein. For example, wheat (lysine-deficient but methionine-rich) may be combined with kidney beans (methionine-poor but lysine-rich) to produce an improved biologic value. Thus, eating foods with different amino acids during the day can result in a dietary combination with a higher biologic value than either of the component proteins (Figure 27.20). [Note: Animal proteins can also complement the biologic value of plant proteins.]

Nitrogen balance occurs when the amount of nitrogen consumed equals that of the nitrogen excreted in the urine, sweat, and feces. Most healthy adults are normally in nitrogen balance.
1. Positive nitrogen balance: This occurs when nitrogen intake exceeds nitrogen excretion. It is observed during situations in which tissue growth occurs, for example, in childhood, pregnancy, or during recovery from an emaciating illness.
2. Negative nitrogen balance: This occurs when nitrogen loss is greater than nitrogen intake. It is associated with inadequate dietary protein, lack of an essential amino acid, or during physiologic stresses, such as trauma, burns, illness, or surgery.

The amount of dietary protein required in the diet varies with its biologic value. The greater the proportion of animal protein included in the diet, the less protein is required. The RDA for protein is computed for proteins of mixed biologic value at 0.8 g/kg of body weight for adults, or about 56 g of protein for a 70-kg individual. People who exercise strenuously on a regular basis may benefit from extra protein to maintain muscle mass; a daily intake of about 1 g/kg has been recommended for athletes. Women who are pregnant or lactating require up to 30 g/day in addition to their basal requirements. To support growth, infants should consume 2 g/kg/day.
Weight for age (% expected), KWASHIORKOR = 60-80. Weight for age (% expected), MARASMUS = <60. Weight for height, KWASHIORKOR = Normal or decreased. Weight for height, MARASMUS = Markedly decreased. Edema, KWASHIORKOR = Present. Edema, MARASMUS = Absent. Mood, KWASHIORKOR = Irritable when picked up; apathetic when left alone. Mood, MARASMUS = Alert, irritable. Appetite, KWASHIORKOR = Poor. Appetite, MARASMUS = Good

Features of protein-energy malnutrition in children.
1. Consumption of excess protein: There is no physiologic advantage to the consumption of more protein than the RDA. Protein consumed in excess of the body's needs is deaminated, and the resulting carbon skeletons are metabolized to provide energy or acetyl coenzyme A for fatty acid synthesis. When excess protein is eliminated from the body as urinary nitrogen, it is often accompanied by increased urinary calcium, increasing the risk of nephrolithiasis and osteoporosis.
2. The protein-sparing effect of carbohydrate: The dietary protein requirement is influenced by the carbohydrate content of the diet. When the intake of carbohydrates is low, amino acids are deaminated to provide carbon skeletons for the synthesis of glucose that is needed as a fuel by the central nervous system. If carbohydrate intake is less than 130 g/day, substantial amounts of protein are metabolized to provide precursors for gluconeogenesis. Therefore, carbohydrate is considered to be "protein-sparing," because it allows amino acids to be used for repair and maintenance of tissue protein rather than for gluconeogenesis.

In developed countries, PEM is most frequently seen in patients with medical conditions that decrease appetite or alter how nutrients are digested or absorbed, or in hospitalized patients with major trauma or infections. [Note: Such highly catabolic patients frequently require intravenous (parenteral) or tube-based (enteral) administration of nutrients.] PEM may also be seen in children or the elderly who are malnourished. In developing countries, an inadequate intake of protein and/or energy is the primary cause of PEM. Affected individuals show a variety of symptoms, including a depressed immune system with a reduced ability to resist infection. Death from secondary infection is common. Two extreme forms of PEM are kwashiorkor and marasmus (Figure 27.21).
1. Kwashiorkor: Kwashiorkor occurs when protein deprivation is relatively greater than the reduction in total calories. Protein deprivation is associated with severely decreased synthesis of visceral protein. Kwashiorkor is frequently seen in children after weaning at about one year of age, when their diet consists predominantly of carbohydrates. Typical symptoms include stunted growth, edema, skin lesions, depigmented hair, anorexia, enlarged fatty liver, and decreased plasma albumin concentration. Edema results from the lack of adequate plasma proteins to maintain the distribution of water between blood and tissues. Edema may mask muscle loss.
2. Marasmus: Marasmus occurs when calorie deprivation is relatively greater than the reduction in protein. It usually occurs in children younger than 1 year of age when breast milk is supplemented with watery gruels of native cereals that are usually deficient in protein and calories. Typical symptoms include arrested growth, extreme muscle wasting (emaciation), weakness, and anemia (Figure 27.22). Victims of marasmus do not show the edema or changes in plasma proteins observed in kwashiorkor.

A.Listless child with kwashiorkor. B.Child suffering with marasmus.
L

Estimated Average Requirement (EAR) is the average daily nutrient intake level estimated to meet the requirement of one half the healthy individuals in a particular life stage and gender group. The Recommended Dietary Allowance (RDA) is the average daily dietary intake level that is sufficient to meet the nutrient requirements of nearly all (97-98%) individuals. Adequate Intake (Al) is set instead of an RDA if sufficient scientific evidence is not available to calculate the RDA. The Tolerable Upper Intake Level (UL) is the highest average daily nutrient intake level that is likely to pose no risk of adverse health effects to almost all individuals in the general population. The energy generated by the metabolism of the macronutrients is used for three energy-requiring processes that occur in the body: resting metabolic rate, thermic effect of food, and physical activity. Acceptable Macronutrient Distribution Ranges (AMDR) are defined as the ranges of intake for a particular macronutrient that is associated with reduced risk of chronic disease while providing adequate amounts of essential nutrients. Adults should consume 45-65% of their total calories from carbohydrates, 20-35% from fat, and 10-35% from protein (Figure 27.23). Elevated levels of cholesterol or LDL cholesterol result in increased risk for cardiovascular disease. In contrast, high levels of HDL cholesterol have been associated with a decreased risk for heart disease. Dietary or drug treatment of hypercholesterolemia is effective in decreasing LDL, increasing HDL, and reducing the risk for cardiovascular events. Consumption of saturated fats is strongly associated with high levels of total plasma and LDL cholesterol. When substituted for saturated fatty acids in the diet, monounsaturated fats lower both total plasma and LDL cholesterol, but increase HDL. Consumption of fats containing w-6 polyunsaturated fatty acids lowers plasma LDL, but HDL, which protect against coronary heart disease, are also lowered. Dietary w-3 polyunsaturated fats suppress cardiac arrhythmias and reduce serum triacylglycerols, decrease the tendency for thrombosis, and substantially reduce the risk of cardiovascular mortality. Carbohydrates provide energy and fiber to the diet. When they are consumed as part of a diet in which caloric intake is equal to energy expenditure, they do not promote obesity. Dietary protein provides essential amino acids. The quality of a protein is a measure of its ability to provide the essential amino acids required for tissue maintenance. Proteins from animal sources, in

general, have a higher-quality protein than that derived from plants. However, proteins from different plant sources may be combined in such a way that the result is equivalent in nutritional value to animal protein. Positive nitrogen balance occurs when nitrogen intake exceeds nitrogen excretion. It is observed in situations in which tissue growth occurs, for example, in childhood, pregnancy, or during recovery from an emaciating illness. Negative nitrogen balance occurs when nitrogen losses are greater than nitrogen intake. It is associated with inadequate dietary protein, lack of an essential amino acid, or during physiologic stresses such as trauma, burns, illness, or surgery. Kwashiorkor is caused by inadequate intake of protein and is characterized by edema. Marasmus results from chronic deficiency of calories.

27.1
Which one of the following statements concerning dietary lipid is correct?, 1 = Which one of the following statements concerning dietary lipid is correct?. A., 1 = Corn oil and soybean oil are examples of fats rich in saturated fatty acids.. B., 1 = Triacylglycerols obtained from plants generally contain less unsaturated fatty acids than those from animals.. C., 1 = Olive oil is rich in saturated fats.. D., 1 = Fatty acids containing double bonds in the trans configuration, unlike the naturally occurring cis isomers, raise plasma cholesterol levels.. E., 1 = Coconut and palm oils are rich in polyunsaturated fats.
Correct answer = D. Trans fatty acids raise plasma cholesterol levels. Corn oil and soybean oil are examples of fats rich in polyunsaturated fatty acids. Triacylglycerols obtained. from plants generally contain more unsaturated fatty acids than those from animals. Olive oil, the staple of the Mediterranean diet, is rich in monounsaturated fats. Coconut and palm oils are unusual plant oils in that they are rich in saturated fats.
Figure 27.23
Key concept map for the macronutrients. *Note: Trans fatty acids are chemically classified as monounsaturated. PEM = protein energy malnutrition.
27.2
27.3
Given the information that a 70-kg man is consuming a daily average of 275 g of carbohydrate, 75 g of protein, and 65 g of lipid, one can draw which of the following conclusions?
A. Total energy intake per day is approximately 3,000
kcal.
B. About 20% of the calories are derived from lipids.
- C. The diet does not contain a sufficient amount of dietary fiber.
D.
The proportions of carbohydrate, protein, and lipid in the diet conform to the recommendations of
academic groups and government agencies.
The individual is in nitrogen balance.
E.
A
sedentary
50-year-old man,
weighing pounds),
requests any
health markable
a
physical problems.
except
The man
80
examination.
Routine for
plasma refuses
kg
He blood
analysis cholesterol
drug terolemia.
Analysis therapy
of
a
(176
denies is
unre- of
280
for his
mg/dl.
hypercholes-
1-day dietary

recall showed
the following:
Kilocalories
3,475 kcal
Protein
102g
Carbohydrate
383g
Fiber crude
6g
Cholesterol
822 mg
Saturated fat
69g
Total Fat
165g
Changes in which one of the following dietary components would have the greatest effect in lowering plasma cholesterol?
A. Cholesterol.
B. Saturated fat.
C. Polyunsaturated fat.
D. Monounsaturated fat.
E. Carbohydrate.
27.4 Which of the findings in the child shown at right would support a diagnosis of kwashiorkor?
A. Shows increased serum albumin.
B. Shows a good appetite.
C. Appears plump due to increased adipose tissue.
D. Displays abdominal and peripheral edema.
E. Has markedly decreased weight for height.
Correct answer = D. The total energy intake is (275 g carbohydrate x 4 kcal/g) + (75 g protein x 4 kcal/g) + (65 g lipid x 9 keal/g) = 1100 + 300 + 585 = 1,985 total kcal/day. The percentage calories from carbohydrate is 1,100/1,985 = 55; percentage calories from protein is 300/1,985 = 15; and percentage calories derived from lipid is 585/1,985 = 30. These are very close to current recommendations. The amount of fiber or nitrogen balance cannot be deduced from the data presented. If the protein is of low biologic value, a negative nitrogen balance is possible.
Correct answer = B. The intake of saturated fat most strongly influences plasma cholesterol in this diet. The patient is consuming a high-calorie, high-fat diet with 40% of the fat as saturated fat. The most important dietary recommendations are: lower total caloric intake, substitute monounsaturated and polyunsaturated fats for saturated fats, and increase dietary fiber. A decrease in dietary cholesterol would be helpful, but is not a primary objective.

The correct answer = D. Kwashiorkor is caused by inadequate protein intake in the presence of fair to good energy (calorie) intake. Typical findings in a patient with Kwashiorkor include abdominal and peripheral edema (note the swollen belly and legs of the child) caused largely by a decreased serum albumin concentration. Anorexia is almost always present. Weight for height can be normal. Treatment includes a diet adequate in calories and protein.

Vitamins are chemically unrelated organic compounds that cannot be synthesized in adequate quantities by humans and, therefore, must be supplied by the diet. Nine vitamins (folic acid, cobalamin, ascorbic acid, pyridoxine, thiamine, niacin, riboflavin, biotin, and pantothenic acid) are classified as water-soluble, whereas four vitamins (vitamins A, D, K, and E) are termed fat-soluble (Figure 28.1). Vitamins are required to perform specific cellular functions, for example, many of the water-soluble vitamins are precursors of coenzymes for the enzymes of intermediary metabolism. In contrast to the water-soluble vitamins, only one fat soluble vitamin (vitamin K) has a coenzyme function. These vitamins are released, absorbed, and transported with the fat of the diet. They are not readily excreted in the urine, and significant quantities are stored in the liver and adipose tissue. In fact, consumption of vitamins A and D in excess of the Dietary Reference Intakes (DRIs) can lead to accumulation of toxic quantities of these compounds.
Figure 28.1
Classification of the vitamins.

Classification of nutritional anemias by cell size. The normal mean corpuscular volume (MCV) for people older than age 18 is between 80 and 100 ums'. [Note: Microcytic anemia is also seen with lead poisoning.]

Folic acid (or folate), which plays a key role in one-carbon metabolism, is essential for the biosynthesis of several compounds. Folic acid deficiency is probably the most common vitamin deficiency in the United States, particularly among pregnant women and alcoholics.

Tetrahydrofolate (reduced folate) receives one-carbon fragments from donors such as serine, glycine, and histidine and transfers them to intermediates in the synthesis of amino acids, purines, and thymidine monophosphate (TMP)—a pyrimidine found in DNA.

Anemia is a condition in which the blood has a lower than normal concentration of hemoglobin, which results in a reduced ability to transport oxygen. Nutritional anemias—those caused by inadequate intake of one or more essential nutrients—can be classified according to the size of the red blood cells or mean corpuscular volume observed in the individual (Figure 28.2). Microcytic anemia, caused by lack of iron, is the most common form of nutritional anemia. The second major category of nutritional anemia, macrocytic, results from a deficiency in folic acid or vitamin Biz. [Note: These macrocytic anemias are commonly called megaloblastic because a deficiency of folic acid or vitamin Bjz2 causes accumulation of large, immature red cell precursors, known as megaloblasts, in the bone marrow and the blood.]
1. Folate and anemia: Inadequate serum levels of folate can be caused by increased demand (for example, pregnancy and lactation), poor absorption caused by pathology of the small intestine, alcoholism, or treatment with drugs that are dihydrofolate reductase inhibitors, for example, methotrexate (Figure 28.3). A folate-free diet

Inhibition of tetrahydrofolate synthesis by sulfonamides and methotrexate.
can cause a deficiency within a few weeks. A primary result of folic acid deficiency is megaloblastic anemia (Figure 28.4), caused by diminished synthesis of purines and TMP, which leads to an inability of cells (including red cell precursors) to make DNA and, therefore, they cannot divide. [Note: It is important to evaluate the cause of the megaloblastic anemia prior to instituting therapy, because vitamin By2 deficiency indirectly causes symptoms of this disorder (see p. 377).]
2. Folate and neural tube defects in the fetus: Spina bifida and anencephaly, the most common neural tube defects, affect approximately 4,000 pregnancies in the United State annually. Folic acid supplementation before conception and during the first trimester has been shown to significantly reduce the defects. Therefore, all women of childbearing age are advised to consume 0.4 mg/day of folic acid to reduce the risk of having a pregnancy affected by neural tube defects. Adequate folate nutrition must occur at the time of conception because critical folate-dependent development occurs in the first weeks of fetal life —at a time when many women are not yet aware of their pregnancy. The U.S. Food and Drug Administration has authorized the addition of folic acid to enriched grain products, resulting in a dietary supplementation of about 0.1 mg/day. It is estimated that this supplementation will allow approximately 50% of all reproductive-aged women to receive 0.4 mg of folate from all sources. However, there is an association of high-dose supplementation with folic acid (>0.8 mg/day) and an increased risk of cancer. Thus, supplementation is not recommended for most middle-aged or older adults.

Vitamin B12 is required in humans for two essential enzymatic reactions: the remethylation of homocysteine to methionine and the isomerization of methylmalonyl coenzyme A (CoA) that is produced during the degradation of some amino acids (isoleucine, valine, threonine, and methionine), and fatty acids with odd numbers of carbon atoms (Figure 28.5). When the vitamin is deficient, unusual fatty acids accumulate and become incorporated into cell membranes, including those of the nervous system. This may account for some of the neurologic manifestations of vitamin B12 deficiency.

Cobalamin contains a corrin ring system that differs from the porphyrins in that two of the pyrrole rings are linked directly rather than through a methene bridge. Cobalt is held in the center of the corrin ring by four coordination bonds from the nitrogens of the pyrrole groups. The remaining coordination bonds of the cobalt are with the nitrogen of 5,6-dimethylbenzimidazole and with cyanide in commercial preparations of the vitamin in the form of cyanocobalamin (Figure 28.6). The coenzyme forms of cobalamin are 5'-deoxyadenosylcobalamin, in which cyanide is replaced with 5'-deoxyadenosine (forming an unusual carbon—cobalt bond), and methylcobalamin, in which cyanide is replaced by a methyl group (see Figure 28.6).

Bone marrow histology in normal and folate-deficient individuals.

Structure of vitamin and its forms and
By2 (cyanocobalamin) coenzyme (methylcobalamin 5'-deoxyadenosyl- cobalamin).

Absorption of vitamin By. IF = intrinsic factor.
Vitamin By2 is synthesized only by microorganisms; it is not present in plants. Animals obtain the vitamin preformed from their natural bacterial flora or by eating foods derived from other animals. Cobalamin is present in appreciable amounts in liver, whole milk, eggs, oysters, fresh shrimp, pork, and chicken.

The effects of cobalamin deficiency are most pronounced in rapidly dividing cells, such as the erythropoietic tissue of bone marrow and the mucosal cells of the intestine. Such tissues need both the N®°,N1°methylene and N'°-formyl forms of tetrahydrofolate for the synthesis of nucleotides required for DNA replication (see pp. 293 and 303). However, in vitamin B,2 deficiency, the utilization of the N°-methyl form of tetrahydrofolate in the By2-dependent methylation of homocysteine to methionine is impaired. Because the methylated form cannot be converted directly to other forms of tetrahydrofolate, folate is trapped in the N°-methyl form, which accumulates. The levels of the other forms decrease. Thus, cobalamin deficiency is hypothesized to lead to a deficiency of the tetrahydrofolate forms needed in purine and TMP synthesis, resulting in the symptoms of megaloblastic anemia.

In contrast to other water-soluble vitamins, significant amounts (4-5 mg) of vitamin By. are stored in the body. As a result, it may take several years for the clinical symptoms of Byi2 deficiency to develop in individuals who have had a partial or total gastrectomy (who, therefore, become intrinsic factor-deficient, see p. 377) and can no longer absorb the vitamin.
1. Pernicious anemia: Vitamin By. deficiency is rarely a result of an absence of the vitamin in the diet. It is much more common to find
deficiencies in patients who fail to absorb the vitamin from the intestine. Malabsorption of cobalamin in the elderly is most often due to reduced secretion of gastric acid and less efficient absorption of vitamin B12 from foods. A severe malabsorption of vitamin B12 leads to pernicious anemia. This disease is most commonly a result of an autoimmune destruction of the gastric parietal cells that are responsible for the synthesis of a glycoprotein called intrinsic factor. Normally, vitamin B12 obtained from the diet binds to intrinsic factor in the intestine (Figure 28.7). The cobalamin-intrinsic factor complex travels through the gut and eventually binds to specific receptors on the surface of mucosal cells of the ileum. The bound cobalamin is transported into the mucosal cell and, subsequently, into the general circulation, where it is carried by By2-binding proteins. Lack of intrinsic factor prevents the absorption of vitamin Bio, resulting in pernicious anemia. Patients with cobalamin deficiency are usually anemic, but later in the development of the disease they show neuropsychiatric symptoms. However, central nervous system (CNS) symptoms may occur in the absence of anemia. The CNS effects are irreversible and occur by mechanisms that appear to be different from those described for megaloblastic anemia. The disease is treated by giving high-dose By orally, or intramuscular (IM) injection of cyanocobalamin. Therapy must be continued throughout the lives of patients with pernicious anemia. Deficiency of vitamin B12 can be measured by the level of methylmalonic acid in blood, which is elevated in individuals with low intake or decreased absorption of the vitamin.

Folic acid can partially reverse the hematologic abnormalities of B12 deficiency and, therefore, can mask a cobalamin deficiency. Thus, therapy of megaloblastic anemia is often initiated with folic acid and vitamin By. until the cause of the anemia can be determined.

The active form of vitamin C is ascorbic acid (Figure 28.8). The main function of ascorbate is as a reducing agent in several different reactions. Vitamin C has a well-documented role as a coenzyme in hydroxylation reactions, for example, hydroxylation of prolyl and lysyl residues of collagen (see p. 47). Vitamin C is, therefore, required for the maintenance of normal connective tissue, as well as for wound healing. Vitamin C also facilitates the absorption of dietary iron from the intestine.

A deficiency of ascorbic acid results in scurvy, a disease characterized by sore and spongy gums, loose teeth, fragile blood vessels, swollen joints, and anemia (Figure 28.9). Many of the deficiency symptoms can be explained by a deficiency in the hydroxylation of collagen, resulting in defective connective tissue.

Structures of vitamin Bg and and the antituberculosis drug isoniazid.
Oo

A. Structure of thiamine and its coenzyme form, thiamine pyrophosphate. B. Structure of intermediate formed in the reaction catalyzed by pyruvate dehydrogenase. C. Structure of intermediate formed in the reaction catalyzed by a-ketoglutarate dehydrogenase.

Vitamin C is one of a group of nutrients that includes vitamin E (see p. 391) and B-carotene (see p. 382), which are known as antioxidants. Consumption of diets rich in these compounds is associated with a decreased incidence of some chronic diseases, such as coronary heart disease and certain cancers. However, clinical trials involving supplementation with the isolated antioxidants have failed to determine any convincing beneficial effects.

Vitamin Bg is a collective term for pyridoxine, pyridoxal, and pyridoxamine, all derivatives of pyridine. They differ only in the nature of the functional group attached to the ring (Figure 28.10). Pyridoxine occurs primarily in plants, whereas pyridoxal and pyridoxamine are found in foods obtained from animals. All three compounds can serve as precursors of the biologically active coenzyme, pyridoxal phosphate. Pyridoxal phosphate functions as a coenzyme for a large number of enzymes, particularly those that catalyze reactions involving amino acids.

Isoniazid (isonicotinic acid hydrazide), a drug frequently used to treat tuberculosis, can induce a vitamin Bg deficiency by forming an inactive derivative with pyridoxal phosphate. Dietary supplementation with Bg is, thus, an adjunct to isoniazid treatment. Otherwise, dietary deficiencies in pyridoxine are rare but have been observed in newborn infants fed formulas low in Bg, in women taking oral contraceptives, and in alcoholics.

Pyridoxine is the only water-soluble vitamin with significant toxicity. Neurologic symptoms (sensory neuropathy) occur at intakes above 200 mg/day, an amount more than 100 times the RDA. Substantial improvement, but not complete recovery, occurs when the vitamin is discontinued.

Thiamine pyrophosphate is the biologically active form of the vitamin, formed by the transfer of a pyrophosphate group from adenosine triphosphate (ATP) to thiamine (Figure 28.11). Thiamine pyrophosphate serves as a coenzyme in the formation or degradation of a-ketols by transketo-
lase (Figure 28.12A), and in the oxidative decarboxylation of «-keto acids (Figure 28.12B).

The oxidative decarboxylation of pyruvate and o-ketoglutarate, which plays a key role in energy metabolism of most cells, is particularly important in tissues of the nervous system. In thiamine deficiency, the activity of these two dehydrogenase-catalyzed reactions is decreased, resulting in a decreased production of ATP and, thus, impaired cellular function. [Note: Thiamine deficiency is diagnosed by an increase in erythrocyte transketolase activity observed on addition of thiamine pyrophosphate.]
1. Beriberi: This is a severe thiamine-deficiency syndrome found in areas where polished rice is the major component of the diet. Signs of infantile beriberi include tachycardia, vomiting, convulsions, and, if not treated, death. The deficiency syndrome can have a rapid onset in nursing infants whose mothers are deficient in thiamine. Adult beriberi is characterized by dry skin, irritability, disordered thinking, and progressive paralysis.
. Wernicke-Korsakoff syndrome: In the United States, thiamine deficiency, which is seen primarily in association with chronic alcoholism, is due to dietary insufficiency or impaired intestinal absorption of the vitamin. Some alcoholics develop WernickeKorsakoff syndrome—a thiamine deficiency state characterized by apathy, loss of memory, ataxia, and a rhythmic to-and-fro motion of the eyeballs (nystagmus). The neurologic consequences of Wernicke's syndrome are treatable with thiamine supplementation.

Reactions that use thiamine pyrophosphate (TPP) as coenzyme. A. Transketolase. B. Pyruvate dehydrogenase and a-ketoglutarate dehydrogenase. Note that TPP is also used by branched-chain a-keto acid dehydrogenase.
Niacin, or nicotinic acid, is a substituted pyridine derivative. The biologically active coenzyme forms are nicotinamide adenine dinucleotide (NAD*) and its phosphorylated derivative, nicotinamide adenine

Structure and biosynthesis of NAD* and NADP'. Note that a metabolite of tryptophan (quinolinate) can also be used in the synthesis of NAD*.
Figure

Reduction of NADt to NADH.
dinucleotide phosphate (NADP*, Figure 28.13). Nicotinamide, a derivative of nicotinic acid that contains an amide instead of a carboxyl group, also occurs in the diet. Nicotinamide is readily deaminated in the body and, therefore, is nutritionally equivalent to nicotinic acid. NAD* and NADP* serve as coenzymes in oxidation-reduction reactions in which the coenzyme undergoes reduction of the pyridine ring by accepting a hydride ion (hydrogen atom plus one electron, Figure 28.14). The reduced forms of NAD* and NADP* are NADH and NADPH, respectively.

Niacin is found in unrefined and enriched grains and cereal, milk, and lean meats, especially liver. [Note: Corn is low in both niacin and tryptophan. Corn-based diets can cause pellagra (see below).]

1.
Deficiency of niacin: A deficiency of niacin causes pellagra, a disease involving the skin, gastrointestinal tract, and CNS. The symptoms of pellagra progress through the three Ds: dermatitis, diarrhea, dementia—and, if untreated, death.
2. Treatment of hyperlipidemia: Niacin (at doses of 1.5 g/day or 100 times the Recommended Dietary Allowance or RDA) strongly inhibits lipolysis in adipose tissue—the primary producer of circulating free fatty acids. The liver normally uses these circulating fatty acids as a major precursor for triacylglycerol synthesis. Thus, niacin causes a decrease in liver triacylglycerol synthesis, which is required for very-low-density lipoprotein (VLDL, see p. 231) production. Low-density lipoprotein (LDL, the cholesterolrich lipoprotein) is derived from VLDL in the plasma. Thus, both plasma triacylglycerol (in VLDL) and cholesterol (in VLDL and LDL) are lowered. Therefore, niacin is particularly useful in the treatment of Type IIb hyperlipoproteinemia, in which both VLDL and LDL are elevated. [Note: Niacin raises HDL levels.]

The two biologically active forms are flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), formed by the transfer of an adenosine

Structure and biosynthesis of flavin mononucleotide and flavin adenine dinucleotide.
monophosphate moiety from ATP to FMN (Figure 28.15). FMN and FAD are each capable of reversibly accepting two hydrogen atoms, forming FMNHz2 or FADH2. FMN and FAD are bound tightly—sometimes covalently—to flavoenzymes that catalyze the oxidation or reduction of a substrate. Riboflavin deficiency is not associated with a major human disease, although it frequently accompanies other vitamin deficiencies. Deficiency symptoms include dermatitis, cheilosis (fissuring at the corners of the mouth), and glossitis (the tongue appearing smooth and purplish).

Biotin is a coenzyme in carboxylation reactions, in which it serves as a carrier of activated carbon dioxide (see Figure 10.3, p. 119, for the mechanism of biotin-dependent carboxylations). Biotin is covalently bound to the e-amino groups of lysine residues in biotin-dependent enzymes (Figure 28.16). Biotin deficiency does not occur naturally because the vitamin is widely distributed in food. Also, a large percentage of the biotin requirement in humans is supplied by intestinal bacteria. However, the addition of raw egg white to the diet as a source of protein induces symptoms of biotin deficiency, namely, dermatitis, glossitis, loss of appetite, and nausea. Raw egg white contains a glycoprotein, avidin, which tightly binds biotin and prevents its absorption from the intestine. With a normal diet, however, it has been estimated that 20 eggs/day would be required to induce a deficiency syndrome. Thus, inclusion of an occasional raw egg in the diet does not lead to biotin deficiency, although eating raw eggs is generally not recommended due to the possibility of salmonella infection.
Multiple carboxylase deficiency results from a defect in the ability to link biotin to carboxylases or to remove it from carboxylases during their degradation. Treatment is biotin supplementation.

Pantothenic acid is a component of coenzyme A (CoA), which functions in the transfer of acyl groups (Figure 28.17). CoA contains a thiol group that carries acyl compounds as activated thiol esters. Examples of such structures are succinyl CoA, fatty acyl CoA, and acetyl CoA. Pantothenic acid is also a component of the acyl carrier protein (ACP) domain of fatty acid synthase (see p. 184). Eggs, liver, and yeast are the most important sources of pantothenic acid, although the vitamin is widely distributed. Pantothenic acid deficiency is not well characterized in humans, and no RDA has been established.

The retinoids, a family of molecules that are related to retinol (vitamin A), are essential for vision, reproduction, growth, and maintenance of epithelial tissues. Retinoic acid, derived from oxidation of dietary retinol, mediates most of the actions of the retinoids, except for vision, which depends on retinal, the aldehyde derivative of retinol.

A. Structure of biotin. B. Biotin covalently bound to a lysyl residue of a biotin-dependent enzyme.

Vitamin A is often used as a collective term for several related biologically active molecules (Figure 28.18). The term retinoids includes both natural and synthetic forms of vitamin A that may or may not show vitamin A activity.
1. Retinol: A primary alcohol containing a B-ionone ring with an unsaturated side chain, retinol is found in animal tissues as a retinyl ester with long-chain fatty acids.
2. Retinal: This is the aldehyde derived from the oxidation of retinol. Retinal and retinol can readily be interconverted.
3. Retinoic acid: This is the acid derived from the oxidation of retinal. Retinoic acid cannot be reduced in the body, and, therefore, cannot give rise to either retinal or retinol.
4. B-Carotene: Plant foods contain B-carotene, which can be oxidatively cleaved in the intestine to yield two molecules of retinal. In humans, the conversion is inefficient, and the vitamin A activity of B-carotene is only about one twelfth that of retinol.

1. Transport to the liver: Retinyl esters present in the diet are hydrolyzed in the intestinal mucosa, releasing retinol and free fatty acids (Figure 28.19). Retinol derived from esters and from the cleavage and reduction of carotenes is re-esterified to long-chain fatty acids in the intestinal mucosa and secreted as a component of chylomicrons into the lymphatic system (see Figure 28.19). Retinyl esters contained in chylomicron remnants are taken up by, and stored in, the liver.
2. Release from the liver: When needed, retinol is released from the liver and transported to extrahepatic tissues by the plasma retinolbinding protein (RBP). The retinol-RBP complex attaches to specific receptors on the surface of the cells of peripheral tissues, permitting retinol to enter. Many tissues contain a cellular retinolbinding protein that carries retinol to sites in the nucleus where the vitamin acts in a manner analogous to that of steroid hormones.

Retinol is oxidized to retinoic acid. Retinoic acid binds with high affinity to specific receptor proteins present in the nucleus of target tissues, such as epithelial cells (Figure 28.20). The activated retinoic acid—receptor complex interacts with nuclear chromatin to regulate retinoid-specific RNA synthesis, resulting in control of the production of specific proteins that mediate several physiologic functions. For example, retinoids control the expression of the gene for keratin in most epithelial tissues of the body. The specific retinoic acid—receptor proteins are part of the superfamily of transcriptional regulators that includes the steroid and thyroid hormones and 1,25-dihydroxycholecalciferol, all of which function in a similar way (see p. 240).

Absorption, transport, and storage of vitamin A and its derivatives. RBP = retinol-binding protein.

Action of retinoids Note: Retinoic acid-receptor complex is a dimer, _ but is shown as monomer for simplicity. [RBP = retinol-binding protein.]

1. Visual cycle: Vitamin A is a component of the visual pigments of rod and cone cells. Rhodopsin, the visual pigment of the rod cells in the retina, consists of 11-cis retinal specifically bound to the protein opsin. When rhodopsin is exposed to light, a series of photochemical isomerizations occurs, which results in the bleaching of the visual pigment and release of all-trans retinal and opsin. This process triggers a nerve impulse that is transmitted by the optic nerve to the brain. Regeneration of rhodopsin requires isomerization of all-trans retinal back to 11-cis retinal. All-trans retinal, after being released from rhodopsin, is reduced to all-trans retinol, esterfied, and isomerized to 11-cis retinol that is oxidized to 11-cis retinal. The latter combines with opsin to form rhodopsin, thus completing the cycle. Similar reactions are responsible for color vision in the cone cells.
2. Growth: Vitamin A deficiency results in a decreased growth rate in children. Bone development is also slowed.
3. Reproduction: Retinol and retinal are essential for normal reproduction, supporting spermatogenesis in the male and preventing fetal resorption in the female. Retinoic acid is inactive in maintaining reproduction and in the visual cycle, but promotes growth and differentiation of epithelial cells; thus, animals given vitamin A only as retinoic acid from birth are blind and sterile.
4. Maintenance of epithelial cells: Vitamin A is essential for normal differentiation of epithelial tissues and mucus secretion.

Liver, kidney, cream, butter, and egg yolk are good sources of preformed vitamin A. Yellow and dark green vegetables and fruits are good dietary sources of the carotenes, which serve as precursors of vitamin A.

The RDA for adults is 900 retinol activity equivalents (RAE) for males and 700 RAE for females. In comparison, 1 RAE = 1 mg of retinol, 12 mg of B-carotene, or 24 mg of other carotenoids.

Although chemically related, retinoic acid and retinol have distinctly different therapeutic applications. Retinol and its precursor are used as dietary supplements, whereas various forms of retinoic acid are useful in dermatology.
- 1. Dietary deficiency: Vitamin A, administered as retinol or retinyl esters, is used to treat patients who are deficient in the vitamin (Figure 28.21). Night blindness is one of the earliest signs of vitamin A deficiency. The visual threshold is increased, making it difficult to see in dim light. Prolonged deficiency leads to an irreversible loss in the number of visual cells. Severe vitamin A deficiency leads to xerophthalmia, a pathologic dryness of the conjunctiva and cornea. If untreated, xerophthalmia results in

Summary of actions of retinoids. Compounds in| boxes] are available as dietary components or as pharmacologic agents.
corneal ulceration and, ultimately, in blindness because of the formation of opaque scar tissue. The condition is most frequently seen in children in developing tropical countries. Over 500,000 children worldwide are blinded each year by xerophthalmia caused by insufficient vitamin A in the diet.
2. Acne and psoriasis: Dermatologic problems such as acne and psoriasis are effectively treated with retinoic acid or its derivatives (see Figure 28.21). Mild cases of acne, Darier disease (keratosis follicularis), and skin aging are treated with topical application of tretinoin (all-trans retinoic acid), as well as benzoyl peroxide and antibiotics. [Note: Tretinoin is too toxic for systemic administration and is confined to topical application.] In patients with severe, recalcitrant, cystic acne unresponsive to conventional therapies, the drug of choice is isotretinoin (13-cis retinoic acid) administered orally. Retinoic acid is also used in the treatment of promyelocytic leukemia.

1. Vitamin A: Excessive intake of vitamin A produces a toxic syndrome called hypervitaminosis A. Amounts exceeding 7.5 mg/day of retinol should be avoided. Early signs of chronic hypervitaminosis A are reflected in the skin, which becomes dry and pru-

Sources of vitamin D.
ritic (due to a decrease in keratin synthesis), the liver, which becomes enlarged and can become cirrhotic, and in the nervous system, where a rise in intracranial pressure may mimic the symptoms of a brain tumor. Pregnant women particularly should not ingest excessive quantities of vitamin A because of its potential for causing congenital malformations in the developing fetus.
- 2. Isotretinoin: The drug is teratogenic and absolutely contraindicated in women with childbearing potential unless they have severe, disfiguring cystic acne that is unresponsive to standard therapies. Pregnancy must be excluded before initiation of treatment, and adequate birth control must be used. Prolonged treatment with isotretinoin leads to hyperlipidemia and an increase in the LDL/HDL ratio, providing some concern for an increased risk of cardiovascular disease.

The D vitamins are a group of sterols that have a hormone-like function. The active molecule, 1,25-dihydroxycholecalciferol (1,25-diOH-Ds), binds to intracellular receptor proteins. The 1,25-diOH-Ds—receptor complex interacts with DNA in the nucleus of target cells in a manner similar to that of vitamin A (see Figure 28.20), and either selectively stimulates gene expression or specifically represses gene transcription. The most prominent actions of 1,25-diOH-Ds are to regulate the plasma levels of calcium and phosphorus.

1. Diet: Ergocalciferol (vitamin Dz), found in plants, and cholecalciferol (vitamin Dg), found in animal tissues, are sources of preformed vitamin D activity (Figure 28.22). Ergocalciferol and cholecalciferol differ chemically only in the presence of an additional double bond and methyl group in the plant sterol.
2. Endogenous vitamin precursor: 7-Dehydrocholesterol, an intermediate in cholesterol synthesis, is converted to cholecalciferol in the dermis and epidermis of humans exposed to sunlight. Preformed vitamin D is a dietary requirement only in individuals with limited exposure to sunlight.

- 1. Formation of 1,25-diOH-D3: Vitamins D2 and Dg are not biologically active, but are converted in vivo to the active form of the D vitamin by two sequential hydroxylation reactions (Figure 28.23). The first hydroxylation occurs at the 25-position, and is catalyzed by a specific hydroxylase in the liver. The product of the reaction, 25-hydroxycholecalciferol (25-OH-Ds, calcidol), is the predominant form of vitamin D in the plasma and the major storage form of the vitamin. 25-OH-Ds is further hydroxylated at the 1 position by 25hydroxycholecalciferol 1-hydroxylase found primarily in the kidney, resulting in the formation of 1,25-diOH-Ds (calcitriol). [Note: This hydroxylase, as well as the liver 25-hydroxylase, are cytochrome P450 (CYP) proteins (see p. 149).]

Metabolism and actions of vitamin D. [Note: Calcitonin, a thyroid hormone, decreases blood calcium by inhibiting mobilization from bone and reabsorption by the kidney.]

Response to low plasma calcium.
2. Regulation of 25-hydroxycholecalciferol 1-hydroxylase: 1,25diOH-Dg is the most potent vitamin D metabolite. Its formation is tightly regulated by the level of plasma phosphate and calcium ions (Figure 28.24). 25-Hydroxycholecalciferol 1-hydroxylase activity is increased directly by low plasma phosphate or indirectly by low plasma calcium, which triggers the release of parathyroid hormone (PTH). Hypocalcemia caused by insufficient dietary calcium thus results in elevated levels of plasma 1,25-diOH-Ds. 1-Hydroxylase activity is also decreased by excess 1,25-diOH-Ds, the product of the reaction.

The overall function of 1,25-diOH-D3 is to maintain adequate plasma levels of calcium. It performs this function by: 1) increasing uptake of calcium by the intestine, 2) minimizing loss of calcium by the kidney, and 3) stimulating resorption of bone when necessary (see Figure 28.23).
1. Effect of vitamin D on the intestine: 1,25-diOH-D3 stimulates intestinal absorption of calcium and phosphate. 1,25-diOH-D3 enters the intestinal cell and binds to a cytosolic receptor. The 1,25-diOH-D3—receptor complex then moves to the nucleus where it selectively interacts with the cellular DNA. As a result, calcium uptake is enhanced by an increased synthesis of a specific calcium-binding protein. Thus, the mechanism of action of 1,25diOH-Ds is typical of steroid hormones (see p. 240).
2. Effect of vitamin D on bone: 1,25-diOH-D3 stimulates the mobilization of calcium and phosphate from bone by a process that requires protein synthesis and the presence of PTH. The result is an increase in plasma calcium and phosphate. Thus, bone is an important reservoir of calcium that can be mobilized to maintain plasma levels.

Vitamin D occurs naturally in fatty fish, liver, and egg yolk. Milk, unless it is artificially fortified, is not a good source of the vitamin. Al for vitamin D is 200 IU to age 50, and 400-600 IU after age 50.

1. Nutritional rickets: Vitamin D deficiency causes a net demineralization of bone, resulting in rickets in children and osteomalacia in adults (Figure 28.25). Rickets is characterized by the continued formation of the collagen matrix of bone, but incomplete mineralization, resulting in soft, pliable bones. In osteomalacia, demineralization of pre-existing bones increases their susceptibility to fracture. Insufficient exposure to daylight and/or deficiencies in vitamin D consumption occur predominantly in infants and the elderly. Vitamin D deficiency is more common in the northern latitudes, because less vitamin D synthesis occurs in the skin as a result of reduced exposure to ultraviolet light. [Note: The recommended intake of 200 IU/day (which corresponds to 5 ug of cholecalciferol) may be insufficient, because higher doses of 800 IU/day have been shown to reduce the inci-
dence of osteoporotic fractures.] Sufficient levels of serum 25hydroxycholecalciferol (>75 nmol/L) have been linked to fall prevention in older people as well as to increased muscle strength and bone mass.
2. Renal osteodystrophy: Chronic renal failure results in decreased ability to form the active form of vitamin D. Supplementation with calcitriol is an effective therapy. [Note: Vitamin D supplementation is accompanied by phosphate reduction therapy to prevent hyperphosphatemia (due to renal failure) and precipitation of calcium phosphate crystals.]
3. Hypoparathyroidism: Lack of parathyroid hormone causes hypocalcemia and hyperphosphatemia. These patients may be treated with calcitriol and calcium supplementation.

Like all fat-soluble vitamins, vitamin D can be stored in the body and is only slowly metabolized. High doses (100,000 IU for weeks or months) can cause loss of appetite, nausea, thirst, and stupor. Enhanced calcium absorption and bone resorption results in hypercalcemia, which can lead to deposition of calcium in many organs, particularly the arteries and kidneys. The UL is 2000 !U/day.

The principal role of vitamin K is in the posttranslational modification of various blood clotting factors, in which it serves as a coenzyme in the carboxylation of certain glutamic acid residues present in these proteins. Vitamin K exists in several forms, for example, in plants as phylloquinone (or vitamin K;), and in intestinal bacterial flora as menaquinone (or vitamin Kz). A synthetic form of vitamin K, menadione, is available.

1. Formation of y-carboxyglutamate (Gla): Vitamin K is required in the hepatic synthesis of prothrombin and blood clotting factors Il, VII, IX, and X. These proteins are synthesized as inactive precursor molecules. Formation of the clotting factors requires the vitamin K—-dependent carboxylation of glutamic acid residues to Gla residues (Figure 28.26). This forms a mature clotting factor that contains Gla and is capable of subsequent activation. The reaction requires O2, COz, and the hydroquinone form of vitamin K. The formation of Gla is sensitive to inhibition by dicumarol, an anticoagulant occurring naturally in spoiled sweet clover, and by warfarin, a synthetic analog of vitamin K.
2. Interaction of prothrombin with platelets: The Gla residues of prothrombin are good chelators of positively charged calcium ions, because of the two adjacent, negatively charged carboxylate groups. The prothrombin-calcium complex is then able to bind to phospholipids essential for blood clotting on the surface of platelets. Attachment to the platelet increases the rate at which the proteolytic conversion of prothrombin to thrombin can occur (Figure 28.27).

Bowed legs of middle-aged man with osteomalacia, a nutritional vitamin D deficiency that results in malformation of the skeleton.

Role of vitamin K in blood coagulation.
3. Role of Gla residues in other proteins: Gla is also present in other proteins (for example, osteocalcin of bone, and in proteins such as protein C involved in limiting the formation of blood clots).

Vitamin K is found in cabbage, kale, spinach, egg yolk, and liver. There is also extensive synthesis of the vitamin by the bacteria in the gut. Al for vitamin K is 120 g/day for adult males and 90 ug for adult females.

1.
Deficiency of vitamin K: A true vitamin K deficiency is unusual because adequate amounts are generally produced by intestinal bacteria or obtained from the diet. If the bacterial population in the gut is decreased, for example, by antibiotics, the amount of endogenously formed vitamin is depressed, and this can lead to hypoprothrombinemia in the marginally malnourished individual, for example, a debilitated geriatric patient. This condition may require supplementation with vitamin K to correct the bleeding tendency. In addition, certain second-generation cephalosporins, for example, cefoperazone, cefamandole, and moxalactam cause hypoprothrombinemia, apparently by a warfarin-like mechanism. Consequently, their use in treatment is usually supplemented with vitamin K.
2. Deficiency of vitamin K in the newborn: Newborns have sterile intestines and so initially lack the bacteria that synthesize vitamin K. Because human milk provides only about one fifth of the daily requirement for vitamin K, it is recommended that all newborns receive a single intramuscular dose of vitamin K as prophylaxis against hemorrhagic disease.

Prolonged administration of large doses of synthetic vitamin K (menadione) can produce hemolytic anemia and jaundice in the infant, due to toxic effects on the membrane of red blood cells; therefore, it is no longer used to treat vitamin K deficiency. No UL has been set for vitamin K.

The E vitamins consist of eight naturally occurring tocopherols, of which a-tocopherol is the most active (Figure 28.28). The primary function of vitamin E is as an antioxidant in prevention of the nonenzymic oxidation of cell components, for example, polyunsaturated fatty acids, by molecular oxygen and free radicals.

Vegetable oils are rich sources of vitamin E, whereas liver and eggs contain moderate amounts. The RDA for a-tocopherol is 15 mg for adults. The vitamin E requirement increases as the intake of polyunsaturated fatty acid increases.

Vitamin E deficiency is almost entirely restricted to premature infants. When observed in adults, it is usually associated with defective lipid absorption or transport. The signs of human vitamin E deficiency include sensitivity of erythrocytes to peroxide, and the appearance of abnormal cellular membranes.

Vitamin E is not recommended for the prevention of chronic disease, such as coronary heart disease or cancer. Clinical trials using vitamin E supplementation have been uniformly disappointing. For example, subjects in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study trial who received high doses of vitamin E not only lacked cardiovascular benefit but also had an increased incidence of stroke.

Vitamin E is the least toxic of the fat-soluble vitamins, and no toxicity has been observed at doses of 300 mg/day.
Populations consuming diets high in fruits and vegetables show decreased incidence of some chronic diseases. However, clinical trials have failed to show a definitive benefit from supplements of vitamins A, C, or E; multivitamins with folic acid; or antioxidant combinations for the prevention of cancer or cardiovascular disease.
The vitamins are summarized in Figure 28.29.

A. Pantothenic acid, FUNCTION = WATER-SOLUBLE Acyl carrier. Vitamin A, OTHER NAMES = Retinol Retinal Retinoic acid β -Carotene. Vitamin A, ACTIVE FORM = Retinol Retinal Retinoic acid. Vitamin A, FUNCTION = FAT-SOLUBLE Maintenance of reproduction Vision Promotion of growth Differentiation and maintenance of epithelial tissues Gene expression. Vitamin D, OTHER NAMES = Cholecalciferol Ergocalciferol. Vitamin D, ACTIVE FORM = 1,25-Dihydroxy- cholecalciferol. Vitamin D, FUNCTION = Calcium uptake. Vitamin K, OTHER NAMES = Menadione Menaquinone Phylloquinone. Vitamin K, ACTIVE FORM = Menadione Menaquinone Phylloquinon. Vitamin K, FUNCTION = γ -Carboxylation of glutamate residue in clotting and other proteins. Vitamin E, OTHER NAMES = α -Tocopherol. Vitamin E, ACTIVE FORM = Any of several tocopherol derivatives. Vitamin E, FUNCTION = Antioxidant
Figure 28.29 (continued on next page)
Summary of vitamins.

SIGNS AND SYMPTOMS = -. Rare, TOXICITY = None. Rare, NOTES = WATER-SOLUBLE -. infertility Night blindness Retardation of growth Xerophthalmia, SIGNS AND SYMPTOMS = Increased visual threshold Dryness of cornea. infertility Night blindness Retardation of growth Xerophthalmia, TOXICITY = Yes. infertility Night blindness Retardation of growth Xerophthalmia, NOTES = FAT-SOLUBLE β -Carotene not acutely toxic, but supplementation is not recommended Excess vitamin A can increase incidence of fractures. Rickets (in children) Osteomalacia (in adults), SIGNS AND SYMPTOMS = Soft, pliable bones. Rickets (in children) Osteomalacia (in adults), TOXICITY = Yes. Rickets (in children) Osteomalacia (in adults), NOTES = Vitamin D is not a true vitamin because it can be synthesized in skin. Application of sunscreen lotions or presence of dark skin color decreases this synthesis.. Newborn Rare in adults, SIGNS AND SYMPTOMS = Bleeding. Newborn Rare in adults, TOXICITY = Rare. Newborn Rare in adults, NOTES = Vitamin K produced by intestinal bacteria. Vitamin K deficiency common in newborns Intramuscular treatment with vitamin K is recommended at birth. Rare, SIGNS AND SYMPTOMS = Red blood cell fragility leads to hemolytic anemia. Rare, TOXICITY = None. Rare, NOTES = Benefits of supplementation not established in controlled trials
Figure 28.29 (continued from previous page)

28.1 Which one of the following statements concerning vitamin By» is correct?
A. The cofactor form is vitamin Bjo itself.
B. It is involved in the transfer of amino groups.
C. It requires a specific glycoprotein for its absorp-
tion.
D. It is present in plant products.
E. Its deficiency is most often caused by a lack of the vitamin in the diet.

A. can be enzymically formed from retinoic acid.
B. in its ester form is transported from the intestine to
the liver in chylomicrons.
C. is the light-absorbing portion of rhodopsin.
D. is phosphorylated and dephosphorylated during the visual cycle.
E. mediates most of the actions of the retinoids.

A. Chronic renal failure requires the oral administration of 1,25-dihydroxycholecalciferol.
B. It is required in the diet of individuals exposed to
sunlight.
C.25-Hydroxycholecalciferol is the active form of the vitamin.
- D.Vitamin D opposes the effect of parathyroid hor-
mone.
- E.A deficiency in vitamin D results in an increased secretion of calcitonin.

A. plays an essential role in preventing thrombosis.
B. increases the coagulation time in newborn infants
with hemorrhagic disease.
C. is present in high concentration in cow or breast milk.
D. is synthesized by intestinal bacteria.
E. is a water-soluble vitamin.
28.5 Deficiency of which vitamin results in: beri-beri; scurvy; pellagra; night blindness; rickets or osteomalacia; pernicious anemia; megaloblastic anemia; bleeding?
Correct answer = C. Vitamin B,2 requires intrinsic factor for its absorption. A deficiency of vitamin By2 is most often caused by a lack of intrinsic factor. However, high does of the vitamin, given orally, are sufficiently absorbed to serve as treatment for pernicious anemia. The cofactor forms are methylcobalamin and deoxyadenosylcobalamin. Vitamin Bg, not vitamin By, is involved in the transfer of amino groups. By. is found in food derived from animal sources.
Correct answer = B. Retinyl esters are incorporated into chylomicrons. Retinoic acid cannot be reduced to retinol. Retinal, the aldehyde form of retinol, is the chromophore for rhodopsin. Retinal is photoisomerized during the visual cycle. Retinoic acid, not retinol, is the most important retinoid.
Correct answer = A. Renal failure results in the decreased ability to form the active form of the vitamin, which must be supplied. The vitamin is not required in individuals exposed to sunlight. 1,25-Dihydroxycholecalciferol is the active form of the vitamin. Vitamin D and parathyroid hormone both increase serum calcium. A deficiency of vitamin D decreases the secretion of calcitonin.
Correct answer = D. Vitamin K is essential for clot formation, decreases coagulation time, and is present in low concentrations in milk. It is one of four fat-soluble vitamins.
Thiamin; vitamin C; niacin; vitamin A, vitamin D; vitamin B12 (due primarily to a deficiency of intrinsic factor), both B12 and folate; vitamin K.

Nucleic acids are required for the storage and expression of genetic information. There are two chemically distinct types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA, see Chapter 30). DNA, the repository of genetic information, is present not only in chromosomes in the nucleus of eukaryotic organisms, but also in mitochondria and the chloroplasts of plants. Prokaryotic cells, which lack nuclei, have a single chromosome, but may also contain nonchromosomal DNA in the form of plasmids. The genetic information found in DNA is copied and transmitted to daughter cells through DNA replication. The DNA contained in a fertilized egg encodes the information that directs the development of an organism. This development may involve the production of billions of cells. Each cell is specialized, expressing only those functions that are required for it to perform its role in maintaining the organism. Therefore, DNA must be able to not only replicate precisely each time a cell divides, but also to have the information that it contains be selectively expressed. Transcription (RNA synthesis) is the first stage in the expression of genetic information (see Chapter 30). Next, the code contained in the nucleotide sequence of messenger RNA molecules is translated (protein synthesis, see Chapter 31), thus completing gene expression. The regulation of gene expression is discussed in Chapter 32.
The flow of information from DNA to RNA to protein is termed the "central dogma" of molecular biology (Figure 29.1), and is descriptive of all organisms, with the exception of some viruses that have RNA as the repository of their genetic information.

A. DNA chain with the nucleotide sequence shown written in the 5'> 3' direction. A 3' -5'-phosphodiester bond is shown highlighted in the blue box, and the deoxyribose-phosphate backbone is shaded in yellow. B. The DNA chain written in a more stylized form, emphasizing the ribose—phosphate backbone. C. A simpler representation of the nucleotide sequence. D. The simplest (and most common) representation, with the abbreviations for the bases written in the conventional 5'3' direction.

DNA is a polymer of deoxyribonucleoside monophosphates covalently linked by 3'+5'-phosphodiester bonds. With the exception of a few viruses that contain single-stranded (ss) DNA, DNA exists as a doublestranded (ds) molecule, in which the two strands wind around each other, forming a double helix. In eukaryotic cells, DNA is found associated with various types of proteins (known collectively as nucleoprotein) present in the nucleus, whereas in prokaryotes, the protein-DNA complex is present in a nonmembrane-bound region known as the nucleoid.

Phosphodiester bonds join the 3'-hydroxyl group of the deoxypentose of one nucleotide to the 5'-hydroxyl group of the deoxypentose of an adjacent nucleotide through a phosphate group (Figure 29.2). The resulting long, unbranched chain has polarity, with both a 5'-end (the end with the free phosphate) and a 3'-end (the end with the free hydroxyl) that are not attached to other nucleotides. The bases located along the resulting deoxyribose—phosphate backbone are, by convention, always written in sequence from the 5'-end of the chain to the 3'-end. For example, the sequence of bases in the DNA shown
in Figure 29.2 is read "thymine, adenine, cytosine, guanine" (5'-TACG-3'). Phosphodiester linkages between nucleotides (in DNA or RNA) can be cleaved hydrolytically by chemicals, or hydrolyzed enzymatically by a family of nucleases: deoxyribonucleases for DNA and ribonucleases for RNA. [Note: Only RNA is cleaved by alkali.]

In the double helix, the two chains are coiled around a common axis called the axis of symmetry. The chains are paired in an antiparallel manner, that is, the 5'-end of one strand is paired with the 3'-end of the other strand (Figure 29.3). In the DNA helix, the hydrophilic deoxyribose—phosphate backbone of each chain is on the outside of the molecule, whereas the hydrophobic bases are stacked inside. The overall structure resembles a twisted ladder. The spatial relationship between the two strands in the helix creates a major (wide) groove and a minor (narrow) groove. These grooves provide access for the binding of regulatory proteins to their specific recognition sequences along the DNA chain. Certain anticancer drugs, such as dactinomycin (actinomycin D), exert their cytotoxic effect by intercalating into the narrow groove of the DNA double helix, thus interfering with DNA and RNA synthesis."
1. Base pairing: The bases of one strand of DNA are paired with the bases of the second strand, so that an adenine is always paired with a thymine and a cytosine is always paired with a guanine. [Note: The base pairs are perpendicular to the axis of the helix (see Figure 29.3).] Therefore, one polynucleotide chain of the DNA double helix is always the complement of the other. Given the sequence of bases on one chain, the sequence of bases on the complementary chain can be determined (Figure 29.4). [Note: The specific base pairing in DNA leads to the Chargaff Rule: In any sample of dsDNA, the amount of adenine equals the amount of thymine, the amount of guanine equals the amount of cytosine, and the total amount of purines equals the total amount of pyrimidines.] The base pairs are held together by hydrogen bonds: two between A and T and three between G and C (Figure 29.5). These hydrogen bonds, plus the hydrophobic interactions between the stacked bases, stabilize the structure of the double helix.

strands of the double helix separate when hydrogen bonds between the paired bases are disrupted. Disruption can occur in the laboratory if the pH of the DNA solution is altered so that the nucleotide bases ionize, or if the solution is heated. [Note: Phosphodiester bonds are not broken by such treatment.] When DNA is heated, the temperature at which one half of the helical structure is lost is defined as the melting temperature (Tm). The loss of helical structure in DNA, called denaturation, can be monitored by measuring its absorbance at 260 nm. [Note: ssDNA has a higher relative absorbance at this wavelength than does dsDNA.] Because there are three hydrogen bonds between G and C but
'See Chapter 39 in Lippincott's Illustrated Reviews: Pharmacology
: . -: : for a discussion of the anticancer drug, actinomycin D.

DNA double helix, illustrating some of its major structural features.

Melting temperatures (Tm) of DNA molecules with different nucleotide compositions. (At a wavelength of 260 nm, single-stranded DNA has a higher relative absorbance than does double-stranded DNA.)
only two between A and T, DNA that contains high concentrations of A and T denatures at a lower temperature than Gand C-rich DNA (Figure 29.6). Under appropriate conditions, complementary DNA strands can reform the double helix by the process called renaturation (or reannealing).
3.
Structural forms of the double helix: There are three major structural forms of DNA: the B form, described by Watson and Crick in 1953, the A form, and the Z form. The B form is a right-handed helix with ten residues per 360° turn of the helix, and with the planes of the bases perpendicular to the helical axis. Chromosomal DNA is thought to consist primarily of B-DNA (Figure 29.7 illustrates a space-filling model of B-DNA). The A form is produced by moderately dehydrating the B form. It is also a right-handed helix, but there are 11 base pairs per turn, and the planes of the base pairs are tilted 20° away from the perpendicular to the helical axis. The conformation found in DNA-RNA hybrids or RNA-RNA double-stranded regions is probably very close to the A form. Z-DNA is a left-handed helix that contains about 12 base pairs per turn (see Figure 29.7). [Note: The deoxyribose—phosphate backbone "zigzags," hence, the name "Z'"-DNA.] Stretches of Z-DNA can occur naturally in regions of DNA that have a sequence of alternating purines and pyrimidines, for example, poly GC. Transitions between the B and Z helical forms of DNA may play a role in regulating gene expression.

Each chromosome in the nucleus of a eukaryote contains one long, linear molecule of dsDNA, which is bound to a complex mixture of proteins (histone and non-histone, see p. 409) to form chromatin. Eukaryotes have closed, circular DNA molecules in their mitochondria, as do plant chloroplasts. A prokaryotic organism typically contains a single, double-stranded, supercoiled, circular chromosome. Each prokaryotic chromosome is associated with non-histone proteins that can condense the DNA to form a nucleoid. In addition, most species of bacteria also contain small, circular, extrachromosomal DNA molecules called plasmids. Plasmid DNA carries genetic information, and undergoes replication that may or may not be synchronized to chromosomal division.?
Plasmids may carry genes that convey antibiotic resistance to the host bacterium, and may facilitate the transfer of genetic information from one bacterium to another.
[Note: The use of plasmids as vectors in recombinant DNA technology is described in Chapter 33.]
See Chapter 7 in Lippincott's Illustrated Reviews: Microbiology for a discussion of plasmids.

When the two strands of the DNA double helix are separated, each can serve as a template for the replication of a new complementary strand. This produces two daughter molecules, each of which contains two DNA strands with an antiparallel orientation (see Figure 29.3). This process is called semiconservative replication because, although the parental duplex is separated into two halves (and, therefore, is not "conserved" as an entity), each of the individual parental strands remains intact in one of the two new duplexes (Figure 29.8). The enzymes involved in the DNA replication process are template-directed polymerases that can synthesize the complementary sequence of each strand with extraordinary fidelity. The reactions described in this section were first known from studies of the bacterium Escherichia coli (E. coli), and the description given below refers to the process in prokaryotes. DNA synthesis in higher organisms is less well understood, but involves the same types of mechanisms. In either case, initiation of DNA replication commits the cell to continue the process until the entire genome has been replicated.

In order for the two strands of the parental double helical DNA to be replicated, they must first separate (or "melt") over a small region, because the polymerases use only ssDNA as a template. In prokaryotic organisms, DNA replication begins at a single, unique nucleotide sequence—a site called the origin of replication (Figure 29.9A). [Note: This is referred to as a consensus sequence, because the order of nucleotides is essentially the same at each site.] This site includes a short sequence composed almost exclusively of AT base pairs that facilitate melting. In eukaryotes, replication begins at multiple sites along the DNA helix (Figure 29.9B). Having multiple origins of replication provides a mechanism for rapidly replicating the great length of the eukaryotic DNA molecules.

As the two strands unwind and separate, they form a "V" where active synthesis occurs. This region is called the replication fork. It moves along the DNA molecule as synthesis occurs. Replication of dsDNA is bidirectional—that is, the replication forks move in opposite directions from the origin, generating a replication bubble (see Figure 29.9).
1. Proteins required for DNA strand separation: Initiation of DNA replication requires the recognition of the origin of replication by a group of proteins that form the prepriming complex. These proteins are responsible for maintaining the separation of the parental strands, and for unwinding the double helix ahead of the advancing replication fork. These proteins include the following:
a. DnaA protein: DnaA protein binds to specific nucleotide sequences at the origin of replication, causing short, tandemly arranged (one after the other) AT-rich regions in the origin to melt. Melting is ATP-dependent, and results in strand separation with the formation of localized regions of ssDNA.
Figure

Replication of DNA: origins and replication forks. A. Small prokaryotic circular DNA. B. Very long eukaryotic DNA.
b. DNA helicases: These enzymes bind to ssDNA near the replication fork, and then move into the neighboring doublestranded region, forcing the strands apart—in effect, unwinding the double helix. Helicases require energy provided by ATP (Figure 29.10). [Note: DnaB is the principal helicase of replication in E. coli. Its binding to DNA requires DnaC.]

Proteins responsible for maintaining the separation of the parental strands and unwinding the double helix ahead of the advancing replication fork.
c. Single-stranded DNA-binding (SSB) proteins: These proteins bind to the ssDNA generated by helicases (see Figure 29.10). They bind cooperatively—that is, the binding of one molecule of SSB protein makes it easier for additional molecules of SSB protein to bind tightly to the DNA strand. The SSB proteins are not enzymes, but rather serve to shift the equilibrium between dsDNA and ssDNA in the direction of the single-stranded forms. These proteins not only keep the two strands of DNA separated in the area of the replication origin, thus providing the single-stranded template required by polymerases, but also protect the DNA from nucleases that degrade ssDNA.
2. Solving the problem of supercoils: As the two strands of the double helix are separated, a problem is encountered, namely, the appearance of positive supercoils (also called supertwists) in the region of DNA ahead of the replication fork (Figure 29.11). The
accumulating positive supercoils interfere with further unwinding of the double helix. [Note: Supercoiling can be demonstrated by tightly grasping one end of a helical telephone cord while twisting the other end. If the cord is twisted in the direction of tightening the coils, the cord will wrap around itself in space to form positive supercoils. If the cord is twisted in the direction of loosening the coils, the cord will wrap around itself in the opposite direction to form negative supercoils.] To solve this problem, there is a group of enzymes called DNA topoisomerases, which are responsible for removing supercoils in the helix.

a. Type | DNA topoisomerases: These enzymes reversibly cut one strand of the double helix. They have both nuclease (strand-cutting) and ligase (strand-resealing) activities. They do not require ATP, but rather appear to store the energy from the phosphodiester bond they cleave, reusing the energy to reseal the strand (Figure 29.12). Each time a transient "nick" is created in one DNA strand, the intact DNA strand is passed through the break before it is resealed, thus relieving ("relaxing") accumulated supercoils. Type | topoisomerases relax negative supercoils (that is, those that contain fewer turns of the helix than relaxed DNA) in E. coli, and both negative and positive supercoils (that is, those that contain fewer or more turns of the helix than relaxed DNA) in eukaryotic cells.
b.
Type Il DNA topoisomerases: These enzymes bind tightly to the DNA double helix and make transient breaks in both strands. The enzyme then causes a second stretch of the DNA double helix to pass through the break and, finally, reseals the break (Figure 29.13). As a result, both negative and positive supercoils can be relieved by this ATP-requiring process. Type Il DNA topoisomerases are also required in both prokaryotes and eukaryotes for the separation of interlocked molecules of DNA following chromosomal replication. DNA gyrase, a Type II topoisomerase found in bacteria and plants, has the unusual property of being able to introduce negative supercoils into relaxed circular DNA using energy from the hydrolysis of ATP. This facilitates the future replication of DNA because the negative supercoils neutralize the positive supercoils introduced during opening of the double helix. It also aids in the transient strand separation required during transcription (see p. 417).
Anticancer agents, such as etoposide, target human topoisomerase II. Bacterial DNA gyrase is a unique target of a group of antimicrobial agents called quinolones, for example, ciprofloxacin.4

AION 8See Chapter 39 in Lippincott's Illustrated Reviews: Pharmacology for a (SRS) discussion of etoposide as an anticancer agent. oe? 4See Chapter 33 in Lippincott's Illustrated Reviews: Pharmacology for a discussion of the quinolones.

The DNA polymerases responsible for copying the DNA templates are only able to "read" the parental nucleotide sequences in the 3'5' direction, and they synthesize the new DNA strands only in the 5'3' (antiparallel) direction. Therefore, beginning with one parental double helix, the two newly synthesized stretches of nucleotide chains must grow in opposite directions—one in the 5'3' direction toward the replication fork and one in the 5'>3' direction away from the replication fork (Figure 29.14). This feat is accomplished by a slightly different mechanism on each strand.
1. Leading strand: The strand that is being copied in the direction of the advancing replication fork is called the leading strand and is synthesized continuously.
2. Lagging strand: The strand that is being copied in the direction away from the replication fork is synthesized discontinuously, with small fragments of DNA being copied near the replication fork. These short stretches of discontinuous DNA, termed Okazaki fragments, are eventually joined (ligated) to become a single, continuous strand. The new strand of DNA produced by this mechanism is termed the lagging strand.

DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a totally single-stranded template. Rather, they require an RNA primer—that is, a short, double-stranded region consisting of RNA base-paired to the DNA template, with a free hydroxyl group on the 3'-end of the RNA strand (Figure 29.15). This hydroxyl group serves as the first acceptor of a deoxynucleotide by action of DNA polymerase. [Note: Recall that glycogen synthase also requires a primer (see p. 126).]
- 1. Primase: A specific RNA polymerase, called primase (DnaG), synthesizes the short stretches of RNA (approximately ten nucleotides long) that are complementary and antiparallel to the DNA template. In the resulting hybrid duplex, the U in RNA pairs with A in DNA. As shown in Figure 29.16, these short RNA sequences are constantly being synthesized at the replication fork on the lagging strand, but only one RNA sequence at the origin of replication is required on the leading strand. The substrates for

Elongation of the leading and lagging strands.
this process are 5'-ribonucleoside triphosphates, and pyrophosphate is released as each ribonucleoside monophosphate is added through formation of a 3'-5' phosphodiester bond. [Note: The RNA primer is later removed as described on p. 405.]
- 2. Primosome: The addition of primase converts the prepriming complex of proteins required for DNA strand separation (see p. 399) to a primosome. The primosome makes the RNA primer required for leading strand synthesis, and initiates Okazaki fragment formation in lagging strand synthesis. As with DNA synthesis, the direction of synthesis of the primer is 5'33'.

Prokaryotic (and eukaryotic) DNA polymerases elongate a new DNA strand by adding deoxyribonucleotides, one at a time, to the 3'end of the growing chain (see Figure 29.16). The sequence of nucleotides that are added is dictated by the base sequence of the template strand with which the incoming nucleotides are paired.
- 1. DNA polymerase Ill: DNA chain elongation is catalyzed by DNA polymerase Ill. Using the 3'-hydroxyl group of the RNA primer as the acceptor of the first deoxyribonucleotide, DNA polymerase III begins to add nucleotides along the single-stranded template that specifies the sequence of bases in the newly synthesized chain. DNA polymerase Ill is a highly "processive" enzyme—that is, it remains bound to the template strand as it moves along, and does not diffuse away and then rebind before adding each new nucleotide. The processivity of DNA polymerase III is the result of

3'55'-Exonuclease activity enables DNA polymerase III to "proofread" the newly synthesized DNA strand.
its B subunit forming a ring that encircles and moves along the template strand of the DNA, thus serving as a sliding DNA clamp. The new strand grows in the 5'-3' direction, antiparallel to the parental strand (see Figure 29.16). The nucleotide substrates are 5'-deoxyribonucleoside triphosphates. Pyrophosphate (PPi) is released when each new deoxynucleoside monophosphate is added to the growing chain (see Figure 29.15). Hydrolysis of PP; to 2P; means that a total of two high-energy bonds are used to drive the addition of each deoxynucleotide.
The production of PP; with subsequent hydrolysis to 2P;, as seen in the synthesis of DNA and RNA, is a common theme in biochemistry. Removal of the PP; product drives a reaction in the forward direction, making it essentially irreversible.
All four deoxyribonucleoside triphosphates (dATP, dTTP, dCTP, and dGTP) must be present for DNA elongation to occur. If one of the four is in short supply, DNA synthesis stops when that nucleotide is depleted.
- 2. Proofreading of newly synthesized DNA: It is highly important for the survival of an organism that the nucleotide sequence of DNA be replicated with as few errors as possible. Misreading of the template sequence could result in deleterious, perhaps lethal, mutations. To ensure replication fidelity, DNA polymerase III has, in addition to its 5'+3' polymerase activity, a "proofreading" activity

(3'45' exonuclease, Figure 29.17). As each nucleotide is added to the chain, DNA polymerase III checks to make certain the added nucleotide is, in fact, correctly matched to its complementary base on the template. If it is not, the 3'+5' exonuclease activity corrects the mistake. [Note: The enzyme requires an improperly basepaired 3'-hydroxy terminus and, therefore, does not degrade correctly paired nucleotide sequences.] For example, if the template base is cytosine and the enzyme mistakenly inserts an adenine instead of a guanine into the new chain, the 3'+5' exonuclease activity hydrolytically removes the misplaced nucleotide. The 53' polymerase activity then replaces it with the correct nucleotide containing guanine (see Figure 29.17). [Note: The proofreading exonuclease activity requires movement in the 3'>5' direction, not 5'3' like the polymerase activity. This is because the excision must be done in the reverse direction from that of synthesis.]

DNA polymerase III continues to synthesize DNA on the lagging strand until it is blocked by proximity to an RNA primer. When this occurs, the RNA is excised and the gap filled by DNA polymerase I.
1.
5'+3' Exonuclease activity: In addition to having the 5'>3' polymerase activity that synthesizes DNA, and the 3'5' exonuclease activity that proofreads the newly synthesized DNA chain like DNA polymerase III, DNA polymerase | also has a 5'>3' exonuclease activity that is able to hydrolytically remove the RNA primer. [Note: These activities are exonucleases because they remove one nucleotide at a time from the end of the DNA chain, rather than cleaving the chain internally as do the endonucleases (Figure 29.18).] First, DNA polymerase | locates the space ("nick") between the 3'-end of the DNA newly synthesized by DNA polymerase III and the 5'-end of the adjacent RNA primer. Next, DNA polymerase | hydrolytically removes the RNA nucleotides "ahead" of itself, moving in the 5'>3' direction (5'>3' exonuclease activity). As it removes the RNA, DNA polymerase | replaces it with deoxyribonucleotides, synthesizing DNA in the 5'-3' direction (5'33' polymerase activity). As it synthesizes the DNA, it also "proofreads" the new chain using 3-45' exonuclease activity. This removal/synthesis/proofreading continues, one nucleotide at a time, until the RNA primer is totally degraded and the gap is filled with DNA (Figure 29.19).

. Differences between 5'->3' and 3'5' exonucleases: The 5'>3' exonuclease activity of DNA polymerase | differs from the 35' exonuclease used by both DNA polymerase | and III in two important ways. First, 53' exonuclease can remove one nucleotide at a time from a region of DNA that is properly base-paired. The nucleotides it removes can be either ribonucleotides or deoxyribonucleotides. Second, 5'>3' exonuclease can also remove groups of altered nucleotides in the 5'3' direction, removing from one to ten nucleotides at a time. This ability is important in the repair of some types of damaged DNA.

Removal of RNA primer and filling of the resulting "gaps" by DNA polymerase I.
pe

The final phosphodiester linkage between the 5'-phosphate group on the DNA chain synthesized by DNA polymerase III and the 3'hydroxyl group on the chain made by DNA polymerase | is catalyzed by DNA ligase (Figure 29.20). The joining of these two stretches of DNA requires energy, which in most organisms is provided by the cleavage of ATP to AMP + PP;.

The process of eukaryotic DNA replication closely follows that of prokaryotic DNA synthesis. Some differences, such as the multiple origins of replication in eukaryotic cells versus single origins of replication in prokaryotes, have already been noted. Eukaryotic single-stranded DNA-binding proteins and ATP-dependent DNA helicases have been identified, whose functions are analogous to those of the prokaryotic enzymes previously discussed. In contrast, RNA primers are removed by RNase H and FEN1 rather than by a DNA polymerase.

; Formation of a phosphodiester bond by DNA ligase. [Note: AMP is linked to ligase then the 5'phosphate and released.]
The events surrounding eukaryotic DNA replication and cell division (mitosis) are coordinated to produce the cell cycle (Figure 29.21). The period preceding replication is called the G; phase (Gap1). DNA replication occurs during the S (synthesis) phase. Following
DNA synthesis, there is another period (G2 phase, or Gap2) before mitosis (M). Cells that have stopped dividing, such as mature neurons, are said to have gone out of the cell cycle into the Go phase. Cells can leave the Go phase and reenter the early G; phase to resume division. [Note: The cell cycle is controlled at a series of "checkpoints" that prevent entry into the next phase of the cycle until the preceding phase has been completed. Two key classes of proteins that control the progress of a cell through the cell cycle are the cyclins and cyclin-dependent kinases (Cdk).]

At least five key eukaryotic DNA polymerases have been identified and categorized on the basis of molecular weight, cellular location, sensitivity to inhibitors, and the templates or substrates on which they act. They are designated by Greek letters rather than by Roman numerals (Figure 29.22).
1. Pol a: Pol a is a multisubunit enzyme. One subunit has primase activity, which initiates strand synthesis on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. The primase subunit synthesizes a short RNA primer that is extended by the pol a 5'>3' polymerase activity, generating a short piece of DNA.
2. Pol e and pol 8: Pol is thought to be recruited to complete DNA synthesis on the leading strand whereas pol 5 elongates the Okazaki fragments of the lagging strand, each using 3'45' exonuclease activity to proofread the newly synthesized DNA. [Note: DNA polymerase e€ associates with the protein, proliferating cell nuclear antigen (PCNA), which serves as a sliding DNA clamp in much the same way the B subunit of DNA polymerase III does in E. coli, thus ensuring high processivity.].
3. Pol B and pol y: Po! B is involved in "gap filling" in DNA repair (see below). Pol yreplicates mitochondrial DNA.

Telomeres are complexes of noncoding DNA plus proteins located at the ends of linear chromosomes. They maintain the structural integrity of the chromosome, preventing attack by nucleases, and allow repair systems to distinguish a true end from a break in dsDNA. In humans, telomeric DNA consists of several thousand tandem repeats of a noncoding hexameric sequence, AG3T2, base-paired to a complementary region of Cs and As. The GT-rich strand is longer than its CA complement, leaving ssDNA a few hundred nucleotides in length at the 3'-end. The single-stranded region is thought to fold back on itself, forming a loop structure that is stabilized by protein.
1. Telomere shortening: Eukaryotic cells face a special problem in replicating the ends of their linear DNA molecules. Following removal of the RNA primer from the extreme 5'-end of the lagging strand, there is no way to fill in the remaining gap with DNA. Consequently, in most normal human somatic cells, telomeres shorten with each successive cell division. Once telomeres are

Activities of eukaryotic DNA polymerases (pols). "3'5' exonuclease activity.
[Yircoc

Mechanism of action of telomerase.
shortened beyond some critical length, the cell is no longer able to divide and is said to be senescent. In germ cells and other stem cells, as well as in cancer cells, telomeres do not shorten and the cells do not senesce. This is a result of the presence of a ribonucleoprotein, telomerase, which maintains telomeric length in these cells.
- 2. Telomerase: This complex contains a protein that acts as a reverse transcriptase, and a short piece of RNA that acts as a template. The CA-rich RNA template base-pairs with the GT-rich, single-stranded 3'-end of telomeric DNA (Figure 29.23). The reverse transcriptase uses the RNA template to synthesize DNA in the usual 5'3' direction, extending the already longer 3'-end. Telomerase then translocates to the newly synthesized end, and the process is repeated. Once the GT-rich strand has been lengthened, primase can use it as a template to synthesize an RNA primer. The RNA primer is extended by DNA polymerase, and the primer is removed.
Telomeres may be viewed as mitotic clocks in that their length in most cells is inversely related to the number of times the cells have divided. The study of telomeres is providing insight into the biology of aging and cancer.

Reverse transcriptases, as seen with telomerase, are RNA-directed DNA polymerases. A reverse transcriptase is involved in the replication of retroviruses, such as human immunodeficiency virus (HIV).° These viruses carry their genome in the form of ssRNA molecules. Following infection of a host cell, the viral enzyme, reverse transcriptase, uses the viral RNA as a template for the 5'—3' synthesis of viral DNA, which then becomes integrated into host chromosomes. Reverse transcriptase activity is also seen with transposons, DNA elements that can move about the genome (see p. 461). In eukaryotes, such elements are transcribed to RNA, the RNA is used as a template for DNA synthesis by a reverse transcriptase encoded by the transposon, and the DNA is randomly inserted into the genome. [Note: Transposons that involve an RNA intermediate are called retrotransposons or retroposons.]

DNA chain growth can be blocked by the incorporation of certain nucleoside analogs that have been modified in the sugar portion of the nucleoside (Figure 29.24).° For example, removal of the hydroxyl
5See Chapter 38 in Lippincott's Illustrated Reviews: Microbiology for a discussion of retroviruses. ®See Chapter 38 in Lippincott's Illustrated Reviews: Pharmacology for a discussion of nucloside analogs.
group from the 3'-carbon of the deoxyribose ring as in 2',3'-dideoxyinosine (ddl, also known as didanosine), or conversion of the deoxyribose to another sugar such as arabinose, prevents further chain elongation. By blocking DNA replication, these compounds slow the division of rapidly growing cells and viruses. Cytosine arabinoside (cytarabine, or araC) has been used in anticancer chemotherapy, whereas adenine arabinoside (vidarabine, or araA) is an antiviral agent. Chemically modifying the sugar moiety, as seen in zidovudine (AZT, ZDV), also terminates DNA chain elongation. [Note: These drugs are generally supplied as nucleosides, which are then converted to the active nucleotides by cellular "salvage" enzymes (see p. 296).]

A typical human cell contains 46 chromosomes, whose total DNA is approximately 1m long! It is difficult to imagine how such a large amount of genetic material can be effectively packaged into a volume the size of a cell nucleus so that it can be efficiently replicated and its genetic information expressed. To do so requires the interaction of DNA with a large number of proteins, each of which performs a specific function in the ordered packaging of these long molecules of DNA. Eukaryotic DNA is associated with tightly bound basic proteins, called histones. These serve to order the DNA into fundamental structural units, called nucleosomes, that resemble beads on a string. Nucleosomes are further arranged into increasingly more complex structures that organize and condense the long DNA molecules into chromosomes that can be segregated during cell division. [Note: The complex of DNA and protein found inside the nuclei of eukaryotic cells is called chromatin.]

There are five classes of histones, designated H1, H2A, H2B, H3, and H4. These small proteins are positively charged at physiologic PH as a result of their high content of lysine and arginine. Because of their positive charge, they form ionic bonds with negatively charged DNA. Histones, along with positively charged ions such as Mg**, help neutralize the negatively charged DNA phosphate groups.
1. Nucleosomes: Two molecules each of H2A, H2B, H3, and H4 form the structural core of the individual nucleosome "beads." Around this core, a segment of the DNA double helix is wound nearly twice, forming a negatively supertwisted helix (Figure 29.25). [Note: The N-terminal ends of these histones can be acetylated, methylated, or phosphorylated. These reversible covalent modifications influence how tightly the histones bind to the DNA, thereby affecting the expression of specific genes (see p. 422).] Neighboring nucleosomes are joined by "linker" DNA approximately 50 base pairs long. Histone H1, of which there are several related species, is not found in the nucleosome core, but instead binds to the linker DNA chain between the nucleosome beads. H1 is the most tissue-specific and species-specific of the histones. It facilitates the packing of nucleosomes into the more compact structures.

Examples of nucleoside analogs that lack a 3'-hydroxyl group. [Note: ddl is converted to its active form (ddATP).]

Organization of human DNA, illustrating the structure of
nucleosomes.

Structural organization of eukaryotic DNA.
2. Higher levels of organization: Nucleosomes can be packed more tightly to form a polynucleosome (also called a nucleofilament). This structure assumes the shape of a coil, often referred to as a 30-nm fiber. The fiber is organized into loops that are anchored by a nuclear scaffold containing several proteins. Additional levels of organization lead to the final chromosomal structure (Figure 29.26).

In order to be replicated, the highly structured and constrained chromatin must be relaxed. Although the nucleosomes are displaced, dissociation of the nucleosome core from the DNA is incomplete, with all the parental histones remaining loosely associated with only one of the parental DNA strands. Synthesis of new histones occurs simultaneously with DNA replication, and nucleosomes containing the newly synthesized histones associate with only one of the new daughter helices. Therefore, the parental histone octamers are conserved.

Despite the elaborate proofreading system employed during DNA synthesis, errors—including incorrect base-pairing or insertion of one to a few extra nucleotides—can occur. In addition, DNA is constantly being subjected to environmental insults that cause the alteration or removal of nucleotide bases. The damaging agents can be either chemicals, for example, nitrous acid, or radiation, for example, ultraviolet light, which
can fuse two pyrimidines adjacent to each other in the DNA, and highenergy ionizing radiation, which can cause double-strand breaks. Bases are also altered or lost spontaneously from mammalian DNA at a rate of many thousands per cell per day. If the damage is not repaired, a permanent change (mutation) is introduced that can result in any of a number of deleterious effects, including loss of control over the proliferation of the mutated cell, leading to cancer. Luckily, cells are remarkably efficient at repairing damage done to their DNA. Most of the repair systems involve recognition of the damage (lesion) on the DNA, removal or excision of the damage, replacement or filling the gap left by excision using the sister strand as a template for DNA synthesis, and ligation. These repair systems thus perform excision repair, with the removal of one to tens of nucleotides. [Note: Repair reduces the error rate from one in ten million bases to one in a billion.]

Sometimes replication errors escape the proofreading function during DNA synthesis, causing a mismatch of one to several bases. In E. coli, mismatch repair is mediated by a group of proteins known as the Mut proteins (Figure 29.27). Homologous proteins are present in humans.
1. Identification of the mismatched strand: When a mismatch occurs, the Mut proteins that identify the mispaired nucleotide(s) must be able to discriminate between the correct strand and the strand with the mismatch. Discrimination is based on the degree of methylation. GATC sequences, which are found approximately once every thousand nucleotides, are methylated on the adenine residue. This methylation is not done immediately after synthesis, so the newly synthesized DNA is hemimethylated (that is, the parental strand is methylated but the daughter strand is not). The methylated parental strand is assumed to be correct, and it is the daughter strand that gets repaired. [Note: The exact mechanism by which the daughter strand is identified in eukaryotes is not yet known.]
2. Repair of damaged DNA: When the strand containing the mismatch is identified, an endonuclease nicks the strand and the mismatched nucleotide(s) is/are removed by an exonuclease. Additional nucleotides at the 5'and 3'-ends of the mismatch are also removed. The gap left by removal of the nucleotides is filled, using the sister strand as a template, by a DNA polymerase. The 3'-hydroxyl of the newly synthesized DNA is joined to the 5'-phosphate of the remaining stretch of the original DNA strand by DNA ligase (see p. 406).
Mutation to the proteins involved in mismatch repair in humans is associated with hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome. With HNPCC, there is an increased risk for developing colon cancer (as well as other cancers), but only about 5% of all colon cancer is the result of mutations in mismatch repair.

Nucleotide excision of dimers in DNA.
Figure pyrimidine
repair E. coli

Exposure of a cell to UV light can result in the covalent joining of two adjacent pyrimidines (usually thymines), producing a dimer. These thymine dimers prevent DNA polymerase from replicating the DNA strand beyond the site of dimer formation. Thymine dimers are excised in bacteria by UvrABC proteins in a process known as nucleotide excision repair as illustrated in Figure 29.28. A related pathway involving XP proteins is present in humans.
1.
Recognition and excision of dimers by UV-specific endonuclease: First, a UV-specific endonuclease (called uvrABC excinuclease) recognizes the dimer, and cleaves the damaged strand on both the 5'-side and 3'-side of the dimer. A short oligonucleotide containing the dimer is released, leaving a gap in the DNA strand that formerly contained the dimer. This gap is filled in using the same process described previously.
2.
UV radiation and cancer: Pyrimidine dimers can be formed in the skin cells of humans exposed to unfiltered sunlight. In the rare genetic disease xeroderma pigmentosum (XP), the cells cannot repair the damaged DNA, resulting in extensive accumulation of mutations and, consequently, early and numerous skin cancers (Figure 29.29). XP can be caused by defects in any of the several genes that code for the XP proteins required for nucleotide excision repair of UV damage in humans.

The bases of DNA can be altered, either spontaneously, as is the case with cytosine, which slowly undergoes deamination (the loss of its amino group) to form uracil, or by the action of deaminating or alkylating compounds. For example, nitrous acid, which is formed by the cell from precursors, such as the nitrosamines, nitrites, and nitrates, is a potent compound that deaminates cytosine, adenine (to hypoxanthine), and guanine (to xanthine). Bases can also be lost spontaneously. For example, approximately 10,000 purine bases are lost this way per cell per day. Lesions involving base alterations or loss can be corrected by base excision repair (Figure 29.30).
1. Removal of abnormal bases: Abnormal bases, such as uracil, which can occur in DNA either by deamination of cytosine or improper use of dUTP instead of dTTP during DNA synthesis, are recognized by specific glycosylases that hydrolytically cleave them from the deoxyribose—phosphate backbone of the strand. This leaves an apyrimidinic site (or apurinic, if a purine was removed), both referred to as AP sites.
2. Recognition and repair of an AP site: Specific AP-endonucleases recognize that a base is missing and initiate the process of excision and gap-filling by making an endonucleolytic cut just to the 5'-side of the AP site. A deoxyribose phosphate lyase removes the single, base-free, sugar phosphate residue. A DNA polymerase and DNA ligase complete the repair process.

High-energy radiation or oxidative free radicals (see p. 148) can cause double-strand breaks in DNA, which are potentially lethal to the cell. Such breaks also occur naturally during gene rearrangements. dsDNA breaks cannot be corrected by the previously described strategy of excising the damage on one strand and using the remaining strand as a template for replacing the missing nucleotide(s). Instead, they are repaired by one of two systems. The first is nonhomologous end-joining repair, in which the ends of two DNA fragments are brought together by a group of proteins that effect their religation. However, some DNA is lost in the process. Consequently, this mechanism of repair is error prone and mutagenic. Defects in this repair system are associated with a predisposition to cancer and immunodeficiency syndromes. The second repair system, homologous recombination repair, uses the enzymes that normally perform genetic recombination between homologous chromosomes during meiosis. This system is much less error prone than nonhomologous end-joining joining because any DNA that was lost is replaced using homologous DNA as a template.

DNA contains many deoxyribonucleoside monophosphates covalently linked by 3'->5'-phosphodiester bonds (Figure 29.31). The resulting long, unbranched chain has polarity, with both a 5'-end and a 3'-end. The sequence of nucleotides is read 5'>3'. DNA exists as a doublestranded molecule, in which the two chains are paired in an antiparallel manner, and wind around each other, forming a double helix. Adenine pairs with thymine and cytosine pairs with guanine. Each strand of the double helix serves as a template for constructing a complementary daughter strand (semiconservative replication). DNA replication occurs in the S phase of the cell cycle and begins at the origin of replication. The strands are separated locally, forming two replication forks. Replication of dsDNA is bidirectional. Helicase unwinds the double helix. As the two strands of the double helix are separated, positive supercoils are produced in the region of DNA ahead of the replication fork. DNA topoisomerases Types | and Il remove supercoils. DNA polymerases synthesize new DNA strands only in the 5'>3' direction. Therefore, one of the newly synthesized stretches of nucleotide chains must grow in the 5'-3' direction toward the replication fork (leading strand), and one in the 5'3' direction away from the replication fork (lagging strand). DNA polymerases require a primer. The primer for de novo DNA synthesis is a short stretch of RNA synthesized by primase. The leading strand only needs one RNA primer, whereas the lagging strand needs many. In E. coli DNA chain elongation is catalyzed by DNA polymerase Ill, using 5'-deoxyribonucleoside triphosphates as substrates. The enzyme "proofreads" the newly synthesized DNA, removing terminal mismatched nucleotides with its 3'—5' exonuclease activity. RNA primers are removed by DNA polymerase I, using its 5'>3' exonuclease activity. This enzyme fills the gaps with DNA, proofreading as it synthesizes. The final phosphodi-

Key concept map for DNA structure, replication, and repair. (continued on next page)
Figure 29.31 (continued from previous page)
Key HR
concept map for DNA structure, replication, and repair. NHEJ = nonhomologous end-joining;
= homologous recombination.

ester linkage is catalyzed by DNA ligase. There are at least five eukaryotic DNA polymerases. Pol « is a multisubunit enzyme, one subunit of which is a primase. Pol « 5'3' polymerase activity adds a short piece of DNA to the RNA primer. Pol ¢ is thought to complete DNA synthesis on the leading strand whereas pol 6 elongates each lagging strand fragment. Both ¢€ and 6 use 3'5' exonuclease activity to proofread. Pol B is involved with DNA repair, and pol y replicates mitochondrial DNA. Nucleoside analogs containing modified sugars can be used to block DNA chain growth. They are useful in anticancer and antiviral chemotherapy. Telomeres are stretches of highly repetitive DNA complexed with protein that protect the ends of linear chromosomes. As most cells divide and age, these sequences are shortened, contributing to senescence. In cells that do not senesce (for example, germline and cancer cells), telomerase employs its enzyme component reverse transcriptase to extend the telomeres, using its RNA as a template. There are five classes of positively charged histone proteins. Two each of histones H2A, H2B, H3, and H4 form a structural core around which DNA is wrapped creating a nucleosome. The DNA connecting the nucleosomes, called linker DNA, is bound to histone H1. Nucleosomes can be packed more tightly to form a nucleofilament. Additional levels of organization create a chromosome. Most DNA damage can be corrected by excision repair involving recognition and removal of the damage by repair proteins, followed by replacement by DNA polymerase and joining by ligase. Ultraviolet light can cause thymine dimers that are recognized and removed by uvrABC proteins of nucleotide excision repair. Defects in the XP proteins needed for thymine dimer repair in humans result in xeroderma pigmentosum. Mismatched bases are repaired by a similar process of recognition and removal by Mut proteins in E. coli. The extent of methylation is used for strand identification in prokaryotes. Defective mismatch repair by homologous proteins in humans is associated with hereditary nonpolyposis colorectal cancer. Abnormal bases (such as uracil) are removed by glycosylases in base excision repair, and the sugar phosphate

at the AP site is cut out. Double-strand breaks in DNA are repaired by nonhomologous end-joining (error prone) and homologous recombination.

29.1 A 10-year-old girl is brought to the dermatologist by her parents. She has many freckles on her face, neck, arms, and hands, and the parents report that she is unusually sensitive to sunlight. Two basal cell carcinomas are identified on her face. Which of the following processes is most likely to be defective in this patient?
A. Repair of double-strand breaks.
B. Removal of mismatched bases from the 3'-end of Okazaki fragments.
C. Removal of pyrimidine dimers from DNA.
D. Removal of uracil from DNA.
29.2 Telomeres are complexes of DNA and protein that protect the ends of linear chromosomes. In most normal human somatic cells, telomeres shorten with each division. In stem cells and in cancer cells, however, telomeric length is maintained. In the synthesis of telomeres:
A. telomerase, a ribonucleoprotein, provides both the RNA and the polymerase needed for synthesis.
B. the RNA of telomerase serves as a primer.
C. the polymerase of telomerase is a DNA-directed
DNA polymerase.
D. the shorter, 3'5' strand gets extended.
E. the direction of synthesis is 3'>5'.
29.3 While studying the structure of a small gene that was
recently sequenced during the Human Genome Project, an investigator notices that one strand of the DNA molecule contains 20 As, 25 Gs, 30 Cs, and 22 Ts. How many of each base is found in the complete double-stranded molecule?
A. A= 40, G= 50, C = 60, T = 44.
B. A= 44, G=60, C = 50, T = 40.
C.A=45, G=45,C = 52, T=52.
D. A=50, G = 47, C= 50, T = 47.
E. A= 42, G=55, C = 55, T = 42.

specifically be
determined by
measuring the
incorpo- ration of radiolabeled:
A. leucine.
B. phosphate.
C. ribose
D. thymidine
E.
uracil
Correct answer = C. The sensitivity to sunlight, extensive freckling on parts of the body exposed to the sun, and presence of skin cancer at a young age indicates that the patient most likely suffers from xeroderma pigmentosum. These patients are deficient in any one of several XP proteins required for nucleotide excision repair of ultraviolet-damaged DNA. Double-strand breaks are repaired by nonhomologous end-joining or homologous recombination. Uracil is removed from damaged DNA molecules by a specific glycosylase.
Correct answer = A. Telomerase is a ribonucleoprotein particle required for telomere maintenance. Telomerase contains an RNA that serves as the template, not the primer, for the synthesis of telomeric DNA by the reverse transcriptase of telomerase. As a reverse transcriptase, it synthesizes DNA using its RNA template and so is an RNA-directed DNA polymerase. The direction of synthesis, as with all DNA synthesis, is 5'3', and it is the already longer 5'—3' strand that gets extended.
Correct answer = E. The two DNA strands are complementary to each other, with A base-paired with T, and G base-paired with C. So, for example, the 20 As on the first strand would be paired with 20 Ts on the second strand, the 25 Gs on the first strand would be paired with 25 Cs on the second strand, and so forth. When these are all added together, the correct numbers of each base are indicated in choice E. Notice that, in the correct answer, A= T and G=C.

Correct answer = D. Thymidine is incorporated only into DNA, and thus would reflect the extent of DNA synthesis. Leucine is an amino acid, not a nucleotide base. Phosphate is not exclusive to DNA. Ribose is found in RNA, whereas deoxyribose is found in DNA. Uracil is normally found only in RNA. If present in DNA, likely as a result of deamination of cytosine, it is removed by uracil glycosylase during base excision repair.

The genetic master plan of an organism is contained in the sequence of deoxyribonucleotides in its deoxyribonucleic acid (DNA). However, it is through the ribonucleic acid (RNA)—the "working copies" of the DNA— that the master plan is expressed (Figure 30.1). The copying process, during which a DNA strand serves as a template for the synthesis of RNA, is called transcription. Transcription produces messenger RNAs that are translated into sequences of amino acids (polypeptide chains or proteins), and ribosomal RNAs, transfer RNAs, and additional small RNA molecules that perform specialized structural, catalytic, and regulatory functions and are not translated, that is, they are noncoding RNAs (ncRNAs). The final product of gene expression, therefore, can be RNA or protein, depending upon the gene. A central feature of transcription is that it is highly selective. For example, many transcripts are made of some regions of the DNA. In other regions, few or no transcripts are made. This selectivity is due, at least in part, to signals embedded in the nucleotide sequence of the DNA. These signals instruct the RNA polymerase where to start, how often to start, and where to stop transcription. A variety of regulatory proteins is also involved in this selection process. The biochemical differentiation of an organism's tissues is ultimately a result of the selectivity of the transcription process. [Note: This selectivity of transcription is in contrast to the "all or none" nature of genomic replication.] Another important feature of transcription is that many RNA transcripts that initially are faithful copies of one of the two DNA strands may undergo various modifications, such as terminal additions, base modifications, trimming, and internal segment removal, which convert the inactive primary transcript into a functional molecule.

There are three major types of RNA that participate in the process of protein synthesis: ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). Like DNA, these three types of RNA are unbranched polymeric molecules composed of nucleoside monophosphates joined together by phosphodiester bonds (see p. 396). However, they differ from DNA in several ways, for example, they are considerably

Expression of genetic information by transcription. [Note: RNAs shown are eukaryotic.] me-'Gppp = 7-methylguanosine triphosphate "cap," AAA = poly-A tail, each described on p. 418.

A. Characteristic tRNA secondary structure. B. Folded (tertiary) tRNA structure found in cells. D = dihydrouracil. '¥ = pseudouracil.
smaller than DNA, contain ribose instead of deoxyribose and uracil instead of thymine, and exist as single strands that are capable of folding into complex structures. The three major types of RNA also differ from each other in size, function, and special structural modifications. [Note: In eukaryotes, additional small ncRNA molecules found in the nucleolus (SnoRNAs) and nucleus (snRNAs) perform specialized functions as described on pp. 425 and 426.]

rRNAs are found in association with several proteins as components of the ribosomes—the complex structures that serve as the sites for protein synthesis (see p. 436). There are three distinct size species of rRNA (23S, 16S, and 5S) in prokaryotic cells (Figure 30.2). In the eukaryotic cytosol, there are four rRNA species (28S, 18S, 5.8S, and 5S). [Note: "S" is the Svedberg unit, which is related to the molecular weight and shape of the compound.] Together, rRNAs make up about 80% of the total RNA in the cell. [Note: Some RNAs function as catalysts, for example, rRNA in protein synthesis (see p. 439). RNA with catalytic activity is termed a "ribozyme".]

tRNAs are the smallest (4S) of the three major types of RNA molecules. There is at least one specific type of tRNA molecule for each of the 20 amino acids commonly found in proteins. Together, tRNAs make up about 15% of the total RNA in the cell. The tRNA molecules contain a high percentage of unusual bases (for example, dihydrouracil, see Figure 22.2, p. 292) and have extensive intrachain base-pairing (Figure 30.3) that leads to characteristic secondary and tertiary structure. Each tRNA serves as an "adaptor" molecule that carries its specific amino acid—covalently attached to its 3'-end—to the site of protein synthesis. There it recognizes the genetic code sequence on an mRNA, which specifies the addition of its amino acid to the growing peptide chain (see p. 432).

mRNA comprises only about 5% of the RNA in the cell, yet is by far the most heterogeneous type of RNA in size and base sequence. The mRNA carries genetic information from the nuclear DNA to the cytosol, where it is used as the template for protein synthesis. If the mRNA carries information from more than one gene, it is said to be polycistronic. Polycistronic mRNA is characteristic of prokaryotes. If the mRNA carries information from just one gene, it is said to be monocistronic and is characteristic of eukaryotes. In addition to the protein coding regions that can be translated, mRNA contains untranslated regions at its 5'and 3'-ends (Figure 30.4). Special structural characteristics of eukaryotic (but not prokaryotic) MRNA include a long sequence of adenine nucleotides (a "poly-A tail") on the 3'-end of the RNA chain, plus a "cap" on the 5'-end consisting of a molecule of 7-methylguanosine attached "backward" (5'—5') through a triphosphate linkage as shown in Figure 30.4. The mechanisms for modifying MRNA to create these special structural characteristics are discussed on p. 425.

The structure of RNA polymerase, the signals that control transcription, and the varieties of modification that RNA transcripts can undergo differ among organisms, and particularly from prokaryotes to eukaryotes. Therefore, the discussions of prokaryotic and eukaryotic transcription are presented separately.

In bacteria, one species of RNA polymerase synthesizes all of the RNA except for the short RNA primers needed for DNA replication (RNA primers are synthesized by a specialized enzyme, primase, see p. 402). RNA polymerase is a multisubunit enzyme that recognizes a nucleotide sequence (the promoter region) at the beginning of a length of DNA that is to be transcribed. It next makes a complementary RNA copy of the DNA template strand, and then recognizes the end of the DNA sequence to be transcribed (the termination region). RNA is synthesized from its 5'-end to its 3'-end, antiparallel to its DNA template strand (see p. 397). The template is copied as it is in DNA synthesis, in which a G on the DNA specifies a C in the RNA, a C specifies a G, a T specifies an A, but an A specifies a U instead of a T (Figure 30.5). The RNA, then, is complementary to the DNA template (antisense) strand and identical to the coding (sense) strand, with U replacing T. Within the DNA molecule, regions of both strands can serve as templates for transcription. For a given gene, however, only one of the two DNA strands can be the template. Which strand is used is determined by the location of the promoter for that gene. Transcription by RNA polymerase involves a core enzyme and several auxiliary proteins:
1. Core enzyme: Four of the enzyme's peptide subunits, 20, 1B, and 1B', are required for enzyme assembly (2a), template binding (f'), and the 5'>3' RNA polymerase activity (B), and are referred to as the core enzyme (Figure 30.6). However, this enzyme lacks specificity, that is, it cannot recognize the promoter region on the DNA template. [Note: The in vivo function of a fifth subunit, Q, is unclear.]
2. Holoenzyme: The o subunit ("sigma factor") enables RNA polymerase to recognize promoter regions on the DNA. The o subunit plus the core enzyme make up the holoenzyme. [Note: Different o factors recognize different groups of genes.]

The process of transcription of a typical gene of E. coli can be divided into three phases: initiation, elongation, and termination. A transcription unit extends from the promoter to the termination region, and the initial product of transcription by RNA polymerase is termed the primary transcript.
- 1. Initiation: Transcription begins with the binding of the RNA polymerase holoenzyme to a region of the DNA known as the promoter, which is not transcribed. The prokaryotic promoter contains

Structure of the prokaryotic promoter region.
characteristic consensus sequences (Figure 30.7). [Note: Consensus sequences are idealized sequences in which the base shown at each position is the base most frequently (but not necessarily always) encountered at that position.] Those that are recognized by prokaryotic RNA polymerase o factors include:
a. -35 sequence: A consensus sequence (5'-TTGACA-3'), centered about 35 bases to the left of the transcription start site (see Figure 30.7), is the initial point of contact for the holoenzyme, and a closed complex is formed. [Note: The regulatory sequences that control transcription are, by convention, designated by the 5'>3' nucleotide sequence on the nontemplate strand. A base in the promoter region is assigned a negative number if it occurs prior to (to the left of, toward the 5'-end of, or "upstream" of) the transcription start site. Therefore, the TTGACA sequence is centered at approximately base —35. The first base at the transcription start site is assigned a position of +1. There is no base designated "0."]
b. Pribnow box: The holoenzyme moves and covers a second consensus sequence (5'-TATAAT-3'), centered at about —10 (see Figure 30.7), which is the site of initial DNA melting (unwinding). Melting of a short stretch (about 14 bases) converts the closed complex to an open one known as a transcription bubble. [Note: A mutation in either the —10 or the —35 sequence can affect the transcription of the gene controlled by the mutant promoter.]

Local unwinding of DNA caused by RNA polymerase and formation of an open initiation complex.
2. Elongation: Once the promoter region has been recognized and bound by the holoenzyme, local unwinding of the DNA helix continues (Figure 30.8), mediated by the polymerase. [Note: Unwinding generates supercoils in the DNA that can be relieved by DNA topoisomerases (see p. 401.] RNA polymerase begins to synthesize a transcript of the DNA sequence, and several short pieces of RNA are made and discarded. The elongation phase is said to begin when the transcript (typically starting with a purine) exceeds ten nucleotides in length. Sigma is then released, and the core enzyme is able to leave ("clear") the promoter and move along the template strand in a processive manner. During transcription, a short DNA-RNA hybrid helix is formed (see Figure 30.8). Like DNA polymerase, RNA polymerase uses nucleoside triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain. As with replication, transcription is always in the 5'3' direction. In contrast to DNA polymerase, RNA polymerase does not require a primer and does not appear to have proofreading activity.
3. Termination: The elongation of the single-stranded RNA chain continues until a termination signal is reached. Termination can be intrinsic (spontaneous) or dependent upon the participation of a protein known as the p (rho) factor.
a. p-Independent termination: Seen with most prokaryotic genes, this requires that a sequence in the DNA template generate a sequence in the nascent (newly made) RNA that is self-complementary (Figure 30.9). This allows the RNA to fold back on itself, forming a GC-rich stem (stabilized by H-bonds) plus a loop. This structure is known as a "hairpin". Additionally, just beyond the hairpin, the RNA transcript contains a string of Us at the 3'-end. The bonding of these Us to the complementary As of the DNA template is weak. This facilitates the separation of the newly synthesized RNA from its DNA template, as the double helix "zips up" behind the RNA polymerase.

b. p-Dependent termination: This requires the participation of an additional protein, rho (p), which is a hexameric adenosine triphosphatase (ATPase) with helicase activity. p binds a C-rich "rho recognition site" near the 3'-end of the nascent RNA and, using its ATPase activity, moves along the RNA until it reaches the RNA polymerase paused at the termination site. The ATPdependent helicase activity of p separates the RNA-DNA hybrid helix, causing the release of the RNA.
4. Action of antibiotics: Some antibiotics prevent bacterial cell growth by inhibiting RNA synthesis. For example, rifampin inhibits the initiation of transcription by binding to the B subunit of prokaryotic RNA polymerase, thus interfering with the formation of the first phosphodiester bond (Figure 30.10). Rifampin is useful in the treatment of tuberculosis.' Dactinomycin (known to biochemists as actinomycin D) was the first antibiotic to find therapeutic application in tumor chemotherapy." It binds to the DNA template and interferes with the movement of RNA polymerase along the DNA.
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a

Rho-independent termination of transcription. A. DNA template sequence generates a selfcomplementary sequence in the nascent RNA. B. Hairpin structure formed by the RNA. "N" represents a noncomplementary base.

Acetylation/deacetylation of a lysine residue in a histone protein. HAT = histone acetyltransferase, HDAC = histone deacetylase.

The transcription of eukaryotic genes is a far more complicated process than transcription in prokaryotes. Eukaryotic transcription involves separate polymerases for the synthesis of rRNA, tRNA, and mRNA. In addition, a large number of proteins called transcription factors (TFs) are involved. TFs bind to distinct sites on the DNA—either within the core promoter region, close (proximal) to it, or some distance away (distal). They are required both for the assembly of a transcription complex at the promoter and the determination of which genes are to be transcribed. [Note: Each eukaryotic RNA polymerase has its own promoters and TFs.] For TFs to recognize and bind to their specific DNA sequences, the chromatin structure in that region must be altered (remodeled) to allow access to the DNA. The role of transcription in the regulation of gene expression is discussed in Chapter 32.

The association of DNA with histones to form nucleosomes (see p. 409) affects the ability of the transcription machinery to access the DNA to be transcribed. Most actively transcribed genes are found in a relatively relaxed form of chromatin called euchromatin, whereas most inactive segments of DNA are found in highly condensed heterochromatin. [Note: The interconversion of these forms is called chromatin remodeling.] A major mechanism by which chromatin is remodeled is through acetylation of lysine residues at the amino terminus of histone proteins (Figure 30.11). Acetylation, mediated by histone acetyltransferases (HATs), eliminates the positive charge on the lysine and thereby decreases the interaction of the histone with the negatively charged DNA. Removal of the acetyl group by histone deacetylases (HDACs) restores the positive charge, and fosters stronger interactions between histones and DNA.

There are three distinct classes of RNA polymerase in the nucleus of eukaryotic cells. All are large enzymes with multiple subunits. Each class of RNA polymerase recognizes particular types of genes.
1. RNA polymerase I: This enzyme synthesizes the precursor of the 288, 18S, and 5.8S rRNA in the nucleolus.
2. RNA polymerase II: This enzyme synthesizes the nuclear precursors of MRNA that are subsequently translated to produce proteins. Polymerase II also synthesizes certain small ncRNAs, such as snRNA (see p. 426), snoRNA (see p. 425) and miRNA (see p. 459).
a. Promoters and transcription factors for RNA polymerase II: In some genes transcribed by RNA polymerase II, a sequence of nucleotides that is nearly identical to that of the Pribnow box (see p. 420) is found centered about 25 nucleotides upstream of the transcription start site. This promoter consensus sequence is called the TATA or Hogness box. Between 70 and

Eukaryotic gene promoter consensus sequences.
80 nucleotides upstream of the transcription start site often is found a second consensus sequence known as the CAAT box (Figure 30.12). In other genes, for example, those that are " vege » : . always ("constitutively") expressed, no TATA box is typically present. Instead, a GC-rich region (GC box) may be found. [Note: No one consensus sequence is found in all core promoters.] Because these sequences are on the same molecule of DNA as the gene being transcribed, they are called cis-acting elements. Such sequences serve as binding sites for TFs, which in turn interact with each other and with RNA polymerase II.
Transcription factors bind DNA through a variety of motifs, such as the helix-loop-helix, zinc fingers, and leucine zippers (see p. 18).]
Because TFs are encoded by different genes, synthesized in the cytosol, and must transit to their sites of action, they are called trans-acting factors. General TFs are the minimal requirements for recognition of the promoter, recruitment of RNA polymerase II to the promoter, and initiation of transcription (Figure 30.13A). [Note: In contrast to the holoenzyme of prokaryotes, eukaryotic RNA polymerase II does not itself recognize and bind the promoter. Instead, TFIID recognizes and binds the TATA box, and TFIIF brings the polymerase to the promoter. The helicase activity of TFIIH melts the DNA and its kinase activity phosphorylates polymerase, allowing it to clear the promoter.] Specific TFs (transcriptional activators) bind to sequences within and outside of the core promoter. They are required to modulate the frequency of initiation, to mediate the response to signals such as hormones (see p. 456), and to regulate which genes are expressed at a given point in time. A typical protein-coding eukaryotic gene has binding sites for many such factors. In addition to binding DNA, specific TFs also bind other proteins ("coactivators"), recruiting them to the transcription complex. [Note: Coactivators include the HAT enzymes involved in chromatin remodeling (see p. 422).]

A. Eukaryotic general transcription factors (CTF, SP1, TFIID) bind to consensus sequences found in promoters for RNA polymerase II . Additional general transcription factors (TFlls) are required for assembly of the initiation complex and recruitment of polymerase. B. Enhancer stimulation of RNA polymerase II.

Posttranscriptional processing of eukaryotic ribosomal RNA by ribonucleases (RNases).
b.
Role of enhancers in eukaryotic gene regulation: Enhancers are special cis-acting DNA sequences that increase the rate of initiation of transcription by RNA polymerase II. Enhancers are on the same chromosome as the gene whose transcription they stimulate (Figure 30.13B). However, they can 1) be located upstream (to the 5'-side) or downstream (to the 3'-side) of the transcription start site; 2) be close to or thousands of base pairs away from the promoter (Figure 30.14); and 3) occur on either strand of the DNA. Enhancers contain DNA sequences called "response elements" that bind specific TFs that function as transcriptional activators. By bending or looping the DNA, these enhancer-binding factors can interact with other transcription factors bound to a promoter and with RNA polymerase II, thereby stimulating transcription (see Figure 30.13B). [Note: Silencers are similar to enhancers in that they act over long distances; however, they reduce gene expression.]
c. Inhibitors of RNA polymerase II: This enzyme is inhibited by oamanitin—a potent toxin produced by the poisonous mushroom Amanita phalloides (sometimes called "death cap" ). a-Amanitin forms a tight complex with the polymerase, thereby inhibiting mRNA synthesis and, ultimately, protein synthesis.
3. RNA polymerase Ill: This enzyme synthesizes tRNA, 5S rRNA, and some snRNA and snoRNA.

Mitochondria contain a single RNA polymerase that more closely resembles bacterial RNA polymerase than the eukaryotic enzyme.

A primary transcript is the initial, linear, RNA copy of a transcription unit—the segment of DNA between specific initiation and termination sequences. The primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are posttranscriptionally modified by cleavage of the original transcripts by ribonucleases. tRNAs are then further modified to help give each species its unique identity. In contrast, prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic MRNA is extensively modified both coand posttranscriptionally.

rRNAs of both prokaryotic and eukaryotic cells are generated from long precursor molecules called pre-rRNAs. The 23S, 16S, and 5S rRNA of prokaryotes are produced from a single pre-rRNA molecule, as are the 28S, 18S, and 5.8S rRNA of eukaryotes (Figure 30.15). [Note: Eukaryotic 5S rRNA is synthesized by RNA polymerase III and modified separately.) The pre-rRNAs are cleaved by ribonucleases to yield intermediate-sized pieces of rRNA, which are further processed (trimmed by exonucleases and modified at some bases and riboses) to produce the required RNA species. [Note: In eukaryotes, rRNA genes are found in long, tandem arrays. rRNA synthesis and processing occur in the nucleolus, with base and sugar modifi-
cations facilitated by small nucleolar RNAs (snoRNA). Some of the proteins destined to become components of the ribosome associate with pre-rRNA prior to and during its modification.]

Both eukaryotic and prokaryotic tRNA are also made from longer precursor molecules that must be modified (Figure 30.16). Sequences at both ends of the molecule are removed and, if present, an intron (see below) is removed from the anticodon loop by nucleases. Other posttranscriptional modifications include addition of a—CCA sequence by nucleotidyltransferase to the 3'-terminal end of tRNA, and modification of bases at specific positions to produce the "unusual bases" characteristic of tRNA (see p. 292).

The collection of all the primary transcripts synthesized in the nucleus by RNA polymerase II is known as heterogeneous nuclear RNA (hnRNA). The pre-mRNA components of hnRNA undergo extensive coand posttranscriptional modification in the nucleus. These modifications usually include:
1. 5' "Capping": This is the first of the processing reactions for premRNA (Figure 30.17). The cap is a 7-methylguanosine attached "backward" to the 5'-terminal end of the MRNA, forming an unusual 5'->5' triphosphate linkage. Creation of the cap requires removal of the y phosphate from the 5'-triphosphate of the premRNA, followed by addition of GMP (from GTP) by the nuclear enzyme guanylyltransferase. Methylation of this terminal guanine occurs in the cytosol, and is catalyzed by guanine-7-methyltransferase. S-adenosylmethionine is the source of the methyl group

A. Primary tRNA transcript. B. Functional tRNA after posttranscriptional modification. Modified bases include D (dihydrouracil), wy (pseudouracil), and ™, which means that the base has been methylated.

Posttranscriptional modification of MRNA showing the 7-methylguanosine cap and poly-A tail.
(see p. 264). Additional methylation steps may occur. The addition of this 7-methylguanosine "cap" helps stabilize the mRNA, and permits initiation of translation (see p. 439). Eukaryotic mRNAs lacking the cap are not efficiently translated.
2.
3.
Addition of a poly-A tail: Most eukaryotic MRNA (with several notable exceptions, including those coding for the histones) have a chain of 40-200 adenine nucleotides attached to the 3'-end (see Figure 30.17). This poly-A tail is not transcribed from the DNA, but rather is added after transcription by the nuclear enzyme, polyadenylate polymerase, using ATP as the substrate. The mRNA is cleaved downstream of a consensus sequence, called the polyadenylation signal sequence (AAUAAA), found near the 3'-end of the RNA, and the poly-A tail is added to the new 3'-end. These tails help stabilize the mRNA, facilitate its exit from the nucleus, and aid in translation. After the mRNA enters the cytosol, the poly-A tail is gradually shortened.
Removal of introns: Maturation of eukaryotic MRNA usually involves the removal of RNA sequences (introns, or intervening sequences), which do not code for protein from the primary transcript. The remaining coding sequences, the exons, are joined together to form the mature MRNA. The process of removing introns and joining exons is called splicing. The molecular complex that accomplishes these tasks is known as the spliceosome. A few eukaryotic primary transcripts contain no introns, for example, those from histone genes. Others contain a few introns, whereas some, such as the primary transcripts for the « chains of collagen, contain more than 50 intervening sequences that must be removed before mature MRNA is ready for translation.

a. Role of snRNAs: In association with proteins, uracil-rich small nuclear RNAs (snRNA) form small nuclear ribonucleoprotein particles (SnRNPs, or "snurps" designated as U1, U2, etc.) that mediate splicing. They facilitate the removal of introns by forming base pairs with the consensus sequences at each end of the intron (Figure 30.18).
Systemic lupus erythematosus, an often fatal inflammatory disease, results from an autoimmune response in which individuals produce antibodies against their own nuclear proteins such as snRNPs.
b.
Mechanism of splicing: The binding of snRNPs brings the sequences of the neighboring exons into the correct alignment for splicing. The 2'-OH group of an adenosine (A) residue (known as the branch site) in the intron attacks the phosphate at the 5'-end of the intron (splice donor site), forming an unusual 2'+5' phosphodiester bond and creating a "lariat" structure (see Figure 30.18). The newly freed 3'-OH of exon 1 attacks the 5'-phosphate at the splice acceptor site, forming a phosphodiester bond that joins exons 1 and 2. The excised intron is released as a lariat, which is typically degraded. [Note: The GU and AG sequences at the beginning and end, respectively, of introns are invariant.] After introns have been removed and exons joined, the mature MRNA molecules leave the nucleus and pass into the cytosol through pores in the nuclear membrane. [Note: The introns in tRNA (see Figure 30.16) are removed by a different mechanism.]
- c. Effect of splice site mutations: Mutations at splice sites can lead to improper splicing and the production of aberrant proteins. It is estimated that 15% of all genetic diseases are a result of mutations that affect RNA splicing. For example, mutations that cause the incorrect splicing of B-globin mRNA are responsible for some cases of B-thalassemia—a disease in which the production of the B-globin protein is defective (see p. 38).

4. Alternative splicing of mRNA molecules: The pre-mRNA molecules from some genes can be spliced in alternative ways in different tissues. This produces multiple variations of the mRNA and, therefore, of its protein product (Figure 30.19). This appears to be a mechanism for producing a diverse set of proteins from a limited set of genes. For example, in eukaryotic cells the mRNA for tropomyosin, an actin filament-binding protein of the cytoskeleton (and of the contractile apparatus in muscle cells), undergoes extensive tissue-specific alternative splicing with production of multiple isoforms of the tropomyosin protein.

There are three major types of RNA that participate in the process of protein synthesis: ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA) (Figure 30.20). They are unbranched polymers of nucleotides, but differ from DNA by containing ribose instead of deoxyribose and uracil instead of thymine. rRNA is a component of the ribosomes. tRNA serves as an "adaptor" molecule that carries a spe-

cific amino acid to the site of protein synthesis. mRNA carries genetic information from the nuclear DNA to the cytosol, where it is used as the template for protein synthesis. The process of RNA synthesis is called transcription, and its substrates are ribonucleoside triphosphates. The enzyme that synthesizes RNA is RNA polymerase, which is a multisubunit enzyme. In prokaryotic cells, the core enzyme has five subunits— 2a, 1B, 18', and 12—and possesses 5'3' polymerase activity that elongates the growing RNA strand. This enzyme requires an additional subunit—sigma (o) factor—that recognizes the nucleotide sequence (promoter region) at the beginning of a length of DNA that is to be transcribed. This region contains characteristic consensus nucleotide sequences that are highly conserved and include the Pribnow box and the -35 sequence. Another protein—rho (p) factor—is required for termination of transcription of some genes. There are three distinct classes of RNA polymerase in the nucleus of eukaryotic cells. RNA polymerase | synthesizes the precursor of large rRNA in the nucleolus. RNA polymerase Il synthesizes the precursors for mRNA and some ncRNAs in the nucleoplasm, and RNA polymerase Ill produces the precursors of tRNA in the nucleoplasm. In both prokaryotes and eukaryotes, RNA polymerase does not require a primer, and has no proofreading activity. Promoters for genes transcribed by RNA polymerase II contain consensus sequences, such as the TATA or Hogness box, the CAAT box, and the GC box. They serve as binding sites for proteins called general transcription factors, which, in turn, interact with each other and with RNA polymerase II. Enhancers are DNA sequences that increase the rate of initiation of transcription by binding specific transcription factors that serve as transcription activators. Eukaryotic transcription requires that the chromatin be accessible. A primary transcript is a linear copy of a transcription unit—the segment of DNA between specific initiation and termination sequences. The primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are posttranscriptionally modified by cleavage of the original transcripts by ribonucleases. rRNA of both prokaryotic and eukaryotic cells are synthesized from long precursor molecules called preribosomal RNA.

These precursors are cleaved and trimmed by ribonucleases, producing the three largest rRNA, and bases and sugars are modified. Eukaryotic 5S rRNA is synthesized by RNA polymerase III , and is modified separately. Prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic mRNA is extensively modified coand posttranscriptionally. For example, a 7-methylguanosine "cap" is attached to the 5'-terminal end of the mRNA through a 5'5' linkage. A long poly-A tail—not transcribed from the DNA—is attached to the 3'-end of most mRNA. Most eukaryotic mRNAs also contain intervening sequences (introns) that must be removed to make the mRNA functional. Their removal, as well as the joining of expressed sequences (exons), requires a spliceosome composed of small, nuclear ribonucleoprotein particles that mediate the process of splicing. Eukaryotic mRNA is monocistronic, containing information from just one gene. Prokaryotic and eukaryotic tRNA are also made from longer precursor molecules. If present, an intron is removed by nucleases, and both ends of the molecule are trimmed by ribonucleases. A 3'-CCA sequence is added, and bases at specific positions are modified, producing "unusual" bases.

30.1
produced gene?
A 1-year-old male with chronic anemia is found to have B-thalassemia. Genetic analysis shows that one of his B-globin genes has a mutation that creates a new splice acceptor site 19 nucleotides upstream of the normal splice acceptor site of the first intron. Which of the following best describes the new mRNA molecule that can be from this mutant
A. Exon 1 will be too short.
B. Exon 1 will be too long.
C. Exon 2 will be too short.
D. Exon 2 will be too long.
E. Exon 2 will be missing.
30.2 The base sequence of the strand of DNA used as the template for transcription is GATCTAC. What is the base sequence of the RNA product? (All sequences are written 5'> 3' according to standard convention.)
A. CTAGATG.
B. GTAGATC.
C. GAUCUAC.
D. CUAGAUG.
E. GUAGAUC.
30.3 A 4-year-old child who becomes easily tired and has trouble walking is diagnosed with Duchenne muscular dystrophy, an X-linked recessive disorder. Genetic analysis shows that the patient's gene for the muscle protein dystrophin contains a mutation in its promoter region. Of the choices listed, which would be the most likely effect of this mutation?
A. Initiation of dystrophin transcription will be defective.
B. Termination of dystrophin transcription will be defective.
C. Capping of dystrophin mRNA will be defective.
D. Splicing of dystrophin mRNA will be defective.
E. Tailing of dystrophin mRNA will be defective.
30.4 A mutation to this sequence in eukaryotic MRNA will affect the process by which the 3'-end poly-A tail is added to the mRNA.
A. CAAT
B. CCA
C. GGGGCG
D. AAUAAA
E. TATAAA

Correct answer = D. Because the mutation creates an additional splice acceptor site (the 3'end) upstream of the normal acceptor site of intron 1, the 19 nucleotides that are usually found at the 3'-end of the excised intron 1 lariat can remain behind as part of exon 2. Exon 2 can, therefore, have these extra 19 nucleotides at its 5'-end. The presence of these extra nucleotides in the coding region of the mutant mRNA molecule will prevent the ribosome from translating the message into a normal B-globin protein molecule. Those mRNA for which the normal splice site is used to remove the first intron will be normal, and their translation will produce normal B-globin protein.
Correct answer = E. The RNA product has a sequence that is complementary to the template strand and identical to the coding strand of DNA. Uracil (U) is found in RNA in place of the thymine (T) in DNA. Thus, the DNA template 5'GATCTAC-3' would produce the RNA product 3'-CUAGAUG-5' or, written correctly in the standard direction, 5'-GUAGAUC-3'.
Correct answer = A. Mutations in the promoter typically prevent formation of the RNA polymerase II transcription complex, resulting in a decrease in the initiation of mRNA synthesis. A deficiency of dystrophin mRNA will result in a deficiency in the production of the dystrophin protein. Capping, splicing and tailing defects are not a consequence of promoter mutations. They can, however, result in MRNA with decreased stability (capping and tailing defects), or a mRNA in which too many or too few introns have been removed (splicing defects)
Correct answer = D. An endonuclease cleaves mRNA just downstream of this polyadenylation signal, creating a new 3'-end to which the pol A polymerase adds the poly-A tail using ATP as the substrate in a template-independent process. CAAT, GGGGCGT, and TATAAA are sequences found in promoters for RNA polymerase II. CCA is added to the 3'-end of tRNA by nucleotidyl transferase.

Genetic information, stored in the chromosomes and transmitted to daughter cells through DNA replication, is expressed through transcription to RNA and, in the case of messenger RNA (mRNA), subsequent translation into proteins (polypeptide chains, Figure 31.1). The pathway of protein synthesis is called translation because the "language" of the nucleotide sequence on the mRNA is translated into the "language" of an amino acid sequence. The process of translation requires a genetic code, through which the information contained in the nucleic acid sequence is expressed to produce a specific sequence of amino acids. Any alteration in the nucleic acid sequence may result in an incorrect amino acid being inserted into the polypeptide chain, potentially causing disease or even death of the organism. Newly made proteins undergo a number of processes to achieve their functional form. They must fold properly, and misfolding can result in degradation of the protein. Many proteins are covalently modified to activate them or alter their activities. Finally, proteins are targeted to their final intraor extracellular destinations by signals present in the proteins themselves.

The genetic code is a dictionary that identifies the correspondence between a sequence of nucleotide bases and a sequence of amino acids. Each individual "word" in the code is composed of three nucleotide bases. These genetic words are called codons.

Codons are presented in the mRNA language of adenine (A), guanine (G), cytosine (C), and uracil (U). Their nucleotide sequences are always written from the 5'-end to the 3'-end. The four nucleotide bases are used to produce the three-base codons. There are, therefore, 64 different combinations of bases, taken three at a time (a triplet code) as shown in Figure 31.2.
1. How to translate a codon: This table (or "dictionary") can be used to translate any codon and, thus, to determine which amino acids are coded for by an mRNA sequence. For example, the codon 5'-AUG-3' codes for methionine (see Figure 31.2). [Note: AUG is

Use of the genetic code table to translate the codon AUG.
the initiation (start) codon for translation.] Sixty-one of the 64 codons code for the 20 common amino acids.

. . ; Fossibie effects of changing a single of an mRNA chain. g reg
- 2. Termination ("stop" or "nonsense") codons: Three of the codons, UAG, UGA, and UAA, do not code for amino acids, but rather are termination codons. When one of these codons appears in an mRNA sequence, synthesis of the polypeptide coded for by that mRNA stops.

Usage of the genetic code is remarkably consistent throughout all living organisms. It is assumed that once the standard genetic code evolved in primitive organisms, any mutation that altered its meaning would have caused the alteration of most, if not all, protein sequences, resulting in lethality. Characteristics of the genetic code include the following:
- 1. Specificity: The genetic code is specific (unambiguous), that is, a particular codon always codes for the same amino acid.
2. Universality: The genetic code is virtually universal, that is, its specificity has been conserved from very early stages of evolution, with only slight differences in the manner in which the code is translated. [Note: An exception occurs in mitochondria, in which a few codons have meanings different than those shown in Figure 31.2, for example, UGA codes for Trp.]
3. Degeneracy: The genetic code is degenerate (sometimes called redundant). Although each codon corresponds to a single amino acid, a given amino acid may have more than one triplet coding for it. For example, arginine is specified by six different codons (see Figure 31.2). Only Met and Trp have just one coding triplet.
4. Nonoverlapping and commaless: The genetic code is nonoverlapping and commaless, that is, the code is read from a fixed starting point as a continuous sequence of bases, taken three at a time. For example, AGCUGGAUACAU is read as AGC/UGG/AUA/CAU without any "punctuation" between the codons.

. . . . . Changing a single nucleotide base on the mRNA chain (a "point mutation") can lead to any one of three results (Figure 31.3):
1. Silent mutation: The codon containing the changed base may code for the same amino acid. For example, if the serine codon UCA is given a different third base—U—to become UCU, it still codes for serine. This is termed a "silent" mutation.
2. Missense mutation: The codon containing the changed base may code for a different amino acid. For example, if the serine codon UCA is given a different first base—C—to become CCA, it will code for a different amino acid, in this case, proline. The substitution of an incorrect amino acid is called a "missense" mutation.
3. Nonsense mutation: The codon containing the changed base may become a termination codon. For example, if the serine codon UCA is given a different second base—A—to become UAA, the new codon causes termination of translation at that point, and the production of a shortened (truncated) protein. The creation of a termination codon at an inappropriate place is called a "nonsense" mutation.
4. Other mutations: These can alter the amount or structure of the protein produced by translation.
a. Trinucleotide repeat expansion: Occasionally, a sequence of three bases that is repeated in tandem will become amplified in number, so that too many copies of the triplet occur. If this happens within the coding region of a gene, the protein will contain many extra copies of one amino acid. For example, amplification of the CAG codon leads to the insertion of many extra glutamine residues in the huntingtin protein, causing the neurodegenerative disorder, Huntington disease (Figure 31.4). The additional glutamines result in unstable proteins that cause the accumulation of protein aggregates. If the trinucleotide repeat expansion occurs in the untranslated regions of a gene, the result can be a decrease in the amount of protein produced as seen, for example, in fragile X syndrome and myotonic dystrophy. [Note: In fragile X syndrome, the most common cause of intellectual disability, the expansion results in gene silencing through DNA hypermethylation (see p. 460).]

Role of tandem triplet repeats in
mRNA causing Huntington
disease and other triplet expansion diseases.

Frame-shift mutations as a result of addition or deletion of a base can cause an alteration in the reading frame of MRNA.

Complementary, antiparallel binding of the anticodon for methionyl-tRNA (CAV) to the mRNA codon for methionine (AUG), the initiation codon for translation.
b. Splice site mutations: Mutations at splice sites (see p. 427) can alter the way in which introns are removed from premRNA molecules, producing aberrant proteins. [Note: In myotonic dystrophy gene silencing is the result of splicing alterations.]
- c. Frame-shift mutations: If one or two nucleotides are either deleted from or added to the coding region of a message sequence, a frame-shift mutation occurs and the reading frame is altered. This can result in a product with a radically different amino acid sequence, or a truncated product due to the creation of a termination codon (Figure 31.5). If three nucleotides are added, a new amino acid is added to the peptide or, if three nucleotides are deleted, an amino acid is lost. Loss of three nucleotides maintains the reading frame, but can result in serious pathology. For example, cystic fibrosis (CF), a hereditary disease that primarily affects the pulmonary and digestive systems, is most commonly caused by deletion of three nucleotides from the coding region of a gene, resulting in the loss of phenylalanine at the 508th position (AF508) in the protein encoded by that gene. This AF508 mutation prevents normal folding of the CF transmembrane conductance regulator (CFTR) protein, leading to its destruction by the proteasome (see p. 247). CFTR normally functions as a chloride channel in epithelial cells, and its loss results in the production of thick, sticky secretions in the lungs and pancreas, leading to lung damage and digestive deficiencies (see p. 248). In over 70% of Caucasians with CF, the AF508 mutation is the cause of the disease.

A large number of components are required for the synthesis of a protein. These include all the amino acids that are found in the finished product, the mRNA to be translated, transfer RNA (tRNA) for each of the amino acids, functional ribosomes, energy sources, and enzymes, as well as protein factors needed for initiation, elongation, and termination steps of polypeptide chain synthesis.

All the amino acids that eventually appear in the finished protein must be present at the time of protein synthesis. [Note: If one amino acid is missing (for example, if the diet does not contain an essential amino acid, see p. 261), translation stops at the codon specifying that amino acid. This demonstrates the importance of having all the essential amino acids in sufficient quantities in the diet to ensure continued protein synthesis.]

At least one specific type of tRNA is required for each amino acid. In humans, there are at least 50 species of tRNA, whereas bacteria contain 30-40 species. Because there are only 20 different amino
acids commonly carried by tRNA, some amino acids have more than one specific tRNA molecule. This is particularly true of those amino acids that are coded for by several codons.
1. Amino acid attachment site: Each tRNA molecule has an attachment site for a specific (cognate) amino acid at its 3'-end (Figure 31.6). The carboxyl group of the amino acid is in an ester linkage with the 3'-hydroxyl of the ribose portion of the adenosine (A) nucleotide in the —CCA sequence at the 3'-end of the tRNA. [Note: When a tRNA has a covalently attached amino acid, it is said to be charged; when it does not, it is said to be uncharged. The amino acid attached to the tRNA molecule is said to be activated.]
2. Anticodon: Each tRNA molecule also contains a three-base nucleotide sequence—the anticodon—that pairs with a specific codon on the mRNA (see Figure 31.6). This codon specifies the insertion into the growing peptide chain of the amino acid carried by that tRNA.

This family of enzymes is required for attachment of amino acids to their corresponding tRNAs. Each member of this family recognizes a specific amino acid and all the tRNAs that correspond to that amino acid (isoaccepting tRNAs). Aminoacyl-tRNA synthetases catalyze a two-step reaction that results in the covalent attachment of the carboxyl group of an amino acid to the 3'-end of its corresponding (cognate) tRNA. The overall reaction requires adenosine triphosphate (ATP), which is cleaved to adenosine monophosphate (AMP) and inorganic pyrophosphate (PPj) (Figure 31.7). The extreme specificity of the synthetase in recognizing both the amino acid and its cognate tRNA contributes to the high fidelity of translation of the genetic message. In addition, the synthetases have a "proofreading" or "editing" activity that can remove amino acids from the enzyme or the tRNA molecule.

The specific mRNA required as a template for the synthesis of the desired polypeptide chain must be present. [Note: Interactions between proteins that bind the 5'-cap (elF-4 proteins) and the 3'-tail (poly-A binding proteins) of eukaryotic MRNA mediate circularization of the mRNA and likely prevent the use of incompletely processed mRNA in translation.]

Ribosomes are large complexes of protein and ribosomal RNA (rRNA, Figure 31.8). They consist of two subunits—one large and one small—whose relative sizes are given in terms of their sedimentation coefficients, or S (Svedberg) values. [Note: Because the S values are determined both by shape as well as molecular mass, their numeric values are not strictly additive. For example, the prokaryotic 50S and 30S ribosomal subunits together form a 70S ribosome. The eukaryotic 60S and 40S subunits form an 80S ribosome.]

Attachment of a specific amino acid to its corresponding tRNA by aminoacyl-tRNA synthetase (E).

Ribosomal composition. (The number of proteins in the eukaryotic ribosomal subunits varies somewhat from species to species.)
Prokaryotic and eukaryotic ribosomes are similar in structure, and serve the same function, namely, as the macromolecular complexes in which the synthesis of proteins occurs.
The small ribosomal subunit binds mRNA and is responsible for the accuracy of translation by ensuring correct base-pairing between the codon in the mRNA and the anticodon of the tRNA. The large ribosomal subunit catalyzes formation of the peptide bonds that link amino acid residues in a protein.
1. Ribosomal RNA: As discussed on p. 418, prokaryotic ribosomes contain three size species of rRNA, whereas eukaryotic ribosomes contain four (see Figure 31.8). The rRNAs are generated from a single pre-rRNA by the action of ribonucleases, and some bases and riboses are modified.
2. Ribosomal proteins: Ribosomal proteins are present in considerably greater numbers in eukaryotic ribosomes than in prokaryotic ribosomes. These proteins play a number of roles in the structure and function of the ribosome and its interactions with other components of the translation system.
3.
- A, P, and E sites on the ribosome: The ribosome has three binding sites for tRNA molecules—the A, P, and E sites—each of which extends over both subunits. Together, they cover three neighboring codons. During translation, the A site binds an incoming aminoacyl-tRNA as directed by the codon currently occupying this site. This codon specifies the next amino acid to be added to the growing peptide chain. The P-site codon is occupied by peptidyl-tRNA. This tRNA carries the chain of amino acids that has already been synthesized. The E site is occupied by the empty tRNA as it is about to exit the ribosome. (See Figure 31.13 for an illustration of the role of the A, P, and E sites in translation.)
4.

Cellular location of ribosomes: In eukaryotic cells, the ribosomes are either "free" in the cytosol or are in close association with the endoplasmic reticulum (which is then known as the "rough" endoplasmic reticulum, or RER). The RER-associated ribosomes are responsible for synthesizing proteins that are to be exported from the cell, as well as those that are destined to become incorporated into plasma, endoplasmic reticulum, or Golgi membranes, or imported into lysosomes (see p. 169 for an overview of the latter process). Cytosolic ribosomes synthesize proteins required in the cytosol itself, or destined for the nucleus, mitochondria, and peroxisomes. [Note: Mitochondria contain their own set of ribosomes and their own unique, circular DNA. Most mitochondrial proteins, however, are encoded by nuclear DNA, synthesized in the cytosol, and posttranslationally targeted to mitochondria.]

Initiation, elongation, and termination (or release) factors are required for peptide synthesis. Some of these protein factors perform a catalytic function, whereas others appear to stabilize the synthetic machinery. [Note: A number of the factors are G proteins, and thus are active when bound to GTP and inactive when bound to GDP]

Cleavage of four high-energy bonds is required for the addition of one amino acid to the growing polypeptide chain: two from ATP in the aminoacyl-tRNA synthetase reaction—one in the removal of PPi, and one in the subsequent hydrolysis of the PP; to inorganic phosphate by pyrophosphatase—and two from GTP—one for binding the aminoacyl-tRNA to the A site and one for the translocation step (see Figure 31.13, p. 440). [Note: Additional ATP and GTP molecules are required for initiation in eukaryotes, whereas an additional GTP molecule is required for termination in both eukaryotes and prokaryotes.]

Correct pairing of the codon in the mRNA with the anticodon of the tRNA is essential for accurate translation (see Figure 31.6). Some tRNAs recognize more than one codon for a given amino acid.

Binding of the tRNA anticodon to the mRNA codon follows the rules of complementary and antiparallel binding, that is, the mRNA codon is "read" 5'3' by an anticodon pairing in the "flipped" (3'-5') orientation (Figure 31.9). [Note: Nucleotide sequences are ALWAYS assumed to be written in the 5' to 3' direction unless otherwise noted.]

The mechanism by which tRNAs can recognize more than one codon for a specific amino acid is described by the "wobble" hypothesis in which the base at the 5'-end of the anticodon (the "first" base of the anticodon) is not as spatially defined as the other two bases. Movement of that first base allows nontraditional (less stringent) base-pairing with the 3'-base of the codon (the "last" base of the codon). This movement is called "wobble" and allows a single tRNA to recognize more than one codon. Examples of these flexible pairings are shown in Figure 31.9. The result of wobble is that there need not be 61 tRNA species to read the 61 codons that code for amino acids.

Wobble: Nontraditional base-pairing between the 5'-nucleotide (first nucleotide) of the anticodon with the 3'-nucleotide (last nucleotide) of the codon. H = hypoxanthine (the product of adenine deamination).

Complementary binding between prokaryotic MRNA Shine-Dalgarno sequence and 16S rRNA.

Generation of the initiator N-formylmethionyl-tRNA (fMet-tRNA).
The process of protein synthesis translates the three-letter alphabet of nucleotide sequences on mRNA into the 20-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5'-end to its 3'-end, producing a protein synthesized from its amino-terminal end to its carboxyl-terminal end. Prokaryotic mRNAs often have several coding regions, that is, they are polycistronic (see p. 418). Each coding region has its own initiation and termination codon and produces a separate species of polypeptide. In contrast, each eukaryotic mRNA has only one coding region, that is, it is monocistronic. The process of translation is divided into three separate steps: initiation, elongation, and termination. Eukaryotic protein synthesis resembles that of prokaryotes in most aspects. Individual differences are noted in the text.
One important difference is that translation and transcription are coupled in prokaryotes, with translation starting before transcription is completed. Coupling is a consequence of the lack of a nuclear membrane in prokaryotes.

Initiation of protein synthesis involves the assembly of the components of the translation system before peptide bond formation occurs. These components include the two ribosomal subunits, the mRNA to be translated, the aminoacyl-tRNA specified by the first codon in the message, GTP (which provides energy for the process), and initiation factors that facilitate the assembly of this initiation complex (see Figure 31.13). [Note: In prokaryotes, three initiation factors are known (IF-1, IF-2, and IF-3), whereas in eukaryotes, there are over ten (designated elF to indicate eukaryotic origin). Eukaryotes also require ATP for initiation.] There are two mechanisms by which the ribosome recognizes the nucleotide sequence (AUG) that initiates translation:
1.
Shine-Dalgarno sequence: In E. coli, a purine-rich sequence of nucleotide bases, known as the Shine-Dalgarno (SD) sequence, is located six to ten bases upstream of the initiating AUG codon on the mRNA molecule—that is, near its 5'-end. The 16S rRNA component of the 30S ribosomal subunit has a nucleotide sequence near its 3'-end that is complementary to all or part of the SD sequence. Therefore, the 5' end of the mRNA and the 3'-end of the 16S rRNA can form complementary base pairs, thus facilitating the positioning of the small (30S) ribosomal subunit on the mRNA in close proximity to the initiating AUG codon (Figure 31.10). Eukaryotic messages do not have SD sequences. In eukaryotes, the 40S ribosomal subunit (aided by members of the elF-4 family of proteins) binds close to the cap structure at the 5-end of the mRNA and moves down the mRNA until it encounters the initiator AUG. This "scanning" process requires ATP.
2.

Initiation codon: The initiating AUG is recognized by a special initiator tRNA. Recognition is facilitated by IF-2-GTP in prokaryotes and elF-2-GTP (plus additional elFs) in eukaryotes. The charged initiator tRNA enters the P site on the small subunit. [Note: The initiator tRNA is the only tRNA recognized by elF-2, and the only tRNA to go directly to the P site.] In bacteria and in mitochondria, the initiator tRNA carries an N-formylated methionine (Figure 31.11). After methionine is attached to the initiator tRNA, the formyl group is added by the enzyme transformylase, which uses N'°-formyl tetrahydrofolate (see p. 267) as the carbon donor. In eukaryotes, the initiator tRNA carries a methionine that is not formylated. [Note: In both prokaryotic and eukaryotic cells, this Nterminal methionine is usually removed before translation is completed.] The large ribosomal subunit then joins the complex, and a functional ribosome is formed with the charged initiating tRNA in the P site, and the A site empty. The GTP on (e)IF-2 gets hydrolyzed to GDP. A guanine nucleotide exchange factor facilitates the reactivation of (e)IF-2-GDP.

Elongation of the polypeptide chain involves the addition of amino acids to the carboxyl end of the growing chain. During elongation, the ribosome moves from the 5'-end to the 3'-end of the mRNA that is being translated. Delivery of the aminoacyl-tRNA whose codon appears next on the mRNA template in the ribosomal A site is facilitated in E. coli by elongation factors EF-Tu-GTP and EF-Ts, and requires GTP hydrolysis. [Note: In eukaryotes, comparable elongation factors are EF-10-GTP and EF-1By. Both EF-Ts and EF-1By function as nucleotide exchange factors.] The formation of the peptide bond is catalyzed by peptidyltransferase, an activity intrinsic to the 23S rRNA found in the large (50S) ribosomal subunit (Figure 31.12). [Note: Because this rRNA catalyzes the reaction, it is referred to as a ribozyme.] After the peptide bond has been formed, what was attached to the tRNA at the P site is now linked to the amino acid on the tRNA at the A site. The ribosome then advances three nucleotides toward the 3'-end of the mRNA. This process is known as translocation and, in prokaryotes, requires the

Formation of a peptide bond. [Note: The tRNA carrying the peptide chain is in the P site of the ribosome, and the tRNA carrying the next amino acid to be incorporated is in the A site.]
Figure 31.13 (Continued on the next page)
Steps in prokaryotic protein synthesis (translation).

A polyribosome consists of several ribosomes simultaneously translating one MRNA. [Note: Eukaryotic mRNA is circularized for translation.]
participation of EF-G-GTP (eukaryotic cells use EF-2-GTP) and GTP hydrolysis. Translocation causes movement of the uncharged tRNA from the P to the E site (before being released), and movement of the peptidyl-tRNA from the A to the P site. The process is repeated until a termination codon is encountered.

Termination occurs when one of the three termination codons moves into the A site. These codons are recognized in E. coli by release factors: RF-1, which recognizes the termination codons UAA and UAG, and RF-2, which recognizes UGA and UAA. The binding of these release factors results in hydrolysis of the bond linking the peptide to the tRNA at the P site, causing the nascent protein to be released from the ribosome. A third release factor, RF-3-GTP then causes the release of RF-1 or RF-2 as GTP is hydrolyzed (see Figure 31.13). [Note: Eukaryotes have a single release factor, eRF, which recognizes all three termination codons. A second factor, eRF-3, is thought to function like the prokaryotic RF-3.] The steps in prokaryotic protein synthesis are summarized in Figure 31.13. The newly synthesized polypeptide may undergo further modification as described below, and the ribosomal subunits, MRNA, tRNA, and protein factors can be recycled and used to synthesize another polypeptide. Some antibiotic inhibitors of protein synthesis are illustrated in Figure 31.13, as is diphtheria toxin. In addition, ricin (from castor beans) is a very potent toxin that exerts its effects by removing an adenine from 28S rRNA, thus inhibiting the function of eukaryotic ribosomes.

Translation begins at the 5'-end of the mRNA, with the ribosome proceeding along the RNA molecule. Because of the length of most mRNAs, more than one ribosome at a time can translate a message (Figure 31.14). Such a complex of one MRNA and a number of ribosomes is called a polysome or polyribosome.
Although most protein synthesis is initiated in the cytoplasm of eukaryotic cells, many proteins are destined to perform their functions extracellularly or within specific cellular organelles. Such proteins usually contain amino acid sequences that direct these proteins to their final locations. For example, proteins destined for secretion from the cell are targeted during their synthesis (cotranslationally) targeted to the RER (see p. 436) by the presence of an N-terminal hydrophobic signal sequence. Proteins targeted after synthesis (posttranslationally) include nuclear proteins, which contain a short, basic "nuclear localization signal," and mitochondrial proteins, which contain an amphipathic, o-helical "mitochondrial entry sequence'

Gene expression is most commonly regulated at the transcriptional level; however, translation may also be regulated. An important mechanism by which this is accomplished in eukaryotes is by covalent modification of elF-2: phosphorylated elF-2 is inactive. In both eukaryotes and prokaryotes, regulation can also be achieved through proteins that bind mRNA and either inhibit its use by blocking translation or extend its use by protecting it from degradation. For a more detailed discussion of the regulation of translation, see p. 454.

Many polypeptide chains are covalently modified, either while they are still attached to the ribosome (cotranslational) or after their synthesis has been completed (posttranslational). These modifications may include removal of part of the translated sequence, or the covalent addition of one or more chemical groups required for protein activity. Some types of posttranslational modifications are listed below.

Many proteins destined for secretion from the cell are initially made as large, precursor molecules that are not functionally active. Portions of the protein chain must be removed by specialized endoproteases, resulting in the release of an active molecule. The cellular site of the cleavage reaction depends on the protein to be modified. Some precursor proteins are cleaved in the endoplasmic reticulum or the Golgi apparatus, others are cleaved in developing secretory vesicles (for example, insulin, see Figure 23.4, p. 309), and still others, such as collagen (see p. 47), are cleaved after secretion. Zymogens are inactive precursors of secreted enzymes (including the proteases required for digestion). They become activated through cleavage when they reach their proper sites of action. For example, the pancreatic zymogen, trypsinogen, becomes activated to trypsin in the small intestine (see Figure 19.5, p. 249). The synthesis of proteases as zymogens protects the cell from being digested by its own products.
Figure 31.15 (continued on next page) Covalent modifications of some amino acid residues.
Figure 31.15 (continued from previous page)
Covalent modifications of some amino acid residues.

Proteins may be activated or inactivated by the covalent attachment of a variety of chemical groups. Examples include (Figure 31.15):
1. Phosphorylation: Phosphorylation occurs on the hydroxyl groups of serine, threonine, or, less frequently, tyrosine residues in a protein. This phosphorylation is catalyzed by one of a family of protein kinases and may be reversed by the action of cellular protein phosphatases. The phosphorylation may increase or decrease the functional activity of the protein. Several examples of these phosphorylation reactions have been previously discussed (for example, see Chapter 11, p. 125, for the regulation of synthesis and degradation of glycogen).
2.
Glycosylation: Many of the proteins that are destined to become part of a plasma membrane or to be secreted from the cell, have carbohydrate chains attached to the amide nitrogen of asparagine (N-linked) or the hydroxyl groups of serine, threonine, or hydroxylysine (O-linked). N-glycosylation occurs in the endoplasmic reticulum and O-glycosyation in the Golgi. (The process of producing such glycoproteins was discussed on p. 165.) Glycosylation is also used to target proteins to the matrix of lysosomes. Lysosomal acid hydrolases are modified by the phosphorylation of mannose residues at carbon 6 (see p. 169).
3. Hydroxylation: Proline and lysine residues of the a chains of collagen are extensively hydroxylated by vitamin C-dependent hydroxylases in the endoplasmic reticulum (see p. 47).
4.

Other covalent modifications: These may be required for the functional activity of a protein. For example, additional carboxyl groups can be added to glutamate residues by vitamin K-dependent carboxylation (see p. 389). The resulting y-carboxyglutamate (Gla) residues are essential for the activity of several of the blood-clotting proteins. Biotin is covalently bound to the eamino groups of lysine residues of biotin-dependent enzymes that catalyze carboxylation reactions, such as pyruvate carboxylase (see p. 119). Attachment of lipids, such as farnesyl groups, can help anchor proteins to membranes. Histone proteins can be reversibly acetylated (see p. 409).

Proteins must fold to assume their functional state. Folding can be spontaneous (as a result of the primary structure), or facilitated by proteins known as "chaperones" (see p. 20).

Proteins that are defective, for example, misfolded, or destined for rapid turnover are often marked for destruction by ubiquitination— the attachment of chains of a small, highly conserved protein, called ubiquitin (see p. 246). Proteins marked in this way are rapidly degraded by a cellular component known as the proteasome, which
is a macromolecular, ATP-dependent, proteolytic system located in the cytosol. [Note: The AF508 mutation seen in CF causes misfolding of the CFTR protein, resulting in its proteasomal degradation.]

Codons are composed of three nucleotide bases presented in the mRNA language of A, G, C, and U. They are always written 5'~3'. Of the 64 possible three-base combinations, 61 code for the 20 common amino acids and three signal termination of protein synthesis (translation). Altering the nucleotide sequence in a codon can cause silent mutations (the altered codon codes for the original amino acid), missense mutations (the altered codon codes for a different amino acid), or nonsense mutations (the altered codon is a termination codon). Characteristics of the genetic code include specificity, universality, and degeneracy, and it is nonoverlapping and commaless. Requirements for protein synthesis include all the amino acids that eventually appear in the finished protein, at least one specific type of tRNA for each amino acid, one aminoacyl-tRNA synthetase for each amino acid, the mRNA coding for the protein to be synthesized, fully competent ribosomes, protein factors needed for initiation, elongation, and termination of protein synthesis, and ATP and GTP as energy sources. tRNA has an attachment site for a specific amino acid at its 3'-end, and an anticodon region that can recognize the codon specifying the amino acid the tRNA is carrying. Ribosomes are large complexes of protein and rRNA. They consist of two subunits. Each ribosome has three binding sites for tRNA molecules—the A, P, and E sites that cover three neighboring codons. The A-site codon binds an incoming aminoacyl-tRNA, the Psite codon is occupied by peptidyl-tRNA, and the E site is occupied by the empty tRNA as it is about to exit the ribosome. Recognition of an mRNA codon is accomplished by the tRNA anticodon. The anticodon binds to the codon following the rules of complementarity and antiparallel binding. (Nucleotide sequences are ALWAYS assumed to be written in the 5' to 3' direction unless otherwise noted.) The "wobble" hypothesis states that the first (5') base of the anticodon is not as spatially defined as the other two bases. Movement of that first base allows nontraditional base-pairing with the last (3') base of the codon, thus allowing a single tRNA to recognize more than one codon for a specific amino acid. For initiation

of protein synthesis, the components of the translation system are assembled, and mRNA associates with the small ribosomal subunit. The process requires initiation factors. In prokaryotes, a purine-rich region of the mRNA (the Shine-Dalgarno sequence) base-pairs with a complementary sequence on 16S rRNA, resulting in the positioning of the small subunit on the MRNA so that translation can begin. The 5'-cap on eukaryotic mRNA is used to position the small subunit on the MRNA. The initiation codon is AUG; N-formylmethionine is the initiating amino acid in prokaryotes, whereas methionine is in eukaryotes. The polypeptide chain is elongated by the addition of amino acids to the carboxyl end of its growing chain. The process requires elongation factors. The formation of the peptide bond is catalyzed by peptidyltransferase, which is an activity intrinsic to the rRNA of the large subunit. Following peptide bond formation, the ribosome advances along the mRNA in the 5'-3' direction to the next codon (translocation). Because of the length of most mRNAs, more than one ribosome at a time can translate a message, forming a polysome. Termination begins when one of the three termination codons moves into the A site. These codons are recognized by release factors. The newly synthesized protein is released from the ribosomal complex, and the ribosome is dissociated from the MRNA. Initiation, elongation, and termination are driven by the hydrolysis of GTP; initiation in eukaryotes also requires ATP for scanning. Numerous antibiotics interfere with the process of protein synthesis. Many polypeptide chains are covalently modified during or after translation. Such modifications include removal of amino acids, phosphorylation, which may activate or inactivate the protein, glycosylation, which plays a role in protein targeting, or hydroxylation such as that seen in collagen. Proteins must fold to achieve their functional form. Folding can be spontaneous or facilitated by chaperones. Proteins that are defective, for example misfolded, or destined for rapid turnover are marked for destruction by the attachment of chains of a small, highly conserved protein called ubiquitin. Ubiquitinated proteins are rapidly degraded by a

31.1
A
20-year-old anemic
man is
mal form
of
B-globin that
is
172
found found
(Hemoglobin amino
acids in
the normal
point mutations
to have
an
Constant long,
rather protein.
is than
Which consistent
abnor-
Spring)
the
141
of with
this the
following abnormality?
A. UAA
>
CAA.
B. UAA
>
UAG.
C. CGA
>
UGA.
D. GAU
>
GAC.
E. GCA
>
GAA.
31.2 A pharmaceutical company is studying a new antibiotic that inhibits bacterial protein synthesis. When this antibiotic is added to an in vitro protein synthesis system that is translating the mRNA sequence AUGUUUUUUUAG, the only product formed is the dipeptide fMet-Phe. What step in protein synthesis is most likely inhibited by the antibiotic?
A. Initiation.
B. Binding of charged tRNA to the ribosomal A site.
C. Peptidyltransferase activity.
D. Ribosomal translocation.
E. Termination.
A
tRNA
molecule
(tRNA%S)
that is
is mischarged,
supposed to
so that
it carry
cysteine actually
carries alanine
(ala-tRNA‘%’).
Assuming no
correction occurs, what will be the fate of this alanine residue during protein synthesis?
A. It will be incorporated into a protein in response to an alanine codon.
B. It will be incorporated into a protein in response to a cysteine codon.
C. It will remain attached to the tRNA, as it cannot be
used for protein synthesis.
D. It will be incorporated randomly at any codon.
E. It will be chemically converted to cysteine by cellular enzymes.
31.4 In a patient with cystic fibrosis caused by the AF508
mutation, the
mutant cystic
fibrosis transmembrane
conductance regulator
rectly.
The patient's
tein by
(CFTR)
cells attaching
protein modify

ubiquitin folds
incor- this
abnormal molecules
to pro-
it.
What is
the fate
of this
modified
CFTR
protein?
A. It performs its normal function, as the ubiquitin largely corrects for the effect of the mutation.
B. It is secreted from the cell.
C. It is placed into storage vesicles.
D. It is degraded by the proteasome.
E. It is repaired by cellular enzymes.
31.3
Correct answer = A. Mutating the normal stop codon for B-globin from UAA to CAA causes the ribosome to insert a glutamine at that point. It will continue extending the protein chain until it comes upon the next stop codon further down the message, resulting in an abnormally long protein. A change from UAA to UAG would simply change one termination codon for another and would have no effect on the protein. The replacement of CGA (arginine) with UGA (stop) would cause the protein to be too short. GAU and GAC both encode aspartate and would cause no change in the protein. Changing GCA (alanine) to GAA (glutamate) would not change the size of the protein product.
Correct answer = D. Because fMet-Phe is made, the ribosomes must be able to complete initiation, bind Phe-tRNA to the A site, and use peptidyltransferase activity to form the first peptide bond. Because the ribosome is not able to proceed any further, ribosomal movement (translocation) is most likely the inhibited step. The ribosome is, therefore, frozen before it reaches the termination codon of this message.
Correct answer = B. Once an amino acid is attached to a tRNA molecule, only the anticodon of that tRNA determines the specificity of incorporation. The mischarged alanine will, therefore, be incorporated in the protein at a position determined by a cysteine codon.
Correct answer = D. Ubiquitination usually marks old, damaged, or misfolded proteins for destruction by the proteasome. There is no known cellular mechanism for repair of damaged proteins.

31.5 Many antimicrobials inhibit protein translation. Which
of the following antimicrobials is correctly paired with
its mechanism of action?
A. Tetracyclines inhibit peptidyltransferase.
B. Diphtheria toxin binds to the 30S ribosomal subunit.
C. Puromycin inactivates EF-2.
D. Clindamycin binds to the 30S ribosomal subunit.
E. Erythromycin binds to the 50S ribosomal subunit.
Translation of a synthetic polyribonucleotide containing the repeating sequence CAA in a cell-free protein-synthesizing system produces three homopolypeptides: polyglutamine, polyasparagine, and polythreonine. If the codon for glutamine and asparagine are CAA and AAC, respectively, which of the following triplets is the codon for threonine?
A. AAC.
B. CAA.
Cc. CAC.
D. CCA.
E. ACA.
31.7 Which of the following is required for both prokaryotic and eukaryotic protein synthesis?
A. Binding of the small ribosomal subunit to the Shine-Dalgarno sequence.
B. fMet~tRNA.
C. Movement of the mRNA out of the nucleus and into the cytoplasm.
D. Recognition of the 5'-cap by initiation factors.
E. Translocation of the peptidyl-tRNA from the A site to the P site.
31.8 Why is the genetic code described as both degener- ate and unambiguous?

«1-Antitrypsin (A1AT) deficiency can result in emphysema, a lung pathology, because the action of elastase, a serine protease, is unopposed. Deficiency of A1AT in the lungs is the consequence of its retention n (rather than secretion from) the liver, the site of its synthesis. Proteins such as A1AT that are designed to be secreted are best characterized by which of the following statements?
A. Their synthesis is initiated on the smooth endoplasmic reticulum.
B. They contain a mannose 6-phosphate targeting signal.
C. They always contain methionine as the N-terminal amino acid.
D. They are produced from translation products that
have an N-terminal hydrophobic signal sequence.
E.
They contain no sugars with O-glycosidic linkages because their synthesis does not involve the Golgi apparatus.

Correct answer = E. Erythromycin and clindamycin both bind the 50S ribosomal subunit. Tetracyclines inhibit the 30S ribosomal subunit (chloramphenicol inhibits peptidyl transferase). Diphtheria toxin binds EF-2, inactivating it through ADP ribosylation. Puromycin has structure similar to that of aminoacyl-tRNA. It is incorporated into the growing chain, and inhibits further elongation of the peptide chain.
Correct answer = E. The synthetic polynucleotide sequence of CAACAACAACAA . . could be read by the in vitro protein synthesizing system starting at the first C, the first A, or the second A. In the first case, the first triplet codon would be CAA, which codes glutamine; in the second case, the first triplet codon would be AAC, which codes for asparagine; in the last case, the first triplet codon would be ACA, which codes for threonine.
Correct answer = E. In both prokaryotes and eukaryotes, continued translation (elongation) requires movement of the peptidyl-tRNA from the A site to the P site to allow the next aminoacyl-tRNA to enter the A site. Only prokaryotes have a Shine-Dalgarno sequence and use fMet, whereas only eukaryotes have a nucleus and coand posttranscriptionally process their mRNA.
A given amino acid can be coded for by more than one codon (degenerate code), but a given codon codes for just one particular amino acid (unambiguous code).

Answer = D. Synthesis of secreted proteins is begun on free (cytosolic) ribosomes. As the Nterminal hydrophobic signal sequence of the peptide emerges from the ribosome, it is bound by the signal recognition particle, taken to the rough endoplasmic reticulum (RER), threaded into the lumen, and removed as translation continues. The proteins move through the RER and the Golgi, and undergo processing such as Nglycosylation (RER) and O-glycosylation (Golgi). In the Golgi, they are packaged in secretory vesicles and released from the cell. The smooth endoplasmic reticulum is associated with synthesis of lipids, not proteins, and has no ribosomes attached. Phosphorylation at carbon 6 of terminal mannose residues in glycoproteins targets these proteins (acid hydrolases) to lysosomes. The N-terminal methionine is removed from most proteins during processing.

Gene expression refers to the multistep process that ultimately results in the production of a functional gene product, either ribonucleic acid (RNA) or protein. The first step in gene expression—the use of deoxyribonucleic acid (DNA) for the synthesis of RNA (transcription)—is the primary site of regulation in both prokaryotes and eukaryotes. In eukaryotes, however, gene expression also involves extensive posttranscriptional and posttranslational processes, as well as actions that influence access to particular regions of the DNA. Each of these steps can be regulated to provide additional control over the kinds and amounts of functional products that are produced.
Not all genes are regulated. For example, genes described as constitutive encode products required for basic cellular functions, and so are continually expressed; they are also known as "housekeeping" genes. Regulated genes, however, are expressed only under certain conditions. They may be expressed in all cells or in only a subset of cells, for example, hepatocytes. The ability to regulate gene expression, that is, to determine if, how much, and when particular gene products will be made, gives the cell control over structure and function. It is the basis for cellular differentiation, morphogenesis, and adaptability of any organism. Control of gene expression is best understood in prokaryotes, but many themes are repeated in eukaryotes. Figure 32.1 lists some common strategies employed in gene regulation.

Regulation of transcription—the initial step in all gene expression—is controlled by regulatory sequences of DNA, usually embedded in the noncoding regions of the genome. The interaction between these DNA segments and regulatory molecules, such as transcription factors, can engage or repress the transcriptional machinery, influencing the kinds and amounts of products that are produced. These DNA sequences flanking a gene are called cis-acting because they influence expression of genes only on the same chromosome (see p. 423). A trans-acting

Cis-acting elements and trans-acting molecules. Pol I= RNA polymerase II.

Zinc finger is a common motif in proteins that bind DNA.
factor is the regulatory molecule itself, which can diffuse through the cell from its site of synthesis to its DNA-binding site (Figure 32.2). For example, a protein transcription factor (a trans-acting molecule) which regulates a gene on chromosome 6 might itself have been transcribed from a gene on chromosome 11. The binding of proteins to DNA is through structural motifs such as the zinc finger (Figure 32.3), leucine zipper, or helix-turn-helix in the protein. [Note: Some trans-acting factors can negatively affect gene expression.]

In prokaryotes such as Escherichia coli (E. coli), regulation of gene expression occurs primarily at the level of transcription and, in general, is mediated by the binding of trans-acting proteins to cis-acting regulatory elements on their single DNA molecule (chromosome). [Note: Regulating the first step in the expression of a gene is an efficient approach, as energy is not wasted making unneeded gene products.] Transcriptional control in prokaryotes can involve the initiation or premature termination of transcription.

In bacteria, the structural genes that code for proteins involved in a particular metabolic pathway are often found sequentially grouped on the chromosome along with the cis-acting regulatory elements that determine the transcription of these genes. The transcription product is a single polycistronic messenger RNA (mRNA) (see p. 418). The genes are thus coordinately controlled, that is, turned on or off as a unit. This entire package is referred to as an operon.

Prokaryotic operons contain an operator—a segment of DNA that regulates the activity of the structural genes of the operon. If the operator is not bound by a repressor molecule, RNA polymerase passes over the operator and reaches the protein-coding genes which it transcribes to MRNA. If a repressor molecule is bound to the operator, the polymerase is blocked and does not produce mRNA. As long as the repressor is bound to the operator, no proteins are made. However, when an inducer molecule is present, it binds to the repressor, causing the repressor to change shape so that it no longer binds the operator. When this happens, the RNA polymerase can proceed with transcription. One of the best-understood examples is the lactose operon of E. coli, which illustrates both positive and negative regulation (Figure 32.4).

The lactose (lac) operon contains the genes that code for three proteins involved in the catabolism of the disaccharide, lactose: The lacZ gene codes for B-galactosidase, which hydrolyzes lactose to galactose and glucose; the lacY gene, which codes for a permease that facilitates the movement of lactose into the cell; and the lacA gene that codes for thiogalactoside transacetylase whose exact physiologic function is unknown. All of these proteins are produced

The lactose operon of E. coli. *[Note: Even when the operon has been turned off by catabolite repression, the repressor transiently dissociates from the operator at a slow rate, allowing a very low level of expression. The synthesis of a few molecules of permease (and -galactosidase) allows the organism to respond rapidly should glucose become unavailable.]

Helix-turn-helix motif.
when lactose is available to the cell but glucose is not. [Note: Bacteria use glucose as a fuel in preference to any other sugar.] The regulatory portion of the operon is upstream of the three structural genes, and consists of the promoter (P) region where RNA polymerase binds, and two additional sites, the operator (O) site and the CAP site, where regulatory proteins bind. The lacZ, lacY, and lacA genes are expressed only when the O site is empty, and the CAP site is bound by a complex of cyclic adenosine monophosphate (cAMP, see p. 94) and the catabolite gene activator protein or CAP (sometimes called the cAMP regulatory protein or CRP, see Figure 32.4). A regulatory gene, the lacl gene, codes for the repressor protein (a trans-acting factor) that binds to the operator site. [Note: The lacl gene has its own promoter.]
1. When glucose is the only sugar available: In this case, the lac operon is repressed (turned off). Repression is mediated by the repressor protein binding via a helix-turn-helix motif (Figure 32.5) to the operator site, which is downstream of the promoter region (see Figure 32.4A). Binding of the repressor interferes with the progress of RNA polymerase, and blocks transcription of the structural genes. This is an example of negative regulation.

When only lactose is available: In this case, the lac operon is induced (maximally expressed or turned on). A small amount of lactose is converted to an isomer, allolactose. This compound is an inducer that binds to the repressor protein, changing its conformation so that it can no longer bind to the operator. In the absence of glucose, adenylyl cyclase is active, and sufficient quantities of cAMP are made and bind to the CAP protein. The cAMP-CAP trans-acting complex binds to the CAP-binding site, causing RNA polymerase to more efficiently initiate transcription at the promoter site (see Figure 32.4B). This is an example of positive regulation. The transcript is a single polycistronic mRNA molecule that contains three sets of start and stop codons. Translation of the mRNA produces the three proteins that allow lactose to be used for energy production by the cell. [Note: In contrast to the inducible lacZ, lacY, and lacA genes, whose expression is regulated, the lacl gene is constitutive. Its gene product, the repressor protein, is active unless the inducer is present.]
- 3. When both glucose and lactose are available: |In this case, transcription of the lac operon is negligible, even if lactose is present at a high concentration. Adenylyl cyclase is deactivated in the presence of glucose—a process known as catabolite repression—so no cAMP-CAP complex forms and the CAP-binding site remains empty. RNA polymerase is, therefore, unable to effectively initiate transcription, even though the repressor may not be bound to the operator region. Consequently, the three structural genes are not expressed (see Figure 32.4C).

The tryptophan (trp) operon codes for five proteins that are required for the synthesis of the amino acid, tryptophan. As with the lac operon, the trp operon is subject to both positive and negative con-

Attenuation of transcription of the trp operon when tryptophan is plentiful.
trol. Negative control includes trp itself binding to the repressor protein and facilitating the binding of the repressor to the operator. Repression by trp is not always complete, however, and so unlike the lac operon, the trp operon is also regulated by a process known as attenuation. With attenuation, transcription is initiated but is terminated well before completion (Figure 32.6). If trp is plentiful, transcription initiation that escaped repression by trp is attenuated (stopped) by the formation at the 5'-end of the mRNA of a hairpin (stem-loop) structure like that seen in p-independent termination (see p. 421). Transcription and translation are coupled processes in prokaryotes (see p. 438), and, therefore, attenuation also results in the formation of a truncated, nonfunctional peptide product that is rapidly degraded. [Note: If trp is scarce, the operon is expressed. The 5'-end of the mRNA contains two adjacent codons for trp. The lack of trp causes ribosomes to stall at these codons, covering regions of the mRNA required for formation of the attenuation hairpin. This prevents attenuation and thus allows transcription to continue.]
Transcriptional attenuation can occur in prokaryotes because translation of an MRNA begins before its synthesis is complete. In eukaryotes this does not occur because, as a result of having a membrane-bound nucleus, transcription and translation are spatially and temporally separate processes.

Whereas transcriptional regulation of mRNA production is primary in bacteria, regulation at the level of ribosomal RNA (rRNA) and protein synthesis also occurs, and plays important roles in the microbe's ability to adapt to environmental stress.

Regulation of transcription by the stringent response to amino acid starvation.

Regulation of translation by an excess of ribosomal proteins.
=
.
Stringent response: E. coli has seven operons that synthesize the rRNA needed for ribosome assembly, and each is regulated in response to changes in environmental conditions. Regulation in response to amino acid starvation is known as the stringent response. The binding of an uncharged transfer RNA (tRNA) to the A site of a ribosome (see p. 436) triggers a series of events that leads to the production of polyphosphorylated guanosine (ppGpp). The synthesis of this unusual phosphorylated derivative of guanosine diphosphate (GDP) is catalyzed by stringent factor (RelA), an enzyme physically associated with ribosomes. Elevated levels of ppGpp result in inhibition of rRNA synthesis (Figure 32.7), but how ppGpp inhibits the rRNA operons is as yet unclear. [Note: In addition to rRNA, tRNA synthesis and some mRNA synthesis are also inhibited; however, MRNAs that code for enzymes that are required for amino acid biosynthesis are not inhibited. Thus, transcriptional control by the stringent response ultimately results in down-regulation of protein synthesis until amino acids are again available.]
2.
Regulatory ribosomal proteins: Operons for ribosomal proteins (r-proteins) can be inhibited by an excess of their own protein products. For each operon, one specific r-protein functions in the repression of translation of the polycistronic mRNA from that operon (Figure 32.8). The r-protein does so by binding to the Shine-Dalgarno (SD) sequence located on the mRNA just upstream of the initiating AUG codon (see p. 439), and acting as a physical impediment to the binding of the small ribosomal subunit to the SD sequence. One r-protein thus inhibits synthesis of all the r-proteins of the operon. This same r-protein also binds to rRNA, and with a higher affinity than for MRNA. If the concentration of rRNA falls, the r-protein then is available to bind its own mRNA and inhibit its translation. This coordinated regulation keeps the synthesis of r-proteins in balance with the transcription of rRNA, so that each is present in appropriate amounts for the formation of ribosomes.

The higher degree of complexity of eukaryotic genomes, as well as the presence of a nuclear membrane, necessitates a wider range of regulatory processes. As with the prokaryotes, the primary site of regulation is at the level of transcription. Again, the theme of trans-acting molecules binding to cis-acting elements is seen. Operons, however, are not found in eukaryotes, which must use alternative strategies to solve the problem of how to coordinately regulate all the genes required for a specific response. In eukaryotes, gene expression is also regulated at multiple levels other than transcription. For example, the major modes of posttranscriptional regulation at the mRNA level are alternative MRNA splicing, control of mRNA stability, and control of translational efficiency. Additional regulation at the protein level occurs by mechanisms that modulate stability, processing, or targeting of the protein.

Specific transcription factors are trans-acting DNA-binding proteins that function as transcriptional activators. They have at least two binding domains: the DNA-binding domain described above (see p. 450), and the transcription activation domain. The transcription activation domain allows the binding of other proteins, such as coactivators (for example, histone acetyltransferases, or HATs, see Figure 30.11 on p. 422). These facilitate formation of the transcription initiation complex (RNA polymerase II plus the general transcription factors, see p. 423) at the promoter, and thus activate transcription (Figure 32.9). Regulation is achieved by the formation of a multiprotein complex bound to DNA, with protein-protein and protein-DNA interactions controlling assembly of the complex. Although activation domains recruit a variety of proteins, the specific effect of any one of them is dependent upon the protein composition of the complex. This is known as combinatorial control. [Note: DNAbinding proteins can also inhibit transcription.]

The need to coordinately regulate a group of genes to effect a particular response is of key importance in multicellular organisms including humans. An underlying theme occurs repeatedly: A protein binds to a regulatory element for each of the genes in the group and coordinately affects the expression of those genes, even if they are on different chromosomes. For example, hormone-response elements (HREs) are cis-acting DNA sequences that bind trans-acting protein factors and regulate gene expression in response to hormonal signals. In general, hormones bind either to intracellular receptors (steroid hormones are an example, see p. 240), or to cellsurface receptors (the peptide hormone, glucagon, is an example, see p. 314).

Transcriptional regulation by intracellular steroid hormone receptors. GRE = glucocorticoid-response element (an example of a hormone response element); GR = glucocorticoid receptor.
1.
Regulatory signals mediated by intracellular receptors: Members of the nuclear receptor superfamily, which includes the glucocorticoid, mineralocorticoid, sex hormone, vitamin D, retinoic acid, and thyroid hormone receptors, all directly influence activity of transcription factors by alteration of the DNA-binding affinity of the factors. This signal transduction pathway is direct, because the receptor and the transcription factor are the same molecule. For example, hydrophobic steroid hormones such as cortisol (a glucocorticoid) bind to soluble receptors (Figure 32.10). Binding of the steroid hormone ligand causes a conformational change in the receptor that activates it. The receptor—ligand complex enters the nucleus, dimerizes, and, in association with coactivators, binds via a zinc finger motif to nuclear DNA at a cis-acting regulatory element, the glucocorticoid-response element (GRE)—an example of an HRE. Each of the cortisol-responsive genes is under the control of its own GRE. Binding of the receptor-hormone complex to the GRE allows coordinate expression of a group of target genes, even when these genes are located on different chromosomes. The GRE can be located upstream or downstream of the genes it regulates, and is able to function at great distances from those genes. The GRE, then, can function as a true enhancer (see p. 424). [Note: If associated with corepressors, hormonereceptor complexes inhibit transcription.]

Transcriptional regulation by receptors located in the cell membrane.
. Regulatory signals mediated by cell-surface receptors: Cell-surface receptors include those for insulin, epinephrine, and glucagon. Glucagon, for example, is a peptide hormone that binds its G protein-coupled plasma membrane receptor. This extracellular signal is then transduced to intracellular cAMP (Figure 32.11; also see Figure 8.7 on p. 95), which can affect protein expression (and activity) through protein kinase A-mediated covalent modification. In response to a rise in cAMP, a trans-acting factor (CAMPresponse element-binding protein, or CREB) is phosphorylated

Alternative splicing produces multiple related proteins, or isoforms, from a single gene.
and activated. Active CREB binds via a leucine zipper to a cis-acting element, the cAMP-response element (CRE), resulting in the transcription of target genes with CREs in their promoters. [Note: The genes for phosphoenolpyruvate carboxykinase and glucose 6-phosphatase, key enzymes of gluconeogenesis (see p. 117), are examples of genes up-regulated by the cAMP/CRE/CREB system.]

Eukaryotic mRNA undergoes several modifications before it is exported from the nucleus to the cytoplasm for use in protein synthesis (see p. 418). Capping at the 5'-end, polyadenylation at the 3'-end, and splicing are essential processing events for the production of a functional eukaryotic messenger from most pre-mRNA (see p. 425), and variations in these events can affect gene expression. In addition, messenger stability also affects gene expression.
1. Splice-site choice: Tissue-specific protein products (protein isoforms) can be made from the same pre-mRNA through differential cotranscriptional processing, particularly the use of alternative splice sites (Figure 32.12). For example, tropomyosin (TM) is an actin filament-binding protein that regulates the functions of actin in both muscle and nonmuscle cells. Its pre-mRNA undergoes differential splicing to yield a number of tissue-specific TM isoforms (see p. 427).
Over 60% percent of the approximately 30,000 genes in the human genome undergo differential splicing. The use of alternative polyadenylation and transcription start sites is also seen in many genes. This explains, at least in part, how 30,000 genes can give rise to over 100,000 proteins.
2.
MRNA editing: Even after mRNA has been fully processed, it may undergo additional posttranscriptional modification in which a base in the mRNA is altered. This is known as RNA editing. An

RNA editing of apo B in the intestine and generation of the apo B-48 protein needed for chylomicron synthesis.
important example in humans occurs with the transcript for apoprotein (apo) B—an essential protein component of chylomicrons (see p. 228) and very low density lipoproteins (VLDL, see p. 231). Apo B mRNA is made in the liver and the small intestine; however, in the intestine only, the C residue in the codon (CAA) for glutamine is deaminated to U, changing the sense codon to a nonsense or stop codon (UAA, Figure 32.13). This results in a shorter protein (apo B-48, representing 48% of the message) being made in the intestine (and incorporated into chylomicrons) than is made in the liver (apo B-100, full-length, incorporated into VLDL).
. MRNA stability: How long an mRNA remains in the cytosol before it is degraded influences how much protein product can be produced from it. Regulation of iron metabolism and the gene-silencing process of RNA interference provide good examples of the importance of mRNA stability in the regulation of gene expression.
a. Iron metabolism: Transferrin is a plasma protein that transports iron. Transferrin binds to cell-surface receptors (transferrin receptors, or TfRs) that are internalized and provide target cells with iron. The mRNA for the TfR has cis-acting iron-responsive elements (IREs) at its 3'-end. The IREs have a stem-loop structure that can be bound by trans-acting iron regulatory proteins (IRPs, Figure 32.14). When the iron concentration in the cell is low, the IRPs bind to the IREs and stabilize the mRNA for TfR, allowing TfR synthesis. When intracellular iron levels are high, the IRPs preferentially bind iron instead of IREs. The lack of IRPs bound to the mRNA hastens its degradation, resulting in decreased TfR synthesis. [Note: The mRNA for apoferritin, a protein of iron storage, also has IREs; however, they are at the 5'-end of the mRNA.

Regulation of transferrin receptor (TfR) synthesis. IRE = iron-responsive element; IRP = iron-responsive element binding protein.
When iron levels in the cell are low, IRPs bind the IREs and prevent the use of the mRNA; thus, less iron is able to be stored, allowing more iron to be transported to cells. As iron accumulates in the cell, however, the IRP protein becomes iron—bound, and loses its affinity for the IRE on mRNA, resulting in production of apoferritin molecules available to store the excess iron.]
. RNA interference (RNAi): RNAi is a mechanism of gene silencing through decreased expression of mRNA, either by repression of translation or by increased degradation. It is thought to play a key role in such fundamental process as cell proliferation, differentiation, and apoptosis. RNAi is mediated by short (~22 bp), noncoding RNAs called microRNAs (miRNAs), which arise from far longer, genomically encoded nuclear transcripts that are partially processed in the nucleus then transported to the cytoplasm. There, an endonuclease (Dicer) completes the processing and generates short, double stranded (ds) miRNA. A single strand (the guide or antisense strand) of the ds miRNA associates with a cytosolic protein complex known as the RNA-induced silencing complex, or RISC. The guide strand hybridizes with a complementary sequence on a full length target mRNA, bringing RISC to the mRNA. This can result in repression of translation of the mRNA or its degradation by an endonuclease (Argonaute/Ago/ Slicer) of the RISC. The extent of complementarity appears to be the determining factor. RNAi can also be triggered by ds short interfering RNAs (siRNAs) introduced into a cell from exogenous sources. [Note: In vertebrates, the function of siRNAs that may arise from endogenous sources is as yet unclear.] The medical and research applications of RNAi are growing rapidly.

1) RNAi therapy: Modulation of gene expression by providing siRNA to trigger RNAi has enormous therapeutic potential. The first clinical trial of RNAi-based therapy involved patients with age-related macular degeneration (AMD), the leading form of adult blindness in the world. AMD is triggered by overproduction of vascular endothelial growth factor (VEGF). In patients with AMD, too much VEGF leads to the sprouting of excess blood vessels behind the retina. The blood vessels leak, clouding and often entirely destroying vision; hence, AMD is also referred to as "wet" macular degeneration. The siRNA drug—a short dsRNA that specifically targets the MRNA of VEGF and promotes its degradation—is injected into the eye. One siRNA molecule can destroy hundreds of MRNA, resulting in the suppression of thousands of VEGF proteins, thus preventing damaging angiogenesis in the retina. [Note: The first clinical trial, involving about two dozen patients, showed promising results; however, subsequent trials were suspended.] Although the list of potential disease targets is extensive, the development of RNAi-based drugs is hindered by the problem of targeted RNAi delivery.

RNA interference or RNAi by cleavage of target MRNA. [Note: RNAi can also result from inhibition of target MRNA translation.]

Regulation of translation initiation in eukaryotes by phosphorylation of elF-2. RER = rough endoplasmic reticulum.

The methylation of cytosine in eukaryotic DNA. SAM = S-adenosylmethionine; SAH = S-adenosyl homocysteine.
4.
Translation of mRNA: Regulation of gene expression can also occur at the level of translation. One mechanism by which translation is regulated is through phosphorylation of the eukaryotic translation initiation factor, elF-2 (Figure 32.16). Phosphorylation of elF-2 inhibits its function and so inhibits translation at the initiation step (see p. 443). Phosphorylation is catalyzed by kinases that are activated in response to environmental conditions such as amino acid starvation, heme deficiency, the presence of dsRNA, and the accumulation of misfolded proteins in the rough endoplasmic reticulum. [Note: Phosphorylation of elF-2 prevents its reactivation by inhibiting GDP—GTP exchange.]

Gene expression in eukaryotes is also influenced by the availability of DNA to the transcriptional apparatus, the amount of DNA, and the arrangement of DNA. [Note: Localized transitions between the B and Z forms of DNA (see p. 398) can also affect gene expression.]

tone and nonhistone proteins to form chromatin (see p. 409). Transcriptionally active, decondensed chromatin (euchromatin) differs from the more condensed, inactive form (heterochromatin) in a number of ways. Active chromatin contains histone proteins that have been covalently modified at their amino terminal ends by acetylation or phosphorylation (see p. 422 for a discussion of histone acetylation/deacetylation by the histone acetyltransferase and histone deacetylase enzymes). Such modifications decrease the positive charge of these basic proteins, thus decreasing the strength of their association with negatively charged DNA. This chromatin remodeling relaxes the nucleosome (see p. 409), allowing transcription factors access to specific regions on the DNA. A second difference between transcriptionally active and inactive chromatin is the extent of methylation of cytosine bases in CGrich regions (CpG islands) in the promoter region of many genes. Methylation is by methyltransferases that use S-adenosylmethionine as the methyl donor (Figure 32.17). Evidence supports the idea that transcriptionally active genes are less methylated (hypomethylated) than their inactive counterparts, suggesting that DNA hypermethylation silences gene expression.
2.

affect the amount of gene product produced. An increase in copy number—gene amplification—has contributed to increased genomic complexity and is still a normal developmental process in certain nonmammalian species. In mammalian cells, however, gene amplification is seen in response to particular chemotherapeutic drugs such as methotrexate, an inhibitor of the enzyme dihydrofolate reductase (DHFR), required for the synthesis of thymidine triphosphate (TTP) in the pyrimidine biosynthetic pathway (see p. 304). TTP is essential for DNA synthesis. Gene amplification results in an increase in the number of DHFR genes and resistance to the drug, allowing TTP to be made.

DNA rearrangements in the generation of immunoglobulins.
3. Arrangement of DNA: The process by which immunoglobulins (antibodies) are produced by B lymphocytes involves permanent rearrangements of the DNA in these cells. The immunoglobulins (for example, IgG) consist of two light and two heavy chains, with each chain containing regions of variable and constant amino acid sequence. The variable region is the result of somatic recombination of segments within both the lightand the heavy-chain genes. During B-lymphocyte development, single variable (V), diversity (D), and joining (J) gene segments are brought together through gene rearrangement to form a unique variable region (Figure 32.18). This process allows the generation of 10°-10"" different immunoglobulins from a single gene, providing the diversity needed for the recognition of an enormous number of antigens. [Note: The shift from the membrane-bound form to the secreted form of immunoglobulins involves polyA-site choice (see p. 426).]
4. Mobile DNA elements: Transposons (Tn) are mobile segments of DNA that move in an essentially random manner from one site to another on the same or a different chromosome. Movement is mediated by transposase, an enzyme encoded by the Tn itself. Movement can be direct, in which transposase cuts out and then inserts the Tn at a new site, or replicative, in which the transposon is copied and the copy inserted elsewhere while the original remains in place. In eukaryotes, including humans, replicative transposition frequently involves an RNA intermediate, in which case the Tn is called a retrotransposon (see p. 408). Transposition has expanded the genome, but also has the potential to alter gene expression and even to cause disease. Although the vast majority of retrotransposons in the human genome have lost the ability to move, a small percentage is still active. Their transposition is thought to be the basis for some rare cases of hemophilia A and Duchenne muscular dystrophy. [Note: The growing problem of antibiotic-resistant bacteria is a consequence, at least in part, of the exchange of plasmids among bacterial cells. If the plasmids contain transposons carrying antibiotic resistance genes, the recipient bacteria gain resistance to one or more antimicrobial drugs.]

Gene expression results in the production of a functional gene product (either RNA or protein) through the processes of replication, transcription, and translation (Figure 32.19). Genes can be either constitutive (always expressed, housekeeping genes) or regulated (expressed only under certain conditions in all cells or in a subset of cells). The ability to appropriately express (positive regulation) or repress (negative regulation) genes is essential in all organisms. Regulation of gene expression occurs primarily at the level of transcription in both prokaryotes and eukaryotes, and is mediated through the binding of trans-acting proteins to cis-acting regulatory elements on the DNA. In eukaryotes, regulation also occurs through modifications to the DNA, as well as through posttranscriptional and posttranslational events. In prokaryotes such as E. coli, the coordinate regulation of genes whose protein products are required for a particular metabolic pathway is achieved through operons (groups of genes sequentially arranged on the chromosome along with the regulatory elements that determine their transcription). The lac operon contains the Z, Y, and A structural genes, the protein products of which are needed for the catabolism of lactose. When glucose is available, the operon is repressed by the binding of the repressor protein (the product of the lacl gene) to the operator, thus preventing transcription. When only lactose is present, the operon is induced by an isomer of lactose (allolactose) that binds the repressor protein, preventing it from binding to the operator. In addition, cAMP binds the CAP protein, and the complex binds the DNA at the CAP site. This increases promoter efficiency and results in the expression of the structural genes through the production of a polycistronic mRNA. When both glucose and lactose are present, glucose prevents formation of cAMP and transcription of these genes is negligible. The trp operon contains genes needed for the synthesis of tryptophan, and like the lac operon, it is regulated by positive and negative control. Unlike the lac operon, it is also regulated by attenuation, in which mRNA synthesis that escaped repression by trp is terminated before completion. Transcription of rRNA and tRNA is selectively inhibited in prokaryotes by the stringent response to amino acid starvation. Translation is also a site of prokaryotic gene regulation: when ribosomal proteins are in excess, they bind the

Shine-Dalgarno sequence on their own polycistronic mRNA, preventing ribosomes from binding. Gene regulation is more complex in eukaryotes. Operons are not present, but coordinate regulation of the transcription of genes located on different chromosomes can be achieved through the binding of trans-acting proteins to cis-acting elements. In multicellular organisms, hormones can cause coordinated regulation, either through the binding of the hormone receptor—hormone complex itself to the DNA (as with steroid hormones) or through the binding of a protein that is activated in response to a second messenger (as with glucagon). In each case, binding to DNA is mediated through structural motifs such as the zinc-finger. Coand posttranscriptional regulation is also seen in eukaryotes, and includes splice-site choice, polyA-site choice, mRNA editing, and variations in mRNA stability as seen with transferrin receptor synthesis and with RNA interference. Regulation at the translational level can be caused by the phosphorylation (inhibition) of elF-2. Gene expression in eukaryotes is also influenced by availability of DNA to the transcriptional apparatus, the amount of DNA, and the arrangement of the DNA.

Choose the one correct answer.
32.1 Which of the following mutations is most likely to result in reduced expression of the lac operon?
A. i (no repressor protein made)
B. O° (operator cannot bind repressor protein)
_ C. Cya" (no adenylyl cyclase made)
D. Functionally impaired glucose transporter
Correct answer = C. In the absence of glucose, adenylyl cyclase makes cAMP, which forms a complex with the CAP protein. The cAMP—CAP complex binds the CAP site on the DNA, causing RNA polymerase to bind more efficiently to the lac operon promoter, thus increasing expression of the operon. With cya" mutations, adenylyl cyclase is not made, and so the operon is unable to be turned on even when glucose is absent and lactose is present. The absence of a repressor protein or decreased ability of the repressor to bind the operator results in constitutive (constant) expression of the lac operon.

Summary of key concepts for the regulation of gene expression.
32.2 Which of the following is best described as being trans-acting?
A. CAP site
B. Operator
C. Promoter
D. Repressor
32.3 Which of the following is the basis for the intestine-
specific expression of apoprotein B-48?
A. DNA rearrangement and loss
B. DNA transposition
C. RNA alternative splicing
D. RNA editing
E. RNA interference
32.4 Which of the following is most likely to be true
in hemochromatosis, a disease of iron accumulation in the body?
A. The mRNA for the transferrin receptor (TfR) is stabilized by the binding of IRPs to 3' stem-loop structures known as IREs.
B. The mRNA for the TfR is not bound by IRPs, and is rapidly degraded.
C. The mRNA for apoferritin is not bound by IRPs at its 5' stem-loop IRE, and is translated.
D. The mRNA for apoferritin is bound by IRPs, and is not translated.
E. Both B and C
32.5 After several weeks of chemotherapy with methotrexate, a cancer patient's tumor begins to show signs of resistance to treatment. Which of the following mechanisms is most likely to explain this resistance to methotrexate?
A. Overproduction of dihydrofolate reductase.
B. Overproduction of xanthine oxidase.
C. Deficiency of PRPP synthase.
D. Deficiency of thymidine kinase.
E. Deficiency of thymidylate synthase.

transcribed if:
A. glucose and lactose are available.
B. cAMP levels are low.
C. the operator is mutated and can't be bound by the repressor.
D. the repressor is mutated and can't be bound by
the inducer.
Correct answer = D. A repressor is a molecule that transits to the DNA, binds, and reduces the expression of that DNA; it is said to be transacting. The CAP site, operator, and promoter are part of the DNA itself, and so are said to be cis-acting.
Correct answer = D. The production of apoB-48 in the intestine and apoB-100 in liver is the result of RNA editing in the intestine, where a sense codon is changed to a nonsense codon by posttranscriptional deamination of C to U. DNA rearrangement and transposition, as well as RNA interference and alternate splicing, do alter gene expression, but are not the basis of apoB-48 tissue-specific production.
Correct answer = E. When iron levels in the body are high, as is seen with hemochromatosis, there is increased synthesis of the iron-storage molecule, apoferritin, and decreased synthesis of the transferrin receptor (TfR) that mediates iron uptake by cells. These effects are the result of trans-acting iron regulatory proteins (IRPs) binding iron rather than binding cis-acting ironresponsive elements (IREs), resulting in degradation of the mRNA for TfR, and increased translation of the mRNA for apoferritin.
Correct answer = A. Methotrexate interferes with folate metabolism by acting as a competitive inhibitor of the enzyme dihydrofolate reductase. This starves cells for tetrahydrofolate, and makes them unable to synthesize purines and dTMP. This is especially toxic to rapidly growing cancer cells. Overproduction of dihydrofolate reductase, usually caused by amplification of its gene, can overcome the inhibition of the enzyme at the methotrexate concentrations used for chemotherapy, and can result in resistance of the tumor to treatment by this drug.

Correct answer = C. The lac operon is negatively regulated by the repressor protein binding to the operator region and preventing RNA polymerase from transcribing the Z, Y, and A genes of the operon. If glucose is present, the operon is off as a result of catabolite repression. It is only when glucose is gone, cAMP levels are increased, and lactose is available that the operon is maximally induced. If the inducer can't bind the repressor, the repressor binds the operator and represses transcription.

In the past, efforts to understand genes and their expression have been confounded by the immense size and complexity of human deoxyribonucleic acid (DNA). The human genome contains approximately three billion (10°) base pairs (bp) that encode 20,000 to 30,000 proteincoding genes located on 23 pairs of chromosomes. It is now possible to determine the nucleotide sequence of long stretches of DNA, and the entire sequence of the human genome has been determined. This effort (called the Human Genome Project) was made possible by several techniques that have already contributed to our understanding of many genetic diseases (Figure 33.1). These include, first, the discovery of restriction endonucleases that permit the dissection of huge DNA molecules into defined fragments. Second, the development of cloning techniques, providing a mechanism for amplification of specific nucleotide sequences. Finally, the ability to synthesize specific probes, which has allowed the identification and manipulation of nucleotide sequences of interest. These and other experimental approaches have permitted the identification of both normal and mutant nucleotide sequences in DNA. This knowledge has led to the development of methods for the diagnosis of genetic diseases, and initial successes in the treatment of patients by gene therapy.

One of the major obstacles to molecular analysis of genomic DNA is the immense size of the molecules involved. The discovery of a special group of bacterial enzymes, called restriction endonucleases (restriction enzymes), which cleave double-stranded (ds) DNA into smaller, more manageable fragments, has opened the way for DNA analysis. Because each enzyme cleaves DNA at a specific nucleotide sequence, restriction enzymes are used experimentally to obtain precisely defined DNA segments called restriction fragments.

Restriction endonucleases recognize short stretches of DNA (four to eight bp) that contain specific nucleotide sequences. These sequences, which differ for each restriction enzyme, are palin-

Recognition sequence of restriction endonuclease EcoR| shows two-fold rotational symmetry.

Specificity of Taq! and Haelll restriction endonucleases.
dromes, that is, they exhibit twofold rotational symmetry (Figure 33.2). This means that, within a short region of the double helix, the nucleotide sequence on the two strands is identical if each is read in the 5'3' direction. Therefore, if you turn the page upside down— that is, rotate it 180 degrees around its axis of symmetry—the sequence remains the same.
In bacteria, restriction endonucleases "restrict" the expression of non-bacterial DNA through cleavage. Bacterial DNA is protected by methylation of bases at the restriction site

A restriction enzyme is named according to the organism from which it was isolated. The first letter of the name is from the genus of the bacterium. The next two letters are from the name of the species. An additional letter indicates the type or strain, and a final number is appended to indicate the order in which the enzyme was discovered in that particular organism. For example, Haelll is the third restriction endonuclease isolated from the bacterium Haemophilus aegyptius.

Restriction enzymes cleave dsDNA so as to produce a 3'-hydroxyl group on one end and a 5'-phosphate group on the other. Some restriction endonucleases, such as Taql, form staggered cuts that produce "sticky" or cohesive ends—that is, the resulting DNA fragments have single-stranded (ss) sequences that are complementary to each other (Figure 33.3). Other restriction endonucleases, such as Haelll, produce fragments that have "blunt" ends that are doublestranded and therefore do not form hydrogen bonds with each other. Using the enzyme DNA ligase (see p. 406), sticky ends of a DNA fragment of interest can be covalently joined with other DNA fragments that have sticky ends produced by cleavage with the same restriction endonuclease (Figure 33.4). Another ligase, encoded by bacteriophage T4, can covalently join blunt-ended fragments.

A DNA sequence that is recognized and cut by a restriction enzyme is called a restriction site. Restriction endonucleases cleave dsDNA into fragments of different sizes. For example, an enzyme that recognizes a specific four-base-pair sequence produces many cuts in the DNA molecule, one every 44 bp. In contrast, an enzyme requiring a unique sequence of six base pairs produces fewer cuts (one every 4° bp) and, hence, longer pieces. Hundreds of these enzymes, having different cleavage specificities (varying in both nucleotide sequences and length of recognition sites), are commercially available as analytic reagents.

Introduction of a foreign DNA molecule into a replicating cell permits the cloning or amplification (that is, the production of many identical copies) of that DNA. In some cases, a single DNA fragment can be isolated and purified prior to cloning. More commonly, to clone a nucleotide sequence of interest, the total cellular DNA is first cleaved with a specific restriction enzyme, creating hundreds of thousands of fragments. Each of the resulting DNA fragments is joined to a DNA vector molecule (referred to as a cloning vector) to form a hybrid or recombinant molecule. Each recombinant DNA molecule conveys its inserted DNA fragment into a single host cell, for example, a bacterium, where it is replicated. [Note: The process of introducing foreign DNA into a cell is called transformation for bacteria and yeast and transfection for higher eukaryotes.] As the host cell multiplies, it forms a clone in which every bacterium carries copies of the same inserted DNA fragment, hence the name "cloning." The cloned DNA is eventually released from its vector by cleavage (using the appropriate restriction endonuclease) and is isolated. By this mechanism, many identical copies of the DNA of interest can be produced. [Note: An alternative to biologic cloning—the polymerase chain reaction—is described on p. 479.]

A vector is a molecule of DNA to which the fragment of DNA to be cloned is joined. Essential properties of a vector include: 1) it must be capable of autonomous replication within a host cell; 2) it must contain at least one specific nucleotide sequence recognized by a restriction endonuclease; and 3) it must carry at least one gene that confers the ability to select for the vector, such as an antibiotic resistance gene. Commonly used vectors include plasmids and viruses.
1.
Prokaryotic plasmids: Prokaryotic organisms typically contain single, large, circular chromosomes. In addition, most species of bacteria also normally contain small, circular, extrachromosomal DNA molecules called plasmids' (Figure 33.5). Plasmid DNA undergoes replication that may or may not be synchronized to chromosomal division. Plasmids may carry genes that convey antibiotic resistance to the host bacterium, and may facilitate the transfer of genetic information from one bacterium to another. Plasmids can be readily isolated from bacterial cells, their circular DNA cleaved at specific sites by restriction endonucleases, and up to 10 kb of foreign DNA (cut with the same restriction enzyme) inserted. The recombinant plasmid can be introduced into a bacterium, and large numbers of copies of the plasmid produced. The bacteria are grown in the presence of antibiotics, thus selecting for cells containing the hybrid plasmids, which provide antibiotic resistance (Figure 33.6). [Note: The experiment is conducted to favor only one DNA fragment being inserted into each plasmid and only one plasmid being taken up by each bacterium.]
Chapter 7 in Lippincott's Illustrated Reviews: Microbiology for a
'See discussion of plasmids.

Formation of recombinant DNA from restriction fragments with "sticky" ends.

A restriction map of plasmid pBR322 indicating the positions of its antibiotic resistance genes and the sites of nucleotide sequences recognized by specific restriction endonucleases.

Summary of gene cloning.
2. Other vectors: The development of improved vectors that can more efficiently accommodate larger DNA segments, or express the passenger genes in different cell types, has aided molecular genetics research. In addition to the prokaryotic plasmids described above, naturally occurring viruses that infect bacteria (bacteriophage A, for example) or mammalian cells (retroviruses, for example), as well as artificial constructs such as cosmids and bacterial or yeast artificial chromosomes (BACs or YACs, respectively), are currently in wide use as cloning vectors. [Note: BACs and YACs can accept DNA inserts of 100-200 kilobases (kb) and 200-500 kb, respectively.]

A DNA library is a collection of cloned restriction fragments of the DNA of an organism. Two kinds of libraries are commonly used: genomic libraries and complementary DNA (cDNA) libraries. Genomic libraries ideally contain a copy of every DNA nucleotide sequence in the genome. In contrast, cDNA libraries contain those DNA sequences that only appear as processed mRNA molecules, and these differ from one cell type to another. [Note: cDNA lacks introns and the control regions of the genes, whereas these are present in genomic DNA.]
1.
Genomic DNA libraries: A genomic library is created by digestion of the total DNA of the organism with a restriction endonuclease and subsequent ligation to an appropriate vector. The recombinant DNA molecules replicate within host bacteria. The amplified DNA fragments thus represent the entire genome of the organism and are called a genomic library. Regardless of the restriction enzyme used, the chances are rather good that the gene of interest contains more than one restriction site recognized by that enzyme. If this is the case, and if the digestion is allowed to go to completion, the gene of interest is fragmented—that is, it is not contained in any one clone in the library. To avoid this usually undesirable result, a partial digestion is performed in which either the amount or the time of action of the enzyme is limited. This results in cleavage occurring at only a fraction of the restriction sites on any one DNA molecule, thus producing fragments of about 20 kb. Enzymes that cut very frequently (that is, those that recognize 4 bp sequences) are generally used for this purpose so that the result is an almost random collection of fragments. This ensures a high degree of probability that the gene of interest is contained, intact, in some fragment.
2.

cDNA libraries: If a protein-coding gene of interest is expressed at a high level in a particular tissue, it is likely that the messenger RNA (mRNA) transcribed from that gene is also present at high concentrations in the cell. For example, reticulocyte mRNA is composed largely of molecules encoding the a-globin and B-globin chains of hemoglobin. This mRNA can be used as a template to make a complementary DNA (cDNA) molecule using the enzyme reverse transcriptase (Figure 33.7). [Note: mRNA is isolated from transfer RNA and ribosomal RNA by the presence of its polyA tail.] The resulting cDNA is thus a double-stranded copy of mRNA. cDNA can be amplified by cloning or by the polymerase chain reaction. It can be used as a probe to locate the gene that coded for the original mRNA (or fragments of the gene) in mixtures containing many unrelated DNA fragments. If the mRNA used as a template is a mixture of many different size species, the resulting cDNA is heterogeneous. These mixtures can be cloned to form a cDNA library. Because cDNA has no intervening sequences, it can be cloned into an expression vector for the synthesis of eukaryotic proteins by bacteria (Figure 33.8). These special plasmids contain a bacterial promoter for transcription of the

Synthesis of cDNA from mRNA using reverse transcriptase. Additional steps (not shown) are required to clone the cDNA.

An expression vector.
cDNA, and a Shine-Dalgarno sequence (see p. 439) that allows the bacterial ribosome to initiate translation of the resulting MRNA molecule. [Note: Therapeutic human insulin is made in bacteria through this technology; however, the extensive coand posttranslational modifications (see p. 443) required for most other human proteins necessitates the use of eukaryotic hosts.]

The base sequence of DNA fragments that have been cloned can be determined. The original procedure for this purpose was the Sanger dideoxy method illustrated in Figure 33.9. The ssDNA to be sequenced is used as the template for DNA synthesis by DNA polymerase. A radiolabeled primer complementary to the 3'-end of the target DNA is added, along with the four deoxyribonucleoside triphosphates (dNTP). The sample is divided into four reaction tubes, and a small amount of one of the four dideoxyribonucleoside triphosphates (ddNTP) is added to each tube. Because it contains no 3'-hydroxyl group, incorporation of a ddNMP terminates elongation at that point. The products of this reaction, then, consist of a mixture of DNA strands of different lengths, each terminating at a specific base. Separation of the various DNA products by size using polyacrylamide gel electrophoresis, followed by autoradiography, yields a pattern of bands from which the DNA base sequence can be read. [Note: The shorter the fragment, the farther it travels on the gel, with the shortest fragment representing that which was made first, that is, the 5'-end.] The Human Genome Project used highly automated variations of this technique to determine the base sequence of the human genome.

Cleavage of large DNA molecules by restriction enzymes produces a bewildering array of fragments. How can the DNA sequence of interest be picked out of a mixture of thousands or even millions of irrelevant DNA fragments? The answer lies in the use of a probe—a short piece of ssDNA, labeled with a radioisotope, such as 32P or with a nonradioactive molecule, such as biotin. The sequence of a probe is complementary to a sequence in the DNA of interest, called the target DNA. Probes are used to identify which band on a gel or which clone in a library contains the target DNA, a process called screening.

The utility of probes hinges on the phenomenon of hybridization (or annealing) in which a probe containing a complementary sequence binds a single-stranded sequence of a target DNA. ssDNA, produced by alkaline denaturation of dsDNA, is first bound to a solid support, such as a nitrocellulose membrane. The immobilized DNA strands are prevented from self-annealing, but are available for hybridization to an exogenous, radiolabeled, ssDNA probe. The extent of hybridization is measured by the retention of radioactivity on the membrane. Excess probe molecules that do not hybridize are removed by washing the filter.

Allele-specific oligonucleotide probe detects hemoglobin (Hb) S allele. [Note: * indicates **P_ radiolabel.]
1.
Detecting the BS-globin mutation: Figure 33.10 shows how an allele-specific oligonucleotide (ASO) probe can be used to detect the presence of the sickle cell mutation in the B-globin gene. DNA, isolated from white blood cells and amplified, is denatured and applied to a filter. A radiolabeled oligonucleotide probe, complementary to the point mutation (GAG —> GTG, glutamate — valine) at position six in patients with the BS gene, is applied to the filter. DNA isolated from a heterozygous individual (sickle cell trait) or a homozygous patient (sickle cell disease) contains a sequence that is complementary to the probe; thus, a double-stranded hybrid forms that can be detected by electrophoresis. In contrast, DNA obtained from normal individuals is not complementary at this positon and, therefore, does not form a hybrid (see Figure 33.10). Use of a pair of such ASO probes (one specific for the normal allele and one specific for the mutant allele) allows all three possible genotypes—homozygous normal, heterozygous, and homozygous mutant (Figure 33.11)—to be distinguished. [Note: ASO probes are useful only if the mutation and its location are known.]

Because the disposal of radioactive waste is becoming increasingly expensive, nonradioactive probes have been developed. One of the most successful is based on the vitamin biotin (see p. 381), which can be chemically coupled to the nucleotides used to synthesize the probe. Biotin was chosen because it binds very tenaciously to avidin—a readily available protein contained in chicken egg whites. Avidin can be attached to a fluorescent dye detectable optically with great sensitivity. Thus, a DNA fragment (displayed, for example, by gel electrophoresis) that hybridizes with the biotinylated probe can be made visible by immersing the gel in a solution of dye-coupled avidin. After washing away the excess avidin, the DNA fragment that binds the probe is fluorescent. [Note: Fluorescent probes can allow detection and localization of DNA (or mRNA) sequences in cell or tissue preparations, a process called in situ hybridization.]

Sometimes no amino acid sequence information is available to guide the synthesis of a probe for direct detection of the DNA of interest. In this case, a gene can be identified indirectly by cloning cDNA in an expression vector that allows the cloned cDNA to be transcribed and translated. A labeled antibody is used to identify which bacterial colony produces the protein and, therefore, contains the cDNA of interest. [Note: A library created using expression vectors is called an expression library.]
Recombinant DNA technology has allowed the production, in nonhuman systems, of human enzymes that have been modified for appropriate targeting to lysosomes. Such recombinant enzyme replacement therapy (ERT) is used in the treatment of lysosomal storage diseases such as Pompe, Gaucher, and Hurler.

Southern blotting is a technique that can detect mutations in DNA. It combines the use of restriction enzymes, electrophoresis, and DNA probes.

This method, named after its inventor, Edward Southern, involves the following steps (Figure 33.12). First, DNA is extracted from cells, for example, a patient's leukocytes. Second, the DNA is cleaved into many fragments using a restriction enzyme. Third, the resulting fragments are separated on the basis of size by electrophoresis. [Note: As the large fragments move more slowly than the smaller ones, the lengths of the fragments, usually expressed as the number of base pairs, can be calculated from comparison of the position of the band relative to standard fragments of known size.] The DNA fragments in the gel are denatured and transferred (blotted) to a nitrocellulose membrane for analysis. If the original DNA represents the individual's entire genome, the enzymic digest contains a million or more fragments. The gene of interest is on only one (or a few if the gene itself was fragmented) of these pieces of DNA. If all the DNA segments were visualized by a nonspecific technique, they would appear as an unresolved blur of overlapping bands. To avoid this, the last step in Southern blotting uses a probe to identify the DNA fragments of interest. The patterns observed on Southern blot analysis depend both on the specific restriction endonuclease and on the probe used to visualize the restriction fragments. [Note: Variants of the Southern blot have been facetiously named "Northern" (electrophoresis of mRNA followed by hybridization with a specific probe), and "Western" (electrophoresis of protein followed by detection with an antibody directed against the protein of interest), neither of which relates to anyone's name or to points of the compass.]

Southern blotting can detect DNA mutations such as the insertion or deletion of nucleotides. It can also detect point mutations (replacement of one nucleotide by another, see p. 433) that cause the loss or gain of restriction enzyme cleavage sites. Such mutations cause the pattern of bands to differ from those seen with a normal gene. Longer fragments are generated if a restriction site is lost. For example, in Figure 33.12, Person 2 lacks a restriction site present in Person 1. Alternatively, the point mutation may create a new cleavage site with the production of shorter fragments. [Note: Most sequence differences at restriction sites represent normal variations present in the DNA.]

Genome variations are differences in the sequence of DNA among individuals. It has been estimated that the genomes of nonrelated people differ at about one in 1,200 DNA bases, or about 0.1% of the genome. These genome variations include both mutations and polymorphisms. A mutation refers to an infrequent, but potentially harmful, genome varia-

Allele-specific oligonucleotide (ASO) probes used to detect the sickle cell mutation and differentiate between sickle cell trait and disease.
Figure 33.12
Southern blotting procedure.
tion that is associated with a specific human disease. In contrast, a polymorphism is a clinically harmless DNA variation that does not affect the phenotype. It is traditionally defined as a sequence variation at a given locus (allele) in more than 1% of a population. Polymorphisms primarily occur in regions of the genome that do not encode proteins, that is, in introns and intergenic regions. [Note: Less than 2% of the human genome actually encodes proteins.] A restriction fragment length polymorphism (RFLP) is a genetic variant that can be observed by cleaving the DNA into fragments (restriction fragments) with a restriction enzyme. The length of the restriction fragments is altered if the genetic variant alters the DNA so as to create or abolish a site of restriction endonuclease cleavage (a restriction site). RFLP can be used to detect human genetic variations, for example, in prospective parents or in fetal tissue.

Two types of DNA variation commonly result in RFLP: single-base changes in the nucleotide sequence, and tandem repeats of DNA sequences.
1. Single base changes in DNA: About 90% of human genome variation comes in the form of single-nucleotide polymorphisms, (SNPs, pronounced "snips"), that is, variations that involve just one base (Figure 33.13). The substitution of one nucleotide at a restriction site can render the site unrecognizable by a particular restriction endonuclease. A new restriction site can also be created by the same mechanism. In either case, cleavage with an endonuclease results in fragments of lengths differing from the normal, which can be detected by DNA hybridization (see Figure 33.12). [Note: The altered restriction site can be either at the site of a disease-causing mutation (rare) or at a site some distance from the mutation, see p. 477.]
2. Tandem repeats: Alternatively, polymorphism in chromosomal DNA can arise from the presence of a variable number of tandem repeats (VNTR, see Figure 33.14). These are short sequences of DNA at scattered locations in the genome, repeated in tandem (one after another). The number of these repeat units varies from person to person, but is unique for any given individual and, therefore, serves as a molecular fingerprint. Cleavage by restriction enzymes yields fragments that vary in length depending on how many repeated segments are contained in the fragment. Variation in the number of tandem repeats can lead to polymorphisms (Figure 33.14). Many different VNTR loci have been identified, and are extremely useful for DNA fingerprint analysis, such as in forensic and paternity identity cases. It is important to emphasize that these polymorphisms, whether SNP or VNTR, are simply markers, which, in most cases, have no known effect on the structure, function, or rate of production of any particular protein. [Note: Microsatellites are a type of VNTR.]

If the DNA of an individual has gained a restriction site by base substitution, then enzymic cleavage yields at least one additional fragment. Conversely, if a mutation results in loss of a restriction site,

Common forms of genetic polymorphism. SNP = single-nucleotide polymorphism.
Restriction fragments
KReleonaD)
—
aa fewer fragments are produced by enzymic cleavage. An individual who is heterozygous for a polymorphism has a sequence variation in the DNA of one chromosome, and not in the DNA of the homologous chromosome. In such individuals, each chromosome can be traced from parent to offspring by determining the presence or absence of the polymorphism.

a . . . . Families with a history of severe genetic disease, such as an affected previous child or near relative, may wish to determine the presence of the disorder in a developing fetus. Prenatal diagnosis, in association with genetic counseling, allows for an informed reproductive choice if the fetus is affected.
1.
Methods available: The available diagnostic methods vary in sensitivity and specificity. Visualization of the fetus, for example, by ultrasound or fiberoptic devices (fetoscopy), is useful only if the genetic abnormality results in gross anatomic defects, for example, neural tube defects. The chemical composition of the amniotic fluid can also provide diagnostic clues. For example, the presence of high levels of o-fetoprotein is associated with neural tube defects. Fetal cells obtained from amniotic fluid or from biopsy of the chorionic villi can be used for karyotyping, which assesses the morphology of metaphase chromosomes. New staining and cell sorting techniques have permitted the rapid identification of trisomies and translocations that produce extra chromosome or chromosomes of abnormal . . lengths. However, molecular analysis of fetal DNA promises to provide the most detailed genetic picture.
- 2. Sources of DNA: DNA may be obtained from white blood cells, amniotic fluid, or chorionic villi (Figure 33.15). For amniotic fluid, in the past, it was necessary to culture the cells in order to have sufficient DNA for analysis. This took 2-3 weeks to grow a sufficient number of cells. The development of the polymerase chain reaction (see below) has dramatically shortened the time needed for a DNA analysis.
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Pattern of fragments varies from one person to another according to where and how many repeated units of the VNTR are located in their genomes.

Restriction fragment length poly- morphism (RFLP) of variable number tandem repeats (VNTR). homoisgce chrownbcomes is shown.
3. Direct diagnosis of sickle cell anemia using RFLP: The genetic disorders of hemoglobin are the most common genetic diseases in humans. In the case of sickle cell anemia (Figure 33.16), the mutation that gives rise to the disease is actually one and the same as the mutation that gives rise to the polymorphism. Direct detection by RFLP of diseases that result from point mutations is, however, limited to only a few genetic diseases.
a. Early efforts to diagnose sickle cell anemia: Prenatal diagnosis of hemoglobinopathies has in the past involved the determination of the amount and kinds of hemoglobin (Hb) synthesized in red cells obtained from fetal blood. For example, the presence of Hb S in hemolysates indicated sickle cell anemia. However, the invasive procedures to obtain fetal blood have a high mortality rate (approximately 5%), and diagnosis cannot be carried out until late in the second trimester of pregnancy when Hb S begins to be produced.
b. RFLP analysis: Sickle cell anemia is an example of a genetic disease caused by a point mutation (see p. 35). The sequence altered by the mutation abolishes the recognition site of the restriction endonuclease Msitll that recognizes the nucleotide sequence CCTNAGG (where N is any nucleotide, see Figure 33.16). Thus, the A-to-T mutation within a codon of the psglobin gene eliminates a cleavage site for the enzyme. Normal DNA digested with Msiil yields a 1.15-kb fragment, whereas a 1.35-kb fragment is generated from the BS gene as a result of the loss of one Msitll cleavage site. Diagnostic techniques for analyzing fetal DNA (from amniotic cells or chorionic villus sampling), rather than fetal blood, have proved valuable because they provide safe, early detection of sickle cell anemia, as well as other genetic diseases. [Note: Genetic disorders caused by the insertion or deletion of nucleotides between two restriction sites, rather than by the creation or loss of cleavage sites, will also display RFLP]

4. Indirect, prenatal diagnosis of phenylketonuria using RFLP: The gene for phenylalanine hydroxylase (PAH), deficient in phenylketonuria (PKU, see p. 270), is located on chromosome 12. It spans about 90 kb of genomic DNA, and contains 13 exons separated by introns (Figure 33.17; see p. 426 for a description of exons and introns). Mutations in PAH usually do not directly affect any restriction endonuclease recognition site. To establish a diagnostic protocol for this disease, one has to analyze DNA of family members of the affected individual. The goal is to identify genetic markers (RFLP) that are tightly linked to the disease trait. Once these markers are identified, RFLP analysis can be used to carry out prenatal diagnosis.
a. Identification of the gene: One can determine the presence of the mutant gene by identifying the polymorphism marker if two conditions are satisfied. First, if the polymorphism is closely linked to a disease-producing mutation, the defective gene can be traced by detection of the RFLP. For example, if one exam-

Sampling of fetal cells. A. Amniotic fluid. B. Chorionic villus.
Figure 33.16
Detection of BS-globin mutation. bp = base pair; Hb = hemoglobin
ines, by restriction enzyme cleavage and Southern blotting, the DNA from a family carrying a disease-producing gene, it is sometimes possible to find an RFLP that is consistently associated with that gene (that is, they show close linkage and are coinherited). It is then possible to trace the inheritance of the disease-producing DNA within a family without knowledge of the nature of the genetic defect or its precise location in the genome. [Note: The polymorphism may be known from the a . . . study of other families with the disorder, or may be discovered to be unique in the family under investigation.] Second, for autosomal recessive disorders, the presence of an affected individ-. . wae . . oe oe ual in the family would aid in the diagnosis. This individual would have the mutation present on both chromosomes, allowing identification of the RFLP associated with the genetic disorder.
b.
RFLP analysis: The presence of abnormal genes for PAH can be shown using DNA polymorphisms as markers to distinguish between normal and mutant genes. For example, Figure 33.18 shows a typical pattern obtained when DNA from the white blood cells of a family is cleaved with an appropriate restriction enzyme and subjected to electrophoresis. The vertical arrows represent the cleavage sites for the restriction enzyme used. The presence of a polymorphic site (see p. 473 for a discussion of polymorphisms) creates fragment "b" in the autoradiogram (after hybridization with a labeled PAH-cDNA probe), whereas the absence of this site yields only fragment "a." Note that Subject Il-2 demonstrates that the polymorphism, as shown by the presence of fragment "b," is associated with the mutant gene. Therefore, in this particular family, the appearance of fragment "b" corresponds to the presence of a polymorphic site that tags the abnormal gene for PAH. The absence of fragment "b" corresponds to having only the normal gene. In Figure 33.18, examination of fetal DNA shows that the fetus inherited two abnormal genes from its parents and, therefore, has PKU.

c. Value of screening: DNA-based screening is useful not only in determining if an unborn fetus is affected, but also in detecting carriers of the mutated gene. PKU, like many inborn errors of amino acid metabolism, is inherited as an autosomal recessive trait. Identification of heterozygotes can aid in future family planning.

The polymerase chain reaction (PCR) is a test tube method for amplifying a selected DNA sequence that does not rely on the biologic cloning method described on p. 467. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. The method can be used to amplify DNA sequences from any source—bacterial, viral, plant, or animal. The steps in PCR are summarized in Figures 33.19 and 33.20.

The gene for phenylalanine hydroxylase showing 13 exons, restriction sites, and some of the mutations resulting in phenylketonuria.

Analysis of restriction fragment length polymorphism in a family with a child affected by phenylketonuria. The molecular defect in the gene for phenylalanine hydroxylase (PAH) in the family is not known. The family wanted to know if the current pregnancy would be affected by phenylketonuria.

PCR uses DNA polymerase to repetitively amplify targeted portions of DNA. Each cycle of amplification doubles the amount of DNA in the sample, leading to an exponential increase in DNA with repeated cycles of amplification. The amplified DNA sequence can then be analyzed by gel electrophoresis, Southern blotting, or direct sequence determination.
1. Primer construction: It is not necessary to know the nucleotide sequence of the target DNA in the PCR method. However, it is necessary to know the nucleotide sequence of short segments on

Steps in one cycle of the polymerase chain reaction.
each side of the target DNA. These stretches, called flanking sequences, bracket the DNA sequence of interest. The nucleotide sequences of the flanking regions are used to construct two, single-stranded oligonucleotides, usually 20-35 nucleotides long, which are complementary to the respective flanking sequences. The 3'-hydroxyl end of each oligonucleotide points toward the target sequence (see Figure 33.19). These synthetic oligonucleotides function as primers in PCR reactions.
2. Denature the DNA: The DNA to be amplified is heated to separate the strands.
double-stranded target DNA into single
3. Annealing of primers to ssDNA: The separated strands are cooled and allowed to anneal to the two primers (one for each strand).
}

Multiple cycles of polymerase chain reaction.
4.
Chain extension: DNA polymerase and deoxyribonucleoside triphosphates (in excess) are added to the mixture to initiate the synthesis of two new strands complementary to the original DNA strands. DNA polymerase adds nucleotides to the 3'-hydroxyl end of the primer, and strand growth extends across the target DNA, making complementary copies of the target. [Note: PCR products can be several thousand base pairs long.] At the completion of one cycle of replication, the reaction mixture is heated again to separate the strands (of which there are now four). Each strand binds a complementary primer, and the cycle of chain extension is repeated. By using a heat-stable DNA polymerase (for example, Taq polymerase from the bacterium, Thermus aquaticus that normally lives at high temperatures), the polymerase is not denatured and, therefore, does not have to be added at each successive cycle. Typically 20-30 cycles are run during this process, amplifying the DNA by a million-fold (22°) to a billion-fold (2°). [Note: Each extension product includes a sequence at its 5'-end that is complementary to the primer (see Figure 33.19). Thus, each newly synthesized strand can act as a template for the successive cycles (see Figure 33.20). This leads to an exponential increase in the amount of target DNA with each cycle hence the name "polymerase chain reaction."] Probes can be made during PCR by adding labeled nucleotides to the last few cycles.

The major advantages of PCR over cloning as a mechanism for amplifying a specific DNA sequence are sensitivity and speed. DNA sequences present in only trace amounts can be amplified to become the predominant sequence. PCR is so sensitive that DNA sequences present in an individual cell can be amplified and studied. Isolating and amplifying a specific DNA sequence by PCR is faster and less technically difficult than traditional cloning methods using recombinant DNA techniques.

PCR has become a very common tool for a large number of applications. These include:
1. Comparison of a normal cloned gene with an uncloned mutant form of the gene: PCR allows the synthesis of mutant DNA in sufficient quantities for a sequencing protocol without laboriously cloning the altered DNA.
. Detection of low-abundance nucleic acid sequences: For example, viruses that have a long latency period, such as human immunodeficiency virus (HIV), are difficult to detect at the early stage of infection using conventional methods. PCR offers a rapid and sensitive method for detecting viral DNA sequences even when only a small proportion of cells is harboring the virus.
. Forensic analysis of DNA samples: DNA fingerprinting by means of PCR has revolutionized the analysis of evidence from crime scenes. DNA isolated from a single human hair, a tiny spot of
blood, or a sample of semen is sufficient to determine whether the sample comes from a specific individual. The DNA markers analyzed for such fingerprinting are most commonly short tandem repeat polymorphisms. These are very similar to the VNTRs described previously (see p. 475), but are smaller in size. [Note: Verification of paternity uses the same techniques.]

- fibrosis is an autosomal recessive genetic disease resulting from mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most common mutation is a three-base deletion that results in the loss of a phenylalanine residue from the CFTR protein (see p. 434). Because the mutant allele is three bases shorter than the normal allele, it is possible to distinguish them from each other by the size of the PCR products obtained by amplifying that portion of the DNA. Figure 33.21 illustrates how the results of such a PCR test can distinguish between homozygous normal, heterozygous (carriers), and homozygous mutant (affected) individuals.
PCR can generate 100 billion identical copies of a specific DNA sequence in an afternoon, using a highly automated system.

The tools of biotechnology not only allow the study of gene structure, but also provide ways of analyzing the products of gene expression— mRNA and proteins.

Messenger RNA levels are usually determined by the hybridization of labeled probes to either MRNA itself or to cDNA produced from mRNA. [Note: Amplification of CDNA made from mRNA by retroviral reverse transcriptase (RT) is referred to as RT-PCR.]
- 1. Northern blots: Northern blots are very similar to Southern blots (see Figure 33.12, p. 474), except that the original sample contains a mixture of MRNA molecules that are separated by electrophoresis, then transferred to a membrane and hybridized to a radiolabeled probe. The bands obtained by autoradiography give a measure of the amount and size of particular mRNA molecules in the sample.

- Microarrays: DNA microarrays contain thousands of immobilized DNA sequences organized in an area no larger than a microscope slide. These microarrays are used to analyze a sample for the presence of gene variations or mutations (genotyping), or to determine the patterns of mRNA production (gene expression analysis), analyzing thousands of genes at the same time. For genotyping analysis, the cellular sample is genomic DNA. For expression analysis, the population of mRNA molecules from a

Genetic testing for cystic fibrosis using PCR. CFTR = cystic fibrosis transmembrane conductance regulator.

Microarray analysis of gene expression.
particular cell type is converted to cDNA and labeled with a fluorescent tag (Figure 33.22). This mixture is then exposed to a gene chip, which is a glass slide or membrane containing thousands of tiny spots of DNA, each corresponding to a different gene. The amount of fluorescence bound to each spot is a measure of the amount of that particular mRNA in the sample. DNA microarrays are often used to determine the differing patterns of gene expression in two different types of cell—for example, normal and cancer cells (see Figure 33.22). Physicians hope to one day be able to tailor treatment regimens to each cancer patient, based on the specific microarray expression patterns exhibited by that patient's individual tumor.

The kinds and amounts of proteins in cells do not always directly correspond to the amounts of MRNA present. Some mRNAs are translated more efficiently than others, and some proteins undergo posttranslational modifications by adding sugars or lipids, or both. When investigating one, or a limited number of gene products, it is convenient to use labeled antibodies to detect and quantify specific proteins. However, when analyzing the abundance and interactions of large numbers of cellular proteins (called proteomics, see below), automated methods employing two-dimensional gel electrophoresis, mass spectrometry, multidimensional liquid chromatography, and bioinformatics are employed.
1. Enzyme-linked immunosorbent assays (ELISA): These assays are performed in the wells of a plastic microtiter dish. The antigen (protein) is bound to the plastic of the dish. The probe used consists of an antibody specific for the particular protein to be measured. The antibody is covalently bound to an enzyme, which will produce a colored product when exposed to its substrate. The amount of color produced can be used to determine the amount of protein (or antibody) in the sample to be tested.
2. Western blots: Western blots (also called immunoblots) are similar to Southern blots, except that protein molecules in the sample are separated by electrophoresis and blotted (transferred) to a membrane. The probe is a labeled antibody, which produces a band at the location of its antigen.
3. Detecting exposure to HIV: ELISA and Western blots are commonly used to detect exposure to HIV by measuring the amount of anti-HIV antibodies present in a patient's blood sample. ELISAs are used as the primary screening tool, because they are very sensitive. These assays sometimes give false positives, however, so Western blots, which are more specific, are often used as a confirmatory test (Figure 33.23). [Note: ELISA and Western blots can only detect HIV exposure after anti-HIV antibodies appear in the bloodstream. PCR-based testing for HIV is more useful in the first few months after exposure.]

4. Proteomics: The study of all proteins expressed by a genome, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules, is known as proteomics. The 20,000 to 30,000 proteincoding genes of the human genome translate into over 100,000 proteins when posttranscriptional and posttranslational modifications are considered. Although a genome remains unchanged, the amounts and types of proteins in any particular cell change dramatically as genes are turned on and off. Proteomics offers the potential of identifying new disease markers and drug targets. Figure 33.24 compares some of the analytic techniques discussed in this chapter.

The goal of gene therapy is to insert the normal, cloned DNA for a gene into the somatic cells of a patient who has a defect in that gene as a result of some disease-causing mutation. The DNA must become permanently integrated into the patient's chromosomes in such a way as to be properly expressed to produce the correct protein. For example, patients with severe combined immunodeficiency disease (SCID) have an immune deficiency as a result of mutations in either the gene for adenosine deaminase (see p. 301) or a gene coding for an interleukin receptor subunit (X-linked severe combined immunodeficiency, or SCIDX1). Patients with both kinds of SCID have been successfully treated by incorporating functional copies of the appropriate gene into their cells (Figure 33.25). [Note: This is often called "gene replacement therapy."] Although gene therapy is an attractive therapeutic strategy for individuals with inherited diseases, the method is not without risks. For example, retrovirus-mediated gene transfer was able to correct SCID-X1 in nine of ten patients. However, leukemias developed in several of the patients, presumably because of activation of a hematopoietic oncogene. Clearly, gene therapy is a work in progress.

Testing for HIV exposure by enzymelinked immunosorbent assays (ELISA) and Westam blots.
Southern blot, SAMPLE ANALYZED = DNA. Southern blot, GEL USED = Yes. Southern blot, PURPOSE = Detects DNA changes. Northern blot, SAMPLE ANALYZED = RNA. Northern blot, GEL USED = Yes. Northern blot, PURPOSE = Measures mRNA amounts and sizes. Western blot, SAMPLE ANALYZED = Protein. Western blot, GEL USED = Yes. Western blot, PURPOSE = Measures protein amounts. ASO, SAMPLE ANALYZED = DNA. ASO, GEL USED = No. ASO, PURPOSE = Detects DNA mutations. Microarray, SAMPLE ANALYZED = cDNA or genomic DNA. Microarray, GEL USED = No. Microarray, PURPOSE = Measures many mRNA levels at once; detects genomic changes. ELISA, SAMPLE ANALYZED = Proteins or antibodies. ELISA, GEL USED = No. ELISA, PURPOSE = Detects proteins (antigens) or antibodies; detects genomic changes. Proteomics, SAMPLE ANALYZED = Proteins. Proteomics, GEL USED = Yes. Proteomics, PURPOSE = Measures abundance, distribution, posttranslational modifications, functions, and interactions of cellular proteins

Techniques used to analyze DNA, RNA, and proteins. ASO = allele-specific oligonucleotides. ELISA = enzyme-linked immunosorbent assay.

Gene therapy for a patient with severe combined immunodeficiency caused by adenosine deaminase deficiency.

Transgenic animals can be produced by injecting a cloned gene into the fertilized egg. If the gene becomes successfully integrated into a chromosome, it will be present in the germline of the resulting animal, and can be passed along from generation to generation. A giant mouse called "Supermouse" was produced in this way by injecting the gene for rat growth hormone into a fertilized mouse egg. In a similar way, transgenic goats and cows can now be designed that produce human proteins, such as blood clotting factors, in their milk. Sometimes, rather than introducing a functional gene into a mouse, a nonfunctional version is inserted. Such genetically engineered animals can be used to produce a colony of "knockout mice" that lack the product of the affected gene. Such animals can then serve as models for the study of a corresponding human disease. [Note: Knock-in mice result if the inserted gene expresses a mutated product or under (over)-expresses a product.]

Restriction endonucleases are bacterial enzymes that cleave doublestranded DNA into smaller fragments. Each enzyme cleaves dsDNA at a specific four to eight-base-long nucleotide sequence, producing DNA segments called restriction fragments. The sequences that are recognized are palindromic. These enzymes form either staggered cuts (sticky ends) or blunt-end cuts on the DNA. A DNA sequence that is recognized by a restriction enzyme is called a restriction site. Bacterial DNA ligases can anneal two DNA fragments from different sources if they have been cut by the same restriction endonuclease. This hybrid combination of two fragments is called a recombinant DNA molecule. Introduction of a foreign DNA molecule into a replicating cell permits the amplification (production of many copies) of the DNA—a process called cloning. A vector is a molecule of DNA to which the fragment of DNA to be cloned is attached. Vectors must be capable of autonomous replication within the host cell, and must contain at least one specific nucleotide sequence recognized by a restriction endonuclease. It must also carry at least one gene that confers the ability to select for the vector, such as an antibiotic resistance gene. Prokaryotic organisms normally contain small, circular, extrachromosomal DNA molecules called plasmids that can serve as vectors. They can be readily isolated from the bacterium, joined with the DNA of interest, and reintroduced into the bacterium which will replicate, thus making multiple copies of the hybrid plasmid. A DNA library is a collection of cloned restriction fragments of the DNA of an organism. A genomic library is a collection of fragments of double-stranded DNA obtained by digestion of the total DNA of the organism with a restriction endonuclease and subsequent ligation to an appropriate vector. It ideally contains a copy of every DNA nucleotide sequence in the

genome. In contrast, complementary DNA (cDNA) libraries contain only those DNA sequences that are complementary to messenger RNA (mRNA) molecules present in a cell, and differ from one cell type to another. Because cDNA has no intervening sequences, it can be cloned into an expression vector for the synthesis of eukaryotic proteins by bacteria. Cloned, then purified, fragments of DNA can be sequenced, for example, using the Sanger dideoxy method. A probe is a small, single-stranded piece of DNA (usually labeled with a radioisotope, such as **P, or another recognizable compound, such as biotin) which has a nucleotide sequence complementary to the DNA molecule of interest (target DNA). Probes can be used to identify which clone of a library or which band on a gel contains the target DNA. Southern blotting is a technique that can be used to detect specific sequences present in DNA. The DNA is cleaved using a restriction endonuclease, and the pieces are separated by gel electrophoresis and are denatured and transferred (blotted) to a nitrocellulose membrane for analysis. The fragment of interest is detected using a probe. The human genome contains many thousands of polymorphisms (DNA sequence variations at a given locus) that do not affect the phenotype of the individual. Polymorphisms can arise from single base changes and from tandem repeats. A polymorphism can serve as a genetic marker that can be followed through families. A restriction fragment length polymorphism is a genetic variant that can be observed by cleaving the DNA into restriction fragments using a restriction enzyme. A base substitution in one or more nucleotides at a restriction site can render the site unrecognizable by a particular restriction endonuclease. A new restriction site also can be created by the same mechanism. In either case, cleavage with the endonuclease results in fragments of lengths differing from the normal that can be detected by DNA hybridization. This technique can be used to diagnose genetic diseases early in the gestation of a fetus. The polymerase chain reaction (PCR), a test tube method for amplifying a selected DNA sequence, does not rely on the biologic cloning method. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. The method

can be used to amplify DNA sequences from any source. Applications of the PCR technique include: 1) efficient comparison of a normal cloned gene with an uncloned mutant form of the gene, 2) detection of low-abundance nucleic acid sequences, 3) forensic analysis of DNA samples, and 4) prenatal diagnosis and carrier detection, for example, of cystic fibrosis. The products of gene expression (MRNA and proteins) can be measured by techniques such as the following: Northern blots are very similar to Southern blots except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radiolabeled probe. Microarrays are used to determine the differing patterns of gene expression in two different types of cells—for example, normal and cancer cells. Enzyme-linked immunosorbent assays and Western blots (immunoblots) are used to detect specific proteins. The goal of gene therapy is the insertion of a normal gene to replace a defective gene. Insertion of a foreign gene into an animal creates a transgenic animal that can produce therapeutic proteins or serve as a model for human diseases.

33.1 Hindlll is a restriction endonuclease commonly used to cut human DNA into pieces before inserting it into
a plasmid. Which of the following is most likely to be the recognition sequence for this enzyme?
A. AAGGAA.
B. AAGAAG.
C. AAGTTC.
D. AAGCTT.
E. AAGAGA.
Correct answer = D. The vast majority of restricMee eases recognize palindromes, and Dene eee eee ee ee CnC aE eee a oe base sequence of the complementary strand. To be a palindrome, both strands must have the same sequence when read in the 5'3' direction. Thus, the complement of 5'-AAGCTT-3' is also 5'-AAGCTT-3'.
33.2
An Ashkenazi Jewish couple brings their 6-month-old son to you for evaluation of listlessness, poor head control, and a fixed gaze. You determine that he has Tay-Sachs disease, an autosomal recessive disorder. The couple also has a daughter. The diagram below shows this family's pedigree, along with Southern blots of an RFLP very closely linked to the hexosaminidase A gene, which is defective in TaySachs. Which of the statements below is most accurate with respect to the daughter?
A. She has a 25% chance of having Tay-Sachs dis-
ease.
B. She has a 50% chance of having Tay-Sachs dis-
ease.
C. She has Tay-Sachs disease.
D. She is a carrier for Tay-Sachs disease.
E. She is homozygous normal.
33.3 A physician would like to determine the global patterns of gene expression in two different types of tumor cells in order to develop the most appropriate form of chemotherapy for each patient. Which of the following techniques would be most appropriate for this purpose?
A. Southern blot.
B. Northern blot.
C. Western blot.
D. ELISA.
E. Microarray.
A
2-week-old infant
is diagnosed
with
a
urea cycle

defect. Enzymic analysis showed no activity for ornithine transcarbamoylase (OTC). Molecular analysis revealed that the mRNA product of the gene for OTC was identical to that of a control. Which of the techniques listed below was most likely used to analyze the size and amount of the mRNA?
A. Dideoxy chain termination.
- B. Northern blot.
C. Polymerase chain reaction.
D. Southern blot
E. Western blot
33.4
Correct answer = E. Both the father and mother must be carriers for this disease. The son must have inherited a mutant allele from each parent. Because he shows only the 3-kb band on the Southern blot, the mutant allele for this disease must be linked to the 3-kb band for both parents. The normal allele must be linked to the 4-kb band in both parents. Because the daughter inherited the 4-kb band from both parents, she must be homozygous normal for the hexosaminidase A gene.
Correct answer = E. Microarray analysis allows the determination of mRNA production (thus, gene expression) from thousands of genes at once. A Northern blot only measures mRNA production from one gene at a time. Western blots and ELISA measure protein production (also gene expression), but only from one gene at a time. Southern blots are used to analyze DNA, not gene expression.
Correct answer = B. Northern blot allows analysis of the mRNA present (expressed) in a particular cell or tissue. Southern blot is used for DNA analysis, whereas Western blot is used for protein analysis. Dideoxy chain termination is used to sequence DNA. Polymerase chain reaction (PCR) is used to generate multiple, identical copies of a DNA sequence in vitro.
A
A, in
Alzheimer’s disease,
21
Abbreviations, for amino acids, 5, 5f
ABCA1,
236
Abetalipoproteinemia, 231
Absorptive state,
321.
See also
Fed state
Acceptable Macronutrient Distribution Ranges

ACE. See Angiotensin-converting enzyme
Acetaldehyde,
317
Acetanilid,
153
Acetate (acetic acid), 6f, 7, 182f in cholesterol synthesis, 220
titration of, 6f, 7
Acetoacetate, 195-196, 196f formation in amino acid catabolism, 261,
262,
266,
266f
Acetoacety!
CoA,
262,
Acetone, formation
266
195,
196f,
262
Acetylation,
422,
422f
Acetylcholinesterase inhibition by insecticides, 62
in membrane protein anchoring, 206

in absorptive/fed
allosteric state,
activation of,
323,
122
amino acids
that form,
266
carboxylation to malonyl CoA, 183-184,

in cholesterol
synthesis, in
citrate synthesis,
220
111-112, conversion
of building
blocks
111f to,
93,
93f cytosolic, production of, 183
decarboxylation of pyruvate to, 96, 105,

in diabetes
mellitus,
197
in fatty acid synthesis, 183-184
formation of
in amino
acid catabolism,
261-262,
266,
266f in fatty acid oxidation, 192, 192f
in gluconeogenesis, 119, 119f, 122
in glycolysis,
96
in ketone
body synthesis,
195-196,
196f oxidation of, 93, 93f
pyruvate dehydrogenase activated by,
119, 119f pyruvate dehydrogenase complex as
323f,
324
in amino
acid,

in tricarboxylic
Acetyl acid
cycle,
109-111,
CoA-ACP
acetyltransferase,
Acetyl
CoA
carboxylase, in
184
183,
185f, absorptive/fed
state, allosteric
activation
324
of, allosteric
inactivation dephosphorylation
183-184, of,
184f
183, of,
in fasting,
330
hormonal regulation
long-term of,
regulation phosphorylation
184f
184
184, of,
184
of,
183-184
short-term regulation
of,
Acetyl
CoA
N*-Acetylcytosine,
183-184, carboxylase-2
(ACC2),
292f
N-Acetyl-D-glucosamine,
142
N-Acetylgalactosamine
(GalNAc),
N-Acetylglucosamindase
160, deficiency,
168
164f
N-Acetylglucosamine
(GIcNAc),
160,
166,
168
N-Acetylglucosamine-6-sulfatase deficiency,
164

synthesis of, 255f, 256
in urea
cycle,
253,
254f
N-Acetylglutamate synthase, 256
N-Acetyllactosamine, 142
CMP-N-Acetylneuraminic acid, 166
N-Acetylneuraminic acid
(NANA),
166
in acidic glycosphingolipids, 209
synthesis of,
160
Acetyl residue, in plasma-activating factor,
202,
202f
Acid/base properties
of amino
acids,
6-9
Henderson-Hasselbalch equation for, 6-9
Acidemia ketone
bodies and,
methylmalonic, 194
Acid hydrolases, 162
Acidic amino acids, 3f, 5
Acidic sugars
in glycosaminoglycans, 157, 157f, 161,
162f synthesis
of,
Acid maltase,
161,
130
Aciduria homogentisic,
274
methylmalonic,
194
orotic,
303,
303f
Acne, retinoic

in citrate
isomerization, inhibition of, 112
Acquired hyperammonemia,
ACTH,
239,
111f,
258
239f
Active sites,
54,
54f,
56,
57
Acute intermittent porphyria, 280, 281f
Acyl carrier protein (ACP), 184
Acyl CoA:cholesterol acyltransferase (ACAT),
177,
233,
234,
234f
Acyl
Acyl
Acyl
Acyl
CoA
dehydrogenase,
76
CoA
derivatives, o-f-unsaturated,
CoA:diacylglycerol
266
acyltransferase
(DGAT),
176
CoA:monoacylglycerol acyltransferase
(MGAT),
176
Acyl
CoA
oxidase,
Acyltransferases,
195
176-177
Added sugars,
365
Adenine, 291, 291f, 292f, 305f, 396, 396f, 397
in codons/genetic
code,
431,
432f acid
for,
385
162f
197
184f
184f
191
190
112
109f
in obesity, 350-351
volume of, 324f, 325

in absorptive/fed state, 324-325
carbohydrate metabolism in, 325, 325f fat metabolism in, 325, 325f
intertissue relationships of, 328f communication with other metabolic
organs,
307,
307f in
diabetes mellitus
type 1, 339, 339f in energy metabolism, 307, 307f
type 2, 343, 344
as
324-325
in energy
storage depot,
fasting,
327,
330f,
331,
331f carbohydrate metabolism in, 331, 331f
fat metabolism
in,
331,
331f intertissue
relationships of,
328f fatty
acids in,
as fuel
reserve,
189
hormones of, and obesity, 352-353
hyperplasia of, 351
hypertrophy of, 351
insulin resistance
and,
343
lipoprotein lipase
in,
228-229
metabolic role of, 307
in obesity,
324-325
subcutaneous,
350
triacylglycerol fate in, 189
visceral,
350

ADP. See Adenosine diphosphate
Adrenal cortex,
cholesterol synthesis
Adrenal cortical steroid hormones

secretion of, 239, 239f
Adrenal medulla, tyrosine hydroxylase in, 286 Adrenergic symptoms, of hyperglycemia, 315 Adrenocorticotropic hormone (ACTH), 239,

Adrenoleukodystrophy, 195, 236
Afferent signals, 352-353, 353f
African Americans lactose intolerance in, 88
sickle cell disease in, 35
Age-related macular degeneration (AMD),

Aggrecan,
158
Aging, telomeres
Alanine, 253
amino group
of dissociation of, 7f, 8
primary,
4f in ammonia transport, 253, 253f
carboxyl group of, dissociation of, 7, 7f catabolism
of,
263,
263f dipolar
form isoelectric
of,
7,
7f form
of,
7f,
8-9
optical properties
of,
5,
5f peptide bond with valine, 13, 14f
pKs of,
8
side chains
of,
2f synthesis of, 267-268, 268f
titration of,
7-9,
7f,
8f transamination of, 263, 263f

in liver
disease,
251-252, mechanism of action, 251
251f and,
408
in,
220

effects of drugs on, 279
in porphyrias, 280
Albinism, 263, 268f, 269f, 273, 273f, 288
oculocutaneous, 272, 272f

of aldosterone, 237
of bile acid, 225
of bilirubin, 282
of free fatty acids, 178, 190
function of, 4

coronary heart disease and, 364-365
in cirrhosis
of liver,
318
in fatty liver, 318
in glycerophospholipids, 202
hypoglycemia and,
317-318,
317f metabolism of, 317-318, 317f
Wernicke-Korsakoff syndrome and, 379
Alcohol dehydrogenase,
317
Aldehyde dehydrogenase, 317
Aldolase,
100
Aldolase
A,
138,
138f
Aldolase
B,
138,
138f deficiency of, 138
Aldose reductase,
139,
140,
141f,
Aldoses, 83, 83f
Aldosterone, 237, 237f, 239, 240f
Alkaline phosphatase, 206
Alkaptonuria, 263, 268f, 269f, 274, 274f
Alkyl group
saturated, in plasma-activating factor,

unsaturated, in plasmalogens, 202, 202f
Allantoin,
298
Allele-specific oligonucleotide (ASO) probe,
472, 4721, 485f
Allolactose, 451f
Allopurinol,
301
Allosteric activators
in absorptive/fed state, 321, 321f in
fasting,
328
in metabolic regulation, 94
Allosteric effectors,
27,
41f,
62-63,
62f in
absorptive/fed state,
321,
321f carboxylation of pyruvate and, 119
328
glucagon levels and, 122

in committed (rate-limiting) step of pathway,
62
effectors or modifiers of, 62-63, 62f, 64f heterotropic,
63
homotropic, negative,
62
positive,
62
sigmoidal kinetics curve of, 57, 62f, 63
Allosteric inhibitors,
in metabolic
regulation,
94,
252
Allysine, in
collagen a-Amanita,
biosynthesis,
RNA
polymerase
424
o4-Antitrypsin deficiency
48,
48f
II
inhibition
(a1-AT)
of,
51f in
emphysema, in
elastin in
50
degradation, lungs,
cells,
313
chains,
a
a
in collagen,
43-44,
43f precursors of (pro-c-chains), 45, 46f
a-helix,
16-17,
16f amino acids disrupting, 16-17
per turn,
16
in myoglobin, 26, 26f
Alpha-Tocopherol, Beta Carotene Cancer
50,
50,
50f by,
62-63
50f
142
Prevention
Trial,
391
Alternative splicing, 457, 457f
Alzheimer disease,
21,
21f,
23f,
231
Amanita phalloides,
251f,
424
Amidation, synthesis of nonessential amino acids by, 268
Amide group, in amino acid side chains, 4
Amine oxidase, 257
Amine(s)
ammonia from, 257
biologically active, 285-287
Amino acid
analyzer,
15,
15f
D-Amino acid
Amino oxidase,
acid pool,
253
246,
246f
Amino acid(s),
1-12
abbreviations and symbols for, 5, 5f absorption
of,
249
in absorptive/fed state, 321
acid/base properties
of,
6-9
acidic,
3f, in
a-helix,
5
16-17
of,

1,
1f, ammonia from, 256
amphoteric properties
attachment to tRNA
enzymes required
site for,
434f,
435
basic,
3f,
5
branched-chain,
326
catabolism of,
266-267, dehydrogenation
4f,
7f, of,
for,
8
9
435,
435f
266f of,
266-267
oxidative decarboxylation
transamination as
of,
266
of,
266
buffers,
6-7,
6f, carbon
skeletons catabolism
8,
9,
Of of,
of,
245,
250
262-267
carboxyl group
of,
1,
1f,
4f,
7
catabolism/degradation of, 245, 249-253,
261-267,
261f in
absorptive/fed acetoacetyl
state,
323f,
CoA
formation
324
in,
266
acetyl
CoA
formation in,
266,
266f fumarate formation in, 263, 263f
a-ketoglutarate formation
in,
250-252,
250f,
252f,
262, oxaloacetate
formation pyruvate
formation succinyl
263f in,
in,
262,
263,
CoA
262f
263f
263-265,
264f classification of, 2-3f, 2-5, 261, 262f
Clinically important,
275f concept map, 11f
conversion to specialized products,
277-290,
277f
D
and forms
of,
5,
5f de novo synthesis of, 245
L
dietary,
245
disulfide bonding by, 4
essential, 261, 262f, 367, 434
free,
1,
1f glucagon secretion and, 313, 313f
glucogenic,
261,
262f,
266
in gluconeogenesis,
118
hydrogen bonding
by,
4,
4f hydrophobic interactions of, 4, 19, 19f

in insulin secretion, 310
ionic interactions
of,
19, ketogenic,
262,
262f,
19f
266
metabolism of,
261f in absorptive/fed state, 323f, 324, 326,

concept map,
275f folic
acid in,
267
metabolic defects in, 270-274
net charge, at neutral pH, 8
nitrogen removal from, 245, 250-253
in urea
cycle,
245f,
253-256,
253f,
254f,
255f nonessential,
261,
262
biosynthesis of, 267-269, 268f nonpolar,
2-4,
2f,
4f location in proteins, 2, 4, 4f
of,
5,
5f oxidative deamination of, 252-253, 252f
allosteric regulation
of,
252
coenzymes in,
252, direction
of peptide
bonds as
252f reactions,
252
of,
1,
1f,
13-14,
14f precursors
of nitrogen-containing
precursors,
277,
277f protein
synthesis, proton
in as
as proton
residue or
sequence acceptors,
donors,
434
5
5
moiety,
14
of,
14-16
45,
45f determination
of,
DNA
analysis
14-15, of,
15f
15-16
of,
1-5,
1f,
2-3f as attachment site for other
compounds,
4
structure of,
substitution
1-5,
1f of
in hemoglobin
in
C
disease, sickle
cell disease,
36,
36f,
36f titration
of,
6f,
7-9,
8f,
Of transamination of, 245, 250-252, 250f,
251f equilibrium of reactions, 251
transport into
cells,
249-250
uncharged polar,
3f,
Amino
4
acid starvation,
stringent

Aminoacyl-tRNA
synthetase,
Amino group,
1, a,
response
435,
435f,
437
1f,
11f transfer
of
(transamination),
250-252,
250f,
251f,
245,
266
dissociation of,
7f,
8
peptide bind to carboxyl group, 13-14, 14f primary,
4,
4f removal
of
(deamination),
252f,
261
secondary,
4,
4f
B-Aminoisobutyrate,
304
5-Aminolevulinic acid (ALA), formation of,
278-279,
278f
6-Aminolevulinic acid
6-aminolevulinic
278
Aminopeptides,

in glycosaminoglycans,
dehydrase, acid
249
synthase-2
157,
161f synthesis
of,
160,
Aminotransferase(s), action
mechanism diagnostic
161f
250,
250f of,
value
251
of, in
liver disease,
251-253,
251-252,
251f
251f in
non-hepatic disease,
252
substrate specificity of, 250-251

from amines,
from
257
amino acids,
from bacterial
disposal
256
action as
urea, in
253, intestines,
257,
257f elevated
levels of,
257-258,
258f from glutamine, 256-257, 257f
332
in liver disease, 258
metabolism of, 245, 256-258, 257f
278f,
279
(ALAS-2),
157f,
160,
245,
252-253,
37
257
targeting DNA gyrase, 401
targeting
RNA
synthesis,
421,
Antibodies, labeled,
Anticancer
472
drugs blocking
DNA
replication,
409,
DNA gyrase as target of, 401
as inhibitors
of deoxyribonucleotide
298
inhibitors as
of purine
293,
294f,
295
as inhibitors of thymidylate synthase,

antiparallel binding with codon, 437, 437f
Antimalarial drugs,
153
Antimycin
A,
76f
Antioxidant reactions, enzymes catalyzing,
148,
148f
Antioxidant(s),
148-149,
Antiparallel binding,
Antiparallel
434f,
B
sheet,
Antipyretics,
377-378,
391
437,
17,
153
AP-endonuclease,
412
apo
B,
457f,
458
apo B-48 protein, 457f, 458
Apoenzyme,
54
Apoferritin,
459
Apolipoprotein B-48, 177, 228
Apolipoprotein B-100, 189, 231
Apolipoprotein
C-ll,
231
E,
231,
Apolipoprotein(s)
classes of,
227
HDL
as reservoir
228
Apoproteins.
See
Apoptosis, mitochondria and, 80
Appetite,
352-353
AraA,
409
Arabinose,
409
AraC,
409
Arachidonic acid, 182, 182f, 183f, 363
in prostaglandin synthesis, 213, 214, 215f
Arginase,
262
Arginine,
5,
262
a-helix disrupted
by,
16-17
catabolism of, 262
cleavage to ornithine and urea, 255
in creatine
287,
288f in
histones, as
410
nitric oxide
synthase
151f side
chains of,
3f transport
of,
250
in urea cycle, 253-255, 254f
Argininosuccinate cleavage
of,
255
255
in urea
cycle,
253-255,
Argininosuccinate
254f lyase,
254
Argonaute synthase,
(Ago),
Aromatase
254f
459
(CYP19),

Arsenic poisoning,
239
101
mechanism of,
Arsenite,
111
Arthritis alkaptonuria and, 274
chronic gouty,
299
Arthrocentesis,
301
Ascorbate, 148
Ascorbic acid,
377-378,
Vitamin C
111
234
437f
17f of,
234
422f
409f substrate,
392f.
See
151, also
deficiency of,
47
Ashkenazi Jews, phospholipid disorders in,
208,
211
Asians, lactose intolerance in, 88
Asites, on
ribosome,
436
Asparaginase, 262

262,
262f side chains of, 3f, 4
synthesis of, 268
Asparagine synthetase, 268
Aspartate transcarbamoylase, 302
Aspartame,
272
Aspartate aminotransferase (AST), 250-253,

diagnostic value
of,
251-253
in jaundice,
Aspartate
284
(aspartic a-helix
acid), disrupted
as atom
as
5,
19f by,
16-17
source for
atom source
302f metabolism
of, oxaloacetate
262,
262f production
as product
of as
amino proton
acid donor,
5
of,
3f, carboxylate
5
group of,
267-268, in
urea cycle,
and,
113
catabolism,
251
5
268f
253-255,

antithrombotic effect
of, as
uncoupler,
AST.
254f
214,
216f
79
See
Aspartate aminotransferase
Asthma,
214
Atherosclerosis, 219, 227
and,
345
homocysteine and, 263
pathogenesis of,
234,
235f premature,
232
Atherosclerotic plaque,
Atorvastatin,
234,
61,
61f,
ATP.
See
235f
224
Adenosine
ATP-ADP
triphosphate transport,
79
ATPase,
232
ATP-binding cassette (ABC) protein, 236
ATP-citrate lyase,
183,
186
ATP-dependent
RNA-DNA
ATP synthase, 78-79
ATP
synthase complexes,
Atractyloside, helicase,
421
74
79
Autoglucosylation, 127
Autoimmune attack,
in mellitus,
338,
Autophosphorylation, type
1
338f
312
Avidin,
381,
472
Axis of
AZT
symmetry,
(zidovudine),

Backward reactions, free energy change in,
70
Bacterial artificial
chromosomes
(BACs),
Bacterial operons, transcription from,
450-452,
451f when glucose is only sugar available, 452
when lactose
is available,
452
Bacteriophage lambda,
468
Barbiturates, porphyria and, 280
Base exchange
reaction, in
phosphatidylserine synthesis, 204f,
205
468
397,
397f
409,
409f purine
ring, for
pyrimidine
292,
293f ring,
302, diabetes
and,
310, simple,
empiric as
structural formula
for, component,
Carbon dioxide
addition of.
as atom
as atom
binding
See
83
83
83-86
Carboxylation source
293f source
302f of,
and oxygen
for purine
ring, for
pyrimidine affinity
hemoglobin,
32
concentration of,
and hemoglobin,
partial oxygen
30
pressure in
of.
See pentose
phosphate
145-146
production of,
in citric
removal of.
Carbonic
See acid
cycle,
Decarboxylation acid,
Carbonic
267
anhydrase,
Carbon monoxide
30
binding, of
hemoglobin, oxygen
32-33, affinity
32f, monoxide
poisoning,
41f
33
monoxyhemoglobin
Carbon
Carbon
Carbon
(HbCO), skeletons
Carbon skeletons,
262-267
of
249
Carbonyl groups,
4f y-Carboxyglutamate,
Carboxyhemoglobin,
B-Carboxyl,
268
y-Carboxyl,
268
Carboxylate group,
Carboxylation of
acetyl
CoA, of
of glutamate,
5
183-184,
389, proteins,
184f
389f
444, of
pyruvate,
Carboxyl
444f
105, group,
106f,

1,
1f,
11f addition
to proteins,
dissociation
443, of,
7,
7f peptide
bond
Cardiac muscle
carnitine deficiency
lipoprotein amino
group, in,
lipase in,
myoglobin in,
skeletal
26
vs.,
326
Cardiac troponin
Cardiolipin,
|
(cTnl),
202, synthesis
of,
Cardiovascular
202f
206
disease,
263,
265
Carnitine deficiencies
of, congenital,
secondary, in
192
192
192
fatty acid
oxidation, functions
of,
190-192,
191f
190-191
sources of,
191
Carnitine-acylcarnitine translocase,
Carnitine acyltransferase
191
|
(CAT-I).
See
Carnitine palmitoyltransferase
palmitoyltransferase
Carnitine
|
|
(CPT-I),
191
deficiency in,
192
II
191,
192
Carnitine
192
shuttle,
190-191,
Cartilage pigmentation,
274
Caspases,
80
Catabolic pathways,
191f in
alkaptonuria,
192
228
66
homocysteine in,
to amino
acids,
389,
32
245,
389f
32
PCO2
pathway,
292, ring,
302, of
affinity of
114f
118-119,
119f
444f
13,
91-93,
93f
14f
(CPT-II),
anabolic pathways vs., 93, 93f as convergent process, 93
conversion of
building blocks
intermediates hydrolysis
of complex
93f oxidation
stages of
of, acetyl
to simple
in,
93,
93f molecules
CoA
91-93, in,
in,
93,
93,
93f
93
Catabolism.
See also

specific pathways
of amino acids, 245, 249-253, 261-267,
261f of
branched-chain amino
acids,
266-267,

Catabolite gene activator protein (CAP), 452
Catalase,
25,
150,
195,
277
Cataracts,
141,
345
Catecholamine(s),
285-287
degradation of, 286, 286f function
of,
285
monoamine oxidase inhibitors, 286-287,

synthesis of, 270f, 285-286, 286f
Catechol-O-methyltransferase (COMT), 286,
286f
Cation-exchange chromatography,
15f
-CCA sequence, 425, 425f
CDP, in phospholipid synthesis, 203, 203f
CDP-choline,
203,
203f
CDP-ethanolamine,
203, cDNA,
469
Cefamandole, 390
Cefoperazone,
390
Celecoxib, 214
Celiac disease
Cell
(celiac cycle,
sprue), eukaryotic,
Cell membrane,
203f
249
406-407,
407f phospholipids
in,
201
Cell(s)
communication between, 94, 94f senescence
in, telomerase
and, signals
within,
94
Cell surface antigenicity, 165, 165f
Cell-surface receptors,
456-457,
456f
Cell surface recognition, 165, 165f
Cellulose,
84
Central dogma of molecular biology, 395,
395f
Central nervous
system.
See also
tyrosine hydroxylase in, 286
Cephalosporins,
390
Ceramidase,
208
Ceramide(s), 203f, 206, 208
Cerebrocan,
158
Cerebrosides,
209
Chaperones,
20,
23f
Chargaff
Rule,
397
Chemical signaling, metabolic regulation via,

chemiosmotic hypothesis, 77-79
Chenodeoxycholic acid, 224, 224f, 225f
Chief cells,
248
Chirality, in amino acids, 5, 5f
Chloramphenicol, 153, 440f
Chocolate cyanosis, 38
Cholecalciferol (vitamin Dg), 386, 386f
Cholecystokinin
(CCK),
176,
248
obesity and, 310, 353
Cholelithiasis,
226-227,
226f
Cholestanol, 224
Cholesterol,
219-224
absorption by intestinal mucosal cells,

bile salts and excretion of, 225
concept map, 243f conversion to pregnenolone, 238, 238f
Brain
14-15,
408,
408f coronary heart disease and, 360-361,
361f,
364-365,
364f degradation of, 224
de novo
deposition
219
of
(plaques), dietary,
219,
219
364,
364f endocytosis
of,
232,
233f effect on cellular homeostasis, 232-234
esterification of,
234-235
excretion of, 224
functions of, 219
hepatic regulation of, 219, 219f as
hydrophobic compound,
219
in lipoproteins,
219,
227,
228f,
232f high density, 232f
low density, 231-234, 232f very
low density,
232f metabolism
of, in
obesity,
219-224
353
plasma,
220,
360-361,
361f coronary heart disease and, 360-361,
361f plasma
lipoproteins, precursor
in as
as
219, of
bile acids,
precursor reverse
of vitamin
transport in
steroid
227,
219
D,
219
of,
235-236, hormone
structure synthesis,
of,
219-220, synthesis
of,
227f
236f
237-238
220f,
237f
220-224, rate-limiting
step
220f, of,
regulation of,
therapy
223
lowering levels
of,
61,
221f,
220-221,
222f
221f
61f,

unesterified,
HDL
uptake of,
Cholesterol-7-a.-hydroxylase,
234,
220,
236f
224,
225f
Cholesterol ester
hydrolase
(cholesterol esterase),
175
Cholesterol ester transfer protein (CETP), 235
Cholesterol gallstones,
226,
226f
Cholesterol side
chain cleavage
enzyme complex (desmolase), 237
Cholesteryl ester(s),
220,
220f

fatty acids
as component
resynthesis of,
227,
228f, of,
181
231,
176-177
secretion from
enterocytes,
177,
231f
177f synthesis of, 234, 234f
transfer from HDL to VLDL, 231, 231f
Cholestyramine,
225
Cholic acid,
224,
224f,
225f
Choline
Dietary Reference Intakes for, 258f as
essential dietary
nutrient,
204
reutilization of,
significance of,
204
synthesis of phosphatidylcholine and,

Chondrodystrophies,
Chondroitin,
163
Chondroitin sulfates,
158, degradation of, 163
162,
163f
Chorionic villi
sampling,
Chromatin,
422,
476,
159f
477f
460
condensed
(heterochromatin), gene
expression,
422
relaxed
(euchromatin), remodeling,
422,
423,
422
460
structure of, 422
Chronic granulomatous disease (CGD), 150
Chromium, Dietary Reference Intakes for, 358f
Chromosome(s)
tracing from parent to offspring, 475-476
Chronic myelogenous leukemia (CML), 298
162
422,
460
Chronic obstructive pulmonary disease, 361f
Chyle,
178
Chylomicron remnants endocytosis of, 230-231
fate of, 178
formation of, 230-231
Chylomicron(s),
177f,
178, assembly
of,
228
composition of, 232f in diabetes mellitus type 1, 339-340, 339f
mellitus type
2,
344
228-231, modification
of
229f nascent,
size and
228
density
Chyme,
178
Chymotrypsin,
56,
249f
Ciprofloxacin,
401

ammonia levels in, 258
a4-antitrypsin deficiency
jaundice in,
284
alcoholic,
318
Cis-acting
DNA
sequences,
449-450,
450f
Cis-acting regulatory elements, 455-456
Cis fatty acids, 363f
Citrate in fatty acid synthesis, 183
isomerization of,
111f,
112
phosphofructokinase inhibition
from acetyl
by,
CoA
and oxaloacetate,
111-112,
111f translocation from mitochondrion to

Citrate synthase,
111,
114,
114f
Citric acid cycle. See Tricarboxylic acid (TCA)
cycle
Citrulline,
257
in nitric oxide synthesis, 151, 151f in urea cycle, 255
CK.
See
Creatine kinase
CkK2 (MB) isoenzyme, 66
Clathrin, in
endocytosis,
232,
Clindamycin,
441f
Cloning,
465,
465f
Cloning,
DNA,
465,
465f,
233f
467-470,
468f libraries
of,
469-470
sequencing of fragments in, 470, 471f vectors,
467-468,
470f expression,
470, plasmid,
470f
467
CMP-NANA
160
Co-activators, 423
Coagulation, vitamin K and, 389, 390f
Cobalamin,
264,
375-377.
See also
Vitamin
Bio
Codons,
431-432,
432f antiparallel binding with anticodon, 437,

initiation,
438f,
439
mutations of, 432f, 433-434, 433f, 434f recognition by tRNA, 437, 437f
termination (stop or nonsense), 432, 432f translation of, 431, 432f
Coenzyme
A,
110,
112,
381,
381f
Coenzyme
Q
(CoQ),
75-76
Coenzyme(s), 54. See also specific enzymes niacin
as,
380
in pyruvate dehydrogenase complex, 110
of vitamin By2, 375-376, 376f
Vitamin
C
as,
377
Cofactors,
54
Colchicine, 301
Colipase,
175
Collagen, 43-49
of,
227,
227f and,
50,
51f
227
99,
112

precursors of
(pro-c-chains), amino acid sequence in, 45, 45f
assembly of, 47
biosynthesis of, 45-48, 46—47f, 51f concept
map,
51f cross-linking of, 45, 48, 48f
degradation of,
48
diseases, 48-49, 48f, 49f, 51f extracellular cleavage of procollagen
molecules,
47
fibril-associated,
44
fibril formation
in, fibril-forming,
44
function of,
43
glycosylation in,
hydroxylation in,
hydroxylysine in,
hydroxyproline
47f,
48
45,
46f,
45,
45,
45f,
45f, in,
45, mechanical
47
46f,
46f,
45f, properties
network-forming,
47
47
46f, of,
44,
45f polypeptide
precursors posttranslational
of,
45-47
modification secretion
of,
47
signal sequence
structural for,
roles of,
45
of, structure
of, triple
helix type
type
43, of,
|,
43
43f,
43f,
43-44,
Il,
45,
45,
44f
43-44,
44f type
Ill,
44,
44f mutations, and Ehlers-Danlos
syndrome,
48
type
IV, type
IX, types
44,
44
of, type
VII, type
XII,
44f
44f
43-44,
44,
44f
44,
44f vitamin
C
and,
Collagenases,
47,
48
Colon cancer,
Colon cancer, hereditary nonpolyposis, 411
Colorimetric test, 85
Combinatorial control, of transcription, 455,
455f
Common intermediate,
for coupling
reactions,
73
Compartmentalization, of enzymes, 55
Compartment for Uncoupling of Receptor and
Ligand

(CURL),
232
Competitive inhibition,
of
Lineweaver-Burke by
statins,
61, enzymes,
plot,
61f
Complementary bases,
397,
397f,
60,
60f,
60-61,
61,
61f
398f
Complementary
DNA
(cDNA),
484
Complementary DNA (cDNA) libraries,
469-470,
469f
Complex oligosaccharides,
Compound
166, heterozygote,
38
Concept map(s),
for
10
metabolism cholesterol,
for
11f of,
275f
243f for
diabetes mellitus,
347f for dietary lipid metabolism, 180f
for
DNA
structure, replication,
and
414—-415f energy
for metabolism,
integration for
enzymes,
67f fatty acid metabolism, for 199f
for feed/fast
cycle,
335f for fructose metabolism, 143f
for galactose
metabolism,
143f for gene expression, regulation of, 463f
166f
60f
51f
46f
47
43
44f
47f
45,
45
46f repair,
of,
319f for gluconeogenesis, 123f
or glycogen
metabolism, for
for or
for or
for for
for or
glycolysis,
107f glycoproteins,
171f glycosaminoglycans,
171f heme
289f macronutrients,
370f monosaccharides,
89f dietary,
143f
NADPH,
155f nitrogen
metabolism, nucleotide
259f metabolism,
305f for
obesity,
355f or oxidative phosphorylation, 81f
for pentose
phosphate pathway,
155f or
protein synthesis,
for
RNA
446f structure
symbols and
used in,
or transcription,
for triacylglycerol
or synthesis,
10f
429f metabolism,
tricarboxylic
199f acid
Congenital adrenal
238f

Congenital disorders
167
Conjugation,
282
Connective tissue proteins, 49. See also
Collagen;
Elastin
Consensus sequences
for
DNA
for replication,
399
RNA
transcription eukaryotic, 423, 423f
prokaryotic, 420, 420f
Constitutive genes,
449
Cooley anemia,
39
Cooperative binding
of oxygen, by hemoglobin, 29, 29f of single-stranded DNA-binding proteins,
400
Cooperativity, of
Copper,
Dietary
Coproporphyria, enzyme
binding
Reference sites,
Intakes hereditary,
Coproporphyrin, for,
280,
277
Coproporphyrinogen
Coprostanol,
224
Core enzyme,
419
Core pentasaccharide, in oligosaccharides,
166,
166f
Core protein, of glycosaminoglycans, 158,
160f,
161
Cori cycle,
118,
118f
Cornea, collagen
in,
43
Coronary artery disease (CAD), 219
Coronary heart
disease alcohol
consumption dietary
plasma and,
fats and,
360-361, lipids
and, soy
protein and,
364-365
361f
360-361,
364
triacylglycerols and,
Corticosteroid-binding
361-362
globulin,
237
Corticosteroids,
237,
237f
Corticotropin-releasing hormone (CRH), 239
Cortisol,
237,
237f action
of,
240f in
hyperglycemia,
315f,
316
prostaglandin synthesis inhibited by, 214
secretion of, 239
in transcriptional regulation, 456
Cosmids,
468
Cosubstrates,
54
Counterregulatory hormones, 313, 316
Coupling reactions
common intermediates for, 72-73
energy, 72-73, 72f
361f
III,
279f oxidase,
281f
281f
62-63

358f cycle,
115f hyperplasias
(CAHs), of
glycosylation
238,
(CDG),
135f
429f
Covalent modification
of enzymes,
63,
64f in absorptive/fed state, 321f, 322
328-329
glucagon of
in,
122, proteins,
122f
443f,
COX-1,
213,
214,
COX-2,
213,
444,
444f
216f
214
C-peptide, in
insulin
C-procollagen synthesis,
peptidases,
Creatine,
287-288
287-288
287,
288f
(CK),
65,
65f in creatine synthesis, 287
Creatine phosphate,
287,
288f
Creatinine,
287,
288f
Creutzfeldt-Jakob disease,
Crigler-Najjar and
22,
II,
282
Cristae,
|
of mitochondrion,
Cyanocobalamin,
74
375,
376f
Cyanogen bromide,
Cyanosis,
16f chocolate,
Cyclic adenosine
38
monophosphate actions
of,
96f glucagon
and,
314
in glycogen degradation, 132-133, 133f glycogen
synthesis inhibition
by,
133-134,
134f hydrolysis
of, as
95-96,
96f second
messenger, in
triacylglycerol
94—96, degradation,
3’,5’-cyclic
291
190,
AMP-dependent protein
190
Cyclic
AMP-independent
PDH
kinase,
190f kinase,
110
Cyclin-dependent kinases (Cdk), 407
Cyclins,
407
Cystathionine
B-synthase, deficiency
in,
265,
273
273
Cystathioninuria,
Cysteine,
4
263

disulfide bonds
of, of,
sulfhydryl
3f, group
synthesis
18f,
269f
19
4
in, of,
Cystic
265, fibrosis
(CF)
ABC
proteins and,
18f,
268
236
mutation causing,
434
pancreatic enzyme deficiency in, 174
prenatal diagnosis
of,
483,
483f steatorrhea in, 177, 248
Cystic fibrosis
transmembrane conductance
regulator (CFTR), 174, 434, 483
Cystine catabolism
of,
263
disulfide bond in, 4, 18f, 19
transport of,
250
Cystinosis,
249
Cystinuria,
249f,
Cytidine,
250,
292,
292f
Cytidine triphosphate
NANA
(CTP)
reaction with,
303,
Cytochrome
a
+
b,
160
303f as,
76
76,
76f c,
25,
25f,
80
Cytochrome oxidase,
76
Cytochrome P450 (CYP), 279
Cytochrome P450 (CYP) mixed-function oxidase,
237
Cytochrome P450 monooxygenase system,
149-150,
149f microsomal,
149-150
268f,
47
268f
19
308,
308f,
309f
23f
(cAMP),
94

in electron transport chain, 75f, 76
Cytoplasm, cholesterol
synthesis in,
220
Cytosine, 291, 291f, 292f, 305f in
codons/genetic code,
431, damage
to and
repair
432f of,
412,
413f pairing
with guanine,
397,
397f,
398f
Cytosine arabinoside (cytarabine, ara C), 409,

Cytosol,
54f cholesterol synthesis in, 220
oxaloacetate transport to, 119, 119f porphyrin synthesis in, 278
transfer of acetyl CoA to, 183, 183f
D
Dactinomycin, 397, 421
Darier disease (keratosis follicularis), 385
dATP,
404
D-configuration of amino acids, 5, 5f

Deamination, oxidative allosteric regulation of, 252
of amino acids, 245, 252-253, 252f coenzymes
in,
252,
252f direction
of reactions,
252
"Debranching enzymes," 128
Decarboxylation of cytosolic oxaloacetate, 119f, 120
of histidine,
287,
287f of
isocitrate,
112
of oxaloacetate,
oxidative of
o-ketoglutarate,
112,
112f thiamine and, 379, 379f
acids, thiamine and, 379f
of pyruvate,
105,
109-111,

7-Dehydrocholesterol,
386,
386f
7-Dehydrocholesterol-7-reductase, 221
Dehydroepiandrosterone,
239
Dehydrogenation, of branched-chain amino

Denaturation of
DNA,
397-398, of
enzymes,
398f
57
of proteins,
20,
23f,
470
Deoxyadenosine, 292f
5'-Deoxyadenosylcobalamin, 375, 376f
Deoxycholic acid,
225
Deoxyhemoglobin,
28
Deoxyribonucleases,
397
Deoxyribonucleoside phosphates, 291. See also Nucleotide(s)
5'-Deoxyribonucleoside triphosphate, 404
Deoxyribonucleoside triphosphates
(dNTPs),
470
in polymerase chain reaction, 482

297-298, regulation
297f of,
297-298,
298f
2-Deoxyribose,
292,
292f
Deoxyribose-phosphate backbone, of DNA,
396-397,
396f,
397f zigzagging of, 398, 399f
Deoxyribose-phosphate lyase, 412
Deoxythymidine,
292
Dephosphorylation of acetyl CoA carboxylase, 183-184
of
119f,
120
110f
266
of enzymes, 63, 63f of fructose 1,6-bisphosphate, 120-121,
120f of
glucose
6-phosphate,
121,
121f of hydroxymethylglutaryl (HMG) CoA
reductase, 223, 223f of
proteins,
95
of pyruvate
kinase,
102
sterol-independent, 223, 223f

Dermatan sulfate, 159f, 163
Desmolase,
237,
238
Desmosine cross-link,
in elastin,
Detoxification, in
cytochrome
49,
P450
monooxygenase dGTP,
system,
404
Diabetes mellitus,
337-348
chronic effects and prevention of, 344-346
347f excessive ketone bodies in, 197
hemoglobin Aj, in, 340, 340f, 346, 346f nutrition
and,
361f sorbitol metabolism in, 345
type
1
(insulin-dependent),
197,
338-341,
347f autoimmune
attack in,
338,
337,
338f diagnosis of, 338
environmental stimulus for, 338, 338f fasting vs., 340
genetic determinant for, 338, 338f intertissue relationships of, 338-339,
339f metabolic changes in, 338-340, 338f
treatment of,
340-341,
340f type
2
(non-insulin-dependent),
337,
341-344,
347f in
children,
337
exercise and
risk of,
genetic determinant
345-346,
346f for,
338
incidence and
prevalence of,
337
intertissue relationships of, 344, 345f metabolic
changes in,
344,
344f obesity and, 342, 342f
progression of
glucose levels
and in,
342-344, insulin
342f,
343f treatment
of,
344
typical progression of, 341-342, 344f type
1
vs.
type
2,
337f
Diabetic ketoacidosis (DKA), 197, 197f, 339
Diabetic nephropathy,
344f,
345
sorbitol metabolism
Diabetic neuropathy,
sorbitol and,
344f, metabolism
Diabetic retinopathy,
140
345
and,
344f,
140
345
and,
140
Diacylglycerolacyltransferase,
176,
(DAG),
189f activation of, 203, 203f

in phospholipid synthesis, 201
in signal
transmission,
205-206
Diarrhea, osmotic,
87
Diastereomers,
84
Dicer,
459
Dicumarol,
389
2',3'-Dideoxyinosine (didanosine, ddl), 409,

Dideoxyribonucleoside triphosphates
(ddNTPs), 470
Diet. See Nutrition
Dietary Reference Intakes (DRIs), 357-358,
357f comparison of components in, 358, 359f
149
49f
177f guide to use, 358, 358f
Dieting, for
weight reduction,
Digestive enzymes
deficiencies of,
diagnosis
88
of,
Dihydrobiopterin, deficiency
88
268
in,
270f
Dihydrofolate
(DHF),
303
Dihydrofolate reductase (DHFR), 267, 460
inhibition of,
293,
294f,
303,
375
Dihydrolipoyl dehydrogenase,
Dihydrolipoyl
110, transacetylase,
110,
110f
110f
Dihydroorotase,
302
Dihydroorotate dehydrogenase, 302
Dihydropteridine (BH2) reductase, 270-271,
270f
Dihydropteridine
(BH2)

Dihydrouracil,
292f
Dihydrouridine,
425f
Dihydroxyacetone, 83, 83f
Dihydroxyacetone phosphate (DHAP), 117,
189
in fructose
138
isomerization of, 101
1,25-Dihydroxycholecalciferol,
240,
382,
386
3,4-Dihydroxyphenylalanine (dopa), 285-286,

N®,N°-Dimethyladenine, 292f
3,3-Dimethylallyl pyrophosphate (DPP), 221
2,4-Dinitrophenol,
78f,
79
1,25-diOH cholecalciferol (calcitriol), 389
Dipalmitoylphosphatidylcholine (DPPC), 204,

Dipeptides, absorption
of,
249
Diphtheria toxin, 441f
Dipolar form, of amino acid, 7, 7f
Disaccharidases,
86
Disaccharide intolerance,
88
Disaccharide(s),
83,
84f,
85
abnormal degradation of, 87-88
dietary, 365
digestion of, 86, 87
in glycosaminoglycans, 157, 157f metabolism
of,
137-144
Dissociation constant
of amino
group,
7f,
8
of carboxyl
group,
7,
7f
Disulfide bonds, 4, 18f, 19, 297, 311-312
Disulfiram,
317
Diverticulosis and
DNA,
395-416
annealing, in

bases of,
396-397, abnormal,
396f removal
alteration hydrogen
pairing of,
of,
410-413
bonds between,
of,
396-397, purine,
291,
396f
291f,
305f pyrimidine,
291,
291f, spontaneous
305f loss
of, unusual,
291-292,

chain elongation
concept
410-413
292f of,
403-405, map,
414—415f denaturation
of,
397-398
in polymerase chain reaction, 481

antiparallel manner
of,
397, axis
of symmetry
for, base
pairing in,
397,
397,
B form of, 398, 399f
397f
397f
397f fiber,
366
synthase,
270-271,
354
chain
412, reaction,
412f
397,
398f
403f
Chargaff's Rules for, 397
complementary nature of, 397, 397f,
398f
A
form of,
398
major (wide) groove of, 397, 397f minor
(narrow)
groove of,
397,
397f strand
separation structural
in,
397-398
forms of,
398,
399f
Z
form of,
398,
399f double-strand breaks in, repair of,

organization of, 409-410, 409f, 410f replication
of,
406-409,
41
4f for
eukaryotic transcription,
information flow
from,
395,
422
395f

melting temperature of, 397-398, 398f nucleoid,
398
nucleoside analog
408-409,
409f
3’
5'-phosphodiester bonds, 396-397,
396f plasmid,
398
polarity of,
396
prokaryotic,
395
See also Prokaryotic DNA
replication of, 399-406
proofreading of,
404—405,
404f regulation
of gene
expression modifications
through to,
460-461,
461f regulatory
sequences renaturation
of,
449-450
(reannealing), repair
of,
410-413,
398
415f base
excision,
412, double-strand
413f breaks,
412-413, homologous
recombination, methyl-directed
413f
413
mismatch nucleotide
excision,
411-412, non-homologous
411f end-joining,
single-stranded
(viral), structure
of,
413
396
396-398, as
template
414f for
RNA,
417.
synthesis
See also
(transcription)
ultraviolet damage
RNA, of
to,
412,
412f variations
in, resulting
in restriction
fragment length polymorphisms,
475, 475f, 476f

DNA analysis, 15-16, 473-485, 485f. See also specific methods
forensic,
482-483
techniques for,
485f

DnaB,
400
DnaC,
400
DnaG,
402
DNA-binding motifs, 423
DNA-binding site,
450,
450f
DNA
cloning,
467-470,
468f libraries of, 469-470
467-468,
470,
470f
DNA fingerprinting, 482-483

blunt ends
of,
466,
466f cloned, sequencing of, 470, 471f
hybridization of probes to, 470
sticky ends of, 466, 466f joining of (recombinant DNA), 467f
460f,
DNA gyrase, 401
DNA helicases, 400, 400f, 406
DNA hybridization, 475
DNA
libraries,
469-470
complementary,
469-470, genomic,
469
DNA
ligase,
406,
406f in
DNA
repair,
412
restriction endonucleases and, 466
DNA
microarrays,
483-484,
484f,
485f
DNA polymerase |, 405, 406, 406f in
DNA
repair,
411
DNA polymerase III, 403-404, 404f, 405,
406
in chain
elongation,
403-405,
404f in proofreading, 404-405, 404f
DNA
polymerase(s),
421
3’5,’
in
DNA
proofreading, chain
elongation
5’~3’
404f, and,
403-404,
DNA
polymerase, in
DNA
repair,
405
404f
404—405,
411,
412
eukaryotic,
407,
407f in polymerase chain reaction, 480-482
sequencing cloned
DNA
fragments

DNA
probes,
465,
465f,
470-472
antibodies,
472
biotinylated,
472
hybridization to DNA fragments, 470
oligonucleotide,
471-472,
472f in
sickle cell
disease,
472,
472f,
473f
DNA sequencing. See also DNA analysis of protein's primary structure, 15-16
DNA
topoisomerase(s),
401
type
I,
401,
421
type
II,
401
Docosahexaenoic acid (DHA), 363
Dolichol,
167
Dolichol-linked oligosaccharide, synthesis of,
167
Domains, of
polypeptides,
19
Dopamine, in catecholamine synthesis,
286,
286f
Dopamine
B-hydroxylase,
Double helix,
286
of
DNA,
395,
397-398, antiparallel
manner of,
397,
397f axis of symmetry for, 397, 397f
base pairing
in,
397,
397f
B
form of,
398,
399f
Chargaff's
Rules for,
397
complementary nature of, 397, 397f
A
form of,
398
major
(wide)
minor groove
of,
(narrow)
groove strand
397,
397f of,
397, in,
397f
397-398
forms of,
398,
399f
Z
form of,
398,
399f
Double-reciprocal plot, of enzyme action,
59,
59f
Down-regulation, 313
Drug absorption, Henderson-Hasselbalch equation
for,
9,
9f dsRNA,
D-sugar,
RNA
interference
84,
84f dTTP,
404
Dubin-Johnson syndrome, 283
Duchenne muscular dystrophy, 461
Duodenun, dietary lipid emulsification in,
175
Dyslipidemia.
See also
Hyperlipidemia/hyperlipoproteine mia
and,
459

404f
469f
397f obesity and, 353
Dystrophin gene, 486
E
EcoRI,
466f
Edman degradation, 15, 15f
Ehlers-Danlos syndrome (EDS), 48, 48f
Eicosanoids,
213
Eicosapentaenoic acid (EPA), 363
Elastase, 249f
Elastase, inhibition
of,
50,
50f deficient, and emphysema, 50
Elastin,
43,
49-51
51f degradation
of,
50
Q4-antitrypsin in,
50,
50f disorders
of,
50,
51f desmosine cross-link in, 49, 49f
43
mechanical structure
properties of,
49,
Electrical
49f gradient,
in oxidative
phosphorylation,
77-79
Electrolyte(s), amphoteric,
Electron acceptors,
Electron
Electron
74,
9
76
donors,
74,
76
transport chain,
ATP
73-77, phosphorylation
75f in,
77-80
coenzyme
Q
in,
75-76
coupled to proton transport, 77-78, 78f cytochromes in, 75f, 76
free energy
release during,
NADH
dehydrogenase
NADH
76-77
in,
75, formation
in,
75, organization
of,
74, reactions
of, site-specific
75f
75f
75f
75-76,
75f, inhibitors
tightly
76f of,
76, coupled
with oxidative
77-78
uncoupling from
78-79
Electrophoresis, in sickle cell disease, 36,
36f
Electrophoretic mobility
of low-density
of lipoproteins,
plasma
227, lipoproteins,
Emphysema,
227, o-antitrypsin
50,
51f
Enalapril,
62

Enantiomers,
84,
84f,
Endergonic reactions,
Endocrine signaling,
Endocytosis of
chylomicrons of
of remnants,
glycosphingolipids,
85f
70
94f
230-231
210
intermediate-density lipoproteins,
231
of low-density lipoproteins, 232, 233f receptor-mediated,
232,
233f
Endoglycosidases,
86
Endonuclease(s), restriction,
465-466,
405,
465f nomenclature
for, restriction
466
sites, specificity
466
of,
“sticky”
465-466, and
“blunt”
467
Endopeptidase,
15,
249
Endoplasmic reticulum
cholesterol synthesis in, 220
rough apolipoprotein synthesis in, 228
collagen precursors in, 45, 46f
466f ends,
466,
228f
228f deficiency
and, of,
43
76f
466f,
glycoprotein synthesis in, 166, 167,
167f,
168f ribosomes
in,
436-437
smooth, cytochrome
P450
monooxygenase system
Endothelium-derived relaxing

End-product inhibition,
293
Energy
ATP
as content
free,
55.
carrier of,
72-73
in food,
359,
See also
of activation,
359f
Free energy
55,
55f change
in,
70-72,
70f standard, change in, 71-72, 71f
production of
in tricarboxylic acid cycle, 113, 113f requirements
in humans,
358-360
use in
body,
359-360,
Energy
Energy
Energy barrier,
359f in
reactions, coupling,
55
72-73,
72f metabolism,
integration
319f
Energy yield
from fatty
acid oxidation,
of,
192,
307,
193f from
104
from oxidation of fatty acids, 189, 192, 193f
Enhancers, in
eukaryotic gene
424,
424f
Enolase,
102
eNOS,
151
Enoyl CoA hydratase, 192
3,2-Enoyl
CoA
isomerase,
Enteral feeding, 369
Enterocytes chylomicron assembly in, 228
enzymes synthesized by, carbohydrate digestion by, 86-87, 87f
lipid absorption
by,
176
lipid secretion
from, monosaccharide
177-178
absorption
Enterohepatic by,
circulation,
Enterohepatic
225-226, urobilinogen

Enteropeptidase
(enterokinase),
Enthalpy,
69,
69f
Entropy, 69, 69f
Environmental factors, in obesity, 352
Enzyme kinetics curve, 57, 57f
Enzyme-linked immunosorbent
assays
(ELISAs),
484,
485f
Enzyme-product (EP) complex, 54
Enzyme replacement
therapy
(ERT),
472
Enzyme(s),
53-68.
See in
absorptive/fed activation
also specific
state, of,
55, active
sites of,
chemistry
163,
301, enzymes
321-322,
55f
54, of,
ionization allosteric,
54f
56
of,
57
57,
67f in
committed
(rate-limiting)
322f step

effectors or
modifiers of,
62-63, sigmoidal kinetics curve of
57,
62f,
63
in alternate
reaction biotin-linked,
pathway,
444,
444f catalytic
efficiency factors
of,
54
in,
56
changes in
fasting,
328-329
in clinical diagnosis, 64-66
cofactors for, 54
of
62f,
87
226f cycle,
283-284,
248
factor,
195
of,
149
151.
See
55
307f,
64f
Enzyme
Epimers,
glucagon secretion
and,
313,
313f glycogen metabolism and, 132, 133f, 134,

in hyperglycemia,
in hypoglycemia,
insulin and,
310, in
integration synthesis
315f,
316
315,
315f
310f of
metabolism, of,
286, in
Epithelial
307
286f degradation,
cells, vitamin
Equilibrium, zero
190f
A
and,
384
Equilibrium constant
change
(Keg), standard
in, relationship
Ergocalciferol,
386,
386f
Erythrocyte-producing cells,
in,
278-279
Erythromycin,
441f
Erythropoietic
Erythropoietin, porphyrias,
280, heme
Escherichia
70
with change,
71-72
heme synthesis
281f synthesis
coli
DNA
replication lactose
in, operon
of, protein
synthesis and,
399-406
450-452, in,
regulation of
gene
Shine-Dalgarno
E
451f
440—441f expression
sequence, sites,
on ribosome,
Eskimos, lactose
88
Essential amino
in in,
450-454
439,
436
intolerance acids,
protein in
261,
262f, synthesis,
Estimated
Average

Estimated
Energy
Estradiol,
367
434
Requirement
Requirement,
237f,
Estrogens,
238f,
237,
Ethanol porphyria
and,
(EAR),
358
239
239,
240f
280
pyruvate reduced
to,
105,
106f secondary hyperuricemia and, 301
Ethanolamine, synthesis
of phosphatidylethanolamine and,
203-204
Ether-linkage in
plasma-activating in
factor, plasmalogens,
202,
Etoposide,
401
Euchromatin,
422,
Eukaryotic
460
cell
Eukaryotic cycle,
406-407,
DNA,
399, chain
elongation organization
400f of,
403-405, of,
409-410, replication
of,
403f
409f,
406-409
therapeutic inhibition

Eukaryotic gene
expression, regulation
410f of,
408-409, of,
449, 449f, 449f, 454-461, 463f
transcription,
422-424
enhancers in,
promoters
424,
424f in,
422-423,
423f mRNA,
425-427,
Eukaryotic translation
460,
426f initiation
460f

development of
type factor
2
diabetes
345-346, energy
weight
Exergonic
346f expenditure
and, reduction
and, reactions,
Exons,
426
Exonuclease,
405
3’~5’
exonuclease,
3’5’-exonuclease,
359f,
354
70
407
404f,
Exopeptidase,
15,
249
Expression library,
Expression vector,
472
470,
405
470f
(elF2), and,
360
202f
454
202,
202f
407f
279
357,
Extracellular matrix (ECM)
collagen in, 43
collagen precursors in, 45
glycosaminoglycans in, 157
Ezetimibe, 176
F
Fabry disease, 211, 212f, 213
FAD. See Flavin adenine dinucleotide
FADHa,
73,
73f,
76
in
193f, in
Familial fatty
195
acid cycle,
112f, oxidation,
113
dysbetalipoproteinemia,
Familial
Familial
Familial
178, hypercholesterolemia,
231
232
deficiency, type
Ill hyperlipoproteinemia,
Farber disease,
Farnesylated
212f protein,
444,
Farnesyl pyrophosphate
Fast/fasting,
444f
(FPP),
327-329
adipose brain
in, enzymic
tissue in,
331,
332,
332f, changes
331f
333f in,
328-329, fuel
stores at
beginning gluconeogenesis
328f of,
327, regulation
glycogen in,
329, glycogen
stores
329f by,
329f and,
hypoglycemia
126, in,
317
intertissue relationships
ketoacidosis of,
and,
197
kidney in
long-term, liver
in,
329-331, pyruvate
332

330f carboxylase
pyruvate in,
131,
334f
196
dehydrogenase resting
skeletal
Fasting muscle
in, in,
blood
Fasting glucose
(FBG), hypoglycemia,
Fat metabolism
196
331-332,
332f
338
317
state adipose
tissue, cerebral,
325,
327,
327f hepatic,
323f,
324
resting skeletal
fasting adipose
tissue, hepatic,
muscle,
325f
326,
331,
331f
330-331,

anatomical differences
biochemical in,
differences depot,
189
dietary,
360-365
acceptable distribution
360f coronary
heart
361f effects
of,
364f energy
content of,
disease
350,
326f
350f in,
350
ranges for,
360, and,
360-361,
359, monounsaturated,
plasma cholesterol
plasma lipids
and, polyunsaturated,
359f
361-362, and,
364
361-364
362-363, saturated,
361,
362f

state,
325f,
326, in
fasting,
323f,
326f,
330-331,
332, neutral,
stored,
Fat-soluble
332
188
mobilization of,
vitamins,
373,
189-195
373f,
392-393f
Fatty acid
a-hydroxylase,
195
Fatty acid CoA synthetases (thiokinases), 189
Fatty acid cyclooxygenase (COX), 213
362f
324,
327,
330f,
325,
327f
331,
331f,
330f
123,
135f acid
in
221
192,
178,
231
362f
229
123f

gene, mutation,
and
Marfan’s
49
tropoelastin interaction
Fibrous proteins,
syndrome, with,
49
43-52.
See
Elastin concept
map, summary,
51f
52
Fight-or-flight reactions,
First-order reactions,
Fischer
285,
59, projection
Fish oil,
59f formula,
363,
363f
Flanking sequences,
Flavin
481
adenine dinucleotide
380-381,
(FAD),
73,
380f

for branched-chain
a-ketoacid dehydrogenase,
266
a-ketoglutarate dehydrogenase
complex,
112
NADPH
oxidase, nitric
oxide
150
synthase,
151
110, complex,
110f fatty
in acid
oxidation, in
mitochondrial reduced
195
matrix, form
of,
FADH2
in
Flavin as
acid
74
76,
380-381.
See cycle,
mononucleotide also
112-113,
(FMN),
380f coenzyme,
151
electron transport
in nitric
oxide oxidation
for nitric
112f
75,
380-381, oxide
chain, synthase,
75, synthesis,
of
NADH
reduced form
in
151
by,
76, of
77f
(FMNHz»), electron
Fluoride,
Dietary
Fluoroacetate, transport
381
chain,
Reference
112
Fluorocitrate,
112
5-Fluorouracil,
303
Fluvastatin,
224
FMN.
See
Flavin mononucleotide
FMNH#p,
381
in electron transport chain, 75, 75f
Foam cells,
234,
235f
Folate trap
Folic acid
hypothesis, analogs
action of,
374-375,
374f dihydrofolate reductase inhibited by, 374f,
375
purine
Folic acid
active in
synthesis inhibited
374f,
375
(folate),
262,
265,
374-375
form of
(tetrahydrofolic one-carbon
in acid),
267, thymidine
monophosphate synthesis,
303-304,
374
amino in
acid as
carrier metabolism,
of
304f,
267
“one-carbon”
267
anemia and,
374-375, units,
374f diagnosis
of,
262

cardiovascular risk and, 265, 265f
Dietary Reference Intakes for, 358f function
of,
374,
392f in
homocystinuria,
273
structural analogs of, 293, 294f supplementation
of,
375
drug
374f inhibiting,
374-375,
267
295,
392f
267f by,
294f,
376
75,
Intakes
75f for,
358f
75f
84,
85f also
Collagen;
307
for for
for for
Fructose
2,6-bisphosphate in
321
in fructose 1,6-bisphosphonate regulation,
120f,
121
glucagon and,
insulin
121
and,
100,
100f in
phosphofructokinase-1
100f
Fructose intolerance,
301
Fructose
1-phosphate cleavage
of,
138,
139f fructose
conversion
Fructose to,
6-phosphate, in
amino
138
147
acid synthesis,
glucokinase regulated
isomerization
161,
161f by,
98-99
of glucose-6-phosphate
99,
99f mannose
conversion in
to,
138
pentose phosphorylation
147f phosphorylation
of,
Fructose
1-phosphate
Fructosuria, essential,
L-Fucose
(Fuc),
Fumarase
Fumarate amino
acids pathway,
99-100,
100f aldolase,
139f
166
(fumarate hydratase),
that hydration
113
form,
263,
263f of,
succinate
112f,
113
oxidation in
Furanose, to,
112f,
253-255,
84
G
Galactocerebroside,
209,
209f
Galactocerebroside
Galactokinase,
140
141f,
D-Galactosamine,

3-sulfate,
210,
210f
142
157
Galactose absorption
attached by
intestinal to
UDP, in
mucosal
140-141, biosynthetic
as cells,
141f reactions,
141
carbon source
for glycolysis
gluconeogenesis, dietary
or
140-141
sources epimers,
of,
140
83-84
as isomer,
83,
84f in
lactose,
142
137f, concept
map,
140-142,
143f disorders
of,
141-142, phosphorylation
of,
Galactosemia classic,
141,
141f galactokinase deficiency and, 141f, 142
Galactose
1-phosphate,
140
Galactose 1-phosphate uridyltransferase, 140,
141,
141f
B-Galactosidase
(lactase), deficiency
of,
140,
141
B-D-Galactosyltransferase,
Gallstones,
226-227,
226f
Gangliosides,
209-210,
Gangliosidosis,
209f
GM,,
212f
Gastric-inhibitory polypeptide
Gastric juice,
248
Gastric lipase,
173,
174
Gastrointestinal hormones.
Cholecystokinin;
See also
Secretin insulin
secretion and,
310
Gaucher disease, 211, 211f, 212f, 213, 472
Gemfibrozil,
226
Gene chip,
484
Gene expression
(GIP),
87,
450
142
140
113
254f
138
to,
141f
141f
310
99,
147,
87
carbohydrate metabolism and, 314
composition of,
313
covalent
319f modification
of

mellitus,
341
fructose
1,6-bisphosphate gluconeogenesis
and,
121
and,
122
glycogen metabolism
and,
133f glycolysis
and, in
105,
105f
HMG
CoA
reductase regulation,
132-133,
223
315f,
316
in hypoglycemia, 315, 315f, 316
induction of
enzyme synthesis
by, insulin
action in
composed integration
by,
313, of
lipid metabolism,
metabolism mechanism
122
319f
307
and,
314
of action,
metabolic effects
314, of,
protein
314
metabolism and,
314f
314
secretion of,
313-314
amino acids and, 313, 313f coordination with insulin secretion,

epinephrine and,
glucose and,
313,
313,
313f
313f inhibition
of,
313f,
314
stimulation of, 313-314
313
in transcriptional
triacylglycerols regulation,
and,
190
Glucoamylase,
87
Glucocerebroside, 209
Glucocorticoid receptors,
456
Glucocorticoid response element (GRE),
456
Glucocorticoids,
237
Glucogenic amino
Glucokinase in
glucose acids,
261,
262f, phosphorylation,
99f in
absorptive/fed hexokinase
vs.,
98
99,
105, state,
105f,
266
98-99,
98f,
322,
323f
310,
313
kinetics of,
98,
98f regulation of, 98-99, 99f, 105, 105f
Gluconeogenesis,
102,
117-124,
117f, in
absorptive/fed allosteric
323-324
of acetyl
119
inhibition concept
map,
Cori
123f cycle
ethanol and,
118,
CoA
by
AMP
and,
118f consumption
and,
329-330,
125
and,
121
317,
317f
330f glucagon
levels and,
glycolysis
121-122
vs., reactions
121
reactions unique
regulation favoring,
to,
118-121
of,
121-122
relationship to
other metabolic
117f substrates
for,
117-118
availability of,
122
UDP-galactose as carbon source for,

Glucopyranose,
84
Glucoregulatory systems,
B-Glucuronidase
315f, deficiency,
316
164f
D-Glucosamine,
157
Glucosamine-N-acetyltransferase deficiency,
164f
Glucose, 365
enzyme
456
activity
117, absorption by intestinal mucosal cells,
87
a-D, in
glycogen, anomeric
state,
126
forms,
85f attached
to uridine
diphosphate
(UDP- glucose), 126-127, 127f
blood, dietary
carbohydrates and,
366f brain
requirements breakdown
for,
327, of.
See in
collagen, conversion
diabetes dietary
45
to fructose,
332
Glycolysis
139-140, to
pyruvate,
140f
96-103
mellitus,
342, carbohydrates
dietary
343f and,
intake of,
365
blood glucose
elevated levels
blood levels
Diabetes
366, and,
of,
366f
366,
337.
mellitus enantiomers,
85f as
energy source,
epimers,
83-84, in
fasting, formation
125
84f
329-331,
330f,
332,
333f of.
See
Gluconeogenesis glucagon
secretion hypoglycemia
and,
313,
313f and,
in insulin
secretion, isomers
of,
83,
84f in
lactose,
142
low blood levels of, 314-318. See also
Hypoglycemia regulatory
systems activated
by,

metabolism of, relation to palmitate synthesis,
186,
187f in
normal weight
and
342,
342f phosphorylation
of,
98-99, in
absorptive/fed glucokinase
obese subjects,
98f state,
in,
98-99,
322
98f,
99f hexokinase
in,
98,
98f in regulation of gene expression, 451f,
452,
452f sources
of,
125
storage of,
125
synthesis of. See Gluconeogenesis in transcription from bacterial operons,
452
transport of
in absorptive/fed state, 325, 325f,
326,
326f into
cells,
96-97
characteristics of,
331,
312,
332f insulin
and,
312,
312f
Nat-independent facilitated
diffusion,
96-97,
97f
Nat*-monosaccharide cotransporter
system of,
96,
Glucose-alanine cycle,
253
Glucose
1,6-bisphosphate,
Glucose
126,
6-phosphatase,
121, deficiency
of,
130f in
fasting,
329,
332
Glucose
1-phosphate conversion to glucose 6-phosphate,
130
Glucose
6-phosphate in absorptive/fed state, 326
dehydrogenation of, in pentose phosphate pathway, 145-146,
146f
97
130
130,
457
314,
310,
315f
310f
366f
See also
321
312f
366,

of,
262
3f,
5
256f,
268
Glutamine:phosphoribosylpyrophosphate amidotransferase,
293,
296
Glutamine synthase,
257,
257
Glutamine synthetase,
253,
253f, y-Glutamylcysteinylglycine, 148
Glutathione,
152
as antioxidant,
148
structure of, 148, 148f
Glutathione peroxidase,
148,
Glutathione reductase,
Gluten
148
(gliadin),
249
Glycation,
168
Glycemic control, in diabetes mellitus,

Glycemic index,
366,
366f
Glycemic load,
366
Glycemic response,
366
Glyceraldehyde,
83,
83f
Glyceraldehyde 3-phosphate, 96, 111, 161
oxidation of,
101,
101f in pentose phosphate pathway, 147
9
lyceraldehyde
3-phosphate dehydrogenase,
101,
103

fate of,
178,
190
as gluconeogenic precursor, 117
117
in phospholipids, 201-202, 201f phosphorylation of, 117
Glycerol kinase,
117,
189,
190
in absorptive/fed state, 325
Glycerol phosphate
in phosphatidic acid synthesis, 189f synthesis
of,
188f,
189
in triacylglycerol
189,
189f
Glycerol 3-phosphate, 311, 324
Glycerol phosphate dehydrogenase, 76, 80,

Glyceroneogenesis,
190,
331
Glycerophosphate shuttle,
Glycerophospholipids,
79f,
80
201-202,
201f antigenic,
202
degradation of, 207-208, 207f synthesis
of,
201f,
202
Glycerylphosphoryl-base,
176
Glycine as atom source for purine ring, 292, 293f
in
B-bends,
17
bile acids
conjugated catabolism
to, of,
263,
263f in
collagen,
45
in creatine synthesis, 287, 288f interconversion
with serine,
263, optical
properties in
porphyrins, of,
278
of,
2f synthesis
of,
268
Glycochenodeoxycholic acid,
Glycocholic acid,
175,
175f,
Glycogen,
84,
85
branches of,
131-132, of
formation of,
128
removal of, 128-130

elongation of,
127
shortening of,
128
degradation of (glycogenolysis), 125, 125f,
128-131,
129-130f
AMP
and,
132
5
225,
263f,
225f
225,
225f
131f
225,
225f
150
268
268
calcium and, 131-132, 132f cAMP-directed
pathway and,
329,
329f lysosomal,
130-131
regulation of, 131-134, 133f in
fasting,
126,
131,
135f during fed state, 126, 131, 135f
as fuel
source for

fuel stores
of, before
fast, function of, 125-126
hepatic,
125-126,
131f hormonal
regulation metabolism
of,
183-134, of,
125-136
muscle,
135f
125-126,
131f storage
of,
125
fluctuations in, 126
125-126,
126f synthesis
of
(glycogenesis),

state, inhibition
323, by
125, muscle,
329,
329f
134f
125f,
326, cAMP-directed
133-134
primer for
initiation regulation
of,
126-127
of,
Glycogenesis.
131-134,
See
Glycogenin,
127,
Glycogen,
127f
Glycogenolysis.
See
Glycogen,
Glycogen phosphorylase,
134
state,
133f,
134f synthesis
of degradation
128,
322, of
128f,
129f,
130,
322f activation
of,
132-133,
133f allosteric control of, 131-132, 131f
AMP
and,
132-133,
133f deficiency
in,
129f in
fasting,
329
inactive b form of, 133, 133f phosphorylation
of,
63
Glycogen phosphorylase
Glycogen kinase,
storage type
diseases, la
type
(Von
Gierke
Il type
V
Glycogen
329
129—130f disease),
(Pompe disease),
(McArdle
121,
129f, syndrome),
synthase,
131
129f
184
state,
323
active a form of, 133, 133f allosteric control of, 131-132, 131f
elongation of glycogen chains by, 127,

in glycogen
inactive synthesis,
b
form inhibition
of,
133, of
glycogen
133-134,
126-127,
127f
133f synthesis
134f phosphorylation
of,
63
Glycolipids,
208-213.
See also
Glycosphingolipids fatty
acids in,
173f
Glycolysis,
91f,
92f, in
96-108
323f,
100,
325, aerobic,
96-102, energy
325f
96f yield
from, anaerobic,
96,
96f,
104
102-104, and,
100f,
323,
104f concept
map,
107f control point and rate-limiting step of,

energy yield
from,
104
enzyme deficiencies in, 102-103
during fasting, 100, 100f, 105
phase of,
of,
101-104,
97-99,
98f,
326f pathway,
132-133
130f
as cell surface receptors, 209
217f degradation of, 210-213
disorders of, 211-213, 211f, 212f neutral, 209, 209f
structure of, 209-210, 209f synthesis
of,
210-213,
211f
Glycosylation in
collagen,
45,
46f,
47
of proteins, 443f, 444
N-Glycosylation, 167
Glycosyl phosphatidylinositol (GPI), 206
Glycosyl residue,
86
Glycosyltransferases, 85, 166-167, 167
225
kinase state,
Glyoxylate,
263
GM;
gangliosidosis,
212
GMP. See Guanosine monophosphate
(GMP)
Golgi apparatus
apolipoprotein synthesis in, 228
glycoprotein synthesis in, 166, 167f,
168f glycoprotein
transport
167f glycosaminoglycan
158
glycosphingolipid synthesis in, 210
Gonad(s).
See also
Ovary(ies);
Testes steroid
hormone
Gout,
299-301, secretion
from,
300f,
301f,
240
305f diagnosis
of,
299,
301f in Lesch-Nyhan syndrome, 296f, 297,
300
primary,
299-300
saturnine,
299
300-301
tophaceous,
299,
301f treatment
of,
300f,
301
GO phase, of cell cycle, 407, 407f
G1
phase, of
cell cycle,
406,
407f
G2
phase, of
407,
407f
G-protein coupled

G-proteins,
94f,
95
Graft rejection, prevention of, 295f, 296
Granulomatosis, chronic,
150
Greek key motif, 18f
Griseofulvin, 279
Ground substance,
157
Growth, vitamin A and, 384
Growth hormone (Gh), in hyperglycemia,

GTP.
See
Guanosine
GTPase,
95
Guanine,
291,
291f,
299, in
codons/genetic damage
triphosphate
305f,
396f code,
to and
repair
431, of,
432f
412, pairing
with cytosine,
397,
413f
397f,
Guanine-7-methyltransferase,
398f
425
Guanine diphosphate-L-fucose,
Guanine
Guanine
166
diphosphate-mannose,
166
monophosphate
(GMP), conversion
IMP
to,
295-296,
295f
Guanosine triphosphate-dependent
regulatory proteins,
95,
95f
Guanosine triphosphate (GTP)
in amino
acid deamination,
in
AMP
295
in protein
437
Guanylate cyclase, 151
Guanyltransferase, 425
receptor, through,
166,
322
252
94f,

H
Haelll restriction endonuclease, 466, 466f
Haemophilus aegyptius,
466
Hairpin
(stem-loop)
Hairpin structure,
turn, in
prokaryotic
453, gene
421,
Hartnup disorder, 250
Haworth projection
formula,
HDL.
See
Health,
High-density obesity
421f
84,
85f lipoproteins
and,
349,
354
Heart.
See also
lactate consumption in, 103
Heart attack.
See
Myocardial infarction
Heart disease
alcohol consumption
dietary and,
fats and,
360-364
isoenzymes and,
66,
66f lipoprotein
(a)
in,
237
plasma cholesterol and, 360-361, 361f soy
364
triacylglycerols and, 361-364
Heat shock
proteins,
20
Heinz bodies,
152,
152f
Helix-turn-helix motif,
450,
452,
452f
Heme,
25,
277
as coenzyme, for nitric oxide synthase,
151
degradation of, 282-284, 282f, 283f,
284f function of, 25
concept
277-278
map,
289f structure
of,
25,
25f,
26f synthesis of, 278-279, 278f, 279f, 280f,

defects in,
278-282,
280f end product inhibition by hemin,
278-279
rate-controlling step of, 278
uroporphyrinogen conversion to, 279,
279
Heme of
group catalase,
25
of cytochromes,
25, of hemoglobin, 25
of myoglobin,
25,
26,
26f
Heme-heme interactions, 29-30
Heme oxygenase
system,
282
Hemeproteins,
277
globular, 25-34. See also Hemoglobin;
Myoglobin
Hemin,
278-279
Hemoglobin
A,
27,
Ao,
29-33,
33-34,
33
33f
Ajc,
33f,
34,
34f in diabetes mellitus type 1, 340, 340f
Bart’s
(Hb
Bart’s)
in a-thalassemia,
in
39,
B-thalassemia,
39f
38f,
C
disease,
F,
39
36f,
33f
H
disease,
Hemoglobinopathies,
39,
39f
35-39,
Hemoglobin(s)
a-helix,
27
carbamino,
32
41f developmental changes in, 33, 33f
embryonic (Hb Gower 1), 33
fetal
(HbF),
33
in
38-39,
42f
37
453f
364-365
25f
38f
function of, 27, 41f

B family, 34f, 35, 35f
6,
35
€,
35
a family, 34, 34f, 35f y,
35
organization of, 34-35, 34f
6,
35

heme-heme interactions
high-oxygen affinity
form low-oxygen
affinity form
in, of,
of, minor, 33-34
normal adult, 33-34, 33f oxygen affinity/binding of, 27-33, 28f
allosteric effectors
and,
27,
29-33
2,3-bisphosphoglycerate and, 31-32,
31f,
32f carbon
dioxide binding
and,
32
carbon monoxide binding and, 32-33,
32f cooperative,
29-30,
PCOz and,
29
PH
and,
30,
POz and,
30f
29, saturation
29f, in,
29f
30
28
oxygen dissociation
curve for,
29,
29f, sigmoidal shape of, significance of,
31
30
oxygen loading
and unloading
by,
30,
30f
R
form
(relaxed)
of,
28,
28f structure of, 27-28, 27f, 28f, 41f
changes with deoxygenation and oxygenation, 26, 27-28, 28f
quaternary,
27-28
T form (deoxy or taut) of, 28, 28f
SC
disease,
38
Hemoglobin S disease. See Sickle cell disease
Hemolytic anemia
glycolytic enzyme
deficiencies and,

G6PD deficiency and, 152, 153
Hemolytic jaundice,
284,
284f,
289f
Hemophilia
A,
461
Hemoproteins,
25-34,
25f.
Hemoglobin;
See also
Myoglobin
Hemosiderosis,
36
Henderson-Hasselbalch equation, 6-9
application of,
7-8
for bicarbonate buffer system, 9, 9f derivation
of,
6
for drug absorption, 9, Of for titration of amino acids, 7-9
Heparan sulfamidase
deficiency,
164f
Heparan sulfate,
159f deficient degradation of, 163, 164f

Hepatic lipase, 228
Hepatic porphyrias, acute, 280, 281f
Hepatic steatosis, 231
Hepatitis alcoholic,
318
jaundice in, 284
Hepatocellular jaundice,
284,
289f
Hepatocytes, 449
Hereditary coproporphyria, 280, 281f
Hereditary fructose intolerance (HFI), 138,
139f
Hereditary hyperammonemia, 258
Hereditary nonpolyposis colorectal cancer
Heterochromatin, 422, 460

29-30
28,
28f
28,
28f
Heterogeneous nuclear RNA (hnRNA), 425
Heterotropic effectors, 63
Hexokinase,
121
state,
326
broad substrate specificity of, 98, 98f in
fructose phosphorylation,
137-138, glucokinase
vs.,
98
in glucose
98
98
in mannose phosphorylation, 138
Hexokinase D, 98
Hexokinase/glucokinase, 229
Hexose monophosphate shunt/pathway. See
Pentose phosphate pathway
D-hexoses, in
oligosaccharides,
165
High altitude
2,3-bisphosphoglycerate levels and, 32, 32f sickle
cell disease
and,
36
High-density lipoproteins
(HDLs),
alcohol consumption and, 365
composition of, 232f coronary heart disease and, 360-361
dietary fats and, 361-364
electrophoretic mobility of, 227, 228f in
esterification of
cholesterol,
234-235
as
“good”
cholesterol
HDLo, 235, 236
HDLs, 235,
236
metabolism of, 234-236, 236f n-6
and,
363
obesity and, 353
for apolipoproteins,
234
reverse transport of cholesterol, 235-236,

size and density of, 227, 227f uptake of unesterified cholesterol by, 234,
236f
VLDL transfer of triacylglycerol to,
231
High fructose
corn
High-mannose syrup
55
and oligosaccharides,
168
Histamine,
287
synthesis of, 287, 287f
Histidase,
262,
263f deficiency
of,
269f
Histidine,
5
by,
17
262,
263f decarboxylation to histamine, 287, 287f
262,
263f distal,
26,
26f in histamine synthesis, 287f
in myoglobin,
26,
26f proximal,
26, side
chains
26f of,
3f,
5
Histidinemia,
268f,
269f
Histone acetyltransferases (HATs), 422, 422f,

Histone deacetylases (HDACs), 422, 422f
Histones,
409-410,
409f acetylation
of,
422
HMG CoA. See 3-Hydroxy-3-methylglutaryl
CoA (HMG CoA)
Hogness box,
423
Holoenzymes,
54,
419
Homocysteine,
263
conversion to cysteine, 264-265, 264f, 268
fate of,
264
methionine conversion to, 263, 264f
42,
365
166,
166f, carrier,
236
234-236,
138f remethylation of, 264
in vascular disease, 263, 265, 265f
Homocysteine-lowering therapy,
265
Homocystinuria,
268f,
269f,
273,
Homogentisic acid, 274
Homogentisic acid oxidase, 274
Homologous recombination repair, 413
Homotropic effectors,
62-63
Homovanillic acid
(HMA),
286
Hormone response elements (HREs), 240,

Hormone(s)
of adipose
tissue,
352-353
leptin,
353,
353f counterregulatory,
313, gastrointestinal,
310
regulation, in
glycolysis regulation,
316
133-134,
134f
104-105,
105f in
HMG
CoA
in lipid regulation, 176, 176f plasma,
307
steroid. See Steroid hormones in triacylglycerol degradation, 190, 190f
Hormone-sensitive lipase
(HSL),
190,
190f in absorptive/fed state, 322, 322f, 325
329
311
Housekeeping genes,
449
Human genome,
465
Human Genome Project, 465, 470
Human immunodeficiency
virus
(HIV), detecting
exposure to,
RNA
Hunter and,
syndrome,
163,
Huntington disease,
Hurler syndrome,
484,
459
164f
433,
164f,
433f
472
Hyaluronic acid,
159f,
163
Hybrid duplex, in DNA replication, 402
Hybridization, in
situ,
472
Hydantoins, 279
Hydratase,
195
Hydration of fumarate, 112f, 113
Hydration therapy, for sickle cell disease, 36
Hydride ions, electron transport via, 76
Hydrochloric acid,
248
Hydrogen atoms,
Hydrogen bonds
in a-helix,
amino
16
acid,
4,
4f, in
protein electron
digestion, transport
19,
19f in
B-sheets,
17,
17f between complementary bases, 397,

reduction of, 148-149, 148f superoxide
conversion to,
150
Hydrolases,
53f

of cAMP, 95-96, 96f in
catabolic pathways,
Hydrophobic interactions,
in hemoglobin,
Hydrophobic
27,
28f side
chains,
93
19,
4
Hydrops fatalis,
39,
39f
Hydroxyacyl-ACP dehydratase, 186
Hydroxyallysine, in
collagen biosynthesis,
3-Hydroxybutyrate,
195-196,
48
196f,
262,
330,
330f
3-Hydroxy-butyrate dehydrogenase, 196
25-Hydroxycholecalciferol, 386
28f
247
via,
76
485f
223
273f
19f
408
25-Hydroxycholecalciferol 1-hydroxylase, 386
regulation of, 386-388
5-Hydroxy-6,8, 11,14 eicosatetraenoic acid (5-

11-B-Hydroxylase deficiency,
238f
17-a-Hydroxylase deficiency, 238f
21-a-Hydroxylase deficiency,
238f
Hydroxylation in collagen, 45, 45f, 46f, 47
of proteins, 444, 444f
Hydroxylysine, in
collagen,
45,
45f,
46f,
Hydroxymethylbilane, 279, 279f
Hydroxymethylbilane synthase, 281f
3-Hydroxy-3-methylglutary] CoA (HMG CoA),

3-Hydroxy-3-methylglutary!
CoA
reductase,
220-221, gene
(HMG
CoA)
232
expression of,
sterol-dependent,

regulation of, 222-224, 223f
3-Hydroxy-3-methylglutaryl CoA (HMG CoA)

220,
220f in
ketone body
196,
196f
Hydroxymethylglutaryl CoA reductase (HMG
CoA
reductase),
61

in collagen, 45, 45f, 46f, 47
49
3-B-Hydroxysteroid dehydrogenase

5-Hydroxytryptamine. See Serotonin
5-Hydroxytryptophan,
287,
287f
Hydroxyurea,
36,
298
hyperammonemia,
257-258,

hereditary,
258
Hyperbilirubinemia,
284,
284f
Hypercalcemia, vitamin D and, 389
Hypercholesterolemia,
224,
226
Hyperglycemia

type 1, 338-339, 339
type
2,
344
glucose phosphorylation
in,
98
140
Hyperlipidemia/hyperlipoproteinemia niacin
for,
380
type
I, type
type
178,
Il,
229
232
Ilb,
380
type
Ill, familial,
231
Hyperoxaluria, primary
Hyperphosphatemia,
Type
389
Hypertension diabetes
mellitus obesity
and,
354f treatment
of,
239

1,
339-340
in type
2,
344
Hyperuricemia,
296,
Hypervitaminosis
A,
Hypocalcemia,
388,
Hypochlorous acid
299
385-386
389
(HOCI),
Hypoglycemia,
314-318,
150
319f alcohol-induced,
317-318, brain
damage in,
characteristics
327
of,
314
and,
258f
1,
346
253
317f
47
concept map, 319f fasting, 317
glucagon and, 315, 315f, 316
glucoregulatory systems in, 315f, 316
insulin-induced, 316, 316f postprandial, 316
symptoms of,
314-315
adrenergic,
314
neuroglycopenic, 314
in type
1
diabetes,
340-341,
341f types of, 316-318
Hypoglycemia unawareness, 341
Hypoglycemic agents, for diabetes mellitus

Hypoparathyroidism,
389
Hypopigmentation, in PKU, 271
Hypoprothrombinemia,
390
Hypoxanthine,
299
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT),
296,
296f,
301
Hypoxia, 2,3-bisphosphoglycerate in, 32, 32f
-cell disease, 169
cterus. See Jaundice
Iduronate sulfatase deficiency, 164f
L-Iduronic acid in glycosaminoglycans, 157, 157f, 161
161
a-L-Iduronidase deficiency,
164f mmunoblots, 484, 485f
mmunodeficiency syndromes. See also
Human immunodeficiency virus
(HIV); Severe combined immunodeficiency disease (SCID)
DNA
repair defects
and,
413

mmunoglobulins, production
MP.
See
Inosine nborn
errors of,
461, monophosphate
of metabolism,
270-274
ncretins,
310
ndomethacin, 214, 301
nfarction, in
nflammatory disease,
processes, nfluenza,
nutrition nitiation
36,
37f in
gout, and,
361f codons,
nitiator
438f, tRNA,
439
438f, nner
membrane iNOS,
439
particles,
151
nosine,
Inosine for
boosting
74
2,3-BPG
32
monophosphate nosine
dehydrogenase, monophosphate
conversion levels
in blood,
295f
(IMP)
to
AMP
and

as parent
purine nucleotide,
293
in purine synthesis, 293-296, 294f synthesis
of,
293
nositol
1,4,5-triphosphate
(IP3), transmission,
205-206, triphosphate
nositol
(ITP), signaling,
nsig in
signal
205f in
intracellular
205, protein,
223
n
situ hybridization,
472
nsulin,
307-313,
353
in absorptive/fed state, 328f in acetyl CoA carboxylase regulation, 184,

anabolic effects of, 308
carbohydrate metabolism and, 311
319f deficiency
of absolute, in type 1 diabetes mellitus,
205f
GMP,
295-296,
299
461f relative,
836, 336F
| in type 2 diabetes mellitus,
341

310, exogenous.
310f,
319f
See
Insulin therapy
fructose
2,6-bisphosphate glucagon
and, opposing
action glucagon
secretion
100, of,
and,
314,
309,
100f
319f
313
glucokinase and,
310,
313
glycerol phosphate synthesis and, 189
glycogen degradation
and,
133
glycolysis and,
105,
105f in
HMG
CoA
reductase hormone-sensitive
regulation, lipase
and, in
hypoglycemia,
190
314-318,
315f hypoglycemia
and alcohol consumption and, 318
in integration
307
lipid metabolism
mechanism and,
311
of action,
membrane effects
of,
311-313,
311f
312, metabolic
effects of,
312f
311
phosphofructokinase and,
protein synthesis
and, secretion
of amino
acids and,
by
B
cells,
311
310
310,
310f coordination
with glucagon
313
secretion

gastrointestinal hormones and, 310
and,
310,
310f inhibition of, 310
during onset of type 1 diabetes mellitus, 338, 338f
309-310,
310f, stimulation
in of,
309-310
signal transduction,
311f, structure
of,
308,
308f synthesis of, 308, 308f
time course of actions, 313
triacylglycerol degradation and, 189, 190f
Insulinase, 308
Insulin-dependent glucose transport (GLUT-4)
proteins,
331
Insulin-induced hypoglycemia, 316, 316f
Insulin receptor,
311-312,
311f
Insulin receptor substrate (IRS) proteins, 311f,
312
Insulin resistance
causes of,
343
obesity and,
342
in type 2 diabetes mellitus, 342-343, 343f
Insulin resistance syndrome, 353
Insulin-sensitive glucose transporters (GLUT-
4),
312
hypoglycemia induced
340-341, for
type by,
316,
340f
1
diabetes standard
for mellitus,
vs.
intensive, type
2
diabetes
Insulitis,
338
Intercellular communication,
Intermediate-density
340,
340, mellitus,
316f,
340f
340f
344
94,
94f lipoproteins
(IDLs),
231
International
Union of
Biochemistry and
Molecular Biology (IUBMB), enzyme nomenclature of, 53, 53f
Intestinal mucosal
cells chylomicron
assembly in,
177f,
228,
229f enzymes
synthesized by,
carbohydrate digestion by, 86-87, 87f
by,
176,
177f lipid secretion by, 177f, 178
313
312
223
monosaccharide absorption by, 87

bacterial action
cholesterol in,
ammonia synthesis
small dietary
dietary in,
220
lipid processing
nucleic in,
175
acid degradation
298,
299f oligopeptide
digestion urobilins
formation vitamin
D
in,
249
in,
283-284
and,
388
Intracellular communication,
205-206,
Intrinsic
Introns, lodine,
94,
94f,
205f factor,
377
removal of,
426-427,
Dietary
Reference lonic
interactions of
of amino
acids, hemoglobin,
lron
Dietary
427f
Intakes
19,
19f
27-28,
Reference in
heme,
28f
Intakes
25,
25f metabolism
of, in
458-459, porphyrins,
lron-deficiency for,
458f
277
anemia, lron-regulatory
458f
Iron responsive
elements lron-sulfur
center,
374,
374f proteins
(IRPs),
458-459,
(IREs), of
NADH
75,
75f
Islets of
Langerhans, lsocitrate
citrate isomerization
to,
111f,
112
oxidation and decarboxylation of, 111f,
112
Isocitrate dehydrogenase,
183
lsoelectric form,
lsoelectric point,
Isoenzymes, of
amino for
112, acid,
amino
65-66, heart
disease
114,
7,
114f,
7f acids,
65f and,
quaternary
66, structure
Isoforms,
21,
231,
Isoleucine in
absorptive/fed a-helix
66f of,
457f state,
disrupted catabolism
65-66
324
by,
17
of,
266-267, formation
of
266f succinyl
hydrophobic
CoA
interactions for
maple syrup
and, and,
urine
265,

19f disease,
of,
2f
Isomaltase,
87
lsomaltase-sucrase deficiency,
Isomerases,
53f
Isomerization of
citrate, of
111f,
112
dihydroxyacetone phosphate,
of glucose-6-phosphate,
Isomers,
101
99,
99f
83-84,
84f
Isoniazid,
377f,
378
lsopentenyl pyrophosphate
lsoprenoids,
Isotretinoin,
221
385,
Isovaleryl
Co
385f,
386
A
266
lsozymes,
21,
213
ITP.
See
Inositol
J
Jaundice,
284-285, bilirubin
284f,
285f concentration
in, determination
of,
285
hemolytic,
284, hepatocellular,
neonatal,
284f,
284f,
289f
284,
289f
285,
285f, deficiency,
273
8-9
307,
458,
458f dehydrogenase,
308f,
313,
316
for,
358f
358f in,
from,
88
(IPP),
289f
257
221
266
lac genes
lacA, lacl,
lacY,
451f,
452
451f,
452
451f,
452
lacZ,
451f,
452
a-Lactalbumin,
142
B-Lactam antibiotics,
Lactase,
87,
88,
140,
Lactate,
96
consumption in
formation
62
141
of,
103
118,
118f in
muscle, in
103
gluconeogenesis, pyruvate
118
reduction to
glycolysis),
Lactate
(anaerobic
103-104, dehydrogenase,
Lacteals,
178
Lactic acidosis,
congenital,
104
111
Lactose,
85,

140, in
365
452
142,
Lactose gene
142f intolerance,
Lactose
88,
88f operon,
450-452,
Lactose synthase,
451f
142
Lactosylceramide,
209
Lagging strand,
402,
Lanosterol,
221
Laparoscopic cholecystectomy,
L-configuration, of
104f
103,
111
451f,
226
LDH.
See
5,
Lactate
LDL.
See
L-dopa
5f dehydrogenase
Low-density lipoproteins
(levodopa),
286
Lead, noncompetitive
inhibition by,
61-62
Leading strand,
Lead
402, poisoning,
Leber
403f
279, hereditary
Lecithin.
281f,
374f optic
neuropathy,
See
Leigh syndrome,
Leishmania,
111
206
Leptin in
Lesch-Nyhan
Leucine,
343
352-353,
352,
353f syndrome,
19f in
262, degradation
of, for
maple
296-297,
296f,
324
266-267,
266, syrup
Leucine
266f urine
of,
2f zipper,
423,
Leukemia,
262
Leukodystrophy globoid
cell,
212f metachromatic,
212f
Leukotriene(s),
213-214
LTAg,
213f,
LTBa,
215f
LTCa,
215f synthesis
of,
Ligandin,
282
Ligases.
See also
classification
214,
215f
215f specific
of,
Lignoceric acid,
53f
182f,
Limit dextrin,
128,
207
129f
Lineweaver-Burk plot,

Lingual lipase,
59,
173, a-1,4
linkages, a-1,6
linkages,
Linker
174
126,
126f
DNA,
410,
Link proteins,
410f in
proteoglycan
158,
Linoleic acid,
160f
182f, aggregates,
214f,
363
126f, disease,
450,
266f
273
457
403f
213, of
ligases
59f,
60,
60f,
61f
127,
128
enzymes
80
297f
a-Linoleic acid,
a-Linolenic
182, acid,
Lipid(s)
complex.
See
363
also
Glycosphingolipids;

absorption by
intestinal cells,
concept coronary
mucosal
176,
177f map,
180f heart
disease
361-364
degradation by
pancreatic

digestion of,
173-178,
174f emulsification
in small
malabsorption intestine,
of,
175
176,
176f of,
177, metabolism
178f of,
in neonates,
processing
173-180,
174
in stomach,
use by
tissues, in
fasting, functions
glucagon
173-174
178
332,
332f of,
173
and, insulin
314
and,
43f plasma
levels
311
of, dietary
361-362, fats
361f secretion
from enterocytes,
177f of,
structure
Lipid
173, storage
Lipoic
173f disease,
acid in
as arsenic
poisoning, coenzyme
266
complex, for
112
complex,
Lipoprotein(a),
110
in heart
Lipoprotein disease,
lipase,
178, in
237
228-230, state,
of,
231
178,
228-229
degradation of
228-22
1, type
2, in
fasting,
339-340
344
331
regulation
311
of,
Lipoprotein(s)
composition
229-230
227,
228f of,
227, concept
map,
243f electrophoretic
mobility in
heart disease,
high
231,
232f of,
237
density.
See
(HDLs)
intermediate density
low
(IDL),
231
density.
See
plasma, size
and very
low
(LDLs)
227-237
density of,
227, density.
227f
See
Very-low-density lipoproteins
Lipoxins,
(VLDLs)
214

Lipoxygenase, 214
227,
228f by,
311
325,
326
211
111
177-178,
180f for
182f and,
enzymes, and,
5-Lipoxygenase,
Lisinopril,
214
62
Liver in
322-324, amino
acid metabolism
323f in,
carbohydrate
323f, metabolism
322-324, fat
324
in,
323f metabolism
in, intertissue
323f, relationships
ammonia transport
bile acid
324
of, to,
253
synthesis bilirubin
uptake in,
149, by,
282, cholesterol
synthesis
224
283f in,
chylomicron remnants
communication
220
in, with
178, other
organs,
207, cytochrome
P450
system
230
metabolic
207f monooxygenase
of,
D-amino
149
acids metabolized
detoxification in,
149
1, type
2, energy
333,
339f
342,
344,
345f metabolism,
fasting,
307,
329-331, by,
253
307f
330f carbohydrate
329-330, fat
in,
330f metabolism
330-331, relationships
fatty,
231
fatty acid
330f of,
334f
183, fructose
metabolism gluconeogenesis
in,
138
in,
117
125-126, heme
degradation heme
126f, in,
synthesis
131,
282, in,
lactate
282f
278
consumption vitamin
in,
103
A
transport/release
383f
Liver disease
aminotransferases

ammonia levels
Liver in,
257-258
parenchymal cells,
in,
L-Methylmalonyl
CoA,
98
194
Long-chain fatty
191f
acyl acids
(LCFA), mitochondria,
181
190-192,
CoA
synthetase
(thiokinase),
Lovastatin,
224
231-234,
190
(LDLs),
232f chemically
modified, uptake
macrophage
227, by
scavenger receptors,
234, composition
of,
235f
231, coronary
232f heart
disease dietary
cholesterol and,
intake
364f electrophoretic
endocytosis mobility
360-361
and, of,
of,
232, functional
233f receptors
232
as negatively
for, deficiency
230f,
231-234, charged
232
n-6
niacin and,
and,
363
380
size and
234, density
235f of,
227,
364,
227,
228f of,
233f glycoproteins,
to,
382, in,
65,
251-252,
324
328f in
in
227f
135f
227f,
VLDL
Lower body
Low-fat obesity,
diet,
Lungs,
362f o14-antitrypsin
to, in
350, plasma,
350f in,
50,
Lung surfactant
phosphatidylcholine in,
in respiratory
50f
204
distress
Luteinizing syndrome,
hormone
Lyases,
53f
Lynch syndrome,
Lysine,
411
266
as carnitine
by, source,
catabolism in
of,
262
collagen,
47,
48, hydroxylation
48f of,
elastin,
49
hemoglobin in
in in
C

histones,
410
ionic bonds
and,
19f of,
3f, transport
of,
250
Lysophosphatidic acid,
189f
Lysophosphatidy!
choline,
Lysophosphoglyceride,
175,
207
Lysophospholipase,
176
Lysophospholipid,
175
Lysosomal degradation,
of glycosaminoglycans,
Lysosomal enzymes,
234
163
247
Lysosome(s),
54f in
chylomicron of
of of
glycogen,
130-131
glycophospholipids,
230
210
glycoproteins,
170
glycosaminoglycans, of
162-163, proteins,
246-247
glycoprotein transport
Lysyl hydroxylase
and
Ehlers-Danlos

hydroxylation in
collagen
Lysyl oxidase
in collagen
tropoelastin biosynthesis,
polypeptides
49
Macrocytic anemia,
Macronutrients,
374,
374f
357
360f caloric
consumption and
calories,
365, concept
map,
370f
Macrophages, nitric
oxide
Macrophage scavenger
chemically
Mad ranges
for,
360, distribution
365f and,
151
receptors, modified
cow disease,
22
Magnesium,
Dietary
Malaria,
Reference resistance
G6PD
to deficiency
sickle and,
cell trait
uptake
LDL
by,
234
Intakes,
152
and,
Malate formation
formation fumarate
from
37,
38f a-ketoglutarate,
of oxaloacetate
112f from,
hydration to,
112f, in
gluconeogenesis, oxaloacetate
119, reduction
113,
113
119f to,
113, in
113f
254f,
Malate-aspartate
255
shuttle,
Malate
79f, dehydrogenase,
113,
119,
80
119
358f of
by,
47
48,
48f deaminated
by, to,
169,
169f
5
45,
45f, disease,
46f,
37
204
239f,
240
17
191
(LH),
231
47
164f
119f of
113f

malate dehydrogenase
Malnutrition, protein-calorie,
369
Malonyl
CoA
carboxylation of acetyl CoA to, 183-184,

carnitine shuttle inhibited by, 191, 191f
Malony! CoA-ACP-transacylase, 184
Maltase, 87
Maltose,
85,
365
Mammary glands, lactating, fatty acid synthesis in, 183
Manganese,
Dietary Reference Intakes for,
358f
Mannose conversion to fructose 6-phosphate, 138
epimers, 83, 84
as isomer, 83, 84f
Mannose 6-phosphate, 138
a-Mannosidosis type 1, 170
Maple syrup urine disease (MSUD), 266,
268f,
269f,
272-273
classification of,
272-273
screening and diagnosis of, 273
273

Marfan's syndrome, 49
Maternal
PKU
syndrome,
272
Maturity onset diabetes of the young

Maximal velocity
(Vmax)
allosteric effectors and, 62-63, 62f of
enzyme-catalyzed reactions,
57,
57f competitive
inhibition and,
60,
60f noncompetitive inhibition and, 61, 61f
McArdle syndrome,
129f
Mediterranean diet,
362,
362f
Medium-chain fatty acids, 192
Medium-chain fatty acyl CoA dehydrogenase

Megaloblastic anemia,
267,
374,
Melanin,
288
in albinism, 273
in phenylketonuria, 271

a-Melanocyte stimulating
353
Melatonin,
287
Melting temperatures
of
DNA,
398,
398f of
fatty acids,
182
Membrane protein anchoring, 206, 206f
Membrane transport systems, 79-80
Menadione, 389
Menaquinone (vitamin Kz), 389
Menkes disease, 48
Mental retardation, in phenylketonuria, 271,

Messenger
RNA
(mRNA)
alternative splicing patterns of, 427, 428f coding
for protein
435
codons of,
431-432,
432f mutations of, 432f, 483-434, 433f,

recognition by
editing,
457-458, tRNA,
457f eukaryotic,
418,
419f,
425-427, posttranscriptional
426f modification
425-427, 426f, 454, 457-460
exons, splicing
of,
426-427, frame-shift
mutation of,
427f of,
434,
434f introns
of,
426
removal of, 426-427, 427f
437,
437f hormone
375,
393f
(a—MSh), levels of, determination of, 483-484, 484f
missense mutation
of,
432f,
433
monocistronic,
438
nonsense mutation
of,
432f,
433
polycistronic, 438, 450
regulatory ribosomal protein and, 454,

silent mutation of, 432f, 433
splice site mutation of, 427
stability of, 458-460, 458f, 460f transcription
of,
450
translation of, 431-443, 431f, 460, 460f components
required for,
434-437
initiation of,
438-439, steps
in,
438-442, trinucleotide
440f
440-441f repeat
expansion

Metabolic acidosis,
195
Metabolic map,
91,
92
Metabolic rate, resting (RMR), 359, 359f
Metabolic syndrome,
353
Metabolism,
73f,
91-93, defined,
91
four major
organs in,
307
communication between, 307, 307f inborn
errors in,
268-274
integration of,
concept
307
map,
319f regulation
of,
93-96
by adenylyl cyclase cascade, 94-96,
95f by
intercellular signals,
94
by intracellular signals, 94
by second
messenger systems,
tricarboxylic acid
Metachromatic cycle
in,
109
leukodystrophy,
Metal ions,
as cofactors,
212f
54
Metalloporphyrins,
277.
Porphyrin(s)
Metformin,
344
Methane,
267
Methanol,
267
Methemoglobinemias, 38
Methemoglobin (Hb M), 38
Methionine as
carnitine source,
191
catabolism of, 263-265, 264f resynthesis
of,
264,
264f side
chains of,
2f synthesis of, 375, 375f

dihydrofolate reductase inhibited by, 304,
375
folic acid absorption and, 375
purine synthesis inhibited by, 293, 294f,
304,
304f
Methylation, of
S-adenosylmethionine,
461
Methylcobalamin, 264, 264f, 375, 376f
N°-N'°-Methylene,
Methyl-directed mismatch repair, 411, 411f
376
N5,N1°-Methylenetetrahydrofolate,
263,
263f
Methyl group,
activated in
SAM,
264
3-Methylcrotonyl
CoA
carboxylase,
7-Methylguanosine cap,
Methylmalonic
266
426,
426f acidemia,
194, aciduria,
194,
D-Methylmalony!
CoA,
377
377
194
Methylmalonyl
CoA
mutase,
268f,
194
269f
CoA
racemase,
194
Mevalonic acid (mevalonate), 220-221, 221f,
Mice, knockout, 486
460,
See also
92f of,
433,
94
Michaelis constant (Km), 59, 59f allosteric
effectors and,
62f, apparent,
competitive
60f noncompetitive
Michaelis-Menten
63
and,
61, equation,
Microarrays,
DNA,
Microcytic
58-59
483-484, anemia,
484f,
374,
374f
Micronutrients,
357.
See also
MicroRNA (miRNA), 459
Microsatellite DNA, 475
Microsomal cytochrome
P450
monooxygenase system, 279
Microsomal TAG transfer protein (MTP),
177-178, 228
Mineralocorticoids,
237
Missense mutation,
Mitchell hypothesis,
432f,
433
77-79
Mitochondrial
DNA,
80

mutations in,
80
shortand medium-chain fatty acid transport into, 192
Mitochondrial entry
sequence,
Mitochondrial myopathies,
443
80,
80f
RNA
polymerase,
Mitochondrion,
54f,
424
74
apoptosis and, 80
cristae of,
74
cytochrome P450 monooxygenase system of,
149
fatty acid
oxidation inner
membrane of
ATP
190-192,
74,
74f cardiolipin
in,
202
electron transport chain in, 74
transport systems of, 79-80, 79f matrix
of,
74,
74f porphyrin
structure synthesis
of,
74,
74f
Mixed micelles,
176,
177,
177f
Molecular biology, central dogma of, 395, 395f
Molybdenum,

2-Monoacylglycerol,
174f,
175-176
Monoacylglycerolacyltransferase, 176, 177
Monoamine oxidase inhibitors, 286-287
Monoamine oxidase (MAO), catecholamine metabolism
and,
286,
286f
Monocistronic mRNA,
438
Monocytes,
150
Monooxygenases (mixed function oxidases),
149
Monosaccharide(s),
83-85, absorption
by intestinal
anomeric forms
of,
84,
83f,
365
mucosal
85f ceramide,
209
classification of, 83, 83f concept
map,
89f cyclization
of,
84-85
dietary,
143f examples of, 83, 83f
glycosidic bonds between, 83, 84f joining
of,
85
137-144
concept map, 143f mutarotation
of,
84,
85f
Monounsaturated fat, 361-362, 362f, 364f
Mortality, obesity
and,
354,
354f
Motifs,
18,
18f,
19
Moxalactam,
390
Msitil restriction endonuclease, 477, 478f
Mucin, 165, 165f in,
278
in,
191f
485f
Vitamin(s)
60,
61f cells,
87
Mucopolysaccharides,
157.
See also
Glycosaminoglycans
Mucopolysaccharidoses, 163, 164f
Multiple carboxylase deficiency, 381
Muscle(s)
cardiac carnitine
192
lipoprotein lipase in, 228
26
skeletal vs.,
326
communication with
organs, in
other
307, diabetes
1, type
2, metabolic
338,
339f
342,
344
role of,
skeletal calcium
and, cardiac
307,
307f
131-132, vs.,
326
132f in,
192

contraction of,
131
exercising, glycogen as fuel source for,
332
125-126,
131f,
126f,
133f lactate
formation lipoprotein
in, lipase
26
resting,
331-332,
332f in
326f in
fasting, fatty
331-332, acids
oxygen
325-326,
332f as
fuel source
consumption smooth,
for, of,
326
and relaxation
150-151,
Muscular
151f dystrophy,
486
Mutarotation,
84,
85f
Mutations,
411,
475
detection of,
frame-shift, using
434, missense,
Southern
434f
432f, nonsense,
432f, silent,
432f,
433
splice site,
427,
434
trinucleotide repeat expansion, 433, 433f
Mut proteins,
411
Mycophenolic acid (MPA), 295f, 296
Myeloperoxidase
(MPO)
system,
150
Myocardial infarction, enzyme markers of, 65f,

a-helical content
of,
26
heme group
of,
25,
26,
26,
26f
26f oxygen
affinity/binding of,
28-29
oxygen dissociation curve for, 28-29, 29f polar
and nonpolar
amino acid
residues in,
26
26,
26f
Myophosphorylase, deficiency in, 129f
Myosin light-chain
kinase,
151
Myotonic dystrophy,
433,
Myristic acid,
361
N-acetylneuraminic acid
(NANA),
166
160
NAD. See Nicotinamide adenine dinucleotide

in alcohol-induced hypoglycemia, 317,
317f
433f
433
433
of,
103, in,
131-132,
118
228
metabolic
307f blotting,
332
473
in conversion of pyruvate to lactate
(anaerobic glycolysis),
97,
103-104,
103f formation
of in
electron in
fatty acid
glycolysis, oxidation
75
192,
193f
101
of,
76, transport
77f of,
to inner
mitochondrial membrane, 79-80, 79f
111,
111f
NADH-cytochrome bs
reductase methemoglobin
NADH
(NADH- reductase),
75,
NADH-dependent
38
75f malate
186
NADP*-dependent malate
152,
186
NADPH
synthesis, competitive
220-221,
221f of
145-146
in
155f cytochrome
P450
monooxygenase system, 149-150, 149f
in fatty
G6PD
186,
186f deficiency
in nitric
in and,
152
oxide synthesis,
pentose phosphate
150-151, pathway,
145-146
in phagocytosis,
150,
150f production
of, in
pentose pathway,
in in
in phosphate
145-146, reduction
of hydrogen
148-149,
146f peroxide,
148f reductive
biosynthesis, sphingomyelin
in
147
synthesis, steroid
hormone structure
206,
207f synthesis,
of,
147f uses
of,
147-151
NADPH-dependent
2,4-dienoyl reductase,
NADPH
237,
CoA
195
oxidase,
150
deficiencies in,
150
Native conformation,
Natural of
killer

(NK)
cells,
Negative regulation,
protein,
23f
301
of gene
453
Neomycin,
255
Neonate(s)
284,
285,
G6PD
285f, deficiency
phototherapy
289f and,
for,
153
285,
285f
PKU
screening in,
270,
271
premature, vitamin E deficiency in, 391
vitamin
K
390
Nephropathy,
345
Nervonic acid,
182f,
Network-forming
207
collagen,
Neural tube
defects,
Neuraminic
44,
44f
265,
375,
393f,
476
acid, in
Neurocan,
158
Neurodegeneration, in
disease,
165
Niemann-Pick
208
Neurofibrillary tangles,
disease, in
Alzheimer
21
Neuroglycopenia,
315
Neuropathy, diabetic,
345
Neuropeptide Y (NPY), 353
Neurotransmitter(s)
in metabolic regulation, 94, 94f, 95
nitric oxide as, 150-151, 151f
Neutral fat, 188
238f
G6PD
by, dehydrogenase,
151f

Nitrous oxide (N20), 151
N-linked glycoproteins,
142
N-linked glycosides, synthesis of, 167-169,
168f
N-linked oligosaccharides,
165,
166
final processing of, 168, 169f

Non-coding RNA (ncRNA), 417
Noncompetitive inhibition, of enzymes, 61-62,

examples of, 61-62
Lineweaver-Burke plot,
61,
61f
Nonessential amino acids, 261, 262f biosynthesis of, 267-269
Nonhomologous end-joining repair, 413
Nonpolar amino
acids,
2-4,
2f,
4f hydrophobic interactions of, 2, 19, 19f
location in
proteins,
2,
4,
4f in
myoglobin,
26
Nonsense codons,
432
Nonsense mutation, 432f, 433
Nonshivering thermogenesis,
79
Non-spherocytic hemolytic
anemia,103,
Norepinephrine catecholamine
synthesis

in energy
metabolism, in fasting, 331
functions of,
285
methyl group
and,
264
Normocytic anemia, 374f
Northern blot, 473, 483, 485f
NO synthase, 151
NPY,
353
N-terminal amino acid, 247
N-terminal residue
in protein degradation, 247
Nuclear localization
signal,
443
Nuclear receptor
superfamily,
456
Nucleic acids,
292.
See also
DNA;
RNA
detection of low-abundance sequences,

dietary, degradation
types of, 395
Nucleofilament,
411
Nucleoid, 398
Nucleolus, RNA synthesis in, 422
Nucleoplasm, in RNA synthesis, 422
Nucleoprotein, 396
Nucleoside analogs,
inhibition of
DNA
synthesis by, 408-409, 409f
Nucleoside diphosphate kinase, 112, 127, 296,

nucleoside monophosphate conversion to,
296,
296f
Nucleoside monophosphate
conversion to
nucleoside triphosphates,
kinases,
296,
(NMP),
292
diphosphates
296,
296f
Nucleoside
5’-phosphate,
292
Nucleoside(s), 292. See also Nucleotide(s)
Nucleoside triphosphate
(NTP),
292
296,
296f

fate during DNA replication, 411
formation of, 409-410, 410f
5’-Nucleotidase,
299
5’-Nucleotide,
292
Nucleotide excision repair, 411, 411f
Nucleotide(s),
292
function of, 291
296f and
of,
298,
299f
153
and,
285-286,
307
high-energy bond of, 292, 293f metabolism of, 291-306
concept map, 305f pentoses added to, 292, 292f
purine degradation
of, parent,
inosine
298-302,
300f,
305f monophosphates
as,
293
purine base conversion to, 296, 296f salvage
pathway for,
296-297,
296f structure
of,
291-292, synthesis
of,
291f,
292f
292-297, committed
step
305f of,
drugs targeting,

degradation of, 304
formation of, 302-303, 303f salvage
of,
304
structure of, 291-292, 291f, 292f synthesis
of,
302-304,
303f,
305f structure
of,
291-292,
291f,
292f synthesis
of,
291
unusual bases of, 291-292, 292f
Nucleotide sequences,
396,
396f alteration of, consequences of, 432f,

mapping of, 449. See also DNA analysis
Nucleotidyltransferase,
425
Nutrients,
357
Acceptable Macronutrient Distribution
Ranges in,
360,
360f availability
of, and
metabolism, classes of, 357, 357f
hepatic distribution of, 322-324

cancer and, 360, 361f
Dietary Reference Intakes in, 357-358,
357f,
358f influence on common causes of death,
361f macronutrients in, 360, 360f
concept map, 370f vitamins in, 372-394
Nutritional anemias,
374,
Nutritional rickets,
Nutrition
388
Facts labels,
Oo

adipose tissue in, 350-353, 353f afferent
signaling molecules
in,
353, assessment of, 349-350
body mass
index in,
349,
349f, carbohydrate intake and, 365
cause of,
351-352
childhood, 349
355f diabetes mellitus and, 349, 354
diseases associated with, relative risk of developing,
349,
354
environmental and behavioral factors in,
352
epidemic in United States, 349
fat deposition
in anatomical
differences in,
350, biochemical differences in, 350
fats cells
in,
351,
351f genetic
factors in,
352
health and,
354
insulin resistance and, 342, 342f, 346f,
353
lower body,
350,
350f metabolic
353
molecules influencing, 352-353
374f
364
94
293,
293,
294f
294f,
305f
353f,
353f
354f
350f mortality and, 354, 354f
pharmacotherapy for,
355
surgical treatment
of,
355
upper body, 350, 350f weight reduction in, 354-355
Obstructive jaundice,
284-285,
289f
Oculocutaneous albinism,
O-glycosides,
86,
272,
86f
O-glycosidic bonds,
86,
86f in glycoproteins, 165
in glycosaminoglycans, 158
in glycosphingolipids, 209, 209f
Oils,
188
Okazaki fragments, 403, 407
Oleic acid,
182f
Oligo-o(1>4)-a(1-4)-glucan transferase,
130
Oligomycin,
78
Oligonucleotide probes
in sickle cell disease, 472, 472f, 473f synthetic,
471-472,
472f
Oligonucleotide(s),
298
Oligopeptides, digestion of, 249
Oligosaccharide(s),
83,
85
carbohydrate-protein linkage in, 165
ceramide,
209

complex,
166,
166f dolichol-linked,
167
synthesis of, 167, 168f in glycoproteins, structure of, 165-166,
166f high-mannose,
166,
166f
N-linked,
165,
166
synthesis of, 167-169, 168f
O-linked,
165
synthesis of, 166-167
Oligosaccharidoses,
169,
170
O-linked glycosides,
synthesis
O-linked oligosaccharides,
Omega-6
of,
165, fatty
acids,
363
Omega
()
oxidation fatty
166-167
166-167
acids,
One-carbon units,
267
folic acid as carrier of, 267
Operators, in
prokaryotic transcription,
Operons bacterial,
450
lactose, 450-452, 451f tryptophan, 452-453, 453f
Opsin,
383f,
384
Optical isomers,
5,
5f
Optical properties
of amino
acids,
5,
5f
Oral tolerance tests, for digestive enzyme

Organic molecules, as enzyme cofactors, 54
Origin of replication, 399, 400f
Orlistat,
175
Ornithine formation from arginine, 262
transport of, 250
in urea
cycle,
255,
255f
Ornithine transcarbamoylase, 254
Ornithine transcarbamoylase
Orotate phosphoribosyltransferase,
Orotic acid,
302
302,
303f
Orotic aciduria,
303,
303f,
304
Orotidine
5’-monophosphate
(OMP),
Orotidylate decarboxylase, 302
Osmotic diarrhea, 87
Osteoblasts, 45
Osteocalcin vitamin K, 390
Osteogenesis imperfecta
(Ol), type
II
(osteogenesis
49,
49f, imperfecta
congenita),
49
type | (osteogenesis imperfecta tarda), 49
195
450
272f
302
51f
258
Osteomalacia, 388, 389f, 393f
Ovary(ies), hormonal secretion by, 237, 240,

NADPH
production in,
183,
183f in citrate synthesis, 111-112, 111f
cytosolic, decarboxylation of, 119f, 120
in amino acid catabolism, 251f in
gluconeogenesis,
118-120,
119f malate
oxidation to,
malate, transport
cytosol, in
to tricarboxylic
acid
111-112

of arachidonic acid, 213, 214f of fatty acids, 189-195
of glyceraldehyde 3-phosphate, 101, 101f of
112
of
LDL,
234,
235f of
malate,
113
of palmitoy! CoA, 192, 193f of
succinate,
112f,
113

of amino
of, acids,
245, coenzymes
252
252-253, in,
252,
252

of a-ketoglutarate,
112, thiamine
and,
379,
112f
379f branched-chain
amino

105,
109-111,
Oxidative phosphorylation,
in catabolic
acids,
252f
266
110f
73, pathways,
93
81f inherited defects of, 80
tightly coupled with electron transport,
77-78
uncoupling from electron transport, 78-79
Oxidative reactions, irreversible, 145-146,
146f
Oxidizing agent (oxidant), 76
Oxidoreductases,
53f
Oxygen hemoglobin affinity for and binding of,

allosteric effectors and, 27, 29-33
2,3-bisphosphoglycerate and,
31-32,
31f,
32f carbon dioxide binding and, 32
29-30,
PCO, and,
29
PH
and,
30,
POz and,
30f
29f, and,
28

reactive intermediates of, 148-149,
148f in steroid hormone synthesis, 237,
238f myoglobin affinity for and binding of,

as nitric oxide synthase substrate, 151
partial pressure of. See PO
Oxygen debt, 104
Oxygen dissociation curve
29f
73f,
113
119,
119, cycle,
119f
119f
109, of
30
109f,
77-80
for hemoglobin, 29, 29f, 31
2,3-bisphosphoglycerate and, 32,
32f sigmoidal
shape
30
for myoglobin,
Oxyhemoglobin,
29,
28
Oxypurinol, 301
P
PABA analogs, 294f
Palindromes,
466
Palmitate
(palmitic acid)
elongation of, 187
184-186,
185f relationship with glucose
186,
Palmitic acid,
Palmitoleic
182f,
361
acid,
Palmitoyl
182f
CoA, of,
energy
192,
193f
Palmitoyl thioesterase,
Pancreas,
307,
308f
Pancreatic enzymes
carbohydrate digestion
yield from
186
by,
86,
187f oxidation
87f deficiency
of, in
cystic fibrosis,
174
dietary nucleic acid degraded by, 298,

by,
176,
176f lipid processing by, 175-176
protein digestion by, 248-249, 249f
Pancreatic islet
cells a,
313f destruction,
in type
mellitus, by,
309,
313,
1
diabetes
338, dysfunction,
in type
mellitus,
338f
2
342, diabetes
342,
344,
344f glucose phosphorylation in, 98
insulin secretion by, 307-308, 308f,
310f
Pancreatic lipase,
175
Pancreatitis, chronic,
248
Pancreozymin. See Cholecystokinin
Pantothenic acid, 54, 184, 381, 392-393f in
coenzyme
A,
381,
381f deficiency
of,
381,
393f dietary
intake of,
381
of,
381,
392f
Parallel
B
sheet,
17,
17f
Parathyroid hormone (PTH), 388
Parenteral feeding,
369
Parietal cells,
248,
377
Parkinson disease,
286
Paroxysmal nocturnal hemoglobinuria, 206
Partial pressure
of carbon
dioxide.
See
PCO2
Partial pressure of oxygen. See PO2
Pathways,
91.
See also
PCO2
oxygen affinity of hemoglobin and, 29,
29f sickle cell disease and, 36
PCR. See Polymerase chain reaction
Pellagra,
380,
393f
Penicillin,
62
Pentasaccharide, core, in oligosaccharides, 166, 166f
Pentavalent arsenic (arsenate), 101
Pentose phosphate pathway, 92f,
145-156,
145f,
146f in absorptive/fed state, 323, 323f, 325,
of,
29f significance
of,
B
concept

map,
155f fatty
acid synthesis
and,
186,
186f irreversible oxidative reactions in,

reversible nonoxidative
Pentoses,
291,
292f
Pentose(s), in
nucleosides,
292, reactions
292f
PEP. See Phosphoenolpyruvate
PEP-carboxykinase (PEPCK), 118, 120, 122
PEP-carboxylase, in
fasting,
329
PEP
carboxylase
(PEPCK),
Pepsin pH optimum for, 58
in protein digestion, 248, 248f
Pepsinogen,
248
Peptide bonds,
1,
1f,
Polypeptide characteristics
of,
14,
13-14,
14f formation of, 13, 14f
naming,
14
partial double-bond character of, 14, 14f polarity of, 14, 14f
rigid and
planar character
of, trans configuration of, 14, 14f

Peptidyltransferase, 439, 440f
Peripheral tissues
ketone body use by, 196, 197f transport of ammonia from, 253, 253f
Peristalsis,
175
Pernicious anemia,
Peroxidase,
377,
213
Peroxisome,
B-oxidation
Peroxynitrite,
151
PEST
247
pH
enzyme denaturation and, 57
optimum, for
enzymes,
58,
58f oxygen affinity of hemoglobin and, 29,
30-31,
30f protons
lowering, source
of,
30,
30f reaction velocity and, 57-58, 58f
sickle cell disease and, 36
Phagocytosis,
150,
150f of extracellular glycosaminoglycans, 163
oxygen-dependent,
150
oxygen-independent,
150
Phenobarbital,
279
Phenolic compounds,
365
Phenylacetate,
271
Phenylacetylglutamine, 258, 258f
Phenylalanine degradation
of,
263,
263f inherited deficiencies in, 263
side chains of, 2f tyrosine
and,
268
Phenylalanine hydroxylase (PAH), 263, 268,

270-271,
270f gene for, mutation of, prenatal detection
of,
477,
479f,
480f
Phenylbutazone,
214
Phenylbutyrate,
258,
258f
Phenylisothiocyanate,
15
Phenylketonuria
(PKU),
263,
268f,
269f,

antenatal diagnosis of, 271-272
characteristics of, 271, 271f maternal,
272
neonatal screening and diagnosis of, 270,
271
prenatal diagnosis of, using RFLPs,
477-479, 479f, 480f treatment of, 272
393f in,
195
14,
14f
14f.
457
in,
See
147
also
Phenyllactate, 271
Phenylpyruvate,
271
Phenylthiohydantoin (PTH), 15
Pheochromocytoma, 286
PH gradient, in oxidative phosphorylation,

Phosphatase, 101
Phosphate addition of. See Phosphorylation
inorganic
(P))
as activator of PRPP synthase, 293,

in
ATP
73
removal of. See Dephosphorylation vitamin
D
and,
388
Phosphatidalcholine,
202
Phosphatidalethanolamine, 202, 202f
Phosphatidic acid,
201,
201f,
202
189f,
203
Phosphatidylcholine,
201f, in
bile,
224
in lung
surfactant,
204
203-204,
204f from phosphatidylserine in liver, 204,

Phosphatidylcholine:cholesterol acyltransferase (PCAT, LCAT),
234-235,
236
Phosphatidylethanolamine, synthesis
of,
203-204
Phosphatidylglycerol, 206
Phosphatidylinositol,
205-206
as arachidonic
acid reservoir,
205
in membrane protein anchoring, 206, 206f in
signal transmission
across membranes,
205-206,
205f synthesis
of,
205-206
Phosphatidylinositol 4,5-bisphosphate (PIP2),

Phosphatidylserine,
201f,
205
synthesis of phosphatidylcholine from,

3’-Phosphoadenosine-5’-phosphosulfate
Phosphodiesterase, 95-96
(PAPS), 162, 210, 210f, 263
3’5’-Phosphodiester bonds,
396-397,
Phosphodiester bonds,
Phosphoenolpyruvate in
RNA,
(PEP), glucagon
and,
122, in
417
102
122f gluconeogenesis,
in
118-120, glycolysis,
102,
102f
Phosphofructokinase in
122, in
glycolysis, hormonal
323
123
102,
121, regulation
138
of, of,
by citrate,
313
Phosphofructokinase-1
(PFK-1)
state, in
321
120,
120f in
99,
100f
Phosphofructokinase-2 (PFK-2), 99
6-Phosphoglucanolactone dehydrogenase,
146
6-Phosphoglucanolactone hydrolase,
Phosphoglucomutase, 126, 128f, 130
6-Phosphogluconate dehydrogenase,
6-Phosphogluconate
146
pathway.
See
Pentose phosphate
pathway
6-Phosphogluconolactone, in
pentose phosphate pathway, 145-146, 146f
Phosphoglucose isomerase, 99
Phosphoglucose isomerase deficiency, 103
2-Phosphoglycerate, dehydration of, 102
146
105,
105f
63,
98,
112
119f
201f,
204
234
396f
classification as
diagnostic elevated
of,
64
tool, levels
65
of,
65
in disease
states,
64f,
65
in normal cell turnover, 64-65, 64f
Plasma hormones,
307
Plasma lipoproteins,
227-237.

Plasmalogens,
202,
202f
Plasmids,
398
467,
Plasminogen,
467f, apo(a)
and,
Plasmodium falciparum,
Platelet-activating
468f
237
37
factor
Platelet aggregation
n-6
and n-3
polyunsaturated
See also
202,
202f fats
and,

nitric oxide as inhibitor of, 150, 151f
Platelet homeostasis, prostaglandins in, 214,
216f
Platelet(s), interaction
of prothrombin
389
Pneumonia, nutrition and, 361f
POz, oxygen
affinity binding
and of
hemoglobin, of
Pol a,
Polar
29, myoglobin,
29,
407,
407f amino
acids in
26
uncharged,
3f,
4
Polar hydroxyl group, in amino acid side chains,
4
Polarity, of
Polar peptide
side
Pol
B,
Pol
Pol
Pol
5,
¢,
407,
407,
407, y,
407, bond,
chains,
3f
407f
407f
407f
407f
Pol
5’3’
407
Polyadenylate polymerase,
Polyadenylation,
426
457
Poly-A
tail, of
mRNA,
Polycistronic
Polydipsia,
426, mRNA,
438, in
diabetes
5’~3’
426f
450, mellitus,
in
DNA
404-405,
Polymerase chain
advantages of,
applications of,
for
452
338,
341
proofreading,
404f reaction
(PCR),
479-483
382
482-483
comparison of
normal cloned
gene with uncloned mutant gene, 482
cystic in
for fibrosis,
483,
483f detection
of low-abundance
acid sequences, 482
for forensic
analysis of
DNA
samples,
482-483
primer construction
steps in,
480-482
Polymorphism(s),
475,
475f.
Restriction in,
480-481,
481f
See also
fragment polymorphism
in phenylketonuria,
for
477-479
prenatal diagnosis,
restriction fragment
short
476-479
length, tandem
in sickle
repeat, cell
473-479
483

disease, single
nucleotide,
476-477,
475
in tandem
repeats,
475,
476f for tracing chromosomes from parent to
offspring,
475-476
Polynucleosome,
411
Polyol,
139
Polypeptide chains,
431,
431f posttranslational modification of,
443-444, 443f
478f length
nucleic
14,
14f
29f
29f with,
(PAF),

amino acid composition of, determination of,
14-15,
15f,
16f amino acid residue or moiety in, 14
B
bends of,
17
B-sheets of, 17, 17f cleavage into smaller fragments, 15, 16f
domains of,
19
elongation of, 439-442, 439f mRNA
translation into,
431-438
posttranslational modification of, 443-444,
443-444f primary structure of, 14-16
secondary structure
of,
16-18
nonrepetitive,
18
sequencing of, from N-terminal end, 15,
15f tertiary
18-20
interactions stabilizing, 19
Polyphagia, in diabetes mellitus, 338, 341
Polyphosphorylated guanosine
(ppGpp),
454
Polysaccharide(s),
83, dietary,
365
digestion of, 86
Polysomes,
442,
442f
Polyubiquitin chain,
247
Polyunsaturated fatty acids (PUFA), 188, 364f
Polyuria, in
338,
341
Pompe disease,
129f,
131,
472
Porphobilinogen,
278,
278f,
279,
279f
Porphyria cutanea tarda, 280, 280f, 281f
Porphyria(s),
279-282
acute intermittent,
280,
281f chronic,
280,
281f clinical manifestations of, 280
erythropoietic,
280,
281f congenital, 280, 281f
hepatic,
280
acute,
280
hereditary coproporphyria, 280, 281f treatment
of,
282
variegate, 280, 281f
Porphyrinogens, 278

degradation of, 282-284, 282f, 283f, 284f metabolism
of,
277-278
of,
277
distribution of, 278, 278f structure of, 277-278, 278f
synthesis of, 278-279, 278f, 279f, 280f,
281f defects
in,
279-282,
278-279
type
|,
278,
278f type Il, 278, 278f
Positive regulation, of gene expression, 452,
453
Postprandial hypoglycemia, 316
Posttranscriptional modification,
of ribosomal
RNA,
424-425, of
transfer
RNA,
425,
425f
Posttranslational modification of
collagen,
45
of polypeptide chains, 443-444, 443f
Pravastatin,
61,
224
Prednisone, 301
Pregnancy folic acid supplementation in, 375
retinoid/vitamin A toxicity in, 386
Pregnenolone, 237f, 238
Premature atherosclerosis,
232
Prenatal diagnosis, 476-479
85
424-427
424f of cystic fibrosis, 483, 483f
DNA
sources,
476,
477f methods,
476
of phenylketonuria,
477-479,
479f,
480f of sickle cell disease, 476-477, 478f
Prenylation, 221
Prepriming complex, for DNA replication, 399,
403
Preproglucagon, 313
Preproinsulin, 308, 308f, 309f
Preribosomal
RNA,
424
Pribnow box,
420,
420f
Primaquine,
153
Primary hyperoxaluria Type 1, 253
Primase,
402-403,
402f
Primosome,
403
Prion disease,
22,
22f
Prion protein (PrP), 22, 22f
Pro-a-chains, in collagen synthesis, 45, 46f
Probenecid,
301
Probes,
DNA,
470-472

472
472
hybridization to
DNA
oligonucleotide, in
Procollagen,
46f, fragments,
471-472,
472f disease,
472,
47
extracellular cleavage
of,
46—47f,
473f
47
Procollagen peptidases, 47, 47f deficiency of, and Ehlers-Danlos
syndrome,
48
N-Procollagen peptidases,
Product inhibition, 64f
Progesterone, 237f, 240f
Progestins,
237
Proinsulin, 308, 308f, 309f
Prokaryotic
DNA,
395
lagging in,
strand of,
leading strand
of,
403-405,
402,
403f
402,
403f proofreading
of,
404-405,
404f proteins required for strand separation in,

replication fork
of,
399-400, replication
of,
399-406
bidirectional,
399,
400f continuous, 402
direction of,
402
discontinuous, 402, 402f origin of, 399, 400f
prepriming complex for, 399, 402
processive,
403
RNA
primer for,
402-403,
402f excision and replacement of, 405,
405f,
406f semiconservative, 399, 399f
supercoil problem in, 400-401, 401f
Prokaryotic gene expression, regulation of,
449f,
450-454,
463f coordination
of transcription
translation, lactose
mRNA
and
453-454, operon
in,
450-452, transcription
454f
451f from
bacterial operons,
450
role of
operators, tryptophan
450, operon
Prokaryotic gene
antibiotics
451f in,
452-453, transcription,
targeting, elongation
hairpin
453f
419-421
421,
422f in,
420-421, turn
420f in,
421,
421f
419-420, regulation
of,
420f
449f,
450-454
termination of,
421,
421f rho-dependent, 421
rho-independent, 421, 421f
400f
47
470
403f
Prokaryotic plasmids, 467, 467f
Prolactin,
142
Proliferating cell nuclear antigen (PCNA), 407
Proline,
4,
4f,
262
a-helix disrupted by, 16-17
in
B-bends,
17
of,
262
collagen,
45
hydroxylation of, 45, 45f, 46f, 47
proteins containing,
247
49
side chains of, 2f, 4, 4f synthesis
of,
268
Prolyl hydroxylase, hydroxylation in collagen

Promoter activation,
hormone response
elements and,
240,
241f
Promoter region, 419-420, 420f
Promoters,
422-423,
423f
Propeptides, in collagen biosynthesis, 47, 47f
Propionate,
277
Propionic acid,
Propionyl
182f
CoA, amino
acid conversion
Propionyl
CoA
265
catabolism to
succinyl forming,
265,
CoA,
265
of,
194-195,
195f carboxylase,
194
Prostacyclin,
213f
Prostaglandin endoperoxide synthase (PGH
synthase),
213
Prostaglandin(s),
213-214
217f fatty acids as precursors of, 181
function of, 215f n-6
and n-3
PGEo,
213f,
PGFo,,
215f
213f,
215f
PGGa,
214f
PG, synthesis of, 213, 214f
PGlo,
213,
213f,
214,
215f in
platelet homeostasis,
214
structure of, 213f synthesis of, 213-214, 215f
214
substrates for,
Prosthetic group,
202
54
Proteases,
50
Proteasomes, 246-247, 444
Protein-calorie malnutrition (PEM), 369
Protein C vitamin K, 390
Protein Digestibility Correct Amino Acid Score
(PDCAAS),
367,
367f
Protein factors,
437
Protein kinase A, 95, 134
Protein kinase A-mediated covalent modification, 456-457
Protein kinase
C,
95,
134,
208
in intracelluar signaling, 205f, 206
G,
151
Protein kinase(s)
cAMP-dependent,
95,
95f glucagon and, 314
in glycogen degradation, 132, 133,
133f in pyruvate kinase regulation, 102,

102f in
63,
63f pyruvate
as inhibitor
of,
111
in pyruvate dehydrogenase complex, 111f
Protein-oligosaccharide transferase,
167
Protein phosphatase
1,
132,
133,
134
Protein phosphatase(s), 95
Protein(s)
a-helix of, 16-17, 16f amino acid sequence, 14-16
fats
266f and,
363
determination
DNA
analysis of,
14-15, of,
15-16
amino acid
sequence in,
14-16
determination of,
14-15,
15f
DNA analysis of, 15-16
484-485,
485f
B-bends of,
17
B-sheets of,
17,
17f carboxylation of, 444, 444f
covalent modification of, 444
246-247,
444
chemical signals
for,
247
ubiquitin-proteasome mechanism for,
246-247,
247f

agents for,
20
reversibility of,
dephosphorylation dietary,
246f,
357,
20
of,
95
357f,
367-369

from animal
sources,
367,
367f deficient intake of, 369, 369f
with differing limiting amino acids, 368,
368f digestion
of,
247-249
abnormalities in, 248-249
by gastric
secretion,
248,
248f by pancreatic enzymes, 248-249,

energy content
excessive of,
intake nitrogen
359,
359f of,
balance from
plant
368
and, sources,
quality of,
368
367f,
367-368, requirement
368
367f, for,
368f
368-369
sparing effect
of

DNA helicases, 400, 400f domains
of,
19
farnesylated,
444, fibrous,
43-52,
444f
51f.
Elastin summary,
52
folding of,
20,
20f chaperones
in,
20
errors or
mutations in
1
y-carboxyglutamate in, 389-390
globular, 18, 25-42. See also Hemoglobin;
Myoglobin glycosylation
of,
443f,
444
hydroxylation of, 444, 444f location of nonpolar side chains in, 2, 4, 4f
metabolism of
332,
332f glucagon and, 314
monomeric,
20
native conformation of, 23f peptide
bonds of,
13-14,
14f phosphorylation
of,
443f,
444
443f,
444
ribosomal,
436
regulatory, 454, 454f single-stranded
DNA-binding

in aqueous
solution, concept
map,
400f
18
23f hierarchy
of,
13,
13f,
23f misfolding, 21-22
primary, 13-16, 13f, 14f, 15f, 23f
See carbohydrates
and,
Collagen;
15f also
360,
400, quaternary,
13f,
20-21, secondary,
13f,
16-18, nonrepetitive,
23f
16f,
18
supersecondary
(motifs), tertiary,
13f,
18-20, interactions
18,
18f,
17f,
18f
19f, stabilizing,
395,
431-448,
20f,
23f
23f
19
440—-441f.
See in
also

components required
323f,
324,
326, for,
446f elongation
in,
439-442, inhibitors of, 442
4838-439, insulin
and, regulation
439f,
434-437
440f
440f
311
of,
443
steps in,
438-443
441f, trimming
of,
443
uncoupling,
79
Protein-sparing effect,
369
Protein targeting, 45, 166, 443
Protein turnover,
246-247,
246f, rate
of,
246
Proteoglycan aggregates,
Proteoglycan monomers,
brush-bottle model
of, structure,
158,
Proteoglycan(s),
160f
157
cartilage,
158, glycoproteins
linkage region
structure
160f vs.,
165
of, of,
158,
Proteolytic enzymes,
Proteomics,
158,
160f
248,
484,
248f
485,
Prothrombin,
389
Proton acceptors,
as,
160f
5
Proton donors, amino acids as, 5
“Proton leak,”
79
Proton pump,
77-78
Proton transport, coupled with electron

Protoporphyria, erythropoietic,
Protoporphyrin,
280,
62,
277
Protoporphyrin
IX,
279f
Protoporphyrinogen oxidase,
PRPP
293,
Pruritus, in
porphyria,
299
280
385
PS
decarboxylase,
Pseudogenes,
204
34
Pseudouridine,
425f
P
sites, on
ribosome,
436
Psoriasis, retinoic acid for, 385
Purine nucleoside
phosphorylase, deficiency
in,
300
Purine nucleotides
diseases
298-302, associated
300f, with,

parent, inosine
purine monophosphates
base conversion
salvage
299
305f
299-302, as,
293
to,
296,
296f pathway
structure for,
296-297, of,
of,
296f
291f,
292f
step
305f of,
Purine ring,
atom
293,
293, sources

bases conversion to nucleotides, 296, 296f
in DNA and RNA, 291-292, 291f, 305f
294f
294f, for,
305f
292,
293f
281f
485f
442
259f
158,
160f
158
158,
160f
281f de novo synthesis of, 291
Puromycin,
440f
Pyranose,
84
Pyridoxal,
377f,
378
Pyridoxal phosphate, 251, 251f, 377f in
glycogen degradation,
128
in histamine
287
in sphingomyelin
206
in transamination of amino acids, 251,

Pyrimidine nucleotides
304
salvage of,
structure
302-303,
304
of, synthesis
291-292, of,
Pyrimidine ring,
in,
302,
302-304, sources
378.
303f
291f,
See
292f
303f,
305f of
individual
302f
Pyrimidine(s)
bases, in
DNA
and

304
de novo synthesis of, 291
304
of,
302-303,
Pyrophosphatase,
291f, atoms
291-292,
291f,
292
303f
126,
296,
Pyrophosphate
(PP),
Pyruvate,
96
alternate fates
404
of, amino
acids that
105
form,
263,
263f carboxylation
to oxaloacetate,

NADPH
production formation
of in
amino in,
186, acid

ATP-producing,
102-103
glucose conversion
to
96-102,
(aerobic
96f glucose
synthesis from
(gluconeogenesis),
118-119, oxidative
119f decarboxylation
of,
105,
106f,

thiamine and,
378f, phosphoenolpyruvate
379,
379f conversion
to,
102
ethanol,
105,
106f reduction to lactate (anaerobic glycolysis),

state,
323
allosteric regulation of, 119
330
Pyruvate carboxylase/PEP carboxykinase,

Pyruvate
Pyruvate decarboxylase,
109-111,
110f coenzymes
in, component
110
enzymes
147
complex, in,
110
111
mechanism of action, 110, 110f regulation
of,
110-111,
111f in tricarboxylic acid cycle, 109-111
Pyruvate dehydrogenase
kinase,
110,
323
Pyruvate dehydrogenase (PDH), 105, 110,

in absorptive/fed state, 323
in arsenic poisoning, 101, 111
106f, glycolysis),
105,
186f
261,
263,
437
378,
RNA, also
118,
120,
105,
106f,
deficiency of, 111
in fasting, 196, 330
inhibition of, by acetyl CoA, 122
irreversibility of
action,
118
Pyruvate dehydrogenase phosphatase,

Pyruvate kinase,
98, in
102,
118
state, of,
102
covalent modification
of, deficiency
in,
102-103
dephosphorylation of,
121, hormonal
102
122f regulation
of,
105
insulin and, 313
mutant forms of, 103
Q

R
Racemases, 84
Random coil,
18
Rate of
reaction,
55
Reactive oxygen species, 148-149, 148f in
280
Receptor-mediated endocytosis,
Recombinant
DNA,
472
joining of sticky ends, 467f
Recommended Dietary Allowance (RDA),

for carbohydrates,
for protein,
368
for vitamin D, 388
for vitamin
E,
391

2,3-bisphoglycerate synthesis in, 101
G6PD
in,
152,
152
deficiency of, 152, 152f hemoglobin
in,
27
NADPH
in,
148
Redox pairs,
76
Reducing agent (reductant), 76
Reducing equivalents, transport of, 79-80,
79f
Reducing sugars,
84-85
Reduction potential, standard (E,), 77, 77f relationship with free energy change, 77
Redundancy,
433
Refsum disease,
195
Regulatory sequences, in transcription,

Release factor (RF), 441f, 442
Renal osteodystrophy, 389
Renal rickets,
389
Renaturation (reannealing), 398
Renin, 239
Replication fork, 399-400, 400f
Reproductive tissues
vitamin
A
and,
384
Respiratory burst,
150
Respiratory control, of energy production, 78
Respiratory distress syndrome, 204, 236
Resting metabolic
rate
(RMR),
359
Restriction endonucleases,
nomenclature
465-466, for,
466
restriction sites,
466
specificity of, 465-466, 466f
“sticky”
and
“blunt”
ends,
466,
466f,
467f
Restriction fragment length polymorphism

close linkage
in,
477
DNA variation resulting in, 475, 475f, 476f
465f
367
232,
233f
323
102
in phenylketonuria, 477-479, 479f for
476-479,
477f in
sickle of
single
476-477,
DNA
of tandem
for base
478f changes,
474, repeats,
475,
476f tracing
chromosome from
offspring, 475-476
Restriction fragment(s), 465-466
sticky and blunt ends of, 266, 266f
Restriction map,
467
Restriction sites,
466
Reticuloendothelial
(RE)
degradation
Retinal, in,
382,
382f,
392f
11-cis,
382f,
383f,
11-cis-Retinal,
384
382f,
383f,
Retinoic acid,
381-382, mechanism
384
382f, of
action, reproduction
and,
392f
382,
384
retinol oxidation
to,
384f in transcriptional regulation, 456
use in
dermatology,
385,
385f,
386
Retinoids,
381-384
clinical indications
for,
384-385
384
mechanism of action, 382, 383f, 384f,

toxicity
382, of,
Retinol,
382f
385-386,
382, as
dietary
393f
382,
392f supplement,
oxidation
384, to
retinoic
391
acid,
Retinol-binding cellular,
protein
382
plasma,
382,
Retinopathy,
384f
140
Retinopathy, diabetic,
Retrotransposons,
345,
408,
Retroviruses,
459,
384f
(RBP),
382
346f
461
468
Reverse transcriptase,
Reyes syndrome,
RFLP.
193
See
Restriction polymorphism
R-group.
See
Side chains,
of amino
Rho-dependent termination, 421
Rhodopsin,
384
Rho factor,
419
Rho-independent termination, 421, 421f
Riboflavin,
54,
380-381,
392-393f active
forms deficiency
of,
380-381, of,
381,
Dietary
380f,
393f
Reference
Intakes function
of,
381,
392f
Ribonucleases, 397, 424
Ribonucleoside 5'-diphosphate (NDP), 293f
Ribonucleoside 5'-monophosphate (NMP),

Ribonucleoside 5'-triphosphate (NTP), 293f
Ribonucleotide reductase,
297,
300f activity
site of,
297-298, reduced,
regeneration regulation
298f of,
of,
297-298, substrate
specificity
297
298f of,
298

as end product of purine synthesis, 293
Ribose,
292,
292f
Ribose
5-phosphate,
145
formation of, in pentose phosphate pathway,
147,
147f
Ribosomal proteins,
436
regulatory, 454, 454f
392f for,
358f
408,
469, fragment
469f length
acids
384f
475
parent to
system, heme
282,
282f
Ribosomal RNA (rRNA), 418, 418f posttranscriptional modification of,
424-425, 424f stringent response and, 454
Ribosomes,
436-437
cellular locations
of, eukaryotic,
436-437
436-437, prokaryotic,
Ribozymes,
436f
436-437,
54,
434,
439
Ribulose
5-phosphate, formation
145, of,
146,
436f
147
146f
Ricin,
442
Rickets,
393f nutritional,
388
renal,
389
Rifampin,
RNA,
421,
395,
422f
417-430
alkali cleavage
of, analysis
of,
485f bases
of purine,
291,
291f, pyrimidine,
396
305f
291,
291f, unusual,
305f
291-292, concept
map,
292f,
429f
DNA
information for,
395, elongation
of,
418
395f
420-421, heterogeneous
420f nuclear,
messenger
(mRNA),
425
417, alternative
splicing

coding for
codons of,
431-432, mutations
432f of,
432f, recognition
by editing,
457-458,
433-434
tRNA,
435
418,
419f posttranscriptional
425-427, modification
426f,
454,
457-460
exons of,
splicing frame-shift
of, mutation
introns of,
426
426-427, levels
of, determination
426-427,
427f of,
434,
434f
427f of,
483-484,

monocistronic, of,
438
non-coding
(ncRNA), nonsense
417
432f, nucleotide
sequence of,
433
alternation

polycistronic,
438,
450, silent
mutation splice
of,
432f, site
mutation
452
433
of, stability
of,
434
458-460, transcription
458f,
460f of,
450
translation of,
431f,
440-441f,
432-443,
460,
460f components
required regulation
of, for,
434-437
443
steps in,
438-444, trinucleotide
expansion

preribosomal,
424
ribosomal (rRNA), 417, 418, 418f posttranscriptional
modification
424-425, in
433, of,
424f regulation
of gene
454,
454f small nuclear (snRNA), 418, 426-427,
427f structure
of,
417-418,
429f synthesis of (transcription), 429f. See also
of, modification
of,
432f,
418,
419f patterns
of,
427,
434
of,
433
of,

antibiotics targeting,
421, from
DNA
palindrome elongation
422f
450-453, and,
466, in,
420-421, eukaryotic,
hairpin turn
initiation
420-422,
466f
420f
454-461
in,
421,
421f, of,
negative positive
453
420f of,
primary transcript
453
452, in
419-421, termination
450-454
of, transfer
(tRNA),
421,
421f
417,
418, as
adaptor
418f molecules,
anticodon of, 435
antiparallel binding
435
with codon,
437
attachment of amino acids to enzymes
435, site
for,
434f,
435
codon recognition
initiator, by,
438,
440f intrachain
of,
418,
418f posttranscriptional modification of, 425,
425f in
ribosomal
435
binding sites
for,
436
synthesis of, 425
unusual bases of, 417f, 418, 425
RNA-induced silencing complex (RISC), 459
RNA
(RNAi),
459,
459f therapy
using,
459-460
RNA polymerase, 417
421,
422f core enzyme of, 419, 419f
DNA
unwinding by,
420-421, in
holoenzyme
420f
422-424,
(sigma factor,

mitochondrial,
424
nuclear, of
eukaryotic in
cells, prokaryotic
450-452, rho
423f unit)
of,
419,
422-424,
423f
419-421,
451f factor
of consensus
nucleotide sequences
recognized by, 419-420, 420f transcription
termination dependent
on,

5’3’
RNA
polymerase in transcription, 419-421, 420f
RNA polymerase |, 422
RNA polymerase II, 422-424, 425
inhibitors of,
424
RNA polymerase III, 424
RNA
primers,
402-403,
402f excision of, 405, 406f

RNA
silencing,
Rosuvastatin,
459
224
Rotenone,
76f
RT-PCR (reverse transcriptase-PCR), 483
Ss
S-adenosylhomocysteine, 264
S-adenosylmethionine (SAM), 204, 263,
425-426
activated methyl group of, 264
as carrier of one-carbon units, 263, 267
hydrolysis of,
264-265,
264f methylation of, 460, 461f
synthesis of, 264, 264f
Salicylates, 282
Salt bridges, 31
419f,
453
435
451f
437,
435f
Salvage enzymes, 409
Sandhoff's disease, 212f
Sanfilippo syndrome, 164f
Sanger dideoxy
method,
470,
Saturated fat,
361,
362f,
Saturnine gout, 299
Scaly dermatitis,
182
Scavenger receptor class A (SR-A), 234
Scavenger receptor class B type 1 (SR-B1),
236
Sclera, blue,
49
yellow,
284
Scrapie,
22
Scurvy,
47,
47f,
377,
Second messenger
Secretin,
377f,
393f systems,
176,
248
Sedimentation coefficients,
of
94
ribosomes,
436
Selenium,
Dietary
Reference
Intakes for,
358f
Semiconservative replication, of DNA, 399,
399f
Senescence, cellular, 408, 408f
Serine,
4,
19f catabolism
of,
263,
263f in cysteine synthesis, 265, 268
interconversion with
glycine, in
phospholipids,
201f,
247
263,
263f,
202
in proteoglycans, 158
of,
3f,
4
in sphingomyelin synthesis, 206, 207f synthesis
of,
268
Serine dehydratase,
263
Serine hydroxymethy! transferase, 268

Serotonin functions of, 287
monoamine oxidase inhibitors and, 287
synthesis of, 270f, 287, 287f
Serum assays,
65
Set point, for body weight, 351
Settling point, for body weight, 351
Severe combined immunodeficiency disease
(SCID)
gene therapy for, 485, 486f
X-linked,
485
Severe combined immunodeficiency (SCID),
300f,
301-302
Sex-hormone binding protein, 237
Sex hormone(s),
237
Shine-Dalgarno sequence, 438f, 439, 454
Short-chain fatty acids, 192
Short tandem repeat polymorphisms (STRs),
483
Sialic acid.
See
N-Acetylneuraminic
Sickle cell
anemia jaundice
and,
284
disease, amino
4,
35-37, acid
substitution anoxia
caused in
by,
42f
HbS
36,
37f diagnosis
of, using
RFLPs, electrophoresis in, 36, 36f
ethnicity and,
35
oligonucleotide probes and, 472, 472f, 473f point
mutation in
B-globin gene
causing,
35,
37f selective advantage of heterozygous state,
37,
38f treatment
of,
36,
298
variables increasing sickling in, 36
Side chains
of amino acids, 1-5, 1f, 2-3f acidic,
3f,
5
as attachment site for other compounds, 4
476-477, and,
36,
36f
478f acid
364f
471f
268
basic,
3f,
5
in disulfide
bonds,
4
hydrogen bonding by, 4, 19, 19f hydrophilic,
18
hydrophobic,
18
hydrophobic interactions of, 4, 19, 19f ionic
interactions of,
19,
19f nonpolar,
2-4,
2f,
polar,

3f,
4
uncharged of
porphyrins, distribution
Sigma polar,
3f,
277-278
of,
278, factor,
419
Signal transmission
ceramides in,
208
insulin in,
311f,
312
phosphatidylinositol in,
4
278f
205-206, second
messengers in,
205f
94-96,
291
sphingosine in, 208, 208f
Sildenafil citrate,
151
Silencers,
424
Silent mutation, 432f, 433
Simvastatin,
61,
224,
224f
Single nucleotide
Single-stranded polymorphisms
400,
(SNPs),
(SSB)
400f
B-Sitosterol,
220
Skeletal muscle
calcium and,
131-132, carnitine
deficiency
475
proteins,
132f in,
contraction of, 131
exercising, glycogen
glycogen as
in,
125-126,
103,
192
fuel
126f, source
for,
332
131-132,
131f,
118
26
resting in
325-326, amino
326f in,
326, metabolism

in,
326,
326f intertissue relationships of, 328f
fasting,
331-332,
332f carbohydrate
332
protein in,
332,
332f metabolism
in,
332, fatty
acids as
332f for,
332
oxygen consumption of, 326
Sly syndrome,
164f
Small intestine
dietary lipid processing in, 175
dietary nucleic acid degradation in, 298,

oligopeptide digestion
Small interfering
Small in,
249
RNA
(siRNA),
459
nuclear ribonucleoprotein
particles
(snRNPs), 427, 427f
Small nuclear RNA (snoRNA), 418, 422,
424-425, 427f
Smith-Lemli-Opitz syndrome (SLOS), 221
Smooth muscle, nitric oxide and relaxation of,

Sodium-dependent glucose co-transporter 1
(SGLT-1),
87
Sodium-glucose linked
transporter
Sodium
(SGLT),
97
(Na*)-independent facilitated
diffusion transport, of glucose, 96-97, 97f
Sodium (Na*)-monosaccharide in,
331,
326f in,
326, in
Sterol
Sterol cotransporter system, 96, 97

and diabetes, 345
to fructose
139-140, 140f hyperglycemia
and,
140
139-140
Sorbitol dehydrogenase,
Southern
139, blotting,
473,
485f detection
of mutations,
experimental
473
procedure, variants
of,
473
Soy protein,
Sperm coronary
cells, fructose
S
phase, of
Sphinganine,
203f
Sphingolipidoses,
210-213,
212
Sphingolipids.
See
Sphingomyelin,
Glycosphingolipids
202-203, degradation
203f of,
208,
208f disorders
of,
208,
208f ratio
with phosphatidylcholine,
in respiratory distress syndrome, 204
206-207,
207f
Sphingomyelinase,
208
Sphingophospholipids,
Sphingosine,
201
Spina bifida,
265,
Spleen, heme
375
Spliceosome, in,
282,
426
Splice site
mutations,

alternative, mechanism
457,
427,
457f of,
427, splice-site
427f choice,
Squalene,
221

dietary intake
of,
365
digestion of, 85
Starvation.
See
Fast/fasting
Statin drugs,
224, competitive
361
Stearic acid,
182f,
Steatorrhea, by,
361
177,
Stercobilin,
283
Steroid,
173f
Steroid hormones,
237-240,
237f action mechanism of, 240, 240f, 241f
adrenal cortical
degradation excretion
239,
238,
239f
238f of,
240
of,
240
gonad secretion
of,
240
240
237-238, cholesterol
238f in,
237-238
rate-limiting step

Steroid nucleus,
219
hydroxylation of, 237
Steroidogenic acute regulatory protein (StAR),
238
Sterol regulatory element-binding protein cleavage
activating protein
(SCAP),
223
regulatory element-binding
protein
(SREBP), 222-223, 223f, 232
regulatory element
(SRE),
222-223,
223f,
232
Sterol(s),
219-220
in HMG CoA reductase regulation,
222-223, 223f of,
237
178f,
248
457
202-203
473,
474f heart
disease metabolism
and, in,
407,
407f
211-213,
364
139-140
211f,
140
434
61, via,
282f
61f
B,

Superoxide catalase,
Superoxide
148
dismutase
(SOD), radicals,
151
Supersecondary structures,
74,
148,
18,
18f
Surfactant,
207
phosphatidylcholine and, 204
Surgery, weight-reduction,
355
Svedberg units,
of rRNA,
418
Symbols, for
amino
Sympathetic acids,
5,
5f ganglia,

Synaptic signaling,
94f
Syndecan,
158
Synthetic uncouplers, 79
Systemic lupus erythematosus, 427
T
TAG
synthase,
176
Tandem repeats, 475, 476f
Tangier disease,
236
Taql, specificity of, 466, 466f
Taq polymerase,
482
TATA
box,
423
Tau protein,
21
Taurine, bile
acids conjugated
Taurochenodeoxycholic acid,
Taurocholic acid,
225
Tay-Sachs disease,
Telomerase,
211,
407-408,
Telomeres,
212f
408f
407-408
Temperature enzyme
denaturation melting
of
DNA, of
and,
398,
398f fatty
acids, reaction
velocity
Termination codons,
182
and,
432,
57
57,
57f
432f
Termination factor,
419
Testes, hormonal secretion by, 237, 239-240,

Testosterone,
237,
237f,
239,
240f
Tetanus toxin, cell surface receptors for, 209
Tetracyclines,
440f
Tetrahydrobiopterin
Tetrahydrofolic acid
in one-carbon
synthesis
(BH4),
151,
(THF).
See metabolism,
of,
268, also
267,
270f
Folic
267f inhibition
in of,
374f, thymidine
303,
375
304f vitamin
By.
Thalassemia(s),
38-39
a,
39,
39f,
42f hemoglobin
H
hydrops fatalis
silent carriers
38-39, disease
and,
376
and, and,
39, of,
38f,
39
42f, major,
39
minor,
Thermogenin,
39
79
Thermus aquaticus,
Thiamine,
482
378-379, active
392-393f form
of,
378,
378f,
392f.
Thiamine clinical
See pyrophosphate
indications as
coenzyme, for,
378, deficiency
of,
379
379f
379,
Dietary
Reference function
393f
Intakes of,
378
378,
378f
Thiamine pyrophosphate
as coenzyme
for a-ketoglutarate
(TPP)
dehydrogenase for,
427
39f
39,
39f also
tyrosine hydroxylase
to,
225,
225,
225f
225f
358f
150
in, acid
complex, 112, 378, 378f, 379f for branched-chain a-ketoacid
dehydrogenase, 266f for pyruvate dehydrogenase complex,

Thiazide diuretics,
299
Thiogalactoside transacetylase, 450
Thiokinases,
176,
189,
190
Thiophorase, 197
Thioredoxin,
297, reduced,
297f regeneration
of,
Thioredoxin reductase,
297,
-35 sequence, 420, 420f
Threonine,
4
catabolism of, 263, 265
degradation of proteins containing, 247
of,
3f,
4
Thrombogenesis n-6 and n-3 polyunsaturated fats and, 363,
364f prostaglandins and, 214
Thromboxane(s),
213-214
n-6 and n-3 polyunsaturated fats and, 363,
364
213-214,
215f
TXAa,
213f,
214,
215f production by activated platelets, 214

304
drug inhibiting,
Thymidine monophosphate
of,
374f
(TMP),
303-304,
304f,
Thymidylate synthase,
Thymine,
291,
303
291f,
305f,
396f damage
to and
repair of,
412
pairing with adenine, 397, 397f, 398f
Thymine dimers,
412
Tightly coupled reactions, electron transport and
oxidative phosphorylation,
78
Tissues, use
of dietary
lipids by,
178
Titration, of
6f,
7-9,
8f
Tocopherol, a-, 391, 392f
Tolerable Upper Intake Level (UL), 358, 359f
Tophaceous gout, 299, 301f
Tophi,
299
Trans-acting elements,
449-450,
450f in eukaryotic transcription, 455
Transaldolase,
147
Transamination, of amino acids, 245, 250-252,

branched-chain, 266
equilibrium of reactions, 251
Transcortin,
237
Transcription, 395, 417, 417f concept
map,
429f eukaryotic,
422-424
combinatorial control of, 455, 455f enhancers in, 424, 424f
promoters in,
422-423, regulation
of,
423f
449f,
454-461

421,
422f elongation in, 420-421, 420f
in,
421,
421f initiation
of,
of, termination
420f
449f,
450-454
of,
421, regulation
of,
449-450
Transcription factors,
422-423,
421f
423f,
Transcription unit, 419
Trans fatty acids, 363-364, 363f
Transfection, 467
synthesis
374
297,
297f
297f
449
Transferases, 53
Transferrin receptor (TfR), synthesis of,
458-459,
458f
Transfer RNA (tRNA), 418, 418f as
adaptor molecules,
435
anticodon of,
435
antiparallel binding with codon, 437, 437f attachment of amino acids to
for,
435, site
for,
434f,
435
by,
435
initiator,
438,
440f posttranscriptional modification of, 425,
425f in
435
ribosomal binding sites for, 436
425
unusual bases
Transformation, of,
467
Transformylase,
439
Transgenic animals,
Transition state,
486
in enzyme-catalyzed
55f,
56
stabilization of, active site and, 56
visualization of,
56,
56f
Transketolase,
Translation, of
146f,
147, mRNA,
379f
431-443,
431f,
460f components required for, 434-437
438-439,
440f steps in, 438-442, 440-441f
Translocation,
439
Transmissible spongiform encephalopathies
(TSEs),
22
Transposons (Tn), 408, 461
Trehalase, 87
Trehalose, 87
Treponema pallidum, 202
Tretinoin,
385,
385f
Triacylglycerol (TAG), 173, 173f in

321
in adipocytes,
350
in chylomicrons, 228-231, 229f, 232f degradation
of,
92f,
175,
181f,
199f in
325
331,
331f hormone regulation of, 190, 190f
311
by lipoprotein lipase, 228-229
type
1, in
type
339,
339f
2,
344,
345f dietary,
361-364
coronary heart disease and, 360-361,
361f elevated
levels of,
344,
345f emulsification
of,
175,
228-229,
224
329,
331,
331f fates of, in liver vs. adipose tissue, 189
fatty acid
component of,
181-183,
188-189
release of,
189-190,
190f insulin
and,
189,
190f metabolism of, 173-178, 180f
niacin and, 380
in obesity, 353
in plasma lipoproteins, 227, 227f processing in stomach, 173-174, 174f
resynthesis of, 176-177
secretion from enterocytes, 177, 177f storage of, 188-189
stored, mobilization of, 189-195
188,
188f synthesis of, 92f, 181f, 188-189, 188f,
339f,
340,
417f,
418,
425
435f in
460,
189f,
199f in
transfer absorptive/fed
state, from
VLDL
324, to
use by
tissues,
178
Tricarboxylic acid
(TCA)
cycle,

as aerobic
pathway,
109
ATP production in, 109, 113, 113f, 114f concept
map,
115f energy produced by, 113-114, 113f
function of, 107, 115f intermediates of, amino acid catabolism
yielding rate limiting steps of, 112
reactions of,
109-113
regulation of, 114, 114f, 115f by activation and inhibition of enzyme
activities,
114,
114f by availability of ADP, 114
Trimethoprim, 293
tetrahydrofolate synthesis inhibition by,
374f
Trimming,
443,
443f
Trinucleotide repeat
expansion,
Triose phosphate
1,4,5-Triphosphate
433,
101
(IP3),
205-206
Triple helix,
43f,
45,
46f
Tropocollagen,
47f,
48
Tropomyosin (TM), 427, 457
Troponin
|
and
T,
66
Trypanosomes,
206
Trypsin,
16f,
443
50
in lipid
digestion,
175
pH
optimum for,
58f in protein digestion, 248, 249f
Trypsinogen,
443
Tryptophan a-helix
by,
17
of,
266
in serotonin
287, side
chains of,
2f
Tryptophan hydroxylase, 271
Tryptophan operon,
452-453,
Tryptophan pyrrolase,
277
TSEs.
See
Transmissible spongiform

TTP, synthesis
of,
460-461
Tuberculosis, isoniazid for, 377f, 378
Tunicamycin,
167
Turnover number
(Kcat),
54
Two-fold rotational symmetry, 466, 466f
Type
1
hyperlipoproteinemia,
229
Type la glycogen storage disease, 121
Type
|
DNA
topoisomerases,
401,
401f
Type
Il
DNA
401,
401f
Type
II
hyperlipidemia,
232
Tyrosinase-negative oculocutaneous albinism,

Tyrosinase (tyrosine hydroxylase)
in catecholamine synthesis, 285
deficiency of, 271
Tyrosine,
4,
4f catabolism
of,
263,
263f disorder
of,
263
in catecholamine synthesis, 285, 286f in
melanin synthesis,
288
metabolic defect in albinism, 273
phenylketonuria and,
272
side chains of, 3f, 4
synthesis of, 268, 270f
Tyrosine kinase,
312
Tyrosinemia type I, 268f, 269f
HDL,
325,
231,
325f
231f
92f,
93,
287f
453f
433f
U
Ubiquinone. See Coenzyme Q
Ubiquitin,
247,
444
Ubiquitination,
444
Ubiquitin-proteasome proteolytic pathway,
246-247, 247f
UCP1
(thermogenin),
79
UDP-galactose,
166
in biosynthetic
as reactions,
141
for glycolysis/gluconeogenesis,
140-141
140,
141f structure
of,
141f
UDP-galactose:glucose galactosyltransferase,
142
UDP-glucose,
166
synthesis
127f of,
126
UDP-glucose pyrophosphorylase,
UDP-glucuronic
126
acid,
161,
162f
UDP-hexose
4-epimerase,
140,
UDP-N-acetylgalactosamine,
141
166
UDP-N-acetylglucosamine,
166
Ultraviolet light, repair of damage caused by,

Uncharged polar
Uncoupling proteins
(UCP),
79
Upper body obesity, 350, 350f
Upstream,
420
Uracil,
291,
291f,
292f,
305f in codons/genetic code, 431, 432f
Urate oxidase,
298

ammonia disposal in, 253, 257, 257f fate
of,
255
Urea cycle,
92f, ammonia
245f, disposal
253-256
in,
253,
256-258
in,
257-258, overall
stoichiometry reactions
258f of,
of,
253-255, regulation
Urease,
255, of,
255-256
254f
255f,
256
257

conversion of dietary nucleic acids to, 298,
299f as
end product
of
298-299, purine
300f formation
of,
298-299
in gout,
299-301,
301f in Lesch-Nyhan syndrome, 296-297, 296f,

galactose attached
to,
140-141, glucose
attached to,
Uridine
126, monophosphate
Uridine monophosphate
deoxy
141f
127f synthase,
(UMP)
form of
(AUMP),
302-303, in
pyrimidine synthesis,

126, synthesis
of,
303,
303f,
Uridyltransferase,
141
Urobilinogen,
283f,
284
284
Urobilins, formation in intestine, 283-284
Urocanic acid,
262,
263f
Urolithiasis, 298
Uronic acid
pathway,
161,
Uronosyl 5-epimerase, 161
Uroporphyrin, 277
302-303
304f
302,
303f
127f
304f degradation,
3f,
4
162f
Uroporphyrin |, 278, 278f
Uroporphyrin
Il,
278,
278f
Uroporphyrinogen,
279
Uroporphyrinogen decarboxylase, deficiency

Uroporphyrinogen III, 279f
Uroporphyrinogen
III
synthase,
II
281f synthase,
279
UTP
diphosphatase
(dUTPase),
303
uvrABC excinuclease, 412
UV-specific endonuclease, 412
Vv

state,
324
by,
17
266-267,
266f glutamate replaced by, in sickle cell

peptide bond with alanine, 13, 14f side
chains of,
2f
Valine therapy, for maple syrup urine disease,

Valyalanine, 14, 14f
Valyglycylleucine, 14
Vanadium, Dietary Reference Intakes for, 358f van den Bergh reaction, 285
Vanillylmandelic acid (VMA), 286
Variable number of tandem repeats (VNTR),

Variegate porphyria,
Vascular disease,
280,
281f homocysteine
265,
265f endothelial
growth factor
459
Vectors,
467-468
cloning, 468
expression, 470, 470f prokaryotic plasmids, 467, 467f
Velocity, of
enzymatic reactions
competitive inhibition and, 60-61, 60f enzyme concentration and, 59
factors affecting, 56-58
hyperbolic kinetics
curve initial,
57,
59
maximal
(Vmax),
57,
57f noncompetitive inhibition and, 61-62, 61f
PH
and,
57-58,
58f substrate
concentration and,

temperature and, 57, 57f
Versican,
158
Very-long-chain fatty
Very-low-density acids
(VLCFA), lipoproteins
57,
57f,
187,
(VLDLs),
195
189,
227,
227f,
228f,
231,
231f,
232f in absorptive/fed state, 323f, 324
circulating, modification of, 231, 231f composition
of,
232f conversion to low-density lipoprotein, in
plasma,
231
1,
339-340,
330f type
2,
344
electrophoretic mobility of, 227, 228f metabolism
of,
230f,
231
380
353
230f,
231
size and density of, 227, 227f
Vibrio cholerae,
94
Vidarabine,
409
Vision, vitamin A and, 382, 383f, 384
Vitamin A, 373, 381-386, 392-393f of,
57, and,
263,
(VEGF),
Vascular
57f absorption of, 382, 383f
for,
384-385, deficiency
of,
384-385, dermatologic
385f
385f,
393f use
of,
385, dietary
intake of
cancer risk and, 391
excessive, 375, 385-386
requirement for,
358f,
384
383f, distribution
of, epithelial
cells functions
384
384
and,
384
of,
382-384, growth
and,
383f,
384
mechanism of
action,
382,
383f,
384f, prevention of chronic disease and,
384-385,
385f reproduction and, 384
383f structure
of,
382, supplementation
382f of,
toxicity of,
384,
385-386, transport
of, vision
and,
Vitamin
B;.
Vitamin
393f
382,
383f
382,
See

Bo.
Vitamin
Bg,
383f,
384
Thiamine
See
378, active
form
Riboflavin
392-393f of,
377f, clinical
378, indications
cysteine
392f for,
synthesis deficiency
of,
378,
378
and,
265
intake of
cardiovascular risk and, 265
of,
392
for homocystinuria,
in homocystinuria,
273
273
377f,
378
supplementation of, 391
378
Vitamin
B12
for homocystinuria, 273
Vitamin
By2,
265,
375-377
absorption of,
active forms
clinical
376f,
377
of,
375, indications
as
376f, for,
392f
376-377
coenzyme,
375,
375f,
376f in succinyl CoA synthesis, 194-195,

375,
377,
393f anemia
and,
374,
377
folate trap hypothesis of, 376

cardiovascular risk
Dietary
Reference sources
of,
376
distribution of,
376
function of, 392f in
273
megaloblastic anemia and, 375
375,
376f supplementation
of,
391
376
Vitamin B12 (deoxyadenosylcobalamin), 194
Vitamin
C,
377-378,
392-393f active form of (ascorbic acid), 377, 377f,

148-149,
377-378,
391
as coenzyme,
377
collagen biosynthesis and, 47, 47f, 377
377,
377f,
intake of
cancer risk and, 378, 391
Dietary Reference Intakes for, 358f and,
265,
Intakes
265f for,
358f
385f,
386
392f
391
385f

prevention of chronic disease and,
377-378
structure of, 377, 377f supplementation
of,
391

bone action
of,
388
386,
388,
388f as
precursor
Clinical indications
219
for,
388-389
of,
388-389,
389f,
intake of
Dietary Reference Intakes for, 358f excessive,
373,
389
386
distribution of, 386, 388
endogenous precursor
of, function
of,
387f, hydroxylation
386
388,
392f of,
intestinal action
149
of,
388
386-388,
387f regulation of, 386-388
389,
393f in transcriptional regulation, 456

active form
of,
392f as
antioxidant,
148-149, clinical
indications deficiency
377, for,
391
of,
391,
intake of
Dietary Reference Intakes for, 358f requirement
for,
391
391
distribution of, 391
391,
392f structure
of,
391, supplementation
391f of,
391
Figure
2.12:
Modified
Grisham,
C.
from
Garrett,
M.
College
Biochemistry.
Publishing,
R.
H.
and
Saunders
1995.
Figure
6.36, p.
193.
Figure
2.13
(panel
C, top):
Abdulla,
S.
Basic mechanisms of protein folding disease.
Nature
Publishing
Group.
Figure owned
3.1A:
Illustration:
by
Not to
Howard be
Irving
Hughes used
without
Figure
3.20:
Geis.
Medical
Rights
Institute.
permission
Photo curtesy
Incorporated,
Hartland, of
Photodyne
WI.

Figure 4.3: Electron micrograph of collagen: Natural Toxin Research Center. Texas A&M University-Kingsville. Collagen molecule modified from Mathews, C. K., van Holde, K. E. and Ahern, K. G. Biochemistry. 3rd Ed., Addison Wesley Longman, Inc. 2000. Figure 6.13, p.175.
Figure 4.4: modified from Yurchenco, P. D., Birk, D. E., and Mecham, R. P., eds. (1994) Extracellular Matrix Assembly and Structure, Academic Press, San Diego, California
Figure 4.8: Kronauer and Buhler, Images in Clinical Medicine, The New England Journal of Medicine, June 15, 1995 , Vol. 332, No. 24, p.1611.
Figure 4.10: Photo from Web site Derma.de
Figure 4.11: Jorde, L. B., Carey, J. C.,
391

Vitamin
K,
373,
389-391, blood
coagulation
Clinical and,
of,
390, in
newborn,
392-393f
389,
390f for,
390
393f
390
dietary intake of requirement
for, sources
of,
390
390
in formation of y-carboxyglutamate, 389,
389f function
of,
389-390,
392f toxicity
of,
391,
393f
Vitamin K-dependent carboxylation, 444
Vitamin(s),
373-394,
392-393f classification
of,
373, coenzymes
and, fat-soluble,
373,
373f
54
373f,
392-393f supplements
of,
391
water-soluble, 373, 373f, 392-393f
VLDL.
See
von
Gierke disease,
130f,
301
Ww
Waist to hip ratio in obesity, 349
Waist size in obesity, 349
Warfarin,
389
Water-soluble vitamins, 373, 373f, 392-393f
Weight. See Body weight, Obesity
Wernicke-Korsakoff syndrome,
110, 379, 393f
Western blot,
473,
484,
485f
Western diet, 362f
White blood cells as DNA source, 476
phagocytosis by, 150, 150f
Wilson disease,
48

Bamshad, M. J. and White, R. L. Medical Genetics, 2nd Ed.
http://medgen.genetics.utah.edu/index.htm
Question
4.3:
from
Berge
LN,
Marton
V,
Tranebjaerg
L,
Kearney
P.
Prenatal
MS, diagnosis
imperfecta.
Kiserud
T, of
osteogenesis
Acta
Obstetricia et
Gynecologica
Scandinavica.
74(4):321-3,
1995
Apr.
Figure 13.11: The Crookston Collection,
University of
Toronto.
Figure
17.13:
Urbana
Illinois
Atlas
College urbana-champaign.
of
Pathology, of
Medicine
Image
Figure
Companion
17.19:
at number
26.
Interactive
Disease.
Figure
18.12:
Study
Case
Robbins
_
Pathologic
Basis of
Custom Medical Stock Photo.
Figure
20.20:
Success in
MRCO
phth.
http:/www.mrcophth.com/iriscases/albinism.ht
Figure
20.22
(top):
Rubin,
E.
and
Farber,
J.
L.
Pathology
194.Figure
(2nd edition).
6-30,
Figure
20.22
and p.
J.
B.
Lippinoctt.
244.
(bottom):
Barness
Gilbert-Barness,
L.
Metabolic
2000.
Figure
E.
Disease.
Eaton
Figure
15,
21.6:
Rich, tarda,
p.42.
M.
W.
Postgraduate
(1999).
Porphyria
Medicine, cutanea
105:
208-214
to
Oian
358f,
390
Wobble hypothesis, 437, 437f
Wolman disease, 232
xX
Xanthine, 299
Xanthine oxidase, 299, 300f, 301

Xenobiotics,
149
Xeroderma pigmentosum, 412, 412f
Xerophthalmia,
384-385,
393f
X-linked adrenoleukodystrophy,
195
X-linked sideroblastic anemia, 278
X-linked severe combined immunodeficiency
(SCID-X),
485
Xylosyltransferase, 162
D-Xylulose 5-phosphate, 161
Y
Yeast artificial chromosomes (YACs), 468
Z
Z-DNA, 398, 399f, 460
Zellweger syndrome, 195
Zero-order reactions, 59, 59f
Zidovudine (AZT), 409, 409f
Zinc, Dietary Reference Intakes for, 358f
Zinc finger motif, 18, 423, 450, 450f
Zwitterion,
7,
7f
Zymogens,
64,
443
activation of, 248
gastric,
248
pancreatic,
248,
249f in protein digestion, 248
Figure
21.5:
Department of
Dermatlogy,
Pittsburgh.
http://www.upmc.edu/dermatology/MedStuden tInfo/introLecture/enlarged/vespct.htm.
Figure
21.10:
Custom
Medical
Stock
Photo,
Inc.
Figure
21.13:
Phototake.
Figure 22.16: Wuthrich, D. A., and Lebowitz.
Tophaceous gout.
Images in
clinical
Medicine.
N
Engl
J
Med,
332:646,
1995.
Figure
22.17:
WebMD
Inc.
http://www.samed.com/sam/forms/index.htm
Figure 22.18 : Corbis.
Figure
23.2:
Childs,
G.
http://www.cytochemistry.net/.
Figure 24.4: Phototake.
Figure
26.6:
Corbis.
Figure 27.20: Joseph E. Armstrong
Illinois
State
University.
Figure 28.4: Matthews, J. H. Queen's
University
Medicine,

Division
Hematology/Oncology,
Figure
29.7:
Nolan,
J.,
Biochemistry,
Tulane
Kingston,
Canada.
Department
University, of
New
Orleans,
LA.
Figure 23.13: Modified from Cryer, P. E.,
Fisher,
J.
N.
and
Shamoon,
H.
Hypoglycemia.
Diabetes Care 17:734-753, 1994.
of

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Professor and Chairman Department of Anesthesiology Virginia Commonwealth University School of Medicine VCU Health System Richmond, Virginia

Professor
Department of Anesthesiology and Perioperative Medicine
University of Texas MD Anderson Cancer Center
Houston, Texas

Steven L. Berk Endowed Chair for Excellence in Medicine
Professor and Chair
Department of Anesthesia
Texas Tech University Health Sciences Center
School of Medicine
Lubbock, Texas
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Chapter Authors
Contributors
Research and Review
Foreword
Preface
1 The Practice of Anesthesiology
SECTION
Anesthetic Equipment & Monitors
- 2 The Operating Room Environment

Charles E. Cowles, Jr., MD, MBA, FASA
Breathing Systems W
The Anesthesia Workstation BR
Cardiovascular Monitoring wo
an Noncardiovascular Monitoring
SECTION

10 Analgesic Agents
11 Neuromuscular Blocking Agents
12 Cholinesterase Inhibitors & Other Pharmacological Antagonists to Neuromuscular Blocking Agents
13 Anticholinergic Drugs
14 Adrenergic Agonists & Antagonists
15 Hypotensive Agents
16 Local Anesthetics
17 Adjuncts to Anesthesia
Pharmacological Principles
Inhalation Anesthetics
Intravenous Anesthetics
SECTION
Anesthetic Management
18, 1 = Preoperative Assessment, Premedication, &Perioperative Documentation. 19, 1 = Airway Management. 20, 1 = Cardiovascular Physiology &Anesthesia. 21, 1 = Anesthesia for Patients with Cardiovascular Disease. 22, 1 = Anesthesia for Cardiovascular Surgery. 23, 1 = Respiratory Physiology &Anesthesia. 24, 1 = Anesthesia for Patients with Respiratory Disease. 25, 1 = Anesthesia for Thoracic Surgery. 26, 1 = Neurophysiology &Anesthesia. 27, 1 = Anesthesia for Neurosurgery. 28, 1 = Anesthesia for Patients with Neurological &Psychiatric Diseases. 29, 1 = Anesthesia for Patients with Neuromuscular Disease. 30, 1 = Kidney Physiology &Anesthesia. 31, 1 = Anesthesia for Patients with Kidney Disease. 32, 1 = Anesthesia for Genitourinary Surgery
33 Hepatic Physiology & Anesthesia
Michael Ramsay, MD, FRCA
34 Anesthesia for Patients with Liver Disease
Michael Ramsay,
MD, FRCA
35 Anesthesia for Patients with Endocrine Disease
36 Anesthesia for Ophthalmic Surgery
37 Anesthesia for Otolaryngology—Head & Neck Surgery
38 Edward
Anesthesia for Orthopedic Surgery
R. Mariano, MD, MAS
39 Anesthesia for Trauma & Emergency Surgery Brian P. McGlinch, MD
40 Maternal & Fetal Physiology & Anesthesia
Michael A. Froélich, MD, MS
41 Obstetric Anesthesia
42 Pediatric Anesthesia
43 Geriatric Anesthesia
44 Ambulatory & Non-Operating Room Anesthesia
SECTION Ea
: : : Regional Anesthesia & Pain
PN
45 Spinal, Epidural, & Caudal Blocks

46 Peripheral Nerve Blocks
Sarah J. Madison, MD and Brian M. Ilfeld, MD, MS (Clinical Investigation)
47 Chronic Pain Management
Bruce M. Vrooman, MD, MS, FIPP and Richard W. Rosenquist, MD
48 Enhanced Recovery Protocols & Optimization of Perioperative Outcomes
Gabriele Baldini, MD, MSc and Timothy Miller, MB ChB FRCA
SECTION
Perioperative & Critical Care Medicine
49 Management of Patients with Fluid & Electrolyte Disturbances
50 Acid-Base Management
51 Fluid Management & Blood Component Therapy
52 Thermoregulation, Hypothermia, & Malignant Hyperthermia
53 Nutrition in Perioperative & Critical Care
54 Anesthetic Complications
55 Cardiopulmonary Resuscitation
N. Martin Giesecke, MD and George W. Williams, MD, FASA, FCCP
56. Postanesthesia Care
57 Common Clinical Concerns in Critical Care Medicine
58 Inhalation Therapy & Mechanical Ventilation in the PACU & ICU
59 Safety, Quality, & Performance Improvement
Index

Associate Professor
Medical Director, Montreal General Hospital Preoperative Centre
McGill University Health Centre
Montreal General Hospital
Montreal, Quebec, Canada

Professor and Chairman
Department of Anesthesiology
Virginia Commonwealth University School of Medicine
VCU Health System
Richmond, Virginia

Associate Professor/Assistant Clinical Director

Professor and Associate Vice Chair for Research
University of Alabama at Birmingham
Birmingham, Alabama

Professor and Vice Chairman for Administrative Affairs
McGovern Medical School
University of Texas Health Science Center at Houston

Professor of Anesthesiology, In Residence
Division of Regional Anesthesia and Pain Medicine
University of California at San Diego
San Diego, California

Department of Anesthesiology, Perioperative & Pain Medicine
Stanford University
Stanford, California

Professor
Stanford University School of Medicine
Chief, Anesthesiology & Perioperative Care Service
Associate Chief of Staff, Inpatient Surgical Services
Veterans Affairs Palo Alto Health Care System
Palo Alto, California

University of Minnesota
Minneapolis, Minnesota
Colonel, Medical Corps, United States Army Reserve
Command Surgeon
84th Training Command
Fort Knox, Kentucky

Chief, Division of General, Vascular and Transplant Anesthesia
Duke University School of Medicine
Durham, North Carolina

Chairman, Department of Anesthesiology
Baylor University Medical Center
Baylor Scott and White Health Care System
Professor
Texas A&M University Health Care Faculty
Dallas, Texas

Chairman, Department of Pain Management
Cleveland Clinic
Cleveland, Ohio

Chief, Section of Pain Medicine
Dartmouth-Hitchcock Medical Center
Associate Professor of Anesthesiology
Geisel School of Medicine at Dartmouth
Lebanon, New Hampshire

Vice Chair for Critical Care Medicine
Associate Professor of Anesthesiology and Neurosurgery
Program Director, Critical Care Medicine Fellowship
University of Texas Health Science Center at Houston—McGovern Medical
School

Professor
West Virginia University School of Medicine
Morgantown, West Virginia

Professor

VCU School of Medicine

St. Luke's Roosevelt Hospital Center and Columbia University College of
Physicians and Surgeons
New York, New York

Duke University Medical Center

UT Southwestern Medical Center

Formerly Resident, Department of Anesthesiology

Texas Tech University Medical Center

St. Luke's Roosevelt Hospital Center

Texas Tech University Medical Center Hospital

When a new residency training program in anesthesia was beginning in Rwanda in 2006, we were looking for a suitable textbook to recommend to the trainees. We chose Clinical Anesthesiology by Morgan and Mikhail. I am happy to state that today, 12 years later, the residents are still making the same choice. Over one third of all copies of the last edition were sold outside of North America thus underlining the popularity of this textbook around the world.
A major change in editors and authors occurred with the 5th edition and it is clear that they stayed true to the ideals of the original editors. Now in 2018, the 6th edition is presented to us. The text continues to be simple, concise, and easily readable. The use of Key Concepts at the beginning of each chapter is very useful and focuses the reader's attention on the important points. The authors have worked hard not to increase the size of the book but to update the material. Expanded chapters on critical care, on enhanced recovery after anesthesia, and on the use of ultrasound will be very useful to readers. This textbook continues to provide a comprehensive introduction to the art and science of anesthesia.
Congratulations to the authors and editors on their fine work.
Angela Enright MB, FRCPC
Past President, World Federation of Societies of Anaesthesiologists (WFSA)
- ¢ We have tried to provide illustrations and images whenever they improve the flow and understanding of the text.
Once again, the goal expressed in the first edition remains unchanged: "to provide a concise, consistent presentation of the basic principles essential to the modern practice of anesthesia." And, once again, despite our best intentions, we fear that errors will be found in our text. We are grateful to the many readers who helped improve the last edition. Please email us at mm6edition@gmail.com when you find errors. This enables us to make corrections in reprints and future editions.
John F. Butterworth, IV, MD
David C. MD
Mackey, John Wasnick,
D. MD, MPH
CHAPTER
1

@ Oliver Wendell Holmes in 1846 was the first to propose use of the term anesthesia to denote the state that incorporates amnesia, analgesia, and narcosis to make painless surgery possible.
@ Ether was used for frivolous purposes ("ether frolics") and was not used as an anesthetic agent in humans until 1842, when Crawford W. Long and William E. Clark independently used it on patients. On October 16, 1846, William T.G. Morton conducted the first publicized demonstration of general anesthesia for surgical operation using ether.
@ The original application of modern local anesthesia is credited to Carl Koller, at the time a house officer in ophthalmology, who demonstrated topical anesthesia of the eye with cocaine in 1884.
@ Curare greatly facilitated tracheal intubation and muscle relaxation during surgery. For the first time, operations could be performed on patients without the requirement that relatively deep levels of inhaled general anesthetic be used to produce muscle relaxation.
@ John Snow, often considered the father of the anesthesia specialty, was the first to scientifically investigate ether and the physiology of general anesthesia.
@ The "captain of the ship" doctrine, which held the surgeon responsible for every aspect of the patient's perioperative care (including anesthesia), is no longer a valid notion when an anesthesiologist is present.
The Greek philosopher Dioscorides first used the term anesthesia in the first century AD to describe the narcotic-like effects of the plant mandragora. The term subsequently was defined in Bailey's An Universal Etymological English
TABLE 1-1 Aspects of the practice of medicine that are included within the scope of anesthesiology.!
Assessment of, consultation for, and preparation of patients for anesthesia.
Relief and prevention of pain during and following surgical, obstetric, therapeutic, and diagnostic procedures.
Monitoring and maintenance of normal physiology during the perioperative or periprocedural period.
Management of critically ill patients.
Diagnosis and treatment of acute, chronic, and cancerrelated pain.
Management of hospice and palliative care.
Clinical management and teaching of cardiac, pulmonary, and neurological resuscitation.
Evaluation of respiratory function and application of respiratory therapy.
Conduct of clinical, translational, and basic science research.

Supervision, teaching, and evaluation of performance of both medical and allied health personnel involved in perioperative or periprocedural care, hospice and palliative care, critical care, and pain management.
Administrative involvement in health care facilities and organizations, and medical schools, as appropriate to the American Board of Anesthesiology's mission.
'Data from the American Board of Anesthesiology Primary Certification Policy Book (Booklet of Information), 2017.
This chapter reviews the history of anesthesia, emphasizing its British and American roots, and considers the current scope of the specialty.

The specialty of anesthesia began in the mid-nineteenth century and became firmly established in the following century. Ancient civilizations had used opium poppy, coca leaves, mandrake root, alcohol, and even phlebotomy (to the point of unconsciousness) to allow surgeons to operate. Ancient Egyptians used the combination of opium poppy (containing morphine) and hyoscyamus (containing scopolamine) for this purpose. A similar combination, morphine and scopolamine, was widely used for premedication until recent times. What passed for regional anesthesia in ancient times consisted of compression of nerve trunks (nerve ischemia) or the application of cold (cryoanalgesia). The Incas may have practiced local anesthesia as their surgeons chewed coca leaves and applied them to operative wounds, particularly prior to trephining for headache.
The evolution of modern surgery was hampered not only by a poor understanding of disease processes, anatomy, and surgical asepsis but also by the lack of reliable and safe anesthetic techniques. These techniques evolved first with inhalation anesthesia, followed by local and regional anesthesia, intravenous anesthesia, and neuromuscular blockers. The development of surgical anesthesia is considered one of the most important discoveries in human history, and it was introduced to practice without a supporting randomized clinical trial.

Because the hypodermic needle was not invented until 1855, the first general anesthetics were destined to be inhalation agents. Diethyl ether (known at the time as "sulfuric ether" because it was produced by a simple chemical reaction between ethyl alcohol and sulfuric acid) was originally prepared in @ 1540 by Valerius Cordus. Ether was used for frivolous purposes ("ether frolics"), but not as an anesthetic agent in humans until 1842, when Crawford W. Long and William E. Clark independently used it on patients for surgery and dental extraction, respectively. However, neither Long nor Clark publicized his discovery. Four years later, in Boston, on October 16, 1846, William T.G. Morton conducted the first publicized demonstration of general anesthesia for surgical operation using ether. The dramatic success of that exhibition led the operating surgeon to exclaim to a skeptical audience: "Gentlemen, this is no humbug!"
Chloroform was independently prepared by Moldenhawer, von Liebig, Guthrie, and Soubeiran around 1831. Although first used by Holmes Coote in 1847, chloroform was introduced into clinical practice by the Scot Sir James Simpson, who administered it to his patients to relieve the pain of labor. Ironically, Simpson had almost abandoned his medical practice after witnessing the terrible despair and agony of patients undergoing operations without anesthesia.
Joseph Priestley produced nitrous oxide in 1772, and Humphry Davy first noted its analgesic properties in 1800. Gardner Colton and Horace Wells are

The medicinal qualities of coca had been recognized by the Incas for centuries before its actions were first observed by Europeans. Cocaine was isolated from coca leaves in 1855 by Gaedicke and was purified in 1860 by Albert Niemann. Sigmund Freud performed ®@ seminal work with cocaine. Nevertheless, the original application of cocaine for anesthesia is credited to Carl Koller, at the time a house officer in ophthalmology, who demonstrated topical anesthesia of the eye in 1884. Later in 1884 William Halsted used cocaine for intradermal infiltration and nerve blocks (including blocks of the facial nerve, brachial plexus, pudendal nerve, and posterior tibial nerve). August Bier is credited with administering the first spinal anesthetic in 1898. He was also the first to describe intravenous regional anesthesia (Bier block) in 1908. Procaine was synthesized in 1904 by Alfred Einhorn and within a year was used clinically as a local anesthetic by Heinrich Braun. Braun was also the first to add epinephrine to prolong the duration of local anesthetics. Ferdinand Cathelin and Jean Sicard introduced caudal epidural anesthesia in 1901. Lumbar epidural anesthesia was described first in 1921 by Fidel Pages and again (independently) in 1931 by Achille Dogliotti. Additional local anesthetics subsequently introduced include dibucaine (1930), tetracaine (1932), lidocaine (1947), chloroprocaine (1955), mepivacaine (1957), prilocaine (1960), bupivacaine (1963), and etidocaine (1972). The most recent additions, ropivacaine (1996) and levobupivacaine (1999), have durations of action similar to bupivacaine but less cardiac toxicity (see Chapter 16). Another, chemically dissimilar local anesthetic, articaine, has been widely applied for dental anesthesia.

Intravenous anesthesia required the invention of the hypodermic syringe and needle by Alexander Wood in 1855. Early attempts at intravenous anesthesia included the use of chloral hydrate (by Oré in 1872), chloroform and ether (Burkhardt in 1909), and the combination of morphine and scopolamine (Bredenfeld in 1916). Barbiturates were first synthesized in 1903 by Fischer and von Mering. The first barbiturate used for induction of anesthesia was diethylbarbituric acid (barbital), but it was not until the introduction of hexobarbital in 1927 that barbiturate induction became popular. Thiopental, synthesized in 1932 by Volwiler and Tabern, was first used clinically by John Lundy and Ralph Waters in 1934 and for many years it remained the most common agent for intravenous induction of anesthesia. Methohexital was first used clinically in 1957 by V.K. Stoelting. Methohexital continues to be very popular for brief general anesthetics for electroconvulsive therapy. After chlordiazepoxide was discovered in 1955 and released for clinical use in 1960, other benzodiazepines—diazepam, lorazepam, and midazolam—came to be used extensively for premedication, conscious sedation, and induction of general anesthesia. Ketamine was synthesized in 1962 by Stevens and first used clinically in 1965 by Corssen and Domino; it was released in 1970 and continues to be popular today, particular when administered in combination with other agents for general anesthesia or when infused in low doses to awake patients for painful conditions. Etomidate was synthesized in 1964 and released in 1972. Initial enthusiasm over its relative lack of circulatory and respiratory effects was tempered by evidence of adrenal suppression, reported after even a single dose. The release of propofol in 1986 (1989 in the United States) was a major advance in outpatient anesthesia because of its short duration of action (see Chapter 9). Propofol is currently the most popular agent for intravenous induction worldwide.

The introduction of curare by Harold Griffith and Enid Johnson in 1942 was a milestone in anesthesia. @ Curare greatly facilitated tracheal intubation and muscle relaxation during surgery. For the first time, operations could be performed on patients without the requirement for relatively deep planes of inhaled general anesthetic to produce muscle relaxation. Such deep planes of general anesthesia often resulted in excessive cardiovascular and respiratory depression as well as prolonged emergence. Moreover, deep planes of inhalation anesthesia often were not tolerated by frail patients. Succinylcholine was synthesized by Bovet in 1949 and released in 1951; it remains a standard agent for facilitating tracheal intubation during rapid sequence induction. Until recently, succinylcholine remained unchallenged in its rapid onset of profound muscle relaxation, but its side effects prompted the search for a comparable substitute. Other neuromuscular blockers (NMBs; discussed in Chapter 11)—gallamine, decamethonium, metocurine, alcuronium, and pancuronium—were subsequently introduced. Unfortunately, these agents were often associated with side effects (see Chapter 11), and the search for the ideal NMB continued. Recently introduced agents that more closely resemble an ideal NMB include vecuronium, atracurium, rocuronium, mivacurium, and cisatracurium.

Morphine, first isolated from opium in between 1803 and 1805 by Sertiirner, was also tried as an intravenous anesthetic. The adverse events associated with opioids in early reports caused many anesthetists to favor pure inhalation anesthesia. Interest in opioids in anesthesia returned following the synthesis and introduction of meperidine in 1939. The concept of balanced anesthesia was introduced in 1926 by Lundy and others and evolved to include thiopental for induction, nitrous oxide for amnesia, an opioid for analgesia, and curare for muscle relaxation. In 1969, Lowenstein rekindled interest in "pure" opioid anesthesia by reintroducing the concept of large doses of opioids as complete anesthetics. Morphine was the first agent so employed, but fentanyl and sufentanil have been preferred by a large margin as sole agents. As experience grew with this technique, its multiple limitations—unreliably preventing patient awareness, incompletely suppressing autonomic responses during surgery, and prolonged respiratory depression—were realized. Remifentanil, an opioid subject to rapid degradation by nonspecific plasma and tissue esterases, permits profound levels of opioid analgesia to be employed without concerns regarding the need for postoperative ventilation, albeit with an increased risk of acute opioid tolerance.

Following its first public demonstration in the United States, ether anesthesia quickly was adopted @ in England. John Snow, often considered the father of the anesthesia specialty, was the first physician to take a full-time interest in this new anesthetic. He was the first to scientifically investigate ether and the physiology of general anesthesia. Of course, Snow was also a pioneer in epidemiology who helped stop a cholera epidemic in London by proving that the causative agent was transmitted by ingestion of contaminated well water rather than by inhalation. In 1847, Snow published the first book on general anesthesia, On the Inhalation of Ether. When the anesthetic properties of chloroform were made known, he quickly investigated and developed an inhaler for that agent as well. He believed that an inhaler should be used in administering ether or chloroform to control the dose of the anesthetic. His second book, On Chloroform and Other Anaesthetics, was published posthumously in 1858. After Snow's death, Dr. Joseph T. Clover took his place as England's leading anesthetist. Clover emphasized continuously monitoring the patient's pulse during anesthesia, a practice that was not yet standard at the time. He was the first to use the jaw-thrust maneuver for relieving airway obstruction, the first to insist that resuscitation equipment always be available during anesthesia, and the first to use a cricothyroid cannula (to save a patient with an oral tumor who developed complete airway obstruction). After Clover, Sir Frederic Hewitt became England's foremost anesthetist in the 1890s. He was responsible for many inventions, including the oral airway. Hewitt also wrote what many consider to be the first true textbook of anesthesia, which went through five editions. Snow, Clover, and Hewitt established the tradition of physician anesthetists in England, but it was Hewitt who made the most sustained and strongest arguments for educating specialists in anesthesia. In 1893, the first organization of physician specialists in anesthesia, the London Society of Anaesthetists, was formed in England by J.F. Silk.
The first elective tracheal intubations during anesthesia were performed in the late nineteenth century by surgeons Sir William MacEwen in Scotland, Joseph O' Dwyer in the United States, and Franz Kuhn in Germany. Tracheal intubation during anesthesia was popularized in England by Sir Ivan Magill and Stanley Rowbotham in the 1920s.

In the United States, only a few physicians had specialized in anesthesia by 1900. The task of providing general anesthesia was often delegated to junior surgical house officers, medical students, or general practitioners.
The first organization of physician anesthetists in the United States was the Long Island Society of Anesthetists, formed in 1905, which, as it grew, was renamed the New York Society of Anesthetists in 1911. The group now known as the International Anesthesia Research Society (IARS) was founded in 1922, and in that same year the I[ARS-sponsored scientific journal Current Researches in Anesthesia and Analgesia (now called Anesthesia and Analgesia) began publication. In 1936, the New York Society of Anesthetists became the American Society of Anesthetists, and later, in 1945, the American Society of Anesthesiologists (ASA). The scientific journal Anesthesiology was first published in 1940.
Harold Griffith and others founded the Canadian Anesthetists Society in 1943, and Griffith (now better known for introducing curare) served as its first president. Twelve years later the journal now known as the Canadian Journal of Anesthesia was first published. Five physicians stand out in the early development of anesthesia in the United States after 1900: James Tayloe

In 1889 Henry Isaiah Dorr, a dentist, was appointed Professor of the Practice of Dentistry, Anaesthetics and Anaesthesia at the Philadelphia College of Dentistry. Thus he was the first known professor of anesthesia worldwide. Thomas D. Buchanan, of the New York Medical College, was the first physician to be appointed Professor of Anesthesia (in 1905). When the American Board of Anesthesiology was established in 1938, Dr. Buchanan served as its first president. Certification of specialists in anesthesia was first available in Canada in 1946. In England, the first examination for the Diploma in Anaesthetics took place in 1935, and the first Chair in Anaesthetics was awarded to Sir Robert Macintosh in 1937 at Oxford University. Anesthesia became an officially recognized specialty in England only in 1947, when the Royal College of Surgeons established its Faculty of Anaesthetists. In 1992 an independent Royal College of Anaesthetists was granted its charter. Momentous changes occurred in Germany during the 1950s, progress likely having been delayed by the isolation of German medical specialists from their colleagues in other countries that began with World War I and continued until the resolution of World War II. First the journal Der Anaesthetist began publication in 1952. The following year, requirements for specialist training in anesthesia were approved and the German Society of Anesthetists was founded.

The practice of anesthesia has changed dramatically since the days of John Snow. The modern anesthesiologist must be both a perioperative consultant and a deliverer of care to patients. In general, anesthesiologists are responsible for nearly all "noncutting" aspects of the patient's medical care in the immediate @ perioperative period. The "captain of the ship" doctrine, which held the surgeon responsible for every aspect of the patient's perioperative care (including anesthesia), is no longer a valid notion when an anesthesiologist is present. The surgeon and anesthesiologist must function together as an effective team, and both are ultimately answerable to the patient rather than to each other.
The modern practice of anesthesia is not confined to rendering patients insensible to pain (Table 1-1). Anesthesiologists monitor, sedate, and provide general or regional anesthesia outside the operating room for various imaging procedures, endoscopy, electroconvulsive therapy, and cardiac catheterization. Anesthesiologists such as Peter Safar have been pioneers in cardiopulmonary resuscitation, and anesthesiologists continue to be integral members of resuscitation teams.
An increasing number of practitioners pursue subspecialty fellowships in anesthesia for cardiothoracic surgery (see Chapter 22), critical care (see Chapter 57), neuroanesthesia (see Chapter 27), obstetric anesthesia (see Chapter 41), pediatric anesthesia (See Chapter 42), palliative care, regional anesthesia, and acute pain management (see Chapters 45, 46, 48) or chronic pain medicine (see Chapter 47). Certification requirements for special competence in critical care, pediatric anesthesia, and pain medicine already exist in the United States. Fellowship programs in Adult Cardiothoracic Anesthesia, Critical Care
Medicine, Pediatric Anesthesiology, Obstetric Anesthesiology, Regional Anesthesia and Acute Pain Management, Sleep Medicine, Palliative Care, and Interventional Pain have specific accreditation requirements. Education and certification in anesthesiology can also be used as the basis for certification in Sleep Medicine or in Palliative Medicine.

Anesthesiologists are actively involved in the administration and medical direction of many ambulatory surgery facilities, operating room suites, intensive care units, and respiratory therapy departments. They have also assumed administrative and leadership positions on the medical staffs of many hospitals and ambulatory care facilities. They serve as deans of medical schools and chief executives of health systems. In the United States they have served in state legislatures, in the U.S. Congress, and as the Surgeon General. The future of the specialty has never looked brighter.

A pressure of 1000 psig indicates an E-cylinder that is approximately half full and represents 330 L of oxygen.
The only reliable way to determine residual volume of nitrous oxide is to weigh the cylinder.
To discourage incorrect cylinder attachments, cylinder manufacturers have adopted a pin index safety system.
A basic principle of radiation safety is to keep exposure "as low as reasonably practical" (ALARP). The principles of ALARP optimize protection from radiation exposure by the use of time, distance, and shielding.
The magnitude of a leakage current is normally imperceptible to touch (<1 mA, and well below the fibrillation threshold of 100 mA). If the current bypasses the high resistance offered by skin, however, and is applied directly to the heart (microshock), current as low as 100 pA may be fatal. The maximum leakage allowed in operating room equipment is 10 pA.
To reduce the chance of two coexisting faults, a line isolation monitor measures the potential for current flow from the isolated power supply to the ground. Basically, the line isolation monitor determines the degree of isolation between the two power wires and the ground and predicts the amount of current that could flow if a second short circuit were to develop.
Almost all surgical fires can be prevented. Unlike medical complications, fires are a product of simple physical and chemistry properties. Occurrence is guaranteed given the proper combination of
factors, but can be almost entirely eliminated through understanding the basic principles of fire risk.
@ The most common risk factor for surgical fire relates to the open delivery of oxygen.
© Administration of oxygen in concentrations of greater than 30% should be guided by the clinical presentation of the patient and not solely by protocols or habits.
@ The sequence of stopping gas flow and removal of the endotracheal tube when fire occurs in the airway is not as important as ensuring that both actions are performed immediately.
@ Before beginning laser surgery, the laser device should be in the operating room, warning signs should be posted on the doors, and protective eyewear should be issued. The anesthesia provider should ensure that the warning signs and eyewear match the labeling on the device, as laser protection is specific to the type of laser.

Anesthesiologists, who spend more time in operating rooms than any other physician specialty, are responsible for protecting patients and operating room personnel from a multitude of dangers during surgery. Some of these threats are unique to the operating room. As a result, the anesthesiologist may be responsible for ensuring proper functioning of the operating room's medical gases, fire prevention and management, environmental factors (eg, temperature, humidity, ventilation, and noise), and electrical safety. Anesthesiologists often coordinate, or assist with, layout and design of surgical and procedural suites, including workflow enhancements. This chapter describes the major operating room features that are of special interest to anesthesiologists and the potential hazards associated with these systems.

Patients often think of the operating room as a safe place where the care given is centered around protecting the patient. Anesthesia providers, surgeons, nurses, and other medical personnel are responsible for carrying out critical tasks safely and efficiently. Unless members of the operating room team remain vigilant, errors can occur that may result in harm to the patient or to members of the operating room team. The best way of preventing serious harm to the patient or to the operating room team is by creating a culture of safety, which identifies and stops unsafe acts before harm occurs.

The medical gases commonly used in operating rooms are oxygen, nitrous oxide, air, and nitrogen. Although technically not a gas, vacuum exhaust for disposal or scavenging of waste anesthetic gas and surgical suction must also be provided, and these are considered integral parts of the medical gas system. Patients are endangered if medical gas systems, particularly oxygen, are misconfigured or malfunction. The anesthesiologist must understand the sources of the gases and the means of their delivery to the operating room to prevent or detect medical gas depletion or supply line misconnection. Estimates of a particular hospital's peak demand determine the type of medical gas supply system required. Design and standards follow National Fire Protection Association (NFPA) 99 in the
United States and HTM 2022 in the United Kingdom.

A reliable supply of oxygen is a critical requirement in any surgical area. Medical grade oxygen (99% or 99.5% pure) is manufactured by fractional distillation of liquefied air. Oxygen is stored as a compressed gas at room temperature or refrigerated as a liquid. Most small hospitals store oxygen in two separate banks of high-pressure cylinders (H-cylinders) connected by a manifold (Figure 2-1). Only one bank is utilized at a time. The number of cylinders in each bank depends on anticipated daily demand. The manifold contains valves that reduce the cylinder pressure (approximately 2000 pounds per square inch [psig]) to line pressure (55 + 5 psig) and automatically switch banks when one group of cylinders is exhausted.
FIGURE 2-1 A bank of oxygen H-cylinders connected by a manifold.
A liquid oxygen storage system (Figure 2—2) is more economical for large hospitals. Liquid oxygen must be stored well below its critical temperature of — 119°C because gases can be liquefied by pressure only if stored below their critical temperature. A large hospital may have a smaller liquid oxygen supply or a bank of compressed gas cylinders that can provide one day's oxygen requirements as a reserve. To guard against a hospital gas-system failure, the anesthesiologist must always have an emergency (E-cylinder) supply of oxygen available during anesthesia.
FIGURE 2-2 liquid storage tank with reserve oxygen tanks in the
A background.

Most anesthesia machines accommodate E-cylinders of oxygen (Table 2-1). As oxygen is expended, the cylinder's pressure falls in proportion @ to its content. A pressure of 1000 psig indicates an E-cylinder that is approximately half full and represents 330 L of oxygen at atmospheric pressure and a temperature of 20°C. If the oxygen is exhausted at a rate of 3 L/min, a cylinder that is half full will be empty in 110 min. Oxygen cylinder pressure should be assessed prior to use and periodically during use. Anesthesia machines usually also accommodate E-cylinders for medical air and nitrous oxide, and may accept cylinders of helium. Compressed medical gases utilize a pin index safety system for these cylinders to prevent inadvertent crossover and connections for different gas types. As a safety feature, oxygen E-cylinders have a "plug" made from Wood's metal. This metallurgic alloy has a low melting point, allowing dissipation of pressure in a fire that might otherwise heat the cylinder to the point of explosion. This pressure-relief "valve" is designed to rupture at 3300 psig, well below the pressure E-cylinder walls should be able to withstand (more than 5000 psig), preventing "overfilling" of the cylinder.
TABLE 2-1 Characteristics of medical gas cylinders.

Oo,, E-Cylinder Capacity’ (L) = 625-700. Oo,, H-Cylinder Capacity’ (L) = 6000-8000. Oo,, Pressure’ (psig at 20°C) = 1800-2200. Oo,, Color (USA) = Green. Oo,, Color (International) = White. Oo,, Form = Gas. Air, E-Cylinder Capacity’ (L) = 625-700. Air, H-Cylinder Capacity’ (L) = 6000-8000. Air, Pressure’ (psig at 20°C) = 1800-2200. Air, Color (USA) = Yellow. Air, Color (International) = White and black. Air, Form = Gas. NO, E-Cylinder Capacity’ (L) = 1590. NO, H-Cylinder Capacity’ (L) = 15,900. NO, Pressure’ (psig at 20°C) = 745. NO, Color (USA) = Blue. NO, Color (International) = Blue. NO, Form = Liquid. N,, E-Cylinder Capacity’ (L) = 625-700. N,, H-Cylinder Capacity’ (L) = 6000-8000. N,, Pressure’ (psig at 20°C) = 1800-2200. N,, Color (USA) = Black. N,, Color (International) = Black. N,, Form = Gas
"Depending on the manufacturer.

Nitrous oxide is almost always stored by hospitals in large H-cylinders connected by a manifold with an automatic crossover feature. Bulk liquid storage of nitrous oxide is economical only in very large institutions.
Because the critical temperature of nitrous oxide (36.5°C) is above room temperature, it can be kept liquefied without an elaborate refrigeration system. If the liquefied nitrous oxide rises above its critical temperature, it will revert to its gaseous phase. Because nitrous oxide is not an ideal gas and is easily compressible, this transformation into a gaseous phase is not accompanied by a great rise in tank pressure. Nonetheless, as with oxygen cylinders, all nitrous oxide E-cylinders are equipped with a Wood's metal plug to prevent explosion under conditions of unexpectedly high gas pressure (eg, unintentional overfilling or during a fire).
Although a disruption in supply is usually not catastrophic, most anesthesia machines have reserve nitrous oxide E-cylinders. Because these smaller cylinders also contain nitrous oxide in its liquid state, the volume remaining in a cylinder is not proportional to cylinder pressure. By the time the liquid nitrous oxide is expended and the tank pressure begins to fall, only about 400 L of nitrous oxide remains. If liquid nitrous oxide is kept at a constant temperature (20°C), it will vaporize at the same rate at which it is consumed and will maintain a constant pressure (745 psig) until the liquid is exhausted.
®@ The only reliable way to determine residual volume of nitrous oxide is to weigh the cylinder. For this reason, the tare weight (TW), or empty weight, of cylinders containing a liquefied compressed gas (eg, nitrous oxide) is often stamped on the shoulder of the cylinder. The pressure gauge of a nitrous oxide cylinder should not exceed 745 psig at 20°C. A higher reading implies gauge malfunction, tank overfill (liquid fill), or a cylinder containing a gas other than nitrous oxide.
Because energy is consumed in the conversion of a liquid to a gas (the latent heat of vaporization), liquid nitrous oxide cools during this process. The drop in temperature results in a lower vapor pressure and lower cylinder pressure. The cooling is so pronounced at high flow rates that there is often frost on the tank, and the pressure regulator may freeze in such circumstances.

The use of air is becoming more frequent in anesthesiology as the popularity of nitrous oxide and unnecessarily high concentrations of oxygen has declined. Cylinder air is medical grade and is obtained by blending oxygen and nitrogen. Dehumidified (but unsterile) air is provided to the hospital pipeline system by compression pumps. The inlets of these pumps must be distant from vacuum exhaust vents and machinery to minimize contamination. Because the critical temperature of air is —140.6°C, it exists as a gas in cylinders whose pressures fall in proportion to their content.

Although compressed nitrogen is not administered to patients, it may be used to drive operating room equipment, such as saws, drills, and surgical handpieces. Nitrogen supply systems incorporate either the use of H-cylinders connected by a manifold or a wall system supplied by a compressor-driven central supply.

A central hospital vacuum system usually consists of independent suction pumps, each capable of handling peak requirements. Traps at every user location prevent contamination of the system with foreign matter. The medical-surgical vacuum system may be used for waste anesthetic gas disposal (WAGD) as long as it does not affect performance of the system. Medical vacuum receptacles are usually black in color with white lettering. A dedicated WAGD vacuum system is required with modern anesthesia machines. The WAGD outlet may incorporate the use of a suction regulator with a float indicator that should be maintained between the designated markings. Excess suction may result in inadequate patient ventilation, and insufficient suction levels may result in the failure to evacuate waste anesthetic gases. WAGD receptacles and tubing are usually lavender in color.

Many surgical procedures are performed using laparoscopic or robotic-assisted techniques requiring insufflation of body cavities with carbon dioxide, an odorless, colorless, nonflammable and slightly acidic gas. Large cylinders containing carbon dioxide, such as M-cylinders or LK-cylinders, are frequently found in the operating room; these cylinders share a common size orifice and thread with oxygen cylinders and can be inadvertently interchanged.

Medical gases are delivered from their central supply source to the operating room through a network of pipes that are sized such that the pressure drop across the whole system never exceeds 5 psig. Gas pipes are usually constructed of seamless copper tubing using a special welding technique. Internal contamination of the pipelines with dust, grease, or water must be avoided. The hospital's gas delivery system appears in the operating room as hose drops, gas columns, or elaborate articulating arms (Figure 2—3). Operating room equipment, including the anesthesia machine, connects to pipeline system outlets by color-coded hoses. Quick-coupler mechanisms, which vary in design with different manufacturers, connect one end of the hose to the appropriate gas outlet. The other end connects to the anesthesia machine through a noninterchangeable diameter index safety system fitting that prevents incorrect hose attachment.
FIGURE 2-3 Typical examples of A: gas columns, B: ceiling hose drops, and C: articulating arms. One end of a color-coded hose connects to the hospital medical gas supply system by way of a quick-coupler mechanism. The other end connects to the anesthesia machine through the diameter index safety system.
E-cylinders of oxygen, nitrous oxide, and air ® attach directly to the anesthesia machine. To discourage incorrect cylinder attachments, cylinder manufacturers have adopted a pin index safety system. Each gas cylinder (sizes A-E) has two holes in its cylinder valve that mate with corresponding pins in the yoke of the anesthesia machine (Figure 2—4). The relative positioning of the pins and holes is unique for each gas. Multiple washers placed between the cylinder and yoke, which prevent proper engagement of the pins and holes, have unintentionally defeated this system and thus must not be used. The pin index safety system is also ineffective if yoke pins are damaged or if the cylinder is filled with the incorrect gas.
FIGURE 2-4 Pin index safety system interlink between the anesthesia machine and gas cylinder.
The functioning of medical gas supply sources and pipeline systems is constantly monitored by central and area alarm systems. Indicator lights and audible signals warn of changeover to secondary gas sources and abnormally high (eg, pressure regulator malfunction) or low (eg, supply depletion) pipeline

Modern anesthesia machines and anesthetic gas analyzers continuously measure the fraction of inspired oxygen (Fio,). Analyzers have a variable threshold setting for the minimal Fio, but must be configured to prevent disabling this alarm. The monitoring of Fio, does not reflect the oxygen concentration distal to the monitoring port and thus should not be used to reference the oxygen concentration within distal devices such as endotracheal tubes. Due to gas exchange, flow rates, and shunting, a marked difference may exist between the indicated Fio, and the actual oxygen concentration at the tissue level.

The temperature in most operating rooms seems uncomfortably cold to many conscious patients and, at times, to anesthesia providers. However, scrub nurses and surgeons stand in surgical garb for hours under hot operating room lights. As a general principle, the comfort of operating room personnel must be reconciled with patient care, and in adult patients temperatures should be maintained between 68°F (20°C) and 75°F (24°C). Hypothermia has been associated with wound infection, impaired coagulation, greater intraoperative blood loss, and prolonged hospitalization (see Chapter 52).

In past decades, static discharges were a feared source of ignition in an operating room filled with flammable anesthetic gas vapors. Now humidity control is more relevant to infection control practices. Humidity levels in the operating room should be maintained between 20% and 60%. Below this range the dry air facilitates airborne mobility of particulate matter, which can be a vector for infection. At high humidity, dampness can affect the integrity of barrier devices such as sterile cloth drapes and pan liners.

A high rate of operating room airflow decreases contamination of the surgical site. These flow rates, usually achieved by blending up to 80% recirculated air with fresh air, are engineered in a manner to decrease turbulent flow and to be unidirectional. Although recirculation conserves energy costs associated with heating and air conditioning, it is unsuitable for WAGD. Therefore, a separate waste anesthetic gas scavenging system must always supplement operating room ventilation. The operating room should maintain a slightly positive pressure to drive away gases that escape scavenging and should be designed so fresh air is introduced through, or near, the ceiling and air return is handled at, or near, floor level. Ventilation considerations must address both air quality and volume changes. The National Fire Protection Agency (NFPA) recommends 20 air volume exchanges per hour to decrease risk of stagnation and bacterial growth. Air quality should be maintained by adequate air filtration using a 90% filter, defined simply as one that filters out 90% of particles presented. Although highefficiency particulate filters (HEPA) are frequently used, these are not required by engineering or infection control standards.

Multiple studies have demonstrated that exposure to noise can have a detrimental effect on human cognitive function, and prolonged exposure may result in hearing impairment. Operating room noise has been measured at 70 to 80 decibels (dB) with frequent sound peaks exceeding 80 dB. As a reference, if the speaking voice has to be raised above conversational level, then ambient noise is approximated at 80 dB. Noise levels in the operating room approach the time-weighted average (TWA) for which the Occupational Safety and Health Administration (OSHA) requires hearing protection. Orthopedic air chisels and neurosurgical drills can approach the noise levels of 125 dB, the level at which most human subjects begin to experience pain.

As use of reliable shielding has increased, the incidence of radiationassociated diseases of sensitive organs has decreased, with the exception of radiation-induced cataracts. Because protective eyewear has not been consistently used to the same degree as other types of personal protection, radiation-induced cataracts are increasing among employees working in interventional radiology suites. Anesthesia providers who work in these environments should consider the use of leaded goggles or glasses to decrease the risk of such problems.

The use of electronic medical equipment subjects patients and health care personnel to the risk of shock and electrocution. Anesthesia providers must have an understanding of electrical hazards and their prevention.
Body contact with two conductive materials at different voltage potentials may complete a circuit and result in an electrical shock. Usually, one point of exposure is a live 110-V or 240-V conductor, with the circuit completed through a ground contact. For example, a grounded person need contact only one live conductor to complete a circuit and receive a shock. The live conductor could be the frame of a patient monitor that has developed a fault to the hot side of the power line. A circuit is now complete between the power line (which is earth grounded at the utility company's pole-top transformer) through the victim and back to the ground (Figure 2-6). The physiological effect of electrical current depends on the location, duration, frequency, and magnitude (more accurately, current density) of the shock.
FIGURE 2-6 The setting for the great majority of electric shocks. An accidentally grounded person simultaneously contacts the hot wire of the electric service, usually via defective equipment that provides a pathway linking the hot wire to an exposed conductive surface. The complete electrical loop originates with the secondary of the pole transformer (the voltage source) and extends through the hot wire, the victim and the victim's contact with a ground, the earth itself, the neutral ground rod at the service entrance, and back to the transformer via the neutral (or ground) wire. (Modified with permission from Bruner J, Leonard PF. Electricity, Safety, and the Patient. St Louis, MO: Mosby Year Book; 1989.)

Leakage current is present in all electrical equipment as a result of capacitive coupling, induction between internal electrical components, or defective insulation. Current can flow as a result of capacitive coupling between two conductive bodies (eg, a circuit board and its casing) even though they are not physically connected. Some monitors are doubly insulated to decrease the effect of capacitive coupling. Other monitors are designed to be connected to a low-impedance ground (the safety ground wire) that should divert the current away from a person touching the instrument's case. The magnitude of @ such leaks is normally imperceptible to touch (<1 mA, and well below the fibrillation threshold of 100 mA). If the current bypasses the high resistance offered by skin, however, and is applied directly to the heart, current as low as 100 pA (microshock) may be fatal. The maximum leakage allowed in operating room equipment is 10 pA.
Cardiac pacing wires and invasive monitoring catheters provide a direct conductive pathway to the myocardium. In fact, blood and normal saline can serve as electrical conductors. The exact amount of current required to produce fibrillation depends on the timing of the shock relative to the vulnerable period of heart repolarization (the T wave on the electrocardiogram). Even small differences in potential between the ground connections of two electrical outlets in the same operating room can place a patient at risk for microelectrocution.

Most patient electrocutions are caused by current flow from the live conductor of a grounded circuit through the body and back to a ground (Figure 2-6). This would be prevented if everything in the operating room were grounded except the patient. Although direct patient grounds should be avoided, complete patient isolation is not feasible during surgery. Instead, the operating room power supply can be isolated from grounds by an isolation transformer (Figure 2—7).

Unlike the utility company's pole-top transformer, the secondary wiring of an isolation transformer is not grounded and provides two live ungrounded voltage lines for operating room equipment. Equipment casing—but not the electrical circuits—is grounded through the longest blade of a three-pronged plug (the safety ground). If a live wire is then unintentionally contacted by a grounded patient, current will not flow through the patient since no circuit back to the secondary coil has been completed (Figure 2-8).
FIGURE 2-8 Even though a person is grounded, no shock results from contact with one wire of an isolated circuit. The individual is in simultaneous contact with two separate voltage sources but does not close a loop including either source. (Modified with permission from Bruner J, Leonard PF. Electricity, Safety, and the Patient. St Louis, MO: Mosby Year Book; 1989.)

Of course, if both power lines are contacted, a circuit is completed and a shock is possible. In addition, if either power line comes into contact with a ground through a fault, contact with the other power line will complete a circuit through a grounded patient. To reduce the chance of two coexisting faults, a line isolation monitor measures the potential for current flow from the isolated power supply to the ground (Figure 2—9). Basically, the line isolation monitor determines the degree of isolation between the two power wires and the ground and predicts the amount of current that could flow if a second short circuit were to develop. An alarm is activated if an unacceptably high current flow to the ground becomes possible (usually 2 mA or 5 mA), but power is not interrupted unless a ground-fault circuit interrupter is also activated. The latter, a common feature in household bathrooms and kitchens, is usually not installed in locations such as operating rooms, where discontinuation of life support systems (eg, cardiopulmonary bypass machine) is more hazardous than @ the risk of electrical shock. The alarm of the line isolation monitor merely indicates that the power supply has partially reverted to a grounded system. In other words, while the line isolation monitor warns of the existence of a single fault (between a power line and a ground), two faults are required for a shock to occur. Since the line isolation monitor alarms when the sum of leakage current exceeds the set threshold, the last piece of equipment added is usually the defective one; however, if this item is life-sustaining, other equipment can be removed from the circuit to evaluate whether the life safety item is truly at fault.

Even isolated power circuits do not provide complete protection from the small currents capable of causing microshock fibrillation. Furthermore, the line isolation monitor cannot detect all faults, such as a broken safety ground wire within a piece of equipment. There are, however, modern equipment designs that decrease the possibility of microelectrocution. These include double insulation of the chassis and casing, ungrounded battery power supplies, and patient isolation from equipment-connected grounds by using optical coupling or transformers.
In the latest edition of the U.S. NPFA 99 Health Care Facilities Code, building system requirements for facilities—including electrical systems—are based upon a risk assessment carried out by facilities personnel with the input of health care providers. The risk levels are categorized in levels as follows:
Category
1—Facility systems
in which
failure of
such equipment
or system
is likely
Category cause
to cause
major
2—Facility injury
systems minor
injury
3—Facility or
death in
which to
patients systems
injury to
of patients
failure or
of or
such caregivers
in which
patients
Category
4—Facility or
systems failure
of caregivers,
but can
in which
failure of
caregivers equipment
cause is
likely to
is not
likely patient
discomfort such
equipment would
have to
no impact
on patient
care
Category 1 locations and systems will have the greatest amount of reliability and redundancy; lesser categories will have less stringent requirements. All codes under the 2012 edition of the NPFA 99 will be determined by the risk assessment category. Under the electrical code, operating rooms are defined as a wet location requiring electrical systems that reduce the risk of electrical shock hazards. If an operating room is used for procedures without liquid exposure, such as rooms used for central line placement or eye procedures, facilities can perform a risk assessment and reclassify the operating room as a nonwet area.

Electrosurgical units (ESUs) generate an ultrahigh-frequency electrical current that passes from a small active electrode (the cautery tip) through the patient and exits by way of a large plate electrode (the dispersal pad, or return electrode). The high current density at the cautery tip is capable of tissue coagulation or cutting, depending on the electrical waveform. Ventricular fibrillation is prevented by the use of ultrahigh electrical frequencies (0.1—3 MHz) compared with line power (50-60 Hz). The large surface area of the low-impedance return electrode avoids burns at the current's point of exit by providing a low current density (the concept of exit is technically incorrect, as the current is alternating rather than direct). The high power levels of ESUs (up to 400 W) can cause inductive coupling with monitor cables, leading to electrical interference. Malfunction of the dispersal pad may result from disconnection from the ESU, inadequate patient contact, or insufficient conductive gel. In these situations, the current will find another place to exit (eg, electrocardiogram pads or metal parts of the operating table), which may result in a burn (Figure 2—10). Precautions to prevent diathermy burns include proper return electrode placement, avoiding prostheses and bony protuberances, and elimination of patient-to-ground contacts. Current flow through the heart may lead to malfunction of an implanted cardiac pacemaker or cardioverter defibrillator. This risk can be minimized by placing the return electrode as close to the surgical field and as far from the implanted cardiac device as practical.

FIGURE 2-10 Electrosurgical burn. If the intended path is compromised, the circuit may be completed through other routes. Because the current is of high frequency, recognized conductors are not essential; capacitances can complete gaps in the circuit. Current passing through the patient to a contact of small area may produce a burn. (A leg drape would not offer protection in the situation depicted.) The isolated output electrosurgical unit (ESU) is much less likely than the ground-referenced ESU to provoke burns at ectopic sites. Ground-referenced in this context applies to the ESU output and has nothing to do with isolated versus grounded power systems. (Modified with permission from Bruner J, Leonard PF. Electricity, Safety, and the Patient. St Louis, MO: Mosby Year Book; 1989.)
Newer ESUs are isolated from grounds using the same principles as the isolated power supply (isolated output versus ground-referenced units). Because this second layer of protection provides ESUs with their own isolated power supply, the operating room's line isolation monitor may not detect an electrical fault. Although some ESUs are capable of detecting poor contact between the return electrode and the patient by monitoring impedance, many older units trigger the alarm only if the return electrode is unplugged from the machine.
Bipolar electrodes confine current propagation to a few millimeters, eliminating the need for a return electrode. Because pacemaker and electrocardiogram interference is possible, pulse or heart sounds should be closely monitored when any ESU is used. Automatic implanted cardioverter defibrillator devices may need to be suspended if monopolar ESU is used, and any implanted cardiac device should be interrogated after use of a monopolar ESU.

Surgical fires are relatively rare, with an incidence of about 1:87,000 cases, which is close to the incidence rate of other events such as retained foreign objects @ after surgery and wrong-site surgery. Almost all surgical fires can be prevented (Figure 2—11). Unlike medical complications, fires are a product of simple physical and chemical properties. Occurrence is guaranteed given the proper combination of factors, but can be almost entirely eliminated by understanding the basic principles of fire risk. @ The most common risk factor for surgical fire relates to the open delivery of oxygen.

chee
Proceed, but frequently reassess for changes TAM S.e
Use room air sedation.
Use delivery device such as a blender or common gas outlet to maintain oxygen below 30%
Secure airway with endotracheal tube or supraglottic device.
FIGURE 2-11 Operating Room Fire Prevention Algorithm. (©Anesthesia Patient Safety Foundation. Used with permission.)
Situations classified as carrying a high risk for a surgical fire are those that involve an ignition source in close proximity to an oxidizer. The simple chemical combination required for any fire is commonly referred to as the fire triad or fire triangle. The triad is composed of fuel, oxidizer, and ignition source (heat). Table 2—2 lists potential contributors to fires and explosions in the operating room. Surgical fires can be managed and possibly avoided completely by incorporating education, fire drills, preparation, prevention, and response into educational programs provided to operating room personnel.

Flammable agents (fuels)
Solutions, aerosols, and ointments
Alcohol
Chlorhexidine
Benzoin
Mastisol
Acetone
Petroleum products
Surgical drapes (paper and cloth)
Surgical gowns
Surgical sponges and packs
Surgical sutures and mesh
Plastic/polyvinyl chloride/latex products
Endotracheal tubes
Masks
Cannulas
Tubing
Intestinal gases
Hair
Gases supporting combustion (oxidizers)
Oxygen
Nitrous oxide
Ignition sources (heat)
Lasers
Electrosurgical units
Fiberoptic light sources (distal tip)
Drills and burrs
External defibrillators
For anesthesia providers, fire prevention education should place a heavy emphasis on the risk relating to the open delivery of oxygen. The Anesthesia
location of fire extinguishers, emergency exits, and fresh gas cutoff valves should be established by the operating room team.
Fires that result in injuries requiring medical treatment or death must be reported to the fire marshal, who retains jurisdiction over the facility. Providers should gain basic familiarity with local reporting standards, which can vary according to location.
Cases in which supplemental oxygen delivery is used and the surgical site is above the xiphoid constitute the most commonly-reported scenario for surgical fires. Frequently, the face or airway is involved, resulting in life-threatening or severely disfiguring injuries. These fires can almost always be avoided by the elimination of the open delivery of oxygen.

For fires not suppressed by initial attempts, or those in which evacuation may be hindered by the location or intensity of the fire, the use of a portable fire extinguisher is warranted. A CO, extinguisher is safe for fires on the patient in the operating room. CO, readily dissipates, is not toxic, and as used in an actual fire is not likely to result in thermal injury. FE-36, a more expensive DuPont product, also can be used.
"A"-rated extinguishers contain water, which makes their use in the operating room problematic because of the presence of electrical equipment. A water mist "AC"-rated extinguisher is excellent but requires time and an adequate volume of mist over multiple attempts to extinguish the fire. Furthermore, these devices are large and difficult to maneuver. Both can be made cheaply in a nonferromagnetic extinguisher, making them the best choice for fires involving magnetic resonance imaging equipment. Halon extinguishers, although very effective, are being phased out because of concerns about depletion of the ozone layer and because of the hypoxic atmosphere that results for rescuers. Halotrons are halon-type extinguishers that may have fewer effects on the ozone layer.

Lasers are commonly used in operating rooms and procedure areas. When lasers are used in the airway or for procedures involving the neck and face, the case should be considered high risk for surgical fire and managed as previously discussed. The type of laser (CO,, neodymium—yttrium aluminum gamet [Nd:YAG], or potassium titanyl phosphate [KTP]), wavelength, and focal length are all important considerations for the safe operation of medical lasers. Without this vital information, operating room personnel cannot adequately protect themselves or the ® patient from harm. Before beginning laser surgery, the laser device should be in the operating room, warning signs should be posted on the doors, and protective eyewear should be issued. The anesthesia provider should ensure that the warning signs and eyewear match the labeling on the device as protection is specific to the type of laser. The American National Standards Institute (ANSI) standards specify that eyewear and laser devices must be labeled for the wavelength emitted or protection offered. Some ophthalmologic lasers and vascular mapping lasers have such a short focal length that protective eyewear is not needed. For other devices, protective goggles should be worn by personnel at all times during laser use, and eye protection in the form of either goggles or protective eye patches should be used on the patient.
Laser endotracheal tube selection should be based on laser type and wavelength. The product insert and labeling for each type of tube should be compared to the type of laser used. Tubes less than 4.0 mm in diameter are not compatible with the Nd:YAG or argon laser, and Nd: YAG-compatible tubes are not available in half sizes. Attempts to wrap conventional endotracheal tubes with foil should be avoided. This obsolete method is not approved by either manufacturers or the U.S. Food and Drug Administration, is prone to breaking or unraveling, and does not confer complete protection against laser penetration. Alternatively, jet ventilation without an endotracheal tube can offer a reduced risk of airway fire.

Crew resource management (CRM) was developed in the aviation industry to promote teamwork and to allow personnel to intervene or call for investigation of any situation thought to be unsafe. Comprising seven principles, its goal is to avoid errors caused by human actions. In the airline model CRM gives any crew member the authority to question situations that fall outside the range of normal practice. Before the implementation of CRM, crew members other that the captain had little or no input on aircraft operations. After CRM was instituted, anyone identifying a safety issue could take steps to ensure adequate resolution of the situation. The benefit of this method in the operating room is clear, given the potential for a deadly mistake to be made.
but quickly eliminate one. In this scenario, tunnel vision can result in catastrophic mistakes. Furthermore, if the sampling line has become dislodged and the capnograph indicates low end-tidal CO,, this finding does not exclude the possibility that at the same time a pulmonary embolus may occur, resulting in decreased end-tidal CO).
If all members of the operating room team apply these seven principles, problems arising from human factors can almost entirely be eliminated. A culture of safety must also exist if the operating room is to be made a safer place. These seven principles serve no purpose when applied in a suppressive operating room environment. Anyone with a concern must be able speak up without fear of repercussion. Chapter 59 provides further discussion of these and other issues relating to patient safety.

In the
United
States, the
Centers for
Medicare and
Medicaid
Services
(CMS)
drive many
of the
Efforts to
mandated reduce
requirement policies
fraudulent of
certification and
claims procedures
and disparities
carried in
of an
accrediting agency
out levels
by facilities.
of such
care as
include the
the
Joint
Commission,
Det
Norske
Veritas/Germanischer
Lloyd
(DNV
GL), and
others.
These accreditation agencies examine processes and procedures and also ensure facilities have appropriate policies in place and that these policies are actually followed. The process may involve a self study submitted by a facility and also a site visit carried out by a team of various medical professionals who inspect a facility, observe operations, and compare observations to the policies and self studies.
Accreditors use laws, codes, and standards to determine if a facility is carrying out care in alignment with current best practices. Anesthesia providers should be apprised that guidelines, recommendations, and advisories typically should not be used by accreditors to determine a best practice. Recommendations, advisories, and guidelines carry a lesser degree of evidence than a standard, and are often educated opinions. Many accreditation agencies maintain policies prohibiting the use of anything other than standards or codes to determine accreditation. Facilities should be cautioned that sometimes the site visitors may issue a citation incorrectly based upon these opinions. All accrediting agencies maintain an appeal process for citations, and if seemingly unwarranted citations are issued, facility administrators may wish to consider

Despite heightened awareness of safety factors and increased educational efforts among operating room personnel, harm to patients still occurs at a rate that most industries and the public deem unacceptably high. Similarly, despite threats of payment withholding, public scoring of medical personnel and hospital systems, provider rating web sites, and punitive legal consequences, the human factors resulting in medical errors have not been entirely eliminated. In the future, safety-engineered designs may assist in the reduction of medical errors. One developing area is the use of interlock devices in the operating room. An interlock device is simply a device that cannot be operated until a defined sequence of events occurs. Anesthesia personnel use interlock technology with anesthesia vaporizers that prevent the use of more than one vaporizer at a time. Expansion of this technology might prevent release of a drug from an automated dispensing device until a barcode is scanned from a patient's hospital armband or, at a minimum, the patient's drug allergies have been entered into the machine's database. Other applications might include an electrosurgical device or laser that could not be used when the Fio, content is higher than 30%, thus minimizing the risk of fire. Similarly, computers, monitors, and other devices could be designed to be inoperable until patient identification is confirmed.

Coordinating the activities of surgical personnel, anesthesia providers, and operating room nurses is essential to the day-to-day running of a surgical suite. Clinical directors in facilities ranging from oneor two-room suites to multiroom centers must accommodate surgical procedures of varying durations, requiring varying degrees of surgical skill and efficiency, while allowing for sudden, unplanned, or emergency operations. The need to monitor workflow and analyze data for optimizing scheduling and staffing prompted the development of software systems that anticipate and record the timing of surgical events; these systems are constantly being refined.
Surgical suites are also being designed to improve workflow by incorporating separate induction areas to decrease nonsurgical time spent in operating rooms. Several models exist for induction room design and staffing. Although uncommon in the United States, induction rooms have long been employed in the United Kingdom.
One induction room model uses rotating anesthesia teams. One team is assigned to the first patient of the day; a second team induces anesthesia for the next patient in an adjacent area while the operating room is being turned over. The second team continues caring for that patient after transfer to the operating room, leaving the first team available to induce anesthesia in the third patient as the operating room is being turned over. The advantage of this model is continuity of care; the disadvantage is the need for two anesthesia teams for every operating room.
Another model uses separate induction and anesthesia teams. The induction team induces anesthesia for all patients on a given day and then transfers care to the anesthesia team, which is assigned to an individual operating room. The advantage of this model is the reduction in anesthesia personnel to staff induction rooms; disadvantages include failure to maintain continuity of care and staffing problems that occur when several patients must undergo induction concurrently. This model can utilize either a separate induction room adjacent to each operating room or one common induction room that services several operating rooms.
The final model uses several staffed operating rooms, one of which is kept open. After the first patient of the day is transferred to the initial room, subsequent patients always proceed to the open room, thus eliminating the wait for room turnover and readiness of personnel. All of these models assume that the increased overhead cost of maintaining additional anesthesia personnel can be justified by the increased surgical productivity.

Many hospitals are exploring methods of applying lean methodology to the surgical environment. Lean examination systems seek to find and eliminate waste and duplicate activity. The most notable company to apply lean methodology is Toyota, which has branded a lean system, the Toyota Production System (TPS), that many health care systems incorporate into their perioperative settings. TPS centers on three concepts: muda, muri, and mura. Muda (from the Japanese term for "waste") is created by muri and mura. Muri is waste created by overburden and production pressure, and mura is the waste created by uneven work patterns or lack of load leveling.
TPS also incorporates a set of five processes, referred to as 5S, into improvement efforts. These key processes, which begin with the letter "S" in the original Japanese, have since been translated into synonymous S-words in English.
¢ Sort—Eliminate excess, remove items that are not in use, remove the unneeded or unwanted items.
¢ Set in Order—Arrange items in use for easy selection and in an organized manner. Make the workflow easier and natural.
¢ Shine—The workplace should be clean.
¢ Standardize—Workstations should be alike and variability should be reduced or eliminated. Every process should have a standard.
¢ Sustain—Work should be goal driven; no one should be told to work, but rather all should do the work without asking.
With the elimination of waste and application of the 5S methodology, daily operations should become safer, standardized, and more efficient.

Radio frequency identification (RFID) technology utilizes a chip with a small transmitter whose signal is read by a reader; each chip yields a unique signal. The technology has many potential applications in the perioperative environment. Using RFID in employee identification (ID) badges could enable surgical control rooms to keep track of nursing, surgical faculty, and anesthesia personnel, obviating the need for paging systems and telephony to establish the location of key personnel. Incorporating the technology in patient ID bands and hospital gurneys could allow a patient's flow to be tracked through an entire facility. The ability to project an identifying signal to hospital systems would offer an additional degree of safety for patients unable to communicate with hospital personnel. Finally, RFID could be incorporated into surgical instruments and sponges, allowing surgical counts to be performed by identification of the objects as they are passed on and off the surgical field. In the event that counts are mismatched, a wand could then be placed over the patient to screen for retained objects.

Monitored Anesthesia Care with Oxygen Supplementation
You are asked to provide monitored anesthesia care for a patient undergoing simple removal of a lesion on the cheek. The patient is morbidly obese and has a history of sleep apnea. He states, "It bothers me when people are working on my face," and indicates that he does not want to remember anything about the surgery. The surgeon assures you the procedure will not last more than 5 minutes. The patient's partner mentions that they are from out of town and have made flight arrangements to return home soon after the procedure.

Patients with a clinical history of obstructive sleep apnea usually have a sensitivity to sedating medications, especially opioids. Administration of even small doses of opioids may lead to upper airway obstruction and hypoventilation, resulting in hypercapnia and hypoxemia. In obese patients, upper airway obstruction, hypoventilation, and decreased functional reserve capacity may result in rapid oxygen desaturation. Most anesthesia providers respond by increasing the amount of oxygen supplementation delivered via face mask or nasal cannula. Open delivery of oxygen in concentrations greater than 30% is one of the elements of the fire triad. Another consideration is the anatomical location of the procedure. A location above the xiphoid process in this patient would place an ignition source (if used) in close proximity to the open delivery of an oxidizer.

There are three strategies that can be implemented to improve safety in this scenario: avoid oxygen supplementation, secure the airway with an endotracheal tube or supraglottic device, or avoid use of an ignition source.
Are there any concerns relating to airway management or selection of the delivery device?
As previously noted, the patient is likely to manifest airway changes associated with obstructive sleep apnea and obesity. Selection of a delivery device should take into consideration the need to prevent the open delivery of oxygen.
How would the length of the procedure affect the management of anesthesia?
Practically speaking, if the patient requires a lengthy procedure, local anesthetics may wear off; the cumulative dose of narcotics provided may exacerbate the patient's obstructive sleep apnea and increase recovery time. Additionally, more complex surgical excision may result in bleeding requiring the use of cautery.
Does the patient's expectation of discharge soon after the

The expectation of an accelerated recovery period may not be feasible if the patient requires general anesthesia or significant amounts of opioids. The American Society of Anesthesiologists (ASA) has published a practice advisory providing direction for the safe postoperative assessment and discharge of patients with obstructive sleep apnea. See www.asahq.org.

The first and most effective means for conflict resolution is to communicate your specific concerns to the surgeon. If this fails, the procedure must not be allowed to proceed as long as any team member has a legitimate safety concern. Many ASA safety-related guidelines and advisories are also endorsed by professional societies such as the American College of Surgeons (ACS). The anesthesiologist should also gain familiarity with a facility's methods of dispute resolution before an event occurs.

Dorsch JA, Dorsch SE. Understanding Anesthesia Equipment. 5th ed. Philadelphia, PA: Williams & Wilkins; 2008. A detailed discussion of compressed gases and medical gas delivery systems. Macdonald MR, Wong A, Walker P, Crysdale WS. Electrocautery-induced ignition of tonsillar packing. J Otolaryngol. 1994;23:426. An examination of factors that can decrease the risk of airway fire including lower oxygen concentration (using a cuffed tracheal tube), completely soaked tonsil packs, and avoidance of contact between electrocautery and bismuth subgallate. National Fire Protection Association (NFPA): Standard for Health Care Facilities. Quincy, MA: NFPA; 2002. An updated version of NFPA 99 standards.

The American National Standards Institute is the reference source for laser standards and many other protective engineering standards. http://www.ansi.org
The Anesthesia Patient Safety Foundation provides resources and a newsletter
precautions. http://www.fuseprogram.org/

@ Because insufflation avoids any direct patient contact, there is no rebreathing of exhaled gases if the flow is high enough. Ventilation cannot be controlled with this technique, however, and the inspired gas contains unpredictable amounts of entrained atmospheric air.
@ Long breathing tubes with high compliance increase the difference between the volume of gas delivered to a circuit by a reservoir bag or ventilator and the volume actually delivered to the patient.
© The adjustable pressure-limiting (APL) valve should be fully open during spontaneous ventilation so that circuit pressure remains negligible throughout inspiration and expiration.
@ Because a fresh gas flow equal to minute ventilation is sufficient to prevent rebreathing, the Mapleson A design is the most efficient Mapleson circuit for spontaneous ventilation.
@ The Mapleson D circuit is efficient during controlled ventilation, because fresh gas flow forces alveolar air away from the patient and toward the APL valve.
@ Increasing the hardness of soda lime by adding silica minimizes the risk of inhalation of sodium hydroxide dust and also decreases resistance of gas flow.
@ Malfunction of either unidirectional valve in a circle system may allow rebreathing of carbon dioxide, resulting in hypercapnia.
© With an absorber, the circle system prevents rebreathing of carbon dioxide at fresh gas flows that are considered low (fresh gas flow <1 L) or even fresh gas flows equal to the uptake of anesthetic gases and oxygen by the patient and the circuit itself (closed-system anesthesia).
© Because of the unidirectional valves, apparatus dead space in a circle
system is limited to the area distal to the point of inspiratory and expiratory gas mixing at the Y-piece. Unlike Mapleson circuits, the circle system tube length does not directly affect dead space.
@ The fraction of inspired oxygen (Fio,) delivered by a resuscitator
breathing system to the patient is directly proportional to the oxygen concentration and flow rate of the gas mixture supplied to the resuscitator (usually 100% oxygen) and inversely proportional to the minute ventilation delivered to the patient.

Breathing systems provide the final conduit for the delivery of anesthetic gases to the patient. Breathing circuits link a patient to an anesthesia machine (Figure 3-1). Many different circuit designs have been developed, each with varying degrees of efficiency, convenience, and complexity. This chapter reviews the most important breathing systems: insufflation, draw-over, Mapleson circuits, the circle system, and resuscitation systems.
FIGURE 3-1 The relationship between the patient, the breathing system, and the anesthesia machine.
Most classifications of breathing systems artificially consolidate functional characteristics (eg, the extent of rebreathing) with physical characteristics (eg, the presence of unidirectional valves). Because these seemingly contradictory classifications (eg, open, closed, semiopen, semiclosed) often tend to confuse rather than aid understanding, they are avoided in this discussion.

The term insufflation usually denotes the blowing of anesthetic gases across a patient's face. Although insufflation is categorized as a breathing system, it is perhaps better considered a technique that avoids direct connection between a breathing circuit and a patient's airway. Because children often resist the placement of a face mask (or an intravenous line), insufflation is particularly valuable during inductions with inhalation anesthetics in children (Figure 3-2). It is useful in other situations as well. Carbon dioxide accumulation under head and neck draping is a hazard of ophthalmic surgery performed with local anesthesia. Insufflation of air across the patient's face at a high flow rate (>10 L/min) avoids this problem, while not increasing the risk of fire from accumulation of oxygen (Figure 3-3). Because insufflation @ avoids any direct patient contact, there is no rebreathing of exhaled gases if the flow is high enough. Ventilation cannot be controlled with this technique, however, and the inspired gas contains unpredictable amounts of entrained atmospheric air.
FIGURE 3-2 Insufflation of an anesthetic agent across a child's face during induction.
FIGURE 3-3 Insufflation of oxygen and air under a head drape.
Insufflation can also be used to maintain arterial oxygenation during brief periods of apnea (eg, during bronchoscopy). Instead of blowing gases across the face, oxygen is directed into the lungs through a device placed in the trachea.

Although open-drop anesthesia is not used in modern medicine, its historic significance warrants a brief description here. A highly volatile anesthetic— historically, ether or chloroform—was dripped onto a gauze-covered mask (Schimmelbusch mask) applied to the patient's face. As the patient inhales, air passes through the gauze, vaporizing the liquid agent, and carrying high concentrations of anesthetic to the patient. The vaporization lowers mask temperature, resulting in moisture condensation and a drop in anesthetic vapor pressure (vapor pressure is proportional to temperature).
A modern derivative of open-drop anesthesia utilizes draw-over vaporizers that depend on the patient's inspiratory efforts to draw ambient air through a vaporization chamber. This technique may be used in locations or situations in which compressed medical gases are unavailable (eg, battlefields).

Draw-over devices have nonrebreathing circuits that use ambient air as the carrier gas, although supplemental oxygen can be used, if available. The devices can be fitted with connections and equipment that allow intermittent positivepressure ventilation (IPPV) and passive scavenging, as well as continuous positive airway pressure (CPAP) and positive end-expiratory pressure (PEEP). In its most basic application (Figure 3—4), air is drawn through a lowresistance vaporizer as the patient inspires. Patients spontaneously breathing room air and a potent halogenated agent often manifest an oxygen saturation (Spo,) less than 90%, a situation treated with IPPV, supplemental oxygen, or both. The fraction of inspired oxygen (Fio,) can be supplemented using an openended reservoir tube of about 400 mL, attached to a T-piece at the upstream side of the vaporizer. Across the clinical range of tidal volume and respiratory rate, an oxygen flow rate of 1 L/min gives an Fio, of 30% to 40%, or with 4 L/min, an Fio, of 60% to 80%. There are several commercial draw-over systems available that share common properties (Table 3-1).

Low resistance to gas flow
Usable with any agent!
Controllable vapor output lyalothane cannot be used with the Epstein Mackintosh Oxford device.
FIGURE 3-4 Schematic diagram of a draw-over anesthesia device/circuit.
The greatest advantage of draw-over systems is their simplicity and portability, making them useful in locations where compressed gases or ventilators are not available.

The insufflation and draw-over systems have several disadvantages: poor control of inspired gas concentration (and, therefore, poor control of depth of anesthesia), mechanical drawbacks during head and neck surgery, and pollution of the operating room with large volumes of waste gas. The Mapleson systems solve some of these problems by incorporating additional components (breathing tubes, fresh gas inlets, adjustable pressure-limiting [APL] valves, and reservoir bags) into the breathing circuit. The relative location of these components determines circuit performance and is the basis of the Mapleson classification (Table 3-2).
TABLE 3-2 Classification and characteristics of Mapleson circuits.
FGI, fresh gas inlet: APL. adjustable pressure-limiting (valve).

Corrugated tubes—made of rubber (reusable) or plastic (disposable)—connect the components of the Mapleson circuit to the patient (Figure 3—5). The large diameter of the tubes (22 mm) creates a low-resistance pathway and a potential reservoir for anesthetic gases. To minimize fresh gas flow requirements, the volume of gas within the breathing tubes in most Mapleson circuits should be at least as great as the patient's tidal volume.
FIGURE 3-5 Components of a Mapleson circuit. APL, adjustable pressurelimiting (valve).
The compliance of the breathing tubes largely determines the compliance of the circuit. (Compliance is defined as the change of volume produced by a @ change in pressure.) Long breathing tubes with high compliance increase the difference between the volume of gas delivered to a circuit by a reservoir bag or ventilator and the volume actually delivered to the patient. For example, if a breathing circuit with a compliance of 8 mL gas/cm H,O is pressurized during delivery of a tidal volume to 20 cm H,O, 160 mL of the tidal volume will be lost to the circuit. The 160 mL represent a combination of gas compression and breathing-tube expansion. This is an important consideration in any circuit delivering positive-pressure ventilation through breathing tubes (eg, circle systems).

Gases (anesthetics mixed with oxygen or air) from the anesthesia machine continuously enter the circuit through the fresh gas inlet. As discussed below, the relative position of the fresh gas inlet is a key differentiating factor among Mapleson circuits.

As anesthetic gases enter the breathing circuit, pressure will rise if the gas inflow is greater than the combined uptake of the patient and the circuit. Gases may exit the circuit through an APL valve, controlling this pressure buildup. Exiting gases enter the operating room atmosphere or, preferably, a waste-gas scavenging system. All APL valves allow a @ variable pressure threshold for venting. The APL valve should be fully open during spontaneous ventilation so that circuit pressure remains negligible throughout inspiration and expiration. Assisted and controlled ventilation require positive pressure during inspiration to expand the lungs. Partial closure of the APL valve limits gas exit, permitting positive circuit pressures during reservoir bag compressions.

Reservoir bags function as a reservoir of anesthetic gas and a method of generating positive-pressure ventilation. They are designed to increase in compliance as their volume increases. Three distinct phases of reservoir bag filling are recognizable (Figure 3—6). After the nominal 3-L capacity of an adult reservoir bag is achieved (phase I), pressure rises rapidly to a peak (phase II). Further increases in volume result in a plateau or even a slight decrease in pressure (phase III). This ceiling effect provides some minimal protection of the patient's lungs against high airway pressures, if the APL valve is unintentionally left in the closed position while fresh gas continues to flow into the circuit.
FIGURE 3-6 The increasing compliance and elasticity of breathing bags as demonstrated by three phases of filling (see text). (Reproduced with permission from Johnstone RE, Smith TC. Rebreathing bags as pressure limiting devices. Anesthesiology. 1973 Feb;38(2):192-194)

Mapleson circuits are lightweight, inexpensive, and simple. Breathing-circuit efficiency is measured by the fresh gas flow required to reduce CO, rebreathing to a negligible value. Because there are no unidirectional valves or CO, absorption in Mapleson circuits, rebreathing is prevented by adequate fresh gas flow into the circuit and venting exhaled gas through the APL valve before inspiration. There is usually some rebreathing in any Mapleson circuit. The total fresh gas flow into the circuit controls the amount. To attenuate rebreathing, high fresh gas flows are required. The APL valve in Mapleson A, B, and C circuits is located near the face mask, and the reservoir bag is located at the opposite end of the circuit.
Reexamine the drawing of a Mapleson A circuit in Figure 3-5. During spontaneous ventilation, alveolar gas containing CO, will be exhaled into the breathing tube or directly vented through an open APL valve. Before inhalation occurs, if the fresh gas flow exceeds alveolar minute ventilation, the inflow of fresh gas will force the alveolar gas remaining in the breathing tube to exit from the APL valve. If the breathing-tube volume is equal to or greater than the patient's tidal volume, the next inspiration will @ contain only fresh gas. Because a fresh gas flow equal to minute ventilation is sufficient to prevent rebreathing, the Mapleson A design is the most efficient Mapleson circuit for spontaneous ventilation.
Positive pressure during controlled ventilation, however, requires a partially closed APL valve. Although some alveolar and fresh gas exits through the valve during inspiration, no gas is vented during expiration, since the exhaled gas stagnates during the expiratory phase of positive-pressure ventilation. As a result, very high fresh gas flows (greater than three times minute ventilation) are required to prevent rebreathing with a Mapleson A circuit during controlled ventilation. Fresh gas flows are conveniently available because the fresh gas inlet is in close proximity to the APL valve in a Mapleson B circuit.

Interchanging the position of the APL valve and the fresh gas inlet transforms a Mapleson A into a @ Mapleson D circuit (Table 3—2). The Mapleson D circuit is efficient during controlled ventilation, since fresh gas flow forces alveolar air away from the patient and toward the APL valve. Thus, simply moving components completely alters the fresh gas requirements of the Mapleson circuits.
The Bain circuit is a coaxial version of the Mapleson D system that incorporates the fresh gas inlet tubing inside the breathing tube (Figure 3—7). This modification decreases the circuit's bulk and retains heat and humidity better than a conventional Mapleson D circuit as a result of partial warming of the inspiratory gas by countercurrent exchange with the warmer expired gases. A disadvantage of this coaxial circuit is the possibility of kinking or disconnection of the fresh gas inlet tubing. Periodic inspection of the inner tubing is mandatory to identify this complication; if unrecognized, either of these mishaps could result in significant rebreathing of exhaled gas.
FIGURE 3-7 A Bain circuit is a Mapleson D circuit design with the fresh gas tubing inside the corrugated breathing tube. APL, adjustable pressure-limiting (valve). (Reproduced with permission from Bain JA, Spoerel WE. Flow requirements for a modified Mapleson D system during controlled ventilation. Can Anaesth Soc J. 1973 Sep;20(5):629-636.)

Although Mapleson circuits overcome some of the disadvantages of the insufflation and draw-over systems, the high fresh gas flows required to prevent rebreathing of CO, result in waste of anesthetic agent, pollution of the operating room environment, and loss of patient heat and humidity (Table 3—3). In an attempt to avoid these problems, the circle system adds more components to the breathing system.
TABLE 3-3 Characteristics of breathing circuits.
Complexity, Insufflation and Open Drop = Very simple. Complexity, Mapleson = Simple. Complexity, Circle = Complex. Control of anesthetic depth, Insufflation and Open Drop = Poor. Control of anesthetic depth, Mapleson = Variable. Control of anesthetic depth, Circle = Good. Ability to scavenge, Insufflation and Open Drop = Very poor. Ability to scavenge, Mapleson = Variable. Ability to scavenge, Circle = Good. Conservation of heat and humidity, Insufflation and Open Drop = No. Conservation of heat and humidity, Mapleson = No. Conservation of heat and humidity, Circle = Yes!. Rebreathing of exhaled gases, Insufflation and Open Drop = No. Rebreathing of exhaled gases, Mapleson = No!. Rebreathing of exhaled gases, Circle = Yes!
'These properties depend on the rate of fresh gas flow.
The components of a circle system include: (1) a CO, absorber containing CO, absorbent; (2) a fresh gas inlet; (3) an inspiratory unidirectional valve and inspiratory breathing tube; (4) a Y-connector; (5) an expiratory unidirectional valve and expiratory breathing tube; (6) an APL valve; and (7) a reservoir (Figure 3-8).
FIGURE 3-8 A circle system. APL, adjustable pressure-limiting (valve).

Rebreathing alveolar gas conserves heat and humidity. However, the CO, in exhaled gas must be eliminated to prevent hypercapnia. CO, chemically combines with water to form carbonic acid. CO, absorbents (eg, soda lime or calcium hydroxide lime) contain hydroxide salts that are capable of neutralizing carbonic acid. Reaction end products include heat (the heat of neutralization), water, and calcium carbonate. Soda lime is an absorbent and is capable of absorbing up to 23 L of CO, per 100 g of absorbent. It consists primarily of calcium hydroxide (80%), along with sodium hydroxide, water, and a small amount of potassium hydroxide. Its reactions are as follows:
(a fast reaction)
Na ,CO3 + Ca(OH), > CaCO; + 2NaOH

snugly between a head and base plate. Together, this unit is called an absorber (Figure 3-9). Although bulky, double canisters permit more complete CO, absorption, require less frequent absorbent changes, and lower gas flow resistance. To ensure complete absorption, a patient's tidal volume should not exceed the air space between absorbent granules, which is roughly equal to 50% of the absorber's capacity. Indicator dye color is monitored through the absorber's transparent walls. Absorbent exhaustion typically occurs first where exhaled gas enters the absorber and along the canister's smooth inner walls. Channeling through areas of loosely packed granules is minimized by a baffle system, which directs gas flow through the center, thereby allowing greater utilization of the absorbent. A trap at the base of the absorber collects dust and moisture.

Unidirectional valves, which function as check valves, contain a ceramic or mica disk resting horizontally on an annular valve seat (Figure 3-10). Forward flow displaces the disk upward, permitting the gas to proceed through the circuit. Reverse flow pushes the disk against its seat, preventing reflux. Valve incompetence is usually due to a warped disk or seat irregularities. The expiratory valve is exposed to the humidity of alveolar gas. Condensation and resultant moisture formation may prevent upward displacement of the disks,

Inhalation opens the inspiratory valve, allowing the patient to breathe a mixture of fresh and exhaled gas that has passed through the CO, absorber. Simultaneously, the expiratory valve closes to prevent rebreathing of exhaled gas that still contains CO,. The subsequent flow of gas away from the patient during exhalation opens the expiratory valve. This gas is vented through the APL valve or rebreathed by the patient after passing through the absorber. Closure of the inspiratory valve during exhalation prevents expiratory gas from mixing with @ fresh gas in the inspiratory limb. Malfunction of either unidirectional valve may allow rebreathing of CO,, resulting in hypercapnia.

Although the major components of the circle system (unidirectional valves, fresh gas inlet, APL valve, CO, absorber, and a reservoir bag) can be placed in several configurations, the following arrangement is preferred (Figure 3-8):
- ¢ Unidirectional valves are relatively close to the patient to prevent backflow into the inspiratory limb if a circuit leak develops. However, unidirectional valves are not placed in the Y-piece, as that makes it difficult to confirm or
maintain proper orientation and intraoperative function.
¢ The fresh gas inlet is placed between the absorber and the inspiratory valve. Positioning it downstream from the inspiratory valve would allow fresh gas to bypass the patient during exhalation and be wasted. Fresh gas introduced between the expiratory valve and the absorber would be diluted by recirculating gas. Furthermore, inhalation anesthetics may be absorbed or released by soda lime granules, thus slowing induction and emergence.
¢ The APL valve is usually placed between the absorber and the expiratory valve and close to the reservoir bag (Figure 3-11). Positioning of the APL valve in this location (ie, before the absorber) helps to conserve absorption capacity and minimizes the venting of fresh gas. The APL valve regulates the flow of gas from the expiratory limb of the circuit into the gas scavenger system.
FIGURE 3-11 Adjustable pressure-limiting (APL) valve. (Reproduced with permission from Rose G, McLarney JT, eds. Anesthesia Equipment Simplified. New York, NY: McGrawHill Education, Inc; 2014.)
- ¢ Resistance to exhalation is decreased by locating the reservoir bag in the expiratory limb.

® With an absorber, the circle system prevents rebreathing of CO, at reduced fresh gas flows (<1 L) or even fresh gas flows equal to the uptake of anesthetic gases and oxygen by the patient and the circuit itself (closed-system anesthesia). At fresh gas flows greater than 5 L/min, rebreathing is so minimal that a CO, absorber is usually unnecessary.
With low fresh gas flows, concentrations of oxygen and inhalation anesthetics can vary markedly between fresh gas (ie, gas in the fresh gas inlet) and inspired gas (ie, gas in the inspiratory limb of the breathing tubes). The latter is a mixture of fresh gas and exhaled gas that has passed through the absorber. The greater the fresh gas flow rate, the less time it will take for a change in fresh gas anesthetic concentration to be reflected in a change in inspired gas anesthetic concentration. Higher flows speed induction and recovery, compensate for leaks in the circuit, and decrease the risks of unanticipated gas mixtures.

That part of a tidal volume that does not undergo alveolar ventilation is referred to as dead space. Thus, any increase in dead space must be accompanied by a corresponding increase in tidal volume, if @ alveolar ventilation is to remain unchanged. Because of the unidirectional valves, apparatus dead space in a circle system is limited to the area distal to the point of inspiratory and expiratory gas mixing at the Y-piece. Unlike Mapleson circuits, the circle system tube length does not affect dead space. Like Mapleson circuits, length does affect circuit compliance and thus the amount of tidal volume lost to the circuit during positive-pressure ventilation. Pediatric circle systems may have both a septum dividing the inspiratory and expiratory gas in the Y-piece and low-compliance breathing tubes to further reduce dead space, and are lighter in weight.

The unidirectional valves and absorber increase circle system resistance, especially at high respiratory rates and large tidal volumes. Nonetheless, even premature neonates can be successfully ventilated using a circle system.

Medical gas delivery systems supply dehumidified gases to the anesthesia circuit at room temperature. Exhaled gas, on the other hand, is saturated with water at body temperature. Therefore, the heat and humidity of inspired gas depend on the relative proportion of rebreathed gas to fresh gas. High flows are accompanied by low relative humidity, whereas low flows allow greater water saturation. Absorbent granules provide a significant source of heat and moisture in the circle system.

The minimal risk of microorganism retention in circle system components could theoretically lead to respiratory infections in subsequent patients. For this reason, bacterial filters are sometimes incorporated into the inspiratory or expiratory breathing tubes or at the Y-piece.

Although most of the problems of Mapleson circuits are solved by the circle system, the improvements have led to other disadvantages: greater size and less portability; increased complexity, resulting in a higher risk of disconnection or malfunction; complications related to use of absorbent; and the difficulty of predicting inspired gas concentrations during low fresh gas flows.

Resuscitation bags (AMBU bags or bag-mask units) are commonly used for emergency ventilation because of their simplicity, portability, and ability to deliver almost 100% oxygen (Figure 3-12). A resuscitator is unlike a Mapleson circuit or a circle system because it contains a nonrebreathing valve. (Remember that a Mapleson system is considered valveless even though it contains an APL valve, whereas a circle system contains unidirectional valves that direct flow through an absorber but allow rebreathing of exhaled gases.)
FIGURE 3-12 The Laerdal resuscitator. (Reproduced with permission from Laerdal Medical Corp.)

An extremely obese but otherwise healthy 5-year-old girl presents for inguinal hernia repair. After uneventful induction of general anesthesia and tracheal intubation, the patient is placed on a ventilator set to deliver a tidal volume of 6 mL/kg at a rate of 16 breaths/min. Despite delivery of high concentrations of sevoflurane in 50% nitrous oxide, tachycardia (145 beats/min) and mild hypertension (144/94 mm Hg) are noted. To increase anesthetic depth, fentanyl (3 mcg/kg) is administered. Heart rate and blood pressure continue to rise and are

What should be considered in the differential diagnosis of this patient's cardiovascular changes?
The combination of tachycardia and hypertension during general anesthesia should always alert the anesthesiologist to the possibility of hypercapnia or hypoxia, both of which produce signs of increased sympathetic activity. These life-threatening conditions should be quickly and immediately ruled out by end-tidal CO, monitoring, pulse oximetry, or arterial blood gas analysis.
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Gas analysis can confirm the delivery of anesthetic gases to the patient. A misconnection of the ventilator could result in hypoxia or hypercapnia. In addition, a malfunctioning unidirectional valve will increase circuit dead space and allow rebreathing of expired CO,. Soda lime exhaustion could also lead to rebreathing in the presence of a low fresh gas flow. Rebreathing of CO, can be detected during the inspiratory phase on a capnograph. If rebreathing appears to be due to an equipment malfunction, the patient should be disconnected from the anesthesia machine and ventilated with a resuscitation bag until repairs are possible.
What are some other consequences of hypercapnia?
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Dobson MB. Anaesthesia for difficult locations—developing countries and military conflicts. In: Prys-Roberts C, Brown BR, eds. International Practice of Anaesthesia. Oxford: Butterworth Heinemann; 1996.
Dorsch JA, Dorsch SE. Understanding Anesthesia Equipment. 5th ed.
Philadelphia, PA: Lippincott, Williams & Wilkins; 2008.
Gegel B. A field expedient Ohmeda Universal Portable Anesthesia Complete Draw-over vaporizer setup. AANA J. 2008;76:185.
Rose, G, McLarmey JT. Anesthesia Equipment Simplified. New York, NY: McGraw-Hill; 2014.

@ Equipment-related adverse outcomes are rarely due to device malfunction or failure; rather, misuse of anesthesia gas delivery systems is three times more prevalent among closed claims. An operator's lack of familiarity with the equipment, an operator's failure to verify machine function prior to use, or both are the most frequent causes. Such mishaps accounted for about 1% of cases in the ASA Closed Claims Project database from 1990 to 2011.
@ The anesthesia machine receives medical gases from a gas supply, controls the flow and reduces the pressure of desired gases to a safe level, vaporizes volatile anesthetics into the final gas mixture, and delivers the gases to a breathing circuit that is connected to the patient's airway. A mechanical ventilator attaches to the breathing circuit but can be excluded with a switch during spontaneous or manual (bag) ventilation.
@ Whereas the oxygen supply can pass directly to its flow control valve, nitrous oxide, air, and other gases must first pass through safety devices before reaching their respective flow control valves. These devices permit the flow of other gases only if there is sufficient oxygen pressure in the safety device and help prevent accidental delivery of a hypoxic mixture in the event of oxygen supply failure.
@ Another safety feature of anesthesia machines is a linkage of the nitrous oxide gas flow to the oxygen gas flow; this arrangement helps ensure a minimum oxygen concentration of 25%.
@ All modern vaporizers are agent specific and temperature corrected, capable of delivering a constant concentration of agent regardless of temperature changes or flow through the vaporizer.
@ Arise in airway pressure may signal worsening pulmonary compliance,

No piece of equipment is more intimately associated with the practice of anesthesiology than the anesthesia machine (Figure 4—1). On the most basic level, the anesthesiologist uses the anesthesia machine to control the patient's ventilation, ensure oxygen delivery, and administer inhalation anesthetics. Proper functioning of the machine is crucial for patient safety. Modern anesthesia machines have become very sophisticated, incorporating many built-in safety features and devices, monitors, and multiple microprocessors that can integrate and monitor all components. Moreover, modular machine designs allow a variety of configurations and features within the same product line. The term anesthesia workstation is therefore often used for modern anesthesia machines. While two manufacturers of anesthesia machines in the United States, GE Healthcare (Datex-Ohmeda) and Drager Medical, have the largest market share, other manufacturers (eg, Mindray, Maquet, Spacelabs) also produce anesthesia delivery systems. Anesthesia providers should be familiar with the operations manuals of all varieties of machines present in their clinical practice.
FIGURE 4-1 Modern anesthesia machine (Datex-Ohmeda Aestiva). A: Front. B: Back.
Much progress has been made in reducing the number of adverse outcomes arising from anesthetic @ gas delivery. Equipment-related adverse outcomes are rarely due to device malfunction or failure; rather, misuse of anesthesia gas
TABLE 4-1 Essential safety features on a modern anesthesia workstation.

"DISS, diameter-index safety system.

Noninterchangeable gas-specific connections to pipeline inlets (DISS)' with pressure gauges, filter, and check valve, Purpose = Prevent incorrect pipeline attachments; detect failure, depletion, or fluctuation. Pin index safety system for cylinders with pressure gauges, and at least one oxygen cylinder, Purpose = Prevent incorrect cylinder attachments; provide backup gas supply; detect depletion. Low oxygen pressure alarm, Purpose = Detect oxygen supply failure at the common gas inlet. Minimum oxygen/nitrous oxide ratio controller device (hypoxic guard), Purpose = Prevent delivery of less than 21% oxygen. Oxygen failure safety device (shut-off or proportioning device), Purpose = Prevent administration of nitrous oxide or other gases when the oxygen supply fails. Oxygen must enter the common manifold downstream to other gases, Purpose = Prevent hypoxia in event of proximal gas leak. Oxygen concentration monitor and alarm, Purpose = Prevent administration of hypoxic gas mixtures in event of a low- pressure system leak; precisely regulate oxygen concentration. Automatically enabled essential alarms and monitors (eg, oxygen concentration), Purpose = Prevent use of the machine without essential monitors. Vaporizer interlock device, Purpose = Prevent simultaneous administration of more than one volatile agent. Capnography and anesthetic gas measurement, Purpose = Guide ventilation; prevent anesthetic overdose; help reduce awareness. Oxygen flush mechanism that does not pass through vaporizers, Purpose = Rapidly refill or flush the breathing circuit. Breathing circuit pressure monitor and alarm, Purpose = Prevent pulmonary barotrauma and detect sustained positive, high peak, and negative airway pressures. Exhaled volume monitor, Purpose = Assess ventilation and prevent hypo- or hyperventilation. Pulse oximetry, blood pressure, and ECG monitoring, Purpose = Provide minimal standard monitoring. Mechanical ventilator, Purpose = Control alveolar ventilation more accurately and during muscle paralysis for prolonged periods. Backup battery, Purpose = Provide temporary electrical power (>30 min) to monitors and alarms in event of power failure. Scavenger system, Purpose = Prevent contamination of the operating room with waste anesthetic gases

@ In its most basic form, the anesthesia machine receives medical gases from a gas supply, controls the flow and reduces the pressure of desired gases to a safe level, vaporizes volatile anesthetics into the final gas mixture, and delivers the gases at the common gas outlet to the breathing circuit connected to the patient's airway (Figures 4—2 and 4-3).

A mechanical ventilator attaches to the breathing circuit but can be excluded with a switch during spontaneous or manual (bag) ventilation. An auxiliary oxygen supply and suction regulator are also usually built into the workstation. In addition to standard safety features (Table 4—1) top-of-the-line anesthesia machines have additional safety features and built-in computer processors that integrate and monitor all components, perform automated machine checkouts, and provide options such as automated record-keeping and networking interfaces to external monitors and hospital information systems. Some machines are designed specifically for mobility, magnetic resonance imaging (MRI) compatibility, or compactness.
Most machines have gas inlets for oxygen, nitrous oxide, and air. Compact models often lack air inlets, whereas other machines may have a fourth inlet for helium, heliox, carbon dioxide, or nitric oxide. Separate inlets are provided for the primary pipeline gas supply that passes through the walls of health care facilities and the secondary cylinder gas supply. Machines therefore have two gas inlet pressure gauges for each gas: one for pipeline pressure and another for cylinder pressure.

Oxygen and nitrous oxide (and often air) are delivered from their central supply source to the operating room through a piping network. The tubing is color coded and connects to the anesthesia machine through a noninterchangeable diameter-index safety system (DISS) fitting that prevents incorrect hose attachment. Noninterchangeability is achieved by making the bore diameter of the body and that of the connection nipple specific for each supplied gas. A filter helps trap debris from the wall supply and a one-way check valve prevents retrograde flow of gases into the pipeline supplies. It should be noted that most modern machines have an oxygen (pneumatic) power outlet that may be used to drive the ventilator or provide an auxiliary oxygen flowmeter. The DISS fittings for the oxygen inlet and the oxygen power outlet are identical and should not be mistakenly interchanged. The approximate pipeline pressure of gases delivered to the anesthesia machine is 50 psig.

Cylinders attach to the machine via hanger-yoke assemblies that utilize a pin index safety system to prevent accidental connection of a wrong gas cylinder. The yoke assembly includes index pins, a washer, a gas filter, and a check valve that prevents retrograde gas flow. The gas cylinders are also color-coded for specific gases to allow for easy identification. In North America, the following color-coding scheme is used: oxygen = green, nitrous oxide = blue, carbon dioxide = gray, air = yellow, helium = brown, nitrogen = black. In the United Kingdom, white is used for oxygen and black and white for air. The E-cylinders attached to the anesthesia machine are a high-pressure source of medical gases and are generally used only as a backup supply in case of pipeline failure. Pressure of gas supplied from the cylinder to the anesthesia machine is 45 psig. Some machines have two oxygen cylinders so that one cylinder can be used while the other is changed. At 20°C, a full E-cylinder contains 600 L of oxygen

Unlike the relatively constant pressure of the pipeline gas supply, the high and variable gas pressure in cylinders makes flow control difficult and potentially dangerous. To enhance safety and ensure optimal use of cylinder gases, machines utilize a pressure regulator to reduce the cylinder gas pressure to 45 to 47 psig.' This pressure, which is slightly lower than the pipeline supply, allows preferential use of the pipeline supply if a cylinder is left open (unless pipeline pressure drops below 45 psig). After passing through pressure gauges and check valves, the pipeline gases share a common pathway with the cylinder gases. A high-pressure relief valve provided for each gas is set to open when the supply pressure exceeds the machine's maximum safety limit (95-110 psig), as might happen with a regulator failure on a cylinder. Some machines also use a second regulator to drop both pipeline and cylinder pressure further (two-stage pressure regulation). A second-stage pressure reduction may also be needed for an auxiliary oxygen flowmeter, the oxygen flush mechanism, or the drive gas to power a pneumatic ventilator.

® Whereas the oxygen supply can pass directly to its flow control valve, nitrous oxide, air (in some machines), and other gases must first pass through safety devices before reaching their respective flow control valves. In other machines, air passes directly to its flow control valve; this allows administration of air even in the absence of oxygen. These devices permit the flow of other gases only if there is sufficient oxygen pressure in the safety device and help prevent accidental delivery of a hypoxic mixture in the event of oxygen supply failure. Thus, in addition to supplying the oxygen flow control valve, oxygen from the common inlet pathway is used to pressurize safety devices, oxygen flush valves, and ventilator power outlets (in some models). Safety devices sense oxygen pressure via a small "piloting pressure" line that may be derived from the gas inlet or secondary regulator. In some anesthesia machine designs (eg, DatexOhmeda Excel), if the piloting pressure line falls below a threshold (eg, 20 psig), the shut-off valves close, preventing the administration of any other gases. The terms fail-safe and nitrous cut-off were previously used for the nitrous oxide

Most modern machines use a proportioning safety device instead of a threshold shut-off valve. These devices, called either an oxygen failure protection device (Drager) or a balance regulator (Datex-Ohmeda), proportionately reduce the pressure of nitrous oxide and other gases except for air. (They completely shut off nitrous oxide and other gas flow only below a set minimum oxygen pressure [eg, 0.5 psig for nitrous oxide and 10 psig for other gases]).
All machines also have an oxygen supply low-pressure sensor that activates alarm sounds when inlet gas pressure drops below a threshold value (usually 20-— 30 psig). It must be emphasized that these safety devices do not protect against other possible causes of hypoxic accidents (eg, gas line misconnections), in which threshold pressure may be maintained by gases containing inadequate or no oxygen.

Once the pressure has been reduced to a safe level, each gas must pass through flow control valves and is measured by flowmeters before mixing with other gases, entering the active vaporizer, and exiting the machine's common gas outlet. Gas lines proximal to flow valves are considered to be in the highpressure circuit, whereas those between the flow valves and the common gas outlet are considered part of the low-pressure circuit of the machine. Touchand color-coded control knobs make it more difficult to turn the wrong gas off or on. As a safety feature the oxygen knob is usually fluted, larger, and protrudes farther than the other knobs. The oxygen flowmeter is positioned furthest to the right, downstream to the other gases; this arrangement helps to prevent hypoxia if there is leakage from a flowmeter positioned upstream.
Flow control knobs control gas entry into the flowmeters by adjustment via a needle valve. Flowmeters on anesthesia machines are classified as either constant-pressure variable-orifice (rotameter) or electronic. In constant-pressure variable-orifice flowmeters, an indicator ball, bobbin, or float is supported by the flow of gas through a tube (Thorpe tube) whose bore (orifice) is tapered. Near the bottom of the tube, where the diameter is small, a low flow of gas will create sufficient pressure under the float to raise it in the tube. As the float rises, the (variable) orifice of the tube widens, allowing more gas to pass around the float. The float will stop rising when its weight is just supported by the difference in pressure above and below it. If flow is increased, the pressure under the float increases, raising it higher in the tube until the pressure drop again just supports the float's weight. This pressure drop is constant regardless of the flow rate or the position in the tube and depends on the float weight and tube cross-sectional area.

Flowmeters are calibrated for specific gases, as the flow rate across a constriction depends on the gas's viscosity at low laminar flows (Poiseuille's law) and its density at high turbulent flows. To minimize the effect of friction with the tube's wall, floats are designed to rotate constantly, which keeps them centered in the tube. Coating the tube's interior with a conductive substance grounds the system and reduces the effect of static electricity. Some flowmeters have two glass tubes, one for low flows and another for high flows (Figure 4— 4A); the two tubes are in series and are still controlled by one valve. A dual taper design can allow a single flowmeter to read both high and low flows (Figure 4— 4B). Causes of flowmeter malfunction include debris in the flow tube, vertical tube misalignment, and sticking or concealment of a float at the top of a tube.
FIGURE 4-3. The anesthesia machine reduces the pressure from the gas supply, vaporizes anesthetic agents, and delivers the gas mixture to the common gas outlet. The oxygen flush line bypasses the vaporizers and directs oxygen directly to the common gas outlet. (Reproduced with permission from Rose G, McLamey JT, eds. Anesthesia Equipment Simplified. New York, NY: McGraw-Hill Education, Inc; 2014.)
Should a leak develop within or downstream from an oxygen flowmeter, a hypoxic gas mixture can be delivered to the patient (Figure 4—5). To reduce this risk, oxygen flowmeters are always positioned downstream to all other flowmeters (nearest to the vaporizer).
Some anesthesia machines have electronic flow control and measurement. In such instances, a backup conventional (Thorpe) auxiliary oxygen flowmeter is provided. Other models have conventional flowmeters but electronic measurement of gas flow along with Thorpe tubes and digital or digital/graphic displays. The amount of pressure drop caused by a flow restrictor is the basis for measurement of gas flow rate in these systems. In these machines oxygen, nitrous oxide, and air each have a separate electronic flow measurement device in the flow control section before they are mixed together. Electronic flowmeters are required if gas flow rate data will be acquired automatically by computerized anesthesia recording systems.

The oxygen flow valves are usually designed to deliver a minimum oxygen flow when the anesthesia machine is turned on. One method involves the use of a minimum flow resistor. This safety feature helps ensure that some oxygen enters the breathing circuit even if the operator forgets to turn on the oxygen flow.

® Another safety feature of anesthesia machines is a linkage of the nitrous oxide gas flow to the oxygen gas flow; this arrangement helps ensure a minimum oxygen concentration of 25%. The oxygen/nitrous oxide ratio controller links the two flow valves either pneumatically or mechanically. To maintain the minimum oxygen concentration, the system (Link-25) in Datex-Ohmeda machines increases the flow of oxygen, whereas the oxygen ratio monitor controller (ORMC) in Drager machines reduces the concentration of nitrous oxide. It should be noted that this safety device does not affect the flow of a third gas (eg, air, helium, or carbon dioxide).

Volatile anesthetics (eg, halothane, isoflurane, desflurane, sevoflurane) must be vaporized before being delivered to the patient. Vaporizers have concentrationcalibrated dials that precisely add volatile anesthetic agents to the combined gas flow from all flowmeters. They must be located between the flowmeters and the common gas outlet. Moreover, unless the machine accepts only one vaporizer at a time, all anesthesia machines should have an interlocking or exclusion device that prevents the concurrent use of more than one vaporizer.

At temperatures encountered in the operating room, the molecules of a volatile anesthetic in a closed container are distributed between the liquid and gaseous phases. The gas molecules bombard the walls of the container, creating the saturated vapor pressure of that agent. Vapor pressure depends on the characteristics of the volatile agent and the temperature. The greater the temperature, the greater the tendency for the liquid molecules to escape into the gaseous phase and the greater the vapor pressure (Figure 4—6). Vaporization requires energy (the latent heat of vaporization), which results in a loss of heat from the liquid. As vaporization proceeds, temperature of the remaining liquid anesthetic drops and vapor pressure decreases unless heat is readily available to enter the system. Vaporizers contain a chamber in which a carrier gas becomes saturated with the volatile agent.
FIGURE 44 Constant-pressure variable-orifice flowmeters (Thorpe type). A:

A liquid's boiling point is the temperature at which its vapor pressure is equal to the atmospheric pressure. As the atmospheric pressure decreases (as in higher altitudes), the boiling point also decreases. Anesthetic agents with low boiling points are more susceptible to variations in barometric pressure than agents with higher boiling points. Among the commonly used agents, desflurane has the lowest boiling point (22.8°C at 760 mm Hg).

The copper kettle vaporizer is no longer used in clinical anesthesia; however, understanding how it works provides invaluable insight into the delivery of volatile anesthetics (Figure 4—7). It is classified as a measured-flow vaporizer (or flowmeter-controlled vaporizer). In a copper kettle, the amount of carrier gas bubbled through the volatile anesthetic is controlled by a dedicated flowmeter. This valve is turned off when the vaporizer circuit is not in use. Copper is used as the construction metal because its relatively high specific heat (the quantity of heat required to raise the temperature of 1 g of substance by 1°C) and high thermal conductivity (the speed of heat conductance through a substance) enhance the vaporizer's ability to maintain a constant temperature. All the gas entering the vaporizer passes through the anesthetic liquid and becomes saturated with vapor. One milliliter of liquid anesthetic yields approximately 200 mL of anesthetic vapor. Because the vapor pressure of volatile anesthetics is greater than the partial pressure required for anesthesia, the saturated gas leaving a copper kettle has to be diluted before it reaches the patient.
FIGURE 4-5 Sequence of flowmeters in a three-gas machine. A: An unsafe sequence. B: Typical Datex-Ohmeda sequence. C: Typical Drager sequence. Note that regardless of sequence a leak in the oxygen tube or further downstream can result in delivery of a hypoxic mixture.
For example, the vapor pressure of halothane is 243 mm Hg at 20°C, so the concentration of halothane exiting a copper kettle at 1 atmosphere would be

243/760, or 32%. If 100 mL of oxygen enters the kettle, roughly 150 mL of gas exits (the initial 100 mL of oxygen plus 50 mL of saturated halothane vapor), one-third of which would be saturated halothane vapor. To deliver a 1% concentration of halothane (MAC 0.75%), the 50 mL of halothane vapor and 100 mL of carrier gas that left the copper kettle have to be diluted within a total of 5000 mL of fresh gas flow. Thus, every 100 mL of oxygen passing through a halothane vaporizer translates into a 1% increase in concentration if total gas flow into the breathing circuit is 5 L/min. Therefore, when total flow is fixed, flow through the vaporizer determines the ultimate concentration of anesthetic. Isoflurane has an almost identical vapor pressure, so the same relationship between copper kettle flow, total gas flow, and anesthetic concentration exists. However, if total gas flow decreases without an adjustment in copper kettle flow (eg, exhaustion of a nitrous oxide cylinder), the delivered volatile anesthetic concentration rises rapidly to potentially dangerous levels.

®@ All modern vaporizers are agent specific and temperature corrected, capable of delivering a constant concentration of agent regardless of temperature changes or flow through the vaporizer. Turning a single calibrated control knob counterclockwise to the desired percentage diverts an appropriate small fraction of the total gas flow into the carrier gas, which flows over the liquid anesthetic in a vaporizing chamber, leaving the balance to exit the vaporizer unchanged (Figure 4—8). Because some of the entering gas is never exposed to anesthetic liquid, this type of agent-specific vaporizer is also known as a variable-bypass vaporizer.

FIGURE 4-7 Schematic of a copper kettle vaporizer. Note that 50 mL/min of halothane vapor is added for each 100 mL/min oxygen flow that passes through the vaporizer.
FIGURE 4-8 Schematic of agent-specific variable-bypass vaporizers. A:

Temperature compensation is achieved by a strip composed of two different metals welded together. The metal strips expand and contract differently in response to temperature changes. When the temperature decreases, differential contraction causes the strip to bend, allowing more gas to pass through the vaporizer. Such bimetallic strips are also used in home thermostats. As the temperature rises differential expansion causes the strip to bend the other way restricting gas flow into the vaporizer. Altering total fresh gas flow rates within a wide range does not significantly affect anesthetic concentration because the same proportion of gas is exposed to the liquid. However, the real output of an agent would be lower than the dial setting at extremely high flow (>15 L/min); the converse is true when the flow rate is less than 250 mL/min. Changing the gas composition from 100% oxygen to 70% nitrous oxide may transiently decrease volatile anesthetic concentration due to the greater solubility of nitrous oxide in volatile agents.
Given that these vaporizers are agent specific, filling them with the incorrect anesthetic must be avoided. For example, unintentionally filling a sevofluranespecific vaporizer with halothane could lead to an anesthetic overdose. First, halothane's higher vapor pressure (243 mm Hg versus 157 mm Hg) will cause a 40% greater amount of anesthetic vapor to be released. Second, halothane is more than twice as potent as sevoflurane (MAC 0.75 versus 2.0). Conversely, filling a halothane vaporizer with sevoflurane will cause an anesthetic underdosage. Modern vaporizers offer agent-specific, keyed, filling ports to prevent filling with an incorrect agent.

Excessive tilting of older vaporizers (Tec 4, Tec 5, and Vapor 19.n) during transport may flood the bypass area and lead to dangerously high anesthetic concentrations. In the event of tilting and spillage, high flow of oxygen with the vaporizer turned off should be used to vaporize and flush the liquid anesthetic from the bypass area. Fluctuations in pressure from positive-pressure ventilation in older anesthesia machines may cause a transient reversal of flow through the vaporizer, unpredictably changing agent delivery. This "pumping effect" is more pronounced with low gas flows. A one-way check valve between the vaporizers and the oxygen flush valve (Datex-Ohmeda) together with some design modifications in newer units limit the occurrence of some of these problems. Variable-bypass vaporizers compensate for changes in ambient pressures (ie, altitude changes maintaining relative anesthetic gas partial pressure). It is the partial pressure of the anesthetic agent that determines its concentrationdependent physiological effects. Thus, there is no need to increase the selected anesthetic concentration when using a variable-bypass vaporizer at altitude because the partial pressure of the anesthetic agent will be largely unchanged. Although at lower ambient pressures gas passing through the vaporizer is exposed to increased vaporizer output, because of Dalton's law of partial pressure the partial pressure of the anesthetic vapor will remain largely unaffected compared with partial pressures obtained at sea level.

Electronically controlled vaporizers must be utilized for desflurane and are used for all volatile anesthetics in some sophisticated anesthesia machines.
1. Desflurane vaporizer—Desflurane's vapor pressure is so high that at sea level it almost boils at room temperature (Figure 4-6). This high volatility, coupled with a potency only one-fifth that of other volatile agents, presents unique delivery problems. First, the vaporization required for general anesthesia produces a cooling effect that would overwhelm the ability of conventional vaporizers to maintain a constant temperature. Second, because it vaporizes so extensively, a tremendously high fresh gas flow would be necessary to dilute the carrier gas to clinically relevant concentrations. These problems have been addressed by the development of special desflurane vaporizers. A reservoir containing desflurane (desflurane sump) is electrically heated to 39°C (significantly higher than its boiling point) creating a vapor pressure of 2 atmospheres. Unlike a variable-bypass vaporizer, no fresh gas flows through the desflurane sump. Rather, pure desflurane vapor joins the fresh gas mixture before exiting the vaporizer. The amount of desflurane vapor released from the sump depends on the concentration selected by turning the control dial and the fresh gas flow rate. Although the Tec 6 Plus maintains a constant desflurane concentration over a wide range of fresh gas flow rates, it cannot automatically compensate for changes in elevation as do the variable-bypass vaporizers. Decreased ambient pressure (eg, high elevation) does not affect the concentration of agent delivered, but decreases the partial pressure of the agent. Thus, at high elevations one must manually increase the desflurane concentration control.

2. Aladin (GE) cassette vaporizer—Gas flow from the flow control is divided into bypass flow and liquid chamber flow. The latter is conducted into an agentspecific, color-coded, cassette (Aladin cassette) in which the volatile anesthetic is vaporized. The machine accepts only one cassette at a time and recognizes the cassette through magnetic labeling. The cassette does not contain any bypass flow channels; therefore, unlike traditional vaporizers, liquid anesthetic cannot escape during handling and the cassette can be carried in any position. After leaving the cassette, the now anesthetic-saturated liquid chamber flow reunites with the bypass flow before exiting the fresh gas outlet. A flow restrictor valve near the bypass flow helps to adjust the amount of fresh gas that flows to the cassette. Adjusting the ratio between the bypass flow and liquid chamber flow changes the concentration of volatile anesthetic agent delivered to the patient. Sensors in the cassette measure pressure and temperature, thus determining agent concentration in the gas leaving the cassette. Correct liquid chamber flow is calculated based on desired fresh gas concentration and determined cassette gas concentration.

In contrast to the multiple gas inlets, the anesthesia machine has only one common gas outlet that supplies gas to the breathing circuit. The term fresh gas outlet is also often used because of its critical role in adding new gas of fixed and known composition to the circle system. Unlike older models, some newer anesthesia machines measure and report common outlet gas flows. An antidisconnect retaining device is used to prevent accidental detachment of the gas outlet hose that connects the machine to the breathing circuit. The oxygen flush valve provides a high flow (35-75 L/min) of oxygen directly to the common gas outlet, bypassing the flowmeters and vaporizers. It is used to rapidly refill or flush the breathing circuit, but because the oxygen may be supplied at a line pressure of 45 to 55 psig, there is a real potential for lung barotrauma to occur. For this reason, the flush valve must be used cautiously whenever a patient is connected to the breathing circuit. Moreover, inappropriate use of the flush valve (or a situation of stuck valve) may result in backflow of gases into the low-pressure circuit, causing dilution of inhaled anesthetic concentration. Some machines use a second-stage regulator to drop the oxygen flush pressure to a lower level. A protective rim around the flush button limits the possibility of unintentional activation

In adults, the breathing system most commonly used with anesthesia machines is the circle system (Figure 4—9); a Bain circuit is occasionally used. The components and use of the circle system were previously discussed (see Chapter 3). It is important to note that gas composition at the common gas outlet can be controlled precisely and rapidly by adjustments in flowmeters and vaporizers. In contrast, gas composition, especially volatile anesthetic concentration, in the breathing circuit is significantly affected by other factors, including anesthetic uptake in the patient's lungs, minute ventilation, total fresh gas flow, volume of the breathing circuit, and the presence of gas leaks. Use of high gas flow rates during induction and emergence decreases the effects of such variables and can diminish the magnitude of discrepancies between fresh gas outlet and circle system anesthetic concentrations. Measurement of inspired and expired anesthetic gas concentration also greatly facilitates anesthetic management.
FIGURE 4-9 Diagram of a typical breathing circuit (Drager Narkomed). Note gas flow during A: spontaneous inspiration, B: manual inspiration ("bagging"), and C: exhalation (spontaneous or bag ventilation).
In most machines, the common gas outlet is attached to the breathing circuit just past the exhalation valve to prevent artificially high exhaled tidal volume measurements. When spirometry measurements are made at the Y-connector, fresh gas flow can enter the circuit on the patient side of the inspiratory valve. The latter enhances CO, elimination and may help reduce desiccation of the CO, absorbent.
Newer anesthesia machines have integrated internalized breathing circuit components (Figure 4-10). The advantages of these designs include reduced probability of breathing circuit misconnects, disconnects, kinks, and leaks. The smaller volume of compact machines can also help conserve gas flow and volatile anesthetics and allow faster changes in breathing circuit gas concentration. Internal heating of manifolds can reduce precipitation of moisture.
A
FIGURE 4-10 Breathing circuit design. A: Conventional external components. B: Compact design that reduces external connections and circuit volume (Drager Fabius GS).

General anesthesia must not be administered without an oxygen analyzer in the breathing circuit. Three types of oxygen analyzers are available: polarographic (Clark electrode), galvanic (fuel cell), and paramagnetic. The first two techniques utilize electrochemical sensors that contain cathode and anode electrodes embedded in an electrolyte gel separated from the sample gas by an oxygen-permeable membrane (usually Teflon). As oxygen reacts with the electrodes, a current is generated that is proportional to the oxygen partial pressure in the sample gas. The galvanic and polarographic sensors differ in the composition of their electrodes and electrolyte gels. The components of the galvanic cell are capable of providing enough chemical energy so that the reaction does not require an external power source.
Although the initial cost of paramagnetic sensors is greater than that of electrochemical sensors, paramagnetic devices are self-calibrating and have no consumable parts. In addition, their response time is fast enough to differentiate between inspired and expired oxygen concentrations.
All oxygen analyzers should have a low-level alarm that is automatically activated by turning on the anesthesia machine. The sensor should be placed into the inspiratory or expiratory limb of the circle system's breathing circuit—but not into the fresh gas line. As a result of the patient's oxygen consumption, the expiratory limb has a slightly lower oxygen partial pressure than the inspiratory limb, particularly at low fresh gas flows. The increased humidity of expired gas does not significantly affect most modern sensors.

Spirometers, also called respirometers, are used to measure exhaled tidal volume in the breathing circuit on all anesthesia machines, typically near the exhalation valve. Some anesthesia machines also measure the inspiratory tidal volume just past the inspiratory valve or the actual delivered and exhaled tidal volumes at the Y-connector that attaches to the patient's airway.
A common method employs a rotating vane of low mass in the expiratory limb in front of the expiratory valve of the circle system (vane anemometer or Wright respirometer, Figure 4—11A).
c
FIGURE 4-11 Spirometer designs. A: Vane anemometer (Datex-Ohmeda). B: Volumeter (Drager). C: Variable-orifice flowmeter (Datex-Ohmeda). D: Fixedorifice flowmeter (Pitot tube).
The flow of gas across vanes within the respirometer causes their rotation, which is measured electronically, photoelectrically, or mechanically. In another variation using this turbine principle, the volumeter or displacement meter is designed to measure the movement of discrete quantities of gas over time (Figure 4—11B).
A pneumotachograph is a fixed-orifice flowmeter that can function as a spirometer. A parallel bundle of small-diameter tubes in chamber (Fleisch pneumotachograph) or mesh screen provides a slight resistance to airflow. The pressure drop across this resistance is sensed by a differential pressure transducer and is proportional to the flow rate. Integration of flow rate over time yields tidal volume. Moreover, analysis of pressure, volume, and time relationships can yield potentially valuable information about airway and lung mechanics. Modifications have been required to overcome inaccuracies due to water condensation and temperature changes. One modification employs two pressuresensing lines in a Pitot tube at the Y-connection (Figure 4—11D). Gas flowing through the Pitot tube (flow sensor tube) creates a pressure difference between the flow sensor lines. This pressure differential is used to measure flow, flow direction, and airway pressure. Respiratory gases are continuously sampled to correct the flow reading for changes in density and viscosity.

A pressure gauge or electronic sensor is always used to measure breathingcircuit pressure somewhere between the expiratory and inspiratory unidirectional valves; the exact location depends on the model of anesthesia machine. Breathing-circuit pressure usually reflects airway pressure if it is measured as close to the patient's airway as possible. The most accurate measurements of both inspiratory and expiratory pressures can be obtained from the @ Yconnection. A rise in airway pressure may signal worsening pulmonary compliance, an increase in tidal volume, or an obstruction in the breathing circuit, tracheal tube, or the patient's airway. A drop in pressure may indicate an improvement in compliance, a decrease in tidal volume, or a leak in the circuit. If circuit pressure is being measured at the CO, absorber, however, it will not always mirror the pressure in the patient's airway. For example, clamping the expiratory limb of the breathing tubes during exhalation will prevent the patient's breath from exiting the lungs. Despite this buildup in airway pressure, a pressure gauge at the absorber will read zero because of the intervening one-way valve.
Some machines have incorporated auditory feedback for pressure changes during ventilator use.

The adjustable pressure-limiting (APL) valve, sometimes referred to as the pressure relief or pop-off valve, is usually fully open during spontaneous ventilation but must be partially closed during manual or assisted bag ventilation. The APL valve often requires fine adjustments. If it is not closed sufficiently excessive loss of circuit volume due to leaks prevents manual ventilation. At the same time, if it is closed too much or is fully closed, a progressive rise in pressure could result in pulmonary barotrauma (eg, pneumothorax) or hemodynamic compromise, or both. As an added safety feature, the APL valves on modern machines act as true pressure-limiting devices that can never be completely closed; the upper limit is usually 70 to 80 cm H,O.

Absolute humidity is defined as the weight of water vapor in 1 L of gas (ie, mg/L). Relative humidity is the ratio of the actual mass of water present in a volume of gas to the maximum amount of water possible at a particular temperature. At 37°C and 100% relative humidity, absolute humidity is 44 mg/L, whereas at room temperature (21°C and 100% humidity) it is 18 mg/L. Inhaled gases in the operating room are normally administered at room temperature with little or no humidification. Gases must therefore be warmed to body temperature and saturated with water by the upper respiratory tract. Tracheal intubation and high fresh gas flows bypass this normal humidification system and expose the lower airways to dry (<10 mg H,O/L), room temperature gases. forced-
Prolonged humidification of gases by the lower respiratory tract leads to dehydration of mucosa, altered ciliary function, and, if excessively prolonged, could potentially lead to inspissation of secretions, atelectasis, and even ventilation/perfusion mismatching, particularly in patients with underlying lung disease. Body heat is also lost as gases are warmed, and even more importantly, as water is vaporized to humidify the dry gases. The heat of vaporization for water is 560 cal/g of water vaporized. Fortunately, this heat loss accounts for about only 5% to 10% of total intraoperative heat loss, is not significant for a short procedure (<1 h), and usually can easily be compensated for with a air warming blanket. Humidification and heating of inspiratory gases may be most important for small pediatric patients and older patients with severe underlying lung pathology, eg, cystic fibrosis.

Humidifiers added to the breathing circuit minimize water and heat loss. The simplest designs are condenser humidifiers or heat and moisture exchanger (HME) units (Figure 4—12). These passive devices do not add heat or vapor but rather contain a hygroscopic material that traps exhaled humidification and heat, which is released upon subsequent inhalation. Depending on the design, they may substantially increase apparatus dead space (more than 60 mL?), which can cause significant rebreathing in pediatric patients. They can also increase breathing-circuit resistance and the work of breathing during spontaneous respirations. Excessive saturation of an HME with water or secretions can obstruct the breathing circuit. Some condenser humidifiers also act as effective filters that may protect the breathing circuit and anesthesia machine from bacterial or viral cross-contamination. This may be particularly important when ventilating patients with respiratory infections or compromised immune systems.
FIGURE 4-12 Heat and moisture exchanger (HME) functions as an "artificial nose" that attaches between the tracheal tube and the right-angle connector of the breathing circuit.

Active humidifiers are more effective than passive ones in preserving moisture and heat. Active humidifiers add water to gas by passing the gas over a water chamber (passover humidifier) or through a saturated wick (wick humidifier), bubbling it through water (bubble-through humidifier), or mixing it with vaporized water (vapor-phase humidifier). Because increasing temperature increases the capacity of a gas to hold water vapor, heated humidifiers with thermostatically controlled elements are most effective.
The hazards of heated humidifiers include thermal lung injury (inhaled gas temperature should be monitored and should not exceed 41°C), nosocomial infection, increased airway resistance from excess water condensation in the breathing circuit, interference with flowmeter function, and an increased likelihood of circuit disconnection. Use of these humidifiers is particularly valuable in children as they help prevent both hypothermia and the plugging of small tracheal tubes by dried secretions. Of course, any design that increases airway dead space should be avoided in pediatric patients. Unlike passive humidifiers, active humidifiers do not filter respiratory gases.

All modern anesthesia machines are equipped with a ventilator. Historically ventilators used in the operating room (OR) were simpler and more compact than their intensive care unit (ICU) counterparts. This distinction has become blurred due to advances in technology together with an increasing need for "ICU-type" ventilators as more critically ill patients come to the OR. The ventilators on some modern machines have almost the same capabilities as those in the ICU. A more complete discussion of mechanical ventilation in relation to ICU practice is contained in Chapter 57.

Ventilators generate gas flow by creating a pressure gradient between the proximal airway and the alveoli. Ventilator function is best described in relation to the four phases of the ventilatory cycle: inspiration, the transition from inspiration to expiration, expiration, and the transition from expiration to inspiration. Although several classification schemes exist, the most common is based on inspiratory phase characteristics and the method of cycling from inspiration to expiration.

During inspiration, ventilators generate tidal volumes by producing gas flow along a pressure gradient. The machine generates either a constant pressure (constant-pressure generators) or constant gas flow rate (constant-flow generators) during inspiration, regardless of changes in lung mechanics (Figure 4-13). Nonconstant generators produce pressures or gas flow rates that vary during the cycle but remain consistent from breath to breath. For instance, a ventilator that generates a flow pattern resembling a half cycle of a sine wave (eg, rotary piston ventilator) would be classified as a nonconstant-flow generator. An increase in airway resistance or a decrease in lung compliance would increase peak inspiratory pressure but would not alter the flow rate generated by this type of ventilator (Figure 4—14).
FIGURE 4-13 Pressure, volume, and flow profiles of different types of ventilators. A: Constant pressure. B: Constant flow. C: Nonconstant generator.

Termination of the inspiratory phase can be triggered by a preset limit of time (fixed duration), a set inspiratory pressure that must be reached, or a predetermined tidal volume that must be delivered. Time-cycled ventilators allow tidal volume and peak inspiratory pressure to vary depending on lung compliance. Tidal volume is adjusted by setting inspiratory duration and inspiratory flow rate. Pressure-cycled ventilators will not cycle from the inspiratory phase to the expiratory phase until a preset pressure is reached. If a large circuit leak decreases peak pressures significantly, a pressure-cycled ventilator may remain in the inspiratory phase indefinitely. On the other hand, a small leak may not markedly decrease tidal volume, because cycling will be delayed until the pressure limit is met. Volume-cycled ventilators vary inspiratory duration and pressure to deliver a preset volume. In reality, modern ventilators overcome the many shortcomings of classic ventilator designs by incorporating secondary cycling parameters or other limiting mechanisms. For example, time-cycled and volume-cycled ventilators usually incorporate a pressure-limiting feature that terminates inspiration when a preset, adjustable safety pressure limit is reached. Similarly, a volume-preset control that limits the excursion of the bellows allows a time-cycled ventilator to function somewhat like a volume-cycled ventilator, depending on the selected ventilator rate and inspiratory flow rate.

The expiratory phase of ventilators normally reduces airway pressure to atmospheric levels or some preset value of positive end-expiratory pressure (PEEP). Exhalation is therefore passive. Flow out of the lungs is determined primarily by airway resistance and lung compliance. Expired gases fill up the bellows; they are then relieved to the scavenging system.

Transition into the next inspiratory phase may be based on a preset time interval or a change in pressure. The behavior of the ventilator during this phase together with the type of cycling from inspiration to expiration determines ventilator mode. During controlled ventilation, the most basic mode of all ventilators, the next breath always occurs after a preset time interval. Thus tidal volume and rate are fixed in volume-controlled ventilation, whereas peak inspiratory pressure and rate are fixed in pressure-controlled ventilation (Figure 4—15).
FIGURE 4-15 Ventilator controls (Datex-Ohmeda). A: Volume control mode. B: Pressure control mode.

@ Traditionally ventilators on anesthesia machines have a double-circuit system design and are pneumatically powered and electronically controlled (Figure 4— 16). Newer machines also incorporate microprocessor controls and sophisticated and precise pressure and flow sensors to achieve multiple ventilatory modes, PEEP, accurate tidal volumes, and enhanced safety features. Some anesthesia machines have ventilators that use a single-circuit piston design.
FIGURE 4-16 Double-circuit pneumatic ventilator design. A: Datex-Ohmeda. B: Drager.

In a double-circuit system design, tidal volume is delivered from a bellows assembly that consists of a bellows in a clear rigid plastic enclosure (Figure 4— 16). A standing (ascending) bellows is preferred as it readily draws attention to a circuit disconnection by collapsing. Hanging (descending) bellows are rarely used and must not be weighted; older ventilators with weighted hanging bellows continue to fill by gravity despite a disconnection in the breathing circuit.
The bellows in a double-circuit design ventilator takes the place of the breathing bag in the anesthesia circuit. Pressurized oxygen or air from the ventilator power outlet (45-50 psig) is routed to the space between the inside wall of the plastic enclosure and the outside wall of the bellows. Pressurization of the plastic enclosure compresses the pleated bellows inside, forcing the gas inside into the breathing circuit and patient. In contrast, during exhalation, the bellows ascend as pressure inside the plastic enclosure drops and the bellows fill up with the exhaled gas. A ventilator flow control valve regulates drive gas flow into the pressurizing chamber. This valve is controlled by ventilator settings in the control box (Figure 4-16). Ventilators with microprocessors also utilize feedback from flow and pressure sensors. If oxygen is used for pneumatic power it will be consumed at a rate at least equal to minute ventilation. Thus, if oxygen fresh gas flow is 2 L/min and a ventilator is delivering 6 L/min to the circuit, a total of at least 8 L/min of oxygen is being consumed. This should be kept in mind if the hospital's medical gas system fails and cylinder oxygen is required. Some anesthesia machines reduce oxygen consumption by incorporating a Venturi device that draws in room air to provide air/oxygen pneumatic power. Newer machines may offer the option of using compressed air for pneumatic power. A leak in the ventilator bellows can transmit high gas pressure to the patient's airway, potentially resulting in pulmonary barotrauma. This may be indicated by a higher than expected rise in inspired oxygen concentration (if oxygen is the sole pressurizing gas). Some machine ventilators have a built-in drive gas regulator that reduces the drive pressure (eg, to 25 psig) for added safety.

Double-circuit design ventilators also incorporate a free breathing valve that allows outside air to enter the rigid drive chamber and the bellows to collapse if the patient generates negative pressure by taking spontaneous breaths during mechanical ventilation.

In a piston design, the ventilator substitutes an electrically driven piston for the bellows, and the ventilator requires either minimal or no pneumatic @ (oxygen) power. The major advantage of a piston ventilator is its ability to deliver accurate tidal volumes to patients with very poor lung compliance and to very small patients.

Whenever a ventilator is used on an anesthesia machine, the circle system's APL valve must be functionally removed or isolated from the circuit. A bag/ventilator switch typically accomplishes this. When the switch is turned to "bag" the ventilator is excluded and spontaneous/manual (bag) ventilation is possible. When it is turned to "ventilator," the breathing bag and the APL are excluded from the breathing circuit. The APL valve may be automatically excluded in some newer anesthesia machines when the ventilator is turned on. The ventilator contains its own pressure-relief (pop-off) valve, called the spill valve, which is pneumatically closed during inspiration so that positive pressure can be generated (Figure 4-16). During exhalation, the pressurizing gas is vented out and the ventilator spill valve is no longer closed. The ventilator bellows or piston refill during expiration; when the bellows is completely filled, the increase in circle system pressure causes the excess gas to be directed to the scavenging system through the spill valve. Sticking of this valve can result in abnormally elevated airway pressure during exhalation.

Peak inspiratory pressure is the highest circuit pressure generated during an inspiratory cycle, and provides an indication of dynamic compliance. Plateau pressure is the pressure measured during an inspiratory pause (a time of no gas flow), and mirrors static compliance. During normal ventilation of a patient without lung disease, peak inspiratory pressure is equal to or only slightly greater than plateau pressure. An increase in both peak inspiratory pressure and plateau pressure implies an increase in tidal volume or a decrease in pulmonary compliance. An increase in peak inspiratory pressure without any change in plateau pressure signals an increase in airway resistance or inspiratory gas flow rate (Table 4—2). Thus, the shape of the breathing-circuit pressure waveform can provide important airway information. Many anesthesia machines graphically display breathing-circuit pressure (Figure 4—17). Airway secretions or kinking of the tracheal tube can be easily ruled out with the use of a suction catheter. Flexible fiberoptic bronchoscopy usually provides a definitive diagnosis.
FIGURE 4-17 Airway pressures (Paw) can be diagrammatically presented as a function of time. A: In normal persons, the peak inspiratory pressure is equal to or slightly greater than the plateau pressure. B: An increase in peak inspiratory pressure and plateau pressure (the difference between the two remains almost constant) can be due to an increase in tidal volume or a decrease in pulmonary compliance. C: An increase in peak inspiratory pressure with little change in plateau pressure signals an increase in inspiratory flow rate or an increase in airway resistance.

Increased tidal volume
Decreased pulmonary compliance
Pulmonary edema
Trendelenburg position
Pleural effusion
Ascites
Abdominal packing
Peritoneal gas insufflation
Tension pneumothorax
Endobronchial intubation

Increased inspiratory gas flow rate
Increased airway resistance
Kinked endotracheal tube
Bronchospasm
Secretions
Foreign body aspiration
Airway compression
Endotracheal tube cuff herniation

Alarms are an integral part of all modern anesthesia @ ventilators. Whenever a ventilator is used "disconnect alarms" must be passively activated. Anesthesia workstations should have at least three disconnect alarms: low peak inspiratory pressure, low exhaled tidal volume, and low exhaled carbon dioxide. The first is always built into the ventilator whereas the latter two may be in separate modules. A small leak or partial breathing-circuit disconnection may be detected by subtle decreases in peak inspiratory pressure, exhaled volume, or end-tidal carbon dioxide before alarm thresholds are reached. Other built-in ventilator alarms include high peak inspiratory pressure, high PEEP, sustained high airway pressure, negative pressure, and low oxygen-supply pressure. Most modern anesthesia ventilators also have integrated spirometers and oxygen analyzers that provide additional alarms.

© From the previous discussion, it is important to appreciate that because the ventilator's spill valve is closed during inspiration, fresh gas flow from the machine's common gas outlet normally contributes to the tidal volume delivered to the patient. For example, if the fresh gas flow is 6 L/min, the inspiratoryexpiratory (I:E) ratio is 1:2, and the respiratory rate is 10 breaths/min, each tidal volume will include an extra 200 mL in addition to the ventilator's output:
(6000 mL/min)(33%)
10 breaths/min
Thus, increasing fresh gas flow increases tidal volume, minute ventilation, and peak inspiratory pressure. To avoid problems with ventilator—fresh gas flow coupling, airway pressure and exhaled tidal volume must be monitored closely and excessive fresh gas flows must be avoided. Current ventilators automatically compensate for fresh gas flow coupling. Piston style ventilators redirect fresh gas flow to the reservoir bag during inspiration thus preventing augmentation of the tidal volume secondary to fresh gas flow.

Intermittent or sustained high inspiratory pressures (>30 mm Hg) during positive-pressure ventilation increase the risk of pulmonary barotrauma (eg, pneumothorax) or hemodynamic compromise, or both, during anesthesia.
may also be measured but are usually not compensated. The actual method of tidal volume compensation or modulation varies according to manufacturer and model. In one design a flow sensor measures the tidal volume delivered at the inspiratory valve for the first few breaths and adjusts subsequent metered drive gas flow volumes to compensate for tidal volume losses (feedback adjustment). Another design continually measures fresh gas and vaporizer flow and subtracts this amount from the metered drive gas flow (preemptive adjustment). Alternately, machines that use electronic control of gas flow can decouple fresh gas flow from the tidal volume by delivery of fresh gas flow only during exhalation. Lastly, the inspiratory phase of the ventilator—fresh gas flow may be diverted through a decoupling valve into the breathing bag, which is excluded from the circle system during ventilation. During exhalation the decoupling valve opens, allowing the fresh gas that was temporarily stored in the bag to enter the breathing circuit.

outlet of the scavenging system may be a direct line to the outside via a ventilation duct beyond any point of recirculation (passive scavenging) or a connection to the hospital's vacuum system (active scavenging). A chamber or reservoir bag accepts waste-gas overflow when the capacity of the vacuum is exceeded. The vacuum control valve on an active system should be adjusted to allow the evacuation of 10 to 15 L of waste gas per minute. This rate is adequate for periods of high fresh gas flow (ie, induction and emergence) yet minimizes the risk of transmitting negative pressure to the breathing circuit during lower flow conditions (maintenance). Unless used correctly the risk of occupational exposure for health care providers is higher with an open interface. Some machines may come with both active and passive scavenger systems.
FIGURE 4-18 Open interface scavenger system. (Reproduced with permission from Rose G, McLarney JT, eds. Anesthesia Equipment Simplified. New York, NY: McGraw-Hill Education, Inc; 2014.)

Misuse or malfunction of anesthesia gas delivery equipment can cause major morbidity or mortality. ® A routine inspection of anesthesia equipment before each use increases operator familiarity and confirms proper functioning. The
U.S. Food and Drug Administration (FDA) has made available a generic checkout procedure for anesthesia gas machines and breathing systems (Table 4-3). This procedure should be modified as necessary, depending on the specific equipment being used and the manufacturer's recommendations. Note that although the entire checkout does not need to be repeated between cases on the same day, the conscientious use of a checkout list is mandatory before each anesthetic procedure. A mandatory check-off procedure increases the likelihood of detecting anesthesia machine faults. Some anesthesia machines provide an automated system check that requires a variable amount of human intervention. These system checks may include nitrous oxide delivery (hypoxic mixture prevention), agent delivery, mechanical and manual ventilation, pipeline pressures, scavenging, breathing circuit compliance, and gas leakage.
TABLE 4-3 Anesthesia apparatus checkout recommendations.!
This checkout, or a reasonable equivalent, should be conducted before administration of anesthesia. These recommendations are valid only for an anesthesia system that conforms to current and relevant standards and includes an ascending bellows ventilator and at least the following monitors: capnograph, pulse oximeter, oxygen analyzer, respiratory volume monitor (spirometer), and breathing-system pressure monitor with highand low-pressure alarms. Users are encouraged to modify this guideline to accommodate differences in equipment design and variations in local clinical practice. Such local modifications should have appropriate peer review. Users should refer to the appropriate operator manuals for specific procedures and precautions.

- "1. Verify backup ventilation equipment is available and functioning

"2. Check O, cylinder supply
a. Open O, cylinder and verify at least half full (about 1000 psig).
b. Close cylinder
*3. Check central pipeline supplies; check that hoses are connected and pipeline gauges read about 50 psig.

*4. Check initial status of low-pressure system
a. Close flow control valves and turn vaporizers off.
b. Check fill level and tighten vaporizers' filler caps.
*5. Perform leak check of machine low-pressure system
a. Verify that the machine master switch and flow control valves are off.
. Attach suction bulb to common (fresh) gas outlet.
Verify bulb stays fully collapsed for at least 10 seconds.
Squeeze bulb repeatedly until fully collapsed. aos
e. Open one vaporizer at a time and repeat steps candd.
f. Remove suction bulb, and reconnect fresh gas hose.
*6. Turn on machine master switch and all other necessary electrical equipment.
*7. Test flowmeters
a. Adjust flow of all gases through their full range, checking for smooth operation of floats and undamaged flowtubes,
b. Attempt to create a hypoxic O,/N,O mixture and verify correct changes in flow and/or alarm.

"8. Adjust and check scavenging system
a. Ensure proper connections between the scavenging system and both APL (pop-off) valve and ventilator relief valve.
b. Adjust waste-gas vacuum (if possible).
c, Fully open APL valve and occlude Y-piece.
d. With minimum O, flow, allow scavenger reservoir bag to collapse completely and verify that absorber pressure gauge reads about zero.
e. Withthe oO, flush activated, allow scavenger reservoir bag to distend fully, and then verify that absorber pressure gauge reads <10cm 4,0.

13, Check, calibrate, and/or set alarm limits of all monitors: capnograph, pulse oximeter, O, analyzer, respiratoryvolume monitor (spirometer), pressure monitor with high and low airway-pressure alarms.
"9. Calibrate O, monitor
a. Ensure monitor reads 21% in room air.
b. Verify low-O, alarm is enabled and functioning.
c. Reinstall sensor in circuit and flush breathing system with O,.
d. Verify that monitor now reads greater than 90%.
10. Check initial status breathing system
a. Set selector switch to Bag mode.
b, Check that breathing circuit is complete, undamaged, and unobstructed.
c. Verify that CO, absorbent is adequate.
d. Install breathing-circuit accessory equipment (eg, humidifier, PEEP valve) to be used during the case.
11. Perform leak check of the breathing system
a. Setall gas flows to zero (or minimum).
b. Close APL (pop-off) valve and occlude Y-piece.
¢, Pressurize breathing system to about 30 cm H,O with O, flush.
d. Ensure that pressure remains fixed for at least 10 seconds.
e. Open APL (pop-off) valve and ensure that pressure decreases.

12. Test ventilation systems and unidirectional valves
a. Place asecond breathing bag on Y-piece.
. Set appropriate ventilator parameters for next patient.
¢, Switch to automatic-ventilation (ventilator) mode.
d. Turn ventilator on and fill bellows and breathing bag with O, flush.
SetO, flow to minimum, other gas flows to zero. ©
Verify that during inspiration bellows deliver appropriate tidal volume and that during expiration bellows fill completely. >
Set fresh gas flow to about 5 L min".
Verify that the ventilator bellows and simulated lungs fill and empty appropriately without sustained pressure at end expiration. ze
i.
Check for
proper action
of unidirectional
valves.
Jj. Exercise breathing circuit accessories to ensure proper function.
k. Turn ventilator off and switch to manual ventilation (Bag/APL) mode.
|. Ventilate manually and ensure inflation and deflation of artificial lungs and appropriate feel of system resistance and compliance.
m. Remove second breathing bag from Y-piece.

14. Check final status of machine
a. Vaporizers off
b. APL valve open
<. Selector switch to Bag mode
d. All flowmeters to zero (or minimum)
€. Patient suction level adequate
f, Breathing system ready to use
"Data from US Food and Drug Administration. US Department of Health and Human Services.
3APL, adjust pressure-limiting; PEEP, positive end-expiratory pressure,
"if an anesthesia provider uses the same machine in successive cases, these steps need not be repeated, or they can be abbreviated after the initial checkout.

After induction of general anesthesia and intubation of a 70-kg man for elective surgery, a standing bellows ventilator is set to deliver a tidal volume of 500 mL at a rate of 10 breaths/min. Within a few minutes, the anesthesiologist notices that the bellows fails to rise to the top of its clear plastic enclosure during expiration. Shortly thereafter, the disconnect alarm is triggered.
Why has the ventilator bellows fallen and the disconnect alarm sounded?
Fresh gas flow into the breathing circuit is inadequate to maintain the circuit volume required for positive-pressure ventilation. In a situation in which there is no fresh gas flow, the volume in the breathing circuit will slowly fall because of the constant uptake of oxygen by the patient (metabolic oxygen consumption) and absorption of expired CO,. An absence of fresh gas flow could be due to exhaustion of the hospital's oxygen supply (remember the function of the fail-safe valve) or failure to turn on the anesthesia machine's flow control valves. These possibilities can be ruled out by examining the oxygen Bourdon pressure gauge and the flowmeters. A more likely explanation is a gas leak that exceeds the rate of fresh gas flow. Leaks are particularly important in closed-circuit anesthesia.

When the rate of fresh gas inflow equals the rate of gas outflow, the circuit's volume will be maintained. Therefore, the size of the leak can be estimated by increasing fresh gas flows until there is no change in the height of the bellows from one expiration to the next. If the bellows collapse despite a high rate of fresh gas inflow, a complete circuit disconnection should be considered. The site of the disconnection must be determined immediately and repaired to prevent hypoxia and hypercapnia. A resuscitation bag must be immediately available and can be used to ventilate the patient if there is a delay in correcting the situation.
Where are the most likely locations of a breathing-circuit disconnection or leak?
Frank disconnections occur most frequently between the right-angle connector and the tracheal tube, whereas leaks are most commonly traced to the base plate of the CO, absorber. In the intubated patient, leaks often occur in the trachea around an uncuffed tracheal tube or an inadequately filled cuff. There are numerous potential sites of disconnection or leak within the anesthesia machine and the breathing circuit, however. Every addition to the breathing circuit, such as a humidifier, provides another potential location for a leak.

Leaks usually occur before the fresh gas outlet (ie, within the anesthesia machine) or after the fresh gas inlet (ie, within the breathing circuit). Large leaks within the anesthesia machine are less common and can be ruled out by a simple test. Pinching the tubing that connects the machine's fresh gas outlet to the circuit's fresh gas inlet creates a back pressure that obstructs the forward flow of fresh gas from the anesthesia machine. This is indicated by a drop in the height of the flowmeter floats. When the fresh gas tubing is released, the floats should briskly rebound and settle at their original height. If there is a substantial leak within the machine, obstructing the fresh gas tubing will not result in any back pressure, and the floats will not drop. A more sensitive test for detecting small leaks that occur before the fresh gas outlet involves attaching a suction bulb at the outlet as described in step 5 of Table 4—3. Correcting a leak within the machine usually requires removing it from service.
Leaks within a breathing circuit not connected to a patient are readily detected by closing the APL valve, occluding the Y-piece, and activating the oxygen flush until the circuit reaches a pressure of 20 to 30 cm H,O. A gradual decline in circuit pressure indicates a leak within the breathing circuit (Table 4—3, step 11).

Any connection within the breathing circuit is a potential site of a gas leak. A quick survey of the circuit may reveal a loosely attached breathing tube or a cracked oxygen analyzer adaptor. Less obvious causes include detachment of the tubing used by the disconnect alarm to monitor circuit pressures, an open APL valve, or an improperly adjusted scavenging unit. Leaks can usually be identified audibly or by applying a soap solution to suspect connections and looking for bubble formation.
Leaks within the anesthesia machine and breathing circuit are usually detectable if the machine and circuit have undergone an established checkout
procedure.
For example,
steps
5
and
11
of the
FDA
recommendations
(Table
4—3)
will reveal
most significant
leaks.
An extremely useful web site of simulations in anesthesia that includes virtual anesthesia machine simulators. http://www.simanest.org/
| Pressure unit conversions: 1 kiloPascal (kP) = kg/m -s? = 1000 N/m? = 0.01 bar = 0.1013 atmospheres = 0.145 psig = 10.2 cm HO = 7.5 mm Hg.

@ The tip of the central venous pressure catheter should not be allowed to migrate into the heart chambers.
Although the PA catheter can be used to guide goal-directed hemodynamic therapy to ensure organ perfusion in shock states, other less invasive methods to determine hemodynamic performance are available, including transpulmonary thermodilution CO measurements, pulse contour analyses of the arterial pressure waveform, and methods based on bioimpedance measurements across the chest.
@ Relative contraindications to pulmonary artery catheterization include left bundle-branch block (because of the concern about complete heart block) and conditions associated with greatly increased risk of arrhythmias.
@ Pulmonary artery pressure should be continuously monitored to detect an overwedged position indicative of catheter migration.
@ Accurate measurements of cardiac output depend on rapid and smooth injection, precisely known injectant temperature and volume, correct entry of the calibration factors for the specific type of pulmonary artery catheter into the cardiac output computer, and avoidance of measurements during electrocautery.
Vigilant perioperative monitoring of the cardiovascular system is one of the primary duties of anesthesia providers. The American Society of Anesthesiologists has established standards for basic anesthesia monitoring. This chapter focuses on the specific monitoring devices and techniques used to monitor cardiac function and circulation in healthy and nonhealthy patients alike.

The rhythmic contraction of the left ventricle, ejecting blood into the vascular system, results in pulsatile arterial pressures. The peak left ventricular endsystolic pressure (in the absence of aortic valve stenosis) approximates the systolic arterial blood pressure (SBP); the lowest arterial pressure during diastolic relaxation is the diastolic blood pressure (DBP). Pulse pressure is the difference between the systolic and diastolic pressures. The time-weighted average of arterial pressures during a pulse cycle is the mean arterial pressure (MAP). MAP can be estimated by application of the following formula:
Arterial blood pressure varies depending upon where within the vasculature the pressure is measured. As a pulse moves peripherally through the arterial tree, wave reflection distorts the pressure waveform, leading to an exaggeration of systolic and pulse pressures (Figure 5—1). For example, radial artery systolic pressure is usually greater than aortic systolic pressure because of its more distal location. In contrast, radial artery systolic pressures often underestimate more "central" pressures following hypothermic cardiopulmonary bypass because of changes in hand vascular resistance. Vasodilating drugs may accentuate this discrepancy. The level of the sampling site relative to the heart affects the measurement of blood pressure because of the effect of gravity (Figure 5—2). In patients with severe peripheral vascular disease, there may be significant differences in blood pressure measurements among the extremities. The greater value should be used in these patients.
FIGURE 5-1 Changes in configuration as a waveform moves peripherally. (Reproduced with permission from Lake CL, Hines RL, Blitt CD. Clinical Monitoring: Practical Applications in Anesthesia and Critical Care Medicine. Philadelphia, PA: WB Saunders; 2001.)
FIGURE 5-2 The difference in blood pressure (mm Hg) at two different sites of measurement equals the height of an interposed column of water (cm H,0) multiplied by a conversion factor (1 cm H,O = 0.74 mm Hg).
Because noninvasive (palpation, Doppler, auscultation, oscillometry, plethysmography) and invasive (arterial cannulation) methods of blood pressure determination differ greatly, they are discussed separately.

The use of any anesthetic is an indication for arterial blood pressure measurement. The techniques and frequency of pressure determination will depend on the patient's condition and the type of surgical procedure. A noninvasive blood pressure measurement every 3 to 5 min is adequate in most cases.

Although some method of blood pressure measurement is mandatory, techniques that rely on a blood pressure cuff are best avoided in extremities with vascular abnormalities (eg, dialysis shunts) or with intravenous lines. It rarely may prove impossible to monitor blood pressure in patients (eg, those who have burns) who have no accessible site from which the blood pressure can be safely recorded.

SBP can be determined by (1) locating a palpable peripheral pulse, (2) inflating a blood pressure cuff proximal to the pulse until flow is occluded, (3) releasing cuff pressure by 2 or 3 mm Hg per heartbeat, and (4) measuring the cuff pressure at which pulsations are again palpable. This method tends to underestimate systolic pressure, however, because of the insensitivity of touch and the delay between flow under the cuff and distal pulsations. Palpation does not provide a diastolic pressure or MAP. The equipment required is simple and inexpensive.

When a Doppler probe is substituted for the anesthesiologist's finger, arterial blood pressure measurement becomes sensitive enough to be useful in obese patients, pediatric patients, and patients in shock (Figure 5—3). The Doppler effect is the shift in the frequency of sound waves when their source moves relative to the observer. For example, the pitch of a train's whistle increases as a train approaches and decreases as it departs. Similarly, the reflection of sound waves off of a moving object causes a frequency shift. A Doppler probe transmits an ultrasonic signal that is reflected by underlying tissue. As red blood cells move through an artery, a Doppler frequency shift will be detected by the probe. The difference between transmitted and received frequency causes the characteristic swishing sound, which indicates blood flow. Because air reflects ultrasound, a coupling gel (but not corrosive electrode jelly) is applied between the probe and the skin. Positioning the probe directly above an artery is crucial, since the beam must pass through the vessel wall. Interference from probe movement or electrocautery is an annoying distraction. Note that only systolic pressures can be reliably determined with the Doppler technique.
FIGURE 5-3 A Doppler probe secured over the radial artery will sense red blood cell movement as long as the blood pressure cuff is below systolic pressure. (Reproduced with permission from Parks Medical Electronics.)
A variation of Doppler technology uses a piezoelectric crystal to detect lateral arterial wall movement to the intermittent opening and closing of vessels between systolic and diastolic pressure. This instrument thus detects both systolic and diastolic pressures. The Doppler effect is routinely employed by perioperative echocardiographers to discern both the directionality and velocity of both blood flow within the heart and the movement of the heart's muscle tissue (tissue Doppler).

Inflation of a blood pressure cuff to a pressure between systolic and diastolic pressures will partially collapse an underlying artery, producing turbulent flow and the characteristic Korotkoff sounds. These sounds are audible through a stethoscope placed under—or just distal to—the distal third of the blood pressure cuff. The clinician measures pressure with an aneroid or mercury manometer. Occasionally, Korotkoff sounds cannot be heard through part of the range from systolic to diastolic pressure. This auscultatory gap is most common in hypertensive patients and can lead to an inaccurate diastolic pressure measurement. Korotkoff sounds are often difficult to auscultate in noisy patient care environments and during episodes of hypotension or marked peripheral vasoconstriction. In these situations, the subsonic frequencies associated with the sounds can be detected by a microphone and amplified to indicate systolic and diastolic pressures. Motion artifact and electrocautery interference limit the usefulness of this method.

Arterial pulsations cause oscillations in cuff pressure. These oscillations are small if the cuff is inflated above systolic pressure. When the cuff pressure decreases to systolic pressure, the pulsations are transmitted to the entire cuff, and the oscillations markedly increase. Maximal oscillation occurs at the MAP, after which oscillations decrease. Because some oscillations are present above and below arterial blood pressure, a mercury or aneroid manometer provides an inaccurate and unreliable measurement. Automated blood pressure monitors electronically measure the pressures at which the oscillation amplitudes change (Figure 5—4). A microprocessor derives systolic, mean, and diastolic pressures using an algorithm. Machines that require identical consecutive pulse waves for measurement confirmation may be unreliable during arrhythmias (eg, atrial fibrillation). Oscillometric monitors should not be used on patients on cardiopulmonary bypass. Nonetheless, the speed, accuracy, and versatility of oscillometric devices have greatly improved, and they have become the preferred noninvasive blood pressure monitors in the United States and worldwide.
FIGURE 5-4 Oscillometric determination of blood pressure.

Arterial tonometry measures beat-to-beat arterial blood pressure by sensing the pressure required to partially flatten a superficial artery that is supported by a bony structure (eg, radial artery). A tonometer consisting of several independent pressure transducers is applied to the skin overlying the artery (Figure 5—5). The contact stress between the transducer directly over the artery and the skin reflects intraluminal pressure. Continuous pulse recordings produce a tracing very similar to an invasive arterial blood pressure waveform. Limitations to this technology include sensitivity to movement artifact and the need for frequent calibration.

FIGURE 5-5 Tonometry is a method of continuous (beat-to-beat) arterial blood pressure determination. The sensors must be positioned directly over the artery.

Adequate oxygen delivery to vital organs must be maintained during anesthesia. Unfortunately, instruments to monitor specific organ perfusion and oxygenation are complex, expensive, and often unreliable, and, for that reason, an adequate arterial blood pressure is assumed to predict adequate organ blood flow. However, flow also depends on vascular resistance:
Even if the pressure is high, when the resistance is also high, flow can be low. Thus, arterial blood pressure should be viewed as an indicator—but not a measure—of organ perfusion.
The accuracy of any method of blood pressure measurement that involves a blood pressure cuff depends on proper cuff size (Figure 5—6). The cuff's bladder should extend at least halfway around the extremity, and the width of the cuff should be 20% to 50% greater than the diameter of the extremity.
FIGURE 5-6 Blood pressure cuff width influences the pressure readings. The narrowest cuff (A) will require more pressure, and the widest cuff (C) less pressure, to occlude the brachial artery for determination of systolic pressure. Too narrow a cuff may produce a large overestimation of systolic pressure. Whereas the wider cuff may underestimate the systolic pressure, the error with a cuff 20% too wide is not as significant as the error with a cuff 20% too narrow. (Reproduced with permission from Gravenstein JS, Paulus DA. Clinical Monitoring Practice. 2nd ed. Philadelphia, PA: Lippincott Williams & Wilkins; 1987.)
Automated blood pressure monitors, using one or a combination of the methods described above, are frequently used in anesthesiology. A self-contained air pump inflates the cuff at set intervals. Incorrect placement or too-frequent cycling of these automated devices has resulted in nerve palsies and extensive extravasation of intravenously administered fluids. In case of equipment failure, an alternative method of blood pressure determination must be immediately available.
2. Invasive Arterial Blood Pressure Monitoring

Indications for invasive arterial blood pressure monitoring by catheterization of an artery include induced current or anticipated hypotension or wide blood pressure deviations, end-organ disease necessitating precise beat-to-beat blood pressure regulation, and the need for multiple arterial blood gas or other blood analyses.

If possible, catheterization should be avoided in smaller end arteries lacking collateral blood flow or in extremities where there is a suspicion of preexisting vascular insufficiency.

Several arteries are available for percutaneous catheterization.

1. The radial artery is commonly cannulated because of its superficial location and substantial collateral flow (in most patients the ulnar artery is larger than the radial and there are connections between the two via the palmar arches). Five percent of patients have incomplete palmar arches and lack adequate collateral blood flow. Allen's test is a simple, but not reliable, method for assessing the safety of radial artery cannulation. In this test, the patient exsanguinates his or her hand by making a fist. While the operator occludes the radial and ulnar arteries with fingertip pressure, the patient relaxes the blanched hand. Collateral flow through the palmar arterial arch is confirmed by flushing of the thumb within 5 s after pressure on the ulnar artery is released. Delayed return of normal color (5—10 s) indicates an equivocal test or insufficient collateral circulation (>10 s). The Allen's test is of such questionable utility that many practitioners routinely avoid it. Alternatively, blood flow distal to the radial artery occlusion can be detected by palpation, Doppler probe, plethysmography, or pulse oximetry. Unlike Allen's test, these methods of determining the adequacy of collateral circulation do not require patient cooperation.
2. Ulnar artery catheterization is usually more difficult than radial catheterization because of the ulnar artery's deeper and more tortuous course. Because of the risk of compromising blood flow to the hand, ulnar catheterization would not normally be considered if the ipsilateral radial artery has been punctured but unsuccessfully cannulated.
3. The brachial artery is large and easily identifiable in the antecubital fossa. Its proximity to the aorta provides less waveform distortion. However, being near the elbow predisposes brachial artery catheters to kinking.
4. The femoral artery is prone to atheroma formation and pseudoaneurysm, but often provides excellent access. The femoral site has been associated with an increased incidence of infectious complications and arterial thrombosis. Aseptic necrosis of the head of the femur is a rare, but tragic, complication of femoral artery cannulation in children.
5. The dorsalis pedis and posterior tibial arteries are some distance from the aorta and therefore have the most distorted waveforms.

6. The axillary artery is surrounded by the axillary plexus, and nerve damage can result from a hematoma or traumatic cannulation. Air or thrombi can quickly gain access to the cerebral circulation during vigorous retrograde flushing of axillary artery catheters.

One technique of radial artery cannulation is illustrated in Figure 5—7. Supination and extension of the wrist optimally position the radial artery. The pressure—tubing—transducer system should be nearby and already flushed with saline to ensure easy and quick connection after cannulation. The radial pulse is palpated, and the artery's course is determined by lightly pressing the tips of the index and middle fingers of the nondominant hand over the area of maximal impulse or by use of ultrasound. After skin cleansing with chlorhexidine (or other prep solution), and using aseptic technique 1% lidocaine is infiltrated in the skin of awake patients, directly above the artery, with a small gauge needle. A larger 18-gauge needle can then be used as a skin punch, facilitating entry of a 20or 22-gauge catheter over a needle through the skin at a 45° angle, directing it toward the point of palpation. Upon blood flashback, a guidewire may be advanced through the catheter into the artery and the catheter advanced over the guidewire. Alternatively, the needle is lowered to a 30° angle and advanced another 1 to 2 mm to make certain that the tip of the catheter is well into the vessel lumen. The catheter is advanced off the needle into the arterial lumen, after which the needle is withdrawn. Applying firm pressure over the artery proximal to the catheter insertion site prevents blood from spurting from the catheter while the tubing is connected. Waterproof tape or suture can be used to hold the catheter in place, and a sterile dressing should be applied over the insertion site.

FIGURE 5-7 Cannulation of the radial artery. A: Proper positioning and palpation of the artery are crucial. After skin preparation, local anesthetic is infiltrated with a 25-gauge needle. B: A 20or 22-gauge catheter is advanced through the skin at a 45° angle. C: Flashback of blood signals entry into the artery, and the catheter—needle assembly is lowered to a 30° angle and advanced 1-2 mm to ensure an intraluminal catheter position. D: The catheter is advanced over the needle, which is withdrawn. E: Proximal pressure with middle and ring fingers prevents blood loss, while the arterial tubing Luer-lock connector is secured to the intraarterial catheter.

Complications of intraarterial monitoring include hematoma, bleeding (particularly with catheter tubing disconnections), vasospasm, arterial thrombosis, embolization of air bubbles or thrombi, pseudoaneurysm formation, necrosis of skin overlying the catheter, nerve damage, infection, necrosis of extremities or digits, and unintentional intraarterial drug injection. Factors associated with an increased rate of complications include prolonged cannulation, hyperlipidemia, repeated insertion attempts, female gender, extracorporeal circulation, the use of larger catheters in smaller vessels, and the use of vasopressors. The risks are reduced with good aseptic techniques, when the ratio of catheter to artery size is small, when saline is continuously infused through the catheter at a rate of 2 to 3 mL/h, and when flushing of the catheter is limited. Adequacy of perfusion can be continually monitored during radial artery cannulation by placing a pulse oximeter on the thumb or index finger.

Because intraarterial cannulation allows continuous beat-to-beat blood pressure measurement, it is considered the optimal blood pressure monitoring technique. The quality of the transduced waveform, however, depends on the dynamic characteristics of the catheter-tubing—transducer system. False readings can lead to inappropriate therapeutic interventions.
A complex waveform, such as an arterial pulse wave, can be expressed as a summation of simple harmonic waves (according to the Fourier theorem). For accurate measurement of pressure, the catheter-tubing—transducer system must be capable of responding adequately to the highest frequency of the arterial waveform (Figure 5-8). Stated another way, the natural frequency of the measuring system must exceed the natural frequency of the arterial pulse (approximately 16-24 Hz).
FIGURE 5-8 An original waveform overlays a four-harmonic reconstruction (left) and an eight-harmonic reconstruction (right). Note that the higher harmonic plot more closely resembles the original waveform. (Reproduced with permission from Saidman LS, Smith WT. Monitoring in Anesthesia. Philadelphia, PA: Butterworth-
Digital readouts of systolic and diastolic pressures are a running average of the highest and lowest measurements within a certain time interval. Because motion or cautery artifacts can result in some very misleading numbers, the arterial waveform should always be monitored. The shape of the arterial wave provides clues to several hemodynamic variables. The rate of upstroke indicates contractility, the rate of downstroke indicates peripheral vascular resistance, and exaggerated variations in size during the respiratory cycle suggest hypovolemia. MAP is calculated by integrating the area under the pressure curve.
Intraarterial catheters also provide access for intermittent arterial blood gas sampling and analysis. The development of fiberoptic sensors that can be inserted through a 20-gauge arterial catheter enables continuous blood gas monitoring. Unfortunately, these sensors are quite expensive and are often inaccurate, so they are rarely used. Analysis of the arterial pressure waveform allows for estimation of cardiac output (CO) and other hemodynamic parameters. These devices are discussed in the section on CO monitoring.

All patients should have intraoperative monitoring of their electrocardiogram (ECG). There are no contraindications.

Lead selection determines the diagnostic sensitivity of the ECG. ECG leads are positioned on the chest and extremities to provide different perspectives of the electrical potentials generated by the heart. At the end of diastole, the atria contract, which provides the atrial contribution to CO, generating the "P" wave. Following atrial contraction, the ventricle is loaded awaiting systole. The QRS complex begins the electrical activity of systole following the 120 to 200 msec atrioventricular (AV) nodal delay. Depolarization of the ventricle proceeds from the AV node through the interventricular system via the His—Purkinje fibers. The normal QRS lasts approximately 120 msec, which can be prolonged in patients with cardiomyopathies and heart failure. The T wave represents repolarization as the heart prepares to contract again. Prolongation of the QT interval secondary to electrolyte imbalances or drug effects can potentially lead to life-threatening arrhythmias (torsades de pointes).
The electrical axis of lead II is approximately 60° from the right arm to the left leg, which is parallel to the electrical axis of the atria, resulting in the largest P-wave voltages of any surface lead. This orientation enhances the diagnosis of arrhythmias and the detection of inferior wall ischemia. Lead Vz lies over the fifth intercostal space at the anterior axillary line; this position is a good compromise for detecting anterior and lateral wall ischemia. A true V; lead is possible only on operating room ECGs with at least five lead wires, but a modified V; can be monitored by rearranging the standard three-limb lead placement (Figure 5—9). Ideally, because each lead provides unique information, leads II and V; should be monitored simultaneously. If only a single-channel machine is available, the preferred lead for monitoring depends on the location of any prior infarction or ischemia and whether arrhythmia or ischemia appears to be the greater concern.

FIGURE 5-9 Rearranged three-limb lead placement. Anterior and lateral ischemia can be detected by placing the left arm lead (LA) at the V; position. When lead I is selected on the monitor, a modified V; lead (CS;) is displayed. Lead II allows detection of arrhythmias and inferior wall ischemia. RA, right arm; LL, left leg.
Electrodes are placed on the patient's body to monitor the ECG (Figure 5— 10). Conductive gel lowers the skin's electrical resistance, which can be further decreased by cleansing the site with alcohol. Needle electrodes are used only if the disks are unsuitable (eg, with an extensively burned patient).
FIGURE 5-10 A cross-sectional view of a silver chloride electrode.

The EGG is a recording of the electrical potentials generated by myocardial cells. Its routine use allows arrhythmias, myocardial ischemia, conduction abnormalities, pacemaker malfunction, and electrolyte disturbances to be detected (Figure 5-11). Because of the small voltage potentials being measured, artifacts remain a major problem. Patient or lead-wire movement, use of electrocautery, 60-Hz interference from nearby alternating current devices, and faulty electrodes can simulate arrhythmias. Monitoring filters incorporated into the amplifier to reduce "motion" artifacts will lead to distortion of the ST segment and may impede the diagnosis of ischemia. Digital readouts of the heart rate (HR) may be misleading because of monitor misinterpretation of artifacts or large T waves—often seen in pediatric patients—as QRS complexes.
FIGURE 5-11 Common ECG findings during cardiac surgery. (Reproduced with permission from Wasnick J, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography. New York, NY: McGraw-Hill; 2011.)
Depending on equipment availability, a preinduction rhythm strip can be printed or frozen on the monitor's screen to compare with intraoperative tracings. To interpret ST-segment changes properly, the ECG must be standardized so that a 1-mV signal results in a deflection of 10 mm on a standard strip monitor. Newer units continuously analyze ST segments for early detection of myocardial ischemia. Automated ST-segment analysis increases the sensitivity of ischemia detection, does not require additional physician skill or vigilance, and may help diagnose intraoperative myocardial ischemia.
Commonly accepted criteria for diagnosing myocardial ischemia require that the ECG be recorded in "diagnostic mode" and include a flat or downsloping STsegment depression exceeding 1 mm, 80 msec after the J point (the end of the

QRS complex), particularly in conjunction with T-wave inversion. ST-segment elevation with peaked T waves can also represent ischemia. Wolff—Parkinson— White syndrome, bundle-branch blocks, extrinsic pacemaker capture, and digoxin therapy may preclude the use of ST-segment information. The audible beep associated with each QRS complex should be loud enough to detect rate and rhythm changes when the anesthesiologist's visual attention is directed elsewhere. Some ECGs are capable of storing aberrant QRS complexes for further analysis, and some can even interpret and diagnose arrhythmias. The interference caused by electrocautery units limits the usefulness of automated arrhythmia analysis in the operating room.

Central venous catheterization is indicated for monitoring central venous pressure (CVP), administration of fluid to treat hypovolemia and shock, infusion of caustic drugs and total parenteral nutrition, aspiration of air emboli, insertion of transcutaneous pacing leads, and gaining venous access in patients with poor peripheral veins. With specialized catheters, central venous catheterization can be used for continuous monitoring of central venous oxygen saturation (Scvo,). Scvo, is used as a measure to assess adequacy of oxygen delivery. Decreased ScvO, (normal >65%) alerts to the possibility of inadequate delivery of oxygen to the tissues (eg, low cardiac output, low hemoglobin, low arterial oxygen saturation, increased oxygen consumption). An elevated Scvo, (>80%) may indicate arterial/venous shunting or impaired cellular oxygen utilization (eg, cyanide poisoning).

Relative contraindications include tumors, clots, or tricuspid valve vegetations that could be dislodged or embolized during cannulation. Other contraindications relate to the cannulation site. For example, subclavian vein cannulation is relatively contraindicated in patients who are receiving anticoagulants (due to the inability to provide direct compression in the event of an accidental arterial puncture). Based on tradition but not science, some clinicians avoid internal jugular vein cannulation on the side of a previous carotid endarterectomy due to concerns about unintentional carotid artery puncture. The presence of other central catheters or pacemaker leads may reduce the number of sites available for central line placement.

FIGURE 5-12 The subclavian and internal jugular veins are both used for central access perioperatively with the sternal notch and ipsilateral nipple in the direction of needle passage for each, respectively. (Reproduced with permission from Wasnick J, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography. New York, NY: McGraw-Hill; 2011.)

The following scenario describes the placement of an internal jugular venous line. The patient is placed in the Trendelenburg position to decrease the risk of air embolism and to distend the internal jugular (or subclavian) vein. Central venous Catheterization requires full aseptic technique, including hand scrub, sterile gloves, gown, mask, hat, bactericidal skin preparation (alcohol-based solutions are preferred), and sterile drapes. The two heads of the sternocleidomastoid muscle and the clavicle form the three sides of a triangle (Figure 5-13A). A 25-gauge needle is used to infiltrate the apex of the triangle with local anesthetic. The internal jugular vein can be located using ultrasound, and we strongly recommend that it be used whenever possible (Figure 5-14). Many institutions mandate the use of ultrasound whenever internal jugular vein cannulation is performed. Alternatively, the vein may be located by advancing the 25-gauge needle—or a 23-gauge needle in heavier patients—along the medial border of the lateral head of the sternocleidomastoid, toward the ipsilateral nipple, at an angle of 30° to the skin, aiming just lateral to the carotid artery pulse. Aspiration of venous blood confirms the vein's location. It is essential that the vein (and not the artery) be cannulated. Cannulation of the carotid artery can lead to hematoma, stroke, airway compromise, and possibly death. An 18-gauge thin-wall needle or an 18-gauge catheter over needle is advanced along the same path as the locator needle (Figure 5—13B), and, with the latter apparatus, the needle is removed from the catheter once the catheter has been advanced into the vein. After free blood flow is achieved we usually confirm central venous versus arterial pressure (using intravenous extension tubing) before introducing a guidewire. (Figure 5-13C). We recommend that correct placement of the guidewire be confirmed using ultrasound. The needle (or catheter) is removed, and a dilator is advanced over the wire. The catheter is prepared for insertion by flushing all ports with saline, and all distal ports are "capped" or clamped, except the one through

which the wire must pass. Next, the dilator is removed, and the final catheter is advanced over the wire (Figure 5-13D). Do not lose control of the proximal tip of the guidewire. The guidewire is removed, with a thumb placed over the catheter hub to prevent aspiration of air until the intravenous catheter tubing is connected to it. The catheter is then secured, and a sterile dressing is applied. Correct location is confirmed with a chest ® radiograph. The tip of the catheter should not be allowed to migrate into the heart chambers. Fluid-administration sets should be changed frequently, per your medical center protocol.
FIGURE 5-14 A: Probe position for ultrasound of the large internal jugular vein with deeper carotid artery and B: corresponding ultrasound image. CA, carotid artery; IJ, internal jugular vein. (Reproduced with permission from Tintinalli JE, Stapczynski J, Ma OJ, et al. Tintinalli's Emergency Medicine: A Comprehensive Study Guide. 7th ed. New York, NY: McGraw-Hill; 2011.)
As mentioned, the likelihood of accidental placement of the vein dilator or catheter into the carotid artery can be decreased by transducing the vessel's pressure from the introducer needle (or catheter, if a catheter over needle has been used) before passing the wire (most simply accomplished by using a sterile intravenous extension tubing as a manometer). Alternatively, one may compare the color or Pao, of the blood with that of an arterial sample. Blood color and pulsatility can be misleading or inconclusive, and more than one confirmation method should be used. In cases where either surface ultrasound or transesophageal echocardiography (TEE) are used, the guidewire can be seen in the jugular vein or right atrium, confirming venous entry (Figure 5—15).
FIGURE 5-15 A wire is seen on this transesophageal echocardiography image of the right atrium.

The risks of central venous cannulation include line infection, bloodstream infection, air or thrombus embolism, arrhythmias (indicating that the catheter tip is in the right atrium or ventricle), hematoma, pneumothorax, hemothorax, hydrothorax, chylothorax, cardiac perforation, cardiac tamponade, trauma to nearby nerves and arteries, and thrombosis.

Normal cardiac function requires adequate ventricular filling. CVP approximates right atrial pressure. Ventricular volumes are related to pressures through compliance. Highly compliant ventricles accommodate volume with minimal changes in pressure. Noncompliant systems have larger swings in pressure with less volume changes. Consequently, any one CVP measurement will reveal only limited information about ventricular volumes and filling. Although a very low CVP may indicate a volume-depleted patient, a moderate to high pressure reading may reflect volume overload, poor ventricular compliance, or both. Changes in CVP associated with volume administration coupled with other measures of hemodynamic performance (eg, stroke volume, cardiac output, blood pressure, HR, urine output) may be a better indicator of the patient's volume responsiveness.
The shape of the central venous waveform corresponds to the events of cardiac contraction (Figure 5-16): a waves from atrial contraction are absent in atrial fibrillation and are exaggerated in junctional rhythms ("cannon" a waves); c waves are due to tricuspid valve elevation during early ventricular contraction; v waves reflect venous return against a closed tricuspid valve; and the x and y descents are probably caused by the downward displacement of the tricuspid valve during systole and tricuspid valve opening during diastole.
FIGURE 5-16 The upward waves (a, c, v) and the downward descents (x, y) of a central venous tracing in relation to the electrocardiogram (ECG).

The pulmonary artery (PA) catheter (or Swan-Ganz catheter) was introduced into routine practice in operating rooms and in coronary and critical care units in the 1970s. It quickly became common for sicker patients undergoing major surgery to be managed with PA catheterization. The catheter provides measurements of both CO and PA occlusion pressures and was used to guide hemodynamic therapy, especially when patients became unstable. Determination of the PA occlusion or wedge pressure permitted (in the absence of mitral stenosis) an estimation of the left ventricular end-diastolic pressure (LVEDP), and, depending upon ventricular compliance, an estimate of ventricular volume. Through its ability to perform measurements of CO, the patient's stroke volume (SV) was also determined.

Consequently, hemodynamic monitoring with the PA catheter attempted to discern why a patient was unstable so that therapy could be directed at the underlying problem.
If the SVR is diminished, such as in states of vasodilatory shock (sepsis), the SV may increase. Conversely, a reduction in SV may be secondary to poor cardiac performance or hypovolemia. Determination of the "wedge" or pulmonary capillary occlusion pressure (PCOP) by inflating the catheter balloon estimates the LVEDP. A decreased SV in the setting of a low PCOP/LVEDP indicates hypovolemia and the need for volume administration. A "full" heart, reflected by a high PCOP/LVEDP and low SV, indicates the need for a positive inotropic drug. Conversely, a normal or increased SV in the setting of hypotension could be treated with the administration of vasoconstrictor drugs to restore SVR in a vasodilated patient.
Although patients can present concurrently with hypovolemia, sepsis, and heart failure, this basic treatment approach and the use of the PA catheter to guide therapy became more or less synonymous with perioperative intensive care and cardiac anesthesia. However, several large observational studies have shown that patients managed with PA catheters had worse outcomes than similar patients who were managed without PA catheters. Other studies seem to indicate that although PA catheter-guided patient management may do no harm, it @ offers no specific benefits. Although the PA catheter can be used to guide goaldirected hemodynamic therapy to ensure organ perfusion in shock states, other less invasive methods to determine hemodynamic performance are available, including transpulmonary thermodilution CO measurements, pulse contour analyses of the arterial pressure waveform, and methods based on bioimpedance measurements across the chest. All these methods permit calculation of the SV as a guide for hemodynamic management. Moreover, right atrial blood oxygen saturation, as opposed to mixed venous saturation (normal is 75%), can be used as an alternative measure to discern tissue oxygen extraction and the adequacy of tissue oxygen delivery.

Despite numerous reports of its questionable utility and the increasing number of alternative methods to determine hemodynamic parameters, the PA catheter is still employed perioperatively more often in the United States than elsewhere. Although echocardiography can readily determine if the heart is full, compressed, contracting, or empty, a trained individual is required to obtain and interpret the images. Alternative hemodynamic monitors have gained wide acceptance in Europe and are increasingly used in the United States, further decreasing the use of PA catheters.
PA catheterization should be considered whenever cardiac index, preload, volume status, or the degree of mixed venous blood oxygenation need to be known. These measurements might prove particularly important in surgical patients at greatest risk for hemodynamic instability or during surgical procedures associated with a greatly increased incidence of hemodynamic complications (eg, thoracic aortic aneurysm repair).

® Relative contraindications to pulmonary artery catheterization include left bundle-branch block (because of the concern about complete heart block) and conditions associated with a greatly increased risk of arrhythmias. A catheter with pacing capability is better suited to these situations. A PA catheter may serve as a nidus of infection in bacteremic patients or thrombus formation in patients prone to hypercoagulation.

Although various PA catheters are available, the most popular design integrates five lumens into a 7.5 FR catheter, 110-cm long, with a polyvinylchloride body (Figure 5-17). The lumens house the following: wiring to connect the thermistor near the catheter tip to a thermodilution CO computer; an air channel for inflation of the balloon; a proximal port 30 cm from the tip for infusions, CO injections, and measurements of right atrial pressures; a ventricular port at 20 cm for infusion of drugs; and a distal port for aspiration of mixed venous blood samples and measurements of PA pressure.
FIGURE 5-17 Balloon-tipped pulmonary artery flotation catheter (Swan—Ganz catheter). RA, right atrium.
Insertion of a PA catheter requires central venous access, which can be accomplished using Seldinger's technique, described earlier. Instead of a central venous catheter, a dilator and sheath are threaded over the guidewire. The sheath lumen accommodates the PA catheter after removal of the dilator and guidewire (Figure 5-18).
FIGURE 5-18 A percutaneous introducer consisting of a vessel dilator and sheath is passed over the guidewire.
Prior to insertion, the PA catheter is checked by inflating and deflating its balloon and filling all three lumens with intravenous fluid. The distal port is connected to a transducer that is zeroed to the patient's midaxillary line.

The PA catheter is advanced through the introducer and into the internal jugular vein. At approximately 15 cm, the distal tip should enter the right atrium, and a central venous tracing that varies with respiration confirms an intrathoracic position. The balloon is then inflated with air according to the manufacturer's recommendations (usually 1.5 mL) to protect the endocardium from the catheter tip and to allow flow through the right ventricle to direct the catheter forward. The balloon is always deflated during withdrawal. During catheter advancement, the ECG should be monitored for arrhythmias. Transient ectopy from irritation of the right ventricle by the balloon and catheter tip is common and rarely requires treatment. A sudden increase in the systolic pressure on the distal tracing indicates a right ventricular location of the catheter tip (Figure 5-19). Entry into the pulmonary artery normally occurs by 35 to 45 cm and is heralded by a sudden increase in diastolic pressure.

FIGURE 5-19 Although their utility is increasingly questioned, pulmonary artery (PA) catheters continue to be a part of perioperative management of the cardiac surgery patient. Following placement of a sheath introducer in the central circulation (panels 1 and 2), the PA catheter is floated. Central line placement should always be completed using rigorous sterile technique, full body draping, and only after multiple, redundant confirmations of the correct localization of the venous circulation. Pressure guidance is used to ascertain the localization of the PA catheter in the venous circulation and the heart. Upon entry into the right atrium (RA; panels 3 and 4), the central venous pressure tracing is noted. Passing through the tricuspid valve (panels 5 and 6) right ventricular pressures are detected. At 35 to 50 cm depending upon patient size, the catheter will pass from the right ventricle (RV) through the pulmonic valve into the pulmonary artery (panels 7 and 8). This is noted by the measurement of diastolic pressure once the pulmonic valve is passed. Lastly, when indicated the balloon-tipped catheter will wedge or occlude a pulmonary artery branch (panels 9, 10, and 11). When this occurs, the pulmonary artery pressure equilibrates with that of the left atrium (LA) which, barring any mitral valve pathology, should be a reflection of left ventricular end-diastolic pressure. IVC, inferior vena cava; SVC, superior vena Cava. (Reproduced with permission from Soni N. Practical Procedures in Anaesthesia and Intensive Care. Philadelphia, PA: Butterworth Heinemann; 1994.)

To prevent catheter knotting, the balloon should be deflated and the catheter withdrawn if pressure changes do not occur at the expected distances. Occasionally, the insertion may require fluoroscopy or TEE for guidance. After the catheter tip enters the PA, minimal additional advancement results in a pulmonary artery occlusion pressure (PAOP) waveform. The PA tracing should reappear when the balloon is deflated. Wedging before maximal balloon inflation signals an overwedged position, and the catheter should be slightly withdrawn (with the balloon down, of course). Because PA rupture from balloon overinflation may cause hemorrhage and mortality, wedge readings should be obtained infrequently. @ PA pressure should be continuously monitored to detect an overwedged position indicative of catheter migration. Correct PA catheter position is confirmed by a chest radiograph. The numerous complications of PA catheterization include all those associated with central venous cannulation plus endocarditis, thrombogenesis, pulmonary infarction, PA rupture, and hemorrhage (particularly in patients taking anticoagulants, elderly or female patients, or patients with pulmonary hypertension), catheter knotting, arrhythmias, conduction abnormalities, and pulmonary valvular damage (Table 5—1). Even trace hemoptysis should not be ignored, as it may herald PA rupture. If the latter is suspected, prompt placement of a double-lumen tracheal tube may maintain adequate oxygenation by the unaffected lung. The risk of complications increases with the duration of catheterization, which usually should not exceed 72 h.
TABLE 5-1 Reported incidence of adverse effects of pulmonary artery catheterization.!

Central venous access, Reported | Incidence (%) = . Arterial puncture, Reported | Incidence (%) = 0.1-13. Bleeding at cut-down site, Reported | Incidence (%) = 5.3. Postoperative neuropathy, Reported | Incidence (%) = 0.3-1.1. Pneumothorax, Reported | Incidence (%) = 0.3-4.5. Air embolism, Reported | Incidence (%) = 0.5. Catheterization, Reported | Incidence (%) = . Minor arrhythmias?, Reported | Incidence (%) = 4.7-68.9. Severe arrhythmias (ventricular tachycardia or fibrillation)?, Reported | Incidence (%) = 0.3-62.7 17. Minor increase in tricuspid, Reported | Incidence (%) = 0.1-4.3. regurgitation, Reported | Incidence (%) = 0-8.5. Right bundle-branch block?, Reported | Incidence (%) = . Catheter residence, Reported | Incidence (%) = . Pulmonary artery rupture?, Reported | Incidence (%) = 0.03-1.5. Positive catheter tip cultures, Reported | Incidence (%) = 14-348. Catheter-related sepsis, Reported | Incidence (%) = 0.7-11.4. Thrombophlebitis, Reported | Incidence (%) = 6.5. Venous thrombosis, Reported | Incidence (%) = 0.5-66.7. Pulmonary infarction?, Reported | Incidence (%) = 0.1-5.6. Mural thrombus, Reported | Incidence (%) = 28-61. Valvular/endocardial vegetations or, Reported | Incidence (%) = 2.2-100. endocarditis?, Reported | Incidence (%) = 0.02-1.5. Deaths?, Reported | Incidence (%) = 
'Adapted with permission from Practice guidelines for pulmonary artery catheterization: An updated report by the American Society of Anesthesiologists Task Force on pulmonary artery catheterization. Anesthesiology. 2003 Oct;99(4):988-1014.
'Complications thought to be more common (or exclusively associated) with pulmonary artery catheterization than with central venous catheterization. LBBB, left bundle-branch block.

The introduction of PA catheters into the operating room revolutionized the intraoperative management of critically ill patients. PA catheters allow more precise estimation of left ventricular preload than either CVP or physical examination (but not as precise as TEE), as well as the sampling of mixed venous blood. Catheters with self-contained thermistors (discussed later in this chapter) can be used to measure CO, from which a multitude of hemodynamic values can be derived (Table 5—2). Some catheter designs incorporate electrodes that allow intracavitary ECG recording and pacing. Optional fiberoptic bundles allow continuous measurement of the oxygen saturation of mixed venous blood.

Cardiac index, Formula = Cardiac output (L/min) Body surface area (m?). Cardiac index, Normal = 2.2-4.2. Cardiac index, Units = U/min/m?. Total peripheral resistance, Formula = (MAP—CVvP)80 Cardiac output (L/min). Total peripheral resistance, Normal = 1200-1500. Total peripheral resistance, Units = dynes +s cm. Pulmonary vascular resistance, Formula = (PA-PAOP) x 80 Cardiac output (L/min). Pulmonary vascular resistance, Normal = 100-300. Pulmonary vascular resistance, Units = dynes +s cm*. Stroke volume, Formula = Cardiac output (L/min)x 1000 Heart rate (beats/min). Stroke volume, Normal = 60-90. Stroke volume, Units = mL/beat. Stroke index (SI), Formula = Stroke volume (mL/beat) Body surface area (m?). Stroke index (SI), Normal = 20-65. Stroke index (SI), Units = ml/beat/m?. Right ventricular stroke-work index, Formula = E0136 PALCULKS|. Right ventricular stroke-work index, Normal = 30-65. Right ventricular stroke-work index, Units = g-m/beat/m?. Left ventricular stroke-work index, Formula = 0.0136 (MAP—PAOP) x SI. Left ventricular stroke-work index, Normal = 46-60. Left ventricular stroke-work index, Units = g-m/beat/m?
g-m, gram meter; MAP, mean arterial pressure; CVP, central venous pressure; PA, mean pulmonary artery pressure; PAOP, pulmonary artery occlusion pressure.

Starling demonstrated the relationship between left ventricular function and left ventricular end-diastolic muscle fiber length, which is usually proportionate to end-diastolic volume (see Chapter 20). If compliance is not abnormally decreased (eg, by myocardial ischemia, overload, ventricular hypertrophy, or pericardial tamponade), LVEDP should reflect fiber length. In the presence of a normal mitral valve, left atrial pressure approaches left ventricular pressure during diastolic filling. The left atrium connects with the right side of the heart through the pulmonary vasculature. The distal lumen of a correctly wedged PA catheter is isolated from right-sided pressures by balloon inflation. Its distal opening is exposed only to capillary pressure, which—in the absence of high airway pressures or pulmonary vascular disease—equals left atrial pressure. In fact, aspiration through the distal port during balloon inflation samples arterialized blood. PAOP is an indirect measure of LVEDP which, depending upon ventricular compliance, approximates left ventricular end diastolic volume. Whereas CVP may reflect right ventricular function, a PA catheter may be indicated if either ventricle is markedly depressed, causing disassociation of rightand left-sided hemodynamics. CVP is poorly predictive of pulmonary capillary pressures, especially in patients with abnormal left ventricular function. Even the PAOP does not always predict LVEDP. The relationship between left ventricular end-diastolic volume (actual preload) and PAOP (estimated preload) can become unreliable during conditions associated with changing left atrial or ventricular compliance, mitral valve function, or pulmonary vein resistance. These conditions are common immediately following major cardiac or vascular surgery and in critically ill patients who are receiving inotropic agents or are suffering from septic shock.
Ultimately, the value of the information provided by the PA catheter is dependent upon its correct interpretation by the patient's caregivers. Thus, the PA catheter is only a tool to assist in goal-directed perioperative therapy. Given the increasing number of less invasive methods now available to obtain similar information, we anticipate that PA catheterization will become mostly of historic interest.

CO measurement to permit calculation of the SV is one of the primary reasons for PA catheterization. Currently, there are a number of alternative, less invasive methods to estimate ventricular function to assist in goal-directed therapy.

The injection of a quantity (2.5, 5, or 10 mL) of fluid that is below body temperature (usually room temperature or iced) into the right atrium changes the temperature of blood in contact with the thermistor at the tip of the PA catheter. The degree of change is inversely proportional to CO: Temperature change is minimal if there is a high blood flow, whereas temperature change is greater when flow is reduced. After injection, one can plot the temperature as a function of time to produce a thermodilution curve (Figure 5—20). CO is determined by a computer program that integrates the area under the curve. @ Accurate measurements of CO depend on rapid and smooth injection, precisely known injectant temperature and volume, correct entry of the calibration factors for the specific type of PA catheter into the CO computer, and avoidance of measurements during electrocautery. Tricuspid regurgitation and cardiac shunts invalidate results because only right ventricular output into the PA is actually being measured. Rapid infusion of the iced injectant has rarely resulted in cardiac arrhythmias.
FIGURE 5-20 Comparison of thermodilution curves after injection of cold saline into the superior vena cava. The peak temperature change arrives earlier when measured in the pulmonary artery (a) than if measured in the femoral artery (b). Thereafter, both curves soon reapproximate baseline. (Reproduced with permission from Reuter D, Huang C, Edrich T, et al. Cardiac output monitoring using indicator dilution techniques: Basics, limits and perspectives. Anesth Analg. 2010 Mar 1;110(3):799-811.)
A modification of the thermodilution technique allows continuous CO measurement with a special catheter and monitor system. The catheter contains a thermal filament that introduces small pulses of heat into the blood proximal to the pulmonic valve and a thermistor that measures changes in PA blood temperature. A computer in the monitor determines CO by cross-correlating the amount of heat input with the changes in blood temperature.

Transpulmonary thermodilution (PiCCO® system) relies upon the same principles of thermodilution, but does not require PA catheterization. A central line and a thermistor-equipped arterial catheter (usually placed in the femoral artery) are necessary to perform transpulmonary thermodilution. Thermal measurements from radial artery catheters have been found to be invalid.
Transpulmonary thermodilution measurements involve injection of cold indicator into the superior vena cava via a central line (Figure 5—21). A thermistor notes the change in temperature in the arterial system following the cold indicator's transit through the heart and lungs and estimates the CO.
FIGURE 5-21 Two methods combined for precise monitoring. (Reproduced with permission from Royal Philips Electronics.)
Transpulmonary thermodilution also permits the calculation of both the global end-diastolic volume (GEDV) and the extravascular lung water (EVLW). Through mathematical analysis and extrapolation of the thermodilution curve, it is possible for the transpulmonary thermodilution computer to calculate both the mean transit time of the indicator and its exponential decay time (Figure 5—22). The intrathoracic thermal volume (ITTV) is the product of the CO and the mean transit time (MTT). The ITTV includes the pulmonary blood volume (PBV), EVLW, and the blood contained within the heart. The pulmonary thermal volume (PTV) includes both the EVLW and the PBV and is obtained by multiplying the CO by the exponential decay time (EDT). Subtracting the PTV from the ITTV gives the GEDV (Figure 5—23). The GEDV is a hypothetical volume that assumes that all of the heart's chambers are simultaneously full in diastole. With a normal index between 640 and 800 mL/m2, the GEDV can assist in determining volume status. An extravascular lung water index of less than 10 mL/kg is normative. The EVLW is the ITTV minus the intrathoracic blood volume (ITBV). The ITBV = GEDV * 1.25.

FIGURE 5-22 The upper curve represents the classic thermodilution curve, showing the concentration of an indicator over time at the site of detection. By extrapolation of the curve (dashed line), potential recirculation phenomena are excluded. Logarithmic illustration (lower curve) allows defining the mean transit time (MTT-+) and the exponential decay time (EDT) of the indicator. (Reproduced with permission from Reuter D, Huang C, Edrich T, et al. Cardiac output monitoring using indicator dilution techniques: Basics, limits and perspectives. Anesth Analg. 2010 Mar 1;110(3):799-811.)
FIGURE 5-23 Assessment of global end-diastolic volume (GEDV) by transcardiopulmonary thermodilution. Upper row: The intrathroacic thermal volume (ITTV) is the complete volume of distribution of the thermal indicator, including the right atrium end-diastolic volume (RAEDV), the right ventricle (RVEDV), the left atrium (LAEDV), the left ventricle (LVEDV), the pulmonary blood volume (PBV), and the extravascular lung water (EVLW). It is calculated by multiplying cardiac output (F) with the mean transit time (MTT*) of the indicator. Middle row: The pulmonary thermal volume (PTV) represents the largest mixing chamber in this system and includes the PBV and the EVLW and is assessed by multiplying F with the exponential decay time (EDT;;) of the thermal indicator. Bottom row: The GEDV, including the volumes of the right and the left heart, now is calculated by subtracting PTV from ITTV. (Reproduced with permission from Reuter D, Huang C, Edrich T, et al. Cardiac output monitoring using indicator dilution techniques: Basics, limits and perspectives. Anesth Analg. 2010 Mar 1;110(3):799-811.)

Thus, EVLW = ITTV — ITBV. An increased EVLW can be indicative of fluid overload. Through mathematical analysis of the transpulmonary thermodilution curve, it is therefore possible to obtain volumetric indices to guide fluid replacement therapy. Moreover, the PiCCO® system calculates SV variation and pulse pressure variation through pulse contour analysis, both of which can be used to determine fluid responsiveness. Both SV and pulse pressure are decreased during positive-pressure ventilation. The greater the variations over the course of positive-pressure inspiration and expiration, the more likely the patient is to improve hemodynamic measures following volume administration. Figure 5—24 demonstrates that patients located on the steeper portion of the curve will be more responsive to volume administration compared with those whose volume status is already adequate. Dynamic measures such as SV and pulse pressure variation assist in the identification of individuals likely to respond to volume administration (Figures 5-25 and 5-26).
Right atrial pressure
FIGURE 5-24 The fluid responder located on the steep portion of the right atrial pressure (RAP)/cardiac output (CO) curve will augment CO with minimal change in RAP when administered a fluid challenge. Conversely, the nonresponder will see little change in CO; however, RAP will likely increase. (Reproduced with permission from Cherpanath T, Aarts L, Groeneveld J, Geerts B. Defining fluid responsiveness: A guide to patient tailored volume titration. J Cardiothorac Vasc Anesth. 2014 Jun;28(3):745-754.)
FIGURE 5-25 Calculation of pulse pressure variation (PPV). PP,,,,,, maximum pulse pressure; PP,,,.a,3 mean pulse pressure; PP,,,;,, minimum pulse pressure. (Reproduced with permission from Scott MC, Mallemat H, eds. Assessing volume status. Emerg Med Clin N Am. 2014 Nov;32(4):811-822.)
FIGURE 5-26 Pulse pressure variation (PPV) decreases as volume is administered. (Reproduced with permission from Ramsingh D, Alexander B, Cannesson M. Clinical review: Does it matter which hemodynamic monitoring system is used? Crit Care. 2013 Mar 5;17(2):208.)

Pulse pressure variation is the change in pulse pressure that occurs throughout the respiratory cycle in patients supported by positive-pressure ventilation. As volume is administered, pulse pressure variation decreases. Variation greater than 12% to 13% is suggestive of fluid responsiveness. Dynamic measures such as pulse pressure variation and stroke volume variation become less reliable when arrhythmias are present. Unfortunately, many of the validation studies using these dynamic measures were performed prior to the routine use of low tidal volume (6 mL/kg) lung protective ventilation strategies during positive-pressure ventilation.

If indocyanine green dye (or another indicator such as lithium) is injected through a central venous catheter, its appearance in the systemic arterial circulation can be measured by analyzing arterial samples with an appropriate detector (eg, a densitometer for indocyanine green). The area under the resulting dye indicator curve is related to CO. By analyzing arterial blood pressure and integrating it with CO, systems that use lithium (LiDCO™) also calculate beatto-beat SV. In the LiDCO™ system, a small bolus of lithium chloride is injected into the circulation. A lithium-sensitive electrode in an arterial catheter measures the decay in lithium concentration over time. Integrating the concentration over time graph permits the machine to calculate the CO. The LiDCO™ device, like the PiCCO® thermodilution device, employs pulse contour analysis of the arterial wave form to provide ongoing beat-to-beat determinations of CO and other calculated parameters. Lithium dilution determinations can be made in patients who have only peripheral venous access. Lithium should not be administered to patients in the first trimester of pregnancy. The dye dilution technique, however, introduces the problems of indicator recirculation, arterial blood sampling, and background tracer buildup, potentially limiting the use of such approaches perioperatively. Nondepolarizing neuromuscular blockers may affect the lithium sensor.

Pulse contour devices use the arterial pressure tracing to estimate the CO and other dynamic parameters, such as pulse pressure and SV variation with mechanical ventilation. These indices are used to help determine if hypotension is likely to respond to fluid therapy.
Pulse contour devices rely upon algorithms that measure the area of the systolic portion of the arterial pressure trace from end diastole to the end of ventricular ejection. The devices then incorporate a calibration factor for the patient's vascular compliance, which is dynamic and not static. Some pulse contour devices rely first on transpulmonary thermodilution or lithium thermodilution to calibrate the machine for subsequent pulse contour measurements. The FloTrac (Edwards Life Sciences) does not require calibration with another measure and relies upon a statistical analysis of its algorithm to account for changes in vascular compliance occurring as a consequence of changed vascular tone.

Esophageal Doppler relies upon the Doppler principle to measure the velocity of blood flow in the descending thoracic aorta. The Doppler principle is integral in perioperative echocardiography. The Doppler effect has been described previously in this chapter. Blood in the aorta is in relative motion compared with the Doppler probe in the esophagus. As red blood cells travel, they reflect a frequency shift, depending upon both the direction and velocity of their movement. When blood flows toward the transducer, its reflected frequency is higher than that which was transmitted by the probe. When blood cells move away from the transducer, the frequency is lower than that which was initially sent by the probe. By using the Doppler equation, it is possible to determine the velocity of blood flow in the aorta. The equation is:
Velocity of blood blow = {frequency change/cosine of angle of incidence between Doppler beam and blood flow} x {speed of sound in tissue/2 (source frequency)}
For Doppler to provide a reliable estimate of velocity, the angle of incidence should be as close to zero as possible, since the cosine of 0 is 1. As the angle approaches 90°, the Doppler measure is unreliable, as the cosine of 90° is 0. The esophageal Doppler device calculates the velocity of flow in the aorta. As the velocities of the cells in the aorta travel at different speeds over the cardiac cycle, the machine obtains a measure of all of the velocities of the cells moving over time. Mathematically integrating the velocities represents the distance that the blood travels. Next, using normograms, the monitor approximates the area of the descending aorta. The monitor thus calculates both the distance the blood travels, as well as the area: area x length = volume.
Consequently, the SV of blood in the descending aorta is calculated. Knowing the HR allows calculation of that portion of the CO flowing through the descending thoracic aorta, which is approximately 70% of total CO. Correcting for this 30% allows the monitor to estimate the patient's total CO. Esophageal Doppler is dependent upon many assumptions and nomograms, which may hinder its ability to accurately reflect CO in a variety of clinical situations.

Changes in thoracic volume cause changes in thoracic resistance (bioimpedance) to low amplitude, high frequency currents. If thoracic changes in bioimpedance are measured following ventricular depolarization, SV can be continuously determined. This noninvasive technique requires six electrodes to inject microcurrents and to sense bioimpedance on both sides of the chest. Increasing fluid in the chest results in less electrical bioimpedance. Mathematical assumptions and correlations are then made to calculate CO from changes in bioimpedance. Disadvantages of thoracic bioimpedance include susceptibility to electrical interference and reliance upon correct electrode positioning. The accuracy of this technique is questionable in several groups of patients, including those with aortic valve disease, previous heart surgery, or acute changes in thoracic sympathetic nervous function (eg, those undergoing spinal anesthesia).

The amount of oxygen consumed by an individual (Vo) equals the difference between arterial and venous (a—v) oxygen content (C) (Cao, and Cvo,) multiplied by CO. Therefore
Mixed venous and arterial oxygen content are easily determined if a PA catheter and an arterial line are in place. Oxygen consumption can also be calculated from the difference between the oxygen content in inspired and expired gas. Variations of the Fick principle are the basis of all indicator—dilution methods of determining CO.

There are no more powerful tools to diagnose and assess cardiac function perioperatively than transthoracic (TTE) and transesophageal echocardiography (TEE). Both TTE and TEE can be employed preoperatively and postoperatively. TTE has the advantage of being completely noninvasive; however, acquiring the "windows" to view the heart can be difficult. In the operating rooms, limited access to the chest makes TEE an ideal option to visualize the heart. Disposable TEE probes are now available that can remain in position in critically ill patients for days, during which intermittent TEE examinations can be performed.
Echocardiography can be employed by anesthesia staff in two ways, depending upon degrees of training and certification. Basic (or hemodynamic) TEE permits the anesthesiologist to discern the primary source of a patient's hemodynamic instability. Whereas in past decades the PA flotation catheter would be used to determine why the patient might be hypotensive, the anesthetist performing TEE is attempting to determine if the heart is adequately filled, contracting appropriately, not externally compressed, and devoid of any grossly obvious structural defects. At all times, information obtained from TEE may be correlated with other information as to the patient's general condition. Anesthesiologists performing advanced (diagnostic) TEE make therapeutic and surgical recommendations based upon their TEE interpretations. Various organizations and boards have been established worldwide to certify individuals in all levels of perioperative echocardiography. More importantly, individuals who perform echocardiography should be aware of the credentialing requirements of their respective institutions.
Echocardiography has many uses, including:
¢ Diagnosis of the source of hemodynamic instability, including myocardial ischemia, systolic and diastolic heart failure, valvular abnormalities, hypovolemia, and pericardial tamponade
¢ Estimation of hemodynamic parameters, such as SV, CO, and intracavitary pressures
¢ Diagnosis of structural diseases of the heart, such as valvular heart disease, shunts, aortic diseases
¢ Guiding surgical interventions, such as mitral valve repair

Various echocardiographic modalities are employed perioperatively by anesthesiologists, including TTE, TEE, epiaortic and epicardiac ultrasound, and three-dimensional echocardiography. Some advantages and disadvantages of the modalities are as follows:
- ¢ TTE has the advantage of being noninvasive and essentially risk free. Limited scope TTE exams are now increasingly common in the intensive care unit (Figure 5—27). Bedside TTE exams such as the FATE or FAST protocols can readily assist in hemodynamic diagnosis. Using pattern recognition, it is possible to identify various common cardiac pathologies perioperatively (Figures 5—28 and 5—29).
FIGURE 5-27 Normal apical four-chamber view. RV, right ventrical; LV, left ventricle; RA, right atrium; LA, left atrium. (Reproduced with permission from Carmody KA, et al. Handbook of Critical Care and Emergency Ultrasound. New York, NY: McGraw-Hill; 2011).

Pos 1: Subcostal 4-chamber
Pos 3: Parasternal long axis
Pos 4: Pleural scanning
Pos 2: Apical 4-chamber
Pos 3: Parasternal LV short axis
FIGURE 5-28 The FATE examination. AO, aorta; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. (Reproduced with permission from UltraSound Airway Breathing Circulation Dolor (USABCD) and Prof. Erik Sloth. http://usabcd.org/node/35.)

Pathology to be considered in particular:
D Post OP cardiac surgery, following cardiac catheterisation, trauma, renal failure, infection.
& Pulmonary embolus, RV infarction, pulmonary hypertension, volume overload.
O Ischemic heart disease, dilated cardiomyopathy, sepsis, volume overload, aorta insufficiency.
@ Aorta stenosis, arterial hypertension, LV outflow tract obstruction, hypertrophic cardiomyopathy, myocardial deposit diseases.
FIGURE 5-29 Important pathological conditions identified with the FATE examination. AO, aorta; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. (Reproduced with permission from UltraSound Airway Breathing Circulation Dolor (USABCD) and Prof. Erik Sloth. http://usabcd.org/node/35.)
- ¢ Unlike TTE, TEE is an invasive procedure with the potential for lifethreatening complications (esophageal rupture and mediastinitis) (Figure 5— 30). The close proximity of the esophagus to the left atrium eliminates the problem of obtaining "windows" to view the heart and permits great detail. TEE has been used frequently in the cardiac surgical operating room over the past decades. Its use to guide therapy in general cases has been limited by both the cost of the equipment and the learning necessary to correctly interpret the images. Both TTE and TEE generate two-dimensional images of the three-dimensional heart. Consequently, it is necessary to view the heart through many two-dimensional image planes and windows to mentally recreate the three-dimensional anatomy. The ability to interpret these images at the advanced certification level requires much training.
FIGURE 5-30 The structures of the heart as seen on a midesophageal fourchamber view, including the right atrium (RA), tricuspid valve (TV), right ventricle (RV), left atrium (LA), mitral valve (MV), and left ventricle (LV). (Reproduced with permission from Wasnick J, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography. New York, NY: McGraw-Hill; 2011.)

- ¢ Epiaortic and epicardiac ultrasound imaging techniques employ an echo probe wrapped in a sterile sheath and manipulated by thoracic surgeons intraoperatively to obtain views of the aorta and the heart. The air-filled trachea prevents TEE imaging of the ascending aorta. Because the aorta is manipulated during cardiac surgery, detection of atherosclerotic plaques permits the surgeon to potentially minimize the incidence of embolic stroke. Imaging of the heart with epicardial ultrasound permits intraoperative echocardiography when TEE is contraindicated because of esophageal or

¢ Three-dimensional echocardiography (TTE and TEE) has become available in recent years (Figure 5—31). These techniques provide a three-dimensional view of the heart's structure. In particular, three-dimensional images can better quantify the heart's volumes and can generate a surgeon's view of the mitral valve to aid in guiding valve repair.
FIGURE 5-31 Three-dimensional echocardiography of the mitral valve demonstrates the anterior leaflet (AML), the posterior leaflet (PML), the anterolateral commissure (ALC), and the posteromedial commissure (PMC). The aortic valve (AV) is also seem. (Reproduced with permission from Wasnick J, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography. New York, NY: McGraw-Hill; 2011.)

Echocardiography employs ultrasound (sound at frequencies greater than normal hearing) from 2 to 10 MHz. A piezoelectrode in the probe transducer converts electrical energy delivered to the probe into ultrasound waves. These waves then travel through the tissues, encountering the blood, the heart, and other structures. Sound waves pass readily through tissues of similar acoustic impedance; however, when they encounter different tissues, they are scattered, refracted, or reflected back toward the ultrasound probe. The echo wave then interacts with the ultrasound probe, generating an electrical signal that can be reconstructed as an image. The machine knows the time delay between the transmitted and the reflected sound wave. By knowing the time delay, the location of the source of the reflected wave can be determined and the image generated. The TEE probe contains myriad crystals generating and processing waves, which then create the echo image. The TEE probe can generate images through multiple planes and can be physically manipulated in the stomach and esophagus, permitting visualization of heart structures (Figure 5-32). These views can be used to determine if the walls of the heart are receiving an adequate blood supply (Figure 5—33). Increasingly it is realized that myocardial blood supply is not demarcated as clearly as in traditional depictions (eg, in Figure 5— 33). In the healthy heart, the walls thicken and move inwardly with each beat. Wall motion abnormalities, in which the heart walls fail to thicken during systole or move in a dyskinetic fashion, can be associated with myocardial ischemia.
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FIGURE 5-32 The echo probe is manipulated by the examiner in multiple ways to create the standard images that constitute the comprehensive perioperative transesophageal echocardiography (TEE) examination. Never force the probe; if resistance is encountered abandon the examination. Echocardiographic information can be provided by intraoperative epicardial and epiaortic examination. Advancing the probe in the esophagus permits the upper, mid, and transgastric examinations (A). The probe can be turned in the esophagus from left to right to examine both leftand right-sided structures (A). Using the button located on the probe permits the echocardiographer to rotate the scan beam through 180°, thereby creating various two-dimensional imaging slices of the three-dimensional heart (B). Lastly, panels (C) and (D) demonstrate manipulation of the tip of the probe to permit the beam to be directed to best visualize the image. (Modified with permission from Shanewise JS, Cheung AT, Aronson S, et al. ASE/SCA guidelines for performing a comprehensive intraoperative multiplane transesophageal echocardiography examination; recommendations of the American Society of Echocardiography Council for Intraoperative Echocardiography and the Society for Cardiovascular Anesthesiologists Task Force for Certification in Perioperative Transesophageal Echocardiography. Anesth Analg. 1999 Oct;89(4):870-884.)

FIGURE 5-33 The midesophageal four-chamber view (A), the midesophageal two-chamber view (B), the midesophageal long-axis vew (C), and the transgastric mid short-axis vew (D) are depicted. The different views provide the opportunity to observe the myocardium supplied by each of the three main coronary vessels, the left circumflex (Cx), the left anterior descending (LAD) and the right coronary artery (RCA). Areas of impaired myocardial perfusion are suggested by the inability of the myocardium to both thicken and move inwardly during systole. Image D is very useful for monitoring in the operating room because left ventricular myocardium supplied by each of the three vessels can be seen in one image. (Modifed with permission from Shanewise JS, Cheung AT, Aronson §, et al. ASE/SCA guidelines for performing a comprehensive intraoperative multiplane transesophageal echocardiography examination; recommendations of the American Society of Echocardiography Council for Intraoperative Echocardiography and the Society for Cardiovascular Anesthesiologists Task Force for Certification in Perioperative Transesophageal Echocardiography. Anesth Analg. 1999 Oct;89(4):870-884.)

The Doppler effect is routinely used in echocardiographic examinations to determine both the direction and the velocity of blood flow and tissue movement. Blood flow in the heart follows the law of the conservation of mass. Therefore, the volume of blood that flows through one point (eg, the left ventricular outflow tract) must be the same volume that passes through the aortic valve. When the pathway through which the blood flows becomes narrowed (eg, aortic stenosis), the blood velocity must increase to permit the volume to pass. The increase in velocity as blood moves toward an esophageal echo probe is detected. The Bernoulli equation (pressure change = 4V°) allows echocardiographers to determine the pressure gradient between areas of different velocity, where v represents the area of maximal velocity (Figure 5-34). Using continuous wave Doppler, it is possible to determine the maximal velocity as blood accelerates through a pathological heart structure. For example, a blood flow of 4 m/s reflects a pressure gradient of 64 mm Hg between an area of slow flow (the left ventricular outflow tract) and a region of high flow (a stenotic aortic valve).
FIGURE 5-34 The time-velocity interval (TVI) of the aortic valve is calculated using continuous wave Doppler, while pulse wave Doppler is useful for measurements at lower blood velocities. This continuous wave Doppler has been aligned parallel to that aortic valve flow as imaged using the deep transgastric view. Of note, the blood velocity across the aortic valve is greater than 4 m/s. (Reproduced with permission from Wasnick J, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography. New York, NY: McGraw-Hill; 2011.)
The Bernoulli equation permits echocardiographers to estimate PA and other intracavitary pressures.
Blood flow proceeds from an area of high pressure P, to an area of low pressure P5.
The pressure gradient = 4V°, where V is the maximal velocity measured in meters per second.
Thus,

Thus, assuming that there is a jet of regurgitant blood flow from the left ventricle into the left atrium and that left ventricular systolic pressure (P,) is the same as systemic blood pressure (eg, no aortic stenosis), it is possible to calculate left atrial pressure (P,). In this manner, echocardiographers can estimate intracavitary pressures when there are pressure gradients, measurable flow velocities between areas of high and low pressure, and knowledge of either P, or P, (Figure 5-35).
FIGURE 5-35 Intracavity pressures can be calculated using known pressures and the Bernoulli equation when regurgitant jets are present. The pulmonary artery (PA) systolic pressure is obtained when tricuspid regurgitation is present and the right atrial pressure known. Assuming no pulmonic valve disease, the right ventricular systolic pressure (RVSP) and the pulmonary systolic pressure are the same. The left atrial pressure can be similarly calculated if mitral regurgitation is present. Again, assuming no valvular disease left ventricular systolic pressure (LVSP) should equal systemic systolic blood pressure. Subtracting 4V* from the LVSP estimates the left atrial pressure (LAP). (Reproduced with permission from Wasnick J, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography. New York, NY: McGraw-Hill; 2011.)
Color flow Doppler is used by echocardiographers to identify areas of abnormal flow. Color flow Doppler creates a visual picture by assigning a color code to the blood velocities in the heart. Blood flow directed away from the echocardiographic transducer is blue, whereas that which is moving toward the probe is red. The higher the velocity of flow, the lighter the color hue (Figure 5— 36). When the velocity of blood flow becomes greater than that which the machine can measure, flow toward the probe is misinterpreted as flow away from the probe, creating images of turbulent flow and "aliasing" of the image. Such changes in flow pattern are used by echocardiographers to identify areas of pathology.

FIGURE 5-36 The color flow Doppler image of the midesophageal aortic valve long-axis view demonstrates measurement of the vena contracta of aortic regurgitation. The vena contracta represents the smallest diameter of the regurgitant jet at the level of the aortic valve. A vena contracta of 6.2 mm grades the aortic regurgitation in this case as severe. (Reproduced with permission from Wasnick J, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography. New York, NY: McGraw-Hill; 2011.)
Doppler can also be used to provide an estimate of SV and CO. Similar to esophageal Doppler probes previously described, TTE and TEE can be used to estimate CO. Assuming that the left ventricular outflow tract is a cylinder, it is possible to measure its diameter (Figure 5—37). Knowing this, it is possible to calculate the area through which blood flows using the following equation:
FIGURE 5-37 The midesophageal long-axis view is employed in this image to measure the diameter of the left ventricular outflow tract (LVOT). Knowing the diameter of the LVOT permits calculation of the LVOT area. (Reproduced with permission from Wasnick J, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography. New York, NY: McGraw-Hill; 2011.)
Area = mr2 = 0.785 x diameter"
Next, the time velocity integral is determined. A Doppler beam is aligned in parallel with the left ventricular outflow tract ( ). The velocities passing through the left ventricular outflow tract are recorded, and the machine integrates the velocity/time curve to determine the distance the blood traveled.

FIGURE 5-38 PW Doppler is employed in this deep transgastric view interrogation of the left ventricular outflow tract (LVOT). Blood is flowing in the LVOT away from the esophagus. Therefore, the flow velocities appear below the baseline. Flow velocity through the LVOT is 46.5 cm/s. This is as expected when there is no pathology noted as blood is ejected along the LVOT. Tracing the flow envelope (dotted lines) identifies the time-velocity interval (TVI). In this example the TVI is 14 cm. (Reproduced with permission from Wasnick J, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography New York, NY: McGraw-Hill; 2011.)

Lastly, Doppler can be used to examine the movement of the myocardial tissue. Tissue velocity is normally 8 to 15 cm/s (much less than that of blood, which is 100 cm/s). Using the tissue Doppler function of the echo machine, it is possible to discern both the directionality and velocity of the heart's movement. During diastolic filling, the lateral annulus myocardium will move toward a TEE probe. Reduced myocardial velocities (<8 cm/s) are associated with impaired diastolic function and higher left ventricular end-diastolic pressures.
Ultimately, echocardiography can provide comprehensive cardiovascular monitoring. Its routine use outside of the cardiac operating room has been hindered by both the costs of the equipment and the training required to correctly interpret the images. It is likely that anesthesia staff will perform an increasing number of echocardiographic examinations perioperatively. All anesthesiology trainees should acquire basic echocardiography skills. When questions arise beyond those related to hemodynamic guidance, interpretation by an individual credentialed in diagnostic echocardiography is warranted.

Hemodynamic Monitoring and Management of a
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Arterial cannulation and monitoring will provide beat-to-beat blood pressure determinations intraoperatively and will also provide for blood gas measurements in a patient likely to be acidotic and hemodynamically unstable. Central venous access is obtained to permit volume resuscitation and to provide a port for delivery of fluid for transpulmonary measurements of CO and SV variation. Alternatively, pulse contour analysis can be employed from an arterial trace to determine volume responsiveness, should the patient become hemodynamically unstable. Echocardiography can be used to determine ventricular function, filling pressures, and CO and to provide surveillance for the development of ischemia-induced wall motion abnormalities.
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The choice of hemodynamic monitors remains with the individual physician and the availability of various monitoring techniques. It is important to also consider which monitors can be used postoperatively to ensure continuation of goal-directed therapy.

Alhasemi J, Cecconi M, della Rocca G, et al. Minimally invasive monitoring of

The American Society of Anesthesiologists (ASA)'s standards for basic anesthesia monitoring can be found on the organization web site.
http://www.asahq.org

@ Capnographs rapidly and reliably detect esophageal intubation—a cause of anesthetic catastrophe—but do not reliably detect mainstem bronchial intubation.
@ Postoperative residual paralysis remains a problem in postanesthesia care, producing potentially injurious airway and respiratory function compromise and increasing length of stay and cost in the postanesthesia care unit (PACU).
The previous chapter reviewed routine hemodynamic monitoring used by anesthesiologists. This chapter examines the vast array of techniques and devices used perioperatively to monitor neuromuscular transmission, neurological condition, respiratory gas exchange, and body temperature.

Prior to the routine availability of gas exchange monitors, anesthesiologists used a precordial or esophageal stethoscope to ensure that the lungs were being ventilated, to monitor for circuit disconnections, and to auscultate heart tones to confirm a beating heart. Although largely supplanted by other modalities, the finger on the pulse and auscultation remain frontline monitors, especially when technology fails. Chest auscultation remains the primary method to confirm bilateral lung ventilation in the operating room, even though detection of an endtidal CO, waveform is definitive to exclude esophageal intubation.

Esophageal stethoscopes and esophageal temperature probes should be avoided in patients with esophageal varices or strictures.

A precordial stethoscope (Wenger chestpiece) is a heavy, bell-shaped piece of metal placed over the chest or suprasternal notch. Although its weight tends to maintain its position, double-sided adhesive disks provide an acoustic seal to the patient's skin. Various chestpieces are available, but the child size works well for most patients. The bell is connected to the anesthesiologist's ear by extension tubing.
The esophageal stethoscope is a soft plastic catheter (8—24FR) with ballooncovered distal openings (Figure 6—1). Although the quality of breath and heart sounds is much better than with a precordial stethoscope, its use is limited to intubated patients. Temperature probes, electrocardiogram (ECG) leads, ultrasound probes, and even atrial pacemaker electrodes have been incorporated into esophageal stethoscopes. Placement through the mouth or nose can occasionally cause mucosal irritation and bleeding. Rarely, the stethoscope slides into the trachea instead of the esophagus, resulting in a gas leak around the tracheal tube cuff.

The information provided by a precordial or esophageal stethoscope includes confirmation of ventilation, quality of breath sounds (eg, stridor, wheezing), regularity of heart rate, and quality of heart tones (muffled tones are associated with decreased cardiac output). The confirmation of bilateral breath sounds after tracheal intubation, however, is best made with a binaural stethoscope.

Pulse oximeters are mandatory monitors for any anesthetic, including cases of moderate sedation. There are no contraindications.

Pulse oximeters combine the principles of oximetry and plethysmography to noninvasively measure oxygen saturation in arterial blood. A sensor containing light sources (two or three light-emitting diodes) and a light detector (a photodiode) is placed across a finger, toe, earlobe, or any other perfused tissue that can be transilluminated. When the light source and detector are opposite one another across the perfused tissue, transmittance oximetry is used. When the light source and detector are placed on the same side of the patient (eg, the forehead), the backscatter (reflectance) of light is recorded by the detector. Oximetry depends on the observation that oxygenated and reduced hemoglobin differ in their absorption of red and infrared light (Lambert—Beer law). Specifically, oxyhemoglobin (Hbo,) absorbs more infrared light (940 nm), whereas deoxyhemoglobin absorbs more red light (660 nm) and thus appears blue, or cyanotic, to the naked eye. The change in light absorption during arterial pulsations is the basis of oximetric determinations (Figure 6—2). The ratio of the absorptions at the red and infrared wavelengths is analyzed by a microprocessor to provide the oxygen saturation (Spo,) of arterial blood based on established norms. The greater the ratio of red to infrared absorption, the lower is the arterial hemoglobin oxygen saturation. Arterial pulsations are identified by plethysmography, allowing corrections for light absorption by nonpulsating venous blood and tissue. Heat from the light source or sensor pressure may, rarely, result in tissue damage if the monitor is not periodically moved. No user calibration is required.
FIGURE 6-2 Oxyhemoglobin and deoxyhemoglobin differ in their absorption of red and infrared light.

In addition to Spos, pulse oximeters provide an indication of tissue perfusion (pulse amplitude) and measure heart rate. Depending on a particular patient's oxygen—hemoglobin dissociation curve, a 90% saturation may indicate a Pao, of less than 65 mm Hg. Clinically detectable cyanosis requires 5 g of desaturated hemoglobin and usually corresponds to Spo, of less than 80%. Mainstem bronchial intubation will usually go undetected by pulse oximetry in the absence of lung disease or low fraction of inspired oxygen concentrations (Fio,).
Because carboxyhemoglobin (COHb) and Hbo, absorb light at 660 nm similarly, pulse oximeters that compare only two wavelengths of light will register a falsely high reading in patients with carbon monoxide poisoning. Methemoglobin has the same absorption coefficient at both red and infrared wavelengths. The resulting 1:1 absorption ratio corresponds to a saturation reading of 85%. Thus, methemoglobinemia causes a falsely low saturation reading when Sao, is actually greater than 85% and a falsely high reading if Sao, is actually less than 85%.
optical spectroscopy). Unlike pulse oximetry, brain oximetry measures venous and capillary blood oxygen saturation in addition to arterial blood saturation. Thus, its oxygen saturation readings represent the average oxygen saturation of all regional microvascular hemoglobin (approximately 70%). Cardiac arrest, cerebral embolization, and severe hypoxia cause a dramatic decrease in rSO). Cerebral oximetry is routinely employed in the perioperative management of patients undergoing cardiopulmonary bypass. (See the section "Neurological System Monitors.")

Determination of end-tidal CO, (ETco,) concentration to confirm adequate ventilation is mandatory during all anesthetic procedures, but particularly so for general anesthesia. Increases in alveolar dead space ventilation (eg, pulmonary thromboembolism, venous air embolism, decreased pulmonary perfusion) produce a decrease in ETco, compared with arterial CO, concentration (Paco3). Generally, ETco, and Paco, increase or decrease depending upon the balance of CO, production and CO, elimination (ventilation). A rapid fall of ETco, is a sensitive indicator of air embolism, in which both an increase in deadspace ventilation and a decrease in cardiac output may occur. There are no contraindications.

Capnography is a valuable monitor of the pulmonary, cardiovascular, and anesthetic breathing systems. Capnographs in common use rely on the absorption of infrared light by CO. As with oximetry, absorption of infrared light by CO, is governed by the Beer—Lambert law.

Nondiverting (mainstream) capnographs measure CO, passing through an adaptor placed in the breathing circuit (Figure 6—3). Infrared light transmission through the gas is measured and CO, concentration is determined by the monitor. Because of problems with drift, older flowthrough models self-zeroed during inspiration. Thus, they were incapable of detecting inspired CO,, such as would occur with a breathing circuit malfunction (eg, absorbent exhaustion, sticking unidirectional valves). The weight of the sensor caused traction on the tracheal tube, and its generation of radiant heat can cause skin burns. Newer designs avoid these problems.
FIGURE 6-3 A nondiverting sensor placed in-line analyzes CO, concentration at the sampling site.

Diverting (sidestream) capnographs continuously suction gas from the breathing circuit into a sample cell within the bedside monitor. CO, concentration is determined by comparing infrared light absorption in the sample cell with a chamber free of CO,. Continuous aspiration of anesthetic gas essentially represents a leak in the breathing circuit that will contaminate the operating room unless it is scavenged or returned to the breathing system. High aspiration rates (up to 250 mL/min) and low-dead-space sampling tubing usually increase sensitivity and decrease lag time. If tidal volumes (VT) are small (eg, pediatric patients), however, a high rate of aspiration may entrain fresh gas from the circuit and dilute ETCo, measurement. Low aspiration rates (<50 mL/min) can retard ETCO, measurement and underestimate it during rapid ventilation.
Diverting units are prone to water precipitation in the aspiration tube and sampling cell that can cause obstruction of the sampling line and erroneous readings. Expiratory valve malfunction is detected by the presence of CO, in inspired gas. Although inspiratory valve failure also results in rebreathing CO,, this is not as readily apparent because part of the inspiratory volume will still be free of CO,, causing the monitor to read zero during part of the inspiratory phase.

@ Capnographs rapidly and reliably detect esophageal intubation—a cause of anesthetic catastrophe—but do not reliably detect mainstem bronchial intubation. Although there may be some CO, in the stomach from swallowed expired air, this should be washed out within a few breaths. Sudden cessation of CO, during the expiratory phase may indicate a circuit disconnection. The increased metabolic rate caused by malignant hyperthermia causes a marked rise in ETCO5.
The gradient between Paco, and ETco, (normally 2-5 mm Hg) reflects alveolar dead space (alveoli that are ventilated but not perfused). Any significant reduction in lung perfusion (eg, air embolism, decreased cardiac output, or decreased blood pressure) increases alveolar dead space, dilutes expired CO,, and lessens ETCO,. True capnographs (as opposed to capnometers) display a waveform of CO, concentration that allows recognition of a variety of conditions (Figure 6-4).
FIGURE 6—4 A: A normal capnograph demonstrating the three phases of expiration: phase I—dead space; phase II—mixture of dead space and alveolar gas; phase IIJ—alveolar gas plateau. B: Capnograph of a patient with severe chronic obstructive pulmonary disease. No plateau is reached before the next inspiration. The gradient between end-tidal CO, and arterial CO, is increased. C: Depression during phase III indicates spontaneous respiratory effort. D: Failure of the inspired CO, to return to zero may represent an incompetent expiratory valve or exhausted CO, absorbent. E: The persistence of exhaled gas during part of the inspiratory cycle signals the presence of an incompetent inspiratory valve.

Analysis of anesthetic gases is essential during any procedure requiring inhalation anesthesia. There are no contraindications to analyzing these gases.

Techniques for analyzing multiple anesthetic gases include mass spectrometry, Raman spectroscopy, infrared spectrophotometry, or piezoelectric crystal
(quartz) oscillation. Most of these methods are primarily of historical interest, as most anesthetic gases are now measured by infrared absorption analysis. Infrared units use a variety of techniques similar to that described for capnography. These devices are all based on the Beer—Lambert law, which provides a formula for measuring an unknown gas within inspired gas because the absorption of infrared light passing through a solvent (inspired or expired gas) is proportional to the amount of the unknown gas. Oxygen and nitrogen do not absorb infrared light. There are a number of commercially available devices that use a singleor dual-beam infrared light source and positive or negative filtering. Because oxygen molecules do not absorb infrared light, their concentration cannot be measured with monitors that rely on infrared technology and, hence, it must be measured by other means (see below).

To measure the Fio, of inhaled gas, manufacturers of anesthesia machines have relied on various technologies.

Galvanic cell (fuel cell) contains a lead anode and gold cathode bathed in potassium chloride. At the gold terminal, hydroxyl ions are formed that react with the lead electrode (thereby gradually consuming it) to produce lead oxide, causing current, which is proportional to the amount of oxygen being measured, to flow. Because the lead electrode is consumed, monitor life can be prolonged by exposing it to room air when not in use. These are the oxygen monitors used on many anesthesia machines in the inspiratory limb.

Oxygen is a nonpolar gas, but it is paramagnetic, and when placed in a magnetic field, the gas will expand, contracting when the magnet is turned off. By switching the field on and off and comparing the resulting change in volume (or pressure or flow) to a known standard, the amount of oxygen can be measured.

A polarographic electrode has a gold (or platinum) cathode and a silver anode, both bathed in an electrolyte, separated from the gas to be measured by a semipermeable membrane. Unlike the galvanic cell, a polarographic electrode works only if a small voltage is applied to two electrodes. When voltage is applied to the cathode, electrons combine with oxygen to form hydroxide ions. The amount of current that flows between the anode and the cathode is proportional to the amount of oxygen present.

Contemporary anesthesia machines measure airway pressures, volume, and flow to calculate resistance and compliance. Measurements of flow and volume are made by mechanical devices that are usually fairly lightweight and are often placed in the inspiratory limb of the anesthesia circuit. The most fundamental detected abnormalities include low peak inspiratory pressure and high peak inspiratory pressure, which indicate either a ventilator or circuit disconnect, or an airway obstruction, respectively. By measuring VT and breathing frequency (f), exhaled minute ventilation (VE) can be calculated, providing some sense of security that ventilation requirements are being met. different,
Spirometric loops are usually displayed as flow versus volume and volume versus pressure (Figure 6—5). There are characteristic changes with obstruction, bronchial intubation, reactive airways disease, and so forth. If a normal loop is observed shortly after induction of anesthesia and a subsequent loop is the observant anesthesiologist is alerted to the fact that pulmonary or airway compliance, or both, may have changed. Mechanical ventilation and ventilators are discussed more completely in Chapter 57.
FIGURE 6-5 A: Normal volume—pressure loop. B: Normal flow—volume loop.

The electroencephalogram (EEG) is occasionally used during cerebrovascular surgery to confirm the adequacy of cerebral oxygenation or during cardiovascular surgery to ensure that burst suppression or an isoelectric signal has been obtained before circulatory arrest. A full 16-lead, 8-channel EEG is not necessary for these tasks, and simpler systems are available. There are no contraindications.

The EEG is a recording of electrical potentials generated by cells in the cerebral cortex. Although standard ECG electrodes can be used, silver disks containing a conductive gel are preferred. Platinum or stainless steel needle electrodes traumatize the scalp and have high impedance (resistance); however, they can be sterilized and placed in a surgical field. Electrode position (montage) is governed by the international 10-20 system (Figure 6—6). Electric potential differences between combinations of electrodes are filtered, amplified, and displayed by an oscilloscope or pen recorder. EEG activity occurs mostly at frequencies between 1 and 30 cycles/sec (Hz). Alpha waves have a frequency of 8 to 13 Hz and are found often in a resting adult with eyes closed. Beta waves at 8 to 13 Hz are found in concentrating individuals, and at times, in individuals under anesthesia. Delta waves have a frequency of 0.5 to 4 Hz and are found in brain injury, deep sleep, and anesthesia. Theta waves (4—7 Hz) are also found in sleeping individuals and during anesthesia. EEG waves are also characterized by their amplitude, which is related to their potential (high amplitude, >50 microV; medium amplitude, 20-50 microV; and low amplitude, <20 microV). Lastly, the EEG is examined for symmetry between the left and right hemispheres.
FIGURE 6-6 International 10—20 system. Montage letters refer to cranial location. F, frontal; C, coronal; T, temporal; O, occipital; Z, middle.
Examination of a multichannel EEG is at times performed intraoperatively to detect areas of cerebral ischemia, such as during carotid endarterectomy. Likewise, it can be used to detect EEG isoelectricity and maximal cerebral protection during hypothermic arrest. The strip chart EEG is cumbersome in the operating room, and often the EEG is processed using power spectral analysis. Frequency analysis divides the EEG into a series of sine waves at different frequencies and then plots the power of the signal at each frequency, allowing for a presentation of EEG activity in a more easily interpreted form than the raw EEG (Figure 6-7).

(EEG) Trace = ree enter rrr
FIGURE 6-7 Patient states, candidate depth of anesthesia devices or approaches, key features of different monitoring approaches, and possible readings at different depths of anesthesia. The readings shown represent examples of possible readings that may be seen in conjunction with each frontal electroencephalography trace. The electroencephalography traces show 3-s epochs (x-axis), and the scale (y-axis) is 50 pV. AAI, A-Line Autoregressive Index (a proprietary method of extracting the midlatency auditory evoked potential from the electroencephalogram); Amp, amplitude of an EEG wave; BIS, bispectral index scale; blinks, eye blink artifacts; BS, burst suppression; BSR, burst suppression ratio; EEG, electroencephalography; ETAG, end-tidal anesthetic gas concentration; f, frequency; y, B, a, 0, 6, EEG waves in decreasing frequencies (y, >30 hertz [Hz]; B, 12-30 Hz; a, 8-12 Hz; 0, 4-8 Hz; 6, 0-4 Hz); K, K complexes; lat, latency between an auditory stimulus and an evoked EEG waveform response; MAC, minimum alveolar concentration; NI, Narcotrend index; SEF9s, spectral edge frequency below which 95% of the EEG frequencies reside; Spindles, sleep spindles. (Reproduced with permission from Mashour GA, Orser BA, Avidan MS. Intraoperative awareness: From neurobiology to clinical practice. Anesthesiology. 2011 May;114(5):1218-1233.)
As inhalational anesthesia progressively deepens, initial beta activation is followed by slowing, burst suppression, and isoelectricity. Intravenous agents, depending on dose and drug used, can produce a variety of EEG patterns.

To reduce the incidence of awareness during general anesthesia, devices have been developed in recent years that process two-channel EEG signals and create a dimensionless variable to indicate level of wakefulness. The bispectral index (BIS) is most commonly used in this regard. BIS monitors examine four components within the EEG that are associated with the anesthetic state: (1) low frequency, as found during deep anesthesia; (2) high-frequency beta activation found during "light" anesthesia; (3) suppressed EEG waves; and (4) burst suppression.
Other devices attempt to include measures of spontaneous muscle activity, as influenced by the activity of subcortical structures not contributing to the EEG to further provide an assessment of anesthetic depth. Various devices, each with its own algorithm to process the EEG or incorporate other variables to ascertain patient wakefulness, may become available in the future (Table 6—1).

state index (cst), Consumable = Ordinary ECG electrode. Cerebral state index (cst), Physiologic Signals = Single channel EEG. Cerebral state index (cst), Recommended Range of Values for Anesthesia = 40-60. Cerebral state index (cst), _Principles of Measurement = CSlis a weig) rween the two and (4) burst sup y an “adaptive newro~ fuzzy
d with permission from Chan MTV, Gi 'ephalogram: ECG. electroca Controversy still exists as to the exact role of £ processed EEG devices in assessing anesthetic depth. Some studies have demonstrated a reduced incidence of awareness when these devices were used, whereas other studies have failed to reveal any advantage over the use of end-tidal inhalational gas measurements to ensure an adequate concentration of volatile anesthetic. Because individual EEG responsiveness to anesthetic agents and level of surgical stimulus are variable, ventional neurophysiologic monitoring. Curr Opin Anaesthesiol, 2004 Oct;! 7(54389-396, evoked potential
EEG monitoring to assess anesthesia depth or titrate anesthetic delivery may not always ensure absence of wakefulness. Moreover, many monitors have a delay, which might only indicate a risk for the patient being aware after he or she had already become conscious (Table 6—2).
TABLE 6-2 Checklist for preventing awareness.!

¥ Check all equipment, drugs, and dosages; ensure that drugs are clearly labeled and that infusions are running into veins.
¥ Consider administering an amnesic premedication.
Y Avoid or minimize the administration of muscle relaxants. Use a peripheral nerve stimulator to guide minimal required dose.
¥ Consider using the isolated forearm technique if intense paralysis is indicated.
¥ Choose potent inhalation agents rather than total intravenous anesthesia, if possible.
¥ Administer at least 0.5-0.7 minimum alveolar concentration (MAC) of the inhalation agent.
¥ Set an alarm fora low anesthetic gas concentration.
¥ Monitor anesthetic gas concentration during cardiopulmonary bypass from the bypass machine.
¥ Consider alternative treatments for hypotension other than decreasing anesthetic concentration.
v lfitis thought that sufficient anesthesia cannot be administered because of concern about hemodynamic compromise, consider the administration of benzodiazepines or scopolamine for amnesia.
¥ Supplement hypnotic agents with analgesic agents such as opioids or local anesthetics, which may help decrease the experience of pain in the event of awareness.
¥ Consider using a brain monitor, such as a raw or processed electroencephalogram but do not try to minimize the anesthetic dose based on the brain monitor because there currently is insufficient evidence to support this practice.
¥ Monitor the brain routinely if using total intravenous anesthesia.
¥ Evaluate known risk factors for awareness, and if specific risk factors are identified consider increasing administered anesthetic concentration.
¥ Re-dose intravenous anesthesia when delivery of inhalation anesthesia is difficult, such as during a long intubation attempt or during rigid bronchoscopy.
'Reproduced with permission from Mashour GA, Orser BA, Avidan MS. Intraoperative awareness: from neurobiology to clinical practice. Anesthesiology. 2011 May;114(S):1218-1233.

To perform a bispectral analysis, data measured by EEG are taken through a number of steps (Figure 6—8) to calculate a single number that correlates with depth of anesthesia/hypnosis.
FIGURE 6-8 Calculation of the bispectral index. EEG, electroencephalogram; BSR, burst suppression ratio; BIS, bispectral index scale. (Reproduced with permission from Rampil IJ: A primer for EEG signal processing in anesthesia. Anesthesiology. 1998 Oct;89(4):9801002.)
BIS values of 65 to 85 have been advocated as a measure of sedation, whereas values of 40 to 65 have been recommended for general anesthesia (Figure 6—9). Bispectral analysis may reduce patient awareness during

FIGURE 6-9 The bispectral index (BIS versions 3.0 and higher) is a dimensionless scale from 0 (complete cortical electroencephalographic suppression) to 100 (awake). BIS values of 65-85 have been recommended for sedation, whereas values of 40-65 have been recommended for general anesthesia. At BIS values lower than 40, cortical suppression becomes discernible in a raw electroencephalogram as a burst suppression pattern. (Reproduced with permission from Johansen JW et al: Development and clinical application of electroencephalographic bispectrum monitoring. Anesthesiology. 2000 Nov;93(5):1337-1344.)
Many of the initial studies of BIS were not prospective, randomized, controlled trials, but were observational in nature. The monitor costs several thousand dollars and the single-use electrodes cost approximately $10 to $15 per anesthetic. Some patients with awareness have been identified as having a BIS of less than 65. However, in other patients with awareness, either there were problems with the recordings, or awareness could not be related to any specific time or BIS value.
Detection of awareness often can minimize its consequences. Use of the Brice questions during postoperative visits can identify a potential awareness event. Ask patients to recall the following:
« What do you remember before going to sleep?
¢ What do you remember right when awakening?
* Do you remember anything in between going to sleep and awakening?
¢ Did you have any dreams while asleep?
Close follow-up and involvement of mental health experts may avoid the traumatic stress that can be associated with awareness events. Increasingly, patients are managed with regional anesthesia and propofol sedation. Patients undergoing such anesthetics should be made aware that they are not having general anesthesia and might recall perioperative events. Clarification of the techniques used may prevent patients so managed from the belief that they "were awake" during anesthesia.
Can BIS predict outcomes? Some investigators have suggested that both hospital stay and mortality are increased in patients experiencing the so called "triple low" of low mean arterial blood pressure, low bispectral index score, and low minimum alveolar concentration of volatile anesthetics. Other investigations have failed to identify such an association.

Indications for intraoperative monitoring of evoked potentials (EPs) include surgical procedures associated with possible neurological injury: spinal fusion with instrumentation, spine and spinal cord tumor resection, brachial plexus repair, thoracoabdominal aortic aneurysm repair, epilepsy surgery, and cerebral tumor resection. Ischemia in the spinal cord or cerebral cortex can be detected by EPs. Auditory EPs have also been used to assess the effects of general anesthesia on the brain. The middle latency auditory EP may be a more sensitive indicator than BIS in regard to anesthetic depth. The amplitude and latency of this signal following an auditory stimulus is influenced by anesthetics.

Although there are no specific contraindications for somatosensory evoked potentials (SEPs), this modality is severely limited by the availability of monitoring sites, equipment, and trained personnel. Sensitivity to anesthetic agents can also be a limiting factor, particularly in children. Motor evoked potentials (MEPs) are contraindicated in patients with retained intracranial metal, skull defects, or implantable devices, as well as after seizures and any major cerebral insult. Brain injury secondary to repetitive stimulation of the cortex and inducement of seizures is a concern with MEPs.

EP monitoring noninvasively assesses neural function by measuring electrophysiological responses to sensory or motor pathway stimulation. Commonly monitored EPs are brainstem auditory evoked responses (BAERs), SEPs, and increasingly, MEPs (Figure 6-10).
A
FIGURE 6-10 Neuroanatomic pathways of somatosensory evoked potential (SEP) and motor evoked potential (MEP). The SEP is produced by stimulation of a peripheral nerve wherein a response can be measured. The electrical volley ascends the spinal cord by the posterior columns and can be recorded in the epidural space and over the posterior cervical spine. It crosses the mid-line after synapsing at the cervicomedullary junction and ascends the lemniscal pathways having a second synapse in the thalamus. From there, it travels to the primary sensory cortex where the cortical response is measured. The MEP is produced by stimulation of the motor cortex leading to an electrical volley that descends to the anterior horn cells of the spinal cord via the corticospinal tract. After synapsing there it travels via a peripheral nerve and crosses the neuromuscular junction (NMJ) to produce a muscle response. The MEP can be measured in the epidural space as D and I waves produced by direct and indirect (via internuncial neurons) stimulation of the motor cortex, respectively. It can also be measured as a compound muscle action potential (CMAP) in the muscle. (Reproduced with permission from Sloan TB, Janik D, Jameson L. Multimodality monitoring of the central nervous system using motor-evoked potentials. Curr Opin Anaesthesiol. 2008 Oct;21(5):560-564.)

For SEPs, a brief electrical current is delivered to a sensory or mixed peripheral nerve by a pair of electrodes. If the intervening pathway is intact, a nerve action potential will be transmitted to the contralateral sensory cortex to produce an EP. This potential can be measured by cortical surface electrodes, but is usually measured by scalp electrodes. To distinguish the cortical response to a specific stimulus, multiple responses are averaged and background noise is eliminated. EPs are represented by a plot of voltage versus time. The resulting waveforms are analyzed for their poststimulus latency (the time between stimulation and potential detection) and peak amplitude. These are compared with baseline tracings. Technical and physiological causes of a change in an EP must be distinguished from changes due to neural damage. Complications of EP monitoring are rare, but include skin irritation and pressure ischemia at the sites of electrode application.

EPs are altered by many variables other than neural damage. The effect of anesthetics is complex and not easily summarized. In general, intravenous anesthetic techniques (with or without nitrous oxide) cause minimal changes, whereas volatile agents (halothane, sevoflurane, desflurane, and isoflurane) are best avoided or used at a constant low concentration. Earlyoccurring (specific) EPs are less affected by anesthetics than are late-occurring (nonspecific) responses. Changes in BAERs may provide a measure of the depth of anesthesia. Physiological (eg, blood pressure, temperature, and oxygen saturation) and pharmacological factors should be kept as constant as possible. Persistent obliteration of EPs is predictive of postoperative neurological deficit. Although SEPs usually identify spinal cord damage, because of their different anatomic pathways, sensory (dorsal spinal cord) EP preservation does not guarantee normal motor (ventral spinal cord) function (false negative). Furthermore, SEPs elicited from posterior tibial nerve stimulation cannot distinguish between peripheral and central ischemia (false positive). Techniques that elicit MEPs by using transcranial magnetic or electrical stimulation of the cortex allow the detection of action potentials in the muscles if the neural pathway is intact. The advantage of using MEPs as opposed to SEPs for spinal cord monitoring is that MEPs monitor the ventral spinal cord, and if sensitive and specific enough, can be used to indicate which patients might develop a postoperative motor deficit. MEPs are more sensitive to spinal cord ischemia than are SEPs. The same considerations for SEPs are applicable to MEPs in that they are reduced in amplitude by volatile inhalational agents, high-dose benzodiazepines, and moderate hypothermia (temperatures <32°C). MEPs require monitoring of the level of neuromuscular blockade. Close communication with a neurophysiologist or monitoring technician is essential prior to the start of any case where these monitors are used to review the optimal anesthetic technique to ensure monitoring integrity.

Cerebral oximetry uses near-infrared spectroscopy (NIRS). Using reflectance spectroscopy near-infrared light is emitted by a probe on the scalp (Figure 6— 11). Receptors are likewise positioned to detect the reflected light from both deep and superficial structures. As with pulse oximetry, oxygenated and deoxygenated hemoglobin absorb light at different frequencies. Likewise, cytochrome absorbs infrared light in the mitochondria. The NIRS saturation largely reflects the absorption of venous hemoglobin, as it does not have the ability to identify the pulsatile arterial component. Regional saturations of less than 40% on NIRS measures, or changes of greater than 25% of baseline measures, may herald neurological events secondary to decreased cerebral oxygenation.
FIGURE 6-11 Principle of the INVOS® near-infrared spectroscopy technique. (Reproduced with permission from Rubio A, Hakami L, Miinch F, et al. Noninvasive control of adequate cerebral oxygenation during low-flow antegrade selective cerebral perfusion on adults and infants in the aortic arch surgery. J Card Surg. 2008 Sep-Oct;23(5):474—479.)
Reduced jugular venous bulb saturation can also provide an indication of increased cerebral tissue oxygen extraction or decreased cerebral oxygen delivery. Direct tissue oxygen monitoring of the brain is accomplished by placement of a probe to determine the oxygen tension in the brain tissue. In addition to maintaining a cerebral perfusion pressure that is greater than 60 mm Hg and an intracranial pressure that is less than 20 mm Hg, neuroanesthesiologists/intensivists attempt to preserve brain tissue oxygenation by intervening when oxygen tissue tension is less than 20 mm Hg. Such interventions center upon improving oxygen delivery by increasing Fio,, augmenting hemoglobin, adjusting cardiac output, decreasing oxygen demand, or a combination of these methods.

The temperature of patients undergoing anesthesia should be monitored during use of all but the shortest anesthetics. Postoperative temperature is increasingly used as a quality anesthesia indicator. Hypothermia is associated with delayed drug metabolism, increased blood glucose, vasoconstriction, impaired coagulation, postoperative shivering accompanied by tachycardia and hypertension, and increased risk of surgical site infections. Hyperthermia can likewise have deleterious effects perioperatively, leading to tachycardia, vasodilation, and neurological injury. Consequently, temperature must be measured and recorded perioperatively.

There are no contraindications, although a particular monitoring site may be unsuitable in certain patients.

Intraoperatively, temperature is usually measured using a thermistor or thermocouple. Thermistors are semiconductors whose resistance decreases predictably with warming. A thermocouple is a circuit of two dissimilar metals joined so that a potential difference is generated when the metals are at different temperatures. Disposable thermocouple and thermistor probes are available for monitoring the temperature of the tympanic membrane, nasopharynx, esophagus, bladder, rectum, and skin. Infrared sensors estimate temperature from the infrared energy that is produced. Tympanic membrane temperatures reflect core body temperature; however, the devices used may not reliably measure the temperature at the tympanic membrane. Complications of temperature monitoring are usually related to trauma caused by the probe (eg, rectal or tympanic membrane perforation).

Each monitoring site has advantages and disadvantages. The tympanic membrane theoretically reflects brain temperature because the auditory canal's blood supply is the external carotid artery. Trauma during insertion and cerumen insulation detract from the routine use of tympanic probes. Rectal temperature probes have a slow response to changes in core temperature. Nasopharyngeal probes are prone to cause epistaxis, but accurately measure core temperature if placed adjacent to the nasopharyngeal mucosa. The thermistor in a pulmonary artery catheter also measures core temperature. There is a variable correlation between axillary temperature and core temperature, depending on skin perfusion. Liquid crystal adhesive strips placed on the skin are inadequate indicators of core body temperature during surgery. Esophageal temperature sensors, often incorporated into esophageal stethoscopes, provide the best combination of economy, performance, and safety. To avoid measuring the temperature of tracheal gases, the temperature sensor should be positioned behind the heart in the lower third of the esophagus. Conveniently, heart sounds are most prominent at this location. For more on the clinical considerations of temperature control, see Chapter 52.

Indications
Urinary bladder catheterization is the most reliable method of monitoring urinary output. Catheterization is routine in some complex and prolonged surgical procedures such as cardiac surgery, aortic or renal vascular surgery, craniotomy, major abdominal surgery, or procedures in which large fluid shifts are expected. Lengthy surgeries and intraoperative diuretic administration are other possible indications. Occasionally, postoperative bladder catheterization is indicated in patients who have difficulty voiding in the recovery room after general or regional anesthesia.

Foley catheters should be removed as soon as feasible to avoid the risk of catheter-associated urinary tract infections.

Bladder catheterization is usually performed by surgical or nursing personnel. The urologist may be needed to catheterize patients with strictures and other abnormal urethral anatomy. A soft rubber Foley catheter is inserted into the bladder transurethrally and connected to a disposable calibrated collection chamber. To avoid urine reflux and minimize the risk of infection, the collection chamber should remain at a level below the bladder. Complications of catheterization include urethral trauma and urinary tract infections. Rapid decompression of a distended bladder can cause hypotension. Suprapubic drainage of the bladder with tubing inserted through a large-bore needle is an uncommon alternative.

An additional advantage of placing a Foley catheter is the ability to include a thermistor in the catheter tip so that bladder temperature can be monitored. As long as urinary output is high, bladder temperature accurately reflects core temperature. An added value with more widespread use of urometers is the ability to electronically monitor and record urinary output and temperature. Urinary output is an imperfect reflection of kidney perfusion and function and of renal, cardiovascular, and fluid volume status. Noninvasive monitors of cardiac function and output (including echocardiography) provide more reliable assessments of the adequacy of intravascular volume. Inadequate urinary output (oliguria) is often arbitrarily defined as urinary output of less than 0.5 mL/kg/h, but actually is a function of the patient's concentrating ability and osmotic load. Urine electrolyte composition, osmolality, and specific gravity aid in the differential diagnosis of oliguria.

Because of the variation in patient sensitivity to neuromuscular blocking agents, the neuromuscular function of all patients receiving intermediateor long-acting neuromuscular blocking agents should be monitored. In addition, peripheral nerve stimulation is helpful in detecting the onset of paralysis during anesthesia inductions or the adequacy of block during continuous infusions with shortacting agents.

There are no contraindications to neuromuscular monitoring, although certain sites may be precluded by the surgical procedure. Additionally, atrophied muscles in areas of hemiplegia or nerve damage may appear refractory to neuromuscular blockade secondary to the proliferation of receptors. Determining the degree of neuromuscular blockade using such an extremity could lead to potential overdosing of competitive neuromuscular blocking agents.

A peripheral nerve stimulator delivers current (60-80 mA) to a pair of either ECG silver chloride pads or subcutaneous needles placed over a peripheral motor nerve. The evoked mechanical or electrical response of the innervated muscle is observed. Although electromyography provides a fast, accurate, and quantitative measure of neuromuscular transmission, visual or tactile observation of muscle contraction is usually relied upon in clinical practice. Ulnar nerve stimulation of the adductor pollicis muscle and facial nerve stimulation of the orbicularis oculi are most commonly monitored (Figure 6—12). Because it is the inhibition of the neuromuscular receptor that needs to be monitored, direct stimulation of muscle should be avoided by placing electrodes over the course of the nerve and not over the muscle itself. To deliver a supramaximal stimulation to the underlying nerve, peripheral nerve stimulators must be capable of generating at least a 50-mA current across a 1000-Q load. This current is uncomfortable for a conscious patient. Complications of nerve stimulation are limited to skin irritation and abrasion at the site of electrode attachment.
FIGURE 6-12 A: Stimulation of the ulnar nerve causes contraction of the adductor pollicis muscle. B: Stimulation of the facial nerve leads to orbicularis oculi contraction. The orbicularis oculi recovers from neuromuscular blockade before the adductor pollicis. (Reproduced with permission from Dorsch JA, Dorsch SE. Understanding Anesthesia Equipment. 4th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 1999.)
Because of concerns of residual neuromuscular blockade, increased attention has been focused on providing quantitative measures of the degree of neuromuscular blockade perioperatively. Acceleromyography uses a piezoelectric transducer on the muscle to be stimulated. Movement of the muscle generates an electrical current that can be quantified and displayed. Indeed, acceleromyography can better predict residual paralysis, compared with routine tactile train-of-four monitoring used in most operating rooms, if calibrated from the beginning of the operative period to establish baselines prior to administration of neuromuscular blocking agents.

The degree of neuromuscular blockade is monitored by applying various patterns of electrical stimulation (Figure 6—13). All stimuli are 200 ps in duration and of square-wave pattern and equal current intensity. A twitch is a single pulse that is delivered from every 1 to every 10 s (1—-0.1 Hz). Increasing block results in decreased evoked response to stimulation.
FIGURE 6-13 Peripheral nerve stimulators can generate various patterns of electrical impulses.
Train-of-four stimulation denotes four successive 200-ps stimuli in 2 s (2 Hz).

Monitoring During Magnetic Resonance Imaging
A50-year-old man with recent onset of seizures is scheduled for magnetic resonance (MR) imaging. A prior MR attempt was unsuccessful because of the patient's severe claustrophobic reaction. The radiologist requests your help in providing either sedation or general anesthesia.
Why does the MR suite pose special problems for the patient and the anesthesiologist?
MR studies tend to be long (often more than 1 h), and many scanners totally surround the body, causing a high incidence of claustrophobia in patients already anxious about their health. Good imaging requires immobility, something that is difficult to achieve in many patients without sedation or general anesthesia.
Because the MR device uses a powerful magnet, no ferromagnetic objects can be placed near the scanner. This includes implanted prosthetic joints, artificial pacemakers, surgical clips, batteries, ordinary anesthesia machines, watches, pens, credit cards, intravenous poles, conventional oxygen cylinders, housekeeping buckets, and lead ankle weights, nearly all of which we have seen drawn in and "captured" by MR devices. Ordinary metal lead wires for pulse oximeters or electrocardiography act as antennas and may attract enough radiofrequency energy to distort the MR image or even cause patient burns. In addition, the scanner's magnetic field causes severe monitor artifact. The more powerful the scanner's magnet, as measured in Tesla units (1 T = 10,000 gauss), the greater is the potential problem. Other obstacles include poor access to the patient during the imaging (particularly the patient's airway), hypothermia in pediatric patients, dim lighting within the patient tunnel, and very loud noise (up to 100 dB).
How have these monitoring and anesthesia machine problems been addressed?
Equipment manufacturers have modified monitors so that they are compatible with the MR environment. These modifications include nonferromagnetic electrocardiographic electrodes, graphite and copper cables, extensive filtering and gating of signals, extra-long blood pressure cuff tubing, and use of fiberoptic technologies. Anesthesia machines with no ferromagnetic components (eg, aluminum gas cylinders) have been fitted with MR-compatible ventilators and long circle systems or Mapleson D breathing circuits.

Although most patients will tolerate an MR study with sedation, headinjured and pediatric patients present special challenges and often require general anesthesia. Because of machine and monitoring limitations, an argument could be made that sedation, when possible, would be a safer choice. On the other hand, loss of airway control from deep sedation could prove catastrophic because of poor patient access and delayed detection. Other important considerations include the monitoring modalities available at a particular facility and the general medical condition of the patient.

The patient should receive at least the same level of monitoring and care in the MR suite as in the operating room for a similarly noninvasive procedure. Thus, the American Society of Anesthesiologists Standards for Basic Anesthetic Monitoring (see following Guidelines section) apply as they would to a patient undergoing general anesthesia.
Continuous auscultation
of breath
sounds with
a
plastic
(not metal)
precordial stethoscope
excessive can
help sedation.
Korotkoff to
identify
Palpation sounds
is circulation
of impractical
depends
a
airway peripheral
obstruction pulse
in this
setting.
on pressure
electrocardiographic monitoring.
cases by
End-tidal connecting
nose if
nasal caused
or listening
Ensuring and
CO, analyzers
the cannula
Because room
technique sampling
with line
a
CO, air
entrainment provides
is planned,
by for
adequacy oscillometric
can to
a
of blood
be adapted
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precludes
a
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near the
channel exact
indicator for
emergency to
sedation patient’s
mouth are
not or
available.
measurements, of
ventilation.
this
Whenever conversion
to general
sedation anesthesia
(eg, tracheal
tubes, resuscitation
bag)
must be
immediately available.
Is the continuous presence of anesthesia personnel required

Absolutely yes. Sedated patients must have continuous monitored anesthesia care to prevent a multitude of unforeseen complications, such as apnea or emesis.

American
Society of Anesthesiologists Standards for Basic Anesthetic Monitoring, July 2011. http://www.asahq.org/For-Members/StandardsGuidelines-and-Statements.aspx. Accessed January 9, 2013.

@ Drug molecules obey the law of mass action. When the plasma concentration exceeds the tissue concentration, the drug moves from the plasma into tissue. When the plasma concentration is less than the tissue concentration, the drug moves from the tissue back to plasma.
@® Most drugs that readily cross the blood—brain barrier (eg, lipophilic drugs like hypnotics and opioids) are avidly taken up in body fat.
© Biotransformation is the chemical process by which the drug molecule is altered in the body. The liver is the primary organ of metabolism for drugs.
@ Small unbound molecules freely pass from plasma into the glomerular filtrate. The nonionized (uncharged) fraction of drug is reabsorbed in the renal tubules, whereas the ionized (charged) portion is excreted in urine.
@ Elimination half-life is the time required for the drug concentration to fall by 50%. For drugs described by multicompartment pharmacokinetics (eg, all drugs used in anesthesia), there are multiple elimination half-lives.
@ The offset of a drug's effect cannot be predicted from half-lives. The context-sensitive half-time is a clinically useful concept to describe the rate of decrease in drug concentration and should be used instead of half-lives to compare the pharmacokinetic properties of intravenous drugs used in anesthesia.
The clinical practice of anesthesiology is directly connected to the science of clinical pharmacology. One would think, therefore, that the study of pharmacokinetics and pharmacodynamics would receive attention comparable to
that given to airway assessment, choice of inhalation anesthetic, neuromuscular blockade, or treatment of sepsis in anesthesiology curricula and examinations. Sadly, the frequent misidentification or misuse of pharmacokinetic principles and measurements suggests that this is not yet the case.

Pharmacokinetics defines the relationships among drug dosing, drug concentration in body fluids and tissues, and time. It consists of four linked processes: absorption, distribution, biotransformation, and excretion.

All venous drainage from the stomach and small intestine flows to the liver. As a result, the bioavailability of highly metabolized drugs may be significantly reduced by first-pass hepatic metabolism. Because the venous drainage from the mouth and esophagus flows into the superior vena cava rather than into the portal system, sublingual or buccal drug absorption bypasses the liver and firstpass metabolism. Rectal administration partly bypasses the portal system, and represents an alternative route in small children or patients who are unable to tolerate oral ingestion. However, rectal absorption can be erratic, and many drugs irritate the rectal mucosa.
Transdermal drug administration can provide prolonged continuous administration for some drugs. However, the stratum corneum is an effective barrier to all but small, lipid-soluble drugs (eg, clonidine, nitroglycerin, scopolamine, fentanyl, and free-base local anesthetics [EMLA]).
Parenteral routes of drug administration include subcutaneous, intramuscular, and intravenous injection. Subcutaneous and intramuscular absorption depend on drug diffusion from the site of injection to the bloodstream. The rate at which a drug enters the bloodstream depends on both blood flow to the injected tissue and the injectate formulation. Drugs dissolved in solution are absorbed faster than those present in suspensions. Irritating preparations can cause pain and tissue necrosis (eg, intramuscular diazepam). Intravenous injections bypass the process of absorption.

Once absorbed, a drug is distributed by the bloodstream throughout the body. Highly perfused organs (the so-called vessel-rich group) receive a disproportionate fraction of the cardiac output (Table 7—1). Therefore, these tissues receive a disproportionate amount of drug in the first minutes following drug administration. These tissues approach equilibration with the plasma concentration more rapidly than less well perfused tissues due to the differences in blood flow. However, less well perfused tissues such as fat and skin may have enormous capacity to absorb lipophilic drugs, resulting in a large reservoir of drug following long infusions.
TABLE 7-1 Tissue group composition, relative body mass, and percentage of cardiac output.

that half-lives provide almost no guidance for predicting emergence times. The offset of a drug's clinical actions is best predicted by computer models using the context sensitive half-time or decrement time. The context-sensitive half-time is the time required for a 50% decrease in plasma drug concentration to occur following a pseudo steady-state infusion (in other words, an infusion that has continued long enough to yield nearly steady-state concentrations). Here the "context" is the duration of the infusion. The context-sensitive decrement time is a more generalized concept referring to any clinically relevant decreased concentration in any tissue, particularly the brain or effect site.
The volume of distribution, V,, is the apparent volume into which a drug has "distributed" (ie, mixed). This volume is calculated by dividing a bolus dose of drug by the plasma concentration at time 0. In practice, the concentration used to define the V, is often obtained by extrapolating subsequent concentrations back to "O time" when the drug was injected (this assumes immediate and complete mixing), as follows:
The concept of a single Vy does not apply to any intravenous drugs used in anesthesia. All intravenous anesthetic drugs are better modeled with at least two compartments: a central compartment and a peripheral compartment. The behavior of many of these drugs is more precisely described using three compartments: a central compartment, a rapidly equilibrating peripheral compartment, and a slowly equilibrating peripheral compartment. The central compartment may be thought of as including the blood and any ultra-rapidly equilibrating tissues such as the lungs. The peripheral compartment is composed of the other body tissues. For drugs with two peripheral compartments, the rapidly equilibrating compartment comprises the organs and muscles, while the slowly equilibrating compartment roughly represents distribution of the drug into fat and skin. These compartments are designated V, (central), V, (rapid distribution), and V3 (slow distribution). The volume of distribution at steady state, V4, is the algebraic sum of these compartment volumes. V, is calculated by the above equation showing the relationship between volume, dose, and concentration. The other volumes are calculated through pharmacokinetic modeling.

Asmall V,,, implies that the drug has high aqueous solubility and will remain largely within the intravascular space. For example, the V;,, of vecuronium is about 200 mL/kg in adult men and about 160 mL/kg in adult women, indicating that vecuronium is mostly present in body water, with little distribution into fat. However, the typical anesthetic drug is lipophilic, resulting in a V,,, that exceeds total body water (approximately 600 mL/kg in adult males). For example, the Vas; for fentanyl is about 350 L in adults, and the V,,, for propofol may exceed 5000 L. V,,, does not represent a real volume but rather reflects the volume into which the administered drug dose would need to distribute to account for the observed plasma concentration.

because the liver removes almost all of the propofol that goes through it, if the hepatic blood flow doubles, then the clearance of propofol doubles. Induction liver enzymes has no effect on propofol clearance, because the liver so efficiently removes all of the propofol that goes through it. Even severe loss of liver tissue, as occurs in cirrhosis, has little effect on propofol clearance. Drugs such as propofol have flow-dependent clearance.
Many extraction ratios are liver. For these drugs, the rate-limiting step is not the flow of blood to the liver, but rather the metabolic capacity of the liver itself. Changes in liver blood flow have little effect on the clearance of such drugs. However, if liver enzymes are induced, then clearance will increase because the liver has more capacity to metabolize the drug. Conversely, if the liver is damaged, then less capacity is available for metabolism and clearance is reduced. Drugs with low hepatic extraction ratios thus have capacity-dependent clearance. The extraction ratios of methadone and alfentanil are 10% and 15% respectively, making these capacitydependent drugs.
of drugs have low hepatic and slowly cleared by the

Some drugs and many drug metabolites are excreted by the kidneys. Renal clearance is the rate of elimination of a drug from the body by kidney excretion. This concept is analogous to hepatic clearance, and similarly, renal clearance can be expressed as the renal blood flow times the renal extraction ratio. @ Small unbound drugs freely pass from plasma into the glomerular filtrate. The nonionized (uncharged) fraction of drug is reabsorbed in the renal tubules, whereas the ionized (charged) portion remains and is excreted in urine. The fraction of drug ionized depends on the pH; thus renal elimination of drugs that exist in ionized and nonionized forms depends in part on urinary pH. The kidney actively secretes some drugs into the renal tubules.
Many drugs and drug metabolites pass from the liver into the intestine via the biliary system. Some drugs excreted into the bile are then reabsorbed in the intestine, a process called enterohepatic recirculation. Occasionally metabolites excreted in bile are subsequently converted back to the parent drug. For example, lorazepam is converted by the liver to lorazepam glucuronide. In the intestine, B-glucuronidase breaks the ester linkage, converting lorazepam glucuronide back to lorazepam.

FIGURE 7-1 Two-compartment model demonstrates the changes in drug concentrations in the distribution phase and the elimination phase. During the distribution phase, the drug moves from the central compartment to the peripheral compartment. In the elimination phase the drug returns from the peripheral compartment to the central compartment and is metabolized and excreted.
As previously noted, in compartmental models the instantaneous concentration at the time of a bolus injection is assumed to be the amount of the bolus divided by the central compartment volume. This is not correct. If the bolus is given over a few seconds, the instantaneous concentration is 0, because the drug is all in the vein, still flowing to the heart. It takes a minute or two for the drug to mix in the central compartment volume. This misspecification is common to conventional pharmacokinetic models. More physiologically based models, sometimes called front-end kinetic models, can characterize the initial delay in concentration. The additional complexity that these models introduce is useful only if the concentrations over the first few minutes are clinically important. After the first few minutes, front-end models resemble conventional compartmental models.
In the first few minutes following initial bolus administration of a drug the concentration drops very rapidly as the drug quickly diffuses into peripheral compartments. Concentrations often decline by an order of magnitude over 10 minutes! For drugs with very rapid hepatic clearance (eg, propofol) or those that are metabolized in the blood (eg, remifentanil), metabolism contributes significantly to the rapid initial drop in concentration. Following this very rapid drop there is a period of slower decrease in plasma concentration. During this period, the rapidly equilibrating compartment is no longer removing drug from the plasma. Instead, drug returns to the plasma from the rapidly equilibrating compartment. The reversed role of the rapidly equilibrating tissues from extracting drug to returning drug accounts for the slower rate of decline in plasma concentration in this intermediate phase. Eventually there is an even slower rate of decrease in plasma concentration, which is log-linear until the drug is completely eliminated from the body. This terminal log-linear phase occurs after the slowly equilibrating compartment shifts from net removal of drug from the plasma to net return of drug to the plasma. During this terminal phase, the organ of elimination (typically the liver) is exposed to the body's entire body drug load, which accounts for the very slow rate of decrease in plasma drug concentration during this final phase.

The mathematical models used to describe a drug with two or three compartments are, respectively:
and
where Cp(t) equals plasma concentration at time t, and a, B, and y are the exponents that characterize the very rapid (ie, very steep), intermediate, and slow (ie, log-linear) portions of the plasma concentration over time, respectively. Drugs described by two-compartment and three-compartment models will have two or three half-lives. Each half-life is calculated as the natural log of 2 (0.693), divided by the exponent. The coefficients A, B, and C represent the contribution of each of the exponents to the overall decrease in concentration over time. The two-compartment model is described by a curve with two exponents and two coefficients, whereas the three-compartment model is described by a curve with three exponents and three coefficients. The mathematical relationships among compartments, clearances, coefficients, and exponents are complex. Every coefficient and every exponent is a function of every volume and every clearance.

@ Elimination half-time is the time required for the drug concentration to fall by 50%. For drugs described by multicompartment pharmacokinetics (eg, fentanyl, sufentanil), there are multiple elimination half-times, in other words the elimination @ half-time is context dependent. The offset of a drug's effect cannot be predicted from half-lives alone. Moreover, one cannot easily determine how rapidly a drug effect will disappear simply by looking at coefficients, exponents, and half-lives. For example, the terminal half-life of sufentanil is about 10 h, whereas that of alfentanil is 2 h. This does not mean that recovery from alfentanil will be faster, because clinical recovery from clinical dosing will be influenced by all half-lives, not just the terminal one. Computer models readily demonstrate that recovery from an infusion lasting several hours will be faster when the drug administered is sufentanil than it will be when the infused drug is alfentanil. The time required for a 50% decrease in concentration depends on the duration or "context" of the infusion. The context-sensitive half-time, mentioned earlier, captures this concept and should be used instead of half-lives to compare the pharmacokinetic properties of intravenous drugs used in anesthesia.

Pharmacodynamics, the study of how drugs affect the body, involves the concepts of potency, efficacy, and therapeutic window. Pharmacokinetic models can range from entirely empirical dose versus response relationships to mechanistic models of ligand-receptor binding. The fundamental pharmacodynamic concepts are captured in the relationship between exposure to a drug and physiological response to the drug, often called the dose-response or concentration—response relationship.

As the body is exposed to an increasing amount of a drug, the response to the drug similarly increases, typically up to a maximal value. This fundamental concept in the exposure versus response relationship is captured graphically by plotting exposure (usually dose or concentration) on the x axis as the independent variable, and the body's response on the y axis as the dependent variable. Depending on the circumstances, the dose or concentration may be plotted on a linear scale (Figure 7—2A) or a logarithmic scale (Figure 7—2B), while the response is typically plotted either as the actual measured response (Figure 7—2A) or as a fraction of the baseline or maximum physiological measurement (Figure 7—2B). For our purposes here, basic pharmacodynamic

FIGURE 7-2 The shape of the dose (or concentration)—response curve depends on whether the dose or plasma concentration is plotted on a linear (A) or logarithmic (B) scale.
The shape of the relationship is typically sigmoidal, as shown in Figure 7-2. The sigmoidal shape reflects the observation that often a certain minimal amount of drug must be present before there is any measurable physiological response. Thus, the left side of the curve is flat until the drug concentration reaches a threshold. The right side is also flat, reflecting the maximum physiological response of the body, beyond which the body simply cannot respond to additional drug. Thus, the curve is flat on both the left and right sides. A sigmoidal curve is required to connect the baseline to the asymptote, which is why sigmoidal curves are ubiquitous when modeling pharmacodynamics.
The sigmoidal relationship between exposure and response is defined by one of two interchangeable relationships:
or
In both cases, Ey is the baseline effect in the absence of drug, C is drug concentration, Cs is the concentration associated with half-maximal effect, and y describes the steepness of the concentration versus response relationship (and is also known as the Hill coefficient). For the first equation, E,,,, is the maximum change from baseline. In the second equation, E,,,,, is the maximum physiological measurement, not the maximum change from baseline.

Once defined in this fashion, each parameter of the pharmacodynamic model speaks to the specific concepts mentioned earlier. E,,,,, is related to the intrinsic efficacy of a drug. Highly efficacious drugs have a large maximum physiological effect, characterized by a large E,,,,. For drugs that lack efficacy, E,,,, will equal Eo. Cs is a measure of drug potency. Highly potent drugs have a low C.,; thus small amounts produce the drug effect. Drugs lacking potency have a high Czo, indicating that a large amount of drug is required to achieve the drug effect. The parameter y indicates steepness of the relationship between concentration and effect. A y value less than 1 indicates a very gradual increase in drug effect with increasing concentration. A y value greater than 4 suggests that once drug effect is observed, small increases in drug concentration produce large increases in drug effect until the maximum effect is reached.
The curve described above represents the relationship of drug concentration to a continuous physiological response. The same relationship can be used to characterize the probability of a binary (yes/no) response to a drug dose:
In this case, the probability (P) ranges from 0 (no chance) to 1 (certainty). Pg max iS the maximum probability, necessarily less than or equal to 1. As before, C is the concentration, Csg is the concentration associated with half-maximal effect, and y describes the steepness of the concentration versus response relationship. Halfmaximal effect is the same as 50% probability of a response when Pp is 0 and P is the probability of a "yes" response in the absence of drug. P max iS 1.

The therapeutic window for a drug is the range between the concentration associated with a desired therapeutic effect and the concentration associated with a toxic drug response. This range can be measured as either the difference between two points on the same concentration versus response curve (when the toxicity represents an exaggerated form of the desired drug response) or the distance between two distinct curves (when the toxicity represents a different response or process from the desired drug response). For a drug such as sodium nitroprusside, a single concentration versus response curve defines the relationship between concentration and decrease in blood pressure. The therapeutic window might be the difference in the concentration producing a desired 20% decrease in blood pressure and a toxic concentration that produces a 60% decrease in blood pressure. However, for a drug such as lidocaine, the therapeutic window might be the difference between the C;, for suppression of ventricular arrhythmias and the C., for lidocaine-induced seizures, the two drug effects being described by separate concentration versus response relationships. The therapeutic index is the C:, for toxicity divided by the Cz, for the desired therapeutic effect. Because of the risk of ventilatory and cardiovascular depression (even at concentrations only slightly greater than those producing anesthesia), most inhaled and intravenous hypnotics are considered to have very low therapeutic indices relative to other drugs.

Drug receptors are macromolecules (typically proteins) that bind a drug (agonist) and mediate the drug response. Pharmacological antagonists reverse the effects of the agonist but do not otherwise exert an effect of their own. Competitive antagonism occurs when the antagonist competes with the agonist for the same binding site, each potentially displacing the other. Noncompetitive antagonism occurs when the antagonist, through covalent binding or another process, permanently impairs the drug's access to the receptor.
The drug effect is governed by the fraction of receptors that are occupied by an agonist. That fraction is based on the concentration of the drug, the concentration of the receptor, and the strength of binding between the drug and the receptor. This binding is described by the law of mass action, which states that the reaction rate is proportional to the concentrations of the reactants:
where [D] is the concentration of the drug, [RU] is the concentration of unbound receptor, and [DR] is the concentration of bound receptor. The rate constant k,, defines the rate of ligand binding to the receptor. The rate constant Kor defines the rate of ligand unbinding from the receptor. According to the law of mass action, the rate of receptor binding, d[DR]/dt is:
Steady state occurs almost instantly. Because the rate of formation at steady state is 0, it follows that:
In this equation, kg is the dissociation rate constant, defined as k,,/Korr. If we define f, fractional receptor occupancy, as:

then we can solve for receptor occupancy as:
The receptors are half occupied when [D] = kg. Thus, kg is the concentration of drug associated with 50% receptor occupancy.
Receptor occupancy is only the first step in mediating drug effect. Binding of the drug to the receptor can trigger myriad subsequent steps, including opening, closing, or inhibition of an ion channel; activation of a G protein; activation of an intracellular kinase; direct interaction with a cellular structure; or direct binding to DNA.
Like the concentration versus response curve, the shape of the curve relating fractional receptor occupancy to drug concentration is intrinsically sigmoidal. However, the concentration associated with 50% receptor occupancy and the concentration associated with 50% of maximal drug effect are not necessarily the same. Maximal drug effect could occur at very low receptor occupancy or (for partial agonists) at greater than 100% receptor occupancy.
Prolonged binding and activation of a receptor by an agonist may lead to hyporeactivity ("desensitization") and tolerance. If the binding of an endogenous ligand is chronically blocked or chronically reduced, then receptors may proliferate resulting in hyperreactivity and increased sensitivity. For example, after spinal cord injury nicotinic acetylcholine receptors are not stimulated by impulses in motor nerves and proliferate in denervated muscle. This can lead to exaggerated responses (including hyperkalemia) to succinylcholine.

Ansari J, Carvalho B, Shafer SL, Flood P. Pharmacokinetics and pharmacodynamics of drugs commonly used in pregnancy and parturition. Anesth Analg. 2016;122:786.
Brunton LL, Knollman BC, eds. Goodman & Gilman's The Pharmacological Basis of Therapeutics. 13th ed. New York, NY: McGraw-Hill; 2017: chap 2.
Bailey JM. Context-sensitive half-times: What are they and how valuable are they in anaesthesiology? Clin Pharmacokinet. 2002;41:793.
Shargel L, Yu ABC, eds. Applied Biopharmaceutics & Pharmacokinetics. 7th ed. New York, NY: McGraw-Hill; 2016.

@ The greater the uptake of anesthetic agent, the greater the difference between inspired and alveolar concentrations, and the slower the rate of induction.
@ Three factors affect anesthetic uptake: solubility in the blood, alveolar blood flow, and the difference in partial pressure between alveolar gas and venous blood.
@ Low-output states predispose patients to overdosage with soluble agents, as the rate of rise in alveolar concentrations will be markedly increased.
@ Many of the factors that speed induction also speed recovery: elimination of rebreathing, high fresh gas flows, low anesthetic-circuit volume, low absorption by the anesthetic circuit, decreased solubility, high cerebral blood flow, and increased ventilation.
© The unitary hypothesis proposes that all inhalation agents share a common mechanism of action at the molecular level. This was previously supported by the observation that the anesthetic potency of inhalation agents correlates directly with their lipid solubility (Meyer— Overton rule). The implication is that anesthesia results from molecules dissolving at specific lipophilic sites; however, the correlation is only approximate.
@ The minimum alveolar concentration (MAC) of an inhaled anesthetic is the alveolar concentration that prevents movement in 50% of patients in response to a standardized stimulus (eg, surgical incision).
@ Prolonged exposure to anesthetic concentrations of nitrous oxide can result in bone marrow depression (megaloblastic anemia) and even neurological deficiencies (peripheral neuropathies).
© "Halothane hepatitis" is extremely rare. Patients exposed to multiple
halothane anesthetics at short intervals, middle-aged obese women, and persons with a familial predisposition to halothane toxicity or a personal history of toxicity are considered to be at increased risk.

© Isoflurane dilates coronary arteries, but not nearly as potently as nitroglycerin or adenosine. Dilation of normal coronary arteries could theoretically divert blood away from fixed stenotic lesions.
@ The low solubility of desflurane in blood and body tissues causes a very rapid induction of and emergence from anesthesia.
@ Rapid increases in desflurane concentration lead to transient but sometimes worrisome elevations in heart rate, blood pressure, and catecholamine levels that are more pronounced than occur with isoflurane, particularly in patients with cardiovascular disease.
@ Nonpungency and rapid increases in alveolar anesthetic concentration make sevoflurane an excellent choice for smooth and rapid inhalation inductions in pediatric and adult patients.
Nitrous oxide, chloroform, and ether were the first universally accepted general anesthetics. Inhalation agents currently in widespread use in clinical anesthesiology include nitrous oxide, halothane, isoflurane, desflurane, and sevoflurane.
The course of a general anesthetic can be divided into three phases: (1) induction, (2) maintenance, and (3) emergence. Inhalation anesthetics, notably halothane and sevoflurane, are particularly useful in the induction of pediatric patients in whom it may be difficult to start an intravenous line. Although adults are usually induced with intravenous agents, the nonpungency and rapid onset of sevoflurane make inhalation induction practical for them as well. Regardless of the patient's age, anesthesia is often maintained with inhalation agents. Emergence depends primarily upon redistribution of the agent from the brain followed by pulmonary elimination. Because of their unique route of administration, inhalation anesthetics have useful pharmacological properties not shared by other anesthetic agents.

Although the mechanism of action of inhalation anesthetics is complex, likely involving multiple membrane proteins and ion channels, it is clear that producing their ultimate effect depends on attainment of a therapeutic tissue concentration in the central nervous system (CNS). There are many steps in between the anesthetic vaporizer and the anesthetic's deposition in the brain (Figure 8-1).
FGF (fresh gas flow) is determined by the vaporizer and flowmeter settings.
Anesthesia machine
Fi (inspired gas concentration) is determined by (1) FGF rate; (2) breathingcircuit volume; and (3) circuit absorption.
Fa (aveolar gas concentration) is determined by (1) uptake (uptake = Ab/g x C(A-V) x Q); (2) ventilation; and (3) the concentration effect and second gas effect: a) concentrating effect b) augmented inflow effect
Fa (arterial gas concentration) is affected by ventilation/perfusion mismatching.
FIGURE 8-1 Inhalation anesthetic agents must pass through many barriers between the anesthesia machine and the brain.

The fresh gas leaving the anesthesia machine mixes with gases in the breathing circuit before being inspired by the patient. Therefore, the patient is not necessarily receiving the concentration set on the vaporizer. The actual composition of the inspired gas mixture depends mainly on the fresh gas flow rate, the volume of the breathing system, and any absorption by the machine or breathing circuit. The greater the fresh gas flow rate, the smaller the breathing system volume, and the lower the circuit absorption, the closer the inspired gas concentration will be to the fresh gas concentration.
FACTORS AFFECTING ALVEOLAR CONCENTRATION (FA)

Nitrous oxide, Blood/ Gas = 0.47. Nitrous oxide, Brain/ Blood = 1.1. Nitrous oxide, Muscle/ Blood = 1.2. Nitrous oxide, Fat/ Blood = 2.3. Halothane, Blood/ Gas = 2.4. Halothane, Brain/ Blood = 2.9. Halothane, Muscle/ Blood = 3.5. Halothane, Fat/ Blood = 60. Isoflurane, Blood/ Gas = 1.4. Isoflurane, Brain/ Blood = 2.6. Isoflurane, Muscle/ Blood = 4.0. Isoflurane, Fat/ Blood = 45. Desflurane, Blood/ Gas = 0.42. Desflurane, Brain/ Blood = 1.3. Desflurane, Muscle/ Blood = 2.0. Desflurane, Fat/ Blood = 27. Sevoflurane, Blood/ Gas = 0.65. Sevoflurane, Brain/ Blood = AN;. Sevoflurane, Muscle/ Blood = 3.1. Sevoflurane, Fat/ Blood = 48
'These values are averages derived from multiple studies and should be used for comparison purposes, not as exact numbers.
The second factor that affects uptake is alveolar blood flow, which—in the absence of pulmonary shunting—is essentially equal to cardiac output. If the cardiac output drops to zero, so will anesthetic uptake. As cardiac output increases, anesthetic uptake increases, the rise in alveolar partial pressure slows, and induction is delayed. The effect of changing cardiac output is less pronounced for insoluble anesthetics, as so little is taken up regardless of alveolar blood ® flow. Low-output states predispose patients to overdosage with soluble agents, as the rate of rise in alveolar concentrations will be markedly increased.

The final factor affecting uptake of anesthetic by the pulmonary circulation is the partial pressure difference between alveolar gas and venous blood. This gradient depends on tissue uptake. If anesthetics did not pass into organs such as the brain, venous and alveolar partial pressures would become identical, and there would be no pulmonary uptake. The transfer of anesthetic from blood to tissues is determined by three factors analogous to systemic uptake: tissue solubility of the agent (tissue/blood partition coefficient), tissue blood flow, and the difference in partial pressure between arterial blood and the tissue.
To better understand inhaled anesthetic uptake and distribution, tissues have been classified into four groups based on their solubility and blood flow (Table 8-2). The highly perfused vessel-rich group (brain, heart, liver, kidney, and endocrine organs) is the first to encounter appreciable amounts of anesthetic. Moderate solubility and small volume limit the capacity of this group, so it is also the first to reach steady state (ie, arterial and tissue partial pressures are equal). The muscle group (skin and muscle) is not as well perfused, so uptake is slower. In addition, it has a greater capacity due to a larger volume, and uptake will be sustained for hours. Perfusion of the fat group nearly equals that of the muscle group, but the tremendous solubility of anesthetic in fat leads to a total capacity (tissue/blood solubility < tissue volume) that would take days to approach steady state. The minimal perfusion of the vessel-poor group (bones, ligaments, teeth, hair, and cartilage) results in insignificant uptake.

Percentage of body weight, Vessel Rich = 10. Percentage of body weight, Muscle = 50. Percentage of body weight, Fat = 20. Percentage of body weight, Vessel Poor = 20. Percentage of cardiac output, Vessel Rich = 75. Percentage of cardiac output, Muscle = 19. Percentage of cardiac output, Fat = 6. Percentage of cardiac output, Vessel Poor = 0. Perfusion (mL/ min/100 g), Vessel Rich = 75. Perfusion (mL/ min/100 g), Muscle = 3. Perfusion (mL/ min/100 g), Fat = 3. Perfusion (mL/ min/100 g), Vessel Poor = 0. Relative solubility, Vessel Rich = 1. Relative solubility, Muscle = 1. Relative solubility, Fat = 20. Relative solubility, Vessel Poor = 0
Anesthetic uptake produces a characteristic curve that relates the rise in alveolar concentration to time (Figure 8—2). The shape of this graph is determined by the uptakes of individual tissue groups (Figure 8—3). The initial steep rise of FA/FI is due to unopposed filling of the alveoli by ventilation. The rate of rise slows as the vessel-rich group—and eventually the muscle group— approach steady-state levels of saturation.
FIGURE 8-2 Fa rises toward FI faster with nitrous oxide (an insoluble agent) than with halothane (a soluble agent). See Figure 8-1 for an explanation of Fa and FI.
FIGURE 8-3 The rise and fall in alveolar partial pressure precedes that of other tissues. (Modified with permission from Cowles AL, Borgstedt HH, Gillies AJ, et al. Uptake and distribution of inhalation anesthetic agents in clinical practice. Anesth Analg. 1968 July-Aug;47(4):404— 414.)

The lowering of alveolar partial pressure by uptake can be countered by increasing alveolar ventilation. In other words, constantly replacing anesthetic taken up by the pulmonary bloodstream results in better maintenance of alveolar concentration. The effect of increasing ventilation will be most obvious in raising the Fa/F! for soluble anesthetics, as they are more subject to uptake. Because the FA/FI very rapidly approaches 1.0 for insoluble agents, increasing ventilation has minimal effect. In contrast to the effect of anesthetics on cardiac output, anesthetics and other drugs (eg, opioids) that depress spontaneous ventilation will decrease the rate of rise in alveolar concentration and create a negative feedback loop.

The slowing of induction due to uptake from alveolar gas can be reduced by increasing the inspired concentration. Interestingly, increasing the inspired concentration not only increases the alveolar concentration, but also increases its rate of rise (ie, increases FA/FI), because of two phenomena (see Figure 8—1) that produce a so-called "concentrating effect." First, if 50% of an anesthetic is taken up by the pulmonary circulation, an inspired concentration of 20% (20 parts of anesthetic per 100 parts of gas) will result in an alveolar concentration of 11% (10 parts of anesthetic remaining in a total volume of 90 parts of gas). On the other hand, if the inspired concentration is raised to 80% (80 parts of anesthetic per 100 parts of gas), the alveolar concentration will be 67% (40 parts of anesthetic remaining in a total volume of 60 parts of gas). Thus, even though 50% of the anesthetic is taken up in both examples, a higher inspired concentration results in a disproportionately higher alveolar concentration. In this example, increasing the inspired concentration four-fold results in a six-fold increase in alveolar concentration. The extreme case is an inspired concentration of 100% (100 parts of 100), which, despite a 50% uptake, will result in an alveolar concentration of 100% (50 parts of anesthetic remaining in a total volume of 50 parts of gas).
The second phenomenon responsible for the concentration effect is the augmented inflow effect. Using the example above, the 10 parts of absorbed gas must be replaced by an equal volume of the 20% mixture to prevent alveolar collapse. Thus, the alveolar concentration becomes 12% (10 plus 2 parts of anesthetic in a total of 100 parts of gas). In contrast, after absorption of 50% of the anesthetic in the 80% gas mixture, 40 parts of 80% gas must be inspired.
This further increases the alveolar concentration from 67% to 72% (40 plus 32 parts of anesthetic in a volume of 100 parts of gas).

The concentration effect is more significant with nitrous oxide than with the volatile anesthetics, as the former can be used in much higher concentrations. Nonetheless, a high concentration of nitrous oxide will augment (by the same mechanism) not only its own uptake, but theoretically that of a concurrently administered volatile anesthetic. The concentration effect of one gas upon another is called the second gas effect, which despite its presence in examination questions is probably insignificant in the clinical practice of anesthesiology.

Normally, alveolar and arterial anesthetic partial pressures are assumed to be equal, but in fact, the arterial partial pressure is consistently less than endexpiratory gas would predict. Reasons for this may include venous admixture, alveolar dead space, and nonuniform alveolar gas distribution. Furthermore, the existence of ventilation/perfusion mismatching will increase the alveolar—arterial difference. Mismatch acts as a restriction to flow: It raises the pressure in front of the restriction, lowers the pressure beyond the restriction, and reduces the flow through the restriction. The overall effect is an increase in the alveolar partial pressure (particularly for highly soluble agents) and a decrease in the arterial partial pressure (particularly for poorly soluble agents). Thus, a bronchial intubation or a right-to-left intracardiac shunt will slow the rate of induction with nitrous oxide more than with sevoflurane.

Recovery from anesthesia depends on lowering the concentration of anesthetic in brain tissue. Anesthetics can be eliminated by biotransformation, transcutaneous loss, or exhalation. Biotransformation usually accounts for a minimal increase in the rate of decline of alveolar partial pressure. Its greatest impact is on the elimination of soluble anesthetics that undergo extensive metabolism (eg, methoxyflurane). The greater biotransformation of halothane compared with isoflurane accounts for halothane's faster elimination, even though it is more soluble. The CYP group of isozymes (specifically CYP 2EI) seems to be important in the metabolism of some volatile anesthetics. Diffusion of anesthetic through the skin is insignificant.
The most important route for elimination of @ inhalation anesthetics is the alveolus. Many of the factors that speed induction also speed recovery: elimination of rebreathing, high fresh gas flows, low anesthetic-circuit volume, low absorption by the anesthetic circuit, decreased solubility, high cerebral blood flow (CBF), and increased ventilation. Elimination of nitrous oxide is so rapid that oxygen and CO, concentrations in alveolar gas are diluted. The resulting diffusion hypoxia is prevented by administering 100% oxygen for 5 to 10 min after discontinuing nitrous oxide. The rate of recovery is usually faster than induction because tissues that have not reached steady state will continue to take up anesthetic until the alveolar partial pressure falls below the tissue partial pressure. For instance, fat will continue to take up anesthetic and hasten recovery until the partial pressure exceeds the alveolar partial pressure. This redistribution is not as useful after prolonged anesthesia (fat partial pressures of anesthetic will have come "closer" to arterial partial pressures at the time the anesthetic was removed from fresh gas)—thus, the speed of recovery also depends on the length of time the anesthetic has been administered.

General anesthesia is an altered physiological state characterized by reversible loss of consciousness, analgesia, amnesia, and some degree of muscle relaxation. The multitude of substances capable of producing general anesthesia is remarkable: inert elements (xenon), simple inorganic compounds (nitrous oxide), halogenated hydrocarbons (halothane), ethers (isoflurane, sevoflurane, desflurane), and complex organic structures (propofol and ketamine). A unifying theory explaining anesthetic action would have to accommodate this diversity of structure. In fact, the various agents probably produce anesthesia by differing sets of molecular mechanisms. Inhalational agents interact with numerous ion channels present in the CNS and peripheral nervous system. Nitrous oxide and xenon are believed to inhibit N-methyl-p-aspartate (NMDA) receptors. NUDA receptors are excitatory receptors in the brain. Other inhalational agents may interact at other receptors (eg, y-aminobutyric acid [GABA]-activated chloride channel conductance) leading to anesthetic effects. Additionally, some studies suggest that inhalational agents continue to act in a nonspecific manner, thereby affecting the membrane bilayer. It is possible that inhalational anesthetics act on multiple protein receptors that block excitatory channels and promote the activity of inhibitory channels affecting neuronal activity, as well as by some nonspecific membrane effects.

There does not seem to be a single macroscopic site of action that is shared by all inhalation agents. Specific brain areas affected by various anesthetics include the reticular activating system, the cerebral cortex, the cuneate nucleus, the olfactory cortex, and the hippocampus; however, to be clear, general anesthetics bind throughout the CNS. Anesthetics have also been shown to depress excitatory transmission in the spinal cord, particularly at the level of the dorsal horn interneurons that are involved in pain transmission. Differing aspects of anesthesia may be related to different sites of anesthetic action. For example, unconsciousness and amnesia are probably mediated by cortical anesthetic action, whereas the suppression of purposeful withdrawal from pain likely relates to subcortical structures, such as the spinal cord or brainstem. One study in rats revealed that removal of the cerebral cortex did not alter the potency of the anesthetic! Indeed, measures of minimum alveolar concentration (MAC), the anesthetic concentration that prevents movement in 50% of subjects or animals, are dependent upon anesthetic effects at the spinal cord and not at the cortex. @ Past understanding of anesthetic action attempted to identify a unitary hypothesis of anesthetic effects. This hypothesis proposes that all inhalation agents share a common mechanism of action at the molecular level. This was previously supported by the observation that the anesthetic potency of inhalation agents correlates directly with their lipid solubility (Meyer—Overton rule). The implication is that anesthesia results from molecules dissolving at specific lipophilic sites. Of course, not all lipid-soluble molecules are anesthetics (some are actually convulsants), and the correlation between anesthetic potency and lipid solubility is only approximate (Figure 8—4).
FIGURE 8-4 There is a good but not perfect correlation between anesthetic potency and lipid solubility. MAC, minimum alveolar concentration. (Modified with permission from Lowe HJ, Hagler K. Gas Chromatography in Biology and Medicine. Phladelphia, PA: Churchill Livingstone; 1969.)

Neuronal membranes contain a multitude of hydrophobic sites in their phospholipid bilayer. Anesthetic binding to these sites could expand the bilayer beyond a critical amount, altering membrane function (critical volume hypothesis). Although this theory is almost certainly an oversimplification, it explains an interesting phenomenon: the reversal of anesthesia by increased pressure. Laboratory animals exposed to elevated hydrostatic pressure develop a resistance to anesthetic effects. Perhaps the pressure is displacing a number of molecules from the membrane or distorting the anesthetic binding sites in the membrane, increasing anesthetic requirements. Studies in the 1980s that demonstrated the ability of anesthetics to inhibit protein actions shifted scientific attention to the numerous ion channels that might affect neuronal transmission
anesthetic delivery, surgery, and AD development has not been sufficiently investigated to draw definitive conclusions.

Although inhalational agents have been suggested as contributing to neurotoxicity, they have also been shown to provide both neurological and cardiac protective effects against ischemia-reperfusion injury. Ischemic preconditioning implies that a brief ischemic episode protects a cell from future, more pronounced ischemic events. Various molecular mechanisms have been suggested to protect cells preconditioned either through ischemic events or secondary to pharmacological mechanisms, such as through the use of inhalational anesthetics. In the heart, preconditioning in part arises from actions at adenosine triphosphate (ATP)-sensitive potassium (Karp) channels. The exact mechanism of anesthetic preconditioning is likely to be multifocal and includes the opening of Karp channels, resulting in less mitochondrial calcium ion concentration and reduction of reactive oxygen species (ROS) production. ROS contributes to cellular injury. Anesthetic preconditioning may be the result of increased production of antioxidants following initial anesthesia exposure. Additionally, excitatory NMDA receptors are linked to the development of neuronal injury. NMDA antagonists, such as the noble anesthetic gas xenon, have been shown to be neuroprotective. Xenon has an anti-apoptotic effect that may be secondary to its inhibition of calcium ion influx following cell injury. As with neurotoxicity, the role of inhalational anesthetics in tissue is the of protection subject ongoing investigation.

@ The MAC of an inhaled anesthetic is the alveolar concentration that prevents movement in 50% of patients in response to a standardized stimulus (eg, surgical incision). MAC is a useful measure because it mirrors brain partial pressure, allows comparisons of potency between agents, and provides a standard for experimental evaluations (Table 8—3). Nonetheless, it should be remembered that this is a median value with limited usefulness in managing individual patients, particularly during times of rapidly changing alveolar concentrations (eg, induction and emergence).

Nitrous oxide, Structure = N=N A. Nitrous oxide, MAC%!' = 105?. Nitrous oxide, Vapor Pressure (mm Hg at 20°C) = _—. Halothane (Fluothane), Structure = F Cl F -¢ —G—H F Br. Halothane (Fluothane), MAC%!' = 0.75. Halothane (Fluothane), Vapor Pressure (mm Hg at 20°C) = 243. Isoflurane (Forane), Structure = E H F H-C-0-¢-C-F Pde. Isoflurane (Forane), MAC%!' = 1.2. Isoflurane (Forane), Vapor Pressure (mm Hg at 20°C) = 240. Desflurane (Suprane), Structure = F Hiee H-G-0-¢-¢-F pore. Desflurane (Suprane), MAC%!' = 6.0. Desflurane (Suprane), Vapor Pressure (mm Hg at 20°C) = 681. Sevoflurane (Ultane), Structure = F F F-G-F H-¢-0-¢ Hl) paces. Sevoflurane (Ultane), MAC%!' = 2.0. Sevoflurane (Ultane), Vapor Pressure (mm Hg at 20°C) = 160
'These minimum alveolar concentration (MAC) values are for 30to 55-year-old human subjects and are expressed as a percentage of 1 atmosphere. High altitude requires a higher inspired concentration of anesthetic to achieve the same partial pressure. 2A concentration greater than 100% means that hyperbaric conditions are required to achieve 1.0 MAC.

The MAC values for different anesthetics are roughly additive. For example, a mixture of 0.5 MAC of nitrous oxide (53%) and 0.5 MAC of halothane (0.37%) produces the same likelihood that movement in response to surgical incision will be suppressed as 1.0 MAC of isoflurane (1.2%) or 1.0 MAC of any other single agent. In contrast to CNS depression, the degree of myocardial depression may not be equivalent at the same MAC: 0.5 MAC of halothane causes more myocardial depression than 0.5 MAC of nitrous oxide. MAC represents only one point on the concentration—response curve—it is the equivalent of a median effective concentration (ECs9). MAC multiples are clinically useful if the concentration—response curves of the anesthetics being compared are parallel, nearly linear, and continuous for the effect being predicted. Roughly 1.3 MAC of any of the volatile anesthetics (eg, for halothane: 1.3 x 0.75% = 0.97%) has been found to prevent movement in about 95% of patients (an approximation of the EDg;); 0.3 to 0.4 MAC is associated with awakening from anesthesia (MAC awake) when the inhaled drug is the only agent maintaining anesthetic (a rare circumstance).
MAC can be altered by several physiological and pharmacological variables
(Table 8-4). One of the most striking is the 6% decrease in MAC per decade of age, regardless of volatile anesthetic. MAC is relatively unaffected by species, sex, or duration of anesthesia. Further, MAC is not altered after spinal cord transection in rats, leading to the hypothesis that the site of anesthetic inhibition of motor responses lies in the spinal cord.
TABLE 84 Factors affecting MAC.!

= L. Benzodiazepines, Comments = . Verapamil, Effect on MAC = L. Verapamil, Comments = . Lithium, Effect on MAC = L. Lithium, Comments = . Sympatholytics, Effect on MAC = . Sympatholytics, Comments = . Methyldopa, Effect on MAC = L. Methyldopa, Comments = . Clonidine, Effect on MAC = L. Clonidine, Comments = . Sympathomimetics, Effect on MAC = . Sympathomimetics, Comments = . Amphetamine Chronic, Effect on MAC = L. Amphetamine Chronic, Comments = . Acute, Effect on MAC = ;. Acute, Comments = . Cocaine, Effect on MAC = ,. Cocaine, Comments = . Ephedrine, Effect on MAC = t. Ephedrine, Comments = 
'These conclusions are based on human and animal studies.
2CSF, cerebrospinal fluid.

Nitrous oxide (NO; laughing gas) is colorless and essentially odorless. Although nonexplosive and nonflammable, nitrous oxide is as capable as oxygen of supporting combustion. Unlike the potent volatile agents, nitrous oxide is a gas at room temperature and ambient pressure. It can be kept as a liquid under pressure because its critical temperature lies above room temperature. Nitrous oxide is a relatively inexpensive anesthetic; however, concerns regarding its safety have led to continued interest in alternatives such as xenon (Table 8-5). As noted earlier, nitrous oxide, like xenon, is an NMDA receptor antagonist.

Inert (probably nontoxic with no metabolism)
Minimal cardiovascular effects
Low blood solubility
Rapid induction and recovery
Does not trigger malignant hyperthermia
Environmentally friendly
Nonexplosive

High cost
Low potency (MAC = 70%)!
IMAC, minimum alveolar concentration.

Nitrous oxide has a tendency to stimulate the sympathetic nervous system. Thus, even though nitrous oxide directly depresses myocardial contractility in vitro, arterial blood pressure, cardiac output, and heart rate are essentially unchanged or slightly elevated in vivo because of its stimulation of catecholamines (Table 8-6). Myocardial depression may be unmasked in patients with coronary artery disease or severe hypovolemia. Constriction of pulmonary vascular smooth muscle increases pulmonary vascular resistance, which results in a generally modest elevation of right ventricular end-diastolic pressure. Despite vasoconstriction of cutaneous vessels, peripheral vascular resistance is not significantly altered.

= . Renal, Isoflurane = . Renal, Desflurane = . Renal, Sevoflurane = . Renal blood flow, Nitrous Oxide = Lt. Renal blood flow, Halothane = W. Renal blood flow, Isoflurane = dL. Renal blood flow, Desflurane = L. Renal blood flow, Sevoflurane = L. Glomerular filtration rate, Nitrous Oxide = dL. Glomerular filtration rate, Halothane = Lt. Glomerular filtration rate, Isoflurane = LL. Glomerular filtration rate, Desflurane = L. Glomerular filtration rate, Sevoflurane = 4. Urinary output, Nitrous Oxide = dL. Urinary output, Halothane = Ll. Urinary output, Isoflurane = LL. Urinary output, Desflurane = L. Urinary output, Sevoflurane = L. Hepatic, Nitrous Oxide = . Hepatic, Halothane = . Hepatic, Isoflurane = . Hepatic, Desflurane = . Hepatic, Sevoflurane = . Blood flow, Nitrous Oxide = L. Blood flow, Halothane = aay. Blood flow, Isoflurane = L. Blood flow, Desflurane = L. Blood flow, Sevoflurane = J. Metabolism*, Nitrous Oxide = 0.004%. Metabolism*, Halothane = 15-20%. Metabolism*, Isoflurane = 0.2%. Metabolism*, Desflurane = <0.1%. Metabolism*, Sevoflurane = 5%
'N/C, no change.
2Controlled ventilation.
*Depolarizing blockage is probably also prolonged by these agents, but this is usually not clinically significant.
"Percentage of absorbed anesthetic undergoing metabolism.

Nitrous oxide increases respiratory rate (tachypnea) and decreases tidal volume as aresult of CNS stimulation and, perhaps, activation of pulmonary stretch receptors. The net effect is a minimal change in minute ventilation and resting arterial CO, levels. Hypoxic drive, the ventilatory response to arterial hypoxia that is mediated by peripheral chemoreceptors in the carotid bodies, is markedly depressed by even small amounts of nitrous oxide.

By increasing CBF and cerebral blood volume, nitrous oxide produces a mild elevation of intracranial pressure. Nitrous oxide also increases cerebral oxygen consumption (CMRO,). Concentrations of nitrous oxide below MAC may provide analgesia in dental surgery, labor, traumatic injury, and minor surgical

In contrast to other inhalation agents, nitrous oxide does not provide significant muscle relaxation. In fact, at high concentrations in hyperbaric chambers, nitrous oxide causes skeletal muscle rigidity. Nitrous oxide is not a triggering agent of malignant hyperthermia.

Nitrous oxide seems to decrease kidney blood flow by increasing renal vascular resistance. This leads to a drop in glomerular filtration rate and urinary output.

Hepatic blood flow probably falls during nitrous oxide anesthesia, but to a lesser extent than with the volatile agents.

Use of nitrous oxide in adults increases the risk of postoperative nausea and vomiting, presumably as a result of activation of the chemoreceptor trigger zone and the vomiting center in the medulla.

During emergence, almost all nitrous oxide is eliminated by exhalation. A small amount diffuses out through the skin. Biotransformation is limited to the less than 0.01% that undergoes reductive metabolism in the gastrointestinal tract by anaerobic bacteria.
By irreversibly oxidizing the cobalt atom in vitamin B,5, nitrous oxide inhibits enzymes that are vitamin B,, dependent. These enzymes include methionine synthetase, which is necessary for myelin formation, and thymidylate synthetase, @ which is necessary for DNA synthesis. Prolonged exposure to anesthetic concentrations of nitrous oxide can result in bone marrow depression (megaloblastic anemia) and even neurological deficiencies (peripheral neuropathies). However, administration of nitrous oxide for bone marrow harvest does not seem to affect the viability of bone marrow mononuclear cells. Because of possible teratogenic effects, nitrous oxide is often avoided in pregnant patients who are not yet in the third trimester. Nitrous oxide may also alter the immunological response to infection by affecting chemotaxis and motility of polymorphonuclear leukocytes.

Although nitrous oxide is insoluble in comparison with other inhalation agents, it is 35 times more soluble than nitrogen in blood. Thus, it tends to diffuse into aircontaining cavities more rapidly than nitrogen is absorbed by the bloodstream. For instance, if a patient with a 100-mL pneumothorax inhales 50% nitrous oxide, the gas content of the pneumothorax will tend to approach that of the bloodstream. Because nitrous oxide will diffuse into the cavity more rapidly than the air (principally nitrogen) diffuses out, the pneumothorax expands until it contains roughly 100 mL of air and 100 mL of nitrous oxide. If the walls surrounding the cavity are rigid, pressure rises instead of volume. Examples of conditions in which nitrous oxide might be hazardous include venous or arterial air embolism, pneumothorax, acute intestinal obstruction with bowel distention, intracranial air (pneumocephalus following dural closure or pneumoencephalography), pulmonary air cysts, intraocular air bubbles, and tympanic membrane grafting. Nitrous oxide will even diffuse into tracheal tube cuffs, increasing the pressure against the tracheal mucosa. Obviously, nitrous oxide is of limited value in patients requiring high inspired oxygen concentrations.

Because the high MAC of nitrous oxide prevents its use as a complete general anesthetic, it is frequently used in combination with the more potent volatile agents. The addition of nitrous oxide decreases the requirements of these other agents (65% nitrous oxide decreases the MAC of the volatile anesthetics by approximately 50%). Although nitrous oxide should not be considered a benign carrier gas, it does attenuate the circulatory and respiratory effects of volatile anesthetics in adults. Nitrous oxide potentiates neuromuscular blockade, but less so than the volatile agents. The concentration of nitrous oxide flowing through a vaporizer can influence the concentration of volatile anesthetic delivered. For example, decreasing nitrous oxide concentration (ie, increasing oxygen concentration) increases the concentration of volatile agent despite a constant vaporizer setting. This disparity is due to the relative solubilities of nitrous oxide and oxygen in liquid volatile anesthetics. The second gas effect was discussed earlier. Nitrous oxide is an ozone-depleting gas with greenhouse effects.

Halothane is a halogenated alkane (see Table 8—3). The carbon—fluoride bonds are responsible for its nonflammable and nonexplosive nature. Thymol preservative and amber-colored bottles retard spontaneous oxidative decomposition. It is rarely used in the United States.

A dose-dependent reduction of arterial blood pressure is due to direct myocardial depression; 2.0 MAC of halothane in patients not undergoing surgery results in a 50% decrease in blood pressure and cardiac output. Cardiac depression—from interference with sodium—calcium exchange and intracellular calcium utilization—causes an increase in right atrial pressure. Although halothane is a coronary artery vasodilator, coronary blood flow decreases, due to the drop in systemic arterial pressure. Adequate myocardial perfusion is usually maintained, as oxygen demand also drops. Normally, hypotension inhibits baroreceptors in the aortic arch and carotid bifurcation, causing a decrease in vagal stimulation and a compensatory rise in heart rate. Halothane blunts this reflex. Slowing of sinoatrial node conduction may result in a junctional rhythm or bradycardia. In infants, halothane decreases cardiac output by a combination of decreased heart rate and depressed myocardial contractility. Halothane sensitizes the heart to the arrhythmogenic effects of epinephrine, so that doses of epinephrine above 1.5 mcg/kg should be avoided. Although organ blood flow is redistributed, systemic vascular resistance is unchanged.

Halothane typically causes rapid, shallow breathing. The increased respiratory rate is not enough to counter the decreased tidal volume, so alveolar ventilation drops, and resting Paco, is elevated. Apneic threshold, the highest Paco, at which a patient remains apneic, also rises because the difference between it and resting Paco, is not altered by general anesthesia. Similarly, halothane limits the increase in minute ventilation that normally accompanies a rise in Paco,. Halothane's ventilatory effects are probably due to central (medullary depression) and peripheral (intercostal muscle dysfunction) mechanisms. These changes are exaggerated by preexisting lung disease and attenuated by surgical stimulation. The increase in Paco, and the decrease in intrathoracic pressure that accompany spontaneous ventilation with halothane partially reverse the depression in cardiac output, arterial blood pressure, and heart rate described above. Hypoxic drive is severely depressed by even low concentrations of halothane (0.1 MAC).
Halothane is considered a potent bronchodilator, as it often reverses asthmainduced bronchospasm. This action is not inhibited by B-adrenergic blocking agents. Halothane attenuates airway reflexes and relaxes bronchial smooth muscle by inhibiting intracellular calcium mobilization. Halothane also depresses clearance of mucus from the respiratory tract (mucociliary function), promoting postoperative hypoxia and atelectasis.

By dilating cerebral vessels, halothane lowers cerebral vascular resistance and increases cerebral blood volume and CBF. Autoregulation, the maintenance of constant CBF during changes in arterial blood pressure, is blunted. Concomitant rises in intracranial pressure can be prevented by establishing hyperventilation prior to administration of halothane. Cerebral activity is decreased, leading to electroencephalographic slowing and modest reductions in metabolic oxygen requirements.

Halothane relaxes skeletal muscle and potentiates nondepolarizing neuromuscular-blocking agents (NMBAs). Like the other potent volatile anesthetics, it is a trigger for malignant hyperthermia.

Halothane reduces renal blood flow, glomerular filtration rate, and urinary output. Part of this decrease can be explained by a fall in arterial blood pressure and cardiac output. Because the reduction in renal blood flow is greater than the reduction in glomerular filtration rate, the filtration fraction is increased. Preoperative hydration limits these changes.

Halothane causes hepatic blood flow to decrease in proportion to the depression of cardiac output. Hepatic artery vasospasm has been reported during halothane anesthesia. The metabolism and clearance of some drugs (eg, fentanyl, phenytoin, verapamil) seem to be impaired by halothane. Other evidence of hepatic cellular dysfunction includes sulfobromophthalein (BSP) dye retention and minor liver transaminase elevations.

Halothane is oxidized in the liver by a particular isozyme of CYP (2EI) to its principal metabolite, trifluoroacetic acid. In the absence of oxygen, reductive metabolism may result in a small amount of hepatotoxic end products that covalently bind to tissue macromolecules. This is more apt to occur following enzyme induction by phenobarbital. Elevated fluoride levels signal significant anaerobic metabolism.
Postoperative hepatic dysfunction has several causes: viral hepatitis, impaired hepatic perfusion, preexisting liver disease, hepatocyte hypoxia, sepsis, hemolysis, benign postoperative intrahepatic @ cholestasis, and drug-induced hepatitis. "Halothane hepatitis" is extremely rare. Patients exposed to multiple halothane anesthetics at short intervals, middle-aged obese women, and persons with a familial predisposition to halothane toxicity or a personal history of toxicity are considered to be at increased risk. Signs are mostly related to hepatic injury, such as increased serum alanine and aspartate transferase, elevated bilirubin (leading to jaundice), and encephalopathy.
The hepatic lesion seen in humans—centrilobular necrosis—also occurs in rats pretreated with an enzyme inducer (phenobarbital) and exposed to halothane under hypoxic conditions (Fio, <14%). This halothane hypoxic model implies hepatic damage from reductive metabolites or hypoxia.
Other evidence points to an immune mechanism. For instance, some signs of the disease indicate an allergic reaction (eg, eosinophilia, rash, fever) and do not appear until a few days after exposure. Furthermore, an antibody that binds to hepatocytes previously exposed to halothane has been isolated from patients with halothane-induced hepatic dysfunction. This antibody response may involve liver microsomal proteins that have been modified by trifluoroacetic acid as the triggering antigens (trifluoroacetylated liver proteins such as microsomal carboxylesterase). Other inhalational agents that undergo oxidative metabolism can likewise lead to hepatitis. However, newer agents undergo little to no metabolism, and therefore do not form trifluroacetic acid protein adducts or produce the immune response leading to hepatitis.

It is prudent to withhold halothane from patients with unexplained liver dysfunction following previous anesthetic exposure.
Halothane, like all inhalational anesthetics, should be used with care (and only in combination with modest hyperventilation) in patients with intracranial mass lesions because of the possibility of intracranial hypertension secondary to increased cerebral blood volume and blood flow.
Hypovolemic patients and some patients with severe reductions in left ventricular function may not tolerate halothane's negative inotropic effects. Sensitization of the heart to catecholamines limits the usefulness of halothane when exogenous epinephrine is administered (eg, in local anesthetic solutions) or in patients with pheochromocytoma.

The myocardial depression seen with halothane is exacerbated by B-adrenergic blocking agents and calcium channel blocking agents. Tricyclic antidepressants and monoamine oxidase inhibitors have been associated with fluctuations in blood pressure and arrhythmias, although neither represents an absolute contraindication. The combination of halothane and aminophylline has resulted in serious ventricular arrhythmias.

Isoflurane is a nonflammable volatile anesthetic with a pungent ethereal odor.

Isoflurane causes minimal left ventricular depression in vivo. Cardiac output is maintained by a rise in heart rate due to partial preservation of carotid baroreflexes. Mild B-adrenergic stimulation increases skeletal muscle blood flow, decreases systemic vascular resistance, and lowers arterial blood pressure. Rapid increases in isoflurane concentration lead to transient increases in heart rate, arterial blood pressure, and @ plasma levels of norepinephrine. Isoflurane dilates coronary arteries, but not nearly as potently as nitroglycerin or adenosine.
Dilation of normal coronary arteries could theoretically divert blood away from fixed stenotic lesions, which was the basis for concern about coronary "steal" with this agent, a concern that has largely been forgotten.

Respiratory depression during isoflurane anesthesia resembles that of other volatile anesthetics, except that tachypnea is less pronounced. The net effect is a more pronounced fall in minute ventilation. Even low levels of isoflurane (0.1 MAC) blunt the normal ventilatory response to hypoxia and hypercapnia. Despite a tendency to irritate upper airway reflexes, isoflurane is considered a good bronchodilator, but may not be as potent a bronchodilator as halothane.

At concentrations greater than 1 MAC, isoflurane increases CBF and intracranial pressure. These effects are thought to be less pronounced than with halothane and are reversed by hyperventilation. In contrast to halothane, the hyperventilation does not have to be instituted prior to the use of isoflurane to prevent intracranial hypertension. Isoflurane reduces cerebral metabolic oxygen requirements, and at 2 MAC, it produces an electrically silent electroencephalogram (EEG).

Isoflurane decreases renal blood flow, glomerular filtration rate, and urinary output.

Total hepatic blood flow (hepatic artery and portal vein flow) may be reduced during isoflurane anesthesia. Hepatic oxygen supply is better maintained with isoflurane than with halothane, however, because hepatic artery perfusion is preserved. Liver function tests are usually not affected.

Isoflurane is metabolized to trifluoroacetic acid. Although serum fluoride fluid levels may rise, nephrotoxicity is extremely unlikely, even in the presence of enzyme inducers. Prolonged sedation (>24 h at 0.1-0.6% isoflurane) of critically ill patients has resulted in elevated plasma fluoride levels (15-50 pmol/L) without evidence of kidney impairment. Similarly, up to 20 MAC-hours of isoflurane may lead to fluoride levels exceeding 50 pmol/L without detectable postoperative kidney dysfunction. Its limited oxidative metabolism also minimizes any possible risk of significant hepatic dysfunction.

Isoflurane presents no unique contraindications. Patients with severe hypovolemia may not tolerate its vasodilating effects. It can trigger malignant hyperthermia.

Epinephrine can be safely administered in doses up to 4.5 mcg/kg. Nondepolarizing NMBAs are potentiated by isoflurane.

The structure of desflurane is very similar to that of isoflurane. In fact, the only difference is the substitution of a fluorine atom for isoflurane's chlorine atom. That "minor" change has profound effects on the physical properties of the drug, however. For instance, because the vapor pressure of desflurane at 20°C is 681 mm Hg, at high altitudes (eg, Denver, Colorado) it boils at room temperature. This problem necessitated the development of a special © desflurane vaporizer. Furthermore, the low solubility of desflurane in blood and body tissues causes a very rapid induction of and emergence from anesthesia. Therefore, the alveolar concentration of desflurane approaches the inspired concentration much more rapidly than with the other volatile agents, giving the anesthesiologist tighter control over anesthetic levels. Wakeup times are approximately 50% less than those observed following isoflurane. This is principally attributable to a blood/gas partition coefficient (0.42) that is even lower than that of nitrous oxide (0.47). Although desflurane is roughly one-fourth as potent as the other volatile agents, it is 17 times more potent than nitrous oxide. A high vapor pressure, an ultrashort duration of action, and moderate potency are the most characteristic

The cardiovascular effects of desflurane seem to be similar to those of isoflurane. An increase in the concentration is associated with a decline in systemic vascular resistance that leads to a fall in arterial blood pressure. Cardiac output remains relatively unchanged or slightly depressed at 1 to 2 MAC. There is a moderate rise in heart rate, central venous pressure, and pulmonary artery pressure that often @ does not become apparent at low doses. Rapid increases in desflurane concentration lead to transient but sometimes worrisome elevations in heart rate, blood pressure, and catecholamine levels that are more pronounced than occur with isoflurane, particularly in patients with cardiovascular disease. These cardiovascular responses to rapidly increasing desflurane concentration can be attenuated by fentanyl, esmolol, or clonidine.

Desflurane causes a decrease in tidal volume and an increase in respiratory rate. There is an overall decrease in alveolar ventilation that causes a rise in resting Paco . Like other modern volatile anesthetic agents, desflurane depresses the ventilatory response to increasing Paco,. Pungency and airway irritation during desflurane induction can be manifested by salivation, breath-holding, coughing, and laryngospasm. Airway resistance may increase in children with reactive airway susceptibility. These problems make desflurane a poor choice for inhalation induction.

Like the other volatile anesthetics, desflurane directly vasodilates the cerebral vasculature, increasing CBF, cerebral blood volume, and intracranial pressure at normotension and normocapnia. Countering the decrease in cerebral vascular resistance is a marked decline in the cerebral metabolic rate of oxygen (CMRO,) that tends to cause cerebral vasoconstriction and moderate any increase in CBF. The cerebral vasculature remains responsive to changes in PacO,, however, so that intracranial pressure can be lowered by hyperventilation. Cerebral oxygen consumption is decreased during desflurane anesthesia. Thus, during periods of desflurane-induced hypotension (mean arterial pressure = 60 mm Hg), CBF is adequate to maintain aerobic metabolism despite a low cerebral perfusion pressure. The effect on the EEG is similar to that of isoflurane. Initially, EEG frequency is increased, but as anesthetic depth is increased, EEG slowing becomes manifest, leading to burst suppression at higher inhaled concentrations.

Desflurane is associated with a dose-dependent decrease in the response to trainof-four and tetanic peripheral nerve stimulation.

There is no evidence of any significant nephrotoxic effects caused by exposure to desflurane. However, as cardiac output declines, decreases in urine output and glomerular filtration should be expected with desflurane and all other anesthetics.

Hepatic function tests are generally unaffected by desflurane, assuming that organ perfusion is maintained perioperatively. Desflurane undergoes minimal metabolism; therefore, the risk of anesthetic-induced hepatitis is likewise minimal. As with isoflurane and sevoflurane, hepatic oxygen delivery is generally maintained.

Desflurane undergoes minimal metabolism in humans. Serum and urine inorganic fluoride levels following desflurane anesthesia are essentially unchanged from preanesthetic levels. There is insignificant percutaneous loss. Desflurane, more than other volatile anesthetics, is degraded by desiccated CO, absorbent (particularly barium hydroxide lime, but also sodium and potassium hydroxide) into potentially clinically important levels of carbon monoxide. Carbon monoxide poisoning is difficult to diagnose under general anesthesia, but the presence of carboxyhemoglobin may be detectable by arterial blood gas analysis or lower than expected pulse oximetry readings (although still falsely high). Disposing of dried out absorbent or use of calcium hydroxide can minimize the risk of carbon monoxide poisoning.

Desflurane shares many of the contraindications of other modern volatile anesthetics: severe hypovolemia, malignant hyperthermia, and intracranial hypertension.

Desflurane potentiates nondepolarizing neuromuscular blocking agents to the same extent as isoflurane. Epinephrine can be safely administered in doses up to 4.5 mcg/kg as desflurane does not sensitize the myocardium to the arrhythmogenic effects of epinephrine. Although emergence is more rapid following desflurane anesthesia than after isoflurane anesthesia, switching from isoflurane to desflurane toward the end of anesthesia does not significantly accelerate recovery, nor does faster emergence translate into faster discharge times from the postanesthesia care unit. Desflurane emergence has been associated with delirium in some pediatric patients.

Like desflurane, sevoflurane is halogenated with fluorine. Sevoflurane's solubility in blood is slightly greater than desflurane Avg 0.65 versus 0.42) (see Table 8-1). @ Nonpungency and rapid increases in alveolar anesthetic concentration make sevoflurane an excellent choice for smooth and rapid inhalation inductions in pediatric and adult patients. In fact, inhalation induction with 4% to 8% sevoflurane in a 50% mixture of nitrous oxide and oxygen can be achieved within 1 min. Likewise, its low blood solubility results in a rapid fall in alveolar anesthetic concentration upon discontinuation and a more rapid emergence compared with isoflurane (although not an earlier discharge from the postanesthesia care unit). Sevoflurane's modest vapor pressure permits the use of a conventional variable bypass vaporizer.

Sevoflurane mildly depresses myocardial contractility. Systemic vascular resistance and arterial blood pressure decline slightly less than with isoflurane or desflurane. Because sevoflurane causes little, if any, rise in heart rate, cardiac output is not maintained as well as with isoflurane or desflurane. Sevoflurane may prolong the QT interval, the clinical significance of which is unknown. QT prolongation may be manifest 60 min following anesthetic emergence in infants.

Sevoflurane depresses respiration and reverses bronchospasm to an extent similar to that of isoflurane.

Similar to isoflurane and desflurane, sevoflurane causes slight increases in CBF and intracranial pressure at normocarbia, although some studies show a decrease in CBF. High concentrations of sevoflurane (>1.5 MAC) may impair autoregulation of CBF, thus allowing a drop in CBF during hemorrhagic hypotension. This effect on CBF autoregulation seems to be less pronounced than with isoflurane. Cerebral metabolic oxygen requirements decrease, and seizure activity has not been reported.

Sevoflurane produces adequate muscle relaxation for intubation following an inhalation induction, although most practitioners will deepen anesthesia with various combinations of propofol, lidocaine, or opioids; administer a neuromuscular blocker prior to intubation; or a combination of these two approaches.

Sevoflurane slightly decreases renal blood flow. Its metabolism to substances associated with impaired renal tubule function (eg, decreased concentrating ability) is discussed below.

Sevoflurane decreases portal vein blood flow, but increases hepatic artery blood flow, thereby maintaining total hepatic blood flow and oxygen delivery. It is generally not associated with immune-mediated anesthetic hepatotoxicity

The liver microsomal enzyme P-450 (specifically the 2E1 isoform) metabolizes

Contraindications include severe hypovolemia, susceptibility to malignant hyperthermia, and intracranial hypertension.

Like other volatile anesthetics, sevoflurane potentiates NMBAs. It does not sensitize the heart to catecholamine-induced arrhythmias.

Xenon is a noble gas that has long been known to have anesthetic properties. It is an inert element that does not form chemical bonds. Xenon is scavenged from the atmosphere through a costly distillation process. It is an odorless, nonexplosive, naturally occurring gas with a MAC of 71% and a blood/gas coefficient of 0.115, giving it very fast onset and emergence parameters. As previously mentioned, xenon's anesthetic effects seem to be mediated by NMDA inhibition by competing with glycine at the glycine binding site. Xenon seems to have little effect on cardiovascular, hepatic, or renal systems and has been found to be protective against neuronal ischemia. Xenon inhalation combined with hypothermia has been suggested as a protective method to prevent cerebral damage following ischemic brain injury. Additionally, xenon anesthesia has been shown to produce less postoperative delirium compared with sevoflurane anesthesia in patients undergoing off-pump coronary artery bypass graft surgery. As a natural element, it has no effect upon the ozone layer compared with another NMDA antagonist, nitrous oxide. Cost and limited availability have prevented its widespread use.

The SmartTots collaborative research initiative focuses on funding research in pediatric anesthesiology. http://smarttots.org/

@ Repetitive administration of highly lipid-soluble barbiturates (eg, infusion of thiopental for "barbiturate coma" and brain protection) saturates the peripheral compartments, minimizing any effect of redistribution and rendering the duration of action more dependent on elimination. This is an example of context sensitivity.
Barbiturates constrict the cerebral vasculature, causing a decrease in cerebral blood flow, cerebral blood volume, and intracranial pressure.
© Although apnea may be relatively uncommon after benzodiazepine induction, even small intravenous doses of these agents have resulted in respiratory arrest.
@ Incontrast to other anesthetic agents, ketamine increases arterial blood pressure, heart rate, and cardiac output, particularly after rapid bolus injections.
@ Induction doses of etomidate transiently inhibit enzymes involved in cortisol and aldosterone synthesis. When used for sedation in the intensive care unit, etomidate was reported to produce consistent adrenocortical suppression.
@ Propofol formulations can support the growth of bacteria, so sterile technique must be observed in preparation and handling. Propofol should be administered within 6 h of opening the ampule.
General anesthesia began with the inhaled agents ether, nitrous oxide, and chloroform, but in current practice, anesthesia can be induced and maintained with drugs that enter the patient through a wide range of routes. Preoperative or procedural sedation is usually accomplished by way of oral or intravenous routes. Induction of general anesthesia is usually accomplished by inhalation or
intravenous drug administration. Alternatively, general anesthesia can be induced and maintained with intramuscular injection of ketamine. General anesthesia is maintained with a total intravenous anesthesia (TIVA) technique, an inhalation technique, or a combination of the two. This chapter focuses on the injectable agents used to produce narcosis (sleep), including barbiturates, benzodiazepines, ketamine, etomidate, propofol, and dexmedetomidine.

Barbiturates depress the reticular activating system in the brainstem, which controls consciousness. Their primary mechanism of action is believed to be through binding to the y-aminobutyric acid type A (GABA,) receptor. Barbiturates potentiate the action of GABA in increasing the duration of openings of a chloride-specific ion channel.

Barbiturates are derived from barbituric acid (Figure 9—1). Substitution at carbon C, determines hypnotic potency and anticonvulsant activity. The phenyl group in phenobarbital is anticonvulsive, whereas the methyl group in methohexital is not. Thus methohexital is useful for providing anesthesia for electroconvulsive therapy when a seizure is desired. Replacing the oxygen at C, (oxybarbiturates) with a sulfur atom (thiobarbiturates) increases lipid solubility. As aresult, thiopental and thiamylal have a greater potency, more rapid onset of action, and shorter durations of action (after a single "sleep dose") than pentobarbital. The sodium salts of the barbiturates are water soluble but markedly alkaline (pH of 2.5% thiopental >10) and relatively unstable (2-week shelf-life for 2.5% thiopental solution).
FIGURE 9-1 Barbiturates share the structure of barbituric acid and differ in the C,, C;, and N, substitutions.

Prior to the introduction of propofol, thiopental, thiamylal, and methohexital were frequently administered intravenously for induction of general anesthesia in adults and children. Rectal methohexital has been used for induction in children.

The duration of induction doses of thiopental, thiamylal, and methohexital is determined by redistribution, not by metabolism or elimination. Thiopental's great lipid solubility and high nonionized fraction (60%) account for rapid brain uptake (within 30 s). If the central compartment is contracted (eg, hypovolemic shock), if the serum albumin is low (eg, severe liver disease or malnutrition), or if the nonionized fraction is increased (eg, acidosis), larger brain and heart concentrations will be achieved for a given dose. Redistribution lowers plasma and brain concentration to 10% of peak levels within 20 to 30 min (Figure 9-2). This pharmacokinetic profile correlates with clinical experience—patients

FIGURE 9-2 Distribution of thiopental from plasma to the vessel-rich group (VRG; brain, heart, liver, kidney, endocrine glands), to the muscle group (MG), and finally to the fat group (FG). Propofol follows the same pattern but on a different time scale. (Modified with permission from Price HL, Kovnat PJ, Safer JN, et al. The uptake of thiopental by body tissues and its relation to the duration of narcosis. Clin Pharmacol Ther. 1960 Jan;1(1):16—-22.)
The minimal induction dose of thiopental will depend on body weight and age. Reduced induction doses are required for elderly patients. In contrast to the rapid initial distribution half-life of a few minutes, elimination of thiopental is prolonged (elimination half-life ranges of 10-12 h). Thiamylal and methohexital have similar distribution patterns, whereas less lipid-soluble barbiturates have much longer distribution half-lives and durations of action @ after a sleep dose. Repetitive administration of highly lipid-soluble barbiturates (eg, infusion of thiopental for "barbiturate coma" and brain protection) saturates the peripheral compartments, minimizing any effect of redistribution and rendering the duration of action more dependent on elimination. This is an example of context sensitivity as is also seen with other lipid-soluble agents (eg, potent inhaled anesthetics, fentanyl, and sufentanil; see Chapter 7).

Barbiturates are principally biotransformed via hepatic oxidation to inactive, water-soluble metabolites. Because of greater hepatic extraction, methohexital is cleared by the liver more rapidly than thiopental. Therefore, full recovery of psychomotor function is also more rapid following methohexital.

Except for the less protein-bound and less lipid-soluble agents such as phenobarbital, renal excretion is limited to water-soluble end products of hepatic biotransformation. Methohexital is excreted in the feces.

The cardiovascular effects of barbiturates vary markedly, depending on rate of administration, dose, volume status, baseline autonomic tone, and preexisting cardiovascular disease. A slow rate of injection with adequate preoperative hydration will attenuate or eliminate these changes in most patients. Intravenous bolus induction doses of barbiturates cause a decrease in blood pressure and an increase in heart rate. Depression of the medullary vasomotor center produces vasodilation of peripheral capacitance vessels, which increases peripheral pooling of blood, mimicking a reduced blood volume. Tachycardia following administration is probably due to a central vagolytic effect and reflex responses to decreases in blood pressure. Cardiac output is often maintained by an increased heart rate and increased myocardial contractility from compensatory baroreceptor reflexes. Sympathetically induced vasoconstriction of resistance vessels (particularly with intubation under light planes of general anesthesia) may actually increase peripheral vascular resistance. However, in situations where the baroreceptor response will be blunted or absent (eg, hypovolemia, congestive heart failure, B-adrenergic blockade), cardiac output and arterial blood pressure may fall dramatically due to uncompensated peripheral pooling of blood and direct myocardial depression. Patients with poorly controlled hypertension are particularly prone to wide swings in blood pressure during anesthesia induction.

Barbiturates depress the medullary ventilatory center, decreasing the ventilatory response to hypercapnia and hypoxia. Apnea often follows an induction dose. During awakening, tidal volume and respiratory rate are decreased. Barbiturates incompletely depress airway reflex responses to laryngoscopy and intubation, and airway instrumentation may lead to bronchospasm (in asthmatic patients) or

® Barbiturates constrict the cerebral vasculature, causing a decrease in cerebral blood flow, cerebral blood volume, and intracranial pressure. Intracranial pressure decreases to a greater extent than arterial blood pressure, so cerebral perfusion pressure (CPP) usually increases. (CPP equals cerebral artery pressure minus the greater of jugular venous pressure or intracranial pressure.) Barbiturates induce a greater decline in cerebral oxygen consumption (up to 50% of normal) than in cerebral blood flow; therefore, the decline in cerebral blood flow is not detrimental. Barbiturate-induced reductions in oxygen requirements and cerebral metabolic activity are mirrored by changes in the electroencephalogram (EEG), which progress from low-voltage fast activity with small doses to high-voltage slow activity, burst suppression, and electrical silence with larger doses. Barbiturates may protect the brain from transient episodes of focal ischemia (eg, cerebral embolism) but probably do not protect from global ischemia (eg, cardiac arrest). Abundant animal data document these effects, but the clinical data are sparse and inconsistent. Furthermore, thiopental doses required to maintain EEG burst suppression or flat line are associated with prolonged awakening, delayed extubation, and the need for inotropic support. The degree of central nervous system depression induced by barbiturates ranges from mild sedation to unconsciousness, depending on the dose administered ('Table 9-1). Some patients relate a taste sensation of garlic, onions, or pizza during induction with thiopental. Barbiturates do not impair the perception of pain. Small doses occasionally cause a state of excitement and disorientation. Barbiturates do not produce muscle relaxation, and some induce involuntary skeletal muscle contractions (eg, methohexital). Relatively small doses of thiopental (50-100 mg intravenously) rapidly (but briefly) control most grand mal seizures.

Thiopental, Use = Induction. Thiopental, Route’ = IV. Thiopental, Concentration (%) = 2.5. Thiopental, Dose (mg/kg) = 3-6. Methohexital, Use = Induction Sedation Induction. Methohexital, Route’ = IV IV Rectal (children). Methohexital, Concentration (%) = 1 1 10. Methohexital, Dose (mg/kg) = 1-2 0.2-0.4 25. Pentobarbital, Use = Premedication. Pentobarbital, Route’ = Oral IM Rectal suppository. Pentobarbital, Concentration (%) = 5. Pentobarbital, Dose (mg/kg) = 2-4 2-4 3
'IV, intravenous; IM, intramuscular.

Barbiturates reduce renal blood flow and glomerular filtration rate in proportion to the fall in blood pressure.

Hepatic blood flow is decreased. Chronic exposure to barbiturates leads to induction of hepatic enzymes and an increased rate of metabolism. On the other hand, binding of barbiturates to the cytochrome P-450 enzyme system interferes with the biotransformation of other drugs (eg, tricyclic antidepressants). Barbiturates promote aminolevulinic acid synthetase, which stimulates the formation of porphyrin (an intermediary in heme synthesis). This may precipitate acute intermittent porphyria or variegate porphyria in susceptible individuals.

Anaphylactic or anaphylactoid allergic reactions are rare. Sulfur-containing thiobarbiturates evoke mast cell histamine release in vitro, whereas oxybarbiturates do not.

Contrast media, sulfonamides, and other drugs that occupy the same proteinbinding sites may displace thiopental, increasing the amount of free drug available and potentiating the effects of a given dose. Ethanol, opioids, antihistamines, and other central nervous system depressants potentiate the sedative effects of barbiturates.

Benzodiazepines bind the same set of receptors in the central nervous system as barbiturates but bind to a different site. Benzodiazepine binding to the GABA, receptor increases the frequency of openings of the associated chloride ion channel. Benzodiazepine-receptor binding by an agonist facilitates binding of GABA to its receptor. Flumazenil (an imidazobenzodiazepine) is a specific benzodiazepine—receptor antagonist that effectively reverses most of the central nervous system effects of benzodiazepines (see Chapter 17).

The chemical structure of benzodiazepines includes a benzene ring and a sevenmember diazepine ring (Figure 9—3). Substitutions at various positions on these rings affect potency and biotransformation. The imidazole ring of midazolam contributes to its water solubility at low pH. Diazepam and lorazepam are insoluble in water so parenteral preparations contain propylene glycol, which can produce venous irritation.
(lipid-soluble), Lorazepam.Midazolam = . (lipid-soluble), Flumazenil. = (water-soluble)
FIGURE 9-3 The structures of commonly used benzodiazepines and their antagonist, flumazenil, share a seven-member diazepine ring. (Modified with permission from White PF. Pharmacologic and clinical aspects of preoperative medication. Anesth Analg. 1986 Sep;65(9):963-974.)

Benzodiazepines are commonly administered orally and intravenously (or, less commonly, intramuscularly) to provide sedation (or, less commonly, to induce general anesthesia) (Table 9—2). Diazepam and lorazepam are well absorbed from the gastrointestinal tract, with peak plasma levels usually achieved in 1 and 2 h, respectively. Intravenous midazolam (0.05—0.1 mg/kg) given for anxiolysis before general or regional anesthesia is nearly ubiquitous. Oral midazolam
(0.25—1 mg/kg), although not approved by the U.S. Food and Drug Administration, is popular for pediatric premedication. Likewise, intranasal (0.2-0.3 mg/kg), buccal (0.07 mg/kg), and sublingual (0.1 mg/kg) midazolam provide effective preoperative sedation.

Diazepam, Use = Premedication Sedation. Diazepam, Route' = Oral IV. Diazepam, Dose (mg/kg) = 0.2-0.5 0.04-0.2. Midazolam, Use = Premedication Sedation Induction. Midazolam, Route' = IM IV IV. Midazolam, Dose (mg/kg) = 0.07-0.15 0.01-0.1 0.1-0.4. Lorazepam, Use = Premedication. Lorazepam, Route' = Oral. Lorazepam, Dose (mg/kg) = 0.05
'NV, intravenous; IM, intramuscular.
Intramuscular injections of diazepam are painful and unreliably absorbed. In contrast, midazolam and lorazepam are well absorbed after intramuscular injection, with peak levels achieved in 30 and 90 min, respectively.

Diazepam is relatively lipid soluble and readily penetrates the blood-brain barrier. Although midazolam is water soluble at reduced pH, its imidazole ring closes at physiological pH, increasing its lipid solubility (see Figure 9-3). The moderate lipid solubility of lorazepam accounts for its slower brain uptake and onset of action. Redistribution is fairly rapid for the benzodiazepines and, like the barbiturates, is responsible for awakening. Although we have used midazolam as an induction agent, none of the benzodiazepines can match the rapid onset and short duration of action of propofol or etomidate. All three benzodiazepines are highly protein bound (90-98%).

The benzodiazepines rely on the liver for biotransformation into water-soluble glucuronidated end products. The phase I metabolites of diazepam are pharmacologically active.
Slow hepatic extraction and a large volume of distribution (V,) result in a long elimination half-life for diazepam (30 h). Although lorazepam also has a low hepatic extraction ratio, its lower lipid solubility limits its Vj, resulting in a shorter elimination half-life (15 h). Nonetheless, the clinical duration of lorazepam is often quite prolonged due to increased receptor affinity. These differences between lorazepam and diazepam underscore the low utility of individual pharmacokinetic half-lives in guiding clinical practice (see Chapter 7). Midazolam shares diazepam's V4, but its elimination half-life (2 h) is the shortest of the group because of its increased hepatic extraction ratio.

The metabolites of benzodiazepines are excreted chiefly in the urine. Enterohepatic circulation produces a secondary peak in diazepam plasma concentration 6 to 12 h following administration.

Benzodiazepines display minimal left-ventricular depressant effects, even at general anesthetic doses, except when they are coadministered with opioids (these agents interact to produce myocardial depression and arterial hypotension). Benzodiazepines given alone decrease arterial blood pressure, cardiac output, and peripheral vascular resistance slightly, and sometimes increase heart rate.

Benzodiazepines depress the ventilatory response to CO,. This depression is usually insignificant unless the drugs are administered intravenously or given ® with other respiratory depressants. Although apnea may be relatively uncommon after benzodiazepine induction, even small intravenous doses of these agents have resulted in respiratory arrest. The steep dose-response curve and delayed onset (compared with propofol or etomidate) necessitate titration to avoid overdosage and apnea, particularly when these agents are used for conscious sedation. Ventilation must be monitored in all patients receiving intravenous benzodiazepines (we recommend end-tidal CO, monitoring), and resuscitation equipment and a practitioner with airway skills must be present.

Benzodiazepines reduce cerebral oxygen consumption, cerebral blood flow, and intracranial pressure but not to the extent the barbiturates do. They are effective in controlling grand mal seizures. Sedative doses often produce anterograde amnesia. The mild muscle-relaxing property of these drugs is mediated at the spinal cord level. The antianxiety, amnestic, and sedative effects seen at lower doses progress to stupor and unconsciousness at anesthetic doses. Compared with propofol or etomidate, induction with benzodiazepines is associated with a slower rate of loss of consciousness and a longer recovery. Benzodiazepines have no direct analgesic properties.

Cimetidine binds to cytochrome P-450 and reduces the metabolism of diazepam. Erythromycin inhibits metabolism of midazolam and causes a twoto threefold prolongation and intensification of its effects.
As previously mentioned, the combination of opioids and benzodiazepines markedly reduces arterial blood pressure and peripheral vascular resistance. This synergistic interaction has often been observed in patients undergoing cardiac surgery who received benzodiazepines for premedication and who received larger doses of opioids during induction of anesthesia.
Benzodiazepines reduce the minimum alveolar concentration of volatile anesthetics as much as 30%. Ethanol, barbiturates, and other central nervous system depressants potentiate the sedative effects of the benzodiazepines.

Ketamine has multiple effects throughout the central nervous system, and it is well recognized to inhibit N-methyl-p-aspartate (NMDA) channels and neuronal hyperpolarization-activated cationic (HCN) channels. Nevertheless, exactly how ketamine produces either anesthesia or analgesia remains controversial. Ketamine functionally "dissociates" sensory impulses from the limbic cortex (which is involved with the awareness of sensation). Clinically, this state of dissociative anesthesia may cause the patient to appear conscious (eg, eye opening, swallowing, muscle contracture) but unable to process or respond to sensory input. Ketamine may have additional actions on endogenous analgesic pathways.
Ketamine has effects on mood, and infusions of this agent are now being widely used to treat severe, treatment-resistant depression, particularly when patients have suicidal ideation. Small infusion doses of ketamine are also being used to supplement general anesthesia and to reduce the need for opioids both during and after the surgical procedure. Low-dose infusions of ketamine have been used for analgesia in postoperative patients and others who are refractory to conventional analgesic approaches. Ketamine has been identified by the World Health Organization as a key necessary agent.

Ketamine (Figure 9-4) is a structural analogue of phencyclidine (a veterinary anesthetic and a drug of abuse). It is one-tenth as potent, yet retains many of phencyclidine's psychotomimetic effects. Ketamine is used for intravenous induction of anesthesia, particularly in settings where its tendency to produce sympathetic stimulation are useful (hypovolemia, trauma). When intravenous access is lacking, ketamine is useful for intramuscular induction of general anesthesia in children and uncooperative adults. Ketamine can be combined with other agents (eg, propofol or midazolam) in small bolus doses or infusions for conscious sedation during procedures such as nerve blocks and endoscopy. Even subanesthetic doses of ketamine may cause hallucinations but usually do not do so in clinical practice, where many patients will have received at least a small dose of midazolam (or a related agent) for amnesia and sedation. The increased anesthetic potency and decreased psychotomimetic side effects of one isomer (S[+] versus R[-]) are the result of stereospecific receptors. The single S(+) stereoisomer preparation is not available in the United States (but widely available elsewhere throughout the world), and it has considerably greater affinity than the racemic mixture for the NMDA receptor as well as several-fold greater potency as a general anesthetic.
Etomidate, Dexmedetomidine = Propofol
FIGURE 9-4 The structures of ketamine, etomidate, propofol, and dexmedetomidine.

Ketamine has been administered orally, nasally, rectally, subcutaneously, and epidurally, but in usual clinical practice it is given intravenously or intramuscularly (Table 9—3). Peak plasma levels are usually achieved within 10 to 15 min after intramuscular injection.
TABLE 9-3 Uses and doses of ketamine, etomidate, and propofol.
Ketamine, Use = Induction Maintenance Analgesia or sedation. Ketamine, Route’ = IV IM IV IV. Ketamine, Dose = 1-2 mg/kg 3-5 mg/kg 10-20 mcg/kg/min 2.5-15 mcg/kg/min. Etomidate, Use = Induction. Etomidate, Route’ = IV. Etomidate, Dose = 0.2-0.5 mg/kg. Propofol, Use = Induction Maintenance infusion Sedation infusion. Propofol, Route’ = IV IV IV. Propofol, Dose = 1-2.5 mg/kg 50-200 mcg/kg/min 25-100 mcg/kg/min. Dexmedetomidine, Use = Induction Maintenance. Dexmedetomidine, Route’ = IV Nasal IV. Dexmedetomidine, Dose = 1 mcg/kg over 10 min 1-2 mcg/kg 0.2-1.4 mcg/kg/h

Ketamine is highly lipid soluble and, along with ketamine-induced increase in cerebral blood flow and cardiac output, results in rapid brain uptake and subsequent redistribution (the distribution half-life is 10-15 min). Awakening is due to redistribution from brain to peripheral compartments.

Ketamine is biotransformed in the liver to several metabolites, one of which (norketamine) retains anesthetic activity. Patients receiving repeated doses of ketamine (eg, for daily changing of dressings on burns) develop tolerance, and this can only be partially explained by induction of hepatic enzymes. Extensive hepatic uptake (hepatic extraction ratio of 0.9) explains ketamine's relatively short elimination half-life (2 h).

End products of ketamine biotransformation are excreted renally.

@ In contrast to other anesthetic agents, ketamine increases arterial blood pressure, heart rate, and cardiac output (Table 9—4), particularly after rapid bolus injections. These indirect cardiovascular effects are due to central stimulation of the sympathetic nervous system and inhibition of the reuptake of norepinephrine after release at nerve terminals. Accompanying these changes are increases in pulmonary artery pressure and myocardial work. For these reasons, ketamine should be administered carefully to patients with coronary artery disease, uncontrolled hypertension, congestive heart failure, or arterial aneurysms. The direct myocardial depressant effects of large doses of ketamine, probably due to inhibition of calcium transients, are unmasked by sympathetic blockade (eg, spinal cord transection) or exhaustion of catecholamine stores (eg, severe endstage shock).

Propofol, Cerebral.icp = . , Cardiovascular.HR = 0. , Cardiovascular.MAP = UW. , Respiratory.Vent = . , Respiratory.Bdil = . , Cerebral.CBF = Wt. , Cerebral.CMRO, = LW. , Cerebral.icp = Wl. Dexmedetomidine, Cardiovascular.HR = 4. Dexmedetomidine, Cardiovascular.MAP = L. Dexmedetomidine, Respiratory.Vent = 0. Dexmedetomidine, Respiratory.Bdil = ?. Dexmedetomidine, Cerebral.CBF = IW. Dexmedetomidine, Cerebral.CMRO, = dt. Dexmedetomidine, Cerebral.icp = W
'HR, heart rate; MAP, mean arterial pressure; Vent, ventilatory drive; B'dil, bronchodilation; CBF, cerebral blood flow; CMRO,, cerebral oxygen consumption; ICP, intracranial pressure; 0, no effect; O/T, no change or mild increase; 1, decrease (mild, moderate, marked); T, increase (mild, moderate, marked); ?, unknown effect.
2Minimal change in CBF and ICP when coadministered with other agents (see text),

Ventilatory drive is minimally affected by induction doses of ketamine, although combinations of ketamine with opioids may produce apnea. Racemic ketamine is a potent bronchodilator, making it a good induction agent for asthmatic patients; however, S(+) ketamine produces minimal bronchodilation. Upper airway reflexes remain largely intact, but partial airway obstruction may occur, and patients at increased risk for aspiration pneumonia ("full stomachs") should be intubated during ketamine general anesthesia (see Case Discussion, Chapter 17). The increased salivation associated with ketamine can be attenuated by premedication with an anticholinergic agent such as glycopyrrolate.

The received dogma about ketamine is that it increases cerebral oxygen consumption, cerebral blood flow, and intracranial pressure. These effects would seem to preclude its use in patients with space-occupying intracranial lesions such as occur with head trauma; however, recent publications offer convincing evidence that when combined with a benzodiazepine (or another agent acting on the same GABA receptor system) and controlled ventilation (in techniques that exclude nitrous oxide), ketamine is not associated with increased intracranial pressure. Myoclonic activity is associated with increased subcortical electrical activity, which is not apparent on surface EEG. Undesirable psychotomimetic side effects (eg, disturbing dreams and delirium) during emergence and recovery are less common in children, in patients premedicated with benzodiazepines, or in those receiving ketamine combined with propofol in a total intravenous anesthesia (TIVA) technique. Of the nonvolatile agents, ketamine comes closest to being a "complete" anesthetic as it induces analgesia, amnesia, and unconsciousness.

Ketamine interacts synergistically (more than additive) with volatile anesthetics but in an additive way with propofol, benzodiazepines, and other GABAreceptor—mediated agents. In animal experiments nondepolarizing neuromuscular blocking agents are minimally potentiated by ketamine (see Chapter 11). Diazepam and midazolam attenuate ketamine's cardiostimulatory effects and diazepam prolongs ketamine's elimination half-life. a-Adrenergic and B-adrenergic antagonists (and other agents and techniques that diminish sympathetic stimulation) unmask the direct myocardial depressant effects of ketamine, which are normally overwhelmed by sympathetic stimulation. Concurrent infusion of ketamine and propofol, often in a fixed infusion (mg:mg) ratio of 1:10, has achieved great popularity for sedation with local and regional anesthesia or intravenous general anesthesia in office-based settings.

Etomidate depresses the reticular activating system and mimics the inhibitory effects of GABA. Specifically, etomidate—particularly the R(+) isomer— appears to bind to a subunit of the GABA, receptor, increasing the receptor's affinity for GABA. Etomidate may have disinhibitory effects on the parts of the nervous system that control extrapyramidal motor activity. This disinhibition offers a potential explanation for the 30% to 60% incidence of myoclonus with induction of etomidate anesthesia.

Etomidate contains a carboxylated imidazole and is structurally unrelated to other anesthetic agents (see Figure 9-4). The imidazole ring provides water solubility in acidic solutions and lipid solubility at physiological pH. Therefore, etomidate is dissolved in propylene glycol for injection. This solution often causes pain on injection that can be lessened by a prior intravenous injection of lidocaine.

Etomidate is available only for intravenous administration and is used primarily for induction of general anesthesia (see Table 9-3). It is sometimes used for brief production of deep (unconscious) sedation such as prior to placement of retrobulbar blocks.

Although it is highly protein bound, etomidate is characterized by a very rapid onset of action due to its great lipid solubility and large nonionized fraction at physiological pH. Redistribution is responsible for decreasing the plasma concentration to awakening levels. Etomidate plasma kinetics are well explained by a two-compartment model.

Hepatic microsomal enzymes and plasma esterases rapidly hydrolyze etomidate to an inactive metabolite.

The end products of etomidate hydrolysis are primarily excreted in the urine.

Etomidate has minimal effects on the cardiovascular system when given by itself. A mild reduction in peripheral vascular resistance is responsible for a decline in arterial blood pressure. Myocardial contractility and cardiac output are usually unchanged. Etomidate does not release histamine. However, etomidate by itself, even in large doses, produces relatively light anesthesia for laryngoscopy, and marked increases in heart rate and blood pressure may be recorded when etomidate provides the only anesthetic depth for intubation.

Ventilation is affected less with etomidate than with barbiturates or benzodiazepines. Even induction doses usually do not result in apnea unless opioids have also been administered.

Etomidate decreases cerebral metabolic rate, cerebral blood flow, and intracranial pressure. Because of minimal cardiovascular effects, cerebral perfusion pressure is well maintained. Although changes on EEG resemble those associated with barbiturates, etomidate (like ketamine) increases the amplitude of somatosensory evoked potentials. Postoperative nausea and vomiting are more common following etomidate than following propofol or barbiturate induction. Etomidate lacks analgesic properties.

@ Induction doses of etomidate transiently inhibit enzymes involved in cortisol and aldosterone synthesis. When infused for sedation in the intensive care unit (ICU), etomidate was reported to produce consistent adrenocortical suppression with an increased mortality rate in critically ill (particularly septic) patients.

Fentanyl increases the plasma level and prolongs the elimination half-life of etomidate. Opioids decrease the myoclonus characteristic of an etomidate induction.

Propofol induction of general anesthesia may involve facilitation of inhibitory neurotransmission mediated by GABA, receptor binding. Propofol allosterically increases binding affinity of GABA for the GABA, receptor. This receptor, as previously noted, is coupled to a chloride channel, and activation of the receptor leads to hyperpolarization of the nerve membrane. Propofol (like most general anesthetics) binds multiple ion channels and receptors. Propofol actions are not reversed by the specific benzodiazepine antagonist flumazenil.

Propofol consists of a phenol ring substituted with two isopropyl groups (see Figure 9-4). Propofol is not water soluble, but a 1% aqueous solution (10 mg/mL) is available for intravenous administration as an oil-in-water emulsion containing soybean oil, glycerol, and egg lecithin. A history of egg allergy does not necessarily contraindicate the use of propofol because most egg allergies involve a reaction to egg white (egg albumin), whereas egg lecithin is extracted from egg yolk. This formulation will often cause pain during injection that can be decreased by prior injection of lidocaine or less effectively by mixing lidocaine with propofol prior to injection @ (2 mL of 1% lidocaine in 18 mL propofol). Propofol formulations can support the growth of bacteria, so sterile technique must be observed in preparation and handling. Propofol should be administered within 6 h of opening the ampule. Sepsis and death have been linked to contaminated propofol preparations. Current formulations of propofol contain 0.005% disodium edetate or 0.025% sodium metabisulfite to help retard the rate of growth of microorganisms; however, these additives do not render the product "antimicrobially preserved" under United States Pharmacopeia standards.

Propofol is available only for intravenous administration for the induction of general anesthesia and for moderate to deep sedation (see Table 9-3).

Propofol has a rapid onset of action. Awakening from a single bolus dose is also rapid due to a very short initial distribution half-life (2-8 min). Recovery from propofol is more rapid and is accompanied by less "hangover" than recovery from methohexital, thiopental, ketamine, or etomidate. This makes it a good anesthetic for ambulatory surgery. A smaller induction dose is recommended in elderly patients because of their smaller Vy. Age is also a key factor determining required propofol infusion rates for TIVA. In countries other than the United
States, a device called the Diprifusor™ is used to provide target (concentration) controlled infusion of propofol. The user must enter the patient's age and weight and the desired target concentration. The device uses these data, a microcomputer, and standard pharmacokinetic parameters to continuously adjust the infusion rate. Unfortunately, this device is not available in the United States.

The clearance of propofol exceeds hepatic blood flow, implying the existence of extrahepatic metabolism. This exceptionally high clearance rate probably contributes to rapid recovery after continuous infusions. Conjugation in the liver results in inactive metabolites that are eliminated by renal clearance. The pharmacokinetics of propofol do not appear to be affected by obesity, cirrhosis, or kidney failure. Use of propofol infusion for long-term sedation of children who are critically ill or young adult neurosurgical patients has been associated with sporadic cases of lipemia, metabolic acidosis, and death, the so-termed propofol infusion syndrome.

Although metabolites of propofol are primarily excreted in the urine, end-stage kidney disease does not affect clearance of the parent drug.

The major cardiovascular effect of propofol is a decrease in arterial blood pressure due to a drop in systemic vascular resistance (inhibition of sympathetic vasoconstrictor activity), preload, and cardiac contractility. Hypotension following induction is usually reversed by the stimulation accompanying laryngoscopy and intubation. Factors associated with propofol-induced hypotension include large doses, rapid injection, and old age. Propofol markedly impairs the normal arterial baroreflex response to hypotension. Rarely, a marked drop in cardiac filling may lead to a vagally mediated reflex bradycardia. Changes in heart rate and cardiac output are usually transient and insignificant in healthy patients, but may be severe in patients at the extremes of age, those receiving B-adrenergic blockers, or those with impaired ventricular function. Although myocardial oxygen consumption and coronary blood flow usually decrease comparably, coronary sinus lactate production increases in some patients, indicating some mismatch between myocardial oxygen supply and

Propofol is a profound respiratory depressant that usually causes apnea following an induction dose. Even when used for conscious sedation in subanesthetic doses, propofol inhibits hypoxic ventilatory drive and depresses the normal response to hypercarbia. As a result, only properly educated and qualified personnel should administer propofol for sedation. Propofol-induced depression of upper airway reflexes exceeds that of thiopental, allowing intubation, endoscopy, or laryngeal mask placement in the absence of neuromuscular blockade. Although propofol can cause histamine release, induction with propofol is accompanied by a lower incidence of wheezing in both asthmatic and nonasthmatic patients compared with barbiturates or etomidate.

Propofol decreases cerebral blood flow, cerebral blood volume, and intracranial pressure. In patients with elevated intracranial pressure, propofol can cause a critical reduction in CPP (<50 mm Hg) unless steps are taken to support mean arterial blood pressure. Propofol and thiopental provide a comparable degree of cerebral protection during experimental focal ischemia. Unique to propofol are its antipruritic properties. Its antiemetic effects provide yet another reason for it to be a preferred drug for outpatient anesthesia. Induction is occasionally accompanied by excitatory phenomena such as muscle twitching, spontaneous movement, opisthotonus, or hiccupping. Propofol has anticonvulsant properties, has been used successfully to terminate status epilepticus, and may safely be administered to epileptic patients. Propofol decreases intraocular pressure. Tolerance does not develop after long-term propofol infusions. Propofol is an uncommon agent of physical dependence or addiction; however, anesthesia personnel, celebrities, and other medically untrained individuals have died while using propofol inappropriately to induce sleep in nonsurgical settings.

Many clinicians administer a small amount of midazolam (eg, 30 mcg/kg) prior to induction with propofol; midazolam can reduce the required propofol dose by more than 10%. Propofol is often combined with either remifentanil or ketamine for TIVA.

Fospropofol is a water-soluble prodrug that is metabolized in vivo to propofol, phosphate, and formaldehyde. It was released in the United States (2008) and other countries based on studies showing that it produces more complete amnesia and better conscious sedation for endoscopy than midazolam plus fentanyl. It has a slower onset and slower recovery than propofol, offering little reason for anesthesiologists to favor it over propofol. The place (if any) of fospropofol relative to other competing agents has not yet been established in clinical practice.

Dexmedetomidine is an a-adrenergic agonist that can be used for anxiolysis, sedation, and analgesia. Strictly speaking, it is not an anesthetic in humans; however, anesthesiologists have used it in combination with other agents to produce anesthesia. It has also been used in combination with local anesthetics to prolong regional blocks. This agent can be used for premedication by nasal (1-2 mcg/kg) or oral (2.5—4 mcg/kg) administration in children where it compares very favorably with oral midazolam.
Most commonly, dexmedetomidine is used for procedural sedation (eg, during awake craniotomy procedures or fiberoptic intubation), ICU sedation (eg, ventilated patients recovering from cardiac surgery), or as a supplement to general anesthesia to reduce the need for intraoperative opioids or to reduce the likelihood of emergence delirium (most often in children) after an inhalation anesthetic. It has also been used to treat alcohol withdrawal and side effects of cocaine intoxication. Typically, intravenous dexmedetomidine sedation in awake adults is initiated with a 1 mcg/kg loading dose given over 5 to 10 min followed by a maintenance infusion of 0.2 to 1.4 mcg/kg/h.
Dexmedetomidine has very rapid redistribution and a relatively short elimination half-life. It is metabolized in the liver by the CYP450 system and through glucuronidation. Nearly all metabolites are excreted in the urine.

Premedication of the Surgical Patient
An extremely anxious patient presents for outpatient surgery. The patient demands to be asleep before going to the operating room and does not want to remember anything.
What are the goals of administering preoperative medication?
Anxiety is a normal response to impending surgery. Diminishing anxiety is usually the main purpose for administering premedication. For many patients, the preoperative interview with the anesthesiologist allays fears more effectively than sedative drugs. Preoperative medication may also provide relief of preoperative pain or perioperative amnesia.
There may also be specific medical indications for certain preoperative medications: prophylaxis against postoperative nausea and vomiting (5HT3s) and against aspiration pneumonia (eg, antacids), or decreasing upper airway secretions (eg, anticholinergics). The goals of preoperative medication depend on many factors, including the health and emotional status of the patient, the proposed surgical procedure, and the anesthetic plan. For this reason, the choice of anesthetic premedication must be individualized and must follow a thorough preoperative evaluation.

Customary levels of preoperative anxiety do not harm most patients; therefore, preoperative sedation is not a requirement for all patients. Some patients dread intramuscular injections, and others find altered states of consciousness more unpleasant than anxiety. If the surgical procedure is brief, the effects of some sedatives may extend into the postoperative period and prolong recovery time. This is particularly troublesome for patients undergoing ambulatory surgery. Specific contraindications for sedative premedication include severe lung disease, hypovolemia, impending airway obstruction, increased intracranial pressure, and depressed baseline mental status. Premedication with sedative drugs should never be given before informed consent has been obtained.
Which patients are most likely to benefit from preoperative medication?
Some patients are quite anxious despite the preoperative interview. Separation of young children from their parents is often a traumatic ordeal for all concerned, particularly if children have undergone multiple prior surgeries. Medical conditions such as coronary artery disease or hypertension may be aggravated by psychological stress.
How does preoperative medication influence the induction of general anesthesia?
Some medications often given preoperatively (eg, opioids) decrease anesthetic requirements and can contribute to a smooth induction. However, intravenous administration of these medications just prior to induction is a more reliable method of achieving the same benefits.
What governs the choice among the preoperative medications commonly administered?
After the goals of premedication have been determined, the clinical effects of the agents dictate choice. For instance, in a patient experiencing preoperative pain from a femoral fracture, the analgesic effects of an opioid (eg, fentanyl, morphine, hydromorphone) will decrease the discomfort associated with transportation to the operating room and positioning on the operating room table. On the other hand, respiratory depression, orthostatic hypotension, and nausea and vomiting may result from opioid premedication.
Benzodiazepines relieve anxiety, often provide amnesia, and are relatively free of side effects; however, they are not analgesics. Diazepam and lorazepam are often administered orally. Intramuscular midazolam has a rapid onset and short duration but intravenous midazolam has an even better pharmacokinetic profile.
Which factors must be considered in selecting the anesthetic premedication for this patient?

First, it must be made clear to the patient that in most centers, lack of necessary equipment and concern for patient safety preclude anesthesia being induced in the preoperative holding room. Long-acting agents such as morphine or lorazepam are poor choices for an outpatient procedure. Diazepam can also affect mental function for several hours. One common approach is to establish an intravenous line in the preoperative holding area and titrate small doses of midazolam using slurred speech as an end point. At that time, the patient can be taken to the operating room. Vital signs— particularly respiratory rate—must be monitored.

@ The accumulation of morphine metabolites (morphine 3-glucuronide and morphine 6-glucuronide) in patients with kidney failure has been associated with narcosis and ventilatory depression.
@ Rapid administration of larger doses of opioids (particularly fentanyl, sufentanil, remifentanil, and alfentanil) can induce chest wall rigidity severe enough to make ventilation with bag and mask nearly impossible.
© Prolonged dosing of opioids can produce "opioid-induced hyperalgesia," in which patients become more sensitive to painful stimuli. Infusion of large doses of (in particular) remifentanil during general anesthesia can produce acute tolerance, in which much larger than usual doses of opioids are required for postoperative analgesia.
@ The neuroendocrine stress response to surgery is measured in terms of the secretion of specific hormones, including catecholamines, antidiuretic hormone, and cortisol. Large doses of opioids inhibit the release of these hormones in response to surgery more completely than volatile anesthetics.
© Aspirin is unique in that it irreversibly inhibits COX-1 by acetylating a serine residue in the enzyme. The irreversible nature of its inhibition underlies the nearly 1-week persistence of its clinical effects (eg, inhibition of platelet aggregation to normal) after drug discontinuation.
Regardless of how expertly surgical and anesthetic procedures are performed, appropriate use of analgesic drugs such as local anesthetics, opioids, ketamine, gabapentinoids, acetaminophen, and cyclooxygenase (COX) inhibitors can make the difference between a satisfied and an unsatisfied postoperative patient.
Moreover, studies have shown that outcomes can be improved when analgesia is provided in a "multimodal" format (typically minimizing opioid use) as one part of a well-organized plan for enhanced recovery after surgery (ERAS; see Chapter 48).

Opioids bind to specific receptors located throughout the central nervous system and other tissues. Four major opioid receptor types have been identified (Table 10-1): mu (p, with subtypes 1, and pl), kappa (x), delta (5), and sigma (0). All opioid receptors couple to G proteins; binding of an agonist to an opioid receptor causes membrane hyperpolarization. Acute opioid effects are mediated by inhibition of adenylyl cyclase (reductions in intracellular cyclic adenosine monophosphate concentrations) and activation of phospholipase C. Opioids inhibit voltage-gated calcium channels and activate inwardly rectifying potassium channels. Opioid effects vary based on the duration of exposure, and opioid tolerance leads to changes in opioid responses.
TABLE 10-1 Classification of opioid receptors.!
UW, Clinical Effect = Supraspinal analgesia (u,) Respiratory depression (u,) Physical dependence Muscle rigidity. UW, Agonists = Morphine | Met-enkephalin? B-Endorphin? Fentanyl. K, Clinical Effect = Sedation Spinal analgesia. K, Agonists = Morphine Nalbuphine Butorphanol Dynorphin? Oxycodone. 6, Clinical Effect = Analgesia Behavioral Epileptogenic. 6, Agonists = Leu-enkephalin? 6-Endorphin?. o, Clinical Effect = Dysphoria Hallucinations Respiratory stimulation. o, Agonists = Pentazocine Nalorphine Ketamine
'Note: The relationships among receptor, clinical effect, and agonist are more complex than indicated in this table. For example, pentazocine is an antagonist at LL receptors, a partial agonist at K receptors, and an agonist at o receptors.
?Endogenous opioid.

Although opioids provide some degree of sedation and in some species can produce general anesthesia when given in large doses, they are principally used to provide analgesia. The clinical actions of opioids depend on which receptor is bound (and in the case of spinal and epidural administration of opioids, the location in the neuraxis where the receptor is located) and the binding affinity of the drug. Agonist—antagonists (eg, nalbuphine, nalorphine, butorphanol, and buprenorphine) have less efficacy than full agonists (eg, fentanyl or morphine), and under some circumstances agonist—antagonists will antagonize the actions of full agonists. Pure opioid antagonists (eg, naloxone or naltrexone) are discussed in Chapter 17. Opioid compounds mimic endorphins, enkephalins, and dynorphins, endogenous peptides that bind to opioid receptors. Opioid receptor activation inhibits the presynaptic release and postsynaptic response to excitatory neurotransmitters (eg, acetylcholine, substance P) released by nociceptive neurons. Transmission of pain impulses can be selectively modified at the level of the dorsal horn of the spinal cord with intrathecal or epidural administration of opioids. Opioid receptors also respond to systemically administered opioids. Modulation through a descending inhibitory pathway from the periaqueductal gray matter to the dorsal horn of the spinal cord may also play a role in opioid analgesia. Although opioids exert their greatest effect within the central nervous system, opioid receptors have also been identified on somatic and sympathetic peripheral nerves. Certain opioid side effects (eg, constipation) are the result of opioid binding to receptors in peripheral tissues (eg, the gastrointestinal tract), and there are now selective antagonists for opioid actions outside the central nervous system (alvimopan and methylnaltrexone). The clinical importance of opioid receptors on primary sensory nerves (if present) remains speculative, despite the persisting practice of compounding of opioids in local anesthetic solutions applied to peripheral nerves.

A chemically diverse group of compounds bind opioid receptors. These agents have common structural characteristics, which are shown in Figure 10-1. Small molecular changes convert an agonist into an antagonist. The levorotatory opioid isomers are generally more potent than the dextrorotatory isomers.
Alfentanil, Meperidine = Naloxone
FIGURE 10-1 Opioid agonists and antagonists share part of their chemical structure, which is outlined in cyan.

Rapid and complete absorption follows intramuscular or subcutaneous injection of hydromorphone, morphine, or meperidine, with peak plasma levels usually reached after 20-60 min. A wide variety of opioids are effective by oral administration, including oxycodone, hydrocodone, codeine, tramadol, morphine, hydromorphone, and methadone. Oral transmucosal fentanyl citrate absorption (fentanyl "lollipop") provides rapid onset of analgesia and sedation in patients who are not good candidates for oral, intravenous, or intramuscular dosing of opioids.
The low molecular weight and high lipid solubility of fentanyl also favor transdermal absorption (the transdermal fentanyl "patch"). The amount of fentanyl absorbed per unit of time depends on the surface area of skin covered by the patch and also on local skin conditions (eg, blood flow). The time required to establish a reservoir of drug in the upper dermis delays by several hours the achievement of effective blood concentrations. Serum concentrations of fentanyl reach a plateau by 14 to 24h of application (with greater delays in elderly than in younger patients) and remain constant for up to 72 h. Continued absorption from the dermal reservoir accounts for persisting fentanyl serum levels many hours after patch removal. Fentanyl patches are most often used for outpatient management of chronic pain and should be reserved for patients who require continuous opioid dosing but cannot use the much less expensive, but equally efficacious and long-acting oral agents.
Fentanyl is often administered in small doses (10-25 mcg) intrathecally with local anesthetics for spinal anesthesia, and adds to the analgesia when included with local anesthetics in epidural infusions. Morphine in doses between 0.1 and 0.5 mg and hydromorphone in doses between 0.05 and 0.2 mg provide 12 to 18h of analgesia after intrathecal administration. Morphine and hydromorphone are commonly included in local anesthetic solutions infused for postoperative epidural analgesia.

Table 10—2 summarizes the physical characteristics that determine distribution and tissue binding of opioid analgesics. After intravenous administration, the distribution half-lives of the opioids are short (5-20 min). The low lipid solubility of morphine delays its passage across the blood-brain barrier, however, so that its onset of action is slow and its duration of action is prolonged. This contrasts with the increased lipid solubility of fentanyl and sufentanil, which are associated with a faster onset and shorter duration of action when administered in small doses. Interestingly, alfentanil has a more rapid onset of action and shorter duration of action than fentanyl following a bolus injection, even though it is less lipid soluble than fentanyl. The high nonionized fraction of alfentanil at physiological pH and its small volume of distribution (V4) increase the amount of drug (as a percentage of the administered dose) available for binding in the brain.

Morphine, Nonionized Fraction = ao. Morphine, Protein Binding = ++. Morphine, Lipid Solubility = +. Meperidine, Nonionized Fraction = +. Meperidine, Protein Binding = +44. Meperidine, Lipid Solubility = pet. Fentanyl, Nonionized Fraction = +. Fentanyl, Protein Binding = +++. Fentanyl, Lipid Solubility = ++++. Sufentanil, Nonionized Fraction = ++. Sufentanil, Protein Binding = +++. Sufentanil, Lipid Solubility = +4+++4. Alfentanil, Nonionized Fraction = +4+4+4+. Alfentanil, Protein Binding = +. Alfentanil, Lipid Solubility = +44. Remifentanil, Nonionized Fraction = +++. Remifentanil, Protein Binding = +++. Remifentanil, Lipid Solubility = epee
'+, very low; + +, low; + + +, high; + + + +, very high.
Significant amounts of lipid-soluble opioids can be retained by the lungs (first-pass uptake); as systemic concentrations fall they will return to the bloodstream. The amount of pulmonary uptake is reduced by prior accumulation of other drugs, increased by a history of tobacco use, and decreased by concurrent inhalation anesthetic administration. Unbinding of opioid receptors and redistribution (of drug from effect sites) terminate the clinical effects of all opioids. After smaller doses of the lipid-soluble drugs (eg, fentanyl or sufentanil), redistribution alone is paramount for reducing blood concentrations, whereas after larger doses biotransformation becomes an important driver in reducing plasma levels below those that have clinical effects. Thus, the time required for fentanyl or sufentanil concentrations to decrease by half (the "halftime") is context sensitive; in other words, the context-sensitive half-time increases as the total dose of drug or duration of exposure, or both, increase (see Chapter 7).

With the exception of remifentanil, all opioids depend primarily on the liver for
FIGURE 10-2 In contrast to other opioids, the time necessary to achieve a 50% decrease in the plasma concentration of remifentanil (its half-time) is very short and is not influenced by the duration of the infusion (it is not context sensitive). (Reproduced with permission from Egan TD. The pharmacokinetics of the new short-acting opioid remifentanil [GI87084B] in healthy adult male volunteers. Anesthesiology. 1993 Nov;79(5):881-892.)

The end products of morphine and meperidine biotransformation are eliminated by the kidneys, with less than 10% undergoing biliary excretion. Because 5% to 10% of morphine is excreted unchanged in the urine, kidney failure prolongs morphine duration of @ action. The accumulation of morphine metabolites (morphine 3-glucuronide and morphine 6-glucuronide) in patients with kidney failure has been associated with prolonged narcosis and ventilatory depression. In fact, morphine 6-glucuronide is a more potent and longer-lasting opioid agonist than morphine. As previously noted, normeperidine at increased concentrations may produce seizures; these are not reversed by naloxone. Renal dysfunction increases the likelihood of toxic effects from normeperidine accumulation. However, both morphine and meperidine have been used safely in patients with kidney failure. Metabolites of sufentanil are excreted in urine and bile. The main metabolite of remifentanil is several thousand times less potent than its parent compound and is unlikely to produce any clinical opioid effects. It is renally excreted.
Effects on Organ Systems

In general,
opioids have
increase heart
rate synthesized
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structurally as
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fentanyl, sufentanil,
nerve—mediated effects
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Intraoperative hypertension during opioid-based intravenous or nitrous oxide— opioid anesthesia is common. Such hypertension is often attributed to inadequate anesthetic depth; thus it is conventionally treated by the addition of other anesthetic agents (benzodiazepines, propofol, or potent inhaled agents). When depth of anesthesia is adequate we recommend treating hypertension with antihypertensives rather than with additional anesthetics.

Opioids depress ventilation, particularly respiratory rate. Thus, respiratory rate and end-tidal CO, tension (in contrast to arterial oxygen saturation) provide simple metrics for the early detection of respiratory depression in patients receiving opioid analgesia. Opioids increase the partial pressure of carbon dioxide (Paco,) and blunt the response to a CO, challenge, resulting in a shift of the CO, response curve downward and to the right (Figure 10—3). These effects result from opioid binding to neurons in the respiratory centers of the brainstem. The apneic threshold—the greatest Paco2 at which a patient remains apneic —rises, and hypoxic drive is decreased. Morphine and meperidine can cause to
histamine-induced bronchospasm in susceptible @ patients. Rapid administration of larger doses of opioids (particularly fentanyl, sufentanil, remifentanil, and alfentanil) can induce chest wall rigidity severe enough to make ventilation with bag and mask nearly impossible. This centrally mediated muscle contraction is effectively treated with neuromuscular blocking agents. This problem is rarely seen now that large-dose opioid anesthesia is no longer a mainstay of cardiovascular anesthesia practice. Opioids can blunt the bronchoconstrictive response to airway stimulation such as occurs during tracheal intubation.
FIGURE 10-3 Opioids depress ventilation. This is graphically displayed by a shift of the CO, curve downward and to the right.

The effects of opioids on cerebral perfusion and intracranial pressure must be separated from any effects of opioids on Paco,. In general, opioids reduce cerebral oxygen consumption, cerebral blood flow, cerebral blood volume, and intracranial pressure, but to a much lesser extent than propofol, benzodiazepines, or barbiturates, provided normocarbia is maintained by artificial ventilation. There are some reports of mild—but transient and almost certainly unimportant
TABLE 10-3 Uses and doses of common opioids.
Morphine, Use = Postoperative analgesia. Morphine, Route’ = IM \v. Morphine, Dose* = 0.05-0.2 mg/kg 0.03-0.15 mg/kg. Hydromorphone, Use = Postoperative analgesia. Hydromorphone, Route’ = IM IV. Hydromorphone, Dose* = 0.02-0.04 mg/kg 0.01-0.02 mg/kg. Fentanyl, Use = Intraoperative anesthesia Postoperative analgesia. Fentanyl, Route’ = iV Vv. Fentanyl, Dose* = 2-50 meg/kq 0.5-1.5 meg/kg. Sufentanil, Use = Intraoperative anesthesia. Sufentanil, Route’ = Vv. Sufentanil, Dose* = 0.25-20 meg/kg. Alfentanil, Use = Intraoperative anesthesia Loading dose Maintenance infusion. Alfentanil, Route’ = Vv Vv. Alfentanil, Dose* = 8-100 mcg/kg 0.5-3 meg/kg/min. Remifentanil, Use = Intraoperative anesthesia Loading dose Maintenance infusion Postoperative analgesia/sedation. Remifentanil, Route’ = VV IV IV. Remifentanil, Dose* = 1 mcg/kg 0.05-2 mcg/kg/min 0.05-0.3 mcg/kg/min.
IM, intramuscular; IV, intravenous.

2Note: The wide range of opioid doses reflects a large therapeutic index and depends upon which other anesthetics are simultaneously administered. For obese patients, dose should be based on ideal body weight or lean body mass, nat total body weight. Tolerance can develop rapidly (ie, within 2 h) during IV infusion of opioids, necessitating higher infusion rates. Dose correlates with other variables besides body weight that need to be considered (eq, age). The relative potencies of fentanyl, sufentanil, and alfentanil are estimated to be 1:9:1/7
Unique among the commonly used opioids, meperidine has minor local anesthetic qualities, particularly when administered into the subarachnoid space. Meperidine's clinical use as a local anesthetic has been limited by its relatively low potency and propensity to cause typical opioid side effects (nausea, sedation, and pruritus) at the doses required to induce local anesthesia. Intravenous meperidine (10—25 mg) is more effective than morphine or fentanyl for decreasing shivering in the postanesthetic care unit and meperidine appears to be the best agent for this indication.

Opioids slow gastrointestinal motility by binding to opioid receptors in the gut and reducing peristalsis. Biliary colic may result from opioid-induced contraction of the sphincter of Oddi. Biliary spasm, which can mimic a common bile duct stone on cholangiography, is reversed with the opioid antagonist naloxone or glucagon. Patients receiving long-term opioid therapy (eg, for cancer pain) usually become tolerant to many of the side effects but rarely to constipation. This is the basis for the development of the peripheral opioid antagonists methylnaltrexone, alvimopan, naloxegol, and naldemedine, which promote gastrointestinal motility in patients with varying indications, such as treatment of opioid bowel syndrome, side effects from opioid treatment of noncancer pain, or reduction of ileus in those receiving intravenous opioids after abdominal surgery.

@ The neuroendocrine stress response to surgery is measured in terms of the secretion of specific hormones, including catecholamines, antidiuretic hormone, and cortisol. Large doses of fentanyl or sufentanil inhibit the release of these hormones in response to surgery more completely than volatile anesthetics. The actual clinical outcome benefit produced by attenuating the stress response with opioids, even in high-risk cardiac patients, remains speculative (and we suspect nonexistent), whereas the many drawbacks of excessive doses of opioids are readily apparent.

Retrospective studies have associated general anesthesia (including opioids) with an increased risk of cancer reoccurrence after surgery as compared to techniques that emphasize opioid-sparing regional anesthetic techniques for analgesia. Ongoing clinical trials will likely clarify whether general anesthesia, opioids, both, or neither influence outcomes after cancer surgery.

There is a well-publicized epidemic of opioid abuse in western democracies, particularly in the United States. Although it comprises less than 5% of the world's population, the United States consumes 80% of the world's prescription opioids (and nearly all of the world's supply of hydrocodone)! Large numbers of patients admit to using prescribed opioids in a recreational fashion, and drug overdosage (most often from prescribed drugs) is the leading cause of accidental death in the United States. Many opioid addicts can trace their addiction to opioids prescribed by a physician for acute or chronic pain. There are many causes of this terrible problem, including excessive and misleading marketing of opioids to physicians, unwise prescribing practices by physicians, inappropriate and misleading assertions by "thought leaders" (many with ties to the pharmaceutical industry) regarding opioids, and well-intended but poorly thought out recommendations for assessment and treatment of pain by certifying agencies. In response the U.S. Centers for Disease Control and Prevention and many other agencies have released guidelines for responsible prescribing of opioids.

The combination of meperidine and monoamine oxidase inhibitors may result in hemodynamic instability, hyperpyrexia, coma, respiratory arrest, or death. The cause of this catastrophic interaction is incompletely understood. (The failure to appreciate this drug interaction in the controversial Libby Zion case led to changes in work hour rules for house officers in the United States.)
Propofol, barbiturates, benzodiazepines, inhaled anesthetics, and other central nervous system depressants can have synergistic cardiovascular, respiratory, and sedative effects with opioids.
The clearance of alfentanil may be impaired and the elimination half-life prolonged following treatment with erythromycin.

that it irreversibly inhibits COX-1 by acetylating a serine residue in the enzyme, resulting in a nearly 1-week persistence of its clinical effects (eg, inhibition of platelet aggregation) after drug discontinuation.
COX inhibitors are most often administered orally. Acetaminophen, ibuprofen, diclofenac, and ketorolac are available for intravenous administration. Unfortunately, intravenous acetaminophen has an acquisition cost several orders of magnitude greater than oral acetaminophen; therefore its use is tightly restricted in many medical centers.
"Multimodal" analgesia typically includes the use of acetaminophen, COX inhibitors, possibly a gabapentinoid, regional or local anesthesia techniques, and other approaches aimed at improving the analgesia while reducing the requirement for opioids in postoperative patients. Multimodal analgesia protocols are best used as part of an enhanced recovery after surgery (ERAS) protocol, a topic that is extensively considered in Chapter 48.

The COX enzyme is inhibited by an unusually diverse group of compounds that can be grouped into salicylic acids (eg, aspirin), acetic acid derivatives (eg, ketorolac), propionic acid derivatives (eg, ibuprofen), heterocyclics (eg, celecoxib), and others. Thus, a conventional discussion of structure to potency (and other factors) is not useful for these chemicals, other than to note that the heterocyclics tend to be the compounds with the greatest selectivity for the COX-2 rather than COX-1 form of the enzyme.

COX inhibitors when taken orally will typically achieve their peak blood concentrations in less than 3 h. Some COX inhibitors are formulated for topical application (eg, as a gel to be applied over joints or as liquid drops to be instilled on the eye). Ketorolac has been widely used as part of a local anesthetic "cocktail" to be injected around the surgical site and joint after arthroplasty.

In blood, COX inhibitors are highly bound by plasma proteins, chiefly albumin. Their lipid solubility allows them to readily permeate the blood-brain barrier to produce a central analgesia and antipyresis, and to penetrate joint spaces to produce (with the exception of acetaminophen) an antiinflammatory effect.

Most COX inhibitors undergo hepatic biotransformation. Acetaminophen at increased doses yields sufficiently large concentrations of N-acetyl-pbenzoquinone imine to produce hepatic failure.

Nearly all COX inhibitors are excreted in urine after biotransformation.

COX inhibitors do not act directly on the cardiovascular system. Any cardiovascular effects result from the actions of these agents on coagulation. Prostaglandins maintain the patency of the ductus arteriosus; thus COX inhibitors have been administered to neonates to promote closure of a persistently patent ductus arteriosus and prostaglandins have been infused to maintain patency of the ductus in neonates awaiting surgery for ductaldependent congenital cardiac lesions.

At appropriate clinical doses, none of the COX inhibitors have effects on respiration or lung function. Aspirin overdosage has very complex effects on acid-base balance and respiration.

The classic complication of COX-1 inhibition is gastrointestinal upset. In its most extreme form this can cause upper gastrointestinal bleeding. Both complications result from direct actions of the drug, in the former case, on protective effects of prostaglandins in the mucosa, and in the latter case, on the combination of mucosal effects and inhibition of platelet aggregation.
Acetaminophen toxicity is a common cause of fulminant hepatic failure and need for hepatic transplantation in western societies; it has replaced viral hepatitis as the most common cause of acute hepatic failure.
There is good evidence that NSAIDs, especially selective COX-2 inhibitors, adversely affect renal function in certain patients. Therefore, NSAIDs are generally avoided in patients with reduced creatinine clearance and in others who are dependent upon renal prostaglandin release for vasodilation to avoid hemodynamically mediated acute kidney injury (eg, patients with hypovolemia, heart failure, cirrhosis, diabetic nephropathy, or hypercalcemia).

Gabapentin was introduced as an antiepileptic agent but was serendipitously discovered to have analgesic properties. It found its earliest applications in the treatment of chronic neuropathic pain and is now licensed for postherpetic neuralgia. It and the closely related compound pregabalin are also widely prescribed for diabetic neuropathy. These agents form part of many multimodal postoperative pain protocols, particularly after total joint arthroplasty. There is no evidence that one agent is predictably more efficacious than the other. Although these agents have been shown to bind to voltage-gated calcium channels and N-methyl-p-aspartate (NMDA) receptors, their exact mechanism of action remains speculative. Despite the structural similarities these agents have to y-aminobutyric acid (GABA) their clinical effects do not appear to arise from binding to GABA receptors.
When used for treatment of chronic pain these agents are generally started at relatively small doses and increased incrementally until side effects of dizziness or sedation appear. An adequate trial of gabapentin can require as much a month to achieve the optimal dosage. Determining the optimal dosage of the more potent pregabalin generally requires less time. When used as part of a multimodal postoperative pain protocol these agents are generally prescribed in a standard dose that is maintained for the several days during which the protocol runs its course.

Angst MS. Intraoperative use of remifentanil for TIVA: Postoperative pain, acute tolerance, and opioid-induced hyperalgesia. J Cardiothorac Vasc Anesth. 2015;29(suppl 1):S16.
Brunton LL, Knollmann BC, eds. Goodman & Gilman's The Pharmacological Basis of Therapeutics. 13th ed. New York, NY: McGraw-Hill; 2018: chaps 18, 34.
Food and Drug Administration. Acetaminophen overdose and liver injury—

@ It is important to realize that muscle relaxation does not ensure unconsciousness, amnesia, or analgesia.
@ Depolarizing muscle relaxants act as acetylcholine (ACh) receptor agonists, whereas nondepolarizing muscle relaxants function as competitive antagonists.
© Because depolarizing muscle relaxants are not metabolized by acetylcholinesterase, they diffuse away from the neuromuscular junction and are hydrolyzed in the plasma and liver by another enzyme, pseudocholinesterase (nonspecific cholinesterase, plasma cholinesterase, or butyrylcholinesterase).
@ Muscle relaxants owe their paralytic properties to mimicry of ACh. For example, succinylcholine consists of two joined ACh molecules.
© Incontrast to patients with low enzyme levels or heterozygous atypical enzyme in whom blockade duration is doubled or tripled, patients with homozygous atypical enzyme will have a very long blockade (eg, 4-8 h) following succinylcholine administration.
@ Succinylcholine is considered relatively contraindicated in the routine management of children and adolescents because of the risk of hyperkalemia, rhabdomyolysis, and cardiac arrest in children with undiagnosed myopathies.
@ Normal muscle releases enough potassium during succinylcholineinduced depolarization to raise serum potassium by 0.5 mEq/L. Although this is usually insignificant in patients with normal baseline potassium levels, a life-threatening potassium elevation is possible in patients with burn injury, massive trauma, neurological disorders, and several other conditions.
@ Pancuronium and vecuronium are partially excreted by the kidneys, and
their action is prolonged in patients with kidney failure.

© Cirrhotic liver disease and chronic kidney failure often result in an increased volume of distribution and a lower plasma concentration for a given dose of water-soluble drugs, such as muscle relaxants. On the other hand, drugs dependent on hepatic or renal excretion may demonstrate prolonged clearance. Thus, depending on the drug, a greater initial dose—but smaller maintenance doses—might be required in these diseases.
@ Atracurium and cisatracurium undergo degradation in plasma at physiological pH and temperature by organ-independent Hofmann elimination. The resulting metabolites (a monoquaternary acrylate and laudanosine) have no intrinsic neuromuscular blocking effects.
@ Hypertension and tachycardia may occur in patients given pancuronium. These cardiovascular effects are caused by the combination of vagal blockade and catecholamine release from adrenergic nerve endings.
@® After long-term administration of vecuronium to patients in intensive care units, prolonged neuromuscular blockade (up to several days), may be present after drug discontinuation, possibly from accumulation of its active 3-hydroxy metabolite, changing drug clearance, or the development of a polyneuropathy.
@® Rocuronium (0.9—-1.2 mg/kg) has an onset of action that approaches succinylcholine (60-90 s), making it a suitable alternative for rapidsequence inductions, but at the cost of a much longer duration of action. The new reversal agent, sugammadex, permits rapid reversal of rocuronium-induced neuromuscular blockade.

Skeletal muscle relaxation can be produced by deep inhalational anesthesia, regional nerve block, or neuromuscular blocking agents (commonly called muscle relaxants). In 1942, Harold Griffith published the results of a study using an extract of curare (a South American arrow poison) during anesthesia. Following the introduction of succinylcholine as a "new approach to muscular relaxation," these agents rapidly became a routine part of the anesthesiologist's drug arsenal. However, as noted by Beecher and Todd in 1954: "[m]Juscle relaxants given inappropriately may provide the surgeon with optimal [operating] conditions in ... a patient [who] is paralyzed but not anesthetized—a state [that] is wholly @ unacceptable for the patient." In other words, muscle relaxation does not ensure unconsciousness, amnesia, or analgesia. This chapter reviews the principles of neuromuscular transmission and presents the mechanisms of action, physical structures, routes of elimination, recommended dosages, and side effects of several muscle relaxants.

Association between a motor neuron and a muscle cell occurs at the neuromuscular junction (Figure 11-1). The cell membranes of the neuron and muscle fiber are separated by a narrow (20-nm) gap, the synaptic cleft. As a nerve's action potential depolarizes its terminal, an influx of calcium ions through voltage-gated calcium channels into the nerve cytoplasm allows storage vesicles to fuse with the terminal plasma membrane and release their contents (acetylcholine [ACh]). The ACh molecules diffuse across the synaptic cleft to bind with nicotinic cholinergic receptors on a specialized portion of the muscle membrane, the motor end-plate. Each neuromuscular junction contains approximately 5 million of these receptors, but activation of only about 500,000 receptors is required for normal muscle contraction.
FIGURE 11-1 The neuromuscular junction. V, transmitter vesicle; M, mitochondrion; ACh, acetylcholine; AChE, acetylcholinesterase; JF, junctional folds. (Reproduced with permission from Drachman DB. Myasthenia gravis (1st of 2 parts). N Engl J

The structure of ACh receptors varies in different tissues and at different times in development. Each ACh receptor in the neuromuscular junction normally consists of five protein subunits: two a subunits, and single B, 6, and € subunits. Only the two identical a subunits are capable of binding ACh molecules. If both binding sites are occupied by ACh, a conformational change in the subunits briefly (1 ms) opens an ion channel in the core of the receptor (Figure 11—2). The channel will not open if ACh binds to only one site. In contrast to the normal (or mature) junctional ACh receptor, another isoform contains a y subunit instead of the e subunit. This isoform is referred to as the fetal or immature receptor because it is in the form initially expressed in fetal muscle. It is also often referred to as extrajunctional because, unlike the mature isoform, it may be located anywhere in the muscle membrane, inside or outside the neuromuscular junction when expressed in adults.
FIGURE 11-2 A: Structure of the ACh receptor. Note the two a subunits that actually bind ACh and the center channel. B: Binding of ACh to receptors on muscle end-plate causes channel opening and ion flux.

Cations flow through the open ACh receptor channel (sodium and calcium in; potassium out), generating an end-plate potential. The contents of a single vesicle, a quantum of ACh (10* molecules per quantum), produce a miniature end-plate potential. The number of quanta released by each depolarized nerve fiber, normally at least 200, is very sensitive to extracellular ionized calcium concentration; increasing calcium concentration increases the number of quanta released. When sufficient receptors are occupied by ACh, the end-plate potential will be strong enough to depolarize the perijunctional membrane. Voltage-gated sodium channels within this portion of the muscle membrane open when a threshold voltage is developed across them, as is true for voltage-gated sodium channels in nerve or heart (Figure 11-3). Perijunctional areas of muscle membrane have a higher density of these sodium channels than other parts of the membrane. The resulting action potential propagates along the muscle membrane and T-tubule system, opening sodium channels and releasing calcium from the sarcoplasmic reticulum. This intracellular calcium allows the contractile proteins actin and myosin to interact, bringing about muscle contraction. The amount of ACh released and the number of receptors subsequently activated with efferent nerve depolarization will normally far exceed the minimum required for the initiation of an action potential in the muscle. The nearly tenfold margin of safety is reduced in Eaton—Lambert myasthenic syndrome (decreased release of ACh) and myasthenia gravis (decreased number of receptors).
FIGURE 11-3 Schematic of the sodium channel. The sodium channel is a transmembrane protein that can be conceptualized as having two gates. Sodium ions pass only when both gates are open. Opening of the gates is time dependent and voltage dependent. The channel therefore possesses three functional states. At rest, the lower gate is open but the upper gate is closed (A). When the muscle membrane reaches threshold voltage depolarization, the upper gate opens and sodium can pass (B). Shortly after the upper gate opens the time-dependent lower gate closes (C). When the membrane repolarizes to its resting voltage, the upper gate closes and the lower gate opens (A).

ACh is rapidly hydrolyzed into acetate and choline by the substrate-specific enzyme acetylcholinesterase. This enzyme is embedded into the motor endplate membrane immediately adjacent to the ACh receptors. After unbinding ACh, the receptors' ion channels close, permitting the end-plate to repolarize. Calcium is resequestered in the sarcoplasmic reticulum, and the muscle cell relaxes.

Neuromuscular blocking agents are divided into two classes: depolarizing and nondepolarizing (Table 11—1). This division reflects distinct differences in the mechanism of action, response to peripheral nerve stimulation, and reversal of block.

Short-acting Succinylcholine, Nondepolarizing = Short-acting Mivacurium Gantacurium! Intermediate-acting Atracurium Cisatracurium Vecuronium Rocuronium Long-acting Pancuronium
'Not yet commercially available in the United States.

Some drugs may interfere with the function of the ACh receptor without acting as an agonist or antagonist. They interfere with normal functioning of the ACh receptor binding site or with the opening and closing of the receptor channel. These may include inhaled anesthetic agents, local anesthetics, and ketamine. The ACh receptor—lipid membrane interface may be an important site of action. Drugs may also cause either closed or open channel blockade. During closed channel blockade, the drug physically plugs up the channel, preventing passage of cations whether or not ACh has activated the receptor. Open channel blockade is "use dependent," because the drug enters and obstructs the ACh receptor channel only after it is opened by ACh binding. The clinical relevance of open channel blockade is unknown. Based on laboratory experiments, one would expect that increasing the concentration of ACh with a cholinesterase inhibitor would not overcome this form of neuromuscular blockade. Drugs that may cause channel block under laboratory conditions include neostigmine, some antibiotics, cocaine and other local anesthetics, and quinidine. Other drugs may impair the presynaptic release of ACh. Prejunctional receptors play a role in mobilizing ACh to maintain muscle contraction. Blocking these receptors can lead to a fading of the train-of-four response.

® Because succinylcholine is not metabolized by acetylcholinesterase, it unbinds the receptor and diffuses away from the neuromuscular junction to be hydrolyzed in the plasma and liver by another enzyme, pseudocholinesterase (nonspecific cholinesterase, plasma cholinesterase, or butyrylcholinesterase). Fortunately, this normally is a fairly rapid process, because no specific agent to reverse a depolarizing blockade is available.
With the
exception of
mivacurium, nondepolarizing
agents are
not metabolized
by their
either blockade
and acetylcholinesterase
depends excretion
agents or
pseudocholinesterase.
on unbinding
the receptor,
of the
relaxant
(eg, cholinesterase
activity.
Because by
the body,
inhibitors)
this inhibition
Reversal redistribution,
metabolism, or
administration that
increases inhibit
of specific
reversal acetylcholinesterase
the amount
enzyme of
ACh that
is available
of at
the neuromuscular
reversal junction
agents clearly
block.
In fact,
and are
of can
compete with
the no
benefit in
reversing by
increasing neuromuscular
inhibiting pseudocholinesterase-induced
cholinesterase nondepolarizing
a
junction agent,
phase
I
depolarizing
ACh metabolism
inhibitors can
prolong concentration
and of
succinylcholine, neuromuscular
succinylcholine.
The only
succinylcholine is
sufficient time
time when
has neostigmine
there passed
is for
blockade produced
reverses neuromuscular
a
phase
II
block the
circulating
(fade by
block of
the after
train-of-four)
concentration of
and succinylcholine
to be
negligible.

Sugammadex, a cyclodextrin, is the first selective relaxant-binding agent; it exerts its reversal effect by forming tight complexes in a 1:1 ratio with steroidal nondepolarizing agents (vecuronium, rocuronium). Investigational neuromuscular blocking agents, such as gantacurium, show promise as ultrashort-acting nondepolarizing agents.

The use of peripheral nerve stimulators to monitor neuromuscular function is discussed in Chapter 6. Four patterns of electrical stimulation with supramaximal square-wave pulses are considered:
Tetany—a sustained
stimulus of
50
to
100
Hz, usually
lasting
5
s
Single twitch—a single pulse 0.2 ms in duration
Train-of-four—a series
of four
twitches in
2
s
(2-Hz long
Double-burst stimulation
stimulations separated
(DBS)—three short
by
a
20-ms interval
frequency),
(0.2
ms)
(50
each
0.2
high-frequency
Hz)
and followed
750
ms ms
later by
two
(DBS3
5)
or three
(DBS3
3)
additional impulses
The occurrence of fade, a gradual diminution of evoked response during prolonged or repeated nerve stimulation, is indicative of a nondepolarizing block (Figure 11-4), or of a phase II block if only succinylcholine has been administered. Fade may be due to a prejunctional effect of nondepolarizing relaxants that reduces the amount of ACh in the nerve terminal available for release during stimulation (blockade of ACh mobilization). Adequate clinical recovery correlates well with the absence of fade. Because fade is more obvious during sustained tetanic stimulation or double-burst stimulation than following a the

Train-of-four, Depolarizing Block.Phase | = Constant but diminished. Train-of-four, Depolarizing Block.Phase Il = Fade. Train-of-four, Depolarizing Block.Nondepolarizing Block = Fade. Tetany, Depolarizing Block.Phase | = Constant but diminished. Tetany, Depolarizing Block.Phase Il = Fade. Tetany, Depolarizing Block.Nondepolarizing Block = Fade. Double-burst stimulation (DBSg 2), Depolarizing Block.Phase | = Constant but diminished. Double-burst stimulation (DBSg 2), Depolarizing Block.Phase Il = Fade. Double-burst stimulation (DBSg 2), Depolarizing Block.Nondepolarizing Block = Fade. Posttetanic potentiation, Depolarizing Block.Phase | = Absent. Posttetanic potentiation, Depolarizing Block.Phase Il = Present. Posttetanic potentiation, Depolarizing Block.Nondepolarizing Block = Present
FIGURE 11—4 Evoked responses during depolarizing (phase I and phase II) and nondepolarizing block.
The ability of tetanic stimulation during a partial nondepolarizing block to increase the evoked response to a subsequent twitch is termed posttetanic potentiation. This phenomenon may relate to a transient increase in ACh mobilization following tetanic stimulation.
In contrast, a phase I depolarization block from succinylcholine does not exhibit fade during tetanus or train-of-four; neither does it demonstrate posttetanic potentiation. With prolonged exposure to succinylcholine, however, the quality of the block will sometimes change to resemble a nondepolarizing block (phase IT block).
Newer quantitative methods of assessment of neuromuscular blockade, such as acceleromyography, permit determination of exact train-of-four ratios, as opposed to subjective interpretations. Acceleromyography may reduce the incidence of unexpected postoperative residual neuromuscular blockade.

The only depolarizing muscle relaxant in clinical use today is succinylcholine.

@ Succinylcholine—also called suxamethonium—consists of two joined ACh molecules (Figure 11—5). This structure underlies succinylcholine's mechanism of action, side effects, and metabolism.
FIGURE 11-5 Chemical structures of neuromuscular blocking agents.

TABLE 11-2 Drugs known to decrease pseudocholinesterase activity.
Echothiophate, Description = Organophosphate use for glaucoma. Neostigmine Pyridostigmine, Description = Cholinesterase inhibitors. Phenelzine, Description = Monoamine oxidase inhibitor. Cyclophosphamide, Description = Antineoplastic agent. Metoclopramide, Description = Antiemetic/prokinetic agent. Esmolol, Description = B-Blocker. Pancuronium, Description = Nondepolarizing muscle relaxant. Oral contraceptives, Description = Various agents
Heterozygote patients
with one
normal and
one abnormal
pseudocholinesterase gene
following may
succinylcholine two
copies produce
have slightly
administration.
of the
most prevalent
an enzyme
the doubling
levels with
or tripling
or prolonged
Far abnormal
little or
(atypical)
block fewer
(20-30
min)
(1
in gene
no of
heterozygous enzyme
will affinity
blockade atypical
have
a
very succinylcholine.
Of dibucaine-resistant
normal affinity
for fluoride-resistant
3000)
patients
(homozygous have
atypical)
for succinylcholine.
duration enzyme,
long seen
that
@
In contrast
in patients
with patients
blockade the
(eg, recognized
with
4—8
abnormal
(variant)
allele, succinylcholine,
and
Dibucaine, low
homozygous enzyme
atypical h)
following administration
pseudocholinesterase which
produces is
the silent
(no activity)
a
local by
80%
but inhibits
individual who
anesthetic, of
genes, the
an enzyme
with most
common.
alleles.
inhibits atypical
enzyme is
heterozygous intermediate
40%
normal activity
for the
to
60%
inhibition.
pseudocholinesterase activity
normal by
atypical
The is
termed pseudocholinesterase
the most
has

1/100
Other variants
of include
only
20%.
Serum from
enzyme percentage
is an
characterized of
the dibucaine
a
common abnormal
dibucaine inhibition
by of
number.
A
number patient
with of
allele dibucaine
number measures
will have
a
80;
a
homozygote dibucaine
pseudocholinesterase number
function, for
of
20.
The not
the amount
an of
to enzyme. Therefore, adequacy of pseudocholinesterase can be determined in the laboratory quantitatively in units per liter (a minor factor) and qualitatively by dibucaine number (the major factor). Prolonged paralysis from succinylcholine caused by abnormal pseudocholinesterase (atypical cholinesterase) should be treated with continued mechanical ventilation and sedation until muscle function returns to normal by clinical signs.

The effects of muscle relaxants can be modified by concurrent drug therapy (Table 11-3). Succinylcholine is involved in two interactions deserving special comment.

succinylcholine. Magnesium sulfate, Effecton Depolarizing Blockade" = +. Magnesium sulfate, Effect on Nondepolarizing Blockade = +. Magnesium sulfate, Comments = Doses used to treat preeclampsia and eclampsia of pregnancy
"2, unknown effect.

Although cholinesterase inhibitors reverse nondepolarizing paralysis, they markedly prolong a depolarizing phase I block by two mechanisms. By inhibiting acetylcholinesterase, they lead to a higher ACh concentration at the nerve terminal, which intensifies depolarization. They also reduce the hydrolysis of succinylcholine by inhibiting pseudocholinesterase. Organophosphate pesticides, for example, cause an irreversible inhibition of acetylcholinesterase and can prolong the action of succinylcholine by 20 to 30 min. Echothiophate eye drops, used in the past for glaucoma, can markedly prolong succinylcholine

In general, small doses of nondepolarizing relaxants antagonize a depolarizing phase I block. Because the drugs occupy some ACh receptors, depolarization by succinylcholine is partially prevented. In the presence of a phase II block, a nondepolarizer will potentiate succinylcholine paralysis.

Because of the rapid onset, short duration, and low cost of succinylcholine, some clinicians believe that it remains a good choice for routine intubation in adults. The usual adult dose of succinylcholine for intubation is 1 to 1.5 mg/kg intravenously. Doses as small as 0.5 mg/kg usually provide acceptable intubating conditions if a defasciculating dose of a nondepolarizing agent is not used. Repeated small boluses (5-10 mg) or a succinylcholine drip (1 g in 500 or 1000 mL, titrated to effect) can be used during surgical procedures that require brief but intense paralysis (eg, otolaryngological endoscopies). Neuromuscular function should be frequently monitored with a nerve stimulator to prevent overdosing and to watch for phase II block. The availability of intermediateacting nondepolarizing muscle relaxants has reduced the popularity of succinylcholine infusions. In the past, succinylcholine infusions were a mainstay of ambulatory practice in the United States. Because succinylcholine is not lipid soluble, it has a small volume of distribution. Per kilogram, infants and neonates have a larger extracellular space than adults. Therefore, on a per-kilogram basis, dosage requirements for pediatric patients are often greater than for adults. If succinylcholine is administered intramuscularly to children, a dose as high as 4 to 5 mg/kg does not always produce complete paralysis.
Succinylcholine should be stored under refrigeration (2—-8°C), and should generally be used within 14 days after removal from refrigeration and exposure to room temperature.

Succinylcholine is a relatively safe drug—assuming that its many potential complications are understood @ and avoided. Because of the risk of hyperkalemia, rhabdomyolysis, and cardiac arrest in children with undiagnosed myopathies, succinylcholine is considered relatively contraindicated in the routine management of children. Some clinicians have also abandoned the routine use of succinylcholine for adults. Succinylcholine is still useful for rapid sequence induction and for short periods of intense paralysis because none of the presently available nondepolarizing muscle relaxants can match its very rapid onset and short duration.

The onset of paralysis by succinylcholine is usually signaled by visible motor unit contractions called fasciculations. These can be prevented by pretreatment with a small dose of nondepolarizing relaxant. Because this pretreatment usually antagonizes a depolarizing block, a larger dose of succinylcholine is required (1.5 mg/kg). Fasciculations are typically not observed in young children and elderly patients.

@ Normal muscle releases enough potassium during succinylcholine-induced depolarization to increase serum potassium by 0.5 mEq/L. Although this is usually insignificant in patients with normal baseline potassium levels, it can be life-threatening in patients with preexisting hyperkalemia. The increase in potassium in patients with burn injury, massive trauma, neurological disorders, and several other conditions (Table 11—4) can be large and catastrophic. Hyperkalemic cardiac arrest can prove to be quite refractory to routine cardiopulmonary resuscitation, requiring calcium, insulin, glucose, bicarbonate, and even cardiopulmonary bypass to support the circulation while reducing serum potassium levels.

Burn injury
Massive trauma
Severe intraabdominal infection
Spinal cord injury
Encephalitis
Stroke
Guillain-Barré syndrome
Severe Parkinson disease
Tetanus
Prolonged total body immobilization
Ruptured cerebral aneurysm
Polyneuropathy
Closed head injury
Hemorrhagic shock with metabolic acidosis
Myopathies (eg, Duchenne dystrophy)
Following denervation injuries (spinal cord injuries, larger burns), the immature isoform of the ACh receptor may be expressed inside and outside the neuromuscular junction (upregulation). These extrajunctional receptors allow succinylcholine to effect widespread depolarization and extensive potassium release. Life-threatening potassium release is not reliably prevented by pretreatment with a nondepolarizer. The risk of hyperkalemia usually seems to peak in 7 to 10 days following the injury, but the exact time of onset and the duration of the risk period vary. The risk of hyperkalemia from succinylcholine is minimal in the first 2 days after spinal cord or burn injury.

Patients who have received succinylcholine have an increased incidence of postoperative myalgia. The efficacy of nondepolarizing pretreatment is controversial. Administration of rocuronium (0.06—0.1 mg/kg) prior to succinylcholine has been reported to be effective in preventing fasciculations and reducing postoperative myalgias. The relationship between fasciculations and postoperative myalgias is also inconsistent. The myalgias are theorized to be due to the initial unsynchronized contraction of muscle groups; myoglobinemia and increases in serum creatine kinase can be detected following administration of succinylcholine. Perioperative use of nonsteroidal antiinflammatory drugs and benzodiazepines may reduce the incidence and severity of myalgias.

Abdominal wall muscle fasciculations increase intragastric pressure, which is offset by an increase in lower esophageal sphincter tone. Therefore, despite being much discussed, there is no evidence that the risk of gastric reflux or pulmonary aspiration is increased by succinylcholine.

Extraocular muscle differs from other striated muscle in that it has multiple motor end-plates on each cell. Prolonged membrane depolarization and contraction of extraocular muscles following administration of succinylcholine transiently raise intraocular pressure and theoretically could compromise an injured eye. However, there is no evidence that succinylcholine leads to worsened outcome in patients with "open" eye injuries. The elevation in intraocular pressure is not always prevented by pretreatment with a nondepolarizing agent.

Succinylcholine transiently increases muscle tone in the masseter muscles. Some difficulty may initially be encountered in opening the mouth because of incomplete relaxation of the jaw. A marked increase in tone preventing laryngoscopy is abnormal and can be a premonitory sign of malignant hyperthermia.

Succinylcholine is a potent triggering agent in patients susceptible to malignant hyperthermia, a hypermetabolic disorder of skeletal muscle (see Chapter 52). Although some of the signs and symptoms of neuroleptic malignant syndrome (NMS) resemble those of malignant hyperthermia, the pathogenesis is completely different and there is no need to avoid use of succinylcholine in patients with NMS.

Patients afflicted with myotonia may develop myoclonus after administration of succinylcholine.

As previously discussed, patients with reduced levels of normal pseudocholinesterase may have a longer than normal duration of action, whereas patients with atypical pseudocholinesterase will experience markedly prolonged paralysis.

Succinylcholine may lead to an activation of the electroencephalogram and slight increases in cerebral blood flow and intracranial pressure in some patients. Muscle fasciculations stimulate muscle stretch receptors, which subsequently increase cerebral activity. The increase in intracranial pressure can be attenuated by maintaining good airway control and instituting hyperventilation. It can also be prevented by pretreating with a nondepolarizing muscle relaxant and administering intravenous lidocaine (1.5—2.0 mg/kg) 2 to 3 min prior to intubation. The effects of intubation on intracranial pressure far outweigh any increase caused by succinylcholine, and succinylcholine is not contraindicated for rapid sequence induction of patients with intracranial mass lesions or other causes of increased intracranial pressure.

Slight histamine release may be observed following succinylcholine in some patients.

In contrast to there being only a single depolarizing muscle relaxant, there is a wide selection of nondepolarizing muscle relaxants (Tables 11—5 and 11-6). Based on their chemical structure, they can be classified as benzylisoquinolinium, steroidal, or other compounds. It is often said that choice of a particular drug depends on its unique characteristics, which are often related to its structure; however, for most patients, the differences among the intermediate-acting neuromuscular blockers are inconsequential. Steroidal compounds can be vagolytic, most notably with pancuronium but inconsequentially with vecuronium or rocuronium. Benzylisoquinolines tend to release histamine. Because of structural similarities, an allergic history to one muscle relaxant strongly suggests the possibility of allergic reactions to other muscle relaxants, particularly those in the same chemical class.

0
'B, benzylisoquinolone; S, steroidal; C, chlorofumarate.
2Onset: +, slow; ++, moderately rapid; +++, rapid
Duration: +, short; ++, intermediate; +++, long.
"Histamine release: 0, no effect; +, slight effect; ++, moderate effect; +++, marked effect.
'Vagal blockade: 0, no effect; +, slight effect; ++, moderate effect.

of the currently available nondepolarizing muscle relaxants equals succinylcholine's rapid onset of action or short duration. However, the onset of nondepolarizing relaxants can be quickened by using either a larger dose or a priming dose. The EDo, of any drug is the effective dose of a drug in 95% of
individuals.
For neuromuscular
blockers, one
often specifies
the dose
that produces
95%
EDgz or
twitch twice
intubation.
depression the
dose
Although duration
of concern
in
50%
a
blockade.
in larger
The regard
to of
95%
intubating twitch
One depression
dose availability
the speeds
onset, to
two times
is usually
it of
steroidal sugammadex
nondepolarizing has
muscle the
used prolongs
for the
largely eliminated
relaxant, this
rocuronium
(see
Chapter
12).
Muscle groups vary in their sensitivity to muscle relaxants. For example, the laryngeal muscles—whose relaxation is important during intubation—recover from blockade more quickly than the adductor pollicis, which is commonly monitored by the peripheral nerve stimulator.

To prevent fasciculations and myalgias, 10% to 15% of a nondepolarizer intubating dose can be administered 5 min before succinylcholine. Similarly, priming with a small dose of a nondepolarizing relaxant (10% of the total dose) a few minutes before intubation can speed the onset of acceptable intubating conditions when followed by the remaining 90% of the drug to be administered. Dysphagia, diplopia, and patient distress will occasionally develop following administration of a priming or defasciculating dose of nondepolarizing muscle relaxants.

Following intubation, muscle paralysis may need to be maintained to facilitate surgery, (eg, abdominal operations), to permit a reduced depth of anesthesia, or to control ventilation. There is great variability among patients in response to muscle relaxants. Monitoring neuromuscular function with a nerve stimulator helps to prevent overand underdosing and to reduce the likelihood of serious residual muscle paralysis in the recovery room. Maintenance doses, whether by intermittent boluses or continuous infusion (Table 11-6), should be guided by the nerve stimulator and clinical signs (eg, spontaneous respiratory efforts or movement). In some instances, clinical signs may precede twitch recovery because of differing sensitivities to muscle relaxants between muscle groups or technical problems with the nerve stimulator. Some return of neuromuscular transmission should be evident prior to administering each maintenance dose, if the patient needs to resume spontaneous ventilation at the end of the anesthetic. When an infusion is used for maintenance, the rate should be adjusted at or just above the rate that allows some return of neuromuscular transmission so that drug effects can be monitored.

Durationof Intubating Dose(min) = 45-90. Vecuronium, Maintenance Dosing by Boluses (mg/kg) = 0.01. Vecuronium, Maintenance Dosing by Infusion (meg/kg/min) = 1-2. Pancuronium, ED,, for Adductor Pollicis During Nitrous Oxide/ Oxygen/Intravenous Anesthesia (mg/kg) = 0.07. Pancuronium, Intubation Dose (mg/kg) = 0.12. Pancuronium, Onset of Actionfor Intubating Dose(min) = 2.0-3.0. Pancuronium, Durationof Intubating Dose(min) = 60-120. Pancuronium, Maintenance Dosing by Boluses (mg/kg) = 0.01. Pancuronium, Maintenance Dosing by Infusion (meg/kg/min) = _-
'Not commercially available in the United States.

Volatile agents decrease nondepolarizer dosage requirements by at least 15%. The actual degree of this postsynaptic augmentation depends on the inhalational anesthetic (desflurane > sevoflurane > isoflurane > halothane > N,O/O,/narcotic > total intravenous anesthesia).

Some combinations of different classes of nondepolarizers (eg, steroidal and benzylisoquinolinium) produce a greater than additive (synergistic)
neuromuscular blockade.

In clinical doses, the nondepolarizers differ in their relative effects on nicotinic and muscarinic cholinergic receptors. Previously used agents (eg, tubocurarine) blocked autonomic ganglia, reducing the ability of the sympathetic nervous system to increase heart contractility and rate in response to hypotension and other intraoperative stresses. In contrast, pancuronium blocks vagal muscarinic receptors in the sinoatrial node, resulting in tachycardia. All newer nondepolarizing relaxants, including atracurium, cisatracurium, mivacurium, vecuronium, and rocuronium, are devoid of significant autonomic effects in their recommended dosage ranges.

Histamine release from mast cells can result in bronchospasm, skin flushing, and hypotension from peripheral vasodilation. Atracurium and mivacurium are capable of triggering histamine release, particularly at higher doses. Slow injection rates and H, and H, antihistamine pretreatment ameliorate these side effects.

Only pancuronium, vecuronium, and rocuronium are metabolized to varying degrees by the liver. Active metabolites likely contribute to their clinical effect. Vecuronium and rocuronium depend heavily on biliary excretion. Clinically, liver failure prolongs blockade. Atracurium, cisatracurium, and mivacurium, although extensively metabolized, depend on extrahepatic mechanisms. Severe liver disease does not significantly affect clearance of atracurium or cisatracurium, but the associated decrease in pseudocholinesterase levels may slow the metabolism of mivacurium.

@ Pancuronium, vecuronium, and rocuronium are partially excreted by the kidneys. The duration of action or pancuronium and vecuronium is prolonged in patients with kidney failure. The elimination of atracurium and cisatracurium is independent of kidney function. The duration of action of rocuronium and mivacurium is not significantly affected by renal dysfunction.

Hypothermia prolongs blockade by decreasing metabolism (eg, mivacurium, atracurium, and cisatracurium) and delaying excretion (eg, pancuronium and vecuronium).

Respiratory acidosis potentiates the blockade of most nondepolarizing relaxants and antagonizes its reversal. This could prevent complete neuromuscular recovery in a hypoventilating postoperative patient. Conflicting findings regarding the neuromuscular effects of other acid—base changes may be due to coexisting alterations in extracellular pH, intracellular pH, electrolyte concentrations, or structural differences between drugs (eg, monoquaternary versus bisquaternary; steroidal versus isoquinolinium).

Hypokalemia and hypocalcemia augment a nondepolarizing block. The responses of patients with hypercalcemia are unpredictable. Hypermagnesemia, as may be seen in preeclamptic patients being managed with magnesium sulfate (or after intravenous magnesium administered in the operating room), potentiates a nondepolarizing blockade by competing with calcium at the motor end-plate.

Neonates have an increased sensitivity to nondepolarizing relaxants because of their immature neuromuscular junctions (Table 11-7). This sensitivity does not necessarily decrease dosage requirements, as the neonate's greater extracellular space provides a larger volume of distribution.
TABLE 11-7 Additional considerations of muscle relaxants in special populations.
Pediatric
Elderly
Obese
Kidney failure
Critically ill
Succinylcholine: should not be used routinely Nondepolarizing agents: faster onset Vecuronium: long-acting in neonates
Decreased clearance: prolonged duration, except with cisatracurium
Dosage 20% more than lean body weight; onset unchanged Prolonged duration, except with cisatracurium
Increased volume of distribution Pancuronium and vecuronium: prolonged elimination due to hepatic metabolism and biliary excretion
Cisatracurium: unchanged Pseudocholinesterase decreased; prolonged action may be seen with succinylcholine in severe disease
Vecuronium: prolonged Rocuronium: relatively unchanged Cisatracurium: safest alternative
Myopathy, polyneuropathy, nicotinic acetylcholine receptor upregulation

As noted earlier, many drugs augment nondepolarizing blockade (see Table 11-— 3). They have multiple sites of interaction: prejunctional structures, postjunctional cholinergic receptors, and muscle membranes.

The presence of neurological or muscular disease can have profound effects on an individual's response @ to muscle relaxants (Table 11—8). Cirrhotic liver disease and chronic kidney failure often result in an increased volume of distribution and a lower plasma concentration for a given dose of water-soluble drugs, such as muscle relaxants. On the other hand, drugs dependent on hepatic or renal excretion may demonstrate prolonged clearance (Table 11-7). Thus, depending on the drug chosen, a greater initial (loading) dose—but smaller maintenance doses—might be required in these diseases.

The onset and intensity of blockade vary among muscle groups. This may be due to differences in blood flow, distance from the central circulation, or different fiber types. Furthermore, the relative sensitivity of a muscle group may depend on the choice of muscle relaxant. In general, the diaphragm, jaw, larynx, and facial muscles (orbicularis oculi) respond to and recover from muscle relaxation sooner than the thumb. Although they are a fortuitous safety feature, persistent diaphragmatic contractions can be disconcerting in the face of complete adductor pollicis paralysis. Glottic musculature is also quite resistant to blockade, as is often confirmed during laryngoscopy. The dose that produces 95% twitch depression in laryngeal muscles is nearly two times that for the adductor pollicis muscle. Good intubating conditions are usually associated with visual loss of the orbicularis oculi twitch response.
Considering the multitude of factors influencing the duration and magnitude of muscle relaxation, it becomes clear that an individual's response to neuromuscular blocking agents should be monitored. Dosage recommendations, including those in this chapter, should be considered guidelines that require modification for individual patients. Wide variability in sensitivity to nondepolarizing muscle relaxants is often encountered in clinical practice.

Like all muscle relaxants, atracurium has a quaternary group; however, a benzylisoquinoline structure is responsible for its unique method of degradation. The drug is a mixture of ten stereoisomers.

Atracurium is so extensively metabolized that its pharmacokinetics are independent of renal and hepatic function, and less than 10% is excreted unchanged by renal and biliary routes. Two separate processes are responsible for metabolism.

This action is catalyzed by nonspecific esterases, not by acetylcholinesterase or pseudocholinesterase.

A spontaneous nonenzymatic chemical breakdown occurs at physiological pH and temperature.

A dose of 0.5 mg/kg is administered intravenously for intubation. After succinylcholine, intraoperative relaxation is achieved with 0.25 mg/kg initially, then in incremental doses of 0.1 mg/kg every 10 to 20 min. An infusion of 5 to 10 mcg/kg/min can effectively replace intermittent boluses. Although dosage requirements do not significantly vary with age, atracurium may be shorter acting in children and infants than in adults.
Atracurium is available as a solution of 10 mg/mL. It must be stored at 2°C to 8°C, as it loses 5% to 10% of its potency for each month it is exposed to room temperature. At room temperature, it should be used within 14 days to preserve potency.

Atracurium triggers dose-dependent histamine release that becomes significant at doses above 0.5 mg/kg.

Cardiovascular side effects are unusual unless doses in excess of 0.5 mg/kg are administered. Atracurium may also cause a transient drop in systemic vascular resistance and an increase in cardiac index independent of any histamine release. A slow rate of injection minimizes these effects.

Atracurium should be avoided in asthmatic patients. Severe bronchospasm is occasionally seen in patients without a history of asthma.

Laudanosine, a tertiary amine, is a breakdown product of atracurium's Hofmann elimination and has been associated with central nervous system excitation, resulting in elevation of the minimum alveolar concentration and even precipitation of seizures. Concerns about laudanosine are probably irrelevant unless a patient has received an extremely large total dose or has hepatic failure. Laudanosine is metabolized by the liver and excreted in urine and bile.

Because of its unique metabolism, atracurium's duration of action can be markedly prolonged by hypothermia and to a lesser extent by acidosis.

Atracurium will precipitate as a free acid if it is introduced into an intravenous line containing an alkaline solution such as thiopental.

Rare anaphylactoid reactions to atracurium have been described. Proposed mechanisms include direct immunogenicity and acrylate-mediated immune activation. Immunoglobulin E-mediated antibody reactions directed against substituted ammonium compounds, including muscle relaxants, have been described. Reactions to acrylate, a metabolite of atracurium and a structural component of some dialysis membranes, have also been reported in patients undergoing hemodialysis.

Cisatracurium is a stereoisomer of atracurium that is four times more potent. Atracurium contains approximately 15% cisatracurium.

© Like atracurium, cisatracurium undergoes degradation in plasma at physiological pH and temperature by organ-independent Hofmann elimination. The resulting metabolites (a monoquaternary acrylate and laudanosine) have no neuromuscular blocking effects. Because of cisatracurium's greater potency, the amount of laudanosine produced for the same extent and duration of neuromuscular blockade is much less than with atracurium. Metabolism and elimination are independent of kidney or liver failure. Minor variations in pharmacokinetic patterns due to age result in no clinically important changes in duration of action.

Cisatracurium produces good intubating conditions following a dose of 0.1 to 0.15 mg/kg within 2 min and results in muscle blockade of intermediate duration. The typical maintenance infusion rate ranges from 1.0 to 2.0 mcg/kg/min. Thus, it is more potent than atracurium.
Cisatracurium should be stored under refrigeration (2—8°C) and should be used within 21 days after removal from refrigeration and exposure to room temperature.

Unlike atracurium, cisatracurium does not produce a consistent, dose-dependent increase in plasma histamine levels following administration. Cisatracurium does not alter heart rate or blood pressure, nor does it produce autonomic effects, even at doses as high as eight times EDgs.
Cisatracurium shares with atracurium the production of laudanosine, pH and temperature sensitivity, and chemical incompatibility.

Mivacurium is a short-acting, benzylisoquinoline, nondepolarizing neuromuscular blocker. It has recently returned to the North American anesthesia market after having been unavailable for a number of years.

Mivacurium, like succinylcholine, is metabolized by pseudocholinesterase. Consequently, patients with low pseudocholinesterase concentration or activity may experience prolonged neuromuscular blockade following mivacurium administration. However, like other nondepolarizing agents, cholinesterase inhibitors will antagonize mivacurium-induced neuromuscular blockade. Edrophonium more effectively reverses mivacurium blockade than neostigmine because neostigmine inhibits plasma cholinesterase activity.

Mivacurium releases histamine to about the same degree as atracurium. The onset time of mivacurium is approximately 2 to 3 min. The main advantage of mivacurium compared with atracurium is its relatively brief duration of action (20-30 min).

Pancuronium consists of a steroid structure on which two modified ACh molecules are positioned (a bisquaternary relaxant). In all of the steroid-based relaxants the steroid "backbone" serves as a "spacer" between the two quaternary amines. Pancuronium resembles ACh enough to bind (but not activate) the nicotinic ACh receptor.

Pancuronium is metabolized (deacetylated) by the liver to a limited degree. Its metabolic products have some neuromuscular blocking activity. Excretion is primarily renal (40%), although some of the drug is cleared by the bile (10%). Not surprisingly, elimination of pancuronium is slowed and neuromuscular blockade is prolonged by kidney failure. Patients with cirrhosis may require a larger initial dose due to an increased volume of distribution but have reduced maintenance requirements because of a decreased rate of plasma clearance.

A dose of 0.08 to 0.12 mg/kg of pancuronium provides adequate relaxation for intubation in 2 to 3 min. Intraoperative relaxation is achieved by administering 0.04 mg/kg initially followed every 20 to 40 min by 0.01 mg/kg. Children may require moderately larger doses of pancuronium. Pancuronium is available as a solution of 1 or 2 mg/mL and is stored at 2°C to 8°C but may be stable for up to 6 months at normal room temperature.

@ These cardiovascular effects are caused by the combination of vagal blockade and sympathetic stimulation. The latter is due to a combination of ganglionic stimulation, catecholamine release from adrenergic nerve endings, and decreased catecholamine reuptake. Large bolus doses of pancuronium should be given with caution to patients in whom an increased heart rate would be particularly detrimental (eg, coronary artery disease, hypertrophic cardiomyopathy, aortic stenosis).

Increased atrioventricular conduction and catecholamine release increase the likelihood of ventricular arrhythmias in predisposed individuals. The combination of pancuronium, tricyclic antidepressants, and halothane has been reported to be particularly arrhythmogenic.

Patients who are hypersensitive to bromides may exhibit allergic reactions to pancuronium (pancuronium bromide).

Vecuronium is pancuronium minus a quaternary methyl group (a monoquaternary relaxant). This minor structural change beneficially alters side effects without affecting potency.

Vecuronium is metabolized to a small extent by the liver. It depends primarily on biliary excretion and secondarily (25%) on renal excretion. Although it is a satisfactory drug for patients with kidney failure, its duration of action will be moderately prolonged. Vecuronium's brief duration of action is explained by its shorter elimination half-life and more rapid ® clearance compared with pancuronium. After long-term administration of vecuronium to patients in intensive care units prolonged neuromuscular blockade (up to several days) may be present after drug discontinuation, possibly from accumulation of its active 3hydroxy metabolite, changing drug clearance, and in some patients, leading to the development of a polyneuropathy. Risk factors seem to include female gender, kidney failure, long-term or high-dose corticosteroid therapy, and sepsis. Thus, these patients must be closely monitored, and the dose of vecuronium carefully titrated. Long-term relaxant administration and the subsequent prolonged lack of ACh binding at the postsynaptic nicotinic ACh receptors may mimic a chronic denervation state and cause lasting receptor dysfunction and paralysis. Tolerance to nondepolarizing muscle relaxants can also develop after long-term use. The best approach is to avoid unnecessary paralysis of patients in critical care units.

Vecuronium is equipotent with pancuronium, and the intubating dose is 0.08 to 0.12 mg/kg. A dose of 0.04 mg/kg initially followed by increments of 0.01 mg/kg every 15 to 20 min provides intraoperative relaxation. Alternatively, an infusion of 1 to 2 mcg/kg/min produces good maintenance of relaxation. Age does not affect initial dose requirements, although subsequent doses are required less frequently in neonates and infants. Women seem to be approximately 30% more sensitive than men to vecuronium, as evidenced by a greater degree of blockade and longer duration of action (this has also been seen with pancuronium and rocuronium). The cause for this sensitivity is likely related to gender-related differences in fat and muscle mass and volume of distribution. The duration of action of vecuronium may be further prolonged in postpartum patients due to alterations in hepatic blood flow or liver uptake.

Even at doses of 0.28 mg/kg, vecuronium is devoid of significant cardiovascular effects. Potentiation of opioid-induced bradycardia may be observed in some patients.

Although it is dependent on biliary excretion, the duration of action of vecuronium is usually not significantly prolonged in patients with cirrhosis unless doses greater than 0.15 mg/kg are given. Vecuronium requirements are reduced during the anhepatic phase of liver transplantation.

This monoquaternary steroid analogue of vecuronium was designed to provide a rapid onset of action.

Rocuronium undergoes no metabolism and is eliminated primarily by the liver and slightly by the kidneys. Its duration of action is not significantly affected by renal disease, but it is modestly prolonged by severe liver failure and pregnancy. Because rocuronium does not have active metabolites, it may be a better choice than vecuronium in the rare patient requiring prolonged infusions in the intensive care unit setting. Elderly patients may experience a prolonged duration of action due to decreased liver mass.
Rocuronium is less potent than most other steroidal muscle relaxants (potency seems to be inversely related to speed of onset). It requires 0.45 to 0.9 mg/kg intravenously for intubation and 0.15 mg/kg boluses for maintenance. Intramuscular rocuronium (1 mg/kg for infants; 2 mg/kg for children) provides adequate vocal cord and diaphragmatic paralysis for intubation, but not until after 3 to 6 min (deltoid injection has a faster onset than quadriceps). The infusion requirements for rocuronium range from 5 to 12 mcg/kg/min. Rocuronium can produce an unexpectedly prolonged duration of action in elderly patients. Initial dosage requirements are modestly increased in patients with advanced liver disease, presumably due to a larger volume of distribution.

® Rocuronium (at a dose of 0.9-1.2 mg/kg) has an onset of action that approaches succinylcholine (60-90 s), making it a suitable alternative for rapidsequence inductions, but at the cost of a much longer duration of action. This intermediate duration of action is comparable to vecuronium or atracurium. Sugammadex permits rapid reversal of dense rocuronium-induced neuromuscular blockade.
Rocuronium (0.1 mg/kg) has been shown to be a rapid (90 s) and effective agent (decreased fasciculations and postoperative myalgias) for precurarization prior to administration of succinylcholine. It has slight vagolytic tendencies.

Gantacurium belongs to a new class of nondepolarizing neuromuscular blockers called chlorofumarates. In preclinical trials, gantacurium demonstrated an ultrashort duration of action, similar to that of succinylcholine. Its pharmacokinetic profile is explained by the fact that it undergoes nonenzymatic degradation by two chemical mechanisms: rapid formation of inactive cysteine adduction product and ester hydrolysis. At a dose of 0.2 mg/kg (EDg;), the onset of action has been estimated to be 1 to 2 min, with a duration of blockade similar to that of succinylcholine. Its clinical duration of action ranged from 5 to 10 min. Recovery can be accelerated by edrophonium, as well as by the administration of exogenous cysteine. Cardiovascular effects suggestive of histamine release were observed following the use of three times the EDg, dosage.
CW002 is another investigational nondepolarizing agent. It is a benzylisoquinolinium fumarate ester-based compound with an intermediate duration of action that undergoes metabolism and elimination similar to that of gantacurium.

A 72-year-old man has undergone general anesthesia for robot-assisted laparoscopic prostatectomy. Twenty minutes after conclusion of the procedure, he is still intubated and shows no evidence of spontaneous respiration or consciousness.

Clues to the solution of complex clinical problems are usually found in a pertinent review of the medical and surgical history, the history of drug ingestions, the physical examination, and laboratory results. In this case, the perioperative anesthetic management should also be considered.
What medical illnesses predispose a patient to delayed awakening or prolonged paralysis?
Chronic hypertension alters cerebral blood flow autoregulation and decreases the brain's tolerance to episodes of hypotension. Liver disease reduces hepatic drug metabolism and biliary excretion, resulting in prolonged drug action. Reduced serum albumin concentrations increase free drug (active drug) availability. Hepatic encephalopathy can alter consciousness. Kidney disease decreases the renal excretion of many drugs. Uremia can also affect consciousness. Diabetic patients are prone to hypoglycemia and hyperosmotic, hyperglycemic, and nonketotic coma. A prior stroke or symptomatic carotid bruit increases the risk of intraoperative cerebral vascular accident. Right-to-left heart shunts, particularly in children with congenital heart disease, allow air emboli to pass directly from the venous circulation to the systemic (possibly cerebral) arterial circulation. A paradoxic air embolism can result in permanent brain damage. Severe hypothyroidism is associated with impaired drug metabolism and, rarely, myxedema coma.
Does an uneventful history of general anesthesia narrow the

Hereditary atypical pseudocholinesterase is ruled out by uneventful prior general anesthesia, assuming succinylcholine was administered. Decreased levels of normal enzyme would not result in postoperative apnea unless the surgery was of very short duration. Malignant hyperthermia does not typically present as delayed awakening, although prolonged somnolence is not unusual. Uneventful prior anesthetics do not, however, rule out malignant hyperthermia. Persons unusually sensitive to anesthetic agents (eg, geriatric patients) may have a history of delayed emergence.
How do drugs that a patient takes at home affect awakening from general anesthesia?
Drugs that decrease minimum alveolar concentration, such as methyldopa, predispose patients to anesthetic overdose. Acute ethanol intoxication decreases barbiturate metabolism and acts independently as a sedative. Drugs that decrease liver blood flow, such as cimetidine, will limit hepatic drug metabolism. Antiparkinsonian drugs and tricyclic antidepressants have anticholinergic side effects that augment the sedation produced by scopolamine. Long-acting sedatives, such as the benzodiazepines, can delay awakening.

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Pupil size is not always a reliable indicator of central nervous system integrity. Fixed and dilated pupils in the absence of anticholinergic medication or ganglionic blockade, however, may be an ominous sign. Response to physical stimulation, such as a forceful jaw thrust, may differentiate somnolence from paralysis. Peripheral nerve stimulation also differentiates paralysis from coma.

Arterial blood gases, plasma glucose, and serum electrolytes may be helpful. Computed tomographic scanning may be necessary if unresponsiveness is prolonged. Increased concentrations of inhalational agent provided by respiratory gas analysis, as well as processed electroencephalogram (EEG) measurements, may assist in determining if the patient is still under the effects of anesthesia. Slow EEG signals can be indicative of both anesthesia and cerebral pathology. Processed EEG awareness monitors can also be employed with the realization that low numbers on the bispectral index can be caused both by anesthetic suppression of the EEG and ischemic brain injury.

Supportive mechanical ventilation should be continued in the unresponsive patient. Naloxone, flumazenil, and physostigmine may be indicated, depending on the probable cause of the delayed emergence, if drug effects are suspected and reversal is considered both safe and desirable.

Brull SJ, Kopman AF. Current status of neuromuscular reversal and monitoring: Challenges and opportunities. Anesthesiology. 2017;126:173. deBacker J, Hart N, Fan E. Neuromuscular blockade in the 21st century management of the critically ill patient. Chest. 2017;151:697.
Heerdt PM, Sunaga H, Savarese JJ. Novel neuromuscular blocking drugs and

@ The primary clinical use of cholinesterase inhibitors is to reverse nondepolarizing neuromuscular blockers.
@® Acetylcholine is the neurotransmitter for the entire parasympathetic nervous system (parasympathetic ganglions and effector cells), parts of the sympathetic nervous system (sympathetic ganglions, adrenal medulla, and sweat glands), some neurons in the central nervous system, and somatic nerves innervating skeletal muscle.
© Neuromuscular transmission is blocked when nondepolarizing muscle relaxants compete with acetylcholine to bind to nicotinic cholinergic receptors. The cholinesterase inhibitors indirectly increase the amount of acetylcholine available to compete with the nondepolarizing agent, thereby reestablishing neuromuscular transmission.
@ Acetylcholinesterase inhibitors prolong the depolarization blockade of succinylcholine.
@ Any prolongation of action of a nondepolarizing muscle relaxant from renal or hepatic insufficiency will probably be accompanied by a corresponding increase in the duration of action of a cholinesterase inhibitor.
@ The time required to fully reverse a nondepolarizing block depends on several factors, including the choice and dose of cholinesterase inhibitor administered, the muscle relaxant being antagonized, and the extent of the blockade before reversal.
@ A reversal agent should be routinely given to patients who have received nondepolarizing muscle relaxants unless full reversal can be demonstrated or the postoperative plan includes continued intubation and ventilation.
@ Newer quantitative methods for assessing recovery from neuromuscular blockade, such as acceleromyography, may further reduce the incidence of undetected residual postoperative neuromuscular paralysis.
Sugammadex exerts its effects by forming tight complexes in a 1:1 ratio with steroidal neuromuscular blocking agents.
Cysteine causes inactivation of gantacurium via metabolic degradation and adduct formation.

Incomplete reversal of neuromuscular blocking agents and residual postprocedure paralysis are associated with morbidity; therefore, careful evaluation of neuromuscular blockade and appropriate pharmacological antagonism are strongly recommended whenever muscle relaxants are administered. The @ primary clinical use of cholinesterase inhibitors is to reverse nondepolarizing neuromuscular blockers. Some of these agents are also used to diagnose and treat myasthenia gravis. More recently, newer agents, such as cyclodextrins and cysteine, with superior ability to reverse neuromuscular blockade from specific agents, are being employed or investigated. This chapter reviews cholinergic pharmacology and mechanisms of acetylcholinesterase inhibition and presents the clinical pharmacology of commonly used cholinesterase inhibitors (neostigmine, edrophonium, pyridostigmine, and physostigmine). It concludes with a brief description and mechanisms of action of new reversal agents.

The term cholinergic refers to the effects of the neurotransmitter acetylcholine. Acetylcholine is synthesized in the nerve terminal by the enzyme cholineacetyltransferase, which catalyzes the reaction between acetylcoenzyme A and choline (Figure 12—1). After its release, acetylcholine is rapidly hydrolyzed by acetylcholinesterase (true cholinesterase) into acetate and choline.

® Acetylcholine is the neurotransmitter for the entire parasympathetic nervous system (parasympathetic ganglions and effector cells), parts of the sympathetic nervous system (sympathetic ganglions, adrenal medulla, and sweat glands), some neurons in the central nervous system, and somatic nerves innervating skeletal muscle (Figure 12-2).
FIGURE 12-2 The parasympathetic nervous system uses acetylcholine as a preganglionic and postganglionic neurotransmitter.
Cholinergic receptors have been subdivided into two major groups based on their reaction to the alkaloids muscarine and nicotine (Figure 12-3). Nicotine stimulates the autonomic ganglia and skeletal muscle receptors (nicotinic receptors), whereas muscarine activates end-organ effector cells in bronchial smooth muscle, salivary glands, and the sinoatrial node (muscarinic receptors). The central nervous system has both nicotinic and muscarinic receptors. Nicotinic receptors are blocked by muscle relaxants (also called neuromuscular blockers), and muscarinic receptors are blocked by anticholinergic drugs, such as atropine. Although nicotinic and muscarinic receptors differ in their response to some agonists (eg, nicotine, muscarine) and some antagonists (eg, vecuronium versus atropine), they both respond to acetylcholine (Table 12—1). Clinically available cholinergic agonists resist hydrolysis by cholinesterase. Methacholine and bethanechol are primarily muscarinic agonists, whereas carbachol has both muscarinic and nicotinic agonist activities. Methacholine by inhalation has been used as a provocative test in asthma, bethanechol is used for bladder atony, and carbachol may be used topically for wide-angle glaucoma.

Location, Nicotinic = Autonomic ganglia Sympathetic ganglia Parasympathetic ganglia Skeletal muscle. Location, Muscarinic = Glands Lacrimal Salivary Gastric Smooth muscle Bronchial Gastrointestinal Bladder Blood vessels Heart Sinoatrial node Atrioventricular. Agonists, Nicotinic = Acetylcholine Nicotine. Agonists, Muscarinic = Acetylcholine Muscarine. Antagonists, Nicotinic = Nondepolarizing relaxants. Antagonists, Muscarinic = Antimuscarinics Atropine Scopolamine Glycopyrrolate
FIGURE 12-3 The molecular structures of nicotine and muscarine. Compare these alkaloids with acetylcholine (Figure 12-1).
When reversing neuromuscular blockade, the goal is to maximize nicotinic transmission with a minimum of muscarinic side effects.

® Normal neuromuscular transmission depends on acetylcholine binding to nicotinic cholinergic receptors on the motor end-plate. Nondepolarizing muscle relaxants act by competing with acetylcholine for these binding sites, thereby blocking neuromuscular transmission. Reversal of blockade depends on diffusion, redistribution, metabolism, and excretion from the body of the nondepolarizing relaxant (spontaneous reversal), often assisted by the administration of specific reversal agents (pharmacological reversal). Cholinesterase inhibitors indirectly increase the amount of acetylcholine available to compete with the nondepolarizing agent, thereby reestablishing normal neuromuscular transmission.
Cholinesterase inhibitors inactivate acetylcholinesterase by reversibly binding to the enzyme. The stability of the bond influences the duration of action. The electrostatic attraction and hydrogen bonding of edrophonium are short-lived; the covalent bonds of neostigmine and pyridostigmine are longer lasting.
Organophosphates, a special class of cholinesterase inhibitors that have

The increase in acetylcholine caused by cholinesterase inhibitors affects more than the nicotinic receptors of skeletal muscle (Table 12—2). Cholinesterase inhibitors can act at cholinergic receptors of several other organ systems, including the cardiovascular and gastrointestinal systems.
TABLE 12-2 Muscarinic side effects of cholinesterase inhibitors.
Cardiovascular, Muscarinic Side Effects = Decreased heart rate, bradyarrhythmias. Pulmonary, Muscarinic Side Effects = Bronchospasm, bronchial secretions. Cerebral, Muscarinic Side Effects = Diffuse excitation!. Gastrointestinal, Muscarinic Side Effects = Intestinal spasm, increased salivation. Genitourinary, Muscarinic Side Effects = Increased bladder tone. Ophthalmological, Muscarinic Side Effects = Pupillary constriction
'Applies only to physostigmine.

Physical Structure
Neostigmine consists of a carbamate moiety and a quaternary ammonium group (Figure 12-4). The former provides covalent bonding to acetylcholinesterase. The latter renders the molecule lipid insoluble, so that it cannot pass through the blood-brain barrier.
FIGURE 12-4 The molecular structures of neostigmine, pyridostigmine, edrophonium, and physostigmine.

The maximum recommended dose of neostigmine is 0.08 mg/kg (up to 5 mg in adults), but smaller amounts often suffice and larger doses have also been given safely (Table 12—3). Neostigmine is most commonly packaged as 10 mL of a 1 mg/mL solution, although 0.5 mg/mL and 0.25 mg/mL concentrations are also available.

Neostigmine, Usual Dose of Cholinesterase Inhibitor = 0.04-0.08 mg/kg. Neostigmine, Recommended Anticholinergic = Glycopyrrolate. Neostigmine, Usual Dose of Anticholinergic per mg of Cholinesterase Inhibitor = 0.2mq. Pyridostigmine, Usual Dose of Cholinesterase Inhibitor = 0.1-0.25 mg/ka. Pyridostigmine, Recommended Anticholinergic = Glycopyrrolate. Pyridostigmine, Usual Dose of Anticholinergic per mg of Cholinesterase Inhibitor = 0.05 mg. Edrophonium, Usual Dose of Cholinesterase Inhibitor = 0.5-1 mg/kg. Edrophonium, Recommended Anticholinergic = Atropine. Edrophonium, Usual Dose of Anticholinergic per mg of Cholinesterase Inhibitor = 0.014mg. Physostigmine’, Usual Dose of Cholinesterase Inhibitor = 0.01-0.03 mg/kg. Physostigmine’, Recommended Anticholinergic = Usually not necessary. Physostigmine’, Usual Dose of Anticholinergic per mg of Cholinesterase Inhibitor = 
'Not used to reverse muscle relaxants.
Clinical Considerations

The effects of neostigmine (0.04 mg/kg) are usually apparent in 5 min, peak at 10 min, and last more than 1 h. In practice, some clinicians use a dose of 0.04 mg/kg (or 2.5 mg) if the preexisting blockade is mild to moderate and a dose of 0.08 mg/kg (or 5 mg) if intense paralysis is being reversed; other clinicians use the "full dose" for all patients. The duration of action is prolonged in geriatric patients. Muscarinic side effects are minimized by prior or concomitant administration of an anticholinergic agent. The onset of action of glycopyrrolate (0.2 mg glycopyrrolate per 1 mg of neostigmine) is similar to that of neostigmine and is associated with less tachycardia than is experienced with atropine (0.4 mg of atropine per 1 mg of neostigmine). It has been reported that neostigmine crosses the placenta, resulting in fetal bradycardia, but there is no evidence that the choice of atropine versus glycopyrrolate makes any difference in newborn outcomes. Neostigmine is also used to treat myasthenia gravis, urinary bladder atony, and paralytic ileus.

Pyridostigmine is structurally similar to neostigmine except that the quaternary ammonium is incorporated into the phenol ring. Pyridostigmine shares neostigmine's covalent binding to acetylcholinesterase and its lipid insolubility.

Pyridostigmine is 20% as potent as neostigmine and may be administered in doses up to 0.25 mg/kg (a total of 20 mg in adults). It is available as a solution of 5 mg/mL.

The onset of action of pyridostigmine is slower (10-15 min) than that of neostigmine, and its duration is slightly longer (>2 h). Glycopyrrolate (0.05 mg per 1 mg of pyridostigmine) or atropine (0.1 mg per 1 mg of pyridostigmine) must also be administered to prevent bradycardia. Glycopyrrolate is preferred because its slower onset of action better matches that of pyridostigmine, again resulting in less tachycardia.

Because it lacks a carbamate group, edrophonium must rely on noncovalent bonding to the acetylcholinesterase enzyme. The quaternary ammonium group limits lipid solubility.

Edrophonium is less than 10% as potent as neostigmine. The recommended dosage is 0.5 to 1 mg/kg.

Edrophonium has the most rapid onset of action (1-2 min) and the shortest duration of effect of any of the cholinesterase inhibitors. Reduced doses should not be used, because longer-acting muscle relaxants may outlast the effects of edrophonium. Higher doses prolong the duration of action to more than 1 h. Edrophonium may not be as effective as neostigmine at reversing intense neuromuscular blockade. In equipotent doses, muscarinic effects of edrophonium are less pronounced than those of neostigmine or pyridostigmine, requiring only half the amount of anticholinergic agent. Edrophonium's rapid onset is well matched to that of atropine (0.014 mg of atropine per 1 mg of edrophonium). Although glycopyrrolate (0.007 mg per 1 mg of edrophonium) can also be used, it should be given several minutes prior to edrophonium to avoid the possibility of bradycardia.

Physostigmine, a tertiary amine, has a carbamate group but no quaternary ammonium. Therefore, it is lipid soluble and is the only clinically available cholinesterase inhibitor that freely passes the blood-brain barrier.

The dose of physostigmine is 0.01—0.03 mg/kg. It is packaged as a solution

The lipid solubility and central nervous system penetration of physostigmine limit its usefulness as a reversal agent for nondepolarizing blockade, but make it effective in the treatment of central anticholinergic actions of scopolamine or overdoses of atropine. In addition, it reverses some of the central nervous system depression and delirium associated with use of benzodiazepines and volatile anesthetics. Physostigmine (0.04 mg/kg) has been shown to be effective in preventing postoperative shivering. It reportedly partially antagonizes morphineinduced respiratory depression, presumably because morphine reduces acetylcholine release in the brain. These effects are transient, and repeated doses may be required. Bradycardia is infrequent in the recommended dosage range, but atropine should be immediately available. Because glycopyrrolate does not cross the blood-brain barrier, it will not reverse the central nervous system effects of physostigmine. Other possible muscarinic side effects include excessive salivation, vomiting, and convulsions. In contrast to other cholinesterase inhibitors, physostigmine is almost completely metabolized by plasma esterases, so renal excretion is not important.

Recovery from neuromuscular blockade is influenced by the depth of block at the time of antagonism, clearance and half-life of the relaxant used, and other factors that affect neuromuscular blockade (Table 12—4), such as drugs and electrolyte disturbances. In addition, some specific agents with the potential of reversing the neuromuscular blocking effects of nondepolarizing muscle relaxants merit brief discussion.

Volatile anesthetics
Antibiotics: aminoglycosides, polymyxin B, neomycin, tetracycline, clindamycin
Dantrolene
Verapamil
Furosemide
Lidocaine

Hypermagnesemia
Hypocalcemia
Hypokalemia
Respiratory acidosis
Temperature
Hypothermia

Besides cholinesterase inhibitors, additional drugs (calabadion and L-cysteine) are currently under investigation, and sugammadex has been newly introduced in the United States. These agents act as selective antagonists of nondepolarizing neuromuscular blockade. Sugammadex is able to reverse aminosteroid-induced neuromuscular blockade, whereas cysteine has been shown to reverse the neuromuscular blocking effects of gantacurium and other fumarates. Calabadion prevents binding to the nicotinic receptor of both benzylisoquinolinium and steroidal nondepolarizing muscle relaxants.

Sugammadex is a novel selective relaxant-binding agent that has recently become available in the United States. Sugammadex is increasingly supplanting neostigmine as the preferred agent for reversal on nondepolarizing neuromuscular blockade. It is a modified y-cyclodextrin (su refers to sugar, and gammadex refers to the structural molecule y-cyclodextrin).

Its three-dimensional structure resembles a hollow truncated cone or doughnut with a hydrophobic @ cavity and a hydrophilic exterior. Hydrophobic interactions trap the drug (eg, rocuronium) in the cyclodextrin cavity (doughnut hole), resulting in tight formation of a water-soluble guest—host complex in a 1:1 ratio. This terminates the neuromuscular blocking action and restrains the drug in extracellular fluid where it cannot interact with nicotinic acetylcholine receptors. Sugammadex is essentially eliminated unchanged via the kidneys. Sugammadex does not require coadministration of an antimuscarinic agent.

Calabadion is a member of the cucurbituril class of "molecular containers" and is capable of reversing both steroidal and benzylisoquinoline neuromuscular blockers. Calabadion prevents muscle relaxant binding to the nicotinic receptor. Calabadion is currently under investigation.

L-cysteine is an endogenous amino acid that is often added to total parenteral nutrition regimens to enhance ® calcium and phosphate solubility. The ultrashort-acting neuromuscular blocker, gantacurium, and other fumarates rapidly combine with L-cysteine in vitro to form less active degradation products (adducts). Exogenous administration of L-cysteine (10-50 mg/kg intravenously)
given to anesthetized monkeys 1 min after these neuromuscular blocking agents abolished the block within 2 to 3 min; this antagonism was found to be superior to that produced by anticholinesterases. This unique method of antagonism by adduct formation and inactivation is still in the investigative stage, especially in terms of its safety and efficacy in humans.

A 66-year-old woman weighing 85 kg is brought to the recovery room following laparoscopic cholecystectomy. The anesthetic technique included the use of isoflurane and vecuronium for muscle relaxation. At the conclusion of the procedure, the anesthesiologist administered 6 mg of morphine sulfate for postoperative pain control and 3 mg of neostigmine with 0.6 mg of glycopyrrolate to reverse any residual neuromuscular blockade. The dose of cholinesterase inhibitor was empirically based on clinical judgment. Although the patient was apparently breathing normally on arrival in the recovery room, her tidal volume progressively diminished. Arterial blood gas measurements revealed a Paco, of 62 mm Hg, a Pao, of 110 mm Hg, and a pH of 7.26 on a fraction of inspired oxygen (Fi0,) of 40%.
Which drugs administered to this patient could explain her hypoventilation?
Isoflurane, morphine sulfate, and vecuronium all interfere with a patient's ability to maintain a normal ventilatory response to an elevated Paco.
Why would the patient's breathing worsen in the recovery room?
Possibilities include the delayed onset of action of morphine sulfate, a lack of sensory stimulation in the recovery area, fatigue of respiratory muscles, the adverse effects of hypoventilation (and hypercarbia) with neuromuscular function, and splinting as a result of upper abdominal pain.

If the dose of neostigmine was not determined by the response to a peripheral nerve stimulator, or if the recovery of muscle function was inadequately tested after the reversal drugs were given, persistent neuromuscular blockade is possible. Assume, for example, that the patient had minimal or no response to initial tetanic stimulation at 100 Hz. Even the maximal dose of neostigmine (5 mg) might not yet have adequately reversed the paralysis. Because of enormous patient variability, the response to peripheral nerve stimulation must always be monitored when muscle relaxants are administered. Even if partial reversal is achieved, paralysis may worsen if the patient hypoventilates. Other factors (in addition to respiratory acidosis) that impair the reversal of nondepolarizing muscle relaxants include intense neuromuscular paralysis, electrolyte disturbances (hypermagnesemia, hypokalemia, and hypocalcemia), hypothermia (temperature <32°C), drug interactions (see Table 11-3), metabolic alkalosis (from accompanying hypokalemia and hypocalcemia), and coexisting diseases (see Table 11—7).

Tetanic stimulation is a sensitive but uncomfortable test of neuromuscular transmission in an awake patient. Because of its shorter duration, double-burst stimulation is tolerated better than tetany by conscious patients. Quantitative measures such as acceleromyography are preferred to assess the adequacy of reversal (train of four >0.9) compared with subjective interpretations of twitch. Many other tests of neuromuscular transmission, such as vital capacity and tidal volume, are insensitive as they may still seem normal when 70% to 80% of receptors are blocked. In fact, 70% of receptors may remain blocked despite an apparently normal response to train-of-four stimulation. The ability to sustain a head lift for 5 s, however, indicates that fewer than 33% of receptors are occupied by muscle relaxant.

Ventilation should be assisted to reduce the respiratory acidosis. Even if diaphragmatic function seems to be adequate, residual blockade can lead to airway obstruction and poor airway protection. More neostigmine (with an anticholinergic) could be administered up to a maximum recommended dose of 5 mg. Since vecuronium was used, sugammadex could be administered. If this does not adequately reverse paralysis, mechanical ventilation and airway protection should be instituted and continued until neuromuscular function is fully restored.

Dandan R, eds. Goodman and Gilman's Pharmacological Basis of Therapeutics. 13th ed. New York, NY: McGraw-Hill; 2018.

@ The ester linkage is essential for effective binding of the anticholinergics to the acetylcholine receptors. This competitively blocks binding by acetylcholine and prevents receptor activation. The cellular effects of acetylcholine, which are mediated through second messengers, are inhibited.
@® Anticholinergics relax the bronchial smooth musculature, which reduces airway resistance and increases anatomic dead space.
© Atropine has particularly potent effects on the heart and bronchial smooth muscle and is the most efficacious anticholinergic for treating bradyarrhythmias.
@ Ipratropium solution (0.5 mg in 2.5 mL) seems to be particularly effective in the treatment of acute chronic obstructive pulmonary disease when combined with a B-agonist drug (eg, albuterol).
@ Scopolamine is a more potent antisialagogue than atropine and causes greater central nervous system effects.
@ Because of its quaternary structure, glycopyrrolate cannot cross the blood-brain barrier and is almost devoid of central nervous system and ophthalmic activity.
One group of cholinergic antagonists has already been discussed: the nondepolarizing neuromuscular blocking agents. These drugs act primarily at the nicotinic receptors in skeletal muscle. This chapter presents the pharmacology of drugs that block muscarinic receptors. Although the classification anticholinergic usually refers to this latter group, a more precise term would be antimuscarinic.
In this chapter, the mechanism of action and clinical pharmacology are
introduced for three common anticholinergics: atropine, scopolamine, and glycopyrrolate. The clinical uses of these drugs in anesthesia relate to their effect on the cardiovascular, respiratory, cerebral, gastrointestinal, and other organ systems (Table 13-1).

Tachycardia, Atropine = +++. Tachycardia, Scopolamine = +. Tachycardia, Glycopyrrolate = ++. Bronchodilatation, Atropine = ++. Bronchodilatation, Scopolamine = +. Bronchodilatation, Glycopyrrolate = ++. Sedation, Atropine = +. Sedation, Scopolamine = +++. Sedation, Glycopyrrolate = 0. Antisialagogue effect, Atropine = ++. Antisialagogue effect, Scopolamine = t+4+. Antisialagogue effect, Glycopyrrolate = +++
'0, no effect; +, minimal effect; ++, moderate effect; +++, marked effect.

Anticholinergics are esters of an aromatic acid @ combined with an organic base (Figure 13-1). The ester linkage is essential for effective binding of the anticholinergics to the acetylcholine receptors. This competitively blocks binding by acetylcholine and prevents receptor activation. The cellular effects of acetylcholine, which are mediated through second messengers, are inhibited. The tissue receptors vary in their sensitivity to blockade. In fact, muscarinic receptors are not homogeneous, and receptor subgroups have been identified, including central nervous system (M, 45), autonomic ganglia and gastric parietal cells (M,), cardiac (M,), and smooth muscle (M3) receptors.
<< SS CH2.——CH———_ CHa t “0 NCH; H— e— O— i c | CH, Aromatic base = Atropine. ———-CH———— CH OQ CH2ZOH | | | | NCH, H—C—O—C—C | | H CH——\CH CHy, Aromatic base = Scopolamine. ° | o—C \ pe N Oo Lie HgC + CHg, Aromatic base = Glycopyrrolate
FIGURE 13-1 Physical structures of anticholinergic drugs.

In normal clinical doses, only muscarinic receptors are blocked by the anticholinergic drugs discussed in this chapter. The extent of the anticholinergic effect depends on the degree of baseline vagal tone.

Blockade of muscarinic receptors in the sinoatrial node produces tachycardia. This effect is especially useful in reversing bradycardia due to vagal reflexes (eg, baroreceptor reflex, peritoneal traction, or oculocardiac reflex). A transient slowing of heart rate in response to smaller intravenous doses of atropine (<0.4 mg) has been reported. The mechanism of this paradoxical response is unclear. Facilitation of conduction through the atrioventricular node shortens the P-R interval on the electrocardiogram and often decreases heart block caused by vagal activity. Atrial arrhythmias and nodal (junctional) rhythms occasionally occur. Anticholinergics generally have little effect on ventricular function or peripheral vasculature because of the paucity of direct cholinergic innervation of these areas despite the presence of cholinergic receptors. Presynaptic muscarinic receptors on adrenergic nerve terminals are known to inhibit norepinephrine release, so muscarinic antagonists may modestly enhance sympathetic activity. Large doses of anticholinergic agents can produce dilation of cutaneous blood vessels (atropine flush).

The anticholinergics inhibit respiratory tract secretions, from the nose to the bronchi, a valuable property during airway endoscopic or surgical procedures. @ Relaxation of the bronchial smooth musculature reduces airway resistance and increases anatomic dead space. These effects are particularly pronounced in patients with chronic obstructive pulmonary disease or asthma.

Anticholinergic medications can cause a spectrum of central nervous system effects ranging from stimulation to depression, depending on drug choice and dosage. Cerebral stimulation may present as excitation, restlessness, or hallucinations. Cerebral depression, including sedation and amnesia, are prominent after scopolamine. Physostigmine, a cholinesterase inhibitor that crosses the blood-brain barrier, promptly reverses anticholinergic actions on the brain.

Salivary secretions are markedly reduced by anticholinergic drugs. Gastric secretions are also decreased, but larger doses are necessary. Decreased intestinal motility and peristalsis prolong gastric emptying time. Lower esophageal sphincter pressure is reduced. Overall, the anticholinergic drugs do not prevent aspiration pneumonia.

Anticholinergics (particularly when dosed topically) cause mydriasis (pupillary dilation) and cycloplegia (an inability to accommodate to near vision); acute angle-closure glaucoma is unlikely but possible following systemic administration of anticholinergic drugs.

Anticholinergics may decrease ureter and bladder tone as a result of smooth muscle relaxation and lead to urinary retention, particularly in elderly men with prostatic hypertrophy.

Inhibition of sweat glands may lead to a rise in body temperature (atropine fever).

Atropine is a tertiary amine. The naturally occurring levorotatory form is active, but the commercial mixture is racemic (Figure 13-1).

As a premedication, atropine is administered intravenously or intramuscularly in a range of 0.01 to 0.02 mg/kg, up to the usual adult dose of 0.4 to 0.6 mg. Larger intravenous doses up to 2 mg may be required to completely block the cardiac vagal nerves in treating severe bradycardia. Atropine sulfate is available in a multitude of concentrations.

® Atropine has particularly potent effects on the heart and bronchial smooth muscle and is the most efficacious anticholinergic for treating bradyarrhythmias. Patients with coronary artery disease may not tolerate the increased myocardial oxygen demand and decreased oxygen supply associated with the tachycardia caused by atropine. A derivative of atropine, ipratropium bromide, is available in a metered-dose inhaler for the treatment of bronchospasm. Its quaternary ammonium structure @ significantly limits systemic absorption. Ipratropium solution (0.5 mg in 2.5 mL) seems to be particularly effective in the treatment of acute chronic obstructive pulmonary disease when combined with a B-agonist

The central nervous system effects of atropine are minimal after the usual doses, even though this tertiary amine can rapidly cross the blood-brain barrier. Atropine has been associated with mild postoperative memory deficits, and toxic doses are usually associated with excitatory reactions. An intramuscular dose of 0.01-0.02 mg/kg reliably provides an antisialagogue effect. Atropine should be used cautiously in patients with narrow-angle glaucoma, prostatic hypertrophy, or bladder-neck obstruction.
Intravenous atropine is used in the treatment of organophosphate pesticide and nerve gas poisoning. Organophosphates inhibit acetylcholinesterase, resulting in overwhelming stimulation of nicotinic and muscarinic receptors that leads to bronchorrhea, respiratory collapse, and bradycardia. Atropine can reverse the effects of muscarinic stimulation but not the muscle weakness resulting from nicotinic receptor activation. Pralidoxime (2-PAM; 1-2 g intravenously) may reactivate acetylcholinesterase.

Scopolamine, a tertiary amine, differs from atropine by the addition of an epoxide to the heterocyclic ring.

@ Scopolamine is a more potent antisialagogue than atropine and causes greater central nervous system effects. Clinical dosages usually result in drowsiness and amnesia, although restlessness, dizziness, and delirium are possible. The sedative effects may be desirable for premedication but can interfere with awakening following short procedures. Scopolamine has the added virtue of preventing motion sickness. The lipid solubility allows transdermal absorption, and transdermal scopolamine (1 mg patch) has been used to prevent postoperative nausea and vomiting. Because of its pronounced mydriatic effects, scopolamine is best avoided in patients with closed-angle glaucoma.

Glycopyrrolate is a synthetic product that differs from atropine in being a quaternary amine and having both cyclopentane and pyridine moieties in the compound.

The usual dose of glycopyrrolate is one-half that of atropine. For instance, the premedication dose is 0.005 to 0.01 mg/kg up to 0.2 to 0.3 mg in adults. Glycopyrrolate for injection is packaged as a solution of 0.2 mg/mL.

@ Because of its quaternary structure, glycopyrrolate cannot cross the blood— brain barrier and is almost devoid of central nervous system and ophthalmic activity. Potent inhibition of salivary gland and respiratory tract secretions is the primary rationale for using glycopyrrolate as a premedication. Heart rate usually increases after intravenous—but not intramuscular—administration. Glycopyrrolate has a longer duration of action than atropine (2-4 h versus 30 min after intravenous administration).

An elderly patient is scheduled for enucleation of a blind, painful eye. Scopolamine, 0.4 mg intramuscularly, is administered as premedication. In the preoperative holding area, the patient becomes agitated and disoriented. The only other medication the patient has received is 1% atropine eye drops.
How many milligrams of atropine are in one drop of a 1% solution?
A 1% solution contains 1 g dissolved in 100 mL, or 10 mg/mL. Eyedroppers vary in the number of drops formed per milliliter of solution, but average 20 drops/mL. Therefore, one drop usually contains 0.5 mg of atropine.

Absorption by vessels in the conjunctival sac is similar to subcutaneous injection. More rapid absorption is possible by the nasolacrimal duct mucosa.
What are the signs and symptoms of anticholinergic poisoning?
Reactions from an overdose of anticholinergic medication involve several organ systems. The central anticholinergic syndrome refers to central nervous system changes that range from unconsciousness to hallucinations. Agitation and delirium are not unusual in elderly patients. Other systemic manifestations include dry mouth, tachycardia, atropine flush, atropine fever, and impaired vision.
What other drugs possess anticholinergic activity that could predispose patients to the central anticholinergic syndrome?
Tricyclic antidepressants, antihistamines, and antipsychotics have antimuscarinic properties that could potentiate the side effects of anticholinergic drugs.
What drug is an effective antidote to anticholinergic overdosage?
Cholinesterase inhibitors indirectly increase the amount of acetylcholine available to compete with anticholinergic drugs at the muscarinic receptor. Neostigmine, pyridostigmine, and edrophonium possess a quaternary ammonium group that prevents penetration of the blood-brain barrier. Physostigmine, a tertiary amine, is lipid soluble and effectively reverses central anticholinergic toxicity. An initial dose of 0.01 to 0.03 mg/kg may have to be repeated after 15 to 30 min.

Brown JH. Muscarinic receptor agonists and antagonists. In: Brunton LL,
Knollmann BC, Hilal-Dandan R, eds. Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 13th ed. New York, NY: McGrawHill; 2018.
Eddleston M, Chowdhury F. Pharmacological treatment of organophosphorous insecticide poisoning: The old and the (possible) new. Br J Clin Pharmacol. 2015;81:462.
Howard J, Wigley J, Rosen G, D'mello J. Glycopyrrolate: It's time to review. J Clin Anesth. 2017;36:51.
Nishtala PS, Salahudeen MS, Hilmer SN. Anticholinergics: Theoretical and clinical overview. Expert Opin Drug Saf. 2016;15:753.

@ Adrenergic agonists can be categorized as direct or indirect. Direct agonists bind to the receptor, whereas indirect agonists increase endogenous neurotransmitter activity.
@ The primary effect of phenylephrine is peripheral vasoconstriction with a concomitant rise in systemic vascular resistance and arterial blood pressure.
Clonidine decreases anesthetic and analgesic requirements and provides sedation and anxiolysis.
Dexmedetomidine has a higher affinity for a-receptors than clonidine. It has sedative, analgesic, and sympatholytic effects that blunt many of the cardiovascular responses seen during the perioperative period.
@ Long-term use of these agents, particularly clonidine and dexmedetomidine, leads to super-sensitization and upregulation of receptors; with abrupt discontinuation of either drug, an acute withdrawal syndrome including hypertensive crisis can occur.
@ Ephedrine is commonly used as a vasopressor during anesthesia. As such, its administration should be viewed as a temporizing measure while the cause of hypotension is determined and remedied.
@ At low doses (0.5-3 mcg/kg/min), dopamine (DA) primarily activates dopaminergic receptors. Stimulation of these receptors (specifically, DA, receptors) vasodilates the renal vasculature and promotes diuresis.
© Labetalol lowers blood pressure without reflex tachycardia because of its combination of a and B effects.
© Esmolol is an ultrashort-acting selective B,-antagonist that reduces heart rate and, to a lesser extent, blood pressure.
@ Abrupt discontinuation of B-blocker therapy for 24 to 48 h may trigger a
withdrawal syndrome characterized by hypertension, tachycardia, and angina pectoris.
Adrenergic agonists and antagonists produce their clinical effects by interacting with the adrenergic receptors (ie, adrenoceptors). The clinical effects of these drugs can be deduced from an understanding of the adrenoceptor physiology and a knowledge of which receptors each drug activates or blocks.

The term adrenergic originally referred to the effects of epinephrine (adrenaline), although norepinephrine (noradrenaline) is the primary neurotransmitter responsible for most of the adrenergic activity of the sympathetic nervous system. With the exception of eccrine sweat glands and some blood vessels, norepinephrine is released by postganglionic sympathetic fibers at end-organ tissues (Figure 14—1). In contrast, acetylcholine is released by preganglionic sympathetic fibers and all parasympathetic fibers.
FIGURE 14-1 The sympathetic nervous system. Organ innervation, receptor type, and response to stimulation. The origin of the sympathetic chain is the thoracoabdominal (T1—-L3) spinal cord, in contrast to the craniosacral distribution of the parasympathetic nervous system. Another anatomic difference is the greater distance from the sympathetic ganglion to the visceral structures.
Norepinephrine is synthesized in the cytoplasm of sympathetic postganglionic nerve endings and stored in the vesicles (Figure 14—2). After release by a process of exocytosis, the action of norepinephrine is primarily terminated by reuptake into the postganglionic nerve ending. Norepinephrine transporter located on neuronal cell membranes facilitates removal of norepinephrine from the synapse. Other transporters facilitate uptake of dopamine and serotonin. Tricyclic antidepressants, cocaine, and amphetamines can inhibit these transporters leading to their clinical effects. Norepinephrine may diffuse from receptor sites where it is taken up by nonneuronal cells and is metabolized by catechol-O-methyltransferase (Figure 14—3). In neurons norepinephrine may be metabolized by monoamine oxidase or repackaged into vesicles. Prolonged adrenergic activation leads to desensitization and hyporesponsiveness to further stimulation.
FIGURE 14-2 The synthesis of norepinephrine. Hydroxylation of tyrosine to dopa is the rate-limiting step. Dopamine is actively transported into storage vesicles. Norepinephrine can be converted to epinephrine in the adrenal medulla.

FIGURE 14-3 Sequential metabolism of norepinephrine and epinephrine. Monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT) produce a common end product, vanillylmandelic acid (VMA).
Adrenergic receptors are divided into two general categories: « and 8. Each of these has been further subdivided into at least two subtypes: a, and a, and B,, B», and B3. The a-receptors have been further divided using molecular cloning techniques into 1,4, Q1p, Op, OA, App, and Qc. These receptors are linked to G proteins (Figure 14—4)—heterotrimeric receptors with a, B, and y subunits. The different adrenoceptors are linked to specific G proteins, each with a unique effector, but each using guanosine triphosphate (GTP) as a cofactor. a, is linked to Gg, which activates phospholipases; ay is linked to G,, which inhibits adenylate cyclase (also termed adenyl or adenylyl cyclase); and is linked to G,, which activates adenylate cyclase.

FIGURE 14-4 Activation and inhibition of adenylyl cyclase by agonists that bind to catecholamine receptors. Binding to B-adrenoceptors stimulates adenylyl cyclase by activating the stimulatory G protein, G,, which leads to the dissociation of its a subunit charged with guanosine triphosphate (GTP). This activated a, subunit directly activates adenylyl cyclase, resulting in an increased rate of synthesis of cAMP. a,-Adrenoceptor ligands inhibit adenylyl cyclase by causing dissociation of the inhibitory G protein, G,, into its subunits; ie, an activated a; subunit charged with GTP and a B-y unit. The mechanism by which these subunits inhibit adenylyl cyclase is uncertain. cAMP binds to the regulatory subunit (R) of cAMP-dependent protein kinase, leading to the liberation of active catalytic subunits (C) that phosphorylate specific protein substrates and modify their activity. These catalytic units also phosphorylate the cAMP response element binding protein (CREB), which modifies gene expression. See text for other actions of Band a-adrenoceptors. (Reproduced with permission from Katzung BG, Trevor AJ, eds. Basic & Clinical Pharmacology. 13th ed. New York, NY: McGraw-Hill Education, Inc; 2015.)

a,-Receptors are postsynaptic adrenoceptors located in smooth muscle throughout the body (in the eye, lung, blood vessels, uterus, gut, and genitourinary system). Activation of these receptors increases intracellular calcium ion concentration, which leads to contraction of smooth muscles. Thus, a,-agonists are associated with mydriasis (pupillary dilation due to contraction of the radial eye muscles), bronchoconstriction, vasoconstriction, uterine contraction, and constriction of sphincters in the gastrointestinal and genitourinary tracts. Stimulation of a,-receptors also inhibits insulin secretion and lipolysis. The myocardium possesses a,-receptors that have a positive inotropic effect, which might play a role in catecholamine-induced arrhythmia. During myocardial ischemia, enhanced a,-receptor coupling with agonists is observed. Nonetheless, the most important cardiovascular effect of a, stimulation is vasoconstriction, which increases peripheral vascular resistance, left ventricular afterload, and arterial blood pressure.

In contrast to oj-receptors, a-receptors are located primarily on the presynaptic nerve terminals. Activation of these adrenoceptors inhibits adenylyl cyclase activity. This decreases the entry of calcium ions into the neuronal terminal, which limits subsequent exocytosis of storage vesicles containing norepinephrine. Thus, a-receptors create a negative feedback loop that inhibits further norepinephrine release from the neuron. In addition, vascular smooth muscle contains postsynaptic aj-receptors that produce vasoconstriction. More importantly, stimulation of postsynaptic a5-receptors in the central nervous system causes sedation and reduces sympathetic outflow, which leads to peripheral vasodilation and lower blood pressure.

B-Adrenergic receptors are classified into B,, B., and B3 receptors.
Norepinephrine and epinephrine are equipotent on B, receptors, but epinephrine is significantly more potent than norepinephrine on B, receptors.
The most important B,-receptors are located on the postsynaptic membranes in the heart. Stimulation of these receptors activates adenylyl cyclase, which converts adenosine triphosphate to cyclic adenosine monophosphate and initiates a kinase phosphorylation cascade. Initiation of the cascade has positive chronotropic (increased heart rate), dromotropic (increased conduction), and inotropic (increased contractility) effects.

B,-Receptors are primarily postsynaptic adrenoceptors located in smooth muscle and gland cells, but are also located in ventricular myocytes. Their relative contribution to the response to intravenous catecholamines increases in patients with chronic heart failure. They share a common mechanism of action with B,receptors: adenylyl cyclase activation. Despite this commonality, B, stimulation relaxes smooth muscle, resulting in bronchodilation, vasodilation, and relaxation of the uterus (tocolysis), bladder, and gut. Glycogenolysis, lipolysis, gluconeogenesis, and insulin release are stimulated by B5-receptor activation. B5Agonists also activate the sodium—potassium pump.

B3-Receptors are found in the gallbladder and brain adipose tissue. Their role in gallbladder physiology is unknown, but they are thought to play a role in lipolysis, thermogenesis in brown fat, and bladder relaxation.

Dopamine
(DA)
receptors are
a
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D, and
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Adrenergic agonists interact with varying specificity (selectivity) at aand Badrenoceptors (Tables 14—1 and 14—2).
TABLE 14-1 Receptor selectivity of adrenergic agonists.!

Dobutamine, DA, = 0. Terbutaline, a, = 0. Terbutaline, a, = 0. Terbutaline, B, = +. Terbutaline, 6, = +++. Terbutaline, DA, = 0. Terbutaline, DA, = 0
'0, no/minimal effect; +, agonist effect (mild, moderate, marked); ?, unknown effect; DA, and DA,, dopaminergic receptors.
"The a, effects of epinephrine, norepinephrine, and dopamine become more prominent at high doses.
'The primary mode of action of ephedrine is indirect stimulation.

= Lt. Dobutamine, HeartRate = T. Dobutamine, MeanArterial Pressure = T. Dobutamine, Cardiac Output = TIT. Dobutamine, Peripheral Vascular Resistance = 4. Dobutamine, Bronchodilation = 0. Dobutamine, Renal Blood Flow = T
- 10, no/minimal effect; 7, increase (mild, moderate, marked); 1, decrease (mild, moderate, marked]; 1/7, variable effect; 1/17, mild-to-moderate increase.
Overlapping of activity complicates the prediction of clinical effects. For example, epinephrine stimulates a,-, a -, B,-, and Bj-adrenoceptors. Its net effect on arterial blood pressure depends on the dose-dependent balance between a,vasoconstriction, aand B5-vasodilation, and B,-inotropic influences (and, to a minor degree, B4-inotropic influences).
® Adrenergic agonists can be categorized as direct or indirect. Direct agonists bind to the receptor, whereas indirect agonists increase endogenous neurotransmitter activity. Mechanisms of indirect action include increased release or decreased reuptake of norepinephrine. The differentiation between direct and indirect mechanisms of action is particularly important in patients who have abnormal endogenous norepinephrine stores, as may occur with use of some antihypertensive medications or monoamine oxidase inhibitors. Intraoperative hypotension in these patients should be treated with direct agonists, as their response to indirect agonists will be unpredictable.

Another feature distinguishing adrenergic agonists from each other is their chemical structure. Adrenergic agonists that have a 3,4-dihydroxybenzene structure (Figure 14—5) are known as catecholamines. These drugs are typically short-acting because of their metabolism by monoamine oxidase and catechol-Omethyltransferase. Patients taking monoamine oxidase inhibitors or tricyclic antidepressants may therefore demonstrate an exaggerated response to catecholamines. The naturally occurring catecholamines are epinephrine, norepinephrine, and DA. Changing the side-chain structure (Rj, Ro, R3) of naturally occurring catecholamines has led to the development of synthetic catecholamines (eg, isoproterenol and dobutamine), which tend to be more receptor specific.
FIGURE 14-5 Adrenergic agonists that have a 3,4-dihydroxybenzene structure are known as catecholamines. Substitutions at the R,, R>, and R3 sites affect activity and selectivity.
Adrenergic agonists commonly used in anesthesiology are discussed individually below. Note that the recommended doses for continuous infusion are expressed as mcg/kg/min for some agents and mcg/min for others. In either case, these recommendations should be regarded only as guidelines, as individual responses are quite variable.

Phenylephrine is a noncatecholamine with selective @ a,-agonist activity. The primary effect of phenylephrine is peripheral vasoconstriction with a concomitant rise in systemic vascular resistance and arterial blood pressure. Reflex bradycardia mediated by the vagus nerve can reduce cardiac output. is also used as a and a
Phenylephrine topically decongestant mydriatic agent. Small intravenous boluses of 50 to 100 mcg (0.5 to 1 mcg/kg) of phenylephrine rapidly reverse reductions in blood pressure caused by peripheral vasodilation (eg, spinal anesthesia). The duration of action is short, lasting approximately 15 min after administration of a single dose. Tachyphylaxis may occur with phenylephrine infusions and require upward titration of the infusion. Phenylephrine must be diluted from a 1% solution (10 mg/1-mL ampule), usually to a 100 mcg/mL solution and titrated to effect.

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Although these agents are adrenergic agonists, they are also considered to be sympatholytic because @ sympathetic outflow is reduced. Long-term use of these agents, particularly clonidine and dexmedetomidine, leads to supersensitization and upregulation of receptors; with abrupt discontinuation of either drug, an acute withdrawal syndrome including hypertensive crisis can occur. Because of the increased affinity of dexmedetomidine for the a-receptor, compared with that of clonidine, this syndrome may manifest after only 48 h of dexmedetomidine use when the drug is discontinued.

Epinephrine is an endogenous catecholamine synthesized in the adrenal medulla. Direct stimulation of B,-receptors of the myocardium by epinephrine raises blood pressure, cardiac output, and myocardial oxygen demand by increasing contractility and heart rate (increased rate of spontaneous phase IV depolarization). «, Stimulation decreases splanchnic and renal blood flow but increases coronary perfusion pressure by increasing aortic diastolic pressure. Systolic blood pressure rises, although B,-mediated vasodilation in skeletal muscle may lower diastolic pressure. 8, Stimulation also relaxes bronchial smooth muscle.
Administration of epinephrine is the principal pharmacological treatment for anaphylaxis and can be used to treat ventricular fibrillation. Complications include cerebral hemorrhage, myocardial ischemia, and ventricular arrhythmias. Volatile anesthetics, particularly halothane, potentiate the arrhythmic effects of epinephrine.
In emergency situations (eg, cardiac arrest and shock), epinephrine is administered as an intravenous bolus of 0.5 to 1 mg, depending on the severity of cardiovascular compromise. In major anaphylactic reactions, epinephrine should be used at a dose of 100 to 500 mcg (repeated, if necessary) followed by infusion. To improve myocardial contractility or heart rate, a continuous infusion is prepared (1 mg in 250 mL [4 mcg/mL]) and run at a rate of 2 to 20 mcg/min
(30-300 ng/kg/min). Epinephrine local infiltration is also used to reduce bleeding from the operative sites. Some local anesthetic solutions containing epinephrine at a concentration of 1:200,000 (5 mcg/mL) or 1:400,000 (2.5 mcg/mL) are characterized by less systemic absorption and a longer duration of action. Epinephrine is available in vials at a concentration of 1:1000 (1 mg/mL) and prefilled syringes at a concentration of 1:10,000 (0.1 mg/mL [100 mcg/mL]). A 1:100,000 (10 mcg/mL) concentration is available for pediatric use.

Direct a, stimulation with limited B, activity (at the doses used clinically) induces intense vasoconstriction of arterial and venous vessels. Increased myocardial contractility from B, effects, along with peripheral vasoconstriction, contributes to a rise in arterial blood pressure. Both systolic and diastolic pressures
usually prevent
any rise,
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Norepinephrine is administered usually as a continuous infusion due to its short half-life at a rate of 2 to 20 mcg/min (30-300 ng/kg/min). Ampules contain 4 mg of norepinephrine in 4 mL of solution.

Isoproterenol is of interest because it is a pure B-agonist. B, effects increase heart rate, contractility, and cardiac output. Systolic blood pressure may increase or remain unchanged, but B, stimulation decreases peripheral vascular resistance and diastolic blood pressure. Myocardial oxygen demand increases while oxygen supply falls, making isoproterenol or any pure B-agonist a poor inotropic choice in most situations.

Dobutamine is a racemic mixture of two isomers with affinity for both B,and B,-receptors, with relatively higher selectivity for B,-receptors. Its primary cardiovascular effect is a rise in cardiac output as a result of increased myocardial contractility. A decline in peripheral vascular resistance caused by B5 activation usually prevents much of a rise in arterial blood pressure. Left ventricular filling pressure decreases, whereas coronary blood flow increases. Dobutamine increases myocardial oxygen consumption and should not be routinely used without specific indications to facilitate separation from cardiopulmonary bypass. It is often employed in pharmacological stress testing. Dobutamine is administered as an infusion at a rate of 2 to 20 mcg/kg/min. It is supplied in 20-mL vials containing 250 mg.

Adrenergic antagonists bind but do not activate adrenoceptors. They act by preventing adrenergic agonist activity. Like the agonists, the antagonists differ in their spectrum of receptor interaction.

Labetalol blocks o,-, B,-, and B-receptors. The ratio of a blockade to B blockade has been estimated to be approximately 1:7 following intravenous administration. This mixed blockade reduces peripheral vascular resistance and arterial blood pressure. Heart rate and cardiac output are usually slightly depressed @ or unchanged. Thus, labetalol lowers blood pressure without reflex tachycardia because of its combination of a and B effects, which is beneficial to patients with coronary artery disease. Peak effect usually occurs within 5 min after an intravenous dose. Left ventricular failure, paradoxical hypertension, and bronchospasm have been reported.
The initial recommended dose of labetalol is 2.5 to 10 mg administered intravenously over 2 min. After the initial dose, and depending on the response, 5 to 20 mg may be given at 10-min intervals until the desired blood pressure response is obtained.

B-Receptor blockers have variable degrees of selectivity for the B,-receptors. Those that are more f, selective have less influence on bronchopulmonary and vascular B5-receptors (Table 14—3). Theoretically, a selective B,-blocker would have less of an inhibitory effect on B,-receptors and, therefore, might be preferred in patients with chronic obstructive lung disease or peripheral vascular disease. Patients with peripheral vascular disease could potentially have a decrease in blood flow if B-receptors, which dilate the arterioles, are blocked. BReceptor blocking agents also reduce intraocular pressure in patients with glaucoma.

Atenolol, Selectivity for B,-Receptors = +. Atenolol, ISA = 0. Atenolol, a-Blockade = 0. Atenolol, Hepatic Metabolism = 0. Esmolol, Selectivity for B,-Receptors = +. Esmolol, ISA = 0. Esmolol, a-Blockade = 0. Esmolol, Hepatic Metabolism = 0. Labetalol, Selectivity for B,-Receptors = . Labetalol, ISA = 0. Labetalol, a-Blockade = +. Labetalol, Hepatic Metabolism = +. Metoprolol, Selectivity for B,-Receptors = +. Metoprolol, ISA = 0. Metoprolol, a-Blockade = 0. Metoprolol, Hepatic Metabolism = ay. Propranolol, Selectivity for B,-Receptors = . Propranolol, ISA = 0. Propranolol, a-Blockade = 0. Propranolol, Hepatic Metabolism = +
'ISA, intrinsic sympathomimetic activity; +, mild effect; 0, no effect.
B-Blockers are also classified by the amount of intrinsic sympathomimetic activity (ISA) they have. Many of the B-blockers have some agonist activity; although they would not produce effects similar to full agonists (such as epinephrine).
B-Blockers can be further classified as those that are eliminated by hepatic metabolism (such as metoprolol), those that are excreted by the kidneys unchanged (such as atenolol), or those that are hydrolyzed in the blood (such as esmolol).

Metoprolol is a selective B,-antagonist with no intrinsic sympathomimetic activity. It is available for both oral and intravenous use. It can be administered intravenously in 1 to 5 mg increments every 2 to 5 min, titrated to blood pressure and heart rate.

Propranolol nonselectively blocks B,and B5-receptors. Arterial blood pressure is lowered by several mechanisms, including decreased myocardial contractility, lowered heart rate, and diminished renin release. Cardiac output and myocardial oxygen demand are reduced. Propranolol slows atrioventricular conduction and slows the ventricular response to supraventricular tachycardia.
Side effects of propranolol include bronchospasm ($5 antagonism), acute congestive heart failure, bradycardia, and atrioventricular heart block (B, antagonism). Concomitant administration of propranolol and verapamil (a calcium channel blocker) can synergistically depress heart rate, contractility, and atrioventricular node conduction.
Propranolol's elimination half-life of 100 min is quite long compared with that of esmolol. Generally, propranolol is titrated to effect in 0.5 mg increments every 3 to 5 min. Total doses rarely exceed 0.15 mg/kg. Propranolol is supplied in 1-mL ampules containing 1 mg.

Nebivolol is a newer generation B-blocker with high affinity for B,-receptors. The drug is unique in its ability to cause direct vasodilation via its stimulatory effect on endothelial nitric oxide synthase.

Carvedilol is a mixed Band a-blocker used in the management of chronic heart failure secondary to cardiomyopathy, left ventricular dysfunction following acute myocardial infarction, and hypertension. Carvedilol dosage is individualized and gradually increased up to 25 mg twice daily, as required and tolerated. Bisoprolol and sustained-release metoprolol are also used in long-term therapy to reduce mortality in patients with reduced ejection fraction heart failure.

Management of fB-blockers perioperatively has been a key anesthesia

Pheochromocytoma
A 45-year-old man with a history of paroxysmal attacks of headache, hypertension, sweating, and palpitations is scheduled for resection of an abdominal pheochromocytoma.

A pheochromocytoma is a vascular tumor of chromaffin tissue (most commonly the adrenal medulla) that produces and secretes norepinephrine and epinephrine. The diagnosis and management of pheochromocytoma are based on the effects of abnormally high circulating levels of these endogenous adrenergic agonists.

Urinary excretion of vanillylmandelic acid (an end product of catecholamine metabolism), norepinephrine, and epinephrine is often markedly increased. Elevated levels of urinary catecholamines and metanephrines (Figure 14—3) provide a highly accurate diagnosis. Fractionated plasma-free metanephrine levels may be superior to urinary studies in making the diagnosis. The location of the tumor can be determined by magnetic resonance imaging or computed tomographic scan with or without contrast.
What pathophysiology is associated with chronic elevations of norepinephrine and epinephrine?
a, Stimulation increases peripheral vascular resistance and arterial blood pressure. Hypertension can lead to intravascular volume depletion (increasing hematocrit), renal failure, and cerebral hemorrhage. Elevated peripheral vascular resistance also increases myocardial work, which predisposes patients to myocardial ischemia, ventricular hypertrophy, and congestive heart failure. Prolonged exposure to epinephrine and norepinephrine may lead to a catecholamine-induced cardiomyopathy. Hyperglycemia results from decreased insulin secretion in the face of increased glycogenolysis and gluconeogenesis. 8, Stimulation increases automaticity and ventricular ectopy.
Which adrenergic antagonists might be helpful in controlling the effects of norepinephrine and epinephrine hypersecretion?
A major regional block—such as an epidural or spinal anesthetic—could block sensory (afferent) nerves and sympathetic (efferent) discharge in the area of the surgical field. However, the catecholamines released from a pheochromocytoma during surgical manipulation would still be able to bind and activate adrenergic receptors throughout the body.

Fleisher LA, Fleischmann KE, Auerbach AD, et al. 2014 ACC/AHA guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery: Executive summary: A report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. Circulation. 2014;130:2215.

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Pharmacological Basis of Therapeutics. 13th ed. New York, NY: McGrawHill Education; 2018.
Gu YW, Poste J, Kunal M, Schwarcz M, Weiss I. Cardiovascular manifestations of pheochromocytoma. Cardiol Rev. 2017;25:215.
Katzung BG, Trevor AJ, eds. Basic and Clinical Pharmacology, 13th ed. New York, NY: McGraw-Hill Education; 2015.
Lother A, Hein L. Pharmacology of heart failure: From basic science to novel therapies. Pharmacol Ther. 2016;166:136.
Nguyen V, Tiemann D, Park E, Salehi A. Alpha-2 agonists. Anesthesiol Clin. 2017;35:233.

@ Inhaled nitric oxide is a selective pulmonary vasodilator that is used in the treatment of reversible pulmonary hypertension.
@ Acute cyanide toxicity is characterized by metabolic acidosis, cardiac arrhythmias, and increased venous oxygen content (as a result of the inability to utilize oxygen). Another early sign of cyanide toxicity is the acute resistance to the hypotensive effects of increasing doses of sodium nitroprusside (tachyphylaxis).
© By dilating pulmonary vessels, sodium nitroprusside may prevent the normal vasoconstrictive response of the pulmonary vasculature to hypoxia (hypoxic pulmonary vasoconstriction).
@ Preload reduction makes nitroglycerin an excellent drug for the relief of cardiogenic pulmonary edema.
@ Hydralazine relaxes arteriolar smooth muscle in multiple ways, including dilation of precapillary resistance vessels via increased cyclic guanosine 3',5'-monophosphate.
@ The body reacts to a hydralazine-induced fall in blood pressure by increasing heart rate, myocardial contractility, and cardiac output. These compensatory responses can be detrimental to patients with coronary artery disease and are minimized by the concurrent administration of a B-adrenergic antagonist.
@ Fenoldopam (infusion rates studied in clinical trials range from 0.01 to 1.6 mcg/kg/min) reduces systolic and diastolic blood pressure in patients with malignant hypertension to an extent comparable to nitroprusside.
© Dihydropyridine calcium channel blockers preferentially dilate arterial vessels, often preserving or increasing cardiac output.
A multitude of drugs are capable of lowering blood pressure, including volatile anesthetics, sympathetic antagonists and agonists, calcium channel blockers, Bblockers, and angiotensin-converting enzyme inhibitors. This chapter examines agents that may be useful to the anesthesiologist for perioperative control of arterial blood pressure.

As patients age, so too does their vasculature. When a pulse wave is generated by ventricular contraction, it is propagated through the arterial system. At branch points of the aorta, the wave is reflected back toward the heart. In younger patients, the reflected wave tends to augment diastole, improving diastolic pressure. In older patients, the wave arrives sooner, being conducted back by the noncompliant vasculature during late systole, which causes an increase in cardiac workload and a decrease in diastolic pressure (Figure 15-1). Thus, older patients develop increased systolic pressure and decreased diastolic pressure. Widened pulse pressures (the difference between systolic and diastolic pressures) have been associated with both increased incidence of postoperative kidney dysfunction and increased risk of cerebral events in patients undergoing coronary bypass surgery.
FIGURE 15-1 Illustration of the influence of increased vascular stiffness on peripheral (radial) and central (aortic) pressures. Note the similarity of peripheral radial pressures in individuals with normal (lower left panel) and increased (upper left panel) vascular stiffness. In young individuals with normal vascular stiffness, central aortic pressures are lower than radial pressures (lower panels). In contrast, in older individuals with increased vascular stiffness, central aortic pressures are increased and can approach or equal peripheral pressures as a result of wave reflection and central wave augmentation during systole (top panels). (Reproduced with permission from Barodka V, Joshi B, Berkowitz D, et al. Implications of vascular aging. Anesth Analg. 2011 May;112(5):1048-1060.)

B-Blocker therapy should be maintained perioperatively in patients who are being treated with B-blockers as a part of their routine medical regimen. The American College of Cardiology/American Heart Association guidelines for Bblocker use perioperatively should be followed (see Chapter 14). B-Blockers (esmolol, metoprolol, and others) were previously discussed for the treatment of transient perioperative hypertension and are routinely used during anesthesia. This chapter discusses antihypertensive agents other than adrenergic antagonists that are used perioperatively. Antihypertensive agents are critically important for managing hypertensive emergencies (blood pressure >180/120 mm Hg) with signs of organ injury (eg, encephalopathy). Excepting patients with acute aortic dissection, mean arterial pressure should be reduced gradually to prevent organ hypoperfusion (eg, 20% decrease in mean arterial pressure or a diastolic blood pressure of 100-110 mm Hg initially). Prompt treatment of hypertension is also advisable following cardiac and intracranial surgery and other procedures where excessive bleeding is a major concern. Perioperative hypertension is often secondary to pain, anxiety, hypoxemia, hypercapnia, distended bladder, and failure to continue baseline antihypertensive medications. These primary etiologies should be considered and addressed when treating perioperative hypertension.
Blood pressure is the product of cardiac output and systemic vascular resistance. Agents that lower blood pressure reduce myocardial contractility or produce vasodilatation of the arterial and venous capacitance vessels, or both. Agents other than B-adrenergic blockers used to lower blood pressure include nitrovasodilators, calcium antagonists, adrenergic agonists, anesthetic agents, and angiotensin-converting enzyme inhibitors.

Sodium nitroprusside (and other nitrovasodilators) relax both arteriolar and venous smooth muscle. Its primary mechanism of action is shared with other nitrates (eg, hydralazine and nitroglycerin). As nitrovasodilators are metabolized, they release nitric oxide, which activates guanylyl cyclase. This enzyme is responsible for the synthesis of cyclic guanosine 3',5'-monophosphate (cGMP), which controls the phosphorylation of several proteins, including some involved in the control of free intracellular calcium and smooth muscle contraction.
Nitric oxide, a naturally occurring potent vasodilator released by endothelial cells (endothelium-derived relaxing factor), plays an important role in regulating vascular tone throughout the body. Its ultrashort half-life (<5 s) provides nimble endogenous @ control of regional blood flow. Inhaled nitric oxide is a selective pulmonary vasodilator that is used in the treatment of reversible pulmonary

Sodium nitroprusside is a potent and reliable antihypertensive. It is usually diluted to a concentration of 100 mcg/mL and administered as a continuous intravenous infusion (0.25-5 mcg/kg/min). Its extremely rapid onset of action (1-2 min) and fleeting duration of action allow precise titration of arterial blood pressure. The potency of this drug requires frequent blood pressure measurements—or, preferably, intraarterial monitoring—and the use of mechanical infusion pumps. Solutions of sodium nitroprusside must be protected from light because of photodegradation.

After parenteral injection, sodium nitroprusside enters red blood cells, where it receives an electron from the iron (Fe**) of oxyhemoglobin. This nonenzymatic electron transfer results in an unstable nitroprusside radical and methemoglobin (Hgb Fe?*), The former moiety spontaneously decomposes into five cyanide ions and the active nitroso (N = O) group.
The cyanide ions can be involved in one of three possible reactions: (1) binding to methemoglobin to form cyanmethemoglobin; (2) undergoing a reaction in the liver and kidney catalyzed by the enzyme rhodanase (thiosulfate + cyanide = thiocyanate); or (3) binding to tissue cytochrome oxidase, which interferes with normal oxygen utilization (Figure 15-2).
SNP + Oxyhemoglobin
CN7 + Methemoglobin
CN7 + Thiosulfate
CN™ + Cytochrome oxidase
———————» (SNP)~ + Methemoglobin (SNP) —————— 5CN™ ——————> Cyanmethemoglobin or Rhodanase, vitamin By z ——-—> Thiocyanate or ————> Cyanide toxicity
FIGURE 15-2 The metabolism of sodium nitroprusside.
®@ The last of these reactions underlies the development of acute cyanide

The combined dilation of venous and arteriolar vascular beds by sodium nitroprusside results in reductions of preload and afterload. Arterial blood pressure falls due to the decrease in peripheral vascular resistance. Although cardiac output is usually unchanged in normal patients, the reduction in afterload may increase cardiac output in patients with congestive heart failure, mitral regurgitation, or aortic regurgitation. In opposition to any favorable changes in myocardial oxygen requirements are reflex-mediated responses to the fall in arterial blood pressure. These include tachycardia and increased myocardial contractility. In addition, dilation of coronary arterioles by sodium nitroprusside may result in an intracoronary steal of blood flow away from ischemic areas that are already maximally dilated.
Sodium nitroprusside dilates cerebral vessels and abolishes cerebral autoregulation. Cerebral blood flow is maintained or increases unless arterial blood pressure is markedly reduced. The resulting increase in cerebral blood volume tends to increase intracranial pressure, particularly in patients with reduced intracranial compliance (eg, brain tumors). This intracranial hypertension can be minimized by slow administration of sodium nitroprusside and institution of hypocapnia.
The pulmonary vasculature also dilates in response to sodium nitroprusside infusion. Reductions in pulmonary artery pressure may decrease the perfusion of some normally ventilated alveoli, @ increasing physiological dead space. By dilating pulmonary vessels, sodium nitroprusside may prevent the normal vasoconstrictive response of the pulmonary vasculature to hypoxia (hypoxic pulmonary vasoconstriction). Both of these effects tend to mismatch pulmonary ventilation to perfusion, increase venous admixture, and decrease arterial oxygenation.
In response to decreased arterial blood pressure, renin and catecholamines are released during administration of nitroprusside. Renin release can be inhibited with B-blockers. Kidney function is fairly well maintained during sodium nitroprusside infusion, despite moderate drops in arterial blood pressure and renal perfusion.

Nitroglycerin relaxes vascular smooth muscle, with venous dilation predominating over arterial dilation. Its mechanism of action is similar to that of sodium nitroprusside: metabolism to nitric oxide, which activates guanylyl cyclase, leading to increased cGMP, decreased intracellular calcium, and vascular smooth muscle relaxation.

Nitroglycerin relieves myocardial ischemia, hypertension, and ventricular failure. Like sodium nitroprusside, nitroglycerin is commonly diluted to a concentration of 100 mcg/mL and administered as a continuous intravenous infusion (0.5-5 mcg/kg/min). In the past glass containers and special intravenous tubing were recommended because of the adsorption of nitroglycerin to polyvinylchloride, but these are now rarely used. Nitroglycerin can also be administered by a sublingual (peak effect in 4 min) or transdermal (sustained release for 24 h) route.

Nitroglycerin (50-100 mcg boluses) has been demonstrated to be an effective (but transient) uterine relaxant that can be beneficial during certain obstetrical procedures if the placenta is still present in the uterus (eg, retained placenta, uterine inversion, uterine tetany, breech extraction, and external version of the second twin). Nitroglycerin therapy has been shown to diminish platelet aggregation.

® Hydralazine relaxes arteriolar smooth muscle in multiple ways, including dilation of precapillary resistance vessels via increased cGMP.
Intraoperative hypertension is sometimes controlled with an intravenous dose of 5 to 20 mg of hydralazine. The onset of action is within 15 min, and the antihypertensive effect usually lasts 2 to 4h. The delayed onset and prolonged duration have made this agent much less attractive than others for perioperative use. Hydralazine can be used to control pregnancy-induced hypertension.
Hydralazine undergoes acetylation and hydroxylation in the liver.

The lowering of peripheral vascular resistance causes @ a drop in arterial blood pressure. The body reacts to a hydralazine-induced fall in blood pressure by increasing heart rate, myocardial contractility, and cardiac output. These compensatory responses can be detrimental to patients with coronary artery disease and are minimized by the concurrent administration of a B-adrenergic antagonist. Conversely, the decline in afterload often proves beneficial to patients in congestive heart failure.
Hydralazine is a potent cerebral vasodilator and inhibitor of cerebral blood flow autoregulation. Unless blood pressure is markedly reduced, cerebral blood flow and intracranial pressure will rise.
Renal blood flow is usually maintained or increased by hydralazine.

Fenoldopam causes rapid vasodilation by selectively activating D,;-dopamine receptors. The R-isomer is responsible for the racemic mixture's biological activity due to its much greater receptor affinity, compared with the S-isomer.

@ Fenoldopam (infusion rates studied in clinical trials range from 0.01 to 1.6 mcg/kg/min) reduces systolic and diastolic blood pressure in patients with malignant hypertension to an extent comparable to nitroprusside. Side effects include headache, flushing, nausea, tachycardia, hypokalemia, and hypotension. The onset of the hypotensive effect occurs within 15 min, and discontinuation of an infusion quickly reverses this effect without rebound hypertension. Some degree of tolerance may develop 48 h after the infusion. Studies are mixed regarding fenoldopam's ability to "protect" and "maintain" kidney function in perioperative patients with hypertension at risk of perioperative kidney injury.

Fenoldopam undergoes conjugation without participation of the cytochrome P450 enzymes, and its metabolites are inactive. Clearance of fenoldopam remains unaltered despite the presence of kidney or hepatic failure, and no dosage adjustments are necessary for these patients.

Fenoldopam decreases systolic and diastolic blood pressure. Heart rate typically increases. Low initial doses (0.03—0.1 mcg/kg/min) titrated slowly have been associated with less reflex tachycardia than higher doses. Tachycardia decreases over time but remains substantial at doses.
As would be expected from a D,-dopamine receptor agonist, fenoldopam markedly increases kidney blood flow. Despite a drop in arterial blood the glomerular filtration rate is well maintained. Fenoldopam increases urinary flow rate, urinary sodium extraction, and creatinine clearance compared with sodium nitroprusside.
higher Fenoldopam can lead to rises in intraocular pressure and should be administered with caution or avoided in patients with a history of glaucoma or intraocular hypertension. pressure,

@ Dihydropyridine calcium channel blockers (nicardipine, clevidipine) are arterial selective vasodilators routinely used for perioperative blood pressure control in patients undergoing cardiothoracic surgery. Clevidipine, prepared as a lipid emulsion, has a short halflife secondary to rapid metabolism by blood esterases, which facilitates its rapid titration. Clevidipine is infused initially at a rate of 1 to 2 mg/h, with the dose doubled until the desired effect is obtained, up to 16 mg/h. Because of its formulation as a lipid emulsion it is contraindicated in patients with soy or egg allergies and those with impaired lipid metabolism. Unlike verapamil and diltiazem, the dihydropyridine calcium channel blockers have minimal effects on cardiac conduction and ventricular contractility. These calcium channel blockers bind to L-type calcium channel and impair calcium entry into the vascular smooth muscle. L-type receptors are more prevalent on arterial than venous capacitance vessels. Consequently, cardiac filling and preload are less affected by these agents than by nitrates, which might dilate both arterial and venous systems. With preload maintained, cardiac output often increases when vascular tone is reduced by use of dihydropyridine calcium blockers. Nicardipine infusion is titrated to effect (5-15 mg/h).
Another intravenous agent that can produce hypotension perioperatively is the intravenous angiotensin-converting enzyme inhibitor enalaprilat (0.625-1.25 mg). The role of enalaprilat as a nondirect-acting agent in the acute treatment of a hypertensive crisis is limited.

A59-year-old man is scheduled for total hip arthroplasty under general anesthesia. The surgeon requests a controlled hypotensive technique.
What is controlled hypotension, and what are its advantages?
Controlled hypotension is the elective lowering of arterial blood pressure. The primary advantages of this technique are minimization of surgical blood loss and better surgical visualization.

The primary methods of electively lowering blood pressure are use of hypotensive anesthetic techniques (eg, neuraxial anesthesia) and the administration of hypotensive drugs. Elevation of the surgical site can selectively reduce the blood pressure at the wound. During general anesthesia the increase in intrathoracic pressure that accompanies positivepressure ventilation impedes venous return to the heart, lowering cardiac output and mean arterial pressure. Numerous pharmacological agents effectively lower blood pressure: volatile anesthetics, spinal and epidural anesthesia, sympathetic antagonists, calcium channel blockers, and the peripheral vasodilators discussed in this chapter.
Which surgical procedures might benefit most from a controlled hypotensive technique?
Controlled hypotension has been successfully used during cerebral aneurysm repair, brain tumor resection, total hip arthroplasty, radical neck dissection, radical cystectomy, major spine surgery, and other operations associated with significant blood loss. Controlled hypotension may allow safer surgery of patients whose religious beliefs prohibit blood transfusions (eg, Jehovah's Witnesses).

Some patients have predisposing illnesses that decrease the margin of safety for adequate organ perfusion: severe anemia, hypovolemia, atherosclerotic cardiovascular disease, renal or hepatic insufficiency, cerebrovascular disease, or uncontrolled glaucoma.

As the preceding list of contraindications suggests, the risks of low arterial blood pressure include cerebral thrombosis, hemiplegia (due to decreased spinal cord perfusion), acute tubular necrosis, massive hepatic necrosis, myocardial infarction, cardiac arrest, and blindness (from retinal artery thrombosis or ischemic optic neuropathy). These complications are more likely in patients with coexisting anemia. Consequently, the use of induced or controlled hypotension continues to decline. Patients requiring beach chair positioning for shoulder surgery or sitting position are at particular risk for cerebral hypoperfusion and perioperative cerebral infarction.

This depends on the patient. Healthy young individuals may tolerate mean arterial pressures as low as 50 to 60 mm Hg without complications. On the other hand, chronically hypertensive patients have altered autoregulation of cerebral blood flow and may tolerate a mean arterial pressure of no more than 20% to 30% lower than baseline. Patients with a history of transient ischemic attacks may not tolerate any decline in cerebral perfusion. Recent studies suggest that the lower limit of cerebral autoregulation may be at a much higher mean arterial pressure than long has been assumed.
What special monitoring is indicated during controlled hypotension?
Intraarterial blood pressure monitoring correctly positioned to determine mean arterial pressure at the brain is suggested. Likewise, cerebral oximetry can be employed if hypotensive anesthesia techniques are to be considered.

Williams-Russo P, Sharrock NE, Mattis S. Randomized trial of hypotensive epidural anesthesia in older adults. Anesthesiology. 1999;91:926. Zhao N, Xu J, Singh B, et al. Nitrates for the prevention of cardiac morbidity and mortality in patients undergoing non cardiac surgery. Cochrane Database Syst Rev. 2016;(8):CD010726.

@ Voltage-gated sodium (Na) channels are membrane-associated proteins that comprising one large a subunit, through which Na ions pass, and one or two smaller 8 subunits. Na channels exist in (at least) three states—resting (nonconducting), open (conducting), and inactivated (nonconducting). Local anesthetics bind and inhibit a specific region of the a subunit, preventing channel activation and the Na influx associated with membrane depolarization.
@ Sensitivity of nerve fibers to inhibition by local anesthetics is influenced by axonal diameter, myelination, and other factors.
@ Clinical local anesthetic potency correlates with octanol solubility and the ability of the local anesthetic molecule to permeate lipid membranes. Potency is increased by adding large alkyl groups to a parent molecule. There is no clinical measurement of local anesthetic potency that is analogous to the minimum alveolar concentration (MAC) of inhalation anesthetics.
@ Onset of action depends on many factors, including lipid solubility and the relative concentration of the nonionized, more lipid-soluble freebase form (B) and the ionized more water-soluble form (BH*), expressed by the pK,. The pK, is the pH at which there is an equal fraction of ionized and nonionized drug. Less potent, less lipid-soluble agents (eg, lidocaine or mepivacaine) generally have a faster onset than more potent, more lipid-soluble agents (eg, ropivacaine or bupivacaine).
@ Duration of action correlates with potency and lipid solubility. Highly lipid-soluble local anesthetics have a longer duration of action, presumably because they more slowly diffuse from a lipid-rich environment to the aqueous bloodstream.
@ Inregional anesthesia local anesthetics are typically applied close to their intended site of action; thus their pharmacokinetic profiles in blood are important determinants of elimination and toxicity and have very little to do with the duration of their desired clinical effect.

@ The rates of local anesthetic systemic absorption and rise of local anesthetic concentrations in blood are related to the vascularity of the site of injection, and generally follow this rank order: intravenous (or intraarterial) > tracheal > intercostal > paracervical > epidural > brachial plexus > sciatic > subcutaneous.
@ Ester local anesthetics are metabolized predominantly by pseudocholinesterase. Amide local anesthetics are metabolized (Ndealkylation and hydroxylation) by microsomal P-450 enzymes in the liver.
@ In awake patients rising local anesthetic concentrations in the central nervous system produce the premonitory signs of local anesthetic intoxication.
@® Major cardiovascular toxicity usually requires about three times the local anesthetic concentration in blood as that required to produce seizures.
@ Unintended intravascular injection of bupivacaine during regional anesthesia may produce severe cardiovascular toxicity, including left ventricular depression, atrioventricular heart block, and life-threatening arrhythmias such as ventricular tachycardia and fibrillation.
® True hypersensitivity reactions (due to IgG or IgE antibodies) to local anesthetics—as distinct from systemic toxicity caused by excessive plasma concentrations—are uncommon. Esters appear more likely to induce an allergic reaction, especially if the compound is a derivative (eg, procaine or benzocaine) of p-aminobenzoic acid, a known allergen.
Local and regional anesthesia and analgesia techniques depend on a group of drugs—local anesthetics—that transiently inhibit some or all of sensory, motor, or autonomic nerve function when the drugs are applied near neural tissue. This chapter presents the mechanism of action, structure—activity relationships, and clinical pharmacology of local anesthetic drugs. The more commonly used regional anesthetic techniques are presented elsewhere (see Chapters 45 and
46).

FIGURE 16-1 Compound Aq, A6, and C fiber action potentials recorded after supramaximal stimulation of a rat sciatic nerve. Note the differing time scale of the recordings. In peripheral nerves, Ad and C fibers have much slower conduction velocities, and their compound action potentials are longer and of less amplitude when compared with those from Aa fibers. (Reproduced with permission from Butterworth JF 4th, Strichartz GR. The alpha2-adrenergic agonists clonidine and guanfacine produce tonic and phasic block of conduction in rat sciatic nerve fibers. Anesth Analg. 1993 Feb;76(2):295-301.)
@ The previously mentioned, voltage-gated Na channels are membraneassociated proteins comprising one large a subunit, through which Na ions pass, and one or two smaller B subunits. Na channels exist in (at least) three states —resting (nonconducting), open (conducting), and inactivated (nonconducting) (Figure 16—2). When local anesthetics bind a specific region of the a subunit, they prevent channel activation and Na influx through the individual channels. Local anesthetic binding to Na channels does not alter the resting membrane potential. With increasing local anesthetic concentrations, an increasing fraction of the Na channels in the membrane bind a local anesthetic molecule and cannot conduct Na ions. As a consequence of more channels binding a local anesthetic, the threshold for excitation and impulse conduction in the nerve increases, the rate of rise and the magnitude of the action potential decreases, and impulse conduction velocity slows. At great enough local anesthetic concentrations (when a sufficient fraction of Na channels has bound a local anesthetic), action potentials can no longer be generated and impulse propagation is abolished. Local anesthetics have a greater affinity for the Na channel in the open or inactivated state than in the resting state. Depolarizations lead to open and inactivated channels; therefore, depolarization favors local anesthetic binding. The fraction of Na channels that bind a local anesthetic increases with frequent depolarization (eg, during trains of impulses). This phenomenon is termed usedependent block. Put another way, local anesthetic inhibition of Na channels is both voltage (membrane potential) and frequency dependent. Local anesthetic binding is greater when nerve fibers are firing and depolarizing frequently than with infrequent depolarizations.

FIGURE 16-2 Voltage-gated sodium (Na,) channels exist in at least three states—resting, open (activated), and inactivated. Resting Na, channels activate and open when they are depolarized, briefly allowing Na ions to pass into the cell down their concentration gradient, then rapidly inactivate. Inactivated Na, channels return to the resting state as the cell membrane repolarizes. In the figure, Na ions are shown on the extracellular side of the cell membrane. Extracellular Na ions conduct only through open Na, channels that have not bound a local anesthetic molecule. The Na, channel binding site for local anesthetics is nearer to the cytoplasmic than the extracellular side of the channel.
Local anesthetics may also bind and inhibit calcium (Ca), K, transient receptor potential vanilloid-1 (TRPV1), and many other channels and receptors. Conversely, other classes of drugs, notably tricyclic antidepressants (amitriptyline), meperidine, volatile anesthetics, Ca channel blockers, a5receptor agonists, and nerve toxins also may inhibit Na channels. Tetrodotoxin and saxitoxin are poisons that specifically bind Na channels at a site on the exterior of the plasma membrane. Human studies are under way with similar toxins to determine whether they might provide effective, prolonged analgesia after local infiltration, particularly when coadministered with local anesthetics.
® Sensitivity of nerve fibers to inhibition by local anesthetics is influenced by axonal diameter, myelination, and other factors. Table 16—1 lists the most commonly used classification for nerve fibers. In comparing nerve fibers of the same type (myelinated versus unmyelinated), smaller diameter associates with increased sensitivity to local anesthetics. Thus, larger, faster-conducting Aa fibers are less sensitive to local anesthetics than smaller, slower-conducting Ad fibers. Larger unmyelinated fibers are less sensitive than smaller unmyelinated fibers. On the other hand, small unmyelinated C fibers are relatively resistant to inhibition by local anesthetics as compared with larger myelinated fibers. In a human peripheral nerve the onset of local anesthetic inhibition generally follow this sequence: autonomic before sensory before motor. But at steady state, if sensory anesthesia is present, usually all modalities are inhibited.

Aa, Modality Served = Motor efferent. Aa, Diameter (mm) = 12-20. Aa, Conduction (m/s) = 70-120. Aa, Myelinated? = Yes. Aa, Modality Served = Proprioception. Aa, Diameter (mm) = 12-20. Aa, Conduction (m/s) = 70-120. Aa, Myelinated? = Yes. AB, Modality Served = Touch, pressure. AB, Diameter (mm) = 5-12. AB, Conduction (m/s) = 30-70. AB, Myelinated? = Yes. Ay, Modality Served = Motor efferent (muscle spindle). Ay, Diameter (mm) = 3-6. Ay, Conduction (m/s) = 15-30. Ay, Myelinated? = Yes. Ad, Modality Served = Pain Temperature Touch. Ad, Diameter (mm) = 2-5. Ad, Conduction (m/s) = 12-30. Ad, Myelinated? = Yes. B, Modality Served = Preganglionic autonomic fibers. B, Diameter (mm) = <3. B, Conduction (m/s) = 3-14. B, Myelinated? = Some. G Dorsal root, Modality Served = Pain Temperature. G Dorsal root, Diameter (mm) = 0.4-1.2. G Dorsal root, Conduction (m/s) = 0.5-2. G Dorsal root, Myelinated? = No. Cc Sympathetic, Modality Served = Postganglionic sympathetic fibers. Cc Sympathetic, Diameter (mm) = 0.3-1.3. Cc Sympathetic, Conduction (m/s) = 0.7-2.3. Cc Sympathetic, Myelinated? = No
'An alternative numerical system is sometimes used to classify sensory fibers.

Local anesthetics consist of a lipophilic group (usually an aromatic benzene ring) separated from a hydrophilic group (usually a tertiary amine) by an intermediate chain that includes an ester or amide linkage. The nature of the intermediate chain is the basis of the classification of local anesthetics as either esters or amides (Table 16—2). Articaine, a popular local anesthetic for dentistry in several European countries, is an amide but it contains a thiophene ring rather than a benzene ring. Local anesthetics are weak bases that at physiological pH usually carry a positive charge at the tertiary amine group. Physicochemical properties of local anesthetics depend on the substitutions in the aromatic ring, the type of linkage in the intermediate chain, and the alkyl groups attached to the amine nitrogen.

Local Anesthetic = . Esters, pK, = . Esters, Protein Binding (%) = . Chloroprocaine (Nesacaine), Structure = . Chloroprocaine (Nesacaine), Relative Lipid Solubility of Unchanged Local Anesthetic = 23. Chloroprocaine (Nesacaine), pK, = 9.1. Chloroprocaine (Nesacaine), Protein Binding (%) = NA’. Cocaine, Structure = . Cocaine, Relative Lipid Solubility of Unchanged Local Anesthetic = . Cocaine, pK, = 87. Cocaine, Protein Binding (%) = 91. Procaine (Novocaine), Structure = . Procaine (Novocaine), Relative Lipid Solubility of Unchanged Local Anesthetic = 0.3. Procaine (Novocaine), pK, = 9.1. Procaine (Novocaine), Protein Binding (%) = . Tetracaine (Pontocaine), Structure = . Tetracaine (Pontocaine), Relative Lipid Solubility of Unchanged Local Anesthetic = . Tetracaine (Pontocaine), pK, = 8.6. Tetracaine (Pontocaine), Protein Binding (%) = 76
*Carbon atom responsible for optical isomerism.
'NA, not available.
® Clinical local anesthetic potency correlates with octanol solubility and the ability of the local anesthetic molecule to permeate lipid membranes. Potency is increased by adding large alkyl groups to a parent molecule (compare tetracaine with procaine, or bupivacaine with mepivacaine). There is no clinical measurement of local anesthetic potency that is analogous to the minimum alveolar concentration (MAC) of inhalation anesthetics. The minimum concentration of local anesthetic that will block nerve impulse conduction is affected by several factors, including fiber size, type, and myelination; pH (an

@ In regional anesthesia local anesthetics are typically applied close to their intended site of action; thus their pharmacokinetic profiles in blood are important determinants of elimination and toxicity and have very little to do with the duration of their desired clinical effect.

Absorption after topical application depends on the site. Most mucous membranes (eg, tracheal or oropharyngeal mucosa) provide a minimal barrier to local anesthetic penetration, leading to a rapid onset of action. Intact skin, on the other hand, requires topical application of a high concentration of lipid-soluble local anesthetic base to ensure permeation and analgesia. EMLA™™ (Eutectic

receptor blockers) will reduce the rate of amide metabolism and potentially predispose patients to having greater blood concentrations and a greater risk of systemic toxicity. Water-soluble local anesthetic metabolites are dependent on renal clearance.
Prilocaine is the only local anesthetic that is metabolized to o-toluidine, which produces methemoglobinemia in a dose-dependent fashion. Classical teaching was that a defined dose of prilocaine (in the range of 10 mg/kg) must be exceeded to produce clinically consequential methemoglobinemia; however, recent studies have shown that younger, healthier patients develop medically important methemoglobinemia after lower doses of prilocaine (and at lower doses than needed in older, sicker patients). Prilocaine currently has limited use in North America, but is more commonly used in other regions. Benzocaine, a common ingredient in topical local anesthetic sprays, can also cause dangerous levels of methemoglobinemia. For this reason, many hospitals no longer permit benzocaine spray during endoscopic procedures. Treatment of medically important methemoglobinemia includes intravenous methylene blue (1-2 mg/kg of a 1% solution over 5 min). Methylene blue reduces methemoglobin (Fe**) to hemoglobin (Fe**).

Because voltage-gated Na channels underlie action potentials in neurons throughout the body as well as impulse generation and conduction in the heart, it is not surprising that local anesthetics in high circulating concentrations could produce systemic toxicity. Although organ system effects are discussed for these drugs as a group, individual drugs differ.
Potency at most toxic side effects correlates with local anesthetic potency at nerve blocks. "Maximum safe doses" are listed in Table 16—3, but it must be recognized that the maximum safe dose depends on the patient, the specific nerve block, the rate of injection, and a long list of other factors. In other words, tables of purported maximal safe doses are nearly nonsensical. Mixtures of local anesthetics should be considered to have additive toxic effects; therefore, injecting a solution combining 50% of a toxic dose of lidocaine and 50% of a toxic dose of bupivacaine likely will produce toxic effects.
TABLE 16-3 Clinical use of local anesthetic agents.

1.5%, 2%, 4%, 5%. Lidocaine (lignocaine), Maximum Dose (mg/kg) = 45 7 (with epinephrine). Lidocaine (lignocaine), Typical Duration of Nerve Blocks’ = Medium. Mepivacaine, Techniques = Epidural, infiltration, peripheral nerve block, spinal. Mepivacaine, Concentrations Available = 1%, 1.5%, 2%, 3%. Mepivacaine, Maximum Dose (mg/kg) = 45 7 (with epinephrine). Mepivacaine, Typical Duration of Nerve Blocks’ = Medium. Prilocaine, Techniques = EMLA (topical), epidural, intravenous regional (outside North America). Prilocaine, Concentrations Available = 0.5%, 2%, 3%, 4%. Prilocaine, Maximum Dose (mg/kg) = 8. Prilocaine, Typical Duration of Nerve Blocks’ = Medium. Ropivacaine, Techniques = Epidural, spinal, infiltration, peripheral nerve block. Ropivacaine, Concentrations Available = 0.2%, 0.5%, 0.75%, 1%. Ropivacaine, Maximum Dose (mg/kg) = 3. Ropivacaine, Typical Duration of Nerve Blocks’ = Long
'Wide variation depending on concentration, location, technique, and whether combined with a vasoconstrictor (epinephrine). Generally, the shortest duration is with spinal anesthesia and the longest with peripheral nerve blocks.
2No longer recommended for topical anesthesia.
2NA, not applicable or not defined.
'Recent literature describes this agent for short-duration spinal anesthesias.

@ The central nervous system is vulnerable to local anesthetic systemic toxicity and there are premonitory signs and symptoms of rising local anesthetic concentrations in blood in awake patients. Such symptoms include circumoral numbness, tongue paresthesia, dizziness, tinnitus, blurred vision, and a feeling of impending doom. Such signs include restlessness, agitation, nervousness, and garrulousness. Muscle twitching precedes tonic-clonic seizures. Still higher blood concentrations may produce central nervous system depression (eg, coma and respiratory arrest). The excitatory reactions are thought to be the result of selective blockade of inhibitory pathways. Potent, highly lipid-soluble local anesthetics produce seizures at lower blood concentrations than less potent agents. Benzodiazepines, propofol, and hyperventilation raise the threshold of local anesthetic-induced seizures. Both respiratory and metabolic acidosis reduce the seizure threshold. Propofol (0.5—2 mg/kg) quickly and reliably terminates seizure activity (as do comparable doses of benzodiazepines or barbiturates). Some clinicians use intravenous lipid to terminate local anesthetic-induced seizures (see below). Maintaining a clear airway with adequate ventilation and oxygenation is most important.

Lidocaine depresses the ventilatory response to low PaO, (hypoxic drive). Apnea can result from phrenic and intercostal nerve paralysis (eg, from "high" spinals)
or depression of the medullary respiratory center following direct exposure to local anesthetic agents (eg, after retrobulbar blocks; see Chapter 36). However, apnea after administration of a "high" spinal or epidural anesthetic is nearly always the result of hypotension and brain ischemia, rather than phrenic block. Local anesthetics relax bronchial smooth muscle. Intravenous lidocaine (1.5 mg/kg) may block the reflex bronchoconstriction sometimes associated with intubation.

® True hypersensitivity reactions (due to IgG or IgE antibodies) to local anesthetics—as distinct from systemic toxicity caused by excessive plasma concentrations—are uncommon. Esters appear more likely to induce an allergic reaction, especially if the compound is a derivative (eg, procaine or benzocaine)
of PABA, a known allergen. Commercial multidose preparations of amides often contain methylparaben, which has a chemical structure vaguely similar to that of PABA. As a consequence, generations of anesthesiologists have speculated whether this preservative may be responsible for most of the apparent allergic responses to amide agents, particularly when skin testing fails to confirm true allergy to the local anesthetic.

When directly injected into skeletal muscle either intentionally (eg, trigger-point injection treatment of myofascial pain) or unintentionally, local anesthetics are mildly myotoxic. Regeneration usually occurs within 4 weeks after the injection. Compounding the local anesthetic with steroid or epinephrine worsens the myonecrosis. When infused into joints for prolonged periods, local anesthetics can produce severe chondromalacia.

Lidocaine mildly depresses normal blood coagulation (reduced thrombosis and decreased platelet aggregation) and enhances fibrinolysis of whole blood as measured by thromboelastography. These actions could underlie the lower incidence of thromboembolic events in patients receiving epidural anesthetics (in older studies of patients not receiving prophylaxis against deep vein thrombosis).

Local laboratory anesthetics potentiate nondepolarizing muscle relaxant blockade in experiments, but this likely has no clinical importance.
As noted earlier, both succinylcholine and ester local anesthetics depend on pseudocholinesterase for metabolism. There is no evidence that this potential competition between ester local anesthetics and succinylcholine for the enzyme has any clinical importance. Dibucaine, an amide local anesthetic, inhibits pseudocholinesterase, and the extent of inhibition by dibucaine defines one form of genetically abnormal pseudocholinesterases (see Chapter 11). Pseudocholinesterase inhibitors (eg, organophosphate poisons) can prolong the metabolism of ester local anesthetics (See Table 11-2).
As noted earlier, drugs that decrease hepatic blood flow (eg, H>-receptor blockers and B-blockers) decrease amide local anesthetic clearance. Opioids potentiate analgesia produced by epidural and spinal local anesthetics. Similarly Q-adrenergic agonists (eg, clonidine) potentiate local anesthetic analgesia produced after epidural or peripheral nerve block injections. Epidural chloroprocaine may interfere with the analgesic actions of neuraxial morphine, notably after cesarean delivery.

An 18-year-old woman in the active stage of labor requests an epidural anesthetic. Immediately following injection of 2 mL and 5 mL test doses of 1.5% lidocaine with 1:200,000 epinephrine through the epidural catheter, the patient complains of lip numbness and becomes very apprehensive. Her heart rate has increased from 85 to 105 beats/min.

Circumoral numbness and apprehension immediately following administration of lidocaine suggest an intravascular injection of local anesthetic. Abrupt tachycardia strongly suggests intravascular injection of epinephrine. These symptoms and signs after relatively small test doses typically will not be followed by a seizure.

The patient should receive supplemental oxygen. She should be closely observed for a possible (but unlikely) seizure and be reassured that the symptoms and signs will soon lapse.

The laboring patient is always considered to be at increased risk for aspiration (see Chapter 41); therefore, the airway should be protected by immediate administration of succinylcholine and tracheal intubation (see Case Discussion, Chapter 17). The succinylcholine will eliminate tonic— clonic activity but will not address the underlying cerebral hyperexcitability. We favor administering an anticonvulsant such as midazolam (1-2 mg) or propofol (20-50 mg) with or before succinylcholine. Thus, wherever conduction anesthetics are administered resuscitation drugs and equipment must be available just as for a general anesthetic.
What could have been expected if a large dose of bupivacaine (eg, 15 mL 0.5% bupivacaine)—instead of lidocaine—had been given intravascularly?
When administered at "comparably anesthetizing" doses, bupivacaine is more likely to produce cardiac toxicity than lidocaine. Acute acidosis (nearly universal after a seizure) tends to potentiate local anesthetic toxicity. Ventricular arrhythmias and conduction disturbances may lead to cardiac arrest and death. Cardiac Na channels unbind bupivacaine more slowly than lidocaine. Amiodarone may be given as treatment for local anestheticinduced ventricular tachyarrhythmias, but we favor immediate administration of lipid emulsion with the onset of seizures and most certainly at the first signs of cardiac toxicity from bupivacaine. Vasopressors may be required. We recommend incremental small (0.5-1 mcg/kg) doses of epinephrine. The reason for the apparent greater susceptibility to local anesthetic cardiotoxicity during pregnancy is unclear. Although total dose (regardless of concentration) of local anesthetic determines toxicity, the Food and Drug Administration recommends against use of 0.75% bupivacaine in pregnant and elderly patients, and in any case this concentration is not needed.

The risk from accidental intravascular injections during attempted epidural anesthesia is reduced by using test doses and administering the local anesthetic dose in smaller, safer aliquots. Finally, one should administer only the minimum required dose for a given regional anesthetic.

Brunton LL, Knollmann BC, Hilal-Dandan R, eds. Goodman and Gilman's The
Pharmacological Basis of Therapeutics. 13th ed. New York, NY: McGrawHill; 2018.
Cousins MJ, Carr DB, Horlocker TT, Bridenbaugh PO, eds. Cousins &
Bridenbaugh's Neural Blockade in Clinical Anesthesia and Pain Medicine. Ath ed. Philadelphia, PA: Lippincott, Williams & Wilkins; 2009.
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This web site provides up-to-date information about the use of lipid for rescue from local anesthetic toxicity. http://www.lipidrescue.org
The American Society of Regional Anesthesia and Pain Medicine (ASRA) web site provides access to all ASRA guidelines (all of which are related to local anesthetics, regional anesthesia, or pain medicine). http://www.asra.com

@ Diphenhydramine is one of a diverse group of drugs that competitively blocks H, receptors. Many drugs with H,-receptor antagonist properties have considerable antimuscarinic, or atropine-like, activity (eg, dry mouth), or antiserotonergic activity (antiemetic).
@ H, blockers reduce the perioperative risk of aspiration pneumonia by decreasing gastric fluid volume and raising the pH of gastric contents.
© Metoclopramide increases lower esophageal sphincter tone, speeds gastric emptying, and lowers gastric fluid volume by enhancing the stimulatory effects of acetylcholine on intestinal smooth muscle.
@ Ondansetron, granisetron, tropisetron, and dolasetron selectively block serotonin 5-HT3 receptors, with little or no effect on dopamine receptors. Located peripherally and centrally, 5-HT3 receptors appear to play an important role in the initiation of the vomiting reflex.
@ Ketorolac is a parenteral nonsteroidal antiinflammatory drug that provides analgesia by inhibiting prostaglandin synthesis.
@ Clonidine is a commonly used antihypertensive agent but in anesthesia it is used as an adjunct for epidural, caudal, and peripheral nerve block anesthesia and analgesia. It is often used in the management of patients with chronic neuropathic pain to increase the efficacy of epidural opioid infusions.
@ Dexmedetomidine is a parenteral selective a-agonist with sedative properties. It appears to be more selective for the a»-receptor than clonidine.
© Selective activation of carotid chemoreceptors by low doses of doxapram stimulates hypoxic drive, producing an increase in tidal
volume and a slight increase in respiratory rate. Doxapram is not a specific reversal agent and should not replace standard supportive therapy (ie, mechanical ventilation).

© Naloxone reverses the agonist activity associated with endogenous or exogenous opioid compounds.
@ Flumazenil is useful in the reversal of benzodiazepine sedation and the treatment of benzodiazepine overdose.
@ Aspiration does not necessarily result in aspiration pneumonia. The seriousness of the lung damage depends on the volume and composition of the aspirate. Patients are at risk if their gastric volume is greater than 25 mL (0.4 mL/kg) and their gastric pH is less than 2.5.
Many drugs are routinely administered perioperatively to protect against aspiration pneumonitis, to prevent or reduce the incidence of perianesthetic nausea and vomiting, or to reverse respiratory depression secondary to narcotics or benzodiazepines. This chapter discusses these agents along with other unique classes of drugs that are often administered as adjuvants during anesthesia or analgesia. Additionally, many nonanesthetic agents are increasingly prescribed perioperatively to provide for enhanced recovery following surgery (see Chapter 48).

Aspiration of gastric contents is a rare and potentially fatal event that can complicate anesthesia. Based on an animal study, it is often stated that aspiration of 25 mL of volume at a pH of less than 2.5 will be sufficient to produce aspiration pneumonia. Many factors place patients at risk for aspiration, including "full" stomach, intestinal obstruction, hiatal hernia, obesity, pregnancy, reflux disease, emergency surgery, and inadequate depth of anesthesia. Many approaches are employed to reduce the potential for aspiration perioperatively. Many of these interventions, such as the holding of cricoid pressure (Sellick maneuver) and rapid sequence induction, may only offer limited protection. Cricoid pressure can be applied incorrectly and fail to occlude the esophagus. Whether it has any beneficial effect on outcomes even when it is applied correctly remains unproven. Anesthetic agents can decrease lower esophageal sphincter tone and decrease or obliterate the gag reflex, theoretically increasing the risk for passive aspiration. Additionally, inadequately anesthetized patients can vomit; if the airway is unprotected, aspiration of gastric contents may occur. Different combinations of premedications have been advocated to reduce gastric volume, increase gastric pH, or augment lower esophageal sphincter tone. These agents include antihistamines, antacids, and metoclopramide.

Histamine is found in the central nervous system, in the gastric mucosa, and in other peripheral tissues. It is synthesized by decarboxylation of the amino acid histidine. Histaminergic neurons are primarily located in the posterior hypothalamus but have wide projections in the brain. Histamine also normally plays a major role in the secretion of hydrochloric acid by parietal cells in the stomach (Figure 17-1). The greatest concentrations of histamine are found in the storage granules of circulating basophils and mast cells. Mast cells tend to be concentrated in connective tissue just beneath epithelial (mucosal) surfaces. Histamine release (degranulation) from these cells can be triggered by chemical, mechanical, or immunological stimulation
FIGURE 17-1 Secretion of hydrochloric acid is normally mediated by gastrininduced histamine release from enterochromaffin-like cells (ECL) in the stomach. Note that acid secretion by gastric parietal cells can also be increased indirectly by acetylcholine (AC) via stimulation of M3 receptors and directly by gastrin through an increase in intracellular Ca** concentration. Prostaglandin E, (PGE,) can inhibit acid secretion by decreasing cyclic adenosine monophosphate (cAMP) activity. ATP, adenosine triphosphate; G,, G inhibitory protein; G,, G stimulatory protein.
Multiple receptors (H,—H,) mediate the effects of histamine. The H, receptor activates phospholipase C, whereas the H, receptor increases intracellular cyclic adenosine monophosphate (cAMP). The H3 receptor is primarily located on histamine-secreting cells and mediates negative feedback, inhibiting the synthesis and release of additional histamine. The H, receptors are present on hematopoietic cells, mast cells, and eosinophils and are active in allergy and inflammation. Histamine-N-methyltransferase metabolizes histamine to inactive metabolites that are excreted in the urine.

Histamine reduces arterial blood pressure but increases heart rate and myocardial contractility. H,-Receptor stimulation increases capillary permeability and enhances ventricular irritability, whereas H,-receptor stimulation increases heart rate and increases contractility. Both types of receptors mediate peripheral arteriolar dilation and some coronary vasodilation.

Histamine constricts bronchiolar smooth muscle via the H, receptor. H5Receptor stimulation may produce mild bronchodilation. Histamine has variable effects on the pulmonary vasculature; the H, receptor appears to mediate some pulmonary vasodilation, whereas the H, receptor may be responsible for histamine-mediated pulmonary vasoconstriction.

Activation of H, receptors in parietal cells increases gastric acid secretion. Stimulation of H, receptors leads to contraction of intestinal smooth muscle.

The classic wheal-and-flare response of the skin to histamine results from increased capillary permeability and vasodilation, primarily via H,-receptor activation.

Histamine is a major mediator of type 1 hypersensitivity reactions. H,;-Receptor stimulation attracts leukocytes and induces synthesis of prostaglandin. In contrast, the Hy receptor appears to activate suppressor T lymphocytes.
1. H,-Receptor Antagonists

@ Diphenhydramine (an ethanolamine) is one of a diverse group of drugs that competitively blocks H, receptors (Table 17—1). Many drugs with H,-receptor antagonist properties have considerable antimuscarinic, or atropine-like, activity (eg, dry mouth), or antiserotonergic activity (antiemetic). Promethazine is a phenothiazine derivative with H,-receptor antagonist activity as well as antidopaminergic and a-adrenergic—blocking properties.

(Zyrtec), Dose (mg) = 5-10. Cetirizine (Zyrtec), Duration (h) = 24. Cetirizine (Zyrtec), Sedation = +. Cetirizine (Zyrtec), Antiemesis = . Cyproheptadine (Periactin), Route = PO. Cyproheptadine (Periactin), Dose (mg) = 4. Cyproheptadine (Periactin), Duration (h) = 6-8. Cyproheptadine (Periactin), Sedation = ++. Cyproheptadine (Periactin), Antiemesis = . Fexofenadine (Allegra), Route = PO. Fexofenadine (Allegra), Dose (mg) = 30-60. Fexofenadine (Allegra), Duration (h) = 12. Fexofenadine (Allegra), Sedation = 0. Fexofenadine (Allegra), Antiemesis = . Meclizine (Antivert), Route = PO. Meclizine (Antivert), Dose (mg) = 12.5-50. Meclizine (Antivert), Duration (h) = 8-24. Meclizine (Antivert), Sedation = +. Meclizine (Antivert), Antiemesis = . Loratadine (Claritin), Route = PO. Loratadine (Claritin), Dose (mg) = 10. Loratadine (Claritin), Duration (h) = 24. Loratadine (Claritin), Sedation = 0. Loratadine (Claritin), Antiemesis = 
10, no effect; ++, moderate activity; +++, marked activity.

Like other H,-receptor antagonists, diphenhydramine has a multitude of therapeutic uses: suppression of allergic reactions and symptoms of upper respiratory tract infections (eg, urticaria, rhinitis, conjunctivitis); vertigo, nausea, and vomiting (eg, motion sickness, Méniére disease); sedation; suppression of cough; and dyskinesia (eg, parkinsonism, drug-induced extrapyramidal side effects). Some of these actions are predictable from an understanding of histamine physiology, whereas others are the result of the drugs' antimuscarinic and antiserotonergic effects (Table 17-1). Although H, blockers prevent the bronchoconstrictive response to histamine, they are ineffective in treating bronchial asthma, which is primarily due to other mediators. Likewise, H, blockers will not completely prevent the hypotensive effect of histamine unless an H, blocker is administered concomitantly.
Although many H, blockers cause significant sedation, ventilatory drive is usually unaffected in the absence of other sedative medications. Promethazine and hydroxyzine were often combined with opioids to potentiate analgesia. Newer (second-generation) antihistamines tend to produce little or no sedation because of limited penetration across the blood-brain barrier. This group of drugs is used primarily for allergic rhinitis and urticaria. They include loratadine, fexofenadine, and cetirizine. Many preparations for allergic rhinitis often also contain vasoconstrictors such as pseudoephedrine. Meclizine and dimenhydrinate are used primarily as an antiemetic, particularly for motion sickness, and in the management of vertigo. Cyproheptadine, which also has significant serotonin antagonist activity, has been used in the management of Cushing disease, carcinoid syndrome, and vascular (cluster) headaches.

The usual adult dose of diphenhydramine is 25 to 50 mg (0.5—1.5 mg/kg) orally, intramuscularly, or intravenously every 3 to 6 h. The doses of other H,-receptor antagonists are listed in Table 17-1.

The sedative effects of H,-receptor antagonists can potentiate other central nervous system depressants such as barbiturates, benzodiazepines, and opioids.

H,-Receptor antagonists include cimetidine, famotidine, nizatidine, and ranitidine (Table 17—2). These agents competitively inhibit histamine binding to H, receptors, thereby reducing gastric acid output and raising gastric pH.
TABLE 17-2 Pharmacology of aspiration pneumonia prophylaxis.!

30-60 min. Nonparticulate antacids (Bicitra, Polycitra), Acidity = Wd. Nonparticulate antacids (Bicitra, Polycitra), Volume = it. Nonparticulate antacids (Bicitra, Polycitra), LES Tone = 0. Metoclopramide (Reglan), Route = Vv PO. Metoclopramide (Reglan), Dose = 10mg 10-15mg. Metoclopramide (Reglan), Onset = 1-3min. Metoclopramide (Reglan), Duration = 1-2h 30-60 min?. Metoclopramide (Reglan), Acidity = 0. Metoclopramide (Reglan), Volume = W. Metoclopramide (Reglan), LES Tone = TT
- 0, no effect; 11, moderate decrease; 11, marked decrease; 7, slight increase; TT, moderate increase; LES, lower esophageal sphincter.
?Oral metoclopramide has a quite variable onset of action and duration of action.

All H,-receptor antagonists are equally effective in the treatment of peptic duodenal and gastric ulcers, hypersecretory states (Zollinger—Ellison syndrome), and gastroesophageal reflux disease (GERD). Intravenous preparations are also used to prevent stress ulceration in critically ill patients. Duodenal and gastric ulcers are usually associated with Helicobacter pylori infection, which is treated with combinations of bismuth, tetracycline, and metronidazole. @ By decreasing gastric fluid volume and hydrogen ion content, H, blockers reduce the perioperative risk of aspiration pneumonia. These drugs affect the pH of only those gastric secretions that occur after their administration. The combination of H,and H,-receptor antagonists provides some protection against drug-induced allergic reactions (eg, intravenous radiocontrast, chymopapain injection for lumbar disk disease, protamine, vital blue dyes used for sentinel node biopsy). Although pretreatment with these agents does not reduce histamine release, it may decrease subsequent hypotension.

Rapid intravenous injection of cimetidine or ranitidine has been rarely associated with hypotension, bradycardia, arrhythmias, and cardiac arrest. H»-Receptor antagonists change the gastric flora by virtue of their pH effects. Complications of long-term cimetidine therapy include hepatotoxicity (elevated serum transaminases), interstitial nephritis (elevated serum creatinine), granulocytopenia, and thrombocytopenia. Cimetidine also binds to androgen receptors, occasionally causing gynecomastia and impotence. Finally, cimetidine has been associated with changes in mental status ranging from lethargy and hallucinations to seizures, particularly in elderly patients. In contrast, ranitidine, nizatidine, and famotidine do not affect androgen receptors and penetrate the blood-brain barrier poorly.

As a premedication to reduce the risk of aspiration pneumonia, H,-receptor antagonists should be administered at bedtime and again at least 2 h before surgery. Because all four drugs are eliminated primarily by the kidneys, the dose should be reduced in patients with significant renal dysfunction.

Cimetidine may reduce hepatic blood flow and binds to the cytochrome P-450 mixed-function oxidases. These effects slow the metabolism of a multitude of drugs, including lidocaine, propranolol, diazepam, theophylline, phenobarbital, warfarin, and phenytoin. Ranitidine is a weak inhibitor of the cytochrome P-450 system, and no significant drug interactions have been demonstrated. Famotidine and nizatidine do not appear to affect the cytochrome P-450 system.

Antacids neutralize the acidity of gastric fluid by providing a base (usually hydroxide, carbonate, bicarbonate, citrate, or trisilicate) that reacts with hydrogen ions to form water.

Common uses of antacids include the treatment of peptic ulcers and GERD. In anesthesiology, antacids provide protection against the harmful effects of aspiration pneumonia by raising the pH of gastric contents. Unlike H,-receptor antagonists, antacids have an immediate effect. Unfortunately, they increase intragastric volume. Aspiration of particulate antacids (aluminum or magnesium hydroxide) produces abnormalities in lung function comparable to those that occur following acid aspiration. Nonparticulate antacids (sodium citrate or sodium bicarbonate) are much less damaging to lung alveoli if aspirated. Furthermore, nonparticulate antacids mix with gastric contents better than particulate solutions. Timing is critical, as nonparticulate antacids lose their effectiveness 30 to 60 min after ingestion.

The usual adult dose of a 0.3 M solution of sodium citrate—Bicitra (sodium citrate and citric acid) or Polycitra (sodium citrate, potassium citrate, and citric acid)—is 15 to 30 mL orally, 15 to 30 min prior to induction (Table 17-2).

Because antacids alter gastric and urinary pH, they change the absorption and elimination of many drugs. The rate of absorption of digoxin, cimetidine, and ranitidine is slowed, whereas the rate of phenobarbital elimination is quickened.

Metoclopramide acts peripherally as a cholinomimetic (ie, facilitates acetylcholine transmission at selective muscarinic receptors) and centrally as a dopamine receptor antagonist. Its action as a prokinetic agent in the upper gastrointestinal (GI) tract is not dependent on vagal innervation but is abolished by anticholinergic agents. It does not stimulate secretions.

® By enhancing the stimulatory effects of acetylcholine on intestinal smooth muscle, metoclopramide increases lower esophageal sphincter tone, speeds gastric emptying, and lowers gastric fluid volume (Table 17—2). These properties account for its efficacy in the treatment of patients with diabetic gastroparesis and GERD, as well as prophylaxis for those at risk for aspiration pneumonia. Metoclopramide does not affect the secretion of gastric acid or the pH of gastric fluid.
Metoclopramide produces an antiemetic effect by blocking dopamine receptors in the chemoreceptor trigger zone of the central nervous system. However, at doses used clinically during the perioperative period, the drug's ability to reduce postoperative nausea and vomiting is negligible.

Rapid intravenous injection may cause abdominal cramping, and metoclopramide is contraindicated in patients with complete intestinal obstruction. It can induce a hypertensive crisis in patients with pheochromocytoma by releasing catecholamines from the tumor. Sedation, nervousness, and extrapyramidal signs from dopamine antagonism (eg, akathisia) are uncommon and reversible. Nonetheless, metoclopramide is best avoided in patients with Parkinson disease. Prolonged treatment with metoclopramide can lead to tardive dyskinesia. Metoclopramide-induced increases in aldosterone and prolactin secretion are probably inconsequential during short-term therapy. Metoclopramide may rarely result in hypotension and arrhythmias.

An adult dose of 10 to 15 mg of metoclopramide (0.25 mg/kg) is effective orally, intramuscularly, or intravenously (injected over 5 min). Larger doses (1-2 mg/kg) have been used to prevent emesis during chemotherapy. The onset of action is much more rapid following parenteral (3-5 min) than oral (30-60 min) administration. Because metoclopramide is excreted in the urine, its dose should be decreased in patients with kidney dysfunction.

Antimuscarinic drugs (eg, atropine, glycopyrrolate) block the GI effects of metoclopramide. Metoclopramide decreases the absorption of orally administered cimetidine. Concurrent use of phenothiazines or butyrophenones (droperidol) increases the likelihood of extrapyramidal side effects.

These agents, including omeprazole (Prilosec), lansoprazole (Prevacid), rabeprazole (AcipHex), esomeprazole (Nexium), and pantoprazole (Protonix), bind to the proton pump of parietal cells in the gastric mucosa and inhibit secretion of hydrogen ions.

Proton pump inhibitors (PPIs) are indicated for the treatment of peptic ulcer, GERD, and Zollinger—Ellison syndrome. They may promote healing of peptic ulcers and erosive GERD more quickly than H,-receptor blockers. There are ongoing questions regarding the safety of PPIs in patients taking clopidogrel (Plavix). These concerns relate to inadequate antiplatelet therapy when these drugs are combined due to inadequate activation of clopidogrel by hepatic enzyme CYP2C19, which is inhibited to varying degrees by PPIs.

PPIs are generally well tolerated, causing few side effects. Adverse side effects primarily involve the GI system (nausea, abdominal pain, constipation, diarrhea). On rare occasions, these drugs have been associated with myalgias, anaphylaxis, angioedema, and severe dermatological reactions. Long-term use of PPIs has also been associated with gastric enterochromaffin-like cell hyperplasia and an increased risk of pneumonia secondary to bacterial colonization in the higher-pH environment.

Recommended oral doses for adults are omeprazole, 20 mg; lansoprazole, 15 mg; rabeprazole, 20 mg; and pantoprazole, 40 mg. Because these drugs are primarily eliminated by the liver, repeat doses should be decreased in patients with severe liver impairment.

PPIs can interfere with hepatic P-450 enzymes, potentially decreasing the clearance of diazepam, warfarin, and phenytoin. Concurrent administration can decrease clopidogrel (Plavix) effectiveness, as the latter medication is dependent on hepatic enzymes for activation.

Without any prophylaxis, PONV occurs in approximately 30% or more of the general surgical population and up to 70% to 80% in patients with predisposing risk factors. The Society for Ambulatory Anesthesia (SAMBA) provides extensive guidelines for the management of PONV. Table 17—3 identifies risks factors for PONV and scores the evidence for assessing risk. When PONV risk is sufficiently great, prophylactic antiemetic medications are administered and strategies to reduce its incidence are initiated. The Apfel score provides a simplified assessment tool to predict risk of PONV (Figures 17—2 and 17-3). (Obesity, anxiety, and reversal of neuromuscular blockade are not independent risk factors for PONV.)
TABLE 17-3 Risk factors for PONV.!3
Positive overall, Risk factors = Female sex (B1) History of PONV or motion sickness (B1) Nonsmoking (B1) Younger age (B1) General versus regional anesthesia (A1) Use of volatile anesthetics and nitrous oxide (A1) Postoperative opiods (A1) Duration of anesthesia (B1) Type of surgery (cholecystectomy, laparoscopic, gynecological) (B1). Conflicting, Risk factors = ASA physical status (B1) Menstrual cycle (B1) Level of anesthetist’s experience (B1) Muscle relaxant antagonists (A2). Disproven or of limited clinical relevance, Risk factors = BMI (B1) Anxiety (B1) Nasogastric tube (A1) Supplemental oxygen (A1) Perioperative fasting (A2) Migraine (B1)
'Reproduced with permission from Gan TJ, Diemunsch P Habib AS, et al. Consensus guidelines for the management of postoperative nausea and vomiting. Anesth Analg. 2014 Jan;118(1):85-113.
"ASA, American Society of Anesthesiolagists; BMI, body mass index; MO, motion sickness; PONV, postoperative nausea and vomiting.

'Risk assessment scoring: Al, randomized trials with supportive meta-analyses; A2, randomized trials but of insufficient number for a meta-analysis; B1, observational studies such as case control or cohort designs.
Female gender, | Points | = . Nonsmoker, | Points | = . History of PONV, | Points | = . Postoperative opioids, | Points | = . Sum =, | Points | = -
FIGURE 17-2 Risk score for PONV in adults. Simplified risk score from Apfel et al to predict the patient's risk for PONV. When 0, 1, 2, 3, and 4 of the risk factors are present, the corresponding risk for PONV is about 10%, 20%, 40%, 60%, and 80%, respectively. PONV, postoperative nausea and vomiting. (Reproduced with permission from Gan TJ, Diemunsch P, Habib A, et al. Consensus guidelines for the management of postoperative nausea and vomiting. Anesth Analg. 2014 Jan;118(1):85-113.)
Surgery = 30 min., | Points | = . Age = 3 years, | Points | = . : Strabismus surgery, | Points | = . History of POV or PONV in relatives, | Points | = . Sum =, | Points | = 
FIGURE 17-3 Simplified risk score for POV in children. Simplified risk score from Eberhart et al to predict the risk for POV in children. When 0, 1, 2, 3, or 4 of the depicted independent predictors are present, the corresponding risk for PONV is approximately 10%, 10%, 30%, 50%, or 70%, respectively. POV, postoperative vomiting; PONV, postoperative nausea and vomiting. (Reproduced with permission from Gan TJ, Diemunsch P, Habib A, et al. Consensus guidelines for the management of postoperative nausea and vomiting. Anesth Analg. 2014 Jan;118(1):85-113.)

Drugs used in the prophylaxis and treatment of PONV include 5-HT3 antagonists, butyrophenones, dexamethasone, neurokinin-1 receptor antagonists (aprepitant); antihistamines and transdermal scopolamine may also be used. Atrisk patients often benefit from one or more prophylactic measures. Because all

FIGURE 17-4 Algorithm for management of postoperative nausea and vomiting. PACU, postanesthesia care unit; PONV, postoperative nausea and vomiting; POV, postoperative vomiting. (Reproduced with permission from Gan TJ,
Diemunsch P, Habib A, et al. Consensus guidelines for the management of postoperative nausea and vomiting. Anesth Analg. 2014 Jan;118(1):85-113.)

Serotonin, 5-hydroxytryptamine (5-HT), is present in large quantities in platelets and the GI tract (enterochromaffin cells and the myenteric plexus). It is also an important neurotransmitter in multiple areas of the central nervous system. Serotonin is formed by hydroxylation and decarboxylation of tryptophan. Monoamine oxidase inactivates serotonin into 5-hydroxyindoleacetic acid (5HIAA). The physiology of serotonin is very complex because there are at least seven receptor types, most with multiple subtypes. The 5-HT3 receptor mediates vomiting and is found in the GI tract and the brain (area postrema). The 5-HT5, receptors are responsible for smooth muscle contraction and platelet aggregation, the 5-HT, receptors in the GI tract mediate secretion and peristalsis, and the 5HT, and 5-HT, receptors are located primarily in the limbic system where they appear to play a role in depression. All except the 5-HT3 receptor are coupled to G proteins and affect either adenylyl cyclase or phospholipase C; effects of the 5-HT3 receptor are mediated via an ion channel.

Except in the heart and skeletal muscle, serotonin is a powerful vasoconstrictor of arterioles and veins. Its vasodilator effect in the heart is endothelium dependent. When the myocardial endothelium is damaged following injury, serotonin produces vasoconstriction. The pulmonary and kidney vasculatures are very sensitive to the arterial vasoconstrictive effects of serotonin. Modest and transient increases in cardiac contractility and heart rate may occur immediately following serotonin release; reflex bradycardia often follows. Vasodilation in skeletal muscle can subsequently cause hypotension. Excessive serotonin can produce serotonin syndrome, characterized by hypertension, hyperthermia, and agitation.

Contraction of smooth muscle increases airway resistance. Bronchoconstriction from released serotonin is often a prominent feature of carcinoid syndrome

Direct smooth muscle contraction (via 5-HT, receptors) and serotonin-induced release of acetylcholine in the myenteric plexus (via 5-HT3 receptors) greatly augment peristalsis. Secretions are unaffected.

@ Ondansetron, granisetron, tropisetron, and dolasetron selectively block serotonin 5-HT3 receptors, with little or no effect on dopamine receptors (5-HT3 receptors, which are located peripherally (abdominal vagal afferents) and centrally (chemoreceptor trigger zone of the area postrema and the nucleus tractus solitarius), appear to play an important role in the initiation of the vomiting reflex. The 5-HT3 receptors of the chemoreceptor trigger zone in the area postrema reside outside the blood-brain barrier (Figure 17—5). The chemoreceptor trigger zone is activated by substances such as anesthetics and opioids and signals the nucleus tractus solitarius, resulting in PONV. Emetogenic stimuli from the GI tract similarly stimulate the development of PONV.
FIGURE 17-5 Neurological pathways involved in pathogenesis of nausea and vomiting (see text). (Reproduced with permission from Krakauer EL, Zhu AX, Bounds BC, et al. Case records of the Massachusetts General Hospital. Weekly clinicopathological exercises. Case 6-2005. A 58-year-old man with esophageal cancer and nausea, vomiting, and intractable hiccups, N Engl J Med. 2005 Feb 24;352(8):817-825.)

5-HT3-Receptor antagonists are generally administered at the end of surgery. All these agents are effective antiemetics in the postoperative period. A new agent, palonosetron has an extended duration of action and may reduce the incidence of postdischarge nausea and vomiting (PDNV). SAMBA guidelines suggest risk factors for PDNV include:
¢ Female sex
« History of PONV
¢ Age 50 years or younger
¢ Use of opioids in the postanesthesia care unit (PACU)
¢ Nausea in the PACU

5-HT3-Receptor antagonists are essentially devoid of serious side effects, even in amounts several times the recommended dose. They do not appear to cause sedation, extrapyramidal signs, or respiratory depression. The most commonly reported side effect is headache. All three drugs can slightly prolong the QT interval on the electrocardiogram. This effect may be more frequent with dolasetron (no longer available in the United States). Nonetheless, these drugs, should be used cautiously in patients who are taking antiarrhythmic drugs or who have a prolonged QT interval.
Ondansetron undergoes extensive metabolism in the liver via hydroxylation and conjugation by cytochrome P-450 enzymes. Liver failure impairs clearance several-fold, and the dose should be reduced accordingly.

Droperidol (0.625—1.25 mg) was previously used routinely for PONV prophylaxis. Given at the end of the procedure, it blocks dopamine receptors that contribute to the development of PONV. Despite its effectiveness, many practitioners no longer routinely administer this medication because of a U.S. Food and Drug Administration (FDA) black box warning related to concerns that doses described in the product labeling ("package insert") may lead to QT prolongation and development of torsades des pointes arrhythmia. However, the doses relevant to the FDA warning, as acknowledged by the FDA, were those used for neurolept anesthesia (5-15 mg), not the much smaller doses employed for PONV. Cardiac monitoring is warranted when large doses of the drug are used. There is no evidence that use of droperidol at the doses routinely employed for PONV management increases the risk of sudden cardiac death in the perioperative population.
As with other drugs that antagonize dopamine, droperidol use in patients with Parkinson disease and in other patients manifesting extrapyramidal signs should be carefully considered.
The phenothiazine, prochlorperazine (Compazine), which affects multiple receptors (histaminergic, dopaminergic, muscarinic), may be used for PONV management. It may cause extrapyramidal and anticholinergic side effects. Promethazine (Phenergan) works primarily as an anticholinergic agent and antihistamine and likewise can be used to treat PONV. As with other agents of this class, anticholinergic effects (sedation, delirium, confusion, vision changes) can complicate the postoperative period.

Dexamethasone (Decadron) in doses as small as 4 mg has been shown to be as effective as ondansetron in reducing the incidence of PONV. Dexamethasone should be given at induction as opposed to the end of surgery, and its mechanism of action is unclear. It may provide analgesic and mild euphoric effects. Dexamethasone can increase postoperative blood glucose concentration, and some practitioners have suggested that dexamethasone could increase the risk of postoperative infection. Nonetheless, most studies have not demonstrated any increase in wound infections following dexamethasone administration for PONV prophylaxis.

Substance P is a neuropeptide that interacts at neurokinin-1 (NK,) receptors. NK, antagonists inhibit substance P at central and peripheral receptors. Aprepitant, an NK, antagonist, has been found to reduce PONV perioperatively and is additive with ondansetron for this indication.

Several other agents and techniques have been employed to reduce the incidence of PONV. Transdermal scopolamine has been used effectively, although it may produce central anticholinergic effects (confusion, blurred vision, and dry mouth). Acupuncture, acupressure, and transcutaneous electrical stimulation of the P6 acupuncture point can reduce PONV incidence and medication requirements.
As no single agent will both treat and prevent PONV, perioperative management centers on identifying patients at greatest risk so that prophylaxis, often with multiple agents, may be initiated. Since systemic opioid administration is associated with PONV, opioid-sparing strategies (eg, use of regional anesthetics and nonopioid analgesics) can markedly reduce the risk of PONV.

® Ketorolac is a parenteral nonsteroidal antiinflammatory drug (NSAID) that provides analgesia by inhibiting prostaglandin synthesis. A peripherally acting drug, it has become a popular alternative to opioids for postoperative analgesia because of its minimal central nervous system side effects.

Ketorolac is indicated for the short-term (<5 days) management of pain, and appears to be particularly useful in the immediate postoperative period. A standard dose of ketorolac provides analgesia equivalent to 6 to 12 mg of morphine administered by the same route. Its time to onset is also similar to morphine, but ketorolac has a longer duration of action (6-8 h). Ketorolac does not cause respiratory depression, sedation, or nausea and vomiting. In fact, ketorolac does not cross the blood-brain barrier to any significant degree. Numerous studies have shown that oral and parenteral NSAIDs have an opioid-sparing effect. They may be most beneficial in patients at increased risk for postoperative respiratory depression or emesis.

As with other NSAIDs, ketorolac inhibits platelet aggregation and prolongs bleeding time. It and other NSAIDs should therefore be used with caution in patients at risk for postoperative hemorrhage. Long-term administration may lead to renal toxicity (eg, papillary necrosis) or GI tract ulceration with bleeding and perforation. Because ketorolac depends on kidney elimination, it should not be given to patients with kidney disease. Ketorolac is contraindicated in patients allergic to aspirin or NSAIDs. Patients with asthma have an increased incidence of aspirin sensitivity (approximately 10%), particularly if they also have a history of nasal polyps (approximately 20%).

Ketorolac has been approved for administration as either a 60 mg intramuscular or 30 mg intravenous loading dose; a maintenance dose of 15 to 30 mg every 6h is recommended. Elderly patients clear ketorolac more slowly and should receive reduced doses.

Aspirin decreases the protein binding of ketorolac, increasing the amount of active unbound drug. Ketorolac does not affect minimum alveolar concentration of inhalation anesthetic agents, and its administration does not alter the hemodynamics of anesthetized patients. It decreases the postoperative requirement for opioid analgesics.

Clonidine is an imidazoline derivative with predominantly a-adrenergic agonist activity. It is highly lipid soluble and readily penetrates the blood-brain barrier and the placenta. Studies indicate that binding of clonidine to receptors is highest in the rostral ventrolateral medulla in the brainstem (the final common pathway for sympathetic outflow), where it activates inhibitory neurons. The overall effect is to decrease sympathetic activity, enhance parasympathetic tone, and reduce circulating catecholamines. There is also evidence that some of clonidine's antihypertensive action may occur via binding to a nonadrenergic (imidazoline) receptor. In contrast, its analgesic effects, particularly in the spinal cord, are mediated entirely via preand possibly postsynaptic a-adrenergic receptors that block nociceptive transmission. Clonidine also has local anesthetic effects when applied to peripheral nerves and is frequently added to local anesthetic solutions to increase duration of action.

@ Clonidine is a commonly used antihypertensive agent that reduces sympathetic tone, decreasing systemic vascular resistance, heart rate, and blood pressure. In anesthesia, clonidine is used as an adjunct for epidural, caudal, and peripheral nerve block anesthesia and analgesia. It is often used in the management of patients with chronic neuropathic pain to increase the efficacy of epidural opioid infusions. When given epidurally, the analgesic effect of clonidine is segmental, being localized to the level at which it is injected or infused. When added to local anesthetics of intermediate duration (eg, mepivacaine or lidocaine) administered for epidural or peripheral nerve block, clonidine will markedly prolong both the anesthetic and analgesic effects. Unlabeled/investigational uses of clonidine include serving as an adjunct in premedication, control of withdrawal syndromes (nicotine, opioids, alcohol, and vasomotor symptoms of menopause), and treatment of glaucoma as well as various psychiatric disorders.

Sedation, dizziness, bradycardia, and dry mouth are common side effects. Less commonly, orthostatic hypotension, nausea, and diarrhea may be observed. Abrupt discontinuation of clonidine following long-term administration (>1 month) can produce a withdrawal phenomenon characterized by rebound hypertension, agitation, and sympathetic overactivity.

Epidural clonidine is usually started at 30 mcg/h in a mixture with an opioid or a local anesthetic. Oral clonidine is readily absorbed, has a 30 to 60 min onset, and lasts 6 to 12 h. In the initial treatment of hypertension, 0.1 mg can be given two times a day and adjusted until the blood pressure is controlled. The maintenance dose typically ranges from 0.1 to 0.3 mg twice daily. Transdermal preparations of clonidine can also be used for maintenance therapy. They are available as 0.1, 0.2, and 0.3 mg/d patches that are replaced every 7 days. Clonidine is metabolized by the liver and excreted by the kidney. Dosages should be reduced for patients with kidney disease.

Clonidine enhances and prolongs sensory and motor blockade from local anesthetics. Additive effects with hypnotic agents, general anesthetics, and sedatives can potentiate sedation, hypotension, and bradycardia. The drug should be used cautiously, if at all, in patients who take B-adrenergic blockers and in those with significant cardiac conduction system abnormalities. Lastly, clonidine can mask the symptoms of hypoglycemia in diabetic patients.

@ Dexmedetomidine is a parenteral selective a, agonist with sedative properties. It appears to be more selective for the a, receptor than clonidine. At higher doses, it loses its selectivity and also stimulates a-adrenergic receptors.

Dexmedetomidine causes dose-dependent sedation, anxiolysis, some analgesia, and blunting of the sympathetic response to surgery and to other stress. Most importantly, it has an opioid-sparing effect and does not significantly depress respiratory drive; excessive sedation, however, may cause airway obstruction. The drug can be used for short-term (<24 h), intravenous sedation of mechanically ventilated patients. Discontinuation after more prolonged use can potentially cause a withdrawal phenomenon similar to that of clonidine. It is also used for intraoperative sedation and as an adjunct to general anesthetics.
The principal side effects are bradycardia, heart block, and hypotension. It may also cause nausea.

The recommended initial loading dose is 1 mcg/kg intravenously over 10 min with a maintenance infusion rate of 0.2 to 0.7 mcg/kg/h. Dexmedetomidine has a rapid onset and terminal half-life of 2 h. The drug is metabolized in the liver and its metabolites are eliminated in the urine. Dosage should be reduced in patients with liver or kidney disease.

Caution should be used when dexmedetomidine is administered with vasodilators, cardiac depressants, and drugs that decrease heart rate. Reduced requirements of hypnotics/anesthetic agents should prevent excessive hypotension.

Gabapentin was initially employed as an anticonvulsant. Gabapentin and pregabalin act by blocking voltage-gated calcium channels, resulting in diminished release of glutamate. Various studies have demonstrated that both drugs may reduce perioperative opioid consumption when included in multimodal pain management. Gabapentin may be given to adults as a 600 mg preemptive dose prior to surgery and continued postoperatively (1200 mg/d in divided doses). These agents are also commonly used for management of chronic (particularly neuropathic) pain syndromes.

Capsaicin is a TRPV1-receptor agonist. It depletes substance P and inhibits pain signal transmission. Infiltration of capsaicin in surgical wounds reduces opioid consumption and improves perioperative analgesia.

Doxapram is a peripheral and central nervous system @ stimulant. Selective activation of carotid chemoreceptors by low doses of doxapram stimulates hypoxic drive, producing an increase in tidal volume and a slight increase in respiratory rate. At larger doses, respiratory centers in the medulla are stimulated.

Doxapram is not a specific reversal agent and should not replace standard supportive therapy (ie, mechanical ventilation). Drug-induced respiratory and central nervous system depression, including that seen immediately postoperatively, can be temporarily overcome. Doxapram will not reverse paralysis caused by muscle relaxants and will not alleviate airway obstruction.

Stimulation of the central nervous system leads to a variety of possible side effects: changes in mental status (confusion, dizziness, seizures), cardiac abnormalities (tachycardia, dysrhythmias, hypertension), and pulmonary dysfunction (wheezing, tachypnea). Vomiting and laryngospasm are of particular concern to the anesthesiologist in the postoperative period. Doxapram should not be used in patients with a history of epilepsy, cerebrovascular disease, acute head injury, coronary artery disease, hypertension, or bronchial asthma.

Bolus intravenous administration (0.5—-1 mg/kg) results in transient increases in minute ventilation (the onset of action is 1 min; the duration of action is 5-12 min). Continuous intravenous infusions (1-3 mg/min) provide longer-lasting effects (the maximum dose is 4 mg/kg).

The sympathetic stimulation produced by doxapram may exaggerate the cardiovascular effects of monoamine oxidase inhibitors or adrenergic agents.

Mechanism of Action
Naloxone is a competitive opioid receptor antagonist. Its affinity for opioid 1 receptors appears to be much greater than for opioid x or 6 receptors Naloxone has no significant agonist activity.

@ Naloxone reverses the agonist activity associated with endogenous (enkephalins, endorphins) or exogenous opioid compounds. A dramatic example is the reversal of unconsciousness that occurs in a patient with opioid overdose who has received naloxone. Thus, naloxone is widely available for first responders and relatives of those who abuse opioids. Perioperative respiratory depression caused by opioids is rapidly antagonized (1-2 min). Some degree of opioid analgesia can often be spared if the dose of naloxone is limited to the minimum required to maintain adequate ventilation (40-80 mcg intravenously in adults, repeated as needed). Small doses of intravenous naloxone reverse the side effects of spinal or epidural opioids without necessarily reversing the analgesia.

Abrupt, complete reversal of opioid analgesia can result in a surge of sympathetic stimulation (tachycardia, ventricular irritability, hypertension, pulmonary edema) caused by severe, acute pain, and an acute withdrawal syndrome in patients who are opioid-dependent.

In postoperative patients experiencing respiratory depression from excessive opioid administration, intravenous naloxone (0.4 mg/mL vial diluted in 9 mL saline to 0.04 mg/mL) can be titrated in increments of 0.5 to 1 mcg/kg every 3 to 5 min until adequate ventilation and alertness are achieved. Doses in excess of 200 mcg are rarely needed. The brief duration of action of intravenous naloxone (30-45 min) is due to rapid redistribution from the central nervous system. A more prolonged effect will be necessary to prevent the recurrence of respiratory depression from longer-acting opioids. Therefore, intramuscular naloxone (twice the required intravenous dose) or a continuous naloxone infusion is recommended. Naloxone may precipitate symptoms of withdrawal in infants of opioid-exposed mothers.

The effect of naloxone on nonopioid anesthetic agents such as nitrous oxide or clonidine is insignificant.

Naltrexone is also a pure opioid antagonist with a high affinity for the 1: receptor, but with a significantly longer half-life than naloxone. Naltrexone is used orally for maintenance treatment of addiction. Chapter 48 reviews the use of the peripherally acting opioid receptor antagonists alvimopan and methylnaltrexone in the management and prevention of postoperative ileus as an element of enhanced perioperative recovery.

Flumazenil, an imidazobenzodiazepine, is a specific and competitive antagonist of benzodiazepines at benzodiazepine receptors.

® Flumazenil is useful in the reversal of benzodiazepine sedation and the treatment of benzodiazepine overdose. Although it promptly (onset <1 min) reverses the hypnotic effects of benzodiazepines, amnesia has proved to be less reliably prevented. Some evidence of respiratory depression may linger despite an alert and awake appearance. Specifically, tidal volume and minute ventilation return to normal, but the slope of the carbon dioxide response curve remains depressed. Effects in elderly patients appear to be particularly difficult to reverse fully, and these patients are more prone to relapse of sedation.

Rapid administration of flumazenil may cause anxiety reactions in previously sedated patients and symptoms of withdrawal in those on long-term benzodiazepine therapy. Flumazenil reversal has been associated with increases in intracranial pressure in patients with head injuries and abnormal intracranial compliance. Flumazenil may induce seizure activity if benzodiazepines have been given as anticonvulsants or in conjunction with an overdose of tricyclic antidepressants. Flumazenil reversal following a midazolam—ketamine anesthetic technique may increase the incidence of emergence dysphoria and hallucinations. Nausea and vomiting are not uncommon following administration of flumazenil. The reversal effect of flumazenil is based on its strong antagonist affinity for benzodiazepine receptors. Flumazenil does not affect the minimum alveolar concentration of inhalation anesthetics.

Gradual titration of flumazenil is usually accomplished by intravenous administration of 0.2 mg/min until reaching the desired degree of reversal. The usual total dose is 0.6 to 1.0 mg. Because of flumazenil's rapid hepatic clearance, repeat doses may be required after 1 to 2 h to avoid re-sedation and premature recovery room or outpatient discharge. Liver failure prolongs the clearance of flumazenil and benzodiazepines.

Management of Patients at Risk for Aspiration Pneumonia A 58-year-old man is scheduled for elective laparoscopic cholecystectomy. His past history reveals a persistent problem with heartburn and passive regurgitation of gastric contents into the pharynx. He has been told by his internist that these symptoms are due to a hiatal hernia.
Why would a history of hiatal hernia concern the

Perioperative aspiration of gastric contents (Mendelson syndrome) is a potentially fatal complication of anesthesia. Hiatal hernia is commonly associated with symptomatic GERD, which is considered a predisposing factor for aspiration. Mild or occasional heartburn may not significantly increase the risk of aspiration. In contrast, symptoms related to passive reflux of gastric fluid, such as acid taste or sensation of refluxing liquid into the mouth, should alert the clinician to a high risk of pulmonary aspiration. Paroxysms of coughing or wheezing, particularly at night or when the patient is flat, may be indicative of chronic aspiration. Aspiration can occur on induction, during maintenance, or upon emergence from anesthesia.

Patients with altered airway reflexes (eg, drug intoxication, general anesthesia, encephalopathy, neuromuscular disease) or abnormal pharyngeal or esophageal anatomy (eg, large hiatal hernia, Zenker diverticulum, scleroderma, pregnancy, obesity, history of esophagectomy) are prone to pulmonary aspiration.

Not necessarily. The seriousness of the lung damage depends on the volume and composition of the aspirate. Traditionally, patients are considered to be at risk if their gastric volume is greater than 25 mL (0.4 mL/kg) and their gastric pH is less than 2.5. Some investigators believe that controlling acidity is more important than volume and that the criteria should be revised to a pH less than 3.5 with a volume greater than 50 mL.
Patients who have eaten immediately prior to emergency surgery are obviously at risk. Traditionally, "NPO after midnight" implied a preoperative fast of at least 6 h. Current opinion allows clear liquids until 2 h before induction of anesthesia. According to the American Society of Anesthesiologists (ASA) guideline, breast milk is permitted up to 4 h before anesthesia. Infant formula, nonhuman milk and a light meal are permitted up to 6 h before induction. Patients consuming a heavy meal including meat, fats, and fried foods should fast for 8 h. Certain patient populations are particularly likely to have large volumes of acidic gastric fluid: patients with an acute abdomen or peptic ulcer disease, children, the elderly, diabetic patients, pregnant women, and obese patients. Furthermore, pain, anxiety, or opioids may delay gastric emptying. Note that pregnancy and obesity place patients in double jeopardy by increasing the chance of aspiration (increased intraabdominal pressure and distortion of the lower esophageal sphincter) and the risk of aspiration pneumonia (increased acidity and volume of gastric contents). Aspiration is more common in patients undergoing esophageal, upper abdominal, or emergency laparoscopic surgery.
Which drugs lower the risk of aspiration pneumonia?
H,-Receptor antagonists decrease gastric acid secretion. Although they will not affect gastric contents already in the stomach, they will inhibit further acid production. Both gastric pH and volume are affected. In addition, the long duration of action of ranitidine and famotidine may provide protection in the recovery room.

Metoclopramide shortens gastric emptying time and increases lower esophageal sphincter tone. It does not affect gastric pH, and it cannot clear large volumes of food in a few hours. Nonetheless, metoclopramide with ranitidine is a good combination for most at-risk patients. Antacids usually raise gastric fluid pH, but, at the same time, they increase gastric volume. Although antacid administration technically removes a patient from the atrisk category, aspiration of a substantial volume of particulate matter will lead to serious physiological damage. For this reason, clear antacids (eg, sodium citrate) are employed. In contrast to H, antagonists, antacids are immediately effective and alter the acidity of existing gastric contents. Thus, they are useful in emergency situations and in patients who have recently eaten.
Anticholinergic drugs, particularly glycopyrrolate, decrease gastric secretions if large doses are administered; however, lower esophageal sphincter tone is reduced. Overall, anticholinergic drugs do not reliably reduce the risk of aspiration pneumonia and can reverse the protective effects of metoclopramide. Proton pump inhibitors are generally as effective as H, antagonists.
The ASA guideline recommends that prophylaxis against gastric content aspiration be undertaken only in at-risk patients.

If the full stomach is due to recent food intake and the surgical procedure is elective, the operation should be postponed. If the risk factor is not reversible (eg, large hiatal hernia) or the case is emergent, proper anesthetic technique can minimize the risk of aspiration pneumonia. Regional anesthesia with minimal sedation should be considered in patients at increased risk for aspiration pneumonia. If local anesthetic techniques are impractical, the patient's airway must be protected. Delivering anesthesia by mask or laryngeal mask airway is contraindicated. As in every anesthetic case, the availability of suction must be confirmed before induction. A rapid-sequence induction (or, depending upon airway examination, an awake intubation) is indicated.

What are the relative contraindications to rapid-sequence inductions?
Rapid-sequence inductions are often associated with increases in intracranial pressure, arterial blood pressure, and heart rate.
Describe the pathophysiology and clinical findings associated with aspiration pneumonia.
The pathophysiological changes depend on the composition of the aspirate. Acid solutions cause atelectasis, alveolar edema, and loss of surfactant. Particulate aspirate will also result in small-airway obstruction and alveolar necrosis. Granulomas may form around food or antacid particles. The earliest physiological change following aspiration is intrapulmonary shunting, resulting in hypoxia. Other changes may include pulmonary edema, pulmonary hypertension, and hypercapnia.
Wheezing, rhonchi, tachycardia, and tachypnea are common physical findings. Decreased lung compliance can make ventilation difficult. Hypotension signals significant fluid shifts into the alveoli and is associated with massive lung injury. Chest roentgenography may not demonstrate diffuse bilateral infiltrates for several hours after the event. Arterial blood gases reveal hypoxemia, hypercapnia, and respiratory acidosis.

As soon as regurgitation is suspected, the patient should be placed in a head-down position so that gastric contents drain out of the mouth instead of into the trachea. The pharynx and, if possible, the trachea should be thoroughly suctioned. The mainstay of therapy in patients who subsequently become hypoxic is positive-pressure ventilation. Intubation and the institution of positive end-expiratory pressure, or noninvasive ventilation, may be required. Bronchoscopy and pulmonary lavage are usually indicated when particulate aspiration has occurred. Use of corticosteroids is generally not recommended, and antibiotics are administered depending upon culture results.
Gan TJ, Diemunsch P, Habib A, et al. Consensus guidelines for the management of postoperative nausea and vomiting. Anesth Analg. 2014;118:85. Practice guidelines for preoperative fasting and the use of pharmacologic agents to reduce the risk of pulmonary aspiration: Application to healthy patients undergoing elective procedures. An updated report by the American Society of Anesthesiologists Committee on Standards and Practice Parameters. Anesthesiology. 2011;114:495.

Young A, Buvanendran A. Multimodal systemic and intra articular analgesics. Int Anesth Clin. 2011;49:117.
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@ The cornerstones of an effective preoperative or preprocedure evaluation are the history and physical examination, which should include a complete and up-to-date listing of all medications taken by the patient in the recent past, all pertinent allergies, and responses and reactions to previous anesthetics.
@ The anesthesiologist should not be expected to provide the risk-versusbenefit discussion for the proposed surgery or procedure; this is the responsibility and purview of the responsible surgeon or "Droceduralist."
© By convention physicians in many countries use the American Society of Anesthesiologists' classification to identify relative risk prior to conscious sedation and surgical anesthesia.
@ In general, the indications for cardiovascular investigations are the same in elective surgical patients as in any other patient with a similar medical condition.
@ Adequacy of long-term blood glucose control can be easily and rapidly assessed by measurement of hemoglobin A,,.
@ Inpatients deemed at high risk for thrombosis (eg, those with certain mechanical heart valve implants or with atrial fibrillation and a prior thromboembolic stroke), chronic anticoagulants should be replaced by intramuscular low-molecular-weight heparins or by intravenous unfractionated heparin.
@ Current guidelines recommend postponing all but mandatory emergency surgery until at least 1 month after any coronary intervention and suggest that treatment options other than a drug-eluting stent (which will require prolonged dual antiplatelet therapy) be used in patients expected to undergo a surgical procedure within 12 months after the intervention.
© There are no good data to support restricting fluid intake (of any kind or any amount) more than 2 h before induction of general anesthesia in healthy patients undergoing elective procedures; moreover, there is strong evidence that nondiabetic patients who drink fluids containing carbohydrates and protein up to 2 h before induction of anesthesia suffer less perioperative nausea and dehydration than those who are fasted longer.
@ To be valuable, preoperative testing must discriminate: There must be an avoidable increased perioperative risk when the results are abnormal (and the risk will remain unknown if the test is not performed), and when testing fails to detect the abnormality (or it has been corrected), there must be reduced risk.

@® The utility of a test depends on its sensitivity and specificity. Sensitive tests have a low rate of false-negative results and rarely fail to identify an abnormality when one is present, whereas specific tests have a low rate of false-positive results and rarely identify an abnormality when one is not present.
Premedication should be given purposefully, not as a mindless routine. 6
Incomplete, inaccurate, or illegible records unnecessarily complicate defending a physician against otherwise unjustified allegations of malpractice. 8

@ The comerstones of an effective preoperative or preprocedure evaluation are the medical history and physical examination, which should include a complete and up-to-date listing of all medications taken by the patient in the recent past, all pertinent allergies, and responses and reactions to previous anesthetics. Additionally, this evaluation may include diagnostic tests, imaging procedures, or consultations from other physicians when indicated. A patient's initial contact with a perioperative surgical home or enhanced recovery after surgery (ERAS)
TABLE 18-1 The anesthetic plan.

General!
Airway management
Induction drugs
Maintenance drugs
Regional
Technique(s)
Agent(s)
Sedation and monitored anesthesia care
Supplemental oxygen
Specific sedative drugs

Nonstandard monitors
Positions other than supine
Relative or absolute contraindications to specific anesthetic drugs
Fluid management
Special techniques
Site (anesthetizing location) concerns

Management of acute pain
Intensive care
Postoperative ventilation
Hemodynamic monitoring
'Including need for (or need for avoidance of) muscle relaxation.
Another purpose of the preoperative evaluation is to provide the patient with an estimate of anesthetic @ risk. However, the anesthesiologist should not be expected to provide the risk-versus-benefit discussion for the proposed surgery or procedure; this is the responsibility and purview of the responsible surgeon or "proceduralist." For example, a discussion of the risks and benefits of robotassisted laparoscopic prostatectomy versus "open" prostatectomy, radiation therapy, or "watchful waiting" requires detailed knowledge of the current medical literature and the capabilities of an individual surgeon. Finally, the preoperative evaluation presents an opportunity for the anesthesiologist to describe the proposed anesthetic plan in the context of the overall surgical and postoperative plan, provide the patient with psychological support, and obtain informed consent for the proposed anesthetic plan from the surgical patient. @By convention, physicians in many countries use the American Society of Anesthesiologists' (ASA) physical status classification to define relative risk prior to conscious sedation or surgical anesthesia (Table 18-2). The ASA physical status classification has many advantages: it is time tested, simple, and reproducible, and, most importantly, it has been shown to be strongly associated with perioperative risk. However, many other risk assessment tools are available, particularly in the area of cardiovascular risk assessment (see Chapter 21).

1, Definition = Normal healthy patient. 2, Definition = Patient with mild systemic disease (no functional limitations). 3, Definition = Patient with severe systemic disease (some functional limitations). a, Definition = Patient with severe systemic disease that is a constant threat to life (functionality incapacitated). 5, Definition = Moribund patient who is not expected to survive without the operation. 6, Definition = Brain-dead patient whose organs are being removed for donor purposes. E, Definition = If the procedure is an emergency, the physical status is followed by “E” (eg, “2E”)
'Data from Committee on Standards and Practice Parameters, Apfelbaum JL, Connis RT, et al. Practice advisory for preanesthesia evaluation: An updated report by the American Society of Anesthesiologists Task Force on Preanesthesia Evaluation, Anesthesiology. 2012 Mar;116(3):522-538.

Patients presenting for elective surgery and anesthesia typically require the recording of a focused medical history emphasizing abnormalities of exercise tolerance; nutritional and functional status; cardiac, pulmonary, endocrine, kidney, or liver function; electrolytes or metabolism; and anatomic issues relevant to airway management or regional anesthesia. How the patient responded to and recovered from previous anesthetics can be helpful. The ASA
and other societies publish and periodically update general guidelines for preoperative assessment (see Guidelines at end of chapter).

Guidelines for preoperative cardiac assessment are regularly updated and available from the American College of Cardiology/American Heart Association and from the European Society of Cardiology (see Guidelines). A more complete discussion of cardiovascular assessment is provided in Chapter 21. The focus of preoperative cardiac assessment should be on determining whether the patient would benefit from further cardiac evaluation or interventions prior to the scheduled surgery. However, the same approach is not appropriate for all patients. The prudent approach to a patient undergoing elective knee arthroplasty will differ from that for a patient needing resection of pancreatic cancer, given the benign results of a delay in the former procedure and the possible deadly effects of a delay in the latter @ procedure. In general, the indications for cardiovascular investigations are the same in elective surgical patients as in any other patient with a similar medical condition. Put another way, the fact that a patient is scheduled to undergo elective surgery does not change the indications for testing to diagnose coronary artery disease.

Perioperative pulmonary complications, most notably postoperative respiratory depression and respiratory failure, are vexing problems associated with obesity and obstructive sleep apnea. A guideline developed by the American College of Physicians identifies patients 60 years of age or older and those with chronic obstructive lung disease, with markedly reduced exercise tolerance, with functional dependence, or with heart failure as potentially requiring preoperative and postoperative interventions to avoid respiratory complications. The risk of postoperative respiratory complications is closely associated with these factors, and with the following: ASA physical status 3 and 4, cigarette smoking, surgeries lasting longer than 4 h, certain types of surgery (abdominal, thoracic, aortic aneurysm, head and neck, and emergency surgery), and general anesthesia (compared with cases in which general anesthesia was not used).
Efforts at prevention of respiratory complications in patients at risk should include cessation of cigarette smoking several weeks before surgery and lung expansion techniques (eg, incentive spirometry) after surgery. Patients with asthma, particularly those receiving suboptimal medical management, have a greater risk for bronchospasm during airway manipulation. Appropriate use of analgesia and monitoring are key strategies for avoiding postoperative respiratory depression in patients with obstructive sleep apnea. Further discussion of this topic appears in Chapter 44.

The appropriate target blood glucose concentration has been the subject of several celebrated clinical trials. "Tight" control of blood glucose, with a target concentration in the "normal" range, was shown in the Diabetes Control and Complications Trial to improve outcomes in ambulatory patients with type 1 diabetes mellitus. Other more recent trials conducted in subjects with critical illness have shown that blood glucose should not be so tightly controlled. The usual practice is to obtain a blood glucose measurement in diabetic patients on the morning of elective surgery. Unfortunately, many diabetic patients presenting for elective surgery do not maintain blood glucose within the desired range. Other patients, who may be unaware that they have type 2 diabetes, present with blood glucose measurements @ above the normal range. Adequacy of long-term blood glucose control can be easily and rapidly assessed by measurement of hemoglobin A,.. In patients with abnormally elevated hemoglobin A,,, referral to a diabetology service for education about the disease and adjustment of diet and medications to improve metabolic control may be beneficial. Elective surgery should be delayed in patients presenting with marked hyperglycemia; in an otherwise well-managed patient with type 1 diabetes, this delay might consist only of rearranging the order of scheduled cases to allow insulin infusion to bring the blood glucose concentration closer to the normal range before surgery. A more complete discussion of diabetes mellitus and other perioperative endocrine concerns is provided in Chapter 35.

Three important coagulation issues that must be addressed during the preoperative evaluation are (1) how to manage patients who are taking warfarin or other long-acting anticoagulants on a long-term basis; (2) how to manage patients with coronary artery disease who are taking clopidogrel or related agents; and (3) whether one can safely provide neuraxial anesthesia to patients who either are receiving long-term anticoagulation therapy or who will receive anticoagulation perioperatively. In the first circumstance, most patients undergoing anything more involved than minor surgery will require discontinuation of anticoagulation in advance of surgery to avoid excessive blood loss. The key issues to be addressed are how far in advance the drug
trimesters, those whose stomachs have not emptied after a recent meal, and those with serious gastroesophageal reflux disease (GERD).
Although there is consensus that pregnant women and those who have recently (within 6 h) consumed a full meal should be treated as if they have "full" stomachs, there is less consensus as to the necessary period of time in which patients must fast before elective surgery. Proof of the lack of consensus is the fact that the ASA's guideline on this topic was voted down by the ASA House of Delegates several years in a row before it was presented in a form that received majority approval. The guideline as approved is more permissive of fluid intake than many anesthesiologists would prefer, and many medical centers have policies that are more restrictive @ than the ASA guideline on this topic. The truth is that there are no good data to support restricting fluid intake (of any kind or any amount) more than 2 h before induction of general anesthesia in healthy patients undergoing elective procedures; moreover, there is strong evidence that nondiabetic patients who drink fluids containing carbohydrates and protein up to 2 h before induction of anesthesia suffer less perioperative nausea and dehydration than those who are fasted longer.

Patients claiming a history of GERD present vexing problems. Some of these patients will be at increased risk for aspiration; others may carry this "selfdiagnosis" based on advertisements or internet searches, or may have been given this diagnosis by a physician who did not follow the standard diagnostic criteria. Our approach is to treat patients who have only occasional symptoms like any other patient without GERD, and to treat patients with consistent symptoms (multiple times per week) with medications (eg, nonparticulate antacids such as sodium citrate) and techniques (eg, tracheal intubation rather than laryngeal mask airway) as if they were at increased risk for aspiration.

The preoperative history and physical examination complement one another: The physical examination may detect abnormalities not apparent from the history, and the history helps focus the physical examination. Examination of healthy asymptomatic patients should include measurement of vital signs (blood pressure, heart rate, respiratory rate, and temperature) and examination of the airway, heart, and lungs using standard techniques of inspection, palpation, percussion, and auscultation. Before administering regional anesthetics or inserting invasive monitors, one should examine the relevant anatomy; infection or anatomic abnormalities near the site may contraindicate the planned procedure (see Chapters 5, 45, and 46). An abbreviated, focused neurological examination serves to document whether any neurological deficits may be present before a regional anesthesia procedure is performed.
The anesthesiologist should examine the patient's airway before every anesthetic is administered. Any loose or chipped teeth, caps, bridges, or dentures should be noted. Poor fit of the anesthesia mask should be expected in edentulous patients and those with significant facial abnormalities. Micrognathia (a short distance between the chin and the hyoid bone), prominent upper incisors, a large tongue, limited range of motion of the temporomandibular joint or cervical spine, or a short or thick neck suggest that difficulty may be encountered in direct laryngoscopy for tracheal intubation (see Chapter 19). The Mallampati score is often recorded.

Routine laboratory testing is not recommended for fit and asymptomatic patients. "Routine" testing is avoidably expensive and rarely alters perioperative management; moreover, inconsequential abnormal values may result in further unnecessary testing, delays, and costs. Nonetheless, despite no evidence of benefit, some physicians request blood tests, an electrocardiogram, and a chest radiograph for all patients, perhaps in the misplaced hope of reducing their exposure to litigation.
Ideally, testing should be guided by the history @ and physical examination. To be valuable, preoperative testing must discriminate: There must be an avoidable increased perioperative risk when the results are abnormal (and the risk will remain unknown if the test is not performed), and when testing fails to detect the abnormality (or it has been corrected), there must be reduced risk. Useful tests have a low rate of false-positive and false-negative ® results (Table 18-3). The utility of a test depends on its sensitivity and specificity. Sensitive tests have a low rate of false-negative results and rarely fail to identify an abnormality when one is present, whereas specific tests have a low rate of false-positive results and rarely identify an abnormality when one is not present.
TABLE 18-3 Calculation of sensitivity and specificity based on presence or absence of disease in the population being tested.
True positives (TP) have both a positive test and the disease for which they are being tested
False positives (FP) have a positive test but do not have the
disease
True negatives (TN) have a negative test and do not have the disease for which they are being tested
False negatives (FN) have a negative test but do have the
disease
Sensitivity = TP/(TP + FN)
Specificity = TN/(TN + FP)
The predictive value of a positive test (PV+) indicates the likelihood that the patient has the disease if they test positive.
(PV+) =TPATP + FP)
The predictive value of a negative test (PV—) indicates the likelihood that the patient is free of the disease if they test negative
(PV-) = TN/(TN + FN)

The prevalence of a disease or of an abnormal test result varies with the population tested. Testing is therefore most effective when sensitive and specific tests are used in patients in whom the abnormality will be detected frequently enough to justify the expense and inconvenience of the test procedure. Accordingly, laboratory testing should be based on the history and physical examination and the nature of the proposed surgery or procedure. Thus, a baseline hemoglobin or hematocrit measurement is desirable in any patient about to undergo a procedure in which extensive blood loss and transfusion are likely, particularly when there is sufficient time to correct anemia preoperatively (eg, with iron supplements).
Testing fertile women and girls for pregnancy is controversial (but done routinely in many centers) and should not be done without the permission of the patient; pregnancy testing involves detection of chorionic gonadotropin in urine or serum. Routine testing for HIV and routine coagulation studies are not indicated. Urinalysis is not cost-effective in asymptomatic healthy patients; nevertheless, a preoperative urinalysis is required by state law in at least one U.S. jurisdiction.

A classic study showed that a preoperative visit from an anesthesiologist resulted in greater reduction in patient anxiety than preoperative sedative drugs. Yet, there was a time when virtually every patient received premedication before arriving in the preoperative area in anticipation of surgery. The belief was that all patients benefitted from preoperative sedation and anticholinergics, often combined with an opioid. With the move to outpatient surgery and "same-day" hospital admission, preoperative sedative-hypnotics or opioids are now almost never administered before patients arrive in the preoperative holding area for elective surgery. Children, especially those aged 2 to 10 years who (along with their parents) likely will experience separation anxiety may benefit from premedication administered in the preoperative holding area. This topic is discussed in Chapter 42. Oral or intravenous midazolam or nasal dexmedetomidine are common methods. Adults often receive intravenous midazolam (2-5 mg) once an intravenous line has been established. If a painful procedure (eg, regional block or a central venous line) will be performed while the patient remains awake, small doses of opioid (typically fentanyl) will often be given. Patients who will undergo airway surgery or extensive airway manipulations benefit from preoperative administration of an anticholinergic agent (glycopyrrolate or atropine) to reduce airway secretions before and during surgery. Patients who are expected to have significant amounts of postoperative pain will often be given "multimodal" analgesia, including various combinations of nonsteroidal antiinflammatory drugs, acetaminophen, gabapentinoids, and anti-nausea drugs in the preoperative @ holding area. The fundamental message here is that premedication should be given purposefully, not as a mindless routine.

Physicians should provide high-quality, safe, and cost-efficient medical care. But they also must document the care that they provide. Adequate documentation provides guidance to those who will encounter the patient in the future. It permits others to assess the quality of the care that was given and to provide risk adjustment of outcomes. Without documentation a physician will not be paid for his or her services; incomplete documentation may not justify the otherwise appropriate "full" payment. Incomplete documentation may render it difficult for a hospital system to recover its costs and may incorrectly lead to the conclusion that a patient's hospitalization was inappropriately prolonged. Finally, adequate and organized documentation (as opposed to inadequate and disorganized documentation) supports a potential defense case should a claim for medical malpractice be filed.

The preoperative assessment note should appear in the patient's permanent medical record and should describe pertinent findings, including the medical history, anesthetic history, current medications (and whether they were taken on the day of surgery), physical examination, ASA physical status, laboratory results, interpretation of imaging, electrocardiograms, and pertinent recommendations of any consultants. A comment is particularly important when a consultant's recommendation will not be followed.
The preoperative note should identify the anesthetic plan, indicating whether regional or general anesthesia (or sedation) will be used, and whether invasive monitoring or other advanced techniques will be employed. It should include a statement regarding the informed consent discussion with the patient (or guardian). Documentation of the informed consent discussion may take the form of a narrative indicating that the plan, alternative plans, and their advantages and disadvantages (including their relative risks) were presented, understood, and accepted by the patient. Some centers include consent for anesthesia within the consent for surgery (or the procedure). Alternatively, the patient may be asked to read and sign a separate anesthesia consent form that contains the same information.
In the United States, the Joint Commission (TJC) requires an immediate preanesthetic "reevaluation" to determine whether the patient's status has changed in the time since the preoperative evaluation was performed. This reevaluation might include a review of the medical record to search for any new laboratory results or consultation reports if the patient was last seen on another date. However, even when the elapsed time is less than a minute, the bureaucracy will not be denied: the "box" must be checked to document that there has been no interval change.

The intraoperative anesthesia record serves many purposes. It functions as documentation of intraoperative monitoring, a reference for future anesthetics for that patient, and a source of data for quality assurance. This record should be terse, pertinent, and accurate. Increasingly, parts of the anesthesia record are generated automatically and recorded electronically. Such anesthesia information management systems (commonly abbreviated AIMS) have many theoretical and practical advantages over the traditional paper record but also introduce all the common pitfalls of computerization, including the potential for unrecognized recording of artefactual data, the possibility that practitioners will find attending to the computer more interesting than attending to the patient, and the inevitable occurrence of device and software shutdowns. Regardless of whether the record is on paper or electronic it should document the anesthetic care in the operating room by including the following elements:
¢ That there has been a preoperative check of the anesthesia machine and other relevant equipment
¢ That there has been a reevaluation of the patient immediately prior to induction of anesthesia (a TJC requirement)
¢ Time of administration, dosage, and route of drugs given intraoperatively
¢ Intraoperative estimates of blood loss and urinary output
¢ Results of laboratory tests obtained during the operation (when there is an AIMS linked to an electronic medical record, such testing may be recorded elsewhere)
¢ Intravenous fluids and any blood products administered
¢ Pertinent procedure notes (eg, for tracheal intubation or insertion of invasive monitors)
¢ Any specialized intraoperative techniques such as hypotensive anesthesia, one-lung ventilation, high-frequency jet ventilation, or cardiopulmonary bypass
¢ Timing and conduct of intraoperative events such as induction, positioning, surgical incision, and extubation
¢ Unusual events or complications (eg, cardiac arrest)
¢ Condition of the patient at the time of "handoff" to the postanesthesia or intensive care unit nurse
By tradition and convention (and, in the United States, according to practice guidelines) arterial blood pressure and heart rate are recorded graphically at no less than 5 min intervals. Data from other monitors are also usually entered graphically, whereas descriptions of techniques or complications are described in text.

Unfortunately, the conventional, handwritten anesthetic record is ill suited for documenting critical incidents, such as a cardiac arrest. In such cases, a separate text note inserted in the patient's medical record may be necessary. Careful recording of the timing of events is needed to avoid discrepancies between multiple simultaneous records (anesthesia record, nurses' notes, cardiopulmonary resuscitation record, and other physicians' entries in the medical record). Such discrepancies are frequently targeted by malpractice attorneys as evidence of ® incompetence, inaccuracy, or deceit. Incomplete, inaccurate, or illegible records unnecessarily complicate defending a physician against otherwise unjustified allegations of malpractice.

After accompanying the patient to the postanesthesia care unit (PACU), the anesthesia provider should remain with the patient until normal vital signs have been measured and the patient's condition is deemed stable. An unstable patient may require being "handed off" to another physician. Before discharge from the PACU, a note should be written by an anesthesiologist to document the patient's recovery from anesthesia, any apparent anesthesia-related complications, the immediate postoperative condition of the patient, and the patient's disposition (discharge to an outpatient area, an inpatient ward, an intensive care unit, or home). In the United States, as of 2009, the Centers for Medicare and Medicaid Services require that certain elements be included in all postoperative notes (Table 18-4). Recovery from anesthesia should be assessed at least once within 48 h after discharge from the PACU in all inpatients. Postoperative notes should document the general condition of the patient, the presence or absence of any anesthesia-related complications, and any measures undertaken to treat such complications. The anesthesiologist's involvement with the patient may continue through the early stages of postoperative recovery when the anesthesiologist is involved in a functioning perioperative surgical home or is providing treatment of postoperative pain (see Chapters 48, 59).
TABLE 18-4 Elements required by the Center for Medicare and Medicaid Services in all postoperative notes.!
Respiratory function, including respiratory rate, airway patency, and saturation
oxygen
Cardiovascular function, including pulse rate and blood pressure
Mental status
Pain
Nausea and vomiting
Postoperative hydration
Data from the Centers for Medicare and Medicaid Services (CMS). Revised Anesthesia Services Interpretive Guidelines, issued December 30, 2009. Available at: http://www.kdheks.gov/bhfr/download/ Appendix_L.pdf (accessed December 15, 2017).

Medical Malpractice (also see Chapter 54)
A healthy 45-year-old man has a cardiac arrest during an elective laparoscopic inguinal hernia repair. Although cardiopulmonary resuscitation is successful, the patient is left with permanent neuropsychological deficits that preclude his return to work. One year later, the patient files a complaint against the anesthesiologist, surgeon, and hospital.
What four elements must be proved by the plaintiff (patient) to establish negligence on the part of the defendant (physician or hospital)?
1. Duty: Once a physician establishes a professional relationship with a patient, the physician owes that patient certain obligations, such as adhering to the "standard of care."
2. Breach of Duty: If these obligations are not fulfilled, the physician has breached his duties to the patient.
3. Injury: An injury must result. The injury may result in general damages (eg, pain and suffering) or special damages (eg, loss of income).
4. Causation: The plaintiff must demonstrate that the breach of duty was the proximate cause of the injury. But for the breach of duty, the injury should not have occurred.

Individual physicians are expected to perform as any prudent and reasonable physician would in similar circumstances. This does not mandate "best" care or optimal care, only care that would meet the minimum standard of a prudent and reasonable physician. As a specialist, the anesthesiologist is held to a higher standard of knowledge and skill with respect to the subject matter of anesthesia than would a general practitioner or a physician in another specialty. Expert witnesses usually provide testimony to define the standard of care in legal proceedings. Medical malpractice cases are governed by the laws of the state or jurisdiction in which the event took place, and these may differ from state to state. For example, some states require that an expert witness have practiced medicine recently in the state or an immediately adjacent state; others have no "residence" requirement for expert witnesses. The specific circumstances pertaining to each individual case are taken into account. The law recognizes that there are differences of opinion and varying schools of thought within the medical profession.

It is usually the plaintiff who bears the burden of proving that the injury would not have occurred "but for" the negligence of the physician, or that the physician's action was a "substantial factor" in causing the injury. An exception is the doctrine of res ipsa loquitur ("the thing speaks for itself"), which permits a finding of negligence based solely on the evidence. For example, if a set of car keys were visualized inside a patient on a chest radiograph after a thoracotomy, the doctrine of res ipsa loquitur would apply. Res ipsa loquitur could not be used in the case under discussion because the plaintiff would have to establish that cardiac arrest could not occur in the absence of negligence and that cardiac arrest could not have been due to something outside the control of the anesthesiologist. An important concept is that causation in civil cases in the United States need only be established by a preponderance of the evidence ("more likely than not")—as opposed to criminal cases, in which all elements of a charged offense must be proved "beyond a reasonable doubt."
What factors influence the likelihood of a malpractice suit?
- 1. The Physician—Patient Relationship: This is particularly important for the anesthesiologist, who usually does not meet the patient until
immediately before the anesthetic is administered. Another problem is that the patient is unconscious while under the anesthesiologist's care. Thus, the preoperative and postoperative visits with the patient are often the only opportunities to establish a good relationship with the patient. Family members should also be included during these meetings with patients (provided the patient does not object), particularly during the postoperative visit if there has been an intraoperative complication.

2. Adequacy of Informed Consent: Rendering care to a competent patient who does not consent constitutes assault and battery. Consent is not enough, however. The patient should be informed of the contemplated procedure, including its reasonably anticipated risks, its possible benefits, and the therapeutic alternatives. The physician may be liable for a complication—even if it is not due to the negligent performance of a procedure—if a jury is convinced that a reasonable person would have refused treatment if properly informed of the possibility of the complication. This does not mean, of course, that a documented consent relieves from liability physicians who violate the standard of care.
3. Quality of Documentation: Careful documentation of the perioperative visits, informed consent, consultations with other specialists, intraoperative events, and postoperative care is essential. The viewpoint of many courts and juries, reinforced by plaintiff's attorneys, is that "if it isn't written, it wasn't done." It goes without saying that medical records should never be intentionally destroyed or altered.

http://www.asahq.org/quality-and-practice-management/standards-andguidelines https://www.asra.com/advisory-guidelines/article/1/anticoagulation-3rd-edition
Fleisher LA, Fleischmann KE, Auerbach AD, et al. American College of
Cardiology; American Heart Association. ACC/AHA guideline on perioperative
cardiovascular evaluation
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undergoing noncardiac
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Horlocker TT, Wedel DJ, Rowlingson JC, et al. Regional anesthesia in the patient receiving antithrombotic or thrombolytic therapy: American Society
J

@ Improper face mask technique can result in continued deflation of the anesthesia reservoir bag despite the adjustable pressure-limiting valve being closed, usually indicating a substantial leak around the mask. In contrast, the generation of high breathing circuit pressures with minimal chest movement and breath sounds implies an obstructed airway or obstructed tubing.
@ The laryngeal mask airway partially protects the larynx from pharyngeal secretions, but not gastric regurgitation.
@ After insertion of an endotracheal tube (ETT), the cuff is inflated with the least amount of air necessary to create a seal during positivepressure ventilation to minimize the pressure transmitted to the tracheal mucosa.
@ Although the persistent detection of CO, by a capnograph is the best confirmation of tracheal placement of an ETT, it cannot exclude bronchial intubation. The earliest evidence of bronchial intubation often is an increase in peak inspiratory pressure.
@ After intubation, the cuff of an ETT should not be felt above the level of the cricoid cartilage, because a prolonged intralaryngeal location may result in postoperative hoarseness and increases the risk of accidental extubation.
@ Unrecognized esophageal intubation can produce catastrophic results. Prevention of this complication depends on direct visualization of the tip of the ETT passing through the vocal cords, careful auscultation for the presence of bilateral breath sounds and the absence of gastric gurgling while ventilating through the ETT, analysis of exhaled gas for the presence of CO, (the most reliable automated method), chest
radiography, airway ultrasonography, or use of fiberoptic bronchoscopy.
@ Clues to the diagnosis of bronchial intubation include unilateral breath sounds, unexpected hypoxia with pulse oximetry (unreliable with high inspired oxygen concentrations), inability to palpate the ETT cuff in the sternal notch during cuff inflation, and decreased breathing bag compliance (high peak inspiratory pressures).
© The large negative intrathoracic pressures generated by a struggling patient in laryngospasm can result in the development of negativepressure pulmonary edema, particularly in healthy patients.

Expert airway management is an essential skill in anesthetic practice. This chapter reviews the anatomy of the upper respiratory tract, describes necessary airway equipment, presents various management techniques, and discusses complications of laryngoscopy, intubation, and extubation. Patient safety depends on a thorough understanding of each of these topics.

The upper airway consists of the pharynx, nose, mouth, larynx, trachea, and mainstem bronchi. The mouth and pharynx are also a part of the upper gastrointestinal tract. The laryngeal structures in part serve to prevent aspiration into the trachea.
There are two openings to the human airway: the nose, which leads to the nasopharynx, and the mouth, which leads to the oropharynx. These passages are separated anteriorly by the palate, but they join posteriorly in the pharynx (Figure 19-1). The pharynx is a U-shaped fibromuscular structure that extends from the base of the skull to the cricoid cartilage at the entrance to the esophagus. It opens anteriorly into the nasal cavity, the mouth, the larynx, and the nasopharynx, oropharynx, and laryngopharynx, respectively. The nasopharynx is separated from the oropharynx by an imaginary plane that extends posteriorly. At the base of the tongue, the epiglottis functionally separates the oropharynx from the laryngopharynx (or hypopharynx). The epiglottis prevents aspiration by covering the glottis—the opening of the larynx —during swallowing. The larynx is a cartilaginous skeleton held together by ligaments and muscle. The larynx is composed of nine cartilages (Figure 19-2): thyroid, cricoid, epiglottic, and (in pairs) arytenoid, corniculate, and cuneiform. The thyroid cartilage shields the conus elasticus, which forms the vocal cords.
FIGURE 19-1 Anatomy of the airway.
FIGURE 19-2 Cartilaginous structures comprising the larynx. (with permission from The Mayo Foundation.)

The sensory supply to the upper airway is derived from the cranial nerves (Figure 19-3). The mucous membranes of the nose are innervated by the ophthalmic division (V,) of the trigeminal nerve anteriorly (anterior ethmoidal nerve) and by the maxillary division (V>) posteriorly (sphenopalatine nerves). The palatine nerves provide sensory fibers from the trigeminal nerve (V.) to the superior and inferior surfaces of the hard and soft palate. The olfactory nerve (cranial nerve I) innervates the nasal mucosa to provide the sense of smell. The lingual nerve (a branch of the mandibular division [V3] of the trigeminal nerve) and the glossopharyngeal nerve (cranial nerve IX) provide general sensation to the anterior two-thirds and posterior one-third of the tongue, respectively. Branches of the facial nerve (VII) and glossopharyngeal nerve provide the sensation of taste to those areas, respectively. The glossopharyngeal nerve also innervates the roof of the pharynx, the tonsils, and the undersurface of the soft palate. The vagus nerve (cranial nerve X) provides sensation to the airway below the epiglottis. The superior laryngeal branch of the vagus divides into an external (motor) nerve and an internal (sensory) laryngeal nerve that provide sensory supply to the larynx between the epiglottis and the vocal cords. Another branch of the vagus, the recurrent laryngeal nerve, innervates the larynx below

PX
V, Ophthalmic division of trigeminal nerve (anterior ethmoidal nerve)
Vz Maxillary division of trigeminal nerve (sphenopalatine nerves)
V3 Mandibular division of trigeminal nerve (lingual nerve)
ix Glossopharyngeal nerve
X Vagus nerve
SL Superior laryngeal nerve
IL Internal laryngeal nerve
RL Recurrent laryngeal nerve

The muscles of the larynx are innervated by the recurrent laryngeal nerve, with the exception of the cricothyroid muscle, which is innervated by the external (motor) laryngeal nerve, a branch of the superior laryngeal nerve. The posterior cricoarytenoid muscles abduct the vocal cords, whereas the lateral cricoarytenoid muscles are the principal adductors.
Phonation involves complex simultaneous actions by several laryngeal muscles. Damage to the motor nerves innervating the larynx leads to a spectrum of speech disorders (Table 19-1). Unilateral denervation of a cricothyroid muscle causes very subtle clinical findings. Bilateral palsy of the superior laryngeal nerve may result in hoarseness or easy tiring of the voice, but airway control is not jeopardized.
TABLE 19-1 The effects of laryngeal nerve injury on the voice.

Routine airway management associated with general anesthesia consists of:
Preanesthetic airway assessment
Preparation and equipment check
Patient positioning
Preoxygenation (denitrogenation)
Bag and mask ventilation
Intubation or placement of a laryngeal mask airway (if indicated)
Confirmation of proper tube or airway placement
Extubation

A preanesthetic airway assessment is mandatory before every anesthetic procedure. Several anatomical and functional maneuvers can be performed to estimate the difficulty of endotracheal intubation; successful ventilation (with or without intubation) must be achieved by the anesthetist if mortality and morbidity are to be avoided. Assessments include:
Mouth opening: an incisor distance of 3 cm or greater is desirable in an adult.
Mallampati classification: a frequently performed test that examines the size
of the tongue in relation to the oral cavity. The more the tongue obstructs the view of the pharyngeal structures, the more difficult intubation may be
(Figure
19-5).
m Class I: The entire palatal arch, including the bilateral faucial pillars, is visible down to the bases of the pillars.
m Class II: The upper part of the faucial pillars and most of the uvula are visible.
m Class III: Only the soft and hard palates are visible.
m Class IV: Only the hard palate is visible.
¢ Thyromental distance: This is the distance between the mentum (chin) and the superior thyroid notch. A distance greater than 3 fingerbreadths is desirable.
¢ Neck circumference: A neck circumference of greater than 17 inches is associated with difficulties in visualization of the glottic opening.
FIGURE 19-5 A: Mallampati classification of oral opening. B: Grading of the laryngeal view. A difficult orotracheal intubation (grade III or IV) may be predicted by the inability to visualize certain pharyngeal structures (class III or IV) during the preoperative examination of a seated patient. (Reproduced with permission from Mallampati SR, Gatt SP, Gugino LD, et al. A clinical sign to predict difficult tracheal intubation: A prospective study. Can Anaesth Soc J. 1985 Jul;32(4):429-434)
Although the presence of these examination findings may not be particularly sensitive for detecting a difficult intubation, the absence of these findings is predictive for relative ease of intubation.

Increasingly, patients present with morbid obesity and body mass indices of 30 kg/m? or greater. Although some morbidly obese patients have relatively normal head and neck anatomy, others have much redundant pharyngeal tissue and increased neck circumference. Not only may these patients prove to be difficult to intubate, but routine ventilation with bag and mask also may be problematic.
Ultrasound examination of the airway has also been suggested to be of assistance in airway assessment and management (Figures 19—6 through 19-8). Ultrasound can be used as an adjunct to confirm ETT placement as well as to assist in identification of the cricothyroid membrane during emergent cricothyroidotomy.
FIGURE 19-6 A transverse view of the trachea with landmarks. The anechoic area posterior to the trachea represents shadowing resulting from an attenuation of the ultrasound beam through the dense cartilage of the rings. (Reproduced with permission from Carmody KA, Moore CL, Feller-Kopman D. Handbook of Critical Care and Emergency Ultrasound. New York, NY: McGraw-Hill Education; 2011.)
FIGURE 19-7 The trachea during intubation as the tube passes underneath the probe. The arrow points to a subtle area of increased echogenicity just distal to the tracheal cartilage. This area is where movement is most often visualized in real time during an intubation. (Reproduced with permission from Carmody KA, Moore CL, Feller-Kopman D. Handbook of Critical Care and Emergency Ultrasound. New York, NY: McGraw-Hill Education; 2011.)
FIGURE 19-8 A transverse view of the trachea and esophagus during an esophageal intubation. In this image the esophagus is visualized posterior and lateral to the trachea. Two parallel echogenic lines are seen in the proximal esophagus, representing the inner and outer walls of the endotracheal (ET) tube as it passes through the lumen of the esophagus. (Reproduced with permission from Carmody KA, Moore CL, Feller-Kopman D. Handbook of Critical Care and Emergency Ultrasound. New York, NY: McGraw-Hill Education; 2011.)

The following equipment should be routinely available for airway management:
¢ An oxygen source
* Capability to ventilate with bag and mask
¢ Laryngoscopes (direct and video)
Several ETTs of different sizes with available stylets and bougies
Other (not ETT) airway devices (eg, oral, nasal, supraglottic airways)
* Suction
¢ Pulse oximetry and CO, detection
¢ Stethoscope
¢ Tape
¢ Blood pressure and electrocardiography (ECG) monitors
¢ Intravenous access
A flexible fiberoptic bronchoscope should be immediately available when difficult intubation is anticipated but need not be present during all routine intubations.

Loss of upper airway muscle tone (eg, weakness of the genioglossus muscle) in anesthetized patients allows the tongue and epiglottis to fall back against the posterior wall of the pharynx. Repositioning the head or a jaw thrust is the preferred technique for opening the airway. To maintain the opening, an artificial airway can be inserted through the mouth or nose to maintain an air passage between the tongue and the posterior pharyngeal wall (Figure 19-9). Awake or lightly anesthetized patients with intact laryngeal reflexes may cough or even develop laryngospasm during airway insertion. Placement of an oral airway is sometimes facilitated by suppressing airway reflexes, and, in addition, sometimes by depressing the tongue with a tongue blade. Adult oral airways typically come in small (80 mm [Guedel No. 3]), medium (90 mm [Guedel No. 4]), and large (100 mm [Guedel No. 5]) sizes.
FIGURE 19-9 A: The oropharyngeal airway in place. The airway follows the curvature of the tongue, pulling it and the epiglottis away from the posterior pharyngeal wall and providing a channel for air passage. B: The nasopharyngeal airway in place. The airway passes through the nose and extends to just above the epiglottis. (Modified with permission from Dorsch JA, Dorsch SE. Face masks and airways. In: Understanding Anesthesia Equipment. 4th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 1999.)

The length of a nasal airway can be estimated as the distance from the nares to the meatus of the ear and should be approximately 2 to 4 cm longer than oral airways. Because of the risk of epistaxis, nasal airways are less desirable in anticoagulated or thrombocytopenic patients. Also, nasal airways (and nasogastric tubes) should be used with caution in patients with basilar skull fractures, as there has been a case report of a nasogastric tube entering the cranial vault. All tubes inserted through the nose (eg, nasal airways, nasogastric catheters, nasotracheal tubes) should be lubricated before being advanced along the floor of the nasal passage.

The use of a face mask can facilitate the delivery of oxygen or an anesthetic gas from a breathing system to a patient by creating an airtight seal with the patient's face (Figure 19-10). The rim of the mask is contoured and conforms to a variety of facial features. The mask's 22-mm orifice attaches to the breathing circuit of the anesthesia machine through a right-angle connector. Several mask designs are available. Transparent masks allow observation of exhaled humidified gas and immediate recognition of vomitus. Retaining hooks surrounding the orifice can be attached to a head strap so that the mask does not have to be continually held in place. Some pediatric masks are specially designed to minimize apparatus dead space (Figure 19-11).

FIGURE 19-11 The Rendell—Baker—Soucek pediatric face mask has a shallow body and minimal dead space.

When manipulating the airway, correct patient positioning is very helpful. Relative alignment of the oral and pharyngeal axes is achieved by having the patient in the "sniffing" position. When cervical spine pathology is suspected, the head must be kept in a neutral position during all airway manipulations. Inline stabilization of the neck must be maintained during airway management in these patients, unless appropriate cervical radiographs have been reviewed and cleared by an appropriate specialist. Patients with morbid obesity should be positioned on a 30° upward ramp (see Figure 41—2), as the functional residual capacity (FRC) of obese patients deteriorates in the supine position, leading to more rapid deoxygenation should ventilation be impaired.

When possible, preoxygenation with face mask oxygen should precede all airway management interventions. Oxygen is delivered by mask for several minutes prior to anesthetic induction. In this way, the functional residual capacity, the patient's oxygen reserve, is purged of nitrogen. Up to 90% of the normal FRC of 2 L following preoxygenation is filled with oxygen. Considering the normal oxygen demand of 200 to 250 mL/min, the preoxygenated patient may have a 5 to 8 min oxygen reserve. Increasing the duration of apnea without desaturation improves safety, if ventilation following anesthetic induction is delayed. Conditions that increase oxygen demand (eg, sepsis, pregnancy) and decrease FRC (eg, morbid obesity, pregnancy, ascites) reduce the apneic period before desaturation ensues. Assuming a patent air passage is present, oxygen insufflated into the pharynx may increase the duration of apnea tolerated by the patient. Because oxygen enters the blood from the FRC at a rate faster than CO, leaves the blood, a negative pressure is generated in the alveolus, drawing oxygen into the lung (apneic oxygenation). With flow of 100% oxygen and a patent airway, arterial saturation can be maintained for a longer period despite no ventilation, permitting multiple airway interventions should a difficult airway be encountered.

Bag and mask ventilation (BMV) is the first step in airway management in most situations, with the exception of patients undergoing rapid sequence intubation or elective awake intubation. Rapid sequence inductions avoid BMV to minimize stomach inflation and to reduce the potential for the aspiration of gastric contents in nonfasted patients and those with delayed gastric emptying. In emergency situations, BMV precedes attempts at intubation in an effort to oxygenate the patient, with the understanding that there is an implicit risk of aspiration.
Effective mask ventilation requires both a gas-tight @ mask fit and a patent airway. Improper face mask technique can result in continued deflation of the anesthesia reservoir bag despite the adjustable pressure-limiting valve being closed, usually indicating a substantial leak around the mask. In contrast, the generation of high breathing circuit pressures with minimal chest movement and breath sounds implies an obstructed airway or obstructed tubing.
If the mask is held with the left hand, the right hand can be used to generate positive-pressure ventilation by squeezing the breathing bag. The mask is held against the face by downward pressure on the mask exerted by the left thumb and index finger (Figure 19-12). The middle and ring finger grasp the mandible to facilitate extension of the atlantooccipital joint. This is a maneuver that is easier to teach with a mannequin or patient than to describe. Finger pressure should be placed on the bony mandible and not on the soft tissues. The little finger is placed under the angle of the jaw and used to thrust the jaw anteriorly, the most important maneuver to open the airway.

In difficult situations, two hands may be needed to provide adequate jaw thrust and to create a mask seal. Therefore, an assistant may be needed to squeeze the bag, or the machine's ventilator can be used. In such cases, the thumbs hold the mask down, and the fingertips or knuckles displace the jaw forward (Figure 19-13). Obstruction during expiration may be due to excessive downward pressure from the mask or from a ball-valve effect of the jaw thrust. The former can be relieved by decreasing the pressure on the mask, and the latter by releasing the jaw thrust during this phase of the respiratory cycle. Positivepressure ventilation using a mask should normally be limited to 20 cm of H,O to avoid stomach inflation.

Most patients' airways can be maintained with a face mask and an oral or nasal airway. Mask ventilation for long periods may result in pressure injury to branches of the trigeminal or facial nerves. Because of the absence of positive airway pressures during spontaneous ventilation, only minimal downward force on the face mask is required to create an adequate seal. If the face mask and mask straps are used for extended periods, the position should be regularly changed to prevent injury. Care should be used to avoid mask or finger contact with the eye, and the eyes should be taped shut as soon as possible to minimize the risk of corneal abrasions.
If the airway is patent, squeezing the bag will result in the rise of the chest. If ventilation is ineffective (no sign of chest rising, no end-tidal CO, detected, no condensation in the clear mask), oral or nasal airways can be placed to relieve airway obstruction secondary to lax upper airway muscle tone or redundant pharyngeal tissues. Difficult mask ventilation is often found in patients with morbid obesity, beards, and craniofacial deformities. It is sometimes difficult to form an adequate mask fit with the cheeks of edentulous patients.
In years past, anesthetics were routinely delivered solely by mask or ETT administration. In recent decades, a variety of supraglottic devices has permitted both airway rescue (when adequate BMV is not possible) and routine anesthetic airway management (when intubation is not necessary).

Supraglottic airway devices (SADs) are used with both spontaneously breathing and ventilated patients during anesthesia. They have also been employed as conduits to aid endotracheal intubation when both BMV and endotracheal intubation have failed. All SADs consist of a tube that is connected to a respiratory circuit or breathing bag, which is attached to a hypopharyngeal device that seals and directs airflow to the glottis, trachea, and lungs. Additionally, these airway devices occlude the esophagus with varying degrees of effectiveness, reducing gas distention of the stomach. Different sealing devices to prevent airflow from exiting through the mouth are also available. Some are equipped with a port to suction gastric contents. offer the protection from aspiration pneumonitis offered by a properly sited, cuffed endotracheal tube.

A laryngeal mask airway (LMA) consists of a wide-bore tube whose proximal end connects to a breathing circuit with a standard 15-mm connector, and whose distal end is attached to an elliptical cuff that can be inflated through a pilot tube. The deflated cuff is lubricated and inserted blindly into the hypopharynx so that, once inflated, the cuff forms a low-pressure seal around the entrance to the larynx. This requires anesthetic depth and muscle relaxation slightly greater than that required for the insertion of an oral airway. Although insertion is relatively simple (Figure 19-14), attention to detail will improve the success rate (Table 19-2). An ideally positioned cuff is bordered by the base of the tongue superiorly, the pyriform sinuses laterally, and the upper esophageal sphincter inferiorly. If the esophagus lies within the rim of the cuff, gastric distention and regurgitation become possible. Anatomic variations prevent adequate functioning in some patients. However, if an LMA is not functioning properly after attempts to improve the "fit" of the LMA have failed, most practitioners will try another LMA one size larger or smaller. The shaft can be secured with @ tape to the skin of the face. The LMA partially protects the larynx from pharyngeal secretions (but not gastric regurgitation), and it should remain in place until the patient has regained airway reflexes. This is usually signaled by coughing and mouth opening on command. The LMA is available in many sizes (Table 19-3).

FIGURE 19-14 A: The laryngeal mask ready for insertion. The cuff should be deflated tightly with the rim facing away from the mask aperture. There should be no folds near the tip. B: Initial insertion of the laryngeal mask. Under direct vision, the mask tip is pressed upward against the hard palate. The middle finger may be used to push the lower jaw downward. The mask is pressed forward as it is advanced into the pharynx to ensure that the tip remains flattened and avoids the tongue. The jaw should not be held open once the mask is inside the mouth. The nonintubating hand can be used to stabilize the occiput. C: By withdrawing the other fingers and with a slight pronation of the forearm, it is usually possible to push the mask fully into position in one fluid movement. Note that the neck is kept flexed and the head extended. D: The laryngeal mask is grasped with the other hand and the index finger withdrawn. The hand holding the tube presses gently downward until resistance is encountered. (Reproduced with permission from LMA North America.)

1. Choose the appropriate size (Table 19-3) and check for leaks before insertion.
2. The leading edge of the deflated cuff should be wrinkle free and facing away from the aperture (Figure 19-14A).
3. Lubricate only the back side of the cuff.
4, Ensure adequate anesthesia before attempting insertion.
5. Place patient's head in sniffing position (Figure 19-14B and Figure 19-26).
6. Use your index finger to guide the cuff along the hard palate and down into the hypopharynx until an increased resistance is felt (Figure 19-14C). The longitudinal black line should always be pointing directly cephalad (ie, facing the patient's upper lip).
7. Inflate with the correct amount of air (Table 19-3).
8. Ensure adequate anesthetic depth during patient positioning.
9. Obstruction after insertion is usually due to a downfolded epiglottis or transient laryngospasm.
10. Avoid pharyngeal suction, cuff deflation, or laryngeal mask removal until the patient is awake (eg, opening mouth on command).
TABLE 19-3 A variety of laryngeal masks with different cuff volumes are available for different sized patients.
1, PatientSize = Infant. 1, Weight(kg) = <6.5. 1, Cuff Volume (mL) = 2-4. 2, PatientSize = Child. 2, Weight(kg) = 65-20. 2, Cuff Volume (mL) = Up to 10. 2%, PatientSize = Child. 2%, Weight(kg) = 20-30. 2%, Cuff Volume (mL) = Up to 15. 3, PatientSize = Small adult. 3, Weight(kg) = >30. 3, Cuff Volume (mL) = Up to 20. 4, PatientSize = Normal adult. 4, Weight(kg) = <70. 4, Cuff Volume (mL) = Up to 30. 5, PatientSize = Largeradult. 5, Weight(kg) = >70. 5, Cuff Volume (mL) = Up to 30

The LMA provides an alternative to ventilation through a face mask or an ETT (Table 19-4). Relative contraindications for the LMA include patients with pharyngeal pathology (eg, abscess), pharyngeal obstruction, aspiration risk (eg, pregnancy, hiatal hernia), or low pulmonary compliance (eg, restrictive airways disease) requiring peak inspiratory pressures greater than 30 cm HO. The LMA may be associated with less frequent bronchospasm than an ETT. Although it is clearly not a substitute for endotracheal intubation, the LMA has proven particularly helpful as a lifesaving, temporizing measure in patients with difficult airways (those who cannot be mask ventilated or intubated) because of its ease of insertion and relatively high success rate (95-99%). It has been used as a conduit for an intubating stylet (eg, gum-elastic bougie), ventilating jet stylet, flexible fiberoptic bronchoscope, or small-diameter (6.0 mm) ETT. Several LMAs are available that have been modified to facilitate placement of a larger ETT, with or without the use of a bronchoscope. Insertion can be performed under topical anesthesia and bilateral superior laryngeal nerve blocks, if the airway must be secured while the patient is awake. Some newer supraglottic devices incorporate a channel to facilitate gastric decompression.
TABLE 19-4 Advantages and disadvantages of the laryngeal mask airway compared with face mask ventilation or tracheal intubation.!

Compared with face mask, Advantages = Hands-free operation Better seal in bearded patients Less cumbersome in ENT surgery Often easier to maintain airway Protects against airway secretions Less facial nerve and eye trauma Less operating room pollution. Compared with face mask, Disadvantages = More invasive More risk of airway trauma Requires new skill Deeper anesthesia required Requires some TMJ mobility N,0 diffusion into cuff Multiple contraindications. Compared with tracheal intubation, Advantages = Less invasive Very useful in difficult intubations Less tooth and laryngeal trauma Less laryngospasm and bronchospasm Does not require muscle relaxation Does not require neck mobility No risk of esophageal or endobronchial intubation. Compared with tracheal intubation, Disadvantages = Increased risk of gastrointestinal aspiration Less safe in prone or jackknife positions Limits maximum PPV Less secure airway Greater risk of gas leak and pollution Can cause gastric distention
"ENT, ear, nose, and throat; TMJ, temporomandibular joint; PPV, positive-pressure ventilation.
Variations in LMA design include:
¢ The ProSeal LMA, which permits passage of a gastric tube to decompress the stomach
¢ The I-Gel, which uses a gel occluder rather than inflatable cuff
¢ The Fastrach intubation LMA, which is designed to facilitate endotracheal intubation through the LMA device
* The LMA CTrach, which incorporates a camera to facilitate passage of an endotracheal tube
Sore throat is a common side effect following SAD use. Injuries to the lingual, hypoglossal, and recurrent laryngeal nerves have been reported. Correct device sizing, avoidance of cuff hyperinflation, adequate lubrication, and gentle movement of the jaw during placement may reduce the likelihood of such injuries.

The esophageal—tracheal Combitube consists of two fused tubes, each with a 15mm connector on its proximal end (Figure 19-15). The longer blue tube has an occluded distal tip that forces gas to exit through a series of side perforations. The shorter clear tube has an open tip and no side perforations. The Combitube is usually inserted blindly through the mouth and advanced until the two black rings on the shaft lie between the upper and lower teeth. The Combitube has two inflatable cuffs, a 100-mL proximal cuff and a 15-mL distal cuff, both of which should be fully inflated after placement. The distal lumen of the Combitube usually comes to lie in the esophagus approximately 95% of the time so that ventilation through the longer blue tube will force gas out of the side perforations and into the larynx. The shorter, clear tube can be used for gastric decompression. Alternatively, if the Combitube enters the trachea, ventilation through the clear tube will direct gas into the trachea.

The King laryngeal tube consists of a tube with a small esophageal balloon and a larger balloon for placement in the hypopharynx (Figure 19-16). Both balloons inflate through one inflation line. The lungs are inflated with gas that exits between the two balloons. A suction port distal to the esophageal balloon is present, permitting decompression of the stomach. If ventilation proves difficult after the King tube is inserted and the cuffs are inflated, the tube is likely inserted too deeply. Slowly withdraw the device until compliance improves.
FIGURE 19-16 King laryngeal tube.

Endotracheal intubation is employed both for the conduct of general anesthesia and to facilitate the ventilator management of the critically ill.

Standards govern ETT manufacturing (in the United States, American National Standard for Anesthetic Equipment; ANSI Z—79). ETTs are most commonly made from polyvinyl chloride. The shape and rigidity of ETTs can be altered by inserting a stylet. The patient end of the tube is beveled to aid visualization and insertion through the vocal cords. Murphy tubes have a hole (the Murphy eye) to decrease the risk of occlusion, should the distal tube opening abut the carina or trachea (Figure 19-17).

Resistance to airflow depends primarily on tube diameter, but is also affected by tube length and curvature. ETT size is usually designated in millimeters of internal diameter, or, less commonly, in the French scale (external diameter in millimeters multiplied by 3). The choice of tube diameter is always a compromise between maximizing flow with a larger size and minimizing airway trauma with a smaller size (Table 19-5).

Full-term infant, Internal Diameter (mm) = 35. Full-term infant, Cut Length (cm) = 12. Child, Internal Diameter (mm) = Age BIg. Child, Cut Length (cm) = Age =e. Adult, Internal Diameter (mm) = . Adult, Cut Length (cm) = . Female, Internal Diameter (mm) = 7.0-7.5. Female, Cut Length (cm) = 24. Male, Internal Diameter (mm) = 7.5-9.0. Male, Cut Length (cm) = 24
Most adult ETTs have a cuff inflation system consisting of a valve, pilot balloon, inflating tube, and cuff (Figure 19-17). The valve prevents air loss after cuff inflation. The pilot balloon provides a gross indication of cuff inflation. The inflating tube connects the valve to the cuff and is incorporated into the tube's wall. By creating a tracheal seal, ETT cuffs permit positive-pressure ventilation and reduce the likelihood of aspiration. Uncuffed tubes are often used in infants and young children; however, in recent years, cuffed pediatric tubes have been increasingly favored.
There are two major types of cuffs: high pressure (low volume) and low pressure (high volume). High-pressure cuffs are associated with more ischemic damage to the tracheal mucosa and are less suitable for intubations of long duration. Low-pressure cuffs may increase the likelihood of sore throat (larger mucosal contact area), aspiration, spontaneous extubation, and difficult insertion (because of the floppy cuff). Nonetheless, because of their lower incidence of mucosal damage, low-pressure cuffs are most frequently employed. Cuff pressure depends on several factors: inflation volume, the diameter of the cuff in relation to the trachea, tracheal and cuff compliance, and intrathoracic pressure (cuff pressures increase with coughing). Cuff pressure may increase during general anesthesia from diffusion of nitrous oxide from the tracheal mucosa into the ETT cuff. have been modified for of specialized applications. Flexible,

ETTs a variety spiral-wound, wire-reinforced ETTs (armored tubes) resist kinking and may prove valuable in some head and neck surgical procedures or in the prone patient. If an armored tube becomes kinked from extreme pressure (eg, an awake patient biting it), however, the lumen will often remain permanently occluded, and the tube will need replacement. Other specialized tubes include microlaryngeal tubes, double-lumen endotracheal tubes (to facilitate lung isolation and one-lung ventilation), ETTs equipped with bronchial blockers (to facilitate lung isolation and one-lung ventilation), metal tubes designed for laser airway surgery to reduce fire hazards, and preformed curved tubes for nasal and oral intubation in head and neck surgery.

A laryngoscope is an instrument used to examine the larynx and to facilitate intubation of the trachea. The handle usually contains batteries to light a bulb on the blade tip (Figure 19-18), or, alternately, to power a fiberoptic bundle that terminates at the tip of the blade. Laryngoscopes with fiberoptic light bundles in their blades can be made magnetic resonance imaging compatible. The Macintosh and Miller blades are the most popular curved and straight designs, respectively, in the United States. The choice of blade depends on personal preference and patient anatomy. Because no blade is perfect for all situations, the clinician should become familiar and proficient with a variety of blade designs (Figure 19-19).

obtained by the operator and adjust their maneuvers accordingly to facilitate intubation or to provide instruction, respectively.

The McGrath laryngoscope is a portable video laryngoscope with a blade length that can be adjusted to accommodate the airway of a child of age 5 years up to an adult (Figure 19-21). The blade can be disconnected from the handle to facilitate its insertion in morbidly obese patients in whom the space between the upper chest and head is reduced. The blade is inserted midline, with the laryngeal structures viewed at a distance to enhance intubation success.

The GlideScope comes with disposable adultand pediatric-sized blades (Figure 19-22). The blade is inserted midline and advanced until glottic structures are identified. The GlideScope has a 60° angle, preventing direct laryngoscopy and necessitating the use of stylet that is similar in shape to the blade.

¢ Airtraq is a single-use optical laryngoscope available in pediatric and adult sizes (Figure 19-23). The device has a channel to guide the endotracheal tube to the glottis. This device is inserted midline. Success is more likely when the device is not positioned too close to the glottis.

¢ Video intubating stylets have a video capability and light source. The stylet is introduced, and the glottis identified. Intubation with a video stylet may result in less cervical spine movement than with other techniques.

In some situations—for example, patients with unstable cervical spines, poor range of motion of the temporomandibular joint, or certain congenital or acquired upper airway anomalies—laryngoscopy with direct or indirect laryngoscopes may be undesirable or impossible. A flexible fiberoptic bronchoscope (FOB) allows indirect visualization of the larynx in such cases or in any situation in which awake intubation is planned (Figure 19-24). Bronchoscopes are constructed of coated glass fibers that transmit light and images by internal reflection (ie, a light beam becomes trapped within a fiber and exits unchanged at the opposite end). The insertion tube contains two bundles of fibers, each consisting of 10,000 to 15,000 fibers. One bundle transmits light from the light source (light source or incoherent bundle), which is either external to the device or contained within the handle (Figure 19-24B), whereas the other provides a high-resolution image (image or coherent bundle). Directional manipulation of the insertion tube is accomplished with angulation wires. Aspiration channels allow suctioning of secretions, insufflation of oxygen, or instillation of local anesthetic. Aspiration channels can be difficult to clean,

FIGURE 19-24 A: Cross section of a fiberoptic bronchoscope. B: A flexible fiberoptic bronchoscope with a fixed light source.

Inserting a tube into the trachea has become a routine part of delivering a general anesthetic. Intubation is not a risk-free procedure, and it is not a requirement for all patients receiving general anesthesia. An ETT is generally placed to protect the airway and for airway access. Intubation is indicated in patients who are at risk of aspiration and in those undergoing surgical procedures involving body cavities, the head and neck, and those who will be positioned so that the airway will be less accessible (eg, those undergoing surgery in the prone position, or whose head is rotated away from the anesthesia workstation). Mask ventilation or ventilation with an LMA is usually satisfactory for short minor procedures such as cystoscopy, examination under anesthesia, inguinal hernia repairs, extremity surgery, and so forth. Nevertheless, the indications for use of supraglottic airway devices during anesthesia continues to expand.

Preparation for intubation includes checking equipment and properly positioning the patient. The ETT should be examined. The tube's cuff can be tested by inflating the cuff using a syringe. Maintenance of cuff pressure after detaching the syringe ensures proper cuff and valve function. Some anesthesiologists cut the ETT to a preset length to decrease the dead space, the risk of bronchial intubation, and the risk of occlusion from tube kinking (Table 19-5). The connector should be pushed firmly into the tube to decrease the likelihood of disconnection. If a stylet is used, it should be inserted into the ETT, which is then bent to resemble a hockey stick (Figure 19-25). This shape facilitates intubation of an anteriorly positioned larynx. The desired blade is locked onto the laryngoscope handle, and bulb function is tested. The light intensity should remain constant even if the bulb is jiggled. A blinking light signals a poor electrical contact, whereas fading indicates depleted batteries. An extra handle, blade, ETT (one size smaller than the anticipated optimal size), stylet, and intubating bougie should be immediately available. A functioning suction unit is needed to clear the airway in case of unexpected secretions, blood, or emesis.
FIGURE 19-25 An endotracheal tube with a stylet bent to resemble a hockey stick.
Adequate glottis exposure during laryngoscopy often depends on correct patient positioning. The patient's head should be level with the anesthesiologist's waist or higher to prevent unnecessary back strain during laryngoscopy.
Direct laryngoscopy displaces pharyngeal soft tissues to create a direct line of vision from the mouth to the glottic opening. Moderate head elevation (5-10 cm above the surgical table) and extension of the atlantooccipital joint place the patient in the desired sniffing position (Figure 19—26). The lower portion of the cervical spine is flexed by resting the head on a pillow or other soft support.
FIGURE 19-26 The sniffing position and intubation with a Macintosh blade. (Modified with permission from Dorsch JA, Dorsch SE. Understanding Anesthesia Equipment: Construction, Care, and Complications. Philadelphia, PA: Lippincott Williams & Wilkins, 1991.)

As previously discussed, preparation for induction and intubation also involves routine preoxygenation. Preoxygenation can be omitted in patients who object to the face mask; however, failing to preoxygenate increases the risk of rapid desaturation following apnea.
Because general anesthesia abolishes the protective corneal reflex, care must be taken during this period not to injure the patient's eyes by unintentionally abrading the cornea. Thus, the eyes are routinely taped shut as soon as possible, often after applying an ophthalmic ointment before manipulation of the airway.

The laryngoscope is held in the left hand. With the patient's mouth opened the blade is introduced into the right side of the oropharynx—with care to avoid the teeth. The tongue is swept to the left and up into the floor of the pharynx by the blade's flange. Successful sweeping of the tongue leftward clears the view for ETT placement. The tip of a curved blade is usually inserted into the vallecula, and the straight blade tip covers the epiglottis. With either blade, the handle is raised up and away from the patient in a plane perpendicular to the patient's mandible to expose the vocal cords (Figure 19-27). Trapping a lip between the teeth and the blade and leverage on the teeth are avoided. The ETT is taken with the right hand, and its tip is passed through the abducted vocal cords. The "backward, upward, rightward, pressure" (BURP) maneuver applied externally moves an anteriorly positioned glottis posterior to facilitate visualization of the glottis. The ETT cuff should lie in the upper trachea, but beyond the larynx. The laryngoscope is withdrawn, again ® with care to avoid tooth damage. The cuff is inflated with the least amount of air necessary to create a seal during positivepressure ventilation to minimize the pressure transmitted to the tracheal mucosa. Overinflation may inhibit capillary blood flow, injuring the trachea. Compressing the pilot balloon with the fingers is not a reliable method of determining whether cuff pressure is either sufficient or excessive.
FIGURE 19-27 Typical view of the glottis during laryngoscopy with a curved blade. (Modified with permission from Barash PG. Clinical Anesthesia. 4th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2001.)

After intubation, the chest and epigastrium are immediately auscultated, and a capnographic tracing (the definitive test) is monitored to ensure intratracheal location (Figure 19-28). If there is doubt as to whether the tube is in the esophagus or trachea, repeat the laryngoscopy to confirm placement. End-tidal CO, will not be produced if there is no cardiac output. FOB through the tube and visualization of the tracheal rings and carina will likewise confirm correct placement. Otherwise, the tube is taped or @ tied to secure its position. Although the persistent detection of CO, by a capnograph is the best confirmation of tracheal placement of an ETT, it cannot exclude bronchial intubation. The earliest evidence of bronchial intubation often is an increase in peak inspiratory pressure. Proper tube location can be reconfirmed by palpating the cuff in the sternal notch while compressing the pilot balloon with the @ other hand. The cuff should not be felt above the level of the cricoid cartilage, because a prolonged intralaryngeal location may result in postoperative hoarseness and increases the risk of accidental extubation. Tube position can also be documented by chest radiography.
FIGURE 19-28 Sites for auscultation of breath sounds at the apices and over the stomach.
The description presented here assumes an unconscious patient. Oral intubation is usually poorly tolerated by awake, fit patients. Intravenous sedation, application of a local anesthetic spray in the oropharynx, regional nerve block, and constant reassurance will improve patient acceptance.

A failed intubation should not be followed by identical repeated attempts. Changes must be made to increase the likelihood of success, such as repositioning the patient, decreasing the tube size, adding a stylet, selecting a different blade, using an indirect laryngoscope, attempting a nasal route, or requesting the assistance of another anesthesia provider. If the patient is also difficult to ventilate with a mask, alternative forms of airway management (eg, second-generation supraglottic airway devices, jet ventilation via percutaneous tracheal catheter, cricothyrotomy, tracheostomy) must be immediately pursued. The guidelines developed by the American Society of Anesthesiologists for the management of a difficult airway include a treatment plan algorithm (Figure 1929).

1. Assess the likelihood and clinical impact of basic management problems:
* Difficulty with patient cooperation or consent
* Difficult mask ventilation
* Difficult supraglottic airway placement
* Difficult laryngoscopy
* Difficult intubation
* Difficult surgical airway access
2. Actively pursue opportunities to deliver supplemental oxygen throughout the process of difficult airway management.
3. Consider the relative merits and feasibility of basic management choices:
* Awake intubation vs. intubation after induction of general anesthesia
* Non-invasive technique vs. invasive techniques for the initial approach to intubation
* Video-assisted laryngoscopy as an initial approach to intubation
* Preservation vs, ablation of spontaneous ventilation
4. Develop primary and alternative strategies:
*Confirm ventilation, tracheal intubation, or SGA placement with exhaled CO,.
FIGURE 19-29 Difficult airway algorithm. Notes: (a) Other options include (but are not limited to): surgery utilizing face mask or supraglottic airway (SGA) anesthesia (eg, laryngeal mask airway [LMA], intubating LMA [ILMA], laryngeal tube), local anesthesia infiltration, or regional nerve blockade. Pursuit of these options usually implies that mask ventilation will not be problematic. Therefore, these options may be of limited value if this step in the algorithm has been reached via the emergency pathway. (b) Invasive airway access includes surgical or percutaneous airway, jet ventilation, and retrograde intubation. (c)

Alternative difficult intubation approaches include (but are not limited to) videoassisted laryngoscopy, alternative laryngoscope blades, SGA (eg, LMA or ILMA) as an intubation conduit (with or without fiberoptic guidance), fiberoptic intubation, intubating stylet or tube changer, light wand, and blind oral or nasal intubation. (d) Consider re-preparation of the patient for awake intubation or canceling surgery. (e) Emergency noninvasive airway ventilation consists of an SGA. (Reproduced with permission from American Society of Anesthesiologists Task Force on the Management of the Difficult Airway. Practice guidelines for management of the difficult airway: An updated report by the American Society of Anesthesiologists Task Force on the Management of the Difficult 2003
Airway. Anesthesiology. May;98(5):1269-1277.)
The Difficult Airway Society (DAS) also provides a useful approach for the management of the unanticipated difficult airway (Figure 19-30).

It in difficulty ———B call for help
Optimise head and neck posit
Preoxygenate Adequate neuromuscular blockade DirectVideo Laryngoscopy (maximum 3+1 attempts) External laryngeal manipulation Bougie Remove cricoid pressure Maintain oxygenation and anaesthesia
|
Succeed tS
——~>
|
Declare failed intubation
2nd generation device recommended Change device or size (maximum 3 attempts) Oxygenate and ventilate
Succeed
|
NT SE Confirm tracheal intubation with capnography
|
|
—————————————e

Options (consider risks and benefits):
1. Wake the patient up
2. Intubate trachea via the SAD.
ed
3. Proceed without intubating the trachea
4. Tracheostomy or cricothyroidotomy
Declare failed SAD ventilation —
Succeed once
OOOO
If facemask ventilation impossible, paralyse Final attempt at facemask ventilation Use 2 person technique and adjuncts
|
Declare CICO
= Scalpel cricothyroidotomy
Wake the patient up
—eEE————————————
SS
Post-operative care
and follow
up
+ Formulate immediate airway management plan
* Monitor for complications
* Complete airway alert form
* Explain to the patient in person and in writing
* Send written report to GP and local database
—————————
This flowchart forms part of the DAS Guidelines for unanticipated difficult intubation in adults 2015 and should be used in conjunction with the text.
FIGURE 19-30 Difficult Airway Society difficult intubation guidelines: overview. CICO, can't intubate can't oxygenate; SAD, supraglottic airway device. (Reproduced with permission from Frerk C, Mitchell V, McNarry A, et al. Difficult Airway Society 2015 guidelines for management of unanticipated difficult intubation in adults. Br J Anaesth. 2015 Dec;115(6):827-848.)
The combined use of a video laryngoscope and an intubation bougie often can facilitate intubation, when the endotracheal tube cannot be directed into the glottis despite good visualization of the laryngeal opening (Figure 19-31). Progression through the DAS plans A to D prevents the anesthetist from unnecessarily repeating the same failed approaches to airway management and maximizes the possibility of preserving patient oxygenation as the airway is secured.

Nasal intubation is similar to oral intubation except that the ETT is advanced through the nose and nasopharynx into the oropharynx before laryngoscopy. The nostril through which the patient breathes most easily is selected in advance and prepared. Phenylephrine (0.5% or 0.25%) or tolazoline nose drops constrict blood vessels and shrink mucous membranes. If the patient is awake, local anesthetic ointment (for the nostril, delivered via an ointment-coated nasopharyngeal airway), spray (for the oropharynx), and nerve blocks can also be utilized.
An ETT lubricated with water-soluble jelly is introduced along the floor of the nose, below the inferior turbinate, at an angle perpendicular to the face. The tube's bevel should be directed laterally away from the turbinates. To ensure that the tube passes along the floor of the nasal cavity, the proximal end of the ETT should be pulled cephalad. The tube is gradually advanced, until its tip can be visualized in the oropharynx. Laryngoscopy, as discussed, reveals the abducted vocal cords. Often the distal end of the ETT can be pushed into the trachea without difficulty. If difficulty is encountered, the tip of the tube may be directed through the vocal cords with Magill forceps, being careful not to damage the cuff. Nasal passage of ETTs, airways, or nasogastric catheters carries greater risk in patients with severe midfacial trauma because of the risk of intracranial placement (Figure 19-32).
FIGURE 19-32 Radiograph demonstrating a 7.0-mm endotracheal tube placed through the cribriform plate into the cranial vault in a patient with a basilar skull fracture.
Although less used today, blind nasal intubation of spontaneously breathing patients can be employed. In this technique, after applying topical anesthetic to the nostril and pharynx, a breathing tube is passed through the nasopharynx. Using breath sounds as a guide, it is directed toward the glottis. When breath sounds are maximal, the anesthetist advances the tube during inspiration in an effort to blindly pass the tube into the trachea.

Fiberoptic intubation (FOI) is routinely performed in awake or sedated patients with problematic airways. FOI is ideal for:
¢ Asmall mouth opening
¢ Minimizing cervical spine movement in trauma or rheumatoid arthritis
¢ Upper airway obstruction, such as angioedema or tumor mass
¢ Facial deformities, facial trauma
FOI can be performed awake or asleep via oral or nasal routes in the following scenarios:
¢« Awake FOI—Predicted inability to ventilate by mask, upper airway obstruction
Asleep FOI—Failed intubation, desire for minimal cervical spine movement in patients who refuse awake intubation, anticipated difficult intubation when ventilation by mask appears easy
¢ Oral FOI—Facial, skull injuries
¢ Nasal FOI—A poor mouth opening
When
FOI
is considered,
careful planning
add to
the anesthesia
time prior
to surgery.
is necessary,
Patients as
should be
it will
otherwise informed
of the
need for
awake intubation
as
a
part of
the informed
consent process.
The airway is anesthetized with a local anesthetic spray, and patient sedation is provided, as tolerated. Dexmedetomidine has the advantage of preserving respiration while providing sedation. Airway anesthesia is discussed in the Case Discussion at the end of this chapter.
If nasal FOI is planned, both nostrils are prepared with vasoconstrictive spray. The nostril through which the patient breathes more easily is identified. Oxygen can be insufflated through the suction port and down the aspiration channel of the FOB to improve oxygenation and blow secretions away from the tip.
Alternatively,
a
large nasal
airway
(eg,
36FR)
can be
inserted in
the contralateral
nostril.
this nasal
airway is
The to
unconscious breathing
circuit administer
and ventilation
100%
not breathing
attempted used,
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ventilation capnography
and into
the
ETT
relatively pulse
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lumen.
straight rotated

connected laryngoscopy.
the to
the
If mouth
single nasal
can be
airway.
oxygenation
When should
The
It is
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(Figure in
one direction,
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the epiglottis
to
19-33)
shaft keep
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As the
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if the
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FIGURE 19-33 Correct technique for manipulating a fiberoptic bronchoscope through an endotracheal tube is shown in the top panel; avoid curvature in the bronchoscope, which makes manipulation difficult.
Having an assistant thrust the jaw forward or apply cricoid pressure may improve visualization in difficult cases. Grasping the tongue with gauze and pulling it forward may also facilitate intubation.
Once in the trachea, the FOB is advanced to within sight of the carina. The presence of tracheal rings and the carina is proof of proper positioning. The ETT is pushed off the FOB. The acute angle around the arytenoid cartilage and epiglottis may prevent easy advancement of the tube. Use of an armored tube usually decreases this problem due to its greater lateral flexibility and more obtusely angled distal end. Proper ETT position is confirmed by viewing the tip of the tube an appropriate distance (3 cm in adults) above the carina before the FOB is withdrawn.
Oral FOI proceeds similarly, with the aid of various oral airway devices to direct the FOB toward the glottis and to reduce obstruction of the view by the tongue.

"Invasive" airways are required when the "can't intubate, can't ventilate" scenario presents and may be performed in anticipation of such circumstances in selected patients. The options include surgical cricothyrotomy, catheter or needle cricothyrotomy, transtracheal catheter with jet ventilation, and retrograde intubation.
Surgical cricothyrotomy refers to surgical incision of the cricothyroid membrane (CTM) and placement of a breathing tube. More recently, several needle/dilator cricothyrotomy kits have become available. Unlike surgical cricothyrotomy, where a horizontal incision is made across the CTM, these kits utilize the Seldinger catheter/wire/dilator technique. A catheter attached to a syringe is inserted across the CTM (Figure 19-34). When air is aspirated, a guidewire is passed through the catheter into the trachea (Figure 19-35). A dilator is then passed over the guidewire, and a breathing tube placed (Figure 19-36).

MD.)
FIGURE 19-35 Cricothyrotomy. Incision at wire entry site. Remove catheter and make incision at the wire entry Site. (Used with permission from Lawrence B. Stack,
FIGURE 19-36 Cricothyrotomy. Insert tracheostomy tube/introducer. Insert both devices over the wire and into the trachea. (Used with permission from Lawrence B. Stack, MD.)
Catheter-based salvage procedures can also be performed. A 16or 14-gauge intravenous cannula is attached to a syringe and passed through the CTM toward the carina. Air is aspirated. If a jet ventilation system is available, it can be attached. The catheter must be secured, otherwise the jet pressure will push the catheter out of the airway, leading to potentially disastrous subcutaneous emphysema. Short (1-s) bursts of oxygen ventilate the patient. Sufficient outflow of expired air must be assured to avoid barotrauma. Patients ventilated in this manner may develop subcutaneous or mediastinal emphysema and may become hypercapneic despite adequate oxygenation. Transtracheal jet ventilation will usually require conversion to a surgical airway or tracheal intubation.
Should a jet ventilation system not be available, a 3-mL syringe can be attached to the catheter and the syringe plunger removed. A 7.0-mm internal diameter ETT connector can be inserted into the syringe and attached to a breathing circuit or an AMBU bag. As with the jet ventilation system, adequate exhalation must occur to avoid barotraumas.
Retrograde intubation is another approach to secure an airway. A wire is passed via a catheter placed in the CTM. The wire is angulated cephalad and emerges either through the mouth or nose. The distal end of the wire is secured with a clamp to prevent it from passing through the CTM. The wire can then be threaded into an FOB with a loaded endotracheal tube to facilitate and confirm placement. Conversely, a small endotracheal tube can be guided by the wire into the trachea. Once placed, the wire is removed.

Following apparently successful intubation, several scenarios may develop that require immediate attention. Anesthesia staff must confirm that the tube is correctly placed with auscultation of bilateral breath sounds immediately following placement. Detection of end-tidal CO, remains the gold standard in this regard, with the caveat that cardiac output must be present for end-tidal CO, production.
Decreases in oxygen saturation can occur following tube placement. This is often secondary to endobronchial intubation, especially in small children and infants. Decreased oxygen saturation perioperatively may be due to inadequate oxygen delivery (oxygen not turned on, patient not ventilated) or to ventilation/perfusion mismatch (almost any form of lung disease). When saturation declines, the patient's chest is auscultated to confirm bilateral tube placement and to listen for wheezes, rhonchi, and rales consistent with lung pathology. The breathing circuit is checked. An intraoperative chest radiograph
administering 1.5 mg/kg of intravenous lidocaine 1-2 min before suctioning and extubation; however, extubation during deep anesthesia may be preferable in patients who cannot tolerate these effects (provided such patients are not at risk of aspiration and/or do not have airways that may be difficult to maintain after removal of the ETT).
Regardless of whether the tube is removed when the patient is deeply anesthetized or awake, the patient's pharynx should be thoroughly suctioned before extubation to decrease the potential for aspiration of blood and secretions. In addition, patients should be ventilated with 100% oxygen in case it becomes difficult to establish an airway after the ETT is removed. Just prior to extubation, the ETT is untaped or untied and its cuff is deflated. The tube is withdrawn in a single smooth motion, and a face mask is applied to deliver oxygen. Oxygen delivery by face mask is maintained during the period of transportation to the postanesthesia care area.

The complications of laryngoscopy and intubation include hypoxia, hypercarbia, dental and airway trauma, tube malpositioning, physiological responses to airway instrumentation, and tube malfunction. These complications can occur during laryngoscopy and intubation, while the tube is in place, or following extubation (Table 19-6).
TABLE 19-6 Complications of intubation.
During laryngoscopy and intubation
Malpositioning
Esophageal intubation
Bronchial intubation
Laryngeal cuff position
Airway trauma
Dental damage
Lip, tongue, or mucosal laceration
Sore throat
Dislocated mandible
Retropharyngeal dissection
Physiological reflexes
Hypoxia, hypercarbia
Hypertension, tachycardia
Intracranial hypertension
Intraocular hypertension
Laryngospasm
Tube malfunction
Cuff perforation
While the tube is in place
Unintentional extubation
Mucosal inflammation and ulceration
Excoriation of nose
Fire/explosion
Obstruction
Following extubation
Edema and stenosis (glottic, subglottic, or tracheal)
Hoarseness (vocal cord granuloma or paralysis)
Laryngeal malfunction and aspiration
Negative-pressure pulmonary edema

Instrumentation with a metal laryngoscope blade and insertion of a stiff ETT
often traumatizes delicate airway tissues. Tooth damage is a common cause of
(relatively small) malpractice claims against anesthesiologists. Laryngoscopy and intubation can lead to a range of complications from sore throat to tracheal stenosis. Most of these are due to prolonged external pressure on sensitive airway structures. When these pressures exceed the capillary—arteriolar blood pressure (approximately 30 mm Hg), tissue ischemia can lead to a sequence of inflammation, ulceration, granulation, and stenosis. Inflation of an ETT cuff to the minimum pressure that creates a seal during routine positive-pressure ventilation (usually at least 20 mm Hg) reduces tracheal blood flow by 75% at the cuff site. Further cuff inflation or induced hypotension can totally mucosal blood flow.
croup or particularly serious in children. The efficacy of corticosteroids (eg, dexamethasone—0.2 mg/kg, up to a maximum of 12 mg) in preventing postextubation airway edema remains controversial, but this approach is often used. Vocal cord paralysis from cuff compression or other trauma to the recurrent laryngeal nerve results in hoarseness and increases the risk of aspiration. The incidence of postoperative hoarseness seems to increase with obesity, difficult intubations, and anesthetics of long duration. Curiously, applying a water-soluble lubricant or a local anesthetic-containing gel to the tip or cuff of the ETT does not decrease the incidence of postoperative sore throat or hoarseness, and, in some studies, actually increased the incidence of these complications. Smaller tubes (size 6.5 in women and size 7.0 in men) are associated with fewer complaints of postoperative sore throat. Repeated attempts at laryngoscopy during a difficult intubation may lead to periglottic edema and the inability to ventilate with a face mask, thus turning a bad situation into a lifethreatening one.
eliminate Postintubation caused by glottic, laryngeal, tracheal edema is

@ Unrecognized esophageal intubation can produce catastrophic results. Prevention of this complication depends on direct visualization of the tip of the ETT passing through the vocal cords, careful auscultation for the presence of bilateral breath sounds and the absence of gastric gurgling while ventilating through the ETT, detection of CO, in exhaled gas (the most reliable automated method), chest radiography, airway ultrasonography, or the use of an FOB. Even though it is confirmed that the tube is in the trachea, it may not be correctly positioned. Overly "deep" insertion usually results in intubation of the right mainstem bronchus because the right bronchus forms a less acute angle with the trachea than the left @ bronchus. Clues to the diagnosis of bronchial intubation include unilateral breath sounds, unexpected hypoxia with pulse oximetry (unreliable with high inspired oxygen concentrations), inability to palpate the ETT cuff in the sternal notch during cuff inflation, and decreased breathing-bag compliance (high peak inspiratory pressures).
In contrast, inadequate insertion depth will position the cuff in the larynx, predisposing the patient to laryngeal trauma. Inadequate depth of insertion can be detected by palpating the cuff over the thyroid cartilage. Because no one technique protects against all possibilities for misplacing an ETT, minimal testing should include chest auscultation, routine capnography, and occasionally cuff palpation.
If the patient is repositioned, tube placement must be reconfirmed. Neck extension or lateral rotation most often moves an ETT away from the carina, whereas neck flexion most often moves the tube toward the carina.
At no time should excessive force be employed during intubation. Esophageal intubations can result in esophageal rupture and mediastinitis. Mediastinitis presents as severe sore throat, fever, sepsis, and subcutaneous air often manifesting as crepitus. Early intervention is necessary to avoid mortality. If esophageal perforation is suspected, consultation with an otolaryngologist or thoracic surgeon is recommended. Vocal cord injury can likewise result from repeated, forceful attempts at endotracheal intubation.

Laryngoscopy and tracheal intubation violate the patient's protective airway reflexes and predictably lead to hypertension and tachycardia when performed under "light" planes of general anesthesia. The insertion of an LMA is typically associated with less hemodynamic change. Hemodynamic changes can be attenuated by intravenous administration of lidocaine, opioids, or B-blockers, or deeper planes of inhalation anesthesia in the minutes before laryngoscopy. Hypotensive agents, including sodium nitroprusside, nitroglycerin, esmolol, nicardipine, and clevidipine, can attenuate the transient hypertensive response associated with laryngoscopy and intubation. Cardiac arrhythmias—particularly ventricular premature beats—sometimes occur during intubation and may indicate light anesthesia.
Laryngospasm is a forceful involuntary spasm of the laryngeal musculature caused by sensory stimulation of the superior laryngeal nerve. Triggering stimuli include pharyngeal secretions or passing an ETT through the larynx during

ETTs do not always function as intended. Polyvinyl chloride tubes may be ignited by cautery or laser in an oxygen/nitrous oxide—enriched environment. Valve or cuff damage is not unusual and should be excluded by careful inspection of the ETT prior to insertion. Obstruction of the ETT can result from kinking, from foreign body aspiration, or from thick or inspissated secretions in the lumen.

Evaluation & Management of a Difficult Airway
A 47-year-old man with a long history of tobacco and alcohol abuse presents for emergency drainage of a right-sided submandibular abscess.
What are some important anesthetic considerations during the

Induction of general anesthesia followed by direct laryngoscopy and oral intubation is dangerous, if not impossible, in several situations. (Table 19— 7). To determine the optimal intubation technique, the anesthesiologist must elicit an airway history and carefully examine the patient's head and neck. Any available prior anesthesia records should be reviewed for previous problems in airway management. If a facial deformity is severe enough to preclude a good mask seal, positive-pressure ventilation may be impossible. Furthermore, patients with hypopharyngeal disease are more dependent on awake muscle tone to maintain airway patency. These two groups of patients should generally not be allowed to become apneic—including induction of anesthesia, sedation, or muscle paralysis—auntil their airway is secured.
TABLE 19-7 Conditions associated with difficult intubations.

Cystic hygroma
Hemangioma
Hematoma'
Infections
Submandibular abscess
Peritonsillar abscess
Epiglottitis
Congenital anomalies
Pierre Robin syndrome
Treacher Collins syndrome
Laryngeal atresia
Goldenhar syndrome
Craniofacial dysostosis
Foreign body
Trauma
Laryngeal fracture Mandibular or maxillary fracture
Inhalation burn
Cervical spine injury
Obesity
Inadequate neck extension
Rheumatoid arthritis?
Ankylosing spondylitis
Halo traction
Anatomic variations
Micrognathia
Prognathism
Large tongue
Arched palate
Short neck
Prominent upper incisors
'Can occur postoperatively in patients who have had any neck surgery. *Also affects arytenoids making them immobile.
If there is an abnormal limitation of the temporomandibular joint that may not improve with muscle paralysis, a nasal approach with an FOB should be considered. Infection confined to the floor of the mouth usually does not preclude nasal intubation. If the hypopharynx is involved to the level of the hyoid bone, however, any translaryngeal attempt will be difficult. Other clues to a potentially difficult laryngoscopy include limited neck extension (<35°), a distance between the tip of the patient's mandible and hyoid bone of less than 7 cm, a sternomental distance of less than 12.5 cm with the head fully extended and the mouth closed, and a poorly visualized uvula during voluntary tongue protrusion. It must be stressed that because no examination technique is foolproof and the signs of a difficult airway may be subtle, the anesthesiologist must always be prepared for unanticipated difficulties.
The anesthesiologist should also evaluate the patient for signs of airway obstruction (eg, chest retraction, stridor) and hypoxia (agitation, restlessness, anxiety, lethargy). Aspiration pneumonia is more likely if the patient has recently eaten or if pus is draining from an abscess into the mouth. In either case, techniques that ablate laryngeal reflexes (eg, topical anesthesia) should be avoided.

Cervical trauma or disease is a factor that should be evaluated prior to direct laryngoscopy. Cervical arthritis or previous cervical fusion may make it difficult for the head to be put in the sniffing position; these patients are candidates for bronchoscopy to secure the airway, as discussed previously. Trauma patients with unstable necks or whose neck has not yet been "cleared" are also candidates for bronchoscopy for tracheal intubation. Alternatively, laryngoscopy with in-line stabilization can be performed (Figure 19-37).
FIGURE 19-37 Technique for airway management of a patient with suspected spinal cord injury. One individual holds the head firmly with the patient on a backboard, the cervical collar left alone if in place, ensuring that neither the head nor neck moves with direct laryngoscopy. A second person applies cricoid pressure and the third performs laryngoscopy and intubation.
In the case under discussion, physical examination reveals swelling below the mandible and trismus that limits the patient's ability to open his mouth. Mask fit does not seem to be impaired. CT of the head and neck suggests that the infection has spread along tissue planes and is displacing the airway to the left.

Oral and nasal intubations can be performed in awake patients. Whether the patient is awake or asleep or whether intubation is to be oral or nasal, it can be performed with direct laryngoscopy, fiberoptic visualization, or video laryngoscopy techniques.
Intubation may be difficult in this patient, however, because of limited mouth opening and distortion/displacement of the glottis. Induction of anesthesia should, therefore, be delayed until after the airway has been secured. Useful alternatives include awake fiberoptic intubation, awake video laryngoscopy, or awake use of optical stylets. The final decision depends on the availability of equipment and the experiences and preferences of the anesthesia caregivers.
Regardless of which alternative is chosen, an emergency surgical airway may be necessary. An experienced team, including a surgeon, should be in the operating room and all necessary equipment should be available and unwrapped. The neck can be prepped and draped.

Any loss of consciousness or interference with airway reflexes could result in airway obstruction or aspiration. Glycopyrrolate would be a good choice of premedication because it minimizes upper airway secretions without crossing the blood-brain barrier. Parenteral sedatives should be very carefully titrated. Dexmedetomidine and ketamine preserve respiratory effort and could be used as sedatives. Psychological preparation of the patient, including explaining each step planned in securing the airway, may improve patient cooperation.
What nerve blocks could be helpful during an awake

The lingual and some pharyngeal branches of the glossopharyngeal nerve that provide sensation to the posterior third of the tongue and oropharynx are easily blocked by bilateral injection of 2 mL of local anesthetic into the base of the palatoglossal arch (also known as the anterior tonsillar pillar) with a 25-gauge spinal needle (Figure 19-38).
FIGURE 19-38 Nerve block. While the tongue is laterally retracted with a tongue blade, the base of the palatoglossal arch is infiltrated with local anesthetic to block the lingual and pharyngeal branches of the glossopharyngeal nerve. Note that the lingual branches of the glossopharyngeal nerve are not the same as the lingual nerve, which is a branch of the trigeminal nerve.
Bilateral superior laryngeal nerve blocks and a transtracheal block would anesthetize the airway below the epiglottis (Figure 19-39). The hyoid bone is located, and 3 mL of 2% lidocaine is infiltrated 1 cm below each greater cornu, where the internal branch of the superior laryngeal nerves penetrates the thyrohyoid membrane.

A transtracheal block is performed by identifying and penetrating the CTM while the neck is extended. After confirmation of an intratracheal position by aspiration of air, 4 mL of 4% lidocaine is injected into the trachea at end expiration. A deep inhalation and cough immediately following injection distribute the anesthetic throughout the trachea. Although these blocks may allow the awake patient to tolerate intubation better, they also obtund protective cough reflexes, depress the swallowing reflex, and may lead to aspiration. Topical anesthesia of the pharynx may induce a transient obstruction from the loss of reflex regulation of airway caliber at the level of the glottis.
A relatively simple alternative to all of these is to allow the patient to breath atomized lidocaine for several minutes prior to instrumentation as is typically done for outpatient bronchoscopy.
Because of this patient's increased risk for aspiration, local anesthesia might best be limited to the nasal passages. Four percent cocaine has no advantages compared with a mixture of 4% lidocaine and 0.25% phenylephrine and can cause cardiovascular side effects. The maximum safe dose of local anesthetic should be calculated—and not exceeded. Local anesthetic is applied to the nasal mucosa with cotton-tipped applicators until a nasal airway that has been lubricated with lidocaine jelly can be placed into the naris with minimal discomfort. Benzocaine spray is frequently used to topicalize the airway, but can produce methemoglobinemia, and for this reason we prefer lidocaine.

Laryngospasm is always a possible complication of intubation in the nonparalyzed patient, even if the patient remains awake. Laryngospasm may make positive-pressure ventilation with a mask impossible. If succinylcholine is administered to break the spasm, the consequent relaxation of pharyngeal muscles may lead to upper airway obstruction and continued inability to ventilate. In this situation, an emergency cricothyrotomy may be lifesaving.

Other possible strategies include the retrograde passage of a long guidewire or epidural catheter through a needle inserted across the CTM. The catheter is guided cephalad into the pharynx and out through the nose or mouth. An ETT is passed over the catheter, which is withdrawn after the tube has entered the larynx. Variations of this technique include passing the retrograde wire through the suction port of a flexible FOB or the lumen of a reintubation stylet that has been preloaded with an ETT. These thicker shafts help the ETT negotiate the bend into the larynx more easily. Obviously, a vast array of specialized airway equipment exists and must be readily available for management of difficult airways (Table 19-8). Either of these techniques would have been difficult in the patient described in this case because of the swelling and anatomic distortion of the neck that can accompany a submandibular abscess.
TABLE 19-8 Suggested contents of the portable storage unit for difficult airway management.
+ Rigid laryngoscope blades of alternate design and size from those routinely used.
+ Endotracheal tubes (ETTs) of assorted size.
+ ETT guides. Examples include (but are not limited to) semirigid stylets with or without a hollow core for jet ventilation, light wands, and forceps designed to manipulate the distal portion of the ETT.
+ Laryngeal mask airways of assorted sizes.
+ Fiberoptic intubation equipment and assorted video and indirect laryngoscopes.
+ Retrograde intubation equipment.
+ Atleast one device suitable for emergency nonsurgical airway ventilation. Examples include (but are not limited to) transtracheal jet ventilator, hollow jet ventilation stylet, and Combitube.
+ Equipment suitable for emergency surgical airway access (eg, cricothyrotomy).
+ Anexhaled CO, detector.
'Modified with permission from the American Society of Anesthesiologists Task Force on Management of the Difficult Airway. Practice guidelines for management of the difficult airway: A report by the American Society of Anesthesiologists Task Force on Management of the Difficult Airway. Anesthesiology. 2003 May;98(5):1269-1277.

- ? The items listed in this table are suggestions. The contents of the portable storage unit should be customized to meet the specific needs, preferences, and skills of the practitioner and healthcare facility.
What are some approaches when the airway is unexpectedly

The unexpected difficult airway can present both in elective surgical patients and also in emergency intubations in intensive care units, the emergency department, or general hospital wards. Should video laryngoscopy fail even after attempts with an intubating bougie, an intubating LMA should be attempted (Figure 19—40). If ventilation is adequate, an FOB can be loaded with an ETT and passed through the LMA into the trachea. Correct tube position is confirmed by visualization of the carina.
FIGURE 19-40 Intubating laryngeal mask airway.
Apfelbaum
J,
Hagberg
C, of
the difficult
airway:
Anesthesiologists
Caplan
RA,
An updated
Task
Force et
al.
Practice report
on the
guidelines by
the
American for
Society
Management of
the of
Difficult
Airway.
Anesthesiology.
2013;118:1.
- Frerk C, Mitchell V, McNarry A, et al. Difficult Airway Society 2015 guidelines for management of unanticipated difficult intubation in adults. Br J Anaesth. 2015;115:827.

@ Incontrast to action potentials in axons, the spike in cardiac action potentials is followed by a plateau phase that lasts 0.2 to 0.3 s. Whereas the action potential for skeletal muscle and nerves is due to the abrupt opening of voltage-gated sodium channels in the cell membrane, in cardiac muscle it is initiated by voltage-gated sodium channels (the spike) and maintained by voltage-gated calcium channels (the plateau).
@ Potent inhalational agents depress sinoatrial (SA) node automaticity. These agents seem to have only modest direct effects on the atrioventricular (AV) node, prolonging conduction time and increasing refractoriness. This combination of effects likely explains the frequent occurrence of junctional tachycardia when an anticholinergic agent is administered for sinus bradycardia during inhalation anesthesia; junctional pacemakers are accelerated more than those in the SA node.
© Studies suggest that volatile anesthetics depress cardiac contractility by decreasing the entry of Ca** into cells during depolarization (affecting Tand L-type calcium channels), altering the kinetics of Ca?* release and uptake into the sarcoplasmic reticulum, and decreasing the sensitivity of contractile proteins to calcium.
@ Because the normal cardiac index (CI) has a wide range, it is a relatively insensitive measurement of ventricular performance. Abnormalities in CI therefore usually reflect gross ventricular impairment.
@ In the absence of hypoxia or severe anemia, measurement of mixed venous oxygen tension (or saturation) provides an estimate of the adequacy of cardiac output.
Patients with reduced ventricular compliance are most affected by loss of a normally timed atrial systole.
Cardiac output in patients with marked right or left ventricular impairment is very sensitive to acute increases in afterload.
© The ventricular ejection fraction, the fraction of the end-diastolic ventricular volume ejected, is the most commonly used clinical measurement of systolic function.
© Left ventricular diastolic function can be assessed clinically by Doppler echocardiography in a transthoracic or transesophageal examination.
@® Because the endocardium is subjected to the greatest intramural pressures during systole, it tends to be most vulnerable to ischemia during decreases in coronary perfusion pressure.

@ The failing heart becomes increasingly dependent on circulating catecholamines. Abrupt withdrawal in sympathetic outflow or decreases in circulating catecholamine levels, such as can occur following induction of anesthesia, may lead to acute cardiac decompensation.
Anesthesiologists must have a thorough understanding of cardiovascular physiology. Anesthetic successes and failures are often directly related to the skill of the practitioner in manipulating cardiovascular physiology. This chapter reviews the physiology of the heart and the systemic circulation and the pathophysiology of heart failure.
The circulatory system consists of the heart, blood vessels, and blood. Its function is to provide oxygen and nutrients to the tissues and to carry away the products of metabolism. The heart propels blood through two vascular systems arranged in series. In the normally low-pressure pulmonary circulation, venous blood flows past the alveolar—capillary membrane, takes up oxygen, and releases CO). In the high-pressure systemic circulation, oxygenated arterial blood is pumped to tissues, and the byproducts of metabolism are taken up for elimination by the lungs, kidneys, or liver.

The heart can be functionally divided into right and left pumps, each consisting of an atrium and a ventricle. The atria serve as both conduits and priming pumps, whereas the ventricles act as the major pumping chambers. The right ventricle receives systemic venous (deoxygenated) blood and pumps it into the pulmonary circulation, whereas the left ventricle receives pulmonary venous (oxygenated) blood and pumps it into the systemic circulation. Four valves normally ensure unidirectional flow through each chamber. The normal pumping action of the heart is the result of a complex series of electrically driven and mechanical events. Electrical events precede mechanical ones.
The heart consists of specialized striated muscle in a connective tissue skeleton. Cardiac muscle can be divided into atrial, ventricular, and specialized pacemaker and conducting cells. The self-excitatory nature of cardiac muscle cells and their unique organization allow the heart to function as a highly efficient pump. Serial low-resistance connections (intercalated disks) between individual myocardial cells allow the rapid and orderly spread of depolarization in each pumping chamber. Electrical activity readily spreads from one atrium to another and from one ventricle to another via specialized conduction pathways. The normal absence of direct connections between the atria and ventricles except through the atrioventricular (AV) node delays ventricular depolarization, enabling the atria to prime the ventricles.

At rest, the myocardial cell membrane is nominally permeable to K*, but is relatively impermeable to Na*. A membrane-bound Na*—K*-adenosine triphosphatase (ATPase) concentrates K* intracellularly and extrudes Na™ out of the cell. Relative to extracellular concentrations intracellular Na* concentration is kept low and intracellular K* concentration is kept high. The relative impermeability of the membrane to calcium also maintains a high extracellular to cytoplasmic calcium gradient. Movement of K* out of the cell and down its concentration gradient results in a net loss of positive charges from inside the cell. An electrical potential is established across the cell membrane, with the inside of the cell negative with respect to the extracellular environment, because anions do not accompany K*. Thus, the resting membrane potential represents the balance between two opposing forces: the movement of K* down its concentration gradient and the electrical attraction of the negatively charged intracellular space for the positively charged K*.
The normal ventricular cell resting membrane potential is -80 to -90 mV. As with other excitable tissues (nerve, skeletal muscle, and some endocrine cells), when the cell membrane potential becomes less negative and reaches a threshold value, a characteristic action potential (depolarization) develops (Figure 20-1 and 'Table 20-1). The action potential transiently increases the membrane potential of the @ myocardial cell to +20 mV. In contrast to action potentials in axons, the spike in cardiac action potentials is followed by a plateau phase that lasts 0.2 to 0.3 s. Whereas the action potential for skeletal muscle and nerves is due exclusively to the opening of voltage-gated sodium channels, in cardiac muscle the action potential is initiated by voltage-gated sodium channels (the spike) and maintained by voltage-gated calcium channels (the plateau). In pacemaker cells depolarization is initiated by voltage-gated calcium channels rather than by sodium channels. The plateau phase is maintained by calcium flow into the cell and potassium flow out of the cell. Voltage-gated sodium channels inactivate and cease to conduct within milliseconds of opening. Subsequent termination of calcium channel permeability and activation of several forms of voltage-gated potassium channels restores the membrane potential to its resting value.

FIGURE 20-1 Cardiac action potentials. A: Note the characteristic contours of action potentials recorded from different parts of the heart. LAF, left anterior fascicle. B: Pacemaker cells in the sinoatrial (SA) node lack the same distinct phases as atrial and ventricular muscle cells and display prominent spontaneous diastolic depolarization. See Table 20—1 for an explanation of the different phases of the action potential. (Modified with permission from Barrett KE. Ganong's Review of Medical Physiology. 24th ed. New York, NY: McGraw-Hill; 2012.)
TABLE 20-1 Cardiac action potential.
0, Name = Upstroke. 0, Event = Activation (opening) of voltage-gated Na* channels. 0, Cellular lon Movement = —_Na* entry and decreased permeability to K~. 1, Name = Early rapid repolarization —_. 1, Event = Inactivation of Nav channel and transient increasein K* permeability. 1, Cellular lon Movement = K~ out (,,). 2, Name = Plateau. 2, Event = Activation of slow calcium channels. 2, Cellular lon Movement = Ca* entry. 3, Name = Final repolarization. 3, Event = Inactivation of calcium channels and increased permeability to K~. 3, Cellular lon Movement = K> out. 4, Name = Resting potential Diastolic repolarization. 4, Event = Normal permeability restored (atrial and ventricular cells) Intrinsic slow leakage of Ca** into cells that spontaneously depolarize. 4, Cellular lon Movement = | Na*-K*-ATPase pumps K’ in and Na* out Ca* in

Following depolarization, the cells are typically refractory to subsequent normal depolarizing stimuli until "phase 4." The absolute refractory period is the minimum interval between two maximal stimuli that will result in an action potential. The relative refractory period is the additional time beyond the absolute refractory period in which a maximal stimulus, but not a stimulus of normal intensity, will produce a depolarization. Table 20—2 lists some of the multiple types of ion channels in cardiac muscle membrane. Some are activated by a change in cell membrane voltage, whereas others open only when bound by ligands. T-type (transient) voltage-gated calcium channels play a role in phase 0 of depolarization. (A more modern nomenclature for voltage-gated calcium channels—Cav1, Cav2, Cav3—is creeping into the scientific literature but remains uncommon in clinical usage.) During the plateau phase (phase 2), Ca?* inflow occurs through slow L-type (long-lasting), voltage-gated calcium channels. Multiple potassium channel forms contribute repolarization, and a full discussion of this topic is beyond the focus of this textbook.
TABLE 20-2 Cardiac ion channels.!2

Na*
Gasn
L Ca’*
Kr
Transient outward
Inward rectifying
Slow (delayed) rectifying

Ca** activated
Na* activated
ATP sensitive
Acetylcholine activated
Arachidonic acid activated
'Reproduced with permission from Ganong WF. Review of Medical Physiology. 21st ed. New York, NY: McGraw-Hill Education; 2003.
2ATP, adenosine triphosphate.

The cardiac impulse normally originates in the sinoatrial (SA) node, a group of specialized pacemaker cells in the sulcus terminalis, located posteriorly at the junction of the right atrium and the superior vena cava. The slow influx of Na* through so-called hyperpolarization-activated cyclic nucleotide-gated (HCN) channels results in a less negative resting membrane potential (50 to -60 mV). This has three important consequences: near constant inactivation of voltagegated sodium channels, an action potential that is primarily due to ion movement across the slow calcium channels and with a threshold of -40 mV, and regular spontaneous depolarizations. During each cycle, Na* ions passing through HCN channels causes the cell membrane to become progressively less negative; when the threshold potential is reached, calcium channels open and an action potential develops. Inactivation of L-type calcium channels and activation of potassium channels returns the cells in the SA node to their normal resting membrane potential.
The impulse generated at the SA node is normally rapidly conducted across the atria and to the AV node. Specialized atrial fibers may speed up conduction to both the left atrium and the AV node. The AV node is located in the septal
and sufentanil, can depress cardiac conduction, increasing the time of AV node conduction and the refractory period and prolonging the duration of the Purkinje fiber action potential.

Local anesthetics have important electrophysiological effects on the heart at blood concentrations that are generally associated with systemic toxicity. In the case of lidocaine, electrophysiological effects at low blood concentrations can be therapeutic. At high blood concentrations, local anesthetics depress conduction; at extremely high concentrations, they also depress the SA node. The most potent local anesthetics—bupivacaine, etidocaine, and to a lesser degree, ropivacaine—seem to have the most potent effects on the heart, particularly on Purkinje fibers and ventricular muscle. Bupivacaine, like all local anesthetics, preferentially binds open or inactivated sodium channels. Bupivacaine dissociates from them more slowly than less toxic agents. It can cause profound sinus bradycardia and sinus node arrest and malignant ventricular arrhythmias; furthermore, it can depress left ventricular contractility. Twenty percent lipid emulsions have been used to treat local anesthetic cardiac toxicity. The mechanisms of action of this therapy are unclear, although most likely the lipid serves as reservoir for circulating bupivacaine, decreasing the amount of bupivacaine in the myocardium.
The clinically used calcium channel blockers are organic compounds that block Ca?* influx through L-type but not T-type channels. In a manner reminiscent of local anesthetics and sodium channels, agents such as verapamil, and to a lesser extent, diltiazem, preferentially bind the calcium channel in its depolarized inactivated state (use-dependent blockade). Calcium channel blockers are employed perioperatively as antihypertensives and as antiarrhythmics.

Myocardial cells contract as a result of the interaction of two overlapping, rigid contractile proteins, actin and myosin. Dystrophin, a large intracellular protein, connects actin to the cell membrane (sarcolemma). Cell shortening occurs when the actin and myosin are allowed to fully interact and slide over one another. This interaction is normally prevented by two regulatory proteins, troponin and tropomyosin; troponin is composed of three subunits (troponin I, troponin C, and troponin T). Troponin is attached to actin at regular intervals, whereas tropomyosin lies within the center of the actin structure. An increase in intracellular Ca** concentration (from about 10-7 to 10-° mol/L) promotes contraction as Ca?* ions bind troponin C. The resulting conformational change in these regulatory proteins exposes the active sites on actin that allow interaction with myosin bridges (points of overlapping). The active site on myosin functions as a magnesium-dependent ATPase whose activity is enhanced by the increase in intracellular Ca** concentration. A series of attachments and disengagements occur as each myosin bridge advances over successive active sites on actin. Adenosine triphosphate (ATP) is consumed during each attachment. Relaxation occurs as Ca?" is actively pumped back into the sarcoplasmic reticulum by a Ca**—Mg?*-ATPase; the resulting drop in intracellular Ca** concentration allows the troponin—tropomyosin complex to again prevent the interaction between actin and myosin.

The quantity of Ca2* ions required to initiate contraction exceeds that entering the cell through L-type calcium channels during phase 2. The small amount that does enter through slow calcium channels triggers the release of much larger amounts of Ca?* stored intracellularly (calcium-dependent calcium release) within cisterns in the sarcoplasmic reticulum.
The action potential of muscle cells depolarizes their T systems, tubular extensions of the cell membrane that transverse the cell in close approximation to the muscle fibrils, via L-type voltage-gated calcium channels. This initial increase in intracellular Ca?* triggers an even greater Ca** inflow across ryanodine receptors, a nonvoltage-dependent calcium channel found in the sarcoplasmic reticulum. The force of contraction is directly dependent on the magnitude of the initial Ca** influx.
During relaxation, when the L-type channels close, a membrane-bound Ca**— Mg?*-ATPase actively transports Ca?* back into the sarcoplasmic reticulum. Thus, relaxation of the heart also requires ATP. Ca** is also extruded extracellularly by an exchange of intracellular Ca?* for extracellular sodium in a 1:3 ratio by the Na*—Ca?* exchanger in the cell membrane (Figure 20-2).
FIGURE 20-2 Excitation—contraction coupling, sarcomere shortening, and relaxation. (Reproduced with permission from Mohrman DE, Heller LJ. Cardiovascular Physiology. 8th ed. New York, NY: McGraw-Hill Education; 2014.)

The quantity of intracellular Ca** available, its rate of delivery, and its rate of removal determine, respectively, the maximum tension developed, the rate of contraction, and the rate of relaxation. Sympathetic stimulation increases the force of contraction by raising intracellular Ca** concentration via a B,adrenergic receptor-mediated increase in intracellular cyclic adenosine monophosphate (cAMP) through the action of a stimulatory G protein. The increase in cAMP recruits additional open calcium channels. Phosphodiesterase inhibitors, such as milrinone, produce similar effects by preventing the breakdown of intracellular cAMP. Milrinone has lusitropic effects, improving relaxation and diastolic function. Digitalis glycosides increase intracellular Ca2* concentration through inhibition of the membrane-bound Na*—K*-ATPase; the resulting small increase in intracellular Na* relatively disfavors the normal pumping of Ca** from the cytoplasm via the Na*—Ca** exchange mechanism, increasing the intracellular Ca** concentration. Glucagon enhances contractility by increasing intracellular cAMP levels via activation of a specific receptor. The newer agent levosimendan is a calcium sensitizer that enhances contractility by binding to troponin C. The investigational drug omecamtiv mecarbil is a myosin activator that increases the duration of cardiac muscle contractility. Istaroxime has both inotropic and lusitropic effects, improving both systolic and diastolic function (Table 20-3).

Digoxin, Mechanism = Na-K pump inhibitor, raises SR calcium. Dopamine, Mechanism = Dose-dependent D,, a,-, and B.- adrendergic receptor agonist. Norepinephrine, Mechanism = B,- and a,-adrenergic receptor agonist. Dobutamine, Mechanism = 6,- and B,-adrenergic receptor agonist. Milrinone, Mechanism = PDE inhibitor, raises SR calcium. Levosimendan, Mechanism = Myofilament calcium sensitizer, PDE-3 inhibitor. Omecamtiv mecarbil, Mechanism = Potentiates the effects of myosin on actin to prolong systole. Istaroxime, Mechanism = Na-K pump inhibitor, PDE inhibitor. SERCA2a gene therapy, Mechanism = Restoration of SERCA2a to improve calcium release and reuptake from the SR
'Modified with permission from Francis GS, Bartos JA, Adatya § et al. Inotropes. J/Am Coll Cardiol. 2014 May 27;63:2069-2078.
*PDE, phosphodiesterase; SERCA, sarco/endoplasmic reticulum Ca?*ATPase; SR, sarcoplasmic reticulum.
In contrast, release of acetylcholine following vagal stimulation depresses contractility through increased cyclic guanosine monophosphate (cGMP) levels and inhibition of adenylyl cyclase; these effects are mediated by an inhibitory G
protein. Acidosis inhibits L-type calcium channels and therefore also depresses cardiac contractility by unfavorably altering intracellular Ca** kinetics (Figure 20-3).

FIGURE 20-3 Diagram of intracellular signaling cascades within cardiomyocytes altered by inotropes Dopamine, Dobutamine, and Norepinephrine that activate the B,-adrenergic receptor, which activates the G protein Gas, which in turn, activates adenylyl cyclase. Adenylyl cyclase converts ATP to cAMP when activated. cAMP can activate PKA, which then phosphorylates the L-type calcium channel, among other targets. cAMP is converted to AMP by PDE. Milrinone inhibits PDE-3, thereby increasing the effective concentration of cAMP. Calcium influx through the L-type calcium channel induces activation of ryanodine receptors, leading to calcium-induced Ca** release. Free intracellular Ca** interacts with troponin C, which changes the binding properties of tropomyosin and allows the interaction between actin and myosin. Levosimendan potentiates the interaction between troponin and calcium. It may also have PDE-3 inhibitor activity. Omecamtiv mecarbil increases the rate of ATP turnover and slows the rate of ADP release, thereby increasing the number of myosin molecules bound to actin at any given time. SERCA is responsible for uptake of calcium into the sarcoplasmic reticulum while the Na*—K*-ATPase participates in resetting the membrane potential of the cell. Istaroxime inhibits Na*—K*-ATPase while also potentiating SERCA.

Digoxin inhibits the Na'"-K*-ATPase. Red arrows denote agonists, whereas black arrows signify antagonists. AC, adenylyl cyclase; ADP, adenosine diphosphate; ATP, adenosine triphosphate; 8, AR, B,-adrenergic receptor; cAMP, cyclic adenosine monophosphate; LTCC, L-type calcium channel; PDE, phosphodiesterase; PKA, protein kinase A; RyR, ryanodine receptor; SERCA, sarco/endoplasmic reticulum Ca**—ATPase. (Reproduced with permission from Francis GS, Bartos JA, Adatya S, et al. Inotropes. J Am Coll Cardiol. 2014 May 27;63:2069-2078.)
® Studies suggest that volatile anesthetics depress cardiac contractility by decreasing the entry of Ca** into cells during depolarization (affecting Tand Ltype calcium channels), altering the kinetics of Ca** release and uptake into the sarcoplasmic reticulum, and decreasing the sensitivity of contractile proteins to Ca?*. Inhalational anesthetics appear to have minimal effect upon early diastolic relaxation in subjects without known diastolic dysfunction. However, they do decrease atrial function perhaps resulting in reduced late diastolic filling. Anesthetic-induced cardiac depression is potentiated by hypocalcemia, Badrenergic blockade, and calcium channel blockers. Nitrous oxide also produces concentration-dependent decreases in contractility by reducing the availability of intracellular Ca** during contraction. The mechanisms of direct cardiac depression from intravenous anesthetics are not well established, but presumably involve similar actions. Of all the typical intravenous induction agents, ketamine seems to have the least depressant effect on contractility due to its central nervous system excitatory effects.

Parasympathetic fibers innervate the atria and conducting tissues. Acetylcholine acts on specific cardiac muscarinic receptors (M,) to produce negative chronotropic, dromotropic, and inotropic effects. In contrast, sympathetic fibers are more widely distributed throughout the heart. Cardiac sympathetic fibers originate in the thoracic spinal cord (T1—-T4) and travel to the heart initially through the cervical (stellate) ganglia and from the ganglia as the cardiac nerves. Norepinephrine release at the heart causes positive chronotropic, dromotropic, and inotropic effects primarily through activation of B,-adrenergic receptors. B5Adrenergic receptors are normally fewer in number and are found primarily in the atria; activation increases heart rate and, to a lesser extent, contractility. The relative fraction of B5to B,-adrenergic receptors increases in heart failure.
Cardiac autonomic innervation has an apparent sidedness, because the right sympathetic and right vagus nerves primarily affect the SA node, whereas the left sympathetic and vagus nerves principally affect the AV node. Vagal effects frequently have a very rapid onset and resolution, whereas sympathetic influences generally have a more gradual onset and dissipation. Sinus arrhythmia is a cyclic variation in heart rate that corresponds to respiration (increasing with inspiration and decreasing during expiration); it is due to cyclic changes in vagal tone.

The cardiac cycle can be defined by both electrical and mechanical events (Figure 20-4). Systole refers to contraction and diastole refers to relaxation. Most diastolic ventricular filling occurs passively before atrial contraction. Contraction of the atria normally contributes 20% to 30% of ventricular filling. Three waves can generally be identified on atrial or central venous pressure tracings (Figure 20—4). The a wave is due to atrial systole. The c wave coincides with ventricular contraction and is said to be caused by bulging of the AV valve into the atrium. The v wave is the result of pressure buildup from venous return before the AV valve opens again. The x descent is the decline in pressure between the c and v waves and is thought to be due to a pulling down of the atrium by ventricular contraction. Incompetence of the AV valve on either side of the heart abolishes the x descent on that side, resulting in a prominent cv wave. The y descent follows the v wave and represents the decline in atrial pressure as the AV valve opens. The notch in the aortic pressure tracing is referred to as the incisura and is said to represent the brief pressure change from transient backflow of blood into the left ventricle just before aortic valve closure.
- 5. Filling
FIGURE 20-4 The normal cardiac cycle. Note the correspondence between electrical and mechanical events. (Modified with permission from Barrett KE. Ganong's Review of Medical Physiology. 25th ed. New York, NY: McGraw-Hill Education; 2016.)

Discussions of ventricular function usually refer to the left ventricle, but the same concepts apply to the right ventricle. Although the ventricles are often thought of as functioning separately, their interdependence has clearly been demonstrated. Moreover, factors affecting systolic and diastolic functions can be differentiated: Systolic function involves ventricular ejection, whereas diastolic function is related to ventricular filling.
Ventricular systolic function is often (erroneously) equated with cardiac output, which can be defined as the volume of blood pumped by the heart per minute. Because the two ventricles function in series, their outputs are normally equal. Cardiac output (CO) is expressed by the following equation:

where SV is the stroke volume (the volume pumped per contraction) and HR is heart rate. To compensate for variations in body size, CO is often expressed in terms of total body surface area:
where CI is the cardiac index and BSA is body surface area. BSA is usually obtained from nomograms based on height and weight. Normal Cl is 2.5 to 4.2 @ L/min/m?. Because the normal CI has a wide range, it is a relatively insensitive measurement of ventricular performance. Abnormalities in CI therefore usually reflect gross ventricular impairment. A more accurate assessment can be obtained if the response of the cardiac output to exercise is evaluated. Under these conditions, failure of the cardiac output to increase and keep up with oxygen consumption is reflected by a decreasing mixed venous oxygen saturation. A decrease in mixed venous oxygen saturation in response to increased demand @ usually reflects inadequate tissue perfusion. Thus, in the absence of hypoxia or severe anemia, measurement of mixed venous oxygen tension (or saturation) provides an estimate of the adequacy of cardiac output.

When stroke volume remains constant, cardiac output is directly proportional to heart rate. Heart rate is an intrinsic function of the SA node (spontaneous depolarization), but is modified by autonomic, humoral, and local factors. The normal intrinsic rate of the SA node in young adults is about 90 to 100 beats/min, but it decreases with age based on the following formula:
Normal intrinsic heart rate = 118 beats/min — (0.57 x Age)
Enhanced vagal activity slows the heart rate via stimulation of M, cholinergic receptors, whereas enhanced sympathetic activity increases the heart rate mainly through activation of 8,-adrenergic receptors and, to lesser extent, B5-adrenergic receptors (see above).

Stroke volume is normally determined by three major factors: preload, afterload, and contractility. This analysis is analogous to laboratory observations on skeletal muscle preparations. Preload is muscle length prior to contraction, whereas afterload is the tension against which the muscle must contract. Contractility is an intrinsic property of the muscle that is related to the force of contraction but is independent of both preload and afterload. Because the heart is a three-dimensional multichambered pump, both ventricular geometric form and valvular dysfunction can also affect stroke volume (Table 20-4).

Preload
Afterload
Contractility
Wall motion abnormalities
Valvular dysfunction

Ventricular preload is end-diastolic volume, which is generally dependent on ventricular filling. The relationship between cardiac output and left ventricular end-diastolic volume was first described by Starling (Figure 20—5). Note that when the heart rate and contractility remain constant, cardiac output increases with increasing preload until excessive end-diastolic volumes are reached. At that point, cardiac output does not appreciably change—or may even decrease. Excessive distention of either ventricle can lead to dilation and incompetence of the AV valves.
FIGURE 20-5 Starling's law of the heart. (Reproduced with permission from Braunwald E, Ross J, Sonnenblick EH. Mechanisms of contraction of the normal and failing heart. N Engl J Med. 1967 Oct 12;277(15):794-800.)

Ventricular filling can be influenced by a variety of factors (Table 20-5), of which the most important is venous return. The heart cannot pump what the heart does not receive; therefore, venous return normally equals cardiac output. Because most of the other factors affecting venous return are usually fixed, vascular capacity is normally its major determinant. Increases in metabolic activity reduce vascular capacity, so that venous return to the heart and cardiac output increase as the volume of venous capacitance vessels decreases. Changes in blood volume and vascular capacity are important causes of intraoperative and postoperative changes in ventricular filling and cardiac output. Any factor that alters the normally small venous pressure gradient favoring blood return to the heart also affects cardiac filling. Such factors include changes in intrathoracic pressure (positive-pressure ventilation or thoracotomy), posture (positioning during surgery), and pericardial pressure (pericardial disease).

Blood volume
Distribution of blood volume
Posture
Intrathoracic pressure
Pericardial pressure
Venous tone
Rhythm (atrial contraction)
Heart rate
The most important determinant of right ventricular preload is venous return. In the absence of significant pulmonary or right ventricular dysfunction, venous return is also the major determinant of left ventricular preload.
Both heart rate and rhythm can affect ventricular preload. Increases in heart rate are associated with proportionately greater reductions in diastole than systole. Ventricular filling therefore progressively becomes impaired at increased heart rates (>120 beats/min in adults). Absent (atrial fibrillation), ineffective (atrial flutter), or altered timing of atrial contraction (low atrial or junctional rhythms) can also reduce ventricular filling by 20% to 30%. @ Patients with reduced ventricular compliance are more affected by the loss of a normally timed atrial systole than are those with normal ventricular compliance.

occlusion pressure) are potential means of estimating left ventricular preload. Changes in central venous pressure can be used as a rough index for changes in right and left ventricular preload in most normal individuals.

Factors affecting ventricular compliance can be separated into those related to the rate of relaxation (early diastolic compliance) and passive stiffness of the ventricles (late diastolic compliance). Hypertrophy (from hypertension or aortic valve stenosis), ischemia, and asynchrony reduce early compliance; hypertrophy and fibrosis reduce late compliance. Extrinsic factors (such as pericardial disease, excessive distention of the contralateral ventricle, increased airway or pleural pressure, tumors, and surgical compression) can also reduce ventricular compliance. Because of its normally thinner wall, the right ventricle is more compliant than the left.

Afterload for the intact heart is commonly equated with either ventricular wall tension during systole or arterial impedance to ejection. Wall tension may be thought of as the pressure the ventricle must overcome to reduce its cavity volume. If the ventricle is assumed to be spherical, ventricular wall tension can be expressed by Laplace's law:
where P is intraventricular pressure, R is the ventricular radius, and H is wall thickness. Although the normal ventricle is usually ellipsoidal, this relationship is still useful. The larger the ventricular radius, the greater the wall tension required to develop the same ventricular pressure. Conversely, an increase in wall thickness reduces ventricular wall tension.
Systolic intraventricular pressure is dependent on the force of ventricular contraction; the viscoelastic properties of the aorta, its proximal branches, and blood (viscosity and density); and systemic vascular resistance (SVR). Arteriolar tone is the primary determinant of SVR. Because viscoelastic properties are generally fixed in any given patient, left ventricular afterload is usually equated clinically with SVR, which is calculated by the following equation:
where MAP is mean arterial pressure in millimeters of mercury, CVP is central venous pressure in millimeters of mercury, and CO is cardiac output in liters per minute. Normal SVR is 900-1500 dyn -s cm~°. Systolic blood pressure may also be used as an approximation of left ventricular afterload in the absence of chronic changes in the size, shape, or thickness of the ventricular wall or acute changes in systemic vascular resistance. Some clinicians prefer to use CI instead of CO in calculating a systemic vascular resistance index (SVRJ), so that SVRI = SVR x BSA (body surface area).
Right ventricular afterload is mainly dependent on pulmonary vascular resistance (PVR) and is expressed by the following equation:
where PAP is mean pulmonary artery pressure and LAP is left atrial pressure. In practice, pulmonary capillary wedge pressure (PCWP) can be substituted as an approximation for LAP. Normal PVR is 50 to 150 dyn: s cm>.

Cardiac output is inversely related to large changes in afterload on the left ventricle; however, small increases or decreases in afterload may have no effect on cardiac output. Because of its thinner wall, the right ventricle is more sensitive to changes @ in afterload than is the left ventricle. Cardiac output in patients with marked right or left ventricular impairment is very sensitive to acute increases in afterload. The latter is particularly true in the presence of drugor ischemia-induced myocardial depression or chronic heart failure.

Cardiac contractility (inotropy) is the intrinsic ability of the myocardium to pump in the absence of changes in preload or afterload. Contractility is related to the rate of myocardial muscle shortening, which is, in turn, dependent on the intracellular Ca** concentration during systole. Increases in heart rate can also enhance contractility under some conditions, perhaps because of the increased availability of intracellular Ca?*.
Contractility can be altered by neural, humoral, or pharmacological influences. Sympathetic nervous system activity normally has the most important effect on contractility. Sympathetic fibers innervate atrial and ventricular muscle, as well as nodal tissues. In addition to its positive chronotropic effect, norepinephrine release also enhances contractility primarily via B,-receptor activation. a-Adrenergic receptors are also present in the myocardium, but seem to have only minor positive inotropic and chronotropic effects that are overwhelmed by the vascular actions of a-adrenergic agonists when these drugs are administered systemically. Sympathomimetic drugs and secretion of epinephrine from the adrenal glands similarly increase contractility via B,-receptor activation.
Myocardial contractility is depressed by hypoxia, acidosis, depletion of catecholamine stores within the heart, and loss of functioning muscle mass as a result of ischemia or infarction. At large enough doses, most anesthetics and antiarrhythmic agents are negative inotropes (ie, they decrease contractility).

Regional wall motion abnormalities cause a breakdown of the analogy between the intact heart and skeletal muscle preparations. Such abnormalities may be due to ischemia, scarring, hypertrophy, or altered conduction. When the ventricular cavity does not collapse symmetrically or fully, emptying becomes impaired. Hypokinesis (decreased contraction), akinesis (failure to contract), and dyskinesis (paradoxic bulging) during systole reflect increasing degrees of contraction abnormalities. Although contractility may be normal or even enhanced in some areas, abnormalities in other areas of the ventricle can impair emptying and reduce stroke volume. The severity of the impairment depends on the size and number of abnormally contracting areas.

Valvular dysfunction can involve any one of the four valves in the heart and can include stenosis, regurgitation (incompetence), or both. Stenosis of an AV valve (tricuspid or mitral) reduces stroke volume primarily by decreasing ventricular preload, whereas stenosis of a semilunar valve (pulmonary or aortic) reduces stroke volume primarily by increasing ventricular afterload. In contrast, valvular regurgitation can reduce stroke volume without changes in preload, afterload, or contractility and without wall motion abnormalities. The effective stroke volume is reduced by the regurgitant volume with every contraction. When an AV valve is incompetent, a significant part of the ventricular end-diastolic volume can flow backward into the atrium during systole; the stroke volume is reduced by the regurgitant volume. Similarly, when a semilunar valve is incompetent, a fraction of end-diastolic volume arises from backward flow into the ventricle during diastole.

Plotting cardiac output or stroke volume against preload can be useful in evaluating pathological states and understanding drug therapy. Normal right and left ventricular function curves are shown in Figure 20—7.
FIGURE 20-7 Function curves for the left and right ventricles.
Ventricular pressure—volume diagrams dissociate contractility from both preload and afterload. Two points are identified on such diagrams: the endsystolic point (ESP) and the end-diastolic point (EDP) (Figure 20-8). ESP is reflective of systolic function, whereas EDP is more reflective of diastolic function. For any given contractile state, all ESPs are on the same line (ie, the relationship between end-systolic volume and end-systolic pressure is fixed). Figure 20—9 describes the events occurring in the left ventricle during each cardiac cycle.
FIGURE 20-8 Ventricular pressure-volume diagrams. A: A single ventricular contraction. Note that stroke volume represents change in volume on the x-axis (difference between end-systolic volume and end-diastolic volume). Note also that the circumscribed area represents external work performed by the ventricle. B: Increasing preload with constant contractility and afterload. C: Increasing afterload with constant preload and contractility. D: Increasing contractility with constant preload and afterload. ESP, end-systolic point; EDP, end-diastolic point.

hemodynamic changes in the left ventricle during a heartbeat and the ejection of one stroke volume (SV). Segment A-B occurs at the beginning of systole after the mitral valve closes. Point A: The left ventricular end-diastolic pressure is noted on the graph at the end of diastolic filling. Pressure in the ventricle gradually builds until point B is reached, when the aortic valve opens and blood is ejected from the ventricle. The aortic valve closes when ejection is complete at end systole. The line at point D identifies end systole. Moving the slope of this line to the reader's right represents a shift to a less contractile state. Moving the slope to the left reflects a more highly contractile ventricle. Segment D-E represents isovolumetric relaxation. Once pressure in the ventricle is reduced the mitral valve again opens (point E) and diastolic filling resumes in preparation for the heart's next systole. Point C reflects peak systolic pressure. ESPV, endsystolic pressure-volume; LVEDP, left ventricular end-diastolic pressure; LVEDV, left ventricular end-diastolic volume. (Reproduced with permission from Hoffman WJ, Wasnick JD. Postoperative Critical Care of the Massachusetts General Hospital. 2nd ed. Boston, MA: Little, Brown and Company; 1992.)

The change in ventricular pressure over time during systole (dP/dt) is defined by the first derivative of the ventricular pressure curve and can be used as a measure of contractility. Contractility is directly proportional to dP/dt, but accurate measurement of this value requires a high-fidelity ("Millar") ventricular catheter. Ventricular systolic function is routinely estimated using echocardiography.

@ The ventricular ejection fraction (EF), the fraction of the end-diastolic ventricular volume ejected, is the most commonly used clinical measurement of systolic function. EF can be calculated by the following equation:
where EDV is left ventricular diastolic volume and ESV is end-systolic volume. Normal EF is approximately 0.67 + 0.08. Measurements can be made preoperatively from cardiac catheterization, radionucleotide studies, or transthoracic (TTE) or transesophageal echocardiography (TEE). Pulmonary artery catheters with fast-response thermistors allow measurement of the right ventricular EF. Unfortunately, when pulmonary vascular resistance increases, decreases in right ventricular EF may reflect afterload rather than contractility. Left ventricular EF is not an accurate measure of ventricular contractility in the presence of mitral insufficiency.
Myocardial deformation analysis provides another measure to quantify ventricular function by assessing the movement of speckles from echocardiographic images. Deformation of the heart occurs along three dimensions: circumferential, radial, and longitudinal (Figure 20—10). Strain is a measure of the change of length between two points. Using strain analysis, echocardiography can determine the percent change in length at different points of the myocardium (Figure 20-11). Longitudinal strain is measured at various segments during systole and diastole; the global strain is -21%, reflecting the average deformation for all segments. Normal estimates for strain under anesthesia are not established; however, —19% to —22% longitudinal strain is a normal range established for healthy patients undergoing transthoracic echocardiography. Longitudinal strain is a negative change because during systole the ventricle shortens, resulting in L being less than Lp. The degree to which estimates of myocardial deformation will be incorporated into perioperative management remains to be determined.
€=(L-Lylp= ALM

FIGURE 20-10 Strain describes fractional change of a myocardial segment length compared with its initial length (at end-diastole). ¢, myocardial strain; L, length at end-systole; Lo, initial length. (Reproduced with permission from Cleveland Clinic Center for Medical Art & Photography © 2013. All Rights Reserved.)
FIGURE 20-11 Longitudinal strain analysis assessed with Cardiac Motion Quantification (CMQ) (Philips Medical Systems, Andover, MA). A: Midesophageal two-chamber echocardiographic view depicting the left ventricle, where the myocardial walls are divided into six segments distinguished by colorcoded labels and dots. Segmental strain measurements are shown adjacent to each segment. The myocardium is colored in shades of red corresponding to percent longitudinal shortening (strain) measured by the red-to-blue scale on the upper right-hand side. B: Longitudinal strain curves, which are color-coded to correspond to myocardial segments in A, with time on the x-axis relative to the cardiac cycle and percent shortening (strain) on the y-axis, demonstrate shortening during systole and returning to baseline at end-diastole. The pink curve, for example, represents the apical anterior wall that experiences peak shortening of -26% during systole (yellow dot identifies peak systolic strain). In contrast, end-systolic strain, measured at time of aortic valve closure, measures —23%, demonstrating that peak strain may differ from end-systolic strain. The dotted white curve represents global longitudinal strain. BA, basal anterior; MA, mid anterior; ApA, apical anterior, BI, basal inferior; MI, mid inferior; Apl, apical inferior myocardial walls; G.L.Strain, global longitudinal strain; AVC, aortic valve closure. (Reproduced with permission from Cleveland Clinic Center for Medical Art & Photography © 2013. All Rights Reserved.)

© Left ventricular diastolic function can be assessed clinically by Doppler echocardiography on a transthoracic or transesophageal examination. Flow velocities are measured across the mitral valve during diastole, or by interrogation of the motion of the mitral valve annulus ("tissue Doppler"). Three patterns of diastolic dysfunction are generally recognized based on isovolumetric relaxation time, the ratio of peak early diastolic flow (E) to peak atrial systolic flow (A), and the deceleration time (DT) of E (DT) (Figure 20-12). Tissue Doppler is frequently used to distinguish "pseudonormal" from normal diastolic function. Tissue Doppler discerns the velocity of movement of the myocardial tissue during the cardiac cycle. During systole the mitral annulus moves toward the apex of the heart, away from the echocardiography probe in the esophagus. A negative deflection s' wave is generated, reflecting systolic movement away from the probe. During diastolic filling the mitral annulus moves toward the transesophageal echocardiography probe, producing a positive e' wave. An e' wave peak velocity of less than 8 cm/s is associated with impaired diastolic function. An E/e' wave ratio that is greater than 15 is consistent with elevated left ventricular end-diastolic pressure (Figures 20—13 and 20-14).
IVRT,  = . IVRT, Impaired relaxation = >100 ms. IVRT, Pseudonormalization = 70-90 ms. IVRT, Restricted = <90 ms. E/A ratio,  = . E/A ratio, Impaired relaxation = <0.8. E/A ratio, Pseudonormalization = 0.8 - 12. E/A ratio, Restricted = >1.2. DT,  = 150 — 300 ms. DT, Impaired relaxation = . DT, Pseudonormalization = 150 — 300 ms. DT, Restricted = <150 ms
FIGURE 20-12 Doppler echocardiography of diastolic flow across the mitral valve. A—-D (from left to right) represents increasing severity of diastolic

FIGURE 20-13 Tissue Doppler. A: Tissue Doppler at the lateral mitral annulus. During diastole the annulus moves toward the transesophageal examination transducer in the esophagus. Thus the e' and a' waves of diastolic filling are positive deflections above the baseline. B: When transesophageal examination is used to measure transmitral diastolic inflow, the E and A waves of early and late filling are below the baseline because flow is moving away from the Doppler probe in the esophagus. Tissue Doppler can be used to distinguish normal from pseudonormal diastolic inflow pattern because the e' wave remains depressed as diastolic dysfunction progresses. (Reproduced with permission from Wasnick JD, Hillel Z, Kramer D, et al. Cardiac Anesthesia and Transesophageal Echocardiography. New York, NY: McGrawHill Education; 2011.)
FIGURE 20-14 The E' wave of early diastolic filling is demonstrated in this tissue doppler tracing. Early filling occurs as the ventricle relaxes during diastole and the mitral valve opens. The A' wave reflects the contribution of atrial contraction to diastolic filling. Lastly, the S' wave demonstrates the movement of the lateral mitral annulus away from the tee probe during systole. (Reproduced with permission from Wasnick JD, Hillel Z, Kramer D, et al. Cardiac Anesthesia and Transesophageal Echocardiography. New York, NY: McGraw-Hill Education; 2011.)

The systemic vasculature can be divided functionally into arteries, arterioles, capillaries, and veins. Arteries are the high-pressure conduits that supply the various organs. Arterioles are the small vessels that directly feed and control blood flow through each capillary bed. Capillaries are thin-walled vessels that allow the exchange of nutrients between blood and tissues. Veins return blood from capillary beds to the heart.
The distribution of blood between the various components of the circulatory system is shown in Table 20-6. Note that most of the blood volume is in the systemic circulation—specifically, within systemic veins. Changes in systemic venous tone allow these vessels to function as a reservoir for blood. Following significant blood or fluid losses, a sympathetically mediated increase in vascular tone reduces the capacity of these vessels and shifts blood into other parts of the vascular system. Conversely, increased capacity ("venodilation") allows these vessels to accommodate increases in blood volume. Sympathetic control of vascular tone is an important determinant of venous return to the heart. Reduced venous tone following induction of anesthesia frequently results in pooling of blood, reduced cardiac output, and hypotension.

Heart, 1 = 7%. Pulmonary circulation, 1 = 9%. Systemic circulation, 1 = . Arterial, 1 = 15%. Capillary, 1 = 5%. Venous, 1 = 64%
Many factors influence blood flow in the vascular tree, including metabolic products, endothelium-derived factors, the autonomic nervous system, and circulating hormones.

Most tissue beds regulate their own blood flow (autoregulation). Arterioles generally dilate in response to reduced perfusion pressure or increased tissue demand. Conversely, arterioles constrict in response to increased pressure or reduced tissue demand. These phenomena are likely due to both an intrinsic response of vascular smooth muscle to stretch and the accumulation of vasodilatory metabolic byproducts. The latter may include K*, H*, CO,, adenosine, and lactate.

The vascular endothelium is metabolically active in elaborating or modifying substances that directly or indirectly play a major role in controlling blood pressure and flow. These include vasodilators (eg, nitric oxide, prostacyclin [PGI,]), vasoconstrictors (eg, endothelins, thromboxane A,), anticoagulants (eg, thrombomodulin, protein C), fibrinolytics (eg, tissue plasminogen activator), and factors that inhibit platelet aggregation (eg, nitric oxide and PGI,). Nitric oxide is synthesized from arginine by nitric oxide synthetase. This substance has a number of functions. It binds guanylate cyclase, increasing cGMP levels and potently produces vasodilation. Endothelially derived vasoconstrictors (endothelins) are released in response to thrombin and epinephrine.

other areas in the brainstem. Areas in the posterolateral medulla receive input from both the vagal and the glossopharyngeal nerves and play an important role in mediating a variety of circulatory reflexes. The sympathetic system normally maintains some tonic vasoconstriction on the vascular tree. Loss of this tone following induction of anesthesia or sympathectomy frequently contributes to perioperative hypotension.

Systemic blood flow is pulsatile in large arteries because of the heart's cyclic activity; when the blood reaches the systemic capillaries, flow is continuous (laminar). The mean pressure falls to less than 20 mm Hg in the large systemic veins that return blood to the heart. The largest pressure drop, nearly 50%, is across the arterioles, and the arterioles account for the majority of SVR. MAP is proportionate to the product of SVR x CO. This relationship is based on an analogy to Ohm's law, as applied to the circulation:

Because CVP is normally very small compared with MAP, the former can usually be ignored. From this relationship, it is readily apparent that hypotension is the result of a decrease in SVR, CO, or both: To maintain arterial blood pressure, a decrease in either SVR or CO must be compensated by an increase in the other. MAP may be estimated by the following formula:
where pulse pressure is the difference between systolic and diastolic blood pressure. Arterial pulse pressure is directly related to stroke volume, but is inversely related to the compliance of the arterial tree. Thus, decreases in pulse pressure may be due to a decrease in stroke volume, an increase in SVR, or both. Increased pulse pressure increases shear stress on vessel walls, potentially leading to atherosclerotic plaque rupture and thrombosis or rupture of aneurysms. Increased pulse pressure in patients undergoing cardiac surgery has been associated with adverse renal and neurological outcomes. Transmission of the arterial pressure wave from large arteries to smaller vessels in the periphery is faster than the actual movement of blood; the pressure wave velocity is 15 times the velocity of blood in the aorta. Moreover, reflections of the propagating waves off arterial walls widen pulse pressure before the pulse wave is completely dampened in very small arteries. Thus, the pulse pressure is generally greater when measured in the femoral or dorsalis pedis arteries than in the aorta.

Arterial blood pressure is regulated by a series of immediate, intermediate, and long-term adjustments that involve complex neural, humoral, and renal mechanisms.

In the course of a few minutes, sustained decreases in arterial pressure, together with enhanced sympathetic outflow, activate the renin—angiotensin—aldosterone system, increase secretion of arginine vasopressin (AVP), and alter normal capillary fluid exchange. Both angiotensin II and AVP are potent arteriolar vasoconstrictors. Their immediate action is to increase SVR. In contrast to formation of angiotensin II, which responds to relatively smaller changes, sufficient AVP secretion to produce vasoconstriction will only occur in response to more marked degrees of hypotension. Angiotensin constricts arterioles via AT, receptors. AVP mediates vasoconstriction via V, receptors and exerts its antidiuretic effect via V> receptors.
Sustained changes in arterial blood pressure can also alter fluid exchange in tissues by their secondary effects on capillary pressures. Hypertension increases interstitial movement of intravascular fluid, whereas hypotension increases reabsorption of interstitial fluid. Such compensatory changes in intravascular volume can reduce fluctuations in blood pressure, particularly in the absence of adequate renal function (see below).

The effects of slower renal mechanisms become apparent within hours of sustained changes in arterial pressure. As a result, the kidneys alter total body sodium and water balance to restore blood pressure to normal. Hypotension results in sodium (and water) retention, whereas hypertension generally increases sodium excretion in normal individuals.

Myocardial blood supply is derived entirely from the right and left coronary arteries (Figure 20-15). Blood flows from epicardial to endocardial vessels. After perfusing the myocardium, blood returns to the right atrium via the coronary sinus and the anterior cardiac veins. A small amount of blood returns directly into the chambers of the heart by way of the thebesian veins.
FIGURE 20-15 Anatomy of the coronary arteries in a patient with a right dominant circulation. A: Right anterior oblique view. B: Left anterior oblique view.
The right coronary artery (RCA) normally supplies the right atrium, most of the right ventricle, and a variable portion of the left ventricle (inferior wall). In 85% of persons, the RCA gives rise to the posterior descending artery (PDA), which supplies the superior—posterior interventricular septum and inferior wall—
a right dominant circulation; in the remaining 15% of persons, the PDA is a branch of the left coronary artery—a left dominant circulation.
The left coronary artery normally supplies the left atrium and most of the interventricular septum and left ventricle (septal, anterior, and lateral walls). After a short course, the left main coronary artery bifurcates into the left anterior descending artery (LAD) and the circumflex artery (CX); the LAD supplies the septum and anterior wall and the CX supplies the lateral wall. In a left dominant circulation, the CX wraps around the AV groove and continues down as the PDA to also supply most of the posterior septum and inferior wall.
The arterial supply to the SA node may be derived from either the RCA (60% of individuals) or the LAD (the remaining 40%). The AV node is usually supplied by the RCA (85-90%) or, less frequently, by the CX (10-15%); the bundle of His has a dual blood supply derived from the PDA and LAD. The anterior papillary muscle of the mitral valve also has a dual blood supply that is fed by diagonal branches of the LAD and marginal branches of the CX. In contrast, the posterior papillary of the mitral valve is usually supplied only by the PDA and is therefore much more vulnerable to ischemic dysfunction.

Coronary perfusion is unique in that it is intermittent rather than continuous, as it is in other organs. During contraction, intramyocardial pressures in the left ventricle approach systemic arterial pressure. The force of left ventricular contraction almost completely occludes the intramyocardial part of the coronary arteries. Coronary perfusion pressure is usually determined by the difference between aortic pressure and ventricular pressure. The left ventricle is perfused almost entirely during diastole. In contrast, the right ventricle is perfused during both systole and diastole (Figure 20-16). Moreover, as a determinant of left heart myocardial blood flow, arterial diastolic pressure is more important than MAP. Therefore, left coronary artery perfusion pressure is determined by the difference between arterial diastolic pressure and left ventricular end-diastolic pressure (LVEDP).
FIGURE 20-16 Coronary blood flow during the cardiac cycle. (Modified with permission from Berne RM, Levy MD, Pappao A, et al. Cardiovascular Physiology. 10th ed. Philadelphia, PA Mosby; 2013.)
Coronary perfusion tae ype = diastolic -LVEDP pressure pressure
Decreases in aortic pressure or increases in ventricular end-diastolic pressure can reduce coronary perfusion pressure. Increases in heart rate also decrease coronary perfusion because of the disproportionately greater reduction in diastolic time ®@ as heart rate increases (Figure 20-17). Because the endocardium is subjected to the greatest intramural pressures during systole, it tends to be most vulnerable to ischemia during decreases in coronary perfusion pressure.
FIGURE 20-17 The relationship between diastolic time and heart rate.

Coronary blood flow normally parallels myocardial metabolic demand. In the average adult man, coronary blood flow is approximately 250 mL/min at rest. The myocardium regulates its own blood flow closely between perfusion pressures of 50 and 120 mm Hg. Beyond this range, blood flow becomes increasingly pressure dependent.
Under normal conditions, changes in blood flow are entirely due to variations in coronary arterial tone (resistance) in response to metabolic demand. Hypoxia —either directly, or indirectly through the release of adenosine—causes coronary vasodilation. Autonomic influences are generally weak. Both a,and B.-adrenergic receptors are present in the coronary arteries. The «,-receptors are primarily located on larger epicardial vessels, whereas the B5-receptors are mainly found on the smaller intramuscular and subendocardial vessels.
Sympathetic stimulation generally increases myocardial blood flow because of an increase in metabolic demand and a predominance of B-receptor activation. Parasympathetic effects on the coronary vasculature are generally minor and weakly vasodilatory.

Myocardial oxygen demand is usually the most important determinant of myocardial blood flow. Relative contributions to oxygen requirements include basal requirements (20%), electrical activity (1%), volume work (15%), and pressure work (64%). The myocardium usually extracts 65% of the oxygen in arterial blood, compared with 25% in most other tissues. Coronary sinus oxygen saturation is usually 30%. Therefore, the myocardium (unlike other tissues) cannot compensate for reductions in blood flow by extracting more oxygen from hemoglobin. Any increases in myocardial metabolic demand must be met by an increase in coronary blood flow. 'Table 20—7 lists the most important factors in myocardial oxygen demand and supply. Note that the heart rate and, to a lesser extent, ventricular end-diastolic pressure are important determinants of both supply and demand.

Heart rate (diastolic filling time)
Coronary perfusion pressure
Aortic diastolic blood pressure
Ventricular end-diastolic pressure
Arterial oxygen content
Arterial oxygen tension
Hemoglobin concentration
Coronary vessel diameter

Basal metabolic requirements
Heart rate
Wall tension
Preload (ventricular radius)
Afterload

Most volatile anesthetic agents are coronary vasodilators. Their effect on coronary blood flow is variable because of their direct vasodilating properties, reduction of myocardial metabolic requirements, and effects on arterial blood pressure.
Volatile agents exert beneficial effects in experimental myocardial ischemia and infarction. They reduce myocardial oxygen requirements and protect against reperfusion injury; these effects are mediated by activation of ATP-sensitive K* (Karp) channels. Some evidence also suggests that volatile anesthetics enhance recovery of the "stunned" myocardium (hypocontractile, but recoverable, myocardium after ischemia). Moreover, although volatile anesthetics decrease myocardial contractility, they can be potentially beneficial in patients with heart failure because most of them decrease preload and afterload.

Systolic heart failure occurs when the heart is unable to pump a sufficient amount of blood to meet the body's metabolic requirements. Clinical manifestations usually reflect the effects of the low cardiac output on tissues (eg, fatigue, dyspnea, oxygen debt, acidosis), the damming-up of blood behind the failing ventricle (dependent edema or pulmonary venous congestion), or both. The left ventricle is most commonly the primary cause, often with secondary involvement of the right ventricle. Isolated right ventricular failure can occur in the setting of advanced disease of the lung parenchyma or pulmonary vasculature. Left ventricular failure is most commonly the result of myocardial dysfunction, usually from coronary artery disease, but may also be the result of viral disease, toxins, untreated hypertension, valvular dysfunction, arrhythmias, or pericardial disease.
Diastolic dysfunction can be present in the absence of signs or symptoms of heart failure, as for example in patients with hypertension or aortic valve stenosis. Symptoms arising from diastolic dysfunction are the result of atrial hypertension and pulmonary congestion (Figure 20-18). Failure of the heart to relax during diastole leads to elevated left ventricular end-diastolic pressure, which is transmitted to the left atrium and pulmonary vasculature. Common causes of diastolic dysfunction include hypertension, coronary artery disease, hypertrophic cardiomyopathy, valvular heart disease, and pericardial disease.
Diastolic dysfunction is not the same as diastolic heart failure. In the patient with systolic heart failure the heart compensates by dilating, which leads to an increase in end-diastolic ventricular volume in an attempt to preserve the stroke volume. In patients with diastolic failure, poor ventricular relaxation leads to a higher LVEDP than would be noted in a patient without diastolic dysfunction for the same end-diastolic volume.
FIGURE 20-18 Ventricular pressure-volume relationships in isolated systolic and diastolic dysfunction.

Diastolic dysfunction is diagnosed echocardiographically. Placing the pulse wave Doppler sample gate at the tips of the mitral valve during left ventricular filling will produce the characteristic diastolic flow pattern (Figure 20-13). In patients with normal diastolic function, the ratio between the peak velocities of the early (E) and the atrial (A) waves is from 0.8 to 2. In the early stages of diastolic dysfunction, the primary abnormality is impaired relaxation. When left ventricular relaxation is delayed, the initial pressure gradient between the left atrium and the left ventricle is reduced, resulting in a decline in early filling, and, consequently, a reduced peak E wave velocity. The A wave velocity is increased relative to the E wave, and the E/A ratio is reduced. As diastolic dysfunction advances, the left atrial pressure increases, restoring the gradient between the left atrium and left ventricle with an apparent restoration of the normal E/A ratio. This pattern is characterized as "pseudonormalized." Using the E/A ratio alone cannot distinguish between a normal and pseudonormalized pattern of diastolic inflow. As diastolic dysfunction worsens further, a restrictive pattern is obtained. In this scenario, the left ventricle is so stiff that pressure builds in the left atrium, resulting in a dramatic peak of early filling and a prominent, tall, narrow E wave. Because the ventricle is so poorly compliant, the atrial contraction contributes little to filling, resulting in a diminished A wave and an E/A ratio greater than

Compensatory mechanisms generally present in patients with heart failure include activation of the sympathetic nervous system and the renin—angiotensin— aldosterone system and increased release of AVP. One result is increased preload (fluid retention). Although these mechanisms can initially compensate for mild to moderate cardiac dysfunction, with increasing severity of dysfunction, they may actually worsen the cardiac impairment. Many of the drug treatments of chronic heart failure serve to counteract these mechanisms.

An increase in ventricular size not only reflects an inability to keep up with an increased circulating blood volume, but also serves to increase stroke volume by moving the heart up the Starling curve (see Figure 20-5). Even when EF is reduced, an increase in ventricular end-diastolic volume can maintain a normal stroke volume. Worsening venous congestion caused by the pooling of blood behind the failing ventricle and excessive ventricular dilation can rapidly lead to clinical deterioration. Left ventricular failure results in pulmonary vascular congestion and progressive transudation of fluid, first into the pulmonary interstitium and then into alveoli (pulmonary edema). Right ventricular failure leads to systemic venous hypertension, which results in peripheral edema, hepatic congestion and dysfunction, and ascites. Dilation of the annulus of either the mitral or tricuspid valves from ventricular dilation leads to valvular regurgitation, further impairing ventricular output.

improved by administration of ACE inhibitors (and/or angiotensin receptor blockers), certain long-acting B-blockers (carvedilol or extended-release metoprolol), and aldosterone inhibitors (spironolactone or eplerenone).
Circulating AVP levels, often markedly increased in patients with severe heart failure, will increase ventricular afterload and are responsible for a defect in free water clearance that is commonly associated with hyponatremia.
Brain natriuretic peptide (BNP) is produced in the heart in response to myocyte distention. Elevated BNP concentration (>500 pg/mL) usually indicates heart failure, and measurement of BNP concentration can be used to distinguish between heart failure and lung disease as a cause of dyspnea. Recombinant BNP was developed as a vasodilator and inhibitor of the renin—angiotensin— aldosterone system for use in patients with severe decompensated heart failure, but outcomes were not improved with its use.

Ventricular hypertrophy can occur with or without dilation, depending on the type of stress imposed on the ventricle. When the heart is subjected to either pressure or volume overload, the initial response is to increase sarcomere length and optimally overlap actin and myosin. With time, ventricular muscle mass begins to increase in response to the abnormal stress.
In the volume-overloaded ventricle, the problem is an increase in diastolic wall stress. The increase in ventricular muscle mass is sufficient only to compensate for the increase in diameter: The ratio of the ventricular radius to wall thickness is unchanged. Sarcomeres replicate mainly in series, resulting in eccentric hypertrophy. Although ventricular EF remains depressed, the increase in end-diastolic volume can maintain normal at-rest stroke volume (and cardiac output).
The problem in a pressure-overloaded ventricle is an increase in systolic wall stress. In this case, sarcomeres mainly replicate in parallel, resulting in concentric hypertrophy: The hypertrophy is such that the ratio of myocardial wall thickness to ventricular radius increases. As can be seen from Laplace's law, systolic wall stress can then be normalized. Ventricular hypertrophy, particularly that caused by pressure overload, usually results in progressive diastolic dysfunction. The most common reasons for isolated left ventricular hypertrophy are hypertension and aortic stenosis.

A 38-year-old man is scheduled for endoscopic sinus surgery following a recent onset of headaches. He gives a history of having passed out at least once during one of these headaches. A preoperative electrocardiogram (ECG) is normal, except for a P-R interval of 0.116 s with normal P-wave morphology.

The P-R interval, which is measured from the beginning of atrial depolarization (P wave) to the beginning of ventricular depolarization (QRS complex), usually represents the time required for depolarization of both atria, the AV node, and the His—Purkinje system. Although the P—R interval can vary with heart rate, it is normally 0.12 to 0.2 s in duration.

Preexcitation usually refers to early depolarization of the ventricles by an abnormal conduction pathway from the atria. Rarely, more than one such pathway is present. The most common form of preexcitation is due to the presence of an accessory pathway (bundle of Kent) that connects one of the atria with one of the ventricles. This abnormal connection between the atria and ventricles allows electrical impulses to bypass the AV node (hence the term bypass tract). The ability to conduct impulses along the bypass tract can be quite variable and may be only intermittent or rate dependent. Bypass tracts can conduct in both directions, retrograde only (ventricle to atrium), or, rarely, anterograde only (atrium to ventricle). The name Wolff— Parkinson—White (WPW) syndrome is often applied to ventricular preexcitation associated with tachyarrhythmias.

In patients with preexcitation, the normal cardiac impulse originating from the SA node is conducted simultaneously through the normal (AV nodal) and anomalous (bypass tract) pathways. Because conduction is more rapid in the anomalous pathway than in the AV nodal pathway, the cardiac impulse rapidly reaches and depolarizes the area of the ventricles where the bypass tract ends. This early depolarization of the ventricle is reflected by a short P-R interval and a slurred initial deflection (6 wave) in the QRS complex. The spread of the anomalous impulse to the rest of the ventricle is delayed because it must be conducted by ordinary ventricular muscle, not by the much faster Purkinje system. The remainder of the ventricle is then depolarized by the normal impulse from the AV node as it catches up with the preexcitation front. Although the P—R interval is shortened, the resulting QRS is slightly prolonged and represents a fusion complex of normal and abnormal ventricular depolarizations.
The P-R interval in patients with preexcitation depends on relative conduction times between the AV nodal pathway and the bypass pathway. If conduction through the former is fast, preexcitation (and the 5 wave) is less prominent, and QRS will be relatively normal. If conduction is delayed in the AV nodal pathway, preexcitation is more prominent, and more of the ventricle will be depolarized by the abnormally conducted impulse. When the AV nodal pathway is completely blocked, the entire ventricle is depolarized by the bypass pathway, resulting in a very short P—R interval, a very prominent 6 wave, and a wide, bizarre QRS complex. Other factors that can affect the degree of preexcitation include interatrial conduction time, the distance of the atrial end of the bypass tract from the SA node, and autonomic tone. The P-R interval is often normal or only slightly shortened with a left lateral bypass tract (the most common location). Preexcitation may be more apparent at fast heart rates because conduction slows through the AV node with increasing heart rates. Secondary ST-segment and T-wave changes are also common because of abnormal ventricular repolarization.

Preexcitation occurs in approximately 0.3% of the general population. Up to 50% of affected persons develop paroxysmal tachyarrhythmias, typically paroxysmal supraventricular tachycardia (PSVT). Although most patients are otherwise normal, preexcitation can be associated with other cardiac anomalies, including Ebstein anomaly, mitral valve prolapse, and cardiomyopathies. Depending on its conductive properties, the bypass tract in some patients may predispose them to tachyarrhythmias and even sudden death. Tachyarrhythmias include PSVT, atrial fibrillation, and, less commonly, atrial flutter. Ventricular fibrillation can be precipitated by a critically timed premature atrial beat that travels down the bypass tract and catches the ventricle at a vulnerable period. Alternatively, very rapid conduction of impulses into the ventricles by the bypass tract during atrial fibrillation can rapidly lead to myocardial ischemia, hypoperfusion, and hypoxia and culminate in ventricular fibrillation.
Recognition of the preexcitation phenomenon is also important because its QRS morphology on the surface ECG can mimic bundle branch block, right ventricular hypertrophy, ischemia, myocardial infarction, and ventricular tachycardia (during atrial fibrillation).
What is the significance of the history of syncope in this patient?
This patient should be evaluated preoperatively with electrophysiological studies, and may possibly require curative radiofrequency ablation of the bypass tract with antiarrhythmic drug therapy. Such studies can identify the location of the bypass tracts, reasonably predict the potential for malignant arrhythmias by programmed pacing, and assess the efficacy of antiarrhythmic therapy if curative ablation is not possible. Ablation is reported to be curative in over 90% of patients. A history of syncope may be ominous because it may indicate the ability to conduct impulses very rapidly through the bypass tract, leading to systemic hypoperfusion and perhaps predisposing the patient to sudden death.

Tachyarrhythmias develop as a result of either abnormal impulse formation or abnormal impulse propagation (reentry). Abnormal impulses result from enhanced automaticity, abnormal automaticity, or triggered activity. Usually, only cells of the SA node, specialized atrial conduction pathways, AV nodal junctional areas, and the His—Purkinje system depolarize spontaneously. Because diastolic repolarization (phase 4) is fastest in the SA node, other areas of automaticity are suppressed. Enhanced or abnormal automaticity in other areas, however, can usurp pacemaker function from the SA node and lead to tachyarrhythmias. Triggered activity is the result of either early after-depolarizations (phase 2 or 3) or delayed after-depolarizations (after phase 3). It consists of small-amplitude depolarizations that can follow action potentials under some conditions in atrial, ventricular, and His—Purkinje tissue. If these after-depolarizations reach threshold potential, they can result in an extrasystole or repetitive sustained tachyarrhythmias. Factors that can promote the formation of abnormal impulses include increased catecholamine levels, electrolyte disorders (hyperkalemia, hypokalemia, and hypercalcemia), ischemia, hypoxia, mechanical stretch, and drug toxicity (particularly digoxin). The most common mechanism for tachyarrhythmias is reentry. Four conditions are necessary to initiate and sustain reentry (Figure 20—19): (1) two areas in the myocardium that differ in conductivity or refractoriness and that can form a closed electrical loop; (2) unidirectional block in one pathway (Figure 20-19A and B); (3) slow conduction or sufficient length in the circuit to allow recovery of the conduction block in the first pathway (Figure 20—19C); and (4) excitation of the initially blocked pathway to complete the loop (Figure 20—19D). Reentry is usually precipitated by a premature cardiac impulse.
FIGURE 20-19 A-—D: The mechanism of reentry. See text for description.
What is the mechanism of PSVT in patients with WPW syndrome?

If the bypass tract is refractory during anterograde conduction of a cardiac impulse, as during a critically timed atrial premature contraction (APC), and the impulse is conducted by the AV node, the same impulse can be conducted retrograde from the ventricle back into the atria via the bypass tract. The retrograde impulse can then depolarize the atrium and travel down the AV nodal pathway again, establishing a continuous repetitive circuit (circus movement). The impulse reciprocates between the atria and ventricles and conduction alternates between the AV nodal pathway and the bypass tract. The term concealed conduction is often applied because the absence of preexcitation during this arrhythmia results in a normal QRS that lacks a 5 wave.
The circus movement less commonly involves anterograde conduction through the bypass tract and retrograde conduction through the AV nodal pathway. In such instances, the QRS has a 6 wave and is completely abnormal; the arrhythmia can be mistaken for ventricular tachycardia.

In addition to the WPW syndrome, PSVT can be caused by AV reentrant tachycardia, AV nodal reentrant tachycardia, and SA node and atrial reentrant tachycardias. Patients with AV reentrant tachycardia have an extranodal bypass tract similar to patients with WPW syndrome, but the bypass tract conducts only retrograde; preexcitation and a 6 wave are absent Functional differences in conduction and refractoriness may occur within the AV node, SA node, or atria; a large bypass tract is not necessary. Thus, the circus movement may occur on a smaller scale within the AV node, SA node, or atria, respectively.
How does atrial fibrillation in patients with WPW syndrome differ from the arrhythmia in other patients?
Atrial fibrillation can occur when a cardiac impulse is conducted rapidly retrograde up into the atria and arrives to find different parts of the atria out of phase in recovery from the impulse. Once atrial fibrillation is established, conduction into the ventricles most commonly occurs through the bypass tract only; because of the accessory pathway's ability to conduct very rapidly (unlike the AV nodal pathway), the ventricular rate is typically very rapid (180-300 beats/min). The majority of QRS complexes are abnormal, but periodic conduction of an impulse through the AV nodal pathway results in occasional normal-looking QRS complexes. Less commonly, impulses during atrial fibrillation are conducted mainly through the AV nodal pathway (resulting in mostly normal QRS complexes) or through both the bypass tract and the AV nodal pathway (resulting in a mixture of normal, fusion, and abnormal QRS complexes). As stated previously, atrial fibrillation in patients with WPW syndrome is a very dangerous arrhythmia.
What anesthetic agents can safely be used in patients with preexcitation?

Few data are available comparing the use of different anesthetic agents or techniques in patients with preexcitation. Almost all the volatile and intravenous agents have been used. Volatile anesthetics increase antegrade refractoriness in both normal and accessory pathways. Propofol, opioids, and benzodiazepines seem to have little direct electrophysiological effects, but can alter autonomic tone, generally reducing sympathetic outflow. Factors that tend to cause sympathetic stimulation and increased cardiac automaticity are undesirable. Light anesthesia, hypercapnia, acidosis, and even transient hypoxia will activate the sympathetic system and are to be avoided. When patients with preexcitation are anesthetized for electrophysiological study and surgical ablation, opioids, propofol, and benzodiazepines may be the agents least likely to alter conduction characteristics.

Most antiarrhythmic agents act by altering myocardial cell conduction (phase 0), repolarization (phase 3), or automaticity (phase 4). Prolongation of repolarization increases the refractoriness of cells. Many antiarrhythmic drugs also exert direct or indirect autonomic effects. Although antiarrhythmic agents are generally classified according to broad mechanisms of action or electrophysiological effects (Table 20—8), the most commonly used classification system is not perfect because some agents have more than one mechanism of action.
TABLE 20-8 Summary of antiarrhythmic drugs.!

Increases K* permeability, K* currents. Potassium, 2 = Slows ectopic pacemakers « slows conduction velocity in heart. Potassium, 3 = Digitalis-induced arrhythmias + arrhythmias associated with hypokalemia. Potassium, 4 = Oral, |V Toxicity: Reentrant arrhythmias, fibrillation or arrestin overdose
'Data from Trevor AJ, Katzung BG, Kruidering-Hall M. Katzung & Trevor's Pharmacology Examination and Board Review. 11th ed. New York, NY: McGraw-Hill Education; 2015.
Selection of an antiarrhythmic agent generally depends on whether the arrhythmia is ventricular or supraventricular and whether acute control or chronic therapy is required. Intravenous agents are usually employed in the acute management of arrhythmias, whereas oral agents are reserved for chronic therapy (Table 20-9).
TABLE 20-9 Clinical pharmacological properties of antiarrhythmic drugs.!

Vernakalant, a.Interval = T. Vernakalant, aie Duration. = . Vernakalant, x Interval. = . Vernakalant, Usefulness in Arrhythmias.Supraventricular = ++. Vernakalant, Usefulness in Arrhythmias.Ventricular = -. Vernakalant, Half-Life. = 2h
'May suppress diseased sinus nodes
*Anticholinergic effect and direct depressant action.
"Especially in Wolff-Parkinson-White syndrome.
Stay be effective in atrial arrhythmias caused by digitalis.
Which agents are most useful for tachyarrhythmias in patients with WPW syndrome?
Cardioversion is the treatment of choice in hemodynamically compromised patients. Small doses of phenylephrine (100 mcg), together with vagal maneuvers (carotid massage if not contraindicated by carotid occlusive disease), help support arterial blood pressure and may terminate the arrhythmia. The most useful pharmacological agents are class Ia drugs (eg, procainamide). Procainamide increases the refractory period and decreases conduction in the accessory pathway. Moreover, class Ia drugs frequently terminate and can suppress the recurrence of PSVT and atrial fibrillation. Amiodarone is not recommended. Adenosine, verapamil, and digoxin are contraindicated during atrial fibrillation or flutter in these patients because they can dangerously accelerate the ventricular response. Both types of agents decrease conduction through the AV node, favoring conduction of impulses down the accessory pathway. The bypass tract is capable of conducting impulses into the ventricles much faster than the AV

nodal pathway. Digoxin may also increase the ventricular response by shortening the refractory period and increasing conduction in accessory pathways. Although verapamil can terminate PSVT, its use in this setting may be hazardous because patients can subsequently develop atrial fibrillation or flutter. Moreover, atrial fibrillation may not be readily distinguishable from ventricular tachycardia in these patients if wide-QRS tachycardia develops.

@ Cardiovascular complications are estimated to account for 25% to 50% of deaths following noncardiac surgery. Perioperative myocardial infarction (MI), pulmonary edema, systolic and diastolic heart failure, arrhythmias, stroke, and thromboembolism are the most common diagnoses in patients with preexisting cardiovascular disease.
@ Regardless of the level of preoperative blood pressure control, many patients with hypertension display an accentuated hypotensive response to induction of anesthesia, followed by an exaggerated hypertensive response to intubation.
© Patients with extensive coronary artery disease, a history of MI, or ventricular dysfunction are at risk of adverse cardiovascular complications.
@ The sudden withdrawal of antianginal medication perioperatively— particularly B-blockers—can precipitate a sudden, rebound increase in ischemic episodes.
@ The overwhelming priority in managing patients with ischemic heart disease is maintaining a favorable myocardial supply—-demand relationship. Autonomic-mediated increases in heart rate and blood pressure should be controlled with deeper planes of general anesthesia or adrenergic blockade, vasodilators, or a combination of these.
@ Intraarterial pressure monitoring is reasonable in most patients with severe coronary artery disease and major or multiple cardiac risk factors who are undergoing any but the most minor procedures. Central venous (or rarely pulmonary artery) pressure can be monitored during prolonged or complicated procedures involving large fluid shifts or

The prevalence of cardiovascular disease increases with advancing age.
Moreover, the number of patients older than 65 years of age is expected to increase by 25% to 35% over the next two decades. @ Cardiovascular complications are estimated to account for 25% to 50% of deaths following noncardiac surgery. Perioperative myocardial infarction (MI), pulmonary edema, systolic and diastolic heart failure, arrhythmias, stroke, and thromboembolism are the most common diagnoses in patients with preexisting cardiovascular disease. The relatively high prevalence of cardiovascular disorders in surgical patients has given rise to attempts to define cardiac risk or the likelihood of intraoperative or postoperative fatal or life-threatening cardiac complications. The ACC/AHA Task Force Report provides guidelines for perioperative cardiovascular evaluation. The guidelines state that the patient's medical history is critical in determining the requirements for preoperative cardiac evaluation and that certain conditions (eg, unstable coronary syndromes and decompensated heart failure) warrant cardiology intervention prior to all but emergency procedures. The preoperative history should also address any past procedures, such as cardioverter defibrillator implants, coronary stents, and other interventions. Additionally, the patient's ability to perform the tasks of daily living should be assessed as a guide to determine functional capacity. A patient with a history of cardiac disease and advanced age, but good exercise tolerance, will likely have a lower perioperative risk than a similar individual with dyspnea after minimal physical activity (Table 21-1).

'Modified with permission from Hlatky MA, Boineau RE, Higginbotham MB, et al. A brief self-administered questionnaire to determine functional capacity (the Duke Activity Status Index). Am J Cardiol. 1989 Sep 15;64(10):651-654.
kph, kilometers per hour; MET, metabolic equivalent; mph, miles per hour.
The patient should be queried about other disease processes that frequently accompany heart disease. Cardiac patients often present with obstructive pulmonary disease, reduced kidney function, and diabetes mellitus.
A physical examination should be performed on all patients, and the heart and should be auscultated. The physical examination is especially useful in lungs
patients with certain conditions. For example, if a harsh systolic murmur suggestive of aortic stenosis is detected in a candidate for elective surgery, additional ultrasound evaluation will likely be warranted, as aortic stenosis substantially increases the risks in patients undergoing noncardiac surgery.
The following conditions are associated with increased risk:
¢ Ischemic heart disease (history of MI, evidence on electrocardiogram [ECG], chest pain)
¢ Congestive heart failure (dyspnea, pulmonary edema)
* Cerebral vascular disease (stroke)
¢ High-risk surgery (vascular, thoracic)
¢ Diabetes mellitus
¢ Preoperative creatinine greater than 2 mg/dL

The ACC/AHA guidelines identify conditions that are a major cardiac risk and warrant intensive management prior to all but emergent surgery. These conditions include unstable coronary syndromes (recent MI, unstable angina), decompensated heart failure, significant arrhythmias, and severe valvular heart disease. The ACC/AHA guidelines identify an MI within 7 days, or one within 1 month with myocardium at risk for ischemia, as "active" cardiac conditions. On the other hand, evidence of past MI with no myocardium thought at ischemic risk is considered a low risk for perioperative infarction after noncardiac surgery. The ACC/AHA guidelines classify recommendations into four categories, as class I (benefit >>> risk), class Ila (benefit >> risk), class IIb (benefit > risk), and class III (no benefit or harm). Additionally, they grade the strength of the evidence upon which the recommendations is based as A (multiple randomized trials), B (limited trials, nonrandomized studies), and C (consensus of experts, case studies).
Class I recommendations are as follows:
* Patients who have a need for emergency noncardiac surgery should proceed to the operating room with perioperative surveillance and postoperative risk factor management
¢ Patients with active cardiac conditions should be evaluated by a cardiologist and treated according to ACC/AHA guidelines
¢ Patients undergoing low-risk procedures should proceed to surgery
¢ Patients with poor exercise tolerance (<4 metabolic equivalents [METs]) and no known risk factors should proceed to surgery
The ACC/AHA guidelines use an algorithmic approach to discern risks of major adverse cardiac events (MACE; eg, perioperative death or myocardial infarction). Risks accrue secondary both to the nature of surgery and because of patient characteristics. The ACC/AHA suggest various risk calculators that are available online (eg, American College of Surgeons risk calculator, www.surgicalriskcalculator.com) to estimate patient risk of perioperative major adverse cardiac events (Figure 21-1).

revascularization depending on the extent of the abnormal test. The patient can then proceed to surgery with GDMT or consider alternative strategies, such as noninvasive treatment of the indication for surgery (eg, radiation therapy for cancer) or palliation. If the test is normal, proceed to surgery according to GDMT (see Section 5.3 of the guidelines). Step 7: If testing will not impact decision making or care, then proceed to surgery according to GDMT or consider alternative strategies, such as noninvasive treatment of the indication for surgery (eg, radiation therapy for cancer) or palliation. MET, metabolic equivalent; NB, No Benefit; NSQIP, National Surgical Quality Improvement Program; STEMI, ST-elevation myocardial infarction; UA/NSTEMI, unstable angina/non—ST-elevation myocardial infarction. (Reproduced with permission from Fleisher LA, Fleischman KE, Auerbach AD, et al. 2014 ACC/AHA guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery: A report of the American College of Cardiology/American Heart Association Task Force on practice guidelines. J Am Coll Cardiol. 2014 Dec 9;64(22):e77-e137.)
The ACC/AHA guidelines also provide specific recommendations regarding various preexistent cardiac conditions (eg, heart failure, valvular heart disease, arrhythmias) likely to be encountered perioperatively. Recommendations regarding supplemental preoperative evaluation are presented in Table 21-2.
TABLE 21-2 Summary of recommendations for supplemental preoperative evaluation.!

No Benefit. Routine screening with noninvasive stress testing is not useful for low-risk noncardiac surgery, LOE = B. Preoperative coronary angiography Routine preoperative coronary angiography is not recommended, COR? = Il: No Benefit. Preoperative coronary angiography Routine preoperative coronary angiography is not recommended, LOE = C
'Reproduced with permission from Fleisher LA, Fleischman KE, Auerbach AD, et al. 2014 ACC/AHA guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery: A report of the American College of Cardiology/American Heart Association Task Force on practice guidelines. J Am Coll Cardiol. 2014 Dec 9;64(22):e77-e137,
2COR, class of recommendation; DSE, dobutamine stress echocardiogram; ECG, electrocardiogram, HF, heart failure; LOE, level of evidence: LF, left ventricular; MPI, myocardial perfusion imaging.

The ACC/AHA guidelines suggest that 60 days or more should elapse after an MI before noncardiac surgery in patients who were not treated with a coronary intervention. Moreover, an MI within 6 months of surgery is associated with increased perioperative mortality. Increased patient age and frailty are likewise associated with greater risk for acute coronary syndromes and stroke. Recently, studies have found a surprising number of asymptomatic patients with elevated levels of troponin after surgery. Such findings are indicative of myocardial injury despite there being no other evidence suggestive of MI. Nevertheless, these patients are at considerably increased risk. The specific management of these patients remains controversial.

hypertension are at some increased risk for cardiovascular complications remains unclear. Accelerated or severe hypertension is defined as a recent, sustained, and progressive increase in blood pressure, usually with diastolic blood pressures in excess of 110 to 119 mm Hg. Kidney dysfunction is often present in such patients. A hypertensive urgency reflects blood pressure elevation of >180/120 mm Hg without signs of organ injury (eg, hypertensive encephalopathy, heart failure). A hypertensive emergency is characterized by severe hypertension (>180/120 mm Hg) often associated with papilledema, encephalopathy, or other organ injury.

Normal, Systolic Pressure (mm Hg) = <120. Normal, Diastolic Pressure (mm Hg) = <80. Prehypertension, Systolic Pressure (mm Hg) = 120-139. Prehypertension, Diastolic Pressure (mm Hg) = 80-89. Hypertension, Systolic Pressure (mm Hg) = . Hypertension, Diastolic Pressure (mm Hg) = . Mild, Systolic Pressure (mm Hg) = 140-159. Mild, Diastolic Pressure (mm Hg) = 90-99. Moderate, Systolic Pressure (mm Hg) = 2160. Moderate, Diastolic Pressure (mm Hg) = 2100. Hypertensive Urgency/ Emergency, Systolic Pressure (mm Hg) = >180. Hypertensive Urgency/ Emergency, Diastolic Pressure (mm Hg) = >120

Hypertension can be either idiopathic (essential) or, less commonly, secondary to other medical conditions such as renal disease, renal artery stenosis, primary hyperaldosteronism, Cushing disease, acromegaly, pheochromocytoma, pregnancy, or estrogen therapy. Essential hypertension accounts for 80% to 95% of cases and may be associated with an abnormal baseline elevation of cardiac output, systemic vascular resistance (SVR), or both. An evolving pattern is commonly seen over the course of the disease, where cardiac output returns to (or remains) normal, but SVR becomes abnormally high. The chronic increase in cardiac afterload results in concentric left ventricular hypertrophy and altered diastolic function. Hypertension also alters cerebral autoregulation, such that normal cerebral blood flow is maintained in the face of high blood pressures; autoregulation limits may be in the range of mean blood pressures of 110 to 180

The mechanisms responsible for the changes observed in hypertensive patients seem to involve vascular hypertrophy, hyperinsulinemia, abnormal increases in intracellular calcium, and increased intracellular sodium concentrations in vascular smooth muscle and renal tubular cells. Sympathetic nervous system overactivity and enhanced responses to sympathetic agonists are present in some patients. Hypertensive patients sometimes display an exaggerated response to vasopressors and vasodilators. Overactivity of the renin—angiotensin—aldosterone system seems to play an important role in patients with accelerated hypertension.

Effective drug therapy reduces the progression of hypertension and the incidence of stroke, congestive heart failure, coronary artery disease (CAD), and kidney damage. Effective treatment can also delay and sometimes reverse concomitant pathophysiological changes, such as left ventricular hypertrophy and altered cerebral autoregulation.
Some patients with mild hypertension require only single-drug therapy, which may consist of a thiazide diuretic, angiotensin-converting enzyme (ACE) inhibitor, angiotensin-receptor blocker (ARB), B-adrenergic blocker, or calcium channel blocker, although guidelines and outcome studies favor the first three options. Concomitant illnesses should guide drug selection. All patients with a prior MI should receive a B-adrenergic blocker and an ACE inhibitor (or ARB) to improve outcomes, irrespective of the presence of hypertension. In many patients, the "guideline-specified" agents will also be more than sufficient to control hypertension.
Patients with moderate to severe hypertension often require two or three drugs for control. The combination of a diuretic with a B-adrenergic blocker and an ACE inhibitor is often effective when single-drug therapy is not. As previously noted, ACE inhibitors (or ARBs) prolong survival in patients with congestive heart failure, left ventricular dysfunction, or a prior MI. Familiarity with the names, mechanisms of action, and side effects of commonly used antihypertensive agents is important for anesthesiologists (Table 21-4).
TABLE 21-4 Summary of drugs used in hypertension.!

Hypertension, heart failure. Losartan, manyothers, 4 = Oral Toxicity: Same as ACE inhibitors but less cough. RENIN INHIBITOR, 1 = RENIN INHIBITOR. RENIN INHIBITOR, 2 = RENIN INHIBITOR. RENIN INHIBITOR, 3 = RENIN INHIBITOR. RENIN INHIBITOR, 4 = RENIN INHIBITOR. Aliskiren, 1 = Inhibits enzyme activity of renin. Aliskiren, 2 = Reduces angiotensin land land aldosterone. Aliskiren, 3 = Hypertension. Aliskiren, 4 = Oral Toxicity: Hyperkalemia, renal impairment Potential teratogen
- 'Reproduced with permission from Katzung BG, Trevor AJ. Basic & Clinical Pharmacology. 13th ed. New York, NY: McGraw-Hill Education; 2015.

A recurring question in anesthetic practice is the degree of preoperative hypertension that is acceptable for patients scheduled for elective surgery. Except for optimally controlled patients, most hypertensive patients present to the operating room with some degree of hypertension. Although data suggest that even moderate preoperative hypertension (diastolic pressure >90-110 mm Hg) is not clearly statistically associated with postoperative complications, other data indicate that the untreated or poorly controlled hypertensive patient is more apt to experience intraoperative episodes of myocardial ischemia, arrhythmias, or hemodynamic instability. Careful intraoperative adjustments in anesthetic depth and use of vasoactive drugs should reduce the incidence of postoperative complications referable to poor preoperative control of hypertension.
Although patients should ideally undergo elective surgery only when rendered normotensive, accomplishing this over the short term is not always feasible or even desirable because hypertensive patients have altered cerebral autoregulation. Excessive reductions in blood pressure can compromise cerebral perfusion. Moreover, the decision to delay or to proceed with surgery should be individualized, based on the severity of the preoperative blood pressure elevation; the likelihood of coexisting myocardial ischemia, ventricular dysfunction, or cerebrovascular or renal complications; and the nature and urgency of the procedure. With rare exceptions, antihypertensive drug therapy should be continued up to the time of surgery. Some clinicians withhold ACE inhibitors and ARBs on the morning of surgery because of their association with an increased incidence of intraoperative hypotension; however, withholding these agents increases the risk of marked perioperative hypertension and the need for parenteral antihypertensive agents. It also requires the surgical team to remember to restart the medication after surgery. The decision to delay elective surgical procedures in patients with sustained preoperative diastolic blood pressures higher than 110 mm Hg should be made when the perceived benefits of delayed surgery exceed the risks. Unfortunately, there are few appropriate studies to guide the decision-making.

The preoperative history should address the severity and duration of the hypertension, the drug therapy currently prescribed, and the presence or absence of hypertensive complications. Symptoms of myocardial ischemia, ventricular failure, impaired cerebral perfusion, or peripheral vascular disease should be elicited, as well as the patient's record of compliance with the drug regimen. The patient should be questioned regarding chest pain, exercise tolerance, shortness of breath (particularly at night), dependent edema, postural lightheadedness, syncope, episodic visual disturbances or episodic neurological symptoms, and claudication. Adverse effects of current antihypertensive drug therapy (Table 21—5) should also be identified.
TABLE 21-5 Adverse effects of long-term antihypertensive therapy.

Diuretics, Adverse Effects = . Thiazide, Adverse Effects = Hypokalemia, hyponatremia, hyperglycemia, hyperuricemia, hypomagnesemia, hyperlipidemia, hypercalcemia. Loop, Adverse Effects = Hypokalemia, hyperglycemia, hypocalcemia, hypomagnesemia, metabolic alkalosis. Potassium sparing, Adverse Effects = Hyperkalemia. Sympatholytics, Adverse Effects = . B-Adrenergic blockers, Adverse Effects = Bradycardia, conduction blockade, myocardial depression, enhanced bronchial tone, sedation, fatigue, depression. a-Adrenergic blockers, Adverse Effects = Postural hypertension, tachycardia, fluid retention. Central a,-agonists, Adverse Effects = Postural hypotension, sedation, dry mouth, depression, decreased anesthetic requirements, bradycardia, rebound hypertension, positive Coombs test and hemolytic anemia (methyldopa), hepatitis (methyldopa). Ganglionic blockers, Adverse Effects = Postural hypotension, diarrhea, fluid retention, depression (reserpine). Vasodilators, Adverse Effects = . Calcium channels blockers, Adverse Effects = Cardiac depression, bradycardia, conduction blockade (verapamil, diltiazem), peripheral edema (nifedipine), tachycardia (nifedipine), enhanced neuromuscular nondepolarizing blockade. ACE inhibitors!, Adverse Effects = Cough, angioedema, reflex tachycardia, fluid retention, renal dysfunction, hyperkalemia, bone marrow depression (captopril). Angiotensin-receptor antagonists, Adverse Effects = Hypotension, renal failure in bilateral renal artery stenosis, hyperkalemia. Direct vasodilators, Adverse Effects = Reflex tachycardia, fluid retention, headache, systemic lupus erythematosus-like syndrome (hydralazine), pleural or pericardial effusion (minoxidil)
'ACE, angiotensin-converting enzyme.

hypertrophy. Echocardiography is a sensitive test for LVH and can be used to evaluate ventricular systolic and diastolic functions in patients with symptoms of heart failure. Chest radiographs are rarely useful in an asymptomatic patient, but may show frank cardiomegaly or pulmonary vascular congestion. Kidney function is typically evaluated by measurement of serum creatinine levels. Serum electrolyte levels (K) should be determined in patients taking diuretics or digoxin or those with kidney impairment. Mild to moderate hypokalemia (3-3.5 mEq/L) is often seen in patients taking diuretics, but does not have adverse outcome effects. Potassium replacement should be undertaken only in patients who are symptomatic or who are also taking digoxin. Hypomagnesemia is often present and may be a cause of perioperative arrhythmias. Hyperkalemia may be encountered in patients who are taking potassium-sparing diuretics or ACE inhibitors, particularly those with impaired renal function.

Mild to moderate preoperative hypertension often resolves following administration of an agent such as midazolam.

The anesthetic for a hypertensive patient should maintain an appropriately stable blood pressure range. Patients with borderline hypertension may be treated as normotensive patients. Those with long-standing or poorly controlled hypertension, however, have altered autoregulation of cerebral blood flow; higher than normal mean blood pressures may be required to maintain adequate cerebral blood flow. Hypertension, particularly in association with tachycardia, can precipitate or exacerbate myocardial ischemia, ventricular dysfunction, or both. Arterial blood pressure should generally be kept within 20% of preoperative levels.

Most hypertensive patients do not require special intraoperative monitors. Direct intraarterial pressure monitoring should be reserved for patients with wide swings in blood pressure and those undergoing major surgical procedures associated with rapid or marked changes in cardiac preload or afterload. Electrocardiographic monitoring should focus on detecting signs of ischemia. Urinary output should generally be monitored with an indwelling urinary catheter in patients with a preexisting kidney impairment who are undergoing procedures expected to last more than 2 h. Ventricular compliance (see Chapter 20) is typically reduced in patients with ventricular hypertrophy. Excessive intravenous fluid administration in patients with decreased ventricular compliance can also result in elevated pulmonary arterial pressures and pulmonary congestion.

Induction of anesthesia and endotracheal intubation are often associated with hemodynamic instability @ in hypertensive patients. Regardless of the level of preoperative blood pressure control, many patients with hypertension display an accentuated hypotensive response to induction of anesthesia, followed by an exaggerated hypertensive response to intubation. Many, if not most, antihypertensive agents and general anesthetics are vasodilators, cardiac depressants, or both. In addition, many hypertensive patients present for surgery in a volume-depleted state. Sympatholytic agents attenuate the normal protective circulatory reflexes, reducing sympathetic tone and unmasking vagal activity. Hypertensive patients may exhibit severe hypertension during airway manipulation. One of several techniques may be used before intubation to attenuate the hypertensive response:
¢ Deepening anesthesia with a potent volatile agent
¢ Administering a bolus of an opioid (fentanyl, 2.5-5 mcg/kg; alfentanil, 15— 25 mcg/kg; sufentanil, 0.5-1.0 mcg/kg; or remifentanil, 0.5-1 mcg/kg)
¢ Administering lidocaine, 1.5 mg/kg intravenously, intratracheally, or topically in the airway
¢ Achieving B-adrenergic blockade with esmolol, 0.3-1.5 mg/kg; metoprolol 1—5 mg; or labetalol, 5-20 mg

The superiority of any one agent or technique over another has not been established. Propofol, barbiturates, benzodiazepines, and etomidate are equally safe for inducing general anesthesia in most hypertensive patients. Ketamine by itself can precipitate marked hypertension; however, it is almost never used as a single agent. When administered with a small dose of another agent, such as a benzodiazepine or propofol, ketamine's sympathetic stimulating properties can be blunted or eliminated.

Anesthesia may be safely maintained with volatile or intravenous agents. Regardless of the primary maintenance technique, addition of a volatile agent or intravenous vasodilator generally allows convenient intraoperative blood pressure control.

Should hypotension develop, a small dose of a direct-acting agent, such as phenylephrine (25-50 mcg), may be beneficial. Patients taking sympatholytics preoperatively may exhibit a decreased response to ephedrine. Vasopressin as a bolus or infusion can also be employed to restore vascular tone in the hypotensive patient.

Intraoperative hypertension not responding to an increase in anesthetic depth (particularly with a volatile agent) can be treated with a variety of parenteral agents (Table 21-6). Readily reversible causes—such as inadequate anesthetic depth, hypoxemia, or hypercapnia—should always be excluded before initiating antihypertensive therapy. Selection of a hypotensive agent depends on the severity, acuteness, and cause of hypertension; the baseline ventricular function; the heart rate; and the presence of bronchospastic pulmonary disease. pBAdrenergic blockade alone or as a supplement is a good choice for a patient with good ventricular function and an elevated heart rate, but is relatively contraindicated in a patient with reactive airway disease. Metoprolol, esmolol, or labetalol is often used intraoperatively. Nicardipine or clevidipine may be preferable to B-blockers for patients with bronchospastic disease. Nitroprusside remains the most rapid and effective agent for the intraoperative treatment of moderate to severe hypertension. Nitroglycerin may be less effective, but is also useful in treating or preventing myocardial ischemia. Fenoldopam, a dopaminergic agonist, is also a useful hypotensive agent; furthermore, it increases renal blood flow. Hydralazine provides sustained blood pressure control, but also has a delayed onset and can cause reflex tachycardia. Reflex tachycardia is not seen with labetalol because of its combined aand Badrenergic blockade.

Nitroprusside, Dosage Range = 0.5-10 mcg/kg/min. Nitroprusside, Onset = 30-60. Nitroprusside, Duration = 1-5 min. Nitroglycerin, Dosage Range = 0.5-10 mcg/kg/min. Nitroglycerin, Onset = min. Nitroglycerin, Duration = 3-5 min. Esmolol, Dosage Range = 0.5 mg/kg over 1 min; 50-300 mcg/kg/min. Esmolol, Onset = 1min. Esmolol, Duration = 12-20 min. Labetalol, Dosage Range = 5-20 mg. Labetalol, Onset = 1-2 min. Labetalol, Duration = 4-8h. Metoprolol, Dosage Range = 2.5-5 mg. Metoprolol, Onset = 1-5 min. Metoprolol, Duration = 5-8h. Hydralazine, Dosage Range = 5-20 mg. Hydralazine, Onset = 5-20 min. Hydralazine, Duration = 4-8h. Clevidipine, Dosage Range = 1-32 mg/h. Clevidipine, Onset = 1-3 min. Clevidipine, Duration = 5-15 min. Nicardipine, Dosage Range = 5-15 mg/h. Nicardipine, Onset = 1-5 min. Nicardipine, Duration = 3-4h. Enalaprilat, Dosage Range = 0.625-1.25 mg. Enalaprilat, Onset = 6-15 min. Enalaprilat, Duration = 4-6h. Fenoldopam, Dosage Range = 0.1-1.6 mg/kg/min. Fenoldopam, Onset = 5 min. Fenoldopam, Duration = Smin

Postoperative hypertension is common and should be anticipated in patients who have poorly controlled baseline blood pressure. Close blood pressure monitoring should be continued in both the postanesthesia care unit and the early postoperative period. Postoperatively, marked sustained elevations in blood pressure can contribute to the formation of wound hematomas and the disruption of vascular suture lines.
Hypertension in the recovery period is often multifactorial and enhanced by respiratory abnormalities, anxiety and pain, volume overload, bladder distention, or any combination of these. Contributing causes should be corrected and parenteral antihypertensive agents given if necessary. Intravenous labetalol is particularly useful in controlling hypertension and tachycardia, whereas vasodilators are useful in controlling blood pressure in the setting of a slow heart rate. When the patient resumes oral intake, preoperative antihypertensive medications should be restarted.

Preoperative Considerations
Myocardial ischemia results from a metabolic oxygen demand that exceeds the oxygen supply. Ischemia can therefore result from increased myocardial metabolic demand, reduced myocardial oxygen delivery, or a combination of both. Common causes include coronary arterial thrombosis or vasospasm; severe hypertension or tachycardia (particularly in the presence of ventricular hypertrophy); severe hypotension, hypoxemia, or anemia; and severe aortic stenosis or regurgitation.
By far, the most common cause of myocardial ischemia is atherosclerosis of the coronary arteries. CAD is responsible for about 25% of all deaths in Western societies and is a major cause of perioperative morbidity and mortality. The overall incidence of CAD in surgical patients is estimated to be between 5% and 10%. Major preoperative risk factors for CAD include hyperlipidemia, hypertension, diabetes, cigarette smoking, increasing age, male sex, and a positive family history. Other risk factors include obesity, a history of cerebrovascular or peripheral vascular disease, menopause, use of high-estrogen oral contraceptives by women who smoke, and a sedentary lifestyle. CAD may be manifested by symptoms, characteristic electrocardiographic or echocardiographic findings, or biochemical evidence of myocardial necrosis (infarction); symptoms (usually angina) or characteristic echocardiographic or electrocardiographic findings of ischemia; or arrhythmias (including sudden death), symptoms (orthopnea, dyspnea on exertion), signs (rales, dependent edema, shock), or echocardiographic changes suggestive of ventricular dysfunction. An ambulatory patient presenting with risk factors for CAD and new symptoms would normally undergo some form of stress testing to confirm the suspected diagnosis.

Unstable angina is defined as (1) an abrupt increase in severity, frequency (more than three episodes per day), or duration of anginal attacks (crescendo angina); (2) angina at rest; or (3) new onset of angina (within the past 2 months) with severe or frequent episodes (more than three per day). Unstable angina may occur following MI or be precipitated by major surgery or by noncardiac medical conditions, including severe anemia, fever, infections, thyrotoxicosis, hypoxemia, and emotional distress in previously stable patients.
Unstable angina, particularly when it is associated with significant STsegment changes at rest, usually reflects severe underlying coronary disease and may be followed by MI. Plaque disruption with platelet aggregates or thrombi and vasospasm are frequent pathological correlates. Critical stenosis in one or more major coronary arteries is present in more than 80% of patients with these symptoms. Patients with unstable angina require evaluation and treatment, which may include admission to a coronary care unit and some form of coronary intervention.

The general approach in treating patients with ischemic heart disease is fivefold:
¢ Correction of risk factors, with the hope of slowing disease progression
¢ Modification of the patient's lifestyle to reduce stress and improve exercise tolerance
* Correction of complicating medical conditions that can exacerbate ischemia (ie, hypertension, anemia, hypoxemia, hyperthyroidism, fever, infection, or adverse drug effects)
¢ Pharmacological manipulation of the myocardial oxygen supply—demand relationship
¢ Anticoagulation
* Correction of coronary lesions by percutaneous coronary intervention (angioplasty [with or without stenting] or atherectomy) or coronary artery bypass surgery
The most commonly used pharmacological agents for stable ischemic heart disease are nitrates, B-blockers, calcium channel blockers, and platelet inhibitors. Those drugs with circulatory effects are compared in Table 21—7.

qT tT. Vasodilatation Coronary Systemic, B-Blockers = tt Ht
'SA, sinoatrial; AV, atrioventricular; f, increases; —, no change; 4, decreases.

These drugs are first-line agents for patients with stable ischemic heart disease. They decrease myocardial oxygen demand by reducing heart rate and contractility, and, in some cases, afterload (via their antihypertensive effect). In contrast to other agents, they increase survival in patients with impaired left ventricular function, they increase survival after MI, and they reduce the likelihood of a subsequent infarction. Optimal blockade results in a resting heart rate between 50 and 60 beats/min and prevents appreciable increases with exercise (<20 beats/min increase during exercise). Available agents differ in receptor selectivity, intrinsic sympathomimetic (partial agonist) activity, and membrane-stabilizing properties (Table 21—8). Membrane stabilization results in antiarrhythmic activity. Agents with intrinsic sympathomimetic properties are better tolerated by patients with mild to moderate ventricular dysfunction. Certain B-blockers (bisoprolol, carvedilol, and extended-duration metoprolol) improve survival in patients with chronic heart failure. Blockade of B>adrenergic receptors also can mask hypoglycemic symptoms in patients with diabetes, delay metabolic recovery from hypoglycemia, and impair the handling of large potassium loads. Cardioselective (B,-receptor-specific) agents, although generally better tolerated than nonselective agents in patients with reactive airways, must still be used cautiously in such patients. The selectivity of cardioselective agents tends to be dose dependent. Patients on long-standing Bblocker therapy should have these agents continued perioperatively. Acute Bblocker withdrawal in the perioperative period places patients at a markedly increased risk of cardiac morbidity and mortality. B-Blocker therapy should be continued perioperatively in patients who take these agents chronically.

B,-Receptor Selectivity = . Nadolol, Half-Life = 10-24h. Nadolol, Sympathomimetic = . Nadolol, a-Receptor Blockade = . Nadolol, Membrane Stabilizing = . Carvedilol, B,-Receptor Selectivity = . Carvedilol, Half-Life = 6-8h. Carvedilol, Sympathomimetic = . Carvedilol, a-Receptor Blockade = +. Carvedilol, Membrane Stabilizing = +
'Also possesses unique antiarrhythmic properties.

These agents are chosen when a patient cannot take a B-blocker or when treatment with a B-blocker is insufficient. The effects and uses of the most commonly used calcium channel blockers are shown in Table 21—9. Calcium channel blockers reduce myocardial oxygen demand by decreasing cardiac afterload and augment myocardial oxygen supply via coronary vasodilation. Verapamil and diltiazem also reduce demand by slowing the heart rate.

Dosage’ = 5-20mg. Felodipine, Half-life = oh. Felodipine, Clinical Use.Angina = . Felodipine, Clinical Use.Hypertension = +. Felodipine, Clinical Use.Cerebral Vasospasm = . Felodipine, Clinical Use.Supraventricular Tachycardia = . Amlodipine, Route = PO. Amlodipine, Dosage’ = 2.5-10 mg. Amlodipine, Half-life = 30-50h. Amlodipine, Clinical Use.Angina = +. Amlodipine, Clinical Use.Hypertension = +. Amlodipine, Clinical Use.Cerebral Vasospasm = . Amlodipine, Clinical Use.Supraventricular Tachycardia = 
Total oral dose per day divided into three doses unless otherwise stated
'Also possesses antiarrhythmic properties.
Nifedipine's potent effects on the systemic blood pressure may precipitate hypotension, reflex tachycardia, or both. Its tendency to decrease afterload generally offsets any negative inotropic effect. Long-acting verapamil, diltiazem, amlodipine, or felodipine are preferred. Nicardipine and clevidipine generally have the same effects as nifedipine but are shorter acting, and clevidipine is particularly useful as a vasodilator infusion. Nimodipine is primarily used in preventing cerebral vasospasm following subarachnoid hemorrhage. All calcium channel blockers potentiate depolarizing and nondepolarizing neuromuscular blocking agents and the circulatory effects of volatile agents. Verapamil and diltiazem can potentiate depression of cardiac contractility and conduction in the atrioventricular (AV) node by volatile anesthetics. Nifedipine and similar agents can potentiate systemic vasodilation by volatile and intravenous agents.

Nitrates decrease venous and arteriolar tone, increase vascular capacitance, and reduce ventricular wall tension. These effects tend to reduce myocardial oxygen demand. Prominent venodilation makes nitrates excellent agents when congestive heart failure is also present.
Additionally, nitrates dilate the coronary arteries. Even minor degrees of dilation at stenotic sites may be sufficient to increase blood flow, because flow is directly related to the fourth power of the radius. Nitrate-induced coronary vasodilation preferentially increases subendocardial blood flow in ischemic areas. This favorable redistribution of coronary blood flow to ischemic areas may be dependent on the presence of collaterals in the coronary circulation.
Nitrates can be used for both the treatment of acute ischemia and prophylaxis against frequent anginal episodes.

Chronic aspirin therapy reduces coronary events in patients with CAD and prevents coronary and ischemic cerebral events in at-risk patients. Other platelet antagonists are generally also included in patients who have undergone percutaneous coronary stenting. Careful review of anticoagulant/antiplatelet medications is a mandatory element of preanesthetic assessment, especially if neuraxial anesthesia is being considered (see Chapter 45).

ACE inhibitors prolong survival in patients with congestive heart failure or left ventricular dysfunction. Antiarrhythmic therapy in patients with complex ventricular ectopy who have significant CAD and left ventricular dysfunction should be guided by an electrophysiological study. Patients with inducible sustained ventricular tachycardia (VT) or ventricular fibrillation are candidates for an automatic internal cardioverter-defibrillator (ICD). Treatment of ventricular ectopy (with the exception of sustained VT) in patients with good ventricular function does not improve survival and may increase mortality. In contrast, ICDs have been shown to improve survival in patients with advanced cardiomyopathy (ejection fraction <30%), even in the absence of demonstrable arrhythmias.

Moderate to severe angina frequently requires combination therapy with two or more classes of agents. Patients with ventricular dysfunction may not tolerate the combined negative inotropic effect of a B-blocker and a calcium channel blocker together; an ACE inhibitor or ARB is better tolerated and seems to improve survival. Similarly, the additive effect of a B-blocker and a calcium channel blocker on the AV node may precipitate heart block in susceptible patients.

TABLE 21-10 Summary of recommendations for perioperative therapy.!

defibrillation equipment should be available. Ensure that ICDs are reprogrammed to active therapy, 1 = |. Patients with ICDs should be ona cardiac monitor continuously during the entire period of inactivation, and external defibrillation equipment should be available. Ensure that ICDs are reprogrammed to active therapy, 2 = Cc
'Reproduced with permission from Fleisher LA, Fleischman KE, Auerbach AD, et al. 2014 ACC/AHA guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery: A report of the American College of Cardiology/American Heart Association Task Force on practice guidelines. J Am Coll Cardiol. 2014 Dec 9;64(22):e77-e137.
2ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blocker; BMS, bare metal stent; CIED, cardiovascular implantable electronic device; COR, class of recommendation; CPG, clinical practice guidelines; DAPT, dual antiplatelet therapy; DES, drug-eluting stent; ERC, Evidence Review Com mittee; ICD, implantable cardioverter-defibrillator; LOE, level of evidence; PCI, percutaneous coronary intervention; RCRI, Revised Cardiac Risk Index SR, systematic review.
'Because of new evidence, this is a new recommendation since the publication of the 2011 PCICPG.
"These recommendations have been designated with a * to emphasize the rigor of support from the ERC's systemic review.

The most important symptoms to elicit include chest pain, dyspnea, poor exercise tolerance, syncope, or near syncope. The relationship between symptoms and activity level should be established. Activity should be described in terms of everyday tasks, such as walking or climbing stairs. Patients may be relatively asymptomatic despite severe CAD if they have a sedentary lifestyle. Patients with diabetes are particularly prone to silent ischemia. Easy fatigability or shortness of breath suggests impaired ventricular function. occurrence
A history of unstable angina or MI should include the time of its and whether it was complicated by arrhythmias, conduction disturbances, or heart failure. Arrhythmias and conduction abnormalities are more common in patients with previous infarction and in those with poor ventricular function. This latter group of patients will often have ICDs.

Evaluation of patients with CAD is similar to that of patients with hypertension. Laboratory evaluation in patients who have a history compatible with recent unstable angina and are undergoing emergency procedures should include cardiac enzymes. Normal serum levels of troponins, creatine kinase (MB isoenzyme), and lactate dehydrogenase (type 1 isoenzyme) are useful in excluding MI.
The baseline ECG is normal in 25% to 50% of patients with CAD but no prior MI. Electrocardiographic evidence of ischemia often becomes apparent only during angina. The most common baseline abnormalities are nonspecific ST-segment and T-wave changes. Prior infarction may be manifested by Q waves or loss of R waves in the leads closest to the infarct. First-degree AV block, bundle-branch block, or hemiblock may be present. Persistent ST-segment elevation following MI may be indicative of a left ventricular aneurysm. A long rate-corrected QT interval (QT, > 0.44 s) may reflect the underlying ischemia, drug toxicity (usually class Ja antiarrhythmic agents, antidepressants, or phenothiazines), electrolyte abnormalities (hypokalemia or hypomagnesemia), autonomic dysfunction, mitral valve prolapse, or, less commonly, a congenital abnormality. Patients with a long QT interval are at risk of developing ventricular arrhythmias—particularly polymorphic VT (torsades de pointes), which can lead to ventricular fibrillation. The long QT interval reflects nonuniform prolongation of ventricular repolarization and predisposes patients to reentry phenomena. In contrast to polymorphic ventricular arrhythmias with a normal QT interval, which respond to conventional antiarrhythmics, polymorphic tachyarrhythmias with a long QT interval generally respond best to pacing or magnesium salts.
The chest radiograph can be used to exclude cardiomegaly or pulmonary vascular congestion secondary to ventricular dysfunction.

Continuous ambulatory electrocardiographic (Holter) monitoring is useful in evaluating arrhythmias, antiarrhythmic drug therapy, and severity and frequency of ischemic episodes. Silent (asymptomatic) ischemic episodes are frequently found in patients with CAD. Frequent ischemic episodes on preoperative Holter monitoring correlate well with intraoperative and postoperative ischemia. Holter monitoring showing no ischemic episodes has an excellent negative predictive value for postoperative cardiac complications.

The usefulness of this test without associated cardiac imaging is limited in patients with baseline ST-segment abnormalities and those who are unable to increase their heart rate (>85% of maximal predicted) because of fatigue, dyspnea, or drug therapy. Overall sensitivity is 65%, and specificity is 90%. Exercise testing is most sensitive (85%) in patients with three-vessel or left main CAD. Disease that is limited to the left circumflex artery may also be missed because ischemia in its distribution may not be evident on the standard surface ECG. A normal test does not necessarily exclude CAD, but suggests that severe disease is not likely. The degree of ST-segment depression, its severity and configuration, the time of onset in the test, and the time required for resolution are important findings. A myocardial ischemic response at low levels of exercise is associated with a significantly increased risk of perioperative complications and long-term cardiac events. Other significant findings include changes in blood pressure and the occurrence of arrhythmias. Exercise-induced ventricular ectopy frequently indicates severe CAD associated with ventricular dysfunction. The ischemia presumably leads to electrical instability in myocardial cells. Given that risk seems to be associated with the extent of potentially ischemic myocardium, testing often includes perfusion scans or echocardiographic assessments; however, in ambulatory patients, exercise ECG testing alone is useful because it estimates functional capacity and detects myocardial ischemia.

C. Myocardial Perfusions Scans and Other Imaging Techniques Myocardial perfusion imaging using thallium-201 or technetium-99m is used in evaluating patients who cannot exercise (eg, peripheral vascular disease) or who have underlying ECG abnormalities that preclude interpretation during exercise (eg, left bundle-branch block). If the patient cannot exercise, images are obtained before and after injection of an intravenous coronary dilator (eg, dipyridamole or adenosine) to produce a hyperemic response similar to exercise. Myocardial perfusion studies following exercise or injection of dipyridamole or adenosine have a high sensitivity, but only fairly good specificity for CAD. They are best for detecting twoor three-vessel disease. These scans can locate and quantitate areas of ischemia or scarring and differentiate between the two. Perfusion defects that fill in on the redistribution phase represent ischemia, not previous infarction. The negative predictive value of a normal perfusion scan is approximately 99%.
Magnetic resonance imaging, positron emission tomography, and computed tomography scans are increasingly being used to define coronary artery anatomy and determine myocardial viability.

This technology provides information about both regional and global ventricular function and may be carried out at rest, following exercise, or with administration of dobutamine. Detectable regional wall motion abnormalities and the derived left ventricular ejection fraction correlate well with angiographic findings. Moreover, dobutamine stress echocardiography seems to be a reliable predictor of adverse cardiac complications in patients who cannot exercise. New or worsening wall motion abnormalities following dobutamine infusion are indicative of significant ischemia. Patients with an ejection fraction of less than 50% tend to have more severe disease and increased perioperative morbidity. Dobutamine stress echocardiography, however, may not be reliable in patients with left bundle-branch block because septal motion may be abnormal, even in the absence of left anterior descending CAD in some patients.

Coronary angiography remains the definitive way to evaluate CAD and is associated with a low complication rate (<1%). The location and severity of occlusions can be defined, and coronary vasospasm may also be observed on angiography. In evaluating fixed stenotic lesions, occlusions greater than 50% to 75% are generally considered significant. The severity of disease is often expressed according to the number of major coronary vessels affected (one-, two-, or three-vessel disease). Significant stenosis of the left main coronary artery is of great concern because disruption of flow in this vessel will have adverse effects on almost the entire left ventricle.
Ventriculography, measurement of the ejection fraction, and measurement of intracardiac pressures, also provide important information. Indicators of significant ventricular dysfunction include an ejection fraction less than 50%, a left ventricular end-diastolic pressure greater than 18 mm Hg, a cardiac index less than 2.2 L/min/m?, and marked or multiple wall motion abnormalities.

Allaying fear, anxiety, and pain preoperatively are desirable goals in patients with CAD. Satisfactory premedication minimizes sympathetic activation, which adversely affects the myocardial oxygen supply—demand balance. Overmedication is equally detrimental and should be avoided because it may result in hypoxemia, respiratory acidosis, and hypotension. Most clinicians now limit premedication to small doses of intravenous midazolam (or the equivalent)
given immediately before invasive procedures or before transporting the patient to the operating theater.
Preoperative medications should generally be @ continued until the time of surgery. The sudden withdrawal of antianginal medication perioperatively— particularly B-blockers—can precipitate a sudden, rebound increase in ischemic episodes. Statins should also be continued in the perioperative period. Prophylactic administration of nitrates intravenously or transdermally to patients with CAD in the perioperative period provides no benefit to patients not previously on long-term nitrate therapy and without evidence of ongoing ischemia. Transdermal absorption of nitroglycerin may be erratic in the perioperative period.

The intraoperative period is regularly associated with factors and events that can adversely affect the myocardial oxygen demand-—supply relationship. Activation of the sympathetic system plays a major role. Hypertension and enhanced contractility increase myocardial oxygen demand, whereas tachycardia increases demand and reduces supply. Although myocardial ischemia is commonly associated with tachycardia, ischemia can occur in the absence of any apparent hemodynamic derangement.

@ The overwhelming priority in managing patients with ischemic heart disease is maintaining a favorable myocardial supply—demand relationship. Autonomicmediated increases in heart rate and blood pressure should be controlled with deeper planes of general anesthesia, adrenergic blockade, vasodilators, or a combination of these. Excessive reductions in coronary perfusion pressure or arterial oxygen content must be avoided. Higher diastolic pressures may be preferable in patients with high-grade coronary occlusions. Excessive increases —such as those caused by fluid overload—in left ventricular end-diastolic pressure should be avoided because they increase ventricular wall tension (afterload) and can reduce subendocardial perfusion (see Chapter 20). Transfusion carries its own risks and consequently there is no set transfusion trigger in patients with CAD; however, most clinicians are reluctant to have hemoglobin levels fall below 7 g/dL. Anemia can lead to tachycardia, worsening the balance between myocardial oxygen supply and demand. The ACC/AHA recommendations for the anesthetic management of the patient with CAD

'Reproduced with permission from Fleisher LA, Fleischman KE, Auerbach AD, et al. 2014 ACC/AHA guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery: A report of the American College of Cardiology/American Heart Association Task Force on practice guidelines, JAm Coll Cardiol. 2014 Dec 9;64(22ke77-€1 37.
2COR, class of recommendation; LOE, level of evidence; Ml, myocardial infarction; TEE, transesophageal echocardiogram.

@ Intraarterial pressure monitoring is reasonable in most patients with severe CAD and major or multiple cardiac risk factors who are undergoing any but the most minor procedures. Central venous (or rarely pulmonary artery) pressure can be monitored during prolonged or complicated procedures involving large fluid shifts or blood loss. Noninvasive methods of cardiac output determination and volume assessment have been previously discussed in this text and we recommend them. Transesophageal echocardiography (TEE) and transthoracic echocardiography (TTE) can provide valuable information, both qualitative and quantitative, on contractility and ventricular chamber size (preload) perioperatively

Early ischemic changes are subtle and involve changes in T-wave morphology, including inversion, tenting, or both (Figure 21—2). More obvious ischemia may be seen in the form of progressive ST-segment depression. Down-sloping and horizontal ST depressions are of greater specificity for ischemia than is upsloping depression. New ST-segment elevations are rare during noncardiac surgery and are indicative of severe ischemia, vasospasm, or infarction.
FIGURE 21-2 Electrocardiographic signs of ischemia. Patterns of ischemia and injury. (Data from Schamroth L. The 12 Lead Electrocardiogram. Oxford, UK: Blackwell; 1989.)
It should be noted that an isolated minor ST elevation in the mid-precordial leads (V3 and V,) can be a normal variant in young patients. Ischemia may also present as an unexplained intraoperative atrial or ventricular arrhythmia or the onset of a new conduction abnormality. The sensitivity of the ECG in detecting ischemia is related to the number of leads monitored. Studies suggest that the V., V4, Il, V2, and V3 leads (in decreasing sensitivity) are most useful. Ideally, at least two leads should be monitored simultaneously. Usually, lead II is monitored for inferior wall ischemia and arrhythmias, and V; is monitored for anterior wall ischemia. When only one lead can be monitored, a modified V; lead provides the greatest sensitivity.
The increasing number of individuals treated with drug-eluting stents can be problematic perioperatively, especially when antiplatelet therapy must be discontinued (eg, emergency spinal surgery). Such patients are at very increased risk of thrombosis and perioperative MI. Anesthesia providers should never for nonsurgical reasons (eg, desire to perform a spinal anesthetic) discontinue antiplatelet or antithrombotic agents perioperatively without first discussing the risks and benefits of the proposed anesthetic requiring suspension of antiplatelet therapy with the patient and his or her cardiologist. The ACC/AHA guidelines offer recommendations on the approach of bringing patients to surgery following percutaneous coronary interventions and the type of interventions suggested when subsequent surgery is expected (Figure 21-3).

FIGURE 21-3 Algorithm for antiplatelet management in patients with percutaneous coronary intervention (PCI) and noncardiac surgery. Colors correspond to the following classes of recommendations: class I, green; class IIa, yellow; class IIb, orange; class III, red. ("Assuming the patient is currently on dual antiplatelet therapy [DAPT].) ASA, acetylsalicylic acid; ASAP, as soon as possible; BMS, bare-metal stent; DES, drug-eluting stent; P2Y, adenosine diphosphate receptor antagonist. (Reproduced with permission from Fleisher LA, Fleischman KE, Auerbach AD, et al. 2014 ACC/AHA guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery: A report of the American College of Cardiology/American Heart Association Task Force on practice guidelines. J Am Coll Cardiol. 2014 Dec 9;64(22):e77-e137.)

The most common hemodynamic abnormalities observed during ischemic episodes are hypertension and tachycardia. They are almost always a cause (rather than the result) of ischemia. Hypotension is a late and ominous manifestation of progressive ventricular dysfunction. TEE readily will demonstrate a dysfunctional ventricle and ventricular wall motion changes associated with myocardial ischemia. Ischemia is frequently, but not always, associated with an abrupt increase in pulmonary capillary wedge pressure; however, it is rare for pulmonary capillary wedge pressure to be measured during general anesthesia.

TEE can be helpful in detecting global and regional cardiac dysfunction, as well as valvular function, in surgical patients. Moreover, detection of new regional wall motion abnormalities is a more sensitive and earlier indication of myocardial ischemia than the ECG. In animal studies in which coronary blood flow is gradually reduced, regional wall motion abnormalities develop before the ECG changes. Although the occurrence of new intraoperative abnormalities correlates with postoperative MIs in some studies, not all such abnormalities are necessarily ischemic. Both regional and global abnormalities can be caused by changes in heart rate, altered conduction, preload, afterload, or drug-induced changes in contractility. Decreased systolic wall thickening may be a more reliable index for ischemia than endocardial wall motion alone.

Electrolyte disorders, heart structure defects, inflammation, myocardial ischemia, cardiomyopathies, and conduction abnormalities can all contribute to the development of perioperative arrhythmias and heart block. Consequently, anesthesia providers must be prepared to treat both chronic and new-onset cardiac rhythm abnormalities.
Supraventricular tachycardias (SVTs) can have hemodynamic consequences secondary to loss of AV synchrony and decreased diastolic filling time. Loss of the "P" wave on the ECG with a fast ventricular response is consistent with SVTs. Most SVTs occur secondary to a reentrant mechanism. Reentrant arrhythmias occur when conduction tissues in the heart depolarize or repolarize at varying rates. In this manner, a self-perpetuating loop of repolarization and depolarization can occur in the conduction pathways or AV node, or both. SVTs producing hemodynamic collapse are treated perioperatively with synchronized cardioversion. Adenosine can likewise be given to slow AV node conduction and potentially disrupt the reentrant loop. SVTs in patients without accessory conduction bundles (Wolff—Parkinson—White [WPW] syndrome) are treated with B-blockers and calcium channel blockers. In patients with known WPW, procainamide or ibutilide can be used to treat SVTs. Use of intravenous amiodarone, adenosine, digoxin, or non-dihydropyridine calcium channel antagonists is considered a class III recommendation by the AHA/ACC as these agents may harmfully increase the ventricular response in patients with preexcitation syndromes such as WPW. At times, SVTs manifest with a broad QRS complex and seem to be similar to VTs. Such rhythms, when they present, should be treated like VT, until proven otherwise.
Atrial fibrillation (AF) can complicate the perioperative period (Figure 21— 4). Up to 35% of cardiac surgery patients develop postoperative AF.

FIGURE 21-4 Posterior view of principal electrophysiological mechanisms of atrial fibrillation. A: Focal activation. The initiating focus (indicated by the dot) often lies within the region of the pulmonary veins. The resulting wavelets represent fibrillatory conduction, as in multiple-wavelet reentry. B: Multiplewavelet reentry. Wavelets (arrows) randomly reenter tissue previously activated by the same or another wavelet. The routes the wavelets travel vary. LA, left atrium; PV, pulmonary vein; ICV, inferior vena cava; SCV, superior vena cava; RA, right atrium. (Reproduced with permission from Konings KT, Kirchhof CJ, Smeets JR, et al. High-density mapping of electrically induced atrial fibrillation in humans. Circulation. 1994 Apr;89(4):1665-1680.)
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When AF develops perioperatively, rate control with B-blockers can often be instituted. Chemical cardioversion can be attempted with amiodarone or procainamide. Of note, if the duration of AF is greater than 48 hours, or unknown, ACC/AHA guidelines recommend anticoagulation for 3 weeks prior to, and 4 weeks following, either electrical or chemical cardioversion. Additionally, TEE can be performed to rule out the presence of left atrial or left atrial appendage thrombus.

Should AF develop postoperatively, ventricular rate response can be controlled with AV nodal blocking agents, unless contraindicated. Should AF result in hemodynamic instability, synchronized cardioversion should be attempted. Patients at high risk of AF following cardiac surgery can be treated with prophylactic amiodarone. Many centers routinely administer B-blockers or amiodarone to all patients undergoing coronary artery surgery to reduce the risk of new onset AF.
AF is most frequently associated with loss of atrial muscle and the development of fibrosis. Fibrosis may contribute to reentrant mechanisms of AF as depolarization/repolarization becomes nonhomogeneous. AF may also develop from a focal source often located in the pulmonary veins. In patients with an accessory bundle, AF can produce rapid ventricular responses and hemodynamic collapse. Drugs that slow conduction across the AV node (eg, digitalis, verapamil, diltiazem) do not slow conduction across the accessory pathway, potentially leading to hemodynamic collapse. The ACC/AHA guidelines likewise recommend caution in the use of B-blockers for AF in patients with preexcitation syndromes.
Ventricular arrhythmias have been the subject of much review by the AHA (Table 21—12). Ventricular premature contractions (VPCs) can appear perioperatively secondary to electrolyte abnormalities (hypokalemia, hypomagnesium, hypocalcemia), acidosis, ischemia, embolic phenomenon, mechanical irritation of the heart from central lines, cardiac manipulation, and drug effects. Correction of the underlying source of any arrhythmia should be addressed. Patients can likewise present with VPCs secondary to various cardiomyopathies.

= VT >30 sin duration and/or requiring termination due to hemodynamic compromise in <30 s.. Bundle-branch reentrant tachycardia, Classification by Clinical Presentation = . Bundle-branch reentrant tachycardia,  = VT due to reentry involving the His-Purkinje system, usually with LBBB morphology; this usually occurs in the setting of cardiomyopathy.. Bidirectional VT, Classification by Clinical Presentation = . Bidirectional VT,  = VT with a beat-to-beat alternans in the ORS frontal plane axis, often associated with digitalis toxicity,. Torsades de pointes, Classification by Clinical Presentation = . Torsades de pointes,  = Characterized by VT associated with along QT or QTc, and electrocardiographically characterized by twisting of the peaks of the ORS complexes around the isoelectric line during the arrhythmia: + “Typical,” initiated following “short-long-short” coupling intervals. + Short coupled variant initiated by normal-short coupling.. Ventricular flutter, Classification by Clinical Presentation = . Ventricular flutter,  = Aregular (cycle length variability <30 ms) ventricular arrhythmia approximately 300 bpm (cycle length 200 ms) with a monomorphic appearance; no isoelectric interval between successive QRS complexes.
Ventricular fibrillation, 1 = Rapid, usually more than 300 bpm/200 ms (cycle length <180 ms), grossly irregular ventricular rhythm with marked variability in QRS cycle length, morphology, and amplitude.. Classification by Disease Entity, 1 = . Chronic coronary heart disease Heart failure Congenital heart disease Neurological disorders Structurally normal hearts Sudden infant death syndrome Cardiomyopathies: Dilated cardiomyopathy Hypertrophic cardiomyopathy Arrhythmogenic right ventricular cardiomyopathy, 1 = 

'Reproduced with permission from Zipes DP, Camm AJ, Borggrefe M, et al. ACC/AHA/ESC 2006 guidelines for management of patients with ventricular arrhythmias and the prevention of sudden cardiac death—executive summary. Circulation, 2006 Sep 5;114(10):1088-1132. ?LBB8, left bundle-branch block; VT, ventricular tachycardia.
The incidence of sudden cardiac death (SCD) is estimated at 1 to 2/1000 per year. Consequently, some patients will experience an unexpected death in the perioperative period. All anesthesia providers must be prepared to resuscitate and manage patients with ventricular arrhythmias, including ventricular tachycardia (VT) (nonsustained and sustained) and ventricular fibrillation.
Nonsustained VT is a short run of ventricular ectopy that lasts <30 s and spontaneously terminates, whereas sustained VT persists longer than 30 s. VT is either monomorphic or polymorphic, depending on the QRS complex. If the QRS complex morphology changes, it is designated as polymorphic VT. Torsades des pointes is a form of VT associated with a prolonged QT interval, producing a sine wave-like VT pattern on the ECG. Ventricular fibrillation requires immediate resuscitative efforts and defibrillation.
Patients presenting with ventricular ectopy and nonsustained runs of VT routinely undergo investigation prior to surgery; however, patients with such rhythm abnormalities may not be at greater risk for nonfatal MI or cardiac death perioperatively. Supraventricular and ventricular arrhythmias constitute active cardiac conditions that warrant evaluation and treatment prior to elective, noncardiac surgery. Electrophysiological studies are undertaken to determine the possibility for catheter-mediated ablation of VTs.
Should VT present perioperatively, cardioversion is recommended whenever hemodynamic compromise occurs. Otherwise, treatment with amiodarone or procainamide can be attempted. At all times, therapy should also be directed at identifying any causative sources of the arrhythmia. B-Blockers are useful in the treatment of VT, especially if ischemia is a suspected causative factor in the

2., 1 = Intraoperative. Determine what electromagnetic interference is likely to present intraoperatively and advise the use of bipolar electrocautery where possible. Assure the availability of temporary pacing and defibrillation equipment and apply pads as necessary. Patients who are pacemaker- dependent can be programmed to an asynchronous mode to mitigate electrical interference. Magnet application to ICDs may disable the antitachycardia function, but not convert to an asynchronous pacemaker. Consultation with the patient's cardiologist and interrogation of the device is often necessary. Most patients will have a card on which the device model and manufacturer are provided. Atelephone call to the device manufacturer can provide information about device performance and the best method for managing the device (eg, reprogramming or applying a magnet) prior to surgery. Alarge number of ICD models are in use; however, most suspend their antitachycardia function in response to a magnet.. 3., 1 = Postoperative . The device must be interrogated to ensure that therapeutic functions have been restored. Patients should be continuously monitored until the antitachycardia functions of the device are restored and its function has been confirmed.. ICDs are particularly problematic intraoperatively when electrocautery is used because the device may (1) interpret cautery as ventricular fibrillation; (2) inhibit pacemaker function due to cautery artifact; (3) increase the pacing rate due to activation of a rate-responsive sensor; or (4) temporarily or permanently reset to a backup or reset mode. Use of bipolar cautery, placement of the grounding pad far from the ICD device, and limiting use of the cautery to only short bursts help to reduce the likelihood of problems, but will not eliminate them., 1 = ICDs are particularly problematic intraoperatively when electrocautery is used because the device may (1) interpret cautery as ventricular fibrillation; (2) inhibit pacemaker function due to cautery artifact; (3) increase the pacing rate due to activation of a rate-responsive sensor; or (4) temporarily or permanently reset to a backup or reset mode. Use of bipolar cautery, placement of the grounding pad far from the ICD device, and limiting use of the cautery to only short bursts help to reduce the likelihood of problems, but will not eliminate them.

An increasing number of patients present for surgery with either systolic or diastolic heart failure. Heart failure may be secondary to ischemia, valvular heart disease, infectious agents, or many forms of cardiomyopathy. Patients may experience heart failure symptoms with both a preserved and reduced ejection fractions. Most heart failure patients seek medical attention for complaints of dyspnea and fatigue. Heart failure develops over time, as symptoms worsen (Figure 21-5). Patients generally undergo echocardiography to diagnose structural heart defects, to detect signs of cardiac "remodeling," to determine the left ventricular ejection fraction, and to assess the heart's diastolic function. Laboratory evaluations of concentration of brain natriuretic peptide (BNP) are likewise obtained to distinguish heart failure from other causes of dyspnea. BNP is released from the heart, and its elevation is associated with impaired ventricular function.

‘surgery as
appropriate
FIGURE 21-5 Stages in the development of heart failure and recommended therapy by stage. ACEI, angiotensin-converting enzyme inhibitor; AF, atrial fibrillation; ARB, angiotensin-receptor blocker; CAD, coronary artery disease; CRT, cardiac resynchronization therapy; DM, diabetes mellitus; EF, ejection fraction; GDMT, guideline-directed medical therapy; HF, heart failure; HFpEF, heart failure with preserved ejection fraction; HFrEF, heart failure with reduced ejection fraction; HRQOL, health-related quality of life; HTN, hypertension; ICD, implantable cardioverter-defibrillator; LV, left ventricular; LVH, left ventricular hypertrophy; MCS, mechanical circulatory support; MI, myocardial infarction. (Reproduced with permission from Yancy C, Jessup M, Bozkurt B, et al. 2013 ACCF/AHA guideline for the management of heart failure: A report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2013 Oct 15;62(16):e147-e239.)
The body attempts to compensate for LV systolic failure through activation of the sympathetic and renin—angiotensin—aldosterone system. Consequently, patients experience salt retention, volume expansion, sympathetic stimulation, and vasoconstriction. The heart dilates to maintain stroke volume in spite of decreased contractility. Over time, compensatory mechanisms fail and contribute to the symptoms associated with heart failure (eg, dependent edema, tachycardia, decreased tissue perfusion). Patients with systolic heart failure are likely to present to surgery having been previously treated with diuretics, B-blockers, ACE inhibitors or ARBs, and possibly aldosterone antagonists. Electrolytes must be measured, as diuretics frequently lead to hypokalemia. ARB or ACE inhibitor use may contribute to hypotension in the surgical patient with heart failure. ACE inhibitors are rarely associated with angioedema requiring emergent airway management.

Myocardial relaxation is a dynamic, not passive, process. The heart with preserved diastolic function accommodates volume during diastole, with minimal increases in left ventricular end-diastolic pressure. Conversely, the heart with diastolic dysfunction relaxes poorly and produces increased left ventricular end-diastolic pressure. The increased left ventricular end-diastolic pressure is transmitted to the left atrium and pulmonary vasculature resulting in symptoms of congestion. Patients with any form of heart failure have increased risk of perioperative morbidity.

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant trait that affects 1 in 500 adults. Many patients are unaware of the condition, and some will present with sudden cardiac death as the initial manifestation. Symptoms include dyspnea, exercise intolerance, palpitations, and chest pain. Clinically, HCM is detected by the murmur of dynamic left ventricular outflow tract (LVOT) obstruction in late systole. Symptomatic patients frequently have a thickened intraventricular septum of 20 to 30 mm. A variety of genetic variants have been identified as causative. The myocardium of the intraventricular septum is abnormal, and many patients can develop diastolic dysfunction without pronounced dynamic obstructive gradients. During systole, the anterior leaflet of the mitral valve abuts the intraventricular septum (Figure 21—6), producing obstruction and a late systolic murmur.
FIGURE 21-6 The midesophageal long axis view is shown. As a consequence of the hypertrophied interventricular septum, flow patterns within the heart are altered so that the anterior leaflet of the mitral valve is drawn during ventricular systole into the left ventricular outflow tract (LVOT), producing obstruction. This is known as systolic anterior motion of the mitral valve. (Reproduced with permission from Wasnick J, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography. New York, NY: McGraw-Hill Education; 2011.)
Perioperative management is aimed at minimizing the degree of LVOT obstruction. This is accomplished by maintaining adequate intravascular volume, avoiding vasodilatation, and reducing myocardial contractility through the use of B-blockers.

1. General Evaluation of Patients
Regardless of the lesion or its cause, preoperative evaluation should be primarily concerned with determining the identity and severity of the lesion and its hemodynamic significance, ventricular function, and the presence of any secondary effects on pulmonary, renal, or hepatic function. Concomitant CAD should not be overlooked, particularly in older patients and those with known risk factors (see earlier discussion). Myocardial ischemia may occur in the absence of significant coronary occlusion in patients with severe aortic stenosis or regurgitation.

One should evaluate exercise tolerance, fatigability, pedal edema, and shortness of breath in general (dyspnea), when lying flat (orthopnea), or at night (paroxysmal nocturnal dyspnea). The New York Heart Association functional classification of heart disease (Table 21—13) is useful for grading the severity of heart failure symptoms and estimating prognosis. Patients should also be questioned about chest pains and neurological symptoms. Some valvular lesions are associated with thromboembolic phenomena. Prior procedures, such as valvotomy or valve replacement and their effects, should also be well documented.

|, Description = Asymptomatic except during severe exertion. lI, Description = Symptomatic with moderate activity. Ml, Description = Symptomatic with minimal activity. IV, Description = Symptomatic at rest
Medications commonly used by valvular heart disease patients include diuretics, vasodilators, ACE inhibitors, B-blockers, antiarrhythmics, and anticoagulants. Preoperative vasodilator therapy may be used to decrease preload, afterload, or both. Excessive vasodilation worsens exercise tolerance and is often first manifested as postural (orthostatic) hypotension.

The most important signs to identify on physical examination are those of congestive heart failure. Left-sided (S3 gallop or pulmonary rales) and rightsided (jugular venous distention, hepatojugular reflux, hepatosplenomegaly, or pedal edema) signs may be present. Auscultatory findings may suggest valvular dysfunction, but echocardiographic studies are more reliable. The ACC/AHA guidelines recommend that transthoracic echocardiography be performed as a class I indication in the initial evaluation of patients suspected of having valvular heart disease. Moreover, the ACC/AHA suggest that any change in symptoms or physical examination findings warrants a repeat transthoracic echocardiography exam. Neurological deficits, usually secondary to embolic phenomena, should be documented.

In addition to the laboratory studies discussed for patients with hypertension and CAD, liver function tests may be useful in assessing hepatic dysfunction caused by passive hepatic congestion in patients with severe or chronic right-sided failure. Adequate reversal of warfarin or heparin anticoagulation may be documented with a prothrombin time and international normalized ratio (INR) or partial thromboplastin time, respectively, prior to surgery. Electrocardiographic findings are generally nonspecific. The chest radiograph is useful to assess cardiac size and pulmonary vascular congestion.

Echocardiography, imaging studies, and cardiac catheterization should only be obtained if the results will change therapy or outcomes. In many instances, noninvasive studies obviate the need for cardiac catheterization, unless there are concerns about CAD. When advanced imaging procedures are performed, they generally address the following questions:
¢ Which valvular abnormality is most important hemodynamically?
¢ What is the severity of an identified lesion?
¢ What degree of ventricular impairment is present?
¢ What is the hemodynamic significance of other identified abnormalities?
¢ Is there any evidence of CAD?
The ACC/AHA have prepared detailed guidelines to assist in the management of patients with valvular heart disease. Although the evaluation of the patient with a heart murmur generally rests with the cardiologist, anesthesia providers will on occasion discover a previously undetected murmur on preanesthetic examination. In particular, anesthetists are concerned that undiagnosed, critical aortic stenosis might be present, which could potentially lead to hemodynamic collapse with either regional or general anesthesia. In the past, most valvular heart diseases were a consequence of rheumatic heart disease; however, with an aging surgical population, increasing numbers of patients have degenerative valve problems. More than one in eight patients older than age 75 years may manifest at least one form of moderate to severe valvular heart disease. A study conducted in the Netherlands reported that the prevalence of aortic stenosis was 2.4% in patients older than age 60 years who were scheduled for elective surgery.
Murmurs occur as a consequence of the accelerated blood flow through narrowed openings in stenotic and regurgitant lesions. Although systolic murmurs may be related to increased blood flow velocity, the ACC/AHA guidelines note that all diastolic and continuous murmurs reflect pathology. When new murmurs are detected in a preoperative evaluation, consultation with the patient's personal physician is helpful to determine the need for echocardiographic evaluation. In many centers, immediate echocardiographic evaluation can be performed in the preoperative area, often by another member of the anesthesia department.

Mitral stenosis almost always occurs as a delayed complication of rheumatic fever. However, mitral stenosis can also occur in dialysis-dependent patients. Two-thirds of patients with mitral stenosis are female. The stenotic process is estimated to begin after a minimum of 2 years following rheumatic heart disease and results from progressive fusion and calcification of the valve leaflets. Symptoms generally develop after 20 to 30 years, when the mitral valve orifice is reduced from its normal 4 to 6 cm? opening to less than 1.5 cm?. Less than 50% of patients have isolated mitral stenosis; the remaining patients also have mitral regurgitation, and up to 25% of patients also have rheumatic involvement of the aortic valve (stenosis or regurgitation).

coronary circulation or acute right ventricular pressure overload. Patients may develop hoarseness as a result of compression of the left recurrent laryngeal nerve by the enlarged left atrium.
Left ventricular function is preserved in the majority of patients with pure mitral stenosis (Figure 21—7); however, impaired left ventricular function may be encountered in up to 25% of patients and presumably represents residual damage from rheumatic myocarditis or coexistent hypertensive or ischemic heart disease.
FIGURE 21-7 Pressure—volume loops in patients with valvular heart disease. A, normal; B, mitral stenosis; C, aortic stenosis; D, mitral regurgitation (chronic); E, aortic regurgitation (chronic). LV, left ventricular. (Reproduced with permission from Jackson JM, Thomas SJ, Lowenstein E. Anesthetic management of patients with valvular heart disease. Semin Anesth. 1982;1:239.)
The left ventricle is chronically underloaded in the patient with mitral stenosis and the stroke volume may be reduced. At the same time, the left atrium, right ventricle, and right atrium are frequently dilated and dysfunctional. Vasodilation that occurs following both neuraxial and general anesthesia can lead to peripheral venous blood pooling and inadequate volume delivery to the left ventricle. This can precipitate hemodynamic collapse.

The time from onset of symptoms to incapacitation averages 5 to 10 years. At that stage, most patients die within 2 to 5 years. Surgical correction is therefore usually undertaken once significant symptoms develop. Percutaneous transseptal balloon valvuloplasty may be used in selected young or pregnant patients, as well as older patients who are poor surgical candidates. Medical management is primarily supportive and includes limitation of physical activity, sodium restriction, and diuretics. Small doses of a B-adrenergic blocking drug may also be useful in controlling heart rate in patients with mild to moderate symptoms. Patients with a history of emboli and those at high risk (age older than 40 years; a large atrium with chronic atrial fibrillation) are usually anticoagulated.

@ The principal hemodynamic goals are to maintain a sinus rhythm (if present preoperatively) and to avoid tachycardia, large increases in cardiac output, and both hypovolemia and fluid overload by judicious administration of intravenous fluids.

Invasive hemodynamic monitoring is often used for major surgical procedures, particularly those associated with large fluid shifts. TEE and noninvasive cardiac output monitors can also be used to help guide perioperative management. Overzealous fluid replacement readily precipitates pulmonary edema in patients with severe disease. Pulmonary capillary wedge pressure measurements in the presence of mitral stenosis reflect the transvalvular gradient and not necessarily left ventricular end-diastolic pressure. Prominent a waves and a decreased y descent are typically present on the pulmonary capillary wedge pressure waveform in patients who are in sinus rhythm. A prominent cv wave on the central venous pressure waveform is usually indicative of secondary tricuspid regurgitation. The ECG typically shows a notched P wave in patients who are in sinus rhythm.

Patients may be very sensitive to the vasodilating effects of spinal and epidural anesthesia. In theory, epidural anesthesia may be easier to manage than spinal anesthesia because of the more gradual onset of sympathetic blockade. There is no "ideal" general anesthetic, and agents should be employed to achieve the desired effects of permitting sufficient diastolic time to adequately load the left ventricle. Vasopressors are often needed to maintain vascular tone following anesthetic induction.
Intraoperative tachycardia may be controlled by deepening anesthesia with an opioid (excluding meperidine) or B-blocker (esmolol or metoprolol). In the presence of atrial fibrillation, ventricular rate should be controlled. Marked hemodynamic deterioration from sudden SVT necessitates cardioversion. Phenylephrine is preferred over ephedrine as a vasopressor because the former lacks B-adrenergic agonist activity. Vasopressin can also be employed to restore vascular tone should hypotension develop secondary to anesthetic induction.

Mitral regurgitation can develop acutely or insidiously as a result of a large number of disorders. Chronic mitral regurgitation is usually the result of rheumatic fever (often with concomitant mitral stenosis); congenital or developmental abnormalities of the valve apparatus; or dilation, destruction, or calcification of the mitral annulus. Acute mitral regurgitation is usually due to myocardial ischemia or infarction (papillary muscle dysfunction or rupture of a chorda tendinea), infective endocarditis, or chest trauma. The ACC/AHA indicate that primary mitral regurgitation is present when one or more of the components of the mitral valve apparatus contribute to valvular incompetence. Correction of the structure of the mitral valve corrects the underlying disease process. In contrast secondary mitral regurgitation, the ACC/AHA notes, is present when ventricular dilation prevents coaptation of the mitral valve leaflets. In this instance, repair of the valve is not curative according to the ACC/AHA's valvular heart disease guidelines because the underlying disease process likewise must be addressed in addition to restoration of valvular competency.

The principal derangement is a reduction in forward stroke volume due to backward flow of blood into the left atrium during systole. The left ventricle compensates by dilating and increasing end-diastolic volume (Figure 21—7). Regurgitation through the mitral valve initially maintains a normal end-systolic volume in spite of an increased end-diastolic volume. However, as the disease progresses the end-systolic volume increases. By increasing end-diastolic volume, the volume-overloaded left ventricle can maintain a normal cardiac output despite blood being ejected retrograde into the atrium. With time, patients with chronic mitral regurgitation eventually develop eccentric left ventricular hypertrophy and progressive impairment in contractility. In patients with severe mitral regurgitation, the regurgitant volume may exceed the forward stroke volume. In time, wall stress increases, resulting in an increased demand for myocardial oxygen supply.
The regurgitant volume passing through the mitral valve is dependent on the size of the mitral valve orifice (which can vary with ventricular cavity size), the heart rate (systolic time), and the left ventricular—left atrial pressure gradient during systole. The last factor is affected by the relative resistances of the two outflow paths from the left ventricle, namely, SVR and left atrial compliance. Thus, a decrease in SVR or an increase in mean left atrial pressure will reduce the regurgitant volume. Atrial compliance also determines the predominant clinical manifestations. Patients with normal or reduced atrial compliance (acute mitral regurgitation) have primarily pulmonary vascular congestion and edema. Patients with increased atrial compliance (long-standing mitral regurgitation resulting in a large dilated left atrium) primarily show signs of a reduced cardiac output. Most patients are between the two extremes and exhibit symptoms of both pulmonary congestion and low cardiac output. Patients with a regurgitant fraction of less than 30% of the total stroke volume generally have mild symptoms. Regurgitant fractions of 30% to 60% generally cause moderate symptoms, whereas fractions greater than 60% are associated with severe disease.

Echocardiography, particularly TEE, is useful in delineating the underlying pathophysiology of mitral regurgitation and guiding treatment. Mitral valve leaflet motion is often described as normal, prolapsing, or restrictive (Figure 21— 8). Excessive motion or prolapse is defined by systolic movement of a leaflet beyond the plane of the mitral valve and into the left atrium (see later section on mitral valve prolapse).
Normal motion
Prolapse (esiliaatve motion)
Restricted motion
Bileaflet prolapse
FIGURE 21-8 Classification of mitral valve leaflet motion (as seen from transesophageal echocardiography). Note that with prolapse, the free edge of the leaflet(s) extends beyond the plane of the mitral annulus, producing an eccentric jet. With restricted motion, the leaflets fail to coapt, resulting in a central jet. LA, left atrium; LV, left ventricle.

Reduction of SVR increases forward stroke volume and decreases the regurgitant volume. Surgical treatment is usually reserved for patients with moderate to severe symptoms. Valvuloplasty or valve repair are performed whenever possible to avoid the problems associated with valve replacement (eg, thromboembolism, hemorrhage, and prosthetic failure). Catheter-mediated valve repairs are continually being refined, potentially reducing the need for "open" surgery. Anesthesiologists skilled in advanced perioperative echocardiography assist in correctly identifying the leaflet(s) to be repaired and determining the repair's success. Three-dimensional echocardiography is increasingly employed to assist in the assessment of the mitral valve (see Figure 5-31).

©@ Anesthetic management should be tailored to the severity of mitral regurgitation as well as the underlying left ventricular function. Factors that exacerbate the regurgitation, such as slow heart rates and acute increases in afterload, should be avoided. Bradycardia can increase the regurgitant volume by increasing left ventricular end-diastolic volume and acutely dilating the mitral annulus. The heart rate should ideally be kept between 80 and 100 beats/min. Acute increases in left ventricular afterload, such as with endotracheal intubation and surgical stimulation under "light" anesthesia, should be treated rapidly.

Monitors are based on the severity of ventricular dysfunction, as well as the procedure. Color-flow Doppler TEE can be invaluable in quantitating the severity of the regurgitation and guiding therapeutic interventions in patients with severe mitral regurgitation. Doppler echocardiography identifies the acceleration of blood as it is ejected through the regurgitant orifice during systole from the left ventricle into the left atrium (Figure 21-9).
FIGURE 21-9 Transesophageal echocardiography using color-flow Doppler demonstrates mitral regurgitation. The left atrium (LA), left ventricle (LV), right ventricle (RV), and ascending aorta (AscAo) are shown. The arrow indicates a jet of mitral regurgitation. (Reproduced with permission from Mathew JP, Swaminathan M, Ayoub CM. Clinical Manual and Review of Transesophageal Echocardiography. 2nd ed. New York, NY: McGraw-Hill Education; 2010.)

Patients with relatively well-preserved ventricular function tend to do well with most anesthetic techniques. Spinal and epidural anesthesia are well tolerated, provided bradycardia is avoided. Patients with compromised ventricular function can likewise be managed with a variety of anesthetic agents and techniques. Invasive monitoring (arterial line, TEE) can be used to guide perioperative management in patients with severe mitral regurgitation and poor ventricular function. Inodilators such as milrinone may be employed to improve ventricular function and reduce systemic resistance in patients with poor ventricular function and severe mitral regurgitation to promote the forward as opposed to regurgitant blood flow.

Mitral valve prolapse (Barlow syndrome) is classically characterized by a midsystolic click, with or without a late apical systolic murmur on auscultation.
It is a relatively common abnormality that is present in up to 1% to 2.5% of the general population. The diagnosis is suggested by auscultatory findings and confirmed by echocardiography, which shows systolic prolapse of mitral valve leaflets into the left atrium. Patients with the murmur often have some element of mitral regurgitation. The posterior mitral leaflet is more commonly affected than the anterior leaflet. The mitral annulus may also be dilated. Pathologically, most patients have redundancy or some myxomatous degeneration of the valve leaflets. Most cases of mitral valve prolapse are sporadic or familial, affecting otherwise normal persons. A high incidence of mitral valve prolapse is found in patients with connective tissue disorders (particularly Marfan syndrome).
The overwhelming majority of patients with mitral valve prolapse are asymptomatic, but in a small percentage of patients, the myxomatous degeneration is progressive. Manifestations, when they occur, can include chest pains, arrhythmias, embolic events, florid mitral regurgitation, infective endocarditis, and, rarely, sudden death. The diagnosis can be made preoperatively by auscultation of the characteristic click, but must be confirmed by echocardiography. The prolapse is accentuated by maneuvers that decrease ventricular volume (preload). Both atrial and ventricular arrhythmias are common. Although bradyarrhythmias have been reported, paroxysmal supraventricular tachycardia is the most commonly encountered sustained arrhythmia. An increased incidence of abnormal AV bypass tracts is reported in patients with mitral valve prolapse. Most patients have a normal life span. About 15% develop progressive mitral regurgitation. A smaller percentage develops embolic phenomena or infective endocarditis. Patients with both a click and a systolic murmur seem to be at greater risk of developing complications. Anticoagulation or antiplatelet agents may be used for patients with a history of emboli, whereas B-adrenergic blocking drugs are commonly used for arrhythmias.

The management of these patients is based on their clinical course. Most patients are asymptomatic and do not require special care. Ventricular arrhythmias may occur intraoperatively, particularly following sympathetic stimulation, and will generally respond to lidocaine or B-adrenergic blocking agents. Mitral regurgitation caused by prolapse is generally exacerbated by decreases in ventricular size. Hypovolemia and factors that increase ventricular emptying or decrease afterload should be avoided. Vasopressors with pure a-adrenergic agonist activity (such as phenylephrine) may be preferable to those that are primarily B-adrenergic agonists.

Valvular aortic stenosis is the most common cause of obstruction to left ventricular outflow. Left ventricular outflow obstruction is less commonly due to hypertrophic cardiomyopathy, discrete congenital subvalvular stenosis, or, rarely, supravalvular stenosis. Valvular aortic stenosis is typically congenital, rheumatic, or degenerative. Abnormalities in the number of cusps (most commonly a bicuspid valve) or their architecture produce turbulence that traumatizes the valve and eventually leads to stenosis. Rheumatic aortic stenosis is rarely isolated; it is more commonly associated with aortic regurgitation or mitral valve disease. In the most common degenerative form, calcific aortic stenosis, wear and tear results in the buildup of calcium deposits on normal cusps, preventing them from opening completely (Figure 21—10).
FIGURE 21-10 A stenotic aortic valve is clearly seen in this midesophageal short axis aortic valve view. Calcification of the aortic valve is usually associated with senile degeneration. However, congenitally abnormal (bicuspid) and rheumatic presentations also occur. (Reproduced with permission from Wasnick J, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography. New York, NY: McGraw-Hill; 2011.)

Left ventricular outflow obstruction caused by valvular aortic stenosis is almost always gradual, allowing the ventricle, at least initially, to compensate and maintain stroke volume. Concentric left ventricular hypertrophy enables the ventricle to maintain stroke volume by generating the needed transvalvular pressure gradient and reducing ventricular wall stress.
Critical aortic stenosis is said to exist when the aortic valve orifice is reduced to 0.5 to 0.7 cm? (normal is 2.5-3.5 cm). With this degree of stenosis, patients generally have a transvalvular gradient of approximately 50 mm Hg at rest (with a normal cardiac output) and are unable to increase cardiac output in response to exertion. Moreover, further increases in the transvalvular gradient do not significantly increase stroke volume. With long-standing aortic stenosis, myocardial contractility progressively deteriorates, compromising left ventricular function.

Classically, patients with advanced (end-stage) aortic stenosis have the triad of heart failure, angina, and syncope. Initial diagnosis in the modern era is usually made earlier in the course of the disease when the more typical complaints are exercise-induced dyspnea, vertigo, or angina. A prominent feature of aortic stenosis is a decrease in left ventricular compliance as a result of hypertrophy. Diastolic dysfunction as a result of an increase in ventricular muscle mass, fibrosis, or myocardial ischemia is to be expected. In contrast to left ventricular end-diastolic volume, which remains normal until very late in the disease, left ventricular end-diastolic pressure is elevated early in the disease. The decreased diastolic pressure gradient between the left atrium and left ventricle impairs ventricular filling, which becomes quite dependent on a normal atrial contraction. Loss of atrial systole can precipitate congestive heart failure or hypotension in patients with aortic stenosis. Cardiac output may be normal in symptomatic patients at rest, but characteristically, it does not appropriately increase with exertion. Patients may experience angina even in the absence of CAD. Myocardial oxygen demand increases because of ventricular hypertrophy, whereas myocardial oxygen supply decreases as a result of the marked compression of intramyocardial coronary vessels caused by high intracavitary systolic pressures (up to 300 mm Hg). Exertional syncope or near-syncope is thought to be due to an inability to tolerate the vasodilation in muscle tissue during exertion. Arrhythmias leading to severe hypoperfusion may also account for syncope and sudden death in some patients.

Once symptoms develop, most patients without valve replacement will die within a few years. Percutaneous balloon valvuloplasty is generally used in younger patients with congenital aortic stenosis; it can also be used in elderly patients with calcific aortic stenosis who are poor candidates for aortic valve replacement. Its efficacy for the latter group is short-lived, however, and restenosis usually occurs within 6 to 12 months. Catheter-delivered aortic valves are increasingly being perfected and deployed in the treatment of aortic valve disease. Surgical replacement of the stenotic aortic valve remains the mainstay of therapy.

® Maintenance of normal sinus rhythm, heart rate, vascular resistance, and intravascular volume is critical in patients with aortic stenosis. Loss of a normally timed atrial systole often leads to rapid deterioration, particularly when associated with tachycardia. The combination of the two (AF with rapid ventricular response) seriously impairs ventricular filling and necessitates immediate cardioversion. Fortunately, AF is uncommon in patients with aortic stenosis. The reduced ventricular compliance also makes the patient very sensitive to abrupt changes in intravascular volume. Many patients behave as though they have a fixed stroke volume in spite of adequate hydration; under these conditions, cardiac output becomes very rate dependent. Extreme bradycardia (<50 beats/min) is therefore poorly tolerated. Heart rates between 60 and 90 beats/min are optimal in most patients.

Monitoring for ischemia is complicated by baseline ST-segment and T-wave abnormalities often seen in the aortic stenosis patient. Intraarterial pressure monitoring is desirable in patients with severe aortic stenosis, as many of these patients do not tolerate even brief episodes of hypotension. Vasodilators should be used cautiously, if at all, because patients are often very sensitive to these agents. TEE can be useful in these patients for monitoring ischemia, ventricular preload, contractility, valvular function, and the effects of therapeutic interventions.

Patients with mild to moderate aortic stenosis (generally asymptomatic) may tolerate spinal or epidural anesthesia. These techniques should be employed very cautiously, however, because hypotension readily occurs as a result of reductions in preload, afterload, or both. A vasoconstrictor medication must be immediately available. Epidural anesthesia may be preferable to single-shot spinal anesthesia because of its slower onset of hypotension, which allows more timely correction. Continuous spinal catheters can similarly be used to gradually increase the level of block and slow the onset of hypotension.
In the patient with severe aortic stenosis the choice of anesthetic agents and techniques is less important than effective management of their hemodynamic effects. Most general anesthetics can produce both vasodilation and hypotension, which require treatment postinduction. If a volatile agent is used, the concentration should be controlled to avoid excessive vasodilation, myocardial depression, or loss of normal atrial systole. Significant tachycardia and severe hypertension, which can precipitate ischemia, should be treated immediately by increasing anesthetic depth or administration of a B-adrenergic blocking agent. Most patients with aortic stenosis tolerate moderate hypertension and are sensitive to vasodilators. Use of vasoconstrictors (eg, vasopressin, phenylephrine, norepinephrine) is often necessary to preserve systemic blood pressure in the anesthetized aortic stenosis patient. Moreover, because of an already precarious myocardial oxygen demand-supply balance, aortic stenosis patients tolerate even mild degrees of hypotension poorly. Intraoperative SVTs with hemodynamic compromise should be treated with immediate synchronized cardioversion. Frequent ventricular ectopy (which often reflects ischemia) is usually poorly tolerated hemodynamically and should be treated.

Aortic regurgitation usually develops slowly and is progressive (chronic), but it can also develop quickly (acute). Chronic aortic regurgitation may be caused by abnormalities of the aortic valve, the aortic root, or both. Abnormalities in the valve are usually congenital (bicuspid valve) or due to rheumatic fever. Diseases affecting the ascending aorta cause regurgitation by dilating the aortic annulus; they include syphilis, annuloaortic ectasia, cystic medial necrosis (with or without Marfan syndrome), ankylosing spondylitis, rheumatoid and psoriatic arthritis, and a variety of other connective tissue disorders. Acute aortic insufficiency most commonly follows infective endocarditis, trauma, or aortic dissection.

Regardless of the cause, aortic regurgitation produces volume overload of the left ventricle. The effective forward stroke volume is reduced because of backward (regurgitant) flow of blood into the left ventricle during diastole. Systemic arterial diastolic pressure and SVR are typically low. The decrease in cardiac afterload helps facilitate ventricular ejection. Total stroke volume is the sum of the effective stroke volume and the regurgitant volume. The regurgitant volume depends on the heart rate (diastolic time) and the diastolic pressure gradient across the aortic valve (diastolic aortic pressure minus left ventricular end-diastolic pressure). Slow heart rates increase regurgitation because of the associated disproportionate increase in diastolic time, whereas increases in diastolic arterial pressure favor regurgitant volume by increasing the pressure gradient for backward flow.
With chronic aortic regurgitation, the left ventricle progressively dilates and undergoes eccentric hypertrophy. Patients with severe aortic regurgitation have the largest end-diastolic volumes of any heart disease. The resulting increase in end-diastolic volume maintains an effective stroke volume. Any increase in the regurgitant volume is compensated by an increase in end-diastolic volume. Left ventricular end-diastolic pressure is usually normal or only slightly elevated, because ventricular compliance initially increases. Eventually, as ventricular function deteriorates, the ejection fraction declines, and impaired ventricular emptying is manifested as gradual increases in left ventricular end-diastolic pressure and end-systolic volume.
Sudden incompetence of the aortic valve does not allow compensatory dilation or hypertrophy of the left ventricle. Effective stroke volume rapidly declines because the normal-sized ventricle is unable to accommodate a sudden large regurgitant volume. The sudden rise in left ventricular end-diastolic pressure is transmitted back to the pulmonary circulation and causes acute pulmonary venous congestion.

Acute aortic regurgitation typically presents as the sudden onset of pulmonary edema and hypotension, whereas chronic regurgitation ultimately manifests as congestive heart failure. Symptoms are generally nil or minimal in the chronic form when the regurgitant volume remains under 40% of stroke volume, but become severe when it exceeds 60%. Angina can occur even in the absence of coronary disease. The myocardial oxygen demand is increased from muscle hypertrophy and dilation; the myocardial blood supply is reduced by low diastolic pressures in the aorta as a result of the regurgitation, and peak myocardial blood flow occurs in systole rather than diastole.

Most patients with chronic aortic regurgitation remain asymptomatic for 10 to 20 years. Once significant symptoms develop, the expected survival time is about 5 years without valve replacement. Diuretics and afterload reduction, particularly with ACE inhibitors, generally benefit patients with advanced chronic aortic regurgitation. The decrease in arterial blood pressure reduces the diastolic gradient for regurgitation. Patients with chronic aortic regurgitation should receive valve replacement before irreversible ventricular dysfunction occurs. Percutaneous aortic valve replacement is increasingly used for high-risk patients with aortic insufficiency.
Patients with acute aortic regurgitation typically require intravenous inotropic and vasodilator therapy. Early intervention is indicated in patients with acute aortic regurgitation; medical management alone is associated with a high mortality rate.

The heart rate should be maintained toward the upper © limits of normal (80100 beats/min). Bradycardia and increases in SVR increase the regurgitant volume in patients with aortic regurgitation, whereas tachycardia can contribute to myocardial ischemia. Excessive myocardial depression should also be avoided. The compensatory increase in cardiac preload should be maintained, but overzealous fluid replacement can readily result in pulmonary edema.

Invasive hemodynamic monitoring should be employed in patients with acute aortic regurgitation or those with severe chronic regurgitation. Premature closure of the mitral valve often occurs during acute aortic regurgitation and may cause pulmonary capillary wedge pressure to give a falsely high estimate of left ventricular end-diastolic pressure. The arterial pressure wave in patients with aortic regurgitation characteristically has a very wide pulse pressure. Pulsus bisferiens may also be present in patients with moderate to severe aortic insufficiency and is thought to result from the rapid ejection of a large stroke volume. Color-flow Doppler TEE can be invaluable in quantitating the severity of the regurgitation and guiding therapeutic interventions (Figure 21-11).
FIGURE 21-11 Transesophageal echocardiography using color-flow Doppler demonstrates aortic regurgitation. The left ventricular outflow tract (LVOT), left atrium (LA), and ascending aorta (ASCAO) are seen. The arrow demonstrates an eccentrically directed jet of aortic regurgitation. (Reproduced with permission from Mathew JP, Swaminathan M, Ayoub CM. Clinical Manual and Review of Transesophageal Echocardiography. 2nd ed. New York, NY: McGraw-Hill Education; 2010.)
Severe aortic regurgitation rapidly raises left ventricular pressure during diastole. Echocardiography can also detect reversal of blood flow in the aorta during diastole in patients with severe aortic regurgitation. The more severe the regurgitation, the further distal in the aorta that diastolic flow reversal is identified.

Most patients with aortic insufficiency tolerate spinal and epidural anesthesia well, provided intravascular volume is maintained. When general anesthesia is required, inhalational agents may be ideal because of the associated vasodilation. Phenylephrine (25-50 mcg) can be used to treat hypotension secondary to excessive anesthetic-induced vasodilatation; however, large doses of phenylephrine may increase SVR (and arterial diastolic pressure) sufficiently to worsen the regurgitation.

Most patients have trace to mild tricuspid regurgitation on echocardiography; the regurgitant volume in these cases is almost always trivial. Clinically significant tricuspid regurgitation, however, is most commonly due to dilation of the right ventricle from pulmonary hypertension that is associated with chronic left ventricular failure. Tricuspid regurgitation can also follow infective endocarditis, rheumatic fever, carcinoid syndrome, or chest trauma or may be due to Ebstein anomaly (downward displacement of the valve because of abnormal attachment of the valve leaflets).

interventricular septum is flattened. These changes can impair left heart function.

Tricuspid regurgitation is generally well tolerated by most patients. Because the underlying disorder is generally more important than the tricuspid regurgitation itself, treatment is aimed at the underlying disease process. Recent studies suggest that correction of significant tricuspid regurgitation with annuloplasty is beneficial when patients with moderate to severe tricuspid regurgitation are brought to surgery for replacement of another valve.

Hemodynamic goals should be directed primarily toward the underlying disorder. Hypovolemia and factors that increase right ventricular afterload, such as hypoxia and acidosis, should be avoided to maintain effective right ventricular stroke volume and left ventricular preload. Positive end-expiratory pressure and high mean airway pressures may also be undesirable during mechanical ventilation because they reduce venous return and increase right ventricular afterload.

Invasive monitoring may be useful. Pulmonary artery catheterization is rarely used and often not feasible; rarely a large regurgitant flow may make passage of a pulmonary artery catheter across the tricuspid valve difficult. Increasing central venous pressure implies worsening right ventricular dysfunction. The x descent is absent, and a prominent cv wave is usually present on the central venous pressure waveform. Thermodilution cardiac output measurements are falsely elevated because of the tricuspid regurgitation. Color-flow Doppler TEE is useful in evaluating the severity of the regurgitation and other associated abnormalities.

The selection of anesthetic agents should be based on the underlying disorder. Most patients tolerate spinal and epidural anesthesia well. Coagulopathy secondary to hepatic dysfunction should be excluded prior to any regional technique.

The ACC/AHA guidelines regarding prophylactic antibiotic regimens in patients with prosthetic heart valves and other structural heart abnormalities have dramatically changed in recent years, decreasing the number of indications for antibiotic administration. The risk of antibiotic administration is often considered greater than the potential for developing perioperative endocarditis. At present, the ACC/AHA guidelines suggest the use of endocarditis prophylaxis in the highest risk patients undergoing dental procedures involving gingival manipulation or perforation of the oral mucosa (class IIa); see Table 21-14. Such conditions include:

Clindamycin. , Regimen: Single Dose 30-60 min Before Procedure.Adults = 600 mg IM or IV. , Regimen: Single Dose 30-60 min Before Procedure.Children = 20 mg/kg IM or IV
Reproduced with permission from Nishimura RA, Carabello BA, Faxon DP, et al. ACC/AHA 2008 guideline update on valvular heart disease: Focused update on infective endocarditis prophylaxis. JAm Colf Cardiol. 2008 Aug 19;52(8):676-685
21M, intramuscular; lV, intravenous.
*Or use other first or second-generation oral cephalosporin in equivalent adult or pediatric dosage.
"Cephalosporins should not be used in an individual with a history of anaphylaxis, angioedema, or urticaria with penicillins or ampicillin,
¢ Patients with prosthetic cardiac valves or prosthetic heart materials
¢ Patients with a past history of endocarditis
¢ Patients with congenital heart disease that is either partially repaired or unrepaired
¢ Patients with congenital heart disease with residual defects following repair
¢ Patients with congenital heart disease within 6 months of a complete repair, whether catheter-based or surgical
¢ Cardiac transplant patients with structurally abnormal valves
Class III recommendations indicate that prophylaxis is not necessary for nondental procedures, including TEE and esophagogastroduodenoscopy, except in the presence of an active infection.
Endocarditis is believed to occur in areas of cardiac endothelial damage, where in cases of bacteremia, bacteria can be deposited and multiply. Areas of increased myocardial blood flow velocity lead to damaged endothelium, providing a template for bacterial adherence and growth. Guidelines are ever changing, and not considered to be "standard of care." Nevertheless, deviation from guidelines often requires explanation as being outside of "evidence-based" practice. Review of the ACC/AHA guidelines is recommended when high-risk patients are encountered.

Patients with mechanical prosthetic heart valves require anticoagulation, which is currently accomplished with warfarin. Aspirin is also indicated in this population, as well as in patients with bioprosthetic valves, to prevent thrombus formation. Warfarin is sometimes also used initially for mitral bioprosthetic valves.
Patients with prosthetic valves often present for noncardiac surgery that will require temporary discontinuation of anticoagulation. The ACC/AHA guidelines indicate that patients at low risk of thrombosis, such as those with bileaflet mechanical valves in the aortic position with no additional problems (eg, no AF or no hypercoagulable state) can discontinue warfarin 48 to 72 hours preoperatively so that the INR falls below 1.5. In patients at greater risk of thrombosis, warfarin should be discontinued and heparin, either unfractionated or low molecular weight, started when the INR falls below 2.0. Heparin can be discontinued 4 to 6 hours prior to surgery and then restarted as soon as surgical bleeding permits, until the patient can be restarted on warfarin therapy. Fresh frozen plasma or prothrombin complex concentrates may be given, if needed, in an emergency situation to interrupt warfarin therapy. Anesthesia providers should always consult with the patient's surgeon and the physician responsible for prescribing the anticoagulation before adjusting anticoagulation or antiplatelet regimens perioperatively.

Congenital heart disease encompasses a seemingly endless list of abnormalities that may be detected in infancy, early childhood, or, less commonly, adulthood.
The incidence of congenital heart disease in all live births approaches 1%. The natural history of some defects is such that patients often survive to adulthood (Table 21—15). Moreover, the number of surviving adults with corrected or palliated congenital heart disease is steadily increasing with advances in surgical and medical treatment. Patients with congenital heart disease may therefore be encountered during noncardiac surgery and obstetric deliveries. Knowledge of the anatomy of the original heart structure defect and of any corrective repairs is essential prior to anesthetizing the patient with congenital heart disease.

Bicuspid aortic valve
Coarctation of the aorta
Pulmonic valve stenosis
Ostium secundum atrial septal defect
Ventricular septal defect
Patent ductus arteriosus

The complex nature and varying pathophysiology of congenital heart defects make classification difficult. A commonly used scheme is presented in 'Table 21-16. Most patients present with cyanosis, congestive heart failure, or an asymptomatic abnormality. Cyanosis is typically the result of an abnormal intracardiac communication that allows unoxygenated blood to reach the systemic arterial circulation (right-to-left shunting). Congestive heart failure is most prominent with defects that either obstruct left ventricular outflow or markedly increase pulmonary blood flow. The latter is usually due to an abnormal intracardiac communication that returns oxygenated blood to the right heart (left-to-right shunting). Whereas right-to-left shunts generally decrease pulmonary blood flow, some complex lesions increase pulmonary blood flow— even in the presence of right-to-left shunting. In many cases, more than one lesion is present. Survival prior to surgical correction with some anomalies (eg, transposition, total anomalous venous return, pulmonary atresia) depends on the simultaneous presence of another shunting lesion (eg, patent ductus arteriosus, patent foramen ovale, ventricular septal defect). Chronic hypoxemia in patients with cyanotic heart disease typically results in erythrocytosis. This increase in red cell mass, which is due to increased erythropoietin secretion from the kidneys, serves to restore tissue oxygen concentration to normal. Unfortunately, blood viscosity can also rise to the point at which it may interfere with oxygen delivery. When tissue oxygenation is restored to normal, the hematocrit is stable (usually <65%), and symptoms of the hyperviscosity syndrome are absent, the patient is said to have compensated erythrocytosis. Patients with uncompensated erythrocytosis do not establish this equilibrium; they have symptoms of hyperviscosity and may be at risk of thrombotic complications, particularly stroke. The risk of stroke is aggravated by dehydration. Children younger than age 4 years seem to be at greatest risk of stroke. Phlebotomy is generally not recommended if symptoms of hyperviscosity are absent and the hematocrit is less than 65%.

Left ventricle
Aortic stenosis
Right ventricle

Atrial septal defect
Endocardial cushion defect
Partial anomalous pulmonary venous return

With decreased pulmonary blood flow
Tetralogy of Fallot
Pulmonary atresia
Tricuspid atresia
With increased pulmonary blood flow
Transposition of the great vessels
Truncus arteriosus
Single ventricle
Double-outlet right ventricle
Total anomalous pulmonary venous return
Hypoplastic left heart
Coagulation abnormalities are common in patients with cyanotic heart disease. Platelet counts tend to be low-normal, and many patients have defects in the coagulation cascade. Hyperuricemia often occurs because of increased urate reabsorption secondary to renal hypoperfusion, and can result in progressively impaired kidney function.
Preoperative echocardiography is invaluable in defining the anatomy of the defect(s) and to confirm or exclude the existence of other lesions or complications, their physiological significance, and the effects of any therapeutic interventions.

This population of patients includes four groups: (1) those who have undergone corrective cardiac surgery and require no further operations, (2) those who have had only palliative surgery, (3) those who have not yet undergone any cardiac surgery, and (4) those whose conditions are inoperable and may be awaiting cardiac transplantation. Although the management of the first group of patients may be the same as that of normal patients (except for consideration of prophylactic antibiotic therapy), the care of others requires familiarity with the complex pathophysiology of these defects (Tables 21-17 and 21-18).

Arrhythmias
Hypoxemia
Pulmonary hypertension
Existing shunts
Paradoxical embolism
Bacterial endocarditis

noncardiac surgery.!
High risk
Pulmonary hypertension, primary or secondary
Cyanotic congenital heart disease
New York Heart Association class III or IV
Severe systemic ventricular dysfunction (ejection fraction less than 35%)
Severe left-sided heart obstructive lesions

Prosthetic valve or conduit
Intracardiac shunt
Moderate left-sided heart obstruction
Moderate systemic ventricular dysfunction lReproduced with permission from Warnes C, Williams R, Bashore T, et al. ACC/AHA 2008 guidelines for the
management of adults with congenital heart disease. Circulation. 2008 Dec 2;118(23):2395-2451.
For the purpose of anesthetic management, congenital heart defects may be divided into obstructive lesions, predominantly left-to-right shunts, or predominantly right-to-left shunts. Shunts can also be bidirectional and may reverse under certain conditions.

Pulmonary valve stenosis obstructs right ventricular outflow and causes concentric right ventricular hypertrophy. Severe obstruction presents in the neonatal period, whereas lesser degrees of obstruction may go undetected until adulthood. The valve is usually deformed and is either bicuspid or tricuspid. Valve leaflets are often partially fused and display systolic doming on echocardiography. The right ventricle undergoes hypertrophy, and poststenotic dilation of the pulmonary artery is often present. Symptoms are those of right ventricular heart failure. Symptomatic patients readily develop fatigue, dyspnea, and peripheral cyanosis with exertion as a result of the limited pulmonary blood flow and increased oxygen extraction by tissues. With severe stenosis, the pulmonic valve gradient exceeds 60 to 80 mm Hg, depending on the age of the patient. Right-to-left shunting may also occur in the presence of a patent foramen ovale or atrial septal defect. Cardiac output is very dependent on an elevated heart rate, but excessive increases in the latter can compromise ventricular filling. Percutaneous balloon valvuloplasty is generally considered the initial treatment of choice in most patients with symptomatic pulmonic stenosis. Anesthetic management for patients undergoing surgery should maintain a normal or slightly high heart rate, augment preload, and avoid factors that increase PVR (such as hypoxemia or hypercarbia).

® The presence of shunt flow between the right and left hearts, regardless of the direction of blood flow, mandates the meticulous exclusion of air bubbles and particulate material from intravenous fluids to prevent paradoxical embolism into the cerebral or coronary circulations.

Ostium secundum atrial septal defects (ASDs) are the most common form and usually occur as isolated lesions in the area of the fossa ovalis. The defect is sometimes associated with partial anomalous pulmonary venous return, most commonly of the right upper pulmonary vein. A secundum ASD may result in single or multiple (fenestrated) openings between the atria. The less common sinus venosus and ostium primum ASDs are typically associated with other cardiac abnormalities. Sinus venosus defects are located in the upper interatrial septum close to the superior vena cava; one or more of the right pulmonary veins often abnormally drains into the superior vena cava. In contrast, ostium primum ASDs are located in the lower interatrial septum and overlie the mitral and tricuspid valves; most patients also have a cleft in the anterior leaflet of the mitral valve, and some have an abnormal septal leaflet in the tricuspid valve. Most children with ASDs are minimally symptomatic; some have recurrent pulmonary infections. Congestive heart failure and pulmonary hypertension are more commonly encountered in adults with ASDs. Patients with ostium primum defects often have large shunts and may also develop significant mitral regurgitation. In the absence of heart failure, anesthetic responses to inhalation and intravenous agents are generally not significantly altered in patients with ASDs. Large increases in SVR should be avoided because they may worsen the left-to-right shunting.

Ventricular septal defect (VSD) is a common congenital heart defect, accounting for up to 25% to 35% of congenital heart disease. The defect is most frequently found in the membranous part of the interventricular septum (membranous or infracristal VSD) in a posterior position and anterior to the septal leaflet of the tricuspid valve. Muscular VSDs are the next most frequent type and are located in the mid or apical portion of the interventricular septum, where there may be a single defect or multiple openings (resembling Swiss cheese). Defects in the subpulmonary (supracristal) septum are often associated with aortic regurgitation because the right coronary cusp can prolapse into the VSD. Septal defects at the ventricular inlet are usually similar in development and location to AV septal defects (see the following section).
The resulting functional abnormality of a VSD is dependent on the size of the defect, PVR, and the presence or absence of other abnormalities. Small VSDs, particularly of the muscular type, may close during childhood. Restrictive defects are associated with only small left-to-right shunts. Patients with small VSDs are treated medically and followed with electrocardiography (for signs of right ventricular hypertrophy) and echocardiography. Surgical closure is usually undertaken in patients with large VSDs before pulmonary vascular disease and Eisenmenger physiology develop. In the absence of heart failure, anesthetic responses to inhalation and intravenous agents are generally not significantly altered. Similarly, increases in SVR worsen the left-to-right shunting. When right-to-left shunting is present, abrupt increases in PVR or decreases in SVR are poorly tolerated.

Endocardial cushion (AV canal) defects produce contiguous atrial and ventricular septal defects, often with very abnormal AV valves. This is a common lesion in patients with Down syndrome. The defect can produce large shunts both at the atrial and ventricular levels. Mitral and tricuspid regurgitation exacerbate the volume overload on the ventricles. Initially, shunting is predominately left to right; however, with increasing pulmonary hypertension, Eisenmenger syndrome with obvious cyanosis develops.

Persistence of the communication between the main pulmonary artery and the aorta can produce restrictive or nonrestrictive left-to-right shunts. This abnormality is commonly responsible for the cardiopulmonary deterioration of premature infants and occasionally presents later in life when it can be corrected thoracoscopically. Anesthetic goals should be similar to atrial and ventricular septal defects.

This defect is present when one or more pulmonary veins drains into the right side of the heart; the anomalous veins are usually from the right lung. Possible anomalous entry sites include the right atrium, the superior or inferior vena cava, and the coronary sinus. The resulting abnormality produces a variable amount of left-to-right shunting. The clinical course and prognosis are usually excellent and similar to that of a secundum ASD. Obstructed total anomalous pulmonary venous return is emergently corrected immediately after birth.

Lesions within this group (some also called mixing lesions) often produce both ventricular outflow obstruction and shunting. The obstruction favors shunt flow toward the unobstructed side. When the obstruction is relatively mild, the amount of shunting is affected by the ratio of SVR to PVR, but increasing degrees of obstruction fix the direction and magnitude of the shunt. Atresia of any one of the cardiac valves represents the extreme form of obstruction. Shunting occurs proximal to the atretic valve and is completely fixed; survival depends on another distal shunt (usually a patent ductus arteriosus [PDA], patent foramen ovale, ASD, or VSD), where blood flows in the opposite direction. This group of defects may also be divided according to whether they increase or decrease pulmonary blood flow.

This anomaly classically includes right ventricular outflow obstruction, right ventricular hypertrophy, and a VSD with an overriding aorta. Right ventricular obstruction in most patients is due to infundibular stenosis, which is due to hypertrophy of the subpulmonic muscle (crista ventricularis). At least 20% to 25% of patients also have pulmonic stenosis, and a small percentage of patients has some element of supravalvular obstruction. The pulmonic valve is often bicuspid, or, less commonly, atretic. Infundibular obstruction may be increased by sympathetic tone and is therefore dynamic; this obstruction is likely responsible for the hypercyanotic spells observed in very young patients. The combination of right ventricular outflow obstruction and a VSD results in ejection of unoxygenated right ventricular blood, as well as oxygenated left ventricular blood into the aorta. The right-to-left shunting across the VSD has both fixed and variable components. The fixed component is determined by the severity of the right ventricular obstruction, whereas the variable component depends on SVR and PVR.
Surgical palliation with a left-to-right systemic shunt or complete correction is usually undertaken. For the former, a modified Blalock-Thomas—Taussig
(systemic—pulmonary artery) shunt is most often used to increase pulmonary blood flow. In this procedure, a synthetic graft is anastomosed between a subclavian artery and an ipsilateral pulmonary artery. Complete correction involves closure of the VSD, removal of obstructing infundibular muscle, and pulmonic valvulotomy or valvuloplasty, when necessary.
(5

® The goals of anesthetic management in patients with tetralogy of Fallot should be to maintain intravascular volume and SVR. Increases in PVR, such as might occur from acidosis or excessive airway pressures, should be avoided. Ketamine (intramuscular or intravenous) is a commonly used induction agent because it maintains or increases SVR and therefore does not aggravate the right-to-left shunting. Patients with milder degrees of shunting generally tolerate inhalation induction. The right-to-left shunting tends to slow the uptake of inhalation anesthetics; in contrast, it may accelerate the onset of intravenous agents. Oxygenation often improves following induction of anesthesia. Muscle relaxants that release histamine should be avoided. Hypercyanotic spells may be treated with intravenous fluid and phenylephrine mecg/kg). B-Blockers (eg, propranolol) may also be effective in relieving infundibular spasm.

With tricuspid atresia, blood can flow out of the right atrium only via a patent foramen ovale (or an ASD). Moreover, a PDA (or VSD) is necessary for blood to flow from the left ventricle into the pulmonary circulation. Cyanosis is usually evident at birth, and its severity depends on the amount of pulmonary blood flow that is achieved. Early survival is dependent on prostaglandin E, infusion (to maintain PDA patency), with or without a percutaneous balloon atrial septostomy. Severe cyanosis requires a modified Blalock-Thomas—Taussig shunt early in life. The preferred surgical management is a modified Fontan procedure, in which the venous drainage is directed to the pulmonary circulation. In some centers, a superior vena cava to the main pulmonary artery (bidirectional Glenn) shunt may be employed before or instead of a Fontan procedure. With both procedures, blood from the systemic veins flows through the pulmonary circulation to the left atrium without the assistance of the right ventricle. Success of the procedure depends on a high systemic venous pressure and maintaining both low PVR and a low left atrial pressure. Heart transplantation may be necessary for a failed Fontan procedure.

In patients with transposition of the great arteries, pulmonary and systemic venous return flows normally back to the right and left atrium, respectively, but the aorta arises from the right ventricle, and the pulmonary artery arises from the left ventricle. Thus, deoxygenated blood returns back into the systemic circulation, and oxygenated blood returns back to the lungs. Survival is possible only through mixing of oxygenated and deoxygenated blood across the foramen ovale and a PDA. The presence of a VSD increases mixing and reduces the level of hypoxemia. Prostaglandin E, infusion is usually necessary. Corrective surgical treatment involves an arterial switch procedure in which the aorta is divided and reanastomosed to the left ventricle, and the pulmonary artery is divided and reanastomosed to the right ventricle. The coronary arteries must also be reimplanted into the old pulmonary artery root. A VSD, if present, is closed. Less commonly, an atrial switch (Senning) procedure may be carried out if an arterial switch is not possible. In this latter procedure, an intraatrial baffle is created from the atrial wall, and blood from the pulmonary veins flows across an ASD to the right ventricle, from which it is ejected into the systemic circulation. Transposition of the great vessels may occur with a VSD and pulmonic stenosis. This combination of defects mimics tetralogy of Fallot; however, the obstruction affects the left ventricle, not the right ventricle. Corrective surgery involves patch closure of the VSD, directing left ventricular outflow into the aorta, ligation of the proximal pulmonary artery, and connecting the right ventricular outflow to the pulmonary artery with a valved conduit (Rastelli procedure).

With a truncus arteriosus defect, a single arterial trunk supplies the pulmonary and systemic circulation. Both ventricles eject into the truncus as it always overrides a VSD. As PVR gradually decreases after birth, pulmonary blood flow increases greatly, resulting in heart failure. If left untreated, PVR increases, and cyanosis develops again, along with Eisenmenger physiology. Surgical correction closes the VSD, separates the pulmonary artery from the truncus, and connects the right ventricle to the pulmonary artery with a conduit (Rastelli repair).

This syndrome describes a group of defects characterized by aortic valve atresia and marked underdevelopment of the left ventricle. The right ventricle is the main pumping chamber for both systemic and pulmonary circulations. It ejects normally into the pulmonary artery, and all (or nearly all) blood flow entering the aorta is usually derived from a PDA. Surgical treatment includes both the Norwood repair and a hybrid approach to palliation. In the Norwood repair, a new aorta is created from the hypoplastic aorta and the main pulmonary artery. Pulmonary blood flow is delivered via a Blalock-Thomas—Taussig shunt. The right ventricle becomes the heart's systemic pumping ventricle. A hybrid approach has also been advocated for the palliation of hypoplastic left heart syndrome. In this approach, the pulmonary arteries are banded to reduce pulmonary blood flow, and the PDA is stented to provide for systemic blood flow.

The number of patients with cardiac transplants is increasing because of both the increasing frequency of transplantation and improved posttransplant survival rates. These patients may present to the operating room early in the postoperative period for mediastinal exploration or retransplantation, or they may appear later for incision and drainage of infections, orthopedic surgery, or unrelated procedures.
© The transplanted heart is totally denervated, so direct autonomic influences are absent. Cardiac impulse formation and conduction are normal, but the absence of vagal influences causes a relatively high resting heart rate (100-120 beats/min). Although sympathetic fibers are similarly interrupted, the response to circulating catecholamines is normal or even enhanced because of denervation sensitivity (increased receptor density). Cardiac output tends to be low-normal and increases relatively slowly in response to exercise because the response is dependent on an increase in circulating catecholamines. Because the Starling relationship between end-diastolic volume and cardiac output is normal, the transplanted heart is also often said to be preload dependent. Coronary autoregulation is preserved.
Preoperative evaluation should focus on evaluating the functional status of the transplanted organ and detecting complications of immunosuppression. Rejection may be heralded by arrhythmias (in the first 6 months) or decreased exercise tolerance from a progressive deterioration of myocardial performance. Periodic echocardiographic evaluations are commonly used to monitor for rejection, but the most reliable technique is endomyocardial biopsy. Accelerated atherosclerosis in the graft is a very common and serious problem that limits the life of the transplant. Moreover, myocardial ischemia and infarction are almost always silent because of the denervation. Because of this, patients must undergo periodic evaluations, including angiography, for assessment of coronary atherosclerosis.
Immunosuppressive therapy may include cyclosporine, tacrolimus, and prednisone. Important side effects include nephrotoxicity, bone marrow suppression, hepatotoxicity, opportunistic infections, and osteoporosis. Hypertension and fluid retention are common and typically require treatment with a diuretic and an ACE inhibitor.

Almost all anesthetic techniques, including regional anesthesia, have been used successfully for transplanted patients. The preload-dependent function of the graft makes maintenance of a normal or high cardiac preload desirable. Moreover, the absence of reflex increases in heart rate can make patients particularly sensitive to rapid vasodilation. Indirect vasopressors, such as ephedrine, are less effective than direct-acting agents because of the absence of catecholamine stores in myocardial neurons. Isoproterenol or epinephrine infusions should be readily available to increase the heart rate if necessary. Electrocardiographic monitoring for ischemia is necessary. The ECG usually demonstrates two sets of P waves, one representing the recipient's own sinoatrial (SA) node (which is left intact), and the other representing the donor's SA node. The recipient's SA node may still be affected by autonomic influences, but it does not affect cardiac function. Direct arterial pressure monitoring should be used for major operations; strict asepsis should be observed during placement.
In a recently transplanted patient, the right ventricle of the transplanted heart may not be able to overcome the resistance of the pulmonary vasculature. Right ventricular failure can occur perioperatively, requiring the use of inhaled nitric oxide, inotropes, and, at times, right ventricular assist devices.
Because the number of transplantable hearts is limited, patients are increasingly treated with left ventricular assist devices (LVADs). LVADs drain blood from the apex of the left ventricular and in a nonpulsatile manner pump oxygenated blood into the aorta, restoring blood delivery to the tissues (Figure
21-12). Patients require anticoagulation to prevent pump thrombosis. Maintenance of right heart function is essential to adequately supply the left side of the heart with sufficient blood for the device to eject. Hypovolemia, pulmonary hypertension, and right heart failure can lead to inadequate loading of the left heart, resulting in reduced LVAD pump flows. LVAD patients presenting for noncardiac procedures are routinely managed by cardiac anesthesiologists familiar with LVAD operations and skilled in advanced perioperative echocardiography.

FIGURE 21-12 Schematic of an implanted LVAD device. (Reproduced with permission from Wasnick JD, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography. New York, NY: McGraw-Hill Education; 2011.)

A 71-year-old woman presents for open reduction and internal fixation of a left hip fracture. She gives a history of two episodes of lightheadedness several days prior to her fall today. When questioned about her fall, she can only recall standing in her bathroom while brushing her teeth and then awakening on the floor with hip pain. The preoperative ECG shows a sinus rhythm with a P-R interval of 220 ms and a right bundle-branch block (RBBB) pattern.
Why should the anesthesiologist be concerned about a history of syncope?
A history of syncope in elderly patients should always raise the possibility of arrhythmias and underlying organic heart disease. Although arrhythmias can occur in the absence of organic heart disease, the two are commonly related. Cardiac syncope usually results from an abrupt arrhythmia that suddenly compromises cardiac output and impairs cerebral perfusion. Lightheadedness and presyncope, may reflect lesser degrees of cerebral impairment. Both bradyarrhythmias and tachyarrhythmias (see Chapter 20) can produce syncope. Table 21—19 lists other cardiac and noncardiac causes of syncope.

Tachyarrhythmias (usually >180 beats/min)
Bradyarrhythmias (usually <40 beats/min)
Impairment of left ventricular ejection
Hypertrophic cardiomyopathy
Massive myocardial infarction
Atrial myxoma
Impairment of right ventricular output
Primary pulmonary hypertension
Pulmonary embolism
Biventricular impairment
Cardiac tamponade

Accentuated reflexes
Vasodepressor reflex (ie, vasovagal syncope)
Carotid sinus hypersensitivity
Neuralgias
Postural hypotension
Hypovolemia
Sympathectomy
Autonomic dysfunction
Sustained Valsalva maneuver
Cerebrovascular disease
Seizures
Metabolic
Hypoxia
Marked hypocapnia
Hypoglycemia

Bradyarrhythmias may arise from either SA node dysfunction or abnormal AV conduction of the cardiac impulse. A delay or block of the impulse can occur anywhere between the SA node and the distal HisPurkinje system). Reversible abnormalities may be due to abnormal vagal tone, electrolyte abnormalities, drug toxicity, hypothermia, or myocardial ischemia. Irreversible abnormalities, which initially may be only
intermittent before they become permanent, reflect either isolated conduction system abnormalities or underlying heart disease (most commonly hypertensive, coronary artery, or valvular heart disease).

Patients with sinus node dysfunction may have a normal baseline 12lead ECG but abrupt pauses in SA node activity (sinus arrest) or intermittent block of conduction of the SA impulse to the surrounding tissue (exit block). Symptoms are usually present when pauses are prolonged (>3 s) or the effective ventricular rate is less than 40 beats/min. Patients may experience intermittent dizziness, syncope, confusion, fatigue, or shortness of breath. Symptomatic SA node dysfunction, or sick sinus syndrome, is often unmasked by B-adrenergic blocking agents, calcium channel blockers, digoxin, or quinidine. The term tachycardia—bradycardia syndrome is often used when patients experience paroxysmal tachyarrhythmias (usually atrial flutter or fibrillation) followed by sinus pauses or bradycardia. The latter, bradycardia, probably represents failure of the SA node to recover normal automaticity following suppression by the tachyarrhythmia.

AV conduction abnormalities are usually manifested by abnormal ventricular depolarization (bundle-branch block), prolongation of the P-R interval (first-degree AV block), failure of some atrial impulses to depolarize the ventricles (second-degree AV block), or AV dissociation (third-degree AV block; also called complete heart block).

The significance of a conduction system abnormality depends on its location, its likelihood for progression to complete heart block, and the likelihood that a more distal pacemaker site will be able to maintain a stable and adequate escape rhythm (>40 beats/min). The His bundle is normally the lowest area in the conduction system that can maintain a stable rhythm (usually 40-60 beats/min). When conduction fails anywhere above it, a normal His bundle can take over the pacemaker function of the heart and maintain a normal QRS complex, unless a distal intraventricular conduction defect is present. When the escape rhythm arises farther down the His-
Purkinje system, the rhythm is usually slower (<40 beats/min) and is often unstable; it results in a wide QRS complex.
What is the significance of isolated bundle-branch block with a normal P-R interval?
A conduction delay or block in the right bundle-branch results in a typical RBBB QRS pattern on the surface ECG (M-shape or rSR' in V,) and may represent a congenital abnormality or underlying organic heart disease. In contrast, a delay or block in the main left bundle-branch results in a left bundle-branch block (LBBB) QRS pattern (wide R with a delayed upstroke in V;) and nearly always represents underlying heart disease. The term hemiblock is often used if only one of the two fascicles of the left bundle-branch is blocked (left anterior or left posterior hemiblock). When the P-R interval is normal—and in the absence of an acute MI—a conduction block in either the left or right bundle rarely leads to complete heart block.
Can the site of an AV block always be determined from a 12lead ECG?

No. A first-degree AV block (PR interval >200 ms) can reflect abnormal conduction anywhere between the atria and the distal HisPurkinje system. Mobitz type I second-degree AV block, which is characterized by progressive lengthening of the P—R interval before a P wave is not conducted (a QRS does not follow the P wave), is usually due to a block in the AV node itself, and can be caused by digitalis toxicity or myocardial ischemia; progression to a third-degree AV block is uncommon. In patients with Mobitz type II second-degree AV block, atrial impulses are periodically not conducted into the ventricle without progressive prolongation of the P-R interval. The conduction block is nearly always in or below the His bundle and frequently progresses to complete (thirddegree) AV block, particularly following an acute anteroseptal MI. The QRS is typically wide.
In patients with a third-degree AV block, the atrial rate and ventricular depolarization rates are independent (AV dissociation) because atrial impulses completely fail to reach the ventricles. If the site of the block is in the AV node, a stable His bundle rhythm will result in a normal QRS complex, and the ventricular rate will often increase following administration of atropine. If the block involves the His bundle, the origin of the ventricular rhythm is more distal, resulting in wide QRS complexes. A wide QRS complex does not necessarily exclude a normal His bundle, as it may represent a more distal block in one of the bundle branches.

Yes. AV dissociation may occur during anesthesia with volatile agents in the absence of AV block and results from sinus bradycardia or an accelerated AV junctional rhythm. During isorhythmic dissociation, the atria and ventricles beat independently at nearly the same rate. The P wave often just precedes or follows the QRS complex, and their relationship is generally maintained. In contrast, interference AV dissociation results from a junctional rhythm that is faster than the sinus rate—such that sinus impulses always find the AV node refractory.

A bifascicular block exists when two of the three major His bundlebranches (right, left anterior, or left posterior) are partially or completely blocked. If one fascicle is completely blocked and the others are only partially blocked, a bundle-branch block pattern will be associated with either first-degree or second-degree AV block. If all three are affected, a trifascicular block is said to exist. A delay or partial block in all three fascicles results in either a prolonged P-R interval (first-degree AV block) or alternating LBBB and RBBB. Complete block in all three fascicles results in third-degree AV block.
What is the significance of the electrocardiographic findings in this patient?
The electrocardiographic findings (first-degree AV block plus RBBB) suggest a bifascicular block. Extensive disease of the conduction system is likely. Moreover, the patient's syncopal and near-syncopal episodes suggest that she may be at risk of life-threatening bradyarrhythmias (third-degree AV block). Intracardiac electrocardiographic recordings would be necessary to confirm the site of the conduction delay.

Cardiac evaluation is required because of the symptomatic bifascicular block. One of two approaches can be recommended, depending on the urgency of the surgery. If the surgery is truly emergent, a temporary transvenous pacing catheter or a transcutaneous pacemaker is indicated prior to induction of general or regional anesthesia. If the surgery can be postponed 24 to 48 h (as in this case), continuous electrocardiographic monitoring, echocardiography, serial 12-lead ECGs, and measurements of cardiac biomarkers can be obtained to exclude myocardial ischemia or infarction, valvular heart disease, or congestive heart failure, in addition to other pathological conditions that might adversely affect the patient's surgical outcome.

Suggested indications include any documented symptomatic bradyarrhythmia, second-degree (type II) AV block, or third-degree AV block and refractory supraventricular tachyarrhythmias. The first three indications generally require ventricular pacing, whereas the fourth requires atrial pacing electrodes and a programmable rapid atrial pulse generator.

Pacing can be established by transvenous, transcutaneous, epicardial, or transesophageal electrodes. The most reliable method is generally via a transvenous pacing electrode in the form of a pacing wire or a balloontipped pacing catheter. A pacing wire should always be positioned fluoroscopically, but a flow-directed pacing catheter can also be placed in the right ventricle under pressure monitoring. If the patient has a rhythm, an intracardiac electrocardiographic recording showing ST-segment elevation when the electrode comes in contact with the right ventricular endocardium confirms placement of either type of electrode. Transcutaneous ventricular pacing is also possible via large stimulating adhesive pads placed on the chest and should be used whenever transvenous pacing is not readily available. Epicardial electrodes are usually used during cardiac surgery. Pacing the left atrium via an esophageal electrode is a simple, relatively noninvasive technique, but it is useful only for symptomatic sinus bradycardias and for terminating some supraventricular tachyarrhythmias.
Once positioned, the pacing electrodes are attached to an electrical pulse generator that periodically delivers an impulse at a set rate and magnitude. Most pacemaker generators can also sense the heart's spontaneous (usually ventricular) electrical activity: when activity is detected, the generator suppresses its next impulse. By altering the generator's sensing threshold, the pacemaker generator can function in a fixed (asynchronous) mode or in a demand mode (by increasing sensitivity). The lowest current through the electrode that can depolarize the myocardium is called the threshold current (usually <2 mA for transvenous electrodes).

Ventricular pacing often reduces cardiac output because the atrial contribution to ventricular filling is lost. When the AV conducting system is diseased, atrial contraction can still be maintained by sequential stimulation by separate atrial and ventricular electrodes. The P—R interval can be varied by adjusting the delay between the atrial and ventricular impulses (usually set at 150—200 ms).

Pacemakers are categorized by a five-letter code, according to the chambers paced, chambers sensed, response to sensing, programmability, and arrhythmia function (Table 21—20). The two most commonly used pacing modes are VVI and DDD (the last two letters are frequently omitted).

O=none, Chamber Sensed = O=none. O=none, Response to Sensing = O-=none. O=none, Programmability = O=none. O=none, Antitachyarrhythmia Function = O=none. A=atrium, Chamber Sensed = A=atrium. A=atrium, Response to Sensing = T=triggered. A=atrium, Programmability = P=simple. A=atrium, Antitachyarrhythmia Function = P =pacing. V=ventricle, Chamber Sensed = V=ventricle. V=ventricle, Response to Sensing = | = inhibited. V=ventricle, Programmability = M=multi- programmable. V=ventricle, Antitachyarrhythmia Function = S=shock. D=dual (atrium and ventricle), Chamber Sensed = D=dual(atriumand ventricle). D=dual (atrium and ventricle), Response to Sensing = —_D =dual (triggered and inhibited). D=dual (atrium and ventricle), Programmability = C = communicating R=rate modulation. D=dual (atrium and ventricle), Antitachyarrhythmia Function = D = dual (pacing and shock)
If a pacemaker is placed in this patient, how can its function be evaluated?
If the patient's underlying rhythm is slower than the rate of a demand pacemaker, pacing spikes should be seen on the ECG. The spike rate should be identical to the programmed (permanent pacemaker—usually 72/min) or set (temporary) pacemaker rate; a slower rate may indicate a low battery. Every pacing spike should be followed by a QRS complex (100% capture). Moreover, every impulse should be followed by a palpable arterial pulse. If the patient has a temporary pacemaker, the escape rhythm can be established by temporarily slowing the pacing rate.
When the patient's heart rate is faster than the set pacemaker rate, pacing spikes should not be observed if the generator is sensing properly. In this instance, ventricular capture cannot be evaluated unless the pacemaker rate increases or the spontaneous heart rate decreases.
What intraoperative conditions may cause the pacemaker to malfunction?

Electrical interference from surgical electrocautery units can be interpreted as myocardial electrical activity and can suppress the pacemaker generator. Problems with electrocautery may be minimized by limiting its use to short bursts, limiting its power output, placing its grounding plate as far from the pacemaker generator as possible, and using bipolar cautery. Moreover, continuous monitoring of an arterial pulse wave (pressure, plethysmogram, or oximetry signal) is mandatory to ensure continuous perfusion during electrocautery.
Both hypokalemia and hyperkalemia can alter the pacing electrodes' threshold for depolarizing the myocardium and can result in failure of the pacing impulse to depolarize the ventricle. Myocardial ischemia, infarction, or scarring can also increase the electrodes' threshold and cause failure of ventricular capture.

If a temporary pacemaker fails intraoperatively, the inspired oxygen concentration should be increased to 100%. All connections and the generator battery should be checked. Most units have a battery-level indicator and a light that flashes with every impulse. The generator should be set into the asynchronous mode, and the ventricular output should be set on maximum. Failure of a temporary transvenous electrode to capture the ventricle is usually due to displacement of the electrode away from the ventricular endocardium; careful slow advancement of the catheter or wire while pacing often results in capture. Pharmacological management (atropine, isoproterenol, or epinephrine) may be useful until the problem is resolved. If an adequate arterial blood pressure cannot be maintained with adrenergic agonists, cardiopulmonary resuscitation should be instituted until another pacing electrode is placed or a new generator box is obtained. Transcutaneous pacing can be employed.
If a permanent pacemaker malfunctions (as with electrocautery), it should generally be converted to an asynchronous mode. Some units will automatically reprogram themselves to the asynchronous mode if malfunction is detected. Other pacemaker units must be reprogrammed by placing either an external magnet, or, preferably, a programming device over the generator. The effect of an external magnet on some pacemakers— particularly during electrocautery—may be unpredictable and should generally be determined prior to surgery.
Which anesthetic agents are appropriate for patients with pacemakers?
All anesthetic agents have been safely used in patients who already have pacemakers. Even volatile agents seem to have no effect on pacing electrode thresholds. Local anesthesia with moderate to deep intravenous sedation is usually used for placement of permanent pacemakers.

Park KW. Preoperative cardiac evaluation. Anesth Clin North Am. 2004;22:199. Wasnick J, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography. New York, NY: McGraw-Hill; 2011.

@ Cardiopulmonary bypass (CPB) diverts venous blood away from the heart (most often via one or more cannulas in the right atrium), adds oxygen, removes CO,, and returns the blood through a cannula in a large artery (usually the ascending aorta or a femoral artery). As a result, nearly all blood bypasses the heart and lungs.
@® The fluid level in the reservoir is critical. If a "roller" pump is used and the reservoir is allowed to empty, air can enter the main pump and be propelled into the patient where it may cause organ damage or fatality.
@ Initiation of CPB is associated with a variable increase in stress hormones and systemic inflammation.
@ Establishing the adequacy of the patient's preoperative cardiac function should be based on exercise (activity) tolerance, measurements of myocardial contractility such as ejection fraction, severity and location of coronary stenoses, ventricular wall motion abnormalities, cardiac end-diastolic pressures, cardiac output, and valvular areas and gradients.
@ Blood should be immediately available for transfusion if the patient has had previous cardiac surgery (a "redo"); when there has been a previous sternotomy, the right ventricle or coronary grafts may be adherent to the sternum and may be accidentally entered during the repeat sternotomy.
@ Transesophageal echocardiography (TEE) provides valuable information about cardiac anatomy and function during surgery. Twodimensional, multiplane TEE can detect regional and global ventricular
adequately drained may cause cardiac tamponade, requiring immediate reopening of the chest.

@ Factors known to increase pulmonary vascular resistance (PVR), such as acidosis, hypercapnia, hypoxia, enhanced sympathetic tone, and high mean airway pressures, are to be avoided for patients with right-to-left shunting; hyperventilation (hypocapnia) with 100% oxygen is usually effective in lowering PVR. Conversely, patients with left-to-right shunting may benefit from systemic vasodilation and increases in PVR.
@ Induction of general anesthesia in patients with cardiac tamponade can precipitate severe hypotension and cardiac arrest.
@ The sudden increase in left ventricular afterload after application of the aortic cross-clamp during aortic surgery may precipitate acute left ventricular failure and myocardial ischemia, particularly in patients with underlying ventricular dysfunction or coronary disease. The period of greatest hemodynamic instability follows the release of the aortic cross-clamp; the abrupt decrease in afterload together with bleeding and the release of vasodilating acid metabolites from the ischemic lower body can precipitate severe systemic hypotension.
@ The emphasis of anesthetic management during carotid surgery is on maintaining adequate perfusion to the brain and heart.
To provide anesthesia for cardiovascular surgery, ideally one will have an understanding of circulatory physiology, pharmacology, and pathophysiology; cardiopulmonary bypass (CPB) pumps, filters, and circuitry; transesophageal echocardiography (TEE); and techniques of myocardial preservation. Because surgical manipulations of the heart and great vessels will often have a profound impact on circulatory function, the anesthesiologist must understand the rationale behind the surgical techniques and follow the progress of the surgery closely, so that he or she can anticipate potential problems associated with each sequential step of the procedure.
This chapter presents an overview of anesthesia for cardiovascular surgery and of the principles, techniques, and physiology of CPB. Surgery on the aorta, the carotid arteries, and the pericardium presents special problems, which are also discussed herein.

of
@ CPB diverts venous blood away from the heart (most often via one or more cannulas in the right atrium), adds oxygen, removes CO3, and returns the blood through a cannula in a large artery (usually the ascending aorta or a femoral artery). As a result, nearly all blood bypasses the heart and lungs. When CPB is fully established, it provides both artificial ventilation and perfusion via the systemic circulation. This technique provides distinctly nonphysiological conditions, because mean arterial pressure is usually less than normal and blood flow is usually nonpulsatile. To minimize organ damage during this stressful period, varying degrees of systemic hypothermia may be employed. Topical hypothermia (an ice-slush solution) and cardioplegia (a chemical solution for arresting myocardial electrical activity) may also be used to protect the heart. The operation of the CPB machine is a complex task requiring the attention a perfusionist—a specialized, certified technician. Optimal results with CPB require close cooperation and clear, continuing communication among the surgeon, anesthesiologist, and perfusionist.

The typical CPB machine has six basic components: a venous reservoir, an oxygenator, a heat exchanger, a main pump, an arterial filter, tubing that conducts venous blood to the venous reservoir, and tubing that conducts oxygenated blood back to the patient (Figure 22—1). Modern CPB machines use a single disposable unit that includes the reservoir, oxygenator, and heat exchanger. Most machines also have separate accessory pumps that can be used for blood salvage (cardiotomy suction), venting (draining) the left ventricle, and administration of cardioplegia solutions. A number of other filters, alarms, and in-line pressure, oxygen-saturation, and temperature monitors are also typically used.

Prior to use, the CPB circuit must be primed with fluid (typically 1200-1800 mL for adults) that is devoid of bubbles. A balanced salt solution, such as lactated Ringer solution, is generally used, but other components are frequently added, including colloid (albumin or starch), mannitol (to promote diuresis), heparin (500-5000 units), and bicarbonate. At the onset of bypass in adults, when using a crystalloid priming solution, hemodilution typically decreases the hematocrit to about 22% to 27%. Blood is included in priming solutions for smaller children and severely anemic adults to prevent excessive hemodilution.

The reservoir of the CPB machine receives blood from the patient via one or two venous cannulas placed in the right atrium, the superior and inferior vena cava, or a femoral vein. With most circuits blood returns to the reservoir by gravity drainage. During extracorporeal circulation the patient's venous pressure is normally low. Thus, the driving force for flow into the pump is directly related to the difference in height between the patient and the reservoir and inversely proportional to the resistance of the cannulas and tubing. An appropriately primed CPB machine draws in blood like a siphon. Entrainment of air in the venous line can produce an air lock that may prevent blood flow. With some circuits (eg, use of an unusually small venous cannula) assisted venous drainage may be required; a regulated vacuum together with a hard shell venous reservoir or centrifugal pump (see below) is used in @ such instances. The fluid level in the reservoir is critical. If a "roller" pump is used and the reservoir is allowed to empty, air can enter the main pump and be propelled into the patient where it may cause organ damage or fatality. A low reservoir level alarm is typically present. Centrifugal pumps will not pump air but have the disadvantage of not impelling a well-defined volume with each revolution of the head (unlike roller pumps).

Blood is drained by gravity from the bottom of the venous reservoir into the oxygenator, which contains a blood—gas interface that allows blood to equilibrate with the gas mixture (primarily oxygen). A volatile anesthetic is frequently added to the oxygenator gas mixture. The blood—gas interface in a modern, membrane-type oxygenator is a very thin, gas-permeable silicone membrane. Arterial CO, tension during CPB is dependent on total gas flow past the oxygenator. A membrane oxygenator permits the perfusion to have independent control of Pao, and Paco, by varying the inspired oxygen concentration and the gas flow rate.

Blood from the oxygenator enters the heat exchanger and can either be cooled or warmed, depending on the temperature of the water flowing through the exchanger; heat transfer occurs by conduction. Because gas solubility decreases as blood temperature rises, a filter or trap is built into the unit to catch any bubbles that may form during rewarming.

Modern CPB machines use either an electrically driven double-arm roller (positive displacement) or a centrifugal pump to propel blood through the CPB circuit.

Roller pumps produce flow by compressing large-bore tubing in the main pumping chamber as the roller heads turn. Subtotal occlusion of the tubing prevents excessive red cell trauma. The rollers pump blood regardless of the resistance encountered, and produce a nearly continuous nonpulsatile flow. Flow is directly proportional to the number of revolutions per minute. In some pumps, an emergency backup battery provides power in case of an electrical power failure. All roller pumps have a hand crank to allow manual pumping, but those who have hand cranked a roller pump head will confirm that this is not a good long-term solution to an electric power failure.

Centrifugal pumps consist of a series of cones in a plastic housing. As the cones spin, the centrifugal forces created propel the blood from the centrally located inlet to the periphery. In contrast to roller pumps, blood flow with centrifugal pumps is pressure sensitive and must be monitored by an electromagnetic flowmeter. Increases in distal pressure will decrease flow and must be compensated for by increasing the pump speed. Because these pumps are nonocclusive, they are less traumatic to blood than roller pumps. Unlike roller pumps, which are placed after the oxygenator (Figure 22-1), centrifugal pumps are normally located between the venous reservoir and the oxygenator. Centrifugal (unlike roller) pumps have the advantage of not being able to pump air into the patient.

Pulsatile blood flow is possible with some roller pumps. Pulsations can be produced by instantaneous variations in the rate of rotation of the roller heads; they can also be added after flow is generated. Pulsatile flow is not available with centrifugal pumps. Although there is no consensus and the data are contradictory, some clinicians believe that pulsatile flow improves tissue perfusion, enhances oxygen extraction, attenuates the release of stress hormones, and results in lower systemic vascular resistance during CPB.

Particulate matter (eg, thrombi, fat globules, tissue debris) may enter the CPB circuit via the cardiotomy suction line. A final, in-line, arterial filter (that passes particles smaller than 27—40 ym) helps to reduce systemic embolism. Once filtered, the propelled blood returns to the patient, usually via a cannula in the ascending aorta, or less commonly in the femoral artery. A competent aortic valve prevents blood from regurgitating into the left ventricle. Arterial inflow pressure is measured before the filter. Arterial inflow pressure is measured before the filter so as to monitor the filter for clogging. The filter is always in parallel with a (normally clamped) bypass limb. The filter is also designed to trap gas bubbles, which can be bled out through a built-in stopcock.

The cardiotomy suction pump aspirates blood from the surgical field during CPB and returns it directly to the main pump reservoir. This is a potential portal for fat and other debris to enter the pump and embolize to organs. A red cell salvage suction device may also be used to aspirate blood from the surgical field, in which case blood is returned to a separate reservoir. When sufficient blood has accumulated (or at the end of the procedure), the salvaged blood is centrifuged, washed, and returned to the patient. Excessive suction pressure can theoretically contribute to red cell trauma. Extensive use of cell salvage suction (instead of cardiotomy suction) during bypass will deplete CPB circuit volume when blood loss is brisk. The high negative pressure of ordinary wall suction devices produces excessive red cell trauma precluding blood salvage from that source.

Over time, even with "total" CPB, blood reaccumulates in the left ventricle as a result of residual pulmonary flow via the bronchial arteries (which arise directly from the aorta or the intercostal arteries) or thebesian vessels (see Chapter 20), or sometimes as a result of aortic valvular regurgitation. Aortic regurgitation can occur as a result of either structural valvular abnormalities or surgical manipulation of the heart. Distention of the left ventricle compromises myocardial preservation (see below) and requires decompression (venting). Surgeons may "vent" the left ventricle by inserting a catheter via the right superior pulmonary vein, through the left atrium, and across the mitral valve into the left ventricle, or by inserting a catheter in the left ventricular apex or across the aortic valve. The blood aspirated by the vent pump normally passes through a filter before being returned to the venous reservoir.

Cardioplegic solutions are most often administered via an accessory pump on the CPB machine. This technique allows optimal control over the infusion pressure, rate, and temperature. A separate heat exchanger ensures control of the temperature of the cardioplegia solution. Less commonly, cardioplegic solutions may be infused from a cold intravenous fluid bag under pressure or by gravity.

Ultrafiltration can be used during CPB to increase the patient's hematocrit without transfusion. Ultrafilters consist of hollow capillary fibers that can function as membranes, allowing separation of the aqueous phase of blood from its cellular and proteinaceous elements. Blood can be diverted to pass through the fibers either from the arterial side of the main pump or from the venous reservoir using an accessory pump. Hydrostatic pressure forces water and electrolytes across the fiber membrane. Effluents of up to 40 mL/min may be removed.

Intentional hypothermia is often used following the initiation of CPB. Core body temperature may be reduced to 20°C to 32°C. In recent years, so-called tepid bypass has been used; this may be accomplished by allowing the patient's temperature to "drift" downward to 30°C to 35°C. Metabolic oxygen requirements are generally halved with each reduction of 10°C in body temperature. Some of the adverse effects of hypothermia include platelet dysfunction, coagulopathy, and depression of myocardial contractility. At the end of the surgical procedure, rewarming via the heat exchanger restores normal body temperature.
For complex repairs, profound hypothermia to temperatures of 15°C to 18°C allows total circulatory arrest for durations of as long as 60 min. During that time, both the heart and the CPB machine are stopped.

Optimal results in cardiac surgery require an expeditious and complete surgical repair with minimal physical trauma to the heart. Meanwhile, several techniques are used to prevent myocardial damage and maintain normal cellular integrity and function during CPB. Nearly all patients sustain at least minimal myocardial injury during cardiac surgery. With good preservation techniques, however, most of the injury is reversible. Although myocardial injury can be related to hemodynamic instability or surgical technique, it most commonly appears to be related to incomplete myocardial preservation during CPB. Injury related to hemodynamic instability results from an imbalance between oxygen demand and supply, producing cell ischemia. Reperfusion following a period of ischemia may produce excess oxygen-derived free radicals, intracellular calcium overload, abnormal endothelial—leukocyte interactions, and myocardial cellular edema. Patients at greatest risk are those with poor preoperative ventricular function (see Table 21-13), ventricular hypertrophy, diffuse severe coronary artery disease, or a combination of these. Inadequate myocardial preservation is usually manifested at the end of bypass as a persistently reduced cardiac output, worsened ventricular function as assessed by TEE, or cardiac arrhythmias. Electrocardiographic signs of myocardial ischemia are often difficult to detect due to frequent use of electrical pacing. Myocardial "stunning," resulting from ischemia and reperfusion, produces systolic and diastolic dysfunction that is reversible with time. The stunned myocardium usually responds to positive inotropic drugs. Myocardial necrosis, on the other hand, produces irreversible injury.

Aortic cross-clamping during CPB completely excludes the coronary arteries from the generalized bypass machine flow to the body, halting coronary blood flow. Although it is difficult to estimate a safe time period for cross-clamping or CPB duration because of differing vulnerabilities among patients and differing techniques for myocardial preservation, CPB times longer than 120 min (while often unavoidable) increase risk. Myocardial ischemia during bypass may occur not only during aortic clamping, but also after release of the cross-clamp. Low arterial pressures, obstruction of the bypass graft ostium, coronary artery embolism (from thrombi, platelets, air, fat, or atheromatous debris), reperfusion injury, coronary artery or bypass graft vasospasm, and contortion of the heart— causing compression or kinking of the coronary vessels—are all possible causes. Areas of the myocardium distal to a high-grade coronary obstruction are at greatest risk.
Ischemia causes depletion of high-energy phosphate compounds and accumulation of intracellular calcium. When coronary blood flow ceases, anaerobic metabolism becomes the principal source of cellular energy, and fatty acid oxidation is impaired. Unfortunately, these high energy phosphate stores are rapidly depleted, producing a progressive acidosis.

Cardioplegic solutions maintain normal cellular integrity and function during CPB by reducing energy expenditure and preserving availability of high-energy phosphate compounds. Although measures directed at increasing or replenishing energy substrates in the form of glucose or glutamate/aspartate infusions are used, the emphasis of myocardial preservation is on reducing cellular energy requirements to minimal levels. This is accomplished initially by the use of potassium cardioplegia (described below). The initial dose of cardioplegic solution may be hypothermic or may start warm ("hot shot") and progress to cold. Maintenance of myocardial protection may be facilitated by systemic and topical cardiac hypothermia (ice slush). Myocardial hypothermia reduces basal metabolic oxygen consumption, and potassium cardioplegia minimizes energy expenditure by arresting both electrical and mechanical activity. Myocardial temperature is often monitored directly; 10°C to 15°C is usually considered desirable. Cardioplegic solutions can be administered either antegrade through a catheter placed in the proximal aorta between the aortic clamp and the aortic valve, or retrograde through a catheter placed through the right atrium into the coronary sinus.
Ventricular fibrillation and distention (previously discussed) are important causes of myocardial damage. Ventricular fibrillation can dangerously increase myocardial oxygen demand, whereas distention not only increases oxygen demand but also reduces oxygen supply by interfering with subendocardial blood flow. The combination of the two is particularly bad. Other factors that might contribute to perioperative myocardial damage include the use of excessive doses of positive inotropes or calcium salts. In open heart procedures, de-airing of cardiac chambers and venting before and during initial cardiac ejection are critically important in preventing cerebral or coronary air embolism (and strokes—see later discussion). Removing air from coronary grafts during bypass procedures is similarly important. Depending on the amount and the location of coronary emboli, even small air bubbles can cause varying degrees of ventricular dysfunction at the end of CPB. Air emboli may preferentially find their way into the right (versus left) coronary ostium because of its superior location on the aortic root in the supine patient.

The most widely used method of arresting myocardial electrical activity is the administration of potassium-rich crystalloid or blood—crystalloid solutions. Following initiation of CPB and aortic cross-clamping, the coronary circulation is perfused intermittently with (usually cold) cardioplegic solutions. The resulting increase in extracellular potassium concentration reduces transmembrane potential. Eventually, the heart is arrested in diastole. Usually, cold cardioplegia must be repeated at intervals (about every 30 min) because of gradual washout and rewarming of the myocardium. The heart is subject to warming by contact with blood in the adjacent descending aorta and by contact with warmer ambient air in the surgical theater. Moreover, multiple doses of cardioplegia solutions may improve myocardial preservation by preventing an excessive accumulation of metabolites that inhibit anaerobic metabolism.

Although the exact recipe varies from center to center, the essential ingredient of the induction dose of cardioplegic solution is the same: an elevated potassium (10-40 mEq/L) concentration in the initial dose. Potassium concentration is kept below 40 mEq/L because higher levels can be associated with an excessive potassium load and excessive potassium concentrations at the end of termination of bypass perfusion. Sodium concentration in cardioplegic solutions is usually less than in plasma (<140 mEq/L) because ischemia tends to increase intracellular sodium content. A small amount of calcium (0.7—1.2 mmol/L) is needed to maintain cellular integrity, whereas magnesium (1.5—-15 mmol/L) is usually added to control excessive intracellular influxes of calcium. A buffer— most commonly bicarbonate—is necessary to prevent excessive buildup of acid metabolites; in fact, alkalotic perfusates are reported to produce better myocardial preservation. Alternative buffers include histidine and tromethamine (also known as THAM). Other components may include hypertonic agents to control cellular edema (mannitol) and agents thought to have membranestabilizing effects (lidocaine or glucocorticoids). Energy substrates are provided as glucose, glutamate, or aspartate. The question of whether to use crystalloid or blood as a vehicle for achieving cardioplegia remains controversial, although blood cardioplegia has become very common in North America. Evidence suggests that at least some groups of high-risk patients may do better with blood cardioplegia. Certainly, oxygenated blood cardioplegia may contain more oxygen than crystalloid cardioplegia.

Because cardioplegia may not reach areas distal to high-grade coronary obstructions (the areas that need it most), many surgeons administer retrograde cardioplegia through a coronary sinus catheter. Some centers have reported that the combination of antegrade plus retrograde cardioplegia is superior to either technique alone. Others have suggested that continuous warm blood cardioplegia is superior to intermittent hypothermic cardioplegia for myocardial preservation, but many surgeons avoid continuous cardioplegia so that they can operate ina "bloodless" surgical field. Cardiac surgery performed with true normothermia (rather than tepid bypass) raises additional concerns about loss of the potentially protective effects of systemic hypothermia against cerebral injury.
As discussed previously, with prolonged myocardial ischemic times (crossclamp time), reperfusion of the myocardium can lead to extensive cell injury, rapid accumulation of intracellular calcium, and potentially irreversible cellular necrosis. This process has long been attributed to accumulation of oxygenderived free radicals. Free radical scavengers, such as mannitol, may help decrease reperfusion injury and are typical constituents of cardioplegic solutions and bypass "priming" solutions. Several steps may help limit reperfusion injury before unclamping of the aorta. Just prior to reperfusion, the heart may be perfused by a reduced potassium cardioplegic solution that serves to wash out accumulated metabolic byproducts. Alternatively, a "hot shot" or warm blood cardioplegic solution may be administered to wash out byproducts and replenish metabolic substrates. Hypercalcemia should be avoided in the immediate reperfusion period. Reperfusion pressures should be controlled closely because of altered coronary autoregulation. Systemic perfusion pressure is reduced just prior to clamp release; it is then brought up initially to about 40 mm Hg before gradually being increased and maintained at about 70 mm Hg. To further minimize metabolic requirement, the heart should have the opportunity to recover and resume contracting in an empty state for some additional time (5-10 min), and acidosis and hypoxia should be corrected before attempting to wean the patient from CPB.

Inadequate myocardial protection or inadequate washout and recovery from cardioplegia can result in asystole, atrioventricular conduction block, or a poorly contracting heart at the end of bypass. Excessive volumes of hyperkalemic cardioplegic solutions may produce persisting systemic hyperkalemia. Although calcium salt administration partially offsets hyperkalemia, excessive calcium can promote and enhance myocardial damage. In the usual patient, myocardial performance improves with time as the contents of the cardioplegia are cleared from the heart.

® Initiation of CPB is associated with a variable increase in stress hormones and systemic inflammation. Elevated concentrations of catecholamines, cortisol, arginine vasopressin, and angiotensin are observed. These neurohormonal responses are variously influenced by depth of anesthesia, blood pressure, type of surgical repair, or presence of pulsatile CPB.
Multiple humoral systems are also activated, including complement, coagulation, fibrinolysis, and the kallikrein system. Contact of blood with the internal surfaces of the CPB system activates complement via the alternate pathway (C3) as well as the classic pathway; the latter also activates the coagulation cascade, platelets, plasminogen, and kallikrein. Mechanical trauma from blood contact with the bypass apparatus also activates platelets and leukocytes. Increased amounts of oxygen-derived free radicals are generated. A systemic inflammatory response similar to that seen with sepsis and trauma can develop. When this response is intense or prolonged, patients can develop the same complications as are seen with sepsis or trauma, including generalized edema, acute respiratory distress syndrome, coagulopathy, and acute kidney failure.
CPB alters and depletes glycoprotein receptors on the surface of platelets. The resulting platelet dysfunction likely increases perioperative bleeding and potentiates other coagulation abnormalities (activation of plasminogen and the inflammatory response previously described).
The inflammatory response to CPB can be modulated by various therapies. Leukocyte depletion reduces inflammation and may similarly reduce complications. Leukocyte-depleted blood cardioplegia has been shown to improve myocardial preservation in some studies. Hemofiltration (ultrafiltration) during CPB, which presumably removes inflammatory cytokines, appears beneficial to pediatric patients. Administration of free radical scavengers such as high-dose vitamins C and E and mannitol has improved outcome in some studies, but remains investigational. Systemic corticosteroids before and during CPB can modulate the inflammatory response during CPB but improved outcome is not well established. Randomized clinical trials failed to find an outcome benefit to the routine use of systemic corticosteroids or statins in patients undergoing CPB.

One once-promising agent, aprotinin, a protease inhibitor, reduced inflammation and surgical bleeding following CPB. Unfortunately, it increased mortality and is no longer available in North America.

Plasma and serum concentrations of most water-soluble drugs (eg, nondepolarizing muscle relaxants) acutely decrease at the onset of CPB, but the change can be minimal and inconsequential for most lipid-soluble drugs (eg, fentanyl and sufentanil). The effects of CPB are complex because of the sudden increase in volume of distribution with hemodilution, decreased protein binding, and changes in perfusion and redistribution between peripheral and central compartments. Some drugs, such as opioids, also bind CPB components, but this is also minimal and inconsequential. Heparin potentially alters protein binding of drugs and ions by releasing and activating lipoprotein lipase, which hydrolyzes plasma triglycerides into free fatty acids; the latter can competitively inhibit drug binding to plasma proteins and bind free calcium ions. With the possible exception of propofol, constant infusion of a drug during CPB, even when adjusted to maintain a constant "effect site" concentration ("target controlled infusion") using data from patients not undergoing CPB, generally causes progressively increasing blood levels as a result of reduced hepatic and renal perfusion (reduced elimination) and hypothermia (reduced metabolism).

The preoperative evaluation and anesthetic management of common cardiovascular diseases are discussed in Chapter 21. The same principles apply whether these patients are undergoing cardiac or noncardiac surgery. An important distinction is that patients undergoing cardiac procedures will by definition @ have advanced disease. Establishing the adequacy of the patient's preoperative cardiac function should be based on exercise (activity) tolerance, measurements of myocardial contractility such as ejection fraction, severity and location of coronary stenoses, ventricular wall motion abnormalities, cardiac end-diastolic pressures, cardiac output, and valvular areas and gradients. Fortunately, unlike noncardiac surgery, cardiac surgery improves cardiac function in the majority of patients, and these patients have usually been extensively evaluated before being offered surgical repair. The anesthetic preoperative evaluation should also include a focus on pulmonary, neurological, and kidney function, as preoperative impairment of these organ systems predisposes patients to myriad postoperative complications.

The prospect of heart surgery is frightening, and relatively "heavy" premedication was often given in the past (see Chapter 21). Benzodiazepine sedative-hypnotics (diazepam, 5-10 mg orally), alone or in combination with an opioid (morphine, 5-10 mg intramuscularly or hydromorphone, 1-2 mg intramuscularly), were often used. However, in current practice most patients receive no sedative-hypnotic premedication until their arrival on the surgical unit, at which time many will receive small doses of intravenous midazolam. Longer acting premedicant agents (eg, lorazepam) are avoided by most practitioners to permit fast-tracking of patients through their recovery.

The best practitioners of cardiac anesthesia formulate a simple anesthetic plan that includes adequate preparations for contingencies. In an emergency one cannot wait for an assistant to search for drugs and equipment. Preparation, organization, and attention to detail permit one to deal more efficiently with unexpected intraoperative problems. The anesthesia machine, monitors, infusion pumps, and blood warmer should all be checked before the patient arrives. Drugs — including anesthetic and vasoactive agents—should be immediately available. Many clinicians prepare one vasoconstrictor and one vasodilator infusion before the start of the procedure.

Cardiac surgery is sometimes associated with large and rapid blood loss, and with the need for multiple drug infusions. Ideally, two large-bore (16-gauge or larger) intravenous catheters should be placed. One of these should be in a large central vein, usually an internal or external jugular or subclavian vein. Central venous cannulations may be accomplished while the patient is awake but sedated or after induction of anesthesia. Studies show no benefit from placing either central venous or pulmonary arterial catheters in awake (versus anesthetized) patients undergoing cardiovascular surgery.
Drug infusions should ideally be given into a central catheter, preferably directly into the catheter or into the injection port closest to the catheter (to minimize dead space). Multilumen central venous catheters and multilumen pulmonary artery catheter introducer sheaths allow for multiple drug infusions with simultaneous measurement of vascular pressures. One intravenous port should be solely for drug infusions; drug and fluid boluses should be administered through another site.
®@ Blood should be immediately available for transfusion if the patient has had previous cardiac surgery (a "redo"); when there has been a previous sternotomy, the right ventricle or coronary grafts may be adherent to the sternum and may be accidentally entered during the repeat sternotomy.

The electrocardiogram (ECG) is continuously monitored with two leads, usually leads II and Vs. Baseline tracings of all leads may be recorded for future reference. Computerized ST-segment analysis and the use of TEE have greatly improved detection of ischemic episodes.

In addition to all basic monitoring, arterial cannulation is always performed either prior to or immediately after induction of anesthesia. Radial arterial catheters may occasionally give falsely low readings following sternal retraction as a result of compression of the subclavian artery between the clavicle and the first rib. They may also provide falsely low values early after CPB due to the opening of atrioventricular shunts in the hand during rewarming. The radial artery on the side of a previous brachial artery cutdown should be avoided, because its use is associated with a greater incidence of arterial thrombosis and wave distortion. Obviously, if a radial artery will be harvested for a coronary bypass conduit, it cannot be used as a site for arterial pressure monitoring. Other useful catheterization sites include the brachial, femoral, and axillary arteries. A backup manual or automatic blood pressure cuff should also be used.

Central venous pressure is not terribly useful for diagnosis of hypovolemia but has been customarily monitored in nearly all patients undergoing cardiac surgery. The pulmonary artery occlusion pressure provides a better measure of left ventricular filling pressure. Pulmonary artery catheterization has declined precipitously in nearly all circumstances except adult cardiac surgery due to lack of evidence of a positive effect on patient outcomes. In theory the decision about whether to use a pulmonary artery catheter should be based on the patient and the procedure; however, in most centers, it is the preferences of the anesthetic, surgical, and critical care teams that really matter. In many centers, either every or almost no cardiac surgery patient receives pulmonary artery catheterization. In general, pulmonary artery catheterization has been most often used in patients with compromised ventricular function, pulmonary hypertension, or those undergoing complicated procedures. The most useful data are pulmonary artery pressures, the pulmonary artery occlusion ("wedge") pressure, and thermodilution cardiac outputs. Specialized catheters provide extra infusion ports, continuous measurements of mixed venous oxygen saturation and cardiac output, and the capability for right ventricular or atrioventricular sequential pacing. Given the risk associated with placing any pulmonary artery catheter, some clinicians opine that it makes sense to insert only those devices that offer these advanced capabilities. Intraoperatively and postoperatively, when pulmonary artery occlusion pressure measurements are not available, left ventricular filling pressures can be measured with a left atrial pressure line inserted by the surgeon during bypass.
The right internal jugular vein is a preferred approach for intraoperative central venous cannulation, particularly when the line will be removed after a day or two. Catheters placed through the other sites, particularly on the left side, are more likely to kink following sternal retraction (as noted earlier) and are less likely to pass into the superior vena cava as those placed through the right internal jugular vein.
Pulmonary artery catheters migrate distally during CPB and may spontaneously wedge without balloon inflation. Inflation of the balloon under these conditions can rupture a pulmonary artery causing lethal hemorrhage. Pulmonary artery catheters should be routinely retracted 2 to 3 cm during CPB and the balloon subsequently inflated slowly. If the catheter wedges with less than 1.5 mL of air in the balloon, it should be withdrawn farther.

Once the patient is anesthetized, an indwelling urinary catheter is placed to monitor the hourly output. Bladder temperature is often monitored as a measure of core temperature but may not track core temperature well with reduced urinary flow. The sudden appearance of reddish urine may indicate excessive red cell hemolysis caused by CPB or a transfusion reaction.

Multiple temperature monitors are usually placed once the patient is anesthetized. Bladder (or rectal), esophageal, and pulmonary artery (blood) temperatures are often simultaneously monitored. Because of the heterogeneity of readings during cooling and rewarming, bladder and rectal readings are generally taken to represent an average body temperature, whereas esophageal represents core temperature. Pulmonary artery temperature provides an accurate estimate of blood temperature, which should be the same as core temperature in the absence of active cooling or warming. Nasopharyngeal and tympanic probes may most closely approximate brain temperature. Myocardial temperature is often measured directly during instillation of cardioplegia.

Laboratory testing is mandatory during cardiac surgery. Blood gases, hemoglobin, potassium, ionized calcium, and glucose measurements should be immediately available. The activated clotting time (ACT) approximates the Lee—White clotting time and is used to monitor heparin anticoagulation and its reversal with protamine. Some centers routinely use thromboelastography (TEG) to identify causes of bleeding after CPB.

One of the most important forms of intraoperative monitoring is inspection of the surgical field. Once the sternum is opened, lung expansion can be observed through the pleura. When the pericardium is opened, the heart (primarily the right ventricle) is visible; thus cardiac rhythm, volume, and contractility can often be judged visually. Blood loss and surgical maneuvers must be closely watched and related to changes in hemodynamics and rhythm.

@ TEE provides valuable information about cardiac anatomy and function during surgery. Two-dimensional, multiplane TEE can detect regional and global ventricular abnormalities, chamber dimensions, valvular anatomy, and the presence of intracardiac air. TEE can also be helpful in confirming cannulation of the coronary sinus for cardioplegia. Multiple views should be obtained from the upper esophagus, mid-esophagus, and transgastric positions in the transverse, sagittal, and in-between planes (Figure 22—2). The two views most commonly used for monitoring during cardiac surgery are the four-chamber view (Figure 22-3) and the transgastric (short-axis) view (Figure 22—4). Three-dimensional echocardiography offers better visualization of complex anatomic features, particularly of cardiac valves. The following represent the most important applications of intraoperative TEE.
D
FIGURE 22-2 Useful views during transesophageal echocardiography. A: The relationship between the angle of the ultrasound beam and image orientation relative to the patient. B—D: Echocardiographic views from the upper midesophagus, lower mid-esophagus, and transgastric position (C). Note that different views can be obtained in each position as the tip of the probe is tilted either upward (anteflexion) or backward (retroflexion) and the angle of the beam is changed from 0° to 180°. The angle of the beam is shown in the upper left- hand comer of each image. The probe is also rotated clockwise or counterclockwise to optimize viewing of the various structures. AO, aorta; AV, aortic valve; CS, coronary sinus; IVC, inferior vena cava; LA, left atrium; LAA, left atrial appendage; LUPV, left upper pulmonary vein; LV, left ventricle; MPA, main pulmonary artery; MV, mitral valve; PA, pulmonary artery; RA, right atrium; RPA, right pulmonary artery; RV, right ventricle; SVC, superior vena cava.

FIGURE 22-3 Transesophageal echocardiogram of the mid-esophageal fourchamber view, showing the right and left atria and ventricles.
FIGURE 22-4 Transesophageal echocardiogram at the lower esophageal/transgastric level looking up at the left ventricle at the level of the papillary muscles.
1. Assessment of valvular function—Valvular morphology can be assessed by multiplane and three-dimensional TEE. Pressure gradients, area and severity of stenosis, and severity of valvular regurgitation can be assessed by Doppler echocardiography and color-flow imaging (Figure 22—5). Colors are usually adjusted so that flow toward the probe is red and flow in the opposite direction is blue. TEE also can detect prosthetic valve dysfunction in the forms of obstruction, regurgitation, paravalvular leak, or vegetations from endocarditis. The TEE images in the upper mid-esophagus at 40° to 60° and 110° to 130° are useful for examining the aortic valve and ascending aorta (Figure 22-6). The diameter of the aortic valve annulus can be estimated accurately. Doppler flow across the aortic valve must be measured looking up from the deep transgastric view (Figure 22—7). The anatomic features of the mitral valve relevant to TEE are shown in Figure 22-8. The mitral valve is examined from the midesophageal position, looking at the mitral valve apparatus with and without color in the 0° through 180° views (Figure 22-9). TEE is an invaluable aid to guide and assess the completeness of mitral valve repair surgery. The commissural view (at about 60°) is particularly helpful because it cuts across many scallops of the mitral valve.
FIGURE 22-5 Transesophageal echocardiography Doppler and color-flow imaging. A: Pulse-wave Doppler recording of mitral valve inflow showing two phases, E (early filling) and A (atrial filling). B: Color-flow imaging demonstrates backward flow (regurgitant jet) across the mitral valve during systole (mitral regurgitation).

FIGURE 22-6 Two views of the aortic valve. A: Between 40° and 60°, all three leaflets are usually visualized. B: Between 110° and 130°, the left ventricular outflow, aortic valve, and ascending aorta are clearly visualized.
FIGURE 22-7 Transesophageal echocardiographic recording of continuouswave Doppler from the transgastric view looking up at the aortic valve, demonstrating severe aortic stenosis. Peak velocity of 409 cm/s indicates a gradient of 66.9 mm Hg.
FIGURE 22-8 The anatomy of the mitral valve and its anatomic relationships to the aortic valve and left circumflex coronary artery. The posterior leaflet has three scallops, P,, P,, and P3. The anterior leaflet is usually divided into A, and Ay regions; in some classifications the anterior leaflet is divided into three areas (A,, Ao, A3), corresponding to the opposing corresponding areas of the posterior leaflet.
Vertical
D
FIGURE 22-9 Multiplane imaging cuts across different segments of the mitral valve apparatus between 0° and 180° (A). Images of the mitral valve at 0°, 71°, and 142° (B, C, and D, respectively).

2. Assessment of ventricular function— Ventricular function can be assessed by global systolic function, estimated by means of ejection fraction (often calculated using Simpson's method of disks) and left ventricular end-diastolic volume; diastolic function (ie, looking for abnormal relaxation and restrictive diastolic patterns by checking mitral flow velocity or by measuring movements of the mitral valve annulus using tissue Doppler techniques); and regional systolic function (by assessing wall motion and thickening abnormalities). Regional wall abnormalities from myocardial ischemia often appear before ECG changes. Regional wall motion abnormalities can be classified into three categories based on severity (Figure 22—10): hypokinesis (reduced wall motion), akinesis (no wall motion), and dyskinesis (paradoxical wall motion). The location of a regional wall motion abnormality can indicate which coronary artery is experiencing reduced flow. The left ventricular myocardium is supplied by three major arteries: the left anterior descending artery, the left circumflex artery, and the right coronary artery (Figure 22—11). The approximate areas of distribution of these arteries on echocardiographic views are shown in Figure 22-12. Increasingly it is recognized that areas classically presented as the distribution of the circumflex artery may receive blood flow from the right coronary artery or the left anterior descending artery. The ventricular short-axis mid view at the mid-papillary muscle level contains all three blood supplies from the major coronary arteries.
FIGURE 22-10 Classification of regional wall motion abnormalities.
FIGURE 22-11 Standard angiographic views of the left (A) and right (B)

coronary arteries. Note the left main coronary artery quickly divides into the left anterior descending and the left circumflex arteries. A: (1) Left anterior descending artery with septal branches; (2) ramus medianus; (3) diagonal artery; (4) first septal branch; (5) left circumflex artery; (6) left atrial circumflex artery; (7) obtuse marginal artery. B: (1) Conus artery; (2) sinoatrial node artery; (3) acute marginal artery; (4) posterior descending artery with septal branches; (5) atrioventricular node artery; (6) posterior left ventricular artery. LAO, left anterior oblique; RAO, right anterior oblique.
FIGURE 22-12 Coronary artery supply of the left and right ventricles in three views: the short-axis view (A), the four-chamber view (B), and the threechamber view (C). Green, right coronary artery; blue, left anterior descending artery; pink, left circumflex artery.
3. Assessment of other cardiac structures and abnormalities—In adults undergoing elective cardiac surgery we have used TEE to diagnose previously undetected congenital defects such as an atrial or ventricular septal defect; pericardial effusions and constrictive pericarditis; and cardiac tumors. Doppler color-flow imaging helps delineate abnormal intracardiac blood flows and shunts. TEE can assess the extent of myomectomy in patients with hypertrophic cardiomyopathy (idiopathic hypertrophic subaortic stenosis). Upper-, mid-, and lower-esophageal views are valuable in diagnosing aortic disease processes such as dissection, aneurysm, and atheroma (Figure 22-13). The extent of dissections in the ascending and descending aorta can be accurately defined; however, airway structures prevent complete visualization of the aortic arch with TEE. The presence of protruding atheroma in the ascending aorta increases the risk of postoperative stroke and should prompt the use of epiaortic scanning to identify

FIGURE 22-13 Upper esophageal views of the aortic arch and descending aorta. The ascending aorta can be visualized in the upper mid-esophagus at 110° to 130° with anteflexion at the aortic valve level (see Figures 22—2B and 22-6B). LAX, long axis; PA, pulmonary artery; SAX, short axis.
4, Examination for residual air—Air is introduced into the cardiac chambers during all "open" heart procedures, such as valve surgery. Residual amounts of air often remain in the left ventricular apex even after the best deairing maneuvers. TEE is helpful in defining the volume of residual air, to determine whether additional surgical maneuvers need to be undertaken to help avoid cerebral or coronary embolism.

Computer-processed electroencephalographic (EEG) recordings can be used to assess anesthetic depth during cardiac surgery, and either the processed or "raw" EEG can be used to ensure complete drug-induced electrical silence (for brain protection) prior to circulatory arrest. These recordings are generally not useful in detecting neurological insults during CPB. Progressive hypothermia (or progressively deepened anesthesia) is typically associated with EEG slowing, burst suppression, and, finally, an isoelectric recording. Most strokes during CPB are due to small emboli that are not likely to produce changes in the EEG. Artifacts from the CPB roller pump may be seen on the raw EEG (due to piezoelectric effects from compression of the pump tubing) but can usually be identified as such by computer processing.

Transcranial Doppler (TCD) provides noninvasive measurements of blood flow velocity in the middle cerebral artery, which is insonated through the temporal bone. TCD and carotid Doppler are useful for detecting cerebral emboli. Increased numbers of emboli detected by TCD or carotid Doppler have been associated with an increased risk of postoperative neurobehavioral dysfunction.

Cerebral oximetry (see Chapter 6) is increasingly employed during cardiac surgery. A baseline value is established for each patient prior to preoxygenation. Decreased cerebral oxygen saturation may be seen when oxygen delivery is impaired secondary to decreased Paco, tension, anemia, decreased arterial oxygen saturation, and diminished cardiac output.

Cardiac operations usually require general anesthesia, endotracheal intubation, and controlled ventilation. Some centers have used thoracic epidural anesthesia alone for minimally invasive surgery without CPB or combined thoracic epidural with light general endotracheal anesthesia for other forms of cardiac surgery. These techniques have never been popular in North America due to concerns about the risk of spinal hematomas following heparinization, the associated medical—legal consequences, and the limited evidence of an outcome benefit. Some centers use a single intrathecal opioid injection to provide postoperative analgesia.
Induction of general anesthesia should be performed in a smooth, controlled (but not necessarily "slow") fashion—often referred to as a "cardiac induction" when it is used for other types of surgery. The principles are discussed in Chapter 21. Selection of anesthetic agents is generally less important than the way they are used. Indeed, studies have failed to show differences in long-term outcome with various anesthetic @ techniques. Anesthetic dose requirements are variable. Severely compromised patients should be given anesthetic agents in incremental, small doses. Patient tolerance of inhaled anesthetics generally declines with declining ventricular function. Blood pressure and heart rate are continuously evaluated following unconsciousness, insertion of an oral airway, urinary catheterization, and tracheal intubation. A sudden increase in heart rate or blood pressure may indicate light anesthesia and the need for more anesthetic prior to the next challenge, whereas a decrease or no change suggests that the patient is ready for the subsequent stimulus. Reductions in blood pressure greater than 20% generally call for administration of a vasopressor (as described later). A series of challenges may be used to judge when anesthetic depth will allow intubation without a marked hypertensive response, while also avoiding hypotension from excessive anesthetic dosing.

The period following intubation is often characterized by a gradual decrease in blood pressure resulting from the anesthetized state (often associated with vasodilation and decreased sympathetic tone) and a lack of surgical stimulation. Patients will usually respond to fluid boluses or a vasoconstrictor. Nevertheless, the administration of large amounts of intravenous fluids prior to the bypass may serve to accentuate the hemodilution associated with CPB (as described below). Small doses of phenylephrine (25-100 mcg), vasopressin (1—3 units), or ephedrine (5S—10 mg) may be useful to avoid excessive hypotension. Following intubation and institution of controlled ventilation; arterial blood gases, hematocrit, serum potassium, and glucose concentrations are measured. The baseline ACT (normal <130 s) is best measured after skin incision.

Anesthetic techniques for cardiac surgery have evolved over the years. Successful techniques range from primarily inhalation anesthesia to high-dose opioid totally intravenous techniques. In recent years, total intravenous anesthesia with short-acting agents and combinations of intravenous and volatile agents have become most popular.

This technique was originally developed to circumvent the myocardial depression associated with older volatile anesthetics, particularly halothane. But pure high-dose opioid anesthesia (eg, fentanyl, 50-100 mcg/kg, or sufentanil, 15-25 mcg/kg) produces prolonged postoperative respiratory depression (12-24 h), is associated with an unacceptably high incidence of patient awareness (recall) during surgery, and often fails to control the hypertensive response to stimulation in many patients with preserved left ventricular function. Other undesirable effects include skeletal muscle rigidity during induction and prolonged postoperative ileus. Moreover, simultaneous administration of benzodiazepines with large doses of opioids can produce hypotension and myocardial depression. Patients anesthetized with sufentanil and other shorter acting agents generally regain consciousness and can be extubated sooner than those anesthetized with comparable doses of fentanyl.

The drive for cost containment in cardiac surgery was a major impetus for development of anesthesia techniques with short-acting agents. Although the drugs may be costlier, large economic benefits resulted from earlier extubation, decreased intensive care unit (ICU) stays, earlier ambulation, and earlier hospital discharge ("fast-track" management). One technique employs induction with propofol (0.5-1.5 mg/kg followed by 25-100 mcg/kg/min), and modest doses of fentanyl (total doses of 5—7 mcg/kg) or remifentanil (0-1 mcg/kg bolus followed by 0.25-1 mcg/kg/min). Target controlled infusion (TCI) employs software and hardware (computerized infusion pump) to deliver a drug and achieve a set concentration at the effect site based on pharmacokinetic modeling. For propofol, the clinician sets only the patient's age and weight, and the desired blood concentration on the Diprifusor™, a TCI device widely available in countries outside North America. During cardiac surgery, a target propofol concentration of 1.5 to 2 mcg/mL is often used. When remifentanil (rather than a longer persisting agent) is used during cardiac anesthesia one must anticipate the need for postoperative analgesia after its discontinuation.

Selection of anesthetic agents is oriented to hemodynamic stability as well as early extubation (1-6 h). Propofol (0.5-1.5 mg/kg) or etomidate (0.1-0.3 mg/kg) is often used for induction. Induction usually follows sedation with small doses of midazolam (0.05 mg/kg). Renewed interest in volatile agents came about following studies demonstrating the protective effects of volatile agents on ischemic myocardium and the utility of these agents for fast-track recovery of cardiac patients. Opioids are given in small doses together with a volatile agent (0.5-1.5 minimum alveolar concentration [MAC]) to maintain anesthesia and to blunt the sympathetic response to stimulation. The opioid may be given by intermittent bolus injections, by continuous infusion, or both (Table 22-1). To facilitate fast-track management, typical total doses of fentanyl and sufentanil generally do not exceed 15 and 5 mcg/kg, respectively, and some clinicians combine smaller doses of fentanyl or sufentanil with an analgesic dose of hydromorphone or morphine administered toward the end of CPB. Some clinicians also administer propofol in a low-dose infusion (25-50 mcg/kg/min) or TCI (1.5-2.0 mcg/mL) for maintenance. The major advantage of volatile agents or infusions of remifentanil or propofol is the ability to change the anesthetic concentration and depth rapidly. Isoflurane, sevoflurane, and desflurane are the most commonly used volatile anesthetics. Early laboratory reports of isoflurane inducing intracoronary steal have been overshadowed by later reports of myocardial protection. Isoflurane remains a commonly used volatile agent. Nitrous oxide is generally not used. Nitrous is particularly disadvantageous during the time interval between cannulation and decannulation, because of its tendency to expand any intravascular air bubbles that may form. Moreover, it cannot be given conveniently during CPB.

Fentanyl, Loading Dose (mcg/kg) = 1-5. Fentanyl, Maintenance Infusion = 1-3 mcg/kg/h. Fentanyl, Boluses (mcg/kg) = 0.5-1. Sufentanil, Loading Dose (mcg/kg) = 0.25-1.25. Sufentanil, Maintenance Infusion = 0.25-0.75 mcg/kg/h. Sufentanil, Boluses (mcg/kg) = 0.125-0.25. Remifentanil, Loading Dose (mcg/kg) = 0.5-1. Remifentanil, Maintenance Infusion = 0.1-1 mcg/kg/min. Remifentanil, Boluses (mcg/kg) = 0.25-1

The combination of ketamine with midazolam (or propofol) for induction and maintenance of anesthesia is a useful technique, particularly in frail patients with hemodynamic compromise. It is associated with stable hemodynamics, reliable amnesia and analgesia, minimal postoperative respiratory depression, and rare (if any) psychotomimetic side effects. For induction, ketamine, 1 to 2 mg/kg, with midazolam, 0.05 to 0.1 mg/kg, is given as a slow intravenous bolus. Anesthesia can then be maintained by infusion of ketamine, 1.3 to 1.5 mg/kg/h, and midazolam, 0.065 to 0.075 mg/kg/h, or more easily with an inhaled agent. Hypertension following intubation or surgical stimulation can be treated with propofol, opioids, B-blockers, or a volatile agent.

Muscle relaxation is helpful for intubation, to facilitate sternal retraction, and to prevent patient movement and shivering. Unless airway difficulties are expected, intubation may be accomplished after administration of a nondepolarizing muscle relaxant. The choice of muscle relaxant in the past was often based on the desired hemodynamic response. Modern, shorter acting agents such as rocuronium, vecuronium, and cisatracurium are commonly used and have almost no hemodynamic side effects of their own. Vecuronium, however, has been reported to markedly enhance bradycardia associated with large doses of opioids, particularly sufentanil. Because of its vagolytic effects, pancuronium was often used in patients with marked bradycardia who were taking B-blocking agents, Succinylcholine remains appropriate for endotracheal intubation, particularly for rapid sequence induction. Judicious dosing, appropriate use of a peripheral nerve stimulator, and reversal (if needed) allow fast-tracking with any of these agents.

Following induction and intubation, the anesthetic course is typically characterized by an initial period of minimal stimulation (skin preparation and draping) that is frequently associated with hypotension, followed by discrete periods of intense stimulation that can produce tachycardia and hypertension. These periods of stimulation include the skin incision, sternotomy and sternal retraction, opening the pericardium, and, sometimes, aortic dissection. The anesthetic agent should be adjusted appropriately in anticipation of these events. Accentuated vagal responses resulting in marked bradycardia and hypotension may occasionally be seen during sternal retraction or opening of the pericardium, perhaps more commonly in patients who have been taking Badrenergic blocking agents or diltiazem.
Myocardial ischemia in the prebypass period is not always associated with hemodynamic perturbations such as tachycardia, hypertension, or hypotension. Prophylactic infusion of nitroglycerin (1-2 mcg/kg/min) has been studied many times and continues to be used in some centers, but it has never been shown to reduce the incidence of ischemia or improve outcomes.

@ Anticoagulation must be established before CPB to prevent acute disseminated intravascular coagulation and formation of clots in the CPB pump. In most centers the adequacy of anticoagulation will be confirmed by measuring the ACT. An ACT longer than 400 to 480 s is considered adequate. Heparin, 300 to 400 units/kg, is usually given before aortic cannulation. Some surgeons prefer to administer the heparin themselves directly into the right atrium. If heparin is administered by the anesthesiologist, it should be given through a reliable (usually central) intravenous line, and the ACT should be measured 3 to 5 min later. If the ACT is less than 400 s, additional heparin (100 units/kg) is given. Some drugs (eg, aprotinin) prolong the celite-activated ACT but not the kaolinactivated ACT; the kaolin-ACT should be used to assess adequacy of anticoagulation in these circumstances. Heparin concentration assays (see Reversal of Anticoagulation, later) measure heparin levels and not necessarily effect; these assays are therefore not reliable for measuring the degree of anticoagulation but can be used as an adjunct. A whole blood heparin concentration of 3 to 4 units/mL is usually sufficient for CPB. The high-dose thrombin time (HiTT) is not influenced by aprotinin but is more complicated to perform than a kaolin-ACT. HiTT cannot provide a preheparin control and does not provide an index for the adequacy of reversal with protamine (as discussed later).
Resistance to heparin is occasionally encountered; many such patients have antithrombin III deficiency (acquired or congenital). Antithrombin III is a circulating serine protease that irreversibly binds and inactivates thrombin (as well as the activated forms of factors X, XI, XII, and XIII). When heparin complexes with antithrombin III, the anticoagulant activity of antithrombin III is enhanced 1000-fold. Patients with antithrombin III deficiency will achieve adequate heparin anticoagulation following infusion of antithrombin III (or fresh frozen plasma). Milder forms of heparin resistance can be managed by administration of a modestly larger than normal dose of heparin.

Patients with a history of heparin-induced thrombocytopenia (HIT) require special consideration. These patients produce heparin-dependent (platelet factor 4) antibodies that agglutinate platelets and produce thrombocytopenia, sometimes associated with thromboembolism. If the history of HIT is remote and antibodies can no longer be demonstrated, heparin may safely be used for CPB. When significant antibody titers are detected, alternative anticoagulants including hirudin, bivalirudin, anacrod, and argatroban may be considered.

Bleeding prophylaxis with antifibrinolytic agents may be initiated before or after anticoagulation. Some clinicians prefer to administer antifibrinolytic agents after heparinization to reduce the possible incidence of thrombotic complications; others fear that delayed administration may reduce antifibrinolytic @ efficacy. Antifibrinolytic therapy may be particularly useful for patients who are undergoing a repeat operation; who refuse blood products (such as Jehovah's Witnesses); who are at high risk for postoperative bleeding because of recent administration of glycoprotein IIb/IIIa inhibitors (abciximab [RheoPro], eptifibatide [Integrilin], or tirofiban [Aggrastat]); who have preexisting coagulopathy; or who are undergoing long and complicated procedures. The antiplatelet effect of abciximab typically lasts 24 to 48 h; those of eptifibatide and tirofiban are 2 to 4h and 4 to 8 h, respectively. The frequent combination of aspirin and the adenosine diphosphate receptor antagonist clopidogrel (Plavix) is also associated with excessive bleeding.
The antifibrinolytic agents currently available, e-aminocaproic acid and tranexamic acid, do not affect the ACT and only rarely induce allergic reactions. e-Aminocaproic acid is usually administered as a 50 to 75 mg/kg loading dose followed by a 20 to 25 mg/kg/h maintenance infusion (some clinicians use a standard 5—10 g loading dose followed by 1 g/h). Tranexamic acid is often dosed at 10 mg/kg followed by 1 mg/kg/h, although pharmacokinetic studies suggest that larger doses may more reliably maintain effective blood concentrations. Intraoperative collection of platelet-rich plasma by pheresis prior to CPB is employed by some centers; reinfusion following bypass may decrease bleeding and reduce transfusion requirements.

Placement of venous and arterial cannulas for CPB is a critical time. After heparinization, aortic cannulation is usually done first because of the hemodynamic problems sometimes associated with venous cannulation and to allow convenient and rapid transfusion from the pump oxygenator. The inflow cannula is most often placed in the ascending aorta. The small opening of most arterial cannulas produces a jet stream that, when not positioned properly, can cause aortic dissection or preferential flow of blood to the innominate artery. The systemic arterial pressure is customarily reduced to 90 to 100 mm Hg systolic during placement of the aortic cannula to reduce the likelihood of dissection. Air bubbles should be absent from the arterial cannula and inflow line, and adequacy of the connection between the arterial inflow line and the patient must be demonstrated before bypass is initiated. Failure to remove all air bubbles will result in air emboli, possibly into the coronary or cerebral circulations, whereas failure of the cannula tip to fully enter the aorta may result in aortic dissection. Some clinicians routinely hand compress the carotid arteries during aortic cannulation to decrease the likelihood of cerebral emboli, but the efficacy of this technique is doubtful.
One or two venous cannulas are placed in the right atrium, usually through the right atrial appendage. One cannula is usually adequate for most coronary artery bypass and aortic valve operations. The single cannula used often has two portals (two-stage); when it is properly positioned, one opening is in the right atrium and the other is in the inferior vena cava.

Separate cannulas in the superior and inferior venae cavae are used for other forms of open-heart procedures (other forms of valve surgery and congenital © repairs). Hypotension from impaired ventricular filling may occur during manipulation of the venae cavae and the heart. Venous cannulation also frequently precipitates atrial or, less commonly, ventricular arrhythmias. Premature atrial contractions and transient bursts of a supraventricular tachycardia are common and need no treatment if they are not sustained. Sustained paroxysmal atrial tachycardia or atrial fibrillation frequently leads to hemodynamic deterioration, which may be treated pharmacologically, electrically, or by immediate initiation of bypass (provided that full anticoagulation has been confirmed). Malpositioning of the venous cannulas can interfere with venous return or impede venous drainage from the head and neck (superior vena cava syndrome). Upon initiation of CPB, the former is manifested as inadequate volume in the venous reservoir, whereas the latter produces engorgement of the head and neck.

Once the cannulas are properly placed and secured, the ACT is acceptable, and the perfusionist is ready, CPB is initiated. The main CPB pump is started and with satisfactory arterial inflow the venous cannula(s) is unclamped. Establishing the adequacy of venous return to the pump reservoir is critical. Normally, the reservoir level rises and CPB pump flow is gradually increased. If venous return is poor, the level of blood in the reservoir will decline, potentially allowing air to enter the pump circuit. When the venous reservoir volume declines the cannulas should be checked for proper placement and for forgotten clamps, kinks, or an air lock. Under these circumstances, pump flow should be slowed until the problem is resolved. Adding volume (blood or colloid) to the reservoir may be necessary. With full CPB and unimpeded venous drainage, the heart should empty; failure to empty or progressive distention may result from malpositioning of the venous cannula or aortic regurgitation. In the rare case of severe aortic insufficiency that limits the extent of peripheral perfusion, immediate aortic cross-clamping (and cardioplegia) may be necessary.

Systemic mean arterial pressure is closely monitored as pump flow is gradually increased to 2 to 2.5 L/min/m?. At the onset of CPB, systemic arterial pressure usually decreases abruptly. Initial mean systemic arterial (radial) pressures of 30 to 40 mm Hg are not unusual. This decrease is usually attributed to abrupt hemodilution, which reduces blood viscosity and effectively lowers SVR. It is often treated with increased flow and vasopressors.
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pump flow is already low, a vasodilator such as clevidipine, nicardipine, or nitroprusside is used.

FIGURE 22-14 Heparin dose—response curve; activated clotting time (ACT) in seconds versus total heparin dose in milligrams per kilogram. (1) Plot the initial ACT on the x-axis (point A). (2) Plot the ACT after heparinization (point B). (3) Draw the line defined by these two points. (4) If additional anticoagulation is needed, find the desired ACT on that line. The amount of additional heparin needed is the difference on the y-axis between the present ACT and the desired ACT (point C). (5) If the third point does not lie on the original line, a new line is drawn originating from the baseline ACT and passing midway between the other two points. (6) For reversal of anticoagulation, the protamine dose is based on the remaining heparin activity, estimated to be the heparin dose corresponding to the latest ACT on the dose-response line.

Moderate (26—32°C) or deep (20—25°C) hypothermia is used routinely for some procedures, particularly those involving the aortic root and great vessels. The lower the temperature, the longer the time required to achieve cooling and rewarming. Lower temperatures, however, permit lower CPB flows to be used safely. At a temperature of 20°C, flows as low as 1.2 L/min/m? may be adequate. Hypothermia produces characteristic changes in the ECG including the Osborne wave, a positive deflection between the QRS and ST segments. Ventricular fibrillation often occurs as the heart is cooled below 28°C to 29°C.
Cardioplegia should be established immediately, as fibrillation consumes highenergy phosphates at a greater rate than slower rhythms. Cardioplegia is achieved by cross-clamping the ascending aorta proximal to the aortic inflow cannula and (as previously described) infusing cardioplegia solution through a small catheter proximal to the cross-clamp or directly into the coronary ostia if the aorta is opened (eg, for aortic valve replacement). Many surgeons routinely employ retrograde cardioplegia via a catheter in the coronary sinus (see earlier discussion). During aortocoronary bypass grafting, cardioplegia solution may also be given through the graft when the surgeon elects to perform the distal anastomosis first.

Ventilation of the lungs is discontinued when adequate pump flows are reached and the heart stops ejecting blood. Following institution of full CPB, ventricular ejection continues briefly until the left ventricular volume reaches a critically low level. Discontinuing ventilation prematurely when there is any remaining pulmonary blood flow acts as a right-to-left shunt that can promote hypoxemia. The importance of this mechanism depends on the relative ratio of remaining pulmonary blood flow to pump flow. At some centers, once ventilation is stopped, oxygen flow is continued in the anesthesia circuit with a small amount of continuous positive airway pressure (5 cm H,0) in the hope of preventing postoperative pulmonary dysfunction. Most centers either stop all gas flow or continue a low flow of oxygen (1-2 L/min) in the anesthesia circuit. Ventilation is resumed at the conclusion of CPB in anticipation of the heart beginning to eject blood.

There formerly was controversy about whether to use temperature-corrected (pH stat) or uncorrected (a-stat) arterial blood gas tensions during hypothermic CPB in adults. The controversy stemmed from the fact that the solubility of a gas increases and the neutral pH (ie, the pH at which concentrations of H* and OH™ ions are the same) of water increases with hypothermia. As a result of the former effect, although total CO, content does not change (in a closed system), the partial pressure of CO, will decrease as blood temperature drops. The problem is most significant for arterial CO, tension because of its effect on arterial pH and cerebral blood flow. As the temperature decreases, the plasma bicarbonate
management appears to preserve cerebral autoregulation of blood flow. Despite the theoretical and observed differences, in most studies comparisons between the two techniques fail to reveal appreciable differences in patient outcomes except in children undergoing circulatory arrest.

@ Hypothermia (<34°C) potentiates general anesthetic potency, but failure to give anesthetic agents, particularly during rewarming on CPB, may result in awareness and recall. With light anesthesia hypertension may be seen and, if muscle paralysis is also allowed to wear off, the patient may move. Consequently, additional doses of anesthetic agents may be necessary during CPB. Reduced concentrations of a volatile agent (eg, 0.5-0.75% isoflurane) via the oxygenator are frequently used. The volatile agent concentration may need to be reduced to a value that does not depress contractility immediately prior to termination of bypass if residual myocardial depression is apparent. Those relying on opioids and benzodiazepines for anesthesia during CPB may need to administer additional doses of these agents or commence a propofol infusion. Some clinicians routinely administer a benzodiazepine (eg, midazolam) when rewarming is initiated. Alternatively, a propofol, opioid, or ketamine—midazolam infusion may be continued throughout CPB. Sweating during rewarming is common and usually indicates a hypothalamic response to perfusion with warm blood (rather than "light" anesthesia). During rewarming, blood temperature should not exceed core temperature by more than 2°C.

The incidence of neurobehavioral deficits after CPB varies widely, depending on how long after surgery the examination is performed and the criteria for diagnosis. In the first week after surgery the incidence may be as high as 80%. Fortunately, in most instances, these deficits are transient. Neurobehavioral deficits detectable 8 weeks or more (20-25%) after operation or strokes (2-6%) are less common. Factors that have been associated with neurological sequelae include increased numbers of cerebral emboli, combined intracardiac (valvular) and coronary procedures, advanced age, and preexisting cerebrovascular disease. During open-heart procedures, deairing of cardiac chambers, assumption of a head-down position, and venting before and during initial cardiac ejection are important in preventing gas emboli. Some centers fill the surgical field with CO,, a gas that if entrained and embolized will more rapidly be reabsorbed. TEE
can detect residual air within the heart and the need for further deairing procedures. During coronary bypass procedures, minimizing the amount of aortic manipulation, the number of aortic clampings, and the number of graft sites on the surface of the aorta, and using sutureless proximal anastomotic devices may help reduce atheromatous emboli. Palpation of the aorta, TEE, and especially epiaortic echocardiography can help identify high-risk patients and guide management. Epiaortic echocardiography is the most sensitive and specific technique.

The relative contributions of emboli versus cerebral hypoperfusion in causing neurological deficits remains unclear. The data are controversial and sparse that prophylactic drug infusions immediately before and during intracardiac (open ventricle) procedures will decrease the incidence and severity of neurological deficits. Prior to circulatory arrest with very deep hypothermia, some clinicians administer a corticosteroid (methylprednisolone, 30 mg/kg, or the equivalent dose of dexamethasone) and mannitol (0.5 g/kg). The head is also covered with ice bags (avoiding the eyes). Surface cooling delays rewarming and may also facilitate adequacy of brain cooling. A long list of drugs has been tested and has failed to improve cerebral outcomes after heart surgery. Human studies during cardiac surgery have not shown improved neurobehavioral outcomes with prophylactic administration of calcium channel blockers (nimodipine), Nmethyl-p-aspartate (NMDA) antagonists (remacemide), free radical scavengers (pegorgotein), sedative-hypnotics (thiopental, propofol, or clomethiazole), or lazaroids (tirilazad).

Discontinuation of bypass is accomplished by a series of necessary procedures and conditions, the first of which is adequate rewarming. The surgeon's decision about when to rewarm is important; adequate rewarming requires time, but rewarming too soon removes the protective effects of hypothermia. Rapid rewarming often results in large temperature gradients between well-perfused organs and peripheral vasoconstricted tissues; subsequent equilibration following separation from CPB decreases core temperature again. An excessive gradient between the infusate temperature and the patient's core temperature can result in deleterious brain hyperthermia. Infusion of a vasodilator drug (eg, isoflurane) allows higher pump flows and will often speeds the rewarming process. Allowing some pulsatile flow (ventricular ejection) may also speed rewarming. Excessively rapid rewarming, however, can result in the formation of gas bubbles in the bloodstream as the solubility of gases rapidly decreases. If the heart fibrillates during rewarming, direct electrical defibrillation (5-10 J) may be necessary. Administration of lidocaine, 100 to 200 mg, and magnesium sulfate, 1 to 2 g, prior to removal of aortic cross-clamping is a common protocol and may decrease the likelihood of fibrillation. Many clinicians advocate a head-down position while intracardiac air is being evacuated to decrease the likelihood of cerebral emboli. Lung inflation facilitates expulsion of (left-sided) intracardiac air by compressing pulmonary vessels and returning blood into the left heart. TEE is useful in detecting residual intracardiac air. Initial reinflation of the lungs requires greater than normal airway pressure and should generally be done under direct visualization of the surgical field because excessive lung expansion can interfere with internal mammary artery grafts.
General guidelines for separation from CPB include the following:

¢ The core body temperature should be at least 37°C.
¢ Astable rhythm must be present. Pacing is often used and confers the benefit of a properly timed atrial systole. Persistence of atrioventricular block should prompt measurement of serum potassium concentration. If hyperkalemia is present, it can be treated with calcium, NaHCOs, furosemide, or glucose and insulin.
¢ The heart rate must be adequate (generally 80-100 beats/min). Slow heart rates are generally treated by pacing. Many inotropic agents will also increase heart rate. Supraventricular tachycardias generally require cardioversion.
¢ Laboratory values must be within acceptable limits. Significant acidosis (pH <7.20), hypocalcemia (ionized), and hyperkalemia (>5.5 mEq/L) should be treated; ideally the hematocrit should exceed 22%; however, a hematocrit <22% should not by itself trigger transfusion of red blood cells at this time. When CPB reservoir volume and flow are adequate, ultrafiltration may be used to increase the hematocrit.
¢ Adequate ventilation with 100% oxygen must have been resumed.
¢ All monitors should be rechecked for proper function and recalibrated if necessary.

CPB should be discontinued as systemic arterial pressure, ventricular volumes and filling pressures, and cardiac function (on TEE) are assessed. Central aortic pressure may be measured directly and compared with the radial artery pressure and cuff pressure (if there is concern about radial artery hypotension). A reversal of the normal systolic pressure gradient, with aortic pressure being greater than radial pressure, is often seen immediately postbypass. This has been attributed to opening of arteriovenous connections in the hand as a consequence of rewarming. Central aortic root pressure can also be estimated by palpation by an experienced surgeon. Right ventricular volume and contractility can be estimated visually, whereas filling pressures are measured directly by central venous, pulmonary artery, or left atrial catheters. Cardiac output can be measured by thermodilution. TEE can define adequacy of end-diastolic volumes, right and left ventricular contractility, and valvular function.
Weaning is typically accomplished by progressively clamping the venous return line (tubing). As the beating heart fills, ventricular ejection resumes. Pump flow is gradually decreased as arterial pressure rises. Once the venous line is completely occluded and systolic arterial pressure is judged to be adequate (>80-90 mm Hg), pump flow is stopped and the patient is evaluated. Some surgeons wean by clamping the venous line and then progressively "filling" the patient with arterial inflow.
Most patients fall into one of four groups when coming off bypass (Table 22-2). Patients with good ventricular function are usually quick to develop good blood pressure and cardiac output and can be separated from CPB immediately. Hyperdynamic patients can also be rapidly weaned. These patients emerge from CPB with a very low SVR, demonstrating good contractility and adequate volume, but have low arterial pressure; their hematocrit is often reduced (<22%). Diuresis (off CPB) or red blood cell transfusions increase arterial blood pressure.

Treatment, Group IIIA: LV Pump Failure = Inotrope;|ABP, LVAD. Treatment, Group IIIB: RV Failure = Inotrope, pulmonary vasodilator; RVAD. Treatment, Group IV: Vasodilated (Hyperdynamic) = —_ Vasoconstrictor, volume
'CPB, cardiopulmonary bypass; LY, left ventricular; RV, right ventricular; |ABP, intraaortic balloon pump; LVAD, left ventricular assist device; RVAD, right ventricular assist device; TEE, transesophageal echocardiography.
Hypovolemic patients include those with normal ventricular function and those with varying degrees of impairment. Those with preserved myocardial function quickly respond to infusion of blood via the aortic cannula. Blood pressure and cardiac output rise with each bolus, and the increase becomes progressively more sustained. Most of these patients maintain good blood pressure and cardiac output with a left ventricular filling pressure below 10 to 15 mm Hg. Ventricular impairment should be suspected (when definitive diagnosis using TEE is not available) in hypovolemic patients whose filling pressures rise during volume infusion without appreciable changes in blood pressure or cardiac output or in those who require filling pressures above 10 to 15 mm Hg. Ventricular dysfunction is easily diagnosed by TEE.

Patients with pump failure emerge from CPB with a sluggish, poorly contracting heart that progressively distends. In such cases, CPB may need to be reinstituted while inotropic therapy is initiated; alternatively, if the patient is less unstable, a positive inotrope (epinephrine, dopamine, dobutamine) can be administered while the patient is observed for improvement. If the patient does not respond to reasonable doses of one of these three agents, milrinone can be added. In patients with poor preoperative ventricular function milrinone may be administered as the first-line agent prior to separation from CPB. In the rare instance that SVR is increased, afterload reduction with nitroprusside or milrinone can be tried. The patient should be evaluated for unrecognized ischemia (kinked graft or coronary vasospasm), valvular dysfunction, shunting, or right ventricular failure (the distention is primarily right sided). TEE will facilitate the diagnosis in these cases.
If drug therapies fail, intraaortic balloon pump (IABP) counterpulsation should be initiated while the heart is "rested" on CPB. The efficacy of IABP depends on proper timing of inflation and deflation of the balloon (Figure 22— 15). The balloon should inflate just after the dicrotic notch is seen on the intraaortic pressure tracing to augment diastolic blood pressure and coronary flow after closure of the aortic valve. Inflation too early increases afterload and exacerbates aortic regurgitation, whereas late inflation reduces diastolic augmentation. Balloon deflation should be timed just prior to left ventricular ejection to decrease its afterload. Early deflation makes diastolic augmentation and afterload reduction less effective. Use of a left or right ventricular assist device (LVAD or RVAD, respectively) may be necessary for patients with refractory pump failure. If myocardial stunning is a major contributor or there are areas of hibernating myocardium, a delayed improvement in contractile function may allow complete weaning from all drugs and support devices only after 12 to 48 h of therapy. Ventricular assist devices can be used as a bridge to cardiac transplantation.

FIGURE 22-15 A central arterial waveform during 1:2 intraaortic balloon pump counterpulsation. Ideally the balloon, which is positioned in the descending aorta just distal to the left subclavian artery, should inflate at the dicrotic notch (1) and be completely deflated just as the left ventricle begins to eject (2). Note the lower end-diastolic pressures after balloon augmentation and slightly lower systolic pressure in the following beat. N, nonaugmented beat; A, augmented beat; B, balloon augmentation.

Many clinicians believe that positive inotropes should not routinely be used in patients coming off CPB because positive inotropes increase myocardial oxygen demand. The routine use of calcium similarly may worsen ischemic injury and may contribute to coronary spasm (particularly in patients who were taking calcium channel blockers preoperatively). Nevertheless, there are centers that administer calcium salts or a positive inotrope (eg, dobutamine), or both, to every patient at the conclusion of CPB. Commonly used positive inotropes and vasopressors are listed in Table 22—3. Epinephrine, dopamine, and dobutamine are the most commonly used agents. Epinephrine is the most potent inotrope and is often effective in increasing both cardiac output and systemic blood pressure when others agents have failed. In lower doses, it has predominantly B-agonist activity. Dobutamine, unlike dopamine, does not increase filling pressures and may be associated with less tachycardia than dopamine; unfortunately, cardiac output often increases without significant changes in blood pressure. On the other hand, dopamine is sometimes more effective in increasing blood pressure than in increasing cardiac output. Interestingly, when infused to increase cardiac output to similar extents, epinephrine is associated with no more increase (and perhaps less) in heart rate than dobutamine. Inamrinone, enoximone, milrinone, and olprinone are selective phosphodiesterase inhibitors, and inotropes with arterial and venous dilator properties. Only inamrinone and milrinone are available in North America. In studies of patients with chronic heart failure inamrinone and milrinone, unlike other inotropes, did not appreciably increase myocardial oxygen consumption. The combination of an inodilator (usually milrinone) and a B-adrenergic agonist results in at least additive (and possibly synergistic) inotropic effects. Norepinephrine is useful for increasing SVR but may compromise splanchnic and renal blood flow at increased doses. Some clinicians use norepinephrine in combination with phosphodiesterase inhibitors to prevent excessive reductions in systemic arterial pressure. Arginine vasopressin may be used in patients with refractory hypotension, a low SVR, and resistance to norepinephrine. There are experimental reports in which doses of methylene blue or vitamin C have

successfully counteracted vasodilation that could not be overcome with norepinephrine, vasopressin, or both. Inhaled nitric oxide and prostaglandin E, (or even inhaled milrinone) may also be helpful for refractory pulmonary hypertension and right ventricular failure (Table 22-4); nitric oxide has the added advantage of not decreasing systemic arterial pressure. Studies have not confirmed outcome benefits to the use of thyroid hormone (T3) or glucose—insulin—potassium infusions for vasoactive/inotropic support after CPB.

AIS ASAIIY.6 = 0. Phenylephrine, Adrenergic Acti BAran AIS ASAIIY.Indirect = 0. Phenylephrine, Phosphodiesterase.Inhibition = 0. Inamrinone, Bolus = 0.5-1.5 mg/kg. Inamrinone, ae.Infusion = 5-10 mcg/kg/min. Inamrinone, Adrenergic Acti BAran AIS ASAIIY.a = 0. Inamrinone, Adrenergic Acti BAran AIS ASAIIY.6 = 0. Inamrinone, Adrenergic Acti BAran AIS ASAIIY.Indirect = 0. Inamrinone, Phosphodiesterase.Inhibition = +44. Milrinone, Bolus = 50 meg/kg. Milrinone, ae.Infusion = 0.375-0.75 mcg/kg/min. Milrinone, Adrenergic Acti BAran AIS ASAIIY.a = 0. Milrinone, Adrenergic Acti BAran AIS ASAIIY.6 = 0. Milrinone, Adrenergic Acti BAran AIS ASAIIY.Indirect = 0. Milrinone, Phosphodiesterase.Inhibition = +++. Vasopressin, Bolus = 1-2 units. Vasopressin, ae.Infusion = 2-8 units/h. Vasopressin, Adrenergic Acti BAran AIS ASAIIY.a = 0. Vasopressin, Adrenergic Acti BAran AIS ASAIIY.6 = 0. Vasopressin, Adrenergic Acti BAran AIS ASAIIY.Indirect = 0. Vasopressin, Phosphodiesterase.Inhibition = °o
"4, mild activity; ++, moderate activity; +++, marked activity.
TABLE 22—4 Vasodilators.

Clevidipine, Dosage = 1-16 mg/h. Fenoldopam, Dosage = 0.03-0.6 mcg/kg/min. Nicardipine, Dosage = 2.5-10 mg/h. Nitric oxide, Dosage = 10-60 ppm (inhaled). Nitroglycerin, Dosage = 0.5-10 mcg/kg/min. Nitroprusside, Dosage = 0.5-10 mcg/kg/min. Prostaglandin E,, Dosage = 0.01-0.2 mcg/kg/min

Following CPB, bleeding is controlled, bypass cannulas are removed, anticoagulation is reversed, and the chest is closed. Systolic arterial pressure is generally maintained at less than 140 mm Hg to minimize bleeding. Checking for bleeding, particularly from the posterior surface of the heart, requires lifting the heart, which can cause periods of precipitous hypotension. Some surgeons will need to be informed of the extent and duration of the hypotension; others have greater situational awareness. The atrial cannula(s) is removed before the aortic cannula in case the latter must be used to rapidly administer volume to the patient. Most patients need additional blood volume after termination of bypass. Administration of blood, colloids, and crystalloid is guided by observation of the left ventricle on TEE, filling pressures, and the postbypass hematocrit. A final hematocrit of 25% to 30% is desirable. Blood remaining in the CPB reservoir can be transfused via the aortic cannula, or it can be washed and processed by a cell-saver device and given intravenously. Frequent ventricular ectopy may reflect electrolyte disturbances or residual ischemia and usually should be treated with amiodarone; hypokalemia or hypomagnesemia should be corrected. Ventricular arrhythmias in this setting can rapidly deteriorate into ventricular tachycardia and fibrillation.

Once hemostasis is judged acceptable and the patient continues to remain stable, heparin activity is reversed with protamine. Protamine is a highly positively charged protein that binds and effectively inactivates heparin (a highly negatively charged polysaccharide). Heparin—protamine complexes are then removed by the reticuloendothelial system. Protamine can be dosed in varying ways, but the results of all techniques should be checked for adequacy by repeating the ACT 3 to 5 min after completion of the protamine infusion. Additional incremental doses of protamine may be necessary.
One dosing technique bases the protamine dose on the amount of heparin initially required to produce the desired ACT; the protamine is then given in a ratio of 1 to 1.3 mg of protamine per 100 units of heparin. A still simpler approach is to give adult patients a defined dose (eg, 3-4 mg/kg) then check for adequacy of reversal. Another approach calculates the protamine dose based on the heparin dose-response curve (Figure 22-14). Automated heparin—protamine titration assays effectively measure residual heparin concentration and can also be used to calculate the protamine dose. The justification for using this methodology is the observation that when protamine is given in excess it may have anticoagulant activity, although this has never been demonstrated in humans. This approach also assumes that administered protamine remains in circulation for a prolonged time (which has been proven false in studies of patients undergoing cardiac surgery). To accomplish the heparin:protamine titration, premeasured amounts of protamine are added in varying quantities to several wells, each containing a blood sample. The well whose protamine concentration best matches the heparin concentration will clot first. Clotting will be prolonged in wells containing either too much or too little protamine. The protamine dose can then be estimated by multiplying the concentration in the tube that clots first by the patient's calculated blood volume. Supplemental protamine (50-100 mg) may be considered after administration of unwashed blood remaining in the pump reservoir after CPB because that blood contains heparin.

® Protamine administration can result in a number of adverse hemodynamic effects, some of which are immunological in origin. Protamine given slowly (5— 10 min) usually has few effects; when given more rapidly it produces a fairly consistent vasodilation that is easily treated with blood from the pump oxygenator and small doses of phenylephrine. Catastrophic protamine reactions often include myocardial depression and marked pulmonary hypertension. Diabetic patients previously maintained on protamine-containing insulin (such as NPH) may be at increased risk for adverse reactions to protamine.

agents has not already being given; the diagnosis should be confirmed by elevated fibrin degradation products (=32 mg/mL), or evidence of clot lysis on thromboelastography. Increasingly, factor VII concentrate or prothrombin complex concentrate are administered as a "last resort" in the setting of coagulopathic bleeding following cardiac surgery.

Unless a continuous intravenous infusion technique is used, additional anesthetic agents are necessary following CPB; the choice may be determined by the hemodynamic response of the patient following CPB. We have found that most patients tolerate modest doses of volatile agents or propofol infusion. Patients with hypertension that is unresponsive to adequate anesthesia with opioids and either a volatile agent or propofol (or both) should receive a vasodilator such as nitroglycerin, nitroprusside, clevidipine, or nicardipine (Table 22-4). Fenoldopam may be used and has the added benefit of increasing renal blood flow, which might possibly improve kidney function in the early postoperative period.
It is common for an opioid (morphine or hydromorphone) and either propofol or dexmedetomidine to be given to provide analgesia and sedation during transfer to the ICU and analgesia (after discontinuation of the propofol or dexmedetomidine) during emergence.

Transporting patients with critical illness from the operating room to the ICU is a consistently nerve-wracking and occasionally hazardous process that is complicated by the possibilities of monitor failure, overdosage, or interruption of drug infusions, and hemodynamic instability en route. Portable monitoring equipment, infusion pumps, and a full oxygen cylinder with a self-inflating bag for ventilation should be readied prior to the end of the operation. Minimum monitoring during transportation includes the ECG, arterial blood pressure, and pulse oximetry. A spare endotracheal tube, laryngoscope, succinylcholine, and emergency resuscitation drugs should also accompany the patient. Upon arrival in the ICU, the patient should be attached to the ventilator, breath sounds should be checked, and an orderly transfer of monitors and infusions (one at a time) should follow. The handoff to the ICU staff should include a brief summary of the procedure, intraoperative problems, current drug therapy, and any expected difficulties. Many centers insist on a standard protocol for the "handoff," and we

Depending on the patient, the type of surgery, and local practices, patients may be mechanically ventilated for 1 to 12 h postoperatively. Sedation may be maintained by a propofol or dexmedetomidine infusion. The emphasis in the first few postoperative hours should be on maintaining hemodynamic stability and monitoring for excessive postoperative ® bleeding. Chest tube drainage in the first 2 h of more than 250 to 300 mL/h (10 mL/kg/h)—in the absence of a hemostatic defect—is excessive and may require surgical reexploration. Subsequent drainage that exceeds 100 mL/h is also worrisome. Intrathoracic bleeding at a site not adequately drained may cause cardiac tamponade, requiring immediate reopening of the chest.
Hypertension despite analgesia and sedation is a common postoperative problem and should generally be treated promptly so as not to exacerbate bleeding or myocardial ischemia. Nitroprusside, nitroglycerin, clevidipine, nicardipine, or esmolol is generally used. Fluid replacement may be guided by filling pressures, echocardiography, or by responses to treatment. Most patients present with relative hypovolemia for several hours following operation. Hypokalemia (from intraoperative diuretics) often develops and requires replacement. Postoperative hypomagnesemia should be expected in patients who receive no magnesium supplementation intraoperatively.
Extubation should be considered only when muscle paralysis has worn off (or been reversed) and the patient is hemodynamically stable. Caution should be exercised in obese and elderly patients and those with underlying pulmonary disease. Cardiothoracic procedures are typically associated with marked decreases in functional residual capacity and postoperative diaphragmatic dysfunction.

The development of advanced epicardial stabilizing devices, such as the Octopus (Figure 22-16), facilitated coronary artery bypass grafting without the use of CPB, also known as off-pump coronary artery bypass (OPCAB). This type of retractor uses suction to stabilize and lift the anastomotic site rather than compress it down, which allows for greater hemodynamic stability. Full (CPB) dose heparinization is usually given and the CPB machine is usually immediately available if needed.
FIGURE 22-16 Schematic illustration of the Octopus retractor for off-pump coronary artery bypass surgery.
Intravenous fluid loading together with intermittent or continuous infusion of a vasopressor may be necessary while the distal anastomoses are sewn. In contrast, a vasodilator may be required to reduce the systolic pressure to 90 to 100 mm Hg during partial clamping of the aorta for the proximal anastomosis. Intravenous nitroglycerin is often used because of its ability to ameliorate myocardial ischemia.
Although OPCAB was initially proposed for "simple" oneor two-vessel bypass grafting in patients with good left ventricular function, it may be the sicker, older patients who benefit most from avoidance of CPB. The surgeon may use an intraluminal shunt to maintain coronary blood flow during sewing of distal anastomoses. Volatile anesthetic agents and morphine provide myocardial protection during prolonged periods of ischemia. Maintenance of anesthesia with a volatile agent may therefore be desirable. When the surgeon is skillful, longterm graft patency may be comparable to procedures done with CPB. Patients with extensive coronary disease, particularly those with poor target vessels, may not be good candidates. OPCAB may decrease the incidence of postoperative neurological complications and the need for transfusion relative to conventional coronary bypass with CPB.

Cardiovascular function in infants and young children differs from that in adults. Stroke volume is relatively fixed, so that cardiac output is primarily dependent on heart rate. The immature hearts of neonates and infants often are less forgiving of pressure or volume overload. Furthermore, the functions of both ventricles are more interdependent, so that failure of one ventricle often precipitates failure of the other (biventricular heart failure). Transition of the neonate from the fetal to the adult circulation is discussed in Chapter 40.

of hypoxemia. Clubbing of the fingers is frequent in children with cyanotic defects. The evaluation should also search for other congenital abnormalities, which are present in up to 30% of patients with congenital heart disease. The results of echocardiography, heart catheterization, electrocardiography, and chest radiography should be reviewed. Laboratory evaluation typically includes a complete blood count (with platelet count), coagulation studies, electrolytes, blood urea nitrogen, and serum creatinine. Measurements of ionized calcium and glucose are also useful in neonates and critically ill children.

Fasting requirements vary according to the patient's age and current guidelines. A preoperative intravenous infusion that provides maintenance fluid requirements should be used in patients susceptible to dehydration, in those with severe polycythemia, and when excessive delays occur prior to surgery.

Premedication varies according to age and cardiac and pulmonary reserves. Atropine, 0.02 mg/kg intramuscularly (minimum dose, 0.15 mg), has by tradition been given to pediatric cardiac patients to counteract enhanced vagal tone. Neonates and infants younger than 6 months of age may receive no premedication or be given only atropine. Sedation is desirable in older patients, particularly those with cyanotic lesions (tetralogy of Fallot), as agitation and crying worsen right-to-left shunting. Patients older than 1 year may be given midazolam orally (0.5—-0.6 mg/kg) or intramuscularly (0.08 mg/kg).

1. Obstructive lesions—Anesthetic management should strive to avoid hypovolemia, bradycardia, tachycardia, and myocardial depression. The optimal heart rate should be selected according to age; slow rates decrease cardiac output, whereas fast rates may impair ventricular filling. Mild cardiac depression may be desirable in some hyperdynamic patients, eg, those with coarctation of the aorta.
2. Shunts—A favorable ratio of pulmonary vascular resistance (PVR) to SVR
should be maintained in the @ presence of shunting. Factors known to increase PVR, such as acidosis, hypercapnia, hypoxia, enhanced sympathetic tone, and high mean airway pressures, are to be avoided in patients with right-to-left shunting; hyperventilation (hypocapnia) with 100% oxygen is usually effective in lowering PVR. Systemic vasodilation also worsens right-to-left shunting and should be avoided; phenylephrine may be used to raise SVR. Inhaled nitric oxide has no effect on systemic arterial pressure. Conversely, patients with left-to-right shunting may benefit from systemic vasodilation and increases in PVR.

Standard intraoperative monitors are generally used, but they may be first applied during the course of an inhaled induction in some patients. A large discrepancy between end-tidal and arterial CO, tensions should be anticipated in patients with large right-to-left shunts because of increased dead space. Following induction, intraarterial and central venous pressure monitoring are employed for thoracotomies and all procedures employing CPB. We recommend sonographic guidance for these cannulations. A 22or 24-gauge catheter is used to enter the radial artery; 24-gauge catheters may be more appropriate for small neonates and premature infants. A cutdown may be necessary in some instances. The internal jugular or subclavian vein is generally used for central venous cannulation; if this approach is unsuccessful, a right atrial catheter may be placed intraoperatively by the surgeon. TEE is invaluable for assessing the surgical repair following CPB. Ever smaller probes are yielding better resolution as the technology advances. Probes are currently available for patients as small as 3 kg. Intraoperative epicardial echocardiography is commonly used either in addition to or instead of TEE.

Venous access is desirable but not always necessary for induction. Agitation and crying are particularly undesirable in patients with cyanotic lesions and can increase right-to-left shunting. Intravenous access can be established after induction but before intubation in most patients. Subsequently, at least two intravenous fluid infusion portals are required; one is typically via a central venous catheter. Caution is necessary to avoid even the smallest air bubbles. Shunting lesions allow the passage of venous air into the arterial circulation; paradoxical embolism can occur through the foramen ovale even in patients without obvious right-to-left shunting. Aspiration prior to each injection prevents dislodgment of any trapped air at stopcock injection ports.

Following induction, opioids or inhalation anesthetics are used for maintenance. Fentanyl and sufentanil are the most commonly used intravenous agents, and isoflurane and sevoflurane the most commonly used inhalation agents. Some clinicians choose the anesthetic according to the patient's hemodynamic responses. Isoflurane and sevoflurane may be more suitable than halothane (the most commonly used inhaled agent in years past) for most patients; in equivalent anesthetic doses, halothane causes more myocardial depression, more slowing of the heart rate, but less vasodilation than sevoflurane or isoflurane. However, one can make a sound theoretical argument in favor of halothane over sevoflurane for patients with tetralogy of Fallot (and similarly obstructive lesions such as hypertrophic subaortic stenosis), where myocardial depression is much preferred over vasodilation.

Many centers use modified ultrafiltration after weaning from CPB to partially correct the hemodilution but remove inflammatory vasoactive substances (cytokines); the technique takes blood from the aortic cannula and venous reservoir, passes it through an ultrafilter, and returns it to the right atrium. Surgical correction of complex congenital lesions often requires a period of complete circulatory arrest under deep hypothermia (deep hypothermic circulatory arrest; DHCA). Following institution of CPB, cooling is accomplished by a combination of surface cooling and a cold perfusate. At a core temperature of 15°C, up to 60 min of complete circulatory arrest may be safe. Ice packing around the head is used to delay rewarming and for surface cooling of the brain. Pharmacological brain protection is often attempted with methylprednisolone, 30 mg/kg, and mannitol, 0.5 g/kg. Following the repair, CPB flow is restarted and rewarming takes place.

Because of the large priming volumes used (often 200—300% of the patient's blood volume), hemostatic defects from dilution of clotting factors and platelets are commonly seen after CPB in infants; in addition to heparin reversal, administration of fresh frozen plasma and platelets is often necessary. Patients undergoing extensive or complicated procedures will generally remain intubated. Extubation may be considered for older, relatively healthy patients undergoing simple procedures such as closure of a patent ductus or atrial septal defect or repair of coarctation of the aorta.

Cardiac transplantation is the treatment of choice for otherwise healthy patients with end-stage heart disease so severe that they are unlikely to survive the next 6 to 12 months. The procedure is generally associated with 80% to 90% postoperative survival at 1 year and 60% to 90% survival at 5 years. Transplantation improves quality of life, allowing most patients to resume a relatively normal lifestyle. Unfortunately, the number of cardiac transplants performed is limited by the supply of donor hearts, which are obtained from brain-dead patients, most commonly following intracranial hemorrhage or head trauma.

Proper timing and coordination are necessary between the donor organ retrieval team and the transplant center. Premature induction of anesthesia unnecessarily prolongs the time under anesthesia for the recipient, whereas delayed induction may jeopardize graft function by prolonging the period of ischemia.
Patients may receive little advance warning of the availability of a suitable organ. Many will have eaten a recent meal and should be considered to have a
refloated into
the patient.
position after
CPB
Right ventricular
and failure
is used
from in
conjunction with
TEE
pulmonary hypertension,
to evaluate
a
common post-CPB
problem, can
be treated
with hyperventilation,
prostaglandin
E,
(0.025-0.2 mcg/kg/min), inhaled nitric oxide (10-60 ppm), milrinone, or an RVAD, if necessary. Bleeding is also a common problem.
Patients will be extubated when they meet criteria, as with other major cardiac operations. The postoperative course may be complicated by acute rejection, renal and hepatic dysfunction, and infections.
Many heart failure patients are treated with long-term LVADs, as they do not qualify for heart transplantation. Moreover, there are insufficient hearts available to meet the needs of the heart failure population. Perioperative management concerns of the LVAD patient are similar to those of the heart failure patient as both procedures are surgical interventions to treat heart failure. Patients are routinely scheduled for LVAD placement as elective procedures. Such patients are frequently managed with home milrinone inotropic therapy and often are treated with furosemide infusions to promote diuresis while awaiting surgical intervention. Ideally, LVAD placement and heart transplantation occur before deterioration in hepatic and kidney function.

TEE examination is required perioperatively to rule out the presence of a patent foramen ovale or other conditions that could lead to a right-to-left shunt (eg, atrial septal defects) following LVAD placement. When activated, the LVAD drains blood from the left ventricle and in a nonpulsatile manner pumps it into the aorta (Figure 22-17). Left-sided heart pressures decrease. If right-sided pressures are greater than those of the left heart, venous blood will flow across an atrial septal defect or patent foramen ovale into the left atrium, decreasing arterial oxygen saturation.
FIGURE 22-17 HeartMate II left ventricular assist device. (HeartMate II and St. Jude Medical are trademarks of St. Jude Medical, LLC or its related companies. Reproduced with permission of St. Jude Medical, ©2018. All rights reserved.)
Additionally, TEE examination is necessary to assess right heart function perioperatively. The right ventricle must sufficiently overcome any pulmonary hypertension to deliver an adequate blood volume to the left heart for the LVAD to pump into the aorta. Should the left heart be inadequately filled, the LVAD device will "suck down" the walls of the left ventricle, resulting in dramatically reduced LVAD pump flow.
A temporary RVAD may be needed perioperatively if right ventricular failure ensues. Pulmonary arterial vasodilators (eg, nitric oxide) are used to reduce pulmonary artery pressure and thus decrease the resistance against which the right ventricle must pump.
Various temporary assist devices are available to transiently support ventricular function. Percutaneous devices can be placed in the cardiac catheterization laboratory to support left ventricular function by pumping blood from the left ventricle and ejecting it past the aortic valve into the aorta. Often these devices are employed during percutaneous coronary artery interventions to support ventricular function. Patients in need of emergency coronary artery bypass surgery following failed percutaneous interventions will routinely present to the operating room supported with a percutaneous ventricular assist device (Figure 22-18).
FIGURE 22-18 Impella percutaneous microaxial blood pump. (Reproduced with permission from Abiomed, Inc, Danvers, MA.)

The parietal pericardium is a fibrous membrane surrounding the heart, to which it normally is not adherent. The pericardium encompasses a relatively fixed intrapericardiac volume that includes a small volume of pericardial fluid (20-50 mL in adults), in addition to the heart and blood. As a result, the pericardium normally limits acute dilation of the ventricles and promotes diastolic coupling of the two ventricles (distention of one ventricle interferes with filling of the other). The latter effect is also due to the interventricular septal wall they share. Moreover, diseases of the pericardium or larger pericardial fluid collections can seriously impair cardiac output.
Pericardial effusions may be due to viral, bacterial, or fungal infections;
malignancies; bleeding after cardiac surgery; trauma; uremia; myocardial infarction; aortic dissection; hypersensitivity or autoimmune disorders; drugs; or myxedema.

Cardiac tamponade exists when increased pericardial pressure impairs diastolic filling of the heart. Cardiac filling is ultimately related to the diastolic transmural (distending) pressure across each chamber, and any increase in pericardial pressure relative to the pressure within the chamber reduces filling. Pressure is applied equally to each cardiac chamber when the problem is a pericardial fluid collection; or, it can be applied "selectively," as for example when an isolated pericardial blood clot compresses the left atrium. In general, the thin-walled atria and the right ventricle are more susceptible to pressure-induced abnormalities of filling than the left ventricle.
Pericardial pressure is normally similar to pleural pressure, varying with respiration between —4 and +4 mm Hg. Elevations in pericardial pressure are most commonly due to increases in pericardial fluid volume (as a consequence of effusions or bleeding). The magnitude of the increased pressure depends on both the volume of fluid and the rate of fluid accumulation; sudden increases exceeding 100 to 200 mL precipitously increase pericardial pressure, whereas very slow accumulations up to 1000 mL allow the pericardium to stretch with minimal increases in pericardial pressure.
The principal hemodynamic features of cardiac tamponade include decreased cardiac output from reduced stroke volume with an increase in central venous pressure. In the absence of severe left ventricular dysfunction, equalization of diastolic pressure occurs throughout the heart (right atrial pressure [RAP] = right ventricular end-diastolic pressure [RVEDP] = left atrial pressure [LAP] = left ventricular end-diastolic pressure [LVEDP]).

The central venous pressure waveform is characteristic in cardiac tamponade. Impairment of both diastolic filling and atrial emptying abolishes the y descent; the x descent (systolic atrial filling) is normal or even accentuated. Reflex sympathetic activation is a prominent compensatory response in cardiac tamponade. The resulting increases in heart rate and contractility help maintain cardiac output. Arterial vasoconstriction (increased SVR) supports systemic blood pressure, whereas sympathetic activation reduces vascular capacitance, having the effect of an autotransfusion. Because stroke volume remains relatively fixed, cardiac output becomes primarily dependent on heart rate. Acute cardiac tamponade usually presents as sudden hypotension, tachycardia, and tachypnea. Physical signs include jugular venous distention, a narrowed arterial pulse pressure, and muffled heart sounds. The patient may complain of an inability to lie flat. A prominent pulsus paradoxus (a cyclic inspiratory decrease in systolic blood pressure of more than 10 mm Hg) is typically present. The latter actually represents an exaggeration of a normal phenomenon related to inspiratory decreases in intrathoracic pressure. (A marked pulsus paradoxus may also be seen with severe airway obstruction or right ventricular infarction.) The heart may appear normal or enlarged on a chest radiograph. Electrocardiographic signs are generally nonspecific and are often limited to decreased voltage in all leads and nonspecific ST-segment and T-wave abnormalities. Electrical alternans (a cyclic alteration in magnitude of the P waves, QRS complex, and T waves) may be seen with large pericardial effusions and is thought to be due to pendular swinging of the heart within the pericardium. Generalized ST-segment elevation may also be seen in two or three limb leads as well as V> to Vg in the early phase of pericarditis. A friction rub may be heard by auscultation. Echocardiography is invaluable in diagnosing and measuring pericardial effusions and cardiac tamponade, and as a guide for accurate needle insertion for pericardiocentesis. Signs of tamponade include diastolic compression or collapse of the right atrium and right ventricle, leftward displacement of the ventricular septum, and an exaggerated increase in right ventricular size with a reciprocal decrease in left

Symptomatic cardiac tamponade requires evacuation of the pericardial fluid, either surgically or by pericardiocentesis. The latter is associated with a risk of lacerating the heart or coronary arteries and of pneumothorax. Traumatic postoperative (following thoracotomy) cardiac tamponade is nearly always treated surgically, whereas tamponade from other causes may more often be amenable to pericardiocentesis. Surgical treatment is also often undertaken for large recurrent pericardial effusions (infectious, malignant, autoimmune, uremic, or radiation induced) to prevent tamponade. Simple needle drainage of pericardial fluid may be achieved through a subxiphoid approach, whereas drainage combined with pericardial biopsy or pericardiectomy may be

Constrictive pericarditis may develop as a sequela of acute or recurrent pericarditis. Pathologically, the pericardium is thickened, fibrotic, and often calcified. The parietal pericardium is typically adherent to the visceral pericardium on the heart, often obliterating the pericardial space. The stiffened parietal pericardium limits diastolic filling of the heart to a fixed and reduced volume. In contrast to acute cardiac tamponade, filling during early diastole is typically accentuated and manifested by a prominent y descent on the central venous pressure waveform.
Patients with constrictive pericarditis display jugular venous distention, hepatomegaly, and often ascites. Liver function may be abnormal. In contrast to acute tamponade, constrictive pericarditis prevents respiratory fluctuations in pericardial pressure; because venous return to the heart does not increase during inspiration, a pulsus paradoxus is uncommon. In fact, venous pressure does not fall or may paradoxically rise during inspiration (Kussmaul sign). The chest radiograph will often reveal pericardial calcification. Low QRS voltage and diffuse T-wave abnormalities are usually present on the ECG. Atrial fibrillation and conduction blocks may be present. Echocardiography may be helpful in making the diagnosis.

Pericardiectomy is usually reserved for patients with moderate to severe disease. The procedure is usually performed through a median sternotomy. It is complicated by the necessity for extensive manipulations of the heart that interfere with cardiac filling and ejection, induce frequent arrhythmias, and risk cardiac perforation. CPB may be required.
Selection of specific anesthetic agents is less important than avoiding excessive cardiac depression, vasodilation, and bradycardia. Cardiac output is generally rate dependent. Adequate large-bore intravenous access and direct arterial and central venous pressure monitoring are usually employed. Although cardiac function usually improves immediately following pericardiectomy, some patients display a persistently low cardiac output and require temporary postoperative inotropic support.

Open surgery on the aorta represents a great challenge for anesthesiologists. Regardless of which part of the vessel is involved, the procedure is complicated by the need to cross-clamp the aorta and by the potential for large intraoperative blood losses. Aortic cross-clamping without CPB acutely increases left ventricular afterload and severely compromises organ perfusion distal to the point of occlusion. Severe hypertension, myocardial ischemia, left ventricular failure, or aortic valve regurgitation may be precipitated. Interruption of blood flow to the spinal cord, kidneys, and intestines can produce paraplegia, kidney failure, or intestinal infarction, respectively. Moreover, emergency aortic surgery is frequently necessary in critically ill patients who are acutely hypovolemic and have a high incidence of coexistent cardiac, renal, and pulmonary disease; hypertension; and diabetes. Advances in surgical techniques now permit many aortic lesions to be managed using stents, thereby avoiding many of the challenges presented by open surgery.
Indications for aortic surgery include aortic dissections, aneurysms, occlusive disease, trauma, and coarctation. Lesions of the ascending aorta lie between the aortic valve and the innominate artery, whereas lesions of the aortic arch lie between the innominate and left subclavian arteries. Disease distal to the left subclavian artery but above the diaphragm involves the descending thoracic aorta; lesions below the diaphragm involve the abdominal aorta.

In an aortic dissection an intimal tear allows blood to track into the aortic wall (the media), creating a new pathway for blood flow. In many cases, a primary degenerative process called cystic medial necrosis predisposes for dissection to occur. Patients with hereditary connective tissue defects such as Marfan syndrome and Ehlers—Danlos syndrome eventually develop cystic medial necrosis and are at risk for aortic dissection. Propagation of the dissection is thought to occur as a result of hemodynamic shear forces acting on the intimal tear; indeed, hypertension is a common finding in patients with aortic dissection. Dissection can also occur from hemorrhage into an atheromatous plaque or at the aortic cannulation site following cardiac surgery.
Dissections may occlude the orifice of any artery arising directly from the aorta; they may extend into the aortic root, producing incompetence of the aortic valve; or may rupture into the pericardium or pleura, producing cardiac tamponade or hemothorax, respectively. TEE plays an important role in diagnosing and characterizing aortic dissections. Dissections are most commonly of the proximal type (Stanford type A, De Bakey types I and II) involving the ascending aorta. Type II dissections do not extend beyond the innominate artery. Distal dissections (Stanford type B, De Bakey type III) originate beyond the left subclavian artery and propagate only distally. Proximal dissections are nearly always treated surgically, whereas distal dissections may be treated medically. In either case, from the time the diagnosis is suspected, measures to reduce systolic blood pressure (usually to 90-120 mm Hg) and aortic wall stress are initiated. These measures usually include intravenous vasodilators (nicardipine or nitroprusside) and B-adrenergic blockade (esmolol or a longer acting agent). The latter is important in reducing the shear forces related to the rate of rise of aortic pressure (dP/dt), which may actually increase with nitroprusside alone.

Atherosclerotic obliteration of the aorta most commonly occurs near the aortic bifurcation (Leriche syndrome). Occlusion results from a combination of atherosclerotic plaque and thrombosis. Atherosclerosis is usually generalized and affects other parts of the arterial system, including the cerebral, coronary, and renal arteries. Treatment may be accomplished by intravascular stenting or by open surgery with an aortobifemoral bypass graft; proximal thromboendarterectomy may also be necessary.

Aortic trauma may be penetrating or nonpenetrating. Both types of injuries can result in massive hemorrhage and require immediate operation. Whereas penetrating injuries are usually obvious, blunt aortic trauma may be easily overlooked if not suspected and the appropriate diagnostic testing performed. Nonpenetrating aortic trauma typically results from sudden high-speed decelerations such as those caused by automobile accidents (eg, in which the driver's chest impacts the steering wheel) and falls. The injury can vary from a partial tear to a complete aortic transection. Because the aortic arch is relatively fixed whereas the descending aorta is relatively mobile, the shear forces are greatest and the site of injury most common just distal to the subclavian artery. The most consistent initial finding is a widened mediastinum on a chest radiograph. Definitive diagnosis can be accomplished with magnetic resonance or computed tomographic imaging or TEE.

This congenital heart defect may be classified according to the position of the narrowed segment relative to the position of the ductus arteriosus. In the preductal (infantile) type, the narrowing occurs proximal to the opening of the ductus. This lesion, which is often associated with other congenital heart defects, is recognized in infancy because of a marked difference in perfusion between the upper and lower halves of the body; the lower half is cyanotic. Perfusion to the upper body is derived from the aorta, whereas perfusion to the lower body is primarily from the pulmonary artery. Postductal coarctation of the aorta may not be recognized until adulthood. The symptoms and hemodynamic significance of this lesion depend on the severity of the narrowing and the extent of collateral circulation that develops to the lower body (internal mammary, subscapular, and lateral thoracic to intercostal arteries). Hypertension in the upper body, with or without left ventricular failure, is usually present. So-called "rib notching" may be present on the chest radiograph as a result of dilated collateral intercostal arteries.

Surgery on the ascending aorta routinely uses median sternotomy and CPB, and may also include DHCA. The conduct of anesthesia is similar to that for cardiac operations involving CPB, but the intraoperative course may be complicated by long aortic cross-clamp times and large intraoperative blood losses. TEE is especially useful. Blood loss can be reduced by administration of ¢-aminocaproic acid or tranexamic acid. Concomitant aortic valve replacement and coronary reimplantation are often necessary (Bentall procedure). The radial artery cannulation site should be guided by the possible need for clamping of either the subclavian or innominate arteries during the procedure. Nicardipine or nitroprusside may be used for precise blood pressure control. B-Adrenergic blockade should also be employed in the presence of an aortic dissection. On the other hand, bradycardia worsens aortic regurgitation and should be avoided. The arterial inflow cannula for CPB is placed in a femoral artery for patients with dissections. In the event that sternotomy may rupture an aneurysm, prior establishment of partial CPB (using the femoral artery and femoral vein) should be considered.

These procedures are usually performed through a median sternotomy with DHCA (following institution of CPB). Additional considerations focus on achieving optimal cerebral protection with systemic and topical hypothermia (as noted earlier). Hypothermia to 15°C, drug infusion to maintain a flat EEG, methylprednisolone or dexamethasone, mannitol, and phenytoin are also commonly administered (but there is vanishingly small evidence for efficacy of these drug treatments). The necessarily long rewarming periods probably contribute to the larger intraoperative blood losses commonly observed after CPB.

Surgery limited to the descending thoracic aorta may be performed through a left thoracotomy without CPB, with or without (so-called "clamp-and—run" technique) a heparin-impregnated left ventricular apex to femoral artery shunt; or using partial right atrium to femoral artery bypass. Alternatively, stenting may obviate the need for complex open surgery. A thoracoabdominal incision is necessary for lesions that also involve the abdominal aorta. One-lung anesthesia greatly facilitates surgical exposure. Correct positioning of the endobronchial tube (even with fiberoptic bronchoscopy) may be difficult because of distortion of the anatomy. A double-lumen tube or a regular endotracheal tube with a bronchial blocker may be necessary.
The aorta must be cross-clamped above and below the lesion. Acute hypertension develops above the clamp, with hypotension below when there is no shunt or partial bypass. Arterial blood pressure should be monitored from the right radial artery, as clamping of the left subclavian artery may be necessary. ® The sudden increase in left ventricular afterload after application of the aortic cross-clamp during aortic surgery may precipitate acute left ventricular failure and myocardial ischemia, particularly in patients with underlying ventricular dysfunction or coronary disease; it can also exacerbate preexisting aortic regurgitation. Cardiac output falls and left ventricular end-diastolic pressure and volume rise. The magnitude of these changes is inversely related to ventricular function. These effects can be ameliorated by the use of shunting or partial bypass. Moreover, the adverse effects of aortic clamping become less pronounced the more distal on the aorta that the clamp is applied. A vasodilator infusion is often needed to prevent excessive increases in blood pressure. In patients with good ventricular function, increasing anesthetic depth just prior to cross-clamping may also be helpful.

Excessive intraoperative bleeding may occur during these procedures. Prophylaxis with antifibrinolytic agents may be helpful. A blood scavenging device (cell saver) for autotransfusion is routinely used. Adequate venous access and intraoperative monitoring are critical. Multiple large-bore (14-gauge) intravenous catheters (preferably with blood warmers) are useful. Intraoperative TEE is often used. The period of greatest hemodynamic instability follows the release of the aortic cross-clamp; the abrupt decrease in afterload together with bleeding and the release of vasodilating acid metabolites from the ischemic lower body can precipitate severe systemic hypotension and less commonly hyperkalemia. Decreasing anesthetic depth, volume loading, and partial or slow release of the cross-clamp are helpful in avoiding severe hypotension. A bolus dose of a vasopressor may be necessary. Sodium bicarbonate is often used, particularly for persistent severe metabolic acidosis (pH <7.20) in association with hypotension. Calcium chloride may be necessary when symptomatic hypocalcemia follows massive transfusion of citrated blood products.

perfusion pressure is mean arterial blood pressure minus CSF pressure; the rise in CSF pressure following experimental cross-clamping of the aorta may explain how a mannitol-induced decrease in CSF pressure could improve spinal cord perfusion pressure during cross-clamping. Any protective effect of drainage of CSF via a lumbar catheter may have a similar mechanism.
The excessive use of vasodilators to control the hypertensive response to cross-clamping may be a contributing factor in spinal cord ischemia, as drug actions also occur distal to the cross-clamp. Excessive reduction in blood pressure above the cross-clamp should therefore be avoided to prevent inadequate blood flow and excessive hypotension below it.

An increased incidence of acute kidney failure following aortic surgery is reported after emergency procedures, prolonged cross-clamping periods, and prolonged hypotension, particularly in patients with preexisting kidney disease. A variety of "cocktails" have been employed in the hope of reducing the risk of kidney failure, including infusion of mannitol (0.5 g/kg) prior to cross-clamping, furosemide, and fenoldopam (or low-dose dopamine); however, there is no convincing evidence that these treatments alter renal outcomes.

Stents are most often placed via catheters inserted in a femoral artery. When an open technique is chosen, either an anterior transperitoneal or an anterolateral retroperitoneal approach can be used to access the abdominal aorta. Depending on the location of the lesion, the cross-clamp can be applied to the supraceliac, suprarenal, or infrarenal aorta. Heparin is usually administered prior to aortic clamping. Intraarterial blood pressure can be monitored from either upper extremity. In general, the more distally the clamp is applied to the aorta, the less the effect on left ventricular afterload. In fact, occlusion of the infrarenal aorta frequently results in minimal hemodynamic changes. In contrast, release of the clamp usually produces hypotension; the same techniques that were described earlier may be used to counteract the effects of unclamping. The large incision and extensive retroperitoneal surgical dissection increase fluid requirements beyond intraoperative blood loss. We recommend colloid to maintain intravascular volume and crystalloid for maintenance fluids. Fluid replacement may be guided by monitoring central venous pressure, or better, noninvasive monitors of stroke volume or TEE.
Clamping of the infrarenal aorta decreases renal blood flow, which may contribute to postoperative kidney failure. The decrease in renal blood flow is not prevented by epidural anesthesia or blockade of the renin—angiotensin system. Some centers use continuous epidural anesthesia combined with general anesthesia for abdominal aortic surgery. This combined technique decreases the general anesthetic requirement and appears to suppress the release of stress hormones. It also provides an excellent route for administering postoperative epidural analgesia. Systemic heparinization during surgery introduces concern regarding the risk of paraplegia secondary to an epidural hematoma; however, all credible studies suggest that when the catheter is placed atraumatically well in advance of heparinization and removed after reversal of anticoagulation there is no increased risk of neuraxial hematoma.

Those undergoing stenting may not require intubation either during or after the procedure. Most patients undergoing open surgery on the ascending aorta, the arch, or the thoracic aorta will remain intubated and ventilated for 1 to 24h postoperatively. As with cardiac surgery, the initial emphasis in their postoperative care should be on hemodynamic stability and monitoring for postoperative bleeding. Patients undergoing open abdominal aortic surgery may be extubated at the end of the procedure.

Ischemic cerebrovascular disease accounts for 80% of strokes; the remaining 20% are due to hemorrhage. Ischemic strokes are usually the result of embolism or (less commonly) thrombosis in one of the blood vessels supplying the brain. Ischemic stroke may follow severe vasospasm after subarachnoid hemorrhage. By convention, a stroke is defined as a neurological deficit that lasts more than 24 h; its pathological correlate is typically focal infarction of brain. Transient ischemic attacks (TIAs), on the other hand, are neurological deficits that resolve within 24 h; they may be due to a low-flow state at a tightly stenotic lesion or to emboli that arise from an extracranial vessel or the heart. When a stroke is associated with progressive worsening of signs and symptoms, it is frequently termed a stroke in evolution. A second distinction is also often made between complete and incomplete strokes, based on whether the territory involved is completely
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Indications for surgical interventions include TIAs associated with ipsilateral severe carotid stenosis (>70% occlusion), severe ipsilateral stenosis in a patient with a minor (incomplete) stroke, and 30% to 70% occlusion in a patient with ipsilateral symptoms (usually an ulcerated plaque). In the past, carotid endarterectomy was recommended for asymptomatic but significantly stenotic lesions (>60%). Currently, stenting would be the recommendation. Operative mortality for open surgery is 1% to 4% and is primarily due to cardiac complications (myocardial infarction). Perioperative morbidity is 4% to 10% and is principally neurological; patients with preexisting neurological deficits have the greatest risk of perioperative neurological events. Studies suggest that age greater than 75 years, symptomatic lesions, uncontrolled hypertension, angina, carotid thrombus, and occlusions near the carotid siphon increase operative risk.

Most patients undergoing carotid endarterectomy are elderly and hypertensive, with generalized arteriosclerosis. Many are also diabetic. Preoperative evaluation and management should focus on defining preexisting neurological deficits as well as optimizing the patient's clinical status in terms of coexisting diseases. Most postoperative neurological deficits appear to be related to surgical technique. Uncontrolled perioperative hyperglycemia can increase morbidity by enhancing ischemic cerebral injury.
Patients should receive their usual cardiac medications on schedule until the may
be time of surgery. Blood pressure and the blood glucose concentration should be controlled. Angina should be stable and controlled, and signs of overt congestive heart failure should be absent. Because most patients are elderly, enhanced sensitivity to premedication should be expected.

lidocaine, but the infiltration itself can induce bradycardia. Arterial CO, tension should be maintained in the normal range because hypercapnia can induce intracerebral steal, whereas extreme hypocapnia decreases cerebral perfusion. Ventilation should be adjusted to maintain normocapnia. Maintenance intravenous fluids should consist of glucose-free solutions because of the potentially adverse effects of hyperglycemia. Heparin (5000—7500 units intravenously) is usually administered prior to occlusion of the carotid artery. Some clinicians routinely use a shunt (as noted below). Protamine is usually given to reverse heparin prior to skin closure.
Rapid emergence from anesthesia is desirable because it allows immediate neurological assessment, but the clinician must be prepared to treat hypertension and tachycardia. Postoperative hypertension may be related to surgical denervation of the ipsilateral carotid baroreceptor. Denervation of the carotid body blunts the ventilatory response to hypoxemia. Following extubation, patients should be observed closely for the development of a wound hematoma. When an expanding wound hematoma compromises the airway, the initial treatment maneuver may require opening the wound to release the hematoma. Transient postoperative hoarseness and ipsilateral deviation of the tongue may be noted; they are due to intraoperative retraction of the recurrent laryngeal or hypoglossal nerves, respectively.

Unless regional anesthesia is used, indirect methods must be relied upon to assess the adequacy of cerebral perfusion during carotid cross-clamping. Some surgeons routinely use a shunt, but this practice may increase the incidence of postoperative neurological deficits; shunt insertion can dislodge emboli. Carotid stump pressure distal to the cross-clamp, EEG, somatosensory evoked potentials (SSEPs), and cerebral oximetry have been used in some centers to determine whether a shunt is needed. A distal stump pressure of less than 50 mm Hg has traditionally been used as an indication for a shunt. Electrophysiological signs of ischemia (or a marked decline in cerebral oxygen saturation) after crossclamping dictate the use of a shunt; changes lasting more than 10 min may be associated with a new postoperative neurological deficit. Although multichannel recordings and computer processing can enhance the sensitivity of the EEG, neither EEG nor SSEP monitoring is sufficiently sensitive or specific to reliably predict the need for shunting or the occurrence of postoperative deficits (See case discussion in Chapter 26). Other techniques, including measurements of regional cerebral blood flow with radioactive xenon-133, transcranial Doppler measurement of middle cerebral artery flow velocity, cerebral oximetry, jugular venous oxygen saturation, and transconjunctival oxygen tension, are also not sufficiently reliable.

Carotid surgery may be performed under regional anesthesia. Blockade of the superficial cervical plexus effectively blocks the C2—C4 nerves and allows the patient to remain comfortably awake during surgery. Deep cervical plexus block is not required. A substantial fraction of patients will require administration of local anesthetic by the surgeon into the carotid sheath (whether or not a deep cervical block is performed). The principal advantage of regional anesthesia (and it is a tremendous advantage) is that the patient can be examined intraoperatively; thus, the need for a temporary shunt can be assessed and any new neurological deficits diagnosed immediately during surgery. In fact, intraoperative neurological examination may be the most reliable method for assessing the adequacy of cerebral perfusion during carotid cross-clamping. The examination minimally consists of level of consciousness, speech, and contralateral handgrip. Experienced clinicians use minimal sedation and "cocktail conversation" with the patient to monitor the neurological status. Some studies also suggest that when compared with general anesthesia, regional anesthesia results in more stable hemodynamics but outcomes appear similar. Regional anesthesia for carotid surgery requires the cooperation of the surgeon and patient.

A 55-year-old man with new-onset atrial fibrillation is scheduled for elective cardioversion.
What are the indications for an elective cardioversion?
Direct current (DC) cardioversion may be used to terminate supraventricular and ventricular tachyarrhythmias caused by reentry. It is
not effective for arrhythmias from enhanced automaticity (multifocal atrial tachycardia) or triggered activity (digitalis toxicity). By simultaneously depolarizing the entire myocardium and possibly prolonging the refractory period, DC cardioversion can terminate atrial fibrillation and flutter, atrioventricular nodal reentry, reciprocating tachycardias from preexcitation syndromes, and ventricular tachycardia or fibrillation.
Specific indications for cardioversion of patients with atrial fibrillation include symptomatic fibrillation, recent onset, and no response to medications. Patients with long-standing fibrillation, a large atrium, chronic obstructive lung disease, congestive heart failure, or mitral regurgitation have a high recurrence rate. A TEE is usually performed shortly before cardioversion to rule out left atrial blood clots. Such clots are typically located in the left atrial appendage and can be embolized by the cardioversion procedure or by sinus rhythm.
Emergency cardioversion is indicated for any tachyarrhythmia associated with hypotension, congestive heart failure, or angina.

Although the procedure is usually performed by cardiologists, the need for immediate cardioversion may arise in the operating room, ICU, or during cardiopulmonary resuscitation. Anesthesiologists must therefore be familiar with the technique. Following deep sedation or light general anesthesia, DC shock is applied by either self-adhesive pads or 8to 13-cm paddles. Larger paddles help reduce any shock-induced myocardial necrosis by distributing the current over a wider area. The energy output should be kept at the minimally effective level to prevent myocardial damage. Placement of the electrodes can be anterolateral or anteroposterior. In the first position, one electrode is placed on the right second intercostal space next to the sternum and the other is placed on the left fifth intercostal space in the midclavicular line. When pads are used for the anteroposterior technique, one is placed anteriorly over the ventricular apex in the fifth intercostal space and the other undermeath the patient in the left infrascapular region.
For supraventricular tachycardias, with the notable exception of atrial fibrillation, energy levels of 25 to 50 J can successfully reestablish normal sinus rhythm. Synchronized shocks should be used for all tachyarrhythmias except ventricular fibrillation. Synchronization times the delivery so that it is given during the QRS complex. If the shock occurs in the ST segment or the T wave (unsynchronized), it can precipitate a more serious arrhythmia, including ventricular fibrillation. All medical personnel should stand clear of the patient and the bed during the shock.
Atrial fibrillation usually requires a minimum of 50 to 100 J, and larger energy levels are often used. Hemodynamically stable ventricular tachycardia can often be terminated with 25 to 50 J, but ventricular fibrillation and unstable ventricular tachycardia require 200 to 360 J. Regardless of the arrhythmia, a higher energy level is necessary when the first shock is ineffective.
The cardiologist wants to do the cardioversion in the postanesthesia care unit (PACU). Is this an appropriate place for cardioversion?

Elective cardioversion can be performed in any setting in which full provisions for cardiopulmonary resuscitation, including cardiac pacing capabilities, are immediately available. A physician skilled in airway management should be in attendance. Cardioversions are commonly performed in an ICU, emergency department, PACU, procedure room, or cardiac catheterization suite.

The patient should be fasted, evaluated, and treated as though he were receiving a general anesthetic in the operating room. An ECG is performed immediately before the procedure to confirm that the arrhythmia is still present; another is performed immediately afterward to confirm the new rhythm. Preoperative laboratory values should be within normal limits because metabolic disorders, particularly electrolyte and acid—base abnormalities, may contribute to the arrhythmia. If not corrected preoperatively, they can reinitiate the tachycardia following cardioversion. An antiarrhythmic agent is often started in patients with atrial fibrillation 1 to 2 days prior to the procedure to help maintain normal sinus rhythm. Patients with atrial fibrillation of longer than a few hours duration likely will have been anticoagulated for a sufficient time prior to cardioversion to reduce the likelihood of a left atrial thrombus. Such clots may embolize upon restoration of a sinus rhythm.
What are the minimum monitors and anesthetic equipment required?
Minimum monitoring consists of the ECG, blood pressure, and pulse oximetry. A precordial stethoscope is useful for monitoring breath sounds. Maintaining continuous verbal contact with the patient may the best method for assessing whether a sufficient amnestic dose of (usually) propofol has been given.
In addition to a DC defibrillator capable of delivering up to 400 J (synchronized or unsynchronized) and transcutaneous pacing, the minimum equipment should include the following:
¢ Reliable intravenous access.
¢ A functional bag-mask device capable of delivering 100% oxygen (see Chapter 3).
¢ An oxygen source from a wall outlet or a full tank.
¢ An airway kit with oral and nasal airways and appropriate laryngoscopes and endotracheal tubes.
¢ A functional suction apparatus.
¢ An anesthetic drug kit that includes at least one sedative-hypnotic as well as succinylcholine.
* A cart that includes all necessary drugs and equipment for cardiopulmonary resuscitation (see Chapter 55).

Premedication is not necessary. Only very brief (1-2 min) light general anesthesia is required. A short-acting agent such as propofol or methohexital is customarily used. Etomidate may be used but may be associated with phonation. Following preoxygenation with 60% to 100% oxygen for 3 to 5 min, the sedative-hypnotic is given in small increments) every 30 to 60 s while maintaining verbal contact with the patient. The shock is delivered when the patient is no longer able to respond verbally; some clinicians use loss of the eyelid reflex as an end point. The shock usually arouses the patient. Transient airway obstruction or apnea may be observed, particularly if more than one shock is necessary.

Complications include transient myocardial depression, postshock arrhythmias, and arterial embolism. Arrhythmias are usually due to inadequate synchronization, but even a properly timed cardioversion can occasionally result in ventricular fibrillation. Most arrhythmias are transient and resolve spontaneously. Although patients may develop ST-segment elevation, serum creatine phosphokinase levels (MB fraction) are usually normal. Embolism of a left-sided clot may be responsible for delayed awakening.
How should the patient be cared for following cardioversion?
Although recovery of consciousness is usually very rapid, patients should be treated like others receiving general anesthesia (See Chapter 56). Recovery also specifically includes monitoring for both recurrence of the arrhythmia and signs of cerebral embolism.

Hiratzka LF, Bakris GL, Beckman JA, et al. 2010
ACCF/AHA/AATS/ACR/ASA/SCA/SCAI/SIR/STS/SVM
diagnosis and
management of
patients with
thoracic aortic
summary:
A
report of
the
American
Foundation/American
American
Association
American
Heart
College
Association for
Thoracic
Stroke of
Cardiology
Task
Surgery,
Association,
Society for
Society
Force on
Practice
American
Guidelines for
the disease:
Executive
Guidelines,
College of
Angiography of
Radiology,
Anesthesiologists, and
Interventions,
Interventional
Society of
Thoracic
Society of
Surgeons, and
Vascular Medicine. Anesth Analg. 2010;111:279.
American Society of Extracorporeal Technology Standards and Guidelines for Perfusion Practice (11/08/2013). http://www.amsect.org/page/standards-andguidelines-1117
See www.guidelines.gov for additional guidelines from multiple organizations related to these topics.

Engelman R, Baker RA, Likosky DS, et al. The Society of Thoracic Surgeons,
The
Society of
The
American
Society of ExtraCorporeal Technology: Clinical Practice Guidelines for
Cardiopulmonary Bypass—Temperature management during cardiopulmonary bypass. J Extra Corpor Technol. 2015;47:145.
Fedorow CA, Moon MC, Mutch WA, Grocott HP. Lumbar cerebrospinal fluid drainage for thoracoabdominal aortic surgery: Rationale and practical
considerations for management. Anesth Analg. 2010;111:46.

@ The trachea serves as a conduit for ventilation and for clearance of tracheal and bronchial secretions and has an average length of 10 to 13 cm. The trachea bifurcates at the carina into the right and left mainstem bronchi. The right mainstem bronchus lies in a more linear arrangement with the trachea, whereas the left mainstem bronchus lies in a more angular orientation with the trachea.
@ The periodic exchange of alveolar gas with the fresh gas from the upper airway reoxygenates desaturated blood and eliminates CO,. This exchange is normally brought about by small cyclic pressure gradients established within the airways. During spontaneous ventilation, these gradients are secondary to variations in intrathoracic pressure; during mechanical ventilation, they are produced by intermittent positive pressure in the upper airway.
@ The lung volume at the end of a normal exhalation is called functional residual capacity (FRC). At this volume, the inward elastic recoil of the lung approximates the outward elastic recoil of the chest (including resting diaphragmatic tone).
@ Closing capacity is normally well below FRC, but it rises steadily with age. This increase is probably responsible for the normal age-related decline in arterial O5 tension.
@ Whereas both forced expiratory volume in the first second of exhalation (FEV,) and forced vital capacity (FVC) are effort dependent, forced midexpiratory flow (FEF>>_750,) is more effort independent and may be a more reliable measure of obstruction.
@ Changes in lung mechanics due to general anesthesia occur shortly after induction. The supine position reduces the FRC by 0.8 to 1.0 L, and

The importance of pulmonary physiology to anesthetic practice is obvious. The commonly used inhalation anesthetics depend on the lungs for uptake and elimination. Both inhalation and intravenously administered anesthetics produce prominent respiratory side effects. Moreover, muscle paralysis, unusual positioning during surgery, and techniques such as one-lung anesthesia and cardiopulmonary bypass profoundly alter normal pulmonary physiology. This chapter reviews the basic pulmonary concepts necessary for understanding and applying anesthetic techniques. Although the pulmonary effects of each of the various anesthetic agents are discussed elsewhere in the book, this chapter also reviews the overall effects of general anesthesia on lung function.

The rib
cage contains
the two
lungs, each
surrounded apex
of the
chest is
small, blood
vessels, whereas
diaphragm—the allowing
the principal
cavity to
descend
Diaphragmatic only
for base
is formed
pulmonary
1.5
to
7
movement volume.
Accessory expansion)
by entry
its own
of the
trachea, by
the diaphragm.
muscle—causes cm
and its
contents normally
respiratory by
their articulates
anteriorly
During intercostal
pleura.
The esophagus,
Contraction the
base
(the lungs)
accounts muscles
action on
for
75%
also to
and of
the of
the thoracic
expand.
of increase
the posteriorly
with to
the sternum.
ribs.
Each
a
vertebra
Upward normal
breathing, muscles,
passive.
With muscles
and the
chest rib
(except is
change volume
in chest
(and for
angulated and
outward the
diaphragm, are
responsible increasing
can be
the last
downward rib
movement and,
to lung
two)
as it
attaches expands
a
lesser extent,
for inspiration;
effort, expiration
the recruited
assist in
elevating displacement
the during
rib sternocleidomastoid,
inspiration.
cage, of
the the
chest.
the external
is generally
scalene, and
The pectoralis
sternocleidomastoid whereas
upper assist
chest expansion
normally passive
position ribs
when the
scalene during
muscles muscles
prevent inward
the in
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supine with
abdominal increased
muscles arms
The are
placed pectoralis
on position,
a
but muscles
fixed becomes
effort.
(rectus
Exhalation abdominis,
may support.
active can
Expiration in
the upright
be external
and facilitated
internal by

the oblique,
and is
transversus) and perhaps the internal intercostal muscles—aiding the downward movement of the ribs.
Although not usually considered respiratory muscles, some pharyngeal muscles are important in maintaining the patency of the airway. Tonic and reflex inspiratory activity in the genioglossus keeps the tongue away from the posterior pharyngeal wall. Tonic activity in the levator palati, tensor palati, palatopharyngeus, and palatoglossus prevents the soft palate from falling back against the posterior pharynx, particularly in the supine position.

@ The trachea serves as a conduit for ventilation and for clearance of tracheal and bronchial secretions. The trachea begins at the lower border of the cricoid cartilage, extends to the carina, and has an average length of 10 to 13 cm. It is composed of C-shaped cartilaginous rings, which form the anterior and lateral walls of the trachea and are connected posteriorly by the membranous wall of the trachea. The cricoid cartilage is the narrowest part of the trachea, with an average diameter of 17 mm in men and 13 mm in women.
The tracheal lumen narrows slightly as it progresses toward the carina, where it bifurcates into the right and left mainstem bronchi at the level of the sternal angle. The right mainstem bronchus lies in a more linear arrangement with the trachea, whereas the left mainstem bronchus lies in a more angular orientation with the trachea. The right mainstem bronchus continues as the bronchus intermedius after the take-off of the right upper lobe bronchus. The distance from the tracheal carina to the take-off of the right upper lobe bronchus is an average of 2.0 cm in men and approximately 1.5 cm in women. One in every 250 individuals in the general population may have an abnormal take-off of the right upper lobe bronchus emerging from above the tracheal carina on the right side. The left mainstem bronchus is longer than the right mainstem bronchus and measures an average of 5.0 cm in men and 4.5 cm in women. The left mainstem bronchus divides into the left upper lobe bronchus and the left lower lobe bronchus.

Humidification and filtering of inspired air are functions of the upper airway (nose, mouth, and pharynx). The tracheobronchial tree serves to conduct gas flow to and from the alveoli. Dichotomous division (each branch dividing into two smaller branches), starting with the trachea and ending in alveolar sacs, is estimated to involve 23 divisions, or generations (Figure 23-1). With each generation, the number of airways is approximately doubled. Each alveolar sac contains, on average, 17 alveoli. An estimated 300 to 500 million alveoli provide an enormous membrane surface area (50-100 m2) for gas exchange in the average adult.
A
FIGURE 23-1 A: Dichotomous division of the airways. (Reproduced with permission from Guyton AC. Textbook of Medical Physiology. 7th ed. Philadelphia, PA: WB Saunders; 1986.) B: The segmental bronchi. (Reproduced with permission from Minnich DJ, Mathisen DJ. Anatomy of the trachea, carina, and bronchi. Thorac Surg Clin. 2007 Nov;17(4):571-585.)
With each successive division, the mucosa makes a gradual transition from ciliated columnar to cuboidal and finally to flat alveolar epithelium. Gas exchange can occur only across the flat epithelium, which begins to appear on respiratory bronchioles (generations 17-19). The wall of the airway gradually loses its cartilaginous support (at the bronchioles) and then its smooth muscle. Loss of cartilaginous support causes the patency of smaller airways to become dependent on radial traction by the elastic recoil of the surrounding tissue; as a corollary, airway diameter becomes dependent on total lung volume.
Cilia on the columnar and cuboidal epithelium normally beat in a synchronized fashion, such that mucus produced by the secretory glands lining the airway (and any associated bacteria or debris) moves toward the mouth.

Alveolar size is a function of both gravity and lung volume. In the upright position, the largest alveoli are at the pulmonary apex, whereas the smallest tend to be at the base. With inspiration, discrepancies in alveolar size diminish. Each alveolus is in close contact with a network of pulmonary capillaries. The walls of each alveolus are asymmetrically arranged (Figure 23-2). Gas exchange occurs primarily on the thin side of the alveolocapillary membrane, which is less than 0.4 pm thick. The thick side (1-2 pm) provides structural support for the alveolus. On the thin side, the alveolar epithelium and capillary endothelium are separated by their respective cellular and basement membranes; on the thick side, where fluid and solute exchange occurs, the pulmonary interstitial space separates alveolar epithelium from capillary endothelium. The pulmonary interstitial space contains mainly elastin, collagen, and nerve fibers.
FIGURE 23-2 The pulmonary interstitial space, with a capillary passing between the two alveoli. The capillary is incorporated into the thin (gasexchanging) side of the alveolus on the right. The interstitial space is incorporated into the thick side of the alveolus on the left. (Reproduced with permission from Nunn JF. Nunn's Applied Physiology. 4th ed. Oxford, UK: Butterworth; 2000.)

The pulmonary epithelium contains at least two cell types. Type I pneumocytes are flat and form tight (1-nm) junctions with one another. These tight junctions are important in preventing the passage of large oncotically active molecules such as albumin into the alveolus. Type II pneumocytes, which are more numerous than type I pneumocytes (but because of their shape occupy <10% of the alveolar space), are round cells that contain prominent cytoplasmic inclusions (lamellar bodies). These inclusions contain surfactant, an important substance necessary for normal pulmonary mechanics. Unlike type I cells, type II pneumocytes are capable of cell division and can produce type I pneumocytes if the latter are destroyed. Other cell types present in the lower airways include pulmonary alveolar macrophages, mast cells, lymphocytes, and amino precursor uptake and decarboxylation (APUD) cells. Neutrophils are also typically present in smokers and patients with pneumonia or acute lung injury.

The lungs are supplied by two circulations, pulmonary and bronchial. The bronchial circulation arises from the left heart and sustains the metabolic needs of the tracheobronchial tree. The bronchial circulation provides a small amount of blood flow (ie, <4% of the cardiac output). Branches of the bronchial artery supply the wall of the bronchi and follow the airways as far as the terminal bronchioles. Along their courses, the bronchial vessels anastomose with the pulmonary arterial circulation and continue as far as the alveolar duct. Below that level, lung tissue is supported by a combination of the alveolar gas and pulmonary circulation.
The pulmonary circulation normally receives the total output of the right heart via the pulmonary artery, which divides into right and left branches to supply each lung. Deoxygenated blood passes through the pulmonary capillaries, where O, is taken up and CO, is eliminated. The oxygenated blood is then returned to the left heart by four main pulmonary veins (two from each lung). Although flows through the systemic and pulmonary circulations are equal, the lower pulmonary vascular resistance results in pulmonary pressures that are less than those in the systemic vessels; as a result, both pulmonary arteries and veins normally have thinner walls than systemic vessels with less smooth muscle. There are connections between the bronchial and the pulmonary circulations. Direct pulmonary arteriovenous communications, bypassing the pulmonary capillaries, are normally insignificant but may become important in certain pathological states. The importance of the bronchial circulation in contributing to the normal venous admixture is discussed below.

Pulmonary capillaries are incorporated into the walls of alveoli. The average diameter of these capillaries (about 10 pm) is barely enough to allow passage of a single red cell. Because each capillary network supplies more than one alveolus, blood may pass through several alveoli before reaching the pulmonary veins. Because of the relatively low pressure in the pulmonary circulation, the amount of blood flowing through a given capillary network is affected by both gravity and alveolar size. Large alveoli have a smaller capillary cross-sectional area and consequently increased resistance to blood flow. In the upright position, apical capillaries tend to have reduced flows, whereas basal capillaries have higher flows.
The pulmonary capillary endothelium has relatively large junctions (5 nm wide), allowing the passage of large molecules such as albumin. As a result, pulmonary interstitial fluid is relatively rich in albumin. Circulating macrophages and neutrophils are able to pass through the endothelium, as well as the smaller alveolar epithelial junctions, with relative ease. Pulmonary macrophages are commonly seen in the interstitial space and inside alveoli; they serve to scavenge bacteria and debris.

Lymphatic channels in the lung originate in the interstitial spaces of large septa and are close to the bronchial arteries. Bronchial lymphatics return fluids, proteins, and various cells that have entered the peribronchovascular interstitium back to the blood circulation. Because of the large endothelial junctions, pulmonary lymph has a relatively high protein content, and total pulmonary lymph flow may be as much as 20 mL/h. Large lymphatic vessels travel upward alongside the airways, forming the tracheobronchial chain of lymph nodes. Lymphatic drainage channels from both lungs communicate along the trachea.

The diaphragm is innervated by the phrenic nerves, which arise from the C3—C5 nerve roots. Unilateral phrenic nerve block or palsy only modestly reduces most indices of pulmonary function (about 25%) in normal subjects. Although bilateral phrenic nerve palsies produce more severe impairment, accessory muscle activity may maintain adequate ventilation in some patients. Intercostal muscles are innervated by their respective thoracic nerve roots. Cervical cord injuries above C5 are incompatible with spontaneous ventilation because both phrenic and intercostal nerves are affected.
The vagus nerves provide sensory innervation to the tracheobronchial tree. Both sympathetic and parasympathetic autonomic innervation of bronchial smooth muscle and secretory glands is present. Vagal activity mediates bronchoconstriction and increases bronchial secretions via muscarinic receptors. Sympathetic activity (T1-T4) mediates bronchodilation and also decreases secretions via B,-receptors. The nerve supply of the larynx is reviewed in Chapter 19.
Both aand B-adrenergic receptors are present in the pulmonary vasculature, but the sympathetic system normally has little effect on pulmonary vascular tone. a, Activity causes vasoconstriction; B, activity mediates vasodilation. Parasympathetic vasodilatory activity seems to be mediated via the release of nitric oxide.

® The periodic exchange of alveolar gas with the fresh gas from the upper airway reoxygenates desaturated blood and eliminates CO,. This exchange is normally brought about by small cyclic pressure gradients established within the airways. During spontaneous ventilation, these gradients are secondary to variations in intrathoracic pressure; during mechanical ventilation, they are produced by intermittent positive pressure in the upper airway. During apneic oxygenation gas exchange depends on mass movement of gases along concentration gradients.

Normal pressure variations during spontaneous breathing are shown in Figure 23-3. The pressure within alveoli is always greater than the surrounding (intrathoracic) pressure unless the alveoli are collapsed. Alveolar pressure is normally atmospheric (zero for reference) at end-inspiration and end-expiration. By convention, in pulmonary physiology, pleural pressure is used as a measure of intrathoracic pressure. Although it may not be entirely correct to refer to the pressure in a potential space, the concept allows the calculation of transpulmonary pressure. Transpulmonary pressure, or P. is then transpulmonary? defined as follows:
FIGURE 23-3 Changes in intrapleural and alveolar pressures during normal breathing. Note that at end inspiration, volume is maximal; flow is zero; and alveolar pressure is atmospheric. (Adapted with permission from West JB. Respiratory Physiology—The Essentials, 6th ed. Philadelphia, PA: Williams & Wilkins; 2000.)
P
P
P
transpulmonary
=
_
Falveolar intrapleural
At end-expiration, intrapleural pressure normally averages about —5 cm H,O, and because alveolar pressure is 0 (no flow), transpulmonary pressure is +5 cm H,O.
Diaphragmatic and intercostal muscle activation during inspiration expands the chest and decreases intrapleural pressure from —5 cm H,O to -8 or —9 cm H,O. As a result, alveolar pressure also decreases and an alveolar—upper airway gradient is established; gas flows from the upper airway into alveoli (Figure 234).
A: End-expiration
B:
During inspiration

FIGURE 23-4 Representation of the interaction of the lung and chest wall. A: At end-expiration, the muscles of respiration are relaxed. The inward elastic recoil of the lung is balanced by the outward elastic recoil of the chest wall. Intrapleural pressure is -5 cm H,O; alveolar pressure is 0. The transmural pressure difference across the alveolus is therefore 0 cm H,O -—(-5 cm H,0), or 5 cm H,O. Since alveolar pressure is equal to atmospheric pressure, no airflow occurs. B: During inspiration, contraction of the muscles of inspiration causes intrapleural pressure to become more negative. The transmural pressure difference increases and the alveoli are distended, decreasing alveolar pressure below atmospheric pressure, which causes air to flow into the alveoli. (Reproduced with permission from Levitzky MG. Pulmonary Physiology. 8th ed. New York, NY: McGraw-Hill Education; 2013.)
During expiration, diaphragmatic relaxation returns intrapleural pressure to — 5 cm H,O. Now the transpulmonary pressure does not support the new lung volume, and the elastic recoil of the lung causes a reversal of the previous alveolar—upper airway gradient; gas flows out of alveoli, and original lung volume is restored.

Most forms of mechanical ventilation intermittently apply positive airway pressure at the upper airway. During inspiration, gas flows into alveoli until pressure in the alveoli reaches that in the upper airway. During the expiratory phase of the ventilator, the positive airway pressure is decreased; the gradient reverses, allowing gas flow out of alveoli.

The movement of the lungs is passive and determined by the impedance of the respiratory system, which can be divided into the elastic resistance of tissues and the gas-liquid interface and the nonelastic resistance to gas flow. Elastic resistance governs lung volume and the associated pressures under static conditions (no gas flow). Resistance to gas flow relates to frictional resistance to airflow and tissue deformation.

Both the lungs and the chest have elastic properties. The chest has a tendency to expand outward, whereas the lungs have a tendency to collapse. When the pleural space is exposed to atmospheric pressure (open pneumothorax), the chest usually expands about 1 L in adults. In contrast, when the pleural space is exposed to atmospheric pressure, the lung collapses completely and all the gas within it is expelled. The recoil properties of the chest are due to structural components that resist deformation and chest wall muscle tone. The elastic recoil of the lungs is due to their high content of elastin fibers, and, even more important, the surface tension forces acting at the air—fluid interface in alveoli.

The gas—fluid interface lining the alveoli causes them to behave as bubbles. Surface tension forces tend to reduce the area of the interface and favor alveolar collapse. Laplace's law can be used to quantify these forces:
2x Surface tension Pressure = - Radius
The pressure derived from the equation is that within the alveolus and it is directly proportional to surface tension. Fortunately, pulmonary surfactant decreases alveolar surface tension in proportion to its concentration within the alveolus. As alveoli become smaller, the surfactant within becomes more concentrated, and surface tension is more effectively reduced. Conversely, when alveoli are overdistended, surfactant becomes less concentrated, and surface tension increases. The net effect is to stabilize alveolar size: small alveoli are prevented from getting smaller, whereas large alveoli are prevented from getting larger.

Elastic recoil is usually measured in terms of compliance (C), which is defined as the change in volume divided by the change in distending pressure. Compliance measurements can be obtained for either the chest, the lung, or both together In the supine position, chest wall compliance (Cw) is reduced because of the weight of the abdominal contents against the diaphragm. Measurements are usually obtained under static conditions, (ie, at equilibrium). (Dynamic lung compliance [Cdyn,L], which is measured during rhythmic breathing, is also dependent on airway resistance.) Lung compliance (Cl) is defined as:
CL is normally 150 to 200 mL/cm H,O. A variety of factors, including lung volume, pulmonary blood volume, extravascular lung water, and pathological processes (eg, inflammation and fibrosis) affect CL,
where transthoracic pressure equals atmospheric pressure minus intrapleural pressure.
Normal chest wall compliance is 200 mL/cm H,O. Total compliance (lung and chest wall together) is 100 mL/cm H,O and is expressed by the following equation:

Lung volumes are important parameters in respiratory physiology and clinical practice (Table 23—1 and Figure 23-5). The sum of all of the named lung volumes equals the maximum to which the lung can be inflated. Lung capacities are clinically useful measurements that represent a combination of two or more volumes.

Tidal volume (V1), Definition = Each normal breath. Tidal volume (V1), Average Adult Values (mL) = 500. Inspiratory reserve volume (IRV), Definition = Maximal additional volume that can be inspired above Vt. Inspiratory reserve volume (IRV), Average Adult Values (mL) = 3000. Expiratory reserve volume (ERV), Definition = Maximal volume that can be expired below Vt. Expiratory reserve volume (ERV), Average Adult Values (mL) = 1100. Residual volume (RV), Definition = Volume remaining after maximal exhalation. Residual volume (RV), Average Adult Values (mL) = 1200. Total lung capacity (TLC), Definition = RV + ERV + V1 +IRV. Total lung capacity (TLC), Average Adult Values (mL) = 5800. Functional residual capacity (FRC), Definition = RV + ERV. Functional residual capacity (FRC), Average Adult Values (mL) = 2300
FIGURE 23-5 Spirogram showing static lung volumes. (Modified with permission from Lumb A. Nunn's Applied Respiratory Physiology. 8th ed. St. Louis, MO: Elsevier; 2017.)

® The lung volume at the end of a normal exhalation is called functional residual capacity (FRC). At this volume, the inward elastic recoil of the lung approximates the outward elastic recoil of the chest (including resting diaphragmatic tone). Thus, the elastic properties of both chest and lung define the point from which normal breathing takes place. FRC can be measured by nitrogen washout, helium washin technique, or body plethysmography. Factors known to alter the FRC include the following:
* Body habitus: FRC is directly proportional to height. Obesity, however, can markedly decrease FRC (primarily as a result of reduced chest compliance).
¢ Sex: FRC is reduced by about 10% in females compared with males. Pregnancy is also associated with a reduction in FRC.
¢ Posture: FRC decreases as a patient is moved from an upright to a supine or prone position. This is the result of reduced chest compliance as the abdominal contents push up against the diaphragm. The greatest change occurs between 0° and 60° of inclination. No further decrease is observed with a head-down position of up to 30°.
¢ Lung disease: Decreased compliance of the lung, chest, or both is characteristic of restrictive pulmonary disorders all of which are associated with a low FRC.
¢ Diaphragmatic tone: This normally contributes to FRC, and its contribution is evident with phrenic nerve paralysis.

As described earlier, small airways lacking cartilaginous support depend on radial traction caused by the elastic recoil of surrounding tissue to keep them open; patency of these airways, particularly in basal areas of the lung, is highly dependent on lung volume. The volume at which these airways begin to close in dependent areas of the lung is called the closing capacity. At lower lung volumes, alveoli in dependent areas continue to be perfused but are no longer ventilated; the resulting intrapulmonary shunting of deoxygenated blood (venous admixture) promotes hypoxemia.
Closing capacity is usually measured with 100% oxygen, which is inhaled near residual volume and then exhaled from total lung capacity. Changes in the nitrogen concentration of the exhaled gas following oxygen inhalation are measured. The nitrogen concentration from alveoli at the base of the lungs is lower than that at the apex because any air remaining in the lungs at the residual volume is in the nondependent alveoli. Closing volume is discerned when the concentration of expired nitrogen rises, indicating that the alveoli from the dependent regions of the lung are no longer contributing to the exhaled gas because their airways have closed (Figure 23-6).
FIGURE 23-6 Expired nitrogen concentration after inhalation of a single breath of 100% oxygen from the residual volume to the total lung capacity. Subject exhales to the residual volume. Phase I: 0% nitrogen from anatomic dead space. Phase II: mixture of gas from anatomic dead space and alveoli. Phase III: "alveolar plateau" gas from alveoli. A steep slope of phase III indicates nonuniform distribution of alveolar gas. Phase IV: closing volume. Take-off point (closing capacity) of phase IV denotes beginning of airway closure in dependent portions of the lung. (Reproduced with permission from Levitzky MG. Pulmonary Physiology. 8th ed. New York, NY: McGraw-Hill Education; 2013.)

®@ Closing capacity is normally well below FRC (Figure 23-7), but rises steadily with age (Figure 23-8). This increase is probably responsible for the normal age-related decline in arterial O, tension. At an average age of 44 years, closing capacity equals FRC in the supine position; by age 66, closing capacity equals or exceeds FRC in the upright position in most individuals. Unlike FRC, closing capacity is unaffected by posture. Closing capacity also approaches or exceeds FRC in morbid obesity.
FIGURE 23-7 The relationship between functional residual capacity, closing volume, and closing capacity. (Reproduced with permission from Lumb A. Nunn's Applied Respiratory Physiology. 8th ed. St. Louis, MO: Elsevier; 2017.)
FIGURE 23-8 The effect of age on closing capacity and its relationship to functional residual capacity (FRC). Note that FRC does not change. (Reproduced with permission from Lumb A. Nunn's Applied Respiratory Physiology. 8th ed. St. Louis, MO: Elsevier; 2017.)

Vital capacity (VC) is the maximum volume of gas that can be exhaled following maximal inspiration. In addition to body habitus, VC is also dependent on respiratory muscle strength and chest—lung compliance. Normal VC is about 60 to 70 mL/kg.

Gas flow in the lung is a mixture of laminar and turbulent flow. Laminar flow can be thought of as consisting of concentric cylinders of gas flowing at different velocities; velocity is greatest in the center and decreases toward the periphery. During laminar flow,
where R,,, is airway resistance.
Turbulent flow is characterized by random movement of the gas molecules down the air passages. Mathematical description of turbulent flow is considerably more complex:
Resistance is not constant but increases in proportion to gas flow. Moreover, resistance is directly proportional to gas density and inversely proportional to the fifth power of the radius. As a result, turbulent gas flow is extremely sensitive to airway caliber.
Turbulence generally occurs at high gas flows, at sharp angles or branching points, and in response to abrupt changes in airway diameter. Whether turbulent or laminar flow occurs can be predicted by the Reynolds number, which results from the following equation:

A low Reynolds number (<1000) is associated with laminar flow, whereas a high value (>1500) produces turbulent flow. Laminar flow normally occurs only distal to small bronchioles (<1 mm). Flow in larger airways is probably turbulent. Of the gases used clinically, only helium has a significantly lower density-to-viscosity ratio, making it useful clinically during severe turbulent flow (as caused by upper airway obstruction). A helium—O, mixture not only is less likely to cause turbulent flow but also reduces airway resistance when turbulent flow is present.
Normal total airway resistance is about 0.5 to 2 cm H,O/L/s, with the largest contribution coming from medium-sized bronchi (before the seventh generation). Resistance in large bronchi is low because of their large diameters, whereas resistance in small bronchi is low because of their large total cross- sectional area. The most important causes of increased airway resistance include bronchospasm, secretions, and mucosal edema as well as volume-related and flow-related airway collapse.

At low lung volumes, loss of radial traction increases the contribution of small airways to total resistance; airway resistance becomes inversely proportional to lung volume (Figure 23-9). Increasing lung volume up to normal with positive end-expiratory pressure (PEEP) can reduce airway resistance.
FIGURE 23-9 The relationship between airway resistance and lung volume. (Reproduced with permission from Lumb A. Nunn's Applied Respiratory Physiology. 8th ed. St. Louis, MO: Elsevier; 2017.)

During forced exhalation, reversal of the normal transmural airway pressure can cause collapse of these airways (dynamic airway compression). Two contributing factors are responsible: generation of a positive pleural pressure and a large pressure drop across intrathoracic airways as a result of increased airway resistance. The latter is in turn due to high (turbulent) gas flow and the reduced lung volume. The terminal portion of the flow/volume curve is therefore considered to be effort independent (Figure 23-10).
FIGURE 23-10 Gas flow (A) during forced exhalation from total lung capacity with varying effort and (B) with maximal effort from different lung volumes. Note that regardless of initial lung volume or effort, terminal expiratory flows are effort independent. (Reproduced with permission from Lumb A. Nunn's Applied Respiratory Physiology. 8th ed. St. Louis, MO: Elsevier; 2017.)
The point along the airways where dynamic compression occurs is called the equal pressure point. It is normally beyond the 11th to 13th generation of bronchioles where cartilaginous support is absent (see earlier discussion). The equal pressure point moves toward smaller airways as lung volume decreases. Emphysema or asthma predisposes patients to dynamic airway compression. Emphysema destroys the elastic tissues that normally support smaller airways. In patients with asthma, bronchoconstriction and mucosal edema intensify airway collapse and promote reversal of transmural pressure gradients across airways. Patients may terminate exhalation prematurely or purse their lips to increase expiratory resistance at the mouth. Premature termination of exhalation may increase FRC above normal, resulting in air trapping and auto-PEEP.

Measuring vital capacity as an exhalation that is as forceful and rapid as possible (Figure 23-11) provides important information about airway resistance. The ratio of the forced expiratory volume in the first second of exhalation (FEV,) to the total forced vital capacity (FVC) is proportional to the degree of airway obstruction. Normally, FEV,/FVC is 80% or greater. Whereas both FEV, and FVC are effort dependent, @ forced midexpiratory flow (FEF ;_750,) is more effort independent and may be a more reliable measurement of obstruction.
FIGURE 23-11 The normal forced exhalation curve. FEF 5. 750, is also called the maximum midexpiratory flow rate (MMF>>_750,). FRC, functional residual capacity; FEV,, forced expiratory volume in 1 s; FVC, forced vital capacity; RV, residual volume; TLC, total lung capacity.

This component of nonelastic resistance is generally underestimated and often overlooked, but may account for up to half of total airway resistance. It seems to be primarily due to viscoelastic (frictional) resistance of tissues to gas flow.

Because expiration is normally entirely passive, both the inspiratory and the expiratory work of breathing is performed by the inspiratory muscles (primarily the diaphragm). Three factors must be overcome during ventilation: the elastic recoil of the chest and lung, frictional resistance to gas flow in the airways, and tissue frictional resistance.
Respiratory work can be expressed as the product of volume and pressure. During inhalation, both inspiratory airway resistance and pulmonary elastic recoil must be overcome; nearly 50% of the energy expended is stored pulmonary elastic recoil. During exhalation, the stored potential energy is released and overcomes expiratory airway resistance. Increases in either inspiratory or expiratory resistance are compensated by increased inspiratory muscle effort. When expiratory resistance increases, the normal compensatory response is to increase lung volume such that VT breathing occurs at an abnormally high FRC. The greater elastic recoil energy stored at a higher lung volume overcomes the added expiratory resistance. Excessive amounts of expiratory resistance also activate expiratory muscles.
Respiratory muscles normally account for only 2% to 3% of O, consumption but operate at about 10% efficiency. Ninety percent of the work is dissipated as heat (due to elastic and airflow resistance). In pathological conditions that increase the load on the diaphragm, muscle efficiency usually progressively decreases, and contraction may become uncoordinated with increasing ventilatory effort; moreover, a point may be reached whereby any increase in O, uptake (because of augmented ventilation) is consumed by the respiratory muscles themselves.
The work required to overcome elastic resistance increases as VT increases, whereas the work required to overcome airflow resistance increases as respiratory rate (and, necessarily, expiratory flow) increases. Faced with either condition, patients minimize the work of breathing by altering the respiratory rate and VT (Figure 23-12). Patients with reduced compliance tend to have rapid, shallow breaths, whereas those with increased airflow resistance have a slow, deep breathing pattern.
FIGURE 23-12 The work of breathing in relation to respiratory rate for normal individuals, patients with increased elastic resistance, and patients with increased airway resistance. (Reproduced with permission from Lumb A. Nunn's Applied Respiratory Physiology. 8th ed. St. Louis, MO: Elsevier; 2017.)

The effects of anesthesia on breathing are complex and relate to changes both in position and anesthetic agent.

@ Changes in lung mechanics due to general anesthesia occur shortly after induction. The supine position reduces the FRC by 0.8 to 1.0 L, and induction of general anesthesia further reduces the FRC by 0.4 to 0.5 L. FRC reduction is a consequence of alveolar collapse and compression atelectasis due to loss of inspiratory muscle tone, change in chest wall rigidity, and upward shift of the diaphragm. The mechanisms may be more complex; for example, only the dependent (dorsal) part of the diaphragm in the supine position moves cephalad. Other factors are likely due to a change in intrathoracic volume secondary to increased blood volume in the lung and changes in chest wall shape (Figure 23— 13). The higher position of the dorsal diaphragm and changes in the thoracic cavity itself decrease lung volumes. This decrease in FRC is not related to anesthetic depth and may persist for several hours or days after anesthesia. Steep head-down (Trendelenburg) position (>30°) may reduce FRC even further as intrathoracic blood volume increases. In contrast, induction of anesthesia in the sitting position seems to have little effect on FRC. Muscle paralysis does not seem to change FRC significantly when the patient is already anesthetized.
FIGURE 23-13 With induction of anesthesia in the supine position, the abdominal contents exert cephalad pressure on the diaphragm. At endexpiration, the dorsal portion of the diaphragm is more cephalad and the ventral portion is more caudal than when awake, the thoracic spine is more lordotic, and the rib cage moves inward, all secondary to loss of motor tone.
The effects of anesthesia on closing capacity are more variable. Both FRC and closing capacity, however, are generally reduced to the same extent under anesthesia. Thus, the risk of increased intrapulmonary shunting under anesthesia is similar to that in the conscious state; it is greatest in the elderly, in obese patients, and in those with underlying pulmonary disease.

The reduction in FRC associated with general anesthesia would be expected to increase airway resistance. Increases in airway resistance are not usually observed, however, because of the bronchodilating properties of the volatile inhalation anesthetics. Increased airway resistance is more commonly due to pathological factors (posterior displacement of the tongue; laryngospasm; bronchoconstriction; or secretions, blood, or tumor in the airway) or equipment problems (small tracheal tubes or connectors, malfunction of valves, or obstruction of the breathing circuit).

Increases in the work of breathing under anesthesia are most often secondary to reduced lung and chest wall compliance and, less commonly, increases in airway resistance (see earlier discussion). The problems of increased work of breathing are usually circumvented by controlled mechanical ventilation.

Regardless of the agent used, light anesthesia often results in irregular breathing patterns; breath holding is common. Breaths become regular with deeper levels of anesthesia. Inhalation agents generally produce rapid, shallow breaths, whereas nitrous—opioid techniques result in slow, deep breaths.

Ventilation is usually measured as the sum of all exhaled gas volumes in 1 min (minute ventilation, or v7).
Minute ventilation = Respiratory rate x Tidal volume
For the average adult at rest, minute ventilation is about 5 L/min.
Not all of the inspired gas mixture reaches alveoli; some of it remains in the airways and is exhaled without being exchanged with alveolar gases. The part of the VT not participating in alveolar gas exchange is known as dead space (VD). Alveolar ventilation (W) is the volume of inspired gases actually taking part in gas exchange in 1 min.
Va = Respiratory rate x Vr — Vp
Dead space is actually composed of gases in nonrespiratory airways (anatomic dead space) and alveoli that are not perfused (alveolar dead space). The sum of the two components is referred to as physiological dead space. In the upright position, dead space is normally about 150 mL for most adults (approximately 2 mL/kg) and is nearly all anatomic. The weight of an individual in pounds is roughly equivalent to dead space in milliliters. Dead space can be affected by a variety of factors (Table 23-2).

Posture, Effect = . Upright, Effect = <-->. Supine of, Effect = . Position airway Neck extension Neck flexion, Effect = €F5. Age, Effect = 3. Artificial airway, Effect = &. Positive-pressure ventilation, Effect = 39. Drugs—anticholinergic, Effect = >. Pulmonary perfusion Pulmonary emboli Hypotension, Effect = °C. Pulmonary vascular disease Emphysema, Effect = 
Because VT in the average adult is approximately 450 mL (6 mL/kg), Vb/VT
is normally 33%. This ratio can be derived by the Bohr equation:
where PACO, is the alveolar CO, tension and PECO, is the mixed expired CO, tension. This equation is useful clinically if arterial CO, tension (Paco,) is used to approximate the alveolar concentration and the CO, tension in expired air gases is the average measured over several minutes.

Regardless of body position, alveolar ventilation is unevenly distributed in the lungs. The right lung receives more ventilation than the left lung (53% vs 47%), and the lower (dependent) areas of both lungs tend to be better ventilated than do the upper areas because of a gravitationally induced gradient in intrapleural pressure (transpulmonary pressure). Pleural pressure decreases about 1 cm H,O (becomes less negative) per 3-cm decrease in lung height. This difference places alveoli from different areas at different points on the pulmonary compliance curve (Figure 23—14). Because of a higher transpulmonary pressure, alveoli in upper lung areas are near-maximally inflated and relatively noncompliant, and they undergo little expansion during inspiration. In contrast, the smaller alveoli in dependent areas have a lower transpulmonary pressure, are more compliant, and undergo greater expansion during inspiration.
FIGURE 23-14 The effect of gravity on alveolar compliance in the upright position.
Airway resistance can also contribute to regional differences in pulmonary ventilation. Final alveolar inspiratory volume is solely dependent on compliance only if inspiratory time is unlimited. In reality, inspiratory time is necessarily limited by the respiratory rate and the time necessary for expiration; consequently, an excessively short inspiratory time will prevent alveoli from reaching the expected change in volume. Moreover, alveolar filling follows an exponential function that is dependent on both compliance and airway resistance. Therefore, even with a normal inspiratory time, abnormalities in either compliance or resistance can prevent complete alveolar filling.

Lung inflation can be described mathematically by the time constant, Tt.
t = Total compliance x Airway resistance
Regional variations in resistance or compliance not only interfere with alveolar filling but can cause asynchrony in alveolar filling during inspiration; some alveolar units may continue to fill as others empty.
Variations in time constants within the normal lung can be demonstrated in normal individuals breathing spontaneously during abnormally high respiratory rates. Rapid shallow breathing reverses the normal distribution of ventilation, preferentially favoring upper (nondependent) areas of the lung over the lower areas.

Of the approximately 5 L/min of blood flowing through the lungs, only about 70 to 100 mL at any one time are within the pulmonary capillaries undergoing gas exchange. At the alveolar—capillary membrane, this small volume forms a 50 to 100 m?-sheet of blood approximately one red cell thick. Moreover, to ensure optimal gas exchange, each capillary perfuses more than one alveolus.
Although capillary volume remains relatively constant, total pulmonary blood volume can vary between 500 mL and 1000 mL. Large increases in either cardiac output or blood volume are tolerated with little change in pressure as a result of passive dilation of open vessels and perhaps some recruitment of collapsed pulmonary vessels. Small increases in pulmonary blood volume normally occur during cardiac systole and with each normal (spontaneous) inspiration. A shift in posture from supine to erect decreases pulmonary blood volume (up to 27%); Trendelenburg positioning has the opposite effect. Changes in systemic capacitance also influence pulmonary blood volume: Systemic venoconstriction shifts blood from the systemic to the pulmonary circulation, whereas vasodilation causes a pulmonary-to-systemic redistribution. In this way, the lung acts as a reservoir for the systemic circulation.
@ Local factors are more important than the autonomic system in influencing pulmonary vascular tone. Hypoxia is a powerful stimulus for pulmonary vasoconstriction (the opposite of its systemic effect). Both pulmonary arterial (mixed venous) and alveolar hypoxia induce vasoconstriction, but the latter is a more powerful stimulus. This response seems to be due to either the direct effect of hypoxia on the pulmonary vasculature or increased production of leukotrienes relative to vasodilatory prostaglandins. Inhibition of nitric oxide production may also play a role. Hypoxic pulmonary vasoconstriction is an important physiological mechanism in reducing intrapulmonary shunting and preventing hypoxemia. Hyperoxia has little effect on the pulmonary circulation in normal individuals. Hypercapnia and acidosis have a constrictor effect, whereas hypocapnia causes pulmonary vasodilation, the opposite of what occurs in the systemic circulation.

Pulmonary blood flow is not uniform. Regardless of body position, dependent areas of the lung receive greater blood flow than nondependent areas. This pattern is the result of a gravitational gradient of 1 cm H,O/cm lung height. The normally low pressures in the pulmonary circulation allow gravity to exert a significant influence on blood flow. Also, in vivo perfusion scanning in normal individuals has shown an "onion-like" layering distribution of perfusion, with reduced flow at the periphery of the lung and increased perfusion toward the hilum.
Although the pulmonary perfusion pressure is not uniform across the lung, the alveolar distending pressure is relatively constant. The interplay of these pressures results in the dividing of the lung into four distinct zones (ie, the West zones; Figure 23-15). In zone 1 (PA > Pa > Pv), alveolar pressure (PA) is greater than both the arterial pulmonary pressure (Pa) and venous pulmonary pressure (Pv), resulting in obstruction of blood flow and creation of alveolar dead space. West zone 1 is fairly small in a spontaneously breathing individual, but can enlarge during positive pressure ventilation. In dependent areas of the lungs, Pa progressively increases due to reduced elevation above the heart. In zone 2 (Pa > Pa > Pv), Pa is greater than Pa, but Pv remains less than both, resulting in blood flow that is dependent on the differential between Pa and Pa. The bulk of the lung is described by zone 3 (Pa > Pv > Pa), where both Pa and Pv are greater than Pa, resulting in blood flow independent of the alveolar pressure. Zone 4, the most dependent part of the lung, is where atelectasis and interstitial pulmonary edema occur, resulting in blood flow that is dependent on the differential between Pa and pulmonary interstitial pressure.

FIGURE 23-15 Pulmonary blood flow distribution relative to the alveolar pressure (PA), the pulmonary arterial pressure (Pa), the pulmonary venous pressure (Pv), and the interstitial pressure (PIs) at various gravitation levels. A: Classic West zones of blood flow distribution in the upright position. (Modified with permission from West JB. Respiratory Physiology: The Essentials. 6th ed. Philadelphia, PA: Williams and Wilkins; 2000.) B: In vivo perfusion scanning illustrating central-to-peripheral, in addition to gravitational, blood flow distribution in the upright position. (Reproduced with permission from Lohser J. Evidence based management of one lung ventilation. Anesthesiol Clin. 2008 June;26(2):241-272.)

@ Because alveolar ventilation (W,) is normally about 4 L/min, and pulmonary capillary perfusion (Q) is 5 L/min, the overall W/Q ratio is about 0.8. W/Q for individual lung units (each alveolus and its capillary) can range from 0 (no ventilation) to infinity (no perfusion); the former is referred to as intrapulmonary shunt, whereas the latter constitutes alveolar dead space. v/iQ normally ranges between 0.3 and 3.0; the majority of lung areas, however, are close to 1.0
(Figure 23—16A). Because perfusion increases at a greater rate than ventilation, nondependent (apical) areas tend to have higher v/Q ratios than do dependent (basal) areas (Figure 23—16B).
FIGURE 23-16 The distribution of \//Q ratios for the whole lung (A) and according to height (B) in the upright position. Note that blood flow increases more rapidly than ventilation in dependent areas. (Reproduced with permission from West JB. Ventilation/Blood Flow and Gas Exchange. 3rd ed. Oxford, UK: Blackwell Science Ltd; 1977.)
The importance of V/Q ratios relates to the efficiency with which lung units resaturate venous blood with O, and eliminate CO,. Pulmonary venous blood (the effluent) from areas with low y/Q ratios has a low O, tension and high CO, tension—similar to systemic mixed venous blood. Blood from these units tends to depress arterial O, tension and elevate arterial CO, tension. Their effect on arterial O, tension is much more profound than that on CO, tension; in fact, arterial CO, tension often decreases from a hypoxemia-induced reflex increase in alveolar ventilation. An appreciable compensatory increase in O, uptake cannot take place in remaining areas where v/Q is normal, because pulmonary end-capillary blood is usually already maximally saturated with O, (see below).

© Shunting denotes the process whereby desaturated, mixed venous blood from the right heart returns to the left heart without being oxygenated in the lungs (Figure 23-17). The overall effect of shunting is to decrease (dilute) arterial O, content; this type of shunt is referred to as right-to-left. Left-to-right shunts (in the absence of pulmonary congestion) do not produce hypoxemia.
capillary
blood
FIGURE 23-17 A three-compartment model of gas exchange in the lungs, showing dead space ventilation, normal alveolar—capillary exchange, and shunting (venous admixture). (Reproduced with permission from Lumb A. Nunn's Applied Respiratory Physiology. 8th ed. St. Louis, MO: Elsevier; 2017.)
Intrapulmonary shunts are often classified as absolute or relative. An absolute shunt refers to anatomic shunts and lung units where V/Q is zero. A relative shunt is an area of the lung with a low v/Q ratio. Clinically, hypoxemia from a relative shunt can usually be partially corrected by increasing the inspired O, concentration; hypoxemia caused by an absolute shunt cannot.

(Qs/Q
Venous admixture is the amount of mixed venous blood that would have to be mixed with pulmonary end-capillary blood to account for the difference in O, tension between arterial and pulmonary end-capillary blood. Pulmonary endcapillary blood is considered to have the same concentrations as alveolar gas. Venous admixture is usually expressed as a fraction of total cardiac output T). The equation for Qs/QT may be derived with the law for the conservation of mass for O, across the pulmonary bed:
Qs = blood flow through the physiologic shunt compartment
Qr = total cardiac output
Qc' = blood flow across normally ventilated pulmonary capillaries
Qr = Qc'+Qs
Cc'o, = oxygen content of ideal pulmonary end-capillary blood
Cao, = arterial oxygen content
Cvo, = mixed venous content
The simplified equation is:
The formula for calculating the O, content of blood is given below.
Qs/Qt can be calculated clinically by obtaining mixed venous and arterial blood
gas measurements; the former requires a pulmonary artery catheter. The alveolar gas equation is used to derive pulmonary end-capillary O, tension.
Pulmonary capillary blood is usually assumed to be 100% saturated for an Fio, of 0.21 or greater.
The calculated venous admixture assumes that all shunting is intrapulmonary and due to absolute shunts (v/Q = 0). In reality, neither is ever the case; nonetheless, the concept is useful clinically. Normal Qs/QT is primarily due to communication between deep bronchial veins and pulmonary veins, the thebesian circulation in the heart, and areas of low v/Q in the lungs (Figure 23— 18). The venous admixture in normal individuals (physiological shunt) is typically less than 5%.
FIGURE 23-18 Components of the normal venous admixture. (Reproduced with permission from Lumb A. Nunn's Applied Respiratory Physiology. 8th ed. St. Louis, MO: Elsevier; 2017.)

Abnormalities in gas exchange during anesthesia are common. They include increased dead space, hypoventilation, and increased intrapulmonary shunting.
There is increased scatter of v/Q ratios. Increases in alveolar dead space are most commonly seen during controlled ventilation, but may also © occur during spontaneous ventilation. General anesthesia commonly increases venous admixture to 5% to 10%, probably as a result of atelectasis and airway collapse in dependent areas of the lung. Inhalation agents also can inhibit hypoxic pulmonary vasoconstriction; for volatile agents, the EDs is about twice the minimum alveolar concentration (MAC). Elderly patients seem to have the largest increases in Qs/QT. Inspired O, tensions of 30% to 40% usually prevent hypoxemia, suggesting anesthesia increases relative shunt. PEEP is often effective in reducing venous admixture and preventing hypoxemia during general anesthesia, as long as cardiac output is maintained. Prolonged administration of high inspired O, concentrations may be associated with atelectasis formation and increases in absolute shunt. Atelectasis in this situation is known as resorption atelectasis and appears in areas with a low v/Q ratio ventilated at an O,-inspired concentration close to 100%. Perfusion results in O5 being transported out of the alveoli at a rate faster than it enters the alveoli, leading to an emptying of the alveoli and collapse.

When dealing with gas mixtures, each gas is considered to contribute separately to total gas pressure, and its partial pressure is directly proportional to its concentration. Air has an O, concentration of approximately 21%; therefore, if the barometric pressure is 760 mm Hg (sea level), the partial pressure of O, (Po,) in air is normally 159.6 mm Hg:

In its general form, the equation may be written as follows:
PIO, = PB x Fio, where Pp = barometric pressure and Fio, = the fraction of inspired O5.
1. Oxygen

With every breath, the inspired gas mixture is humidified at 37°C in the upper airway. The inspired tension of O, (PI0,) is therefore reduced by the added water vapor. Water vapor pressure is dependent upon temperature and is 47 mm Hg at 37°C. In humidified air, the normal partial pressure of O, at sea level is 150 mm Hg:
The general equation is:
where PH,0 = the vapor pressure of water at body temperature.
In alveoli, the inspired gases are mixed with residual alveolar gas from previous breaths, O, is taken up, and CO, is added. The final alveolar O, tension (PAO>) is therefore dependent on all of these factors and can be estimated by the following equation:
where Paco, = arterial CO, tension and RQ = respiratory quotient.
@ RQ is usually not measured. Note that large increases in Paco (>75 mm Hg) readily produce hypoxia (Pao, <60 mm Hg) at room air, but not at high inspired O, concentrations.

For all practical purposes, pulmonary end-capillary O, tension (Pc'0,) may be considered identical to PAO; the PAo,—Pc'o, gradient is normally minute. Pc'o, is dependent on the rate of O, diffusion across the alveolar—capillary membrane, as well as on pulmonary capillary blood volume and transit time. The large capillary surface area in alveoli and the 0.4 to 0.5 y:m thickness of the alveolar— capillary membrane greatly facilitate O, diffusion. Enhanced O, binding to hemoglobin at saturations above 80% also augments O, diffusion (see below).
Capillary transit time can be estimated by dividing pulmonary capillary blood volume by cardiac output (pulmonary blood flow); thus, normal capillary transit time is 70 mL + 5000 mL/min (0.8 s). Maximum Pc'o, is usually attained after only 0.3 s, providing a large safety margin.
® The binding of O, to hemoglobin seems to be the principal rate-limiting factor in the transfer of O, from alveolar gas to blood. Therefore, pulmonary diffusing capacity reflects not only the capacity and permeability of the alveolar— capillary membrane, but also pulmonary blood flow. Moreover, O, uptake is normally limited by pulmonary blood flow, not O, diffusion across the alveolar— capillary membrane; the latter may become significant during exercise in normal individuals at high altitudes and in patients with extensive destruction of the alveolar—capillary membrane.
O, transfer across the alveolar—capillary membrane is expressed as O, diffusing capacity (DLO,):
Because Pc'o, cannot be measured accurately, measurement of carbon monoxide diffusion capacity (DLCO) is used instead to assess gas transfer across the alveolar—capillary membrane. Because carbon monoxide has a very high affinity for hemoglobin, there is little or no carbon monoxide in pulmonary capillary blood, so that even when it is administered at low concentration, Pc'co can be considered zero. Therefore,

Reductions in Dico imply an impediment in gas transfer across the alveolar— capillary membrane. Such impediments may be due to abnormal V/Q ratios, extensive destruction of the gas alveolar—capillary membrane, or very short capillary transit times. Abnormalities are accentuated by increases in O, consumption and cardiac output, such as occurs during exercise.

Pao, cannot be calculated like Pao, but must be measured at room air. The alveolar-to-arterial O, partial pressure gradient (A—a gradient) is normally less than 15 mm Hg, but progressively increases with age up to 20 to 30 mm Hg. Arterial O, tension can be approximated by the following formula (in mm Hg):
The range is 60 to 100 mm Hg. Decreases are probably the result of a progressive increase in closing capacity relative to FRC (see earlier discussion). Table 23-3 lists the mechanisms of hypoxemia (Pao, <60 mm Hg).

Low alveolar oxygen tension
Low inspired oxygen tension
Low fractional inspired concentration
Alveolar hypoventilation
Diffusion hypoxia
Increased oxygen consumption
Increased alveolar—arterial gradient
Right-to-left shunting
Increased areas of low y/Q! ratios
Low mixed venous oxygen tension
Decreased cardiac output
Decreased hemoglobin concentration
wiq, ventilation/perfusion.
right-
The most common mechanism for hypoxemia is an increased alveolar— arterial gradient. The A—a gradient for O, depends on the amount of to-left shunting, the amount of v/Q scatter, and the mixed venous O, tension (see below). The last depends on cardiac output, O consumption, and hemoglobin concentration.
The A-a gradient for O, is directly proportional to shunt, but inversely proportional to mixed venous O, tension. The effect of each variable on PAO, (and consequently the A—a gradient) can be determined only when the other variables are held constant. Figure 23-19 shows the effect of different degrees of ® shunting on Pao,. It should also be noted that the greater the shunt, the less likely the possibility that an increase in Fio, will correct hypoxemia. Moreover, isoshunt lines seem to be most useful for O, concentrations between 35% and 100%. Lower O, concentrations require modification of isoshunt lines to account for the effect of V/Q scatter.
FIGURE 23-19 Isoshunt curves showing the effect of varying amounts of shunt on Pao,. Note that there is little benefit in increasing inspired oxygen concentration in patients with very large shunts. (Modified with permission from Benatar SR, Hewlett AM, Nunn JF. The use of isoshunt lines for control of oxygen therapy. Br J Anaesth. 1973 July;45(7):711-718.)
The effect of cardiac output on the A—a gradient (Figure 23-20) is due not only to its secondary effects on mixed venous O, tension but also to a direct relationship between cardiac output and intrapulmonary shunting. As can be seen, a low cardiac output tends to accentuate the effect of shunt on Pao». A

reduction in venous admixture may be observed with low-normal cardiac outputs secondary to accentuated pulmonary vasoconstriction from a lower mixed venous O, tension. On the other hand, high cardiac outputs can increase venous admixture by elevating mixed venous O, tension, which in turn inhibits hypoxic pulmonary vasoconstriction.
FIGURE 23-20 The effect of cardiac output on the alveolar—arterial Po, difference with varying degrees of shunting. Vo, = 200 mL/min and Pao, = 180 mm Hg.) (Reproduced with permission from Lumb A. Nunn's Applied Respiratory Physiology. 8th ed. St. Louis, MO: Elsevier; 2017.)
O, consumption and hemoglobin concentration can also affect Pao, through their secondary effects on mixed venous O, tension (below). High O5 consumption rates and low hemoglobin concentrations can increase the A—a gradient and depress Pao,.

Normal mixed venous O, tension (PVO,) is about 40 mm Hg and represents the overall balance between O, consumption and O, delivery ('Table 23-4). A true mixed venous blood sample contains venous drainage from the superior vena cava, the inferior vena cava, and the heart; it must therefore be obtained from a pulmonary artery catheter.
TABLE 23-4 Alterations in mixed venous oxygen tension (and saturation).
Decreased Pvo,
Increased O, consumption
Fever
Shivering
Exercise
Thyroid storm
Decreased O, delivery
Hypoxia
Abnormal hemoglobin
Increased Pvo,
Left-to-right shunting
High cardiac output
Impaired tissue uptake
Cyanide poisoning
Decreased oxygen consumption
Combined mechanisms
Sepsis
Sampling error
Wedged pulmonary artery catheter

dioxide is a by product of aerobic metabolism in mitochondria.
there are small continuous gradients for CO, tension from
Carbon Therefore, mitochondria to cell cytoplasm, extracellular fluid, venous blood, and alveoli,
where the CO, is finally eliminated.

Normal mixed venous CO, tension (PVCO 5) is about 46 mm Hg and is the end result of mixing of blood from tissues of varying metabolic activity. Venous CO, tension is lower in tissues with low metabolic activity (eg, skin), but higher in blood from those with relatively high activity (eg, heart).

Alveolar CO, tension (PACO>) is generally considered to represent the balance between total CO, production (Vco ,) and alveolar ventilation (elimination):
where V4 is alveolar ventilation (Figure 23—21). During periods of acute hypoventilation or hypoperfusion, body content of carbon dioxide increases. Clinically, PACO, is more dependent on variations in alveolar ventilation than in CO, production, because CO, production does not vary appreciably under most circumstances. However, conditions such as malignant hyperthermia can lead to dramatic increases in CO, production, which can overwhelm the body's buffering system (see below).
FIGURE 23-21 The effect of alveolar ventilation on alveolar Pco, at two rates of CO, production.
(Reproduced with permission from Lumb A. Nunn's Applied Respiratory
Physiology. 8th ed. St. Louis, MO: Elsevier; 2017.)

Pulmonary end-capillary CO, tension (Pc'co>) is virtually identical to Paco, for the same reasons as those discussed in the section about O5. In addition, the diffusion rate for CO, across the alveolar—capillary membrane is 20 times that of Op.

Arterial CO, tension (Paco), which is readily measurable, is identical to Pc'coy and, necessarily, PACO. Normal Paco, is 38 + 4mm Hg (5.1 + 0.5 kPa); in practice, 40 mm Hg is usually considered normal.
Although low V/Q ratios tend to increase Paco, whereas high V/Q ratios tend to decrease it, significant arterial-to-alveolar gradients for CO, develop only in the presence of marked V/Q abnormalities (>30% venous admixture); even then the gradient is relatively small (2-3 mm Hg). Moreover, small increases in the gradient appreciably increase CO, output into alveoli with relatively normal V/Q. Even moderate to severe disturbances usually fail to appreciably alter arterial CO, because of a reflex increase in ventilation from concomitant hypoxemia.

Because end-tidal gas is primarily alveolar gas and PACO, is virtually identical to PacO5, end-tidal CO, tension (PETCO>) is used clinically as an estimate of Paco5. The PACO,—PETCO, gradient is normally less than 5 mm Hg and represents dilution of alveolar gas with CO,-free gas from nonperfused alveoli (alveolar dead space).

1. Oxygen
O, is carried in blood in two forms: dissolved in solution and in reversible association with hemoglobin.

The amount of O, dissolved in blood can be derived from Henry's law, which states that the concentration of any gas in solution is proportional to its partial pressure. The mathematical expression is as follows:

where a = the gas solubility coefficient for a given solution at a given temperature.
The solubility coefficient for O at normal body temperature is 0.003 mL/dL/mm Hg. Even with a Pao, of 100 mm Hg, the maximum amount of O, dissolved in blood is very small (0.3 mL/dL) compared with that bound to hemoglobin.

Hemoglobin is a complex molecule consisting of four heme and four protein subunits. Heme is an iron—porphyrin compound that is an essential part of the O,-binding sites; only the divalent form (+2 charge) of iron can bind O,. The normal hemoglobin molecule (hemoglobin A,) consists of two a and two B chains (subunits); the four subunits are held together by weak bonds between the amino acid residues. Each gram of hemoglobin can theoretically carry up to 1.39 mL of O,.

Each hemoglobin molecule binds up to four O, molecules. The complex interaction between the hemoglobin subunits results in nonlinear (an elongated S shape) binding with O, (Figure 23—22). Hemoglobin saturation is the amount of O, bound as a percentage of its total O>-binding capacity. Four separate chemical reactions are involved in binding each of the four O, molecules. The change in molecular conformation induced by the binding of the first three molecules greatly accelerates binding of the fourth O, molecule. The last reaction is responsible for the accelerated binding between 25% and 100% saturation. At about 90% saturation, the decrease in available O, receptors flattens the curve until full saturation is reached.
FIGURE 23-22 The normal adult hemoglobin—oxygen dissociation curve. (Modified with permission from West JB. Respiratory Physiology—The Essentials. 6th ed. Philadelphia, PA: Williams & Wilkins; 2000.)

Clinically important factors altering O, binding include hydrogen ion concentration, CO, tension, temperature, and 2,3-diphosphoglycerate (2,3-DPG) concentration. Their effect on hemoglobin—O, interaction can be expressed by Pz, the O, tension at which hemoglobin is 50% saturated (Figure 23—23). Each factor shifts the dissociation curve either to the right (increasing P.,) or to the left (decreasing P9). ® A rightward shift in the oxygen—hemoglobin dissociation curve lowers O, affinity, displaces O, from hemoglobin, and makes more O, available to tissues; a leftward shift increases hemoglobin's affinity for O,, reducing its availability to tissues. The normal Psg in adults is 26.6 mm Hg.
FIGURE 23-23 The effects of changes in acid—base status, body temperature, and 2,3-DPG concentration on the hemoglobin—oxygen dissociation curve.
An increase in blood hydrogen ion concentration reduces O, binding to hemoglobin (Bohr effect). Because of the shape of the hemoglobin dissociation curve, the effect is more important in venous blood than arterial blood (see Figure 23-23); the net result is facilitation of O, release to tissue with little impairment in O, uptake (unless severe hypoxia is present).
The influence of CO, tension on hemoglobin's affinity for O, is important physiologically and is secondary to the associated rise in hydrogen ion concentration when CO, tension increases. The high CO, content of venous capillary blood, by decreasing hemoglobin's affinity for O., facilitates the release of O, to tissues; conversely, the lower CO, content in pulmonary capillaries increases hemoglobin's affinity for O, again, facilitating O uptake from alveoli.
2,3-DPG is a byproduct of glycolysis and accumulates during anaerobic metabolism. Although its effects on hemoglobin under these conditions are theoretically beneficial, its physiological importance normally seems minor. 2,3DPG levels may, however, play an important compensatory role in patients with chronic anemia and may significantly affect the O,-carrying capacity of blood transfusions.

Carbon monoxide, cyanide, nitric acid, and ammonia can combine with hemoglobin at O,-binding sites. They can displace O, and shift the saturation curve to the left. Carbon monoxide is particularly potent, having 200 to 300 times the affinity of O, for hemoglobin, combining with it to form carboxyhemoglobin. Carbon monoxide decreases hemoglobin's O,-carrying capacity and impairs the release of O, to tissues.
Methemoglobin results
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Nitrates, nitrites,
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as methylene
blue or
ascorbic acid.
Abnormal hemoglobins can also result from variations in the protein subunit composition. Each variant has its own O,-saturation characteristics. These include fetal hemoglobin, hemoglobin A,, and sickle hemoglobin.

The total O, content of blood is the sum of that in solution plus that carried by hemoglobin. In reality, O, binding to hemoglobin never achieves the theoretical maximum, but is closer to 1.31 mL O,/dL blood per millimeter of mercury. Total O, content is expressed by the following equation:
O, content = ([0.003 mL O,/dL blood per mm Hg] x Pos) + (Sos x Hb x 1.31

where Hb is hemoglobin concentration in g/dL blood, and So, is hemoglobin saturation at the given Po5.
Using this formula and a hemoglobin of 15 g/dL, the normal O, content for both arterial and mixed venous blood and the arteriovenous difference can be calculated as follows:

O, transport is dependent on both respiratory and circulatory function. Total O, delivery (Do,) to tissues is the product of arterial O5 content and cardiac output:
Note that arterial O, content is dependent on Pao, as well as hemoglobin concentration. As a result, deficiencies in O, delivery may be due to a low Pao2, a low hemoglobin concentration, or an inadequate cardiac output. Normal O, delivery can be calculated as follows:
The Fick equation expresses the relationship between O, consumption, O, content, and cardiac output:
Rearranging the equation:
Consequently, the arteriovenous difference is a good measure of the overall adequacy of O, delivery.
As calculated above, the arteriovenous difference (CaO, -CVO.) is about 5 mL O,/dL blood (20 mL O,/dL — 15 mL O,/dL). Note that the normal extraction fraction for O[(Cao, -C¥Oz)/Cao,] is 5 mL + 20 mL, or 25%; thus, the body normally consumes only 25% of the O, carried on hemoglobin. When O5 demand exceeds supply, the extraction fraction exceeds 25%. Conversely, if O5 supply exceeds demand, the extraction fraction falls below 25%. When D'o, is even moderately reduced, V0, usually remains normal because of increased O, extraction (mixed venous O, saturation decreases); V'0> remains independent of delivery. With further reductions in D'05, however, a critical point is reached beyond which Vo, becomes directly proportional to D'05. This state of supply-dependent O, is typically associated with progressive lactic acidosis caused by cellular hypoxia.

concept is the basis for preoxygenation prior to induction of anesthesia.

Carbon dioxide is transported in blood in three forms: dissolved in solution, as bicarbonate, and with proteins in the form of carbamino compounds (Table 23-— 5). The sum of all three forms is the total CO, content of blood (routinely reported with electrolyte measurements).

Mixed venous whole blood, Plasma = Mixed venous whole blood. Mixed venous whole blood, Erythrocytes = . Mixed venous whole blood, Combined = . Mixed venous whole blood, Contribution (%) = . Dissolved CO,, Plasma = 0.76. Dissolved CO,, Erythrocytes = 0.51. Dissolved CO,, Combined = 1.27. Dissolved CO,, Contribution (%) = 5.5. Bicarbonate, Plasma = 14.41. Bicarbonate, Erythrocytes = 5.92. Bicarbonate, Combined = 20.33. Bicarbonate, Contribution (%) = 87.2. Carbamino CO,, Plasma = Negligible. Carbamino CO,, Erythrocytes = 1.70. Carbamino CO,, Combined = 1.70. Carbamino CO,, Contribution (%) = 7. Total CO,, Plasma = 15.17. Total CO,, Erythrocytes = 8.13. Total CO,, Combined = 23.30. Total CO,, Contribution (%) = . Arterial whole blood, Plasma = Arterial whole blood. Arterial whole blood, Erythrocytes = . Arterial whole blood, Combined = . Arterial whole blood, Contribution (%) = . Dissolved CO,, Plasma = 0.66. Dissolved CO,, Erythrocytes = 0.44. Dissolved CO,, Combined = 1.10. Dissolved CO,, Contribution (%) = 5.1. Bicarbonate, Plasma = 13.42. Bicarbonate, Erythrocytes = 5.88. Bicarbonate, Combined = 19.30. Bicarbonate, Contribution (%) = 89.9. Carbamino CO,, Plasma = Negligible. Carbamino CO,, Erythrocytes = 1.10. Carbamino CO,, Combined = 1.10. Carbamino CO,, Contribution (%) = 5.1. Total co,, Plasma = 14.08. Total co,, Erythrocytes = 742. Total co,, Combined = 21.50. Total co,, Contribution (%) = 

'Data from Nunn JF. Nunn's Applied Physiology. 4th ed. Philadelphia, PA: Butterworth; 2000.
Values are expressed in millimoles, except where indicated otherwise.

Carbon dioxide is more soluble in blood than O,, with a solubility coefficient of 0.031 mmol/L/mm Hg (0.067 mL/dL/mm Hg) at 37°C.

In aqueous solutions, CO, slowly combines with water to form carbonic acid and bicarbonate, according to the following reaction:
In plasma, although less than 1% of the dissolved CO, undergoes this reaction, the presence of the enzyme carbonic anhydrase within erythrocytes and endothelium greatly accelerates the reaction. ® As a result, bicarbonate represents the largest fraction of the CO, in blood (see Table 23-5).
Administration of acetazolamide, a carbonic anhydrase inhibitor, can impair CO, transport between tissues and alveoli.
On the venous side of systemic capillaries, CO, enters red blood cells and is converted to bicarbonate, which diffuses out of red cells into plasma; chloride ions move from plasma into red cells to maintain electrical balance. In the pulmonary capillaries, the reverse occurs: chloride ions move out of red cells as bicarbonate ions reenter them for conversion back to CO5, which diffuses out into alveoli. This sequence is referred to as the chloride or Hamburger shift.

Carbon dioxide can react with amino groups on proteins, as shown by the following equation:
At physiological pH, only a small amount of CO, is carried in this form, mainly as carbaminohemoglobin. Deoxygenated hemoglobin (deoxyhemoglobin) has a greater affinity (3.5 times) for CO, than does oxyhemoglobin. As a result, venous blood carries more CO, than does arterial blood (Haldane effect; see Table 23-5). Pco, normally has little effect on the fraction of CO, carried as carbaminohemoglobin.

The buffering action of hemoglobin also accounts for part of the Haldane effect. Hemoglobin can act as a buffer at physiological pH because of its high content of histidine. Moreover, the acid—base behavior of hemoglobin is influenced by its oxygenation state:
Removal of O, from hemoglobin in tissue capillaries causes the hemoglobin molecule to behave more like a base; by taking up hydrogen ions, hemoglobin shifts the CO,—bicarbonate equilibrium in favor of greater bicarbonate formation:
a carried
As direct result, deoxyhemoglobin also increases the amount of CO, that is in venous blood as bicarbonate. As CO, is taken up from tissue and converted to bicarbonate, the total CO, content of blood increases (see Table 23— 5).
In the lungs, the reverse is true. Oxygenation of hemoglobin favors its action as an acid, and the release of hydrogen ions shifts the equilibrium in favor of greater CO, formation:
Bicarbonate concentration decreases as CO, is formed and eliminated, so that the total CO, content of blood decreases in the lungs. Note that there is a difference between CO, content (concentration per liter) of whole blood (see Table 23-5) and plasma (Table 23-6).

Dissolved CO,, Arterial = 1.2. Dissolved CO,, Venous = 1.4. Bicarbonate, Arterial = 24.4. Bicarbonate, Venous = 26.2. Carbamino CO,, Arterial = Negligible. Carbamino CO,, Venous = Negligible. Total CO,, Arterial = 25.6. Total CO,, Venous = 27.6
*Values are expressed in millimoles, except where indicated otherwise.
Carbon Dioxide Dissociation Curve
A CO, dissociation curve can be constructed by plotting the total CO, content of blood against Pco. The contribution of each form of CO, can also be quantified in this manner (Figure 23-24).
FIGURE 23-24 The CO, dissociation curve for whole blood. (Reproduced with permission from Lumb A. Nunn's Applied Respiratory Physiology. 8th ed. St. Louis, MO: Elsevier; 2017.)
Carbon Dioxide Stores
Carbon dioxide stores in the body are large (approximately 120 L in adults) and primarily in the form of dissolved CO, and bicarbonate. When an imbalance occurs between production and elimination, establishing a new CO, equilibrium requires 20 to 30 min (compared with less than 4 to 5 min for O.; see above). Carbon dioxide is stored in the rapid-, intermediate-, and slow-equilibrating compartments. Because of the larger capacity of the intermediate and slow compartments, the rate of rise in arterial CO, tension is generally slower than its fall following acute changes in ventilation.

Spontaneous ventilation is the result of rhythmic neural activity in respiratory centers within the brainstem. This activity regulates respiratory muscles to maintain normal tensions of Oj and CO, in the body. The basic neuronal activity is modified by inputs from other areas in the brain, volitional and autonomic, as well as various central and peripheral receptors (sensors).

The basic breathing rhythm originates in the medulla. Two medullary groups of neurons are generally recognized: a dorsal respiratory group, which is primarily active during inspiration, and a ventral respiratory group, which is active during inspiration and expiration.
Two pontine areas influence the dorsal (inspiratory) medullary center. A lower pontine (apneustic) center is excitatory, whereas an upper pontine (pneumotaxic) center is inhibitory. The pontine centers appear to fine-tune respiratory rate and rhythm.

The most important of these sensors are chemoreceptors that respond to changes in hydrogen ion concentration. @ Central chemoreceptors are thought to lie on the anterolateral surface of the medulla and respond primarily to changes in cerebrospinal fluid (CSF) [H*]. This mechanism is effective in regulating Paco,, because the blood-brain barrier is permeable to dissolved CO,, but not to bicarbonate ions. Acute changes in Pacos, but not in arterial [HCO], are reflected in CSF; thus, a change in CO, must result in a change in [H*]:

Over the course of a few days, CSF [HCO3_] can compensate to match any change in arterial [HCO, ].
Increases in Paco, elevate CSF hydrogen ion concentration and activate the chemoreceptors. Secondary stimulation of the adjacent respiratory medullary centers increases alveolar ventilation (Figure 23—25) and reduces Paco, back to normal. Conversely, decreases in CSF hydrogen ion concentration secondary to reductions in Paco, reduce alveolar ventilation and elevate Paco,. Note that the relationship between Paco, and minute volume is nearly linear. Also note that very high arterial Paco, tensions depress the ventilatory response (CO, narcosis). The Paco, at which ventilation is zero (x-intercept) is known as the apneic threshold. Spontaneous respirations are typically absent under anesthesia when Paco, falls below the apneic threshold. (In the awake state, cortical influences prevent apnea, so apneic thresholds are not ordinarily seen.) In contrast to peripheral chemoreceptors (see below), central chemoreceptor activity is depressed by hypoxia.
FIGURE 23-25 The normal relationship between Paco, and minute ventilation. (Reproduced
WB
with permission from Guyton AC. Textbook of Medical Physiology. 7th ed. Philadelphia, PA: Saunders; 1986.)

Peripheral chemoreceptors include the carotid bodies (at the bifurcation of the common carotid arteries) and the aortic bodies (surrounding the aortic arch). The carotid bodies are the principal peripheral chemoreceptors in humans and are sensitive to changes in PaO», Paco, pH, and arterial perfusion pressure. They interact with central respiratory centers via the glossopharyngeal nerves, producing reflex increases in alveolar ventilation in response to reductions in PaOy, arterial perfusion, or elevations in [H*] and Paco,. Peripheral chemoreceptors are also stimulated by cyanide, doxapram, and large doses of nicotine. In contrast to central chemoreceptors, which respond primarily to Paco, (more specifically, [H*]), the carotid bodies are most sensitive to Pao, (Figure 23—26). Note that receptor activity does not appreciably increase until Pao, decreases below 50 mm Hg. Cells of the carotid body (glomus cells) are thought to be primarily dopaminergic neurons. Anti-dopaminergic drugs (such as phenothiazines), most general anesthetics, and bilateral carotid surgery abolish the peripheral ventilatory response to hypoxemia.
FIGURE 23-26 The relationship between Pao, and minute ventilation at rest and with a normal PacO5. (Data from Weil JV, Byrne-Quinn E, Sodal IE, et al. Hypoxic ventilatory drive in normal man. J Clin Invest. 1970;49:1061-1072; Dripps RD, Comroe JH. The effect of the inhalation of high and low oxygen concentration on respiration, pulse rate, ballistocardiogram and arterial oxygen saturation (oximeter) of normal individuals. Am J Physiol. 1947;149:277-291; Cormac RS, Cunningham DJC, Gee JBL. The effect of carbon dioxide on the respiratory response to want of oxygen in man. Q J Exp Physiol. 1957;42:303-316.)

Impulses from these receptors are carried centrally by the vagus nerve. Stretch receptors are distributed in the smooth muscle of airways; they are responsible for inhibition of inspiration when the lung is inflated to excessive volumes (Hering—Breuer inflation reflex) and shortening of exhalation when the lung is deflated (deflation reflex). Stretch receptors normally play a minor role in humans. In fact, bilateral vagal nerve blocks have a minimal effect on the normal respiratory pattern.
Irritant receptors in the tracheobronchial mucosa react to noxious gases, smoke, dust, and cold gases; activation produces reflex increases in respiratory rate, bronchoconstriction, and coughing. Juxtacapillary (J) receptors are located in the interstitial space within alveolar walls; these receptors induce dyspnea in response to expansion of interstitial space volume and various chemical mediators following tissue damage.

These include various muscle and joint receptors on pulmonary muscles and the chest wall. Input from these sources is probably important during exercise and in pathological conditions associated with decreased lung or chest compliance.

The most important effect of most general anesthetics on breathing is a tendency to promote hypoventilation. The mechanism is probably dual: central depression of the chemoreceptor and depression of external intercostal muscle activity. The magnitude of the hypoventilation is generally proportional to @ anesthetic depth. With increasing depth of anesthesia, the slope of the Paco,/minute ventilation curve decreases, and the apneic threshold increases (Figure 23-27). This effect is at least partially reversed by surgical stimulation.
FIGURE 23-27 The effect of volatile agents (halothane) on the PETCO,— ventilation response curve (see text). (Data from Munson ES, Larson CP, Babad AA, et al. The effects of halothane, fluroxene and cyclopropane on ventilation: A comparative study in man. Anesthesiology. 1966 Nov-Dec;27(6):716-728.)
The peripheral response to hypoxemia is even more sensitive to anesthetics than the central CO, response and is nearly abolished by even subanesthetic doses of most inhalation agents (including nitrous oxide) and many intravenous agents.

The unique in-series position of the pulmonary capillaries within the circulation allows them to act as a filter for debris in the bloodstream. The high content of heparin and plasminogen activator in the lungs facilitates the breakdown of entrapped fibrin debris. Although pulmonary capillaries have an average diameter of 7 ym, larger particles have been shown to pass through to the left heart.

The role of the pulmonary circulation as a reservoir for the systemic circulation was discussed earlier.

The lungs are metabolically very active organs. In addition to surfactant synthesis, pneumocytes account for a major portion of extrahepatic mixed-
function oxidation.
Neutrophils and
macrophages in
the lung
produce
O,-derived free
radicals in
response variety
of to
infection.
vasoactive bradykinin,
compounds, and
a
variety epinephrine
are
The pulmonary
including of
endothelium norepinephrine,
prostaglandins generally
not and
metabolized metabolizes
a
serotonin, leukotrienes.
in the
Histamine lungs;
in fact,
and the
lungs can
major site
of histamine
synthesis and
release during
allergic reactions.
The lungs are also responsible for converting angiotensin I to its physiologically active form, angiotensin II. The enzyme responsible, angiotensin-converting enzyme, is bound on the surface of the pulmonary endothelium.

A 67-year-old man with carcinoma is undergoing laparoscopic hemicolectomy under general anesthesia. His history includes an old anterior myocardial infarction and heart failure treated with enalapril, carvedilol, furosemide, and spironolactone. Arterial and central venous catheters are placed preoperatively for monitoring during surgery. Following a smooth induction and an atraumatic intubation, anesthesia is maintained with 40% oxygen, sevoflurane, and vecuronium. Thirty minutes into the operation, the surgeon asks for steep Trendelenburg position to facilitate surgical exposure. The pulse oximeter, which had been reading 99% saturation, suddenly drops and remains at 93%. The pulse oximeter's signal strength and waveform are unchanged. Auscultation of the lungs reveals diminished breath sounds over the left be
a

Unilaterally diminished breath sounds under anesthesia are most commonly caused by accidental placement or migration of the tracheal tube into one of the two main bronchi. As a result, only one lung is ventilated. Other causes of unilaterally diminished breath sounds (such as pneumothorax, a large mucus plug, lobar atelectasis, or undiagnosed bullae) are less easily diagnosed, but are fortunately less common during anesthesia.
The Trendelenburg (head-down) position typically causes the tip of the tracheal tube to advance 1 to 2 cm relative to the carina. In this case, the tube was apparently placed just above the carina with the patient in the supine position, but migrated into the right bronchus when the Trendelenburg position was imposed. The diagnosis is confirmed by drawing the tube back 1 to 2 cm at a time as the chest is auscultated. Breath sounds will become equal again when the tip of the tube reenters the trachea. Following initial placement, tracheal tubes should be routinely checked for correct positioning by auscultating the chest, ascertaining depth of tube insertion by the markings on the tube (normally 20-24 cm at the teeth for an adult), and feeling for the cuff in the suprasternal notch. Tube position relative to the carina can also be quickly confirmed with a flexible fiberoptic bronchoscope.

In most cases of unintentional bronchial intubation, the tracheal tube enters the right bronchus because the latter diverges away from the trachea at a less acute angle than does the left bronchus.

Failure to ventilate one lung as it continues to be perfused creates a large intrapulmonary shunt. Venous admixture increases and tends to depress Pao, and hemoglobin saturation.

No; if both lungs continued to have equal blood flow, venous admixture should have theoretically increased to 50%, resulting in severe hypoxemia and very low hemoglobin saturation. Fortunately, hypoxic pulmonary vasoconstriction is a powerful compensatory response that tends to reduce flow to the hypoxic lung and reduces the expected venous admixture. In fact, if the patient has been receiving a higher inspired O, concentration (50—100%), the drop in arterial tension may not be detectable by the pulse oximeter due to the characteristics of the normal hemoglobin saturation curve. For example, bronchial intubation in a patient inspiring 50% O, might drop Pao, from 250 mm Hg to 95 mm Hg; the resulting change in pulse oximeter readings (100-99 to 98-97) would hardly be noticeable.
Arterial and mixed venous blood gas tensions are obtained with the

Pao, = 69 mm Hg; Paco, = 42 mm Hg; Sao, = 93%; Pvo, = 40 mm Hg; and Svo, = 75%. Hemoglobin concentration is 15 g/dL.

In this case, Pc'0, = Pao, = ([760 — 47] x 0.4) — 42 = 243 mm Hg.
Therefore, Cc'0, = (15 x 1.31 x 1.0) + (243 x 0.003) = 20.4 mL/dL.
Cao, = (15 x 1.31 x 0.93) + (69 x 0.003) = 18.5 mL/dL
Cv—0, = (15 x 1.31 x 0.75) + (40 x 0.003) = 14.8 mL/dL
Q's/Q'T = (20.4 — 18.5)/(20.4 — 14.8) = 34%
How does bronchial intubation affect arterial and end-tidal CO

PacoO, is typically not appreciably altered as long as the same minute ventilation is maintained (see One-Lung Ventilation, Chapter 25).
Clinically, the Paco,—PETCO, gradient often widens, possibly because of increased alveolar dead space (overdistention of the ventilated lung). Thus, PETCO, may decrease or remain unchanged.

Baumgardner JE, Hedenstierna G. Ventilation/perfusion distributions revisited. Curr Opin Anaesthesiol. 2016;29:2.
Campos J. Update on tracheobronchial anatomy and flexible fiberoptic bronchoscopy in thoracic anesthesia. Curr Opin Anaesthesiol. 2009;22:4.
Hedenstierna G, Edmark L. Effects of anesthesia on the respiratory system. Best

@ Ina patient with an acute asthma attack, a normal or high Paco, indicates that the patient can no longer maintain the work of breathing and is often a sign of impending respiratory failure. A pulsus paradoxus and electrocardiographic signs of right ventricular strain (ST-segment changes, right axis deviation, and right bundle-branch block) are also indicative of severe airway obstruction.
@ Asthmatic patients with active bronchospasm presenting for emergency surgery should be treated aggressively. Supplemental oxygen, aerosolized B-agonists, and intravenous glucocorticoids can dramatically improve lung function in a few hours.
© Intraoperative bronchospasm is usually manifested as wheezing, increasing peak airway pressures (plateau pressure may remain unchanged), decreasing exhaled tidal volumes, or a slowly rising waveform on the capnograph.
@ Other causes can simulate bronchospasm: These include obstruction of the tracheal tube from kinking, secretions, or an overinflated balloon; bronchial intubation; active expiratory efforts (straining); pulmonary edema or embolism; and pneumothorax.
@ Chronic obstructive pulmonary disease (COPD) is currently defined as a disease state characterized by airflow limitation that is not fully reversible. The chronic airflow limitation of this disease is due to a mixture of small and large airway disease (chronic bronchitis/bronchiolitis) and parenchymal destruction (emphysema), with the representation of these two components varying from patient

Cessation of smoking is the long-term intervention that has been shown to reduce the rate of decline in lung function.
Preoperative interventions in patients with COPD aimed at correcting
hypoxemia, relieving bronchospasm, mobilizing and reducing secretions, and treating infections may decrease the incidence of postoperative pulmonary complications. Patients at greatest risk of complications are those with preoperative pulmonary function measurements less than 50% of predicted.
@ Restrictive pulmonary diseases are characterized by decreased lung compliance. Lung volumes are typically reduced, with preservation of normal expiratory flow rates. Thus, both forced expiratory volume in the first second of exhalation (FEV,) and forced vital capacity (FVC) are reduced, but the FEV,/FVC ratio is normal.
© Intraoperative pulmonary embolism usually presents as sudden
cardiovascular collapse, hypoxemia, or bronchospasm. A decrease in end-tidal CO, concentration is also suggestive of pulmonary embolism, but is not specific.
The effects of preexisting pulmonary disease during anesthesia and in the postoperative period are predictable: Greater degrees of preoperative pulmonary impairment are associated with more marked intraoperative alterations in respiratory function and higher rates of postoperative pulmonary complications. Failure to recognize patients who are at increased risk may result in patients not receiving appropriate perioperative care. This chapter examines pulmonary risk in general and then reviews the anesthetic approach for patients with the most common types of respiratory disease.

Certain risk factors (Table 24—1) may predispose patients to postoperative pulmonary complications. Atelectasis, pneumonia, pulmonary embolism, and respiratory failure are common following surgery, but the incidence varies widely (from 6% to 60%), depending on the patient population studied and the surgical procedures performed. In the general surgery population, the incidence of postoperative pulmonary complications ranges from 2.0% to 5.6%. The two strongest predictors of complications seem to be operative site and a history of dyspnea, the latter of which correlates with the degree of preexisting pulmonary disease.
TABLE 24-1 Risk factors for postoperative pulmonary complications.!
. Patient-Related Factors?
Procedure-Related Factors?

Advanced age
Aortic aneurysm repair
ASA’ class =2
Thoracic surgery
Congestive heart failure
Abdominal surgery
Functional dependency
Upper abdominal
surgery Neurosurgery Prolonged surgery Head and neck surgery Emergency surgery Vascular
Chronic obstructive
pulmonary disease
surgery
Use of general anesthesia

Weight loss
Impaired sensorium
Cigarette use
Alcohol use
Abnormal chest exam

Well-controlled asthma
Hip
Obesity surgery Genitourinary/gynecologic surgery

Obstructive sleep apnea* Poor exercise capacity
Esophageal surgery
'Data from Smetana GW, Lawrence VA, Cornell JE, et al. Preoperative pulmonary risk stratification for noncardiothoracic surgery: Systematic review for the American College of Physicians. Ann Intern Med. 2006 Apr 18;144(8):581-595,
*Within each evidence category, risk factors are listed according to strength of evidence, with the first factor listed having the strongest evidence.
3ASA, American Society of Anesthesiologists.
'Subsequent evidence indicates that this is a probable risk factor.
Perioperative transfusion
FIGURE 24-1 Evaluation of dyspnea. ABGs, arterial blood gases; BNP, brain natriuretic peptide; BUN, blood urea nitrogen; COPD, chronic obstructive pulmonary disease; CT, computed tomography; PFTs, pulmonary function tests. (Reproduced with permission from Sweitzer BJ, Smetana GW. Identification and evaluation of the patient with lung disease. Anesthesiol Clin. 2009 Dec;27(4):673-686.)

Obstructive and restrictive diseases are the two most common abnormal patterns as determined by PFTs. The former are by far the more common. Obstructive diseases include asthma, emphysema, chronic bronchitis, cystic fibrosis, bronchiectasis, and bronchiolitis. The primary characteristic of these disorders is resistance to airflow. An MMEF of less than 70% (forced expiratory flow [FEF><_750,]) is often the only abnormality early in the course of these disorders. Values for FEF. 750, in adult males and females are normally greater than 2.0 L/s and 1.6 L/s, respectively. As the disease progresses, both forced expiratory volume in the first second of exhalation (FEV,) and the FEV,/FVC (forced vital capacity) ratio are less than 70% of the predicted values.
Elevated airway resistance and air trapping increase the work of breathing; respiratory gas exchange is impaired because of ventilation/perfusion (V/Q) imbalance. The predominance of expiratory airflow resistance results in air trapping; residual volume and total lung capacity (TLC) increase. Wheezing is a common finding and represents turbulent airflow. It is often absent with mild obstruction that may be manifested initially only by prolonged exhalation. Progressive obstruction typically results first in expiratory wheezing only, and then in both inspiratory and expiratory wheezing. With marked obstruction, wheezing may be absent when airflow has nearly ceased.

Asthma is a common disorder, affecting 5% to 7% of the population. Its primary characteristic is airway (bronchiolar) inflammation and hyperreactivity in response to a variety of stimuli. Clinically, asthma is manifested by episodic attacks of dyspnea, cough, and wheezing. Airway obstruction, which is generally reversible, is the result of bronchial smooth muscle constriction, edema, and increased secretions. Classically, the obstruction is precipitated by a variety of airborne substances, including pollens, animal dander, dusts, pollutants, and various chemicals. Some patients also develop bronchospasm following ingestion of aspirin, nonsteroidal antiinflammatory agents, sulfites, or other compounds. Exercise, cold air, emotional excitement, and viral infections also precipitate bronchospasm in many patients. Asthma is classified as acute or chronic. Chronic asthma is further classified as intermittent (mild) and mild, moderate, and severe persistent disease.
The terms extrinsic (allergic) asthma (attacks related to environmental exposures) and intrinsic (idiosyncratic) asthma (attacks usually occurring without provocation) were used in the past, but these classifications were imperfect; many patients show features of both forms. Moreover, overlap with chronic bronchitis (see later discussion) is common.

The pathophysiology of asthma involves the local release of various chemical mediators in the airway, and, possibly, overactivity of the parasympathetic

Drugs used to treat asthma include B-adrenergic agonists, methylxanthines, glucocorticoids, anticholinergics, leukotriene modifiers, and mast-cell— stabilizing agents. Although devoid of any bronchodilating properties, cromolyn sodium and nedocromil are effective in preventing bronchospasm by blocking the degranulation of mast cells.

The emphasis in evaluating patients with asthma should be on determining the severity and recent course of the disease, as well as on ascertaining whether the patient is in optimal condition. Patients with poorly controlled asthma or wheezing at the time of anesthesia induction have a greater risk of perioperative

The most critical time for asthmatic patients undergoing anesthesia is during
distribution of gas flow to both lungs and may help avoid air trapping. The Paco, may increase, which is acceptable if there is no contraindication from a cardiovascular or neurologic perspective.

® Intraoperative bronchospasm is usually manifested as wheezing, increasing peak airway pressures (plateau pressure may remain unchanged), decreasing exhaled tidal volumes, or a slowly rising @ waveform on the capnograph. Other causes can simulate bronchospasm: These include obstruction of the tracheal tube from kinking, secretions, or an overinflated balloon; bronchial intubation; active expiratory efforts (straining); pulmonary edema or embolism; and pneumothorax. Bronchospasm should be treated by increasing the concentration of the volatile agent and administering an aerosolized bronchodilator. Infusion of low-dose epinephrine may be needed if bronchospasm is refractory to other interventions.
Intravenous hydrocortisone can be given, particularly in patients with a history of responding to glucocorticoid therapy. At the completion of surgery, the patient should ideally be free of wheezing. Reversal of nondepolarizing neuromuscular blocking agents with anticholinesterase agents generally does not precipitate bronchoconstriction if preceded by the appropriate dose of an anticholinergic agent. Sugammadex avoids the issue of increasing acetylcholine concentration; however, cases of allergic reaction to sugammadex have been reported. Deep extubation (before the return of airway reflexes) reduces the risk of bronchospasm on emergence. Lidocaine as a bolus (1.5—2 mg/kg) may help to obtund airway reflexes upon emergence.

COPD is the most common pulmonary disorder encountered in anesthetic practice,
and its
prevalence increases
with age.
The disorder
is strongly
associated with
cigarette currently
smoking defined
fully reversible.
as and
a
disease
The of
small and
has
a
male state
chronic
@
characterized airflow
large parenchymal
airway by
limitation predominance.
COPD
airflow limitation
of disease
destruction components
varying
Most
(chronic this
disease that
is due
to bronchitis/bronchiolitis)
(emphysema), from
patients show
with patient
with expiratory
obstruction is
is not
a
mixture and
representation of
these to
patient.
COPD
airflow are
asymptomatic obstruction
has an
element shown
by improvement
advancing or
only mildly
upon
PFTs.
of reversibility,
in response
disease,
In many
presumably to
two symptomatic,
patients, from
administration maldistribution
but the
bronchospasm of
of results
in areas
of low
(V/Q)
ratios high (V/Q) ratios (dead space).

The clinical diagnosis of chronic bronchitis is defined by the presence of a productive cough on most days of 3 consecutive months for at least 2 consecutive years. In addition to cigarette smoking, exposure to air pollutants, occupational exposure to dusts, recurrent pulmonary infections, and familial factors may be responsible. Secretions from hypertrophied bronchial mucous glands and mucosal edema from inflammation of the airways produce airflow obstruction. Recurrent pulmonary infections (viral and bacterial) are common and often associated with bronchospasm. RV is increased, but TLC is often normal. and are common.
Intrapulmonary shunting hypoxemia
In patients
with
COPD, chronic
hypoxemia leads
to erythrocytosis,
and eventually
right ventricular
failure
(cor pulmonale);
this combination
of findings
is often
referred to
as the
“blue bloater
syndrome, but less than 5% of patients with COPD fit this description (Table both
ventilation
(intrapulmonary
a
bronchodilator).
and
(as
With pulmonary
blood shunt),
as well
as flow
areas of
29
24—2). In the course of disease progression, patients gradually develop chronic CO, retention; the normal ventilatory drive becomes less sensitive to arterial CO, tension and may be depressed by oxygen administration (see below).

Cough, Chronic Bronchitis = Frequent. Cough, Emphysema = With exertion. Sputum, Chronic Bronchitis = Copious. Sputum, Emphysema = Scant. Hematocrit, Chronic Bronchitis = Elevated. Hematocrit, Emphysema = Normal. Paco, (mm Hg), Chronic Bronchitis = Often elevated (>40). Paco, (mm Hg), Emphysema = Usually normal or <40. Chest radiograph, Chronic Bronchitis = Increased lung markings. Chest radiograph, Emphysema = Hyperinflation. Elastic recoil, Chronic Bronchitis = Normal. Elastic recoil, Emphysema = Decreased. Airway resistance, Chronic Bronchitis = Increased. Airway resistance, Emphysema = Normal to slightly increased. Cor pulmonale, Chronic Bronchitis = Early. Cor pulmonale, Emphysema = Late

Emphysema is a pathological disorder characterized by irreversible enlargement of the airways distal to terminal bronchioles and destruction of alveolar septa. The diagnosis can be reliably made with computed tomography (CT) of the chest. Mild apical emphysematous changes are a normal, clinically insignificant consequence of aging. Significant emphysema is more frequently related to cigarette smoking. Less commonly, emphysema occurs at an early age and is associated with a homozygous deficiency of a,-antitrypsin. This is a protease inhibitor that prevents excessive activity of proteolytic enzymes (mainly elastase) in the lungs; these enzymes are produced by pulmonary neutrophils and macrophages in response to infection and pollutants. Emphysema associated with smoking may similarly be due to a relative imbalance between protease and antiprotease activities in susceptible individuals.
Emphysema may exist in a centrilobular or panlobular form. The centrilobular (or centriacinar) form results from dilation or destruction of the respiratory bronchioles, is more closely associated with tobacco smoking, and has predominantly an upper lobe distribution. The panlobular (or panacinar) form results in a more even dilation and destruction of the entire acinus, is associated with a,-antitrypsin deficiency, and has predominantly a lower lobe distribution.
Loss of
the elastic
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normally supports
small airways
by radial
traction allows
premature limitation
with collapse
air trapping
characteristically have
Disruption of
during exhalation,
and hyperinflation
increases leading
(see
Table in
RV, the
lead to
decreased gas
exchange.
FRC,
TLC, alveolar—capillary
diffusion structure
lung capacity,
Also, hyperinflated
normal portions
mismatch.
Due preserved
to expiratory
24—2).
and the
Patients
RV/TLC
and
(V/Q)
parenchyma of
the to
the until
occurs slowly
blood
(V/Q)
loss of
mismatch, may
lung, become
resulting higher
diffusibility ratio.
the acinar
structure and

impairment compressed
in
a
further of
abnormalities and
generally gas
analysis.
CO,, become
results of
by the
increase in
its elimination
severe.
is
Chronic in
Arterial oxygen
a
CO, compensated
tension respiratory
is usually
the
(V/Q)
well retention
acidosis normal
or on
slightly reduced.
Acute
CO, retention
is
a
sign of
impending respiratory
failure.
Destruction of pulmonary capillaries in the alveolar septa leads to the development of mild to moderate pulmonary hypertension. When dyspneic, patients with emphysema often purse their lips to delay closure of the small airways, which accounts for the term "pink puffers" that is often used. However, as mentioned above, most patients diagnosed with COPD have a combination of bronchitis and emphysema.

@ Treatment for COPD is primarily supportive. Cessation of smoking is the long-term intervention that has been shown to reduce the rate of decline in lung function. Various guidelines have been suggested to aid in the primary medical management of patients with COPD. In general, spirometry is employed to assess the severity of airflow reduction characteristic of obstruction, and whether there is a response to bronchodilators. For bronchodilator-responsive patients, flow
short-acting bronchodilators are recommended when FEV, is greater than 80% of predicted and longer-acting bronchodilators and inhaled corticosteroids are suggested as FEV, and patient symptoms worsen. Inhaled B,-adrenergic agonists, glucocorticoids, and ipratropium are routinely employed. Hypoxemia is treated with supplemental oxygen. Patients with chronic hypoxemia (PaO, <55 mm Hg) and pulmonary hypertension require low-flow oxygen therapy (1-2 L/min). CO, retention may be exacerbated in patients with reduced hypoxic ventilatory drive. Consequently, oxygen therapy is targeted to a hemoglobin oxygen saturation of 90%.
Pulmonary rehabilitation may improve the functional status of the patient by improving physical symptoms and exercise capacity.

Patients with COPD should be prepared prior to elective surgical procedures in the same way as patients with asthma (see earlier discussion). They should be questioned about recent changes in dyspnea, sputum, and wheezing. Patients with an FEV, less than 50% of predicted (1.2—1.5 L) usually have dyspnea on exertion, whereas those with an FEV, less than 25% (<1 L in men) typically have dyspnea with minimal activity. The latter finding, in patients with predominantly chronic bronchitis, is also often associated with CO, retention and pulmonary hypertension. PFTs, chest radiographs, and arterial blood gas measurements, if available, should be reviewed carefully. The presence of bullous changes on the radiograph should be noted. Many patients have concomitant cardiac disease and should also receive a careful cardiovascular evaluation.
@ In contrast to asthma, only limited improvement in respiratory function may be seen after a short period of intensive preoperative preparation. Nonetheless, preoperative interventions in patients with COPD aimed at correcting hypoxemia, relieving bronchospasm, mobilizing and reducing secretions, and treating infections may decrease the incidence of postoperative pulmonary complications. Patients at greatest risk of complications are those with preoperative pulmonary function measurements less than 50% of predicted. The possibility that postoperative ventilation may be necessary in high-risk patients should be discussed with both the patient and the surgeon.
Smoking should be discontinued for at least 6 to 8 weeks before the operation to decrease secretions and to reduce pulmonary complications. Cigarette smoking increases mucus production and decreases clearance. Both gaseous and particulate phases of cigarette smoke can deplete glutathione and vitamin C and may promote oxidative injury to tissues. Cessation of smoking for as little as 24 h has theoretical beneficial effects on the oxygen-carrying capacity of hemoglobin; acute inhalation of cigarette smoke releases carbon monoxide, which increases carboxyhemoglobin levels, as well as nitric oxide, and nitrogen dioxide, which can lead to formation of methemoglobin. Long-acting bronchodilators and mucolytics should be continued, including on the day of surgery. COPD exacerbations should be treated aggressively. Preoperative chest physiotherapy and lung expansion interventions with incentive spirometry, deep breathing exercises, cough, chest percussion, and postural drainage may be beneficial in decreasing postoperative pulmonary complications.

Although regional anesthesia is often considered preferable to general anesthesia, high spinal or epidural anesthesia can decrease lung volumes, restrict the use of accessory respiratory muscles, and produce an ineffective cough, leading to dyspnea and retention of secretions. Loss of proprioception from the chest and positions such as lithotomy or lateral decubitus may accentuate dyspnea in awake patients. Concerns about hemidiaphragmatic paralysis may make interscalene blocks a less attractive option in the lung disease patient. Preoxygenation prior to induction of general anesthesia prevents the rapid oxygen desaturation often seen in these patients. The selection of anesthetic agents and general intraoperative management must be tailored to the specific needs and goals of every patient. Unfortunately, the use of bronchodilating anesthetics improves only the reversible component of airflow obstruction; significant expiratory obstruction may still present, even under deep anesthesia. Expiratory airflow limitation, especially under positive pressure ventilation, may lead to air trapping, dynamic hyperinflation, and elevated intrinsic positive endexpiratory pressure (IPEEP). Dynamic hyperinflation may result in volutrauma to the lungs, hemodynamic instability, hypercapnia, and acidosis. Interventions to mitigate air trapping include: (1) allowing more time to exhale by decreasing both the respiratory rate and inspiratory/expiratory (1:E) ratio; (2) allowing permissive hypercapnia; (3) applying low levels of extrinsic PEEP; and (4) aggressively treating bronchospasm.
Intraoperative causes of hypotension in these patients include (in addition to
but shallow, breathing pattern. Respiratory gas exchange is usually maintained until the disease process is advanced.

Acute intrinsic pulmonary disorders include pulmonary edema (including the acute respiratory distress syndrome [ARDS]), infectious pneumonia, and aspiration pneumonitis.

Reduced lung compliance in these disorders is primarily due to an increase in extravascular lung water, from an increase in either pulmonary capillary pressure or pulmonary capillary permeability. Increased pressure occurs with left ventricular failure, whereas fluid overload and increased permeability are present with ARDS. Localized or generalized increases in permeability also occur following aspiration or infectious pneumonitis.

Patients with acute pulmonary disease should be spared elective surgery. In preparation for emergency procedures, oxygenation and ventilation should be optimized preoperatively to the greatest extent possible. Fluid overload should be treated with diuretics; heart failure may also require treatment. Large pleural effusions may require drainage before anesthesia. Similarly, massive abdominal distention should be relieved by nasogastric suction or drainage of ascites. Persistent hypoxemia may require mechanical ventilation.

Selection of anesthetic agents should be tailored to each patient. Surgical patients with acute pulmonary disorders, such as ARDS, cardiogenic pulmonary edema, or pneumonia, are critically ill; anesthetic management should be a continuation of their preoperative intensive care. Increased inspired oxygen concentrations and PEEP may be required. The decreased lung compliance results in high peak inspiratory pressures during positive-pressure ventilation and increases the risk of barotrauma and volutrauma. Tidal volumes for these patients should be reduced to 4 to 6 mL/kg, with a compensatory increase in the ventilatory rate (14-18 breaths/min), even if the result is an increase in end-tidal CO,. Airway pressure should generally not exceed 30 cm H,O. Right ventricular function may be challenged due to to increases in pulmonary vascular resistance secondary to permissive hypercapnia.

Chronic intrinsic pulmonary disorders are also often referred to as interstitial lung diseases. Regardless of etiology, the disease process is generally characterized by an insidious onset, chronic inflammation of alveolar walls and perialveolar tissue, and progressive pulmonary fibrosis. The latter can eventually interfere with gas exchange and ventilatory function. The inflammatory process may be primarily confined to the lungs or may be part of a generalized multiorgan process. Causes include hypersensitivity pneumonitis from occupational and environmental pollutants, drug toxicity (bleomycin and nitrofurantoin), radiation pneumonitis, idiopathic pulmonary fibrosis, autoimmune diseases, and sarcoidosis. Chronic pulmonary aspiration, oxygen toxicity, and severe ARDS can also produce chronic fibrosis.

Patients typically present with dyspnea on exertion and sometimes a nonproductive cough. Symptoms of cor pulmonale are present only with advanced disease. Physical examination may reveal fine (dry) crackles over the lung bases, and, in late stages, evidence of right ventricular failure. The chest radiograph progresses from a "ground-glass" appearance to prominent reticulonodular markings, and, finally, to a "honeycomb" appearance. Arterial blood gases usually show mild hypoxemia with normocarbia. PFTs are typical of a restrictive ventilatory defect (see above), and carbon monoxide diffusing capacity is reduced.
Treatment is directed at abating the disease process and preventing further exposure to the causative agent (if known). If the patient has chronic hypoxemia, oxygen therapy may be started to prevent, or attenuate, right ventricular failure.

Preoperative evaluation should focus on the underlying disease process and the degree of pulmonary impairment. A history of dyspnea on exertion (or at rest) should be evaluated further with PFTs and arterial blood gas analysis. A vital capacity of less than 15 mL/kg is indicative of severe dysfunction (normal is >70 mL/kg). A chest radiograph is helpful in assessing disease severity.

The management of these patients is complicated by their predisposition to hypoxemia and their need for controlled ventilation to ensure optimum gas exchange. The reduction in FRC (and oxygen stores) predisposes these patients to rapid hypoxemia following induction of anesthesia. Because these patients may be more susceptible to oxygen-induced toxicity, particularly patients who have received bleomycin, the inspired fractional concentration of oxygen should be kept to the minimum concentration compatible with acceptable oxygenation (Spo, of >90%). Protective ventilation strategies employed in ventilated patients in the intensive care unit should be continued through to the operating room. Nitric oxide may be used to reduce pulmonary vascular resistance and reduce the work of the right ventricle.
Extracorporeal membrane oxygenation (ECMO) is increasingly used in the management of acute respiratory failure. Following anticoagulation, blood is drained from venous cannulae and delivered to a membrane oxygenator. Oxygenated blood can then either be returned either to the venous system, if cardiac function is preserved, or pumped into the arterial circulation, bypassing the heart and lungs. Consequently, ECMO can provide transient support for both cardiac and pulmonary failure.

Extrinsic restrictive pulmonary disorders alter gas exchange by interfering with normal lung expansion. They include pleural effusions, pneumothorax, mediastinal masses, kyphoscoliosis, pectus excavatum, neuromuscular disorders, and increased intraabdominal pressure from ascites, pregnancy, or bleeding. Marked obesity also produces a restrictive ventilatory defect. Anesthetic considerations are similar to those discussed for intrinsic restrictive disorders.

Pulmonary embolism results from the entry of blood clots, fat, tumor cells, air, amniotic fluid, or foreign material into the venous system. Clots from the lower extremities, pelvic veins, or, less commonly, the right side of the heart are usually responsible. Venous stasis or hypercoagulability is often contributory (Table 24—3). Pulmonary embolism can also occur intraoperatively.

Prolonged bed rest
Postpartum state
Fracture of the lower extremities
Surgery on the lower extremities
Carcinoma
Heart failure
Obesity
Surgery lasting more than 30 min
Hypercoagulability
Antithrombin III deficiency
Protein C deficiency
Protein S deficiency
Factor V Leiden mutation

Embolic occlusions in the pulmonary circulation increase dead space, and, if minute ventilation does not change, this increase in dead space should theoretically increase Paco,. However, in practice, hypoxemia is more often seen. Pulmonary emboli acutely increase pulmonary vascular resistance by reducing the cross-sectional area of the pulmonary vasculature, causing reflex and humoral vasoconstriction. Localized or generalized reflex bronchoconstriction further increases areas with low (V/Q) ratios. The net effect is an increase in (V/Q) mismatch and hypoxemia. The affected area loses its surfactant within hours and may become atelectatic within 24 to 48 h.
Pulmonary infarction occurs if the embolus involves a large vessel and collateral blood flow from the bronchial circulation is insufficient for that part of the lung (incidence <10%). In previously healthy persons, occlusion of more than 50% of the pulmonary circulation (massive pulmonary embolism) is necessary before sustained pulmonary hypertension is seen. Patients with preexisting cardiac or pulmonary disease can develop acute pulmonary hypertension with occlusions of lesser magnitude. A sustained increase in right ventricular afterload can precipitate acute right ventricular failure and hemodynamic collapse. If the patient survives acute pulmonary thromboembolism, the thrombus usually begins to resolve within 1 to 2 weeks.

Clinical manifestations of pulmonary embolism include sudden tachypnea, dyspnea, chest pain, or hemoptysis. The latter generally implies lung infarction. Symptoms are often absent or mild and nonspecific unless massive embolism has occurred. Wheezing may be present on auscultation. Arterial blood gas analysis typically shows mild hypoxemia with respiratory alkalosis (the latter due to an increase in ventilation). The chest radiograph is commonly normal, but may show an area of oligemia (radiolucency), a wedge-shaped density with an infarct, atelectasis with an elevated diaphragm, or an asymmetrically enlarged proximal pulmonary artery with acute pulmonary hypertension. Cardiac signs include tachycardia and wide fixed splitting of the S, heart sound; hypotension with elevated central venous pressure is usually indicative of right ventricular failure. The electrocardiogram frequently shows tachycardia and may show signs of acute cor pulmonale, such as new right axis deviation, right bundle-branch block, and tall peaked T waves. Ultrasound studies of the lower extremities also may be helpful in demonstrating deep venous thrombosis (DVT). The diagnosis of embolism is more difficult to make intraoperatively (see below).
Computed tomography angiography is performed emergently when pulmonary embolism is suspected. Echocardiography can also be used to assist in the diagnosis under emergent conditions in unstable patients perioperatively. Right ventricular overload is seen following significant pulmonary embolism. Sometimes clot can be seen in the right heart and pulmonary artery confirming the diagnosis. At other times, only the signs of right ventricular overload are seen (eg, tricuspid regurgitation, right ventricular dilation). The left ventricle may be relatively under-loaded secondary to the inadequate delivery of blood across the pulmonary circulation as a consequence of the embolus.

The best treatment for perioperative pulmonary embolism is prevention. Various regimens for DVT prophylaxis are employed, including heparin (unfractionated heparin 5000 U subcutaneously every 12 h begun preoperatively or immediately postoperatively in high-risk patients), enoxaparin, fondaparinux, and, most importantly, early ambulation after surgery. Patients at risk for thrombophilia are treated with warfarin. Newer anticoagulants such as factor X, inhibitors (eg, rivaroxaban, apixaban) and the direct thrombin inhibitor dabigatran will likely assume a greater role in DVT prophylaxis. The use of intermittent pneumatic compression of the legs may decrease the incidence of venous thrombosis in the legs, but not in the pelvis or the heart.
After a pulmonary embolism, parenteral anticoagulation prevents the formation of new blood clots or the extension of existing clots. Low-molecularweight heparin (LMWH) or fondaparinux are now preferred over intravenous unfractionated heparin for initial anticoagulation following a pulmonary embolism for most patients. All patients should start warfarin therapy concurrent with starting parenteral therapy, and the two should overlap for a minimum of 5 days. The international normalized ratio should also be within the therapeutic range (>2.0) for at least 24 h before discontinuation of parenteral DVT prophylaxis. Warfarin should be continued for 3 to 12 months. Thrombolytic therapy is indicated in patients with massive pulmonary embolism and hypotension. Recent surgery and active bleeding are contraindications to anticoagulation and thrombolytic therapy. In these cases, an inferior vena cava filter may be placed to prevent recurrent pulmonary emboli. Pulmonary embolectomy may be lifesaving for hemodynamically unstable patients with massive embolism in whom thrombolytic therapy is contraindicated or ineffective.

Patients with acute pulmonary embolism may present in the operating room for placement of an inferior vena cava filter, or, rarely, for pulmonary embolectomy. In most instances, the patient will have a history of pulmonary embolism and presents for unrelated surgery; in this group of patients, the risk of interrupting anticoagulant therapy perioperatively is unknown. If the acute episode is more than 1 year old, the risk associated with temporarily stopping anticoagulant therapy is probably small. Moreover, except in the case of chronic recurrent pulmonary emboli, pulmonary function has usually returned to normal. The emphasis in the perioperative management of these patients should be in preventing new episodes of embolism (see earlier discussion).

Vena cava filters are usually placed percutaneously under local anesthesia with sedation.
Patients presenting for emergency pulmonary embolectomy are critically ill. They are usually already intubated, but tolerate positive-pressure ventilation poorly. Inotropic support is usually necessary until they are placed on cardiopulmonary bypass to facilitate clot removal. Inotropic support may be required to separate from cardiopulmonary bypass.

Significant pulmonary embolism is rare during anesthesia. Diagnosis requires a high index of suspicion. Air emboli are common but are often overlooked unless large amounts are entrained. Fat embolism, as well as embolism of microthrombi and bone debris, can occur during orthopedic procedures; amniotic fluid embolism is a rare, unpredictable, and often fatal, complication of late pregnancy and obstetrical delivery. Thromboembolism may occur intraoperatively during prolonged procedures. The clot may have been present prior to surgery or may form intraoperatively; surgical manipulations or a change in the patient's position may then dislodge the venous thrombus. Manipulation of tumors with intravascular extension (eg, renal cell carcinoma invading the vena cava) can similarly produce pulmonary embolism.
© Intraoperative pulmonary embolism usually presents as sudden cardiovascular collapse, hypoxemia, or bronchospasm. A decrease in end-tidal CO, concentration is also suggestive of pulmonary embolism but is not specific. Invasive monitoring may reveal elevated central venous pressure. Depending on the type and location of an embolism, a transesophageal echocardiogram may be helpful; this may not reveal the embolus but will often demonstrate right heart distention and dysfunction. If air is identified in the right atrium, or if it is suspected, emergent central vein cannulation and aspiration of the air may be lifesaving. For all other emboli, treatment is supportive, with intravenous fluids and inotropes. Placement of a vena cava filter may be considered postoperatively.

A 45-year-old woman is scheduled for a laparoscopic cholecystectomy. Known medical problems include morbid obesity and a history of smoking.
What are the advantages of laparoscopic cholecystectomy compared with open cholecystectomy?
Laparoscopic techniques have rapidly increased in popularity because of the multiple benefits associated with much smaller incisions than with traditional open techniques. These benefits include decreased postoperative pain, less postoperative pulmonary impairment, a reduction in postoperative ileus, shorter hospital stays, earlier ambulation, and smaller surgical scars. Thus, laparoscopic surgery can provide substantial medical and economic advantages.
How does laparoscopic surgery affect intraoperative pulmonary function?
The hallmark laparoscopy is the creation a pneumoperitoneum with pressurized CO). The resulting increase in intraabdominal pressure displaces the diaphragm cephalad, causing a decrease in lung compliance and an increase in peak inspiratory pressure. Atelectasis, diminished FRC, ventilation/perfusion mismatch, and pulmonary shunting contribute to a decrease in arterial oxygenation. These changes should be exaggerated in this obese patient with a long history of tobacco use.
The high solubility of increases systemic absorption by the vasculature of the peritoneum. This, combined with smaller tidal volumes because of poor lung compliance, leads to increased arterial CO, levels and decreased
of of CO, pH.

A head-down (Trendelenburg) position causes a cephalad shift in abdominal viscera and the diaphragm. FRC, total lung volume, and pulmonary compliance will be decreased. Although these changes are usually well tolerated by healthy patients, this patient's obesity and presumed preexisting lung disease increase the likelihood for hypoxemia. A head-down position also tends to shift the trachea upward, so that a tracheal tube anchored at the mouth may migrate into the right mainstem bronchus. This tracheobronchial shift may be exacerbated during insufflation of the abdomen.
After insufflation, the patient's position is usually changed to a steep head-up position (reverse Trendelenburg) to facilitate surgical dissection. The respiratory effects of the head-up position are the opposite of the headdown position.

Moderate insufflation pressures usually leave heart rate, central venous pressure, and cardiac output unchanged or slightly elevated. This seems to result from increased effective cardiac filling because blood tends to be forced out of the abdomen and into the chest. Higher insufflation pressures (>25 cm H2O or 18 mm Hg), however, tend to collapse the major abdominal veins (particularly the inferior vena cava), which decreases venous return and leads to a drop in preload and cardiac output in some patients.
Hypercarbia will stimulate the sympathetic nervous system and increase blood pressure, heart rate, and the risk of arrhythmias. Attempting to compensate by increasing the tidal volume or respiratory rate will increase the mean intrathoracic pressure, further hindering venous return and increasing mean pulmonary artery pressures. These effects can prove particularly challenging in patients with restrictive lung disease, impaired cardiac function, or intravascular volume depletion.
Although the Trendelenburg position increases preload, mean arterial pressure and cardiac output usually either remain unchanged or decrease. These seemingly paradoxical responses may be explained by carotid and aortic baroreceptor-mediated reflexes. The reverse Trendelenburg position decreases preload, cardiac output, and mean arterial pressure.
Describe the advantages and disadvantages of alternative anesthetic techniques for this patient.
Anesthetic approaches to laparoscopic surgery have in the past included infiltration of local anesthetic with an intravenous sedative, epidural or spinal anesthesia, or general anesthesia. Experience with local anesthesia has been largely limited to brief gynecological procedures (laparoscopic tubal sterilization, intrafallopian transfers) in young, healthy, and motivated patients. Although postoperative recovery is rapid, patient discomfort and suboptimal visualization of intraabdominal organs preclude the use of this local anesthesia technique for laparoscopic cholecystectomy.
Epidural or spinal anesthesia represents another rarely chosen alternative for laparoscopic surgery. A high level is required for complete muscle relaxation and to prevent diaphragmatic irritation caused by gas insufflation and surgical manipulations. An obese patient with lung disease may not be able to increase spontaneous ventilation to maintain normocarbia in the face of a T2 level regional block during insufflation and a 20° Trendelenburg position. Another disadvantage of a regional technique is the occasional occurrence of referred shoulder pain from diaphragmatic irritation.

Tracheal intubation with positive-pressure ventilation is usually favored for many reasons: the risk of regurgitation from increased intraabdominal pressure during insufflation; the necessity for controlled ventilation to prevent hypercapnia; the relatively high peak inspiratory pressures required because of the pneumoperitoneum; the need for neuromuscular blockade during surgery to allow lower insufflation pressures, provide better visualization, and prevent unexpected patient movement; and the placement of a nasogastric tube and gastric decompression to minimize the risk of visceral perforation during trocar introduction and optimize visualization. The obese patient presented here would benefit from intubation to decrease the likelihood of hypoxemia, hypercarbia, and aspiration. In lower risk patients, secondgeneration supraglottic airway devices are increasingly employed for a variety of surgical procedures, including those performed laparoscopically.

Monitoring end-tidal CO, normally provides an adequate guide for determining the minute ventilation required to maintain normocarbia. This assumes a constant gradient between arterial CO, and end-tidal CO, which is generally valid in healthy patients undergoing laparoscopy. This assumption would not apply if alveolar dead space changes during surgery. For example, any significant reduction in lung perfusion increases alveolar dead space and therefore increases the gradient between arterial and endtidal CO,. This may occur during laparoscopy if cardiac output drops because of high inflation pressures, the reverse Trendelenburg position, or gas embolism. Furthermore, abdominal distention lowers pulmonary compliance. Large tidal volumes are usually avoided because they are associated with high peak inspiratory pressures and can cause considerable movement of the surgical field.

Surgical complications include hemorrhage, if a major abdominal vessel is lacerated, or peritonitis, if a viscus is perforated during trocar introduction. Significant intraoperative hemorrhage may go unrecognized because of the limitations of laparoscopic visualization. Fulguration has been associated with bowel burns and bowel gas explosions. The use of pressurized gas introduces the possibility of extravasation of CO, along tissue planes, resulting in subcutaneous emphysema, pneumomediastinum, or pneumothorax. Nitrous oxide should be discontinued and insufflating pressures decreased as much as possible. Patients with this complication may benefit from the continuation of mechanical ventilation into the immediate postoperative period.
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Even though laparoscopic procedures are associated with less muscle trauma and incisional pain than open surgery, pulmonary dysfunction can persist for at least 24 h postoperatively. For example, FEV,, FVC, and forced expiratory flow are reduced by approximately 25% following laparoscopic cholecystectomy, compared with a 50% reduction following open cholecystectomy. The cause of this dysfunction may be related to diaphragmatic tension during the pneumoperitoneum.

Guyatt G, Akl E, Crowther M, et al. Antithrombotic therapy and prevention of thrombosis: 9th ed: American College of Chest Physicians evidence based clinical practice guidelines. Chest. 2012;141(suppl):7s. Qaseem A, Snow V, Fitterman N, et al. Risk assessment for and strategies to reduce perioperative pulmonary complication for patients undergoing noncardiothoracic surgery: a guideline from the American College of Physicians. Ann Intern Med. 2006;144:576. for from
See www.guidelines.gov additional guidelines multiple organizations on deep vein thrombosis prophylaxis and pulmonary embolism.

Canet
J,
Gallart
L,
Gomar
C, et
al.
Prediction complications
in
a
population based
of surgical
postoperative pulmonary
cohort.
2010;113:1338.
Cox J, Jablons D. Operative and perioperative pulmonary emboli. Thorac Surg Clin. 2015;15:289.
Gallart L, Canet J. Post-operative pulmonary complications: Understanding definitions and risk assessment. Best Pract Res Clin Anaesthesiol. 2015;29:315.
Hedenstierna G, Edmark L. Effects of anesthesia on the respiratory system. Best Pract Res Clin Anaesthesiol. 2015;29:273.
- Henzler T, Schoenberg S, Schoepf U, Fink C. Diagnosing acute pulmonary embolism: Systematic review of evidence base and cost effectiveness of imaging tests. J Thorac Imaging. 2012;27:304.
Hurford
WE.
The bronchospastic
patient.
Int
Anesthesiol
Clin.
2000;38:77.
Lakshminarasimhachar
A,
Smetana
G.
Preoperative evaluation:
Estimation of

@ During one-lung ventilation, the mixing of unoxygenated blood from the collapsed upper lung with oxygenated blood from the stillventilated dependent lung widens the alveolar-to-arterial (A-a) O5 gradient and often results in hypoxemia.
@ There are certain clinical situations in which the use of a right-sided double-lumen tube is recommended: (1) distorted anatomy of the left main bronchus by an intrabronchial or extrabronchial mass; (2) compression of the left main bronchus due to a descending thoracic aortic aneurysm; (3) left-sided pneumonectomy; (4) left-sided single lung transplantation; and (5) left-sided sleeve resection.
© If epidural or intrathecal opioids are to be used for postoperative pain control, intravenous opioids should be limited during surgery to prevent excessive postoperative respiratory depression.
@ Postoperative hemorrhage complicates about 3% of thoracotomies and may be associated with up to 20% mortality. Signs of hemorrhage include increased chest tube drainage (>200 mL/h), hypotension, tachycardia, and a falling hematocrit.
@ Bronchopleural fistula presents as a sudden large air leak from the chest tube that may be associated with an increasing pneumothorax and partial lung collapse.
@ Acute herniation of the heart into the operative hemithorax can occur through the pericardial defect that is left following a radical pneumonectomy.
@ Nitrous oxide is contraindicated in patients with cysts or bullae because it can expand the air space and cause rupture. The latter may be signaled by sudden hypotension, bronchospasm, or an abrupt rise in
peak inflation pressure and requires immediate placement of a chest tube.
@ Following transplantation, peak inspiratory pressures should be
maintained at the minimum pressure compatible with good lung
expansion, and
the inspired
oxygen concentration
should be
maintained as
close to
room air
as allowed
by
a
Pao, greater
than
60
mm
Hg.
- © Regardless of the procedure, a common anesthetic concern for patients with esophageal disease is the risk of pulmonary aspiration.

Indications and techniques for thoracic surgery continually evolve. Common indications now include thoracic malignancies (mainly of the lungs and esophagus), chest trauma, esophageal disease, and mediastinal tumors. Diagnostic procedures such as bronchoscopy, mediastinoscopy, and open-lung biopsies are also common. Anesthetic techniques for providing lung separation have allowed the refinement of surgical techniques to the point that many procedures are increasingly performed thoracoscopically.

Thoracic surgery presents a unique set of physiological problems for the anesthesiologist. These include physiological derangements caused by placing the patient in the lateral decubitus position, opening the chest (open pneumothorax), and the need for one-lung ventilation.

The lateral decubitus position provides optimal access for most operations on the lungs, pleura, esophagus, the great vessels, other mediastinal structures, and vertebrae. Unfortunately, this position may significantly alter the normal pulmonary ventilation/perfusion relationships. These derangements are further accentuated by anesthetics, mechanical ventilation, neuromuscular blockade, opening the chest, and surgical retraction. Although perfusion continues to favor the dependent lung (under anesthesia in the supine position the dependent part of the lung is toward the back, rather than toward the feet, as would be true in an awake, standing patient), ventilation progressively favors the less perfused, superior part of the lung. The resulting ventilation/perfusion mismatch increases the risk of hypoxemia.

When a supine patient assumes the lateral decubitus position, ventilation/perfusion matching is preserved during spontaneous ventilation. The dependent (lower) lung receives more perfusion than does the upper lung due to gravitational influences on blood flow distribution in the pulmonary circulation. The dependent lung also receives more ventilation because (1) contraction of the dependent hemidiaphragm is more efficient compared with the nondependent (upper) hemidiaphragm, and (2) the dependent lung is on a more favorable part of the compliance curve (Figure 25—1). However, spontaneous ventilation in this position is the exception, not the rule.
FIGURE 25-1 The effect of the lateral decubitus position on lung compliance.

The decrease in functional residual capacity (FRC) with induction of general anesthesia moves the upper lung to a more favorable part of the compliance curve, but moves the lower lung to a less favorable position (Figure 25-2). Asa result, the upper lung is ventilated more than the dependent lower lung;
ventilation/perfusion mismatching occurs because the dependent lung continues to have greater perfusion.
FIGURE 25-2 The effect of anesthesia on lung compliance in the lateral decubitus position. The upper lung assumes a more favorable position, and the lower lung becomes less compliant.

Controlled positive-pressure ventilation favors the upper lung in the lateral position because it is more compliant than the lower lung. Neuromuscular blockade enhances this effect by allowing the abdominal contents to rise up further against the dependent hemidiaphragm and impede ventilation of the lower lung. Using a rigid "bean bag" to maintain the patient in the lateral decubitus position further restricts movement of the dependent hemithorax. Finally, opening the nondependent side of the chest further accentuates differences in compliance between the two sides because the upper lung is now less restricted in movement. All of these effects worsen ventilation/perfusion mismatching and predispose the patient to hypoxemia.

The lungs are normally kept expanded by a negative pleural pressure—the net result of the tendency of the lung to collapse and the chest wall to expand. When one side of the chest is opened, the negative pleural pressure is lost, and the elastic recoil of the lung on that side tends to collapse it. Spontaneous ventilation with an open pneumothorax in the lateral position results in paradoxical respirations and mediastinal shift. These two phenomena can cause progressive hypoxemia and hypercapnia, but, fortunately, their effects are overcome by the use of positive-pressure ventilation during general anesthesia and thoracotomy.

During spontaneous ventilation in the lateral position, inspiration causes pleural pressure to become more negative on the dependent side, but not on the side of the open pneumothorax. This results in a downward shift of the mediastinum during inspiration and an upward shift during expiration (Figure 25-3). The major effect of the mediastinal shift is to decrease the contribution of the dependent lung to the tidal volume.
EXPIRATION
FIGURE 25-3 Mediastinal shift in a spontaneously breathing patient in the lateral decubitus position. (Reproduced with permission from Tarhan S, Moffitt EA. Principles of thoracic anesthesia. Surg Clin North Am. 1973 Aug;53(4):813-826.)

Spontaneous ventilation in a patient with an open pneumothorax also results in to-and-fro gas flow between the dependent and nondependent lung (paradoxical respiration [pendeluft]). During inspiration, the pneumothorax increases, and gas flows from the upper lung across the carina to the dependent lung. During expiration, the gas flow reverses and moves from the dependent to the upper lung (Figure 25-4).
INSPIRATION
Pneumothorax
FIGURE 25-4 Paradoxical respiration in spontaneously breathing patients on their side. (Reproduced with permission from Tarhan S, Moffitt EA. Principles of thoracic anesthesia. Surg Clin North Am. 1973 Aug;53(4):813-826.)

Intentional collapse of the lung on the operative side facilitates most thoracic procedures, but complicates anesthetic management. Because the collapsed lung continues to be perfused and is deliberately no longer ventilated, the patient develops a large right-to-left @ intrapulmonary shunt (20-30%). During onelung ventilation, the mixing of unoxygenated blood from the collapsed upper lung with oxygenated blood from the still-ventilated dependent lung widens the alveolar-to-arterial (A-a) O, gradient and often results in hypoxemia. Fortunately, blood flow to the nonventilated lung is decreased by hypoxic pulmonary vasoconstriction (HPV) and possibly surgical compression of the upper lung. The surgeon can also clamp the pulmonary arterial supply to the collapsed lung when all else fails.
Factors known to inhibit HPV (thereby increasing venous admixture), and thus worsen the right-to-left shunting, include pulmonary hypertension; hypocapnia alkalosis; increased cardiac output and increased mixed venous Po5; hypothermia; vasodilators such as nitroglycerin, nitroprusside, and nitric oxide; phosphodiesterase inhibitors (milrinone, enoximone, inamrinone), B-adrenergic agonists; calcium channel blockers; and inhalation anesthetics.
Factors that decrease blood flow to the ventilated lung can be equally detrimental; they counteract the effect of HPV by indirectly increasing blood flow to the collapsed lung. Such factors include (1) high mean airway pressures in the ventilated lung due to high positive end-expiratory pressure (PEEP), hyperventilation, or high peak inspiratory pressures; (2) a low Fio,, which produces hypoxic pulmonary vasoconstriction in the ventilated lung; (3)

vasoconstrictors that may have a greater effect on normoxic vessels than hypoxic ones; and (4) intrinsic PEEP that develops due to inadequate expiratory times. Elimination of CO, is usually unchanged by one-lung ventilation, provided that minute ventilation is unchanged; arterial CO, tension is usually not appreciably altered. One-lung ventilation can result in injury to both ventilated and nonventilated lungs. The dependent, ventilated lung is subject to hyperperfusion as well as ventilator-induced trauma secondary to large tidal volume ventilation. The nonventilated, nondependent lung is exposed to both surgical trauma and ischemia reperfusion injuries. We recommend the use of tidal volumes of no more than 4 to 5 mL/kg of predicted body weight, rather than earlier recommendations to use the same tidal volume as during two-lung ventilation.

One-lung ventilation can also be utilized to isolate a lung or to facilitate ventilatory management under certain conditions (Table 25—1). Three techniques can be employed: (1) placement of a double-lumen bronchial tube; (2) use of a single-lumen tracheal tube in conjunction with a bronchial blocker; or (3) insertion of a conventional endotracheal tube into a mainstem bronchus. Doublelumen tubes are most often used.

Confine infection to one lung
Confine bleeding to one lung
Separate ventilation to each lung
Bronchopleural fistula
Tracheobronchial disruption
Large lung cyst or bulla
Severe hypoxemia due to unilateral lung disease
Procedure-related
Repair of thoracic aortic aneurysm
Lung resection
Pneumonectomy
Lobectomy
Segmental resection
Thoracoscopy
Single-lung transplantation
Anterior approach to the thoracic spine
Bronchoalveolar lavage

The principal advantages of double-lumen tubes are relative ease of placement, the ability to ventilate one or both lungs, and the ability to suction either lung. All double-lumen tubes share the following characteristics:
A longer bronchial lumen that enters either the right or left main bronchus and another shorter tracheal lumen that terminates in the lower trachea
A preformed curve that when properly "aimed" allows preferential entry into a bronchus
¢ A bronchial cuff
¢ A tracheal cuff
Ventilation can be delivered to only one lung by clamping the tube delivering gas to either the bronchial or tracheal lumen with both cuffs inflated; disconnecting the appropriate connection distal to the clamp site allows the ipsilateral lung to collapse. Because of differences in bronchial anatomy between the two sides, tubes are designed specifically for either the right or left bronchus. A right-sided double-lumen tube incorporates a modified cuff and a proximal portal on the endobronchial side for ventilation of the right upper lobe. The most commonly used double-lumen tubes are available in several sizes: 35, 37, 39, and 41FR.

On average, the adult trachea is 11 to 13 cm long. It begins at the level of the cricoid cartilage (C6) and bifurcates at the level of the carina behind the sternomanubrial joint (T5). Major differences between the right and left main bronchi are as follows: (1) the larger diameter right bronchus diverges away from the trachea at a less acute angle in relation to the trachea, whereas the left bronchus diverges at a more horizontal angle (Figure 25—5); (2) the right bronchus has upper, middle, and lower lobe branches, whereas the left bronchus divides into only upper and lower lobe branches; and (3) the orifice of the right upper lobe bronchus is typically about 1 to 2.5 cm from the carina, whereas the bifurcation of the left main bronchus is typically about 5 cm distal to the carina. There is considerable anatomic variation: for example, the right upper lobe bronchus will occasionally arise from the trachea itself.
FIGURE 25-5 Anatomy of the tracheobronchial tree. Note bronchopulmonary segments (1-10) as numbered. (Adapted with permission from Gothard JWW, Branthwaite MA. Anesthesia for Thoracic Surgery. Oxford, UK: Blackwell; 1982.)

As previously noted, right-sided double-lumen tubes must have a portal through the bronchial cuff for ventilating the right upper lobe (Figure 25-6). Anatomic variations among individuals in the distance between the carina and the right upper lobe orifice will occasionally result in difficulty ventilating that lobe with right-sided tubes. Either a left-sided or right-sided double-lumen tube can be used in most surgical procedures, irrespective of the operative side; for simplicity, many practitioners prefer to use left-sided tubes for nearly every case. ®@ There are certain clinical situations in which the use of a right-sided doublelumen tube is recommended: (1) distorted anatomy of the left main bronchus by an intrabronchial or extrabronchial mass; (2) compression of the left main bronchus due to a descending thoracic aortic aneurysm; (3) left-sided pneumonectomy; (4) left-sided single lung transplantation; and (5) left-sided sleeve resection. Finally, despite concerns about right upper lobe atelectasis and potentially difficult placement, studies have failed to detect differences in clinical performance of rightand left-sided double-lumen tubes when used clinically.
FIGURE 25-6 Correct position of a leftand right-sided double-lumen tube.

Laryngoscopy with a curved (MacIntosh) blade usually provides better intubating conditions than does a straight blade because the curved blade typically provides more room for manipulation of the large double-lumen tube. Video laryngoscopy can also be employed to facilitate tube placement. The double-lumen tube is passed with the distal curvature concave anteriorly and is rotated 90° (toward the side of the bronchus to be intubated) after the tip passes the vocal cords and enters the larynx (Figure 25-7). At this point, the operator has two options: the tube can be advanced until resistance is felt (the average depth of insertion is about 29 cm [at the teeth]), or alternatively, the fiberoptic bronchoscope can be inserted through the bronchial limb and advanced into the desired bronchus. The double-lumen tube can be advanced over the bronchoscope into the desired bronchus. Correct tube placement should be established using a preset protocol (Figure 25-8 and Table 25—2) and confirmed by flexible fiberoptic bronchoscopy. When problems are encountered in intubating the patient with the double-lumen tube, placement of a single-lumen endotracheal tube should be attempted; once positioned in the trachea, the latter can be exchanged for the double-lumen tube by using a specially designed catheter guide ("tube exchanger"). We have found that double-lumen tubes placed in this way will often, as expected, find their way into the right main stem bronchus.
FIGURE 25-7 Placement of a left-sided double-lumen tube. Note that the tube is turned 90° as soon as it enters the larynx. A: Initial position. B: Rotated 90°. C: Final position.
FIGURE 25-8 Results of unilateral clamping of the bronchial lumen tube when the double-lumen tube is in the correct position.
TABLE 25-2 Protocol for checking placement of a left-sided double-lumen tube.

1. Inflate the tracheal cuff (5-10 mL of air).
2. Check for bilateral breath sounds. Unilateral breath sounds indicate that the tube is too far down (tracheal opening is bronchial).
Inflate the bronchial cuff (1-2 mL). see
Clamp the tracheal lumen.
5. Check for unilateral left-sided breath sounds.
a. Persistence of right-sided breath sounds indicates that the bronchial opening is still in the trachea (tube should be advanced).
b. Unilateral right-sided breath sounds indicate incorrect entry of the tube in the right bronchus.
c. Absence of breath sounds over the entire right lung and the left upper lobe indicates that the tube is too far down the left bronchus.
6. Unclamp the tracheal lumen and clamp the bronchial lumen.
7. Check for unilateral right-sided breath sounds. Absence or diminution of breath sounds indicates that the tube is not far enough down and that the bronchial cuff is occluding the distal trachea.

Most double-lumen tubes easily accommodate bronchoscopes with a 3.6to 4.2-mm outer diameter. When the bronchoscope is introduced into the tracheal lumen and advanced through the tracheal orifice, the carina should be visible (Figure 25-9), and the bronchial limb of the tube should be seen entering the respective bronchus; additionally, the top of the bronchial cuff (usually colored blue) should be visible, but should not extend above the carina. If the bronchial cuff of a left-sided double-lumen tube is not visible, the bronchial limb may have been inserted sufficiently far to allow the bronchial cuff to obstruct the orifice of the left upper or lower lobe; the tube should be withdrawn until the cuff can be identified distal to the carina. The optimal position of a right-sided double-lumen tube is confirmed by placing the fiberoptic scope through the endobronchial lumen, which should show alignment of the endobronchial side portal with the opening of the right upper lobe bronchus. The bronchial cuff should be inflated only to the point at which the audible leak from the open tracheal lumen disappears while ventilating only through the bronchial lumen.
FIGURE 25-9 The view of the carina looking down the tracheal lumen of a properly positioned left double-lumen bronchial tube.

Tube position should be reconfirmed after the patient is positioned for surgery because the tube may move relative to the carina as the patient is turned into the lateral decubitus position. Malpositioning of a double-lumen tube within the tracheobronchial tree may lead to failure of the operative lung to collapse, apparent poor lung compliance, and low exhaled tidal volume. Problems with left-sided double-lumen tubes are usually related to one of three possibilities: (1) the tube tip is too distal; (2) the tube tip is too proximal; or (3) the tube is in the right bronchus (the wrong side). If the tube tip is located too distally, the bronchial cuff can obstruct the left upper or the left lower lobe orifice, and the bronchial lumen can be inserted into the orifice of the left lower or left upper lobe bronchus, respectively. When the tube is not advanced distally enough, the inflated bronchial cuff may be above the carina and also occlude the tracheal lumen. In both instances, deflation of the bronchial cuff improves ventilation to the lung and helps to identify the problem. In some patients, the bronchial lumen may be within the left upper or left lower lobe bronchus but with the tracheal opening remaining above the carina; this situation is suggested by collapse of only one of the left lobes when the bronchial lumen is clamped. In the same situation, if the surgical procedure is in the right thorax, clamping of the tracheal lumen will lead to ventilation of only the left upper or left lower lobe; hypoxia usually develops rapidly.

Right-sided double-lumen tubes can be accidentally inserted into the left mainstem bronchus, inserted too distally or too proximally, or have misalignment of the endobronchial side portal with the opening of the right upper lobe bronchus. If the tube enters the wrong bronchus, the fiberoptic bronchoscope can be used to redirect it into the correct side: (1) the bronchoscope is passed through the bronchial lumen to the tip of the tube; (2) under direct vision, the tube and the bronchoscope are withdrawn together into the trachea just above the carina; (3) the bronchoscope alone is then advanced into the correct bronchus; and (4) the double-lumen tube is gently advanced over the bronchoscope, which functions as a stylet to guide the bronchial lumen into the correct bronchus.

Major complications of double-lumen tubes include (1) hypoxemia due to tube malplacement, tube occlusion, or excessive degrees of venous admixture with one-lung ventilation; (2) traumatic laryngitis; (3) tracheobronchial rupture resulting from traumatic placement or overinflation of the bronchial cuff; and (4) inadvertent suturing of the tube to a bronchus during surgery (detected as the inability to withdraw the tube during attempted extubation).

Bronchial blockers are inflatable devices that are passed alongside or through a single-lumen tracheal tube to selectively occlude a bronchial orifice. The bronchial blocker must be advanced, positioned, and inflated under direct visualization via flexible bronchoscope.
The major advantage of a single-lumen tube with a bronchial blocker is that, unlike a double-lumen tube, it does not need to be replaced with a conventional tracheal tube if the patient will remain intubated postoperatively (see later discussion). Its major disadvantage is that the "blocked" lung collapses slowly (and sometimes incompletely) because of the small size of the channel within the blocker catheter.
There are several types of bronchial blockers. They come in different sizes (7FR and 9FR) and have a 1.4-mm diameter inner lumen. Bronchial blockers have a high-volume low-pressure cuff with either an elliptical or spherical shape. The spherical shape of the cuff facilitates adequate blockade of the right mainstem bronchus. The spherical or the elliptical cuff can be used for the left mainstem bronchus. The inner lumen contains a nylon wire, which exits the

Lung resections are usually carried out for the diagnosis and treatment of pulmonary tumors, and, less commonly, for traumatic lung injury, bullae, complications of necrotizing pulmonary infections, or bronchiectasis.
1.
Tumors
Pulmonary tumors can be either benign or malignant, and, with the widespread use of bronchoscopic sampling (often guided by endobronchial ultrasound), diagnosis is usually available prior to surgery. Hamartomas account for 90% of benign tumors; they are usually peripheral pulmonary lesions and represent disorganized normal pulmonary tissue. Bronchial adenomas are usually central pulmonary lesions that are typically benign, but occasionally may be locally invasive and rarely metastasize. These tumors include pulmonary carcinoids, cylindromas, and mucoepidermoid adenomas. They often obstruct the bronchial lumen and cause recurrent pneumonia distal to the obstruction in the same area. Primary pulmonary carcinoids may secrete multiple hormones, including adrenocorticotropic hormone (ACTH) and arginine vasopressin; however, manifestations of the carcinoid syndrome are uncommon and are more likely with metastatic disease.
Malignant pulmonary tumors are divided into small ("oat") cell and non— small cell carcinomas. The latter group includes squamous cell (epidermoid) tumors, adenocarcinomas, and large cell (anaplastic) carcinomas. Epidermoid and small cell carcinomas usually present as central masses with bronchial lesions; adenocarcinoma and large cell carcinomas are more typically peripheral lesions that often involve the pleura.

Symptoms may include cough, hemoptysis, wheezing, weight loss, productive sputum, dyspnea, or fever. Pleuritic chest pain or pleural effusion suggests pleural extension. Involvement of mediastinal structures is suggested by hoarseness that results from compression of the recurrent laryngeal nerve, Horner's syndrome caused by involvement of the sympathetic chain, an elevated hemidiaphragm caused by compression of the phrenic nerve, dysphagia caused by compression of the esophagus, or the superior vena cava syndrome caused by compression or invasion of the superior vena cava. Pericardial effusion or cardiomegaly suggests cardiac involvement. Extension of apical (superior sulcus) tumors can result in either shoulder or arm pain, or both, because of involvement of the C7—T2 roots of the brachial plexus (Pancoast syndrome). Distant metastases most commonly involve the brain, bone, liver, and adrenal glands.
Lung carcinomas—particularly small cell—can produce remote effects that are not related to malignant spread (paraneoplastic syndromes). Mechanisms include ectopic hormone production and immunological cross-reactivity between the tumor and normal tissues. Cushing's syndrome, hyponatremia (syndrome of inappropriate antidiuretic hormone secretion [SIADH]), and hypercalcemia may be encountered, resulting from ectopic secretion of ACTH, arginine vasopressin, and parathyroid hormone, respectively. Lambert—Eaton (myasthenic) syndrome is characterized by a proximal myopathy in which muscle strength increases with repeated effort (in contrast to myasthenia gravis). Other paraneoplastic syndromes include peripheral neuropathy and migratory thrombophlebitis.

Surgery is the treatment of choice to reduce the tumor burden in nonmetastatic lung cancer. Various perioperative chemotherapy and radiation treatments are likewise employed, but there is wide variation among tissue types in their sensitivity to chemotherapy and radiation.

Resectability is determined by the anatomic stage of the tumor, whereas operability is dependent on the interaction between the extent of the procedure required for cure and the physiological status of the patient. Anatomic staging is accomplished using chest radiography, computed tomography (CT) or magnetic resonance (MR) imaging, bronchoscopy, and (sometimes) mediastinoscopy. The extent of the surgery should maximize the chances for a cure but still allow for adequate residual pulmonary function postoperatively. Lobectomy via a posterior thoracotomy, through the fifth or sixth intercostal space, or (more commonly) using video-assisted thoracoscopic surgery (VATS), is the procedure of choice for most lesions. Segmental or wedge resections may be performed for initial diagnosis or for definitive treatment of small peripheral lesions. Pheumonectomy is necessary for curative treatment of lesions involving the left or right main bronchus or when the tumor extends toward the hilum. A sleeve resection may be employed for patients with proximal lesions and limited pulmonary reserve as an alternative to pneumonectomy; in such instances, the involved lobar bronchus, together with part of the right or left main bronchus, is resected, and the distal bronchus is reanastomosed to the proximal bronchus or the trachea. Sleeve pneumonectomy may be considered for tumors involving the trachea. The incidence of pulmonary complications after thoracotomy and lung resection is about 30% and is related not only to the amount of lung tissue resected, but also to the disruption of chest wall mechanics due to the thoracotomy. Postoperative pulmonary dysfunction seems to be less after VATS than The rate for is
"open" thoracotomy. mortality pneumonectomy generally more than twice that for a lobectomy. Mortality is greater for right-sided than left-sided pneumonectomy, possibly because of greater loss of lung tissue.

A comprehensive preoperative assessment is necessary to assess and modify perioperative risk; minimize perioperative complications, hospital length-of-stay, and hospital readmission risk; and optimize outcomes. Preoperative assessment of respiratory function may include determinations of respiratory mechanics, gas exchange, and cardiorespiratory interaction. Spirometry and diffusion capacity are initially assessed and can be used to predict postoperative values. In a fit, younger patient undergoing VATS for a peripheral "coin" lesion, not all of these tests will be needed. For example:
Postoperative FEV, = preoperative FEV, = (1 — the percentage of functional lung tissue removed divided by 100)
Removal of
extensively diseased
lung
(nonventilated but
perfused)
does not
necessarily adversely
oxygenation.
affect
Mortality
FEV, is
less pulmonary
and than
be
30%
characterized by
DLCO
correlates interface.
function morbidity
and may
are to
40%
significantly of
normative diffusion
with
Predictive as
postoperative
60%, lung
actually improve
increased
FEV,.
Gas capacity
the total
functioning postoperative
FEV).
the patient
for carbon
surface
DLCO
can
If both
the is
generally exercise
testing is
if exchange
monoxide area
postoperative will
sometimes
(DLCO).
of the
be alveolar—capillary
calculated predicted
at warranted
Ventilation/perfusion lower
DLCO
risk for
when either
(V/Q)
in and
FEV, lung
resection.
of scintigraphy
each lobe
to overall
pulmonary predicted
indicated postoperative
the tests
provides function
the same
fashion are
greater than
Cardiopulmonary is
less than
the and
lung function
surgical procedure
in and
there is
30%.
relative may
further patients
contribution of
refine when
concern the
assessment pneumonectomy
about is
the whether
a
single lung
will be
adequate to
support life.

Patients considered at greater risk of perioperative complications (predicted FEV, or DLCO between 60% and 30%) based on standard spirometry testing and calculation of postoperative function should undergo exercise testing for evaluation of cardiopulmonary interaction. Stair climbing is the easiest way to assess exercise capacity and cardiopulmonary reserve. Patients capable of climbing two or three flights of stairs have decreased mortality and morbidity.
of
Conversely, the ability to climb less than two flights of stairs is associated with increased perioperative risk. The gold standard for evaluating cardiopulmonary interaction is by cardiopulmonary exercise testing (CPET) and measurement of maximal minute oxygen consumption. Vo i of greater than 20 mL/kg is not associated with a significant increase in perioperative mortality or morbidity, whereas minute consumption of less than 10 mL/kg is associated with an increased perioperative risk.
A combination of tests to evaluate the patient for thoracotomy and major pulmonary resection is recommended by the American College of Chest Physicians (Figure 25-10).
Algorithm for
Thoracotomy and
Major
Anatomic
Resection
(Lobectomy or
Greater)
Ei
Actual risks
affected by
parameters defined
here and:
+
Patient
Factors:
comorbidities, age
* Structural Aspects: center (volume, specialization)
* Process Factors: management of complications
* Surgical Access: thoracotomy vs minimally invasive
FIGURE 25-10 Physiological evaluation resection algorithm. (a) For pneumonectomy candidates, we suggest using Q scan to calculate predicted postoperative (PPO) FEV, or DLCO values (PPO values = preoperative values = [1 — fraction of total perfusion for the resected lung]), where the preoperative values are taken as the best measured postbronchodilator values. For lobectomy patients, segmental counting is indicated to calculate PPO FEV, or DLCO values (PPO values = preoperative values x [1 — y/z]), where the preoperative values are taken as the best measured postbronchodilator value and the number of functional or unobstructed lung segments to be removed is y and the total

Pulmonary infections may present as a solitary nodule or cavitary lesion (necrotizing pneumonitis). VATS may be carried out to exclude malignancy and diagnose the infectious agent. Lung resection is also indicated for cavitary lesions that are refractory to antibiotic treatment, are associated with refractory empyema, or result in massive hemoptysis. Responsible organisms include both bacteria and fungi.

Bronchiectasis is a permanent dilation of bronchi. It is usually the end result of severe or recurrent inflammation and obstruction of bronchi. Causes include cigarette smoking; a variety of viruses, bacteria, nontuberculous mycobacteria, and fungi; as well as inhalation of toxic gases, aspiration of gastric acid, and defective mucociliary clearance (cystic fibrosis and disorders of ciliary dysfunction). Bronchial muscle and elastic tissue are typically replaced by very vascular fibrous tissue. The latter predisposes to bouts of hemoptysis. Pulmonary resection is usually indicated for massive hemoptysis when conservative measures have failed and the disease is localized. Patients with diffuse bronchiectasis have a chronic obstructive ventilatory defect.

The majority of patients undergoing pulmonary resections have underlying lung disease. It should be emphasized that smoking is a risk factor for both chronic obstructive pulmonary disease and coronary artery disease; both disorders commonly coexist in patients presenting for thoracotomy. Echocardiography is useful for assessing baseline cardiac function and may suggest evidence of cor pulmonale (right ventricular enlargement or hypertrophy) in patients with poor exercise tolerance. Stress echocardiography (exercise or dobutamine) may be useful in diagnosing coronary artery disease when indicated by suggestive signs and symptoms.
Patients with tumors should be queried regarding signs and symptoms of local extension of the tumor and paraneoplastic syndromes (see above). Preoperative chest radiographs and CT or MR images should be reviewed. Tracheal or bronchial deviation can make tracheal intubation and proper positioning of bronchial tubes much more difficult. Moreover, airway compression can lead to difficulty in ventilating the patient following induction of anesthesia. Pulmonary consolidation, atelectasis, and large pleural effusions predispose to hypoxemia. The location of any bullous cysts or abscesses should be noted.
Patients undergoing thoracic procedures are at increased risk of postoperative pulmonary and cardiac complications. Perioperative arrhythmias, particularly supraventricular tachycardias, are common and thought to result from surgical manipulations or distention of the right atrium following reduction of the pulmonary vascular bed. The incidence of arrhythmias increases with age and the amount of pulmonary resection.

As with anesthesia for cardiac surgery, optimal preparation may help to prevent potentially catastrophic problems. Limited pulmonary reserve, anatomic abnormalities, or compromise of the airways, and the need for one-lung ventilation predispose these patients to the rapid onset of hypoxemia. A wellthought-out plan to deal with potential difficulties is necessary. Moreover, in addition to items for basic airway management, specialized and properly functioning equipment—such as multiple sizes of singleand double-lumen tubes, a flexible (pediatric) fiberoptic bronchoscope, a small-diameter "tube exchanger" of adequate length to accommodate a double lumen tube, a continuous positive airway pressure (CPAP) delivery system, and an anesthesia circuit adapter for administering bronchodilators—should be immediately available.
Patients undergoing open-lung resections (segmentectomy, lobectomy, and pneumonectomy) often receive postoperative thoracic epidural analgesia, unless there is a contraindication. However, patients are increasingly being treated with numerous antiplatelet and anticoagulant medications, which may preclude epidural catheter placement. Opioid-sparing, multimodal analgesia regimens, including paravertebral blocks, local injection of liposomal bupivacaine, and wound infusion catheters, are increasingly a part of enhanced recovery programs for thoracic surgery patients.

At least one large-bore (14or 16-gauge) intravenous line is mandatory for all open thoracic surgical procedures. A blood warmer and a rapid infusion device are also desirable if extensive blood loss is anticipated.

Direct monitoring of arterial pressure is indicated for resections of large tumors (particularly those with mediastinal or chest wall extension), and any procedure performed in patients who have limited pulmonary reserve or significant cardiovascular disease. Central venous access (preferably on the side of the thoracotomy to avoid the risk of pneumothorax on the side that will be ventilated intraoperatively) with monitoring of central venous pressure (CVP) is commonly used for pneumonectomies and resections of large tumors; however, less invasive measures of cardiac output through use of thoracic bioimpedance, pulse contour analysis, or transpulmonary thermodilution provide better estimates of cardiac function and volume responsiveness (see Chapter 5). Pulmonary artery catheters are very rarely used. In patients with significant coronary artery disease or pulmonary hypertension, intraoperative diagnosis of hypovolemia or reduced right or left ventricular performance can be easily accomplished with transesophageal echocardiography.

The selection of an induction agent should be based on the patient's preoperative status. All patients should receive adequate preoxygenation before induction. An adequate depth of anesthesia will help prevent reflex bronchospasm and exaggerated cardiovascular pressor responses to laryngoscopy. This may be accomplished by incremental doses of the induction agent, an opioid, or deepening the anesthesia with a volatile inhalation agent (the latter is particularly useful in patients with reactive airways). Moreover, volatile anesthetic agents may protect the lung from injury during one-lung ventilation. Tracheal intubation with a single-lumen tracheal tube (or with a laryngeal mask airway [LMA]) may be necessary, if the surgeon performs diagnostic bronchoscopy (see below) prior to surgery. Once the bronchoscopy is completed, the single-lumen tracheal tube (or LMA) can be replaced with a double-lumen bronchial tube (see above). Controlled positive-pressure ventilation helps prevent atelectasis, paradoxical breathing, and mediastinal shift; it also allows control of the operative field to facilitate the surgery.

Following induction, intubation, and confirmation of correct tracheal or bronchial blocker position, additional venous access and monitoring may be obtained before the patient is positioned for surgery. Most lung resections are performed with the patient in the lateral decubitus position. Proper positioning avoids injuries and facilitates surgical exposure. The lower arm is flexed and the upper arm is extended in front of the head, pulling the scapula away from the operative field (Figure 25-11). Pillows are placed between the arms and legs, and an axillary (chest) roll is usually positioned just beneath the dependent axilla to reduce pressure on the inferior shoulder (it is assumed, but not proven, that the axillary roll helps protect the brachial plexus); care is taken to avoid pressure on the eyes and the dependent ear.
FIGURE 25-11 Proper positioning for a lateral thoracotomy. (Reproduced with permission from Gothard JWW, Branthwaite MA. Anesthesia for Thoracic Surgery. Oxford, UK: Blackwell; 1982.)

All current anesthetic techniques have been successfully used for thoracic surgery, but the ideal techniques must provide the ability to administer high concentrations of inspired oxygen and all must permit rapid adjustments in anesthetic depth. Potent halogenated agents (isoflurane, sevoflurane, or desflurane) are often used in North American practice. Advantages of the halogenated agents versus total intravenous techniques include potent, doserelated bronchodilation and consistent depression of airway reflexes. Halogenated agents generally have minimal effects on HPV in doses less than 1 minimum alveolar concentration (MAC). Advantages of opioids include (1) generally minimal hemodynamic effects; (2) depression of airway reflexes; and (3) residual @ postoperative analgesia. If epidural or intrathecal opioids are to be used for postoperative analgesia, intravenous opioids should be minimized during surgery to prevent excessive postoperative respiratory depression. Maintenance of neuromuscular blockade with a nondepolarizing neuromuscular blocker (NMB) during surgery facilitates rib spreading as well as anesthetic management. Excessive fluid administration in thoracic surgical patients has been associated with acute lung injury in the postoperative period. Excessive fluid administration in the lateral decubitus position may promote a "lower lung syndrome" (ie, gravity-dependent transudation of fluid into the dependent lung). The latter increases intrapulmonary shunting and promotes hypoxemia, particularly during one-lung ventilation. While we manage lung resections with a relative fluid restriction, we acknowledge that definitive studies on an ideal goal-directed fluid management strategy are lacking.
The collapsed lung may be prone to acute lung injury due to surgical retraction during the procedure and possible ischemia—reperfusion injury. During lung resections, the bronchus (or remaining lung tissue) is usually divided with an automated stapling device. The bronchial stump is then tested for an air leak under water by transiently sustaining 30 cm of positive pressure to the airway. Prior to completion of chest closure, all remaining lung segments should be fully expanded manually under direct vision. Controlled mechanical ventilation is then resumed and continued until thoracostomy tubes are connected to suction.

End-tidal CO, measurement is useful as a trend monitor but may not be accurate due to increased dead-space and an unpredictable gradient between the arterial and end-tidal CO, partial pressure.

Hypoxemia during one-lung anesthesia requires one or more of the following interventions:, 1 = Hypoxemia during one-lung anesthesia requires one or more of the following interventions:. 1., 1 = Adequate position of the bronchial tube (or bronchial blocker) must be confirmed, as its position relative to the carina can change as a result of surgical manipulations or traction; repeat fiberoptic bronchoscopy through the tracheal lumen can quickly detect this problem. Both lumens of the tube should also be suctioned to exclude excessive secretions or obstruction as a factor.. 2., 1 = Increase Fi O 2 to 1.0.. 3., 1 = Recruitment maneuvers on the dependent, ventilated lung may eliminate atelectasis and improve shunt.. 4., 1 = Ensure that there is sufficient (but not excessive) PEEP to the dependent, nonoperative lung to eliminate atelectasis.. 5., 1 = CPAP or blow-by oxygen to the operative lung will decrease shunting and improve oxygenation. However, uncontrolled inflation of the operative lung during VATS will make identification and visualization of the lung structures difficult for the surgeon; therefore, such maneuvers should be applied carefully and cautiously.. 6., 1 = Two-lung ventilation should be instituted for severe hypoxemia. If possible, pulmonary artery clamp can also be placed during pneumonectomy to eliminate shunt.. 7., 1 = In patients with chronic obstructive lung disease, one should always be suspicious of pneumothorax on the dependent, ventilated side as a cause of severe hypoxemia. This complication requires immediate detection and treatment by aborting the surgical procedure, reexpanding the operative lung, and immediately inserting a chest tube in the contralateral chest.. 8., 1 = If other causes and maneuvers have failed to improve oxygen saturation and one-lung anesthesia is required to complete the anesthetic, one can discontinue all medications known to inhibit HPV (inhalation anesthetics, vasodilators, β-agonists, etc) in favor of alternative drugs and techniques (eg,

Ventilation can be stopped for short periods if 100% oxygen is insufflated at a rate greater than oxygen consumption (apneic oxygenation) into an unobstructed tracheal tube. Adequate oxygenation can often be maintained for prolonged periods, but progressive respiratory acidosis limits the use of this technique to 10 to 20 min in most patients. Arterial Pco, rises 6 mm Hg in the first minute, followed by a rise of 3 to 4 mm Hg during each subsequent minute. High-frequency positive-pressure ventilation and high-frequency jet ventilation have been used during thoracic procedures as alternatives to one-lung ventilation. A standard tracheal tube may be used with either technique. Small tidal volumes (<2 mL/kg) allow decreased lung excursion, which may facilitate the surgery but still allow ventilation of both lungs. Unfortunately, mediastinal "bounce"—a to-and-fro movement—often interferes with the surgery.

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Patients are observed in the postanesthesia care unit, and, in most instances, at least overnight in a monitored or intensive care unit. Atelectasis and shallow breathing ("splinting") from incisional pain commonly lead to hypoxemia and respiratory acidosis. Gravity-dependent transudation of fluid into the intraoperative dependent lung may also be contributory. Reexpansion edema of the collapsed nondependent lung can also occur.
®@ Postoperative hemorrhage complicates about 3% of thoracotomies and may be associated with up to 20% mortality. Signs of hemorrhage include increased chest tube drainage (>200 mL/h), hypotension, tachycardia, and a falling hematocrit. Postoperative supraventricular tachyarrhythmias are common and usually require immediate treatment. Routine postoperative care should include semiupright (>30°) positioning, supplemental oxygen (40-50%), incentive spirometry, electrocardiographic and hemodynamic monitoring, a postoperative chest radiograph (to confirm proper position of all thoracostomy tube drains and central lines and to confirm expansion of both lung fields), and adequate analgesia.

The importance of adequate pain management in the thoracic surgical patient cannot be overstated. Inadequate pain control in these high-risk patients will result in splinting, poor respiratory effort, and the inability to cough and clear secretions, with an end result of airway closure, atelectasis, shunting, and hypoxemia. Irrespective of the modality used, there must be a comprehensive plan for pain management.
A balance between comfort and respiratory depression in patients with marginal lung function is difficult to achieve with parenteral opioids alone. Patients who have undergone thoracotomy clearly benefit from the use of other techniques (described later) that may reduce the need for parenteral opioids. If parenteral opioids are used alone, they are best administered via a patientcontrolled analgesia device.
In the absence of an epidural catheter, intercostal or paravertebral nerve blocks with long-acting local anesthetics may facilitate extubation and contribute to postoperative analgesia, but have a limited duration of action, so additional means of pain management must be employed. Alternatives to epidural, intercostal, or paravertebral techniques include infusion of local anesthetic through a catheter placed in the surgical wound during closure or injection of liposomal bupivacaine into the wound, which will markedly reduce the requirement for parenteral opioids and improve the overall quality of analgesia relative to parenteral opioids alone.
Epidural analgesia provides excellent pain relief and continuous therapy and avoids side effects associated with administration of systemic opioids. On the other hand, epidural techniques require around-the-clock attention from the acute pain team for the duration of the infusion and subject the patient to the long list of epidural-related side effects and complications Most practitioners use a combination of opioid (fentanyl, morphine, hydromorphone) and local anesthetic (bupivacaine or ropivacaine), with the epidural catheter placed at a thoracic level. Gabapentin and low-dose ketamine and lidocaine intravenous infusions have all been employed as a part of multimodal analgesia regimens following thoracotomy. Oral or intravenous acetaminophen and oral or intravenous nonsteroidal antiinflammatory agents are likewise routinely used in combination with other modalities to reduce or eliminate opioid use for postoperative analgesia.

elevation of the ipsilateral hemidiaphragm together with difficulty in weaning the patient from the ventilator. Large chest wall resections may include part of the diaphragm, causing a similar problem, in addition to a flail chest. If an epidural catheter has been placed, any loss of motor function or unexplained back pain should immediately trigger imaging to rule out epidural hematoma.

Pulmonary cysts or bullae may be congenital or acquired as a result of emphysema. Large bullae can impair ventilation by compressing the surrounding lung. These air cavities often behave as if they have a one-way valve, predisposing them to progressively enlarge. Lung resection may be undertaken for progressive dyspnea or recurrent pneumothorax. The greatest risk of anesthesia is rupture of the air cavity during positive-pressure ventilation, resulting in tension pneumothorax; the latter may occur on either side prior to thoracotomy or on the nonoperative side during the lung resection. Induction of anesthesia with maintenance of spontaneous ventilation is desirable until the side with the cyst or bullae is isolated with a double-lumen tube, or until a chest tube is placed; most patients have a large increase in dead space, so assisted ventilation is necessary @ to avoid excessive hypercarbia. Nitrous oxide is contraindicated in patients with cysts or bullae because it can expand the air space and cause rupture. The latter may be signaled by sudden hypotension, bronchospasm, or an abrupt rise in peak inflation pressure and requires immediate placement of a chest tube.

Lung abscesses result from primary pulmonary infections, obstructing pulmonary neoplasms (see earlier discussion), or, rarely, hematogenous spread of systemic infections. The two lungs should be isolated to prevent contamination of the healthy lung. A rapid-sequence intravenous induction with tracheal intubation with a double-lumen tube is generally recommended, with the affected lung in a dependent position. As soon as the double-lumen tube is placed, both bronchial and tracheal cuffs should be inflated. The bronchial cuff should make a tight seal before the patient is turned into the lateral decubitus position, with the diseased lung in a nondependent position. The diseased lung should be frequently suctioned during the procedure to decrease the likelihood of contaminating the healthy lung.

Bronchopleural fistulas occur following lung resection (usually pneumonectomy), rupture of a pulmonary abscess into a pleural cavity, pulmonary barotrauma, or spontaneous rupture of bullae. The majority of patients are treated (and cured) conservatively; patients come to surgery when chest tube drainage has failed. Anesthetic management may be complicated by the inability to effectively ventilate the patient with positive pressure because of a large air leak, the potential for a tension pneumothorax, and the risk of contaminating the other lung if an empyema is present. The empyema is usually drained, prior to closure of the fistula.
A correctly placed double-lumen tube greatly simplifies anesthetic management by isolating the fistula and allowing one-lung ventilation to the normal lung. The patient should be extubated as soon as possible after the repair.

Tracheal resection is most commonly performed for tracheal stenosis, tumors, or, less commonly, congenital abnormalities. Tracheal stenosis can result from penetrating or blunt trauma, as well as tracheal intubation and tracheostomy. Squamous cell and adenoid cystic carcinomas account for the majority of tumors. Progressive compromise of the tracheal lumen produces dyspnea. Wheezing or stridor may be evident only with exertion. The dyspnea may be worse when the patient is lying down, with progressive airway obstruction. Hemoptysis can also complicate tracheal tumors. CT or MR imaging is valuable in localizing the lesion. Measurement of flow—volume loops confirms the location of the obstruction and aids the clinician in evaluating the severity of the lesion (Figure 25-12).
FIGURE 25-12 A—D: Flow—volume loops.

Little premedication is given, as most patients presenting for tracheal resection have moderate to severe airway obstruction. Use of an anticholinergic agent to dry secretions is controversial because of the theoretical risk of inspissation. Monitoring should include direct arterial pressure measurements.
An inhalation induction (in 100% oxygen) is carried out in patients with severe obstruction. Sevoflurane is preferred because it is the potent anesthetic that is least irritating to the airway. Spontaneous ventilation is maintained throughout induction. NMBs are generally avoided because of the potential for complete airway obstruction following neuromuscular blockade. Laryngoscopy is performed only when the patient is judged to be under deep anesthesia. Intravenous lidocaine (1-2 mg/kg) can deepen the anesthesia without depressing respirations. The surgeon may then perform rigid bronchoscopy to evaluate and possibly dilate the lesion. Following bronchoscopy, the patient is intubated with a tracheal tube small enough to be passed distal to the obstruction whenever possible.
A collar incision is utilized for high tracheal lesions. The surgeon divides the trachea in the neck and advances a sterile armored tube into the distal trachea, passing off a sterile connecting breathing circuit to the anesthesiologist for ventilation during the resection. Following the resection and completion of the posterior part of the reanastomosis, the armored tube is removed, and the original tracheal tube is advanced distally, past the anastomosis (Figure 25-13). Alternatively, high-frequency jet ventilation may be employed during the anastomosis by passing the jet cannula past the obstruction and into the distal trachea (Figure 25-14). Return of spontaneous ventilation and early extubation at the end of the procedure are desirable. Patients should be positioned with the neck flexed immediately after the operation to minimize tension on the suture line (Figure 25-15).
FIGURE 25-14 Tracheal resection using high-frequency jet ventilation. A: The catheter is advanced past the obstruction, and the cuff is deflated when jet ventilation is initiated. B: The catheter is advanced distally by the surgeon. Jet ventilation can be continued without interruption during resection and reanastomosis.

FIGURE 25-15 Position of the patient before (A) and after (B) tracheal resection and reanastomosis with the patient's neck flexed for the first 24 to 48 h.
Surgical management of low tracheal lesions requires a median sternotomy or right posterior thoracotomy. Anesthetic management may include more complicated techniques, such as high-frequency ventilation or even cardiopulmonary bypass (CPB) in complex congenital cases.

VATS is now used for most lung resections. Most procedures are performed through three or more small incisions in the chest, with the patient in the lateral decubitus position. Anesthetic management is similar to that for open procedures, except that one-lung ventilation is required (as opposed to being

Rigid bronchoscopy for removal of foreign bodies or tracheal dilation is usually performed under general anesthesia. These procedures are complicated by the need to share the airway with the surgeon or pulmonologist; fortunately, these procedures are often brief. After a standard intravenous induction, anesthesia is often maintained with total intravenous anesthesia, and a shortor intermediateacting NMB. One of three techniques can then be used during rigid bronchoscopy: (1) apneic oxygenation using a small catheter positioned alongside the bronchoscope to insufflate oxygen (see above); (2) conventional ventilation through the side arm of a ventilating bronchoscope (when the proximal window of this instrument is opened for suctioning or biopsies, ventilation must be interrupted); or (3) jet ventilation through an injector-type bronchoscope.

Mediastinoscopy, much more commonly employed in the past than at present, provides access to the mediastinal lymph nodes and is used to establish either the diagnosis or the resectability of intrathoracic malignancies (see above). Preoperative CT or MR imaging is useful for evaluating tracheal distortion or compression.
Mediastinoscopy is performed under general tracheal anesthesia with neuromuscular paralysis. Venous access with a large-bore (14to 16-gauge) intravenous catheter is mandatory because of the risk of bleeding and the difficulty in controlling bleeding when it occurs. Because the innominate artery may be compressed during the procedure, blood pressure should be measured in the left arm.
Complications associated with mediastinoscopy include (1) vagally mediated reflex bradycardia from compression of the trachea or the great vessels; (2) excessive hemorrhage; (3) cerebral ischemia from compression of the innominate artery (detected with a right radial arterial line or pulse oximeter on the right hand); (4) pneumothorax (usually presents postoperatively); (5) air embolism (because of a 30° head elevation, the risk is greatest during spontaneous ventilation); (6) recurrent laryngeal nerve damage; and (7) phrenic nerve injury.

Bronchoalveolar lavage may be employed for patients with pulmonary alveolar proteinosis. These patients produce excessive quantities of surfactant and fail to clear it. They present with dyspnea and bilateral consolidation on the chest radiograph. In such patients, bronchoalveolar lavage may be indicated for severe hypoxemia or worsening dyspnea. Often, one lung is lavaged, allowing the patient to recover for a few days before the other lung is lavaged; the "sicker" lung is therefore lavaged first.
Unilateral bronchoalveolar lavage is performed under general anesthesia with a double-lumen bronchial tube. The cuffs on the tube should be properly positioned and should make a watertight seal to prevent spillage of fluid into the other side. The procedure is normally done in the supine position; although lavage with the lung in a dependent position helps to minimize contamination of the other lung, this position can cause severe ventilation/perfusion mismatch. Warm normal saline is infused into the lung to be treated and is drained by gravity At the end of the procedure, both lungs are well suctioned, and the double-lumen tracheal tube is replaced with a single-lumen tracheal tube.

Lung transplantation is indicated for end-stage pulmonary parenchymal disease or pulmonary hypertension. Candidates are functionally incapacitated by dyspnea and have a poor prognosis. Criteria vary according to the primary disease process. Common etiologies are listed in Table 25-3. Lung transplantation (as is true for all solid organ transplants) is limited by the availability of suitable organs, not by the availability of recipients. Patients typically have dyspnea at rest or with minimal activity and resting hypoxemia (Pao, <50 mm Hg) with increasing oxygen requirements. Progressive CO, retention is also very common. Patients may be ventilator dependent or may be supported by extracorporeal membrane oxygenation (ECMO).

Bronchiectasis
Obstructive
Chronic obstructive pulmonary disease a,-Antitrypsin deficiency
Pulmonary lymphangiomatosis
Restrictive
Idiopathic pulmonary fibrosis

Effective coordination between the organ-retrieval team and the transplant team minimizes graft ischemia time and avoids unnecessary prolongation of pretransplant anesthesia time. These procedures are performed on an emergency basis; therefore, patients may have little time to fast for surgery. Oral cyclosporine also may be given preoperatively. Administration of a clear antacid, an H, blocker, or metoclopramide should be considered. Any premedication is usually administered only in the operating room when the patient is directly attended and monitored. Immunosuppressants and antibiotics are also administered after induction and prior to surgical incision.

Strict asepsis should be observed for invasive monitoring procedures. Central venous access might be accomplished only after induction of anesthesia because patients may not be able to lie flat while awake. Patients with a patent foramen ovale are at risk of paradoxical embolism because of potentially high right atrial pressures. Transesophageal echocardiography is used to assess right ventricular function, integrity of the intraatrial septum, and pulmonary vein flow following anastomosis.

Induction with ketamine, etomidate, an opioid, or a combination of these agents is employed, avoiding precipitous drops in blood pressure. An NMB is used to facilitate laryngoscopy. Permissive hypercapnia and protective lung ventilation strategies are employed. Inspired oxygen concentration is adjusted to maintain Sa0, above 92%. Tidal volumes are maintained below 6 mL/kg using pressurecontrolled ventilation. Inhalational agents are administered as tolerated for anesthesia and to provide a possible lung-protective effect.
Hypercarbia and acidosis may lead to pulmonary vasoconstriction and acute right heart failure, and hemodynamic support with inotropes may be required for these patients.

Whether to employ CPB (see Chapter 22) or ECMO during transplantation of one lung is based on the patient's response to collapsing the lung to be replaced and clamping its pulmonary artery as well as institutional practices. Persistent arterial hypoxemia (Spo, <88%) or right heart failure may require institution of CPB. Drugs such as milrinone may be used for inotropic support, and inhaled nitric oxide can be delivered to dilate the pulmonary vasculature. After the recipient lung is removed, the pulmonary artery, left atrial cuff (with the pulmonary veins), and bronchus of the donor lung are anastomosed. Flexible bronchoscopy is used to examine the bronchial suture line after its completion.

A "clamshell" transverse sternotomy can be used for double-lung transplantation. Heart—lung transplantation is performed through median sternotomy with CPB.

After anastomosis of the donor organ or organs, @ ventilation to both lungs is resumed. Following transplantation, peak inspiratory pressures should be maintained at the minimum pressure compatible with good lung expansion, and the inspired oxygen concentration should be maintained as close to room air as allowed by a Pao, greater than 60 mm Hg. If transplantation has been performed on CPB, the patient is separated from CPB. Pulmonary vasodilators, inhaled nitric oxide, and inotropes (see earlier discussion) may be necessary.
Transesophageal echocardiography is helpful in identifying right or left ventricular dysfunction, as well as in evaluating blood flow in the pulmonary vessels after transplantation.
Transplantation disrupts the neural innervation, lymphatic drainage, and bronchial circulation of the transplanted lung. The respiratory pattern is unaffected, but the cough reflex is abolished below the carina. Bronchial hyperreactivity is observed in some patients. Hypoxic pulmonary vasoconstriction remains normal. Loss of lymphatic drainage increases extravascular lung water and predisposes the transplanted lung to pulmonary edema. Consequently, fluid overload should be avoided. Loss of the bronchial circulation predisposes to ischemic breakdown of the bronchial suture line.

Patients are extubated after surgery as soon as is feasible. A thoracic epidural catheter may be employed for postoperative analgesia when coagulation studies are normal. The postoperative course may be complicated by acute rejection, infections, and renal and hepatic dysfunction. Deteriorating lung function may result from rejection or reperfusion injury. Occasionally, temporary ECMO may be necessary. Frequent bronchoscopy with transbronchial biopsies and lavage are necessary to differentiate between rejection and infection. Nosocomial gramnegative bacteria, cytomegalovirus, Candida, Aspergillus, and Pneumocystis jiroveci are common pathogens. Other postoperative surgical complications include damage to the phrenic, vagus, and left recurrent laryngeal nerves.

Common indications for esophageal surgery include tumors, gastroesophageal reflux, and motility disorders (achalasia). Surgical procedures include simple endoscopy, esophageal dilation, cervical esophagomyotomy, open or thoracoscopic distal esophagomyotomy, insertion or removal of esophageal stents, and open or minimally invasive esophagectomy. Squamous cell carcinomas account for the majority of esophageal tumors; adenocarcinomas are less common, whereas benign tumors (leiomyomas) are rare. Most tumors occur in the distal esophagus. Operative treatment may be palliative or curative. After esophageal resection, the stomach is pulled up into the thorax, or the esophagus is functionally replaced with part of the colon (colonic interposition). Gastroesophageal reflux is treated surgically when the esophagitis is refractory to medical management or results in complications such as stricture, recurrent pulmonary aspiration, or a Barrett esophagus (columnar epithelium). A variety of antireflux operations may be performed (Nissen, Belsey, Hill, or Collis—Nissen) via thoracic or abdominal approaches, often laparoscopically. Achalasia and systemic sclerosis (scleroderma) account for the majority of surgical procedures performed for motility disorders. The former usually occurs as an isolated finding, whereas the latter is part of a generalized collagen— vascular disorder. Cricopharyngeal muscle dysfunction can be associated with a variety of neurogenic or myogenic disorders and often results in a Zenker diverticulum.

® Regardless of the procedure, a common anesthetic concern in patients with esophageal disease is the risk of pulmonary aspiration. This may result from obstruction, altered motility, or abnormal sphincter function. In fact, most patients typically complain of dysphagia, heartburn, regurgitation, coughing, or wheezing when lying flat. Dyspnea on exertion may also be prominent when chronic aspiration results in pulmonary fibrosis. Patients with malignancies may present with anemia and weight loss. Esophageal cancer patients usually have a history of cigarette smoking and alcohol consumption, so patients should be evaluated for coexisting chronic obstructive pulmonary disease, coronary artery disease, and liver dysfunction. Patients with systemic sclerosis (scleroderma) should be evaluated for involvement of other organs, particularly the kidneys, heart, and lungs; Raynaud phenomena is also common.
In patients with reflux, consideration should be given to administering one or more of the following preoperatively: metoclopramide, an H,-receptor blocker, sodium citrate, or a proton-pump inhibitor. In such patients, a rapid-sequence induction should be used. A double-lumen tube is used for procedures involving thoracoscopy or thoracotomy. The anesthesiologist may be asked to pass a largediameter bougie into the esophagus as part of the surgical procedure; great caution must be exercised to help avoid pharyngeal or esophageal injury.
Transhiatal (blunt) and thoracic esophagectomies deserve special consideration. The former requires an upper abdominal incision and a left cervical incision, whereas the latter requires posterolateral thoracotomy, an abdominal incision, and, finally, a left cervical incision. Parts of the procedure may be performed using laparoscopy or VATS. Direct monitoring of arterial pressure is indicated. During the transhiatal approach to esophagectomy, substernal and diaphragmatic retractors can interfere with cardiac function. Moreover, as the esophagus is freed up blindly from the posterior mediastinum by blunt dissection, the surgeon's hand transiently interferes with cardiac filling and produces profound hypotension. The dissection can also induce marked vagal stimulation.

Colonic interposition involves forming a pedicle graft of the colon and passing it through the posterior mediastinum up to the neck to take the place of the esophagus. This procedure is lengthy, and maintenance of an adequate blood pressure, cardiac output, and hemoglobin concentration is necessary to ensure graft viability. Graft ischemia may be heralded by a progressive metabolic acidosis. Both too much and too little fluid administration can negatively impact outcome. Goal-directed fluid therapy using hemodynamic measures (eg, stroke volume variation) may be helpful in perioperative fluid management of the esophagectomy patient.
Lung protective ventilation and multimodal perioperative analgesia should be used postoperatively.

A 9-year-old boy with mediastinal lymphadenopathy seen on a chest radiograph presents for biopsy of a cervical lymph node.
What is the most important preoperative consideration?
Is there any evidence of airway compromise? Tracheal compression may produce dyspnea (proximal obstruction) or a nonproductive cough (distal obstruction).
Asymptomatic compression is also common and may be evident only as tracheal deviation on physical or radiographic examinations. A CT scan of the chest provides invaluable information about the presence, location, and severity of airway compression. Flow—volume loops will also detect subtle airway obstruction and provide important information regarding the location and functional importance of the obstruction (see earlier discussion).

No. Severe airway obstruction can occur following induction of anesthesia in these patients even in the absence of any preoperative symptoms. This mandates that the chest radiograph and CT scan be reviewed for evidence of asymptomatic airway obstruction. The point of obstruction is typically distal to the tip of the tracheal tube. Moreover, loss of spontaneous ventilation can precipitate complete airway obstruction.

Superior vena cava syndrome is the result of progressive enlargement of a mediastinal mass and compression of mediastinal structures, particularly the vena cava. Lymphomas are most commonly responsible, but primary pulmonary or mediastinal neoplasms can also produce the syndrome. Superior vena cava syndrome is often associated with severe airway obstruction and cardiovascular collapse on induction of general anesthesia. Caval compression produces venous engorgement and edema of the head, neck, and arms. Direct mechanical compression, as well as mucosal edema, severely compromise airflow in the trachea. Most patients favor an upright posture, as recumbency worsens the airway obstruction. Cardiac output may be severely depressed due to impeded venous return from the upper body, direct mechanical compression of the heart, and (with malignancies) pericardial invasion. An echocardiogram is useful in evaluating cardiac function and detecting pericardial fluid.
What is the anesthetic of choice for a patient with superior vena cava syndrome?
The absence of signs or symptoms of airway compression or superior vena cava syndrome does not preclude potentially life-threatening complications following induction of general anesthesia. Therefore, biopsy of a peripheral node (usually cervical or scalene) under local anesthesia is safest whenever possible. Although establishing a diagnosis is of prime importance, the presence of significant airway compromise or the superior vena cava syndrome may dictate empiric treatment with corticosteroids prior to tissue diagnosis at surgery (cancer is the most common cause); preoperative radiation therapy or chemotherapy may also be considered. The patient can usually safely undergo surgery with general anesthesia once

in patients with reduced cardiac output); (2) inhalational induction with a volatile agent (usually sevoflurane); or (3) incremental small doses of propofol or etomidate. Positive-pressure ventilation can precipitate severe hypotension, and volume loading prior to induction may partly offset impaired ventricular
filling secondary to caval obstruction.
5. Maintenance of anesthesia: The technique selected should be tailored to the patient's hemodynamic status. Following intubation, neuromuscular blockade prevents coughing or straining.
6. Extubation: At the end of the procedure, patients should be left intubated until the airway obstruction has resolved, as determined by flexible bronchoscopy or the presence of an air leak around the tracheal tube when the tracheal cuff is deflated.

@ Cerebral perfusion pressure is the difference between mean arterial pressure and intracranial pressure (or central venous pressure, whichever is greater).
@ The cerebral autoregulation curve is shifted to the right in patients with chronic arterial hypertension.
© The most important extrinsic influences on cerebral blood flow (CBF) are respiratory gas tensions—particularly Paco,. CBF is directly proportionate to Paco, between tensions of 20 and 80 mg Hg. Blood flow changes approximately 1 to 2 mL/100 g/min per mm Hg change in PacO5.
@ CBF changes 5% to 7% per 1°C change in temperature. Hypothermia decreases both cerebral metabolic rate and CBF, whereas pyrexia has the reverse effect.
@ The movement of a given substance across the blood-brain barrier is governed simultaneously by its size, charge, lipid solubility, and degree of protein binding in blood.
@ The blood-brain barrier may be disrupted by severe hypertension, tumors, trauma, strokes, infection, marked hypercapnia, hypoxia, and sustained seizure activity.
@ The cranial vault is a rigid structure with a fixed total volume, consisting of brain (80%), blood (12%), and cerebrospinal fluid (8%). Any increase in one component must be offset by an equivalent decrease in another to prevent a rise in intracranial pressure.
© With the exception of ketamine, all intravenous agents either have little effect on or reduce cerebral metabolic rate and CBF.
© With normal autoregulation and an intact blood-brain barrier,
vasopressors increase CBF only when mean arterial blood pressure is below 50 to 60 mm Hg or above 150 to 160 mm Hg.
© The brain is very vulnerable to ischemic injury because of its relatively high oxygen consumption and near-total dependence on aerobic glucose metabolism.
@ Hypothermia is the most effective method for protecting the brain during focal and global ischemia.
Anesthetic agents may have profound effects on cerebral metabolism, blood flow, cerebrospinal fluid (CSF) dynamics, and cerebral blood volume and pressure. In some instances, these alterations are deleterious, whereas in others they may be beneficial. This chapter reviews important physiological concepts and discusses the cerebral effects of commonly used anesthetics.

The brain normally consumes 20% of total body oxygen. Most cerebral oxygen consumption (60%) is used to generate adenosine triphosphate (ATP) to support neuronal electrical activity (Figure 26—1). The cerebral metabolic rate (CMR) is usually expressed in terms of oxygen consumption (CMRo,) and averages 3 to 3.8 mL/100 g/min (50 mL/min) in adults. CMRo, is greatest in the gray matter of the cerebral cortex and generally parallels cortical electrical activity. Because of the rapid oxygen consumption and the absence of significant oxygen reserves, interruption of cerebral perfusion usually results in unconsciousness within 10 s. If blood flow is not reestablished within 3 to 8 min under most conditions, ATP stores are depleted, and irreversible cellular injury occurs. The more rostral "higher" brain regions (cortex, hippocampus) are more sensitive to hypoxic injury than the brainstem.

Neuronal cells normally utilize glucose as their primary energy source. Brain glucose consumption is approximately 5 mg/100 g/min, of which more than 90% is metabolized aerobically. CMRo, therefore normally parallels glucose consumption. This relationship is not maintained during starvation, when ketone bodies (acetoacetate and B-hydroxybutyrate) also become major energy substrates. Although the brain can also take up and metabolize lactate, cerebral function is normally dependent on a continuous supply of glucose. Acute sustained hypoglycemia is injurious to the brain. Paradoxically, hyperglycemia can exacerbate global and focal hypoxic brain injury by accelerating cerebral acidosis and cellular injury. Adequate control of perioperative blood glucose concentration is advocated in part to prevent adverse effects of hyperglycemia during ischemia; however, overzealous blood glucose control can likewise produce injury through iatrogenic hypoglycemia.

Cerebral blood flow (CBF) varies with metabolic activity. There are a variety of methods available to directly measure CBF, including positron emission tomography, xenon washout, and computed tomography perfusion scans. Except in research environments, these methods do not lend themselves to bedside monitoring of CBF. Regional CBF parallels metabolic activity and can vary from 10 to 300 mL/100 g/min. For example, motor activity of a limb is associated with a rapid increase in regional CBF of the corresponding motor cortex. Similarly, visual activity is associated with an increase in regional CBF of the corresponding occipital visual cortex.
Although overall CBF averages 50 mL/100 g/min at a Paco, of 40 mmHg,

® Cerebral perfusion pressure (CPP) is the difference between mean arterial pressure (MAP) and intracranial pressure (ICP) (or central venous pressure [CVP], if it is greater than ICP). MAP — ICP (or CVP) = CPP. CPP is normally
80 to 100 mm Hg. Moreover, because ICP is normally less than 10 mm Hg, CPP is primarily dependent on MAP.
Moderate to severe increases in ICP (>30 mm Hg) can compromise CPP and CBF, even in the presence of a normal MAP. Patients with CPP values less than 50 mm Hg often show slowing on the EEG, whereas those with a CPP between 25 and 40 mm Hg typically have a flat EEG. Sustained perfusion pressures less than 25 mm Hg may result in irreversible brain damage.

Much like the heart and kidneys, the brain normally tolerates a wide range of blood pressure, with little change in blood flow. The cerebral vasculature rapidly (10-60 s) adapts to changes in CPP. Decreases in CPP result in cerebral vasodilation, whereas elevations induce vasoconstriction. In normal individuals, CBF remains nearly constant between MAPs of about 60 and 160 mm Hg (Figure 26—2), although the lower limit of this autoregulation may be increased in some patients. Outside these limits, blood flow becomes pressure dependent. Pressures above 150 to 160 mm Hg can disrupt the blood-brain barrier (see below) and may result in cerebral edema and hemorrhage.
FIGURE 26-2 Normal cerebral autoregulation curve.
® The cerebral autoregulation curve (Figure 26—2) is shifted to the right in patients with chronic arterial hypertension. Both upper and lower limits are shifted. Flow becomes more pressure dependent at low "normal" arterial pressures in return for cerebral protection at higher arterial pressures. Studies suggest that long-term antihypertensive therapy can restore cerebral autoregulation limits toward normal. Myogenic, neurogenic, and metabolic mechanisms may contribute to cerebral autoregulation.

® The most important extrinsic influences on CBF are respiratory gas tensions— particularly Paco,. CBF is directly proportionate to Paco, between tensions of 20 and 80 mm Hg (Figure 26—3). Blood flow changes approximately 1 to 2 mL/100 g/min per millimeter of mercury change in Paco>. This effect is almost immediate and is thought to be secondary to changes in the pH of CSF and cerebral tissue. Because ions do not readily cross the blood-brain barrier (see below) but CO, does, acute changes in Paco, but not HCO; affect CBF. Thus, acute metabolic acidosis has little effect on CBF because hydrogen ions (H*) cannot readily cross the blood-brain barrier. After 24 to 48 h, CSF HCO," ion concentration adjusts to compensate for the change in Paco, so that the effects of hypocapnia and hypercapnia are diminished. Marked hyperventilation (Paco, <20 mm Hg) shifts the oxygen—hemoglobin dissociation curve to the left, and, with changes in CBF, may result in EEG changes suggestive of cerebral impairment, even in normal individuals.
FIGURE 26-3 The relationship between cerebral blood flow and arterial respiratory gas tensions.
Only marked changes in Pao, alter CBF. Whereas hyperoxia may be associated with only minimal decreases (—10%) in CBF, severe hypoxemia (Pao, <50 mm Hg) greatly increases CBF (Figure 26-3).

@ CBF changes 5% to 7% per 1°C change in temperature. Hypothermia decreases both CMR and CBF, whereas hyperthermia has the reverse effect. Between 17°C and 37°C, the Q10 for humans is approximately 2—that is, for every 10° increase in temperature, the CMR doubles. Conversely, the CMR decreases by 50% if the temperature of the brain falls by 10°C (eg, from 37°C to 27°C) and another 50% if the temperature decreases from 27°C to 17°C. At 20°C, the EEG is isoelectric, but further decreases in temperature continue to reduce CMR throughout the brain. Hyperthermia (above 42°C) may result in neuronal cell injury.

The most important determinant of blood viscosity is hematocrit. A decrease in hematocrit decreases viscosity and can improve CBF; unfortunately, a reduction in hematocrit also decreases the oxygen-carrying capacity and thus can potentially impair oxygen delivery. Elevated hematocrit, as may be seen with marked polycythemia, increases blood viscosity and can reduce CBF. Some studies suggest that optimal cerebral oxygen delivery may occur at hematocrits of approximately 30%.

Intracranial vessels are innervated by the sympathetic (vasoconstrictive) and parasympathetic (vasodilatory) systems, Intense sympathetic stimulation induces vasoconstriction in these vessels, which can limit CBF. Autonomic innervation may also play an important role in cerebral vasospasm following brain injury and stroke.

becomes dependent on hydrostatic pressure rather than osmotic gradients.

FIGURE 26—4 The flow of cerebrospinal fluid in the central nervous system, per classic dogma. As noted in the text, unidirectional flow of CSF is now questioned by investigators. (Reproduced with permission from Waxman SG. Correlative Neuroanatomy. 24th ed. New York, NY: McGraw-Hill Education; 2000.)
CSF formation involves active secretion of sodium in the choroid plexuses. The resulting fluid is isotonic with plasma despite lower potassium, bicarbonate, and glucose concentrations. Its protein content is limited to the very small amounts that leak into perivascular fluid. Carbonic anhydrase inhibitors (acetazolamide), corticosteroids, spironolactone, furosemide, isoflurane, and vasoconstrictors decrease CSF production.
Absorption of CSF involves the translocation of fluid from the arachnoid granulations into the cerebral venous sinuses. Smaller amounts are absorbed at nerve root sleeves and by meningeal lymphatics. Because the brain and spinal cord lack lymphatics, absorption of CSF is also the principal means by which perivascular and interstitial protein is returned to the blood.

@ The cranial vault is a rigid structure with a fixed total volume, consisting of brain (80%), blood (12%), and CSF (8%). Any increase in one component must be offset by an equivalent decrease in another to prevent a rise in ICP. By convention, ICP means supratentorial CSF pressure measured in the lateral ventricles or over the cerebral cortex and is normally 10 mm Hg or less. Minor variations may occur, depending on the site measured, but, in the lateral recumbent position, lumbar CSF pressure normally approximates supratentorial pressure.
Intracranial elastance is determined by measuring the change in ICP in response to a change in intracranial volume. Normally, small increases in volume of one component are initially well compensated (Figure 26—5). A point is eventually reached, however, at which further increases produce precipitous rises in ICP. Major compensatory mechanisms include (1) an initial displacement of CSF from the cranial to the spinal compartment, (2) an increase in CSF absorption, (3) a decrease in CSF production, and (4) a decrease in total cerebral blood volume (primarily venous).

The concept of total intracranial compliance is useful clinically, even though compliance probably varies in the different compartments of the brain and is affected by arterial blood pressure and Paco,. Cerebral blood volume is estimated to increase 0.05 mL/100 g of brain per 1 mm Hg increase in Paco,. Blood pressure effects upon cerebral blood volume are dependent on the autoregulation of CBF.
Sustained elevations in ICP (when isolated to the intracranial space) can lead to catastrophic herniation of the brain. Herniation may occur at one of four sites (Figure 26-6): (1) the cingulate gyrus under the falx cerebri, (2) the uncinate gyrus through the tentorium cerebelli, (3) the cerebellar tonsils through the foramen magnum, or (4) any area beneath a defect in the skull (transcalvarial).
FIGURE 26-6 Potential sites of brain herniation. (Reproduced with permission from Fishman RA. Brain edema. N Engl J Med. 1975 Oct 2;293(14):706-711.)

Overall, most general anesthetics have a favorable effect on brain energy consumption by reducing electrical activity. The effects of the specific agents are complicated by concomitant administration of other drugs, surgical stimulation, intracranial compliance, blood pressure, and CO, tension. For example, hypocapnia blunts the increases in CBF and ICP that usually occur with ketamine and volatile agents.
This section describes the changes generally associated with each drug when given alone. Table 26—1 summarizes and compares the effects of the various anesthetics. The effects of vasoactive agents and neuromuscular blocking agents are also discussed.
TABLE 26-1 Comparative effects of anesthetic agents on cerebral physiology.!

?. Propofol, cBV = 4. Propofol, icp = i. Benzodiazepines, CMR = LW. Benzodiazepines, CBF = L. Benzodiazepines, CSF Production = +. Benzodiazepines, CSF Absorption = tT. Benzodiazepines, cBV = a. Benzodiazepines, icp = 1. Ketamine, CMR = +. Ketamine, CBF = TT. Ketamine, CSF Production = +. Ketamine, CSF Absorption = L. Ketamine, cBV = TT. Ketamine, icp = TT. Opioids, CMR = +. Opioids, CBF = +. Opioids, CSF Production = £. Opioids, CSF Absorption = if. Opioids, cBV = +. Opioids, icp = +. Lidocaine, CMR = dW. Lidocaine, CBF = Lt. Lidocaine, CSF Production = ?. Lidocaine, CSF Absorption = . Lidocaine, cBV = . Lidocaine, icp = 
- 'T, increase; J, decrease; +, little or no change; ?, unknown; CMR, cerebral metabolic rate; CBF, cerebral blood flow; CSF, cerebrospinal fluid; CBV, cerebral blood volume; ICP, intracranial pressure.

Halothane, desflurane, sevoflurane, and isoflurane produce concentrationdependent decreases in CMR. Isoflurane produces the greatest maximal depression (up to 50% reduction), whereas halothane has the least effect (<25% reduction). The effects of desflurane and sevoflurane are nearly the same as those of isoflurane. No further reduction in CMR is produced by doses of anesthetics or other drugs greater than the doses that render the EEG isoelectric.

At normocarbia, volatile anesthetics dilate cerebral vessels and impair autoregulation in a concentration-dependent manner (Figure 26—7). Halothane has the greatest effect on CBF; at concentrations greater than 1%, it nearly abolishes cerebral autoregulation. Moreover, the increase in blood flow is generalized throughout all parts of the brain. At an equivalent minimum alveolar concentration (MAC) and blood pressure, halothane increases CBF up to 200%, compared with 20% for isoflurane or desflurane. Sevoflurane produces the least cerebral vasodilation. The effect of volatile agents on CBF also seems to be time dependent, because, with continued administration (2-5 h), blood flow begins to return to normal.
FIGURE 26-7 Dose-dependent depression of cerebral autoregulation by the volatile anesthetics.
The response of the cerebral vasculature to CO, is generally retained with all volatile agents. Hyperventilation (hypocapnia) can therefore abolish or blunt the initial effects of these agents on CBF. With halothane, the timing of the hyperventilation is important. Only if hyperventilation is initiated prior to the administration of halothane will halothane-induced increases in CBF be prevented. In contrast, simultaneous hyperventilation with administration of the other can increases in CBF and ICP.
agents prevent
Increases in cerebral blood volume (10—12%) generally parallel increases in CBF, but the relationship is not necessarily linear. Expansion of cerebral blood volume can markedly elevate ICP in patients with reduced intracranial compliance. Hypocapnia can blunt the increase in cerebral blood volume associated with volatile anesthetic administration.

As is apparent from the preceding discussion, volatile agents alter, but do not uncouple, the normal relationship of CBF and CMR. The combination of a decrease in neuronal metabolic demand with an increase in CBF (metabolic supply) has been termed Juxury perfusion. In contrast to this potentially beneficial effect during global ischemia, a detrimental circulatory steal phenomenon is possible with volatile anesthetics in the setting of focal ischemia. Volatile agents can increase blood flow in normal areas of the brain, but not in ischemic areas, where arterioles are already maximally vasodilated. The end result may be a redistribution ("steal") of blood flow away from ischemic to normal areas.

Volatile anesthetics affect both formation and absorption of CSF. Halothane impedes absorption of CSF, but only minimally retards formation. Isoflurane, on the other hand, facilitates absorption and is therefore an agent with favorable effects on CSF dynamics.

The net effect of volatile anesthetics on ICP is the result of immediate changes in cerebral blood volume, delayed alterations on CSF dynamics, and arterial CO, tension.

The effects of nitrous oxide are influenced by other agents or changes in CO, tension. Thus, when combined with intravenous agents, nitrous oxide has minimal effects on CBF, CMR, and ICP. Adding this agent to a volatile anesthetic, however, can in theory further increase CBF. When given alone, nitrous oxide causes cerebral vasodilation and can potentially increase ICP.

@ With the exception of ketamine, all intravenous agents either have little effect on or reduce CMR and CBF. Moreover, with some exceptions, changes in blood flow generally parallel those in metabolic rate. Cerebral autoregulation and CO, responsiveness are preserved with all agents.

Barbiturates have four major actions on the CNS: (1) hypnosis, (2) depression of CMR, (3) reduction of CBF due to increased cerebral vascular resistance, and (4) anticonvulsant activity. Barbiturates produce dose-dependent decreases in CMR and CBF until the EEG becomes isoelectric. At that point, maximum CMR reductions of nearly 50% are observed; additional barbiturate dosing does not further reduce CMR. Unlike isoflurane, barbiturates reduce metabolic rate uniformly throughout the brain. CMR is depressed slightly more than CBF, such that metabolic supply exceeds metabolic demand (as long as CPP is maintained). Because barbiturate-induced cerebral vasoconstriction occurs only in normal areas, these agents tend to redistribute blood flow from normal to ischemic areas in the brain. The cerebral vasculature in ischemic areas remains maximally dilated because of ischemic vasomotor paralysis. Barbiturates also seem to facilitate absorption of CSF. The resultant reduction in CSF volume, combined with decreases in CBF and cerebral blood volume, makes barbiturates highly effective in lowering ICP. Their anticonvulsant properties are also advantageous in neurosurgical patients who are at increased risk of seizures.

Opioids generally have minimal effects on CBF, CMR, and ICP, unless Paco, rises secondary to respiratory depression. Increases in ICP have been reported in some patients with intracranial tumors following administration of opioids. The mechanism seems to be a precipitous drop in blood pressure; reflex cerebral vasodilation likely increases intracranial blood volume and potentially ICP. Significant decreases in blood pressure can adversely affect CPP, regardless of the opioid selected.

Etomidate decreases the CMR, CBF, and ICP in much the same way as barbiturates. Its effect on CMR is nonuniform, affecting the cortex more than the brainstem. Etomidate also decreases production and enhances absorption of CSF. Induction with etomidate is associated with a frequent incidence of myoclonic movements, but no seizure activity on the EEG in normal individuals. Reports of seizure activity following etomidate suggest that the drug is best avoided in patients with a history of epilepsy.

Propofol reduces CBF and CMR, similar to barbiturates and etomidate. Although it has been associated with dystonic and choreiform movements, propofol seems to have significant anticonvulsant activity. Its short elimination half-life makes it a useful agent for neuroanesthesia. Propofol infusion is commonly used for maintenance of total intravenous anesthesia in patients with or at risk of intracranial hypertension. Propofol is by far the most common induction agent for neuroanesthesia.

Benzodiazepines lower CBF and CMR, but to a lesser extent than barbiturates, etomidate, or propofol. Benzodiazepines also have useful anticonvulsant properties. Midazolam is the benzodiazepine of choice in neuroanesthesia because of its short half-life. Midazolam used as an induction agent may cause decreases in blood pressure and CPP and may result in prolonged emergence.

Ketamine is the only intravenous anesthetic that dilates the cerebral vasculature and increases CBF (50-60%). Selective activation of certain areas (limbic and reticular) is partially offset by depression of other areas (somatosensory and auditory) such that total CMR does not change. Seizure activity in thalamic and limbic areas is also described. Ketamine may also impede absorption of CSF without affecting formation. Increases in CBF, cerebral blood volume, and CSF volume can potentially increase ICP markedly in patients with decreased intracranial compliance. However, ketamine administration does not increase ICP in neurologically impaired patients under controlled ventilation with concomitant administration of propofol or a benzodiazepine. Additionally, ketamine may offer neuroprotective effects, according to some investigations. Ketamine's blockade of the N-methyl-p-aspartate (NMDA) receptor during periods of increased glutamate concentrations, as occurs during brain injury, may be protective against neuronal cell death (Figure 26—8). In spite of theoretical concerns regarding ketamine's ability to increase ICP in patients with traumatic injury, it has been used in brain-injured patients without deleterious effects upon ICP.

FIGURE 26-8 Pharmacological effects reported for racemic and S(+)ketamine, which are presumed to be relevant for neuroprotection. After onset of brain injury, blockade of excessive stimulation of N-methyl-p-aspartate (NMDA) receptors by ketamine reduces calcium influx through the receptor channel (1). This attenuates supraphysiological increases in the assembly and interaction of NMDA receptor subunits, postsynaptic density proteins, and other intracellular signaling systems such as protein kinases (2). Thus, several kinase transduction cascades become less activated. This improves preservation of metabolism and maintenance of the mitochondrial transmembrane potential (3). This, in turn, reduces pathological activation of transcription factors (4). Proteins involved in apoptosis are less activated, which is associated with less DNA fragmentation (5). A better preservation of synaptic proteins occurs, and the expression of growth proteins indicating regeneration in adult neurons is enhanced (6, 7). The prevention of pathological amplification of NMDA receptor signaling finally results in increased cellular survival, preserved cellular and synaptic integrity, and regenerative efforts (8). *Superiority of effects induced by S(+)-ketamine, only. (Reproduced with permission from Himmelseher S, Durieux ME. Revising a dogma: ketamine for patients with neurological injury? Anesth Analg. 2005 Aug;101(2):524-534.)
Intravenous lidocaine decreases CMR, CBF, and ICP, but to a lesser degree than other agents. Its principal advantage is that it decreases CBF (by increasing cerebral vascular resistance) without causing other significant hemodynamic effects. Lidocaine may also have neuroprotective effects. Lidocaine infusions are used in some centers as a supplement to general anesthesia to reduce emergence delirium and the requirement for opioids.
Droperidol has little or no effect on CMR and minimally reduces CBF. Droperidol and opioids were once mainstays of neuroanesthesia. Droperidol's prolongation of the QT interval and risk of fatal arrhythmia have retarded its use. Reversal of opioids or benzodiazepines with naloxone or flumazenil, respectively, can reverse any beneficial reductions in CBF and CMR. Reversal of narcotics or benzodiazepines in chronic users can lead to symptoms of substance withdrawal.

® With normal autoregulation and an intact blood-brain barrier, vasopressors increase CBF only when mean arterial blood pressure is below 50 to 60 mm Hg or above 150 to 160 mm Hg. In the absence of autoregulation, vasopressors increase CBF by their effect on CPP. Changes in CMR generally parallel those in blood flow. B-Adrenergic agents seem to have a greater effect on the brain when the blood-brain barrier is disrupted; central B,-receptor stimulation increases CMR and blood flow. B-Adrenergic blockers generally have no direct effect on CMR or CBF. Excessive elevations in blood pressure with any agent can disrupt the blood-brain barrier.

In the absence of hypotension, most vasodilators induce cerebral vasodilation and increase CBF in a dose-related fashion. When these agents decrease blood pressure, CBF is usually maintained and may even increase. The resultant increase in cerebral blood volume can elevate ICP in patients with decreased intracranial compliance.

Neuromuscular blockers (NMBs) lack direct action on the brain but can have important secondary effects. Hypertension and histamine-mediated cerebral vasodilation increase ICP, whereas systemic hypotension (from histamine release or ganglionic blockade) lowers CPP. Succinylcholine can increase ICP to a generally minimal and clinically unimportant extent. Moreover, a small (defasciculating) dose of a nondepolarizing NMB seems to blunt the increase, although this appears to be a dated practice. In the majority of instances, increases in ICP following administration of an NMB are the result of a hypertensive response due to light anesthesia during laryngoscopy and tracheal intubation. Acute elevations in ICP will also be seen if hypercapnia or hypoxemia results from prolonged apnea.

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@ Hypothermia is a suggested method for protecting the brain during focal and global ischemia. Indeed, profound hypothermia is often used for up to 1 h of total circulatory arrest. Unlike anesthetic agents, hypothermia decreases both basal and electrical metabolic requirements throughout the brain; metabolic requirements continue to decrease even after complete electrical silence. Additionally, hypothermia reduces free radicals and other mediators of ischemic injury. Induced hypothermia has shown benefit following cardiac arrest and is a routine part of most post-arrest protocols for comatose patients.

Barbiturates, etomidate, propofol, isoflurane, desflurane, and sevoflurane can produce burst suppression, and all but desflurane and sevoflurane can produce complete electrical silence of the brain and eliminate the metabolic cost of electrical activity. Unfortunately, these agents have no effect on basal energy requirements. Furthermore, with the exception of barbiturates, their effects are nonuniform, affecting different parts of the brain to variable extents.
Ketamine may also have a protective effect because of its ability to block the actions of glutamate at the NMDA receptor.
No anesthetic agent has consistently been shown to be protective against global ischemia. Studies highlighting the potential neurotoxicity of anesthetics (especially in infants) also question the role of volatile anesthetics in neuroprotection.

Nimodipine plays a role in the in the treatment of vasospasm associated with subarachnoid hemorrhage.

Maintenance of a satisfactory CPP is critical. Hypotension, increases in venous pressure, and increases in ICP should be avoided. Oxygen-carrying capacity should be maintained and normal arterial oxygen tension preserved. Hyperglycemia aggravates neurological injuries following either focal or global ischemia, and blood glucose should be maintained at less than 180 mg/dL. Normocarbia should be maintained, as both hypercarbia and hypocarbia have no beneficial effect on cerebral ischemia; hypocarbia-induced cerebral vasoconstriction may aggravate the ischemia, whereas hypercarbia may induce a steal phenomenon (with focal ischemia) or worsen intracellular acidosis.

Electrophysiological monitors are used to assess the functional integrity of the CNS. The most commonly used monitor during neurosurgical procedures is evoked potentials. EEG is less commonly used. Proper application of these monitoring modalities is critically dependent on recognizing anesthetic-induced changes. Both monitoring modalities are described in Chapter 6.
The effects of anesthetic agents on the EEG are summarized in Table 26-2.

Inhalational agents (subanesthetic), Depression = Inhalation agents (1-2 MAC). Barbiturates (small doses), Depression = Barbiturates. Benzodiazepines (small doses), Depression = Opioids. Etomidate (small doses), Depression = Propofol. Nitrous oxide, Depression = Etomidate. Ketamine, Depression = Hypocapnia. Mild hypercapnia, Depression = Marked hypercapnia. Sensory stimulation, Depression = Hypothermia. Hypoxia (early), Depression = Hypoxia (late) Ischemia

EEG monitoring is useful for assessing the adequacy of cerebral perfusion during carotid endarterectomy (CEA), as well as anesthetic depth (most often with processed EEG). EEG changes can be simplistically described as either activation or depression. EEG activation (a shift to predominantly highfrequency and low-voltage activity) is seen with light anesthesia and surgical stimulation, whereas EEG depression (a shift to predominantly low-frequency and high-voltage activity) occurs with deep anesthesia or cerebral compromise. Most anesthetics produce activation (at subanesthetic doses) followed by dose-dependent depression of the EEG.

Isoflurane, desflurane, and sevoflurane produce a burst suppression pattern at high doses (>1.2-1.5 MAC). Nitrous oxide is unusual in that it increases both frequency and amplitude (high-amplitude activation).

Benzodiazepines can produce both activation and depression of the EEG. Barbiturates, etomidate, and propofol produce a similar pattern and are the only commonly used intravenous agents capable of producing burst suppression and electrical silence at high doses. Opioids characteristically produce only dosedependent depression of the EEG. Lastly, ketamine produces an unusual activation consisting of rhythmic high-amplitude theta activity followed by very high-amplitude gamma and low-amplitude beta activities.

Somatosensory evoked potentials test the integrity of the spinal dorsal columns and the sensory cortex and may be useful during resection of spinal tumors, instrumentation of the spine, and carotid artery and aortic surgery. The adequacy of perfusion of the spinal cord during aortic surgery is better assessed with motor evoked potentials (which assess the anterior part of the spinal cord). Brainstem auditory evoked potentials test the integrity of the eighth cranial nerve and the auditory pathways above the pons and are used for surgery in the posterior fossa. Visual evoked potentials may be used to monitor the optic nerve and occipital cortex during resections of large pituitary tumors.
Interpretation of evoked potentials is more complicated than that of the EEG. Evoked potentials have poststimulus latencies that are described as short, intermediate, and long. Short-latency evoked potentials arise from the nerve stimulated or the brainstem. Intermediateand long-latency evoked potentials are primarily of cortical origin. In general, short-latency potentials are least affected by anesthetic agents, whereas long-latency potentials are affected by even subanesthetic levels of most agents. Visual evoked potentials are most affected by anesthetics, whereas brainstem auditory evoked potentials are least affected.
Intravenous agents in clinical doses generally have less marked effects on evoked potentials than do volatile agents, but, in high doses, can also decrease amplitude and increase latencies (see Chapter 6). Ketamine generally increases the amplitude of short-latency signals.

A 62-year-old man has undergone a right carotid endarterectomy (CEA). Immediately following surgery, in the recovery room, he is noted to be weak on the contralateral side.

Patients with carotid stenosis are at very increased risk of coronary artery and peripheral arterial disease. It would be unusual for a patient to have carotid stenosis who did not have evidence of atherosclerosis elsewhere. Patients undergoing CEA, therefore, require a preoperative cardiac evaluation, according to American College of Cardiology/American Heart Association guidelines.
With respect to patient risk factors, the guidelines provide algorithms for how patients should be evaluated and managed intraoperatively. As part of this patient's preoperative evaluation, a thorough neurological examination should have been performed with special attention paid to motor function. This patient may well have been weak on the left side prior to surgery, in which case the hemiparesis might be due to a preexisting condition. If this is a new finding, it requires aggressive management.
Is general or regional anesthesia the optimal anesthetic technique for managing patients undergoing CEA?
For the past several decades, the majority of patients undergoing CEAs in the United States have had general anesthesia. General anesthesia was chosen because many surgeons operating in the neck area felt more comfortable if the airway was controlled, and the patient was completely anesthetized should evidence of cerebral ischemia develop.
More recently, regional anesthesia has been advocated as providing an adequate surgical field, a comfortable and relaxed patient (if done with monitored anesthesia care), stable hemodynamics, and ideal monitoring of cerebral function during crossclamping because an awake patient provides the best evidence of adequate cerebral perfusion. The patient can indicate or be observed for evidence of aphasia, facial droop, or hemiparesis. Regional anesthesia is usually performed with superficial cervical plexus blocks.
How should cerebral function be monitored intraoperatively in this patient?
When the carotid is cross-clamped, the ability to identify inadequate cerebral circulation in the ipsilateral hemisphere is critical, as there is a window of opportunity for immediate intervention and correction of any deficit.
Global and focal neurological status can continuously be assessed in awake patients if they are mildly sedated when undergoing regional anesthesia. In such a situation, practical assessment consists of frequent (every 2-5 min) examination of strength using the contralateral handgrip and maintenance of constant verbal contact ("cocktail conversation") with the patient to assess level of consciousness.

In patients undergoing general anesthesia, indirect cerebral monitoring techniques have been used to assess the adequacy of the cerebral circulation. These techniques include stump bleeding, stump pressure, jugular venous oxygen saturation, EEG, and transcranial Doppler (TCD). Back bleeding of the distal carotid artery following crossclamp and incision of the artery suggests reasonable collateral circulation above the clamp. It is very subjective and nonquantitative.
To better qualify and quantify the adequacy of collateral perfusion (Figure 26-9), stump pressure measurements can be used. Some surgeons believe that a shunt should be used in all patients with a previous cerebrovascular accident, independent of stump pressure, and for any patient whose stump pressure is less than 25 mm Hg. However, this is controversial, as many neurosurgeons and vascular surgeons use 50 mm Hg as a cutoff. The reliability of stump pressure to predict the need for selective shunting has also been questioned. Some surgeons routinely shunt all patients, some shunt no patients, and others use selective shunting. Outcome data have not identified the best surgical approach.
The EEG is sometimes used for monitoring patients undergoing CEA
under general anesthesia. In such a circumstance, inhalation or intravenous anesthesia can influence the EEG, but gross changes associated with carotid clamping can be detected. However, analyzing subtleties of the EEG is labor and technology intensive.
Evoked potentials have also been employed during CEA. Should neurophysiological studies identify cerebral ischemia, the surgeon can place a vascular shunt during the surgical repair to provide for ipsilateral cerebral perfusion.

During carotid clamping and immediately afterward in the recovery room, patients are often hemodynamically labile. Bradycardia can develop during surgical manipulation of the carotid sinus because of the direct stimulation of the vagus nerve. Tachycardia may develop as a result of stress or pain or as a direct result of manipulation of the carotid sinus with release of catecholamines into the circulation.
Hypotension is also observed because of the direct vasodilating and negative ionotropic effects of anesthetic agents. Hypotension following carotid unclamping is common, particularly in patients with more severe carotid stenosis. This could be due to a cerebral protective process. Cerebral autoregulation protects the brain from reperfusion by reducing production of renin, vasopressin, and norepinephrine, which results in hypotension. Hypertension is also a frequent finding in patients undergoing CEA. Many patients have hypertension as a comorbid condition, which is often further exacerbated by the surgical stress and manipulation of the carotid body, which causes release of catecholamines and sympathetic stimulation.
Invasive arterial pressure monitoring and suitable venous access to infuse vasoactive medications are necessary during carotid surgery.

This patient most likely has had a cerebrovascular accident due to an arterio-to-arterial embolus; more than 95% of such patients will fit into this category. Weakness can also develop as a result of a hyperperfusion syndrome, which occurs in patients with severe carotid stenosis who have now reestablished flow to the affected cerebral hemisphere. Such patients usually have a greater than 95% carotid stenosis with a less than 1-mm channel in the affected carotid artery. Typically, the syndrome does not develop in the postoperative anesthesia care unit (PACU), but several hours afterward when the patient begins complaining of a headache, and, in severe cases, develops hemiparesis.
Because a cerebrovascular accident is most likely, when the anesthesiologist is called to see such a patient in the PACU, a thorough neurological examination quantifying any cranial nerve involvement and the degree of weakness on the contralateral side should be performed. Any hemodynamic changes need to be treated immediately, with assurance of adequate hemoglobin and oxygenation levels. The surgeon needs to be notified at once, and ultrasonic evaluation of the carotid artery is frequently required to determine whether there might be problems with the intimal suture line. It may be necessary to return to the operating room to explore the carotid artery.

- Rabinstein A. Elucidating the value of continuous brain oxygenation monitoring. Neurocrit Care. 2010;12:144.
Saqqur M, Zygun D, Demchuk D. Role of transcranial Doppler in neurocritical care. Crit Care Med. 2007;35:S216.
- Todd M. Outcomes after neuroanesthesia and neurosurgery: What makes a difference? Anesthesiol Clin. 2012;30:399.

@ Regardless of the cause, intracranial masses present symptoms and signs according to growth rate, location, and intracranial pressure. Slowly growing masses are frequently asymptomatic for long periods (despite relatively large size), whereas rapidly growing ones may present when the mass remains relatively small.
@ Computed tomography and magnetic resonance imaging scans should be reviewed for evidence of brain edema, midline shift greater than 0.5 cm, or ventricular displacement or compression.
@ Operations in the posterior fossa can injure vital circulatory and respiratory brainstem centers, as well as cranial nerves or their nuclei.
@ Venous air embolism can occur when the pressure within an open vein is subatmospheric. These conditions may exist in any position and during any procedure whenever the wound is above the level of the heart.
@ Optimal recovery of air following venous air embolism is provided by a multiorificed catheter positioned at the junction between the right atrium and the superior vena cava. Confirmation of correct catheter positioning can be accomplished by intravascular electrocardiography, radiography, or transesophageal echocardiography.
@ Ina patient with head trauma, correction of hypotension and control of any bleeding take precedence over radiographic studies and definitive neurosurgical treatment because systolic arterial blood pressures of less than 80 mm Hg predict a poor outcome.
@ Sudden, massive blood loss from injury to the great vessels can occur intraoperatively with adjacent thoracic or lumbar spine procedures.
Anesthetic techniques must be modified in the presence of intracranial hypertension and marginal cerebral perfusion. In addition, many neurosurgical procedures require patient positions (eg, sitting, prone) that further complicate management. This chapter applies the principles developed in Chapter 26 to the anesthetic care of neurosurgical patients.

Intracranial hypertension is defined as a sustained increase in intracranial pressure (ICP) above 15 mm Hg. Intracranial hypertension may result from an expanding tissue or fluid mass, a depressed skull fracture if it compresses a venous sinus, inadequate absorption of cerebrospinal fluid (CSF), excessive cerebral blood volume (CBV), or systemic disturbances promoting brain edema (see next section). Multiple factors may be present. For example, tumors in the posterior fossa usually not only are associated with some degree of brain edema and mass effect, but also readily obstruct CSF outflow by compressing the fourth ventricle (obstructive hydrocephalus).
Although many patients with increased ICP are initially asymptomatic, they typically develop characteristic symptoms and signs, including headache, nausea, vomiting, papilledema, focal neurological deficits, and altered consciousness. When ICP exceeds 30 mm Hg, cerebral blood flow (CBF) progressively decreases, and a vicious circle is established: ischemia causes brain edema, which in turn, increases ICP, resulting in more ischemia. If left unchecked, this cycle continues until the patient dies of progressive neurological damage or catastrophic herniation. Periodic increases in arterial blood pressure with reflex slowing of the heart rate (Cushing response) can be correlated with abrupt increases in ICP (plateau waves) lasting 1 to 15 min. This phenomenon is the result of autoregulatory mechanisms periodically decreasing cerebral vascular resistance and increasing arterial blood pressure in response to cerebral ischemia. Eventually, severe ischemia and acidosis completely abolish autoregulation (vasomotor paralysis).

An increase in brain water content can be produced by several mechanisms. Disruption of the blood-brain barrier (vasogenic edema) is most common and allows the entry of plasma-like fluid into the brain. Increases in blood pressure enhance the formation of this type of edema. Common causes of vasogenic edema include mechanical trauma, high altitudes, inflammatory lesions, brain tumors, hypertension, and infarction. Cerebral edema following metabolic insults (cytotoxic edema), such as hypoxemia or ischemia, results from failure of brain cells to actively extrude sodium, causing progressive cellular swelling. Interstitial cerebral edema is the result of obstructive hydrocephalus and entry of CSF into brain interstitium. Cerebral edema can also be the result of intracellular movement of water secondary to acute decreases in serum osmolality (water intoxication).

Hypertonic saline (3% NaCl) is sometimes used to reduce cerebral edema and
ICP. Hypertonic saline should be administered with care to avoid central pontine myelinolysis or osmotic demyelination syndrome in hyponatremic patients (see Chapter 49). Serum sodium concentration and osmolality should be frequently monitored. In patients with traumatic brain injury, interventions in addition to mannitol to lower intracranial pressure include head elevation, CSF drainage via ventriculostomy, moderate hypocapnia, and metabolic suppression with barbiturates. Decompressive craniectomy has been shown to decrease mortality in patients with sustained increases in ICP (> 25 mm Hg) following traumatic brain injury.

Intracranial masses may be congenital, neoplastic (benign or malignant), infectious (abscess or cyst), or vascular (hematoma or arteriovenous malformation). Craniotomy is commonly undertaken for neoplasms of the brain. Primary tumors usually arise from glial cells (astrocytoma, oligodendroglioma, or glioblastoma), ependymal cells (ependymoma), or supporting tissues (meningioma, schwannoma, or choroidal papilloma). Childhood tumors include medulloblastoma, neuroblastoma, and astrocytoma.
® Regardless of the cause, intracranial masses present symptoms and signs according to growth rate, location, and ICP. Slowly growing masses are frequently asymptomatic for long periods (despite relatively large size), whereas rapidly growing ones may present when the mass remains relatively small. Common presentations include headache, seizures, a general decline in cognitive or specific neurological functions, and focal neurological deficits. Symptoms typical to supratentorial masses include seizures, hemiplegia, or aphasia, whereas symptoms typical of infratentorial masses may include cerebellar dysfunction (ataxia, nystagmus, and dysarthria) or brainstem compression (cranial nerve palsies, altered consciousness, or abnormal respiration).

The preoperative evaluation for patients undergoing craniotomy should attempt to establish the presence or @ absence of intracranial hypertension. Computed tomography (CT) and magnetic resonance imaging (MRI) scans should be reviewed for evidence of brain edema, midline shift greater than 0.5 cm, or ventricular displacement or compression. Imaging studies typically will be performed before the patient receives dexamethasone, so the mass effect may be less acute when patients who have already received dexamethasone present in the operating room. The neurological examination should document mental status and any sensory or motor deficits. Medications should be reviewed with special reference to corticosteroid, diuretic, and anticonvulsant therapy. Laboratory evaluation should rule out corticosteroid-induced hyperglycemia, electrolyte disturbances due to diuretics, or abnormal secretion of antidiuretic hormone. Anticonvulsant blood concentrations may be measured, particularly when seizures are not well controlled.

Sedative or opioid premedication is best avoided, particularly when intracranial hypertension is suspected. Hypercapnia secondary to respiratory depression increases ICP. Corticosteroids and anticonvulsant therapy should be continued until the time of surgery.

Induction of anesthesia and tracheal intubation are critical periods for patients with compromised intracranial pressure to volume relationships, particularly if there is an elevated ICP. Intracranial elastance can be improved by osmotic diuresis or removal of small volumes of CSF via a ventriculostomy drain. The goal of any technique should be to induce anesthesia and intubate the trachea without increasing ICP or compromising CBF. Arterial hypertension during induction increases CBV and promotes cerebral edema. Sustained hypertension can lead to marked increases in ICP, decreasing CPP and risking herniation. Excessive decreases in arterial blood pressure can be equally detrimental by compromising CPP.
The most common induction technique employs propofol together with modest hyperventilation to reduce ICP and blunt the noxious effects of laryngoscopy and intubation. All patients receive controlled ventilation once the propofol has been injected. A neuromuscular blocker (NMB) is given to facilitate ventilation and prevent straining or coughing, both of which can abruptly increase ICP. An intravenous opioid given with propofol blunts the sympathetic response, particularly in young patients. Esmolol (0.5—-1.0 mcg/kg) is effective in preventing tachycardia associated with intubation in lightly anesthetized patients.
The actual induction technique can be varied according to individual patient responses and coexisting diseases. Succinylcholine may theoretically increase

ICP, particularly if intubation is attempted before deep anesthesia is established. Succinylcholine, however, remains the agent of choice for rapid sequence induction or when there are concerns about a potentially difficult airway, as hypoxemia and hypercarbia are much more detrimental than any effect of succinylcholine to the patient with intracranial hypertension. Hypertension during induction can be treated with B,-blockers or by deepening the anesthetic with additional propofol. Modest concentrations of volatile agents (eg, sevoflurane) may also be used, provided that hyperventilation is also used. Sevoflurane best preserves autoregulation of CBF and produces limited vasodilation; it may be the preferred volatile agent in patients with elevated ICP. Because of their potentially deleterious effect on CBV and ICP, vasodilators (eg, nicardipine, nitroprusside, nitroglycerin, and hydralazine) are avoided until the dura is opened. Hypotension is generally treated with incremental doses of vasopressors (eg, phenylephrine).

Frontal, temporal, and parietooccipital craniotomies are performed in the supine position. The head is elevated 15° to 30° to facilitate venous and CSF drainage. The head may also be turned to the side to facilitate exposure. Excessive flexion or rotation of the neck impedes jugular venous drainage and can increase ICP. Before and after positioning, the tracheal tube should be secured and position verified, and all breathing circuit connections checked. The risk of unrecognized disconnections is increased because the patient's airway cannot be easily assessed after surgical draping; moreover, the operating table is usually turned 90° or 180° away from the anesthesia provider.

Anesthesia can be maintained with inhalation anesthesia, total intravenous anesthesia techniques (TIVA), or a combination of an opioid and intravenous hypnotic (most often propofol) with a low-dose inhalation agent. Even though periods of stimulation are few, neuromuscular blockade is recommended— unless neurophysiological monitoring contradicts its use—to prevent straining, bucking, or other movement. Increased anesthetic requirements can be expected during the most stimulating periods: laryngoscopy—intubation, skin incision, dural opening, periosteal manipulations, including Mayfield pin placement and closure. TIVA with remifentanil and propofol facilitates rapid emergence and immediate neurological assessment. Likewise, the a)-agonist dexmedetomidine can be employed during both asleep and awake craniotomies to similar effect.
Hyperventilation should be continued intraoperatively to maintain Paco, at roughly 30 to 35 mm Hg. Lower Paco, tensions provide little additional benefit and may be associated with cerebral ischemia and impaired oxygen dissociation from hemoglobin. Ventilatory patterns resulting in high mean airway pressures (a low rate with large tidal volumes) should be avoided because of a potentially adverse effect on ICP by increasing central venous pressure and the potential for lung injury. Hypoxic patients may require positive end-expiratory pressure (PEEP) and increased mean airway pressure; in such patients, the effect of PEEP on ICP is variable.
Intravenous fluid replacement should be limited to glucose-free isotonic crystalloid. Hyperglycemia is common in neurosurgical patients and has been implicated in increasing ischemic brain injury. Hyperglycemia should be corrected preoperatively. Neurosurgical procedures are often associated with occult blood loss (underneath surgical drapes or on the floor). Hypotension and hypertension should both be expeditiously corrected.

Most patients undergoing elective craniotomy can be extubated at the end of the procedure. Patients who will remain intubated should be sedated to prevent agitation. Extubation in the operating room requires special handling during emergence. Straining or "bucking" on the tracheal tube may precipitate intracranial hemorrhage or worsen cerebral edema. As the skin is being closed, the patient may resume breathing spontaneously. Should the patient's head be secured in a Mayfield pin apparatus, care must be taken to avoid any patient motions (eg, bucking on the tube), which could promote neck or cranial injuries. After the head dressing is applied and full access to the patient is regained (the table is turned back to its original position as at induction), any anesthetic agents are discontinued and the neuromuscular blockade is reversed. Rapid awakening facilitates immediate neurological assessment and is generally expected. Delayed awakening may be seen following opioid or sedative overdose, when the end-tidal concentration of the volatile agent remains greater than 0.2 minimum alveolar concentration (MAC), or when there is a metabolic derangement or a perioperative neurological injury. Patients may need to be transported to the CT scanner directly from the operating room for evaluation when they do not respond as predicted. Immediate reexploration may be required. Most patients are taken to the intensive care unit postoperatively for close monitoring of neurological function.

Craniotomy for a mass in the posterior fossa presents a unique set of potential problems: obstructive hydrocephalus, possible injury to vital brainstem centers, pneumocephalus, and, when these procedures are performed with the patient in the sitting position, an increased risk of postural hypotension and venous air embolism.

Infratentorial masses can obstruct CSF flow through the fourth ventricle or the cerebral aqueduct of Sylvius. Small but critically located lesions can markedly increase ICP. In such cases, a ventriculostomy is often performed under local anesthesia to decrease ICP prior to induction of general anesthesia.

® Operations in the posterior fossa can injure vital circulatory and respiratory brainstem centers, as well as cranial nerves or their nuclei. Such injuries may occur as a result of direct surgical trauma or ischemia from retraction or other interruptions of the blood supply. Damage to respiratory centers is said to nearly always produce circulatory changes; therefore, abrupt changes in blood pressure, heart rate, or cardiac rhythm should alert the anesthesia provider to the possibility of such an injury. Such changes should be communicated to the surgeon. Isolated damage to respiratory centers may rarely occur without premonitory circulatory signs during operations in the floor of the fourth ventricle. Historically, some clinicians have employed spontaneous ventilation during these procedures as an additional monitor of brain function. At completion of the surgery, brainstem injuries may present as an abnormal respiratory pattern or an inability to maintain a patent airway following extubation. Monitoring brainstem auditory evoked potentials may be useful in preventing eighth nerve damage during resections of acoustic neuromas. Electromyography is also used to avoid injury to the facial nerve but requires incomplete neuromuscular blockade intraoperatively.

Although most explorations of the posterior fossa can be performed with the patient in either a modified lateral or prone position, the sitting position may be preferred by some surgeons.
The patient is actually semirecumbent in the standard sitting position (Figure 27-1); the back is elevated to 60°, and the legs are elevated with the knees flexed. The head is fixed in a three-point holder with the neck flexed; the arms remain at the sides with the hands resting on the lap.
Figure 27-1 The sitting position for craniotomy.
Careful positioning and padding help avoid injuries. Pressure points, such as the elbows, ischial spines, heels, and forehead, must be protected. Excessive neck flexion has been associated with swelling of the upper airway (due to venous obstruction), and, rarely, quadriplegia (due to compression of the cervical spinal cord). Preexisting cervical spinal stenosis probably predisposes patients to the latter injury.

The sitting position increases the likelihood of pneumocephalus. In this position, air readily enters the subarachnoid space as CSF is lost during surgery. In patients with cerebral atrophy, drainage of CSF is marked; air can replace CSF on the surface of the brain and in the lateral ventricles. Expansion of a pneumocephalus following dural closure can compress the brain. Postoperative pneumocephalus can cause delayed awakening and continued impairment of neurological function. Because of these and other concerns, nitrous oxide is rarely used for sitting craniotomies (see further discussion that follows).

ventilation but decreased perfusion). Conversely, major hemodynamic manifestations, such as sudden hypotension, can occur well before hypoxemia is noted. Moreover, large amounts of intracardiac air impair tricuspid and pulmonic valve function and can produce sudden circulatory arrest by obstructing right ventricular outflow.
Paradoxic air embolism can result in a stroke or coronary occlusion, which may be apparent only postoperatively. Paradoxic air emboli are more likely to occur in patients with right-to-left intracardiac shunts, particularly when the normal transatrial (left > right) pressure gradient is consistently reversed.

A properly positioned central venous catheter can be used to aspirate entrained air, but there is only limited evidence that this influences outcomes after venous air embolism. Some clinicians have considered right atrial catheterization mandatory for sitting craniotomies, but this is a minority viewpoint. @ Optimal recovery of air following venous air embolism is provided by a multiorificed catheter positioned at the junction between the right atrium and the superior vena cava. Confirmation of correct catheter positioning can be accomplished by intravascular electrocardiography, radiography, or transesophageal echocardiography (TEE), with the latter being simplest and easiest. Intravascular electrocardiography is accomplished by using the saline-filled catheter as a "V" lead. Correct positioning near the cavoatrial junction is indicated by the appearance of a maximally biphasic P wave. If the catheter is advanced farther into the heart, the P wave changes from a biphasic to a undirectional deflection. A right ventricular or pulmonary artery waveform may also be observed when the catheter is connected to a pressure transducer and advanced too far, but pressure waveforms do not identify the cavoatrial junction.

The most sensitive detectors available should be used. Detecting even small amounts of venous air emboli is important because it allows surgical control of the entry site before additional air is entrained. Currently, the devices capable of detecting the smallest volumes of air are TEE and precordial Doppler sonography. These monitors can detect air bubbles as small as 0.25 mL. TEE has the added benefit of detecting the volume of the bubbles and any transatrial passage through a patent foramen ovale, as well as evaluating any effect venous air embolism may have on cardiac function. Doppler methods employ a probe over the right atrium (usually to the right of the sternum and between the third and sixth ribs). Interruption of the regular swishing of the Doppler signal by sporadic roaring sounds indicates venous air embolism. Changes in end-tidal respiratory gas concentrations are less sensitive but are important monitors that can also detect venous air embolism before overt clinical signs are present. Venous air embolism causes a sudden decrease in end-tidal CO, tension in proportion to the increase in pulmonary dead space; however, decreases can also be seen with hemodynamic changes unrelated to venous air embolism, such as decreased cardiac output. A reappearance (or increase) of nitrogen in expired gases may also be seen with venous air embolism. Changes in blood pressure and heart sounds ("mill wheel" murmur) are late manifestations of venous air embolism.

1., 1 = The surgeon should be notified so that he or she can flood the surgical field with saline or pack it with wet gauzes and apply bone wax to the skull edges until the entry site is identified and occluded.. 2., 1 = Nitrous oxide (if used) should be discontinued, and the patient ventilated with 100% oxygen.. 3., 1 = If a central venous catheter is present, it should be aspirated in an attempt to retrieve the entrained air.. 4., 1 = Intravascular volume infusion should be given to increase central venous pressure.. 5., 1 = Vasopressors should be given to treat hypotension.. 6., 1 = Bilateral jugular vein compression, by increasing intracranial venous pressure, may slow air entrainment and cause back bleeding, which might help the surgeon identify the entry point of the embolus.. 7., 1 = Some clinicians advocate PEEP to increase cerebral venous pressure; however, reversal of the normal transatrial pressure gradient may promote paradoxical embolism in a patient with incomplete closure of the foramen ovale.. 8., 1 = If the previously listed measures fail, the patient should be placed in a head- down position, and the wound should be closed quickly.. 9., 1 = Persistent circulatory arrest necessitates the supine position and institution of resuscitation efforts using advanced cardiac life support algorithms.

27-1 Advantages and disadvantages of drugs used for conscious
TABLE sedation.!?
GABA receptor agonists, Advantages = . GABA receptor agonists, Disadvantages = . Benzodiazepines, Advantages = Anxiolysis. Benzodiazepines, Disadvantages = Large dose abolishes MER Alters the threshold for stimulation Induces dyskinesia. Propofol, Advantages = Widely used Short acting Predictable emergence profile. Propofol, Disadvantages = Abolishes tremors Attenuation of MER Unpredictable dosing in patients with Parkinson disease Induces dyskinesia Tendency to cause sneezing. Opioids, Advantages = . Opioids, Disadvantages = . Fentanyl Remifentanil, Advantages = ? Minimal effect on MER Short acting. Fentanyl Remifentanil, Disadvantages = Rigidity Suppression of tremors. a,-Agonist, Advantages = . a,-Agonist, Disadvantages = . Dexmedetomidine, Advantages = Non-GABA-mediated action Less effect on MER Anxiolysis and analgesic effects Sedation: easily arousable Does not ameliorate clinical signs of parkinsonism Maintains hemodynamic stability Preserves respiration. Dexmedetomidine, Disadvantages = High doses can abolish MER Hypotension, bradycardia
'Reproduced with permission from Venkatraghavan L, Luciano M, Manninen P, Anesthetic management of patients undergoing deep brain stimula: tor insertion. Anesth Analg. 2010 Apr 1;110(4):1138-1145.
2GABA, y-aminobutyric acid; MER, microelectrode recording,

Eye opening, Score = . Spontaneous, Score = 4. To speech, Score = 3. To pain, Score = 2. Nil, Score = 1. Best motor response, Score = . To verbal command, Score = . Obeys, Score = Oo. To pain, Score = . Localizes, Score = . Withdraws, Score = bw. Decorticate flexion, Score = . Extensor response, Score = —-NwW. Nil, Score = . Best verbal response, Score = . Oriented, Score = . Confused conversation, Score = bu. Inappropriate words, Score = . Incomprehensible sounds, Score = =—~NW. Nil, Score = 
Specific lesions include skull fractures, subdural and epidural hematomas, brain contusions (including intracerebral hemorrhages), penetrating head injuries, and traumatic vascular occlusions and dissections. The presence of a skull fracture greatly increases the likelihood of an intracranial lesion. Linear skull fractures are commonly associated with subdural or epidural hematomas. Basilar skull fractures may be associated with CSF rhinorrhea, pneumocephalus, cranial nerve palsies, or even a cavernous sinus—carotid artery fistula. Depressed skull fractures often present with an underlying brain contusion. Contusions may be limited to the surface of the brain or may involve hemorrhage in deeper hemispheric structures or the brainstem. Rapid deceleration injuries often produce both coup (frontal) and contrecoup (occipital) lesions. Epidural and subdural hematomas can occur as isolated lesions, as well as in association with cerebral contusions (more commonly with subdural than epidural lesions).
Operative treatment is usually elected for depressed skull fractures; evacuation of epidural, subdural, and some intracerebral hematomas; and debridement of penetrating injuries. Decompressive craniectomy is used to provide room for cerebral swelling. The cranium is subsequently reconstructed following resolution of cerebral edema.

ICP monitoring is usually indicated in patients with lesions associated with intracranial hypertension: large contusions, mass lesions, intracerebral hemorrhage, or evidence of edema on imaging studies. ICP monitoring should also be considered in patients with signs of intracranial hypertension who are undergoing nonneurological procedures. Acute intracranial hypertension leading to herniation should be treated with hyperventilation, osmolar therapy, and barbiturates. Immediate neurosurgical intervention is mandated. Multiple studies have found that sustained increases in ICP of greater than 60 mm Hg result in severe disability or death. Randomized trials have failed to detect the efficacy of early use of large doses of glucocorticoids in patients with head trauma. Hypothermia has likewise failed to improve survival following traumatic brain injury.

Anesthetic care of patients with severe head trauma begins in the emergency department. Measures to ensure patency of the airway, adequacy of ventilation and oxygenation, stabilization of the cervical spine, and correction of systemic hypotension should proceed simultaneously with neurological and trauma surgical evaluation. Airway obstruction and hypoventilation are common. Up to 70% of such patients have hypoxemia, which may be complicated by pulmonary contusion, fat emboli, or neurogenic pulmonary edema. The latter is attributed to marked systemic and pulmonary hypertension secondary to intense sympathetic nervous system activity. Supplemental oxygen should be given to all patients while the airway and ventilation are evaluated. Many patients will have substance intoxication. All patients must be assumed to have a cervical spine injury (up to 10% incidence) until the contrary is proven radiographically. Patients with obvious hypoventilation, an absent gag reflex, or a persistent score below 8 on the GCS (Table 27—2) require tracheal intubation. All other patients should be carefully observed for deterioration.

All patients should be regarded as having a full stomach and should have cricoid pressure (Sellick maneuver) applied during ventilation and tracheal intubation. Nevertheless, the effectiveness of this maneuver in preventing aspiration is questionable. In-line stabilization should be used during airway manipulation to maintain the head in a neutral position, unless radiographs confirm that there is no cervical spine injury. Following preoxygenation, the adverse effects of intubation on ICP are blunted by prior administration of propofol, 1.5 to 3.0 mg/kg, and a rapid-onset NMB. Succinylcholine may produce mild and transient increases in ICP in patients with closed head injury; however, the necessity for expeditious airway management trumps these theoretical concerns. Rocuronium is often used to facilitate intubation. The presence of a hard collar for cervical spine stabilization will increase intubation difficulty. Video laryngoscopy performed with in-line stabilization generally permits neutral position intubation of the trauma patient. An intubating bougie should be available to facilitate tube placement. If a difficult intubation is encountered with video laryngoscopy, fiberoptic or other techniques (eg, intubating LMA) can be attempted. If airway attempts are unsuccessful, a surgical airway should be obtained. Blind nasal intubation or blind passage of a nasogastric tube should be avoided in the presence of a basilar skull fracture due to the possibility of passing tubes directly through the fracture into the brain. The diagnosis of basilar skull fracture is suggested by CSF rhinorrhea or otorrhea, hemotympanum, or ecchymosis into periorbital tissues (raccoon sign) or behind the ear (Battle sign).

Hypotension in the setting of head trauma is nearly always related to other associated injuries (often intraabdominal). Profuse bleeding from scalp lacerations may cause hypovolemic hypotension in children. Hypotension may be seen with spinal cord injuries because of the sympathectomy associated @ with spinal shock. In a patient with head trauma, correction of hypotension and control of any bleeding take precedence over radiographic studies and definitive neurosurgical treatment because systolic arterial blood pressures of less than 80 mm Hg predict a poor outcome. Glucose-containing or hypotonic solutions should not be used (see earlier discussion). Otherwise, crystalloids and blood products can be administered as necessary. Massive blood loss in the patient with multiple injuries should result in activation of a massive transfusion protocol to provide a steady supply of platelets, fresh frozen plasma, and packed red blood cells. Invasive monitoring of arterial pressure, central venous pressure, and ICP are valuable, but should not delay diagnosis and treatment. Arrhythmias and electrocardiographic abnormalities in the T wave, U wave, ST segment, and QT
interval are common following head injuries, but are not necessarily associated with cardiac injury; they likely represent altered autonomic function.

The choice between operative and medical management of head trauma is based on radiographic and clinical findings. Patients should be stabilized prior to any CT or other imaging studies. Critically ill patients must be closely monitored during such studies. Restless or uncooperative patients may require general anesthesia for imaging. Sedation in such cases without control of the airway should be avoided because of the risk of further increases in ICP from hypercapnia or hypoxemia and because of the risk of aspiration.

and neurogenic pulmonary edema may also be responsible for deteriorating lung function. When PEEP is used, ICP monitoring can be useful to confirm an adequate CPP. Diabetes insipidus, characterized by inappropriately dilute polyuria, is frequently seen following brain trauma, especially with injuries to the pituitary. Other likely causes of polyuria should be excluded and the diagnosis confirmed by measurement of urine and serum osmolality prior to treatment with fluid restriction and vasopressin (see Chapter 49). Gastrointestinal bleeding from stress ulceration is common in patients not receiving prophylaxis.
The decision whether to extubate the trachea at the conclusion of the surgical procedure depends on the severity of the injury, the presence of concomitant abdominal or thoracic injuries, preexisting illnesses, and the preoperative level of consciousness. Young patients who were conscious preoperatively may be extubated following the removal of a localized lesion, whereas patients with diffuse brain injury should remain intubated. Moreover, persistent intracranial hypertension requires continued paralysis, sedation, CSF drainage, and elevated head position. Protracted hyperventilation may be employed should other interventions fail to correct ICP; however, cerebral blood flow may be deleteriously affected, leading to brain ischemia.

Saccular aneurysms and AVMs are common causes of nontraumatic intracranial hemorrhages. Surgical or interventional neuroradiological treatment may be undertaken either electively to prevent hemorrhage or emergently to prevent further complications once hemorrhage has taken place. Other nontraumatic hemorrhages (eg, from hypertension, sickle cell disease, or vasculitis) are usually treated medically.

Cerebral aneurysms typically occur at the bifurcation of the large arteries at the base of the brain; most are located in the anterior circle of Willis. Approximately 10% to 30% of patients have more than one aneurysm. The general incidence of saccular aneurysms in some estimates is reported to be 5%, but only a minority of those with aneurysms will have complications. Rupture of a saccular aneurysm is the most common cause of subarachnoid hemorrhage (SAH). The acute mortality following rupture is approximately 10%. Of those who survive the initial hemorrhage, about 25% die within 3 months from delayed complications. Moreover, up to 50% of survivors are left with neurological deficits. As a result, the emphasis in management is on prevention of rupture. Unfortunately, most patients present only after rupture has already occurred.

Patients may present with prodromal symptoms and signs suggesting progressive enlargement. The most common symptom is headache, and the most common physical sign is a third-nerve palsy. Other manifestations could include brainstem dysfunction, visual field defects, trigeminal nerve dysfunction, cavernous sinus syndrome, seizures, and hypothalamic—pituitary dysfunction. The most commonly used techniques to diagnose an aneurysm are MRI, angiography, and helical CT angiography. Following diagnosis, patients are brought to the operating room, or more likely the "hybrid" suite, for coiling or clipping of the aneurysm. Most patients are in the 40to 60-year-old age group and in otherwise good health.

Ruptured aneurysms usually present acutely as SAH. Patients typically complain of a sudden severe headache without focal neurological deficits, but often associated with nausea and vomiting. Transient loss of consciousness may occur and may result from a sudden rise in ICP and precipitous drop in CPP. If ICP does not decrease rapidly after the initial sudden increase, death usually follows. Large blood clots can cause focal neurological signs in some patients. Minor bleeding may cause only a mild headache, vomiting, and nuchal rigidity. The severity of SAH is graded according to the Hunt and Hess scale (Table 27—3), as well as the World Federation of Neurological Surgeons grading scale of SAH (Table 27-4). The Fisher grading scale, which uses CT to assess the amount of blood detected, gives the best indication of the likelihood of the development of cerebral vasospasm and patient outcome (Table 27-5).
TABLE 27-3 Hunt and Hess grading scale for SAH.!
|, Clinical Description = Asymptomatic or minimal headache and slight nuchal rigidity. II, Clinical Description = Moderate to severe headache, nuchal rigidity, no neurological deficit other than cranial nerve palsy. ll, Clinical Description = Drowsiness, confusion, or mild focal deficit. IV, Clinical Description = Stupor, moderate to severe hemiparesis, and possibly early decerebrate rigidity and vegetative disturbances. Vv, Clinical Description = Deep coma, decerebrate rigidity, and moribund appearance
'Reproduced with permission from Priebe H-J. Aneurysmal subarachnoid haemorrhage and the anaesthetist. BrJ Anaesth. 2007 July;99(1):102-118.

|, GCS Score? = 15. |, Motor Deficit? = Absent. II, GCS Score? = 13 0r 14. II, Motor Deficit? = Absent. ll, GCS Score? = 13 or 14. ll, Motor Deficit? = Present. IV, GCS Score? = 7-12. IV, Motor Deficit? = Present or absent. V, GCS Score? = 3-6. V, Motor Deficit? = Present or absent
'Reproduced with permission from Priebe H-J. Aneurysmal subarachnoid haemorrhage and the anaesthetist. Br J Anaesth. 2007 July;99(1):102-118.
2GCS, Glasgow Coma Scale,
Excludes cranial neuropathies, but includes dysphasia.
TABLE 27-5 Fisher grading scale of cranial computerized tomography
1, Findings on CCT = No subarachnoid blood detected. 2, Findings on CCT = Diffuse or vertical layers < 1mm. 3, Findings on CCT = Localized clot and/or vertical layer >1 mm. 4, Findings on CCT = Intracerebral or intraventricular clot with diffuse or no subarachnoid haemorrhage
reviews have questioned the role for hypervolemia, recommending maintenance of euvolemia while acknowledging that hypertension may be most beneficial in the management of DCI. Refractory vasospasm may be treated with catheterdelivered vasodilators, angioplasty, or both. However, radiological improvement in the vessel diameter does not necessarily correlate with an improvement in clinical status.

In addition to assessing and documenting neurological findings, the preoperative evaluation should include a search for coexisting diseases, such as hypertension and renal, cardiac, or ischemic cerebrovascular disease. Electrocardiographic abnormalities are commonly seen in patients with SAH, but do not necessarily reflect underlying heart disease. However, increases of cardiac troponin during SAH are associated with myocardial injury and may herald a poor outcome. Stress-induced cardiomyopathy may also be present. Most conscious patients with normal ICP are sedated following rupture to prevent rebleeding; such sedation should be continued until induction of anesthesia. Patients with persistent elevation in ICP should receive little or no premedication to avoid hypercapnia.

Aneurysm surgery can result in exsanguinating hemorrhage as a consequence of rupture or rebleeding. Blood should be available prior to the start of these operations.
Regardless of the anesthetic technique employed, anesthetic management should focus on preventing rupture (or rebleeding) and avoiding factors that promote cerebral ischemia or vasospasm. Intraarterial pressure monitoring is useful. Sudden increases in blood pressure with tracheal intubation or surgical stimulation should be avoided. Judicious intravascular volume loading permits surgical levels of anesthesia without excessive decreases in blood pressure. Because calcium channel blockers, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors cause systemic vasodilation and reduce systemic vascular resistance, patients receiving these agents preoperatively may be particularly prone to hypotension.
The great majority of cerebral aneurysms are addressed via an endovascular approach. The anesthetic concerns of patients taken for coiling in the neurointerventional suite are similar to those of patients undergoing craniotomy.
General anesthesia is often employed. Patients require heparin anticoagulation and radiological contrast. Communication with the surgeon or neurointerventionalist as to the desired activated clotting time and need for protamine reversal is essential. Moreover, anesthesia staff in the neuroradiology suite must be prepared to manipulate and monitor the blood pressure, as with an open surgical procedure.
For the less common situation in which open craniotomy is required, once the dura is opened, mannitol is often given to facilitate surgical exposure and reduce the need for surgical retraction. Rapid decreases in ICP prior to dural opening are avoided as they may promote rebleeding by removing a tamponading effect on the aneurysm.

Elective (controlled) hypotension has been used in aneurysm surgery. Decreasing mean arterial blood pressure reduces the transmural tension across the aneurysm, making rupture (or rebleeding) less likely and facilitating surgical clipping. Controlled hypotension can also decrease blood loss and improve surgical visualization in the event of bleeding. The combination of a slightly head-up position with a volatile anesthetic enhances the effects of any of the commonly used hypotensive agents. Should accidental rupture of the aneurysm occur, the surgeon may request transient hypotension to facilitate control of the bleeding. Neurophysiological monitoring may be employed during aneurysm surgery to identify potential ischemia during clip application. Rarely, hypothermic circulatory arrest is used for large basilar artery aneurysms.
Depending on neurological condition, most patients should be extubated at the end of surgery. Extubation should be handled similarly to other craniotomies (see earlier discussion). A rapid awakening allows neurological evaluation in the operating room, prior to transfer to the intensive care unit.

AVMs cause intracerebral hemorrhage more often than SAH. These lesions are developmental abnormalities that result in arteriovenous fistulas; they typically grow in size with time. AVMs may present at any age, but bleeding is most common between 10 and 30 years of age. Other common presentations include headache and seizures. The combination of high blood flow with low vascular resistance can rarely result in high-output cardiac failure. In most cases an endovascular approach to occlude the vessels feeding the AVM will be attempted in the "hybrid" operating room or neurointerventional suite. This may provide definitive therapy or may render the AVM more amenable to surgical excision.
Neuroradiological embolization employs various coils, glues, and balloons to obliterate the AVM. Risks include embolization into cerebral arteries feeding the normal brain, as well as systemic or pulmonary embolism.
Anesthetic management of patients undergoing resection of AVMs may be complicated by extensive blood loss. Venous access with multiple large-bore cannulas is necessary. Hyperventilation and mannitol may be used to facilitate surgical access. Hyperemia and swelling can develop following resection, possibly because of altered autoregulation in the remaining normal brain. Emergence hypertension is typically controlled with agents that do not induce increases in CBF, such as B-blockers and clevidipine.

Acute ischemic strokes are treated with thrombolysis (with tissue plasminogen activator [tPA]), or endovascular clot removal and stenting, or both. Multiple well-performed randomized clinical trials reported in 2015 have confirmed that immediate endovascular intervention greatly improves outcomes relative to tPA alone in patients with occlusions of proximal large cerebral arteries. The mantra in neurology and neurosurgery is "time is brain." The goal is to have the patient revascularized as soon as possible. Endovascular treatment should not be delayed for placement of arterial lines, etc. These patients are at immediate risk of death and disability without treatment and certainly meet the criteria for an American Society of Anesthesiologists physical status of 5E! The benefit from endovascular treatment of acute ischemic stroke is even greater (a smaller number of patients need to be treated to have a patient with outcome benefit) than that for immediate revascularization for myocardial infarctions with elevated ST segments (STEMIs). However, the only patients who will benefit from endovascular treatment will have a significant amount of the ischemic brain tissue that remains "recoverable" as assessed in imaging studies by measures of CBF, CBV, and tissue transit time. In infarcted brain, all are abnormal. In recoverable brain tissue, at a minimum the CBV may be maintained or even increased.
Several post hoc analyses of the original clinical trials have suggested an association between the use of general anesthesia (versus sedation and monitoring) and worse outcomes in patients undergoing endovascular embolectomy. Nevertheless, general anesthesia remains the preference in many centers and will be required for many patients. We have observed that patients with acute left middle cerebral artery occlusion and aphasia may not hold still no matter how loudly they are asked!
The goals of anesthesia for endovascular treatment of acute ischemic stroke are to maintain the blood pressure less than 180 mm Hg if tPA has been given. If tPA has not been given, relative hypertension may be preferable to maintain cerebral perfusion pending clot retrieval and stenting. Once the occluded vessel has been reopened, we recommend tight control of blood pressure, in most cases keeping it at 140/90 mm Hg or less.

Spinal surgery is most often performed for symptomatic nerve root or cord compression secondary to trauma or degenerative disorders. Compression may occur from protrusion of an intervertebral disk or osteophytic bone (spondylosis) into the spinal canal or an intervertebral foramen. Prolapse of an intervertebral disk often occurs at either the fourth or fifth lumbar or the fifth or sixth cervical levels in adults. Spondylosis tends to affect the lower cervical spine more than the lumbar spine and typically afflicts older patients. Operations on the spinal column can help correct deformities (eg, scoliosis), decompress the cord, and fuse the spine if disrupted by trauma or degenerative conditions. Spinal surgery may also be performed to resect a tumor or vascular malformation or to drain an abscess or hematoma.

Preoperative evaluation should focus on any anatomic abnormalities and limited neck movements (from disease, traction, "collars," or other devices) that might complicate airway management. Neurological deficits should be documented. Neck mobility should be assessed. Patients with unstable cervical spines can be managed with either awake fiberoptic intubation or intubation after induction with in-line stabilization.

Spinal operations involving multiple levels, fusion, and instrumentation are also complicated by the potential for large intraoperative blood loss; a red cell salvage device is often used. Excessive distraction during spinal instrumentation (Harrington rod or pedicle screw fixation) can injure the spinal cord. Transthoracic approaches to the spine require one-lung ventilation. Anterior/posterior approaches require the patient to be repositioned in the middle

POVL occurs secondary to:
¢ Ischemic optic neuropathy
¢ Perioperative glaucoma
* Cortical hypotension and embolism
Prolonged surgery in a head-down position, major blood loss, relative hypotension, diabetes, obesity, and smoking all put patients at greater risk of POVL following spine surgery.
Airway and facial edema can likewise develop after prolonged "head-down" positioning. Reintubation, if required, will likely present more difficulty than the intubation at the start of surgery.
Specialized head positioning pillows (eg, Covidien, ProneView) are often used when patients are placed in the prone position, and the face must be checked periodically to verify that the eyes, nose, and ears are free of pressure. Even foam cushions can exert pressure over time on the chin, orbit, and maxilla. Turning the head is not easily accomplished when the head is positioned on a cushion; therefore, if prolonged procedures are planned, the head can be secured with pins keeping the face free from any pressure.

When major blood loss is anticipated or the patient has preexisting cardiac disease, intraarterial pressure monitors should be considered prior to "positioning" @ or "turning." Sudden, massive blood loss from injury to the great vessels can occur intraoperatively with adjacent thoracic or lumbar spine procedures. of the the ability intraoperatively detect
Instrumentation spine requires to spinal cord injury. Intraoperative wake-up techniques employing nitrous oxidenarcotic or total intravenous anesthesia allow the testing of motor function following distraction. Once preservation of motor function is established, the patient's anesthetic can be deepened. Continuous monitoring of somatosensory evoked potentials and motor evoked potentials provides alternatives that avoid the need for intraoperative awakening. These monitoring techniques require substitution of propofol, opioid, or ketamine infusions for deep levels of inhalation anesthetics and avoidance of neuromuscular paralysis.

A 41-year-old woman presents to the operating room for resection of a 10-mm pituitary tumor. She complains of amenorrhea and galactorrhea, and had recently noticed a decrease in her visual acuity, with bitemporal hemianopsia.

Functionally and anatomically, the pituitary is divided into two parts: anterior and posterior. The latter is part of the neurohypophysis, which also includes the pituitary stalk and the median eminence.
The anterior pituitary is composed of several cell types, each secreting a specific hormone. Anterior pituitary hormones include adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), growth hormone (GH), the gonadotropins (follicle-stimulating hormone [FSH] and luteinizing hormone [LH]), and prolactin (PRL). Secretion of each of these hormones is regulated by hypothalamic peptides (releasing hormones) that are transported to the adenohypophysis by a capillary portal system. The secretion of FSH, LH, ACTH, TSH, and their respective releasing hormones is also under negative feedback control by the products of their target organs. For example, an increase in circulating thyroid hormone inhibits the secretion of TSH-releasing factor and TSH.
The posterior pituitary secretes antidiuretic hormone (ADH, also called vasopressin) and oxytocin. These hormones are actually formed in supraoptic and paraventricular neurons, respectively, and are transported down axons that terminate in the posterior pituitary. Hypothalamic osmoreceptors, and, to a lesser extent, peripheral vascular stretch receptors, regulate secretion of ADH.

ACTH stimulates the adrenal cortex to secrete glucocorticoids. Unlike production of mineralocorticoids, production of glucocorticoids is dependent on ACTH secretion. TSH accelerates the synthesis and release of thyroid hormone (thyroxine). Normal thyroid function is dependent on production of TSH. The gonadotropins FSH and LH are necessary for normal production of testosterone and spermatogenesis and cyclic ovarian function. GH promotes tissue growth and increases protein synthesis as well as fatty acid mobilization. Its effects on carbohydrate metabolism are to decrease cellular glucose uptake and utilization and increase insulin secretion. PRL functions to support breast development during pregnancy. Dopamine receptor antagonists are known to increase secretion of PRL. Through its effect on water permeability in renal collecting ducts, ADH regulates extracellular osmolarity and blood volume. Oxytocin acts on areolar myoepithelial cells as part of the milk letdown reflex during suckling and enhances uterine activity during labor.

The pituitary gland is attached to the brain by a stalk and extends downward to lie in the sella turcica of the sphenoid bone. Anteriorly, posteriorly, and inferiorly, it is bordered by bone. Laterally, it is bordered by the cavernous sinus, which contains cranial nerves III, IV, V;, and VI, as well as the cavernous portion of the carotid artery. Superiorly, the diaphragma sella, a thick dural reflection, usually tightly encircles the stalk and forms the roof of the sella turcica. In close proximity to the stalk lie the optic nerves and chiasm. The hypothalamus lies contiguous and superior to the stalk.
Tumors less than 10 mm in diameter are usually approached via the transsphenoidal route, whereas larger tumors and those with significant suprasellar extension are approached via a bifrontal craniotomy. With the use of prophylactic antibiotics, morbidity and mortality rates are significantly less with the transsphenoidal approach; the operation is carried out with the aid of a microscope through an incision in the gingival mucosa beneath the upper lip. The surgeon enters the nasal cavity, dissects through the nasal septum, and finally penetrates the roof of the sphenoid sinus to enter the floor of the sella turcica.

Problems include (1) the need for mucosal injections of epinephrinecontaining solution to reduce bleeding, (2) the accumulation of blood and tissue debris in the pharynx and stomach, (3) the risk of hemorrhage from accidental entry into the cavernous sinus or the internal carotid artery, (4) cranial nerve damage, and (5) pituitary hypofunction. Prophylactic administration of glucocorticoids is routinely used in most centers. Diabetes insipidus develops postoperatively in up to 40% of patients but is usually transient. Less commonly, the diabetes insipidus presents intraoperatively. The supine and slightly head-up position used for this procedure may also predispose to venous air embolism.

Tumors in or around the sella turcica account for 10% to 15% of intracranial neoplasms. Pituitary adenomas are most common, followed by craniopharyngiomas and then parasellar meningiomas. Primary malignant pituitary and metastatic tumors are rare. Pituitary tumors that secrete hormones (functional tumors) usually present early, when they are still relatively small (<10 mm). Other tumors present late, with signs of increased ICP (headache, nausea, and vomiting) or compression of contiguous structures (visual disturbances or pituitary hypofunction). Compression of the optic chiasm classically results in bitemporal hemianopia. Compression of normal pituitary tissue produces progressive endocrine dysfunction. Failure of hormonal secretion usually progresses in the order of gonadotropins, GH, ACTH, and TSH. Diabetes insipidus can also be seen preoperatively. Rarely, hemorrhage into the pituitary results in acute panhypopituitarism (pituitary apoplexy) with signs of a rapidly expanding mass, hemodynamic instability, and hypoglycemia. This patient has the most common type of secretory adenoma, producing hyperprolactinemia.

Adenomas secreting ACTH (Cushing disease) produce classic manifestations of Cushing syndrome: truncal obesity, moon facies, abdominal striae, proximal muscle weakness, hypertension, and osteoporosis. Glucose tolerance is typically impaired, but frank diabetes is less common (<20%). Hirsutism, acne, and amenorrhea are also commonly seen in women.
Adenomas that secrete GH are often large and result in either gigantism (prepubertal patients) or acromegaly (adults). Excessive growth prior to epiphyseal fusion results in massive growth of the entire skeleton. After epiphyseal closure, the abnormal growth is limited to soft tissues and acral parts: hands, feet, nose, and mandible. Patients develop osteoarthritis, which often affects the temporomandibular joint and spine. Diabetes, myopathies, and neuropathies are common. Cardiovascular complications include hypertension, premature coronary disease, and cardiomyopathy in some patients. The most serious anesthetic problem encountered in these patients is difficulty in intubating the trachea.

Monitoring should be carried out in somewhat the same way as for craniotomies. Visual evoked potentials may be employed with large tumors that involve the optic nerves. Precordial Doppler sonography may be used for detecting venous air embolism. Venous access with large-bore catheters

What modifications, if any, are necessary in the anesthetic technique?
The same principles discussed for craniotomies apply; however, patients rarely have evidence of increased ICP. Intravenous antibiotic prophylaxis and glucocorticoid coverage (hydrocortisone, 100 mg) are usually given prior to induction. Many clinicians avoid nitrous oxide to prevent problems with a postoperative pneumocephalus (see earlier discussion). Effective neuromuscular blockade is important to prevent movement while the surgeon is using the microscope. A lumbar drain is often placed to reduce ICP, facilitate surgical exposure, and reduce the likelihood of CSF leaks after closure of the dura.

Induction of anesthesia in patients receiving long-term levodopa therapy may result in either marked hypotension or hypertension.
In patients with multiple sclerosis, increases in body temperature cause exacerbation of symptoms.
The major risk of anesthesia in patients with autonomic dysfunction is severe hypotension, compromising cerebral and coronary blood flow.
Autonomic hyperreflexia should be expected in patients with spinal cord lesions above T6 and can be precipitated by surgical manipulations.
The most important interaction between anesthetic agents and tricyclic antidepressants is an exaggerated response to both indirect-acting vasopressors and sympathetic stimulation.
Patients with vascular and nonvascular neurological diseases or psychiatric disorders are frequently encountered by anesthesia providers. Anesthesiologists must have a basic understanding of the major neurological and psychiatric disorders and their drug therapy. Failure to recognize potential adverse anesthetic interactions may result in avoidable perioperative morbidity.

result from emboli of fibrin-platelet aggregates that form on plaques in extracranial vessels. Unilateral visual impairment, numbness or weakness of an extremity, or aphasia is suggestive of carotid disease, whereas bilateral visual impairment, dizziness, ataxia, dysarthria, bilateral weakness, or amnesia is suggestive of vertebral—basilar disease. Patients with TIAs have a 30% to 40% chance of developing a frank stroke within 5 years; 50% of these strokes occur within the first year. Patients with TIAs should not undergo any elective surgical procedure without an adequate medical evaluation that generally includes at least noninvasive (Doppler) flow and imaging studies. The presence of an ulcerated plaque of greater than 60% occlusion is generally an indication for endovascular intervention or less commonly an open carotid thromboendarterectomy.

Preoperative assessment requires neurological and cardiovascular evaluations. The type of stroke, the presence of neurological deficits, and the extent of residual impairment should be determined. Thromboembolic strokes usually occur in patients with generalized atherosclerosis. Most patients are elderly and have comorbid conditions, such as hypertension, hyperlipidemia, and diabetes. Coexisting coronary artery disease and renal impairment are common. Following nonhemorrhagic strokes or TIAs, many patients are placed on long-term anticoagulant or antiplatelet therapy, or both. Management of antiplatelet therapy and antithrombotic therapy should be reviewed by the anesthesia and surgical teams, often guided by the physician who prescribed the therapy, to determine the risks and benefits of discontinuation or maintenance of such therapy perioperatively. Other systemic diseases, such as diabetes, hypertension, coronary artery disease, heart failure, and chronic obstructive lung disease, frequently manifest in the patient with cerebrovascular disease.

Patients may present for surgery following embolic, thrombotic, and hemorrhagic strokes.
Management of the patient following acute embolic stroke is directed toward the embolic source. Cardiac surgery is performed to remove atrial myxomas. Systemic emboli can also be produced from endocarditic vegetations, as well as from degenerated heart valves and intracardiac thrombus.
Patients with acute strokes secondary to carotid and intracranial occlusive arterial disease present for carotid endarterectomy and endovascular procedures.
When an awake procedure is undertaken, the patient serves as a monitor of the adequacy of cerebral blood flow during application of vessel clamps or placement of a stent. When general anesthesia is used, electroencephalography, evoked potentials, carotid stump pressure, near-infrared cerebral oximetry, or transcranial Doppler may be used to estimate the adequacy of cerebral oxygen delivery. During open carotid thromboendarterectomy the surgeon may place a shunt to deliver blood to the brain around the cross-clamped vessel. Despite adequate cerebral blood flow, perioperative stroke can occur during carotid surgery secondary to emboli.
Management of patients following thrombotic or hemorrhagic stroke for nonneurological surgery must be individualized. Cerebral autoregulation of blood flow may fail, leaving flow directly dependent upon cerebral perfusion pressure (Figure 28—1). The penumbra of potentially salvageable neurological tissue may therefore be very sensitive to injury from the effects of both hypotension and hypertension (Figure 28—2). We advocate tight control of blood pressure in these patients.
Figure 28—1 Cerebral autoregulation in a normal person, cerebral ischemia, and chronic hypertension. (Reproduced with permission from Shaikh S. Anesthesia considerations for the patient with acute ischemic stroke. Semin Cardiothorac Vasc Anesth. 2010 Mar;14(1):62-63.)
Figure 28—2 Penumbra. (Reproduced with permission from Shaikh S. Anesthesia considerations for the patient with acute ischemic stroke. Semin Cardiothorac Vasc Anesth. 2010 Mar;14(1):62-63.)

Patients taken to surgery following administration of thrombolytic therapy are at increased risk of cerebral hemorrhage, and tight blood pressure control may reduce the likelihood of cerebral bleeding.
Patients with major intracerebral hemorrhage or epidural or subdural hematomas after trauma typically undergo craniotomy and evacuation of hematoma. In these patients invasive arterial pressure monitoring is helpful given that cerebral autoregulation is deranged (see Figure 28-1). Hypertension is frequently treated with intravenous vasodilators and B-blockers. Subarachnoid hemorrhage is discussed in Chapter 27.

Patients with intracranial mass lesions frequently present to their primary care physicians with complaints of headache, visual disturbances, or seizures. Families may have noticed behavioral changes in patients with frontal lobe masses. Imaging studies confirm the presence of a mass, and initial treatment with dexamethasone is aimed at decreasing cerebral edema. Electrolytes should be reviewed perioperatively in all patients undergoing cranial surgery, as both hyponatremia and hypernatremia can develop secondary to cerebral salt wasting, inappropriate antidiuretic hormone secretion, or central diabetes insipidus (Table 28-1; see also Chapter 49). Patients with altered mentation preoperatively may likewise be dehydrated. Hyperglycemia secondary to steroid use is frequently seen.

SIADH, Serum Sodium Concentration = Low. SIADH, Plasma Volume = Normal or increased. SIADH, Serum Osmolality = Low. SIADH, Urine Sodium —_ Concentration = High. SIADH, Urine Osmolality = High. SIADH, Treatment = Fluid restriction. CSWS, Serum Sodium Concentration = Low. CSWS, Plasma Volume = Decreased. CSWS, Serum Osmolality = Normal or high. CSWS, Urine Sodium —_ Concentration = — High. CSWS, Urine Osmolality = Normalorhigh. CSWS, Treatment = Isotonic or hypertonic saline. Dl, Serum Sodium Concentration = High. Dl, Plasma Volume = Decreased. Dl, Serum Osmolality = High. Dl, Urine Sodium —_ Concentration = Normal. Dl, Urine Osmolality = Low. Dl, Treatment = Hypotonic saline + vasopressin
Reproduced with permission from Reddy U, Amin Y. Preoperative assessment of neurosurgical patients. Anaesth intensive Care Med. 2010 Sep;11{9):357-362. 2CSWS, cerebral salt wasting; DI, diabetes insipidus; SIADH, syndrome of inappropriate antidiuretic hormone secretion.

Seizures represent abnormal synchronized electrical activity in the brain. They may be a manifestation of an underlying central nervous system disease, a systemic disorder, or idiopathic. Up to 2% of the population may experience a seizure in their lifetime. Epilepsy is a disorder characterized by recurrent paroxysmal seizure activity. Healthy individuals who experience an isolated nonrecurrent seizure are not considered to have epilepsy. Seizure activity may be localized to a specific area in the brain or may be generalized. Moreover, initially localized (focal) seizures can subsequently spread, becoming generalized. A simple classification scheme is presented in Table 28-2. Partial seizures (also called focal) are clinically manifested by motor, sensory, autonomic, or psychiatric symptoms, depending on the brain region affected. Focal seizures associated with impairment in consciousness are termed "complex partial" (psychomotor or temporal lobe) seizures. Generalized seizures characteristically produce bilaterally symmetric electrical activity without local onset. They may result in abnormal motor activity, loss of consciousness, or both. Absence (petit mal) seizures produce generalized activity resulting in isolated, transient lapses in consciousness. Other generalized seizures are usually classified according to the type of motor activity. Tonic— clonic (grand mal) seizures are most common and are characterized by a loss of consciousness followed by clonic and then tonic motor activity.

Partial (focal)
Simple
Complex
Secondarily generalized tonic-clonic
Generalized
Absence (petit mal)
Myoclonic
Clonic
Tonic
Tonic—clonic (grand mal)
Atonic

Anesthetic evaluation should focus primarily on the cause and type of seizure activity and the drugs with which the patient is being treated. Seizures in adults are most commonly due to structural brain lesions (head trauma, tumor, degeneration, or stroke) or metabolic abnormalities (uremia, hepatic failure, hypoglycemia, hypocalcemia, drug toxicity, or drug/alcohol withdrawal). Idiopathic seizures occur more often in children than in adults. Grand mal seizures are serious complicating factors in surgical patients and should be treated promptly to prevent musculoskeletal injury, hypoventilation, hypoxemia, and aspiration of gastrointestinal contents. If a seizure occurs, maintaining an open airway and adequate oxygenation are the first priorities. Propofol (50-100 mg) or a benzodiazepine such as diazepam (5—10 mg) or midazolam (1-5 mg) can be given intravenously to terminate the seizure. Most patients with seizure disorders receive antiepileptic drugs preoperatively (Table 28-3). Antiseizure medications should be continued throughout the perioperative period to maintain therapeutic levels.
TABLE 28-3 Commonly used antiepileptic drugs, mechanisms of action, and common side effects.'

Reduces GABA. Gabapentin, Major Side Effects = Somnolence, fatigue Ataxia. Gabapentin, Comments = Used in intractable complex partial seizures
- 'Reproduced with permission from Veenith T, Burnstein RM. Management of patients with neurological and psychiatric disorders. Surgery. 2010 Sep;28(9):441-445,
2AMPA, c-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid: GABA, y-amino butyric acid; GHB, y-hydroxybutyric acid; NMDA, N-methyl-o-aspartate.

In selecting general anesthetic agents, we avoid those few drugs with consistent propensity to induce seizures, (eg, enflurane) when there are reasonable alternatives. Theoretically, ketamine and methohexital (in small doses) could precipitate seizure activity. Hypothetically, large doses of atracurium, cisatracurium, or meperidine may be relatively contraindicated because of the reported epileptogenic potential of their respective metabolites, laudanosine, and normeperidine. Hepatic microsomal enzyme induction should be expected from chronic antiseizure therapy. Enzyme induction may increase the dose requirement and frequency of intravenous anesthetics and nondepolarizing neuromuscular blockers (NMBs) and may increase the risk for hepatotoxicity from halothane.

stimulation is currently the most common approach. Stimulation of the subthalamic nucleus improves all of the primary symptoms of PD and results in a greater decrease in the amount of medication required for symptom relief, when compared with stimulation of the globus pallidus pars internus. However, globus pallidus pars internus stimulation may produce greater improvement in dyskinesia as compared with stimulation of the subthalamic nucleus.
Neurodegenerative diseases often lead to dementia. The incidence of dementia varies by population but seems to double every decade of life, starting at age 60. Alzheimer disease (AD) is the most common neurodegenerative disease, causing more than half of all cases of dementia; dementia with Lewy bodies (most often associated with PD) and vascular dementia comprise most of the rest. Slow, progressive impairment of memory, judgment, and decision making, as well as emotional lability, are hallmarks of AD. Late in the course of the disease, severe extrapyramidal signs, apraxias, and aphasia are often present. Patients with AD usually show marked cortical atrophy with ventricular enlargement; the pathological hallmarks of AD seen at necropsy include neurofibrillary tangles that contain the phosphorylated microtubular protein tau, neuritic plaques, and excessive brain concentrations of amyloid B-peptides.

Anesthetic management of patients with moderate to severe dementia is often complicated by disorientation and uncooperativeness. New onset of temporary cognitive impairment is frequent in elderly patients and often persists for 1 to 3 days following surgery. Such patients require repeated reassurance and explanation. Legally incompetent patients cannot provide informed consent for anesthesia or surgery. We avoid premedication if at all possible. Centrally acting anticholinergics, such as atropine and scopolamine, may contribute to postoperative confusion. Glycopyrrolate, which does not cross the blood-brain barrier, may be the preferred agent when an anticholinergic is required. Patients having dementia with Lewy bodies often will have PD and thus the anesthetic issues with PD that were addressed earlier in the chapter would apply. Animal studies have shown that conventional general anesthetic agents are associated with neuronal injury and cell death. The outcome implications of general anesthesia in both the elderly and small children are currently the subject of much investigation and debate. Apoptotic neurodegeneration has been linked to the use of GABA receptor modulators and N-methyl-p-aspartic acid receptor antagonists, mechanisms by which common general anesthetics produce their effects. Moreover, increased B-amyloid production is associated with anesthetic exposure; tau protein phosphorylation increases after general anesthesia; and hypothermia transiently increases tau protein phosphorylation. Despite concerns about anesthetic exposure in patients with or at risk for AD, current studies do not support avoiding needed surgery in this group.

The effect of stress, anesthesia, and surgery on the course of MS is controversial. Overall, the effect of anesthesia is unpredictable. Elective surgery should be avoided during relapse, regardless of the anesthetic technique employed. The preoperative consent record should document counseling of the patient to the effect that the stress of surgery and anesthesia might worsen symptoms. Spinal anesthesia has been associated with exacerbation of the disease; however, the entire surgery/delivery/anesthetic process may likewise lead to exacerbations. Peripheral nerve blocks are less of a concern because MS is a disease of the central nervous system; however, patients may have preexisting peripheral neuropathies. Epidural and other regional techniques seem to have no adverse effect on the course of the disease. No specific interactions with general anesthetics are recognized. Patients with advanced disease may have a labile cardiovascular system due to autonomic dysfunction. In the setting of paresis or paralysis, succinylcholine should be avoided because of possible hyperkalemia. Increases in body temperature should be avoided. Irrespective of anesthetic technique, patients may experience a worsening of symptoms perioperatively and should be counseled accordingly.

Motor neuron disease is another common neurodegenerative disease, with amyotrophic lateral sclerosis (ALS) being the most prevalent. The cause of ALS is unknown, although small numbers of patients with the familial form of the disease have a defect in the superoxide dismutase-1 gene. ALS is a rapidly progressive disorder of both upper and lower motor neurons. Patients often present in the fifth or sixth decade of life with muscular weakness, atrophy, fasciculation, and spasticity. The disease may initially be asymmetric, but over the course of 2 to 3 years becomes generalized, involving all skeletal and bulbar muscles. Progressive respiratory muscle weakness makes the patient susceptible to aspiration and eventually leads to death from ventilatory failure. Although the heart is unaffected, autonomic dysfunction can be seen. There is no specific treatment for ALS.
The primary emphasis in management is judicious respiratory care. As with other patients with lower motor neuron disease, succinylcholine is contraindicated because of the risk of hyperkalemia. Adequacy of ventilation should be carefully assessed both intraoperatively and postoperatively; an awake extubation is desirable. Difficulty in weaning patients from mechanical ventilation postoperatively may be anticipated in patients with advanced disease.

Autonomic dysfunction, or dysautonomia, may be due to generalized or segmental disorders of the central or peripheral nervous system. Symptoms can be generalized, segmental, or focal. These disorders may be congenital, familial, or acquired. Common manifestations include impotence; bladder and gastrointestinal dysfunction; abnormal regulation of body fluids; decreased sweating, lacrimation, and salivation; and orthostatic hypotension. The latter can be the most serious manifestation of the disorder.
Acquired autonomic dysfunction can be isolated (pure autonomic failure), part of a more generalized degenerative process (Shy—Drager syndrome, PD, olivopontocerebellar atrophy), part of a segmental neurological process (MS, syringomyelia, reflex sympathetic dystrophy, or spinal cord injury), or a manifestation of disorders affecting peripheral nerves (GBS, diabetes, chronic alcoholism, amyloidosis, or porphyria).
There are at least three forms of hereditary sensory and autonomic neuropathies, each with its own underlying genetic mutation(s). Autonomic dysfunction is prominent and is associated with generalized diminished sensation and emotional lability. Moreover, patients are predisposed to dysautonomic crises triggered by stress and characterized by marked hypertension, tachycardia, abdominal pain, diaphoresis, vomiting, and the risk of dehydration.

® The major risk of anesthesia in patients with autonomic dysfunction is severe hypotension, compromising cerebral and coronary blood flow. Marked hypertension can be equally deleterious. Most patients are chronically hypovolemic, and if volume deficits are not corrected the vasodilatory effects of spinal and epidural anesthesia are poorly tolerated. The vasodilatory and cardiac depressant effects of most general anesthetic agents combined with positive airway pressure can be equally problematic. Continuous intraarterial blood pressure monitoring is useful. Hypotension should be treated with fluids and direct-acting vasopressors (in preference to indirect-acting agents). The dose— response relationship for vasopressors may be abnormal due to denervation. Blood loss also is usually poorly tolerated. Body temperature should be monitored closely. Patients with anhidrosis are particularly susceptible to hyperpyrexia.

Syringomyelia results in progressive cavitation of the spinal cord. In many cases, obstruction of cerebrospinal fluid outflow from the fourth ventricle seems to be contributory. Many patients have craniovertebral abnormalities, particularly the Armold-Chiari malformation. Increased pressure in the central canal of the spinal cord produces enlargement or diverticulation to the point of cavitation.
Syringomyelia typically affects the cervical spine, producing sensory and motor deficits in the upper extremities, and, frequently, thoracic scoliosis. Extension upward into the medulla (syringobulbia) leads to cranial nerve deficits. Syringoperitoneal shunting and other decompressive procedures have variable success in arresting the disease.
Anesthetic evaluation should focus on defining existing neurological deficits and any pulmonary impairment due to scoliosis. Autonomic instability should be expected in patients with extensive lesions. Succinylcholine should be avoided when muscle wasting is present because of the risk of hyperkalemia. As always, adequacy of ventilation and reversal of nondepolarizing NMBs should be achieved prior to extubation. Spinal puncture is contraindicated with intracranial hypertension. Epidural anesthetics have been used successfully for labor analgesia in patients with Amold—Chiari malformations, with and without syringomyelia. Risks of cerebral herniation, worsening nerve injury, and infection must be weighed against potential benefits.

descending inhibitory impulses in the cord results in autonomic hyperreflexia. Cutaneous or visceral stimulation below the level of injury can induce intense autonomic reflexes: sympathetic discharge produces hypertension and vasoconstriction below the transection and a baroreceptor-mediated reflex bradycardia and vasodilation above the transection. Cardiac arrhythmias are common.
Emergent surgical management is undertaken whenever there is reversible compression of the spinal cord due to dislocation of a vertebral body or bony fragment. Operative treatment is also indicated for spinal instability to prevent further injury.

Anesthetic management depends on the age of the injury. In the early care of acute injuries, the emphasis should be on preventing further spinal cord damage during patient movement, airway manipulation, and positioning. High-dose corticosteroid therapy (methylprednisolone) has been traditionally given for the first 24 h following injury to possibly improve neurological outcome. Airway management of the patient with an unstable cervical spine is discussed in Chapter 19. Patients with high transections often have impaired airway reflexes and are further predisposed to hypoxemia because of a decrease in functional residual capacity and atelectasis. Spinal shock can lead to hypotension and bradycardia prior to any anesthetic administration. Direct arterial pressure monitoring is helpful. An intravenous fluid bolus and the use of ketamine for anesthesia may help to prevent further decreases in blood pressure; vasopressors may also be required. Succinylcholine can be used safely in the first 24 h but should not be used thereafter because of the risk of hyperkalemia. The latter can occur within the first week following injury and is due to excessive release of potassium secondary to the proliferation of acetylcholine receptors beyond the neuromuscular synaptic cleft.

Anesthetic management of patients with nonacute transections is complicated by the possibility of autonomic hyperreflexia and the risk of hyperkalemia. ® Autonomic hyperreflexia should be expected in patients with spinal cord lesions above T6 and can be precipitated by surgical manipulations. Regional anesthesia and deep general anesthesia are effective in preventing hyperreflexia. Many clinicians, however, are reluctant to administer spinal and epidural anesthesia in these patients because of the difficulties encountered in determining anesthetic level, exaggerated hypotension, and technical problems resulting from deformities. Severe hypertension can result in pulmonary edema, myocardial ischemia, or cerebral hemorrhage and should be treated promptly. Direct arterial vasodilators should be readily available. Nondepolarizing muscle relaxants may be used. Body temperature should be monitored carefully, particularly in patients with transections above T1, because chronic vasodilation and loss of normal reflex cutaneous vasoconstriction predispose to hypothermia. Many patients with a long-standing spinal cord injury have a long history of undergoing surgery without hyperreflexia. These patients can often be managed with monitoring and sedation.

Various forms of encephalitis can present secondary to infectious or autoimmune mechanisms. Patients with encephalitis are managed with the normal care given any patient with potentially increased intracranial pressure at risk of cerebral hypoperfusion.

Depression is a very common mood disorder characterized by sadness and pessimism. Its cause is multifactorial, but pharmacological treatment is based on the presumption that its manifestations are due to a brain deficiency of dopamine, norepinephrine, and serotonin or altered receptor activities. Up to 50% of patients with major depression hypersecrete cortisol and have abnormal circadian secretion. Current pharmacological therapy utilizes drugs that increase brain levels of these neurotransmitters: tricyclic antidepressants, selective serotonin reuptake inhibitors (SSRIs), MAO inhibitors, and atypical antidepressants. The mechanisms of action of these drugs result in some potentially serious anesthetic interactions. Electroconvulsive therapy (ECT) is increasingly used for refractory severe cases and may be continued prophylactically after the patient's mood recovers. The use of general anesthesia for ECT is largely responsible for its safety and widespread acceptance.

SSRIs include fluoxetine, sertraline, and paroxetine, which some clinicians consider first-line agents of choice for depression. A surprisingly large fraction of patients undergoing elective surgery will be receiving one of these agents. These agents have little or no anticholinergic activity and do not generally affect cardiac conduction. Their principal side effects are headache, agitation, and insomnia. Other agents include the norepinephrine—dopamine reuptake inhibitors and the serotonin—norepinephrine reuptake inhibitors.

Tricyclic antidepressants are used for the treatment of depression and chronic pain syndromes. All tricyclic antidepressants work at nerve synapses by blocking neuronal reuptake of catecholamines, serotonin, or both. Desipramine and nortriptyline are used because they are less sedating and tend to have fewer side effects. Other agents are generally more sedating and include amitriptyline, imipramine, protriptyline, amoxapine, doxepin, and trimipramine. Clomipramine is used in the treatment of obsessive—compulsive disorders. Most tricyclic antidepressants also have significant anticholinergic (antimuscarinic) actions: dry mouth, blurred vision, prolonged gastric emptying, and urinary retention. Quinidine-like cardiac effects include tachycardia, T-wave flattening or inversion, and prolongation of the PR, QRS, and QT intervals. Amitriptyline has the most marked anticholinergic effects, whereas doxepin has the fewest cardiac effects.

St. John's wort is being used with increased frequency as an over-the-counter therapy for depression. Because it induces hepatic enzymes, blood levels of other drugs may decrease, sometimes with serious complications. During the preoperative evaluation, the use of all over-the-counter medications should be reviewed.

Antidepressant drugs are generally continued perioperatively. Increased anesthetic requirements, presumably from enhanced brain catecholamine activity, have been reported with these agents. Potentiation of centrally acting anticholinergic agents (atropine and scopolamine) may increase the likelihood of postoperative sedation, confusion, delirium, blurred vision, and urinary retention. The most @ important interaction between anesthetic agents and tricyclic antidepressants is an exaggerated response to both indirect-acting vasopressors and sympathetic stimulation. Chronic therapy with tricyclic antidepressants is reported to deplete cardiac catecholamines, theoretically potentiating the cardiac depressant effects of anesthetics. If hypotension occurs, small doses of a direct-acting vasopressor should be used instead of an indirectacting agent. Amitriptyline's anticholinergic action may occasionally contribute to postoperative delirium.

hyperthermia, tremor, acidosis, and autonomic instability. Treatment is supportive, along with the administration of a 5-HT antagonist (eg, cyproheptadine).

Mania is a mood disorder characterized by elation, hyperactivity, and flight of ideas. Manic episodes may alternate with depression in patients with a bipolar (formerly manic—depressive) disorder. Mania is thought to be related to excessive norepinephrine activity in the brain. The most common agents used for maintenance therapy for this condition are lithium, valproate, quetiapine, and lamotrigine. Aripiprazole, olanzapine, and risperidone are used if the first group fails.
The mechanism of action of lithium is poorly understood. It has a narrow therapeutic range, with a desirable blood concentration between 0.8 and 1.0 mEq/L. Side effects include reversible T-wave changes, mild leukocytosis, and, on rare occasions, hypothyroidism or a vasopressin-resistant diabetes insipiduslike syndrome. Toxic blood concentrations produce confusion, sedation, muscle weakness, tremor, and slurred speech. Still higher concentrations result in of the QRS atrioventricular block, and widening complex, hypotension, seizures. Although lithium is reported to decrease minimum alveolar concentration and prolong the duration of some NMBs, clinically these effects seem to be minor. Blood levels should be checked perioperatively. Sodium depletion (secondary to loop or thiazide diuretics) decreases renal excretion of lithium and can lead to lithium toxicity. Fluid restriction and overdiuresis should be avoided. Lithium dilution cardiac output measurements are contraindicated in patients on lithium therapy.

Patients with schizophrenia display delusions, hallucinations, disorganized or withdrawn behavior, disorganized speech, and severe emotional withdrawal. The diagnoses of schizoaffective disorder, bipolar disorder, and severe depression will need to be excluded. Schizophrenia is thought to result from an excess of dopaminergic activity in the brain.
The most commonly used antipsychotics include phenothiazines, thioxanthenes, phenylbutylpiperidines, dihydroindolones, dibenzapines, benzisoxazoles, and butyrophenones. There are numerous trade names for these drugs. First-generation antipsychotic medications had strong dopamine but
antagonistic effects, leading to extrapyramidal side effects (eg, muscle rigidity and progression to tardive dyskinesia). Second-generation agents have less dopamine antagonism and reduced extrapyramidal effects. The antipsychotic effect of these agents seems to be due to dopamine antagonist activity. Most cause weight gain and sedation and are mildly anxiolytic. Mild a-adrenergic blockade and anticholinergic activity are also observed. Side effects include orthostatic hypotension, acute dystonic reactions, and parkinsonism-like manifestations. Risperidone and clozapine have little extrapyramidal activity, the latter is associated with a significant incidence of granulocytopenia. T-wave flattening, ST segment depression, and prolongation of the PR and QT intervals may be seen, increasing the risk of les torsades des pointes.
Continuing antipsychotic medication perioperatively is desirable. Reduced anesthetic requirements may be observed in some patients, and some patients may experience perioperative hypotension.

Neuroleptic malignant syndrome is a rare and life-threatening complication of antipsychotic therapy that may occur hours or weeks after drug administration. It has also accompanied abrupt withdrawal of medication for PD. Meperidine and metoclopramide can also precipitate the disorder. The mechanism is related to dopamine blockade in the basal ganglia and hypothalamus and impairment of thermoregulation. In its most severe form, the presentation is similar to that of malignant hyperthermia. Muscle rigidity, hyperthermia, rhabdomyolysis, autonomic instability, and altered consciousness are seen. Creatine kinase levels are often high. The mortality rate approaches 20% to 30%, with deaths occurring primarily as a result of kidney failure or arrhythmias. Treatment begins with stopping the offending agent and initiating supportive care. Dantrolene and bromocriptine have been used, but there is no strong evidence for consistent efficacy. Differential diagnoses include serotonin syndrome, malignant hyperthermia, malignant catatonia, and some other acute intoxications (eg, cocaine).

Behavioral disorders from abuse of psychotropic (mind-altering) substances may involve a socially acceptable drug (alcohol), a medically prescribed drug (eg, an opioid or diazepam), or an illegal substance (eg, cocaine or "ecstasy"). With chronic abuse, patients develop tolerance to the drug and varying degrees of
TABLE 28-4 Effect of acute and chronic substance abuse on anesthetic requirements.!
Opioids, 1 = . Opioids, 2 = ili. Barbiturates, 1 = . Barbiturates, 2 = iP. Alcohol, 1 = . Alcohol, 2 = T. Marijuana, 1 = . Marijuana, 2 = 0. Benzodiazepines, 1 = . Benzodiazepines, 2 = T. Amphetamines, 1 = T?. Amphetamines, 2 = L. Cocaine, 1 = qT?. Cocaine, 2 = 0). Phencyclidine, 1 = L. Phencyclidine, 2 = ?
'|, decreases; T, increases; 0, no effect; ?, unknown.
"Associated with marked sympathetic stimulation.

A 64-year-old man with depression refractory to drug therapy is scheduled for electroconvulsive therapy (ECT).

The electroconvulsive shock is applied to one or both cerebral hemispheres to induce a seizure. Variables include stimulus pattern, amplitude, and duration. The goal is to produce a therapeutic generalized seizure 30 to 60 s in duration. Electrical stimuli are usually administered until a therapeutic seizure is induced. A good therapeutic effect is generally not achieved until a total of 400 to 700 seizure seconds have been induced. Because only one treatment is given per day, patients are usually scheduled for a series of treatments, generally two or three a week. Progressive memory loss often occurs with an increasing number of treatments, particularly when electrodes are applied bilaterally.

When the efficacy of ECT was discovered, enthusiasm was tempered in the medical community because drugs were not used to control the violent seizures caused by the procedure, thus engendering a relatively high incidence of musculoskeletal injuries. Moreover, when an NMB was used alone, patients sometimes recalled being paralyzed and awake just prior to the shock. The routine use of general anesthesia to ensure amnesia and neuromuscular blockade to prevent injuries has renewed interest in ECT. The current mortality rate for ECT is estimated to be one death per 10,000 treatments.

Seizure activity is characteristically associated with an initial parasympathetic discharge followed by a more sustained sympathetic discharge. The initial phase is characterized by bradycardia and increased secretions. Marked bradycardia (<30 beats/min) including transient asystole, is occasionally seen. The hypertension and tachycardia that follow are typically sustained for several minutes. Transient autonomic imbalance can produce arrhythmias and T-wave abnormalities on the flow and electrocardiogram. Cerebral blood and ICP, intragastric pressure,

Contraindications are a recent myocardial infarction (usually <3 months), a recent stroke (usually <1 month), an intracranial mass or aneurysm, or increased ICP from any cause. More relative contraindications include angina, poorly controlled heart failure, significant pulmonary disease, bone fractures, severe osteoporosis, pregnancy, glaucoma, and retinal detachment.

Amnesia is required only for the brief period (1-5 min) from when the NMB is given to when a therapeutic seizure has been successfully induced. The seizure itself usually results in a brief period of anterograde amnesia, somnolence, and often confusion. Consequently, only a short-acting induction agent is necessary. Moreover, because most induction agents (barbiturates, etomidate, benzodiazepines, and propofol) have anticonvulsant properties, small doses must be used. Seizure threshold is increased and seizure duration is decreased by all of these agents. Following adequate preoxygenation, methohexital (0.5—1 mg/kg) is most commonly employed. Propofol (1-1.5 mg/kg) may be used, but higher doses reduce seizure duration. Benzodiazepines raise the seizure threshold and decrease duration. Ketamine increases seizure duration but is generally not used because it also increases the incidence of delayed awakening, nausea, and ataxia and is also associated with hallucinations during emergence. Use of etomidate also prolongs recovery. Short-acting opioids, such as alfentanil, are not given alone because they do not consistently produce amnesia. However, alfentanil can be a useful adjunct when very small doses of methohexital (10-20 mg) are required in patients with a high seizure threshold. In very small doses, methohexital may actually enhance seizure activity. Increases in seizure threshold are often observed with each subsequent ECT.
Neuromuscular blockade is required from the time of electrical stimulation until the end of the seizure. A short-acting agent, such as succinylcholine (0.25—0.5 mg/kg), is most often selected. Controlled mask ventilation, using a self-inflating bag device or an anesthesia circle system, is required until spontaneous respirations resume.

Hyperventilation can increase seizure duration and is routinely employed in some centers. Intravenous caffeine (125-250 mg), given slowly, has also been reported to increase seizure duration.

Monitoring should be similar to what is appropriate with the use of any other general anesthetic. Seizure activity is sometimes monitored by an unprocessed electroencephalogram. It can also be monitored in an isolated limb: a tourniquet is inflated around one arm prior to injection of succinylcholine, preventing entry of the NMB and allowing observation of convulsive motor activity in that arm.
How can the adverse hemodynamic effects of the seizure be controlled in patients with limited cardiovascular reserve?
Exaggerated parasympathetic effects should be treated with atropine. In fact, premedication with glycopyrrolate is desirable both to prevent the profuse secretions associated with seizures and to attenuate bradycardia. Nitroglycerin, nifedipine, and aand B-adrenergic blockers have all been employed successfully to control sympathetic manifestations. High doses of B-adrenergic blockers (esmolol, 200 mg), however, are reported to decrease seizure duration.

Patients with pacemakers may safely undergo electroconvulsive treatments, but a magnet should be readily available to convert the pacemaker to a fixed mode, if necessary.

Bao FP, Zhang HG, Zhu SM. Anesthetic considerations for patients with acute cervical spinal cord injury. Neural Regen Res. 2017;12:499. Bornemann-Cimenti Toft al. Neuraxial anesthesia
H, Sivro N, F, et in patients with multiple sclerosis—a systematic review. Rev Bras Anesthesiol. 2017;67:404.

@ Weakness associated with myasthenia gravis is due to autoimmune destruction or inactivation of postsynaptic acetylcholine receptors at the neuromuscular junction, leading to reduced numbers of receptors and degradation of their function, and to complement-mediated damage to the postsynaptic membrane.
@ Patients who have myasthenia gravis with respiratory muscle or bulbar involvement are at increased risk for pulmonary aspiration.
@ Many patients with myasthenia gravis are exquisitely sensitive to nondepolarizing neuromuscular blockers (NMBs).
@ Patients who have myasthenia gravis are at risk for postoperative respiratory failure. Disease duration of more than 6 years, concomitant pulmonary disease, a peak inspiratory pressure of less than —25 cm H,O (ie, —-20 cm H,0), a vital capacity less than 4 mL/kg, anda pyridostigmine dose greater than 750 mg/d are predictive of the need for postoperative ventilation following thymectomy.
© Patients with Lambert—Eaton myasthenic syndrome and other paraneoplastic neuromuscular syndromes are very sensitive to both depolarizing and nondepolarizing NMBs.
@ Respiratory muscle degeneration in patients with muscular dystrophy interferes with an effective cough mechanism and leads to retention of secretions and frequent pulmonary infections.
@ Degeneration of cardiac muscle in patients with muscular dystrophy is also common, but results in dilated or hypertrophic cardiomyopathy in only 10% of patients.
@ Succinylcholine should be avoided in patients with Duchenne or Becker muscular dystrophies because of unpredictable response and the risk of inducing severe hyperkalemia or triggering malignant hyperthermia.
© Anesthetic management in patients with periodic paralysis is directed toward preventing attacks. Perioperative management must include frequent determinations of plasma potassium concentration and correction of abnormal values, with careful electrocardiographic monitoring to detect arrhythmias.
@ In patients with periodic paralysis, the response to NMBs is unpredictable, and neuromuscular function should be carefully monitored during their use. Increased sensitivity to nondepolarizing NMBs is particularly apt to be encountered in patients with hypokalemic periodic paralysis.

Neuromuscular diseases are disorders that adversely affect muscle function either primarily or via nerve or neuromuscular junction abnormalities. They include myasthenia gravis; Lambert—Eaton syndrome; amyotrophic lateral sclerosis (ALS, or Lou Gehrig disease); Becker, Duchenne, facioscapulohumeral, and myotonic muscular dystrophies; Charcot—Marie—Tooth disease; polymyositis; and a large number of other pathological conditions. Although relatively uncommon, patients with these diseases will present to the operating room and to non—operating room procedure areas for diagnostic studies, treatment of complications, or procedural management of related or unrelated disorders, and may also be evaluated and managed by anesthesia providers in the emergency department, intensive care unit, and on hospital wards. Overall debility, with diminished respiratory muscle strength and increased sensitivity to neuromuscular blockers (NMBs), predisposes these patients to postoperative ventilatory failure and pulmonary aspiration, and may slow their post-procedure recovery because of difficulty with ambulation and increased risk of falling. Cardiac involvement may include cardiomyopathy or dysrhythmias. A basic understanding of the major disorders and their potential interaction with anesthetic agents is necessary to minimize the risk of perioperative morbidity. In addition, inherited or acquired neuromuscular pathology must be considered in the differential diagnosis of any patient with unexplained acute respiratory failure (Table 29-1).
TABLE 29-1 Differential diagnosis of acute neuromuscular respiratory failure.!
Myasthenia gravis
West Nile myelopathy
Organophosphate or sarin poisoning
Paraneoplastic neuropathy
Motor neuron disease
Endocrine myopathies
Hypophosphatemia
Hypokalemia or hyperkalemia
Mitochondrial myopathies
Acid maltase deficiencies
Tick paralysis
Botulism
Fish poisoning (tetrodotoxin and ciguatera)
Snake bite
Vasculitis
Acute porphyria
'Adapted with permission from Wijdicks EFM, Kramer AH: Handbook of Clinical Neurology, Vol 140 (3rd series) Critical Care Neurology, Part |. Philadelphia, PA: Elsevier; 2017.

Myasthenia gravis is an autoimmune disorder characterized by weakness and easy fatigability of skeletal muscle. It is classified according to disease distribution and severity (Table 29-2). The prevalence is estimated at 50 to 200 per million population. The incidence is highest in women during their third decade, and men exhibit two peaks, one in the third decade and another in the sixth decade.
TABLE 29-2 Myasthenia Gravis Foundation of America clinical classification of myasthenia gravis.!
, Definition = Any ocular muscle weakness May have weakness of eye closure All other muscle strength is normal. , Definition = Mild weakness affecting other than ocular muscles May also have ocular muscle weakness of any severity. lla, Definition = Predominantly affecting limb, axial muscles, or both May also have lesser involvement of oropharyngeal muscles. IIb, Definition = Predominantly affecting oropharyngeal, respiratory muscles, or both May also have lesser or equal involvement of limb, axial muscles, or both. , Definition = Moderate weakness affecting other than ocular muscles May also have ocular muscle weakness of any severity
Illa, 1 = Predominantly affecting limb, axial muscles, or both May also have lesser involvement of oropharyngeal muscles. IIIb, 1 = Predominantly affecting oropharyngeal, respiratory muscles, or both May also have lesser or equal involvement of limb, axial muscles, or both. , 1 = Severe weakness affecting other than ocular muscles May also have ocular muscle weakness of any severity. Va, 1 = Predominantly affecting limb or axial muscles, or both May also have lesser involvement of oropharyngeal muscles. IVb, 1 = Predominantly affecting oropharyngeal, respiratory muscles, or both May also have lesser or equal involvement of limb, axial muscles, or both. , 1 = Defined by intubation, with or without mechanical ventilation, except when employed during routine postoperative management. The use of a feeding tube without intubation places the patient in class IVb
"Reproduced with permission from Jaretzki Ill A, Barohn RJ. Myasthenia gravis: Recommendations for clinical research standards. Neurology. 2000 July 12;55(1):16-23.

@ Weakness associated with myasthenia gravis is due to autoimmune destruction or inactivation of postsynaptic acetylcholine receptors at the neuromuscular junction, leading to reduced numbers of receptors and degradation of their function, and to complement-mediated damage to the postsynaptic end-plate. IgG antibodies against the nicotinic acetylcholine receptor in neuromuscular junctions are found in 85% to 90% of patients with generalized myasthenia gravis and up to 50% to 70% of patients with ocular myasthenia. Among patients
TABLE 29-3 Differential diagnosis of myasthenia gravis.!

Diabetes
Intracranial aneurism
Trauma (eg, orbital factures)
Congenital (eg, Dwayne syndrome)
Infections (eg, basilar meningitis)
Inflammation (eg, cavernous sinus syndromes)
Neoplasm (eg, basilar meningioma)
Horner syndrome

Myotonic muscular dystrophy
Oculopharyngeal muscular dystrophy Mitochondrial myopathies (eg, chronic progressive external ophthalmoplegia)

Metabolic disease (eg, thyroid disease)
'Reproduced with permission from Mahadeva B, Phillips IIL, Juel VC. Autoimmune disorders of neuromuscular transmission. Semin Neurol. 2008 Apr;28(2):212-217.
The course of myasthenia gravis is marked by exacerbations and remissions, which may be partial or complete. The weakness can be asymmetric, confined to one group of muscles, or generalized. Ocular muscles are most commonly affected, resulting in fluctuating ptosis and diplopia. With bulbar involvement, laryngeal and pharyngeal muscle weakness can result in dysarthria, difficulty in chewing and swallowing, problems clearing secretions, or pulmonary aspiration. Severe disease is usually also associated with proximal muscle weakness (primarily in the neck and shoulders) and involvement of respiratory muscles. As with other neuromuscular diseases, active management of potential respiratory complications is a critical element of disease therapy (Table 29-4). Muscle strength characteristically improves with rest but deteriorates rapidly with exertion. Infection, stress, surgery, and pregnancy have unpredictable effects on the disease but often lead to exacerbations. A number of medications may exacerbate the signs and symptoms of myasthenia gravis (Table 29-5).

Low upper airway muscle tone, Respiratory Complication = Upper airway obstruction Obstructive sleep apnea. Low upper airway muscle tone, Treatment = Consideration of tonsillectomy or adenoidectomy, or both Noninvasive ventilation. Abnormal chest wall compliance, Respiratory Complication = Lung restriction Atelectasis Scoliosis. Abnormal chest wall compliance, Treatment = Lung volume recruitment Scoliosis surgery. Weak inspiratory muscles, Respiratory Complication = Lung restriction Atelectasis Ineffective airway clearance. Weak inspiratory muscles, Treatment = Respiratory muscle training Lung volume recruitment Airway clearance therapies
Dysphagia, 1 = Pulmonary aspiration Recurrent respiratory infections Bronchiectasis and fibrosis Impaired oxygenation. Dysphagia, 2 = Feeding therapy Enteric tube feeding Airway clearance therapies. Respiratory insufficiency, 1 = Acute or chronic respiratory failure. Respiratory insufficiency, 2 = Noninvasive ventilation Tracheostomy tube placement and invasive ventilation
'Reproduced with permission from Buu MC: Respiratory complications, management and treatments for neuromuscular disease in children.
Curr Opin Pediatr. 2017 Jun;29(3):326-333.

Ampicillin
Azithromycin
Ciprofloxacin
Clarithromycin
Erythromycin
Gentamycin
Neomycin
Streptomycin
Sulfonamides
Tetracycline
Tobramycin

lodinated radiocontrast agents
Magnesium
Nondepolarizing neuromuscular blockers
'Data from Mahadeva B, Phillips II L, Juel VC: Autoimmune disorders of neuromuscular transmission. Semin Neurol. 2008;28:212; and Matney 5, Huff D. Diagnosis and treatment of myasthenia gravis. Consult Pharm. 2007;22:239.
Anticholinesterase drugs are used most commonly to treat the muscle weakness of this disorder. These drugs increase the amount of acetylcholine at the neuromuscular junction through inhibition of end-plate acetylcholinesterase. Pyridostigmine is prescribed most often; when given orally, it has an effective duration of 2 to 4h. Excessive administration of an anticholinesterase may precipitate cholinergic crisis, which is characterized by increased weakness and excessive muscarinic effects, including salivation, diarrhea, miosis, and bradycardia. An edrophonium (Tensilon) test may help differentiate a cholinergic from a myasthenic crisis. Increased weakness after administration of up to 10 mg of intravenous edrophonium indicates cholinergic crisis, whereas increasing strength implies myasthenic crisis. If this test is equivocal or if the patient clearly has manifestations of cholinergic hyperactivity, all cholinesterase drugs should be discontinued and the patient should be monitored in an intensive care unit or close-observation area. Anticholinesterase drugs are often the only agents used to treat patients with mild disease. Moderate to severe disease is treated with a combination of an anticholinesterase drug and immunomodulating therapy. Corticosteroids are usually tried first, followed by other agents (Table 29-6). Plasmapheresis is reserved for patients with dysphagia or respiratory failure, or to normalize muscle strength preoperatively in patients undergoing a surgical procedure, including thymectomy. Up to 85% of patients younger than 55 years of age show clinical improvement following thymectomy even in the absence of a tumor, but improvement may be delayed up to several years.

neuropsychiatric problems. Tacrolimus, Risks and Contraindications = Liver and kidney toxicity. Cyclophosphamide, Mode of Action = — Suppression of B and T cells. Cyclophosphamide, Side Effects = Nausea, vomiting, alopecia, discoloration of nails and skin, infections. Cyclophosphamide, Risks and Contraindications = Leukopenia. Intravenous immune globulin, Mode of Action = Suppression of B and T cells, neutralization of autoantibodies. Intravenous immune globulin, Side Effects = Nausea, headache, fever, hypotension or hypertension, local skin reactions. Intravenous immune globulin, Risks and Contraindications = IgA deficiency, allergic reactions
'Adapted with permission from Gillhus, NE. Myesthenia gravis. N Engi J Med. 2016 Dec 29;375(26):2570-2581

Patients with myasthenia gravis may present for thymectomy or for unrelated surgical or obstetric procedures, and medical management of their condition should be optimized prior to the intended procedure. Myasthenic patients with respiratory and oropharyngeal weakness should be treated preoperatively with intravenous immunoglobulin or plasmapheresis. If strength normalizes, the incidence of postoperative respiratory complications should be similar to that of a nonmyasthenic patient undergoing a similar surgical procedure. Patients scheduled for thymectomy may have deteriorating muscle strength, whereas those undergoing other elective procedures may be well controlled or in remission. Adjustments in anticholinesterase medication, immunosuppressants,

@ Patients who have myasthenia gravis are at risk for postoperative respiratory failure. Disease duration of more than 6 years, concomitant pulmonary disease, peak inspiratory pressure of less than —-25 cm H,O (ie, —20 cm H,0), vital capacity less than 4 mL/kg, and pyridostigmine dose greater than 750 mg/d are predictive of the need for postoperative ventilation following thymectomy.
Women with myasthenia can experience increased weakness in the last trimester of pregnancy and in the early postpartum period. Epidural anesthesia is generally preferable for these patients because it avoids potential problems with respiratory depression and NMBs related to general anesthesia. Excessively high levels of motor blockade, however, can also result in hypoventilation. Infants of myasthenic mothers may show transient myasthenia for 1 to 3 weeks following birth, induced by transplacental transfer of acetylcholine receptor antibodies, which may necessitate intubation and mechanical ventilation.

Paraneoplastic syndromes are immune-mediated diseases associated with an underlying cancer in which organ or tissue dysfunction or damage occurs distant from the primary or metastatic tumor. Myasthenia gravis is often considered a paraneoplastic syndrome because it is an autoimmune disorder associated with thymic hyperplasia, including thymoma. Other neurological or neuromuscular paraneoplastic syndromes include Lambert—Eaton myasthenic syndrome, limbic encephalitis, neuromyotonia, stiff person syndrome, myotonic dystrophy, and polymyositis.

The Lambert—Eaton myasthenic syndrome (LEMS) is a paraneoplastic syndrome characterized by proximal muscle weakness that typically begins in the lower extremities but may spread to involve upper limb, bulbar, and respiratory muscles. Dry mouth, male impotence, and other manifestations of autonomic dysfunction are also common. LEMS is usually associated with small cell carcinoma of the lung but may also be seen with other malignancies or as an idiopathic autoimmune disease. The disorder results from a presynaptic defect of neuromuscular transmission in which antibodies to voltage-gated calcium channels on the nerve terminal markedly reduce the quantal release of acetylcholine at the motor end-plate. Small cell lung carcinoma cells express identical voltage-gated calcium channels, serving as a trigger for the autoimmune response in patients with paraneoplastic LEMS. In contrast to myasthenia gravis, muscle weakness associated with LEMS improves with repeated effort and is improved less dramatically by anticholinesterase drugs. Guanidine hydrochloride and 3,4-diaminopyridine (3,4DAP), which increase the presynaptic release of acetylcholine, often produce significant improvement in LEMS. Corticosteroid or other immunosuppressive medications, or plasmapheresis, may also be of benefit.

Limbic encephalitis is a degenerative central nervous system disorder characterized by personality changes, hallucinations, seizures, autonomic dysfunction, varying degrees of dementia, and asymmetric loss of sensation in the extremities. It may involve the brain, brainstem, cerebellum, and spinal cord. In approximately 60% of cases, limbic encephalitis is paraneoplastic. There is a strong association with small cell lung carcinoma, and neurological dysfunction often precedes the cancer diagnosis. Therapy includes treatment of the underlying cancer, if present, and administration of immunosuppressive medications.

Neuromyotonia is a condition of peripheral nerve hyperexcitability that is frequently associated with an underlying cancer but may also be inherited or associated with diabetic, drugor toxin-induced, or other acquired neuropathies. Its features include myokymia (a continuous undulating movement of muscles described as being like a "bag of worms"), stiffness, impaired muscle relaxation, painful muscle cramping, hyperhidrosis, and muscle hypertrophy. Treatment includes immunoglobulin therapy, plasma exchange, and administration of anticonvulsants.

Stiff person syndrome is a progressive disorder characterized by axial stiffness and rigidity that may subsequently involve the proximal limb muscles. In advanced cases, paraspinal rigidity may cause marked spinal deformities, and the patient may have difficulty with ambulation and a history of frequently falling. Although stiff person syndrome is rare, when it occurs it is frequently associated with cancer. Therapy includes treatment of the underlying cancer, if present, and administration of immunoglobulin and benzodiazepines.

Polymyositis is an inflammatory myopathy of skeletal musculature, especially proximal limb muscles, characterized by weakness and easy fatigability. Patients are prone to aspiration and frequent pneumonias because of thoracic muscle weakness and dysphagia secondary to oropharyngeal muscle involvement. They may also exhibit cardiac dysrhythmias due to conduction defects. Therapy includes treatment of the underlying neoplasm, if present; plasma exchange; and administration of immunoglobulin, corticosteroids, and immunomodulators such as methotrexate, cyclosporine, and tumor necrosis factor-a inhibitors.

® Patients with LEMS and other neuromuscular paraneoplastic syndromes are very sensitive to both depolarizing and nondepolarizing NMBs. Volatile agents alone are often sufficient to provide muscle relaxation for both intubation and most surgical procedures. NMBs should be given only in small increments and with careful neuromuscular monitoring. Because these patients frequently exhibit marked debility, benzodiazepines, opioids, and other medications with sedative effects should be administered with caution, if at all.

Muscular dystrophies are a heterogeneous group of hereditary disorders characterized by muscle fiber necrosis and regeneration, leading to muscle degeneration and progressive weakness. Anticipated anesthetic risk is increased by the patient's overall debilitated status, which may impede clearance of secretions and postoperative ambulation, as well as by increased risk of respiratory failure and pulmonary aspiration. Duchenne muscular dystrophy is the most common and most severe form of muscular dystrophy. Other muscular dystrophy variants include Becker, myotonic, facioscapulohumeral, and limbgirdle dystrophies.

An X-linked recessive disorder, Duchenne muscular dystrophy affects males almost exclusively. It has an incidence of approximately 1 to 3 cases per 10,000 live male births and most commonly presents between 3 and 5 years of age. Affected individuals produce abnormal dystrophin, a protein found on the sarcolemma of muscle fibers. Patients characteristically develop symmetric proximal muscle weakness that is manifested as a gait disturbance. Fatty infiltration typically causes enlargement (pseudohypertrophy) of muscles, particularly the calves. Progressive weakness and contractures eventually result in kyphoscoliosis. Many patients are confined to wheelchairs by age 12. Disease progression may be delayed by up to 2 to 3 years with glucocorticoid therapy in some patients. Intellectual impairment is common but generally nonprogressive. Plasma creatine kinase (CK) levels are 10 to 100 times normal even early in the disease and may reflect an abnormal increase in the permeability of muscle cell membranes. Female genetic carriers often also have high plasma CK levels, variable degrees of muscle weakness, and, rarely, cardiac involvement. Plasma myoglobin concentration may also be elevated. The diagnosis is confirmed by muscle biopsy.
@ Respiratory muscle degeneration in patients with muscular dystrophy interferes with an effective cough mechanism and leads to retention of secretions and frequent pulmonary infections. The combination of marked kyphoscoliosis and muscle wasting may produce a severe restrictive ventilatory defect. Pulmonary hypertension is common with @ disease progression. Degeneration of cardiac muscle in patients with muscular dystrophy is also common, but results in dilated or hypertrophic cardiomyopathy in only 10% of patients. Mitral regurgitation secondary to papillary muscle dysfunction is also found in up to 25% of patients. Electrocardiogram abnormalities include P—R interval prolongation, QRS and ST-segment abnormalities, and prominent R waves over the right precordium with deep Q waves over the left precordium. Atrial arrhythmias are common. Death at a relatively young age is usually due to recurrent pulmonary infections, respiratory failure, or cardiac failure.

Becker muscular dystrophy is, like Duchenne, an X-linked recessive disorder, but it is less common (1:30,000 male births). Manifestations are nearly identical to those of Duchenne muscular dystrophy except that they usually present later in life (adolescence) and progress more slowly. Mental retardation is less common. Patients often reach the fourth or fifth decade, although some may survive into their 80s. Death is usually from respiratory complications. Cardiomyopathy may occur in some cases and may precede severe skeletal weakness.

Myotonic dystrophy is a multisystem disorder that is the most common cause of myotonia, a slowing of relaxation after muscle contraction in response to electrical or percussive stimuli. The disease is autosomal dominant, with an incidence of 1:8000, and usually becomes clinically apparent in the second to third decade of life, but it has also been reported as a paraneoplastic disorder in association with thymoma. Myotonia is the principal early manifestation; muscle weakness and atrophy become more prominent as the disease progresses. This weakness and atrophy usually affect cranial muscles (orbicularis oculi and oris, masseter, and sternocleidomastoid), and in contrast to most myopathies, distal muscles more than proximal muscles. Plasma CK levels are normal or slightly elevated.
Multiple organ systems are involved in myotonic dystrophy, as evidenced by presenile cataracts, premature frontal baldness, hypersomnolence with sleep apnea, and endocrine dysfunction leading to pancreatic, adrenal, thyroid, and gonadal insufficiency. Respiratory involvement leads to decreased vital capacity, and chronic hypoxemia may cause cor pulmonale. Gastrointestinal hypomotility may predispose patients to pulmonary aspiration. Uterine atony can prolong labor and increase the incidence of retained placenta. Cardiac manifestations, which are often present before other clinical symptoms appear, may include cardiomyopathy, atrial arrhythmias, and varying degrees of heart block.
The myotonia is usually described by patients as a "stiffness" that may lessen with continued activity—the so-called "warm-up" phenomenon. Patients often report that cold temperatures worsen stiffness. Antimyotonic treatment may include mexiletine, phenytoin, baclofen, dantrolene, or carbamazepine. A cardiac pacemaker may be placed in patients with significant conduction defect, even if they are asymptomatic.

Facioscapulohumeral dystrophy, an autosomal dominant disorder with an incidence of approximately 1 to 3 cases per 100,000, affects both sexes, although more females than males are asymptomatic. Patients usually present in the second or third decade of life with weakness that is confined primarily to the muscles of the face and the shoulder girdle. Muscles in the lower extremities are less commonly affected, and respiratory muscles are usually spared. The disease is slowly progressive with a variable course. Plasma CK levels are usually normal or only slightly elevated. Cardiac involvement is rare, but loss of atrial electrical activity with an inability to atrially pace the heart has been reported; ventricular pacing is still possible in these patients. Longevity is minimally affected.

Limb-girdle muscular dystrophy is a heterogeneous group of genetic neuromuscular diseases. Limb-girdle syndromes include severe childhood autosomal recessive muscular dystrophy and other incompletely defined autosomal recessive syndromes such as Erb (scapulohumeral type) and Leyden— Mobius (pelvifemoral type) dystrophies. Most patients present between childhood and the second or third decade of life with slowly progressive muscle weakness that may involve the shoulder girdle, the hip girdle, or both. Plasma CK levels are usually elevated. Cardiac involvement is relatively uncommon but may present as frequent arrhythmias or congestive heart failure. Respiratory complications, such as hypoventilation and recurrent respiratory infections, may occur.

The anesthetic management of these patients is complicated not only by muscle weakness but also by cardiac and pulmonary manifestations. An association with malignant hyperthermia has been suggested but is unproven. Preoperative premedication with sedatives or opioids should be avoided because of increased aspiration risk due to respiratory muscle weakness, gastric hypomotility, or both. Intraoperative positioning may be complicated by kyphoscoliosis or by flexion contractures of the @ extremities or neck. Succinylcholine should be avoided in patients with Duchenne or Becker muscular dystrophies because of unpredictable response and the risk of inducing severe hyperkalemia or triggering malignant hyperthermia. Inhalational anesthetics have been associated with rhabdomyolysis and hyperkalemia in patients with Duchene muscular dystrophy in cases where no succinylcholine was employed. Although some patients exhibit a normal response to nondepolarizing NMBs, others may be very sensitive. Marked respiratory and circulatory depression may be seen with volatile anesthetics in patients with advanced disease, and regional or local anesthesia may be preferable in these patients. Perioperative morbidity is usually due to respiratory complications. Patients with vital capacities less than 30% of predicted are at greatest risk and often require temporary postoperative mechanical ventilation.

Patients with myotonic dystrophy are at increased risk for perioperative respiratory and cardiac complications. Most perioperative problems arise in patients with severe weakness and in those cases in which surgeons and anesthesiologists are unaware of the diagnosis. The diagnosis of myotonic dystrophy has been made in some patients in the course of investigating prolonged apnea following general anesthesia.
Patients with myotonic dystrophy are often sensitive to even small doses of opioids, sedatives, and inhalation and intravenous anesthetic agents, all of which may cause sudden and prolonged apnea. Premedication should therefore be avoided. Succinylcholine is relatively contraindicated because it may precipitate intense myotonic contraction of the diaphragm, chest wall, or laryngeal muscles, making ventilation difficult or impossible. Other drugs that act on the motor endplate, such as neostigmine, and physostigmine, can aggravate myotonia. Regional anesthesia may be preferentially employed but does not always prevent myotonic contractions.
The response to nondepolarizing NMBs is reported to be normal; however, they do not consistently prevent or relieve myotonic contractions. As reversal of nondepolarizing NMBs can induce myotonic contractions, the use of shortacting nondepolarizing agents is recommended. Postoperative shivering may induce myotonic contractions in the recovery room, and doses of meperidine can often prevent such shivering and may preempt myotonic contractions.
Induction of anesthesia without complications has been reported with a number of agents including inhalation agents and propofol. Neuromuscular blockade, if needed, should employ short-acting nondepolarizing NMBs. An association between myotonic dystrophy and malignant hyperthermia has been suggested but not established. Nitrous oxide and inhalation agents can be used as maintenance anesthesia. Reversal with anticholinesterases should be avoided, if possible.

The principal postoperative complications of myotonic dystrophy are prolonged hypoventilation, atelectasis, aspiration, and pneumonia. Patients undergoing upper abdominal surgery or those with severe proximal weakness are more likely to experience pulmonary complications. Close postoperative monitoring for arrhythmias should be accompanied by aggressive pulmonary hygiene with physical therapy and incentive spirometry. Aspiration prophylaxis is indicated.

Patients with facioscapulohumeral and limb-girdle muscular dystrophy generally have normal responses to anesthetic agents. Nevertheless, because of the great variability and overlap among the various forms of muscular dystrophy, sedative-hypnotics, opioids, and nondepolarizing NMBs should be used cautiously, and succinylcholine should be avoided.

Myotonia congenita is a disorder manifested early in life with generalized myotonia. Both autosomal dominant (Thomsen) and recessive (Becker) forms exist. The disease is confined to skeletal muscle, and weakness is minimal or absent. Many patients have very well-developed musculature due to near constant muscle contraction. Antimyotonic therapy includes phenytoin, mexiletine, quinine sulfate, or procainamide. Other medications that have been used include tocainide, dantrolene, prednisone, acetazolamide, and taurine. There is no cardiac involvement in myotonia congenita, and a normal life span is expected.
Paramyotonia congenita is a very rare autosomal dominant disorder characterized by transient stiffness (myotonia) and, occasionally, weakness after exposure to cold temperatures. The stiffness worsens with activity, in contrast to true myotonia, thus the term paramyotonia. Serum potassium concentration may rise following an attack similar to hyperkalemic periodic paralysis (discussed next). Medications that have been used to block the cold response include

Anesthetic management of patients with myotonia congenita and paramyotonia is complicated by an abnormal response to succinylcholine, intraoperative myotonic contractions, and the need to avoid hypothermia. NMBs may paradoxically cause generalized muscle spasms, including trismus, leading to difficulty with intubation and ventilation.
Infiltration of muscles in the operative field with a dilute local anesthetic may alleviate refractory myotonic contraction. Among patients with these types of myotonia, none have been reported with positive in vitro tests for malignant hyperthermia. Excised muscle in these patients does, however, display a prolonged myotonic contraction when exposed to succinylcholine. Excessive muscle contraction during anesthesia, therefore, likely represents aggravation of myotonia and not malignant hyperthermia.

Periodic paralysis is a group of disorders characterized by spontaneous episodes of transient muscle weakness or paralysis. Symptoms usually begin in childhood, with episodes lasting a few hours and typically sparing respiratory muscle involvement. The weakness usually lasts less than 1 h but can last several days, and frequent attacks may lead to progressive, long-term weakness in some patients. Hypothermia exacerbates the frequency and severity of episodes. Muscle strength and serum potassium concentrations are usually normal between attacks. The episodes of weakness are due to a loss of muscle fiber excitability secondary to partial depolarization of the resting potential. This partial depolarization prevents the generation of action potentials and thereby precipitates weakness.
Periodic paralysis is classified into primary genetic channelopathies and secondary acquired forms. The genetic types are due to dominantly inherited mutations in the voltage-gated sodium, calcium, or potassium ion channels. Classifications have been based on clinical differences, but these have not been shown to relate to specific ion channels. Different defects in the same channel can cause different clinical pictures, whereas mutations in different channels may have similar clinical pictures. However, the clinical classifications remain useful as guides to prognosis and therapy.
Hypokalemic periodic paralysis is typically associated with low serum potassium levels, and hyperkalemic periodic paralysis with elevated serum potassium levels, during episodes of weakness. In these defects, muscle membranes are inexcitable to both direct and indirect stimulation due to either decreased potassium conductance or increased sodium conductance, respectively. Both defects are associated with fluid and electrolyte shifts.
Thyrotoxic periodic paralysis occurs most commonly in Asian men and is characterized by episodes of marked weakness associated with increased thyroid hormones, low thyroid-stimulating hormone, and hypokalemia. Primary therapy is treatment of the underlying hyperthyroid state.
Secondary hypokalemic paralysis can also develop if there are marked losses of potassium through the kidneys or the gastrointestinal tract. The associated weakness is, at times, episodic, and potassium levels are much lower than in other variants of hypokalemic periodic paralysis. Management of the primary disease with potassium replacement and treatment of acidosis or alkalosis are is important in preventing attacks.
Patients who consume large amounts of barium salts, which block potassium channels, can also develop hypokalemic periodic paralysis. This condition is treated by stopping the barium salts and administering oral potassium.

Potassium levels that exceed 7 mEq/L between episodes of weakness suggest a secondary form of hyperkalemic periodic paralysis. Treatment is targeted toward the primary disease and involves restriction of potassium.

® Anesthetic management of patients with periodic paralysis is directed toward preventing attacks. Perioperative management must include frequent determinations of plasma potassium concentration and correction of abnormal values, with careful electrocardiographic monitoring to detect arrhythmias. Because of the potential for glucose and alkalosis to lower the plasma potassium concentration, glucose-containing intravenous solutions and hyperventilation should be avoided in patients with hypokalemic paralysis, including thyrotoxic periodic paralysis, and use of drugs such as insulin and epinephrine that lower serum potassium should be minimized. Tachycardia associated with thyrotoxic periodic paralysis is treated with nonselective © B-blockade. The response to NMBs is unpredictable, and neuromuscular function should be carefully monitored during their use. Increased sensitivity to nondepolarizing NMBs is particularly apt to be encountered in patients with hypokalemic periodic paralysis. Succinylcholine is contraindicated in hyperkalemic paralysis and perhaps other variants as well because of the risk of hyperkalemia. Intraoperative maintenance of core temperature is important because shivering and

The choice of anesthesia should be based on both patient and procedural requirements. Most muscle biopsies can be performed under local or regional anesthesia with supplemental intravenous sedation, using small doses of midazolam. Spinal or epidural anesthesia may be utilized. A femoral nerve block can provide excellent anesthesia for biopsy of the quadriceps muscle; a separate injection may be necessary for the lateral femoral cutaneous nerve to anesthetize the anterolateral thigh. General anesthesia should be reserved for uncooperative patients or for times when local or regional anesthesia is inadequate. The anesthesiologist must therefore always be prepared with a plan for general anesthesia.

Major goals include preventing pulmonary aspiration, avoiding excessive respiratory or circulatory depression, avoiding NMBs if possible, and perhaps avoiding agents known to trigger malignant hyperthermia. A normal response to a previous general anesthetic in the patient or a family member may be reassuring but does not guarantee the same response subsequently. General anesthesia may be induced and maintained with a combination of a benzodiazepine, propofol, and a short-acting opioid with or without nitrous oxide. Patients at increased risk for aspiration should be intubated. When an NMB is necessary, a short-acting nondepolarizing agent should be used. Succinylcholine should generally be avoided because of the unknown risk of an unusual response (myotonic contractions, prolonged duration, or phase II block), of inducing severe hyperkalemia, or of triggering malignant hyperthermia.

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@ The combined blood flow through both kidneys normally accounts for 20% to 25% of total cardiac output.
® Autoregulation of renal blood flow normally occurs between mean arterial blood pressures of 80 and 180 mm Hg and is principally due to intrinsic myogenic responses of the afferent glomerular arterioles to blood pressure changes.
© Renal synthesis of vasodilating prostaglandins (PGD,, PGE>, and PGI,) is an important protective mechanism during periods of systemic hypotension and kidney ischemia.
@ Dopamine and fenoldopam dilate afferent and efferent arterioles via D,receptor activation.
@ Reversible decreases in renal blood flow, glomerular filtration rate, urinary flow, and sodium excretion occur during both neuraxial and general anesthesia. Acute kidney injury is less likely to occur if adequate intravascular volume and normal blood pressure are maintained.
@ The endocrine response to surgery and anesthesia is at least partly responsible for the transient fluid retention often seen postoperatively.
@ Compound A, a breakdown product of sevoflurane, causes acute kidney injury in laboratory animals. Low fresh gas flow rates promote its accumulation in the anesthesia machine breathing circuit. No clinical study has detected significant kidney injury in humans as a consequence of sevoflurane anesthesia; nonetheless, some authorities recommend a fresh gas flow of at least 2 L/min with sevoflurane to minimize the risk of this theoretical problem.
@ Pneumoperitoneum produced during laparoscopy causes an abdominal
compartment syndrome-like state. The increase in intraabdominal pressure often produces oliguria or anuria that is generally proportional to insufflation pressure. Mechanisms include vena cava and renal vein compression; kidney parenchymal compression; decreased cardiac output; and increases in plasma levels of renin, aldosterone, and antidiuretic hormone.

The kidneys play a vital role in regulating the volume and composition of body fluids, eliminating toxins, and elaborating hormones, including renin, erythropoietin, and the active form of vitamin D. Factors directly and indirectly related to operative procedures and to anesthetic management frequently have a physiologically significant impact on kidney physiology and function, and may lead to perioperative fluid overload, hypovolemia, and acute kidney injury, which are major causes of perioperative morbidity and mortality.
Diuretics are frequently used in the perioperative period. They are commonly administered on a chronic basis to patients with hypertension or chronic heart failure, and to patients with liver or kidney disease. Diuretics may be used intraoperatively, typically during neurosurgical, cardiac, major vascular, ophthalmic, and urological procedures. Familiarity with the various types of diuretics and their mechanisms of action, side effects, and potential anesthetic interactions is therefore essential.

Each kidney is made up of approximately 1 million functional units called nephrons. Anatomically, a nephron consists of a tortuous tubule with at least six specialized segments. In the renal corpuscle, a structure at its proximal end composed of a glomerulus and a Bowman's capsule, an ultrafiltrate of blood is formed which flows through the nephron's tubules. During this process, the ultrafiltrate's volume and composition are modified by both reabsorption and secretion of solutes, and the collected final product is eliminated as urine.
Nephrons are classified as cortical or juxtamedullary, and the renal corpuscles of all nephrons are located in the renal cortex. In addition to the renal corpuscle, the other major anatomical and functional divisions of the nephron are the proximal convoluted tubule, the loop of Henle, the distal renal tubule, the collecting tubule, and the juxtaglomerular apparatus (Figure 30—1 and Table 30-1).
Distal
FIGURE 30-1 Major anatomical divisions of the nephron. (Reproduced with permission from Ganong WEF. Review of Medical Physiology. 24th ed. New York, NY: McGraw-Hill Education; 2012.)
TABLE 30-1 Functional divisions of a nephron.!
Renal corpuscle (glomerulus, Bowman's capsule), 1 = Ultrafiltration of blood. Proximal tubule, 1 = Reabsorption Sodium? chloride Water Bicarbonate Glucose, protein, amino acids Potassium, magnesium, calcium Phosphates,’ uric acid, urea Secretion Organic anions Organic cations Ammonia production. Loop of Henle, 1 = Reabsorption Sodium, chloride Water Potassium, calcium, magnesium Countercurrent multiplier
Distal tubule
Reabsorption
Sodium‘ chloride
Water
Potassium
Calcium?
Bicarbonate
Secretion
Hydrogen ion*
Potassium*
Calcium
Collecting tubule
Sodium** chloride
Water®’
Potassium
Secretion
Ammonia production
Juxtaglomerular apparatus
Secretion of renin

'Adapted with permission from Rose BD. Clinical Physiology of Acid-Base and Electrolyte Disorders. 3rd ed. New York, NY: McGraw-Hill Education; 1989,
?Partially augmented by angiotensin Il.
4Inhibited by parathyroid hormone.
*At least partly aldosterone mediated.
'Augmented by parathyroid hormone.
'Inhibited by atrial natriuretic peptide.
?Antidiuretic hormone mediated.

Each renal corpuscle contains a glomerulus, which is composed of tufts of capillaries that jut into Bowman's capsule, providing a large surface area for blood filtration. Blood enters by a single afferent arteriole and departs by a single efferent arteriole. The glomerular endothelial cells are separated from the epithelial cells of Bowman's capsule only by their fused basement membranes.
The endothelial cells are perforated with relatively large fenestrae (70-100 nm), but the epithelial cells interdigitate tightly with one another, leaving relatively small filtration slits (approximately 25 nm). The two cell types with their basement membranes provide an effective filtration barrier to cells and largemolecular-weight substances. This barrier has multiple anionic sites that give it a net negative charge, favoring filtration of cations relative to anions. A third cell type, called intraglomerular mesangial cells, is located between the basement membrane and epithelial cells near adjacent capillaries. These contractile cells regulate glomerular blood flow and also exhibit phagocytic activity. Mesangial cells contract, reducing glomerular filtration, in response to angiotensin II, vasopressin, norepinephrine, histamine, endothelins, thromboxane A,, leukotrienes (C, and D,), prostaglandin F,, and platelet-activating factor. They relax, thereby increasing glomerular filtration, in response to atrial natriuretic peptide (ANP), prostaglandin E,, and dopaminergic agonists.
Glomerular filtration pressure (approximately 60 mm Hg) is normally approximately 60% of mean arterial pressure and is opposed by both plasma oncotic pressure (approximately 25 mm Hg) and renal interstitial pressure (approximately 10 mm Hg). Afferent and efferent arteriolar tone are both important in determining glomerular filtration pressure: filtration pressure is directly proportional to efferent arteriolar tone but inversely proportional to afferent tone. Approximately 20% of plasma is normally filtered into the Bowman's capsule as blood passes through the glomerulus.

Of the ultrafiltrate formed in Bowman's capsule, 65% to 75% is normally reabsorbed isotonically (proportional amounts of water and sodium) in the proximal renal tubule (Figure 30—2). To be reabsorbed, most substances must first traverse the tubular (apical) side of the cell membrane, and then cross the basolateral cell membrane into the renal interstitium before entering peritubular capillaries. The major function of the proximal tubule is Na* reabsorption. Sodium is actively transported out of proximal tubular epithelial cells at their capillary side by membrane-bound Na*—K*-adenosine triphosphatase (Na*—K*ATPase) (Figure 30-3). The resulting low intracellular concentration of Na* allows passive movement of Na* down its gradient from tubular fluid into the tubular epithelial cells. Angiotensin II and norepinephrine enhance Na* reabsorption in the early proximal tubule. In contrast, dopamine and fenoldopam decrease the proximal reabsorption of sodium via D,-receptor activation.
FIGURE 30-2 Sodium reabsorption in the nephron. Numbers represent the percentage of the filtered sodium reabsorbed at each site. (Reproduced with permission from Cogan MG. Fluid and Electrolytes: Physiology and Pathophysiology. New York, NY: Appleton & Lange; 1991.)
FIGURE 30-3 Reabsorption of solutes in proximal tubules. Note that Na*—K*ATPase supplies the energy for reabsorption of most solutes by maintaining a low intracellular concentration of sodium.

Sodium reabsorption is coupled with the reabsorption of other solutes and the secretion of H* (Figure 30-3). Specific carrier proteins use the low concentration of Na' inside cells to transport phosphate, glucose, and amino acids. The net loss of intracellular positive charges, the result of Na*—K*-ATPase activity (exchanging 3Na* for 2K*), favors the absorption of other cations (K*, Ca**, and Mg?**). Thus, the Na*—K*-ATPase at the basolateral side of the renal tubular epithelial cells provides the energy for the reabsorption of most solutes. Sodium reabsorption at the luminal membrane is also coupled with countertransport
(secretion) of H*. The latter mechanism is responsible for reabsorption of 90% of the filtered bicarbonate ions (see Figure 50-3). Unlike other solutes, chloride can traverse the tight junctions between adjacent tubular epithelial cells, and accordingly, is passively resorbed via its concentration gradient. Active chloride reabsorption may also take place as a result of a K*—Clcotransporter that extrudes both ions at the capillary side of the cell membrane (Figure 30-3). Water moves passively out the proximal tubule along osmotic gradients. Apical membranes of epithelial cells contain specialized water channels, composed of a membrane protein called aquaporin-1, that facilitate water movement. proteins,
The proximal tubules are capable of secreting organic cations and anions. Creatinine excretion can be inhibited by other organic cations (eg, trimethoprim or pyrimethamine), leading to increases in serum creatinine concentration. Organic anions such as urates, ketoacids, penicillins, cephalosporins, diuretics, salicylates, and most radiocontrast dyes also share common secretory mechanisms and can compete with one another. Low-molecular-weight which are filtered by glomeruli, are normally reabsorbed by proximal tubular epithelial cells, to be metabolized intracellularly.

The loop of Henle consists of descending and ascending portions. They are responsible for maintaining a hypertonic medullary interstitium and also indirectly for provide the collecting tubules with the ability to concentrate urine. The thin descending segment is a continuation of the proximal tubule and descends from the renal cortex into the renal medulla. In the medulla, the descending portion acutely turns back upon itself and rises back up toward the cortex as the ascending portion. The ascending portion consists of a functionally distinct thin ascending limb, a medullary thick ascending limb, and a cortical thick ascending limb (Figure 30-1). Cortical nephrons have relatively short loops of Henle that extend only into the more superficial regions of the renal medulla and often lack a thin ascending limb. Juxtamedullary nephrons, which have renal corpuscles located near the renal medulla, possess loops of Henle that project deeply into the renal medulla. Cortical nephrons outnumber juxtamedullary nephrons by approximately 7:1.
Only 25% to 35% of the ultrafiltrate formed in Bowman's capsule normally reaches the loop of Henle, where 15% to 20% of the filtered sodium load is normally reabsorbed. With the notable exception of the ascending thick medullary and cortical segments, solute and water reabsorption in the loop of
Henle is passive and follows concentration and osmotic gradients. In the ascending thick segment, however, Na* and CI are reabsorbed in excess of water; Na* reabsorption in this part of the nephron is directly coupled to both K* and CI reabsorption (Figure 30—4), and [CI'] in tubular fluid appears to be the rate-limiting factor. Active Na* reabsorption still results from Na*—K*-ATPase activity on the capillary side of tubular epithelial cells.
FIGURE 30-4 Sodium and chloride reabsorption in the thick ascending loop of Henle. All four sites on the luminal carrier protein must be occupied for transport to occur. The rate-limiting factor appears to be chloride concentration in tubular fluid.

Unlike the descending limb and the thin ascending limb of the loop of Henle, the thick parts of the ascending limb are impermeable to water. As a result, tubular fluid flowing out of the loop of Henle is hypotonic (100-200 mOsm/L) and the interstitium surrounding the loop of Henle is therefore hypertonic. A countercurrent multiplier mechanism is established such that both the tubular fluid and medullary interstitium become increasingly hypertonic with increasing depth into the medulla (Figure 30—5). Urea concentrations also increase within the medulla and contribute to the hypertonicity. The countercurrent mechanism includes the loop of Henle, the cortical and medullary collecting tubules, and adjacent capillaries (vasarecta).
FIGURE 30-5 The countercurrent multiplier mechanism. This mechanism is dependent on differential permeability and transport characteristics between the descending and ascending limbs. The descending limb and the thin ascending limb are permeable to water, Na*, Cl, and urea. The thick ascending limb is impermeable to water and urea, actively reabsorbs Na* and CI, and therefore can generate an osmotic gradient. This figure depicts from "time zero," a progressive 200-mOsm/kg gradient between the descending and ascending limbs. Note that as urine flows, the gradient remains unchanged but the osmolality progressively increases at the bottom of the loop. (Reproduced with permission from Pitts RF. Physiology of the Kidney and Body Fluids. 3rd ed. Philadelphia, PA: Year Book; 1974.)
The thick ascending loop of Henle is also an important site for calcium and magnesium reabsorption, and parathyroid hormone augments calcium reabsorption at this location.

The collecting tubule can be divided into cortical and medullary portions, and together, they normally account for the reabsorption of 5% to 7% of the filtered sodium load.

This part of the nephron consists of two cell types: (1) principal cells that primarily secrete potassium and participate in aldosterone-stimulated Na* reabsorption, and (2) intercalated cells that are responsible for acid—base regulation. Because principal cells reabsorb Na* via an electrogenic pump, either Cl must also be reabsorbed or K* must be secreted to maintain electroneutrality. Increased intracellular [K*] favors K* secretion. Aldosterone enhances Na*—K*ATPase activity in this part of the nephron by increasing the number of open K* and Na* channels in the luminal membrane. Aldosterone also enhances the H*secreting ATPase on the luminal border of I cells (Figure 30-6). Intercalated cells additionally have a luminal K'Y-H*-ATPase pump, which reabsorbs K* and

FIGURE 30-6 Secretion of hydrogen ions and reabsorption of bicarbonate and potassium in the cortical collecting tubule.

The medullary collecting tubule courses down from the cortex through the hypertonic medulla before joining collecting tubules from other nephrons to form a single ureter in each kidney. This part of the collecting tubule is the principal site of action for antidiuretic hormone (ADH), also called vasopressin or arginine vasopressin (AVP). Vasopressin stimulates the expression of a water channel protein, aquaporin-2, in the cell membrane. The permeability of the luminal membrane to water is entirely dependent on the presence of vasopressin (see Chapter 49). Dehydration increases vasopressin secretion, rendering the luminal membrane permeable to water. As a result, water is osmotically drawn out of the collecting tubule fluid passing through the medulla, resulting in production of concentrated urine (up to 1400 mOsm/L). Conversely, adequate hydration suppresses vasopressin secretion, allowing fluid in the collecting tubules to pass through the medulla relatively unchanged and to remain hypotonic (100-200 mOsm/L). This part of the nephron is responsible for acidifying urine; the hydrogen ions secreted are excreted in the form of titratable acids (phosphates) and ammonium ions (see Chapter 50).

Differences in permeability to urea in the cortical and medullary collecting tubules account for up to half the hypertonicity of the renal medulla. Cortical collecting tubules are freely permeable to urea, whereas medullary collecting tubules are normally impermeable. In the presence of vasopressin, the innermost part of the medullary collecting tubules becomes even more permeable to urea. Thus, when vasopressin is secreted, water moves out of the collecting tubules and the urea becomes highly concentrated. Urea can then diffuse out deeply into the medullary interstitium, increasing its tonicity.

This small organ within each nephron consists of a specialized segment of the afferent arteriole, containing juxtaglomerular cells within its wall, and the end of the thick, ascending cortical segment of the loop of Henle, the macula densa (Figure 30-7). Juxtaglomerular cells contain the enzyme renin and are innervated by the sympathetic nervous system. Release of renin depends on B,adrenergic sympathetic stimulation, changes in afferent arteriolar wall pressure (see Chapter 49), and changes in chloride flow past the macula densa. Renin released into the bloodstream catalyzes the conversion of angiotensinogen, a protein synthesized by the liver, to angiotensin I. This inert decapeptide is then rapidly converted, primarily in the lungs, by angiotensin-converting enzyme (ACE) to form the octapeptide angiotensin II. Angiotensin II plays a major role in blood pressure regulation (see Chapter 15) and aldosterone secretion (see Chapter 49). Proximal renal tubular cells have ACE as well as angiotensin II
receptors. Moreover, intrarenal formation of angiotensin II enhances sodium reabsorption in proximal tubules. Extrarenal production of renin and angiotensin II also takes place in the vascular endothelium, the adrenal glands, and the brain.

Renal function is intimately related to renal blood flow (RBF). In fact, the kidneys are the only organs for which oxygen consumption is determined by @ blood flow; the reverse is true in other organs. The combined blood flow through both kidneys normally accounts for 20% to 25% of total cardiac output. Approximately 80% of RBF normally goes to cortical nephrons, and only 10% to 15% goes to juxtamedullary nephrons. The renal cortex extracts relatively little oxygen, having an oxygen tension of approximately 50 mm Hg, because the relatively high blood flow principally serves the filtration function. In contrast, the renal medulla maintains high metabolic activity because of solute reabsorption and requires low blood flow to maintain high osmotic gradients. It has an oxygen tension of approximately 15 mm Hg and is relatively vulnerable to ischemia.
Redistribution of RBF away from cortical nephrons with short loops of Henle to larger juxtamedullary nephrons with long loops occurs under conditions including sympathetic stimulation, increased levels of catecholamines and angiotensin II, and heart failure, and this redistribution of RBF to the medulla is associated with sodium retention.
In most individuals, each kidney is supplied by a single renal artery arising from the aorta. The renal artery divides at the renal pelvis into interlobar arteries, which in turn give rise to arcuate arteries at the junction between the renal cortex and medulla (Figure 30-8). Arcuate arteries further divide into interlobular branches that eventually supply each nephron via a single afferent arteriole. Blood from each glomerular capillary tuft is drained via a single efferent arteriole and then travels alongside adjacent renal tubules in a second peritubular system of capillaries. In contrast to the glomerular capillaries, which favor filtration, peritubular capillaries are primarily "reabsorptive." Venules draining this second capillary plexus return blood to the inferior vena cava via a single renal vein from each kidney.
FIGURE 30-8 The renal circulation. (Reproduced with permission from Leaf A, Cotran RS.

The concept of clearance is frequently used in measurements of RBF and the glomerular filtration rate (GFR). The renal clearance of a substance is defined as the volume of blood that is completely cleared of that substance per unit of time (usually, per minute).

Renal plasma flow (RPF) is commonly measured by p-aminohippurate (PAH) clearance. PAH at low plasma concentrations can be assumed to be completely cleared from plasma by filtration and secretion in one passage through the kidneys. Consequently,
where [PAH ]y is the urinary concentration of PAH and [PAH ]p is the plasma PAH concentration.
If the hematocrit (measured as a decimal rather than as a percent) is known, then
RPF and RBF are normally about 660 and 1200 mL/min, respectively.

The GFR, the volume of fluid filtered from the glomerular capillaries into Bowman's capsule per unit time, normally approximates 20% of RPF. Clearance of inulin, a fructose polysaccharide that is completely filtered but is neither secreted nor reabsorbed, is a good measure of GFR. Normal values for GFR are approximately 120 + 25 mL/min in men and 95 + 20 mL/min in women. Although less accurate than measuring inulin clearance, creatinine clearance is a much more practical measurement of GFR. Creatinine clearance tends to overestimate GFR because some creatinine is normally secreted by renal tubules (see Chapter 30). Creatinine is a product of phosphocreatine breakdown in muscle. Creatinine clearance is calculated as follows:
Creatinine clearance =
([Creatinine],, x Urinary flow rate)
—————____—_———— [Creatinine], where [creatinine], is the creatinine concentration in urine and [creatinine]p is the creatinine concentration in plasma.
The ratio of GFR to RPF is called the filtration fraction (FF) and is normally 20%. GFR is dependent on the relative tones of both the afferent and efferent arterioles (discussed earlier). Afferent arteriolar dilation or efferent arteriolar vasoconstriction can increase the FF and maintain GFR, even when RPF decreases. Afferent arteriolar tone is responsible for maintaining a relatively constant GFR over a wide range of blood pressures.

Regulation of RBF represents a complex interplay between intrinsic autoregulation, tubuloglomerular balance, and hormonal and neuronal influences on the kidney and on systemic blood pressure (Figure 30-9).
FIGURE 30-9 The role of the renin-angiotensin-aldosterone system in regulation of blood pressure and fluid balance. AGT, angiotensinogen; Ang I, angiotensin I; Ang II, angiotensin II; ACE, angiotensin-converting enzyme. (Reproduced with permission from Rastogi A, Arman F, Alipourfetrati S. New agents in treatment of hyperkalemia: An opportunity to optimize use of RAAS inhibitors for blood pressure control and organ protection in patients with chronic kidney disease. Curr Hypertens Rep. 2016 Jul;18(7):55.)

® Autoregulation of RBF normally occurs between mean arterial blood pressures of 80 and 180 mm Hg and is principally due to intrinsic smooth muscle response of afferent glomerular arterioles to blood pressure changes. Within these limits, RBF and GFR are kept relatively constant by afferent arteriolar vasoconstriction or vasodilation. Outside the autoregulation limits, RBF becomes pressure dependent. Glomerular filtration generally ceases when mean systemic arterial pressure is less than 40 to 50 mm Hg.

Tubuloglomerular feedback plays an important role in maintaining constant GFR over a wide range of perfusion pressures. Increased tubular flow tends to result in reduced GFR; conversely, decreased tubular flow tends to result in increased GFR. Although the mechanism is poorly understood, the macula densa appears to be responsible for tubuloglomerular feedback by inducing reflex changes in afferent arteriolar tone and possibly glomerular capillary permeability. Angiotensin II probably plays a permissive role in this mechanism. Local release of adenosine, which occurs in response to volume expansion, may inhibit renin release and dilate the afferent arteriole.

Decreases in afferent glomerular arteriolar pressure, increases in sympathetic nervous system activity, and decreases in distal tubule sodium load stimulate prorenin (the precursor to renin) and renin release, resulting in the subsequent formation of angiotensin II, which causes generalized arterial vasoconstriction and secondarily reduces RBF. Both afferent and efferent glomerular arterioles are constricted, but because the efferent arteriole is smaller, its resistance becomes relatively greater than that of the afferent arteriole; GFR therefore tends to be relatively preserved. Very high levels of angiotensin II constrict both arterioles and can markedly decrease GFR. Epinephrine and norepinephrine directly and preferentially increase afferent arteriolar tone but usually do not cause marked decreases in GFR because these agents also increase renin release and angiotensin II formation. Relative preservation of GFR during increased aldosterone or catecholamine secretion appears at least partly to be mediated by angiotensin-induced prostaglandin synthesis because it can be blocked by ® inhibitors of prostaglandin synthesis such as nonsteroidal antiinflammatory drugs (NSAIDs). Renal synthesis of vasodilating prostaglandins (PGD,, PGE,, and PGI,) is an important protective mechanism during periods of systemic hypotension and kidney ischemia.
Atrial naturiuetic protein (ANP) is released from atrial myocytes in response to atrial distention and is a direct smooth muscle dilator that antagonizes the vasoconstrictive action of norepinephrine and angiotensin II. It preferentially dilates the afferent glomerular arteriole, constricts the efferent glomerular arteriole, and relaxes mesangial cells, effectively increasing GFR (see Chapter 49). ANP also inhibits both the release of renin and angiotensin-induced secretion of aldosterone, and antagonizes the action of aldosterone in the distal and collecting tubules.

Sympathetic outflow from the spinal cord at the level of T4—L1 reaches the kidneys via the celiac and renal plexuses. Sympathetic nerves innervate the juxtaglomerular apparatus (B,) as well as the renal vasculature (a,), and this innervation is largely responsible for sympathetic-mediated reductions in RBF (see later discussion). «,-Adrenergic receptors enhance sodium reabsorption in proximal tubules, whereas a, receptors decrease sodium reabsorption and promote ®@ water excretion. Dopamine and fenoldopam dilate afferent and efferent arterioles via D,-receptor activation. Unlike dopamine, fenoldopam is selective for the D,-receptor. Although these agents are commonly administered for "renal preservation" during catecholamine infusions, there is no clinical evidence that they are effective. Activation of Dj-receptors on presynaptic postganglionic sympathetic neurons can also vasodilate arterioles through inhibition of norepinephrine secretion (negative feedback). Dopamine is formed extraneuronally in the proximal tubule cells from circulating L-3,4dihydroxyphenylalanine (L-dopa) and is released into the tubule where it can bind dopaminergic receptors to reduce proximal reabsorption of Na*.

Acute kidney injury (AKI) is a common and underappreciated perioperative problem, occurring in 1% to 5% of all hospitalized patients, and it is a major contributor to increased hospital length of stay, markedly increasing morbidity, mortality, and cost of care. Patients may develop AKI and kidney failure secondary to intrinsic kidney disease (Table 30—2), and risk factors for AKI in the perioperative setting include preexisting renal impairment, diabetes mellitus, cardiovascular disease, hypovolemia, and use of potentially nephrotoxic medication by elderly patients. The risk index in Table 30—3 identifies

Vascular Effects Hemodynamic effects Acute kidney failure (eg, in elderly patients and those taking nonsteroidal antiinflammatory drugs [NSAIDs]) Contrast agent-induced (producing renal vasoconstriction and avid sodium retention) Hepatorenal syndrome Cirrhosis (producing intense renal vasoconstriction and sodium retention) Impaired renal perfusion and autoregulation Angiotensin-converting enzyme inhibitors, NSAIDs plus Atherosclerotic renal vascular disease or hypovolemia Abdominal compartment syndrome Postoperative abdominal exploration Tense ascites Atheroembolism (“cholesterol embolism”) Angiography Anticoagulation Thrombolysis Renal embolism Endocarditis Cardiac thrombus Renal vein thrombosis Malignancy Preexisting nephritis syndrome, Renal Parenchymal Effects = Glomerular diseases Rapidly progressive glomerulonephritis (systemic vasculitis, Goodpasture disease, systemic lupus erythematosus, other forms of glomerulonephritis) Hemolytic uremic syndrome Cryoglobulinemia Malignant hypertension Untreated primary (“essential”) hypertension Chronic glomerulonephritis Acute tubular necrosis Surgery (general, cardiac, vascular) Obstetric complications Sepsis Acute heart failure Burns Rhabdomyolysis Post-crush injury Drug overdose Status epilepticus Osmotic damage to proximal tubular cells Sucrose-containing intravenous immunoglobulin solutions Acute pyelonephritis Infection (eg, in patients with diabetes and partial obstruction from papillary necrosis) Myeloma
'Data from Armitage AJ, Tomson C. Acute renal failure. Medicine. 2003 June 1;31(6):43-48.
30-3 Acute kidney injury risk index for patients undergoing
TABLE general surgery.)2

+ Age =56y
+ Male sex
+ Active congestive heart failure
« Ascites
+ Hypertension
+ Emergency surgery
+ Intraperitoneal surgery
+ Renal insufficiency—mild or moderate'
+ Diabetes mellitus—oral or insulin therapy
'Reproduced with permission from Kheterpal S, Tremper KK, Heung M, et al. Development and validation of an acute kidney injury risk index for patients undergoing general surgery. Results from a national data set. Anesthesiology. 2009 Mar;110(3):505-515.
*Risk Index classification is based on the number of risk factors present: class | (0-2 risk factors), class lI! (3 risk factors), class Ill (4 risk factors), class IV (5 risk factors), class V (26 risk factors).
3Preoperative serum creatinine >1.2 mg/dL.
Clinical studies attempting to define the effects of anesthetic agents on renal function are complicated and difficult. However, several conclusions can be stated:
@ 1. Reversible decreases in RBF, GFR, urinary flow, and sodium excretion occur during both neuraxial and general anesthesia.
2. Such changes are usually less pronounced during neuraxial anesthesia.
3. Most of these changes are indirect and are mediated by autonomic and hormonal responses to surgery and anesthesia.
4. AKT is less likely to occur when an adequate intravascular volume and a normal blood pressure are maintained.
5. There is no evidence that currently utilized vapor anesthetic agents cause AKI in humans. However, studies have reported that compound A, a breakdown product of sevoflurane, produces renal toxicity when sevoflurane is administered with reduced fresh gas flow rates in laboratory animals.

Most inhalation and intravenous anesthetics produce concentration-dependent decreases in systemic blood pressure via cardiac depression and vasodilation. Depending on the level of sympathetic blockade, spinal or epidural anesthesia may cause a drop in systemic blood pressure secondary to decreased cardiac output as a result of decreased sympathetic tone. This leads to increased venous pooling of blood and decreased systemic vascular resistance, decreased heart rate and contractility, and decreased cardiac output. Decreases in blood pressure below the limits of autoregulation reduce RBF, GFR, urinary flow, and sodium excretion, and this adverse impact on renal function can be reversed by administration of pressor agents and intravenous fluids.

Increased sympathetic tone commonly occurs in the perioperative period as a result of anxiety, pain, light anesthesia, and surgical stimulation. Heightened sympathetic activity increases renal vascular resistance and activates several hormonal systems, reducing RBF, GFR, and urine output.

Endocrine changes during sedation and general anesthesia are a component of the stress response induced by factors that may include anxiety, pain, surgical stimulation, circulatory depression, hypoxia, acidosis, and hypothermia. Increases in epinephrine and norepinephrine, renin, angiotensin II, aldosterone, ADH, adrenocorticotropic hormone, and cortisol are common. Catecholamines, ADH, and angiotensin II all reduce RBF by inducing renal arterial constriction. Aldosterone enhances sodium reabsorption in the distal tubule and collecting tubule, resulting in sodium retention and expansion of the extracellular fluid compartment. Nonosmotic release of ADH also favors water retention and @ may result in hyponatremia. The endocrine response to surgery and anesthesia is at least partly responsible for the transient fluid retention often seen postoperatively in many patients.

The direct effects of anesthetics on renal function are minor compared with the secondary effects described earlier.

Halothane, isoflurane, sevoflurane, and desflurane @ decrease renal vascular resistance. As previously noted, compound A, a breakdown product of sevoflurane, causes acute kidney injury in laboratory animals. Low fresh gas flow rates promote its accumulation in the anesthesia machine breathing circuit. No clinical study has detected kidney injury in humans as a consequence of sevoflurane anesthesia; nonetheless, some authorities recommend a fresh gas flow of at least 2 L/min with sevoflurane to minimize the risk of this theoretical problem, especially when anesthetizing laboratory rats!

Opioids and propofol exhibit minor, if any, effects on the kidney when used alone. Ketamine minimally affects kidney function and may, relative to other anesthetic agents, preserve kidney function during hemorrhagic hypovolemia. Agents with a-adrenergic blocking activity may prevent catecholamine-induced redistribution of RBF. Drugs with antidopaminergic activity—such as metoclopramide, phenothiazines, and droperidol—may impair the renal response to dopamine. Inhibition of prostaglandin synthesis by NSAIDs such as ketorolac prevents renal production of vasodilatory prostaglandins in patients with high levels of angiotensin II and norepinephrine; attenuation of prostaglandin synthesis in this setting may promote AKI. ACE inhibitors block the protective effects of angiotensin II and may result in reductions in GFR during anesthesia. The effects of intravenous fluids on kidney function are reviewed in Chapter 31.

Many medications, including radiocontrast agents, used in the perioperative period can adversely affect kidney function, especially in the setting of preexisting renal dysfunction (Table 30-4). Mechanisms of injury include vasoconstriction, direct tubular injury, drug-induced immunological and inflammatory responses, and renal microvascular or tubular obstruction. Radiocontrast agents are probably the most common cause of AKI in the acute care setting. In addition to intravenous hydration, pretreatment with Nacetylcysteine (600 mg orally every 12 h in four doses beginning prior to contrast administration) has been shown to decrease the risk of radiocontrast agent—induced AKI in patients with preexisting renal dysfunction. NAcetylcysteine's protective action may be due to free radical scavenging or sulfhydryl donor reducing properties. Fenoldopam, mannitol, loop diuretics, and low-dose dopamine infusion do not help maintain renal function or confer protection against AKI, and N-acetylcysteine has not been shown to be protective in the perioperative setting except in patients who receive radiocontrast dyes.

Decreased renal perfusion, Drug or Toxin = Nonsteroidal antiinflammatory drugs (NSAIDs), angiotensin-converting enzyme inhibitors, radiocontrast agents, amphotericin B, cyclosporine, tacrolimus. Direct tubular injury, Drug or Toxin = Aminoglycosides, radiocontrast agents, amphotericin B, methotrexate, cisplatin, foscarnet, pentamidine, heavy metals, myoglobin, hemoglobin, intravenous immunoglobulin, HIV protease inhibitors. Intratubular obstruction, Drug or Toxin = Radiocontrast agents, methotrexate, acyclovir, sulfonamides, ethylene glycol, uric acid, cocaine, lovastatin. Immunological- Inflammatory, Drug or Toxin = _ Penicillin, cephalosporins, allopurinol, NSAIDs, sulfonamides, diuretics, rifampin, ciprofloxacin, cimetidine, proton pump inhibitors, tetracycline, phenytoin
'Reproduced with permission from Anderson RJ, Barry DW. Clinical and laboratory diagnosis of acute renal failure. Best Pract Res Clin Anaesthesioll, 2004 Mar;18(1):1-20,

In addition to the physiological changes associated with the neuroendocrine stress response to surgery, certain surgical procedures can significantly alter @
kidney physiology. Pneumoperitoneum produced during laparoscopy creates an abdominal compartment syndrome-—like state. The increase in intraabdominal pressure often produces oliguria or anuria that is proportional to insufflation pressure. Mechanisms include vena cava and renal vein compression; kidney parenchymal compression; decreased cardiac output; and increases in plasma levels of renin, aldosterone, and ADH. Abdominal compartment syndrome can also be produced by a number of comorbid problems, with similar adverse impact on kidney function via the same mechanisms (Table 30—5; see Chapters 31 and 39).

Acute respiratory failure, especially mechanical ventilation with high mean airway pressure (ie, high positive end-expiratory pressure)
Abdominal surgery with primary fascial or tight closure Major trauma/burns
Prone positioning or head of bed >30°
High BMI, central obesity
Abdominal wall edema

Gastroparesis lleus
Colonic pseudo-obstruction
Bowel obstruction

Hemoperitoneum/pneumoperitoneum
Ascites (from any mechanism)
Space-occupying mass (ie, malignancy)
Intraabdominal abscess or other infection
Peritoneal dialysis

Acidosis (pH <7.2)
Hypotension
Hypothermia (core temperature <33°)
Polytransfusion (>10 units of blood in 24 h)
Coagulopathy (INR > 1.5, platelets <55,000, PTT
> 2 times normal)
Massive fluid resuscitation (>5 L/24 h)
Pancreatitis
Oliguria
Sepsis
Damage control laparotomy
'Reproduced with permission from Patel DM, Connor Jr, MJ. Intraabdominal hypertension and abdominal compartment syndrome: An underappreciated cause of acute kidney injury. Adv Chronic Kidney Dis. 2016 May;23(3):160-166.
*BMI, body mass index; INR, international normalized ratio; PTT, partial thromboplastin time.
Other surgical procedures that can impair kidney function and may also increase the risk of AKI include cardiopulmonary bypass (see Chapter 22), cross-clamping of the aorta (see Chapter 22), and dissection near the renal arteries (see Chapter 32). The potential effects of neurosurgical procedures on ADH physiology are discussed in Chapters 27 and 49.

Diuretics increase urinary output by decreasing reabsorption of Na* and water. Although classified according to their mechanism of action, many diuretics have more than one such mechanism; hence this classification system is imperfect. Only major mechanisms will be reviewed here.
The majority of diuretics exert their action on the luminal cell membrane from within the renal tubules. Because nearly all diuretics are highly protein bound, relatively little of the free drug enters the tubules by filtration. Most diuretics must therefore be secreted by the proximal tubule (usually via the organic anion pump) to exert their action. Impaired delivery into the renal tubules accounts for resistance to diuretics in patients with decreased kidney function.
OSMOTIC DIURETICS (MANNITOL)
Osmotically active diuretics are filtered at the glomerulus and undergo limited or no reabsorption in the proximal tubule. Their presence in the proximal tubule limits passive water reabsorption that normally follows active sodium reabsorption. Although their major effect is to increase water excretion, in large doses, osmotically active diuretics also increase electrolyte excretion. The same mechanism also impairs water and solute reabsorption in the loop of Henle. Mannitol is a six-carbon sugar that is the most commonly used osmotic diuretic. It also increases RBF in addition to its diuretic effect, which can promote washing out some of the medullary hypertonicity and thus interfere with renal concentrating ability. Mannitol activates intrarenal synthesis of vasodilating prostaglandins and may be a free radical scavenger.

Many clinicians continue to administer mannitol for kidney protection and, less frequently, to convert oliguric acute kidney failure to nonoliguric kidney failure, with the goal of lowering associated morbidity and mortality. However, there is no clinical evidence that such use of mannitol provides kidney protection, lessens the severity of AKI, or lessens the morbidity or mortality associated with AKI when compared with correction of hypovolemia and preservation of adequate kidney perfusion alone. In addition, high-dose mannitol can be nephrotoxic, especially in patients with impaired kidney function.

Mannitol will augment urinary output in the setting of hypovolemia but will have little effect in the presence of severe glomerular or tubular injury. The optimal initial approach to evaluation of acute oliguria is to correct hypovolemia and optimize cardiac output and kidney perfusion.
C. Acute Reduction of Intracranial Pressure & Cerebral Edema See Chapter 27.
D. Acute Reduction of Intraocular Pressure in the Perioperative Period
See Chapter 36.

The intravenous dose for mannitol is 0.25 to 1 g/kg of ideal body weight.

Mannitol solutions are hypertonic and acutely raise plasma and extracellular osmolality. A rapid intracellular to extracellular shift of water can transiently increase intravascular volume and precipitate cardiac decompensation and pulmonary edema in patients with limited cardiac reserve. Transient hyponatremia and reductions in hemoglobin concentration are also common and represent acute hemodilution resulting from rapid movement of water out of cells; a small and transient increase in plasma potassium concentration may also be observed. It is also important to note that the initial hyponatremia does not represent hypoosmolality but reflects the presence of mannitol (see Chapter 49). If fluid and electrolyte losses are not replaced following diuresis, mannitol administration can result in hypovolemia, hypokalemia, and hypernatremia. The hypernatremia occurs because water is lost in excess of sodium. As previously noted, high-dose mannitol can be nephrotoxic, especially in patients with impaired kidney function.

The loop diuretics include furosemide (Lasix), bumetanide (Bumex), ethacrynic acid (Edecrin), and torsemide (Demadex). All loop diuretics inhibit Na* and Clreabsorption in the thick ascending limb. Sodium reabsorption at that site requires that all four sites on the Na*—K*—2CI luminal carrier protein be occupied. Loop diuretics compete with CI for its binding site on the carrier protein (see Figure 30—4). With a maximal effect, they can promote excretion of 15% to 20% of the filtered sodium load. Both urinary concentrating and urinary diluting capacities are impaired. The large amounts of Na* and Cl presented to the distal nephron overwhelm its limited reabsorptive capability. The resulting urine remains hypotonic due to rapid urinary flow rates that prevent equilibration with the hypertonic renal medulla and due to interference with the action of ADH on the collecting tubules. A marked increase in diuresis may occur when a loop diuretic is combined with a thiazide diuretic, especially metolazone (Mykrox, Zaroxolyn, Zytanix).
Loop diuretics also increase urinary calcium and magnesium excretion. Ethacrynic acid is the only loop diuretic that is not a sulfonamide derivative, and thus may be the diuretic of choice in patients allergic to sulfonamide drugs. Torsemide may have an antihypertensive action independent of its diuretic effect.

These disorders include heart failure, cirrhosis, nephrotic syndrome, and renal insufficiency. When given intravenously, these loop diuretics can rapidly reverse cardiac and pulmonary manifestations of fluid overload.

Loop diuretics may be used as adjuncts to other hypotensive agents, particularly when thiazides alone are ineffective (see later discussion).

The optimal initial approach to acute oliguria is to correct hypovolemia and optimize cardiac output and renal perfusion.

As with mannitol, discussed earlier, many clinicians continue to administer loop diuretics for kidney protection and to convert oliguric acute kidney failure to nonoliguric kidney failure, despite lack of evidence that such use provides kidney protection, lessens the severity of AKI, or lessens the morbidity or mortality associated with AKI, when compared with correction of hypovolemia and preservation of adequate kidney perfusion alone.

The intravenous doses are furosemide, 10 to 100 mg; bumetanide, 0.5 to 1 mg; ethacrynic acid, 50 to 100 mg; and torsemide 10 to 100 mg.

Increased delivery of Na* to the distal and collecting tubules increases K* and H* secretion at those sites and thus produce hypokalemia and metabolic alkalosis. Marked Na' losses will also lead to hypovolemia and prerenal azotemia; secondary hyperaldosteronism often accentuates the hypokalemia and metabolic alkalosis. Urinary calcium and magnesium loss promoted by loop diuretics may result in hypocalcemia or hypomagnesemia, or both. Hypercalciuria can promote urolithiasis. Hyperuricemia may result from increased urate reabsorption and from competitive inhibition of urate secretion in the proximal tubule. Reversible and irreversible hearing loss has been reported with loop diuretics, especially furosemide and ethacrynic acid.

This group of agents includes thiazides, containing a benzothiadiazine molecular structure, and also thiazide-like drugs with similar actions but without the benzothiadiazine structure, including chlorthalidone (Thalitone), quinethazone (Hydromox), metolazone, and indapamide (Lozol). These diuretics act at the distal tubule, including the connecting segment, and inhibition of sodium reabsorption at this site impairs diluting but not concentrating ability. They compete for the Cl site on the luminal Na*—CI carrier protein. When given alone, thiazide and thiazide-like diuretics increase Na* excretion to only 3% to 5% of the filtered load because of enhanced compensatory Na' reabsorption in the collecting tubules. They also possess carbonic anhydrase—inhibiting activity in the proximal tubule, which is usually masked by sodium reabsorption in the loop of Henle and which is probably responsible for the marked diuresis often seen when they are combined with loop diuretics. In contrast to their effects on sodium excretion, thiazide and thiazide-like diuretics augment Ca** reabsorption in the distal tubule. Indapamide has some vasodilating properties and is the only thiazide or thiazide-like diuretic with significant hepatic excretion.

Thiazide and thiazide-like diuretics are often selected as first-line agents in the treatment of hypertension (see Chapter 21), and they have been shown to improve long-term outcomes in this disorder.

These drugs are used to treat mild to moderate edema and congestive heart failure related to mild to moderate sodium overload.

Thiazide and thiazide-like diuretics are often used to decrease calcium excretion in patients who form calcium-containing kidney stones.

The efficacy of these agents in this disorder is based upon their ability to impair diluting capacity and increase urine osmolality (see Chapter 49).

Although thiazide and thiazide-like diuretics deliver less sodium to the collecting tubules than loop diuretics, the increase in sodium excretion is enough to enhance K* secretion and frequently results in hypokalemia. Enhanced H* secretion can also occur, resulting in metabolic alkalosis. Impairment of renal diluting capacity may produce hyponatremia. Hyperuricemia, hyperglycemia, hypercalcemia, and hyperlipidemia may also be seen.

These are weak diuretic agents and characteristically do not increase potassium excretion. Potassium-sparing diuretics inhibit Na* reabsorption in the collecting tubules and therefore can maximally excrete only 1% to 2% of the filtered Na* load. They are usually used in conjunction with more potent diuretics for their potassium-sparing effect.
1. Aldosterone Antagonists (Spironolactone & Eplerenone)
Spironolactone (Aldactone) and eplerenone (Inspra) are direct antagonists of collecting tubule aldosterone receptor. They inhibit aldosterone-mediated Na* reabsorption and K* secretion. Both agents have been shown to improve survival in patients with chronic heart failure.

These agents may be used as adjuvants in the treatment of refractory edematous states associated with secondary hyperaldosteronism (see Chapter 49). Spironolactone is particularly effective in patients with ascites related to advanced liver disease. Eplerenone is commonly used in the management of chronic heart failure, where it may improve outcomes.

These agents can result in hyperkalemia in patients with high potassium intake or renal insufficiency and in those receiving B-blockers or ACE inhibitors. Metabolic acidosis may also be seen. Eplerenone lacks spironolactone's side effects of gynecomastia and sexual dysfunction.

Triamterene (Dyrenium) and amiloride (Midamor) inhibit Na* reabsorption and K* secretion by decreasing the number of open sodium channels in the luminal membrane of collecting tubules and are not dependent upon aldosterone activity Amiloride may also inhibit Na*—K*-ATPase activity in the collecting tubule.

In patients with hypertension, these agents are often combined with a thiazide or similar diuretic to minimize hypokalemia produced by the other agent. They have been added to more potent loop diuretics in congestive heart failure patients with marked potassium wasting. These agents are only given orally.
Amiloride and triamterene can cause hyperkalemia and metabolic acidosis similar to that seen with spironolactone (see above). Both can also cause nausea, vomiting, and diarrhea. Amiloride is generally associated with fewer side effects, but paresthesias, depression, muscle weakness, and cramping may occasionally be seen. Triamterene on rare occasions has resulted in renal stones and is potentially nephrotoxic, particularly when combined with nonsteroidal antiinflammatory agents.

Carbonic anhydrase inhibitors such as acetazolamide (Diamox) interfere with Na' reabsorption and H* secretion in proximal tubules. They are weak diuretics because the former effect is limited by the reabsorptive capacities of more distal segments of nephrons. Nonetheless, these agents significantly interfere with H* secretion in the proximal tubule and impair HCO3" reabsorption.

Carbonic anhydrase inhibitors often potentiate the effects of other diuretics.

Alkalinization enhances urinary excretion of weakly acidic compounds such as uric acid.

Inhibition of carbonic anhydrase in the ciliary processes reduces the formation of aqueous humor and, secondarily, intraocular pressure. Carbonic anhydrase inhibitors, including oral or intravenous acetazolamide, oral methazolamide (Neptazane), and ophthalmic topical brinzolamide (Azopt) and dorzolamide (Trusopt), are often used to treat glaucoma.

Carbonic anhydrase inhibitors generally produce only a mild hyperchloremic metabolic acidosis because of an apparently limited effect on the distal nephron. Large doses of acetazolamide have been reported to cause drowsiness, paresthesias, and confusion. Alkalinization of the urine can interfere with the excretion of amine drugs, such as quinidine. Acetazolamide is frequently used for prophylaxis against altitude sickness.

These agents may increase GFR by elevating cardiac output or arterial blood pressure, thereby increasing RBF. Drugs in this category are not primarily classified as diuretics because of their other major actions. They include methylxanthines (theophylline), cardiac glycosides (digitalis), fenoldopam (Corlopam), inotropes (dopamine, dobutamine), and intravenous crystalloid and colloid infusions. Methylxanthines also appear to decrease sodium reabsorption in both the proximal and distal renal tubules.

A 58-year-old woman is undergoing radical hysterectomy under general anesthesia. She was in good health prior to the diagnosis of uterine carcinoma. An indwelling urinary catheter is placed following induction of general anesthesia. Total urinary output was 60 mL for the first 2 h of surgery. After the third hour of surgery, only 5 mL of urine is noted in the drainage reservoir.

Decreases in urinary output during anesthesia are very common. Although decreases may be expected owing to the physiological effects of surgery and anesthesia, a urinary output of less than 20 mL/h in adults generally requires evaluation.
What issues should be addressed?
The following questions should be answered:
1. Is there a problem with the urinary catheter and drainage system?
2. Are hemodynamic parameters compatible with adequate renal function?
3. Could the decrease in urinary output be directly related to surgical manipulations?
How can the urinary catheter and drainage system be evaluated intraoperatively?
Incorrect catheter placement is not uncommon and should be suspected if there has been a total absence of urine flow since the time of catheter insertion. The catheter may be inadvertently placed and inflated in the urethra in men or the vagina in women. Catheter displacement, kinking, obstruction, or disconnection from the reservoir tubing can all present with features similar to this case, with complete or near-complete cessation of urinary flow. The diagnosis of such mechanical problems requires retracing and inspecting the path of urine (often under the surgical drapes) from the catheter to the collection reservoir. Obstruction of the catheter can be confirmed by an inability to irrigate the bladder with saline through the catheter.

Decreased urinary output during surgery is most commonly the result of hormonal and hemodynamic changes. In many instances, a decrease in intravascular volume (hypovolemia), cardiac output, or mean arterial blood pressure is responsible. Redistribution of renal blood flow from the renal cortex to the medulla may also play a role.
Intravascular volume depletion can rapidly develop when intravenous fluid replacement does not match intraoperative blood loss and insensible fluid loss. Oliguria requires careful assessment of intravascular volume to exclude hypovolemia. An increase in urinary output following an intravenous fluid bolus is highly suggestive of hypovolemia. In contrast, oliguria in patients with a history of congestive heart failure may require inotropes, vasodilators, or diuretics. Intravascular volume status is often difficult to optimize, and goal-directed hemodynamic and fluid therapy utilizing arterial pulse contour analysis (eg, LIDCO Rapid, Vigileo FloTrak), esophageal Doppler, or transesophageal echocardiography should be considered when accurate determination of hemodynamic and fluid volume
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with central
venous access
procedures and
with pulmonary
artery catheter
placement and
use.
When mean arterial blood pressure drops below the lower limit of renal autoregulation (80 mm Hg), urinary flow may become blood pressure dependent. The latter may be particularly true in patients with chronic systemic hypertension, in whom renal autoregulation has shifted to a higher range of mean arterial blood pressure. Reductions in anesthetic depth, intravenous fluid boluses, or the administration of a vasopressor or inotrope will usually increase blood pressure and urinary output in such instances.

Otherwise normal patients may exhibit decreased urinary output in spite of normal intravascular volume, cardiac output, and mean arterial blood pressure. A small dose of a loop diuretic (eg, furosemide, 5-10 mg) usually restores urinary flow in such instances but is not necessary since such therapy does not convey protection against acute kidney injury.

In addition to the neuroendocrine response to surgery, mechanical factors related to the surgery itself can alter urinary output. This is particularly true during pelvic surgery, when compression of the bladder by retractors, unintentional cystotomy, and ligation or severing of one or both ureters can dramatically affect urinary output. Retractor compression combined with a head-down (Trendelenburg) position commonly impedes emptying of the bladder. Excessive pressure on the bladder will often produce hematuria. Excessive insufflation pressure during laparoscopy may result in abdominal compartment syndrome, with decreased or absent urine output, as previously described.
When mechanical problems with the urinary catheter drainage system and hemodynamic factors are excluded, a surgical explanation should be sought. The surgeon should be notified so that the position of the retractors can be checked, the ureters identified, and their path retraced in the operative area. Intravenous methylene blue or indigo carmine dyes (excreted in urine) are useful in identifying the site of an unintentional cystotomy or the end of a severed ureter. Note that the appearance of dye in the urinary drainage reservoir does not exclude unilateral ligation of one ureter. Methylene blue and, to a much lesser extent, indigo carmine, can transiently give falsely low pulse oximeter readings (see Chapter 6). In the case of laparoscopic surgery, the surgeon should be asked to verify and to minimize insufflation pressure.

After the integrity of the urinary catheter and drainage system was checked, 1 L of Plasma-Lyte along with 500 mL of 5% albumin and 10 mg of furosemide were administered intravenously but failed to significantly increase urinary output. Indigo carmine was given intravenously, and the proximal end of a severed left ureter was subsequently identified. A urologist was called and the ureter was reanastomosed.

@ The utility of a single serum creatinine measurement as an indicator of glomerular filtration rate (GFR) is limited in critical illness: The rate of creatinine production, and its volume of distribution, may be abnormal in the critically ill patient, and the serum creatinine concentration often does not accurately reflect GFR in the physiological disequilibrium of acute kidney injury (AKI).
@® Creatinine clearance measurement is the most accurate method available for clinically assessing overall kidney function.
© The accumulation of morphine and meperidine metabolites has been reported to prolong respiratory depression in patients with kidney failure.
@ Succinylcholine can be safely used in patients with kidney failure in the absence of hyperkalemia at the time of induction.
® Extracellular fluid overload from sodium retention, in association with increased cardiac demand imposed by anemia and hypertension, makes patients with end-stage kidney disease particularly prone to congestive heart failure and pulmonary edema.
@ Delayed gastric emptying secondary to kidney disease—associated autonomic neuropathy may predispose patients to perioperative aspiration.
@ Controlled ventilation should be considered for patients with kidney failure under general anesthesia. Inadequate spontaneous or assisted ventilation with progressive hypercarbia under anesthesia can result in respiratory acidosis that may exacerbate preexisting acidemia, lead to potentially severe circulatory depression, and dangerously increase
serum potassium concentration.
© Correct anesthetic management of patients with renal insufficiency is as critical as management of those with frank kidney failure, especially during procedures associated with a relatively high incidence of postoperative kidney failure, such as cardiac and aortic reconstructive surgery.
© Intravascular volume depletion, sepsis, obstructive jaundice, crush injuries, and renal toxins, such as radiocontrast agents, certain antibiotics, angiotensin-converting enzyme inhibitors, and nonsteroidal antiinflammatory drugs, are major risk factors for acute deterioration in kidney function.
@ Kidney protection with adequate hydration and maintenance of renal blood flow is especially important for patients at high risk for AKI and kidney failure undergoing cardiac, major aortic reconstructive, and other surgical procedures associated with significant physiological trespass. The use of mannitol, low-dose dopamine infusion, loop diuretics, or fenoldopam for kidney protection is controversial and without proof of efficacy.

Acute kidney injury (AKI) is a common problem, with an incidence of up to 5% in all hospitalized patients and up to 8% in critical illness. Postoperative AKI may occur in 1% or more of general surgery patients, and up to 30% of patients undergoing cardiothoracic and vascular procedures. Perioperative AKI is a markedly underappreciated problem that greatly increases perioperative morbidity, mortality, and costs. It is a systemic disorder that can include fluid and electrolyte derangements, respiratory failure, major cardiovascular events, weakened immunocompetence leading to infection and sepsis, altered mental status, hepatic dysfunction, and gastrointestinal hemorrhage. It is also a major cause of chronic kidney disease. Preoperative risk factors for perioperative AKI include preexisting kidney disease, hypertension, diabetes mellitus, liver disease, sepsis, trauma, hypovolemia, multiple myeloma, and age greater than 55 years. The risk of perioperative AKI is also increased by exposure to nephrotoxic agents such as nonsteroidal antiinflammatory drugs (NSAIDs), radiocontrast agents, and antibiotics (see Table 30-4). The clinician must possess a thorough understanding of the risks of AKI, its differential diagnosis, and its evaluation strategy (Figure 31-1).
AKI
FIGURE 31-1 Differential diagnosis and evaluation of acute kidney injury (AKI). ANA, antinuclear antibody; ANCA, antineutrophil cytoplasmic antibody; Anti-ds-DNA, anti—double stranded DNA; Anti-GMB, anti-glomerular basement membrane; C3, complement component 3; C4, complement component 4; CK, creatine kinase; CK-MB, creatine kinase MB fraction; ENA, extractable nuclear antigen; HIV, human immunodeficiency virus; HUS, hemolytic uremic syndrome; LDH, lactate dehydrogenase; NT-proBNP, Nterminal pro-brain natriuretic peptide; TTP, thrombotic thrombocytopenic purpura. (Reproduced with permission from Ostermann M, Joannidis M. Acute kidney injury 2016: Diagnosis and diagnostic workup. Crit Care. 2016 Sep 27;20(1):299.)

Impaired kidney function may be due to glomerular dysfunction, tubular dysfunction, or urinary tract obstruction. Accurate clinical assessment of kidney function is often difficult and relies heavily on laboratory determinations of glomerular filtration rate (GFR), including creatinine clearance, and other evaluations (Tables 31—1 and 31—2). Even small postoperative increases in serum creatinine are associated with increased morbidity and mortality, although many factors may confound its measurement (Figure 31—2). Systems employed in defining and staging the degree of kidney dysfunction include the Acute Dialysis Quality Initiative Risk, Injury, Failure, Loss, End-Stage (RIFLE) criteria and the Acute Kidney Injury Network (AKIN) staging system. These systems were merged into the Kidney Disease Improving Global Outcomes (KDIGO)
classification (Table 31—3). Thus, the traditional diagnosis of AKI, based upon serum creatinine and urine output, has been refined into an increase of serum creatinine of 0.3 mg/dL or more within 48 h or a 1.5-fold or greater increase in baseline within 7 days. Since AKI is a systemic disorder, it is important to recall that the kidney excretory function assessed via serum creatinine and urine output ignores endocrine, metabolic, and immunological kidney functions. A great deal of research is currently evaluating plasma and urine biomarkers associated with AKI, such as cystatin C, neutrophil gelatinase—associated lipocalin, interleukin18, and kidney injury molecule-1, and several are now commercially available (Figure 31-3). It is likely that biomarkers will play an increasingly prominent role in the near future for diagnosis, staging, and prognostic assessment of AKI.

TABLE 31-1 Severity of kidney injury according to glomerular function.

Normal, Creatinine Clearance (mL/min) = 100-120. Decreased kidney reserve, Creatinine Clearance (mL/min) = 60-100. Mild kidney impairment, Creatinine Clearance (mL/min) = 40-60. Moderate kidney insufficiency, Creatinine Clearance (mL/min) = 25-40. Kidney failure, Creatinine Clearance (mL/min) = <25. End-stage kidney disease!, Creatinine Clearance (mL/min) = <10
"This term applies to patients with chronic kidney failure.
TABLE 31-2 Laboratory evaluation of kidney impairment.!2
Serum creatinine, 1 = Easily available Low cost. Serum creatinine, 2 = Not renal-specific Late marker after renal injury Serum levels confounded by muscle mass, drugs, laboratory technique, fluid status. Blood urea nitrogen, 1 = Easily available Low cost. Blood urea nitrogen, 2 = Not renal-specific Serum levels confounded by liver disease, gastrointestinal bleed, and hypovolemia. FeNa, 1 = Easily available Low cost. FeNa, 2 = Difficult to interpret in patients with chronic kidney disease Confounded by diuretic treatment. Urine microscopy, 1 = Noninvasive Low cost Can provide very valuable information if done properly, (i.e., red cell casts in case of glomerulonephritis). Urine microscopy, 2 = Operator-dependent Requires training and experience. Renal histology, 1 = Can provide very valuable information about cause of AKI and degree of chronic changes. Renal histology, 2 = Invasive Requires competency Bleeding complications. Novel AK! biomarkers, 1 = Opportunity to diagnose AKI before creatinine rise May provide additional diagnostic and prognostic information. Novel AK! biomarkers, 2 = Costs Significant confounders. Techniques to measure real-time GFR, 1 = Opportunity to monitor GFR in real time and to diagnose AKI early. Techniques to measure real-time GFR, 2 = Costs Not yet available in clinical practice Requires training and experience

'Reproduced with permission from Ostermann M. Diagnosis of acute kidney injury: Kidney Disease Improving Global Outcomes criteria and beyond. Curr Opin Crit, 2014 Dec;20(6):581-587.
2AKI, acute kidney injury; FeNa, fractional excretion of sodium; GFR, glomerular filtration rate.
Acute rise
in creatinine:
*
Dietary creatine
intake
—ameat meal
* Increased creatinine generation — thabdomyolysis
* Decreased glomerular filtration AKI
+ Reduced tubular secretion — trimethoprim and cimetidine

- + Jaffe assay interference
—
hyperglycemia and
DKA
— delayed centrifugation
— other: hemolysis; high total protein
+ Enzymatic assay interference
— high total protein, lidocaine

- * Reduced creatinine generation — sepsis
*
Increased volume
of distribution
—
edematous states*
- —acute fluid overload
FIGURE 31-2 Factors affecting serum creatinine interpretation in acute kidney injury. *Edematous states: cirrhosis, nephrotic syndrome, heart failure. DKA, diabetic ketoacidosis; eGFR, estimated glomerular filtration rate. (Reproduced with permission from Thomas MD, Blaine C, Dawnay A, et al. The definition of acute kidney injury and its use
Factors having
~
affecting baseline
generate creatinine
Chronic
‘elevation’
*
Increased effect
on creatinine
and rise
to during
of creatinine
—
muscular eGFR
ability creatinine:
generation body
habitus
—
Afro-Caribbean
Decreased
*
ethnicity glomerular
filtration chronic
kidney disease

*
Jaffe assay
— hyperbilirubinemia
* Enzymatic interference
— hyperbilirubinaemia, hemolysis
Chronic "reduction" in creatinine:
* Low dietary protein (cooked meat) intake
* Reduced creatinine generation with lower muscle mass
-old age and female sex
—muscle-wasting conditions
—
amputation
- — malnutrition and critical illness
AKI
a
chronic

RRT irrespective of the stage at the time of RRT. Stage Ill or RIFLE failure, 3 = Rise in serum creatinine three times from baseline, or increase in serum creatinine to 2353.6 pmol/L, or initiation of RRT irrespective of serum creatinine. Stage Ill or RIFLE failure, 4 = <0.3 mL/kg/h for 24h or more, or anuria for12h. RIFLE loss, 1 = Complete loss of kidney function for >4 wk. RIFLE loss, 2 = . RIFLE loss, 3 = . RIFLE loss, 4 = . End-stage kidney disease, 1 = End-stage kidney disease for >3 mo. End-stage kidney disease, 2 = . End-stage kidney disease, 3 = . End-stage kidney disease, 4 = 
'Reproduced with permission from Ostermann M. Diagnosis of acute kidney injury: Kidney Disease Improving Global Outcomes and beyond. Curr Opin Crit Care, 2014 Dec;20{6):581-587,
*AKI, acute kidney injury; AKIN, Acute Kidney Injury Network; GFR, glomerular filtration rate; KDIGO, Kidney Disease Improving Global Outcomes; RIFLE, Risk, Injury, Failure, Loss, End-Stage; RRT, renal replacement therapy.

FIGURE 31-3 AKI biomarkers. a-GST, a-glutathione S-transferase; AAP, alanine aminopeptidase; ALP, alkaline phosphatase; y-GT, y-glutamyl transpeptidase; n-GST, n-glutathione S-transferase; HGF, hepatocyte growth factor; IGFBP-7, insulin-like growth factor binding protein 7; IL-18, inteleukin18; KIM-1, kidney injury molecule-1; L-FABP, liver fatty acid-binding protein; NAG, N-acetyl-B-p-glucosaminidase; NGAL, neutrophil gelatinase-associated lipocalin; RBP, retinol-binding protein; TIMP-2, tissue inhibitor metalloproteinase-2. (Reproduced with permission from Ostermann M, Joannidis M. Acute kidney injury 2016: Diagnosis and diagnostic workup. Crit Care. 2016 Sep 27;20(1):299.)

The primary source of urea in the body is the liver. During protein catabolism, ammonia is produced from the deamination of amino acids. Hepatic conversion of ammonia to urea prevents the buildup of toxic ammonia levels:
2NH; + CO, — H)N -CO -NH, + H,O
Blood urea nitrogen (BUN) is therefore directly related to protein catabolism and inversely related to glomerular filtration. As a result, BUN is not a reliable indicator of the GFR unless protein catabolism is normal and constant. Recall that 40% to 50% of the urea filtrate is normally reabsorbed passively by the renal tubules; hypovolemia increases this fraction.
The normal BUN concentration is 10 to 20 mg/dL. Lower values may be seen with starvation or liver disease; elevations usually result from decreases in GFR or increases in protein catabolism. The latter may be due to a high catabolic state (trauma or sepsis), degradation of blood either in the gastrointestinal tract or in a large hematoma, or a high-protein diet. BUN concentrations greater than 50 mg/dL are generally associated with impaired kidney function.

Creatine is a product of muscle metabolism that is nonenzymatically converted to creatinine. Daily creatinine production in most people is relatively constant and related to muscle mass, averaging 20 to 25 mg/kg in men and 15 to 20 mg/kg in women. Creatinine is then filtered (and to a minor extent secreted) but not reabsorbed in the kidneys. Serum creatinine concentration is therefore directly related to body muscle mass and inversely related to glomerular filtration (Figure 31-4). Because body muscle mass is usually relatively constant, serum creatinine measurements are generally reliable indices of GFR @ in the ambulatory patient. However, the utility of a single serum creatinine measurement as an indicator of GFR is limited in critical illness: The rate of creatinine production, and its volume of distribution, may be abnormal in the critically ill patient, and a single serum creatinine measurement often will not accurately reflect GFR in the physiological disequilibrium of AKI.
FIGURE 31-4 The relationship between the serum creatinine concentration and the glomerular filtration rate.
The normal serum creatinine concentration is 0.8 to 1.3 mg/dL in men and 0.6 to 1 mg/dL in women. Note from Figure 31-4 that each doubling of the serum creatinine represents a 50% reduction in GFR. As previously noted, many factors may affect serum creatinine measurement.
GFR declines with increasing age in most individuals (5% per decade after age 20), but because muscle mass also declines, the serum creatinine remains relatively normal; creatinine production may decrease to 10 mg/kg. Thus, in elderly patients, small increases in serum creatinine may represent large changes in GFR. Using age and lean body weight (in kilograms), GFR can be estimated by the following formula for men:
For women, this equation must be multiplied by 0.85 to compensate for a smaller muscle mass.
The serum creatinine concentration requires 48 to 72 h to equilibrate at a new level following acute changes in GFR.

® Creatinine clearance measurement is the most accurate method available for clinically assessing overall kidney function (actually, GFR). Although measurements are usually performed over 24 h, 2-h creatinine clearance determinations are reasonably accurate and easier to perform. Mild impairment of kidney function generally results in creatinine clearances of 40 to 60 mL/min. Clearances between 25 and 40 mL/min produce moderate kidney dysfunction and nearly always cause symptoms. Creatinine clearances less than 25 mL/min are indicative of overt kidney failure.
Later stage kidney disease leads to increased creatinine secretion in the proximal tubule. As a result, with declining kidney function the creatinine clearance progressively overestimates the true GFR. Moreover, relative preservation of GFR despite progressive kidney disease may result from compensatory hyperfiltration in the remaining nephrons and increases in glomerular filtration pressure. It is therefore important to look for other signs of deteriorating kidney function such as hypertension, proteinuria, or abnormalities in urine sediment.

Low renal tubular flow rates enhance urea reabsorption but do not affect creatinine excretion. As a result, the ratio of BUN to serum creatinine increases to more than 10:1. Decreases in tubular flow can be caused by decreased kidney perfusion or obstruction of the urinary tract. BUN:creatinine ratios greater than 15:1 are therefore seen in volume depletion and in edematous disorders associated with decreased tubular flow (eg, congestive heart failure, cirrhosis, nephrotic syndrome) as well as in obstructive uropathies. Increases in protein catabolism can also increase this ratio.

Urinalysis continues to be routinely performed for evaluating kidney function. Although its utility and cost-effectiveness for this purpose are questionable, urinalysis can be helpful in identifying some disorders of renal tubular dysfunction as well as some nonrenal disturbances. A routine urinalysis typically includes pH; specific gravity; detection and quantification of glucose, protein, and bilirubin content; and microscopic examination of the urinary sediment. Urinary pH is helpful only when arterial pH is also known. A urinary pH greater than 7.0 in the presence of systemic acidosis is suggestive of renal tubular acidosis (see Chapter 50). Specific gravity is related to urinary osmolality; 1.010 usually corresponds to 290 mOsm/kg. A specific gravity greater than 1.018 after an overnight fast is indicative of adequate renal concentrating ability. A lower specific gravity in the presence of hyperosmolality in plasma is consistent with diabetes insipidus.
Glycosuria is the result of either a reduced tubular threshold for glucose (normally 180 mg/dL) or hyperglycemia. Proteinuria detected by routine urinalysis should be evaluated by means of 24-h urine collection. Urinary protein excretions greater than 150 mg/d are significant. Elevated levels of bilirubin in the urine are seen with biliary obstruction.
Microscopic analysis of the urinary sediment detects the presence of red or white blood cells, bacteria, casts, and crystals. Red cells may be indicative of bleeding due to tumor, stones, infection, coagulopathy, or trauma (commonly, urinary catheterization). White cells and bacteria are generally associated with infection. Disease processes at the level of the nephron produce tubular casts. Crystals may be indicative of abnormalities in oxalic acid, uric acid, or cystine metabolism.

Most drugs commonly employed during anesthesia (other than volatile anesthetics) are at least partly dependent on renal excretion for elimination. In the presence of kidney impairment, dosage modifications may be required to prevent accumulation of the drug or its active metabolites. Moreover, the systemic effects of AKI can potentiate the pharmacological actions of many of these agents. This latter observation may be the result of decreased protein binding of the drug, greater brain penetration due to some breach of the blood— brain barrier, or a synergistic effect with the toxins retained in kidney failure.

The pharmacokinetics of both propofol and etomidate are minimally affected by impaired kidney function. Decreased protein binding of etomidate in patients with hypoalbuminemia may enhance its pharmacological effects.

Patients with kidney disease often exhibit increased sensitivity to barbiturates during induction, even though pharmacokinetic profiles appear to be unchanged. The mechanism appears to be an increase in free circulating barbiturate from decreased protein binding. Acidosis may also favor a more rapid entry of these agents into the brain by increasing the nonionized fraction of the drug (see Chapter 26).

Ketamine pharmacokinetics are minimally altered by kidney disease. Some active hepatic metabolites are dependent on renal excretion and can potentially accumulate in kidney failure.

Benzodiazepines undergo hepatic metabolism and conjugation prior to elimination in urine. Because they are highly protein bound, increased benzodiazepine sensitivity may be seen in patients with hypoalbuminemia. Diazepam and midazolam should be administered cautiously in the presence of kidney impairment because of a potential for the accumulation of active metabolites.

Most opioids used in anesthetic practice (morphine, meperidine, fentanyl, sufentanil, and alfentanil) are inactivated by the liver; some of these metabolites are then excreted in urine. Remifentanil pharmacokinetics are unaffected by kidney function due to rapid ester hydrolysis in blood. With the exception of morphine and meperidine, significant accumulation of active metabolites generally does not occur @ with these agents. Accumulation of morphine (morphine-6-glucuronide) and meperidine (normeperidine) metabolites may prolong respiratory depression in patients with kidney failure, and increased levels of normeperidine are associated with seizures. The pharmacokinetics of the most commonly used opioid agonist—antagonists (butorphanol, nalbuphine, and buprenorphine) are unaffected by kidney failure.

In doses used for premedication, atropine and glycopyrrolate can generally be used safely in patients with kidney impairment. Because up to 50% of these drugs and their active metabolites are normally excreted in urine, however, the potential for accumulation exists following repeated doses. Scopolamine is less dependent on renal excretion, but its central nervous system effects can be enhanced by decreased kidney function.

Most phenothiazines, such as promethazine, are metabolized to inactive compounds by the liver. Droperidol may be partly dependent on the kidneys for excretion. Although their pharmacokinetic profiles are not appreciably altered by kidney impairment, potentiation of the central depressant effects of the effects of disease occur.
phenothiazines by systemic kidney may All H,-receptor blockers are dependent on kidney excretion, and their dose must be reduced for patients with kidney disease. Proton pump inhibitor dosage does not need to be reduced for patients with kidney disease. Metoclopramide is partly excreted unchanged in urine and will accumulate in kidney failure. Although up to 50% of dolasetron is excreted in urine, no dosage adjustments are recommended for any of the 5-HT3 blockers in patients with kidney disease.

Volatile anesthetic agents are ideal for patients with kidney disease because of lack of dependence on the kidneys for elimination, ability to control blood pressure, and minimal direct effects on kidney blood flow. Although patients with mild to moderate kidney impairment do not exhibit altered uptake or distribution, accelerated induction and emergence may be seen in severely anemic patients (hemoglobin <5 g/dL) with chronic kidney failure; this observation may be explained by a decrease in the blood:gas partition coefficient or by a decrease in minimum alveolar concentration. Some clinicians avoid sevoflurane (with <2 L/min gas flows) for patients with kidney disease who undergo lengthy procedures (see Chapters 8 and 30).

Some clinicians omit entirely or limit the use of nitrous oxide (or air) to maintain an Fio, of 50% or greater in severely anemic patients with end-stage kidney disease in an attempt to increase arterial oxygen content. This may be justified in patients with hemoglobin less than 7 g/dL, in whom even a small increase in the dissolved oxygen content may represent a significant percentage of the arterial to venous oxygen difference (see Chapter 23).

® Succinylcholine can be safely used in patients with kidney failure, in the absence of hyperkalemia at the time of induction. When the serum potassium is known to be increased or is in doubt, succinylcholine should be avoided. Although decreased plasma cholinesterase levels have been reported in uremic patients following dialysis, significant prolongation of neuromuscular blockade is rarely seen.

Cisatracurium and atracurium are degraded by plasma ester hydrolysis and nonenzymatic Hofmann elimination. These agents are often the drugs of choice for muscle relaxation in patients with kidney failure, especially in clinical situations where neuromuscular function monitoring is difficult or impossible.

The elimination of vecuronium is primarily hepatic, but up to 20% of the drug is eliminated in urine. The effects of large doses of vecuronium (>0.1 mg/kg) are only modestly prolonged in patients with kidney disease. Rocuronium primarily undergoes hepatic elimination, but prolongation in patients with severe kidney disease has been reported. In general, with appropriate neuromuscular monitoring, these two agents can be used with few problems in patients with severe kidney disease.

Elimination of d-tubocurarine is dependent on both kidney and biliary excretion; 40% to 60% of a dose of curare is normally excreted in urine. Increasingly prolonged effects are observed following repeated doses in patients with decreased kidney function. Smaller doses and longer dosing intervals are therefore required for maintenance of optimal muscle relaxation.

Pancuronium is primarily dependent on renal excretion (60-90%). Although pancuronium is metabolized by the liver into less active intermediates, its elimination half-life is still primarily dependent on renal excretion (60-80%). Neuromuscular function should be closely monitored if these agents are used in patients with abnormal renal function.

Renal excretion is the principal route of elimination for edrophonium, neostigmine, and pyridostigmine. The half-lives of these agents in patients with renal impairment are therefore prolonged at least as much as any of the above relaxants, and problems with inadequate reversal of neuromuscular blockade are usually related to other factors (see Chapter 11). Thus, "recurarization" due to inadequate duration of reversal agent is unlikely. Sugammadex is a steroidal muscle relaxant encapsulator drug that, even after binding vecuronium or rocuronium, is rapidly and entirely eliminated (along with the neuromuscular blocker) in its unmetabolized form by the kidney (see Chapter 11). Early studies suggest that onset of sugammadex muscle relaxant reversal may be delayed and that the sugammadex—muscle relaxant complex may persist for several days in the plasma of patients with decreased kidney function. Because of the potential patient safety implications of prolonged sugammadex—muscle relaxant complex exposure in this situation, the use of sugammadex is not recommended at this time in patients with low creatine clearance (<30 mL/min) or on renal replacement therapy (RRT).

This syndrome is a rapid deterioration in kidney function that results in retention of nitrogenous waste products (azotemia). These substances, many of which behave as toxins, are byproducts of protein and amino acid metabolism. Impaired kidney metabolic activity may contribute to widespread organ dysfunction (see Chapter 30).
Kidney failure can be classified as prerenal, renal, and postrenal, depending on its cause(s), and the initial therapeutic approach varies accordingly (see Figure 31-1 and Table 31-4). Prerenal kidney failure results from an acute decrease in renal perfusion; intrinsic kidney failure is usually due to underlying kidney disease, kidney ischemia, or nephrotoxins; and postrenal failure is the result of urinary collecting system obstruction or disruption. Both prerenal and postrenal forms of kidney failure are readily reversible in their initial stages but with time both progress to intrinsic kidney failure. Most adult patients with kidney failure first develop oliguria. Nonoliguric patients with kidney failure (urinary outputs >400 mL/d) continue to form urine that is qualitatively poor; these patients tend to have greater preservation of GFR. Although glomerular filtration and tubular function are impaired in both cases, abnormalities tend to be less severe in nonoliguric kidney failure.

¢ Search for and correct prerenal and postrenal causes
¢ Review medications and patient-administered substances and stop any potential nephrotoxins
¢ Administer medications in doses appropriate for their clearance
* Optimize cardiac output and renal blood flow
¢ Monitor fluid intake and output; measure body weight daily
¢ Search for and treat acute complications (hyperkalemia, hyponatremia, acidosis, hyperphosphatemia, pulmonary edema)
¢ Search for and aggressively treat infections and sepsis
¢ Provide early nutritional support
¢ Provide expert supportive care (management of catheter and skin care; pressure sore and deep venous thromboembolic prophylaxis; psychological support).
TReproduced with permission from Lameire N, Van Biesen W, Vanholder R. Acute renal failure. Lancet. 2005 Jan 29-Feb 4;365(9457):417-430.
The course of intrinsic acute kidney failure varies widely, but the oliguria typically lasts for 2 weeks and is followed by a diuretic phase marked by a progressive increase in urinary output. This diuretic phase often results in very large urinary outputs and is usually absent in nonoliguric kidney failure. Kidney function improves over the course of several weeks but may not return to normal for up to 1 year, and subsequent chronic kidney disease is common. The course of prerenal and postrenal kidney failure is dependent upon promptness in diagnosis and correction of the causal condition. Diagnostic ultrasound, including point-of-care ultrasound, is increasingly used to rapidly and noninvasively evaluate possible obstructive uropathy.

The most common causes of chronic kidney disease (CKD) are hypertensive nephrosclerosis, diabetic nephropathy, chronic glomerulonephritis, and polycystic kidney disease. The uncorrected manifestations of this syndrome (Table 31-5) are usually seen only after GFR decreases below 25 mL/min. Patients with GFR less than 10 mL/min are dependent upon RRT for survival, in the form of hemodialysis, hemofiltration, or peritoneal dialysis.

Peripheral neuropathy
Autonomic neuropathy
Muscle twitching
Encephalopathy
Asterixis
Myoclonus
Lethargy
Confusion
Seizures
Coma

Fluid overload
Hypertension
Pericarditis
Arrhythmia
Conduction blocks
Vascular calcification
Accelerated atherosclerosis

Hyperventilation
Interstitial edema
Alveolar edema

Anorexia
Delayed gastric emptying
Hyperacidity
Mucosal ulcerations
Hemorrhage
Adynamic ileus

Metabolic acidosis
Hyperkalemia
Hyponatremia
Hyperphosphatemia
Hyperuricemia
Hypoalbuminemia

Glucose intolerance
Secondary hyperparathyroidism
Hypertriglyceridemia

Hyperpigmentation
Ecchymosis
Pruritus
The generalized effects of severe CKD can usually be controlled by RRT. The majority of patients with end-stage kidney disease who do not undergo renal transplantation receive RRT three times per week. There are complications directly related to RRT itself (Table 31-6). Hypotension, neutropenia, hypoxemia, and disequilibrium syndrome are generally transient, if they occur, and resolve within hours after RRT. Factors contributing to hypotension during dialysis include the vasodilating effects of acetate dialysate solutions, autonomic neuropathy, and rapid removal of fluid. The interaction of white cells with cellophane-derived dialysis membranes can result in neutropenia and leukocyte- mediated pulmonary dysfunction leading to hypoxemia. Dialysis disequilibrium syndrome (DDS) is most frequently seen following aggressive dialysis and is characterized by transient alterations in mental status and focal neurological deficits that are secondary to cerebral edema.

Anemia
Transient neutropenia
Residual anticoagulation
Hypocomplementemia

Multiple metabolic abnormalities, including hyperkalemia, hyperphosphatemia, hypocalcemia, hypermagnesemia, hyperuricemia, and hypoalbuminemia, typically develop in patients with kidney failure. Water and sodium retention can result in worsening hyponatremia and extracellular fluid overload, respectively. Failure to excrete nonvolatile acids produces an increased anion gap metabolic acidosis (see Chapter 50). Hypernatremia and hypokalemia are uncommon complications.
Hyperkalemia is a potentially lethal consequence of kidney failure (see Chapter 49). It usually occurs in patients with creatinine clearances of less than 5 mL/min, but it can also develop rapidly in patients with higher clearances in the setting of large potassium loads (eg, trauma, hemolysis, infections, or potassium administration).
Hypermagnesemia is generally mild unless magnesium intake is increased
(commonly from magnesium-containing antacids). Hypocalcemia is secondary to resistance to parathyroid hormone, decreased intestinal calcium absorption secondary to decreased kidney synthesis of 1,25-dihydroxycholecalciferol, and hyperphosphatemia-associated calcium deposition into bone. Symptoms of hypocalcemia rarely develop unless patients are also alkalotic.
Patients with kidney failure also rapidly lose tissue protein and readily develop hypoalbuminemia. Anorexia, protein restriction, and dialysis are contributory.

Anemia is nearly always present when the creatinine clearance is below 30 mL/min. Hemoglobin concentrations are generally 6 to 8 g/dL due to decreased erythropoietin production, red cell production, and red cell survival. Additional factors may include gastrointestinal blood loss, hemodilution, bone marrow suppression from recurrent infections, and blood loss for laboratory testing. Even with transfusions, it is often difficult to maintain hemoglobin concentrations greater than 9 g/dL. Erythropoietin administration may partially correct the anemia. Increased levels of 2,3-diphosphoglycerate (2,3-DPG), which facilitates the unloading of oxygen from hemoglobin (see Chapter 23), develop in response to the decrease in blood oxygen-carrying capacity. The metabolic acidosis associated with CKD also favors a rightward shift in the hemoglobin-oxygen dissociation curve. In the absence of symptomatic heart disease, most CKD patients tolerate anemia well.
Both platelet and white cell function are impaired in patients with kidney failure. Clinically, this is manifested as a prolonged bleeding time and increased susceptibility to infections, respectively. Most patients have decreased platelet factor III activity as well as decreased platelet adhesiveness and aggregation. Patients who have recently undergone hemodialysis may also have residual anticoagulant effects from heparin.

Cardiac output increases in kidney failure to maintain oxygen delivery due to decreased blood oxygen-carrying capacity. Sodium retention and abnormalities in the renin—angiotensin system result in systemic arterial hypertension. Left ventricular hypertrophy is a common finding in CKD. Extracellular @ fluid overload from sodium retention, in association with increased cardiac demand imposed by anemia and hypertension, makes CKD patients prone to congestive heart failure and pulmonary edema. Increased permeability of the alveolar—
capillary membrane may also be a predisposing factor for pulmonary edema associated with CKD (see later discussion). Arrhythmias, including blocks, are common, and may be related to metabolic abnormalities and to deposition of calcium in the conduction system. Uremic pericarditis may develop in some patients, who may be asymptomatic, may present with chest pain, or may present with cardiac tamponade. Patients with CKD also characteristically develop accelerated peripheral vascular and coronary artery atherosclerotic disease.
Intravascular volume depletion may occur in high-output acute kidney failure if fluid replacement is inadequate. Hypovolemia may occur secondary to excessive fluid removal conduction during dialysis.

Without RRT or bicarbonate therapy, CKD patients may be dependent on increased minute ventilation as compensation for metabolic acidosis (see Chapter 50). Pulmonary extravascular water is often increased in the form of interstitial edema, resulting in a widening of the alveolar to arterial oxygen gradient and predisposing to hypoxemia. Increased permeability of the alveolar— capillary membrane in some patients can result in pulmonary edema even with normal pulmonary capillary pressures.

Abnormal glucose tolerance is common in CKD, usually resulting from peripheral insulin resistance (type 2 diabetes mellitus is one of the most common causes of CKD). Secondary hyperparathyroidism in patients with chronic kidney failure can produce metabolic bone disease, predisposing to fractures. Abnormalities in lipid metabolism frequently lead to hypertriglyceridemia and contribute to accelerated atherosclerosis. Increased circulating levels of proteins and polypeptides normally degraded by the kidneys are often present, including parathyroid hormone, insulin, glucagon, growth hormone, luteinizing hormone, and prolactin.

Anorexia, nausea, vomiting, and ileus are commonly associated with uremia. Hypersecretion of gastric acid increases the incidence of peptic ulceration and gastrointestinal hemorrhage, which occurs in 10% to 30% @ of patients. Delayed gastric emptying secondary to kidney disease—associated autonomic neuropathy may predispose patients to perioperative aspiration. Patients with CKD also have an increased incidence of hepatitis B and C, often with associated hepatic dysfunction.

Asterixis, lethargy, confusion, seizures, and coma are manifestations of uremic encephalopathy, and symptoms usually correlate with the degree of azotemia. Autonomic and peripheral neuropathies are common in patients with CKD. Peripheral neuropathies are typically sensory and involve the distal lower extremities.

Most perioperative patients with acute kidney failure are critically ill, and their kidney failure is frequently associated with trauma or perioperative medical or surgical complications. They are typically in a state of metabolic catabolism. Optimal perioperative management is dependent upon RRT. Hemodialysis is more effective than peritoneal dialysis and can be readily accomplished via a temporary internal jugular, subclavian, or femoral dialysis catheter. Continuous renal replacement therapy (CRRT) is often used when patients are too hemodynamically unstable to tolerate intermittent hemodialysis. Indications for RRT are listed in Table 31—7.

Severe acidosis
Metabolic encephalopathy
Coagulopathy
Refractory gastrointestinal symptoms
Drug toxicity
Patients with chronic kidney failure commonly present to the operating room for creation or revision of an arteriovenous dialysis fistula under local or regional anesthesia. Preoperative dialysis on the day of surgery or on the previous day is typical. However, regardless of the intended procedure or the anesthetic employed, one must be certain that the patient is in optimal medical condition; potentially reversible manifestations of uremia (see Table 31-5) should be addressed.
The history and physical examination should address both cardiac and respiratory function. Signs of fluid overload or hypovolemia should be sought. Patients are often relatively hypovolemic immediately following dialysis. A comparison of the patient's current weight with previous predialysis and postdialysis weights may be helpful. Hemodynamic data and a chest radiograph, if available, are useful in confirming clinical suspicion of volume overload. Arterial blood gas analysis is useful in evaluating oxygenation, ventilation, hemoglobin level, and acid—base status in patients with dyspnea or tachypnea. The electrocardiogram should be examined for signs of hyperkalemia or hypocalcemia (see Chapter 49) as well as ischemia, conduction block, and ventricular hypertrophy. Echocardiography can assess cardiac function, ventricular hypertrophy, wall motion abnormalities, and pericardial fluid. A friction rub may not be audible on auscultation of patients with a pericardial effusion.
Preoperative red blood cell transfusions are usually administered only for severe anemia as guided by the patient's clinical status. Bleeding time and coagulation studies (or perhaps a thromboelastogram) may be advisable, particularly if neuraxial anesthesia is being considered. Serum electrolyte, BUN, and creatinine measurements can assess the adequacy of dialysis. Glucose measurements guide the potential need for perioperative insulin therapy.
Drugs with significant renal elimination should be avoided if possible (Table 31-8). Dosage adjustments and measurements of blood levels (when available) are necessary to minimize the risk of drug toxicity.

Pancuronium
Anticholinergics
Atropine
Glycopyrrolate
Metoclopramide
H,-receptor antagonists
Cimetidine
Ranitidine
Digitalis
Diuretics
Calcium channel antagonists
Diltiazem
Nifedipine

Captopril
Clonidine
Enalapril
Hydralazine
Lisinopril
Nitroprusside (thiocyanate)

Bretylium
Disopyramide
Encainide (genetically determined)
Procainamide
Tocainide
Bronchodilators
Terbutaline
Psychiatric
Lithium
Antibiotics
Aminoglycosides
Cephalosporins
Penicillins
Vancomycin

Alert patients who are stable can be given reduced doses of a benzodiazepine, if needed. Chemoprophylaxis for patients at risk for aspiration is reviewed in Chapter 17. Preoperative medications—particularly antihypertensive agents— should be continued until the time of surgery (see Chapter 21). The management of diabetic patients is discussed in Chapter 35.

Patients with kidney disease and failure are at increased risk for perioperative complications, and their general medical condition and the planned operative procedure dictate monitoring requirements. Because of the risk of thrombosis, blood pressure should not be measured by a cuff on an arm with an arteriovenous fistula. Continuous invasive or noninvasive blood pressure monitoring may be indicated in patients with poorly controlled hypertension.

Patients with nausea, vomiting, or gastrointestinal bleeding should undergo rapid-sequence induction. The dose of the induction agent should be reduced for debilitated or critically ill patients, or for patients who have recently undergone hemodialysis and who remain relative hypovolemic. Propofol, 1 to 2 mg/kg, or etomidate, 0.2 to 0.4 mg/kg, is often used. An opioid, B-blocker (esmolol), or lidocaine may be used to blunt the hypertensive response to airway instrumentation and intubation. Succinylcholine, 1.5 mg/kg, can be used to facilitate endotracheal intubation in the absence of hyperkalemia. Rocuronium (1 mg/kg), vecuronium (0.1 mg/kg), cisatracurium (0.15 mg/kg), or propofol— lidocaine induction without a relaxant may be considered for intubation in patients with hyperkalemia.

The ideal anesthetic maintenance technique should control hypertension with minimal deleterious effect on cardiac output, because increased cardiac output is the principal compensatory mechanism for tissue oxygen delivery in anemia. Volatile anesthetics, propofol, fentanyl, sufentanil, alfentanil, and remifentanil are satisfactory maintenance agents. Meperidine should be avoided because of accumulation of its metabolite normeperidine. Morphine may be used, but prolongation of its effects may occur.
@ Controlled ventilation should be considered for patients with kidney failure under general anesthesia. Inadequate spontaneous ventilation with progressive hypercarbia under anesthesia can result in respiratory acidosis that may exacerbate preexisting acidemia, lead to potentially severe circulatory depression, and dangerously increase serum potassium concentration (see Chapter 50). On the other hand, respiratory alkalosis may also be detrimental because it shifts the hemoglobin dissociation curve to the left, can exacerbate preexisting hypocalcemia, and may reduce cerebral blood flow.

Superficial procedures involving minimal physiological trespass require replacement of insensible fluid losses only. In situations requiring significant fluid volume for maintenance or resuscitation, isotonic crystalloids, colloids, or both may be used (see Chapter 51). Current evidence suggests balanced crystalloids such as Plasma-Lyte or lactated Ringer's solution are preferable in such circumstances to chloride-rich crystalloids such as 0.9% saline because of the deleterious effects of hyperchloremia on kidney function. However, 0.9% saline is preferable to balanced crystalloids in patients with alkalosis and hypochloremia. Lactated Ringer's solution should be avoided in hyperkalemic patients when large fluid volumes are required, because it contains potassium 4 mEq/L. Glucose-free solutions should generally be used because of the glucose

The kidney normally possesses large functional reserve. GFR, as determined by creatinine clearance, can decrease from 120 to 60 mL/min without clinical signs or symptoms of diminished kidney function. Even patients with creatinine clearances of 40 to 60 mL/min usually are asymptomatic. These patients have only mild kidney impairment but should still be thought of as having decreased kidney reserve. Preservation of remaining kidney function is paramount, and best accomplished by maintaining normovolemia and normal kidney perfusion. When creatinine clearance decreases to 25 to 40 mL/min, kidney impairment is moderate, and patients are said to have renal insufficiency. Azotemia is always present, and hypertension and anemia are @ common. Correct anesthetic management of this group of patients is as critical as management of those with frank kidney failure, especially during procedures associated with a relatively high incidence of postoperative kidney failure, such as cardiac @ and aortic reconstructive surgery. Intravascular volume depletion, sepsis, obstructive jaundice, crush injuries, and renal toxins such as radiocontrast agents, certain antibiotics, angiotensin-converting enzyme inhibitors, and NSAIDs (see Table 30-4) are additional major risk factors for acute deterioration in kidney function. Hypovolemia and decreased kidney perfusion are particularly important causative factors in the development of acute postoperative kidney failure. The emphasis in management of these patients is on prevention, because the mortality rate of postoperative kidney failure may surpass 50%. The combination of diabetes and preexisting kidney disease markedly increases perioperative risk of kidney function deterioration and of kidney failure.
© Kidney protection with adequate hydration and maintenance of renal blood flow is indicated for patients at high risk for kidney injury and kidney failure. The use of mannitol, low-dose dopamine or fenoldopam infusion, loop diuretics, or bicarbonate infusion for kidney protection is controversial and without proof of efficacy (see earlier discussion). N-acetylcysteine, when given prior to the administration of radiocontrast agents, reduces the risk of radiocontrast agent— induced AKI (see Chapter 30).

The American Society of Anesthesiologists' basic monitoring standards are used for procedures involving minimal fluid losses. For procedures associated with significant blood or fluid loss, close monitoring of hemodynamic performance and urinary output is important (see Chapter 51). Although maintenance of urinary output does not ensure preservation of kidney function, urinary outputs greater than 0.5 mL/kg/h are preferable. Continuous invasive blood pressure monitoring is also important if rapid changes in blood pressure are anticipated, such as in patients with poorly controlled hypertension and in those undergoing procedures associated with abrupt changes in sympathetic stimulation or in cardiac preload or afterload.

Selection of an induction agent is not as important as ensuring an adequate intravascular volume prior to induction; induction of anesthesia in hypovolemic patients with impaired kidney function frequently results in hypotension. Unless a vasopressor is administered, such hypotension typically resolves only following intubation or surgical stimulation. Kidney perfusion, which may already be compromised by preexisting hypovolemia, may then deteriorate further, first as a result of hypotension, and subsequently from sympathetically or pharmacologically mediated renal vasoconstriction. If sustained, the decrease in renal perfusion may contribute to postoperative kidney impairment or failure. Preoperative hydration usually prevents this sequence of events.

All anesthetic maintenance agents are acceptable, with the possible exception of sevoflurane administered with low gas flows over a prolonged time period (see Chapter 30). Intraoperative deterioration in kidney function may result from adverse effects of the operative procedure (hemorrhage, vascular occlusion, abdominal compartment syndrome, arterial emboli) or anesthetic (hypotension secondary to myocardial depression or vasodilation), from indirect hormonal effects (sympathoadrenal activation or antidiuretic hormone secretion), or from impeded venous return secondary to positive-pressure ventilation. Many of these effects are avoidable or reversible when adequate intravenous fluids are given to maintain a normal or slightly expanded intravascular volume. The administration of large doses of predominantly a-adrenergic vasopressors (phenylephrine and norepinephrine) may also be detrimental to preservation of kidney function. Small, intermittent doses, or brief infusions, of vasoconstrictors may be useful in maintaining renal blood flow until other measures (eg, transfusion) are undertaken to correct hypotension.

As reviewed earlier, appropriate fluid administration is important in managing patients with preexisting AKI or kidney failure or who are at risk for AKI. We find guidance from noninvasive monitors of stroke volume and cardiac output useful. Concern over fluid overload is justified, but acute problems are rarely encountered in such patients with normal urinary outputs if rational fluid administration guidelines and appropriate monitoring are employed (see Chapter 51). Moreover, the adverse consequences of excessive fluid overload are far easier to treat than those of AKI and kidney failure.

A Patient with Uncontrolled Hypertension
A 59-year-old man with a recent onset of hypertension is scheduled for stenting of a stenotic left renal artery. His preoperative blood pressure is 180/110 mm Hg.
What is the likely cause of this patient's hypertension?
Renovascular hypertension is one of the few forms of hypertension that can be corrected with surgery or mechanical intervention. Others include coarctation of the aorta, pheochromocytoma, Cushing disease, and primary hyperaldosteronism.
Most studies suggest that renovascular hypertension accounts for 2% to 5% of all cases of hypertension. Characteristically it manifests as a relatively sudden onset of hypertension in a person younger than 35 or older than 55 years of age. Renal artery stenosis can also be responsible for the development of accelerated or malignant hypertension in previously hypertensive persons of any age.

Unilateral or bilateral stenosis of the renal artery decreases the perfusion pressure to the kidney(s) distal to the obstruction. Activation of the juxtaglomerular apparatus and release of renin increase circulating levels of angiotensin II and aldosterone, resulting in peripheral vascular constriction and sodium retention, respectively. The resulting systemic arterial hypertension is often severe.
In nearly two thirds of patients, the stenosis results from an atheromatous plaque in the proximal renal artery. These patients are typically men over the age of 55 years. In the remaining one third of patients, the stenosis is more distal and is due to malformations of the arterial wall, commonly referred to as fibromuscular hyperplasia (or, dysplasia). This latter lesion most commonly presents in women younger than 35 years. Bilateral renal artery stenosis is present in 30% to 50% of patients with renovascular hypertension. Less common causes of stenosis include dissecting aneurysms, emboli, polyarteritis nodosa, radiation, trauma, extrinsic compression from retroperitoneal fibrosis or tumors, and hypoplasia of the renal arteries.
What clinical manifestations other than hypertension may be present?
Signs of secondary hyperaldosteronism can be prominent. These include sodium retention in the form of edema, metabolic alkalosis, and hypokalemia. The latter can cause muscle weakness, polyuria, and even tetany.
How is the diagnosis made?
The diagnosis is suggested by the clinical presentation previously described. A midabdominal bruit may also be present, but the diagnosis requires laboratory and radiographic confirmation. A definitive diagnosis is made by renal arteriography, and percutaneous balloon angioplasty with stenting may be performed at the same time. The functional significance of the restrictive lesion(s) may be evaluated by selective catheterization of both renal veins and subsequent measurement of plasma renin activity in blood from each kidney. Restenosis rates following angioplasty are estimated to be less than 15% after 1 year. Patients who are not candidates for angioplasty and stenting are referred for surgery.
Should this patient undergo intervention or surgical correction given his present blood pressure?

Optimal medical therapy is important in preparing these patients for operation. Relative to patients with well-controlled hypertension, those with poorly controlled hypertension have an increased incidence of intraoperative problems, including marked hypertension, hypotension, myocardial ischemia, and arrhythmias. Ideally, arterial blood pressure should be well controlled prior to surgery. Patients should be evaluated for preexisting kidney dysfunction, and metabolic disturbances such as hypokalemia should be corrected. Patients should also be evaluated as indicated for the presence and severity of coexisting atherosclerotic disease, according to current American College of Cardiology/American Heart Association (ACC/AHA) guidelines (see Chapter 21).
What antihypertensive agents are most useful for controlling blood pressure perioperatively in these patients?
B-Adrenergic blocking drugs are frequently utilized for blood pressure control in the perioperative period. They are particularly effective because secretion of renin is partly mediated by B,-adrenergic receptors. Although parenteral selective B,-blocking agents such as metoprolol and esmolol would be expected to be most effective, nonselective agents appear equally effective. Esmolol may be the intraoperative B,-blocking agent of choice because of its short half-life and titratability. Direct vasodilators and nicardipine are also useful in controlling intraoperative hypertension (see Chapter 15).
ACE inhibitors and angiotensin-converting enzyme receptor blockers are contraindicated in bilateral renal artery stenosis or in unilateral renal artery stenosis where there is only one functioning kidney because they can precipitate kidney failure.

Open surgical revascularization of a kidney is a major procedure, with the potential for major blood loss, fluid shifts, and hemodynamic changes. One of several procedures may be performed, including transaortic renal endarterectomy, aortorenal bypass (using a saphenous vein, synthetic graft, or segment of the hypogastric artery), a splenic to (left) renal artery bypass, a hepatic or gastroduodenal to (right) renal artery bypass, or excision of the stenotic segment with reanastomosis of the renal artery to the aorta. Rarely, nephrectomy may be performed. Isolated stenosis of a renal artery may be corrected with stenting performed with local anesthesia and sedation.
With all "open" procedures, an extensive retroperitoneal dissection often necessitates relatively large volumes of intravenous fluid replacement. Large-bore intravenous access is mandatory because of the potential for extensive blood loss. Heparinization contributes to increased blood loss. Depending on the surgical technique, aortic cross-clamping, with its associated hemodynamic consequences, often complicates anesthetic management (see Chapter 22). Continuous intraarterial blood pressure monitoring will nearly always be used, and central venous pressure monitoring is often helpful. Goal-directed hemodynamic and fluid therapy utilizing arterial pulse contour analysis, esophageal Doppler, or transesophageal echocardiography should be considered for patients with poor ventricular function and may be advisable in most patients to guide fluid management (see Chapter 51). The choice of anesthetic technique is generally determined by the patient's cardiovascular function.
Generous hydration and maintenance of adequate cardiac output and blood pressure are important to protect both the affected and the normal kidney against acute ischemic injury. Topical cooling of the affected kidney during the anastomosis may also be employed.

Although in most patients hypertension is ultimately cured or significantly improved, arterial blood pressure is often quite labile in the early postoperative period. Close hemodynamic monitoring should be continued well into the postoperative period. Reported operative mortality rates range from 1% to 6%, and most deaths are associated with myocardial infarction. The latter probably reflects the relatively high prevalence of coronary artery disease in older patients with renovascular hypertension.

approach.
Sem
Nephrol.
2016;36:53.
patients requiring volume resuscitation. A systematic review and metaanalysis. JAMA. 2013;309:678.
Zazzeron L, Gattinoni L, Caironi P. Role of albumin, starches and gelatins versus crystalloids in volume resuscitation of critically ill patients. Curr Opin Crit Care. 2016;22:428.

@ Next to the supine position, the lithotomy position is the most commonly used position for patients undergoing urological and gynecological procedures. Failure to properly position and pad the patient can result in pressure sores, nerve injuries, or compartment syndromes.
@ The lithotomy position is associated with major physiological alterations. Functional residual capacity decreases, predisposing patients to atelectasis and hypoxia. Elevation of the legs drains blood into the central circulation acutely. Mean blood pressure often increases, but cardiac output does not change significantly. Conversely, rapid lowering of the legs from the lithotomy or Trendelenburg position acutely decreases venous return and can result in hypotension. Blood pressure measurement should be taken immediately after the legs are lowered.
@ Because of the short duration (15-20 min) and outpatient setting of most cystoscopies, general anesthesia is often chosen, commonly employing a laryngeal mask airway.
@ Both epidural and spinal blockade with a T10 sensory level provide excellent anesthesia for cystoscopy.
@ Manifestations of TURP (transurethral resection of the prostate) syndrome are primarily those of circulatory fluid overload, water intoxication, and, occasionally, toxicity from the solute in the irrigating fluid.
@ Absorption of TURP irrigation fluid is dependent on the duration of the resection and the pressure of the irrigation fluid.
@ When compared with general anesthesia, regional anesthesia for TURP may reduce the incidence of postoperative venous thrombosis. It is also
less likely to mask symptoms and signs of TURP syndrome or bladder perforation.

@ Patients with a history of cardiac arrhythmias and those with a pacemaker or implantable cardioverter defibrillator (ICD) may be at risk for developing arrhythmias induced by shock waves during extracorporeal shock wave lithotripsy (ESWL). Shock waves can damage the internal components of pacemaker and ICD devices.
@ Patients who are undergoing retroperitoneal lymph node dissection and who have received bleomycin preoperatively are at increased risk for developing postoperative pulmonary insufficiency. These patients may be particularly at risk for oxygen toxicity and fluid overload, and for developing acute respiratory distress syndrome postoperatively.
@ For patients undergoing kidney transplantation, the preoperative serum potassium concentration should be below 5.5 mEq/L and existing coagulopathies should be corrected. Hyperkalemia has been reported after release of the vascular clamp following completion of the arterial anastomosis, particularly in pediatric and other small patients. Release of potassium contained in the preservative solution has been implicated as the cause of this phenomenon.
Urological procedures range in impact from simple outpatient cystoscopy to surgical procedures with increased risk of perioperative complications, such as radical cystectomy and nephrectomy for renal cell carcinoma with vena caval thrombosis. Patients undergoing genitourinary procedures may be of any age, but many are elderly with coexisting medical illnesses, including kidney dysfunction. The impact of anesthesia on kidney function is discussed in Chapter 31. This chapter reviews the anesthetic management of common urological procedures. Use of lithotomy and steep head-down (Trendelenburg) positions complicates many of these procedures. Moreover, advances in preoperative patient optimization, perioperative management, and postoperative rehabilitation allow more patients with coexisting disease to be considered acceptable candidates for kidney transplantation, extensive tumor debulking operations, and reconstructive genitourinary procedures.

Cystoscopy is a very commonly performed urological procedure, and indications for this investigative or therapeutic operation include hematuria, recurrent urinary infections, renal calculi, and urinary obstruction. Bladder biopsies, retrograde pyelograms, transurethral resection of bladder tumors, extraction or laser lithotripsy of kidney stones, and placement or manipulation of ureteral catheters (stents) are also commonly performed through the cystoscope. Anesthetic management varies with the age and gender of the patient and the purpose of the procedure. General anesthesia is usually necessary for children. Viscous lidocaine topical anesthesia with or without sedation provides satisfactory anesthesia for diagnostic cystoscopy in most women because of the short urethra. Men generally require regional or general anesthesia for diagnostic cystoscopy. Operative cystoscopies involving biopsies, cauterization, or manipulation of ureteral catheters require regional or general anesthesia, regardless of patient gender.

extremities are inappropriately positioned (eg, hyperextension at the axilla). Compartment syndrome of the lower extremities with rhabdomyolysis has been reported with prolonged time in the lithotomy position, after which lower extremity nerve damage is also more likely.
FIGURE 32-1 The lithotomy position. A: Strap stirrups. B: Bier—Hoff stirrups. C: Allen stirrups. (Reproduced with permission from Martin JT. Positioning in Anesthesia. Philadelphia, PA: WB Saunders; 1988.)
® The lithotomy position is associated with major physiological alterations. Functional residual capacity decreases, predisposing patients to atelectasis and hypoxia. This effect is amplified by steep Trendelenburg positioning (30—45°), which is commonly utilized in combination with the lithotomy position. Elevation of the legs drains blood into the central circulation acutely. Mean blood pressure and cardiac output may increase. Conversely, rapid lowering of the legs from the lithotomy or Trendelenburg position acutely decreases venous return and cardiac output and can result in hypotension. Vasodilation from either general or regional anesthesia potentiates the hypotension in this situation, and for this reason, blood pressure measurement should be taken immediately after the legs are lowered.

1.
General anesthesia—Any
may be
utilized.
anesthetic
Because of
most of
the cystoscopies,
laryngeal general
mask
@
technique short
duration anesthesia
airway.
obese or
elderly
Oxygen patients,
(15-20
is suitable
for min)
often saturation
or those
outpatients and
chosen, should
with marginal
outpatient setting
commonly be
closely employing
monitored pulmonary
a
when reserve,
are placed
in the
lithotomy or
Trendelenburg position.
@ 2. Regional anesthesia—Both epidural and spinal blockade provide satisfactory anesthesia for cystoscopy. However, when neuraxial regional anesthesia is chosen most anesthesiologists prefer spinal anesthesia because onset of satisfactory sensory blockade may require 15 to 20 min for epidural anesthesia compared with 5 min or less for spinal anesthesia. Studies fail to demonstrate that immediate elevation of the legs into lithotomy position following administration of hyperbaric spinal anesthesia either increases the dermatomal extent of anesthesia to a clinically significant degree or increases the likelihood of severe hypotension. A T10 sensory level block provides excellent anesthesia for all cystoscopic procedures.

Benign prostatic hyperplasia (BPH) frequently leads to bladder outlet obstruction in men older than 60 years. Although most patients are treated medically, some require surgical intervention. Transurethral resection of the prostate (TURP) may be performed for bladder outlet obstruction due to BPH. Indications for TURP include obstructive uropathy, bladder calculi, and recurrent episodes of urinary retention, urinary tract infections, and hematuria. Patients with prostate cancer who are not candidates for radical prostatectomy may also benefit from TURP to relieve urinary obstruction.
TURP requires regional or general anesthesia. Despite advanced age and prevalence of significant comorbidity, perioperative mortality and medical morbidity (most frequently myocardial infarction, pulmonary edema, and kidney failure) for this procedure are both less than 1%.
The most common surgical complications of TURP are clot retention, failure to void, uncontrolled hematuria requiring surgical revision, urinary tract infection, and chronic hematuria, although other complications may include TURP syndrome, bladder perforation, sepsis, hypothermia, and disseminated intravascular coagulation (DIC). A blood type and screen (see Chapter 51) is adequate for most patients, although crossmatched blood should be available for anemic patients and for patients with a large prostate in which extensive resection is contemplated. Prostatic bleeding can be difficult to control through the cystoscope.

TURP is conventionally performed by passing a monopolar electrical loop through a special cystoscope (resectoscope). Using continuous irrigation and direct visualization, prostatic tissue is resected by applying a cutting current to the loop. Because of the characteristics of the prostate and the large amounts of irrigation fluid often used, TURP can be associated with serious complications (Table 32-1).

Clot retention
Failure to void
Uncontrolled acute hematuria
Urinary tract infection
Chronic hematuria

TURP syndrome
Bladder perforation
Sepsis
Disseminated intravascular coagulation

Transurethral prostatic resection, now a relatively uncommon procedure, often opens the extensive network of venous sinuses in the prostate, potentially allowing systemic absorption of the irrigating fluid. The absorption of large amounts of fluid (2 L or more) results in a constellation of symptoms and signs commonly referred to as TURP syndrome @ (Table 32—2). The manifestations are primarily those of circulatory fluid overload, water intoxication, and, occasionally, toxicity from the solute in the irrigating fluid. The incidence of TURP syndrome is less than 1%. This syndrome presents intraoperatively or postoperatively as headache, restlessness, confusion, cyanosis, dyspnea, arrhythmias, hypotension, seizures, or a combination of these, and it can be rapidly fatal. TURP syndrome is most commonly associated with large-volume prostate resection and use of large volumes of irrigation fluid, and has also been much less commonly reported with cystoscopy, arthroscopy, transurethral resection of bladder tumors, and transcervical resection of the endometrium.

Hypoosmolality
Hemolysis
Solute toxicity
Hyperglycinemia (glycine)
Hyperammonemia (glycine)
Hyperglycemia (sorbitol)
Intravascular volume expansion (mannitol)
TURP, transurethral resection of the prostate.
Electrolyte solutions cannot be used for irrigation during monopolar TURP because they disperse the electrocautery current. Water provides excellent visibility because its hypotonicity lyses red blood cells, but significant water absorption can readily result in acute water intoxication. Water irrigation is generally restricted to transurethral resection of bladder tumors only. For monopolar TURP, slightly hypotonic nonelectrolyte irrigating solutions such as glycine 1.5% (230 mOsm/L) or a mixture of sorbitol 2.7% and mannitol 0.54% (195 mOsm/L) are most commonly used. Less commonly used solutions include sorbitol 3.3%, mannitol 3%, dextrose 2.5% to 4%, and urea 1%. Because all these fluids are still hypotonic, significant absorption of water can occur. Fluid absorption rate is also influenced by irrigation fluid pressure, and high pressures (high bottle or bag height) increases the rate of fluid absorption.
@ Absorption of TURP irrigation fluid is dependent on the duration of the resection and the pressure of the irrigation fluid. Pulmonary edema can readily result from the absorption of large amounts of irrigation fluid, particularly in patients with limited cardiac reserve. The hypotonicity of these fluids also results in acute hyponatremia and hypoosmolality, which can lead to serious neurological manifestations. Symptoms of hyponatremia usually do not develop until the serum sodium concentration decreases below 120 mEq/L. Marked hypotonicity in plasma ([Na*] <100 mEq/L) may also result in acute intravascular hemolysis.

Toxicity may also arise from absorption of the solutes in these fluids. Marked hyperglycinemia has been reported with glycine solutions and may contribute to circulatory depression and central nervous system toxicity. Glycine has been implicated in rare instances of transient blindness following TURP. Hyperammonemia, presumably from the degradation of glycine, has also been documented in a few patients with marked central nervous system toxicity following TURP.
Treatment of TURP syndrome depends on early recognition and should be based on the severity of the symptoms. The absorbed water must be eliminated, and hypoxemia and hypoperfusion treated. Most patients can be managed with fluid restriction and intravenous administration of furosemide. Symptomatic hyponatremia resulting in seizures or coma should be treated with hypertonic saline (see Chapter 49). Seizure activity can be terminated with small doses of midazolam (2—4 mg). Endotracheal intubation may be considered to prevent aspiration until the patient's mental status normalizes. The amount and rate of hypertonic saline solution (3% or 5%) needed to correct the hyponatremia to a safe level should be based on the patient's serum sodium concentration (see Chapter 49).
Recently, additional procedural methods for treatment of BPH have included bipolar TURP, laser and radiofrequency ablation, photodynamic and thermal therapy, and cryotherapy. Low-voltage bipolar TURP allows use of isotonic saline irrigation fluid, thus avoiding TURP syndrome with the exception of the persisting risk of fluid overload, and because the procedure simultaneously cauterizes as it resects, it decreases the risk of clot retention.

Large volumes of irrigating fluids at room temperature can be a major source of heat loss in patients. Irrigating solutions should be warmed to body temperature prior to use to prevent hypothermia. Postoperative shivering associated with hypothermia may dislodge clots and promote postoperative bleeding.

The incidence of bladder perforation during TURP is less than 1% and may result from either the resectoscope going through the bladder wall or from overdistention of the bladder with irrigation fluid. Most bladder perforations are extraperitoneal and are signaled by poor return of the irrigating fluid. Awake patients will typically complain of nausea, diaphoresis, and retropubic or lower abdominal pain. Large extraperitoneal, and most intraperitoneal, perforations are usually even more obvious, presenting as sudden unexplained hypotension or hypertension, and with generalized abdominal pain in awake patients. Regardless of the anesthetic technique employed, perforation should be suspected in settings of sudden hypotension or hypertension, particularly with acute, vagal-mediated bradycardia.

DIC following TURP may result from the release of thromboplastins from prostate tissue during the procedure. Rarely, patients with metastatic carcinoma of the prostate develop a coagulopathy from primary fibrinolysis due to secretion of a fibrinolytic enzyme. Coagulopathy may be suspected from diffuse, uncontrollable bleeding, but must be defined with laboratory tests. Primary fibrinolysis should be treated with e-aminocaproic acid (Amicar) or tranexamic acid. Treatment of DIC in this setting may require heparin in addition to replacement of clotting factors and platelets, and consultation with a hematologist should be considered.

The prostate is often colonized with bacteria and may harbor chronic infection. Extensive surgical resection with the opening of venous sinuses can allow entry of organisms into the bloodstream. Bacteremia following transurethral surgery is common. Prophylactic antibiotic therapy (most commonly gentamicin, levofloxacin, or cefazolin) prior to TURP is often administered.

Either spinal or epidural anesthesia with a T10 sensory level, or general anesthesia, provides excellent anesthesia and good operating conditions for TURP. @ When compared with general anesthesia, regional anesthesia may reduce the incidence of postoperative venous thrombosis. It is also less likely to mask symptoms and signs of TURP syndrome or bladder perforation. Clinical studies have failed to show any differences in blood loss, postoperative cognitive function, and mortality between regional and general anesthesia. Acute hyponatremia from TURP syndrome may delay or prevent emergence from general anesthesia.

Evaluation of mental status in the awake or moderately sedated patient is the best monitor for detection of early signs of TURP syndrome and bladder perforation. Blood loss is particularly difficult to assess during TURP because of the use of irrigating solutions, so it is necessary to rely on clinical signs of hypovolemia (see Chapter 51). Blood loss averages approximately 3 to 5 mL/min of resection (usually 200-300 mL total) but is rarely life-threatening. Transient, postoperative decreases in hematocrit may simply reflect hemodilution from absorption of irrigation fluid. Very few patients will require intraoperative blood transfusion.

The treatment of kidney stones has evolved from primarily open surgical procedures to less invasive or entirely noninvasive techniques. Cystoscopic procedures, including flexible ureteroscopy with stone extraction, stent placement, and intracorporeal lithotripsy (laser or electrohydraulic), along with medical expulsive therapy (MET), have become first-line therapy. Extracorporeal shock wave lithotripsy (ESWL) is also utilized, primarily for 4mm to 2-cm intrarenal stones, and percutaneous and laparoscopic nephrolithotomy for larger or impacted stones. MET has become the treatment of choice among many clinicians for acute episodes of urolithiasis: for stones up to 10 mm in diameter, administration of the a-blockers tamsulosin (Flomax), doxazosin (Cardura), or terazosin (Hytrin) or the calcium channel blocker nifedipine (Procardia, Adalat) increases the likelihood of stone expulsion. During ESWL, repetitive high-energy shocks (sound waves) are generated and focused on the stone, causing it to fragment. Water or (more commonly) a conducting gel couples the generator to the patient. The change in acoustic impedance at the tissue—stone interface creates shear and tear forces on the stone, fragmenting it sufficiently to allow its passage in small pieces down the urinary tract. Ureteral stents are often placed prior to the procedure. Contraindications to the procedure include the inability to position the patient so that lung and intestine are away from the sound wave focus, urinary obstruction below the stone, untreated infection, a bleeding diathesis, and pregnancy. The presence of a nearby aortic aneurysm or an orthopedic prosthetic device is a relative contraindication.

Electrohydraulic, electromagnetic, or piezoelectric shock wave generators may be used for ESWL. With older electrohydraulic units, the patient is placed in a hydraulic chair and immersed in a heated water bath, which conducts the shock waves to the patient. Modern lithotriptors generate shock waves either electromagnetically or from piezoelectric crystals. The generator is enclosed in a water-filled casing and comes in contact with the patient via a conducting gel on a plastic membrane (Figure 32—2). In the case of electromagnetic machines, the vibration of a metallic plate in front of an electromagnet produces the shock waves. With piezoelectric models, the waves are the result of changes in the external dimensions of ceramic crystals when electric current is applied.
FIGURE 32-2 Schematic representation of tubless lithotripsy unit.

@ Patients with a history of cardiac arrhythmias and those with a pacemaker or implantable cardioverter defibrillator (ICD) may be at greater risk for arrhythmias during ESWL. Synchronization of the shock waves with the electrocardiogram (ECG) R wave decreases the incidence of arrhythmias during ESWL. The shock waves are usually timed to be 20 ms after the R wave to correspond with the ventricular refractory period, although studies suggest that asynchronous delivery of shocks may be safe in patients without heart disease. Shock waves can damage the internal components of implanted cardiac devices. The manufacturer should be contacted as to the best method for managing the device (eg, reprogramming or applying a magnet).
Intraoperative Considerations
Anesthetic considerations for ureteroscopy, stone manipulation, and laser lithotripsy are similar to those for cystoscopic procedures. ESWL requires special considerations, particularly when older lithotriptors requiring the patient to be immersed in water are used.

Immersion into a heated water bath (36—37°C) initially results in vasodilation that can transiently lead to hypotension. Arterial blood pressure, however, subsequently rises as venous blood is redistributed centrally due to the hydrostatic pressure of water on the legs and abdomen. Systemic vascular resistance (SVR) rises and cardiac output may decrease. Moreover, the increase in intrathoracic blood volume reduces functional residual capacity and may predispose some patients to hypoxemia.

Pain during lithotripsy is from dissipation of a small amount of energy as shock waves enter the body through the skin. The pain is therefore localized to the skin and is proportionate to the shock wave intensity. Older water bath lithotripsy units require 1000 to 2400 relatively high-intensity shock waves, which most patients cannot tolerate without either regional or general anesthesia. In contrast, newer lithotripsy units that are coupled directly to the skin utilize 2000 to 3000 lower-intensity shock waves that usually require only light sedation.

Continuous epidural anesthesia is commonly employed when ESWL utilizes older water bath lithotriptors. A T6 sensory level ensures adequate anesthesia, as renal innervation is derived from T10 to L2. When using the loss of resistance technique for placement of the epidural catheter, saline should be used instead of air during epidural catheter insertion as air in the epidural space can dissipate shock waves and may promote injury to neural tissue. Foam tape should not be used to secure the epidural catheter as this type of tape has been shown to dissipate the energy of the shock waves when it is in their path. Spinal anesthesia can also be used satisfactorily but offers less control over the sensory level and an uncertain duration of surgery; for this reason, epidural anesthesia is usually preferred.
Disadvantages of regional anesthesia or sedation include the inability to control diaphragmatic movement (excessive diaphragmatic excursion can move the stone out of the wave focus and may prolong the procedure) and bradycardia
(this will prolong the procedure when shock waves are coupled to the ECG). Glycopyrrolate may be administered to accelerate the ESWL procedure.

General endotracheal anesthesia allows control of diaphragmatic excursion during lithotripsy using older water bath lithotriptors. The procedure is complicated by the inherent risks associated with placing a supine anesthetized patient in a chair, elevating and then lowering the chair into a water bath to shoulder depth, and then reversing the sequence at the end. A light general anesthetic technique in conjunction with a muscle relaxant is preferable. The muscle relaxant ensures patient immobility and control of diaphragmatic movement.

Monitored anesthesia care with intravenous midazolam and fentanyl is usually adequate for modern low-energy lithotripsy. Deeper sedation may also be used.

Standard anesthesia monitoring must be used for conscious or deep sedation, or for general anesthesia. Supraventricular arrhythmias may occur even with R wave synchronized shocks. With immersion lithotripsy, ECG pads should be attached securely with waterproof dressing. The temperature of the bath and the patient should be monitored to prevent hypothermia or hyperthermia.

Intravenous fluid therapy is typically generous. Following an initial intravenous crystalloid fluid bolus, an additional 1000 to 2000 mL is often given with a small dose of furosemide to maintain brisk urinary flow and flush stone debris and blood clots. Patients with impaired cardiac reserve require more conservative fluid therapy.

Laparoscopic urological procedures, including partial and total nephrectomy, live donor nephrectomy, nephrolithectomy, and pyeloplasty are increasingly utilized because of advantages that include less perioperative pain, shorter function.
hospital length of stay, and relatively rapid convalescence and return to Both transperitoneal and retroperitoneal approaches have been developed. A hand-assisted technique employs an additional larger incision that allows the surgeon to insert one hand for tactile sensation and facilitation of dissection. Anesthetic management is similar to that for any laparoscopic procedure.
Open procedures for kidney stones in the upper ureter and renal pelvis, and nephrectomies for nonmalignant disease, are often carried out in the "kidney rest" or lateral flexed position. With the patient in a full lateral position, the dependent leg is flexed and the other leg is extended. An axillary roll is placed beneath the dependent upper chest to minimize the risk of brachial plexus injury. The operating table is then extended to achieve maximal separation between the iliac crest and the costal margin on the operative side, and the kidney rest (a bar in the groove where the table bends) is elevated to raise the nondependent iliac crest higher and improve surgical exposure.
The lateral flexed position is associated with adverse respiratory and circulatory effects. Functional residual capacity is reduced in the dependent lung but may increase in the nondependent lung. In the anesthetized patient receiving controlled ventilation, ventilation/perfusion mismatching occurs because the dependent lung receives greater blood flow than the nondependent lung, whereas the nondependent lung receives greater ventilation, predisposing the patient to atelectasis in the dependent lung and to shunt-induced hypoxemia. The arterial to end-tidal gradient for carbon dioxide progressively increases during general anesthesia in this position, indicating that dead space ventilation also increases in the nondependent lung. Moreover, elevation of the kidney rest can significantly decrease cardiac output in some patients by compressing the inferior vena cava. Venous pooling in the legs potentiates anesthesia-induced vasodilation. of the potential for large blood loss and limited

Because access to major vascular structures in the lateral flexed position, initial placement of at least one large-bore intravenous catheter is advisable. Arterial catheters are often utilized. Endotracheal tube placement may be altered during positioning; thus proper endotracheal tube placement must again be verified following final patient positioning prior to skin preparation and surgical draping. Intraoperative pneumothorax may occur as a result of surgical entry into the pleural space.

Improved survival rates for patients with urological cancer following radical surgical resections have resulted in an increase in the number of procedures performed for prostatic, bladder, testicular, and renal cancer. The desire for accelerated, less-complicated recovery with smaller, less painful incisions has prompted development of laparoscopic pelvic and abdominal operations, including radical prostatectomy, cystectomy, pelvic lymph node dissection, nephrectomy, and adrenalectomy. Robotic-assisted technology has increasingly been applied to these procedures over the past decade.
Many urological procedures are carried out with the patient in a hyperextended supine position to facilitate exposure of the pelvis during pelvic lymph node dissection, retropubic prostatectomy, or cystectomy (Figure 32-3). The patient is positioned supine with the iliac crest over the break in the operating table, and the table is extended such that the distance between the iliac crest and the costal margin increases maximally. Care must be taken to avoid putting excessive strain on the patient's back. The operating room table is also tilted head-down to make the operative field horizontal. In the frog-leg position, a variation of the hyperextended supine position, the knees are also flexed and the hips are abducted and externally rotated.
FIGURE 32-3 The hyperextended position. (Reproduced with permission from Skinner DG, Lieskovsky G. Diagnosis and Management of Genitourinary Cancer. Philadelphia, PA: WB Saunders; 1988.)

Adenocarcinoma of the prostate is the most common nonskin cancer in men and is second only to lung cancer as the most common cause of cancer deaths in men older than 55 years. Approximately one in six men will be diagnosed with prostate cancer in their lifetime. Management varies from surveillance to radical surgery. Important variables include the grade and stage of the malignancy, the patient's age, the concentration of prostate-specific antigen (PSA), and the presence of medical comorbidity. Transrectal ultrasound is used to guide transrectal biopsies. Clinical staging is based on the Gleason score of the biopsy specimens, magnetic resonance imaging (MRI) to determine whether there is tumor migration to regional lymph nodes, and bone scan.

Patients with prostate cancer may present to the operating room for open radical retropubic prostatectomy with lymph node dissection, robot-assisted laparoscopic prostatectomy with pelvic lymph node dissection, salvage prostatectomy (following failure of radiation therapy), cryoablation, or bilateral orchiectomy for hormonal therapy.

Radical retropubic prostatectomy is usually performed with pelvic lymph node dissection through a lower midline abdominal incision. It may be curative for localized prostate cancer or occasionally used as a salvage procedure after failure of radiation. The prostate is removed en bloc with the seminal vesicles, ejaculatory ducts, and part of the bladder neck. A "nerve-sparing" technique may be used to help preserve sexual function. Following prostatectomy, the remaining bladder neck is anastomosed directly to the urethra over an indwelling urinary catheter. The surgeon may ask for intravenous administration of indigo carmine for visualization of the ureters, and this dye can be associated with hypertension or hypotension.
Radical retropubic (open) prostatectomy may be accompanied by significant operative blood loss. Most centers use direct arterial blood pressure monitoring, and central venous pressure monitoring may also be employed. Other centers routinely utilize noninvasive cardiac output monitoring (eg, LiDCOrapid or FloTrac/Vigileo). Operative blood loss varies considerably from center to center, with mean values less than 500 mL common. Factors influencing blood loss include prostate size, duration of operation, and the skill and experience of the surgeon. Blood loss and operative morbidity and mortality are similar in patients receiving general anesthesia and those receiving regional anesthesia. Neuraxial anesthesia requires a T6 sensory level, but these patients typically do not tolerate regional anesthesia without deep sedation because of the hyperextended supine position. The combination of a prolonged Trendelenburg position together with administration of large amounts of intravenous fluids may rarely produce edema of the upper airway. The risk of hypothermia should be minimized by utilizing a forced-air warming blanket and an intravenous fluid warmer.

Postoperative complications include hemorrhage, deep venous thrombosis (perhaps with pulmonary embolus), injuries to the obturator nerve, ureter, and rectum, and urinary incontinence and impotence. Extensive surgical dissection around the pelvic veins increases the risk of intraoperative venous air embolism and postoperative thromboembolic complications. An enhanced recovery approach to perioperative care should be standard. Although epidural anesthesia may reduce the incidence of postoperative deep venous thrombosis following open prostatectomy, this beneficial effect may be negated by the routine use of warfarin or fractionated heparin prophylaxis postoperatively, and in the era of enhanced recovery is less often used. Ketorolac and acetaminophen can be used as analgesic adjuvants and have been reported to decrease opioid requirements, improve analgesia, and promote earlier return of bowel function.

Robot-assisted laparoscopic radical prostatectomy with pelvic lymph node dissection differs from most other laparoscopic procedures by the frequent use of steep (>30°) Trendelenburg position for surgical exposure. Patient positioning, duration of procedure, need for abdominal distention, and desirability of increasing minute ventilation require use of general endotracheal anesthesia. Nitrous oxide is usually avoided to prevent bowel distention. Most laparoscopic prostatectomies are performed with robotic assistance, and the majority of radical prostatectomies in the United States are now performed via robot-assisted laparoscopy. When compared with open retropubic prostatectomy, laparoscopic robot-assisted prostatectomy is associated with a longer procedure time but with less blood loss and fewer blood transfusions, lower postoperative pain scores and lower opioid requirements, less postoperative nausea and vomiting, and shorter hospital length of stay. The steep Trendelenburg position can lead to head and neck tissue edema and to increased intraocular pressure. Complications reported to be associated with such positioning include upper airway edema and postextubation respiratory distress, postoperative visual loss involving ischemic optic neuropathy or retinal detachment, and brachial plexus injury. The surgeon should be routinely advised as to the length of time during which steep Trendelenburg positioning is maintained, and some centers have abandoned the routine use of this positioning entirely.
Most clinicians use a single large-bore intravenous catheter. The risk of hypothermia should be minimized by utilizing a forced-air warming blanket and an intravenous fluid warmer. Adequate postoperative analgesia is provided by ketorolac or acetaminophen, or both, and supplemented as needed with opioids. Postoperative epidural analgesia is not warranted because of relatively low postoperative pain scores and because patients may be discharged 24 h after surgery.

Bilateral orchiectomy may be performed for hormonal control of metastatic prostate cancer. The procedure is relatively short (20-45 min) and is performed through a single midline scrotal incision. Although bilateral orchiectomy can be performed with local or regional anesthesia, most patients and many clinicians prefer general anesthesia, usually administered via a laryngeal mask airway, or spinal anesthesia.

Bladder cancer occurs at an average patient age of 65 years with a 3:1 male to female ratio. Transitional cell carcinoma of the bladder is second to prostate adenocarcinoma as the most common malignancy of the male genitourinary tract. The association of cigarette smoking with bladder carcinoma results in coexistent coronary artery and chronic obstructive pulmonary disease in many of these patients. Underlying kidney impairment, when present, may be age related or secondary to urinary tract obstruction. Staging includes cystoscopy and computed tomography (CT) or MRI scans. Intravesical chemotherapy is used for superficial tumors, and transurethral resection of bladder tumors (TURBT) is carried out via cystoscopy for low-grade, noninvasive bladder tumors. Some patients may receive preoperative radiation to shrink the tumor before radical cystectomy. Urinary diversion is usually performed immediately following cystectomy.

Bladder tumors may occur at various sites within the bladder, and laterally located tumors may lie in proximity to the obturator nerve. In such cases, if spinal anesthesia is administered or if general anesthesia is administered without use of a muscle relaxant, use of the cautery resectoscope may result in stimulation of the obturator nerve and adduction of the legs. Urologists rarely derive amusement from having their ear struck by the patient's knee; thus, in contrast to TURP, TURBT procedures are more commonly performed with general anesthesia and neuromuscular blockade. TURBT, unlike TURP, is rarely associated with absorption of significant amounts of irrigating solution.

Urinary diversion (ie, implanting the ureters into a segment of bowel) is usually performed immediately following radical cystectomy. The selected bowel segment is either left in situ, such as in ureterosigmoidostomy, or divided with its mesenteric blood supply intact and attached to a cutaneous stoma or urethra. Moreover, the isolated bowel can either function as a conduit (eg, ileal conduit) or be reconstructed to form a continent reservoir (neobladder). Conduits may be formed from ileum, jejunum, or colon.
Major anesthetic goals for urinary diversion procedures include keeping the patient well hydrated and maintaining a brisk urinary output once the ureters are opened. Neuraxial anesthesia often produces unopposed parasympathetic activity due to sympathetic blockade, which results in a contracted, hyperactive bowel that makes construction of a continent ileal reservoir technically difficult. Papaverine (100-150 mg as a slow intravenous infusion over 2-3 h), glycopyrrolate (1 mg), or glucagon (1 mg) may alleviate this problem.
Prolonged contact of urine with bowel mucosa (slow urine flow) may produce significant metabolic disturbances. Hyponatremia, hypochloremia, hyperkalemia, and metabolic acidosis can occur following construction of jejunal conduits. In contrast, colonic and ileal conduits may be associated with hyperchloremic metabolic acidosis. The use of temporary ureteral stents and maintenance of high urinary flow help alleviate this problem in the early postoperative period.

Testicular tumors are classified as either seminomas or nonseminomas. The initial treatment for all tumors is radical (inguinal) orchiectomy, and subsequent management depends on tumor histology. Retroperitoneal lymph node dissection (RPLND) plays a major role in the staging and management of patients with nonseminomatous germ cell tumors. Low-stage disease is managed with RPLND or in some instances by surveillance. High-stage disease is usually treated with chemotherapy followed by RPLND.
In contrast to other tissue types, seminomas are very radiosensitive tumors that are primarily treated with retroperitoneal radiotherapy. Chemotherapy is used for patients who relapse after radiation. Patients with large bulky seminomas or those with increased a-fetoprotein levels (usually associated with nonseminomas) are treated primarily with chemotherapy. Chemotherapeutic agents commonly include cisplatin, vincristine, vinblastine, cyclophosphamide, dactinomycin, bleomycin, and etoposide. RPLND is usually undertaken for patients with residual tumor after chemotherapy.
Patients undergoing RPLND for testicular cancer are typically young (15-35 years old) but are at increased risk for morbidity from the residual effects of preoperative chemotherapy and radiation therapy. In addition to bone marrow suppression, specific organ toxicity may be encountered, such as impaired kidney function following cisplatin, pulmonary fibrosis following bleomycin, and neuropathy following vincristine.

Inguinal orchiectomy can be carried out with regional or general anesthesia. Anesthetic management may be complicated by reflex bradycardia from traction on the spermatic cord.

The retroperitoneum is usually accessed through a midline incision, but regardless of the surgical approach, all lymphatic tissue between the ureters from the renal vessels to the iliac bifurcation is removed. With the standard RPLND, all sympathetic fibers are disrupted, resulting in loss of normal ejaculation and infertility. A modified technique that may help preserve fertility limits the dissection below the inferior mesenteric artery to include lymphatic tissue only on the ipsilateral side of the testicular tumor.
® Patients receiving bleomycin preoperatively may be particularly at risk for oxygen toxicity and fluid overload. Excessive intravenous fluid administration may promote pulmonary insufficiency or acute respiratory distress syndrome postoperatively and should be avoided. Anesthetic management should include use of the lowest inspired concentration of oxygen compatible with oxygen saturation above 90%. Positive end-expiratory pressure (5-10 cm H,O) may help optimize oxygenation.
Evaporative and redistributive fluid losses with open RPLND can be considerable as a result of the large wound and the extensive surgical dissection. Retraction of the inferior vena cava during surgery often results in transient arterial hypotension.
Postoperative pain associated with open RPLND incisions is severe, and continuous epidural analgesia, intrathecal morphine or hydromorphone, or TAP block should be considered. Because ligation of intercostal arteries during leftsided dissections has rarely resulted in paraplegia, it may be prudent to document normal motor function postoperatively prior to institution of epidural analgesia. The arteria radicularis magna (artery of Adamkiewicz), which is supplied by these vessels and is responsible for most of the arterial blood to the lower half of the spinal cord, arises on the left side in most individuals. It should be noted that unilateral sympathectomy following modified RPLND usually results in the ipsilateral leg being warmer than the contralateral one. Patients who have undergone RPLND frequently complain of severe bladder spasm pain in the postanesthesia care unit.

Renal cell carcinoma is the cause of approximately 3% of all adult cancers and 95% of all kidney cancers. It has a peak incidence between the fifth and sixth decades of life, with 2:1 male to female ratio. It is commonly discovered as an incidental finding in the course of evaluating a supposedly unrelated medical problem, such as in an MRI performed for evaluation of low back pain. The classic triad of hematuria, flank pain, and palpable mass occurs in only 10% of patients, and the tumor often causes symptoms only after it has grown considerably in size. Renal cell carcinoma is frequently associated with paraneoplastic syndromes, such as erythrocytosis, hypercalcemia, hypertension, and nonmetastatic hepatic dysfunction. Tumors confined to the kidney may be treated by open or laparoscopic partial or total nephrectomy, or by percutaneous cryoablation or radiofrequency ablation. Palliative surgical treatment may involve more extensive tumor debulking. In approximately 5% to 10% of patients, the tumor extends into the renal vein and inferior vena cava as a thrombus (Figure 32-4), and in some cases approaches or enters the right atrium. Staging includes CT or MRI scans and an arteriogram. Preoperative arterial embolization may shrink the tumor mass and reduce operative blood loss.

FIGURE 32-4 Mayo classification of venous thrombus invasion in renal cell carcinoma. Level I: Tumor thrombus is either at the entry of the renal vein or within the inferior vena cava (IVC) <2 cm from the confluence of the renal vein and the IVC. Level II: Thrombus extends within the IVC >2 cm above the confluence of the renal vein and IVC but still remains below the hepatic veins. Level III: Thrombus involves the intrahepatic IVC. The size of the thrombus ranges from a narrow tail that extends into the IVC to one that fills the lumen and enlarges the IVC. Level IV: Thrombus extends above the diaphragm or into the right atrium. (Reproduced with permission from Morita Y, Ayabe K, Nurok M, et al. Perioperative anesthetic management for renal cell carcinoma with venal caval thrombus extending into the right atrium: case series. J Clin Anesth. 2017 Feb;36:39-46.)
Preoperative evaluation of the patient with renal carcinoma should focus on tumor staging, kidney function, the presence of coexisting systemic diseases, and anesthetic management needs dictated by the scope of anticipated surgical resection. Preexisting kidney function impairment depends upon tumor size in the affected kidney as well as coexisting systemic disorders such as hypertension, diabetes, and coronary artery disease. Smoking is a wellestablished risk factor for renal cell carcinoma, and these patients have a high incidence of underlying coronary artery and chronic obstructive lung disease. Although some patients present with erythrocytosis, the majority are anemic.

Relatively small kidney tumors without metastasis are commonly ablated by interventional radiologists using percutaneous cryoprobes or radiofrequency probes with ultrasonography or CT guidance. This may be performed on an outpatient or 23-h stay basis. Routine American Society of Anesthesiologists (ASA) monitors are used, and general endotracheal anesthesia with muscle relaxation is usually employed to minimize the risk of patient movement during the procedure. An indwelling urinary catheter is typically used if the procedure duration is anticipated to be more than approximately 2 to 3 h. Precautions must be taken for patients with pacemakers or ICDs who are undergoing radiofrequency ablation (see Chapter 21). The patient is typically placed in the lateral decubitus or prone position. The patient may experience significant postoperative pain of limited duration requiring intravenous analgesia.

The operation may be carried out via an anterior subcostal, flank, or (rarely)
midline incision. Hand-assisted laparoscopic technique is often utilized for partial or total nephrectomy associated with a smaller tumor mass. Many centers prefer a thoracoabdominal approach for large tumors, particularly when a tumor thrombus is present. The kidney, adrenal gland, and perinephric fat are removed en bloc with the surrounding (Gerota) fascia. General endotracheal anesthesia is used, often in combination with epidural anesthesia.
This operation has the potential for extensive blood loss because these tumors are very vascular and often very large. Two large-bore intravenous lines with an indwelling peripheral arterial catheter are typically used. Transesophageal echocardiography (TEE), esophageal Doppler, or peripheral pulse wave analysis (Lidco or Vigileo) are often used for hemodynamic monitoring. TEE should be used for all patients with vena cava thrombus. Retraction of the inferior vena cava may be associated with transient arterial hypotension. Only brief periods of controlled hypotension should be used to reduce blood loss because of the potential for acute kidney injury in the contralateral kidney. Reflex vasoconstriction in the unaffected kidney can also result in postoperative kidney dysfunction.
If combined general—epidural anesthesia is employed, administration of epidural local anesthetic may be postponed until the risk of significant operative blood loss has passed. As with all lengthy operative procedures, the risk of hypothermia should be minimized by utilizing core temperature monitoring, a forced-air warming blanket and intravenous fluid warming. The postoperative course of subcostal, flank, or midline incision for open nephrectomy is extremely painful, and epidural analgesia is very useful in minimizing discomfort and accelerating convalescence.

Because of the degree of physiological trespass and potential for major blood loss associated with this operation, the anesthetic management can be challenging. A thoracoabdominal approach allows the use of cardiopulmonary bypass when necessary.
Surgery can significantly prolong and improve quality of life, and in some patients, metastases may regress after resection of the primary tumor. A preoperative ventilation-perfusion scan may detect preexisting pulmonary embolization of the thrombus. Intraoperative TEE is helpful in determining whether the uppermost margin of the tumor thrombus extends to the diaphragm, above the diaphragm, into the right atrium, or even across the tricuspid valve. TEE can also be used to confirm the absence of tumor in the vena cava, right atrium, and right ventricle after successful surgery.
The presence of a large thrombus (level II, III, or IV) complicates anesthetic management. Problems associated with massive blood transfusion should be anticipated (see Chapter 51). Central venous catheterization should be performed cautiously to prevent dislodgement and embolization of tumor thrombus extending into the right atrium. An increased central venous pressure is typical with significant caval thrombus and reflects the degree of venous obstruction. Pulmonary artery catheters risk dislodgement of right atrial tumor thrombus, and provide no useful information that cannot be obtained from TEE. Complete obstruction of the inferior vena cava markedly increases operative blood loss because of dilated venous collaterals. Patients are also at significant risk for potentially catastrophic intraoperative pulmonary embolization of the tumor. Tumor embolization may be heralded by sudden supraventricular arrhythmias, arterial desaturation, and profound systemic hypotension. TEE is critical in this situation. Cardiopulmonary bypass may be used when the tumor cannot be pulled back from the right atrium into the cava. Heparinization and hypothermia greatly increase surgical blood loss.

The success of renal transplantation, which is largely due to advances in immunosuppressive therapy, has greatly improved the quality of life for patients with end-stage kidney disease. With modern immunosuppressive regimens, cadaveric transplants have achieved almost the same 80% to 90% 3-year graft survival rate as living-related donor grafts.

Current organ preservation techniques allow ample time (24—48 h) for preoperative dialysis of cadaveric kidney recipients. Living-related transplants are performed electively with simultaneous donor and @® recipient operations. The recipient's serum potassium concentration should be below 5.5 mEq/L, and existing coagulopathies should be corrected.

Kidney transplantation is carried out by placing the donor kidney retroperitoneally in the iliac fossa and anastomosing the renal vessels to the iliac vessels and the ureter to the bladder. Heparin is administered prior to temporary clamping of the iliac vessels. Intravenous mannitol administered to the recipient helps establish an osmotic diuresis following reperfusion. Immunosuppression is initiated on the day of surgery with a combination of medications that may include corticosteroids, cyclosporine or tacrolimus, azathioprine or mycophenolate mofetil, antithymocyte globulin, monoclonal antibodies directed against specific subsets of T lymphocytes (OKT3), and interleukin-2 receptor antibodies (daclizumab or basiliximab). The anesthesia provider should discuss in advance with the surgery team the timing and dosage of any immunosuppressive agents that will need to be given perioperatively. Recipient nephrectomy for a failed transplant may be performed for intractable hypertension or chronic infection.

Most kidney transplants are performed with general anesthesia, although spinal and epidural anesthesia are also utilized. All general anesthetic agents have been employed without any apparent detrimental effect on graft function. Cisatracurium may be the muscle relaxant of choice, as it is not dependent upon renal excretion for elimination. With careful neuromuscular monitoring other relaxants can be used safely.

Aurinary catheter is placed preoperatively in addition to routine monitors, and brisk urine flow following the arterial anastomosis generally indicates good graft function. If the graft ischemic time was prolonged, an oliguric phase may precede the diuretic phase, in which case intravenous fluid therapy must be appropriately adjusted. Administration of furosemide or additional mannitol may be indicated in such cases. Hyperkalemia has been reported after release of the vascular clamp following completion of the arterial anastomosis, particularly in pediatric and other small patients, and release of potassium contained in the preservative solution has been implicated as the cause of this phenomenon. Donor kidney washout of the preservative solution with ice-cold lactated Ringer's solution just prior to the vascular anastomosis may help avoid this problem. Serum electrolyte concentrations (particularly potassium) should be monitored closely after completion of the anastomosis. Hyperkalemia may be suspected from peaking of the T wave on the ECG.

A 79-year-old man with a history of an inferior myocardial infarction was admitted to the recovery room following TURP under general anesthesia. The procedure took 90 min and was reported to be uncomplicated. On admission, the patient is extubated but still unresponsive, and vital signs are stable. Twenty minutes later, he is noted to be awake but restless. He begins to shiver intensely, his blood pressure decreases to 80/35 mm Hg, and his respirations increase to 40 breaths/min. The bedside monitor shows a sinus tachycardia of 140 beats/min and an oxygen saturation of 92%.

The differential diagnosis of hypotension following TURP should always include (1) hemorrhage, (2) TURP syndrome, (3) bladder perforation, (4) myocardial infarction or ischemia, (5) septicemia, and (6) disseminated intravascular coagulation (DIC).
Other possibilities (see Chapter 56) are less likely in this setting but should always be considered, particularly when the patient fails to respond to appropriate measures (see below).

A diagnosis cannot be made with reasonable certainty at this point, and the patient requires further evaluation. Nonetheless, the hypotension and shivering must be treated rapidly because of the history of coronary artery disease. The hypotension seriously compromises coronary perfusion, and the shivering markedly increases myocardial oxygen demand (see Chapter 21).

A quick examination of the patient is extremely useful in narrowing down the possibilities. Hemorrhage from the prostate should be apparent from effluent of the continuous bladder irrigation system placed after the procedure. Relatively little blood in the urine makes it look pink or red; brisk hemorrhage is often apparent as grossly bloody drainage. Occasionally, the drainage may be scant because of clots blocking the drainage catheter; irrigation of the catheter is indicated in such cases.
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The abdomen should be examined for signs of perforation. A rigid and tender or distended abdomen is very suggestive of perforation and should prompt immediate surgical evaluation. When the abdomen is soft and nontender, perforation can reasonably be excluded.

Further evaluation requires laboratory measurements, an ECG, a chest radiograph, and possibly a transthoracic echocardiogram. Blood should be immediately obtained for arterial blood gas analysis and measurements of hematocrit, hemoglobin, electrolytes, glucose, platelet count, and prothrombin and partial thromboplastin tests. If DIC is suggested by diffuse oozing, fibrinogen and fibrin split product measurements will confirm the diagnosis. A 12-lead ECG should be evaluated for evidence of ischemia or evolving myocardial infarction. A chest radiograph should be obtained to search for evidence of pulmonary congestion, aspiration, pneumothorax, or cardiomegaly. An echocardiogram helps determine end-diastolic volume and systolic function (particularly the presence or absence of regional wall motion abnormalities), and can detect valvular abnormalities; comparison to prior studies would be invaluable. Sonography of the abdomen can be used to detect extravasation.
While laboratory measurements are being performed, what therapeutic and diagnostic measures should be undertaken?
Immediate measures aimed at avoiding hypoxemia and hypoperfusion should be instituted. Supplemental oxygen should be administered, and endotracheal intubation is indicated if significant hypoventilation or respiratory distress is present. Frequent blood pressure measurements should be obtained. If signs of fluid overload are absent, a diagnostic fluid challenge with 300 to 500 mL of crystalloid or 250 mL of colloid is helpful. A favorable response, as indicated by an increase in blood pressure and a decrease in heart rate (or an increase in cardiac output as measured using a noninvasive monitor) supports a diagnosis of hypovolemia and may indicate the need for additional fluid boluses. Obvious bleeding in the setting of anemia and hypotension necessitates blood transfusion. The absence of a quick response to intravenous fluid volume challenge should prompt further evaluation. Administration of an inotrope is appropriate should ventricular dysfunction be detected by echocardiography. Direct intraarterial pressure measurement is invaluable in this setting.
If signs of fluid overload are present, intravenous furosemide in addition to an inotrope is indicated.

No. Anesthesia is commonly associated with altered temperature regulation. Moreover, correlation between axillary and core temperatures is quite variable (see Chapter 52). A high index of suspicion is therefore required to diagnose sepsis. Leukocytosis is common following surgery and is not a reliable indicator of sepsis in this setting.
The mechanism of shivering in patients recovering from anesthesia is poorly understood. Although shivering is common in patients who become hypothermic during surgery (and presumably functions to raise body temperature back to normal), its relation to body temperature is inconsistent. Anesthetics probably alter the normal behavior of hypothalamic thermoregulatory centers. In contrast, infectious agents, circulating toxins, or immune reactions cause the release of cytokines (interleukin-1 and tumor necrosis factor) that stimulate the hypothalamus to synthesize prostaglandin (PG) E>. The latter, in turn, activates neurons responsible for heat production, resulting in intense shivering.

Regardless of its cause, shivering has the undesirable effects of markedly increasing metabolic oxygen demand (100—200%) and CO, production. Both cardiac output and minute ventilation must therefore increase, and these effects are often poorly tolerated by patients with limited cardiac or pulmonary reserve. Although the ultimate therapeutic goal is to correct the underlying problem, additional measures are indicated in this patient. Supplemental oxygen therapy addresses hypoxemia. Meperidine in small doses (12.5—25 mg intravenously) frequently terminates shivering regardless of the cause. Shivering associated with sepsis and immune reactions can also be moderated or abolished by prostaglandin synthatase inhibitors (aspirin, acetaminophen, and nonsteroidal antiinflammatory agents), of which only acetaminophen would likely be appropriate until hemorrhage was ruled out because it does not affect platelet function.

Examination of the patient reveals warm extremities with a good pulse, even with the low blood pressure. The abdomen is soft and nontender. The irrigation fluid from the bladder is only slightly pink. A diagnosis of probable sepsis is made. Blood cultures are obtained and antibiotic therapy is initiated to cover gram-negative organisms and enterococci, the most common pathogens. The patient receives empiric antibiotic coverage and a dopamine infusion is initiated. In settings of redistributive, vasodilatory shock, additional vasoconstrictors (eg, vasopressin) may be needed. The shivering ceases following administration of meperidine, 12.5 mg intravenously. The blood pressure increases to 110/60 mm Hg and the heart rate slows to 90 beats/min following a 1000 mL intravenous fluid bolus and initiation of a 5-mcg/kg/min dopamine infusion. The serum sodium concentration was found to be 130 mEq/L. Four hours later, dopamine was no longer needed and was discontinued. The patient's subsequent recovery was uneventful.

Xu W, Daneshmand S, Bazargani S, et al. Postoperative pain management after radical cystectomy: Comparing traditional versus enhanced recovery protocol pathway. J Urol. 2015;194:1209.

The hepatic artery supplies about 25% of the blood supply but 45% to 50% of the liver's oxygen requirements, and the portal vein supplies 75% of the blood supply and the remaining 50% to 55% of oxygen
@ requirements.
@ All coagulation factors, with the exception of factor VIII and von Willebrand factor, are produced by the liver. Vitamin K is a necessary cofactor in the synthesis of prothrombin (factor II) and factors VII, IX, and X.
@® Many "liver function" tests, such as serum transaminase measurements, reflect hepatocellular integrity more than hepatic function. Liver tests that measure hepatic synthetic function include serum albumin, prothrombin time (PT) or international normalized ratio (INR), serum cholesterol, and plasma pseudocholinesterase.
@ Albumin values less than 2.5 g/dL are generally indicative of chronic liver disease, acute stress, or severe malnutrition. Increased losses of albumin in the urine (nephrotic syndrome) or the gastrointestinal tract (protein-losing enteropathy) can also produce hypoalbuminemia.
@ The PT, which normally ranges between 11 and 14 s, depending on the control value, measures the activity of fibrinogen, prothrombin, and factors V, VII, and X. A prolonged INR reflects a patient with a dysfunctional liver. The effect on coagulation will depend on the balance between coagulation and anticoagulation factors. If production of protein C, protein S, and antithrombin 3, are affected more than the coagulation factors a normal or hypercoaguable state might exist. The INR was developed to monitor the effect of warfarin and that does not
affect the anticoagulant factors.

@ Operative procedures near the liver can reduce hepatic blood flow up to 60%. Although the mechanisms are not clear, they most likely involve sympathetic activation, local reflexes, and direct compression of vessels in the portal and hepatic circulations.
@ The neuroendocrine stress response to surgery and trauma is characterized by elevated circulating levels of catecholamines, glucagon, and cortisol and results in mobilization of carbohydrate stores and proteins, causing hyperglycemia and a negative nitrogen balance (catabolism).
@ All opioids can potentially cause spasm of the sphincter of Oddi and increase biliary pressure.
© When liver tests are abnormal postoperatively, the usual cause is underlying liver disease or the surgical procedure itself.

The liver is the heaviest organ in the body, weighing approximately 1500 g in adults. It is separated by the falciform ligament into right and left anatomic lobes; the larger right lobe has two additional smaller lobes at its posterior— inferior surface, the caudate and quadrate lobes.
In contrast, surgeons describe the liver based on its blood supply. Thus, the right and left surgical lobes are defined by anatomists by the point of bifurcation of the hepatic artery and portal vein (porta hepatis); the falciform ligament therefore divides the left surgical lobe into medial and lateral segments. Surgical anatomy defines a total of eight segments.
The liver is made up of 50,000 to 100,000 discrete anatomic units called lobules. Each lobule is composed of plates of hepatocytes arranged cylindrically around a centrilobular vein (Figure 33-1). Four to five portal tracts, composed of hepatic arterioles, portal venules, bile canaliculi, lymphatics, and nerves, surround each lobule.

In contrast to a lobule, an acinus, the functional unit of the liver, is defined by a portal tract in the middle and centrilobular veins at the periphery. Cells closest to the portal tract (zone 1) are well oxygenated; those closest to centrilobular veins (zone 3) receive the least oxygen and are therefore most susceptible to ischemic injury.
Blood from hepatic arterioles and portal venules comingle in the sinusoidal channels, which lie between the cellular plates and serve as capillaries. These channels are lined by endothelial cells and by macrophages known as Kupffer cells. The Kupffer cells remove bacterial endotoxins, viruses, proteins, and particulate matter from the blood. The space of Disse lies between the sinusoidal capillaries and the hepatocytes. Venous drainage from the central veins of hepatic lobules coalesces to form the hepatic veins (right, middle, and left), which empty into the inferior vena cava (Figure 33—2). The caudate lobe is usually drained by its own set of veins.
FIGURE 33-2 Hepatic blood flow. (Modified with permission from Guyton AC. Textbook of Medical Physiology. 7th ed. Philadelphia, PA: WB Saunders; 1986.)
Bile canaliculi originate between hepatocytes within each plate and join to form bile ducts. An extensive system of lymphatic channels also forms within the plates and is in direct communication with the space of Disse.
The liver is supplied by T6—-T11 sympathetic nerve fibers, right and left vagal nerve parasympathetic fibers, and right phrenic nerve fibers. Some autonomic fibers synapse first in the celiac plexus, whereas others reach the liver directly via splanchnic nerves and vagal branches before forming the hepatic plexus. The majority of sensory afferent fibers travel with sympathetic fibers.

Normal hepatic blood flow is 25% to 30% of the cardiac output and is provided by the hepatic artery and @ portal vein. The hepatic artery supplies about 25% of the blood supply but 45% to 50% of the liver's oxygen requirements, and the portal vein supplies 75% of the blood supply and the remaining 50% to 55%
(Figure 33-2). Hepatic arterial flow is dependent on metabolic demand (autoregulation), whereas flow through the portal vein is dependent on blood flow to the gastrointestinal tract and the spleen. A reciprocal, though somewhat limited, mechanism exists, such that a decrease in either hepatic arterial or portal venous flow results in a compensatory increase in the other. The hepatic artery has a,-adrenergic vasoconstriction receptors as well as B5adrenergic, dopaminergic (D,), and cholinergic vasodilator receptors. The portal vein has only a,-adrenergic and dopaminergic (D,) receptors. Sympathetic activation results in vasoconstriction of the hepatic artery and mesenteric vessels, decreasing hepatic blood flow. B-Adrenergic stimulation vasodilates the hepatic artery; B-blockers reduce blood flow and, therefore, decrease portal pressure.

Portal vein pressure is normally only about 7 to 10 mm Hg, but the low resistance of the hepatic sinusoids allows relatively large blood flows through the portal vein. Small changes in hepatic venous tone and hepatic venous pressure thus can result in large changes in hepatic blood volume, allowing the liver to act as a blood reservoir (Figure 33—3). A decrease in hepatic venous pressure, as occurs during hemorrhage, shifts blood from hepatic veins and sinusoids into the central venous circulation and augments circulating blood volume. Blood loss can be reduced during liver surgery by lowering the central venous pressure, thereby reducing hepatic venous pressure and hepatic blood volume. In patients with congestive heart failure, the increase in central venous pressure is transmitted to the hepatic veins and causes congestion of the liver that can adversely affect liver function.
FIGURE 33-3 The role of the liver as a blood reservoir. (Modified with permission from Lautt WW, Greenway CV. Hepatic venous compliance and role of liver as a blood reservoir. Am J Physiol. 1976 Aug;231(2):292-295.)

The abundance of enzymatic pathways in the liver allows it to play a key role in the metabolism of carbohydrates, fats, proteins, and other substances (Figure 33-4 and Table 33-1). The final products of carbohydrate digestion are glucose, fructose, and galactose. With the exception of the large amount of fructose that is converted by the liver to lactate, hepatic conversion of fructose and galactose into glucose makes glucose metabolism the final common pathway for most carbohydrates.
FIGURE 33-4 Important metabolic pathways in hepatocytes. Although small amounts of adenosine triphosphate (ATP) are derived directly from some intermediary reactions, the overwhelming majority of ATP produced is the result of oxidative phosphorylation of the reduced forms of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH).

Creation and secretion of bile
Nutrient metabolism
Amino acids
Monosaccharides (sugars)
Lipids (fatty acids, cholesterol, phospholipids, lipoproteins)
Vitamins
Phase I and II biotransformation
Toxins
Drugs
Hormones (steroids)

Albumin, a,-antitrypsin, proteases
Clotting factors
Acute phase proteins
Plasma cholinesterase
Immune function
Kupffer cells
All cells utilize glucose to produce energy in the form of adenosine triphosphate (ATP), either aerobically via the citric acid cycle or anaerobically via glycolysis. The liver and adipose tissue can also utilize the phosphogluconate pathway, which provides energy and fatty acid synthesis. Most of the glucose absorbed following a meal is normally stored as glycogen, which only the liver and muscle are able to store in significant amounts. When glycogen storage capacity is exceeded, excess glucose is converted into fat. Insulin enhances glycogen synthesis, and epinephrine and glucagon enhance glycogenolysis. Because glucose consumption averages 150 g/d, and hepatic glycogen stores are normally depleted after 24 h of fasting. After this period of fasting, gluconeogenesis, the de novo synthesis of glucose, is necessary to provide an uninterrupted supply of glucose for other organs.
The liver and kidney are unique in their capacity to form glucose from lactate, pyruvate, amino acids (mainly alanine), and glycerol (derived from fat metabolism). Hepatic gluconeogenesis is vital in the maintenance of a normal blood glucose concentration. Glucocorticoids, catecholamines, glucagon, and thyroid hormone greatly enhance gluconeogenesis, whereas insulin inhibits it.
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fatty acids that have been synthesized by the liver as an energy source.
enzyme that hydrolyzes esters, including ester local anesthetics and some muscle relaxants, including succinylcholine. Other important proteins formed by the liver include protease inhibitors (antithrombin III, a,-antiplasmin, and a,antitrypsin), transport proteins (transferrin, haptoglobin, and ceruloplasmin), complement, a,-acid glycoprotein, C-reactive protein, and serum amyloid A.

|,  = Fibrinogen. |, Approximate Half-Life (h) = 100. ll,  = Prothrombin. ll, Approximate Half-Life (h) = 80. Il,  = Tissue thromboplastin. Il, Approximate Half-Life (h) = —. lV,  = Calcium. lV, Approximate Half-Life (h) = —. Vv,  = Proaccelerin. Vv, Approximate Half-Life (h) = 18. Vil,  = Proconvertin. Vil, Approximate Half-Life (h) = 6. Vill,  = Antihemophilic factor. Vill, Approximate Half-Life (h) = 10. IX,  = Christmas factor. IX, Approximate Half-Life (h) = 24. x,  = Stuart factor. x, Approximate Half-Life (h) = 50. xl,  = Plasma thromboplastin antecedents. xl, Approximate Half-Life (h) = 25. XII,  = Hageman factor. XII, Approximate Half-Life (h) = 60. XIill,  = Fibrin-stabilizing factor. XIill, Approximate Half-Life (h) = 90
FIGURE 33-5 The intrinsic and extrinsic coagulation pathways. HMWK, highmolecular-weight kininogen.

Bile (Table 33-3) plays an important role in absorption of fat and excretion of bilirubin, cholesterol, and many drugs. Hepatocytes continuously secrete bile salts, cholesterol, phospholipids, conjugated bilirubin, and other substances into

97% water
<1% bile salts
Pigments
Inorganic salts
Lipids
Fatty acids
Lecithin
Alkaline phosphatase
Bile ducts from hepatic lobules join and eventually form the right and left hepatic ducts. These ducts, in turn, combine to form the hepatic duct, which together with the cystic duct from the gallbladder becomes the common bile duct (Figure 33-6). The gallbladder serves as a reservoir for bile. The bile acids formed by hepatocytes from cholesterol are essential for emulsifying the insoluble components of bile and facilitating the intestinal absorption of lipids. Defects in the formation or secretion of bile salts interfere with the absorption of fats and fat-soluble vitamins (A, D, E, and K). Because of limited stores of vitamin K, a deficiency of this fat-soluble vitamin can develop within a few days. Vitamin K deficiency is manifested as a coagulopathy due to impaired formation of prothrombin and of factors VII, [X, and X.
FIGURE 33-6 The biliary system. (Modified with permission from Guyton AC. Textbook of Medical Physiology. 7th ed. Philadelphia, PA: WB Saunders; 1986.)
Bilirubin is primarily the end product of hemoglobin metabolism, and it is formed from degradation of the heme ring in Kupffer cells. Bilirubin is then released into blood, where it readily binds to albumin. Hepatic uptake of bilirubin from the circulation is passive, but binding to intracellular proteins traps the bilirubin inside hepatocytes. Bilirubin is conjugated by the hepatocytes, primarily with glucuronide, and actively excreted into bile canaliculi.

The most commonly performed liver tests are neither sensitive nor specific. No one laboratory test evaluates overall hepatic function, reflecting instead one aspect of hepatic function that must be interpreted in conjunction with other tests and clinical assessment of the patient.
® Many "liver function" tests, such as serum transaminase measurements, reflect hepatocellular integrity more than hepatic function. Liver tests that measure hepatic synthetic function include serum albumin, prothrombin time (PT) or international normalized ratio (INR), serum cholesterol, and plasma pseudocholinesterase. Moreover, because of the liver's large functional reserve, substantial cirrhosis may be present with few or no laboratory abnormalities evident.
Liver abnormalities can often be divided into either parenchymal disorders or obstructive disorders based on laboratory tests (Table 33—4). Obstructive disorders primarily affect biliary excretion of substances, whereas parenchymal disorders result in generalized hepatocellular dysfunction.

AST (SGOT), Parenchymal (Hepatocellular) Dysfunction = Tto TTT. AST (SGOT), Biliary Obstruction or Cholestasis = tT. ALT (SGPT), Parenchymal (Hepatocellular) Dysfunction = Tto TTT. ALT (SGPT), Biliary Obstruction or Cholestasis = iti. Albumin, Parenchymal (Hepatocellular) Dysfunction = Otolll. Albumin, Biliary Obstruction or Cholestasis = 0). Prothrombin time, Parenchymal (Hepatocellular) Dysfunction = oto TTT. Prothrombin time, Biliary Obstruction or Cholestasis = Oto TT. Bilirubin, Parenchymal (Hepatocellular) Dysfunction = Oto TTT. Bilirubin, Biliary Obstruction or Cholestasis = OtoTTT. Alkaline phosphatase, Parenchymal (Hepatocellular) Dysfunction = F. Alkaline phosphatase, Biliary Obstruction or Cholestasis = Tto TTT. 5’-Nucleotidase, Parenchymal (Hepatocellular) Dysfunction = OtoT. 5’-Nucleotidase, Biliary Obstruction or Cholestasis = T to TTT. y-Glutamyl transpeptidase, Parenchymal (Hepatocellular) Dysfunction = Tto TTT. y-Glutamyl transpeptidase, Biliary Obstruction or Cholestasis = TTT
'Adapted with permission from Wilson JD, Braunwald E, Isselbacher KJ et al. Harrison's Principles of Internal Medicine. 12th ed. New York, NY: McGraw-Hill Education; 1991.
"AST, aspartate aminotransferase; SGOT, serum glutamic-oxaloacetic transaminase; ALT, alanine aminotransferase; SGPT, serum glutamic pyruvic-transferase.
- 37, increases; 0, no change; 1, decreases.
"Usually corrects with vitamin K.

is 1.5
The normal total bilirubin concentration, composed of conjugated (direct), water-soluble and unconjugated (indirect), lipid-soluble forms, less than mg/dL (<25 mmol/L) and reflects the balance between bilirubin production and excretion. Jaundice is usually clinically obvious when total bilirubin exceeds 3 mg/dL. A predominantly conjugated hyperbilirubinemia (>50%) is associated with increased urinary urobilinogen and may reflect hepatocellular dysfunction, congenital (Dubin—Johnson or Rotor syndrome) or acquired intrahepatic cholestasis, or extrahepatic biliary obstruction. Hyperbilirubinemia that is primarily unconjugated may be seen with hemolysis or with congenital (Gilbert or Crigler—Najjar syndrome) or acquired defects in bilirubin conjugation. Unconjugated bilirubin is neurotoxic, and high levels may produce encephalopathy.

These enzymes are released into the circulation as a result of hepatocellular injury or death. Two aminotransferases are most commonly measured: aspartate aminotransferase (AST), also known as serum glutamic-oxaloacetic transaminase (SGOT); and alanine aminotransferase (ALT), also known as serum glutamic pyruvic-transferase (SGPT).

Alkaline phosphatase is produced by the liver, bone, small bowel, kidneys, and placenta and is excreted into bile. Normal serum alkaline phosphatase activity is 25 to 85 IU/L; children and adolescents have much higher levels, reflecting active growth. Most circulating alkaline phosphatase is normally derived from bone; however, with biliary obstruction, more hepatic alkaline phosphatase is synthesized and released into the circulation.

The normal serum albumin concentration is 3.5 to 5.5 g/dL. Because its half-life is approximately 2 to 3 weeks, albumin concentration may initially be @ normal with acute liver disease. Albumin values less than 2.5 g/dL are generally indicative of chronic liver disease, acute stress, or severe malnutrition. Increased losses of albumin in the urine (nephrotic syndrome) or the gastrointestinal tract (protein-losing enteropathy) can also produce hypoalbuminemia.

Significant elevations of blood ammonia levels usually reflect disruption of hepatic urea synthesis. Normal whole blood ammonia levels are 47 to 65 mmol/L (80-110 mg/dL). Marked elevations usually reflect severe hepatocellular damage and may cause encephalopathy.

TABLE 33-5 Coagulation test abnormalities.
Advanced liver disease, PT = T. Advanced liver disease, PTT = T. Advanced liver disease, TT = Nor?. Advanced liver disease, Fibrinogen = Nord. DIC, PT = T. DIC, PTT = fi. DIC, TT = T. DIC, Fibrinogen = L. Vitamin K deficiency, PT = TT. Vitamin K deficiency, PTT = 1). Vitamin K deficiency, TT = N. Vitamin K deficiency, Fibrinogen = N. Warfarin therapy, PT = TT. Warfarin therapy, PTT = 7. Warfarin therapy, TT = N. Warfarin therapy, Fibrinogen = N. Heparin therapy, PT = t. Heparin therapy, PTT = Tt. Heparin therapy, TT = A. Heparin therapy, Fibrinogen = N. Hemophilia Factor VIII deficiency Factor IX deficiency, PT = N N. Hemophilia Factor VIII deficiency Factor IX deficiency, PTT = iy T. Hemophilia Factor VIII deficiency Factor IX deficiency, TT = N N. Hemophilia Factor VIII deficiency Factor IX deficiency, Fibrinogen = N N. Factor Vil deficiency, PT = T. Factor Vil deficiency, PTT = N. Factor Vil deficiency, TT = N. Factor Vil deficiency, Fibrinogen = N. Factor XIll deficiency, PT = N. Factor XIll deficiency, PTT = N. Factor XIll deficiency, TT = N. Factor XIll deficiency, Fibrinogen = N
- 'PT, prothrombin time; PTT, partial thromboplastin time; TT, thrombin time; N, normal; DIC, disseminated intravascular coagulation.

This technology provides a "real time" assessment of the coagulation status and utilizes thromboelastography (TEG), rotation thromboelastometry (ROTEM), or Sonoclot analysis to assess global coagulation via the viscoelastic properties of whole blood (Figure 33-7). A clear picture is provided of the global effect of balance between the procoagulant and anticoagulant systems and the profibrinolytic and antifibrinolytic systems and the resultant clot tensile strength, allowing precise management of hemostatic therapy. The rate of clot formation, the strength of the clot, and the impact of any clot lysis can be observed. The presence of disseminated intravascular coagulation can be evaluated, as can the effect of heparin or heparinoid activity. In addition, platelet function can be assessed, including the effects of platelet inhibition. Viscoelastic coagulation monitoring is particularly important when assessing the coagulation and thromboembolic risk of the patient who has a prolonged INR from liver dysfunction and has undergone, or is about to undergo, procedural care. Coagulopathy is a balance of the procoagulant and anticoagulant factors produced by the liver, and the INR only examines the procoagulant side. A patient with an INR of 3, for example, may have also have anticoagulant factors so reduced that in fact the patient is in a hypercoagulable state and thereby actually at increased risk for venous thromboembolism. This risk can be readily evaluated by viscoelastic testing.
FIGURE 33-7 Examples of typical thromboelastograph tracings. A: Normal. B: Hypercoagulation. C: Hypocoagulation (eg, thrombocytopenia). D: Fibrinolysis. (Reproduced with permission from Johansson PI, Stissing T, Bochsen L, et al. Thrombelastography and thromboelastometry in assessing coagulopathy in trauma. Scand J Trauma Resusc Emerg Med. 2009 Sep 23:17:45.)

Hepatic blood flow usually decreases during regional and general anesthesia, and multiple factors are responsible, including both direct and indirect effects of anesthetic agents, the type of ventilation employed, and the type of surgery being performed.
Decreases in cardiac output reduce hepatic blood flow. The hemodynamic effects of ventilation can also have a significant impact on hepatic blood flow. Controlled positive-pressure ventilation with high mean airway pressures reduces venous return and cardiac output, and can compromise hepatic blood flow. The former increases hepatic venous pressure, whereas the latter can reduce blood pressure and increase sympathetic tone. Positive end-expiratory pressure (PEEP) further accentuates these effects.
B-Adrenergic blockers, a-adrenergic agonists, H,-receptor blockers, and vasopressin reduce hepatic blood flow. Dopamine infusions (0.5—2.5 mcg/kg/min) may increase liver blood flow.

The effects of the various anesthetic agents on hepatic metabolism are poorly defined. An endocrine stress response secondary to fasting and surgical @ generally observed. The neuroendocrine and trauma is stress response to surgery trauma is characterized by elevated circulating levels of catecholamines, glucagon, and cortisol and results in the mobilization of carbohydrate stores and protein, causing hyperglycemia and negative nitrogen balance (catabolism). The neuroendocrine stress response may be at least partially blunted by regional anesthesia, deep general anesthesia, or pharmacological blockade of the sympathetic system, with regional anesthesia having the most salutary effect @ on catabolism. All opioids can potentially cause spasm of the sphincter of Oddi and increase biliary pressure. Naloxone and glucagon may relieve opioidinduced spasm.
Procedures in close proximity to the liver frequently result in modest elevations in lactate dehydrogenase and transaminase concentrations regardless of the anesthetic agent or technique employed. @ When liver function tests are abnormal postoperatively, the usual cause is underlying liver disease or the surgical procedure itself. Persistent abnormalities in liver tests may be indicative of viral hepatitis, sepsis, idiosyncratic drug reactions, or surgical complications. Postoperative jaundice can result from a variety of factors (Table 33-6), but the most common cause is overproduction of bilirubin because of resorption of a large hematoma or red cell breakdown following transfusion. Nonetheless, all other causes should be considered. Correct diagnosis requires a careful review of preoperative liver function and of intraoperative and postoperative events, such as transfusions, sustained hypotension or hypoxemia, and drug exposure. Desflurane, sevoflurane, and isoflurane have minimal, if any, direct adverse effect upon hepatocytes.

Resorption of hematomas
Hemolytic anemia transfusion
Senescent red cell breakdown
Hemolytic reactions
Hepatic (hepatocellular dysfunction)
Preexisting liver disease
Ischemic or hypoxemic injury
Drug-induced
Gilbert syndrome
Intrahepatic cholestasis
Halothane
Posthepatic (biliary obstruction)
Postoperative cholecystitis
Postoperative pancreatitis
Retained common bile duct stone
Bile duct injury
Miscellaneous

Coagulopathy in a Patient with Liver Disease (also see
Chapter 51)
A 52-year-old man with a long history of alcohol abuse presents for a splenorenal shunt after three major episodes of upper gastrointestinal hemorrhage from esophageal varices. Coagulation studies reveal a PT of 17 s (control: 12 s), INR of 1.7, and a partial PT of 43 s (control: 29 s). The platelet count is 75,000/pL.
What factors can contribute to excessive bleeding during and following surgery?
Hemostasis following trauma or surgery is dependent on three major processes: (1) vascular spasm, (2) formation of a platelet plug (primary hemostasis), and (3) coagulation of blood (secondary hemostasis) in addition to adequate surgical control of bleeding sites. The first two are nearly immediate (seconds), whereas the third is delayed (minutes). A defect in any of these processes can lead to a bleeding diathesis and increased blood loss.
Outline the mechanisms involved in primary hemostasis.
Injury to smaller blood vessels normally causes localized spasm as a result of the release of humoral factors from platelets and local myogenic reflexes. Sympathetic-mediated vasoconstriction is also operative in medium-sized vessels. Exposure of circulating platelets to the damaged endothelial surface causes them to undergo a series of changes that results in the formation of a platelet plug. If the break in a vessel is small, the plug itself can often completely stop bleeding. If the break is large, however, coagulation of blood is also necessary to stop the bleeding.

Formation of the platelet plug can be broken down into three stages: (1) adhesion, (2) release of platelet granules, and (3) aggregation. Following injury, circulating platelets adhere to subendothelial collagen via specific glycoprotein (GP) receptors on their membrane. This interaction is stabilized by a circulating GP called von Willebrand factor (vWF), which forms additional bridges between subendothelial collagen and platelets via GPIb. Collagen (as well as epinephrine and thrombin) activates platelet membrane-bound phospholipases A and C, which, in turn, results in the formation of thromboxane A, (TXA>) and platelet degranulation. TXA, is a potent vasoconstrictor that also promotes platelet aggregation. Platelet granules contain a large number of substances, including adenosine diphosphate (ADP), factor V, vWF, fibrinogen, and fibronectin. These factors attract and activate additional platelets. ADP alters platelet membrane GPIIb/IIIa, which facilitates the binding of fibrinogen to activated platelets.

Coagulation, often referred to as secondary hemostasis, involves formation of a fibrin clot, which usually binds and strengthens a platelet plug. Fibrin can be formed via one of two pathways (extrinsic or intrinsic; see Figure 33-5) that involve calcium and activation of soluble coagulation precursor proteins in blood (see Table 33-2). Regardless of which pathway is activated, the coagulation cascade ends in the conversion of fibrinogen to fibrin. The extrinsic pathway of the coagulation cascade is triggered by the release of a tissue lipoprotein, thromboplastin, from the membranes of injured cells and is likely the more important pathway. The intrinsic pathway can be triggered by the interaction between subendothelial collagen with circulating Hageman factor (XII), high-molecular-weight kininogen, and prekallikrein. The latter two substances are also involved in the formation of bradykinin.
Thrombin plays a central role in coagulation because it not only activates platelets, but also accelerates conversion of factors V, VIII, and XIII to their active forms. Conversion of prothrombin to thrombin is markedly accelerated by activated platelets. Thrombin then converts fibrinogen to soluble fibrin monomers that polymerize on the platelet plug. The crosslinking of fibrin polymers by factor XIII is necessary to form a strong, insoluble fibrin clot. Finally, retraction of the clot, which requires platelets, expresses fluid from the clot and helps pull the walls of the damaged blood vessel together.

The coagulation process is limited to injured areas by localization of platelets to the injured area and by maintenance of normal blood flow in uninjured areas. Normal endothelium produces prostacyclin (prostaglandin I,, PGI,), which is a potent vasodilator that also inhibits platelet activation and helps to confine the primary hemostatic process to the injured area. Normal blood flow is important in clearing activated coagulation factors, which are taken up by the monocyte—macrophage scavenger system. Multiple inhibitors of coagulation are normally present in plasma, including antithrombin III, protein C, protein S, and tissue factor pathway inhibitor. Antithrombin III complexes with and inactivates circulating coagulation factors (with the notable exception of factor VII), and protein C specifically inactivates factors V and VIII. Heparin exerts its anticoagulant activity by augmenting the activity of antithrombin III. Protein S enhances the activity of protein C, and deficiencies of protein C and protein S lead to hypercoagulability. Tissue factor pathway inhibitor antagonizes the action of activated factor VII.

The fibrinolytic system is normally activated simultaneously with the coagulation cascade and functions to maintain the fluidity of blood during coagulation. It is also responsible for clot lysis once tissue repair begins. When a clot is formed, a large amount of the protein plasminogen is incorporated. Plasminogen is then activated by tissue plasminogen activator (tPA), which is usually released by endothelial cells in response to thrombin, and by Hageman factor (XII). The resulting formation of plasmin degrades fibrin and fibrinogen, as well as other coagulation factors. Urokinase (found in urine) and streptokinase (a product of bacteria) are also potent activators of plasminogen to plasmin. The action of tPA is localized because (1) it is absorbed into the fibrin clot, (2) it activates plasminogen more effectively on the clot, (3) free plasmin is rapidly neutralized by a circulating a-antiplasmin, and (4) circulating tPA is cleared by the liver. Plasmin degrades fibrin and fibrinogen into small fragments. These fibrin degradation products possess anticoagulant activity because they compete with fibrinogen for thrombin; they are normally cleared by the monocyte— macrophage system. The drugs e-aminocaproic acid (EACA) and tranexamic acid inhibit the conversion of plasminogen to plasmin. Endothelium also normally secretes a plasminogen activator inhibitor (PAI1) that antagonizes tPA.

Multifactorial coagulopathy often develops in patients with advanced liver disease. Three major causes are usually responsible: (1) vitamin K deficiency due to dietary deficiency or to impaired absorption or storage, (2) impaired hepatic synthesis of coagulation factors, and (3) splenic sequestration of platelets resulting from hypersplenism. To complicate matters further, patients with cirrhosis typically have multiple potential bleeding sites (esophageal varices, gastritis, peptic ulcers, and hemorrhoids) and frequently require multiple blood transfusions. With severe liver disease, patients may also have decreased synthesis of coagulation inhibitors and may fail to clear activated coagulation factors and fibrin split products because of impaired Kupffer cell function; the resultant coagulation defect resembles, and becomes indistinguishable from, disseminated intravascular coagulation (DIC).

In DIC, the coagulation cascade is activated by the release of endogenous tissue thromboplastin or thromboplastin-like substances, or by direct activation of factor XII by endotoxin or foreign surfaces. Widespread deposition of fibrin in the microcirculation results in consumption of coagulation factors, secondary fibrinolysis, acute severe thrombocytopenia, and a microangiopathic hemolytic anemia. Diffuse bleeding, and, in some cases, thromboembolic phenomena, usually follows. Treatment is generally aimed at the underlying cause. Supportive measures include transfusion of coagulation factors and platelets. Heparin therapy is controversial but may benefit patients with thromboembolic phenomena.

This bleeding disorder is due to uncontrolled fibrinolysis. Patients may have a deficiency of a,-antiplasmin or impaired clearance of tPA. The latter may be common in patients with severe liver disease and during the anhepatic phase of liver transplantation. The disorder may occasionally be encountered in patients with carcinoma of the prostate. Diagnosis is often difficult, but is suggested by a bleeding diathesis with a low fibrinogen level but relatively normal coagulation tests and platelet count (as noted next). Treatment includes fresh frozen plasma or cryoprecipitate and possibly either EACA or tranexamic acid.

The diagnosis of coagulation abnormalities can be facilitated by measurement of the activated partial thromboplastin time (aPTT), PT, thrombin time (TT), fibrin degradation products, and fibrinogen level (see Table 33-5). The aPTT measures the intrinsic pathway (factors I, II, V, VIII, IX, X, XI, and XII). The whole blood clotting time and activated clotting time (ACT) also measure the intrinsic pathway. In contrast, the PT measures the extrinsic pathway (factors I, II, V, and VII). The TT specifically measures conversion of fibrinogen to fibrin (factors I and II). The normal plasma fibrinogen level is 200 to 400 mg/dL (5.9-11.7 umol/L). Because heparin therapy primarily affects the intrinsic pathway, in low doses it usually prolongs the aPTT only. In high doses, heparin also prolongs the PT. In contrast, warfarin primarily affects vitamin Kdependent factors (II, VII, IX, and X), so the PT is prolonged at usual doses, and the aPTT is prolonged only at high doses. In vivo plasmin activity can be evaluated by measuring circulating levels of peptides cleaved from fibrin and fibrinogen by plasmin, namely fibrin degradation products (FDPs) and D-dimers. Patients with primary fibrinolysis usually have elevated FDPs, but normal p-dimer levels.
What tests are most helpful in evaluating inadequate primary hemostasis?

The most commonly performed tests include a platelet count and a bleeding time, but also include TEG, ROTEM, and Sonoclot analysis (see Figure 33-7 and Chapter 51). Patients with normally functioning platelets and platelet counts above 100,000/y1L have normal primary hemostasis. The normal platelet count is 150,000 to 450,000/pL, and the bleeding time is generally not affected by the platelet count when the latter is greater than 100,000/p.L. When the platelet count is 50,000/yL or greater, excessive bleeding generally occurs only with severe trauma or extensive surgery. In contrast, patients with platelet counts less than 20,000/pL often develop significant bleeding following even minor trauma. However, patients with liver cirrhosis may be thrombocytopenic but have increased levels of vWF, resulting in very active platelets that may compensate for the low count. Thrombocytopenia usually results from one of three mechanisms: (1) decreased platelet production, (2) splenic sequestration of platelets, or (3) increased platelet destruction. The third mechanism may fall under one of two categories of destruction: immune or nonimmune. Nonimmune destruction includes vasculitis or DIC.
A prolonged bleeding time with a normal platelet count implies a qualitative platelet defect. Although the bleeding time is somewhat dependent on the technique employed, values longer than 10 min are generally considered abnormal. Significant intraoperative and postoperative bleeding may be expected when the bleeding time exceeds 15 min. Specialized testing is required to diagnose specific platelet functional defects.

The most common platelet defect is due to inhibition of TXA, production by aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs). In contrast to aspirin, which irreversibly acetylates and inactivates cyclooxygenase for the life of the platelet (up to 8 days), enzyme inhibition by other NSAIDs is reversible and generally lasts only 24 h. Increasingly patients with cardiac stents are treated with a variety of antiplatelet agents such as clopidogrel, which impair platelet function for the life of the platelet. Assays of platelet function are available to determine the degree to which platelet function is inhibited.

The most common inherited bleeding disorder (1:800—1000 patients) is von Willebrand disease. Patients with this disorder produce either a defective vWF or low levels of a normal vWF (normal: 5-10 mg/L). Most patients are heterozygous and have relatively mild hemostatic defects that become clinically apparent only when they are subjected to major surgery or trauma or following ingestion of NSAIDs. In addition to helping link platelets, vWF serves as a carrier for coagulation factor VIII. As a result, these patients typically have a prolonged bleeding time, decreased plasma vWE concentration, and decreased factor VIII activity. Acquired forms of von Willebrand disease may be encountered in patients with some immune disorders and those with tumors that absorb vWF onto their surface. At least three forms of the disease are recognized, ranging in severity from mild to severe.
Treatment with desmopressin (DDAVP) can raise vWF levels in some patients with mild von Willebrand disease (as well as normal individuals). The drug is usually administered at a dose of 0.3 mcg/kg intravenously 30 min prior to surgery. Patients who do not respond to DDAVP should receive cryoprecipitate or factor VIII concentrates, both of which are rich in VWF; prophylactic infusions are generally recommended before and after surgery twice a day for 2 to 4 days to help assure surgical hemostasis.
What other hereditary hemostatic defects may be encountered in anesthetic practice?

The most common inherited secondary hemostasis defect is factor VIII deficiency (hemophilia A), an X-linked abnormality estimated to affect 1:10,000 males. Disease severity is generally inversely related to factor VII activity. Most symptomatic patients experience hemarthrosis, bleeding into deep tissues, and hematuria, and usually have less than 5% of normal factor VIII activity. Classically, patients present with a prolonged aPTT, but a normal PT and bleeding time. The diagnosis is confirmed by measuring factor VIII activity in blood. Affected patients generally do not experience increased bleeding during surgery when factor VIII levels are more than 30% of normal, but most clinicians recommend increasing factor VIII levels to more than 50% of normal prior to surgery. Normal (fresh frozen) plasma, by definition, is considered to have 1 U of factor VIII activity per milliliter. In contrast, cryoprecipitate has 5 to 10 U/mL, whereas factor VIII concentrates have approximately 40 U/mL. Each unit of factor VIII transfused is estimated to raise factor VIII levels 2% per kilogram of body weight. Twice-a-day transfusions are generally recommended following surgery because of the relatively short half-life of factor VIII (8-12 h). Administration of DDAVP can raise factor VIII levels twoto threefold in some patients. EACA or tranexamic acid may also be used as adjuncts. Recombinant factor VIII is now undergoing clinical trials.
Hemophilia B (also known as Christmas disease) is the result of an Xlinked hereditary deficiency of factor IX. It is very similar to hemophilia A, but much less common (1:100,000 males). Measurement of factor IX levels establishes the diagnosis. Perioperative administration of fresh frozen plasma is generally recommended to maintain factor IX activity at more than 30% of normal. Recombinant or monoclonal purified factor IX is available.
Factor XIII deficiency is extremely rare, but notable in that the aPTT, PT, TT, and bleeding times are normal. The diagnosis requires measurement of factor XIII levels. Because only 1% of normal factor XIII activity is generally required, patients are treated by a single transfusion of fresh frozen plasma.

A bleeding diathesis may exist even in the absence of gross abnormalities on routine laboratory tests. Some hemostatic defects are often not detected by routine testing, but require additional specialized tests. A history of excessive bleeding after dental extractions, childbirth, minor surgery, minor trauma, or even during menstruation suggests a hemostatic defect. Conversely, there may be no excess bleeding despite abnormal laboratory testing. A family history of a bleeding diathesis may suggest an inherited coagulation defect, but such history is often absent because the increased bleeding is often minor and goes unnoticed.
Hemostatic defects can often be differentiated by their clinical presentation. Bleeding in patients with primary hemostatic defects usually immediately follows minor trauma, is confined to superficial sites (skin or mucosal surfaces), and often can be controlled by local compression. Small pinpoint hemorrhages from capillaries in the dermis (petechiae) are typically present on examination. Bleeding into subcutaneous tissues (ecchymosis) from small arterioles or venules is also common in patients with platelet disorders. In contrast, bleeding that results from secondary hemostatic defects is usually delayed following injury, is typically deep (subcutaneous tissues, joints, body cavities, or muscles), and is often difficult to stop even with compression. Hemorrhages may be palpable as hematomas or may go unnoticed when located deeper (retroperitoneal). Coagulation may be impaired by systemic hypothermia or by subnormal temperature at the site of bleeding, even when coagulation test results (PT, aPTT, bleeding time) are normal and there is no history of hemostatic defects. Most laboratory tests are carried out at body temperature and may not reflect the effects of hypothermia.

@ Because of increased perioperative risk, patients with acute hepatitis should have any elective surgery postponed until the acute hepatitis has resolved, as indicated by the normalization of liver tests.
@ Isoflurane and sevoflurane are the volatile agents of choice for patients with significant liver disease because they preserve hepatic blood flow and oxygen delivery. Factors known to reduce hepatic blood flow, such as hypotension, excessive sympathetic activation, and high mean airway pressures during controlled ventilation, should be avoided.
© In evaluating patients for chronic hepatitis, laboratory test results may show only a mild elevation in serum aminotransferase activity and often correlate poorly with disease severity.
@ Liver cirrhosis refers to the damaging effects to the liver of inflammation, hepatocellular injury, and the resulting fibrosis and regeneration of hepatocytes.
© Liver cirrhosis leads to portal hypertension, varices, and widespread endothelial damage from toxins not cleared by the liver that may cause multiorgan dysfunction.
@ Massive bleeding from gastroesophageal varices is a major cause of morbidity and mortality in patients with liver disease, and, in addition to the cardiovascular effects of acute blood loss, the absorbed nitrogen load from the breakdown of blood in the gastrointestinal tract can precipitate hepatic encephalopathy.
@ Cardiovascular changes observed in cirrhotic patients are usually those
of hyperdynamic circulation, although clinically significant cirrhotic cardiomyopathy is often present and not recognized.

@ The effects of hepatic cirrhosis on pulmonary arterioles may result in vasodilation, causing shunts and chronic hypoxemia, or conversely lead to pulmonary vasoconstriction and medial hyperplasia, causing an increase in vascular resistance and pulmonary hypertension.
© Hepatorenal syndrome is a functional renal defect in patients with cirrhosis that usually follows gastrointestinal bleeding, aggressive diuresis, sepsis, or major surgery. It is characterized by progressive oliguria with avid sodium retention, azotemia, intractable ascites, and a very high mortality rate.
© Factors known to precipitate hepatic encephalopathy in patients with cirrhosis include gastrointestinal bleeding, increased dietary protein intake, hypokalemic alkalosis from vomiting or diuresis, infections, worsening liver function, and drugs with central nervous system depressant activity.
@ Following the removal of large amounts of ascitic fluid, aggressive intravenous fluid replacement is often necessary to prevent profound hypotension and acute kidney injury or failure.
The prevalence of liver disease is increasing. Cirrhosis, the terminal pathology of most liver diseases, has a general population incidence as high as 5% in some autopsy series. It is a major cause of death in men in their fourth and fifth decades of life, and mortality rates are increasing. Ten percent of the patients with liver disease undergo operative procedures during the final 2 years of their lives. The liver has remarkable functional reserve, and thus overt manifestations of hepatic disease are often absent until extensive damage has occurred. When patients with little hepatic reserve come to the operating room, effects from anesthesia and the surgical procedure can precipitate hepatic decompensation and frank hepatic failure.

The hemostatic changes that occur with liver disease may cause hypercoagulation and thrombosis, as well as an increased risk of bleeding. The causes of excessive bleeding primarily involve thrombocytopenia, endothelial dysfunction, portal hypertension, kidney failure, and sepsis (see Chapters 31 and 51). Clot breakdown may be enhanced by an imbalance of the fibrinolytic

Chronic liver disease is characterized by the impaired synthesis of coagulation factors, resulting in prolongation of the prothrombin time (PT) and international normalized ratio (INR) (Table 34-1). The INR was designed to monitor the anticoagulant effect of warfarin, not the anticoagulant effect of liver dysfunction. In liver dysfunction, the anticoagulant factors (protein C, antithrombin, and tissue factor pathway inhibitor) are also reduced and may balance out any effect of a prolonged PT. This may be confirmed by assessing thrombin generation in the presence of endothelial-produced thrombomodulin. Adequate thrombin production requires an adequate number of functioning platelets. If the platelet count is greater than 60,000/L, coagulation may well be normal in a patient with severe cirrhosis. In fact, more recently it has been shown that an increased von Willebrand factor (VWF) in liver disease may result in activated platelets allowing normal or increased coagulation despite a much lower platelet count. It is important to use point-of-care global viscoelastic coagulation testing to fully understand the state of coagulation (see later discussion).

Advanced liver disease, PT = T. Advanced liver disease, PTT = T. Advanced liver disease, TT = Nor?. Advanced liver disease, Fibrinogen = Nord. DIC, PT = T. DIC, PTT = T. DIC, TT = T. DIC, Fibrinogen = L. Vitamin K deficiency, PT = TT. Vitamin K deficiency, PTT = T. Vitamin K deficiency, TT = N. Vitamin K deficiency, Fibrinogen = N. Warfarin therapy, PT = a. Warfarin therapy, PTT = fj. Warfarin therapy, TT = N. Warfarin therapy, Fibrinogen = N. Heparin therapy, PT = T. Heparin therapy, PTT = tin. Heparin therapy, TT = it. Heparin therapy, Fibrinogen = N. Hemophilia Factor VIII deficiency Factor IX deficiency, PT = N N. Hemophilia Factor VIII deficiency Factor IX deficiency, PTT = fi tT. Hemophilia Factor VIII deficiency Factor IX deficiency, TT = N N. Hemophilia Factor VIII deficiency Factor IX deficiency, Fibrinogen = N N. Factor VII deficiency, PT = T. Factor VII deficiency, PTT = N. Factor VII deficiency, TT = N. Factor VII deficiency, Fibrinogen = N. Factor XIll deficiency, PT = N. Factor XIll deficiency, PTT = N. Factor XIll deficiency, TT = N. Factor XIll deficiency, Fibrinogen = N
'PT, prothrombin time; PTT, partial thromboplastin time; TT, thrombin time; N, normal; DIC, disseminated intravascular coagulation.
Cirrhotic patients will typically have hyperfibrinolysis. However, individual laboratory tests may not give a true picture of the state of fibrinolysis. Thromboelastography (TEG), rotational thromboelastometry (ROTEM), and Sonoclot viscoelastic coagulation testing technologies are the optimal methods of demonstrating the global state of the coagulation system at a specific moment in time in any patient with liver disease (see Chapter 51).

Acute hepatitis is usually the result of a viral infection, drug reaction, or exposure to a hepatotoxin. The illness represents acute hepatocellular injury with a variable degree of cellular necrosis. Clinical manifestations depend on the severity of the inflammatory reaction and on the extent of necrosis. Mild inflammatory reactions may present merely as asymptomatic elevations in the serum transaminases, whereas massive hepatic necrosis presents as acute fulminant hepatic failure.

Viral hepatitis is most commonly due to hepatitis A, B, or C viral infection. At least two other hepatitis viruses have also been identified: hepatitis D (delta virus) and hepatitis E (enteric non-A, non-B). Hepatitis types A and E are transmitted by the fecal—oral route, whereas hepatitis types B and C are transmitted primarily percutaneously and by contact with body fluids. Hepatitis D is unique in that it may be transmitted by either route and requires the presence of hepatitis B virus in the host to be infective. Other viruses may also cause hepatitis, including Epstein—Barr, herpes simplex, cytomegalovirus, and coxsackieviruses.
Patients with viral hepatitis often have a 1to 2-week mild prodromal illness (fatigue, malaise, low-grade fever, or nausea and vomiting) that may or may not be followed by jaundice. The jaundice typically lasts 2 to 12 weeks, but complete recovery, as evidenced by serum transaminase measurements, usually takes 4 months. Serological testing is necessary to determine the causative viral agent. The clinical course tends to be more complicated and prolonged with hepatitis B and C viruses relative to other types of viral hepatitis. Cholestasis (see later discussion) may be a major manifestation. Rarely, fulminant hepatic failure (massive hepatic necrosis) can develop.

The incidence of chronic active hepatitis is 3% to 10% following infection with hepatitis B virus and at least 50% following infection with hepatitis C virus. A small percentage of patients (mainly immunosuppressed patients and those on long-term hemodialysis regimens) become asymptomatic infectious carriers following infection with hepatitis B virus, and up to 30% of these patients remain infectious with the hepatitis B surface antigen (HBsAg) persisting in their blood. Most patients with chronic hepatitis C infection seem to have very low, intermittent, or absent circulating viral particles and are therefore not highly infective. Approximately 0.5% to 1% of patients with hepatitis C infection become asymptomatic infectious carriers, and infectivity correlates with the detection of hepatitis C viral RNA in peripheral blood. Such infectious carriers pose a major health hazard to operating room personnel. In addition to "universal precautions" for avoiding direct contact with blood and secretions (gloves, mask, protective eyewear, and not recapping needles), immunization of health care personnel is highly effective against hepatitis B infection. A vaccine for hepatitis C is not available; moreover, unlike hepatitis B infection, hepatitis C infection does not seem to confer immunity to subsequent exposure. Postexposure prophylaxis with hyperimmune globulin is effective for hepatitis B, but not hepatitis C. Effective therapies now exist for hepatitis C, but continued hepatocellular carcinoma surveillance remains important for these patients.

Drug-induced hepatitis (Table 34—2) can result from direct, dose-dependent toxicity of a drug or drug metabolite, an idiosyncratic drug reaction, or a combination of these two causes. The clinical course often resembles viral hepatitis, making diagnosis difficult. Alcoholic hepatitis is probably the most common form of drug-induced hepatitis, but the etiology may not be obvious from the history. Chronic alcohol ingestion can also result in hepatomegaly from fatty infiltration of the liver, which reflects impaired fatty acid oxidation, increased uptake and esterification of fatty acids, and diminished lipoprotein synthesis and secretion. Acetaminophen ingestion of 25 g or more usually results in fatal fulminant hepatotoxicity. A few drugs, such as chlorpromazine and oral contraceptives, may cause cholestatic-type reactions (see later discussion). Ingestion of potent hepatotoxins, such as carbon tetrachloride and certain species of mushrooms (Amanita, Galerina), also may result in fatal hepatotoxicity.

Alcohol
Acetaminophen
Salicylates
Tetracyclines
Trichloroethylene
Vinyl chloride
Carbon tetrachloride
Yellow phosphorus
Poisonous mushrooms (Amanita, Galerina)

Volatile anesthetics (halothane)
Phenytoin
Rifampin
Indomethacin

Chlorpromazine
Cyclosporine
Oral contraceptives
Anabolic steroids
Erythromycin estolate
Methimazole
® Because of increased perioperative risk, patients with acute hepatitis should have elective surgery postponed until the illness has resolved, as indicated by the normalization of liver tests. In addition, acute alcohol toxicity greatly complicates anesthetic management, and acute alcohol withdrawal during the perioperative period may be associated with a mortality rate as high as 50%. Only emergent surgery should be considered for patients presenting in acute alcohol withdrawal. Patients with hepatitis are at risk of deterioration of hepatic function and the development of complications from hepatic failure, such as encephalopathy, coagulopathy, or hepatorenal syndrome.

The goal of intraoperative management is to preserve existing hepatic function. Some patients with viral hepatitis may exhibit increased central nervous system sensitivity to anesthetics, whereas alcoholic patients will often display crosstolerance to both intravenous and volatile anesthetics. Alcoholic patients also require close cardiovascular monitoring because the cardiac depressant effects of alcohol are additive to those of anesthetics; moreover, alcoholic cardiomyopathy may be present in alcoholic patients.
Inhalation anesthetics are generally preferable to intravenous agents because most of the latter are dependent on the liver for metabolism, elimination, or both. Standard induction doses of intravenous induction agents can generally be used because their action is terminated by redistribution rather than metabolism or excretion. A prolonged duration of action, however, may be encountered with large or repeated doses of intravenous agents, particularly @ opioids. Isoflurane and sevoflurane are the volatile agents of choice for patient with significant liver disease because they preserve hepatic blood flow and oxygen delivery. Factors known to reduce hepatic blood flow, such as hypotension, excessive sympathetic activation, and high mean airway pressures during controlled ventilation, should be avoided. Regional anesthesia, including major conduction blockade, may be employed in the absence of coagulopathy, provided hypotension is avoided.

Chronic hepatitis is defined as persistent hepatic inflammation for longer than 6 months, as evidenced by elevated serum aminotransferases. Patients can usually be classified as having one of three distinct syndromes based on a liver biopsy: chronic persistent hepatitis, chronic lobular hepatitis, or chronic active hepatitis. Patients with chronic active hepatitis have chronic hepatic inflammation with destruction of normal cellular architecture "piecemeal necrosis" on the biopsy. Evidence of cirrhosis is either present initially or eventually develops in 20% to 50% of patients. Although chronic active hepatitis seems to have many causes, it occurs most commonly as a sequela of hepatitis B or hepatitis C. Other causes include medications (methyldopa, isoniazid, and nitrofurantoin) and autoimmune disorders. Both immunological factors and a genetic predisposition may be responsible in most cases. Patients usually present with a history of fatigue and recurrent jaundice; extrahepatic manifestations, such as arthritis and serositis, are not uncommon. Manifestations of cirrhosis eventually predominate in patients with progressive disease. In @ evaluating patients for chronic hepatitis, laboratory test results may show only a mild elevation in serum aminotransferase activity and often correlate poorly with disease severity. Patients without chronic hepatitis B or C infection usually have a favorable response to immunosuppressants and are treated with long-term corticosteroid therapy with

Patients with chronic persistent or chronic lobular hepatitis should be treated similarly to those with acute hepatitis. In contrast, those with chronic active hepatitis should be assumed to already have cirrhosis and should be treated accordingly (as discussed next). Patients with autoimmune chronic active hepatitis may also present with problems related to other autoimmune manifestations (such as diabetes or thyroiditis) or to the long-term corticosteroid therapy that they have likely received.

of spontaneous bacterial peritonitis, and some patients eventually develop hepatocellular carcinoma.
A few diseases can produce hepatic fibrosis without hepatocellular necrosis or nodular regeneration, resulting in portal hypertension and its associated complications with hepatocellular function often preserved. These disorders include schistosomiasis, idiopathic portal fibrosis (Banti syndrome), and congenital hepatic fibrosis. Obstruction of the hepatic veins or inferior vena cava (Budd—Chiari syndrome) can also cause portal hypertension. The latter may be the result of venous thrombosis (hypercoagulable state), a tumor thrombus (eg, renal carcinoma), or occlusive disease of the sublobular hepatic veins.

The detrimental effects of anesthesia and surgery on hepatic blood flow are discussed later in this section. Patients with cirrhosis are at increased risk of deterioration of liver function because of limited functional reserve. Successful anesthetic management of these patients is dependent on recognizing the multisystem nature of cirrhosis (Table 34—3) and controlling or preventing its complications.

Portal hypertension
Ascites
Esophageal varices
Hemorrhoids
Gastrointestinal bleeding

Hyperdynamic state (high cardiac output)
Systemic arteriovenous shunts
Low systemic vascular resistance
Cirrhotic cardiomyopathy; pulmonary hypertension

Increased intrapulmonary shunting; hepatopulmonary syndrome
Decreased functional residual capacity
Pleural effusions
Restrictive ventilatory defect
Respiratory alkalosis

Increased proximal reabsorption of sodium
Increased distal reabsorption of sodium
Impaired free water clearance
Decreased renal perfusion
Hepatorenal syndrome

Hyponatremia and hypernatremia
Hypokalemia and hypocalcemia
Hypomagnesemia

Portal hypertension leads to the development of extensive portosystemic venous collateral channels. Four major collateral sites are generally recognized:
gastroesophageal, hemorrhoidal, periumbilical, and retroperitoneal. Portal hypertension is often apparent preoperatively, as evidenced by dilated abdominal
@ wall veins (caput medusae). Massive bleeding from gastroesophageal varices is a major cause of morbidity and mortality in patients with liver disease, and, in addition to the effects of acute blood loss, the absorbed nitrogen load from the breakdown of blood in the gastrointestinal tract can precipitate hepatic encephalopathy.
The treatment of variceal bleeding is primarily supportive, but frequently involves endoscopic procedures for identification of the bleeding site(s) and therapeutic maneuvers, such as injection sclerosis of varices, monopolar and bipolar electrocoagulation, or application of hemoclips or bands. In addition to the risks posed by a patient who is physiologically fragile and acutely hypovolemic and hypotensive, anesthesia for such endoscopic procedures frequently involves the additional challenges of an encephalopathic and uncooperative patient and a stomach full of food and blood. Endoscopic unipolar electrocautery may adversely affect implanted cardiac pacing and defibrillator devices.

Blood loss should be replaced with intravenous fluids and blood products. Nonoperative treatment includes vasopressin, somatostatin, propranolol, and balloon tamponade with a Sengstaken—Blakemore tube. Vasopressin, somatostatin, and propranolol reduce the rate of blood loss. High doses of vasopressin can result in congestive heart failure or myocardial ischemia; concomitant infusion of intravenous nitroglycerin may reduce the likelihood of these complications and bleeding. Placement of a percutaneous transjugular intrahepatic portosystemic shunt (TIPS) can reduce portal hypertension and subsequent bleeding, but may increase the incidence of encephalopathy. When the bleeding fails to stop or recurs, emergency surgery may be indicated. Perioperative risk correlates with degree of hepatic impairment, based on clinical and laboratory findings. Child's classification for evaluating hepatic reserve is shown in Table 34—4. Shunting procedures are generally performed on low-risk patients, whereas ablative surgery, esophageal transection, and gastric devascularization are reserved for high-risk patients.

Bilirubin (mg/dL), A = <2.0. Bilirubin (mg/dL), B = 2.0-3.0. Bilirubin (mg/dL), c = >3.0. Serum albumin (g/dL), A = >3.5. Serum albumin (g/dL), B = 3.0-3.5. Serum albumin (g/dL), c = <3.0. Ascites, A = . Ascites, B = Controlled. Ascites, c = Poorly controlled. Encephalopathy, A = Absent. Encephalopathy, B = Minimal. Encephalopathy, c = Coma. Nutrition, A = Excellent. Nutrition, B = Good. Nutrition, c = Poor. Mortality rate (%), A = 2-5. Mortality rate (%), B = 10. Mortality rate (%), c = 50
Adapted with permission from Child CG. The Liver and Portal Hypertension. Philadelphia, PA: WB Saunders; 1964.

Anemia, thrombocytopenia, and, less commonly, leukopenia may be present. The cause of the anemia is usually multifactorial and includes blood loss, increased red blood cell destruction, bone marrow suppression, and nutritional deficiencies. Congestive splenomegaly secondary to portal hypertension is largely responsible for the thrombocytopenia and leukopenia. Coagulation factor deficiencies arise as a result of decreased hepatic synthesis. Enhanced fibrinolysis secondary to decreased clearance of activators of the fibrinolytic system may also contribute to the coagulopathy.
The need for preoperative blood transfusions should be balanced against the obligatory increase in nitrogen load. Protein breakdown from excessive blood transfusions can precipitate encephalopathy. Nevertheless, coagulopathy should be corrected before surgery. Clotting factors should be replaced with appropriate blood products, such as fresh frozen plasma and cryoprecipitate. Platelet transfusions should be considered immediately prior to surgery for platelet counts less than 75,000/pL. Assessment of integrity of the coagulation system by viscoelastic technology will provide specific management information.

End-stage liver disease, and in particular, cirrhosis of the liver may be associated with disorders of all major organ systems (Tables 34—3 and 34-5). Cardiovascular changes observed in cirrhotic patients @ are usually those of a hyperdynamic circulation, although clinically significant cirrhotic cardiomyopathy is often present and not recognized (Table 34—6). There may be a reduced cardiac contractile response to stress, altered diastolic relaxation, downregulation of B-adrenergic receptors, and electrophysiological changes as a result of cirrhotic cardiomyopathy.

Chronic obstructive pulmonary disease
Asthma
Restrictive lung disease
Pneumonia

Alcoholic liver disease with alcoholic cardiomyopathy
Hemochromatosis with iron overload cardiomyopathy a,-Antitrypsin deficiency with panacinar emphysema
Primary biliary cirrhosis with fibrosing alveolitis

Increased cardiac output
Increased heart rate
Decreased systemic vascular resistance
Increased circulating volume
Coronary artery disease
Cirrhotic cardiomyopathy (often unrecognized)
Low systemic vascular resistance conceals poor left ventricular function
Reduced responsiveness to B-agonists
Echocardiographic examination of cardiac function may initially be interpreted as normal because of significant afterload reduction caused by low systemic vascular resistance. However, both systolic and diastolic dysfunction are often present. Noninvasive stress imaging is frequently used to assess coronary artery disease in patients older than age 50 years and in those with cardiac risk factors.

@ The effects of hepatic cirrhosis on pulmonary vascular resistance (PVR)
vessels may result in vasodilation, causing shunts and chronic hypoxemia, or conversely lead to pulmonary vasoconstriction and medial hyperplasia, causing an increase in vascular resistance and pulmonary hypertension. Hepatopulmonary syndrome (HPS; Table 34—7) is found in approximately 30% of liver transplant candidates and is characterized by a triad of decreased oxygen saturation in the presence of advanced liver disease and intrapulmonary arteriolar dilation. Intrapulmonary vascular dilation causes intrapulmonary rightto-left shunting and an increase in the alveolar-to-arterial oxygen gradient.

Cyanosis
Digital clubbing
Cutaneous telangiectasia
Orthodeoxia (oxygen desaturation on sitting or standing)
Platypnea (breathing more easily while lying flat)
Dyspnea

Presence of liver disease, usually with portal hypertension and cirrhosis
An alveolar to arterial oxygen gradient of >15 mm Hg
Pulmonary arteriovenous connections demonstrated by:
¢ A delayed contrast-enhanced (agitated saline) echocardiogram showing contrast in the left heart chambers 4—6 heartbeats after contrast appears in the right heart chambers
¢ Brain uptake >6% following technetium-99m macroaggregated albumin lung perfusion scan

Liver transplantation is the only therapy that will cure hepatopulmonary syndrome
Pulse oximetry may be used to screen for HPS, and room air Spo, less than 96% requires investigation. There is no medical treatment for HPS, which is progressive, but it is reversed over 6 months to 2 years by liver transplantation.

Pulmonary vascular remodeling may occur in association with chronic liver disease, involving vascular smooth muscle proliferation, vasoconstriction, intimal proliferation, and eventual fibrosis, all presenting as obstruction causing an increased resistance to pulmonary blood flow. These pathological changes may result in pulmonary hypertension, and if associated with portal hypertension, the condition is termed portopulmonary hypertension (POPH; Table 34-8).

Increased pulmonary vascular resistance: vasoconstriction, structural vascular remodeling, and eventual fibrosis
Mean pulmonary artery pressure >25 mm Hg with normal pulmonary capillary wedge pressure
Right ventricular overload
Right heart failure
Hepatic congestion
Increased liver transplantation mortality risk, especially if mean pulmonary artery pressure is >35 mm Hg
The diagnostic criteria for POPH include a mean pulmonary artery pressure
(mPAP)
25
mm
Hg at
rest, and
a
PVR
240
dyn-s-cm™.
The transpulmonary
pulmonary arteriolar
and distinguishes
the gradient
of greater
occlusion pressure
contribution than
12
mm
[PAOP])
of volume
Hg
(mPAP
reflects and
minus the
obstruction resistance
to to
the increase
flow in
mPAP.
POPH may be classified as mild (mPAP 25-35 mm Hg), moderate (mPAP >35 and <45 mm Hg), and severe (mPAP >45 mm Hg). Mild POPH is not associated with increased mortality at liver transplantation, although the immediate recovery period may be challenging if there is significant increase in cardiac output after reperfusion of the new graft. Moderate and severe POPH are associated with significant mortality at transplantation. However, the key factor is not mPAP, but rather right ventricular (RV) function.
The success of liver transplantation will depend on the right ventricle maintaining good function during and after the transplant procedure despite increases in cardiac output, volume, and PVR. If RV dysfunction or failure occurs, graft congestion with possible graft failure and possible mortality may ensue. Assessment of the right ventricle using transesophageal echocardiography (TEE) is often helpful.

The role of liver transplantation in the management of POPH is not well defined. In some patients, pulmonary hypertension will reverse quickly after transplant; however, other patients may require months or years of ongoing vasodilator therapy. Yet other patients may continue to progress and eventually develop RV failure. Some patients with HPS develop pulmonary hypertension after liver transplantation, suggesting that both pathologies may occur simultaneously. Liver transplantation offers the best outcome in patients with POPH that is responsive to vasodilator therapy.

Disturbances in pulmonary gas exchange and ventilatory mechanics are often present. Hyperventilation is common and results in a primary respiratory alkalosis. As previously noted, hypoxemia is frequently present and is due to right-to-left shunting of up to 40% of cardiac output. Shunting is due to an increase in both pulmonary arteriovenous communications (absolute) and ventilation/perfusion mismatching (relative). Elevation of the diaphragm from ascites decreases lung volumes, particularly functional residual capacity, and predisposes to atelectasis. Moreover, large amounts of ascites produce a restrictive ventilatory defect that increases the work of breathing. Severe hydrothorax is also frequently found.
Review of the chest radiograph and arterial blood gas measurements is useful preoperatively because atelectasis and hypoxemia are usually not evident on clinical examination. Paracentesis should be considered in patients with massive ascites and pulmonary compromise, but should be performed with caution because excessive fluid removal can lead to circulatory collapse.

Derangements of fluid and electrolyte balance may manifest as ascites, edema, electrolyte disturbances, and hepatorenal syndrome (see below). Important mechanisms responsible for ascites include (1) portal hypertension, which increases hydrostatic pressure and favors transudation of fluid across the intestine into the peritoneal cavity; (2) hypoalbuminemia, which decreases plasma oncotic pressure and favors fluid transudation; (3) seepage of proteinrich lymphatic fluid from the serosal surface of the liver secondary to distortion and obstruction of lymphatic channels in the liver; and (4) avid renal sodium and

Hepatic encephalopathy is characterized by mental status alterations with fluctuating neurological signs (asterixis, hyperreflexia, or inverted plantar reflex) and characteristic electroencephalographic changes (symmetric high-voltage, slow-wave activity). This is associated with accumulation of neurotoxins, including ammonia and short-chain fatty acids. Some patients also have elevated intracranial pressure. Metabolic encephalopathy seems to be proportionately related to both the amount of hepatocellular damage present and the degree of shunting of portal blood away from the liver and directly into the systemic circulation. The accumulation of substances originating in the gastrointestinal tract (but normally metabolized by the liver) has been implicated. Factors © known to precipitate hepatic encephalopathy include gastrointestinal bleeding, increased dietary protein intake, hypokalemic alkalosis from vomiting or diuresis, infections, worsening liver function, and drugs with central nervous system depressant activity.
Hepatic encephalopathy should be aggressively treated preoperatively. Precipitating causes should be corrected. Oral lactulose (30-50 mL every 8 h) or neomycin (500 mg every 6 h) is useful in reducing intestinal ammonia absorption. Lactulose acts as an osmotic laxative, and, like neomycin, likely inhibits ammonia production by intestinal bacteria. Sedatives, especially benzodiazepines, should be avoided as they may precipitate hepatic coma.

Patients with postnecrotic cirrhosis due to hepatitis B or hepatitis C who are carriers of the virus may be infectious. Universal precautions are always indicated in preventing contact with blood and body fluids from all patients.

The response to anesthetic agents is unpredictable in patients with cirrhosis. Changes in central nervous system sensitivity, volumes of distribution, protein binding, drug metabolism, and drug elimination are common. An increase in the volume of distribution for highly ionized drugs, such as neuromuscular blockers (NMBs), is due to the expanded extracellular fluid compartment; an apparent resistance may be observed, requiring larger than normal loading doses. However, smaller than normal maintenance doses of NMBs dependent on hepatic elimination (pancuronium, rocuronium, and vecuronium) are needed. The duration of action of succinylcholine may be prolonged because of reduced levels of pseudocholinesterase, but this is rarely of clinical consequence.

The cirrhotic liver is very dependent on hepatic arterial perfusion because of reduced portal venous blood flow. Preservation of hepatic arterial blood flow and avoidance of agents with potentially adverse effects on hepatic function are critical. Regional anesthesia may be used in patients without thrombocytopenia or coagulopathy, but hypotension must be avoided. A propofol induction followed by isoflurane or sevoflurane with an oxygen—air mixture is commonly employed for general anesthesia. Opioid supplementation reduces the dose of the volatile agent required, but the half-lives of opioids are often significantly prolonged, which may cause postoperative respiratory depression. Cisatracurium may be the NMB of choice because of its nonhepatic metabolism.
Preoperative nausea, vomiting, upper gastrointestinal bleeding, and abdominal distention due to massive ascites require a well-planned anesthetic induction. Preoxygenation and rapid-sequence induction/intubation with cricoid pressure are often performed. For unstable patients and those with active bleeding, either an awake intubation or a rapid-sequence induction using ketamine or etomidate and succinylcholine is suggested.

Pulse oximetry should be supplemented with arterial blood gas measurements to monitor acid-base status. Patients with large right-to-left intrapulmonary shunts may not tolerate the addition of nitrous oxide and may require positive endexpiratory pressure (PEEP) to treat ventilation/perfusion inequalities and associated hypoxemia. Patients receiving vasopressin infusions should be monitored for myocardial ischemia due to coronary vasoconstriction. Continuous intraarterial pressure monitoring is often used because hemodynamic instability frequently occurs as a result of excessive bleeding and operative manipulations. Intravascular volume status is often difficult to optimize, and goal-directed hemodynamic and fluid therapy utilizing esophageal Doppler, arterial waveform analysis, pleth variability index (PVI), or TEE should be considered. Such approaches may be helpful in preventing acute kidney injury. Urinary output must be followed closely; mannitol may be considered for persistently low urinary outputs despite adequate intravascular fluid replacement.

Most patients are sodium-restricted preoperatively, but preservation of intravascular volume and urinary output takes priority intraoperatively. The use of predominantly colloid intravenous fluids (albumin) may be preferable to avoid sodium overload and to increase plasma oncotic pressure. Intravenous fluid replacement should take into account the excessive bleeding and fluid shifts that often occur in these patients during abdominal procedures. Venous engorgement from portal hypertension, varices, lysis of adhesions from previous surgery, and coagulopathy lead to excessive bleeding during surgical procedures, whereas evacuation of ascites and prolonged surgical procedures result in large fluid shifts. @ Following the removal of large amounts of ascitic fluid, aggressive intravenous fluid replacement is often necessary to prevent profound hypotension and acute kidney injury or failure. Liberal use of crystalloid solutions may result in widespread edema because of the low serum albumin, and colloid solutions are usually more appropriate.
Most preoperative patients are anemic and coagulopathic, and perioperative red blood cell transfusion may lead to hypocalcemia from impaired citrate metabolism in the cirrhotic liver. Citrate, the anticoagulant in stored red blood cell preparations, binds with plasma calcium, producing hypocalcemia. Intravenous calcium may be necessary (see Chapter 51).

Common hepatic procedures include repair of lacerations, drainage of abscesses, and resection of primary or metastatic neoplasms, and up to 80% to 85% of the liver can be resected in many patients. In addition, liver transplantation is performed in many centers. The perioperative care of patients undergoing hepatic surgery is often challenging because of coexisting medical problems and debilitation found in many patients with intrinsic liver disease, and because of the potential for significant operative blood loss. Hepatitis and cirrhosis greatly complicate anesthetic management and increase perioperative mortality. Multiple large-bore intravenous catheters and fluid blood warmers are necessary; rapid infusion devices facilitate management when massive blood transfusion is anticipated. Continuous intraarterial pressure monitoring is typically utilized. Hemodynamic optimization is often complicated by the conflict between the need to maintain sufficient intravascular volume to ensure adequate hepatic perfusion and the need to keep central venous pressure low to minimize liver engorgement and surgical bleeding. Central venous pressure measurement is not an accurate monitor of volume status; we suggest goal-directed fluid therapy utilizing esophageal Doppler, arterial waveform analysis, PVI, or TEE. Care should be taken in placing an esophageal Doppler or TEE probe in a patient with

Hypotensive anesthesia should be avoided because of its potentially deleterious effects on liver function. Administration of antifibrinolytics, such as €-aminocaproic acid or tranexamic acid, may reduce operative blood loss, especially if fibrinolysis can be demonstrated by viscoelastic coagulation monitoring. Hypoglycemia, coagulopathy, and sepsis may occur following large liver resections. Drainage of an abscess or cyst may be complicated by peritoneal contamination. In the case of a hydatid cyst, spillage can cause anaphylaxis due to the release of Echinococcus antigens.
Postoperative complications include hepatic dysfunction, sepsis, and blood loss secondary to coagulopathy or surgical bleeding. Postoperative pain from the surgical incision may hinder postoperative mobilization and convalescence, but perioperative coagulopathy may limit the use of epidural analgesia. Transversus abdominis plane (TAP) blocks may be very effective. Postoperative mechanical ventilation may be necessary in patients who have undergone extensive resections or who are markedly debilitated.

When a center opens a liver transplantation program, a credentialed director should be appointed who should be an anesthesiologist with experience and training in liver transplantation anesthesia. A dedicated team of anesthesiologists should be assembled to manage the perioperative course of all liver transplantation patients. This team should have a thorough understanding of the indications for, and contraindications to, liver transplantation (Tables 34—9 and 34-10), as well as the perioperative implications of associated comorbidities such as coronary artery disease, cirrhotic cardiomyopathy, portopulmonary hypertension, HPS, hepatorenal syndrome, and hepatic encephalopathy and cerebral edema. It has been demonstrated that such an approach improves outcomes, as measured by reduced blood transfusions, the need for postoperative mechanical ventilation, and the intensive care unit length of stay.
TABLE 34-9 Indications for liver transplantation.
Congenital hepatic fibrosis Alagille disease Biliary atresia a,-Antitrypsin deficiency Byler disease Metabolic disorders Wilson disease Tyrosinemia Glycogen storage diseases Crigler—-Najjar disease Hemophilia Lysosomal storage diseases Protoporphyria Familial hypercholesterolemia Primary hyperoxaluria, Adult = Primary biliary cirrhosis Primary sclerosing cholangitis Autoimmune hepatitis Cryptogenic cirrhosis Viral hepatitis with cirrhosis Alcoholic cirrhosis Primary hepatocellular malignancies Nonalcoholic steatohepatitis Fulminant hepatitis Hepatic vein thrombosis Familial amyloid polyneuropathy Chronic viral hepatitis

Active sepsis Active substance or alcohol abuse Advanced cardiac disease Extrahepatic malignancy Metastatic malignancy Cholangiocarcinoma, Relative = Severe obesity Severe pulmonary hypertension Severe cardiomyopathy High viral load HIV

FIGURE 34-1 Relationship between Model for End-stage Liver Disease (MELD) score and 3-month mortality in patients with cirrhotic liver disease. (Reproduced with permission from Wiesner RH, McDiarmid SV, Kamath PS, et al. MELD and PELD: Application of survival models to liver allocation. Liver Transpl. 2001 July;7(7):567-580.)
The MELD score = 0.957 x log [serum creatinine (mg/dL)] + 0.378 x log [total serum bilirubin (mg/dL)] + 1.120 x log [INR]
Multiply the resulting value by 10, and round to nearest whole number. The minimum for all values is 1.0; the maximum value for creatinine is 4.0. Most liver transplant candidates have high MELD scores and present with jaundice, kidney failure, and coagulopathy. They may also be emaciated and have massive ascites, and some may have encephalopathy, HPS, cirrhotic cardiomyopathy, and POPH. These patients often have an increased cardiac index and reduced systemic vascular resistance.
Significant blood loss may be anticipated, and large-bore intravenous catheters should be placed for access. A rapid infusion pump should be available. Routine hemodynamic monitoring should include intraarterial pressure monitoring. TEE is routinely utilized in many centers. Pulmonary artery catheterization, once routine, has now been replaced by a central venous catheter at many centers, except when there is concern for POPH or cirrhotic

The immediate availability of intraoperative continuous venovenous hemodialysis (CVVHD) may be very helpful for volume and electrolyte management in the patient with marginal or no kidney function. In patients with significant electrolyte abnormalities, serum sodium and potassium can be closely managed by adjusting the CVVHD dialysate solution.

Patients who undergo liver transplantation are often severely debilitated and malnourished and have multiorgan dysfunction. They will need careful support and continuous monitoring until they have recovered. Early extubation is appropriate in patients who are comfortable, cooperative, and not excessively coagulopathic. Immunosuppression must be carefully managed to minimize risk of sepsis. A close watch of graft function must be maintained, with a low threshold for checking hepatic artery patency and flow. Postoperative bleeding, biliary leaks, and vascular thromboses may require surgical reexploration.

Patients with elevated intracranial pressure (ICP) and those at risk of its development should have ICP monitoring in place, if possible, to enable the appropriate management of CPP. The management of patients who are at risk of or have elevated ICP should include the following:
¢ ICP less than 20 mm Hg
* CPP greater than 50 mm Hg
¢ Mean arterial pressure greater than 60 mm Hg
* Proper bed position (elevate the head of the bed by 20—25°)
* Controlled airway and ventilation
* Controlled sedation (eg, propofol)
¢ Vasopressor support (eg, vasopressin, norepinephrine) when necessary
* Controlled hypothermia (32—33°C)
¢ Glycemic control
¢ Aggressive treatment of metabolic acidosis and coagulopathy
* CVVHD

Selected pediatric centers report 1-year survival rates of 90%. The use of reduced-size and living donor grafts has increased the organ availability in this patient population.

The use of living donors has increased the pool of organs available for transplantation. However, this procedure does expose healthy individuals to morbidity and mortality risks. Informed consent from the donor must be obtained with the understanding that there is often a great deal of emotional pressure on family members to donate, and that consent must be freely given without coercion.
In most donor anesthesia protocols, maintenance of a CVP less than 5 cm H,O is utilized to reduce intraoperative blood loss. Adequate postoperative analgesia is required so that comfortable donor patients may be extubated at the end of the procedure. TAP blocks meet this need. Complications of this surgery for the donor patient include transient hepatic dysfunction, wound infection, postoperative bleeding, portal vein thrombosis, biliary leaks, and, rarely, death. An increased incidence of perioperative brachial plexus injury has been reported in donor patients.

A 23-year-old woman develops fulminant hepatic failure after ingesting wild mushrooms. She is not expected to survive without a liver transplant.

Orthotopic liver transplantation is usually performed in patients with end-stage liver disease who begin to experience life-threatening complications, especially when such complications become unresponsive to medical or nontransplant surgery. Transplantation is also carried out in patients with fulminant hepatic failure (from viral hepatitis or a hepatotoxin) when survival with medical management alone is judged unlikely. The MELD score is used to assess urgency for transplantation. The most common indications for liver transplantation in children, in order of decreasing frequency, are biliary atresia, inborn errors of metabolism (usually a,-antitrypsin deficiency, Wilson disease, tyrosinemia, and Crigler—Najjar type I syndrome), and postnecrotic cirrhosis.
The most common indications in adults are postnecrotic (nonalcoholic) cirrhosis, primary biliary cirrhosis, and sclerosing cholangitis, and, less commonly, primary malignant tumors in the liver.
What factors have contributed to the recent success of liver transplantation?
One-year survival rates for liver transplantations exceed 80% to 85% in some centers. Currently, 5-year survival rates are 50% to 60%. The success of this procedure owes much to the use of cyclosporine and tacrolimus for immunosuppressant therapy. These drugs selectively suppress the activities of helper T cells (CD4 lymphocytes) by inhibiting production of interleukin-2 (IL-2) and other cytokines. IL-2 is required for the generation and
proliferation activating
B
cells
Cyclosporine of
cytotoxic responsible
T
cells for
is usually
initially agents
(eg, responsible
for
T
cell-dependent combined
graft rejection
and humoral
responses.
with mycophenolate
effective and
corticosteroids azathioprine).
in cyclosporine-resistant
to cyclosporine
OKT-3,
Tacrolimus and
other has
proved rejection
and is
the as
the primary
preferred immunosuppressant
a
monoclonal antibody
agent.
alternative
The directed
against use
lymphocytes, of
anti- has
been extremely
useful in

treating steroid-resistant
acute rejection.
Additional factors influencing the improvement in liver transplantation outcome include a greater understanding and experience with transplantation and improved assessment and monitoring with echocardiography.

These procedures can be divided into three phases: a dissection (preanhepatic) phase, an anhepatic phase, and a neohepatic phase.
1. Dissection (preanhepatic) phase: Through a hockey stick incision, the liver is dissected so that it remains attached only by the inferior vena cava and portal vein. The hepatic artery, and common bile duct are ligated. Large abdominal varices may prolong the duration of, and increase the blood loss associated with, this phase.
. Anhepatic phase: Once the donor liver graft is ready, the portal vein is clamped, followed by the inferior vena cava above and below the liver. The liver is then lifted out of the abdomen. Venovenous bypass maybe employed during this phase. The donor liver is then anastomosed to the supraand infrahepatic inferior venae cavae and the portal vein.
. Revascularization and biliary reconstruction (neohepatic or postanhepatic) phase: Following completion of the venous anastomoses, venous clamps are removed from the cava, allowing venous blood to return to the heart. Next, the portal vein is slowly opened, allowing blood to flush out preservation fluid and other substances accumulated in the liver during its ischemic time. This reperfusion may result in hypotension, arrhythmias, or cardiac arrest—a scenario termed the reperfusion syndrome. The circulation to the new liver is completed by anastomosing the hepatic artery. Lastly, the common bile duct of the donor liver is then usually anastomosed to the recipient using the recipient's common bile duct or a Roux-en-Y choledochojejunostomy.
for

Potential problems include multiorgan dysfunction caused by cirrhosis, massive blood loss, hemodynamic instability from clamping and unclamping the inferior vena cava and portal vein, metabolic consequences of the anhepatic phase, and air embolism and hyperkalemia. Preoperative coagulation defects, thrombocytopenia, and previous abdominal surgery greatly increase blood loss. Extensive venous collaterals between the portal and systemic venous circulations also contribute to increased bleeding from the abdominal wall. Potential complications of massive blood transfusion include hypothermia, coagulopathies, hyperkalemia, citrate intoxication (hypocalcemia), and the potential transmission of infectious agents. Blood salvaging techniques are useful in reducing donor red blood cell transfusion.

Bleeding is a recurring problem during each phase of liver transplantation. Adequate venous access is paramount in anesthetic management. Several large-bore (14-gauge or larger) intravenous catheters should be placed above the diaphragm. Specialized 8.5FR catheters can be placed in antecubital veins and used in conjunction with rapid infusion devices. Most centers place a large-bore central venous catheter. Efforts to minimize the risk of hypothermia should include the use of fluid warming and forced-air surface warming devices.

All patients require direct intraarterial pressure monitoring. A central venous catheter should be available. Goal-directed hemodynamic and fluid management utilizing arterial pulse wave analysis, esophageal Doppler, PVI, or TEE is common. Urinary output should be monitored throughout surgery via an indwelling urinary catheter.
Laboratory measurements constitute an important part of intraoperative monitoring. Serial hematocrit measurements guide red blood cell replacement. Similarly, frequent measurements of arterial blood gases, serum electrolytes, serum ionized calcium, and serum glucose are necessary to detect and appropriately treat metabolic derangements. Coagulation can be monitored by measuring PT, activated partial thromboplastin time, fibrinogen level, and platelet counts, and by point-of-care viscoelastic coagulation analysis—TEG, ROTEM, or Sonoclot analysis. These latter modalities not only assess overall clotting and platelet function, but can also detect fibrinolysis.

Most patients should be considered as having a "full stomach," often because of marked abdominal distention or recent upper gastrointestinal bleeding. General anesthesia is usually induced via a rapid sequence induction. The semiupright (back up) position during induction prevents rapid oxygen desaturation and facilitates ventilation until the abdomen is open. Hyperventilation should be avoided unless there is increased intracranial pressure. Anesthesia is generally maintained with a volatile agent (usually isoflurane or sevoflurane), and an intravenous opioid (usually fentanyl or sufentanil). The concentration of the volatile agent should be limited to less than 1 minimum alveolar concentration in patients with severe encephalopathy. Nitrous oxide is usually avoided. Many patients are routinely transferred to the intensive care unit intubated and mechanically ventilated at the end of the operative procedure, although immediate postoperative extubation may be considered if the patient is comfortable, cooperative, physiologically stable, and not hemorrhaging significantly.

When the liver is removed, the large citrate load from blood products is no longer metabolized and results in hypocalcemia and secondary myocardial depression. Periodic calcium chloride administration (1 g) is necessary, but should be guided by ionized calcium concentration measurements to avoid hypercalcemia. Progressive acidosis is also encountered because acid metabolites from the intestines and lower body are not cleared by the liver. Sodium bicarbonate therapy may be necessary and should similarly be guided by arterial blood gas analysis. Excessive administration of sodium bicarbonate results in hypernatremia, hyperosmolality, and accentuation of the metabolic alkalosis that typically follows massive blood transfusions. Tromethamine should be considered when large amounts of alkali therapy are necessary. Although hypoglycemia can occur during the anhepatic phase, hyperglycemia is a more common occurrence following reperfusion.
Pulmonary and systemic (paradoxic) air embolism can occur when the circulation is fully reestablished to the donor liver because air often enters hepatic sinusoids after harvesting. Systemic air embolism probably reflects the fact that many of these patients have extensive arteriovenous communications. The anhepatic phase ends when the three venous clamps are removed and the donor liver is perfused. Thromboembolic phenomena are also possible following reperfusion.

Perfusion of the donor liver by the recipient's blood often results in transiently increased serum potassium concentration of up to 1 to 2 mEq/L and increased systemic acidosis. Reperfusion releases potassium from any remaining preservative solution (115-120 mEq/L of potassium) still within the liver, as well as potassium released from tissues distal to venous clamps. Unclamping may also release a large acid load from ischemic tissue in the lower body (particularly without venovenous bypass); preemptive administration of sodium bicarbonate is advocated by some.
When the
circulation to
the new
liver is
established, the
sudden increase
in blood
or, more
volume, acidosis,
commonly, sodium
and hyperkalemia
bradyarrhythmias.
bicarbonate,
Hyperfibrinolysis can
produce
inotropic support
is is
increase commonly
in tissue
plasminogen activator
inhibitor
Fibrinolysis also
present tachyarrhythmias,
to calcium
often and
activator and
a-antiplasmin can
be detected
analysis.
seems and
chloride and
required.
to be
a
due decrease
during by
point-of-care e-Aminocaproic
formation acid
of plasmin,
may or
be tranexamic
indicated to
a
marked in
plasminogen the
anhepatic viscoelastic
phase.
coagulation acid,
in which
those inhibit
the instances,
but should
used prophylactically.

Patients often have an uncomplicated postoperative course and, after a sufficient period of observation in the intensive care unit, may be transferred directly to the nursing unit designed for liver transplant patients. Problems to anticipate include graft dysfunction or failure, persistent hemorrhage, fluid overload, metabolic abnormalities (particularly metabolic not
be

@ Diabetic autonomic neuropathy may limit the patient's ability to compensate (with tachycardia and increased peripheral resistance) for intravascular volume changes and may predispose the patient to cardiovascular instability (eg, postinduction hypotension) and even sudden cardiac death.
@ Temporomandibular joint and cervical spine mobility should be assessed preoperatively in diabetic patients to reduce the likelihood of unanticipated difficult intubation. Difficult intubation has been reported in as many as 30% of persons with type 1 diabetes.
© Sulfonylureas and metformin have long half-lives and many clinicians will discontinue them 24 to 48 h before surgery. They can be started postoperatively when the patient resumes oral intake.
@ Incompletely treated hyperthyroid patients may be chronically hypovolemic and prone to an exaggerated hypotensive response to induction of anesthesia.
@ Clinically hypothyroid patients are more susceptible to the hypotensive effect of anesthetic agents because of diminished cardiac output, blunted baroreceptor reflexes, and decreased intravascular volume.
@ Patients with glucocorticoid deficiency must receive adequate steroid replacement therapy during the perioperative period.
@ Inpatients with a pheochromocytoma, drugs or techniques that indirectly stimulate or promote the release of catecholamines (eg, ephedrine, hypoventilation, or bolus doses of ketamine), potentiate the arrhythmic effects of catecholamines (halothane), or consistently release histamine (eg, large doses of atracurium or morphine sulfate)
may precipitate hypertension and are best avoided.
@ Obese patients may be difficult to intubate as a result of limited mobility of the temporomandibular and atlantooccipital joints, a narrowed upper airway, and a shortened distance between the mandible and sternal fat pads.
© The key to perioperative management of patients with carcinoid syndrome is to avoid anesthetic and surgical techniques or agents that could cause the tumor to release vasoactive substances.

The underproduction or overproduction of hormones can have life-threatening consequences. Therefore, it is not surprising that endocrinopathies affect anesthetic management. This chapter briefly reviews normal physiology and pathophysiology of four endocrine organs: the pancreas, the thyroid, the parathyroids, and the adrenal glands. It also considers obesity and carcinoid syndrome.

Adults normally secrete approximately 50 units of insulin each day from the B cells of the islets of Langerhans in the pancreas. The rate of insulin secretion is primarily determined by the plasma glucose concentration. Insulin, the most important anabolic hormone, has multiple metabolic effects, including facilitating glucose and potassium entry into adipose and muscle cells; increasing glycogen, protein, and fatty acid synthesis; and decreasing glycogenolysis, gluconeogenesis, ketogenesis, lipolysis, and protein catabolism. In general, insulin stimulates anabolism and weight gain, whereas lack of insulin is associated with catabolism, a negative nitrogen balance, and weight loss (Table 35-1).

Anabolic
Promotes glycogenesis
Increases synthesis of triglycerides, cholesterol, and VLDL?
Increases protein synthesis
Promotes glycolysis
Anticatabolic
Inhibits glycogenolysis
Inhibits ketogenesis
Inhibits gluconeogenesis

Anabolic
Increases amino acid transport
Increases glucose transport
Enhances activity of glycogen synthetase
Inhibits activity of glycogen phosphorylase

Promotes triglyceride storage
Induces lipoprotein lipase, making fatty acids available for absorption into fat cells
Increases glucose transport into fat cells, thus increasing availability of a- glycerol phosphate for triglyceride synthesis
Inhibits intracellular lipolysis lReproduced with permission from Gardner DG, Shoback D. Greenspan's Basic & Clinical Endocrinology. 9th ed. New York, NY: McGraw-Hill Education; 2011.
2VLDL, very low-density lipoprotein.

Diabetes mellitus is characterized by hyperglycemia and glycosuria arising from impairment of carbohydrate metabolism. The cause is an absolute or relative deficiency of insulin or of insulin responsiveness. The diagnosis is based on an elevated fasting plasma glucose greater than 126 mg/dL or glycated hemoglobin
(HbA,,) of 6.5% or greater. Values are sometimes reported for blood glucose, which runs 12% to 15% lower than plasma glucose. Even when testing whole blood, newer glucose meters calculate and display plasma glucose. Long-term complications of diabetes include retinopathy, kidney disease, hypertension, coronary artery disease, peripheral and cerebral vascular disease, and peripheral and autonomic neuropathies.
Diabetes is classified in multiple ways (Table 35—2). Type 1 (insulinrequiring due to endogenous insulin deficiency) and type 2 (insulin-resistant) diabetes are the most common and well known.

Fasting
126 mg/dL (7.0 mmol/L)
Glucose tolerance test
200 mg/dL (11.1 mmol/L)

Type 1 (juvenile)
Absolute insulin deficiency secondary to immune- mediated or idiopathic causes Onset in childhood or adulthood secondary to insulin resistance (relative insulin insensitivity) Onset of disease during pregnancy; may or may not persist postpartum
Type 2
Gestational
There are three life-threatening acute complications of diabetes and its treatment—diabetic ketoacidosis (DKA), hyperosmolar nonketotic coma, and hypoglycemia—in addition to other acute medical problems (such as sepsis) in which the presence of diabetes makes treatment more difficult. Decreased insulin activity allows the catabolism of free fatty acids into ketone bodies (acetoacetate and B-hydroxybutyrate), some of which are weak acids (see Chapter 50). Accumulation of these organic acids results in DKA, an anion-gap metabolic acidosis. DKA can easily be distinguished from lactic acidosis; lactic acidosis is identified by elevated plasma lactate (>6 mmol/L) and the absence of urine and plasma ketones. However, both DKA and starvation ketosis may occur concurrently with lactic acidosis, underscoring the need for measurement of lactate. DKA is associated with type 1 diabetes mellitus, but a rare individual
status and seizures. Severe hyperglycemia causes a factitious hyponatremia: each 100 mg/dL increase in plasma glucose lowers plasma sodium concentration by 1.6 mEq/L. Treatment includes fluid resuscitation with normal saline, relatively small doses of insulin, and potassium supplementation.

Hypoglycemia in the diabetic patient is the result of an absolute or relative excess of insulin relative to carbohydrate intake and exercise. Furthermore, diabetic patients are incompletely able to counter hypoglycemia despite secreting glucagon or epinephrine (counterregulatory failure). The dependence of the brain on glucose as an energy source makes it the organ most susceptible to episodes of hypoglycemia. If hypoglycemia is not treated, mental status changes can progress from anxiety, lightheadedness, headache, or confusion to convulsions and coma. Systemic manifestations of hypoglycemia result from catecholamine discharge and include diaphoresis, tachycardia, and nervousness. Most of the signs and symptoms of hypoglycemia will be masked by general anesthesia. Although the lower boundary of normal plasma glucose levels is illdefined, medically important hypoglycemia is present when plasma glucose is less than 50 mg/dL. The treatment of hypoglycemia in anesthetized or critically ill patients consists of intravenous administration of 50% glucose (each milliliter of 50% glucose will raise the blood glucose of a 70-kg patient by approximately 2 mg/dL). Awake patients can be treated orally with fluids containing glucose or sucrose.

Abnormally elevated hemoglobin A,, concentrations identify patients who have maintained poor control of blood glucose over time. These patients are at greater risk for perioperative hyperglycemia, perioperative complications, adverse outcomes, and increased costs. The perioperative morbidity of diabetic patients is related to their preexisting end-organ damage. Unfortunately, many surgical patients with type 2 diabetes mellitus may be unaware of their condition. A preoperative chest radiograph is not routinely indicated in diabetic patients. Diabetic patients have an increased incidence of ST-segment and T-wavesegment abnormalities on preoperative electrocardiograms (ECGs). Myocardial ischemia or old infarction may be evident on an ECG despite a negative history. Diabetic patients with hypertension have a 50% likelihood of coexisting diabetic autonomic neuropathy (Table 35—3). Reflex dysfunction of the autonomic nervous system may be increased by old age, diabetes of longer than @ 10 years'
duration, coronary artery disease, or B-adrenergic blockade. Diabetic autonomic neuropathy may limit the patient's ability to compensate (with tachycardia and increased peripheral resistance) for intravascular volume changes and may predispose the patient to cardiovascular instability (eg, postinduction hypotension) and even sudden cardiac death. Autonomic dysfunction contributes to delayed gastric emptying (diabetic gastroparesis). Premedication with a nonparticulate antacid and metoclopramide is often used in obese diabetic patients with signs of cardiac autonomic dysfunction. However, autonomic dysfunction of the gastrointestinal tract may be present without any signs of cardiac involvement. Diabetic neuropathy may also lead to silent (painless) myocardial ischemia.

Painless myocardial ischemia
Orthostatic hypotension
Lack of heart rate variability!
Reduced heart rate response to atropine and propranolol
Resting tachycardia
Early satiety
Neurogenic bladder
Lack of sweating
Impotence
Normal heart rate variability during voluntary deep breathing (6 breaths/min) should be >10 beats/min.
Diabetic renal dysfunction is manifested first by proteinuria and later by elevated serum creatinine. By these criteria, most patients with type 1 diabetes have evidence of kidney disease by 30 years of age. Chronic hyperglycemia leads to glycosylation of tissue proteins and reduced mobility of joints. Temporomandibular @ joint and cervical spine mobility should be assessed preoperatively in diabetic patients to reduce the likelihood of unanticipated difficult intubations. Difficult intubation has been reported in as many as 30% of persons with type 1 diabetes.

The goal of intraoperative blood glucose management is to avoid hypoglycemia

Preoperative, Bolus Administration = D.W(1.5 mL/ kg/h) NPH! insulin (half usual am dese). Preoperative, Continuous Infusion = D.W (1 mU/kg/h) Regular insulin: Units/h= Plasma glucose 180. Intraoperative, Bolus Administration = Regularinsulin (as per sliding scale). Intraoperative, Continuous Infusion = Sameas preoperative. Postoperative, Bolus Administration = Same as intraoperative. Postoperative, Continuous Infusion = Same as preoperative
'NPH, neutral protamine Hagedorn.
A better method, feasible in all but short procedures, is to administer regular insulin as a continuous infusion. The advantage of this technique is more precise control of insulin delivery than can be achieved with a subcutaneous or intramuscular injection of NPH insulin, particularly in conditions associated with poor skin and muscle perfusion. Regular insulin can be added to normal saline in a concentration of 1 unit/mL and the infusion begun at 0.1 unit/kg/h or less. As blood glucose fluctuates, the insulin infusion can be adjusted up or down as required. Dedication of a small-gauge intravenous line for the dextrose infusion prevents interference with other intraoperative fluids and drugs. Supplemental dextrose can be administered if the patient becomes hypoglycemic (<100 mg/dL). It must be stressed that these doses are approximations and do not apply to patients in catabolic states (eg, sepsis, hyperthermia).
The dose required may be approximated by the following formula:
A reasonable target for the intraoperative maintenance of blood glucose is less than 180 mg/dL and greater than 85 mg/dL.
When administering an intravenous insulin infusion to surgical patients, adding some (eg, 20 mEq) KCI to each liter of maintenance fluid may be useful, as insulin causes an intracellular potassium shift. Because individual insulin needs can vary dramatically, any formula should be considered only a crude guideline. Periodic glucose measurements are required.

type 1 diabetes can leave the pump programmed to deliver "basal" amounts of regular insulin (or insulin glargine) when fasting. By definition, the basal rate is the amount of insulin required during fasting. Patients can safely undergo short outpatient surgery with the pump on the basal setting. If more extensive inpatient procedures are required, these patients will normally suspend their pumps and be managed with intravenous insulin infusions and periodic blood glucose measurements, as described earlier.

Close monitoring of blood glucose must continue postoperatively. There is considerable patient-to-patient variation in onset and duration of action of insulin preparations (Table 35-5). For example, the onset of action of subcutaneous regular insulin is less than 1 h, but in rare patients its duration of action may continue for 6 h. NPH insulin typically has an onset of action within 2 h, but the action can last longer than 24 h.

Short-acting, Insulin Type = Lispro Regular Semilente, Semitard. Short-acting, Onset = 10-20 min 15-30 min 30-60 min. Short-acting, Peak Action = 30-90 min 1-3h 4-6h. Short-acting, Duration = 4-6h 5-7h 12-16h. Intermediate-acting, Insulin Type = Lente, Lentard, NPH*. Intermediate-acting, Onset = 2-4h. Intermediate-acting, Peak Action = 8-10h. Intermediate-acting, Duration = 18-24h. Long-acting, Insulin Type = Ultralente, Glargine, Insulatard. Long-acting, Onset = 4-Sh. Long-acting, Peak Action = 8-14h. Long-acting, Duration = 25-36h
There is considerable patient-to-patient variation, Not al! formulations are available in every country.
- NPH, neutral protamine Hagedorn

Dietary iodine is absorbed by the gastrointestinal tract, converted to iodide ion, and actively transported into the thyroid gland. Once in the gland, iodide is oxidized back to iodine, which is bound to the amino acid tyrosine. The end result is two hormones—triiodothyronine (T3) and thyroxine (T,)—which are bound to proteins and stored within the thyroid. Although the gland releases more T, than T3, the latter is more potent and less protein bound. Of all circulating T3, most is formed peripherally from partial deiodination of T,. An elaborate feedback mechanism controls thyroid hormone synthesis and involves the hypothalamus (thyrotropin-releasing hormone [TRH]), the anterior pituitary
(thyroid-stimulating hormone [TSH]), autoregulation, and the adequacy of iodine intake.
Thyroid hormone (T3) increases carbohydrate and fat metabolism and is an important factor in determining growth and metabolic rate. An increase in metabolic rate is accompanied by an increase in oxygen consumption and CO, production. Heart rate and contractility are also increased, presumably from direct actions of thyroid on contractile proteins and other internal protein alterations.

All elective surgical procedures, including subtotal thyroidectomy, should be postponed until the patient is rendered clinically and chemically euthyroid with medical treatment. The patient should have normal T3 and T, concentrations, and should not have resting tachycardia. Antithyroid medications and Badrenergic antagonists are continued through the momming of surgery. Administration of propylthiouracil and methimazole is particularly important because of their relatively short half-lives.
Patients with larger goiters or thyroid masses will often have preoperative CT or MR imaging studies to rule out extension into the mediastinum. Such extension might mandate sternotomy for complete resection.

Cardiovascular function and body temperature should be closely monitored in patients with a history of hyperthyroidism. When emergency surgery must proceed despite clinical hyperthyroidism, the hyperdynamic circulation can be controlled intraoperatively with an esmolol infusion. The exophthalmos of Graves disease increases the risk of corneal abrasion or ulceration.
Ketamine, indirect-acting adrenergic agonists (ephedrine), and other drugs that stimulate the sympathetic nervous system are best avoided in patients with current or recently corrected hyperthyroidism because of the possibility of exaggerated elevations @ in blood pressure and heart rate. Incompletely treated hyperthyroid patients may be hypovolemic and prone to an exaggerated hypotensive response to induction of anesthesia. Adequate anesthetic depth must be obtained before laryngoscopy or surgical stimulation to avoid tachycardia, hypertension, and ventricular arrhythmias.
Thyrotoxicosis is associated with an increased incidence of myopathies and myasthenia gravis; therefore, neuromuscular blocking agents (NMBs) should be administered cautiously. Hyperthyroidism does not increase anesthetic requirements; that is, there is no increase in minimum alveolar concentration.

The most serious threat to a hyperthyroid patient undergoing surgery is thyroid storm, which is characterized by hyperpyrexia, tachycardia, altered consciousness (eg, agitation, delirium, coma), and hypotension. Thyroid storm is a medical emergency that requires aggressive management and monitoring (see Case Discussion, Chapter 56). The onset is usually 6 to 24 h after surgery but can occur intraoperatively, mimicking malignant hyperthermia. Unlike malignant hyperthermia, thyroid storm is not associated with muscle rigidity, elevated creatine kinase, or a marked degree of metabolic (lactic) and respiratory acidosis. Treatment includes hydration and cooling, an esmolol infusion or another intravenous B-blocker (with a target of maintaining heart rate <100/min), propylthiouracil (250-500 mg every 6 h orally or by nasogastric tube) followed by sodium iodide (1 g intravenously over 12 h), and correction of any precipitating cause (eg, infection). Hydrocortisone (100-200 mg every 8 h) or the equivalent is recommended to prevent complications from coexisting adrenal gland suppression.
Thyroidectomy is associated with several potential surgical complications. Recurrent laryngeal nerve palsy will result in hoarseness (unilateral) or aphonia and stridor (bilateral). Vocal cord function can be evaluated by laryngoscopy immediately following "deep extubation"; however, this is rarely necessary. Immobility of one or both cords may require reintubation and exploration of the wound. Hematoma formation may cause airway compromise from collapse of the trachea, particularly in patients with tracheomalacia. Dissection of the hematoma into the compressible soft tissues of the neck may distort the airway anatomy and may make intubation difficult. Immediate treatment includes opening the neck wound and evacuating the clot, then reassessing the need for reintubation. Anesthesia staff in the postoperative setting must be prepared to open the surgical wound and relieve airway compression if the surgeon is unavailable.
Hypoparathyroidism from unintentional removal of all four parathyroid glands will cause acute hypocalcemia within 12 to 72 h (see the section on Clinical Manifestations under Hypoparathyroidism). Pneumothorax is a rare complication of neck exploration.

Hypothyroidism can be caused by autoimmune disease (eg, Hashimoto thyroiditis), thyroidectomy, radioactive iodine, antithyroid medications, iodine deficiency, or failure of the hypothalamic—pituitary axis (secondary hypothyroidism). Hypothyroidism during neonatal development results in cretinism, a condition marked by physical and mental retardation. Clinical manifestations of hypothyroidism in the adult are usually subtle and include

Patients with
uncorrected severe
hypothyroidism or
myxedema coma
must not
undergo elective
surgery.
Such patients
should be
T3
intravenously prior to urgent or emergency surgery. Although a euthyroid state is ideal, mild to moderate hypothyroidism is not an absolute contraindication to necessary surgery, for example, urgent coronary bypass surgery.
Symptomatic hypothyroid patients should receive minimal preoperative sedation given that they are prone to drug-induced respiratory depression. In addition, they may fail to respond to hypoxia with increased minute ventilation.
Patients who have been rendered euthyroid may receive their usual dose of thyroid medication on the morning of surgery; however, most commonly used preparations have long half-lives (the half-life of T, is about 8 days), and omission of a single dose should have no medical importance.

@ Clinically hypothyroid patients are more susceptible to the hypotensive effect of anesthetic agents because of diminished cardiac output, blunted baroreceptor reflexes, and decreased intravascular volume. In this circumstance, ketamine or etomidate can be recommended for induction of anesthesia. The possibility of coexistent primary adrenal insufficiency should be considered in cases of refractory hypotension. Other potential coexisting conditions include hypoglycemia, anemia, hyponatremia, difficulty during intubation because of a large tongue, and hypothermia from a low basal metabolic rate.

Recovery from general anesthesia may be delayed in hypothyroid patients by hypothermia, respiratory depression, or slowed drug biotransformation; thus, these patients may require mechanical ventilation. Because hypothyroidism increases vulnerability to respiratory depression, a multimodal approach to postoperative pain management, rather than strict reliance on opioids is appropriate.

Parathyroid hormone (PTH) is the principal regulator of calcium homeostasis. It increases serum calcium concentrations directly by promoting resorption of bone and teeth, limiting renal excretion of calcium, and indirectly by stimulating vitamin D synthesis in the kidney to enhance gastrointestinal absorption. PTH decreases serum phosphate by increasing renal excretion. Two other recently identified hormones, fibroblast growth factor 23 (FGF23) and klotho, have effects on phosphate metabolism. FGF23 arises from bone and acts on the kidney to induce phosphaturia and to reduce production of vitamin D3. FGF23 actions require that klotho activate the FGF receptor.
The effects of PTH on calcium serum levels are countered in lower animals by calcitonin, a hormone excreted by parafollicular C cells in the thyroid. Although calcitonin is effective given as a pharmaceutical in humans, its role in normal human physiology is not clear (Table 35-6). Of total body calcium, 99% is in the skeleton. Of the calcium in the blood, 40% is bound to proteins and 60% is ionized or complexed to organic ions. Unbound ionized calcium is physiologically the most important fraction.

Parathyroid hormone (PTH), Bone = —_ Increases resorption of calcium and phosphate. Parathyroid hormone (PTH), Kidney = Increases reabsorption of calcium; decreases reabsorption of phosphate; increases conversion of 25-OHD, to 1,25(OH), D.;' decreases reabsorption of bicarbonate. Parathyroid hormone (PTH), Intestines = No direct effects; increases renal production of vitamin D.. Calcitonin, Bone = Inhibits osteoclastic resorption. Calcitonin, Kidney = Decreases reabsorption of calcium and phosphate. Calcitonin, Intestines = Inhibits reabsorption of phosphate; increases renal excretion of sodium and calcium. Vitamin D, Bone = Maintains Ca** homeostasis. Vitamin D, Kidney = Decreases reabsorption of calcium (probably less important than PTH). Vitamin D, Intestines = Increases absorption of calcium
25-OHD, 25-hydroxyvitamin D,; 1,25(0H),D,, 1,25-dihydroxyvitamin D,
The remaining major player in this system, vitamin D, is a steroid hormone that can be absorbed via the gastrointestinal tract from food or synthesized from cholesterol derivatives. Conversion of 7-dehydrocalciferol to vitamin D3 is facilitated by exposure to ultraviolet light. Hydroxylation in the liver and kidneys yields 1,25(OH) D3 (1,25-dihydroxyvitamin D3), the active molecule that binds vitamin D receptors (VDRs) and produces its physiological actions. VDRs are located in the nucleus where, after binding vitamin D, they regulate expression of specific genes. 1,25(OH).D3 promotes normal growth and remodeling of bone, in addition to helping to regulate calcium and phosphate concentrations.

Causes of primary hyperparathyroidism include parathyroid adenomas, hyperplasia of the parathyroid gland, and certain carcinomas. Secondary hyperparathyroidism is an adaptive response to hypocalcemia produced by conditions such as end-stage kidney disease or intestinal malabsorption syndromes. Ectopic hyperparathyroidism is due to production of PTH by rare tumors outside the parathyroid gland. Overall, the most common cause of hypercalcemia in hospitalized patients is malignancy. Parathyroid hormone— related peptide may cause significant hypercalcemia when secreted by a tumor (eg, bronchogenic [lung] carcinoma or hepatoma). Bone invasion with osteolytic hypercalcemia may complicate multiple myeloma, lymphoma, or leukemia. Nearly all clinical manifestations of hyperparathyroidism are due to hypercalcemia (Table 35—7). Rarer causes of hypercalcemia include bone metastases of solid organ tumors, vitamin D intoxication, milk-alkali syndrome, lithium therapy, sarcoidosis, and prolonged immobilization. The treatment of hyperparathyroidism depends on the cause, but surgical removal of all four glands is often required in the setting of parathyroid hyperplasia. When there is a single adenoma, its removal cures many patients with sporadic primary hyperparathyroidism.

Ventricular arrhythmias
ECG! changes (shortened QT interval,” widened T wave)

Polyuria
Impaired renal concentrating ability
Kidney stones
Hyperchloremic metabolic acidosis
Dehydration
Polydipsia

Constipation
Anorexia
Peptic ulcer disease

Mental status change (eg, delirium, psychosis, coma)
lace, electrocardiogram.
2The QT interval may be prolonged at serum calcium concentrations >16 mg/dL.

In patients with hypercalcemia due to hyperparathyroidism, hydration with normal saline and diuresis facilitated by furosemide will usually decrease serum calcium to acceptable values (<14 mg/dL, 7 mEq/L, or 3.5 mmol/L). More aggressive therapy with the intravenous bisphosphonates pamidronate (Aredia) or etidronate (Didronel) may be necessary for patients with hypercalcemia of malignancy. Plicamycin (Mithramycin), glucocorticoids, calcitonin, or dialysis may be necessary when intravenous bisphosphonates are not sufficient or are contraindicated. Hypoventilation should be avoided, as acidosis increases ionized calcium. Elevated calcium levels can cause cardiac arrhythmias. The response to NMBs may be altered in patients with preexisting muscle weakness caused by the effects of calcium at the neuromuscular junction. Osteoporosis worsened by hyperparathyroidism predisposes patients to vertebral compression and bone fractures during anesthetic procedures, positioning, and transport. The notable postoperative complications of parathyroidectomy are similar to those for subtotal thyroidectomy.

Hypoparathyroidism is usually due to deficiency of PTH following parathyroidectomy. Clinical manifestations of hypoparathyroidism are a result of hypocalcemia (Table 35-8), which can also be caused by kidney failure, hypomagnesemia, vitamin D deficiency, and acute pancreatitis (see Chapter 49). Hypoalbuminemia decreases total serum calcium (a 1-g/dL drop in serum albumin causes a 0.8-mg/dL decrease in total serum calcium), but ionized calcium, the active entity, is unaltered. The archetypical presentation of hypocalcemia is tetany, classically diagnosed by the Chvostek sign (painful twitching of the facial musculature following tapping over the facial nerve) or the Trousseau sign (carpal spasm following inflation of an arm tourniquet above systolic blood pressure for 3 min). Treatment of symptomatic hypocalcemia consists of intravenous administration of calcium salts.

ECG! changes (prolonged QT interval)

Neuromuscular irritability (eg, laryngospasm, inspiratory stridor, tetany, seizures)
Perioral paresthesia
Mental status changes (eg, dementia, depression, psychosis)
lec, electrocardiogram.
Mild hypocalcemia is common following cardiopulmonary bypass or infusion of albumin solutions. In many adult patients, this need not be treated as the response of the PTH—vitamin D axis will usually be sufficient to restore ionized calcium to normal values and mild hypocalcemia will usually have no hemodynamic consequences.

Serum calcium must be normalized in any patient who presents with cardiac manifestations of severe hypocalcemia. Alkalosis from hyperventilation or sodium bicarbonate therapy will further decrease ionized calcium. Although citrate-containing blood products usually do not lower serum calcium significantly, they should be administered cautiously in patients with preexisting hypocalcemia. Other considerations include avoiding the bolus administration of albumin solutions (which bind and reduce ionized calcium concentrations) and being mindful of the possibility of hypocalcemia-induced coagulopathy.

A diet deficient in vitamin D combined with a lack of sun (ultraviolet light)
exposure will lead to rickets in children and osteomalacia in adults. Decreased concentrations of vitamin D, more common in the elderly, those who live in far northern latitudes, and those too frail or ill to spend time outdoors, have been associated with a great many conditions and diseases. Causal linkages have been hard to identify. Despite the great numbers of articles that have been written on this topic, we lack proper evidence regarding the vitamin D concentrations below which vitamin D replacement is mandatory, how best to replace vitamin D in those who require replacement, and indeed whether vitamin D replacement improves outcomes in those conditions in which reduced vitamin D concentrations have been associated with worse outcomes. The data are even murkier regarding vitamin D replacement in patients undergoing surgery.

utilization. These actions tend to raise blood glucose and worsen diabetic control. Glucocorticoids are required for vascular and bronchial smooth muscle to respond to catecholamines. Because glucocorticoids are structurally related to aldosterone, most tend to promote sodium retention and potassium excretion (a mineralocorticoid effect). ACTH released by the anterior pituitary is the principal regulator of glucocorticoid secretion. Basal secretion of ACTH and glucocorticoids exhibits a diurnal rhythm. Stressful conditions promote secretion of ACTH and cortisol, while circulating glucocorticoids inhibit ACTH and cortisol secretion. Under nonstressed conditions, endogenous production of cortisol, the most important endogenous glucocorticoid, averages 20 mg/d. The structure, biosynthesis, physiological effects, and metabolism of catecholamines are discussed in Chapter 14. Epinephrine constitutes 80% of adrenal catecholamine output in humans. Catecholamine release is regulated mainly by sympathetic cholinergic preganglionic fibers that innervate the adrenal medulla. Stimuli include exercise, hemorrhage, surgery, hypotension, hypothermia, hypoglycemia, hypercapnia, hypoxemia, pain, and fear.

Hypersecretion of aldosterone by the adrenal cortex (primary aldosteronism) can be due to a unilateral adenoma (aldosteronoma or Conn syndrome), bilateral hyperplasia, or in very rare cases carcinoma of the adrenal gland. Some disease states stimulate aldosterone secretion by affecting the renin—angiotensin system. For example, congestive heart failure, hepatic cirrhosis with ascites, nephrotic syndrome, and some forms of hypertension (eg, renal artery stenosis) can cause secondary hyperaldosteronism. Although both primary and secondary hyperaldosteronism are characterized by increased levels of aldosterone, only the latter is associated with increased renin activity. The usual clinical manifestations of mineralocorticoid excess include hypokalemia and hypertension, and an increased ratio of aldosterone—plasma renin activity has been noted in laboratory studies.

Fluid and electrolyte disturbances can be corrected preoperatively using spironolactone. This aldosterone antagonist is a potassium-sparing diuretic with antihypertensive properties. Intravascular volume can be assessed preoperatively by testing for orthostatic hypotension.

Atrophy or destruction of both adrenal glands results in a combined deficiency of mineralocorticoids and glucocorticoids (see the section on Glucocorticoid Deficiency). Isolated deficiency of mineralocorticoid activity almost never occurs.

Glucocorticoid excess may be due to exogenous administration of steroid hormones, intrinsic hyperfunction of the adrenal cortex (eg, adrenocortical adenoma), ACTH production by a nonpituitary tumor (ectopic ACTH syndrome), or hypersecretion by a pituitary adenoma (Cushing disease). Regardless of the cause, an excess of corticosteroids produces Cushing syndrome, characterized by muscle wasting and weakness, osteoporosis, central obesity, abdominal striae, glucose intolerance, menstrual irregularity, hypertension, and mental status changes.

Patients with Cushing syndrome may be volume overloaded and have hypokalemic metabolic alkalosis resulting from the mineralocorticoid activity of glucocorticoids. These abnormalities should be corrected preoperatively in the manner previously described. Patients with osteoporosis are at risk for fracture during positioning. If the cause of Cushing syndrome is exogenous glucocorticoids, the patient's adrenal glands may not be able to respond to perioperative stresses, and supplemental steroids are indicated (see the section on Glucocorticoid Deficiency). Likewise, patients undergoing adrenalectomy require intraoperative glucocorticoid replacement (in adults, intravenous hydrocortisone succinate, 100 mg every 8 h has been the traditional stress dose). Although many adrenal tumors are removed uneventfully during laparoscopic surgery, complications of adrenalectomy may include major blood loss and unintentional pneumothorax.

Primary adrenal insufficiency (Addison disease), caused by destruction of the adrenal gland, results in a combined mineralocorticoid and glucocorticoid deficiency. Clinical manifestations are due to aldosterone deficiency (hyponatremia, hypovolemia, hypotension, hyperkalemia, and metabolic acidosis) and cortisol deficiency (weakness, fatigue, hypoglycemia, hypotension, and weight loss).
Secondary adrenal insufficiency is a result of inadequate ACTH secretion by the pituitary. The most common cause of secondary adrenal insufficiency is prior administration of exogenous glucocorticoids. Because mineralocorticoid secretion is usually adequate in secondary adrenal insufficiency, fluid and electrolyte disturbances are not present. Acute adrenal insufficiency (addisonian crisis), however, can be triggered in steroid-dependent patients who do not receive appropriate glucocorticoid doses during periods of stress (eg, infection, trauma, surgery) and in patients who receive infusions of etomidate. The clinical features of this medical emergency include fever, abdominal pain, orthostatic hypotension, and hypovolemia that may progress to circulatory shock unresponsive to resuscitation.

@ Patients with glucocorticoid deficiency must receive adequate steroid replacement therapy during the perioperative period. Patients who have received potentially suppressive doses of steroids (eg, the daily equivalent of 5 mg of prednisone) by any route of administration (topical, inhalational, or oral) for a period of more than 2 weeks any time in the previous 12 months may be unable to respond appropriately to surgical stress and should receive perioperative glucocorticoid supplementation.
What represents adequate steroid coverage is controversial, and there are those who advocate variable dosing based on the extent of the surgery. Although adults normally secrete 20 mg of cortisol daily, this may increase to more than 300 mg under conditions of maximal stress. Thus, a traditional recommendation was to administer 100 mg of hydrocortisone every 8 h beginning on the morning of surgery. An alternative low-dose regimen (25 mg of hydrocortisone at the time of induction followed by an infusion of 100 mg during the subsequent 24 h) maintains plasma cortisol levels equal to or higher than those reported in healthy patients undergoing similar elective surgery. This second regimen might be particularly appropriate for diabetic patients, in whom glucocorticoid administration often interferes with control of blood glucose.

Pheochromocytoma is a catecholamine-secreting tumor that consists of cells originating from the embryonic neural crest. This tumor accounts for 0.1% of all cases of hypertension. Although the tumor is usually localized in a single adrenal gland, 10% to 15% are bilateral or extraadrenal. Approximately 10% of tumors are malignant. The cardinal manifestations of pheochromocytoma are paroxysmal hypertension, headache, sweating, and palpitations. Unexpected intraoperative hypertension and tachycardia during manipulation of abdominal structures may occasionally be the first indications of an undiagnosed pheochromocytoma. The pathophysiology, diagnosis, and treatment of these tumors require an understanding of catecholamine metabolism and of the pharmacology of adrenergic agonists and antagonists. The Case Discussion in Chapter 14 examines these aspects of pheochromocytoma management.

Overweight and obesity are classified using the body mass index (BMI). Overweight is defined as a BMI of 24 kg/m? or higher, obesity as a BMI of 30 or higher, and extreme obesity (formerly termed "morbid obesity") as a BMI of more than 40. BMI is calculated by dividing the weight (in kilograms) by the height (in meters) squared. A great many BMI calculators are available online or as apps for smartphones. Health risks increase with the degree of obesity and with increased abdominal distribution of weight. Men with a waist measurement of 40 in. or more and women with a waist measurement of 35 in. or more are at increased health risk. For a patient 1.8 m tall and weighing 70 kg, the BMI would be as shown in the following formula:
= 21.6 kg/m?

Obesity is associated with many diseases, including type 2 diabetes mellitus, hypertension, coronary artery disease, obstructive sleep apnea, degenerative joint disease (osteoarthritis), and cholelithiasis. Even in the absence of obvious coexisting disease, however, extreme obesity has profound physiological consequences. Oxygen demand, CO, production, and alveolar ventilation are elevated because metabolic rate is proportional to body weight. Excessive adipose tissue over the thorax decreases chest wall compliance even though lung compliance may remain normal. Increased abdominal mass forces the diaphragm cephalad, yielding lung volumes suggestive of restrictive lung disease. Reductions in lung volumes are accentuated by the supine and Trendelenburg positions. In particular, functional residual capacity may fall below closing capacity. If this occurs, some alveoli will close during normal tidal volume ventilation, causing a ventilation/perfusion mismatch.
Whereas obese patients are often hypoxemic, only a few are hypercapnic, which when present should be a warning of impending complications. Obstructive sleep apnea (OSA) is a complication of extreme obesity characterized by hypercapnia, cyanosis-induced polycythemia, right-sided heart failure, and somnolence. These patients appear to have blunted respiratory drive and often suffer from loud snoring and upper-airway obstruction during sleep. OSA patients often report dry mouths and daytime somnolence; bed partners frequently describe apneic pauses. OSA has also been associated with increased perioperative complications, including hypertension, hypoxia, arrhythmias, myocardial infarction, pulmonary edema, stroke, and death. The potential for difficult mask ventilation and difficult intubation, followed by upper airway obstruction during recovery, should be anticipated.
OSA patients are vulnerable during the postoperative period, particularly when sedatives or opioids have been given. OSA patients positioned supine are unusually susceptible to upper airway obstruction. For patients with known or suspected OSA, postoperative continuous positive airway pressure (CPAP) should be considered until the anesthesiologist can be sure that the patient can protect his or her airway and maintain spontaneous ventilation without evidence of obstruction. Both the American Society of Anesthesiologists and the Society of Ambulatory Anesthesia offer guidelines on perioperative management of the patient with OSA (see Chapter 44).

An OSA patient's heart has an increased workload, as cardiac output and blood volume increase to perfuse additional fat stores. Arterial hypertension leads to left ventricular hypertrophy. Elevations in pulmonary blood flow and pulmonary artery vasoconstriction from persistent hypoxia can lead to pulmonary hypertension and cor pulmonale.
Obesity is also associated with hiatal hernia, gastroesophageal reflux disease, delayed gastric emptying, and hyperacidic gastric fluid, as well as with an increased risk of gastric cancer. Fatty infiltration of the liver also occurs and may be associated with abnormal liver tests, but the extent of infiltration does not correlate well with the degree of liver test abnormality.

For the
reasons outlined
above, obese
patients are
at an
increased risk
for developing
aspiration pneumonia.
Pretreatment with
nonparticulate antacid,
antagonists, and metoclopramide should be considered. Premedication with respiratory depressant drugs must be avoided in patients with OSA.
Before anesthetizing
extremely obese
patients for
major surgery
one should
attempt to
such items
signs assess
cardiopulmonary as
chest of
cardiac taken
with reserve.
radiograph,
ECG, failure
a
cuff intraarterial
Preoperative and
may be
of arterial
difficult the
appropriate access
should
Obscured landmarks,
may make
to size.
be checked
difficult regional
testing blood
identify.
may gas
include analysis.
Blood
Potential sites
in pressures
Physical must
for anticipation
positioning, anesthesia
techniques.
Obese be
intravenous of
technical and
extensive difficult
mobility of
may be
the standard
difficult temporomandibular
airway, and
a
shortened and
difficulties.
layers of
equipment to
intubate adipose
tissue
@
as and
a
result atlantooccipital
distance between

joints, the
mandible and
of limited
a
narrowed and
sternal upper
fat pads.
H,
Because of the risks of aspiration and hypoventilation, morbidly obese patients are often intubated for all but short general anesthetics. If intubation appears possibly to be difficult, we recommend using either video laryngoscopy or fiberoptic bronchoscopy. Positioning the patient on an intubating ramp is helpful. Auscultation of breath sounds may prove difficult. Even with controlled ventilation these patients may require increased inspired oxygen concentrations to prevent hypoxia, particularly in the lithotomy, Trendelenburg, or prone positions. Subdiaphragmatic abdominal laparotomy packs can cause further deterioration of pulmonary function and a reduction of arterial blood pressure by increasing the resistance to venous return. Volatile anesthetics may be metabolized more extensively in obese patients. Increased metabolism may explain the increased incidence of halothane hepatitis observed in obese patients. Obese patients may have a prolonged induction and emergence from inhaled anesthetics.
Theoretically, greater fat stores would increase the volume of distribution for lipid-soluble drugs (eg, benzodiazepines, opioids) relative to a lean person of the same body weight. However, the volume of distribution of, for example, fentanyl or sufentanil is so large that obesity has minimal influence. Water-soluble drugs (eg, NMBs) have small volumes of distribution, which are minimally increased by body fat. Therefore, the dosing of water-soluble drugs should be based on ideal body weight to avoid overdosage.
Although dosage requirements for epidural and spinal anesthesia are difficult to predict, obese patients typically require 20% to 25% less local anesthetic per blocked segment because of epidural fat and distended epidural veins. Continuous epidural anesthesia has the usual advantages of providing pain relief and potentially decreasing respiratory complications in the postoperative period. Regional nerve blocks, particularly when combined with multimodal pain control, have the additional advantages of not interfering with the postoperative deep vein thrombosis prophylaxis, rarely producing hypotension, and of reducing the need for opioids (see Chapter 48).

Respiratory failure is a major postoperative problem of morbidly obese patients. The risk of postoperative hypoxia is increased in these patients, especially when there is preoperative hypoxia, and with surgery involving the thorax or upper abdomen. An obese patient should remain intubated until there is no doubt that an adequate airway and tidal volume will be maintained, NMBs are completely reversed, and the patient is awake. This does not mean that all obese patients need be ventilated overnight in an intensive care unit. If the patient is extubated in the operating room, supplemental oxygen should be provided during transportation to the postanesthesia care unit. A 45° modified sitting position will improve ventilation and oxygenation. The risk of hypoxia extends for several days into the postoperative period, and providing supplemental oxygen or CPAP, or both, should be routinely considered. Other common postoperative complications in obese patients include wound infection, deep venous thrombosis, and pulmonary embolism. Morbidly obese and OSA patients may be candidates for outpatient surgery provided they are adequately monitored and assessed postoperatively before discharge to home, and provided the surgical procedure will not require large doses of opioids for postoperative pain control. It is hard to conceive of a better indication for multimodal analgesia.

Carcinoid syndrome results from the secretion of vasoactive substances (eg, serotonin, kallikrein, histamine) from enteroepinephrine tumors (carcinoid tumors). Most of these tumors are located in the gastrointestinal tract, so their metabolic products are released into the portal circulation and largely metabolized by the liver before they can cause systemic effects. However, the products of nonintestinal tumors (eg, pulmonary, ovarian) or hepatic metastases bypass the portal circulation and can cause a variety of clinical manifestations. Many patients undergo surgery for resection of carcinoid tumors; most such patients have not experienced carcinoid syndrome.

The most common manifestations of carcinoid syndrome are cutaneous flushing, bronchospasm, profuse diarrhea, dramatic swings in arterial blood pressure (usually hypotension), and supraventricular arrhythmias (Table 35-9). Carcinoid syndrome is associated with right-sided heart disease caused by valvular and myocardial plaque formation and, in some cases, implantation of tumors on the tricuspid and pulmonary valves. The diagnosis of carcinoid syndrome is confirmed by detection of serotonin metabolites in the urine (5hydroxyindoleacetic acid) or plasma or suggested by elevated plasma levels of chromogranin A. Treatment varies depending on tumor location but may include surgical resection, symptomatic relief, or specific serotonin and histamine antagonists. Somatostatin, an inhibitory peptide, reduces the release of

Serotonin, 1 = Vasoconstriction (coronary artery spasm, hypertension), increased intestinal tone, water and electrolyte imbalance (diarrhea), tryptophan deficiency (hypoproteinemia, pellagra). Kallikrein, 1 = Vasodilation (hypotension, flushing), bronchoconstriction. Histamine, 1 = —_- Vasodilation (hypotension, flushing), arrhythmias, bronchoconstriction

® The key to perioperative management of patients with carcinoid syndrome is to avoid anesthetic and surgical techniques or agents that could cause the tumor to release vasoactive substances. Regional anesthesia may limit release of stress hormones perioperatively. Large bolus doses of histamine-releasing drugs (eg, morphine and atracurium) should be avoided. Surgical manipulation of the tumor can cause a massive release of hormones. Monitoring likely will include an arterial line. If there are concerns about hemodynamic instability or intrinsic heart disease caused by carcinoid syndrome, transesophageal echocardiography may be helpful. Alterations in carbohydrate metabolism may lead to unsuspected hypoglycemia or hyperglycemia. Consultation with an endocrinologist may help clarify the role of antihistamine, antiserotonin drugs (eg, methysergide), octreotide (a long-acting somatostatin analogue), or antikallikrein drugs (eg, corticosteroids) in specific patients.

An isolated thyroid nodule is discovered during physical examination of a 36-year-old woman complaining of diarrhea and headaches. Workup of the tumor reveals hypercalcemia and an elevated calcitonin level, which leads to the diagnosis of medullary cancer of the thyroid and primary hyperparathyroidism. During induction of general anesthesia for total thyroidectomy, the patient's blood pressure rises to 240/140 mm Hg and her heart rate approaches 140 beats/min, with frequent premature ventricular contractions. The operation is canceled, an arterial line is inserted, and the patient is treated with intravenous esmolol and nicardipine.
What could be the cause of this patient's hypertensive crisis during induction of general anesthesia?
Multiple endocrine neoplasia (MEN) is characterized by tumors in several endocrine organs. MEN type 1 consists of pancreatic (gastrinomas, insulinomas), pituitary, and parathyroid tumors. MEN type 2 consists of medullary thyroid carcinoma, pheochromocytoma, and hyperparathyroidism (type 2a) or multiple mucosal neuromas (type 2b or type 3). The hypertensive episode in this case may be due to a previously undiagnosed pheochromocytoma. The pheochromocytoma in MEN may consist of small multiple tumors. These patients are typically young adults with strong family histories of MEN. If multiple surgeries are planned, pheochromocytoma resection will usually be scheduled first.
What is calcitonin, and why is it associated with medullary cancer?
Calcitonin is a polypeptide manufactured by the parafollicular cells (C cells) in the thyroid gland. It is secreted in response to increases in plasma ionic calcium and tends to lower calcium levels by affecting kidney and bone function. Therefore, it acts as an antagonist of parathyroid hormone (see Table 35-6).
Why is this patient hypercalcemic if calcitonin lowers serum calcium?
An excess or deficiency of calcitonin has minor effects in humans compared with the effects of parathyroid disorders. This patient's hypercalcemia is most likely due to coexisting primary hyperparathyroidism (MEN type 2a).
Are headache and diarrhea consistent with the diagnosis of MEN?

The history of headaches suggests the possibility of pheochromocytoma, whereas diarrhea may be due to calcitonin or one of the other peptides often produced by medullary thyroid carcinoma (eg, ACTH, somatostatin, Bendorphin).

Because of the life-threatening hemodynamic changes associated with pheochromocytoma, this entity must be medically controlled before any surgery can be considered (see Case Discussion, Chapter 14). Because MEN syndromes are hereditary, family members should be screened for early signs of pheochromocytoma, thyroid cancer, and hyperparathyroidism.

Practice guidelines for the perioperative management of patients with obstructive sleep apnea: An updated report by the American Society of Anesthesiologists Task Force on Perioperative Management of patients with obstructive sleep apnea. Anesthesiology. 2014;120:268. Society for Ambulatory Anesthesia Consensus Statement on Selection of
Patients With Obstructive Sleep Apnea Undergoing Ambulatory Surgery. http://www.sambahq.org/main/clinical-practice-guidelines/

Agus MS, Wypij D, Hirshberg EL, et al; HALF-PINT Study Investigators and the PALISI Network. Tight glycemic control in critically ill children. N Engl J Med. 2017;376:729.
Arlt W, Allolio B. Adrenal insufficiency. Lancet. 2003;361:1881.
Azim S, Kashyap SR. Bariatric surgery: Pathophysiology and outcomes.
Endocrinol Metab Clin North Am. 2016;45:905.
Blau JE, Collins MT. The PTH-vitamin D-FGF23 axis. Rev Endocr Metab Disord. 2015;16:165.
- El-Menyar A, Mekkodathil A, Al-Thani H. Traumatic injuries in patients with diabetes mellitus. J Emerg Trauma Shock. 2016;9:64.
Jones GC, Macklin JP, Alexander WD. Contraindications to the use of metformin. Evidence suggests that it is time to amend the list. BMJ.

@ Any factor that increases intraocular pressure in the setting of an open globe may cause drainage of aqueous or extrusion of vitreous through the wound, serious complications that can permanently worsen vision.
@ Succinylcholine increases intraocular pressure by 5 to 10 mm Hg for 5 to 10 min after administration, principally through prolonged contracture of the extraocular muscles. However, in studies of hundreds of patients with open eye injuries, no patient experienced extrusion of ocular contents after administration of succinylcholine. Thus, succinylcholine is not contraindicated in cases of open eye injuries.
© Traction on extraocular muscles, pressure on the eyeball, administration of a retrobulbar block, and trauma to the eye can elicit a wide variety of cardiac arrhythmias ranging from bradycardia and ventricular ectopy to sinus arrest or ventricular fibrillation.
@ Complications involving the intraocular expansion of gas bubbles injected by the ophthalmologist can be avoided by discontinuing nitrous oxide at least 15 min prior to the injection of air or sulfur hexafluoride, or by avoiding the use of nitrous oxide entirely.
@ Medications applied topically to mucosa are absorbed systemically at a rate intermediate between absorption following intravenous and subcutaneous injection.
@ Echothiophate is an irreversible cholinesterase inhibitor used in the treatment of glaucoma. Topical application leads to systemic absorption and an inhibition of plasma cholinesterase activity. Because succinylcholine is metabolized by this enzyme, echothiophate will prolong its duration of action.
@ The key to inducing anesthesia in a patient with an open eye injury is controlling intraocular pressure with a smooth induction. Coughing and
gagging during intubation is avoided by first achieving a deep level of anesthesia and profound paralysis.
@ The postretrobulbar block apnea syndrome is probably due to injection of local anesthetic into the optic nerve sheath, with spread into the cerebrospinal fluid.
© Regardless of the anesthetic technique, American Society of
Anesthesiologists standards for basic monitoring must be employed, and equipment and drugs necessary for airway management and resuscitation must be immediately available.

Ophthalmic surgery poses unique problems, including regulation of intraocular pressure, control of intraocular gas expansion, prevention of the oculocardiac reflex and management of its consequences, and management of systemic effects of ophthalmic drugs. Mastery of general and sedation anesthesia techniques for ophthalmic surgery and a thorough understanding of potentially complicating issues—including the comorbidities of an increasing geriatric patient population —are necessary for optimal perioperative outcomes. In addition, the majority of ophthalmic procedures are performed under topical or regional anesthesia. The anesthesiologist must be familiar with their potential complications, including those of the accompanying sedation, even if not personally administering the topical anesthetic or the block.

The eye can be considered a hollow sphere with a rigid wall. If the contents of the sphere increase, the normal intraocular pressure of 12 to 20 mm Hg will rise. For example, glaucoma is caused by an obstruction to aqueous humor outflow. Similarly, intraocular pressure will rise if the volume of blood within the globe is increased. A rise in venous pressure will increase intraocular pressure by decreasing aqueous drainage and increasing choroidal blood volume. Any event that alters arterial blood pressure or ventilation (eg, laryngoscopy, intubation, airway obstruction, coughing, Trendelenburg position) can also affect intraocular pressure (Table 36-1).
TABLE 36-1 The effect of cardiac and respiratory variables on intraocular pressure (IOP).!
Central venous pressure, Effecton 10P = . Increase, Effecton 10P = T. Decrease, Effecton 10P = Lu. Arterial blood pressure, Effecton 10P = . increase, Effecton 10P = . Decrease, Effecton 10P = . Paco,, Effecton 10P = . Increase (hypoventilation), Effecton 10P = Tt. Decrease (hyperventilation), Effecton 10P = . Pad,, Effecton 10P = . increase, Effecton 10P = Qo. Decrease, Effecton 10P = '
"i decrees A
moder, matte
_inereaie
(mild, moderate,
Alternatively, compressing the globe without a proportional change in the volume of its contents will increase intraocular pressure. Pressure on the eye from a malpositioned mask, improper prone positioning, or retrobulbar hemorrhage can lead to a marked increase in intraocular pressure, possible eye pain, and temporary or permanent visual changes.
Intraocular pressure helps to maintain the shape and the optical properties of the eye. Temporary variations in pressure are normally well tolerated. For example, blinking raises intraocular pressure by 5 mm Hg, and squinting (forced contraction of the orbicularis oculi muscles) may transiently increase intraocular pressure greater than 50 mm Hg. However, even brief episodes of increased intraocular pressure in patients with underlying low ophthalmic artery pressure (eg, from systemic hypotension, arteriosclerotic involvement of the retinal artery) may cause retinal ischemia.

When the globe is opened by surgical incision (Table 36—2) or traumatic perforation, intraocular pressure approaches atmospheric pressure. Any factor @ that increases intraocular pressure in the setting of an open globe may cause drainage of aqueous or extrusion of vitreous through the wound, serious complications that can permanently worsen vision.

Cataract extraction
Corneal laceration repair
Corneal transplant (penetrating keratoplasty)
Peripheral iridectomy
Removal of foreign body
Ruptured globe repair
Secondary intraocular lens implantation
Trabeculectomy (and other filtering procedures)

Most anesthetic drugs either reduce intraocular pressure or have no effect (Table 36-3). Intraocular pressure decreases with inhalational anesthetics in proportion to anesthetic depth. There are multiple causes for this: A drop in blood pressure reduces choroidal volume, relaxation of the extraocular muscles lowers wall tension, and pupillary constriction facilitates aqueous outflow. Intravenous anesthetics also decrease intraocular pressure, with the exception of ketamine, which usually raises arterial blood pressure and does not relax extraocular muscles.

Inhaled anesthetics, Effect on lOP = . Volatile agents, Effect on lOP = Ly. Nitrous oxide, Effect on lOP = 4. Intravenous anesthetics, Effect on lOP = . , Effect on lOP = Lt. Benzodiazepines, Effect on lOP = u. Ketamine, Effect on lOP = ?. Oploids, Effect on lOP = L. Muscle relaxants, Effect on lOP = . Suceinylcholine, Effect on lOP = tT. Nondepolarizers, Effect on lOP = orl
incon
Topically administered anticholinergic drugs result in pupillary dilation (mydriasis), which may precipitate or worsen angle-closure glaucoma. Systemically administered atropine or glycopyrrolate for premedication are not associated with intraocular hypertension, even in patients with glaucoma.
® Succinylcholine increases intraocular pressure by 5 to 10 mm Hg for 5 to 10 min after administration, principally through prolonged contracture of the extraocular muscles. However, in studies of hundreds of patients with open eye injuries, no patient experienced extrusion of ocular contents after administration of succinylcholine. Thus, succinylcholine is not contraindicated in cases of open eye injuries. Nevertheless, dogma often trumps data and ophthalmic surgeons may request that it not be administered in certain circumstances. Unlike other skeletal muscle, extraocular muscles contain myocytes with multiple neuromuscular junctions, and depolarization of these cells by succinylcholine causes prolonged contracture. The resulting increase in intraocular pressure may have several effects: it will cause spurious measurements of intraocular pressure during examinations under anesthesia in glaucoma patients, potentially leading to unnecessary surgery, and prolonged contracture of the extraocular muscles may result in an abnormal forced duction test, a maneuver utilized in strabismus surgery to evaluate the cause of extraocular muscle imbalance and to determine the type of surgical correction. Nondepolarizing neuromuscular blockers (NMBs) do not increase intraocular pressure, and we advocate that succinylcholine be reserved for rapid-sequence induction.

A gas bubble may be injected by the ophthalmologist into the posterior chamber during vitreous surgery. Intravitreal air injection will tend to flatten a detached retina and facilitate anatomically correct healing. Nitrous oxide administration is contraindicated in this circumstance: The bubble will increase in size if nitrous oxide is administered because nitrous oxide is 35 times more soluble than nitrogen in blood (see Chapter 8). Thus, it tends to diffuse into an air bubble more rapidly than nitrogen (the major component of air) is absorbed by the bloodstream. If the bubble expands after the globe is closed, intraocular pressure will rise.
Sulfur hexafluoride is an inert gas that is less soluble in blood than is nitrogen —and much less soluble than nitrous oxide. Its longer duration of action (up to 10 days) compared with an air bubble can provide a therapeutic advantage. The bubble size doubles within 24 h after injection, because nitrogen from inhaled air enters the bubble more rapidly than the sulfur hexafluoride diffuses into the bloodstream. Even so, unless high volumes of pure sulfur hexafluoride are injected, the slow bubble expansion does not typically raise intraocular pressure. If the patient is breathing nitrous oxide, however, the bubble will rapidly increase in size and may lead to intraocular hypertension. A 70% inspired nitrous oxide concentration will almost triple the size of a 1-mL bubble and may double the pressure in a closed eye within 30 min. Subsequent discontinuation of nitrous oxide will lead to reabsorption of the bubble, which has become a mixture of nitrous oxide and sulfur hexafluoride. The consequent fall in intraocular pressure may precipitate another retinal detachment.
@ Complications involving the intraocular expansion of gas bubbles can be avoided by discontinuing nitrous oxide at least 15 min prior to the injection of air or sulfur hexafluoride, or by avoiding the use of nitrous oxide entirely. Nitrous oxide should be avoided until the bubble is absorbed (5 days after air and 10 days after sulfur hexafluoride injection). Avoiding nitrous oxide seems the simplest approach in these patients.

Topically applied eye drops are systemically absorbed by vessels in the conjunctival sac and the nasolacrimal duct mucosa (see Case Discussion, Chapter 13). One drop (typically, approximately 1/20 mL) of 10% phenylephrine contains approximately 5 mg of drug. Compare this dose with the intravenous dose of phenylephrine (0.05—0.1 mg) used to treat an adult patient with acute hypotension. Medications applied @ topically to mucosa are absorbed systemically at a rate intermediate between absorption following intravenous and subcutaneous injection. The two patient populations most likely to require eye surgery, pediatric and geriatric, are at particular risk of the toxic effects of topically applied medications and should receive at most a 2.5% phenylephrine solution (Table 36-4).

Acetylcholine, Mechanise of Acthon = Cholinergic agonins (miosis). Acetylcholine, Effect = Bronchospasm, bradycardia, kypotension:. Acetarclamide, Mechanise of Acthon = Carbon anhydrase inhibitor (decreases KOPF"). Acetarclamide, Effect = Dhuress, hypokalemic metabolic ackdosts. Atropine, Mechanise of Acthon = Anticholinerpic (enydeiars}. Atropine, Effect = Central antichotinergic sysdroene!. Cytlopentolite, Mechanise of Acthon = Aarticholinen gic (enpdirianst). Cytlopentolite, Effect = Dhorkentation, prychors, commubsons.. Echothiophate, Mechanise of Acthon = (Cholinetterste intibtor (miosis, decreates OP). Echothiophate, Effect = Profengation of suceinyichoting amd mivacuriuns paralysis, beonchospasm.. Epinephiine, Mechanise of Acthon = Syenparisesic agonist (mydriasis, Geceoases OP). Epinephiine, Effect = Hypertension, bradycarcka, tachycardia, headache. Phenylephrine, Mechanise of Acthon = Adkenengit aqoelit inrydriasis, vasoconatietion). Phenylephrine, Effect = © Hypertension, tachycardia, dysrhythodas. Seopolanine-, Mechanise of Acthon = Anbicholinengic (enrdesa, vasoconstriction}. Seopolanine-, Effect = Cenanal anticholinergic symdroene”. Tenotat, Mechanise of Acthon = B-Adrenergic blocking agent (decreases KP). Tenotat, Effect = Brodycardia, asthma, congestive heart failure

The choice between general and local anesthesia should be made jointly by the patient, anesthesiologist, and surgeon. Patients may refuse to consider local anesthesia due to fear of being awake during the operation, fear of the eye block procedure, or unpleasant recall of a previous eye block or local eye procedure. General anesthesia is indicated in children and uncooperative patients, as even small head movements can prove disastrous during microsurgery.

Patients undergoing eye surgery may be apprehensive; however, premedication must be administered with caution and only after careful consideration of the patient's medical status. Adult patients are often elderly, with systemic illnesses such as hypertension, diabetes mellitus, and coronary artery disease, and pediatric patients may have associated congenital disorders.

The choice of induction technique for eye surgery usually depends more on the patient's other medical problems than on the patient's eye disease or the specific operation contemplated. One exception is the patient with a ruptured globe. The key to inducing @ anesthesia in a patient with an open eye injury is controlling intraocular pressure with a smooth induction. Specifically, coughing during intubation must be avoided by first achieving a deep level of anesthesia and profound paralysis. The intraocular pressure response to laryngoscopy and endotracheal intubation can be moderated by prior administration of intravenous lidocaine (1.5 mg/kg), an opioid (eg, remifentanil 0.5—1 mcg/kg or alfentanil 20 mcg/kg), or esmolol (0.5-1.5 mg/kg). A nondepolarizing muscle relaxant or succinylcholine may be used. Many patients with open globe injuries have full stomachs and require a rapid-sequence induction technique to avoid aspiration (see the later Case Discussion); despite theoretical concerns, succinylcholine does not increase the likelihood of vitreous loss with open eye injuries.

Eye surgery often necessitates positioning the anesthesia provider away from the patient's airway, making close monitoring of pulse oximetry and the capnograph particularly important. Endotracheal tube kinking, breathing circuit disconnection, and unintentional extubation may be more likely because of the surgeon working near the airway. Kinking and obstruction can be minimized by using a preformed oral RAE (Ring-Adair-Elwyn) endotracheal tube (Figure 36— 1). The possibility of arrhythmias caused by the oculocardiac reflex increases the importance of closely monitoring the electrocardiogram. In contrast to most other types of pediatric surgery, infant body temperature may rise during ophthalmic surgery because of head-to-toe draping and minimal body surface exposure. End-tidal CO, analysis helps to differentiate this situation from malignant hyperthermia.
FIGURE 36-1 An oral RAE endotracheal tube has a preformed right-angle bend at the level of the teeth so that it exits the mouth away from the surgical field during ophthalmic or nasal surgery.
The pain and stress evoked by eye surgery are considerably less than during a major surgical procedure. "Lighter" anesthesia might be attractive if the consequences of patient movement were not so potentially catastrophic. The lack of cardiovascular stimulation inherent in most eye procedures combined with the need for adequate anesthetic depth can result in hypotension in elderly individuals. This problem is usually avoided by ensuring adequate intravenous hydration and by administering small doses of intravenous vasoconstrictors. Administration of nondepolarizing muscle relaxants to avoid patient movement is often used in such circumstances in order to allow reduced depth of general anesthesia.
Emesis caused by vagal stimulation is a common postoperative problem following eye surgery, particularly with strabismus repair. The Valsalva effect and the increase in central venous pressure that accompany vomiting can be detrimental to the surgical result. Intraoperative intravenous administration of drugs that prevent postoperative nausea and vomiting is strongly recommended.

A smooth emergence from general anesthesia is important in order to minimize the risk of postoperative wound dehiscence. Coughing or gagging due to stimulus from the endotracheal tube can be minimized by extubating the patient at a moderately deep level of anesthesia. As the time of extubation approaches intravenous lidocaine (1.5 mg/kg) may be given to blunt cough reflexes temporarily. Extubation proceeds 1 to 2 min after the lidocaine administration and during spontaneous respiration with 100% oxygen. Proper airway maintenance is crucial until the patient's cough and swallowing reflexes return. Severe discomfort is unusual following eye surgery. Scleral buckling procedures, enucleation, and ruptured globe repair are the most painful operations. Modest incremental doses of intravenous opioid usually provide sufficient analgesia. The surgeon should be alerted if severe pain is noted following emergence from general anesthesia, as it may signal intraocular hypertension, corneal abrasion, or other surgical complications.

Options for local anesthesia for eye surgery include topical application of local anesthetic or placement of a retrobulbar, peribulbar, or sub-Tenon (episcleral) block. All of these techniques are commonly combined with intravenous sedation. Local anesthesia is preferred to general anesthesia for eye surgery because local anesthesia involves less physiological trespass and is less likely to be associated with postoperative nausea and vomiting. However, eye block procedures have potential complications and may not provide adequate ophthalmic akinesia or analgesia. Some patients may be unable to lie perfectly still for the duration of the surgery. For these reasons, appropriate equipment and qualified personnel required to treat the complications of local anesthesia and to induce general anesthesia must be readily available.

In this technique, local anesthetic is injected behind the eye into the cone formed by the extraocular muscles (Figure 36—2), and a facial nerve block is utilized to prevent blinking (Figure 36-3). A blunt-tipped 25-gauge needle penetrates the lower lid at the junction of the middle and lateral one-third of the orbit (usually 0.5 cm medial to the lateral canthus). Awake patients are instructed to stare supranasally as the needle is advanced toward the apex of the muscle cone. Commonly, patients undergoing such eye blocks will receive a brief period of deep sedation or general anesthesia during the block (using such agents as etomidate, propofol, or remifentanil). After aspiration to preclude intravascular injection, 2 to 5 mL of local anesthetic is injected, and the needle is removed. Choice of local anesthetic varies, but lidocaine 2% or bupivacaine (or ropivacaine) 0.75% are common. Addition of epinephrine may reduce bleeding and prolong the anesthesia. A successful retrobulbar block is accompanied by anesthesia, akinesia, and abolishment of the oculocephalic reflex (ie, a blocked eye does not move during head turning).
FIGURE 36-2 A: During administration of a retrobulbar block, the patient looks supranasally as a needle is advanced 1.5 cm along the inferotemporal wall of the orbit. B: The needle is then redirected upward and nasally toward the apex of the orbit and advanced until its tip penetrates the muscle cone.

Complications of retrobulbar injection of local anesthetics include retrobulbar hemorrhage, perforation of the globe, optic nerve injury, intravascular injection with resultant convulsions, oculocardiac reflex, trigeminal nerve block, respiratory arrest, and, rarely, acute neurogenic pulmonary edema. Forceful injection of local anesthetic into the ophthalmic artery causes retrograde flow toward the brain and may result in an instantaneous seizure. @ The postretrobulbar block apnea syndrome is probably due to injection of local anesthetic into the optic nerve sheath, with spread into the cerebrospinal fluid. The central nervous system is exposed to high concentrations of local anesthetic, leading to mental status changes that may include unconsciousness. Apnea occurs within 20 min and resolves within an hour. Treatment is supportive, with positive-pressure ventilation to prevent hypoxia, bradycardia, and cardiac arrest. Adequacy of ventilation must be constantly monitored in patients who have received retrobulbar anesthesia.
The adjuvant hyaluronidase is frequently added to local anesthetic solutions used in eye blocks to enhance the spread and density of the block. Patients may rarely experience an allergic reaction to hyaluronidase. Retrobulbar hemorrhage, cellulitis, occult injury, and contact allergy to topical eye drops must be ruled out in the differential diagnosis. Retrobulbar injection is usually not performed in patients with bleeding disorders or receiving anticoagulation therapy because of the risk of retrobulbar hemorrhage, extreme myopia because the elongated globe increases the risk of perforation, or an open eye injury because the pressure from injecting fluid behind the eye may cause extrusion of intraocular contents through the wound.

In contrast to retrobulbar blockade, in the peribulbar blockade technique, the needle does not penetrate the cone formed by the extraocular muscles. Advantages of the peribulbar technique include less risk of penetration of the globe, optic nerve, and artery, and less pain on injection. Disadvantages include a slower onset and an increased likelihood of ecchymosis. Both techniques will have success at akinesia of the equal producing eye.
The peribulbar block is performed with the patient supine and looking directly ahead (or possibly under a brief period of deep sedation). After topical anesthesia of the conjunctiva, one or two transconjunctival injections are administered (Figure 36—4). As the eyelid is retracted, an inferotemporal injection is given halfway between the lateral canthus and the lateral limbus. The needle is advanced under the globe, parallel to the orbital floor; when it passes the equator of the eye, it is directed slightly medial (20°) and cephalad (10°), and 5 mL of local anesthetic is injected. To ensure akinesia, a second 5-mL injection may be given through the conjunctiva on the nasal side, medial to the caruncle, and directed straight back parallel to the medial orbital wall, pointing slightly cephalad (20°).
FIGURE 36-4 Anatomic landmarks for the introduction of a needle or catheter in most frequently employed eye blocks: (1) medial canthus peribulbar anesthesia, (2) lacrimal caruncle, (3) semilunaris fold of the conjunctiva, (4) medial canthus episcleral anesthesia, and (5) inferior and temporal peribulbar anesthesia.

Tenon's fascia surrounds the globe and extraocular muscles. Local anesthetic injected beneath it into the episcleral space spreads circularly around the sclera and to the extraocular muscle sheaths (Figure 36-4). A special blunt curved cannula is used for a sub-Tenon block. After topical anesthesia, the conjunctiva is lifted along with Tenon's fascia in the inferonasal quadrant with forceps. A small nick is then made with blunt-tipped scissors, which are then slid underneath to create a path in Tenon's fascia that follows the contour of the globe and extends past the equator. While the eye is still fixed with forceps, the cannula is inserted, and 3 to 4 mL of local anesthetic is injected. Complications with sub-Tenon blocks are significantly less than with retrobulbar and peribulbar techniques. Globe perforation, hemorrhage, cellulitis, permanent visual loss, and local anesthetic spread into cerebrospinal fluid have been reported.

A facial nerve block prevents squinting of the eyelids during surgery and allows placement of a lid speculum. There are several techniques of facial nerve block: van Lint, Atkinson, and O'Brien (Figure 36-3). The major complication of these blocks is subcutaneous hemorrhage. The Nadbath technique blocks the facial nerve as it exits the stylomastoid foramen under the external auditory canal, in close proximity to the vagus and glossopharyngeal nerves. This block is not recommended because it has been associated with vocal cord paralysis, laryngospasm, dysphagia, and respiratory distress.

Simple topical local anesthetic techniques have been used for anterior chamber (eg, cataract) and glaucoma operations, and, increasingly, the trend has been to eliminate local anesthetic injections entirely. A typical regimen for topical local anesthesia consists of application of 0.5% proparacaine (also known as proxymetacaine) local anesthetic drops, repeated at 5-min intervals for five applications, followed by topical application of a local anesthetic gel (lidocaine plus 2% methyl-cellulose) with a cotton swab to the inferior and superior conjunctival sacs. Ophthalmic 0.5% tetracaine may also be utilized. Topical anesthesia is not appropriate for posterior chamber surgery (eg, retinal detachment repair with a buckle), and it works best for faster surgeons with a gentle surgical technique that does not require akinesia of the eye.

Many techniques of intravenous sedation are available for eye surgery, and the particular drug used is less important than the dose. Deep sedation, although sometimes used during placement of ophthalmic nerve blocks, is almost never used intraoperatively because of the risks of apnea, aspiration, and unintentional patient movement during surgery. An intraoperative light sedation regimen that includes small doses of midazolam, with or without fentanyl or sufentanil, is recommended. Doses vary considerably among patients but should be administered in small increments.
Patients may find administration of eye blocks uncomfortable, and many anesthesia providers will administer small incremental doses of propofol to produce a brief state of unconsciousness during the regional block. Some will substitute a bolus of opioid (remifentanil 0.1-0.5 mcg/kg or alfentanil 375-500
mcg) to produce a brief period of intense analgesia during the eye block procedure.
Administration of an antiemetic should be considered if an opioid is used. Regardless of the @ anesthetic technique, American Society of Anesthesiologists standards for basic monitoring must be employed, and equipment and drugs necessary for airway management and resuscitation must be immediately available.

A 12-year-old boy is brought to the emergency department after being shot in the eye with a pellet gun. A brief examination by the ophthalmologist reveals intraocular contents presenting at the wound. The boy is scheduled for emergency repair of the ruptured globe.
What should be emphasized in the preoperative evaluation of this patient?
Aside from taking a medical history and performing a physical examination, the time of last oral intake before or after the injury should be established. The patient must be considered to have a full stomach if the injury occurred within 8 h after the last meal, even if the patient did not eat for several hours after the injury: Gastric emptying is delayed by the pain and anxiety that follow trauma.
What is the significance of a full stomach in a patient with an open globe injury?
Managing patients who have sustained penetrating eye injuries provides a challenge because of the need to deal with at least two conflicting objectives: (1) preventing further damage to the eye by avoiding increases in intraocular pressure, and (2) preventing pulmonary aspiration in a patient with a full stomach. However, many of the common strategies used to achieve these objectives are in conflict with one another (Tables 36—5 and 36-6). For example, although regional anesthesia (eg, retrobulbar block)
minimizes the risk of aspiration pneumonia, it is relatively contraindicated in patients with penetrating eye injuries because injecting local anesthetic behind the globe increases intraocular pressure and may lead to expulsion of intraocular contents. Therefore, these patients require general anesthesia —despite the increased risk of aspiration pneumonia.

Avoid direct pressure on the globe
Patch eye with Fox shield
No retrobulbar or peribulbar injections
Careful face mask technique
Avoid increases in central venous pressure
Prevent coughing during induction and intubation
Ensure a deep level of anesthesia and relaxation prior to laryngoscopy!
Avoid head-down positions
Extubate under deep anesthesia!
Avoid pharmacological agents that increase IOP
1 These strategies are not recommended in patients with full stomachs.

Regional anesthesia with minimal sedation!
Premedication
Histamine H,-receptor antagonists
Nonparticulate antacids
Evacuation of gastric contents
Nasogastric tube!
Rapid-sequence induction
Cricoid pressure
Rapid induction with rapid onset of paralysis
Avoidance of positive-pressure ventilation via mask
Intubation as soon as possible

| These strategies are not recommended for patients with penetrating eye injuries.

One clearly will want to minimize the risk of aspiration pneumonia by decreasing gastric volume and acidity (see Case Discussion, Chapter 17). The risk of aspiration in patients with eye injuries is reduced by proper selection of drugs and anesthetic techniques. Evacuation of gastric contents with a nasogastric tube may lead to coughing, retching, and other responses that can dramatically increase intraocular pressure.
Metoclopramide increases lower esophageal sphincter tone, speeds gastric emptying, lowers gastric fluid volume, and exerts an antiemetic effect. It should be given intravenously (10 mg) as soon as possible and repeated every 2 to 4h until surgery.
Ranitidine (50 mg intravenously), cimetidine (300 mg intravenously), and famotidine (20 mg intravenously) are H,-receptor antagonists that inhibit gastric acid secretion. Because they have no effect on the pH of gastric secretions present in the stomach prior to their administration, they have limited value in patients presenting for emergency surgery.
Unlike
H,-receptor antagonists,
antacids have
an immediate
effect.
Unfortunately, they
increase
(preparations of
effectiveness intragastric
sodium within
citrate,
30
to volume.
potassium
60
min citrate,
and should
and be
citric acid)
given lose
immediately prior
induction
(15-30
mL
orally).

The ideal induction agent for patients with full stomachs would provide a rapid onset of action in order to minimize the risk of regurgitation. Propofol and etomidate have essentially equally rapid onsets of action and lower intraocular pressure. Although investigations of the effects of ketamine on intraocular pressure have provided conflicting results, ketamine is not recommended in penetrating eye injuries, owing to the increased risk of blepharospasm and nystagmus.
Although etomidate
may prove
valuable in
some patients
with cardiac
disease, it
is associated
with an
incidence of
myoclonus ranging
from
10%
to
painlessly to allow the placement of an intravenous line, yet maintain a level of consciousness adequate to protect airway reflexes. However, the most prudent strategy is to do everything reasonable to avoid aspiration— even at the cost of further eye damage.
Are there special considerations during extubation and emergence?
Patients at risk of aspiration during induction are also at risk during extubation and emergence. Therefore, extubation must be delayed until the patient is awake and has intact airway reflexes (eg, spontaneous swallowing and coughing on the endotracheal tube). Deep extubation increases the risk of vomiting and aspiration. Intraoperative administration of antiemetic medication and nasogastric or orogastric tube suctioning may decrease the incidence of emesis during emergence, but they do not guarantee an empty stomach.

surgery. J Cataract Refract Surg. 2013;39:789.
Riad
W,
Akbar
F.
Ophthalmic regional
blockade complication
rate:
A
single center
audit of
33,363
ophthalmic operations.
J
Clin
Anesth.
2012;24:193.
- Rogers A, Cox RG. Anesthetic management for pediatric strabismus surgery: Continuing professional development. Can J Anesth. 2010;57:602.
Spiteri N, Sidaras G, Czanner G, et al. Assessing the quality of ophthalmic anesthesia. J Clin Anesth. 2015;27:285.
Vachon C, Warner D, Bacon D. Succinylcholine and the open globe. Anesthesiology. 2003;99:220.

@ The anesthetic goals for laryngeal endoscopy include an immobile surgical field and adequate masseter muscle relaxation for introduction of the suspension laryngoscope (typically profound muscle paralysis will be sought), adequate oxygenation and ventilation, and cardiovascular stability despite periods of rapidly varying surgical stimulation.
@ During jet ventilation, chest wall motion must be monitored and sufficient exhalation time allowed in order to avoid air trapping and barotrauma.
© The greatest concern during laser airway surgery is an airway tube fire. This risk can be moderated by minimizing the fraction of inspired oxygen (FiO, <30% if tolerated by the patient) and can be eliminated when there is no combustible material (eg, flammable endotracheal tube, catheter, or dry cotton pledget) in the airway.
@ Techniques to minimize intraoperative blood loss include topical vasoconstriction with cocaine or an epinephrine-containing local anesthetic for vasoconstriction, maintaining a slightly head-up position, and providing a mild degree of controlled hypotension.
@ If there is serious preoperative concern regarding potential airway problems, intravenous induction may be avoided in favor of awake direct or fiberoptic laryngoscopy (cooperative patient) or direct or fiberoptic intubation following an inhalational induction, maintaining spontaneous ventilation (uncooperative patient). In any case, the appropriate equipment and qualified personnel required for emergent tracheostomy must be immediately available.
@ The surgeon may request the omission of neuromuscular blockers during neck dissection, thyroidectomy, or parotidectomy to allow nerve identification (eg, spinal accessory, facial nerves) by direct nerve stimulation and thereby facilitate their preservation.
@ Manipulation of the carotid sinus and stellate ganglion during radical neck dissection has been associated with wide swings in blood pressure, bradycardia, arrhythmias, sinus arrest, and prolonged QT intervals. Infiltration of the carotid sheath with local anesthetic will usually moderate these problems. Bilateral neck dissection may result in postoperative hypertension and loss of hypoxic drive due to denervation of the carotid sinuses and bodies.

© Patients undergoing maxillofacial reconstruction or orthognathic surgical procedures often pose airway challenges. If there are any anticipated signs of problems with mask ventilation or tracheal intubation, the airway should be secured prior to induction of general anesthesia.
© If there is risk of postoperative edema involving structures that could obstruct the airway (eg, tongue, pharynx), the patient should be closely observed and perhaps kept intubated.
@® Nitrous oxide is either entirely avoided during tympanoplasty or discontinued prior to graft placement.
Cooperation and communication between surgeon and anesthesia provider are critical elements of all surgery within or adjacent to the airway. Establishing, maintaining, and protecting an airway in the face of abnormal anatomy and simultaneous surgical intervention are demanding tasks. An understanding of airway anatomy (see Chapter 19) and an appreciation of common otorhinolaryngologic and maxillofacial procedures are invaluable in handling these anesthetic challenges.

Endoscopy includes laryngoscopy (diagnostic and operative) and microlaryngoscopy (laryngoscopy aided by an operating microscope) for
conditions including vocal cord cysts and polyps and upper airway papillomatosis and malignancy, esophagoscopy, and bronchoscopy (discussed in
Chapter 25). Endoscopic procedures may be accompanied by laser surgery.

Patients presenting for endoscopic surgery are often being evaluated for voice disorders (often presenting as hoarseness), stridor, or hemoptysis. Possible diagnoses include foreign body aspiration, trauma to the aerodigestive tract, papillomas, tracheal stenosis, tumors, or vocal cord dysfunction. Thus, a preoperative medical history and physical examination, with particular attention to potential airway problems, must precede any decisions regarding the anesthetic plan. In some patients, flow-volume loop (see Chapter 6), radiographic, computed tomography, ultrasound, or magnetic resonance imaging studies may be available for review. Many patients will have undergone preoperative indirect laryngoscopy or fiberoptic nasopharyngoscopy, and the information gained from these procedures may be of critical importance. Important initial questions that must be answered are whether adequate positive-pressure ventilation via face or laryngeal mask is feasible, and the patient can be intubated using conventional direct or video laryngoscopy. If, 1 = . whether the answer to either question is 'no,' the patient's airway should be secured prior to induction using an alternative technique such as fiberoptic bronchoscope or tracheostomy under local anesthesia (see Case Discussion, Chapter 19)., 1 = . However, even the initial securing of an airway with tracheostomy does not prevent intraoperative airway obstruction due to surgical manipulation, a foreign, 1 = . Sedative premedication should be avoided in a patient with threatening upper airway obstruction. Glycopyrrolate (0.2-0.3 mg) works more effectively and persistently when given intramuscularly rather than intravenously 1 h before surgery and may prove helpful by minimizing secretions, thereby facilitating, 1 = . body,, 1 = . or hemorrhage., 1 = . , 1 = airway visualization.

@ The anesthetic goals for laryngeal endoscopy include an immobile surgical field and adequate masseter muscle relaxation for introduction of the suspension laryngoscope (typically profound muscle paralysis will be sought), adequate oxygenation and ventilation, and cardiovascular stability despite rapidly varying levels of surgical stimulation.

Intraoperative muscle relaxation can be achieved by intermittent boluses or infusion of intermediate-duration nondepolarizing neuromuscular blocking agents (NMBs) (eg, rocuronium, vecuronium, cisatracurium), or with a succinylcholine infusion. Rapid recovery is important, as endoscopy is often an outpatient procedure. Given that profound muscle relaxation is often needed until the very end of the operative procedure, endoscopy remains one of the few remaining indications for succinylcholine infusions. Use of sugammadex (Bridion) to reverse profound degrees of rocuronium or vecuronium neuromuscular blockade is an alternative approach.

Several methods have successfully been used to provide oxygenation and ventilation during endoscopy, while simultaneously minimizing interference with the operative procedure. Most commonly, the patient is intubated with a small-diameter endotracheal tube through which conventional positive-pressure ventilation is administered. Standard endotracheal tubes of smaller diameters, however, are designed for pediatric patients, and therefore are too short for the adult trachea and have a low-volume cuff that will exert increased pressure against the tracheal mucosa. A 4.0-, 5.0-, or 6.0-mm specialized microlaryngeal endotracheal tube (Mallinckrodt MLT) is the same length as an adult tube, has a disproportionately large high-volume low-pressure cuff, and is stiffer and less prone to compression than is a conventional endotracheal tube of the same diameter. The advantages of intubation in endoscopy include protection against aspiration and the ability to administer inhalational anesthetics and to continuously monitor end-tidal CO.
In some cases (eg, those involving the posterior commissure or vocal cords), intubation with an endotracheal tube may interfere with the surgeon's visualization or performance of the procedure. A simple alternative is insufflation of high flows of oxygen through a small catheter placed in the trachea. Although oxygenation may be maintained in patients with good lung function, ventilation will be inadequate for longer procedures unless the patient is allowed to breathe spontaneously.
Another option is the intermittent apnea technique, in which positive-pressure ventilation with oxygen by face mask or endotracheal tube is alternated with periods of apnea, during which the surgical procedure is performed. The duration of apnea, usually 2 to 3 min, is determined by how well the patient maintains oxygen saturation, as measured by pulse oximetry. Risks of this technique include hypoventilation with hypercarbia, failure to reestablish the airway, and pulmonary aspiration.

Another attractive alternative approach involves manual jet ventilation via laryngoscope side port. During inspiration (1-2 s), a high-pressure (30-50 psi) jet of oxygen is directed through the glottic opening and entrains a mixture of oxygen and room air into the lungs (Venturi effect). Expiration (4-6 s duration) ® is passive. Chest wall motion must be monitored and sufficient exhalation time allowed in order to avoid air trapping and barotrauma. This technique requires total intravenous anesthesia. A variation of this technique is highfrequency jet ventilation, which utilizes a small cannula or tube in the trachea, through which gas is injected 80 to 300 times per minute (see Chapter 58). Capnography will not provide an accurate estimate of end-tidal CO, during jet ventilation due to constant and sizable dilution of alveolar gases.

Blood pressure and heart rate often fluctuate markedly during endoscopic procedures for two reasons. First, some of the patients undergoing these procedures are elderly and have a long history of heavy tobacco and alcohol use that predisposes them to cardiovascular disease. In addition, the endoscopic procedure is, in essence, a series of physiologically stressful laryngoscopies and interventions, separated by varying periods of minimal surgical stimulation. Attempting to maintain a constant level of anesthesia invariably results in alternating intervals of hypertension and hypotension. Providing a modest baseline level of anesthesia allows supplementation with short-acting anesthetics (eg, propofol, remifentanil) or sympathetic antagonists (eg, esmolol), or both, as needed during periods of increased stimulation. Alternatively, some anesthesia providers utilize regional nerve block of the glossopharyngeal nerve and superior laryngeal nerve to help minimize intraoperative swings in blood pressure (see Case Discussion, Chapter 19).

Laser light differs from ordinary light in three ways: It is monochromatic (possesses one wavelength), coherent (oscillates in the same phase), and collimated (exists as a narrow parallel beam). These characteristics offer the surgeon excellent precision and hemostasis with minimal postoperative edema or pain. Unfortunately, lasers introduce several major hazards into the operating room environment.
The uses and side effects of a laser vary with its wavelength, which is determined by the medium in which the laser beam is generated. For example, a CO, laser produces a long wavelength (10,600 nm), whereas yttrium—
TABLE 37-1 Advantages and disadvantages of various tracheal tubes for laser airway surgery.
Polyvinyl chloride, Advantages = Inexpensive, nonreflective. Polyvinyl chloride, Disadvantages = Low melting point, highly combustible’. Red rubber, Advantages = Puncture-resistant, maintains structure, nonreflective. Red rubber, Disadvantages = Highly combustible’. Silicone rubber, Advantages = Nonreflective. Silicone rubber, Disadvantages = Combustible,' turns to toxic ash. Metal, Advantages = Combustion- resistant,' kink-resistant. Metal, Disadvantages = Thick-walled flammable cuff, transfers heat, reflects laser, cumbersome
'Combustibility depends on fraction of inspired oxygen and laser energy.
Although specialized, laser-resistant endotracheal tubes may be used, it must be emphasized that no endotracheal tube or currently available endotracheal tube protection device is reliably laser-proof. Therefore, whenever laser airway surgery is being performed with an endotracheal tube in place, the following precautions should be observed:
¢ Inspired oxygen concentration should be as low as possible by utilizing air in the inspired gas mixture (many patients tolerate an Fio, of 21%).

¢ Nitrous oxide supports combustion and must be avoided.
¢ The endotracheal tube cuffs should be filled with saline. Some practitioners add methylene blue to the saline to make cuff rupture more obvious. A wellsealed, cuffed endotracheal tube will minimize oxygen concentration in the pharynx.
¢ Laser intensity and duration should be limited as much as possible.
¢ Saline-saturated pledgets, although potentially flammable, should be placed in the airway to limit risk of endotracheal tube ignition and damage to adjacent tissue.
¢ Asource of water (eg, water-filled 60-mL syringe and basin) should be immediately available in case of fire.
These precautions limit, but do not eliminate, the risk of an airway fire; anesthesia providers must proactively address the hazard of fire whenever laser or electrocautery is utilized near the airway (Table 37-3).

1. Stop ventilation and remove tracheal tube.
2. Turn off oxygen and disconnect circuit from machine.
3. Submerge tube in water.
4. Ventilate with face mask and reintubate.
5. Assess airway damage with bronchoscopy, serial chest x-rays, and arterial blood gases.
6. Consider bronchial lavage and steroids.
If an airway fire should occur, all air/oxygen should immediately be turned off at the anesthesia gas machine, and burning combustible material (eg, an endotracheal tube) should be removed from the airway. The fire can be extinguished with saline, and the patient's airway should be examined to be certain that all foreign body fragments have been removed.

Common nasal and sinus surgeries include polypectomy, endoscopic sinus surgery, maxillary sinusotomy (Caldwell—Luc procedure), rhinoplasty, and septoplasty.

Patients undergoing nasal or sinus surgery may have a considerable degree of preoperative nasal obstruction caused by polyps, a deviated septum, or mucosal congestion from infection. This may make face mask ventilation difficult, particularly if combined with other causes of difficult ventilation (eg, obesity, maxillofacial deformities).
Nasal polyps are often associated with allergic disorders, such as asthma. Patients who also have a history of allergic reactions to aspirin should not be given any nonsteroidal antiinflammatory drugs (including ketorolac) for postoperative analgesia. Nasal polyps are a common feature of cystic fibrosis. Because of the rich vascular supply of the nasal mucosa, the preoperative interview should concentrate on questions concerning medication use (eg, aspirin, clopidogrel) and any history of bleeding problems.

Many nasal procedures can be satisfactorily performed under local anesthesia with sedation. The anterior ethmoidal nerve and sphenopalatine nerves (see Figure 19-3) provide sensory innervation to the nasal septum and lateral walls. Both can be blocked by packing the nose with gauze or cotton-tipped applicators soaked with local anesthetic. The topical anesthetic should be allowed to remain in place at least 10 min before instrumentation is attempted. Supplementation with submucosal injections of local anesthetic is often required. Use of an epinephrine-containing or cocaine solution will shrink the nasal mucosa and potentially decrease intraoperative blood loss. Intranasal cocaine (maximum dose, 3 mg/kg) is rapidly absorbed, reaching peak levels in 30 min, and may be associated with cardiovascular side effects (see Chapter 16).
General anesthesia is often preferred for nasal surgery because of the discomfort and incomplete block that may accompany topical anesthesia. Special considerations during and shortly following induction include using an oral airway during face mask ventilation to mitigate the effects of nasal obstruction, intubating with a reinforced or preformed Mallinckrodt oral RAE (Ring—Adair— Elwyn) endotracheal tube (Figure 36-1), and tucking the patient's padded arms, with protection of the fingers, to the side. Because of the proximity of the surgical field, it is important to tape the patient's eyes closed to avoid a corneal abrasion. One exception to this occurs during dissection in endoscopic sinus surgery, when the surgeon may wish to periodically check for eye movement because of the close proximity of the sinuses and orbit (Figure 37-1); nonetheless, the eyes should remain protected until the surgeon is ready to observe them. NMBs are often utilized because of potential neurological or ophthalmic injury that may occur if the patient moves during sinus instrumentation.
A
B
FIGURE 37-1 Orbital fracture is a risk of endoscopic sinus surgery because of the proximity of the sinuses to the orbit (A, frontal view; B, coronal section). (Modified with permission from Snell RS, Katz J. Clinical Anatomy for Anesthesiologists. New York, NY: Appleton & Lange; 1988.)

@ Techniques to minimize intraoperative blood loss include topical vasoconstriction with cocaine or an epinephrine-containing local anesthetic, maintaining a slightly head-up position, and providing a mild degree of controlled hypotension. A posterior pharyngeal pack is often placed to limit the risk of aspiration of blood. Despite these precautions, the anesthesiologist must be prepared for major blood loss, particularly during the resection of vascular tumors (eg, juvenile nasopharyngeal angiofibroma).
Coughing or straining during emergence from anesthesia and extubation should be avoided, as these events will increase venous pressure and increase postoperative bleeding. However, relatively deep extubation strategies that are commonly and appropriately utilized to accomplish this goal also may increase the risk of aspiration.

Surgery for cancer of the head and neck includes laryngectomy, glossectomy, pharyngectomy, parotidectomy, hemimandibulectomy, and radical neck dissection. An endoscopic examination following induction of anesthesia often precedes these surgical procedures. Timing of a tracheostomy, if planned, depends upon the patient's preoperative airway compromise. Some procedures may include extensive reconstructive surgery, such as the transplantation of a free microvascular muscle flap, with long surgical time duration.

The typical patient presenting for head and neck cancer surgery is older and often has had many years of heavy tobacco and alcohol use. Common coexisting medical conditions include chronic obstructive pulmonary disease, coronary artery disease, hypertension, diabetes, alcoholism, and malnutrition. These patients often greatly benefit from an enhanced recovery after surgery program that includes preoperative nutritional repletion over the course of days and hydration with a carbohydrate—protein drink during the 24 h prior to surgery Airway management may be complicated by abnormal airway anatomy, an obstructing lesion, or by preoperative radiation therapy that has fibrosed, immobilized, and distorted the patient's airway @ structures. If there is concern regarding potential airway problems, intravenous induction may be avoided in favor of awake direct or fiberoptic laryngoscopy (cooperative patient) or direct or fiberoptic intubation following an inhalational induction, maintaining spontaneous ventilation (uncooperative patient). Elective tracheostomy under local anesthesia prior to induction of general anesthesia is often a prudent option. In any case, the appropriate equipment and qualified personnel required for emergent tracheostomy must be immediately available.

Because many of these procedures are lengthy and associated with substantial blood loss, and because of the prevalence of coexisting cardiopulmonary disease, arterial cannulation may be utilized for blood pressure monitoring and frequent laboratory analyses. If central venous access is deemed necessary, the surgeon should be consulted to ascertain that planned internal jugular or subclavian venous access will not interfere with the intended surgical procedures; antecubital or femoral veins are reasonable alternatives. Arterial lines and intravenous cannulas should not be placed in the operative arm if a radial forearm flap is planned. A minimum of two large-bore intravenous lines and a urinary catheter (preferably with temperature-monitoring capability) should be placed. A forced-air warming blanket should be used to help maintain normal body temperature. Intraoperative hypothermia and consequent vasoconstriction can be detrimental to perfusion of a microvascular free flap.
Intraoperative nerve monitoring is increasingly utilized by surgeons in anterior neck operations to help preserve the superior laryngeal, recurrent laryngeal, and vagus nerves (Figure 37—2), and the anesthesia provider may be asked to place a specialized nerve integrity monitor endotracheal tube (Medtronic Xomed NIM endotracheal tube) to facilitate this process (Figure 37— 3).

FIGURE 37-2 The vagus nerve (cranial nerve X) originates in the medulla oblongata and then ramifies in the superior and inferior vagal ganglia in the neck. Its first major branch is the pharyngeal plexus of the vagus. The superior laryngeal nerve divides into the external and internal laryngeal nerves. The internal branch supplies sensory innervation of the laryngeal mucosa above the vocal cords, and the external branch innervates the inferior pharyngeal constrictor muscles and the cricothyroid muscle of the larynx. Cricothyroid muscle contraction increases the voice pitch by lengthening, tensing, and adducting the vocal folds. The superior laryngeal nerve is at risk of damage during operations of the anterior neck, especially thyroid surgery, and injury to this nerve may result in hoarseness and loss of vocal volume. The next branch of the vagus is the recurrent laryngeal nerve, which innervates all of the muscles of the larynx except the cricothyroid, and is responsible for phonation and glottic opening. The recurrent laryngeal nerve runs immediately behind the thyroid gland and thus is the nerve of greatest risk for injury during thyroid surgery. Unilateral recurrent laryngeal nerve damage may result in vocal changes or hoarseness, and bilateral nerve damage may result in aphonia and respiratory distress. Inferior to this nerve, the vagus nerve provides autonomic motor and sensory nerve fibers to the thoracic and abdominal viscera. (Reproduced with permission from Dillon FX. Electromyographic (EMG) neuromonitoring in otolaryngology-head and neck surgery. Anesthesiol Clin. 2010 Sep;28(3):423-442.)
FIGURE 37-3 A: The Medtronic Xomed NIM electromyographic (EMG) nerve integrity monitoring endotracheal tube. Succinylcholine (or no relaxant at all) should be used for intubation, and the endotracheal tube should be secured in the midline. If lubricant is used, it must not contain local anesthetics. B: A

slightly larger endotracheal tube size should be used to facilitate mucosal contact with the electrodes, and the blue band of the NIM tube must be positioned at the level of the vocal cords. C: Nerve integrity is continuously monitored via EMG activity (Medtronic Xomed NIM-Response 3.0 Nerve Integrity Monitor). Nondepolarizing muscle relaxants are contraindicated because they prevent EMG monitoring. (Used with permission from Medtronic Xomed.)

Head and neck cancer surgery often includes tracheostomy. Immediately prior to surgical entry into the trachea, the endotracheal tube and hypopharynx should be thoroughly suctioned to limit the risk of aspiration of blood and secretions. If electrocautery is used during the surgical dissection, the Fio, should be lowered to 30% or less, if possible, in order to minimize the risk of fire as the trachea is surgically entered. In any case, the easiest way to minimize airway fire risk in this circumstance is for the surgeon not to use the cautery to enter the trachea. After dissection down to the trachea, the tracheal tube cuff is deflated to avoid perforation by the scalpel. When the tracheal wall is transected, the endotracheal tube is withdrawn so that its tip is immediately cephalad to the incision. Ventilation during this period is difficult because of the large leak through the tracheal incision. A sterile, cuffed tracheostomy tube is placed in the trachea, the cuff is inflated, and the tube is connected to a sterile breathing circuit. As soon as correct position is confirmed by capnography and bilateral chest auscultation, the original endotracheal tube is removed. An increase in peak inspiratory pressure immediately after tracheostomy usually indicates a malpositioned endotracheal tube, bronchospasm, debris or secretions in the trachea, or, rarely, pneumothorax.

@ The surgeon may request the omission of NMBs during neck dissection, thyroidectomy, or parotidectomy to allow nerve identification (eg, spinal accessory, facial nerves) by direct nerve stimulation and thereby facilitate their preservation. If a nerve integrity monitor endotracheal tube is utilized, succinylcholine (or propofol with no relaxant) may be used to facilitate intubation. Moderate controlled hypotension may be helpful in limiting blood loss; however, cerebral perfusion may be compromised with moderate hypotension when a tumor invades the carotid artery or jugular vein (the latter may increase cerebral venous pressure). If head-up tilt is utilized, it is important that the arterial blood pressure transducer be zeroed at the level of the brain
(external auditory meatus) in order to most accurately determine cerebral perfusion pressure. In addition, head-up tilt increases the risk of venous air embolism.
Following reanastomosis of a microvascular free flap, blood pressure should be maintained at the patient's baseline level. The use of vasoconstrictive agents (eg, phenylephrine) should be minimized because of potential decrease in flap perfusion due to vasoconstriction. Similarly, the use of vasodilators (eg, sodium nitroprusside or hydralazine) should be avoided in order to minimize any decrease in graft perfusion pressure.

Transfusion decisions must balance the patient's immediate surgical risks with the possibility of an increased cancer recurrence rate resulting from transfusioninduced immune suppression. Rheological factors make a relatively low hematocrit (eg, 27-30%) desirable when microvascular free flaps are performed. Excessive diuresis should be avoided during microvascular free-flap surgery in order to optimize graft perfusion in the postoperative period.

@ Manipulation of the carotid sinus and stellate ganglion during radical neck dissection has been associated with wide swings in blood pressure, bradycardia, arrhythmias, sinus arrest, and prolonged QT intervals. Infiltration of the carotid sheath with local anesthetic will usually moderate these problems. Bilateral neck dissection may result in postoperative hypertension and loss of hypoxic drive due to denervation of the carotid sinuses and carotid bodies.

The principal postoperative complications associated with head and neck cancer surgery include hypocalcemia secondary to acute hypoparathyroidism, and threats to airway integrity secondary to hemorrhage, hematoma formation, and bilateral vocal cord palsy with stridor resulting from bilateral recurrent laryngeal nerve injury (see Chapter 35). Postoperative hypoparathyroidism is a common condition, resulting from injury to the parathyroid glands or their blood supply during thyroidectomy or neck dissection, or from unintentional or intentional removal of all four parathyroid glands. It may be either symptomatic or asymptomatic, and occurs transiently in up to 49% of thyroidectomy patients, and is permanent in up to 33% of thyroidectomy patients. Symptoms and signs depend upon the rate of onset and severity of hypocalcemia. Clinical signs of acute, severe hypocalcemia include laryngospasm, bronchospasm, QT prolongation-related arrhythmias, and congestive heart failure. Neurological symptoms and signs range from circumoral paresthesias, distal extremity numbness, and carpopedal spasm to confusion, delirium, and seizure activity. Symptomatic hypocalcemia is a medical emergency and should be treated with intravenous calcium salts, whereas asymptomatic hypocalcemia may be treated with oral calcium preparations (see Chapter 49).

Maxillofacial reconstruction is often required to correct the effects of trauma (eg, fractures of the mandible or maxilla) or developmental malformations, or for radical cancer surgeries (eg, maxillectomy or mandibulectomy). Orthognathic procedures (eg, LeFort osteotomies, mandibular osteotomies) for skeletal malocclusion share many of the same surgical and anesthetic techniques.

® Patients undergoing maxillofacial reconstruction or orthognathic surgical procedures often pose airway challenges. Particular attention should be focused on jaw opening, mask fit, neck mobility, micrognathia, retrognathia, maxillary protrusion (overbite), macroglossia, dental pathology, nasal patency, and the existence of any intraoral lesions or debris. If there are any anticipated signs of problems with mask ventilation or endotracheal intubation, the airway should be secured prior to induction of general anesthesia. This may involve fiberoptic nasal intubation, fiberoptic oral intubation, or tracheostomy with local anesthesia facilitated with cautious sedation. Nasal intubation with a straight tube with a flexible angle connector (Figure 37—4A) or a preformed nasal RAE (Figure 37— 4B) tube is usually preferred in dental and oral surgery. The endotracheal tube can then be directed cephalad over the patient's forehead. With any nasal intubation, care should be taken to prevent the endotracheal tube from putting pressure on the tissues of the nasal opening, as this situation may result in local tissue pressure necrosis in the setting of a lengthy surgical procedure. Nasal intubation should be considered with caution in LeFort II and III fractures because of the possibility of a coexisting basilar skull fracture (Figure 37-5).
FIGURE 37-4 A: A regular straight endotracheal tube can be cut at the level of the nares, and a flexible connector attached. B: Alternatively, a nasal RAE endotracheal tube has a preformed right-angle bend at the level of the nose so that the tube is directed over the forehead.
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FIGURE 37-5 Diagrammatic representation of LeFort I, II, and III fractures. LeFort II and III fractures may coexist with a basilar skull fracture, a contraindication to nasal intubation.

Maxillofacial reconstructive and orthognathic surgeries can be lengthy and involve substantial blood loss. An oropharyngeal ("throat") pack is often placed to minimize the amount of blood and other debris reaching the larynx and trachea, and one must remember to remove the pack at the end of surgery before the jaws are wired shut! Strategies to minimize bleeding include a slight head-up position, controlled hypotension, and local infiltration with epinephrine solutions. Because the patient's arms are typically tucked at the side, two intravenous lines may be established prior to surgery. An arterial line may be placed. As previously noted, if head-up tilt is utilized, it is important that the arterial blood pressure transducer be zeroed at the level of the brain (external auditory meatus) in order to most accurately determine cerebral perfusion pressure. In addition, the anesthesia provider must be alert to the increased risk of venous air embolism in the of tilt.

setting head-up Because of proximity of the airway to the surgical field, positioning of surgical team personnel, and positioning of the patient's head often 90° or 180° away from the anesthesia provider, there is an increased risk of critical intraoperative airway problems, such as endotracheal tube kinking, disconnection, or perforation by a surgical instrument. Monitoring of end-tidal COs, peak inspiratory pressures, and breath sounds via an esophageal stethoscope assume greater importance in such cases. If the operative procedure is near the airway, the use of electrocautery or laser increases the risk of fire. At the end of surgery, the oropharyngeal pack must be @ removed and the pharynx suctioned. If there is risk of postoperative tissue edema involving structures that could potentially obstruct the airway (eg, tongue, pharynx), the patient should be closely observed and perhaps kept intubated. In such uncertain situations, extubation may be performed over an endotracheal tube exchanger (eg, Cook Airway Exchange Catheter with Rapi-Fit Adapter, Cook Medical), which can facilitate reintubation and provide oxygenation in the setting of immediate postextubation respiratory obstruction. In addition, the operating team should be prepared for emergent tracheotomy or cricothyrotomy. Otherwise, extubation can be attempted once the patient is fully awake and there are no signs of continued bleeding. Patients with intermaxillary fixation (eg, maxillomandibular wiring) must have suction and appropriate wire cutting tools continuously at the bedside in case of vomiting or other airway emergencies. Extubating a patient whose jaws are wired shut and whose oropharyngeal pack has not been removed can lead to life-threatening airway obstruction. "Has the throat pack been removed?" should be asked before intermaxillary fixation is initiated and again before removing the endotracheal tube.

Frequently performed ear surgeries include stapedectomy or stapedotomy, tympanoplasty, and mastoidectomy. Myringotomy with insertion of tympanostomy tubes is the most common pediatric surgical procedure and is discussed in Chapter 42.

Nitrous oxide is not often used in anesthesia for ear surgery. Because nitrous oxide is more soluble than nitrogen in blood, it diffuses into air-containing cavities more rapidly than nitrogen (the major component of air) can be absorbed by the bloodstream (see Chapter 8). Normally, changes in middle ear pressures caused by nitrous oxide are well tolerated as a result of passive venting through the eustachian tube. However, patients with a history of chronic ear problems such as otitis media or sinusitis often suffer from obstructed eustachian tubes and may, on rare occasion, experience hearing loss or tympanic membrane rupture from administration of nitrous oxide anesthesia.
During tympanoplasty, the middle ear is open to the atmosphere, and there is no pressure buildup. However, once the surgeon has placed a tympanic membrane graft, the middle ear becomes a closed space, and if nitrous oxide is allowed to diffuse into any gas remaining in this space, middle ear pressure will rise and the graft may be displaced. Conversely, discontinuing nitrous oxide after graft placement will create a negative middle ear pressure that could also cause graft dislodgment. Therefore, © nitrous oxide is either entirely avoided during tympanoplasty or discontinued prior to graft placement. Obviously, the exact amount of time required to wash out the nitrous oxide depends on many factors, including alveolar ventilation and fresh gas flows (see Chapter 8), but 15 to 30 min is usually recommended.

As with any form of microsurgery, even tiny amounts of blood can obscure the operating field. Techniques to minimize blood loss during ear surgery include mild (15°) head elevation, infiltration or topical application of epinephrine (1:50,000—1:200,000), and moderate controlled hypotension. Because coughing on the endotracheal tube during emergence (particularly during neck movement associated with head bandaging) will increase venous pressure and may cause bleeding and increased middle ear pressure, deep extubation is often utilized.

Preservation of the facial nerve is an important consideration during some ear procedures, such as resection of a glomus tumor or acoustic neuroma. During such cases, intraoperative paralysis with NMBs will make identification of the facial nerve by direct nerve stimulation impossible. Thus, intraoperative paralysis should not be employed unless requested by the surgeon.

Because the inner ear is intimately involved with the sense of balance, ear surgery may cause postoperative dizziness (vertigo) and postoperative nausea and vomiting (PONV). Induction and maintenance with propofol have been shown to decrease PONV in patients undergoing middle ear surgery. Prophylaxis with decadron prior to induction, and a 5-HT; blocker prior to emergence, should be considered. Patients undergoing ear surgery should be carefully assessed for vertigo postoperatively, and their ambulation closely monitored, in order to minimize the risk of falling.

Most minor oral surgical procedures are performed in a clinic or office setting utilizing local anesthesia, augmented with varying degrees of sedation. If intravenous sedation is employed, or if the procedure is complex, a qualified anesthesia provider should be present. A qualified anesthesia provider must be present to administer deep sedation or general anesthesia if either is utilized. Typically, a bite block and an oropharyngeal throat pack protect the airway. For light to moderate levels of sedation, the oropharyngeal pack prevents irrigating fluids and dental debris from entering the airway. Deep sedation and general anesthesia require an increased level of airway management by a qualified anesthesia provider. Regardless of whether deep sedation or general anesthesia is inadvertent or intended, appropriate equipment, supplies, and medications must be immediately available to help ensure that any anticipated or unexpected anesthesia-related problem occurring in an office or clinic environment can be safely addressed with the same standard of care that is required in the hospital or ambulatory surgery center setting.
Minor oral surgical procedures, such as dental extractions, typically last no longer than 1 h. Nerve block or local anesthetic infiltration is typically utilized. In adults, most oral surgeons use 2% lidocaine with 1:100,000 epinephrine or 0.5% bupivacaine with 1:200,000 epinephrine in quantities no greater than 12 mL and 8 mL, respectively. Articaine is commonly used in Europe. The anesthesia provider must be informed by the surgeon of the local anesthetic used and its concentration and volume injected so that the allowed dosage based on patient weight is not exceeded. Pediatric patients are particularly at risk of local anesthesia toxicity due to excess local anesthetic dose administration or accidental intravascular injection.
Intravenous sedation during oral surgical procedures greatly increases the patient's comfort and facilitates surgery. Small doses of fentanyl and midazolam are usually adequate for adults prior to injection of the local anesthetic. The sedation can be further augmented by additional small dosages of fentanyl, midazolam, or propofol. Incremental doses of propofol, 20 to 30 mg for adults, are often used if the surgeon requires a brief episode of deep sedation or general anesthesia.

These techniques require a high level of cooperation and participation by both the surgeon and anesthesia provider. If there is the possibility of increased risk due to preexisting medical conditions, less than ideal airway, or extent of contemplated surgical procedure, it is safer to perform the procedure in a hospital or ambulatory surgery center setting with general endotracheal anesthesia.

A 50-year-old man has a paroxysm of coughing in the recovery room while awakening following uneventful endoscopic sinus surgery. Immediately afterward, his respirations seem labored with a loud inspiratory stridor.

The acute onset of inspiratory stridor in a postoperative patient may be due to laryngospasm, laryngeal edema, foreign body aspiration, or vocal cord dysfunction. Laryngospasm, an involuntary spasm of the laryngeal musculature, may be triggered by blood or secretions stimulating the superior laryngeal nerve (see Chapter 19). Laryngeal edema may be caused by an allergic drug reaction, hereditary or iatrogenic angioedema, or a traumatic intubation. Vocal cord dysfunction could be due to residual muscle relaxant effect, hypocalcemic alkalotic tetany, intubation trauma, or paradoxical vocal cord motion.
Another paroxysm of coughing is accompanied by hemoptysis. What is your immediate management?
Bleeding after nose or throat surgery can be very serious. Patients who are not fully awake may continue to gag and cough on the secretions, increasing venous pressure and worsening the bleeding. Furthermore, they may aspirate blood and other secretions. Fortunately, because of its physiological pH, aspiration of blood is not as serious as aspiration of acidic gastric contents. Nonetheless, the airway should be immediately secured in the obtunded patient. This may be accomplished with an awake intubation or a rapid-sequence induction.
If the patient is awake and alert enough to cough and swallow and does not seem to be aspirating blood, the first priority should be to decrease the bleeding as quickly as possible. Immediate measures that should be considered include raising the head of the bed to decrease venous and arterial pressures at the site of bleeding and aggressively treating any degree of systolic hypertension with intravenous antihypertensive agents. Sedation should be avoided so that airway reflexes are not compromised.
Despite these measures, the bleeding continues, and surgical intervention seems to be necessary. Describe your strategy for induction of anesthesia in this patient.

Before induction of general anesthesia in bleeding patient, a hypovolemia should be corrected with isotonic crystalloid or with colloid. The degree of hypovolemia may be difficult to assess because much of the blood may be swallowed, but it can be estimated by changes in vital signs, postural hypotension, and hematocrit. Cross-matched blood should be readily available, and a second large-bore intravenous line secured. It must be appreciated that from an anesthetic standpoint, this is an entirely different patient than the one who presented for surgery initially: the patient now has a full stomach, is hypovolemic, and may be more difficult to intubate.
The preferred technique in this patient is a rapid-sequence induction. Induction drug choice (eg, ketamine, etomidate) and dosage should anticipate the possibility of hypotension from persistent hypovolemia. Qualified personnel and appropriate equipment for an emergency tracheostomy should be immediately available. An orogastric tube should be passed to decompress the stomach.

The arterial supply of the nose is provided by the internal maxillary artery and the anterior ethmoid artery. These may have to be ligated in uncontrollable epistaxis.

Because this patient is still at risk of aspiration, extubation should not be attempted until the patient has fully awakened and regained airway reflexes. Although it is desirable to limit coughing and "bucking" on the endotracheal tube during emergence, this may be difficult to achieve in the awakening patient. Intravenous lidocaine or dexmedetomidine may be helpful in this situation.

Acharya K. Rigid bronchoscopy in airway foreign bodies: Value of the clinical

@
Clinical manifestations of bone cement implantation syndrome include hypoxia (increased pulmonary shunt), hypotension, arrhythmias (including heart block and sinus arrest), pulmonary hypertension (increased pulmonary vascular resistance), and decreased cardiac output.
@ Use of a pneumatic tourniquet on an extremity creates a bloodless field that greatly facilitates surgery. However, tourniquets can produce potential problems of their own, including hemodynamic changes, pain, metabolic alterations, arterial thromboembolism, and pulmonary embolism.
©® Fat embolism syndrome classically presents within 72 h following longbone or pelvic fracture, with the triad of dyspnea, confusion, and petechiae.
@ Deep vein thrombosis and pulmonary embolism can cause morbidity and mortality following orthopedic operations on the pelvis and lower extremities.
@ Neuraxial anesthesia alone or combined with general anesthesia may reduce thromboembolic complications by several mechanisms, including sympathectomy-induced increases in lower extremity venous blood flow, systemic antiinflammatory effects of local anesthetics, decreased platelet reactivity, attenuated postoperative increase in factor VIII and von Willebrand factor, attenuated postoperative decrease in antithrombin III, and alterations in stress hormone release.
@ For patients receiving prophylactic low-molecular-weight heparin once daily, neuraxial techniques may be performed (or neuraxial catheters
removed) 10 to 12 h after the previous dose, with a 4-h delay before administering the next dose.

@ Flexion and extension lateral radiographs of the cervical spine should be obtained preoperatively in patients with rheumatoid arthritis severe enough to require steroids, immune therapy, or methotrexate. If atlantoaxial instability is present, intubation should be performed with inline stabilization utilizing video or fiberoptic laryngoscopy.
@ Effective communication between the anesthesia provider and surgeon is essential during bilateral hip arthroplasty. If major hemodynamic instability occurs during the first hip replacement procedure, the second arthroplasty should be postponed.
© Adjuvants such as opioids, clonidine, ketorolac, and neostigmine, when added to local anesthetic solutions for intraarticular injection, have been used in various combinations to extend the analgesia duration following knee arthroscopy.
@® Effective postoperative multimodal analgesia facilitates early physical rehabilitation to maximize postoperative range of motion and prevent joint adhesions following knee replacement.
@ Interscalene brachial plexus block with or without a perineural catheter is ideally suited for shoulder procedures. Even when general anesthesia is employed, a peripheral nerve or brachial plexus block can supplement anesthesia and provide effective postoperative analgesia.
Orthopedic surgery challenges the anesthesia provider. The comorbidities of these patients vary widely based on age group. Patients may present as neonates with congenital limb deformities, as teenagers with sports-related injuries, as adults for procedures ranging from excision of minor soft-tissue mass to joint replacement, or at any age with bone cancer. This chapter focuses on perioperative care issues specific to patients undergoing common orthopedic surgical procedures. For example, patients with long bone fractures are predisposed to fat embolism syndrome. Patients are at increased risk for venous thromboembolism following pelvic, hip, and knee operations. Use of bone cement during arthroplasties can cause hemodynamic instability. Limb tourniquets limit blood loss but introduce additional risks. Perioperative care of patients undergoing cervical, thoracic, and lumbar spine procedures is reviewed in Chapter 27.

Neuraxial and other regional anesthetic techniques play an important role in decreasing the incidence of perioperative thromboembolic complications, providing postoperative analgesia, and facilitating early rehabilitation and hospital discharge. Advances in surgical techniques, such as minimally invasive approaches to knee and hip replacement, are necessitating modifications in anesthetic and perioperative management to facilitate overnight or even sameday discharge of patients who formerly required days of hospitalization. It is impossible to cover the anesthetic implications of diverse orthopedic operations in one chapter; hence, the focus here is on perioperative management considerations and strategies for the anesthetic management of patients undergoing select orthopedic surgical procedures. Anesthesia for surgery on the spine is discussed in Chapter 27.

femur to relieve intramedullary pressure, performing high-pressure lavage of the femoral shaft to remove debris (potential microemboli), or using a femoral component that does not require cement.
Another source of concern related to the use of cement is the potential for gradual loosening of the prosthesis over time. Newer cementless implants are made of a porous material that allows natural bone to grow into them. Cementless prostheses generally last longer and may be advantageous for younger, active patients; however, healthy active bone formation is required and recovery may be longer compared to cemented joint replacements. Therefore, cemented prostheses are preferred for older (>80 years) and less active patients who often have osteoporosis or thin cortical bone. Practices continue to evolve regarding selection of cemented versus cementless implants, depending on the joint affected, patient, and surgical technique.

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Some degree of fat embolism probably occurs with all long-bone fractures. Fat embolism syndrome is less frequent but potentially fatal (10-20% mortality). @ It classically presents within 72 h following long-bone or pelvic fracture, with the triad of dyspnea, confusion, and petechiae. This syndrome can also be seen following cardiopulmonary resuscitation, parental feeding with lipid infusion, and liposuction. The most popular theory for its pathogenesis holds that fat globules are released by the disruption of fat cells in the fractured bone and enter the circulation through tears in medullary vessels. An alternative theory proposes that the fat globules are chylomicrons resulting from the aggregation of circulating free fatty acids caused by changes in fatty acid metabolism. Regardless of their source, the increased free fatty acid levels can have a toxic effect on the capillary—alveolar membrane leading to the release of vasoactive amines and prostaglandins and the development of acute respiratory distress syndrome (ARDS; see Chapter 58). Neurological manifestations (eg, agitation, confusion, stupor, or coma) are the probable result of capillary damage in the cerebral circulation and cerebral edema. These signs may be exacerbated by hypoxia.
The diagnosis of fat embolism syndrome is suggested by petechiae on the chest, upper extremities, axillae, and conjunctiva. Fat globules occasionally may be observed in the retina, urine, or sputum. Coagulation abnormalities such as thrombocytopenia or prolonged clotting times are occasionally present. Serum lipase activity may be elevated but does not predict disease severity. Pulmonary involvement typically progresses from mild hypoxia and a normal chest radiograph to severe hypoxia or respiratory failure with radiographic findings of diffuse pulmonary opacities. Most of the classic signs and symptoms of fat embolism syndrome occur 1 to 3 days after the precipitating event. During general anesthesia, signs may include a decline in Erco, and arterial oxygen saturation and a rise in pulmonary artery pressures. Electrocardiography may show ischemic-appearing ST-segment changes and a pattern of right-sided heart strain.

Management of fat embolism syndrome involves preemption and support. Early stabilization of the fracture decreases the likelihood of fat embolism syndrome and, in particular, the risk of pulmonary complications. Supportive treatment consists of oxygen therapy with continuous positive airway pressure ventilation to prevent hypoxia and with specific ventilator strategies in the event of ARDS. Systemic hypotension will require appropriate pressor support, and vasodilators may aid the management of pulmonary hypertension. High-dose corticosteroid therapy is not supported by randomized clinical trials.

@ Deep vein thrombosis (DVT) and pulmonary embolism (PE) can cause morbidity and mortality following orthopedic operations on the pelvis and lower extremities. Risk factors include obesity, age greater than 60 years, procedures lasting more than 30 min, use of a tourniquet, lower extremity fracture, and immobilization for more than 4 days. Patients at greatest risk include those undergoing hip surgery or knee replacement or major operations for lower extremity trauma. Such patients will experience DVT rates of 40% to 80% without prophylaxis. The incidence of clinically important PE following hip surgery in some studies is reported to be as high as 20%, whereas that of fatal PE may be 1% to 3%. Underlying pathophysiological mechanisms include venous stasis with hypercoagulable state due to local and systemic inflammatory responses to surgery.
Pharmacological prophylaxis and the routine use of mechanical devices such as intermittent pneumatic compression (IPC) have been shown to decrease the incidence of DVT and PE. While mechanical thromboprophylaxis should be considered for every patient, the use of pharmacological anticoagulants must be balanced against the risk of bleeding. For patients at increased risk for DVT but having "normal" bleeding risk, low-dose subcutaneous unfractionated heparin (LUFH), warfarin, or low-molecular-weight heparin (LMWH) may be employed in addition to mechanical prophylaxis. Patients at significantly increased risk of bleeding may be managed with mechanical prophylaxis alone until bleeding risk decreases. In general, anticoagulants are started the day of surgery in patients without indwelling epidural catheters. Warfarin may be started the night before surgery depending on the particular orthopedic surgeon's routine. Newer direct oral anticoagulants that inhibit factor Xa or thrombin have a fast onset of action, are renally excreted, and deserve special consideration in the context of regional anesthesia.

@ Neuraxial anesthesia alone or combined with general anesthesia may reduce thromboembolic complications by several mechanisms, including sympathectomy-induced increases in lower extremity venous blood flow, systemic antiinflammatory effects of local anesthetics, decreased platelet reactivity, attenuated postoperative increases in factor VIII and von Willebrand factor, attenuated postoperative decreases in antithrombin III, and alterations in stress hormone release.
According to the Third Edition of the American Society of Regional Anesthesia and Pain Medicine Evidence-Based Guidelines on regional anesthesia and anticoagulation, patients currently receiving antiplatelet agents (eg, ticlopidine, clopidogrel, and intravenous glycoprotein IIb/IIa inhibitors), thrombolytics, fondaparinux, direct thrombin inhibitors, or therapeutic regimens of LMWH present an unacceptable risk for spinal or epidural hematoma following neuraxial anesthesia. Performance of neuraxial block (or removal of a neuraxial catheter) is not contraindicated with subcutaneous LUFH when the total daily dose is 10,000 units or less; there are no data on the safety of neuraxial anesthesia @ when larger doses are given. For patients receiving prophylactic LMWH, the guidelines vary based on regimen. With once-daily dosing, neuraxial techniques may be performed (or neuraxial catheters removed) 10 to 12 h after the previous dose, with a 4-h delay before administering the next dose. With twice-daily dosing, neuraxial catheters should not be left in situ and should be removed 2 or more hours before the first dose of LMWH. Patients on warfarin therapy should not receive a neuraxial block unless the international normalized ratio (INR) is normal, and catheters should be removed when the INR is 1.5 or lower. The Third Edition of these guidelines also suggests that these recommendations be applied to deep peripheral nerve and plexus blocks and catheters (see Suggested Reading). Revisions to these guidelines occur regularly.

Common hip procedures performed in adults include repair of hip fracture, total hip arthroplasty (THA), and closed reduction of hip dislocation.

Most patients presenting with femoral neck fractures are frail and elderly. An occasional young patient will have sustained major trauma to the femur or pelvis. Studies have reported mortality rates following hip fracture of up to 10% during the initial hospitalization and over 25% within 1 year. Many of these patients have concomitant diseases such as coronary artery disease, cerebrovascular disease, chronic obstructive pulmonary disease, or diabetes. Patients presenting with hip fractures are frequently dehydrated from inadequate oral intake. Depending on the site of the hip fracture, occult blood loss may be significant, further compromising intravascular volume. In general, intracapsular (subcapital, transcervical) fractures are associated with less blood loss than extracapsular (base of the femoral neck, intertrochanteric, subtrochanteric) fractures (Figure 38-1). A normal or borderline-low preoperative hematocrit may be deceiving when hemoconcentration masks occult blood loss.
FIGURE 38-1 Blood loss from hip fracture depends on the location of the fracture (subtrochanteric, intertrochanteric > base of femoral neck > transcervical, subcapital) because the capsule restricts blood loss by acting like a tourniquet.
Another characteristic of hip fracture patients is the frequent presence of preoperative hypoxia that may, at least in part, be due to fat embolism; other factors can include bibasilar atelectasis from immobility, pulmonary congestion (and effusion) from congestive heart failure, or consolidation due to infection.

The choice between regional (spinal or epidural) and general anesthesia has been extensively evaluated for hip fracture surgery. A meta-analysis of 15 randomized clinical trials showed a decrease in postoperative DVT and 1-month mortality with regional anesthesia, but these advantages do not persist beyond 3 months. A large database study involving over 50,000 patients treated for hip fracture in New York State also did not show a difference in 30-day mortality based on anesthetic technique but did show a slightly shorter length of stay for patients who received regional anesthesia. The incidence of postoperative delirium and cognitive dysfunction may be lower following regional anesthesia if intravenous sedation can be minimized. A large clinical trial addressing this important question is currently underway.
A neuraxial anesthetic technique, with or without concomitant general anesthesia, provides the additional advantage of postoperative pain control. If a spinal anesthetic is planned, hypobaric or isobaric local anesthesia facilitates positioning since the patient can remain in the same position for both block placement and surgery. Intrathecal opioids such as morphine can extend postoperative analgesia but require close postoperative monitoring for delayed respiratory depression.
Consideration should also be given to the type of reduction and fixation to be used. This is dependent on the fracture site, degree of displacement, preoperative functional status of the patient, and surgeon preference. Undisplaced fractures of the proximal femur may be treated with percutaneous pinning or cannulated screw fixation with the patient in the supine position. A hip compression screw and side plate are most often employed for intertrochanteric fractures. Displaced intracapsular fractures may require internal fixation, hemiarthroplasty, or THA (Figure 38-2). Surgical treatment of extracapsular hip fractures is accomplished with either an extramedullary implant (eg, sliding screw and plate) or intramedullary implant (eg, Gamma nail).

Hemiarthroplasty and THA are longer, more invasive operations than most other orthopedic procedures. They are usually performed with patients in the lateral decubitus position, are associated with greater blood loss, and, potentially, result in greater hemodynamic changes, particularly if cement is used. One should secure sufficient venous access to permit rapid transfusion.

Most patients undergoing THA suffer from osteoarthritis (degenerative joint disease), hip fracture, avascular necrosis, or autoimmune conditions such as rheumatoid arthritis (RA). Osteoarthritis is a degenerative disease affecting the articular surface of joints (commonly the hips and knees). The etiology of osteoarthritis appears to involve repetitive joint trauma. Because osteoarthritis may also involve the spine, neck manipulation during tracheal intubation should be minimized to avoid nerve root compression or disc protrusion. RA is characterized by immune-mediated joint destruction with chronic and progressive inflammation of synovial membranes, as opposed to the articular wear-and-tear of osteoarthritis. RA is a systemic disease affecting multiple organ systems (Table 38-1). RA often affects the small joints of the hands, wrists, and feet causing severe deformity.

Cardiovascular, Abnormalities = _—_ Pericardial thickening and effusion, myocarditis, coronary arteritis, conduction defects, vasculitis, cardiac valve fibrosis (aortic regurgitation). Pulmonary, Abnormalities = Pleural effusion, pulmonary nodules, interstitial pulmonary fibrosis. Hematopoietic, Abnormalities = Anemia, eosinophilia, platelet dysfunction (from aspirin therapy), thrombocytopenia. Endocrine, Abnormalities = Adrenal insufficiency (from glucocorticoid therapy), impaired immune system. Dermatological, Abnormalities = Thin and atrophic skin from the disease and immunosuppressive drugs
Extreme cases of RA involve almost all synovial membranes, including those in the cervical spine and temporomandibular joint. Atlantoaxial subluxation,
which can be diagnosed radiologically, may lead to protrusion of the odontoid process into the foramen magnum during intubation, compromising vertebral blood flow and compressing the spinal @ cord or brainstem (Figure 38-3). Flexion and extension lateral radiographs of the cervical spine should be obtained preoperatively in patients with RA severe enough to require steroids, immune therapy, or methotrexate. If atlantoaxial instability is present, intubation should be performed with inline stabilization utilizing video or fiberoptic laryngoscopy. Involvement of the temporomandibular joint can limit jaw mobility and range of motion to such a degree that conventional orotracheal intubation may be impossible. Hoarseness or inspiratory stridor may signal a narrowing of the glottic opening caused by cricoarytenoid arthritis. This condition may lead to postextubation airway obstruction even when a smaller diameter tracheal tube has been used.
FIGURE 38-3 Because instability of the cervical spine may be asymptomatic, lateral radiographs are mandatory in patients with severe rheumatoid arthritis. A: Radiograph of a normal lateral cervical spine. B: Lateral cervical spine of a patient with rheumatoid arthritis; note the severe C1—C2 instability.

Patients with RA or osteoarthritis commonly re-ceive nonsteroidal antiinflammatory drugs (NSAIDs) for pain management. These drugs can have serious side effects such as gastrointestinal bleeding, kidney toxicity, and platelet dysfunction.

THA involves several surgical steps, including positioning of the patient (usually in the lateral decubitus position), dislocation and removal of the femoral head, reaming of the acetabulum and insertion of a prosthetic acetabular cup (with or without cement), and reaming of the femur and insertion of a femoral component (femoral head and stem) into the femoral shaft with or without cement. THA is associated with three potentially life-threatening complications: bone cement implantation syndrome, intraand postoperative hemorrhage, and venous thromboembolism. Thus, invasive arterial monitoring may be justified for select patients undergoing these procedures. General anesthesia, neuraxial anesthesia, or a combination of techniques can provide suitable operating conditions. The use of neuraxial anesthesia with or without general anesthesia may be associated with decreased incidence of postoperative complications such as all-cause infections, acute kidney injury, and need for mechanical ventilation. Neuraxial administration of opioids such as morphine in the perioperative period extends the duration of postoperative analgesia. Tranexamic acid may be administered intraoperatively to reduce blood loss.

The increasing number of younger patients presenting for hip arthroplasty and of other patients who require revision of standard (metal-on-polyethylene) THA implants has led to redevelopment of hip resurfacing arthroplasty techniques. Compared with traditional hip arthroplasty implants, hip resurfacing maintains patients' native bone to a greater degree. Metal-on-metal hybrid implants are usually employed. Surgical approaches can be anterior or posterior, with the posterior approach most commonly used due to better visualization. Since the femoral head remains intact, its dislocation and repositioning during surgery may theoretically compromise its own blood supply. With the posterior approach, patients are placed in the lateral decubitus position similar to traditional hip arthroplasty.
Outcomes data related to hip resurfacing versus traditional THA are controversial. Prospective studies have not shown a difference in gait or postural balance at 3 months postoperatively. One meta-analysis favored resurfacing in terms of functional outcome and blood loss despite comparable results for postoperative pain scores and patient satisfaction. Of particular concern is the finding that patients who undergo resurfacing are nearly twice as likely to require revision surgery as those receiving traditional hip arthroplasty. There is a greater incidence of aseptic component loosening (possibly from metal hypersensitivity) and femoral neck fracture, particularly in women. Finally, the presence of metal debris in the joint space (from metal-on-metal contact) has led to a marked narrowing of indications for the prostheses and the procedure. Monitoring of serum cobalt and chromium levels has been suggested for these patients. Adverse local tissue reactions, including abnormal fluid collections and pseudotumors, may also occur as a result of metal debris and are best detected early to avoid permanent damage to muscle, bone, and soft tissues.

Bilateral hip arthroplasty can be safely performed in fit patients as a combined procedure, assuming the absence of significant pulmonary embolization after insertion of the first femoral component. @ Monitoring may include echocardiography. Effective communication between the anesthesia provider and surgeon is essential. If major hemodynamic instability occurs during the first hip replacement procedure, the second arthroplasty should be postponed.

Revision of a prior hip arthroplasty may be associated with much greater blood loss than in the initial procedure. Blood loss depends on many factors, including the experience and skill of the surgeon. Some studies suggest that blood loss may be decreased during hip surgery if a regional anesthesia technique is used (eg, spinal or epidural anesthesia) compared with general anesthesia even at similar mean arterial blood pressures. The mechanism is unclear. Because the likelihood of perioperative blood transfusion is increased, preoperative autologous blood donation and intraoperative blood salvage should be considered. Preoperative administration of vitamins B,, and K and iron can treat mild forms of chronic anemia. Alternatively, intravenous iron infusion can be used. Finally, and most expensively, recombinant human erythropoietin (600 IU/kg subcutaneously weekly beginning 21 days before surgery and ending on the day of surgery) may also decrease the need for perioperative allogeneic blood transfusion. Erythropoietin increases red blood cell production by stimulating the division and differentiation of erythroid progenitors in the bone marrow. Maintaining normal body temperature during hip replacement surgery reduces blood loss.

Computer-assisted surgery (CAS) has been proposed to improve surgical outcomes and promote early rehabilitation through minimally invasive techniques employing cementless implants, although this approach is not considered mainstream. Computer software can accurately reconstruct threedimensional images of bone and soft tissue based on radiographs, fluoroscopy, computed tomography, or magnetic resonance imaging data. The computer matches preoperative images or planning information to the position of the patient on the operating room table. Tracking devices are attached to target bones (Figure 38—4) and instruments used during surgery, and the navigation system utilizes optical cameras and infrared light-emitting diodes to sense their positions. CAS thus allows accurate placement of implants through small incisions, and the resulting reduction in tissue and muscle damage may lead to less pain and early rehabilitation. The lateral approach utilizes a single incision with the patient in the lateral decubitus position (Figure 38—4) while the anterior approach utilizes two small incisions (one for the acetabular component and another for the femoral component) with the patient supine. The evidence to date does not demonstrate outcome advantages of CAS.
FIGURE 38-4 Minimally invasive total hip arthroplasty: lateral approach. Note the small 3-in. incision and tracking devices for the CAS navigation system.
E. Hip Arthroscopy
In recent years, hip arthroscopy has increased in popularity as a minimally invasive alternative to open arthrotomy for a variety of surgical indications such as femoroacetabular impingement (FAI), acetabular labral tears, loose bodies, and osteoarthritis. At present, there is some evidence in the published literature to support hip arthroscopy for FAI, but evidence is lacking for other indications.

There is a 3% incidence of hip dislocation following primary hip arthroplasty and a 20% incidence following total hip revision arthroplasty. Because less force is required to dislocate a prosthetic hip, patients with hip implants require special precautions during positioning for subsequent surgical procedures. Extremes of hip flexion, internal rotation, and adduction increase the risk of dislocation. Hip dislocations may be corrected with closed reduction facilitated by use of a brief intravenous general anesthetic, often performed in a monitored setting outside the operating room (eg, emergency department). Temporary paralysis can be provided by succinylcholine, if necessary, to facilitate the reduction when the hip musculature is severely contracted. Successful reduction should be confirmed radiologically.

The two most frequently performed knee surgeries are arthroscopy and total or partial arthroplasty.

Arthroscopy has revolutionized surgery of many joints, including the hip, knee, shoulder, ankle, elbow, and wrist. Joint arthroscopies are usually performed as outpatient procedures. Although the typical patient undergoing knee arthroscopy is often thought of as being a healthy young athlete, knee arthroscopies may also be performed in elderly patients with multiple medical problems.

A bloodless field facilitates arthroscopic surgery. Knee surgery lends itself to the use of a pneumatic tourniquet, although its use is optional. The surgery is performed as an outpatient procedure. Alternative anesthetic techniques include general anesthesia, neuraxial anesthesia, peripheral nerve blocks, periarticular injections, or intraarticular injections employing local anesthetic solutions with or without adjuvants combined with intravenous sedation analgesia. Comparing neuraxial anesthesia techniques, success and patient satisfaction appear to be equal between epidural and spinal anesthesia. However, for ambulatory surgery, time to discharge following neuraxial anesthesia may be prolonged compared with general anesthesia.

Successful outpatient recovery depends on early ambulation, adequate analgesia, and minimal sedation and nausea and vomiting. Techniques that avoid large doses of systemic opioids have obvious appeal. Intraarticular bupivacaine or ropivacaine usually provide satisfactory @ analgesia for several hours postoperatively. Adjuvants such as opioids, clonidine, ketorolac, epinephrine, and neostigmine, when added to local anesthetic solutions for intraarticular injection, have been used in various combinations to extend analgesia duration. Other multimodal pain management strategies include systemic NSAIDs, gabapentinoids, and single or continuous peripheral nerve blocks for arthroscopic ligament reconstruction.

Patients presenting for total knee arthroplasty (TKA) (Figure 38—5) have similar comorbidities to those undergoing THA.
FIGURE 38-5 Total (A) and partial (B) knee replacement.
Intraoperative Management
FIGURE 38-6 A "block room" can be located in a preoperative holding area, induction room, or postanesthesia care unit and should offer standard monitoring (as outlined by the American Society of Anesthesiologists) and ample storage for regional anesthesia supplies and equipment.
Unicompartmental or patellofemoral partial knee replacement and minimally invasive knee arthroplasty with muscle-sparing approaches have been described. With proper patient selection, these techniques may reduce quadriceps muscle damage, facilitating earlier achievement of range-of-motion and ambulation goals, and may allow for discharge within 24 h following surgery in select situations. Anesthetic management and postoperative analgesia should both accommodate and facilitate an accelerated, enhanced recovery program. Single or continuous peripheral nerve blocks, alone or in combination, can provide target-specific pain control and facilitate early rehabilitation. Continuous peripheral nerve block catheters with subsequent perineural local anesthetic infusions have been shown to decrease time to meet discharge criteria for TKA. The management of perineural catheters takes a hands-on team approach with 24/7/365 coverage. Among the complications of lower extremity perineural local anesthetic infusions, those involving patient falls are of greatest concern, and comprehensive fall prevention programs need to be in place whenever these techniques are employed. Spinal anesthesia with intrathecal morphine or hydromorphone is also widely used and compatible with enhanced recovery programs. Finally, spinal anesthesia with postoperative analgesia using local injection in and around the knee joint of a "cocktail" containing ketorolac and dilute local anesthetic is increasingly popular and apparently very effective.

Procedures on the upper extremities include those for disorders of the shoulder (eg, subacromial impingement or rotator cuff tears), traumatic fractures, nerve entrapment syndromes (eg, carpal tunnel syndrome), and autoimmune or degenerative joint diseases.

surgeon may insert a subacromial catheter to provide continuous infusion of local anesthetic for postoperative analgesia. Direct placement of intraarticular catheters into the glenohumeral joint with infusion of bupivacaine has been associated with glenohumeral chondrolysis in human and animal studies and is not recommended. Multimodal analgesia, including systemic NSAIDs (if no contraindications) and local anesthetic infusions in the perioperative period, can help reduce postoperative opioid requirements.

Distal upper extremity surgical procedures generally take place on an outpatient basis. Minor soft tissue operations of the hand (eg, carpal tunnel release) of short duration may be performed with local infiltration or with intravenous regional anesthesia (IVRA, or Bier block). The limiting factor with IVRA is tourniquet tolerance.
For operations lasting more than 1 h or more invasive procedures involving bones or joints, a brachial plexus block is the preferred regional anesthetic technique. Multiple approaches can be used to anesthetize the brachial plexus for distal upper extremity surgery (see Chapter 46). Selection of brachial plexus block technique should take into account the planned surgical site and location of the pneumatic tourniquet, if applicable. Continuous peripheral nerve blocks may be appropriate for inpatient and select outpatient procedures to extend the duration of analgesia further into the postoperative period or facilitate physical therapy, or both. Brachial plexus blocks do not anesthetize the intercostobrachial nerve distribution (arising from the dorsal rami of T1 and sometimes T2); hence, subcutaneous infiltration of local anesthetic may be required for procedures involving the medial upper arm or cases in which a tourniquet is applied to the upper arm.
Anesthetic considerations for distal upper extremity surgery should include patient positioning and use of a pneumatic tourniquet. Most procedures can be performed with the patient supine; the operative arm abducted 90° and resting on a hand table; and the operating room table rotated 90° to position the operative arm in the center of the room. Exceptions to this rule often involve surgery around the elbow, and certain operations may require the patient be in lateral decubitus or even prone position. Because patients are often scheduled for sameday discharge, perioperative management should focus on ensuring rapid recovery without severe pain or nausea (see Chapter 44).

A 58-year-old male patient who is a Jehovah's Witness presents for hemipelvectomy to resect a malignant bone tumor (osteogenic sarcoma). The patient has received chemotherapy over the past 2 months with multiple drugs, including doxorubicin. He has no other medical problems, and the preoperative hematocrit is 47%.
How does the care of patients who are Jehovah's Witnesses particularly challenge the anesthesiologist?
Jehovah's Witnesses, a fellowship of more than 1 million Americans, object to the administration of blood for any indication. This objection stems from their interpretation of the Bible ("to keep abstaining from ... blood," Acts 15:28,29) and not for medical reasons (eg, the fear of hepatitis). Physicians should honor the principle of autonomy: Patients have final authority over what is done to them. Witnesses typically sign a waiver releasing physicians from liability for any consequences of blood refusal.

Witnesses abstain from blood and blood products (eg, packed red blood cells, fresh frozen plasma, platelets) but not non—blood-containing solutions. They accept crystalloids, hetastarch, and dextran replacement solutions. Witnesses often view albumin, erythropoietin (because of the use of albumin), immune globulins, and hemophiliac preparations as a gray area that requires a personal decision by the believer.

According to their religion, any blood that is removed from the body should be discarded ("You should pour it out upon the ground as water," Deuteronomy 12:24) and not stored. Thus, the usual practice of autologous preoperative collection and storage would not be allowed. Techniques of acute normovolemic hemodilution and intraoperative blood salvage have been accepted by some Witnesses, however, as long as their blood maintains continuity with their circulatory systems at all times. For example, up to 4 units of blood could be drawn from the patient immediately before surgery and kept in anticoagulant-containing bags that maintain a constant link to the patient's body. The blood could be replaced by an acceptable colloid or crystalloid solution then reinfused as needed during surgery.

Hemipelvectomy involves radical resection that can lead to massive blood loss. This is particularly true for large tumors removed using the more invasive internal approach. Intraarterial and central venous catheter placement would be indicated in most patients undergoing this procedure. Techniques that minimize intraoperative blood loss (eg, controlled hypotension, aprotinin) should be considered. In a Jehovah's Witness patient, the management of life-threatening anemia (Hb <5 g/dL) may be improved by optimizing cardiac output, oxygen delivery, and oxygen consumption.

Assuming the maintenance of normovolemia and the absence of preexisting major end-organ dysfunction, most patients tolerate severe anemia surprisingly well. Decreased blood viscosity and vasodilation lower systemic vascular resistance and increase blood flow. Augmentation of stroke volume increases cardiac output, allowing arterial blood pressure and heart rate to remain relatively unchanged. Coronary and cerebral blood flows increase in the absence of coronary artery disease and carotid artery stenosis. A decrease in venous oxygen saturation reflects an increase in tissue oxygen extraction. Oozing from surgical wounds as a result of dilutional coagulopathy may accompany extreme degrees of anemia.

This anthracycline chemotherapeutic agent has well-recognized cardiac side effects, ranging from transient arrhythmias and electrocardiographic changes (eg, ST-segment and T-wave abnormalities) to irreversible cardiomyopathy and congestive heart failure. The risk of cardiomyopathy appears to increase with a cumulative dose greater than 550 mg/m2, prior radiotherapy, and concurrent cyclophosphamide treatment. Mild degrees of cardiomyopathy can be detected preoperatively with endomyocardial biopsy, echocardiography, or exercise radionuclide angiography. The other important toxicity of doxorubicin is myelosuppression manifesting as thrombocytopenia, leukopenia, and anemia.
Are there any special considerations regarding postoperative pain management in the Jehovah's Witness patient?
Witnesses generally refrain from any mind-altering drugs or medications, although opioids prescribed by a physician for severe pain are accepted by some believers. Insertion of an epidural catheter can provide acceptable analgesia with local anesthetics, with or without opioids.

Horlocker TT, Wedel DJ, Rowlingson JC, et al. Regional Anesthesia in the Patient Receiving Antithrombotic or Thrombolytic Therapy: American Society of Regional Anesthesia and Pain Medicine EvidenceBased Guidelines. 3rd ed. Pittsburgh, PA: American Society of Regional Anesthesia; 2010. http://www.asra.com/publications-anticoagulation-3rdedition-2010.php.

Ward B, Lubowitz J. Basic knee arthroscopy. Part 1: Patient positioning. Arthrosc Tech. 2013;2:e497.
- Webb C, Mariano E. Best multimodal analgesic protocol for total knee arthroplasty. Pain Manag. 2015;5:185.
Zaric D, Boysen K, Christiansen C, et al. A comparison of epidural analgesia with combined continuous femoral-sciatic nerve blocks after total knee replacement. Anesth Analg. 2006;102:1240.

All trauma patients should be presumed to have a full stomach and thereby be at increased risk of pulmonary aspiration.
@ Cervical spine injury is presumed in any trauma patient presenting with neck pain or any suggestion of neurologic injury as well as those with loss of consciousness, significant head injury and/or intoxication.
@ In the patient with blunt or penetrating injury, providers should maintain a high level of suspicion for pulmonary injury that could evolve into a tension pneumothorax when mechanical ventilation is initiated. No trauma patient should die without having potential tension pneumothorax relieved.
@ Inup to 25% of trauma patients, trauma-induced coagulopathy (TIC) is present shortly after injury and before any resuscitative efforts have been initiated.
@ Balanced administration of red blood cell, fresh frozen plasma, and platelet units (1:1:1) is termed damage control resuscitation (DCR). Administering blood products in equal ratios early in resuscitation has become an accepted approach for preventing or correcting TIC.
@ Transfusion-associated circulatory overload (TACO) is the greatest risk to trauma patients from DCR. The incidence of transfusion-related acute lung injury (TRALI) has decreased markedly with restriction of plasma and platelet donation to donors who are male, or who are female and who have either never been pregnant or who have been tested and found to be anti-HLA negative.
@ Damage control surgery is a surgical intervention intended to stop hemorrhage and limit gastrointestinal contamination of the abdominal compartment in severely injured and bleeding patients. An emergent exploratory laparotomy is performed in a start—stop fashion, attempting to discover and control bleeding injuries, while affording the anesthesia provider opportunities for resuscitation and preventing prolonged hypotension and hypothermia between surgical interventions.
@ Any trauma patient with an altered level of consciousness must be considered to have a traumatic brain injury (TBI) until proven otherwise. Presence or suspicion of TBI mandates attention to maintaining cerebral perfusion pressure and oxygenation during all aspects of care. The most reliable clinical assessment tool in determining the significance of TBI in a nonsedated, nonparalyzed patient is the Glasgow Coma Scale. Acute subdural hematoma is the most common brain injury warranting emergency neurosurgical intervention and is associated with the highest mortality.

© Systemic hypotension (systolic blood pressures <90 mm Hg), hypoxia (Pao, <60 mm Hg), hypercapnia (Paco, >50 mm Hg), and hyperthermia (temperature >38.0°C) have a negative impact on morbidity and mortality following head injuries, likely because of their contributions to increasing cerebral edema and intracranial pressure (ICP).
® Current Brain Trauma Foundation guidelines recommend maintaining cerebral perfusion pressure between 50 and 70 mm Hg and ICP at less than 20 mm Hg for patients with severe head injury.
@ Maintaining supranormal mean arterial blood pressure to help ensure adequate spinal cord perfusion in areas of otherwise reduced blood flow due to cord compression or vascular compromise is likely to be of more benefit than steroid administration.
@® Major burns (a secondor third-degree burn involving =>20% total body surface area [TBSA]) induce a unique hemodynamic response. Cardiac output declines abruptly by up to 50% within 30 minutes of injury due to massive vasoconstriction, inducing a state of normovolemic hypoperfusion (burn shock).
® In contrast to blunt and penetrating trauma, where crystalloid fluids are discouraged, burn fluid resuscitation emphasizes the use of balanced crystalloid fluids in preference to albumin, hydroxylethyl starch, normal or hypertonic saline, or blood.
@ The differential diagnosis for altered mental status following burn injury, smoke inhalation, or both, includes carbon monoxide and cyanide poisoning.
@ Beyond 48 h after significant burn injury, succinylcholine can produce lethal hyperkalemia.
@ The elderly represent the fastest-growing trauma population. A progressive decrease in trauma survivability is first seen beginning around age 50. Significant underlying medical conditions contribute to increased trauma-related morbidity and mortality after even modest injuries.
@ Insituations of mass casualty incidents, point-of-care ultrasound technology may provide timely, critical information related to triaging patients for surgical and nonsurgical therapies using the CAVEAT (chest, abdomen, vena cava, and extremities) assessment.

Trauma is a leading cause of morbidity and mortality in all age groups and a leading cause of death in both the young (under 20 years old) and elderly (over 70 years old). All aspects of trauma care, from that provided at the scene, through transport, resuscitation, surgery, intensive care, and rehabilitation, must be coordinated if trauma patients are to have the greatest opportunity for full recovery. The Advanced Trauma Life Support (ATLS) program developed by the American College of Surgeons Committee on Trauma has provided training and recommendations leading to increasingly consistent approaches for trauma resuscitation. The development of criteria for Level 1 Trauma Centers has also improved trauma care by directing severely injured patients to facilities with appropriate trauma care resources. Although trauma anesthesia is sometimes thought of as a unique topic, many of the principles for managing trauma patients are relevant to any unstable or hemorrhaging patient. Thus, many common issues encountered in typical anesthesia practice are addressed in this chapter.

Emergency Medical Technician-Paramedics (EMT-P), Critical Care flight nurses and Emergency Medicine physicians have airway management training for the prehospital and hospital settings. As a result, the anesthesiologist's role in providing initial trauma airway interventions has declined significantly in North America. This trend also means that when called upon for assistance in airway management in the emergency department, anesthesia providers must expect a challenging airway since routine airway management techniques have likely proved unsuccessful.
There are three important aspects of airway management in the initial evaluation of a trauma patient: (1) the need for basic life support intervention; (2) the presumed presence of a cervical spinal cord injury until proven otherwise; and (3) the potential for failed endotracheal intubation. Effective basic life support, by improving oxygenation and reducing hypercarbia in the unresponsive trauma patient, may be sufficiently effective in improving a patient's level of consciousness to remove the need for endotracheal intubation. In those with persistent unresponsiveness, effective basic life support skills improve preoxygenation and reduce the risk for hypoxia during @ airway management interventions. All trauma patients should be presumed to have a full stomach and thereby be at increased risk of pulmonary aspiration.
® Cervical spine injury is presumed in any trauma patient presenting with neck pain or any suggestion of neurologic injury as well as those with loss of consciousness, significant head injury and/or intoxication. Cervical collar ("Ccollar") application before transport protects the cervical spinal cord by limiting cervical extension, and first-responders should utilize well-designed "hard" collars (eg, Aspen, Miami-J, Philadelphia) for cervical spine stabilization. Traditional "soft" cervical collars provide essentially no useful cervical spine stabilization. Hard collar cervical spine stabilization negatively impacts positioning for direct laryngoscopy and tracheal intubation, and alternative airway management devices (eg, video laryngoscope, fiberoptic bronchoscope) must be immediately available. If tracheal intubation is required, the front part of the C-collar can be removed as long as the head and neck are maintained in neutral position by a designated assistant maintaining manual in-line stabilization. This is usually performed with the assistant standing by the torso or kneeling at the head of the bed, holding the patient's head at the level of the ears, allowing the patient's mouth to open during laryngoscopy.

Alternative supraglottic devices for airway management (eg, King supralaryngeal device) may be used if direct laryngoscopy has failed in any environment (prehospital to intensive care unit). These devices, blindly placed into the airway, isolate the glottis opening between a large inflatable cuff positioned at the base of the tongue and the distal cuff that most likely rests in the proximal esophagus (Figure 39-1). The prolonged presence of supraglottic devices in the airway has been associated with tongue engorgement resulting from the large, proximal cuff obstructing venous outflow from the tongue, and in some cases, tongue engorgement has been sufficiently severe to warrant tracheostomy prior to its removal.
FIGURE 39-1 The King LT supralaryngeal device. The glottic opening lies between the large cuff positioned at the base of the tongue and the smaller balloon positioned in the proximal esophagus. The airway is not secured but rather isolated between the oropharynx and the proximal esophagus. (Reproduced with permission from King Systems Corporation, KLTD/KLTSD Disposable Supralaryngeal Airways Inservice Program, August 23, 2006.)
Limited evidence exists that prehospital airway management in trauma patients improves outcomes. However, failed endotracheal intubation in the prehospital environment certainly exposes patients to significant morbidity. Failed intubation attempts often result in systemic hypoxemia, and repeated hypoxemic events after even modest neurological injury further exacerbate the initial neurological insult (the second hit phenomenon).
Airway management in trauma patients is uneventful in most circumstances. Cricothyroidotomies and tracheostomies are rarely required for securing trauma airways. However, when trauma significantly alters or distorts the facial or upper airway anatomy to the point of preventing effective mask ventilation, or when hemorrhage into the airway precludes the patient from lying supine, elective cricothyroidotomy or tracheostomy should be considered before any attempts are made at sedating or paralyzing the patient for oral intubation.

Pulmonary injury may not be immediately apparent @ upon the trauma patient's arrival to the hospital. In the patient with blunt or penetrating injury, providers should maintain a high level of suspicion for pulmonary injury that could evolve into a tension pneumothorax when mechanical ventilation is initiated. Peak inspiratory pressure and tidal volumes should be monitored throughout the initial resuscitation. Abrupt cardiovascular collapse shortly after instituting mechanical ventilation may signal the presence of a pneumothorax. Any trauma-related cardiovascular collapse is managed by disconnecting the patient from mechanical ventilation and performing bilateral needle thoracostomies. This intervention is accomplished by inserting a 14-gauge intravenous catheter into the second intercostal space in the midclavicular line followed by larger, more effective thoracostomy tube insertion placed in the midaxillary line. No trauma patient should die without having potential tension pneumothorax relieved.

Signs of a pulse and blood pressure are sought during the primary trauma patient survey. Unless the trauma patient arrives at the hospital other than by ambulance, the resuscitation team will likely have received information about the patient's vital signs from the prehospital first responders. The absence of a pulse following trauma is associated with dismal chances of survival. An emergent ultrasound evaluation of the chest and abdomen is indicated for any patient arriving after trauma in cardiac arrest, as is bilateral needle chest decompression. The ultrasound evaluation will focus on the presence of an empty heart or massive blood collections in the chest or abdomen, which are indications of lethal injury.
The American College of Surgeons Committee on Trauma no longer endorses the use of emergency thoracotomy in treating patients without blood pressure or palpable pulse following blunt trauma, given the lack of evidence supporting survival following this intervention. In victims of penetrating trauma without a palpable pulse or blood pressure, but with organized cardiac rhythm, a resuscitative thoracotomy may offer some survivability but mortality remains exceedingly high.
Tourniquet use remains underutilized for compressible hemorrhage. Any extremity with significant vascular injury should have a tourniquet applied at the earliest possible moment ("stop the bleed"). The fear of tourniquet-induced limb ischemia often distracts first responders from making prompt, effective interventions in controlling hemorrhage with a tourniquet. Hemorrhage, not limb ischemia and limb function, is the most pressing threat to life, and it should be controlled by any effective measure at the earliest possible opportunity.

Once the presence of circulation is confirmed, a brief neurological examination is conducted. Level of consciousness, pupillary size and reaction, lateralizing signs suggesting intracranial or extracranial injuries, and indications of potential spinal cord injury are quickly evaluated. As noted earlier, hypercarbia often causes depressed neurological responsiveness following trauma, and it is effectively corrected with basic life support airway interventions. Additional causes of depressed neurological function (eg, alcohol/drug intoxication, effects of illicit or prescribed medications, hypoglycemia, hypoperfusion, brain or spinal cord injury) must also be addressed. Mechanisms of injury must be considered as well as exclusion of other factors in determining the risk for central nervous system trauma. Persistently depressed levels of consciousness should be considered a result of central nervous system injury until disproved by emergent diagnostic studies (eg, computed tomography scan).

The patient must be fully exposed and examined in order to adequately assess the extent of injury. This physical exposure increases the risk of hypothermia, which is associated with increased bleeding in the trauma patient. The resuscitation bay and operating room must be maintained near body temperature (uncomfortably warm), all intravenous fluids and blood products (except platelets) should be warmed during administration, and under-body forced air patient warmers should be utilized. While these interventions are important in addressing hypothermia, trauma team efficiency in identifying life-threatening injuries is critical for patient survival. In most urban trauma centers, the initial major trauma evaluation is completed within 20 min of patient arrival.

The FAST (Focused Assessment with Sonography for Trauma) examination utilizes ultrasonography at the trauma victim's bedside, performed by surgeons or emergency medicine physicians, for detecting the presence or absence of free fluid in the perihepatic and perisplenic spaces, pericardium, and pelvis. Patients with free fluid in these areas, as well as two of the following—penetrating injury, systolic blood pressures less than 90 mm Hg, or heart rate over 120 beats per minute—are likely to have a high mortality, trauma-induced coagulopathy, and require a massive transfusion. These critical findings have been validated in numerous trauma studies and warrant immediate surgical intervention for hemorrhage control.

Certain trauma terminology must be understood and utilized by the trauma care team in order to effectively communicate during trauma resuscitation or procedures in which blood loss is occurring. Hemorrhage classifications I through IV, damage control resuscitation, and damage control surgery are terms that quickly convey critical information, using a common understanding of the various interventions that may be required for resuscitating a trauma or surgical patient experiencing life-threatening bleeding. The American College of Surgeons identifies four classes of hemorrhage:
*
Class I hemorrhage is the volume of blood that can be lost without hemodynamic consequence. The heart rate does not change and the blood pressure does not decrease with this volume of blood loss. In most circumstances, this amount of blood represents less than 15% of circulating blood volume. The typical adult has a blood volume equivalent of 70 mL/kg. For a 100-kg patient, the circulating blood volume is nearly 7 L. Children are considered to have 80 mL/kg blood volume and infants, 90 mL/kg. Intravenous resuscitation is not required if the bleeding is controlled promptly, as in minor elective surgical procedures.
* Class II hemorrhage is the volume of blood that, when lost, prompts sympathetic responses to maintain perfusion, and represents the loss of 15% to 30% of circulating blood volume. The diastolic blood pressure will increase due to vasoconstriction and the heart rate will increase to maintain cardiac output. Intravenous fluid replacement is indicated for blood loss of this volume. Transfusions may be required if the bleeding continues, suggesting a progression to class III hemorrhage.
* Class III hemorrhage represents loss of 30% to 40% of circulating blood volume, which consistently results in decreased blood pressure. Compensatory mechanisms of vasoconstriction and tachycardia are not
sufficient for maintaining tissue perfusion to meet metabolic demand, and metabolic acidosis will be detected on arterial blood gas analysis. Blood transfusion is necessary to restore adequate tissue perfusion and oxygenation. The other trauma team members must be notified when this pattern of fluid dependence develops, and discussion must be initiated regarding the possible need for damage control intervention (discussed later) for hemorrhage control.

- Life-threatening class [V hemorrhage represents more than 40% of circulating blood volume loss. The patient will be unresponsive and profoundly hypotensive, and rapid control of bleeding and aggressive blood-based resuscitation (damage control resuscitation) are required to prevent death. Patients experiencing this degree of hemorrhage will have some element of trauma-induced coagulopathy (TIC), require massive blood transfusion (more than 10 units of red blood cells in a 24-h period), and are at high risk of death. The response to hemorrhage of this consequence must be damage control resuscitation and damage control surgery (see later discussion).

Coagulation abnormalities are common following major trauma, and traumainduced coagulopathy (TIC) coagulopathy is an independent risk factor for @ death. Recent prospective clinical studies suggest that in up to 25% of major trauma patients, TIC is present shortly after injury and before any resuscitative efforts have been initiated. This means that the coagulopathy cannot be attributed to dilutional effects of resuscitative fluids. In one report, TIC was only related to the presence of a severe metabolic acidosis (base deficits >6 mEq/L) and appeared to have a dose-dependent relationship with the degree of tissue hypoperfusion; 2% of patients with base deficits less than 6 mEq/L developed coagulopathy compared with 20% of patients with base deficits greater than 6 mEq/L. Although injury severity scores were likely higher in the coagulopathic patients, only the presence of metabolic acidosis correlated to developing TIC. Global tissue hypoperfusion appears to play a key role in the development of TIC. During hypoperfusion, the endothelium releases thrombomodulin and activated protein C which, at the microcirculation level, prevents thrombosis. Thrombomodulin binds thrombin, thereby preventing thrombin from cleaving fibrinogen to fibrin. The thrombomodulin—thrombin complex activates protein C, which then inhibits the extrinsic coagulation pathway through effects on cofactors V and VIII (Figure 39—2). Activated protein C also inhibits plasminogen activator inhibitor-1 proteins, which increases tissue plasminogen activator, resulting in hyperfibrinolysis (Figure 39-3). One prospective clinical study found the following effects of hypoperfusion on coagulation parameters: (1) progressive coagulopathy as base deficit increases; (2) increasing plasma thrombomodulin and falling protein C (indicating activation of the protein levels with increasing base deficit), supporting the argument that the anticoagulant effects of these proteins in the presence of hypoperfusion are related to the prolongation of prothrombin and partial thromboplastin times; and (3) earlyonset TIC and increased mortality.

Thrombin is generated primarily via the 'extrinsic' pathway with multiple feed-forward loops. When thrombomodulin (TM) is presented by the endothelium, it complexes thrombin which is no longer available to cleave fibrinogen. This anticoagulent thrombin activates protein C (PC) which reduces further thrombin generation through inhibition of cofactors V and VIII.
FIGURE 39-2 Mechanism of trauma-induced coagulopathy. During periods of tissue hypoperfusion, thrombomodulin (TM) released by the endothelium complexes with thrombin. The thrombin—TM complexes prevent cleavage of fibrinogen to fibrin and also activate protein C (PC), reducing further thrombin generation through cofactors V and VIII. (Reproduced with permission from Brohi K, Cohen MJ, Davenport RA. Acute coagulopathy of trauma: mechanism, identification and effect. Curr Opin Crit Care. 2007 Dec;13(6):680-685.)
FIGURE 39-3 Mechanism of hyperfibrinolysis in tissue hypoperfusion. Tissue plasminogen activator (tPA) released from the endothelium during hypoperfusion states cleaves plasminogen to initiate fibrinolysis. Activated protein C (aPC) consumes plasminogen activator inhibitor-1 (PAI-1) when present in excess, and reduced PAI-1 leads to increased tPA activity and hyperfibrinolysis. FDPs, fibrin degradation products; PC, protein C; TM, thrombomodulin. (Reproduced with permission from Brohi K, Cohen MJ, Davenport RA. Acute coagulopathy of trauma: Mechanism, identification and effect. Curr Opin Crit Care. 2007 Dec;13(6):680685.)

TIC is not solely related to impaired clot formation. As previously noted, fibrinolysis is an equally important component as a result of plasmin activity on an existing clot. Tranexamic acid administration is associated with decreased bleeding during cardiac and orthopedic surgeries, presumably because of its antifibrinolytic properties. A randomized control study involving 20,000 trauma patients with or at risk of significant bleeding found a significantly reduced risk for death from hemorrhage when tranexamic acid therapy (loading dose, 1 g over 10 min, followed by an infusion of 1 g over 8 h) was initiated within the first 3 h following major trauma (the CRASH-II study). Figure 39-4 demonstrates the benefit of initiating this therapy in relation to the time of injury. Although this study resulted in widespread use of tranexamic acid in major trauma, its shortcomings (eg, a very small number of its 20,000 patients required transfusion or were transfused) have more recently resulted in reconsideration of tranexamic acid administration in trauma care.
FIGURE 39-4 Influence of tranexamic acid in preventing death from bleeding. Outcomes ratios (OR) of tranexamic acid with 95% confidence interval (green area) on the x-axis and time (h) to treatment on the y-axis demonstrate improved survival if tranexamic acid therapy is initiated within 3 h of injury. The area of the curve to the left of OR 1.0 demonstrates the benefits of therapy, while that to the right demonstrates harm from intervention. (Reproduced with permission from Roberts I, Shakur H, Afolabi A, et al. The importance of early treatment with tranexamic acid in bleeding trauma patients: An exploratory analysis of the CRASH-2 randomised controlled trial. Lancet. 2011 Mar 26;377(9771):1096-1101.)

Early coagulopathy of trauma is associated with increased mortality. Military experience treating combat-wounded soldiers and civilians has provided great insight into trauma resuscitation and TIC. Battlefield resuscitation protocols have utilized whole blood for decades. Whole blood resuscitation is instituted in circumstances where casualty load exceeds available blood resources, usually in remote or forward bases near combat. The process requires about an hour to collect, process, and then deliver blood between soldiers. The blood is warm, and clotting factors and platelets are at optimum temperature and pH. The use of whole blood transfusions in these settings is lifesaving. However, in most circumstances, the U.S. Department of Defense utilizes more conventional blood banking techniques and utilization of blood products in combat theaters, making the need for whole blood transfusions infrequent.
Military conflicts in the 2000s have provided ample opportunities for developing updated transfusion protocols. Retrospective analysis of severely wounded service members found improved survival when fresh frozen plasma was administered early in @ trauma resuscitations. In an attempt to recreate whole blood, balanced administration of red blood cell, fresh frozen plasma, and platelet units (1:1:1) became the standard trauma transfusion protocol in military settings, and was promptly adopted thereafter by major civilian trauma centers, which also noted improved patient survival. This approach to transfusion is termed damage control resuscitation (DCR).
Administering blood products in equal ratios early in resuscitation has become an accepted approach for preventing or correcting TIC. Although this combination of blood products attempts replication of whole blood, the resultant fluid is pancytopenic, with only a fraction of whole blood's hematocrit and coagulation factor concentration. Red blood cells will improve oxygen delivery to hypoperfused, ischemic tissues. Fresh frozen plasma provides clotting factors V and VIII along with fibrinogen, which improves clotting, possibly due to overwhelming of the thrombin—thrombomodulin complex. Platelets and cryoprecipitate, although included in the 1:1:1 DCR protocol, are probably not necessary in the initial phase of resuscitation, given the normal platelet and fibrinogen levels noted in early coagulopathy. The use of crystalloid fluids in early trauma resuscitation has markedly decreased with the increased emphasis upon early blood product administration.

Most trauma centers have early-release type O-negative blood available for immediate transfusion to patients with severe hemorrhage. Depending on the urgency of transfusion need, blood product administration typically progresses from O-negative to type-specific, then to cross-matched units as the acute need decreases. Patients administered uncrossmatched, O-negative blood are those at high risk of requiring massive transfusion. As the amount of uncrossmatched blood administered increases beyond eight units, resuscitation should persist using O-negative blood without attempting to switch to the patient's native blood type. Reverting to the patient's native blood type in this situation risks transfusion reactions that will further complicate resuscitation and survival.
Most clinical studies evaluating DCR have been retrospective. However, a prospective, randomized, multi-institutional massive transfusion study from ten U.S. level 1 trauma centers (the PROMTT study) was conducted and published in 2013, confirming that the greater the injury and concomitant hemorrhagic shock, the greater the likelihood of TIC requiring massive transfusion, and the greater the risk of death. The contribution of activated protein C to TIC and the benefits of blood-based, rather than crystalloid-based, resuscitation for hemorrhagic shock were also demonstrated.
Point-of-care functional clotting studies are extremely useful for guiding specific blood product use. Thromboelastography (TEG) and rotational elastometry (ROTEM) allow more specific use of blood components by addressing the specific deficiencies rather than relying solely on the 1:1:1 transfusion ratio DCR approach. Both TEG and ROTEM demonstrate the rate of clot formation and clot stability, reflecting the interactions between the coagulation cascades, platelets, and the fibrinolytic system. Figure 39-5 demonstrates the pattern seen with TEG. The end results of this technology in trauma resuscitations are reduced blood product use (with less exposure to potential infection and less expense) as well as recognition of fibrinolysis.

FIGURE 39-5 Thromboelastograph (TEG). The graph begins as a straight line until clot formation begins (the enzymatic stage of clotting). As a clot forms, increasing resistance develops on the strain gauge, creating a splaying of the graph. The pattern of the graph suggests the status of fibrinogen stores (a angle) and platelet function (maximum amplitude, MA). Eventually, fibrinolysis will occur as demonstrated by decreasing MA. Deficiencies of various clotting components will affect each phase of the TEG, whereas increased fibrinolysis will be demonstrated by an earlier decline in the maximum amplitude. ACT, activated clotting time; EPL, Ly30, K, R, values related to rate of clot breakdown. (Reproduced with permission from Kashuk JL, Moore EE, Sawyer M, et al. Postinjury coagulopathy management: Goal directed resuscitation via POC thrombelastography. Ann Surg. 2010 Apr;251(4):604-614.)
Administration of blood products must be done with consideration for potential hazards that may result from aggressive administration during the resuscitation phase. Although blood-borne diseases such as acquired immunodeficiency syndrome, hepatitis B, and hepatitis C are usually cited as significant transfusion-related risks, modern blood bank donor screening has decreased the incidence of such @ infections by as much as 10,000-fold. The incidence of transfusion-related acute lung injury (TRALI), until recently the leading cause of transfusion-related death, has also markedly declined. With the recognition that the presence of HLA antibodies in donor plasma is the principal TRALI risk factor, most blood banks now accept plasma and platelet donations only from males, or from females who have either never been pregnant or who have been tested and found to be anti-HLA negative. The greatest transfusion risk patients now face during trauma resuscitation is transfusion-associated circulatory overload (TACO), which occurs when blood products are administered at a rate greater than the patient's cardiac output. This is most likely to occur when the provider administering blood products has not recognized that the source of bleeding has been successfully controlled by the surgeon or proceduralist. Communication in this situation is critical between those team members resuscitating the patient with blood products those attempting to control the hemorrhage.

Delay in obtaining blood products other than red blood cells is a potential problem for both military and civilian trauma resuscitations. Clinical evidence supports the need for, and benefit of, established massive transfusion protocols (MTPs), allowing the blood bank to assemble blood products in prescribed ratios in supporting blood-based trauma resuscitations. With MTPs in place, hemostatic resuscitation can continue until the demand for blood products stops. An MTP-driven, blood-based resuscitation, rather than a crystalloid-based resuscitation, improves survival from trauma, reduces total blood product utilization in the first 24 h following injury, reduces acute infectious complications (severe sepsis, septic shock, and ventilator-associated pneumonia), and decreases postresuscitation organ dysfunction (an 80% decrease in odds of developing multisystem organ failure).
MTPs benefit both the patient (improved survival and fewer complications) and the institution (more efficient and effective processes for utilizing critical blood bank resources). Most hospitals performing complex surgeries, transplants, or trauma resuscitations now have MTPs in place, although one must recognize that the transfusion needs of transplant, cancer, and cardiac patients may differ from those of trauma patients. Establishing which personnel are empowered to initiate MTP use during trauma resuscitation is of great importance, given the expense and implications for the blood bank in terms of blood product inventory, personnel training and availability, and disruption of routine blood bank duties. Annual reviews of MTPs are necessary for ensuring that the most current knowledge, technology, and medications are included in the protocols that will optimize utilization of blood products.

The initial history and physical examination, emergency procedures, and evaluations used to determine extent of injury, need for resuscitation, and need for surgical intervention all occur outside the operating room and at times before an anesthesia provider has been alerted. However, critical initial issues impacting anesthetic management of trauma patients include adequacy of airway and vascular access, ability of the patient to tolerate anesthesia, prevention of hypothermia, access to robust blood bank supplies, and avoidance of crystalloids and vasopressors until hemorrhage is controlled. Therefore, anesthesiologist participation in the earliest assessment of potentially severely injured trauma patients in the emergency room should be encouraged.

Severely injured, conscious, and oriented trauma patients arriving for emergency surgery should have an abbreviated interview and examination, including emphasis on consent for blood transfusions and advice that intraoperative awareness may occur during emergency surgery. As always, such discussions should be documented in the patient's record.
The operating room should be as warm as practical. Intravenous fluid warmers and rapid infusion devices should be prepared and ready for use. As previously noted, all patients arriving for trauma surgery should be presumed to have full stomachs with increased risk for aspiration of gastric contents, and the presence of a C-collar for cervical spine stabilization may increase intubation difficulty. Alternative airway devices (eg, fiberoptic bronchoscope,
Fluid management in major trauma resuscitations emphasizes blood products rather than crystalloid fluids, as previously noted. An MTP should be requested and followed, with the blood immediately available upon the arrival of the patient to the operating room. All fluids should be warmed, except for platelets. When blood products are rapidly infused, ionized calcium quickly declines and must be replaced. Vasopressors should not be used, if possible, until the source of bleeding is controlled. Studies suggest that raising the blood pressure with vasopressors during hemorrhage disrupts fresh clots, resulting in more bleeding. An arterial line is helpful but not mandatory in the initial resuscitation of the trauma victim. Even with the assistance of ultrasonography, cannulating an artery in the presence of profound hypotension may prove difficult. Attempts at placing invasive monitors can continue as the patient is prepared for incision, to include gowning and gloving the person attempting arterial line placement on the surgical side of the drape, if necessary. Although arterial line placement may be a challenge, surgical incision cannot be delayed. Surgical control of bleeding and DCR are the top priorities in trauma resuscitation, not arterial line placement. Patients in this degree of hemodynamic compromise can be presumed to have TIC and be in need of massive transfusion. Attempts for arterial line placement can resume, and are more likely to be successful, as blood pressure improves from operative hemostasis and resuscitative transfusion.

the surgical procedure to allow resuscitation. Pausing surgery results in the surgeon compressing or packing an area of bleeding during times of profound hypotension until transfusion restores acceptable systolic blood pressure (80-90 mm Hg). If this interruption of surgery is unsuccessful in improving blood pressure, the surgeon can directly compress the aorta. This intervention provides the surgeon direct feedback as to the effectiveness of transfusion—a soft aorta suggests profound hypovolemia, whereas the return of a firm, pulsatile aorta suggests a more acceptable circulating blood volume. A brief episode of bradycardia/asystole may accompany direct aortic compression. When transfusions are ineffective maintaining perfusion, the operation should be interrupted, the bleeding areas packed, and a decision should be made between the surgeon and anesthesia team as to whether the patient can be transferred to the interventional radiology suite to treat bleeding from surgically-inaccessible sites or to the intensive care unit where rewarming, correction of coagulopathy and hemodynamic stabilization may occur.
A key component of DCS is planned re-operation once the patient is more stable. At a later time, bowel continuity can be restored or a colostomy can be performed. The abdominal fascia is often not definitively closed after DCS. The wound may be covered with an occlusive dressing over a wound vacuum sponge. Bowel edema in the setting of a closed abdomen following massive transfusion risks abdominal compartment syndrome, respiratory compromise, and multisystem organ failure.
The interventional radiology suite is increasingly utilized as part of the DCS sequence. Interventional radiology techniques can reach essentially any bleeding vessel and deposit coils or foam that may control hemorrhage, most notably in liver, kidney, and retroperitoneal injuries. Hemorrhage from pelvic ring fractures or major thoracic or abdominal vascular injuries are also potentially controlled by such intravascular interventions. In addition, patients are often taken to interventional radiology following DCS to assess blood flow and hemostasis in organs either injured by the initial trauma or potentially compromised as part of the DCS.

@ Any trauma patient with an altered level of consciousness must be considered to have a traumatic brain injury (TBI) until proven otherwise (see Chapter 27). Presence or suspicion of TBI mandates attention to maintaining cerebral perfusion pressure and oxygenation during all aspects of care. The most reliable clinical assessment tool in determining the significance

In the absence of an intracranial clot requiring surgical evacuation, medical interventions are the primary means of treating elevated ICP following head trauma. Normal cerebral perfusion pressure (CPP), the difference between mean arterial pressure (MAP) and ICP, is approximately 80 to 100 mm Hg (MAP — ICP = CPP; see Chapter 26). ICP monitoring is not required for conscious and alert patients. In addition, patients who are intentionally anticoagulated or who have bleeding diathesis in response to trauma should not have ICP monitoring. However, an ICP monitor should be placed when serial neurological examinations and additional clinical assessments reveal impairment, or when there is an increased risk for elevated ICP (Table 39-1). Interventions for reducing ICP are indicated when readings are higher than 20 to 25 mm Hg. Multiple studies have evaluated interventions aimed at improving CPP and managing ICP without finding obvious outcome benefits for any treatment scheme. © Current Brain Trauma Foundation guidelines recommend maintaining CPP between 50 and 70 mm Hg and ICP at less than 20 mm Hg for patients with severe head injury.

Severe head injury (defined as GCS score <8 after cardiopulmonary resuscitation) plus
(a) Abnormal admitting head CT scan or
(b) Normal CT scan plus =2 of: age >40 y, systolic blood pressure >90 mm Hg, decerebrate or decorticate position
Sedated patients; patient in induced coma after severe TBI
Multisystem injury with altered level of consciousness
Patient receiving treatment that increases risk of increased ICP, eg, high- volume intravenous fluids
Postoperatively after removal or intracranial mass
Abnormal values
in noninvasive
ICP
monitoring, simulated
values, or
velocity abnormal
waveform
(increased increased
shapes in
transcranial pulsatility)
dynamics
Doppler with
exclusion blood
hypotension and
hypocapnia
TReproduced with permission from Li LM, Timofeev I, Czosnyka M, et al. Review article: The surgical of
flow

The normal spine structure comprises three columns: anterior, middle, and
parameters (eg, avoiding hypotension) and surgical stabilization of the spine to prevent extension of existing injury and exacerbation of existing neurological deficits.
Surgical decompression and stabilization of spinal fractures is indicated when a vertebral body loses more than 50% of its normal height or the spinal canal is narrowed by more than 30% of its normal diameter. Methylprednisolone is often administered for spinal cord injury in this situation. The antiinflammatory properties of this steroid potentially reduce spinal cord edema within the tight confines of a compromised spinal canal. Despite outcome studies from animal models of traumatic spinal cord injury demonstrating benefit from early surgical intervention, steroid therapy, or both, current human studies have failed to demonstrate benefit from either intervention. The presence of a decompressible lesion in the area of an incomplete spinal cord transection is not an indication for early operative intervention unless other, more life-threatening, conditions are present.
® Maintaining supranormal mean arterial blood pressure to help ensure adequate spinal cord perfusion in areas of otherwise reduced blood flow due to cord compression or vascular compromise is likely to be of more benefit than steroid administration. Hypotension must be avoided during induction of anesthesia, throughout operative decompression and stabilization of the spine injury, and extending into the postoperative phase.
The elderly are at greater risk for spinal cord injury due to decreased mobility and flexibility, greater incidence of spondylosis and osteophytes, and decreased space within the spinal canal to accommodate spinal cord edema following trauma. The incidence of spinal injury from falls in the elderly is rapidly approaching that of spinal cord injury from motor vehicle accidents in younger patients. Mortality following spinal cord injury in the elderly, particularly those over the age of 75 years, is greater than that in younger patients with similar injury.

The unique injury pattern of penetrating spinal cord injury warrants separate consideration. Unlike blunt spinal trauma, penetrating trauma of the spinal cord due to bullets and shrapnel is unlikely to cause an unstable spine. As a result, Ccollar or long-board immobilization may not be indicated in an isolated penetrating spinal cord injury. The C-collar placement in the presence of a cervical spine penetrating injury may actually hinder observation of soft tissue swelling, tracheal deviation, or other anatomic indications of imminent airway compromise. Unlike, blunt trauma, penetrating injuries of the spinal cord induce damage at the moment of injury without risk of subsequent exacerbation of the injury. Like other spinal cord injuries, however, maintenance of spinal cord perfusion using supranormal mean arterial pressure is indicated until spinal cord function can be more thoroughly evaluated.

fluids according to recommended protocols (see below). This intense hemodynamic response may be poorly tolerated by patients with significant underlying medical conditions. If adequate intravenous fluid therapy is provided, cardiac function returns to normal within 48 h of injury, then typically progresses to a hyperdynamic physiology as the metabolic challenge of healing begins. Plasma volume and urine output are reduced early on, after major burn injuries.
® In contrast to fluid management of blunt and penetrating trauma, where crystalloid fluids are discouraged, burn fluid resuscitation emphasizes the use of balanced crystalloid fluids (see Chapter 51) in preference to albumin, hydroxylethyl starch, normal or hypertonic saline, or blood. Following burn injuries, acute kidney failure is more common when hypertonic saline is used during initial fluid resuscitation, death is more likely when blood is administered, and outcomes are unchanged when albumin (rather than crystalloid) is used in resuscitation.
Fluid resuscitation is continuous over the first 24 h following burn injury. Two formulas are commonly used in guiding burn injury fluid resuscitation; the Parkland and the modified Brooke. Both require an understanding of the Rule of Nines (Figure 39-6) to calculate resuscitation fluid volumes. The adult Parkland protocol recommends 4 mL/kg/% burned TBSA to be given in the first 24 h, with half the volume given in the first 8 h and the remaining volume over the following 16 h. The adult modified Brooke protocol recommends 2 mL/kg/% burned TBSA, with half the calculated volume beginning in the first 8 h and the remainder over the following 16 h. Both formulas use urine output as a reliable indicator of fluid resuscitation adequacy, targeting adult urine production of 0.5 to 1.0 mL/kg/h as indicators of adequate circulating volume. If adult urine output exceeds 1.0 mL/kg/h, the infusions are slowed. In both protocols, an amount equal to half the volume administered in the first 24 h is infused in the second 24-h period following injury. The goal of maintaining adult urine output at 0.5 to 1.0 mL/kg/h continues throughout the initial phase of resuscitation.

FIGURE 39-6 The Rule of Nines, utilized to estimate burned surface area as a percentage of total body surface area (TBSA). (Reproduced with permission from American College of Surgeons. ATLS: Advanced Trauma Life Support for Doctors (Student Course Manual). 9th ed. Chicago, IL: ACS; 2012.)
When pediatric burn patients are encountered, the fluid resuscitation protocols are the same as for adults. Children weighing less than 30 kg should receive 5% dextrose in their intravenous fluids and the target urine output is 1.0 mL/kg/h. The target urine output for infants younger than 1 year of age is 1 to 2 mL/kg/h.

The Parkland and modified Brooke protocols both use urine output as an indicator for adequate fluid resuscitation. However, circumstances may arise in which the volume of fluid administered exceeds the original volume goal. For example, initial fluid resuscitation volumes may be miscalculated if first-degree burns are mistakenly incorporated into the TBSA value. Prolonged use of sedation may result in hypotension, prompting administration of additional fluids rather than vasoconstrictors. The phenomenon of fluid creep occurs when intravenous fluid therapy volumes are increased beyond intended calculations in response to hemodynamic changes related to issues other than circulating volumes. Fluid creep is also associated with abdominal compartment syndrome and pulmonary complications, often leading to resuscitation-related morbidity.

Abdominal compartment syndrome (ACS) is a risk for pediatric and adult patients with circumferential abdominal burns and patients receiving intravenous fluid volumes greater than 6 mL/kg/% burned TBSA. Intraabdominal pressure can be determined by measuring intraluminal bladder pressure using a Foley catheter connected to a pressure transducer. The transducer is connected to a three-way stopcock at the point where the Foley catheter connects to the drainage tube. After the transducer is zeroed at the pelvic brim, 20 mL of fluid is instilled into the bladder. Intraabdominal pressure readings are taken 60 s after infusing fluid into the bladder, which allows the bladder to relax. Intraabdominal pressures exceeding 20 mm Hg warrant abdominal cavity decompression. However, an abdominal surgical procedure places the burn patient at increased risk for intraabdominal Pseudomonas infection, particularly if the laparotomy incision is near burned tissue. Early and frequent assessment of intraabdominal pressure and consideration of potential etiologies of hypotension in the burn patient other than hypovolemia are important preventative measures for ACS.

Excessive fluid resuscitation volumes are associated with an increased incidence of pneumonia. Patients with severe burns frequently have pulmonary injury related to the burn. Decreased tracheal ciliary activity, the presence of resuscitation-induced pulmonary edema, reduced immunocompetence, and tracheal intubation predispose burn patients to pneumonia. ACS can have an adverse impact on pulmonary function. Intravenous fluid administration volumes must be monitored closely and documented to be consistent with American Burn Association recommendations (ie, the Parkland or modified Brooke protocol). Fluid administration that exceeds recommendations warrants careful review of the rationale.

@The differential diagnosis for altered mental status following burn injury and smoke inhalation includes carbon monoxide and cyanide poisoning (see Chapter 57). Endotracheal intubation and mechanical ventilation with high inspired oxygen concentration are indicated in this situation. Carbon monoxide binds hemoglobin with an affinity approximately 250 times that of oxygen. The resultant carboxyhemoglobin (HbCO) leaves less hemoglobin available for binding oxygen and shifts the O,—-Hb dissociation curve to the left, both causing impaired delivery of oxygen to tissues. Pulse oximetry provides a falsely elevated indication of oxygen saturation in the setting of carbon monoxide exposure because of its inability to distinguish oxygenated hemoglobin (HbO, ) from HbCO. Arterial or venous blood gas analysis can directly measure HbCO. Clinically significant carbon monoxide poisoning is seen when HbCO levels exceed 10% (those who regularly smoke tobacco have HbCO levels of up to 10%). If HbCO exceeds 20%, intubation and mechanical ventilation is indicated in order to improve local tissue oxygenation and accelerate carbon monoxide elimination. Death from carbon monoxide occurs when HbCO levels exceed 60%. Hyperbaric oxygen therapy is indicated for carbon monoxide poisonings from any etiology. Multiple hyperbaric oxygen sessions are required to reduce the long-term consequences of carbon monoxide poisoning.

A primary characteristic of all burn patients is an inability to regulate temperature. The resuscitation environment must be maintained near body temperature through the use of radiant warming, forced air warming devices, and fluid warming devices. All burn care environments must be maintained near 40°C.
Assessment of the burn patient begins with inspection of the airway. Although the face may be burned (singed facial hair, nasal vibrissae), facial burns are not an indication for tracheal intubation. The need for urgent airway management, mechanical ventilation, and oxygen therapy is indicated by hoarse voice, dyspnea, tachypnea, or altered level of consciousness. Arterial blood gas determination should be obtained early in the treatment process for assessment of HbCO level. Mechanical ventilation should be adjusted to achieve adequate oxygen saturation (based upon measured oxygen levels rather than pulse oximetry) at the lowest tidal volumes.
Tracheal intubation in the early period following burn injury (up to the first 48 h) can be facilitated with succinylcholine for muscle relaxation. In patients with significant burns (>20% TBSA), injury and disruption of neuromuscular end plates occurs, followed by upregulation of acetylcholine receptors. ® Beyond 48 h after significant burn injury, succinylcholine can produce lethal hyperkalemia. This risk for succinylcholine-induced hyperkalemia persists for up to 2 years following burn injury.
Analgesia for burn patients is challenging. Considerations and concerns regarding opioid tolerance and psychosocial complications of burn therapy are commonplace. Multimodal approaches are often advantageous. Regional analgesia may provide benefit, although in the early post-burn period this technique may mask the symptoms of compartment syndrome or other clinically significant signs and symptoms related to the primary burn injury.

@ The elderly represent the fastest-growing trauma population seen in trauma centers across the world. The aged are at greater risk for serious complications and death after even modest trauma. Preexisting, significant conditions that negatively impact physiological reserve and ability to recover from trauma contribute more to the higher complication and death rate after trauma than age itself. Although "geriatric" is not a specific age, with respect to "geriatric" trauma outcomes, several studies have observed increased morbidity and mortality after trauma beginning between the ages of 45 and 55 years, suggesting decreased physiological reserve following trauma may start at ages far earlier than previously appreciated. With advancing age, several studies found the risk for death after trauma doubled for trauma victims older than 65 years, when injury severity was matched for those younger than 65 years. Falling from standing height is responsible for 90% of injuries in patients over the age of 65 years. Fall-related intracranial hemorrhage, skeletal fractures, and major thoracic or intraabdominal vascular injuries increase morbidity and mortality in this patient population. Patients taking oral anticoagulants have lower GCS scores and higher morbidity than elderly patients experiencing similar injuries who do not use oral anticoagulants. Use of oral anticoagulants in this patient population is an independent predictor of 30-day mortality after falling. Preventing falls in the elderly is an emerging public health issue.

in need of emergency surgery, 20% will require urgent surgery within 8 h, and 30% will require nonemergent interventions. The key is identifying the nature and likely number of victims involved. Second, victims of mass casualty events often arrive at hospitals unannounced. Passers-by or law enforcement personnel begin transporting victims to the nearest hospital, even if the nearest hospital is not intended for trauma care, and may arrive without notice. This creates a need for transporting patients between facilities when emergency first-responder resources are likely already overwhelmed by the initial incident. Finally, resources must be reserved for salvageable victims in this setting. Only patients with pulses should be allowed access to surgical resources and intensive care beds in mass casualty incidents, and only those patients should be transfused.

A 22-year-old, previously healthy, 70-kg man is brought to the emergency department (ED) at 9 am by his mother after fainting at home earlier that morning. The previous evening, he was involved in an altercation in a bar during which he was kicked repeatedly in the stomach. The patient is pale, tachycardic, and lethargic. His pulse is 140 beats/min and blood pressure is 60/34 mm Hg. A FAST (focused assessment with sonography for trauma) examination in the ED demonstrates free fluid in the abdomen. Two 18-gauge antecubital intravenous lines are placed and a blood sample is sent to the blood bank for type and cross-matching. A massive transfusion protocol (MTP) is initiated. The patient is brought to the operating room within 16 min of his arrival in the ED.
What measures should be taken prior to induction of anesthesia?
The patient has life-threatening injuries. Although surgical intervention is necessary, the interval between anesthetic induction and surgical incision must be as short as possible. Preparing the patient for incision for a major trauma surgical intervention warrants skin preparation extending from the patient's chin to his toes, with warm skin preparation solution. The operating room should be as warm as possible until the patient is draped.
Avoiding hypothermia is of critical importance. The trauma surgeons should be prepared to operate immediately in case hemodynamic collapse occurs with anesthetic induction. This is why the skin must be prepared for incision prior to, or during, anesthetic induction. Delaying surgical incision for the placement of arterial line or central venous access is not indicated in this scenario. Red blood cells and other blood products should be available prior to anesthetic induction.
Warm intravenous fluids are infused quickly to prevent hemodynamic collapse upon anesthetic induction. While the fluids are infusing, standard ASA monitors are applied while the patient is preoxygenated. The surgical nurse is painting the patient from chin to toes with antiseptic solution. The surgeons are gowning and gloving. Immediate release, non—cross-matched O-negative packed red blood cells (RBCs) are brought to the room. The operating room temperature is uncomfortably warm. Invasive hemodynamic monitoring catheters and ultrasound devices are present and immediately available.

The patient is lethargic. Cerebral hypoperfusion, traumatic brain injury, or intoxication cannot be ruled out. The mechanism of injury is that of blunt violence, obligating the need for cervical spinal cord immobilization until a more thorough neurological evaluation can occur. The choice of anesthetic induction agent must be chosen with appreciation for the need to prevent hemodynamic collapse. Benzodiazepines, opioids, and propofol each attenuate sympathetic tone, and their administration may be life-threatening in this scenario. Since all trauma patients should be considered to have full stomachs, rapid sequence anesthetic induction/intubation is indicated. If sufficient staff is available, someone from the anesthesia or surgical team should be gowned and gloved, working next to the operating surgeons to place arterial and central venous lines, using ultrasound technology, if needed, at the time of surgical incision.
With the patient draped, the surgeons ready for incision and arrival of the uncross-matched blood in the operating room. Scopolamine, 0.4 mg, is injected intravenously followed immediately by succinylcholine, 100 mg intravenously. With inline cervical spine immobilization maintained, the endotracheal tube is passed with the assistance of a video laryngoscope. Once endotracheal tube position is confirmed and secured, the surgeons initiate the incision and proceed with damage control surgical intervention. The surgeons make a full laparotomy incision while an anesthesia colleague places a left subclavian central venous catheter. Hemodynamic collapse occurs as blood spills from the surgical site. The arterial line is not yet in place, but central venous access via the subclavian vein is now present. The first MTP blood products arrive in the operating room.
What are the implications of damage control surgery (DCS) and damage control resuscitation (DCR)?
When confronted with hemodynamic instability during life-threatening hemorrhage, the surgeons intervene to stop the hemorrhage until the hemodynamic status can be stabilized, and in a case of abdominal trauma, to limit gastrointestinal contamination of the abdominal compartment. This represents DCS. In this case, they pack off the abdomen until sufficient blood-based resuscitation restores systolic blood pressures to 80 to 90 mm Hg, which should allow the surgery to proceed.

DCR is an aggressive transfusion protocol utilized during lifethreatening hemorrhage that seeks to mimic whole blood (ie, RBC, fresh frozen plasma [FFP], and platelet units in a ratio of 1:1:1). Emphasizing warmed blood product transfusion during hemorrhage reduces hypothermia and acidosis while attenuating trauma-induced coagulopathy (providing intravenous crystalloid administration is kept to a minimum). Blood products in this scenario, with the exception of platelets, must be infused through a fluid warmer capable of delivering large volumes of infusate for extended periods of time.
The surgeon packs the abdomen and compresses the aorta. Cardiopulmonary resuscitation (CPR) is not useful in blood loss scenarios; aortic compression is the most effective and prudent intervention in hemorrhage-related hemodynamic collapse. Blood is administered through a rapid infusion device in a 1:1:1 ratio. An increased end-tidal carbon dioxide tracing is now observed, and the surgeon notes the aorta is now more firm. The noninvasive blood pressure cuff shows a systolic pressure of 82 mm Hg. The surgeon removes an abdominal pack and begins exploring the abdomen. Although DCR continues, surgical exploration is repeatedly interrupted due to hemodynamic instability. Eventually, a brachial artery monitoring line is placed with ultrasound guidance.
What technology is available for more closely tailoring blood

DCR emphasizes blood product use during hemorrhage resuscitation. Experience with this lifesaving intervention suggests that blood product utilization can be more precise if functional coagulation tests (eg, thromboelastography [TEG] or rotational thromboelastometry [ROTEM]) are utilized. These technologies assess the functional status of clot formation, with results available within 5 min of sampling. Patterns of clot formation can guide the administration of platelets, fibrinogen, and plasma. Thrombolysis is also detected with this technology, providing evidence of the need for antithrombolytic therapies.
A blood gas sample demonstrates metabolic acidosis and hemoglobin of 7.0 g/dL. The functional coagulation study (TEG or ROTEM) demonstrates a hypercoagulable pattern. As a result, the anesthesiologist alters the transfusion ratios to 3 RBC:1 FFP:1 platelet units. The surgeon now indicates that the sources for bleeding—a ruptured spleen and lacerated kidney—are controlled. The anesthesiologist notifies the team that blood products will no longer need to be rapidly infused unless hemodynamic instability recurs.

The question of when to initiate vasopressors during trauma resuscitation has not been defined. So-called permissive hypotension during active surgical hemostasis and damage control resuscitation is optimal to prevent disruption of clot formation. Systolic blood pressures in the 80 to 90 mm Hg range reduce blood loss and transfusion needs. However, once the source of blood loss is surgically controlled, no guidelines exist as to when vasopressors should be initiated, rather than continued transfusion of blood products. This is an active area of investigation in trauma practices. As the surgeons continue assessing the abdominal cavity for any as yet undetected relevant trauma, the patient's systolic blood pressure gradually declines to 70 mm Hg. The patient's core temperature is 35.5°C, the blood gas shows an improvement of the metabolic acidosis, with a base excess of —4 mmol and hemoglobin of 10.0 g/dl. The TEG demonstrates normal coagulation patterns. Ionized calcium is within normal limits. The decision to initiate low-dose vasopressin and epinephrine infusions, rather than transfuse additional blood products, is made; the patient was otherwise healthy prior to this injury, and transfusion of additional blood products increases the risk of transfusion-associated circulatory overload (TACO).
The patient's blood pressure and heart rate stabilize to within normal limits, the abdomen is temporarily closed, and he is transferred to the surgical intensive care unit while remaining intubated and sedated.

This typical trauma scenario broadly addresses the common resuscitation and management decisions required for major trauma resuscitation. At this time, applying these resuscitation concepts to intraoperative surgery-related hemorrhage is not supported. Unlike trauma, where the patient is typically hypotensive for an extended period of time (frequently more than an hour), intraoperative hemorrhage is typically recognized promptly and addressed rapidly. Such patients usually do not become profoundly acidotic prior to initiating resuscitation measures and transfusion. Unlike trauma coagulopathy, intraoperative coagulopathy in the setting of surgical hemorrhage is more likely dilutional, rather than endothelium-derived (thrombolytic), as in the trauma setting. However, the basic concepts of trauma resuscitation remain relevant: Allow lower systolic blood pressures until the source for bleeding is identified and controlled, limit administration of intravenous crystalloids during hemorrhage in deference to administration of blood products, and utilize TEG or ROTEM to provide functional assessment of clotting for guidance of blood product administration. These concepts are accepted and defendable interventions in nontrauma resuscitation, where research is ongoing to clarify best practices.

Adams SD, Holcomb JB. Geriatric trauma. Curr Opin Crit Care. 2015;21:520.
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@ The minimum alveolar concentration (MAC) progressively decreases during pregnancy—at term, by as much as 40%—for all general anesthetic agents; MAC returns to normal by the third day after delivery.
@ Pregnant patients display enhanced sensitivity to local anesthetics during regional anesthesia and analgesia, and neural blockade occurs at reduced concentrations of local anesthetics; dose requirements may be reduced as much as 30%.
© Obstruction of the inferior vena cava by the enlarging uterus distends the epidural venous plexus and and increases epidural blood volume.
@ Approximately 5% of women at term develop the supine hypotension syndrome, which is characterized by hypotension associated with pallor, sweating, or nausea and vomiting.
© The reduction in gastric motility and gastroesophageal sphincter tone place the parturient at high risk for regurgitation and pulmonary aspiration.
@ Ephedrine, which has considerable B-adrenergic activity, has traditionally been considered the vasopressor of choice for hypotension during pregnancy. However, clinical studies suggest that the aadrenergic agonist phenylephrine is more effective in treating hypotension in pregnant patients and is associated with less fetal acidosis than ephedrine.
@ Volatile inhalational anesthetics decrease blood pressure and,
potentially, uteroplacental blood flow. In concentrations of less than 1 MAC, however, their effects are generally minor, consisting of dosedependent uterine relaxation and minor reductions in uterine blood flow.
©@ The greatest strain on the parturient's heart occurs immediately after delivery, when intense uterine contraction and involution suddenly relieve inferior vena caval obstruction and increase cardiac output as much as 80% above late third-trimester values.
© Current techniques employing dilute combinations of a local anesthetic (eg, bupivacaine, <0.125%) and an opioid (eg, fentanyl, <5 mcg/mL) for epidural or combined spinal—epidural (CSE) analgesia do not appear to prolong the first stage of labor or increase the likelihood of an operative delivery.
This chapter reviews the normal physiological changes associated with pregnancy, labor, and delivery. It concludes with a description of the physiological transition from fetal to neonatal life.

Pregnancy affects most organ systems (Table 40—1). Many of these physiological changes appear to be adaptive and useful to the mother in tolerating the stresses of pregnancy, labor, and delivery.
TABLE 40-1 Average maximum physiological changes associated with pregnancy.!
Neurological, Change = —40%. MAC, Change = . Respiratory, Change = . Oxygen consumption, Change = +20 to 50%. Airway resistance, Change = -35%. FRC, Change = -20%. Minute ventilation, Change = +50%. Tidal volume, Change = +40%. Respiratory rate, Change = +15%. Pao,, Change = +10%. Paco,, Change = -15%. HCO,, Change = -15%. Cardiovascular, Change = . Blood volume, Change = +35%. Plasma volume, Change = +55%. Cardiac output, Change = +40%. Stroke volume, Change = +30%. Heart rate, Change = +20%. Systolic blood pressure, Change = -5%. Diastolic blood pressure, Change = -15%. Peripheral resistance, Change = -15%. Pulmonary resistance, Change = -—30%. Hematological, Change = . Hemoglobin, Change = -20%. Platelets, Change = -10%. Clotting factors?, Change = +30 to 250%. Renal, Change = . GFR, Change = +50%
'MAC, minimum alveolar concentration; FRC, functional residual capacity; GFR, glomerular filtration rate.
- *Varies with each factor.

@ The minimum alveolar concentration (MAC) progressively decreases during pregnancy—at term, by as much as 40%—for all general anesthetic agents; MAC returns to normal by the third day after delivery. Progesterone, which is sedating when given in pharmacological doses, increases up to 20 times normal at term and is at least partly responsible for this phenomenon. A surge in Bendorphin levels during labor and delivery also likely plays a major role. ® Pregnant patients display enhanced sensitivity to local anesthetics during regional anesthesia and analgesia, and neural blockade occurs at reduced concentrations of local anesthetics. The term minimum local analgesic concentration (MLAC) is used in obstetric anesthesia to compare the relative potencies of local anesthetics and the effects of additives; MLAC is defined as the local analgesic concentration leading to satisfactory analgesia in 50% of patients (EC.,). Local anesthetic dose requirements during epidural anesthesia may be reduced as much as 30%, a phenomenon that appears to be hormonally mediated but may also be related to engorgement of the @ epidural venous plexus. Obstruction of the inferior vena cava by the enlarging uterus distends the epidural venous plexus and increases epidural blood volume. The latter has three major effects: (1) decreased spinal cerebrospinal fluid volume, (2) decreased potential volume of the epidural space, and (3) increased epidural (space) pressure. The first two effects enhance the cephalad spread of local anesthetic solutions during spinal and epidural anesthesia (see Chapter 45). Bearing down during labor further accentuates all these effects. Positive (rather than the usual negative) epidural pressures have been recorded in parturients. Engorgement of the epidural veins also increases the likelihood of placing an epidural needle or catheter in a vein, resulting in an unintentional intravascular injection.

Oxygen consumption and minute ventilation increase progressively during pregnancy. Tidal volume and, to a lesser extent, respiratory rate and inspiratory reserve volume also increase. By term, both oxygen consumption and minute ventilation have increased up to 50%. Paco, decreases to 28 to 32 mm Hg; significant respiratory alkalosis is prevented by a compensatory decrease in plasma bicarbonate concentration. Hyperventilation may also increase Pao, slightly. Elevated levels of 2,3-diphosphoglycerate offset the effect of hyperventilation on hemoglobin's affinity for oxygen (see Chapter 23). The Ps for hemoglobin increases from 27 to 30 mm Hg; the combination of the latter with an increase in cardiac output (see next section on Cardiovascular Effects) enhances oxygen delivery to tissues.
The maternal respiratory pattern changes as the uterus enlarges. In the third trimester, elevation of the diaphragm is compensated by an increase in the anteroposterior diameter of the chest; diaphragmatic motion, however, is not restricted. Both vital capacity and closing capacity are minimally affected, but functional residual capacity (FRC) decreases up to 20% at term; FRC returns to normal within 48 h after delivery. This decrease is principally due to a reduction in expiratory reserve volume as a result of larger than normal tidal volumes. Flow—volume loops are unaffected, and airway resistance decreases. Physiological dead space decreases but intrapulmonary shunting increases toward term. A chest film may show prominent vascular markings due to increased pulmonary blood volume and an elevated diaphragm. Pulmonary vasodilation prevents pulmonary pressures from rising.
The combination of decreased FRC and increased oxygen consumption promotes rapid oxygen desaturation during periods of apnea. Preoxygenation (denitrogenation) prior to induction of general anesthesia is therefore mandatory to avoid hypoxemia in pregnant patients. Closing volume exceeds FRC in some pregnant women at term when they lie supine. Under these conditions, atelectasis and hypoxemia readily occur. The decrease in FRC coupled with the increase in minute ventilation accelerates the uptake of all inhalational anesthetics. The reduction in dead space narrows the arterial endtidal CO, gradient.

Engorgement of the respiratory mucosa during pregnancy predisposes the upper airways to trauma, bleeding, and obstruction. Gentle laryngoscopy and smaller endotracheal tubes (6—6.5 mm) should be employed during general anesthesia.

Cardiac output and blood volume increase to meet increased maternal and fetal metabolic demands. In the first trimester, there is a substantial decrease in peripheral vascular resistance with a nadir during the middle of the second trimester and a subsequent plateau or slight increase for the remainder of the pregnancy. An increase (55%) in plasma volume in excess of an increase in red cell mass (45%) produces dilutional anemia and reduces blood viscosity. Hemoglobin concentration usually remains greater than 11 g/dL. Moreover, the reduction in hemoglobin concentration is offset by the increase in cardiac output and the rightward shift of the hemoglobin dissociation curve (see the section on Respiratory Effects) to maintain oxygen delivery to tissues.
At term, blood volume has increased by 1000 to 1500 mL in most women, allowing them to easily tolerate the blood loss associated with delivery; total blood volume reaches 90 mL/kg. Average blood loss during vaginal delivery is 400 to 500 mL, compared with 800 to 1000 mL for a cesarean section. Blood volume does not return to normal until 1 to 2 weeks after delivery.
The increase in cardiac output (40% at term) is due to increases in both heart rate (20%) and stroke volume (30%). Cardiac chambers enlarge and myocardial hypertrophy is often noted on echocardiography. Pulmonary artery, central venous, and pulmonary artery occlusion pressures remain unchanged. Most of these effects are observed in the first and, to a lesser extent, the second trimester. In the third trimester, cardiac output does not appreciably rise, except during labor. The greatest increases in cardiac output are seen during labor and immediately after delivery (see the section on Effect of Labor on Maternal Physiology). Cardiac output often does not return to normal until 2 weeks after delivery.

Decreases in cardiac output can occur in the supine position after week 20 of pregnancy. Such decreases have been shown to be secondary to impeded venous return to the heart as the enlarging uterus compresses the inferior vena cava. @ Approximately 5% of women at term develop the supine hypotension syndrome (aortocaval compression), which is characterized by hypotension associated with pallor, sweating, or nausea and vomiting. The cause of this syndrome appears to be compression of the inferior vena cava by the gravid uterus. When combined with the hypotensive effects of regional or general anesthesia, aortocaval compression can readily produce fetal asphyxia. Turning the patient on her side typically restores venous return from the lower body and corrects the hypotension in such instances. This maneuver is conveniently accomplished by placing a wedge (>15°) under the right hip. The gravid uterus also compresses the aorta in most parturients when they are supine. This latter effect decreases blood flow to the lower extremities and, more importantly, to the uteroplacental circulation. Uterine contraction reduces caval compression but exacerbates aortic compression.
Chronic partial caval obstruction in the third trimester predisposes to venous stasis, phlebitis, and edema in the lower extremities. Moreover, compression of the inferior vena cava below the diaphragm distends and increases blood flow through the paravertebral venous plexus (including the epidural veins), and to a minor degree, the abdominal wall.
Lastly, elevation of the diaphragm shifts the heart's position in the chest, resulting in the appearance of an enlarged heart on a plain chest film and in left axis deviation and T wave changes on the electrocardiogram. Physical examination often reveals a systolic ejection flow murmur (grade I or IT) and exaggerated splitting of the first heart sound (S,); a third heart sound (S3) may be audible. A few patients develop small, asymptomatic pericardial effusion.

Renal plasma flow and the glomerular filtration rate increase during pregnancy; as a result, serum creatinine and blood urea nitrogen may decrease to 0.5 to 0.6 mg/dL and 8 to 9 mg/dL, respectively. A decreased renal tubular threshold for glucose and amino acids is common and often results in mild glycosuria (1-10 g/d) or proteinuria (<300 mg/d), or both. Plasma osmolality decreases by 8 to 10 mOsm/kg.
@ Gastroesophageal reflux and esophagitis are common during pregnancy. Gastric motility is reduced, and upward and anterior displacement of the stomach by the uterus promotes incompetence of the gastroesophageal sphincter. These factors place the parturient at high risk for regurgitation and pulmonary aspiration. However, neither gastric acidity nor gastric volume changes significantly during pregnancy. Opioids and anticholinergics reduce lower esophageal sphincter pressure, may facilitate gastroesophageal reflux, and delay gastric emptying.

Overall hepatic function and blood flow are unchanged; minor elevations in serum transaminases and lactic dehydrogenase levels may be observed in the third trimester. Mild elevations in serum alkaline phosphatase are due to its secretion by the placenta. A mild decrease in serum albumin is due to an expanded plasma volume, and as a result, colloid oncotic pressure is reduced. A 25% to 30% decrease in serum pseudocholinesterase activity is also present at term but rarely produces significant prolongation of paralysis by succinylcholine. The metabolism of ester local anesthetics is not appreciably altered. Pseudocholinesterase activity may not return to normal until up to 6 weeks postpartum. High progesterone levels appear to inhibit the release of cholecystokinin, resulting in incomplete emptying of the gallbladder. The latter, together with altered bile acid composition, can predispose to the formation of cholesterol gallstones during pregnancy.

Pregnancy is associated with a hypercoagulable state that may be beneficial in limiting blood loss at delivery. Fibrinogen and concentrations of factors VII, VIII, IX, X, and XII all increase; only factor XI levels may decrease. Accelerated fibrinolysis can be observed late in the third trimester. In addition to the dilutional anemia (see the section on Cardiovascular Effects), leukocytosis (up to 21,000/pL) and a 10% decrease in platelet count may be encountered during the third trimester. Because of fetal utilization, iron and folate deficiency anemias readily develop if supplements of these nutrients are not taken.

Complex metabolic and hormonal changes occur during pregnancy. Altered carbohydrate, fat, and protein metabolism favors fetal growth and development. These changes resemble starvation, because blood glucose and amino acid levels are low whereas free fatty acid, ketone, and triglyceride levels are high. Nonetheless, pregnancy is a diabetogenic state; insulin levels steadily rise during pregnancy. Secretion of human placental lactogen, also called human chorionic somatomammotropin, by the placenta is probably responsible for the relative insulin resistance associated with pregnancy. Pancreatic beta cell hyperplasia occurs in response to an increased demand for insulin secretion.
Secretion of human chorionic gonadotropin and elevated levels of estrogens promote hypertrophy of the thyroid gland and increase thyroid-binding globulin; although thyroxine (T,) and triiodothyronine (T3) levels are elevated, free Ty, free T3, and thyrotropin (thyroid-stimulating hormone) remain normal. Serum calcium levels decrease, but ionized calcium concentration remains normal.

Elevated levels of relaxin throughout pregnancy help prepare for delivery by softening the cervix, inhibiting uterine contractions, and relaxing the pubic symphysis and pelvic joints. Ligamentous laxity of the spine increases the risk of back injury. The latter may contribute to the relatively frequent occurrence of back pain during pregnancy.

A normal uteroplacental circulation (Figure 40-1) is critical in the development and maintenance of a healthy fetus. Uteroplacental insufficiency is an important cause of intrauterine fetal growth retardation, and when severe, can result in fetal demise. The integrity of this circulation is, in turn, dependent on both adequate uterine blood flow and normal placental function.
FIGURE 40-1 The uteroplacental circulation. (Reproduced with permission from Shnider S, Levinson G. Anesthesia for Obstetrics. 2nd ed. Philadelphia, PA: Williams & Wilkins; 1987.)
Uterine Blood Flow

The fetus is dependent on the placenta for respiratory gas exchange, nutrition, and waste elimination. The placenta is formed by both maternal and fetal tissues and derives a blood supply from each. The resulting exchange membrane has a functional area of about 1.8 m2.

The placenta (Figure 40—2) is composed of projections of fetal tissue (villi) that lie in maternal vascular spaces (intervillous spaces). As a result of this arrangement, the fetal capillaries within villi readily exchange substances with the maternal blood that bathes them. Maternal blood in the intervillous spaces is derived from spiral branches of the uterine artery and drains into the uterine veins. Fetal blood within villi is derived from the umbilical cord via two umbilical arteries and returns to the fetus via a single umbilical vein.

Placental exchange can occur by one of six mechanisms:
1. Diffusion—Respiratory gases and small ions are transported by diffusion. Most drugs used in anesthesia have molecular weights well under 1000 and consequently can readily diffuse across the placenta.
2. Osmotic and hydrostatic pressure (bulk flow)—Water moves across by osmotic and hydrostatic pressures. Water enters the fetal circulation in quantities greater than any other substance.
3. Facilitated diffusion—Glucose enters the fetal circulation down the concentration gradient (no energy is consumed) facilitated by a specific transporter molecule.
4. Active transport—Amino acids, vitamin B,5, fatty acids, and some ions (calcium and phosphate) utilize this mechanism.
- 5. Vesicular transport—Large molecules, such as immunoglobulins, are transported by pinocytosis. Iron also enters the fetal circulation in this way, facilitated by ferritin and transferrin.
6. Breaks—Breaks in the placental membrane may permit mixing of maternal and fetal blood. This probably underlies Rh sensitization (see Chapter 51). Rh sensitization occurs most commonly during delivery.

At term, fetal oxygen consumption averages about 7 mL/min/kg of fetal body weight. Fortunately, because of multiple adaptive mechanisms, the normal fetus at term can survive 10 min or longer instead of the expected 2 min in a state of total oxygen deprivation. Partial or complete oxygen deprivation can result from umbilical cord compression, umbilical cord prolapse, placental abruption, severe maternal hypoxemia, or hypotension. Compensatory fetal mechanisms include redistribution of blood flow primarily to the brain, heart, placenta, and adrenal gland; decreased oxygen consumption; and anaerobic metabolism.
Transfer of oxygen across the placenta is dependent on the ratio of maternal uterine blood flow to fetal umbilical blood flow. The reserve for oxygen transfer is small even during normal pregnancy. Normal fetal blood from the placenta has a Pao, of only 30 to 35 mm Hg. To aid oxygen transfer, the fetal hemoglobin oxygen dissociation curve is shifted to the left such that fetal hemoglobin has greater affinity for oxygen than does maternal hemoglobin (whose curve is already shifted to the right; see the section on Respiratory Effects). In addition, fetal hemoglobin concentration is usually 15 g/dL (compared with approximately 12 g/dL in the mother).
Carbon dioxide readily diffuses across the placenta. Maternal hyperventilation (see the section on Respiratory Effects) increases the gradient for the transfer of carbon dioxide from the fetus into the maternal circulation.
Fetal hemoglobin has less affinity for carbon dioxide than do adult forms of hemoglobin. Carbon monoxide readily diffuses across the placenta, and fetal hemoglobin has greater affinity for carbon monoxide than do adult forms.

Transfer of a drug across the placenta is reflected by the ratio of its fetal umbilical vein to maternal venous concentrations (UV/MV), whereas its uptake by fetal tissues can be correlated with the ratio of its fetal umbilical artery to umbilical vein concentrations (UA/UV). Fetal effects of drugs administered to parturients depend on multiple factors, including route of administration (oral, intramuscular, intravenous, epidural, or intrathecal), dose, timing of administration (both relative to delivery as well as contractions), and maturity of the fetal organs (brain and liver). Thus, a drug given hours before delivery or as a single intravenous bolus during a uterine contraction just prior to delivery (when uterine blood flow is maximally reduced) is unlikely to produce high fetal levels. Fortunately, current-anesthetic techniques for labor and delivery generally have minimal fetal effects despite significant placental transfer of anesthetic agents and adjuncts.

All inhalational agents and most intravenous agents freely cross the placenta. Inhalational agents generally produce little fetal depression when they are given in limited doses (<1 MAC) and delivery occurs within 10 min of induction. Ketamine, propofol, and benzodiazepines readily cross the placenta and can be detected in the fetal circulation. Fortunately, when these agents (with the exception of benzodiazepines) are administered in usual induction doses, drug distribution, metabolism, and possibly placental uptake may limit fetal effects. Although most opiates readily cross the placenta, their effects on neonates at delivery vary considerably. Newborns appear to be more sensitive to the respiratory depressant effect of morphine compared with other opioids. Although meperidine produces respiratory depression, peaking 1 to 3 h after administration, it produces less than morphine; butorphanol and nalbuphine produce even less respiratory depression but still may have significant neurobehavioral depressant effects. Although fentanyl readily crosses the placenta, it appears to have minimal neonatal effects unless larger intravenous doses (>1 mcg/kg) are given immediately before delivery. Epidural or intrathecal fentanyl, sufentanil, and, to a lesser extent, morphine, generally produce minimal neonatal effects. Alfentanil causes neonatal depression similar to meperidine. Remifentanil also readily crosses the placenta and has the potential to produce respiratory depression in newborns. Fetal blood concentrations of remifentanil are generally about half those of the mother just prior to delivery. The UA/UV ratio is about 30%, suggesting fairly rapid metabolism of remifentanil in the neonate. The highly ionized nature of muscle relaxants impedes placental transfer, resulting in minimal effects on the fetus.

Local anesthetics are weakly basic drugs that are principally bound to a,-acid glycoprotein. Placental transfer depends on three factors: (1) pK, (see Chapter 16), (2) maternal and fetal pH, and (3) degree of protein binding. Except for chloroprocaine, fetal acidosis increases fetal-to-maternal drug ratios because binding of hydrogen ions to the nonionized form causes trapping of the local anesthetic in the fetal circulation. Highly protein-bound agents diffuse slowly across the placenta; thus, greater protein binding of bupivacaine and ropivacaine, compared with that of lidocaine, likely accounts for their lower fetal blood levels. Chloroprocaine has the least placental transfer because it is rapidly hydrolyzed by plasma cholinesterase in the maternal circulation.
Most commonly used anesthetic adjuncts also readily cross the placenta. Thus, maternally administered ephedrine, B-adrenergic blockers (such as labetalol and esmolol), vasodilators, phenothiazines, antihistamines (H, and H)), and metoclopramide are transferred to the fetus. Atropine and scopolamine, but not glycopyrrolate, cross the placenta; the latter's quaternary ammonium (ionized) structure results in only limited transfer.

Intravenous anesthetic agents have variable effects on uteroplacental blood flow. Propofol and barbiturates are typically associated with small reductions in uterine blood flow due to dose-dependent decreases in maternal blood pressure. A small induction dose, however, can produce greater reductions in blood flow as a result of sympathoadrenal activation (due to light anesthesia). Ketamine in doses of less than 1.5 mg/kg does not appreciably alter uteroplacental blood flow; its hypertensive effect typically counteracts any vasoconstriction. Uterine hypertonus may occur with ketamine at doses of more than 2 mg/kg. Etomidate likely has minimal effects, but its actions on uteroplacental circulation have not been well described.
@ Volatile inhalational anesthetics decrease blood pressure and, potentially, uteroplacental blood flow. In concentrations of less than 1 MAC, however, their effects are generally minor, consisting of dose-dependent uterine relaxation and minor reductions in uterine blood flow. Nitrous oxide has minimal effects on uterine blood flow when administered with a volatile agent. In animal studies, nitrous oxide alone can vasoconstrict the uterine arteries.
High blood levels of local anesthetics—particularly lidocaine—cause uterine arterial vasoconstriction. Such levels are seen only with unintentional intravascular injections and occasionally following paracervical blocks (in which the injection site is in close proximity to the uterine arteries), and local absorption or injection into these vessels cannot be ruled out). Spinal and epidural anesthesia typically do not decrease uterine blood flow except when arterial hypotension occurs. Moreover, uterine blood flow during labor may actually improve in preeclamptic patients following epidural anesthesia; a reduction in circulating endogenous catecholamines likely decreases uterine vasoconstriction. The addition of dilute concentrations of epinephrine to local anesthetic solutions does not appreciably alter uterine blood flow. Intravascular uptake of the epinephrine from the epidural space may result in only minor systemic B-adrenergic effects.

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True labor begins when the sporadic Braxton Hicks contractions increase in strength (25-60 mm Hg), coordination, and frequency (15-20 min apart). Amniotic membranes may rupture spontaneously prior or subsequent to the onset of true labor. Following progressive cervical dilation, the contractions propel first the fetus and then the placenta through the pelvis and perineum. By convention, labor is divided into three stages. The first stage is defined by the onset of true labor and ends with complete cervical dilation. The second stage begins with full cervical dilation, is characterized by fetal descent, and ends with complete delivery of the fetus. Finally, the third stage extends from the birth of the baby to the delivery of the placenta.

Based on the rate of cervical dilation, the first stage is further divided into a slow latent phase followed by a faster active phase (Figure 40—3). The latent phase is characterized by progressive cervical effacement and minor dilation (2— 4 cm). The subsequent active phase is characterized by more frequent contractions (3-5 min apart) and progressive cervical dilation up to 10 cm. The first stage usually lasts 8 to 12 h in nulliparous patients and about 5 to 8 h in multiparous patients.
FIGURE 40-3 The course of normal labor. (Reproduced with permission from DeCherney AH, Pernoll ML. Current Obstetric & Gynecologic Diagnosis & Treatment. 9th ed. New York, NY: McGraw-Hill; 2001.)
Contractions during the second stage occur 1.5 to 2 min apart and last 1 to 1.5 min. Although contraction intensity does not appreciably change, the parturient, by bearing down, can greatly augment intrauterine pressure and facilitate expulsion of the fetus. The second stage usually lasts 15 to 120 min and the third stage typically 15 to 30 min.
The course of labor is monitored by uterine activity, cervical dilation, and fetal descent. Uterine activity refers to the frequency and magnitude of uterine contractions. The latter may be measured directly, with a catheter inserted through the cervix, or indirectly, with a tocodynamometer applied externally around the abdomen. Cervical dilation and fetal descent are assessed by pelvic examination. Fetal station refers to the level of descent (in centimeters) of the presenting part relative to the ischial spines (eg, —1 or +1).

During intense painful contractions, maternal minute ventilation may increase up to 300%. Oxygen consumption also increases by an additional 60% above thirdtrimester values. With excessive hyperventilation, Paco, may decrease below 20 mm Hg. Marked hypocapnia can cause periods of hypoventilation and transient maternal and fetal hypoxemia between contractions. Excessive maternal hyperventilation also reduces uterine blood flow and promotes fetal acidosis. Each contraction places an additional burden on the heart by displacing 300 to 500 mL of blood from the uterus into the central circulation (analogous to an autotransfusion). Cardiac output rises 45% over @ third-trimester values. The greatest strain on the parturient's heart, however, occurs immediately after delivery, when intense uterine contraction and involution suddenly relieve inferior vena caval obstruction and increase cardiac output as much as 80% above late third-trimester values.

Sevoflurane, desflurane, isoflurane, and halothane depress uterine activity equally at equipotent doses; all cause dose-dependent uterine relaxation. Low doses (<0.75 MAC) of these agents, however, do not interfere with the effect of oxytocin on the uterus. Higher doses can result in uterine atony and increase blood loss at delivery. Nitrous oxide has minimal, if any, effects.

Opioids minimally decrease the progression of labor; ketamine, in doses of less than 2 mg/kg, appears to have little effect.

The administration of epidural analgesia is usually based upon the patient's choice, and it is often utilized for patients with maternal or fetal factors that increase the likelihood of prolonged labor or cesarean delivery (Table 40-2).
Current evidence indicates @ that dilute combinations of a local anesthetic (eg, bupivacaine, <0.125%) and an opioid (eg, fentanyl, <5 mcg/mL) for epidural or combined spinal—epidural (CSE) analgesia do not prolong labor or increase the likelihood of operative delivery.
TABLE 40-2 Factors that prolong labor, increase the likelihood of cesarean section, and often cause patients to request an epidural.
Primigravida
Prolonged labor
High parenteral analgesic requirements
Use of oxytocin
Large baby
Small pelvis
Fetal malpresentation
When greater concentrations of local anesthetic (eg, bupivacaine, 0.25%) are used for continuous epidural analgesia, the second stage of labor may be prolonged by approximately 15 to 30 min. Intense regional analgesia/anesthesia can remove the urge to bear down during the second stage (Ferguson reflex), and motor weakness can impair expulsive efforts, often prolonging the second stage of delivery. Use of dilute local anesthetic-opioid mixtures can preserve motor function and allow effective pushing. Intravenous fluid loading (crystalloid boluses) is often used to reduce the severity of hypotension following an epidural or subarachnoid injection, and the prophylactic infusion of phenylephrine started at the time of intrathecal local anesthetic injection is effective in preventing post-spinal hypotension. Fluid loading before a block in a euvolemic patient does not reduce the incidence of hypotension and has been shown to reduce endogenous oxytocin secretion from the pituitary and transiently decrease uterine activity. Epinephrine-containing local anesthetic solutions could theoretically prolong the first stage of labor if absorption of epinephrine from the epidural space results in significant systemic B-adrenergic effects. Prolongation of labor is generally not clinically observed with very dilute (eg, 1:400,000) epinephrine-containing local anesthetics.

Uterine muscle has both a and B receptors. a,-Receptor stimulation causes uterine contraction, whereas B,-receptor stimulation produces relaxation. Large doses of a-adrenergic agents, such as phenylephrine, in addition to causing uterine arterial constriction, can produce tetanic uterine contractions. Small doses of phenylephrine (40 mcg) may increase uterine blood flow in normal parturients by raising arterial blood pressure. In contrast, ephedrine has little effect on uterine contractions.

Oxytocin (Pitocin) is administered intravenously to induce or augment uterine contractions or to maintain uterine tone postpartum. It has a half-life of 3 to 5 min. Induction doses for labor are 0.5 to 8 mU/min. Complications include fetal distress due to hyperstimulation, uterine tetany, and, less commonly, maternal water retention (antidiuretic effect). Rapid intravenous infusion can cause transient systemic hypotension due to relaxation of vascular smooth muscle; reflex tachycardia may also occur. Uterine is the of atony most common cause severe postpartum hemorrhage. Immediate administration of oxytocin after delivery is a standard measure to prevent this complication. Despite this practice, uterine atony complicates 4% to 6% of pregnancies. The concentration of volatile anesthetics should be reduced to 0.5 MAC in obstetric patients undergoing general anesthesia for cesarean delivery to avoid the uterine-relaxing effects of these drugs. Second-line oxytocics are methylergonovine (Methergine) and carboprost tromethamine (Hemabate).

Methylergonovine causes intense and prolonged uterine contractions. It is therefore given only after delivery (postpartum) to treat uterine atony. Moreover, because it also constricts vascular smooth muscle and can cause severe hypertension if given as an intravenous bolus, it is usually administered only as a single 0.2 mg dose intramuscularly or in dilute form as an intravenous infusion over 10 minutes.

Carboprost tromethamine (Hemabate, prostaglandin F,,) is a synthetic analogue of prostaglandin F., that stimulates uterine contractions. It is often used to treat refractory postpartum hemorrhage. An initial dose of 0.25 mg intramuscularly may be repeated every 15 to 90 min to a maximum of 2 mg. Common side effects include nausea, vomiting, bronchoconstriction, and diarrhea. It is contraindicated in patients with asthma. Prostaglandin E, (Cytotec, rectal suppository) or E, (Dinoprostone, vaginal suppository) is sometimes administered and has no bronchoconstricting effect.

Magnesium is used in obstetrics both to stop premature labor (tocolysis) and to prevent eclamptic seizures. It is usually administered as a 4 g intravenous loading dose (over 20 min) followed by a 2 g/h infusion. Therapeutic serum levels are considered to be 6 to 8 mg/dL. Serious side effects include hypotension, heart block, muscle weakness, and sedation. Magnesium in these doses and concentrations intensifies neuromuscular blockade from nondepolarizing agents.

The B,-adrenergic agonists ritodrine and terbutaline inhibit uterine contractions and are used to treat premature labor.

The placenta, which receives nearly half the fetal cardiac output, is responsible for respiratory gas exchange. The fetal lungs receive little blood flow and the pulmonary and systemic circulations are parallel instead of in series, as in the adult (Figures 40—4 and 40-5). This arrangement is made possible by two cardiac shunts—the foramen ovale and the ductus arteriosus:
1. Well-oxygenated blood from the placenta (approximately 80% oxygen saturation) mixes with venous blood returning from the lower body (25% oxygen saturation) and flows via the inferior vena cava into the right atrium.
2. Right atrial anatomy preferentially directs blood flow from the inferior vena cava (67% oxygen saturation) through the foramen ovale into the left atrium.
3. Left atrial blood is then pumped by the left ventricle to the upper body (mainly the brain and the heart).
4. Poorly oxygenated blood from the upper body returns via the superior vena cava to the right atrium.
5. Right atrial anatomy preferentially directs flow from the superior vena cava

6. Right ventricular blood is pumped into the pulmonary artery.
7. Because of high pulmonary vascular resistance, 95% of the blood ejected from the right ventricle (60% oxygen saturation) is shunted across the ductus arteriosus, into the descending aorta, and back to the placenta and lower body.
FIGURE 40-4 The fetal circulation before and after birth. (Reproduced with permission from Ganong WEF. Review of Medical Physiology. 24th ed. New York, NY: McGraw-Hill; 2012.)
FIGURE 40-5 A schematic comparison of fetal and neonatal circulation. (Reproduced with permission from Danforth DN, Scott JR. Obstetrics and Gynecology. 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 1986.)
The parallel circulation results in unequal ventricular flows; the right ventricle ejects two-thirds of the combined ventricular outputs, whereas the left ventricle ejects only one-third.
Up to 50% of the well-oxygenated blood in the umbilical vein can pass directly to the heart via the ductus venosus, bypassing the liver. The remainder of the blood flow from the placenta mixes with blood from the portal vein (via the portal sinus) and passes through the liver before reaching the heart. The latter may be important in allowing relatively rapid hepatic degradation of drugs (or toxins) that are absorbed from the maternal circulation.
In contrast to the fetal circulation, which is established very early during intrauterine life, maturation of the lungs lags behind. Extrauterine survival is not possible until after 22 to 24 weeks of gestation, when pulmonary capillaries are formed and come to lie in close approximation to an immature alveolar epithelium. At 30 weeks, the cuboidal alveolar epithelium flattens out and begins to produce pulmonary surfactant. This substance provides alveolar stability and is necessary to maintain normal lung expansion after birth (see Chapter 23). Sufficient pulmonary surfactant is usually present after 34 weeks of gestation. Administration of glucocorticoids to the mother may accelerate fetal surfactant production.

The most profound adaptive changes at birth involve the circulatory and respiratory systems. Failure to make this transition successfully results in fetal death or permanent neurological damage.
At term, the fetal lungs are developed but contain about 90 mL of a plasma ultrafiltrate. During expulsion of the fetus at delivery, this fluid is normally squeezed from the lungs by the forces of the pelvic muscles and the vagina acting on the fetus (the vaginal squeeze). Any remaining fluid is reabsorbed by the pulmonary capillaries and lymphatics. Small (preterm) neonates and neonates delivered via cesarean section do not benefit from the vaginal squeeze and thus typically have greater difficulty in maintaining respirations (transient tachypnea of the newborn). Respiratory efforts are normally initiated within 30 s after birth and become sustained within 90 s. Mild hypoxia and acidosis as well as sensory stimulation—cord clamping, pain, touch, and noise—help initiate and sustain respirations, whereas the outward recoil of the chest at delivery aids in filling the lungs with air.
Lung expansion increases both alveolar and arterial oxygen tensions and decreases pulmonary vascular resistance. The increase in oxygen tension is a potent stimulus for pulmonary arterial vasodilation. The resultant increase in pulmonary blood flow and augmented flow to the left heart elevates left atrial pressure and functionally closes the foramen ovale. The increase in arterial oxygen tension also causes the ductus arteriosus to contract and functionally close. Other chemical mediators that may play a role in ductal closure include acetylcholine, bradykinin, and prostaglandins. The overall result is elimination of right-to-left shunting and establishment of the adult circulation (Figure 40-5). Anatomic closure of the ductus arteriosus does not usually occur until about 2 to 3 weeks, whereas closure of the foramen ovale takes months if it occurs at all. right-to-
Hypoxia or acidosis during the first few days of life can prevent or reverse these physiological changes, resulting in persistence of (or return to) the fetal circulation, or persistent pulmonary hypertension of the newborn. The left shunting promotes hypoxemia and acidosis, which in turn promote more shunting (Figure 40-6). Right-to-left shunting may occur across the foramen ovale, the ductus arteriosus, or both.

FIGURE 40-6 Pathophysiology of persistent pulmonary hypertension of the newborn (persistent fetal circulation).

A 36-year-old woman is scheduled for bilateral tubal ligation 12 h after delivery of a healthy baby.
Is this patient still at increased risk for pulmonary aspiration?
Controversy exists over when the increased risk for pulmonary aspiration diminishes following pregnancy. Certainly, many factors contributing to delayed gastric emptying are alleviated shortly after delivery: mechanical distortion of the stomach is relieved, labor pains cease, and the circulating progesterone level rapidly declines. Gastric volume and acidity usually do not differ in pregnant compared with nonpregnant women, although 30% to 60% of pregnant patients have a gastric volume greater than 25 mL or a gastric fluid pH less than 2.5. In these parturients, gastric volume and gastric fluid pH (see the section on Renal & Gastrointestinal Effects) normalize within 24 h after delivery. Therefore, most clinicians still consider the immediate postpartum patient to be at increased risk for pulmonary aspiration and take appropriate precautions (see Chapters 17 and 41). It is not known when the risk returns to the level associated with elective surgical patients. Although some physiological changes associated with pregnancy may require up to 6 weeks for resolution, the increased risk of pulmonary aspiration probably returns to "normal" well before that time.
Other than aspiration risk, what factors determine the "optimal" time for postpartum sterilization?
The decision about when to perform postpartum tubal ligation (or laparoscopic fulguration) is complex and varies according to patient and obstetrician preferences as well as local practices. Factors influencing the decision include whether the patient had a vaginal or cesarean delivery and whether an anesthetic was administered for labor (epidural anesthesia) or delivery (epidural or general anesthesia).
Postpartum tubal ligation or fulguration may be (1) performed immediately following delivery of the baby and repair of the uterus during a cesarean section, (2) delayed 8 to 48 h following delivery to allow an elective fasting period, or (3) deferred until after the postpartum period (generally 6 weeks). Sterilization is technically easier to perform in the immediate postpartum period because of the enlargement of the uterus and tubes. Postpartum sterilizations following natural vaginal delivery are generally performed within 48 h of delivery.
What factors determine selection of an anesthetic technique for postpartum sterilization?

When continuous epidural anesthesia is administered for labor and vaginal delivery, the epidural catheter may be left in place up to 48 h for subsequent tubal ligation. The delay allows a period of elective fasting. A T4_—5 sensory level with regional anesthesia is usually necessary to ensure a pain-free anesthetic experience. Lower sensory levels (as low as T10) may be adequate but sometimes fail to prevent pain caused by surgical traction on viscera.
When the patient has not had anesthesia for delivery, postpartum sterilization may be performed under either regional or general anesthesia. Because of the increased risk of pulmonary aspiration, regional anesthesia usually is preferred for bilateral tubal ligation via a minilaparotomy. Many clinicians prefer spinal over epidural anesthesia in this setting because of its greater speed of onset and reliability (see Chapter 45). In addition, the incidence of postdural puncture headache is as low as 1% when a 25-gauge or smaller pencil-point needle is used. Dosage requirements for regional anesthesia generally return to normal within 24 to 36 h after delivery. Bupivacaine (8-12 mg) or lidocaine (60—75 mg) may be used for spinal anesthesia. For epidural anesthesia, 15 to 30 mL of lidocaine 1.5% to 2% or chloroprocaine 3% is most commonly used.
In contrast, when laparoscopic tubal fulguration is planned, general endotracheal anesthesia is preferred. Insufflation of gas during laparoscopy impairs pulmonary gas exchange and predisposes the patient to nausea, vomiting, and possibly pulmonary aspiration. Endotracheal intubation generally ensures adequate ventilation and protects the airway.
What considerations are important for postpartum patients undergoing general anesthesia?

Preoperative concerns include the persistent increased risk of pulmonary aspiration. Anemia is nearly always present as a result of the physiological effects of pregnancy combined with blood loss during and following delivery. Hemoglobin concentrations are usually greater than 9 g/dL, but levels as low as 7 g/dL are generally considered safe. Fortunately, sterilization procedures are rarely associated with significant blood loss. The risk of pulmonary aspiration is diminished by a minimum of 8 h of fasting, premedication with an H, blocker (ranitidine), a clear antacid (sodium citrate), or metoclopramide (see Chapters 17 and 41). In addition, induction of anesthesia should employ a rapid-sequence technique prior to endotracheal intubation, and the patient should be extubated only when she is awake. Decreased plasma cholinesterase levels persist after delivery (see

Chestnut DH, Polley LS, Tsen LC, et al. Chestnut's Obstetric Anesthesia:
Principals and Practice. 5th ed. St Louis, MO: Mosby; 2014.
Suresh M. Shnider and Levinson's Anesthesia for Obstetrics. 5th ed.
Philadelphia, PA: Lippincott, Williams & Wilkins; 2013.
Wanderer JP, Rathmell JP. Infographics in anesthesiology: Anesthesia &
breastfeeding: More often than not, they are compatible. Anesthesiology.
2017;127(4). Available at http://anesthesiology.pubs.asahq.org/article.aspx? articleid=2654616

This chapter focuses on the practice of obstetric anesthesia. Techniques for analgesia and anesthesia during labor, vaginal delivery, and cesarean section are presented. The chapter concludes with a review of neonatal resuscitation.
Although the majority of women of childbearing age would be considered to be at minimal operative risk, pregnancy, certain maternal—fetal factors, and preexisting medical conditions significantly increase surgical and obstetric risks.

Maternal mortality is usually presented as the number of women who die while pregnant or within 42 days of pregnancy termination after excluding accidents and unrelated causes. This number is often indexed to the total number of live births. The maternal mortality index has decreased nearly 100-fold since 1900. Likely due to better reporting, it rose slightly in the United States to 21 deaths per 100,000 live births in 2010. The world average is 400 deaths per 100,000 live births. Of all maternal deaths worldwide, 99% occur in Africa, Asia, Latin America, and the Caribbean.
In the United States, overall mortality risk is greater for women older than 35 years of age, African-American women, and women who do not receive prenatal care. The leading causes of death associated with a live birth in 2010 were cardiovascular diseases (14%), cardiomyopathy (13%), hemorrhage (12%), noncardiovascular diseases (12%), hypertensive disorders of pregnancy (11%), infection/sepsis (11%), thrombotic pulmonary embolism (6%), amniotic fluid embolism (6%), cerebrovascular accidents (5%) and anesthesia-related complications (<1%). Of all maternal deaths, only 34% of patients die within 24 h of delivery, whereas 55% die between 1 and 42 days, and another 11% die between 43 days and 1 year. Direct causes of maternal deaths are more completely described in Canadian data, which show that, in addition to pulmonary embolism and preeclampsia/pregnancy-induced hypertension (PIH), amniotic fluid embolism and intracranial hemorrhage are also important additional causes of death.
Severe obstetric morbidity may be a more useful measure of peripartum outcome than maternal mortality. Data from the United Kingdom suggest that incidence of severe obstetric morbidity is 12 per 1000 deliveries, 100 times more common than mortality. Risk factors include age greater than 34 years, nonwhite ethnicity, multiple pregnancy, history of hypertension, previous postpartum hemorrhage, and emergency cesarean delivery. Table 41—1 lists the estimated incidence of the most common causes of severe morbidity; thromboembolic disease was deliberately excluded because of the difficulty in @ making the diagnosis in nonfatal cases. By far the most common morbidities encountered in obstetrics are severe hemorrhage and severe preeclampsia.

Severe hemorrhage, Incidence per 1000 = 6.7. Severe preeclampsia, Incidence per 1000 = 3.9. HELLP syndrome?’, Incidence per 1000 = 0.5. Severe sepsis, Incidence per 1000 = 0.4. Eclampsia, Incidence per 1000 = 0.2. Uterine rupture, Incidence per 1000 = 0.2
'Data from Waterstone M, Bewley S, Wolfe C. Incidence and predictors of severe obstetric morbidity: Case-control study. BMJ. 2001 May 5;322(7294):1089-1093.
*Note thromboembolic disease was excluded.
5HELLP syndrome consists of hemolysis, elevated liver enzymes, and low platelet count.

Data collected between 1985 and 1990 suggested a maternal mortality of 32 deaths per 1,000,000 live births due to general anesthesia and 1.9 deaths per 1,000,000 live births due to regional anesthesia. More recent data between 2006 and 2010 suggest a lower overall maternal mortality from anesthesia (an estimated 0.9% of pregnancy-related deaths), possibly due to greater use of regional anesthesia for labor and cesarean delivery. Most deaths occur during or after cesarean section. Moreover, the risk of an adverse outcome is much greater with emergent than with elective cesarean sections.

Obstetric anesthesia care accounts for approximately 12% of the American Society of Anesthesiologists (ASA) Closed Claims database claims. A comparison of obstetric anesthesia claims from 1990 to 2003 with pre-1990 claims shows a decrease in maternal deaths, as well as a decrease in airwayrelated events (aspiration, difficult intubation, esophageal intubation, and inadequate oxygenation/ventilation). Although newborn deaths and brain damage also decreased over this period, they remained a leading cause of obstetric anesthesia malpractice claims. Maternal nerve injury was more common in claims reported after 1990 compared with earlier years.

entering the
obstetric suite
potentially require
anesthesia
Patients requiring
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care evaluation
health for
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history, or
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® Regardless of the time of last oral intake, all patients are considered to have a full stomach and to be at risk for pulmonary aspiration. Because the duration of labor is often prolonged, guidelines usually allow small amounts of oral clear liquid during uncomplicated labor. The minimum fasting period for elective cesarean section remains controversial but is typically recommended to be 6 h for light meals and 8 h for heavy meals. Prophylactic administration of a clear antacid (15-30 mL of 0.3 M sodium citrate orally) every 30 min prior to a cesarean section may help maintain gastric pH greater than 2.5 and may decrease the likelihood of severe aspiration pneumonitis. An H,-blocking drug (eg, ranitidine, 100-150 mg orally or 50 mg intravenously) or metoclopramide, 10 mg orally or intravenously, should also be considered in high-risk patients and in those expected to receive general anesthesia. H, blockers reduce both gastric volume and pH but have no effect on the gastric contents already present. Metoclopramide accelerates gastric emptying, decreases gastric volume, and increases lower esophageal sphincter tone. The supine position should be avoided unless a left uterine displacement device (>15° wedge) is placed under the right hip.

The pain of labor arises from contraction of the myometrium against the resistance of the cervix and perineum, progressive dilation of the cervix and lower uterine segment, and stretching and compression of pelvic and perineal structures. Discomfort during the first stage of labor is primarily visceral pain resulting from uterine contractions and cervical dilation. It is usually initially confined to the T11—-T12 dermatomes during the latent phase, but eventually involves the T10-L1 dermatomes as labor enters the active phase. The visceral afferent fibers responsible for labor pain travel with sympathetic nerve fibers first to the uterovaginal plexus, then through the inferior hypogastric plexus, before entering the spinal cord with the T10—-L1 nerve roots. The pain is initially perceived in the lower abdomen but may increasingly be referred to the lumbosacral area, gluteal region, and thighs as labor progresses. Pain intensity also increases with progressive cervical dilation and with increasing intensity and frequency of uterine contractions. Nulliparous women typically experience greater pain during the first stage of labor.
The onset of perineal pain at the end of the first stage signals the beginning of fetal descent and the second stage of labor. Stretching and compression of pelvic and perineal structures intensifies the pain. Sensory innervation of the perineum is provided by the pudendal nerves (S2-4), so pain during the second stage of labor involves the T10-S4 dermatomes.

Psychological analgesic techniques include those of Bradley, Dick-Read, Lamaze, and LeBoyer. Patient education and positive conditioning about the birthing process are central to such techniques. Pain during labor tends to be accentuated by fear of the unknown or by previous unpleasant experiences. The Lamaze technique, one of the most popular, coaches the parturient to take a deep breath at the beginning of each contraction followed by rapid, shallow breathing for the duration of the contraction. The parturient also concentrates on an object in the room and attempts to focus her thoughts away from the pain. Less common nonpharmacological techniques include hypnosis, transcutaneous electrical nerve stimulation, biofeedback, and acupuncture. The success of all these techniques varies considerably from patient to patient, and many patients require additional forms of analgesia.

® Nearly all parenteral opioid analgesics and sedatives readily cross the placenta and can affect the fetus. Concern regarding fetal depression limits the use of these agents to the early stages of labor or to situations in which regional
antiinflammatory agents, such as ketorolac, are not recommended as antepartum therapy because they suppress uterine contractions and promote closure of the fetal ductus arteriosus.
Small doses (up to 2 mg intravenously) of midazolam (Versed) may be administered in combination with a small dose of fentanyl (up to 100 mcg intravenously) in healthy parturients at term to facilitate the analgesic effect of neuraxial blockade. At this dose, maternal amnesia has not been observed. Chronic administration of the longer-acting benzodiazepine diazepam (Valium) has been associated with fetal depression.
Low-dose intravenous ketamine is a powerful analgesic. In doses of 10 to 15 mg intravenously, good analgesia can be obtained in 2 to 5 min without loss of consciousness. Large boluses of ketamine (>1 mg/kg) can be associated with hypertonic uterine contractions. Low-dose ketamine is most useful just prior to delivery or as an adjuvant to regional anesthesia (see Chapter 9).
In the past, reduced concentrations of volatile anesthetic agents (eg, methoxyflurane) in oxygen were sometimes used for relief of milder labor pain. Inhalation of nitrous oxide—oxygen remains in common use for relief of mild labor pain. As previously noted, nitrous oxide has minimal effects on uterine blood flow or uterine contractions.

Pudendal nerve blocks are often combined with perineal infiltration of local anesthetic to provide perineal anesthesia during the second stage of labor when other forms of anesthesia are not employed or prove to be inadequate. Paracervical plexus blocks are no longer used because of their association with a relatively high rate of fetal bradycardia; the close proximity of the injection site to the uterine artery may result in uterine arterial vasoconstriction, uteroplacental insufficiency, and increased levels of the local anesthetic in the fetal blood. During a pudendal nerve block, a special needle (Koback) or guide (Iowa trumpet) is used to place the needle transvaginally underneath the ischial spine on each side; the needle is advanced 1 to 1.5 cm through the sacrospinous ligament, and 10 mL of 1% lidocaine or 2% chloroprocaine is injected following negative needle aspiration. The needle guide is used to limit the depth of injection and protect the fetus and vagina from the needle. Other potential complications include intravascular injection, retroperitoneal hematoma, and retropsoas or subgluteal abscess.

Epidural or intrathecal techniques, alone or in combination, are currently the most popular methods of pain relief during labor and delivery. They can provide excellent analgesia while allowing the @ mother to be awake and cooperative during labor. Although spinal opioids or local anesthetics alone can provide adequate analgesia, techniques that combine the two have proved to be the most satisfactory in most parturients. Moreover, the synergy between opioids and local anesthetics decreases dose requirements and provides excellent analgesia with few maternal side effects and little or no neonatal depression.

Opioids may be given intrathecally as a single injection or intermittently via an epidural or intrathecal catheter (Table 41—2). Relatively large doses are required for analgesia during labor when epidural or intrathecal opioids are used alone. For example, the EDs, during labor is 124 mcg for epidural fentanyl and 21 mcg for epidural sufentanil. The higher doses may be associated with a high risk of side effects, most importantly respiratory depression. For this reason, combinations of local anesthetics and opioids are most commonly used (see later discussion). Pure opioid techniques are most useful for high-risk patients who may not tolerate the functional sympathectomy associated with spinal or epidural anesthesia (see Chapter 45). This group includes patients with hypovolemia or significant cardiovascular disease such as moderate to severe aortic stenosis, tetralogy of Fallot, Eisenmenger syndrome, or pulmonary hypertension. With the exception of meperidine, which has local anesthetic properties, spinal opioids alone do not produce motor blockade or sympathectomy. Thus, they do not impair the ability of the parturient to "push." Disadvantages include less complete analgesia, lack of perineal relaxation, and side effects such as pruritus, nausea, vomiting, sedation, and respiratory depression. Side effects may be ameliorated with low doses of naloxone (0.1—0.2 mg/h intravenously).
TABLE 41-2 Spinal opioid dosages for labor and delivery.
Morphine, Intrathecal = 0.1-0.5 mg. Morphine, Epidural = 5mg. Meperidine, Intrathecal = 10-15 mg. Meperidine, Epidural = 50-100 mg. Fentanyl, Intrathecal = 10-25 mcg. Fentanyl, Epidural = 50-150 mcg. Sufentanil, Intrathecal = 3-10 mcg. Sufentanil, Epidural = 10-20 mcg

Intrathecal morphine in doses of 0.1 to 0.3 mg may produce satisfactory and prolonged (4-6 h) analgesia during the first stage of labor. Unfortunately, the onset of analgesia is slow (45-60 min), and these doses may not be sufficient in many patients. However, higher doses are associated with a relatively high incidence of side effects. Morphine is therefore rarely used alone. The combination of morphine, 0.1 to 0.25 mg, and fentanyl, 12.5 mcg (or sufentanil, 5 mcg), may result in a more rapid onset of analgesia (5 min). Intermittent boluses of 10 to 15 mg of meperidine, 12.5 to 25 mcg of fentanyl, or 3 to 10 mcg of sufentanil via an intrathecal catheter can also provide satisfactory analgesia for labor. Early reports of fetal bradycardia following intrathecal opioid injections (eg, sufentanil) have not been confirmed by subsequent studies. Hypotension following administration of intrathecal opioids for labor may be due to the resultant analgesia and decreased circulating catecholamine levels.

Relatively large doses (=>7.5 mg) of epidural morphine are required for satisfactory labor analgesia but are not recommended because of the increased risk of delayed respiratory depression and because the resultant analgesia is effective only in the early first stage of labor. Onset may take 30 to 60 min but analgesia lasts up to 12 to 24 h (as does the risk of delayed respiratory depression). Epidural meperidine, 50 to 100 mg, provides good, but relatively brief, analgesia (1 to 3 h). Epidural fentanyl, 50 to 150 mcg, or sufentanil, 10 to 20 mcg, usually produces analgesia within 5 to 10 min with few side effects, but it has a short duration (1-2 h). Although "single-shot" epidural opioids do not appear to cause significant neonatal depression, caution should be exercised following repeated administrations. Combinations of a lower dose of morphine,
2.5 mg, with fentanyl, 25 to 50 mcg (or sufentanil, 7.5-10 mcg), may result in a more rapid onset and prolongation of analgesia (4—5 h) with fewer side effects.
intravenous fluids, ephedrine, atropine, propofol, and succinylcholine. The ability to monitor blood pressure and heart rate is mandatory. A pulse oximeter and capnograph should be readily available, and ready availability of equipment such as a video laryngoscope or an intubating laryngeal mask airway device, for use with a difficult airway, is advisable.

Epidural analgesia for labor may be administered in early labor after the patient has been evaluated by @ her obstetrician. Epidural analgesia does not increase the rate of operative delivery and has little if any effect on the progress of labor when dilute mixtures of a local anesthetic and an opioid are used. Concerns that regional analgesia will increase the likelihood of oxytocin augmentation, operative (eg, forceps) delivery, or cesarean section, are unjustified. It is often advantageous to place an epidural catheter early in labor, when the patient is less uncomfortable and can be positioned more easily. Moreover, should an urgent or emergent cesarean section become necessary, the presence of a functioning epidural catheter makes it possible to avoid general anesthesia.

for
Parturients may be positioned on their sides or in the sitting position for the procedure. The sitting position often makes it easier to identify the midline and spine, particularly in obese patients. When epidural anesthesia is being given vaginal delivery (second stage of labor), the sitting position may promote sacral spread.

Identification of the epidural space can be difficult, and unintentional dural puncture will sometimes occur even in experienced hands; the incidence of "wet taps" in obstetric patients is 0.25% to 9%, depending on clinician experience. Many practitioners add a compressible air bubble to the saline syringe and bounce the plunger to ensure that it moves freely and does not stick to the syringe wall (Figure 41—-1A and C). Most clinicians advocate the midline approach, whereas a minority favors the paramedian approach. For the placement of a lumbar epidural catheter in the obstetric patient, most anesthesiologists advance the epidural needle with the left hand, which is braced against the patient's back, while applying continuous pressure to the plunger of a glass syringe filled with sterile saline (Figure 41—1A and C). Alternatively, some make use of the "wings" of the Weiss epidural needle by advancing it with both hands a few millimeters at a time (Figure 41—1B). A change of tissue resistance is then tested continuously using tactile feedback when advancing the needle and by intermittently applying pressure to the air-filled loss-of-resistance syringe. The later technique allows for precise control of needle advancement and may allow a better distinction of various tissue densities. If air is used for detecting loss of resistance, the amount injected should be limited; injection of larger volumes of air (>2—3 mL) in the epidural space has been associated with patchy or unilateral analgesia and headache. The average depth of the lumbar epidural space in obstetric patients is 6 cm from the skin. Insertion of the epidural catheter at the L3—-4 or L4—5 interspace is generally optimal for achieving a T10-S5 neural blockade. Ultrasound guidance has recently been offered as tool in assisting with the placement of an epidural catheter. This technique allows the practitioner to judge the depth of the epidural space and estimate the best angle of needle insertion. The potential benefit of this technique is most obvious in obese patients with poor anatomic landmarks. However, the technique is highly user-dependent, and few practitioners have adopted it.

FIGURE 41-1 A: One-handed needle advancement; continuous pressure technique. The operator applies continuous pressure to the plunger of a loss-ofresistance syringe filled with saline and an air bubble while advancing the needle with the left hand braced against the patient's back. B: Bimanual needle advancement; intermittent pressure technique. The operator advances the lossof-resistance syringe with both hands 2 to 3 mm at a time while appreciating the resistance encountered by the needle. C: In between bimanual advancements of the needle, the operator tests the tissue resistance of the needle tip by bouncing the plunger of the air-filled loss-of-resistance syringe. Many practitioners add a compressible air bubble to a saline-filled syringe and bounce the plunger to ensure that the plunger is moving freely and not sticking to the syringe barrel wall.
If unintentional dural puncture occurs, the anesthetist has two choices: (1) place the epidural catheter in the subarachnoid space for continuous spinal (intrathecal) analgesia and anesthesia (see discussion that follows), or (2) remove the needle and attempt placement at another spinal level. The intrathecally placed epidural catheter may be used as continuous spinal anesthetic. If used in this fashion, an infusion of 0.0625% to 0.125% bupivacaine with fentanyl, 2 to 3 mcg/mL starting at 1-3 mL/h, is a typical choice.

Many clinicians advocate use of a multi-orifice catheter instead of a singleorifice catheter for obstetric anesthesia. Use of a multi-orifice catheter may be associated with fewer unilateral blocks and greatly reduces the incidence of false-negative aspiration when assessing for intravascular or intrathecal catheter placement. Advancing a multi-orifice catheter 4 to 6 cm into the epidural space appears to be optimal for obtaining adequate sensory levels. A single-orifice catheter need only be advanced 3 to 5 cm into the epidural space. Shorter insertion lengths in the epidural space (<5 cm) may favor dislodgment of the catheter out of the epidural space in obese patients following flexion/extension movements of the spine. Longer epidural insertion lengths in the epidural space may increase the risk of unilateral analgesia or epidural vein insertion. Spiral wire-reinforced catheters are very resistant to kinking. A spiral or spring tip, particularly when used without a stylet, is associated with fewer, less intense paresthesias.

The addition of opioids to local anesthetic solutions for epidural anesthesia has dramatically changed the practice of obstetric anesthesia. The synergy between epidural opioids and local anesthetic solutions reflects separate sites of action: opioid receptors and neuronal axons, respectively. When the two are combined, very low concentrations of both local anesthetic and opioid can be used with excellent effect. In addition, the incidence of adverse side effects, such as hypotension and drug toxicity, is reduced. Although local anesthetics can be used alone, there is rarely a reason to do so. Moreover, when an opioid is omitted, the greater concentration of local anesthetic required (eg, bupivacaine, 0.25%, and ropivacaine, 0.2%) for adequate analgesia can impair the parturient's ability to push effectively as labor progresses. The most common local anesthetic/opioid combination used for labor analgesia is bupivacaine or ropivacaine in concentrations of 0.0625% to 0.125% with either fentanyl, 2 to 3 mcg/mL, or sufentanil, 0.3 to 0.5 mcg/mL. In general, the lower the concentration of the local anesthetic the greater the concentration of opioid that is required. Very dilute local anesthetic mixtures (0.0625%) generally do not produce motor blockade and may allow some patients to ambulate ("walking" or "mobile" epidural). The long duration of action of bupivacaine makes it a popular agent for labor. Ropivacaine may be preferable because of its reduced potential for cardiotoxicity (see Chapter 16). At equi-analgesic doses, ropivacaine and bupivacaine appear to produce the same degree of motor block. The effect of epinephrine-containing solutions on the course of labor is somewhat controversial. Many clinicians use epinephrine-containing solutions only for intravascular test doses because of concern that the solutions may slow the progression of labor or adversely affect the fetus; others use only very dilute concentrations of epinephrine such as 1:800,000 or 1:400,000. However, studies comparing these various agents have failed to find any differences in neonatal Apgar scores, acid—base status, or neurobehavioral evaluations.

Initial epidural injections may be done either before or after the catheter is placed. Administration through the needle can facilitate catheter placement, whereas administration through the catheter provides assurance that the catheter is functioning properly. The following sequence is suggested for epidural activation:
1. Test for unintentional subarachnoid or intravascular placement of the needle or catheter using a 3-mL test dose of a local anesthetic with 1:200,000 epinephrine Many clinicians test with lidocaine 1.5% because it results in less toxicity following unintentional intravascular injection and more rapid onset of spinal anesthesia following unintentional intrathecal injection than with bupivacaine or ropivacaine. The test dose should be injected between contractions to help reduce false-positive signs of an intravascular injection (ie, tachycardia due to a painful contraction).
2. If after 5 min signs of intravascular or intrathecal injection are absent, with the patient supine and left uterine displacement, administer 10 mL of the local anesthetic—opioid mixture in 5-mL increments, waiting 1 to 2 min between doses, to achieve a T10—L1 sensory level. The initial bolus is usually composed of 0.1% to 0.2% ropivacaine or 0.0625% to 0.125% bupivacaine combined with either 50 to 100 mcg of fentanyl or 10 to 20 mcg of sufentanil.
3. Monitor with frequent assessment of vital signs for 20 to 30 min, or until the patient is stable. Pulse oximetry should also be used. Oxygen is administered via face mask if significant decrease in blood pressure or oxygen saturation occurs.

4. Repeat steps 2 and 3 when pain recurs until the first stage of labor is completed. Alternatively, a continuous epidural infusion technique may be employed using bupivacaine or ropivacaine in concentrations of 0.0625% to 0.125% with either fentanyl, 1 to 5 mcg/mL, or sufentanil, 0.2 to 0.5 mcg/mL, at a rate of 10 mL/h, which subsequently is adjusted to the patient's analgesic requirements (range: 5-15 mL/h). A third choice would be to use patient-controlled epidural analgesia (PCEA). Some studies suggest that total drug requirements may be less and patient satisfaction greater with PCEA when compared with other epidural analgesia techniques. Traditional PCEA settings are typically a 5-mL bolus dose with a 5 to 10 min lockout and 0 to 12 mL/h basal rate; a 1-h limit of 15 to 25 mL is typically used. Recent evidence also suggests that, compared with an epidural baseline infusion, programmed intermittent bolus techniques (eg, 6 mL bupivacaine 0.0625% every 30 min without baseline infusion) may improve patient satisfaction. Migration of the epidural catheter into a blood vessel during a continuous infusion technique may be heralded by loss of effective analgesia; a high index of suspicion is required because overt signs of systemic toxicity may be absent. Erosion of the catheter through the dura results in a slowly progressive motor blockade of the lower extremities and a rising sensory blockade level.

Epidural analgesia administration for the second stage of labor should extend the block to include the S2—4 dermatomes. Whether a catheter is already in place or epidural anesthesia is just being initiated, the following steps should be undertaken:
1. If the patient does not already have a catheter in place, establish epidural space access while the patient is in a sitting position. A patient who already has an epidural catheter in place should be placed in a semiupright or sitting position prior to injection.
2. Give a 3-mL test dose of local anesthetic (eg, lidocaine 1.5%) with 1:200,000 epinephrine. As previously noted, the injection should be completed between contractions.
3. If, after 5 min, signs of intravascular or intrathecal injection are absent, administer 10 to 15 mL of additional local anesthetic-opioid mixture at a rate not faster than 5 mL every 1 to 2 min.
4. Lay the patient supine with left uterine displacement and monitor vital signs every 1 to 2 min for the first 15 min, then every 5 min thereafter.

Safe administration of epidural analgesia and anesthesia is dependent on avoiding unintentional intrathecal @ or intravascular injection. Unintentional intravascular or intrathecal placement of an epidural needle or catheter is possible even when aspiration fails to yield blood or cerebrospinal fluid (CSF). The incidence of unintentional intravascular or intrathecal placement of an epidural catheter is 5% to 15% and 0.5% to 2.5%, respectively. Even a properly placed catheter can subsequently erode into an epidural vein or an intrathecal position. This possibility should be considered each time local anesthetic is injected through an epidural catheter.
Test doses of lidocaine (45-60 mg), bupivacaine (7.5—-10 mg), ropivacaine (6-8 mg), or chloroprocaine (100 mg) can be given to exclude unintentional intrathecal placement. Signs of sensory and motor blockade usually become apparent within 2 to 3 min and 3 to 5 min, respectively, if the injection is intrathecal.
In patients not receiving B-adrenergic antagonists, the intravascular injection of a local anesthetic solution with 15 to 20 mcg of epinephrine consistently increases the heart rate by 20 to 30 beats/min within 30 to 60 s if the epidural catheter or needle tip is intravascular. This technique is not always reliable in parturients because they often have marked spontaneous baseline variations in heart rate with contractions. Alternative methods of detecting unintentional intravascular catheter placement are based on eliciting signs of CNS toxicity, including tinnitus, dizziness, perioral numbness, or metallic taste. The use of dilute local anesthetic solutions and slow injection rates of no more than 5 mL at a time may also enhance detection of unintentional intravascular injections before catastrophic complications develop.

® 1. Hypotension—Generally defined as a greater than 20% decrease in the patient's baseline systolic blood pressure, or a systolic blood pressure less than 100 mm Hg, hypotension is a common side effect of neuraxial anesthesia. It is primarily due to decreased sympathetic tone and is greatly accentuated by aortocaval compression and an upright or semiupright position. Treatment should consist of intravenous boluses of phenylephrine (40-120 mcg), supplemental oxygen, left uterine displacement, and an intravenous fluid bolus. Although the routine use of a crystalloid fluid bolus prior to dosing an epidural catheter is not effective in the prevention of hypotension, ensuring proper intravenous hydration of the pregnant patient is important. Use of the head-down (Trendelenburg) position is controversial because of its potentially detrimental effects on pulmonary gas exchange.
2. Unintentional intravascular injection—Early recognition of intravascular injection, facilitated by the use of small, repeated doses of local anesthetic instead of a large bolus, may prevent more serious local anesthetic toxicity, such as seizures or cardiovascular collapse. Intravascular injections of toxic doses of lidocaine or chloroprocaine usually present as seizures. Propofol, 20 to 50 mg, will terminate seizure activity. Maintenance of a patent airway and adequate oxygenation are critical; however, administration of succinylcholine and endotracheal intubation are rarely necessary. Intravascular injections of bupivacaine can cause rapid cardiovascular collapse as well as seizure activity. Cardiac resuscitation may be exceedingly difficult and is aggravated by acidosis and hypoxia. An immediate infusion of 20% Intralipid combined with incremental doses of epinephrine has shown efficacy in reversing bupivacaineinduced cardiac toxicity. Amiodarone is the agent of choice for treating local anesthetic—induced ventricular arrhythmias.

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phenylephrine and
intravenous fluids. Moderate to profound hypotension may require the administration of epinephrine (10-50 mcg) or vasopressin (0.4—2.0 U intravenously). A high spinal level can also result in diaphragm and intercostal muscle paralysis, which necessitates intubation and ventilation with 100% oxygen. Delayed onset of a very high and often patchy or unilateral block may be due to unrecognized subdural injection (see Chapter 45), which is managed similarly.

4. Postdural puncture headache (PDPH)—There are many causes of headache after regional anesthesia in obstetrics. Caffeine-withdrawal and migraine headaches are not uncommon. Unintended dural puncture with a large epidural needle will commonly result in a PDPH, as a consequence of decreased intracranial pressure with compensatory cerebral vasodilation (See Chapter 45). Bed rest, hydration, oral analgesics, and caffeine sodium benzoate (5 00 mg added to 1000 mL intravenous fluids administered at 200 mL/h) may be effective in patients with mild headaches and as temporary treatment. Intravenous gabapentin, hydrocortisone, and oral theophylline have been shown to be successful in some studies. Patients with moderate to severe headaches usually require an epidural blood patch (10—20 mL; see Chapter 45). Prophylactic epidural blood patches are not recommended, as 25% to 50% of patients may not require a blood patch following dural puncture. Delaying a blood patch for 2 4h increases its efficacy, although supine bed rest for a 24 h while awaiting the epidural blood patch is both inconvenient and impractical for the new mother. Intracranial subdural hematoma has been reported as a rare complication 1 to 6 weeks following unintentional dural puncture in obstetric patients.
5. Maternal fever—Maternal fever is often interpreted as chorioamnionitis and may trigger evaluation for neonatal sepsis. Contrary to reports by some authors, there is no clear evidence that epidural anesthesia affects maternal temperature or that neonatal sepsis is increased with epidural analgesia. An elevation in maternal temperature is associated with a high body mass index and with nulliparity in women and prolonged labor.

@ Techniques using CSE analgesia and anesthesia may especially benefit patients with severe pain early in labor and those who receive analgesia/anesthesia immediately prior to delivery. Intrathecal opioid and local anesthetic are injected through the spinal needle, after which an epidural catheter is placed via the epidural needle. The intrathecal drugs provide essentially immediate pain control and have minimal effects on the early progress of labor, whereas the epidural catheter provides a route for subsequent analgesia for labor and delivery or anesthesia for cesarean section. The addition of a small dose of local anesthetic agent to the intrathecal opioid injection greatly potentiates the opioid efficacy and can significantly reduce opioid requirement. Thus, many clinicians will inject preservative-free bupivacaine, 2.5 mg, or ropivacaine, 3 to 4 mg, with intrathecal opioid for analgesia in the first stage of labor. Typical intrathecal opioid doses for CSE are fentanyl, 10 to 12.5 mcg, or sufentanil, 5 mcg. Some studies suggest that CSE techniques may be associated with greater patient satisfaction and lower incidence of PDPH than epidural analgesia alone. A 24to 27-gauge pencil-point spinal needle (Whitacre, Sprotte, or Gertie Marx) is used to minimize the incidence of PDPH.

For CSE techniques, the spinal and epidural needles may be placed at separate interspaces, but most clinicians use a needle-through-needle technique at the same interspace. Use of saline for identification of the epidural space may potentially cause confusion of saline for CSF. With the needle-through-needle technique, the epidural needle is placed in the epidural space and a long spinal needle is then introduced through it and advanced farther into the subarachnoid space. A distinct pop is felt as the needle penetrates the dura. The needle-besideneedle technique typically employs a specially designed epidural needle that has a channel for the spinal needle. After the intrathecal injection and withdrawal of the spinal needle, the epidural catheter is threaded into position and the epidural needle is withdrawn. The risk of advancing the epidural catheter through the dural hole created by the spinal needle is negligible when a 25-gauge or smaller spinal needle is used. The epidural catheter, however, should be aspirated carefully prior to use and local anesthetic should always be given slowly and in small increments to avoid unintentional intrathecal injections. Moreover, epidural drugs should be titrated carefully because the dural hole may facilitate entry of epidural drugs into intrathecal space and thereby potentiate their effects.

Spinal anesthesia given just prior to delivery—also known as saddle block— provides profound anesthesia for operative vaginal delivery. Use of a 22-gauge or smaller, pencil-point spinal needle (Whitacre, Sprotte, or Gertie Marx) decreases the likelihood of PDPH. Hyperbaric tetracaine, 3-4 mg, bupivacaine, 2.5—-5 mg, or lidocaine, 20-40 mg, usually provides excellent perineal anesthesia. Addition of fentanyl, 12.5-25 mcg, or sufentanil, 5-7.5 mcg, significantly potentiates the block. A T10 sensory level can be obtained with slightly larger amounts of local anesthetic. Three minutes after intrathecal injection of the hyperbaric solution, the patient is placed in the lithotomy position with left uterine displacement.

Because of the increased risk of aspiration, general anesthesia for vaginal delivery is avoided except for a true emergency. If an epidural catheter is already in place and time permits, rapid-onset regional anesthesia can be obtained with alkalinized lidocaine 2% or chloroprocaine 3%. Table 41-3 lists indications for general anesthesia during vaginal delivery. These indications are rare, and most include the need for urgent uterine relaxation.

Fetal distress during the second stage
Tetanic uterine contractions
Breech extraction
Version and extraction
Manual removal of a retained placenta
Replacement of an inverted uterus

Common indications for cesarean section are listed in Table 41—4. The choice of anesthesia for cesarean section is determined by multiple factors, including the indication for operative delivery, its urgency, patient and obstetrician preferences, and the skills of the anesthetist. In a given country, cesarean section rates may vary as much as twofold between institutions. In some countries, cesarean delivery is seen as preferable to labor and rates are much greater than those in the United States, where rates vary between @ 15% and 35% from hospital to hospital. In the United States, most elective cesarean sections are performed under spinal anesthesia. Regional anesthesia has become the preferred technique because general anesthesia is associated with a greater risk of maternal morbidity and mortality, greater hemodynamic fluctuation during anesthetic induction, and the need for additional analgesia during anesthetic recovery. Deaths associated with general anesthesia are generally related to airway problems, such as inability to intubate, inability to ventilate, or aspiration pneumonitis. However, most of the studies showing greater risk of general anesthesia were conducted before the arrival of video laryngoscopy and other advanced airway techniques. Deaths associated with regional anesthesia are generally related to excessive dermatomal spread of blockade or to local anesthetic
toxicity.

Increased risk of uterine rupture
Previous classic cesarean section
Previous extensive myomectomy or uterine reconstruction
Increased risk of maternal hemorrhage
Central or partial placenta previa
Abruptio placentae
Previous vaginal reconstruction

Abnormal fetopelvic relations
Fetopelvic disproportion
Abnormal fetal presentation
Transverse or oblique lie
Breech presentation
Dysfunctional uterine activity

Fetal distress
Umbilical cord prolapse with fetal bradycardia
Maternal hemorrhage
Genital herpes with ruptured membranes
Impending maternal death
Additional advantages of regional anesthesia include (1) less neonatal exposure to potentially depressant drugs, (2) a decreased risk of maternal pulmonary aspiration, (3) an awake mother who can experience the birth of her child, and (4) the option of using spinal opioids for postoperative pain relief.
Continuous epidural anesthesia allows ®@ better continuing control over the sensory level than "single-shot" spinal anesthesia techniques. Conversely, spinal anesthesia has a more rapid, predictable onset; may produce a denser (more complete) block; and lacks the potential for serious systemic drug toxicity because of the smaller local anesthetic dose employed. Regardless of the regional technique chosen, one must be prepared to administer a general anesthetic at any time during the procedure. Moreover, administration of a nonparticulate antacid within 30 min of surgery should be considered.
General anesthesia offers (1) a very rapid and reliable onset, (2) control over the airway and ventilation, (3) greater comfort for extremely anxious parturients, and (4) potentially less hypotension than regional anesthesia. General anesthesia also facilitates management in the event of severe hemorrhagic complications such as placenta accreta. Its principal disadvantages are the risk of pulmonary aspiration, the potential inability to intubate or ventilate the patient, and druginduced fetal depression. Present anesthetic techniques, however, limit the dose of intravenous agents such that fetal depression is usually not clinically significant with general anesthesia when delivery occurs within 10 min of induction of anesthesia. Regardless of the type of anesthesia, neonates delivered more than 3 min after uterine incision have lower Apgar scores and pH values.

Cesarean section requires that dermatomes up to and including T4 be anesthetized. Because of the associated sympathetic blockade, patients should receive an appropriate intravenous bolus of crystalloid such as lactated Ringer's (typically 1000-1500 mL) solution at the time of neural blockade. Such boluses will not consistently prevent hypotension but can virtually eliminate preexisting hypovolemia. Concomitant with local anesthetic injection, phenylephrine may be titrated to maintain a blood pressure within 20% of baseline. An approximate 10% decrease in blood pressure is expected. Some studies suggest that phenylephrine produces less neonatal acidosis than ephedrine, although administration of ephedrine, 5 to 10 mg intravenously, may be necessary in the hypotensive patient with reduced heart rate.
After spinal anesthetic injection, the patient is placed supine with left uterine displacement; supplemental oxygen (40-50%) is given; and blood pressure is measured every 1 to 2 min until it stabilizes. Hypotension following epidural anesthesia administration typically has a slower onset. Slight Trendelenburg positioning facilitates achieving a T4 sensory level and may also help prevent severe hypotension. Extreme degrees of Trendelenburg may interfere with

Epidural anesthesia for cesarean section is typically performed using a catheter, which allows supplementation of anesthesia, if necessary, and provides an excellent route for postoperative opioid administration. @ After negative aspiration and a negative test dose, a total volume of 15 to 35 mL of local anesthetic is injected slowly in 5-mL increments in order to minimize the risk of systemic local anesthetic toxicity. Lidocaine 2% (typically with 1:200,000 epinephrine) or chloroprocaine 3% are most commonly used in the United

The technique for CSE is described in the earlier section on Combined Spinal & Epidural Analgesia for labor and vaginal delivery. For cesarean section, CSE combines the benefit of rapid, reliable, intense blockade of spinal anesthesia with the flexible utility of an epidural catheter. The catheter also allows supplementation of anesthesia and can be used for postoperative analgesia. As mentioned previously, drugs administered epidurally should be titrated carefully because the dural hole created by the spinal needle may facilitate movement of epidural drugs into CSF and thereby potentiate their effects.

Pulmonary aspiration of gastric contents and failed endotracheal intubation are
FIGURE 41-2 Optimal positioning for obese patients with a short neck. A: The normal supine position often prevents extension of the head and makes endotracheal intubation difficult. B: Elevation of the shoulder allows some neck flexion with more optimal extension of the head at the atlantooccipital joint, facilitating intubation.
When a difficult airway is suspected, alternatives to the standard rapidsequence induction with conventional laryngoscopy, such as regional anesthesia or awake fiberoptic techniques, should be considered. We have found that videoassisted laryngoscopy has greatly reduced the likelihood of difficult or failed tracheal intubation at our institutions. Moreover, a clear plan should be formulated for a failed endotracheal intubation following induction of anesthesia
(Figure 41-3). In the absence of fetal distress, the patient should be awakened, and an awake intubation with regional or topical local anesthesia should be initiated. In the presence of fetal distress, if spontaneous or positive-pressure ventilation by mask or LMA with cricoid pressure is possible, delivery of the fetus should be initiated. In such instances, a potent volatile agent with oxygen is employed for general anesthesia, but once the fetus is delivered, nitrous oxide may be added to reduce the concentration of the volatile agent; sevoflurane may be the best volatile agent because it may be least likely to depress ventilation. The inability to intubate the patient or ventilate the patient via mask or LMA will require transtracheal jet ventilation or immediate cricothyrotomy or tracheostomy.
FIGURE 41-3 An algorithm for a difficult intubation in obstetric patients.

1. The patient is placed supine with a wedge under the right hip for left uterine displacement.
2. Denitrogenation is accomplished with 100% oxygen for 3 to 5 min while monitors are applied.

TABLE 41-5 Signs of fetal distress.
Nonreassuring fetal heart rate pattern
Repetitive late decelerations
Loss of fetal beat-to-beat variability associated with late or deep decelerations
Sustained fetal heart rate <80 beats/min
Fetal scalp pH <7.20
Meconium-stained amniotic fluid
Intrauterine growth restriction

Prolapse of the umbilical cord complicates 0.2% to 0.6% of deliveries. Umbilical cord compression following prolapse can rapidly lead to fetal asphyxia. Predisposing factors include excessive cord length, malpresentation, low birth weight, grand parity (more than five pregnancies), multiple gestations, and artificial rupture of membranes. The diagnosis is suspected after sudden fetal bradycardia or profound decelerations and is confirmed by physical examination. Treatment includes immediate steep Trendelenburg or knee—chest position and manual pushing of the presenting fetal part back up into the pelvis until immediate cesarean section under general anesthesia can be performed. If the fetus is not viable, vaginal delivery is allowed to continue.

A prolonged latent phase by definition exceeds 20 h in a nulliparous parturient and 14h in a multiparous patient. The cervix usually remains at 4 cm or less but is completely effaced. The etiology is likely ineffective contractions without a dominant myometrial pacemaker. Arrest of dilation is present when the cervix undergoes no further change after 2 h in the active phase of labor. A protracted active phase refers to slower than normal cervical dilation, defined as less than
1.2 cm/h in a nulliparous patient and less than 1.5 cm/h in a multiparous parturient. A prolonged deceleration phase occurs when cervical dilation slows markedly after 8 cm. The cervix becomes very edematous and appears to lose effacement. A prolonged second stage (disorder of descent) is defined as a descent of less than 1 cm/h and 2 cm/h in nulliparous and multiparous parturients, respectively. Failure of the head to descend 1 cm in station after adequate pushing is referred to as arrest of descent.
Oxytocin is usually the treatment of choice for uterine contractile abnormalities. The drug is given intravenously at 1 to 6 mU/min and increased in increments of 1 to 6 mU/min every 15 to 40 min, depending on the protocol. Use of amniotomy is controversial. Treatment is usually expectant management, as long as the fetus and mother are tolerating the prolonged labor. Operative vaginal delivery or cesarean section is indicated when a trial of oxytocin is unsuccessful or when malpresentation or cephalopelvic disproportion is also present.

Breech presentations complicate 3% to 4% of deliveries and significantly increase both maternal and fetal morbidity and mortality rates. Breech presentations increase neonatal mortality and the incidence of cord prolapse more than tenfold. External cephalic version may be attempted after 34 weeks of gestation and prior to the onset of labor; however, the fetus may spontaneously return to the breech presentation before the onset of labor. Some obstetricians may administer a tocolytic agent at the same time. External version can be facilitated, and its success rate improved, by providing epidural analgesia with 2% lidocaine and fentanyl. Although an external version is successful in 75% of patients, it can cause placental abruption and umbilical cord compression necessitating immediate cesarean section.
Because the shoulders or head can become trapped after vaginal delivery of the body, some obstetricians employ cesarean section for all breech presentations. If vaginal delivery is elected, manual or forceps-assisted partial breech extraction is usually necessary. The need for breech extraction does not appear to be increased when epidural anesthesia is used for labor—if labor is well established prior to epidural activation. Moreover, epidural anesthesia may decrease the likelihood of a trapped head, because the former relaxes the perineum. Nonetheless, the fetal head can become trapped in the uterus even during cesarean section under regional anesthesia; rapid induction of general endotracheal anesthesia and administration of a volatile agent may be attempted in such instances to relax the uterus. Alternatively, nitroglycerin, 50 to 100 mcg intravenously, can be administered.

When the fetal occiput fails to spontaneously rotate anteriorly, a persistent occiput posterior presentation results in a more prolonged and painful labor. Manual, vacuum, or forceps rotation is usually necessary but increases the likelihood of maternal and fetal injuries. Regional anesthesia can be used to provide perineal analgesia and pelvic relaxation, facilitating manual or forceps rotation followed by forceps delivery.
A face presentation occurs when the fetal head is hyperextended and generally requires cesarean section. A compound presentation occurs when an extremity enters the pelvis along with either the head or the buttocks. Vaginal delivery is usually still possible because the extremity often withdraws as the labor progresses.
Shoulder dystocia, or impaction of a shoulder against the pubic symphysis, complicates 0.2% to 2% of deliveries and is one of the major causes of birth injuries. Shoulder dystocias are often difficult to predict, and the most important risk factor is fetal macrosomia.. Several obstetric maneuvers can be used to relieve shoulder dystocia, but a prolonged delay in the delivery could result in fetal asphyxia. Induction of general anesthesia may be necessary if an epidural catheter is not already in place.

Multiple gestations account for approximately 1 in 150 births and are commonly complicated by breech presentation, prematurity, or both. Anesthesia may be necessary for version, extraction, or cesarean section. The second infant (and any subsequent ones) is often more depressed and asphyxiated than the first. Regional anesthesia provides effective pain relief during labor, minimizes the need for sedative and analgesic medication, and may shorten the interval between the birth of the first and second infants. Some studies suggest that the acid-base status of the second twin is improved when epidural anesthesia is used. Patients with multiple gestations, are more prone to develop hypotension from aortocaval compression, particularly after regional anesthesia.

© Maternal hemorrhage is one of the most common severe morbidities complicating obstetric anesthesia. Causes include uterine atony, placenta previa, abruptio placentae, and uterine rupture.

Premature separation of a normal placenta, abruptio placentae, complicates approximately 1% to 2% of pregnancies. Most abruptions are mild (grade I), but up to 25% are severe (grade III). Risk factors include hypertension, trauma, a short umbilical cord, multiparity, prolonged premature rupture of membranes, alcohol abuse, cocaine use, and an anatomically abnormal uterus. Patients usually experience painful vaginal bleeding and exhibit tenderness to palpation. Abdominal ultrasonography can help in the diagnosis. Factors in the choice between regional and general anesthesia include urgency for delivery, maternal hemodynamic stability, and presence of coagulopathy. Hemorrhage may remain concealed inside the uterus, contributing to underestimation of blood loss. Severe abruptio placentae can cause coagulopathy, particularly following fetal demise. Fibrinogen levels are mildly reduced (150-250 mg/dL) with moderate abruptions, but are typically less than 150 mg/dL with fetal demise. The coagulopathy is thought to be due to activation of circulating plasminogen (fibrinolysis) and the release of tissue thromboplastins that precipitate disseminated intravascular coagulation (DIC). Platelet count and factors V and VIII are low, and fibrin split products are elevated. Severe abruption is a lifethreatening emergency that necessitates an emergency cesarean section. The need for massive blood transfusion, including replacement of coagulation factors and platelets, should be considered.

Uterine rupture is relatively uncommon (1:1000-—3000 deliveries) but can occur during labor as a result of (1) dehiscence of a scar from a previous (usually classic) cesarean section (such a trial of labor is termed vaginal birth after cesarian [VBAC]); extensive myomectomy or uterine reconstruction; (2) intrauterine manipulations or use of forceps (iatrogenic); or (3) spontaneous rupture following prolonged labor in patients with hypertonic contractions (particularly with oxytocin infusions), fetopelvic disproportion, or a very large, thin, and weakened uterus. Uterine rupture can present as frank hemorrhage, fetal distress, loss of uterine tone, hypotension with occult bleeding into the abdomen, or a combination of these. Even when epidural anesthesia is employed for labor, uterine rupture is often heralded by the abrupt onset of continuous abdominal pain and hypotension. Treatment requires volume resuscitation and immediate laparotomy, usually under general anesthesia. Ligation of the internal iliac (hypogastric) arteries, with or without hysterectomy, may be necessary to control hemorrhage.

Premature rupture of membranes (PROM) is present when leakage of amniotic fluid occurs before the onset of labor. Confirmation of this diagnosis frequently involves the nitrazine test: The mildly alkaline pH of amniotic fluid (>7.1) causes nitrazine paper to change color from orange to blue in contrast to normal vaginal secretions, which are acidic pH <6). PROM complicates 10% of all

Preterm labor by definition occurs between 20 and 37 weeks of gestation and is the most common complication of the third trimester. Approximately 8% of liveborn infants in the United States are delivered before term. Important contributory maternal factors include extremes of age, inadequate prenatal care, unusual body habitus, increased physical activity, infections, prior preterm labor, multiple gestation, and other medical illnesses or complications during pregnancy.
Because of their small size and incomplete development, preterm infants— particularly those less than 30 weeks of gestational age or weighing less than 1500 g—experience a greater number of complications than term infants. PROM complicates one-third of premature deliveries; the combination of PROM and premature labor increases the likelihood of umbilical cord compression, resulting in fetal hypoxemia and asphyxia. Preterm infants with a breech presentation are particularly prone to prolapse of the umbilical cord during labor. Moreover, inadequate production of pulmonary surfactant frequently leads to idiopathic respiratory distress syndrome (hyaline membrane disease) after delivery since surfactant levels are generally adequate only after week 35 of gestation. Lastly, a soft, poorly calcified cranium predisposes these neonates to intracranial hemorrhage during vaginal delivery.

When preterm labor occurs before 35 weeks of gestation, bed rest and tocolytic therapy are usually initiated, with the goal of delaying birth to allow for the maternal administration of glucocorticoid (betamethasone) to enhance fetal lung maturity. The risk of respiratory distress syndrome is markedly reduced when the amniotic fluid lecithin/sphingomyelin ratio is greater than 2. Treatment successfully delays birth by 48 hours in 75% of patients; however, little evidence exists that the eventual preterm delivery is prevented. The most commonly used tocolytics are Bj-adrenergic agonists (ritodrine or terbutaline) and magnesium (6 g intravenously over 30 min followed by 2-4 g/h intravenously). Ritodrine, 100 to 350 mcg/min intravenously, and terbutaline, 2.5 to 5 mg orally every 4 to 6 h, also have some B,-adrenergic receptor activity, which accounts for some of their side effects. Maternal side effects include tachycardia, arrhythmias, myocardial ischemia, mild hypotension, hyperglycemia, hypokalemia, and, rarely, pulmonary edema. Other tocolytic agents include calcium channel blockers (nifedipine), prostaglandin synthetase inhibitors, oxytocin antagonists (atosiban), and, possibly, nitric oxide. Fetal ductal constriction can occur after 32 weeks of gestation with administration of nonsteroidal antiinflammatory drugs (NSAIDs) such as indomethacin, but is usually transient and resolves after discontinuation of the drug; NSAID-associated acute kidney injury in the fetus may cause

Anesthesia often becomes necessary when tocolytic therapy fails to arrest labor. The goal during vaginal delivery of a preterm fetus is a slow, controlled delivery with minimal pushing by the mother. An episiotomy and low forceps assistance are often employed. Spinal or epidural anesthesia promotes complete pelvic relaxation. Cesarean section is performed for fetal distress, breech presentation, intrauterine growth retardation, or failure of labor to progress. Residual effects from B-adrenergic agonists may complicate general anesthesia. The half-life of ritodrine may be as long as 3 h. Ketamine, ephedrine, and halothane should be used cautiously due to interaction with tocolytics. Hypokalemia is usually due to an intracellular uptake of potassium and rarely requires treatment; however, it may increase sensitivity to muscle relaxants. Magnesium therapy potentiates muscle relaxants and may predispose to hypotension secondary to vasodilation. Residual effects from tocolytics interfere with uterine contraction following delivery. Lastly, preterm newborns are often depressed at delivery and frequently need resuscitation; therefore, preparations for resuscitation should be completed prior to delivery.

Hypertension during pregnancy can be classified as pregnancy-induced hypertension (PIH, often also referred to as preeclampsia), chronic hypertension that preceded pregnancy, or chronic hypertension with superimposed preeclampsia. Preeclampsia is usually defined as a systolic blood pressure greater than 140 mm Hg or diastolic pressure greater than 90 mm Hg on two occasions at least 4 h apart after the 20th week of gestation in a woman with previously normal blood pressure, accompanied by proteinuria (>300 mg/d) or protein/creatinine ratio greater than 0.3, and resolving within 48 h after delivery. When seizures occur, the syndrome is termed eclampsia. HELLP syndrome describes preeclampsia associated with hemolysis, elevated liver enzymes, and a low platelet count. In the United States, preeclampsia complicates approximately 7% to 10% of pregnancies; eclampsia is much less common, occurring in 1 of 10,000 to 15,000 pregnancies. Severe preeclampsia causes or contributes to 20% to 40% of maternal deaths and 20% of perinatal deaths. Maternal deaths are usually due to stroke, pulmonary edema, hepatic necrosis or rupture, or a combination of these complications.

The pathophysiology of preeclampsia is related to vascular dysfunction of the placenta, resulting in abnormal prostaglandin metabolism. Patients with preeclampsia have elevated production of thromboxane A, (TXA,) and decreased production of prostacyclin (PGI,). TXA, is a potent vasoconstrictor and promoter of platelet aggregation, whereas PGI, is a potent vasodilator and inhibitor of platelet aggregation. Endothelial dysfunction may reduce production of nitric oxide and increase production of endothelin-1. The latter is also a potent vasoconstrictor and activator of platelets. Marked vascular reactivity and endothelial injury reduce placental perfusion and can lead to widespread systemic manifestations.
Severe preeclampsia substantially increases both maternal and fetal morbidity and mortality. Features of severe preeclampsia include the standard features of preeclampsia in association with any of the following: blood pressure greater than 160/110 mm Hg, thrombocytopenia (<100,000/yL), proteinuria greater than 5 g/d, impaired liver function, progressive kidney insufficiency (serum creatinine concentration greater than 1.1 mg/d oliguria, pulmonary edema, and CNS signs or symptoms (headache or visual disturbances; 'Table 41-6). Hepatic rupture may occur in patients with the HELLP syndrome.

Headache
Visual disturbances
Hyperexcitability
Seizures
Intracranial hemorrhage
Cerebral edema

Decreased intravascular volume
Increased arteriolar resistance

Proteinuria
Sodium retention
Decreased glomerular filtration
Renal failure

Thrombocytopenia
Platelet dysfunction
Prolonged partial thromboplastin time
Microangiopathic hemolysis
Patients with severe preeclampsia or eclampsia have widely differing hemodynamic profiles. Most patients have low-normal cardiac filling pressures with increased systemic vascular resistance, but cardiac output may be reduced, normal, or increased.

Treatment of preeclampsia consists of bed rest, sedation, repeated doses of antihypertensive drugs (usually labetalol, 5-10 mg, or hydralazine, 5 mg intravenously), and magnesium sulfate (4 g loading followed by 1-3 g/h intravenously) to treat hyperreflexia and prevent convulsions. Therapeutic magnesium levels are 4 to 6 mEq/L. It is recommended that corticosteroids be given if the fetus is viable and 33 weeks of gestation or less.
Invasive arterial and central venous monitoring are indicated in patients with severe hypertension, pulmonary edema, refractory oliguria, or a combination of these; in such patients an intravenous vasodilator infusion may be necessary. Definitive treatment of preeclampsia is delivery of the fetus and placenta.

magnesium potentiates muscle relaxants, doses of nondepolarizing muscle relaxants should be reduced in patients receiving magnesium therapy and should be guided by a peripheral nerve stimulator. The patient with suspected magnesium toxicity, manifested by hyporeflexia, excessive sedation, blurred vision, respiratory compromise, and cardiac depression, can be treated with intravenous administration of calcium gluconate (1 g over 10 min).

Amniotic fluid embolism is a rare (1:20,000 deliveries) but often lethal complication (86% mortality rate in some series) that can occur during labor, delivery, or cesarean section, or postpartum. Mortality may exceed 50% in the first hour. Entry of amniotic fluid into the maternal circulation can occur through any break in the uteroplacental membranes. Such breaks may occur during

Postpartum hemorrhage is the leading cause of maternal mortality in developing countries, and it is diagnosed when postpartum blood loss exceeds 500 mL. Up to 4% of parturients may experience postpartum hemorrhage, which is often associated with a prolonged third stage of labor, preeclampsia, multiple gestations, and forceps delivery. Common causes include uterine atony, a retained placenta, obstetric lacerations, uterine inversion, and use of tocolytic agents prior to delivery. Atony is often associated with uterine overdistention (multiple gestation and polyhydramnios). Less commonly, a clotting defect may be responsible.
The anesthesiologist may be consulted to assist in venous access or fluid and blood resuscitation, as well as to provide anesthesia for careful examination of the vagina, cervix, and uterus. Perineal lacerations can usually be repaired with local anesthetic infiltration or pudendal nerve blocks. Residual anesthesia from prior epidural or spinal anesthesia facilitates examination of the patient; however, supplementation with an opioid, nitrous oxide, or both may be required. Induction of spinal or epidural anesthesia should be avoided in the presence of marked hypovolemia. General anesthesia is usually required for manual extraction of a retained placenta, reversion of an inverted uterus, or repair of a major laceration. Uterine atony should be treated with oxytocin (a slow 0.3-1 IU intravenous bolus of oxytocin over 1 min, followed by an infusion of 5-10 IU/h), methylergonovine (0.2 mg in 100 mL of normal saline administered over 10 min intravenously), and prostaglandin F,, (0.25 mg intramuscularly). Emergency laparotomy and hysterectomy may be necessary in rare instances. Early ligation of the internal iliac (hypogastric) arteries may help avoid hysterectomy and reduce blood loss.

Resuscitation of the neonate starts during labor. Any compromise of the uteroplacental circulation readily @ produces fetal asphyxia. Intrauterine asphyxia during labor is the most common cause of neonatal depression. Fetal monitoring throughout labor is helpful in identifying which fetuses may be at risk, detecting fetal distress, and evaluating the effect of acute interventions. These include correcting maternal hypotension with fluids or vasopressors, providing supplemental oxygen, and decreasing uterine contraction (stopping oxytocin or administering tocolytics). Some studies suggest that the normal fetus can compensate for up to 45 min of relative hypoxia, a period termed fetal stress; the latter is associated with a marked redistribution of blood flow primarily to the heart, brain, and adrenal glands. With time, however, progressive lactic acidosis and asphyxia produce increasing fetal distress that necessitates

Monitoring of fetal heart rate (FHR) is presently the most useful technique in assessing fetal well-being, although alone it has a 35% to 50% false-positive rate of predicting fetal compromise. Because of this, the term fetal distress in the context of FHR monitoring has been largely replaced with nonreassuring FHR. Correct interpretation of heart rate patterns is crucial. Three parameters are evaluated: baseline heart rate, baseline variability, and the relationship to uterine contractions (deceleration patterns). Monitoring of heart rate is most accurate when fetal scalp electrodes are used, but this may require rupture of the membranes and is not without complications (eg, amnionitis or fetal injury). Based on concerns about a lack of consistency in interpretation and management of FHR, a three-tier FHR system was developed. Category I tracings are normal. Category II tracings are indeterminate and do not predict abnormal fetal acid base status. Category III tracings are abnormal and include either absent baseline variability with recurrent late or variable decelerations or bradycardia, or the presence of a sinusoidal pattern. They predict abnormal fetal acid—base status.

The mature fetus normally has a baseline heart rate of 110 to 160 beats/min. An increased baseline heart rate may be due to prematurity, mild fetal hypoxia, chorioamnionitis, maternal fever, maternally administered drugs (anticholinergics or B-agonists), or, rarely, hyperthyroidism. A decreased baseline heart rate may be due to a postterm pregnancy, fetal heart block, or fetal asphyxia.

The healthy mature fetus normally displays a baseline beat-to-beat (R-wave to R-wave) variability that can be classified as minimal (<5 beats/min), moderate (6-25 beats/min), or marked (>25 beats/min). Baseline variability, which is best assessed with scalp electrodes, has become an important sign of fetal well-being and represents a normally functioning autonomic system. Sustained decreased baseline variability is a prominent sign of fetal asphyxia. CNS depressants (opioids, barbiturates, volatile anesthetics, benzodiazepines, or magnesium sulfate) and parasympatholytics (atropine) also decrease baseline variability, as do prematurity, fetal arrhythmias, and anencephaly. A sinusoidal pattern that resembles a smooth sine wave is associated with fetal depression (hypoxia, drugs, and anemia secondary to Rh isoimmunization).

Accelerations of FHR are defined as increases of 15 beats/min or more lasting for more than 15 s. Periodic accelerations in FHR reflect normal oxygenation and are usually related to fetal movements and to responses to uterine pressure. Such accelerations are generally considered reassuring. By 32 weeks, fetuses display periodic increases in baseline heart rate that are associated with fetal movements. Normal fetuses have 15 to 40 accelerations/h, and the mechanism is thought to involve increases in catecholamine secretion with decreases in vagal tone. Accelerations diminish with fetal sleep, some drugs (opioids, magnesium, and atropine), as well as fetal hypoxia. Accelerations to fetal scalp or vibroacoustic stimulation are considered a reassuring sign of fetal well-being. The absence of both baseline variability and accelerations is nonreassuring and may be an important sign of fetal compromise.

Early deceleration (usually 10-40 beats/min) (Figure 41—4A) is thought to be a vagal response to compression of the fetal head or stretching of the neck during uterine contractions. The heart rate forms a smooth mirror image of the contraction. Early decelerations are generally not associated with fetal distress and occur during descent of the head.
FIGURE 41-4 Periodic changes in fetal heart rate related to uterine contraction. A: Early (type I) decelerations. B: Late (type II) decelerations. C: Variable (type III) decelerations. (Reproduced with permission from Danforth DN, Scott JR. Obstetrics and Gynecology. 5th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 1986.)

Late decelerations (Figure 41—4B) are associated with placental insufficiency and fetal compromise and are characterized by a decrease in heart rate at or following the peak of uterine contractions. Late decelerations may be subtle (as few as 5 beats/min) and are thought to represent the impact of decreased arterial oxygen tension on atrial chemoreceptors. Late decelerations with normal variability may be observed following acute insults (maternal hypotension or hypoxemia) and are usually reversible with treatment. Late decelerations with decreased variability are associated with prolonged asphyxia and may be an indication for fetal scalp sampling (see section on Other Monitoring). Complete abolition of variability in this setting is an ominous sign signifying severe decompensation and the need for immediate delivery.

The most common type of deceleration is variable (Figure 41-4C). These decelerations are variable in onset, duration, and magnitude (often >30 beats/min). They are typically abrupt in onset and are thought to be related to umbilical cord compression and acute intermittent decreases in umbilical blood flow. Variable decelerations are typically associated with fetal asphyxia when fetal heart rate declines to less than 60 beats/min, fetal bradycardia lasts more than 60 s, or recurrent bradycardia occurs in a pattern that persists for more than 30 min.

Other less commonly used monitors include fetal scalp pH measurements, scalp lactate concentration, fetal pulse oximetry, and fetal ST-segment analysis. Clinical experience is limited with all of these modalities except fetal scalp pH measurements. Unfortunately, fetal scalp pH measurement is associated with a small but significant incidence of false negatives and false positives. Fetal blood can be obtained and analyzed via a small scalp puncture once the membranes are ruptured. A fetal scalp pH higher than 7.20 is usually associated with a vigorous neonate, whereas a pH less than 7.20 is often, but not always, associated with a depressed neonate and necessitates prompt (typically operative) delivery. Because of wide overlap, fetal blood sampling can be interpreted correctly only in conjunction with heart rate monitoring.

Treatment of intrauterine fetal asphyxia is aimed at preventing fetal demise or permanent neurological damage. All interventions are directed at restoring an adequate uteroplacental circulation. Aortocaval compression, maternal hypoxemia or hypotension, or excessive uterine activity (during oxytocin infusions) must be corrected. Changes in maternal position, supplemental oxygen, and intravenous ephedrine or fluid, or adjustments in an oxytocin infusion often correct the problem. Failure to relieve fetal stress, as well as progressive fetal acidosis and asphyxia, necessitate immediate delivery.

FIGURE 41-5 An algorithm for resuscitation of the newly born infant.
(Reproduced with permission from Wyckoff MH, Aziz K, Escobedo MB, et al. Part 13: Neonatal Resuscitation: 2015 American Heart Association Guidelines Update for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Circulation. 2015 Nov 3;132(18 Suppl 2):S543-S560.)
In addition to respirations and heart rate, color, tone, and reflex irritability should be evaluated. The Apgar score (Table 41—7), recorded at 1 min and again at 5 min after delivery, remains the most valuable assessment of the neonate. The 1-min score correlates with survival, whereas the 5-min score has limited relationship to neurological outcome.

Heart rate (beats/min), Points.0 = Absent. Heart rate (beats/min), Points.7 = <100. Heart rate (beats/min), Points.2 = >100. Respiratory effort, Points.0 = Absent. Respiratory effort, Points.7 = Slow, irregular. Respiratory effort, Points.2 = Good, crying. Muscle tone, Points.0 = Flaccid. Muscle tone, Points.7 = Some flexion. Muscle tone, Points.2 = Active motion. Reflex irritability, Points.0 = Noresponse. Reflex irritability, Points.7 = Grimace. Reflex irritability, Points.2 = Crying. Color, Points.0 = Blue or pale. Color, Points.7 = Body pink, extremities blue. Color, Points.2 = All pink
Neonates with Apgar scores of 8 to 10 are vigorous and may require only gentle stimulation (flicking the foot, rubbing the back, and additional drying). A catheter should first be gently passed through each nostril to rule out choanal atresia, and then through the mouth to suction the stomach and rule out esophageal atresia.

The presence or absence of meconium in the amniotic fluid (approximately 10% to 12% of deliveries) changes the immediate management of the neonate at birth.
Fetal distress, particularly after 42 weeks of gestation, is often associated with release of thick meconium into the amniotic fluid. Fetal gasping during stress results in entry of a large amount of meconium-containing amniotic fluid into the lungs. When the neonate initiates respiration at birth, the meconium moves from the trachea and large airways down toward the periphery of the lung. Thick or particulate meconium may obstruct small airways and cause severe respiratory distress in 15% of meconium-stained neonates. Moreover, these infants can develop persistent fetal circulation.
Unless the neonate has absent or depressed respirations, thin watery meconium does not require suctioning beyond careful bulb suctioning of the oropharynx when the head emerges from the perineum (or from the uterus at cesarean section). When thick meconium is present in the amniotic fluid, however, some clinicians intubate and suction the trachea immediately after delivery but before the first breath is taken. However, recent evidence has challenged the benefit of tracheal suctioning even in the depressed infant. Tracheal suctioning of the thick meconium is accomplished by a special suctioning device attached to the endotracheal tube as the tube is withdrawn. If meconium is aspirated from the trachea, the procedure should be repeated until no meconium is obtained—but no more than three times, after which it is usually of no further benefit. The infant should then be given supplemental oxygen by face mask and observed closely. The stomach should also be suctioned to prevent passive regurgitation of any meconium. Newborns with meconium aspiration have an increased incidence of pneumothorax (10% compared with 1% for all vaginal deliveries).

Approximately 6% of newborns require some form of advanced life support. Resuscitation of these depressed neonates, most of whom weigh less than 1500 g, requires two or more caregivers—one to manage the airway and ventilation and another to perform chest compressions, if necessary. A third caregiver greatly facilitates placement of intravascular catheters and administration of fluids or drugs, or both. The anesthesiologist caring for the mother can render only brief assistance and only when it does not jeopardize the mother; other are, therefore, for neonatal resuscitation.
personnel usually responsible Because the most common cause of neonatal depression is intrauterine asphyxia, the emphasis in resuscitation is on respiration and oxygenation. Hypovolemia is also a contributing factor in a significant number of neonates. Factors associated with hypovolemia include early clamping of the umbilical cord, holding the neonate above the introitus prior to clamping, prematurity, maternal hemorrhage, placental transection during cesarean section, sepsis, and twin-to-twin transfusion.
Failure of the neonate to quickly respond to respiratory resuscitative efforts mandates vascular access and blood gas analysis; pneumothorax (1% incidence) and congenital anomalies of the airway, including tracheoesophageal fistula (1:3000-5000 live births), and congenital diaphragmatic hernia (1:2000-4000 live births) should be considered.
Grouping by the 1-min Apgar score greatly facilitates resuscitation: (1) mildly asphyxiated neonates (Apgar score of 5—7) usually need only stimulation while 100% oxygen is blown across the face; (2) moderately asphyxiated neonates (Apgar score of 3-4) require temporary assisted positive-pressure ventilation with mask and bag; and (3) severely depressed neonates (Apgar score of 0-2) should be immediately intubated, and chest compressions may be required.

and >2 kg, respectively). Correct endotracheal tube size is indicated by a small leak with 20 cm H,O pressure. Right endobronchial intubation should be excluded by chest auscultation. The correct depth of the endotracheal tube ("tip to lip") is usually 6 cm plus the weight in kilograms. Oxygen saturation can usually be measured by a pulse oximeter probe applied to the palm. Capnography should be used to confirm endotracheal intubation. Transcutaneous oxygen sensors are useful for measuring tissue oxygenation but require time for initial equilibration. Use of a laryngeal mask airway (LMA#1) has been reported in neonates weighing more than 2.5 kg and may be useful if endotracheal intubation is difficult (eg, Pierre Robin syndrome).
FIGURE 41-6 Intubation of the neonate. The head is placed in a neutral position, and the laryngoscope handle is held with the thumb and index finger as the chin is supported with the remaining fingers. Pressure applied over the hyoid bone with the little finger will bring the larynx into view. A straight blade such as a Miller 0 or 1 usually provides the best view.

Indications for chest compressions are a heart rate that is less than 60 beats/min or 60 to 80 beats/min and not rising after 30 s of adequate ventilation with 100% oxygen.
Cardiac compressions should be provided at a rate of 120/min. The two thumb/encircling hands technique (Figure 41—7) is generally preferred because it appears to generate higher peak systolic and coronary perfusion pressures. Alternatively, the two-finger technique can be used (Figure 41—8). The depth of compressions should be approximately one third of the anterior—posterior diameter of the chest and sufficient to generate a palpable pulse.
FIGURE 41-7 Chest compressions in the neonate. The neonate is held with both hands as each thumb is placed just beneath a line connecting the nipples and the remaining fingers encircle the chest. The sternum is compressed 1/3 to % in. (1 cm) at a rate of 120/min. (Reproduced with permission from Rudolph CD, Rudolph AM, Lister GE, et al. Rudolph's Pediatrics. 22nd ed. New York, NY: McGraw-Hill; 2011.)
FIGURE 41-8 The alternative technique for neonatal chest compressions: two fingers are placed on the lower third of the sternum at right angles to the chest. The chest is compressed approximately 1 cm at a rate of 120/min.
Compressions should be interposed with ventilation in a 3:1 ratio, such that 90 compressions and 30 ventilations are given per minute. The heart rate should be checked periodically. Chest compressions should be stopped when the spontaneous heart rate exceeds 80 beats/min.

Cannulation of the umbilical vein with a 3.5F or 5F umbilical catheter is easiest and the preferred technique. The tip of the catheter should be just below skin level and allow free backflow of blood; further advancement may result in infusion of hypertonic solutions directly into the liver. A peripheral vein or even the endotracheal tube can be used as an alternate route for drug administration. Cannulation of one of the two umbilical arteries allows measurement of blood pressure and facilitates blood gas measurements but may be more difficult. Specially designed umbilical artery catheters allow continuous Pao, or oxygen saturation monitoring as well as blood pressure. Care must be taken not to introduce any air into either the artery or the vein.

Nearly two-thirds of premature infants requiring resuscitation are hypovolemic at birth. Diagnosis is based on physical examination and a poor response to resuscitation. Neonatal blood pressure generally correlates with intravascular volume and should therefore routinely be measured. Normal blood pressure depends on birth weight and varies from 50/25 mm Hg for neonates weighing 1 to 2 kg to 70/40 mm Hg for those weighing over 3 kg. A low blood pressure and pallor suggest hypovolemia. Volume expansion may be accomplished with 10 mL/kg of lactated Ringer's injection, normal saline, or type O-negative blood cross-matched with maternal blood. Less common causes of hypotension include hypocalcemia, hypermagnesemia, and hypoglycemia.

Epinephrine, 0.01 to 0.03 mg/kg (0.1-0.3 mL/kg of a 1:10,000 solution), should be given for asystole or a spontaneous heart rate of less than 60 beats/min in spite of adequate ventilation and chest compressions. It may be repeated every 3 to 5 min. Epinephrine may be given in 1 mL of saline via the endotracheal tube when venous access is not available.

Naloxone, 0.1 mg/kg intravenously or 0.2 mg/kg intramuscularly, may be given to reverse the respiratory depressant effect of opioids given to the mother in the last 4 h of labor. Withdrawal symptoms may be precipitated in babies of mothers who chronically consume opioids.

Other drugs may be indicated only in specific settings. Sodium bicarbonate (2 mEq/kg of a 0.5 mEq/mL 4.2% solution) should usually be given only for a severe metabolic acidosis documented by blood gas measurements and when ventilation is adequate. It may also be administered during prolonged (>5 min) resuscitation—particularly if blood gas measurements are not readily available. The infusion rate should not exceed 1 mEq/kg/min to avoid hypertonicity and intracranial hemorrhage. As previously noted, in order to prevent hypertonicityinduced hepatic injury, the tip of the umbilical vein catheter should not be in the liver. Calcium gluconate 100 mg/kg (CaCl,, 30 mg/kg) should be given only to neonates with documented hypocalcemia or those with suspected magnesium intoxication (from maternal magnesium therapy); these neonates are usually hypotensive, hypotonic, and appear vasodilated. Glucose (8 mg/kg/min of a 10%
solution) is given only for documented hypoglycemia because hyperglycemia worsens hypoxic neurological deficits. Blood glucose should be measured because up to 10% of neonates may have hypoglycemia (glucose <35 mg/dL), particularly those delivered by cesarean section. Dopamine may be started at 5 mcg/kg/min to support arterial blood pressure. Lastly, surfactant may be given through the endotracheal tube to premature neonates with respiratory distress syndrome.

A 31-year-old woman with a 24-week gestation presents for an appendectomy.
How does pregnancy complicate the management of this patient?
Approximately 1% to 2% of pregnant patients require surgery during their pregnancy. The most common procedure during the first trimester is laparoscopy; appendectomy (1:1500 pregnancies) and cholecystectomy (1:2000-10,000 pregnancies) are the most commonly performed general surgical procedures. Cervical cerclage may be necessary in some patients for cervical incompetence. The physiological effects of pregnancy can alter the manifestations of disease process and make diagnosis difficult. Patients may therefore present with advanced or complicated disease. The physiological changes associated with pregnancy (see Chapter 40) further predispose the patient to increased morbidity and mortality. Moreover, both the operation and the anesthesia can adversely affect the fetus.
What are the potentially detrimental effects of surgery and anesthesia on the fetus?
The procedure can have both immediate and long-term adverse effects on the fetus. Maternal hypotension, hypovolemia, severe anemia, hypoxemia, and marked increases in sympathetic tone can seriously compromise the transfer of oxygen and other nutrients across the uteroplacental circulation and promote intrauterine fetal asphyxia. The stress of the operative procedure and the underlying surgical disease process may also precipitate preterm labor, which often follows intraabdominal surgery near the uterus. Laparoscopy may be safely performed. Mild to moderate maternal hyperventilation and limiting both insufflation pressure and duration of the procedure limit the degree of fetal acidosis. Long-term detrimental effects relate to possible teratogenic effects on the developing fetus.

Three stages of susceptibility are generally recognized. In the first 2 weeks of intrauterine life, teratogens have either a lethal effect or no effect on the embryo. The third to eighth weeks are the most critical period, when organogenesis takes place; drug exposure during this period can produce major developmental abnormalities. From the eighth week onward, organogenesis is complete, and organ growth takes place. Teratogen exposure during this last period usually results in only minor morphological abnormalities but can produce significant physiological abnormalities and growth retardation. Although the teratogenic influences of anesthetic agents have been extensively studied in animals, retrospective human studies have been inconclusive. Past concerns about possible teratogenic effects of nitrous oxide and benzodiazepines do not appear to be justified. Nonetheless, exposure to all anesthetic agents should be kept to a minimum. We tend to administer only those agents that are required for the pregnant patient—and in our practice, nitrous oxide is never required and benzodiazepines are only rarely needed.

Toward the end of the second trimester (after 20-24 weeks of gestation), most of the major physiological changes associated with pregnancy have taken place. Regional anesthesia, when feasible, is preferable to general anesthesia in order to decrease the risks of pulmonary aspiration and failed intubation and to minimize drug exposure to the fetus. The patient should be maintained with left lateral uterine displacement when supine. Total drug exposure is least with spinal anesthesia. Moreover, spinal anesthesia may be preferable to epidural anesthesia because it is not associated with unintentional intravascular injection or the potential for accidental intrathecal injection of large epidural doses of local anesthetic. On the other hand, general anesthesia guarantees patient comfort and, when a volatile agent is used, may even suppress preterm labor (see Chapter 40). Nitrous oxide without concomitant administration of a halogenated anesthetic is reported to reduce uterine blood flow.
Although regional anesthesia is preferable in most instances, the choice between regional and general anesthesia must be individualized according to the patient, the anesthesiologist, and the type of surgery. Spinal anesthesia is usually satisfactory for open appendectomies, whereas general anesthesia is appropriate for laparoscopic procedures.

In addition to standard monitors, fetal heart rate and uterine activity should be monitored with a Doppler and tocodynamometer immediately prior to surgery and during anesthesia recovery in a woman who is 24 weeks or more pregnant. When regular organized uterine activity is detected, early treatment with a B-adrenergic agonist such as ritodrine usually aborts the preterm labor. Magnesium sulfate and oral or rectal indomethacin may also be used as tocolytics.

All elective operations should be postponed until 6 weeks after delivery. Only emergency procedures that pose an immediate threat to the mother or fetus should be routinely performed. The timing of semielective procedures, such as those for cancer, valvular heart disease, or intracranial aneurysms, must be individualized and must balance the threat to maternal health versus fetal well-being. Controlled (deliberate) hypotensive anesthesia has been utilized to reduce blood loss during extensive cancer operations; nitroprusside, nitroglycerin, and hydralazine have been used during pregnancy without apparent fetal compromise. Nonetheless, large doses and prolonged infusions of nitroprusside should be avoided because the immature liver of the fetus may have a limited ability to metabolize the cyanide breakdown product. Cardiopulmonary bypass has been employed in pregnant patients successfully without adverse fetal outcome. Elective use of circulatory arrest during pregnancy is not recommended.

Neumar RW, Shuster M, Callaway CW, et al. Part 1: Executive summary 2015 American Heart Association guidelines update for cardiopulmonary resuscitation and emergency cardiovascular care. Circulation.
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@ Neonates and infants have fewer and smaller alveoli, reducing lung compliance; in contrast, their cartilaginous rib cage makes their chest wall very compliant. The combination of these two characteristics promotes chest wall collapse during inspiration and relatively low residual lung volumes at expiration. The resulting decrease in functional residual capacity (FRC) limits oxygen reserves during periods of apnea (eg, intubation attempts) and readily predisposes them to atelectasis and hypoxemia.
@ Compared with older children and adults, neonates and infants have a proportionately larger head and tongue, narrower nasal passages, an anterior and cephalad larynx, a longer epiglottis, and a shorter trachea and neck. These anatomic features make neonates and young infants obligate nasal breathers until about 5 months of age. The cricoid cartilage is the narrowest point of the airway in children younger than 5 years of age.
© Cardiac stroke volume is relatively fixed by the immature, noncompliant left ventricle in neonates and infants. The cardiac output is therefore very sensitive to changes in heart rate.
@ Thin skin, low fat content, and a greater surface area relative to weight promote greater heat loss to the environment in neonates. Heat loss can be made worse by prolonged exposure to an inadequately warmed operating room environment, administration of room-temperature intravenous fluid, and dehumidified anesthetic gases, and the effects of anesthetic agents on temperature regulation. Hypothermia has been associated with delayed awakening from anesthesia, cardiac arrhythmias, respiratory depression, increased pulmonary vascular resistance, and increased susceptibility to anesthetics and other agents.

Pediatric anesthesia involves more than simply adjusting drug doses and equipment for smaller patients. Neonates (0O—1 months), infants (1-12 months), toddlers (12-24 months), and young children (2-12 years of age) have differing anesthetic requirements. A safe anesthetic requires attention to the physiological, anatomic, and pharmacological characteristics of each group ('Table 42-1). Risk is generally inversely proportional to age, and infants are at much greater risk of anesthetic morbidity and mortality than older children. In addition, pediatric patients are prone to illnesses that require unique surgical and anesthetic strategies.

Heart-rate-dependent cardiac output
Reduced blood pressure
Increased respiratory rate
Increased metabolic rate
Reduced lung compliance
Increased chest wall compliance
Reduced functional residual capacity
Increased ratio of body surface area to body weight
Increased total body water content

Noncompliant left ventricle
Residual fetal circulation
Difficult venous and arterial cannulation
Relatively larger head and tongue
Narrower nasal passages
Anterior and cephalad larynx
Relatively longer epiglottis
Shorter trachea and neck
More prominent adenoids and tonsils
Weaker intercostal and diaphragmatic muscles
Greater resistance to airflow

Immature hepatic biotransformation
Decreased blood protein for drug binding
More rapid rise in FA/FI' and more rapid induction and recovery from inhaled anesthetics
Increased minimum alveolar concentration
Relatively larger volume of distribution for water-soluble drugs
Immature neuromuscular junction lpA/FI, fractional alveolar concentration/fractional inspired concentration.

The transition from fetal to neonatal physiology is reviewed in Chapter 40. Compared with older children and adults, neonates and infants have weaker intercostal muscles and weaker diaphragms (due to a paucity of type I fibers) and less efficient ventilation, more horizontal and pliable ribs, and protuberant abdomens. The alveoli are fully mature by about 8 years of age. Respiratory rate is increased in neonates and gradually falls to adult values by adolescence. Tidal volume and dead space per kilogram are nearly constant during development. The presence of fewer, smaller airways produces increased airway resistance. The work of breathing is increased and respiratory muscles easily fatigue. @ Neonates and infants have fewer and smaller alveoli, reducing lung compliance; in contrast, their cartilaginous rib cage makes their chest wall very compliant. The combination of these two characteristics promotes chest wall collapse during inspiration and relatively low residual lung volumes at expiration. The resulting decrease in functional residual capacity (FRC) limits oxygen reserves during periods of apnea (eg, intubation attempts) and predisposes neonates and infants to atelectasis and hypoxemia. These effects of reduced FRC may be exaggerated by the relatively higher rate of oxygen consumption of neonates and infants, 6 to 8 mL/kg/min versus 3 to 4 mL/kg/min in adults. Moreover, hypoxic and hypercapnic ventilatory drives are not fully developed in neonates and infants. In, contrast to adults, hypoxia and hypercapnia may depress respiration in these patients.
® Neonates and infants have, compared with older children and adults, a proportionately larger head and tongue, narrower nasal passages, an anterior and cephalad larynx (the glottis is at a vertebral level of C4 versus C6 in adults), a longer epiglottis, and a shorter trachea and neck (Figure 42-1). These anatomic features make neonates and young infants obligate nasal breathers until about 5 months of age. The cricoid cartilage is the narrowest point of the airway in children younger than 5 years of age; in adults, the narrowest point is the glottis (vocal cords). One millimeter of mucosal edema will produce a greater decline in tracheal cross-sectional area and gas flow in children because of their smaller tracheal diameters.
Mandible

Hyoid cartilage
Thyroid cartilage
Cricoid cartilage
Manubrium sterni
FIGURE 42-1 Sagittal section of the adult (A) and infant (B) airway. (Reproduced with permission from Snell RS, Katz J. Clinical Anatomy for Anesthesiologists. New York, NY: Appleton & Lange; 1988.)

® Cardiac stroke volume is relatively fixed by the immature, noncompliant left ventricle in neonates and infants. The cardiac output is therefore very sensitive to changes in heart rate (see Chapter 20). Although basal heart rate is greater in neonates and infants than in adults (Table 42—2), in these children activation of the parasympathetic nervous system, anesthetic overdose, or hypoxia can quickly trigger bradycardia and profound reductions in cardiac output. Sick infants undergoing emergency or prolonged surgical procedures appear particularly prone to episodes of bradycardia that can lead to hypotension, asystole, and intraoperative death. The sympathetic nervous system and baroreceptor reflexes are not fully mature. The infant cardiovascular system displays a blunted response to exogenous catecholamines. The immature heart is more sensitive to depression by volatile anesthetics and to opioid-induced bradycardia. Infants are less able to respond to hypovolemia with compensatory vasoconstriction. Intravascular volume depletion in neonates and infants may be signaled by hypotension without tachycardia.

Neonate, ..Respiratory Rate = 40. Neonate, Heart.Rate = 140. Neonate, Arterial Blood Pressure.Systolic = 65. Neonate, Arterial Blood Pressure.——H———__ Diastolic = 40. 12months, ..Respiratory Rate = 30. 12months, Heart.Rate = 120. 12months, Arterial Blood Pressure.Systolic = 95. 12months, Arterial Blood Pressure.——H———__ Diastolic = 65. 3 years, ..Respiratory Rate = 25. 3 years, Heart.Rate = 100. 3 years, Arterial Blood Pressure.Systolic = 100. 3 years, Arterial Blood Pressure.——H———__ Diastolic = 70. 12 years, ..Respiratory Rate = 20. 12 years, Heart.Rate = 80. 12 years, Arterial Blood Pressure.Systolic = 110. 12 years, Arterial Blood Pressure.——H———__ Diastolic = 60
Values are mean averages derived from numerous sources. Normal ranges may include measurements that deviate from these as much as 25-50%.

Pediatric patients have a larger surface area per kilogram than adults (smaller body-mass index). Metabolism and its associated parameters (oxygen consumption, CO, production, cardiac output, and alveolar ventilation) correlate better with surface area than with weight.
@ Thin skin, low fat content, and a greater surface area relative to weight promote greater heat loss to the environment in neonates. This problem can be made worse by prolonged exposure to an inadequately warmed operating room environment, administration of room temperature intravenous or irrigation fluid, and dehumidified anesthetic gases. There are also effects of anesthetic agents on temperature regulation (see Chapter 52). Even mild degrees of hypothermia can cause delayed awakening from anesthesia, cardiac arrhythmias, respiratory depression, increased pulmonary vascular resistance, and increased susceptibility to anesthetics, neuromuscular blockers, and other agents. Neonates produce heat by metabolism of brown fat (nonshivering thermogenesis) and by shifting hepatic oxidative phosphorylation to a more thermogenic pathway. Yet, metabolism of brown fat is severely limited in premature infants and in sick neonates, who are deficient in fat stores. Furthermore, volatile anesthetics inhibit this process.

Kidney function usually approaches normal values (corrected for size) by 6 months of age, but this may be delayed until the child is 2 years old. Premature neonates often demonstrate renal immaturity with one or more of the following: decreased creatinine clearance, impaired sodium retention, impaired glucose excretion, impaired bicarbonate reabsorption, reduced diluting ability and reduced concentrating ability. These abnormalities underscore the importance of appropriate fluid administration in neonates.
immature
Neonates also have an increased likelihood of gastroesophageal reflux. The liver conjugates drugs and other molecules less readily.

Neonates have relatively reduced glycogen stores, predisposing them to hypoglycemia. In general, the neonates at greatest risk for hypoglycemia are either premature or small for gestational age, receiving total parenteral nutrition, or had diabetic mothers.

Pediatric drug dosing is typically adjusted on a per-kilogram basis for convenience (Table 42-3), although there are strong advocates for allometric dosing, in which adjustments for weight are not made in a linear fashion. In early childhood a patient's weight can be approximated based on age in years:
TABLE 42-3 Pediatric drug dosages.

Deep block. , 0.2-0.5 mg/kg = 4mg/kg. Sufentanil, Pain relief (IV) = Premedication (intranasal). Sufentanil, 0.2-0.5 mg/kg = 2mceg/kg. , Pain relief (IV) = Anesthetic adjunct (IV). , 0.2-0.5 mg/kg = 0.5-1 meg/kg. , Pain relief (IV) = Maintenance infusion. , 0.2-0.5 mg/kg = 05-2 meg/kg/h. , Pain relief (IV) = Main anesthetic (IV). , 0.2-0.5 mg/kg = 10-15 mcg/kg. Thiopental, Pain relief (IV) = Induction (IV). Thiopental, 0.2-0.5 mg/kg = 5-6 mg/kg. Trimethoprim/sulfamethoxazole, Pain relief (IV) = Vv. Trimethoprim/sulfamethoxazole, 0.2-0.5 mg/kg = 4-5 mg/kg. Vancomycin, Pain relief (IV) = J. Vancomycin, 0.2-0.5 mg/kg = 20 mg/kg. Vecuronium, Pain relief (IV) = Nv. Vecuronium, 0.2-0.5 mg/kg = 0.1 mg/kg. Verapamil, Pain relief (IV) = Vv. Verapamil, 0.2-0.5 mg/kg = 0.1-0.3 mg/kg
- 'This drug is not yet approved for pediatric administration.

In contrast to weight-adjustment of drug dosing, allometric drug dose calculations take into account age-related physiological differences such as the disproportionately larger pediatric intravascular and extracellular fluid compartments, the immaturity of hepatic biotransformation pathways, the increased organ blood flow, the decreased protein for drug binding, and the higher metabolic rate.
Neonates and infants have a proportionately greater total water content (7075%) than adults (50-60%). Total body water content decreases while fat and muscle content increase with age. As a result, the volume of distribution for many intravenous drugs (eg, neuromuscular blockers) is disproportionately greater in neonates, infants, and young children, and the optimal dose (per kilogram) is usually greater than in older children and adults. A disproportionately smaller fat and muscle mass in neonates prolongs the clinical duration of action (by delaying redistribution) of lipid-soluble drugs such as propofol and fentanyl. Neonates also have a relatively decreased glomerular filtration rate, decreased hepatic blood flow, impaired renal tubular function, and immature hepatic enzyme systems. Increased intraabdominal pressure and abdominal surgery may further reduce hepatic blood flow. All of these factors can impair renal drug handling, hepatic metabolism, and biliary excretion of drugs in neonates and young infants. Neonates also have decreased drug binding to proteins, notably for local anesthetics and many antibiotics. In the case of bupivacaine, an increase in free drug likely increases the risk of systemic toxicity.

® Neonates, infants, and young children have relatively greater alveolar ventilation and reduced FRC compared with older children and adults. This greater minute ventilation-to-FRC ratio contributes to a rapid increase in alveolar anesthetic concentration that, combined with relatively greater blood flow to the brain, speeds inhalation induction. Furthermore, the blood/gas coefficients of volatile anesthetics are reduced in neonates compared with adults, contributing to faster induction times and potentially increasing the risk of accidental overdosage.
@ The minimum alveolar concentration (MAC) for halogenated agents is greater in infants than in neonates and adults (Table 42—4). In contrast to other agents, no increase in the MAC of sevoflurane can be demonstrated between neonates and infants. Nitrous oxide does not appear to reduce the MAC of desflurane or sevoflurane in children to the same extent as it does for other agents.

Halothane, Neonates = 0.90. Halothane, Infants = 17-12. Halothane, Small Children = 09. Halothane, Adults = 0.75. Sevoflurane, Neonates = 3.2. Sevoflurane, Infants = 3.2. Sevoflurane, Small Children = 2.5. Sevoflurane, Adults = 2. Isoflurane, Neonates = 1.6. Isoflurane, Infants = 1.8-1.9. Isoflurane, Small Children = 1.3-1.6. Isoflurane, Adults = 1.2. Desflurane, Neonates = 8-9. Desflurane, Infants = 9-10. Desflurane, Small Children = 7-8. Desflurane, Adults = 6
"MAC, minimum alveolar concentration.
*Values are derived from various sources.

The blood pressure of neonates and infants appears to be especially sensitive to volatile anesthetics. This clinical observation has been attributed to less-well-developed compensatory mechanisms (eg, vasoconstriction, tachycardia) and greater sensitivity of the immature myocardium to myocardial depressants. Halothane (now rarely used) sensitizes the heart to catecholamines; thus, the maximum recommended dose of epinephrine in local anesthetic solutions during halothane anesthesia is reduced. Cardiovascular depression, bradycardia, and arrhythmias are less frequent with sevoflurane than with halothane. Sevoflurane and halothane are less likely than other volatile agents to irritate the airway or cause breath holding or laryngospasm during induction (see Chapter 8). In general, volatile anesthetics appear to depress ventilation more in infants than in older children. Sevoflurane appears to produce the least respiratory depression. Halothane-induced hepatic dysfunction is much rarer in prepubertal children than adults. There are no reported instances of renal toxicity attributed to fluoride production during sevoflurane anesthesia in children. Sevoflurane is the preferred agent for inhaled induction in pediatric anesthesia. Emergence is fast following desflurane or sevoflurane, but both agents are associated with agitation or delirium upon emergence, particularly in young children. Because of the latter, some clinicians switch to isoflurane for maintenance anesthesia following a sevoflurane induction (see later discussion).

Dexmedetomidine has been used widely for sedation and as a supplement to general anesthesia in children. In patients without an intravenous line, dexmedetomidine can be given intranasally (1-2 mcg/kg) for sedation.

TABLE 42-5 Approximate EDg; for muscle relaxants in infants and children.!
Succinylcholine, Infants ED... (mg/kg) = 0.7. Succinylcholine, Children ED,. (mg/kg) = 04. Atracurium, Infants ED... (mg/kg) = 0.25. Atracurium, Children ED,. (mg/kg) = 0.35. Cisatracurium, Infants ED... (mg/kg) = 0.05. Cisatracurium, Children ED,. (mg/kg) = 0.06. Rocuronium, Infants ED... (mg/kg) = 0.25. Rocuronium, Children ED,. (mg/kg) = 0.4. Vecuronium, Infants ED... (mg/kg) = 0.05. Vecuronium, Children ED,. (mg/kg) = 0.08. Pancuronium, Infants ED... (mg/kg) = 0.07. Pancuronium, Children ED,. (mg/kg) = 0.09
'Average values during nitrous oxide/oxygen anesthesia.
The response of neonates to nondepolarizing muscle relaxants is variable. Popular explanations for this include "immaturity of the neuromuscular junction" (in premature neonates), tending to increase sensitivity (unproven), counterbalanced by a disproportionately larger extracellular compartment, reducing drug concentrations (proven). The relative immaturity of neonatal hepatic function prolongs the duration of action for drugs that depend primarily on hepatic metabolism (eg, pancuronium, vecuronium, and rocuronium). Atracurium and cisatracurium do not depend on hepatic biotransformation and behave as muscle relaxants.
reliably intermediate-acting

@ Children are more susceptible than adults to cardiac arrhythmias, hyperkalemia, rhabdomyolysis, myoglobinemia, masseter spasm, and malignant hyperthermia (see Chapter 52) associated with succinylcholine. When a child experiences cardiac arrest following administration of succinylcholine, immediate treatment for hyperkalemia should be instituted. Prolonged resuscitative efforts (potentially including cardiopulmonary bypass) may be required. Thus, succinylcholine has long been avoided for routine, elective paralysis for intubation in children and @ adolescents. Children may have profound bradycardia and sinus node arrest following the first dose of succinylcholine without atropine pretreatment. Therefore, atropine (0.1 mg minimum) is customarily administered prior to succinylcholine in children. Generally accepted indications for intravenous succinylcholine in children include rapid sequence induction with a "full" stomach and laryngospasm that does not respond to positive-pressure ventilation. When rapid muscle relaxation is required prior to intravenous access (eg, with inhaled inductions in patients with full stomachs), intramuscular succinylcholine (4-6 mg/kg) can be used. Intramuscular atropine (0.02 mg/kg) should be administered with intramuscular succinylcholine to reduce the likelihood of bradycardia. Some clinicians advocate intralingual administration (2 mg/kg in the midline) as an alternate emergency route for succinylcholine.

Many clinicians consider rocuronium (0.6 mg/kg intravenously) the drug of choice (when an intravenous relaxant will be used) for routine intubation in pediatric patients because it has the fastest onset of nondepolarizing neuromuscular blocking agents (see Chapter 11). Larger doses of rocuronium (0.9-1.2 mg/kg) may be used for rapid sequence induction if a prolonged duration (up to 90 min) is not a concern. Rocuronium is the only nondepolarizing neuromuscular blocker that has been adequately studied for intramuscular administration (1.0-1.5 mg/kg), but this approach requires 3 to 4 min for onset. Atracurium or cisatracurium may be preferred in young infants, particularly for short procedures, because these drugs consistently display short to intermediate duration.
As with adults, the effect of muscle relaxants should be monitored with a peripheral nerve stimulator. Sensitivity can vary significantly between patients. Nondepolarizing blockade can be reversed with neostigmine (0.03—-0.07 mg/kg) or edrophonium (0.5—1 mg/kg) along with an anticholinergic agent (glycopyrrolate, 0.01 mg/kg, or atropine, 0.01-0.02 mg/kg). Sugammadex, a specific antagonist for rocuronium and vecuronium, was recently released in the United States and will likely change the typical choices and doses of neuromuscular blockers given its effectiveness, even in the face of neuromuscular blockade that could not be reversed with conventional cholinesterase inhibitors.

The Pediatric Perioperative Cardiac Arrest (POCA) Registry provides a useful database for assessing pediatric anesthetic risk. This registry includes reports derived from millions of pediatric anesthetics administered since 1994. Case records of children experiencing cardiac arrests or death during the administration of or recovery from anesthesia were investigated regarding any possible relationship with anesthesia. Nearly all patients received general anesthesia alone or combined with regional anesthesia. In a preliminary analysis that included 289 cases of cardiac arrest, anesthetic care was judged to have contributed to 150 arrests. Thus, the risk of cardiac arrest in pediatric anesthetic cases would appear to be approximately 1.4 in 10,000. Thirty-three percent of patients who suffered a cardiac arrest were classified as American Society of Anesthesiologists (ASA) physical status 1 or 2. Infants accounted for 55% of all anesthesia-related arrests in children, with those younger than 1 month of age (ie, neonates) having the greatest risk. After cardiac arrest, mortality was 26%. Six percent suffered permanent injury, but the majority (68%) had either no or only temporary injury. Mortality was 4% in patients classified as ASA physical status 1 and 2 compared with 37% in those with ASA physical status 3 to 5. As with adults, two major predictors of mortality were ASA physical status 3 to 5 and emergency surgery.
Most cardiac arrests occurred during induction of anesthesia; bradycardia, hypotension, and a low Spo, frequently preceded arrest. The most common mechanism of arrest was judged to be medication related (Figure 42—2). Cardiovascular depression from halothane, alone or in combination with other drugs, was believed to be responsible in two-thirds of all medication-related arrests. Another 9% were due to intravascular injection of a local anesthetic, most often following a negative aspiration test during attempted caudal injection. Presumed cardiovascular mechanisms most often had no clear etiology; in more than 50% of those cases the patient had congenital heart disease. Where a cardiovascular mechanism could be identified, it was most often related to hemorrhage, transfusion, or inadequate or inappropriate fluid therapy. These studies await replication in the modern, virtually "halothane-free," era in which regional techniques (and, presumably, the attendant risks) are markedly more prevalent.
FIGURE 42-2 Mechanisms of cardiac arrest in pediatric patients, based on

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Children are at greater risk than adults of developing malignant hyperthermia. This complex and important topic is covered in depth in Chapter 52.

Depending on age, past experiences, and maturity, children present with varying degrees of fright (even terror) when faced with the prospect of surgery or other procedures requiring anesthesia. Unlike adults, who are usually most concerned about the possibility of injury or death, children, when they verbalize their concerns, worry about pain and separation from their parents. Presurgical preparation programs—such as age-appropriate brochures and videos, or tours— can help prepare both children and parents. When time permits, one can demystify the process of anesthesia and surgery by explaining in age-appropriate terms what lies ahead. For example, the anesthesiologist might bring an anesthesia mask for the child to play with during the interview and describe it as like something the astronauts use. Alternatively, in some centers, someone the child trusts (eg, a parent, nurse, another physician) may be allowed to be in attendance during preanesthetic preparations and induction of anesthesia. This can have a particularly calming influence on children undergoing repeated procedures (eg, administration of intrathecal chemotherapy). Unfortunately, outpatient and "same day admit" surgery, coupled with a busy operating room schedule, often make it nearly impossible to adequately reassure parents and patients. Thus, premedication (discussed below) often can be helpful. Some pediatric hospitals have induction rooms adjacent to their operating rooms to permit parental attendance and a quieter, less startling environment for anesthetic inductions.

Children frequently present for surgery with signs and symptoms—a runny nose with fever, cough, or sore throat—of a viral upper respiratory tract infection (URI). Attempts should be made to differentiate between an infectious cause of rhinorrhea and an @ allergic or vasomotor cause. A viral infection within 2 to 4 weeks before general anesthesia and endotracheal intubation appears to place the child at an increased risk for perioperative pulmonary complications, such as wheezing (10-fold), laryngospasm (5-fold), hypoxemia, and atelectasis. This is particularly likely if the child has a severe cough, high fever, or a family history of reactive airway disease. On the other hand, children can have mild URIs on a nearly monthly basis and it can become nearly impossible to schedule them for anesthesia at a time when they neither currently have, nor are recovering from, a URI. The decision to anesthetize children with URIs remains controversial and should be based on the severity of URI symptoms, the urgency of the surgery, and the presence of other coexisting illnesses, When anesthesia will be provided to a child with a URI, one may consider premedicating with an anticholinergic or a By-agonist (eg, albuterol), avoiding intubation (if feasible), and humidifying inspired gases. A longer-than-usual stay in the postanesthesia recovery area may be required.

Few, if any, preoperative laboratory tests are cost effective. Some pediatric centers require no preoperative laboratory tests in healthy children undergoing minor procedures. Obviously, this places responsibility on the anesthesiologist, surgeon, and pediatrician to correctly identify those patients who should have preoperative testing for specific surgical procedures.
Most asymptomatic patients with cardiac murmurs do not have significant cardiac pathology. Innocent murmurs may occur in more than 30% of normal children. These are typically soft, short systolic ejection murmurs that are best heard along the left upper or left lower sternal border and that do not radiate. Innocent murmurs at the left upper sternal border typically are due to flow across the pulmonic valve (pulmonic ejection) whereas those at the lower left border typically are due to flow from the left ventricle to the aorta (Still's vibratory murmur). The pediatrician should carefully evaluate patients with a newly diagnosed murmur, particularly in infancy. Consultation with a pediatric cardiologist, echocardiography, or both, should be obtained if the patient is symptomatic (eg, poor feeding, failure to thrive, or easy fatigability). Murmurs that are loud, "harsh," holosystolic, diastolic, or that radiate widely—or pulses that are either bounding or markedly diminished—require further evaluation and diagnosis.

Because children are more prone to dehydration than adults, their preoperative fluid restriction has always been more lenient. Several studies, however, have documented low gastric pH (<2.5) and relatively high residual volumes in pediatric patients scheduled for surgery, suggesting that children may be at a greater risk for aspiration than was previously thought. The incidence of aspiration is reported to be approximately 1:1000. There is no evidence that prolonged fasting decreases the risk of aspiration. In fact, several studies have demonstrated lower residual volumes and higher gastric pH in pediatric patients who received clear fluids a few hours before induction (see Chapter 53). The guideline on preoperative fasting produced by the American Society of Anesthesiologists specifies that infants may be fed breast milk up to 4 h before induction, and formula or liquids and a "light" meal may be given up to 6h before induction. Clear fluids are offered until 2 h before induction. These recommendations are for healthy neonates, infants, and children without risk factors for decreased gastric emptying or aspiration. In any case, there is almost no clinical evidence for the recommendations.

There is great variation in the premedication of pediatric patients. Sedative premedication is generally omitted for neonates and sick infants. Children who appear likely to exhibit uncontrollable separation anxiety may be given a sedative, such as midazolam (0.3-0.5 mg/kg, 15 mg maximum). The oral route is generally preferred because it is less traumatic than intramuscular injection, but it requires 20 to 45 min for effect. Smaller doses of midazolam have been used in combination with oral ketamine (4-6 mg/kg) for inpatients. For uncooperative patients, intramuscular midazolam (0.1—-0.15 mg/kg, 10 mg maximum) or ketamine (2-3 mg/kg) with atropine (0.02 mg/kg) may be helpful. Rectal midazolam (0.5—1 mg/kg, 20 mg maximum) or rectal methohexital (25— 30 mg/kg of 10% solution) may also be administered in such cases while the child is in the parent's arms. Some clinicians administer dexmedetomidine (1-2 mcg/kg) or midazolam premedication intranasally. Fentanyl can also be administered as a lollipop (Actiq, 5-15 mcg/kg); however, fentanyl levels continue to rise intraoperatively and can contribute to postoperative analgesia. In the past, anesthesiologists routinely premedicated young children with anticholinergic drugs to reduce the likelihood of bradycardia. Atropine reduces the incidence of hypotension during induction in neonates and in infants younger than 3 months. Atropine can also prevent accumulation of secretions that can block small airways and endotracheal tubes. Secretions can be particularly troublesome for children with URIs or those who have been given ketamine. Atropine may be administered orally (0.05 mg/kg), intramuscularly, or occasionally rectally. In current practice, most prefer to administer atropine intravenously during induction.

Monitoring requirements for infants and children are generally similar to those for adults with some minor modifications. Alarm limits should be appropriately adjusted. Smaller electrocardiographic electrode pads may be necessary so that they do not encroach on sterile surgical areas. Blood pressure cuffs must be properly fitted. Noninvasive blood pressure monitors have proved to be reliable in infants and children. A precordial or esophageal stethoscope provides an inexpensive means of monitoring heart rate, quality of heart sounds, and airway patency. Finally, monitors may sometimes need to be first attached (or induction of anesthesia in less reattached) following cooperative patients. Pulse oximetry and capnography assume an even more important role in infants and small children because hypoxia from inadequate ventilation remains a common cause of perioperative morbidity and mortality. In neonates, the pulse oximeter probe should preferably be placed on the right hand or earlobe to measure preductal oxygen saturation. As in adult patients, end-tidal CO, analysis allows assessment of adequacy of ventilation, changes in cardiac output, confirmation of endotracheal tube placement, and early warning of malignant hyperthermia. Flow-through (mainstream) analyzers are usually less accurate in patients weighing less than 10 kg. Even with aspiration (sidestream)

General anesthesia is usually induced by an intravenous or inhalational technique. Induction with intramuscular ketamine (5—10 mg/kg) is reserved for specific situations, such as those involving combative, particularly mentally challenged, or autistic patients. Intravenous induction is usually preferred when the patient comes to the operating room with a functional intravenous catheter or will allow awake venous cannulation. Prior application of EMLA (eutectic mixture of local anesthetic) cream (see Chapter 16) may render intravenous cannulation less painful for the patient, and less stressful for the parent and anesthesiologist. However, EMLA cream is neither a perfect nor a complete solution. Some children become anxious at the sight of a needle, particularly those who have had multiple needle punctures in the past, with or without EMLA. Furthermore, it can be difficult to anticipate in which extremity intravenous cannulation will prove to be successful. Finally, to be effective, EMLA cream must remain in contact with the skin for at least 30 to 60 min. Awake or sedated-awake intubation with topical anesthesia should be considered for emergency procedures in neonates and small infants when they are critically ill or a potential difficult airway is present.

The same induction sequence can be used as in adults: propofol (2-3 mg/kg) followed by a nondepolarizing muscle relaxant (eg, rocuronium, cisatracurium, atracurium), or succinylcholine. We recommend that atropine be given routinely prior to succinylcholine. The advantages of an intravenous technique include availability of intravenous access if emergency drugs need to be administered and rapidity of induction in the child at risk for aspiration. Alternatively (and very commonly in pediatric practice), intubation can be accomplished after the combination of propofol, lidocaine, and an opiate, with or without an inhaled agent, avoiding the need for a paralytic agent. Finally, paralytic agents are not needed for placement of LMAs, which are commonly used in pediatric anesthesia.

Many children do not arrive in the operating room with an intravenous line in place and nearly all dread the prospect of being stuck with a needle. Fortunately, sevoflurane can render small children unconscious within minutes. We find this easier in children who have been sedated (most often with oral midazolam) prior to entering the operating room and who are sleepy enough to be anesthetized without ever knowing what has happened (steal induction). One can also insufflate the anesthetic gases over the face, place a drop of food flavoring on the inside of the mask (eg, oil of orange), and allow the child to sit during the early stages of induction. Specially contoured masks minimize dead space (see Figure 19-11).
There are many differences between adult and pediatric anatomy that influence mask ventilation and intubation. Equipment appropriate for age and size should be selected (Table 42-6). Neonates and most young infants are obligate nasal breathers and obstruct easily. Oral airways will help displace an oversized tongue; nasal airways, so useful in adults, can traumatize small nares or prominent adenoids in small children. Compression of submandibular soft tissues should be avoided during mask ventilation to prevent upper airway obstruction.

Infant = °o. Mask size, Toddler = 1. Mask size, Small Child = 2. Mask size, Large Child = 3. Oral airway, Premature = 000-00. Oral airway, Neonate = 00. Oral airway, Infant = 0(40 mm). Oral airway, Toddler = 1(450mm). Oral airway, Small Child = =2(70 mm). Oral airway, Large Child = 3 (80mm). Laryngeal mask airway (LMA#), Premature = — —. Laryngeal mask airway (LMA#), Neonate = 1. Laryngeal mask airway (LMA#), Infant = 1. Laryngeal mask airway (LMA#), Toddler = 2. Laryngeal mask airway (LMA#), Small Child = 25. Laryngeal mask airway (LMA#), Large Child = 3
*ET, endotracheal tube.
Typically, the child can be coaxed into breathing an odorless mixture of nitrous oxide (70%) and oxygen (30%). Sevoflurane (or halothane) can be added to the gas mixture in 0.5% increments every few breaths. As previously discussed, we favor sevoflurane in most situations. Desflurane and isoflurane are avoided for inhalation induction because they are pungent and associated with more coughing, breath-holding, and laryngospasm. We use a single (sometimes two) breath induction technique with sevoflurane (7—-8% sevoflurane in 60% nitrous oxide) to speed the induction in cooperative patients. After an adequate depth of anesthesia has been achieved, an intravenous line can be started and propofol and an opioid (or a muscle relaxant) administered to facilitate intubation. Patients typically pass through an excitement stage during which any stimulation can induce laryngospasm. Breath-holding must be distinguished from laryngospasm. Steady application of 10 cm of positive end-expiratory pressure will usually overcome laryngospasm.

Alternatively, the anesthesiologist can deepen the level of anesthesia, by increasing the concentration of volatile anesthetic, and place an LMA or intubate the patient under "deep" sevoflurane anesthesia. Because of the greater anesthetic depth required for tracheal intubation, the risk of cardiac depression, bradycardia, or laryngospasm occurring without intravenous access detracts from this latter technique. Intramuscular succinylcholine (4 mg/kg, not to exceed 150 mg) and atropine (0.02 mg/kg, not to exceed 0.4 mg) should be available if laryngospasm or bradycardia occurs before an intravenous line is established. Positive-pressure ventilation during mask induction and prior to intubation sometimes causes gastric distention, with impairment of lung expansion. Suctioning with an orogastric or nasogastric tube will decompress the stomach, but it must be done without traumatizing fragile mucous membranes.

Intravenous cannulation in infants can be a vexing ordeal. This is particularly true for infants who have spent weeks in a neonatal intensive care unit and have few intact veins. Even healthy 1-year-old children can prove a challenge because of extensive subcutaneous fat. Venous cannulation usually becomes easier after 2 years of age. The saphenous vein has a consistent location at the ankle and an experienced practitioner can usually cannulate it even if it is not visible or palpable. Transillumination of the hands or ultrasonography will often reveal previously hidden cannulation sites. Twenty-four-gauge over-the-needle catheters are adequate in neonates and infants when blood transfusions are not anticipated. All air bubbles should be removed from the intravenous line to reduce the risk of paradoxical air embolism from occult patent foramen ovale. In emergency situations where intravenous access is impossible, fluids can be effectively infused through an 18-gauge needle inserted into the medullary sinusoids within the tibial bone. This intraosseous infusion can be used for all medications normally given intravenously, with almost as rapid results (see Chapter 55), and is considered part of the standard trauma resuscitation, advanced cardiac life support (ACLS), and pediatric advanced life support (PALS) protocols when intravenous access cannot be obtained.

One hundred percent oxygen should be administered prior to intubation to increase patient safety during the obligatory period of apnea prior to and during intubation. The choice of muscle relaxant has been discussed earlier in this chapter. For awake intubations in neonates or infants, adequate preoxygenation and continued oxygen insufflation during laryngoscopy may help prevent hypoxemia.
The infant's prominent occiput tends to place the head in a flexed position prior to intubation. This is easily corrected by slightly elevating the shoulders on towels and placing the head on a doughnut-shaped pillow. In older children, prominent tonsillar tissue can obstruct visualization of the larynx. Straight laryngoscope blades aid intubation of the anterior larynx in neonates, infants, and young children (Table 42-6). Endotracheal tubes that pass through the glottis may still impinge upon the cricoid cartilage, which is the narrowest point of the airway in children younger than 5 years of age. Mucosal trauma from trying to force a tube through the cricoid cartilage can cause postoperative edema, stridor, croup, and airway obstruction.
The appropriate diameter inside the endotracheal tube can be estimated by a formula based on age:

For example, a 4-year-old child would be predicted to require a 5-mm uncuffed tube. This formula provides only a rough guideline, however. Exceptions include premature neonates (2.5—-3 mm tube) and full-term neonates (3-3.5 mm tube). Alternatively, the practitioner can remember that a newborn takes a 2.5or 3-mm tube, and a 5-year-old takes a 5-mm tube. It should not be that difficult to identify which of the three sizes of tube between 3 and 5 mm is required in small children. In larger children, small (S—6 mm) cuffed tubes can be used either with or without the cuff inflated to minimize the need for precise sizing. Endotracheal tubes 0.5 mm larger and smaller than predicted should be readily available in or on the anesthetic cart. In the past, uncuffed endotracheal tubes were selected for children aged 5 years or younger in the hope of decreasing the risk of postintubation croup. Currently, many anesthesiologists no longer use size 4.0 or larger uncuffed tubes. The leak test will minimize the likelihood that an excessively large tube has been inserted. Correct tube size and appropriate cuff inflation is confirmed by easy passage into the larynx and the development of a gas leak at 15 to 25 cm H,O pressure. No leak indicates an oversized tube or overinflated cuff that should be replaced or deflated to prevent postoperative edema, whereas an excessive leak may preclude adequate ventilation and contaminate the operating room with anesthetic gases. As previously noted, many clinicians use a down-sized cuffed tube in younger patients at high risk for aspiration; minimal inflation of the cuff can stop any air leak. There is also a formula to estimate endotracheal length:

Again, this formula provides only a guideline, and the result must be confirmed by auscultation and clinical judgment. To avoid endobronchial intubation, the tip of the endotracheal tube should pass only 1 to 2 cm beyond an infant's glottis. Alternatively, one can intentionally advance the tip of the endotracheal tube into the right mainstem bronchus and then withdraw it until breath sounds are equal over both lung fields.

Ventilation is almost always controlled during anesthesia of neonates and infants when using a conventional semiclosed circle system. During spontaneous ventilation, even the low resistance of a circle system can become a significant obstacle for a sick neonate to overcome. Unidirectional valves, breathing tubes, and carbon dioxide absorbers account for most of this resistance. For patients weighing less than 10 kg, some anesthesiologists prefer the Mapleson D circuit or the Bain system because of their low resistance and light weight (see Chapter 3). Nonetheless, because breathing-circuit resistance is easily overcome by positive-pressure ventilation, the circle system can be safely used in patients of all ages if ventilation is controlled. Monitoring of airway pressure may provide early evidence of obstruction from a kinked endotracheal tube or accidental advancement of the tube into a mainstem bronchus.

Many anesthesia ventilators on older machines are designed for adult patients and cannot reliably provide the reduced tidal volumes and rapid rates required by neonates and infants. Unintentional delivery of large tidal volumes to a small child can generate excessive peak airway pressures and cause barotrauma. Pressure control ventilation, which is found on nearly all newer anesthesia ventilators, should be used for neonates, infants, and toddlers. Small tidal volumes can also be manually delivered with greater ease with a 1-L breathing bag than with a 3-L adult bag. For children less than 10 kg, adequate tidal volumes are achieved with peak inspiratory pressures of 15 to 18 cm H,O. For larger children the volume control ventilation may be used and tidal volumes may be set at 6 to 8 mL/kg. Many spirometers are less accurate at lower tidal volumes. In addition, the gas lost in long, compliant adult breathing circuits becomes large relative to a child's small tidal volume. For this reason, pediatric breathing circuits are usually shorter, lighter, and stiffer (less compliant). Nevertheless, one should recall that the additional dead space contributed by the tube and circle system consists only of the volume of the distal limb of the Yconnector and that portion of the endotracheal tube that extends beyond (proximal to) the airway. In other words, the dead space is unchanged by switching from adult to pediatric breathing circuits. Condenser humidifiers or heat and moisture exchangers (HMEs) can add considerable dead space; depending on the size of the patient, they either should not be used or an appropriately sized, pediatric HME should be employed. Anesthesia be maintained in pediatric patients with the in

can same agents as adults. Some clinicians switch to isoflurane following a sevoflurane induction in the hope of reducing the likelihood of emergence agitation or postoperative delirium (see earlier discussion). Administration of an opioid (eg, fentanyl, 1-1.5 mcg/kg) or dexmedetomidine (0.5 mcg/kg, given slowly with heart rate monitoring) 15 to 20 min before the end of the procedure can reduce the incidence of emergence delirium and agitation if the surgical procedure is likely to produce postoperative pain. Although the MAC is greater in children than in adults (see Table 42-4), neonates may be particularly susceptible to the cardiacdepressing effects of general anesthetics and may not tolerate the concentrations of volatile agents required when the volatile agent alone is used to maintain good surgical operating conditions.

@ Meticulous attention to fluid intake and loss is required in younger pediatric patients because these patients have limited margins for error. A programmable infusion pump or a buret with a microdrip chamber is useful for accurate measurements. Drugs can be flushed through low dead-space tubing to minimize unnecessary fluid administration. Fluid overload is diagnosed by prominent veins, flushed skin, increased blood pressure, decreased serum sodium, and a loss of the folds in the upper eyelids.
Fluid therapy can be divided into maintenance, deficit, and replacement requirements.

Maintenance requirements for pediatric patients can be determined by the "4:2:1 rule": 4 mL/kg/h for the first 10 kg of weight, 2 mL/kg/h for the second 10 kg, and 1 mL/kg/h for each remaining kilogram. The choice of maintenance fluid remains controversial. A solution such as D-% NS with 20 mEq/L of potassium chloride provides adequate dextrose and electrolytes at these maintenance infusion rates. D;4 NS may be a better choice in neonates because of their limited ability to handle sodium loads. Children up to the age of 8 years require 6 mg/kg/min of glucose to maintain euglycemia (40-125 mg/dL); premature neonates require 6-8 mg/kg/min. Euglycemia is normally well maintained in older children and adults by hepatic glycogenolysis and gluconeogenesis despite administration of glucose-free solutions. Both hypoglycemia and hyperglycemia should be avoided; however, the amount of hepatic glucose production is widely variable during major surgery and critical illness. Thus glucose infusion rates during longer surgeries, particularly in neonates and infants, should be adjusted based on blood glucose measurements.

In addition to a maintenance infusion, any preoperative fluid deficits must be replaced. For example, if a 5-kg infant has not received oral or intravenous fluids for 4h prior to surgery, a deficit of 80 mL has accrued (5 kg x 4 mL/kg/h x 4h). In contrast to adults, infants respond to dehydration with decreased blood pressure and without increased heart rate. Preoperative fluid deficits are often administered with hourly maintenance requirements in aliquots of 50% in the first hour and 25% in the second and third hours. In the example above, a total of 60 mL would be given in the first hour (80/2 + 20) and 40 mL in the second and third hours (80/4 + 20). Bolus administration of dextrose-containing solutions should be avoided to prevent hyperglycemia. Preoperative fluid deficits are usually replaced with a balanced salt solution (eg, lactated Ringer's injection) or YANS. Glucose is omitted to prevent hyperglycemia. Compared with lactated Ringer's injection, normal saline has the disadvantage of promoting hyperchloremic acidosis.

Replacement can be subdivided into blood loss and third-space loss.
1. Blood loss—The blood volume of premature neonates (100 mL/kg), full-term neonates (85-90 mL/kg), and infants (80 mL/kg) is proportionately larger than
Regional Anesthesia and Analgesia
The primary uses of regional techniques in pediatric anesthesia have been to supplement and reduce general anesthetic requirements and to provide better postoperative pain relief. Blocks range in complexity from relatively simple peripheral nerve blocks (eg, penile block, ilioinguinal block); to brachial plexus, sciatic nerve, femoral nerve, and transversus abdominis plane (TAP) blocks; to major conduction blocks (eg, spinal or epidural techniques). Regional blocks in children (as in adults) are often facilitated by ultrasound guidance, sometimes with nerve stimulation.
Caudal blocks have proved useful following a variety of surgeries, including circumcision, inguinal herniorrhaphy, hypospadias repair, anal surgery, clubfoot repair, and other subumbilical procedures. Contraindications include infection around the sacral hiatus, coagulopathy, or anatomic abnormalities. The patient is usually lightly anesthetized or sedated and placed in the lateral position.
For pediatric caudal anesthesia, a short-bevel 22-gauge needle can be used. If the loss-of-resistance technique is used, the glass syringe should be filled with saline, not air, because of the latter's possible association with air embolism. After the characteristic pop that signals penetration of the sacrococcygeal membrane, the needle angle of approach is reduced and the needle is advanced only a few more millimeters to avoid entering the dural sac or the anterior body of the sacrum. Aspiration is used to check for blood or cerebrospinal fluid; local anesthetic can then be slowly injected; failure of a 2-mL test dose of local anesthetic with epinephrine (1:200,000) to produce tachycardia helps exclude intravascular placement.

Many anesthetic agents have been used for caudal anesthesia in pediatric patients, with 0.125% to 0.25% bupivacaine (up to 2.5 mg/kg) or 0.2% ropivacaine being most common. Ropivacaine, 0.2%, can provide analgesia similar to bupivacaine but with less motor blockade. Ropivacaine appears to have less cardiac toxicity than bupivacaine when compared milligram to milligram. Addition of epinephrine to caudal solutions tends to increase the degree of motor block. Clonidine, either by itself or combined with local anesthetics, has also been widely used. Morphine sulfate (25 mcg/kg) or hydromorphone (6 mcg/kg) may be added to the local anesthetic solution to prolong postoperative analgesia for inpatients, but will increase the risk of delayed postoperative respiratory depression. The volume of local anesthetic required depends on the level of blockade desired, ranging from 0.5 mL/kg for a sacral block to 1.25 mL/kg for a midthoracic block. Single-shot injections generally last 4 to 12 h. Placement of 20-gauge caudal catheters with continuous infusion of local anesthetic (eg, 0.125% bupivacaine or 0.1% ropivacaine at 0.2—
Sedation for Procedures In & Out of the Operating

Pediatric patients are particularly vulnerable to two common postanesthetic complications: laryngospasm and postintubation croup. As with adult patients, postoperative pain requires close, careful attention. Pediatric anesthesia practice varies widely, particularly in regard to extubation following a general anesthetic. In some pediatric hospitals, all children who will be extubated after a general anesthetic arrive in the postanesthesia care unit (PACU) with the tube or LMA still in place. They are subsequently extubated by the PACU nurse when defined criteria are reached. In other centers, nearly all children are extubated in the operating room before arriving in the PACU. High quality and safety are reported at centers following either protocol.

Laryngospasm is a forceful, involuntary spasm of the laryngeal musculature caused by stimulation of the superior laryngeal nerve (see Chapter 19). It may occur at induction, emergence, or any time in between without an endotracheal tube. Presumably, it can also occur when a tube is in place, but its occurrence will not be recognized. Laryngospasm is more common in young pediatric patients (almost 1 in 50 anesthetics) than in adults, and is most common @® in infants 1 to 3 months old. Laryngospasm at the end of a procedure can usually be avoided by extubating the patient either while awake (opening the eyes) or while deeply anesthetized (spontaneously breathing but not swallowing or coughing); both techniques have advocates, and despite strong opinions, evidence is lacking as to which is the better approach. Extubation during the interval between these extremes, however, is generally recognized as more hazardous. Recent URI or exposure to secondhand tobacco smoke predisposes children to laryngospasm on emergence. Treatment of laryngospasm includes gentle positive-pressure ventilation, forward jaw thrust, deepening of the anesthetic with intravenous propofol, intravenous lidocaine (1-1.5 mg/kg), or paralysis with intravenous succinylcholine (0.5—1 mg/kg), or rocuronium (0.4 mg/kg) and controlled ventilation. Intramuscular succinylcholine (4-6 mg/kg) remains an acceptable alternative in patients without intravenous access and in whom conservative measures have failed. Laryngospasm is usually an immediate postoperative event but may occur in the recovery room as the patient wakes up and chokes on pharyngeal secretions. For this reason, recovering pediatric patients should be positioned in the lateral position so that oral secretions pool and drain away from the vocal cords. When the child begins to regain consciousness, having the parents at the bedside may reduce his or her anxiety.

Croup is due to glottic or tracheal edema. Because the narrowest part of the pediatric airway is the cricoid cartilage, this is the most susceptible area. Croup is less common with properly sized endotracheal tubes that are small enough to allow a slight gas leak at 10 to 25 cm H,O. Postintubation croup is associated with early childhood (age 1-4 years), repeated intubation attempts, overly large endotracheal tubes, prolonged surgery, head and neck procedures, and excessive movement of the tube (eg, coughing with the tube in place, moving the patient's head). Intravenous dexamethasone (0.25—0.5 mg/kg) may prevent formation of edema, and inhalation of nebulized racemic epinephrine (0.25—0.5 mL of a 2.25% solution in 2.5 mL normal saline) is an often-effective treatment. Although postintubation croup occurs later than laryngospasm, it will almost always appear within 3 h after extubation.

Pain in pediatric patients has received considerable attention in recent years, and over that time the use of regional anesthetic and analgesic techniques (as previously above) has greatly increased. Commonly used parenteral opioids include fentanyl (1-2 mcg/kg), morphine (0.05—0.1 mg/kg), and hydromorphone (15 mcg/kg). A multimodal technique incorporating ketorolac (0.5-0.75 mg/kg) and intravenous dexmedetomidine will reduce opioid requirements. Oral, rectal, or intravenous acetaminophen will also reduce opioid requirements and can be a helpful substitute for ketorolac.
Patient-controlled analgesia (see Chapter 48) can also be successfully used in patients as young as 5 years old, depending on their maturity and on preoperative preparation. Commonly used opioids include morphine and hydromorphone. With a 10-min lockout interval, the recommended interval dose is either morphine, 20 mcg/kg, or hydromorphone, 5 mcg/kg. As with adults, continuous infusions increase the risk of respiratory depression; typical continuous infusion doses are morphine, 0 to 12 mcg/kg/h, or hydromorphone, 0 to 3 mcg/kg/h. The subcutaneous route may be used with morphine. Nurse-controlled and parentcontrolled analgesia remain controversial but widely used techniques for pain control in children.
As with adults, epidural infusions for postoperative analgesia often consist of a local anesthetic combined with an opioid. Bupivacaine, 0.1% to 0.125%, or ropivacaine, 0.1% to 0.2%, are often combined with fentanyl, 2 to 2.5 mcg/mL (or equivalent concentrations of morphine or hydromorphone). Recommended infusion rates depend on the size of the patient, the final drug concentration, and the location of the epidural catheter, and range from 0.1 to 0.4 mL/kg/h. Local anesthetic infusions can also be used with continuous nerve block techniques, but this is less common than in adults.

Prematurity is defined as birth before 37 weeks of gestation. This is in contrast to small for gestational age, which describes an infant (full-term or premature) whose age-adjusted weight is less than the fifth percentile. The multiple medical problems of premature neonates are usually due to immaturity of major organ systems or to intrauterine asphyxia. Pulmonary complications include hyaline membrane disease, apneic spells, and bronchopulmonary dysplasia. Exogenous pulmonary surfactant has proved to be an effective treatment for respiratory distress syndrome in premature infants. A patent ductus arteriosus leads to shunting and may possibly lead to pulmonary edema and congestive heart failure. Persistent hypoxia or shock may result in ischemic gut and necrotizing enterocolitis. Prematurity increases susceptibility to infection, hypothermia, intracranial hemorrhage, and kernicterus. Premature neonates also have an increased incidence of congenital anomalies.

The small size (often <1000 g) and fragile medical condition of premature neonates demand that special attention be paid to airway control, fluid management, and temperature regulation. The problem of retinopathy of prematurity, a fibrovascular proliferation overlying the retina that may lead to progressive visual loss, deserves special consideration. Recent evidence suggests that fluctuating oxygen levels may be more damaging than increased oxygen tension. Moreover, other major risk factors, such as respiratory distress, apnea, mechanical ventilation, hypoxia, hypercarbia, acidosis, heart disease, bradycardia, infection, parenteral nutrition, anemia, and multiple blood transfusions, must be present. Nonetheless, oxygenation should be continuously

Malrotation of the intestines is a developmental abnormality that permits spontaneous abnormal rotation of the midgut around the mesentery (superior mesenteric artery). The incidence of malrotation is estimated to be about 1:500 live births. Most patients with malrotation of the midgut present during infancy with symptoms of bowel obstruction. Coiling of the duodenum with the ascending colon can produce complete or partial duodenal obstruction. The most serious complication of malrotation, a midgut volvulus, can rapidly compromise intestinal blood supply causing infarction. Midgut volvulus is a true surgical emergency that most commonly occurs in infancy, with up to one third occurring in the first week of life. The mortality rate is high (up to 25%). Typical symptoms are bilious vomiting, progressive abdominal distention and tenderness, metabolic acidosis, and hemodynamic instability. Bloody diarrhea may be indicative of bowel infarction. Abdominal ultrasonography or upper gastrointestinal imaging confirms the diagnosis.

Surgery provides the only definitive treatment of malrotation and midgut volvulus. If obstruction is present but obvious volvulus has not yet occurred, preoperative preparation may include stabilization of any coexisting conditions, insertion of a nasogastric (or orogastric tube) to decompress the stomach, broadspectrum antibiotics, and fluid and electrolyte replacement before prompt transport to the operating room.
These patients are at increased risk for pulmonary aspiration. Depending on the size of the patient, rapid sequence induction (or awake intubation) should be employed. Patients with volvulus are usually hypovolemic and acidotic, and may be prone to hypotension. Postoperative ventilation will often be necessary, making an opioid-based anesthetic a reasonable choice. Fluid resuscitation, likely including blood products, with correction of acidosis is usually necessary. Arterial and central venous lines are helpful. Surgical treatment includes reducing the volvulus, freeing the obstruction, and resecting any obviously necrotic bowel. Bowel edema can complicate abdominal closure and has the potential to produce an abdominal compartment syndrome. The latter can impair ventilation, hinder venous return, and produce acute kidney injury; delayed fascial closure or temporary closure with a Silastic "silo" may be necessary. A second-look laparotomy may be required 24 to 48 h later to ensure viability of the remaining bowel and close the abdomen.

During fetal development, the gut can herniate into the thorax through one of three diaphragmatic defects: the left or right posterolateral foramina of
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Treatment is aimed at immediate stabilization with sedation, paralysis, and moderate hyperventilation. Pressure-limited ventilation is used. Some centers employ permissive hypercapnia (postductal Paco, < 65 mm Hg) and accept mild hypoxemia (preductal Spo, > 85%) in an effort to reduce pulmonary barotrauma. High-frequency oscillatory ventilation (HFOV) can improve ventilation and oxygenation with less barotrauma. Inhaled nitric oxide may be used to lower pulmonary artery pressures, but it does not appear to improve survival. If the pulmonary hypertension stabilizes and there is little right-to-left shunting, early surgical repair may be undertaken. If the patient fails to stabilize, venoarterial extracorporeal membrane oxygenation (ECMO) may be undertaken. Treatment with prenatal intrauterine surgery has not improved outcomes.

Gastric distention must be minimized by placement of a nasogastric tube and avoidance of high levels of positive-pressure ventilation. The neonate is preoxygenated and typically intubated without the aid of muscle relaxants. Anesthesia is maintained with low concentrations of volatile agents or opioids, muscle relaxants, and oxygen-enriched air. Hypoxia and expansion of air in the bowel contraindicate use of nitrous oxide. If possible, peak inspiratory airway pressures should be less than 30 cm H,O. A sudden fall in lung compliance, blood pressure, or oxygenation may signal a contralateral (usually rightsided) pneumothorax and necessitate placement of a chest tube. Arterial blood gases are monitored by sampling a preductal artery if an umbilical artery catheter is not already in place. Surgical repair is performed via a subcostal the
incision of the affected side; the bowel is reduced into the abdomen and the diaphragm is closed. Aggressive attempts at expansion of the ipsilateral lung following surgical decompression are detrimental. The extent of pulmonary hypoplasia and the presence of other congenital defects determine the prognosis.

There are several types of tracheoesophageal fistulae (Figure 42—3). The most common (type IIIB) is the combination of an upper esophagus that ends in a blind pouch and a lower esophagus that connects to the trachea. Breathing results in gastric distention, whereas feeding leads to choking, coughing, and cyanosis (three Cs). The diagnosis is suspected by failure to pass a catheter into the stomach and confirmed by visualization of the catheter coiled in a blind, upper esophageal pouch. Aspiration pneumonia and the coexistence of other congenital anomalies (eg, cardiac) are common. These may include the association of vertebral defects, anal atresia, tracheoesophageal fistula with esophageal atresia, and radial dysplasia, known as the VATER syndrome. The VACTERL variant also includes cardiac and limb anomalies. Preoperative management is directed at identifying all congenital anomalies and preventing aspiration pneumonia. This may include maintaining the patient in a head-up position, using an oral-esophageal tube, and avoiding feedings. In some instances gastrostomy may be performed under local anesthesia. Definitive surgical treatment is usually postponed until any pneumonia clears or improves with antibiotic therapy.
FIGURE 42-3 Of the five types of tracheoesophageal fistula, type IIIB represents 90% of cases.

These neonates tend to have copious pharyngeal secretions that require frequent suctioning before and during surgery. Positive-pressure ventilation is avoided prior to intubation, as the resulting gastric distention may interfere with lung expansion. Intubation is often performed awake and without muscle relaxants. These neonates are often dehydrated and malnourished due to poor oral intake. The key to successful management is correct endotracheal tube position. Ideally, the tip of the tube lies distal to the fistula and proximal to the carina, so that anesthetic gases pass into the lungs instead of the stomach. This is impossible if the fistula connects to the carina or a mainstem bronchus. In these situations, intermittent venting of a gastrostomy tube may permit positivepressure ventilation without excessive gastric distention. Suctioning of the gastrostomy tube and upper esophageal pouch tube helps prevent aspiration pneumonia. Surgical division of the fistula and esophageal anastomosis is performed via a right extrapleural thoracotomy with the patient in the left lateral position. A precordial stethoscope should be placed in the dependent (left) axilla, since obstruction of the mainstem bronchus during surgical retraction is not uncommon. A drop in oxygen saturation indicates that the retracted lung needs to be reexpanded. Surgical retraction can also compress the great vessels, trachea, heart, and vagus nerve. Blood pressure should be continuously monitored with an arterial line. These infants often require ventilation with 100% oxygen. Blood should be immediately available for transfusion. Postoperative complications include gastroesophageal reflux, aspiration pneumonia, tracheal compression, and anastomotic leakage. Most patients must remain intubated and receive positive-pressure ventilation in the immediate postoperative period. Neck extension and instrumentation (eg, suctioning) of the esophagus may disrupt the surgical repair and should be avoided.

Gastroschisis and omphalocele are congenital disorders characterized by defects in the abdominal wall that allow external herniation of viscera. Omphaloceles occur at the base of the umbilicus, have a hernia sac, and are often associated with other congenital anomalies such as trisomy 21, diaphragmatic hernia, and cardiac and bladder malformations. In contrast, the gastroschisis defect is usually lateral to the umbilicus, does not have a hernia sac, and is often an isolated finding. Antenatal diagnosis by ultrasound can be followed by elective cesarean section at 38 weeks and immediate surgical repair. Perioperative management focuses on preventing hypothermia, infection, and dehydration. These problems are usually more serious in gastroschisis, as the protective hernial sac is absent.

The stomach is decompressed with a nasogastric tube before induction. Intubation can be accomplished with the patient awake or anesthetized and with or without muscle relaxation. Nitrous oxide should be avoided. Muscle relaxation is required for replacing the bowel into the abdominal cavity. A onestage closure (primary repair) is often not advisable, as it can cause an abdominal compartment syndrome. A staged closure with a temporary Silastic "silo" may be necessary, followed by a second procedure a few days later for complete closure. Suggested criteria for a staged closure include intragastric or intravesical pressure greater than 20 cm H,O, peak inspiratory pressure greater than 35 cm H,O, or an end-tidal CO, greater than 50 mm Hg. The neonate remains intubated after the procedure and is weaned from the ventilator over the next 1 to 2 days in the ICU.

Hypertrophic pyloric stenosis impedes emptying of gastric contents. Persistent vomiting depletes potassium, chloride, hydrogen, and sodium ions, causing hypochloremic metabolic alkalosis. Initially, the kidney tries to compensate for the alkalosis by excreting sodium bicarbonate in the urine. Later, as hyponatremia and dehydration worsen, the kidneys must conserve sodium even at the expense of hydrogen ion excretion (paradoxic aciduria). Correction of the volume and ion deficits and metabolic alkalosis is an indication for hydration with a sodium chloride (rather than lactated Ringer's) solution supplemented with potassium chloride.

Surgery should be delayed until fluid and electrolyte abnormalities have been corrected. Operation for correction of pyloric stenosis is never an emergency.
The stomach should be emptied with a nasogastric or orogastric tube; the tube should be suctioned with the patient in the supine and lateral positions. Diagnosis often requires contrast radiography, and all contrast media must be suctioned from the stomach before induction. Techniques for intubation and induction vary, but in all cases the patient's increased risk of aspiration must be considered. Experienced clinicians have variously advocated awake intubation, rapid sequence intravenous induction, and even careful inhalation induction in selected patients. Pyloromyotomy typically is a short procedure that may require muscle relaxation. These neonates may be at increased risk for respiratory depression and hypoventilation in the recovery room because of persistent metabolic (measurable in arterial blood) or cerebrospinal fluid alkalosis (despite neutral arterial pH).

Croup is obstruction of the airway characterized by a barking cough. One type of croup, postintubation croup, has already been discussed. Another type is due to viral infection. Infectious croup usually follows a viral URI in children aged 3 months to 3 years. The airway below the epiglottis is involved (laryngotracheobronchitis). Infectious croup progresses slowly and rarely requires intubation. Foreign body aspiration is typically encountered in children aged 6 months to 5 years. Commonly aspirated objects include peanuts, coins, small batteries, screws, nails, tacks, and small pieces of toys. Onset is typically acute and the obstruction may be supraglottic, glottic, or subglottic. Stridor is prominent with the first two, whereas wheezing is more common with the latter. A clear history of an aspiration may be absent. Acute epiglottitis is a bacterial infection (most commonly Haemophilus influenzae type B) classically affecting 2to 6-year-old children but also occasionally appearing in older children and adults. It rapidly progresses from a sore throat to dysphagia and complete airway obstruction. The term supraglottitis has been suggested because the inflammation typically involves all supraglottic structures. Endotracheal intubation and antibiotic therapy can be lifesaving. Epiglottitis has increasingly become a disease of adults because of the widespread use of H influenzae vaccines in children.

Pathophysiology
Lymphoid hyperplasia can lead to upper airway obstruction, obligate mouth breathing, and even pulmonary hypertension with cor pulmonale. Although these extremes of pathology are unusual, all children undergoing tonsillectomy or adenoidectomy should be considered to be at increased risk for perioperative airway problems.

Surgery should be postponed if there is evidence of acute infection or suspicion of a clotting abnormality. Administration of an anticholinergic agent will decrease pharyngeal secretions. A history of airway obstruction or apnea suggests an inhalational induction without paralysis until the ability to ventilate with positive pressure is established. A reinforced or preformed endotracheal tube (eg, RAE tube) may decrease the risk of kinking by the surgeon's selfretaining mouth gag. Blood transfusion is rarely necessary, but one must be wary of occult blood loss. Gentle inspection and suctioning of the pharynx precede extubation. Although deep extubation decreases the chance of laryngospasm and may prevent blood clot dislodgment from coughing, an awake extubation is generally preferred to reduce the likelihood of aspiration. Postoperative vomiting is common and gastric suctioning is usually performed prior to extubation. One must be alert in the recovery room for postoperative bleeding, signs of which may include restlessness, pallor, tachycardia, or hypotension. If reoperation is necessary to control bleeding, intravascular volume must first be restored. Evacuation of stomach contents with a nasogastric tube is followed by a rapidsequence induction. Because of the possibility of bleeding and airway obstruction, children younger than 3 years old may be hospitalized for the first postoperative night. Sleep apnea and recent infection increase the risk of postoperative complications and may necessitate admission.

Children presenting for myringotomy and insertion of tympanostomy tubes have a long history of URIs that have spread through the eustachian tube, causing repeated episodes of otitis media. Causative organisms are usually bacterial and include pneumococcus, H influenzae, Streptococcus, and Mycoplasma pneumoniae. Myringotomy, a radial incision in the tympanic membrane, releases any fluid that has accumulated in the middle ear. Tympanostomy tubes provide long-term drainage. Because of the chronic and recurring nature of this illness, it is not surprising that these patients often have symptoms of a URI on the day of scheduled surgery.

These are typically very short (10-15 min) outpatient procedures. Inhalational induction is a common technique. Unlike tympanoplasty surgery, nitrous oxide diffusion into the middle ear is not a concern during myringotomy because of the brief period of anesthetic exposure before the middle ear is vented. Because most of these patients are otherwise healthy and there is no blood loss, intravenous access is usually not necessary. Ventilation with a face mask or LMA minimizes the risk of perioperative respiratory complications (eg, laryngospasm) associated with intubation.

An additional chromosome 21—part or whole—results in the most common pattern of congenital human malformation: Down syndrome. Characteristic abnormalities of interest to the anesthesiologist include a short neck, atlantooccipital instability, irregular dentition, mental retardation, hypotonia, and a large tongue. Associated abnormalities include congenital heart disease in 40% of patients (particularly endocardial cushion defects and ventricular septal defect), subglottic stenosis, tracheoesophageal fistula, chronic pulmonary infections, and seizures. These neonates are often premature and small for their gestational age. Later in life many patients with Down syndrome undergo multiple procedures requiring general anesthesia.

Because of anatomic differences, these patients often have difficult airways, particularly during infancy. The size of the endotracheal tube required is typically smaller than that predicted by age. Respiratory complications such as postoperative stridor and apnea are common. Neck flexion during laryngoscopy and intubation may result in atlantooccipital dislocation because of the congenital laxity of these ligaments. The possibility of associated congenital diseases must always be considered. As in all pediatric patients, care must be taken to avoid air bubbles in the intravenous line because of possible right-to-left shunts and paradoxical air emboli.

Cystic fibrosis is a genetic disease of the exocrine glands primarily affecting the pulmonary and gastrointestinal systems. Abnormally thick and viscous secretions coupled with decreased ciliary activity lead to pneumonia, wheezing, and bronchiectasis. Pulmonary function studies reveal increased residual volume and airway resistance with decreased vital capacity and expiratory flow rate. Malabsorption syndrome may lead to dehydration and electrolyte abnormalities.

Anticholinergic drugs have been used without ill effects, and the choice whether to use them appears to be inconsequential. Induction with inhalational anesthetics may be prolonged in patients with severe pulmonary disease. Intubation should not be performed until the patient is deeply anesthetized to avoid coughing and stimulation of mucus secretions. The patient's lungs should be suctioned during general anesthesia and before extubation to minimize the accumulation of secretions. Outcome is favorably influenced by preoperative and postoperative respiratory therapy that includes bronchodilators, incentive spirometry, postural drainage, and pathogen-specific antibiotic therapy.

Scoliosis is lateral rotation and curvature of the vertebrae and a deformity of the rib cage. It can have many etiologies, including idiopathic, congenital, neuromuscular, and traumatic. Scoliosis can affect cardiac and respiratory function. Elevated pulmonary vascular resistance from chronic hypoxia causes pulmonary hypertension and right ventricular hypertrophy. Respiratory abnormalities include reduced lung volumes and chest wall compliance. Pao, is reduced as a result of ventilation/perfusion mismatching, whereas an increased

Preoperative evaluation may include pulmonary function tests, arterial blood gases, and electrocardiography. Corrective surgery is complicated by prone positioning, and by the possibility of major blood loss and of paraplegia. Spinal cord function can be assessed by neurophysiological monitoring (somatosensory and motor evoked potentials, see Chapters 6 and 26) or by awakening the patient intraoperatively to test lower limb muscle strength. Patients with severe respiratory disease may remain ® intubated postoperatively. Patients with scoliosis due to muscular dystrophy are predisposed to malignant hyperthermia, cardiac arrhythmias, and untoward effects of succinylcholine (hyperkalemia, myoglobinuria, and sustained muscular contractures).

Bhananker SM, Ramamoorthy C, Geiduschek JM, et al. Anesthesia-related
Smart Tots. http://www.smarttots.org/.

@ In the absence of coexisting disease, resting systolic cardiac function seems to be preserved, even in octogenarians. Increased vagal tone and decreased sensitivity of adrenergic receptors lead to a decline in heart rate.
@ Elderly patients undergoing echocardiographic evaluation for surgery have an increased incidence of diastolic dysfunction compared with younger patients.
@® Diminished cardiac reserve in many elderly patients may be manifested as exaggerated decreases in blood pressure during induction of general anesthesia. A prolonged circulation time delays the onset of intravenous drugs, but speeds induction with inhalational agents.
@ Aging decreases the elasticity of lung tissue, allowing overdistention of alveoli and collapse of small airways. Residual volume and the functional residual capacity increase with aging. Airway collapse increases residual volume and closing capacity. Even in normal persons, closing capacity exceeds functional residual capacity at age 45 years in the supine position and age 65 years in the sitting position.
@ The neuroendocrine response to stress seems to be largely preserved or, at most, only slightly decreased in healthy elderly patients. Aging is associated with a decreasing response to B-adrenergic agents.
@ Impairment of Na* handling, concentrating ability, and diluting capacity predispose elderly patients to both dehydration and fluid overload.
@ Liver mass and hepatic blood flow decline with aging. Hepatic function declines in proportion to the decrease in liver mass.
© Aging produces both pharmacokinetic and pharmacodynamic changes. Disease-related changes and wide variations among individuals in
similar populations prevent convenient generalizations.
@ The principal pharmacodynamic change associated with aging is a reduced anesthetic requirement, represented by a reduced minimum alveolar concentration (MAC).
@ Elderly patients display a lower dose requirement for propofol, etomidate, opioids, benzodiazepines, and barbiturates.
The elderly patient typically presents for surgery with multiple chronic medical conditions, in addition to any acute surgical illness. Age is not a contraindication to anesthesia and surgery; however, perioperative morbidity and mortality are greater in elderly than younger surgical patients.

As with pediatric patients, optimal anesthetic management of geriatric patients depends upon an understanding of the normal changes in physiology, anatomy, and response to pharmacological agents that accompany aging. In fact, there are many similarities between elderly and pediatric patients (Table 43-1). Individual genetic polymorphisms and lifestyle choices can modulate the inflammatory response, which contributes to the development of many systemic diseases. Consequently, chronologic age may not fully reflect an individual patient's true physical condition. The relatively high frequency of serious physiological abnormalities in elderly patients demands a particularly careful preoperative evaluation.

Decreased ability to increase heart rate in response to hypovolemia, hypotension, or hypoxia
Decreased lung compliance
Decreased arterial oxygen tension
Impaired ability to cough
Decreased renal tubular function
Increased susceptibility to hypothermia
Elderly patients are frequently treated with B-blockers. B-Blockers should be continued perioperatively, if patients are taking such medications chronically, to avoid the effects of B-blocker withdrawal. A careful review of patients' often extensive medication lists can reveal the routine use of oral hypoglycemic agents, angiotensin-converting enzyme inhibitors or angiotensin receptor blockers, antiplatelet agents, statins, and anticoagulants. Because elderly patients frequently take multiple drugs for multiple conditions, they often benefit from an evaluation before the day of surgery, even when scheduled for outpatient surgery. Preoperative laboratory studies should be guided by patient condition and history. Patients who have cardiac stents requiring antiplatelet therapy present particularly vexing problems. Their management should be closely coordinated among the surgeon, cardiologist, and anesthesiologist and adhere to appropriate management guidelines (see Chapter 21). At no time should the anesthesia staff discontinue antiplatelet/anticoagulant therapy without discussing the plan with the patient's primary physicians.

To promote quality improvement in geriatric surgical care, extensive best practice guidelines have been issued by the American College of Surgeons National Surgical Quality Improvement Program (NSQIP) and the American Geriatrics Society (AGS). These guidelines provide a systematic approach to perioperative geriatric care. In particular they require the care team to confirm that the patient's wishes regarding treatment preferences and advanced directives are understood and documented. A checklist to ensure optimal preoperative assessment in the geriatric surgical patient is suggested (Table 43-2).

In addition to conducting a complete history and physical examination of the patient, the following assessments are strongly recommended:
a Assess the patient's cognitive ability and capacity to understand the anticipated surgery.
a Screen the patient for depression.
a Identify the patient's risk factors for developing postoperative delirium.
a Screen for alcohol and other substance abuse/dependence.
a
Perform a preoperative cardiac evaluation according to the American College of Cardiology/American Heart Association algorithm for patients undergoing noncardiac surgery.
Identify the patient's risk factors for postoperative pulmonary complications and implement appropriate strategies for prevention.
a Document functional status and history of falls.
a Determine baseline frailty score.
a Assess patient's nutritional status and consider preoperative interventions if the patient is at severe nutritional risk.
a Take an accurate and detailed medication history and consider appropriate perioperative adjustments. Monitor for polypharmacy.
a Determine the patient's treatment goals and expectations in the context of the possible treatment outcomes.
a Determine patient's family and social support system.
a Order appropriate preoperative diagnostic tests focused on elderly patients.
lReproduced with permission from Chow W, Rosenthal R, Merkow R, et al. Optimal preoperative assessment of the geriatric surgical patient: A best practice guideline from the American College of Surgeons National Surgical Quality Improvement Program and the American Geriatrics Society. J Am Coll Surg. 2012 Oct;215(4):453-466.
A cognitive assessment such as the Mini Cog examination is recommended for patients who do not have a history of dementia or cognitive impairment (Figure 43-1). In addition to screening for cognitive impairment, depression screening should also be conducted. Frailty reflects a decrease in functional reserve capacity and an inability to respond to the physiological challenges presented by the stress of surgery. Frailty can be assessed using one of several scoring systems available. A simple scoring system from Makary et al is presented in 'Table 43-3.

Look directly at person and say, "Please listen carefully. | am going to say three words that | want you to repeat back to me now and try to remember. The words are [select a list of words from the versions below]. Please say them for me now." If the person is unable to repeat the words after three attempts, move on to Step 2 (clock drawing).
The following and other word lists have been used in one or more clinical studies.'* For repeated administrations, use of an alternative word list is recommended.
Version 1 Banana Sunrise Chair
Version 2 Leader Season Table
Version 3 Village Kitchen Baby
Version 4 River Nation Finger
Version 5 Captain Garden Picture
Version 6 Daughter Heaven Mountain

Say. "Next, | want you to draw a clock for me. First, put in all of the numbers where they go." When that is completed, say. "Now, set the hands to 10 past 11."
Use preprinted circle (see next page) for this exercise. Repeat instructions as needed as this is not a memory test. Move to Step 3 if the clock is not complete within three minutes.

Ask the person to recall the three words you stated in Step 1. Say. "What were the three words | asked you to remember?" Record the word list version number and the person's answers below.
Word List Version:
______
Person's Answers:

Word Recall:
(0-3 points)
1 point for each word spontaneously recalled without cueing.
Normal clock = 2 points. A normal clock has all numbers placed in the cor- rect sequence and approximately correct position (e.g., 12, 3,6 and 9 are in anchor positions) with no missing or duplicate numbers. Hands are point- ing to the 11 and 2 (11:10). Hand length is not scored. Inability or refusal to draw a clock (abnormal) = 0 points.
. ______(Oor2 points)
Clock Draw:
Total score = Word Recall score + Clock Draw score.
Total Score:
(0-5 points)
Accut point of <3 on the Mini-Cog™ has been validated for dementia screening, but many individuals with clinically meaningful cognitive impairment will score higher. When greater sensitivity is desired, a cut point of <4 is recom- mended as it may indicate a need for further evaluation of cognitive status.

1. Borson S, Scanlan JM, Chen PJ et al. The Mini-Cog as a screen for dementia: Validation in a population based sample. J Am Geriatr Soc 2003;51:1451-1454.
2. Borson S, Scanlan JM, Watanabe J et al. Improving identification of cognitive impairment in primary care. Int J Geriatr Psychiatry 2006;21: 349-355.
3. Lessig M, Scanlan J et al. Time that tells: Critical clock-drawing errors for dementia screening. Int Psychogeriatr. 2008 June; 20(3): 459-470.
FIGURE 43-1 Cognitive assessment with the Mini-Cog: 3-item recall, clock draw, and interpretation. (Mini-Cog™ © S. Borson. All rights reserved. Reprinted with permission of the author solely for clinical and educational purposes. May not be modified or used for commercial, marketing, or research purposes without permission of the author (soob@uw.edu). v. 01.19.16.)
TABLE 43-3 Frailty score (operational definition).1
Shrinkage, Definition = Unintentional weight loss =10 past year. Weakness, Definition = Decreased grip strength. Exhaustion, Definition = Self-reported poor energy and endurance. Low physical activity, Definition = | Low weekly energy expenditure. Slowness, Definition = Slow walking
'Data from Makary MA, Segev DL, Pronovost PJ, et al. Frailty asa predictor of surgical outcomes in older patients. J Am Coll Surg. 2010 Jun;210(6):901-908.
*The patient receives 1 point for each criterion met: 0-1, not frail; 2-3, intermediate frail (pre-frail); 4-5, frail.
Optimal preoperative assessment provides the care team with information to identify patients at high risk for adverse perioperative outcomes and to enact strategies to mitigate those risks where possible. Moreover, such assessment permits realistic discussions of likely patient outcomes when considering surgical interventions.

Cardiovascular diseases are more prevalent in the geriatric than general population. Still, it is important to distinguish between changes in physiology that normally accompany aging and the pathophysiology of diseases common in the geriatric population (Table 43-4). For example, atherosclerosis is pathological—it is not present in healthy elderly patients. On the other hand, a reduction in arterial elasticity caused by fibrosis of the media is part of the normal aging process. Changes in the cardiovascular system that accompany aging include decreased vascular and myocardial compliance and autonomic responsiveness. In addition to myocardial fibrosis, calcification of the valves can occur. Elderly patients with systolic murmurs should be suspected of having @ aortic stenosis. However, in the absence of coexisting disease, resting systolic cardiac function seems to be preserved, even in octogenarians. Functional capacity of less than 4 metabolic equivalents (METS) is associated with potential adverse outcomes (see Chapter 21). Increased vagal tone and decreased sensitivity of adrenergic receptors lead to a decline in heart rate; maximal heart rate declines by approximately 1 beat/min per year of age over 50. Fibrosis of the conduction system and loss of sinoatrial node cells increase the incidence of dysrhythmias, particularly atrial fibrillation and flutter. Preoperative risk assessment and evaluation of the patient with cardiac disease were previously reviewed in this text (see Chapters 18, 20, and 21). Age per se does not mandate any particular battery of tests or evaluative tools, although there is a long tradition of routinely requesting tests such as 12-lead electrocardiography in patients who are older than a defined age. Additionally, the NSQIP/AGS guidelines recommend that patient hemoglobin, kidney function tests, and albumin be determined (Table 43-5).

Cardiovascular Decreased arterial elasticity Elevated afterload Elevated systolic blood pressure Left ventricular hypertrophy Decreased adrenergic activity Decreased resting heart rate Decreased maximal heart rate, Common Pathophysiology = Atherosclerosis Coronary artery disease Essential hypertension Congestive heart failure Cardiac arrhythmias Aortic stenosis. Respiratory, Common Pathophysiology = . Decreased pulmonary elasticity Decreased alveolar surface area Increased residual volume Increased closing capacity Ventilation/perfusion mismatching Decreased arterial oxygen tension Increased chest wall rigidity Decreased muscle strength Decreased cough Decreased maximal breathing capacity Blunted response to hypercapnia and hypoxia, Common Pathophysiology = Emphysema Chronic bronchitis Pneumonia. Renal, Common Pathophysiology = . Decreased renal blood flow Decreased renal plasma flow Decreased glomerular filtration rate Decreased renal mass Decreased tubular function Impaired sodium handling Decreased concentrating ability Decreased diluting capacity Impaired fluid handling Decreased drug excretion Decreased renin-aldosterone responsiveness Impaired potassium excretion, Common Pathophysiology = Diabetic nephropathy Hypertensive nephropathy Prostatic obstruction Congestive heart failure

Hemoglobin, Indications = Recommended for all geriatric surgical patients, especially those: Undergoing operations with anticipated clinically significant blood loss or transfusion requirement. With suspected or known severe anemia. Renal function tests (blood urea nitrogen, creatinine), Indications = Recommended for all geriatric surgical patients, especially those: Undergoing major surgery” With diabetes, hypertension, cardiovascular disease, or who use medications that affect renal function ACE inhibitors, NSAID®. Serum albumin, Indications = Recommended for all geriatric surgical patients, especially those: With known liver disease, multiple serious chronic illnesses, and recent major illness Undergoing major surgery Likely to have malnutrition
"Reproduced with permission from Chow W, Rosenthal R, Merkow R, et al. Optimal preoperative assessment of the geriatric surgical patient: A best practice guideline from the American College of Surgeons National Surgical Quality Improvement Program and the American Geriatrics Society. JAm Coll Surg. 2012 Oct;215(4):453-466
2Major surgery includes cardiac, vascular, thoracic, and abdominal operations.
ACE, angiotensin-converting enzyme; NSAID, nonsteroidal antiinflammatory drug.
FIGURE 43-2 A: In this Doppler study of diastolic inflow, the E wave is seen with a peak velocity of 90.9 cm/s. This Doppler study reflects the velocity of blood as it fills the left ventricle early in diastole. B: In tissue Doppler, the velocity of the movement of the lateral annulus of the mitral valve is measured. The E' wave in this image is 6.95 cm/s. This corresponds to the movement of the myocardium during diastole. (Reproduced with permission from Wasnick J, Hillel Z, Kramer D, et al. Cardiac Anesthesia & Transesophageal Echocardiography. New York, NY: McGraw-Hill; 2011.)

Marked diastolic dysfunction may be seen with systemic hypertension, coronary artery disease, cardiomyopathies, and valvular heart disease (particularly aortic stenosis), all of which are more common in older than younger patients. Patients may be asymptomatic or complain of exercise intolerance, dyspnea, cough, or fatigue. Diastolic dysfunction results in relatively large increases in ventricular end-diastolic pressure, with small changes of left ventricular volume; the atrial contribution to ventricular filling becomes even more important than in younger patients. Atrial enlargement predisposes patients to atrial fibrillation and flutter. Patients are at increased risk of developing congestive heart failure. The elderly patient with diastolic dysfunction may poorly tolerate perioperative fluid administration, resulting in elevated left ventricular end-diastolic pressure and pulmonary congestion. Diminished cardiac reserve in many elderly patients may be manifested as exaggerated decreases in blood pressure during induction of general anesthesia. A prolonged circulation time delays the onset of intravenous drugs, but speeds induction with inhalational agents. Like infants, elderly patients have less ability to respond to hypovolemia, hypotension, or hypoxia with an increase in heart rate. Ultimately, cardiovascular diseases, including heart failure, stroke, arrhythmias, and hypertension, contribute to an increased risk of morbidity, mortality, increased cost of care, and frailty in elderly patients.

@ Aging decreases the elasticity of lung tissue, allowing overdistention of alveoli and collapse of small airways. Residual volume and the functional residual capacity increase with aging. Airway collapse increases residual volume and closing capacity. Even in normal persons, closing capacity exceeds functional residual capacity at age 45 years in the supine position and age 65 years in the sitting position. When this happens, some airways close during part of normal tidal breathing, resulting in a mismatch of ventilation and perfusion. The additive effect of these changes variably decreases arterial oxygen tension. Both anatomic and physiological dead space increase. Other pulmonary effects of aging are summarized in Table 43-4.
Decreased respiratory muscle function/mass, a less compliant chest wall, and intrinsic changes in lung function can increase the work of breathing and make it more difficult for elderly patients to muster a respiratory reserve in settings of acute illness (eg, infection). Many patients also present with obstructive or restrictive lung diseases. In patients who have no intrinsic pulmonary disease, gas exchange is unaffected by aging.
Measures to prevent perioperative hypoxia in elderly patients include a longer preoxygenation period prior to induction, increased inspired oxygen concentrations during anesthesia, positive end-expiratory pressure, and pulmonary toilet. Aspiration pneumonia is a potentially life-threatening complication in elderly patients. Ventilatory impairment in the recovery room is more common in elderly than younger patients. Factors associated with an increased risk of postoperative pulmonary complications include older age, chronic obstructive pulmonary disease, sleep apnea, malnutrition, and abdominal or thoracic surgical incisions.

Basal and maximal oxygen consumption declines with age. After reaching peak weight at about age 60 years, most men and women begin losing weight; the average elderly man and woman weigh less than their younger counterparts. Heat production decreases, heat loss increases, and hypothalamic temperatureregulating centers may reset at a lower level.
Diabetes affects approximately 15% of patients older than age 70 years. Its impact on numerous organ systems can complicate perioperative management. Diabetic neuropathy and autonomic dysfunction are particular problems for the elderly.
Increasing insulin resistance leads to a progressive decrease in the ability to avoid hyperglycemia with glucose loads. Institutions typically have their own protocols on how to manage increased blood glucose perioperatively, and these protocols reflect the changing literature on appropriate blood glucose targets. Attempts to maintain blood glucose within a strictly normal range during surgery, anesthesia, or critical illness may lead to hypoglycemia and adverse outcomes. Anesthesia practitioners are advised to be aware of changing performance benchmarks related to this measure.

Kidney blood flow and kidney mass (eg, glomerular number and tubular length) decrease with age. Kidney function, as determined by glomerular filtration rate and creatinine clearance, is reduced (Table 43-4). The serum creatinine level is unchanged because of a decrease in muscle mass and creatinine production, whereas blood urea nitrogen gradually @ increases with aging. Impairment of Na'* handling, concentrating ability, and diluting capacity predispose elderly patients to both dehydration and fluid overload. The response to antidiuretic hormone and aldosterone is reduced. The ability to reabsorb glucose is decreased. The combination of reduced kidney blood flow and decreased nephron mass in elderly patients increases the risk of acute kidney failure in the postoperative period, particularly when patients are exposed to nephrotoxic drugs and techniques.
As kidney function declines, so does the kidney's ability to excrete drugs. The decreased capacity to handle water and electrolyte loads makes proper fluid management more critical; elderly patients are more predisposed to developing hypokalemia and hyperkalemia. This is further complicated by the common use of diuretics in the elderly population. The search is ongoing for drugs that might protect the kidney perioperatively, as well as for specific genetic profiles of patients at greater risk of perioperative kidney injury.

@ Liver mass and hepatic blood flow decline with aging. Hepatic function declines in proportion to the decrease in liver mass. Thus, the rate of biotransformation and albumin production decreases. Plasma cholinesterase levels are reduced in elderly men. Malnutrition is associated with adverse surgical outcomes. Nutritional screening should be a part of preoperative assessment. NSQIP/AGS guidelines, in particular, note a severe nutritional risk is present when:
1. Body mass index (BMI) is less than 18.5 kg/m?
2. Serum albumin is less than 3 g/dL
3. Unintended weight loss is greater than 10% within 6 months

Brain mass decreases with age; neuronal loss is prominent in the cerebral cortex, particularly the frontal lobes. Cerebral blood flow also decreases about 10% to 20% in proportion to neuronal losses. It remains tightly coupled to metabolic rate, and autoregulation is intact. Neurons lose complexity of their dendritic tree and the number of synapses. The synthesis of neurotransmitters is reduced. Serotonergic, adrenergic, and y-aminobutyric acid (GABA) binding sites are also
TABLE 43-6 Predisposing and precipitating factors for delirium after surgery.!
'Reproduced with permission from Rudoph J, Marcantonio E. Postoperative delirium: acute change with long term implications, Anesth Analg. 2011 May;) 12(5):1202-1211.
TABLE 43-7 Prevention of delirium after surgery.!
Cognitive stimulation, Postoperative Intervention = Orientation (clock, calendar, orientation board) Avoid cognitively active medications. Improve sensory input, Postoperative Intervention = Glasses Hearing aids/amplifiers. Mobilization, Postoperative Intervention = Early mobilization and rehabilitation. Avoidance of psychoactive medication, Postoperative Intervention = Elimination of unnecessary medications Pain management protocol. Fluid and nutrition, Postoperative Intervention = Fluid management Electrolyte monitoring and repletion Adequate nutrition protocol. Avoidance of hospital complications, Postoperative Intervention = Bowel protocol Early removal of urinary catheters Adequate central nervous system O, delivery, including supplemental O, and transfusion for very low hematocrit Postoperative complication monitoring protocol
'Reproduced with permission from Rudoph J, Marcantonio E. Postoperative delirium: acute change with long term implications. Anesth Analg. 2011 May;112(5):1202-1211.
Additionally, the AGS has developed guidelines for the prevention and treatment of postoperative delirium in older adults. These guidelines suggest that nonopioid analgesia techniques be employed where possible to reduce the incidence of postoperative delirium. Additionally, they recommend avoidance of meperidine, drugs with anticholinergic effects, and benzodiazepines. Some studies have suggested that lighter planes of general anesthesia are associated with a reduction in postoperative delirium.

Elderly patients often take more time to recover from the central nervous system effects of general anesthesia, especially if they were confused or disoriented preoperatively. This is important in outpatient surgery when the lack of a caretaker at home may necessitate that patients assume a higher level of self-care. In the absence of disease, any perioperative decrease in cognitive function is normally modest. Short-term memory seems to be most affected. Continued physical and intellectual activity seems to have a positive effect on preservation of cognitive functions.
The etiology of POCD is likely multifactorial and includes drug effects, pain, underlying dysfunction, hypothermia, and metabolic disturbances. Elderly patients are particularly sensitive to centrally acting anticholinergic agents, such as scopolamine and atropine. Some patients experience prolonged or permanent POCD after surgery and anesthesia. Some studies suggest that POCD can be detected in 10% to 15% of patients older than age 60 years up to 3 months following major surgery. In some settings (eg, following cardiac and major orthopedic procedures), intraoperative arterial emboli may be contributory. Animal studies suggest that anesthesia without surgery can impair learning for weeks, particularly in older animals. Elderly inpatients seem to have a significantly higher risk of POCD than elderly outpatients.

Muscle mass is reduced in elderly patients. With aging, skin atrophies and is more susceptible to trauma from removal of adhesive tape, electrocautery pads, and electrocardiographic electrodes. Veins are often frail and easily ruptured by intravenous infusions. Arthritic joints may interfere with positioning or regional anesthesia. Degenerative cervical spine disease can limit neck extension, potentially making intubation difficult.

@ Aging produces both pharmacokinetic (the relationship between drug dose and plasma concentration) and pharmacodynamic (the relationship between plasma concentration and clinical effect) changes. Disease-related changes and wide variations among individuals in similar populations prevent generalizations.
A progressive decrease in muscle mass and increase in body fat (particularly in older women) results in decreased total body water. The reduced volume of distribution for water-soluble drugs can lead to greater plasma concentrations; conversely, an increased volume of distribution for lipid-soluble drugs could theoretically reduce their plasma concentration. Any change in volume of distribution sufficient to significantly change concentrations will influence the elimination time. Because renal and hepatic functions decline with age, reductions in clearance the duration of action of
Distribution and elimination are also by any altered plasma protein concentrations. Albumin binds acidic drugs (eg, barbiturates, benzodiazepines, opioid agonists). a,-Acid glycoprotein binds basic drugs (eg, local anesthetics). Concentrations of these binding proteins may vary depending upon diseases associated with aging.
prolong many drugs. affected
® The principal pharmacodynamic change associated with aging is a reduced anesthetic requirement, represented by a reduced MAC. Careful titration of anesthetic agents helps to avoid adverse side effects and unexpected, prolonged duration; short-acting agents, such as propofol, desflurane, remifentanil, and succinylcholine, may be particularly useful in elderly patients. Drugs that are not significantly dependent on hepatic or renal function or blood flow, such as atracurium or cisatracurium, are useful.

The MAC for inhalational agents is reduced by 4% per decade of age over 40 years. Onset of action is faster if cardiac output is depressed, whereas it is delayed if there is a significant ventilation/perfusion abnormality. Recovery from anesthesia with a volatile anesthetic may be prolonged because of an increased volume of distribution (increased body fat) and decreased pulmonary gas exchange. Decreased hepatic function is of less importance as modern inhalational agents undergo little metabolism. Agents that are rapidly eliminated (eg, desflurane) are good choices for speeding emergence in the elderly patient.

@ In general, elderly patients display a lower dose requirement for propofol, etomidate, opioids, benzodiazepines, and barbiturates. The typical octogenarian will require a smaller induction dose of propofol than that required by a 20-yearold patient.
Although propofol may be close to an ideal induction agent in elderly patients because of its rapid elimination, it is more likely to cause apnea and hypotension than in younger patients. Both pharmacokinetic and pharmacodynamic factors are responsible for this enhanced sensitivity. Elderly patients require nearly 50%
lower blood levels of propofol for anesthesia than do younger patients.
Moreover, both the rapidly equilibrating peripheral compartment and systemic clearance for propofol are significantly reduced in elderly patients. The initial volume of distribution for etomidate significantly decreases with aging: lower doses are required to achieve the same electroencephalographic endpoint in elderly patients (compared with young patients).
Enhanced sensitivity to fentanyl, alfentanil, and sufentanil is primarily pharmacodynamic. Pharmacokinetics for these opioids are not significantly affected by age. Dose requirements for the same EEG endpoint using fentanyl and alfentanil are 50% lower in elderly patients.
Aging increases the volume of distribution for all benzodiazepines, which effectively prolongs their elimination half-lives. Enhanced pharmacodynamic sensitivity to benzodiazepines is also observed. Midazolam requirements are generally 50% less in elderly patients, and its elimination half-life is prolonged by about 50%.
Anticholinergic drugs and benzodiazepines are associated with an increased risk of postoperative delirium. Conversely, a single bolus of ketamine (0.5 mg/kg) has been suggested in one trial to reduce the incidence of delirium. Use of sedative and antinausea agents with anticholinergic and antidopaminergic properties may produce adverse effects in patients with Parkinson disease.

The response to succinylcholine is not altered by aging. Decreased cardiac output and reduced muscle blood flow, however, may cause up to a two-fold prolongation in the onset of neuromuscular blockade in elderly patients. Recovery from nondepolarizing muscle relaxants that depend on kidney excretion (eg, pancuronium) may be delayed due to decreased drug clearance. Likewise, decreased hepatic excretion from a loss of liver mass prolongs the elimination half-life and duration of action of rocuronium and vecuronium. The pharmacological profiles of atracurium and cisatracurium are not significantly affected by age.

How should this patient be evaluated for the risk of perioperative morbidity?
Anesthetic risk correlates much better with the presence of coexisting disease than chronological age. Therefore, preanesthetic evaluation should concentrate on the identification of age-related diseases (Table 43-4) and an estimation of physiological reserve. There is a tremendous physiological difference between a patient who walks three blocks to a grocery store on a regular basis and one who is bedridden, even though both may be the same age. Obviously, any condition that may be amenable to preoperative therapy (eg, bronchodilator administration) must be identified and addressed. On the other hand, lengthy delays may compromise surgical repair and increase overall morbidity.
What factors might influence the choice between regional and general anesthesia?

Advancing age is not a contraindication for either regional or general anesthesia. Each technique, however, has its advantages and disadvantages in the elderly population. For hip surgery, regional anesthesia can be achieved with a spinal or epidural block extending to the T10 sensory level. Both of these blocks require patient cooperation and the ability to lie still for the duration of the surgery. A paramedian approach may be helpful when optimal positioning is not possible Unless regional anesthesia is accompanied by heavy sedation, postoperative confusion and disorientation may be less troublesome than after general anesthesia. Cardiovascular changes are usually limited to a decrease in arterial blood pressure as sympathetic block is established. Although this decrease can be treated with fluid loading, a patient with borderline heart function may develop congestive heart failure when the block dissipates and sympathetic tone returns. Vasoconstrictors can be used to support the blood pressure during periods of sympathetic blockade. Reduced peripheral vascular resistance can result in profound hypotension and cardiac arrest in patients with aortic stenosis, a common valvular lesion in the elderly population. Likewise, patients can become profoundly hypotensive secondary to reduced peripheral vascular resistance accompanying neuraxial anesthesia. Invasive arterial pressure monitoring is at times useful when taking the elderly patient to surgery. Monitors of hemodynamic function using pulse contour analysis that estimate stroke volume variation in addition to transesophageal echocardiography can all be employed to guide fluid therapy. The benefits of transesophageal echocardiography must be considered in the context of the risks of esophageal rupture and mediastinitis in the elderly.
Are there any specific advantages or disadvantages to a regional technique in elderly patients having hip surgery?
A major advantage in regional anesthesia—particularly for hip surgery— is a lower incidence of postoperative thromboembolism. This is presumably due to peripheral vasodilation and maintenance of venous blood flow in the lower extremities. Many anesthesiologists believe that regional anesthesia maintains respiratory function better than general anesthesia. Unless the anesthetic level involves the intercostal musculature, ventilation and the cough reflex are well maintained. Studies conflict as to whether regional anesthesia offers a mortality advantage in the management of surgical treatment for fractures of the hip, and a randomized clinical trial is underway currently to answer this important question.

Technical problems associated with regional anesthesia in the elderly include obscured landmarks as a result of degeneration of the vertebral column and the difficulty of obtaining adequate patient positioning secondary to pain related to the fracture. To avoid having the patient lie on the fracture, a hypobaric or isobaric solution can be injected intrathecally. Postpuncture headache is less of a problem in the elderly population.
If the patient refuses regional anesthesia, is general anesthesia acceptable?
General anesthesia is an acceptable alternative to regional block. One advantage is that the patient can be induced in bed and moved to the operating room table after intubation, avoiding the pain of positioning.
What specific factors should be considered during induction and maintenance of general anesthesia with this patient?
It is important to remember that because a subtrochanteric fracture can be associated with more than 1 L of occult blood loss, induction with propofol may lead to an exaggerated decrease in arterial blood pressure. Initial hypotension may be replaced by hypertension and tachycardia during laryngoscopy
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@ Non-operating room anesthesia requires the anesthesia provider to work in remote locations in a hospital, where ease of access to the patient and anesthesia equipment may both be limited; furthermore, the support staff at these locations may be unfamiliar with the requirements for safe anesthetic delivery.
@ Anesthesia providers should confirm that both the infrastructure and operational policies are consistent with acceptable anesthesia practice standards before providing anesthesia in such settings.
© In general, ambulatory procedures should be of a complexity and duration such that one could reasonably assume that the patient will make an expeditious recovery and not require post-procedure hospital admission.
@ Factors considered in selecting patients for ambulatory procedures include systemic illnesses and their current management, airway management problems, sleep apnea, morbid obesity, previous adverse anesthesia outcomes (eg, malignant hyperthermia), allergies, and the patient's social network (eg, availability of someone to be responsive to the patient for 24 h).
Outpatient/ambulatory anesthesia is the subspecialty of anesthesiology that deals with the preoperative, intraoperative, and postoperative anesthetic care of patients undergoing elective, same-day surgical procedures. Patients undergoing ambulatory surgery rarely require admission to a hospital and are fit enough to be discharged from the surgical facility less than 24 hours after the procedure.

In recent years, patients with increasing burdens of comorbid conditions have presented for ambulatory procedures. Each patient must be considered in the context of his or her comorbidities, the type of surgery to be performed, and the expected response to @ anesthesia. In general, ambulatory procedures should be
patient in question. Likewise, procedures suitable for ambulatory surgery should have a minimal risk of perioperative hemorrhage, airway compromise, and no particular requirement for specialized postoperative care. Based on risk identification, the anesthesiologist should be able to reduce the likelihood of unforeseen AEs. Although current evidence-based medicine can provide recommendations for some high-risk ambulatory issues, evidence is lacking for most such situations.

Obesity is associated with many concomitant disease states, such as hypertension, diabetes, hyperlipidemia, the combination of the preceding three disease states (metabolic syndrome), and obstructive sleep apnea (OSA). The physiological derangements that accompany these conditions include changes in oxygen demand, carbon dioxide production, alveolar ventilation, and cardiac output. There is no precise "cutoff" body mass index (BMI) for patients who may or may not undergo ambulatory surgery. However, Joshi and colleagues suggest that patients with a BMI less than 40 kg/m? tolerate ambulatory surgery adequately, assuming control of comorbidities. Conversely, patients with a BMI greater than 50 kg/m? are thought to be at greater risk in the ambulatory surgical care environment. Patients with obesity and OSA are at increased risk of postoperative respiratory complications, such as prolonged airway obstruction and apnea, particularly if they will receive opioids postoperatively. Scores for predicting the probability of these complications can aid in the preoperative assessment and referral to a hospital setting (Tables 44—1 and 44—2). However, the ASA guidelines note that the literature is insufficient to offer guidance as to which OSA patients can be safely managed on an inpatient versus an outpatient basis. Although a sleep study is the standard way to diagnose sleep apnea, many patients with OSA have never been identified as having OSA. Consequently, an anesthesiologist may be the first physician to detect the presence or risk of sleep apnea. Preoperative initiation of continuous positive airway pressure (CPAP) may reduce the incidence of postoperative cardiac complications, according to ASA guidelines. Avoidance of respiratory depressants to the degree possible through the use of opioid-sparing multimodal analgesia, neuraxial, and regional anesthetic techniques is likewise suggested when appropriate. In addition to the usual discharge criteria, the ASA also recommends that patients at increased risk from OSA not be discharged to an unmonitored setting until they no longer are at risk for perioperative respiratory depression. Other ASA recommendations include:

* Return of room air oxygen saturation to baseline level prior to discharge
* Observation of respiratory function when unstimulated, such as when sleeping
* Consideration of CPAP or noninvasive positive-pressure ventilation (NIPPV) if frequent airway obstruction or hypoxemia develops postoperatively
* A possible prolonged period of postoperative observation to ensure that patients with OSA are not at increased risk from postoperative respiratory depression compared with non-OSA patients undergoing similar procedures
TABLE 44-1 Identification and assessment of obstructive sleep apnea: example.!?

A. Clinical signs and symptoms suggesting the possibility of OSA
1, Predisposing physical characteristics
BMI 35 kg/m? [95th percentile for age and gender)'
Neck circumference 17 inches (men) or 16 inches (women) ry
Craniofacial abnormalities affecting the airway
Tonsils nearly touching or touching in the midline
Anatomical nasal obstruction pen
2. History of apparent airway obstruction during sleep (two or more of the following are present; if patient lives alone or sleep is not observed by another person, then only one of the following needs to be present)
|. Snoring (loud enough to be heard through closed door)
Observed pauses in breathing during sleep
Frequent snoring ose
Awakens from sleep with choking sensation
{Intermittent vocalization during sleep]
Frequent arousals from sleep >pa
|. {Parental report of restless sleep, difficulty breathing, or struggling respiratory efforts during sleep]*
somnolence (one or more of the following is present) no a
Frequent somnolence or fatigue despite adequate "sleep"
Falls asleep easily in a nonstimulating environment (e.g., watching TV, reading, riding in or driving a car) despite adequate "sleep" oe
¢. [Parent or teacher comments that child appears sleepy during the day, is easily distracted, is overly aggressive, or has difficulty concentrating)'
d. [Child often difficult to arouse at usual awakening time]?
If a patient has signs or symptoms in two or more of the above categories, there is a significant probability that he or she has OSA. The severity of OSA may be determined by sleep study (see below). If a sleep study is not available, such patients should be treated as though they have moderate sleep apnea unless one or more of the signs or symptoms above is severely abnormal (eg, markedly increased BMI or neck circumference, respiratory pauses that are frightening to the observer, patient regularly falls asleep within minutes after being left unstimulated), in which case they should be treated as though they have severe sleep apnea.

B. Ifasleep study has been done, the results should be used to determine the perioperative anesthetic management of a patient. However, because sleep laboratories differ in their criteria for detecting episodes of apnea and hypopnea, the Task Force believes that the sleep laboratory's assessment (none, mild, moderate, or severe) should take precedence over the actual AHI (the number of episodes of sleep-disordered breathing per hour). If the overall severity is not indicated, it may be determined by using the table below:
, 1 = 0-5. , 2 = 0. Mild OSA, 1 = 6-20. Mild OSA, 2 = 5. Moderate OSA, 1 = 21-40. Moderate OSA, 2 = 6-10. Severe OSA, 1 = >40. Severe OSA, 2 = >10
"Reproduced with permission from Gross JB, Bachenberg KL, Benumof JL, et al. Practice guidelines for the perioperative management of patients with obstructive sleep apnea: A report by the American Society of Anesthesiologists Task Force on Perioperative Management of patients with obstructive sleep apnea. Anesthesiology. 2006 May;104(5):1081.
*AHI, apnea-hypopnea index; BMI, body mass index OSA, obstructive sleep apnea; TV, television.
items in brackets refer to pediatric patients.
TABLE 44-2 Obstructive sleep apnea scoring system: example.)

A.,  = Severity of sleep apnea based on sleep study (or clinical indicators if sleep study not available). Point score (0-3)??. A., Points = . ,  = Severity of OSA (Table 44-1). , Points = . ,  = . , Points = 0. ,  = Mild. , Points = 1. ,  = Moderate. , Points = 2. ,  = Severe. , Points = 3. B.,  = Invasiveness of surgery and anesthesia. Point score (0-3). B., Points = . ,  = Type of surgery and anesthesia. , Points = . ,  = Superficial surgery under local or peripheral nerve block anesthesia without sedation. , Points = ). ,  = Superficial surgery with moderate sedation or general anesthesia. , Points = 1. ,  = Peripheral surgery with spinal or epidural anesthesia (with no more than moderate sedation). , Points = 1. ,  = Peripheral surgery with general anesthesia. , Points = ho. ,  = Airway surgery with moderate sedation. , Points = WWwWhN. ,  = Major surgery, general anesthesia. , Points = . ,  = Airway surgery, general anesthesia. , Points = 
C., 1 = Requirement for postoperative opioids. Point score (0-3) Opioid requirement. , 1 = Low-dose oral opioids. , 1 = High-dose oral opioids, parenteral or neuraxial opioids. D., 1 = Estimation of perioperative risk. Overall score = the score for A plus the greater of the score
'Reproduced with permission from Gross JB, Bachenberg KL, Benumof JL, et al. Practice guidelines for the perioperative management of patients with obstructive sleep apnea: A report by the American Society of Anesthesiologists Task Force on Perioperative Management of patients with obstructive sleep apnea. Anesthesiology. 2006 May;104(5):1081.
2A scoring system similar to this table may be used to estimate whether a patient is at increased perioperative risk of complications from obstructive sleep apnea (OSA). This example, which has not been clinically validated, is meant only as a guide, and clinical judgment should be used to assess the risk of an individual patient.

3One point may be subtracted if a patient has been on continuous positive airway pressure (CPAP) or noninvasive positive-pressure ventilation (NIPPV) before surgery and will be using his or her appliance consistently during the postoperative period.
"One point should be added if a patient with mild or moderate OSA also has a resting arterial carbon dioxide tension (PaCO,) greater than 50 mm Hg.
'Patients with score of 4 may be at increased perioperative risk from OSA; patients with a score or 5 or 6 may be at significantly increased perioperative risk from OSA,
The literature is insufficient to offer guidance regarding an appropriate time to discharge patients with OSA from the surgical facility.
The Society for Ambulatory Anesthesia has issued its own consensus statement regarding the management of OSA perioperatively (Table 44—3). This statement recommends the use of the STOP-Bang criteria for preoperative OSA screening. Additionally, the consensus statement provides a decision tree to assist in determining which known and presumed OSA patients are candidates for ambulatory surgery (Figure 44—1). The Society of Anesthesia and Sleep Medicine has also issued guidelines to assist in the screening for OSA. Their recommendations are summarized in Table 44—4. Perioperative complications associated with OSA are increasingly the subject of malpractice litigation.
Difficult airway management and cardiopulmonary arrest associated with death or brain injury are often the basis of such claims.

S = Snoring. Do you snore loudly (louder than talking or loud enough to be heard through closed doors)?
T = Tiredness. Do you often feel tired, fatigued, or sleepy during daytime?
O = Observed apnea. Has anyone observed you stop breathing during your sleep?
P = Pressure. Do you have or are you being treated for high blood pressure?
B = BMI > 35 kg/m?
A= Age > 50 years
N = Neck circumference > 40 cm
G = Male gender
Ipata from Chung F, Yegneswaran B, Liao P, et al. STOP questionnaire: A tool to screen patients for obstructive sleep apnea. Anesthesiology. 2008 May;108(5):812-821.
Fewer than 3 questions positive: low risk of OSA; 3 or more questions positive: high risk of OSA; 5 to 8 questions positive: high probability of moderate-to-severe OSA.

* Comorbid conditions include hypertension, arrhythmias, heart failure, cerebrovascular disease, and metabolic syndrome.
* If OSA is suspected during the preoperative evaluation, one could proceed with a presumptive diagnosis of OSA albeit with caution
* Educate surgeon, patient and family

- + Exercise caution in OSA patients who develop prolonged and frequent severe respiratory events (eg, sedation analgesic mismatch, desaturation, and apneic episodes) in the postoperative period
FIGURE 44-1 Decision making in preoperative selection of a patient with obstructive sleep apnea (OSA) scheduled for ambulatory surgery. CPAP, continuous positive airway pressure. (Reproduced with permission from Joshi G, Ankichetty S, Gan T, Chung F. Society for Ambulatory Anesthesia consensus statement on preoperative selection of adult patients with obstructive sleep apnea scheduled for ambulatory surgery. Anesth Analg. 2012 Nov;115(5):1060-1068.)

a Patients with obstructive sleep apnea (OSA) undergoing procedures under anesthesia are at increased risk for perioperative complications compared with patients without the disease diagnosis. Identifying patients at high risk for OSA before surgery for targeted perioperative precautions and interventions may help to reduce perioperative patient complications.
a Screening tools help to risk stratify patients with suspected OSA with reasonable accuracy. Practice groups should consider making OSA screening part of standard preanesthetic evaluation.
a There is insufficient evidence in the current literature to support canceling or delaying surgery for a formal diagnosis (laboratory or
lReproduced with permission from Chung F, Memtsoudis S, Ramachandran §S, et al. Society of Anesthesia and Sleep Medicine guidelines on preoperative screening and assessment of adult patients with obstructive sleep apnea. Anesth Analg. 2016 Aug;123(2):452-473.

need to have intraoperative hypotension or postoperative hypertension, or both, corrected in either case. The ASAguidelines recommend that patients presenting with a pacemaker or ICD should not leave a monitored setting until the device is interrogated, if electrocautery was employed; however, we believe that this ASArecommendation is overly conservative, if bipolar cautery is used at a distance of greater than 15 cm from the device or if unipolar cautery is used below the umbilicus and the ground pad is on the leg. Likewise, if an ICD is, 2 = . and there is anticipated electromagnetic interference the device's, 1 = . and there is anticipated electromagnetic interference the device's, 2 = . present,, 1 = . present,, 2 = . antitachycardia, 1 = . antitachycardia, 2 = . features should be inhibited perioperatively ( Figures 44-2, 1 = . features should be inhibited perioperatively ( Figures 44-2, 2 = . and, 1 = . and, 2 = . ). An external defibrillation capability should always be available, 1 = . ). An external defibrillation capability should always be available, 2 = . whenever, 1 = . whenever, 2 = . 44-3, 1 = . 44-3, 2 = . the ICD's, 1 = . the ICD's, 2 = . , 1 = antitachycardia. , 2 = features are inactivated.
FIGURE 44-2 Preoperative considerations in a patient with an implanted cardioverter defibrillator. EMI, electromagnetic interference; ICD, implanted cardioverter defibrillator. (Reproduced with permission from Joshi GP. Perioperative management of outpatients with implantable cardioverter defibrillators. Curr Opin Anaesthesiol. 2009 Dec;22(6):701704.)
FIGURE 44-3 Postoperative considerations in a patient with an implanted cardioverter defibrillator. EMI, electromagnetic interference; ICD, implanted cardioverter defibrillator. (Reproduced with permission from Joshi GP. Perioperative management of outpatients with implantable cardioverter defibrillators. Curr Opin Anaesthesiol. 2009 Dec;22(6):701704.)

In a consensus statement on perioperative glucose control, the Society for Ambulatory Anesthesia found insufficient evidence to make strong recommendations about glucose management in ambulatory patients, and thus management suggestions parallel those of the inpatient population; however, the panel recommends a target intraoperative blood glucose concentration of less than 180 mg/dL.

Patients with a history of malignant hyperthermia can be safely given nontriggering anesthetics and discharged as ambulatory patients. Prophylactic dantrolene should not be administered.

Intraoperative management in the ambulatory patient undergoing surgery is aimed at providing rapid emergence, good post-procedure analgesia, minimal postoperative nausea and vomiting (PONV), and rapid return of fitness for discharge while creating acceptable operating conditions. Often these goals compete with one another. Although inhalational anesthesia with sevoflurane may speed emergence, compared with total intravenous anesthesia (TIVA), the likelihood of PONV may be greater if an additional prophylactic drug is not administered. Numerous studies have shown how regional anesthesia can speed discharge time, compared with general anesthetics, in the ambulatory population —in part, by potentially reducing the incidence of PONV and the need for opioid analgesia. Nitrous oxide increases the likelihood of PONV, but this effect can be overcome by adding a prophylactic agent. Likewise, multimodal perioperative analgesia can be approached using a variety of drugs, including local anesthetics, acetaminophen, and nonsteroidal anti-inflammatory agents (NSAIDs) to reduce the use of opioids, which contribute to PONV risk.
Thromboembolism remains a risk after ambulatory and office-based surgery, as with inpatient surgery. Pneumatic compression devices and pharmacological thromboprophylaxis should be used in patients at increased risk. During monitored anesthesia care, supplemental oxygen can contribute to operating room fires by creating an oxygen-rich environment that facilitates ignition by cautery devices. During head and neck surgery, anesthesia providers must be especially vigilant not to create an environment where fire becomes more likely. When oxygen is administered via a nasal cannula or face mask, the minimal amount of supplemental oxygen should be delivered, if any, and tenting of the drapes around the patient's head should be prevented.

Managing a patient's emergence, postoperative pain, and PONV is critical to expediting discharge. A plan to handle complications, such as postoperative pain and PONV, should be in place preoperatively to standardize and streamline management as much as possible.
The entire anesthetic experience of the ambulatory surgery patient should be focused on minimizing complications, especially postoperative pain and PONV, and facilitating readiness for discharge. Multimodal approaches are advised; see Chapter 17 for a discussion of PONV prophylaxis and management. Use of a combination of agents (eg, ondansetron, dexamethasone, and droperidol) has shown greater efficacy than monotherapy (eg, ondansetron alone) in patients at high risk of PONV. Likewise, analgesia regimens that minimize opioid use reduce PONV.
Pain management is centered on the combined use of regional techniques, opioids, and NSAIDSs (multimodal analgesia). Gabapentinoids (gabapentin, pregabalin) may have beneficial effects as part of a multimodal pain regimen. Likewise, oral, rectal, or intravenous acetaminophen or NSAIDs, or both, can be useful in the ambulatory setting.

Scoring systems have been devised to facilitate timely and safe postanesthesia care unit (PACU) discharge and assess home readiness after ambulatory surgery. The Aldrete scoring system, which includes activity, respiration, circulation, consciousness, and oxygen saturation, helps guide recovery from the PACU in the ambulatory surgery unit. Scoring systems and guidelines that standardize patient discharge from the ambulatory surgery center to home are also available (Tables 44—5 through 44-8)

Early recovery, Clinical Definition = Awakening and recovery of vital reflexes. Intermediate recovery, Clinical Definition = Immediate clinical recovery Home readiness. Late recovery, Clinical Definition = Full recovery Psychological recovery
'Data from Steward DJ, Volgyesi G. Stabilometry: A new tool for measuring recovery following general anaesthesia. Can Anaesth Soc J. 1978 Jan;25(1):4-6,
TABLE 44-6 The modified Aldrete scoring system for determining when patients are ready for discharge from the postanesthesia care unit.!~%
Activity: able to move voluntarily or on command, 1 = . 4 extremities, 1 = 2. 2 extremities, 1 = 1. 0 extremities, 1 = 0. Respiration, 1 = . Able to deep breathe and cough freely, 1 = 2. Dyspnea, shallow or limited breathing, 1 = 1. Apneic, 1 = 0. Circulation, 1 = . BP + 20 mm of preanesthetic level, 1 = 2. BP + 20-50 mm of preanesthesia level, 1 = 1. BP + 50 mm of preanesthesia level, 1 = 0. Consciousness, 1 = . Fully awake, 1 = 2. Arousable on calling, 1 = 1. Not responding, 1 = 0. O, saturation, 1 = . Able to maintain 0, saturation >92% on room air, 1 = 2. Needs O, inhalation to maintain O, saturation >90%, 1 = 1. O, saturation <90% even with O, supplementation, 1 = 0
'Reproduced with permission from Aldrete AL. The post anesthesia recovery score revisited (letter). Clin Anesth. 1995 Feb;7(1):89-91.
"BP, blood pressure.
3A score =9 was required for discharge.

More than minimal nausea and vomiting
Excessive pain
Bleeding
The patient must be discharged by both the person who administered anaesthesia and the person who performed surgery, or by their designates.
Written instructions for the postoperative period at home, including a contact place and person, must be reinforced.
The patient must have a responsible, "vested" adult escort them home and stay with them at home.
lReproduced with permission from Korttila K. Recovery from outpatient anaesthesia, factors affecting outcome. Anaesthesia. 1995 Oct;50(suppl):22-28.

Vital signs must be stable and consistent with age and preoperative baseline
BP and pulse within 20% of preoperative baseline
BP and pulse 20%-40% of preoperative baseline
BP and pulse >40% of preoperative baseline aS)
0

Patient must be able to ambulate at preoperative level
Steady gait, no dizziness, or meets preoperative 2 level
Requires assistance
Unable to ambulate
0
=

The patient should have minimal nausea and vomiting
before discharge
Minimal: successfully treated with PO medication
Moderate: successfully treated with IM
medication
Severe: continues after repeated treatment
2
1
0

The patient should have minimal or no pain before discharge
The level of pain that the patient has should be acceptable to the patient
Pain should
be controllable
by oral
analgesics
The location,
type, be
and intensity
consistent with
discomfort
Acceptability
Yes
No

Postoperative bleeding should be consistent with expected blood loss for the procedure
Minimal: does not require dressing change 2 Moderate: up to two dressing changes required 1 Severe: more than three dressing changes 0 required
'Reproduced with permission from Marshall SI, Chung F. Assessment of "home readiness": Discharge criteria and postdischarge complications. Curr Opin Anesthiol. 1997 Dec;10(6):445-450.
"BP, blood pressure; PO, per os; |M, intramuscular.
3Maximal score = 10; patients scoring =9 are fit for discharge.
Criteria for discharge generally require that the patient:

* Have stable vital signs
¢ Have pain controlled by oral analgesics, liposomal bupivacaine, or peripheral nerve block
* Have nausea or emesis controlled
* Be able to walk without dizziness
¢ Have no unexpected bleeding from the operative site
* Be able to take oral fluids and void
¢ Have discharge instructions and prescriptions from the surgeon and anesthesiologist
of anticipated
pain should
2
]
- * Accept readiness for discharge

Increasingly, patients are not being required to drink or void before discharge from ambulatory surgery centers (ASCs). Such patients require plans and instructions for follow-up care to provide for possible rehydration and bladder catheterization, if required.

Occasionally an ambulatory surgery patient may require emergent transfer to a nearby hospital. Some surgical complications cannot be repaired in the ambulatory operating suite. Inadequately controlled pain and postoperative nausea and vomiting are the two most frequent causes of unplanned hospital admission from ASCs. Accreditation agencies mandate that office-based operating rooms have appropriate emergency equipment, drugs, and protocols for patient transfer to a nearby hospital. In addition to advanced cardiac life support medications, dantrolene and intravenous lipid emulsion should be available to treat malignant hyperthermia and local anesthetic systemic toxicity, respectively. Additionally, surgeons operating in an office-based practice must have admitting privileges at a nearby hospital or arrangements with an accepting physician to provide for patient transfer.

Off-site anesthesia (NORA) encompasses all sedation/anesthesia provided by anesthesiology services outside of the operating room environment. Our experience has been that requests for these services in remote locations have been steadily increasing, and in some hospitals today, more anesthetics are routinely administered for procedures off-site than in the operating room suite. We and others assign "block time" for such procedures just as we do for the various surgeons and surgical services. We and other institutions have constructed procedural suites where bronchoscopy, gastrointestinal endoscopy, and other procedures can be performed in a centralized area for increased safety and efficiency. The same basic standards for anesthesia care must be met, regardless of the location. Furthermore, the challenges of unfamiliar environments that are far removed from the surgical suite require advance planning for the off-site anesthesia provider.
In contrast to patients undergoing office-based or ambulatory surgery center procedures, out of the operating room patients are frequently among the sickest of inpatients. Often such locations as the endoscopy suite, cardiac catheterization laboratory, electrophysiology laboratory, radiology suite, or radiotherapy unit were constructed without anticipation that anesthesia would be provided there. Consequently, anesthesia work space is routinely constrained, and access to the patient is limited. Moreover, the procedure physicians and ancillary staff in these areas often fail to understand what is required to safely deliver anesthesia (hence the frequent request to "give them a squirt" of propofol) and do not know how to assist the anesthesia provider when difficulty arises. As noted in the ASA guidelines, the standard of care for NORA is the same as in the operating room (Table 44-9).

Reliable O, source with backup, 1 = Sufficient space for anesthesia personnel, equipment. Suction apparatus, 1 = Emergency cart, defibrillator, drugs, etc. Waste gas scavenging, 1 = Reliable means for two-way communication. Adequate monitoring equipment, 1 = Applicable facility, safety codes met. Safe electrical outlets, 1 = Appropriate postanesthesia management. Adequate illumination, battery backup, 1 = 
'Data from American Society of Anesthesiologists guidelines for nonoperating room anesthetizing locations (2008), Committee of Origin: Standards and Practice Parameters (approved by the ASA House of Delegates on October 15, 2003 and amended on October 22, 2008.
Basic principles for NORA can be broadly classified into three categories: patient factors, environmental issues, and procedure-related aspects. Patient factors include comorbidity, airway assessment, fasting status, and monitoring. Environmental issues include anesthesia equipment, emergency equipment, and magnetic and radiation hazards. Procedure-related aspects include duration, level of discomfort, patient position, and surgical support.
The ASA Closed Claims Database has demonstrated that claims related to NORA care have a greater severity of injury than closed claims related to operating room anesthesia care. Monitored anesthesia care was the primary technique in more than half of the claims reviewed. Many of these closed claims arose from injuries related to inadequate oxygenation/ventilation during endoscopy.

Increasingly, personnel in the endoscopy suite and the emergency department who are not qualified anesthesia providers provide sedation with a variety of agents, including propofol and ketamine. In fact, some reports indicate that nonanesthesia providers administer sedation and analgesia for almost 40% of the procedures performed in the United States. The ASA guidelines and the Joint Commission have described the continuum of depth of sedation, ranging from minimal sedation to general anesthesia (Table 44—10). The Centers for Medicare and Medicaid Services has mandated that all sedation in a hospital be under the direction of a physician—generally, the anesthesia service chief. Consequently, anesthesiologists must not only from time to time provide anesthesia in a non— operating room setting, but must also develop policies and quality assurance review mechanisms for non—anesthesia providers to safely and legally provide sedation. Such policies should be focused on assuring that the "sedationist" has the necessary skills to provide for patient rescue should mild or moderate sedation unintentionally progress to deep sedation or general anesthesia.

1, Type = Minimal. 1, Responsiveness = Normal to verbal stimulation. 1, Airway = Unaffected. 1, Spontaneous Ventilation = Unaffected. 1, Cardiovascular Function = Unaffected. 2, Type = Moderate. 2, Responsiveness = Purposeful response to verbal or tactile stimulation. 2, Airway = No intervention required. 2, Spontaneous Ventilation = Adequate. 2, Cardiovascular Function = Usually maintained. 3, Type = Deep. 3, Responsiveness = Purposeful after repeated or painful stimulus. 3, Airway = Intervention may be required. 3, Spontaneous Ventilation = May be inadequate. 3, Cardiovascular Function = Usually maintained. 4, Type = General anesthesia. 4, Responsiveness = Unarousable to painful stimulus. 4, Airway = Intervention often required. 4, Spontaneous Ventilation = Often inadequate. 4, Cardiovascular Function = May be impaired
Data from American Society of Anesthesiologists
Risks associated with sedation/analgesia are highlighted in Table 44-11. Sedation providers should know how to reverse benzodiazepines and opioids and provide bag/mask airway support and to be facile in the use of airway adjuvants.
A mechanism to ensure the timely arrival of anesthesia personnel capable of airway rescue must likewise be incorporated into such policies.
TABLE 44-11 Complications associated with sedation and analgesia.!

Airway obstruction
Aspiration
Regurgitation
Dental/soft tissue injury

Undesirable patient movement
Drug interactions
Adverse reactions
Unanticipated admission
IData from American Society of Anesthesiologists.

Anesthesia services are requested at various locations throughout the hospital facility; some of these are delineated in Table 44—12. Post-procedure disposition (whether discharge or admission) needs appropriate coordination by the anesthesiologist for postanesthesia care or safe transport from the remote unit, or both.
TABLE 44-12 Common locations for non—operating room anesthesia.'*

Neurointerventional radiology
Vascular radiology
MRI/CT
PET scan

Tracheostomy, percutaneous gastrostomy
Intracranial and other catheter placement
Abdominal/pelvic explorations

Cardiac catheterization lab
Cardioversion
Electrophysiology suite
Radiation Therapy
Emergency Medicine Suite

Electroconvulsive therapy suite
Urology
Lithotripsy
Dental Surgery
2cT, computed tomography; MRI, magnetic resonance imaging; PET, positron emission tomography.
Patients presenting to the gastrointestinal endoscopy suite include healthy individuals for routine diagnostic screenings, as well as patients with fulminant
cholangitis, sepsis, or gastrointestinal hemorrhage. As always, the patient's current illness and comorbidities, as well as the specific diagnostic/therapeutic procedure, determine both the anesthetic techniques (sedation or general anesthesia) and the monitoring required.
Patients undergoing cardiac catheterization are routinely sedated by cardiologists without involvement of an anesthesiologist. Occasionally, a patient with significant comorbidities (eg, morbid obesity) requires the presence of a qualified anesthesia provider. General anesthesia is sometimes required for placement of aortic stents, which are increasingly performed by cardiologists in the cardiac catheterization laboratory. Specially built hybrid surgical suites enable both open and catheter-based vascular repairs. Increasingly, patients for transcatheter aortic valve replacement are managed with local anesthesia and sedation rather than general anesthesia. Institutional protocols and patient characteristics determine the anesthetic/sedation management of these patients. General anesthesia is frequently administered to patients in the neurointerventional suite for treatment of cerebral aneurysms and ischemic strokes.

Patients requiring electrophysiology procedures for catheter-mediated arrhythmia ablation often need general anesthesia. Such patients may have both systolic and diastolic heart failure, leading to potential hemodynamic difficulties perioperatively. Sudden hypotension can herald the development of pericardial tamponade secondary to catheter perforation of the heart. Other patients require sedation for the placement of ICDs. Once placed, the device will be tested by inducing ventricular fibrillation. During testing, deeper levels of sedation are required, as the defibrillation shock can be frightening and very uncomfortable. Likewise, anesthesia staff are called upon to provide anesthesia for cardioversion of patients in atrial fibrillation. These patients usually have associated cardiac diseases and require brief intravenous anesthetics to facilitate cardioversion. Oftentimes, a transesophageal echocardiogram must be performed prior to cardioversion to rule out clot in the left atrial appendage. In such cases, anesthesia staff may also provide sedation for this procedure. Determination as to whether a patient needs sedation or general anesthesia with or without intubation is dependent upon routine patient assessment.
Children and some adults (ie, those who are claustrophobic, developmentally disabled, or have conditions that prevent them from remaining still or lying flat) require anesthesia or sedation for MRI and computed tomography (CT) imaging. Additionally, painful CT-guided biopsies may require anesthesia management. Anesthetic technique is dependent upon patient comorbidities.
MRI creates numerous problems for anesthesia staff. First, all ferromagnetic materials must be excluded from the area of the magnet. Most institutions have policies and training protocols to prevent catastrophes (eg, oxygen tanks flying into the scanner). Second, all anesthetic equipment must be compatible with the magnet in use. Third, patients must be free of implants that could interact with the magnet, such as pacemakers, vascular clips, ICDs, and infusion pumps. As with all NORA, the exact choice of technique is dependent upon the patient's comorbidities. Both deep sedation and general anesthesia approaches with intubation or supraglottic airways can be used, depending on practitioner preference and patient requirements.

Patients often require general anesthesia and tight blood pressure control to facilitate coiling and embolization of cerebral aneurysms, arteriovenous malformations, or stenting and clot removal for acute strokes. Patients taken to the radiology suite for relief of portal hypertension (see Chapters 33 and 34) via creation of a transjugular intrahepatic portosystemic shunt (TIPS) are frequently intravascularly depleted, with profound ascites, and are at risk of esophageal variceal bleeding and aspiration. General anesthesia with intubation is preferred for management of the TIPS procedure.
Anesthesia for electroconvulsive therapy is often provided in a separate suite in the psychiatry unit or a monitored area in the hospital (eg, PACU). Patient comorbidity, drug interactions with various psychotropic medications, multiple anesthetic procedures, and effects of anesthetic agents on the quality of electroconvulsive therapy also need to be considered (see Chapter 28).
Anesthesia providers are at times called to deliver anesthesia in the intensive care unit (ICU) for bedside tracheostomy or emergent chest and abdominal exploration in patients considered too critically ill to tolerate transport to the operating room. In most of these cases, the anesthesia staff generally employ the ICU ventilator and monitors. Intravenous agents are typically used along with muscle relaxants. When performing anesthesia for bedside tracheostomy, it is important that the endotracheal tube not be withdrawn from the trachea until end-tidal CO, is measured from the newly placed tracheostomy tube.

Acute Hypoxia After TIPS Procedure in the Radiology Suite
A 58-year-old Caucasian woman with decompensated cryptogenic

What does a TIPS procedure entail? What are its indications and contraindications?
TIPS (transjugular intrahepatic portosystemic shunt) involves the passage of a catheter, usually inserted through the internal jugular vein and directed into the liver, which creates a low-resistance conduit between a portal vein and a hepatic vein by deployment of an intrahepatic expandable stent. Hemodynamically, this allows immediate decompression of portal hypertension by partial or complete diversion of portal flow from hepatic sinusoids into the inferior vena cava and the systemic circulation.
Indications for the TIPS procedure include variceal bleeding not controlled by endoscopic or medical therapy, intractable ascites, hepatic hydrothorax, Budd—Chiari syndrome, hepatorenal syndrome and hepatopulmonary syndrome, and bridge to liver transplantation. Some contraindications of TIPS are primary prevention of variceal hemorrhage, congestive heart failure, severe pulmonary hypertension and tricuspid regurgitation, severe hepatic failure, hepatocellular carcinoma, active intrahepatic or systemic infection, and severe coagulopathy or thrombocytopenia.

TIPS can be performed under moderate sedation, monitored anesthesia care, or general anesthesia. Given the usual need for long immobilization, potential risk of aspiration, and significant comorbidity, general anesthesia is often the recommended anesthetic plan.
Preoperative considerations
include risk
of aspiration,
gastrointestinal bleeding,
decreased functional
effusions, coagulopathy,
Intraoperative residual
capacity thrombocytopenia,
considerations monitoring
(usually from
ascites, and
should include
via arterial
blood gases,
electrolytes, pharmacokinetics
catheter)
hepatic pleural
encephalopathy.
careful and
and coagulation
of anesthetic
hemodynamic frequent
measurement parameters.
agents from
Altered liver
failure should
also of
be kept
in mind.
The patient is induced with etomidate, fentanyl, and succinylcholine, using rapid sequence induction; atraumatic intubation is accomplished uneventfully. Prior to placement of the TIPS, the radiologist evacuates approximately 8 L of ascitic fluid.
What are your concerns about this paracentesis? How would you balance these hemodynamic fluid shifts?
Large volume paracentesis is believed to be a relatively safe and effective procedure; however, it can lead to paracentesis-induced circulatory dysfunction (PICD). PICD is characterized by a marked activation of the renin—angiotensin axis, as well as accentuation of the arteriolar vasodilation that is present in cirrhotic patients. Administration of albumin may mitigate the hemodynamic effects of large volume paracentesis.

@ Neuraxial anesthesia greatly expands the anesthesiologists' armamentarium, in many cases providing alternatives to general anesthesia. Neuraxial anesthesia may also be used simultaneously with general anesthesia or afterward for postoperative analgesia. Neuraxial blocks can be performed as a single injection or with a catheter to allow intermittent boluses or continuous infusions.
@ Performing a lumbar (subarachnoid) spinal puncture below L1 in an adult (L3 in a child) usually avoids potential needle trauma to the spinal cord.
© The principal site of action for neuraxial blockade is believed to be the nerve root, at least during initial onset of block.
@ Differential blockade typically results in sympathetic blockade (judged by temperature sensitivity) that may be two segments or more cephalad than the sensory block (pain, light touch), which, in turn, is usually several segments more cephalad than the motor blockade.
© Interruption of efferent autonomic transmission at the spinal nerve roots during neuraxial blocks produces sympathetic blockade.
@ Neuraxial blocks typically produce variable decreases in blood pressure that may be accompanied by a decrease in heart rate.
@ Deleterious cardiovascular effects should be anticipated and steps undertaken to minimize the degree of hypotension. However, volume loading with 10 to 20 mL/kg of intravenous fluid in a healthy patient before initiation of the block has been shown repeatedly to fail to prevent hypotension (in the absence of preexisting hypovolemia).
@ Excessive or symptomatic bradycardia should be treated with atropine, and hypotension should be treated with vasopressors.
© Major contraindications to neuraxial anesthesia include lack of consent, coagulation abnormalities, severe hypovolemia, elevated intracranial pressure, and infection at the site of injection.
© For epidural anesthesia, a sudden loss of resistance (to injection of air or saline) is encountered as the needle passes through the ligamentum flavum and enters the epidural space. For spinal anesthesia, the needle is advanced through the epidural space and penetrates the dura— subarachnoid membranes, as signaled by freely flowing cerebrospinal fluid.

@ Continuous epidural anesthesia is a neuraxial technique offering a range of applications wider than single dose spinal anesthesia. An epidural block can be performed at the lumbar, thoracic, or cervical level.
Epidural techniques are widely used for surgical anesthesia, obstetric analgesia, postoperative pain control, and chronic pain management.
Epidural anesthesia is slower in onset (10-20 min) and may not be as dense as spinal anesthesia, a feature that can be useful clinically.
The quantity (volume and concentration) of local anesthetic needed for epidural anesthesia is larger than that needed for spinal anesthesia. Toxic side effects are almost guaranteed if a "full epidural dose" is injected intrathecally or intravascularly.
@ Caudal epidural anesthesia is a common regional technique in pediatric patients.
Spinal, caudal, and epidural blocks were first used for surgical procedures at the turn of the twentieth century. These central blocks were widely used worldwide until reports of permanent neurological injury appeared, most prominently in the United Kingdom. However, a large-scale epidemiological study conducted in the 1950s proved that complications were rare when these blocks were performed skillfully, with attention to asepsis, and when newer, safer local anesthetics were used. Today, neuraxial blocks are routinely employed for labor analgesia, cesarian delivery, orthopedic surgery, perioperative analgesia, and chronic pain management. However, they are still associated with various complications, and much literature has examined the incidence of complications following neuraxial blocks associated with different disease states.
- @ Neuraxial anesthesia greatly expands the anesthesiologists' armamentarium, in many cases providing alternatives to general anesthesia. Neuraxial anesthesia may be used simultaneously with general anesthesia or
afterward for postoperative analgesia. Neuraxial blocks can be performed as a single injection or with a catheter to allow intermittent boluses or continuous infusions.
Adverse reactions and complications associated with regional anesthesia range from self-limited back soreness to debilitating permanent neurological deficits and even death. The practitioner must therefore be thoroughly familiar with the anatomy involved and the pharmacology and toxic dosages of the agents employed. The practitioner must diligently employ sterile techniques and quickly address physiological derangements arising from neuraxial techniques.

Some studies suggest that postoperative morbidity may be reduced when neuraxial blockade is used either alone or in combination with general anesthesia. Some less convincing studies suggest that neuraxial blocks are associated with reduced perioperative mortality. Neuraxial blocks may reduce the incidence of venous thrombosis and pulmonary embolism, cardiac complications in high-risk patients, bleeding and transfusion requirements, vascular graft occlusion, and pneumonia and respiratory depression following upper abdominal or thoracic surgery in patients with chronic lung disease. Neuraxial blocks may also allow earlier return of gastrointestinal function following surgery. Proposed mechanisms (in addition to avoidance of larger doses of anesthetics and opioids) include reducing the hypercoagulable state associated with surgery, increasing tissue blood flow, improving oxygenation from decreased splinting, enhancing peristalsis, and suppressing the neuroendocrine stress response to surgery. Reduction of parenteral opioid administration may decrease the incidence of atelectasis, hypoventilation, and aspiration pneumonia and reduce the duration of ileus. Postoperative epidural analgesia may also significantly reduce both the need for mechanical ventilation and the time until extubation after major abdominal or thoracic surgery.

Anesthesiologists are all too familiar with situations in which a consultant "clears" a sick elderly patient with significant cardiac disease for surgery "under spinal anesthesia." A spinal anesthetic with little or no intravenous sedation may reduce the likelihood of postoperative delirium or cognitive dysfunction, which is sometimes seen in the elderly. Unfortunately, many patients will require some sedation during the procedure, either for comfort or to facilitate cooperation. Is spinal anesthesia always safer in a patient with severe coronary artery disease or with a decreased ejection fraction? Ideally, an anesthetic technique should produce neither hypotension (which decreases myocardial perfusion pressure) nor hypertension and tachycardia (which increase myocardial oxygen consumption). Spinal anesthesia may produce both hypotension and bradycardia. Administration of large intravenous volumes may lead to fluid overload in the elderly patient with diastolic dysfunction, especially after the sympathetic block resolves postoperatively. General anesthesia, on the other hand, also poses potential problems for patients with cardiac compromise. Most general anesthetics are cardiac depressants, and many cause vasodilation. Deep anesthesia can readily cause hypotension, whereas light anesthesia relative to the level of stimulation causes hypertension and tachycardia. Research is ongoing to discern if neuraxial techniques offer survival and other benefits to patients compared with general anesthetics for major operations such as open reduction and internal fixation of femoral neck fractures. To date, the results are conflicting.

Currently, epidural anesthesia is widely used for analgesia in women in labor and during vaginal delivery. Cesarean delivery is most commonly performed under spinal or epidural anesthesia. Both blocks allow a mother to remain awake and experience the birth of her child. Large population studies in Great Britain and the United States have shown that regional anesthesia for cesarean delivery is associated with less maternal morbidity and mortality than is general anesthesia. This may be largely due to a reduction in the incidence of pulmonary aspiration and failed intubation when neuraxial anesthesia is employed. Fortunately, the increased availability of video laryngoscopes may also reduce the incidence of adverse outcomes related to airway difficulties associated with general anesthesia for cesarean delivery.

The spine is composed of the vertebral bones and intervertebral disks (Figure
45-1). There are 7 cervical (C), 12 thoracic (T), and 5 lumbar (L) vertebrae (Figure 45-2). The sacrum is a fusion of 5 sacral (S) vertebrae, and there are small rudimentary coccygeal vertebrae. The spine as a whole provides structural support for the body and protection for the spinal cord and nerves and allows a degree of mobility in several spatial planes. At each vertebral level, paired spinal nerves exit the central nervous system (Figure 45-2).
FIGURE 45-1 A: Sagittal section through lumbar vertebrae. B, C: Common features of vertebrae.
FIGURE 45-2 The vertebral column. (Adapted with permission from Waxman SG. Correlative Neuroanatomy. 24th ed. New York, NY: McGraw-Hill; 2000.)

Vertebrae differ in shape and size at the various levels. The first cervical vertebra, the atlas, lacks a body and has unique articulations with the base of the skull and with the second vertebra. The second vertebra, called the axis, consequently has atypical articulating surfaces. All 12 thoracic vertebrae articulate with their corresponding rib. Lumbar vertebrae have a large anterior cylindrical vertebral body. A hollow ring is defined anteriorly by the vertebral body, laterally by the pedicles and transverse processes, and posteriorly by the lamina and spinous processes (Figure 45—-1B and C). The laminae extend between the transverse processes and the spinous processes, and the pedicle extends between the vertebral body and the transverse processes. When stacked vertically, the hollow rings become the spinal canal in which the spinal cord and its coverings sit. The individual vertebral bodies are connected by the intervertebral disks. There are four small synovial joints at each vertebra, two articulating with the vertebra above it and two with the vertebra below. These are the facet joints, which are adjacent to the transverse processes (Figure 45-1C). The pedicles are notched superiorly and inferiorly, these notches forming the intervertebral foramina from which the spinal nerves exit. Sacral vertebrae normally fuse into one large bone, the sacrum, but each one retains discrete anterior and posterior intervertebral foramina. The laminae of S5 and all or part of S4 normally do not fuse, leaving a caudal opening to the spinal canal, the sacral hiatus (Figure 45-3).

The spinal column normally forms a double C, being convex anteriorly in the cervical and lumbar regions (Figure 45—2). Ligamentous elements provide structural support, and, together with supporting muscles, help to maintain the unique shape. Ventrally, the vertebral bodies and intervertebral disks are connected and supported by the anterior and posterior longitudinal ligaments (Figure 45-1A). Dorsally, the ligamentum flavum, interspinous ligament, and supraspinous ligament provide additional stability. Using the midline approach, a needle passes through these three dorsal ligaments and through an oval space between the bony lamina and spinous processes of adjacent vertebra (Figure 45— 4).
FIGURE 45-4 Lumbar epidural anesthesia; midline approach.

The spinal canal contains the spinal cord with its coverings (the meninges), fatty tissue, and a venous plexus (Figure 45—5). The meninges are composed of three layers: the pia mater, the arachnoid mater, and the dura mater; all are contiguous with their cranial counterparts (Figure 45—6). The pia mater is adherent to the spinal cord, whereas the arachnoid mater is usually adherent to the thicker and denser dura mater. Cerebrospinal fluid (CSF) is contained between the pia and arachnoid maters in the subarachnoid space. The spinal subdural space is generally a poorly demarcated, potential space that exists between the dura and arachnoid membranes. The epidural space is a better defined potential space within the spinal canal that is bounded by the dura and the ligamentum flavum (Figures 45-1 and 45-5).
FIGURE 45-5 Exit of the spinal nerves. (Adapted with permission from Waxman SG. Correlative Neuroanatomy. 24th ed. New York, NY: McGraw-Hill; 2000.)
FIGURE 45-6 The spinal cord.

The spinal cord normally extends from the foramen magnum to the level of L1 in adults (Figure 45—7). In children, the spinal cord ends at L3 and moves up with age. The anterior and posterior nerve roots at each spinal level join one another and exit the intervertebral foramina, forming spinal nerves from C1 to S5 (Figure 45—2). At the cervical level, the nerves arise above their respective vertebrae, but starting at T1, exit below their vertebrae. As a result, there are eight cervical nerve roots, but only seven cervical vertebrae. The cervical and upper thoracic nerve roots emerge from the spinal cord and exit the vertebral foramina nearly at the same level (Figure 45—2). But, because the spinal cord normally ends at L1, lower nerve roots course some distance before exiting the intervertebral foramina. These lower spinal nerves form the cauda equina ("horse's tail"; Figure 45-2). @ Therefore, performing a lumbar (subarachnoid) puncture below L1 in an adult (L3 in a child) usually avoids potential needle trauma to the spinal cord; damage to the cauda equina is unlikely, as these nerve roots float in the dural sac below L1 and tend to be pushed away (rather than pierced) by an advancing needle.
FIGURE 45-7 Sagittal view through the lumbar vertebrae and sacrum. Note the end of the spinal cord rises with development from approximately L3 to L1. The dural sac normally ends at S2.

A dural sheath invests most nerve roots for a small distance, even after they exit the spinal canal (Figure 45-5). Nerve blocks close to the intervertebral foramen therefore carry a risk of subdural or subarachnoid injection. The dural sac and the subarachnoid and subdural spaces usually extend to S2 in adults and often to S3 in children, important considerations in avoiding accidental dural puncture during caudal anesthesia. An extension of the pia mater, the filum terminale, penetrates the dura and attaches the terminal end of the spinal cord (conus medullaris) to the periosteum of the coccyx (Figure 45—7). The blood supply to the spinal cord and nerve roots is derived from a single anterior spinal artery and paired posterior spinal arteries (Figure 45—8). The anterior spinal artery is formed from the vertebral artery at the base of the skull and courses down along the anterior surface of the cord. The anterior spinal artery supplies the anterior two-thirds of the cord, whereas the two posterior spinal arteries supply the posterior one-third. The posterior spinal arteries arise from the posterior inferior cerebellar arteries and course down along the dorsal surface of the cord medial to the dorsal nerve roots. The anterior and posterior spinal arteries receive additional blood flow from the intercostal arteries in the thorax and the lumbar arteries in the abdomen. One of these radicular arteries is typically large, the artery of Adamkiewicz, or arteria radicularis magna, arising from the aorta (Figures 45—8A). It is typically unilateral and nearly always arises on the left side, providing the major blood supply to the anterior, lower twothirds of the spinal cord. Injury to this artery can result in the anterior spinal artery syndrome (see Chapter 22).
FIGURE 45-8 Arterial supply to the spinal cord. A: Anterior view showing principal sources of blood supply. B: Cross-sectional view through the spinal cord showing paired posterior spinal arteries and a single anterior spinal artery. (Adapted with permission from Waxman SG. Correlative Neuroanatomy. 24th ed. New York, NY: McGraw-Hill; 2000.)

® The principal site of action for neuraxial blockade is believed to be the nerve root, at least during initial onset of block. Local anesthetics may also have actions on structures within the spinal cord during epidural and spinal anesthesia. Local anesthetic is injected into CSF (spinal anesthesia) or the epidural space (epidural and caudal anesthesia) and bathes the nerve root in the subarachnoid space or epidural space, respectively. Direct injection of local anesthetic into CSF for spinal anesthesia allows a relatively small dose and volume of local anesthetic to achieve dense sensory and motor blockade. In contrast, neuraxial block is achieved only with much larger volumes and quantities of local anesthetic molecules during epidural and caudal anesthesia. The injection site (level) for epidural anesthesia is ideally sited in the middle of the nerve roots that must be anesthetized. Blockade of neural transmission (conduction) in the posterior nerve root fibers interrupts somatic and visceral sensation, whereas blockade of anterior nerve root fibers prevents efferent motor and autonomic outflow.

By interrupting the afferent transmission of painful stimuli and abolishing the efferent impulses responsible for skeletal muscle tone, neuraxial blocks provide excellent operating conditions. Sensory blockade interrupts both somatic and visceral painful stimuli. The mechanism of action of local anesthetic agents is discussed in Chapter 16. Smaller and myelinated fibers are generally more easily blocked than larger and unmyelinated ones. The size and character of the fiber types, and the fact that the concentration of local anesthetic decreases with increasing distance from the level of injection, explains the phenomenon of differential blockade during neuraxial ® anesthesia. Differential blockade typically results in sympathetic blockade (judged by temperature sensitivity) that may be two segments or more cephalad than the sensory block (pain, light touch), which, in turn, is usually several segments more cephalad than the motor

@ Interruption of efferent autonomic transmission at the spinal nerve roots during neuraxial blocks produces sympathetic blockade. Sympathetic outflow from the spinal cord may be described as thoracolumbar, whereas parasympathetic outflow is craniosacral. Sympathetic preganglionic nerve fibers (small, myelinated B fibers) exit the spinal cord with the spinal nerves from T1— L2 and may course many levels up or down the sympathetic chain before synapsing with a postganglionic cell in a sympathetic ganglion. In contrast, parasympathetic preganglionic fibers exit the spinal cord with the cranial and sacral nerves. Neuraxial anesthesia does not block the vagus nerve (tenth cranial nerve). The physiological responses to neuraxial blockade therefore result from decreased sympathetic tone or unopposed parasympathetic tone, or both.

@ Neuraxial blocks produce variable decreases in blood pressure that may be accompanied by a decrease in heart rate. These effects generally increase with more cephalad dermatomal levels and more extensive sympathectomy. Vasomotor tone is primarily determined by sympathetic fibers arising from T5 to L1, innervating arterial and venous smooth muscle. Blocking these nerves causes vasodilation of the venous capacitance vessels and pooling of blood in the viscera and lower extremities, thereby decreasing the effective circulating blood volume and often decreasing cardiac output. Arterial vasodilation may also decrease systemic vascular resistance. The effects of arterial vasodilation may be minimized by compensatory vasoconstriction above the level of the block, particularly when the extent of sensory anesthesia is limited to the lower thoracic dermatomes. A high sympathetic block not only prevents compensatory vasoconstriction but may also block the sympathetic cardiac accelerator fibers that arise at T1 to T4. Profound hypotension may result from arterial dilation and venous pooling combined with bradycardia. These effects are exaggerated if venous pooling is further augmented by a head-up position or the weight of a gravid uterus. Unopposed vagal tone may explain the sudden cardiac arrest sometimes seen with spinal anesthesia.

@ Deleterious cardiovascular effects should be anticipated and steps undertaken to minimize the degree of hypotension. However, volume loading with 10 to 20 mL/kg of intravenous fluid in a healthy patient before initiation of the block has been shown repeatedly to fail to prevent hypotension (in the absence of preexisting hypovolemia). Left uterine displacement in the third trimester of pregnancy helps to minimize physical obstruction to venous return in some patients. Despite these efforts, hypotension may still occur and should be treated promptly. Autotransfusion may be accomplished by placing the patient in a head-down position. A bolus of intravenous fluid (5-10 mL/kg) may be helpful in patients who have adequate cardiac and renal function to be able to "handle" the fluid load @ after the block wears off. Excessive or symptomatic bradycardia should be treated with atropine, and hypotension should be treated with vasopressors. Direct a-adrenergic agonists (such as phenylephrine) primarily produce vasoconstriction, increasing systemic vascular resistance, and may reflexively increase bradycardia. The "mixed" agent ephedrine has direct and indirect B-adrenergic effects that increase heart rate and contractility, and indirect effects that also produce vasoconstriction. Much like ephedrine, small doses of epinephrine (2-5 mcg boluses) are particularly useful in treating spinal anesthesia—induced hypotension. If profound hypotension or bradycardia persists, vasopressor infusions may be required.

pneumonia and respiratory failure, improving oxygenation, and decreasing the duration of mechanical ventilatory support.

Neuraxial block—induced sympathectomy allows vagal "dominance" with a small, contracted gut and active peristalsis. This can improve operative conditions during intestinal surgery when used as an adjunct to general anesthesia. Postoperative epidural analgesia with local anesthetics and minimal systemic opioids hastens the return of gastrointestinal function after open abdominal procedures.
Hepatic blood flow will decrease with reductions in mean arterial pressure from any anesthetic technique, including neuraxial anesthesia.

Renal blood flow is maintained through autoregulation, and there is little effect of neuraxial anesthesia on kidney function. Neuraxial anesthesia at the lumbar and sacral levels blocks both sympathetic and parasympathetic control of bladder function. Loss of autonomic bladder control results in urinary retention until the block wears off. If no urinary catheter is placed perioperatively, it is prudent to use the regional anesthetic of shortest duration sufficient for the surgical procedure and to administer the minimal safe volume of intravenous fluid. Patients with urinary retention should be checked for bladder distention after neuraxial anesthesia.

Surgical trauma produces a systemic neuroendocrine stress response via activation of somatic and visceral afferent nerve fibers, in addition to a localized inflammatory response. This systemic response includes increased concentrations of adrenocorticotropic hormone, cortisol, epinephrine, norepinephrine, and vasopressin levels, as well as activation of the renin— angiotensin—aldosterone system. Clinical manifestations include intraoperative and postoperative hypertension, tachycardia, hyperglycemia, protein catabolism, suppressed immune responses, and altered renal function. Neuraxial blockade can partially suppress (during major invasive abdominal or thoracic surgery) or totally block (during lower extremity surgery) the neuroendocrine stress response. To maximize this blunting of the neuroendocrine stress response, neuraxial block should precede incision and continue postoperatively.

Neuraxial blocks may be used alone or in conjunction with general anesthesia for many procedures below the neck. As a primary anesthetic, neuraxial blocks have proved most useful in lower abdominal, inguinal, urogenital, rectal, and lower extremity surgery. Lumbar spinal surgery may also be performed under spinal anesthesia. Upper abdominal procedures (eg, gastrectomy) have been performed with spinal or epidural anesthesia, but because it can be difficult to safely achieve a sensory level adequate for patient comfort, these techniques are less commonly used.
If a neuraxial anesthetic is being considered, the risks and benefits must be discussed with the patient, and informed consent should be obtained. The patient must be mentally prepared for neuraxial anesthesia, and neuraxial anesthesia must be appropriate for the type of surgery. Patients should understand that they will have little or no lower extremity motor function until the block resolves. Procedures that require maneuvers that might compromise respiratory function (eg, pneumoperitoneum or pneumothorax) or those operations that are unusually of long duration are typically performed with general anesthesia, with or without neuraxial blockade.

@ Major contraindications to neuraxial anesthesia include lack of consent, coagulation abnormalities, severe hypovolemia, elevated intracranial pressure (particularly with an intracranial mass), and infection at the site of injection. Other relative contraindications include severe aortic or mitral stenosis and severe left ventricular outflow obstruction (hypertrophic obstructive cardiomyopathy); however, with close monitoring and control of the anesthetic level, neuraxial anesthesia can be performed safely in patients with stenotic valvular heart disease, particularly if extensive dermatomal spread of anesthesia is not required (eg, "saddle" block spinal anesthetics).
Relative and controversial contraindications are also shown in Table 45-1.
Inspection and palpation of the back can reveal surgical scars, scoliosis, skin lesions, and whether or not the spinous processes can be identified. Although preoperative screening tests are not required in healthy patients undergoing neuraxial blockade, appropriate testing should be performed if the clinical history suggests a coagulation abnormality. Neuraxial anesthesia in the presence of sepsis or bacteremia could theoretically predispose patients to hematogenous spread of the infectious agents into the epidural or subarachnoid space.

Infection at the site of injection
Lack of consent
Coagulopathy or other bleeding diathesis
Severe hypovolemia
Increased intracranial pressure

Sepsis
Uncooperative patient
Preexisting neurological deficits
Demyelinating lesions
Stenotic valvular heart lesions
Left ventricular outflow obstruction (hypertrophic obstructive cardiomyopathy)
Severe spinal deformity

Prior back surgery at the site of injection
Complicated surgery
Prolonged operation
Major blood loss
Maneuvers that compromise respiration
Patients with preexisting neurological deficits or demyelinating diseases may report worsening symptoms following a neuraxial block. It may be impossible to discern effects or complications of the block from preexisting deficits or unrelated exacerbation of preexisting disease. For these reasons, some riskaverse practitioners argue against neuraxial anesthesia in such patients. A preoperative neurological examination should thoroughly document any deficits. In a retrospective study examining the records of 567 patients with preexisting neuropathies, 2 of the patients developed new or worsening neuropathy following neuraxial anesthesia. Although this finding indicates a relatively low risk of further injury, study investigators suggest that an injured nerve is vulnerable to additional injury, increasing the likelihood of poor neurological outcomes. However, a history of preexisting neurological deficits or demyelinating disease is at best a relative contraindication, and the balance of perioperative risks in this patient population may favor neuraxial anesthesia in certain select patients.
Regional anesthesia requires at least some degree of patient cooperation. This may be difficult or impossible for patients with dementia, psychosis, or emotional instability. The decision must be individualized. Unsedated young children may not be suitable for pure regional techniques; however, regional anesthesia is frequently used with general anesthesia in children.

Whether a block should be performed in the setting of anticoagulants and antiplatelet agents can be problematic. The American Society of Regional Anesthesia and Pain Medicine (ASRA) has issued guidelines on this subject. Because guidelines are frequently revised and updated, practitioners are advised to seek the most recent edition. Fortunately, the incidence of epidural hematoma is reported to be infrequent (1 in 150,000 epidurals). The use of anticoagulant and antiplatelet medications continues to increase, placing an ever-larger number of patients at potential risk of epidural hematomas. However, because of the rarity of epidural hematomas, most guidelines are based on expert opinion and case series reviews, as Clinical trials are not feasible.

If neuraxial anesthesia is to be used in patients receiving warfarin therapy, a normal prothrombin time and international normalized ratio usually will be documented prior to the block, unless the drug has been discontinued for weeks. Anesthesia staff should always consult with the patient's primary physicians whenever considering the discontinuation of antiplatelet or antithrombotic therapy. New agents such as the direct thrombin inhibitor dabigatran and the factor X, inhibitors rivaroxaban and apixaban are increasingly encountered by anesthesia staff (Figure 45-9).
FIGURE 45-9 Sites of action of anticoagulant drugs. Clotting factors are indicated by Roman numerals. Warfarin reduces production of factors VII, IX, X, and prothrombin. Heparin and LMWH inhibit factor Xa and thrombin. Fondaparinux, rivaroxaban, and apixaban are direct factor X, inhibitors. Dabigatran is a direct thrombin inhibitor. HMWK, high-molecular-weight kininogen; LMWH, low-molecular-weight heparin. (Reproduced with permission from Benzon H, Avram M, Green D, Bonow R. New oral anticoagulants and regional anesthesia. Br J Anaesth. 2013 Dec;111(Suppl 1):i96-i113.)

Two drug half-life intervals have been suggested as the time from drug discontinuation until neuraxial procedures are performed. However, depending upon patient factors two half-life intervals may not be sufficient to mitigate the increased risk of bleeding following neuraxial procedures. A period from drug discontinuation of up to five to six drug half-life intervals may be necessary to avoid increased bleeding risk. Thrombin clotting time assays can be used to detect the effects of dabigatran. Likewise, factor X, inhibitors can be assessed through assays of factor X, inhibition. Anesthesia providers planning neuraxial procedures should consult closely with the patient's primary providers to discern if suspension of anticoagulation is advised when considering a neuraxial technique. Bridging therapy with low-molecular-weight heparin can be considered during the time that oral anticoagulation is suspended if there is increased thrombotic risk. It has been suggested that new oral anticoagulants can be resumed 24 to 48 h following a neuraxial procedure or removal of an epidural catheter.

By themselves, aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) drugs do not increase the risk of spinal hematoma from neuraxial anesthesia procedures or epidural catheter removal. This assumes a normal patient with a normal coagulation profile who is not receiving other medications that might affect clotting mechanisms. In contrast, more potent agents should be stopped, and neuraxial blockade should generally be administered only after their effects have worn off. The waiting period depends on the specific agent: for ticlopidine it is 14 days; clopidogrel, 7 days; prasugrel, 7 to 10 days; ticagrelor, 5 days; abciximab, 48 h; and eptifibatide, 8 h. Neuraxial techniques should be avoided in patients receiving antiplatelet medications until platelet function has been recovered. Metabolites of clopidogrel and prasugrel block the P2Y12 receptor, impeding platelet aggregation. Ticagrelor directly inhibits the P2Y12 receptor. Both prasugrel and ticagrelor have greater platelet inhibition compared with clopidogrel. In patients with a recently placed cardiac stent, discontinuation of antiplatelet therapy can result in stent thrombosis and acute ST-segment elevation myocardial infarction. Risks versus benefits of a neuraxial technique should be discussed with the patient and the patient's primary physicians.

"Minidose" subcutaneous heparin prophylaxis is not a contraindication to neuraxial anesthesia or epidural catheter removal. In patients who are to receive systemic heparin intraoperatively, blocks may be performed 1 h or more before heparin administration. A bloody epidural or spinal catheter placement does not necessarily require cancellation of surgery, but discussion of the risks with the surgeon and careful postoperative monitoring is needed. Removal of an epidural catheter should occur 1 h prior to, or 4 h following, subsequent heparin dosing. Neuraxial anesthesia should be avoided in patients on therapeutic doses of intravenous heparin and with increased partial thromboplastin time. If the patient is started on heparin after the placement of an epidural catheter, the catheter should be removed only after discontinuation or interruption of heparin infusion and evaluation of the coagulation status. The risk of spinal hematoma is unclear in the setting of the anticoagulation required for cardiac surgery, and we have used epidural techniques for pain control after such procedures as off-pump coronary bypass only in the rare instances where the anticipated benefits exceed the perceived risks. Prompt diagnosis and evacuation of symptomatic epidural hematomas increase the likelihood that neuronal function will be preserved.

A number of cases of spinal hematoma were reported associated with neuraxial anesthesia after patients began receiving low-molecular-weight heparin (LMWH) enoxaparin (Lovenox) in the United States in 1993. Many of these cases involved intraoperative or early postoperative LMWH use, and several patients were receiving concomitant antiplatelet medication. The ASRA guideline provides useful recommendations for risk reduction in such circumstances. Some of the draft recommendations include that if an unusually bloody needle or catheter placement occurs, LMWH should be delayed until 24 h postoperatively, because this trauma may increase the risk of spinal hematoma. If postoperative LMWH thromboprophylaxis will be utilized, epidural catheters should be removed 4 h prior to the first LMWH dose.

Neuraxial anesthesia should not be performed if a patient has received fibrinolytic or thrombolytic therapy.
ASRA has developed a smartphone application (ASRA Coags Regional app) to assist in the perioperative management of patients taking drugs that affect coagulation.

Should lumbar neuraxial anesthesia, when used in conjunction with general anesthesia, be performed after induction of general anesthesia? This is controversial. The major arguments for having the patient asleep are that (1) most patients, if given a choice, would prefer to be asleep, and (2) the possibility of sudden patient movement causing injury is markedly diminished. The major argument in favor of neuraxial blockade administration only while the patient is still awake is that the patient can alert the clinician to paresthesias and pain on injection in this circumstance, both of which have been associated with postoperative neurological deficits. Although many clinicians are comfortable performing lumbar epidural or spinal puncture in anesthetized or deeply sedated adults, there is greater consensus, although not unanimous opinion, that thoracic and cervical punctures should, except under unusual circumstances, only be performed in awake, responsive patients. Pediatric neuraxial blocks, particularly caudal and epidural blocks, are usually performed under general anesthesia.

Neuraxial blocks must be performed only in a facility in which all the equipment and drugs needed for intubation, resuscitation, and general anesthesia are immediately available. Regional anesthesia is greatly facilitated by adequate patient premedication. Nonpharmacological patient preparation is also very helpful. The patient should be told what to expect so as to minimize anxiety. This is particularly important in situations in which premedication is not used, as is typically the case in obstetric anesthesia. Supplemental oxygen via a face mask or nasal cannula may be required to avoid hypoxemia when sedation is used. Minimum monitoring requirements include blood pressure and pulse oximetry for labor analgesia. Monitoring for blocks rendered in surgical anesthesia is the same as that in general anesthesia.

Spinous processes are usually palpable and help to define the midline. Ultrasound can be used when landmarks are not palpable (Figure 45-10). The spinous processes of the cervical and lumbar spine are nearly horizontal, whereas those in the thoracic spine slant in a caudal direction and can overlap significantly (Figure 45-2). Therefore, when performing a lumbar or cervical epidural block (with maximum spinal flexion), the needle is directed with only a slight cephalad angle, if at all, whereas for a thoracic block, the needle must be angled significantly more cephalad to enter the thoracic epidural space. In the cervical area, the first palpable spinous process is that of C2, but the most prominent one is that of C7 (vertebra prominens). With the arms at the side, the spinous process of T7 is usually at the same level as the inferior angle of the scapulae (Figure 45-11). A line drawn between the highest points of both iliac crests (Tuffier's line) usually crosses either the body of L4 or the L4—L5 interspace. Counting spinous processes up or down from these reference points identifies other spinal levels. A line connecting the posterior superior iliac spine crosses the S2 posterior foramina. In slender persons, the sacrum is easily palpable, and the sacral hiatus is felt as a depression just above or between the gluteal clefts and above the coccyx, defining the point of entry for caudal blocks.
Cephalad
FIGURE 45-10 A: Transducer position to image paramedian epidural space at the lumbar spine, longitudinal view. B: Corresponding ultrasound image. Post. Long. Lig., posterior longitudinal ligament; Lig. Flavum, ligamentum flavum; Ant. Dura Mater, anterior dura mater; Post. Dura Mater, posterior dura mater. (Reproduced with permission from Hadzic, A. Peripheral Nerve Blocks and Anatomy for UltrasoundGuided Regional Anesthesia. 2nd ed. New York, NY: McGraw-Hill; 2012.)
FIGURE 45-11 Surface landmarks for identifying spinal levels.

The anatomic midline is often easier to identify when the patient is sitting than when the patient is in the lateral decubitus position (Figure 45-12). This is particularly true with obese patients. Patients sit with their elbows resting on their thighs or a bedside table, or they can hug a pillow. Flexion of the spine (arching the back "like an angry cat") maximizes the "target" area between adjacent spinous processes and brings the spine closer to the skin surface (Figure 45-13).
FIGURE 45-12 Sitting position for neuraxial blockade. Note an assistant helps in obtaining maximal spinal flexion.
Extension
FIGURE 45-13 The effect of flexion on adjacent vertebrae. A: Posterior view. B: Lateral view. Note the target area (interlaminar foramen) for neuraxial blocks increases in size with flexion.

Many clinicians prefer the lateral position for neuraxial blocks (Figure 45-14). Patients lie on their side with their knees flexed and pulled high against the abdomen or chest, assuming a "fetal position." An assistant can help the patient assume and hold this position.
FIGURE 45-14 Lateral decubitus position for neuraxial blockade. Note again the assistant helping to provide maximal spine flexion.

This position may be used for anorectal procedures utilizing an isobaric or hypobaric anesthetic solution (see later discussion). The advantage is that the block is done in the same position as the operative procedure, so that the patient does not have to be moved following the block. The disadvantage is that CSF will not freely flow through the needle, so that correct subarachnoid needle tip placement will need to be confirmed by CSF aspiration. A prone position is typically used when fluoroscopic guidance is required.

The spine is palpated, and the patient's body positioned so that a needle passed parallel to the floor will stay midline as it courses deeper (Figure 45—4). The depression between the spinous processes of the vertebrae above and below the level to be used is palpated; this will be the needle entry site. A sterile field is established with an appropriate antibacterial solution. A fenestrated sterile drape is applied. After the preparation solution has dried, a skin wheal is raised at the level of the chosen interspace with local anesthetic using a small (25-gauge) needle. A longer needle can be used for deeper local anesthetic infiltration.
Next, the procedure needle is introduced in the midline. Remembering that the spinous processes course caudad from their origin at the spine, the needle will be directed slightly cephalad. The subcutaneous tissues offer little resistance to the needle. As the needle courses deeper, it will enter the supraspinous and interspinous ligaments, felt as an increase in tissue resistance. The needle also feels more firmly implanted in the back (like "an arrow in a target"). If bone is contacted superficially, a midline needle is likely hitting the lower spinous process. Contact with bone at a deeper level usually indicates that the needle is in the midline and hitting the upper spinous process, or that it is lateral to the midline and hitting a lamina. In either case, the needle must be redirected. As the needle penetrates the ligamentum flavum, an obvious increase in resistance is encountered. At this point, the procedures for spinal and epidural anesthesia differ.
© For epidural anesthesia, a sudden loss of resistance (to injection of air or saline) is encountered as the needle passes through the ligamentum flavum and enters the epidural space. For spinal anesthesia, the needle is advanced through the epidural space and penetrates the dura—subarachnoid membranes, as signaled by freely flowing CSF.

The paramedian technique may be selected, particularly if epidural or subarachnoid block is difficult, particularly in patients who cannot be positioned easily (eg, severe arthritis, kyphoscoliosis, or prior spine surgery) (Figure 45— 15). Many clinicians routinely use the paramedian approach for thoracic epidural puncture. After skin preparation and sterile draping (as previously described), the skin wheal for a paramedian approach is raised 2 cm lateral to the inferior aspect of the superior spinous process of the desired level. Because this approach is lateral to most of the interspinous ligaments and penetrates the paraspinous muscles, the needle may encounter little resistance initially and may not seem to be in firm tissue. The needle is directed and advanced at a 10° to 25° angle toward the midline. If bone is encountered at a shallow depth with the paramedian approach, the needle is likely in contact with the medial part of the lower lamina and should be redirected mostly upward and perhaps slightly more laterally. On the other hand, if bone is encountered deeply, the needle is usually in contact with the lateral part of the lower lamina and should be redirected only slightly craniad, more toward the midline (Figure 45-16).
FIGURE 45-15 Paramedian approach.
FIGURE 45-16 Paramedian approach. A needle that encounters bone at a shallow depth (a) is usually hitting the medial lamina, whereas one that encounters bone deeply (b) is farther lateral from the midline. A: Posterior view. B: Parasagittal view.

With knowledge of the sensory dermatomes (see appendix), the extent of sensory block can be assessed by a blunted needle or a piece of ice.

Although it has not, as of yet, transformed the practice of neuraxial blockade in the same manner as it has for other procedures, ultrasound guidance can facilitate neuraxial blockade in patients with poorly palpable landmarks. As with other uses of ultrasound, specific training is required for practitioners to identify correctly the landmarks and interspaces necessary for neuraxial blockade.

Spinal needles are commercially available in an array of sizes lengths, and bevel and tip designs (Figure 45-17). All should have a tightly fitting, removable stylet that completely occludes the lumen to avoid tracking epithelial cells into the subarachnoid space. Broadly, they can be divided into either sharp (cutting)tipped or blunt-tipped needles. The Quincke needle is a cutting needle with end injection. The introduction of blunt tip (pencil-point) needles has markedly decreased the incidence of postdural puncture headache. The Whitacre and other pencil-point needles have rounded points and side injection. The Sprotte is a side-injection needle with a long opening. It has the advantage of more vigorous CSF flow compared with similar gauge needles. However, this can lead to a failed block if the distal part of the opening is subarachnoid (with free flow CSF), the proximal part is not past the dura, and the full dose of medication is not delivered. In general, the smaller the gauge needle (along with use of a blunttipped needle), the lower will be the incidence of headache.

Larger catheters designed for epidural use are frequently employed for continuous spinal anesthesia following accidental dural puncture during performance of epidural anesthesia. Catheters must be carefully labeled as being subarachnoid, as opposed to epidural, to avoid the potential for inadvertent dosage.

The midline or paramedian approaches, with the patient positioned in the lateral decubitus, sitting, or prone positions, can be used for spinal anesthesia. As previously discussed, the needle is advanced from skin through the deeper structures until two "pops" are felt. The first is penetration of the ligamentum flavum, and the second is penetration of the dura—arachnoid membrane. Successful dural puncture is confirmed by withdrawing the stylet to verify free flow of CSF. With small-gauge needles (<25 gauge), aspiration may be necessary to detect CSF. If free flow occurs initially, but CSF cannot be aspirated after attaching the syringe, the needle likely will have moved. Persistent paresthesias or pain with injection of drugs should prompt the clinician to withdraw and redirect the needle.

Table 45-2 lists factors that have been shown to affect the level of neural blockade following spinal anesthesia. The most important determinants are baricity of the local anesthetic solution, position of the patient during and immediately after injection, and drug dosage. In general, the larger the dosage or more cephalad the site of injection, the more cephalad the level of anesthesia that will be obtained. Moreover, migration of the local anesthetic cephalad in CSF depends on its density relative to CSF (baricity). CSF has a specific gravity of 1.003 to 1.008 at 37°C. Table 45—3 lists the specific gravity of anesthetic solutions. A hyperbaric solution of local anesthetic is denser (heavier) than CSF, whereas a hypobaric solution is less dense (lighter) than CSF. The local anesthetic solutions can be made hyperbaric by the addition of glucose or hypobaric by the addition of sterile water or fentanyl. Thus, with the patient in a head-down position, a hyperbaric solution spreads cephalad, and a hypobaric anesthetic solution moves caudad. A head-up position causes a hyperbaric solution to settle caudad and a hypobaric solution to ascend cephalad. Similarly, when a patient remains in a lateral position, a hyperbaric spinal solution will have a greater effect on the dependent (down) side, whereas a hypobaric solution will achieve a higher level on the nondependent (up) side. An isobaric solution tends to remain at the level of injection. Anesthetic agents lacking glucose may be mixed with CSF (at least 1:1) to make their solutions isobaric. Other factors affecting the level of neural blockade include the level of injection and the patient's height and vertebral column anatomy. The direction of the needle bevel or injection port may also play a role; higher levels of anesthesia are achieved if the injection is directed cephalad than if the point of injection is oriented laterally or caudad.

Baricity of anesthetic solution
Position of the patient
During injection
Immediately after injection
Drug dosage
Site of injection

Age
Cerebrospinal fluid
Curvature of the spine
Drug volume
Intraabdominal pressure
Needle direction
Patient height
Pregnancy
TABLE 45-3 Specific gravities of some spinal anesthetic agents.
Bupivacaine, Specific Gravity = . 0.5% in 8.25% dextrose, Specific Gravity = 1.0227-1.0278. 0.5% plain, Specific Gravity = 0.9990-1.0058. Lidocaine, Specific Gravity = . 2% plain, Specific Gravity = 1.0004-1.0066. 5% in 7.5% dextrose, Specific Gravity = 1.0262-1.0333. Tetracaine, Specific Gravity = . 0.5% in water, Specific Gravity = 0.9977-0.9997. 0.5% in D.W, Specific Gravity = 1.0133-1.0203
Hyperbaric solutions tend to move to the most dependent area of the spine (normally T4—T$8 in the supine position).
With normal spinal anatomy, the apex of the thoracolumbar curvature is T4 (Figure 45-18). In the supine position, this should limit a hyperbaric solution to produce a level of anesthesia at or below T4. Abnormal curvatures of the spine, such as scoliosis and kyphoscoliosis, have multiple effects on spinal anesthesia. Placing the block becomes more difficult because of the rotation and angulation of the vertebral bodies and spinous processes. Finding the midline and the interlaminar space may be difficult. The paramedian approach to lumbar puncture may be preferable in patients with severe scoliosis and kyphoscoliosis. Reviewing radiographs of the spine before attempting the block may be useful. Spinal curvature affects the ultimate level by changing the contour of the subarachnoid space. Previous spinal surgery can similarly result in technical difficulties in placing a block. Correctly identifying the interspinous and interlaminar spaces may be difficult at the levels of previous laminectomy or spinal fusion. The paramedian approach may be easier, or a level above the surgical site can be chosen. The block may be incomplete, or the level may be different than anticipated, due to postsurgical anatomic changes.

FIGURE 45-18 The position of the spinal canal in the supine position (A) and lateral decubitus position (B). Note the lowest point is usually between T5 and T7, where a hyperbaric solution tends to settle once the patient is placed supine.
Lumbar CSF volume inversely correlates with the dermatomal spread of spinal anesthesia. Increased intraabdominal pressure or other conditions that cause engorgement of the epidural veins, thus decreasing CSF volume, are associated with greater dermatomal spread for a given volume of injectate. This would include conditions such as pregnancy, ascites, and large abdominal tumors. In these clinical situations, higher levels of anesthesia are achieved with a given dose of local anesthetic than would otherwise be expected. For spinal anesthesia on a term parturient, some clinicians reduce the dosage of anesthetic by one-third compared with a nonpregnant patient, particularly when the block will be initiated with the patient in the lateral position. Age-related decreases in CSF volume are likely responsible for the higher anesthetic levels achieved in the elderly for a given dosage of spinal anesthetic. Severe kyphosis or kyphoscoliosis can also be associated with a decreased volume of CSF and often results in a higher than expected level, particularly with a hypobaric technique or rapid injection.

Many local anesthetics have been used for spinal anesthesia in the past, but only a few are currently in use (Table 45-4). Only preservative-free local anesthetic solutions are used. Addition of vasoconstrictors (a-adrenergic agonists, epinephrine [0.1—0.2 mg]) and opioids enhance the quality or prolong the duration of spinal anesthesia, or both. Vasoconstrictors seem to delay the uptake of local anesthetics from CSF and may have weak spinal analgesic properties. Opioids and clonidine can likewise be added to spinal anesthetics to improve both the quality and duration of the subarachnoid block.

= 0.5%, 0.75%. Ropivacaine, Dose.(mg) = 18-225. Ropivacaine, Procedures = TBlevel. Ropivacaine, Duration (h).Plain = 3-4. Ropivacaine, Duration (h).Epinephrine = . Ropivacaine, Preparation = 1% + 10% dextrose (equal volumes D., and ropivacaine). Ropivacaine, Dose.(mg) = 18-225. Ropivacaine, Procedures = T4level. Ropivacaine, Duration (h).Plain = 1.5-2. Ropivacaine, Duration (h).Epinephrine = 
Tetracaine, 1 = 1% + 10% dextrose (0.5% hyperbaric). Tetracaine, 2 = 4-8 10-12 10-16. Tetracaine, 3 = Perineum/lower extremities Lower abdomen Upper abdomen. Tetracaine, 4 = 15-2 35-4. Adjuvant Fentanyl, 1 = Dose (meg) 10-25. Adjuvant Fentanyl, 2 = . Adjuvant Fentanyl, 3 = Duration (h) 1-2. Adjuvant Fentanyl, 4 = Comments/Side Effects Itching; nausea; urinary retention;. Epinephrine, 1 = 100-200. Epinephrine, 2 = . Epinephrine, 3 = . Epinephrine, 4 = Prolongs nerve exposure to local anesthetic + a-adrenergic modulation. Phenylephrine, 1 = 1000-2000. Phenylephrine, 2 = . Phenylephrine, 3 = . Phenylephrine, 4 = Hypotension. Prolongs tetracaine but not bupivacaine. Extends tetracaine better than epinephrine does. May cause TNS. Clonidine, 1 = 15-150. Clonidine, 2 = . Clonidine, 3 = . Clonidine, 4 = Hypotension. Sedation. Prolongs motor and sensory block

"Reproduced with permission from Atchabahian A, Gupta R, eds. The Anesthesia Guide. New York, NY: McGraw-Hill Education, Inc; 2013.
THA, total hip arthroplasty; TKA, total knee arthroplasty; TNS, transient neurological symptoms; TURP. transurethral resection of the prostate; ORIF, open reduction and internal fixation.
Used as an alternative to lidocaine, but TNS also occur with mepivacaine.
"Each change of 15 mg prolongs or hastens ambulatory milestones by 20-30 min. Fentanyl, 10 mcg, extends surgical block but not ambulatory recovery times and should probably be added if using 30 mg dose to ensure adequate duration
Nery low dose (4-5 mg) works well for ambulatory, unilateral, knee surgery. Keep patient lateral, affected side down, for 6 min after block.
Until very recently in North America, hyperbaric spinal anesthesia was more commonly used than hypobaric or isobaric techniques. The level of anesthesia is then dependent on the patient's position during and immediately following the injection. In the sitting position, "saddle block" can be achieved by keeping the patient sitting for 3 to 5 min following injection, so that only the lower lumbar nerves and sacral nerves are blocked. If the patient is moved from a sitting position to a supine position immediately after injection, the agent will move more cephalad to the dependent region defined by the thoracolumbar curve. Hyperbaric anesthetics injected intrathecally with the patient in a lateral decubitus position are useful for unilateral lower extremity procedures. The patient is placed laterally, with the extremity to be operated on in a dependent position. If the patient is kept in this position for about 5 min following injection, the block will tend to be denser and achieve a higher level on the operative dependent side.
If regional anesthesia is chosen for surgical procedures involving hip or lower extremity fracture, hypobaric or isobaric spinal anesthesia can be useful because the patient need not lie on the fractured extremity.

® Continuous epidural anesthesia is a neuraxial technique offering a range of applications wider than single-dose spinal anesthesia. An epidural block can be performed at the lumbar, thoracic, or cervical level. Sacral epidural anesthesia is referred to as a caudal block and is described at the end of this chapter. @ Epidural techniques are widely used for surgical anesthesia, obstetric analgesia, postoperative pain control, and chronic pain management. Epidurals can be used as a single shot technique or with a catheter that allows intermittent boluses or continuous infusion, or both. The motor block can range from none to complete. All of these variables are controlled by the choice of drug, concentration, dosage, and level of injection.
The epidural space surrounds the dura mater posteriorly, laterally, and anteriorly. Nerve roots travel in this space as they exit laterally through the foramen and course outward to become peripheral nerves. Other contents of the lumbar epidural space include fatty connective tissue, lymphatics, and a rich venous (Batson's) plexus. Fluoroscopic studies have demonstrated the presence of septa or connective tissue bands within the epidural space, possibly explaining the occasional one-sided epidural block. ® Epidural anesthesia is slower in onset (10-20 min) and may not be as dense as spinal anesthesia, a feature that can be useful clinically. For example, by using relatively dilute concentrations of a local anesthetic combined with an opioid, an epidural provides analgesia without motor block. This is commonly employed for labor and postoperative analgesia. Moreover, a segmental block is possible because the anesthetic can be confined close to the level at which it was injected. A segmental block is characterized by a well-defined band of anesthesia at certain nerve roots; leaving nerve roots above and below unblocked. This can be seen with a thoracic epidural that provides upper abdominal anesthesia while sparing cervical and lumbar nerve roots.

Epidural anesthesia is most often performed in the lumbar region. The midline (Figure 45—4) or paramedian approaches (Figure 45—15) can be used. Lumbar epidural anesthesia can be used for any procedure below the diaphragm. Because the spinal cord typically terminates at the L1 level, there is an extra measure of safety in performing the block in the lower lumbar interspaces, particularly if an accidental dural puncture occurs (see "Complications," later).
Thoracic epidural blocks are technically more difficult to accomplish than are lumbar blocks because of greater angulation and the overlapping of the spinous processes at the vertebral level (Figure 45-19). Moreover, the potential risk of spinal cord injury with accidental dural puncture, although exceedingly small with good technique, may be greater than that at the lumbar level. Thoracic epidural blocks can be accomplished with either a midline or paramedian approach. The thoracic epidural technique is more commonly used for postoperative analgesia than as a primary anesthetic. Single shot or catheter techniques are used for the management of chronic pain. Infusions via an epidural catheter are useful for providing prolonged durations of analgesia and may obviate or shorten postoperative ventilation in patients with underlying lung disease and following chest surgery.
FIGURE 45-19 Angulation of the epidural needle at the cervical (A), thoracic (B), and lumbar (C) levels. Note that an acute angulation (30—50°) is required for a thoracic epidural block, whereas only a slight cephalad orientation is usually required for cervical and lumbar epidural blocks.
Cervical blocks are usually performed with the patient sitting, with the neck flexed, using the midline approach. They are used most often for the management of acute and chronic pain.

The standard epidural needle is typically 17 to 18 gauge, 3 or 3.5 inches long, and has a blunt bevel with a gentle curve of 15° to 30° at the tip. The Tuohy needle is most commonly used (Figure 45—20). The blunt, curved tip theoretically helps to push away the dura after passing through the ligamentum flavum instead of penetrating it. Straight needles without a curved tip (Crawford needles) may have a greater incidence of dural puncture. Needle modifications include winged tips and introducer devices set into the hub designed for guiding catheter placement.

Placing a catheter into the epidural space allows for continuous infusion or intermittent bolus techniques. In addition to extending the duration of the block, catheter techniques may allow a lower total dose of anesthetic to be used. Epidural catheters are useful for intraoperative epidural anesthesia and postoperative analgesia. Typically, a 19or 20-gauge catheter is introduced through a 17or 18-gauge epidural needle. When using a curved tipped needle, the bevel opening is directed either cephalad or caudad, and the catheter is advanced 2 to 6 cm into the epidural space. The shorter the distance the catheter is advanced, the more likely it is to become dislodged. Conversely, the further the catheter is advanced, the greater the chance of a unilateral block (due to the catheter tip either exiting the epidural space via an intervertebral foramen or coursing into the anterolateral recesses of the epidural space) and the greater chance of penetration of an epidural vein occurring. After advancing the catheter the desired depth, the needle is removed, leaving the catheter in place. The catheter can be taped or otherwise secured along the back. Catheters that will remain in place for prolonged times (eg, >1 week) may be tunneled under the skin. Catheters have either a single port at the distal end or multiple side ports close to a closed tip. Some have a stylet for easier insertion or to help steer the catheter passage in the epidural space with fluoroscopic guidance. Spiral wirereinforced catheters are very resistant to kinking. The spiral or spring tip is associated with fewer, less intense paresthesias and may be associated with a lower incidence of inadvertent intravascular insertion.

Using the midline or paramedian approaches detailed previously, the epidural needle is passed through the skin and the ligamentum flavum. The needle must stop short of piercing the dura. Two techniques make it possible to determine when the tip of the needle has entered the potential (epidural) space: the "loss of resistance" and "hanging drop" techniques.
The loss of resistance technique is preferred by most clinicians. The needle is advanced through the subcutaneous tissues with the stylet in place until the interspinous ligament is entered, as noted by an increase in tissue resistance. The stylet or introducer is removed, and a glass syringe filled with saline or air is attached to the hub of the needle. If the tip of the needle is within the ligament, gentle attempts at injection are met with resistance, and injection is not possible. The needle is then slowly advanced, millimeter by millimeter, with either continuous or rapidly repeating attempts at injection. As the tip of the needle just enters the epidural space, there is a sudden loss of resistance, and injection is easy.
Once the interspinous ligament has been entered and the stylet has been removed, the hanging drop technique requires that the hub of the needle be filled with solution so that a drop hangs from its outside opening. The needle is then slowly advanced deeper. As long as the tip of the needle remains within the ligamentous structures, the drop remains "hanging." However, as the tip of the needle enters the epidural space, it creates negative pressure, and the drop of fluid is sucked into the needle. If the needle becomes plugged, the drop will not be drawn into the hub of the needle, and accidental dural puncture may occur. Some clinicians prefer to use this technique for the paramedian approach and for cervical epidurals. Skilled "epiduralists" rely on either the loss of resistance or hanging drop as confirmation (rather than as the primary test) that the needle has entered the epidural space. Successful "epiduralists" will generally have sensed the "give" in their hands as the epidural needle tip passes through the ligamentum flavum.

© The quantity (volume and concentration) of local anesthetic needed for epidural anesthesia is larger than that needed for spinal anesthesia. Toxic side effects are almost guaranteed if a "full epidural dose" is injected intrathecally or intravascularly. Safeguards against toxic epidural side effects include test dosing and incremental dosing. These safeguards apply whether the injection is through the needle or through an epidural catheter.
A test dose is designed to detect both subarachnoid and intravascular injection. The classic test dose combines local anesthetic and epinephrine, typically 3 mL of 1.5% lidocaine with 1:200,000 epinephrine (0.005 mg/mL). The 45 mg of lidocaine, if injected intrathecally, will produce spinal anesthesia that should be rapidly apparent. Some clinicians have suggested the use of lower doses of local anesthetic, as an unintended injection of 45 mg of intrathecal lidocaine, can be difficult to manage in areas such as labor rooms. The 15 mcg dose of epinephrine, if injected intravascularly, should produce a noticeable increase in heart rate (20% or more), with or without hypertension. Unfortunately, epinephrine as a marker of intravenous injection is not ideal. False positives (a uterine contraction causing pain or an increase in heart rate

Factors affecting the level of epidural anesthesia may not be as predictable as with spinal anesthesia. In adults, 1 to 2 mL of local anesthetic per segment to be blocked is a generally accepted guideline. For example, to achieve a T4 sensory level from an L4—L5 injection would require about 12 to 24 mL. For segmental or analgesic blocks, less volume is needed.
The dose required to achieve the same level of anesthesia decreases with age. This is probably a result of age-related decreases in the size or compliance of the epidural space. Although there is little correlation between body weight and epidural dosage requirements, patient height affects the extent of cephalad spread. Thus, shorter patients may require only 1 mL of local anesthetic per segment to be blocked, whereas taller patients generally require 2 mL per segment. Although less dramatic than with hyperbaric or hypobaric spinal anesthesia, spread of epidural local anesthetics tends to be partially affected gravity. The lateral decubitus, Trendelenburg, and reverse Trendelenburg positions can be used to help achieve blockade in the desired dermatomes.
by
Additives to the local anesthetic, particularly opioids, tend to have a greater effect on the quality of epidural anesthesia than on the duration of the block. Epinephrine in concentrations of 5 mcg/mL prolongs the effect of epidural lidocaine, mepivacaine, and chloroprocaine more than that of bupivacaine, levobupivacaine, or ropivacaine. In addition to prolonging the duration and improving the quality of block, epinephrine delays vascular absorption and reduces peak systemic blood levels of all epidurally administered local anesthetics.

The epidural agent is chosen based on the desired clinical effect, whether it is to be used as a primary anesthetic, supplementation of general anesthesia, or analgesia. The anticipated duration of the procedure may call for a shortor long-acting single shot anesthetic or the insertion of a catheter (Table 45-5). Commonly used shortto intermediate-acting agents for surgical anesthesia include chloroprocaine, lidocaine, and mepivacaine. Longer acting agents include bupivacaine, levobupivacaine, and ropivacaine.

Chloroprocaine, Concentration = 2% 3%. Chloroprocaine, Onset = Fast Fast. Chloroprocaine, Sensory Block = Analgesic Dense. Chloroprocaine, Motor Block = Mild to moderate Dense. Lidocaine, Concentration = <1% 1.5% 2%. Lidocaine, Onset = Intermediate Intermediate Intermediate. Lidocaine, Sensory Block = Analgesic Dense Dense. Lidocaine, Motor Block = Minimal Mild to moderate Dense. Mepivacaine, Concentration = 19% 2-3%. Mepivacaine, Onset = Intermediate Intermediate. Mepivacaine, Sensory Block = Analgesic Dense. Mepivacaine, Motor Block = Minimal Dense. Bupivacaine, Concentration = 0.25% 0.5% 0.75%. Bupivacaine, Onset = Slow Slow Slow. Bupivacaine, Sensory Block = Analgesic Dense Dense. Bupivacaine, Motor Block = Minimal Mild to moderate Moderate to dense. Ropivacaine, Concentration = 0.2% 0.5% 0.75-1.0%. Ropivacaine, Onset = Slow Slow Slow. Ropivacaine, Sensory Block = Analgesic Dense Dense. Ropivacaine, Motor Block = Minimal Mild to moderate Moderate to dense
Following the initial 1 to 2 mL per segment bolus (in fractionated doses), repeat doses delivered through an epidural catheter are done on a fixed time interval (either as a bolus or continuous infusion), based on the practitioner's experience with the agent, or only redosed when the block demonstrates some degree of regression. Once some regression in sensory level has occurred, onethird to one-half of the initial activation dose can generally safely be reinjected in incremental doses.

It should be noted that chloroprocaine, an ester with rapid onset, short duration, and extremely low toxicity, may interfere with the analgesic effects of epidural opioids. Previous chloroprocaine formulations with preservatives, specifically bisulfite and ethylenediaminetetraacetic acid (EDTA), produced cauda equine syndrome when accidentally injected in a large volume intrathecally. Bisulfite preparations of chloroprocaine were believed to be associated with neurotoxicity, whereas EDTA formulations were associated with severe back pain (presumably due to localized hypocalcemia). Most current preparations of chloroprocaine are preservative-free and without these complications.
Surgical anesthesia is obtained with a 0.5% bupivacaine formulation. The 0.75% formulation of bupivacaine is no longer used in obstetrics, as its use in cesarean delivery has been associated with reports of cardiac arrest after accidental intravenous injection Very dilute concentrations of bupivacaine (eg, 0.0625%) are commonly combined with fentanyl and used for analgesia for labor and postoperative pain. Compared with bupivacaine, ropivacaine may produce less motor block at similar concentrations while maintaining a satisfactory sensory block.

Local anesthetic solutions have an acidic pH for chemical stability and bacteriostasis. Local anesthetic solutions that are formulated with epinephrine by the manufacturer are more acidic than the "plain" solutions that do not contain epinephrine. Because they are weak bases, they exist primarily in the ionic form in commercial preparations. The onset of neural block requires permeation of lipid barriers by the uncharged form of the local anesthetic. Increasing the pH of the solutions increases the fraction of the uncharged form of the local anesthetic. Addition of sodium bicarbonate (1 mEq/10 mL of local anesthetic) immediately before injection may therefore accelerate the onset of the neural blockade. Sodium bicarbonate is typically not added to bupivacaine, which precipitates above a pH of 6.8.

®
Caudal epidural anesthesia is a common regional technique in pediatric patients. It may also be used for anorectal surgery in adults. The caudal space is the sacral portion of the epidural space. Caudal anesthesia involves needle or catheter penetration of the sacrococcygeal ligament covering the sacral hiatus that is created by the unfused S4 and S5 laminae. The hiatus may be felt as a groove or notch above the coccyx and between two bony prominences, the sacral cornua (Figure 45-3). Its anatomy is very easily appreciated in infants and children (Figure 45—21). The posterior superior iliac spines and the sacral hiatus define an equilateral triangle (Figure 45-14). Calcification of the sacrococcygeal ligament may make caudal anesthesia difficult or impossible in older adults. As previously noted, the dural sac extends to the first sacral vertebra in adults and to about the third sacral vertebra in infants, making accidental intrathecal injection a common concern in infants.
FIGURE 45-21 Positioning an anesthetized child for caudal block and palpation for the sacral hiatus. An assistant gently helps flex the spine.

In children, caudal anesthesia is typically combined with general anesthesia for intraoperative supplementation and postoperative analgesia. It is commonly used for procedures below the diaphragm, including urogenital, rectal, inguinal, and lower extremity surgery. Pediatric caudal blocks are most commonly performed after the induction of general anesthesia. However, regional techniques are increasingly used for surgical anesthesia in infants and young children because of concerns about the possible neurotoxic effects of general anesthesia in that population. The patient is placed in the lateral or prone position with one or both hips flexed, and the sacral hiatus is palpated. After sterile skin preparation, a needle or intravenous catheter (18—23 gauge) is advanced at a 45° angle cephalad until a pop is felt as the needle pierces the sacrococcygeal ligament. The angle of the needle is then flattened and advanced (Figure 45—22). Aspiration for blood and CSF is performed, and, if negative, injection can proceed. Some clinicians recommend test dosing as with other epidural techniques, although many simply rely on incremental dosing with frequent aspiration. Tachycardia (if epinephrine is used) or increasing size of the T waves on electrocardiography may indicate intravascular injection. Complications are fortunately infrequent but include total spinal and intravenous injection, causing seizure or cardiac arrest. Ultrasound has also been employed in the performance of caudal blocks.
FIGURE 45-22 Caudal block. Note the sacrococcygeal ligament is penetrated with the needle almost at 90° and then must be angled down and advanced to enter the sacral hiatus.
A dosage of 0.5 to 1.0 mL/kg of 0.125% to 0.25% bupivacaine (or ropivacaine), with or without epinephrine, can be used. Opioids may also be added (eg, 30-40 mcg/kg of morphine). Clonidine is often included, as well. The analgesic effects of the block extend for hours into the postoperative period.

may In adults undergoing anorectal procedures, caudal anesthesia can provide dense sacral sensory blockade with limited cephalad spread. Furthermore, the injection can be given with the patient in the prone jackknife position, which is used for surgery (Figure 45—23). A dose of 15 to 20 mL of 1.5% to 2.0% lidocaine, with or without epinephrine, is usually effective. Fentanyl, 50 to 100 mcg, may also be added. This technique should be avoided in patients with pilonidal cysts because the needle may pass through the cyst track and can potentially introduce bacteria into the caudal epidural space.
FIGURE 45-23 The prone jackknife position often used for anorectal surgery can also be used for caudal anesthesia in adults. (Reproduced with permission from Lambert DH, Covino BG. Hyperbaric, hypobaric, and isobaric spinal anesthesia. Res Staff Phys. 1987 Sep;10(33):79-87.)

The complications of epidural, spinal, or caudal anesthetics range from mild to crippling and life-threatening (Table 45-6). Broadly, complications from neuraxial techniques are secondary to excessive physiological effects of an appropriately injected drug, injury from needle or catheter placement, and local anesthetic systemic toxicity.
TABLE 45-6 Complications of neuraxial anesthesia.

Urinary retention
High block
Total spinal anesthesia
Cardiac arrest
Anterior spinal artery syndrome

Backache
Dural puncture/leak
Postdural puncture headache
Diplopia
Tinnitus
Neural injury
Nerve root damage
Spinal cord damage
Cauda equina syndrome
Bleeding
Intraspinal/epidural hematoma
Misplacement
No effect/inadequate anesthesia
Subdural block
Inadvertent subarachnoid block!
Inadvertent intravascular injection
Catheter shearing/retention
Inflammation
Arachnoiditis
Infection
Meningitis
Epidural abscess

Cauda equina syndrome lor epidural block only.
A very large survey of regional anesthetics provides an indication of the relatively low incidence of serious complications from spinal and epidural anesthesia (Table 45—7). The American Society of Anesthesiologists (ASA) Closed Claims Project helps to identify the most common causes of liability claims involving regional anesthesia in the operating room setting. In a 20-year period (1980-1999), regional anesthesia accounted for 18% of all liability claims. In the majority of these claims, the injuries were judged as temporary or nondisabling (64%). Serious injuries in the remaining claims included death (13%), permanent nerve injury (10%), permanent brain damage (8%), and other permanent injuries (4%). The majority of regional anesthesia claims involved either lumbar epidural anesthesia (42%) or spinal anesthesia (34%) and tended to occur mostly in obstetric patients.

Spinal (n = 40,640), CardiacArrest = 26. Spinal (n = 40,640), Death = 6. Spinal (n = 40,640), Seizure = 0. Spinal (n = 40,640), CaudaEquinaSyndrome = 5. Spinal (n = 40,640), Paraplegia = 0. Spinal (n = 40,640), Radiculopathy = 19. Epidural (n = 30,413), CardiacArrest = 3. Epidural (n = 30,413), Death = 0. Epidural (n = 30,413), Seizure = 4. Epidural (n = 30,413), CaudaEquinaSyndrome = QO. Epidural (n = 30,413), Paraplegia = 1. Epidural (n = 30,413), Radiculopathy = 5
'Data from Auroy Y, Narchi P. Messiah A, et al. Serious complications related to regional anesthesia: Results of a prospective survey in France. Anesthesiology. 1997 Sep;87(3):479-486.
Complications Associated with Excessive Responses to Appropriately Placed Drug

Exaggerated dermatomal spread of neural blockade can occur readily with either spinal or epidural anesthesia. Administration of an excessive dose, failure to reduce standard doses in selected patients (eg, the elderly, pregnant, obese, or very short), or unusual sensitivity or spread of local anesthetic may be responsible. Patients may complain of dyspnea and have numbness or weakness in the upper extremities. Nausea often precedes hypotension. Once exaggerated spread of anesthesia is recognized, patients should be reassured, oxygen supplementation may be required, and bradycardia and hypotension should be treated.
Spinal anesthesia ascending into the cervical levels causes severe hypotension, bradycardia, and respiratory insufficiency. Unconsciousness, apnea, and hypotension resulting from high levels of spinal anesthesia are referred to as a "high spinal," or when the block extends to cranial nerves, as a "total spinal." These conditions can also occur following attempted epidural or caudal anesthesia if there is accidental intrathecal injection (see later discussion). Apnea is more often the result of severe sustained hypotension and medullary hypoperfusion than a response to phrenic nerve paralysis from anesthesia of C3— C5 roots. Anterior spinal artery syndrome has been reported following neuraxial anesthesia, presumably due to prolonged severe hypotension together with an increase in intraspinal pressure.
Treatment of an excessively high neuraxial block involves maintaining adequate arterial oxygenation and ventilation and supporting the circulation. When respiratory insufficiency becomes evident, in addition to supplemental oxygen and assisted ventilation, intubation and mechanical ventilation may be necessary. Hypotension can be treated with intravenous vasopressors and rapid administration of intravenous fluids. Bradycardia can be treated early with atropine. Ephedrine or epinephrine can also increase heart rate and arterial blood pressure.

Examination of data from the ASA Closed Claim Project identified several cases of cardiac arrest during spinal anesthesia. Because many of these cases predated the routine use of pulse oximetry, oversedation and unrecognized hypoventilation and hypoxia may have contributed. However, large prospective studies continue to report a relatively high incidence (perhaps as high as 1:1500) of cardiac arrest in patients having received a spinal anesthetic. Many of these cardiac arrests were preceded by bradycardia, and many occurred in young healthy patients. To prevent this from occurring, hypovolemia should be corrected. Prompt drug treatment of hypotension and bradycardia are recommended.

Local anesthetic block of S2—S4 root fibers decreases urinary bladder tone and inhibits the voiding reflex. Epidural opioids can also interfere with normal voiding.

As with other regional anesthesia techniques, neuraxial blocks are associated with a small, but measureable, failure rate that is usually inversely proportional to the clinician's experience. Failure may still occur, even when CSF is obtained during spinal anesthesia. Movement of the needle during injection, incomplete entry of the needle opening into the subarachnoid space, subdural injection, or injection of the local anesthetic solution into a nerve root sleeve may be responsible. Causes for failed epidural blocks were discussed earlier (see "Failed Epidural Blocks").

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Total spinal anesthesia can occur following attempted epidural or caudal anesthesia if there is accidental intrathecal injection. Onset is usually rapid, because the amount of anesthetic required for epidural and caudal anesthesia is 5 to 10 times that required for spinal anesthesia. Careful aspiration, use of a test dose, and incremental injection techniques (remember, "every dose is a test dose") during epidural and caudal anesthesia can help avoid this complication.

Because of the larger amount of local anesthetic administered, accidental subdural injection of local anesthetic during attempted epidural anesthesia is much more serious than during attempted spinal anesthesia. A subdural injection of epidural doses of local anesthetic produces a clinical presentation similar to that of high spinal anesthesia, with the exception that the onset may be delayed for 15 to 30 min and the block may be "patchy." The spinal subdural space is a potential space between the dura and the arachnoid that extends intracranially, so that anesthetic injected into the spinal subdural space can ascend to higher levels than epidural medications. As with high spinal anesthesia, treatment is supportive and may require intubation, mechanical ventilation, and cardiovascular support. The effects generally last from one to several hours.

As a needle passes through skin, subcutaneous tissues, muscle, and ligaments it causes varying degrees of tissue trauma. Bruising and a localized inflammatory response with or without reflex muscle spasm may be responsible for postoperative backache. One should remember that up to 25% to 30% of patients receiving general anesthesia also complain of backache postoperatively, and a significant percentage of the general population has chronic back pain. Postoperative back soreness or ache is usually mild and self-limited, although it may last for a number of weeks. If treatment is sought, acetaminophen or NSAIDs should suffice. Although backache is usually benign, it may be an important clinical sign of much more serious complications, such as epidural hematoma and abscess (see later discussion).

CSF. Caffeine further helps by vasoconstricting intracranial vessels as cerebral vasodilation is thought to be a response to the intracranial hypotension secondary to the CSF leak. Stool softeners and soft diet are used to minimize Valsalva straining. Headache may persist for days, despite conservative therapy. An epidural blood patch is an effective treatment for PDPH. It involves injecting 15 to 20 mL of autologous blood into the epidural space at, or one interspace below, the level of the dural puncture. It is believed to stop further leakage of CSF by either mass effect or coagulation. The effect is usually immediate and complete, but may take some hours as CSF production slowly rebuilds intracranial pressure. Approximately 90% of patients will respond to a single blood patch, and 90% of initial nonresponders will obtain relief from a second injection. We do not recommend prophylactic blood patching after a wet tap. Not all patients will develop PDPH following a wet tap, and in any case, prophylaxis has not proven terribly effective. When evaluating patients with presumed PDPH, other sources of headache, including migraine, caffeine withdrawal, meningeal infection, and subarachnoid hemorrhage, should be considered in the differential diagnosis.

Perhaps no complication is more perplexing or distressing than persistent neurological deficits following an apparently routine neuraxial block. An epidural hematoma or abscess must be ruled out. Either nerve roots or spinal cord may be injured. The latter may be avoided if the neuraxial blockade is performed below the termination of the conus (L1 in adults and L3 in children). Postoperative peripheral neuropathies can be due to direct physical trauma to nerve roots. Although most resolve spontaneously, some are permanent. Any sustained paresthesia during neuraxial anesthesia/analgesia should alert the clinician to redirect the needle. Injections should be immediately stopped and the needle withdrawn, if injection is associated with pain. Direct injection into the spinal cord can cause paraplegia. Damage to the conus medullaris may cause isolated sacral nerve dysfunction. Not all neurological deficits that are reported after a regional anesthetic are the direct result of the block. Postpartum neurological deficits, including lateral femoral cutaneous neuropathy and foot drop, were recognized as complications before the era of routine epidural anesthesia/analgesia.

Needle or catheter trauma to epidural veins often causes minor bleeding in the spinal canal, although this usually has no consequences. The incidence of spinal hematomas has been estimated to be about 1:150,000 for epidural blocks and 1:220,000 for spinal anesthetics. The vast majority of reported cases have occurred in patients with abnormal coagulation secondary to either disease or drugs. Some hematomas have occurred immediately following removal of an epidural catheter. Thus, both insertion and removal of an epidural catheter can lead to epidural hematoma formation.
Diagnosis and treatment must be prompt, if permanent neurological sequelae secondary to neuronal ischemia are to be avoided. The onset of symptoms is typically more sudden than with epidural abscess. Symptoms include sharp back and leg pain with a motor weakness or sphincter dysfunction, or both. When hematoma is suspected, imaging (magnetic resonance [MR] or computed tomography [CT]) and neurosurgical consultation must be obtained immediately. In many cases, good neurological recovery has occurred in patients who have undergone prompt surgical decompression.
Neuraxial anesthesia should be avoided in patients with coagulopathy, significant thrombocytopenia, platelet dysfunction, or those who have received fibrinolytic or thrombolytic therapy. Practice guidelines should be reviewed when considering neuraxial anesthesia in such patients, and the risk versus benefit of these techniques should be weighed and delineated in the informed consent process.

Infection of the subarachnoid space can follow neuraxial blocks as the result of contamination of the equipment or injected solutions, or as a result of organisms tracked in from the skin. Indwelling catheters may become colonized with skin organisms.
Strict sterile technique should be employed. Careful attention is particularly warranted in the labor room, where family members are often curious to see what is being done to mitigate the parturient's pain. If hospital policy permits their presence during epidural placement, such individuals should be advised to avoid contaminating the tray. Family members should also wear a mask to prevent contamination of the epidural tray with oral flora.

Spinal epidural abscess (EA) is a rare but potentially devastating complication of neuraxial anesthesia. The reported incidence varies widely, from 1:6500 to 1:500,000 epidurals. Most reported anesthesia-related cases involve epidural catheters. In one reported series, there was a mean of 5 days from catheter insertion to the development of symptoms, although presentation can be delayed for weeks.
There are four classic clinical stages of EA, although progression and time course can vary. Initially, symptoms include back pain that is intensified by percussion over the spine. Second, nerve root or radicular pain develops. The third stage is marked by motor or sensory deficits, or sphincter dysfunction. Paraplegia or paralysis marks the fourth stage. Ideally, the diagnosis is made in the early stages. Prognosis has consistently been shown to correlate to the degree of neurological dysfunction at the time the diagnosis is made. Back pain and fever after epidural anesthesia should alert the clinician to consider EA. Radicular pain or neurological deficit heightens the urgency to investigate. Once EA is suspected, the catheter should be removed (if still present) and the tip cultured. The injection site is examined for evidence of infection; if pus is expressed, it is sent for culture. Blood cultures should be obtained. If suspicion is high and cultures have been obtained, anti-Staphylococcus coverage can be instituted, as the most common organisms causing EA are Staphylococcus aureus and Staphylococcus epidermidis. MR or CT imaging should be performed to confirm or rule out the diagnosis. We recommend prompt consultation with specialists in neurosurgical and infectious disease. In addition to antibiotics, treatment of EA usually involves decompression (laminectomy), although percutaneous drainage with fluoroscopic or CT guidance has been used. Suggested strategies for guarding against the occurrence of EA include (1) minimizing catheter manipulations and maintaining a closed system when possible; (2) using a micropore (0.22-1m) bacterial filter; and (3) removing an epidural catheter or at least changing the catheter, filter, and solution after a defined time interval (eg, some clinicians replace or remove all epidurals after 4 days).

There is a risk of neuraxial catheters sheering and breaking off inside of tissues if they are withdrawn through the needle. If a catheter must be withdrawn while the needle remains in situ, both must be carefully withdrawn together. If a catheter breaks off within the epidural space, many experts suggest leaving it and observing the patient. If, however, the breakage occurs in superficial tissues, the catheter should be surgically removed.
Complications Associated with Drug Toxicity

Absorption of excessive amounts of local anesthetics can produce toxic blood levels (see "Intravascular Injection"). Excessive absorption from epidural or caudal blocks is rare when appropriate doses of local anesthetic are used.

First described in 1993, transient neurological symptoms (TNS), also referred to as transient radicular irritation, are characterized by back pain radiating to the legs without sensory or motor deficits, occurring after the resolution of spinal anesthesia and resolving spontaneously within several days. It is most commonly associated with hyperbaric lidocaine (incidence up to 12%), but has also been reported with tetracaine (2%), bupivacaine (1%), mepivacaine, prilocaine, procaine, and subarachnoid ropivacaine. There are also case reports of TNS following epidural anesthesia. The incidence of this syndrome is greatest among outpatients, particularly males undergoing surgery in the lithotomy position, and least among inpatients undergoing surgery in positions other than lithotomy. The pathogenesis of TNS is believed to represent concentration-dependent neurotoxicity of local anesthetics.

A 56-year-old man presents for cystoscopy and stent placement for a large kidney stone. The patient has a long history of spinal problems and has undergone fusion of the cervical spine (C3—C6) and laminectomy with fusion of the lower lumbar spine (L3-L5). On examination, he has no neck flexion or extension and has a Mallampati class IV airway.
What types of anesthesia are appropriate for this patient?
Cystoscopy and stent placement usually require general or neuraxial anesthesia. Selection of the type of anesthesia, as always, should be based on patient preference after informed consent. This patient presents potential difficulties for both general and regional anesthesia. The limited excursion of the cervical spine, together with the anatomy of a class IV airway, makes difficulty in intubation and possibly ventilation almost certain. Induction of general anesthesia would be safest after the airway is secured with an awake fiberoptic intubation.
Regional anesthesia also presents a problem in that the patient has had previous back surgery in the lumbar area, where neuraxial anesthesia is most commonly performed. Postoperative distortion of the anatomy makes the block technically challenging and may increase the likelihood of a failure, inadvertent dural puncture during epidural anesthesia, paresthesias, and unpredictable spread of the local anesthetics.
If the patient chooses to have neuraxial anesthesia, would spinal or epidural anesthesia be more appropriate?
The associated sympathectomy and subsequent drop in blood pressure are more gradual after epidural anesthesia than that following spinal anesthesia. With either type of anesthesia, significant hypotension should be treated with vasoconstrictors and fluids, and bradycardia should be treated with anticholinergics.
After an explanation of the options, the patient seems to understand the risks of both types of anesthesia and desires epidural anesthesia. Placement of an epidural catheter is attempted at the L1-L2 interspace, but accidental dural puncture occurs. What options are now available?

Options include injecting a spinal dose of local anesthetic through the epidural needle to induce spinal anesthesia, passing an epidural catheter into the subarachnoid space to perform a continuous spinal anesthetic, or proceeding with an awake fiberoptic intubation in advance of general anesthesia. If a spinal dose of local anesthetic is to be injected, the syringe and needle should be kept in place for a few moments to prevent significant back leakage of anesthetic through the large dural hole. Threading an epidural catheter through the needle into the subarachnoid space allows subsequent redosing and may reduce the incidence of dural puncture headache. When a catheter is advanced in the subarachnoid space well below L2, it should not be advanced more than 2 to 3 cm to avoid injury to the cauda equina.
How might a dural puncture affect subsequent epidural or

A potential hazard of epidural anesthesia at a level adjacent to a large dural puncture is the possibility that some local anesthetic might pass through the dural puncture into the subarachnoid space. This could result in a higher than expected level of sensory and motor blockade.

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@ In addition to potent analgesia, regional anesthesia may lead to reductions in the stress response, systemic analgesic requirements, opioid-related side effects, general anesthesia requirements, and, possibly, the development of chronic postoperative pain.
@ Regional anesthetics must be administered in an area where standard anesthetic monitors, supplemental oxygen, and resuscitative medications and equipment are immediately available.
© Local anesthetic may be deposited at any point along the brachial plexus, depending on the desired block effects: interscalene for shoulder and proximal humerus surgical procedures; and supraclavicular, infraclavicular, and axillary for surgeries distal to the mid-humerus.
@ A properly performed interscalene block almost invariably blocks the ipsilateral phrenic nerve, so careful consideration must be given to patients with severe pulmonary disease or preexisting contralateral phrenic nerve palsy.
@ Brachial plexus block at the level of the cords provides excellent anesthesia for procedures at or distal to the elbow. The upper arm and shoulder are not anesthetized with this approach. As with other brachial plexus blocks, the intercostobrachial nerve (T2 dermatome) is spared.
@ The axillary, musculocutaneous, and medial brachial cutaneous nerves branch from the brachial plexus proximal to where local anesthetic is deposited for an axillary brachial plexus block, and thus are usually spared.
@ Often it is necessary to anesthetize a single terminal nerve, either for

An understanding of regional anesthesia anatomy and techniques is required of the well-rounded anesthesiologist. Although anatomic relationships have not changed over time, our ability to identify them has improved. From the paresthesia-seeking techniques described by Winnie, to the popularization of the nerve stimulator, to the introduction of ultrasound guidance, anesthesiologists and their patients have benefitted from evolving and improving techniques. The field of regional anesthesia has accordingly expanded to one that addresses not only the intraoperative concerns of the anesthesiologist, but also longer term perioperative pain management @ and acceleration of convalescence. In addition to potent analgesia, regional anesthesia may lead to reductions in the stress response, systemic analgesic requirements, opioid-related side effects, general anesthesia requirements, and, possibly, the development of chronic postoperative pain.

The selection of a regional anesthetic technique is a process that begins with a thorough history and physical examination. As with any medical procedure a risk—benefit analysis must be performed. The risk—benefit ratio often favors regional anesthesia in patients with multiple comorbidities for whom a general anesthetic carries a greater risk. In addition, patients intolerant of opioids (eg, those with obstructive sleep apnea or at high risk for nausea) may benefit from the opioid-sparing effects of a regional analgesic. Patients with chronic pain and opioid tolerance may receive optimal analgesia with a continuous peripheral nerve block (perineural local anesthetic infusion).
A comprehensive knowledge of regional anatomy and an understanding of the planned surgical procedure are important for selection of the appropriate regional anesthetic technique. If possible, review of various considerations (tourniquet placement, bone grafting, projected surgical duration) should be undertaken with the surgeon. Also, knowing the anticipated course of recovery and anticipated level of postoperative pain will often influence specific decisions regarding a regional anesthetic technique (eg, a single injection versus continuous peripheral nerve block).

Patient cooperation is key to the success and safety of every regional anesthetic

The decision about which local anesthetic to employ for a particular nerve block depends on the desired onset, duration, and relative blockade of sensory and motor fibers. Potential for systemic toxicity should be considered, as well as sitespecific risks. A detailed discussion of local anesthetics is provided elsewhere (see Chapter 16).

® Regional anesthetics must be administered in an area where standard anesthetic monitors, supplemental oxygen, and resuscitative medications and equipment are immediately available. Patients should be monitored with pulse oximetry, noninvasive blood pressure, and electrocardiography; measurement of end-tidal CO, and fraction of inspired oxygen (Fio,) should also be available. Positioning should be ergonomically favorable for the practitioner and comfortable for the patient. Intravenous premedication may be employed to allay anxiety and minimize discomfort. A relatively short-acting benzodiazepine and opioid are most often used and should be titrated for comfort while ensuring that patients can respond to verbal cues. Sterile technique should be strictly observed.

A field block is a local anesthetic injection that targets terminal cutaneous nerves (Figure 46-1). Field blocks are used commonly by surgeons to minimize incisional pain and may be used as a supplementary technique or as a sole anesthetic for minor, superficial procedures. Anesthesiologists often use field blocks to anesthetize the superficial cervical plexus for procedures involving the neck or shoulder; the intercostobrachial nerve for surgery involving the medial upper extremity proximal to the elbow (in combination with a brachial plexus nerve block); and the saphenous nerve for surgery involving the medial leg or ankle joint (in combination with a sciatic nerve block). Field blocks may be undesirable in cases where they obscure or distort the operative anatomy or where local tissue acidosis from infection prevents effective local anesthetic function.
FIGURE 46-1 A field block targets terminal cutaneous nerves, such as the intercostobrachial nerve.

Formerly the mainstay of regional anesthesia, this technique is now rarely used for nerve localization. Using known anatomic relationships and surface landmarks as a guide, a block needle is advanced toward the target nerve or plexus. When a needle makes direct contact with a sensory nerve, a paresthesia (abnormal sensation) is elicited in its area of sensory distribution.

For this technique, an insulated needle concentrates electrical current at the needle tip, while a wire attached to the needle hub connects to a nerve stimulator —a battery-powered machine that emits a small amount (0-5 mA) of electric current at a set frequency (usually 1 or 2 Hz). A grounding electrode is attached to the patient to complete the circuit (Figure 46—2). When the insulated needle tip is placed in proximity to a motor nerve, specific muscle contractions are induced, and local anesthetic is injected. Although it is common to redirect the block needle until muscle contractions occur at a current less than 0.5 mA, there is scant evidence to support this specific current in all cases. Similarly, although some have suggested that muscle contraction with current less than 0.2 mA implies intraneural needle placement, there is little evidence to support this specific cutoff. Nonetheless, most practitioners inject local anesthetic when current between 0.2 and 0.5 mA results in a muscle response. For most blocks using this technique in adults, 30 to 40 mL of anesthetic is usually injected with gentle aspiration between divided doses.
FIGURE 46-2 A nerve stimulator delivers a small amount of electrical current to the block needle to facilitate nerve localization.

Ultrasound for peripheral nerve localization is becoming increasingly popular, and it may be used either alone or combined with other modalities such as nerve stimulation. Ultrasound uses high-frequency (1-20 MHz) sound waves emitted from piezoelectric crystals that travel at different rates through tissues of different densities, returning a signal to the transducer. Depending on the amplitude of signal received, the crystals deform to create an electronic voltage that is converted into a two-dimensional grayscale image. The degree of efficiency with which sound passes through a substance determines its echogenicity. Structures and substances through which sound passes easily are described as hypoechoic and appear dark or black on the ultrasound screen. In contrast, structures reflecting more sound waves appear brighter—or white—on the ultrasound screen, and are termed hyperechoic.
The optimal transducer varies depending upon the depth of the target nerve and approach angle of the needle relative to the transducer (Figure 46—3). Highfrequency transducers provide a high-resolution picture with a relatively clear image but offer poor tissue penetration and are therefore used predominantly for more superficial nerves. Low-frequency transducers provide an image of poorer quality but have better tissue penetration and are therefore used for deeper structures. Transducers with a linear array offer an undistorted image and are therefore often the first choice among practitioners. However, for deeper target nerves that require a more acute angle between the needle and long-axis of the transducer, a curved array (curvilinear) transducer will maximize returning ultrasound waves, providing the optimal needle image (Figure 46—3). Nerves are best imaged in cross-section, where they have a characteristic honeycomb appearance (short-axis). Needle insertion can pass either parallel (in-plane) or not parallel (out-of-plane) to the plane of the ultrasound waves (Figure 46-4). Unlike nerve stimulation alone, ultrasound guidance allows for a variable volume of local anesthetic to be injected, with the final amount determined by what is observed under direct ultrasound vision. This technique usually results in a far smaller injected volume (10-30 mL) of local anesthetic.
FIGURE 46-3 A linear probe offers higher resolution with less penetration. A curvilinear probe provides better penetration with lower resolution.
FIGURE 46-4 In-plane (A) and out-of-plane (B) ultrasound approaches.

Also termed perineural local anesthetic infusion, continuous peripheral nerve blocks involve the placement of a percutaneous catheter adjacent to a peripheral nerve, followed by local anesthetic administration to prolong a nerve block (Figure 46-5). Potential advantages appear to depend on successfully improving analgesia and include reductions in resting and dynamic pain, supplemental analgesic requirements, opioid-related side effects, and sleep disturbance. In some cases, patient satisfaction, ambulation, and functioning may be improved, accelerated resumption of passive joint range-of-motion realized, and reduced time until discharge-readiness as well as actual discharge from the hospital or rehabilitation center achieved. Recent evidence suggests that continuous sciatic, femoral, and paravertebral perineural local anesthetic infusions in the immediate postoperative period may decrease the risk of persistent ("chronic") postsurgical pain.
FIGURE 46-5 Placement of a percutaneous catheter adjacent to a peripheral nerve.
There are many types of perineural catheters, including nonstimulating and stimulating, flexible and relatively rigid, through-the-needle and over-the-needle. Currently, there is little evidence that a single design results in superior effects. Long-acting local anesthetics (eg, ropivacaine) are more commonly used, as they provide a more favorable sensory-to-motor block ratio (maximizing analgesia while minimizing motor block). In an attempt to further minimize any induced motor block, dilute local anesthetic (0.1-0.2%) is often infused; however, recent evidence suggests that it is the total mass of drug, and not concentration alone, that determines the majority of block effects. Unlike single-injection peripheral nerve blocks, no adjuvant added to a perineural local anesthetic infusion has been demonstrated to be of benefit. The local anesthetic may be administered exclusively as repeated bolus doses or a basal infusion, or as a combination of the two methods. Using a small, portable infusion pump (Figure 46-6), continuous peripheral nerve blocks may be provided on an ambulatory basis.

As with all medical procedures, there are potential risks associated with continuous peripheral nerve blocks. Therefore, these infusions are usually reserved for patients having procedures expected to result in postoperative pain that is difficult to control with oral analgesics and will not resolve in less time than the duration of a single-injection peripheral nerve block. Serious complications, which are relatively rare, include systemic local anesthetic toxicity, catheter retention, nerve injury, infection, and retroperitoneal hematoma formation. In addition, a perineural infusion affecting the femoral nerve increases the risk of falling, although to what degree and by what specific mechanism (eg, sensory, motor, or proprioception deficits) remain unknown.

The brachial plexus is formed by the union of the anterior primary divisions (ventral rami) of the fifth through the eighth cervical nerves and the first thoracic nerve. Contributions from C4 and T2 are often minor or absent. As the nerve roots leave the intervertebral foramina, they converge, successively forming trunks, divisions, cords, branches, and then finally terminal nerves. The three distinct trunks formed between the anterior and middle scalene muscles are termed superior, middle, and inferior based on their vertical orientation. As the trunks pass over the lateral border of the first rib and under the clavicle, each trunk divides into anterior and posterior divisions. As the brachial plexus emerges below the clavicle, the fibers combine again to form three cords that are named according to their relationship to the axillary artery: lateral, medial, and posterior. At the lateral border of the pectoralis minor muscle, each cord gives off a large branch before ending as a major terminal nerve. The lateral cord gives off the lateral branch of the median nerve and ® terminates as the musculocutaneous nerve; the medial cord gives off the medial branch of the median nerve and terminates as the ulnar nerve; and the posterior cord gives off the axillary nerve and terminates as the radial nerve. Local anesthetic may be deposited at any point along the brachial plexus, depending on the desired block effects (Figure 46—7): interscalene for shoulder and proximal humerus surgical procedures; and supraclavicular, infraclavicular, and axillary for surgeries distal to the mid-humerus.
FIGURE 46-7 The location of local anesthetic deposition along the brachial

An interscalene brachial plexus block is indicated for procedures involving the shoulder and upper arm (Figure 46-8). Roots C5—7 are most densely blocked with this approach; and the ulnar nerve originating from C8 and T1 may be spared. Therefore, interscalene blocks are not ideal for surgery at or distal to the elbow. For complete surgical anesthesia of the shoulder, the C3 and C4 cutaneous branches may need to be supplemented with a superficial cervical plexus block or local infiltration.
FIGURE 46-8 An interscalene block is appropriate for shoulder and proximal humerus procedures. The ventral rami of C5—C8 and T1 form the brachial plexus.
Contraindications to an interscalene block include local infection, severe coagulopathy, local ® anesthetic allergy, and patient refusal. A properly performed interscalene block almost invariably blocks the ipsilateral phrenic nerve (completely for nerve stimulation techniques, but less so with certain lowvolume ultrasound techniques), so careful consideration must be given to patients with severe pulmonary disease or preexisting contralateral phrenic nerve
FIGURE 46-9 The brachial plexus passes between the anterior and middle scalene muscles at the level of the cricoid cartilage, or C6.

A relatively short (5-cm) insulated needle is usually employed. The interscalene groove is palpated using the nondominant hand, pressing firmly to stabilize the skin against the underlying structures (Figure 46—10). After the skin is anesthetized, the block needle is inserted at a slightly medial and caudad angle and advanced to optimally elicit a motor response of the deltoid or biceps muscles (suggesting stimulation of the superior trunk). A motor response of the diaphragm indicates that the needle is placed in too anterior a direction; a motor response of the trapezius or serratus anterior muscles indicates that the needle is placed in too posterior a direction. If bone (transverse process) is contacted, the needle should be redirected more anteriorly. Aspiration of arterial blood should raise concern for vertebral or carotid artery puncture; the needle should be withdrawn, pressure held for 3 to 5 min, and landmarks reassessed.
FIGURE 46-10 Interscalene block using nerve stimulation.

A needle in-plane or out-of-plane technique may be used, and an insulated needle attached to a nerve stimulator can be used to confirm the accuracy of the targeted structure. For both techniques, after identification of the sternocleidomastoid muscle and interscalene groove at the approximate level of C6, a high-frequency linear transducer is placed perpendicular to the course of the interscalene muscles (short axis; Figure 46—11). The brachial plexus and anterior and middle scalene muscles should be visualized in cross-section (Figure 46—12). The brachial plexus at this level appears as three to five hypoechoic circles. The carotid artery and internal jugular vein may be seen lying anterior to the anterior scalene muscle; the sternocleidomastoid is visible superficially as it tapers to form its lateral edge.
FIGURE 46-11 Ultrasound-guided interscalene block (in-plane technique).
FIGURE 46-12 Interscalene block. Ultrasound image of the brachial plexus in the interscalene groove. ASM, anterior scalene muscle; MSM, middle scalene muscle; SCM, sternocleidomastoid; N, brachial plexus nerve roots in crosssection.
For an out-of-plane technique, the block needle is inserted just cephalad to the transducer and advanced in a caudal direction toward the visualized plexus. After careful aspiration for nonappearance of blood, local anesthetic (hypoechoic) spread should be noted adjacent to, and usually surrounding, the plexus.

For an in-plane technique, the needle is inserted just posterior to the ultrasound transducer in a direction exactly parallel to the ultrasound beam. A longer block needle (8 cm) is usually necessary. It may be helpful to have the patient turn slightly laterally with the affected side up to facilitate manipulation of the needle. The needle is advanced through the middle scalene muscle until it has passed through the fascia anteriorly into the interscalene groove. The needle tip and shaft should be visualized during the entire block performance. Depending on visualized spread relative to the target nerve(s), a lower volume (10 mL) may be employed for postoperative analgesia, whereas a larger volume (20-30 mL) is commonly used for surgical anesthesia.

Once described as the "spinal of the arm" due to its relatively rapid onset and reliability, a supraclavicular block offers dense anesthesia of the brachial plexus for surgical procedures at or distal to the elbow (Figure 46—13). Historically, the supraclavicular block fell out of favor due to the relatively increased incidence of complications (namely, pneumothorax) that occurred with paresthesia and nerve stimulator techniques. It has seen a resurgence in recent years with the use of ultrasound guidance. The supraclavicular block does not reliably anesthetize the axillary and suprascapular nerves, and thus is not ideal for shoulder surgery. Sparing of distal branches, particularly the ulnar nerve, may occur. Supraclavicular perineural catheters provide inferior analgesia compared with infraclavicular perineural catheter infusion and are more often displaced with movement.
FIGURE 46-13 A supraclavicular block can provide dense anesthesia for procedures at or distal to the elbow. Light blue shading indicates regions of variable blockade; purple shading indicates regions of more reliable blockade.
Many of the same precautions that are taken with patient selection for an interscalene block should be exercised with a supraclavicular block. Nearly half of patients undergoing supraclavicular block will experience ipsilateral phrenic nerve palsy, although ultrasound guidance may decrease this incidence by allowing use of minimal local anesthetic volume. Horner syndrome and recurrent laryngeal nerve palsy may also occur. Risks of pneumothorax and subclavian artery puncture remain, although they are theoretically less likely with ultrasound guidance.

The patient should lie supine with the head turned 30° toward the contralateral side. A linear, high-frequency transducer is placed in the supraclavicular fossa superior to the clavicle and angled slightly toward the thorax (Figure 46—14). The subclavian artery should be easily identified. The brachial plexus appears as multiple hypoechoic disks just superficial and lateral to the subclavian artery (Figure 46-15). The first rib should also be identified as a hyperechoic line just deep to the artery. Pleura may be identified adjacent to the rib and can be distinguished from bone by its movement with breathing.
FIGURE 46-14 Ultrasound probe placement for supraclavicular block (inplane technique).
FIGURE 46-15 Supraclavicular block. Ultrasound image of the brachial plexus in the supraclavicular fossa. SA, subclavian artery; R, rib; N, brachial plexus in cross-section.
For an out-of-plane technique, a short, 22-gauge blunt-tipped needle is used. The skin is anesthetized, and the needle inserted just cephalad to the ultrasound transducer in a posterior and caudad direction. After careful aspiration for the nonappearance of blood, 30 to 40 mL of local anesthetic is injected in 5-mL increments while visualizing local anesthetic spread around the brachial plexus.
For an in-plane technique, a longer needle may be necessary. The needle is inserted lateral to the transducer in a direction parallel to the ultrasound beam. The needle is advanced medially toward the subclavian artery until the tip is visualized near the brachial plexus just lateral and superficial to the artery. Local anesthetic spread should be visualized surrounding the plexus after careful aspiration and incremental injection, which often requires injections in multiple locations with a highly variable volume (20-30 mL).

@ Brachial plexus block at the level of the cords provides excellent anesthesia for procedures at or distal to the elbow (Figure 46—16). The upper arm and shoulder are not anesthetized with this approach. As with other brachial plexus blocks, the intercostobrachial nerve (T2 dermatome) is spared. Site-specific risks of the infraclavicular approach include vascular puncture and pneumothorax, although less common than with supraclavicular block. It is often prudent to use an alternative approach for patients with an indwelling vascular catheter in the subclavian region, or for patients with an ipsilateral transvenous pacemaker.
FIGURE 46-16 Infraclavicular block coverage and anatomy. Light blue shading indicates regions of variable blockade; purple shading indicates regions of more reliable blockade.
As the brachial plexus traverses beyond the first rib and into the axilla, the cords are arranged around the axillary artery according to their anatomic position: medial, lateral, and posterior.

The patient is positioned supine with the head turned to the contralateral side, and the coracoid process is identified (a bony prominence of the scapula that can be palpated between the acromioclavicular joint and the deltopectoral groove). The subclavian artery and brachial plexus run deep to the coracoid process and can be found approximately 2 cm medial and 2 cm caudad to it, about 4 to 5 cm deep in the average patient (Figure 46-17). A relatively long (8-cm) insulated needle is placed perpendicular to the skin and advanced directly posterior until a motor response is elicited. An acceptable motor response is finger flexion or extension at a current less than 0.5 mA, but not elbow flexion/extension.
FIGURE 46-17 Infraclavicular block using nerve stimulation: coracoid technique.

With the patient in the supine position, a small curvilinear transducer is placed in the parasagittal plane over the point 2 cm medial and 2 cm caudad to the coracoid process (Figure 46—18A). (Abducting the arm 90° improves axillary artery imaging.) A high-frequency linear transducer may be used but will often provide inadequate needle visualization due to the relatively acute needle-totransducer angle. The axillary artery and vein are identified in cross-section (Figure 46—18B). The medial, lateral, and posterior cords appear as hyperechoic bundles positioned caudad, cephalad, and posterior to the artery, respectively. A long (10-cm) needle is inserted 2 to 3 cm cephalad to the transducer. Optimal needle positioning is between the axillary artery and the posterior cord, where a single 30-mL injection is as effective as individual cord injections. Insertion of a perineural catheter should be in the same location posterior to the axillary artery.
Infraclavicular perineural infusion has been shown to provide superior analgesia to both supraclavicular and axillary infusions.
FIGURE 46-18 Infraclavicular block. A: Use a small curvilinear probe in a parasagittal plane to visualize the brachial plexus. B: Ultrasound image of the brachial plexus surrounding the axillary artery. AA, axillary artery; N, medial, lateral, and posterior cords of the brachial plexus; AV, axillary vein; PMa, pectoralis major muscle; PMi, pectoralis minor muscle. The red dot indicates the location of local anesthetic deposition.

At the lateral border of the pectoralis minor muscle, the cords of the brachial plexus form large terminal @ branches. The axillary, musculocutaneous, and medial brachial cutaneous nerves branch from the brachial plexus proximal to where local anesthetic is deposited for an axillary brachial plexus block, and thus are usually spared (Figure 46-19). At this level, the major terminal nerves often are separated by fascia; therefore, multiple injections (10-mL each) may be required to reliably produce anesthesia of the entire arm distal to the elbow (Figure 46-20).
FIGURE 46-19 Axillary block. The axillary, musculocutaneous, and medial brachial cutaneous nerves are usually spared with an axillary approach.
FIGURE 46-20 A: Patient positioning and needle angle for axillary brachial plexus block. B: A multiple injection technique is more effective because of fascial separation between nerves.
There are few contraindications to axillary brachial plexus blocks. Local infection, neuropathy, and bleeding risk must be considered. Because the axilla is highly vascularized, there is a risk of local anesthetic uptake through small veins traumatized by needle placement. The axilla is also a suboptimal site for perineural catheter placement because of greatly inferior analgesia versus an infraclavicular infusion, as well as theoretically increased risks of infection and catheter dislodgement.
All of the numerous axillary block techniques require the patient to be positioned supine, with the arm abducted 90° and the head turned toward the contralateral side (Figure 46-20). The axillary artery pulse should be palpated and its location marked as a reference point.

This technique has fallen out of favor due to the trauma of twice purposefully penetrating the axillary artery along with a theoretically increased risk of inadvertent intravascular local anesthetic injection. The nondominant hand is used to palpate and immobilize the axillary artery, and a 22-gauge needle is inserted high in the axilla (Figure 46—20) until bright red blood is aspirated. The needle is then slightly advanced until blood aspiration ceases. Injection can be performed both posterior and anterior to the artery. A total of 30 to 40 mL of local anesthetic is typically used.

The nondominant hand is used to palpate and immobilize the axillary artery. With the patient's arm abducted and externally rotated, the terminal nerves usually lie in the following positions relative to the artery (Figure 46-21, although variations are common): median nerve superior (wrist flexion, thumb opposition, forearm pronation); ulnar nerve inferior (wrist flexion, thumb adduction, fourth/fifth digit flexion); and radial nerve inferior—posterior (digit/wrist/elbow extension, forearm supination). The musculocutaneous nerve (elbow flexion) is most often separate and deep within the coracobrachialis muscle, which is more superior (lateral) in this position and, as a consequence, is generally not anesthetized unless specifically targeted (Figure 46—21). A 2-in., 22-gauge insulated needle is inserted proximal to the palpating fingers to elicit muscle twitches in the hand. Once an appropriate muscle response is identified, and after reducing the stimulation current to less than 0.5 mA, careful aspiration is performed and local anesthetic is injected. Although a single injection of 40 mL may be used, greater success will be achieved with multiple nerve stimulations (ie, two or three nerves) and divided doses of local anesthetic.
FIGURE 46-21 Positioning of terminal nerves about the axillary artery (variations are common).

Using a high-frequency linear array ultrasound transducer, the axillary artery and vein are visualized in cross-section. The brachial plexus can be identified surrounding the artery (Figure 46—22). The needle is inserted superior (lateral) to the transducer and advanced inferiorly (medially) toward the plexus under direct visualization. Ten milliliters of local anesthetic is then injected around each nerve (including the musculocutaneous nerve, if indicated).
FIGURE 46-22 Ultrasound image of axillary brachial plexus block. AA, Axillary artery; AV, axillary vein; U, ulnar nerve; M, median nerve; MC, musculocutaneous nerve; R, radial nerve; CB, coracobrachialis muscle; TM, triceps muscle; BM, biceps muscle.

@ Often it is necessary to anesthetize a single terminal nerve, either for minor surgical procedures with a limited field or as a supplement to an incomplete brachial plexus block. Terminal nerves may be anesthetized anywhere along their course, but the elbow and the wrist are the two most favored sites.

The median nerve is derived from the lateral and medial cords of the brachial plexus. It enters the arm and runs just medial to the brachial artery (Figure 46— 23). As it enters the antecubital space, it lies medial to the brachial artery near the insertion of the biceps tendon. Just distal to this point, it gives off numerous motor branches to the wrist and finger flexors and follows the interosseous membrane to the wrist. At the level of the proximal wrist flexion crease, it lies directly behind the palmaris longus tendon in the carpal tunnel.

To block the median nerve at the elbow, the brachial artery is identified in the antecubital crease just medial to the biceps insertion. A short 22-gauge insulated needle is inserted just medial to the artery and directed toward the medial epicondyle until wrist flexion or thumb opposition is elicited (Figure 46—24); 3 to 5 mL of local anesthetic is then injected. If ultrasound is used, the median nerve may be identified in cross-section just medial to the brachial artery and local anesthetic injected to surround it (Figure 46—25).
FIGURE 46-24 Median nerve block at the elbow.
FIGURE 46-25 Cross-sectional anatomy of median nerve at the elbow.
To block the median nerve at the wrist, the palmaris longus tendon is first identified by asking the patient to flex the wrist against resistance. A short 22gauge needle is inserted adjacent to the palmaris longus tendon and advanced about 0.5 cm (a "pop" can usually be felt as the needle traverses the flexor retinaculum), and 3 to 5 mL of local anesthetic is injected (Figure 46—26). With ultrasound, the median nerve may be identified at the level of the mid-forearm between the muscle bellies of the flexor digitorum profundus, flexor digitorum superficialis, and flexor pollicis longus (transducer faces perpendicular to the trajectory of the nerves).

The ulnar nerve is the continuation of the medial cord of the brachial plexus and maintains a position medial to the axillary and brachial arteries in the upper arm (Figure 46—27). At the distal third of the humerus, the nerve moves more medially and passes under the arcuate ligament of the medial epicondyle. The nerve is frequently palpable just proximal to the medial epicondyle. In the midforearm, the nerve lies between the flexor digitorum profundus and the flexor carpi ulnaris. At the wrist, it is lateral to the flexor carpi ulnaris tendon and medial to the ulnar artery.

To block the ulnar nerve at the level of the elbow, an insulated 22-gauge needle is inserted approximately one fingerbreadth proximal to the arcuate ligament (Figure 46-28), and advanced until fourth/fifth digit flexion or thumb adduction is elicited; 3 to 5 mL of local anesthetic is then injected. To block the ulnar nerve at the wrist, the ulnar artery pulse is palpated just lateral to the flexor carpi ulnaris tendon. The needle is inserted just medial to the artery (Figure 46—

FIGURE 46-28 Ulnar nerve block at the elbow with region of anesthesia illustrated on the hand.
FIGURE 46-29 Ulnar nerve block at the wrist.

The radial nerve—the terminal branch of the posterior cord of the brachial plexus—courses posterior to the humerus, innervating the triceps muscle, and enters the spiral groove of the humerus before it moves laterally at the elbow (Figure 46—30). Terminal sensory branches include the lateral cutaneous nerve of the arm and the posterior cutaneous nerve of the forearm. After exiting the spiral groove as it approaches the lateral epicondyle, the radial nerve separates into superficial and deep branches. The deep branch remains close to the periosteum and innervates the postaxial extensor group of the forearm. The superficial branch becomes superficial and follows the radial artery across the "anatomic snuff box" to innervate the radial aspects of the dorsal wrist and the dorsal aspect of the lateral three digits and half of the fourth.

To block the radial nerve at the elbow, the biceps tendon is identified in the antecubital fossa. A short 22-gauge insulated needle is inserted just lateral to the tendon and directed toward the lateral epicondyle (Figure 46—31) until wrist or finger extension is elicited; 5 mL of local anesthetic is then injected. With ultrasound, the radial nerve can be identified in cross-section just proximal to the antecubital fossa between the biceps and brachioradialis muscles.

At the wrist, the superficial branch of the radial nerve lies just lateral to the radial artery, which can be easily palpated lateral to the flexor carpi radialis tendon (Figure 46—32). Using a short 22-gauge needle, 3 to 5 mL of local anesthetic is injected lateral to the artery. Ultrasound may be used at the level of the wrist or mid-forearm to identify the radial nerve just lateral to the radial artery. Alternatively, the local anesthetic can be injected as a field block in the dorsal subcutaneous tissue proximal to the base of the thumb and index finger.
FIGURE 46-32 Radial nerve block at the wrist.

A musculocutaneous nerve block is essential to complete the anesthesia for the forearm and wrist and is commonly included when performing an axillary brachial plexus block. The musculocutaneous nerve is the terminal branch of the lateral cord and the most proximal of the major nerves to emerge from the brachial plexus (Figure 46—33). This nerve innervates the biceps and brachialis muscles and distally terminates as the lateral antebrachial cutaneous nerve, supplying sensory input to the lateral aspect of the forearm and wrist.

To target the musculocutaneous nerve following an axillary block, the needle is redirected superior and proximal to the artery (see Figure 46—21), the coracobrachialis muscle is pierced, and 5 to 10 mL of local anesthetic is injected after nerve-stimulated elbow flexion. (Alternatively, simple infiltration may be used, although the success rate using this technique is questionable.) Ultrasound may be used to confirm the location of the musculocutaneous nerve in the coracobrachialis muscle or between this muscle and the biceps (see Figure 46— 22). Alternatively, the block can be performed at the elbow as the nerve courses superficially at the interepicondylar line. The insertion of the biceps tendon is identified, and a short 22-guage needle is inserted 1 to 2 cm laterally; 5 to 10 mL of local anesthetic is then injected as a field block.

Digital nerve blocks are used for minor operations on the fingers and to supplement incomplete brachial plexus and terminal nerve blocks. Sensory innervation of each finger is provided by four small digital nerves that enter each digit at its base in each of the four corners (Figure 46—34). A small-gauge needle is inserted at the medial and lateral aspects of the base of the selected digit, and 2 to 3 mL of local anesthetic is injected. Addition of a vasoconstrictor (epinephrine) to local anesthetics reduces blood flow to the digit; however, there are no case reports of necrosis following use of epinephrine with lidocaine or other modern local anesthetics. Nevertheless, it seems prudent to include epinephrine only as a surgical decision to avoid the need for a tourniquet.
FIGURE 46-34 Sensory innervation of the fingers is provided by the digital nerves.

The intercostobrachial nerve originates in the upper thorax (T2) and becomes superficial on the medial upper arm. It supplies cutaneous innervation to the medial aspect of the proximal arm and is not anesthetized with a brachial plexus block (Figure 46—35). The patient should be supine with the arm abducted and externally rotated. Starting at the deltoid prominence and proceeding inferiorly, a field block is performed in a linear fashion using 5 mL of local anesthetic, extending to the most inferior aspect of the medial arm (Figure 46-36).
FIGURE 46-35 Intercostobrachial nerve cutaneous innervation.

® Intravenous regional anesthesia, also called a Bier block, can provide surgical anesthesia for relatively short (45-60 min) surgical procedures on an extremity (eg, carpal tunnel release). An intravenous catheter is usually inserted on the dorsum of the hand (or foot) and a double pneumatic tourniquet is placed on the forearm or arm, or below the knee or on the thigh. The extremity is elevated and exsanguinated by tightly wrapping an Esmarch elastic bandage from a distal to proximal direction. The proximal tourniquet is inflated, the Esmarch bandage removed, and 0.5% lidocaine (25 mL for a forearm or ankle, 50 mL for an arm or below the knee, and 100 mL for a thigh tourniquet) injected over 2 to 3 min through the catheter, which is subsequently removed (Figure 46—37). Anesthesia is usually established after 5 to 10 min. Tourniquet pain usually develops after 20 to 30 min, at which time the distal tourniquet is inflated and the proximal tourniquet subsequently deflated. Patients usually tolerate the distal tourniquet for an additional 15 to 20 min because it is inflated over an anesthetized area. Even for surgical procedures of a very short duration, the tourniquet must be left inflated for a total of at least 15 to 20 min to avoid a rapid intravenous bolus of local anesthetic resulting in systemic toxicity. Slow deflation is also recommended to provide an additional margin of safety.
FIGURE 46-37 Intravenous regional anesthesia provides surgical anesthesia for procedures of short duration.

The lumbosacral plexus provides innervation to the lower extremities (Figure 46-38). The lumbar plexus is formed by the ventral rami of L1—4, with occasional contribution from T12. It lies within the psoas muscle with branches descending into the proximal thigh. Three major nerves from the lumbar plexus make contributions to the lower limb: the femoral (L2-4), lateral femoral cutaneous (L1-3), and obturator (L2—4). These provide motor and sensory innervation to the anterior portion of the thigh and sensory innervation to the medial leg. The sacral plexus arises from L4—5 and S1—4. The posterior thigh and most of the leg and foot are supplied by the tibial and peroneal portions of the sciatic nerve. The posterior femoral cutaneous nerve (S1-3), and not the sciatic nerve, provides sensory innervation to the posterior thigh; it travels with the sciatic nerve as it emerges around the piriformis muscle.
FIGURE 46-38 The ventral rami of L1—-5 and S14 form the lumbosacral plexus, which provides innervation to the lower extremities.

The femoral nerve innervates the main hip flexors and knee extensors, and provides much of the sensory innervation of the hip and thigh (Figure 46—39). Its most medial branch is the saphenous nerve, which innervates much of the skin of the medial leg and ankle joint. The term 3-in-1 block refers to anesthetizing the femoral, lateral femoral cutaneous, and obturator nerves with a single injection below the inguinal ligament; this term has largely been abandoned as evidence accumulated demonstrating the failure of most single injections to consistently affect @ all three nerves. A femoral nerve block alone will seldom provide adequate surgical anesthesia, but it is often used to provide postoperative analgesia for hip, thigh, knee, and ankle (via the saphenous nerve)
procedures. Femoral nerve blocks have a relatively low rate of complications and few contraindications. Local infection, previous vascular grafting, and local adenopathy should be carefully considered in patient selection.
FIGURE 46-39 The femoral nerve provides sensory innervation to the hip and thigh, and to the medial leg via its terminal branch, the saphenous nerve.

With the patient positioned supine, the femoral artery pulse is palpated at the level of the inguinal ligament. A short (5-cm) insulated needle is inserted at a 45° angle to the skin in a cephalad direction (Figure 46—40) until a distinct quadriceps twitch is elicited with a current less than 0.5 mA (look for patella motion).
FIGURE 46-40 Femoral block using nerve stimulation.

A high-frequency linear ultrasound transducer is placed over the area of the inguinal crease parallel to the crease itself, or slightly more transverse (Figure 46-41). The femoral artery and femoral vein are visualized in cross-section, with the overlying fascia iliaca. Just lateral to the artery and deep to the fascia iliaca, the femoral nerve appears in cross-section as a spindle-shaped structure with a "honeycomb" texture (Figure 46—42).
FIGURE 46-42 Femoral nerve block. Ultrasound image of the femoral nerve. FA, femoral artery; FV, femoral vein; FN, femoral nerve; SM, sartorius muscle; IM, iliacus muscle.
For an out-of-plane technique, the block needle is inserted just lateral to where the femoral nerve is seen, and directed cephalad at an angle approximately 45° to the skin. The needle is advanced until it is seen penetrating the fascia iliaca or (if using concurrent electrical stimulation) until an appropriate motor response is elicited. Following careful aspiration for the nonappearance of blood, 30 to 40 mL of local anesthetic is injected.
For an in-plane technique, a longer needle may be used. The needle is inserted parallel to the ultrasound transducer just lateral to the outer edge. The needle is advanced through the sartorius muscle, deep to the fascia iliaca, until it is visualized just lateral to the femoral nerve. Local anesthetic is injected while observing its hypoechoic spread deep to the fascia iliaca and around the nerve.

The goal of a fascia iliaca block is similar to that of a femoral nerve block, but the approach is slightly different. A relatively reliable level of anesthesia may be attained simply with anatomic landmarks and tactile sensation, although ultrasound may be used to facilitate proper needle placement. Once the inguinal ligament and femoral artery pulse are identified, the length of the inguinal ligament is divided into thirds (Figure 46—43). Two centimeters distal to the junction of the middle and outer thirds, a short, blunt-tipped needle is inserted in a slightly cephalad direction. As the needle passes through the two layers of fascia in this region (fascia lata and fascia iliaca), two "pops" will be felt. Once the needle has passed through the fascia iliaca, careful aspiration is performed and 30 to 40 mL of local anesthetic is injected. This block usually anesthetizes both the femoral nerve and lateral femoral cutaneous nerves, since the local anesthetic is deposited under the fascia iliaca between the two nerves that run in the same plane between the fascia and underlying muscle.

The lateral femoral cutaneous nerve provides sensory innervation to the lateral thigh (see Figure 46-39). It may be anesthetized as a supplement to a femoral nerve block or as an isolated block for limited anesthesia of the lateral thigh. As there are few vital structures in proximity to the lateral femoral cutaneous nerve, complications with this block are exceedingly rare. The lateral femoral cutaneous nerve (L2—3) departs from the lumbar plexus, traverses laterally from the psoas muscle, and courses anterolaterally along the iliacus muscle (see Figure 46-38). It emerges inferior and medial to the anterior superior iliac spine (ASIS) to supply the cutaneous sensory innervation of the lateral thigh.
The patient is positioned supine or lateral, and the point 2 cm medial and 2 cm distal to the ASIS is identified. A short 22-gauge block needle is inserted and directed laterally, observing for a "pop" as it passes through the fascia lata. A field block is performed with 10 to 15 mL of local anesthetic, which is deposited above and below the fascia (Figure 46-44). Ultrasound may be used to guide needle placement.
FIGURE 46-44 Lateral femoral cutaneous nerve block.

A block of the obturator nerve is usually required for complete anesthesia of the knee and is most often performed in combination with femoral and sciatic nerve blocks for this purpose. The obturator nerve contributes sensory branches to the hip and knee joints, a variable degree of sensation to the medial thigh, and innervates the adductors of the hip (Figure 46-45). This nerve exits the pelvis and enters the medial thigh through the obturator foramen, which lies beneath the superior pubic ramus. After identification of the pubic tubercle, a long (10cm) block needle is inserted 1.5 cm inferior and 1.5 cm lateral to the tubercle. The needle is advanced posteriorly until bone is contacted (Figure 46—46). Redirecting laterally and caudally, the needle is advanced an additional 2 to 4 cm until a motor response (thigh adduction) is elicited and maintained below 0.5 mA. Following careful aspiration for the nonappearance of blood, 15 to 20 mL of local anesthetic is injected.
FIGURE 46—45 Obturator nerve innervation.
FIGURE 46-46 Obturator nerve block. Contact pubic tubercle (1), then redirect laterally and caudally (2) until a motor response is elicited.
Posterior Lumbar Plexus (Psoas Compartment) Block @ Posterior lumbar plexus blocks are useful for surgical procedures involving areas innervated by the femoral, lateral femoral cutaneous, and obturator nerves (Figure 46—47). These include procedures on the hip, knee, and anterior thigh. Complete anesthesia of the knee can be attained with a proximal sciatic nerve block. The lumbar plexus is relatively close to multiple sensitive structures (Figure 46—48) and reaching it requires a very long needle. Hence, the posterior lumbar plexus block has one of the highest complication rates of any peripheral nerve block; these include retroperitoneal hematoma, intravascular local anesthetic injection with toxicity, intrathecal and epidural injections, and renal capsular puncture with subsequent hematoma.
FIGURE 46-47 Lumbar plexus blocks provide anesthesia to the femoral, lateral femoral cutaneous, and obturator nerves.

FIGURE 46-48 The lumbar plexus lies in close proximity to several important structures.
Lumbar nerve roots emerge into the body of the psoas muscle and travel within the muscle compartment before exiting as terminal nerves (see Figure 46— 38). Modern posterior lumbar plexus blocks deposit local anesthetic within the body of the psoas muscle. The patient is positioned in lateral decubitus with the side to be blocked in the nondependent position (Figure 46—49). The midline is palpated, identifying the spinous processes if possible. A line is first drawn through the lumbar spinous processes, and both iliac crests are identified and connected with a line to approximate the level of L4. The posterior superior iliac spine is then palpated and a line is drawn cephalad, parallel to the first line. If available, ultrasound imaging of the transverse process may be helpful to estimate lumbar plexus depth. A long (10to 15-cm) insulated needle is inserted at the point of intersection between the transverse (intercristal) line and the intersection of the lateral and middle thirds of the two sagittal lines. The needle is advanced in an anterior direction until a femoral motor response is elicited (quadriceps contraction). If the transverse process is contacted, the needle should be withdrawn slightly and "walked off" the transverse process in a caudal direction, maintaining the needle in the parasagittal plane. The needle should never be inserted more than 3 cm past the depth at which the transverse process was contacted. Local anesthetic volumes greater than 20 mL will increase the risk of bilateral spread and contralateral limb involvement.
Curvilinear array
ultrasound transducer
FIGURE 46-49 Patient positioning and surface landmarks for posterior lumbar plexus block.

The adductor canal block is used for analgesia of the knee and medial leg; single injection or continuous techniques can be used. The quadriceps muscles are affected to a lesser degree by an adductor canal block than by a femoral block, which may facilitate ambulation following knee surgery. Bounded by the sartorius muscle and vasto-adductor membrane medially, the vastus medialis anteriorly, and the adductor muscles posteriorly, the adductor canal contains several nerves that provide sensory innervation to the knee. Most notable is the saphenous nerve, and a block within the adductor canal will likely affect the posterior division of the obturator nerve and the nerve to the vastus medialis as well.

The patient is positioned supine with the knee slightly bent and the leg externally rotated. A high-frequency linear transducer is positioned in a transverse orientation over the medial thigh, halfway between the ASIS and the superior patellar pole (Figure 46—50). The femoral artery and vein are visualized deep to the sartorius muscle, and the saphenous nerve lies just lateral to the artery (Figure 46-51). A long (10-cm) block needle is placed 2 to 3 cm lateral to the transducer and advanced in-plane to the triangular space deep to the sartorius muscle and vasto-adductor membrane and lateral to the artery. After careful aspiration for nonappearance of blood, 15 to 20 mL of local anesthetic is injected.
FIGURE 46-50 Positioning for adductor canal block. The patient is positioned supine with the leg externally rotated. A high-frequency linear transducer is placed in a transverse orientation at the mid-thigh.
FIGURE 46-51 Ultrasound image of adductor canal block. Femoral artery (A) and vein (V) lie deep to sartorius muscle (SM) with saphenous nerve (SN) just lateral to the artery.

The saphenous nerve is the most medial branch of the femoral nerve and innervates the skin over the medial leg and the ankle joint (see Figure 46-39). Therefore, this block is used mainly in conjunction with a sciatic nerve block to provide complete anesthesia/analgesia below the knee.

The saphenous nerve may be accessed proximal to the knee, just deep to the sartorius muscle. A high-frequency linear probe is used to identify the junction between the sartorius, vastus medialis, and adductor muscles in cross-section just distal to the adductor canal. A long needle is inserted from medial to lateral (inplane) or angled cephalad (out-of-plane) and 5 to 10 mL of local anesthetic deposited within this fascial plane.

A short block needle is inserted 2 cm distal to the tibial tuberosity and directed medially, infiltrating 5 to 10 mL of local anesthetic as the needle passes toward the posterior aspect of the leg (Figure 46-52). Ultrasound may be used to identify the saphenous vein near the tibial tuberosity and facilitate a perivascular technique, with infiltration about the vein.

The medial malleolus is identified, infiltrating 5 mL of local anesthetic in a line running anteriorly around the ankle (see "Ankle Block," below).

The sciatic nerve originates from the lumbosacral trunk and is composed of nerve roots L4—5 and S1—3 @ (see Figure 46-38). Blockade of the sciatic nerve may occur anywhere along its course and is indicated for surgical procedures involving the hip, thigh, knee, lower leg, and foot. The posterior femoral cutaneous nerve is variably anesthetized as well, depending on the approach. If sacral plexus or posterior femoral cutaneous nerve anesthesia is required, the parasacral approach is used (a technique that is beyond the scope of this chapter).

The patient is positioned laterally with the side to be blocked in the nondependent position. The patient is asked to bend the knee of the affected leg and tilt the pelvis slightly forward (Sim's position; Figure 46-53). The greater trochanter, posterior superior iliac spine (PSIS), and sacral hiatus are then identified. A line is drawn from the greater trochanter to the PSIS, its midpoint identified, and a perpendicular line extended in a caudal direction. Next, a line is drawn from the greater trochanter to the sacral hiatus and the intersection point is marked; this is the initial needle insertion point. A long (10-cm) insulated needle is inserted at an angle perpendicular to all planes to the skin (Figure 46— 53). The needle is advanced through the gluteal muscles (a motor response of these muscles may be encountered) until plantaror dorsiflexion is elicited (plantarflexion or foot inversion is preferred for surgical anesthesia). A local anesthetic volume of 25 mL provides surgical anesthesia.
FIGURE 46-53 Patient positioning, surface landmarks, and needle positioning for proximal sciatic nerve block (classic approach).

After leaving the sciatic notch, the sciatic nerve descends behind the lesser trochanter to a position posterior to the femur. It can be accessed from the anterior thigh just medial to the lesser trochanter. Lateral or prone positioning may present a challenge for some patients requiring a sciatic nerve block (ie, elderly patients, pediatric patients under general anesthesia). An anterior approach can be technically challenging but offers an alternative path to the sciatic nerve. Before proceeding with this block, which carries a risk of vascular puncture (femoral artery and vein), patient-specific risks should be considered (eg, coagulopathy and vascular grafting). In addition, if combining this block with the femoral nerve block in an unanesthetized patient, performing the sciatic block first is recommended to avoid passing the block needle through a previously anesthetized femoral nerve. A local anesthetic volume of 25 mL provides surgical anesthesia.
1. Nerve stimulation—With the patient positioned supine, a line is drawn along the inguinal ligament, from the ASIS to the pubic tubercle (Figure 46—54). A second line is drawn parallel to the first that traverses the greater trochanter (intertrochanteric line). Next, these two lines are connected with a third line drawn from the point between the medial one-third and lateral two-thirds of the first line, at a 90° angle, and extended caudally to intersect with the intertrochanteric line. A long (10to 15-cm) needle is inserted through this intersection and directly posterior until foot inversion or plantarflexion is elicited (dorsiflexion is acceptable for postoperative analgesia). Often with this approach, the femur is contacted before the needle reaches the sciatic nerve. When this occurs, the needle should be withdrawn 2 to 3 cm, the patient should be asked to internally rotate the leg, and then the needle should be advanced. If the femur is contacted again, the landmarks may require reassessment. A local anesthetic volume of 25 mL provides surgical anesthesia.
FIGURE 46-54 Anatomy and surface landmarks for anterior sciatic nerve block.

2. Ultrasound—wWith the patient positioned supine and the leg externally rotated, a low-frequency curvilinear transducer is placed transversely over the medial thigh, approximately at the level of the lesser trochanter. The femur, femoral vessels, adductor muscles, and gluteus maximus are identified in crosssection. The elliptical, hyperechoic sciatic nerve is found in the fascial plane between adductors and gluteus muscles, posterior to the femur. Using a long (10cm) needle, the nerve is approached in-plane (anterior to posterior) or out-ofplane (cephalad to caudad), taking care to avoid femoral vessels, until the needle tip lies in this muscle plane and a local anesthetic injection can be observed as hypoechoic spread surrounding the sciatic nerve.

A subgluteal approach to the sciatic nerve is a useful alternative to the traditional posterior approach because the landmarks are often more easily identified and less tissue is traversed. With the sciatic nerve at a more superficial location, the exclusive use of ultrasound becomes far more practical, as well. If sciatic nerve block is being combined with a femoral block and ambulation is desired within the local anesthetic duration, consider a popliteal approach (see next section) that will not affect the hamstring muscles to the same degree, allowing knee flexion to lift the foot with the use of crutches.
1. Nerve stimulation—With the patient in the Sim's position, the greater trochanter and ischial tuberosity are identified and a line drawn between them (Figure 46—55). From the midpoint of this line, a second line is drawn perpendicularly and extended caudally 4 cm. Through this point a long (10-cm) insulated needle is inserted directly slightly cephalad until foot plantarflexion or inversion is elicited (dorsiflexion is acceptable for analgesia). A local anesthetic volume of 25 mL provides surgical anesthesia.
FIGURE 46-55 Patient positioning and surface landmarks for subgluteal sciatic block.
2. Ultrasound—Using the same positioning and landmarks (Figure 46-55), a linear or low-frequency curvilinear (best) ultrasound transducer is placed over the midpoint between the ischial tuberosity and the greater trochanter in a transverse orientation. Both bony structures should be visible in the ultrasound field simultaneously. Gluteal muscles are identified superficially, along with the fascial layer defining their deep border. The triangular sciatic nerve should be visible in cross-section just deep to this layer in a location approximately midway between the ischial tuberosity and the greater trochanter, superficial to the quadratus femoris muscle.

For an out-of-plane ultrasound-guided sciatic block, the block needle is inserted just caudad to the ultrasound transducer and advanced in an anterior and cephalad direction. Once the needle passes through the gluteus muscles with the tip next to sciatic nerve, careful aspiration for the nonappearance of blood is performed and local anesthetic is injected, visualizing spread around the nerve. For an in-plane technique, the block needle is inserted just lateral to the ultrasound transducer near the greater trochanter. It is advanced through the field of the ultrasound beam until the tip is visible deep to the gluteus maximus, next to the sciatic nerve. Again, local anesthetic spread around the nerve should be observed.

® Popliteal nerve blocks provide excellent coverage for foot and ankle surgery, while sparing much of the hamstring muscles, allowing lifting of the foot with knee flexion, thus facilitating ambulation. All sciatic nerve blocks fail to provide complete anesthesia for the cutaneous medial leg and ankle joint capsule, but when a saphenous (or femoral) block is added, complete anesthesia below the knee is provided. The major site-specific risk of a popliteal block is vascular puncture, owing to the sciatic nerve's proximity to the popliteal vessels at this location.
The sciatic nerve divides into the tibial and common peroneal nerves within or just proximal to the popliteal fossa (Figure 46—56). The upper popliteal fossa is bounded laterally by the biceps femoris tendon and medially by the semitendinosus and semimembranosus tendons. Cephalad to the flexion crease of the knee, the popliteal artery is immediately lateral to the semitendinosus tendon. The popliteal vein is lateral to the artery, and the tibial and common peroneal nerves are just lateral to the vein and medial to the biceps tendon, 2 to 6 cm deep to the skin. The tibial nerve continues deep behind the gastrocnemius muscle, and the common peroneal nerve exits the popliteal fossa between the head and neck of the fibula to supply the lower leg. The sciatic nerve is approached by either a posterior or a lateral approach. For posterior approaches, the patient is usually positioned prone with the knee slightly flexed by propping the ankle on pillows or towels. For lateral approaches, the patient may be in the lateral or supine position.
FIGURE 46-56 The sciatic nerve divides into tibial and peroneal branches just proximal to the popliteal fossa and provides sensory innervation to much of the lower leg.

1. Nerve stimulation (posterior approach)—With the patient in the prone position, the apex of the popliteal fossa is identified. The hamstring muscles are palpated to locate the point where the biceps femoris (lateral) and the semimembranosus/semitendinosus complex (medial) join (Figure 46—57). Having the patient flex the knee against resistance facilitates recognition of these structures. The needle entry point is 1 cm caudad from the apex. An insulated needle (5-10 cm) is advanced until foot plantarflexion or inversion is elicited (dorsiflexion is acceptable for analgesia). A volume of 30 to 40 mL of local anesthetic is often required for single-injection popliteal—sciatic nerve block.
Proximal
FIGURE 46-57 Anatomy and sonoanatomy of the sciatic nerve in the popliteal fossa. PA, popliteal artery; PV, popliteal vein; N, sciatic nerve; BFM, biceps femoris muscle; F, femur.
FIGURE 46-58 Patient positioning, probe, and needle orientation for popliteal block.
If surgical anesthesia is desired, local anesthetic should be seen surrounding all sides of the nerve, which usually requires multiple needle tip placements with incremental injections. For analgesia alone, a single injection of local anesthetic is acceptable. Injection into the nerve or within the paraneural sheath hastens onset and prolongs duration, but may place the nerve at risk for injury. Ultrasound-guided popliteal sciatic blocks may be performed with the patient in the lateral or supine positions (the latter with leg up-raised on several pillows). These maneuvers are often more technically challenging.

For surgical procedures of the foot, an ankle block is a fast, low-technology, low-risk means of providing anesthesia. Excessive injectate volume and use of vasoconstrictors such as epinephrine must be avoided to minimize the risk of ischemic complications. Since this block includes five separate injections, it is often uncomfortable for patients, and adequate sedation and analgesia premedication is advised.
Five nerves supply sensation to the foot (Figure 46—59). The saphenous nerve is a terminal branch of the femoral nerve and the only innervation of the foot that is not a part of the sciatic system. It supplies superficial sensation to the anteromedial foot and is most constantly located just anterior to the medial malleolus. The deep peroneal nerve runs in the anterior leg after branching off the common peroneal nerve, entering the ankle between the extensor hallucis longus and the extensor digitorum longus tendons (Figure 46—60), just lateral to the dorsalis pedis artery. It provides innervation to the toe extensors and sensation to the first dorsal webspace. The superficial peroneal nerve, also a branch of the common peroneal nerve, descends toward the ankle in the lateral compartment, giving motor branches to the muscles of eversion. It enters the ankle just lateral to the extensor digitorum longus and provides cutaneous sensation to the dorsum of the foot and toes. The posterior tibial nerve is a direct continuation of the tibial nerve and enters the foot posterior to the medial malleolus, branching into calcaneal, lateral plantar, and medial plantar nerves. It is located behind the posterior tibial artery at the level of the medial malleolus and provides sensory innervation to the heel, the medial sole, and part of the lateral sole of the foot, as well as the tips of the toes. The sural nerve is a branch of the tibial nerve and enters the foot between the Achilles tendon and the lateral malleolus to provide sensation to the lateral foot.
FIGURE 46-60 Tibial and common peroneal nerve courses.

® A complete ankle block requires a series of five nerve blocks, but the process may be streamlined to minimize needle insertions (Figure 46-61). All five injections are required to anesthetize the entire foot; however, surgical procedures rarely require that all terminal nerves be blocked. In addition, unlike a sciatic nerve block, an ankle block provides no analgesia for (below-the-knee) tourniquet pain, nor does it allow for perineural catheter insertion. To block the deep peroneal nerve, the groove between the extensor hallucis longus and extensor digitorum longus tendons is identified. The dorsalis pedis pulse is often palpable here. A short, small-gauge block needle is inserted perpendicular to the skin just lateral to the pulse, bone is contacted, and 5 mL of local anesthetic is infiltrated as the needle is withdrawn. Continuing from this insertion site, a subcutaneous wheal of 5 mL of local anesthetic is extended toward the lateral malleolus to target the superficial peroneal nerve. The needle is withdrawn and redirected from the same location in a medial direction, infiltrating 5 mL of local anesthetic toward the medial malleolus to target the saphenous nerve. The posterior tibial nerve may be located by identifying the posterior tibial artery pulse behind the medial malleolus. A short, small-gauge block needle is inserted just posterior to the artery and 5 mL of local anesthetic is distributed in the pocket deep to the flexor retinaculum. To target the sural nerve, 5 mL of local anesthetic is injected subcutaneously posterior to the lateral malleolus.
FIGURE 46-61 Needle placement for ankle block.

The superficial cervical plexus block provides cutaneous analgesia for surgical procedures on the neck, anterior shoulder, and clavicle. The cervical plexus is formed from the anterior rami of C1—4, which emerge from the platysma muscle posterior to the sternocleidomastoid (Figure 46—62). It supplies sensation to the jaw, neck, occiput, and areas of the chest and shoulder.

The patient is positioned supine with the head turned away from the side to be blocked. It is helpful to identify and avoid the external jugular vein. The sternocleidomastoid muscle is identified and its lateral edge marked. Typically, the nerve passes over the sternocleidomastoid very near to where the external jugular vein crosses the muscle, and this can be the starting point for injection of local anesthetic. Alternatively, at the junction of the upper and middle thirds, a short (5-cm) block needle is inserted, directed cephalad toward the mastoid process, and 10 mL of local anesthetic is injected in a subcutaneous plane. The needle is turned to advance it in a caudad direction, maintaining a path along the posterior border of sternocleidomastoid. An additional 10 mL of local anesthetic is infiltrated subcutaneously. A dilute concentration of local anesthetic (eg, 0.25% bupivacaine) is appropriate, as this is essentially a field block.

Intercostal blocks provide analgesia following thoracic and upper abdominal surgery, and relief of pain associated with rib fractures, herpes zoster, and cancer. These blocks require individual injections delivered at the intercostal nerves that innervate the area of ® body wall to be anesthetized. Intercostal blocks result in the highest blood levels of local anesthetic per local anesthetic dose injected of any nerve block procedure, and if multiple blocks will be performed care must be taken to avoid toxic levels of local anesthetic. The intercostal block has one of the highest complication rates of any peripheral nerve block due to the close proximity of the intercostal artery and vein (intravascular local anesthetic injection), as well as underlying pleura (pneumothorax). In addition, duration is impressively short due to the high vascular flow, and placement of a perineural catheter is tenuous, at best. With the advent of ultrasound guidance, the paravertebral approach is rapidly replacing the intercostal approach, with exception of specific indications such as providing anesthesia prior to placement of a chest tube.
The intercostal nerves arise from the dorsal and ventral rami of the thoracic spinal nerves. They exit from the spine at the intervertebral foramen and enter a groove on the underside of the corresponding rib, running with the intercostal artery and vein; the nerve is generally the most inferior structure in the neurovascular bundle (Figure 46-63). Branches are given off for sensation in a single dermatome from the midline dorsally all the way to across the midline ventrally.
FIGURE 46-63 Anatomy and needle positioning for intercostal nerve block.
With the patient in the lateral decubitus or supine position, the level of each rib in the mid and posterior axillary line is palpated and marked. A small-gauge needle is inserted at the inferior edge of each of the selected ribs, bone is contacted, and the needle is then "walked off" inferiorly (Figure 46-62). The needle is advanced approximately 0.25 cm. Following aspiration, observing for blood or air, 3 to 5 mL of local anesthetic is injected at each desired level.

FIGURE 46-64 Paravertebral anatomy and traditional approach. Surface anatomy: spinous processes are identified by circles and needle insertion sites for transverse processes are identified by dots. Needle is first inserted perpendicular to the plane of the skin to contact the transverse process (1), then redirected caudally (2) and advanced 1 cm.
With the patient seated and vertebral column flexed, each spinous process is palpated, counting from the prominent C7 for thoracic blocks, and the iliac crests as a reference for lumbar levels. From the midpoint of the superior aspect of each spinous process, a point 2.5 cm laterally is measured and marked. In the thorax, the target nerve is located lateral to the spinous process above it, due to the steep angulation of thoracic spinous processes (eg, the T4 nerve root is located lateral to the spinous process of T3).

A 20-gauge Tuohy needle is inserted at each point and advanced perpendicular to the skin (Figure 46-64). Upon contact with the transverse process, the needle is withdrawn slightly and redirected caudally an additional 1 cm (0.5 cm for lumbar placement). A "pop" or loss of resistance may be felt as the needle passes through the costotransverse ligament. Some practitioners use a loss-of-resistance syringe to guide placement; others prefer use of a nerve stimulator with chest wall motion for the end point. Inject 5 mL of local anesthetic at each level. The difficulty with this technique is that the depth of the transverse process is simply estimated; thus, the risk of pneumothorax is relatively high. Using ultrasound to gauge transverse process depth prior to needle insertion theoretically decreases the risk of pneumothorax.

An ultrasound transducer with a curvilinear array is used, with the beam oriented in a parasagittal or transverse plane. The transverse process, head of the rib, costotransverse ligament, and pleura are identified. The paravertebral space may be approached from a caudal-to-cephalad direction (parasagittal) or a lateral-tomedial direction (transverse). It is helpful to visualize the needle in-plane as it passes through the costotransverse ligament and observe a downward displacement of the pleura as local anesthetic is injected. At each level 5 mL of local anesthetic is injected.

The Pecs block is emerging as a useful alternative to paravertebral block for surgery involving the chest wall. It provides analgesia to the anterior chest wall via the medial and lateral pectoral nerves and is administered further from the neuraxis—an important consideration in the coagulopathic patient. A Pecs block can be achieved by depositing local anesthetic in the plane between the pectoralis major and pectoralis minor muscles at the level of the third rib. In addition, local anesthetic can be deposited between the pectoralis minor and around the serratus anterior, which targets the T2—4 spinal nerves as well as the long thoracic nerve.

A high-frequency linear transducer is placed at the mid-clavicular line and angled inferolaterally. The pectoralis major, pectoralis minor, and axillary vessels are identified. Tracing the muscles toward their insertion, the serratus anterior muscle may then be identified deep to the pectoralis muscles and superficial to the third and fourth ribs (Figure 46-65). A long (10-cm) needle is inserted lateral to the transducer and advanced in plane to target the interfascial plane between the pectoralis major and minor muscles. An additional injection may be performed after advancing the needle through the pectoralis minor muscle to surround the serratus anterior.
FIGURE 46-65 Ultrasound image of Pecs block. Pectoralis major (PMM) and pectoralis minor (PmM) overlie serratus anterior (SAM). Local anesthetic may be injected between the pectoralis major and minor, deep to the pectoralis minor, or around the serratus anterior.

The transversus abdominis plane (TAP) block is most often used to provide surgical anesthesia for minor, superficial procedures on the lower abdominal wall, or postoperative analgesia for procedures below the umbilicus. For hernia surgeries, intravenous or local supplementation may be necessary to provide anesthesia during peritoneal traction. Potential complications include violation of the peritoneum with or without bowel perforation, and the use of ultrasound is highly recommended to minimize this risk.
@ The subcostal (T12), ilioinguinal (L1), and iliohypogastric (L1) nerves are targeted in the TAP block, providing anesthesia to the ipsilateral lower abdomen below the umbilicus (Figure 46-66). For part of their course, these three nerves travel in the muscle plane between the internal oblique and transversus abdominis muscles. Needle placement should be between the two fascial layers of these muscles, with local anesthetic filling the transversus abdominis plane. The patient is ideally positioned in lateral decubitus, but if mobility is limited the block may be performed in the supine position.
FIGURE 46-66 Transversus abdominis plane (TAP) anatomy.

With a linear or curvilinear array transducer oriented parallel to the inguinal ligament, the layers of the external oblique, internal oblique, and transversus abdominis muscles are identified just superior to the ASIS (Figure 46-67). Muscles appear as striated hypoechoic structures with hyperechoic layers of fascia at their borders. A long (10-cm) needle is inserted in-plane just lateral (posterior) to the transducer and advanced, noting tactile feedback from fascial planes, to the hyperechoic effacement of the deep border of internal oblique and the superficial border of transversus abdominis. Following careful aspiration for the nonappearance of blood, 20 mL of local anesthetic is injected, observing for an elliptical separation between the two fascial layers (Figure 46-67).
FIGURE 46-67 Ultrasound image of TAP block. SQ, subcutaneous tissue; EO, external oblique; IO, internal oblique; TA, transversus abdominis; TAP, transversus abdominis plane.

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@ Pain may be classified according to pathophysiology (eg, nociceptive or neuropathic pain), etiology (eg, arthritis or cancer pain), or the affected area (eg, headache or low back pain).
@ Nociceptive pain is caused by activation or sensitization of peripheral nociceptors, specialized receptors that transduce noxious stimuli. Neuropathic pain is the result of injury or acquired abnormalities of peripheral or central neural structures.
@ Acute pain is caused by noxious stimulation due to injury, a disease process, or the abnormal function of muscle or viscera. It is almost always nociceptive.
@ Chronic pain is pain that persists beyond the usual course of an acute disease or after a reasonable time for healing to occur, typically 1 to 6 months. Chronic pain may be nociceptive, neuropathic, or mixed.
@ Modulation of pain occurs peripherally at the nociceptor, in the spinal cord, or in supraspinal structures. This modulation can either inhibit (suppress) or facilitate (intensify) pain.
@ At least three mechanisms are responsible for central sensitization in the spinal cord: (1) wind-up and sensitization of second-order wide dynamic range neurons; (2) dorsal horn neuron receptor field expansion; and (3) hyperexitability of flexion reflexes.
@ Chronic pain may be caused by a combination of peripheral, central, and psychological mechanisms.
@ Moderate to severe acute pain, regardless of site, can affect the function of nearly every organ and may adversely influence perioperative morbidity, mortality, and convalescence.
The evaluation of any patient with pain should include several key components. Information about location, onset, and quality of pain, as well as alleviating and exacerbating factors, should be obtained along with a pain history that includes previous therapies and changes in symptoms over time.
Psychosocial evaluation is useful whenever medical evaluation fails to reveal an apparent cause for pain, pain intensity is disproportionate to disease or injury, or when psychological or social issues are apparent.
Myofascial pain syndromes are common disorders characterized by aching muscle pain, muscle spasm, stiffness, weakness, and, occasionally, autonomic dysfunction.

Ninety percent of disc herniations occur at L5—S1 or L4Q—L5. Symptoms usually develop following flexion injuries or heavy lifting and may be associated with bulging, protrusion, or extrusion of the disc.
Back pain caused by spinal stenosis usually radiates into the buttocks, thighs, and legs. Termed pseudoclaudication or neurogenic claudication, this pain is characteristically worse with exercise and relieved by rest, particularly sitting with the spine flexed.
88 Diabetic neuropathy is the most common type of neuropathic pain. Complex regional pain syndrome (CRPS) is a neuropathic pain disorder with significant autonomic features that is usually subdivided into two variants: CRPS 1, formerly known as reflex sympathetic dystrophy (RSD), and CRPS 2, formerly known as causalgia. The major difference between the two is the absence or presence, respectively, of documented nerve injury.
Trigeminal neuralgia (tic douloureux) is classically unilateral and usually located in the V2 or V3 distribution of the trigeminal nerve. It has an electric shock quality, with episodes lasting from seconds to minutes, and is often provoked by contact with a discrete trigger.
Antidepressants are most useful for patients with neuropathic pain and demonstrate an analgesic effect that occurs at a dose lower than needed for antidepressant activity.
Anticonvulsant medications are useful for patients with neuropathic pain, especially trigeminal neuralgia and diabetic neuropathy.
Patients who experience opioid tolerance require escalating doses of opioid to maintain the same analgesic effect. Physical dependence manifests in opioid withdrawal when the opioid medication is either abruptly discontinued or the dose is abruptly and significantly
decreased. Psychological dependence, characterized by behavioral changes focusing on drug craving, is rare in cancer patients.

€ Complications of stellate block include intravascular and subarachnoid injection, hematoma, pneumothorax, epidural anesthesia, brachial plexus block, hoarseness due to blockade of the recurrent laryngeal nerve, and, rarely, osteomyelitis or mediastinitis.
Ganglion impar block is effective for patients with visceral or sympathetically maintained pain in the perineal area.
Neurolytic blocks are indicated for patients with severe, intractable cancer pain in whom more conventional therapy proves inadequate or conventional analgesic modalities are accompanied by unacceptable side effects.
& Spinal cord stimulation may be most effective for neuropathic pain; accepted indications include sympathetically mediated pain, spinal cord lesions with localized segmental pain, phantom limb pain, ischemic lower extremity pain due to peripheral vascular disease, adhesive arachnoiditis, peripheral neuropathies, post-thoracotomy pain, intercostal neuralgia, postherpetic neuralgia, angina, visceral abdominal pain, and visceral pelvic pain.
@ Patients with pathological or osteoporotic vertebral compression fracture may benefit from vertebral augmentation with polymethylmethacrylate cement. Vertebroplasty involves injection of the cement through the trocar needle. Kyphoplasty involves inflation of a balloon inserted through a percutaneously placed trocar needle, with subsequent injection of cement.
& Acupuncture can be a useful adjunct for patients with chronic pain, particularly that associated with chronic musculoskeletal disorders and headaches.
Pain is the most common symptom that brings patients to see a physician, and the symptom may have a wide variety of causes ranging from relatively benign conditions to acute injury, myocardial ischemia, degenerative changes, or malignancy. In most cases, after a diagnosis is made, conservative measures are prescribed and the patient responds successfully. In others, referral to a pain medicine specialist for evaluation and treatment improves outcomes and conserves health care resources. In still other situations, pain persists and patients develop chronic pain, the cause of which remains obscure after preliminary investigations have excluded serious and life-threatening illnesses and, if warranted, surgical intervention has either failed to relieve pain or has produced a new pain syndrome.

The term pain management in a general sense applies to the entire discipline of anesthesiology, but its modern usage more specifically involves management of pain throughout the perioperative period as well as nonsurgical pain in both inpatient and outpatient settings. Pain medicine practice may be broadly divided into acute and chronic pain management. The former primarily deals with patients recovering from surgery or with acute medical conditions in a hospital or ambulatory surgery center setting (see Chapter 48), whereas the latter includes diverse groups of patients almost always seen in the outpatient setting. Unfortunately, this distinction is artificial and considerable overlap exists; a good example is the patient with cancer who frequently requires shortand long-term pain management in both inpatient and outpatient settings.
The contemporary practice of pain management is not limited to anesthesiologists but is often team based and includes other physicians (physiatrists, surgeons, internists, oncologists, psychiatrists, and neurologists) and nonphysicians (psychologists, physical therapists, acupuncturists, and hypnotists). The most effective approaches are multidisciplinary, in which the patient is evaluated by one or more physicians who conduct an initial examination, make a diagnosis, and formulate a treatment plan, and where subsequent evaluation and use of the services and resources of other health care providers is typical.
Anesthesiologists trained in pain management are in a unique position to coordinate multidisciplinary pain management centers because of their broad training in dealing with a wide variety of patients from surgical, obstetric, pediatric, and medical subspecialties and their expertise in clinical pharmacology and applied neuroanatomy, including the use of peripheral and central nerve blocks.

Like other conscious sensations, normal pain perception depends on specialized neurons that function as receptors, detecting a noxious stimulus and then transducing and conducting it to the central nervous system. Sensation is often described as either protopathic (noxious) or epicritic (nonnoxious). Epicritic sensations (light touch, pressure, proprioception, and temperature discrimination) are characterized by low-threshold receptors and are generally conducted by large myelinated nerve fibers. In contrast, protopathic sensations (pain) are detected by high-threshold receptors and conducted by smaller myelinated (AS) and unmyelinated (C) nerve fibers.

TABLE 47-1 Terms used in pain management.
Allodynia, Description = Perception of an ordinarily nonnoxious stimulus as pain. Analgesia, Description = Absence of pain perception. Anesthesia, Description = Absence of all sensation. Anesthesia dolorosa, Description = Pain in an area that lacks sensation. Dysesthesia, Description = Unpleasant or abnormal sensation with or without a stimulus. Hypalgesia (hypoalgesia), Description = Diminished response to noxious stimulation (eg, pinprick). Hyperalgesia, Description = Increased response to noxious stimulation. Hyperesthesia, Description = Increased response to mild stimulation. Hyperpathia, Description = Presence of hyperesthesia, allodynia, and hyperalgesia usually associated with overreaction, and persistence of the sensation after the stimulus. Hypesthesia (hypoesthesia), Description = Reduced cutaneous sensation (eg, light touch, pressure, or temperature). Neuralgia, Description = Pain in the distribution of a nerve ora group of nerves. Paresthesia, Description = Abnormal sensation perceived without an apparent stimulus. Radiculopathy, Description = Functional abnormality of one or more nerve roots
® Pain may also be classified according to pathophysiology (eg, nociceptive or neuropathic pain), etiology (eg, arthritis or cancer pain), or the affected area (eg, headache or low back pain). Such classifications are useful in the selection of treatment @ modalities and drug therapy. Nociceptive pain is caused by activation or sensitization of peripheral nociceptors, specialized receptors that transduce noxious stimuli. Neuropathic pain is the result of injury or acquired abnormalities of peripheral or central neural structures.

frequently associated with abnormal sympathetic or parasympathetic activity causing nausea, vomiting, sweating, and changes in blood pressure and heart rate. Parietal pain is typically sharp and often described as a stabbing sensation that is either localized to the area around the organ or referred to a distant site (Table 47—2). The phenomenon of visceral or parietal pain referred to cutaneous areas results from patterns of embryological development and migration of tissues, and the convergence of visceral and somatic afferent input into the central nervous system. Thus, pain associated with disease processes involving the peritoneum or pleura over the central diaphragm is frequently referred to the neck and shoulder, whereas pain from disease processes affecting the parietal surfaces of the peripheral diaphragm is referred to the chest or upper abdominal wall.
TABLE 47-2 Patterns of referred pain.
Central diaphragm, Cutaneous Dermatome = C4. Lungs, Cutaneous Dermatome = T2-T6. Aorta, Cutaneous Dermatome = T1-L2. Heart, Cutaneous Dermatome = T1-T4. Esophagus, Cutaneous Dermatome = 73-18. Pancreas and spleen, Cutaneous Dermatome = T5-T10. Stomach, liver, and gallbladder, Cutaneous Dermatome = T6-19. Adrenals, Cutaneous Dermatome = T8-L1. Small intestine, Cutaneous Dermatome = T9-111. Colon, Cutaneous Dermatome = T10-L1. Kidney, ovaries, and testes, Cutaneous Dermatome = T10-L1. Ureters, Cutaneous Dermatome = T10-T12. Uterus, Cutaneous Dermatome = T11-L2. Bladder and prostate, Cutaneous Dermatome = S2-S4. Urethra and rectum, Cutaneous Dermatome = $2-S4

@ Chronic pain persists beyond the usual course of an acute disease or after a reasonable time for healing to occur, typically 1 to 6 months. Chronic pain may be nociceptive, neuropathic, or mixed, and psychological mechanisms or environmental factors, or both, frequently play a major role. Patients with chronic pain often have attenuated or absent neuroendocrine stress responses and have prominent sleep and affective (mood) disturbances. Neuropathic pain is classically paroxysmal and lancinating, has a burning quality, and is associated with hyperpathia. When it is also associated with loss of sensory input (eg, amputation) into the central nervous system, it is termed deafferentation pain. When the sympathetic system plays a major role, it is often termed sympathetically maintained pain.
The most common forms of chronic pain include those associated with musculoskeletal disorders, chronic visceral disorders, lesions of peripheral nerves, nerve roots, or dorsal root ganglia (including diabetic neuropathy, causalgia, phantom limb pain, and postherpetic neuralgia), lesions of the central nervous system (stroke, spinal cord injury, and multiple sclerosis), and cancer pain. The pain of most musculoskeletal disorders (eg, rheumatoid arthritis and osteoarthritis) is primarily nociceptive, whereas pain associated with peripheral or central neural disorders is primarily neuropathic. The pain associated with some disorders, eg, cancer and chronic back pain (particularly after surgery), is often mixed.

Pain is conducted along three neuronal pathways that transmit noxious stimuli from the periphery to the cerebral cortex (Figure 47—1). The cell bodies of primary afferent neurons are located in the dorsal root ganglia, which lie in the vertebral foramina at each spinal cord level. Each neuron has a single axon that bifurcates, sending one end to the peripheral tissues it innervates and the other into the dorsal horn of the spinal cord. In the dorsal horn, the primary afferent neuron synapses with a second-order neuron whose axon crosses the midline and ascends in the contralateral spinothalamic tract to reach the thalamus. Secondorder neurons synapse in thalamic nuclei with third-order neurons, which in turn send projections through the internal capsule and corona radiata to the postcentral gyrus of the cerebral cortex (Figure 47—2).

Central sulcus
(fissure of
Rolando)
FIGURE 47-2 Lateral (A) and coronal (B) views of the brain show the location of the primary sensory cortex. Note the cortical representation of body parts, the sensory homunculus (B).

The majority of first-order neurons send the proximal end of their axons into the spinal cord via the dorsal (sensory) spinal root at each cervical, thoracic, lumbar, and sacral level. Some unmyelinated afferent (C) fibers have been shown to enter the spinal cord via the ventral nerve (motor) root, accounting for observations that some patients continue to feel pain even after transection of the dorsal nerve root (rhizotomy) and report pain following ventral root stimulation. Once in the dorsal horn, in addition to synapsing with second-order neurons, the axons of first-order neurons may synapse with interneurons, sympathetic neurons, and ventral horn motor neurons.
Pain fibers originating from the head are carried by the trigeminal (V), facial (VID, glossopharyngeal (IX), and vagal (X) nerves. The gasserian ganglion contains the cell bodies of sensory fibers in the ophthalmic, maxillary, and mandibular divisions of the trigeminal nerve. Cell bodies of first-order afferent neurons of the facial nerve are located in the geniculate ganglion; those of the glossopharyngeal nerve lie in its superior and petrosal ganglia; and those of the vagal nerve are located in the jugular ganglion (somatic) and the ganglion nodosum (visceral). The proximal axonal processes of the first-order neurons in these ganglia reach the brainstem nuclei via their respective cranial nerves, where they synapse with second-order neurons in brainstem nuclei.

As afferent fibers enter the spinal cord, they segregate according to size, with large, myelinated fibers becoming medial, and small, unmyelinated fibers becoming lateral. Pain fibers may ascend or descend one to three spinal cord segments in Lissauer's tract before synapsing with second-order neurons in the gray matter of the ipsilateral dorsal horn. In many instances, they communicate with second-order neurons through interneurons.
Spinal cord gray matter was divided by Rexed into ten laminae (Figure 47—3 and Table 47—3). The first six laminae, which make up the dorsal horn, receive all afferent neural activity and represent the principal site of modulation of pain by ascending and descending neural pathways. Second-order neurons are either nociceptive-specific or wide dynamic range (WDR) neurons. Nociceptivespecific neurons serve only noxious stimuli, but WDR neurons also receive nonnoxious afferent input from AB, Ad, and C fibers. Nociceptive-specific neurons are arranged somatotopically in lamina I and have discrete, somatic receptive fields; they are normally silent and respond only to high-threshold noxious stimulation, poorly encoding stimulus intensity. WDR neurons are the most prevalent cell type in the dorsal horn. Although they are found throughout the dorsal horn, WDR neurons are most abundant in lamina V. During repeated stimulation, WDR neurons characteristically increase their firing rate exponentially in a graded fashion (wind-up), even with the same stimulus intensity. They have large receptive fields compared with nociceptive-specific neurons.
FIGURE 47-3 Rexed's spinal cord laminae. Note the termination of the different types of primary afferent neurons.

|, Predominant Function = Somatic nociception thermoreception. |, Input = AG, C. |, Name = Marginal layer. i, Predominant Function = Somatic nociception thermoreception. i, Input = CAS. i, Name = Substantia gelatinosa. i, Predominant Function = Somatic mechanoreception. i, Input = AB, Ad. i, Name = Nucleus proprius. Vv, Predominant Function = Mechanoreception. Vv, Input = AB, Ad. Vv, Name = Nucleus proprius. Vv, Predominant Function = Visceral and somatic nociception and mechanoreception. Vv, Input = AB, AS, (C). Vv, Name = Nucleus proprius WOR neurons!. vi, Predominant Function = Mechanoreception. vi, Input = AB. vi, Name = Nucleus proprius. Vil, Predominant Function = Sympathetic. Vil, Input = . Vil, Name = Intermediolateral column. vill, Predominant Function = . vill, Input = AB. vill, Name = Motor horn. IX, Predominant Function = Motor. IX, Input = AB. IX, Name = Motor horn. x, Predominant Function = . x, Input = AB, (AS). x, Name = Central canal
"WOR, wide dynamic range.

Most nociceptive C fibers send collaterals to, or terminate on, second-order neurons in laminae I and II, and, to a lesser extent, in lamina V. In contrast, nociceptive Aé fibers synapse mainly in laminae I and V, and, to a lesser degree, in lamina X. Lamina I responds primarily to noxious (nociceptive) stimuli from cutaneous and deep somatic tissues. Lamina II, also called the substantia gelatinosa, contains many interneurons and plays a major role in processing and modulating nociceptive input from cutaneous nociceptors. It is a major site of action for opioids. Laminae III and IV primarily receive nonnociceptive sensory input. Laminae VIII and [IX make up the anterior (motor) horn. Lamina VII is the intermediolateral column and contains the cell bodies of preganglionic sympathetic neurons. Visceral afferents terminate primarily in lamina V, and, to a lesser extent, in lamina I. These two laminae represent points of central convergence between somatic and visceral inputs. Lamina V responds to both noxious and nonnoxious sensory input and receives both visceral and somatic pain afferents. The phenomenon of convergence between visceral and somatic sensory input is manifested clinically as referred pain (see Table 47-2). Compared with somatic fibers, visceral nociceptive fibers are fewer in number, more widely distributed, proportionately activate a larger number of spinal neurons, and are not organized somatotopically.

The axons of most second-order neurons cross the midline close to their dermatomal level of origin (at the anterior commissure) to the contralateral side of the spinal cord before they form the spinothalamic tract and send their fibers to the thalamus, the reticular formation, the nucleus raphe magnus, and the periaqueductal gray. The spinothalamic tract, which is classically considered the major pain pathway, lies anterolaterally in the white matter of the spinal cord (Figure 47-4). This ascending tract can be divided into a lateral and a medial tract. The lateral spinothalamic (neospinothalamic) tract projects mainly to the ventral posterolateral nucleus of the thalamus and carries discriminative aspects of pain, such as location, intensity, and duration. The medial spinothalamic (paleospinothalamic) tract projects to the medial thalamus and is responsible for mediating the autonomic and unpleasant emotional perceptions of pain. Some spinothalamic fibers also project to the periaqueductal gray and thus may be an important link between the ascending and descending pathways. Collateral fibers also project to the reticular activating system and the hypothalamus; these are likely responsible for the arousal response to pain.
Ventral spinothalamic tract
FIGURE 47-4 A cross section of the spinal cord showing the spinothalamic and other ascending sensory pathways. Note the spatial distribution of fibers from different spinal levels: cervical (C), thoracic (T), lumbar (L), and sacral (S).

As with epicritic sensation, pain fibers ascend diffusely, ipsilaterally, and contralaterally; some patients continue to perceive pain following ablation of the contralateral spinothalamic tract, and therefore other ascending pain pathways are also important. The spinoreticular tract is thought to mediate arousal and autonomic responses to pain. The spinomesencephalic tract may be important in activating antinociceptive, descending pathways, because it has some projections to the periaqueductal gray. The spinohypothalamic and spinotelencephalic tracts activate the hypothalamus and evoke emotional behavior. The spinocervical tract ascends uncrossed to the lateral cervical nucleus, which relays the fibers to the contralateral thalamus; this tract is likely a major alternative pathway for pain. Lastly, some fibers in the dorsal columns (which mainly carry light touch and proprioception) are responsive to pain; they ascend medially and ipsilaterally.

Somatic and visceral afferents are fully integrated with the skeletal motor and sympathetic systems in the spinal cord, brainstem, and higher centers. Afferent dorsal horn neurons synapse both directly and indirectly with anterior horn motor neurons. These synapses are responsible for the reflex muscle activity— whether normal or abnormal—that is associated with pain. In a similar fashion, synapses between afferent nociceptive neurons and sympathetic neurons in the intermediolateral column result in reflex sympathetically mediated vasoconstriction, smooth muscle spasm, and the release of catecholamines, both locally and from the adrenal medulla.

Third-order neurons are located in the thalamus and send fibers to somatosensory areas I and II in the postcentral gyrus of the parietal cortex and the superior wall of the sylvian fissure, respectively. Perception and discrete localization of pain take place in these cortical areas. Although most neurons from the lateral thalamic nuclei project to the primary somatosensory cortex, neurons from the intralaminar and medial nuclei project to the anterior cingulate gyrus and are likely involved in mediating the suffering and emotional components of pain.

Nociceptors are characterized by a high threshold for activation and encode the intensity of stimulation by increasing their discharge rates in a graded fashion. Following repeated stimulation, they characteristically display delayed adaptation, sensitization, and afterdischarges.
Noxious sensations can often be broken down into two components: a fast, sharp, and well-localized sensation ("first pain"), which is conducted with a short latency (0.1 s) by Aé fibers (tested by pinprick); and a slower onset, duller, and often poorly localized sensation ("second pain"), which is conducted by C fibers. In contrast to well-localized epicritic sensation, which may be transduced by specialized end organs on the afferent neuron (eg, pacinian corpuscle for touch), less well-localized protopathic sensation is transduced mainly by free nerve endings nociceptors that sense heat and mechanical and chemical tissue damage. Nociceptor types include (1) mechanonociceptors, which respond to pinch and pinprick, (2) silent nociceptors, which respond only in the presence of inflammation, and (3) polymodal mechanoheat nociceptors. The last are most prevalent and respond to excessive pressure, extremes of temperature (>42°C and <40°C), and noxious substances such as bradykinin, histamine, serotonin (5hydroxytryptamine or 5-HT), H*, K*, some prostaglandins, capsaicin, and possibly adenosine triphosphate. At least two nociceptor receptors (containing ion channels in nerve endings) have been identified, TRPV1 and TRPV2. Both respond to high temperatures. Capsaicin stimulates the TRPV1 receptor. Polymodal nociceptors are slow to adapt to strong pressure and display heat sensitization.

Nociceptors are present in both somatic and visceral tissues. Primary afferent neurons reach tissues by traveling along spinal somatic, sympathetic, or parasympathetic nerves. Somatic nociceptors include those in skin (cutaneous) and deep tissues (muscle, tendons, fascia, and bone), whereas visceral nociceptors include those in internal organs. The cornea and tooth pulp are unique in that they are almost exclusively innervated by nociceptive Aé and C fibers.

Deep somatic nociceptors are less sensitive to noxious stimuli than cutaneous nociceptors but are easily sensitized by inflammation. The pain arising from them is characteristically dull and poorly localized. Specific nociceptors exist in muscles and joint capsules, and they respond to mechanical, thermal, and chemical stimuli.

Visceral organs are generally insensitive tissues that mostly contain silent nociceptors. Some organs appear to have specific nociceptors, such as the heart, lung, testis, and bile ducts. Most other organs, such as the intestines, are innervated by polymodal nociceptors that respond to smooth muscle spasm, ischemia, and inflammation. These receptors generally do not respond to the cutting, burning, or crushing that occurs during surgery. A few organs, such as the brain, lack nociceptors altogether; however, the brain's meningeal coverings do contain nociceptors.
Like somatic nociceptors, those in the viscera are the free nerve endings of primary afferent neurons whose cell bodies lie in the dorsal horn. These afferent nerve fibers, however, frequently travel with efferent sympathetic nerve fibers to reach the viscera. Afferent activity from these neurons enters the spinal cord between T1 and L2. Nociceptive C fibers from the esophagus, larynx, and trachea travel with the vagus nerve to enter the nucleus solitarius in the brainstem. Afferent pain fibers from the bladder, prostate, rectum, cervix and urethra, and genitalia are transmitted into the spinal cord via parasympathetic nerves at the level of the S2—S4 nerve roots. Though relatively few compared with somatic pain fibers, fibers from primary visceral afferent neurons enter the cord and synapse more diffusely with single fibers, often synapsing with multiple dermatomal levels and often crossing to the contralateral dorsal horn.

Several neuropeptides and excitatory amino acids function as neurotransmitters for afferent neurons subserving pain (Table 47—4). Many, if not most, of these neurons contain more than one neurotransmitter, which are simultaneously released. The most important of these peptides are substance P and calcitonin gene-related peptide (CGRP). Glutamate is the most important excitatory amino acid.
TABLE 47-4 Major neurotransmitters mediating or modulating pain.

Substance P, Receptor’ = Neurokinin-1. Substance P, Effect on Nociception = Excitatory. Calcitonin gene- related peptide, Receptor’ = . Calcitonin gene- related peptide, Effect on Nociception = Excitatory. Glutamate, Receptor’ = NMDA, AMPA, kainate, quisqualate. Glutamate, Effect on Nociception = Excitatory. Aspartate, Receptor’ = NMDA, AMPA, kainate, quisqualate. Aspartate, Effect on Nociception = Excitatory. Adenosine triphosphate (ATP), Receptor’ = Paes. Adenosine triphosphate (ATP), Effect on Nociception = Excitatory. Somatostatin, Receptor’ = . Somatostatin, Effect on Nociception = Inhibitory. Acetylcholine, Receptor’ = Muscarinic. Acetylcholine, Effect on Nociception = Inhibitory. Enkephalins, Receptor’ = , 5, K. Enkephalins, Effect on Nociception = Inhibitory. 6-Endorphin, Receptor’ = u, 5, K. 6-Endorphin, Effect on Nociception = Inhibitory. Norepinephrine, Receptor’ = a,. Norepinephrine, Effect on Nociception = Inhibitory. Adenosine, Receptor’ = A,. Adenosine, Effect on Nociception = Inhibitory. Serotonin, Receptor’ = 5-HT, (5-HT,). Serotonin, Effect on Nociception = Inhibitory. y-Aminobutyric acid (GABA), Receptor’ = A,B. y-Aminobutyric acid (GABA), Effect on Nociception = Inhibitory. Glycine, Receptor’ = . Glycine, Effect on Nociception = Inhibitory

'NMDA, N-methyl-p-aspartate; AMPA, 2-(aminomethyl)phenylacetic acid; 5-HT, 5-hydroxytryptamine.
Substance P is an 11-amino-acid peptide that is synthesized and released by first-order neurons both peripherally and in the dorsal horn. Also found in other parts of the nervous system and the intestines, it facilitates transmission in pain pathways via neurokinin-1 receptor activation. In the periphery, substance P neurons send collaterals that are closely associated with blood vessels, sweat glands, hair follicles, and mast cells in the dermis. Substance P sensitizes nociceptors, degranulates histamine from mast cells and 5-HT from platelets, and is a potent vasodilator and chemoattractant for leukocytes. Substance P— releasing neurons also innervate the viscera and send collateral fibers to paravertebral sympathetic ganglia; intense stimulation of viscera, therefore, can cause direct postganglionic sympathetic discharge.
Both opioid and a,-adrenergic receptors have been described on or near the terminals of unmyelinated peripheral nerves. Although their physiological role is not clear, the latter may explain the observed analgesia of peripherally applied opioids, particularly in the presence of inflammation.

® Modulation of pain occurs peripherally at the nociceptor, in the spinal cord, and in supraspinal structures. This modulation can either inhibit (suppress) or facilitate (intensify) pain.

Nociceptors and their neurons display sensitization following repeated stimulation. Sensitization may be manifested as an enhanced response to noxious stimulation or a newly acquired responsiveness to a wider range of stimuli, including nonnoxious stimuli.

Sensitization of nociceptors results in a decrease in threshold, an increase in the frequency response to the same stimulus intensity, a decrease in response latency, and spontaneous firing even after cessation of the stimulus (afterdischarges). Such sensitization, termed primary hyperalgesia, commonly occurs with injury and following application of heat and is mediated by the release of noxious substances from damaged tissues. Histamine is released from mast cells, basophils, and platelets, whereas serotonin is released from mast cells and platelets. Bradykinin is released from tissues following activation of factor XII. Bradykinin activates free nerve endings via specific B1 and B2 receptors. Prostaglandins are produced following tissue damage by the action of phospholipase A, on phospholipids released from cell membranes to form arachidonic acid (Figure 47—5). The cyclooxygenase (COX) pathway then converts the latter into endoperoxides, which in turn are transformed into prostacyclin, prostaglandins and thromboxanes. The lipoxygenase pathway converts arachidonic acid into hydroperoxy compounds, which are subsequently converted into leukotrienes. Acetylsalicylic acid (aspirin), nonsteroidal antiinflammatory drugs (NSAIDs), and likely also acetaminophen produce analgesia by inhibition of COX. The analgesic effect of corticosteroids is likely the result of inhibition of prostaglandin production through blockade of phospholipase A, activation.
FIGURE 47-5 Phospholipase C (PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP) to produce inositol triphosphate (IP) and diacylglycerol (DAG). Protein kinase C (PKC) is also important. Phospholipase A, (PLA,) catalyzes the conversion of phosphatidylcholine (PC) to arachidonic acid (AA).

Neurogenic inflammation, also called secondary hyperalgesia, plays an important role in peripheral sensitization following injury. It is manifested by the triple response of Lewis: a red flush around the site of injury (flare), local tissue edema, and sensitization to noxious stimuli. Secondary hyperalgesia is primarily due to antidromic release of substance P (and probably CGRP). Substance P degranulates histamine and 5-HT, vasodilates blood vessels, causes tissue edema, and induces the formation of leukotrienes. The neural origin of this response is supported by the following findings: (1) it can be produced by electrical stimulation of a sensory nerve, (2) it is not observed in denervated skin, and (3) it is diminished by injection of a local anesthetic. Capsaicin applied topically in a gel, cream, or patch depletes substance P, diminishes neurogenic inflammation, and is useful for some patients with postherpetic neuralgia.

@ At least three mechanisms are responsible for central sensitization in the spinal cord:
1. Wind-up and sensitization of second-order neurons. WDR neurons increase their frequency of discharge with the same repetitive stimuli and exhibit prolonged discharge, even after afferent C fiber input has stopped.
2. Receptive field expansion. Dorsal horn neurons increase their receptive fields such that adjacent neurons become responsive to stimuli (whether noxious or not) to which they were previously unresponsive.
3. Hyperexcitability of flexion reflexes. Enhancement of flexion reflexes is observed both ipsilaterally and contralaterally.
Neurochemical mediators of central sensitization include substance P, CGRP, vasoactive intestinal peptide (VIP), cholecystokinin (CCK), angiotensin, and galanin, as well as the excitatory amino acids L-glutamate and L-aspartate. These substances trigger changes in membrane excitability by interacting with G protein-coupled membrane receptors on neurons (Figure 47—5).
Glutamate and aspartate play important roles in wind-up, via activation of Nmethyl-p-aspartate (NMDA) and other receptor mechanisms, and in the induction and maintenance of central sensitization. Activation of NUDA receptors also induces nitric oxide synthetase, increasing formation of nitric oxide. Both prostaglandins and nitric oxide facilitate the release of excitatory amino acids in the spinal cord. Thus, COX inhibitors such as acetylsalicylic acid (ASA) and NSAIDs have important analgesic actions in the spinal cord.

Transmission of nociceptive input in the spinal cord can be inhibited by segmental activity in the cord itself, as well as by descending neural activity from supraspinal centers.
- 1. Segmental inhibition—Activation of large afferent fibers subserving
hyperpolarize primary afferent neurons and inhibit the release of substance P; they also appear to cause some postsynaptic inhibition. Exogenous opioids preferentially act postsynaptically on the second-order neurons or interneurons in the substantia gelatinosa.

@ Chronic pain may be caused by a combination of peripheral, central, and psychological mechanisms. Sensitization of nociceptors plays a major role in the origin of pain associated with peripheral mechanisms, such as chronic musculoskeletal and visceral disorders.
Neuropathic pain involves peripheral—central and central neural mechanisms that are complex and generally associated with partial or complete lesions of peripheral nerves, dorsal root ganglia, nerve roots, or more central structures (Table 47-5). Peripheral mechanisms include spontaneous discharges, sensitization of receptors (to mechanical, thermal, and chemical stimuli), and upregulation of adrenergic receptors. Neural inflammation may also be present. Systemic administration of local anesthetics and anticonvulsants has been shown to suppress the spontaneous firing of sensitized or traumatized neurons, and this laboratory observation is reinforced by the clinical efficacy of agents such as lidocaine, mexiletine, and carbamazepine in many patients with neuropathic pain. Central mechanisms include loss of segmental inhibition, wind-up of WDR neurons, spontaneous discharges in deafferentated neurons, and reorganization of neural connections.

Spontaneous self-sustaining neuronal activity in the primary afferent neuron (such as a neuroma).
Marked mechanosensitivity associated with chronic nerve compression.
Short-circuits between pain fibers and other types of fibers following demyelination, resulting in activation of nociceptive fibers by nonnoxious stimuli at the site of injury (ephaptic transmission).
Functional reorganization of receptive fields in dorsal horn neurons such that sensory input from surrounding intact nerves emphasizes or intensifies any input from the area of injury.
Spontaneous electrical activity in dorsal horn cells or thalamic nuclei.
Release of segmental inhibition in the spinal cord.
Loss of descending inhibitory influences that are dependent on normal sensory input.
Lesions of the thalamus or other supraspinal structures.
The sympathetic nervous system appears to play a major role in some patients with chronic pain. The efficacy of sympathetic nerve blocks in some of these patients supports the concept of sympathetically maintained pain. Painful disorders that often respond to sympathetic blocks include complex regional pain syndrome, deafferentation syndromes due to nerve avulsion or amputations, and postherpetic neuralgia. However, the simplistic theory of heightened sympathetic activity resulting in vasoconstriction, edema, and hyperalgesia fails to account for the warm and erythematous phase observed in some patients. Similarly, clinical and experimental observations do not satisfactorily support the theory of ephaptic transmission between pain fibers and demyelinated sympathetic fibers. Psychological mechanisms or environmental factors are rarely the sole mechanisms for chronic pain but are commonly seen in combination with other mechanisms ('Table 47-6).

Psychophysiological mechanisms in which emotional factors act as the initiating cause (eg, tension headaches)
Learned or operant behavior in which chronic behavior patterns are rewarded (eg, by attention of a spouse) following an often minor injury
Psychopathology such as major affective disorders (depression), schizophrenia, and somatization disorders (conversion hysteria) in which the patient has an abnormal preoccupation with bodily functions
Pure psychogenic mechanisms (somatoform pain disorder), in which suffering is experienced despite absence of nociceptive input

Systemic Responses to Acute Pain
Acute pain is typically associated with a systemic neuroendocrine stress response that is proportional to pain intensity. The pain pathways mediating the afferent limb of this response are discussed above. The efferent limb is mediated by the sympathetic nervous and endocrine systems. Sympathetic activation increases efferent sympathetic tone to all viscera and releases catecholamines from the adrenal medulla. The hormonal response results from increased sympathetic @ tone and from hypothalamically-mediated reflexes. Moderate to severe acute pain, regardless of site, may adversely affect perioperative morbidity, mortality, and convalescence.

Cardiovascular effects of acute pain often include hypertension, tachycardia, enhanced myocardial irritability, and increased systemic vascular resistance. Cardiac output increases in most normal patients but may decrease in patients with compromised ventricular function. Because of the increase in myocardial oxygen demand, pain can worsen or precipitate myocardial ischemia.

An increase in total body oxygen consumption and carbon dioxide production necessitates a concomitant increase in minute ventilation. The latter increases the work of breathing, particularly in patients with underlying lung disease. Pain due to abdominal or thoracic incisions further compromises pulmonary function because of guarding (splinting). Decreased movement of the chest wall reduces tidal volume and functional residual capacity, promoting atelectasis, intrapulmonary shunting, hypoxemia, and, less commonly, hypoventilation. Reductions in vital capacity impair coughing and clearing of secretions.

Enhanced sympathetic tone increases sphincter tone and decreases intestinal and urinary bladder motility, promoting ileus and urinary retention. Hypersecretion of gastric acid can promote stress ulceration. Nausea, vomiting, and constipation are common. In addition, systemic opioids used to treat postoperative pain (and also administered as a component of the operative anesthetic) are a common cause of postoperative ileus and urinary retention.

Stress increases release of catabolic hormones (catecholamines, cortisol, and glucagon) and inhibits release of anabolic hormones (insulin and testosterone).
Patients develop a negative nitrogen balance, carbohydrate intolerance, and increased lipolysis. The increase in cortisol, renin, angiotensin, aldosterone, and antidiuretic hormone results in sodium retention, water retention, and secondary expansion of the extracellular space.

The neuroendocrine stress response to acute pain may increase platelet adhesiveness, reduce fibrinolysis, and promote a hypercoagulability state.

The neuroendocrine stress response produces leukocytosis and may predispose patients to infection. Worsening carbohydrate intolerance with sustained hyperglycemia also increases the risk of infection. Stress-related enhance tumor and metastasis.
immunodepression may growth

Anxiety and sleep disturbances are common reactions to acute pain. With prolonged duration of the pain, depression is not unusual. Some patients react with frustration and anger that may be directed at family, friends, and the medical staff.

The neuroendocrine stress response in the setting of chronic pain is generally observed only in patients with severe recurring pain due to peripheral (nociceptive) mechanisms and in patients with prominent central mechanisms such as pain associated with paraplegia. It is attenuated or absent in most patients with chronic pain. Sleep and affective disturbances, particularly depression, are often prominent. Many patients also experience significant decreases or increases in appetite and psychological stress related to social relationships.

@ The evaluation of any patient with pain should include several key components. Information about location, onset, and quality of pain, as well as alleviating and exacerbating factors, should be obtained, along with a pain history that includes previous therapies and changes in symptoms over time. In addition to physical symptoms, chronic pain usually involves a psychological component that should be addressed. Questionnaires, diagrams, and pain scales are useful tools that help patients adequately describe the characteristics of their pain and how it affects their quality of life. Information gathered during the physical examination can help distinguish pain location, type, and systemic sequelae, if any. Imaging studies such as plain radiographs, computed tomography (CT), magnetic resonance imaging (MRI), and bone scans can often help delineate physiological causes. All components are necessary for a comprehensive evaluation of the pain patient prior to determining appropriate treatment options.

A reliable measurement of pain severity would help determine therapeutic interventions and evaluate the efficacy of treatments. This is a challenge, however, because pain is a subjective experience that is influenced by psychological, social, cultural, and other variables. Clear definitions are necessary, as pain may be described in terms of tissue destruction or bodily or emotional reaction.
The numerical rating scale, Wong-Baker FACES rating scale, visual analog scale (VAS), and McGill Pain Questionnaire (MPQ) are most commonly used. In the numerical scale, 0 corresponds to no pain and 10 is intended to reflect the worst possible pain. The Wong-Baker FACES pain scale, designed for children 3 years of age and older, is useful in patients with whom communication may be difficult. The patient is asked to point to various facial expressions ranging from a smiling face (no pain) to an extremely unhappy one that expresses the worst possible pain. The VAS is a 10-cm horizontal line labeled "no pain" at one end and "worst pain imaginable" on the other end. The patient is asked to mark on this line where the intensity of the pain lies. The distance from "no pain" to the patient's mark numerically quantifies the pain. The VAS is a simple and efficient method that correlates well with other reliable methods.
The MPQ is a checklist of words describing symptoms. Unlike other pain rating methods that assume pain is one-dimensional and describe intensity but not quality, the MPQ attempts to define the pain in three major dimensions: (1) sensory—discriminative (nociceptive pathways), (2) motivational—affective (reticular and limbic structures), and (3) cognitive—evaluative (cerebral cortex). It contains 20 sets of descriptive words that are divided into four major groups: 10 sensory, 5 affective, 1 evaluative, and 4 miscellaneous. The patient selects the sets that apply to his or her pain and circles the words in each set that best describe the pain.

TABLE 47-7 Emotional and related disorders commonly associated with chronic pain.
Somatization disorder, Brief Description = Physical symptoms of a medical condition that cannot be explained, resulting in involuntary distress and physical impairment. Conversion disorder, Brief Description = Symptoms of voluntary motor or sensory deficits that suggest a medical condition; symptoms cannot be medically explained but are associated with psychological factors and are not intentionally feigned. Hypochondriasis, Brief Description = Prolonged (>6 months) preoccupation with the fear of having a serious illness despite adequate medical evaluation and reassurance. Malingering, Brief Description = Intentional production of physical or psychological symptoms that is motivated by external incentives (eg, avoiding work or financial compensation). Substance- related disorders, Brief Description = Habitual misuse of prescribed or illicit substances that often precedes and drives complaints of pain and drug- seeking behavior

Electromyography and nerve conduction studies complement one another and are useful for confirming the diagnosis of entrapment syndromes, radicular syndromes, neural trauma, and polyneuropathies. They can often distinguish between neurogenic and myogenic disorders. Patterns of abnormalities can localize a lesion to the spinal cord, nerve root, limb plexus, or peripheral nerve. In addition, they may also be useful in clarifying the contribution of "organic"
disorders when psychogenic pain or a "functional" syndrome is suspected. Electromyography employs needle electrodes to record potentials in individual muscles. Muscle potentials are recorded first while the muscle is at rest and then as the patient is asked to move the muscle. Abnormal findings suggestive of denervation include persistent insertion potentials, the presence of positive sharp waves, fibrillary activity, or fasciculation potentials. Abnormalities in muscles produce changes in amplitude and duration as well as polyphasic action potentials. Peripheral nerve conduction studies employ supramaximal stimulations of motor or mixed sensorimotor nerve, whereas muscle potentials are recorded over the appropriate muscle. The time between the onset of the stimulation and the onset of the muscle potential (latency) is a measurement of the fastest conducting motor fibers in the nerve. The amplitude of the recorded potential indicates the number of functional motor units, whereas its duration reflects the range of conduction velocities in the nerve. Conduction velocity can be obtained by stimulating the nerve from two points and comparing the latencies. When a pure sensory nerve is evaluated, the nerve is stimulated while action potentials are recorded either proximally or distally (antidromic conduction).
Nerve conduction studies distinguish between mononeuropathies (due to trauma, compression, or entrapment) and polyneuropathies. The latter includes acute or chronic neuropathies that are widespread and symmetrical (eg, related to diabetes, alcohol abuse, malnutrition, toxins, or drugs such as chemotherapeutic agents), or that are focal but random (eg, mononeuropathy multiplex).

Neural compression may occur wherever a nerve courses through an anatomically narrowed passage, and entrapment neuropathies can involve sensory, motor, or mixed nerves. Genetic factors and repetitive macrotrauma or microtrauma are likely involved, and adjacent tenosynovitis is often responsible. Table 47—8 lists the most commonly recognized entrapment syndromes. When a sensory nerve is involved, patients complain of pain and numbness in its distribution distal to the site of entrapment; occasionally, a patient may complain of pain referred proximal to the site of entrapment. Entrapment of the sciatic nerve can mimic a herniated intervertebral disc. Entrapment of a motor nerve produces weakness in the muscles it innervates. Even entrapments of "pure"
motor nerves can produce a vague pain that may be mediated by afferent fibers from muscles and joints. The diagnosis can usually be confirmed by electromyography and nerve conduction studies. Neural blockade of the nerve with local anesthetic, with or without corticosteroid, may be diagnostic and may provide temporary pain relief. Treatment is usually symptomatic, with oral analgesics and temporary immobilization, but may eventually include operative decompression. Development of complex regional pain syndrome may respond to sympathetic blocks.

@ Myofascial pain syndromes are common disorders characterized by aching muscle pain, muscle spasm, stiffness, weakness, and, occasionally, autonomic dysfunction. Patients have discrete areas (trigger points) of marked tenderness in one or more muscles or the associated connective tissue. Palpation of the involved muscles may reveal tight, ropy bands over trigger points. Signs of autonomic dysfunction (vasoconstriction or piloerection) in the overlying muscles may be present. The pain characteristically radiates in a fixed pattern that does not follow dermatomes.
Gross trauma or repetitive microtrauma may play a major role in initiating myofascial pain syndromes. Trigger points develop following acute injury; stimulation of these active trigger points produces pain, and the ensuing muscle spasm sustains the pain. When the acute episode subsides, the trigger points become latent (tender, but not pain-producing) only to be reactivated at a later time by subsequent stress. The pathophysiology is poorly understood.
The diagnosis of a myofascial pain syndrome is suggested by the character of the pain and by palpation of discrete trigger points that reproduce it. Common syndromes produce trigger points in the levator scapulae, masseter, quadratus lumborum, and gluteus medius muscles. The latter two syndromes produce low back pain and should be considered in all patients with back pain; moreover, gluteal trigger points can mimic S, radiculopathy.
Although myofascial pain may spontaneously resolve without sequelae, many patients continue to have latent trigger points. When trigger points are active, treatment is directed at regaining muscle length and elasticity. Analgesia may be provided utilizing local anesthetic (1-3 mL) trigger point injections. Topical cooling with either an ethyl chloride or fluorocarbon (fluoromethane) spray can also induce reflex muscle relaxation, facilitating massage ("stretch and spray") and ultrasound therapy. Physical therapy is important in establishing and maintaining normal range of motion for affected muscles, and biofeedback may be helpful.

The American College of Rheumatology recently identified three criteria that, if met, suggest the diagnosis of fibromyalgia:
1. Widespread Pain Index (WPI) score of 7 or higher, and Symptom Severity (SS) scale score of 5 or higher, or WPI of 3 to 6 and SS scale score of 9 or higher.
Symptoms present at a similar level for at least 3 months.
3. Absence of another disorder that would otherwise explain the pain.
Treatment of fibromyalgia includes cardiovascular conditioning, strength training, improving sleep hygiene, cognitive—behavioral therapy, patient education, and pharmacotherapy. Medications approved by the U.S. Food and Drug Administration (FDA) for the treatment of fibromyalgia include pregabalin
(Lyrica), duloxetine (Cymbalta), and milnacipran (Savella).

Back pain is a common complaint and a major cause of disability. Lumbosacral strain, degenerative disc disease, and myofascial syndromes are the most common causes. Low back pain, with or without associated leg pain, may also have congenital, traumatic, degenerative, inflammatory, infectious, metabolic, psychological, and neoplastic causes. Moreover, back pain can be due to disease processes in the abdomen and pelvis, particularly those affecting retroperitoneal structures (pancreas, kidneys, ureters, and aorta), the uterus and adnexa, the prostate, and the rectosigmoid colon. Disorders of the hip can also mimic back disorders. A positive Patrick's sign (or Patrick's test)—ie, the elicitation of pain in the hip or sacroiliac joint when the examiner places the ipsilateral heel of the supine patient on the contralateral knee and presses down on the ipsilateral knee —helps identify back pain due to hip or sacroiliac joint disorders. This sign is also referred to by the acronym FABERE (sign), because the movement of the leg involves flexion, abduction, external rotation, and extension.

The back can be described in terms of anterior and posterior elements. The anterior elements consist of cylindrical vertebral bodies interconnected by intervertebral discs and supported by anterior and posterior longitudinal ligaments. The posterior elements are bony arches extending from each vertebral body, consisting of two pedicles, two transverse processes, two laminae, and a spinous process. The transverse and spinous processes provide points of attachment for the muscles that move and protect the spinal column. Adjacent vertebrae also articulate posteriorly by two gliding facet joints.
Spinal structures are innervated by the sinuvertebral branches and posterior rami of spinal nerves. The sinuvertebral nerve arises before each spinal nerve divides into anterior and posterior rami, and reenters the intervertebral foramen to innervate the posterior longitudinal ligament, the posterior annulus fibrosus, periosteum, dura, and epidural vessels. Paraspinal structures are supplied by the posterior primary ramus. Each facet joint is innervated by the medial branch of the posterior primary rami of the spinal nerves above and below the joint.
As lumbar spinal nerve roots exit the dural sac, they travel down 1 to 2 cm laterally before exiting through their respective intervertebral foramina; thus, for example, the L5 nerve root leaves the dural sac at the level of the L4—L5 disc
(where it is more likely to be compressed) but leaves the spinal canal beneath the LS pedicle opposite the L5—-S1 disc.

Approximately 80% to 90% of low back pain is due to sprain or strain associated with lifting heavy objects, falls, or sudden abnormal movements of the spine. The term sprain is generally used when the pain is related to a well-defined acute injury, whereas strain is used when the pain is more chronic and is likely related to repetitive minor injuries.
Injury to paravertebral muscles and ligaments results in reflex muscle spasm, which may or may not be associated with trigger points. The pain is usually dull and aching, and occasionally radiates down the buttocks or hips. Sprain is a selflimited benign process that resolves in 1 to 2 weeks. Symptomatic treatment consists of rest and oral analgesics.
The sacroiliac joint is particularly vulnerable to rotational injuries. It is one of the largest joints in the body and functions to transfer weight from the upper body to the lower extremities. Acute or chronic injury can cause slippage, or subluxation, of the joint. Pain originating from this joint is characteristically located along the posterior ilium and radiates down the hips and posterior thigh to the knees. The diagnosis is suggested by tenderness on palpation, particularly on the medial aspect of the posterior superior iliac spine, and by compression of the joints. Pain relief following injection of the joint with local anesthetic (3 mL) is diagnostic and may also be therapeutic. Injection of intraarticular steroid medication may be considered. For potentially longer duration of analgesia, radiofrequency ablation may be performed at the dorsal ramus of L5 as well as the lateral branches of the S1, S2, and S3 nerves if the patient initially responded well to local anesthetic injections of the sacroiliac joint or to diagnostic injections of these nerves.

Buttock pain may be due to several different factors and can be quite debilitating. Coccydynia may result from trauma to the coccyx or surrounding ligaments. It may resolve by means of physical therapy, coccygeal nerve blocks to the lateral aspects of the coccyx, or ablative or neuromodulatory techniques. Piriformis syndrome presents as pain in the buttock, which can be accompanied by numbness and tingling in distribution of the sciatic nerve. The nerve may or may not be entrapped. Injection of local anesthetic into the belly of this muscle or into trigger points located at the origin and insertion of the muscle may help relieve the pain.

has shown mixed degrees of success. The evaluation and treatment of discogenic pain is an area of significant controversy and ongoing research.

Weakness and degeneration of the annulus fibrosus and posterior longitudinal ligament can cause herniation of the nucleus pulposus posteriorly into @ the spinal canal. Ninety percent of disc herniations occur at L5—S1 or L4—L5. Symptoms usually develop following flexion injuries or heavy lifting and may be associated with bulging, protrusion, or extrusion of the disc. Disc herniations usually occur posterolaterally and often result in compression of adjacent nerve roots, producing pain that radiates along that dermatome (radiculopathy, or radicular pain). Sciatica describes pain along the sciatic nerve due to compression of the lower lumbar nerve roots. When disc material is extruded through the annulus fibrosus and posterior longitudinal ligament, free fragments can become wedged in the spinal canal or the intervertebral foramina. Less commonly a large disc bulges or large fragments extrude posteriorly, compressing the cauda equina in the dural sac; in these instances, patients can experience bilateral pain, urinary retention, or, less commonly, fecal incontinence.
Pain associated with disc disease is aggravated by bending, lifting, prolonged sitting, or anything that increases intraabdominal pressure, such as sneezing, coughing, or straining. It is usually relieved by lying down. Numbness or weakness is indicative of radiculopathy (Table 47-9). Bulging of the disc through the posterior longitudinal ligament can also produce low back pain that radiates to the hips or buttocks. Straight leg-raising tests may be used to assess nerve root compression. With the patient supine and the knee fully extended, the leg on the affected side is raised and the angle at which the pain develops is noted; dorsiflexion of the ankle with the leg raised typically exacerbates the pain by further stretching the lumbosacral plexus. Pain while raising the contralateral leg is an even more reliable sign of nerve compression.

Pain distribution, Disk Level.13-L4 (L4 Nerve) = Anterolateral thigh, anteromedial calf to the ankle. Pain distribution, Disk Level.L4-L5 (L5 Nerve) = Lateral thigh, anterolateral calf, medial dorsum of foot, especially between the first and second toes. Pain distribution, Disk Level.L5-S1(S1 Nerve) = Gluteal region, posterior thigh, posterolateral calf, lateral dorsum and undersurface of the foot, particularly between fourth and fifth toes. Weakness, Disk Level.13-L4 (L4 Nerve) = Quadriceps femoris. Weakness, Disk Level.L4-L5 (L5 Nerve) = Dorsiflexion of the foot. Weakness, Disk Level.L5-S1(S1 Nerve) = Plantar flexion of foot. Reflex affected, Disk Level.13-L4 (L4 Nerve) = Knee. Reflex affected, Disk Level.L4-L5 (L5 Nerve) = . Reflex affected, Disk Level.L5-S1(S1 Nerve) = Ankle
considered. Of note, back supports should be discouraged because they may weaken paraspinal muscles.

Spinal stenosis is a disease of advancing age. Degeneration of the nucleus pulposus reduces disc height and leads to osteophyte formation (spondylosis) at the endplates of adjoining vertebral bodies. In conjunction with facet joint hypertrophy and with ligamentum flavum hypertrophy and calcification, this process leads to progressive narrowing of the neural foramina and spinal canal. Neural compression may cause radiculopathy that mimics a herniated disc. Extensive osteophyte formation may compress multiple nerve roots and cause bilateral ® pain. The back pain usually radiates into the buttocks, thighs, and legs. It is characteristically worse with exercise and relieved by rest, particularly sitting with the spine flexed (shopping cart sign). The terms pseudoclaudication and neurogenic claudication are used to describe such pain that develops with prolonged standing or ambulation. The diagnosis is suggested by the clinical presentation and is confirmed by MRI, CT, or myelography. Electromyography and nerve conduction studies may be useful in evaluating the degree of neurological compromise.
Patients with mild to moderate stenosis and radicular symptoms may obtain benefit from epidural steroid injections via a transforaminal, interlaminar, or caudal approach, which may help these individuals tolerate physical therapy. Those with moderate to severe stenosis may be candidates for the minimally invasive lumbar decompression (MILD) procedure, which involves percutaneously sculpting the lamina and ligamentum flavum to reduce central canal compression. Severe, multilevel symptoms may warrant surgical decompression.

Degenerative changes in the facet (zygapophyseal) joints may also produce back pain. Pain may be near the midline; may radiate to the gluteal region, thigh, and knee; and may be associated with muscle spasm. Hyperextension and lateral rotation of the spine usually exacerbate the pain. The diagnosis may be confirmed if pain relief is obtained following intraarticular injection of local anesthetic solution into affected joints or by blockade of the medial branch of the posterior division (ramus) of the spinal nerves that innervate them. Long-term studies suggest that medial branch nerve blocks are more effective than facet joint injections. Medial branch rhizotomy may provide long-term analgesia for patients with facet joint disease.

Although most spine-related pain due to disc disease, spinal stenosis, or degenerative changes in the zygapophyseal joints is felt in the low back and lower extremities, patients may have cervical pain attributed to these processes. A key anatomic difference is that the cervical nerve roots, unlike those in the thoracic and lumbar spine, exit the foramina above the vertebral bodies for which they are named. This occurs until the level of C7, where the extra cervical nerve roots, C8, exit below the pedicles of C7, thus transitioning to the nomenclature of the thoracicand lumbar-level vertebral bodies and nerve root denominations. The clinical examination may help to identify the nerve root that is affected with confirmation by a selective nerve root block. Risks inherent with percutaneous cervical procedures include accidental intravascular injection of local anesthetic or steroid. Particulate steroid injections in the neck have been associated with devastating outcomes and should be avoided.
For primarily axial pain in the neck with extension into the head or to the shoulders, cervical medial branch blocks may clarify the diagnosis. Long-term analgesia may be obtained with radiofrequency ablation of the medial branches innervating the zygapophyseal joints.

Congenital abnormalities of the back may be asymptomatic and remain occult for many years. Abnormal spinal mechanics can make the patient prone to back pain, and in some instances, progressive deformities. Relatively common anomalies include sacralization of L5 (the vertebral body is fused to the sacrum), lumbarization of S1 (it functions as a sixth lumbar vertebra), spondylolysis (disruption of the pars interarticularis), spondylolisthesis (displacement anteriorly of one vertebral body on the next due to disruption of the posterior elements, usually the pars interarticularis), and spondyloptosis (subluxation of one vertebral body on another resulting in one body in front of the next). These diagnoses are made with radiographic imaging studies. Spinal fusion may be necessary in patients with progressive symptoms and spinal instability.
A wide range of benign and malignant tumors may lead to back pain. When diagnosed by imaging these tumors will be managed by neurosurgeons, radiotherapists, and or medical oncologists, not by pain specialists.

Bacterial infections of the spine usually begin as a discitis before progressing to osteomyelitis, and can be due to pyogenic as well as tuberculous organisms. Patients may present with chronic back pain without fever or leukocytosis (eg, spinal tuberculosis). Those with acute discitis, osteomyelitis, or epidural abscess present with acute pain, fever, leukocytosis, elevated sedimentation rate, and elevated C-reactive protein, warranting immediate initiation of antibiotics. Urgent surgical intervention is indicated when the patient also suffers from acute weakness.

Ankylosing spondylitis is a familial disorder that is associated with the HLA-B27 antigen. It typically presents as low back pain associated with early morning stiffness in a young male patient. The pain has an insidious onset and may initially improve with activity. After a few months to years, the pain gradually intensifies and is associated with progressively restricted movement of the spine. Diagnosis may be difficult early in the disease, but radiographic evidence of sacroiliitis is usually present. As the disease progresses, the spine develops a characteristic "bamboo-like" radiographic appearance. Some patients develop arthritis of the hips and shoulders, as well as extraarticular inflammatory manifestations. Treatment is primarily directed at functional preservation of posture. NSAIDs are effective analgesics that reduce the early morning stiffness. Anti-tumor necrosis factor-a agents, including infliximab (Remicade), etanercept (Enbrel), adalimumab (Humira), and golimumab (Simponi), decrease the progression of ankylosing spondylitis when administered early in the course of therapy. However, patients treated with these medications may be at increased risk for infection and for the development of lymphoma. with syndrome, arthritis, inflammatory bowel for
Patients Reiter psoriatic or disease may also present with low back pain, but extraspinal manifestations are usually more prominent. Rheumatoid arthritis usually spares the spine except the zygapophyseal joints of the cervical spine.

Neuropathic pain tends to be paroxysmal and sometimes lancinating with a burning quality, and is usually accompanied by hyperpathia. It is associated with diabetic neuropathy, causalgia, phantom limbs, postherpetic neuralgia, stroke, spinal cord injury, and multiple sclerosis. Cancer pain and chronic low back pain may also have prominent neuropathic components. Mechanisms of neuropathic pain are reviewed earlier in this chapter. modalities
Neuropathic pain is often difficult to treat, and multiple therapeutic may be needed. Treatment options include anticonvulsants (eg, gabapentin, pregabalin), antidepressants (tricyclic antidepressants or serotoninnorepinephrine reuptake inhibitors), antiarrhythmics (mexiletine), a-adrenergic agonists (clonidine), topical agents (lidocaine or capsaicin), and analgesics (NSAIDs and opioids). Of note, tricyclic antidepressants may have significant anticholinergic side effects that may limit their tolerability. Secondary amines, such as nortriptyline or desipramine, may have fewer or less severe anticholinergic side effects than tertiary amines such as amitriptyline or imipramine. Sympathetic blocks are effective in selected disorders (see later discussion). Spinal cord stimulation may be effective for patients who do not tolerate or respond to other treatments. Spinal opioids may be effective and appropriate for selected patients.

® Diabetic neuropathy is the most common type of neuropathic pain and is a major cause of morbidity and disability. Its pathophysiology may be related to microangiopathy and to abnormal activation of glycation of proteins as a consequence of chronic hyperglycemia. Diabetic neuropathy may be symmetric (generalized), focal, or multifocal, affecting peripheral (sensory or motor), cranial, or autonomic nerves.
The most common diabetic neuropathy syndrome is peripheral polyneuropathy, which results in symmetric numbness ("stocking-and-glove" distribution), paresthesias, dysesthesias, and pain. The pain varies in intensity, may be severe, and is often worst at night. Loss of proprioception may lead to gait disturbances, and sensory deficits can lead to repetitive traumatic injuries. Isolated mononeuropathies affecting individual nerves may lead to wrist or foot drop or to cranial nerve palsy. Mononeuropathies typically have a sudden onset and are self-limiting, lasting a few weeks. Autonomic neuropathy typically affects the gastrointestinal tract, causing diarrhea, delayed gastric emptying, and esophageal motility disorders. Orthostatic hypotension and other forms of autonomic dysfunction are common.
Treatment of diabetic neuropathy is symptomatic and directed at optimal glycemic control to slow progression. Acetaminophen and NSAIDs are usually ineffective for moderate to severe pain. Risks associated with opioids limit their use. Adjuvant drugs play a major role. The combination of an antiepileptic drug and a tricyclic antidepressant may be particularly effective.

During an initial childhood chickenpox infection, the varicella-zoster virus

Headache is a common complaint that affects nearly everyone at some time in life. In the vast majority of cases, headaches are not of sufficient severity or frequency for the individual to seek medical attention and do not reflect a serious underlying disorder. However, as with other complaints of pain, the possibility of a Clinically significant underlying disorder should always be considered. The practitioner should solicit other associated symptoms or clinical findings that suggest serious underlying pathology. Table 47—10 lists important causes of headache. Disorders in which the primary complaint is headache are considered in the following discussion. As will become apparent, there is significant variability in clinical presentation and overlap in the symptoms of the major headache syndromes, particularly between tension and migraine headaches.

Classic headache syndromes
Migraine
Tension
Cluster
Vascular disorders
Temporal arteritis
Stroke
Venous thrombosis
Trigeminal
Glossopharyngeal
Occipital
Intracranial pathology
Tumor
Cerebrospinal fluid leak
Pseudomotor cerebri
Aneurysm
Eye disorders
Glaucoma
Optic neuritis
Sinus disease
Allergic
Bacterial
Temporomandibular joint disease
Dental disorders
Acute ingestion
Withdrawal (eg, caffeine and alcohol)
Systemic disorders
Viral (eg, influenza)
Bacterial
Fungal
Metabolic
Hypoxemia
Hypercarbia
Trauma
Cold stimulus (swallowing cold liquid)

Tension headaches are classically described as tight, bandlike pain or discomfort that is often associated with neck muscle tightness. The headache may be frontal, temporal, or occipital, and is more often bilateral than unilateral. Intensity typically builds gradually and fluctuates, lasting hours to days. They may be associated with emotional stress or depression. Treatment is symptomatic and most commonly consists of NSAIDs or acetaminophen, or both, with caffeine often combined with both of these agents in a single medication.

Migraine headaches are typically described as throbbing or pounding and are often associated with photophobia, scotoma, nausea and vomiting, and localized transient neurological dysfunction (aura). The latter may be sensory, motor, visual, or olfactory. Classic migraines by definition are preceded by an aura, whereas common migraines are not. The pain lasts 4 to 72 h and is usually unilateral, but can be bilateral with a frontotemporal location. Migraines primarily affect children (both sexes equally) and young adults (predominantly females). A family history is often present. Provocation by odors, certain foods (eg, red wine), menses, and sleep deprivation is common. Sleep characteristically relieves the headache. The pathophysiology is complex and may include vasomotor, autonomic (serotonergic brainstem systems), and trigeminal nucleus dysfunction. Treatment is both abortive and prophylactic. Rapid abortive treatment includes oxygen, sumatriptan (6 mg subcutaneously), dihydroergotamine (1 mg intramuscularly or subcutaneously), intravenous lidocaine (100 mg), nasal butorphanol (1-2 mg), and sphenopalatine ganglion block. Other abortive options include zolmitriptan nasal spray, dihydroergotamine nasal spray, or an oral serotonin 5-HTjp,;p-receptor agonist (almotriptan, frovatriptan, naratriptan, rizatriptan, eletriptan, or sumatriptan). Prophylactic treatment may include B-adrenergic blockers, calcium channel blockers, valproic acid, amitriptyline, and onabotulinumtoxinA (Botox) injections.

Cluster headaches are classically unilateral and periorbital, occurring in clusters of one to three attacks a day over a 4to 8-week period. The pain is described as a burning or drilling sensation that may awaken the patient from sleep. Each episode lasts 30 to 120 min. Remissions lasting a year at a time are common. Red eye, tearing, nasal stuffiness, ptosis, and Horner syndrome are classic findings. The headaches are typically episodic but can become chronic without remissions. Cluster headaches primarily affect males (90%). Abortive treatments include oxygen and sphenopalatine block. Lithium, a short course of steroid medication, and verapamil may be used for prophylaxis.

Temporal arteritis is an inflammatory disorder of extracranial arteries. The headache can be bilateral or unilateral and is located in the temporal area in at least 50% of patients. The pain develops over a few hours, is usually dull in quality but may be lancinating at times, and is often worse at night and in cold weather. Scalp tenderness is usually present. Temporal arteritis is often accompanied by polymyalgia rheumatica, fever, and weight loss. It is a relatively common disorder of older patients (>55 years), with an incidence of about 1 in 10,000 per year and a slight female predominance. Early diagnosis and treatment with steroids is important because progression can lead to blindness through involvement of the ophthalmic artery.

® Trigeminal neuralgia (or tic douloureux) is classically unilateral and usually located in the V2 or V3 distribution of the trigeminal nerve. It has an electric shock quality, with episodes lasting from seconds to minutes at a time and is often provoked by contact with a discrete trigger. Facial muscle spasm may be present. Patients are middle-aged and elderly, with a 2:1 female-to-male ratio. Common causes of trigeminal neuralgia include compression of the nerve by the superior cerebellar artery as it exits the brainstem, multiple sclerosis, and cerebellopontine angle tumor. The drug of choice for treatment is carbamazepine, although it carries a risk of agranulocytosis. Phenytoin or baclofen may be added, particularly if patients do not tolerate the required doses of carbamazepine. More invasive treatments for patients who do not respond to drug therapy include glycerol injection, radiofrequency ablation, balloon compression of the gasserian ganglion, and microvascular decompression of the trigeminal nerve.

Chronic abdominal pain has a variety of causes, and it is useful to distinguish between somatosensory, visceral, and centralized pain origins. A differential epidural block may help in elucidating the primary source but is time consuming and may be difficult to interpret. A transversus abdominis plane (TAP) blockade with ultrasound guidance may help treat somatosensory pain and thus may be potentially both diagnostic and therapeutic (see Chapter 46). The patient with pain of visceral origin may benefit from a celiac or splanchnic block.

Cancer-related pain may be due to the primary cancer lesion itself, metastatic disease, complications such as neural compression or infection, or treatment such as surgery, chemotherapy, or radiation therapy. In addition, the cancer patient may have acute or chronic pain that is entirely unrelated to the cancer. The pain manager must therefore have a thorough understanding of the nature of the cancer, its stage, the presence of metastatic disease, and treatments. Cancer pain can be managed with oral analgesics in most patients. The World Health Organization recommends a progressive, three-step approach: (1) nonopioid analgesics such as aspirin, acetaminophen, or an NSAID for mild pain, (2) "weak" oral opioids such as codeine or oxycodone for moderate pain, and (3) stronger opioids such as morphine or hydromorphone for severe pain (Tables 47-11 and 47-12). Parenteral therapy is necessary when patients have refractory pain, cannot take medication orally, or have poor enteral absorption. Regardless of the agent selected, in most instances drug therapy should be provided on a fixed time schedule rather than as needed. Adjuvant drug therapy, including antidepressants and anticonvulsants, may be used to both increase analgesia quality and decrease opioid-related side effects (Tables 47—13 and 47— 14). Intrathecal drug delivery systems may improve analgesia and, via a drugsparing effect, also help decrease side effects associated with oral or intravenous agents. Numerous intrathecal agents have been studied, and opioids have been utilized both alone and in combination with other medications. Ziconotide is a direct-acting N-type calcium-channel blocker that may be helpful for refractory pain or as a first-line agent. It acts by decreasing the release of substance P from the presynaptic nerve terminal in the dorsal horn of the spinal cord. Dosedependent side effects include auditory hallucinations and worsening of preexisting depression or psychosis. It does not lead to significant withdrawal symptoms if abruptly discontinued.

. COX-2 Inhibitors, Dosing Interval (h) = . COX-2 Inhibitors, Maximum Daily Dosage (mg) = . Celecoxib (Celebrex), Onset (h) = 3. Celecoxib (Celebrex), Dose (mg) = 100-200. Celecoxib (Celebrex), Dosing Interval (h) = 12. Celecoxib (Celebrex), Maximum Daily Dosage (mg) = 400
TABLE 47-12 Oral opioids.

05-1. Morphine solution‘ (Roxanol), 2 = 1. Morphine solution‘ (Roxanol), 3 = 10. Morphine solution‘ (Roxanol), 4 = 3-4. Morphine sustained-release* (MS Contin, Oramorph SR), 1 = 1. Morphine sustained-release* (MS Contin, Oramorph SR), 2 = 1. Morphine sustained-release* (MS Contin, Oramorph SR), 3 = 15. Morphine sustained-release* (MS Contin, Oramorph SR), 4 = 8-12. (Kadian), 1 = 1. (Kadian), 2 = 1. (Kadian), 3 = 10-20. (Kadian), 4 = 12-24. (Avinza), 1 = 1. (Avinza), 2 = 1. (Avinza), 3 = 30. (Avinza), 4 = 24
'Preparations also contain acetaminophen (Hycet, Lorcet, Lortab, Norco, Vicodin, others).
?Preparations may contain acetaminophen (Percocet) or aspirin (Percodan).
'Some preparations contain acetaminophen (Darvocet).
"Used primarily for cancer pain.

750-1250. Valproic acid (Depakene), Therapeutic Level’ (meg/mL) = 50-100
'Efficacy in pain management may not correlate with blood level.
Surgery, radiation therapy, chemotherapy, and comprehensive palliative care may prolong survivorship for patients with cancer. However, survivorship may be accompanied by therapy-related acute or chronic pain, including radiation fibrosis or chemotherapy-induced peripheral neuropathy, or both.

Interventional pain therapy options include pharmacological treatment, nerve blocks with local anesthetics and steroid or a neurolytic solution, radiofrequency ablation, neuromodulatory techniques, or multidisciplinary treatment (psychological interventions, physical or occupational therapy, or modalities such as acupuncture).

Pharmacological interventions in pain management include acetaminophen, cyclooxygenase (COX) inhibitors, antidepressants, neuroleptic agents, anticonvulsants, corticosteroids, systemic administration of local anesthetics, and opioids.

Acetaminophen (paracetamol) is an oral analgesic and antipyretic agent that is also available as an intravenous preparation (Ofirmev). It inhibits prostaglandin synthesis but lacks significant antiinflammatory activity. Acetaminophen has few side effects but is hepatotoxic at high doses. The recommended adult maximum daily limit is 3000 mg/d, reduced from a previously recommended limit of 4000 mg/d. Isoniazid, zidovudine, and barbiturates may potentiate acetaminophen toxicity.

Nonopioid oral analgesics include salicylates, acetaminophen, and NSAIDs (see Table 47-11). NSAIDs inhibit prostaglandin synthesis by inhibiting COX activity. Prostaglandins sensitize and amplify nociceptive input, and blockade of their synthesis results in the analgesic, antipyretic, and antiinflammatory properties characteristic of NSAIDs. At least two types of COX are recognized. COX-1 is found throughout body tissues, with functions including promotion of gastric mucus production, which protects the stomach lining, and stimulation of thromboxane A, production in platelets, which induces platelet aggregation. COX-2 is induced primarily with inflammation. COX inhibitors likely have important peripheral and central nervous system actions. Their analgesic action is limited by side effects and toxicity at higher doses, especially peptic ulceration and kidney failure. Selective COX-2 inhibitors, such as celecoxib, have a lower risk of peptic ulceration but are associated with an increased risk of thrombotic events, including myocardial infarction. The COX-2 inhibitor rofecoxib (Vioxx) has been taken off the market in the United States for this reason. Some types of pain, particularly pain that follows orthopedic and gynecological surgery, respond very well to COX inhibitors.
All of the nonopioid oral analgesic agents are well absorbed enterally. Food delays absorption but otherwise has no effect on bioavailability. Because most of these agents are highly protein bound (>80%), they can displace other highly bound drugs such as warfarin. All undergo hepatic metabolism and are excreted by the kidney. Dosages should therefore be reduced, or alternative medications selected, in patients with hepatic or kidney impairment.
The most common side effects of aspirin (acetylsalicylic acid, ASA) and other NSAIDs are stomach upset, heartburn, nausea, and ulceration of the gastric mucosa. Diclofenac is available as both an oral preparation and a topical gel or patch that may be less likely to contribute to gastric distress.

Other side effects of NSAIDs include dizziness, headache, and drowsiness. With the exception of selective COX-2 inhibitors, all COX inhibitors decrease platelet aggregation. Aspirin irreversibly inhibits platelet aggregation for 1 to 2 weeks, whereas the antiplatelet effect of other NSAIDs is reversible and lasts approximately five elimination half-lives (24—96 h). This inhibition of platelet aggregation does not appreciably increase the incidence of postoperative hemorrhage following most outpatient procedures. NSAIDs may exacerbate bronchospasm in patients with the triad of nasal polyps, rhinitis, and asthma. ASA should not be used in children with varicella or influenza infections because it may precipitate Reye syndrome. NSAIDs may cause acute kidney injury, particularly in patients with underlying kidney dysfunction.

® Antidepressants are most useful for patients with neuropathic pain and demonstrate an analgesic effect that occurs at a dose lower than that needed for antidepressant activity. Both of these actions are due to blockade of presynaptic reuptake of serotonin, norepinephrine, or both. Older tricyclic agents appear to be more effective analgesics than selective serotonin reuptake inhibitors (SSRIs). Serotonin and norepinephrine reuptake inhibitors (SNRIs) may provide the most favorable balance between analgesic efficacy and side effects. Antidepressants potentiate the action of opioids and frequently help normalize sleep patterns.
All antidepressant medications undergo extensive first-pass hepatic metabolism and are highly protein bound. Most are highly lipophilic and have large volumes of distribution. Elimination half-lives of most of these medications vary between 1 and 4 days, and many have active metabolites. Available agents differ in their side effects (see Table 47-13), which include antimuscarinic effects (dry mouth, impaired visual accommodation, urinary retention, and constipation), antihistaminic effects (sedation and increased gastric pH), a-adrenergic blockade (orthostatic hypotension), and a quinidinelike effect (atrioventricular block, QT prolongation, torsades de pointes).

The SNRIs milnacipran (Ixel, Savella, Dalcipran, Toledomin) and duloxetine (Cymbalta) have been approved in the United States for the treatment of fibromyalgia. Milnacipran has an elimination half-life of 8 h, is minimally metabolized by the liver, and is primarily excreted unchanged in the urine. Duloxetine (Cymbalta) is also useful in the treatment of neuropathic pain and depression. It has a half-life of 12 h and is metabolized by the liver, and most of its metabolites are excreted in the urine.
Absolute and relative contraindications for the use of SNRIs include known hypersensitivity, usage of other drugs that act on the central nervous system (including monoamine oxidase inhibitors), hepatic and kidney impairment, uncontrolled narrow-angle glaucoma, and suicidal ideation. Common side effects include nausea, headache, dizziness, constipation, insomnia, hyperhydrosis, hot flashes, vomiting, palpitations, dry mouth, and hypertension.

Neuroleptic medications may occasionally be useful for patients with refractory neuropathic pain, and may be most helpful in patients with marked agitation or psychotic symptoms. The most commonly used agents are fluphenazine (Prolixin), haloperidol (Haldol), chlorpromazine (Thorazine, Largactil), and perphenazine (Trilafon). Their therapeutic action appears to be due to blockade of dopaminergic receptors in mesolimbic sites. Unfortunately, the same action in nigrostriatal pathways can produce undesirable extrapyramidal side effects, such as masklike facies, a festinating gait, cogwheel rigidity, and bradykinesia. Some patients also develop acute dystonic reactions such as oculogyric crisis and torticollis. Long-term side effects include akathisia (extreme restlessness) and tardive dyskinesia (involuntary choreoathetoid movements of the tongue, lip smacking, and truncal instability). Like antidepressants, many of these drugs also have antihistaminic, antimuscarinic, and a-adrenergic—blocking effects.
Antispasmodics & Muscle Relaxants

Glucocorticoids are extensively used in pain management for their antiinflammatory and possibly analgesic actions. They may be given topically, orally, or parenterally (intravenously, subcutaneously, intrabursally, intraarticularly, or epidurally). 'Table 47—15 lists the most commonly used agents, which differ in potency, relative glucocorticoid and mineralocorticoid activities, and duration of action. Large doses or prolonged administration result in significant side effects. Excess glucocorticoid activity can produce hypertension, hyperglycemia, increased susceptibility to infection, peptic ulcers, osteoporosis, aseptic necrosis of the femoral head, proximal myopathy, cataracts, and, rarely, psychosis. Patients with diabetes may have elevated blood glucose levels after corticosteroid injections. Patients can also develop the physical features characteristic of Cushing syndrome. Excess mineralocorticoid activity causes sodium retention and hypokalemia, and may precipitate congestive heart failure.
TABLE 47-15 Selected corticosteroids.!

Glucocorticoid Activity = 25. Betamethasone (Celestone), Mineralocorticoid Activity = 0. Betamethasone (Celestone), Equivalent Dose(mg) = 0.75. Betamethasone (Celestone), Half-Life (h) = 36-72. Dexamethasone (Decadron), RoutesGiven? = OLT. Dexamethasone (Decadron), Glucocorticoid Activity = 25. Dexamethasone (Decadron), Mineralocorticoid Activity = 0. Dexamethasone (Decadron), Equivalent Dose(mg) = 0.75. Dexamethasone (Decadron), Half-Life (h) = 36-72
'Data from Goodman LS, Gilman AG. The Pharmacologic Basis of Therapeutics. 8th ed. New York, NY: Pergamon, 1990.
20, oral; |, injectable; T, topical,
Many corticosteroid preparations are suspensions, rather than solutions, and the relative particulate size of a given glucocorticoid suspension may affect the risk of neural damage due to arterial occlusion when accidental arterial injection occurs. Because of the relatively small size of its suspension particles, dexamethasone is becoming the preferred corticosteroid for injection procedures involving relatively vascular areas, such as the head and neck region.

@ Anticonvulsant medications are useful for patients with neuropathic pain, especially trigeminal neuralgia and diabetic neuropathy. These agents can suppress the spontaneous neural discharges that play a major role in these disorders. The most commonly utilized agents are phenytoin (Dilantin), carbamazepine (Tegretol), valproic acid (Depakene, Stavzor), clonazepam (Klonopin), and gabapentin (Neurontin) (see Table 47-14). Pregabalin (Lyrica) has been approved for the treatment of diabetic peripheral neuropathy and fibromyalgia, but is widely prescribed for all forms of neuropathic pain. Lamotrigine (Lamictal) and topiramate (Topamax) may also be effective. All are highly protein-bound and have relatively long half-lives. Carbamazepine (Carbatrol, Equetro, Tegretol) has a slow and unpredictable absorption, which requires monitoring of blood levels for optimal efficacy. Phenytoin may be effective, but is associated with the side effect of gum hyperplasia. Levetiracetam (Keppra) and oxcarbazepine (Trileptal) have been used as adjuvant pain therapies. Gabapentin and pregabalin may also be effective adjuvants for the treatment of acute postoperative pain.

Systemic infusion of local anesthetic medication produces sedation and central analgesia and is occasionally used in the treatment of patients with neuropathic pain. The resultant analgesia may outlast the pharmacokinetic profile of the local anesthetic and break the so-called pain cycle. Lidocaine and procaine are the most commonly used agents. They are given either as a slow bolus or by continuous infusion. Lidocaine is given by infusion over 5 to 30 min for a total of 1 to 5 mg/kg. Procaine, 200 to 400 mg, can be given intravenously over the course of 1 to 2 h. Monitoring by qualified medical personnel should include electrocardiography, blood pressure, respiration, pulse oximetry, and mental status, and full resuscitation equipment must be immediately available. Signs of toxicity, such as tinnitus, slurring of speech, excessive sedation, or nystagmus, necessitate slowing or discontinuing the infusion to avoid the progression to seizures. Patients who do not respond satisfactorily to anticonvulsants but respond to intravenous local anesthetics may benefit from chronic oral antiarrhythmic therapy. Mexiletine (Mexitil, 150-300 mg every 6-8 h) is a class 1B antiarrhythmic that is commonly used and generally well tolerated. Topical lidocaine preparations, in concentrations up to 5%, may be helpful in the treatment of some neuropathic pain conditions. A 5% lidocaine transdermal patch (Lidoderm) containing 700 mg of lidocaine has been approved for the treatment of PHN. One to three patches may be applied to dry, intact skin, alternating 12 h on, then 12 h off.

The primary effect of a-adrenergic agonists is activation of descending inhibitory pathways in the spinal cord dorsal horn. Epidural and intrathecal a>adrenergic agonists are particularly effective in the treatment of neuropathic pain and opioid tolerance. Clonidine (Catapres), a direct-acting a-adrenergic agonist, is effective as an adjunctive medication in the treatment of severe pain. The oral dosage is 0.1 to 0.3 mg twice daily; a transdermal patch (0.1—0.3 mg/d) is also available and is usually applied for 7 d. When used in combination with a local anesthetic or opioid in an epidural or intrathecal infusion, clonidine may contribute to a synergistic or prolonged analgesic effect, or both, especially for neuropathic pain.

The most commonly prescribed oral opioid agents are codeine, oxycodone, and hydrocodone. They are easily absorbed, but hepatic first-pass metabolism limits systemic delivery. Like other opioids, they undergo hepatic biotransformation and conjugation before renal elimination. Codeine is transformed by the liver into morphine. The side effects of orally administered opioids are similar to those of systemic opioids. When prescribed on a fixed schedule, stool softeners or laxatives are often indicated. Tramadol (Rybix, Ryzolt, Ultram) is a synthetic oral opioid that also blocks neuronal reuptake of norepinephrine and serotonin. It appears to have the same efficacy as the combination of codeine and acetaminophen but is associated with significantly less respiratory depression and has little effect on gastric emptying.
Moderate to severe cancer pain is usually treated with an immediate-release oral morphine preparation (eg, liquid morphine, Roxanol, 10-30 mg every 1-4 h). These preparations have an effective half-life of 2 to 4h. Once the patient's daily requirements are determined, the same dose can be given in the form of a sustained-release morphine preparation (MS Contin or Oramorph SR), which is dosed every 8 to 12 h. The immediate-release preparation is then used only as needed for breakthrough pain. Oral transmucosal fentanyl lozenges (Actiq, 200— 1600 mcg) can also be used for breakthrough pain but are very expensive. Excessive sedation can be treated with dextroamphetamine (Dexedrine, ProCentra) or methylphenidate (Ritalin), 5 mg in the morning and 5 mg the early afternoon. Most patients require a stool softener. Nausea may be treated with transdermal scopolamine, oral meclizine, or metoclopramide. Hydromorphone (Dilaudid) is an excellent alternative to morphine, particularly in elderly patients because of fewer side effects, and in patients with impaired kidney function. Methadone (Dolophine) has a half-life of 15 to 30 h, but clinical duration is shorter and quite variable (usually 6-8 h).

® Patients who experience opioid tolerance require escalating doses of opioid to maintain the same analgesic effect. Physical dependence manifests in opioid withdrawal when the opioid medication is either abruptly discontinued or the dose is abruptly and significantly decreased. Psychological dependence, characterized by behavioral changes focusing on drug craving, is rare in cancer patients. The development of opioid tolerance is highly variable but results in some desirable effects such as decreased opioid-related sedation, nausea, and respiratory depression. Unfortunately, many patients continue to suffer from constipation. Physical dependence occurs in all patients receiving large doses of opioids for extended periods. Opioid withdrawal phenomena can be precipitated by the administration of opioid antagonists. Concomitant use of peripheral opioid antagonists that do not cross the blood-brain barrier, such as methylnaltrexone (Relistor) and alvimopan (Entereg), may help reduce systemic opioid side effects without significantly affecting analgesia.
Tapentadol (Nucynta), a p!-opioid receptor agonist that also has norepinephrine reuptake inhibition properties, has been introduced for the management of acute and chronic pain. This opioid may be associated with less nausea and vomiting and less constipation. It should not be used concomitantly with monoamine oxidase inhibitors due to potentially elevated levels of norepinephrine.
Propoxyphene with and without acetaminophen (Darvocet and Darvon) was withdrawn from the U.S. market in 2010 due to the risk of cardiac toxicity.

Intravenous, intraspinal (epidural or intrathecal), or transdermal routes of opioid administration may be utilized when the patient fails to adequately respond to, or is unable to tolerate, oral regimens. However, when the patient's pain increases significantly, or changes markedly in quality, it is equally important to reevaluate the patient for adequacy of pain diagnosis and for the potential of disease progression. In patients with cancer, adjunctive treatments such as surgery, radiation, chemotherapy, hormonal therapy, and neurolysis may be helpful. Intramuscular opioid administration is rarely optimal because of variability in systemic absorption and resultant delay and variation in clinical effect.

Parenteral opioid therapy is usually best accomplished by intermittent or continuous intravenous infusion, or both, but can also be given subcutaneously. Modern portable infusion devices have patient-controlled analgesia (PCA) capability, allowing the patient to self-treat for breakthrough pain.

The use of intraspinal opioids is an excellent alternative for patients obtaining poor relief with other analgesic techniques or who experience unacceptable side effects. Epidural and intrathecal opioids offer pain relief with substantially lower total doses of opioid and fewer side effects. When compared with intermittent boluses, continuous intraspinal infusion techniques reduce drug requirements, minimize side effects, and decrease the likelihood of catheter occlusion. Myoclonic activity may be occasionally observed with intrathecal morphine or hydromorphone.
Epidural or intrathecal catheters can be placed percutaneously or implanted to provide long-term effective pain relief. Epidural catheters can be attached to lightweight external pumps that can be worn by ambulatory patients. A temporary catheter must be inserted first to assess the potential efficacy of the technique. Correct placement of the permanent catheter should be confirmed using fluoroscopy with contrast dye. Completely implantable intrathecal catheters with externally programmable pumps can also be used for continuous infusion (Figure 47-6). The reservoir of the implanted pump (Figure 47-7) is periodically refilled percutaneously. Implantable systems are most appropriate for patients with a life expectancy of several months or longer, whereas tunneled epidural catheters are appropriate for patients expected to live only weeks. Formation of an inflammatory mass (granuloma) at the tip of the intrathecal catheter may occur and may reduce efficacy.
FIGURE 47-6 Placement of an implanted intrathecal drug delivery system. With the patient in right lateral decubitus position, access to the intrathecal space and to the anterior abdominal wall is optimized. After the posterior incision is made, a needle is advanced through the incision into the intrathecal space, and a catheter is advanced through the needle into the posterior intrathecal space. After the proximal catheter end is anchored, the distal end of the catheter is tunneled around the flank, beneath the costal margin to the anterolateral aspect of the abdominal wall.
FIGURE 47-7 Fluoroscopic image showing an intrathecal drug pump implanted in the anterolateral abdomen wall. The catheter connecting the pump to the intrathecal space is tunneled around the flank.

The most frequently encountered problem associated with intrathecal opioids is tolerance, which is usually, but not always, a slowly developing phenomenon. Adjuvant therapy may be used to address this problem, and may include intermittent local anesthetics or a mixture of opioids with local anesthetics (bupivacaine or ropivacaine 2—24 mg/d), clonidine (2-4 mcg/kg/h or 48-800 mceg/d, respectively), or the GABA agonist baclofen. Clonidine is particularly useful for neuropathic pain. In high doses, it is more likely to be associated with hypotension and bradycardia.
Complications of spinal opioid therapy include local skin infection, intrathecal pump pocket infection, meningitis, other infections, and death or permanent injury related to pump programming, pump refill or drug dilution errors. Superficial infections can be reduced by the use of a silver-impregnated cuff close to the exit site. Other complications of spinal opioid therapy include epidural hematoma, which may become clinically apparent either immediately following catheter placement or several days later, and respiratory depression. Respiratory depression secondary to spinal opioid overdose can be treated by decreasing the pump infusion rate to its lowest setting and initiating a naloxone intravenous infusion.

Transdermal fentanyl (Duragesic patch) is an alternative to sustained-release oral morphine and oxycodone preparations, particularly when oral medication is not possible. Currently available patches are constructed as a drug reservoir that is separated from the skin by a microporous rate-limiting membrane and an adhesive polymer. A very large quantity of fentanyl (10 mg) provides a large force for transdermal diffusion. Transdermal fentanyl patches are available in 25, 50, 75, and 100 mcg/h sizes that provide drug for 2 to 3 days. The largest patch is equivalent to 60 mg/d of intravenous morphine. The major obstacle to fentanyl absorption through the skin is the stratum corneum. Because the dermis acts as a secondary reservoir, fentanyl absorption continues for several hours after the patch is removed. The transdermal route avoids hepatic first-pass metabolism. Major disadvantages of the transdermal route are its slow rate of drug delivery onset and the inability to rapidly change dosage in response to changing opioid requirements. Blood fentanyl levels rise and reach a plateau in 12 to 40 h, providing average concentrations of 1, 1.5, and 2 ng/mL for the 50, 75, and 100 mcg/h patches, respectively. Large interpatient variability results in actual delivery rates ranging from 50 to 200 mcg/h. Transdermal fentanyl patches are often diverted for illicit use, resulting in numerous accidental fatalities.

Opioid medications have been identified as having greater risks to patients than were previously considered, including dependence, addiction, overdose, endocrine changes, immunological compromise, hyperalgesia, and respiratory depression that may be life threatening. The efficacy and appropriateness of opioid treatment of chronic benign pain has increasingly come into question as well. For these reasons, risks may outweigh potential or real benefits. In 2016 the U.S. Centers for Disease Control and Prevention published guidelines for prescribing opioids for chronic pain to help mitigate risks (see Guidelines at end of chapter).

OnabotulinumtoxinA (Botox) injection has been increasingly utilized in the treatment of pain syndromes. Studies support its use in the treatment of conditions associated with involuntary muscle contraction (eg, focal dystonia and spasticity), and it is approved by the U.S. FDA for prophylactic treatment of chronic migraine headache. This toxin blocks acetylcholine released at the synapse in motor nerve endings but not sensory nerve fibers. Proposed mechanisms of analgesia include improved local blood flow, relief of muscle spasms, and release of muscular compression of nerve fibers

Local anesthetic nerve blocks are useful in delineating pain mechanisms, and they play a major role in the management of patients with acute or chronic pain. Pain relief following diagnostic nerve blockade often carries favorable prognostic implications for a subsequent therapeutic series of blocks. This technique can identify patients exhibiting a placebo response and those with psychogenic mechanisms. In selected patients, "permanent" neurolytic nerve blocks may be appropriate.
The efficacy of nerve blocks is due to interruption of afferent nociceptive activity, which may be in addition to, or in combination with, blockade of afferent and efferent limbs of abnormal reflex activity involving sympathetic nerve fibers and skeletal muscle innervation. The pain relief frequently outlasts the known pharmacological duration of the agent employed by hours or up to several weeks. Selection of the type of block depends on the location of pain, its presumed mechanism, and the skills of the treating physician. Local anesthetic solutions may be infiltrated locally or injected at specific peripheral nerve, somatic plexus, sympathetic ganglia, or nerve root sites. The local anesthetic may also be applied centrally in the neuraxis.

The use of ultrasound in interventional pain medicine has increased dramatically over the past decade due to its utility in precisely visualizing vascular, neural, and other anatomic structures, its role as an alternative to the use of fluoroscopy and radiocontrast agents, and progressive improvements in technology leading to better visual images and greater simplicity of use. Procedures that may benefit from ultrasound guidance include trigger point injections, nerve blocks, and joint injections.

Fluoroscopy is highly effective for visualizing bony structures and observing the spread of radiopaque contrast agents. Live fluoroscopy with contrast agent may be used to minimize the risk of intravascular injection of therapeutic agents. Care should be taken to avoid excessive radiation dosage and to employ appropriate radiation shielding, given the risks of ionizing radiation to the patient and to the health care team in the fluoroscopy suite.

The two principal indications for trigeminal nerve block are trigeminal neuralgia and intractable facial cancer pain. Depending on the site of pain, these blocks may be performed on the gasserian ganglion itself, on one of the major divisions (ophthalmic, maxillary, or mandibular), or on one of their smaller branches.

The rootlets of cranial nerve V arise from the brainstem and join one another to form a crescent-shaped sensory (gasserian) ganglion in Meckel's cave. Most of the ganglion is invested with a dural sleeve. The three subdivisions of the trigeminal nerve arise from the ganglia and exit the cranium separately (Figure 47-8A).

F: Lingual and inferior alveolar nerve block
E: Mandibular nerve block

1. Gasserian ganglion block—Fluoroscopic guidance is mandatory for the performance of this procedure (Figure 47—8B). An 8to 10-cm 22-gauge needle is inserted approximately 3 cm lateral to the angle of the mouth at the level of the upper second molar. The needle is then advanced posteromedially and angled superiorly to bring it into alignment with the pupil in the anterior plane and with the mid-zygomatic arch in the lateral plane. Without entering the mouth, the needle should pass between the mandibular ramus and the maxilla, and lateral to the pterygoid process to enter the cranium through the foramen ovale. After a negative aspiration for cerebrospinal fluid and blood, local anesthetic is injected. 2. Blocks of the ophthalmic nerve and its branches—In this procedure, to avoid denervation-related keratitis, only the supraorbital branch is blocked in most cases (Figure 47—8C); the ophthalmic division itself is not blocked. The nerve is easily located and blocked with local anesthetic at the supraorbital notch, which is located on the supraorbital ridge above the pupil. The supratrochlear branch can also be blocked with local anesthetic at the superior medial corner of the orbital ridge.
the mouth. Local anesthetic is injected once paresthesias are elicited or the needle is felt to enter the foramen.

Complications of a gasserian ganglion block include accidental intravascular injection, subarachnoid injection, Horner syndrome, and motor block of the muscles of mastication. The potential for serious hemorrhage is greatest for blockade of the maxillary nerve. The facial nerve may be unintentionally blocked during blocks of the mandibular division.

Blockade of the facial nerve is occasionally indicated to relieve spastic contraction of the facial muscles, to treat herpes zoster involving the facial nerve, and to facilitate certain surgical procedures involving the eye.

The facial nerve can be blocked where it exits the cranium through the stylomastoid foramen. A small sensory component supplies special sensation (taste) to the anterior two thirds of the tongue and general sensation to the tympanic membrane, external auditory meatus, soft palate, and part of the pharynx.

The entry point is just anterior to the mastoid process, beneath the external auditory meatus, and at the midpoint of the mandibular ramus. The nerve is approximately 1 to 2 cm deep and is blocked with local anesthetic just below the stylomastoid process.

If the needle is inserted too deeply past the level of the styloid bone, the glossopharyngeal and vagal nerves may also be blocked. Careful aspiration is necessary because of the proximity of the facial nerve to the carotid artery and the internal jugular vein.

Glossopharyngeal nerve block may be used for patients with pain due to cancer involving the base of the tongue, the epiglottis, or the palatine tonsils. It can also be used to distinguish glossopharyngeal neuralgia from trigeminal and geniculate neuralgia.

The nerve exits from the cranium via the jugular foramen medial to the styloid process and courses anteromedially to supply the posterior third of the tongue, pharyngeal muscles, and mucosa. The vagus and spinal accessory nerves also exit the cranium via the jugular foramen and descend alongside the glossopharyngeal nerve in close proximity to the internal jugular vein.

The block is performed using a 5-cm 22-gauge needle inserted just posterior to the angle of the mandible (Figure 47—9). The nerve is approximately 3 to 4 cm deep; therefore, use of a nerve stimulator facilitates correct placement of the needle. An alternative approach is from a point over the styloid process, midway between the mastoid process and the angle of the mandible; the nerve is located just anteriorly.
FIGURE 47-9 Glossopharyngeal nerve block.

Complications include dysphagia and vagal blockade resulting in ipsilateral vocal cord paralysis and tachycardia. Block of the accessory nerve and hypoglossal nerves causes ipsilateral paralysis of the trapezius muscle and the tongue, respectively. Careful aspiration is necessary to prevent intravascular injection.

Occipital nerve block is useful diagnostically and therapeutically in patients with occipital headaches and neuralgias.

The greater occipital nerve is derived from the dorsal primary rami of the C2 and C3 spinal nerves, whereas the lesser occipital nerve arises from the ventral rami of the same roots.

The greater occipital nerve is blocked approximately 3 cm lateral to the occipital prominence at the level of the superior nuchal line (Figure 47—10); the nerve is just medial to the occipital artery, which is often palpable. The lesser occipital nerve is blocked 2 to 3 cm more laterally along the nuchal ridge. Ultrasound guidance may be employed to help identify the nerves. For patients who have responded well but temporarily to occipital nerve blocks, implantation of an occipital nerve stimulator may provide prolonged relief.

This block is useful for painful conditions arising from the shoulder, including arthritis, bursitis, and myofascial pain.

The suprascapular nerve is the major sensory nerve of the shoulder joint. It arises from the brachial plexus (C4—C6) and passes over the upper border of the scapula in the suprascapular notch to enter the suprascapular fossa.

The nerve is blocked at the suprascapular notch, which is located at the junction of the lateral and middle thirds of the superior scapular border (Figure 47-11). Correct placement of the needle is determined by paresthesia, ultrasound, or the use of a nerve stimulator.

Pneumothorax is possible if the needle is advanced too far anteriorly. Paralysis of the supraspinatus and infraspinatus muscles will result in impaired shoulder abduction.

Cervical paravertebral nerve blocks can be useful diagnostically and therapeutically for patients with cervical disc displacement, cervical foraminal stenosis, or cancer-related pain originating from the cervical spine or shoulder.

The cervical spinal nerves lie in the sulcus of the transverse process of their respective vertebral levels. As noted earlier in this chapter, unlike thoracic and lumbar nerve roots, those in the cervical spine exit the foramina above the vertebral bodies for which they are named.

The lateral approach is most commonly used to block C2—C7 (Figure 47-12). Patients are asked to turn the head to the opposite side while in a sitting or supine position. A line is then drawn between the mastoid process and the tubercle of the C6 transverse process (Chassaignac's tubercle). A series of injections are made with a 5-cm 22-gauge needle along a second parallel line 0.5 cm posterior to the first line. In the case of diagnostic blocks, a smaller injectate volume may be helpful in order to minimize local anesthetic spread to adjacent structures and thereby increase block specificity. Because the transverse process of C2 is usually difficult to palpate, the injection for this level is placed 1.5 cm beneath the mastoid process. The other transverse processes are usually interspaced 1.5 cm apart and are 2.5 to 3 cm deep. Fluoroscopy is useful in identifying specific vertebral levels during diagnostic blocks. This block may also be performed with ultrasound guidance.

Unintentional intrathecal or epidural anesthesia at this level rapidly causes respiratory paralysis and hypotension. Injection of even small volumes of local anesthetic into the vertebral artery causes unconsciousness and seizures. Other complications include Horner syndrome and blockade of the recurrent laryngeal and phrenic nerves.
Embolic cerebrovascular and spinal cord complications have resulted from injection of particulate steroid with this block. Particulate steroid should not be used with cervical paravertebral nerve blocks because of possible anomalous vertebral artery anatomy in this region.

This technique may be used to block the upper thoracic dermatome segments because the scapula interferes with the intercostal technique at these levels. Unlike an intercostal nerve block, a thoracic paravertebral nerve block anesthetizes both the dorsal and ventral rami of spinal nerves. It is therefore useful in patients with pain originating from the thoracic spine, thoracic cage, or abdominal wall, including compression fractures, proximal rib fractures, and acute herpes zoster. This block is also frequently utilized for intraoperative anesthesia and for postoperative pain management in breast surgery and is described in detail in Chapter 46.

Lumbar paravertebral nerve blocks may be useful in evaluating pain due to disorders involving the lumbar spine or spinal nerves.

The lumbar spinal nerves enter the psoas compartment after exiting the intervertebral foramina beneath the pedicles and transverse processes. This compartment is formed by the psoas fascia anteriorly, the quadratus lumborum fascia posteriorly, and the vertebral bodies medially.

The approach to lumbar spinal nerves is essentially the same as for thoracic paravertebral blockade (Figure 47-13). An 8-cm 22-gauge needle is usually used, and radiographic confirmation of the correct level is helpful. For diagnostic blocks, only 2 mL of local anesthetic is injected at any one level, because larger volumes may block more than one level. Larger volumes of local anesthetic are used for therapeutic blocks, or to produce complete somatic and sympathetic block of the lumbar nerves.

Complications are primarily those of unintentional intrathecal or epidural anesthesia. Patients may experience paresthesias if inadvertent nerve injury occurs during needle placement. Some physicians advocate the use of a blunttipped needle to (theoretically) decrease the risk of accidental intraneural injection. Digital subtraction angiography with radiopaque contrast may lessen the risk of intravascular injection of local anesthetic or steroid medication.

These blocks may be utilized in patients with back pain to assess the contribution of lumbar facet (zygapophyseal) joint disease. Corticosteroids are commonly injected with the local anesthetic when the intraarticular technique is chosen. The cervical, thoracic, or lumbar facet joints may be injected for diagnostic and potentially therapeutic purposes.

Each facet joint is innervated by the medial branches of the posterior primary division of the spinal nerves above and below the joint (Figure 47-14). Thus, every joint is supplied by two or more adjacent spinal nerves. Each medial branch crosses the upper border of the lower transverse process running in a groove between the root of the transverse process and the superior articular process.
FIGURE 47-14 Lumbar medial branch nerve and facet blocks. A: Posterior view; B: 30° oblique posterior view.

These blocks are performed under fluoroscopic guidance with the patient in a prone position, or in some cases, the lateral position for cervical procedures. A posterior—anterior view facilitates visualization of the spine for lumbar medial branch blocks. A 10-cm 22-gauge needle is inserted 3 to 4 cm lateral to the spinous process at the desired level and directed anteriorly toward the junction of the transverse process and the superior articular process to block the medial branch of the posterior division of the spinal nerve (Figures 47—15 through 47— 17).
FIGURE 47-15 Anatomy of the lumbar facet joint and location for blocking the medial branch of the posterior primary division of the lumbar spinal nerves above and below the joint.
FIGURE 47-16 Fluoroscopic image of a cervical medial branch blockade. A: Anteroposterior view; B: Lateral view. The lateral view reveals the needles at C4, C5, and C6 advanced toward the trapezoid of the articular pillar at each level. Note the "waist" of the vertebrae. Spinal needles may be advanced to come into contact with the medial branch of the nerve.

Alternatively, local anesthetic with or without corticosteroid may be directly injected into the facet joint. Positioning the patient prone and using an oblique fluoroscopic view facilitates identification of the joint space. Correct placement of the needle may be confirmed by injecting radiopaque contrast prior to injection of local anesthetic. Total injection volumes should ideally be limited to less than 1 mL in order to prevent rupture of the joint capsule.

Injection into a dural sleeve results in a subarachnoid block, whereas injection near the spinal nerve root results in sensory and motor block at that level. Because the joint normally has a small volume, larger injections can cause rupture of the joint capsule.
If a patient achieves improved pain control after a diagnostic block, he or she may be considered for radiofrequency ablation of the medial branch. There is debate about whether a second, confirmatory diagnostic block should be performed prior to radiofrequency ablation. Injection of steroid may be considered before or after radiofrequency ablation to theoretically decrease the chance for postprocedural neuritis.

This technique is useful in the diagnosis and treatment of pelvic and perineal pain. In addition, blockade of the $1 spinal root can help define its role in back pain.

The five paired sacral spinal nerves and one pair of coccygeal nerves descend in the sacral canal, and each nerve then exits through its respective intervertebral foramen. The S5 and coccygeal nerves exit through the sacral hiatus.

While the patient is prone, the sacral foramina are identified with a needle along a line drawn 1.5 cm medial to the posterior superior iliac spine and 1.5 cm lateral to the ipsilateral sacral cornu (Figure 47—18). Correct positioning requires entry of the needle into the posterior sacral foramen and usually produces paresthesias. The S1 nerve root is usually 1.5 cm above the level of the posterior superior iliac spine along this imaginary line. Blockade of the S5 and coccygeal nerves can be accomplished by injection at the sacral hiatus.
FIGURE 47-18 Trans-sacral nerve block.

Complications are rare but include nerve damage and intravascular injection.
Sagittal

Pudendal nerve block is useful in evaluating patients with perineal somatosensory pain.

The pudendal nerve arises from S2—S4 and courses between the sacrospinous and the sacrotuberous ligaments to reach the perineum.

This block is usually performed transperineally with the patient in the lithotomy position (Figure 47—19) although it may be performed via a posterior approach in the prone position. Injection of anesthetic is carried out percutaneously just posterior to the ischial spine at the attachment of the sacrospinous ligament. The ischial spine can be palpated transrectally or transvaginally. Alternatively, this procedure may be performed in the prone position with a 22-gauge needle directed toward the base of the ischial spine. Patients should be advised that they may have numbness of the genitalia for hours after this procedure is performed.

Potential complications include unintentional sciatic blockade and intravascular injection.

Sympathetic blockade can be accomplished by a variety of techniques, including intrathecal, epidural, and paravertebral blocks. Unfortunately, these approaches usually block both somatic and sympathetic fibers. Problems with differential spinal and epidural techniques are discussed later in this section. The following techniques specifically block sympathetic fibers and can be used to define the role of the sympathetic system in a patient's pain and possibly also provide longterm pain relief. The most common indications for sympathetic nerve blocks include reflex sympathetic dystrophy, visceral pain, acute herpetic neuralgia, postherpetic pain, and peripheral vascular disease. Isolated sympathetic blockade to a region is characterized by loss of sympathetic tone, as evidenced by increased cutaneous blood flow and cutaneous temperature, and by unaltered somatic sensation. Other tests include loss of the skin conductance (sympathogalvanic reflex) and sweat response (ninhydrin, cobalt blue, or starch tests) following a painful stimulus.

This block is often used for patients with head, neck, arm, and upper chest pain. It is commonly referred to as a stellate block but usually blocks the upper thoracic as well as all cervical ganglia. Injection of larger volumes of anesthetic often extends the block to the T5 ganglia. Stellate blocks may also be used for vasospastic disorders of the upper extremity.

Sympathetic innervation of the head, neck, and most of the arm is derived from four cervical ganglia, the largest being the stellate ganglion. The latter usually represents a fusion of the lower cervical and first thoracic ganglia. Some sympathetic innervation of the arm (T1) as well as innervation of all of the thoracic viscera derives from the five upper thoracic ganglia. The sympathetic supply to the arm in some individuals may also originate from T2—T3 via anatomically distinct nerves (Kuntz's nerves) that join the brachial plexus high in the axilla. These nerves may be missed by a stellate block but not an axillary block. The point of injection is at the level of the stellate ganglion, which lies posterior to the origin of the vertebral artery from the subclavian artery, anterior to the longus colli muscle and the first rib, anterolateral to the prevertebral fascia, and medial to the scalene muscles. The sympathetic supply to the arm in some individuals may also originate from T2—T3 via anatomically distinct nerves (Kuntz's nerves) that join the brachial plexus high in the axilla. These nerves may be missed by a stellate block but not an axillary block.

The paratracheal technique is most commonly used (Figure 47—20), although an oblique or posterior approach may also be taken. With the patient's head extended, a 4to 5-cm 22-gauge needle is inserted at the medial edge of the sternocleidomastoid muscle just below the level of the cricoid cartilage at the level of the transverse process of C6 (Chassaignac's tubercle) or C7 (3-5 cm above the clavicle). The nonoperative hand should be used to retract the muscle together with the carotid sheath prior to needle insertion. The needle is advanced to the transverse process and withdrawn 2 to 3 mm prior to injection. Aspiration must be carried out in two planes before a 1-mL test dose is used to exclude unintentional intravascular injection into the vertebral or subclavian arteries or subarachnoid injection into a dural sleeve. A total of 5 to 10 mL of local anesthetic may be injected. Fluoroscopy or ultrasound may be used to visualize the anatomy and decrease the risk of inadvertent intravascular injection.

Correct placement of the needle is usually followed promptly by an increase in the skin temperature of the ipsilateral arm and the onset of Horner syndrome. The latter consists of ipsilateral ptosis, meiosis, enophthalmos, nasal congestion, and anhydrosis of the neck and face.

® In addition to intravascular and subarachnoid injection, other complications of stellate block include hematoma, pneumothorax, epidural anesthesia, brachial plexus block, hoarseness due to blockade of the recurrent laryngeal nerve, and, rarely, osteomyelitis or mediastinitis following esophageal puncture, particularly if a left-sided approach is taken. The posterior approach may have the highest incidence of pneumothorax.

The thoracic sympathetic ganglia lie just lateral to the vertebral bodies and anterior to the spinal nerve roots, but this block is generally not used because of a significant risk of pneumothorax.

Three groups of splanchnic nerves (greater, lesser, and least) arise from the lower seven thoracic sympathetic ganglia on each side and descend alongside the vertebral bodies to communicate with the celiac ganglia. Although similar to celiac plexus block, the splanchnic nerve block may be preferred because it is less likely to block the lumbar sympathetic chain and because it requires less anesthetic.
The needle is inserted 6 to 7 cm from the midline at the lower end of the T11 spinous process, and advanced under fluoroscopic guidance to the anterolateral surface of T12. Ten milliliters of local anesthetic is injected on each side. The needle should maintain contact with the vertebral body at all times to avoid a pneumothorax. Other complications may include hypotension and possible injury to the azygos vein on the right or to the hemiazygos vein and the thoracic duct on the left.
If a patient's pain lessens after a splanchnic nerve block, the procedure may be repeated to ensure that this result was not due to placebo effect. In addition, if the patient obtained pain relief from the initial block, he or she may subsequently benefit from radiofrequency ablation of the splanchnic nerves at T11 and T12, with potentially longer duration of analgesia. Performing the procedure on one side initially, and then the other side on a subsequent day, is advised due to the risk of pneumothorax.

A celiac plexus block is indicated for patients with pain arising from the abdominal viscera, particularly intraabdominal cancers.

The celiac ganglia vary in number (one to five), form, and position. They are generally clustered at the level of the body of L1, posterior to the vena cava on the right, just lateral to the aorta on the left, and posterior to the pancreas.

The patient is placed in a prone position and a 15-cm 22-gauge needle is used to inject 15 to 20 mL of local anesthetic (Figure 47—21). Under fluoroscopic guidance, each needle is inserted 7 to 8 cm from the midline at the inferior edge of the spinous process of L1 and advanced under radiographic guidance toward the midline. The needle passes under the edge of the 12th rib and should be positioned anterior to the body of L1 in the lateral radiographic view and close to the midline overlying the same vertebral body in the anteroposterior view. When CT guidance is used, the tip of the needle should come to lie anterolateral to the aorta at a level between the celiac and superior mesenteric arteries.

The celiac plexus block may be performed from multiple approaches, including a posterior retrocrural approach, a posterior anterocrural approach, a posterior transaortic approach, and an anterior approach. These blocks may be facilitated with fluoroscopic, CT, or ultrasound guidance.

The most common complication is postural hypotension from block of visceral sympathetic innervation and resultant vasodilation. For this reason, patients should be adequately hydrated intravenously prior to this block and precautions taken to minimize the risk of orthostatic lightheadedness or syncope. Accidental intravascular injection into the vena cava is more likely to produce a severe systemic reaction than accidental intraaortic injection. Other, less common, complications include pneumothorax, retroperitoneal hemorrhage, injury to the kidneys or pancreas, sexual dysfunction, or, rarely, paraplegia (due to spasm of, or injury to, the lumbar artery of Adamkiewicz). Blocking the sympathetic chain may result in relatively unopposed parasympathetic activity that may lead to increased gastrointestinal motility and diarrhea. Back pain is another common side effect of a celiac plexus block.

Lumbar sympathetic block may be indicated for painful conditions involving the pelvis or the lower extremities, and possibly for some patients with peripheral vascular disease.

The lumbar sympathetic chain contains three to five ganglia and is a continuation of the thoracic chain. It also supplies sympathetic fibers to the pelvic plexus and ganglia. The lumbar sympathetic chain ganglia are in a more anteromedial position to the vertebral bodies than the thoracic ganglia, and are anterior to the psoas muscle and fascia. The lumbar chain is usually posterior to the vena cava on the right but is just lateral to the aorta on the left.

A single-needle technique at the L3 level on either side is most commonly employed with the patient either prone or in a lateral position (Figure 47—22). The needle is inserted at the upper edge of the spinous process and is directed above or just lateral to the transverse process of the vertebrae (depending on the distance from the midline). Fluoroscopic or ultrasound guidance is often used.

Complications include intravascular injection into the vena cava, aorta, or lumbar vessels and somatic nerve block of the lumbar plexus. In particular, the genitofemoral nerve may be blocked.

This procedure is indicated for pelvic pain that is unresponsive to lumbar or caudal epidural blocks. The hypogastric plexus contains visceral sensory fibers that bypass the lower spinal cord. This block is usually appropriate for patients with cancer of the cervix, uterus, bladder, prostate, or rectum, and may also be effective for some women with chronic noncancer pelvic pain.

The hypogastric plexus contains not only postganglionic fibers derived from the lumbar sympathetic chain, but also visceral sensory fibers from the cervix, uterus, bladder, prostate, and rectum. The superior hypogastric plexus usually lies just to the left of the midline at the L5 vertebral body and beneath the bifurcation of the aorta. The fibers of this plexus divide into left and right branches and descend to the pelvic organs via the left and right inferior hypogastric and pelvic plexuses. The inferior hypogastric plexus additionally receives preganglionic parasympathetic fibers from the S2-S4 spinal nerve roots.

The patient is positioned prone, and a 15-cm needle is inserted approximately 7 cm lateral to the L4—L5 spinal interspace. The needle is directed medially and caudally under fluoroscopic or ultrasound guidance so that it passes by the transverse process of LS. In its final position, the needle should lie anterior to the intervertebral disc between L5 and S1 and within 1 cm of the vertebral bodies in the anteroposterior view. When fluoroscopy is used, injection of radiopaque contrast confirms correct position of the needle in the retroperitoneal space; 8 to 10 mL of local anesthetic is then injected. The superior hypogastric plexus block may also be performed using a transdiscal approach, though there is a risk of discitis associated with this procedure.

Complications include intravascular injection and transient bowel and bladder dysfunction.

@ Ganglion impar block is effective for patients with visceral or sympathetically maintained pain in the perineal area.

The ganglion impar (ganglion of Walther) is the most caudal part of the sympathetic trunks. The two lowest pelvic sympathetic ganglia often fuse forming one ganglion in the midline just anterior to the coccyx.

The patient may be positioned in the prone, lateral decubitus, or lithotomy position. A 22-gauge needle is advanced through the sacrococcygeal ligament and the rudimentary disc into a position just anterior to the coccyx. This procedure can be facilitated with fluoroscopy or ultrasound. Radiofrequency ablation, or in some cases a neurolytic injection, may provide longer duration of analgesia for this sympathetically mediated pain.

Intravascular injection and transient bowel or bladder dysfunction are possible. Alternative approaches involve placement of the needle through the anococcygeal ligament, although these may have higher potential to perforate the rectum.

A Bier block (see Chapter 46) utilizing local anesthetic solution with or without adjuvants can be used to interrupt sympathetic innervation to an extremity. A total volume of 50 mL of 0.5% lidocaine is typically injected, either alone or in combination with clonidine (150 mcg) and in some cases ketorolac (15-30 mg). The technique is described in Chapter 46.

Epidural steroid injections (Figure 47—23) are used for symptomatic relief of radicular pain associated with nerve root compression. Pathological studies often demonstrate local inflammatory changes following disc herniation, and clinical improvement appears to be correlated with resolution of resultant nerve root edema. Epidural steroid injections are clearly superior to local anesthetics alone. They are most effective when given within 2 weeks of pain onset but appear to be of little benefit in the absence of neural compression or irritation. Long-term studies have failed to show any persistent benefit after 3 months, and these injections may change the time course of pain relief without changing long-term outcomes.

The two most commonly used agents are methylprednisolone acetate (40-80 mg) and triamcinolone diacetate (40-80 mg). Dexamethasone is being used with increased frequency due to its smaller particulate size (smaller than an erythrocyte). Intravascular injection of steroid suspension with larger particulate size may lead to embolic complications. The steroid may be injected with diluent (saline) or local anesthetic in volumes of 6 to 10 mL or 10 to 20 mL for lumbar and caudal injections, respectively. Simultaneous injection of opioids offers no added benefit and may significantly increase risks. The epidural needle should be cleared of the steroid prior to its withdrawal to prevent formation of a fistula tract or skin discoloration. Injection of local anesthetic along with the steroid can be helpful if the patient has significant muscle spasm, but it is associated with risks of intrathecal or subdural injection resulting in lower extremity weakness and risk of patient fall, and of intravascular injection. The presenting pain is often transiently intensified following injection, and the local anesthetic provides immediate pain relief until the steroidal antiinflammatory effects take place, usually within 12 to 48 h.

Epidural steroid injections may be most effective when the injection is at the site of injury. Only a single injection is given if complete pain relief is achieved. If there is a good but temporary response, a second injection may be given 2 to 4 weeks later. Larger or more frequent doses increase the risk of adrenal suppression and systemic side effects. Most pain practitioners utilize fluoroscopy for epidural injection and confirm correct placement with injection of radiopaque contrast (Figures 47—24 through 47—26). A transforaminal epidural steroid injection may be more effective than the standard interlaminar epidural technique, especially for radicular pain. The needle is directed under fluoroscopic guidance into the foramen of the affected nerve root; contrast is then injected to confirm spread into the epidural space and absence of intravascular injection prior to steroid injection. This technique differs from a selective nerve root block (SNRB) in two important ways; with an SNRB, the needle does not enter the foramen and the injected solution tracks along the nerve but not into the epidural space. The SNRB may be helpful as a diagnostic procedure for the surgeon who is considering a foraminotomy at a particular affected level based upon imaging, clinical presentation, and the results of the SNRB.
FIGURE 47-24 Fluoroscopic image of a C7—T1 epidural steroid injection; anteroposterior view. Note the Tuohy needle advanced just to the right of midline for treatment of degenerative disc disease and right radicular pain.
FIGURE 47-25 Fluoroscopic image of a C7—T1 epidural steroid injection with contrast; lateral view. Note radiopaque contrast confirmation of the needle in the epidural space. Live fluoroscopy is used to minimize the risk of inadvertent intravascular injection.
FIGURE 47-26 Lumbar epidural steroid injection, anteroposterior view. The epidural injection of contrast followed by local anesthetic and steroid solution results in spread at multiple levels of the epidural space and through the neuroforamen.

Caudal injection may be used in patients with previous back surgery when scarring and anatomic distortion make lumbar epidural injections more difficult. Unfortunately, migration of the steroid to the site of injury may not be optimal due to anatomic distortion of the epidural space. The use of a catheter to more precisely direct the injection within the sacral and epidural canal may improve outcome. However, above the level of S2, there is a risk of thecal perforation with a stylet-guided catheter. Intrathecal steroid injections are not recommended because the ethylene glycol preservative in the suspension has been implicated in arachnoiditis following unintentional subarachnoid injections.

Percutaneous radiofrequency ablation (RFA) relies on the heat produced by current flow from an active electrode that is incorporated at the tip of a special

@ Neurolytic blocks are indicated for patients with severe, intractable cancer pain in whom more conventional therapy proves inadequate or conventional analgesic modalities are accompanied by unacceptable side effects. The most common chemical neurolytic techniques utilized for cancer patients are celiac plexus, lumbar sympathetic chain, hypogastric plexus, and ganglion impar blocks. Chemical neurolysis may also occasionally be used in patients with refractory benign neuralgia and, rarely, in patients with peripheral vascular disease. These blocks can be associated with considerable morbidity (loss of motor and sensory function), so patients must be selected carefully, and only after thorough consideration of alternative analgesic modalities. Moreover, although the initial result may be excellent, the original pain may recur or new (deafferentation or central) pain develop in a majority of patients within weeks to months.
Temporary destruction of nerve fibers or ganglia can be accomplished by injection of alcohol or phenol. These neurolytic agents are not selective, affecting visceral, sensory, and motor fibers equally. Ethyl alcohol 50% to 100% causes extraction of membrane phospholipids and precipitation of lipoproteins in axons and Schwann cells, whereas phenol 6% to 12% coagulates proteins. Alcohol causes severe pain on injection; thus local anesthetic is usually administered first. For peripheral nerve blocks, alcohol may be given undiluted, but for sympathetic blocks in which large volumes are injected, it is given in a 1:1 mixture with bupivacaine. Phenol is usually painless when injected either as a 6% or an 8% aqueous solution or in glycerol; a 12% phenol solution can be prepared in radiopaque contrast solution.

Neurolytic celiac plexus or splanchnic nerve blocks may be effective for painful intraabdominal neoplasms, especially pancreatic cancer. Lumbar sympathetic, hypogastric plexus, or ganglion impar neurolytic blocks can be used for pain secondary to pelvic neoplasms. Neurolytic saddle block can provide pain relief for patients with refractory pain from pelvic malignancy; however, bowel and bladder dysfunction should be expected. Neurolytic intercostal blocks can be helpful for patients with painful rib metastases. Additional neurodestructive procedures, such as pituitary adenolysis and cordotomy, may be useful in end-oflife palliative care.
When considering any neurolytic technique, at least one diagnostic block with a local anesthetic solution alone should be used initially to confirm the pain pathway(s) involved and to assess the potential efficacy and morbidity of the planned neurolysis. Local anesthetic solution should again be injected immediately prior to the neurolytic agent under fluoroscopic guidance. Following injection of any neurolytic agent, the needle must be cleared with air saline prior to withdrawal to prevent damage to superficial structures.
or Many clinicians prefer alcohol for celiac plexus block and phenol for lumbar sympathetic block. For subarachnoid neurolytic techniques, very small amounts of neurolytic agent (0.1 mL) are injected. Alcohol is hypobaric, whereas phenol in glycerin is hyperbaric; the patient undergoing subarachnoid neurolysis is carefully positioned so that the solution travels to the appropriate level and is confined to the dorsal horn region following subarachnoid administration. Cancer patients frequently receive anticoagulation therapy if they are at elevated risk for venous thromboembolic phenomena. When such a patient has discontinued anticoagulant medication in preparation for a diagnostic local anesthetic block, it may be more practical to obtain consent for a neurolytic procedure in advance and to follow the diagnostic block immediately with chemical neurolysis if the diagnostic procedure has resulted in pain relief.

Pharmacological or anatomic differential neural blockade has been advocated as a method of distinguishing somatic, sympathetic, and psychogenic pain mechanisms. The procedure is controversial owing to the challenges of interpreting the data and the inability to define exactly which nerve fibers or pathways are blocked. Theoretically, the pharmacological approach relies on the differential sensitivity of nerve fibers to local anesthetics. Preganglionic sympathetic (B) fibers are reported to be most sensitive, closely followed by pain (AS) fibers, somatosensory (AB) fibers, motor fibers (Aa), and finally C fibers. By using different concentrations of local anesthetic, it may be possible to selectively block certain types of fibers while preserving the function of others. The challenge is that the critical concentration needed to block sympathetic fibers can vary considerably between patients, and conduction block by local anesthetics is dependent not only on fiber size but also on the duration of contact and frequency of impulses conducted. Many clinicians have therefore abandoned the use of pharmacological differential neural blocks in favor of anatomic differential blockade.
Stellate ganglion blocks can be used to selectively block sympathetic fibers to the head, neck, and arm. Celiac plexus, hypogastric plexus, and lumbar paravertebral sympathetic blocks can be used for sympathetic blocks of the abdomen, pelvis, and leg, respectively. Selective nerve root, intercostal, cervical plexus, brachial plexus, or lumbosacral plexus blocks may be used for somatic nerve blockade.
Differential epidural blocks may be used for thoracic pain when the techniques for sympathetic blockade carry a significant risk of pneumothorax (Table 47—16). After each epidural injection, the patient is evaluated for pain relief, signs of sympathetic blockade (a decrease in blood pressure), sensation to pinprick and light touch, and motor function. If the pain disappears after the saline injection, the patient either has psychogenic pain (usually a profound long-lasting effect) or is displaying a placebo effect (usually short lasting). If pain relief coincides with isolated signs of sympathetic blockade, it is likely mediated by sympathetic fibers. If pain relief only follows somatosensory blockade, it is likely mediated by somatic fibers. Lastly, if the pain persists even after signs of motor blockade, the pain is either central (supraspinal) or psychogenic.

Placebo, Epidural’ = Saline. Sympatholytic, Epidural’ = 0.5% lidocaine. Somatic, Epidural’ = 1% lidocaine. All fibers, Epidural’ = 2% lidocaine
'Chloroprocaine may be used instead.
The differential epidural block carries the risk of any neuraxial block, and the possibility of hypotension and blocking cardiac accelerator fibers at T1-T4. The level should not extend above the T5 dermatome due to these risks. Following catheter insertion, injections should be administered with the patient in a monitored setting for the rest of this procedure.
Although differential epidural blockade has limitations, it may be helpful to identify primarily centralized pain when a patient continues to have a significant level of pain despite multilevel dermatomal blockade over the painful region. It is unlikely that a subsequent nerve block would help to treat the painful condition.
When it is thought that a patient may have abdominal pain from the anterior abdominal wall, a TAP block may be performed using ultrasound guidance. This may offer potential shortor long-term relief and can be considered as an alternative to differential epidural blockade. If no relief is obtained, the pain may have a visceral origin or a central cause. Visceral pain may best respond to a celiac or splanchnic nerve block and possibly to subsequent splanchnic RFA. Patients with pain that is primarily of a central origin may respond to multidisciplinary therapy, including counseling and biofeedback training.

Electrical stimulation of the nervous system can produce analgesia in patients with acute and chronic pain. Current may be applied transcutaneously, epidurally, or by electrodes implanted into the central nervous system.

TENS is thought to produce analgesia by stimulating large afferent fibers. It may have a role for patients with mild to moderate acute pain and those with chronic low back pain, arthritis, and neuropathic pain. The gate theory of pain processing suggests that the afferent input from large epicritic fibers competes with that from the smaller pain fibers. An alternative theory proposes that at high rates of stimulation, TENS causes conduction block in small afferent pain fibers. With conventional TENS, electrodes are applied to the same dermatome as the pain and are stimulated periodically by direct current from a generator (usually for 30 min several times a day). A current of 10 to 30 mA with a pulse width of 50 to 80 ps is applied at a frequency of 80 to 100 Hz. Some patients whose pain is refractory to conventional TENS respond to low-frequency TENS (acupuncturelike TENS), which employs stimuli with a pulse width greater than 200 ps at frequencies less than 10 Hz (for 5-15 min). Unlike conventional TENS, lowfrequency stimulation is at least partly reversed by naloxone, suggesting a role for endogenous opioids. This technique is also called dorsal column stimulation because it was thought to produce analgesia by directly stimulating large AB fibers in the dorsal columns of the spinal cord.

® Spinal cord stimulation (SCS) may be effective for neuropathic pain; accepted indications include sympathetically mediated pain, spinal cord lesions with localized segmental pain, phantom limb pain, ischemic lower extremity pain due to peripheral vascular disease, adhesive arachnoiditis, peripheral neuropathies, post-thoracotomy pain, intercostal neuralgia, postherpetic neuralgia, angina, visceral abdominal pain, and visceral pelvic pain. Patients with persisting pain after back surgery, which is typically a mixed nociceptive—neuropathic disorder, also appear to benefit from SCS.
Temporary electrodes are initially placed in the posterior epidural space and connected to an external generator to evaluate efficacy in a 5to 7-day trial (Figures 47—27 and 47—28). The trial may be extended, particularly if it allows a patient, such as one with CRPS, to tolerate more aggressive physical therapy. If a favorable response is obtained, a fully implantable system is inserted. Unfortunately, the effectiveness of the technique decreases with time in some patients. Complications include infection, lead migration, and lead breakage.
FIGURE 47-27 Patient positioning for insertion of a spinal cord stimulator (SCS) with fluoroscopic guidance.
FIGURE 47-28 Two-lead SCS placement. A: Anteroposterior view. The right contact lead has been advanced to its final position at the top of T10. The left lead is advanced through the Tuohy needle. B: Lateral view. The first lead is in position, with the left lead entering the epidural space.
Dorsal root ganglion (DRG) stimulation has been shown to be an effective therapy and alternative to SCS to help treat CRPS in the lower extremities and allow for improved tolerance of physical therapy (Figure 47—29). Complications of SCS and DRG stimulation include infection, lead migration, and lead breakage.
FIGURE 47-29 Dorsal root ganglion (DRG) stimulation. A: Anteroposterior view. Dorsal root ganglion stimulation at right T11 and bilateral T12. Note Tuohy needle entry into the lateral aspect of the epidural space one level below the stimulator exiting the contralateral foramen. B: Lateral view reveals one DRG stimulator at T11 and two at T12.

Peripheral nerve stimulation (PNS) differs from SCS in that leads are placed in close anatomic proximity to an injured peripheral nerve. The leads may be placed percutaneously, with or without ultrasound guidance, or surgically under direct vision of the nerve. Occipital nerve stimulators are one form of peripheral nerve stimulator that may be helpful in treating occipital neuralgia and migraine headache (Figure 47—30).
FIGURE 47-30 Occipital nerve stimulator placement, anteroposterior view. Following placement of right occipital nerve stimulator lead below the nuchal ridge, a left occipital nerve stimulator lead has been advanced through the introducer needle.

Deep brain stimulation (DBS) is used for intractable cancer pain and for intractable nonmalignant neuropathic pain. Electrodes are implanted stereotactically into the periaqueductal and periventricular gray areas for nociceptive pain, usually in patients with cancer or chronic low back pain. For neuropathic pain, the electrodes are frequently implanted into the ventral posterolateral and ventral posteromedial thalamic nuclei. DBS may also be helpful for patients with movement disorders, headache, and neuropsychiatric disorders. The most serious complications are intracranial hemorrhage and infection.
9. Vertebral Augmentation
® Patients with pathological or osteoporotic vertebral compression fractures may benefit from vertebral augmentation with polymethylmethacrylate (PMMA) cement. Vertebroplasty involves injection of the cement through the trocar needle. Kyphoplasty involves inflation of a balloon inserted through a percutaneously placed trocar needle, with subsequent injection of cement (Figure 47—31). Anteroposterior and lateral fluoroscopic views facilitate optimal placement of the cement. For patients with a sacral insufficiency fracture, cement sacroplasty may help stabilize the fracture. Risks of vertebral augmentation include direct nerve injury (due to placement of the trocar needle), hemorrhage, cement extravasation, and embolic events.
FIGURE 47-31 Kyphoplasty. A: Anteroposterior view reveals inflation of balloons at L2 prior to deflation and subsequent injection of polymethylmethacrylate (PMMA) at that level. Previously injected PMMA can be seen at L3 into the cavity created by bilateral balloon inflation and deflation. B: Lateral view. The balloons are inflated above the introducers at L2. The previously injected PMMA can be seen in the L3 vertebral body.
Kiva VCF vertebral augmentation has become available as an alternative approach to increase vertebral height after a recent compression fracture. The Kiva procedure involves placing a coil into the vertebral body over which a polyetheretherketone (PEEK) implant may be inserted. PMMA cement may be injected subsequently into the PEEK implant (Figure 47-32).

FIGURE 47-32 Kiva VCF vertebral augmentation. A: Anteroposterior view reveals appropriate width of coil to allow for subsequent polyetheretherketone (PEEK) implant deployment. B: Lateral view reveals advancing coil to create a cavity and allow for advancing and deployment of a PEEK implant and subsequent injection of PMMA.

Psychological techniques, including cognitive therapy, behavioral therapy, biofeedback, relaxation techniques, and hypnosis, are widely used as part of a multidisciplinary approach to pain control. Cognitive interventions are based on the assumption that a patient's attitude toward pain can influence the perception of pain. Maladaptive attitudes contribute to suffering and disability. The patient is taught skills for coping with pain either individually or in group therapy. The most common techniques include attention diversion and imagery. Behavioral (operant) therapy is based on the premise that behavior in patients with chronic pain is determined by consequences of the behavior. Positive reinforcers (such as attention from a spouse) tend to enable or intensify the pain, whereas negative reinforcers reduce pain. The therapist's role is to guide behavior modification with the aid of family members and medical providers in order to nurture negative reinforcers and minimize positive reinforcers. Relaxation techniques teach the patient to alter the arousal response and the increase in sympathetic tone associated with pain. The most commonly employed technique is a progressive muscle relaxation exercise. Biofeedback and hypnosis are closely related interventions. All forms of biofeedback are based on the principle that patients can be taught to control involuntary physiological parameters. Once proficient in the technique, the patient may be able to induce a relaxation response and more effectively apply coping skills to control physiological factors (eg, muscle tension) that worsen pain. The most commonly utilized physiological parameters in biofeedback are muscle tension (electromyographic biofeedback) and temperature (thermal biofeedback). The effectiveness of hypnosis varies considerably among individuals. Hypnotic techniques teach patients to alter pain perception by having them focus on other sensations, localize the pain to another site, and dissociate themselves from a painful experience through imagery. Patients with chronic headaches and musculoskeletal disorders benefit most from these relaxation techniques.

Heat and cold can provide pain relief by alleviating muscle spasm. In addition, heat decreases joint stiffness and increases blood flow, and cold vasoconstricts and can reduce tissue edema. The analgesic action of heat and cold may at least partially be explained by the gate theory of pain processing.
Superficial heating modalities include conductive (hot packs, paraffin baths, fluidotherapy), convective (hydrotherapy), and radiant (infrared) techniques. Techniques for application of deep heat include ultrasound as well as shortwave and microwave diathermy. These modalities are more effective for pain involving deep joints and muscles. Cold is most effective for pain associated with acute injuries and edema. When applied selectively, cold can also relieve muscle spasm. Application may take the form of cold packs, ice massage, or vapocoolant sprays (ethyl chloride or fluoromethane).
Exercise should be part of any rehabilitation program for chronic pain. A graded exercise program prevents joint stiffness, muscle atrophy, and contractures, all of which can contribute to the patient's pain and functional disabilities. McKenzie exercises are particularly helpful for patients with lumbar disc displacement. Patients may state that physical therapy has not helped in the past. The efficacy of previous physical therapy techniques should be assessed, and the appropriateness of current physical therapy sessions and of the home exercise program should also be evaluated. By facilitating increased range of motion and providing constant resistance, aquatherapy may be particularly helpful for patients who may not be able to tolerate other forms of therapy.

® Acupuncture can be a useful adjunct for patients with chronic pain, particularly that associated with chronic musculoskeletal disorders and headaches. The technique involves insertion of needles into discrete anatomically defined points, called meridians. Stimulation of the needle after insertion takes the form of twirling or of application of a mild electrical current. Insertion points appear to be unrelated to the conventional anatomy of the nervous system. Although the scientific literature concerning the mechanism of action and role of acupuncture in pain management is controversial, some studies suggest that acupuncture stimulates the release of endogenous opioids, as its effects can be antagonized by naloxone.

Deer TR, Pope JE, Hyek SM, et al. The Polyanalgesic Consensus Conference (PACC): Recommendations on intrathecal drug infusion systems best practices and guidelines. Neuromodulation. 2017;20:96. Dowell D, Haegerich TM, Chou R. CDC guidelines for prescribing opioids for chronic pain—United States, 2016. MMWR Recomm Rep. 2016;65:1. Narouze S, Benzon HT, Provenzano DA, et al. Interventional spine and pain procedures in patients on antiplatelet and anticoagulant medications: Guidelines from the American Society of Regional Anesthesia and Pain Medicine, the European Society of Regional Anaesthesia and Pain Therapy, the American Academy of Pain Medicine, the International Neuromodulation Society, the North American Neuromodulation Society, and the World Institute of Pain. Reg Anesth Pain Med. 2015;40:182. Rathmell JP, Benzon HT, Dreyfuss T, et al. Safeguards to prevent neurologic complications after epidural steroid injections: Consensus opinions from a multidisciplinary working group and national organizations. Anesthesiology. 2015;122:974.

evidence-
@ A well-functioning enhanced recovery program (ERP) uses based practices that ensure continuity of care, decrease variation in clinical management, minimize organ dysfunction, decrease postoperative complications, and accelerate convalescence.
@ The perioperative surgical home (PSH) is defined by interdisciplinary, team-based global management of the surgical patient throughout the surgical care continuum. PSH management begins following the initial surgical diagnosis and patient preparation, and ends when the patient is returned to the care of his or her primary health care provider after full recovery. It includes several perioperative enhanced recovery program (ERP) elements that are adapted to the local clinical care environment.
© Persistent postsurgical pain (chronic pain that continues beyond the typical healing period of 1 to 2 months following surgery, or well past the normal period for postoperative follow-up) is increasingly acknowledged as a common and significant problem following surgery.
@ The magnitude of the surgical stress response is related to the intensity of the surgical stimulus, hypothermia, and psychological stress. It can be moderated by perioperative interventions, including neural blockade and reduction in procedural invasiveness.
@ Neuraxial blockade of nociceptive stimuli by epidural and spinal local anesthetics has been shown to blunt the metabolic, inflammatory, and

is often a source of considerable patient discomfort, and is one of the most common causes of prolonged postoperative hospital length of stay and preventable hospitalization costs. Nasogastric tubes should be discouraged whenever possible or used for only a very short period of time, even with gastrointestinal surgery. The opioid-sparing effects of multimodal analgesia shorten the duration of postoperative ileus or may preempt it entirely.
- @ Because either excessive, or excessively restricted, perioperative fluid therapy increases the incidence and severity of postoperative ileus, a goal-directed fluid administration strategy may be beneficial, especially in patients undergoing major surgery associated with large fluid shifts or patients at high risk of developing postoperative gastrointestinal complications.

Major advances in surgical and anesthetic management have progressively decreased risk-adjusted perioperative mortality and morbidity. Continued improvement in perioperative outcomes, including accelerated postoperative convalescence and decreased perioperative complications, depends upon the continued evolution of integrated, multidisciplinary team approaches to perioperative care. The goal of team-based care is to combine individual evidence-based elements of perioperative care (eg, analgesic regimens, nutritional interventions, and physical therapy), each of which may have modest benefits when used in isolation into a tightly coordinated effort that has significant synergistic, beneficial effects upon surgical outcomes (the theory of aggregation of marginal gains).
Such coordinated, multidisciplinary perioperative care programs are termed enhanced @ recovery programs (ERPs), fast-track surgery, or enhanced recovery after surgery (ERAS) (Figure 48-1). A well-functioning ERP uses evidence-based practices that ensure continuity of care, decrease variation in clinical management, minimize organ dysfunction, decrease postoperative complications, and accelerate convalescence (Figure 48—2). The success of ERPs relies upon the additive or synergistic effects of the interventions included in these programs. Adherence to ERP pathways is associated with better postoperative outcomes, accelerated convalescence, and lower costs.
FIGURE 48-1 Perioperative elements contributing to enhanced recovery after surgery (ERAS). CHO, carbohydrate; DVT, deep vein thrombosis; PONV, postoperative nausea and vomiting. (Reproduced with permission from Fearon KC, Ljungqvist O, Von Meyenfeldt M, et al. Enhanced recovery after surgery: A consensus review of clinical care for patients undergoing colonic resection. Clin Nutr. 2005 June;24(3):466-477.)

response. (Reproduced with permission from Kehlet H, Wilmore DW. Evidence-based surgical care and the evolution of fast-track surgery. Ann Surg. 2008 Aug;248(2):189-198.)
Assessing outcomes following ERP adoption is critical. Hospital length of stay is a commonly used measure of success, although in many health care systems the timing of hospital discharge is more directly related to administrative and organizational issues than to discrete surgical and medical milestones in the patient's postoperative recovery. Little research has been undertaken to define the process of postoperative recovery, and few outcome measures are currently available to confirm that postoperative recovery has been accomplished for a given surgical disease. Other measures of successful implementation of ERPs include reduced rates of hospital readmission and reduced rates and severity of perioperative complications. Promising data suggest that ERPs may improve oncologic outcomes after surgery.
Anesthetic interventions that reduce pain, facilitate earlier postoperative mobilization, and allow earlier resumption of oral feeding accelerate the pace of perioperative recovery. In this context, the anesthesia provider must not only provide ideal anesthetic management throughout the procedure, but must also help improve overall perioperative care. These goals are achieved through optimizing the patient's preoperative medical condition, avoiding prolonged fasting, moderating the adverse effects of the intraoperative neuroendocrine stress response, and providing multimodal pain and symptom management to accelerate postoperative recovery.
Initially developed for patients undergoing colorectal and cardiac surgery, ERPs have also been developed for patients undergoing esophageal, bariatric, pancreatic, liver, head and neck, breast, orthopedic, bariatric, and pediatric abdominal surgery. Many ERP guidelines and consensus statements are available from the various specialty societies.

® The perioperative surgical home (PSH) has been defined as a "patient centered, innovative model of delivering health care during the entire patient surgical/procedural experience; from the time of the decision for surgery until the patient has recovered and returned to the care of his or her patient centered medical home or primary health care provider." The PSH "provides coordination of care throughout all of the clinical microsystems of care." PSH programs can be considered an evolution of ERPs, because they include several perioperative ERP elements but are adapted to the local clinical environment. Based on their unique clinical skills, anesthesiologists are called to play a pivotal, coordinating care role in the PSH. To accomplish this, anesthesia providers must integrate with the surgical and medical teams with whom they jointly provide perioperative care. Accordingly, anesthesiology training programs must expand their curricula to include the entire continuum of perioperative care.
® Persistent postsurgical pain (chronic pain that continues beyond the typical healing period of 1-2 months following surgery) is increasingly acknowledged as a common and significant issue following surgery. The incidence of persistent postsurgical pain may exceed 30% after some operations, especially amputations, thoracotomy, mastectomy, and inguinal herniorrhaphy. Although the cause is unclear, several risk factors have been identified (Figure 48-3). Multimodal perioperative pain control is often suggested as a fundamental preemptive strategy to reduce the incidence of persistent postsurgical pain (see Chapter 47).
FIGURE 48-3 Risk factors for persistent postsurgical pain. (Reproduced with permission from Wu CL, Raja SN. Treatment of acute postoperative pain. Lancet. 2011;377(9784):22152225.)

Cooperation from the patient and family is essential if an ERP is to be effectively implemented. Preoperative teaching must use plain language and avoid medical jargon. Well-designed print, video, and online materials presented in the patient's native language are useful to introduce ERPs. Smartphone text messaging and patient navigation apps are increasingly utilized to help organize and coordinate the patient's perioperative care continuum.
Preoperative Risk Assessment & Optimization of

Reducing the likelihood of perioperative complications improves surgical recovery. Preoperative assessment is discussed in detail in Chapter 18. Although international guidelines evaluating the risk for developing cardiovascular, respiratory, or metabolic complications have been extensively reviewed and published, less attention has been given to assessment and optimization of preoperative functional and physiological status. Nonetheless, some recommendations can be made. For example, perioperative B-blockers should be continued in patients already receiving this therapy. Perioperative statins appear to decrease postoperative cardiovascular complications and should not be abruptly discontinued perioperatively. Several procedure-specific scoring systems based on patient comorbidity, type of surgery, and biochemical data are being used to predict postoperative mortality and morbidity. In addition, riskadjusted scoring systems, such as the American College of Surgeons' National Surgical Quality Improvement Program (NSQIP) and the Society of Thoracic Surgeons' National Database, can be used to compare outcomes among institutions.

Preoperative evaluation of surgical risk and optimization of medical comorbidities also provides opportunity to modify habits that may significantly impact the patient's short-term and long-term health and quality of life. Smoking, drug abuse, and excessive alcohol use are risk factors for the development of postoperative complications. Perioperative interventions aimed at risk modification can reduce risks of complications, accelerate surgical recovery, and reduce perioperative costs. A recent meta-analysis found that preoperative smoking cessation, for any type of surgery, reduced postoperative complications by 41%, especially those related to wound healing and the lungs. Intense preoperative smoking cessation programs of 3 to 4 weeks' duration that include pharmacological interventions (eg, nicotine replacement therapy) and patient counseling produce better results than brief and isolated preoperative smoking cessation interventions. Many psychological and pharmacological strategies are also available to help patients stop excessive alcohol consumption and reduce the risk of alcohol withdrawal.

Antithrombotic prophylaxis reduces perioperative venous thromboembolism risk and related morbidity and mortality. Both pneumatic compression devices and anticoagulant medications are now commonly used. Because neuraxial anesthesia techniques are commonly employed for many patients undergoing major abdominal, vascular, thoracic, and orthopedic procedures, appropriate timing and administration of antithrombotic agents in these cases is of critical importance in order to avoid the risk of epidural hematoma. International recommendations on the management of anticoagulated patients receiving regional anesthesia are discussed in Chapter 45.

Appropriate selection and timing of preoperative antibiotic prophylaxis reduces the risk of surgical site infections. Antibiotics should be administered intravenously within 1 h before skin incision and, based on their plasma half-life and estimated blood loss, should be repeated during lengthy surgeries to ensure adequate tissue concentrations. Recent data from large national databases have demonstrated that administration of oral antibiotics 24 h prior to colorectal surgery in patients receiving mechanical bowel preparation (MBP) reduces the risk of surgical site infections, when compared with patients receiving MBP alone or those not receiving MBP. Antibiotic prophylaxis for surgical site infections should be discontinued within 24 h following surgery, although current guidelines permit cardiothoracic patients to receive antibiotics for 48 h following surgery.

The surgical stress response is characterized by neuroendocrine, metabolic, inflammatory, and immunological changes initiated by the physiological trespass of the surgical incision and subsequent invasive procedures. The stress response can adversely affect organ function and perioperative outcomes, and may include induction of a catabolic state as well as a transient, but reversible, state of insulin resistance, characterized by decreased peripheral glucose uptake and increased endogenous @ glucose production. The magnitude of the surgical stress response is related to the intensity of the surgical stimulus, hypothermia, and psychological stress. It can be moderated by perioperative interventions, including neural blockade and reduction in procedural invasiveness. Much recent effort has focused on developing surgical and anesthetic techniques that reduce the surgical stress response, with the goal of lowering the risk of stress-related organ dysfunction and perioperative complications. An overview of several techniques that have proved effective in ERP protocols follows.

It is well established that minimally invasive surgical procedures are associated with significantly less surgical stress when compared with corresponding "open" procedures. Published data highlight the safety of minimally invasive procedures in the hands of adequately trained and experienced surgeons. Moreover, a longer term salutary impact is achieved when laparoscopic techniques are included in ERPs. For example, laparoscopic procedures are associated with a reduced incidence of surgical complications, especially surgical site infections, when compared with the same procedures performed in "open" fashion. A laparoscopic approach is also associated with less postoperative surgical pain, better postoperative respiratory function, and less morbidity in elderly surgical patients. Over the past 15 years, further advancements in surgical care, such as robotic surgery, natural orifice specimen extraction during laparoscopic surgery, endoscopic surgical approaches, and minimally invasive orthopedic surgery, have further moderated the impact of surgery on surgical stress, and such progress is expected to continue.

A variety of fast-track surgical procedures have taken advantage of the beneficial clinical and metabolic effects of regional anesthesia/analgesia techniques @ (Table 48-1). Neuraxial blockade of nociceptive stimuli by epidural and spinal local anesthetics has been shown to blunt the metabolic, inflammatory, and neuroendocrine stress responses to surgery. In major open abdominal and thoracic procedures, thoracic epidural blockade with local anesthetic is a recommended anesthetic component of a postoperative ERP, providing excellent analgesia, facilitating mobilization and physical therapy, and decreasing the incidence and severity of ileus. However, the advantages of epidural blockade in such cases are not as evident when minimally invasive surgical techniques are used, and in certain cases, epidural blockade has even been shown to delay recovery and prolong hospital stay. Lumbar epidural anesthesia/analgesia should be discouraged for abdominal surgery because it often does not provide adequate segmental analgesia for an abdominal incision. In addition, it frequently causes urinary retention and lower limb sensory and motor blockade, increasing the need for urinary drainage catheters (with accompanying increased risk of urinary tract infection), delaying mobilization and recovery, and increasing the risk of falls.

Nephrectomy, Length of Stay = 2-4d. Hysterectomy, Incision = Laparotomy, laparoscopy. Hysterectomy, Regional Anesthesia /Analgesia Techniques = TAP. Hysterectomy, Length of Stay = 1-2d. Radical cystectomy, Incision = Laparotomy. Radical cystectomy, Regional Anesthesia /Analgesia Techniques = TEA, wound infusion of local anesthetic. Radical cystectomy, Length of Stay = 5-7d. Arthroplasty (hip, knee), Incision = Open. Arthroplasty (hip, knee), Regional Anesthesia /Analgesia Techniques = CPNB (femoral and sciatic), high-volume periarticular infiltration. Arthroplasty (hip, knee), Length of Stay = 1-3d
"TEA, thoracic epidural analgesia; TAP, transversus abdominus plane block; ICB, intercostal block; INB, ilioinguinal nerve block; CPNB, continuous peripheral nerve block.
@ Epidural blockade using a solution of local anesthetic and low-dose opioid provides better postoperative analgesia at rest and with movement than systemic opioids (Figure 48—4 and Table 48—2). By sparing opioid use and minimizing the incidence of systemic opioid-related side effects, epidural analgesia facilitates both earlier mobilization and earlier resumption of oral nutrition, expediting exercise activity and attenuating loss of body mass. It minimizes postoperative insulin resistance by attenuating the postoperative hyperglycemic response and by facilitating utilization of exogenous glucose, thereby preventing postoperative loss of amino acids and conserving lean body mass.
FIGURE 48-4 Optimal regions for placing an epidural catheter in the adult spine when administering epidural anesthesia/analgesia for thoracic and abdominal procedures. (Reproduced with permission from Manion SC, Brennan TJ. Thoracic epidural analgesia and acute pain management. Anesthesiology. 2011 July;115(1):181-188.)
TABLE 48-2 Options for composition of thoracic epidural infusion analgesia solutions.!

*Reproduced with permission from Manion SC, Brennan TJ. Thoracic epidural analgesia and acute pain management. Anesthesiology. 2011 July;115(1}181

Bupivacaine, 0.125%, Opioid = . Bupivacaine, 0.125%, Advantages = 4 Nausea/Vomiting 4 Pruritus 4 Sedation 4 Respiratory depression. Bupivacaine, 0.125%, Disadvantages = T Hypotension T Motor blockage. Bupivacaine, 0.1%, Opioid = Hydromorphone, 5-10 mcg/mL or Fentanyl, 2-5 meg/mL. Bupivacaine, 0.1%, Advantages = 4 Both hemodynamic and opioid side effects. Bupivacaine, 0.1%, Disadvantages = . Bupivacaine, 0.05%, Opioid = Hydromorphone, 5-10 mcg/mL or Fentanyl, 2-5 mcg/mL. Bupivacaine, 0.05%, Advantages = 4 Both hemodynamic and opioid side effects. Bupivacaine, 0.05%, Disadvantages = . Bupivacaine, 0.05%, Opioid = Hydromorphone, 20 mcg/mL or Fentanyl, S-10 meg/mL. Bupivacaine, 0.05%, Advantages = 4 Both hemodynamic and opioid side effects. Bupivacaine, 0.05%, Disadvantages = . , Opioid = Hydromorphone, 20-40 meg/mL. , Advantages = 4 Hypotension 1 Motor blockade. , Disadvantages = 7 Nausea/Vomiting 7 Pruritus T Sedation T Respiratory depression

If spinal anesthesia is used for fast-track (and especially ambulatory) surgery, attention must be paid to delayed recovery due to prolonged motor blockade. The use of smaller doses of intrathecal local anesthetics (lidocaine, 30-40 mg; bupivacaine, 3-10 mg; or ropivacaine, 5-10 mg) with lipophilic intrathecal opioids (fentanyl, 10-25 mcg, or sufentanil, 5-10 mcg) can prolong postoperative analgesia and minimize motor block without delaying recovery from anesthesia. The introduction of ultra-short-acting intrathecal agents such as 2-chloroprocaine (still controversial at present) may further speed the fast-track process. Spinal opioids are associated with side effects such as nausea, pruritus, and postoperative urinary retention. Adjuvants such as clonidine are effective alternatives to intrathecal opioids, with the goal of avoiding opioid side effects that may delay hospital discharge. For example, intrathecal clonidine added to spinal local anesthetic provides effective analgesia with less urinary retention than intrathecal morphine. In a recent study, lower cortisol and glucose levels were observed in colorectal patients receiving spinal anesthesia with intrathecal local anesthetic and morphine compared with patients receiving systemic opioids; however, the inflammatory response did not differ between the two analgesic techniques. Further studies are needed to define the safety and efficacy of regional anesthesia techniques in fast-track cardiac surgery. Although some cardiac surgery studies have shown that spinal analgesia with intrathecal morphine decreases extubation time, decreases length of stay in the intensive care unit (ICU), reduces pulmonary complications and arrhythmias, and provides analgesia with less respiratory depression, other studies have shown no benefit to this approach.
@ Peripheral nerve blocks (PNBs) with local anesthetics (single-shot or continuous infusion) block afferent nociceptive pathways and are an excellent way to minimize the need for systemic opioids and thereby reduce the incidence of opioid-related side effects and facilitate recovery (see Chapter 46). The choice of local anesthetic, dosage, and concentration should be made with the goal of avoiding prolonged motor blockade and delayed mobilization and discharge.

@ Lidocaine (intravenous bolus of 100 mg or 1.5—2 mg/kg, followed by continuous intravenous infusion of 1.5—-3 mg/kg/h or 2-3 mg/min) has analgesic, antihyperalgesic, and antiinflammatory properties. In patients undergoing colorectal and radical retropubic prostate surgeries, intravenous lidocaine has been shown to reduce requirements for opioids and general anesthetic agents, provide satisfactory analgesia, facilitate early return of bowel function, and accelerate hospital discharge. Although lidocaine infusion potentially may replace neuraxial blockade and regional anesthesia in some circumstances, more studies are needed to confirm the advantage of this technique in the context of ERPs. The most effective dose and duration of infusion for various surgical procedures remains to be determined; even short duration of lidocaine infusion may have benefit.

B-Blockers have been used to blunt the sympathetic response during laryngoscopy and intubation and to attenuate the surgical stress-induced increase in circulating catecholamines. They also have been shown to prevent perioperative cardiovascular events in at-risk patients undergoing noncardiac surgery and to help maintain hemodynamic stability during the intraoperative period and during emergence from anesthesia. They possess anticatabolic properties, which may be explained by reduced energy requirements associated with decreased adrenergic stimulation. Intravenous esmolol reduces the requirement of volatile anesthetic agents and decreases minimum alveolar concentration values; it has also been shown to reduce postoperative pain intensity, opioid consumption, and PONV. A positive protein balance has been reported in critically ill patients when B-blockade is combined with parenteral nutrition. However, the precise role of B-blockers as adjuvant analgesics in the context of ERPs remains undetermined.

Both clonidine and dexmedetomidine have anesthetic and analgesic properties. Clonidine decreases postoperative pain, reduces opioid consumption and opioidrelated side effects, and prolongs neuraxial and peripheral nerve local anesthetic blockade. In patients undergoing cardiovascular fast-track surgery, spinal morphine with clonidine decreases extubation time, provides effective analgesia, and improves quality of recovery. The role of dexmedetomidine in ERP pathways has not been extensively studied.

Intravenous propofol is the deep sedation and general anesthesia induction agent of choice for most surgical procedures. Propofol total intravenous anesthesia (TIVA) is often used as part of a multimodal regimen for patients at high risk for PONV.

When compared with other volatile anesthetic agents, desflurane and sevoflurane may shorten anesthesia emergence, reduce postanesthesia care unit (PACU) length of stay, and decrease recovery-associated costs. There is evidence that avoidance of deep general anesthesia by use of bispectral index (BIS) monitoring may improve outcomes, including a reduction of the incidence of postoperative delirium and cognitive dysfunction. Nitrous oxide, because of its anestheticand analgesic-sparing effects, rapid pharmacokinetic profile, and low cost, is sometimes administered with other inhalation agents. However, routine administration of nitrous oxide has declined recently, as it may cause bowel distention and impair the laparoscopic surgeon's view of anatomic structures, and may increase the risk of PONV (see Chapter 8).

Short-acting opioids such as fentanyl, alfentanil, and remifentanil are commonly used during fast-track surgery in combination with inhalation agents or propofol, and with regional or local anesthesia/analgesia techniques. However, intraoperative administration of remifentanil to patients who will experience extensive postoperative pain has been associated with opioid-induced hyperalgesia, acute opioid tolerance, and increased analgesic requirements during the postoperative period. There is increasing evidence that the use of opioids should be minimized in all phases of the perioperative course as part of a multimodal analgesia technique to reduce opioid side effects and optimize recovery. Opioid-free anesthesia has been shown to reduce PONV and postoperative opioid use when compared with opioid-based anesthesia and may be an alternative technique, especially in patients at high risk for PONV, sleep apnea, or respiratory depression.

The inhibitory effects of anesthetic agents on thermoregulation, exposure to the relatively cool surgical environment, and intraoperative heat loss can lead to hypothermia in all patients undergoing surgical procedures. The duration and extent of the surgical procedure directly correlates with hypothermia risk. Perioperative hypothermia increases cardiovascular morbidity and wound infection risk by increasing sympathetic discharge and inhibiting the immune cellular response. A decrease in core body temperature of 1.9°C triples the incidence of surgical wound infection. The risk of bleeding and blood transfusion requirement are also increased with hypothermia. Furthermore, by impairing the metabolism of many anesthetic agents, hypothermia may significantly prolong anesthesia recovery (see Chapter 52).

Surgical stress leads to impaired pulmonary function and to peripheral vasoconstriction, resulting in arterial and local tissue hypoxemia, respectively. Perioperative hypoxia can increase cardiovascular and cerebral complications, and many strategies should be adopted during the perioperative period to prevent its development.
Maintenance of adequate perioperative oxygenation by oxygen supplementation has been associated with the improvement of some clinically relevant outcomes. Intraoperative and postoperative (for 2 h) inspired oxygen concentration of 80% increases arterial and subcutaneous oxygen tension and may decrease the rate of wound infection and lower the incidence of PONV, without increasing potential complications associated with high inspired oxygen fraction, such as atelectasis and hypercapnia. Regional anesthesia techniques decrease vascular resistance and may improve peripheral tissue perfusion and oxygenation. Finally, early mobilization and avoidance of bedrest improve postoperative central and peripheral tissue oxygenation.

PONV is a frequent complication that delays early feeding and postoperative recovery. Preemptive strategies minimizing the risk of PONV are strongly advocated for any type of surgery, and consensus guidelines for prevention and management of PONV are available in the current literature (see Chapters 17 and 56).

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The type of fluid infused is also important: isotonic balanced crystalloid should be used to replace extracellular losses, whereas iso-oncotic colloids are commonly used to replace intravascular volume (Table 48-3).
TABLE 48-3 Physiologically based first-line fluid replacement for goaldirected therapy.!
3

Extracellular, Replace with = . Extracellular, Amount = . Insensible perspiration, Replace with = Crystalloids*. Insensible perspiration, Amount = . Closed abdomen, Replace with = . Closed abdomen, Amount = 0.5 mL/kg/h. Open abdomen, Replace with = . Open abdomen, Amount = 1 mL/kg/h. Urine production, Replace with = _—__Crystalloids. Urine production, Amount = Measured output*. Intravascular, Replace with = . Intravascular, Amount = . Blood loss, Replace with = Colloids?. Blood loss, Amount = Estimated losses. Further preload deficit, Replace with = Colloids. Further preload deficit, Amount = According to clinical estimation?
"Reproduced with permission from Chappell D, Jacob M. Influence of non-ventilatory options on postoperative outcome. Best Pract Res Clin Anaesthesiol. 2010 June;24(2):267-281.
*Crystalloids should be given in an isotonic balanced form.
Colloids should be given in an iso-oncotic form in balanced solutions. 'First-line approach in healthy kidneys.
- 'If possible use extended monitoring (eg, PiCCO system, esophageal Doppler, etc).

The primary cause of postoperative shivering is perioperative hypothermia, although other, non-thermoregulatory, mechanisms may be involved. Postoperative shivering can greatly increase oxygen consumption, catecholamine release, cardiac output, heart rate, blood pressure, and intracranial and intraocular pressure. It increases cardiovascular morbidity, especially in elderly patients, and increases PACU length of stay and cost. Shivering is uncommon in elderly and hypoxic patients: the efficacy of thermoregulation decreases with aging, and hypoxia can directly inhibit shivering. Many drugs, notably meperidine, clonidine, and tramadol, can be used to reduce postoperative shivering; however, prevention via strategies intended to minimize thermal loss is optimal (see Chapter 52).

Pharmacological treatment of PONV should be promptly initiated once medical or surgical causes of PONV have been ruled out (see Chapters 17 and 56).

® Multimodal analgesia combines different classes of medications that have different (multimodal) pharmacological mechanisms of action, resulting in additive or synergistic effects to reduce postoperative pain and its sequelae. Such an approach may achieve desired analgesic effects while reducing analgesic dosage and associated side effects. Multimodal pain management often includes utilization of regional analgesic techniques, including local anesthetic wound infusion, epidural/intrathecal analgesia, or single-shot/continuous peripheral nerve blockade. Multimodal analgesia is routinely utilized in ERPs to improve postoperative outcomes. Discussion here focuses on principal analgesic interventions used in perioperative multimodal analgesia regimens.
@ 1. NSAIDs—The addition of nonsteroidal antiinflammatory drugs (NSAIDs) to systemic opioid analgesia diminishes postoperative pain intensity, reduces opioid requirements, and decreases opioid-related side effects such as PONV, sedation, and urinary retention. However, NSAIDs may increase the risk of gastrointestinal and wound bleeding, decrease kidney function, and impair wound healing. There is also a concern that NSAIDs may have a detrimental effect on anastomotic healing of the gastrointestinal tract and increase the risk of anastomotic leak, although this is controversial and further research is needed.
Perioperative administration of selective cyclooxygenase-2 (COX-2) inhibitor NSAIDs likewise reduces postoperative pain and decreases both opioid consumption and opioid-related side effects, and although their use reduces the incidence of NSAID-related platelet dysfunction and gastrointestinal bleeding, the potential adverse effects of COX-2 inhibitors on kidney function remain controversial. Concerns have also been raised, primarily with rofecoxib and valdecoxib, regarding COX-2 safety for patients undergoing cardiovascular surgery. Increased cardiovascular risk associated with the perioperative use of celecoxib or valdecoxib in patients with minimal cardiovascular risk factors and

6. Opioids—Despite the increasing use of new, nonopioid analgesic medications and adjuvants and of regional anesthesia and analgesia techniques intended to minimize opioid requirements and opioid-related side effects (Table 48—4), the use of systemic opioids remains a commerstone in the management of surgical pain. Parenteral opioids are frequently prescribed in the postoperative period ® during the transitional phase to oral analgesia. Opioid administration by patientcontrolled analgesia (PCA) provides better pain control, greater patient satisfaction, and fewer opioid side effects when compared to nurse-administered, on-request (PRN) parenteral opioid administration. Oral administration of opioids, such as oxycodone or hydrocodone, in combination with NSAIDs or acetaminophen, or both, is common in the perioperative period.

MgSO,, 1 = Cardiac Cholecystectomy Lower limb orthopedic Gynecological Ambulatory. MgSO,, 2 = + ‘ + + +. MgSO,, 3 = 30-50 mg/kg. followed by 8-15 mg/kg/h Cl. MgSO,, 4 = Vv. MgSO,, 5 = Pre, Intra. MgSO,, 6 = CNS (somnolence), neuromuscular function, respiratory depression, cardiovascular (bradycardia). Steroids, 1 = Hip arthroplasty Brea:. Steroids, 2 = + + +. Steroids, 3 = Dexamethasone: 8-16 m Methylprednisolone: 125mg. Steroids, 4 = v. Steroids, 5 = Pre. Steroids, 6 = Glycemia, Gi bleeding, wound healing. a,-Agonist Clonidine Dexmedetomidine, 1 = PO Abdominal Total knee arthroplasty Hysterectomy Prostatectomy WV Cholecystectomy Abdominal Spine Thoracotomy Abdominal Hysterectomy Bariatric. a,-Agonist Clonidine Dexmedetomidine, 2 = + + = + + + + +. a,-Agonist Clonidine Dexmedetomidine, 3 = PO 3-5 meg/kg IV 150meq Loading dose 0.5-1 megikg, followed by 0.2-0.4 meg/kg/h Cl. a,-Agonist Clonidine Dexmedetomidine, 4 = POV Vv. a,-Agonist Clonidine Dexmedetomidine, 5 = Pre Intra, Post (PCAN Pre, Intra, Post ca. a,-Agonist Clonidine Dexmedetomidine, 6 = CNS’ (level of sedation), cardiovascular (hypotension, bradycardia)
N, OF both; or opioid side effects; or all three.
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7. Epidural analgesia—In addition to providing excellent analgesia, epidural blockade blunts the stress response associated with surgery, decreases postoperative morbidity, attenuates catabolism, and accelerates postoperative functional recovery. Compared with systemic opioid analgesia, thoracic epidural analgesia provides better static and dynamic pain relief. However, these benefits have mainly been observed in patients undergoing open abdominal and thoracic surgery; its usefulness in patients undergoing minimally invasive abdominal and thoracic surgery is questionable, as recent trials have suggested it may actually prolong in-hospital recovery in such cases. Long-acting local anesthetics such as ropivacaine (0.2%), bupivacaine (0.0625—0.125%), and levobupivacaine (0.1— 0.125%) are commonly administered together with lipophilic opioids by continuous epidural infusion or by patient-controlled epidural analgesia (PCEA). As previously noted, administering low doses of local anesthetic via thoracic epidural infusion instead of lumbar levels avoids lower extremity motor blockade that may delay postoperative mobilization and recovery and will increase the risk of patient falls. Adding opioids to epidural local anesthetics improves the quality of postoperative analgesia without delaying recovery of bowel function.
8. Paravertebral nerve blocks—Paravertebral nerve blocks provide similar parietal analgesia to epidural blockade but without the risk of epidural-related side effects. However, they have been poorly studied in the context of ERPs.
@ 9. Peripheral nerve blockade—Single-shot and continuous peripheral nerve blockade is frequently utilized for fast-track ambulatory and inpatient orthopedic

Instillation and nebulization of intraperitoneal local anesthetic decreases pain intensity and decreases opioid consumption following open abdominal and laparoscopic surgery. However, the precise roles of these techniques in multimodal management remain to be determined.

The multidisciplinary aspect of postoperative care should bring together the surgeon, anesthesia care team, nurses, nutritionist, physiotherapist, pharmacist, and case manager/social worker in an interdisciplinary team effort to optimize each patient's care based upon standardized, procedure-specific protocols. Comfortable chairs and walkers should be made readily available near each patient bed to encourage patients to sit, stand, and walk. Routine bedrest after surgery should be avoided. Patients should be encouraged to sit in a chair the afternoon or evening following surgery, with ambulation starting the same or next day. If patients are unable to get out of bed, they should be instructed and encouraged to perform physical and deep breathing exercises in their beds.

A well-organized, well-trained, acute pain service (APS) utilizing procedurespecific clinical protocols to optimally manage analgesia and related side effects helps drive ERPs. The quality of pain relief and symptom control heavily influences postoperative recovery; optimal mobilization and dietary intake depend upon adequate analgesia with minimal analgesic-related side effects. The surgeon and the APS must identify and employ optimal analgesic techniques tailored to the specific surgical procedure, and the quality of analgesia and risk of analgesic-related side effects must be closely and continuously assessed. The goal is not to achieve "zero pain," but to make patients comfortable while walking and performing physiotherapy, with minimal side effects such as lightheadedness, sedation, nausea and vomiting, urinary retention, ileus, and leg weakness. It should be noted that opioid-related PONV is the most common cause of unplanned hospital admission following ambulatory surgery, and opioid-related ileus is one of the most common causes of extended hospital length of stay. Both of these problems significantly increase perioperative costs.

® Postoperative ileus delays postoperative resumption of enteral feeding, is often a source of considerable patient discomfort, and is one of the most common causes of prolonged postoperative hospital length of stay and preventable hospitalization costs. Because early enteral nutrition is associated with decreased postoperative morbidity, interventions and strategies aimed at minimizing the risk and severity of postoperative ileus are essential for patients in an ERP. Four main mechanisms contribute to ileus: sympathetic inhibitory reflexes, local inflammation initiated by the operative procedure, intraoperativelyand postoperatively-administered systemic opioids, and bowel edema caused by administration of excess intravenous fluid. Nasogastric tubes, frequently inserted after abdominal surgery, do not speed the recovery of bowel function and may increase pulmonary morbidity by increasing the risk of aspiration. Therefore, nasogastric tubes should be discouraged whenever possible or used for only a very short period of time, even with gastrointestinal surgery.
The opioid-sparing effects of multimodal analgesia with minimal or non— opioid analgesia techniques shorten the duration of postoperative ileus or may preempt it entirely. Minimally invasive surgical techniques are associated with less surgical stress and inflammation than open procedures, resulting in more rapid return of bowel function. For open abdominal procedures, thoracic epidural local anesthetic infusions not only provide superior analgesia, but also speed recovery of bowel function by suppressing inhibitory sympathetic spinal cord reflexes that promote the development and severity of postoperative ileus. Epidural analgesia does not appear to have the same impact following laparoscopic procedures. Laxatives, such as milk of magnesia and bisacodyl, reduce postoperative ileus duration. Prokinetic medications such as metoclopramide are ineffective. Neostigmine increases peristalsis but may also increase the incidence of PONV.

Postoperative chewing gum, by stimulating gastrointestinal reflexes, may decrease ileus duration. Although its effect has not been evaluated in ERP patients, postoperative chewing gum may be included in multimodal interventions to decrease postoperative ileus because of its safety and low cost. Peripheral opioid pt-receptor antagonists methylnaltrexone (Relistor) and alvimopan (Entereg) minimize the adverse effects of opioids on bowel function without antagonizing systemic opioid analgesia because of their limited ability to cross the blood-brain barrier. In laparotomy patients receiving high-dose intravenous morphine analgesia, alvimopan decreases the duration of postoperative ileus by 16 to 18 h, decreases the incidence of nasogastric tube reinsertion, shortens hospital length of stay, and lowers hospital readmission rates, especially in patients undergoing bowel resection. Nevertheless, recovery of postoperative bowel function in such alvimopan-treated, high-dose systemic morphine analgesia patients remains slower than that of patients utilizing opioidsparing, multimodal ERPs.
Excessive perioperative fluid administration commonly causes bowel mucosal edema and delays postoperative return of bowel function. Because ® either excessive, or excessively restricted, perioperative fluid therapy increases the incidence and severity of postoperative ileus, a goal-directed fluid administration strategy may be beneficial, especially in patients undergoing major surgery associated with large fluid shifts and patients at high risk of developing postoperative gastrointestinal complications (see Chapter 51). However, results from a recent randomized double-blind study of liberal versus restricted fluid administration showed no differences with regard to recovery of bowel function in patients undergoing fast-track abdominal surgery.

The success of ERPs depends upon the ability and willingness of perioperative team stakeholders to reach evidence-based interdisciplinary consensus. Many traditional aspects of perioperative care, such as use of drains, dietary and activity restrictions, excessive or excessively restrictive fluid management, and bedrest, must be extensively revised in ERPs. Patient involvement and patient and family expectations are fundamentally important, but frequently overlooked, aspects of these programs. New surgical techniques, such as transverse incisions or minimally invasive surgery, may require surgeons to acquire and perfect new skills. Similarly, the emphasis on thoracic epidural blockade or peripheral nerve blocks, pharmacological modulation of the neuroendocrine stress response to surgery, goal-directed fluid and hemodynamic therapy, and integral involvement of a well-organized and managed APS requires a substantial expansion of the traditional roles of anesthesiologists and of the anesthesia care team. Aggressive analgesia and symptom management, early ambulation and physiotherapy, early nutrition protocols, and early removal or total avoidance of urinary drainage catheters significantly change the way patients are cared for in the postanesthesia recovery unit and on the surgical unit and require a well-organized, highly trained, highly motivated nursing staff.
Although there are published studies of successful ERPs, there are no "off- the-shelf" protocols with universal application: Local differences in goals, expertise, experience, resources, and politics markedly influence the development, implementation, and management of ERPs for each institution or health care system. Each family of similar surgical procedures requires a standardized interdisciplinary clinical protocol or pathway, with specialized input from a team with experience in caring for those patients. Such an interdisciplinary team should include representation from surgery, anesthesiology, nursing, pharmacy, physiotherapy, nutrition, and administration, and it should be responsible not only for each clinical protocol's creation, but also for continuously monitoring its efficacy and cost, and for instituting continuous improvement-related protocol modifications and provider feedback as indicated by outcomes data (Figure 48-5).
FIGURE 48-5 Stepwise process for initiating and implementing an enhanced recovery program. (Reproduced with permission from Kehlet H, Wilmore DW. Evidence-based surgical care and the evolution of fast-track surgery. Ann Surg. 2008 Aug;248(2):189-198.)

Optimal perioperative care requires the anesthesia provider to be an integral part of the perioperative surgical care team leadership and management. The skill sets of the anesthesiologist and anesthesia care team are essential for the success of ERPs and have potential benefits for surgical care delivery on a global basis, from initial surgical diagnosis, preoperative evaluation, and presurgical preparation through postoperative recovery and return of the patient to his or her primary healthcare provider. The perioperative surgical home concept represents an accumulation of developments derived from ambulatory, minimally invasive, fast-track, enhanced recovery, and interdisciplinary, team-based surgical care and allows both qualityand cost-related variables to be analyzed and optimized from the patient's perspective (eg, incidence and severity of perioperative complications, hospital length of stay, hospital readmission rate, return to work, and return to chemotherapy). By optimizing these many variables, the PSH contributes to delivering a higher value surgical experience to the patient. This will require new standards for clinical education and training.
consensus statement
pathway on
for
Perioper colorectal
optimal analgesia
surgery:
Med
(Lond).
Thiele
RH,
Raghunathan
Recovery within
Part
2017;6:7.
K,
(ASER)
and consensus
statement
Brudney
CG,
Perioperative on
2—from an
enhanced
PACU
et al.
recovery to
the transition
American
Quality perioperative
Society home.
for
Initiative
Enhanced
(POQI)
fluid management
recovery pathway
for colorectal
surgery.
Periop joint
within
Med
(Lond).
an enhanced
2016;5:24.

Osmotic pressure is generally dependent only on the number of nondiffusible solute particles. This is because the average kinetic energy of particles in solution is similar regardless of their mass.
Potassium is the most important determinant of intracellular osmotic pressure, whereas sodium is the most important determinant of extracellular osmotic pressure.
Fluid exchange between the intracellular and interstitial spaces is governed by the osmotic forces created by differences in nondiffusible solute concentrations.
@ Serious manifestations of hyponatremia are generally associated with plasma sodium concentrations less than 120 mEq/L.
@ Excessively rapid correction of hyponatremia has been associated with demyelinating lesions in the pons (central pontine myelinolysis), and more generally in both pontine and extrapontine central nervous system structures (osmotic demyelination syndrome), resulting in both temporary and permanent neurological sequelae.
A major hazard of increased extracellular volume is impaired gas exchange due to pulmonary interstitial edema, alveolar edema, and/or large collections of pleural or ascitic fluid.
Intravenous replacement of potassium chloride is usually reserved for patients with, or at risk for, significant cardiac manifestations or severe muscle weakness.
Because of its lethal potential, hyperkalemia exceeding 6 mEq/L should always be corrected.
© Symptomatic hypercalcemia requires rapid treatment. The most effective initial treatment is rehydration followed by a brisk diuresis (urinary output 200-300 mL/h) utilizing intravenous saline infusion and a loop diuretic to accelerate calcium excretion.
@ Symptomatic hypocalcemia is a medical emergency and should be treated immediately with intravenous calcium chloride (3-5 mL of a 10% solution) or calcium gluconate (10-20 mL of a 10% solution).
@ Some patients with severe hypophosphatemia may require mechanical ventilation postoperatively because of muscle weakness.
Severe hypermagnesemia can lead to respiratory and cardiac arrest. 8
Isolated hypomagnesemia should be corrected prior to elective procedures because of its potential for causing cardiac arrhythmias. 8

Fluid and electrolyte disturbances associated with surgical and coexisting medical disease are extremely common in the perioperative period. Moreover, large volumes of intravenous fluids and blood components are frequently required to correct fluid deficits and compensate for blood loss during surgery. Major disturbances in fluid and electrolyte balance can rapidly alter cardiovascular, neurological, and neuromuscular functions, and anesthesia providers must have a clear understanding of normal water and electrolyte physiology. This chapter examines the body's fluid compartments and common water and electrolyte derangements, their treatment, and anesthetic implications. Acid-base disorders and intravenous fluid and blood therapy are discussed in Chapters 50 and 51.

The system of international units (SI) has still not gained universal acceptance in clinical practice, and many older expressions of concentration remain in common use. Thus, for example, the quantity of a solute in a solution may be expressed in grams, moles, or equivalents. To complicate matters further, the concentration of a solution may be expressed either as quantity of solute per volume of solution or quantity of solute per weight of solvent.

One mole (mol) of a substance represents 6.02 x 10*? molecules. Molarity is the standard SI unit of concentration that expresses the number of moles of solute per liter of solution (mol/L, or M). Molality is an alternative term that expresses moles of solute per kilogram of solvent. Equivalency is also commonly used for substances that ionize: The number of equivalents of an ion in solution is the number of moles multiplied by its charge (valence). Thus, a 1 M solution of MgCl, yields 2 equivalents of magnesium per liter and 2 equivalents of chloride per liter.

Osmosis is the net movement of water across a semipermeable membrane as a result of a difference in nondiffusible solute concentrations between the two sides. Osmotic pressure is the pressure that must be applied to the side with more solute to prevent a net movement of water across the membrane to dilute the solute.
@ Osmotic pressure is generally dependent only on the number of nondiffusible solute particles. This is because the average kinetic energy of particles in solution is similar regardless of their mass. One osmole (Osm) equals 1 mol of nondissociable substances. For substances that ionize, however, each mole results inn Osm, where n is the number of ionic species produced. Thus, 1 mol of a highly ionized substance such as NaCl dissolved in solution should produce 2 Osm; in reality, ionic interaction between the cation and anion reduces the effective activity of each such that NaCl behaves as if it is only 75% ionized. A difference of 1 mOsm/L between two solutions results in an osmotic pressure of 19.3 mm Hg. The osmolarity of a solution is equal to the number of osmoles per liter of solution, whereas its osmolality equals the number of osmoles per kilogram of solvent. Tonicity, a term that is often used interchangeably with osmolarity and osmolality, refers to the effect a solution has on cell volume. An isotonic solution has no effect on cell volume, whereas hypotonic and hypertonic solutions increase and decrease cell volume, respectively.

Body water is distributed between two major fluid compartments separated by cell membranes: intracellular fluid (ICF) and extracellular fluid (ECF). The latter can be further subdivided into intravascular and interstitial compartments. The interstitium includes all fluid that is both outside cells and outside the vascular endothelium. The relative contributions of each compartment to total body water

Intracellular, Fluid as Percent Body Weight (%) = 40. Intracellular, TotalBody Water (%) = 67. Intracellular, Fluid Volume (L) = 28. Extracellular Interstitial Intravascular, Fluid as Percent Body Weight (%) = 15. Extracellular Interstitial Intravascular, TotalBody Water (%) = 25. Extracellular Interstitial Intravascular, Fluid Volume (L) = 10.5. , Fluid as Percent Body Weight (%) = 5. , TotalBody Water (%) = 8. , Fluid Volume (L) = 3.5. Total, Fluid as Percent Body Weight (%) = 60. Total, TotalBody Water (%) = 100. Total, Fluid Volume (L) = 42
The volume of fluid (water) within each compartment is determined by its solute composition and concentrations (Table 49—2). Differences in solute concentrations are largely due to the characteristics of the physical barriers that separate compartments. The osmotic forces created by "trapped" solutes govern the distribution of water between compartments and ultimately each compartment's volume.

Gram-Molecular Weight = . Protein (g/dL), Intracellular (mEq/L) = 16. Protein (g/dL), Extracellular.Intravascular (mEq/L) = 7. Protein (g/dL), Extracellular.Interstitial (mEq/L) = 2
'PO, is95g.

The outer membrane of cells plays an important role in regulating intracellular volume and composition. A membrane-bound adenosine triphosphate (ATP)— dependent pump exchanges Na™ for K* in a 3:2 ratio. Because cell membranes are relatively impermeable to sodium and, to a lesser extent, potassium ions, potassium is concentrated intracellularly, whereas sodium is concentrated extracellularly. @ As a result, potassium is the most important determinant of intracellular osmotic pressure, whereas sodium is the most important determinant of extracellular osmotic pressure.
The impermeability of cell membranes to most proteins results in a high intracellular protein concentration. Because proteins act as nondiffusible solutes (anions), the unequal exchange ratio of 3 Na* for 2 K* by the cell membrane pump is critical in preventing relative intracellular hyperosmolality. Interference with Na*—K*-ATPase activity, as occurs during ischemia or hypoxia, results in progressive swelling of cells.

The principal function of ECF is to provide a medium for delivery of cell nutrients and electrolytes and for removal of cellular waste products. Maintenance of a normal extracellular volume—particularly the circulating component (intravascular volume)—is critical. For the reasons described earlier, sodium is quantitatively the most important extracellular cation and the major determinant of extracellular osmotic pressure and volume. Changes in ECF volume are therefore related to changes in total body sodium content. The latter is a function of sodium intake, renal sodium excretion, and extrarenal sodium losses (see later discussion).

Very little interstitial fluid is normally in the form of free fluid. Most interstitial water is in chemical association with extracellular proteoglycans, forming a gel. Interstitial fluid pressure is generally thought to be negative (approximately —5 mm Hg). Increases in extracellular volume are normally proportionately reflected in intravascular and interstitial volume. However, as interstitial fluid volume progressively increases, interstitial pressure also rises and eventually becomes positive. When the latter occurs, free fluid in the interstitial gel matrix increases rapidly and the result is expansion only of the interstitial fluid compartment (Figure 49-1). In this way, the interstitial compartment acts as an overflow reservoir for the intravascular compartment, as seen clinically in tissue edema.
FIGURE 49-1 The relationship between blood volume and extracellular fluid volume. (Modified with permission from Guyton AC. Textbook of Medical Physiology. 7th ed. Philadelphia, PA: WB Saunders; 1986.)
Because only small quantities of plasma proteins can normally cross capillary clefts, the protein content of interstitial fluid is relatively low (2 g/dL). Protein entering the interstitial space is returned to the vascular system via the lymphatic system.

Intravascular fluid, commonly referred to as plasma, is restricted to the intravascular space by the vascular endothelium. Most electrolytes (small ions) freely pass between plasma and the interstitium, resulting in nearly identical electrolyte composition. However, the tight intercellular junctions between adjacent endothelial cells impede the passage of plasma proteins to outside the intravascular compartment. As a result, plasma proteins (mainly albumin) are the only osmotically active solutes in fluid not normally exchanged between plasma and interstitial fluid.

Diffusion is the random movement of molecules due to their kinetic energy and is responsible for the majority of fluid and solute exchange between compartments. The rate of diffusion of a substance across a membrane depends upon (1) the permeability of that substance through that membrane; (2) the concentration difference for that substance between the two sides; (3) the pressure difference between either side, because pressure imparts greater kinetic energy; and (4) the electrical potential across the membrane for charged substances.

Diffusion between interstitial fluid and ICF may take place by one of several mechanisms: (1) directly through the lipid bilayer of the cell membrane, (2) through protein channels within the membrane, or (3) by reversible binding to a carrier protein that can traverse the membrane (facilitated diffusion). Oxygen, CO), water, and lipid-soluble molecules penetrate the cell membrane directly. Cations such as Na*, K*, and Ca** penetrate the lipid membrane poorly and can diffuse only through specific protein channels. Passage through these channels is dependent on membrane voltage and the binding of ligands (such as acetylcholine) to the membrane receptors. Glucose and amino acids diffuse with the help of membrane-bound carrier proteins. governed
® Fluid exchange between the intracellular and interstitial spaces is by the osmotic forces created by differences in nondiffusible solute concentrations. Relative changes in osmolality between the intracellular and interstitial compartments result in a net water movement from the hypoosmolar to the hyperosmolar compartment.

Capillary walls are typically 0.5 pm thick, consisting of a single layer of endothelial cells with their basement membrane. Intercellular clefts, 6 to 7 nm wide, separate each cell from its neighbors. Oxygen, CO,, water, and lipidsoluble substances can penetrate directly through both sides of the endothelial cell membrane. Only low-molecular-weight, water-soluble substances such as sodium, chloride, potassium, and glucose readily cross intercellular clefts. Highmolecular-weight substances such as plasma proteins penetrate the endothelial clefts poorly, with the exception of the liver and the lungs, where the clefts are larger.
Fluid exchange across capillaries differs from that across cell membranes in that it is governed by significant differences in hydrostatic pressures in addition to osmotic forces (Figure 49-2). These forces are operative on both arterial and venous ends of capillaries, with a tendency for fluid to move out of capillaries at the arterial end and back into capillaries at the venous end. Moreover, the magnitude of these forces differs between the various tissue beds. Arterial capillary pressure is determined by precapillary sphincter tone. Thus, capillaries that require a high pressure, such as glomeruli, have low precapillary sphincter tone, whereas the normally low-pressure capillaries of muscle have high precapillary sphincter tone. Normally, all but 10% of the fluid filtered is reabsorbed back into capillaries. What is not reabsorbed (about 2 mL/min) enters the interstitial fluid and is then returned by lymphatic flow to the intravascular compartment.
FIGURE 49-2 Capillary fluid exchange. The numbers in this figure are in mm Hg and indicate the pressure gradient for the respective pressures. "Net" refers to the net pressure at either end of the capillary, ie, 13 mm Hg at the arterial and 7 mm Hg at the venous end of the capillary.

The human body at birth is approximately 75% water by weight. By 1 month this value decreases to 65%, and by adulthood to 60% for males and 50% for females. The higher fat content in females decreases water content. For the same reason, obesity and advanced age further decrease water content.

The normal adult daily water intake averages 2500 mL, which includes approximately 300 mL as a byproduct of the metabolism of energy substrates.
Daily water
loss averages
2500
mL
and is
typically accounted
for by
1500
mL
urine,
400
mL
tract evaporation,
400
mL
in skin
evaporation, in
100
mL in sweat, and 100 mL in feces. Evaporative loss is very important in thermoregulation because this mechanism normally accounts for 20% to 25% of heat loss.
Both ICF and ECF osmolalities are tightly regulated to maintain normal water content in tissues. Changes in water content and cell volume may induce significant impairment of function, particularly in the brain (see later discussion).

The osmolality of ECF is equal to the sum of the concentrations of all dissolved solutes. Because Na'® and its anions account for nearly 90% of these solutes, the following approximation is valid:
Plasma osmolality = 2 x Plasma sodium concentration
Moreover, because ICF and ECF are in osmotic equilibrium, plasma sodium concentration [Na*]plasma generally reflects total body osmolality:
Total body osmolality
Extracellular solutes * Intracellur solutes
=
TEW
Because sodium and potassium are the major intraand extracellular solutes, respectively:

Using these principles, the effect of isotonic, hypotonic, and hypertonic fluid loads on compartmental water content and plasma osmolality can be calculated (Table 49-3). The potential importance of intracellular potassium concentration is readily apparent from this equation. Thus, significant potassium losses may contribute to hyponatremia.
TABLE 49-3 Effect of different fluid loads on extracellular and intracellular water contents.!

Total body solute = 280 mOsm/kg x 42 kg = 11,760 mOsm Intracellular solute = 280 mOsm/kg x 25 kg = 7000 mOsm Extracellular solute = 280 mOsm/kg x 17 kg = 4760 mOsm
Extracellular sodium concentration = 280 + 2= 140 mEq/L
Osmolality, Intracellular = . Osmolality, Extracellular = . Volume (L), Intracellular = 25. Volume (L), Extracellular = 17. Net water gain, Intracellular = 0. Net water gain, Extracellular = 0

Total body solute = 280 mOsm/kg x 44 kg = 12,320 mOsm
Intracellular solute = 280 mOsm/kg x 25 kg = 7000 mOsm
Extracellular solute = 280 mOsm/kg x 19 kg = 5320 mOsm
Osmolality, Intracellular = 280. Osmolality, Extracellular = 280. Volume (L), Intracellular = 25. Volume (L), Extracellular = 19. Net water gain, Intracellular = 0. Net water gain, Extracellular = 2
Net effect: Fluid remains in extracellular compartment.

New body water =42+2=44kg
New body osmolality = 11,760 mOsm + 44 kg = 267
mOsm/kg
New intracellular volume = 7000 mOsm + 267 mOsm/kg = 26.2 kg
New extracellular sodium concentration = 267 +2 = 133 mEq/L
Osmolality, Intracellular = 267.0. Osmolality, Extracellular = 267.0. Volume (L), Intracellular = 26.2. Volume (L), Extracellular = 178. Net water gain, Intracellular = +1.2. Net water gain, Extracellular = +08
Net effect: Fluid distributes between both compartments.

Total body solute = 11,760 + 600 = 12,360 mOsm/kg New body osmolality = 12,360 mOsm/kg + 42 kg = 294 mOsm
New extracellular solute = 600 + 4760 = 5360 mOsm New extracellular volume = 5360 mOsm + 294 mOsm/kg =18.2kg
New intracellular volume = 42 -18.2 = 23.8 kg
New extracellular sodium concentration = 294 + 2 = 147 mEq/L
Osmolality, Intracellular = 294.0. Osmolality, Extracellular = 294.0. Volume (L), Intracellular = 23.8. Volume (L), Extracellular = 18.2. Net water gain, Intracellular = -1.2. Net water gain, Extracellular = +1.2
Net effect: An intracellular to extracellular movement of water.
'Based on a 70-kg adult male.
In pathological states, glucose and—to a much lesser extent—urea can contribute significantly to extracellular osmolality. A more accurate approximation of plasma osmolality is therefore given by the following equation:
where [Na'] is expressed as mEq/L and blood urea nitrogen (BUN) and glucose as mg/dL. Urea is an ineffective osmole because it readily permeates cell membranes and is therefore frequently omitted from this calculation:

Plasma osmolality normally varies between 280 and 290 mOsm/L. Plasma sodium concentration decreases approximately 1 mEq/L for every 62 mg/dL increase in glucose concentration. A discrepancy between the measured and calculated osmolality is referred to as an osmolal gap. Significant osmolal gaps indicate a high concentration of an abnormal osmotically active molecule in plasma such as ethanol, mannitol, methanol, ethylene glycol, or isopropyl alcohol. Osmolal gaps may also be seen in patients with chronic kidney failure (attributed to retention of small solutes), patients with ketoacidosis (as a result of a high concentration of ketone bodies), and those receiving large amounts of glycine (as during transurethral resection of the prostate). Lastly, osmolal gaps may also be present in patients with marked hyperlipidemia or hyperproteinemia. In such instances, the protein or lipid part of plasma contributes significantly to plasma volume; although plasma [Na'] is decreased, [Na*] in the water phase of plasma (true plasma osmolality) remains normal. The water phase of plasma is normally only 93% of its volume; the remaining 7% consists of plasma lipids and proteins.

Plasma osmolality is closely regulated by the hypothalamus, which controls both the secretion of antidiuretic hormone (ADH) and the thirst mechanism. Plasma osmolality is therefore maintained within relatively narrow limits by control of both water intake and water excretion.

Specialized neurons in the supraoptic and paraventricular nuclei of the hypothalamus are sensitive to changes in extracellular osmolality. When ECF osmolality increases, these cells shrink and release ADH from the posterior pituitary. ADH markedly increases water reabsorption in renal collecting tubules (see Chapter 30), which tends to reduce plasma osmolality back to normal. Conversely, a decrease in extracellular osmolality causes osmoreceptors to swell and suppresses the release of ADH. Decreased ADH secretion allows a water diuresis, which tends to increase osmolality to normal. Peak diuresis occurs once circulating ADH is metabolized (90-120 min). With complete suppression of ADH secretion, the kidneys can excrete up to 10 to 20 L of water per day.

Carotid baroreceptors (volume receptors), as well as low-pressure volume receptors in the atria, vena cavae, and pulmonary arteries, also influence ADH release. A fall in wall tension results in a reflex increase of ADH secretion from the posterior pituitary. Increased stretch of these receptors not only suppresses ADH secretion, but the increased atrial volume receptor stretch also increases secretion of atrial natriuretic peptide (ANP; see later discussion), which promotes renal excretion of sodium and water. Increased sympathetic activity associated with conditions such as pain, emotional stress, and hypoxia also promotes ADH release.

Activation of osmoreceptors in the lateral preoptic area of the hypothalamus neurons by increases in ECF osmolality induces thirst, stimulating the individual to drink water. Conversely, hypoosmolality suppresses thirst. Thirst is the major defense mechanism against hyperosmolality and hypernatremia, because it is the only mechanism that increases water intake.

Hyperosmolality occurs whenever total body solute content increases relative to TBW and is usually, but not always, associated with hypernatremia ([Na*] > 145 mEq/L). Hyperosmolality without hypernatremia may be seen during marked hyperglycemia or following the accumulation of abnormal osmotically active substances in plasma (see earlier discussion). In the latter two instances, plasma sodium concentration may actually decrease as water is drawn from the intracellular to the extracellular compartment. For every 100 mg/dL increase in plasma glucose concentration, plasma sodium decreases approximately 1.6 mEq/L.
Hypernatremic patients may be hypovolemic, euvolemic, or hypervolemic (Table 49-4). However, hypernatremia is nearly always the result of either a relative loss of water in excess of sodium (hypotonic fluid loss) or the retention of large quantities of sodium. Even when kidney concentrating ability is impaired, thirst is normally highly effective in preventing hypernatremia. Hypernatremia is therefore most commonly seen in debilitated patients who are unable to drink, the very aged, the very young, and patients with altered consciousness. Much of the total body sodium is stored in the skin, bone, and cartilage, which serves as a reservoir for the rest of the body, and patients with dysnatremias may have a low, normal, or high total body sodium content

Body fluid loss (eg, burns, sweating)
Diuretic use
Gastrointestinal loss (eg, vomiting, diarrhea, fistulas)
Heat injury
Osmotic diuresis (eg, hyperosmolar nonketotic coma, enteral feeding)
Post-obstructive diuresis

Central diabetes insipidus
Nephrogenic diabetes insipidus
Fever
Hyperventilation/mechanical ventilation
Hypodipsia
Medications (eg, amphotericin aminoglycosides, lithium, phenytoin)
Sickle cell disease
Suprasellar and infrasellar tumors

Cushing syndrome
Hemodialysis
Hyperaldosteronism
Iatrogenic (eg, salt tablet or salt water ingestion, saline infusions, saline enemas, intravenous bicarbonate, enteral feedings)
1Data from Braun MM, Barstow CH, Pyzocha NJ. Diagnosis and management of sodium disorders:
Hyponatremia and hypernatremia. Am Fam Phys. 2015 Mar 1;91(5):299-307.
FIGURE 49-3 Solute and water balance and the plasma sodium concentration. Plasma sodium concentration is determined according to the ratio of sodium and potassium to total body water. This concentration is altered by net external intake/output balances of sodium, potassium, and water and by internal exchanges between sodium that is free in solution and sodium that is bound to polyanionic proteoglycans in bone, cartilage, and skin. (Reproduced with permission from Sterns RH. Disorders of plasma sodium—Causes, consequences, and correction. N Engl J Med. 2015 Jan 1;372(1):55-65.)

These patients have lost both sodium and water, but the water loss is in relative excess to that of the sodium loss. Hypotonic losses can be renal (osmotic diuresis) or extrarenal (diarrhea or sweat). In either case, patients usually manifest signs of hypovolemia (see Chapter 51). Urinary sodium concentration is generally greater than 20 mEq/L with renal losses and less than 10 mEq/L with extrarenal losses.

This group of patients generally manifests signs of water loss without overt hypovolemia unless the water loss is massive. Total body sodium content is generally normal. Nearly pure water losses can occur via the skin, respiratory tract, or kidneys. Occasionally transient hypernatremia is observed with movement of water into cells following exercise, seizures, or rhabdomyolysis. The most common cause of hypernatremia in conscious patients with normal total body sodium content is diabetes insipidus. Diabetes insipidus (DI) is characterized by marked impairment in renal concentrating ability that is due either to decreased ADH secretion (central DI) or failure of the renal tubules to respond normally to circulating ADH (nephrogenic DI). Rarely, essential hypernatremia may be encountered in patients with central nervous system disorders. These patients appear to have "reset" osmoreceptors that function at a higher baseline osmolality.

Lesions in or around the hypothalamus and the pituitary stalk frequently produce DI. DI often develops with brain death. Transient DI is also commonly seen following neurosurgical procedures and head trauma. The diagnosis is suggested by a history of polydipsia, polyuria (often >6 L/d), and the absence of hyperglycemia. In the perioperative setting, the diagnosis of DI is suggested by marked polyuria without glycosuria and a urinary osmolality lower than plasma osmolality. The absence of thirst in unconscious individuals leads to marked water losses and can rapidly produce hypovolemia. The diagnosis of central DI is confirmed by an increase in urinary osmolality following the administration of exogenous ADH. Aqueous vasopressin (5-10 units subcutaneously or intramuscularly every 4-6 h) is the treatment of choice for acute central DI. Vasopressin in oil (0.3 mL intramuscularly every day) is longer lasting but is more likely to cause water intoxication. Desmopressin (DDAVP), a synthetic analogue of ADH with a 12to 24-h duration of action, is available as an intranasal preparation (10-40 mcg/d either as a single daily dose or divided into two doses) that can be used in both ambulatory and perioperative settings.

Nephrogenic DI can be congenital but is more commonly secondary to other disorders, including chronic kidney disease, hypokalemia, hypercalcemia, sickle cell disease, and hyperproteinemias. Nephrogenic DI can also be secondary to the side effects of some drugs (amphotericin B, lithium, demeclocycline, ifosfamide, mannitol). ADH secretion in nephrogenic DI is normal, but the kidneys fail to respond to ADH and urinary concentrating ability is therefore impaired. The diagnosis is confirmed by failure of the kidneys to produce hypertonic urine following the administration of exogenous ADH. Treatment is generally directed at the underlying illness and ensuring an adequate fluid intake. Volume depletion by a thiazide diuretic can paradoxically decrease urinary output by reducing water delivery to collecting tubules. Sodium and protein restriction can similarly reduce urinary output.

This condition most commonly results from the administration of large quantities of hypertonic saline solutions (3% NaCl or 7.5% NaHCO3). Patients with primary hyperaldosteronism and Cushing syndrome may also have elevations in serum sodium concentration along with signs of increased sodium retention.

Neurological manifestations predominate in patients with symptomatic hypernatremia, and restlessness, lethargy, and hyperreflexia can progress to seizures, coma, and ultimately death. Symptoms correlate more closely with the rate of movement of water out of brain cells than with the absolute level of hypernatremia. Rapid decreases in brain volume can rupture cerebral veins and result in focal intracerebral or subarachnoid hemorrhage. Seizures and serious neurological damage are common, particularly in children with acute hypernatremia when plasma [Na*] exceeds 158 mEq/L. Chronic hypernatremia is usually better tolerated than the acute form. After 24 to 48 h, intracellular osmolality begins to rise as a result of increases in intracellular inositol and amino acid concentrations, and brain intracellular water content slowly returns to normal.

The treatment of hypernatremia is aimed at restoring plasma osmolality to normal and correcting the underlying cause. Water deficits should generally be corrected over 48 h, as rapid correction (or overcorrection) can cause cerebral edema. Enteral free water administration is preferable when feasible, but a hypotonic intravenous solution such as 5% dextrose in water can also be used (see later discussion). Abnormalities in extracellular volume must also be corrected (Figure 49-4). Hypernatremic patients with decreased total body sodium should be given isotonic fluids to restore plasma volume to normal prior to treatment with a hypotonic solution. Hypernatremic patients with increased total body sodium should be treated with a loop diuretic along with intravenous 5% dextrose in water. The treatment of DI is discussed in the preceding section.

Rapid correction of hypernatremia can result in seizures, brain edema, permanent neurological damage, and even death. Serial Na* osmolalities should be obtained during treatment. In general, decreases in plasma sodium concentration should not proceed at a rate faster than 0.5 mEq/L/h.
Example: A 70-kg man is found to have a plasma [Na*] of 160 mEq/L. What is his water deficit?
If one assumes that hypernatremia in this case represents water loss only, then total body osmoles are unchanged. Thus, assuming a normal [Na] of 140 mEq/L and TBW content that is 60% of body weight:
Normal TBW x 140 = Present TBW x[Na" ]yjasma OF (70 0.6) x 140 = Present TBW x 160

Present TBW = 36.7 L
Water deficit = Normal TBW — Present TBW or (70 x 0.6) — 36.7 =5.3 L
To replace this deficit over 48 h, it is necessary to administer 5.3 L enteral free water in small amounts over 48 h, or, 5% dextrose in water intravenously, 5300 mL over 48 h, or 110 mL/h.
Note that this method ignores any coexisting isotonic fluid deficits, which if present should be replaced with an isotonic solution.

Hypernatremia has been demonstrated to increase the minimum alveolar concentration for inhalation anesthetics in animal studies, but its clinical significance is more closely related to the associated fluid deficits. Hypovolemia accentuates any vasodilation or cardiac depression from anesthetic agents and predisposes to hypotension and hypoperfusion of tissues. Decreases in the volume of distribution for drugs necessitate dose reductions for most intravenous agents, whereas decreases in cardiac output enhance the uptake of inhalation anesthetics.
Even mild serum sodium elevation is associated with increased perioperative morbidity, mortality, and hospital length of stay, and thus hypernatremia must not be ignored. Elective anesthetics should be postponed in patients with significant hypernatremia (>150 mEq/L) until the cause is established and total body sodium or TBW, or both, corrected.

Hypoosmolality is nearly always associated with hyponatremia ([Na*] <135 mEq/L). 'Table 49-5 lists rare instances in which hyponatremia does not necessarily reflect hypoosmolality (pseudohyponatremia). Routine measurement of plasma osmolality in hyponatremic patients rapidly excludes pseudohyponatremia.

Asymptomatic
Marked hyperlipidemia
Marked hyperproteinemia
Symptomatic
Marked glycine absorption during transurethral surgery
Hyponatremia with an elevated plasma osmolality

Administration of mannitol
Hyponatremia invariably reflects water retention from either an absolute increase in TBW or a loss of sodium in relative excess to loss of water. The kidneys' normal capacity to produce dilute urine with an osmolality as low as 40 mOsm/kg (specific gravity 1.001) allows them to excrete over 10 L of free water per day if necessary. Because of this tremendous reserve, hyponatremia is nearly always the result of a defect in urinary diluting capacity (urinary osmolality >100 mOsm/kg or specific gravity >1.003—ie, limited ability of the kidneys to excrete free water). Rare instances of hyponatremia without an abnormality in renal diluting capacity (urinary osmolality <100 mOsm/kg) are generally attributed to primary polydipsia or reset osmoreceptors; the latter two conditions can be differentiated by water restriction.
Clinically, hyponatremia is best classified according to total body sodium content (Table 49-6). Hyponatremia associated with transurethral resection of the prostate is discussed in Chapter 32.

Diuretics
Mineralocorticoid deficiency
Salt-losing nephropathies
Osmotic diuresis (glucose, mannitol)
Renal tubular acidosis
Extrarenal
Vomiting
Diarrhea
Integumentary loss (sweating, burns)

Primary polydipsia
Syndrome of inappropriate antidiuretic hormone
Glucocorticoid deficiency
Hypothyroidism

Progressive losses of both sodium and water eventually lead to extracellular volume depletion. As the intravascular volume deficit approaches 5% to 10%, nonosmotic ADH secretion is activated (see earlier discussion). With further volume depletion, the stimuli for nonosmotic ADH release overcome any hyponatremia-induced suppression of ADH. and preservation of circulatory volume takes place at the expense of plasma osmolality. Fluid losses resulting in hyponatremia may be renal or extrarenal in origin. Renal losses are most commonly related to thiazide diuretics and result in a urinary [Na*] greater than 20 mEq/L. Extrarenal losses are typically gastrointestinal and usually are associated with a urinary [Na] of less than 10 mEq/L. A major exception to the latter is hyponatremia due to vomiting, which can result in a urinary [Na*] greater than 20 mEq/L. In this situation, renal compensation for the associated metabolic alkalosis results in bicarbonaturia, with concomitant excretion of Na* with HCO to maintain electrical neutrality in the urine; urinary chloride concentration, however, is usually less than 10 mEq/L.

Edematous disorders are characterized by an increase in both total body sodium and TBW. When the increase in TBW is relatively greater than the increase in total body sodium, hyponatremia occurs. Edematous disorders include congestive heart failure, cirrhosis, kidney failure, and nephrotic syndrome. Hyponatremia in these settings results from progressive impairment of renal free water excretion and generally parallels underlying disease severity. Pathophysiological mechanisms include nonosmotic ADH release and decreased delivery of fluid to nephron distal diluting segments (see Chapter 30).

Hyponatremia in the absence of edema or hypovolemia may be seen with glucocorticoid insufficiency, hypothyroidism, drug therapy, and the syndrome of inappropriate antidiuretic hormone secretion (SIADH, also referred to as the syndrome of inappropriate diureses [SIAD]; 'Table 49—7). The hyponatremia associated with adrenal hypofunction may be due to co-secretion of ADH with corticotropin-releasing factor (CRF). Diagnosis of SIADH requires exclusion of other causes of hyponatremia and the absence of hypovolemia, edema, and adrenal, renal, or thyroid disease. Various malignant tumors, pulmonary diseases, and central nervous system disorders are commonly associated with SIADH. In most such instances, plasma ADH concentration is not elevated but is inadequately suppressed relative to the degree of hypoosmolality in plasma; urine osmolality is usually greater than 100 mOsm/kg and urine sodium concentration is greater than 40 mEq/L.

Stimulate AVP release
Chlorpropamide
Clofibrate
Carbamazepine
Vincristine
SSRIs
MDMA
Ifosfamide
Antipsychotics
Opioids
Potentiate action of AVP
NSAIDs
Cyclophosphamide
AVP analogues
Desmopressin
Oxytocin
Vasopressin
Cerebral salt wasting (CSW) is a syndrome of inappropriate renal sodium wasting and hyponatremia with polyuria and hypovolemia that may be seen with intracranial disease, including brain tumors, subarachnoid hemorrhage, subdural hematoma, meningitis, and head trauma. Proposed mechanisms for this disorder include excess secretion of natriuretic peptides and altered sympathetic stimulation to the kidney. Both SIADH and CSW are characterized by elevated urine sodium concentration, low serum osmolality, and high urine osmolality. However, patients with SIADH are usually euvolemic or mildly hypervolemic, whereas patients with CSW are hypovolemic, and thus treatments for these two disorders are very different. The treatment of SIADH is free water restriction, and the treatment of CSW is volume and sodium replacement with normal or hypertonic saline.

Symptoms of hyponatremia are primarily neurological and result from an increase in intracellular water. Their severity is generally related to the rapidity with which extracellular hypoosmolality develops. Patients with mild to moderate hyponatremia ([Na*] >125 mEq/L) are frequently asymptomatic. Early symptoms are typically nonspecific and may include anorexia, nausea, and weakness. Progressive cerebral edema, however, results in lethargy, confusion, @ seizures, coma, and finally death. Serious manifestations of hyponatremia are generally associated with plasma sodium concentrations less than 120 mEq/L. Patients with slowly developing or chronic hyponatremia are generally less symptomatic, probably because the gradual compensatory loss of intracellular solutes (primarily Na*, K*, and organic osmolytes) restores cell volume to near normal. Neurological symptoms in patients with chronic hyponatremia may be related more closely to changes in cell membrane potential (due to a low extracellular [Na*]) than to changes in cell volume.

As with hypernatremia, the treatment of hyponatremia (Figure 49—5) is directed at correcting both the underlying disorder as well as the plasma [Na'*]. Isotonic saline is generally the treatment of choice for hyponatremic patients with decreased total body sodium content. Once the ECF deficit is corrected, spontaneous water diuresis returns plasma [Na*] to normal. Conversely, water restriction is the primary treatment for hyponatremic patients with normal or increased total body sodium. More specific treatments such as hormone replacement in patients with adrenal or thyroid hypofunction and measures aimed at improving cardiac output in patients with heart failure may also be indicated. Demeclocycline (Declomycin, Declostatin), a tetracycline antibiotic that antagonizes ADH activity at the renal tubules, is often used as an adjunct in the treatment of SIADH when water restriction alone is insufficient.

Acute, symptomatic hyponatremia requires prompt treatment. In such instances, correction of plasma [Na'] to greater than 125 mEq/L is usually sufficient to alleviate symptoms and signs. The amount of NaCl necessary to raise plasma [Na'*] to the desired value, the Na* deficit, can be estimated by the following formula:

@ Excessively rapid correction of hyponatremia has been associated with demyelinating lesions in the pons (central pontine myelinolysis) and more generally in both pontine and extrapontine central nervous system structures (osmotic demyelination syndrome), resulting in both temporary and permanent neurological sequelae. The rapidity with which hyponatremia is corrected should be tailored to the severity of symptoms. The following correction rates have been suggested: for mild symptoms, 0.5 mEq/L/h or less; for moderate symptoms, 1 mEq/L/h or less; and for severe symptoms, 1.5 mEq/L/h or less.
Example: An 80-kg woman is lethargic and is found to have plasma [Na*] of 118 mEq/L. How much NaCl must be given to raise her plasma [Na*] to 130 mEq/L?
Na' deficit = TBW x (130 — 118)
TBW is approximately 50% of body weight in females:
Na' deficit = 80 x 0.5 x (130 — 118) = 480 mEq

Because normal (isotonic) saline contains 154 mEq/L, the patient should receive 480 mEq + 154 mEd/L, or 3.12 L of normal saline. For a correction rate of 0.5 mEq/L/h, this amount of saline should be given over 24 h (130 mL/h). Note that this calculation does not take into account any coexisting isotonic fluid deficits, which, if present, should also be replaced. More rapid correction of hyponatremia can be achieved by giving a loop diuretic to induce water diuresis while replacing urinary Na* losses with isotonic saline. Even more rapid corrections can be achieved with intravenous hypertonic saline (3% NaCl). Hypertonic saline may be indicated in markedly symptomatic patients with plasma [Na'] less than 110 mEq/L. Three percent NaCl should be administered with caution, as it can precipitate pulmonary edema, hypokalemia, hyperchloremic metabolic acidosis, and transient hypotension; bleeding associated with prolongation of the prothrombin time and activated partial thromboplastin time has been reported.

Hyponatremia is the most common electrolyte disorder, and SIADH is its most common cause. Hyponatremia, in association with its underlying disorder(s), increases both perioperative morbidity and mortality. A plasma sodium concentration greater than 130 mEq/L is usually considered safe for patients undergoing general anesthesia. In most circumstances, plasma [Na*] should be corrected to greater than 130 mEq/L for elective procedures, even in the absence of neurological symptoms. Lower concentrations may result in significant cerebral edema that can be manifested intraoperatively as a decrease in minimum alveolar concentration or postoperatively as agitation, confusion, or somnolence. Patients undergoing transurethral resection of the prostate can absorb significant amounts of water from irrigation fluids (as much as 20 mL/min) and are at high risk for rapid development of profound acute water intoxication (see Chapter

ECF volume is directly proportionate to total body sodium content. Variations in ECF volume result from changes in total body sodium content. A positive sodium balance increases ECF volume, whereas a negative sodium balance decreases ECF volume. Extracellular (plasma) Na*™ concentration is more indicative of water balance than total body sodium content.

Net sodium balance is equal to total sodium intake (adults average 170 mEq/d) minus both renal sodium excretion and extrarenal sodium losses. (One gram of sodium yields 43 mEq of Na'* ions, whereas 1 g of sodium chloride yields 17 mEq of Na* ions.) The kidneys' ability to vary urinary Na* excretion from less than 1 mEq/L to more than 100 mEq/L allows them to play a critical role in sodium balance (see Chapter 30).

Because of the relationship between ECF volume and total body sodium content, regulation of one is intimately tied to the other. This regulation is achieved via sensors that detect changes in the most important component of ECF, namely, the "effective" intravascular volume. The latter correlates more closely with the rate of perfusion in renal capillaries than with measurable intravascular fluid (plasma) volume. Indeed, with edematous disorders (heart failure, cirrhosis, and kidney failure), "effective" intravascular volume can be independent of the measurable plasma volume, ECF volume, and even cardiac output. ECF volume and total body sodium content are ultimately controlled by appropriate adjustments in renal Na* excretion. In the absence of kidney disease, diuretic therapy, and renal ischemia, urinary Na* concentration reflects "effective" intravascular volume. A low urine Na' concentration (<10 mEq/L) is therefore generally indicative of a low "effective" intravascular fluid volume and reflects secondary retention of Na* by the kidneys.

The multiple mechanisms involved in regulating ECF volume and sodium balance normally complement one another but can function independently.

Baroreceptors are the principal volume receptors in the body. Significant changes in intravascular volume (preload) not only affect cardiac output but also affect arterial blood pressure. The baroreceptors at the carotid sinus and afferent renal arterioles (juxtaglomerular apparatus) indirectly function as sensors of intravascular volume. Changes in blood pressure at the carotid sinus modulate sympathetic nervous system activity and nonosmotic ADH secretion, whereas changes at the afferent renal arterioles modulate the renin—angiotensin— aldosterone system (RAAS). As previously noted, stretch receptors in both atria are affected by changes in intravascular volume, and the degree of atrial distention modulates the release of atrial natriuretic hormone and ADH.

Regardless of the mechanism, effectors of volume change ultimately alter urinary Na' excretion. Decreases in "effective" intravascular volume decrease urinary Na' excretion, whereas increases in the "effective" intravascular volume increase urinary Na* excretion. These mechanisms include the following:
1.
Renin—angiotensin—aldosterone—Renin secretion
increases the
angiotensin
II.
The latter
secretion effect
in enhancing
II
is also
Na*
a
potent reabsorption
in direct
norepinephrine.
of aldosterone
the and
proximal vasoconstrictor
Secretion and
renal tubules.
potentiates enhances
has
a
direct
Angiotensin the
Na*
actions of
reabsorption in
the distal
nephron
(see
Chapter
30)
and is
a
major determinant
of urinary
Na*
excretion.
2. Atrial natriuretic peptide (ANP)—This peptide is normally released from both right and left atrial cells in response to atrial distention. ANP appears to have two major actions: arterial vasodilation and increased urinary sodium and water excretion in the renal collecting tubules. Na*-mediated afferent arteriolar dilation and efferent arteriolar constriction can also increase glomerular filtration rate (GFR). Other effects include the inhibition of both renin and aldosterone secretion and antagonism of ADH.
3. Brain natriuretic peptide (BNP)—ANP, BNP, and C-type natriuretic peptide

Osmoregulation protects the normal ratio of solutes to water, whereas extracellular volume regulation preserves absolute solute and water content

Purpose, Volume Regulation = Control extracellular volume. Purpose, Osmoregulation = Control extracellular osmolality. Mechanism, Volume Regulation = Vary renal Na* excretion. Mechanism, Osmoregulation = Vary water intake Vary renal water excretion. Sensors, Volume Regulation = Afferent renal arterioles Carotid baroreceptors Atrial stretch receptors. Sensors, Osmoregulation = Hypothalamic osmoreceptors. Effectors, Volume Regulation = Renin-angiotensin- aldosterone Sympathetic nervous system Tubuloglomerular balance Renal pressure natriuresis Atrial natriuretic peptide Antidiuretic hormone Brain natriuretic peptide. Effectors, Osmoregulation = Thirst Antidiuretic hormone
'Adapted with permission from Rose RD. Clinical Physiology of AcidBase and Electrolyte Disorders, 3rd ed. New York, NY: McGraw-Hill; 1989.
Anesthetic Implications
Problems related to altered sodium balance (dysnatremia) result from its direct manifestations as well as those of its the underlying disorder(s). Although patients with disorders of sodium balance may be euvolemic, they usually present with either hypovolemia (sodium deficit) or hypervolemia (sodium excess); see Tables 49-4 and 49-6. Both disturbances should be corrected prior to elective surgical procedures. Cardiac, liver, and kidney function should also be carefully evaluated in the presence of sodium excess (typically manifested as tissue edema).
Hypovolemic patients are sensitive to the vasodilating and negative inotropic effects of vapor anesthetics, propofol, and drug-induced histamine release. Dosage requirements for other drugs may be reduced to compensate for decreases in their volume of distribution. Hypovolemic patients are particularly sensitive to sympathetic blockade from spinal or epidural anesthesia. If a general anesthesia must be administered prior to adequate correction of hypovolemia, etomidate or ketamine may be the induction agents of choice.
Hypervolemia may be corrected preoperatively @ with diuretics. A major hazard of increased extracellular volume is impaired gas exchange due to pulmonary interstitial edema, alveolar edema, and/or large collections of pleural or ascitic fluid.

Potassium plays a major role in regulating membrane potential as well as in carbohydrate and protein synthesis (see later discussion). Intracellular potassium concentration is estimated to be 140 mEq/L, whereas normal extracellular potassium concentration is approximately 4 mEq/L.

Dietary potassium intake averages 80 mEq/d in adults (range, 40-140 mEq/d). About 70 mEq of that amount is normally excreted in urine, whereas the remaining 10 mEq is lost through the gastrointestinal tract.
Renal excretion of potassium can vary from as little as 5 mEq/L to over 100 mEq/L. Nearly all the potassium filtered in glomeruli is normally reabsorbed in the proximal tubule and the loop of Henle. As a component of the RAAS, the mineralocorticoid aldosterone is secreted by the adrenal cortex in response to stimulation by angiotensin II. Acting on mineralocorticoid receptors in the distal tubules and collecting ducts of the kidney, aldosterone promotes sodium resorption directly coupled to potassium secretion. Thus, through this influence on kidney reabsorption/loss of sodium, and the resultant regulatory influence on kidney water reabsorption/loss and blood volume and blood pressure, aldosterone also directly influences potassium balance (see earlier discussion and Chapter 30). Mineralocorticoid receptor antagonists (MRAs) block the sodium retention/potassium loss effects of aldosterone. The potassium-sparing diuretics spironolactone and eplerenone are MRAs that exemplify these effects by promoting sodium and water loss along with potassium retention. Amiloride and triamterene also antagonize the sodium retention/potassium loss effects of aldosterone, but do so by blocking sodium channels in renal collecting tubule epithelial cells.

Extracellular potassium concentration is determined by cell membrane Na*—K*ATPase activity and plasma [K*], and is influenced by the balance of total body potassium intake and excretion. Cell membrane Na*—K*-ATPase activity regulates the distribution of potassium between cells and ECF, whereas plasma [K*], nephron Na* load, and aldosterone are the major determinants of urinary potassium excretion.

Intercompartmental shifts of potassium are known to occur following changes in extracellular pH (see Chapter 50), circulating insulin levels, circulating catecholamine activity, and plasma osmolality. Insulin and catecholamines directly affect Na*-K*-ATPase activity and decrease plasma [K*]. Exercise may transiently increase plasma [K*] as a result of the release of K* by muscle cells; the increase in plasma [K*] (0.3—2 mEq/L) is proportionate to the intensity and duration of muscle activity. Intercompartmental potassium shifts are also likely to be responsible for changes in plasma [K*] in syndromes of periodic paralysis (see Chapter 29).
Because the ICF may buffer up to 60% of an acid load (see Chapter 50), changes in extracellular hydrogen ion concentration (pH) directly affect extracellular [K*]. In the setting of acidosis, extracellular hydrogen ions enter

Hypokalemia, defined as plasma [K*] less than 3.5 mEq/L, can occur as a result of (1) an intercompartmental shift of K*, (2) increased potassium loss, or (3) an inadequate potassium intake (Table 49-9). Plasma potassium concentration typically correlates poorly with the total potassium deficit. A decrease in plasma [K*] from 4 mEq/L to 3 mEq/L usually represents a 100to 200-mEq total body deficit, whereas plasma [K*] below 3 mEq/L can represent a deficit of 200 mEq to 400 mEq.

Mineralocorticoid excess
Primary hyperaldosteronism (Conn syndrome)
Glucocorticoid-remediable hyperaldosteronism
Renin excess
Renovascular hypertension
Bartter syndrome
Liddle syndrome
Diuresis
Chronic metabolic alkalosis
Carbenicillin
Gentamicin
Amphotericin B
Distal, gradient-limited
Proximal
Ureterosigmoidostomy

Vomiting
Diarrhea, particularly secretory diarrheas
ECF — ICF shifts
Acute alkalosis
Hypokalemic periodic paralysis
Barium ingestion
Vitamin Bj therapy
Thyrotoxicosis (rarely)
Inadequate intake

Hypokalemia due to the intracellular movement of potassium occurs with alkalosis, insulin therapy, B,-adrenergic agonists, and hypothermia and during attacks of hypokalemic periodic paralysis. Cellular K* uptake by red blood cells (and platelets) also accounts for the hypokalemia seen in patients recently treated with folate or vitamin B,» for megaloblastic anemia.

Excessive potassium loss is usually either renal or gastrointestinal. Renal wasting of potassium is most commonly the result of diuresis or enhanced mineralocorticoid activity. Other renal causes include hypomagnesemia, renal tubular acidosis (see Chapter 30), ketoacidosis, salt-wasting nephropathies, and some drug therapies (amphotericin B). Increased gastrointestinal loss of potassium is most commonly due to nasogastric suctioning or to persistent vomiting or diarrhea. Other gastrointestinal causes include losses from fistulae, laxative abuse, villous adenomas, and pancreatic tumors secreting vasoactive intestinal peptide.
Chronic increased sweat formation occasionally causes hypokalemia when potassium intake is limited. Dialysis with a low-potassium-containing dialysate solution can also cause hypokalemia. Uremic patients may actually have a total body potassium deficit (primarily intracellular) despite a normal or even high plasma concentration; the absence of hypokalemia in these instances is probably due to acidosis-induced intercompartmental shifts. Dialysis in these patients unmasks the total body potassium deficit and often results in hypokalemia.
Urinary [K*] less than 20 mEq/L is generally indicative of increased extrarenal K* losses, whereas concentrations greater than 20 mEq/L suggest renal wasting of K*.

Because of the kidney's ability to decrease urinary potassium excretion to as low as 5 to 20 mEq/L, marked reductions in potassium intake are required to produce hypokalemia. Low potassium intake, however, often accentuates the effects of increased loss.
potassium

Hypokalemia can produce widespread organ dysfunction (Table 49-10). Most patients are asymptomatic until plasma [K*] falls below 3 mEq/L. Cardiovascular effects are most prominent and include an abnormal electrocardiogram (ECG; Figure 49-6), arrhythmias, decreased cardiac contractility, and a labile arterial blood pressure due to autonomic dysfunction. ECG manifestations are primarily due to delayed ventricular repolarization and include T-wave flattening and inversion, an increasingly prominent U wave, STsegment depression, increased P-wave amplitude, and prolongation of the P—R interval. Increased myocardial cell automaticity and delayed repolarization promote both atrial and ventricular arrhythmias.

Polyuria (nephrogenic diabetes insipidus)
Increased ammonia production
Increased bicarbonate reabsorption

Decreased insulin secretion
Decreased aldosterone secretion
Metabolic

with permission from Schrier RW. Renal and Electrolyte Disorders. 3rd ed. Philadelphia, PA:
Brown and 1986.
1 adapted Little, Company;
FIGURE 49-6 Electrocardiographic effects of acute hypokalemia. Note progressive flattening of the T wave, an increasingly prominent U wave, increased amplitude of the P wave, prolongation of the P—R interval, and STsegment depression.
Neuromuscular effects of hypokalemia include skeletal muscle weakness, hyporeflexia, muscle cramping, ileus, and, rarely, flaccid paralysis or rhabdomyolysis. Hypokalemia induced by diuretics is often associated with metabolic alkalosis; as the kidneys absorb sodium to compensate for intravascular volume depletion and in the presence of diuretic-induced hypochloremia, bicarbonate is absorbed and potassium is excreted. The end result is hypokalemia and hypochloremic metabolic alkalosis. Renal dysfunction is seen due to impaired concentrating ability (resistance to ADH, resulting in polyuria) and increased production of ammonia resulting in impairment of urinary acidification. Increased ammonia production represents intracellular acidosis; hydrogen ions move intracellularly to compensate for intracellular potassium losses. The resulting metabolic alkalosis, together with increased ammonia production, can precipitate encephalopathy in patients with advanced liver disease.

The treatment of hypokalemia depends on the presence and severity of any associated organ dysfunction. Significant ECG changes such as ST-segment changes or arrhythmias mandate continuous ECG monitoring, particularly during intravenous K* replacement. Digoxin therapy—as well as the hypokalemia itself—sensitizes the heart to changes in potassium ion concentration.
In most circumstances, the safest method by which to correct a potassium deficit is oral replacement @ over several days (60-80 mEq/d). Intravenous replacement of potassium chloride is usually reserved for patients with, or at risk for, significant cardiac manifestations or severe muscle weakness. The goal of intravenous therapy is to remove the patient from immediate danger, not to correct the entire potassium deficit. Because of potassium's irritative effect on peripheral veins, peripheral intravenous replacement should not exceed 8 mEq/h. More rapid intravenous potassium replacement (10—20 mEq/h) requires central venous administration and close ECG monitoring. Intravenous replacement should generally not exceed 240 mEq/d. Dextrose-containing solutions should be avoided because the resulting hyperglycemia and secondary insulin secretion may worsen the low plasma [K*].
Potassium chloride is the preferred potassium salt when a metabolic alkalosis is also present because it also corrects the chloride deficit discussed earlier. Potassium bicarbonate or equivalent (K* acetate or K* citrate) is preferable for patients with metabolic acidosis. Potassium phosphate is a suitable alternative with concomitant hypophosphatemia (eg, diabetic ketoacidosis).

Hypokalemia is a common preoperative finding. The decision to proceed with elective surgery is often based on lower plasma [K*] limits somewhere around 3 mEq/L. The decision, however, should also be based on the rate at which the hypokalemia developed as well as the presence or absence of secondary organ dysfunction. In general, chronic mild hypokalemia (3-3.5 mEq/L) without ECG changes does not increase anesthetic risk. The exception is patients receiving digoxin, who risk developing digoxin toxicity from the hypokalemia; plasma [K*] values above 4 mEq/L are desirable in such patients.
The intraoperative management of hypokalemia requires vigilant ECG monitoring. Intravenous potassium should be given if atrial or ventricular arrhythmias develop. Glucose-free intravenous solutions should be used and hyperventilation avoided to prevent further decreases in plasma [K*]. Increased sensitivity to neuromuscular blockers (NMBs) may occur.

Hyperkalemia exists when plasma [K*] exceeds 5.5 mEq/L, and rarely occurs in normal individuals because of the kidney's capability to excrete large potassium loads. When potassium intake is increased slowly, the kidneys can excrete as much as 500 mEq of K* per day. The sympathetic nervous system and insulin secretion also play important roles in preventing acute increases in plasma [K*] following acquired potassium loads.
Hyperkalemia can result from (1) an intercompartmental shift of potassium ions, (2) decreased urinary excretion of potassium, or, rarely, (3) an increased potassium intake or increased release from a formerly ischemic organ (Table 49-11). Measurements of plasma potassium concentration can be spuriously elevated if red cells hemolyze in a blood specimen. In vitro release of potassium from blood specimen leukocytes can also falsely indicate increased levels in the measured plasma [K*] when the leukocyte count exceeds 70,000 x 109/L. A similar release of potassium from platelets may occur when the platelet count exceeds 1,000,000 x 109/L.

Acidosis
Hypertonicity
Rhabdomyolysis
Excessive exercise
Periodic paralysis
Succinylcholine

Decreased mineralocorticoid activity and impaired Na* reabsorption
Acquired immunodeficiency syndrome
Potassium-sparing diuretics
Spironolactone
Eplerenone
Amiloride
Triamterene
ACE! inhibitors
Nonsteroidal antiinflammatory drugs
Pentamidine
Trimethoprim
Enhanced CI reabsorption
Gordon syndrome
Increased potassium intake
Salt substitutes
lace, angiotensin-converting enzyme.

Movement of K* out of cells can be seen with acidosis, cell lysis following chemotherapy, hemolysis, rhabdomyolysis, massive tissue trauma, hyperosmolality, digitalis overdoses, during episodes of hyperkalemic periodic paralysis, and with administration of succinylcholine, B-adrenergic blockers, and arginine hydrochloride. The average increase in plasma [K*] of 0.5 mEq/L following succinylcholine administration can be exaggerated in patients with large burns or severe muscle trauma and in those with muscle denervation, and its use in these settings should be avoided.

Decreased renal excretion of potassium can result from (1) marked reductions in glomerular filtration, (2) decreased aldosterone activity, or (3) a defect in potassium secretion in the distal nephron.
Glomerular filtration rates less than 5 mL/min are nearly always associated with hyperkalemia. Patients with lesser degrees of kidney impairment can also readily develop hyperkalemia when faced with increased potassium loads (dietary, catabolic, or iatrogenic). Uremia may also impair Na*—K*-ATPase

Hyperkalemia due to decreased aldosterone activity can result from a primary defect in adrenal hormone synthesis or a defect in the RAAS. Patients with primary adrenal insufficiency (Addison disease) and those with isolated 21hydroxylase adrenal enzyme deficiency have marked impairment of aldosterone synthesis. Patients with the syndrome of isolated hypoaldosteronism (also called hyporeninemic hypoaldosteronism, or type IV renal tubular acidosis) are usually diabetics with renal impairment; they have an impaired ability to increase aldosterone secretion in response to hyperkalemia. These patients develop hyperkalemia when they increase their potassium intake or when given potassium-sparing diuretics. They also often have varying degrees of Na* wasting and a hyperchloremic metabolic acidosis.
Drugs interfering with the RAAS have the potential to cause hyperkalemia, particularly in the presence of kidney disease. Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit prostaglandin-mediated renin release. Angiotensinconverting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) interfere with angiotensin II—mediated release of aldosterone. Heparin used in thromboprophylaxis regimens may cause hyperkalemia (heparin—induced hyperkalemia [HIH]) by interfering with aldosterone secretion and antagonizing the activity of angiotensin II receptors. Potassium-sparing diuretics antagonize aldosterone activity in the kidney, impairing potassium excretion (see earlier discussion).
Decreased renal excretion of potassium may also occur as a result of an intrinsic or acquired defect in the distal nephron's ability to secrete potassium. Such defects may occur even in the presence of normal renal function and are characteristically unresponsive to mineralocorticoid therapy. The kidneys of patients with pseudohypoaldosteronism display an intrinsic resistance to aldosterone. Acquired defects have been associated with systemic lupus erythematosus, sickle cell anemia, obstructive uropathies, and cyclosporine nephropathy in transplanted kidneys.

Increased potassium loads rarely cause hyperkalemia in normal individuals unless large amounts are given rapidly and intravenously. Hyperkalemia, however, may be seen when potassium intake is increased in patients receiving B-blockers or in patients with impaired kidney function. Unrecognized sources of potassium include potassium penicillin, sodium substitutes (primarily potassium salts), and transfusion of stored whole blood. The plasma [K*] in a unit of whole blood can increase to 30 mEq/L after 21 days of storage. The risk of hyperkalemia from multiple transfusions (transfusion-associated hyperkalemia) is reduced, although not eliminated, by minimizing the volume of plasma given through the use of packed red blood cell transfusions or by using washed red blood cells (see Chapter 51).

The most important effects of hyperkalemia are on skeletal and cardiac muscle. Skeletal muscle weakness is generally not seen until plasma [K*] is greater than 8 mEq/L. Cardiac manifestations (Figure 49—7) are primarily due to delayed depolarization and are consistently present when plasma [K*] is greater than 7 mEq/L. ECG changes characteristically progress sequentially from symmetrically peaked T waves (often with a shortened QT interval) — widening of the QRS complex = prolongation of the P—R interval = loss of the P wave — loss of R-wave amplitude — ST-segment depression (occasionally elevation) — an ECG that resembles a sine wave, before progressing to ventricular fibrillation and asystole. Contractility may be relatively well preserved until late in the course of progressive hyperkalemia. Hypocalcemia, hyponatremia, and acidosis accentuate the cardiac effects of hyperkalemia.
FIGURE 49-7 Electrocardiographic effects of hyperkalemia.
Electrocardiographic changes characteristically progress from symmetrically peaked T waves, often with a shortened QT interval, to widening of the QRS complex, prolongation of the P—R interval, loss of the P wave, loss of R-wave amplitude, and ST-segment depression (occasionally elevation)—to an ECG that resembles a sine wave—before final progression into ventricular fibrillation or asystole.

be used to treat urgent or emergent hyperkalemia because of its delayed onset of action.

Elective surgery should not be undertaken in patients with significant hyperkalemia. Anesthetic management of hyperkalemic perioperative patients is directed at both lowering the plasma potassium concentration and preventing any further increases, with treatment approach dependent upon situational acuity. The ECG should be carefully monitored. Succinylcholine is contraindicated, as is the use of potassium-containing intravenous solutions. The avoidance of metabolic or respiratory acidosis is critical to prevent further increases in plasma [K*]. Ventilation should be controlled under general anesthesia, and mild hyperventilation may be desirable. Lastly, neuromuscular function should be monitored closely, as hyperkalemia can accentuate the effects of NMBs.

Although 98% of total body calcium is in bone, maintenance of a normal extracellular calcium concentration is critical to homeostasis. Calcium ions are involved in nearly all essential biological functions, including muscle contraction, the release of neurotransmitters and hormones, blood coagulation, and bone metabolism, and abnormalities in calcium balance can result in profound physiological derangements.

Calcium intake in adults averages 600 to 800 mg/d, and calcium absorption occurs primarily in the proximal small bowel. Calcium is also secreted into the intestinal tract, a phenomenon that appears to be constant and independent of absorption. Up to 80% of the daily calcium intake is normally lost in feces. The kidneys are responsible for most calcium excretion. Renal calcium excretion averages 100 mg/d but may vary from as low as 50 mg/d to more than 300 mg/d. Normally, 98% of the filterable calcium is reabsorbed. Calcium reabsorption parallels that of sodium in the proximal renal tubules and the ascending loop of Henle. In the distal tubules, however, calcium reabsorption is dependent on parathyroid hormone (PTH) secretion, whereas sodium reabsorption is dependent on aldosterone secretion. Increased PTH levels enhance distal calcium reabsorption and thereby decrease urinary calcium excretion.

The normal plasma calcium concentration is 8.5 to 10.5 mg/dL (2.1—2.6 mmol/L). Approximately 50% is in the free, ionized form, 40% is protein bound (mainly to albumin), and 10% is complexed with anions such as citrate and amino acids. The free, ionized calcium concentration ([Ca?*]) is physiologically most important. Plasma [Ca2*] is normally 4.75 to 5.3 mg/dL (1.19-1.33 mmol/L). Changes in plasma albumin concentration affect the total, but not ionized, calcium concentration: For each increase or decrease of 1 g/dL in albumin, the total plasma calcium concentration increases or decreases approximately 0.8 to 1.0 mg/dL, respectively.
Changes in plasma pH directly affect the degree of protein binding and thus ionized calcium concentration. Ionized calcium increases approximately 0.16 mg/dL for each decrease of 0.1 unit in plasma pH and decreases by the same amount for each 0.1 unit increase in pH.

Calcium normally enters ECF by either absorption from the intestinal tract or resorption of bone; only 0.5% to 1% of calcium in bone is exchangeable with ECF. In contrast, calcium normally leaves the extracellular compartment by (1) deposition into bone, (2) urinary excretion, (3) secretion into the intestinal tract, and (4) sweat formation. Extracellular [Ca**] is closely regulated by three hormones—PTH, vitamin D, and calcitonin—that act primarily on bone, the distal renal tubules, and the small bowel. Hormonal control of calcium is described in Chapter 35.

Hypercalcemia can occur as a result of a variety of disorders (Table 49-12). In primary hyperparathyroidism, secretion of PTH is inappropriately increased in relation to [Ca**]. In contrast, in secondary hyperparathyroidism (eg, chronic kidney failure or malabsorption syndromes), PTH levels are elevated in response to chronic hypocalcemia. Prolonged secondary hyperparathyroidism, however, can occasionally result in autonomous secretion of PTH, resulting in a normal or elevated [Ca?*] (tertiary hyperparathyroidism).

Hyperparathyroidism
Malignancy
Excessive vitamin D intake
Paget disease of bone
Granulomatous disorders (sarcoidosis, tuberculosis)
Chronic immobilization
Milk-alkali syndrome
Adrenal insufficiency
Thiazide diuretics
Lithium
Patients with cancer can present with hypercalcemia whether or not bone metastases are present. Most often this is due to direct bony destruction, or secretion of humoral mediators of hypercalcemia (PTH-like substances, cytokines, or prostaglandins), or both. Hypercalcemia due to increased turnover of calcium from bone can also be encountered in patients with benign conditions such as Paget disease and chronic immobilization. Increased gastrointestinal absorption of calcium can lead to hypercalcemia in patients with the milk-alkali syndrome (marked increase in calcium intake), excessive vitamin D intake, or granulomatous diseases (enhanced sensitivity to vitamin D).

Hypercalcemia often produces anorexia, nausea, vomiting, weakness, and polyuria. Ataxia, irritability, lethargy, or confusion can rapidly progress to coma. Hypertension is often present initially before hypovolemia supervenes. ECG signs include a shortened ST segment and shortened QT interval. Hypercalcemia increases cardiac sensitivity to digitalis. Hypercalcemia may promote pancreatitis, peptic ulcer disease, and kidney failure.

® Symptomatic hypercalcemia requires rapid treatment. The most effective initial treatment is rehydration followed by a brisk diuresis (urinary output 200300 mL/h) utilizing intravenous saline infusion and a loop diuretic to accelerate calcium excretion. Premature diuretic therapy prior to rehydration may aggravate the hypercalcemia by exacerbating volume depletion. Renal loss of potassium and magnesium usually occurs during diuresis, mandating laboratory monitoring and intravenous replacement as necessary. Although hydration and diuresis may remove the potential risk of cardiovascular and neurological complications of hypercalcemia, the serum calcium level usually remains elevated above normal. Additional therapy with a bisphosphonate or calcitonin may be required to further lower the serum calcium level. Severe hypercalcemia (>15 mg/dL) usually requires additional therapy after saline hydration and furosemide calciuresis. Bisphosphonates or calcitonin are preferred agents. Intravenous administration of pamidronate (Aredia) or etidronate (Didronel) is often utilized in this setting. Hemodialysis is very effective in correcting severe hypercalcemia and may be necessary in the presence of kidney or heart failure. Additional treatment depends on the underlying cause of the hypercalcemia and may include glucocorticoids in the setting of vitamin D—induced hypercalcemia such as granulomatous disease states.
It is necessary to look for the underlying etiology and direct appropriate treatment toward the cause of the hypercalcemia once any critical hypercalcemia threat has been addressed. Approximately 90% of all hypercalcemia is due to either malignancy or hyperparathyroidism. The best laboratory test for discriminating between these two main categories of hypercalcemia is the PTH assay. The serum PTH concentration is usually suppressed in malignancy states and elevated in hyperparathyroidism.

Significant hypercalcemia is a medical emergency and should be corrected before any elective anesthetic. Ionized calcium levels should be monitored closely. If surgery must be performed, saline diuresis should be continued intraoperatively with care to avoid hypovolemia; appropriate goal-directed hemodynamic and fluid management therapy (see Chapter 51) should be utilized, especially for patients with cardiac or kidney impairment. Serial measurements of [K*] and [Mg?*] are obtained in anticipation of diuresis-related hypokalemia and hypomagnesemia. Responses to anesthetic agents and NMBs are not predictable. Ventilation should be controlled under general anesthesia. Acidosis should be avoided so as to not worsen the elevated plasma [Ca2*].

Hypocalcemia should be diagnosed only on the basis of the plasma ionized calcium concentration. When direct measurements of plasma [Ca**] are not available, the total calcium concentration must be corrected for decreases in plasma albumin concentration (see earlier discussion). Causes of hypocalcemia are listed in Table 49-13.

Hypoparathyroidism
Pseudohypoparathyroidism
Vitamin D deficiency
Nutritional
Malabsorption
Postsurgical (gastrectomy, short bowel)
Inflammatory bowel disease
Altered vitamin D metabolism

Precipitation of calcium
Fat embolism

Multiple rapid red blood transfusions or rapid infusion of large amounts of albumin
Hypocalcemia due to hypoparathyroidism is a relatively common cause of symptomatic hypocalcemia. Hypoparathyroidism may be surgical (see Chapter 37), idiopathic, part of multiple endocrine defects (most often with adrenal insufficiency), or associated with hypomagnesemia. Magnesium deficiency may impair the secretion of PTH and antagonize the effects of PTH on bone. Sepsis is often accompanied by suppressed PTH release, resulting in hypocalcemia.

Manifestations of hypocalcemia include paresthesias, confusion, laryngeal stridor (laryngospasm), carpopedal spasm (Trousseau sign), masseter spasm (Chvostek sign), and seizures. Biliary colic and bronchospasm have also been described. ECG may reveal cardiac irritability or QT interval prolongation, which may not correlate in severity with the degree of hypocalcemia. Decreased cardiac contractility may result in heart failure, hypotension, or both. Decreased responsiveness to digoxin and B-adrenergic agonists may also occur.

© Symptomatic hypocalcemia is a medical emergency and should be treated immediately with intravenous calcium chloride (3-5 mL of a 10% solution) or calcium gluconate (10-20 mL of a 10% solution). Ten mL of 10% CaCl, contains 272 mg of Ca**, whereas 10 mL of 10% calcium gluconate contains 93 mg of Ca**. To avoid precipitation, intravenous calcium should not be given with bicarbonateor phosphate-containing solutions. Serial ionized calcium monitoring is mandatory. Repeat intravenous boluses or a continuous infusion (Ca** 1-2 mg/kg/h) may be necessary. Plasma magnesium concentration should be checked to exclude hypomagnesemia. In chronic hypocalcemia, oral calcium (CaCO3) and vitamin D replacement are usually adequate.

Significant hypocalcemia should be corrected preoperatively. Serial ionized calcium levels should be monitored intraoperatively in patients with a history of hypocalcemia. Alkalosis should be avoided to prevent further decreases in [Ca?*]. Intravenous calcium may be necessary following rapid transfusions of citrated blood products or large volumes of albumin solutions (see Chapter 51). Potentiation of the negative inotropic effects of anesthetics should be expected. Responses to NMBs are inconsistent and require nerve stimulator monitoring.

Phosphorus is an important intracellular constituent. Its presence is required for the synthesis of (1) the phospholipids and phosphoproteins in cell membranes and intracellular organelles, (2) the phosphonucleotides involved in protein synthesis and reproduction, and (3) ATP used for the storage of energy. Only 0.1% of total body phosphorus is in ECF, with 85% in bone and 15% intracellular.

Phosphorus intake averages 800 to 1500 mg/d in adults, and 80% of that amount is normally absorbed in the proximal small bowel. Vitamin D increases intestinal absorption of phosphorus. The kidneys are the major route for phosphorus excretion and are responsible for regulating total body phosphorus content. Urinary excretion of phosphorus depends on both intake and plasma concentration. Secretion of PTH promotes urinary phosphorus excretion by inhibiting proximal tubular reabsorption. The latter effect may be offset by PTHinduced release of phosphate from bone.

Plasma phosphorus exists in both organic and inorganic forms. Organic phosphorus is mainly in the form of phospholipids. Of the inorganic phosphorus fraction, 80% is filterable in the kidneys and 20% is protein bound. The majority of inorganic phosphorus is in the form of H,PO,and HPO," in a 1:4 ratio. By convention, plasma phosphorus is measured as milligrams of elemental phosphorus. Normal plasma phosphorus concentration is 2.5 to 4.5 mg/dL (0.8— 1.45 mmol/L) in adults and up to 6 mg/dL in children. Plasma phosphorus concentration is usually measured during fasting, because recent carbohydrate intake transiently decreases plasma phosphorus concentration. Hypophosphatemia increases vitamin D production, whereas hyperphosphatemia depresses it. The latter plays an important role in the genesis of secondary hyperparathyroidism in patients with chronic kidney failure (see Chapter 31).

Hyperphosphatemia may be seen with increased phosphorus intake (abuse of phosphate laxatives or excessive potassium phosphate administration), decreased phosphorus excretion (chronic kidney disease), or tumor lysis syndrome (see earlier discussion).

Although hyperphosphatemia per se does not appear to be directly responsible for any functional disturbances, significant hyperphosphatemia may produce hypocalcemia via phosphate chelation with plasma [Ca?*] and may also produce acute kidney injury via parenchymal and tubular deposits of calcium-phosphate salts. Hyperphosphatemia is associated with increased mortality in chronic kidney disease and kidney failure patients, and is managed in this patient population by dietary restriction, the use of phosphate binders, dialysis, or a combination of these methods.

Hyperphosphatemia is generally treated with phosphate-binding antacids such as aluminum hydroxide or aluminum carbonate.

Although specific interactions between hyperphosphatemia and anesthesia have not been described, kidney function should be assessed and hypocalcemia should be excluded.

Hypophosphatemia is usually the result of either a negative phosphorus balance or cellular uptake of extracellular phosphorus (an intercompartmental shift). Intercompartmental shifts of phosphorus can occur during alkalosis and following carbohydrate ingestion or insulin administration. Large doses of aluminumor magnesium-containing antacids, severe burns, insufficient phosphorus supplementation during total parenteral nutrition, diabetic ketoacidosis, alcohol withdrawal, and prolonged respiratory alkalosis can each produce negative phosphorus balance and lead to severe hypophosphatemia (<0.3 mmol/dL or <1.0 mg/dL). In contrast to respiratory alkalosis, metabolic alkalosis rarely leads to severe hypophosphatemia.

Mild to moderate hypophosphatemia (1.5—2.5 mg/dL) is generally asymptomatic. In contrast, severe hypophosphatemia (<1.0 mg/dL) is associated with increased morbidity and mortality in critically ill patients. Cardiomyopathy, impaired oxygen delivery (decreased 2,3-diphosphoglycerate levels), hemolysis, impaired leukocyte function, platelet dysfunction, encephalopathy, arrhythmia, skeletal myopathy, respiratory failure, rhabdomyolysis, skeletal demineralization, metabolic acidosis, and hepatic dysfunction have all been associated with severe hypophosphatemia. However, at this time it is uncertain whether hypophosphatemia is a direct and independent contributor to these major morbidities or to mortality, or is merely a marker of illness severity.

Oral phosphorus replacement is generally preferable to parenteral replacement because of the increased risk of phosphate precipitation with calcium, resulting in hypocalcemia, and also because of the increased risks of hyperphosphatemia, hypomagnesemia, and hypotension. Accordingly, intravenous replacement therapy is usually reserved for instances of symptomatic hypophosphatemia and extremely low phosphate levels (<0.32 mmol/L). In situations where oral phosphate replacement is utilized, vitamin D is required for intestinal phosphate absorption.

Anesthetic management of patients with hypophosphatemia requires familiarity with its potential complications (see earlier discussion). Hyperglycemia and respiratory alkalosis should be avoided to prevent further decreases in plasma phosphorus concentration. Neuromuscular function must be @ monitored carefully when NMBs are given. Some patients with severe hypophosphatemia may require mechanical ventilation postoperatively because of muscle weakness.

Magnesium functions as a cofactor in many enzyme pathways. Only 1% to 2% of total body magnesium stores is present in the ECF compartment, with 67% contained in bone and the remaining 31% intracellular. Magnesium decreases anesthetic requirements, attenuates nociception, blunts the cardiovascular response to laryngoscopy and intubation, and potentiates NMBs. Suggested mechanisms of action include altering central nervous system neurotransmitter release, moderating adrenal medullary catecholamine release, and antagonizing the effect of calcium on vascular smooth muscle. Magnesium impairs the calcium-mediated presynaptic release of acetylcholine and may also decrease motor end-plate sensitivity to acetylcholine and alter myocyte membrane potential.
In addition to the treatment of magnesium deficiency, magnesium is used to treat preeclampsia and eclampsia, torsades de pointes, and digoxin-induced cardiac tachyarrhythmias.

Magnesium intake averages 20 to 30 mEq/d (240-370 mg/d) in adults. Of that amount, only 30% to 40% is absorbed, mainly in the distal small bowel. Renal excretion is the primary route for elimination, averaging 6 to 12 mEd/d. Magnesium reabsorption by the kidneys is very efficient. Twenty-five percent of filtered magnesium is reabsorbed in the proximal tubule and 50% to 60% is reabsorbed in the thick ascending limb of the loop of Henle. Factors known to increase magnesium reabsorption in the kidneys include hypomagnesemia, PTH, hypocalcemia, ECF depletion, and metabolic alkalosis. Factors known to increase renal excretion include hypermagnesemia, acute volume expansion, aldosterone, hypercalcemia, ketoacidosis, diuretics, phosphate depletion, and alcohol ingestion.

Plasma [Mg**] is closely regulated between 1.7 and 2.1 mEq/L (0.7-1 mmol/L or 1.7—2.4 mg/dL) through interaction of the gastrointestinal tract (absorption), bone (storage), and the kidneys (excretion). Approximately 50% to 60% of plasma magnesium is unbound and diffusible.

Increases in plasma [Mg*] are nearly always due to excessive intake (magnesium-containing antacids or laxatives: magnesium hydroxide, Milk of Magnesia), kidney impairment (GFR <30 mL/min), or both. Less common causes include adrenal insufficiency, hypothyroidism, rhabdomyolysis, and lithium administration. Magnesium sulfate therapy for preeclampsia and eclampsia can result in maternal and fetal hypermagnesemia.

Symptomatic hypermagnesemia typically presents with neurological, neuromuscular, and cardiac manifestations, including hyporeflexia, sedation, muscle weakness, and respiratory depression. Vasodilation, bradycardia, and myocardial depression may cause hypotension. ECG signs may include prolongation of the P—-R interval and widening of the QRS complex. ® Severe hypermagnesemia can lead to respiratory and cardiac arrest.

With relatively mild hypermagnesemia, all that is usually necessary is to discontinue source(s) of magnesium intake (most often antacids or laxatives). In cases of relatively high [Mg**], and especially in the presence of clinical signs of magnesium toxicity, intravenous calcium can temporarily antagonize most of the effects of clinical toxicity. Forced diuresis with a loop diuretic and intravenous fluid replacement enhances urinary magnesium excretion in patients with adequate renal function. When diuretic administration with intravenous infusion is used to enhance magnesium excretion in urgent or emergent cases of magnesium toxicity, serial measurements of [Ca**] and [Mg?*] should be obtained, a urinary catheter is required, and goal-directed hemodynamic and fluid management should be considered. Dialysis will be necessary in such patients with significant kidney impairment or kidney failure. Ventilatory or circulatory support, or both, may be necessary.

Hypermagnesemia requires close monitoring of the ECG, blood pressure, and neuromuscular function. Potentiation of the vasodilatory and negative inotropic properties of anesthetics should be expected. Dosages of nondepolarizing NMBs should be reduced.

Hypomagnesemia is a common problem, particularly in critically ill patients, and is often associated with deficiencies of other intracellular components such as potassium and phosphorus. It is commonly found in patients undergoing major cardiothoracic or abdominal operations, and its incidence among patients in intensive care units may exceed 50%. Deficiencies of magnesium are generally the result of inadequate intake, reduced gastrointestinal absorption, increased renal excretion, or a combination of these factors (Table 49-14). Drugs that cause renal wasting of magnesium include ethanol, theophylline, diuretics, cisplatin, aminoglycosides, cyclosporine, amphotericin B, pentamidine, and granulocyte colony-stimulating factor. Hypomagnesemia has also been associated with long-term proton pump inhibitor (PPI) therapy, with such cases ascribed to impaired intestinal absorption of magnesium. However, the prevalence of hypomagnesemia among patients with chronic use of PPIs is low, probably less than 1%. Hypomagnesemia is common after cardiopulmonary bypass unless patients receive intraoperative supplementation, due to hemodilution and the frequent use of albumin, transfusion, and other magnesium-lowering constituents in the priming solution.

Malabsorption syndromes
Small bowel or biliary fistulas
Prolonged nasogastric suctioning
Severe vomiting or diarrhea
Chronic laxative abuse
Chronic proton pump inhibitor (PPI) use

Diuresis
Diabetic ketoacidosis
Nephrotoxic drugs
Postobstructive diuresis

Chronic alcoholism
Protein—calorie malnutrition
Hyperthyroidism
Burns

Most patients with hypomagnesemia are asymptomatic, but weakness, fasciculation, paresthesias, confusion, ataxia, and seizures may be encountered.
Hypomagnesemia is frequently associated with both hypocalcemia (impaired
PTH secretion) and hypokalemia (due to renal K* wasting). Cardiac manifestations include arrhythmias and potentiation of digoxin toxicity; both are
worsened by hypokalemia. Hypomagnesemia is associated with an increased
incidence of atrial fibrillation. Prolongation of the P—R and QT intervals may also be present.

Asymptomatic hypomagnesemia can be treated orally or intramuscularly. Serious manifestations such as seizures should be treated with intravenous magnesium sulfate, 1 to 2 g (8-16 mEq or 4-8 mmol) given over 10 to 60 min.

Although no specific anesthetic interactions are described, coexistent electrolyte disturbances such as hypokalemia, hypophosphatemia, and hypocalcemia are often present and should be corrected prior ® to surgery. Isolated hypomagnesemia should be corrected prior to elective procedures because of its potential to cause arrhythmias. Moreover, magnesium appears to have intrinsic antiarrhythmic properties and possibly cerebral protective effects (see Chapter 26). It is frequently administered preemptively to lessen the risk of postoperative atrial fibrillation in patients undergoing cardiac surgery.

A 70-year-old man with carcinoma of the bladder presents for radical cystectomy and ileal loop urinary diversion. He weighs 70 kg and has a 20-year history of hypertension. Preoperative laboratory measurements revealed normal plasma electrolyte concentrations and a BUN of 20 mg/dL with a serum creatinine of 1.5 mg/dL. The operation lasts 4 h and is performed under general anesthesia. The estimated blood loss is 900 mL. Fluid replacement consists of 3500 mL of lactated Ringer's injection and 750 mL of 5% albumin.
One hour after admission to the postanesthesia care unit, the patient is awake, his blood pressure is 130/70 mm Hg, and he appears to be breathing well (18 breaths/min, Fio, = 0.4). Urinary output has been only
20
mL
in the
last hour.
Laboratory measurements
are as
follows:
Hb,
10.4
g/dL;
plasma
Nat+,
133
mEq/L;
K+,
3.8
mEq/L;
Cl,
104
mEq/L; total CO,, 20 mmol/L; Pao,, 156 mm Hg; arterial blood pH, 7.29; PacO, 38 mm Hg; and calculated HCO3-, 18 mEq/L. (See Table 49-15 for normal ranges or common electrolytes.)

hypocalcemia and hypokalemia. Anion, Normal Values (mmol/L) = . Anion, Effect of Excess = . Anion, Effect of Deficit = . Chloride, Normal Values (mmol/L) = 95-105. Chloride, Effect of Excess = Possible acute renal impairment. Chloride, Effect of Deficit = Unknown-/related to associated abnormality. Phosphate, Normal Values (mmol/L) = 0.8-1.5. Phosphate, Effect of Excess = Symptoms of acute hypocalcaemia, acute tubular necrosis, ectopic calcification. Phosphate, Effect of Deficit = Below 0.32 mmol/L: respiratory muscle dysfunction, left shift of oxyhemoglobin dissociation curve, myocardial dysfunction, arrhythmias, myopathy, encephalopathy, irritability, seizures, coma, rhabdomyolysis, hemolytic anemia
'Reproduced with permission from Tan SC, Freebairn R. Electrolyte disorders in the critically ill. Anaesth Intens Care Med. 2017 Niar;18(3)-133-137.

Multiple factors tend to promote hyponatremia postoperatively, including nonosmotic ADH secretion due to surgical stress, hypovolemia, or pain; large evaporative and functional fluid losses (tissue sequestration); and the administration of hypotonic intravenous fluids. Hyponatremia is particularly common postoperatively in patients who have received relatively large amounts of lactated Ringer's injection ([Na*] 130 mEq/L); the postoperative plasma [Na'] generally approaches 130 mEq/L in such patients. (Fluid replacement in this patient was appropriate considering basic maintenance requirements, blood loss, and the additional fluid losses usually associated with this type of surgery.)
Why is the patient hyperchloremic and acidotic (normal arterial

ileal conduits is often transient and usually due to urinary stasis. Persistent or more severe acidosis requires treatment with sodium bicarbonate. Potassium replacement may also be required if hypokalemia is present.
Are electrolyte abnormalities seen with other types of urinary diversion?
Procedures employing bowel as a conduit (ileal or colonic) are less likely to result in hyperchloremic metabolic acidosis than those in which bowel functions as a reservoir. The incidence of hyperchloremic metabolic acidosis approaches 80% following ureterosigmoidostomies.

Al Dhaybi O, Bakris G. Mineralocorticoid antagonists in chronic kidney disease. Curr Opin Nephrol Hypertens. 2017;26:50.
Arampatzis S, Frauchiger B, Fiedler G-M, et al. Characteristics, symptoms, and outcome of severe dysnatremias present on hospital admission. Am J Med. 2012;125:1125.e1.
Awad
S,
Allison
SP,
Lobo
DN.
The history
of
0.9%
saline.
Clin
Nutr.
2008;27:179.
Aylwin
S,
Burst
V,
Peri
A, et
al.
“Dos and
don’ts”
in the
hyponatremia.
Curr
Med
Res
Opin.
2015;31:1755.
Ball SG, Iqbal Z. Diagnosis and treatment of hyponatraemia. Best Pract Res Clin Endocrinol Metabol. 2016;30:161.
Bhaskar
B,
Fraser
JF,
Mullaney
D.
Lest we
forget:
Heparin-induced hyperkalemia.
J
Cardiothorac
Braun
MM,
Barstow
Vasc
CH,
Pyzocha
Anesth.
2012;26:106.
NJ.
Diagnosis and
sodium disorders:
Hyponatremia and
hypernatremia.
Am
Fam
Phys.
2015;91:299.
Buffington MA, Abreo K. Hyponatremia: A review. J Intensive Care Med. 2016;31:223.
Campese VM, Adenuga G. Electrophysiological and clinical consequences of hyperkalemia. Kidney Int Suppl. 2016;6:16.
Chatzizisis
YS,
Misirli
G,
Hatzitolios
AI, et
al.
The syndrome
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Complications and
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Cooper MS, Gittoes NJL. Diagnosis and management of hypocalcaemia. Brit Med J. 2008;336:1298.

- @ Changes in temperature affect Pco, PO», and pH. Both Pco, and Po,
decrease during
hypothermia, but
pH
increases because
temperature does not appreciably alter [HCO3 ] and the dissociation of water
decreases (decreasing H* and increasing pH).
Nearly all biochemical reactions in the body are dependent on maintenance of a physiological hydrogen ion concentration, and alterations beyond normal hydrogen ion concentration are associated with widespread organ dysfunction. This regulation—usually referred to as acid-base balance—is of prime importance in critical illness. Changes in ventilation and perfusion, as well as infusion of electrolyte-containing solutions, are common during anesthesia and can rapidly alter acid—base balance.
Our understanding of acid-base balance is evolving. In the past, we focused on the concentration of hydrogen ions [H*], CO, balance, and the base excess/deficit. We now understand that the @strong ion difference (SID), Pco,, and total weak acid concentration (Ayoy) best explain acid-base balance in physiological systems.
This chapter examines acid-base physiology and the perioperative care implications of common disturbances. Clinical measurements of blood gases and their interpretation are reviewed.

In any aqueous solution, water molecules reversibly dissociate into hydrogen and hydroxide ions:
H,O « H* + OH"
This process is described by the dissociation constant, Kw:
Kw = [H*] + [OH] = 10-4
The concentration of water is omitted from the denominator of this expression because it does not vary appreciably and is already included in the constant. Therefore, given [H*] or [OH7], the concentration of the other ion can be readily calculated.
Example: If [H*] = 10-8 nEq/L, then [OH-] = 10-4 + 10-8 = 10-6 nEq/L.
Arterial [H*] is normally 40 nEq/L, or 40 x 10-9 mol/L. Hydrogen ion concentration is more commonly expressed as pH, which is defined as the negative logarithm (base 10) of [H*] (Figure 50—1). Normal arterial pH is therefore —log (40 x 10~°) = 7.40. Hydrogen ion concentrations between 16 and 160 nEq/L (pH 6.8—7.8) are compatible with life.
FIGURE 50-1 The relationship between pH and [H*]. Note that between a pH of 7.10 and 7.50, the relationship between pH and [H*] is nearly linear. (Reproduced with permission from Narins RG, Emmett M. Simple and mixed acid-base disorders: A practical approach. Medicine. 1980 May;59(3):161-187.)
Like most dissociation constants, Kw is affected by changes in temperature. Thus, the electroneutrality point for water occurs at a pH of 7.0 at 25°C, but at about a pH of 6.8 at 37°C; temperature-related changes may be important during hypothermia (see Chapter 22).
Because physiological fluids are complex aqueous solutions, SID, the Pco,, and Ayor are other factors that affect the dissociation of water into H* and OH.

An acid is usually defined as a chemical species that can act as a proton (H*) donor, whereas a base is a species that can act as a proton acceptor (Brénsted— Lowry definitions). In physiological solutions, it is probably better to use Arrhenius' definitions: An acid is a compound that contains hydrogen and reacts with water to form hydrogen ions. A base is a compound that produces hydroxide ions in water. Using these definitions, the SID becomes important, as other ions in solutions (cations and anions) will affect the dissociation constant for water, and, therefore, the hydrogen ion concentration. A strong acid is a substance that readily and almost irreversibly gives up an H™ and increases [H*], whereas a strong base avidly binds H* and decreases [H*]. In contrast, weak acids reversibly donate H*, whereas weak bases reversibly bind H*; both weak acids and bases tend to have less of an effect on [H*] (for a given concentration of the parent compound) than do strong acids and bases. Biological compounds are either weak acids or weak bases.
For a solution containing the weak acid HA, where
a dissociation constant, K, can be defined as follows:
The negative logarithmic form of the latter equation is called the Henderson— Hasselbalch equation:
From this equation, it is apparent that the pH of this solution is related to the ratio of the dissociated anion to the undissociated acid.
This approach works well with pure water: The concentration of [H*] must equal [OH]. But physiological solutions are far more complex. Even in such a complex solution, the [H*] can be predicted using three variables: the SID, the PcO,, and A7qor.

The SID is the sum of all the strong, completely or almost completely dissociated cations (Na*, K*, Ca2*, Mg*) minus the strong anions (CI, lactate", etc; Figure 50—2). Although we can calculate the SID, because the laws of electroneutrality must be observed, if there is a SID, other unmeasured ions must be present. Pco, is an independent variable, assuming ventilation is ongoing. The conjugate base of HA is A" and is composed mostly of phosphates and proteins that do not change independent of the other two variables. AT plus AH is an independent variable because its value is not determined by any other variable. Note that [H*] is not a strong ion (water does not completely dissociate), but it can, does, and must change in response to any change in SID, PCcO5, or Ayoz to comply with the laws of electroneutrality and conservation of mass. Hydrogen ions are created or consumed based on changes in the dissociation of water.
FIGURE 50-2 The strong ion difference (SID). SIDa, apparent strong ion difference. SIDe, effective strong ion difference. The strong ion gap (SIG) is the difference between SIDa and SIDe and represents the anion gap. (Reproduced with permission from Greenbaum J, Nirmalan M. Acid-base balance: Stewart's physiochemical approach. Curr
Anaesth Crit Care. 2005 June; 16(3):133-135.)

As previously discussed, when the weak acid HA is in solution, HA can act as an acid by donating an H*, and A™ can act as a base by taking up H*. A7 is therefore often referred to as the conjugate base of HA. A similar concept can be applied for weak bases. Consider the weak base B, where
B+H* s BH*
BH' is therefore the conjugate acid of B.
A buffer is a solution that contains a weak acid and its conjugate base or a weak base and its conjugate acid (conjugate pairs). Buffers minimize any change in [H*] by readily accepting or giving up hydrogen ions. It is readily apparent that buffers are most efficient in minimizing changes in the [H*] of a solution (ie, [A~] = [HA]) when pH = pK. Moreover, the conjugate pair must be present in significant quantities in solution to act as an effective buffer.

A clear understanding of acid-base disorders and compensatory physiological responses requires precise terminology (Table 50—1). The suffix "-osis" is used here to denote any pathological process that alters arterial pH. Thus, any disorder that tends to reduce pH to a less than normal value is an acidosis, whereas one tending to increase pH is termed an alkalosis. If the disorder primarily affects [HCO ], it is termed metabolic. If the disorder primarily affects Pacos, it is termed respiratory. Secondary compensatory responses (discussed in the next section) should be referred to as just that and not as an "-osis." For example, one might refer to a metabolic acidosis with respiratory compensation.
TABLE 50-1 Defining acid-base disorders.
Respiratory Acidosis Alkalosis, Primary Change = T Paco, 4 Paco,. Respiratory Acidosis Alkalosis, Compensatory Response = T HCO, 1 HCO,. Metabolic Acidosis Alkalosis, Primary Change = 4 HCO, T HCO,. Metabolic Acidosis Alkalosis, Compensatory Response = 1 Paco, T Paco,
When only one pathological process occurs by itself, the acid-base disorder is considered to be simple (Figure 50—3). The presence of two or more primary processes indicates a mixed acid—base disorder.

The suffix "-emia" is used to denote the net effect of all primary processes and compensatory physiological responses (described next) on arterial blood pH. Because arterial blood pH is normally between 7.35 and 7.45 in adults, the term acidemia signifies a pH less than 7.35, whereas alkalemia signifies a pH greater than 7.45.

Physiological responses to changes in [H*] are characterized by three phases: (1) immediate chemical buffering, (2) respiratory compensation (whenever possible), and (3) a slower, but more effective, renal compensatory response that may nearly normalize arterial pH even if the underlying pathological process remains present.

Although in the strictest sense, the bicarbonate buffer consists of H»CO3 and HCO; , CO, tension (Pco,) may be substituted for HyCO3 because:
H,0 + CO, « H,CO, « H* + HCO,
This hydration of CO, is catalyzed by carbonic anhydrase. If adjustments are made in the dissociation constant for the bicarbonate buffer and if the solubility coefficient for CO, (0.03 mEq/L) is taken into consideration, the Henderson— Hasselbalch equation for bicarbonate can be written as follows:

Note that its pK' is well removed from the normal arterial pH of 7.40, which means that bicarbonate would not be expected to be an efficient extracellular buffer (see earlier discussion). The bicarbonate system is, however, important for two reasons: (1) Bicarbonate (HCO;,) is present in relatively high concentrations in extracellular fluid, and (2) more importantly, Paco, and plasma [HCO ] are closely regulated by the lungs and the kidneys, respectively. The ability of these two organs to alter the [HCO3 ]/Paco, ratio allows them to exert important influences on arterial pH.
A simplified and more practical derivation of the Henderson—Hasselbalch equation for the bicarbonate buffer is as follows:
This equation is very useful clinically because pH can be readily converted to [H*] (Table 50-2). Note that below 7.40, [H*] increases 1.25 nEq/L for each 0.01 decrease in pH; above 7.40, [H*] decreases 0.8 nEq/L for each 0.01 increase in pH.
TABLE 50-2 The relationship between pH and [H'].
6.80, [H*] nEq/L = 158. 6.90, [H*] nEq/L = 126. 7.00, [H*] nEq/L = 100. 7.10, [H*] nEq/L = 79. 7.20, [H*] nEq/L = 63. 7.30, [H*] nEq/L = 50. 7.40, [H*] nEq/L = 40. 7.50, [H*] nEq/L = 32. 7.60, [H*] nEq/L = 25. 7.70, [H*] nEq/L = 20
Example: If arterial pH = 7.28 and Paco, = 24 mm Hg, what should the plasma [HCO 3] be?

@It should be emphasized that the bicarbonate buffer is effective against metabolic but not respiratory acid-base disturbances. If 3 mEq/L of a strong nonvolatile acid, such as HCl, is added to extracellular fluid, the following reaction takes place:
Note that HCO; reacts with H* to produce CO,. Moreover, the CO, generated is normally eliminated by the lungs such that Paco, does not change. Consequently, [H*] = 24 x 40 + 21 = 45.7 nEd/L, and pH = 7.34. Furthermore, the decrease in [HCO; ] reflects the amount of nonvolatile acid added.
In contrast, an increase in CO, tension (volatile acid) has a minimal effect on [HCO3]. If, for example, Paco, increases from 40 to 80 mm Hg, the dissolved CO, increases only from 1.2 mEq/L to 2.2 mEq/L. Moreover, the equilibrium constant for the hydration of CO, is such that an increase of this magnitude minimally drives the reaction to the left:
If the valid assumption is made that [HCO3 ] does not appreciably change, then
[H*] therefore increases by 40 nEq/L, and because HCO; is produced in a 1:1 ratio with H*, [HCO; ] also increases by 40 nEq/L. Thus, extracellular [HCO3 ] increases negligibly, from 24 mEq/L to 24.000040 mEq/L. Therefore, the bicarbonate buffer is not effective against increases in Paco2, and changes in [HCO3-] do not reflect the severity of a respiratory acidosis.

Hemoglobin is rich in histidine, which is an effective buffer from pH 5.7 to 7.7 (pK, 6.8). Hemoglobin is the most important noncarbonic buffer in intravascular fluid. Simplistically, hemoglobin may be thought of as existing in red blood cells in equilibrium as a weak acid (HHb) and a potassium salt ® (KHb). In contrast to the bicarbonate buffer, hemoglobin is capable of buffering both carbonic (CO,) and noncarbonic (nonvolatile) acids:

Changes in alveolar ventilation responsible for the respiratory compensation of Paco, are mediated by chemoreceptors within the brainstem and the carotid and aortic bodies (see Chapter 23). These receptors respond to changes in cerebrospinal spinal fluid pH. Minute ventilation increases 1 to 4 L/min for every (acute) 1 mm Hg increase in Pacos. In fact, the lungs are responsible for eliminating the approximately 15 mEq of CO, produced every day as a byproduct of carbohydrate and fat metabolism. Respiratory compensatory responses are also important in defending against marked changes in pH during metabolic disturbances.

Decreases in arterial blood pH stimulate medullary respiratory centers. The resulting increase in alveolar ventilation lowers Paco, and tends to restore arterial pH toward normal. The respiratory response to lower the Paco, occurs rapidly but may not reach a predictably steady state until 12 to 24 h; pH is never completely restored to normal. Paco, normally decreases 1 to 1.5 mm Hg below 40 mm Hg for every 1 mEq/L decrease in plasma [HCO ].

Increases in arterial blood pH depress respiratory centers. The resulting alveolar hypoventilation tends to elevate Paco, and restore arterial pH toward normal. The respiratory response to metabolic alkalosis is generally less predictable than the respiratory response to metabolic acidosis. Hypoxemia, as a result of progressive hypoventilation, eventually activates oxygen-sensitive chemoreceptors; the latter stimulates ventilation and limits the compensatory respiratory response. Consequently, Paco, usually does not increase above 55 mm Hg in response to @ metabolic alkalosis. As a general rule, Paco, can be expected to increase 0.25 to 1 mm Hg for each 1 mEq/L increase in [HCO3 ].

The ability of the kidneys to control the amount of HCO; reabsorbed from filtered tubular fluid, form new HCO,,, and eliminate H* in the form of titratable acids and ammonium ions (see Chapter 30) allows them to exert a major influence on pH during both metabolic and respiratory acid—base disturbances. The kidneys are responsible for eliminating the approximately 1 mEq/kg per day of sulfuric acid, phosphoric acid, uric acid, and incompletely oxidized organic acids that are normally produced by the metabolism of dietary and endogenous proteins, nucleoproteins, and organic phosphates (from phosphoproteins and phospholipids). Incomplete metabolisms of fatty acids and glucose produces keto acids and lactic acid. Endogenous bases are produced during the metabolism of some anionic amino acids (eg, glutamate and aspartate) and other organic compounds (eg, citrate, acetate, and lactate), but the quantity is insufficient to offset the endogenous acid production.

@ The renal response to acidemia is three-fold: (1) increased reabsorption of the filtered HCO,,, (2) increased excretion of titratable acids, and (3) increased production of ammonia. Although these mechanisms are probably activated immediately, their effects are generally not appreciable for 12 to 24h and may not be maximal for up to 5 days.

Bicarbonate reabsorption is shown in Figure 50—4. CO, within renal tubular cells combines with water in the presence of carbonic anhydrase. The carbonic acid (H,CO3) formed rapidly dissociates into H* and HCO; . Bicarbonate ion then enters the bloodstream while the H* is secreted into the renal tubule, where it reacts with filtered HCO," to form H,CO3. Carbonic anhydrase associated with the luminal brush border catalyzes the dissociation of HyCO3 into CO, and H,O. The CO, thus formed can diffuse back into the renal tubular cell to replace the CO, originally consumed. The proximal tubules normally reabsorb 80% to 90% of the filtered bicarbonate load along with sodium, whereas the distal tubules are responsible for the remaining 10% to 20%. Unlike the proximal H* pump, the H* pump in the distal tubule is not necessarily linked to sodium reabsorption and is capable of generating steep H* gradients between tubular fluid and tubular cells. Urinary pH can decrease to as low as 4.4 (compared with a pH of 7.40 in plasma).
FIGURE 50-4 Reclamation of filtered HCO," by the proximal renal tubules.

After all of the HCO3 in tubular fluid is reclaimed, the H* secreted into the tubular lumen can combine with HPO,?to form HPO," (Figure 50-5); the latter is not readily reabsorbed because of its charge and is therefore eliminated in urine. The net result is that H* is excreted from the body as HjPO,,, and the HCO; that is generated in the process can enter the bloodstream. With a pK of 6.8, the H,PO,-/HPO,* pair is normally an ideal urinary buffer. When urinary pH approaches 4.4, however, all of the phosphate reaching the distal tubule is in the H,PO,form; HPO,*ions are no longer available for eliminating H*.

After complete reabsorption of HCO; and consumption of the phosphate buffer, the NH,/NH," pair becomes the most important urinary buffer (Figure 50-6). Deamination of glutamine within the mitochondria of proximal tubular cells is the principal source of NH3 production in the kidneys. Acidemia markedly increases renal NH; production. The ammonia formed is then able to passively cross the cell's luminal membrane, enter the tubular fluid, and react with H* to form NH,*. Unlike NH3, NH," does not readily penetrate the luminal membrane and is therefore trapped within the tubules. Thus, excretion of NH," in urine effectively eliminates H*.
FIGURE 50-6 Formation of ammonia in urine.

The tremendous amount of HCO;" normally filtered and subsequently reabsorbed allows the kidneys to rapidly excrete large amounts of bicarbonate, if necessary (see Chapter 49). As a result, the kidneys are highly effective in protecting against metabolic alkalosis, which therefore generally occurs only in association with concomitant sodium deficiency or mineralocorticoid excess. Sodium depletion decreases extracellular fluid volume and enhances Na* reabsorption in the proximal tubule. To maintain neutrality, the Na* ion is brought across with a Cl ion. As Cl ions decrease in number (<10 mEq/L of urine), HCO; must be reabsorbed. Increased H* secretion in exchange for augmented Na' reabsorption favors HCO; formation with metabolic alkalosis. Similarly, increased mineralocorticoid activity augments aldosterone-mediated Na' reabsorption in exchange for H* secretion in the distal tubules. The resulting increase in HCO, formation can initiate or propagate metabolic alkalosis. Metabolic alkalosis is commonly associated with increased mineralocorticoid activity, even in the absence of sodium and chloride depletion.

Base excess is defined as the amount of acid or base (expressed in mEq/L) that must be added for blood pH to return to 7.40 and Paco, to return to 40 mm Hg at full O, saturation and 37°C. Moreover, it adjusts for noncarbonic buffering in the blood. Simplistically, base excess represents the metabolic component of an acid-base disturbance. A positive value indicates metabolic alkalosis, whereas a negative value reveals metabolic acidosis. Base excess is usually derived from a nomogram and requires measurement of hemoglobin concentration.

Biochemical reactions are very sensitive to changes in [H*]. [H*] is strictly regulated (36-43 nmol/L), as H* ions have high charge densities and "large" electric fields that can affect the strength of hydrogen bonds that are present on most physiological molecules. The overall effects of acidemia represent the balance between the direct biochemical effects of H+ and the effects of acidemia-induced sympathoadrenal activation. With severe acidosis (pH <7.20), direct myocardial and smooth muscle depression reduces cardiac contractility and peripheral vascular resistance, resulting in progressive hypotension. Severe acidosis can lead to tissue hypoxia, despite a rightward shift in hemoglobin affinity for oxygen. Both cardiac and vascular smooth muscle become less responsive to endogenous and exogenous catecholamines, and the ventricular fibrillation threshold is decreased. The movement of K* out of cells in exchange for increased extracellular H* results in hyperkalemia that is also potentially lethal. Plasma [K+] increases approximately 0.6 mEq/L for each 0.10 decrease in pH.
Central nervous system depression is more prominent with respiratory acidosis than with metabolic acidosis. This effect is often termed CO, narcosis. Unlike CO,, H* ions do not readily penetrate the blood-brain barrier.

Respiratory acidosis is defined as a primary increase in Paco5. This increase drives the reaction
to the right, leading to an increase in [H*] and a decrease in arterial pH. For the reasons described above, [HCO 3 ] is minimally affected.
Paco, represents the balance between CO, production and CO, elimination:
CO, is a byproduct of fat and carbohydrate metabolism—and muscle activity, body temperature, and thyroid hormone activity can all have major influences on CO, production. Because CO, production does not appreciably vary under most circumstances, respiratory acidosis is usually the result of alveolar hypoventilation (Table 50—3). In patients with a limited capacity to increase alveolar ventilation, however, increased CO, production can precipitate respiratory acidosis.
TABLE 50-3 Differential diagnosis of respiratory acidosis.

Central nervous system depression
Sleep disorders
Obesity hypoventilation (Pickwickian) syndrome
Cerebral ischemia
Cerebral trauma
Neuromuscular disorders
Myopathies
Neuropathies
Chest wall abnormalities
Flail chest
Kyphoscoliosis
Pleural abnormalities
Upper airway
Tumor
Lower airway
Severe asthma
Tumor
Parenchymal lung disease
Cardiogenic
Noncardiogenic
Pulmonary emboli
Pneumonia
Interstitial lung disease
Ventilator malfunction

Large caloric loads
Intensive shivering
Prolonged seizure activity
Extensive thermal injury (burns)

The compensatory response to acute (6-12 h) elevations in Paco, is limited. Buffering is primarily provided by hemoglobin and the exchange of extracellular H* for Na* and K* from bone and the intracellular fluid compartment (see earlier discussion). The renal response to retain more bicarbonate is acutely very limited. As a result, plasma [HCO ] increases only about 1 mEq/L for each 10 mm Hg increase in Paco, above 40 mm Hg.

Renal compensation in respiratory acidosis is appreciable only after 12 to 24h and may not be maximal @ until 3 to 5 days have elapsed. During chronic respiratory acidosis, plasma [HCO3 ] increases approximately 4 mEq/L for each 10 mm Hg increase in Paco, above 40 mm Hg.

Respiratory acidosis is treated by reversing the imbalance between CO, production and alveolar ventilation. In most instances, this is accomplished by increasing alveolar ventilation. Measures aimed at reducing CO, production are useful only in specific instances (eg, dantrolene for malignant hyperthermia, muscle paralysis for status epilepticus, antithyroid medication for thyroid storm, or reduced caloric intake in patients receiving excessive enteral or parenteral nutrition). Potential temporizing measures aimed at improving alveolar ventilation (in addition to controlled mechanical ventilation) include bronchodilation, reversal of narcosis, or improving lung compliance via diuresis. Severe acidosis (pH <7.20), CO, narcosis, and respiratory muscle fatigue are indications for mechanical ventilation. An increased inspired oxygen concentration is also usually necessary, as coexistent hypoxemia is common. Intravenous NaHCO; is rarely necessary, unless pH is less than 7.10 and HCO3;— is less than 15 mEq/L. Sodium bicarbonate therapy will transiently increase Paco,:
Buffers that do not produce CO,, such as Carbicarb or tromethamine
(THAM), are
theoretically attractive
alternatives;
however, there
is almost
evidence showing
is
a
mixture that
they of
have
0.3
by this
mixture
Tromethamine
M
greater sodium
mainly efficacy
bicarbonate produces
has the
than bicarbonate.
and
0.3
M
sodium sodium
bicarbonate added
advantage no
Carbicarb™
carbonate;
instead of
lacking sodium
buffering of
CO5.
and may
be
a
more effective
intracellular buffer.

Patients with chronic respiratory acidosis require special consideration. When such patients develop acute ventilatory failure, the goal of therapy should be to return Paco, to the patient's "normal" baseline. Normalizing the patient's Paco, to 40 mm Hg will produces the equivalent of a respiratory alkalosis (see later discussion). Oxygen therapy must also be carefully controlled, because the respiratory drive in these patients may be dependent on hypoxemia, not Paco). "Normalization" of Paco, or relative hyperoxia can precipitate severe hypoventilation.

Metabolic acidosis is defined as a primary decrease in [HCO ]. Pathological processes can initiate metabolic acidosis by one of three mechanisms: (1) consumption of HCO; by a strong nonvolatile acid, (2) renal or gastrointestinal wasting of bicarbonate, or (3) rapid dilution of the extracellular fluid compartment with a bicarbonate-free fluid.
A fall in plasma [HCO3 ] without a proportionate reduction in Paco, decreases
arterial pH.
compensatory response
in
a
simple metabolic
Paco, to
acidosis
(see preceding
a
level that
discussion)
completely marked
characteristically normalizes
hyperventilation
Table pH
(Kussmaul’s
50-4
lists disorders
differential diagnosis
does but
nevertheless respiration).
that can
of metabolic
cause metabolic
acidosis acidosis.
may be
facilitated not
reduce can
Note by
the anion
gap.
TABLE 50-4 Differential diagnosis of metabolic acidosis.
produce that
calculation of

Increased production of endogenous nonvolatile acids
Ketoacidosis
Diabetic
Starvation
Lactic acidosis
Mixed
Nonketotic hyperosmolar coma
Alcoholic
Inborn errors of metabolism
Ingestion of toxin
Salicylate
Methanol
Ethylene glycol
Paraldehyde
Toluene
Sulfur

Increased gastrointestinal losses of HCO,
Diarrhea
Anion exchange resins (cholestyramine)
Ingestion of CaCl,, MgCl,
Fistulas (pancreatic, biliary, or small bowel)
Ureterosigmoidostomy or obstructed ileal loop
Increased renal losses of HCO,
Carbonic anhydrase inhibitors
Hypoaldosteronism
Dilutional
Large amount of bicarbonate-free fluids (eg, 0.9%
NaCl)
Total parenteral nutrition (Cl salts of amino acids)
Increased intake of chloride-containing acids
Ammonium chloride
Lysine hydrochloride
Arginine hydrochloride

The anion gap in plasma is most commonly defined as the difference between the major measured cations and the major measured anions:
Some practitioners also include plasma kK" in the calculation. Using normal values,
In reality, an anion gap cannot exist because electroneutrality must be maintained in the body; the sum of all anions must equal the sum of all cations. Therefore,

"Unmeasured cations" include K*, Ca**, and Mg?*, whereas "unmeasured anions" include all organic anions (including plasma proteins), phosphates, and sulfates. Plasma albumin normally accounts for the largest fraction of the anion gap (approximately 11 mEq/L). The anion gap decreases by 2.5 mEq/L for every 1 g/dL reduction in plasma albumin concentration. Any process that increases "unmeasured anions" or decreases "unmeasured cations" will increase the anion gap. Conversely, any process that decreases "unmeasured anions" or increases "unmeasured cations" will decrease the anion gap.
Mild elevations of plasma anion gap up to 20 mEq/L may not be helpful diagnostically during acidosis, but values greater than 30 mEq/L usually indicate the presence of a high anion gap acidosis. Metabolic alkalosis can also produce a high anion gap because of extracellular volume depletion, an increased charge on albumin, and a compensatory increase in lactate production. A low plasma anion gap may be encountered with hypoalbuminemia, bromide or lithium intoxication, and multiple myeloma.

Metabolic acidosis with an increased anion gap is characterized by an increase in relatively strong nonvolatile acids. These acids dissociate into H* and their respective anions; the H* consumes HCO; to produce CO;, whereas their anions (conjugate bases) accumulate and take the place of HCO3" in extracellular fluid (hence the anion gap increases). Nonvolatile acids can be endogenously produced or ingested.

Endogenously produced organic acids are normally eliminated by the kidneys in urine (as described earlier). Glomerular filtration rates below 20 mL/min (kidney injury or failure) typically result in progressive metabolic acidosis from the accumulation of these acids.

Severe tissue hypoxia following hypoxemia, hypoperfusion (ischemia), or an inability to utilize oxygen (cyanide poisoning) can result in lactic acidosis. Lactic acid is the end product of the anaerobic metabolism of glucose (glycolysis) and can rapidly accumulate under these conditions. Decreased utilization of lactate by the liver, and, to a lesser extent by the kidneys, is less commonly responsible for lactic acidosis; causes include hypoperfusion, alcoholism, and liver disease. Lactate levels can be readily measured and are normally 0.3 to 1.3 mEq/L. Acidosis resulting from D-lactic acid, which is not recognized by a-lactate dehydrogenase (and not measured by routine assays), may be encountered in patients with short bowel syndromes; D-lactic acid is formed by colonic bacteria from dietary glucose and starch and is absorbed systemically.
An absolute or relative lack of insulin can result in hyperglycemia and progressive ketoacidosis from the accumulation of B-hydroxybutyric and acetoacetic acids (diabetic ketoacidosis). Ketoacidosis may also be seen following starvation or alcoholic binges. The pathophysiology of the acidosis often associated with severe alcoholic intoxication and nonketotic hyperosmolar coma is complex and may represent a buildup of lactic, keto, or other unknown acids.
Some inborn errors of metabolism, such as maple syrup urine disease, methylmalonic aciduria, propionic acidemia, and isovaleric acidemia, produce a high anion gap metabolic acidosis as a result of accumulation of abnormal amino acids.

Ingestion of large amounts of salicylates may result in metabolic acidosis. Salicylic acid and other acid intermediates rapidly accumulate and produce a high anion gap acidosis. Because salicylates also produce direct respiratory stimulation, most adults develop mixed metabolic acidosis with superimposed respiratory alkalosis. Ingestion of methanol (methyl alcohol) frequently produces acidosis and retinitis. Symptoms are typically delayed until the slow oxidation of methanol by alcohol dehydrogenase produces formic acid, which is highly toxic to the retina. The high anion gap represents the accumulation of many organic acids, including acetic acid. The toxicity of ethylene glycol is also the result of the action of alcohol dehydrogenase to produce glycolic acid. Glycolic acid, the principal cause of the acidosis, is further metabolized to form oxalic acid, which may be deposited in the renal tubules and produce acute kidney injury.

Metabolic acidosis associated with a normal anion gap is typically characterized by hyperchloremia. Plasma [CI] increases to take the place of the HCO3" ions that are lost. Hyperchloremic metabolic acidosis most commonly results from abnormal gastrointestinal or renal losses of HCO;,, or from excessive intravenous administration of 0.9% NaCl solution.
Calculation of the anion gap in urine can be helpful in diagnosing a normal anion gap acidosis.

The urine anion gap is normally positive or close to zero. The principal unmeasured urinary cation is normally NH,*, which should increase (along with Cl) during a metabolic acidosis; the latter results in a negative urinary anion gap. Impairment of H* or NH," secretion, as occurs in kidney failure or renal tubular acidosis (discussed below), results in a positive urine anion gap despite systemic acidosis.

@ Diarrhea is a common cause of hyperchloremic metabolic acidosis. Diarrheal fluid contains 20 to 50 mEq/L of HCO;-. Small bowel, biliary, and pancreatic fluids are all rich in HCO,~. Loss of large volumes of these fluids can lead to hyperchloremic metabolic acidosis. Patients with ureterosigmoidostomies and those with ileal loop neobladders that are too long or that become partially obstructed frequently develop hyperchloremic metabolic acidosis. The ingestion of chloride-containing anion-exchange resins (cholestyramine) or large amounts of calcium or magnesium chloride can result in increased absorption of chloride and loss of bicarbonate ions. The nonabsorbable resins bind bicarbonate ions, whereas calcium and magnesium combine with bicarbonate to form insoluble salts within the intestines.

Renal wasting of HCO; can occur as a result of failure to reabsorb filtered HCO, or to secrete adequate amounts of H* in the form of titratable acid or ammonium ion. These defects are encountered in patients taking carbonic anhydrase inhibitors, such as acetazolamide, and in those with renal tubular acidosis.
Renal tubular acidosis (RTA) is a disease of systemic acidosis resulting from inadequate renal compensation for systemic acid production. The kidneys are unable to adequately acidify the urine, and urinary pH is inappropriately high relative to the systemic acidemia. Kidney function is otherwise normal. RTA involves a defect in distal renal tubular H* secretion (type 1 RTA), proximal renal tubular reabsorption of filtered HCO," (type 2 RTA), or both (type 3 RTA). Type 4 RTA is the result of hypoaldosteronism or renal insensitivity to aldosterone.

Dilutional hyperchloremic acidosis may occur when extracellular volume is rapidly expanded with a bicarbonate-free, chloride-rich fluid such as normal saline. Plasma [HCO3 ] decreases in proportion to the amount of fluid infused as extracellular HCO3 is diluted, and this fall in [HCO37] is compensated by a rise in [CI]. This is a reason to prefer balanced salt solutions over 0.9% saline for fluid resuscitation. Amino acid infusions (parenteral hyperalimentation) contain organic cations in excess of organic anions and can produce hyperchloremic metabolic acidosis because chloride is commonly used as the anion for the cationic amino acids. Lastly, the administration of excessive quantities of chloride-containing acids, such as ammonium chloride or arginine hydrochloride (usually given to treat a metabolic alkalosis), can cause hyperchloremic metabolic acidosis.

The bicarbonate space is defined as the volume to which HCO3° will distribute when administered intravenously. Although this theoretically should equal the extracellular fluid space (approximately 25% of body weight), in reality, it ranges anywhere between 25% and 60% of body weight, depending on the severity and duration of the acidosis. This variation is at least partly related to the amount of intracellular and bone buffering that has taken place.
Example: Calculate the amount of NaHCO; necessary to correct a base deficit (BD) of -10 mEq/L for a 70-kg man with an estimated HCO;" space of 30%:
NaHCO; = BD x 30% x body weight in L
NaHCO; = —10 mEq/L x 30% x 70 L = 210 mEq
In practice, only 50% of the calculated dose (eg., 105 mEq) is usually given, after which another blood gas is measured.

Acidemia can potentiate the depressant effects of most sedatives and anesthetic agents on the central nervous and circulatory systems. Because most opioids are weak bases, acidosis can increase the fraction of the drug in the nonionized form and facilitate opioid penetration into the brain, potentiating its sedative effect. The circulatory depressant effects of both volatile and intravenous anesthetics can also be exaggerated. Moreover, any agent that rapidly decreases sympathetic tone can potentially allow unopposed circulatory depression in the setting of acidosis. Halothane is more arrhythmogenic in the presence of acidosis. Succinylcholine should generally be avoided in acidotic patients with hyperkalemia to prevent further increases in plasma [K*].

Alkalosis increases the affinity of hemoglobin for oxygen and shifts the oxygen dissociation curve to the left, making it more difficult for hemoglobin to give up oxygen to tissues. Movement of H™ out of cells in exchange for the movement of extracellular K* into cells can produce hypokalemia. Alkalosis increases the number of anionic binding sites for Ca2* on plasma proteins and can therefore decrease ionized plasma [Ca**], leading to circulatory depression and neuromuscular irritability. Respiratory alkalosis reduces cerebral blood flow. In the lungs, respiratory alkalosis increases bronchial smooth muscle tone (bronchoconstriction), but decreases pulmonary vascular resistance.

Respiratory alkalosis is defined as a primary decrease in Paco,. The mechanism is usually an inappropriate increase in alveolar ventilation relative to CO, production. Table 50—5 lists the most common @ causes of respiratory alkalosis. The distinction between acute and chronic respiratory alkalosis is not always made, because the compensatory response to chronic respiratory alkalosis is quite variable: Plasma [HCO ; ] usually decreases 2 to 5 mEq/L for each 10 mm Hg decrease in Paco, below 40 mm Hg.
TABLE 50-5 Differential diagnosis of respiratory alkalosis.

Pain
Anxiety
Ischemia
Stroke
Tumor
Infection
Fever
Progesterone (pregnancy)
Analeptics (doxapram)

Hypoxemia
Pulmonary disease
Noncardiogenic pulmonary edema
Asthma
Severe anemia

Correction of the underlying process is the only treatment for respiratory alkalosis. For severe alkalemia (arterial pH >7.60), intravenous hydrochloric acid, arginine chloride, or ammonium chloride may be indicated (see later discussion).

Metabolic alkalosis is defined as a primary increase in plasma [HCO3 ]. Most cases of metabolic alkalosis can be divided into (1) those associated with NaCl deficiency and extracellular fluid depletion, often described as chloridesensitive, and (2) those associated with enhanced mineralocorticoid activity, commonly referred to as chloride-resistant (Table 50-6).

Gastrointestinal
Vomiting
Gastric drainage
Chloride diarrhea
Villous adenoma
Renal
Diuretics
Posthypercapnic
Low chloride intake
Sweat

Increased mineralocorticoid activity
Primary hyperaldosteronism
Edematous disorders (secondary hyperaldosteronism)
Licorice ingestion
Severe hypokalemia

Massive blood transfusion
Acetate-containing colloid solutions
Alkaline administration with renal insufficiency
Alkali therapy
Combined antacid and cation-exchange resin therapy
Hypercalcemia
Bone metastases
Sodium penicillins
Glucose feeding after starvation
Chloride-Sensitive Metabolic Alkalosis

Increased mineralocorticoid activity commonly results in metabolic alkalosis, even when it is not associated with extracellular volume depletion. Inappropriate increases in mineralocorticoid activity cause sodium retention and expansion of extracellular fluid volume. Increased H* and k* secretion takes place to balance enhanced mineralocorticoid-mediated sodium reabsorption, resulting in metabolic alkalosis and hypokalemia. Urinary chloride concentrations are typically greater than 20 mEq/L in such cases.

Metabolic alkalosis is rarely encountered in patients given even large doses of NaHCO; unless renal excretion of HCO;" is impaired. The administration of large amounts of blood products and some plasma protein—containing colloid solutions frequently results in metabolic alkalosis because citrate, lactate, and acetate contained in these fluids are converted by the liver into HCO;~. Patients receiving high doses of sodium penicillin (particularly carbenicillin) can develop metabolic alkalosis. Because penicillins act as nonabsorbable anions in the renal tubules, increased H* (or K*) secretion must accompany sodium absorption. For reasons that are not clear, hypercalcemia that results from nonparathyroid causes (milk-alkali syndrome and bone metastases) is also often associated with metabolic alkalosis.

As with other acid—base disorders, correction of metabolic alkalosis is never complete until the underlying disorder is corrected. When ventilation is controlled, any respiratory component contributing to alkalemia should be corrected by decreasing minute ventilation to normalize Paco,. The treatment of choice for chloride-sensitive metabolic alkalosis is administration of intravenous saline (NaCl) and potassium (KCl). H-blocker therapy is useful when excessive loss of gastric fluid is a factor. Acetazolamide may also be useful in edematous patients. Alkalosis associated with primary increases in mineralocorticoid activity readily responds to aldosterone antagonists (spironolactone). When arterial blood pH is greater than 7.60, treatment with intravenous hydrochloric acid (0.1 mol/L), ammonium chloride (0.1 mol/L), arginine hydrochloride, or hemodialysis should be considered.

Cerebral ischemia can occur from marked reduction in cerebral blood flow during respiratory alkalosis, particularly during hypotension. © The combination of alkalemia and hypokalemia can precipitate severe atrial and ventricular arrhythmias. Reports of effects of alkalemia on neuromuscular blockers are inconsistent.

Interpretation of acid-base status from analysis of blood gases requires a systematic approach. A recommended approach follows (see Figure 50-3):
1. Examine arterial pH: Is acidemia or alkalemia present?
2. Examine Paco;: Is the change in Paco, consistent with a respiratory component?
3. Ifthe change in Paco, does not explain the change in arterial pH, does the change in [HCO ] indicate a metabolic component?
4. Make a tentative diagnosis (see Table 50-1).
5. Compare the change in [HCO3 _] with the change in Paco . Does a
exist
(Table
50—7)?
Because arterial
pH
is related
the ratio
of
Paco, to
[HCO3
], both
respiratory and
renal compensatory
mechanisms are always such that Paco, and [HCO3 ] change in the same direction. A change in opposite directions implies a mixed acid—base disorder.
TABLE 50-7 Normal compensatory responses in acid-base disturbances.
to
Respiratory acidosis, Response = . Respiratory acidosis, Expected Change = . Acute, Response = T [HCO,]. Acute, Expected Change = 1 mEq/L/10 mm Hg increase in Paco,. Chronic, Response = T[HCO,]. Chronic, Expected Change = 4m€Eq/L/10 mm Hg increase in Paco,. Respiratory alkalosis, Response = . Respiratory alkalosis, Expected Change = . Acute, Response = L{HCO,]. Acute, Expected Change = 2mEq/L/10 mm Hg decrease in Paco,. Chronic, Response = L{HCO,]. Chronic, Expected Change = 4m€Eq/L/10 mmHg decrease in Paco,. Metabolic acidosis, Response = 1 Paco,. Metabolic acidosis, Expected Change = 1.2 x the decrease in ([HCO,]. Metabolic alkalosis, Response = Th Paco,. Metabolic alkalosis, Expected Change = 0.7 x the increase in {HCO,]

6. If the compensatory response is more or less than expected, by definition, a mixed acid-base disorder exists.
7. Calculate the plasma anion gap in the case of metabolic acidosis.
8. Measure urinary chloride concentration in the case of metabolic alkalosis.
An alternative approach that is rapid, though less precise, is to correlate changes in pH with changes in CO, or HCO3. For a respiratory disturbance, every 10 mm Hg change in CO, should change arterial pH by approximately 0.08 U in the opposite direction. During metabolic disturbances, every 6 mEq change in HCO; also changes arterial pH by 0.1 in the same direction. If the change in pH is greater or less than predicated, a mixed acid—base disorder is likely to be present.

Values obtained by routine blood gas measurement include oxygen and carbon dioxide tensions (Po, and Pco,), pH, [HCO37], base excess, hemoglobin, and the percentage oxygen saturation of hemoglobin. As a rule, only Po», PcO,, and pH are measured. Hemoglobin and percentage oxygen saturation are measured with a cooximeter. [HCO3 ] is derived using the Henderson—Hasselbalch equation and base excess from the Siggaard—Andersen nomogram.

Arterial blood samples are most commonly utilized clinically, though capillary or venous blood can be used if the limitations of such samples are recognized. Oxygen tension in venous blood (normally 40 mm Hg) reflects tissue extraction, not pulmonary function. Venous PCO, is usually 4 to 6 mm Hg higher than Paco,. Consequently, venous blood pH is usually 0.05 U lower than arterial blood pH. Despite these limitations, venous blood is often useful in determining acid-base status. Capillary blood represents a mixture of arterial and venous blood, and the values obtained reflect this fact. Samples are usually collected in heparin-coated syringes and should be analyzed as soon as possible. Air bubbles should be eliminated, and the sample should be capped and placed on ice to prevent significant uptake of gas from blood cells or loss of gases to the atmosphere. Although heparin is highly acidic, excessive heparin in the sample syringe usually lowers pH only minimally, but decreases Pco, in direct proportion to percentage dilution and has a variable effect on Pop.

@ Changes in temperature affect Pco5, Po», and pH. Decreases in temperature lower the partial pressure of a gas in solution—even though the total gas content does not change—because gas solubility is inversely proportionate to temperature. Both Pco, and Po, therefore decrease during hypothermia, but pH increases because temperature does not appreciably alter [HCO ] and the dissociation of water decreases (decreasing H* and increasing pH). Because blood gas tensions and pH are always measured at 37°C, controversy exists over whether to correct the measured values to the patient's actual temperature. "Normal" values at temperatures other than 37°C are not known. Many clinicians use the measurements at 37°C directly (a-stat), regardless of the patient's actual temperature (see Chapter 22).

A 1-month-old male infant with an anorectal malformation undergoes anoplasty. Postoperatively, he is found to be in congestive heart failure resulting from coarctation of the aorta. He is noted to have tachypnea, decreased urinary output, poor peripheral perfusion, hepatomegaly, and cardiomegaly. Following tracheal intubation, the infant is placed on a ventilator (pressure support ventilation, fraction of inspired oxygen [Fio,] = 1.0). Initial arterial blood gas, hemoglobin, and

Paco, = 11 mm pH = 7.47
Pao, = 209 mm Hg
Calculated [HCO3 ] = 7.7 mEq/L
Base deficit (BD) = -14.6 mEq/L
Hemoglobin (Hb) = 9.5 g/Dl
[Na*] = 135 mEq/L
[Cl] = 95 mEq/L
[K*] = 5.5 mEq/L
[Total CO,] = 8 mEq/L
Note that the [total CO,] normally measured with electrolytes includes both plasma [HCO3-] and dissolved CO, in plasma.

Using the approach described earlier, the patient clearly has an alkalosis (pH >7.45), which is at least partly respiratory in origin (Paco, <40 mm Hg). Because Paco, has decreased by nearly 30 mm Hg, we would expect [HCO3 ] to be 18 mEq/L:
In fact, the patient's [HCO ] is nearly 10 mEq/L less than that! The patient therefore also has a mixed acid-base disturbance: primary respiratory alkalosis and primary metabolic acidosis. Note that the difference between the patient's [HCO ] and the [HCO; ] expected for a pure respiratory alkalosis roughly corresponds to the base excess.

The respiratory alkalosis is probably the result of congestive heart failure, whereas the metabolic acidosis results from lactic acidosis secondary to poor perfusion. The latter is suggested by the calculated plasma anion gap:
Anion gap = 135 — (95 + 8) = 32 mEq/L
The lactate level was in fact measured and found to be elevated at 14.4 mEq/L. It is probable that fluid overload precipitated the congestive heart failure.

Treatment should be directed at the primary process (ie, the congestive failure). The patient was treated with diuresis and inotropes.
heart Following diuresis, the patient's tachypnea has improved, but perfusion still seems to be poor. Repeat laboratory measurements are as follows (FiO, = 0.5):
Paco, = 23 mm Hg pH = 7.52
Pao, = 136 mm Hg
Calculated [HCO3_] = 18 mEq/L
BD =-3.0 mEq/L
Hb = 10.3 g/dL
[Na*] = 137 mEq/L
[Cl] = 92 mEq/L
[K*] = 3.9 mEq/L
[Total CO,] = 18.5 mEq/L
What is the acid—base disturbance?
Respiratory alkalosis is still present, whereas the BD has improved. Note that the hemoglobin concentration has increased slightly, but [K*] has decreased as a result of the diuresis. With the new Paco, the expected [HCO ] should be 20.6 mEq/L:
Therefore, the patient still has metabolic acidosis because the [HCO3 ] is 2 mEq/L less. Note again that this difference is close to the given BD and that the anion gap is still high:
The repeat lactate measurement is now 13.2 mEq/L.
The high anion gap and lactate level explain why the patient is still not doing well and indicate that a new process is masking the severity of the metabolic acidosis (which is essentially unchanged).
Given the clinical course, it is likely that the patient now has a triple acid-base disorder: respiratory alkalosis, metabolic acidosis, and now metabolic alkalosis. The latter probably resulted from hypovolemia secondary to excessive diuresis (chloride-sensitive metabolic alkalosis). Note also that the metabolic alkalosis is nearly equal in magnitude to the metabolic acidosis.
The patient was subsequently given packed red blood cells in saline, and within 24 h all three disorders began to improve:
Paco, = 35 mm Hg pH = 7.51

Pao, = 124 mm Hg
Calculated [HCO3_] = 26.8 mEq/L
Base excess = +5.0 mEq/L
Hb = 15 g/dL
[Na*] = 136 mEq/L
[Cl] = 91 mEq/L
[K*] = 3.2 mEq/L
[Total CO,] = 27 mEq/L
Lactate = 2.7 mEq/L

The respiratory alkalosis and the metabolic acidosis have now resolved, and the metabolic alkalosis is now most prominent.
Intravenous KCl replacement and a small amount of saline were judiciously given, followed by complete resolution of metabolic alkalosis.
The patient subsequently underwent surgical correction of the coarctation.

management of acid-base and electrolyte imbalances in acute kidney injury. Adv Chron Kidney Dis. 2016;23:203.

@ Although the intravascular half-life of a crystalloid solution is 20 to 30 min, most colloid solutions have intravascular half-lives between 3 and 6h.
@ Patients with a normal hematocrit should generally be transfused only after losses greater than 10% to 20% of their blood volume. The timing of transfusion initiation is based on the patient's surgical procedure, comorbid conditions, and rate of blood loss.
@ The most severe transfusion reactions are due to ABO incompatibility; naturally acquired antibodies can react against the transfused (foreign) antigens, activate complement, and result in intravascular hemolysis.
@ In anesthetized patients, an acute hemolytic reaction is manifested by a rise in temperature, unexplained tachycardia, hypotension, hemoglobinuria, diffuse oozing in the surgical field, or a combination of these findings.
@ Allogeneic transfusion of blood products may diminish immunoresponsiveness and promote inflammation.
@ Immunocompromised and immunosuppressed patients (eg, premature infants, organ transplant recipients, and cancer patients) are particularly susceptible to severe transfusion-related cytomegalovirus (CMV) infections. Ideally, such patients should receive only CMV-negative units.
@ The most common cause of nonsurgical bleeding following massive blood transfusion is dilutional thrombocytopenia.
@ Clinically important hypocalcemia, causing cardiac depression, will not
occur in most normal patients unless the transfusion rate exceeds 1 unit every 5 min, and intravenous calcium salts should rarely be required in the absence of measured hypocalcemia.
- © Once normal tissue perfusion is restored, any metabolic acidosis typically resolves, and metabolic alkalosis commonly occurs as citrate and lactate contained in transfusions and resuscitation fluids are converted to bicarbonate by the liver.
Almost all patients undergoing surgical procedures require venous access for administration of intravenous fluids and medication, and some patients will require transfusion of blood components. The anesthesia provider should be able to assess intravascular volume with sufficient accuracy to correct existing fluid or electrolyte deficits and replace ongoing losses. Errors in fluid and electrolyte replacement or transfusion may result in morbidity or death.

Intravascular volume can be estimated using patient history, physical examination, and laboratory analysis, often with the aid of sophisticated hemodynamic monitoring techniques. Regardless of the method employed, serial evaluations are necessary to confirm initial impressions and to guide fluid, electrolyte, and blood component therapy. Multiple modalities complement one another, because all parameters are indirect, nonspecific measures of volume; reliance upon any one parameter may lead to erroneous conclusions.

The patient history may reveal recent oral intake, persistent vomiting or diarrhea, gastric suction, significant blood loss or wound drainage, intravenous fluid and blood administration, and recent hemodialysis if the patient has kidney failure.

Indications of hypovolemia include abnormal skin turgor, dehydration of mucous membranes, thready peripheral pulses, increased resting heart rate and decreased blood pressure, orthostatic heart rate and blood pressure changes from the supine to sitting or standing positions, and decreased urinary flow rate (Table 51-1).
Unfortunately, many medications administered during anesthesia, as well as the neuroendocrine stress response to operative procedures, frequently alter these signs and render them unreliable in the immediate postoperative period. Intraoperatively, in addition to heart rate and blood pressure, the fullness of a peripheral pulse, urinary flow rate, and indirect signs, such as the blood pressure response to positive-pressure ventilation and to vasodilating or negative inotropic effects of anesthetics, are often used for guidance.

Mucous membranes, Fluid Loss (Expressed as Percentage of Body Weight).5% = Dry. Mucous membranes, Fluid Loss (Expressed as Percentage of Body Weight).10% = Very dry. Mucous membranes, Fluid Loss (Expressed as Percentage of Body Weight).15% = Parched. Sensorium, Fluid Loss (Expressed as Percentage of Body Weight).5% = Normal. Sensorium, Fluid Loss (Expressed as Percentage of Body Weight).10% = Lethargic. Sensorium, Fluid Loss (Expressed as Percentage of Body Weight).15% = Obtunded. Orthostatic changes In heart rate In blood pressure, Fluid Loss (Expressed as Percentage of Body Weight).5% = . Orthostatic changes In heart rate In blood pressure, Fluid Loss (Expressed as Percentage of Body Weight).10% = Present. Orthostatic changes In heart rate In blood pressure, Fluid Loss (Expressed as Percentage of Body Weight).15% = Marked >15 bpm 7! >10 mm Hg L. Urinary flow rate, Fluid Loss (Expressed as Percentage of Body Weight).5% = Mildly decreased. Urinary flow rate, Fluid Loss (Expressed as Percentage of Body Weight).10% = Decreased. Urinary flow rate, Fluid Loss (Expressed as Percentage of Body Weight).15% = Markedly decreased. Pulse rate, Fluid Loss (Expressed as Percentage of Body Weight).5% = Normal orincreased. Pulse rate, Fluid Loss (Expressed as Percentage of Body Weight).10% = Increased >100 bpm. Pulse rate, Fluid Loss (Expressed as Percentage of Body Weight).15% = Markedly increased >120 bpm. Blood pressure, Fluid Loss (Expressed as Percentage of Body Weight).5% = Normal. Blood pressure, Fluid Loss (Expressed as Percentage of Body Weight).10% = Mildly decreased with respiratory variation. Blood pressure, Fluid Loss (Expressed as Percentage of Body Weight).15% = Decreased
"bpm, beats per minute.

Pitting edema—presacral in the bedridden patient or pretibial in the ambulatory patient—and increased urinary flow are signs of excess extracellular water and likely hypervolemia in patients with normal cardiac, hepatic, and renal function. Late signs of hypervolemia in settings such as congestive heart failure may include tachycardia, tachypnea, elevated jugular pulse pressure, pulmonary crackles, wheezing, cyanosis, and pink, frothy pulmonary secretions.

Several laboratory measurements may be used as surrogates of intravascular volume and adequacy of tissue perfusion, including serial hematocrits, arterial blood pH, urinary specific gravity or osmolality, urinary sodium or chloride concentration, serum sodium, and the blood urea nitrogen (BUN)-to-serum creatinine ratio. However, because these measurements are only indirect indices of intravascular volume, they are affected by many perioperative factors, and because laboratory results are often delayed, they often cannot be relied upon intraoperatively. Laboratory signs of dehydration may include rising hematocrit and hemoglobin, progressive metabolic acidosis (including lactic acidosis), urinary specific gravity greater than 1.010, urinary sodium less than 10 mEq/L, urinary osmolality greater than 450 mOsm/L, hypernatremia, and BUN-to- creatinine ratio greater than 10:1. The hemoglobin and hematocrit are usually unchanged in patients with acute hypovolemia secondary to acute blood loss because there is insufficient time for extravascular fluid to shift into the intravascular space. Ultrasound can reveal a nearly collapsed vena cava or incompletely filled cardiac chambers. Radiographic indicators of volume overload include increased pulmonary vascular and interstitial markings (Kerley "B" lines), diffuse alveolar infiltrates, or both.

Hemodynamic monitoring is discussed in Chapter 5. Central venous pressure (CVP) monitoring has been used when volume status is difficult to assess by other means or when rapid or major alterations are expected. However, static CVPreadings do not provide accurate or reliable indication of volume status. Pulmonary artery pressure monitoring has been used in settings where readings do not correlate with the clinical assessment or when the patient has primary or secondary right ventricular dysfunction; the latter is usually due to pulmonary or left ventricular disease, respectively. Pulmonary artery pressure (PAOP) readings of less than 8 mmHg may indicate hypovolemia in patients with normal left ventricular compliance; however, values less than 15 mmHg may be associated with relative hypovolemia in patients with poor ventricular compliance. PAOP measurements greater than 18 mmHg are elevated and may imply left ventricular volume overload. The normal relationship between PAOP and left ventricular end-diastolic volume is by the presence of mitral valve disease, severe aortic stenosis, or a left atrial, 1 = . occlusion altered or thrombus, as well as by increased thoracic and pulmonary airway pressures (see Chapters 5, 20, 21, and 22). All PAOP measurements should be obtained at end expiration and interpreted in the context of the clinical setting. Finally, one should recognize that multiple studies have failed to show that, 1 = . CVP myxoma, 1 = . artery pressure monitoring leads to improved outcomes in critically, 1 = . pulmonary ill patients, and that echocardiography provides a much more accurate and, 1 = . less invasive estimate of cardiac filling and function. Intravascular volume status is often difficult to assess, and noninvasive fluid, 1 = . guidance utilizing arterial pulse contour analysis and estimation of volume variation (eg, LIDCOunity, Vigileo FloTrak), esophageal, 1 = . therapy, 1 = . echocardiography, or transthoracic echocardiography) should, 1 = . Doppler, be, 1 = . stroke, 1 = . transesophageal when accurate determination of hemodynamic and fluid status is, 1 = . considered, 1 = . , 1 = important. Stroke volume variation (SVV) is calculated as follows:

The maximum, minimum, and mean SV are calculated for a set period of time by the various measuring devices. During spontaneous ventilation the blood pressure decreases on inspiration. During positive pressure ventilation the opposite occurs. Normal SVV is less than 10% to 15% for patients on controlled ventilation. Patients with greater degrees of SVV are likely to be responsive to fluid therapy. In addition to providing more accurate assessment of volume and hemodynamic status than that obtained with CVP monitoring, these noninvasive modalities avoid multiple risks associated with central venous and pulmonary artery catheters. Consequently, we infrequently employ pulmonary artery catheters to guide hemodynamic therapy.

Intravenous fluid therapy may consist of infusions of crystalloids, colloids, or a combination of both. Crystalloid solutions are aqueous solutions of ions (salts) with or without glucose, whereas colloid solutions also contain high-molecularweight substances such as proteins or large glucose polymers. Colloid solutions help maintain plasma colloid oncotic pressure (see Chapter 49) and for the part remain intravascular, whereas crystalloid solutions rapidly equilibrate with and distribute throughout the entire extracellular fluid space.
Controversy remains regarding the use colloid versus crystalloid surgical patients. Proponents of colloids justifiably argue that by maintaining plasma oncotic pressure, colloids are more efficient (ie, a smaller volume of colloids than crystalloids is required to produce the same effect) in restoring normal intravascular volume and cardiac output. Crystalloid proponents, on the other hand, maintain that the crystalloid solutions are equally effective when given in appropriate amounts. Concerns that colloids may enhance the formation of pulmonary edema fluid in patients with increased pulmonary capillary permeability are unfounded (see Chapter 23). Several generalizations can be made:
most of fluids for
1. Crystalloids, when given in sufficient amounts, are just as effective as colloids in restoring intravascular volume.
2. Replacing an intravascular volume deficit with crystalloids generally requires three to four times the volume needed when using colloids.
3. Surgical patients may have an extracellular fluid deficit that exceeds the

4. Severe intravascular fluid deficits can be more rapidly corrected using colloid solutions.
5. The rapid administration of large amounts of crystalloids (>4—5 L) is often associated with tissue edema.
Tissue edema secondary to excessive fluid administration can impair oxygen transport, tissue healing, and return of bowel function following surgery and may increase the risk of surgical site infection.

Crystalloids are often considered as the initial resuscitation fluid in patients with hemorrhagic and septic shock, in burn patients, in patients with head injury (to maintain cerebral perfusion pressure), and in patients undergoing plasmapheresis and hepatic resection. Colloids may be included in resuscitation efforts following initial administration of crystalloid solutions depending upon anesthesia provider preferences and institutional protocols.
A wide variety of solutions is available, and choice is according to the type of fluid loss being replaced. For losses primarily involving water, replacement is with hypotonic solutions, and if losses involve both water and electrolytes, replacement is with isotonic electrolyte solutions. Glucose is provided in some solutions to maintain tonicity, or prevent ketosis and hypoglycemia due to fasting, or based on tradition. Children are prone to developing hypoglycemia (<50 mg/dL) following 4to 8-h fasts.
Because most intraoperative fluid losses are isotonic, isotonic crystalloid solutions such as normal saline or balanced electrolyte solutions (low-[Cl"] crystalloids, which have preserved ionic "balance" by replacing Cl with lactate, gluconate, or acetate) such as lactated Ringer's solution or PlasmaLyte are most commonly used for replacement (Table 51—2). Normal saline, when given in large volumes, produces hyperchloremic metabolic acidosis because of its high chloride content and lack of bicarbonate (see Chapter 50). In addition, chloride-rich crystalloids such as normal saline may contribute to perioperative acute kidney injury. Therefore, we prefer balanced salt solutions for most intraoperative uses. Normal saline is the preferred solution for hypochloremic metabolic alkalosis and for diluting packed red blood cells prior to transfusion. Five percent dextrose in water (D;W) is used for replacement of pure water deficits and as a maintenance fluid for patients on sodium restriction. Hypertonic 3% saline is employed in therapy of severe symptomatic hyponatremia (see Chapter 49). Hypotonic solutions must be administered slowly to avoid inducing hemolysis.

(mmol/L), 0,9% Sodium Chloride = Qo. Gluconate (mmol/L), Hartmann’s = 9. Gluconate (mmol/L), Ringer's Lactate = 0. Gluconate (mmol/L), Ringer's Acetate = 0. Gluconate (mmol/L), Plasma- Lyte148 = 23. Gluconate (mmol/L), Plasma- LyteA pH7.4 = 23. Gluconate (mmol/L), Sterofundin/ Ringerfundin = 0. Maleate (mmol/L), Human Plasma = 0. Maleate (mmol/L), 0,9% Sodium Chloride = 0. Maleate (mmol/L), Hartmann’s = . Maleate (mmol/L), Ringer's Lactate = 0. Maleate (mmol/L), Ringer's Acetate = . Maleate (mmol/L), Plasma- Lyte148 = 0. Maleate (mmol/L), Plasma- LyteA pH7.4 = 0. Maleate (mmol/L), Sterofundin/ Ringerfundin = 5. Na:CI ratio, Human Plasma = 1.21:1 to 1541. Na:CI ratio, 0,9% Sodium Chloride = 1:1. Na:CI ratio, Hartmann’s = 1.18:1. Na:CI ratio, Ringer's Lactate = 1.19:1. Na:CI ratio, Ringer's Acetate = 1.16:1. Na:CI ratio, Plasma- Lyte148 = 1.43:1. Na:CI ratio, Plasma- LyteA pH7.4 = 1.43:1. Na:CI ratio, Sterofundin/ Ringerfundin = 1,14:1
'Reproduced with permission from Lobo DN, Awad S. Should chloride-rich crystalloids remain the mainstay of fluid resuscitation to prevent "prerenal" acute kidney injury?: Con. Kidney Int. 2014 Dec;86(6):1096-1105.

The osmotic activity of high-molecular-weight substances in colloids tends to maintain these solutions @ intravascularly. Although the intravascular half-life of a crystalloid solution is 20 to 30 min, most colloid solutions have intravascular half-lives between 3 and 6 h. The relatively greater cost and occasional complications associated with colloids may limit their use. Generally accepted indications for colloids include (1) fluid resuscitation in patients with severe intravascular fluid deficits (eg, hemorrhagic shock) prior to the arrival of blood for transfusion, and (2) fluid resuscitation in the presence of severe hypoalbuminemia or conditions associated with large protein losses such as burns. For burn patients, colloids are not included in most initial resuscitation protocols, but may be considered following initial resuscitation with more extensive burn injuries during subsequent operative procedures.
Many clinicians also use colloid solutions in conjunction with crystalloids
down by amylase. Hetastarch is highly effective as a plasma expander and is less expensive than albumin. Allergic reactions are rare, but anaphylactoid and anaphylactic reactions have been reported. Hetastarch can decrease von Willebrand factor antigen levels, may prolong the prothrombin time, and has been associated with hemorrhagic complications. It is potentially nephrotoxic and should not be administered to patients at risk for acute kidney injury, including those who are elderly, critically ill, or have a history of kidney disease. However, its perioperative use in patients who are not critically ill or are not at increased risk of acute kidney injury remains controversial and is subject to a great deal of current debate.

Perioperative fluid therapy includes replacement of normal losses (maintenance requirements), of preexisting fluid deficits, and of surgical wound losses including blood loss.

In the absence of oral intake, fluid and electrolyte deficits can rapidly develop as a result of continued urine formation, gastrointestinal secretions, sweating, and insensible losses from the skin and lungs. Normal maintenance requirements can be estimated from Table 51-3.

For the first 10 kg, Rate = 4 mL/kg/h. For the next 10 kg, Rate = Add 2 mL/kg/h. For each kg above 20 kg, Rate = Add 1 mL/kg/h
'Example: What are the maintenance fluid requirements for a 25-kq child?
Answer: 40 + 20+ 5=65 mL/h.

Patients presenting for surgery after a traditional overnight fast without any fluid intake will have a preexisting deficit proportionate to the duration of the fast. The deficit can be estimated by multiplying the normal maintenance rate by the length of the fast. For the average 70-kg person fasting for 8 h, this amounts to (40 + 20 + 50) mL/h x 8 h, or 880 mL. In fact, the real deficit is less as a result of renal conservation—after all, how many of us would feel the need to consume nearly 1 L of fluid upon awakening after 8 h of sleep? However, current anesthesia practice often allows oral fluids up to 2 h prior to an elective procedure, and the preoperative regimen may include preoperative carbohydrate fluid loading (see Chapters 18 and 48). Such patients will present for surgical or procedural care with essentially no fluid deficit, as will the hospitalized patient who has received intravenous maintenance fluids preoperatively.
Abnormal fluid losses frequently contribute to preoperative deficits. Preoperative bleeding, vomiting, nasogastric suction, diuresis, and diarrhea are often contributory. Losses (actually, redistribution; see next section) due to fluid sequestration by traumatized or infected tissues, coagulopathy-related occult hematoma formation, or ascites can also be substantial. Increased insensible losses due to hyperventilation, fever, and sweating are often overlooked.
Ideally, deficits should be replaced preoperatively in surgical patients, and the fluids administered should be similar in composition to the fluids lost (Table 51— 4).

One of the most important, yet difficult, tasks in anesthesia practice is to monitor and estimate blood loss. Although estimates are complicated by occult bleeding into the wound or under the surgical drapes, accuracy is important to guide fluid therapy and transfusion.
The most commonly used method for estimating blood loss is measurement of blood in the surgical suction container and visual estimation of the blood on surgical sponges ("4 by 4's") and laparotomy pads ("lap sponges"). A fully soaked "4 x 4" is generally considered to hold 10 mL of blood, whereas a soaked "lap" may hold 100 to 150 mL. More accurate estimates are obtained if sponges and "laps" are weighed before and after use, which is especially important during pediatric procedures. Use of irrigating solutions complicates estimates, but their volume should be noted and an attempt made to compensate. Serial hematocrits or hemoglobin concentrations reflect the ratio of RBCs to plasma, not necessarily blood loss, and rapid fluid shifts and intravenous replacement affect such measurements.

Many surgical procedures are associated with obligatory losses of fluids other than blood. Such losses are due mainly to evaporation and internal redistribution of body fluids. Evaporative losses are most significant with large wounds, especially burns, and are proportional to the surface area exposed and to the duration of the surgical procedure.
Internal redistribution of fluids—often called third-spacing—can cause massive fluid shifts and severe intravascular depletion in patients with peritonitis, burns, and similar situations characterized by inflamed or infected tissue. Traumatized, inflamed, or infected tissue can sequester large amounts of fluid in the interstitial space and can translocate fluid across serosal surfaces (ascites) or into bowel lumen. Shifting of intravascular fluid into the interstitial space (edema) is especially important; protein-free fluid shift across an intact vascular barrier into the interstitial space is exacerbated by hypervolemia (water and sodium excess), and pathological alteration of the vascular barrier allows protein-rich fluid shift.

Intraoperative fluid therapy should include supplying basic fluid requirements and replacing residual preoperative deficits as well as intraoperative losses (blood loss, fluid redistribution, and evaporation). Selection of the type of intravenous solution depends on the surgical procedure and the expected blood loss. For minor procedures involving minimal or no blood loss, minimal or no fluid is often administered other than for drug delivery and for maintenance of intravenous line patency. For all other procedures, a balanced crystalloid such as lactated Ringer's solution or Plasmalyte is generally used for maintenance requirements.

The concept of goal-directed fluid therapy (GDFT) arose from a 1983 study by Shoemaker and colleagues that demonstrated lower mortality in critically ill patients in whom tissue oxygen delivery was optimized through "physiological goals" related to cardiac output and fluid administration. The current concept of GDFT has many variations but in general assumes no need for maintenance fluid administration, instead using hemodynamic variables such as stroke volume, cardiac output, cardiac index, and mean arterial blood pressure to determine volume responsiveness and to guide fluid administration by bolus. Some anesthesiologists also incorporate the use of inotropes and vasopressors in their GDFT regimens. GDFT has been widely incorporated into enhanced recovery protocols, but studies of GDFT have been inconsistent to date, with some investigators reporting fewer postoperative complications and shorter hospital lengths of stay. Inconsistent reports of GDFT results may be due to inconsistent GDFT regimens, or to the possibility that not all operations (eg, those with less physiological trespass, such as laparoscopic or robotic procedures) require GDFT. The precise role of GDFT in perioperative care related to various surgical procedures is currently the subject of intense scrutiny.

Ideally, blood loss should be replaced with sufficient crystalloid or colloid solutions to maintain normovolemia until the danger of anemia outweighs the risks of transfusion. At that point, further blood loss is replaced with transfusion of red blood cells to maintain hemoglobin concentration (or hematocrit) at an acceptable level. There are no mandatory transfusion triggers. The point where the benefits of transfusion outweigh its risks must be considered on an individual basis.
Below a hemoglobin concentration of 7 g/dL, the resting cardiac output increases to maintain a normal oxygen delivery. An increased hemoglobin concentration may be appropriate for older and sicker patients with cardiac or pulmonary disease, particularly when there is clinical evidence (eg, a reduced mixed venous oxygen saturation and a persisting tachycardia) that transfusion

In settings other than massive trauma, most clinicians administer lactated Ringer's solution or Plasmalyte in approximately three to four times the volume of the blood lost, or colloid in a 1:1 ratio, until the transfusion trigger point is reached. At that time, blood is replaced unit-for-unit as it is lost, with reconstituted packed red blood cells (see Chapter 39).
The transfusion point can be determined preoperatively from the hematocrit and by estimating @ blood volume (Table 51-5). Patients with a normal hematocrit should generally be transfused only after losses greater than 10% to 20% of their blood volume. The timing of transfusion initiation is based on the patient's surgical procedure, comorbid conditions, and rate of blood loss. The amount of blood loss necessary for the hematocrit to fall to 30% can be calculated as follows:

Neonates, Blood Volume = . Premature, Blood Volume = 95 mL/kg. Full-term, Blood Volume = 85 mL/kg. Infants, Blood Volume = 80 mL/kg. Adults, Blood Volume = . Men, Blood Volume = 75 mL/kg. Women, Blood Volume = 65 mL/kg
1. Estimate blood volume from Table 51-5.
2. Estimate the red blood cell volume (RBCV) at the preoperative hematocrit (RBCV cop):
3. Estimate RBCV at a hematocrit of 30% (RBCV390,), assuming normal blood volume is maintained.
4. Calculate the RBCV lost when the hematocrit is 30%; RBCV),4 =
RBCV
—
peop
RBCV
30%.
- 5. Allowable blood loss = RBCV),,; * 3.
Example: An 85-kg woman has a preoperative hematocrit of 35%. How much blood loss will decrease her hematocrit to 30%?
Estimated blood volume = 65 mL/kg x 85 kg
= 5525 mL.
RBCV,,.,. = 5525 x 35% = 1934 mL.
RBCV,,,,
= 5525 x 30% = 1658 mL.
Red cell loss at 30% = 1934 - 1658 = 276 mL.
Allowable blood loss = 3 x 276 mL = 828 mL.
Therefore, transfusion should be considered only when this patient's blood loss exceeds 800 mL. Increasingly, transfusions are not recommended until the hematocrit decreases to 24% or lower (hemoglobin <8.0 g/dL), but it is necessary to take into account the potential for further blood loss, rate of blood loss, and comorbid conditions (eg, cardiac disease).
Clinical guidelines for transfusion commonly used include the following: (1) transfusing 1 unit of red blood cells will increase hemoglobin 1 g/dL and the hematocrit 2% to 3% in adults; and (2) a 10-mL/kg transfusion of red blood cells will increase hemoglobin concentration by 3 g/dL and the hematocrit by 10%.

Because redistributive and evaporative losses are primarily related to wound size and the extent of surgical dissections and manipulations, procedures can be classified according to the degree of tissue trauma. These additional fluid losses can be replaced according to Table 51-6, based on whether tissue trauma is minimal, moderate, or severe. These values are only guidelines, and actual needs vary considerably from patient to patient. Fluid replacement can also be guided by a GDFT regimen.
TABLE 51-6 Redistribution and evaporative surgical fluid losses.
Minimal (eg, herniorrhaphy), Additional Fluid Requirement = 0-2 mL/kg. Moderate (eg, open cholecystectomy), Additional Fluid Requirement = 2-4 mL/kg. Severe (eg, open bowel resection), Additional Fluid Requirement = 4-8 mL/kg

Human red cell membranes are estimated to contain at least 300 different antigenic determinants, and at least 20 separate blood group antigen systems are known. Fortunately, only the ABO and the Rh systems are important in the majority of blood transfusions. Individuals often produce antibodies (alloantibodies) to the alleles they lack within each system. Such antibodies are responsible for the most serious reactions to transfusions. Antibodies may occur spontaneously or in response to sensitization from a previous transfusion or pregnancy.

ABO blood group typing is determined by the presence or absence of A or B red blood cell (RBC) surface antigens: Type A blood has A RBC antigen, type B blood has B RBC antigen, type AB blood has both A and B RBC antigens, and type O blood has neither A nor B RBC antigen present. Almost all individuals not having A or B antigen "naturally" produce antibodies, mainly immunoglobulin (Ig) M, against those missing antigens within the first year of life.

There are approximately 46 Rhesus group red cell surface antigens, and patients with the D Rhesus antigen are considered Rh-positive. Approximately 85% of the white population and 92% of the black population has the D antigen, and individuals lacking this antigen are called Rh-negative. In contrast to the ABO groups, Rh-negative patients usually develop antibodies against the D antigen only after an Rh-positive transfusion or with pregnancy, in the situation of an Rh-negative mother delivering an Rh-positive baby.

Other red cell antigen systems include Lewis, P, li, MNS, Kidd, Kell, Duffy, Lutheran, Xg, Sid, Cartright, YK, Ss, and Chido Rodgers. Fortunately, with some exceptions (Kell, Kidd, Duffy, and Ss), alloantibodies against these antigens rarely cause serious hemolytic reactions.

The purpose of compatibility testing is to predict and to prevent antigen— antibody reactions resulting from red cell transfusions.

® The most severe transfusion reactions are due to ABO incompatibility; naturally acquired antibodies can react against the transfused (foreign) antigens, activate complement, and result in intravascular hemolysis. The patient's red cells are tested with serum known to have antibodies against A and against B to determine blood type. Because of the almost universal prevalence of natural ABO antibodies, confirmation of blood type is then made by testing the patient's serum against red cells with a known antigen type.
The patient's red cells are also tested with anti-D antibodies to determine Rh status. If the subject is Rh-negative, the presence of anti-D antibody is checked by mixing the patient's serum against Rh-positive red cells. The probability of developing anti-D antibodies after a single exposure to the Rh antigen is 50% to 70%.

The purpose of this test is to detect in the serum the presence of the antibodies that are most commonly associated with non-ABO hemolytic reactions. The test (also known as the indirect Coombs test) requires 45 min and involves mixing the patient's serum with red cells of known antigenic composition; if specific antibodies are present, they will coat the red cell membrane, and subsequent addition of an antiglobulin antibody will result in red cell agglutination. Antibody screens are routinely done on all donor blood and are frequently done for a potential recipient instead of a crossmatch (described next).

A crossmatch mimics the transfusion: Donor red cells are mixed with recipient serum. Crossmatching serves three functions: (1) it confirms ABO and Rh typing, (2) it detects antibodies to the other blood group systems, and (3) it detects antibodies in low titers or those that do not agglutinate easily.

In the situation of negative antibody screen without crossmatch, the incidence of serious hemolytic reaction with ABOand Rh-compatible transfusion is less than 1:10,000. Crossmatching, however, assures optimal safety and detects the presence of less common antibodies not usually tested for in a screen. Because of the expense and time involved (45 min), crossmatches are often now performed before the need to transfuse only when the patient's antibody screen is positive, when the probability of transfusion is high, or when the patient is considered at risk for alloimmunization.

When a patient is exsanguinating, the urgent need to transfuse may arise prior to completion of a crossmatch, screen, or even blood typing. If the patient's blood type is known, an abbreviated crossmatch, requiring less than 5 min, will confirm ABO compatibility. If the recipient's blood type and Rh status are not known with certainty and transfusion must be started before determination, type O Rh-negative (universal donor) red cells may be used. Red blood cells, fresh frozen plasma, and platelets are often transfused in a balanced ratio (1:1:1) in massive transfusion protocols and in trauma damage control resuscitation (see later discussion and Chapter 39).

Blood donors are screened to exclude medical conditions that might adversely affect the donor or the recipient. Once the blood is collected, it is typed, screened

Blood transfusions should usually be given as PRBCs, which allows optimal utilization of blood bank resources. Surgical patients require volume as well as red cells, and crystalloid or colloid can be infused simultaneously through a second intravenous line for volume replacement.
Prior to transfusion, each unit should be carefully checked against the blood bank slip and the recipient's identity bracelet. The transfusion tubing should contain a 170-um filter to trap any clots or debris. Blood for intraoperative transfusion should be warmed to 37°C during infusion, particularly when more than 2 to 3 units will be transfused; failure to do so can result in profound hypothermia. The additive effects of hypothermia and the typically low levels of 2,3-diphosphoglycerate (2,3-DPG) in stored blood can cause a marked leftward shift of the hemoglobin—oxygen dissociation curve (see Chapter 23) and, at least theoretically, promote tissue hypoxia.

Fresh frozen plasma (FFP) contains all plasma proteins, including most clotting factors. Transfusions of FFP are indicated in the treatment of isolated factor deficiencies, the reversal of warfarin therapy, and the correction of coagulopathy associated with liver disease. Each unit of FFP generally increases the level of each clotting factor by 2% to 3% in adults. The initial therapeutic dose is usually 10 to 15 mL/kg. The goal is to achieve 30% of the normal coagulation factor concentration. Administration of FFP and platelets in treatment of coagulopathy is optimally guided by point-of-care coagulation analysis, such as thromboelastography (TEG) or rotational thromboelastometry (ROTEM). FFP may also be used in patients who have received massive blood transfusions (see later discussion and Chapter 39) and continue to bleed following platelet transfusions. Patients with antithrombin III deficiency or thrombotic thrombocytopenic purpura also benefit from FFP transfusions.
Each unit of FFP carries the same infectious risk as a unit of whole blood. In addition, occasional patients may become sensitized to plasma proteins. ABOcompatible units are usually given but are not mandatory. As with red cells, FFP should generally be warmed to 37°C prior to transfusion.

Platelet transfusions should be given to patients with thrombocytopenia or dysfunctional platelets in the presence of bleeding. Prophylactic platelet transfusions are also indicated in patients with platelet counts below 10,000 to
20,000 x 10°/L because of an increased risk of spontaneous hemorrhage.
Platelet counts
less than
50,000
x
109/L
are associated
with increased
loss during
surgery, prophylactic
and platelet
Vaginal delivery
such thrombocytopenic
transfusions and
with normal
platelet
Administration of
patients often
prior to
surgery minor
surgical function
a
platelet count
by procedures
and or
invasive may
counts single
unit
5000
of to
10,000
greater platelets
x
blood receive
procedures.
be performed
than may
be
10°/L, and
50,000
x
10°/L.
expected with
to increase
administration the
of
a
platelet apheresis
unit, by
30,000
to
60,000
x
10°/L.
ABO-compatible platelet transfusions are desirable but not necessary. Transfused platelets generally survive only 1 to 7 days following transfusion. ABO compatibility may increase platelet survival. Rh sensitization can occur in Rh-negative recipients due to the presence of a few red cells in Rh-positive platelet units. Moreover, anti-A or anti-B antibodies in the 70 mL of plasma in each platelet unit can cause a hemolytic reaction against the recipient's red cells when a large number of ABO-incompatible platelet units is given. Administration of Rh immunoglobulin to Rh-negative individuals can protect against Rh sensitization following Rh-positive platelet transfusions.

Granulocyte transfusions, prepared by leukapheresis, may be indicated in neutropenic patients with bacterial infections not responding to antibiotics. Transfused granulocytes have a very short circulatory life span, so that daily transfusions of 10!° granulocytes are usually required. Irradiation of these units decreases the incidence of graft-versus-host reactions, pulmonary endothelial damage, and other problems associated with transfusion of leukocytes (see next section), but may adversely affect granulocyte function. The availability of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) has greatly reduced the need for granulocyte transfusions.

Blood products can be misused in surgical settings. Use of a transfusion algorithm, particularly for components such as plasma, platelets, and cryoprecipitate, and particularly when the algorithm is guided by appropriate laboratory testing, will reduce unnecessary transfusion of these precious and costly (but dangerous) resources (see Chapter 22). Following military experience, major civilian trauma care units commonly transfuse blood products in equal ratios early in severe trauma resuscitation efforts in order to preempt or correct trauma-induced coagulopathy. This balanced approach to transfusion of blood products, 1:1:1 (one unit of FFP and one unit of platelets with each unit of PRBCs), is termed damage control resuscitation (see Chapter 39).

Immune complications following blood transfusions are primarily due to sensitization of the recipient to donor red cells, white cells, platelets, or plasma proteins. Less commonly, the transfused cells or serum may mount an immune response against the recipient.

Hemolytic reactions usually involve specific destruction of the transfused red cells by the recipient's antibodies. Less commonly, hemolysis of a recipient's red cells occurs as a result of transfusion of red cell antibodies. Incompatible units of platelet concentrates, FFP, clotting factor concentrates, or cryoprecipitate may contain small amounts of plasma with anti-A or anti-B (or both) alloantibodies. Transfusions of large volumes of such units can lead to intravascular hemolysis. Hemolytic reactions are commonly classified as either acute (intravascular) or delayed (extravascular).

Acute intravascular hemolysis is usually due to ABO blood incompatibility, and the reported frequency is approximately 1:38,000 transfusions. The most common cause is misidentification of a patient, blood specimen, or transfusion unit, a risk that is not abolished with autologous blood transfusion. These reactions are often severe, and may occur after infusion of as little as 10 to 15 mL of ABO-incompatible blood. The risk of a fatal hemolytic reaction is about 1 in 100,000 transfusions. In awake patients, symptoms include chills, fever, nausea, and chest and flank pain. @ In anesthetized patients, an acute hemolytic reaction may be manifested by a rise in temperature, unexplained tachycardia, hypotension, hemoglobinuria, diffuse oozing in the surgical field, or a combination of these findings. Disseminated
intravascular coagulation,
shock, and
kidney acute
failure can
develop
The severity
of
a
reaction often
depends upon
the volume
rapidly.
of incompatible
blood that
has been
administered.
Management of hemolytic reactions can be summarized as follows:
1. If ahemolytic reaction is suspected, the transfusion should be stopped immediately and the blood bank should be notified.
2. The unit should be rechecked against the blood slip and the patient's identity bracelet.
3. Blood should be drawn to identify hemoglobin in plasma, to repeat compatibility testing, and to obtain coagulation studies and a platelet count.
4. Aurinary bladder catheter should be inserted, and the urine should be checked for hemoglobin.
5. Forced diuresis should be initiated with mannitol and intravenous fluids, and with a loop diuretic if necessary.

A delayed hemolytic reaction—also called extravascular hemolysis—is generally mild and is caused by antibodies to non-D antigens of the Rh system or to foreign alleles in other systems such as the Kell, Duffy, or Kidd antigens. Following an ABO and Rh D-compatible transfusion, patients have a 1% to 1.6% chance of forming antibodies directed against foreign antigens in these other systems. By the time significant amounts of these antibodies have formed (weeks to months), the transfused red cells have been cleared from the circulation. Moreover, the titer of these antibodies subsequently decreases and may become undetectable. Reexposure to the same foreign antigen during a subsequent red cell transfusion, however, triggers an anamnestic antibody response against the foreign antigen. The hemolytic reaction is therefore typically delayed 2 to 21 days after transfusion, and symptoms are generally mild, consisting of malaise, jaundice, and fever. The patient's hematocrit typically fails to rise, or rises only transiently, in spite of the transfusion and the absence of bleeding. The serum unconjugated bilirubin increases as a result of hemoglobin breakdown.
Diagnosis of delayed antibody-mediated hemolytic reactions may be facilitated by the antiglobulin (Coombs) test. The direct Coombs test detects the presence of antibodies on the membrane of red cells. In this setting, however, this test cannot distinguish between recipient antibodies coated on donor red cells and donor antibodies coated on recipient red cells. The latter requires a more detailed reexamination of pretransfusion specimens from both the patient and the donor.
The treatment of delayed hemolytic reactions is primarily supportive. The frequency of delayed hemolytic transfusion reactions is estimated to be approximately 1:12,000 transfusions. Pregnancy (exposure to fetal red cells) can also be responsible for the formation of alloantibodies to red cells.

Nonhemolytic immune reactions are due to sensitization of the recipient to the donor's white cells, platelets, or plasma proteins; the risk of these reactions may be minimized by the use of leukoreduced blood products.

White cell or platelet sensitization is typically manifested as a febrile reaction. Such reactions are relatively common (1—3% of transfusion episodes) and are characterized by an increase in temperature without evidence of hemolysis. Patients with a history of repeated febrile reactions should receive leukoreduced transfusions only.

Urticarial reactions are usually characterized by erythema, hives, and itching without fever. They are relatively common (1% of transfusions) and are thought to be due to sensitization of the patient to transfused plasma proteins. Urticarial reactions can be treated with antihistaminic drugs (H, and perhaps H, blockers) and steroids.

Anaphylactic reactions are rare (approximately 1:150,000 transfusions). These severe reactions may occur after only a few milliliters of blood has been given, typically in IgA-deficient patients with anti-IgA antibodies who receive IgAcontaining blood transfusions. The prevalence of IgA deficiency is estimated to be 1:600 to 1:800 in the general population. Such reactions require treatment with epinephrine, fluids, corticosteroids, and H, and H, blockers. Patients with IgA deficiency should receive thoroughly washed packed red cells, deglycerolized frozen red cells, or IgA-free blood units.

Transfusion-related acute lung injury (TRALI) presents as acute hypoxia and noncardiac pulmonary edema occurring within 6 h of blood product transfusion. It may occur as frequently as 1:5000 transfused units, and with transfusion of any blood component, but especially platelets and FFP. Treatment is similar to that for acute respiratory distress syndrome (see Chapter 58), with the important difference that TRALI may resolve within a few days with supportive therapy. The incidence of TRALI, until recently the leading cause of transfusion-related death, has markedly declined with the recognition that the presence of HLA antibodies in donor plasma is the principal TRALI risk factor (see Chapter 39).

Transfusion-associated circulatory overload (TACO) occurs when blood products are administered at a rate greater than the patient's cardiac output, usually in a massive hemorrhage resuscitation scenario. This is most likely to occur when the provider administering blood products has not recognized that the source of bleeding has been successfully controlled. Communication in this situation is critical between the team members resuscitating the patient with blood products and those attempting to control the hemorrhage (see Chapter 39). TACO has replaced TRALI as the leading transfusion-related risk for trauma patients.

This type of reaction may be seen in immunocompromised patients. Cellular blood products contain lymphocytes capable of mounting an immune response against the compromised recipient. Use of special leukocyte filters alone does not reliably prevent graft-versus-host disease; irradiation of red cell, granulocyte, and platelet transfusions effectively eliminates lymphocytes without altering the efficacy of such transfusions.

Post-transfusion purpura is a potentially fatal thrombocytopenic disorder that occurs, rarely, following blood or platelet transfusion. It results from development of platelet alloantibodies that destroy the patient's own platelets. The platelet count typically drops precipitously 5 to 10 days following transfusion. Treatment may include intravenous IgG and plasmapheresis.

@ Allogeneic transfusion of blood products may diminish immunoresponsiveness and promote inflammation. Post-transfusion immunosuppression is clearly evident in kidney transplant recipients, in whom preoperative blood transfusion improves graft survival. Recent studies suggest that perioperative transfusion may increase the risk of postoperative bacterial infection, cancer recurrence, and mortality, all of which emphasize the need to avoid unnecessary blood product administration.

The incidence of post-transfusion viral hepatitis varies greatly, from approximately 1:200,000 transfusions (for hepatitis B) to approximately 1:1,900,000 (for hepatitis C). Most acute cases are anicteric. Hepatitis C is the more serious infection; most cases progress to chronic hepatitis, with cirrhosis developing in 20% of chronic carriers and hepatocellular carcinoma developing in up to 5% of chronic carriers.

All blood is tested for the presence of anti-HIV-1 and anti-HIV-2 antibodies. The requirement for donor blood testing by the U.S. Food and Drug Administration (FDA) has decreased the risk of transfusion-transmitted HIV to approximately 1:1,900,000 transfusions.

Cytomegalovirus (CMV) and Epstein-Barr virus usually cause asymptomatic or mild systemic illness. Some individuals infected with these viruses become asymptomatic infectious carriers; the white cells in blood units from such donors are capable of transmitting @ either virus. Immunocompromised and immunosuppressed patients (eg, premature infants, organ transplant recipients, and cancer patients) are particularly susceptible to severe transfusion-related CMV infections. Ideally, such patients should receive only CMV-negative units. However, recent studies indicate that the risk of CMV transmission from transfusion of leukoreduced blood products is equivalent to CMV test-negative units. Human T-cell lymphotropic viruses 1 and 2 (HTLV-1 and HTLV-2) are leukemia and lymphoma viruses, respectively, that have been reported to be transmitted by blood transfusion; the former has also been associated with myelopathy. Parvovirus transmission has been reported following transfusion of coagulation factor concentrates and can result in transient aplastic crises in immunocompromised hosts. West Nile virus infection may result in encephalitis with a fatality rate of up to 10%, and transmission of this virus by transfusion has been reported.

Parasitic diseases that can be transmitted by transfusion include malaria, toxoplasmosis, and Chagas disease. Such cases are very rare in developed countries.

Bacterial contamination of blood products is the second leading cause of transfusion-associated mortality. The prevalence of positive bacterial cultures in blood products ranges from 1:2000 for platelets to 1:7000 for PRBCs and may be due to transient donor bacteremia or to inadequate antisepsis during phlebotomy. The prevalence of sepsis due to blood transfusion ranges from 1:25,000 for platelets to 1:250,000 for PRBCs. Both gram-positive (Staphylococcus) and gram-negative (Yersinia and Citrobacter) bacteria can contaminate blood transfusions and transmit disease. To avoid the possibility of significant bacterial contamination, blood products should be administered over a period shorter than 4 h. Specific bacterial diseases rarely transmitted by blood transfusions from donors include syphilis, brucellosis, salmonellosis, yersiniosis, and various rickettsioses.

Massive transfusion is most often defined as the need to transfuse the patient's total estimated blood volume in less than 24 h, or one-half the patient's total estimated blood volume in 1 h. For most adult patients, the total estimated blood volume is the equivalent of 10 to 20 units. The approach to massive transfusion (and to lesser degrees of transfusion) after trauma injury has been greatly influenced by recent military experience in which outcomes have improved with concurrent transfusion of packed red cells, FFP, and platelets to avoid dilutional coagulopathy (see Chapter 39).

@ The most common cause of nonsurgical bleeding following massive blood transfusion is dilutional thrombocytopenia, although clinically significant dilution of coagulation factors may also occur. Point-of-care coagulation studies and platelet counts should guide platelet and FFP transfusion. Although most clinicians will be familiar with "routine" coagulation tests (eg, prothrombin time [PT], activated partial thromboplastin time [aPTT], international normalized ratio [INR], platelet count, fibrinogen), multiple studies show that viscoelastic analysis of whole blood clotting (thromboelastography, rotation thromboelastometry, or Sonoclot analysis) is more useful in resuscitation, liver transplantation, and cardiac surgical settings.

Calcium binding by the citrate preservative can rise in importance following transfusion of large volumes @ of blood or blood products. Clinically important hypocalcemia, causing cardiac depression, will not occur in most normal patients unless the transfusion rate exceeds 1 unit every 5 min, and intravenous calcium salts should rarely be required in the absence of measured hypocalcemia. Because citrate metabolism is primarily hepatic, patients with hepatic disease or dysfunction (and possibly hypothermic patients) may demonstrate hypocalcemia and require calcium infusion during massive transfusion, as may small children and others with relatively impaired parathyroid—vitamin D function.

Massive blood transfusion is an absolute indication for warming all blood products and intravenous fluids to normal body temperature. Ventricular arrhythmias progressing to fibrillation often occur at temperatures close to 30°C, and hypothermia will hamper cardiac resuscitation. The customary use of rapid infusion devices with efficient heat transfer capability has decreased the incidence of transfusion-related hypothermia.

Although stored blood is acidic due to the citric acid anticoagulant and accumulation of red cell metabolites (carbon dioxide and lactic acid), metabolic acidosis due to transfusion is uncommon because citric acid and lactic acid are rapidly metabolized to bicarbonate by the normal liver. However, in the situation of massive blood transfusion, acid-base status is largely dependent upon tissue perfusion, rate of @ blood transfusion, and citrate metabolism. Once normal tissue perfusion is restored, any metabolic acidosis typically resolves, and metabolic alkalosis commonly occurs as citrate and lactate contained in transfusions and resuscitation fluids are converted to bicarbonate by the liver.

The extracellular concentration of potassium in stored blood steadily increases with time. The amount of extracellular potassium transfused with each unit is typically less than 4 mEq per unit. Hyperkalemia can develop regardless of the age of the blood when transfusion rates exceed 100 mL/min. The treatment of hyperkalemia is discussed in Chapter 49. In contrast, hypokalemia is commonly encountered postoperatively, particularly in association with metabolic alkalosis (see Chapters 49 and 50).

Patients undergoing some elective surgical procedures with a high probability for transfusion can donate their own blood for use during that surgery. Collection is usually started 4 to 5 weeks prior to the procedure. The patient is usually allowed to donate a unit as long as the hematocrit is at least 34% or hemoglobin at least 11 g/dL. A minimum of 72 h is required between donations to ensure plasma volume has returned to normal. With iron supplementation and erythropoietin therapy, at least 3 or 4 units can usually be collected prior to operation. Autologous blood transfusions probably do not adversely affect survival in patients undergoing operations for cancer. Although autologous transfusions likely reduce the risk of infection and transfusion reactions, they are not risk-free. Risks include those of immunological reactions due to clerical errors in collection, labeling, and administration; bacterial contamination; and improper storage. Allergic reactions can occur due to allergens (eg, ethylene oxide) that dissolve into the blood from collection and storage equipment.

This technique is used widely during cardiac, major vascular, and orthopedic surgery (see Chapter 22). The shed blood is aspirated intraoperatively into a reservoir and mixed with heparin. After a sufficient amount of blood is collected, the red cells are concentrated and washed to remove debris and anticoagulant and then reinfused into the patient. The concentrates obtained usually have hematocrits of 50% to 60%. To be used effectively, this technique requires blood losses greater than 1000 to 1500 mL. Contraindications to blood salvage and reinfusion include septic contamination of the wound and perhaps malignancy. Newer, simpler systems allow reinfusion of shed blood without centrifugation.

Acute normovolemic hemodilution relies on the premise that if the concentration of red cells is decreased, total red cell loss is reduced when large amounts of blood are shed; moreover, cardiac output remains normal because intravascular volume is maintained. One or 2 units of blood are typically removed just prior to surgery from a large-bore intravenous catheter and replaced with crystalloid and colloids so that the patient remains normovolemic but has a hematocrit of 21% to 25%. The blood that is removed is stored in a CPD bag at room temperature (up to 6 h) to preserve platelet function; the blood is given back to the patient after the blood loss, or sooner if necessary. The use of normovolemic hemodilution is now rare because of progressive improvements in transfusion safety.

Patients can request donated blood from family members or friends known to be ABO compatible. Most blood banks discourage this practice and generally require donation at least 7 days prior to surgery in order to process the donated blood and confirm compatibility. Studies comparing the safety of donor-directed units to that of random donor units have found either no difference or that random units from blood banks are safer than directed units.

A 24-year-old woman with a history of sickle cell anemia presents with abdominal pain and is scheduled for cholecystectomy.

Sickle cell anemia is a hereditary hemolytic anemia resulting from the formation of an abnormal hemoglobin (HbS). HbS differs structurally from the normal adult hemoglobin (HbA) only in the substitution of valine for glutamic acid at the sixth position of the B chain. Functionally, sickle hemoglobin has less affinity for oxygen (P;,. = 31 mm Hg) as well as less solubility. Upon deoxygenation, HbS readily polymerizes and precipitates inside RBCs, causing them to sickle. Sickle cell patients produce variable amounts (2—20%) of fetal hemoglobin (HbF), and it is likely that cells with large amounts of HbF are somewhat protected from sickling. The continuous destruction of irreversibly sickled cells leads to anemia, and hematocrits are typically 18% to 30% due to the extravascular hemolysis. RBC survival is reduced to 10 to 15 days, compared with up to 120 days in normal individuals.
What is the difference between sickle cell anemia and sickle cell trait?
When the genetic defect for adult hemoglobin is present on both the maternally and paternally derived chromosomes (No. 11), the patient is homozygous for HbS and has sickle cell anemia (HbSS). When only one chromosome has the sickle gene, the patient is heterozygous and has sickle cell trait (HbAS). Patients with the sickle trait produce variable amounts of HbA (55-60%) and HbS (35-40%). Unlike those with HbSS, they are generally not anemic, are asymptomatic, and have a normal life span. Sickling occurs only under extreme hypoxemia or in low-flow states. Sickling is particularly apt to occur in the renal medulla; indeed, many patients with the sickle trait have impaired renal concentrating ability. Patients with HbAS may have renal medullary, splenic, and pulmonary infarcts.
Sickle cell anemia is primarily a disease of individuals of Central African ancestry. Approximately 0.2% to 0.5% of African Americans are homozygous for the sickle gene and approximately 8% to 10% are heterozygous. Sickle cell anemia is found less commonly in patients of Mediterranean ancestry.

Conditions favoring the formation of deoxyhemoglobin (eg, hypoxemia, acidosis, intracellular hypertonicity or dehydration, increased 2,3-DPG levels, or increased temperature) can precipitate sickling in patients with HbSS. Hypothermia may also be detrimental because of associated vasoconstriction (see below). Intracellular polymerization of HbS distorts red cells, makes them less pliable and more "sticky," increasing blood viscosity. Sickling may initially be reversible but eventually becomes irreversible in some RBCs. Formation of red cell aggregates in capillaries can obstruct tissue microcirculation, and a vicious cycle is established in which circulatory stasis leads to localized hypoxia, which, in turn, causes more sickling.
With what symptoms do patients with sickle cell anemia usually present?
Patients with HbSS generally first develop symptoms in infancy, when levels of HbF decline. The disease is characterized by both acute episodic crises and chronic and progressive features (Table 51—7). Children display retarded growth and have recurrent infections. Recurrent splenic infarction leads to splenic atrophy and functional asplenism by adolescence. Patients usually die from recurrent infections or kidney failure. Chronic abdominal, bone, and joint pain is typical, often complicated by repeated, acutely painful sickling crises, and patients frequently require chronic opioid therapy. Because of issues related to analgesic tolerance and addiction, these patients often benefit from management by pain specialists. Crises are often precipitated by infection, cold weather, dehydration, or other forms of stress. Crises may be divided into three types:

Vitreous hemorrhage
Retinal infarcts
Proliferative retinopathy
Retinal detachment

Increased intrapulmonary shunting
Pleuritis
Recurrent pulmonary infections
Pulmonary infarcts

Cor pulmonale

Cholelithiasis (pigmented stones)
Cholecystitis
Hepatic infarcts
Hepatic abscesses
Hepatic fibrosis

Anemia
Aplastic anemia
Recurrent infections
Splenic infarcts
Splenic sequestration
Functional asplenia

Hematuria
Renal papillary necrosis
Nephrotic syndrome
Renal insufficiency
Priapism

Synovitis
Arthritis
Aseptic necrosis of femoral head
Small bone infarcts in hand and feet (dactylitis)
Biconcave ("fishmouth") vertebrae
Osteomyelitis

Chronic ulcers
1. Vasoocclusive crises: Depending on the vessels involved, these acute episodes can result in microor macroinfarctions. Most painful crises are thought to be due to microinfarcts in various tissues. Clinically, they present as acute abdominal, chest, back, or joint pain. Differentiation between surgical and nonsurgical causes of abdominal pain is difficult. Most patients form pigmented gallstones by adulthood, and many present with acute cholecystitis. Vasoocclusive phenomena in larger vessels can produce thromboses resulting in splenic, cerebral, pulmonary, hepatic, renal, and, less commonly, myocardial infarctions.
2. Aplastic crisis: Profound anemia (Hb 2—3 g/dL) can rapidly occur when red cell production in the bone marrow is exhausted or suppressed. Infections and folate deficiency may play a major role. Some patients also develop leukopenia.
3. Splenic sequestration crisis: Sudden pooling of blood in the spleen can occur in infants and young children and can cause life-threatening hypotension. The mechanism is thought to be partial or complete occlusion of venous drainage from the spleen.

RBCs from patients with sickle cell anemia readily sickle following addition of an oxygen-consuming reagent (metabisulfite) or a hypertonic ionic solution (solubility test). Confirmation requires hemoglobin electrophoresis.
What would be the best way to prepare patients with sickle cell anemia for surgery?
Optimal preoperative preparation includes the following: patients should be well hydrated, infections should be controlled, and the hemoglobin concentration should be at an acceptable level. Preoperative transfusion must be individualized for the patient and to the surgical procedure. Partial exchange transfusions before major surgical procedures are usually advocated, which decrease blood viscosity, increase blood oxygen-carrying capacity, and decrease likelihood of sickling. The goal of such transfusions is generally to achieve a hematocrit of 35% to 40% with 40% to 50% normal hemoglobin (HbA). Chronic management of sickle cell disease has been revolutionized by the introduction of hydroxyurea.

Conditions that might promote hemoglobin desaturation or low-flow states should be avoided. Every effort must be made to avoid hypothermia, hyperthermia, acidosis, and even mild degrees of hypoxemia, hypotension, or hypovolemia, and generous hydration and a relatively high (>50%) inspired oxygen tension are important. The principal compensatory mechanism for impaired tissue oxygen delivery in these patients is increased cardiac output, which should be maintained intraoperatively. GDFT may be useful. Mild alkalosis may help avoid sickling, but even moderate degrees of respiratory alkalosis may have an adverse effect on cerebral blood flow. Tourniquet use, other than brief, should be avoided.

Most perioperative deaths occur in the postoperative period, and the same management principles applied intraoperatively should be utilized following surgery. Hypoxemia and pulmonary complications (particularly acute chest syndrome) are major risk factors. Supplemental oxygen; optimal hemodynamic, fluid, and pain and symptom management; and pulmonary physiotherapy with early mobilization all help minimize the risk of these complications.

Thalassemia is a hereditary defect in the production of one or more of the normal subunits of hemoglobin. Patients with thalassemia may be able to produce normal HbA but have reduced amounts of aor B-chain production; the severity of this defect depends on the subunit affected and the degree to which hemoglobin production is affected. Symptoms range from absent to severe. Patients with a-thalassemia produce reduced amounts of the a subunit, whereas patients with B-thalassemia produce reduced amounts of the B subunit. The formation of hemoglobins with abnormal subunit composition can alter the red cell membrane and lead to variable degrees of hemolysis as well as ineffective hematopoiesis. The latter can result in hypertrophy of the bone marrow and often an abnormal skeleton. Maxillary hypertrophy may make tracheal intubation difficult. Thalassemias are most common in patients of Southeast Asian, African, Mediterranean, and Indian ancestry.
What is the significance of sickle cell anemia and thalassemia

The combination of HbS and thalassemia, most commonly sickle Bthalassemia, has a variable and unpredictable effect on disease severity.

Substitution of lysine for glutamic acid at position 6 on the B subunit results in hemoglobin C (HbC). Approximately 0.05% of African Americans carry the gene for HbC. Patients homozygous for HbC generally have only a mild hemolytic anemia and splenomegaly and rarely develop significant complications. The tendency for HbC to crystallize in hypertonic environments is probably responsible for the hemolysis and characteristically produces target cells on the peripheral blood smear.

Nearly 0.1% of African Americans are simultaneously heterozygous for both HbS and HbC (HbSC). These patients generally have a mild to moderate hemolytic anemia. Some patients occasionally have painful crises, splenic infarcts, and hepatic dysfunction. Eye manifestations similar to those associated with HbSS disease are particularly prominent. Women with HbSC have a high rate of complications during the third trimester of pregnancy and delivery.

Hemoglobin E is the result of a single substitution on the B chain and is the second most common hemoglobin variant worldwide. It is most often encountered in patients from Southeast Asia. Although oxygen-binding affinity is normal, the substitution impairs production of B chains (similar to B-thalassemia). Homozygous patients have marked microcytosis and prominent target cells, but are not usually anemic and lack any other manifestations.
What is the hematologic significance of glucose-6-phosphate dehydrogenase (G6PD) deficiency?
RBCs are normally well-protected against oxidizing agents. The sulfhydryl groups on hemoglobin are protected by reduced glutathione. The latter is regenerated by NADPH (reduced nicotinamide adenine dinucleotide phosphate), which itself is regenerated by glucose metabolism in the hexose monophosphate shunt. G6PD is a critical enzyme in this pathway. A defect in this pathway results in an inadequate amount of reduced glutathione, which can potentially result in the oxidation and precipitation of hemoglobin in red cells (seen as Heinz bodies) and hemolysis.
Abnormalities in G6PD are relatively common, with over 400 variants described. Clinical manifestations are variable, depending on the functional significance of the enzyme abnormality. Up to 15% of African American males have the common, clinically significant, AT variant. A second variant is common in individuals of eastern Mediterranean ancestry, and a third in individuals of Chinese ancestry. Because the locus for the enzyme is on the X chromosome, abnormalities are X-linked traits, with males being primarily affected. G6PD activity decreases as RBCs age; therefore, aging red cells are most susceptible to oxidation. This decay is markedly accelerated in patients with the Mediterranean variant, but only moderately so in patients with the A™ variant.
Most patients with G6PD deficiency are not anemic but can develop hemolysis following stresses such as viral and bacterial infections, or after administration of certain drugs (Table 51-8). Hemolytic episodes can be precipitated by metabolic acidosis (eg, diabetic ketoacidosis) and may present with hemoglobinuria and hypotension. Such episodes are generally self-limited because only the older population of RBCs is destroyed. Mediterranean variants may be associated with chronic hemolytic anemia of varying severity and may include the classic feature of marked sensitivity to fava beans.

Antimalarial drugs
Nitrofurantoin
Nalidixic acid
Probenecid
Aminosalicylic acid
Phenacetin
Acetanilid
Ascorbic acid (in large doses)
Vitamin K
Methylene blue
Quinine?
Quinidine?
Chloramphenicol
Penicillamine
Dimercaprol

Prilocaine
Nitroprusside
'G6PD, glucose-6-phosphate dehydrogenase,
?May be safe in patients with A> variant.
*Should be avoided because of potential to cause methemoglobinemia.
Management of G6PD deficiency is primarily preventive, avoiding factors known to promote or exacerbate hemolysis. Measures aimed at preserving kidney function (see above) are indicated in patients who develop hemoglobinuria.

Ahn HJ, Kim JA, Lee AR, et al. The risk of acute kidney injury from fluid restriction and hydroxyethyl starch in thoracic surgery. Anesth Analg.
2016;122:186.
Albrecht FW, Glas M, Rensing H, et al. A change of colloid from hydroxyethyl starch to gelatin does not reduce rate of renal failure or mortality in surgical critical care patients: Results of a retrospective cohort study. J Crit Care.

@ When there is no attempt to actively warm an anesthetized patient, core temperature usually decreases 1°C to 2°C during the first hour of general anesthesia (phase one), followed by a more gradual decline during the ensuing 3 to 4 h (phase two), eventually reaching a point of steady state.
@ In the normal, unanesthetized patient the hypothalamus maintains core body temperature within very narrow tolerances, termed the interthreshold range, with the threshold for sweating and vasodilation at one extreme and the threshold for vasoconstriction and shivering at the other.
© Anesthetics inhibit central thermoregulation by interfering with these hypothalamic reflex responses.
@ Postoperative hypothermia should be treated with a forced-air warming device, if available; alternately (but less satisfactorily) warming lights or heating blankets can be used to restore body temperature to normal.
@ Nearly 50% of patients who experience an episode of malignant hyperthermia (MH) have had at least one previous uneventful exposure to anesthesia during which they received a recognized triggering agent. Why MH fails to occur after every exposure to a triggering agent is unclear.
@ The earliest signs of MH during anesthesia include muscle rigidity, tachycardia, unexplained hypercarbia, and increased temperature.
@ Susceptibility to MH may be increased in several musculoskeletal diseases. These include central-core disease, multi-minicore myopathy, and King—Denborough syndrome.
© Treatment of an MH episode is directed at terminating the episode and treating complications such as hyperthermia and acidosis. The mortality rate for MH, even with prompt treatment, ranges from 5% to 30%. First and most importantly, the triggering agent must be stopped; second, dantrolene must be given immediately.
© Dantrolene, a hydantoin derivative, directly interferes with muscle contraction by inhibiting calcium ion release from the sarcoplasmic reticulum. The dose is 2.5 mg/kg intravenously every 5 min until the episode is terminated (upper limit, 10 mg/kg). Dantrolene should be continued for 24 h after initial treatment.

© Propofol, etomidate, benzodiazepines, ketamine, thiopental, methohexital, opiates, droperidol, nitrous oxide, nondepolarizing muscle relaxants, and all local anesthetics are safe for use in MHsusceptible patients.

Anesthesia and surgery predispose patients to hypothermia, usually defined as a body temperature less than 36°C. Unintentional perioperative hypothermia is more common in patients at the extremes of age, and in those undergoing abdominal surgery or procedures of long duration, especially with cold ambient operating room temperatures; it will occur in nearly every such patient unless steps are taken to prevent this complication.
Hypothermia (in the absence of shivering) reduces metabolic oxygen requirements and can be protective during cerebral or cardiac ischemia. Nevertheless, hypothermia has multiple deleterious physiological effects (Table 52-1). In fact, unintended perioperative hypothermia has been associated with an increased mortality rate.
TABLE 52-1 Deleterious effects of hypothermia.
Cardiac arrhythmias and ischemia
Increased peripheral vascular resistance
“Left shift" of the hemoglobin—oxygen saturation curve
Reversible coagulopathy (platelet dysfunction)
Increased postoperative protein catabolism and stress
response
Altered mental status
Impaired renal function
Delayed drug metabolism
Impaired wound healing
Increased risk of infection
Core temperature is normally the same as the central venous blood temperature (except during periods of relatively rapid temperature change as can occur during and after extracorporeal perfusion). @ When there is no attempt to actively warm an anesthetized patient, core temperature usually decreases 1°C to 2°C during the first hour of general anesthesia (phase one), followed by a more gradual decline during the ensuing 3 to 4 h (phase two), eventually reaching a point of steady state (phase three). With general, epidural, or spinal anesthesia most of the initial decrease in temperature during phase one is explained by redistribution of heat from warm "central" compartments (eg, abdomen, thorax) to cooler peripheral tissues (eg, arms, legs) from anesthetic-induced vasodilation. Actual heat loss from the patient to the environment is a minor contributor. Continuous heat loss to the environment is the primary driver for the slower decline during phase two. At steady state, heat loss equals metabolic heat production (Figure 52-1).

FIGURE 52-1 Unintentional hypothermia during general anesthesia follows a typical pattern: a steep drop in core temperature during the first hour (phase one, redistribution), followed by a gradual decline during the next 3 to 4 h (phase two, heat loss), eventually reaching a steady state (phase three).
® In the normal unanesthetized patient the hypothalamus maintains core body temperature within very narrow tolerances, termed the interthreshold range, with the threshold for sweating and vasodilation at one extreme and the threshold for vasoconstriction and shivering at the other. Increasing core temperature a fraction of a degree induces sweating and vasodilation, whereas a minimally reduced core temperature triggers vasoconstriction ® and shivering. Anesthetic agents inhibit central thermoregulation by interfering with these hypothalamic reflex responses. For example, isoflurane produces a dose-dependent decrease in the threshold temperature that triggers vasoconstriction (3°C decrease for each percent of inhaled isoflurane). Both general and neuraxial anesthetics increase the interthreshold range, albeit by different mechanisms. Spinal and epidural anesthetics, like general anesthetics, lead to hypothermia by causing vasodilation and internal redistribution of heat. The thermoregulatory impairment from conduction anesthesia that allows continued heat loss is likely also due to altered perception by the hypothalamus of temperature in the anesthetized dermatomes.

A cold ambient temperature in the operating room, prolonged exposure of a large wound, and the use of large amounts of room-temperature intravenous fluids or high flows of unhumidified gases can contribute to hypothermia. Prewarming the patient for half an hour with convective forced-air warming blankets prevents phase one hypothermia by eliminating the central—peripheral temperature gradient. Methods to minimize phase two hypothermia from heat loss during anesthesia include use of forced-air warming blankets and warmwater blankets, heated humidification of inspired gases, warming of intravenous fluids, and increasing ambient operating room temperature. Passive insulators such as heated cotton blankets or so-called space blankets have limited utility unless virtually the entire body is covered.

Shivering can occur in postanesthesia care units (PACUs) or critical care units as a result of actual hypothermia or neurological aftereffects of general anesthetic agents. Shivering is also common immediately postpartum. Shivering in such instances represents the body's effort to increase heat production and raise body temperature and may be associated with intense vasoconstriction. Emergence from even brief general anesthesia is sometimes also associated with shivering. Although shivering can be part of nonspecific neurological signs (posturing, clonus, or the Babinski sign) sometimes observed during emergence, shivering is typically associated with hypothermia and volatile anesthetics. Shivering appears to be more common after longer durations of surgery and the use of greater concentrations of a volatile agent. Occasionally, shivering is intense enough to cause hyperthermia (38-39°C) and metabolic acidosis, both of which promptly resolve when the shivering stops. Both spinal and epidural anesthesia lower the shivering threshold and vasoconstrictive response to hypothermia; shivering may also be encountered in the PACU following regional anesthesia. Other causes of shivering should be excluded, such as sepsis, drug allergy, or a transfusion reaction. Intense shivering may increase oxygen consumption, CO, production, and cardiac output. These physiological effects are often poorly tolerated by patients with preexisting cardiac or pulmonary impairment. Postoperative shivering increase consumption much may oxygen as as fivefold, may decrease arterial oxygen saturation, and may be associated with an increased risk of myocardial ischemia. Although postoperative shivering can be effectively treated with small intravenous doses of meperidine (12.5—25 mg) in adults, the better option is to reduce the likelihood of shivering by maintaining normothermia. Shivering in intubated and mechanically ventilated patients can
also be controlled with sedation and a muscle relaxant pending normothermia and dissipation of all effects of anesthesia. @ Postoperative hypothermia should be treated with a forced-air warming device, if available; alternately (but less satisfactorily) warming lights or heating blankets can be used. In addition to an increased incidence of myocardial ischemia, hypothermia has been associated with arrhythmias, increased transfusion requirements, and increased duration of muscle relaxant effects, the latter of which can be especially harmful in the recently extubated patient.

Malignant hyperthermia (MH) is a rare (1:15,000 in pediatric patients and 1:40,000 in adult patients) genetic hypermetabolic muscle disease, the characteristic phenotypical signs and symptoms of which most commonly appear with exposure to inhaled general anesthetics or succinylcholine (triggering agents). MH may occasionally present more than an hour after emergence from an anesthetic, and rarely may occur without exposure to known triggering agents. Most cases have been reported in young males; almost none have been reported in infants, and few have been reported in the elderly. Nevertheless, all ages and both sexes may be affected. The incidence of MH varies greatly from country to country and even among different geographic localities within the same country, reflecting varying gene pools. The upper Midwest appears to have the greatest incidence of MH in the United States.

A halogenated anesthetic agent alone may trigger an episode of MH (Table 52— 2). In many of the early reported cases, both succinylcholine and a halogenated anesthetic agent were used and so-called masseter muscle rigidity was observed. However, succinylcholine is less frequently used in modern practice, and about half of the cases in the past decade were associated with a volatile anesthetic as the only triggering agent. Whether succinylcholine is a trigger in the absence of a volatile agent is now controversial. Nearly 50% of patients who experience an episode of MH have had at least one previous uneventful exposure to anesthesia during which they received a recognized triggering agent. Why MH fails to occur after every exposure to a triggering agent is unclear. Investigations into the biochemical causes of MH reveal an uncontrolled increase in intracellular calcium in skeletal muscle. The sudden release of calcium from sarcoplasmic reticulum removes the inhibition of troponin, resulting in sustained muscle contraction. Markedly increased adenosine triphosphatase activity results in an uncontrolled hypermetabolic state with significantly increased oxygen consumption and CO, production, producing severe lactic acidosis and hyperthermia.

Ether
Halothane
Methoxyflurane
Enflurane
Isoflurane
Desflurane
Sevoflurane

Most patients with an episode of MH have relatives who have had a similar episode or who have an abnormal halothane—caffeine contracture test (see later discussion). The complexity of genetic inheritance patterns in families reflects the fact that MH can be associated with a variety of different mutations. The major focus of investigations into the genetic mechanisms of MH has been on the gene for the ryanodine (Ryr,) receptor, located on chromosome 19. Ryr, is a calcium channel responsible for calcium release from the sarcoplasmic reticulum and it plays an important role in muscle depolarization. Most investigators believe that MH results from abnormalities in this channel. The great majority of families in which there is susceptibility to MH harbor one of the known mutations of Ryry.

@ The earliest signs of MH during anesthesia include muscle rigidity, tachycardia, unexplained hypercarbia, and increased temperature (Table 52-3). Two or more of these signs greatly increase the likelihood that the clinical signs are the result of MH. The hypercarbia (due to increased CO, production) results in tachypnea when muscle relaxants are not used. Overactivity of the sympathetic nervous system produces tachyarrhythmias, hypertension, and mottled cyanosis. Hyperthermia may be an early sign, and when it occurs, core temperature can rise as much as 1°C every 5 min. Generalized muscle rigidity is not always present. Hypertension may be rapidly followed by hypotension if cardiac depression occurs. Dark-colored urine reflects myoglobinemia and myoglobinuria.

Increased production
CO, oxygen
Reduced mixed venous tension
Cyanosis
Mottling

Masseter spasm
Generalized rigidity
Increased serum creatine kinase
Hypernatremia
Myoglobinemia
Myoglobinuria

Fever
Sweating
Laboratory testing typically reveals mixed metabolic and respiratory acidosis with a marked base deficit, hyperkalemia, hypermagnesemia, and reduced mixed-venous oxygen saturation. Some case reports describe isolated respiratory acidosis early in the course of an episode of MH. Serum ionized calcium concentration is variable: It may initially increase before a later decrease. Patients typically have increased serum myoglobin, creatine kinase (CK), lactic dehydrogenase, and aldolase levels. When peak serum CK levels (the maximum is usually measured 12-18 h after anesthesia) exceed 20,000 IU/L the diagnosis is strongly suspected. It should be noted that succinylcholine administration to some normal patients without MH may cause serum myoglobin and CK levels to increase markedly.
Much of the problem in diagnosing MH arises from its variable presentation. An unanticipated doubling or tripling of end-tidal CO, (in the absence of a ventilatory change) is an early and sensitive indicator of MH. If the patient survives the initial presentation, acute kidney failure and disseminated intravascular coagulation (DIC) can rapidly ensue. Other complications of MH include cerebral edema with seizures and hepatic failure. Most MH deaths are due to DIC and organ failure due to delayed or no treatment with dantrolene. @ Susceptibility to MH may be increased in several musculoskeletal diseases. These include central-core disease, multi-minicore myopathy, and King— Denborough syndrome. Duchenne and other muscular dystrophies, nonspecific myopathies, heat stroke, and osteogenesis imperfecta have been associated with MH-like symptoms in some reports; however, their association with MH is controversial. Other possible clues to susceptibility include a family history of anesthetic complications, or a history of unexplained fevers or muscular cramps. There are several reports of MH episodes occurring in patients with a history of exercise-induced rhabdomyolysis. Prior uneventful anesthetics and absence of a positive family history are notoriously unreliable predictors of lack of susceptibility to MH. Any patient who develops masseter muscle rigidity during induction of anesthesia should be considered potentially susceptible to MH.

@Treatment of an MH episode is directed at terminating the episode and treating complications such as hyperthermia and acidosis. The mortality rate for MH, even with prompt treatment, ranges from 5% to 30%. Table 52-4 illustrates a standard protocol for management of MH.
TABLE 52-4 Protocol for immediate treatment of malignant hyperthermia.!
1. Discontinue volatile anesthetic and succinylcholine. Notify the surgeon. Call for help.
2. Mix dantrolene sodium with sterile distilled water and administer 2.5 mg/kg intravenously as soon as possible.
3. Administer bicarbonate for metabolic acidosis.
4. Institute cooling measures (lavage, cooling blanket, cold intravenous solutions).
5. Treat severe hyperkalemia with dextrose, 25-50 g intravenously, and regular insulin, 10-20 units intravenously (adult dose).
6. Administer antiarrhythmic agents if needed despite correction of hyperkalemia and acidosis.
7. Monitor end-tidal CO, tension, electrolytes, blood gases, creatine kinase, serum myoglobin, core temperature, urinary output and color, and coagulation status.
8. If necessary, consult on-call physicians at the 24-hour MHAUS hotline, 1-800-644-9737.
'Data from the MHAUS protocol available at https://www.mhaus.org/ healthcare-professionals/mhaus-recommendations/.

First and most importantly, volatile agents and succinylcholine must be discontinued immediately. Even trace amounts of anesthetics released from soda lime, breathing tubes, and breathing bags may be detrimental. The patient should be hyperventilated with 100% oxygen to counteract the effects of uncontrolled CO, production and increased oxygen consumption.

The mainstay of therapy for MH is immediate administration of intravenous dantrolene. Its safety and efficacy mandate its immediate use in this potentially ® life-threatening situation. Dantrolene, a hydantoin derivative, directly interferes with muscle contraction by binding the Ryr, receptor channel and inhibiting calcium ion release from the sarcoplasmic reticulum. The dose is 2.5 mg/kg intravenously every 5 min until the episode is terminated (upper limit, 10 mg/kg). "Conventional" dantrolene is packaged as 20 mg of lyophilized powder to be dissolved in 60 mL of sterile water, thus, reconstitution of the initial dose can be unavoidably time consuming. A new, more costly formulation is available in which 250 mg can be reconstituted in 5 mL, making it an attractive option for the "initial" dose of dantrolene given as an emergency treatment when MH is first diagnosed (Table 52-5). The effective half-life of dantrolene is about 6 h.

One vial contains 20 mg, Ryanodex = One vial contains 250 mg. Mix with 60 mL sterile water, Ryanodex = Mix with 5 mL sterile water. Contains 3000 mg mannitol, Ryanodex = Contains 125 mg mannitol. Solution pH ~9.5, Ryanodex = Solution pH ~10.3. Shelf life 3 y, Ryanodex = Shelf life 2 y. Cost $62/vial!, Ryanodex = Cost $2581/vial'. Remains on formulary for subsequent treatment doses, Ryanodex = Formulary restriction limiting use to initial dose
'Personal Communication from Rodney Stiltner, VCU Health System, Richmond, VA; 23 Apr 2018.
After initial control of symptoms, 1 mg/kg of dantrolene intravenously is recommended every 6 h for 24 to 48 h to prevent relapse (MH can recur within 24 h of an initial episode). Dantrolene is a relatively safe drug that is also used to decrease temperature in patients with thyroid "storm" and neuroleptic malignant syndrome. Although its use in chronic therapy for spastic disorders has been associated with hepatic dysfunction, the most serious complication following acute administration is generalized muscle weakness that may result in respiratory insufficiency or aspiration pneumonia. Dantrolene can cause phlebitis in small peripheral veins and should be given through a central venous line if one is available. Following administration of dantrolene, most patients revert to normal acid-base status promptly and no further pharmacological treatment is necessary.

Persisting metabolic acidosis should be treated with intravenous sodium bicarbonate, recognizing that this treatment will worsen the hypercarbia.
Hyperkalemia should be treated with glucose, insulin, and diuresis. There is no useful role for intravenous calcium salts in treatment of MH. Antiarrhythmic agents, vasopressors, and inotropes should be administered if indicated. Calcium channel blockers should not be given to patients receiving dantrolene because this combination appears to promote hyperkalemia. Furosemide may be used to establish diuresis and prevent acute kidney failure, which may develop as a consequence of myoglobinuria. Dantrolene contains a considerable amount of mannitol (3 g per 20-mg bottle); thus furosemide or bumetanide should be used in preference to mannitol for diuresis.

If fever is present, cooling measures should be instituted immediately. Surface cooling with ice packs over major arteries, cold air convection, and cooling blankets are used. Iced saline lavage of the stomach and any open body cavities (eg, in patients undergoing abdominal surgery) should also be instituted. Use of hypothermic cardiopulmonary bypass may be appropriate if other measures fail.

Masseter muscle rigidity, or trismus, is a forceful contraction of the jaw musculature that prevents full mouth opening. This must be distinguished from incomplete jaw relaxation due to inadequate dosing or inadequate delay after dosing of muscle relaxants. Both myotonia and MH can cause masseter spasm. The two disorders can be differentiated by the medical history, neurological examination, and electromyography. The historical incidence of masseter muscle rigidity following administration of succinylcholine with halothane to pediatric patients was 1% or more; fortunately, only a small fraction of these patients actually developed MH. Isolated masseter muscle rigidity is now of mostly historical interest given the rare combination of halothane and succinylcholine in current pediatric practice. When this occurs and there is no other sign of MH, most anesthesiologists would allow surgery to continue using nontriggering anesthetic agents. Elevated serum CK levels after an episode of masseter muscle rigidity may indicate an underlying myopathy.

including fever, tachypnea, tachycardia, and metabolic acidosis (see Chapter 57). Sepsis can be difficult to diagnose if there is no obvious primary site of infection.

Neuroleptic malignant syndrome
Thyroid Sepsis
storm
Drug-induced hyperthermia
Serotonin syndrome
latrogenic hyperthermia
Brainstem/hypothalamic injury
Transfusion reaction
Less commonly, drug-induced hyperthermia may be encountered in the perioperative period. In these cases, the drugs appear to markedly increase serotonin activity in the brain, causing hyperthermia, confusion, shivering, diaphoresis, hyperreflexia, and myoclonus. Drug combinations associated with this serotonin syndrome include monoamine oxidase inhibitors (MAOIs) and meperidine, and MAOIs and selective serotonin reuptake inhibitors (SSRIs). Hyperthermia can also be caused by some illicit drugs, including 3,4methylenedioxymethamphetamine (MDMA or "ecstasy'), "crack" cocaine, amphetamines, phencyclidine (PCP), and lysergic acid diethylamine (LSD). Iatrogenic hyperthermia is also a possibility, particularly in pediatric patients. Common sources of excessive heat in the operating room include humidifiers on ventilators, warming blankets, heat lamps, and increased ambient temperature. Injuries to the brainstem, hypothalamus, or nearby regions can be associated with marked hyperthermia.
2. Neuroleptic malignant syndrome (NMS)—NMS is characterized by hyperthermia, muscle rigidity with extrapyramidal signs (dyskinesia), altered consciousness, and autonomic lability in patients receiving antidopaminergic agents. The syndrome is caused by an imbalance of neurotransmitters in the central nervous system. It can occur either during drug therapy with antidopaminergic agents (eg, phenothiazines, butyrophenones, thioxanthenes, or metoclopramide) or less commonly following the withdrawal of dopaminergic agonists (levodopa or amantadine) in patients with Parkinson disease. Thus, it appears to involve abnormal central dopaminergic activity, as opposed to the altered peripheral calcium release seen in MH. These differing mechanisms probably explain why nondepolarizing relaxants reverse the rigidity of NMS, but not the rigidity associated with MH.

NMS does not appear to be inherited and typically takes hours to weeks to develop; the majority of episodes develop within 2 weeks of a dose adjustment. Hyperthermia generally tends to be mild, and appears to be proportional to the amount of rigidity. Autonomic dysfunction results in tachycardia, labile blood pressure, diaphoresis, increased secretions, and urinary incontinence. Muscle rigidity can produce dyspnea and respiratory distress and, together with the increased secretions, can promote aspiration pneumonia. CK levels are typically elevated; some patients may develop rhabdomyolysis resulting in myoglobinemia, myoglobinuria, and kidney failure.
Mild forms of NMS promptly resolve after withdrawal of the causative drug (or reinstitution of antiparkinsonian therapy). Initial treatment of more severe forms of NMS should include oxygen therapy and endotracheal intubation for respiratory distress or altered consciousness. Marked muscle rigidity can be controlled with muscle paralysis, dantrolene, or a dopaminergic agonist (amantadine, bromocriptine, or levodopa), depending on the severity and acuity of the syndrome. Resolution of the muscle rigidity usually decreases body temperature.
This syndrome is considered a separate entity from MH; nevertheless, some clinicians believe that NMS may predispose patients to MH and recommend that patients with NMS should not receive succinylcholine or a volatile anesthetic. In contrast to patients with NMS, patients susceptible to MH can safely receive phenothiazines.

© Propofol, etomidate, benzodiazepines, ketamine, thiopental, methohexital, opiates, droperidol, nitrous oxide, nondepolarizing muscle relaxants, and all local anesthetics are safe for use in MH-susceptible patients. An adequate supply of dantrolene should always be available wherever general anesthesia is provided. Prophylactic administration of intravenous dantrolene to susceptible patients is not appropriate if a nontriggering anesthetic is administered. For MH-susceptible patients, the consensus is that the vaporizers should be removed from the anesthesia workstation (or fixed in an "off" position) and the

Association of Anaesthetists of Great Britain & Ireland. http://www.aagbi.org/ Malignant Hyperthermia Association of the United States. http://www.mhaus.org/

@ The fit, previously well-nourished patient undergoing elective surgery could be fasted for up to a week postoperatively without apparent adverse effect on outcomes, provided that fluid and electrolyte needs are met. On the other hand, it is well established in multiple studies that malnourished patients benefit from nutritional repletion via either enteral or parenteral routes prior to surgery.
@ The indications for total parenteral nutrition (TPN) are narrow, including those patients who cannot absorb enteral solutions (small bowel obstruction, short gut syndrome, etc); partial parenteral nutrition may be indicated to supplement enteral nutrition (EN) when EN cannot fully provide for nutritional needs.
@ TPN will generally require a venous access line with its catheter tip in the superior vena cava. The line or port through which the TPN solution will be infused should be dedicated to this purpose, if at all possible, and strict aseptic techniques should be employed for insertion and care of the catheter.
@ Inthe patient with critical illness, discontinuing an EN infusion may require multiple potentially dangerous adjustments in insulin infusions and maintenance of intravenous fluid rates. Meanwhile, the evidence is sparse that EN infusions delivered through an appropriately sited gastrointestinal feeding tube increases the risk of aspiration pneumonitis.
@ Regardless of whether the TPN infusion is continued, reduced, replaced with 10% dextrose, or stopped, blood glucose monitoring will be needed during all but short, minor surgical procedures.
Issues related to nutrition tend to be far removed from the usual concerns of the surgical anesthesiologist, other than those related to whether patient listed for elective surgery has fasted for whatever interval one's institution or colleagues insist upon (this highly controversial issue is also considered in Chapter 19). On the other hand, appropriate nutritional support has been recognized to be of key importance for favorable outcomes in patients with critical illness, a large fraction of whom will require procedural services. Severe malnutrition causes widespread organ dysfunction and increases the risk of perioperative morbidity and mortality. Nutritional repletion may improve wound healing, restore immune competence, and reduce morbidity and mortality rates in critically ill patients. Nutritional support is a key element of an enhanced recovery program (these issues are dealt with in Chapter 48).

This chapter does not provide a complete review of nutrition in the patient undergoing surgery or with critical illness, but rather offers the framework for providing basic nutritional support in such patients. We consider, for example, whether enteral nutrition (EN) or parenteral nutrition (PN) will best meet the needs of an individual patient. This chapter also briefly reviews the conditions under which the ongoing nutritional needs of patients may come into conflict with anesthetic preferences and dogmas, such as the duration that patients must not receive EN before undergoing general anesthesia.

Maintenance of
normal body
mass, composition,
intake of
water, that
cannot energy
substrates, be
synthesized
Relatively few
compounds and
from structure,
specific nutrients.
other essential
nutrients that
Energy make
up are
the
2
fatty is
and protein.
nutrients and
function
Ions and
are required
requires compounds
characterized to
body.
acids, normally
Known
13
form as
“essential.”
the thousands
essential vitamins,
derived
Metabolic triphosphate
and from
dietary breakdown
required for
carbohydrates of
these normal
cellular normally
proteins supply
provide amino
exceeds requirements,
metabolic most
acids nutrients
of include
approximately
8
to
16
or endogenous
substrates minerals.
carbohydrates, yields
function.
of the
Dietary body’s
synthesis;
also pathways
of that
some carbohydrate,
interconversions function
the adenosine
fats and
energy requirements.
however, as
fat, can
occur and
when their
energy substrates.
amino
(see
Figure acid
substrates
33-4).
Dietary supply
The overlap,
Excess amino
acids
10
fats, such
can be converted to carbohydrate or fatty acid precursors. Excess carbohydrates are stored as glycogen in the liver and skeletal muscle. When glycogen stores are saturated (200-400 g in adults), excess carbohydrate is converted to fatty acids and stored as triglycerides, primarily in fat cells.

During starvation, the protein content of essential tissues is spared. As blood glucose concentration begins to fall during fasting, insulin secretion decreases and counterregulatory hormones (eg, glucagon) increase. Hepatic and, to a lesser extent, renal glycogenolysis and gluconeogenesis are enhanced. As glycogen supplies are depleted (within 24 h), gluconeogenesis from amino acids becomes increasingly important. Only neural tissue, renal medullary cells, and erythrocytes continue to utilize glucose—in effect, sparing tissue proteins. Lipolysis is enhanced, and fats become the principal energy source. Glycerol from triglycerides enters the glycolytic pathway, and fatty acids are broken down to acetylcoenzyme A (acetyl-CoA). Excess acetyl-CoA results in the formation of ketone bodies (ketosis). Some fatty acids can contribute to gluconeogenesis. If starvation is prolonged, the brain, kidneys, and muscle also begin to utilize ketone bodies efficiently.
@ The previously well-nourished patient undergoing elective surgery could be fasted for up to a week postoperatively, provided fluid and electrolyte needs are met. Whether early postoperative nutritional support influences outcomes likely relates to the degree of preoperative malnutrition, number of nutrient deficiencies, and severity of the illness, injury, or surgical procedure. The optimal timing and amount of nutrition support following acute illness remain unknown. On the other hand, malnourished patients likely benefit from nutritional repletion prior to and after surgery.
Modern surgical practice has evolved to an expectation of an accelerated ("enhanced") recovery. Enhanced recovery programs generally include early enteral feeding, even in patients undergoing surgery on the gastrointestinal tract, so prolonged periods of postoperative starvation are no longer common practice. Such protocols often specify a carbohydrate drink the night before surgery and again shortly before operation. Previously well-nourished patients should receive nutritional support after no more than 5 days of postsurgical starvation, and those with ongoing critical illness or severe malnutrition should be given nutritional support immediately. The healing of wounds requires energy, protein, lipids, electrolytes, trace elements, and vitamins. Depletion of any of these substrates may delay wound healing and predispose to complications, such as infection. Nutrient depletion may also delay optimal muscle function, which is important for supporting increased respiratory demands and early mobilization of the

The resting metabolic rate can be measured (but often inaccurately) using indirect calorimetry (known as a metabolic cart) or by estimating energy expenditure using standard nomograms (such as the Harris—Benedict equation) to approximate the daily energy requirements. Alternatively, a simple and practical approach assumes that patients require 25 to 30 kcal/kg daily. The weight is usually taken as the ideal body weight or adjusted body weight. One determines the daily requirements to ensure that patients are not unnecessarily overfed, recognizing that nutritional requirements can increase greatly above basal levels with certain conditions (eg, burns). Thus, obese patients require an estimation of ideal body weight to prevent overfeeding.

ill
There was a time when nearly every physician who took care of critically patients was in the position of frequently ordering TPN for patients. This is no longer the case, given that EN is now so much more widely employed. As a consequence, many hospitals and health systems insist that a nutrition support team take responsibility for those rarer patients who require TPN.
In general, patients with critical illness should undergo whatever initial hemodynamic resuscitation they require before initiation of nutritional support (either EN or PN). Absorption, distribution, and metabolism of nutrients require tissue blood flow, oxygen, and carbon dioxide removal. Adequate tissue blood flow requires an adequately resuscitated patient. Patients with critical illness who require EN will usually require placement of a feeding tube. Feeding tubes may be placed into the stomach in patients with adequate gastric emptying and low risk of aspiration. In patients with delayed gastric emptying or those at high risk of aspiration, feeding tubes are best placed into the small intestine. Ideally, the tip of such tubes will be sited within the small intestine, either by transpyloric placement of a nasoenteral tube or directly into the jejunum (via a percutaneous route) during abdominal surgery, reducing the likelihood of gastric distention and regurgitation. Patients who are unable to eat, but require EN over long periods of time, will often undergo percutaneous endoscopic placement of gastrostomy (PEG) tubes (the tips of such tubes can be sited distal to the pylorus). One should confirm that the tips of all feeding tubes are appropriately located before initiating feeds to reduce the likelihood that EN solutions will be accidentally infused, say, into the tracheobronchial tree or abdominal cavity. @ TPN will generally require that a venous access line be placed with the catheter tip in the superior vena cava. PN can be given through a peripheral intravenous catheter but will require larger volumes of fluid (to accommodate the requirement for reduced osmolarity) and will carry an increased risk of phlebitis. The catheter or port through which the TPN solution will be infused should be dedicated to this purpose, if at all possible, and strict aseptic techniques should be employed for insertion and care of the catheter.

Diarrhea is a common problem with EN and may be related to either hyperosmolarity of the solution or lactose intolerance. Gastric distention is another complication that increases the risk of regurgitation and pulmonary aspiration; the use of duodenal or jejunostomy tubes should decrease the likelihood of gastric distention. Complications of TPN are either metabolic or related to central venous access (Table 53—1). Bloodstream infections associated with central and peripheral venous lines remain a major concern, particularly in patients with critical illness and immunocompromised states.

Hemothorax
Chylothorax
Hydrothorax
Air embolism
Thrombosis of central vein
Bloodstream infection

Azotemia
Hepatic dysfunction
Cholestasis
Hyperosmolar coma
Excessive CO, production
Hypoglycemia (due to interruption of infusion)
Metabolic acidosis or alkalosis
Hyperlipidemia
Fat embolism syndrome
Anemia
lron
Vitamin D, K, or B,, deficiency
Essential fatty acid deficiency
Hypervitaminosis A
Hypervitaminosis D
Overfeeding with excess amounts of glucose can increase energy requirements and production of carbon dioxide; the respiratory quotient can be greater than 1 because of lipogenesis. Overfeeding can lead to reversible cholestatic jaundice. Mild elevations of serum transaminases and alkaline phosphatase may reflect fatty infiltration of the liver as a result of overfeeding.

Certain nutrients have been associated with improved outcomes. Surgery and anesthesia are well-recognized inducers of inflammation, producing changes in local (near the wound) and plasma concentrations of neurohormones, cytokines, and other mediators. Many investigators have hypothesized that adverse neurohormonal and inflammatory responses to surgery and anesthesia can be ameliorated through specific diets. Several clinical trials (and a recent metaanalysis) suggest that the addition of "immunomodulating" nutrients (specifically arginine and "fish" oil) to EN may reduce the risk of infection and reduce the length of hospital stay in high-risk surgical patients. Similarly, current guidelines for perioperative PN also advocate the inclusion of n-3 fatty acids. There is some evidence that inclusion of long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs), long-chain monounsaturated fatty acids (found in olive oil), or medium-chain fatty acids may be preferable to the use of solutions (such as soybean-derived lipids) that are rich in longer chain n-6 PUFA. However, such solutions (although widely available outside of the United States) are not approved for use in the United States.
In the past, it was customary to individualize TPN solutions for each patient. Currently, there is little evidence that this is necessary, except in patients who cannot handle a sodium load (eg, those with severe heart failure). Adjustments may also be made in patients requiring renal replacement therapy; however, in most cases this is not necessary. Similarly, except in patients who are already suffering from hepatic encephalopathy, most patients with liver disease can safely receive standard amino acid solutions. Thus, most patients receiving EN and PN can be safely managed with standardized, "off-the-shelf" nutritional formulations. Both EN and PN standardized formulations are available in readyto-use formats that decrease preparation times, reduce contamination risks during formulation, and are associated with lower costs and similar outcomes to compounded solutions.
ENTERAL NUTRITION & NIL PER OS RULES PRIOR TO ELECTIVE SURGERY

blood glucose monitoring will be needed during all but short, minor surgical procedures.

American
Dietetic
Association.
Critical illness
evidence-based guideline.
http://www.guidelines.gov/content.aspx?
id=12818&search=ada+critical+illness+nutrition
American Society for Parenteral and Enteral Nutrition.
http://www. nutritioncare.org/Guidelines_and_Clinical_Resources/Clinical_Gu
Braga
M,
Ljungqvist
O,
Soeters
P, et
al.
ESPEN
guidelines on
parenteral nutrition: Surgery. Clin Nutr. 2009;28:378.
European
Clinical
Nutrition and
Metabolism.
http://www.espen.org/education/espen-guidelines
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Ji,
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A,
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DB, et
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surgery metabolic,
patient—2013
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Clinical and
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Cosponsored
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American
Obesity
Society,
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@ The likelihood of anesthetic complications will never be zero. All anesthesia practitioners, irrespective of their experience, abilities, diligence, and best intentions, will participate in anesthetics that are associated with patient injury.
@ Malpractice occurs when four requirements have been met: (1) the practitioner must have a duty to the patient; (2) there must have been a breach of duty (deviation from the standard of care); (3) the patient (plaintiff) must have suffered an injury; and (4) the proximate cause of the injury must have been the practitioner's deviation from the standard of care.
@ Anesthetic mishaps can be categorized as preventable or unpreventable. Of the preventable incidents, most involve human error, as opposed to equipment malfunctions.
@ The relative decrease in death attributed to respiratory rather than cardiovascular damaging events has been attributed to the increased use of pulse oximetry and capnometry.
@ Many anesthetic fatalities occur only after a series of coincidental circumstances, misjudgments, and technical errors combine (mishap chain).
@ Despite differing mechanisms, anaphylactic and anaphylactoid reactions are typically clinically indistinguishable and equally life threatening.
@ True anaphylaxis due to anesthetic agents is rare; anaphylactoid reactions are much more common.
© Patients with spina bifida, spinal cord injury, and congenital abnormalities of the genitourinary tract have a markedly increased incidence of latex allergy. The incidence of latex anaphylaxis in children is estimated to be 1 in 10,000.
© There is no clear evidence that exposure to trace amounts of anesthetic agents presents a health hazard to operating room personnel; however, the U.S. Occupational Health and Safety Administration continues to set maximum acceptable trace concentrations of less than 25 ppm for nitrous oxide and 0.5 ppm for halogenated anesthetics (2 ppm if the halogenated agent is used alone).
@® Hollow (hypodermic) needles pose a greater risk than do solid (surgical) needles because of the potentially larger inoculum. The use of gloves, needleless systems, or protected needle devices may decrease the incidence of some (but not all) types of injury.
Anesthesiology is a high-risk medical specialty for substance abuse. 8

The three most important methods of minimizing radiation doses are limiting total exposure time during procedures, using proper barriers, and maximizing one's distance from the source of radiation. @
® The likelihood of anesthetic complications will never be zero. All anesthesia practitioners, irrespective of their experience, abilities, diligence, and best intentions, will participate in anesthetics that are associated with patient injury. Moreover, unexpected adverse perioperative outcomes can lead to litigation, even if those outcomes did not directly arise from anesthetic mismanagement. This chapter reviews management approaches to complications secondary to anesthesia and discusses medical malpractice and legal issues from an American (U.S.) perspective. Readers based in other countries may not find this section to be as relevant to their practices.

All anesthesia practitioners will have patients with adverse outcomes, and in the United States most anesthesiologists will at some point in their career be involved to one degree or another in malpractice litigation. Consequently, anesthesia staff should expect litigation to be a part of their professional lives and maintain adequate medical malpractice liability insurance with coverage appropriate for the community in which they practice.
When unexpected events occur, anesthesia staff must generate an appropriate differential diagnosis, seek any necessary consultation, and execute a treatment plan to mitigate (to the greatest degree possible) any patient injury. Appropriate documentation in the patient record is helpful, as adverse outcomes may be reviewed by quality assurance and performance improvement authorities.
Deviations from acceptable practice will likely be noted in the practitioner's quality assurance file. Should an adverse outcome lead to litigation, the medical record documents the practitioner's actions at the time of the incident. Years may pass before litigation proceeds to the point where the anesthesia provider is asked about the case in question. Although memories fade, a clear and complete anesthesia record can provide evidence that a complication was recognized and appropriately treated.
It is often difficult to predict which cases will be pursued by plaintiffs! Litigation may ensue when it is clear (at least to the defense team) that the anesthesia care conformed to standards, and, conversely, a suit may not be filed when there is obvious anesthesia culpability. That said, anesthetics that are followed by unexpected death, paralysis, or brain injury of young, economically productive individuals are particularly attractive to plaintiff's lawyers. When a patient has an unexpectedly poor outcome, one should expect litigation irrespective of one's "positive" relationship with the patient or the injured patient's family or guardians.
®
Malpractice occurs
when four
requirements are
met:
(1)
the practitioner
must have
a
duty to
from the
the patient;
standard injury;
and
(4)
(2)
there of
care);
(3)
the practitioner’s
proximate deviation
practitioner has
must have
cause from
an
The obligation
practitioner’s
Injuries been
a
breach
(plaintiff)
of the
to must
injury must
of provide
be of
have have
duty
(deviation suffered
an been
the care.

care to
execute physical,
for the
breach claim
of that
emotional, duty,
is the
meritorious, the
A
duty is
established
(physician—patient duty
when relationship).
constitutes or
a
breach financial.
patient would
tort system
Causation not
have attempts
of duty.
is established
experienced to
compensate if
the injury.
the injured
but
When patient
or family
members, or
both, by
awarding them
monetary damages.
Being sued is stressful, regardless of the perceived "merits" of the claim. Preparation for defense begins before an injury has occurred. The patient grants informed consent following a discussion of the risks and benefits of the anesthesia options that are available and reasonable. Informed consent does not consist of handing the patient a form to sign. Informed consent requires that the patient understand the choices being presented. As previously noted, appropriate documentation of patient care activities, differential diagnoses, and therapeutic interventions helps to provide a defensible record of the care that was provided, resistant to the passage of time and the stress of the litigation experience.
When an adverse outcome occurs, the hospital or practice risk management group, or both, should be immediately notified. Likewise, one's liability the
a
literature. The increasing number of guidelines proffered by the numerous anesthesia and other societies and their frequent updating can make it difficult for clinicians to stay abreast of the changing nature of practice. This is a particular problem when two societies produce conflicting guidelines on the same topic using the same data. Likewise, the information upon which guidelines are based can range from randomized clinical trials to the opinion of "experts" in the field. Consequently, guidelines do not hold the same weight as standards. Guidelines produced by reputable societies will generally include an appropriate disclaimer based on the level of evidence used to generate the guideline. Nonetheless, plaintiff's attorneys will attempt to use guidelines to establish a "standard of care," when, in fact, clinical guidelines are prepared to assist in guiding the delivery of therapy. However, if deviation from guidelines is required for good patient care, the rationale for such actions should be documented on the anesthesia record, as plaintiff's attorneys may attempt to use the guideline as a de facto standard of care.

1999 and 2005 (Figure 54—1). These results suggest a 97% decrease in anesthesia mortality since the 1940s. However, a 2002 study reported an estimated rate of 1 death per 13,000 anesthetics. Due to differences in methodology, there are discrepancies in the literature as to how well anesthesiology is doing in achieving safe practice. In a 2008 study of 815,077 patients (ASA class 1, 2, or 3) who underwent elective surgery at U.S. Department of Veterans Affairs hospitals, the mortality rate was 0.08% on the day of surgery. The strongest association with perioperative death was the type of surgery (Figure 54—2). Other factors associated with increased risk of death included dyspnea, reduced albumin concentrations, increased bilirubin, and increased creatinine concentrations. A subsequent review of the 88 deaths that occurred on the surgical day noted that 13 of the patients might have benefitted from better anesthesia care, and estimates suggest that death might have been prevented by better anesthesia practice in 1 of 13,900 cases. Additionally, this study reported that the immediate postsurgical period tended to be the time of unexpected mortality. Indeed, often missed opportunities for improved anesthetic care occur following complications when "failure to rescue" contributes to patient demise.
FIGURE 54-1 Annual in-hospital anesthesia-related deaths rates per million hospital surgical discharges and 95% confidence intervals by age, United States, 1999-2005. (Reproduced with permission from Li G, Warner M, Lang B, et al. Epidemiology of anesthesia-related mortality in the United States 1999-2005. Anesthesiology. 2009 Apr;110(4):759-765.)
FIGURE 54-2 Total number of deaths by type of surgery in Veterans Affairs hospitals. (Reproduced with permission from Bishop M, Souders J, Peterson C, et al. Factors associated with unanticipated day of surgery deaths in Department of Veterans Affairs hospitals. Anesth Analg. 2008 Dec;107(6):1924-1935.)

The goal of the ASA Closed Claims Project is to identify common events leading to claims in anesthesia, patterns of injury, and strategies for injury prevention. It is a collection of closed malpractice claims that provides a "snapshot" of anesthesia liability rather than a study of the incidence of anesthetic complications, as only events that lead to the filing of a malpractice claim are considered. The Closed Claims Project consists of trained physicians who review claims against physician anesthesiologists represented by some U.S. malpractice insurers. The number of claims in the database continues to rise each year as new Claims are closed and reported. The claims are grouped according to specific damaging events and complication type. Closed Claims Project analyses have been reported for airway injury, nerve injury, awareness, and so forth. These analyses provide insights into the circumstances that result in claims; however, the incidence of a complication cannot be determined from closed claim data, because we know neither the actual incidence of the complication (some with the complication may not file suit), nor how many anesthetics were performed for which the particular complication might possibly develop. Other similar analyses have been performed in the United Kingdom, where National Health Service (NHS) Litigation Authority claims are reviewed.

® Anesthetic mishaps can be categorized as preventable or unpreventable. Examples of the latter include fatal idiosyncratic drug reactions or any poor outcome that occurs despite proper management. However, studies of anestheticrelated deaths or near misses suggest that many accidents are preventable. Of these preventable incidents, most involve human error (Table 54—1), as opposed to equipment malfunctions (Table 54—2). Unfortunately, some rate of human error is inevitable,. During the 1990s, the top three causes for claims in the ASA Closed Claims Project were death (22%), nerve injury (18%), and brain damage (9%). In a 2009 report based on an analysis of NHS litigation records, anesthesia-related claims accounted for 2.5% of total claims filed and 2.4% of the value of all NHS claims. Moreover, regional and obstetrical anesthesia were responsible for 44% and 29%, respectively, of anesthesia-related claims filed. The authors of the latter study noted that there are two ways to examine data related to patient harm: critical incident and closed claim analyses. Clinical (or critical) incident data consider events that either cause harm or result in a "nearmiss." Comparison between clinical incident datasets and closed claims analyses demonstrates that not all critical events generate claims and that claims may be filed in the absence of negligent care. Consequently, closed claims reports must always be considered in this context.

Unrecognized breathing circuit disconnection
Mistaken drug administration
Airway mismanagement
Anesthesia machine misuse
Fluid mismanagement
Intravenous line disconnection

Breathing circuit
Monitoring device
Ventilator
Laryngoscope

Trends in anesthesia-related death and brain damage have been tracked for many years. In a Closed Claims Project report examining claims in the period between 1975 and 2000, there were 6750 claims (Figure 54—3A and B), 2613 of which were for brain injury or death. The proportion of claims for brain injury or death was 56% in 1975, but had decreased to 27% by 2000. The primary pathological mechanisms by which these outcomes occurred were related to cardiovascular or respiratory problems. Early in the study period, respiratory-related damaging events were responsible for more than 50% of brain injury/death claims, whereas cardiovascular-related damaging events were responsible for 27% of such claims; however, by the late 1980s, the percentage of damaging events related to respiratory issues had decreased, with both respiratory and cardiovascular events being equally likely to contribute to severe brain injury or death. Respiratory damaging events included difficult airway, esophageal intubation, and unexpected extubation. Cardiovascular damaging events were usually multifactorial. Closed claims reviewers found that anesthesia care was substandard in 64% of claims in which respiratory complications contributed to brain injury or death, but in only 28% of cases in which the primary mechanism of patient injury was cardiovascular in nature. Esophageal intubation, premature extubation, and inadequate ventilation were the primary mechanisms by which less than optimal anesthetic care was thought to have contributed to patient injury related to respiratory events. @ The relative decrease in causes of death being attributed to respiratory rather cardiovascular damaging events during the review period was attributed to the increased use of pulse oximetry and capnometry.
FIGURE 54-3 A: The total number of claims by the year of injury. Retrospective data collection began in 1985. Data in this analysis include data collected through December 2003. B: Claims for death or permanent brain damage as percentage of total claims per year by year of injury. (Reproduced with permission from Cheney FW, Domino KB, Caplan RA, Posner KL. Nerve injury associated with anesthesia: A closed claims analysis. Anesthesiology. 1999 Apr; 90(4):1062-1069.)

A 2010 study examining the NHS Litigation Authority dataset noted that airway-related claims led to higher awards and poorer outcomes than did non— airway-related claims. Indeed, airway manipulation and central venous catheterization claims in this database were most associated with patient death. Trauma to the airway also generates significant claims if esophageal or tracheal rupture occur. Postintubation mediastinitis should always be considered whenever there are repeated unsuccessful airway manipulations, as early intervention presents the best opportunity to mitigate any injuries incurred.

Claims related to central venous access in the ASA database were associated with patient death 47% of the time and represented 1.7% of the 6449 claims reviewed. Complications secondary to guidewire or catheter embolism, tamponade, bloodstream infections, carotid artery puncture, hemothorax, and pneumothorax all contributed to patient injury. Although guidewire and catheter embolisms were associated with generally lower level patient injuries, these complications were generally attributed to substandard care. Tamponade following line placement often led to a claim for patient death. The authors of a 2004 closed claims analysis recommended reviewing the chest radiograph following line placement and repositioning lines found in the heart or at an acute angle to reduce the likelihood of vascular perforation and tamponade. Brain damage and stroke are associated with claims secondary to carotid cannulation. Multiple confirmatory methods including ultrasound should be used to ensure that the internal jugular and not the carotid artery is cannulated.
Claims related to peripheral vascular cannulation in the ASA database accounted for 2% of 6849 claims, 91% of which were for complications secondary to the extravasation of fluids or drugs from peripheral intravenous catheters that resulted in extremity injury (Figure 54—4). Air embolisms, infections, and vascular insufficiency secondary to arterial spasm or thrombosis also resulted in claims. Of interest, intravenous catheter claims in patients who had undergone cardiac surgery formed the largest cohort of claims related to peripheral intravenous catheters, most likely due to the usual practice of tucking the arms alongside the patient during the procedure, placing them out of view of the anesthesia providers. Radial artery catheters seem to generate few closed claims; however, femoral artery catheters can lead to greater complications and potentially increased liability exposure.
FIGURE 54—4 Injuries related to IV catheters (n = 127). (Reproduced with permission from Bhananker S, Liau D, Kooner P, et al. Liability related to peripheral venous and arterial catheterization: A closed claims analysis. Anesth Analg. 2009 July;109(1):124-129.)

hemorrhage, and high neuraxial blockade. The most common claim associated with obstetrical anesthesia was related to nerve injury following regional anesthesia. Nerve injury can be secondary to neuraxial anesthesia and analgesia, but also due to obstetrical causes. Early neurological consultation to identify the source of nerve injury is suggested to discern if injury could be secondary to obstetrical rather than anesthesia interventions.

In a 2007 study reviewing 532 claims in pediatric patients younger than 16 years of age in the ASA Closed Claims database from 1973 to 2000 (Figure 54-5), a decrease in the proportion of claims for death and brain damage was noted over the three decades. Likewise, the percentage of claims related to respiratory events also was reduced. Compared with before 1990, the percentage of claims secondary to respiratory events decreased during the years 1990 to 2000, accounting for only 23% of claims in the latter study years compared with 51% of claims in the 1970s. Moreover, the percentage of claims that could be avoided by better monitoring decreased from 63% in the 1970s to 16% in the 1990s. Death and brain damage constitute the major complications for which claims are filed. In the 1990s, cardiovascular events joined respiratory complications in sharing the primary causes of pediatric anesthesia litigation. In the previously mentioned study, better monitoring and newer airway management techniques may have reduced the incidence of respiratory events leading to litigationgenerating complications in the latter years of the review period. Additionally, the possibility of a claim being filed secondary to death or brain injury is greater in children who are in ASA classes 3, 4, or 5.
better monitoring. Years are grouped for illustration. (Reproduced with permission from Jimenez N, Posner K, Cheney F, et al. An update on pediatric anesthesia liability: A closed claims analysis. Anesth Analg. 2007 Jan;104(1):147-129.)
In a review of the Pediatric Perioperative Cardiac Arrest Registry, which collects information from about 80 North American institutions that provide pediatric anesthesia, 193 arrests were reported in children between 1998 and 2004. During the study period, 18% of the arrests were "drug related," compared with 37% of all arrests during the years 1994 to 1997. Cardiovascular arrests occurred most often (41%), with hypovolemia and hyperkalemia being the most common causes. Respiratory arrests (27%) were most commonly associated with laryngospasm. Central venous catheter placement with resultant vascular injury also contributed to some perioperative arrests. Arrests from cardiovascular causes occurred most frequently during surgery, whereas arrests from respiratory causes tended to occur after surgery.

A review of data from the Pediatric Perioperative Cardiac Arrest Registry with a focus on children with congenital heart disease found that such children were more likely to arrest perioperatively secondary to a cardiovascular cause. In particular, children with a single ventricle were at increased risk of perioperative arrest. Children with aortic stenosis and cardiomyopathy were similarly found to be at increased risk of cardiac arrest perioperatively.
Increasingly, obese children present for anesthesia and surgery. These children are at particular risk for obstructive sleep apnea, increased sensitivity to opioids, and respiratory arrest. In particular, death and neurological injury have been reported following tonsillectomy in children at risk for sleep apnea. At-risk children require an extended period of postoperative monitoring to prevent death from perioperative apnea.

Review of the ASA Closed Claims Project database indicates that anesthesia at remote (non—operating room) locations poses a risk to patients secondary to hypoventilation and excessive sedation. Remote location anesthesia care was more likely than operating room anesthesia care to involve a claim for death (54% vs 29%, respectively). The endoscopy suite and cardiac catheterization laboratory were the most frequent locations from which claims were generated. Monitored anesthesia care (MAC) was the most common technique employed in these claims. Overwhelmingly, adverse respiratory events were most frequently

An analysis of the ASA Closed Claims Project database focusing on MAC likewise revealed that oversedation and respiratory arrest frequently led to claims. Claims for burn injuries suffered in operating room fires were also found in the database. Supplemental oxygen, draping, pooling of flammable antiseptic preparatory solutions, and surgical cautery combine to produce the potential for operating room fires (see Chapter 2).

"Equipment problems" is probably a misnomer; the ASA Closed Claims Project review of 72 claims involving gas delivery systems found that equipment misuse was three times more common than equipment malfunction. Anesthesia gas delivery claims have decreased in recent years, accounting for just 1% of claims in the new millennium. Provider equipment misuse was associated with severe patient injuries.
Errors in drug administration also typically involve human error. It has been suggested that as many as 20% of the drug doses given to hospitalized patients are incorrect. Errors in drug administration account for 4% of cases in the ASA Closed Claims Project. Errors resulting in claims were most frequently due to either incorrect dosage or unintentional administration of the wrong drug (syringe swap). In the latter category, accidental administration of epinephrine proved particularly dangerous.
Another type of human error occurs when the most critical problem is ignored because the anesthesia provider's attention is inappropriately focused on a less important problem or an incorrect solution (fixation error). Serious anesthetic mishaps are often associated with distractions and other factors (Table 54—3). The harmful impact of most equipment failures can be decreased or avoided when the problem is identified during the mandatory preoperative anesthesia workstation inspection and checkout. @ Many anesthetic fatalities occur only after a series of coincidental circumstances, misjudgments, and technical errors combine (mishap chain).
TABLE 54-3 Factors associated with human errors and equipment misuse.
Inadequate preparation, Example = No machine checkout or preoperative evaluation; haste and carelessness; production pressure. Inadequate experience and training, Example = Unfamiliarity with anesthetic technique or equipment. Environmental limitations, Example = Inability to visualize surgical field: poor communication with surgeons. Physical and emotional factors, Example = Fatigue; distraction caused by personal problems

To minimize errors in drug administration, drug syringes and ampules in the workspace should be restricted to those needed for the current specific case. Drugs should be consistently diluted to the same concentration in the same way for each use, and they should be clearly labeled. Computer systems for scanning bar-coded drug labels are available that may help to reduce medication errors. The conduct of all anesthetics should follow a predictable pattern by which the anesthetist actively surveys the monitors, the surgical field, and the patient on a recurrent basis. In particular, patient positioning should be frequently reassessed to avoid the possibility of compression or stretch injuries. Increasingly, protocols or algorithms, or both, are provided in anesthetizing locations to ensure compliance with preprocedural checklists, to standardize responses to adverse events, and to minimize errors related to transfer of patient care between clinicians.

The daily insertion of endotracheal tubes (particularly with stylets), laryngeal mask airways, oral/nasal airways, gastric tubes, transesophageal echocardiogram (TEE) probes, esophageal (bougie) dilators, and emergency airways all involve the risk of airway damage. Common complaints, such as sore throat and dysphagia, are usually self-limiting, but they may also be nonspecific symptoms of more ominous complications.
The most common persisting airway injury is dental trauma. In a retrospective study of 600,000 surgical cases, the incidence of injury requiring dental intervention and repair was approximately 1 in 4500. In most cases, laryngoscopy and endotracheal intubation were involved, and the upper incisors were the most frequently injured. Major risk factors for dental trauma included tracheal intubation, preexisting poor dentition, and patient characteristics associated with difficult airway management (including limited neck motion, previous head and neck surgery, craniofacial abnormalities, and a history of difficult intubation).
Laryngeal injuries included vocal cord paralysis, granuloma, and arytenoid dislocation. Most tracheal injuries were associated with emergency surgical tracheotomy, but a few were related to endotracheal intubation. Some injuries occurred during seemingly easy, routine intubations. Proposed mechanisms include excessive tube movement in the trachea and excessive cuff inflation, leading to pressure necrosis. Esophageal perforations contributed to death in 5 of 13 patients reviewed. Esophageal perforation often presents with delayed-onset subcutaneous emphysema or pneumothorax, unexpected febrile state, and sepsis. Pharyngoesophageal perforation is associated with difficult intubation, age over 60 years, and female gender. As in tracheal perforation, signs and symptoms are often delayed in onset. Initial sore throat, cervical pain, and cough progress to fever, dysphagia, and dyspnea, as mediastinitis, abscess, or pneumonia develop. Mortality rates of up to 50% have been reported after esophageal perforation, with better outcomes attributable to rapid detection and treatment.

Minimizing the risk of airway injury begins with the preoperative assessment. Documentation of current dentition (including dental work) should be included. The ASA algorithm for difficult airway management is a useful guide to have immediately available in every anesthetizing location.

The most commonly injured peripheral nerve is the ulnar nerve (Figure 54—6). In a retrospective study of over 1 million patients, ulnar neuropathy (persisting for more than 3 months) occurred in approximately 1 in 2700 patients. Of interest, initial symptoms were most frequently noted more than 24 h after a surgical procedure. Risk factors included male gender, hospital stay greater than 14 days, and very thin or obese body habitus. More than 50% of these patients regained full sensory and motor function within 1 year. Anesthetic technique was not implicated as a risk factor; 25% of patients with ulnar neuropathy underwent monitored care or lower extremity regional technique. Many ulnar neuropathies occurred despite notation of extra padding over the elbow area.
FIGURE 54-6 A: Pronation of the forearm can cause external compression of the ulnar nerve in the cubital tunnel. B: Forearm supination avoids this problem. (Modified with permission from Wadsworth TG. The cubital tunnel and the external compression syndrome. Anesth Analg. 1974 Mar-April;53(2):303-308.)

External pressure on a nerve may compromise its perfusion and disrupt its cellular integrity, resulting in edema, ischemia, and necrosis. Pressure injuries are particularly likely when nerves pass through closed compartments or take a superficial course (eg, the peroneal nerve around the fibula). Postoperative lower extremity neuropathies, particularly those involving the peroneal nerve, have been associated with such factors as extreme degrees (high) and prolonged (greater than 2 h) durations of the lithotomy position. Other risk factors for postoperative lower extremity neuropathy include hypotension, thin body habitus, older age, vascular disease, diabetes, and cigarette smoking. An axillary (chest) "roll" is commonly used to reduce pressure on the inferior shoulder of patients in the lateral decubitus position. This roll should be located caudad to the axilla to prevent direct pressure on the brachial plexus and should be large enough to relieve any pressure from the mattress on the lower shoulder. The data are convincing that some postoperative peripheral nerve injuries are not preventable. The risk of peripheral neuropathy should be included in discussions leading to informed consent. When reasonable, patients with contractures (or other causes of limited flexibility) can be positioned before induction of anesthesia to check for feasibility and discomfort. Final positioning should be evaluated prior to draping. In most circumstances, the head and neck should be kept in a neutral position. Shoulder braces to support patients maintained in a steep Trendelenburg position should be avoided if possible, and shoulder abduction and lateral rotation should be minimized. The upper extremities should not be extended greater than 90°. (There should be no continuous external compression on the knee, ankle, or heel.) Although injuries may still occur, additional padding may be helpful in vulnerable areas. Documentation should include information on positioning, including the presence of padding. Finally, patients who complain of sensory or motor dysfunction in the postoperative period should be reassured that this is usually a temporary condition. Motor and sensory function should be documented. When symptoms persist for more than 24 h, the patient should be referred to a neurologist, physiatrist, or hand surgeon who is knowledgeable about perioperative nerve damage for evaluation. Physiological testing, such as nerve conduction and electromyographic studies, can be useful to document whether nerve damage is a new or chronic condition. In the latter case, fibrillations will be observed in chronically denervated muscles.

Fibrillations should not be present in the first few days after an acute nerve injury.

Changes of body position have physiological consequences that can be exaggerated in disease states. General and regional anesthesia may limit the cardiovascular response to such a change. Even positions that are safe for short periods may eventually lead to complications in persons who are not able to move in response to pain. Regional and general anesthesia abolish protective reflexes and predispose patients to injury.
Many complications, including air embolism, blindness from sustained pressure on the globe, and finger amputation following a crush injury, can be caused by improper patient positioning (Table 54—4). These complications are best prevented by evaluating the patient's postural limitations during the preanesthetic visit; padding pressure points, susceptible nerves, and any area of the body that will possibly be in contact with the operating table or its attachments; avoiding flexion or extension of a joint to its limit; having an awake patient assume the position to ensure comfort; and understanding the potential complications of each position. Monitors must often be disconnected during patient repositioning, making this a time of greater risk for unrecognized hemodynamic derangement or hypoventilation.

Venous air embolism, Position = Sitting, prone, reverse Trendelenburg. Venous air embolism, Prevention = Maintain adequate venous pressure; ligate “open” veins. Alopecia, Position = Supine, lithotomy, Trendelenburg. Alopecia, Prevention = Avoid prolonged hypotension, padding, and occasional head turning. Backache, Position = Any. Backache, Prevention = Lumbar support, padding, and slight hip flexion. Extremity compartment syndromes, Position = Especially lithotomy. Extremity compartment syndromes, Prevention = Maintain perfusion pressure and avoid external compression. Corneal abrasion, Position = Any, but especially prone. Corneal abrasion, Prevention = Taping and/or lubricating eye. Digit amputation, Position = Any. Digit amputation, Prevention = Check for protruding digits before changing table configuration. Nerve palsies, Position = . Nerve palsies, Prevention = . Brachial plexus Common peroneal Radial UInar, Position = Any Lithotomy, lateral Any Any. Brachial plexus Common peroneal Radial UInar, Prevention = Avoid stretching or direct compression at neck, shoulder, or axilla sustained pressure on lateral aspect of upper fibula compression of lateral humerus Avoid sustained pressure on ulnar groove Avoid pressure on globe. , Position = decubitus. , Prevention = Avoid. , Position = . , Prevention = Avoid. Retinal ischemia, Position = Prone, sitting. Retinal ischemia, Prevention = . Skin necrosis, Position = Any. Skin necrosis, Prevention = Avoid sustained pressure over bony prominences
Compartment syndromes can result from hemorrhage into a closed space following a vascular puncture or prolonged venous outflow obstruction, particularly when associated with hypotension. In severe cases, this may lead to muscle necrosis, myoglobinuria, and renal damage, unless the pressure within the extremity compartment is relieved by surgical decompression (fasciotomy) or in the abdominal compartment by laparotomy.

A
continuing series
of media
reports have
imprinted the
fear of
awareness under
general anesthesia
and into
helplessness while
undergoing awareness
does occur,
to posttraumatic
stress the
psyche of
paralyzed the
general have
population.
made general
Accounts unconsciousness
patients disorder
When may
exhibit
(eg, of
recall
a
primary concern
unintended intraoperative
symptoms ranging
sleep disturbances,
from mild
anxiety nightmares,
and social
difficulties).
Although the incidence is difficult to measure, approximately 2% of the closed claims in the ASA Closed Claims Project database relate to awareness
similar monitors may be helpful, although randomized clinical trials have failed to demonstrate a reduced incidence of awareness with use of BIS when compared with appropriate concentrations of volatile agents. Finally, if there is evidence of intraoperative awareness during postoperative rounds, the practitioner should obtain a detailed account of the experience, answer patient questions, be very empathetic, and refer the patient for psychological counseling if appropriate.

Sudden cardiac arrest during an otherwise routine administration of spinal anesthetics is an uncommon complication. The initial published report was a closed claims analysis of 14 patients who experienced cardiac arrest during spinal anesthesia. The cases primarily involved young (average age 36 years), relatively healthy (ASA physical status I-II) patients who were given appropriate doses of local anesthetic that produced a high dermatomal level of block prior to arrest (T4 level). Respiratory insufficiency with hypercarbia due to sedatives was thought to be a potential contributing factor. The average time from induction of

Perioperative hearing loss is usually transient and often goes unrecognized. The incidence of low-frequency hearing loss following dural puncture may be as high as 50%. It seems to be due to cerebrospinal fluid leak and, if persistent, can be relieved with an epidural blood patch. Hearing loss following general anesthesia can be due to a variety of causes and is much less predictable. Mechanisms include middle ear barotrauma, vascular injury, and ototoxicity of drugs (aminoglycosides, loop diuretics, nonsteroidal antiinflammatory drugs, and antineoplastic agents).

Hypersensitivity (or allergic) reactions are exaggerated immunological responses to antigenic stimulation in previously sensitized persons. The antigen, or allergen, may be a protein, polypeptide, or smaller molecule. Moreover, the allergen may be the substance itself, a metabolite, or a breakdown product. Patients may be exposed to antigens through the respiratory tract, gastrointestinal tract, eyes, skin and from previous intravenous, intramuscular, or peritoneal exposure.
Anaphylaxis occurs when inflammatory agents are released from basophils
TABLE 54-5 Hypersensitivity reactions.
Type | (immediate)
Atopy
Urticaria—angioedema
Anaphylaxis
Type II (cytotoxic)
Hemolytic transfusion reactions
Autoimmune hemolytic anemia
Heparin-induced thrombocytopenia
Type Ill (immune complex)
Arthus reaction
Serum sickness
Acute hypersensitivity pneumonitis
Type IV (delayed, cell-mediated)
Contact dermatitis
Tuberculin-type hypersensitivity
Chronic hypersensitivity pneumonitis

Initial exposure of a susceptible person to an antigen induces CD4* T cells to lymphokines that activate and transform specific B lymphocytes into plasma cells, producing allergen-specific IgE antibodies (Figure 54—7). The Fc portion of these antibodies then associates with high affinity receptors on the cell surface of tissue mast cells and circulating basophils. During subsequent reexposure to the antigen, it binds the Fab portion of adjacent IgE antibodies on the mast cell surface, inducing degranulation and release of inflammatory lipid mediators and additional cytokines from the mast cell. The end result is the release of histamine, tryptase, proteoglycans (heparin and chondroitin sulfate), and carboxypeptidases. An elevated tryptase concentration in the setting of clinical signs of hypersensitivity signals mast cell activation and is the diagnostic test of choice for anaphylactic reactions. The combined effects of these mediators can produce arteriolar vasodilation, increased vascular permeability, increased mucus secretion, smooth muscle contraction, and other clinical manifestations of type I reactions.
FIGURE 54-7 A: Induction of IgE-mediated allergic sensitivity to drugs and other allergens. B: Response of IgE-sensitized cells to subsequent exposure to allergens. Ig, immunoglobulin. (Reproduced with permission from Katzung BG, ed. Basic & Clinical Pharmacology. 8th ed. New York, NY: McGraw-Hill; 2001.)

Type I hypersensitivity reactions are classified as atopic or nonatopic. Atopic disorders typically affect the skin or respiratory tract and include allergic rhinitis, atopic dermatitis, and allergic asthma. Nonatopic hypersensitivity disorders include urticaria, angioedema, and anaphylaxis; when these reactions are mild, they are confined to the skin (urticaria) or subcutaneous tissue (angioedema), but when they are severe, they become generalized and a life-threatening medical emergency (anaphylaxis). Urticarial lesions are characteristically wellcircumscribed skin wheals with raised erythematous borders and blanched centers; they are intensely pruritic. Angioedema presents as deep, nonpitting cutaneous edema from marked vasodilation and increased permeability of subcutaneous blood vessels. When angioedema is extensive, it can be associated with large fluid shifts; when it involves the pharyngeal or laryngeal mucosa, it can rapidly compromise the airway.
Angioedema can lead to airway compromise and is frequently the cause for anesthesiology airway management consultation in the emergency department. Angioedema is secondary to increased capillary permeability secondary either to activation of mast cells or through kinin mediation. Patients taking angiotensinconverting enzyme (ACE) inhibitors may experience kinin-mediated angioedema. Bradykinin is inactivated by ACE and consequently it may accumulate when ACE is inhibited. Hereditary angioedema can occur in patients with ineffective amounts of complement inhibitor (C1-INH). Treatment for angioedema first focuses upon airway management as needed. Fresh frozen plasma can be given to increase ACE if ACE inhibition is thought to be contributing to angioedema. The bradykinin receptor antagonist icatibant can likewise be administered if available. C1-INH replacement protein is also available to inhibit kinin synthesis. The kallikrein inhibitor ecallantide can also be used to decrease bradykinin production (Figure 54—8).

FIGURE 54-8 Treatment of acute attacks of angioedema based on underlying etiology. ACE-I, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; C1INHRP, C1 inhibitor replacement protein; FFP, fresh frozen plasma; IV, intravenous; NSAID, nonsteroidal anti-inflammatory drug. (Reproduced with permission from Barbara D, Ronan K, Maddox D, et al. Perioperative angioedema; background,

TABLE 54-6 Clinical manifestations of anaphylaxis.
Cardiovascular, Signsand Symptoms = | Hypotension,’ tachycardia, arrhythmias. Pulmonary, Signsand Symptoms = Bronchospasm,' cough, dyspnea, pulmonary edema, laryngeal edema, hypoxia. Dermatological, Signsand Symptoms = Urticaria,' facial edema, pruritus
'Key signs during general anesthesia.
Anaphylactoid reactions resemble anaphylaxis but do not depend on IgE antibody interaction with antigen. A drug can directly release histamine from mast cells (eg, urticaria following high-dose morphine @ sulfate) or activate complement. Despite differing mechanisms, anaphylactic and anaphylactoid reactions typically are clinically indistinguishable and equally life threatening. 'Table 54—7 lists common causes of anaphylactic and anaphylactoid reactions.

Anaphylactic reactions against polypeptides, 1 = Venoms (Hymenoptera, fire ant, snake, jellyfish) Airborne allergens (pollen, molds, danders) Foods (peanuts, milk, egg, seafood, grain) Enzymes (trypsin, streptokinase, chymopapain, asparaginase) Heterologous serum (tetanus antitoxin, antilymphocyte globulin, antivenin) Human proteins (insulin, corticotropin, vasopressin, serum and seminal proteins) Latex. Anaphylactic reactions against hapten carrier, 1 = Antibiotics (penicillin, cephalosporins, sulfonamides) Disinfectants (ethylene oxide, chlorhexidine) Local anesthetics (procaine). Anaphylactoid reactions, 1 = Polyionic solutions (radiocontrast medium, polymyxin B) Opioids (morphine, meperidine) Hypnotics (propofol, thiopental) Muscle relaxants (rocuronium, succinylcholine, cisatracurium) Synthetic membranes (dialysis) Nonsteroidal antiinflammatory drugs Preservatives (sulfites, benzoates) Protamine Dextran Steroids Exercise Idiopathic
'Adapted with permission from Bochner BS, Lichtenstein LM. Anaphylaxis, N Engi J Med. 1991 June 20;324(25):1786-1799.
Serum tryptase measurement is helpful in confirming the diagnosis of an anaphylactic reaction. Treatment must be immediate and tailored to the severity of the reaction (Table 54-8).

Discontinue drug administration
Administer 100%
oxygen mg
Epinephrine (0.01-0.5 mg IV or IM)!
Consider intubation
Intravenous fluid bolus
Diphenhydramine (50-75 mg IV)
Ranitidine (150 IV)
Hydrocortisone (up to 200 mg IV) or methylprednisolone (1-2 mg/kg)
"The dose and route of epinephrine depend on the severity of the reaction. An infusion of 1-5 meg/min may be necessary in adults.

@ True anaphylaxis due to anesthetic agents is rare; anaphylactoid reactions are much more common. Risk factors associated with hypersensitivity to anesthetics include female gender, atopic history, preexisting allergies, and previous anesthetic exposures. An estimated 1 in 6500 patients has an allergic reaction to a muscle relaxant. In many instances, the patient had no previous exposure to the agent. Investigators suggest that over-the-counter drugs, cosmetics, and food products, many of which contain tertiary or quaternary ammonium ions, can sensitize susceptible individuals.
The incidence of anaphylaxis for thiopental and propofol is 1 in 30,000 and 1 in 60,000, respectively. Allergic reactions to etomidate, ketamine, and benzodiazepines are exceedingly rare. True anaphylactic reactions due to opioids are far less common than nonimmune histamine release. Similarly, anaphylactic reactions to local anesthetics are much less common than vasovagal reactions, toxic reactions to accidental intravenous injections, and side effects from absorbed or intravenously injected epinephrine. IgE-mediated reactions to certain ester-type local anesthetics (eg, procaine and benzocaine), however, are well described secondary to reaction to the metabolite, para-aminobenzoic acid. In contrast, true anaphylaxis due to amide-type local anesthetics is very rare; in some instances, the preservative (paraben or methylparaben) was believed to be responsible for an apparent anaphylactoid reaction to a local anesthetic. Moreover, the cross-reactivity between amide-type local anesthetics seems to be low. Volatile anesthetics are not likely to initiate anaphylaxis.

Many true drug allergies in surgical patients are due to antibiotics, mainly Blactam antibiotics, such as penicillins and cephalosporins. Although 1% to 4% of B-lactam administrations result in allergic reactions, only 0.004% to 0.015% of these reactions result in anaphylaxis. Up to 2% of the general population is

Risk management and continuous quality improvement programs at the departmental level may reduce anesthetic morbidity and mortality rates, and decrease perioperative costs, by addressing monitoring standards, equipment, practice guidelines, continuing education, care variation and quality of care, and staffing and "system" issues. Specific responsibilities of peer review committees include identifying (and, ideally, preventing) potential problems, formulating and periodically revising departmental policies, ensuring the availability of properly functioning anesthetic equipment, enforcing standards required for clinical privileges, and evaluating the appropriateness and quality of patient care. A
quality improvement system impartially and continuously reviews complications, compliance with standards, and quality indicators (see Chapter 59).
Health care purchasers seek maximal health care value.
Consequently, anesthesia providers must consistently deliver high-quality results while minimizing expenses. To achieve value, hospitals and providers alike have looked to adopt principles of continuous improvement borrowed from industry. Adapting the work of W. Edwards Deming, health care organizations often employ the PDSA (Plan-Do-Study-Act) cycle to promote ongoing improvement, to standardize results, and to reduce waste (see Figure 54—9). So-called lean management strategies are used to achieve maximal health care value by continually attempting to improve processes to minimize variability so to ensure optimal results with minimal waste.
FIGURE 54-9 Plan-Do-Study-Act (PDSA) cycle for improvement. (Reproduced with permission from Moriates C, Arora V, Shah N, eds. Understanding Value-Based Healthcare. New York, NY: McGraw-Hill Education; 2015.)

Anesthesia providers spend much of their workday exposed to anesthetic gases, low-dose ionizing radiation, electromagnetic fields, blood products, and workplace stress. Each of these can contribute to negative health effects. A 2000 paper compared the mortality risks of anesthesiologists and internists. Death from heart disease or cancer did not differ between the groups; however, anesthesiologists had an increased rate of suicide and illicit drug-related death (Table 54-9). Anesthesiologists also had a greater chance of death from external causes, such as boating, bicycling, and aeronautical accidents compared with internists. Nevertheless, both anesthesiologists and internists had lower mortality than the general population, likely due to their higher socioeconomic status. Anesthesiologists' access to parenteral opioids possibly contributes to a 2.21 relative risk for drug-related deaths compared with that of internists.

All drug-related deaths,  = <1987. All drug-related deaths, Anesthesiologists (N} = 36. All drug-related deaths, Internists = 14. All drug-related deaths, RR? = 2.65. All drug-related deaths, 95% Cl = 1.42-4.91. ,  = 21987. , Anesthesiologists (N} = 55. , Internists = 19. , RR? = 2.87. , 95% Cl = 1.71-4.84. Drug-related suicides,  = <1987. Drug-related suicides, Anesthesiologists (N} = 16. Drug-related suicides, Internists = 11. Drug-related suicides, RR? = 1.48. Drug-related suicides, 95% Cl = 0.69-3.20. ,  = 21987. , Anesthesiologists (N} = 32. , Internists = 11. , RR? = 2.88. , 95% Cl = 1.45-5.71. Suicides,  = <1987. Suicides, Anesthesiologists (N} = 41. Suicides, Internists = 33. Suicides, RR? = 1.25. Suicides, 95% Cl = 0.79-1.97. ,  = 21987. , Anesthesiologists (N} = 62. , Internists = 38. , RR? = 1.60. , 95% Cl = 1.07-2.39
'Reproduced with permission from Alexander B, Checkoway H, Nagahama S, Domino K. Cause-specific mortality risks of anesthesiologists. Anesthesiology. 2000 Oct;93(4):922-930.
- 2Cl, confidence interval.
3Relative Ratio (RR) of anesthesiologists compared with internists for that time period, RR is adjusted for age, gender, and race.

® There is no clear evidence that exposure to trace amounts of anesthetics presents a health hazard to operating room personnel. However, because previous studies examining this issue have yielded flawed but conflicting results, the U.S. Occupational Health and Safety Administration (OSHA) continues to set maximum acceptable trace concentrations of less than 25 ppm for nitrous oxide and 0.5 ppm for halogenated anesthetics (2 ppm if the halogenated agent is used alone). Achieving these low levels depends on efficient scavenging equipment, adequate operating room ventilation, and conscientious anesthetic technique. Most people cannot detect the odor of volatile agents at a concentration of less than 30 ppm. If there is no functioning scavenging system, operating room anesthetic gas concentrations reach 3000 ppm for nitrous oxide and 50 ppm for volatile agents.

Narcotics Anonymous), random urine testing, and oral naltrexone therapy (a long-acting opioid antagonist). Effective prevention strategies are difficult to formulate; "better" control of drug availability is unlikely to deter a determined individual. It is unlikely that education about the severe consequences of substance abuse will bring new information to the potential drug-abusing physician. There remains controversy regarding the rate at which anesthesia staff will experience recidivism. Many experts argue for a "one strike and you're out" policy for anesthesiology residents who abuse injectable drugs. The decision as to whether a fully trained and certified physician who has been discovered to abuse injectable drugs should return to anesthetic practice after completing a rehabilitation program varies and depends on the rules and traditions of the practice group, the medical center, the relevant medical licensing board, and the perceived likelihood of recidivism. Physicians returning to practice following successful completion of a program must be carefully monitored over the long term, as relapses can occur years after apparent successful rehabilitation. Alcohol abuse is a common problem among physicians and nurses, and anesthesia personnel are no exception. Interventions for alcohol abuse, as is true for injectable drug abuse, must be carefully orchestrated. Guidance from the employee health program, local medical society, or licensing authority is highly recommended.

The use of imaging equipment (eg, fluoroscopy) during surgery and interventional radiological procedures exposes the anesthesiologist to the potential ® risks of ionizing radiation. The three most important methods of minimizing radiation doses are limiting total exposure time during procedures, using proper barriers, and maximizing one's distance from the source of radiation. Anesthesiologists who routinely perform fluoroscopic image-guided invasive procedures should consider wearing protective eyewear incorporating radiation shielding. Lead glass partitions or lead aprons with thyroid shields are mandatory for all personnel who are exposed to ionizing radiation. The inverse square law states that the dosage of radiation varies inversely with the square of the distance. Thus, the exposure at 4 m will be one-sixteenth that at 1 m. The maximum recommended occupational whole-body exposure to radiation is 5 rem/y. This can be monitored with an exposure badge. The health impact of exposure to electromagnetic radiation remains unclear.

A 73-year-old man is scheduled for emergency relief of an intestinal obstruction from a sigmoid volvulus. The patient had a myocardial infarction 1 month earlier that was complicated by congestive heart failure. His blood pressure is 160/90 mm Hg, pulse 110 beats/min, respiratory rate 22 breaths/min, and temperature 38.8°C.

Strangulation of the bowel begins with venous obstruction, but can quickly progress to arterial occlusion, ischemia, infarction, and perforation. Acute peritonitis could lead to severe dehydration, sepsis, shock, and multiorgan failure.

Because of the history of recent myocardial infarction and congestive heart failure, an arterial line would be useful. TEE and pulse contour analysis monitors of cardiac output could be used. Pulmonary arterial flotation catheters have often been used in the past, but they are associated with significant complications and their use does not improve patient outcomes. Large fluid shifts should be anticipated. Furthermore, information regarding myocardial oxygen supply (diastolic blood pressure) and demand (systolic blood pressure, left ventricular wall stress, and heart rate) should be continuously available.

Severe tachycardia or extremes in arterial blood pressure should be avoided. During the laparotomy, gradual increases in heart rate and blood pressure are noted. ST-segment elevations appear on the electrocardiogram. A nitroglycerin infusion is started. The heart rate is now 130 beats/min, and the blood pressure is 220/140 mm Hg. The concentration of volatile anesthetic is increased, and metoprolol is administered intravenously in 1mg increments. This results in a decline in heart rate to 115 beats/min, with no change in blood pressure. Suddenly, the rhythm converts to ventricular tachycardia, with a profound drop in blood pressure. As amiodarone is being administered and the defibrillation unit prepared, the rhythm degenerates into ventricular fibrillation.

A differential diagnosis of pronounced tachycardia and hypertension might include pheochromocytoma, malignant hyperthermia, or thyroid storm. In this case, further inspection of the nitroglycerin infusion reveals a labeling error: although the tubing was labeled "nitroglycerin," the infusion bag was labeled "epinephrine."

Metoprolol is a B;-adrenergic antagonist. It inhibits epinephrine's B,stimulation of heart rate, but does not antagonize a-induced vasoconstriction. The net result is a decrease in heart rate, but a sustained increase in blood pressure.

An overdose of epinephrine can cause life-threatening ventricular arrhythmias. In addition, if a central venous catheter was malpositioned, with its tip in the right ventricle, the catheter tip could have stimulated ventricular arrhythmias.
What other factors may have contributed to this anesthetic mishap?
Multiple factors will often combine to create an anesthetic misadventure. Incorrect drug labels are but one example of errors that can result in patient injury. Inadequate preparation, technical failures, knowledge deficits, and practitioner fatigue or distraction can all contribute to adverse outcomes. Careful adherence to hospital policies, checklists, patient identification procedures, and surgical and regional block timeouts can all help to prevent iatrogenic complications.

Institute for Healthcare Improvement. PDSA cycles.
http://www.ihi.org/resources/pages/tools/plandostudyactworksheet.aspx.

Practice advisory for the prevention of perioperative peripheral neuropathies: A report by the American Society of Anesthesiologists Task Force on prevention of peripheral neuropathies. Anesthesiology. 2011;114:1.

Cardiopulmonary resuscitation and emergency cardiac care (CPRECC) should be considered any time an individual cannot adequately oxygenate or perfuse vital organs—not only following cardiac or respiratory arrest.
Regardless of which transtracheal jet ventilation system is chosen, it must be readily available, use low-compliance tubing, and have secure connections.
Chest compressions should not be delayed; intubation may take place during CPR or the pulse check.
Attempts at intubation should not interrupt ventilation for more than 10 Ss.
Chest compressions should begin prior to the delivery of breaths in the pulseless patient.
Whether adult resuscitation is performed by a single rescuer or by two rescuers, two breaths are administered every 30 compressions (30:2), allowing 3 to 4s for each two breaths. The cardiac compression rate should be 100/min regardless of the number of rescuers.
Health care personnel working in hospitals and ambulatory care facilities must be able to provide early defibrillation to patients with ventricular fibrillation as soon as possible. Shock should be delivered within 3 to 4 min of arrest.
@ If intravenous cannulation is difficult, an intraosseous infusion can provide emergency vascular access in children and adults.
© Lidocaine, epinephrine, atropine, naloxone, and vasopressin (but not sodium bicarbonate) can be delivered via a catheter whose tip extends
past the
endotracheal tube.
Dosages
2
to
2%
times higher
than recommended for intravenous use, diluted in 10 mL of normal saline or distilled water, are recommended for adult patients.
@ Because carbon dioxide, but not bicarbonate, readily crosses cell membranes and the blood-brain barrier, arterial hypercapnia causes intracellular tissue acidosis.
@ A wide QRS complex following a pacing spike signals electrical capture, but mechanical (ventricular) capture must be confirmed by an improving pulse or blood pressure.

® Cardiopulmonary resuscitation and emergency cardiac care (CPR-ECC) should be considered any time an individual cannot adequately oxygenate or perfuse vital organs—not only following cardiac or respiratory arrest. This chapter presents an overview of the 2015 American Heart Association (AHA) Guidelines Update for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care, which provides revised recommendations for establishing and maintaining the "CABDs" of cardiopulmonary resuscitation: Circulation, Airway, Breathing, and Defibrillation (Table 55-1, Figures 55—1 and 55—2). The 2015 CPR-ECC guidelines have been updated with new evidence-based recommendations. Points of particular importance for the layperson are that the pulse should not be checked, and chest compression without ventilation may be as effective as compression with ventilation for the first several minutes. If a second lay rescuer is unavailable to perform mouth-to-mask ventilation, chest compressions alone are preferred to the primary rescuer attempting to do both. For the health care provider, defibrillation using biphasic electrical current works best and endotracheal tube (ETT) placement should be confirmed with a quantitative capnographic waveform analysis. More importantly, in the new guidelines, emphasis has been placed on the quality and adequacy of compressions, minimizing interruption time of compressions and preshock pause (the time taken from the last compression to the delivery of shock).
TABLE 55-1 Emergency cardiac care (ECC).
AnNnRWN=
Recognition of impending event
Activation of emergency response system
Basic life support
Defibrillation
Ventilation
Pharmacotherapy
FIGURE 55-1 Universal algorithm for adult emergency cardiac care. ACS, acute coronary syndrome; BLS, basic life support; CPR, cardiopulmonary resuscitation; IV, intravenous; OD, overdose; VF/VT, ventricular fibrillation and pulseless ventricular tachycardia.
FIGURE 55-2 Comprehensive emergency cardiac care algorithm. BLS, basic life support; CPR, cardiopulmonary resuscitation; IO, intraosseous; IV, intravenous; PEA, pulseless electrical activity; VF/VT, ventricular fibrillation and pulseless ventricular tachycardia.

The sequence of steps in resuscitation has been changed since the 2010 guidelines from ABC (airway and breathing first, before compression) to CAB (compression first, with airway and breathing treated later). Based on the 2015 guidelines, titrating resuscitative efforts to physiological parameters

Before CPR is initiated, unresponsiveness is established and the emergency response system is activated. During low blood flow states such as cardiac arrest, oxygen delivery to the heart and brain is limited by blood flow rather than by arterial oxygen content; thus, current guidelines place greater emphasis on immediate initiation of chest compressions than on rescuer breaths.
The patient is positioned supine on a firm surface. After initiation of chest compressions, the airway is evaluated. The airway is most commonly obstructed by posterior displacement of the tongue or epiglottis. If there is no evidence of cervical spine instability, a head-tilt chin-lift should be tried first (Figure 55-3). One hand (palm) is placed on the patient's forehead applying pressure to tilt the head back while lifting the chin with the forefinger and index finger of the opposite hand. The jaw-thrust may be more effective in opening the airway and is executed by placing both hands on either side of the patient's head, grasping the angles of the jaw, and lifting. Basic airway management is discussed in detail in Chapter 19, and the trauma patient is considered in Chapter 39,
FIGURE 55-3 Loss of consciousness is often accompanied by loss of submandibular muscle tone (A). Occlusion of the airway by the tongue can be relieved by a head-tilt chin-lift (B) or a jaw-thrust (C). In patients with possible cervical spine injury, the angles of the jaw should be lifted anteriorly without hyperextending the neck.
Any vomitus or foreign body visible in the mouth of an unconscious patient should be removed. If the patient is conscious or if the foreign body cannot be removed by a finger sweep, the Heimlich maneuver is recommended. This subdiaphragmatic abdominal thrust elevates the diaphragm, expelling a blast of air from the lungs that displaces the foreign body (Figure 55—4). Complications of the Heimlich maneuver include rib fracture, trauma to internal viscera, and regurgitation with aspiration. A combination of back blows and chest thrusts is recommended to clear foreign body obstruction in infants (Table 55—2).

FIGURE 55-4 The Heimlich maneuver can be performed with the victim standing (A) or lying down (B). The hands are positioned slightly above the navel and well below the xiphoid process and then pressed into the abdomen with a quick upward thrust. The maneuver may need to be repeated.
TABLE 55-2 Summary of recommended basic life support techniques.
'Decrease to 8-10 breaths/min if the airway is secured with an endotracheal tube.

Breathing rate, Infant (1-12 mo) = 20 breaths/min. Breathing rate, Child (>12mo) = 20 breaths/min. Breathing rate, Adult = 10-12 breaths/min’. Pulse check, Infant (1-12 mo) = Brachial. Pulse check, Child (>12mo) = Carotid. Pulse check, Adult = Carotid. Compression rate, Infant (1-12 mo) = >100/min. Compression rate, Child (>12mo) = 100/min. Compression rate, Adult = 100/min. Compression method, Infant (1-12 mo) = Two or three fingers. Compression method, Child (>12mo) = Heel of one hand. Compression method, Adult = Hands interlaced. Compression/ventilation ratio, Infant (1-12 mo) = 30:2. Compression/ventilation ratio, Child (>12mo) = 30:2. Compression/ventilation ratio, Adult = 30:2. Foreign body obstruction, Infant (1-12 mo) = Back blows and chest thrusts. Foreign body obstruction, Child (>12mo) = Heimlich maneuver. Foreign body obstruction, Adult = Heimlich maneuver
If after opening the airway there is no evidence of adequate breathing, the rescuer should initiate assisted ventilation by inflating the victim's lungs with each breath using a bag-mask device (see Chapter 19). Breaths are delivered slowly (inspiratory time of %—1 s) at a rate of about 10 breaths/min, with smaller tidal volumes [VT] so as to minimize adverse effect on cardiac preload. Compressions (100—120/min) should not be suspended during two-person CPR to permit ventilation unless ventilation is not possible during compressions.

With positive-pressure ventilation, even with a small VT, gastric inflation with subsequent regurgitation and aspiration is possible. Therefore, as soon as feasible, the airway should be secured with an ETT, or, if that is not possible, an alternative airway should be inserted. There is inadequate evidence to support the optimal timing of the placement of an advanced airway (supraglottic device, ETT); however, chest compressions should not be more than minimally interrupted to place any advanced airway device. The benefit of an advanced airway must be considered in light of the risk of potential interruption in compressions. Advanced airways include the esophageal—tracheal Combitube (ETC), laryngeal mask airway (LMA), pharyngotracheal lumen airway, King laryngeal tube, and cuffed oropharyngeal airway. The ETC and LMA, along with oral and nasopharyngeal airways, face masks, laryngoscopes, and ETTs, are discussed in Chapter 19. Of these, the LMA is increasingly preferred for inhospital arrests. The choice of bag-mask ventilation versus placement of an advanced airway is dependent upon the skills of the providers. Studies conflict as to optimal use of advanced airway management techniques in comparison to bag-mask ventilation.
Independent of which airway adjunct is used, the guidelines state that rescuers must confirm ETT placement with a PETCO, detector—an indicator, a capnograph, or a capnometric device. The preferred choice for confirmation of ETT placement is continuous capnographic waveform analysis. All confirmation devices are considered adjuncts to clinical conformation techniques (eg, auscultation). Once an artificial airway is successfully placed, it must be carefully secured with a tie or tape (25% of airways are displaced during transportation).

Some causes of airway obstruction may not be relieved by conventional methods. Furthermore, endotracheal intubation may be technically impossible to perform (eg, severe facial trauma), or repeated attempts may be unwise (eg, cervical spine trauma). In these circumstances, cricothyrotomy or tracheotomy may be necessary. Cricothyrotomy involves placing a large intravenous catheter or a commercially available cannula into the trachea through the midline of the cricothyroid membrane (Figure 55—5). Proper location is confirmed by aspiration of air. A 12or 14-gauge catheter requires a driving pressure of 50 psi to generate sufficient gas flow for transtracheal jet ventilation. The catheter must be adequately secured to the skin, as the jet ventilation pressure can otherwise easily propel the catheter out of the trachea and, if the tip remains beneath the skin, lead to massive subcutaneous emphysema.
FIGURE 55-5 Percutaneous cricothyrotomy with a 14-gauge over-the-needle intravenous catheter. A: Locate the cricothyroid membrane. B: Puncture the membrane at the midline while stabilizing the trachea with the other hand. Proper location is confirmed by easy aspiration of air. C: Advance the catheter and withdraw the needle. Attach syringe to catheter and aspirate again after the catheter is advanced in order to confirm that the catheter remains in the tracheal lumen.
Various systems are available that connect a high-pressure source of oxygen (eg, central wall oxygen, tank oxygen, or the anesthesia machine fresh gas outlet) to the catheter (Figure 55—6). A hand-operated jet injector or the oxygen flush valve of an anesthesia machine controls ventilation. The addition of a pressure regulator minimizes the risk of barotrauma.

FIGURE 55-6 A, B: Two systems for transtracheal jet ventilation after cricothyrotomy (see Figure 55-5). A jet ventilator and pressure regulator (as shown in A) provide better control of the inspiratory cycle. Both systems use low-compliance tubing and a high-pressure source of oxygen. C: The hub from a 7.0 ETT may be adapted to connect with a 14-gauge catheter and 3 mL syringe in order to provide jet ventilation with oxygen when outside the operating room or when jet ventilation is not available.
@ Regardless of which transtracheal jet ventilation system is chosen, it must be readily available, use low-compliance tubing, and have secure connections. Direct connection of a 12or 14-gauge intravenous catheter to the anesthesia circle system does not allow adequate ventilation because of the high compliance of the corrugated breathing tubing and breathing bag. Similarly, one cannot reliably deliver acceptable ventilation through a 12or 14-gauge catheter with a self-inflating resuscitation bag.
Adequacy of ventilation—particularly expiration—is judged by observation of chest wall movement and auscultation of breath sounds. Acute complications include pneumothorax, subcutaneous emphysema, mediastinal emphysema, bleeding, esophageal puncture, aspiration, and respiratory acidosis. Long-term complications include tracheomalacia, subglottic stenosis, and vocal cord changes. Cricothyrotomy is not generally recommended in children younger than 10 years of age.
Tracheotomy can be performed in a more controlled fashion after oxygenation has been restored by cricothyrotomy (see Chapter 19).

Endotracheal intubation by competent personnel should be attempted as soon as practical. @ Attempts at endotracheal intubation should not interrupt ventilation for more than 10 s. After intubation, the patient can be ventilated with high oxygen concentrations. A rate of 8 to 10 breaths/min should be maintained, as higher ventilatory rates may impede cardiac output during CPR in a cardiac arrest situation.
The ratio of physiological dead space to tidal volume (VD/VT) reflects the efficiency of CO, elimination. VD/VT increases during CPR as a result of low pulmonary blood flow and high alveolar pressures. Thus, minute ventilation may need to be increased by 50% to 100% once circulation is restored as CO, from the periphery is brought back to the lungs.

Circulation takes precedence over airway interventions and breathing in the cardiac arrest @ patient. As previously noted, chest compressions should begin prior to the delivery of breaths. Subsequent actions to assess circulation may then vary depending on whether the responder is a lay person or health care provider. Although lay rescuers should assume that an unresponsive patient is in cardiac arrest and need not check the pulse, health care providers should assess for or absence of a
If the patient has an adequate pulse (carotid artery in an adult or child, brachial or femoral artery in an infant) or blood pressure, breathing is at 10 to 12 breaths/min for an adult or a child older than 8 years, and 20 breaths/min for an infant or a child younger than 8 years of age (Table 55—2). If the patient is pulseless or severely hypotensive, the circulatory system must be supported by a combination of external chest compressions, intravenous drug administration, and defibrillation when appropriate. Initiation of chest compressions is mandated by the inadequacy of peripheral perfusion, and drug choices and defibrillation energy levels often depend on electrocardiographic diagnosis of arrhythmias.
presence pulse. continued

Chest compressions force blood to flow either by increasing intrathoracic pressure (thoracic pump) or by directly compressing the heart (cardiac pump). During CPR of short duration, the blood flow is created more by the cardiac pump mechanism; as CPR continues, the heart becomes less compliant and the thoracic pump mechanism becomes more important. As important as the rate and force of compression are for maintaining blood flow, effective perfusion of the heart and brain is best achieved when chest compression consumes 50% of the duty cycle, with the remaining 50% devoted to the relaxation phase (allowing blood return into the chest and heart).
To perform chest compressions in the unresponsive or pulseless patient, the xiphoid process is located and the heel of the rescuer's hand is placed over the lower half of the sternum. The other hand is placed over the hand on the sternum with the fingers either interlaced or extended, but off the chest. The rescuer's shoulders should be positioned directly over the hands with the elbows locked into position and arms extended, so that the weight of the upper body is used for compressions. With a straight downward thrust, the sternum is depressed approximately 2 in. (5 cm) in adults, 1 to 1% in. (2-4 cm) in children, and then allowed to return to its normal position. For an infant, compressions % to 1 in. (1%-2% cm) in depth are made with the middle and ring fingers on the sternum one finger-breadth below the nipple line. Compression and release times should be equal.
@ Whether adult resuscitation is performed by a single rescuer or by two rescuers, two breaths are administered every 30 compressions (30:2), allowing 3 to 4s for the two breaths. The cardiac compression rate should be 100/min regardless of the number of rescuers. A slightly higher compression rate of more than 100/min is suggested for infants, with two breaths delivered every 30 compressions.

Adequacy of cardiac output can be estimated by monitoring PETCO, (>10 mm Hg), Scvo, (>30%), or arterial pulsations (with arterial diastolic relaxation pressure >20 mm Hg). Arterial pulsations during chest compressions are not a good measure of adequate chest compression; however, spontaneous arterial pulsations are an indicator of ROSC. Physiological parameters, such as PETCO>, SCVO5, and diastolic arterial pressure, may aid in assessing the adequacy of chest compressions but cannot be used exclusively to determine if CPR should be discontinued.
1. Petco2—In an intubated patient, a PETCO, greater than 10 mm Hg indicates good-quality chest compressions; a PETCO, less than 10 mm Hg has been shown
to be a predictor of poor outcomes of CPR (decreased chance of ROSC) in CPR of more than 20 min duration. A transient increase in PETCO, may be seen with administration of sodium bicarbonate; however, an abrupt and sustained rise of PETCO, is an indicator of ROSC.
2. Coronary perfusion pressure (CPP)—This is the difference between the aortic diastolic pressure and the right atrial diastolic pressure. Arterial diastolic pressure in the radial, brachial, or femoral artery is a good indicator of CPP. Arterial diastolic pressure greater than 20 mm Hg is an indicator of adequate chest compressions.
3. SCVO2—An SCV0, less than 30% in the jugular vein is associated with poor outcomes. If the Scvo,j is less than 30%, attempts to improve the quality of CPR, either by improving the quality of compressions or through administration of medications, should be considered.

Ventricular fibrillation is found most commonly in adults who experience nontraumatic cardiac arrest. The time from collapse to defibrillation is the most important determinant of survival. The chances for survival decline 7% to 10% for every minute without defibrillation (Figure 55-7). Therefore, patients who have cardiac arrest should be defibrillated @ at the earliest possible moment. Health care personnel working in hospitals and ambulatory care facilities must be able to provide early defibrillation to collapsed patients with ventricular fibrillation as soon as possible. Shock should be delivered within 3 to 4 min of arrest.
FIGURE 55-7 Success of defibrillation versus time. The chance of successful defibrillation of a patient in ventricular fibrillation decreases 7% to 10% per minute.
There is no definite relationship between the energy requirement for successful defibrillation and body size. A shock with too low an energy level will not successfully defibrillate; conversely, too high an energy level may result in myocardial injury. Defibrillators deliver energy in either monophasic or biphasic waveforms. Biphasic waveforms are recommended for cardioversion as they achieve the same degree of success but with less energy and theoretically less myocardial damage; newly manufactured defibrillators use biphasic waveforms.

In many institutions, automated external defibrillators (AEDs) are available. Such devices are increasingly being used throughout communities by police, firefighters, security personnel, sports marshals, ski patrol members, and airline flight attendants, among others. They are placed in any public location where 20,000 or more people pass by every day. AEDs are technologically advanced, microprocessor-based devices that are capable of electrocardiographic analysis with very high specificity and sensitivity in differentiating shockable from nonshockable rhythms. All AEDs manufactured today deliver some type of biphasic waveform shock. Compared with monophasic shocks, biphasic shocks deliver energy in two directions with equivalent efficacy at lower energy levels and possibly with less myocardial injury. These devices deliver impedancecompensating shocks employing either biphasic truncated exponential (BTE) or rectilinear (RBW) morphology. Biphasic shocks delivering low energy for defibrillation (120-200 joule [J]) have been found to be as or more effective than 200 to 360 J monophasic damped sine (MDS) waveform shocks. When using AEDs, one electrode pad is placed beside the upper right sternal border, just below the clavicle, and the other pad is placed just lateral to the left nipple, with the top of the pad a few inches below the axilla.
A decrease in time delay between the last compression and the delivery of a shock (the preshock pause) has received special emphasis in the new guidelines. Stacking shocks (delivering two or more shocks in immediate succession without intervening compressions) increases the time to next compression, and it has been noted that the first shock is usually associated with a 90% efficacy. Thus, stacked shocks have been replaced by a recommendation for a single shock, followed by immediate resumption of chest compressions. For cardioversion of atrial fibrillation (Table 55—3), 120-200 J can be used initially with escalation if needed. For atrial flutter or paroxysmal supraventricular tachycardia (PSVT), an initial energy level of 50 to 100 J is often adequate. All monophasic shocks should start with 200 J. Manufacturer instructions typically provide the recommended starting shock energy level specific to the device.

Unstable atrial fibrillation, Shocks (J) = 120-200. Unstable atrial flutter/tachycardia, Shocks (J) = 50-100. Monomorphic ventricular tachycardia, Shocks (J) = 100. Polymorphic ventricular tachycardia or ventricular fibrillation, Shocks (J) = 120-200
'Data from Advanced Cardiovascular Life Support (ACLS) Provider Manual 2015 Guidelines. American Heart Association; 2016.
Ventricular tachycardia, particularly monomorphic ventricular tachycardia, responds well to shocks at initial energy levels of 100 J. For polymorphic ventricular tachycardia or for ventricular fibrillation, initial energy can be set at 120 to 200 J, depending upon the type of biphasic waveform being used.
Stepwise increases in energy levels can be used if the first shock fails, although some AEDs operate with a fixed-energy protocol of 150 J with very high success in terminating ventricular fibrillation (Table 55-3).
Cardioversion should be synchronized with the QRS complex and is recommended for hemodynamically stable, wide-complex tachycardia requiring cardioversion, PSVT, atrial fibrillation, and atrial flutter. Polymorphic ventricular tachycardia should be treated as ventricular fibrillation with shocks.
unsynchronized

Thoracotomy and open-chest cardiac massage are not part of routine CPR because of the frequent incidence of complications. Nonetheless, these invasive techniques can be helpful in specific life-threatening circumstances that preclude effective closed-chest massage. Possible indications include cardiac arrest associated with penetrating or blunt chest trauma, penetrating abdominal trauma, severe chest deformity, pericardial tamponade, or pulmonary embolism. Extracorporeal membrane oxygenation is increasing employed when the cause of arrest (eg, local anesthetic systemic toxicity) is reversible.

Even though establishing reliable intravenous access is a high priority, it should not take precedence over initial chest compressions, airway management, or defibrillation. A preexisting internal jugular or subclavian catheter is ideal for venous access during resuscitation. If there is no central line access, an attempt should be made to establish peripheral intravenous access in either the antecubital or the external jugular vein. Peripheral intravenous sites are associated with a significant delay of 1 to 2 min between drug administration and delivery to the heart, as peripheral blood flow is drastically reduced during CPR. Administration of drugs given through a peripheral intravenous line should be followed by an intravenous flush (eg, a 20-mL fluid bolus in adults) or elevation of the extremity for 10 to 20 s, or both. Establishing central vein access can potentially cause interruption of CPR but should be considered if an inadequate response is seen to peripherally-administered drugs. Olf intravenous cannulation is difficult, an intraosseous infusion can provide emergency vascular access in children and adults. A rigid 18-gauge spinal needle with a stylet or a small bone marrow trephine needle can be inserted into the distal femur or proximal tibia. If the tibia is chosen, a needle is inserted 2 to 3
FIGURE 55-8 Intraosseous infusions provide emergency access to the venous circulation in pediatric patients by way of the large medullary venous channels. The needle is directed away from the epiphyseal plate to minimize the risk of injury.

Successful pharmacological and electrical treatment of cardiac arrest (Figure 55-9) depends on definitive identification of the underlying arrhythmia. Interpreting rhythm strips in the midst of a resuscitation situation is frequently complicated by artifacts and variations in monitoring techniques (eg, lead systems, equipment).

* Push hard (at least 2 inches (5 cm) and fast (100-120/min) and allow complete chest recoil.
'* Minimize interruptions in
* Rotate compressor every 2 minutes, or sooner if fatigued.
Ifno advanced airway, 30:2 compression-ventilation ratio.
* Quantitative waveform capnography
~ If PeTCO, <10 mm Hg, attempt to improve CPR quality.
* Intra-arterial pressure
~ If relaxation phase (diastolic) pressure <20 mm Hg, attempt to improve CPR quality.

Biphasic: Manufacturer recommendation (eg, initial dose of 120-200 J); if unknown, use maximum available. Second and subsequent doses should be equivalent, and higher doses may be considered.
* Monophasic: 360 J

Epinephrine IV/O dose: 11mg every 3-5 minutes
+ Amiodarone IVAO dose: First dose: 300 mg bolus. Second dose: 150 mg.

* Endotracheal intubation or supraglottic advanced airway
* Waveform capnography or capnometry to confirm and monitor ET tube placement
* Once advanced airway in place, give 1 breath every 6 seconds (10 breaths/min) with continuous chest compressions

* Pulse and blood pressure
* Abrupt sustained increase in PETCO, (typically 240 mm Hg)
* Spontaneous arterial pressure waves with intra-arterial monitoring

xia
* Hydrogen ion (acidosis)
* Hypo-/hyperkalemia
* Hypothermia
* Tension pneumothorax
* Tamponade, cardiac
* Toxins
* Thrombosis, pulmonary
* Thrombosis, coronary
FIGURE 55-9 Adult cardiac arrest algorithm—2015 update. Algorithm for treating ventricular fibrillation and pulseless ventricular tachycardia (VF/VT). Pulseless ventricular tachycardia should be treated in the same way as ventricular fibrillation. Note: This figure and Figures 55-1 and 55—2 emphasize the concept that rescuers and health care providers must assume that all unmonitored adult cardiac arrests are due to VF/VT. In each figure, the flow of the algorithm assumes that the arrhythmia is continuing. CPR, cardiopulmonary resuscitation; I[V/IO, intravenous or intraosseous; PEA, pulseless electrical activity. (Reprinted with permission from Advanced Cardiovascular Life Support Provider Manual

Many of the drugs administered during CPR have been described elsewhere in this text. Table 55—4 summarizes the cardiovascular actions, indications, and dosages of drugs commonly used during resuscitation.

or children (0.1-1 meg/kg/min)}. Administration down a tracheal tube requires higher doses (2-2.5 mg in adults, 0.1 mg/kg in chikiren). High-dose epinephrine (0.1 mg/kg) in adults is recommended only after standard therapy has failed.. Lidocaine, 1 = Decreases rate of phase 4 depolarization (decreases. automaticity); depresses conduction in reentry. pathways. Elevates VE threshold. Reduces: disparity in action potential duration between normal and ischemic tissue. Reduces action potential and effective refractory period duration. Lidocaine, 2 = Vi that has responded to defibrillation; premature ventricular contractions. Use only after ROSC: found to be less _ effective than amiodarone in VF or pulseless VT following HCA. Lidocaine, 3 = 1-15 mg/kg. Lidocaine, 4 = 1 mg/kg. Lidocaine, 5 = Doses of 0.5~1.5 mg/kg can be repeated every 5-10 min toa total dose of 3 mg/kg After infarction o successful resuscitation, a continuous infusion (eg, 1 gin 500 mL D.W, 2mg/mL) should be run at a rate of 20-50 mcg/ kg/min (2-4 mg/min in most adults). Therapeutic blood levels are usually 15-6 meg/ml_
, Nonadrenergic peripheral = . , Seek = . , Oe = mg/kg at 10 ma/min {IV use not approved. , Not = . , era inien ie sar maybe = ‘Should notbe used in patients with impaired LV function, or when coronary artery disease is suspected.

Atropine is not included as a drug for pulseless electrical activity/asystole in the new CPR-ECC guidelines; however, its use is retained for symptomatic bradycardia. Infusions of chronotropic drugs (eg, dopamine, epinephrine, isoproterenol) can be considered as an alternative to pacing if atropine is ineffective in the setting of symptomatic bradycardia. Calcium chloride, sodium bicarbonate, and bretylium are conspicuously absent from this table. Calcium (2-4 mg/kg of the chloride salt) is helpful in the treatment of documented hypocalcemia, hyperkalemia, hypermagnesemia, or a calcium channel blocker overdose. When used, 10% calcium chloride can be given at 2 to 4 mg/kg every 10 min. Sodium bicarbonate (0.5-1 mEq/kg) is not recommended in the guidelines and should be considered only in specific situations such as preexisting metabolic acidosis or hyperkalemia, or in the treatment of tricyclic antidepressant or barbiturate overdose. Sodium bicarbonate elevates plasma pH by combining with hydrogen ions to @ form carbonic acid, which readily dissociates into carbon dioxide and water. Because carbon dioxide, but not bicarbonate, readily crosses cell membranes and the blood-brain barrier, arterial hypercapnia causes intracellular tissue acidosis. Although successful defibrillation is not related to arterial pH, increased intramyocardial carbon dioxide may reduce the possibility of successful cardiac resuscitation. Furthermore, bicarbonate administration can lead to detrimental alterations in osmolality and the oxygen—hemoglobin dissociation curve. Therefore, effective alveolar hyperventilation and adequate tissue perfusion are the treatments of choice for the respiratory and metabolic acidosis that accompany resuscitation.
Intravenous fluid therapy with either colloid or balanced salt solutions is indicated in patients with intravascular volume depletion (eg, acute blood loss, diabetic ketoacidosis, thermal burns). Dextrose-containing solutions may lead to a hyperosmotic diuresis and may worsen neurological outcome. They should be avoided unless hypoglycemia is suspected. Likewise, administration of free water (eg, D;-W) may lead to cerebral edema.

Transcutaneous cardiac pacing (TCP) is a noninvasive method of rapidly treating arrhythmias caused by conduction disorders or abnormal impulse. TCP is not routinely recommended in cardiac arrest. TCP use may be considered to treat asystole, bradycardia caused by heart block, or tachycardia from a reentrant mechanism. If there is concern about the use of atropine in high-grade block, TCP is always appropriate. If the patient is unstable with marked bradycardia, TCP should be implemented immediately while awaiting treatment response to drugs. A pacemaker unit has become a built-in feature of some defibrillator models. Disposable pacing electrodes are usually positioned on the patient in an anterior—posterior manner. The placement of the negative electrode corresponds to a V, electrocardiograph position, whereas the positive electrode is placed on the left posterior chest beneath the scapula and lateral to the spine. Note that this positioning does not interfere with paddle placement during defibrillation. Failure to capture may be due to electrode misplacement, poor electrode-to-skin contact, or increased transthoracic impedance (eg, barrel-shaped chest, pericardial effusion). Current output is slowly increased until the pacing stimuli obtain electrical and mechanical capture. A wide @ QRS complex following a pacing spike signals electrical capture, but mechanical (ventricular) capture must be confirmed by an improving pulse or blood pressure. Conscious patients may require sedation to tolerate the discomfort of skeletal muscle contractions. Transcutaneous pacing can provide effective temporizing therapy until transvenous pacing or other definitive treatment can be initiated. TCP has many advantages over transvenous pacing because it can be used by almost all acute care providers and can be initiated quickly at the bedside.

The precordial thump is to be considered only in witnessed, monitored, pulseless ventricular tachycardia when a defibrillator is not immediately available. It provides only 5 to 10 joules of mechanical energy to the heart. Recent studies suggest the precordial thump rarely results in ROSC and usually results in either no change in rhythm or deterioration into ventricular fibrillation or asystole. The latter situation may represent the phenomenon known as commotio cordis, where blunt impact to the chest without structural trauma results in ventricular arrhythmias or asystole.

A resuscitation team leader integrates the assessment of the patient, including electrocardiographic diagnosis, with the electrical and pharmacological therapy (Table 55—5). This person must have a firm grasp of the guidelines for cardiac arrest presented in the CPR-ECC algorithms (Figures 55—9 to 55-13).
TABLE 55-1 Steps for synchronized cardioversion.1

charge button.. Clear the patient when the defibrillator is charged. (See “Foundational Facts: Clearing for Defibrillation” in the VF/Pulseless VT Case.), 1 = Clear the patient when the defibrillator is charged. (See “Foundational Facts: Clearing for Defibrillation” in the VF/Pulseless VT Case.). Press the shock button(s)., 1 = Press the shock button(s).. Check the monitor. If tachycardia persists, increase the energy level (joules) according to the Electrical Cardioversion Algorithm., 1 = Check the monitor. If tachycardia persists, increase the energy level (joules) according to the Electrical Cardioversion Algorithm.. (see Supplementary Materials on the ACLS Student Website; www.heart.org/eccstudent)., 1 = (see Supplementary Materials on the ACLS Student Website; www.heart.org/eccstudent).. Activate the sync mode after delivery of each synchronized shock. Most defibrillators default back to the unsynchronized mode after delivery of a synchronized shock. This default allows an immediate shock if cardioversion produces VF., 1 = Activate the sync mode after delivery of each synchronized shock. Most defibrillators default back to the unsynchronized mode after delivery of a synchronized shock. This default allows an immediate shock if cardioversion produces VF.
'Reprinted with permission from Advanced Cardiovascular Life Support Provider Manual ©2016 American Heart Association, Inc.
? SVT, supraventricular tachycardia; VF, ventricular fibrillation, VT, ventricular tachycardia.
FIGURE 55-10 Pulseless electrical activity (PEA) algorithm. ACS, acute coronary care; IV/IO, intravenous or intraosseous; OD, overdose; PETCO3, endtidal carbon dioxide; VF/VT, ventricular fibrillation and pulseless ventricular tachycardia.
FIGURE 55-11 Asystole: The silent heart algorithm. DP, diastolic pressure; IV/IO, intravenous or intraosseous; PETCO,, end-tidal carbon dioxide; Scvo,, central venous oxygen saturation; VF/VT, ventricular fibrillation and pulseless ventricular tachycardia.

FIGURE 55-12 Adult bradycardia with a pulse algorithm. AV, atrioventricular; EGG, electgrocardiogram; IV, intravenous. (Reprinted with permission from Advanced Cardiovascular Life Support Provider Manual ©2016 American Heart Association, Inc.)
FIGURE 55-13 Adult tachycardia with a pulse algorithm. CHF, congestive heart failure; ECG, electrocardiogram; IV, intravenous; VT, ventricular tachycardia; WPW, Wolff-Parkinson-White syndrome. (Reprinted with permission from Advanced Cardiovascular Life Support Provider Manual ©2016 American Heart Association, Inc.)

A 16-year-old boy is rushed to the operating room for emergency laparotomy and thoracotomy after suffering multiple abdominal and thoracic stab wounds. In the field, paramedics intubated the patient, started two large-bore intravenous lines, began fluid resuscitation, and inflated a pneumatic antishock garment. Upon arrival in the operating room, the patient's blood pressure is unobtainable, heart rate is 128 beats/min (sinus tachycardia), and respirations are being controlled by a bag-valve device.

Cardiopulmonary resuscitation must be initiated immediately: external chest compressions should be started as soon as the arterial blood pressure is found to be inadequate for vital organ perfusion. Because the patient is already intubated, the location of the endotracheal tube should be confirmed with chest auscultation and quantitative waveform capnography (if available, to assist in both confirmation of tube placement as well as to assess the adequacy of CPR) and 100% oxygen should be delivered.

Pulselessness in the presence of sinus rhythm suggests severe hypovolemia, cardiac tamponade, ventricular rupture, dissecting aortic aneurysm, tension pneumothorax, profound hypoxemia and acidosis, or pulmonary embolism. Epinephrine, 1 mg, should be administered intravenously.

The presence of multiple stab wounds strongly suggests hypovolemia. Point-of-care abdominal ultrasonography can rapidly identify a collapsed vena cava, which is pathognomonic of hypovolemia. Warmed fluids should be rapidly administered. Additional venous access can be sought as other members of the operating room team administer fluid through blood pumps or other rapid infusion devices. Five percent albumin or lactated Ringer's solution is acceptable until blood products are available. Activation of a massive transfusion protocol is indicated.
What are the signs of tension pneumothorax and pericardial tamponade?
The signs of tension pneumothorax—the presence of air under pressure in the pleural space—include increasing peak inspiratory pressures, tachycardia and hypotension (decreased venous return), hypoxia (atelectasis), distended neck veins, unequal breath sounds, tracheal deviation, and mediastinal shift away from the pneumothorax. Point-of-care ultrasonography can also be used for identification of tension pneumothorax (and for diagnosis of pericardial tamponade), but should not interrupt chest compressions.
Pericardial tamponade—cardiac compression from pericardial contents —should be suspected in any patient with narrow pulse pressure, pulsus paradoxus (>10 mm Hg drop in systolic blood pressure with inspiration), elevated central venous pressure with neck vein distention, distant heart sounds, tachycardia, hypotension, and equalization of central venous, atrial, and ventricular end-diastolic pressures. Many of these signs may be masked by concurrent hypovolemic shock.
Fluid administration and properly performed external cardiac compressions do not result in satisfactory carotid or femoral pulsations. What else should be done?

Because external chest compressions are often ineffective in trauma patients, an emergency thoracotomy should be performed as soon as possible to clamp the thoracic aorta, relieve a tension pneumothorax or pericardial tamponade, identify possible intrathoracic hemorrhage, and perform open-chest cardiac compressions. Cross-clamping of the thoracic aorta increases brain and heart perfusion and decreases subdiaphragmatic hemorrhage. Lack of response to cross-clamping is a good predictor of demise.
What is the function of the pneumatic antishock garment, and how should it be removed?
Inflation of the bladders within a pneumatic antishock garment increases arterial blood pressure by elevating peripheral vascular resistance. Functionally, the suit has the same effect as thoracic aorta cross-clamping by decreasing blood flow and hemorrhage in the lower half of the body. Complications of inflating the abdominal section of the pneumatic antishock garment include kidney dysfunction, altered lung volumes, and visceral injury during external chest compressions. The suit should be deflated only after restoration of hemodynamic parameters. Even then, deflation should be gradual, as it may be accompanied by marked hypotension and by metabolic acidosis caused by reperfusion of ischemic tissues.

Link MS, Berkow LC, Kudenchuk PJ, et al. Part 7: Adult advanced cardiovascular life support: 2015 American Heart Association guidelines update for cardiopulmonary resuscitation and emergency cardiovascular care. Circulation. 2015;132(suppl 2):S444

Neumar RW, Shuster M, Callaway CW, et al. Part 1: Executive summary. 2015 American Heart Association guidelines update for cardiopulmonary resuscitation and emergency cardiovascular care. Circulation. 2015(suppl 2);132:S315.

@ Patients emerging from anesthesia should not leave the operating room until they have a patent airway, have adequate ventilation and oxygenation, and are hemodynamically stable; qualified anesthesia personnel must attend the transfer to the postanesthesia care unit (PACU).
@ Before the recovering patient is fully responsive, pain is often manifested as postoperative restlessness or agitation. Significant systemic disturbances (eg, hypoxemia, respiratory or metabolic acidosis, hypotension), bladder distention, or a surgical complication (eg, occult intraabdominal hemorrhage) must also be considered in the differential diagnosis of postoperative restlessness or agitation.
© Postoperative nausea and vomiting (PONV; see Chapter 17) is the most common significant complication following general anesthesia, occurring in approximately 30% or more of all patients.
@ Intense shivering causes precipitous rises in oxygen consumption, CO, production, and cardiac output. These physiological effects may be poorly tolerated by patients with cardiac or pulmonary impairment.
@ Respiratory problems are the most frequently encountered serious complications in the PACU. The overwhelming majority are related to airway obstruction, hypoventilation, hypoxemia, or a combination of these problems.
@ Hypoventilation in the PACU is most commonly due to the residual depressant effects of anesthetic agents on respiratory drive, often made worse by preexisting obstructive sleep apnea.
@ Hypoventilation with obtundation, circulatory depression, and severe acidosis (arterial blood pH < 7.15) is an indication for immediate and
decisive ventilatory and hemodynamic intervention, including airway and inotropic support as needed.

@ Following naloxone administration, patients should be observed closely for recurrence of opioid-induced respiratory depression ("renarcotization"), as naloxone has a shorter duration of action than many opioids.
© Increased intrapulmonary shunting from a decreased functional residual capacity relative to closing capacity is the most common cause of hypoxemia following general anesthesia.
@ The possibility of a postoperative pneumothorax should always be considered following central line placement, supraclavicular or intercostal blocks, abdominal or chest trauma (including rib fractures), neck dissection, thyroidectomy (especially if thyroid dissection extends into the thorax), tracheostomy, nephrectomy, or other retroperitoneal or intraabdominal procedures (including laparoscopy), especially if the diaphragm may have been penetrated or disrupted.
@ Hypovolemia is the most common cause of hypotension in the PACU and can result from inadequate fluid replacement, wound draining, or hemorrhage.
@® Noxious stimulation from incisional pain, endotracheal intubation, bladder distention, or preoperative discontinuation of antihypertensive medication is usually responsible for postoperative hypertension.
Historically, emphasis on specialized nursing care during the immediate postoperative period was prompted by the realization that many preventable early postoperative deaths occurred immediately after anesthesia and surgery. The World War II experience of providing surgical care to large numbers of battle casualties contributed to the postwar trend of centralization of immediate postoperative care in recovery rooms, where skilled nurses could pay close attention to several acute postoperative patients simultaneously. Recently, the practice of caring for selected postoperative patients overnight in a postanesthesia care unit (PACU), or the equivalent, has been a response to increasingly complex surgical procedures performed on higher-acuity patients, when there is a shortage of surgical intensive care beds.

Another recent transformation in postanesthesia care is related to the shift from inpatient to outpatient surgery. Now, more than 70% of surgical procedures in the United States are performed on an outpatient basis. Two phases of recovery may be recognized for outpatient surgery. Phase 1 is the immediate care for patients during emergence and awakening from anesthesia that continues until standard PACU discharge criteria are met (see Discharge Criteria, later in the chapter). Phase 2 is a lower-level care that continues until the patient is ready to go home. "Fast-tracking" of appropriately managed outpatients may allow them to safely bypass phase 1 recovery and go directly to the phase 2 level of care.
In many institutions, the PACU also functions as a more intensely monitored location for perioperative and chronic pain patients undergoing procedures such as single-shot nerve blocks and placement of epidural and peripheral nerve catheters, and for patients undergoing other invasive procedures such as central line placement, electroconvulsive therapy, thoracentesis, paracentesis, or cardioversion. The PACU must be appropriately staffed and equipped to manage such patients and their potential complications. For example, in areas where regional and epidural blocks are administered, Intralipid should be stocked in anticipation of treating systemic local anesthetic toxicity.
This chapter reviews the essential components of a modern PACU, the general care of patients acutely recovering from anesthesia and surgery, and the respiratory and circulatory complications most commonly encountered in the PACU.

At the conclusion of any procedure requiring anesthesia, after anesthetic agents are discontinued, monitors are disconnected and the emerging patient is taken to the PACU by one or more qualified anesthesia providers, often assisted by other personnel. During transport, supplemental oxygen is given by nasal cannulae or mask and the patient is monitored with pulse oximetry. Following general anesthesia, if an endotracheal tube or laryngeal mask airway (LMA) was utilized, the endotracheal tube or LMA will usually be removed prior to transport. Patients are also routinely observed in the PACU following regional anesthesia and monitored anesthesia care (local anesthesia with sedation). Most facilities have protocols that require patients to be admitted to the PACU following any type of anesthesia, except by specific order of the attending anesthesiologist. After a brief "hand off" report to the PACU nurse, the patient will remain in the PACU until the major effects of anesthesia have worn off and any anesthesiaor surgery-related complications of significance have been adequately addressed. This period is characterized by a frequent incidence of potentially life-threatening respiratory and circulatory complications.
The delivery of anesthesia services in areas remote from the main operating room, such as gastrointestinal and pulmonary endoscopy, interventional radiology, and magnetic resonance imaging suites, is common. Patients recovering from anesthesia delivered in these areas must receive the same standard of care as surgical patients recovering from anesthesia. Some institutions have "satellite" PACUs to serve each of these remote areas individually and others send patients from their various procedural areas to one centralized PACU; however, the standard of postanesthesia care must be the same for all PACUs within a given institution.

The PACU should be located near the operating rooms and remote procedure areas. A central location in the operating room area itself is desirable, as it ensures that the patient can be urgently transported back to surgery, if needed, or that members of the operating room team can quickly respond to urgent or emergent patient care issues in the PACU. Proximity to radiographic, laboratory, and other intensive care facilities on the same floor is also advantageous. Prolonged transfers subject critically ill patients to increased jeopardy from urgent problems that may arise along the way. An open-ward design facilitates observation of multiple patients simultaneously. However, individually enclosed patient care spaces are required for patients needing isolation for infection control. Many institutions constructing new PACUs choose to fully enclose all the PACU patient beds for infection control and privacy. A ratio of 1.5 PACU beds per operating room is customary, although this ratio will vary depending on the respective operating room suite's case type and volume, average case duration, and patient acuity. Each patient space should be well lit and large enough to allow easy access to patients for intravenous infusion pumps, ventilators, and radiographic equipment. Construction guidelines typically specify a minimum of 7 ft between beds and 120 sq ft per patient. Multiple electrical outlets, including at least one with backup emergency power, and at least one outlet each for oxygen and suction, should be present at each bed space.

Inadequate monitoring in the PACU can lead to serious morbidity or mortality. Pulse oximetry (Spo,), electrocardiogram (ECG), and automated noninvasive blood pressure (NIBP) monitors are mandatory for each patient. Although ECG,
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The PACU must have its own supplies of basic and emergency equipment, separate from that of the operating room, including airway equipment and supplies, such as oxygen cannulas, a selection of masks, oral and nasal airways, laryngoscopes, endotracheal tubes, LMAs, a cricothyrotomy kit, and selfinflating bags for ventilation. Respiratory therapy equipment for aerosol bronchodilator treatments, continuous positive airway pressure (CPAP), and ventilators should be in close proximity to the recovery room. A difficult airway equipment and supplies cart with a bronchoscope and a video laryngoscope must be immediately available.

A readily available supply of catheters for venous, arterial, and central venous cannulation is mandatory in an inpatient setting. A defibrillation device with transcutaneous pacing capabilities, and an emergency cart with drugs and supplies for advanced life support (see Chapter 55) and infusion pumps, must be present and periodically inspected according to accreditation standards. Transvenous pacing catheters; pulse generators; and tracheostomy, chest tube, and vascular cut-down trays are typically present, depending on the surgical patient population. Point-of-care ultrasonography equipment is increasingly available for central line and perineural catheter placement, assessment of hemodynamic status, endotracheal tube placement, gastric and bladder volume, and detection of pleural effusion, pneumothorax, and other pulmonary pathology.

Inadequate staffing is often cited as a major contributing factor in PACU mishaps. The PACU should be staffed by nurses specifically trained in the care of patients emerging from anesthesia. They should have expertise in airway management and advanced cardiac life support, as well as in problems commonly encountered in surgical patients relating to wound care, drainage catheters, and postoperative hemorrhage.
Patients in the PACU should be under the medical direction of a physician, usually an anesthesiologist, who must be immediately available to respond to urgent or emergent patient care problems. High-volume tertiary care surgical institutions often have an anesthesiologist assigned full time to the PACU. The management of the patient in the PACU should reflect a coordinated effort involving qualified anesthesia providers, surgeons, nurses, respiratory therapists, and appropriate consultants. The anesthesia team emphasizes management of analgesia, airway, cardiac, pulmonary, and metabolic problems, whereas the surgical team generally manages any problems directly related to the surgical procedure itself. A ratio of one recovery nurse for two patients is generally satisfactory; however, staffing for nursing care should be tailored to the unique requirements of each patient and each facility. If the operating room schedule regularly includes pediatric patients or frequent short procedures, a ratio of one nurse to one patient is often needed. A charge nurse should be assigned to ensure optimal staffing resource management at all times, including the appropriate response to urgent or emergent patient care problems.

Emergence from general anesthesia should ideally be characterized by a smooth and gradual awakening in a controlled environment. However, problems such as airway obstruction, shivering, agitation, delirium, pain, nausea and vomiting, hypothermia, and autonomic lability are frequently encountered. Patients receiving spinal or epidural anesthesia may experience decreases in blood pressure during transport or recovery; the sympatholytic effects of major conduction blocks may prevent compensatory reflex vasoconstriction when patients are moved or when they sit up.
Following an inhalational-based anesthetic, the speed of emergence is directly proportional to alveolar ventilation, but inversely proportional to the agent's blood solubility (see Chapter 8). Hypoventilation delays emergence from inhalational anesthesia. As the duration of anesthesia increases, emergence also becomes increasingly dependent on total tissue uptake, which is a function of agent solubility, the average concentration used, and the duration of exposure to the anesthetic. Emergence from an intravenous anesthetic is a function of drug pharmacokinetics. Recovery from most intravenous agents is dependent primarily on redistribution rather than metabolism and elimination. As the total administered dose increases, however, cumulative effects become clinically apparent in the form of prolonged emergence; the termination of action becomes increasingly dependent on the metabolism or elimination. This is the basis for the concept of a context-sensitive half-time (see Chapter 7). Advanced age or kidney or liver disease can prolong emergence (see Chapter 9). Shortand ultrashort-acting anesthetic agents, such as propofol and remifentanil, significantly shorten emergence and time to discharge. Use of a bispectral index (BIS) monitor (see Chapter 6) may reduce total drug dosage and shorten recovery and time to discharge. The speed of emergence can also be influenced by preoperative medications. Premedication with agents that outlast the procedure (eg, lorazepam) may be expected to prolong emergence. The short duration of action of midazolam makes it a suitable premedication agent for short procedures.

The most frequent cause of delayed emergence (when the patient fails to regain consciousness within an expected period of time after general anesthesia) is residual drug effect. Delayed emergence may occur as a result of absolute or relative drug overdose. The effects of preoperative sleep deprivation or drug ingestion (alcohol, sedatives) can be additive to those of anesthetic agents in producing prolonged emergence. Intravenous naloxone (in 80 mcg increments in adults) and flumazenil (in 0.2 mg increments in adults) will readily reverse the effects of an opioid and benzodiazepine, respectively. Intravenous physostigmine (1-2 mg) may partially reverse the effect of other agents. A nerve stimulator can be used to exclude persisting neuromuscular blockade in poorly responsive patients on a mechanical ventilator who have inadequate spontaneous tidal volumes.
Less common causes of delayed emergence include hypothermia, marked metabolic disturbances, and perioperative stroke. A core temperature of less than 33°C has an anesthetic effect and greatly potentiates the actions of central nervous system depressants. Forced-air warming devices are most effective in raising body temperature. Hypoxemia and hypercarbia are readily excluded by pulse oximetry, capnography, and blood gas analysis. Hypercalcemia, hypermagnesemia, hyponatremia, and hypoglycemia and hyperglycemia are rare causes of delayed emergence that require laboratory measurements for diagnosis. Perioperative stroke is rare, except after neurological, cardiac, and cerebrovascular surgery (see Chapter 28); diagnosis is facilitated by neurological evaluation and radiological imaging.

Airway patency, vital signs, oxygenation, and level of consciousness must be assessed immediately upon PACU arrival. Subsequent blood pressure, heart rate, and respiratory rate measurements are routinely made at least every 5 min for 15 min or until stable, and every 15 min thereafter. Pulse oximetry and ECG are monitored continuously in all patients. In awake PACU patients, neuromuscular function should be assessed clinically (eg, head-lift and grip strength). At least one temperature measurement must also be obtained. Additional monitoring

Patients who are heavily sedated or hemodynamically unstable following regional anesthesia should also receive supplemental oxygen in the PACU. Sensory and motor levels should be periodically recorded following regional anesthesia to document regression of the block. Precautions in the form of padding or repeated warning may be necessary to prevent self-injury from uncoordinated arm movements following brachial plexus blocks. Blood pressure should be closely monitored following spinal and epidural anesthesia. Bladder catheterization may be necessary in patients who have had spinal or epidural anesthesia.

psychological dependence. Consultation with a pain specialist is often extremely helpful in these situations. If liposomal bupivacaine (Exparel) wound infiltration is used, appropriate written and verbal communication must be employed to preempt use of additional local anesthetics that could lead to systemic local anesthetic toxicity.
Analgesic effects of intravenous opioids usually peak within minutes of administration, although maximal respiratory depression, particularly with morphine and hydromorphone, may not occur until 20 to 30 min later. Patientcontrolled analgesia can be instituted for inpatients when they are fully awake. Intramuscular administration of opioids is discouraged because delayed and variable onset (10-20 min or longer) and delayed respiratory depression (up to 1 h).
When an epidural catheter is used, epidural bolus administration of fentanyl (50-100 mcg) or sufentanil (20-30 mcg) with 5 to 10 mL of 0.1% bupivacaine can provide excellent pain relief in adults. Epidural morphine (3-5 mg) may also be used, but delayed respiratory depression with epidural administration of this opioid mandates close monitoring for 24 h afterward (see Chapter 48).

® Before the recovering patient is fully responsive, pain is often manifested as postoperative restlessness or agitation. Significant systemic disturbances (eg, hypoxemia, respiratory or metabolic acidosis, hypotension), bladder distention, or a surgical complication (eg, occult intraabdominal hemorrhage) must also be considered in the differential diagnosis of postoperative restlessness or agitation. Marked agitation may necessitate arm and leg restraints to avoid self-injury, particularly in children. When serious physiological disturbances have been excluded in children, cuddling and kind words from a sympathetic attendant or, preferably, the parents, often calms the pediatric patient. Other contributory factors include marked preoperative anxiety and fear, as well as adverse drug effects (large doses of central anticholinergic agents, phenothiazines, or ketamine). Physostigmine, 1 to 2 mg intravenously (0.05 mg/kg in children), is most effective in treating delirium due to atropine and scopolamine. If serious systemic disturbances and pain are excluded, persistent agitation may require sedation with intermittent intravenous doses of midazolam, 0.5 to 1 mg (0.05 mg/kg in children).

likelihood of PONV. Unfortunately, droperidol carries a U.S. Food and Drug Administration "black box" warning, indicating that large (5-15 mg) doses can prolong the QT interval and have been associated with fatal cardiac arrhythmias. Nonpharmacological prophylaxis against PONV includes ensuring adequate hydration (20 mL/kg) after fasting, and stimulation of the P6 acupuncture point (wrist). The latter may include application of pressure, electrical current, or injections.

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Controversy exists regarding routine PONV prophylaxis for all patients. Because of the cost of treatment of established PONV, it may be cost-effective to provide prophylaxis to all patients in certain populations (eg, outpatients). Clearly, patients with multiple risk factors should receive prophylaxis. In addition, the use of two or three agents that act on differing receptors is more effective than single-agent prophylaxis.

TABLE 56-2 Postanesthetic Aldrete recovery score.!2
Color, Modified Criteria = Oxygenation. Color, Point Value = . Pink, Modified Criteria = Spo, >92% on room air. Pink, Point Value = 2. Pale or dusky, Modified Criteria = Spo, >90% on oxygen. Pale or dusky, Point Value = 1. Cyanotic, Modified Criteria = Spo, <90% on oxygen. Cyanotic, Point Value = 0. Respiration, Modified Criteria = . Respiration, Point Value = . Can breathe deeply and cough, Modified Criteria = Breathes deeply and coughs freely. Can breathe deeply and cough, Point Value = 2. Shallow but adequate exchange, Modified Criteria = Dyspneic, shallow, or limited breathing. Shallow but adequate exchange, Point Value = 1. Apnea or obstruction, Modified Criteria = Apnea. Apnea or obstruction, Point Value = 0. Circulation, Modified Criteria = . Circulation, Point Value = . Blood pressure within 20% of normal, Modified Criteria = Blood pressure + 20 mm Hg of normal. Blood pressure within 20% of normal, Point Value = 2. Blood pressure within 20% to 50% of normal, Modified Criteria = Blood pressure + 20-50 mm Hg of normal. Blood pressure within 20% to 50% of normal, Point Value = 1. Blood pressure deviating >50% from normal, Modified Criteria = Blood pressure more than + 50 mm Hg of normal. Blood pressure deviating >50% from normal, Point Value = 0. Consciousness, Modified Criteria = . Consciousness, Point Value = . Awake, alert, and oriented, Modified Criteria = Fully awake. Awake, alert, and oriented, Point Value = 2. Arousable but readily drifts back to sleep, Modified Criteria = Arousable on calling. Arousable but readily drifts back to sleep, Point Value = 1. No response, Modified Criteria = Not responsive. No response, Point Value = 0. Activity, Modified Criteria = . Activity, Point Value = . Moves all extremities, Modified Criteria = Same. Moves all extremities, Point Value = 2. Moves two extremities, Modified Criteria = Same. Moves two extremities, Point Value = 1. No movement, Modified Criteria = Same. No movement, Point Value = 0

'Data from Aldrete JA, Kronlik D. A postanesthetic recovery score. Anesth Analg. 1970;49:924; and Aldrete JA. The post-anesthesia recovery score revisited. J Clin Anesth. 1995 Feb;7(1):89.
ideally, the patient should be discharged when the total score is 10, but a minimum of 9 |s required.
In addition to the preceding criteria, patients receiving regional anesthesia should also be assessed for regression of both sensory and motor blockade. Complete resolution of the block prior to PACU dismissal avoids inadvertent injuries due to motor weakness or sensory deficits; however, many institutions have protocols that allow earlier discharge to appropriately monitored areas, and patients may be discharged with peripheral nerve blocks from single-shot or continuous perineural catheter infusions for the purpose of regional analgesia. Documenting regression of a block is important. Failure of a spinal or epidural block to resolve 6 h after the last dose of local anesthetic raises the possibility of spinal subdural or epidural hematoma, which should be excluded by prompt radiological imaging and neurologic evaluation. A major goal of most anesthetics should be rapid, comfortable emergence with minimal risk of PONV and postoperative pain in order to minimize the time needed in recovery and facilitate transfer to the next stage of recovery. Outpatients who meet the discharge criteria when they exit the operating room may be "fast-tracked," bypassing the PACU and proceeding directly to the phase 2 recovery area. Similarly, inpatients who meet the same criteria may be transferred directly from the operating room to their ward, if appropriate staffing and monitoring are present.

In addition to emergence and awakening, recovery from anesthesia following outpatient procedures includes two additional stages: home readiness (phase 2
recovery) and complete psychomotor recovery. A scoring system has been developed to help assess home readiness discharge (Table 56—3). Recovery of proprioception, sympathetic tone, bladder function, and motor strength are additional criteria following regional anesthesia. For example, intact proprioception of the big toe, minimal orthostatic blood pressure or heart rate changes, and normal plantar flexion of the foot are important signals of recovery following spinal anesthesia. Urination and drinking or eating before discharge are usually no longer required; exceptions include patients with a history of urinary retention and those with diabetes.
TABLE 56-3 Postanesthesia discharge scoring system (PADS).!2
Vital signs, Points = . Within 20% of preoperative baseline, Points = 2. Within 20% to 40% of preoperative baseline, Points = 1. >40% of preoperative baseline, Points = 0. Activity level, Points = . Steady gait, no dizziness, at preoperative level, Points = 2. Requires assistance, Points = 1. Unable to ambulate, Points = 0. Nausea and vomiting, Points = . Minimal, treated with oral medication, Points = 2. Moderate, treated with parenteral medication, Points = 1. Continues after repeated medication, Points = 0. Pain: minimal or none, acceptable to patient, controlled with oral medication, Points = . Yes, Points = 2. No, Points = 1. Surgical bleeding, Points = . Minimal: no dressing change required, Points = 2. Moderate: up to two dressing changes, Points = 1. Severe: three or more dressing changes, Points = 0
'Modified with permission from Marshall SI, Chung F, Discharge criteria and complications after ambulatory surgery. Anesth Analg. 1999 Mar;88(3):508-517.
*Score =9 is required for discharge.

All outpatients must be discharged home in the company of a responsible adult who will stay with them overnight. Patients and their adult accompaniment must be provided with written postoperative instructions covering both routine follow-up care and urgent/emergent assistance. The assessment of home readiness is the responsibility of a qualified anesthesia provider who is preferably already familiar with the patient, although authority to discharge a patient home can be delegated to a nurse if approved discharge criteria are

Home readiness does not imply that the patient has the ability to make important decisions, to drive, or to return to work. These activities require complete psychomotor recovery, which is often not achieved until 24 to 72h postoperatively. All outpatient centers must use some system of postoperative follow-up, preferably phone contact or smartphone/web app the day after discharge.

@ Respiratory problems are the most frequently encountered serious complications in the PACU. The overwhelming majority are related to airway obstruction, hypoventilation, hypoxemia, or a combination of these problems. Because hypoxemia is the final common pathway to serious morbidity and mortality, routine monitoring of pulse oximetry in the PACU leads to earlier recognition of these complications and fewer adverse outcomes.

Airway obstruction in unconscious patients is most commonly due to the tongue falling back against the posterior pharynx and is often seen in patients with obstructive sleep apnea (see Chapter 19). Other causes include laryngospasm, glottic edema, aspirated vomitus, a retained throat pack, secretions or blood in the airway, or external pressure on the trachea (eg, from a neck hematoma). Partial airway obstruction usually presents as sonorous respiration. Near-total or total obstruction causes cessation of airflow and an absence of breath sounds and may be accompanied by paradoxic (rocking) movement of the chest. The abdomen and chest should normally rise together during inspiration; however, with airway obstruction, the chest descends as the abdomen rises during each inspiration (paradoxic chest movement). Patients with airway obstruction should receive supplemental oxygen while corrective measures are undertaken. A combined jaw-thrust and head-tilt maneuver pulls the tongue forward and opens the airway, and insertion of an oral or nasal airway often alleviates the problem. Nasal airways may be better tolerated than oral airways by patients emerging from anesthesia, especially when lidocaine-containing lubricant is used, and may decrease the likelihood of

acidosis can result when these factors are superimposed on an impaired ventilatory reserve due to preexisting pulmonary, neuromuscular, or neurological disease.

Treatment should generally be directed at the underlying cause, but marked hypoventilation always requires assisted or controlled ventilation until causal factors @ are identified and corrected. Hypoventilation with obtundation, circulatory depression, and severe acidosis (arterial blood pH <7.15) is an indication for immediate and decisive ventilatory and hemodynamic intervention, including airway and inotropic support as needed. If intravenous naloxone is used to reverse opioid-induced respiratory depression, titration in small increments (80 mcg in adults) usually avoids complications and minimizes reversal of analgesia. Antagonism of opioid-induced depression with large doses of naloxone often results in sudden pain and marked increase in sympathetic tone. The latter can precipitate a hypertensive crisis, pulmonary edema, and myocardial ischemia or infarction. @ Following naloxone administration, patients should be observed closely for recurrence of opioidinduced respiratory depression ("renarcotization"), as naloxone has a shorter duration of action than many opioids. If residual muscle paralysis is present, sugammadex (if rocuronium or vecuronium has been administered) or an additional cholinesterase inhibitor may be administered. Inadequate reversal in spite of a full dose of sugammadex or a cholinesterase inhibitor necessitates controlled ventilation under close observation until adequate recovery of muscle strength occurs. Hypoventilation due to pain and splinting following upper abdominal or thoracic procedures should be treated with multimodal analgesia, including intravenous or intraspinal opioid administration, intravenous acetaminophen, or NSAIDs, or with regional anesthesia techniques.

Mild hypoxemia is common in patients recovering from anesthesia when supplemental oxygen is not given. Mild to moderate hypoxemia (Pao, 50-60 mm Hg) in young healthy patients may be well tolerated initially, but with increasing duration or severity, the initial sympathetic stimulation often seen is replaced with progressive acidosis and circulatory depression. Cyanosis may not be detected if the hemoglobin concentration is reduced. Hypoxemia may also be suspected from restlessness, agitation, tachycardia, or atrial or ventricular dysrhythmias. Obtundation, bradycardia, hypotension, and cardiac arrest are late signs. Pulse oximetry facilitates early detection of hypoxemia and must be routinely utilized in the PACU. Arterial blood gas measurements may be performed to confirm the diagnosis and guide therapy.

Hypoxemia in the PACU is usually caused by hypoventilation with or without obstruction, increased right-to-left intrapulmonary shunting, or both. A decrease in cardiac output or an increase in oxygen consumption (as with shivering) will accentuate hypoxemia. Diffusion hypoxia (see Chapter 8) is an uncommon cause of hypoxemia when recovering patients are given supplemental oxygen. Hypoxemia due exclusively to hypoventilation without obstruction is also unusual in patients receiving supplemental oxygen, unless marked hypercapnia or a concomitant increase in intrapulmonary shunting is present. @ Increased intrapulmonary shunting from a decreased functional residual capacity (FRC) relative to closing capacity is the most common cause of hypoxemia following general anesthesia. The greatest reductions in FRC occur following upper abdominal and thoracic surgery. The loss of lung volume is often attributed to microatelectasis, as atelectasis is often not identified on a chest radiograph. A semi-upright position helps maintain FRC. Marked right-to-left intrapulmonary shunting (QJ Q, >15%) is usually caused by atelectasis from prolonged intraoperative hypoventilation with low tidal volumes, unintentional endobronchial intubation, lobar collapse from bronchial obstruction by secretions or blood, pulmonary aspiration, pulmonary edema, large pleural effusion, or pneumothorax. Postoperative pulmonary edema may present with wheezing or less commonly with pink frothy fluid in the airway. Pulmonary edema may be due to left ventricular failure (cardiogenic), acute respiratory distress syndrome, or relief of prolonged airway obstruction (negative-pressure pulmonary edema). In contrast to wheezing associated with pulmonary edema, wheezing due to primary obstructive lung disease, which also often results in large increases in intrapulmonary shunting, is not associated with edema fluid in the airway or infiltrates on the chest ® radiograph. The possibility of a postoperative pneumothorax should always be considered following central line placement, supraclavicular or intercostal blocks, abdominal or chest trauma (including rib fractures), neck dissection, thyroidectomy (especially if the thyroid dissection extends into the thorax), tracheostomy, nephrectomy, or other retroperitoneal or intraabdominal procedures (including

laparoscopy), especially if the diaphragm may have been penetrated or disrupted. Patients with subpleural blebs or large bullae can also develop pneumothorax during positive-pressure ventilation.

The most common circulatory disturbances in the PACU are hypotension, hypertension, and arrhythmias. The possibility that the circulatory abnormality is secondary to an underlying respiratory disturbance should always be considered before any other intervention, especially in children.

Hypotension is usually due to hypovolemia, left ventricular @ dysfunction, or excessive arterial vasodilatation. Hypovolemia is the most common cause of hypotension in the PACU, and can result from inadequate fluid replacement, wound drainage, or hemorrhage. Vasoconstriction from hypothermia may mask hypovolemia until the patient's temperature begins to rise again; subsequent peripheral vasodilation during rewarming unmasks the hypovolemia and results in hypotension. Spinal and epidural anesthesia produce hypotension from a combination of arterial vasodilation and venous pooling of blood. Venodilators such as nitroglycerine (or spinal anesthesia) produce venous pooling and reduce the effective circulating blood volume, despite an otherwise normal intravascular volume (see Chapter 45). Hypotension associated with sepsis and allergic reactions is usually the result of both hypovolemia and vasodilation. Hypotension from a tension pneumothorax or cardiac tamponade is the result of impaired venous return to the right atrium. Removal of more than 500 to 1000 mL of ascites fluid during surgical procedure or paracentesis may result in subsequent hypotension as additional fluid migrates from the intravascular space into the abdomen.
Left ventricular dysfunction in previously healthy persons is unusual unless associated with severe metabolic disturbances (hypoxemia, acidosis, or sepsis). Hypotension due to ventricular dysfunction is primarily encountered in patients with underlying coronary artery or valvular heart disease or congestive heart failure and is usually precipitated by fluid overload, myocardial ischemia, acute increases in afterload, or arrhythmias.

Mild hypotension during recovery from anesthesia is common and typically does not require intensive treatment. Significant hypotension is often defined as a
20% to 30% reduction in blood pressure below the patient's baseline level and usually requires correction. Treatment depends on the ability to assess intravascular volume. An increase in blood pressure following a fluid bolus (250-500 mL crystalloid or 100-250 mL colloid) generally confirms hypovolemia. With severe hypotension, a vasopressor or inotrope (dopamine or epinephrine) may be necessary to increase arterial blood pressure until the intravascular volume deficit can be at least partially corrected. Signs of cardiac dysfunction should be sought in patients with known heart disease or cardiac risk factors. Failure of a patient with severe hypotension to promptly respond to initial treatment mandates echocardiographic examination or invasive hemodynamic monitoring (see Chapter 5). Manipulations of cardiac preload, contractility, and afterload are at times all required to restore the blood pressure to acceptable levels. The presence of a tension pneumothorax, as suggested by hypotension with unilaterally decreased breath sounds, hyperresonance, and tracheal deviation, is an indication for immediate pleural aspiration, even before ultrasound or radiographic confirmation. Similarly, hypotension due to cardiac tamponade, usually following chest trauma or thoracic surgery, often necessitates immediate pericardiocentesis or surgical exploration.

Postoperative hypertension is common in the PACU and typically occurs within the first 30 min after @ admission. Noxious stimulation from incisional pain, endotracheal intubation, bladder distention, or preoperative discontinuation of antihypertensive medication, is usually responsible. Postoperative hypertension may also reflect the neuroendocrine stress response to surgery or increased sympathetic tone secondary to hypoxemia, hypercapnia, or metabolic acidosis. Patients with a history of hypertension are more likely to develop hypertension in the PACU. Fluid overload or intracranial hypertension may also occasionally present as postoperative hypertension.

Mild hypertension generally does not require treatment, but a reversible cause should be sought. Marked hypertension can precipitate postoperative bleeding, myocardial ischemia, heart failure, or intracranial hemorrhage. Although decisions to treat postoperative hypertension should be individualized, in general, elevations in blood pressure greater than 20% to 30% of the patient's baseline, or those associated with adverse effects such as myocardial ischemia, heart failure, or bleeding, should be treated. After pain and potential bladder distention have been addressed, mild to moderate elevations can be treated with intravenous labetalol; an angiotensin-converting enzyme inhibitor, such as enalapril; or a calcium channel blocker, such as nicardipine. Hydralazine and sublingual nifedipine (when administered to patients not receiving B-blockers) may cause tachycardia and myocardial ischemia and infarction. Marked hypertension in patients with limited cardiac reserve requires direct intraarterial pressure monitoring and should be treated with an intravenous infusion of nitroprusside, nitroglycerin, nicardipine, clevidipine, or fenoldopam. The endpoint for treatment should be consistent with the patient's own normal blood pressure.

Respiratory disturbances, particularly hypoxemia, hypercarbia, and acidosis, will commonly be associated with cardiac arrhythmias. Residual effects from anesthetic agents, increased sympathetic nervous system activity, other metabolic abnormalities, and preexisting cardiac or pulmonary disease also predispose patients to arrhythmias in the PACU.
Bradycardia often represents the residual effects of cholinesterase inhibitors, opioids, or B-adrenergic blockers. Tachycardia in the PACU is most commonly caused by pain, hypovolemia, or fever, but may also be due to the effects of an anticholinergic agent, a B-agonist such as albuterol, or reflex tachycardia from hydralazine. Anesthetic-induced depression of baroreceptor function makes heart rate an unreliable monitor of intravascular volume in the PACU.
Premature atrial and ventricular beats may be the result of hypokalemia, hypomagnesemia, increased sympathetic tone, or, less commonly, myocardial ischemia. The latter can be diagnosed with a 12-lead ECG. Premature atrial or ventricular beats noted in the PACU without discernable cause will often also be found on the patient's preoperative ECG, if one is available. Supraventricular tachyarrhythmias, including paroxysmal supraventricular tachycardia, atrial flutter, and atrial fibrillation, are typically encountered in patients with a history of these arrhythmias and are more commonly encountered following thoracic surgery. The management of arrhythmias is discussed in Chapters 21 and 55.

A 19-year-old man sustained a closed fracture of the femur in a motor vehicle accident. He was placed in traction for 3 days prior to surgery. During that time, a persistent low-grade fever (37.5—38.7°C orally), mild hypertension (150—170/70-90 mm Hg), and tachycardia (100-126 beats/min) were noted. His hematocrit remained at 30%. Broadspectrum antibiotic coverage was initiated.
He is scheduled for open reduction and internal fixation of the fracture. When the patient is brought into the operating room, vital signs are as follows: blood pressure 160/95 mm Hg, pulse 150 beats/min, respirations 20 breaths/min, and oral temperature 38.1°C. He is sweating and is anxious. On close examination, he is noted to have a slightly enlarged thyroid gland.

The proposed operation is elective; therefore, significant abnormalities should be diagnosed and properly treated preoperatively, if possible, to prepare the patient optimally for surgery. If the patient had an open fracture, the risk of infection would clearly mandate immediate operation. Even with a closed femoral fracture, cancellations or delays should be avoided because nonoperative treatment potentiates the risks associated with bed rest, including atelectasis, pneumonia, deep venous thrombosis, and potentially lethal pulmonary thromboembolism. In deciding whether to proceed with the surgery, the anesthesia provider must ask the following questions:
1. What are the most likely causes of the abnormalities based on the clinical presentation?
2. What, if any, additional investigations or consultations might be helpful?
3. How would these or other commonly associated abnormalities affect anesthetic management?
4. Are the potential anesthetic interactions serious enough to delay surgery until a suspected cause is conclusively excluded? The tachycardia of 150 beats/min and the low-grade fever therefore require further evaluation prior to surgery.

These two abnormalities may reflect one process or separate entities (Tables 56—4 and 56-5). Moreover, although multiple factors can often be simultaneously identified, their relative contribution is often not readily apparent. Fever commonly follows major trauma; contributory factors can include the inflammatory reaction to the tissue trauma, superimposed infection (most commonly wound, pulmonary, or urinary), antibiotic therapy (drug reaction), or thrombophlebitis. Infection must be seriously considered in this patient because of the risk of bacteria seeding and infecting the metal fixation device placed during surgery. Although tachycardia is commonly associated with a low-grade fever, it is usually not of this magnitude in a 19-year-old patient. Moderate to severe pain, anxiety, hypovolemia, or anemia may be other contributory factors. Pulmonary embolism should also be considered in any patient with long bone fracture, particularly when hypoxemia, tachypnea, or mental status changes are present. Lastly, the possibly enlarged thyroid gland, sweating, and anxious appearance, together with both fever and tachycardia, suggest thyrotoxicosis.
TABLE 56-4 Perioperative causes of tachycardia.
Anxiety
Pain
Fever (see Table 56-5)
Respiratory
Hypoxemia
Hypercapnia
Circulatory
Anemia
Thromboembolism
Antimuscarinic agents
6-Adrenergic agonists
Vasodilators
Allergy
Drug withdrawal
Metabolic disorders
Thyrotoxicosis
Adrenal (addisonian) crisis
Carcinoid syndrome

Immunologically mediated processes
Drug reactions
Blood reactions
Tissue destruction (rejection)
Connective tissue disorders
Granulomatous disorders
Tissue damage
Trauma
Infarction
Thrombosis
Neoplastic disorders
Thyroid storm (thyroid crisis)
Acute gout

Arterial blood gas measurements, echocardiography and a chest radiograph would be helpful in the identification of pulmonary embolism. A repeat hematocrit or hemoglobin concentration measurement would exclude worsening anemia; significant tachycardia may be expected when the hematocrit is below 25% to 27% (hemoglobin <8 g/dL) in most patients. The response to an intravenous fluid challenge with 250 to 500 mL of a colloid or crystalloid solution may be helpful; a decrease in heart rate after the fluid bolus would be strongly suggestive of hypovolemia. Similarly, response of the heart rate to sedation and additional opioid analgesia would be helpful in excluding anxiety and pain, respectively, as causes. Although a tentative diagnosis of hyperthyroidism can be made based on clinical grounds, confirmation requires measurement of serum thyroid hormones. Signs of infection—such as increased inflammation or purulence in a wound, purulent sputum, an infiltrate on the chest film, pyuria, or leukocytosis with premature white cells on a blood smear ("shift to the left")—-should prompt cultures and a delay of surgery until the results are obtained and correct antibiotic coverage is confirmed.
The patient is transferred to the PACU for further evaluation. A 12-lead ECG confirms sinus tachycardia of 150 beats/min. A chest film is normal. Arterial blood gas measurements on room air are normal (pH 7.44, Paco, 41 mm Hg, Pao, 87 mm Hg, and HCO; 27 mEq/L). The hemoglobin concentration is found to be 11 g/dL. Blood for thyroid function tests is sent to the laboratory. The patient is sedated intravenously with midazolam (2 mg) and fentanyl (50 mcg) and is given 500 mL of 5% albumin. He seems to be relaxed and pain free, but the heart rate decreases only to 144 beats/min. The decision is made to proceed with surgery using continuous lumbar epidural anesthesia with 2% lidocaine. Esmolol is administered slowly until his pulse decreases to 120 beats/min, and a continuous esmolol infusion is administered at a rate of 300 mcg/kg/min.

The procedure is completed in 3 h. Although the patient did not complain of any pain during the procedure and was given only minimal additional sedation (midazolam, 2 mg), he is delirious upon admission to the PACU. The esmolol infusion is continuing at a rate of 500 mcg/kg/min. Estimated blood loss was 500 mL, and fluid replacement consisted of 2 units of packed red blood cell, 500 mL of 5% albumin solution, and 2000 mL of lactated Ringer's injection. Vital signs are as follows: blood pressure 105/40 mm Hg, pulse 124 beats/min, respirations 30 breaths/min, and rectal temperature 38.8°C. Arterial blood gas measurements are reported as follows: pH 7.37, Paco, 37 mm Hg, Pao, 91 mm Hg, and HCO; 22 mEq/L.

The patient appears to be in a hypermetabolic state manifested by excessive adrenergic activity, fever, and a worsening mental status. The absence of major metabolic acidosis and lack of exposure to a known triggering agent exclude malignant hyperthermia (see Chapter 52). Other possibilities include a transfusion reaction, sepsis, or an undiagnosed pheochromocytoma. The sequence of events makes the first two unlikely, and the decreasing prominence of hypertension (now replaced with relative hypotension) and increasing fever also make the latter unlikely. The clinical presentation now suggests thyroid storm. He has also received a very large dose of esmolol for several hours and this may be contributing to the relatively low blood pressure despite aggressive fluid administration.
Emergency consultation is obtained with an endocrinologist, who concurs with the diagnosis of thyroid storm and assists with its management. How is thyroid storm managed?
Thyroid storm (crisis) is a medical emergency that carries a 10% to 50% mortality rate. It is usually encountered in patients with poorly controlled or undiagnosed Graves disease. Precipitating factors include (1) the stress of surgery and anesthesia, (2) labor and delivery, (3) severe infection, and, rarely, (4) thyroiditis 1 to 2 weeks following administration of radioactive iodine. Manifestations usually include mental status changes (irritability, delirium, or coma), fever, tachycardia, and hypotension. Both atrial and ventricular arrhythmias are common, particularly atrial fibrillation. Congestive heart failure develops in 25% of patients. Hypertension that often precedes hypotension, heat intolerance with profuse sweating, nausea and vomiting, and diarrhea may be prominent initially. Hypokalemia is present in up to 50% of patients. Levels of thyroid hormones are high in plasma, but correlate poorly with the severity of the crisis. The sudden exacerbation of thyrotoxicosis may represent a rapid shift of the hormone from the protein-bound to the free state or increased responsiveness to thyroid hormones at the cellular level.

Treatment is directed toward reversing the crisis and its complications. Large doses of corticosteroids inhibit the synthesis, release, and peripheral conversion of thyroxine (T,) to the more active triiodothyronine (T3). Corticosteroids also prevent relative adrenal insufficiency secondary to the hypermetabolic state. Propylthiouracil is administered to inhibit synthesis of thyroid hormone, and iodide is given to inhibit release of thyroid hormones from the gland. Propranolol not only antagonizes the peripheral effects of the thyrotoxicosis, but may also inhibit the peripheral conversion of T,. Combined B,and B,-blockade is preferable to selective B,antagonism (esmolol or metoprolol) because excessive B5-receptor activity is responsible for the metabolic effects. B.-Receptor blockade also reduces muscle blood flow and may decrease heat production. Supportive measures include surface cooling (cooling blanket), acetaminophen (aspirin is not recommended because it may displace thyroid hormone from plasma carrier proteins), and generous intravenous fluid replacement. Vasopressors are often necessary to support arterial blood pressure.
Ventricular rate control is indicated in patients with atrial fibrillation. Transthoracic echocardiography, transesophageal echocardiography, and hemodynamic monitoring may facilitate management of patients with signs of congestive heart failure or persistent hypotension. B-Adrenergic blockade is contraindicated in patients with congestive heart failure.
Propranolol, dexamethasone, propylthiouracil, and sodium iodide are given; the patient is admitted to the intensive care unit, where treatment is continued. Over the next 3 days, his mental status markedly improves. The T; and total T, levels on the day of surgery were both elevated to 250 ng/dL and 18.5 ng/dL, respectively. He was discharged home 6 days later on a regimen of propranolol and propylthiouracil, with a blood pressure of 124/80 mm Hg, a pulse of 92 beats/min, and an oral temperature of 37.3°C.

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@ Pulmonary edema usually results from either an increase in the net hydrostatic pressure across the capillaries (hemodynamic or cardiogenic pulmonary edema) or an increase in the permeability of the alveolar—capillary membrane (increased permeability edema or noncardiogenic pulmonary edema).
@ Reduced tidal volumes are associated with the greatest improvement in outcome after ARDS of any intervention subjected to a randomized clinical trial to date.
© Early elective tracheal intubation is advisable when there are obvious signs of heat injury to the airway. Patients with hoarseness and stridor require immediate tracheal intubation or a surgical airway.
@ The criteria developed by the Acute Kidney Injury Network are now most often used to stage acute kidney injury (AKI). AKI is diagnosed by documenting an increase in serum creatinine of more than 50%, or an absolute increase of 0.3 mg/dL, and a reduction in urine output to less than 0.5 mL/kg/h for 6 h or longer, with all findings developing over 48 h or less.
@ Septic shock is defined as acute circulatory failure in a patient with sepsis or, more specifically, systolic blood pressure less than 90 mm Hg that is not responsive to volume resuscitation and requiring vasopressors for life support.
@ Critically ill patients frequently have abnormal host defenses from advanced age, malnutrition, drug therapy, loss of integrity of mucosal and skin barriers, and underlying diseases. Thus, age greater than 70 years, corticosteroid therapy, chemotherapy of malignancy, prolonged
use of invasive devices, respiratory failure, kidney failure, head trauma, and burns are established risk factors for nosocomial infections.
- @ Systemic pooling of blood and transudation of fluid into tissues result in relative hypovolemia in patients with sepsis.

Respiratory failure may be defined as a disorder of gas exchange severe enough to require acute therapeutic intervention. Definitions based on arterial blood gas measurements may not apply to patients with chronic pulmonary diseases. For example, dyspnea and progressive respiratory acidosis may be present in patients with chronic CO, retention. Arterial blood gases typically follow one of several patterns in patients with respiratory failure (Figure 57-1). At one extreme, the derangement primarily affects oxygen transfer from the alveoli into blood, giving rise to hypoxemia (hypoxic respiratory failure); in these patients CO, elimination is typically normal or even enhanced unless severe ventilation/perfusion mismatching is present. At the other extreme, pure ventilatory failure primarily affects CO, elimination, resulting in hypercapnia; mismatching of ventilation to perfusion is typically absent or minimal. Hypoxemia, however, can occur with pure ventilatory failure when arterial CO, tension reaches 75 to 80 mm Hg in patients breathing room air (see the alveolar gas equation in Chapter 23). Few patients with respiratory failure display a pattern as "pure" as these extreme examples.
FIGURE 57-1 Arterial gas tension (room air) patterns during acute respiratory failure.

Regardless of the disorder, the treatment of respiratory failure is primarily supportive while the reversible components of underlying disease(s) are treated. Hypoxemia is treated with oxygen therapy and positive airway pressure (if functional residual capacity is decreased), whereas hypercarbia (ventilatory failure) is treated with mechanical ventilation when there is no effective pharmacological countermeasure (eg, naloxone for milder degrees of opioid overdosage). Other general measures may include using aerosolized bronchodilators, intravenous antibiotics, and diuretics for fluid overload, therapy to improve cardiac function, and nutritional support.

Pulmonary edema results from transudation of fluid, first from pulmonary capillaries into interstitial spaces and then from the interstitial spaces into alveoli. Fluid within the interstitial space and alveoli is collectively referred to as extravascular lung water. The movement of water across the pulmonary capillaries is similar to what occurs in other capillary beds and can be expressed by the Starling equation:

in which Q is net flow across the capillary; Pc' and Pi are capillary and interstitial hydrostatic pressures, respectively; mc' and mi are capillary and interstitial oncotic pressures, respectively; K is a filtration coefficient related to effective capillary surface area per mass of tissue; and o is a reflection coefficient that expresses the permeability of the capillary endothelium to albumin. Albumin is particularly important in this context because more water will be lost to the interstitium when albumin is also lost to the interstitium. A o with a value of 1 implies that the endothelium is completely impermeable to albumin, whereas a value of 0 indicates free passage of albumin and other particles/molecules. The pulmonary endothelium normally is partially permeable to albumin, such that interstitial albumin concentration is approximately one-half that of plasma; therefore, under normal conditions mi must be about 14 mm Hg (one-half that of plasma). Pulmonary capillary hydrostatic pressure is gravity dependent and thus depends on vertical height in the lung. It normally varies from 0 to 15 mm Hg (average, 7 mm Hg). Because Pi is thought to be normally about —4 to -8 mm Hg, the forces favoring transudation of fluid (Pc', Pi, and ti) are usually almost balanced by the forces favoring reabsorption (mc'). The net amount of fluid that normally moves out of pulmonary capillaries is small (about 10-20 mL/h in adults) and is rapidly removed by pulmonary lymphatics, which return it into the central venous system.
The alveolar epithelial membrane is usually permeable to water and gases but is impermeable to albumin (and other proteins). A net movement of water from the interstitium into alveoli occurs only when the normally negative Pi becomes positive (relative to atmospheric pressure). Fortunately, because of the lung's unique ultrastructure and its capacity to increase lymph flow, the pulmonary interstitium usually accommodates large increases in capillary transudation before Pi becomes positive. When this reserve capacity is exceeded, pulmonary edema develops.

@ Pulmonary edema usually results from either an increase in the net hydrostatic pressure across the capillaries (hemodynamic or cardiogenic pulmonary edema) or an increase in the permeability of the alveolar—capillary membrane (increased permeability edema or noncardiogenic pulmonary edema). If a pulmonary artery catheter is present, the distinction can be based on the pulmonary artery occlusion pressure, which if greater than 18 mm Hg indicates that hydrostatic pressure is involved in forcing fluid across the capillaries into the interstitium and alveoli. However, the pulmonary artery catheter may provide incorrect guidance regarding etiology: In the case of "flash" pulmonary edema the pulmonary artery occlusion pressure may now be normal despite it having been elevated at the point in time when the pulmonary edema was induced. The protein content of the edema fluid can also help differentiate the two. Fluid due to hemodynamic edema has a low protein content, whereas that due to permeability edema has a high protein content.
Less common causes of edema include prolonged severe airway obstruction (negative pressure pulmonary edema), sudden reexpansion of a collapsed lung, high altitude, pulmonary lymphatic obstruction, and severe head injury, although the same mechanisms (ie, changes in hemodynamic parameters or capillary permeability) also account for these diagnoses. Pulmonary edema associated with airway obstruction may result from an increase in the transmural pressure across pulmonary capillaries associated with a markedly negative interstitial hydrostatic pressure. Neurogenic pulmonary edema appears to be related to a marked increase in sympathetic tone, which causes severe pulmonary hypertension and disruption of the alveolar—capillary membrane.

Significantly increased Pc' may increase extravascular lung water and result in pulmonary edema. As can be seen from the Starling equation, a decrease in 1c' may accentuate the effects of any increase in Pc'. Two major mechanisms increase Pc'; namely, pulmonary venous hypertension and a markedly increased pulmonary blood flow. Any elevation of pulmonary venous pressure is transmitted passively backward to the pulmonary capillaries and secondarily increases Pc'. Pulmonary venous hypertension may result from left ventricular failure, mitral stenosis, or left atrial obstruction. Increases in pulmonary blood flow that exceed the capacity of the pulmonary vasculature will also raise Pc'. Marked increases in pulmonary blood flow can be the result of large left-to-right cardiac or peripheral shunts, hypervolemia (fluid overload), or extremes of anemia or exercise.

Management of cardiogenic pulmonary edema involves decreasing the pressure in the pulmonary capillaries. Generally, this includes measures to improve left ventricular function, correct fluid overload with diuretics, or reduce pulmonary blood flow. Pharmacological treatment of acute cardiogenic pulmonary edema has included oxygen, morphine, diuretics (especially loop diuretics), vasodilators such as nitrates, and inotropes such as dobutamine or milrinone. By reducing left atrial pressure, pulmonary congestion is relieved; by reducing systemic vascular resistance, cardiac output may be improved. Positive airway pressure therapy is also a useful adjunct for improving oxygenation. When pulmonary edema is a consequence of left ventricular failure from acute coronary ischemia, intraaortic balloon counterpulsation, acute revascularization, or other assist devices may be needed.
2. Increased Permeability Pulmonary Edema (Noncardiogenic Pulmonary Edema): Acute Lung Injury & ARDS
Extravascular lung water increases in patients with pulmonary edema from enhanced permeability or disruption of the capillary—alveolar membrane. The protective effect of plasma oncotic pressure is lost as increased amounts of albumin "leak" into the pulmonary interstitium; normal—or even low—capillary hydrostatic pressures are unopposed and result in transudation of fluid into the lungs. Permeability edema is seen with acute lung injury (PaO,:Fio, ratio <300) and is often associated with sepsis, trauma, and pulmonary aspiration; when severe (PaO :Fi0, ratio <200), it is referred to as the acute respiratory distress syndrome (ARDS).

Acute lung injury and ARDS represent the pulmonary manifestation of the systemic inflammatory response syndrome (SIRS) as a consequence of severe injury of the capillary—alveolar membrane. Regardless of the original cause of SIRS, the lung appears to respond similarly to the ensuing inflammatory response. The released secondary mediators increase pulmonary capillary permeability, induce pulmonary vasoconstriction, and inhibit hypoxic pulmonary vasoconstriction. Destruction of alveolar epithelial cells is prominent. Alveolar flooding, with decreased surfactant production (due to loss of type II pneumocytes), leads to alveolar collapse. The exudative phase of ARDS will persist for a varying period, and is often followed by a fibrotic phase (fibrosing alveolitis), which may lead to permanent scarring.

The diagnosis of acute lung injury or ARDS requires a PaO,:Fio, ratio <300 (acute lung injury) or <200 (ARDS), and the presence of diffuse infiltrates on chest radiograph, while also excluding significant left ventricular dysfunction. The lung is often affected in a nonhomogeneous pattern, although dependent areas tend to be most affected.
Acute lung injury and ARDS are commonly seen in association with sepsis or trauma. Patients present with severe dyspnea and labored respirations. Hypoxemia due to intrapulmonary shunting is universal. Although dead space ventilation is increased, arterial CO, tension is typically decreased because of a marked increase in minute ventilation. Ventilatory failure may be seen in severe cases due to respiratory muscle fatigue or marked destruction of the capillary— alveolar membrane. Pulmonary hypertension and low or normal left ventricular

Forceful inspiration against a closed glottis or an obstructed airway may lead to negative pressure pulmonary edema. In adults this is often the consequence of laryngospasm during or after a general anesthetic, whereas in children it is often a consequence of airway obstruction from infection (eg, epiglottitis) or tumor. Other potential causes include endotracheal tube obstruction or occlusion, and virtually any other cause of airway obstruction. In adults the incidence seems greater in healthier (American Society of Anesthesiologists class 1 or 2) than sicker patients, and greater in males than females, possibly a consequence of more forceful inspiratory efforts by healthy male patients. The onset is extremely rapid and with appropriate treatment the recovery may also be much more rapid than for other forms of pulmonary edema.

Head injury, intracranial bleeding, and abrupt reversal of opioid overdosage with naloxone can all precipitate pulmonary edema. Although the precise pathophysiology of this condition is not well understood, it has long been assumed that an abrupt, large increase in sympathetic tone is the precipitating event. Often, neurogenic pulmonary edema presents very rapidly after the inciting event and then dissipates over 24 h or so. In other cases, the presentation may be delayed by 12 to 18 h. Major neurological injuries may produce effects on the heart, so distinguishing between neurogenic and cardiogenic pulmonary edema is not always easy, and the two conditions may overlap.

Drowning is asphyxia and death while submerged in water. Near-drowning is suffocation while submerged with (at least temporary) survival. Survival depends on the intensity and duration of the hypoxia and on the water temperature.

Both drowning and near-drowning can occur whether or not inhalation (aspiration) of water occurs. If water does not enter the airways, the patient primarily suffers from asphyxia; however, if the patient inhales water, marked intrapulmonary shunting also takes place. Ninety percent of drowned patients aspirate fluid: fresh water, seawater, brackish water, or other fluids. Although the amount of liquid aspirated is generally small, marked ventilation/perfusion mismatching can result from fluids in the lung, reflex bronchospasm, and loss of pulmonary surfactant. Aspiration of gastric contents can also complicate drowning before or after loss of consciousness or during resuscitation.
The hypotonic water aspirated following fresh water drowning is rapidly absorbed by the pulmonary circulation; water cannot usually be recovered from the airways. If a significant amount is absorbed (>800 mL in a 70-kg adult), transient hemodilution, hyponatremia, and even hemolysis may occur. In contrast, aspiration of salt water (which is hypertonic) draws out water from the pulmonary circulation into the alveoli, flooding them, and sometimes also causing hemoconcentration and hypernatremia. Hypermagnesemia and hypercalcemia have also been reported following near-drowning in salt water. Patients who suffer from cold water near-drowning lose consciousness when core body temperature decreases below 32°C. Ventricular fibrillation occurs at about 28°C to 30°C, but relative to normothermic near-drowning, hypothermia has a protective effect on the brain and may improve outcome provided that resuscitation is successful.

Nearly all patients with a true near-drowning episode will have hypoxemia, hypercarbia, and metabolic acidosis. Patients may also suffer from other injuries, such as spine fractures following diving accidents. Brain damage is generally related to duration of submersion and severity of asphyxia. Cerebral edema often complicates prolonged asphyxia. Acute lung injury and ARDS develop in many patients following successful resuscitation.

Initial treatment of near-drowning is directed at restoring ventilation, perfusion, oxygenation, and acid-base balance as quickly as possible. Immediate measures include establishing a secure and unobstructed airway, administering oxygen, and initiating cardiopulmonary resuscitation. In-line stabilization of the cervical spine is necessary when intubating patients who suffer from near-drowning following a dive. Institution of cardiopulmonary resuscitation should not be delayed by attempts to drain salt water from the lungs. Resuscitation efforts should not be suspended for futility until the patient is rewarmed following cold water near-drowning. Complete recovery is possible in such instances even after prolonged periods of asphyxia. After the initial resuscitation, further management will usually include positive-pressure ventilation and PEEP. Bronchospasm should be treated with bronchodilators, electrolyte abnormalities corrected, and acute lung injury and ARDS treated as previously discussed.

Smoke inhalation is the leading cause of death from fires. Affected persons may or may not have sustained a burn. Burn victims who suffer from smoke inhalation have a mortality rate significantly greater than other comparably burned patients without smoke inhalation. Any exposure to smoke in a fire requires a presumptive diagnosis of smoke inhalation until proved otherwise. A suggestive history might include loss of consciousness or disorientation in a patient exposed to a fire, or a burn acquired in a closed space.

The consequences of smoke inhalation are complex because they can involve three types of injuries: heat injury to the airways, exposure to toxic gases, and a chemical burn with deposition of carbonaceous particulates in the lower airways. The pulmonary response to smoke inhalation is equally complex and depends on the duration of the exposure, composition of the material that burned, and presence of any underlying lung disease. Combustion of synthetic materials may produce toxic gases such as carbon monoxide, hydrogen cyanide, hydrogen sulfide, hydrogen chloride, ammonia, chlorine, benzene, and aldehydes. When these gases react with water in the airways, they can produce hydrochloric, acetic, formic, and sulfuric acids. Carbon monoxide and cyanide poisoning are common.
After smoke inhalation direct mucosal injury may result in edema, inflammation, and sloughing. Loss of ciliary activity impairs the clearance of mucus and bacteria. Manifestations of acute lung injury and ARDS typically appear 2 to 3 days after the injury and seem related to the delayed development of SIRS rather than the acute smoke inhalation itself.

Patients initially may have minimal symptoms after smoke inhalation. Suggestive physical findings include facial or intraoral burns, singed nasal hairs, cough, carbonaceous sputum, and wheezing. The diagnosis is confirmed when

Acute myocardial infarction (AMI) is a dreaded complication of ischemic heart disease, with an overall mortality rate of 25%. More than one half of these deaths occur shortly after onset, usually due to arrhythmias (ventricular fibrillation). With recent advances in interventional cardiology, the in-hospital mortality rate has been reduced to less than 10%.
Diagnosis of AMI can be made with combinations of increased troponin concentrations in blood, ECG findings, and clinical history. Perioperative diagnosis can be vexing and may depend primarily on troponin measurements. AMIs are divided into those associated with ST-segment elevation (STEMI) and those that are not associated with ST-segment elevation (NSTEMI). These latter are usually diagnosed by elevations of biomarkers, most commonly troponin. Another classification scheme divides AMI into five types (type 1, from a spontaneous event such as plaque rupture; type 2, from increased demand or decreased oxygen supply; type 3, sudden cardiac death associated with symptoms or postmortem evidence of myocardial ischemia; type 4, AMI in association with percutaneous coronary interventions; type 5, AMI in association with coronary artery bypass surgery).

hypokinesis or akinesis is observed in the setting of severe chronic ischemia, the myocardium in these noninfarcted but poorly contractile areas may be said to be "hibernating." This diagnosis can be confirmed by observing viable tissue with positron emission tomography, or by showing that the hypocontractile myocardium responds to dobutamine.

amiodarone. Patients with paroxysmal supraventricular tachycardia, whose ejection fraction is preserved, should be treated with a calcium channel blocker, a B-blocker, or direct current (DC) cardioversion. Unstable hypotensive patients should be cardioverted. Patients with ectopic or multifocal atrial tachycardia should not receive DC cardioversion; instead they should be treated with calcium channel blockers, a 8 blocker, or amiodarone.

Acute kidney injury (AKI) is a rapid deterioration in renal function that is not immediately reversible by normalizing blood pressure, intravascular volume, or cardiac output, or relieving urinary obstruction. The hallmark of AKI is azotemia, frequently accompanied by oliguria. Azotemia may be classified as prerenal, renal, or postrenal. The diagnosis of renal azotemia is one of exclusion: Prerenal and postrenal causes must @ always be excluded. However, not all patients with acute azotemia have kidney failure. Likewise, urine output of more than 500 mL/d does not imply that renal function is normal. Basing the diagnosis of AKI on creatinine levels or an increase in blood urea nitrogen (BUN) is also problematic because creatinine clearance is not always a good measure of glomerular filtration rate. The criteria developed by the Acute Kidney Injury Network are now most often used to stage AKI (see Chapter 30). AKI is diagnosed by documenting an increase in serum creatinine of more than 50%, or an absolute increase of 0.3 mg/dL, and a reduction in urine output to less than 0.5 mL/kg/h for 6 h or longer, with all findings developing over 48 h or less.

Prerenal azotemia results from hypoperfusion of the kidneys; if untreated, it progresses to AKI. Renal hypoperfusion is typically the result of decreased arterial perfusion pressure, markedly increased venous pressure, or renal vasoconstriction (Table 57-1). Decreased perfusion pressure is usually associated with the release of norepinephrine, angiotensin I, and vasopressin. These hormones constrict cutaneous muscle and splanchnic vasculature and promote salt and water retention. The synthesis of vasodilating prostaglandins (prostacyclin and PGE,) and nitric oxide in the kidneys and the intrarenal action of angiotensin II help maintain glomerular filtration. In the setting of marked prerenal azotemia the use of cyclooxygenase inhibitors (eg, ketorolac for postoperative pain control) or ACE inhibitors can precipitate AKI. The diagnosis of prerenal azotemia is usually suspected from the clinical setting and confirmed by urinary laboratory indices (Table 57—2). Treatment of prerenal azotemia includes correcting intravascular volume deficits, improving cardiac function, restoring a normal blood pressure, and reversing increases in renal vascular resistance. The hepatorenal syndrome is discussed in Chapter 33.

Reduced cardiac output
Abdominal compartment syndrome
Increased renal vascular resistance
Neural
Humoral/pharmacological
Thromboembolic

Urethral obstruction
Bladder outlet obstruction
Bilateral ureteral obstruction
Intrinsic
Calculi
Tumor
Blood clots
Papillary necrosis
Extrinsic
Abdominal or pelvic tumor
Retroperitoneal fibrosis
Postsurgical (ligation)
TABLE 57-2 Urinary indices in azotemia.
Specific gravity, Prerenal = >1.018. Specific gravity, Renal = <0.012. Specific gravity, Postrenal = Variable. Osmolality (mmol/kg), Prerenal = >500. Osmolality (mmol/kg), Renal = <350. Osmolality (mmol/kg), Postrenal = Variable. Urine/plasma urea nitrogen ratio, Prerenal = >8. Urine/plasma urea nitrogen ratio, Renal = <3. Urine/plasma urea nitrogen ratio, Postrenal = Variable. Urine/plasma creatinine ratio, Prerenal = >40. Urine/plasma creatinine ratio, Renal = <20. Urine/plasma creatinine ratio, Postrenal = Variable. Urine/sodium (mEq/L), Prerenal = <10. Urine/sodium (mEq/L), Renal = >40. Urine/sodium (mEq/L), Postrenal = Variable. Fractional excretion of sodium (%), Prerenal = <1. Fractional excretion of sodium (%), Renal = >3. Fractional excretion of sodium (%), Postrenal = Variable. Renal failure index, Prerenal = <1. Renal failure index, Renal = >1. Renal failure index, Postrenal = Variable

Azotemia due to urinary tract obstruction is termed postrenal azotemia. Obstruction of urinary flow from both kidneys is usually necessary for azotemia and oliguria/anuria in these conditions. Complete obstruction eventually develops into AKI and kidney failure, whereas prolonged partial obstruction leads to chronic renal impairment. Rapid diagnosis and relief of acute obstruction usually restore normal renal function, often accompanied by a postobstructive diuresis. Obstruction may be diagnosed by a physical examination (the upper margin of the bladder can be percussed) or ultrasound (showing a distended bladder) or suggested by a radiograph of the abdomen (revealing bilateral renal calculi), but is definitively diagnosed by demonstrating dilation of the urinary tract proximal to the site of obstruction on imaging studies. Treatment depends on the site of obstruction. Obstruction at the bladder outlet can be relieved with catheterization of the bladder or suprapubic cystostomy, whereas ureteral obstruction requires nephrostomy or ureteral stents.

In addition to physical diagnosis and imaging, diagnosis and treatment may be facilitated of urine Table in by analysis (see 57—2); urinary composition
postrenal azotemia is variable and depends on the duration and severity of obstruction. In prerenal azotemia, tubular concentrating ability is preserved and reflected by a low urinary sodium concentration and high urine/serum creatinine ratio. Calculation of the fractional excretion of filtered sodium (FENa") may also be extremely useful in the setting of oliguria:
Fenat Urine sodium/serum sodium wf = Urine creatinine/serum creatinine
FeNa' is less than 1% in oliguric patients with prerenal azotemia but typically exceeds 3% in patients with oliguric AKI. Values of 1% to 3% may be present in patients with nonoliguric AKI. The renal failure index, which is the urinary sodium concentration divided by the urine/plasma creatinine ratio, is a sensitive index for diagnosing kidney failure. The use of diuretics increases urinary sodium excretion and invalidates indices that rely on urinary sodium concentration as a measure of tubular function. Moreover, intrinsic kidney diseases that primarily affect renal vasculature or glomeruli may not affect tubular function and therefore are associated with indices that are similar to prerenal azotemia. Measurement of creatinine clearance can estimate the residual glomerular filtration rate but may underestimate the degree of renal impairment when the serum creatinine concentration is still rising.

Causes of AKI are listed in Table 57—3. Up to 50% of cases follow major trauma or surgery; in the majority of instances, ischemia and nephrotoxins are responsible. AKI associated with ischemia has been termed acute tubular necrosis. Postischemic AKI follows certain surgical procedures more frequently than others: open abdominal aortic aneurysm resection, cardiac surgery with cardiopulmonary bypass, and operations to relieve obstructive jaundice. Aminoglycosides, amphotericin B, radiographic contrast dyes, cyclosporine, and cisplatin are the most commonly implicated exogenous nephrotoxins. Amphotericin B, contrast dyes, and cyclosporine also appear to produce direct intrarenal vasoconstriction. Hemoglobin and myoglobin are potent nephrotoxins when they are released during intravascular hemolysis and rhabdomyolysis, respectively. Nonsteroidal antiinflammatory drugs may play an important role in some patients. Inhibition of prostaglandin synthesis by the latter group of agents decreases prostaglandin-mediated renal vasodilation, allowing unopposed renal vasoconstriction. Other factors predisposing to AKI include preexisting renal impairment, advanced age, atherosclerotic vascular disease, diabetes, and dehydration.

Impaired cardiac output

Endogenous pigments
Hemoglobulin (hemolysis)
Myoglobin (rhabdomyolysis from crush injury
and burns)
Radiographic contrast agents
Drugs
Antibiotics (aminoglycosides, amphotericin)
Chemotherapeutic agents (cisplatin, methotrexate)
Tubular crystals
Uric acid
Oxalate
Heavy metal poisoning
Organic solvents
Myeloma protein

The sensitivity of the kidneys to injury may be explained by their very high metabolic rate and ability to concentrate potentially toxic substances. The pathogenesis of AKI is complex and probably has both a vascular endothelial and a renal epithelial (tubular) basis. Inadequate oxygen deliver to the kidney is the likely triggering event, leading to afferent arteriolar constriction, decreased glomerular permeability, increased vascular permeability, altered coagulation, inflammation, leukocyte activation, direct epithelial cell injury, and tubular obstruction. All can decrease glomerular filtration. A leak of filtered solutes through damaged portions of renal tubules may allow reabsorption of creatinine, urea, and other nitrogenous wastes.

AKI is often classified as nonoliguric (urinary volume >400 mL/d), oliguric (urinary volume <400 mL/d), or anuric (urinary volume <100 mL/d). Nonoliguric AKI accounts for up to 50% of cases. Urinary sodium concentrations in patients with nonoliguric AKI are typically lower than those in oliguric patients. For years it was speculated that one could convert oliguric AKI into nonoliguric AKI by administering mannitol, furosemide, "renal" doses of dopamine (1-2 mcg/kg/min), or fenoldopam. Theoretically, the resulting increase in urinary output might be therapeutic by preventing tubular obstruction. However, recent studies have found increased mortality in patients with AKI who received diuretics, and a meta-analysis showed no improvement in mortality or decrease in need for dialysis; therefore, diuretics should not be routinely administered in AKI.

jugular, subclavian, or femoral vein is usually used. The high morbidity and mortality rates associated with AKI would seem to argue for early dialysis, but dialysis does not appear to hasten recovery and may aggravate kidney injury if hypotension occurs or too much fluid is removed.
Because of concern that intermittent hemodialysis associated with hypotension may perpetuate renal injury, continuous renal replacement therapy (CRRT; continuous venovenous hemofiltration or continuous venovenous hemodialysis, which removes fluid and solutes at a slow, controlled rate) has been used in critically ill patients with uremic AKI who do not tolerate the hemodynamic effects of intermittent "standard" hemodialysis. The main problem associated with CRRT is the expense, as the membrane is prone to clot formation and, therefore, must be periodically replaced. Despite this limitation, many experts believe CRRT is the best way to manage uremic ICU patients with AKI. The indications for CRRT are being expanded from oliguria and uremia to metabolic acidosis, fluid overload, and hyperkalemia. Nevertheless, we note that recent clinical trials have failed to show benefit of continuous technique over intermittent hemodialysis in these critically ill patients.
The nutritional management of AKI with uremia continues to evolve, and there is now consensus among nephrologists, intensivists, and nutritionists that nutrition should be provided, and 1.0 to 1.5 g/kg/d of protein can be given, particularly for patients on CRRT.

The systemic inflammatory response to infection, termed sepsis syndrome (Figure 57—2), does not necessarily indicate the presence of bacteremia. Moreover, the inflammatory response is not unique to severe infections: Similar manifestations may be encountered with noninfectious illnesses. While the use of the term systemic inflammatory response syndrome (SIRS) was suggested by the Society of Critical Care Medicine (SCCM), European Society of Intensive Care Medicine (ESICM), American College of Chest Physicians (ACCP), American Thoracic Society (ATS), and Surgical Infection Society (SIS), these and other groups are now moving away from an emphasis on SIRS in the identification and treatment of patients with sepsis (see the ESICM's and SCCM's Third International Consensus Definitions for Sepsis and Septic Shock [Sepsis-3]) (Table 57-4).
FIGURE 57-2 The relationship among infection, sepsis, and the systemic inflammatory response syndrome (SIRS). (Modified with permission from American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med. 1992 Jun;20(6):864-874.)
TABLE 57-4 Diagnostic criteria for sepsis.!~3
Infection,‘ documented or suspected, and some of the following:
General variables
Fever (core temperature >38.3°C)
Hypothermia (core temperature <36°C)
Heart rate >90/min or >2 SD above the normal value for age
Tachypnea
Significant edema or positive fluid balance (>20 mL/kg over 24 h)
Hyperglycemia (plasma glucose >120 mg/dL or 7.7 mmol/L) in the absence of diabetes
Inflammatory variables
Leukocytosis (WBC count >12,000/pL)
Leukopenia (WBC count <4000/pL)
Normal WBC count with >10% immature forms
Plasma C-reactive protein >2 SD above the normal value
Plasma procalcitonin >2 SD above the normal value
Hemodynamic variables

Arterial hypotension® (SBP <90 mm Hg, MAP <70, or an SBP decrease >40 mm Hg in adults or <2 SD below normal value for age)
Sto, >70%*
Cardiac index >3.5 L/min per m*
Organ dysfunction variables
Arterial hypoxemia (Pao,/Fio, < 300)
Acute oliguria (urine output <0.5 mL/kg/h or 45 mmol/L for at least 2 h)
Creatinine increase >0.5 mg/dL
Coagulation abnormalities (INR >1.5 or aPTT >60 s)
Ileus (absent bowel sounds)
Thrombocytopenia (platelet count <100,000/1L)
Hyperbilirubinemia (plasma total bilirubin >4 mg/dL or 70 mmol/L)
Tissue perfusion variables
Hyperlactatemia (>1 mmol/L)
Decreased capillary refill or mottling
"Reproduced with permission from Levy MM, Fink MP, Marshall JC, et al. 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit Care Med, 2003 Apr;31 (4):1250-1256.
2SD, standard deviation; WBC, white blood cell; SBP, systolic blood pressure; MAP, mean arterial blood pressure; Svo,, mixed venous oxygen saturation; INR, international normalized ratio; aP TT, activated partial thromboplastin time.
'Diagnostic criteria for sepsis in the pediatric population are signs and symptoms of inflammation plus infection with hyperor hypothermia (rectal temperature >38.4°C or <35°C), tachycardia (may be absent in hypothermia patients), and at least one of the following indications of altered organ function: altered mental status, hypoxemia, increased serum lactate level, or bounding pulses.
"infection defined as a pathological process induced by a microorganism.

- 5 $¥0, >70% (normally, 75-80%) and cardiac index 3.5-5.5 are normal in children; therefore, neither should be used as a sign of sepsis in newborns or children
Sepsis may be classified based on predisposition, insult, infection, response, and organ dysfunction. Severe sepsis exists when these features are associated with @ organ dysfunction. The term multiple organ dysfunction syndrome (MODS) has been suggested to describe dysfunction of two or more organs associated with sepsis. Septic shock is defined as acute circulatory failure in a patient with sepsis or, more specifically, systolic blood pressure less than 90 mm Hg that is not responsive to volume resuscitation and requiring vasopressors for life support.

A mild systemic inflammatory response to an injury, infection, or other bodily insult may normally have salutatory effects. However, a marked or prolonged response, such as that associated with severe infections, can result in widespread organ dysfunction. Although gram-negative organisms account for most cases of infection-related SIRS, many other infectious agents are capable of inducing the same syndrome. These organisms either elaborate toxins or stimulate release of substances that trigger this response. The most commonly recognized initiators are the lipopolysaccharides, which are released by gram-negative bacteria. Lipopolysaccharide is composed of an O polysaccharide, a core, and lipid A. Lipid A, an endotoxin, is responsible for the compound's toxicity, which can affect nearly every organ.
The central mechanism in initiating SIRS appears to be the abnormal secretion of cytokines. These low-molecular-weight peptides and glycoproteins function as intercellular mediators regulating such biological processes as local and systemic immune responses, inflammation, wound healing, and hematopoiesis. The resulting inflammatory response includes release of potentially harmful phospholipids, attraction of neutrophils, and activation of the complement, kinin, and coagulation cascades.

TABLE 57-5 Pathogens commonly associated with serious infections in ICU patients.
Pneumonia, 1 = . Community-acquired (nonimmunocompromised host), 1 = Streptococcus pneumoniae Haemophilus influenzae Moraxella catarrhalis Mycoplasma pneumoniae Legionella pneumophila Chlamydia pneumoniae Methicillin-resistant Staphylococcus aureus (MRSA) Influenza virus. Health care-associated, 1 = MRSA Pseudomonas aeruginosa Klebsiella pneumoniae Acinetobacter species Stenotrophomonas species. Immunocompromised host, 1 = . Neutropenia, 1 = Any pathogen listed above Aspergillus species Candida species. Human immunodeficiency virus, 1 = Any pathogen listed above Pneumocystis jirovecii (formerly P. carinii) Mycobacterium tuberculosis Histoplasma capsulatum Other fungi Cytomegalovirus. Solid organ transplant or bone marrow transplant, 1 = Any pathogen listed above (Can vary depending on timing of infection to transplant). Cystic fibrosis, 1 = H. influenzae (early) S. aureus P. aeruginosa Burkholderia cepacia. Lung abscess, 1 = Bacteroides species Peptostreptococcus species Fusobacterium species Nocardia (immunocompromised patients) Amebic (when suggestive by exposure). Empyema, 1 = . Usually acute, 1 = S. aureus S. pneumoniae Group A streptococci H. influenzae. Usually subacute or chronic, 1 = Anaerobic bacteria Enterobacteriaceae M. tuberculosis

Meningitis, 1 = . , 1 = 5. pneumoniae Neisseria meningitidis Listeria monocytogenes H. influenzae. Neonates, 1 = Escherichia coli Group B streptococci. Postsurgical or post-trauma, 1 = S. aureus Enterobacteriaceae P. aeruginosa. Brain abscess, 1 = . , 1 = Streptococci Bacteroides species. Postsurgical or post-trauma, 1 = Enterobacteriaceae 5S. aureus. Immunocompromised or HIV infected, 1 = Nocardia Toxoplasma gondii. Encephalitis, 1 = . , 1 = West Nile virus Herpes simplex virus Arbovirus Rabies virus Bartonella henselae. Endocarditis, 1 = . , 1 = Streptococcus viridans Enterococcus species 5S. aureus Streptococcus bovis. Intravenous drug user, prosthetic valves, 1 = MRSA. Prosthetic valve, 1 = Candida species. Catheter-associated bacteremia, 1 = . , 1 = Candida species 5S. aureus Enterococcus species Enterobacteriaceae P. aeruginosa. Pyelonephritis, 1 = . , 1 = Enterobacteriaceae E.coli Enterococcus species. (This group catheter-associated, postsurgical), 1 = P. aeruginosa Acinetobacter species

Primary or spontaneous,  = Enterobacteriaceae S. pneumoniae Enterococcus species Anaerobic bacteria (rare). Secondary (bowel perforation),  = Enterobacteriaceae Bacteroides species Enterococcus species P. aeruginosa (uncommon). Tertiary (bowel surgery, hospitalized on antibiotics),  = P. aeruginosa MRSA Acinetobacter species Candida species. Skin structure infections,  = . Cellulitis,  = Group A streptococci 5. aureus Enterobacteriaceae (diabetic patients). Decubitus ulcer,  = Polymicrobial Streptococcus pyogenes Enterococcus species Enterobacteriaceae Anaerobic streptococci P. aeruginosa S. aureus. Necrotizing fasciitis,  = Streptococcus species Clostridia species Mixed aerobic/anaerobic bacteria. Muscle infection,  = . Myonecrosis (gas gangrene),  = Clostridium perfringens Other Clostridia species. Pyomyositis,  = 5. aureus Group A streptococci Anaerobic bacteria Gram-negative bacteria (rare). Septic shock,  = . Community-acquired,  = S. pneumoniae N. meningitidis H. influenzae E.coli Capnocytophaga (with splenectomy). Health care-associated,  = MRSA P. aeruginosa Acinetobacter species. Toxic shock syndrome,  = S. aureus Streptococcus species. Regional illness or special circumstances,  = Rickettsial species Ehrlichia species Babesia species 8. henselae (immunocompromised hosts) Yersinia pestis Francisella tularensis Leptospira Salmonelia enteritidis
'Reproduced with permission from Gabrielli A, Layon AJ, Yu M. Civetta, Taylor & Kirby's Critical Care. 4th ed, Philadelphia, PA: Lippincott Williams & Wilkins; 2009,

@ Critically ill patients frequently have abnormal host defenses from advanced age, malnutrition, drug therapy, loss of integrity of mucosal and skin barriers, and underlying diseases. Thus, age greater than 70 years, corticosteroid therapy, chemotherapy of malignancy, prolonged use of invasive devices, respiratory failure, kidney failure, head trauma, and burns are established risk factors for nosocomial infections. Patients with burns involving more than 40% of body surface area have significantly increased risk of mortality from infections. After burn injury, early removal of the necrotic eschar followed by skin grafting and wound closure appears to reverse immunological defects and reduce infection risk.
Nosocomial pneumonias are usually caused by gram-negative organisms. Preservation of gastric acidity inhibits overgrowth of gram-negative organisms in the stomach and their subsequent migration into the oropharynx. Tracheal intubation does not provide complete protection because patients may aspirate gastric fluid containing bacteria despite a properly functioning cuff; nebulizers and humidifiers can also be sources of infection. Selective decontamination of the gut with nonabsorbable antibiotics may reduce the incidence of infection but does not change outcome. Elevating the head of the bed 30° or more will reduce the likelihood of ventilator-associated pneumonia.
Wounds are common sources of sepsis in postoperative and trauma patients; restricting antibiotic prophylaxis to the immediate perioperative time appears to decrease the incidence of postoperative infections. Intraabdominal infections due to perforated ulcer, diverticulitis, appendicitis, and acalculous cholecystitis can develop in critically ill patients whether or not they are recovering from a surgical procedure. Intravascular catheter-related infections are most commonly caused by Staphylococcus epidermidis, Staphylococcus aureus, streptococci, Candida species, and gram-negative rods. Bacterial sinusitis may be an unrecognized source of sepsis in patients ventilated through nasotracheal tubes. The widespread administration of antibiotics has led to the development of microorganisms that are resistant to most standard antimicrobials. Organisms that were formerly rare such as Clostridium difficile ("c. diff.") and multidrugresistant organisms are now vexing problems for those who care for sick patients in tertiary hospitals.

The SCCM/ESICM/ACCP/ATS/SIS Consensus Conference defined septic shock as sepsis associated with hypotension (systolic blood pressure <90 mm Hg, mean arterial pressure <60 mm Hg, or systemic blood pressure <40 mm Hg from baseline) despite adequate fluid resuscitation.
Septic shock is usually characterized by inadequate tissue perfusion and cellular dysfunction. In contrast to other forms of shock widespread (hypovolemic, cardiogenic, neurogenic, or anaphylactic), cellular dysfunction in
septic shock is not necessarily related to hypoperfusion. Instead, metabolic blocks at the cellular and microcirculation levels may contribute to impaired cellular oxidation. A different definition chosen by the Third International Consensus Definitions for Sepsis and Septic Shock was reported in 2016. This publication deemphasized the role of systemic inflammation in making the diagnosis of sepsis while emphasizing the role of organ dysfunction per se. At the present time it is unclear who will win the debate regarding systemic inflammation! Meanwhile, the SOFA (quick Sepsis-related Organ Failure Assessment) score using only altered mental status (a Glasgow Coma Scale score of <15), tachypnea (respiratory rate of >20/min), and hypotension (systolic blood pressure of <100 mm Hg) for a maximum score of 3 and a minimal score of 0 is increasingly used to identify those patients with suspected infection who are at greater risk for a poor outcome.

An infectious process that induces severe or protracted systemic inflammation can result in septic shock. In hospitalized patients septic shock most commonly follows gram-negative infections in either the genitourinary tract or the lungs. In up to 50% of cases of severe sepsis no organisms can be cultured from blood. Hypotension is due to a decreased circulating intravascular volume resulting from a diffuse capillary leak. Patients may also have myocardial depression. Activation of platelets and coagulation can lead to the formation of fibrinplatelet aggregates, which further compromise tissue blood flow. Hypoxemia from ARDS accentuates tissue hypoxia. The release of vasoactive substances and formation of microthrombi in the pulmonary circulation increase pulmonary vascular resistance.

The circulation in patients with septic shock is often described as either hyperdynamic or hypodynamic. In reality, both represent the same process, but their expression depends on preexisting cardiac function and intravascular volume and the patient's response. @ Systemic pooling of blood and transudation of fluid into tissues result in relative hypovolemia in patients with sepsis. Hyperdynamic septic shock is characterized by normal or elevated cardiac output and profoundly reduced systemic vascular resistance. Decreased myocardial contractility is often demonstrable by echocardiography even in patients with markedly increased cardiac output. Mixed venous oxygen saturation is characteristically increased in the absence of hypoxemia and likely reflects the increased cardiac output and the cellular metabolic defect in oxygen utilization. Using an operational definition, patients with septic shock require vasopressor support to maintain a mean arterial pressure of 65 mm Hg or greater despite adequate circulating blood volume, and they have serum lactate levels greater than 2 mmol/L.
It used to be accepted wisdom that decreased cardiac output with low or normal systemic vascular resistance was usually seen later in the course of shock. This view is false. It is more likely to be seen in severely hypovolemic patients and in those with underlying cardiac disease. Myocardial depression is prominent. Mixed venous oxygen saturation is reduced in these patients. Pulmonary hypertension is often prominent and may contribute to right ventricular dysfunction.

Clinical manifestations of septic shock appear to be primarily related to host response rather than the infective agent. Septic shock classically presents following an abrupt onset of chills, fever, nausea (and often vomiting), with decreased mental status, tachypnea, hypotension, and tachycardia. The patient may appear flushed and feel warm (hyperdynamic) or pale with cool and often cyanotic extremities (hypodynamic). In old, debilitated patients and in infants, the diagnosis often is less obvious and hypothermia may be seen. Leukocytosis with a leftward shift to premature cell forms is typical, but leukopenia, an ominous sign, can be seen with overwhelming sepsis. Progressive metabolic (usually lactic) acidosis may be partially compensated by a concomitant respiratory alkalosis. Elevated lactate levels reflect both increased production resulting from poor tissue perfusion and decreased uptake by the liver and kidneys. Hypoxemia may herald the onset of ARDS. Hypovolemia and hypotension may lead to oliguria that will often progress to AKI. Elevations in serum aminotransferases and bilirubin are due to hepatic dysfunction. Insulin resistance is uniformly present and produces hyperglycemia. Thrombocytopenia is common and is often an early sign of sepsis. Laboratory evidence of disseminated intravascular coagulation (DIC) is often present but is rarely associated with bleeding. The latter responds only to control of the sepsis. Stress ulceration of gastric mucosa is common. Respiratory and kidney failure are the leading causes of death in septic patients.
Neutropenic patients (absolute neutrophil count 500/p.L) may develop skin lesions that can ulcerate and become gangrenous (ecthyma gangrenosum). These lesions are commonly associated with Pseudomonas septicemia but can be caused by other organisms. Perirectal abscesses can develop very quickly in neutropenic patients with few external signs; conscious patients may complain only of perirectal pain.

Acute gastrointestinal hemorrhage is a common reason for admission to the ICU. Older age (>60 years), comorbid illnesses, hypotension, marked blood loss (>5 units), and recurrent hemorrhage are associated with increased mortality. Management consists of stabilizing the patient with rapid identification of the site of bleeding. The clinician should differentiate between upper and lower gastrointestinal bleeding. A history of hematemesis indicates bleeding proximal to the ligament of Treitz. Melena often indicates bleeding proximal to the cecum. Hematochezia (bright red blood from the rectum) indicates either very brisk upper gastrointestinal bleeding (likely to be associated with hypotension) or more commonly lower gastrointestinal bleeding. The presence of maroon stools usually localizes the bleeding to the area between the distal small bowel and the right colon.
At least one large-bore intravenous cannulas should be placed, and blood should be sent for laboratory analysis and crossmatch. Fluid resuscitation guidelines are discussed in Chapter 51. Serial hemoglobin or hematocrit measurements may not accurately reflect acute blood loss during ongoing hemorrhage. Placement of a nasogastric tube may help identify an upper gastrointestinal source if bright red blood or "coffee grounds"—appearing material can be aspirated; inability to aspirate blood, however, does not rule out an upper gastrointestinal source.

or those receiving large-dose corticosteroid therapy.
Once bleeding has begun, there is generally no specific therapy other than embolization or coagulation. Interventional endoscopy or interventional angiography reduce blood transfusions, rebleeding, hospital stay, and the need for urgent surgery. Balloon tamponade (Sengstaken—Blakemore, Minnesota, or Linton tubes) may be used as adjunctive therapy for variceal bleeding but usually requires concurrent tracheal intubation to protect the airway against aspiration.

Common causes of lower gastrointestinal bleeding include diverticulosis, angiodysplasia, neoplasms, inflammatory bowel disease, ischemic colitis, infectious colitis, and anorectal disease (hemorrhoids, fissure, or fistula). Inspection and sigmoidoscopy can usually diagnose the more distal lesions. Colonoscopy usually allows definitive diagnosis (particularly for more proximal bleeding sites) and is often useful therapeutically. Interventional endoscopy or interventional angiography are the usual first choices for persistent bleeding. Surgical treatment is reserved for severe or recurrent hemorrhage.

In the past, patients with critical illness and delirium were typically mechanically restrained and given sedative or paralytic drugs, or both. All of these approaches have been associated with adverse consequences. Restraints are inhumane unless used only as a last resort for patient protection. Prolonged use of propofol has led to "propofol infusion syndrome," particularly in children. Prolonged use of neuromuscular blockers has been associated with critical illness myopathy. Fortunately, improved drugs for sedation (eg, dexmedetomidine) are more effective and have fewer adverse consequences.

Both surviving patients and caregivers find being in the critical care unit stressful. Increasing clinical and research attention is being paid to these concerns.

An Obtunded Young Woman
A 23-year-old woman is admitted to the hospital obtunded with slow respirations (7 breaths/min). Blood pressure is 90/60 mm Hg and the pulse is 90 beats/min. She was found at home in bed with empty bottles of diazepam, acetaminophen with hydrocodone, and fluoxetine lying next to her.

The presumptive diagnosis of a drug overdose usually must be made from the history, circumstantial evidence, and any witnesses. Signs and symptoms may not be helpful. Confirmation of a suspected drug overdose or poison ingestion usually requires delayed laboratory testing for the suspected agent in body fluids. Intentional overdoses (self-poisoning) are the most common mechanism and typically occur in young adults who are depressed. Ingestion of multiple drugs is common. Benzodiazepines, antidepressants, aspirin, acetaminophen, and alcohol are the most commonly ingested agents.
Accidental overdoses frequently occur in intravenous drug abusers and children. Commonly abused substances include opioids, stimulants (cocaine and methamphetamine), and hallucinogens (ketamine and phencyclidine [PCP]). Younger children occasionally accidentally ingest caustic household alkali (eg, drain cleaner), acids, and hydrocarbons (eg, petroleum products), in addition to unsecured medications of all types. Organophosphate poisoning (parathion and malathion) usually occurs in adults following agricultural exposure. Overdoses and poisoning less commonly occur as an attempted homicide.

Regardless of the type of drug or poison ingested, the principles of initial supportive care are the same. Airway patency with adequate ventilation and oxygenation must be obtained. Unless otherwise contraindicated, oxygen therapy (100%) should be administered. Hypoventilation and obtunded airway reflexes require tracheal intubation and mechanical ventilation. Many clinicians routinely administer naloxone (up to 2 mg), dextrose 50% (50 mL), and thiamine (100 mg) intravenously to all obtunded or comatose patients until a diagnosis is established; this may help exclude or treat opioid overdose, hypoglycemia, and Wernicke—Korsakoff syndrome, respectively. The dextrose can be omitted when a glucose measurement indicates it is not necessary. In this patient, intubation should be performed prior to naloxone because the respiratory depression is likely due to both the hydrocodone and the diazepam.
Blood, urine, and gastric fluid specimens should be obtained and sent for drug screening. Blood is also sent for routine hematological and chemistry studies (including liver function). Urine is usually obtained after bladder catheterization, and gastric fluid can be aspirated from a nasogastric tube; the latter should be placed after intubation to avoid pulmonary aspiration. emesis material be tested for in conscious
Alternatively, may drugs persons.
Hypotension should generally be treated with intravenous fluids unless the patient is obviously in pulmonary edema; an inotrope or vasopressor may be necessary in some instances. Seizure activity may be the result of hypoxia or a pharmacological action of a drug (tricyclic antidepressants) or poison. Seizure activity is unlikely in this patient because she ingested diazepam, a potent anticonvulsant.

Flumazenil should generally not be administered to patients who overdose on both a benzodiazepine and an antidepressant and those who have a history of seizures. Reversal of the benzodiazepine's anticonvulsant action can precipitate seizure activity in such instances. Moreover, as is the case with naloxone and opioids, the half-life of flumazenil is shorter than that of benzodiazepines. Thus, it is often preferable to ventilate the patient until the benzodiazepine effect dissipates, the patient regains consciousness, and the respiratory depression resolves.

Because the patient also ingested an unknown quantity of acetaminophen (paracetamol) administration of N-acetylcysteine (NAC; Mucomyst) should be considered. Acetaminophen toxicity is due to depletion of hepatic glutathione, resulting in the accumulation of toxic metabolic intermediates. Hepatic toxicity is usually associated with ingestion of more than 140 mg/kg of acetaminophen. NAC prevents hepatic damage by acting as a sulfhydryl donor and restoring hepatic glutathione levels. If the patient is suspected of having ingested a toxic dose of acetaminophen, an initial dosage of NAC (140 mg/kg orally or by nasogastric tube) should be administered even before plasma acetaminophen levels are obtained; additional doses are given according to the measured plasma level. If the patient cannot tolerate oral or gastric administration of NAC, if the patient is pregnant, or if the risk of hepatotoxicity is high, NAC should be given intravenously.

Toxicity might be reduced by decreasing drug absorption or enhancing elimination. Gastrointestinal absorption of an ingested substance can be reduced by emptying stomach contents and administering activated charcoal. Both methods can be effective up to 12 h following ingestion. If the patient is intubated, the stomach is lavaged carefully to avoid pulmonary aspiration. Emesis may be induced in conscious patients with syrup of ipecac 30 mL (15 mL in a child). Gastric lavage and induced emesis are generally contraindicated for patients who ingest caustic substances or hydrocarbons because of a high risk of aspiration and worsening mucosal injury.
Activated charcoal, 1 to 2 g/kg, is administered orally or by nasogastric tube with a diluent. The charcoal irreversibly binds most drugs and poisons in the gut, allowing them to be eliminated in stools. In fact, charcoal can create a negative diffusion gradient between the gut and the circulation, allowing the drug or poison to be effectively removed from the body. Alkalinization of the serum with sodium bicarbonate for tricyclic antidepressant overdose is beneficial because, by increasing pH, protein binding is enhanced; if seizures occur, the alkalinization prevents acidosisinduced cardiotoxicity.

The easiest method of increasing drug elimination is forced diuresis. Unfortunately, this method is of limited use for drugs that are highly protein bound or have large volumes of distribution. Mannitol or furosemide with saline may be used. Concomitant administration of alkali (sodium bicarbonate) enhances the elimination of weakly acidic drugs such as salicylates and barbiturates; alkalization of the urine traps the ionized form of these drugs in the renal tubules and enhances urinary elimination. Hemodialysis is usually reserved for patients with severe toxicity who continue to deteriorate despite aggressive supportive therapy.

Quick Sepsis-Related Organ Function Assessment. http://www.qsofa.org/ SOFA calculator. http://clincalc.com/IcuMortality/SOFA.aspx Surviving Sepsis campaign. http://www.survivingsepsis.org/

Hyperoxia and hypoxia are risk factors for but not the primary causes of retinopathy of prematurity (ROP). Neonates' risk of ROP increases with low birth weight and complexity of comorbidities (eg, sepsis).
The disadvantage of conventional PCV is that tidal volume (VT) is not guaranteed (although there are modes in which the consistent delivered pressure of PCV can be combined with a predefined volume delivery).
Pressure control ventilation (PCV) is similar to pressure support ventilation in that peak airway pressure is controlled but is different in that a mandatory rate and inspiratory time are selected. As with pressure support, gas flow ceases when the pressure level is reached; however, the ventilator does not cycle to expiration until the preset inspiration time has elapsed.
@ Both nasotracheal and orotracheal intubation appear to be relatively safe for at least 2 to 3 weeks.
@ When left in place for more than 2 to 3 weeks, both orotracheal and nasotracheal tubes predispose patients to subglottic stenosis. If longer periods of mechanical ventilation are necessary, the tracheal tube should generally be replaced by a cuffed tracheostomy tube.
The major effect of positive end-expiratory pressure (PEEP) on the lungs is to increase functional residual capacity (FRC). In patients with decreased lung volume, appropriate levels of either PEEP or continuous positive airway pressure (CPAP) will increase FRC and tidal ventilation above closing capacity. This will improve lung compliance and will correct ventilation/perfusion abnormalities.
Compared with a VT of 12 mL/kg, a VT of 6 mL/kg and plateau
pressure (P,,,) less than 30 cm H,O have been associated with reduced mortality in patients with acute respiratory distress syndrome.
@ A higher incidence of pulmonary barotrauma is observed with excessive PEEP or CPAP at levels greater than 20 cm H,O0.
@ Maneuvers that produce sustained maximum lung inflation, such as the use of an incentive spirometer, can be helpful in inducing cough as well as preventing atelectasis and preserving normal lung volume.

Respiratory care includes both the delivery of pulmonary therapy and performance of diagnostic tests. Respiratory therapists' scope of practice encompasses medical gas therapy, delivery of aerosolized medications, airway management, mechanical ventilation, positive airway pressure therapy, critical care monitoring, cardiopulmonary rehabilitation, and the application of various techniques collectively termed chest physiotherapy. The latter includes administering aerosols, clearing pulmonary secretions, reexpansion of atelectatic lung, and preserving normal lung function postoperatively or during illness. Diagnostic services may include pulmonary function testing, arterial blood gas analysis, and evaluation of sleep-disordered breathing. These procedures and services are well described in clinical practice guidelines developed by the American Association for Respiratory Care.

Therapeutic medical gases include oxygen at ambient or hyperbaric pressure, helium—oxygen mixtures (heliox), and nitric oxide. Oxygen is made available in high-pressure cylinders, via pipeline systems, from oxygen concentrators, as well as in liquid form. Heliox is occasionally used to reduce the increased work of breathing caused by partial upper airway obstruction. Nitric oxide is administered as a direct, selective pulmonary vasodilator.
The primary goal of oxygen therapy is to prevent or correct hypoxemia or tissue hypoxia. Table 58—1 identifies classic categories of hypoxia. Having a patient inhale an increased concentration of oxygen alone may not correct either hypoxemia or hypoxia. Continuous positive airway pressure (CPAP) or positive end-expiratory pressure (PEEP) may be required to recruit collapsed alveoli. Patients with profound hypercapnia may require ventilatory assistance. Increased concentrations of oxygen (possibly at hyperbaric pressures) may be indicated for conditions requiring removal of entrapped gas (eg, nitrogen) from body cavities or vessels. The short-term inhalation of increased concentrations of oxygen is free of

Hypoxic hypoxia, Pathophysiological Category = Y Pavrcen OF 4 Fio, (<0.21) Alveolar hypoventilation Pulmonary diffusion defect Pulmonary V/Q mismatch R—Lshunt. Hypoxic hypoxia, Clinical Example = Altitude, O, equipment error Drug overdose, COPD exacerbation Emphysema, pulmonary fibrosis Asthma, pulmonary emboli Atelectasis, cyanotic congenital heart disease. Circulatory hypoxia, Pathophysiological Category = Reduced cardiac output Microvascular dysfunction. Circulatory hypoxia, Clinical Example = Severe heart failure, dehydration Sepsis, SIRS. Hemic hypoxia, Pathophysiological Category = Reduced hemoglobin content Reduced hemoglobin function. Hemic hypoxia, Clinical Example = Anemias Carboxyhemoglobinemia, methemoglobinemia. Demand hypoxia Histotoxic hypoxia, Pathophysiological Category = T Oxygen consumption Inability of cells to utilize oxygen. Demand hypoxia Histotoxic hypoxia, Clinical Example = Fever, seizures Cyanide toxicity, TTNF, late sepsis
P,__,» barometric pressure; COPD, chronic obstructive pulmonary disease; Fio,, fraction of inspired oxygen: V/Q, ventilation/perfusion; R~ L, right to left; SIRS, systemic inflammatory response syndrome; TNF, tumor necrosis factor.

Supplemental oxygen is indicated for adults, children, and infants (older than 1 month) when Pao, is less than 60 mm Hg (8 kPa) or Sao, or Spo, is less than 90% while breathing room air. In neonates, therapy is recommended if Pao, is less than 50 mm Hg (6.7 kPa) or Sa0z is less than 88% (or capillary Po, is less than 40 mm Hg [5.3 kPa]). Therapy may be indicated for patients when hypoxemia or hypoxia is suspected based on a medical history and physical examination. Supplemental oxygen is given during the perioperative period because general anesthesia commonly causes a decrease in Pao, secondary to increased pulmonary ventilation/perfusion mismatching and decreased functional residual capacity (FRC). Supplemental oxygen should be provided before procedures such as tracheal suctioning or bronchoscopy, which commonly cause arterial desaturation. There is evidence that supplemental oxygen is effective in prolonging survival of patients with chronic obstructive pulmonary disease (COPD) whose resting Pao, is lower than 60 mm Hg at sea level. Supplemental oxygen therapy also appears to have a mild beneficial effect on the mean pulmonary arterial pressure and subjective indices of patients' dyspnea.

Oxygen given alone or in a gas can be mixed with air as a partial supplement to patients' tidal volume or serve as the entire source of the inspired volume. The devices or systems used for this are classified based on their maximal flow rates and a range of fractions of inspired oxygen (Fio,). Other considerations in selecting an oxygen delivery technique include patient compliance, the presence and type of artificial airway, and the need for humidification or an aerosol delivery system.

Oxygen (usually 100%) is supplied at a fixed flow that is only a portion of inspired gas. Such devices (eg, nasal "prongs") are usually intended for patients with stable breathing patterns. As ventilatory demands change, variable amounts of room air will dilute the oxygen flow. Low-flow systems are adequate for patients with:
¢ Minute ventilation less than ~8 to 10 L/min
¢ Breathing frequencies less than ~20 breaths/min
¢ Tidal volumes (VT) less than ~0.8 L
¢ Normal inspiratory flow (10-30 L/min)

Inspired gas at a preset Fio, is supplied continuously at high flow or by providing a sufficiently large reservoir of premixed gas. Ideally, the delivered Fio, is not affected by variations in ventilatory level or breathing pattern. Highflow systems are indicated for patients who require:

The nasal cannula is available as either a single-ended soft plastic tube with an over-the-ear head-elastic or dual-flow (to both nares) with under-the-chin lariat adjustment. Sizes appropriate for adults, children, and infants are available. Cannulas are connected to flowmeters with small-bore tubing. The tubing should be adjusted to avoid pressure sores on the ears, cheeks, and nose. Patients receiving long-term oxygen therapy most commonly use a nasal cannula. The appliance is usually well tolerated, allowing unencumbered speech, eating, and drinking. Cannulas can be combined with spectacle frames for convenience or to improve cosmesis. Since oxygen flows continuously, approximately 80% of the gas is wasted during expiration.
The actual Fio, delivered to adults with nasal cannulas is determined by oxygen flow, nasopharyngeal volume, and the patient's inspiratory flow (the latter depends both on VT and inspiratory time) (see Table 58—2). Oxygen from the cannula can fill the nasopharynx after exhalation, yet with inspiration, oxygen and entrained air are drawn into the trachea. The inspired percent oxygen in inspired air increases by approximately 2% (above 21%) per liter of oxygen flow with quiet breathing in adults; thus, cannulas can be expected to provide inspired oxygen concentrations up to 30% with normal breathing and oxygen flows of 4 L/min. Levels of 40% or greater can be attained with oxygen flows of 10 L/min or greater; however, flows greater than 5 L/min are poorly tolerated because of the discomfort of gas jetting into the nasal cavity and because of drying and crusting of the nasal mucosa.
TABLE 58-2 Oxygen delivery devices and systems.

33). , Fio, Range = 0.50
Data from "normal-breathing subjects" may not be accurate for acutely ill tachypneic patients. Increasing VT and reducing inspiratory time will dilute the small flow of oxygen. Different proportions of mouth-only versus nose-only breathing and varied inspiratory flow can alter Fio, by up to 40%. In clinical practice, flow should be titrated according to vital signs and pulse oximetry (or arterial blood gas measurements). Some patients with COPD tend to hypoventilate with even modest oxygen flows, yet are hypoxemic on room air. They may do well with cannula flows of less than less than 2 L/min. Special cannulas allow infants to nurse and produce less trauma of the face and nose than oxygen masks. Because of the reduced minute ventilation of infants, flow through the cannula must be proportionately reduced. This generally requires a pressure-compensated flowmeter accurate at delivering oxygen flows in the less than 3 L/min range. Hypopharyngeal oxygen sampling from infants breathing with cannulas has demonstrated mean Fio, of 0.35, 0.45,
0.6, and 0.68 with flows of 0.25, 0.5, 0.75, and 1.0 L/min, respectively.

The nasal mask is a hybrid of the nasal cannula and a face mask. The lower edge of the mask's flanges rests on the upper lip, surrounding the external nose. Nasal masks provide supplemental oxygen equivalent to the nasal cannula under lowflow conditions in adult patients. Many patients find the nasal mask more comfortable than a nasal cannula. The nasal mask does not produce sores around the external nares and dry oxygen is not "jetted" into the nasal cavity. The nasal mask should be considered if it improves patient comfort and compliance.

Incorporating a gas reservoir expands the applicability of the simple mask. Two types of reservoir mask are commonly used: the partial rebreathing mask and the nonrebreathing mask. Both are disposable, lightweight, transparent plastic underthe-chin reservoirs. The difference between the two relates to use of valves on the mask and between the mask and the bag reservoir. Mask reservoirs commonly hold less than 600 mL of gas volume. The phrase "partial rebreather" is used because "part" of the patient's expired VT refills the bag. Because expired gas is largely from "dead space ventilation," significant rebreathing of carbon dioxide usually does not occur.
The nonrebreather uses the same basic system as the partial rebreather but incorporates flap-type valves between the bag and mask and on at least one of the mask's exhalation ports. Inboard leaking is common, and room air will enter during brisk inspiratory flows, even when the bag contains gas. The lack of a complete facial seal and a relatively small reservoir limit the delivered oxygen concentration. The key factor in successful application of the mask is to use a sufficiently high flow of oxygen, so that the reservoir bag is at least partially full during inspiration. Typical minimum flows of oxygen are 10 to 15 L/min. Wellfitting partial rebreathing masks provide a range of Fio, from 0.35 to 0.60 with oxygen flows up to 10 L/min. With inlet flows of 15 L/min or more and ideal breathing conditions, Fio, may approach 1.0. Either style of mask is indicated for patients with significant hypoxemia, but relatively normal spontaneous minute ventilation.

Profoundly dyspneic patients with gasping respiration may be served by a fixedperformance, high-flow oxygen system.

Self-inflating bags consist of a roughly 1.5 L bladder, usually with an oxygen inlet reservoir. Anesthesia bags are 1-, 2-, or 3-L non-self-inflating reservoirs with a tailpiece gas inlet. Masks are designed to provide a comfortable leak-free seal for manual ventilation. The inspiratory/expiratory valve systems may vary. The flow to the reservoir should be kept high so that the bags do not deflate substantially. When using an anesthesia bag, operators may frequently have to adjust the oxygen flow and exhaust valve to respond to changing breathing patterns or demands, particularly when maintaining a complete seal between the mask and face is difficult.
The most common systems for disposable and permanent self-inflating resuscitation bags use a unidirectional gas flow. Although these devices offer the potential for a constant Fio, greater than 0.9, tailpiece inlet valves will not open for a spontaneously breathing patient. Opening the valves requires negativepressure bag recoil after compression. If this situation is not recognized, clinicians might be misled into thinking the patient is receiving a greater concentration of oxygen than is the case.
There are limits to the ability of each system to maintain its fixedperformance characteristics. Delivered Fio, can approach 1.0 with either anesthesia or self-inflating bags. Spontaneously breathing patients are allowed to breathe only the contents of the system if the mask seal is tight and the reservoir is adequately maintained.
Failure to maintain an adequate oxygen supply in the reservoir and inlet flow is a concern. The spring-loaded valve of anesthesia bags must be adjusted to prevent overdistention of the bag. Self-inflating bags look the same whether or not oxygen flow to the unit is adequate, and they will entrain room air into the bag, thus lowering the delivered Fio,.

The gas delivery approach with air-entraining masks is different than with an oxygen reservoir. The goal is to create an open system with high flow about the nose and mouth, with a fixed Fio. Oxygen is directed by small-bore tubing to a mixing jet; the final oxygen concentration depends on the ratio of air drawn in through entrainment ports. Manufacturers have developed both fixed and adjustable entrainment selections over an Fio, range. Most provide instructions for the operator to set a minimum flow of oxygen. Table 58-3 identifies total flow at various inlet flows and Fio,.
TABLE 58-3 Air-entrainment mask input flow versus total flow at varying Fio,.!
0.24, Inlet Oxygen Flow (Minimum) = 4. 0.24, Total Flow — (L/min) = 97. 0.28, Inlet Oxygen Flow (Minimum) = 6. 0.28, Total Flow — (L/min) = 68. 0.3, Inlet Oxygen Flow (Minimum) = 6. 0.3, Total Flow — (L/min) = 54. 0.35, Inlet Oxygen Flow (Minimum) = 8. 0.35, Total Flow — (L/min) = 45. 0.4, Inlet Oxygen Flow (Minimum) = 12. 0.4, Total Flow — (L/min) = 50. 0.5, Inlet Oxygen Flow (Minimum) = 12. 0.5, Total Flow — (L/min) = 33. 0.7, Inlet Oxygen Flow (Minimum) = 12. 0.7, Total Flow — (L/min) = 19. 0.8, Inlet Oxygen Flow (Minimum) = 12. 0.8, Total Flow — (L/min) = 16. 1.0, Inlet Oxygen Flow (Minimum) = 12. 1.0, Total Flow — (L/min) = 12
'Fio,, fraction of inspired oxygen.

Despite the high-flow concept, Fio, can vary up to 6% from the anticipated setting. The air-entraining masks are a logical choice for patients who require greater Fio, than can be provided by devices such as the nasal cannula. Patients with COPD who tend to hypoventilate with a moderate Fio, are candidates for the Venturi mask. Clinicians providing oxygen therapy with Venturi masks should be aware of the previously mentioned problems involving the mask itself. Fio, can increase if the air entrainment ports are obstructed by the patient's hands, bed sheets, or water condensate. Clinicians should encourage the patient and caregivers to keep the mask on the face continuously: Interruption of oxygen delivery may be life threatening in unstable patients with hypoxemia. Accurate analysis of the delivered Fio, during air-entrainment mask breathing is difficult. Pulse oximetry (or arterial blood gas analysis) and the patient's respiratory rate should guide clinicians as to whether the patient's demands are being met by the mask's flow.

Large-volume, high-output or "all-purpose" nebulizers have been used in

Dual air—oxygen flowmeters and air—oxygen blenders are commonly used for oxygen administration as well as freestanding CPAP and "add-on" ventilator systems. These systems differ from the air-entraining nebulizers, as their total output flows do not diminish at Fio, greater than 0.4. With these high-flow systems, the total flow to the patient and Fio, can be set independently to meet patient needs. This can be done using a large reservoir bag or constant flows in the range of 50 to more than 100 L/min. Clinicians can use a variety of appliances with these systems, including aerosol masks, face tents, or well-fitted nonrebreathing system masks with air-oxygen blenders. Face-sealing mask systems can also be constructed with a reservoir bag and a safety valve to allow breathing if the blender fails. The high flows of gas require use of heated humidifiers of the type commonly used on mechanical ventilators. Humidification offers an advantage for patients with reactive airways. Because of the high flows, such systems are used to apply CPAP or bilevel positive airway pressure (BiPAP) for spontaneously breathing patients.

Many infants and neonates will not tolerate facial appliances. Oxygen hoods cover only the head, allowing access to the child's lower body while still permitting use of a standard incubator or radiant warmer. The hood is ideal for relatively short-term oxygen therapy for newborns and inactive infants. However, for mobile infants requiring longer term therapy, the nasal cannula, face mask, or full-bed enclosure allow for greater mobility.
Normally, oxygen and air are premixed by an air—oxygen blender and passed through a heated humidifier. Nebulizers should be avoided. Most pneumatic jettype nebulizers create noise levels (>65 dB) that may cause newborn hearing loss. Hoods come in different sizes. Some are simple Plexiglas boxes; others have elaborate systems for sealing the neck opening. There is no attempt to completely seal the system, as a constant flow of gas is needed to remove carbon dioxide (minimum flow >7 L/min). Hood inlet flows of 10 to 15 L/min are adequate for a majority of patients.

Helium—oxygen (heliox) mixtures have notable medical applications. Helium is premixed with oxygen in several standard blends. The most popular mixture is 79%/21% helium—oxygen, which has a density that is 40% that of pure oxygen. Helium—oxygen mixtures are available in large-sized compressed gas cylinders. In anesthetic practice, pressures needed to ventilate patients with smalldiameter tracheal tubes can be substantially reduced when the 79%/21% mixture is used. Heliox can provide patients with upper airway—obstructing lesions (eg, subglottic edema, foreign bodies, and tracheal tumors) with relief from acute distress until more definitive care can be delivered. The benefit is less convincing in treating lower airway obstruction from COPD or acute asthma. Helium mixtures may also be used as the driving gas for small-volume nebulizers in bronchodilator therapy for asthma. However, with heliox, the nebulizer flow needs to be increased to 11 L/min versus the usual 6 to 8 L/min with oxygen. Patients' work of breathing can be reduced with heliox as compared to a conventional oxygen/nitrogen gas mixture.

Hyperbaric oxygen therapy uses a pressurized chamber to expose the patient to oxygen tensions exceeding ambient barometric pressure (at sea level the ambient pressure is 760 mm Hg). With a one-person hyperbaric chamber, 100% oxygen is usually used to pressurize the chamber. Larger chambers allow for the simultaneous treatment of multiple patients and for the presence of medical personnel in the chamber with patients. Multiplace chambers use air to pressurize the chamber, whereas patients receive 100% oxygen by mask, hood, or tracheal tube. Common indications for hyperbaric oxygen where outcome benefits have been shown include decompression sickness (the "bends"), certain forms of gas embolism, gas gangrene, anaerobic bacterial infections, carbon monoxide poisoning, and treatment of certain wounds.

Oxygen therapy can result in both respiratory and nonrespiratory toxicity. Important factors include patient susceptibility, the Fio,, and duration of therapy.

This complication is often seen in patients with COPD who have chronic CO, retention or in patients receiving opioids. Patients who retain CO, have a respiratory drive that becomes at least partly dependent on the maintenance of relative hypoxemia. Elevation of arterial oxygen tension to "normal" can therefore cause severe hypoventilation in these patients. Oxygen therapy can be indirectly hazardous for patients being monitored with pulse oximetry while receiving opioids for pain. Opioid-induced hypoventilation may not cause arterial oxygen desaturation, despite markedly reduced respiratory rates. Thus, supplemental oxygen renders pulse oximetry a poor monitor for opioid-induced respiratory depression.

High concentrations of oxygen can cause pulmonary atelectasis in areas of low V'/Q' ratios. As nitrogen is "washed out" of the lungs, the lowered gas tension in pulmonary capillary blood results in increased uptake of alveolar gas and absorption atelectasis. If the area remains perfused but nonventilated, the resulting intrapulmonary shunt with increasing "venous admixture" can lead to progressive widening of the alveolar-to-arterial (A—a) gradient.

Retinopathy of prematurity (ROP), formerly termed retrolental fibroplasia, is a neovascular retinal disorder that develops in the great majority of premature survivors born at less than 28 weeks' gestation. ROP may include disorganized vascular proliferation and fibrosis and may lead to retinal detachment and blindness. ROP resolves in approximately 80% of these cases without visual loss from retinal detachments or scars. ROP was very common in the 1940s—1950s when unmonitored high (>0.5 Fio,) oxygen was often administered to premature infants. @ However, it is now known that hyperoxia and hypoxia are risk factors for but not the primary causes of ROP. Neonates' risk of ROP increases with low birth weight and complexity of comorbidities (eg, sepsis). In contrast to pulmonary toxicity, ROP correlates better with arterial than with alveolar O, tension. The recommended arterial concentrations for premature infants receiving oxygen are 50 to 80 mm Hg (6.6—10.6 kPa). If an infant requires arterial O, saturations of 96% to 99% for cardiopulmonary reasons, fear about causing or worsening ROP is not a reason to withhold the oxygen.

The increased inspired O, tensions associated with hyperbaric O, therapy greatly accelerate O, toxicity. The risk and expected degree of toxicity are directly related to the pressures used as well as the duration of exposure. Prolonged exposure to O, partial pressures in excess of 0.5 atmosphere can cause pulmonary O, toxicity. This may present initially with retrosternal burning, cough, and chest tightness. There will be progressive impairment of pulmonary function with continued exposure. Patients exposed to O, at 2 atmospheres or greater are also at risk for central nervous system toxicity that may be expressed as behavior changes, nausea, vertigo, muscular twitching, or convulsions.

Oxygen vigorously supports combustion. The potential for uncontained oxygen enriched gas mixtures to promote fires and explosions is discussed in Chapter 2.

Patients with critical illness often require mechanical ventilation. Mechanical ventilation can replace or supplement normal spontaneous ventilation. In some instances, the problem is primarily that of impaired CO, elimination (ventilatory failure). In other instances, mechanical ventilation may be used as an adjunct (usually to positive-pressure therapy, as discussed next) in the treatment of hypoxemia. The decision to initiate mechanical ventilation is made on clinical grounds, but certain parameters have been suggested as guidelines (Table 58-4).

Of the two available techniques, positive-pressure ventilation and negative- ventilation (iron lung), the former has much wider applications and is universally used. Although negative-pressure ventilation does not require intubation, it cannot overcome substantial increases in airway resistance decreases in pulmonary compliance, and it also limits access to the patient. During positive-pressure ventilation, lung inflation is achieved by applying positive pressure to the upper airway through a tight-fitting (noninvasive mechanical ventilation) or through a tracheal or tracheostomy Increased airway resistance and decreased lung compliance can be by manipulating inspiratory gas flow and pressure. The major of positive-pressure ventilation are altered ventilation-to-perfusion circulatory effects, and risk of pulmonary, 1 = Of the two available techniques, positive-pressure ventilation and negative- ventilation (iron lung), the former has much wider applications and is universally used. Although negative-pressure ventilation does not require intubation, it cannot overcome substantial increases in airway resistance decreases in pulmonary compliance, and it also limits access to the patient. During positive-pressure ventilation, lung inflation is achieved by applying positive pressure to the upper airway through a tight-fitting (noninvasive mechanical ventilation) or through a tracheal or tracheostomy Increased airway resistance and decreased lung compliance can be by manipulating inspiratory gas flow and pressure. The major of positive-pressure ventilation are altered ventilation-to-perfusion circulatory effects, and risk of pulmonary. pressure almost tracheal or periodically mask tube. overcome disadvantages relationships, potentially adverse barotrauma and volutrauma. Positive-pressure physiological dead space because, 1 = 
compliant, nondependent areas of the lungs, whereas blood flow (influenced by gravity) favors dependent areas. Reductions in cardiac output are primarily due to impaired venous return to the heart from increased intrathoracic pressure. Barotrauma is closely related to repetitive high peak inflation pressures and underlying lung disease, whereas volutrauma is related to the repetitive collapse and reexpansion of alveoli.

Positive-pressure ventilators periodically create a pressure gradient between the machine circuit and alveoli that results in inspiratory gas flow. Exhalation occurs passively. Ventilators and their control mechanisms can be powered pneumatically (by a pressurized gas source), electrically, or by both mechanisms. Gas flow is either derived directly from the pressurized gas source or produced by the action of a rotary or linear piston. This gas flow then either goes directly to the patient (single-circuit system) or, as commonly occurs with operating room ventilators, compresses a reservoir bag or bellows that is part of the patient circuit (double-circuit system). All ventilators have four phases: the changeover from inspiration, inspiration to expiration (cycling), expiration, and the changeover from expiration to inspiration (trigger) (see Chapter 4). These phases are defined by VT, ventilatory rate, inspiratory time, inspiratory gas flow, and expiratory time.

Modern ventilators are complicated and defy simple classification. Incorporation of microprocessors into ventilators has further complicated this task. Complexity of ventilator nomenclature combined with proprietary naming schema for similar ventilator functionality has led to calls for uniform taxonomy for ventilator modes. Chatburn provides one such nomenclature, and its salient points are used to frame the following discussion on ventilator modes.

The breathing period can be divided into four phases: (1) change from expiration to inspiration (trigger), (2) inspiration (target), (3) change from inspiration to expiration (cycle), and (4) expiration. The trigger variable starts inspiration when it (pressure, volume, flow, or time) reaches a preset value. When time is the trigger, breaths are initiated on a defined frequency regardless of patient effort
(see Figure 58-1). Alternatively, pressure, flow, or volume triggers initiate a breath when the ventilator detects a change in pressure, flow, or volume, respectively caused by patient effort.
FIGURE 58-1 Trigger and synchronization windows. If a patient signal occurs within the trigger window, inspiration is patient triggered. If a patient signal occurs within a synchronization window, inspiration is ventilator triggered (or cycled if at the end of inspiration) and patient synchronized. Note that, in general, a trigger window is used with continuous mandatory ventilation, and a synchronization window is used with intermittent mandatory ventilation. (Reproduced with permission from Chatburn RL, Khatib M, Mireles-Cabodevila E. A taxonomy for mechanical ventilation: 10 fundamental maxims. Resp Care. 2014 Nov;59(11):1747-1763.)
The target variable (pressure, volume, or flow) must reach a specified level before inspiration ends. This used to be called "limit" but nomenclatures have changed. The target variable does not define the end of inspiration, but only the upper boundary for each breath. When the cycle variable is reached, inspiration ends. Options include pressure, volume, flow, or time.

A ventilator mode is a combination of control variable, breath sequence, and target scheme. Additionally, to understand ventilator mechanics one must consider the phase variables (introduced earlier).

The control variable is the independent variable in the ventilator mode (see Figure 58-2). Choice are pressure, volume, and flow. In pressure-controlled ventilation (PCV), pressure is independent variable, and pressure waveform is specified (eg, rectangular waveform). In volume-controlled ventilation (VCV), similarly a volume waveform is defined. In common terminology, flowcontrolled ventilation is not commonly used because flow is derivative of volume. When one directly controls volume, one indirectly controls flow.
Examples:
Intrapulmonary percussive ventilation
elastance; P, pressure; P,,,,, airway pressure; R, resistance; SIMV, synchronized intermittent mandatory ventilation; V, volume; V', inspiratory flow; VT, tidal volume. (Reproduced with permission from Mandu Press. Cleveland, Ohio.)

Targeting scheme is a feedback control design to deliver a specific pattern. A type of targeting scheme called set point targeting is the most basic. One sets a value, and ventilator seeks to deliver it. For VCV, set points would be VT and flow. For PCV, commonly it would be inspiratory pressure and inspiratory time.

Breath sequence is the pattern of mandatory or spontaneous breaths in a ventilator mode, or both (see Figure 58—3). A spontaneous breath is defined as a breath in which a patient determines both timing and size of the breath. It is patient triggered and patient cycled. A mandatory breath is any breath that is not spontaneous. An assisted breath is a breath in which the ventilator does some of the work for a patient-initiated breath.
FIGURE 58-3 Rubric for determining the breath sequence of a mode. APRV, airway pressure release ventilation; C, compliance; CMV, continuous mandatory ventilation; CSV, continuous spontaneous ventilation; HFV, high-frequency ventilation; IMV, intermittent mandatory ventilation; P,,,,,, ventilatory muscle pressure; R, resistance. (Reproduced with permission from Mandu Press. Cleveland, Ohio.)
There are three possible breath sequences. Continuous spontaneous ventilation (CSV) is a sequence in which all breaths are spontaneous. Intermittently mandatory ventilation (IMV) is a sequence in which spontaneous breaths are permitted in between mandatory breaths. If a mandatory breath is triggered by the patient, it a "synchronized" mandatory breath. In continuous mandatory ventilation (CMV), all breaths (including those by patient effort) are mandatory.
Combining the two types of control variables (pressure control [PC] and volume control [VC]) with three breath sequences gives us five breathing patterns: VC-CMV, VC-IMV, PC-CMV, PC-IMV, and PC-CSV. The sixth, VCCSV, would mean that patient would specify time and size of breath; however, in VC mode, a patient could not specify the size of breath.

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Synchronized intermittent mandatory ventilation (SIMV) times the mechanical breath, whenever possible, to coincide with the beginning of a spontaneous effort. Proper synchronization prevents superimposing (stacking) a mechanical breath in the middle of a spontaneous breath, which might otherwise result in a very large VT. As with CMV and assist-control (AC) ventilation (discussed next), settings to limit inspiratory pressure guard against pulmonary barotrauma. The great advantage of SIMV over IMV is that it provides for increased patient comfort. When IMV or SIMV are used for weaning, the machine breaths provide a backup if the patient becomes fatigued. However, if the rate is set too low (4 breaths/min), the backup may be insufficient, particularly for weak patients who may not be able to overcome the added work of breathing during spontaneous breaths.
IMV circuits provide a continuous flow of gas for spontaneous ventilation between mechanical breaths. Modern ventilators incorporate SIMV into their design, but older models required modification with a parallel circuit, a continuous flow system, or a demand flow valve. Regardless of the system, proper functioning of one-way valves and sufficient gas flow are necessary to prevent an increase in the patient's work of breathing, particularly when PEEP is also used.

Thus far the discussion of IMV has assumed this to be a volume-limited format; however, IMV can also be provided in pressure-limited format if desired (as described next).

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With PCV, pressure and inspiratory time are preset, whereas flow and volume are variable and dependent on the patient's resistance and compliance. With volume ventilation, on the other hand, inspiratory time is also preset but flow and VT are also preset, and with increased resistance or reduced compliance the inspiratory pressure can be greatly increased.
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pressure expiration
until the

In this mode, the ventilator cycles from expiration to inspiration after a fixed time interval. The interval determines the ventilatory rate. Typical settings on this mode provide a fixed VT and fixed rate (and, therefore, minute ventilation) regardless of patient effort, because the patient cannot breathe spontaneously. Settings to limit inspiratory pressure guard against pulmonary barotrauma, and indeed CMV can be provided in a pressure-limited (rather than volume-limited) way. Controlled ventilation is best reserved for patients capable of little or no ventilatory effort. Awake patients with active ventilatory effort require sedation, possibly with muscle paralysis, to safely receive CMV.

Incorporation of a pressure sensor in the breathing circuit of AC ventilators permits the patient's inspiratory effort to be used to trigger inspiration. A sensitivity control allows selection of the inspiratory effort required. The ventilator can be set for a fixed ventilatory rate, but each patient effort of sufficient magnitude will trigger the set Vt. If spontaneous inspiratory efforts are not detected, the machine functions as if in the control mode. Most often, AC ventilation is used in a volume-limited format, but it can also be provided in a pressure-limited way (as discussed later).

PSV was designed to augment the VT of spontaneously breathing patients and overcome any increased inspiratory resistance from the tracheal tube, breathing circuit (tubing, connectors, and humidifier), and ventilator (pneumatic circuitry and valves). Microprocessor-controlled machines have this mode, which delivers sufficient gas flow with every inspiratory effort to maintain a predetermined positive pressure throughout inspiration. When inspiratory flow decreases to a predetermined level, the ventilator's feedback (servo) loop cycles the machine into the expiratory phase, and airway pressure returns to baseline (Figure 58-4). The only setting in this mode is inspiratory pressure. The patient determines the respiratory rate and VT varies according to inspiratory gas flow, lung mechanics, and the patient's own inspiratory effort. Low levels of PSV (5—10 cm H,0) are usually sufficient to overcome any added resistance imposed by the breathing apparatus. Higher levels (10-40 cm HO) can function as a standalone ventilatory mode if the patient has sufficient spontaneous ventilatory drive and stable lung mechanics. The principal advantages of PSV are its ability to augment spontaneous VT, decrease the work of breathing, and increase patient comfort. However, if the patient fatigues or lung mechanics change, VT may be inadequate, and there is no backup rate if the patient's intrinsic respiratory rate decreases (eg, after opioid dosing). Note that one can add "pressure support" to IMV breathing breath sequences (Figure 58—5). The IMV machine mandatory breaths provide backup, and a low level of pressure support is used to offset the increased work of breathing resulting from the breathing circuit and machine during the spontaneous breaths.
FIGURE 58—4 Pressure support ventilation. The patient initiates a breath; the machine is set to deliver 15 cm H,O pressure (above 5 cm H,O of continuous positive airway pressure [CPAP]). When flow ceases, the machine cycles into the expiratory mode.
FIGURE 58-5 Intermittent mechanical ventilation with pressure support. M = machine breath — set tidal volume (VT) delivered. S = spontaneous breath, 15 cm of pressure support over 5 cm of positive end-expiratory pressure. VT depends on patient effort and lung mechanics. V, flow; P,,,, partial airway pressure.

IRV reverses the normal I:E time ratio of 1:3 or greater to a ratio of greater than 1:1 (eg, 1.5:1). This may be achieved by adding an end-inspiratory pause, by decreasing peak inspiratory flow during volume-controlled ventilation (CMV), or by setting an inspiratory time such that inspiration is longer than expiration during PCV (PC-IRV). Intrinsic PEEP may be produced during IRV and is caused by air trapping or incomplete emptying of the lung to the baseline pressure prior to the initiation of the next breath. This air trapping increases FRC until a new equilibrium is reached. This mode does not allow spontaneous breathing and requires heavy sedation or neuromuscular blockade. IRV with PEEP is effective for improving oxygenation in patients with decreased FRC.

Airway pressure release ventilation (APRV), or bilevel ventilation, is a mode in which a relatively high PEEP is used, despite the patient being allowed to breathe spontaneously. Intermittently, the PEEP level decreases to help augment the elimination of CO, (Figure 58-6). The inspiratory and expiratory times, high and low PEEP levels, and spontaneous respiratory activity determine minute ventilation. Initial settings include a minimum PEEP of 10 to 12 cm H,O anda release level of 5 to 10 cm H,O. Commonly, 10 to 12 releases are chosen as a starting point, along with a time at low PEEP to allow only 50% to 70% of expiratory flow (to provide "autoPEEP"). Advantages of APRV appear to be less circulatory depression and pulmonary barotrauma as well as less need for sedation. This technique appears to be an attractive alternative to PC-IRV for overcoming problems with high peak inspiratory pressures in patients with reduced lung compliance.

Most modern ventilators behave like flow generators. Constant flow generators deliver a constant inspiratory gas flow regardless of airway circuit pressure. Constant flow is produced by the use of either a solenoid (on-off) valve with a high-pressure gas source (5—50 psi) or via a gas injector (Venturi) with a lowerpressure source. Machines with high-pressure gas sources allow inspiratory gas flow to remain constant despite large changes in airway resistance or pulmonary compliance. Ventilator performance varies with airway pressure for gas injectors. Nonconstant flow generators consistently vary inspiratory flow with each inspiratory cycle (such as by a rotary piston); a sine wave pattern of flow is typical.
Constant-pressure generators maintain airway pressure constant throughout inspiration and irrespective of inspiratory gas flow. Gas flow ceases when airway pressure equals the set inspiratory pressure. Pressure generators typically operate at low gas pressures (just above peak inspiratory pressure).

Time-cycled ventilators cycle to the expiratory phase once a predetermined interval elapses from the start of inspiration. VT is the product of the set inspiratory time and inspiratory flow rate. Time-cycled ventilators are commonly used for neonates and in the operating room.
Volume-cycled ventilators terminate inspiration when a preselected volume is delivered. Many adult ventilators are volume cycled but also have secondary limits on inspiratory pressure to guard against pulmonary barotrauma. If inspiratory pressure exceeds the pressure limit, the machine cycles into expiration even if the selected volume has not been delivered.
Pressure-cycled ventilators cycle into the expiratory phase when airway pressure reaches a predetermined level. VT and inspiratory time vary, being related to airway resistance and pulmonary and circuit compliance. A significant leak in the patient circuit can prevent the necessary rise in circuit pressure and machine cycling. Conversely, an acute increase in airway resistance, or decrease in pulmonary compliance, or circuit compliance (kink) causes premature cycling and decreases the delivered VT. Pressure-cycled ventilators have been most often used for short-term indications (transport).
Flow-cycled ventilators have pressure and flow sensors that allow the ventilator to monitor inspiratory flow at a preselected fixed inspiratory pressure; when this flow reaches a predetermined level (usually 25% of the initial peak mechanical inspiratory flow rate), the ventilator cycles from inspiration into expiration (see the earlier sections on pressure support and pressure-controlled ventilation).

These versatile machines can be set to function in any one of a variety of inspiratory flow and cycling patterns. Microprocessor-controlled ventilators are the norm in modern critical care units and on newer anesthesia machines.

High-frequency ventilation is sufficiently different from conventional modes of mechanical ventilation that its mechanics require separate mention. Three forms of high-frequency ventilation (HFV) are available. High-frequency positivepressure ventilation involves delivering a small "conventional" VT at a rate of 60 to 120 breaths/min. High-frequency jet ventilation (HFJV) utilizes a small cannula at or in the airway through which a pulsed jet of high-pressure gas is delivered at a set frequency of 120 to 600 times/min (2-10 Hz). The jet of gas may entrain air (Bernoulli effect), which may augment VT. High-frequency oscillation employs a driver (usually a piston) that creates to-and-fro gas movement in the airway at rates of 180 to 3000 times/min (3-50 Hz).
These forms of ventilation all produce VT at or below anatomic dead space. The exact mechanism of gas exchange is unclear but is probably a combination of effects. Jet ventilation has found widest use in the operating room. It may be used for laryngeal, tracheal, and bronchial procedures and can be lifesaving in emergency airway management when tracheal intubation and conventional positive-pressure ventilation are unsuccessful (see Chapter 19). In the intensive care unit (ICU), HFJV may be useful in managing some patients with bronchopleural and tracheoesophageal fistulas when conventional ventilation has failed. Occasionally, HFJV or high-frequency oscillation is used in patients with ARDS to try to improve oxygenation. Inadequate heating and humidification of inspired gases during prolonged HFV, however, can be a problem. Initial settings for HFJV are typically a rate of 120 to 240 breaths/min, an inspiratory time of 33%, and a drive pressure of 15 to 30 psi. Mean airway pressure should be measured in the trachea at least 5 cm below the injector to avoid an artifactual error from gas entrainment. Carbon dioxide elimination is generally increased by increasing the drive pressure, whereas adequacy of oxygenation relates to the mean airway pressure. An intrinsic PEEP effect is seen during HFJV at high drive pressures and inspiratory times greater than 40%.

This technique, also referred to as independent lung ventilation, may be used in patients with severe unilateral lung disease or those with bronchopleural fistulae. Use of conventional positive-pressure ventilation and PEEP in such instances can aggravate ventilation/perfusion mismatching or, in patients with fistula, result in inadequate ventilation of the unaffected lung. In patients with restrictive disease of one lung, overdistention of the normal lung can lead to worsening hypoxemia or barotrauma. After separation of the lungs with a double-lumen tube, positive-pressure ventilation can be applied to each lung independently using two ventilators. When two ventilators are used, the timing of mechanical breaths is often synchronized, with one ventilator, the "master," setting the rate for the "slave" ventilator.

Tracheal intubation is most commonly undertaken in ICU patients to manage

Depending on the type of pulmonary failure, mechanical ventilation is used to

Sedation and paralysis may be necessary in patients who become agitated and "fight" the ventilator. Repetitive coughing ("bucking") and straining can have adverse hemodynamic effects, can interfere with gas exchange, and may predispose to pulmonary barotrauma and self-inflicted injury. Sedation with or without paralysis may also be desirable when patients continue to be tachypneic despite high mechanical respiratory rates (> 16-18 breaths/min).
Commonly used sedatives include opioids (morphine or fentanyl), benzodiazepines (usually midazolam), propofol, and dexmedetomidine. These agents may be used alone or in combination and are often administered by continuous infusion. Propofol is uncommonly used for prolonged sedation due to concerns about propofol infusion syndrome (see Chapter 9). Nondepolarizing paralytic agents are used in combination with sedation when ventilation cannot otherwise be accomplished.

There are two phases to discontinuing mechanical ventilation. In the first, "readiness testing," so-called weaning parameters and other subjective and objective assessments are used to determine whether the patient can sustain progressive withdrawal of mechanical ventilator support. The second phase,
"weaning" or "liberation," describes the way in which mechanical support is removed.
Readiness testing should include determining whether the process that necessitated mechanical ventilation has been reversed or controlled. Complicating factors such as bronchospasm, heart failure, infection, malnutrition, increased CO, production due to increased carbohydrate loads, acid-base derangements, anemia, altered mental status, and sleep deprivation should be adequately treated. Chronic lung disease, respiratory muscle wasting from prolonged disuse, and heart failure may lead to complicated (or failed) weaning.
Weaning from mechanical ventilation should be considered when patients no longer meet criteria for mechanical ventilation (see Table 58—4). In general, this occurs when patients have a pH greater than 7.25, show adequate arterial oxygen saturation while receiving Fio, less than 0.5, are able to spontaneously breathe, are hemodynamically stable, and have no current signs of myocardial ischemia. Additional mechanical indices have also been suggested (Table 58—5). Useful weaning parameters include arterial blood gas tensions, respiratory rate, and rapid shallow breathing index (RSBJ). Intact airway reflexes are mandatory and a cooperative patient is helpful for successful weaning and extubation, unless the patient will retain a cuffed tracheostomy tube. Similarly, adequate oxygenation (arterial oxygen saturation >90% on 40-50% O, with <5 cm H,O of PEEP) is mandatory prior to extubation. When the patient is weaned from mechanical ventilation and extubation is planned, the RSBI is frequently used to help predict who can be successfully weaned from mechanical ventilation and extubated. With the patient breathing spontaneously on a T-piece, the VT (in liters) and respiratory rate (f) are measured:
TABLE 58-5 Mechanical criteria for weaning/extubation.

Inspiratory pressure, Measurement = <-25 cm H,O. Tidal volume, Measurement = >5 mL/kg. Vital capacity, Measurement = >10 mL/kg. Minute ventilation, Measurement = <10 mL. Rapid shallow breathing index, Measurement = <100
Most patients with an RSBI less than 105 can be successfully extubated. Those with an RSBI greater than 120 should retain some degree of mechanical ventilator support.
The common techniques to wean a patient from the ventilator include SIMV, pressure support, or periods of spontaneous breathing alone on a T-piece or on low levels of CPAP. Many institutions use "automated tube compensation" to provide just enough pressure support to compensate for the resistance of breathing through an endotracheal tube. Newer mechanical ventilators have a setting that will automatically adjust gas flows to make this adjustment. In practice in adults breathing through conventionally sized tubes (7.5—8.5), the adjustment will typically amount to pressure support of 5 cm H,O and PEEP of 5 cm HO.

With SIMV the number of mechanical breaths is progressively decreased (by 1— 2 breaths/min) as long as the arterial CO, tension and respiratory rate remain acceptable (generally <45-50 mm Hg and <30 breaths/min, respectively). If pressure support is concomitantly used, it should generally be reduced to 5 to 8 cm H,O. In patients with acid-base disturbances or chronic CO, retention, arterial blood pH (>7.35) is more useful than CO, tension. Blood gas measurements can be checked after a minimum of 15 to 30 min at each setting. When an IMV of 2 to 4 breaths is reached, mechanical ventilation is discontinued if arterial oxygenation remains acceptable.

Weaning with PSV alone is accomplished by gradually decreasing the pressure support level by 2 to 3 cm H,O while VT, arterial blood gas tensions, and respiratory rate are monitored (using the same criteria as for IMV). The goal is to try to ensure a VT of 4 to 6 mL/kg and an f of less than 30 with acceptable Pao, and Paco. When a pressure support level of 5 to 8 cm H,O is reached, the patient is considered weaned.

T-piece trials allow observation while the patient breathes spontaneously without any mechanical breaths. The T-piece attaches directly to the tracheal tube or tracheostomy tube and has corrugated tubing on the other two limbs. A humidified oxygen—air mixture flows into the proximal limb and exits from the distal limb. Sufficient gas flow must be given in the proximal limb to prevent the mist from being completely drawn back at the distal limb during inspiration; this ensures that the patient is receiving the desired oxygen concentration. The patient is observed closely during this period; obvious new signs of fatigue, chest retractions, tachypnea, tachycardia, arrhythmias, or hypertension or hypotension should terminate the trial. If the patient appears to tolerate the trial period and the RSBI is less than 100, mechanical ventilation can be discontinued permanently. If the patient can also protect and clear the airway, the tracheal tube can be removed.
If the patient has been intubated for a prolonged period or has severe underlying lung disease, sequential T-piece trials may be necessary. Periodic trials of 10 to 30 min are initiated and progressively increased, typically by 5 to 10 min per trial as long as the patient appears comfortable, maintains acceptable arterial saturation, and does not become hypercarbic.
Many patients develop progressive atelectasis during prolonged T-piece trials. This may reflect the absence of a normal "physiological" PEEP when the larynx is bypassed by a tracheal tube. If this is a concern, spontaneous breathing trials on low levels (5 cm H,O) of CPAP can be tried. The CPAP helps maintain FRC and prevent atelectasis.

Positive airway pressure therapy can be used in patients who are breathing spontaneously as well as those who are mechanically ventilated. The principal indication for positive airway pressure therapy is a decrease in FRC resulting in absolute or relative hypoxemia. By increasing transpulmonary distending pressure, positive airway pressure therapy can increase FRC, improve (increase) lung compliance, and reverse ventilation/perfusion mismatching. Improvement in the latter parameter will show as a decrease in venous admixture and an improvement in arterial O, tension.

Application of positive pressure during expiration as an adjunct to a mechanically delivered breath is referred to as PEEP. The ventilator's PEEP valve provides a pressure threshold that allows expiratory flow to occur only when airway pressure exceeds the selected PEEP level.

Application of a positive-pressure threshold during both inspiration and expiration with spontaneous breathing is referred to as CPAP. Constant levels of pressure can be attained only if a high-flow (inspiratory) gas source is provided. When the patient does not have an artificial airway, tightly fitting full-face masks, nasal masks, or nasal "pillows" can be used. Because of the risks of gastric distention and regurgitation, CPAP masks should be used only on patients with intact airway reflexes and with CPAP levels less than 15 cm H,O (less than lower esophageal sphincter pressure in normal persons). Expiratory pressures greater than 15 cm H,O should only be administered by tracheal or tracheostomy tube.

The distinction between PEEP and CPAP is often blurred in the clinical setting because patients may breathe with a combination of mechanical and spontaneous breaths. Therefore, the two terms are often used interchangeably. In the strictest sense, "pure" PEEP is provided as a ventilator-cycled breath. In contrast, a "pure" CPAP system provides only sufficient continuous or "on-demand" gas flows (60-90 L/min) to prevent inspiratory airway pressure from falling perceptibly below the expiratory level during spontaneous breaths (Figure 58— 7). Some ventilators with demand valve—based CPAP systems may not be adequately responsive and result in increased inspiratory work of breathing. This situation can be corrected by adding low levels of (inspiratory) PSV if ina volume-targeted mode or changing to a pressure-targeted mode. In clinical practice, controlled ventilation, PSV, and CPAP/PEEP support can be delivered by most modern ICU ventilators. Manufacturers have also developed specific devices to deliver bilevel inspiratory positive airway pressure (IPAP) with expiratory positive airway pressure (EPAP) in either a spontaneous or timecycled fashion. The term bilevel positive airway pressure (BiPAP) has become a commonly used phrase, adding to the confusion of airway pressure terminology.
FIGURE 58-7 Airway pressure during positive end-expiratory pressure (PEEP) and continuous positive airway pressure (CPAP). Note that by increasing inspiratory gas flows, PEEP progressively becomes CPAP.

@ The major effect of PEEP and CPAP on the lungs is to increase FRC. In patients with decreased lung volume, appropriate levels of either PEEP or CPAP will increase FRC and tidal ventilation above closing capacity, will improve lung compliance, and will correct ventilation/perfusion abnormalities. The resulting decrease in intrapulmonary shunting improves arterial oxygenation. The principal mechanism of action for both PEEP and CPAP appears to be expansion of partially collapsed alveoli. Recruitment (reexpansion) of collapsed alveoli occurs at PEEP or CPAP levels above the inflection point, defined as the pressure level on a pressure—volume curve at which collapsed alveoli are recruited (open); with small changes in pressure there are large changes in volume (Figure 58-8). Although neither PEEP nor CPAP decreases total extravascular lung water, studies suggest that they do redistribute extravascular lung water from the interstitial space between alveoli and endothelial cells toward peribronchial and perihilar areas. Both effects can potentially improve arterial oxygenation.
FIGURE 58-8 Pressure—volume curve for pulmonary system (eg, lung, thoracic). Inflection point (IP) above which the majority of alveoli are recruited. E, result of excessive pressure when alveoli are overdistended and pulmonary compliance decreases.
Excessive PEEP or CPAP, however, can overdistend alveoli (and bronchi), increasing dead space ventilation and reducing lung compliance; both effects can significantly increase the work of breathing. By compressing alveolar capillaries, overdistention of normal alveoli can also increase pulmonary vascular resistance and right ventricular afterload.
@ A higher incidence of pulmonary barotrauma is observed with PEEP or CPAP at levels greater than 20 cm H,O. Disruption of alveoli allows air to track interstitially along bronchi into the mediastinum (pneumomediastinum). From the mediastinum, air can then rupture into the pleural space (pneumothorax) or the pericardium (pneumopericardium) or can dissect along tissue planes

The goal of positive-pressure therapy is to increase oxygen delivery to tissues, while avoiding the adverse sequelae of excessively increased (>0.5) Fio,. The need for excessive Fio, is most easily avoided with an adequate cardiac output and hemoglobin concentration. Ideally, mixed venous oxygen tensions should be followed. As we have repeatedly emphasized, the salutary effect of PEEP (or CPAP) on arterial oxygen tension must be balanced against any detrimental effect on cardiac output. Volume infusion or inotropic support may be necessary. At optimal PEEP the benefits of PEEP exceed any risks. Practically, PEEP is usually added in increments of 2 to 5 cm H,O until the desired therapeutic endpoint is reached. The most commonly suggested endpoint is an arterial oxygen saturation of hemoglobin of greater than 88% to 90% on a nontoxic inspired oxygen concentration (<50%). Many clinicians favor reducing the inspired oxygen concentration to 50% or less because of the potentially adverse effect of greater oxygen concentrations on the lung. Alternatively, PEEP may be titrated to the mixed venous artery oxygen saturation (SVO, >50-60%).

Other respiratory care techniques, including administration of aerosolized water or bronchodilators and clearing of pulmonary secretions, preserve or improve pulmonary function.
An aerosol mist is a gas or gas mixture containing a suspension of liquid particles. Aerosolized water may be administered to loosen inspissated secretions and facilitate their removal from the tracheobronchial tree. Aerosol mists are also used to administer bronchodilators, mucolytic agents, or vasoconstrictors (metered-dose inhalers are preferred for administration of bronchodilators). A normal cough requires an adequate inspiratory capacity, an intact glottis, and adequate muscle strength (abdominal muscles and Aerosol mists with or without bronchodilators may induce cough as well as loosen secretions. Instillation of hypertonic saline has been used as a mucolytic and to induce cough. Additional effective measures include chest percussion or vibration therapy and postural drainage of the various lung lobes. @ Maneuvers that produce sustained maximum lung inflation, such as the use of an incentive spirometer, can be helpful in inducing cough as well as preventing atelectasis and preserving normal lung volume. Patients should be instructed to inhale maximally and to hold their breath for 2 to 3 s before exhalation.
diaphragm). When thick, copious secretions are associated with atelectasis and hypoxemia, more aggressive measures may be indicated. These include suctioning the patient with a catheter or fiberoptic bronchoscope. When there is atelectasis without retention of secretions, a brief period of CPAP by mask or positive-pressure ventilation through a tracheal tube is often effective.

@ Inthe 1980s, anesthesiology was recognized as the first medical specialty to adopt mandatory safety-related clinical practice guidelines. Adoption of these guidelines, describing standards for basic monitoring during general anesthesia, was associated with a reduction in the number of patients suffering brain damage or death secondary to ventilation mishaps during general anesthesia.
@ In 1999 the Institute of Medicine of the (U.S.) National Academy of Sciences summarized available safety information in its report, To Err Is Human: Building a Safer Healthcare System, which highlighted many opportunities for improved quality and safety.
© It has long been recognized that quality and safety are closely related to consistency and reduction in practice variation.
@ In manufacturing and in medicine, there is a natural tendency to assume that errors can be prevented by better education or better management of individual workers (ie, to look at errors as individual failures made by individual workers rather than as failures of a system or a process). To reduce errors, one changes the system or process to reduce unwanted variation so that random errors are less likely.

As a profession, anesthesiology has spearheaded efforts to improve patient safety. Some of the first studies to evaluate safety of care focused on provision and sequelae of anesthesia. When spinal anesthesia was virtually abandoned in the United Kingdom (as a response to two patients developing subsequent paraplegia), Drs Robert Dripps and Leroy Vandam helped prevent spinal anesthesia from being abandoned in North America by carefully reporting outcomes of 10,098 patients who received this technique. They determined that only one patient (who proved to have a previously undiagnosed spinal meningioma) developed severe, long-term neurological sequelae.
After halothane was introduced into clinical practice in 1954, concerns arose about whether it might be associated with an increased risk of hepatic injury. The National Halothane Study, a very early clinical outcomes study, was performed (long before the term outcomes research gained widespread use), demonstrating the remarkable safety of the then relatively new agent compared with the alternatives. It failed, however, to settle the question of whether "halothane hepatitis" actually existed.
@ In the 1980s, anesthesiology was recognized as the first medical specialty to adopt mandatory safety-related clinical practice guidelines. Adoption of these guidelines was not without controversy, given that for the first time the American Society of Anesthesiologists (ASA) was "dictating" how physicians should practice. Adoption of standards for basic monitoring during general anesthesia (that included detection of carbon dioxide in exhaled gas) was associated with a reduction in the number of patients suffering brain damage or death secondary to ventilation mishaps. A fortunate associated result was that the cost of medical liability insurance coverage for American anesthesiologists also declined.

In 1984, Dr Ellison Pierce, president of the ASA, created its Patient Safety and Risk Management Committee. The Anesthesia Patient Safety Foundation (APSF), which celebrated its 30th anniversary in 2016, was also Dr Pierce's creation. The APSF continues to spearhead efforts to make anesthesia and perioperative care safer for patients and practitioners. Similarly, through its guidelines, statements, advisories, and practice parameters, the ASA continues to promote safety and provide guidance to clinicians. As Dr Pierce noted, "Patient safety is not a fad. It is not a preoccupation of the past. It is not an objective that has been fulfilled or a reflection of a problem that has been solved. Patient safety is an ongoing necessity. It must be sustained by research, training, and daily application in the workplace."
Meanwhile, other specialties of medicine began to place greater emphasis on quality and safety. @ In 1999 the Institute of Medicine (IOM) of the (U.S.) National Academy of Sciences summarized available safety information in a report entitled To Err Is Human: Building a Safer Healthcare System. That document highlighted many opportunities for improved quality and safety in the U.S. health care system. A subsequent IOM report, Crossing the Quality Chasm: A New Health System for the 21st Century, explored the way that variation in medical practice reduced quality and safety of health care system. More recently, the Institute for Healthcare Improvement has been "motivating and building the will for change; identifying and testing new models of care in partnership with both patients and health care professionals; and ensuring the broadest possible adoption of best practices and effective innovations," as described on its web site. Meanwhile, the ASA has continued its involvement in quality improvement by funding and sponsoring the Anesthesia Quality Institute, which allows individual departments and even individual physicians to compare their performance on a long list of outcomes and other metrics to those in a national database of tens of millions of anesthetics.

® It has long been recognized that quality and safety are closely related to consistency and reduction in process variation. The quality and safety movement(s) in medicine have their origins in the work of Walter Shewhart and his associate W. Edwards Deming, who popularized the use of statistics and control charts in improving the reliability of a process. In manufacturing (where these ideas were initially applied), reducing an error rate reduces the frequency of defective products, reduces the need for product inspections, and increases the customer's satisfaction with the product and the manufacturer. In medicine, reducing the error rate (for everything from accurate timing and delivery of prophylactic antibiotics to ensuring "correct side and site" surgery or regional anesthetic blocks) reduces preventable injuries to patients, eliminates the additional costs resulting from those errors, and improves quality.

@ In manufacturing and in medicine, there is a natural tendency to assume that errors can be prevented by better education, better performance, or better management of individual workers. In other words, there is a tendency to look at errors as individual failures made by individual workers, rather than as failures of a system or a process. This is exemplified by the so-called "root cause analysis." But there often are multiple factors, not a single "root cause," that lead to a system failure. As was first stated by Arthur Jones and then popularized by Paul Batalden, "All organizations are perfectly designed to get the results they get!" "Accidents" are included among the results about which he speaks! Therefore, in taking an organizational or systems point of view (as advocated by Deming) to reduce errors, one changes the system or process so that random errors will be less likely. An outstanding example of this is aadoption of the "universal protocol" prior to invasive procedures. Adherence to this protocol ensures that the correct procedure is performed on the correct part of the correct patient by the correct physician, that the patient has given informed consent, that all needed equipment and images are available, and that (if needed) the correct prophylactic antibiotic was given at the correct time.
A related example of a simple approach to improve safety and quality of a procedure is the use of a cognitive aid, such as a standardized checklist, as described in the popular press by Dr Atul Gawande. The importance of checklists is addressed elsewhere in this text, for example, in Chapter 2 in the context of developing a culture of safety in the operating room. Such checklists provide the "script" for the preprocedure universal protocol (Figure 59-1). Studies have shown that the incidence of catheter-related bloodstream infections can be reduced when central venous catheters are inserted after adequate cleansing and disinfection of the operator's hands by an operator wearing a surgical hat and mask, sterile gown, and gloves; using chorhexidine (rather than povidone iodide) skin preparation of the insertion site; and with sterile drapes of adequate size to maintain a sterile field. Individual elements in this central line "bundle" are much less likely to be omitted when a checklist is required prior to every central line insertion; a sample checklist is shown in Figure 59-2.

= > Availability of Implants/special equipment confirmed Signature & Printed Name or ID of Provider Performing Procedure. > Availability of Implants/special equipment confirmed Signature & Printed Name or ID of Provider Performing Procedure, Pre-Procedure = . > Availability of Implants/special equipment confirmed Signature & Printed Name or ID of Provider Performing Procedure, and Time Documentation = Date. > Availability of Implants/special equipment confirmed Signature & Printed Name or ID of Provider Performing Procedure,  = Time. > Availability of Implants/special equipment confirmed Signature & Printed Name or ID of Provider Performing Procedure,  = 
FIGURE 59-1 The "time out" checklist used at the Virginia Commonwealth University Health System before all regional anesthesia procedures. There is space for two separate time outs. An additional time out is performed whenever a patient's position is changed for a second regional block (most commonly for lower extremity surgery). For convenience, the regional anesthesia time out checklist is printed on the reverse of the Consent for Anesthesia acknowledgment form. (Reproduced with permission from Virginia Commonwealth University Health
System.)
ESEENLNSME
MRN
(or PATIENT LABEL)

Purpose: To work as a team to decrease patient harm from catheter-related blood stream When: During all central venous catheter insertion or re-wirings Who: Assistant to complete this form during catheter insertion
infections
1. Date:
Time:
a.m.
p.m.
2. Procedure Site:
O New
O Re-wiring
3. Procedure is:
O Elective
O Emergent
4. Before procedure, did person(s) performing procedure:
>» Wash hands immediately prior?
O Yes
O No
> Sterilize procedure site (chlorhexidine)?
O Yes
O No
> Drape entire patient in sterile fashion?
O Yes
O No
5. During procedure, did personnel performing procedure:
> Wear sterile gloves?
Q Yes
O No
> Wear hat and mask?
Q Yes
QO No
> Wear sterile gown?
O Yes
QO No
> Maintain a sterile field?
O Yes
O No
6. Did all personnel assisting with procedure follow the policy?
O Yes
O No
7. Procedure stopped at any time due to break in sterile field?
O Yes
O No
If yes, Corrective Actions Taken:
O Person performing procedure applied appropriate barrier, re-prepped and draped the pateint
O New checklist initiated
O Complete new set up: staff barriers, prep, drape new line
0 Attending/designee paged; problem corrected
O Attending/designee paged; problem not corrected
8. After procedure, were:
> Sterile dressings applied to the site?
O Yes
O No
> New IV bag & tubing set up?
O Yes
O No
> New stopcocks and access devices used?
O Yes
O No
> All ports closed with sterile dead enders?
O Yes
O No
9. Comments- Please note any additional corrective actions taken:
The assistant should STOP any procedure that does not meet this standard of care. The procedure should not continue until everyone is in compliance. The assistant will contact unit or division leadership immediately for anyone refusing to comply with this policy.
—
Name of Person Performing Procedure (& ID #) PRINT NAME
Assistant Completing Checklist PRINT NAME

FIGURE 59-2 Mandatory checklist for insertion of central venous catheters in patients who are not currently undergoing anesthesia and surgery at the Virginia Commonwealth University Health System. A similar electronic document is contained within the Anesthesia Information Management electronic record for central venous lines. (Reproduced with permission from Virginia Commonwealth University Health System.)
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Second, team participation in a checklist reminds every member of the surgical team that everyone has a stake in patient safety and good surgical outcomes. The team member who records the checklist "results" is usually not a physician but has the implicit authority to enforce adherence to the checklist. On poorly functioning teams in which there is excessive deference to authority figures, team members may feel that their opinions are not wanted or valued, or may be afraid to bring up safety concerns for fear of retaliation. On wellfunctioning teams, there is a "flattening" of the hierarchy such that every team member has the authority and every team member feels an obligation to halt the proceedings to prevent potential patient harm. The Toyota Corporation is famous for allowing any team member to shut down the automotive production line when a problem is suspected.

In surgery, there are well-recognized indicators of good quality, such as a very low incidence of surgical site infections or of perioperative mortality. However, at present there is no consensus as to the important measurements that can be used to assess quality of anesthesia care. Nevertheless, surrogate anesthesia indicators have been monitored by a variety of well-meaning agencies. Examples include selection and timing of preoperative antibiotics and temperature of patients in the postanesthesia care unit after colorectal surgery.
a
The Physician Quality Reporting System (PQRS) is a quality reporting program that encourages individual eligible professionals and practice groups to submit information on their quality of care to the U.S. Medicare program. PQRS was initiated by the Centers for Medicare and Medicaid Services in 2006 as a way to increase quality in healthcare. This program began as a "pay for performance" program that paid bonuses to those who reported quality measures. Since 2015, PQRS no longer pays out bonuses for quality reporting, but issues a 0.5% penalty for those who do not report these measures. Since 2013, anesthesiologists, pain physicians, and certified registered nurse anesthetists must all report Medicare PQRS quality measures.
Mindful of the importance of having accurate and relevant outcome measures, the ASA charged the Anesthesia Quality Institute (in 2009) with developing and collecting valid quality indicators for anesthetic care that can be used for quality improvement programs. Aggregation of the large amounts of data required for statistical validity is dependent on widespread adoption of electronic medical records (EMRs) and anesthesia information management systems (AIMS) (discussed in Chapter 18). It is our hope that as their use becomes more widespread, the data and indicators that are collected and aggregated will provide greater insight into how quality of anesthesia care may influence clinical outcomes that are important to patients.
In any individual department or medical center there should be myriad opportunities to improve the quality of care; members of the anesthesiology department need to be involved with many of them. The Accreditation Council for Graduate Medical Education now specifies that house officers participate in quality improvement exercises. This is a very welcome development, but only a limited number of anesthesiologists are familiar with the methodology. The Institute for Health Care Improvement (IHI) offers a variety of teaching tools to facilitate performance improvement in health care.

We strongly recommend that every medical center use a standard format for planning and conducting such projects. Some medical centers favor the so-called "6 sigma" program. This is a reasonable approach, but it does mandate more specialized training. We favor Dr Shewhart's (as adapted by Dr Deming and also by the THI) plan-do-study-act (PDSA) cycles, as being simple, straightforward, and readily accomplished without need for a specialized training program. Regardless of the rubric, success requires proper planning. Specifically, one cannot improve quality if one does not define a goal and define how success toward achieving that goal will be measured. On the other hand, a group cannot allow itself to be frozen in a perpetual state of planning. After "doing" the change and studying the results, the group can act on them in deciding how best to evolve the current process improvement plan. In a true "continuous quality improvement" program, unless the PDSA cycles continue indefinitely there will be an inevitable "drift" toward poorer performance.

American Society of Anesthesiologists standards, guidelines, statements, and other documents. https://www.asahq.org/quality-and-practicemanagement/standards-and-guidelines
Anesthesia Business Consultants, "PQRS Refresher for Anesthesiologist,
CRNAs and Pain Physicians." http://www.anesthesia.llc
Center for Medicare and Medicaid Services, "About PQRS."
http://www.cms.gov
Institute for Healthcare Improvement. http://www. ihi.org.
The W. Edwards Deming Institute. https://www.deming.org/
Please note that index links point to page beginnings from the print edition. Locations are approximate in e-readers, and you may need to page down one or more times after clicking a link to get to the indexed material.
Note: Page numbers followed by f and t indicate figures and tables, respectively.
A
Abdominal compartment syndrome, 835-837
Abdominal pain, 1075
Absence seizures, 624
Absolute refractory period, 346
Absolute shunt, 516
Absorbent granules, 42
Absorption, 140
ACC/AHA guidelines, 384, 408, 415, 425
Acceleromyography, 134
Accreditation Council of Graduate Medical Education, 1358
Acetaminophen, 1075, 1078
ingestion, 736
intravenous, 287
Acetylcholine chemical structure of, 206f
parasympathetic nervous system, 223f synthesis and hydrolysis, 222f
Acetylcholinesterase (ACh) receptors, 201, 201f
Acetyl-CoA, 718-719, 1224
a,-Acid glycoprotein (AAG), 141
Acid-base balance, 1169
Acid—base management, 1169
acid—base disorders, 1184-1185
acidemia, 1176-1177
acidosis, 1176-1177
acids/bases, 1170-1171
alkalemia, 1184
alkalosis physiological effects, 1182
respiratory alkalosis, 1182-1183
anesthetic considerations, 1182
blood gas tensions/pH, measurement of sample source/collection, 1185

temperature correction, 1185
body buffers bicarbonate buffer, 1172—1174
hemoglobin as a buffer, 1174
case discussion, 1185-1187
clinical disorders, 1171-1172
compensatory mechanisms, 1172-1176
conjugate pairs/buffers, 1171
definitions of, 1170-1172
high anion gap metabolic acidosis, 1179-1180
exogenous nonvolatile acids, ingestion of, 1180
failure to excrete endogenous nonvolatile acids, 1179
increased endogenous nonvolatile acid production, 1179-1180
hydrogen ion concentration, 1170
hyperchloremic acidosis, causes of, 1181
metabolic acidosis anion gap, 1178-1179
treatment of, 1181-1182
metabolic alkalosis causes of, 1184
chloride-resistant metabolic alkalosis, 1183-1184
chloride-sensitive metabolic alkalosis, 1183
treatment of, 1184
normal anion gap metabolic acidosis description of, 1180-1181
HCO3, 1180-1181

renal compensation, 1174-1176
during acidosis, 1175-1176
alkalosis, 1176
ammonia, 1176
base excess, 1176
HCOs,
1174-1176
titratable acids, 1175-1176
respiratory acidosis, 1177-1178
acute, 1177-1178
chronic, 1178
treatment of, 1178
respiratory compensation, 1174
strong ion difference (SID), 1171, 1171f
Acidemia, 1172
Acidic drugs, 141
Acidosis, 1176-1177
Acinus, 716
Acquired immunodeficiency syndrome (AIDS), 1203
Action potentials, 262, 263f
Activated clotting time (ACT), 451
Active humidifiers, 66
Acupuncture, 1109
Acute gastrointestinal hemorrhage, 1325
Acute herpes zoster/postherpetic neuralgia, 1073
Acute intravascular hemolysis, 1201
Acute ischemic stroke, 615-616
Acute kidney failure, 684
Acute kidney injury (AKD, 663, 676
azotemia associated with, 1313-1314, 1314t biomarkers of, 680f
causes of, 663t description of, 1313
differential diagnosis, 676f drugs and toxins, 665t
etiology of, 1315, 1315t nonoliguric, 1315-1316
nutritional management of, 1316
oliguric, 1315-1316
pathogenesis of, 1315
postischemic, 1315
renal replacement therapy for, 1316
RIFLE criteria for, 679t risk index, 664t
severity of, 677t treatment of, 1316
Acute kidney injury network (AKIN) staging system, 677, 679t
Acute lung injury (ALI), 568-569, 1308, 1311
Acute myocardial infarction (AMI), 1312-1313
Acute respiratory distress syndrome (ARDS), 1308-1309, 1311
Acute subdural hematoma, 831
Acute tubular necrosis, 1315

Adaptability/flexibility crew resource management (CRM), 26
Adductor canal block, 1030-1031, 1032f
Adenosine, 376t, 378t, 1274t
Adenosine triphosphate (ATP), 348, 584, 718
Adenylyl cyclase, 242
Adjustable pressure-limiting (APL) valves, 38, 43, 43f, 63, 65
Adrenal gland, 764
catecholamine excess, 766—767
glucocorticoids deficiency of, 765—766
excess of, 765
mineralocorticoid deficiency, 765
mineralocorticoid excess anesthetic considerations, 765
clinical manifestations, 764—765
physiology, 764
Adrenergic agonists, 244-248. See also specific agents

case discussion on, 251—252
Adrenergic agonists (Cont.)
dobutamine, 248
dopamine, 247-248
effects of, 245t ephedrine, 246-247
epinephrine, 246-247
fenoldopam, 248
isoproterenol, 248
norepinephrine, 247
phenylephrine, 245-246
pheochromocytoma case discussion, 251-252
selectivity of, 244t structure of, 245f
Adrenergic antagonists a-blockers (phentolamine), 248-249
-blockers, 249
carvedilol, 250
case discussion on pheochromocytoma, 251-252
esmolol, 250
metoprolol, 250
mixed antagonists (labetalol), 249
nebivolol, 250
perioperative beta-blocker therapy, 251
propranolol, 250
a-Adrenergic vasopressors, 690
&-Adrenergic blocking agents comparison of, 397t
description of, 165, 182, 396-398
Adrenoceptors (adrenergic receptors), 240-244
a, 242
A, 242-243
&,, 243
&,, 244

dopamine (DA) receptors, 244
epinephrine synthesis, 242f norepinephrine synthesis, 241f, 242f
physiology of, 240-244
in sympathetic nervous system, 239, 240f transmembrane structure of, 242f
Adrenocortical suppression, 181
Adrenocorticotropic hormone (ACTH), 563, 764
Aerosol mist, 1351
Agent-specific variable-bypass vaporizers, 58f
Age-related physiological changes, 933
Q>-Agonists, 246
clinical considerations, 246
dosing, 246

medical gas cylinders, 12t in medical gas systems, 13
Airtraq optical laryngoscope, 323f
Airway. See also Airway management anatomy, 308f, 308-310
assessment, 310-312
dichotomous division of, 498f difficult, 315f
algorithm, 328f case discussion on, 336-339
intubating laryngeal mask, 339f nerve block in, 338f
portable storage unit for, 339t positioning in, 314
superior laryngeal nerve block and transtracheal block in, 338f gas-flow resistance, 507-508
flow-related airway collapse, 507-508, 509f forced vital capacity, 508, 510f
volume-related airway collapse, 507, 508f instrumentation, physiological responses, 335
nasal, 313
oral, 313
oropharyngeal, 313f patency, 1289
sensory nerve supply of, 309f trauma, 334
Airway equipment, 912t
Airway management, 307-341
bag and mask ventilation (BMV), 314-315
direct/indirect laryngoscopy and intubation techniques, 324—331
endotracheal intubation, 319-320
equipment in, 312-314
extubation techniques, 333
face mask design and technique, 313-314
of high tracheal lesion, 575f after intubation, 332-333
King laryngeal tubes (LTs), 319
laryngoscope, 321
laryngoscopy and intubation, complications of, 333-335
oral and nasal airways, 313
oral opening, Mallampati classification of, 311f preoxygenation, 314
routine, 310
surgical airway techniques, 331-332
technique for, 337f video laryngoscopes, 321-324
Airway pressures, 73f, 1347
Aladin cassette vaporizer, 59
Alanine aminotransferase (ALT), 723
Albumin, 1307
Alcohol-based skin preparations, 24
Aldosterone, 764, 1150
antagonists, 669
intravenous dosage, 669
side effects, 669

hypersecretion of, 764—765
Aldrete recovery score, 1294t
Alfentanil
biotransformation of, 195
characteristics of, 190t
chemical structure of, 189f
Aliskiren, 390t
Alkalemia, 1172
Alkaline phosphatase, 736
Alkalosis
physiological effects, 1182
Allen’s test, 87
Allergic reactions, 1246
anaphylactic reactions, 1248-1249
to anesthetic agents, 1249-1250
to antibiotics, 1251
to atracurium, 216
immediate hypersensitivity reactions, 1246-1248
latex allergy, 1250-1251
to pancuronium, 217

Alveolar concentration (FA)
of anesthetics inhalation, 150-154
concentration of, 154
uptake in, 150-152, 151t, 153f
ventilation in, 152, 154
Alveolar dead space, 512, 515
Alzheimer disease (AD), 627
Ambient oxygen therapy equipment, 1331-1337
Ambulatory anesthesia and surgery, 944-945
Ambulatory surgery
cardiac conditions, 945-947
glucose control, 947
malignant hyperthermia, 948
inhalation. See Inhalation anesthesia
intravenous. See Intravenous anesthesia
physiologic considerations in, 554-557
lateral decubitus position in, 554f, 554-555, 555f
open pneumothorax in, 555-556
providers, 10
scope of, 6-7
sedation and, outside operating room, 953t—954t
Anesthesia, adjuncts to, 275-294, 593-594
aspiration of, 276-281
antacids, 279-280
histamine-receptor antagonists, 276-279
metoclopramide, 280
pneumonia prophylaxis, pharmacology of, 279t
proton pump inhibitors, 280-281
case discussion on aspiration pneumonia, 291-293
drugs used as adjuvants to, 286-291
clonidine, 287-288
dexmedetomidine, 288-289
doxapram, 289
flumazenil, 290-291
ketorolac, 286-287
naloxone, 289-290
naltrexone, 290
postoperative nausea and vomiting (PONV), 281-286
butyrophenones, 285-286
dexamethasone, 286
5-HT3 receptor antagonists, 282-285
neurokinin-1 receptor antagonist, 286
risk factors for, 281t
strategies of, 286
Anesthesia dolorosa, 1050t
Anesthesia information management systems (AIMS), 302, 1358
Anesthesia airway pressures, 73f
machines, 38, 47-79, 124. See also Emergency (E-cylinder) cylinder

basics of, 50t, 51
breathing circuit in, 59-66, 60f, 62f. See also Breathing circuits case discussion on, 77—78
checkout list for, 76t-77t, 77
flow control circuits in, 52-59. See also Flow control circuits gas supply in, 51-52
gases, vapor pressure, 56f heat and moisture exchanger (HME), 67f
modern machine, 49f overview of, 51
safety features of, 50t, 51
schematic of, 52f agent-specific variable-bypass vaporizers, 58f
copper kettle vaporizer, 56f functional, 51f
spirometer designs, 64f—65f ventilators in, 66-75. See also Ventilators
waste-gas scavengers, 75—77
Anesthesia ventilators excessive positive pressure, 74
problems associated with, 74—75
tidal volume discrepancies, 74—75
ventilator—fresh gas flow coupling, 74
Anesthesiologist, 913, 944
Anesthesiology clinical practice, 139
practice of, 1-7, 2t
Anesthesiology Assistant programs, 6
Anesthetic agents, 594, 681-684
choice of, 393-394
induction agents, 393
inhalation agents, 683
nitrous oxide, 683
volatile anesthetic agents, 683
intravenous agents, 682
anticholinergic agents, 682
barbiturates, 682
benzodiazepines, 682
H,-receptor blockers, 682
ketamine, 682
opioids, 682
phenothiazines, 682
propofol and etomidate, 682
maintenance agents, 393-394
muscle relaxants, 683-684
cisatracurium and atracurium, 683
curare (d-tubocurarine), 683
pancuronium, 683
reversal agents, 683-684
succinylcholine, 683
vasopressors, 394
vecuronium and rocuronium, 683
Anesthetic complications adverse anesthetic outcomes
ASA Closed Claims Project, 953, 989, 1234-1235, 1237

causes, 1235
human errors, 1235t incidence, 1233-1234
airway injury, 1240-1241
allergic reactions, 1246
awareness, 1243-1244
brain injury, 1235-1236
cardiopulmonary arrest during spinal anesthesia, 1245
case discussion, 1255-1256
equipment problems, 1240
prevention, 1240
eye injury, 1244-1245
hearing loss, 1245
litigation, 1230-1233
mortality/brain injury, 1235-1236
equipment malfunctions, 1235t
IV catheters, injuries, 1237f
retrospective data collection, 1236f
obstetric anesthesia, 1237-1238
occupational hazards, 1252-1255
anesthetic gases, chronic exposure, 1253
chronic exposure, 1253
ionizing radiation exposure, 1254-1255
substance abuse, 1254
out of operating room, 1239-1240
pediatric anesthesia, 1238-1239
peripheral nerve injury, 1241-1243
positioning, complications, 1242-1243, 1243t
positioning, role of, 1241-1242
quality management, 1251-1252
regional anesthesia, 1238
vascular cannulation, 1236-1237
Anesthetic gas analysis, 123-124
clinical considerations, 124
galvanic cell, 124
indications in, 123
paramagnetic analysis, 124
piezoelectric analysis, 123
spirometry, 124
techniques for, 123-124
Anesthetic management-related factors contributing to enhanced recovery
programs (ERPs), 1116

course of, 150
general. See also specific agents
inhalation, 2—3. See also specific agents
agents, 151f
clinical pharmacology of, 159-169, 162t desflurane, 166—167
halothane, 163-165
isoflurane, 165-166

nitrous oxide, 159t, 159-163
pharmacodynamics of, 155-159
anesthetic neuroprotection and cardiac preconditioning, 158
anesthetic neurotoxicity, 157-158
minimum alveolar concentration, 156f, 158-159, 159t, 160¢t theories of anesthetic action in, 155—157
pharmacokinetics of, 150-155
alveolar concentration (F,), 150-154
arterial concentration (F,), 154
elimination in, 154-155
inspiratory concentration (FI), 150
partition coefficients in, 151t, 151-152
sevoflurane, 167-169
xenon, 169
intravenous, 4-5, 171-184. See also specific agents barbiturates, 171-175
benzodiazepines, 175-177
case discussion on, 183-184
etomidate, 180-181
ketamine, 177-180
propofol, 181-183
mishaps, 1235
Anesthetics, local, 261-273. See also specific agents agents of, 267
amides, 268
clinical pharmacology, 267-272
clinical use of, 269t drug interactions, 272
effects on organ systems, 268-272
cardiovascular, 270-271
hematological, 272
immunological, 271
musculoskeletal, 271-272
neurological, 269-270
respiratory, 270
esters, 268
mechanisms of action, 262-264, 263f nerve fiber classification of, 264t
neurotoxicity, 157-158
overdose, case discussion on, 272—273
pharmacokinetics of, 267-268
absorption, 267
biotransformation and excretion, 268
distribution, 267-268
physicochemical properties of, 265t site of injection of, 267
structure-activity relationships, 264-267
tissue mass, 268
tissue perfusion, 268
tissue/blood partition coefficient, 268
vasoconstrictor, presence of, 267
voltage-gated sodium (Na) channels, 263f

chronic stable, 395-396
unstable, 395
Angioedema, 1248, 1248f
Angiotensin-converting enzyme (ACE), 1155
Angiotensin-converting enzyme inhibitors, 371, 387, 391t
Angle-closure glaucoma, 235
Anhepatic phase, 746-747
Ankle block, 1038-1040
needle placement for, 1040f
Ankylosing spondylitis, 1071
Antacids clinical uses, 279-280
dosage, 280
drug interactions, 280
mechanism of action, 279
Anterior spinal artery syndrome, 966
Antianginal agents, 396t
Antiarrhythmic drugs, 375t—378t
Anticholinergic drugs, 233-237, 539, 682
atropine, 235-236
central anticholinergic syndrome, case discussion on, 236-237
clinical pharmacology, 234-235
general pharmacological characteristics, 234t, 234-235
cardiovascular, 234—235
cerebral, 235
gastrointestinal, 235
genitourinary, 235
ophthalmic, 235
respiratory, 235
thermoregulation, 235
mechanisms of action, 233-234
physical structures, 234f scopolamine, 236
Anticholinesterases, 222, 640-641
Anticoagulants, 399
Anticonvulsants, 1078t, 1080-1081
Antidepressants, 631-632, 1077t, 1079
Antidiuretic hormone (ADH), 657, 1140, 1149
Antiepileptic drugs, 625t
Antifibrinolytic agents, 463
Antihistamines, 278
Antihypertensives. See Hypotensive agents
Antimuscarinics, 233
Antispasmodics, 1079-1080
Antithrombotic therapy, 970
a,-Antitrypsin, 542
Aortic arch, 459f
Aortic cross-clamping, 446
Aortic lesions, 484—485
aneurysms in, 484
coarctation of, 485
dissection of, 484

occlusive disease of, 484-485
trauma in, 485
Aortic regurgitation, 423-424
anesthetic management, 424
choice of agents, 424
monitoring, 424
objectives, 424
pathophysiology, 423
preoperative considerations, 423
treatment, 423-424
Aortic stenosis, 421-423
pathophysiology, 421-422
preoperative considerations, 421
Aortic valve, 455f
anesthetic management, 422-423
choice of agents, 422—423
monitoring, 422
objectives, 422
treatment, 422
Apgar score, 891t
Apixaban, 970
Apneic oxygenation, 314, 569
Apneic threshold, 163, 529
Apneustic center, 529
Aprepitant, 1292
Arachnoiditis, 993
Arginine, 1226
Arginine vasopressin (AVP), 365, 657
Arnold—Chiari malformation, 630
Arrest of descent, 879
antiarrhythmic drugs for, 375t-378t
pancuronium as cause of, 217
Arterial blood gases, 1306, 1306f
Arterial blood pressure. See Blood pressure, arterial
Arterial concentration (F,)
of anesthetics inhalation, 154
ventilation/perfusion mismatch, 154
Arterial hypertension, 768
Arteriovenous malformations (AVMs), 602, 615-616
Arthritides, 1071
"As low as reasonably practical" (ALARP) principles, 16-17
Ascites, 742
Aspartate, 1060
Aspartate aminotransferase (AST), 723

drowning-related, 1309-1310
fetal, 889
intrauterine, 891
Aspiration, 195, 399
Aspiration pneumonia, 279t, 783t
Aspirin, 1313
Assertiveness, 26
Asthma, 538-541
anesthetic considerations, 539-541
intraoperative management, 540-541, 541f pathophysiology of, 538-539
preoperative considerations, 538-539
sympathomimetic agents for, 539
treatment of, 539
Asystole, 1280f
Atlantoaxial subluxation, 809
Atmospheric pressure, 55
Atracurium, 216, 683, 770, 908
characteristics of, 211t, 212t chemical structure of, 206f
metabolism and excretion ester hydrolysis, 214
Hofmann elimination, 214
physical structure, 214
side effects and clinical considerations, 215-216
allergic reactions, 216
bronchospasm, 215
chemical incompatibility, 216
hypotension and tachycardia, 215
laudanosine toxicity, 215
temperature and pH sensitivity, 216
Atrial natriuretic peptide (ANP), 654, 1149
Atrial septal defects (ASDs), 429-430
Atrioventricular septal defects, 430
Atropine, 235-236, 1272, 1274t clinical considerations, 235-236
dosage and packaging, 235
physical structure, 235

for arterial blood pressure monitoring, 84
sites for, 327f
Automated external defibrillators (AEDs), 1270
Autonomic blockade, at spinal nerve roots cardiovascular manifestations, 967-968
gastrointestinal manifestations, 968
metabolic/endocrine manifestations, 968—969
pulmonary manifestations, 968
urinary tract manifestations, 968
Autonomic dysfunction, 629
anesthetic considerations, 629
preoperative considerations, 629
Autoregulation, 164
of circulation, 363
Axillary artery blood pressure monitoring via, 87
positioning of terminal nerves, 1015f terminal nerves, positioning of, 1015f
Axillary brachial plexus block, 1014f-1015f
Azathioprine, 641t
Azotemia, 1313-1314, 1314t

Babinski sign, 1215
Back pain syndromes. See Low back pain syndromes
"Backward, upward, rightward, pressure" (BURP), 326
Baclofen, 1080
Bacterial contamination, 44
Bacterial infections, 1070
Baffle system, 42
Bag and mask ventilation (BMV), 314-315
esophageal—tracheal combitube design and technique, 317-319
supraglottic airway devices (SADs), 316-319
Bag-mask rescue breathing, 1268
Bain circuit, 39, 59
Balanced anesthesia, 4—5
Banti syndrome, 738
Barbiturate coma, 173, 831
Barbiturates, 4, 171-175, 591, 682, 722. See also specific agents dosages of, 174t
drug interactions, 174-175
effects on organ systems, 173-174
cardiovascular, 173
cerebral, 174
hepatic, 174
immunological, 174
renal, 174
respiratory, 173
mechanisms of action, 171
pharmacokinetics, 172-173
absorption, 172
biotransformation, 173
distribution, 172-173, 173f excretion, 173
structure of, 172f
structure—activity relationships, 172
Barbituric acid, 172f
Barium hydroxide, 41
Barlow syndrome, 420
Baroreceptor reflex, 364
Barotrauma, 65, 1337
Basal/maximal oxygen consumption, 936
Base, 1170
Becker muscular dystrophy, 644-645
Beer—Lambert law, 124
Benign prostatic hyperplasia (BPH), 698
Benzocaine, 268, 269t
Benzodiazepines, 175-177, 591, 682, 722, 1220
chemical structure of, 175f
doses of, 176t
drug interactions, 177
effects on organ systems, 176-177
cardiovascular, 176
cerebral, 177
respiratory, 176-177
mechanisms of action, 175
pharmacokinetics, 175-177
absorption, 175-176
biotransformation, 176—177
distribution, 176
excretion, 176

structure—activity relationships, 175
Bernoulli equation, 112-113, 114f
Betamethasone, 1080t
Bethanechol, 222
Bier block, 1022
Bilateral orchiectomy, 705
Bile canaliculi, 717
Bilevel positive airway pressure (BiPAP), 1350
Biliary system, 722f
Bilirubin, 736
Biotransformation, 142-143
Biphasic truncated exponential (BTE), 1270
Bipolar disease, 632-633
Bispectral analysis, 127
Bispectral index (BIS), 127, 1289
calculation, 129f scale, 130f
Bleomycin, 707
Block room, preoperative holding area, 815f a-Blockers, 248-249, 390t
-Blockers, 249, 254, 390t
American College of Cardiology/American Heart Association guidelines, pharmacology of, 249t

respiratory gases, transport of, 523-528
carbon dioxide, 526-528
oxygen, 523-526
volume, normal distribution of, 363t
Blood component therapy, 1189-1214
hemodynamic monitoring, 1191
intravascular volume estimations, 1189-1191
laboratory evaluation, 1190-1191
patient history, 1190
physical examination, 1190
Blood glucose, 756
Blood pressure, 789
arterial immediate control, 364—365
intermediate control, 365
long-term control, 365
monitoring of. See Blood pressure monitoring, arterial peripheral movement of pulse on, 81, 82f
site of measurement on, 82, 83f classification of, 389t
254

Blood pressure monitoring, arterial, 81-90, 82f, 83f invasive, 86-90 cannulation in complications of, 87-89 of radial artery, 87, 88f selection of artery for, 86-87 clinical considerations in, 89-90 indications and contraindications for, 86-89 noninvasive, 82-86 arterial tonometry in, 84, 85f auscultation in, 84 automatic, 86 clinical considerations in, 84-86, 86f Doppler probe in, 83-84, 84f Blood pressure monitoring, arterial (Cont.) indications and contraindications to, 82 oscillometry in, 84, 85f palpation in, 82 Blood transfusion, 1200-1205. See also Transfusion autologous transfusion, 1205 case discussion, 1205-1209 donor-directed, 1205 immune complications of, 1200-1203 hemolytic reactions, 1201-1202 infectious complications of bacterial infections, 1203-1204 parasitic infections, 1203 viral infections, 1203 massive transfusion acid—base balance, 1204 citrate toxicity, 1204 coagulopathy, 1204 hypothermia, 1204
serum potassium concentration, 1204—1205
nonhemolytic immune reactions
anaphylactic reactions, 1202
febrile reactions, 1202
graft-versus-host disease, 1202
post-transfusion purpura, 1203
transfusion-related acute lung injury, 1202
transfusion-related immunomodulation, 1203
urticarial reactions, 1202
normovolemic hemodilution, 1205
salvage/reinfusion, 1205
Blood urea nitrogen (BUN), 678-679, 1190
Blood-brain barrier, 586-587
“Blue bloater” syndrome, 542
Body mass index (BMI), 767, 945
Body surface area (BSA) nomograms, 353
Bone cement implantation syndrome, 804—805
Botox. See Botulinum toxin
Botulinum toxin, 1084
Bougie, 329f
Brachial artery, 87
Brachial plexus, 1004, 1007f-1008f
Bradycardia
algorithm, 1281f

description of, 209, 1300
Bradykinin, 1058
Brain
abscess of, 1320t
cerebral oximetry and monitors of, 131
herniation of, 588f
oxygen requirements of, 584f
Brain natriuretic peptide (BNP), 371, 412, 1149
Brain protection, 593-595
anesthesia on electrophysiological monitoring and, 595
cerebral ischemia in, 593-594
electroencephalography in, 595
evoked potentials in, 595-596
inhalation anesthetics in, 595
intravenous agents in, 595
strategies for, 594
Brain tissue oximetry, 585
Brainstem auditory evoked responses (BAERs), 129
Braxton Hicks contractions, 851
Breathing, 529-530
anesthesia effects on, 530, 531f central respiratory centers, 529
central sensors in, 529, 529f mechanisms of, 500-502
mechanical ventilation, 502
spontaneous ventilation, 500-502, 501f peripheral sensors in, 529-530, 530f
lung receptors, 530
peripheral chemoreceptors, 529-530
Breathing circuits, 34, 59-66, 60f, 62f adjustable pressure-limiting (APL) valve, 38, 43, 43f, 63, 65
characteristics of, 40t circuit pressure, 63
humidifiers, 66
oxygen analyzers, 40t spirometers, 61, 63
Breathing systems, 33-46, 34f-35f case discussion on, 45-46
circle system, 40t, 40-43, 41f. See also Circle system classification and characteristics of, 37t
draw-over anesthesia in, 35-36, 36f, 36t insufflation in, 34f-35f, 34-35, 35f
Mapleson circuits in, 36-40, 37t, 38f-40f open-drop anesthesia in, 35
resuscitation, 44-45, 45f
Breathing tubes, 38
Bronchitis, chronic, 541-542
Bronchoalveolar lavage, 576-577
Bronchospasm, 215

Bubble-through humidifier, 66
Budd-Chiari syndrome, 738
Buffer, 1171
Bupivacaine, 265t, 269t, 271, 985t
Bupropion, 1077t
Burn shock, 835
Burns, 834-837
abdominal compartment syndrome, 835-837
anesthetic considerations, 837
carbon monoxide poisoning, 837, 1311
fluid creep, 835
pulmonary complications of, 837
Rule of Nines for, 835, 836f smoke inhalation associated with, 1310-1312
Buttock pain, 1067
Butyrophenones, 285-286
C
Calcitonin, 762, 1159
Calcitonin gene-related peptide (CGRP), 1057
Calcium, 1156
antagonists, 258-259
hormones that regulate, 762t
Calcium channel blockers, 348, 398, 398t
Calcium disorders extracellular ionized calcium concentration, 1158
calcitonin, 1159
vitamin D, 1159
hypercalcemia anesthetic considerations, 1159
causes of, 1158t clinical manifestations, 1158
treatment of, 1158-1159
hypocalcemia, 1159-1160
anesthetic considerations, 1160
causes of, 1159t clinical manifestations, 1160

normal plasma calcium concentration, 1157-1158
Cancer-related pain, 1075-1077
Cannulas, 1331-1332
Cannulation, 94f artery selection for, 86-87
catheter-tubing-transducer system, 89
complications of, 87-89
digital readouts vs. arterial waveform in, 90
radial artery, 87, 88f right internal jugular, Seldinger's technique of, 94, 94f
subclavian and internal jugular veins, 93f, 94
Capillary fluid exchange, 1138f
Capnograph, 123f
Capnography, 121-123, 789
clinical considerations, 122—123
complications of, 122
diverting (sidestream) capnographs, 122
indications and contraindications, 121—122
nondiverting (mainstream) capnographs, 122
techniques for, 122
Capsaicin, 289, 1059
Caput medusae, 739
Carbamazepine, 1078t
Carbohydrate drinks, 1116
Carbon dioxide (CO,)
absorbent, 40-42, 42f absorber, 40-42, 42f
absorption spectrum for, 122
accumulation, 34
bicarbonate, 527
blood respiratory gases, transport of, 526t, 526-528
carbamino compounds, 527
dissociation curve, 528, 528f dissolved carbon dioxide, 527
hemoglobin buffering effect on transport, 527, 527t in medical gas systems, 13
stores in, 528
Carbon dioxide tension alveolar tension, 522, 522f
arterial tension, 522
end-tidal tension, 523
mixed venous tension, 521
pulmonary end-capillary tension, 522
Carbon monoxide poisoning, 167, 837, 1311
Carbon—fluoride bonds, 163
Carbonic anhydrase, 527
Carbonic anhydrase inhibitors, 669-670
alkalinization of urine, 670
intraocular pressure reduction using, 670
intravenous dosage, 670
metabolic alkalosis correction in edematous patients using, 670
side effects, 670

Carboprost tromethamine, 854
Carboxyhemoglobin (COHb), 121, 837, 1311
Carcinoid syndrome, 769t, 769-770
Cardiac action potentials, 344-346, 345f, 346t
Cardiac contraction, 96
Cardiac cycle, 350-351, 352f, 367f
Cardiac function, 740
Cardiac impulse, 346-348
Cardiac ion channels, 346t
Cardiac manifestations, 1156
Cardiac Motion Quantification (CMQ) Systems, 360f
Cardiac output (CO), 90, 101-115, 173
calculation of, 108
dye dilution, 106
echocardiography, 108-115
esophageal Doppler, 107
Fick principle, 107-108
indications of, 101
pregnancy-related changes in, 845
pulse contour devices, 106-107
techniques and complications, 101-115
thermodilution, 101-106
thoracic bioimpedance, 107
Cardiac surgery, 449-478. See also Cardiovascular surgery; specific surgeries anesthetic management of, in adults, 449-475
bypass period in, 464-468
anesthesia in, 467
cerebral protection in, 467-468
flow and pressure in, 464-465
hypothermia and cardioplegia in, 465
initiation in, 464
monitoring in, 465
respiratory gas management in, 466-467
ventilation in, 465-466
postbypass period in, 472-474
anesthesia in, 473-474
anticoagulation, reversal of, 472-473
persistent bleeding in, 473
transportation in, 474
postoperative period in, 474
prebypass period in, 462-464
anticoagulation in, 462-463
bleeding prophylaxis, 463
cannulation, 463-464
preinduction period in, 450-462
anesthetic agents in, 460-462
"high-dose" opioid anesthesia, 460-461
mixed intravenous/inhalation anesthesia, 461
muscle relaxation, 462

total intravenous anesthesia (TIVA), 461
induction in, 460
monitoring in, 450-460
arterial blood pressure, 450
central venous and pulmonary artery pressure, 451
electrocardiography, 450
electroencephalography, 458-459
laboratory parameters, 451
surgical field, 452
temperature, 451
transcranial Doppler (TCD), 459
transesophageal echocardiography, 452-458
urinary output, 451
premedication in, 450
preparation in, 450
venous access in, 450
termination of bypass in, 468-472
hemodynamic subgroups, 469t weaning in, 469-472
Cardiac surgery, in children cardiopulmonary bypass in, 477-478
induction for, 476-477
hemodynamic anesthetic goals, 476
monitoring, 476
route of, 477
venous access in, 476-477
maintenance in, 477
postbypass period in, 478
preinduction period in, 476
fasting requirements in, 476
premedication in, 476
preoperative evaluation in, 475-476
Cardiac transplantation, 478-481
anesthetic management in, 479-481
pericardial disease in, 481-483
preoperative considerations in, 478-479
Cardiogenic pulmonary edema, 1307-1308
Cardioplegia pump, 445
Cardiopulmonary bypass (CPB), 573
pumps, 442
Cardiopulmonary resuscitation and emergency cardiac care (CPR-ECC), 1259
Cardiopulmonary resuscitation (CPR), 1259-1260
airway, 1263-1266
adult emergency cardiac care algorithm, 1261f
basic life support techniques, 1265t
Heimlich maneuver, 1264, 1264f
loss of consciousness, 1263f
asystole, 1280f
bradycardia algorithm, 1281f
breathing, 1267-1268
percutaneous cricothyrotomy, 1266f
transtracheal jet ventilation after cricothyrotomy, 1267f
case studies, 1283-1284

circulation, 1268-1269
chest compression, 1268-1269
defibrillation, 1269-1279
arrhythmia, 1272
drug administration, 1272
emergency pacemaker therapy, 1272, 1278-1279
intravenous access, 1271-1272
invasive cardiopulmonary resuscitation, 1270-1271
emergency cardiac care (ECC), 1260¢t
pulseless electrical activity algorithm, 1279f
recommended protocols for, 1279, 1279f-1282f
resuscitation drugs, cardiovascular effects of, 1274t-1277t
synchronized cardioversion, steps, 1278t
tachycardia algorithm, 1282f
ventricular fibrillation/pulseless ventricular tachycardia (VF/VT) treatment

Cardiotomy suction pump, 445
Cardiovascular diseases, anesthesia with, 381-439, 933
case discussion on, 434—438
congenital heart disease, 427—432
energy requirements for, 383t
noncardiac surgery, 383-414, 401/-402f
transplanted heart, patient with, 432-434
valvular heart disease, 414—427
Cardiovascular monitors, 81-117
arterial blood pressure, 81-90. See also Blood pressure monitoring, arterial
cardiac output, 101-115
case discussion on, 115-116
central venous catheterization, 92-96
electrocardiography, 90-92, 92f
pulmonary artery catheterization, 96-101, 98f, 100f, 101t, 102¢. See also
Pulmonary artery catheterization
Cardiovascular physiology
case discussion of, 371-378
circulation in, 361-371. See also Circulation, systemic
coronary circulation in, 365-368
Cardiovascular receptors, 225
Cardiovascular surgery
anesthesia for, 441—493
cardiac surgery, 449-478. See also Cardiac surgery
cardiac transplantation, 478-481
cardiopulmonary bypass, 443-449
basic circuit in, 443-446
accessory pumps and devices, 445-446
arterial filter, 445
heat exchanger, 444
main pump, 444—445
oxygenator, 444
reservoir, 444
hypothermia in, systemic, 446
myocardial preservation in, 446-448
potassium cardioplegia in, 447-448
physiological effects of, 448-449
effects on pharmacokinetics, 449
hormonal, humoral, and immunological responses, 448-449
cardioversion in, case discussion on, 490-492
off-pump coronary artery bypass surgery, 474-475. See also Coronary artery
bypass surgery, off-pump
with pericardial disease, 481-483
vascular surgery, 483-490. See also Vascular surgery
Cardioversion, 490-492, 1270, 1313
Carina, 310f
Carotid artery surgery, 488-490
general anesthesia, 489

monitoring cerebral function, 489-490
preoperative anesthetic evaluation and management, 488-489
preoperative considerations, 488
regional anesthesia, 490
Carotid bodies, 529
Carotid endarterectomy (CEA), 595
Carvedilol, 250
Catabolism, 726
Catheter balloon-tipped pulmonary artery flotation (Swan—Ganz), 98f
central venous, 94—95, 96f pulmonary artery, 98, 100f
Catheter-associated bacteremia, 1320t
Catheterization, 133
Catheter—tubing—transducer system, 89
Cauda equina, 965
Caudal anesthesia, 987—989
Caudal blocks, 916, 987, 988f
Caudal injection, 1101
C-collar, 821, 834
Cefazolin, 700
Celiac plexus block, 1097-1098, 1098f anatomy, 1097, 1098f
complication, 1098
indications, 1097
technique, 1097-1098
Cellulitis, 1321t
Central diabetes insipidus, 1142
Central nervous system (CNS)
depression of, 1177
description of, 150, 587
Central venous catheterization, 92-96
clinical considerations, 96
indications and contraindications, 92—93
techniques and complications, 93-96
Central venous oxygen saturation (SCVO,), 92, 1269
Central venous pressure (CVP)
description of, 567, 746
monitoring of, 1191
Centrally acting sympathoplegics, 390t
Centrifugal pumps, 444-445
Centrilobular vein, 716, 717f
Cerebral aneurysms, craniotomy and anesthesia for, 612-615
intraoperative management in, 614-615
preoperative considerations in, 612-613
preoperative management in, 614
ruptured, 613
unruptured, 613
Cerebral autoregulation, 623f curve, 585f
dose-dependent depression of, 590f

Cerebral blood flow (CBF), 155, 584-585, 602
age-related decreases in, 937
arterial carbon dioxide and, 832
arterial respiratory gas tensions and, 586f autoregulation, 585
cerebral perfusion pressure, 585
extrinsic mechanisms, 586
regulation of, 585-586
Cerebral circulation, 598f
Cerebral edema, 602
Cerebral metabolic rate (CMR), 584
Cerebral metabolism, 584
Cerebral oxygen consumption (CMRO,), 161
Cerebral perfusion pressure (CPP), 174, 585, 604, 746, 832
Cerebral physiology, 584-589
anesthetic agents' effect on, 589t, 589-593

anesthetic adjuncts, 592-593
induction agents, 590-592
neuromuscular blockers (NMBs), 593
vasodilators, 593
vasopressors, 593
Cerebral receptors, 225
Cerebral salt wasting (CSW), 1145-1146
Cerebrospinal fluid (CSF), 583, 587f, 587-588, 963, 978-979
Cerebrovascular disease intracranial mass lesions, 623
intraoperative management, 622-623
preoperative considerations, 621-622
Cervical collar, 821, 834
Cervical dilation, 852
Cervical medial branch blockade, 1094f
Cervical pain, 1070
Cervical paravertebral nerve blocks, 1090-1091, 1091f anatomy, 1090
complications, 1090-1091
technique, 1090
Cervical spine illustration of, 810f
injuries to, 821, 833
Cervicothoracic block, 1096-1097
anatomy, 1096
complications, 1097
indications, 1096
paratracheal technique, 1096
Cesarean section, 853t, 873-874
indications for, 873t
Cetirizine, 277t
Chassaignac's tubercle, 1096
Chemical incompatibility, 216

Chest compressions, 892-893, 1268-1269 Chest physiotherapy, 1330 Chloroform anesthetic properties, 5 history of, 2 Chlorofumarates, 219 Chloroprocaine, 265t, 269t, 851, 985t Chlorpheniramine, 277t Cholestasis, 735 Cholinergic antagonists, 233 Cholinergic crisis, 640 Cholinergic receptors, 222 Cholinesterase inhibitors clinical use, 222 dose of, 227t Chromogranin A, 769 Chronic obstructive pulmonary disease (COPD), 541-544 signs and symptoms of, 542t treatment for, 542-543 Chronic pain management, 1049, 1051, 1061t, 1064t acute pain, 1049-1051 somatic pain, 1050 visceral pain, 1051 definitions, 1049 terms used in, 1050t Chvostek sign, 763, 1160 Cimetidine, 279t Circle system, 40t, 40-43, 41f bacterial contamination, 44 carbon dioxide absorber and absorbent, 40-42, 42f components of, 40-43 dead space, 44 disadvantages of, 44 fresh gas requirement, 43-44 humidity and heat conservation, 44
optimization of, 43
performance characteristics of, 44
resistance, 44
unidirectional valves, 42-43, 43f
Circuit pressure, 63
Circulation
coronary, 365-368
anatomy in, 365-367
coronary perfusion in, 367
myocardial oxygen balance in, 368
renal, 659-662, 660f
systemic, 361-371
arterial blood pressure in, 364—365
autonomic control of systemic vasculature in, 363-364
autoregulation of, 363
endothelium-derived factors, 363
Circulatory steal phenomenon, 590
Circulatory system, 344
Circumflex artery (CX), 365
Cirrhosis, 734
Cisatracurium, 216, 683
characteristics of, 211t, 212t
dosage, 216
metabolism and excretion, 217
physical structure, 216
side effects and clinical considerations, 217
Citalopram, 1077t
Clark electrode, 61
Clevidipine, 394, 394t, 398

Clomipramine, 632, 1077t
Clonazepam, 1078t
Clonidine (Catapres, Duraclon), 246, 287-288, 1081, 1121, 1126¢t
clinical uses, 287-288
dosage, 288
drug interactions, 288
mechanism of action, 287
side effects, 288
Clopidogrel, 299
Coagulopathy, 746
Cocaine, 265t, 269t, 270-271
Coccydynia, 1067
Codeine, 191
Color-flow Doppler, 420
Combination therapy, 399
Combitube, 319f
Common bile duct, 722
Commotio cordis, 1279
Communication, 26
Community-acquired pneumonia, 1319t
Compartment models, 143-145
Compartment syndromes, 697, 1243
Compensatory mechanisms, in heart failure, 370-371
Complex regional pain syndrome (CRPS), 1072
Compound A, 42
Computed tomography (CT), 603
Computer-assisted surgery (CAS), 811
Concentration effect, 154
Concentration—response relationship, 145
Conductive gel, 91
Congenital heart disease (CHD), 427-432
anesthetic management, 428
atrial septal defects, 429-430
atrioventricular septal defects, 430
classification of, 427t
hypoplastic left heart syndrome, 432
obstructive lesions, 428-429
partial anomalous venous return, 430-431
patent ductus arteriosus, 430
in patients, 427t
perioperative risk for, 428t
predominantly left-to-right (simple) shunts, 429-431
predominantly right-to-left (complex) shunts, 431-432
preoperative considerations, 427-428
pulmonic stenosis, 428-429
survivors of surgery, 428t tetralogy of Fallot, 431
transposition of great arteries, 432
tricuspid atresia, 431-432
truncus arteriosus, 432
ventricular septal defect, 430
Connecting segment, 656
Constant-flow generators, 67

Constant-pressure generators, 67
Constant-pressure variable-orifice flowmeters, 54f
Context-sensitive half-time, 1288
Continuous peripheral nerve blocks (CPNBs), 1120
Continuous positive airway pressure (CPAP), 36, 768, 945, 1349
delivery system, 567
PEEP versus, 1349-1350
adverse nonpulmonary effects, 1351
optimum use of, 1351
pulmonary effects of, 1350-1351
Continuous pressure technique, 868f
Continuous renal replacement therapy (CRRT), 687, 1316
Continuous venovenous hemodialysis (CVVHD), 745-746
Continuous-wave Doppler, 456f
Contraction mechanism, 348-350
Controlled respiration, 6
Conversion disorder, 1064t
Copper kettle vaporizer, 55-56, 56f
Coronary arteries anatomy of, 366f
angiographic views of, 458f to right and left ventricles, 366f
supply of left and right ventricles in, 459f
Coronary artery bypass surgery, off-pump, 443f, 474-475
Coronary artery disease (CAD), 384, 395
Coronary perfusion pressure (CPP), 367, 1269
Cortical spreading depolarizations (CSDs), 613
Corticosteroids, 1080t
Corticotropin-releasing factor (CRF), 1145
Countercurrent multiplier mechanism, 656, 657f
Counterregulatory failure, 755
COX-1 inhibition, 196
COX inhibitors, 195-196, 1078
CPDA-1, 1199
Cranial computerized tomography (CCT), 613t
Craniotomy, for mass lesions, 603-605
emergence in, 605
induction in, 604
intraoperative management of, 603-605
maintenance of anesthesia in, 605
monitoring in, 604—605
positioning in, 604—605
premedication in, 603
preoperative management of, 603
Creatine kinase, 644
Crew resource management (CRM), 26
assertiveness, 26

principles of, 26
Cricoid cartilage, 899
Cricothyroid membrane (CTM), 310
Cricothyrotomy, 331, 1265, 1266f
Critical care medicine acute kidney injury
azotemia associated with, 1313-1314, 1314t description of, 1313

acute myocardial infarction, 1312-1313
ambient oxygen therapy equipment, 1331-1337
case discussion on, 1326-1328
delirium, 1326
drowning, 1309-1310
end-of-life care, 1326
gastrointestinal hemorrhage, 1325-1326
infections, 1316-1322, 1319t-1322t
Critical care medicine (Cont.)
near-drowning, 1309-1310
post-traumatic stress disorder, 1326
pulmonary edema cardiogenic, 1307-1308
causes of, 1307
negative pressure, 1309
neurogenic, 1307, 1309
noncardiogenic, 1308-1309
pathophysiology of, 1306-1307
treatment of, 1308-1309
respiratory failure, 1306, 1306f scope of conditions in, 1305-1306
sepsis, 1316, 1318t septic shock, 1321t, 1322-1325
smoke inhalation, 1310-1312
Critical volume hypothesis, 157
Cryoanalgesia, 1102
Cryoneurolysis, 1102
Crystalloid solutions, 1191-1192, 1193t
Cuff deflation, 805
Curare (d-tubocurarine), 683
Curved array transducer, 1001
Curvilinear probe, 1002f
Cushing response, 602
Cushing's disease, 278
Cyanide toxicity, 255-256, 1311-1312
Cyanmethemoglobin, 255
Cyclic adenosine monophosphate (cAMP), 276, 349, 539
Cyclic guanosine monophosphate (cGMP), 255, 349, 538
Cyclooxygenase (COX) inhibitors, 187
effects on organ systems, 191-193
cardiovascular, 196
gastrointestinal, 196
respiratory, 196
mechanisms of action, 195
pharmacokinetics, 195-196
absorption, 195
biotransformation, 196
distribution, 196
excretion, 196
structure—activity relationships, 196

Cyclooxygenase (COX) pathway, 1058
Cyclophosphamide, 641t
Cycloplegia, 235
Cyclosporine, 641t
Cylinder pressure, 11
Cyproheptadine, 277t
Cystic fibrosis, 926, 1319t
Cytochrome P-450 (CYP) enzymes description of, 190-191, 720, 722
mixed-function oxidases, 279
Cytomegalovirus (CMV), 1203

Damage control resuscitation (DCR), 826
Damage control surgery (DCS), 830
Dantrolene, 1218, 1218t
Datex-Ohmeda balance regulator, 54f
Datex-Ohmeda Tec 7, 58f
D,-dopamine receptors, 258
Dead space, 44
Desipramine, 1077t
Desmopressin (DDAVP), 473, 1142
Dexamethasone (Decadron), 267, 286, 1080t, 1100
Dexmedetomidine (Precedex), 246, 288-289, 330, 1126t characteristics of, 183
in children, 907, 915
clinical uses of, 183, 288
dosage, 178t, 288
drug interactions, 288-289
mechanism of action, 288
pharmacokinetics of, 183
Dextran, 1194
Diabetes Control and Complications Trial, 298
Diabetes insipidus, 612, 1141-1142
Diabetes mellitus, 754-758
diagnosis and classification, 754t perioperative insulin management, 757t
Diabetic autonomic neuropathy, 756, 756t
Diabetic ketoacidosis (DKA), 754-755, 1180
Diabetic neuropathy, 1071-1072
Diabetic renal dysfunction, 756
Diacetylcholine. See Succinylcholine
Dialysis, 687t
Dialysis disequilibrium syndrome, 685
Diameter-index safety system (DISS), 51
3,4-Diaminopyridine (DAP), 643
Diaphragmatic herniation, 921
Diarrhea, 1226
Diastole, 350
Diastolic blood pressure (DBP), 81
Diastolic dysfunction, 369, 933-934
Diastolic function, 361
Diazepam chemical structure of, 175f
uses and doses of, 176t
Dibucaine, 272
Dibucaine number, 207
Diffuse neuronal injury, 831
Diffusing capacity, 530
Diffusion hypoxia, 155, 1297
Diffusion lung capacity for carbon monoxide (DLCO), 564
Digital nerve blocks, 1020

Dihydropyridine calcium channel blockers, 258
Diltiazem, 378t, 1276t
Dilutional hyperchloremic acidosis, 1181
Dimenhydrinate, 277t
Diphenhydramine (ethanolamine), 277, 277t, 626
2,3-Diphosphoglycerate, 523, 525, 845
Diprifusor, 182
Direct and indirect laryngoscopy and intubation, 324—331
direct laryngoscopy, 324-326
intubation, 324
nasotracheal intubation, 329-330
orotracheal intubation, 326-329
Direct Coombs test, 1201
Direct current (DC) cardioversion, 1313
Direct myocardial depressant, 179
Discharge criteria, 951-952
Discogenic pain, 1067
Discography, 1068
Disopyramide, 378t
Disorder of descent, 879
Disseminated intravascular coagulation (DIC), 612, 881, 885, 1217
Dissociative anesthesia, 177
Distribution, 140-142
Diuretics, 390t, 667-669
carbonic anhydrase inhibitors, 669-670
correction of metabolic alkalosis in edematous patients, 670
reduction of intraocular pressure, 670

loop, 667-668
edematous states (sodium overload), 667
evaluation of acute oliguria, 667
hypercalcemia, treatment of, 668
hypertension, 667
hyponatremia correction, 668
intravenous dosages, 668
oliguric kidney failure to nonoliguric failure, conversion of, 668
side effects, 668
osmotic, 666-667
potassium-sparing, 669
aldosterone antagonists (spironolactone and eplerenone), 669
noncompetitive potassium-sparing diuretics, 669
therapy, 1183
thiazide/thiazide-like, 668-669
edematous disorders (sodium overload), 668
hypercalciuria, 668
hypertension, 668
nephrogenic diabetes insipidus, 668
Diverting (sidestream) capnographs, 122
DNA synthesis, 162
Dobutamine, 248, 471, 1276t
Documentation, 301-304
American Society of Anesthesiologists' physical status classification, 297t anesthetic plan, 297
intraoperative anesthesia record in, 302-303
postoperative notes in, 303, 303t preoperative note in, 301-302
Dofetilide, 376t, 378t
Dopamine, 247-248
Dopamine (DA) receptors, 244
Doppler device, 107
Doppler echocardiography, 361f
Doppler effect, 83
Doppler principle, 114
Doppler probe, for arterial blood pressure monitoring, 83-84, 84f
Doppler technology, 83-84
Dorsal column stimulation, 1105
Dorsal root ganglion (DRG) stimulation, 1105, 1106f
Dorsalis pedis artery, blood pressure monitoring via, 87
Dorsolateral funiculus, 1060
Dose-response relationship, 144, 146f
Double effect principle, 1326
Double-burst stimulation (DBS), 136, 204
Double-circuit design ventilators, 69-71, 71f

Double-circuit system, 69-71, 71f
Double-lumen bronchial tubes
complications of, 562
description of, 558-562, 560f-561f, 561¢
placement of, 559-562
Down syndrome, 925-926
Doxapram (Dopram), 289
clinical uses, 289
dosage, 289
drug interactions, 289
mechanism of action, 289
side effects, 289
Doxepin, 1077t
Drager oxygen failure protection device (OFPD), 54f
Draw-over anesthesia, 35-36, 36f, 36t
Dronedarone, 378t
Droperidol, 285, 1220
Drowning, 1309-1310
Drugs. See also specific drug
classes of, 275
molecules, 141
pediatric dosages for, 901t-905t
receptors, 147-148
toxicity, complications of, 994
Dual air—oxygen flowmeters, 1334-1335
Duloxetine, 1079
Duragesic patch, 1084
Dye dilution, 106
Dye indicator curve, 106
Dysesthesia, 1050t
E

intraoperative management
facial nerve identification, 799
hemostasis, 798
nausea/vomiting, 799
nitrous oxide, 798
postoperative vertigo, 799
oral surgical procedures, 798-799
Eaton—Lambert myasthenic syndrome, 202
Echo probe, 112f
Echocardiography, 108-115, 109f-114f, 933-934
Echothiophate, 776
Ectopic hyperparathyroidism, 762
Edrophonium, 227
clinical considerations, 227
dosage and packaging, 227, 227t
molecular structures, 226f
physical structure, 227
Edrophonium test, 640
Ejection fraction (EF), 357—360
Elderly. See also Geriatric anesthesia
&-blockers in, 930
preoperative assessment in, 930-933, 935t
trauma in, 838
Electric potential, 125
Electrical safety
electrical shock protection in, 17—20, 18f
electrocution risk in, 17
isolation transformer and monitor, circuit diagram, 19f, 20f
line isolation monitor, 21f
surgical diathermy in, 20-22
Electrocardiograms (ECGs), 119, 756
Electrocardiography (ECG), 90-92, 92f, 96f
during cardiac surgery, 92f
clinical considerations, 91-92
hypokalemia findings, 1153f indications and contraindications, 90
techniques and complications, 90-92
Electroconvulsive therapy (ECT), 631
Electrode, silver chloride ECG, 91f
Electroencephalogram (EEG), 124, 127, 174, 193
Electroencephalography (EEG), 124-127
clinical considerations, 127
indications and contraindications, 124-125
techniques and complications, 125-127
Electrolyte disorders, 406, 1134
extracellular fluid, 1135-1136
fluid compartments, 1134-1135
interstitial fluid, 1136
intracellular fluid, 1135
intravascular fluid, 1136
nomenclature of solutions, 1134
molarity/molality/equivalency, 1134

osmolality, 1134
osmosis, 1134
tonicity, 1134
Electromyography, 1063-1064
Electronic flowmeters, 54
Electronic medical equipment, 17
Electronic sensor, 63
Electronic vaporizers, 57, 59
desflurane vaporizer, 57, 59
Electrosurgical burn, 21f
Electrosurgical units (ESUs), 20
Emergency cardiac care (ECC), 1259, 1262f
Emergency (E-cylinder) cylinder of oxygen, 11, 12t
Emesis, 778
EMLA, 267, 912
Emphysema, 542
Employee identification (ID) badges, 29
Empyema, 1319t
Enalaprilat, 259, 394t
Encephalitis, 631, 1320t
End-diastolic point (EDP), 357
Endocarditis, 426t, 1320t
Endocrine disease adrenal gland, 764
glucocorticoid deficiency, 765-766
glucocorticoid excess, 765
Endocrine disease (Cont.)
carcinoid syndrome, 769-770
case discussion, 770
obesity, 767—769
pancreas diabetes mellitus, 754-758
physiology, 754
parathyroid glands hyperparathyroidism, 762—763
hypoparathyroidism, 763—764
physiology, 761-762

hyperthyroidism, 759-760
hypothyroidism, 760—761
physiology, 758-759
End-of-life care, 1326
Endoscopy, 788
intraoperative management, 788—789
laser precautions, 789-791
preoperative considerations, 788
sinus surgery, orbital fracture, 792f
Endothelium-derived factors, 363. See also specific factors
Endothelium-derived relaxing factor, 255
Endotracheal intubation, 319-320
flexible fiberoptic bronchoscopes, 323-324
tracheal tubes, 319-320
Endotracheal tube, 797f
End-plate depolarization, 203
End-plate potential, 201
End-systolic point (ESP), 357
Energy-consuming sodium—potassium pump, 262
Enhanced recovery after surgery (ERAS), 296, 1112
Enhanced recovery programs (ERPs)
anesthetic management-related factors, 1116
evolution of, 1112-1115
implementation, 1129-1130, 1130f intraoperative period, 1117
antibiotic prophylaxis, 1117
antithrombotic prophylaxis, 1117
surgical stress response, 1117-1121
perioperative elements, 1113f preoperative period
food/fluid intake, guidelines, 1116-1117
patient education, 1116
risk assessment, 1116
smoking/alcohol cessation, 1116
Enteral nutrition (EN), 1223, 1227
Enterohepatic recirculation, 143
Enzymatic pathways, 717
Ephedrine, 247
Epidural anesthesia, 975, 980-987
activating, 984
agents for, 985t, 985-986
catheters used in, 983
factors affecting level of, 984-985
failed epidural blocks, 986-987
local anesthetic pH adjustment, 986
needles for, 983
techniques for, 983-984
Epidural blockade, 1104t
Epidural catheters, 983, 1119f
Epidural hematoma, 623, 831, 970
Epidural injection, 1102f

Epidural morphine, 875
Epidural needle, 982f
Epidural steroid injections, 1101
Epiglottitis, acute, 924
Epinephrine, 246-247, 471, 893, 968, 1274t
desflurane and, 167
eye drops, 777
synthesis, 242f
Eplerenone, 669
Epstein—Barr virus, 1203
Equilateral triangle, 987
Equivalency, 1134
Escitalopram, 1077t
Esmolol, 250, 378t, 394t
Esophageal Doppler, 107
Esophageal perforations, 1241
Esophageal stethoscopes, 119-120, 120f
Esophageal surgery, 578-579
Esophageal—tracheal Combitube (ETC), 317-319
Esophagogastroduodenoscopy (EGD), 1325
Essential hypernatremia, 1142
Ethylenediaminetetraacetic acid (EDTA), 986
Etidocaine, 265t
Etomidate, 180-181, 591, 682, 1220
doses of, 178t
drug interactions, 181
effects on organ systems, 180-181
cardiovascular, 180
cerebral, 181
endocrine, 181
respiratory, 181
mechanisms of action, 180
pharmacokinetics, 180-181
absorption, 180
biotransformation, 180
distribution, 180
excretion, 180
structure of, 177f structure—activity relationships, 180
Evoked potentials (EPs), 127-130
clinical considerations, 130
indications and contraindications, 127, 129
motor-evoked potentials (MEPs), 129
somatosensory-evoked potentials (SEPs), 129-130
techniques and complications, 129-130
Excitation—contraction coupling, 348-350
Excretion, 143
Exocytosis, 240
Expiratory positive airway pressure (IPAP), 1350
Exposure-response relationships, 145-146
Extracorporeal membrane oxygenation (ECMO), 546, 1260

Extracorporeal shock wave lithotripsy (ESWL), 701-702
Extraction ratio, 143
Extravascular lung water (EVLW), 103-104
Extrinsic asthma, 538
Extubation techniques, 333
Eye surgery, 778
F
FABERE sign, 1066
Face mask clear adult, 313f
design and technique, 313-314
Rendell—Baker—Soucek pediatric, 314f use of, 313-314
Facet syndrome, 1070
Facial nerve block anatomy, 1088
complications, 1088
indications, 1088
techniques, 780f, 781
Facilitated diffusion, 1136
Facioscapulohumeral dystrophy, 645
Falciform ligament, 716
Falls, 838
False negatives, 300t
False positives, 300t
Famotidine, 279t
Fascia iliaca block, 1027f
Fasting, 910
Fast-track surgery programs, 1118t
Fat embolism syndrome, 806
Fatty acids, 718
Femoral artery, blood pressure monitoring via, 87
Femoral nerve, 1024f
Femoral nerve block description of, 1023-1025
ultrasound, 1026f using nerve stimulation, 1025f
Femoroacetabular impingement (FAI), 812
Fenoldopam, 248, 258, 1324
clinical considerations, 248
clinical uses, 258
dosing and packaging, 248
effects on organ systems, 258
hypertension treated with, 394, 394t mechanism of action, 258
metabolism, 258
Fentanyl, 5, 181, 189f, 190, 190t, 869, 919, 1291. See also Opioids transdermal, 1084
Ferguson reflex, 853
Fetal asphyxia, 889
Fetal circulation, 855f
Fetal distress, 879t
Fetal heart rate (FHR)
monitoring of, 886-887
periodic changes in, 888f
Fetal physiology, 854-857
Fetal resuscitation accelerations, 887
baseline heart rate, 887
baseline variability, 887

deceleration patterns, 887-888
fetus, treatment, 889
monitoring of fetal heart rate (FHR), 886-887
Fetal/neonatal circulation, 856f
Fetal/neonatal resuscitation, 886-894
Fetus, at birth, 857
Fever, 1301t
Fexofenadine, 277t
Fiberoptic bronchoscope, 324f, 331f
Fiberoptic intubation (FOI), 330
Fibroblast growth factor 23 (FGF23), 761
Fick equation, 525
Field block, 1000f
Filtration fraction (FF), 660

crew resource management, 26
extinguishers, 25
laser safety, 25
in operating rooms, 22, 24t prevention algorithm for, 23f
radio frequency identification (RFID) technology, 29
safety interlock technology, 27—28
workflow design, 28
prevention and preparation, 22-25
tracheal tube, 24t
Fire triad, 22
Fire triangle, 22
First-degree burns, 834
First-order neurons, 1052
Fish oil, 1226
Fixation error, 1240
Flecainide, 375t, 378t, 1276t
FloTrac, 107
Flow control circuits, 52—59
common (fresh) gas outlet, 59
flow valves and meters, 53-55
oxygen supply failure protection devices, 53
pressure regulator, 52
vaporizers, 55—59
Flow valves and meters, 53-55
minimum oxygen flow, 55
oxygen/nitrous oxide ratio controller, 55
Flow-cycled ventilators, 1339
Flowmeters, 53
malfunction, 54
sequence of, 54f
Flow—volume loops, 125f, 574f
Fluid and electrolyte disorders, 624t
Fluid compartments, 1135t
Fluid creep, 835
Fluid disturbances, 1134
antidiuretic hormone, 1140
calcium disorders. See Calcium disorders
fluid compartments, exchange, 1136
diffusion through capillary endothelium, 1137
diffusion through cell membranes, 1136-1137
hyperosmolality/hypernatremia
anesthetic considerations, 1143-1144, 1147-1148
central diabetes insipidus, 1142
clinical manifestations, 1142, 1146
Fresh gas inlet, 38
Fresh gas outlet, 59
Front-end kinetic models, 144
Fuel cell, 124. See also Galvanic fuel cell
Functional neurosurgery, 608
Functional residual capacity (FRC), 314, 503-504, 536, 555, 845, 898, 1330
body habitus, 504
diaphragmatic tone, 504
lung disease, 504
posture, 504
sex, 504

G proteins, 241
Gabapentin, 196, 289, 1078t, 1124, 1125t
Gabapentinoids, 1124, 1125t
Galvanic fuel cell, 61, 124
Gamma-aminobutyric acid (GABA), 626
activated chloride channel, 157
binding sites, 937
receptor system, 180
Ganglion impar block anatomy, 1099
complications, 1099
indications, 1099
technique, 1099
Gantacurium, 211t-212t, 219
Gas gangrene, 1321t
Gas reservoirs, 1332-1333
Gas supply, in anesthesia machines, 51-52
cylinder inlets, 51-52
pipeline inlets, 51
Gas systems, medical, 10-13
air, 13
carbon dioxide, 13
cylinders, characteristics of, 12t nitrogen, 13
nitrous oxide, 12—13
oxygen, 10-12, 11f, 12f vacuum system, 13

exchange, effects of anesthesia, 518-519
vapor pressure, 56f
Gasserian ganglion, 1052
Gasserian ganglion block, 1085, 1086f
Gastric distention, 921
Gastroesophageal reflux disease (GERD), 278, 299, 579, 846
Gastrointestinal hemorrhage, 1325-1326
Gastrointestinal receptors, 225
Gauze-covered mask. See Schimmelbusch mask
Gelatins, 1194
Generalized seizures, 624
Genitourinary surgery, 696
case discussion, 711—713

intraoperative considerations, lithotomy position, 696-698
preoperative considerations, 696
lithotripsy, 701-703
prostate, transurethral resection of intraoperative considerations, 698-701
preoperative considerations, 698
Genitourinary surgery (Cont.)
renal transplantation, 710-711
upper ureter/kidney, noncancer surgery of, 703
urological malignancies, surgery for, 703-710

Geriatric anesthesia, 929. See also Elderly age-related anatomic/physiological changes
cardiovascular system, 933-935
common diseases, 934t gastrointestinal function, 937
metabolic/endocrine function, 936
musculoskeletal, 939
nervous system, 937-938
renal function, 936
respiratory system, 936
age-related pharmacological changes inhalational anesthetics, 939-940
muscle relaxants, 940
nonvolatile anesthetic agents, 940
case discussion, 940-942
delirium prevention, 939t
Doppler study of diastolic inflow, 935f elderly people and infants, 930t
predisposing/precipitating factors for delirium, 938t
Gerota fascia, 708
Glasgow Coma Scale (GCS) score, 610, 610t
Gleason score, 704
Glidescope, 323f
Global tissue hypoperfusion, 824
Global-end diastolic volume (GEDV), 103
Glomerular filtration pressure, 654
Glomerular filtration rate (GFR), 659-660, 676, 1149
Glossopharyngeal block, 1088, 1089f
Glossopharyngeal nerve, 308
Glottic edema, 1295-1296
Glucocorticoids, 539, 764, 765
Gluconeogenesis, 718
Glucose homeostasis, 901
Glutamate, 1060
Glycolic acid, 1180
Glycopyrrolate, 788
clinical considerations, 236
dosage and packaging, 236
physical structure, 236
Goal-directed fluid therapy (GDFT), 1196, 1324
G6PD deficiency, 1209t
Granulocyte colony-stimulating factor (G-CSF), 1200, 1324
Granulocyte transfusions, 1200
Granulocyte-macrophage colony-stimulating factor (GM-CSF), 1200, 1324
Graves’ disease, 759
Great arteries, transposition of, 432
Guanethidine, 390t

Guanosine triphosphate (GTP), 242
Guest-host complex, 228
Guillain-Barré syndrome (GBS), 629

Hageman factor, 1249
Halothane biotransformation and toxicity, 164-165
chemical structure of, 159t clinical pharmacology of, 163-165
contraindications, 165
drug interactions, 165
effects on organ systems cardiovascular, 163
cerebral, 164
hepatic, 164
neuromuscular, 164
renal, 164
respiratory, 163-164
hypoxic model, 164
minimum alveolar concentration of, 158, 159t physical properties of, 163
vapor pressure of, 56
vaporizer, 57
Halothane hepatitis, 164
Halothane—caffeine contracture test, 1216
Harris—Benedict equation, 1224
Head trauma, 611
Glasgow Coma Scale (GCS) score, 610, 610t surgery for, 609-612
diagnostic studies in, 611
intraoperative management in, 611-612
intubation in, 611
preoperative management in, 610-611
Headache, 1073-1075
classification of, 1074t cluster, 1074
migraine, 1074
temporal arteritis, 1074-1075
tension, 1073-1074
trigeminal neuralgia, 1075
Head/neck cancer surgery intraoperative management, 793-796
preoperative considerations, 792—793
Health care-associated pneumonia, 1319t

cardiac action potentials, 344-346
cardiac cycle in, 350-351
cardiac impulse, initiation and conduction, 346-348
innervation of, 350
Starling's law, 354f ventricular function assessments, 357-361
ventricular performance in, 353-357

New York Association functional classification, 414t pressure—volume loops in, 416f
Heart failure, 412
biventricular, 475
pathophysiology of, 368-370
treatment of, 413f
Heart rate, 353. See also Arrhythmias diastolic time and, 367f
Heat and moisture exchangers (HMEs), 915
schematic diagram of, 67f units, 66
Heat conservation, 44
Helicobacter pylori infection, 278
Helium—oxygen (heliox) mixtures, 1335
Hematochezia, 1325
Hemiarthroplasty, 808-809
Hemoglobin dissociation curve, 523, 524f
Hemolytic reactions, 1201-1202
acute, 1201
delayed, 1201-1202
Hemoptysis, 416
Henderson-Hasselbalch equation, 1173
Henry's law, 523
Heparin, 547
dose-response curve, 466f
Heparin-induced hyperkalemia, 1155
Heparin-induced thrombocytopenia (HIT), 463
Heparinization, 710
Hepatic artery, 717
Hepatic blood flow, 717f, 968
Hepatic encephalopathy, 742
Hepatic enzymes, 178
Hepatic function tests, 167
Hepatic physiology bile, 722t, 722-723
biliary system, 722f case discussion, 726—730
coagulation factors, 720t coagulation test abnormalities, 724t
drug metabolism, 720-722
functional anatomy, 716-717
hepatic blood flow, 717, 717f hepatic function, anesthesia effects on, 725-726
hepatic lobule, 716f hepatic venous compliance, 718f
hepatocytes, 719f intrinsic/extrinsic coagulation pathways, 721f
liver tests, 723-725
abnormalities in, 724t
blood ammonia, 724
point-of-care viscoelastic coagulation monitoring, 725
prothrombin time, 724
serum albumin, 724

serum alkaline phosphatase, 723-724
serum aminotransferases, 723
serum bilirubin, 723
metabolic functions, 717-720, 720t
postoperative jaundice, 726t
reservoir function, 717
thromboelastograph tracings, 725f
Hepatic surgery, 744—747
Child’s classification for, 739t
Hepatitis, 735

drug-induced, 735-737
intraoperative considerations, 737
viral, 735
chronic, anesthetic management, 737
hepatitis C, 1253-1254
post-transfusion viral infections, 1203
Hepatitis B surface antigen (HBsAg), 735, 1253
Hepatopulmonary syndrome, 741t
Hepatorenal syndrome, 742
Hering—Breuer inflation reflex, 530
Hetastarch, 1194
“High-dose” opioid anesthesia, 460-461
High-efficiency particulate filters (HEPA), 16
High-frequency jet ventilation, 789
High-frequency ventilation (HFV), 1345
Hip arthroplasty
intraoperative management, 809-812
preoperative considerations, 809
Hip arthroscopy, 812
Hip dislocation, 812
Hip fracture
blood loss, 808f intraoperative management, 808-809
preoperative considerations, 807
Histamine (H,) receptor, 272
Histamine-receptor antagonists, 276-279
H,-, 277-278
H,-, 278-279
physiology of, 276-277
cardiovascular, 277
dermal, 277
gastrointestinal, 277
immunological, 277
respiratory, 277
History, of anesthesia, 2-6
inhalation, 2—3
intravenous anesthesia, 4—5
induction agents, 4
neuromuscular blocking agents, 4
opioids, 4-5
local and regional, 3-4
specialty, evolution of, 5-6
American origins, 5—6
British origins, 5
official recognition, 6
Hoarseness/inspiratory stridor, 809
Horner syndrome, 1088
Hospital information systems, 51
Hot-wire anemometer, 63
H,-receptor antagonists, 277-278
clinical uses, 278
dosage, 278
drug interactions, 278
mechanism of action, 277—278
properties of, 277t
H,-receptor antagonists, 278-279, 682
dosage, 279
drug interactions, 279
mechanism of action, 278
side effects, 278-279

Human immunodeficiency virus, 1319t
Humidifiers, 66
Humidity, 44
in operating rooms, 16
Hyaluronidase, 780
Hydralazine, 257-258, 394t
Hydrochloric acid, 276f
Hydrocortisone, 766, 1080t
Hydromorphone, 707, 1082
5-Hydroxyindoleacetic acid (5-HIAA), 284
5-Hydroxytryptamine (5-HT)3 receptor antagonists, 282-285
clinical uses, 284
mechanism of action, 284
serotonin physiology, 282, 284
cardiovascular, 284
gastrointestinal, 284
hematological, 284
respiratory, 284
side effects, 284—285
Hydroxyzine, 277t
Hyperalgesia, 1050t
Hyperammonemia, 699
Hyperbaric oxygen therapy, 1335
Hyperbaric solutions, 978
Hypercalcemia, 1289
Hyperchloremic metabolic acidosis, 1180
Hyperechoic, 1001
Hyperglycemia, 756
chronic, 756
Hyperglycinemia, 699
Hyperkalemia, 686
anesthetic considerations, 1157
causes of, 1154t clinical manifestations, 1156
decreased renal excretion, 1155
electrocardiographic effects, 1156f extracellular movement of potassium, 1155
increased potassium intake, 1155-1156
succinylcholine as cause of, 209-210
treatment of, 1156-1157
Hyperkalemic cardiac arrest, 209
Hyperkalemic paralysis, 647
Hypermagnesemia, 686, 1289
Hypernatremia, 1140-1144
increased total body sodium content, 1142, 1145
low total body sodium content, 1141, 1144-1145
nephrogenic diabetes insipidus, 1142
normal total body sodium content, 1141-1142, 1145-1146
treatment of, 1142-1143, 1146-1147

Hyperosmolar nonketotic coma, 755
Hyperparathyroidism, 763t
Hyperpathia, 1050t
Hyperphosphatemia, 1159, 1161
Hypersensitivity reactions, type I, 1246t, 1246-1248
Hypertension, 385-389
complications of, 385
intraoperative management, 393-394
anesthetic agents, choice of, 393-394
induction of, 393
monitoring, 393
objectives, 393
long-term treatment, 387-388
parenteral agents for, 394t pathophysiology, 385-389
postoperative management, 394—395
during pregnancy, 883
preoperative management, 389-393
history of, 389
physical examination and laboratory evaluation, 391-392
premedication, 393
treatment of, 394t
Hypertensive emergency, 385
Hyperthermia, 132
Hyperthyroidism, 759
Hypertonic solution, 1134
Hypertrophic cardiomyopathy (HCM), 413-414, 969
Hypertrophied interventricular septum, 414f
Hypervolemia, 1150
Hypesthesia, 1050t
Hypoalbuminemia, 736
Hypoalgesia, 1050t
Hypobaric solution, 978
Hypochondriasis, 1064t
Hypoglycemia, 736, 755
Hypokalemia, 1151-1154, 1152t, 1153f anesthetic considerations, 1154
clinical manifestations, 1152—1153
decreased potassium intake, 1152
increased potassium losses, 1152
intracellular movement of, 1152
treatment of, 1153-1154
Hypokalemic periodic paralysis, 647
Hypokinesis, 356
Hypomagnesemia, 393, 1163-1164
Hyponatremia, 1144, 1144t, 1147f
Hypoosmolal hyponatremia, 1144t
Hypoparathyroidism, 760, 763t, 763-764
Hypoplastic left heart syndrome, 432
Hypotension, 365, 871
atracurium as cause of, 216
in head trauma surgery, 611

Hypotensive agents, 253-260. See also specific agents calcium antagonists, 258-259
case discussion on controlled hypotension, 259-260
diastolic pressure, 254f fenoldopam, 258
hydralazine, 257-258
nitroglycerin, 256-257
sodium nitroprusside, 255-256
Hypothermia, 132, 207, 594, 1214-1215, 1292-1293
deleterious effects of, 1214, 1214f, 1214t differential diagnosis of, 1219-1220
postoperative considerations, 1215
unintentional, during general anesthesia, 1214f
Hypotonic solution, 1134
Hypoventilation, 1296-1297
Hypovolemia, 1190t, 1298
Hypoxemia description of, 1297-1298
interventions, 569
Hypoxia, 857
acute, 956
management of, 569
Hypoxic pulmonary vasoconstriction (HPV), 519, 556-557
I
Ibutilide, 378t, 1277t
Ileal conduit, 706
Imidazobenzodiazepine. See Flumazenil
Imipramine, 1077t
Immunoglobulin E (IgE), 538, 1247f
Immunoglobulin G (IgG) antibodies, 638
Immunomodulating nutrients, 1226
Implantable cardioverter-defibrillators (ICDs)
description of, 409, 411, 945, 947
preoperative considerations, 950f
Incisura, 351
Induction agents. See also specific agents
barbiturates, 591
benzodiazepines, 591
cerebral physiology affected by, 590-592
etomidate, 591
history of, 4
ketamine, 591-592, 592f
opioids, 591
propofol, 591
Induction technique, 604
Infectious croup, 924
Inferior alveolar nerve block, 1087f
Infraclavicular block, 1010-1012, 1011f
coverage and anatomy, 1011f
using nerve stimulation, 1012f
Infusion pumps, 1004f

portable, 1004f
Inhalation anesthesia. See Anesthetics, inhalation; specific agents
Inhalational-based anesthetic, 1288
Inhibitory adrenergic pathways, 1060
Inotropic agents, 471t
Inspiratory concentration (FI), 150
Inspiratory positive airway pressure (IPAP), 1350
Institute for Health Care Improvement (IH), 1358
Insufflation, 34f/-35f, 34-35, 35f
Insulin, 754
bioavailability characteristics, 759t
effects of, 754t
Intensive care unit (ICU), 66
Intercostal nerve block, 1042f
Intercostobrachial nerve
cutaneous innervation, 1021f
description of, 1000f
Intercostobrachial nerve block, 1022f
Intermittent pneumatic compression (IPC), 806
Intermittent positive-pressure ventilation (IPPV), 36
Internal cardioverter-defibrillator (ICD), 399
International 10-20 system, 125f
International Anesthesia Research Society (IARS), 5
International normalized ratio (INR), 415, 807
Interscalene block, 1006f
using nerve stimulation, 1006/—1007f
Interscalene groove, 1007
Interthreshold range, 1214
Interventional pain therapy, 1077
acetaminophen, 1077
Q5-adrenergic agonists, 1081
anticonvulsants, 1080-1081
antidepressants, 1079
antispasmodics/muscle relaxants, 1079-1080
botulinum toxin, 1084
corticosteroids, 1080
local anesthetics, 1081
neuroleptics, 1079
nonsteroidal antiinflammatory drugs (NSAIDs), 1078-1079
opioids, 1081-1084
Intestines, malrotation of, 920-921
Intraaortic balloon counterpulsation, 470f, 1313
Intraaortic balloon pump (IABP), 470
Intraarterial blood pressure monitoring, 884
Intracerebral hemorrhage, 623
Intracoronary steal, 256
Intracranial hypertension, 601-603
cerebral edema, 602

treatment of, 602-603
Intracranial pressure (ICP)
description of, 585, 588f, 588-589, 601, 831
monitoring of, 747, 832t
Intraglomerular mesangial cells, 653
Intraocular pressure (IOP), 774
anesthetic agents, effect of, 775t cardiac/respiratory variables, effect of, 774t
strategies to prevent, 782t
Intraoperative bronchospasm, 541
Intraoperative hypertension, 192
Intraoperative nerve monitoring, 793
Intraosseous infusions, 1271f
Intrapulmonary shunt, 515
Intrapulmonary shunting, 505
Intrathecal morphine, 866
Intrathoracic blood volume (ITBV), 104
Intrathoracic drug deliver system, 1083
Intrathoracic thermal volume (ITTV), 104
Intrauterine asphyxia, 891
Intravenous anesthesia. See also specific agents; specific surgeries

opioids, 4—5
Intravenous bisphosphonates, 762
Intravenous fluid therapy, 1191-1192, 1272
colloid solutions, 1192-1194
crystalloid solutions, 1191-1192, 1193t
Intravenous heparin, 299
Intravenous immune globulin, 641t
Intravenous regional anesthesia, 1022f
Intravenous regional block, 1099-1100
Intrinsic asthma, 538
Intubation complications of, 334t
conditions associated with, 336t
Invasive monitoring, 911
Ionized (charged) drugs, 140
Ionizing radiation in operating rooms, 16—17
radiation safety, principle of, 16
Iowa trumpet, 865
Ischemia, 406f
Ischemic heart disease, 395-399
arrhythmias, pacemakers, and internal cardioverter-defibrillator management,

chronic stable angina, 395-396
intraoperative management, 403-404
monitoring, 404—406
electrocardiography, 404-405
hemodynamic monitoring, 405
transesophageal echocardiography, 404—406
preoperative considerations, 395
preoperative management, 399-403
coronary angiography, 403
echocardiography, 403
exercise electrocardiography, 400, 402
history of, 400
Holter monitoring, 400
myocardial perfusions scans and imaging techniques, 402-403
physical examination and routine laboratory evaluation, 400
premedication, 403
specialized studies, 400-403
treatment of, 396—399
&-adrenergic blocking agents, 396-398
calcium channel blockers, 398
combination therapy, 399
nitrates, 399
unstable angina, 395
Ischemic optic neuropathy (ION), 1244-1245
Ischemic preconditioning, 158
Isoflurane biotransformation and toxicity, 165
chemical structure of, 159t clinical pharmacology of, 165-166
contraindications, 166
minimum alveolar concentration of, 158, 159t drug interactions, 166 effects on organ systems cardiovascular, 165 cerebral, 165 hepatic, 165 neuromuscular, 165 renal, 165 respiratory, 165 physical properties of, 165 Isolation transformer, 18, 19f
Isoproterenol, 248
Isotonic solution, 1134
Istaroxime, 349
J
Jugular venous bulb saturation, 131
Juxtamedullary nephrons, 656

Ketamine, 177-180, 431, 591-592, 592f, 682, 760, 1220
cardiac tamponade uses of, 483
doses of, 178t drug interactions, 180
effects on organ systems, 178-180
cardiovascular, 178-179
cerebral, 179-180
respiratory, 179
in elderly, 940
hypertension caused by, 393
labor applications of, 865
mechanisms of action, 177
perioperative uses of, 1125t pharmacokinetics, 178
absorption, 178
biotransformation, 178
distribution, 178
excretion, 178
structure of, 177f structure—activity relationships, 177-178
Ketorolac, 286-287
clinical uses, 286
dosage, 287
drug interactions, 287
mechanism of action, 286
NSAID adjuvant drugs, 286-287
side effects, 287

donor, 710
function, 900. See also Renal function physiology of. See Renal physiology
Kidney disease, 675-693, 677t anesthetic agents, 681-684
case discussion on, 690-692
kidney failure, 684-689
mild to moderate renal impairment, 689-690
renal function evaluation, 676-681
Kidney failure, 487, 684-689. See also Acute kidney injury

management priorities in, 684t anesthesia maintenance, 688-689
fluid therapy, 689
induction, 688
intraoperative considerations, 688-689
manifestations of, 685-687
cardiovascular, 686
endocrine, 687
gastrointestinal, 687
hematological, 686
metabolic, 685-686
neurological, 687
pulmonary, 686
monitoring, 688
premedication, 688
preoperative considerations, 684-688
preoperative evaluation, 687-688
King laryngeal tubes (LTs), 319, 319f supralaryngeal device, 821, 822f
King—Denborough syndrome, 1217
Kiva VCF vertebral augmentation, 1107, 1108f
Klotho, 761
Knee arthroscopy, 812
Knee replacement, 814f
Knee surgery intraoperative management, 812-813
postoperative pain management, 813
preoperative considerations, 812
Kupffer cells, 716, 723
Kyphoplasty, 1107
L
Labetalol, 394t
Labor and delivery. See also Pregnancy anesthetic agents, 853
&,-adrenergic agonists, 854
ergot alkaloids, 854
inhalational agents, 853
magnesium, 854
oxytocin, 853-854
parenteral agents, 853
prostaglandins, 854
regional anesthesia, 853
vasopressors, 853
course of, 852f physiology of, 851-852

spinal opioid dosages for, 866t
Laerdal resuscitator, 45f
Lambert—Beer law, 120
Lambert—Eaton myasthenic syndrome (LEMS), 563, 643
Lamotrigine, 1078t
Laparoscopic radical prostatectomy, 705
Laplace’s law, 355, 503
Laryngeal mask
illustration of, 317f
insertion of, 318t
Laryngeal mask airway (LMA), 316-317, 318t, 567, 608, 876, 907, 944, 1265,

advantages and disadvantages, 318t
variations in design, 316
Laryngoscope, 321
blades, assortment of, 322f
rigid, 321f

complications of, 333-335
airway instrumentation, physiological responses, 335
tracheal tube malfunction, 336
tracheal tube positioning errors, 334-335
direct, 324—336
glottis, 326f
indirect, 321-323
Laryngospasm, 335, 336, 1295-1296

cartilaginous structures, 309f
laryngeal nerve injury, effects of, 310t
Laser airway surgery, 24

Laser endotracheal tube selection, 25
Laser precautions, 780—791
Lateral decubitus position, 554f, 554-555, 555f
awake state, 554f, 554-555
induction of anesthesia, 555, 555f
positive-pressure ventilation, 555
Lateral femoral cutaneous nerve block, 1027f
Lateral spinothalamic tract, 1055
Lateral thoracotomy, 568f
Laudanosine toxicity, 216
L-Cysteine, 228
Leadership, 26
Leakage current, 17
Lean methodology, 28-29
Lecithin/sphingomyelin ratio, 882
LeFort I, 797f
LeFort osteotomies, 796
Left anterior descending artery (LAD), 365
Left ventricular assist device (LVAD), 480, 480f
Left ventricular end-diastolic pressure (LVEDP), 97, 355, 367
Left ventricular hypertrophy (LVH), 385
Left ventricular outflow tract (LVOT), 413-414
Left ventricular systolic pressure (LVSP), 114f
Left ventricular vent, 445
Leukocyte-depleted blood cardioplegia, 449
Leukocytosis, 1323
Leukoreduction, 1199
Levofloxacin, 700
Leyden—Mobius dystrophies, 645
LiDCO™ system, 106
Lidocaine, 265t, 269t, 270, 375t, 378t, 778, 874, 985t, 1081, 1120, 1124, 1125¢,

Ligand-gated ion channels, 157
Light-emitting diode, 121
Limb-girdle muscular dystrophy, 645
Limbic encephalitis, 643
Lindegaard ratio, 614
Line isolation monitor, 18, 21f
electrical safety, 21f
Line pressure, 11
Lingual nerve block, 1087f
Lipid-soluble barbiturates, 173
Lipid-soluble opioids, 190
Liquid oxygen storage system, 11, 12f
Lissauer's tract, 1052
Lithotomy position, 697f
Liver abnormalities, 723
failure due to vecuronium, 217-218
metabolic functions of, 720t physiology of. See Hepatic physiology
in protein metabolism, 719

cardiopulmonary dysfunction, 740
cirrhosis, 737—744, 738t, 740t coagulation, 734
coagulation test abnormalities, 734t drugs and substances, 736t
enzymatic pathways, 717
hepatic surgery, 744—747
Child's classification for, 739t

acute drug-induced, 735-737
viral, 735
hepatopulmonary syndrome, 741t
Model for End-stage Liver Disease (MELD) score, 745, 746f portopulmonary hypertension, 741t
Liver tests, 723-725
abnormalities in, 724t blood ammonia, 724
Liver transplantation, 744—747
case discussion on, 748—751
contraindications, 745t indications for, 745t
living donor, 727
pediatric, 727
Living donor transplantation, 727
Local anesthetic deposition, 1005f
Local anesthetic nerve blocks, 1084—1085
Long-term antihypertensive therapy, 392t
Loop diuretics, 390t, 667-668
hypercalcemia treated with, 668
hyponatremia treated with, 668

Low back pain syndromes, 1066
applied anatomy of, 1066-1067
arthritides, 1071
bacterial infections, 1070
buttock pain, 1067
cervical pain, 1070
congenital abnormalities, 1070
degenerative disc disease, 1067-1068
from facet syndrome, 1070
herniated/prolapsed intervertebral disc, 1068-1070
lumbar disc radiculopathies, 1068t lumbosacral joint sprain/strain, 1067
paravertebral muscle, 1067
spinal stenosis, 1069-1070
tumors, 1070
Low-dose intravenous ketamine, 865
Lower extremity peripheral nerve blocks femoral nerve block, 1023-1025
lateral femoral cutaneous nerve block, 1025-1026
lumbar/sacral plexus anatomy, 1022
obturator nerve block, 1026
posterior lumbar plexus block, 1028-1030
Lower gastrointestinal bleeding, 1325-1326
Low-molecular-weight heparin (LMWH), 547, 807, 867, 971-972
Lumbar epidural anesthesia, 964f
Lumbar epidural steroid injection, 1102f
Lumbar medial branch blockade, left, 1094f
Lumbar medial branch blocks anatomy, 1093
complications, 1093
indications, 1091
technique, 1093
Lumbar nerve roots, 1028
Lumbar neuraxial anesthesia awake/asleep, 972
Lumbar paravertebral nerve blocks, 1091
anatomy, 1091
complications, 1091, 1093
technique, 1091
Lumbar plexus, 1030f blocks, 1029f
Lumbar sympathetic block, 1098, 1099f anatomy, 1098
complications, 1098
indications, 1098
technique, 1098
Lumbar vertebrae, 962f, 965f
Lumbosacral plexus, 1023f
ventral rami, 1023f
Lung(s), 531
abscess of, 1319t
filtration and reservoir function, 531

metabolism, 531
nonrespiratory functions of, 531
Lung compliance, 503
Lung mechanics, 502-507
compliance in, 503
elastic resistance in, 503
lung volumes in, 503-507, 504f, 504t, 512f
closing capacity, 505, 505f-506f
functional residual capacity, 503-504
vital capacity (VC), 507
nonelastic resistances in, 507-508
tissue resistance, 508
pulmonary mechanics, effects of
on airway resistance, 511-512
anesthesia, 511-512
lung volumes and compliance, 511
respiratory pattern, 512
work of breathing, 512
surface tension forces, 503
work of breathing in, 508-510, 511f
Lung resection, 563-573
bronchiectasis, 565
bronchopleural fistulas, 572-573
considerations, 571-572
infection in, 565
intraoperative management, 565-570
induction of anesthesia, 567
maintenance of anesthesia, 568
monitoring, 567
positioning, 567-568
preparation, 565, 567
venous access, 567
lung abscesses, 572
massive pulmonary hemorrhage, 571-572
postoperative management, 570-571
general care, 570
postoperative analgesia, 570-571
postoperative complications, 571
preoperative management, 565
pulmonary cyst and bulla, 572
tumors treated with, 563-565
clinical manifestations, 563
evaluation for, 564—565
resectability and operability, 564
treatment of, 563—564
Lung transplantation, 577-578
indications for, 577t intraoperative management, 577-578
double-lung transplantation, 578
induction and maintenance, 577-578
monitoring, 577
posttransplantation management, 578
single-lung transplantation, 578
postoperative management, 578
preoperative considerations, 577
preoperative management, 577
Lymphoid hyperplasia, 925

Macintosh and Miller blades, 321
Macintosh blade, 325f
Magnesium, 377t, 1162, 1277t
Magnesium balance disorders, 1162-1164
case discussion, 1164-1166
hypermagnesemia, 1163
anesthetic considerations, 1163 causes of, 1163 clinical manifestations of, 1163 treatment of, 1163 hypomagnesemia, 1163-1164 anesthetic considerations, 1164 clinical manifestations, 1164, 1 = . Magnetic resonance imaging (MRI) scans, 603, 1 = . Malignant hyperthermia (MH), 1215-1221 clinical manifestations, 1216-1217 drugs that cause, 1216 t intraoperative considerations, 1217-1219 pathophysiology, 1216 postoperative considerations, 1219-1221 signs of, 1216 t, 1 = . Malingering, 1064 t, 1 = . Mandibular nerve, 1087-1088 1087-1088, 1 = . Mapleson circuits, 36-40, 37 t, 38 f -40 f, 44 adjustable pressure-limiting valve (pressure-relief valve, pop-off breathing tubes, 38 classification and characteristics of, 37 t components of, 36, 38 f, 38-39 fresh gas inlet, 38 performance characteristics of, 39-40 reservoir bag (breathing bag), 38-39, 39 f, 1 = . D circuit, 37 t, 39, 1 = . 602, 1 = . Mandibular nerve block, 1087 f, Mannitol,, 1 = . Malpositioned sensor, 121, 1 = . Mapleson systems, 36, 1 = . , 1 = Mapleson
Masks, 1332-1334
Mass casualty incidents, 838
Mass spectrometry, 123
Masseter muscle rigidity, 1218-1219
Massive hemoptysis, 571
Massive transfusion, 1204—1205
Master alarm panel, 15f
Maternal fever, 872
Maxillary nerve block, 1085-1087, 1087f
Maxillofacial reconstruction, 796—798
Maximal midexpiratory flow (MMEF) rates, 536
McGill Pain Questionnaire (MPQ), 1063
McGrath laryngoscope, 323f

Mean arterial pressure (MAP), 81, 364, 585
Mean transit time (MTT), 104
Mechanical ventilation, 1337
acute respiratory distress syndrome treated with, 1309
classification of, 1338t microprocessor-controlled ventilators, 1345
time-cycled ventilator cycle, 1339
discontinuing, 1347-1349
PSV, 1348-1349
SIMV, 1348
T-piece/CPAP, 1349
indicators of, 1337t patient care on
initial ventilator settings, 1346-1347
monitoring, 1347
sedation and paralysis, 1347
tracheal intubation, 1346
positive airway pressure therapy, 1349-1351
continuous positive airway pressure, 1349
CPAP vs. PEEP, 1349-1350
PEEP/CPAP, 1350-1351
positive-pressure ventilators, 1337-1346
respiratory care techniques, 1351-1352
ventilatory modes airway pressure release ventilation (APRV), 1344, 1344f
assist-control (AC) ventilation, 1343
continuous mandatory ventilation (CMV), 1343
differential lung ventilation, 1346
high-frequency ventilation (HFV), 1345
intermittent mandatory ventilation IMV), 1342, 1344f inverse I:E ratio ventilation (IRV), 1344
pressure control ventilation (PCV), 1342-1343
pressure support ventilation (PSV), 1343-1344
weaning from, 1348, 1348t
Mechanical ventilator, 51
Meclizine, 277t
Medial spinothalamic tract, 1055
Median nerve block at elbow, 1016f-1017f
at wrist, 1017f
Median nerve course, 1016f
Medical expulsive therapy (MET), 701
Medical gas systems. See also Gas systems, medical delivery of, 13-15, 14f
master alarm panel, 15f pin index safety system, 15f
sources of, 10—13
Medical malpractice, 303-304
Medical providers, 10
Medtronic Xomed NIM electromyographic (EMG) nerve, 795f
Melena, 1325

Meningitis, 993, 1320t
Meperidine, 191
characteristics of, 190t chemical structure of, 189f
labor applications of, 864
postoperative shivering, 1215
Mepivacaine, 265t, 269t, 985t
Metabolic acidosis, 1178, 1185t
Metabolic alkalosis, 1185t
Metabolic biotransformation, 142
Metabolic equivalents (METS), 933
Methacholine, 222
Methemoglobin, 525
Methemoglobinemia, 121
Methohexital, 4, 172f, 173, 174t, 1220
Methotrexate, 641t
Methylene blue, 268
Methylergonovine, 854
Methylparaben, 271
Methylprednisolone, 834, 1080t, 1100
Metoclopramide, 279t, 280, 1292
Metoprolol, 250, 394t
Mexiletine, 378t
Meyer—Overton rule, 156
Microelectrode recording (MER), 609
Micrognathia, 300
Microshock, 17
Midazolam, 175f, 176t, 906, 910
Midazolam—ketamine anesthetic technique, 290
Midesophageal aortic valve, 115f
Midgut volvulus, 920
Milk-alkali syndrome, 1158
Mineralocorticoid receptor antagonists, 1150
Mini Cog, 930, 931f-932f
Minimally invasive arthroplasty, 811, 812f
Minimally invasive lumbar decompression (MILD), 1069
Minimum alveolar concentration (MAC), 155, 266, 519, 540, 605, 844, 937
of inhalation anesthetics, 158—159
Minimum local analgesic concentration (MLAC), 844
Minnesota Multiphasic Personality Inventory (MMPI), 1063
Mitral regurgitation, 417-420
anesthetic management, 418-420
choice of agents, 420
monitoring, 420
objectives, 418-419
pathophysiology, 418
preoperative considerations, 417-418
treatment, 418
Mitral stenosis, 415-417

anesthetic management, 417
choice of agents, 417
monitoring, 417
objectives, 417
pathophysiology, 416-417
preoperative considerations, 415-416
treatment, 417

anatomy of, 456f
apparatus, 457f
leaflet motion, 419f
prolapse of, 420-421
Mivacurium, 212t
Mixed antagonists (labetalol), 249
Mixing lesions, 431
Modern anesthesia machine, 49f
Modern conventional vaporizer, 56-57
Modified Brooke formula, 835
Molality, 1134
Molarity, 1134
Monitored Anesthesia Care (MAC), 1239
Monoamine oxidase inhibitors (MAOIs), 626, 1220
Monophasic amped sine (MDS), 1270
Morphine, 770. See also Opioids
history of, 4-5
Morton, William T.G., 2
Motor-evoked potentials (MEPs), 129, 131f
Multimodal analgesia, 195
Multiple organ dysfunction syndrome (MODS), 1317
Multiple sclerosis, 627-628
anesthetic considerations, 628
preoperative considerations, 627-628
Murphy tracheal tube, 320f
Muscarine molecular structures, 224f
receptors, blockade of, 234
side effects, 225t
Muscle infection, 1321t
Muscle pains, 210
Muscle relaxants, 200, 683-684. See also specific agents depolarizing, 205-211
evoked responses, 205f succinylcholine, 205-211
diseases with, 215t nondepolarizing. See Nondepolarizing muscle relaxants; specific agents
Musculocutaneous nerve, 1012
block, 1018-1019
course, 1021f
Myasthenia gravis, 638-642
anesthetic considerations, 641-642
women, 642
Myasthenia gravis crisis, 638
Mycophenolate mofetil, 641t
Mydriasis, 235
Myocardial cells, 348
Myocardial contractility, 356
Myocardial infarction (MI), 383, 1312-1313
Myocardial ischemia, 395, 756
Myocardial oxygen balance, 368, 368t
Myocardial stunning, 1312

Myofascial pain syndrome, 1065-1066
Myokymia, 643
Myonecrosis, 1321t
Myotonia, 645
Myotonic dystrophy, 645
Myringotomy, 925

NabotulinumtoxinA. See Botulinum toxin
N-acetylcysteine, 665
Nadbath technique, 781
Naloxone (Narcan), 893 chemical structure of, 189f clinical uses, 289-290 dosage, 290 drug interactions, 290 mechanism of action, 289 side effects, 290 Naltrexone, 290 Nasal cannula, 1331-1332 Nasal mask, 1332 Nasal polyps, 791 Nasal/sinus surgery airway fire protocol, 791t intraoperative management, 791—792 cardiovascular stability, 789 oxygenation, 789 ventilation, 789 laser airway surgery, tracheal tubes for, 790t preoperative considerations, 791 Nasopharyngeal probes, 132 National Fire Protection Agency (NFPA), 10, 16 National Institute for Occupational Safety and Health (NIOSH), 75 Nausea and vomiting neurological pathways involved in, 285f postoperative, 799, 948, 1116, 1122-1123, 1291-1292 ND:YAG-compatible tubes, 25 Near infrared spectroscopy (NIRS), 131, 585 Near-drowning, 1309-1310 Near-infrared cerebral oximetry (NIRS), 459-460
Nebivolol, 250
Necrotizing fasciitis, 1321t
Nefazodone, 1077t
Negative-pressure pulmonary edema, 1298, 1309
Neobladder, 706
Neohepatic phase, 746-747
Neonatal resuscitation chest compressions, guidelines, 892-893
depressed neonate, 891
drug therapy, 893-894
epinephrine, 893
naloxone, 893
general care of, 889
meconium-stained neonates, 889-891
vascular access, 893
ventilation, guidelines, 891-892
volume resuscitation, 893

chest compressions, 892f alternative technique, 893f
intubation, 892f
Neostigmine, 226f clinical considerations, 227
dosage and packaging, 226f, 227t molecular structures, 226f
physical structure, 226
Nephrogenic diabetes insipidus, 1142

anatomic divisions of, 652f collecting tubule in, 657-658
distal tubule, 656
functional divisions of, 653t juxtaglomerular apparatus, 658f, 658-659
loop of Henle, 655-656
proximal tubule in, 654-655
reabsorption of solutes, 655f renal corpuscle in, 653-654
sodium reabsorption in, 654f
Nephrotic syndrome, 724
Nerve blocks, 1014f efficacy of, 1084-1085
Nerve injuries, 1238
Nerve localization, 1002f
Nerve stimulation, 1033-1034
infraclavicular block, 1011f interscalene block using, 1007f
Nerve stimulator, 1001f
Nerve-sparing technique, for radical retropubic prostatectomy, 704—705
Neuralgia, 1050t
Neuraxial anesthesia, 807, 808, 960-961, 994-995
anatomy spinal cord, 963-966
vertebral column, 961—963
mechanism of action, 966—967
autonomic blockade, 967-969
somatic blockade, 967
obstetric patient, 961
sick elderly patient, 961
spinal cord, 963-966
Neuraxial block-induced sympathectomy, 968
Neuraxial blocks, 961, 968
anatomic approach, 974-976
Buie's/Jackknife position, 974
complications of, 989t, 989-994
cardiac arrest during spinal anesthesia, 990
high neural blockade, 989-990
needle/catheter insertion arachnoiditis, 993
backache, 991
epidural catheter, sheering of, 994
inadequate anesthesia/analgesia, 990
intravascular injection, 990-991
meningitis, 993
neurological injury, 992-993
postdural puncture headache, 991-992
spinal epidural abscess (EA), 993-994
spinal/epidural hematoma, 993

subdural injection, 991
total spinal anesthesia, 991
spinal anesthesia, 990
urinary retention, 990
lateral decubitus, 974, 975f
paramedian approach, 975-976, 976f
patient positioning, 972-974
sitting position, 972-973
surface anatomy, 972
ultrasound-guided, 976
Neuraxial techniques, 960
Neurogenic claudication, 1069
Neurogenic inflammation, 1059
Neurogenic pulmonary edema, 1307, 1309
Neurokinin-1 receptor antagonist, 286
Neuroleptic malignant syndrome (NMS), 633, 1220
Neurological diseases
cerebrovascular, 621-623
degenerative and demyelinating diseases, 626-630
amyotrophic lateral sclerosis (ALS), 628
autonomic dysfunction, 629
multiple sclerosis (MS), 627-628
Parkinson disease (PD), 626-627
spinal cord injury, 630-631
syringomyelia, 630
seizure disorders, 623-625
Neurological system monitors, 124-131
brain, cerebral oximetry and monitors of, 131
electroencephalography, 124-127
evoked potentials, 127-130
Neurolytic nerve blocks, 1084-1085
Neuromuscular blockade, 228t
Neuromuscular blocking agents, 133, 136, 164, 199-220
case discussions delayed recovery from general anesthesia, 219-220
respiratory failure, 229-230
chemical structures, 206f cholinergic pharmacology, 222-226
clinical pharmacology, 224-226
general pharmacological characteristics, 224—226
mechanism of action, 223—226
cholinesterase inhibitors, 226—231
edrophonium, 227
neostigmine, 226-227
physostigmine, 227-228
pyridostigmine, 226-227
considerations, 228
depolarizing muscle relaxants, 205-211
succinylcholine, 205-211

depolarizing vs. nondepolarizing blockade, 202-205, 203t, 205f mechanisms of action of, 202—203
response to peripheral nerve stimulation of, 204—205
reversal of actions of, 204
description of, 593, 604, 626, 743
L-cysteine, 228
neuromuscular transmission, 200—202
nonclassic reversal agents, 228
nondepolarizing muscle relaxants, 211-219
atracurium, 214-216
cisatracurium, 216
pancuronium, 217
rocuronium, 218-219
vecuronium, 218
pharmacologic antagonists to, 221-231
potentiation and resistance, 208t sugammadex, 228-229
Neuromuscular diseases, 639t anesthetic considerations, 641-642
case discussion, 648-649
differential diagnosis of, 639t drugs, 640t
muscular dystrophies, 646
Duchenne muscular dystrophy, 644
Duchenne/Becker muscular dystrophies, 645-646
facioscapulohumeral dystrophy, 645
limb-girdle muscular dystrophy, 645
myotonic dystrophy, 645, 646
preoperative considerations, 644
myasthenia gravis, 638-642
women, 642
myotonias, 646-647
paramyotonia congenita, 646-647
paraneoplastic neuromuscular syndromes, 642-644
anesthetic considerations, 644
Lambert—Eaton myasthenic syndrome (LEMS), 643
limbic encephalitis, 643
neuromyotonia, 643
polymyositis, 643-644
stiff person syndrome, 643
periodic paralysis, 647-648
Neuromuscular junction, 200f
Neuromuscular transmission, 200—202
Neuropathic pain, 1050, 1061, 1061t, 1071

acute herpes zoster/postherpetic neuralgia, 1073
complex regional pain syndrome (CRPS), 1072
diabetic neuropathy, 1071-1072
headache, 1073-1075
sympathetically maintained/sympathetically independent pain, 1072-1073
Neurophysiology, 583-599
blood-brain barrier, 586—587
brain protection in, 593-595
electroencephalography in, 595, 595t evoked potentials in, 595-596
case discussion on postoperative hemiplegia, 596-599
cerebral. See also Cerebral physiology anesthetic agents' effect on, 589-593
description of, 584-589
cerebral blood flow (CBF), 584-585
autoregulation, 585
cerebral perfusion pressure (CPP), 585
cerebral metabolism, 584
cerebrospinal fluid, 587-588
intracranial pressure, 588-589
Neurosurgery, 601-620
case discussion on, 617-619
craniotomy and anesthesia in for arteriovenous malformation, 615-616
for intracranial aneurysms, 612-615
for mass lesions, 603-605. See also Craniotomy, for mass lesions head trauma, 609-612
intracranial hypertension, 601-603
posterior fossa, 605-608
spine surgery, 616-617
stereotactic, 608-609
Neutral protamine Hagedorn, 757—758
Neutropenia, 1319t, 1323
Newly born infant, 890f
Nicardipine, 394, 394t, 767
Nicotine, 224f
Nicotinic receptors, 222
Nifedipine, 398
Nitrates, 399
Nitric oxide, 255

Nitrogen
medical gas cylinders, 12t
in medical gas systems, 13
Nitroglycerin, 256-257
clinical uses, 257
mechanism of action, 256—257
metabolism, 257
Nitroglycerin, 394t
Nitroprusside, 394t
Nitrous oxide, 590, 1220
biotransformation and toxicity, 161-162
clinical pharmacology of, 153f, 159t, 159-163
contraindications, 162—163
critical temperature, 12
drug interactions, 163
effects on organ systems, 161
cardiovascular, 161
cerebral, 161
gastrointestinal, 161
hepatic, 161
neuromuscular, 161
renal, 161
respiratory, 161
history of, 3
in medical gas systems, 12-13
physical properties of, 159, 161
Nizatidine, 279t
N-Methyl-D-aspartate (NMDA) receptors
antagonists of, 1124
description of, 155, 177, 196, 592

anatomy brain, lateral/coronal views, 1053f
first-order neurons, 1052
pain pathways, 1051-1052, 1052f
Rexed's spinal cord laminae, 1054f second-order, 1052-1056, 1054f
alternate pain pathways, 1056
spinal cord, cross section of, 1056f spinothalamic tract, 1055-1056
sympathetic/motor systems, integration, 1056
central modulation facilitation, 1059-1060
inhibition, 1060
chemical mediators of, 1057—1058
chronic pain, 1060-1061
fibromyalgia, 1066
modulation of, 1058—1060
primary hyperalgesia, 1058-1059
secondary hyperalgesia, 1059
myofascial pain syndromes, 1065-1066
nociceptors, 1056-1057
deep somatic, 1057
visceral nociceptors, 1057
pain syndromes, selected, 1064-1065
entrapment neuropathies, 1065t entrapment syndromes, 1064-1065
patient evaluation, 1062-1063
electromyography/nerve conduction studies, 1063-1064
pain measurement, 1063
psychological evaluation, 1063
spinal cord lamina, 1055t systemic responses, to acute pain, 1061-1062
cardiovascular effects, 1062
endocrine effects, 1062
gastrointestinal/urinary effects, 1062
hematological effects, 1062
immune effects, 1062
psychological effects, 1062
respiratory effects, 1062
systemic responses, to chronic pain, 1062
third-order, 1056
Nociceptive pain, 1050
Nociceptors, 1056-1057
Noise, in operating rooms, 16
Noncardiac surgery
coronary artery disease, 384
heart failure, 412
hypertension, 385-389
ischemic heart disease, 395-399
perioperative evaluation and preparation, 383-414, 388t
Noncardiogenic pulmonary edema, 1308-1309
Noncardiovascular monitoring, 119-138

awareness, checklist for prevention, 129t
case discussion on, 136-137
neurological system monitors, 124-131
peripheral nerve stimulation, 133-136
respiratory gas exchange monitors, 119-124
temperature in, 132-133
urinary output in, 133
Nonclassic reversal agents, 228
Noncompetitive potassium-sparing diuretics, 669
Nondepolarizing muscle relaxants, 211-219, 223, 1220
clinical characteristics of, 212t
evoked responses, 205f
pharmacological characteristics of, 211-214, 213-214
acid—base balance, 213
age, 213, 214t
autonomic side effects, 213
concurrent disease, 214
drug interactions, 213
electrolyte abnormalities, 213
hepatic clearance, 213
histamine release, 213
maintenance relaxation, 212
muscle groups, 214
potentiation by inhalational anesthetics, 212
potentiation by other nondepolarizers, 212
preventing fasciculations, suitability for, 212
renal excretion, 213
suitability for intubation, 211-212
temperature, 213
pharmacology of, 211t
vecuronium, 217-218
Nondiverting (mainstream) capnographs, 122
Nondiverting sensor, 122f
Noninvasive blood pressure (NIBP), 1287
Noninvasive brain oximetry monitors, 121
Noninvasive stress imaging, 740
Nonionized (uncharged) drugs, 140
Non-operating room anesthesia, 943, 952-953
common locations for, 954t
locations, 953t—954t
Nonrebreathing valve, 44
Nonshivering thermogenesis, 900
Nonsteroidal antiinflammatory drugs (NSAIDs), 195, 196, 286, 662, 676
Non-ST-segment elevation myocardial infarction (NSTEMI), 1312-1313
Norepinephrine, 240, 247, 471
clinical considerations, 247
dosing and packaging, 247
synthesis, 241f, 242f

Nortriptyline, 1077t
Nosocomial infection, 1317-1318
Noxious sensations, 1057
Nucleus raphe magnus (NRM), 1060
Nutrients, 1226-1227
Nutrition
basic needs for, 1224-1225
enteral, 1227
Nutritional repletion, 1223
Nutritional support, 1226

Obesity/obese patients psychological/nonpharmacological techniques, 864
combined spinal and epidural (CSE) analgesia, 872
local anesthetic/local mixtures, 867
pulmonary disease, 537-544
algorithm for, 877f description of, 767-769 optimal positioning for, 876, 876f Obstetric anesthesia, 862 anesthetic risk anesthetic mortality, 863 maternal mortality, 862-863 obstetric anesthesia closed claims, 863 cesarean section, 873-874 for emergency, 878-879 general anesthesia, 875-879 regional anesthesia, 874-875 general anesthesia, 873 general approach, 863 labor/vaginal delivery, 864 pain pathways, 864 parenteral agents, 864-865 pudendal nerve block, 865 regional anesthetic techniques, 865-866 epidural opioids, 866-867 intrathecal opioids, 866 anesthetic-opioid lumbar epidural analgesia, 867-872 spinal anesthesia, 872 spinal opioids, 866 Obstetric morbidity, 863¢t Obstructive lesions, 428-429 Obstructive asthma, 538-541. See also Asthma
chronic, 541-544, 542t chronic bronchitis, 541-542
emphysema, 542
intraoperative management, 543-544
preoperative management, 543
treatment for, 542-543
Obstructive sleep apnea (OSA), 944, 945t perioperative risk from, 947t
scoring system, 947t
Obturator nerve block, 1029f
Obturator nerve innervation, 1028f
Occipital nerve block, 1089, 1089f anatomy, 1089
complications, 1089
technique, 1089
Occipital nerve stimulator placement, 1107f
Occupational Safety and Health Administration (OSHA), 16
Octopus retractor, 475f
Oculocephalic reflex, 780
Office-based anesthesia, 943, 944-945
Off-site anesthesia, 952
intercostal block, 1040-1041

paravertebral anatomy, 1043f paravertebral block, 1041-1043, 1043f
superficial cervical plexus block, 1040
transversus abdominis plane (TAP) block, 1044, 1045f
Olfactory nerve, 308
OnabotulinumtoxinA, 1084
Ondansetron, 284—286
One-handed face mask technique, 315f
One-handed needle advancement, 868f
One-lung ventilation, 556-563
alternatives to, 569-570
anatomic considerations in, 559, 559f double-lumen bronchial tubes in
description of, 558-562, 560f-561f, 561t placement of, 559-562
indications for, 557t management of, 568-569
single-lumen tracheal tubes with bronchial blocker in, 562—563
Open pneumothorax, 555-556
mediastinal shift, 556, 556f paradoxical respiration, 556, 557f
Open-drop anesthesia, 35

case discussion on, 29-30
electrical safety in, 17-22. See also Electrical safety environmental factors in, 15-17
fires and explosions in, 22-25, 23f future design of, 27-29
locations, special considerations, 954-956
medical gas systems in, 10-15. See also Gas systems, medical radiofrequency identification (RFID) technology, 29
safety culture, 10

case discussion, 782—784
eye blocks, needle/catheter, 781f facial nerve block techniques, 780f
general anesthesia for extubation/emergence, 778-779
induction, 777—778
monitoring/maintenance, 778
premedication, 777
intraocular gas expansion, 776
intraocular pressure dynamics anesthetic drugs, effect of, 774-775
intraocular pressure, physiology of, 774
oculocardiac reflex, 775-776
open-eye surgical procedures, 774t ophthalmic drugs, systemic effects of, 776-777
oral RAE endotracheal tube, 778f regional anesthesia
eye, topical anesthesia, 781
facial nerve block, 781
intravenous sedation, 782
peribulbar blockade, 780-781
retrobulbar blockade, 779-780
sub-Tenon's block, 781
retrobulbar block, 779f systemic effects of, 777t
Ophthalmoscopy, 391
Opiates, 1220
Opioid receptors, 188t
Opioid-induced hyperalgesia, 193
Opioids, 591, 634, 682, 737, 866, 906
agonists, 189f antagonists, 189f
characteristics of, 1076t chemical structure, 189f
dosing of, 194t, 461t drug interactions, 194
cardiovascular, 191-192
cerebral, 192-193
endocrine, 193
gastrointestinal, 193
respiratory, 192
mechanisms of action, 187-188
pharmacokinetics, 188-191
absorption, 188, 190
biotransformation, 190-191
distribution, 190
excretion, 191
physical characteristics, 190¢t receptor types, 187
sparing strategies, 1290
structure—activity relationships, 188t uses of, 194t
ventilation depression caused by, 192f
Optical intubating stylet, 322f
Oral antihypertensive agents, 390t-391t
Oral drug administration, 140
Oral lactulose, 742
Oral tracheal tube, 320t

Organophosphates, 207, 224, 236
Orthognathic surgery, 796-798
Orthopedic surgery, anesthesia for bone cement, 804—805
case discussion, 816-817
deep vein thrombosis (DVT), 806-807
fat embolism syndrome, 806
hip surgery. See Hip fracture pneumatic tourniquets, 805-806
pulmonary embolism (PE), 806-807
Oscillometry, for arterial blood pressure monitoring, 84
Osmolal gap, 1139
Osmotic diuretics (mannitol), 666-667
side effects, 667
therapy, 832
Osteomalacia, 764
Otorhinolaryngologic surgery case discussion, 799-800
ear surgery intraoperative management
endoscopy, 788
head/neck cancer surgery intraoperative management, 793—796
maxillofacial reconstruction, 796-798
nasal/sinus surgery intraoperative management, 791—792
preoperative considerations, 791
orthognathic surgery, 796—798
Outside operating room, anesthesia/sedation, 953t-954t

administration of, 24
blood respiratory gases, transport of, 523-526
content of, 525
dissolved oxygen, 523
emergency (E-cylinder) cylinder, 11, 12t hemoglobin, 523
hemoglobin dissociation curve, 523, 524f factors influencing, 523-525, 524f
hemoglobins, abnormal ligands and abnormal forms of, 525
high-pressure cylinders, 11, 11f liquid oxygen storage system, 11, 12f
in medical gas systems, 10-12, 11f, 12f stores in, 526
transport of, 525-526
Oxygen analyzers, 40¢t, 61
Oxygen consumption, 845
Oxygen mask, 1332
Oxygen ratio monitor controller (ORMC), 55
Oxygen supply failure protection devices, 53
Oxygen tension alveolar tension, 519
arterial tension, 520t, 520-521, 521f mixed venous tension, 521, 522t
pulmonary end-capillary tension, 519-520
Oxygen therapy, 1298, 1330
Oxygen therapy equipment, 1331
device/system, 1332t
hazards of, 1335-1336
absorption atelectasis, 1336
fire hazard, 1337
hyperbaric oxygen toxicity, 1336-1337
hypoventilation, 1336
pulmonary toxicity, 1336
retinopathy of prematurity (ROP), 1336
high-flow/fixed-performance equipment, 1331
air-entraining nebulizers, 1334
air-entraining Venturi masks, 1333-1334
air—oxygen systems, 1334-1335
anesthesia bag/bag-mask-valve systems, 1333
helium—oxygen therapy, 1335
hyperbaric oxygen therapy, 1336-1337
oxygen hoods, 1335
variable-performance equipment, 1331
masks, with gas reservoirs, 1332-1333
nasal cannula, 1331-1332
nasal mask, 1332
simple oxygen mask, 1332
Oxygen—-hemoglobin dissociation curve, 121
Oxyhemoglobin, 120f

Packed red blood cells (PRBCs), 1199
Pain management, chronic. See also Interventional pain therapy multidisciplinary treatment
acupuncture, 1109
physical therapy, 1109
psychological interventions, 1107-1109
procedural therapy, 1084
chemical neurolysis, 1103
cryoneurolysis, 1102
diagnostic/therapeutic blocks, 1084-1085
differential neural blockade, 1103-1104
epidural injections, 1100-1102
neuromodulation, 1104-1107
radiofrequency ablation (RFA), 1102
somatic nerve blocks, 1085-1095
sympathetic nerve blocks, 1095-1100
vertebral augmentation, 1107
Pain modulation, 1058-1060
Palpation, for arterial blood pressure monitoring, 82
Pancoast syndrome, 563
Pancreas insulin, effects of, 754t
Pancuronium, 217, 683
dosage, 217
physical structure, 217
allergic reactions, 217
arrhythmias, 217
hypertension and tachycardia, 217
Paradoxical air embolism, 607
Paralysis, succinylcholine-related, 210
Paramagnetic analysis, 124
Paramagnetic sensors, 61
Paramyotonia congenita, 646-647
Paraneoplastic syndromes, 642-643
Paraplegia, 486-487
Parasympathetic nervous system, 223f
Parasympathetic system, 363
Parathyroid glands
hyperparathyroidism, 762—763
Parathyroid hormone (PTH), 761, 1157
Paravertebral anatomy, 1043f
Paravertebral nerve blocks, 1127
Parenteral nutrition (PN), 1223
Paresthesia, 1050t
Parietal cells, 276

Parkland formula, 835
Paroxetine, 1077t
Partial anomalous venous return, 430-431
Partition coefficients, 151t, 151-152
Passive humidifiers, 66
Passover humidifier, 66
Patent ductus arteriosus, 430
Patient safety issues, 1353-1354. See also Performance improvement issues
Patient-controlled analgesia, 919
Patiromer, 1157
Patrick’s sign, 1066
Pecs block, 1043-1044, 1044f
Pediatric anesthesia
anatomic/physiological development, 898
cardiovascular system, 899
glucose homeostasis, 901
metabolism/temperature regulation, 899-900
renal/gastrointestinal function, 900-901
respiratory system, 898-899
inhalational anesthetics, 905-906
MAC values, 906t
muscle relaxants, 907-908
nonvolatile anesthetics, 906
pharmacological differences, 901-908
risk, 908-909
maintenance, 915
replacement requirements, 916
prematurity anesthetic considerations, 920
pathophysiology, 919
premedication, 910
preoperative fasting, 910
preoperative interview, 909
regional anesthesia and analgesia, 916-917
sagittal section of, 900f scoliosis, 926
sedation for procedures, 917-918
tonsillectomy/adenoidectomy, 925
tracheal intubation, 913-914
tracheoesophageal fistula anesthetic considerations, 922—923
pathophysiology, 922
tracheoesophageal fistula, types of, 922f trisomy 21 syndrome, 925-926
tympanostomy tubes, myringotomy/insertion of, 925
upper respiratory tract infection (URI), 909
Pediatric drug dosages, 901t-905t
Pediatric liver transplantation, 727
Pediatric perioperative cardiac arrest (POCA), 908
PEEK implant, 1107, 1108f
Pentobarbital, 172f, 174t
Penumbra, 623f

Percutaneous catheter, placement of, 1003f
Percutaneous coronary intervention (PCI)
management of patients with, 407f treatment for, 407f
Percutaneous cricothyrotomy, 1265, 1266f
Percutaneous cryoablation, 708
Percutaneous endoscopic placement of gastrostomy (PEG) tubes, 1225
Percutaneous introducer, 98, 99f
Performance improvement issues central venous catheter insertion checklist, 1357f
quality assurance measures, 1355-1358
standardized checklists, 1355
strategies to performance errors reduction, 1354-1355
time out checklist, 1356f
Peribulbar/sub-Tenon's (episcleral) block, 779
Pericardial disease, 481-483
cardiac tamponade in, 481-483
anesthetic considerations, 482-483
preoperative considerations, 478-479
constrictive pericarditis in, 483
anesthetic considerations, 483
preoperative considerations, 483
Perineural local anesthetic infusion, 998
Perioperative beta-blocker therapy, 251
Perioperative fluid therapy intraoperative fluid therapy, 1196-1197
replacing blood loss, 1196-1197
replacing redistributive/evaporative losses, 1197
normal maintenance requirements, 1194
preexisting deficits, 1194-1195
surgical fluid losses blood loss, 1195
third-space fluid loss, 1195
Perioperative mortality, 1233
Perioperative pain management adequate tissue oxygenation, maintenance, 1122
goal-directed fluid/hemodynamic therapy, 1122-1123
intraoperative period, 1117-1123
normothermia, maintenance of, 1122
postoperative nausea and vomiting (PONV) prophylaxis, 1122
use of inhalational anesthetics, 1121
intravenous anesthetics, 1121
opioids, 1121-1122
Perioperative stroke, 621
Perioperative surgical home, 1114
Perioperative vision loss (POVL), 617

Peripheral nerve block techniques, 1000-1004. See also Lower extremity peripheral nerve
blocks; Upper extremity continuous, 1002-1004
field block technique, 1000
nerve stimulation technique, 1001
paresthesia technique, 1000-1001
ultrasound technique, 1001-1002
blocks, continuous, 1002-1004
conduction, 1064
intercostal nerve block, 1042f local anesthetic, 999
localization of, ultrasound for, 1001
lower extremity ankle block, 1038-1040
femoral nerve block, 1023-1025
lateral femoral cutaneous nerve, 1025—1026
saphenous nerve block, 1031-1032
sciatic nerve block, 1032—1038
patient selection, 998-999
percutaneous catheter adjacent to, 1003f preparation, 999-1000
risks and contraindications, 999
stimulators, 135f, 225
Peripheral nerve stimulation (PNS), 133-136, 1105
clinical considerations, 134-136
indications and contraindications, 133-134
techniques and complications, 134
Perirectal abscess, 1324
Peritonitis, 1321t
Peritubular system, 659
Persistent postsurgical pain, 1115
Persistent pulmonary hypertension, 857f pH
indicator dye, 41
sensitivity due to atracurium, 216
stat management, 467
Pharmacodynamics, 145-148. See also specific agents drug receptors, 147-148
exposure—response relationships, 145-146
Pharmacokinetics, 139-145. See also specific agents absorption, 140
biotransformation, 142-143
compartment models, 143-145
definition, 139
distribution, 140-142
excretion, 143
Pharmacological principles, 139-148
pharmacodynamics, 145-148
pharmacokinetics. See Pharmacokinetics
Pharynx, 308, 308f
Phenelzine, 632
Phenobarbital, 172f
Phenothiazines, 682

Phenylephrine, 245-246, 417, 796
Phenytoin, 1078t
Pheochromocytoma, 766
Phospholipase C, 1059f
Phosphorus disorders, 1160-1162
hyperphosphatemia anesthetic considerations, 1161
clinical manifestations, 1161
treatment of, 1161
hypophosphatemia, 1161-1162
anesthetic considerations, 1162
clinical manifestations, 1162

normal plasma phosphorus concentration, 1161
Physician Quality Reporting System (PQRS), 1355, 1358
Physiological dead space, 1268
Physostigmine, 227-228, 1291
clinical considerations, 228
Piezoelectric analysis, 123
Pin index safety system, 14, 51
in medical gas systems, 15f
“Pink puffers,” 542
Piriformis syndrome, 1067
Piston ventilator, 71
Pitot tube, 63
Placenta, 849f
Placenta previa, 880-881
Plan-do-study-act cycle, 1358
Plasma glucose, 758
Plasma osmolality, 1139-1140
Plateau pressure, 72
Platelet transfusions, 740
Plicamycin (Mithramycin), 763
Pneumonia, 1319t
Pneumoperitoneum, 969
Pneumotachograph, 63
Pneumotaxic center, 529
Poiseuille’s law, 53
Polymethylmethacrylate, 804
Pop-off valves. See Adjustable pressure-limiting (APL) valves
Porphyrin, 174
Porta hepatis, 716
Portal hypertension, 739
Portopulmonary hypertension (POPH), 741
Positive end-expiratory pressure (PEEP), 36, 69, 508, 605, 743, 1309, 1330,

Post anesthesia recovery/discharge, 949-951
Postanesthesia care, 1286
case studies, 1300-1303
circulatory complications arrhythmias, 1300
hypertension, 1299-1300
hypotension, 1298-1299
discharge criteria outpatients, 1294-1295
PACU, 1293-1294
emergence from general anesthesia delayed emergence, 1289
operating room to PACU, 1289
respiratory complications, 1295-1298
airway obstruction, 1295-1296
hypoventilation, 1296-1297
hypoxemia, 1297-1298

agitation, 1291
general anesthesia, 1289-1290
nausea and vomiting, 1291-1292
pain control, 1290-1291
regional anesthesia, 1290
shivering and hypothermia, 1292-1293
Postanesthesia care units (PACUs), 303, 918, 951t, 1215, 1286-1288
design, 1287
equipment, 1287-1288
staffing, 1288
Postanesthesia discharge scoring system (PDSS), 952t, 1295t
Postdural puncture headache (PDPH), 872, 991-992
Posterior descending artery (PDA), 365
Posterior fossa surgery, 605-608
complications of brainstem injury, 606
obstructive hydrocephalus, 606
pneumocephalus, 606
positioning in, 606, 606f venous air embolism, 607
for obstructive hydrocephalus, 606
pneumocephalus in, 606
positioning in, 606, 606f
Posterior lumbar plexus block, 1028-1030, 1031f
Posterior superior iliac spine (PSIS), 1033
Posterior tibial artery, blood pressure monitoring via, 87
Posterior tibial nerve, 1038
Posthypercapnic alkalosis, 1183
Postintubation croup, 334
Postoperative cognitive dysfunction (POCD), 937-938
Postoperative hypothermia, 1215
Postoperative nausea and vomiting (PONV), 799, 948, 1116, 1122-1123, 1291-

Postoperative pain management acetaminophen (paracetamol), 1124
analgesia to facilitate functional recovery, 1128
epidural analgesia, 1124, 1127
local anesthetic wound infusion, 1127
minimize postoperative ileus, 1128-1129
multidisciplinary surgical care, organization, 1127-1128
multimodal analgesia, 1123-1127
nonsteroidal antiinflammatory drugs (NSAIDs), 1123-1124
opioids, 1124
peripheral nerve block, 1127
PONV treatment, 1123
shivering minimization, strategies, 1123
Postpartum tubal ligation, 858-859
Postrenal azotemia, 1314
Postretrobulbar block apnea syndrome, 780
Postsynaptic membranes, 243
Posttetanic potentiation, 204
Post-transfusion purpura, 1203
Post-traumatic stress disorder, 1326
Potassium, 377t, 1150-1151
Potassium chloride, 1154
Potassium disorders hyperkalemia

anesthetic considerations, 1154
intercompartmental shifts, 1151
urinary potassium excretion, 1151
normal balance, 1150
Potassium-sparing diuretics, 669, 1150
Preanhepatic phase, 746
Precordial stethoscopes, 119-120
contraindications in, 119
indications in, 119
techniques and complications, 119-120
Precordial thump, 1278-1279
Prednisolone, 641t, 1080t
Prednisone, 641t, 1080t
Predominantly left-to-right (simple) shunts, 429-431
Predominantly right-to-left (complex) shunts, 431-432
Preeclampsia, complications, 884t
Preexcitation, 371-372
Pregabalin, 196, 289, 1078t, 1125t
Pregnancy. See also Labor and delivery
amniotic fluid embolism, 885-886
antepartum hemorrhage, 880
abruptio placentae, 881
placenta previa, 880-881
uterine rupture, 881
appendicitis in, 894-895
case discussion, 894-895
chorioamnionitis, 881-882
dystocia/abnormal fetal presentations/positions in
abnormal vertex presentations, 880
breech presentation, 879-880
primary dysfunctional labor, 879
heart disease, 885
hypertensive disorders, 883-885
anesthetic management, 884-885
pathophysiology/manifestations, 883
treatment, 883-884
multiple gestations, 880
physiological changes during, 844t
cardiovascular system, 845-846
central nervous system, 844
hematological system, 846-847
hepatic system, 846

metabolic system, 847
musculoskeletal system, 847
renal/gastrointestinal system, 846
respiratory system, 844-845
postpartum hemorrhage, 886
premature rupture of membranes (PROM), 881-882
preterm labor, 882-883
umbilical cord prolapse, 879
Pregnancy Mortality Surveillance System, 1237
Prehypertension, 385
Prejunctional receptors, 204

in compensatory mechanisms, 370
in diastolic function and ventricular compliance, 355
ventricular, 354t ventricular filling in, 354-355
Premature atrial/ventricular beats, 1300
Premature rupture of membranes (PROM), 881-882
Premedication, 301. See also specific agents and surgeries
Preoperative evaluation, 296-301
cardiovascular issues, 297—298
coagulation issues, 298-299
endocrine and metabolic issues, 298
gastrointestinal issues, 299-300
laboratory evaluation in, 300-301
physical examination in, 300
preoperative history in, 297—300
pulmonary issues, 298
Preoxygenation, 314
Prerenal azotemia, 1313-1314
Pressure regulator, 52
Pressure support ventilation (PSV), 1343
Pressure transducers, 89-90
Pressure-controlled ventilation (PCV), 1339
Pressure-relief valves. See Adjustable pressure-limiting (APL) valves
Pressure—volume loop, 359f
Pressurized oxygen, 69
Prilocaine, 265t, 268, 269t
Primary brain injuries, 831
Principal electrophysiological mechanisms, 408f
Procainamide, 375t, 378t, 1275t
Procaine, 265t, 269t
Prochlorperazine, 286
Promethazine, 277t, 865
Prone jackknife position, 988f, 989
Propafenone, 378t, 1277t
Propofol, 181-183, 591, 682, 1220
anaphylaxis caused by, 1249
in children, 906
doses of, 178t drug interactions, 183
effects on organ systems, 182-183
cardiovascular, 182
cerebral, 182-183
respiratory, 182
mechanisms of action, 181
pharmacokinetics of, 181-182
absorption, 181
biotransformation, 182
distribution, 181-182
excretion, 182
structure of, 177f structure—activity relationships, 181
Propofol infusion syndrome, 182, 906, 1326
Propranolol, 250
characteristics of, 375t, 378t clinical considerations, 250

dosing and packaging, 250
Prostacyclin (PGI,), 883
Prostaglandins, 196, 1058
Prostate, transurethral resection of intraoperative considerations, 698-701
anesthesia, choice of, 700
bladder perforation, 700
coagulopathy, 700
hypothermia, 700
monitoring, 700—701
septicemia, 700
TURP syndrome, 698-700
Protamine, 472-473
Protein-losing enteropathy, 724
Prothrombin time (PT), 724
Proton pump inhibitors (PPIs), 280-281
clinical uses, 281
dosage, 281
drug interactions, 281
mechanism of action, 280
side effects, 281
Proximal saphenous nerve block, 1033f
Proximal sciatic nerve block, 1033f
Pseudocholinesterase, 204, 207t
Pseudoclaudication, 1069
Pseudohyponatremia, 1144, 1144t
Psoas compartment block. See Posterior lumbar plexus block
Psychiatric disorders, 631-634
bipolar disease, 632-633
case discussion on, 634—636
depression, 631-632
neuroleptic malignant syndrome, 633
schizophrenia, 633
substance abuse, 633-634
Pudendal nerve block, 865, 1095f
anatomy, 1094
complications, 1095
indications, 1094
technique, 1095
Pulmonary artery catheterization, 96-101, 98f, 100f, 101t, 102t
clinical considerations, 99-101
contraindications, 97-98
incidence of, 100f
techniques and complications, 98f, 98-99
Pulmonary artery occlusion pressure (PAOP), 98, 1191
Pulmonary capillary occlusion pressure (PCOP), 97
Pulmonary capillary wedge pressure (PCWP), 356
Pulmonary edema, 1297
cardiogenic, 1307-1308

Pulmonary embolism, 546t, 546-548
diagnosis of, 547
intraoperative, 548
intraoperative management, 548
pathophysiology of, 546
preoperative management, 547-548
treatment and prevention, 547
Pulmonary function tests (PFTs), 536
Pulmonary gas exchange, 741
Pulmonary perfusion, 514-515
distribution of, 514-515, 515f ventilation/perfusion ratios, 515-516, 516f
Pulmonary receptors, 225
Pulmonary thermal volume (PTV), 104
Pulmonary vascular resistance (PVR), 356, 416, 429, 476, 741
Pulmonic stenosis, 428-429
Pulsatile blood flow, 445
Pulse contour devices, 106-107
Pulse oximeters, 120-121, 121
Pulse oximetry, 120-121, 743, 911
clinical considerations, 121
indications and contraindications, 120
techniques and complications, 120
Pulsus bisferiens, 424
Pumping effect, 57
Purkinje fibers, 348
Pyelonephritis, 1320t
Pyomyositis, 1321t
Pyridostigmine, 226-227, 640, 641t clinical considerations, 227
dosage and packaging, 227, 227t molecular structures, 226f
physical structure, 226-227

Quadriceps contraction, 1029
Quality of care, 1354-1358
Quinidine, 378t

blood pressure monitoring via, 86-87
cannulation of, 87, 88f
Radial nerve block at elbow, 1018
at wrist, 1021f
Radial nerve course, 1020f
Radical cystectomy, 706
Radical nephrectomy, 708-710
Radical orchiectomy, 707
Radical retropubic prostatectomy, 704—705
Radicular irritation, 270
Radiculopathy, 1050t, 1068
Radiofrequency ablation (RFA), 708, 1102
Radiofrequency identification (RFID) technology, 29
RAE tube, 925
Raman spectroscopy, 123
Ranitidine, 279t
Reactive oxygen species (ROS) production, 158
Receptor occupancy, 148
Recovery, stages of, 951t
Rectal midazolam, 910
Recurrent laryngeal nerve, 308, 794f
Reentry, 373f
Referred pain, 1051t, 1055
Reflex bronchospasm, 540
Reflex sympathetic dystrophy (RSD), 1072
Regional wall motion abnormalities, 457f
Relative humidity, 66
Relative refractory period, 346
Relative shunt, 516
Remifentanil. See also Opioids characteristics of, 190t
chemical structure of, 189f ester structure, 191
history of, 5
plasma concentration of, 191f
Renal blood flow, 659, 936
hormonal regulation, 662
intrinsic regulation, 660, 662
neuronal and paracrine regulation, 662
tubuloglomerular balance and feedback, 662
Renal blood flow (RBF), 659
Renal cell carcinoma, 707, 709f
Renal clearance, 659
Renal corpuscle, 653-654
Renal function, 662—666
blood urea nitrogen, 678-679
blood urea nitrogen:creatinine ratio, 681
creatinine clearance, 681
direct anesthetic effects, 664-665
drugs, 665
intravenous agents, 665
volatile agents, 664-665
direct surgical effects, 665-666
evaluation, 676-681

indirect effects, 664
cardiovascular, 664
endocrine, 664
neurologic, 664
serum creatinine, 679-681, 680f
urinalysis, 681
Renal impairment
drugs with potential for, 688¢
fluid therapy, 690
induction, 690
intraoperative considerations, 690
maintenance of anesthesia, 690
mild to moderate, 689-690
monitoring, 690
preoperative considerations, 689-690
Renal physiology, 651-673. See also Diuretics
anesthesia and surgery on, 662—666
case discussion on, 670-672
circulation in, 659-662
nephron in, 653-659. See also Nephron
Renal plasma flow (RPF), 659, 846
Renal replacement therapy (RRT), 685, 687t, 1316
Renal tubular acidosis, 1181
Rendell—Baker—Soucek pediatric face mask, 314f
Renin inhibitors, 390t
Renin—angiotensin—aldosterone system, 371, 387, 661f, 1148-1149
Resectoscope, 698
Reserpine, 390t
Reservoir bag (breathing bag), 38-39, 39f
Resistance, 44
Respiratory acidosis, 1178, 1185t
Respiratory alkalosis, 1182t, 1182-1183, 1185t
Respiratory anatomy, functional, 496-500
innervation, 500
pulmonary circulation and lymphatics, 498-500
pulmonary capillaries, 499-500
pulmonary lymphatics, 500
rib cage and muscles of respiration, 496-497
tracheobronchial tree, 497-498, 498f
alveoli, 497-498, 499f

hypoxia, 1330t medical gas therapy, 1330-1331
Respiratory disease, 535-551
case discussion on, 548-550
obstructive pulmonary disease, 537-544
asthma, 538-541. See also Asthma chronic, 541-544
pulmonary embolism, 546-548
pulmonary risk factors and, 536t, 536-537, 537f restrictive pulmonary disease, 544-546
acute intrinsic, 544-545
chronic intrinsic, 546-547
extrinsic, 546
Respiratory failure, 769, 1306, 1306f
Respiratory gas exchange monitors, 119-124
anesthetic gas analysis, 123-124
capnography, 121-123
esophageal stethoscopes, 119-120, 120f precordial stethoscopes, 119-120
pulse oximetry, 120-121
Respiratory muscle degeneration, 644
Respiratory physiology, 495-533
blood respiratory gases, transport of, 523-528
breathing, 500-502
spontaneous ventilation, 500-502, 501f carbon dioxide tension
alveolar tension, 522, 522f arterial tension, 522
case discussion on, 531—533
control of breathing, 529-530
central respiratory centers, 529
central sensors, 529, 529f effects of anesthesia, 530, 531f
peripheral sensors, 529-530, 530f lung receptors, 530
gas exchange, effects of anesthesia, 518-519

oxygen tension alveolar tension, 519
pulmonary perfusion, 514-515
shunts, 516-517, 517f venous admixture, 517-518, 518f
ventilation, 512-514, 513t distribution of, 513f, 513-514
time constants of, 514
Respirometers. See Spirometers
Restrictive pulmonary disease, 544-546
Resuscitation, 44-45, 45f

Resuscitation breathing systems description of, 41f, 44-45
Resuscitator breathing systems, 44
Retinopathy of prematurity (ROP), 920, 1336
Retrobulbar block, 779
Retrobulbar injection, 779—780
Retroperitoneal lymph node dissection (RPLND), 707
Revision arthroplasty, 811
Reynolds number, 507
Rheumatoid arthritis (RA), 809, 809t
Rhodanese, 1312
Rickets, 764
Right coronary artery (RCA), 365
Right ventricular systolic pressure (RVSP), 114f
Rigid bronchoscopy apneic oxygenation, 575
conventional ventilation, 575
jet ventilation, 575
Ringer's injection, 915
Ringer's solution, 710
Rituximab, 641t
Rivaroxaban, 970
Robot-assisted radical prostatectomy, 705
Rocuronium, 211t, 218-219, 683
characteristics of, 212t chemical structure of, 206f
in children, 908
dosage, 218
metabolism and excretion, 218
physical structure, 218
side effects and clinical considerations, 218-219
Roentgen equivalent in man (REM), 16
Roller pumps, 444
Ropivacaine, 265t, 269t, 271, 985t, 989
Rotational thromboelastometry (ROTEM), 725, 827
Routine airway management, 310
Rule of Nines, 835, 836f
Ryanodine receptor, 1216
Safety interlock technology in operating rooms, 27—28
Safety-related clinical practice guidelines, 1353
Saphenous nerve, 1038
Saphenous nerve block distal saphenous technique, 1032
proximal saphenous technique, 1032
trans-sartorial technique, 1031-1032
Scavenging system, 75
Schimmelbusch mask, 35
Schizophrenia, 633
Sciatic nerve, 1036f anatomy of, 1034f, 1037f

in popliteal fossa, 1037f sonoanatomy of, 1037f
subgluteal, 1035f surface of, 1034f
into tibial and peroneal branches, 1036f
Sciatic nerve block, 1032-1038
anterior, 1033-1034, 1034f popliteal approach, 1035-1038
posterior/classic/labat approach, 1032-1033
subgluteal approach, 1034-1035
Sciatica, 1068
Scopolamine, 236, 1292
Secobarbital, 172f
Second hit phenomenon, 821
Second-degree burns, 834
Seizure disorders, 623-625
classification of, 624t intraoperative management, 625
preoperative considerations, 623-624
preoperative management, 624-625
Seldinger's technique, of right internal jugular cannulation, 94, 94f, 98
Selective nerve root block (SNRB), 1101
Selective serotonin reuptake inhibitors (SSRIs), 631, 1079
Sellick maneuver, 276
Sensitivity, 300, 300t
Sensory innervation, of fingers, 1021f
Sepsis, 1316, 1318t
Sepsis syndrome, 1316, 1317f
Septic shock, 1321t, 1322-1325
Serotonin and norepinephrine reuptake inhibitors (SNRIs), 1079
Serotonin syndrome, 284, 632, 1220
Sertraline, 1077t
Serum calcium, 763
Serum creatinine, 679-681, 680f
Serum glutamic-oxaloacetic transaminase (SGOT), 723
Serum potassium concentration, 647
Set point targeting, 1339
Sevoflurane biotransformation and toxicity, 168-169
chemical structure of, 159t in children, 906
clinical pharmacology of, 167-169
contraindications, 169
drug interactions, 169
effects on organ systems cardiovascular, 168
cerebral, 168
hepatic, 168
neuromuscular, 168
renal, 168
respiratory, 168
physical properties of, 167-168
popularity of, 3

Shivering, 1215, 1292-1293
Short P—R interval, 371-378
Shoulder dystocia, 880
Shunts, 516-517, 517f venous admixture, 517-518, 518f
Shy—Drager syndrome, 629
Sickle cell anemia, 1207t
Simple oxygen mask, 1332
Sinoatrial (SA) node, 346-347
Sinus surgery. See Nasal/sinus surgery
Sinusoidal channels, 716
Situational awareness, 26
Skin structure infections, 1321t
Sleeve pneumonectomy, 564
Smoke inhalation, 1310-1312
Society for Ambulatory Anesthesia (SAMBA), 281
Soda lime, 41
Sodium bicarbonate, 893
Sodium channel, 202f
Sodium disorders, 1148-1150
extracellular fluid volume regulation
anesthetic implications, 1149-1150
control mechanisms, 1148-1149
osmoregulation vs. volume regulation, 1149
hypernatremia. See Hypernatremia
Sodium nitroprusside, 255-256
clinical uses, 255
effects on organ systems, 256
mechanism of action, 255
metabolism, 255f, 255-256
Somatic nociceptors, 1057
Somatization disorder, 1064t
Somatosensory-evoked potentials (SEPs), 129-130, 131f
Sotalol, 378t, 1277t
Specificity, 300, 300¢t
Speech disorders, 310, 310¢t
Spinal anesthesia
description of, 874
intrathecal, 990
specific technique for, 978
spinal anesthetic agents, 980
spinal block, factors influencing level, 978-980
spinal catheters, 977-978
spinal needles, 976-977
Spinal anesthetic agents, 978t, 981t
Spinal canal, 979f, 1068
Spinal column, 963
Spinal cord, 963-966, 966f
Spinal cord injury, 833-834
anesthetic considerations, 630-631
acute transection, 630-631

chronic transection, 631
encephalitis, 631
preoperative considerations, 630
Spinal cord stimulation (SCS), 1105, 1106f
Spinal cord stimulator, 1105f
Spinal epidural abscess (EA), 993-994
Spinal needles, 976-977, 977f
Spinal stenosis, 1069-1070
Spinal/epidural anesthesia, 967
Spinal/epidural blocks
contraindications, 969t, 969-970
indications for, 969
neuraxial blockade, 970
antiplatelet drugs, 971
fibrinolytic/thrombolytic therapy, 972
low-molecular-weight heparin (LMWH), 971-972
oral anticoagulants, 970-971
standard/unfractionated heparin, 971
Spine surgery, 616-617
intraoperative management in, 616-617
monitoring in, 617
positioning in, 616-617
preoperative management in, 616
Spinothalamic tract, 1052, 1055
Spinous processes, 972
Spiral wire-reinforced catheters, 983
Spirometers, 61, 63
designs, 64f—65f
Spirometric loops, 124
Spirometry, 124
Spironolactone, 669
Splanchnic nerve block, 1097
Spondylolisthesis, 1070
Spondylolysis, 1070
Spondyloptosis, 1070
Spondylosis, 1069
Spontaneous ventilation, 39
Sprotte, 977
St. John’s wort, 632
Starling equation, 1307
Starling’s law, 354f
Starvation, 1224
a-stat management, 467
Steal induction, 912
Stellate block, 1096f. See Cervicothoracic block
Stellate ganglion blocks, 1104
Stenotic aortic valve, 421f
Stereotactic surgery, 608-609, 609t
Steroid injection, fluoroscopic image of, 1101f
Stiff person syndrome, 643

Strain gauge pressure transducers, 90
Stroke, acute ischemic, 615-616
Stroke in evolution, 488
Stroke volume (SV), 97, 353-357
afterload in, 355-356
cardiac, 353t
in children, 899
contractility in, 356
preload in, 353-355
diastolic function and ventricular compliance, 355
ventricular filling in, 354-355
valvular dysfunction in, 357
wall motion abnormalities in, 356
Stroke volume variation (SVV), 1191
Strong acid, 1170
Strong ion difference (SID), 1170, 1171, 1171f
ST-segment elevation myocardial infarction (STEMI), 1312
Subarachnoid hemorrhage (SAH), 612
Hunt and Hess grading scale for, 613t
neurological surgeons grading scale for, 613t
Subdural hematoma, 623, 831
Subgluteal sciatic block, 1035f
Subluxation, 1067
Substance abuse, 633-634, 634t
Substance P, 1058-1059
Succinylcholine, 204, 205-211, 683, 775, 1157
acetylcholinesterase inhibitors’ effect on, 224
characteristics of, 212t
chemical structure of, 206f
in children, 907
dosage, 208
drug interactions, 207-208
cholinesterase inhibitors, 207-208
nondepolarizing relaxants, 207-208
metabolism and excretion, 206-207
physical structure, 205-206
side effects and clinical considerations, 208—211
cardiovascular, 209
fasciculations, 209
generalized contractions, 210
histamine release, 211
hyperkalemia, 209-210
intracranial pressure, 210-211
intragastric pressure elevation, 210
intraocular pressure elevation, 210
malignant hyperthermia, 210
masseter muscle rigidity, 210
muscle pains, 210
prolonged paralysis, 210
Succinylcholine-induced hyperkalemia, 209t
Sudden cardiac death (SCD), 409

Sufentanil, 4, 189f, 190t. See also Opioids
Sugammadex, 204, 228-229, 541, 683-684, 789, 908
Sulfobromophthalein (BSP) dye, 164
Sulfur hexafluoride, 776
Superficial cervical plexus, 1041f
Superior hypogastric plexus block
anatomy, 1099
Supine hypotension syndrome, 846
Supraclavicular block
supraclavicular block, 1009f
ultrasound probe placement, 1010f
Supraglottic airway devices (SADs), 316-319
Supraorbital nerve block, 1086f
Suprascapular nerve block, 1090, 1090f
anatomy, 1090
complications, 1090
Supraventricular tachycardias (SVTs), 406, 408
Sural nerve, 1038
Surfactant, 1308
Surgical airway techniques, 331-332
Surgical cricothyrotomy, 331
Surgical stress response, 1117-1121
beta blockers, 1121
intravenous a,-agonist therapy, 1121
lidocaine infusion, intravenous, 1120-1121
minimally invasive surgery, 1117-1118
multimodal interventions, 1114f
regional anesthesia/analgesia techniques, 1118-1120
Suxamethonium. See Succinylcholine
Sympathetic nerve blocks, 1095-1100
Sympathetic tone, in heart failure, 370-371
Sympathetically maintained pain, 1051
Sympathetic-induced vasoconstriction, 363
Syncope, 435t
Syndrome of inappropriate antidiuretic hormone (SIADH), 1145
Syphilitic aneurysms, 484
Syringomyelia, 630
Systemic circulation, 361-371. See also Circulation, systemic
Systemic inflammatory response syndrome (SIRS), 1308, 1316-1317
Systemic vascular resistance index (SVRI), 356

Systemic vascular resistance (SVR), 355-356, 385, 429, 464
Systole, 350
Systolic arterial blood pressure (SBP), 81
Systolic function, 357-360

T lymphocytes, 710
Tachycardia algorithm for, 1282f
atracurium as cause of, 215
perioperative causes of, 1301t
Tacrolimus, 641t
Tapentadol, 1082
Tau protein, 158
Temperature in noncardiovascular monitoring, 132-133
indications and contraindications, 132
techniques and complications, 132
in operating rooms, 15
Tenon's fascia, 781
Tensilon test, 640
Terminal nerve blocks, 1015-1022
digital nerve block, 1020
intravenous regional anesthesia, 1022
median nerve block, 1016-1017
musculocutaneous nerve block, 1018-1019
radial nerve block, 1018
ulnar nerve block, 1017-1018
Testicular tumors, 706—707
Tetany, 204
Tetracaine, 265t, 269t
Tetralogy of Fallot, 431
Therapeutic window, 147
Thermodilution, 101-106
Thermodilution curve, 101, 102f, 104f
Thermoregulation, 1214-1215
Thiamylal, 172f
Thiazide diuretics, 390t
Thiazide/thiazide-like diuretics, 668—669
Thiocyanate, 256
Thiopental, 172f, 174t, 1220, 1249
Third-degree burns, 834
"Third-space" loss, 916
Thoracic bioimpedance, 107
Thoracic epidural blocks, 982
Thoracic paravertebral nerve block, 1091
Thoracic surgery, 553-582. See also specific surgeries bronchoscopy for, 573-575
case discussion on, 580-581
diagnostic thoracic procedures, 573-577
esophageal surgery, 578-579

lung resection, 563-573. See also Lung resection lung transplantation, 577-578
mediastinoscopy, 575-576
one-lung ventilation in, 556-563
anatomic considerations in, 559
double-lumen bronchial tubes in complications of, 562
description of, 558-562
single-lumen tracheal tubes with bronchial blocker in, 562-563
physiologic considerations in anesthesia for, 554-557
lateral decubitus position in, 554f, 554-555, 555f open pneumothorax in, 555—556
tracheal resection, 573. See also Tracheal resection video-assisted thoracoscopic surgery (VATS), 573
Thoracic sympathetic chain block, 1097
Three-limb lead placement, rearranged, 90, 91f
Thrombocytopenia, 867
Thromboelastography (TEG), 451, 725, 827, 828f
Thromboembolism, 948
Thrombomodulin (TM), 824
Thromboxane A, (TXA,), 883
Thyroid disorders hyperthyroidism, 759-760
Thyroid hormone, 759
Thyroid storm, 760
Thyroidectomy, 760
Thyromental distance, 311
Thyrotoxicosis, 647, 760
Thyrotropin-releasing hormone (TRH), 758
Thyroxine (T,), 722, 758
Tibial nerve, 1039f
Tibial/common peroneal nerve courses, 1039f
Tidal volume, 1268
Time-cycled ventilators, 68

Time-weighted average (TWA), 16
Timolol, 777
Tissue Doppler, 362f
Tissue group composition, 140t
Tissue hypoperfusion, 825f
Tissue plasminogen activator (tPA), 615-616
Tonometry, for arterial blood pressuring monitoring, 84
Topiramate, 1078t
Torsades des pointes, 409
Total hip arthroplasty illustration of, 812f
uncemented, 808f
Total intravenous anesthesia (TIVA) technique, 171, 180, 461, 605, 948
Total knee replacement intraoperative management, 813-814
preoperative considerations, 813
Total lung capacity (TLC), 538
Total parenteral nutrition (TPN), 1225-1226
infusions, 1227
Tourniquets, 822
Toxic shock syndrome, 1322t
Toyota Production System (TPS), 28-29
Tracheal lumen, 561f
Tracheal resection, 573, 575f, 576f anesthetic considerations, 573
preoperative considerations, 573
Tracheal tube (TT)
description of, 325f, 1260, 1264
disadvantages of wrapping, 790t
Tracheobronchial tree, 559f
Tracheostomy, 793
Tracheotomy, 1266
Train-of-four stimulation, 134, 204
Tramadol, 1081
Tranexamic acid, 825, 826f
Transcardiopulmonary thermodilution, 105f
Transcranial Doppler (TCD), 459
Transcutaneous cardiac pacing (TCP), 1272
Transcutaneous electrical nerve stimulation (TENS), 1072, 1104-1105
Transdermal fentanyl, 1084
Transesophageal echocardiography (TEE), 96, 96f, 108-109, 357, 404-406, 442,
452f-456f, 607, 708, 741
probes, 1240
Transfusion. See also Blood transfusion blood bank practices, 1199
blood groups, 1197-1198
ABO system, 1197
red blood cell antigen systems, 1198

Rh system, 1198
compatibility testing
ABO-Rh testing, 1198
antibody screen, 1198
crossmatch, 1198
crossmatching, 1198
emergency, 1198-1199
intraoperative, 1199-1200
fresh frozen plasma (FFP), 1199-1200
granulocyte transfusions, 1200
packed red blood cells, 1199
platelet transfusions, 1200
procoagulant transfusions, 1200
Transfusion-associated circulatory overload (TACO), 827, 1202
Transfusion-associated hyperkalemia, 1156
Transfusion-related acute lung injury (TRALD, 827, 1202
Transient ischemic attacks (TIAs), 488, 621
Transient neurological symptoms (TNS), 994
Transient receptor potential vanilloid 1 (TRPV1), 264
Transjugular intrahepatic portosystemic shunt (TIPS), 739, 955
Transmittance oximetry, 120
Transplanted heart anesthetic management, 432-434
patient with, 432-434
preoperative considerations, 432-433
Transpulmonary pressure, 501
Transpulmonary thermodilution, 102-103
Trans-sacral nerve block anatomy, 1093
indications, 1093
technique, 1094
Transthoracic echocardiography (TTE), 108-109, 357, 404
Transtracheal jet ventilation, 1265
Transurethral bladder resection, 705-706
Transurethral resection of the prostate (TURP)
absorption of irrigation fluid, 699
intraoperative considerations, 698—701
manifestations of, 699t preoperative considerations, 698
surgical complications, 699t
Transversus abdominis plane (TAP), 916
Transversus abdominis plane (TAP) block, 1044, 1045f, 1075, 1105

Trauma anesthesia, 820
Trauma/emergency surgery airway, 820-821
breathing, 821-822
case discussion on, 839-841
circulation, 822
definitive trauma interventions anesthetic induction/maintenance, 829-830
damage control surgery, 830
in elderly, 838
head. See Head trauma injury assessment, 823
mass casualty incidents, 838
neurological function, 823
resuscitation hemorrhage, 823-824
hemostatic resuscitation, 826-827
massive transfusion protocols, 827-828
trauma-induced coagulopathy, 824-826
Trauma-induced coagulopathy, 825f
Traumatic brain injury (TBI), 830-833
diffuse neuronal injury, 831
Glasgow Coma Scale for, 610t intracranial pressure, 831
intraparenchymal injuries, 831
primary brain injuries, 831
secondary brain injuries, 831
Trazodone, 1077t
Trendelenburg positions, 696-697
Triamcinolone, 1080t
Tricuspid atresia, 431-432
Tricuspid regurgitation, 424—425
anesthetic management choice of agents, 425
monitoring, 425
objectives, 425
pathophysiology, 425
preoperative considerations, 424—425
treatment, 425
Trifluoroacetic acid, 164
Trigeminal nerve block anatomy, 1085
gasserian ganglion block, 1085, 1086f illustration of, 1086f
indications, 1085
mandibular nerve, 1087f, 1087-1088
maxillary nerve, 1085-1087, 1087f ophthalmic nerve, 1085
Triiodothyronine (T3), 722, 758
Triple response of Lewis, 1059
Trismus. See Masseter muscle rigidity
Tromethamine, 447, 1178, 1181
Troponin, 348
Trousseau sign, 763, 1160
True negatives, 300t
True positives, 300t
Truncus arteriosus, 432

Trunk, peripheral nerve blocks intercostal block, 1040-1041
paravertebral block, 1041-1043
Pecs block, 1043-1044, 1044f superficial cervical plexus block, 1040
transversus abdominis plane (TAP) block, 1044
ultrasound image of, 1045f
Tubeless lithotripsy unit, 701f
Tubuloglomerular balance, 1149
Tubuloglomerular feedback, 662
Tuffier's line, 972
Tumor lysis syndrome, 1160
Tumors, 1070
Tuohy needle, 1042
Two-compartment model, 144, 144f
Tympanoplasty, 798
U
Ulnar artery, blood pressure monitoring via, 87
Ulnar nerve course, 1018f

Ulnar nerve block, 1017-1018
at elbow, 1019f at wrist, 1019f
Ultrafiltration, 445-446
Ultrasonic flow sensors, 63
Ultrasound-guided interscalene block, 1008f
Ultrasound-guided procedures, 1085
Unanticipated hospital admission, following ambulatory surgery, 952
Unidirectional valves, 42-43, 43f
Unstable angina, 395
Upper extremity axillary block, 1012-1015
brachial plexus anatomy, 1004
infraclavicular block, 1010-1012
interscalene block, 1005-1008
peripheral nerve blocks, 1004—1022
supraclavicular block, 1008-1010
surgery of distal upper extremity surgery, 815-816
shoulder surgery, 815
terminal nerves, blocks, 1015-1022
Upper gastrointestinal bleeding, 1325
Upper respiratory tract infection (URI), 909
Urinary diversion, 706

clinical considerations, 133
indications and contraindications, 133
in noncardiovascular monitoring, 133
techniques and complications, 133
U.S. Food and Drug Administration (FDA), 77, 285
Use-dependent block, 264
Uterine activity, 853-854
Uterine atony, 854
Uterine muscle, 853
Uteroplacental circulation, 847, 848f anesthetic agents, placental transfer of, 850-851
placental function, 848-849
physiological anatomy, 848
placental exchange, 849
respiratory gas exchange, 849-850
uterine blood flow, 847-848
uteroplacental blood flow, anesthetic agents effect, 851

Vacuum system, in medical gas systems, 13
Vaginal birth after cesarean (VBAC), 867, 881
Vaginal delivery, general anesthesia for, 873t
Vagus nerve, 500, 794f
Valproic acid, 1078t
Valvular aortic stenosis, 421
Valvular heart disease, 414-427, 415-427
anticoagulation, 426-427
aortic regurgitation, 423-424
aortic stenosis, 421-423
endocarditis prophylaxis, 425-426
general evaluation of patients, 414-415
history of, 414
laboratory evaluation, 415
mitral regurgitation, 417-420
mitral stenosis, 415-417
mitral valve prolapse, 420-421
physical examination, 414-415
special studies, 415
tricuspid regurgitation, 424-425
Vancomycin, 1251
Vaporization, 55
Vaporizers, 55-59
copper kettle, 55-56
electronic, 57, 59
modern conventional, 56—57
Vaporizing chamber, 57
Vapor-phase humidifier, 66
Vascular surgery, 483-490
anesthetic management of, 485-488
kidney failure, 487
paraplegia, 486-487
postoperative considerations, 488
surgery involving aortic arch, 485-486
surgery involving descending thoracic aorta, 486-487
surgery on abdominal aorta, 487-488
surgery on ascending aorta, 485
aorta/aortic lesions, 484—485
occlusive disease of, 484—485
carotid artery surgery, 488-490
Vasoconstriction, 1299
Vasodepressor, 363
Vasodilators, 391t, 472t, 593

Vasopressin, 739, 1275t
Vasopressors, 421, 471t, 593, 1324
Vecuronium, 217-218, 683
metabolism and excretion, 217-218
side effects and clinical considerations cardiovascular, 218
liver failure, 218
Venlafaxine, 1077t
Venous air embolism, 606-608
central venous catheterization, 607
monitoring for, 607-608
treatment of, 608
Ventilation, 512-514, 513t
distribution of, 513f, 513-514
mechanical. See Mechanical ventilation
spontaneous. See Spontaneous ventilation
Ventilation-perfusion (V/Q) scintigraphy, 564
Ventilators, 66-75
alarms, 72
anesthesia ventilators, problems associated with, 74—75
circuit design, 69—72
double-circuit system ventilators, 69-71, 71f
piston ventilators, 71
spill valve, 72
expiratory phase, 68-69
function, 67
inspiratory phase, 67-68
mechanics of, 1344-1345
overview, 67-69
pressure and volume monitoring, 72
transition phase from expiration to inspiration, 68
transition phase from inspiration to expiration, 68
ventilator alarms, 72
ventilator circuit design, 69-72
double-circuit system ventilators, 69-71
Ventricular arrhythmias, 409, 410t—411t
Ventricular compliance, 355f
Ventricular function curves, 357, 357f
Ventricular hypertrophy, 371
Ventricular pressure-volume diagrams, 358F
Ventricular pressure-volume relationships, 369f
Ventricular septal defect (VSD), 430
Ventricular systolic function, 353
Ventricular tachycardia (VT), 399, 1270

Verapamil, 376t, 378t, 1276t
Vernakalant, 378t
Vertebra prominens, 972
Vertebrae, 962
Vertebral column, 963f
Vertebroplasty, 1107
Vessel-rich group, 140
Video laryngoscopes, 321-324
Video-assisted thoracoscopic surgery (VATS), 564, 573
Viral DNA, 1253
Vitamin D, 762, 764, 1159
Vitamin D receptors, 762
Vitamin K, 720, 722
Volatile anesthetics, 55, 159, 540, 590. See also Desflurane; Halothane;
Isoflurane; Sevoflurane cerebral autoregulation, dose-dependent depression of, 590f
cerebral blood flow and volume affected by, 589-590
cerebral metabolic rate affected by, 589
cerebral metabolic rate and blood flow, altered coupling of, 590
cerebrospinal fluid dynamics affected by, 590
intracranial pressure affected by, 590
Volume-controlled ventilation (VCV), 1339
Volume-cycled ventilators, 68, 1339
Volume-—pressure loop, 125f von Willebrand factor (VWF), 734

Waste anesthetic gas disposal (WAGD), 13, 16
Waste-gas scavengers, 75—77
Weak base, 1170
Wenger chestpiece. See Precordial stethoscope
West zones, 515
Wick humidifier, 66
Wide dynamic range (WDR) neurons, 1054
Widened pulse pressures, 253
Widespread Pain Index (WPI), 1066
Wind-up, 1055
Withdrawal of treatment, 1326
Wolff—Parkinson—White (WPW) syndrome, 373-374, 408
Wong-Baker FACES rating scale, 1063
Wood's metal, 11
World Health Organization (WHO), 10
Wound-related sepsis, 1322
Wright respirometer, 61, 64f

advantages and disadvantages of, 161t anti-apoptotic effect of, 158
clinical pharmacology of, 169

Ziconotide, 1075
Zollinger—Ellison syndrome, 278
Zygapophyseal joints, 1070

M.Sc., Ph.D., F.I.C., F.A.C.B.
Professor of Biochemistry & Director (Research)
Dr. Pinnamaneni Siddhartha Institute Medical Sciences of (Dr. NTR University of Health Sciences) Chinaoutpalli, Gannavaram (Mdl) Krishna (Dist), A.P., India
Dr. U. Chakrapani
M.B.B.S., M.S., D.N.B.
Books & Allied Pvt. Ltd.

A division of
Reed Elsevier India Private Limited
Mosby, Saunders, Churchill Livingstone, Butterworth-Heinemann and Hanley & Belfus are the Health Science imprints of Elsevier.
© 2013 Dr. U, Satyanarayana
First Published: March 1999
Revised Reprint: August 2000
Second Revised Edition: June 2002
Revised Reprint: 2004, 2005
Third Revised Edition (multicolour): 2006
Revised Reprint: 2007, 2010
Fourth Revised Edition: 2013
All rights are reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise without the prior permission of the publishers.

Medical knowledge is constantly changing. As new information becomes available, changes in treatment, procedures, equipment and the use of drugs become necessary. The author, editors, contributors and the publisher have, as far as it is possible, taken care to ensure that the information given in this text is accurate and up-to-date. However, readers are strongly advised to confirm that the information, especially with regard to drug dose/usage, complies with current legislation and standards of practice. Please consult full prescribing information before issuing prescriptions for any product mentioned in this publication.
This edition of Biochemistry, 4e by Dr. U. Satyanarayana and Dr. U. Chakrapani is co-published by an arrangement with Elsevier, a division of Reed Elsevier India Private Limited and Books and Allied (P) Ltd.

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This book 'Biochemistry' has undoubtedly become one of the most preferred text books (in India and many other countries) by the students as well as teachers in medical, biological and other allied sciences. It is certainly a book of choice and a true companion to all learning biochemistry, hence appropriately regarded by many as 'Bible of Biochemistry'. This book has undergone three editions, several reprints, and revised reprints in a span of 13 years.
The advances in biochemistry are evergrowing due to exponential growth of the subject. Further, the critical comments, frank opinions and constructive suggestions by teachers and students need to be seriously considered. All this necessitates frequent revision of the book.
In this fourth edition, a thorough revision and update of each chapter with latest advances has been done. The main emphasis of this edition is an improved orientation and treatment of human biochemistry in health and disease. A wide variety of case studies with relevant biochemical profiles (along with diagnosis and discussion) are newly added as an appendix. In addition, several newer aspects of biochemistry are covered in this edition, some of them are listed below.
e Triacylgylcerol/fatty acid cycle
e w-fatty acid
e@ Metabolic syndrome
@ Glucose toxicity
e Estimated average glucose
e Peptide nucleic acids
e@ Pseudogenes
e Recombinant ribozymes
e Epigenetic regulation of gene expression
e@ Metagenomics
e@ Therapeutic diets
e Atkins diet
e Dietary antioxidants
e Soluble and insoluble fiber
e
Trans fatty acids
- e Nutrigenomics
e
Detailed information on antivitamins
e
Dental caries
- e Amino acids as neurotransmitters
e
Disorders of membrane transport
Diagnostic importance of various body fluids and tissues
e e Enzyme patterns in diseases
e Cystatin C
e Pleural fluid
e High fructose corn syrups
e High sensitive CRP

It is a fact that I represent a selected group of individuals authoring books, having some time at disposal, besides hard work, determination and dedication. I consider myself as an eternal reader and a regular student of biochemistry. However, it is beyond my capability to keep track of the evergrowing advances in biochemistry due to exponential growth of the subject. And, this makes me nervous whenever I think of revising the book. I honestly and frankly admit that I have to depend on mature readers for subsequent editions of this book.

I have to admit that it is not all the time possible for me to meet the readers individually and get their feedback. I sincerely invite the readers, my well wishers and experts in biochemistry subject to feel free and write to me (Email ID: uppalasatya@yahoo.com) expressing their frank opinions, critical comments and constructive suggestions. And this will help me to further improve the book in subsequent revisions.

I owe a deep debt of gratitude to my parents, the late Sri U. Venkata Subbaiah, and Smt. Vajramma, for cultivating in me the habit of early rising. The writing of this book would never have been possible without this healthy habit. I am grateful to Dr. B. S. Narasinga Rao (former Director, National Institute of Nutrition, Hyderabad) for disciplining my professional life, and to my eldest brother Dr. U. Gudaru (former Professor of Power Systems, Walchand College of Engineering, Sangli) for disciplining my personal life.
My elder son, U. Chakrapani (MBBS) deserves a special place in this book. He made a significant contribution at every stage of its preparation—writing, verification, proof-reading and what not. I had the rare privilege of teaching my son as he happened to be a student of our college. And a major part of this book was written while he was learning biochemistry. Thus, he was the first person to learn the subject of biochemistry from my handwritten manuscript. The student-teacher relation (rather than the father-son) has helped me in receiving constant feedback from him and restructure the book in a way an undergraduate student would expect a biochemistry textbook to be.
Next, I thank Dr. G. Pitcheswara Rao (former Professor of Anatomy, SMC, Vijayawada) for his constructive criticism and advice, and Dr. B. Sivakumar (Director, National Institute of Nutrition, Hyderabad) for his helpful suggestions on the microfigures.
Last but not least, I thank my wife Krishna Kumari and my younger son, Amrutpani, without whose cooperation and encouragement this book could never have been written. The manuscript was carefully nurtured like a new born baby and the book has now become a full-pledged member of our family.

I am grateful to a large number of faculty members, students, friends and pen friends who directly or indirectly helped me to revise and improve the content and quality of the book. I have individually and personally thanked all of them (who number a few hundreds!). I once again express my gratitude to them.
I thank Dr (Mrs) U.B. Vijaya Lakshmi, MD, Associate Professor of Biochemistry at our college who participated to comprehensively prepare case studies with biochemical correlations, besides improving the biomedical/ clinical aspects in some chapters. My special thanks goes to one student, and an ardent fan of my books, Mr. Y. Nagendra Sastry (Ph.D), who has been studying my books regularly for over 7-8 years. His constant feedback and suggestions have certainly contributed to improve this book. I express my gratitude to Mr. M.S.T. Jagan Mohan (my former colleague), who has helped me with his frequent interactions to revise the book, and make it more student-friendly.
I express my sincere thanks to Mr Arunabha Sen, Director, Books & Allied (P) Ltd, Kolkata for his whole hearted support and constant encouragement in revising the book, and taking all pains to bring it out to my satisfaction. I thank Mr. Shyamal Bhattacharya for his excellent page making and graphics-work in the book. I am grateful to Mr. Abhijit Ghosal for his help in the cover design.
I thank my wife, Krishna Kumari, my younger son Amrut Pani and my daughter-in law Oohasri for their constant support and encouragement. My special thanks to my grand daughter Maahe (2 years) whose ever smiling face, sweet words and deeds infuse energy into my academic activities. I am grateful to Uppala Author-Publisher interlinks, Vijayawada for sponsoring and supporting me to bring out this edition.
Dr. U. SATYANARAYANA

The term Biochemistry was introduced by Carl Neuberg in 1903. Biochemistry broadly deals with the chemistry of life and living processes. There is no exaggeration in the statement, 'The scope of biochemistry is as vast as life itself !' Every aspect of life-birth, growth, reproduction, aging and death, involves biochemistry. For that matter, every movement of life is packed with hundreds of biochemical reactions. Biochemistry is the most rapidly developing and most innovative subject in medicine. This becomes evident from the fact that over the years, the major share of Nobel Prizes earmarked for Medicine and Physiology has gone to researchers engaged in biochemistry.
The discipline of biochemistry serves as a torch light to trace the intricate complexicities of biology, besides unravelling the chemical mysteries of life. Biochemical research has amply demonstrated that all living things are closely related at the molecular level. Thus biochemistry is the subject of unity in the diversified living kingdom.
Advances in biochemistry have tremendous impact on human welfare, and have largely benefited mankind and their living styles. These include the application of biochemistry in the laboratory for the diagnosis of diseases, the products (insulin, interferon, growth hormone etc.) obtained from genetic engineering, and the possible use of gene therapy in the near future.

This textbook, comprising 43 chapters, is organized into seven sections in the heirarchical order of learning biochemistry.
e Section I deals with the chemical constituents of life—carbohydrates, lipids, proteins and amino acids, nucleic acids and enzymes.
e Section II physiological chemistry includes digestion and absorption, plasma proteins, hemoglobin and prophyrins, and biological oxidation.
e Section III incorporates all the metabolisms (carbohydrates, lipids, amino acids, nucleotides, minerals)
e Section IV covers hormones, organ function tests, water, electrolyte and acid-base balance, tissue proteins and body fluids, and nutrition.
e Section V is exclusively devoted to molecular biology and biotechnology (DNA-replication, recombination, and repair, transcription and translation, regulation of gene expression, recombinant DNA and biotechnology)
e Section VI gives relevant information on current topics such as human genome project, gene therapy, bioinformatics, prostaglandins, diabetes, cancer, AIDS etc.
e Section VII deals with the basic aspects for learning and understanding biochemistry (bioorganic chemistry, biophysical chemistry, tools of biochemistry, genetics, immunology).
Each chapter in this book is carefully crafted with colour illustrations, headings and subheadings to facilitate quick understanding. The important applications of biochemistry to human health and disease are put together as biomedical/clinical concepts. Icons are used at appropriate places to serve as 'landmarks'.
The origins of biochemical words, confusables in biochemistry, practical biochemistry and clinical biochemistry laboratory, case studies with biochemical correlations, given in the appendix are novel features. The book is so organized as to equip the readers with a comprehensive knowledge of biochemistry.

16, 1 = ➤. 16, 2 = Integration of metabolism. 16, 3 = 380. 17, 1 = ➤. 17, 2 = Metabolism of nucleotides. 17, 3 = 387. 18, 1 = ➤. 18, 2 = Mineral metabolism. 18, 3 = 403. SECTION FOUR, 1 = SECTION FOUR. SECTION FOUR, 2 = SECTION FOUR. SECTION FOUR, 3 = . Clinical Biochemistry and Nutrition, 1 = Clinical Biochemistry and Nutrition. Clinical Biochemistry and Nutrition, 2 = Clinical Biochemistry and Nutrition. Clinical Biochemistry and Nutrition, 3 = Clinical Biochemistry and Nutrition. 19, 1 = ➤. 19, 2 = Hormones. 19, 3 = 427. 20, 1 = ➤. 20, 2 = Organ function tests. 20, 3 = 453. 21, 1 = ➤. 21, 2 = Water, electrolyte and. 21, 3 = . acid-base balance, 1 = acid-base balance. acid-base balance, 2 = acid-base balance. acid-base balance, 3 = 468. 22, 1 = ➤. 22, 2 = Tissue proteins and body fluids. 22, 3 = 487. 23, 1 = ➤. 23, 2 = Nutrition. 23, 3 = 502
__

Molecular Biology and Biotechnology
24
>» DNA-eplication, recombination and repair
25
26
>»
Transcription and translation
>»
Regulation of gene expression
27
>
Recombinant DNA and biotechnology

28
>
Human genome project
29
30
31
32
33
34
35
36
37
38
>
>
>
»
Gene therapy
Bioinformatics
Metabolism of xenobiotics (detoxification)
Prostaglandins and related compounds
>»
Biological membranes and transport
» Free radicals and antioxidants
>»
Environmental biochemistry
>
>
>
Insulin, glucose homeostasis, and diabetes mellitus
Cancer
Acquired immunodeficiency syndrome (AIDS)

Basics to Learn Biochemistry
39
»
Introduction to
bioorganic chemistry
40
41
42
>
>
>
Overview of biophysical chemistry
Tools of biochemistry
Immunology
43
>
Genetics

Answers to Self-assessment Exercises
|
Abbreviations used in this book
I
Ill
IV
V_
Origins of important biochemical words
Common confusables in biochemistry
Practical biochemistry—principles
Clinical biochemistry laboratory
VI
Case studies with biochemical correlations
INDEX
523
542
566
578
619
625
634
638
644
650
655
662
669
685
695
703
708
719
732
737
745
751
756
759
763
769
772
779

Chapter
he living matter is composed of mainly six elements—carbon, hydrogen, oxygen, nitrogen, phosphorus and sulfur. These elements together constitute about 90% of the dry weight of the human body. Several other functionally important elements are also found in the cells. These include Ca, K, Na, Cl, Mg, Fe, Cu, Co, I, Zn, F, Mo and Se.

Carbon is the most predominant and versatile element of life. It possesses a unique property to form infinite number of compounds. This is attributed to the ability of carbon to form stable covalent bonds and C—C chains of unlimited length. It is estimated that about 90% of compounds found in living system invariably contain carbon.

Life is composed of lifeless chemical molecules. A single cell of the bacterium, Escherichia coli contains about 6,000 different organic compounds. It is believed that man may contain about 100,000 different types of molecules although only a few of them have been characterized.

The organic compounds such as amino acids, nucleotides and monosaccharides serve as the monomeric units or building blocks of complex biomolecules—proteins, nucleic acids (DNA and RNA) and_ polysaccharides, respectively. The important biomolecules (macromolecules) with their respective building blocks and major functions are given in Table 1.1. As regards lipids, it may be noted that they are not biopolymers in a strict sense, but majority of them contain fatty acids.

The macromolecules (proteins, lipids, nucleic acids and polysaccharides) form supramolecular assemblies (e.g. membranes) which in turn organize into organelles, cells, tissues, organs and finally the whole organism.

1. Protein, Building block (repeating unit) = Amino acids. 1. Protein, Major functions = Fundamental basis of structure and function of cell (static and dynamic functions).. 2. Deoxyribonucleic acid (DNA), Building block (repeating unit) = Deoxyribonucleotides. 2. Deoxyribonucleic acid (DNA), Major functions = Repository of hereditary information.. 3. Ribonucleic acid (RNA), Building block (repeating unit) = Ribonucleotides. 3. Ribonucleic acid (RNA), Major functions = Essentially required for protein biosynthesis.. 4. Polysaccharide (glycogen), Building block (repeating unit) = Monosaccharides (glucose). 4. Polysaccharide (glycogen), Major functions = Storage form of energy to meet short term demands.. 5. Lipid, Building block (repeating unit) = Fatty acids, glycerol. 5. Lipid, Major functions = Storage form of energy to meet long term demands; structural components of membranes.

The chemical composition of a normal man, weighing 65 kg, is given in Table 1.2. Water is the solvent of life and contributes to more than 60% of the weight. This is followed by protein (mostly in muscle) and lipid (mostly in adipose tissue). The carbohydrate content is rather low which is in the form of glycogen.

The cell is the structural and functional unit of life. It may be also regarded as the basic unit of biological activity.
The concept of cell originated from the contributions of Schleiden and Schwann (1838). However, it was only after 1940, the complexities of cell structure were exposed.

Water, Percent (%) = 61.6. Water, Weight (kg) = 40. Protein, Percent (%) = 17.0. Protein, Weight (kg) = 11. Lipid, Percent (%) = 13.8. Lipid, Weight (kg) = 9. Carbohydrate, Percent (%) = 1.5. Carbohydrate, Weight (kg) = 1. Minerals, Percent (%) = 6.1. Minerals, Weight (kg) = 4
The cells of the living kingdom be categories
may divided into two
1. Prokaryotes (Greek : pro — before; karyon — nucleus) lack a well defined nucleus and possess relatively simple structure. These include the various bacteria.
2. Eukaryotes (Greek : eu-—true; karyon — nucleus) possess a well defined nucleus and are more complex in their structure and function. The higher organisms (animals and plants) are composed of eukaryotic cells.
A comparison of the characteristics between and eukaryotes is listed in Table 1.3.
prokaryotes

The human body is composed of about 10!4 cells. There are about 250 types of specialized cells in the human body e.g. erythrocytes, nerve cells, muscle cells, B cells of pancreas. An eukaryotic cell is generally 10 to 100 um in diameter. A diagrammatic representation of a typical rat liver cell is depicted in Fig.1.1.
The plant cell differs from an animal cell by possessing
a
rigid cell
wall
(mostly composed
of cellulose)
and chloroplasts.
sites of photosynthesis.
The latter
are the

Nucleus is the largest cellular organelle, surrounded by a double membrane nuclear envelope. The outer membrane is continuous with the membranes of endoplasmic reticulum. At certain intervals, the two nuclear membranes have nuclear pores with a diameter of about 90 nm. These pores permit the free passage of the products synthesized in the nucleus into the surrounding cytoplasm.
Fig. 1.1 : Diagrammatic representation of a rat liver cell.
Nucleus contains DNA, the repository of genetic information. Eukaryotic DNA is associated with basic protein (histones) in the ratio of 1 : 1, to form nucleosomes. An assembly of nucleosomes constitutes chromatin fibres of chromosomes (Greek: chroma — colour; soma — body). Thus, a single human chromosome is composed of about a million nucleosomes. The number of chromosomes is a_ characteristic feature of the species. Humans have 46 chromosomes, compactly packed in the nucleus. The nucleus of the eukaryotic cell contains a dense body known as nucleolus. It is rich in RNA, particularly the ribosomal RNA which enters the cytosol through nuclear pores.
The ground material of the nucleus is often referred to as nucleoplasm. It is rich in enzymes such as DNA polymerases and RNA polymerases.
Hutchinson-Gilford progeria__ syndrome (HGPS) is a rare condition of aging beginning at birth (incidence | in 5 million births). HGPS occurs as a result of distortion of nuclear envelope due to accumulation of abnormal protein namely lamina A.

The mitochondria (Greek: mitos — thread; chondros — granule) are the centres for the cellular respiration and energy metabolism. They are regarded as the power houses of the cell with variable size and shape. Mitochondria are rod-like or filamentous bodies, usually with dimensions of 1.0 x 3 um. About 2,000 mitochondria, occupying about '/;th of the total cell volume, are present in a typical cell.
The mitochondria are composed of a double membrane system (Refer Fig.11.5). The outer membrane is smooth and completely envelops the organelle. The inner membrane is folded to form cristae (Latin—crests) which occupy a larger surface area. The internal chamber of mitochondria is referred to as matrix or mitosol.
The components of electron transport chain and oxidative phosphorylation (flavoprotein, cytochromes b, cy, c, a and a3 and coupling factors) are buried in the inner mitochondrial membrane. The matrix contains several enzymes concerned with the energy metabolism of carbohydrates, lipids and amino acids (e.g., citric acid cycle, B-oxidation). The matrix enzymes also participate in the synthesis of heme and urea. Mitochondria are the principal producers of ATP in the aerobic cells. ATP, the energy currency, generated in mitochondria is exported to all parts of the cell to provide energy for the cellular work.
The mitochondrial matrix contains a circular double stranded DNA (mtDNA), RNA = and ribosomes. Thus, the mitochondria are equipped with an independent protein synthesizing machinery. It is estimated that about 10% of the mitochondrial proteins are produced in the mitochondria.
The structure and functions of mitochondria closely resemble prokaryotic cells. \t is hypothesized that mitochondria have evolved from aerobic bacteria. Further, it is believed that during evolution, the aerobic bacteria developed a symbiotic relationship with — primordial anaerobic eukaryotic cells that ultimately led to the arrival of aerobic eukaryotes.

The network of membrane enclosed spaces that extends throughout the cytoplasm constitutes endoplasmic reticulum (ER). Some of these thread-like structures extend from the nuclear pores to the plasma membrane.
A large portion of the ER is studded with ribosomes to give a granular appearance which is referred to as rough endoplasmic reticulum. Ribosomes are the factories of protein biosynthesis. During the process of cell fractionation, rough ER is disrupted to form small vesicles known as microsomes. It may be noted that microsomes as such do not occur in the cell.
The smooth endoplasmic reticulum does not contain ribosomes. It is involved in the synthesis of lipids (triacylglycerols, phospholipids, sterols) and metabolism of drugs, besides supplying Ca? for the cellular functions.

Eukaryotic cells contain a unique cluster of membrane vesicles known as_ dictyosomes
which, in turn, constitute Golgi apparatus (or Golgi complex). The newly synthesized proteins are handed over to the Golgi apparatus which catalyse the addition of carbohydrates, lipids or sulfate moieties to the proteins. These chemical modifications are necessary for the transport of proteins across the plasma membrane.
Certain proteins and enzymes are enclosed in membrane vesicles of Golgi apparatus and secreted from the cell after the appropriate signals. The digestive enzymes of pancreas are produced in this fashion.
Golgi apparatus are also involved in the membrane _ synthesis, particularly for the formation of intracellular organelles (e.g. peroxisomes, lysosomes).

Lysosomes are spherical vesicles enveloped by a single membrane. Lysosomes are regarded as the digestive tract of the cell, since they are actively involved in digestion of cellular substances—namely proteins, lipids, carbohydrates and nucleic acids. Lysosomal enzymes are categorized as hydrolases. These include the enzymes (with substrate in brackets)— a-glucosidase (glycogen), cathepsins (proteins), lipases (lipids), ribonucleases (RNA).
by diffusion, for reutilization by the cell. Sometimes, however, certain residual products, rich in lipids and proteins, collectively known as lipofuscin accumulate in the cell. Lipofuscin is the age pigment or wear and tear pigment which has been implicated in ageing process. As the cell dies, the lysosomes rupture and release hydrolytic enzymes that results in post-morteum autolysis.
The digestive enzymes of cellular compounds are confined to the lysosomes in the best interest of the cell. Escape of these enzymes into cytosol will destroy the functional macromolecules of the cell and result in many complications. The occurrence of several diseases (e.g. arthritis, muscle diseases, allergic disorders) has been partly attributed to the release of lysosomal enzymes.
Inclusion cell (I-cell) desease is a_ rare condition due to the absence of certain hydrolases in lysosomes. However, these enzyme are syntherized and found in the circulation. I-cell disease is due to a defect in protein targetting, as the enzymes cannot reach lysosomes.

The lysosomal enzymes are responsible for maintaining the cellular compounds in a dynamic state, by their degradation and recycling. The degraded products leave the lysosomes, usually
Peroxisomes, also known as microbodies, are single membrane cellular organelles. They are spherical or oval in shape and contain the enzyme catalase. Catalase protects the cell from the toxic effects of H,O, by converting it to HO and O;. Peroxisomes are also involved in the oxidation of long chain fatty acids (> Cis), and synthesis of plasmalogens and glycolipids. Plants contain glyoxysomes, a specialized type of

a functions. A living cell is a true representative of life with its own organization and specialized
+ Accumulation of lipofuscin, a pigment rich in lipids and proteins, in the cell has been implicated in ageing process.
+ Leakage of lysosomal enzymes into the cell degrades several functional macromolecules
and this may lead to certain disorders (e.g. arthritis).
+ Zellweger syndrome is a rare disease characterized by the absence of functional peroxisomes.
peroxisomes,
which
are
involved
in
the
glyoxylate
pathway.
Peroxisome biogenesis disorders (PBDs), are a group of rare diseases involving the enzyme activities of peroxisomes. The biochemical abnormalities associated with PBDs include increased levels of very long chain fatty acids (Cy4 and Cy¢) and decreased concentrations of plasmalogens. The most severe form of PBDs is Zellweger syndrome, a condition characterized by the absence of functional peroxisomes. The victims of this disease may die within one year after birth.

The cellular matrix is collectively referred to as cytosol. Cytosol is basically a compartment containing several enzymes, metabolites and salts in an aqueous gel like medium. More recent studies however, indicate that the cytoplasm actually contains a complex network of protein filaments, spread throughout, that constitutes cytoskeleton. The cytoplasmic filaments are of three types — microtubules, actin filaments and intermediate filaments. The filaments which are polymers of proteins are responsible for the structure, shape and organization of the cell.

The eukaryotic cells perform a wide range of complex reactions/functions to maintain tissues, and for the ultimate well-being of the whole organism. For this purpose, the various intracellular processes and biochemical reactions are tightly controlled and integrated. Division of a cell into two daughter cells is good example of the orderly occurrence of an integrated series of cellular reactions.
Apoptosis is the programmed cell death or cell suicide. This occurs when the cell has fulfilled its biological functions. Apoptosis may be regarded as a natural cell death and it differs from the cell death caused by injury due to radiation, anoxia etc. Programmed cell death is a highly regulated process.

. Life is composed of lifeless chemical molecules. The complex biomolecules, proteins, nucleic acids (DNA and RNA), polysaccharides and lipids are formed by the monomeric units amino acids, nucleotides, monosaccharides and fatty acids, respectively.
. The cell is the structural and functional unit of life. The eukaryotic cell consists of well defined subcellular organelles, enveloped in a plasma membrane.
. The nucleus contains DNA, the repository of genetic information. DNA, in association with proteins (histones), forms nucleosomes which, in turn, make up the chromosomes.
. The mitochondria are the centres for energy metabolism. They are the principal producers of ATP which is exported to all parts of the cell to provide energy for cellular work.
. Endoplasmic reticulum (ER) is the network of membrane enclosed spaces that extends throughout the cytoplasm. ER studded with ribosomes, the factories of protein biosynthesis, is referred to as rough ER. Golgi apparatus are a cluster of membrane vesicles to which the newly synthesized proteins are handed over for further processing and export.
. Lysosomes are the digestive bodies of the cell, actively involved in the degradation of cellular compounds. Peroxisomes contain the enzyme catalase that protects the cell from the toxic effects of H,O,. The cellular ground matrix is referred to as cytosol which, in fact, is composed of a network of protein filaments, the cytoskeleton.
.
The
eukaryotic
cells
perform
a
wide
range
of
complex
functions
in
a
well
coordinated
and
integrated
fashion.
Apoptosis
is
the
process
of
programmed
cell
death
or
cell
suicide.

arbohydrates are the most abundant organic eer in nature. They are primarily composed of the elements carbon, hydrogen and oxygen. The name carbohydrate literally means 'hydrates of carbon'. Some of the carbohydrates possess the empirical formula (C.H2O), where n <3, satisfying that these carbohydrates are in fact carbon hydrates. However, there are several non-carbohydrate compounds (e.g. acetic acid, CyH4Oo; lactic acid, C3HgO3) which also appear as hydrates of carbon. Further, some of the genuine carbohydrates (e.g. rhamnohexose, CgH 120s; deoxyribose, C5;H;gO,4) do not satisfy the general formula. Hence carbohydrates cannot be always considered as hydrates of carbon.
Carbohydrates may be defined as polyhydroxyaldehydes or ketones or compounds which produce them on hydrolysis. The term 'sugar' is applied to carbohydrates soluble in water and sweet to taste.

Carbohydrates participate in a wide range of functions
- 1. They are the most abundant dietary source
of energy (4 Cal/g) for all organisms.
- 2. Carbohydrates are precursors for many
organic compounds (fats, amino acids).
3. Carbohydrates (as glycoproteins and glycolipids) participate in the structure of cell membrane and cellular functions such as cell growth, adhesion and fertilization.
4.
They are
structural components
of many
organisms.
These include
plants, the
exoskeleton of
fiber
(cellulose)
some insects
of and
the cell
wall of
microorganisms.
5. Carbohydrates also serve as the storage form of energy (glycogen) to meet the immediate demands of the energy body.

Carbohydrates are often referred to as saccharides (Greek: sakcharon-sugar). They are broadly classified into three major groups— monosaccharides, oligosaccharides and_poly-saccharides. This categorization is based on the
Trioses (C 3 H 6 O 3 ), T ABLE 2.1 Classification of monosaccharides with selected examples.Aldose = Glyceraldehyde. Trioses (C 3 H 6 O 3 ), T ABLE 2.1 Classification of monosaccharides with selected examples.Ketose = Dihydroxyacetone. Tetroses (C 4 H 8 O 4 ), T ABLE 2.1 Classification of monosaccharides with selected examples.Aldose = Erythrose. Tetroses (C 4 H 8 O 4 ), T ABLE 2.1 Classification of monosaccharides with selected examples.Ketose = Erythrulose. Pentoses (C 5 H 10 O 5 ), T ABLE 2.1 Classification of monosaccharides with selected examples.Aldose = Ribose. Pentoses (C 5 H 10 O 5 ), T ABLE 2.1 Classification of monosaccharides with selected examples.Ketose = Ribulose. Hexoses (C 6 H 12 O 6 ), T ABLE 2.1 Classification of monosaccharides with selected examples.Aldose = Glucose. Hexoses (C 6 H 12 O 6 ), T ABLE 2.1 Classification of monosaccharides with selected examples.Ketose = Fructose. Heptoses (C 7 H 14 O 7 ), T ABLE 2.1 Classification of monosaccharides with selected examples.Aldose = Glucoheptose. Heptoses (C 7 H 14 O 7 ), T ABLE 2.1 Classification of monosaccharides with selected examples.Ketose = Sedoheptulose
number of sugar units. Mono-and_ oligosaccharides are sweet to taste, crystalline in character and soluble in water, hence they are commonly known as sugars.

liberated on hydrolysis. Based on the number of monosaccharide units present, the oligosaccharides are further subdivided to disaccharides, trisaccharides etc.

Monosaccharides (Greek : mono-one) are the simplest group of carbohydrates and are often referred to as simple sugars. They have the general formula C,(H O),, and they cannot be further hydrolysed. The monosaccharides are divided into different categories, based on the functional group and the number of carbon atoms
Aldoses : When the functional group in
H
|
monosaccharides is
an aldehyde
Lio)
they are known as aldoses e.g. glyceraldehyde, glucose.
Ketoses : When the functional group is a keto
|
group,
Cd
=o}
they are
as ketoses
e.g. dihydroxyacetone, fructose.
Based on the number of carbon atoms, the monosaccharides are regarded as trioses (3C), tetroses (4C), pentoses (5C), hexoses (6C) and heptoses (7C). These terms along with functional groups are used while naming monosaccharides. For instance, glucose is an aldohexose while fructose is a ketohexose (Table 2.1).
The common monosaccharides and disaccharides of biological importance are given in the Table 2.2.

Oligosaccharides (Greek: oligo-few) contain 2-10 monosaccharide molecules which are

Polysaccharides (Greek: poly-many) are polymers of monosaccharide units with high molecular weight (up to a million). They are usually tasteless (non-sugars) and form colloids with water. The polysaccharides are of two types — homopolysaccharides and heteropolysaccharides.

Stereoisomerism is an important character of monosaccharides. | Stereoisomers are the compounds that have the same structural formulae but differ in their spatial configuration.
A carbon is said to be asymmetric when it is attached to four different atoms or groups. The number of asymmetric carbon atoms (n) determines the possible isomers of a given compound which is equal to 2". Glucose contains 4 asymmetric carbons, and thus has 16 isomers.

Glyceraldehyde (triose) is the simplest monosaccharide with one asymmetric carbon atom. It exists as two stereoisomers and has been chosen as the reference carbohydrate to represent the structure of all other carbohydrates.

2.2 Monosaccharides and disaccharides of biological importance.Biochemical importance = Biochemical importance. Sucrose, T ABLE 2.2 Monosaccharides and disaccharides of biological importance.Occurrence = As a constituent of cane sugar and beet sugar, pineapple. Sucrose, T ABLE 2.2 Monosaccharides and disaccharides of biological importance.Biochemical importance = Most commonly used table sugar supplying calories. Lactose, T ABLE 2.2 Monosaccharides and disaccharides of biological importance.Occurrence = Milk sugar. Lactose, T ABLE 2.2 Monosaccharides and disaccharides of biological importance.Biochemical importance = Exclusive carbohydrate source to breast fed infants. Lactase deficiency (lactose intolerance) leads to diarrhea and flatulence. Maltose, T ABLE 2.2 Monosaccharides and disaccharides of biological importance.Occurrence = Product of starch hydrolysis, occurs in germinating seeds. Maltose, T ABLE 2.2 Monosaccharides and disaccharides of biological importance.Biochemical importance = An important intermediate in the digestion of starch
Fig. 2.1 : D-and-Lforms of glucose compared with
Dand Lglyceraldehydes (the reference carbohydrate).

The D and L isomers are mirror images of each other. The spatial orientation of —H and —OH groups on the carbon atom (Cs for glucose) that is adjacent to the terminal primary alcohol carbon determines whether the sugar is Dor L-isomer. If the —OH group is on the right side, the sugar is of D-series, and if on the left side, it belongs to L-series. The structures of Dand L-glucose based on the reference monosaccharide, Dand L-glyceraldehyde (glycerose) are depicted in Fig.2.1.
It may be noted that the naturally occurring monosaccharides in the mammalian tissues are mostly of D-configuration. The enzyme machinery of cells is specific to metabolise D-series of monosaccharides.

Optical activity is a characteristic feature of compounds with asymmetric carbon atom. When a beam of polarized light is passed through a solution of an optical isomer, it will be rotated either to the right or left. The term dextrorotatory (d+) and levorotatory (|-) are used to compounds that respectively rotate the plane of polarized light to the right or to the left. An optical isomer may be designated as
D(+), DO), L(+) and L-) based on its structural relation with glyceraldehyde. It may be noted that the Dand L-configurations of sugars are primarily based on the © structure of glyceraldehyde, the optical activities however, may be different.
Racemic mixture : If dand /-isomers are present in equal concentration, it is known as racemic mixture or dl mixture. Racemic mixture does not exhibit any optical activity, since the dextroand levorotatory activities cancel each other.
In the medical practice, the term dextrose is used for glucose in solution. This is because of the dextrorotatory nature of glucose.

The configuration of possible D-aldoses starting from D-glyceraldehyde is depicted in Fig.2.2. This is a representation of KillianiFischer synthesis, by increasing the chain length of an aldose, by one carbon at a time. Thus, starting with an aldotriose (3C), aldotetroses (4C), aldopentoses (5C) and aldohexoses (6C) are formed. Of the 8 aldohexoses, glucose, mannose and galactose are the most familiar. Among these, D-glucose is the only aldose monosaccharide that predominantly occurs in nature.

Starting from dihydroxyacetone (triose), there are five keto-sugars which are physiologically important. Their structures are given in Fig.2.3.

If two monosaccharides differ from each other in their configuration around a single specific carbon (other than anomeric) atom, they are referred to as epimers to each other (Fig.2.4). For instance, glucose and galactose are epimers with regard to carbon 4 (C4-epimers). That is, they differ in the arrangement of —OH group at C4. Glucose and mannose are epimers with regard to carbon 2 (Cy-epimers).
The interconversion of epimers (e.g. glucose to galactose and vice versa) is known as
Fig. 2.2 : The structural relationship between D-aldoses shown in Fischer projection. (The configuration around C, (red) distinguishes the members of each pair).
epimerization, and
a
group of
enzymes—
namely—epimerases
The term diastereomers is used to represent the stereoisomers that are not mirror images of one another.

Enantiomers stereoisomers
each other.
are that
The two
catalyse this
reaction.
a_
special are
type mirror
members are
D- and
L-sugars.
Enantiomers of
images designated
glucose of
of as
are

Majority of the sugars in the higher animals
(including man) are of D-type (Fig.2.5).

For a_ better understanding of glucose structure, let us consider the formation of hemiacetals and hemiketals, —_ respectively produced when an aldehyde or a ketone reacts with alcohol.
Fig. 2.3 : Structures of ketoses of physiological importance.

The hydroxyl group of monosaccharides can react with its own aldehyde or keto functional group to form hemiacetal and hemiketal. Thus, the aldehyde group of glucose at C; reacts with alcohol group at Cs to form two types of cyclic hemiacetals namely a and £, as depicted in Fig.2.6. The configuration of glucose is conveniently represented either by Fischer formulae or by Haworth projection formulae.

Haworth projection formulae are depicted by a six-membered ring pyranose (based on pyran) or a five-membered ring furanose (based on furan). The cyclic forms of glucose are known as a-D-glucopyranose and a-D-glucofuranose (Fig.2.7).
Fig. 2.4 : Structures of epimers (glucose and galactose are C,-epimers while glucose and mannose are
C,-epimers).
The a and f cyclic forms of D-glucose are known as anomers. They differ from each other in the configuration only around C, known as anomeric carbon (hemiacetal carbon). In case of a anomer, the —OH group held by anomeric carbon is on the opposite side of the group —CH,OH of sugar ring. The reverse is true for B-anomer. The anomers differ in certain physical and chemical properties.
Mutarotation : The a and 6 anomers of glucose have different optical rotations. The specific optical rotation of a freshly prepared glucose (% anomer) solution in water is +112.2° which gradually changes and_ attains an equilibrium with a constant value of +52.7°. In the presence of alkali, the decrease in optical rotation is rapid. The optical rotation of B-glucose is +18.7°. Mutarotation is defined as the change in the specific optical rotation representing the interconversion of « and f
Fig. 2.5 : Enantiomers (mirror images) of glucose.
Fig.
2.6
:
Mutarotation of
glucose representing
a
and
B
anomers
(A)
Fischer projections
(B)
Haworth projections.
1% open chain form. In aqueous solution, the B form is more predominant due to its stable conformation. The « and B forms of glucose are interconvertible which occurs through a linear form. The latter, as such, is present in an insignificant quantity.

Mutarotation of fructose Fructose also exhibits mutarotation. In case of fructose, the pyranose ring (six-membered) is converted to furanose (five-membered) ring, till an equilibrium is attained. And fructose has a specific optical rotation of —92° at equilibrium.
The conversion of dextrorotatory (+) sucrose to levorotatory fructose is explained under inversion of sucrose (see later in this chapter).

The process of shifting a hydrogen atom from one carbon atom to another to produce enediols is known as tautomerization. Sugars possessing anomeric carbon atom undergo tautomerization in alkaline solutions.
When glucose is kept in alkaline solution for several hours, it undergoes isomerization to form
Fig. 2.8 : Formation of a common enediol from glucose, fructose and mannose (R corresponds to the end 3 carbon common structure).
D-fructose and D-mannose. This reaction— known as the Lobry de Bruyn-von Ekenstein transformation—results in the formation of a common intermediate—namely enediol—for all the three sugars, as depicted in Fig.2.8.
The enediols are highly reactive, hence sugars in alkaline solution are powerful reducing agents.

The classified reducing sugars are as or nonreducing. The reducing property is attributed to the free aldehyde or keto group of anomeric carbon.
In the laboratory, many tests are employed to identify the reducing action of sugars. These include Benedict's test, Fehling's test, Barfoed's test etc. The reduction is much more efficient in the alkaline medium than in the acid medium.
The enediol forms (explained above) or sugars reduce cupric ions (Cu2+) of copper sulphate to cuprous ions (Cut), which form a yellow precipitate of cuprous hydroxide or a red precipitate of cuprous oxide as shown next.
It may be noted that the reducing property of sugars cannot help for a specific identification of any one sugar, since it is a general reaction.

Depending on the oxidizing agent used, the terminal aldehyde (or keto) or the terminal alcohol or both the groups may be oxidized. For instance, consider glucose :
1. Oxidation of aldehyde group (CHO —> COOH) results in the formation of gluconic acid.
2. Oxidation of terminal alcohol group (CH,OH —> COOH) leads to the production of glucuronic acid.

When treated with reducing agents such as sodium amalgam, the aldehyde or keto group of monosaccharide is reduced to corresponding alcohol, as indicated by the general formula :
The important monosaccharides and_ their corresponding alcohols are given below.
D-Sorbitol
D-Dulcitol
D-Mannitol
D-Mannitol + D-Sorbitol
D-Ribitol
D-Glucose
D-Galactose
—>
D-Mannose
——>
D-Fructose
D-Ribose
—5
—5
—5
Sorbitol and dulcitol when accumulate in tissues in large amounts cause strong osmotic effects leading to swelling of cells, and certain pathological conditions. e.g. cataract, peripheral neuropathy, nephropathy. Mannitol is useful to reduce intracranial tension by forced diuresis.

When treated with concentrated sulfuric acid, monosaccharides undergo dehydration with an elimination of 3 water molecules. Thus hexoses give hydroxymethyl furfural while pentoses give furfural on dehydration (Fig.2.9). These furfurals can condense with phenolic compounds (a-naphthol) to form coloured products. This is the chemical basis of the popular Molisch test. In case of oligoand polysaccharides, they are first hydrolysed to monosaccharides by acid, and this is followed by dehydration.
Bial's test : Pentoses react with strong HCI to form furfural derivatives which in turn react with orcinol to form green coloured complex. Bial's test is useful for detection of xylose in urine in essential pentosuria.
Mucic acid test : Galactose when treated with nitric acid forms insoluble mucic acid crystals.

Phenylhydrazine in acetic acid, when boiled with reducing sugars, forms osazones in a reaction summarized in Fig.2.10.
As is evident from the reaction, the first two carbons (C, and C) are involved in osazone formation. The sugars that differ in their configuration on these two carbons give the same type of osazones, since the difference is masked by binding with phenylhydrazine. Thus glucose, fructose and mannose give the same type (needle-shaped) osazones.
Reducing disaccharides also give osazones— maltose sunflower-shaped, and lactose powderpuff shaped.

The alcoholic groups of monosaccharides may be esterified by non-enzymatic or enzymatic reactions. Esterification of carbohydrate with phosphoric acid is a common reaction in metabolism. Glucose 6-phosphate and glucose 1-phosphate are good examples. ATP donates the phosphate moiety in ester formation.

Glycosides are formed when the hemiacetal or hemiketal hydroxyl group (of anomeric carbon) of a carbohydrate reacts with a hydroxyl group of another carbohydrate or a noncarbohydrate (e.g. methyl alcohol, phenol, glycerol). The bond so formed is known as glycosidic bond and the non-carbohydrate moiety (when present) is referred to as aglycone.
Fig. 2.10 : A summary of osazone formation (R represents C, to C, of glucose).
The monosaccharides are held together by glycosidic bonds to result in di-, oligoor polysaccharides (see later for structures).
Naming of glycosidic bond The nomenclature of glycosidic bonds is based on the linkages between the carbon atoms and the status of the anomeric carbon (a or §). For instance, lactose—which is formed by a bond between C, of B-galactose and Cy of glucose— is named as B(1 > 4) glycosidic bond. The other glycosidic bonds are described in the structure of diand polysaccharides.

1. Glucovanillin (vanillin-D-glucoside) is a natural substance that imparts vanilla flavour. 2. Cardiac glycosides (steroidal glycosides) : Digoxin and digitoxin contain the aglycone steroid and they stimulate muscle contraction. 3. Streptomycin, an antibiotic used in the treatment of tuberculosis is a glycoside. 4. Ouabain inhibits Nat—K+ ATPase and blocks the active transport of Nat. 5. Phlorhizin produces renal experimental animals. damage _ in

There are several derivatives of monosaccharides, some of which are physiologically important (Fig.2.11)
1. Sugar acids : Oxidation of aldehyde or primary alcohol group in monosaccharide results in sugar acids. Gluconic acid is produced from glucose by oxidation of aldehyde (C; group) whereas glucuronic acid is formed when primary alcohol group (Cg) is oxidized.
2. Sugar alcohols (polyols) They are produced by reduction of aldoses or ketoses. For instance, sorbitol is formed from glucose and mannitol from mannose.
3. Alditols The monosaccharides, on reduction, yield polyhydroxy alcohols, known as alditols. Ribitol is a constituent of flavin coenzymes; glycerol and myo-inositol are components of lipids. Xylitol is a sweetener used in sugarless gums and candies.
4. Amino sugars When one or more hydroxyl groups of the monosaccharides are replaced by amino groups, the products formed are amino sugars e.g. D-glucosamine, D-galactosamine. They are present as constituents of heteropolysaccharides.
N-Acetylneuraminic acid (NANA) is a derivative of N-acetylmannose and pyruvic acid. It is an important constituent of glycoproteins and glycolipids. The term sialic acid is used to include NANA and its other derivatives.
Certain antibiotics contain amino sugars which may be involved in the antibiotic activity e.g. erythromycin.
5. Deoxysugars : These are the sugars that contain one oxygen less than that present in the parent molecule. The groups —CHOH and —CH,OH become —CH) and —CH3 due to the absence of oxygen. D-2-Deoxyribose is the most important deoxysugar since it is a structural constituent of DNA (in contrast to D-ribose in RNA). Feulgen staining can specifically detect deoxyribose, and thus DNA in tissues. Fucose is a deoxy L-galactose found in blood group antigens, and certain glycoproteins.

6. L-Ascorbic acid (vitamin C) : This is a water-soluble vitamin, the structure of which closely resembles that of a monosaccharide.

Among the oligosaccharides, disaccharides are the most common (Fig.2.12). As is evident from the name, a disaccharide consists of two monosaccharide units (similar or dissimilar) held together by a glycosidic bond. They are crystalline, water-soluble and sweet to taste. The disaccharides are of two types
1. Reducing disaccharides with free aldehyde or keto group e.g. maltose, lactose.
2. Non-reducing disaccharides with no free aldehyde or keto group e.g. sucrose, trehalose.

Maltose is composed of two a-D-glucose units held together by a (1 — 4) glycosidic bond. The free aldehyde group present on C; of second glucose answers the reducing reactions, besides
Fig. 2.11 : Structures of monosaccharide derivatives (selected examples).
the osazone formations (sunflower-shaped). Maltose can be hydrolysed by dilute acid or the enzyme maltase to liberate two molecules of a-D-glucose.

In isomaltose, the glucose units are held together by & (1 > 6) glycosidic linkage.
Cellobiose is another disaccharide, identical in structure with maltose, except that the former has B (1 > 4) glycosidic linkage. Cellobiose is formed during the hydrolysis of cellulose.

Sucrose (cane sugar) is the sugar of commerce, mostly produced by sugar cane and sugar beets. Sucrose is made up of a@-D-glucose and pD-fructose. The two monosaccharides are held together by a glycosidic bond (a, > B»), between C, of a-glucose and C, of f-fructose. The reducing groups of glucose and fructose are involved in glycosidic bond, hence sucrose is a non-reducing sugar, and it cannot form osazones.
Sucrose is an important source of dietary carbohydrate. It is sweeter than most other common sugars (except fructose) namely glucose, lactose and maltose. Sucrose is employed as a sweetening agent in food industry. The intestinal enzyme—sucrase—hydrolyses sucrose to glucose and fructose which are absorbed.
Sucrose, as such is dextrorotatory (+66.5°). But, when hydrolysed, sucrose becomes levorotatory (-28.2°). The process of change in optical rotation from dextrorotatory (+) to levorotatory (-) is referred to as inversion. The hydrolysed mixture of sucrose, containing glucose and fructose, is known as invert sugar. The process of inversion is explained below.
Hydrolysis of sucrose by the enzyme sucrase (invertase) or dilute acid liberates one molecule each of glucose and fructose. It is postulated that sucrose (dextro) is first split into o-Dglucopyranose (+52.5°) and B-D-fructofuranose, both being dextrorotatory. However, B-Dfructofuranose is less stable and immediately gets converted to {-D-fructopyranose which _ is strongly levorotatory (-92°). The overall effect is that dextro sucrose (+66.5°) on inversion is converted to levo form (—28.2°).

Lactose is more commonly known as milk sugar since it is the disaccharide found in milk. Lactose is composed of B-D-galactose and B-Dglucose held together by B (1 — 4) glycosidic bond. The anomeric carbon of C, glucose is free, hence lactose exhibits reducing properties and forms osazones (powder-puff or hedgehog shape).
Fig.
2.12
:
Structures of
disaccharides
—maltose, sucrose
and lactose.
Lactose of milk is the most important carbohydrate in the nutrition of young mammals. It is hydrolysed by the intestinal enzyme lactase to glucose and galactose.

Lactulose is a synthetic dissccharide containing galactose and fructose. It is neither digested nor absorbed in the inestine. Lactulose is useful for the treatment of hepatic encephalopathy, a disorder characterized by elevated plasma ammonium levels. Lactulose converts ammonia (NH3) in the lumen to ammonium ion (NH). This results in a reduction in the plasma NH3, since NHj ions are not easily absorbed.

Polysaccharides (or simply glycans) consist of repeat units of monosaccharides or their derivatives, held together by glycosidic bonds. They are primarily concerned with two important functions-structural, and storage of energy.
Polysaccharides are linear as well as branched polymers. This is in contrast to structure of proteins and nucleic acids which are only linear polymers. The occurrence of branches in polysaccharides is due to the fact that glycosidic linkages can be formed at any one of the hydroxyl groups of a monosaccharide.
Polysaccharides are of two types
1. Homopolysaccharides on hydrolysis yield only a single type of monosaccharide. They are named based on the nature of the monosaccharide. Thus, glucans are polymers of glucose whereas fructosans are polymers of fructose.
2. Heteropolysaccharides on hydrolysis yield a mixture of a few monosaccharides or their derivatives.

Starch is the carbohydrate reserve of plants which is the most important dietary source for higher animals, including man. High content of starch is found in cereals, roots, tubers, vegetables etc. Starch is a homopolymer composed of D-glucose units held by a-glycosidic bonds. It is known as or

Starch consists of two polysaccharide components-water soluble amylose (15-20%) and a water insoluble amylopectin (80-85%). Chemically, amylose is a long unbranched chain with 200-1,000 D-glucose units held by « (1 > 4) glycosidic linkages. Amylopectin, on the other hand, is a branched chain with @ (1 > 6) glycosidic bonds at the branching points and a (1 — 4) linkages everywhere else (Fig.2.13). Amylopectin molecule containing a few thousand glucose units looks like a branched tree (20-30 glucose units per branch).
21
Fig. 2.13 : Structure of starch (a-amylose and amylopectin).
Starches are hydrolysed by amylase (pancreatic or salivary) to liberate dextrins, and finally maltose and glucose units. Amylase acts specifically on a (1 > 4) glycosidic bonds.

Dextrins are the breakdown products of starch by the enzyme amylase or dilute acids. Starch is sequentially hydrolysed through different dextrins and, finally, to maltose and glucose. The various intermediates (identified by iodine colouration) are soluble starch (blue), amylodextrin (violet), erythrodextrin (red) and achrodextrin (no colour).

Dextrans are polymers of glucose, produced by microorganisms. They are used as plasma volume expanders in transfusion, and chromatography (e.g. gel filtration).
Inulin is a polymer of fructose i.e., fructosan. It occurs in dahlia bulbs, garlic, onion etc. It is a low molecular weight (around 5,000) polysaccharide easily soluble in water. Inulin is not utilized by the body. It is used for assessing kidney function through measurement — of glomerular filtration rate (GFR).

Glycogen is the carbohydrate reserve in animals, hence often referred to as animal starch. It is present in high concentration in liver, followed by muscle, brain etc. Glycogen is also found in plants that do not possess chlorophyll (e.g. yeast, fungi).
The structure of glycogen is similar to that of amylopectin with more number of branches. Glucose is the repeating unit in glycogen joined together by a (1 — 4) glycosidic bonds, and
Fig. 2.14 : Structure of glycogen (A) General structure (B) Enlarged at a branch point.
a (1 — 6) glycosidic bonds at branching points (Fig.2.14). The molecular weight (up to 1 x 108) and the number of glucose units (up to 25,000) vary in glycogen depending on the source from which glycogen is obtained.

Cellulose occurs exclusively in plants and it is the most abundant organic substance in plant kingdom. It is a predominant constituent of plant cell wall. Cellulose is totally absent in

Cellulose is composed of {-D-glucose units linked by B (1 — 4) glycosidic bonds (Fig.2.15). Cellulose cannot be digested by mammals— including man—due to lack of the enzyme that cleaves B-glycosidic bonds (a amylase breaks a bonds only). Certain ruminants and herbivorous animals contain microorganisms in the gut which produce enzymes that can cleave f-glycosidic bonds. Hydrolysis of cellulose yields a disaccharide cellobiose, followed by B-D-glucose.
Cellulose, though not digested, has great importance in human nutrition. It is a major constituent of fiber, the non-digestable carbohydrate. The functions of dietary fiber include decreasing the absorption of glucose and cholesterol from the intestine, besides increasing the bulk of feces. (For details, Chapter 23)

Chitin is composed of N-acetyl Dglucosamine units held together by B (1 — 4) glycosidic bonds. It is a structural polysaccharide found in the exoskeleton of some invertebrates e.g. insects, crustaceans.

When the polysaccharides are composed of different types of sugars or their derivatives, they are referred to as heteropolysaccharides or heteroglycans.

Mucopolysaccharides are heteroglycans made up of repeating units of sugar derivatives, namely amino sugars and uronic acids. These are more commonly known as _— glycosaminoglycans (GAG). Acetylated amino groups, besides sulfate and carboxyl groups are generally present in GAG structure. The presence of sulfate and carboxyl groups contributes to acidity of the molecules, making them acid mucopolysaccharides.
Some of the mucopolysaccharides are found in} combination with proteins to form mucoproteins or mucoids or proteoglycans (Fig.2.16). Mucoproteins may contain up to 95% carbohydrate and 5% protein.
Mucopolysaccharides are essential components of
tissue structure.
The extracellular
spaces of
tissue
(particularly connective
_
tissue-cartilage, skin,
blood vessels,
tendons)
consist of
collagen and elastin fibers embedded in a matrix or ground
substance.
The ground
substance is
predominantly composed of GAG.
The important mucopolysaccharides include hyaluronic acid, chondroitin 4-sulfate, heparin, . dermatan sulfate and keratan sulfate (Fig.2.17).

Hyaluronic acid is an important GAG found in the ground substance of synovial fluid of joints
and vitreous humor of eyes. It is also present as
a
ground substance
in connective
tissues, and
forms
a
gel around
the
Fig. 2.16 : Diagrammatic representation of a proteoglycan complex.
serves as
a
lubricant joints.

+ Glucose is the most important energy source of carbohydrates to the mammals (except ruminants). The bulk of dietary carbohydrate (starch) is digested and finally absorbed as glucose into the body.
+ Dextrose (glucose in solution in dextrorotatory form) is frequently used in medical practice.
+ Fructose is abundantly found in the semen which is utilized by the sperms for energy.
+ Several diseases are associated with carbohydrates e.g., diabetes mellitus, glycogen storage diseases, galactosemia.
+ Accumulation of sorbitol and dulcitol in the tissues may cause certain pathological conditions e.g. cataract, nephropathy.
+ Inulin, a polymer of fructose, is used to assess renal function by measuring glomerular filtration rate (GFR).
+ The non-digestible carbohydrate cellulose plays a significant role in human nutrition. These include decreasing the intestinal absorption of glucose and cholesterol, and increasing bulk of feces to avoid constipation.
+ The mucopolysaccharide hyaluronic acid serves as a lubricant and shock absorbant in joints.
+ The enzyme hyaluronidase of semen degrades the gel (contains hyaluronic acid) around
the
ovum.
This
allows
effective
penetration
of
sperm
into
the
ovum.
+ The mucopolysaccharide heparin is an anticoagulant (prevents blood clotting).
+ The survival of Antarctic fish below —2°C is attributed to the antifreeze glycoproteins.
+ Streptomycin is a glycoside employed in the treatment of tuberculosis.
ovum.
Hyaluronic and
shock acid
absorbant in
Hyaluronic acid is composed of alternate units of D-glucuronic acid and N-acetyl D-glucosamine. These two molecules form disaccharide units held together by B (1 > 3) glycosidic bond (Fig.2.16). Hyaluronic acid contains about 250-25,000 disaccharide units (held by B 1 > 4 bonds) with a molecular weight up to 4 million.

Hyaluronidase is an enzyme that breaks (8 14 linkages) hyaluronic acid and other GAG. This enzyme is present in high concentration in testes, seminal fluid, and in certain snake and insect venoms. Hyaluronidase of semen is assigned an important role in fertilization as this enzyme clears the gel (hyaluronic acid) around the ovum allowing a better penetration of sperm into the ovum. Hyaluronidase of bacteria helps their invasion into the animal tissues.

Chondroitin 4-sulfate (Greek: chondroscartilage) is a major constituent of various mammalian tissues (bone, cartilage, tendons, heart, valves, skin, cornea etc.). Structurally, it is comparable with hyaluronic acid. Chondroitin 4-sulfate consists of repeating disaccharide units composed of D-glucuronic acid and N-acetyl D-galactosamine 4-sulfate (Fig.2.17).

Heparin is an anticoagulant (prevents blood clotting) that occurs in blood, lung, liver, kidney, spleen etc. Heparin helps in the release of the enzyme lipoprotein lipase which helps in clearing the turbidity of lipemic plasma.
Heparin is composed of alternating units of N-sulfo D-glucosamine 6-sulfate and glucuronate 2-sulfate (Fig.2.17).

Mostly found in skin, dermatan sulfate is structurally related to chondroitin 4-sulfate. The only difference is that there is an inversion in the configuration around Cs of D-glucuronic acid to form L-iduronic acid
(Fig.2.17).
Hyaluronic acid
Chondroitin 4-sulfate
Heparin
Dermatan sulfate
Keratan sulfate
Fig. 2.17 : Structures of common glycosaminoglycans — the disaccharides as repeating units.

Hyaluronic acid, Composition = D-Glucuronic acid, N-acetylglucosamine. Hyaluronic acid, Tissue distribution = Connective tissue, synovial fluid, vitrous humor. Hyaluronic acid, Function(s) = Serves as a lubricant, and shock absorber. Promotes wound healing. Chondroitin sulfate, Composition = D-Glucuronic acid, N-acetylgalactosamine 4-sulfate. Chondroitin sulfate, Tissue distribution = Cartilage, bone, skin, blood vessel walls. Chondroitin sulfate, Function(s) = Helps to maintain the structure and shapes of tissues. Heparin, Composition = D-Glucuronate 2-sulfate, N-sulfoglucosamine 6-sulfate. Heparin, Tissue distribution = Blood, lung, liver, kidney, spleen. Heparin, Function(s) = Acts as an anticoagulant. Dermatan sulfate, Composition = L-Iduronic acid, N-acetyl- galactosamine 4-sulfate. Dermatan sulfate, Tissue distribution = Blood vessel valves, heart valves, skin. Dermatan sulfate, Function(s) = Maintains the shapes of tissues. Keratan sulfate, Composition = D-Galactose, N-acetyl- glucosamine 6-sulfate. Keratan sulfate, Tissue distribution = Cartilage, cornea, connective tissues. Keratan sulfate, Function(s) = Keeps cornea transparent

Sometimes the
term mucoprotein
is used
for
It is a heterogeneous GAG with a variable sulfate content, besides small amounts of mannose, fructose, sialic acid etc. Keratan sulfate essentially consists of alternating units of D-galactosamine and N-acetylglucosamine 6-sulfate.

Agar, mostly found in sea weeds, is a polymer of galactose sulfate and glucose. Since agar is not digested, it serves as a dietary fiber (Refer Chapter 23). Agarose (with galactose and anhydrogalactose) is useful in the laboratory as a major component of microbial culture media, and in electrophoresis.
Pectins, found in apples and citrus fruits, contain galactouronate and rhamnose. Pectins, being non-digestible, are useful as dietary fiber. They are also employed in the preparation of jellies.

Several
proteins
are
covalently
bound
to
carbohydrates
which
are
referred
to
as
glyco-
proteins.
The
content
of
glycoprotein
varies
from
1%
to
90%
by
weight.
glycoprotein with
more than
4%.
distributed functions.
hormones, carbohydrate
concentration
Glycoproteins in
the are
very cells
and widely
perform
transport and
receptors.
and their
proteins,
A
selected their
variety of
role as
enzymes, structural
list major
The functions
carbohydrates include
proteins of
is glycoproteins
given found
mannose, in
in
Table
2.4.
glycoproteins galactose,
N-acetyl- glucosamine,
N-acetylgalactosamine, xylose,

Glycoprotein(s), 1 = Major function(s). Collagen, 1 = Structure. Hydrolases, proteases, glycosidases, 1 = Enzymes. Ceruloplasmin, 1 = Transport. Immunoglobulins, 1 = Defense against infection. Synovial glycoproteins, 1 = Lubrication. Thyrotropin, erythropoietin, 1 = Hormones. Blood group substances, 1 = Antigens. Fibronectin, laminin, 1 = Cell-cell recognition and adhesion. Intrinsic factor, 1 = Absorption of vitamin B 12. Fibrinogen, 1 = Blood clotting
L-fucose
and
N-acetylneuraminic
acid
(NANA).
NANA is an important sialic acid (See Fig.2.11).
Antifreeze glycoproteins : The Antarctic fish
live below —2°C, a temperature at which the
blood
would
freeze.
It
is
now
known
that
these
fish
contain
antifreeze
which
lower
the
freezing
point
of
water
and
interfere
with
the
crystal
formation
of
ice.
Antifreeze
glycoproteins
consist
of
50
repeating
units
of
the
tripeptide,
alanine-alanine-threonine.
Each
__
threonine
residue
is
bound
to
P-galactosyl
(13)
a
N-acetylgalactosamine.

The
blood
group
antigens
(of
erythrocyte
membrane)
contain
as
_
glyco-
proteins
or
glycolipids.
N-Acetylgalactosamine,
galactose,
fucose,
sialic
acid
etc.
are
found
in
the
blood
group
substances.
The
content
also
plays
a
determinant
role
in
blood
grouping.

1. Carbohydrates are the polyhydroxyaldehydes or ketones, or compounds which produce them on hydrolysis. The term sugar is applied to carbohydrates soluble in water and sweet to taste. Carbohydrates are the major dietary energy sources, besides their involvement in cell structure and various other functions.
2. Carbohydrates are broadly classified into 3 groups—monosaccharides, oligosaccharides and polysaccharides. The monosaccharides are further divided into different categories based on the presence of functional groups (aldoses or ketoses) and the number of carbon atoms (trioses, tetroses, pentoses, hexoses and heptoses).
3.
Glyceraldehyde
(triose)
is
the
simplest
and
is
chosen
as
a
reference
to
write
the
configuration
of
all
other
monosaccharides
(D-
and
L-
forms).
If
two
differ
in
their
structure
around
a
single
carbon
atom,
they
are
known
as
epimers.
Glucose
and
galactose
are
C4-
epimers.
4.
D-Glucose
is
the
most
important
naturally
occurring
aldose/monosaccharide.
Glucose
exists
as
a
and
B
anomers
with
different
optical
rotations.
The
interconversion
of
aand
B
anomeric
forms
with
change
in
the
optical
rotation
is
known
as
mutarotation.
5. Monosaccharides participate in several reactions. These include oxidation, reduction, dehydration, osazone formation etc. Formation of esters and glycosides by monosaccharides is of special significance in biochemical reactions.
6. Among the oligosaccharides, disaccharides are the most common. These include the reducing disaccharides namely lactose (milk sugar) and maltose (malt sugar) and the non-reducing sucrose (cane sugar).

7. Polysaccharides are the polymers of monosaccharides or their derivatives, held together by glycosidic bonds. Homopolysaccharides are composed of a single monosaccharide (e.g., starch, glycogen, cellulose, inulin). Heteropolysaccharides contain a mixture of few monosaccharides or their derivatives (e.g., mucopolysaccharides).
8. Starch and glycogen are the carbohydrate reserves of plants and animals respectively. Cellulose, exclusively found in plants, is the structural constituent. Inulin is utilized to assess kidney function by measuring glomerular filtration rate (GFR).
9. Mucopolysaccharides (glycosaminoglycans) are the essential components of tissue structure. They provide the matrix or ground substance of extracellular tissue spaces in which collagen and elastin fibers are embedded. Hyaluronic acid, chondroitin 4-sulfate, heparin, are among the important glycosaminoglycans.
10.
Glycoproteins are a group of biochemically important compounds with a variable composition of carbohydrate (1-90%), covalently bound to protein. Several enzymes, hormones, structural proteins and cellular receptors are in fact glycoproteins.

1. Define and classify carbohydrates with suitable examples. Add a note on the functions of carbohydrates.
2. Describe the structure and functions of mucopolysaccharides.
3. Give an account of the structural configuration of monosaccharides, with special reference to glucose.
4. Discuss the structure and functions of 3 biochemically important disaccharides.
5. Define polysaccharides and describe the structure of 3 homopolysaccharides.

(a) Epimers, (b) Mutarotation, (c) Osazone formation, (d) Glycosidic bond, (e) Sugar derivatives, (f) Anomers, (g) Enediol, (h) Amino sugars, (i) Inversion of sucrose, (j) Deoxysugars.

1. Name a non-reducing
disaccharide
2. The carbohydrate that is taken as a reference for writing the configuration of others
3. If two monosaccharides differ in configuration around a single carbon atom, they are known as
. The @ and B cyclic forms of D-glucose are referred to as
. The non-carbohydrate moiety found in glycosides is known as . Give an example of a glycoside antibiotic nn
. The glycosidic bonds at the branching points in the structure of starch are on . The polysaccharide employed for the assessment of kidney function
9. The glycosaminoglycan that serves as a lubricant and shock absorbant of joints
10. Name the sialic acid, mostly found in the structure of glycoproteins and glycolipids

11. Ribose and deoxyribose differ in structure around a single carbon, namely
(a) C; (b) Cy ©) C, A) Cy.
12. One of the following is not an aldose
(a) Glucose (b) Galactose (c) Mannose (d) Fructose.
13. The glycosaminoglycan that serves as an anticoagulant
(a) Heparin (b) Hyaluronic acid (c) Chondroitin sulfate (d) Dermatan sulfate.
14. The following polysaccharide is composed of
B-glycosidic bonds
(a) Starch (b) Glycogen (c) Dextrin (d) Cellulose.
15. The carbon atoms involved in the osazone formation
(a) 1 and 2 (b) 2 and 3 (c) 3 and 4 (d) 5 and 6.

(ee (Greek: lipos-fat) are of great importance to the body as the chief concentrated storage form of energy, besides their role in cellular structure and various other biochemical functions. As such, lipids are a heterogeneous group of compounds and, therefore, it is difficult to define them precisely.
Lipids may be regarded as organic substances relatively insoluble in water, soluble in organic solvents (alcohol, ether etc.), actually or potentially related to fatty acids and utilized by the living cells.
Unlike the polysaccharides, proteins and nucleic acids, lipids are not polymers. Further, lipids are mostly small molecules.

Lipids are broadly classified (modified from Bloor) into simple, complex, derived and miscellaneous lipids, which are further subdivided into different groups
1. Simple lipids: Esters of fatty acids with alcohols. These are mainly of two types
(a) Fats and oils (triacylglycerols) : These are esters of fatty acids with glycerol. The difference between fat and oil is only physical. Thus, oil is a liquid while fat is a solid at room temperature.
(b) Waxes: Esters of fatty acids (usually long chain) with alcohols other than glycerol. These alcohols may be aliphatic or alicyclic. Cetyl alcohol is most commonly found in waxes. Waxes are used in the preparation of candles, lubricants, cosmotics, ointments, polishes etc.
2. Complex (or compound) lipids: These are esters of fatty acids with alcohols containing additional groups such as___ phosphate, nitrogenous base, carbohydrate, protein etc. They are further divided as follows
(a) Phospholipids : They contain phosphoric acid and frequently a nitrogenous base. This is in addition to alcohol and fatty acids.

(i) Glycerophospholipids : These phospholipids contain glycerol as the alcohol e.g., lecithin, cephalin.
(ii) Sphingophospholipids : Sphingosine is the alcohol in this group of phospholipids e.g., sphingomyelin.
Ss Glycolipids : These lipids contain a fatty acid, carbohydrate and nitrogenous base. The alcohol is sphingosine, hence they are also called as glycosphingolipids. Glycerol and phosphate are absent e.g., cerebrosides, gangliosides.
(c Lipoproteins : Macromolecular complexes of lipids with proteins.
(d) Other complex lipids : Sulfolipids, aminolipids and lipopolysaccharides are among the other complex lipids.
3. Derived lipids : These are the derivatives obtained on the hydrolysis of group 1 and group 2 lipids which possess the characteristics of lipids. These include glycerol and other alcohols, fatty acids, monoand diacylglycerols, lipid (fat) soluble vitamins, steroid hormones, hydrocarbons and ketone bodies.
4. Miscellaneous lipids: These include a large number of compounds possessing the characteristics of lipids e.g., carotenoids, squalene, hydrocarbons such as pentacosane (in bees wax), terpenes etc.
NEUTRAL LIPIDS: The lipids which are uncharged are referred to as neutral lipids. These are mono-, di-, and triacylglycerols, cholesterol and cholesteryl esters.

Lipids perform several important functions
1. They are the concentrated fuel reserve of the body (triacylglycerols).
2. Lipids are the constituents of membrane structure and regulate the membrane permeability (phospholipids and cholesterol).
3.
They serve
as
a
source of
fat soluble
vitamins
(A,
D,
E
and kK).
4. Lipids are important as cellular metabolic regulators (steroid hormones and prostaglandins).
5. Lipids protect the internal organs, serve as insulating materials and give shape and smooth appearance to the body.

Fatty acids are carboxylic acids with hydrocarbon side chain. They are the simplest form of lipids.

Fatty acids mainly occur in the esterified form as major constituents of various lipids. They are also present as free (unesterified) fatty acids. Fatty acids of animal orgin are much simpler in structure in contrast to those of plant origin which often contain groups such as epoxy, keto, hydroxy and cyclopentane rings.

Most of the fatty acids that occur in natural lipids are of even carbons (usually 14C — 20C). This is due to the fact that biosynthesis of fatty acids mainly occurs with the sequential addition of 2 carbon units. Palmitic acid (16C) and stearic acid (18C) are the most common. Among the odd chain fatty acids, propionic acid (3C) and valeric acid (5C) are well known.

Saturated fatty acids do not contain double bonds, while unsaturated fatty acids contain one or more double bonds. Both saturated and unsaturated fatty acids almost equally occur in the natural lipids. Fatty acids with one double bond are monounsaturated, and those with 2 or more double bonds are collectively known as polyunsaturated fatty acids (PUFA).

The naming of a fatty acid (systematic name) is based on the hydrocarbon from which it is derived. The saturated fatty acids end with a suffix -anoic (e.g., octanoic acid) while the unsaturated fatty acids end with a suffix -enoic

Depending on the length of carbon chains, fatty acids are categorized into 3 groups—short chain with less than 6 carbons; medium chain with 8 to 14 carbons and long chain with 16 to 24 carbons.

Instead of writing the full structures, biochemists employ shorthand notations (by numbers) to represent fatty acids. The general rule is that the total number of carbon atoms are written first, followed by the number of double bonds and finally the (first carbon) position of

Arachidonic acid, T ABLE 3.1 Selected examples of biochemically important fatty acids.Structure = CH 3 (CH 2 ) 4 CH = CHCH 2 CH = CHCH 2 CH = CHCH 2 CH = CH(CH 2 ) 3 COOH. * Total number of carbon atoms, followed by the number of double bonds and the first carbon position of the double bond(s)., T ABLE 3.1 Selected examples of biochemically important fatty acids.Systematic name = * Total number of carbon atoms, followed by the number of double bonds and the first carbon position of the double bond(s).. * Total number of carbon atoms, followed by the number of double bonds and the first carbon position of the double bond(s)., T ABLE 3.1 Selected examples of biochemically important fatty acids.Abbreviation * = * Total number of carbon atoms, followed by the number of double bonds and the first carbon position of the double bond(s).. * Total number of carbon atoms, followed by the number of double bonds and the first carbon position of the double bond(s)., T ABLE 3.1 Selected examples of biochemically important fatty acids.Structure = * Total number of carbon atoms, followed by the number of double bonds and the first carbon position of the double bond(s).
double bonds, starting from the carboxyl end. Thus, saturated fatty acid, palmitic acid is written as 16 : 0, oleic acid as 18 : 1; 9, arachidonic acid as 20 : 4; 5, 8, 11, 14.
There are other conventions of representing the double bonds. A9 indicates that the double bond is between 9 and 10 of the fatty acid. w 9 represents the double bond position (9 and 10) from the @ end. Naturally occurring unsaturated fatty acids belong to m 9, ® 6 and w 3 series.
@3series
Linolenic acid
(18
:
3;
9,
12,
15)
@6
series
Linoleic acid
arachidonic
(18
acid
2;
(20
11,
14)
@9
series
Oleic acid (18 : 1; 9)
The biochemically important saturated and unsaturated fatty acids are given in the Table 3.1.

The fatty acids that cannot be synthesized by the body and, therefore, should be supplied in the diet are known as essential fatty acids (EFA). Chemically, they are polyunsaturated fatty acids, namely linoleic acid (18 : 2; 9, 12) and linolenic acid (18 : 3; 9, 12, 15). Arachidonic acid (20 : 4; 5, 8, 11, 14) becomes essential, if its precursor linoleic acid is not provided in the diet in sufficient amounts. The structures of EFA
are given in the Table 3.1.
Biochemical basis for essentiality : Linoleic acid and linolenic acid are essential since humans lack the enzymes that can introduce double bonds beyond carbons 9 to 10.
Functions of EFA: Essential fatty acids are required for the membrane structure and function, transport of cholesterol, formation of lipoproteins, prevention of fatty liver etc. (Chapter 23). They are also needed for the synthesis of another important group of compounds, namely eicosanoids (Chapter 32).
Deficiency of EFA: The deficiency of EFA results in phrynoderma_ or toad _ skin, characterized by the presence of horny eruptions
:
9,
:
12)
and
4;
5,
8,
on the posterior and lateral parts of limbs, on the back and buttocks, loss of hair and poor wound healing.

Unsaturated fatty acids exhibit geometric isomerism depending on the orientation of the groups around the double bond axis.
If the atoms or acyl groups are present on the same side of the double bond, it is a cis configuration. On the other hand, if the groups occur on the opposite side, it is a trans configuration. Thus oleic acid is a cis isomer while elaidic acid is a trans isomer, as depicted in Fig.3.1. Cis isomers are less stable than trans isomers. Most of the naturally occurring unsaturated fatty acids exist as cis isomers.
In the
cis isomeric
form, there
is
a
molecular binding at the double bond. Thus, oleic acid exists in an L-shape while elaidic acid is a straight chain. Increase in the number of double bonds will cause more bends (kinks) and arachidonic acid with 4 double bonds will have a U-shape. It is believed that cis isomers of fatty acids with their characteristic bonds will compactly pack the membrane structure.
Hydroxy fatty acids : Some of the fatty acids are hydroxylated. B-Hydroxybutyric acid, one of the ketone bodies produced in metabolism, is a simple example of hydroxy fatty acids. Cerebronic acid and recinoleic acid are long chain hydroxy fatty acids.
Cyclic fatty acids: Fatty acids with cyclic structures are rather rare e.g., chaulmoogric acid found in chaulmoogra oil (used in leprosy treatment) contains cyclopentenyl ring.

(For palmitoyl
R
=
C15H31;
for stearoyl
R
=
C17H35;
For linoleoyl

Triacylglycerols (formerly triglycerides) are the esters of glycerol with fatty acids. The fats and oils that are widely distributed in both plants and animals are chemically triacylglycerols. They are insoluble in water and non-polar in character and commonly known as neutral fats. Fats as stored fuel: Triacylglycerols are the most abundant group of lipids that primarily function as fuel reserves of animals. The fat reserve of normal humans (men 20%, women 25% by weight) is sufficient to meet the body's caloric requirements for 2-3 months.
Fats primarily occur in adipose tissue : Adipocytes of adipose tissue—predominantly found in the subcutaneous layer and in the abdominal cavity—are specialized for storage of triacylglycerols. The fat is stored in the form of globules dispersed in the entire cytoplasm. And surprisingly, triacylglycerols are not the structural components of biological membranes.
Structures of acylglycerols: ©Monoacylglycerols, diacylglycerols and_triacylglycerols, respectively consisting of one, two and three molecules of fatty acids esterified to a molecule
R
=
Cy7H31)
of glycerol, are known (Fig.3.2). Among these, triacylglycerols are the most important biochemically.
Simple triacylglycerols contain the same type of fatty acid residue at all the three carbons e.g., tristearoyl glycerol or tristearin.
Mixed triacylglycerols
are more
common.
They contain 2 or 3 different types of fatty acid residues. In general, fatty acid attached to C, is saturated, that attached to Cy is unsaturated while that on C3 can be either. Triacylglycerols are named according to placement of acyl radical on glycerol e.g., 1,3-palmitoyl 2-linoleoyl glycerol.
Triacylglycerols of plants, in general, have higher content of unsaturated fatty acids compared to that of animals.

The structure of glycerol gives an impression that carbons 1 and 3 are identical. This is not true in a 3-dimensional structure. In order to represent the carbon atoms of glycerol in an unambiguous manner, biochemists adopt a_ stereospecific numbering (sn) and prefix glycerol with sn.
It should be noted that C; and C3 are different. Cells possess enzymes that can distinguish these two carbons. — Thus glycerokinase phosphorylates sn-3 (and not sn-1) glycerol to give sn-glycerol 3-phosphate.

A few important properties of triacylglycerols, which have' biochemical relevance, are discussed below
1. Hydrolysis: Triacylglycerols undergo stepwise enzymatic hydrolysis to finally liberate free fatty acids and glycerol. The process of hydrolysis, catalysed by lipases is important for digestion of fat in the gastrointestinal tract and fat mobilization from the adipose tissues.
2. Saponification : The hydrolysis of triacylglycerols by alkali to produce glycerol and soaps is known as saponification.
Triacylglycerol + 3 NaOH
——>
Glycerol + 3 R-COONa (soaps)
3. Rancidity : Rancidity is the term used to represent the deterioration of fats and oils resulting in an unpleasant taste. Fats containing unsaturated fatty acids are more susceptible to rancidity.
Rancidity occurs when fats and oils are exposed to air, moisture, light, bacteria etc. Hydrolytic rancidity occurs due to partial hydrolysis of triacylglycerols by bacterial enzymes. Oxidative rancidity is due to oxidation of unsaturated fatty acids. This results in the formation of unpleasant products such as dicarboxylic acids, aldehydes, ketones etc. Rancid fats and oils are unsuitable for human consumption.
Antioxidants : The substances which can prevent the occurrence of oxidative rancidity are known as antioxidants. Trace amounts of antioxidants such as tocopherols (vitamin E), hydroquinone, gallic acid and a-naphthol are added to the commercial preparations of fats and oils to prevent rancidity. Propyl gallate, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are the antioxidants used in food preservation.
4. Lipid peroxidation in vivo: In the living cells, lipids undergo oxidation to produce peroxides and free radicals which can damage the tissue. The free radicals are believed to cause inflammatory diseases, ageing, cancer, atherosclerosis etc. It is fortunate that the cells possess antioxidants such as vitamin E, urate and superoxide dismutase to prevent in vivo lipid peroxidation (Chapter 34).

Adulteration of fats and oils is increasing day by day. Several tests are employed in the laboratory to check the purity of fats and oils. Some of them are discussed hereunder lodine number : It is defined as the grams (number) of iodine absorbed by 100 g of fat or oil. lodine number is useful to know the relative unsaturation of fats, and is directly proportional to the content of unsaturated fatty acids. Thus lower is the iodine number, less is the degree of unsaturation. The iodine numbers of common oils/fats are given below.
Coconut oil, Iodine number = 7 - 10. Butter, Iodine number = 25 - 28. Palm oil, Iodine number = 45 - 55. Olive oil, Iodine number = 80 - 85. Groundnut oil, Iodine number = 85 - 100. Cottonseed oil, Iodine number = 100 - 110. Sunflower oil, Iodine number = 125 - 135. Linseed oil, Iodine number = 175 - 200
Determination of iodine number will help to know the degree of adulteration of a given oil.
Saponification number : It is defined as the mg (number) of KOH required to hydrolyse (saponify) one gram of fat or oil. Saponification number is a measure of the average molecular size of the fatty acids present. The value is higher for fats containing short chain fatty acids. The saponification numbers of a few fats and oils are given below
Human fat:
195-200
Butter
:
230-240
Coconut oil
:
250-260
Reichert-MeissI (RM) number : It is defined as the number of ml 0.1 N KOH required to completely neutralize the soluble volatile fatty acids distilled from 5 g fat. RM number is useful in testing the purity of butter since it contains a good concentration of volatile fatty acids (butyric acid, caproic acid and caprylic acid). This is in contrast to other fats and oils which have a negligible amount of volatile fatty acids. Butter has a RM number in the range 25-30, while it is less than 1 for most other edible oils. Thus any adulteration of butter can be easily tested by this sensitive RM number.

Acid number : It is defined as the number of mg of KOH required to completely neutralize free fatty acids present in one gram fat or oil. In normal circumstances, refined oils should be free from any free fatty acids. Oils, on decomposition—due to chemical or bacterial contamination—yield free fatty acids. Therefore, oils with increased acid number are unsafe for human consumption.

These are complex or compound lipids containing phosphoric acid, in addition to fatty acids, nitrogenous base and alcohol (Fig.3.3).
There are two classes of phospholipids
1. Glycerophospholipids (or phosphoglyce-
rides) that contain glycerol as the alcohol.
2. Sphingophospholipids (or sphingomyelins)
that contain sphingosine as the alcohol.

Glycerophospholipids are the major lipids that occur in biological membranes. They consist of glycerol 3-phosphate esterified at its C; and Cy with fatty acids. Usually, C; contains a saturated fatty acid while C) contains an unsaturated fatty acid.
1. Phosphatidic acid: This is the simplest phospholipid. It does not occur in good concentration in the — tissues. Basically, phosphatidic acid is an intermediate in the synthesis of triacylglycerols and phospholipids.
The other glycerophospholipids containing different nitrogenous bases or other groups may be regarded as the derivatives of phosphatidic acid.
2. Lecithins (phosphatidylcholine): These are the most abundant group of phospholipids in the cell membranes. Chemically, lecithin (Greek : lecithos—egg yolk) is a phosphatidic acid with choline as the base. Phosphatidylcholines the form of choline.
represent storage body's

+ Lipids are important to the body as constituents of membranes, source of fat soluble (A, D, E and k) vitamins and metabolic regulators (steroid hormones and prostaglandins).
+ Triacylglycerols (fats) primarily stored in the adipose tissue are concentrated fuel
reserves of the body. Fats found in the subcutaneous tissue and around certain organs serve as thermal insulators.
+ The unsaturated fatty acids-linoleic and linolenic acid—are essential to humans, the
of
which
causes
phrynoderma
or
toad
skin.
+ The cyclic fatty acid, namely chaulmoogric acid,is employed in the treatment of leprosy.
+ Fats and oils on exposure to air, moisture, bacteria etc. undergo rancidity (deterioration). This can be prevented by the addition of certain antioxidants (vitamin E, hydroquinone, gallic acid).
+ In food preservation, antioxidants—namely propyl gallate, butylated hydroxyanisole and butylated hydroxytoluene—are commonly used.
Chapter
3
:
LIPIDS
- (a) Dipalmitoyl lecithin is an important phosphatidylcholine found in lungs. It is a surface active agent and prevents the adherence of inner surface of the lungs due to surface tension. Respiratory distress syndrome in infants is a disorder characterized by the absence of dipalmitoy| lecithin.

(b) Lysolecithin is formed by removal of fatty acid either at C, or C, of lecithin.
3. Cephalins (phosphatidylethanolamine) : Ethanolamine is the nitrogenous base present in cephalins. Thus, lecithin and cephalin differ with regard to the base.
4. Phosphatidylinositol: The steroisomer myo-inositol is attached to phosphatidic acid to give phosphatidylinositol(Pl). This is an important component of cell membranes. The action of certain hormones (e.g. oxytocin, vasopressin) is mediated through PI.
5. Phosphatidylserine: The amino acid serine is present in this group of glycerophospholipids. Phosphatidylthreonine is also found in certain tissues.
6. Plasmalogens: When a fatty acid is attached by an ether linkage at C, of glycerol in the — glycerophospholipids, the resultant compound is plasmalogen. | Phosphatidalethanolamine is the most important which is similar in structure to phosphatidylethanolamine but for the ether linkage (in place of ester). An unsaturated fatty acid occurs at C;. Choline, inositol and serine may substitute ethanolamine to give other plasmalogens.
7. Cardiolipin : It is so named as it was first isolated from heart muscle. Structurally, a cardiolipin consists of two molecules of phosphatidic acid held by an additional glycerol through phosphate groups. It is an important component of inner mitochondrial membrane and essential for mitrochondrial function. Decreased cardiolipin levels may result in mitochondrial dysfunction, aging, — hypothyroidism, cardioskeletal myopathy (Barth syndrome). Cardiolipin is the only phosphoglyceride that possesses antigenic properties.

Sphingosine is an amino alcohol present in sphingomyelins (sphingophospholipids). They do not contain glycerol at all. Sphingosine is attached by an amide linkage to a fatty acid to produce ceramide. The alcohol group of sphingosine is bound to phosphorylcholine in sphingomyelin structure (Fig.3.3). Sphingomyelins are important constituents of myelin and are found in good quantity in brain and nervous tissues.
Ceramide, acts as a second messenger (signaling molecule) by regulating programmed cell death (apoptosis), cell cycle and cell differentiation. A ceramide containing a 30-carbon fatty acid is a major component of skin, and it regulates skin's water permeability.

Phospholipids constitute an important group of compound lipids that perform a wide variety of functions
1. In association with proteins, phospholipids form the structural components of membranes and regulate membrane permeability.
2. Phospholipids (lecithin, cephalin and cardiolipin) in the mitochondria maintain the conformation of electron transport chain components, and thus cellular respiration.
3. Phospholipids participate in the absorption of fat from the intestine.
4. Phospholipids are essential for the synthesis of different lipoproteins, and thus participate in the transport of lipids.
5. Accumulation of fat in liver (fatty liver) can be prevented by phospholipids, hence they are regarded as lipotropic factors.
6. Arachidonic acid, an unsaturated fatty acid liberated from phospholipids, serves as a precursor for the synthesis of eicosanoids (prostaglandins, prostacyclins, thromboxanes etc.).
7. Phospholipids participate in the reverse cholesterol transport and thus help in the removal of cholesterol from the body.
8. Phospholipids act as surfactants (agents lowering surface tension). For _ instance, dipalmitoyl phosphatidylcholine is an important
7) Fig. 3.4 : Structure of galactosylceramide (R = H). For sulfagalactosylceramide R is a sulfatide (R = SOZ>).
lung surfactant. Respiratory distress syndrome in infants is associated with insufficient production of this surfactant.
- 9. Cephalins, an important group of phospho-
lipids participate in blood clotting.
10.
Phosphatidylinositol is
the second
messengers—inositol diacylglyceol,
source triphosphate
that are
involved some
of and
in the
action horomones.

Glycolipids (glycosphingolipids) are important constituents of cell membrane and nervous tissues (particularly the brain). Cerebrosides are the simplest form of glycolipids. They contain a ceramide (sphingosine attached to a fatty acid) and one or more sugars. Galactocerebroside (galactosylceramide) and glucocerebroside are the most important glycolipids. The structure of galactosylceramide is given in Fig.3.4. It contains the fatty acid cerebronic acid. Sulfagalactosylceramide is the sulfatide derived from galactosylceramide.
Gangliosides : These are predominantly found in ganglions and are the most complex form of glycosphingolipids. They are the derivatives of cerebrosides and contain one or more molecules of N-acetylneuraminic acid (NANA), the most important sialic acid. The structure of NANA is given in carbohydrate chemistry (Refer Fig.2.11).
The most important gangliosides present in the brain are GM,, GM»), GD, and GT, (G represents ganglioside while M, D and T indicate mono-, dior trisialic acid residues, and the number denotes the carbohydrate

Lipoproteins are molecular complexes of lipids with proteins. They are the transport vehicles for lipids in the circulation. There are five types of lipoproteins, namely chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL), high density lipoproteins (HDL) and free fatty acidalbumin complexes. Their structure, separation, metabolism and diseases are discussed together (Chapter 14).

Steroids are the compounds containing a cyclic steroid nucleus (or ring) namely cyclopentanoperhydrophenanthrene (CPPP). |t consists of a phenanthrene nucleus (rings A, B and C) to which a cyclopentane ring (D) is attached.
The structure and numbering of CPPP are shown in Fig.3.5. The steroid nucleus represents saturated carbons, unless specifically shown as of
Fig. 3.5 : Structures of steroids (A, B, C—Perhydrophenanthrene; D-Cyclopentane).
double bonds. The methyl! side chains (19 & 18) attached to carbons 10 & 13 are shown as single bonds. At carbon 17, steroids usually contain a side chain.
There are several steroids in the biological system. These include cholesterol, bile acids, vitamin D, sex hormones, adrenocortical hormones, sitosterols, cardiac glycosides and alkaloids. If the steroid contains one or more hydroxyl groups it is commonly known as sterol (means solid alcohol).

Cholesterol, exclusively found in animals, is the most abundant animal sterol. It is widely distributed in all cells and is a major component of cell membranes and lipoproteins. Cholesterol (Greek : chole—-bile) was first isolated from bile. Cholesterol literally means 'solid alcohol from bile.'

The structure of cholesterol (Cj7H4¢O) is depicted in Fig.3.5. It has one hydroxy! group at C3 and a double bond between Cs and Cg. An 8 carbon aliphatic side chain is attached to C7. Cholesterol contains a total of 5 methyl groups.
Due to the presence of an —OH group, cholesterol is weakly amphiphilic. As a structural component of plasma membranes, cholesterol is an important determinant of membrane permeability properties. The occurrence of cholesterol is much higher in the membranes of sub-cellular organelles.
Cholesterol is found in association with fatty acids to form cholesteryl esters (esterification occurs at the OH group of C3).
Properties and reactions : Cholesterol is an yellowish crystalline solid. The crystals, under the microscope, show a _ notched (C4) appearance. Cholesterol is insoluble in water and soluble in organic solvents such as chloroform, benzene, ether etc.
Several reactions given by cholesterol are useful for its qualitative identification and quantitative estimation. These include Salkowski's test, Liebermann-Burchard reaction and Zak's test.
Functions of cholesterol: Cholesterol is a poor conductor of heat and electricity, since it has a high dielectric constant. It is present in abundance in nervous tissues. It appears that cholesterol functions as an insulating cover for the transmission of electrical impulses in the nervous tissue. Cholesterol performs several other biochemical functions which include its role in membrane structure and function, in the synthesis of bile acids, hormones (sex and cortical) and vitamin D (for details, Refer Chapters 7 and 19).

Ergosterol occurs in plants. It is also found as a structural constituent of membranes in yeast and fungi. Ergosterol (Fig.3.5) is an important precursor for vitamin D. When exposed to light,
Non-polar tail
Polar head
(C)
Amphipathic lipid
Fig. 3.6 : Summary of amphipathic lipids in the formation of micelle and lipid bilayer.
the ring B of ergosterol opens and it is converted to ergocalciferol, a compound containing vitamin D activity.
The other sterols present in plant cells include stigmasterol and [-sitosterol.

As per definition, lipids are insoluble (hydrophobic) in water. This is primarily due to the predominant presence of hydrocarbon groups. However, some of the lipids possess polar or hydrophilic groups which tend to be soluble in water. Molecules = which contain both hydrophobic and hydrophilic groups are known as amphipathic (Greek : amphi-both, pathos— passion).
Examples of amphipathic lipids : Among the lipids, fatty acids, phospholipids, sphingolipids, bile salts and cholesterol (to some extent) are amphipathic in nature.
Phospholipids have a hydrophilic head (phosphate group attached to choline, ethanolamine, inositol etc.) and a long hydrophobic tail. The general structure of an amphipathic lipid may be represented as a polar or hydrophilic head with a non-polar or hydrophobic tail (Fig.3.6).
Fatty acids contain a hydrocarbon chain with a carboxyl (COO-) group at physiological pH. The carboxyl group is polar in nature with affinity to water (hydrophilic) while hydrocarbon chain of fatty acid is hydrophobic.
Orientation of amphipathic lipids : When the amphipathic lipids are mixed in water (aqueous phase), the polar groups (heads) orient themselves towards aqueous phase while the non-polar (tails) orient towards the opposite directions. This leads to the formation of micelles (Fig.3.6).
Micelles are primarily molecular aggregates of amphipathic lipids. Micelle formation, facilitated by bile salts, is very important for lipid digestion and absorption (Chapter 8).

+ The phospholipid-dipalmitoyl lecithin—prevents the adherence of inner surface of the lungs, the absence of which is associated with respiratory distress syndrome in infants.
+ Cephalins participate in blood clotting.
+ The action of certain hormones is mediated through phosphatidylinositol.
+ Phospholipids are important for the synthesis and transport of lipoproteins and reverse transport of cholesterol.
+ Cholesterol is essential for the synthesis of bile acids, hormones (sex and cortical) and vitamin D.
+ Lipoproteins occur in the membrane structure, besides serving as a means of transport vehicles for lipids.
+ Lipids are associated with certain disorders—obesity, atherosclerosis, and diabetes mellitus.
+ Liposomes are used for administration of a variety of therapeutic substances (drugs, proteins, nucleic acids) in order to target specific organs or tissues.

In case of biological membranes, a bilayer of lipids is formed orienting the polar heads to the outer aqueous phase on either side and the nonpolar tails into the interior (Fig.3.6). The formation of a lipid bilayer is the basis of membrane structure.
Liposomes : They are produced when amphipathic lipids in aqueous medium are subjected to sonification. They have intermittent aqueous phases in the lipid bilayer. Liposomes, in combination with tissue specific antigens, are used as carriers of drugs to target tissues.
Emulsions : These are produced when nonpolar lipids (e.g. triacylglycerols) are mixed with water. The particles are larger in size and stabilized by emulsifying agents (usually amphipathic lipids), such as bile salts and phospholipids.

Soaps are sodium or potassium salts of fatty acids. They are produced by saponification of fats. Sodium soaps are hard that result in bar soaps. Soaps serve as cleansing agents since they can emulsify oils and remove the dirt.

Detergents are synthetic cleansing agents e.g. sodium lauryl sulfate. Detergents are superior in their cleansing action compared to soaps, and are used in washing clothes, and in tooth paste.

1. Write an account of classification of lipids with suitable examples.
2. Describe the structure and functions of phospholipids.
3. Discuss the saturated and unsaturated fatty acids of biological importance, along with their structures.
4. Describe the structure of steroids. Add a note on the functions of cholesterol.
5 . Discuss the biological importance of amphipathic lipids.

(a) Structure of triacylglycerols, (b) Glycolipids, (c) Essential fatty acids, (d) Cis-trans isomerism,
(e) Rancidity, (f) lodine number, (g) Phosphatidylinositol, (h) Sphingomyelins, (i) Steroid nucleus, Micelles.
(j)

1. The lipids that function as fuel reserve in animals
. The isomerism associated with unsaturated fatty acids NY
. The cyclic fatty acid employed in the treatment of leprosy RW
. The lipids that are not the structural components of biological membranes
. The prefix sn used to represent glycerol, sn stands for DUH
. The number of mg of KOH required to hydrolyse 1 g fat or oil is known as
. The phospholipid that prevents the adherence of inner surfaces of lungs OWN
. The phospholipid that produces second messengers in hormonal action
.
. Name the glycolipids containing N-acetylneuraminic acid oO
= . The steroids contain a cyclic ring known as

11. The nitrogenous base present in lecithin
(a) Choline (b) Ethanolamine (c) Inositol (d) Serine.
12. The number of double bonds present in arachidonic acid
(a) 1 (b) 2 (c) 3 (d) 4.
13. One of the following is an amphipathic lipid
(a) Phospholipids (b) Fatty acid (c) Bile salts (d) All of the above.
14. Esterification of cholesterol occurs at carbon position
15. Name the test employed to check the purity of butter through the estimation of volatile fatty acids
(a) lodine number (b) Reichert-Meiss! number (c) Saponification number (d) Acid number.
roteins are the most abundant organic molecules of the living system. They occur in every part of the cell and constitute about 50% of the cellular dry weight. Proteins form the fundamental basis of structure and function of life.
Structural functions : Certain proteins perform brick and mortar roles and are primarily responsible for structure and strength of body. These include collagen and elastin found in bone matrix, vascular system and other organs and a-keratin present in epidermal tissues.

The term protein is derived from a Greek word proteios, meaning holding the first place. Berzelius (Swedish chemist) suggested the name proteins to the group of organic compounds that are utmost important to life. Mulder (Dutch chemist) in 1838 used the term proteins for the high molecular weight nitrogen-rich and most abundant substances present in animals and plants.

Proteins perform a great variety of specialized and essential functions in the living cells. These functions may be broadly grouped as. static (structural) and dynamic.
Dynamic functions : The dynamic functions of proteins are more diversified in nature. These include proteins acting as enzymes, hormones, blood clotting factors, immunoglobulins, membrane receptors, storage proteins, besides their function in genetic control, muscle contraction, respiration etc. Proteins performing dynamic functions are appropriately regarded as the working horses of cell.

Proteins are predominantly constituted by five major elements in the following proportion.
Carbon
50
-
55%
Hydrogen
6
-
7.3%
Oxygen
:
19
—
24%
Nitrogen
:
13
-
19%
Sulfur
:
0
-
4%
Besides the above, proteins may also contain other elements such as P, Fe, Cu, |, Mg, Mn, Zn etc. The content of nitrogen, an_ essential component of proteins, on an average is 16%. Estimation of nitrogen in the laboratory (mostly by Kjeldahl's method) is also used to find out the amount of protein in biological fluids and foods.

Proteins on
complete hydrolysis
(with concen-
trated
HCl acids.
This for
several hours)
is
a
yield common
proteins.
Therefore,
L-a-amino property
all the
are the
polymers
L-c-amino acids.

As many as 300 amino acids occur in nature— Of these, only 20—known as standard amino acids are repeatedly found in the structure of proteins, isolated from different forms of life— animal, plant and microbial. This is because of the universal nature of the genetic code available for the incorporation of only 20 amino acids when the proteins are synthesized in the cells. The process in turn is controlled by DNA, the genetic material of the cell. After the synthesis of proteins, some of the incorporated amino acids undergo modifications to form their derivatives.

Amino acids are a group of organic compounds containing two functional groups— amino and carboxyl. The amino group (—NH)) is basic while the carboxyl group (—COOH) is acidic in nature.

The amino acids are termed as o-amino acids, if both the carboxyl and amino groups are attached to the same carbon atom, as depicted below
General structure
of
The a-carbon atom binds to a side chain represented by R which is different for each of the 20 amino acids found in proteins. The amino acids mostly exist in the ionized form in the biological system (shown above).

If a carbon atom is attached to four different groups, it is asymmetric and therefore exhibits optical isomerism. The amino acids (except glycine) possess four distinct groups (R, H, COO-, NH3) held by a-carbon. Thus all the amino acids (except glycine where R = H) have optical isomers.
The structures of Land D-amino acids are written based on the configuration of Land D-glyceraldehyde as shown in Fig.4.1. The proteins are composed of L-a-amino acids.

There are different ways of classifying the amino acids based on the structure and chemical nature, nutritional requirement, metabolic fate etc.
A. Amino acid classification based on the structure : A comprehensive classification of amino acids is based on their structure and chemical nature. Each amino acid is assigned a 3 letter or 1 letter symbol. These symbols are commonly used to represent the amino acids in protein structure. The 20 amino acids found in proteins are divided into seven distinct groups.
In Table 4.1, the different groups of amino acids, their symbols and structures are given. The salient features of different groups are described next

Name
Symbol
Structure
Special
group
present
3
letters
1
letter
I.
Amino
acids
with
aliphatic
side
chains
1. Glycine
Gly
G
eee
+
NH3
2. Alanine
Ala
A
eee
NHS
H3C_
3. Valine
Val
V
oS
GH
COON
Branched
chain
HsC
HE
H3C_
4. Leucine
Leu
L
4
oe
Oe
Branched
chain
3
NH3
CH3
“CHe
_
5.
Isoleucine
lle
|
eee)
Branched
chain
H3C
NHt

6. Serine
Ser
S
CH2—CH~-COO™
Hydroxyl
OH
NHs
7. Threonine
Thr
T
MgC
SCHS
CH
SCOOS
Hydroxyl
OH
NH3
Tyrosine
Tyr
Y
See
under
aromatic
Hydroxyl
Table 4.1 contd. next page

acids, Structure. = V. Basic amino acids. , Name Symbol.3 letters = 14. Lysine Lys. , Name Symbol.1 letter = K. , Structure. = D E J G CH 2 CH 2 CH 2 CH 2 CH COO H NH 3 + + NH 3. , Name Symbol.3 letters = 15. Arginine Arg. , Name Symbol.1 letter = R. , Structure. = NH 3 + NH 2 + C NH 2 NH CH 2 CH 2 CH 2 CH COO. , Name Symbol.3 letters = 16. Histidine His. , Name Symbol.1 letter = H. , Structure. = HN N NH 3 + 2
page
Table
4.1
contd.
next

red), Special group present. = ( Note : R group is shown in red)
1. Amino acids with aliphatic side chains : These are monoamino monocarboxylic acids. This group consists of the most simple amino acids—glycine, alanine, valine, leucine and isoleucine. The last three amino acids (Leu, Ile, Val) contain branched aliphatic side chains, hence they are referred to as branched chain amino acids.
2. Hydroxyl group containing amino acids : Serine, threonine and_ tyrosine are hydroxyl group containing amino acids. Tyrosine—being aromatic in nature—is usually considered under aromatic amino acids.
3. Sulfur containing amino acids : Cysteine with sulfhydryl group and methionine with thioether group are the two amino acids incorporated during the course of
protein synthesis.
Cystine,
|
another importa
nt is
sulfur containing
formed amino
by condensation
acid, of
two molecules
of cysteine.
. Acidic amino acids and their amides : Aspartic acid and glutamic acids are dicarboxylic monoamino acids while asparagine and glutamine are their respective amide derivatives. All these four amino acids possess distinct codons for their incorporation into proteins.
. Basic amino acids : The three amino acids lysine, arginine (with guanidino group) and histidine (with imidazole ring) are dibasic monocarboxylic acids. They are highly basic in character.
. Aromatic amino acids : Phenylalanine, tyrosine and tryptophan (with indole ring)
are aromatic amino acids. Besides these, histidine may also be considered under this category.
7. Imino acids : Proline containing pyrrolidine ring is a unique amino acid. It has an imino group (=NH), instead of an amino group (—NH),) found in other amino acids. Therefore, proline is an a-imino acid.
Heterocyclic amino acids : Histidine, tryptophan and proline.

B. Classification of amino acids based on polarity : Amino acids are classified into 4 groups based on their polarity. Polarity is important for protein structure.
1. Non-polar amino acids : These amino acids are also referred to as hydrophobic (water hating). They have no charge on the ‘R’ group. The amino acids included in this group are—alanine, leucine, isoleucine, valine, methionine, phenyl- alanine, tryptophan and proline.
2. Polar amino acids with no charge on ‘R’ group : These amino acids, as such, carry no charge on the ‘R’ group. They however possess groups such as__ hydroxyl, sulfhydryl and amide and participate in hydrogen bonding of protein structure. The simple amino acid glycine (where R =H) is also considered in this category. The amino acids in this group are— glycine, serine, threonine, cysteine, glutamine, asparagine and tyrosine.
3. Polar amino acids with positive ‘R’ group : The three amino acids lysine, arginine and histidine are included in this group.
4. Polar amino acids with negative ‘R’ group : The dicarboxylic monoamino acids— aspartic acid and glutamic acid are considered in this group.
C. Nutritional classification of amino acids : The 20 amino acids (Table 4.1) are required for the synthesis of variety proteins, besides other biological functions. However, all these 20 amino acids need not be taken in the diet. Based on the nutritional requirements, amino acids are grouped into two classes—essential and non- essential.
- 1. Essential or indispensable amino acids : The amino acids which cannot be synthesized by the body and, therefore, need to be supplied through the diet are called essential amino acids. They are required for proper growth and maintenance of the individual. The ten amino acids listed below are essential for humans (and also rats) :
Arginine, Valine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan.

[The code A.V. HILL, MP., T. T. (first letter of each amino acid) may be memorized to recall essential amino acids. Other useful codes are H. VITTAL, LMP; PH. VILLMA, TT, PVT TIM HALL and MATTVILPhLy.]
The two amino acids namely arginine and histidine can be synthesized by adults and not by growing children, hence these are considered as semi-essential amino acids (remember Ah, to recall). Thus, 8 amino acids are absolutely essential while 2 are semi-essential.
- 2. Non-essential or dispensable amino acids : The body can synthesize about 10 amino acids to meet the biological needs, hence they need not be consumed in the diet. These are—glycine, alanine, serine, cysteine, aspartate, asparagine, glutamate, glutamine, tyrosine and proline.
D. Amino acid classification based on their metabolic fate : The carbon skeleton of amino acids can serve as a precursor for the synthesis of glucose (glycogenic) or fat (ketogenic) or both. From metabolic view point, amino acids are divided into three groups (for details, Refer Chapter 15).
1. Glycogenic amino acids : These amino acids can serve as precursors for the formation of glucose or glycogen. e.g. alanine, aspartate, glycine, methionine etc.
2. Ketogenic amino acids : Fat can be synthesized from these amino acids. Two amino acids leucine and lysine are exclusively ketogenic.
- 3. Glycogenic and ketogenic amino acids : The four amino acids isoleucine, phenylalanine, tryptophan, tyrosine are precursors for synthesis of glucose as well as fat.

As already stated, 20 amino acids are commonly found in proteins. In recent years, a 21st amino acid namely selenocysteine has been added. It is found at the active sites of certain enzymes/proteins (selenoproteins). e.g. glutathione peroxidase, glycine reductase, 5'-deiodinase, thioredoxin reductase. Selenocysteine is an unusual amino acid containing the trace element selenium in place of the sulfur atom of cysteine.
CH2~CH—~COOT
CH2~CH—COOT
SH
NH3
SeH
NH3
Cysteine
Selenocysteine
Incorporation of selenocysteine into the proteins during translation is carried out by the codon namely UGA. It is interesting to note that UGA is normally a stop codon that terminates protein biosynthesis. Another unique feature is that selenocysteine is enzymatically generated from serine directly on the tRNA (selenocysteinetRNA), and then incorporated into proteins.
Pyrrolysine -the 22nd amino acid? : In the year 2002, some researchers have described yet another amino acid namely pyrrolysine as the 22nd amino acid present in protein. The stop codon UAG can code for pyrrolysine.

The amino acids differ in their physico— chemical properties which ultimately determine the characteristics of proteins.

1. Solubility : Most of the amino acids are usually soluble in water and insoluble in organic solvents.
2. Melting points: Amino acids generally melt at higher temperatures, often above 200°C.
3. Taste: Amino acids may be sweet (Gly, Ala, Val), tasteless (Leu) or bitter (Arg, Ile). Monosodium glutamate (MSG; ajinomoto) is used as a flavoring agent in food industry, and Chinese foods to increase taste and flavor. In some individuals intolerant to MSG, Chinese restaurant syndrome (brief and reversible flulike symptoms) is observed.
4. Optical properties: All the amino acids except glycine possess optical isomers due to the presence of asymmetric carbon atom. Some amino acids also have a second asymmetric carbon e.g. isoleucine, threonine. The structure of Land D-amino acids in comparison with glyceraldehyde has been given (See Fig.4.1).
5. Amino acids as ampholytes : Amino acids contain both acidic (—COOH) and_ basic (—NH)) groups. They can donate a proton or accept a proton, hence amino acids are regarded as ampholytes.
Zwitterion or dipolar ion: The name zwitter is derived from the German word which means hybrid. Zwitter ion (or dipolar ion) is a hybrid molecule containing positive and negative ionic groups.
The amino acids rarely exist in a neutral form with free carboxylic (~COOH) and free amino (—NHb>) groups. In strongly acidic pH (low pH), the amino acid is positively charged (cation) while in strongly alkaline pH (high pH), it is negatively charged (anion). Each amino acid has a characteristic pH (e.g. leucine, pH 6.0) at which it carries both positive and negative charges and exists as zwitterion (Fig.4.2).
Isoelectric pH
(symbol pl)
is defined
as the
pH at which a molecule exists as a zwitterion or dipolar ion and carries no net charge. Thus, the molecule is electrically neutral.

The pl value can be calculated by taking the average pKa values corresponding to the ionizable groups. For instance, leucine has two ionizable groups, and its pl can be calculated as follows.
Fig.
4.2
:
Existence of
an amino
acid as
cation, anion
and zwitterion.
Leucine exists as cation at pH below 6 and anion at pH above 6. At the isoelectric pH (pl = 6.0), leucine is found as zwitterion. Thus the pH of the medium determines the ionic nature of amino acids.
For the calculation of pl of amino acids with more than two ionizable groups, the pKas for all the groups have to be taken into account.
Titration of amino acids : The existence of different ionic forms of amino acids can be more easily understood by the titration curves. The graphic representation of leucine titration is depicted in Fig.4.3. At low pH, leucine exists in a fully protonated form as cation. As the titration proceeds with NaOH, leucine loses its protons and at isoelectric pH (pl), it becomes a zwitterion. Further titration results in the formation of anionic form of leucine.
Some more details on isoelectric pH are discussed under the properties of proteins (p. 60).

The general reactions of amino acids are mostly due to the presence of two functional
groups namely
carboxyl
(~COOH)
amino
(—NHg)
group.

1.
form salts
(-COONa)
bases and
esters
(—COOR’)
Decarboxylation group
and with
with alcohols.
:
Amino decarboxylation
acids undergo
to amines.
R-CH-COO
produce corresponding
+
NH3
—+R-CH,
COz
NH3
This reaction assumes significance in the living cells due to the formation of many biologically important amines. These include histamine, tyramine and y-amino butyric acid (GABA) from the amino acids histidine, tyrosine — and glutamate, respectively.
Reaction with
ammonia:
The carboxyl
group of
dicarboxylic with
NH,
Aspartic amino
to form
amide acid
Glutamic
+
NH, acid
0.5
+
——>
NH;
1.0
—>
1.5
acids reacts
Asparagine
Glutamine
2.0
+
Equivalents of
NaOH
——>

(R
=
(CH,),—CH—CH,—;
pK,
=
Dissociation for
constant
COOH;
pK,
=
pl
=
Isoelectric
Dissociation pH;
constant for
NH).
2.
3.

4. The amino groups behave as bases and combine with acids (e.g. HCl) to form salts (-NH}CI).
5. Reaction with ninhydrin: The o-amino acids react with ninhydrin to form a purple, blue or pink colour complex (Ruhemann’s purple).
Amino acid + Ninhydrin —> Keto acid + NH3 + CO2 + Hydrindantin
Hydrindantin
+
NH3
+
Ninhydrin
—>
Ruhemann’s
purple
Ninhydrin reaction is effectively used for the quantitative determination of amino acids and proteins. (Note : Proline and hydroxyproline give yellow colour with ninhydrin).
6. Colour reactions of amino acids : Amino acids can be identified by specific colour reactions (See Table 4.3).
7. Transamination: Transfer of an amino group from an amino acid to a keto acid to form a new amino acid is a very important reaction in amino acid metabolism (details given in Chapter 15).
8. Oxidative deamination : The amino acids undergo oxidative deamination to liberate free ammonia (Refer Chapter 15).

Besides the 20 standard amino acids (described above) present in the protein structure, there are several other amino acids which are biologically important. These include the amino acid derivatives found in proteins, non-protein amino acids performing specialized functions and the D-amino acids.
A. Amino acid derivatives in proteins : The 20 standard amino acids can be incorporated into proteins due to the presence of universal genetic code. Some of these amino acids undergo specific modification after the protein synthesis occurs. These derivatives of amino acids are very important for protein structure and functions. Selected examples are given hereunder.
1 Collagen—the most abundant protein in mammals—contains 5-hydroxylysine. 4-hydroxyproline and
| Histones—the proteins found in association with DNA—contain many methylated, phosphorylated or acetylated amino acids.
1 y-Carboxyglutamic acid is found in certain plasma proteins involved in blood clotting.
1 Cystine is formed by combination of two cysteines. Cystine is also considered as derived amino acid.
B. Non-protein amino acids : These amino acids, although never found in proteins, perform several biologically important functions. They may be either a-or non-a-amino acids. A selected list of these amino acids along with their functions is given in Table 4.2.
C. D-Amino acids : The vast majority of amino acids isolated from animals and plants are of L-category. Certain D-amino acids are also found in the antibiotics (actinomycin-D, valinomycin, gramicidin-S). D-serine and D-aspartate are found in brain tissue. DGlutamic acid and D-alanine are present in bacterial cell walls.

There a certain non-standard amino acids that are used as drugs.
| D-Penicillamine — (D-dimethylglycine), a metabolite of penicillin, is employed in the chelation therapy of Wilson’s disease. This is possible since D-penicillamine can effectively chelate copper.
|
N-Acetylcysteine
is
used
in
cystic
fibrosis,
and
chronic
renal
insufficiency,
as
it
can
function
as
an
antioxidant.
| Gabapentin (y-aminobutyrate linked to cyclohexane) is used as an anticonvulsant.

polypeptide chains referred to as subunits. The spatial arrangement of these subunits is known as quaternary structure.
Proteins are
the polymers
of
L-a-amino acids.
The structure of proteins is rather complex which can
be divided
into
4
levels of
organization
(Fig.4.4)
:
1. Primary structure : The linear sequence of amino acids forming the backbone of proteins (polypeptides).
2. Secondary structure: The _ spatial arrangement of protein by twisting of the polypeptide chain.

structure of a functional protein.
the more Some of two. or 4. Quaternary — structure: proteins are composed of
[The structural hierarchy of proteins is comparable with the structure of a building. The amino acids may be considered as the bricks, the wall as the primary structure, the twists in a wall as the secondary structure, a full-fledged self-contained room as the tertiary structure. A building with similar and dissimilar rooms will be the quaternary structure].
The term protein is generally used for a polypeptide containing more than 50 amino acids. In recent years, however, some authors have been using 'polypeptide' even if the number of amino acids is a few hundreds. They prefer to use protein to an assembly of polypeptide chains with quaternary structure.

Each protein has a unique sequence of amino acids which is determined by the genes contained in DNA. The primary structure of a protein is largely responsible for its function. A vast majority of genetic diseases are due to abnormalities in the amino acid sequences of proteins i.e. changes associated with primary structure of protein.
double bond in character. It generally exists in trans configuration. Both —C=O and —NH groups of peptide bonds are polar and are involved in hydrogen bond formation.
The amino acid composition of a protein determines its physical and chemical properties.

The amino acids are held together in a protein by covalent peptide bonds or linkages. These bonds are rather strong and serve as the cementing material between the individual amino acids (considered as bricks).
Formation of a peptide bond: When the amino group of an amino acid combines with the carboxyl group of another amino acid, a peptide bond is formed (Fig.4.5). Note that a dipeptide will have two amino acids and one peptide (not two) bond. Peptides containing more than 10 amino acids (decapeptide) are referred to as polypeptides.
Characteristics of peptide bonds: The peptide bond is rigid and planar with partial
Writing of peptide structures : Conventionally, the peptide chains are written with the free amino end (N-terminal residue) at the left, and the free carboxyl end (C-terminal residue) at the right. The amino acid sequence is read from N-terminal end to C-terminal end. Incidentally, the protein biosynthesis also starts from the N-terminal amino acid.
Shorthand to read peptides: The amino acids in a peptide or protein are represented by the 3-letter or one letter abbreviation. This is the chemical shorthand to write proteins.
Naming of
peptides:
For naming
peptides, the
-ine
(glycine),
(glutamate)
are the
exception acid.
Thus amino
acid suffixes
-an
(tryptophan), changed
-ate to
-yl of
C-terminal
a
terminal tripeptide
glutamate, with
amino composed
H,IN—glutamate—cysteine—glycine—COO-
E
-—
Cc
—
G
Glu
—
Cys
—
Gly
in a peptide
One letter symbols
Three letter symbols
Glutamyl—
cysteinyl
—
glycine
Peptide name
Fig. 4.6 : Use of symbols in representing a peptide
(Note : A tripeptide with 3 amino acids and two peptide bonds is shown; Free —NH3 is on the left while free —COO is on the right).

of an
N-
2.
Degradation of
protein or
a
cysteine and
a
C-terminal glycine
is called
glutamyl-cysteinyl-glycine.
In the Fig.4.6, the naming and representation of a tripeptide are shown.
Dimensions of a_ peptide chain: The dimensions of a fully extended polypeptide chain are depicted in Fig.4.7. The two adjacent a-carbon atoms are placed at a distance of 0.36 nm. The interatomic distances and bond angles are also shown in this figure.

The primary structure comprises the identification of constituent amino acids with regard to their quality, quantity and sequence in a protein structure. A pure sample of a protein or a polypeptide is essential for the determination of primary structure which involves 3 stages :
into smaller
fragments.
3.
Determination of
the amino
1. Determination of amino acid composition in a protein : The protein or polypeptide is completely hydrolysed to liberate the amino acids which are quantitatively estimated. The hydrolysis may be carried out either by acid or alkali treatment or by enzyme hydrolysis. Treatment with enzymes, however results in smaller peptides rather than amino acids.
mixture that Pronase is a proteolytic enzymes hydrolysis of proteins. of non-specific causes complete
1. Determination of amino acid composition.
Separation and estimation of amino acids : The mixture of amino acids liberated by protein hydrolysis can be determined by chromatographic techniques. The reader must refer Chapter 41 for the separation and quantitative determination of amino acids. Knowledge on
Fig. 4.7 : Dimensions of a fully extended polypeptide chain.
(The distance between two adjacent a-carbon atoms is 0.36 nm).
Fig. 4.8 : Sanger's reagent (1-fluoro 2,4-dinitrobenzene) and Edman's reagent (Phenyl isothiocyanate) in the determination of amino acid sequence of a protein (AA-Amino acid).
primary structure of proteins will be incomplete without a thorough understanding of chromatography.
2. Degradation of protein into smaller fragments : Protein is a large molecule which is sometimes composed of individual polypeptide chains. Separation of polypeptides is essential before degradation.

(c) Breakdown of polypeptides _ into fragments: Polypeptides are degraded into smaller peptides by enzymatic or chemical methods.
(a) Liberation of polypeptides: Treatment with urea or guanidine hydrochloride disrupts the non-covalent bonds and dissociates the protein into polypeptide units. For cleaving the disulfide linkages between the polypeptide units, treatment with performic acid is necessary.
(b) Number of polypeptides : The number of polypeptide chains can be identified by treatment of protein with dansyl chloride. It specifically binds with N-terminal amino acids to form dansyl polypeptides which on hydrolysis yield N-terminal dansyl amino acid. The number of dansyl amino acids produced is equal to the number of polypeptide chains in a protein.
Enzymatic cleavage : The proteolytic enzymes such as trypsin, chymotrypsin, pepsin and elastase exhibit specificity in cleaving the peptide bonds (Refer Fig.8.7). Among these enzymes, trypsin is most commonly used. It hydrolyses the peptide bonds containing lysine or arginine on the carbonyl (—C=O) side of peptide linkage.
Chemical cleavage: Cyanogen bromide (CNBr) is commonly used to split polypeptides into smaller fragments. CNBr specifically splits peptide bonds, the carbonyl side of which is contributed by the amino acid methionine.
3. Determination of amino acid sequence : The polypeptides or their smaller fragments are conveniently utilized for the determination of sequence of amino acids. This is done in a stepwise manner to finally build up the order of amino acids in a protein. Certain reagents are employed for sequence determination (Fig.4.8).

Sanger's reagent: Sanger used 1-fluoro 2, 4-dinitrobenzene (FDNB) to determine insulin structure. FDNB_ specifically binds with N-terminal amino acid to form a dinitrophenyl (DNP) derivative of peptide. This on hydrolysis yields DNP-amino acid (N-terminal) and free amino acids from the rest of the peptide chain. DNP-amino acid can be identified by chromatography.
Sanger's reagent has limited use since the peptide chain is hydrolysed to amino acids.
Edman's reagent: Phenyl isothiocyanate is the Edman's reagent. It reacts with the Nterminal amino acid of peptide to form a phenyl thiocarbamy| derivative. On treatment with mild acid, phenyl thiohydantoin (PTH)—amino acid, a cyclic compound is liberated. This can be identified by chromatography (Fig.4.8).
Edman's reagent has an advantage since a peptide can be sequentially degraded liberating N-terminal amino acids one after another which can be identified. This is due to the fact that the peptide as a whole is not hydrolysed but only releases PTH-amino acid.
Sequenator : This is an automatic machine to determine the amino acid sequence in a polypeptide (with around 100 residues). It is based on the principle of Edman's degradation (described above). Amino acids are determined sequentially from N-terminal end. The PTHamino acid liberated is identified by highperformance liquid chromatography (HPLC). Sequenator takes about 2 hours to determine each amino acid.

In the determination of primary structure of protein, several methods (enzymatic or chemical) are simultaneously employed. This results in the formation of overlapping peptides. This is due to the specific action of different agents on different sites in the polypeptide. Overlapping peptides are very useful in determining the amino acid sequence.

It is the genetic material (chemically DNA) which ultimately determines the sequence of amino acids in a polypeptide chain. By analysing the nucleotide sequence of DNA that codes for protein, it is possible to translate the nucleotide sequence into amino acid sequence. This technique, however, fails to identify the disulfide bonds and changes that occur in the amino acids after the protein is synthesized (post-translational modifications).

The conformation of polypeptide chain by twisting or folding is referred to as secondary structure. The amino acids are located close to each other in their sequence. Two types of secondary structures, ot-helix and f-sheet, are mainly identified.
Indian scientist Ramachandran made a significant contribution in understanding the spatial arrangement of polypeptide chains.

a-Helix is the most common spiral structure of protein. It has a rigid arrangement of polypeptide chain. a-Helical structure was proposed by Pauling and Corey (1951) which is regarded as one of the milestones in the biochemistry research. The salient features of a-helix (Fig.4.9) are given below
1. The a-helix is a tightly packed coiled structure with amino acid side chains extending outward from the central axis.
2. The a-helix is stabilized by extensive hydrogen bonding. |t is formed between H atom attached to peptide N, and O atom attached to peptide C. The hydrogen bonds are individually weak but collectively, they are strong enough to stabilize the helix.
3. All the peptide bonds, except the first and last in a polypeptide chain, participate in hydrogen bonding.
4. Each turn of o-helix contains 3.6 amino acids and travels a distance of 0.54 nm. The spacing of each amino acid is 0.15 nm.
5. a-Helix is a stable conformation formed spontaneously with the lowest energy.
6. The right handed a-helix is more stable than left handed helix (a right handed helix turns in the direction that the fingers of right hand curl when its thumb points in the direction the helix rises).
7. Certain amino acids (particularly proline) disrupt the a-helix. Large number of acidic (Asp,
Glu) or basic (Lys, Arg, His) amino acids also interfere with o-helix structure.

This is the second type of structure (hence B after a) proposed by Pauling and Corey. B-Pleated sheets (or simply f-sheets) are composed of two or more segments of fully extended peptide chains (Fig.4.10). \In the B-sheets, the hydrogen bonds are formed between the neighbouring segments of polypeptide chain(s).

The polypeptide chains in the B-sheets may be arranged either in parallel (the same direction) or anti-parallel (opposite direction). This is illustrated in Fig.4.10.
B-Pleated sheet may be formed either by separate polypeptide chains (H-bonds are interchain) or a single polypeptide chain folding back on to itself (H-bonds are intrachain).
Fig. 4.11 : Diagrammatic representation of a protein containing a-helix and B-pleated sheet (blue).
Occurrence of f-sheets: Many proteins contain B-pleated sheets. As such, the o-helix and B-sheet are commonly found in the same protein structure (Fig.4.11). In the globular proteins, B-sheets form the core structure.
Other types of secondary structures : Besides the a-and f-structures described above, the B-bends and nonrepetitive (less organised structures) secondary structures are also found in proteins.

The three-dimensional arrangement of protein structure is referred to as_ tertiary structure. It is a compact structure with hydrophobic side chains held interior while the hydrophilic groups are on the surface of the protein molecule. This type of arrangement ensures stability of the molecule.
Bonds of tertiary structure: Besides the hydrogen bonds, disulfide bonds (—S—S), ionic interactions (electrostatic bonds), hydrophobic interactions and van der Waals forces also contribute to the tertiary structure of proteins.
Domains: The term domain is used to represent the basic units of protein structure (tertiary) and function. A polypeptide with 200 amino acids normally consists of two or more domains.

A great majority of the proteins are composed of single polypeptide chains. Some of the proteins, however, consist of two or more polypeptides which may be identical or unrelated. Such proteins are termed as oligomers and possess quaternary structure. The individual polypeptide chains are known as monomers, protomers or subunits. A dimer consits of two polypeptides while a tetramer has four.
Bonds in quaternary structure: The monomeric subunits are held together by nonconvalent bonds namely hydrogen bonds, hydrophobic interactions and ionic bonds.
Importance of oligomeric proteins: These proteins play a significant role in the regulation of metabolism and cellular function.
Examples of oligomeric proteins: Hemoglobin, aspartate transcarbomylase, _ lactate dehydrogenase.

Protein structure is stabilized by two types of bonds—covalent and non-covalent.
1. Covalent bonds : The peptide and disulfide bonds are the strong bonds in protein structure. The formation of peptide bond and_ its chracteristics have been described.
Disulfide bonds: A disulfide bond (—S—S) is formed by the sulfhydryl groups (—SH) of two cysteine residues, to produce — cystine (Fig.4.12A). The disulfide bonds may be formed in a single polypeptide chain or between different polypeptides. These bonds contribute to the structural conformation and _ stability of proteins.
four
2. Non-covalent bonds : There are, mainly, types of non-covalent bonds.
(a) Hydrogen bonds: The hydrogen bonds are formed by sharing of hydrogen atoms between the nitrogen and carbonyl oxygen of different peptide bonds (Fig.4.12B). Each hydrogen bond is weak but collectively they are strong. A large number of hydrogen bonds significantly contribute to the protein structure.
(b) Hydrophobic bonds : The non-polar side chains of neutral amino acids tend to be
Fig. 4.12 : Major bonds in protein structure (A) Disulfide bond (B) Hydrogen bonds (C) Hydrophic bonds (D) Electrostatic bond. (Note : See Fig. 4.5 for peptide bond).
(c)
Electrostatic bonds:
These bonds
are formed
by negatively
_
interactions charged
groups between
(e.g.
COO-)
of

Structure of human insulin: Insulin consists of two polypeptide chains, A and B (Fig.4.13). The A chain has glycine at the N-terminal end and asparagine at the C-terminal end. The B chain has phenylalanine and alanine at the Nand C-terminal ends, respectively. Originally, insulin is synthesized as a single polypeptide preproinsulin which undergoes _ proteolytic processing to give proinsulin and finally insulin. The structural aspects of hemoglobin and collagen are respectively given in Chapters 10 and 22.

For the determination of secondary and tertiary protein structures, X-ray crystallography is most commonly used. Nuclear magnetic resonance (NMR) spectra of proteins provides structural and functional information on the atoms and groups present in the proteins.
Fig. 4.13 : Diagrammatic representation of human insulin structure (Note : A and B polypeptide chains are held by two disulfide linkages).

Several methods are employed to isolate and purify proteins. Initially, proteins are fractionated by using different concentrations of ammonium sulfate or sodium sulfate. Protein fractionation may also be carried out by ultracentrifugation.
Protein separation is achieved by utilizing electrophoresis, isoelectric focussing, immunoelectrophoresis, ion-exchange chromatography, gel-filtration, high performance liquid chromatography (HPLC) etc. The details of these techniques are described in Chapter 41.

1. Solubility: Proteins form colloidal solutions instead of true solutions in water. This is due to huge size of protein molecules.
2. Molecular weight: The proteins vary in their molecular weights, which, in turn, is dependent on the number of amino acid residues. Each amino acid on an average contributes to a molecular weight of about 110. Majority of proteins/polypeptides may be composed of 40 to 4,000 amino acids with a molecular weight ranging from 4,000 to 440,000. A few proteins with their molecular weights are listed below :
Insulin-5,700; Myoglobin-17,000; Hemoglobin64,450; Serum albumin-69,000.
3. Shape: There is a wide variation in the protein shape. It may be globular (insulin), oval (albumin) fibrous or elongated (fibrinogen).
4. Isoelectric pH: Isoelectric pH (pl) as a property of amino acids has been described. The nature of the amino acids (particularly their ionizable groups) determines the pl of a protein. The acidic amino acids (Asp, Glu) and basic amino acids (His, Lys, Arg) strongly influence the pl. At isoelectric pH, the proteins exist as zwitterions or dipolar ions. They are electrically neutral (do not migrate in the electric field) with minimum. solubility, maximum precipitability and least buffering capacity. The isoelectric pH(pl) for some proteins are given here
Pepsin-1.1; Casein-4.6; Human albumin-4.7; Urease-5.0; Hemoglobin-6.7; Lysozyme-11.0.
5. Acidic and basic proteins: Proteins in which the ratio (e Lys + € Arg)/(e Glu + € Asp) is greater than 1 are referred to as basic proteins. For acidic proteins, the ratio is less than 1.
6. Precipitation of proteins : Proteins exist in colloidal solution due to hydration of polar groups (—COO-, —NH3, —OH). Proteins can be precipitated by dehydration or neutralization of polar groups.

Precipitation at pl: The proteins in general are least soluble at isoelectric pH. Certain proteins (e.g. casein) get easily precipitated when the pH is adjusted to pl (4.6 for casein). Formation of curd from milk is a marvellous example of slow precipitation of milk protein, casein at pl. This occurs due to the lactic acid produced by fermentation of bacteria which lowers the pH to the pl of casein.
Precipitation by salting out: The process of protein precipitation by the additional of neutral salts such as ammonium sulfate or sodium sulfate is known as salting out. This phenomenon is explained on the basis of dehydration of protein molecules by salts. This causes increased proteinprotein interaction, resulting in molecular aggregation and precipitation.
The amount of salt required for protein precipitation depends on the size (molecular weight) of the protein molecule. In general, the higher is the protein molecular weight, the lower is the salt required for precipitation. Thus, serum globulins are precipitated by half saturation with ammonium sulfate while albumin is precipitated by full saturation. Salting out procedure is conveniently used for separating serum albumins from globulins.
The addition of small quantities of neutral salts increases the solubility of proteins. This process called as salting in is due to the diminished protein-protein interaction at low salt concentration.
Precipitation by salts of heavy metals : Heavy metal ions like Pb2+, Hg2+, Fe2+, Zn2+, Cd2+ cause precipitation of proteins. These metals
being positively charged, when added to protein solution (negatively charged) in alkaline medium results in precipitate formation. Based on the principle of precipitation, raw egg-white (protein-albumin) is sometimes used to overcome the toxicity of mercury.

Precipitation by anionic or alkaloid reagents : Proteins can be precipitated by trichloroacetic acid, sulphosalicylic acid, phosphotungstic acid, picric acid, tannic acid, phosphomolybdic acid etc. By the addition of these acids, the proteins existing as cations are precipitated by the anionic form of acids to produce proteinsulphosalicylate, protein-tungstate, protein-picrate etc. Industrial tanning of leather is based on the principle of protein precipitation by tannic acid.
Precipitation by organic solvents : Organic solvents such as alcohol are good _ protein precipitating agents. They dehydrate the protein molecule by removing the water envelope and cause precipitation. The use of surgical spirit (about 20% alcohol) as a disinfectant is based on the precipitation of proteins and the death of bacteria.
7. Colour reactions of proteins : The proteins give several colour reactions which are often useful to identify the nature of the amino acids present in them (Table 4.3).
Biuret reaction : Biuret is a compound formed
possessing at least two peptide linkages i.e., tripeptides (with 3 amino acids) give positive biuret test. Histidine is the only amino acid that answers biuret test. The principle of biuret test is conveniently used to detect the presence of proteins in biological fluids. The mechanism of biuret test is not clearly known. It is believed that the colour is due to the formation of a copper co-ordinated complex, as shown below.
The presence of magnesium and ammonium ions interfere in the biuret test. This can be overcome by using excess alkali.

The phenomenon of disorganization of native protein structure is known as denaturation. Denaturation results in the loss of secondary, tertiary and quaternary structure of proteins. This involves a change in physical, chemical and biological properties of protein molecules.
1., T ABLE 4.3 Colour.Reaction = Biuret reaction. 1., reactions of proteins/amino acids.Specific group or amino acid = Two peptide linkages. 2., T ABLE 4.3 Colour.Reaction = Ninhydrin reaction. 2., reactions of proteins/amino acids.Specific group or amino acid = D -Amino acids. 3., T ABLE 4.3 Colour.Reaction = Xanthoproteic reaction. 3., reactions of proteins/amino acids.Specific group or amino acid = Benzene ring of aromatic amino acids (Phe, Tyr, Trp). 4., T ABLE 4.3 Colour.Reaction = Milllons reaction. 4., reactions of proteins/amino acids.Specific group or amino acid = Phenolic group (Tyr). 5., T ABLE 4.3 Colour.Reaction = Hopkins-Cole reaction. 5., reactions of proteins/amino acids.Specific group or amino acid = Indole ring (Trp). 6., T ABLE 4.3 Colour.Reaction = Sakaguchi reaction. 6., reactions of proteins/amino acids.Specific group or amino acid = Guanidino group (Arg). 7., T ABLE 4.3 Colour.Reaction = Nitroprusside reaction. 7., reactions of proteins/amino acids.Specific group or amino acid = Sulfhydryl groups (Cys). 8., T ABLE 4.3 Colour.Reaction = Sulfur test. 8., reactions of proteins/amino acids.Specific group or amino acid = Sulfhydryl groups (Cys). 9., T ABLE 4.3 Colour.Reaction = Pauly's test. 9., reactions of proteins/amino acids.Specific group or amino acid = Imidazole ring (His). 10., T ABLE 4.3 Colour.Reaction = Folin-Coicalteau's test. 10., reactions of proteins/amino acids.Specific group or amino acid = Phenolic groups (Tyr)

Physical agents: Heat, violent X-rays, UV radiation. shaking,
Chemical agents : Acids, alkalies, organic solvents (ether, alcohol), salts of heavy metals (Pb, Hg), urea, salicylate, detergents (e.g. sodium dodecyl sulfate).

1. The native helical structure of protein is lost (Fig.4.14).
2. The primary structure of a protein with peptide linkages remains intact i.e., peptide bonds are not hydrolysed.
3. The protein loses its biological activity.
4. Denatured protein becomes insoluble in the solvent in which it was originally soluble.
5 The viscosity of denatured protein (solution) increases while its surface tension decreases.
6. Denaturation is associated with increase in
ionizable and
sulfhydryl groups
of protein.
This is
due to
loss of
hydrogen and
disulfide bonds.
- 7. Denatured protein is more easily digested.
This is
due to
increased exposure
of peptide
bonds to enzymes. Cooking causes protein denaturation and, therefore, cooked food (protein) is more easily digested. Further, denaturation of dietary protein by gastric HCl enchances protein digestion by pepsin.

instance, omelet
can be
prepared from
an egg
(protein-albumin)
but the
reversal is
not possible.
9. Careful denaturation is sometimes reversible (known as_ renaturation). Hemoglobin undergoes denaturation in the presence of salicylate. By removal of salicylate, hemoglobin is renatured.
- 10. Denatured protein cannot be crystallized.
Coagulation : The term 'coagulum' refers to a semi-solid viscous precipitate of protein. Irreversible denaturation results in coagulation. Coagulation is optimum and requires lowest temperature at isoelectric pH. Albumins and globulins (to a lesser extent) are coagulable proteins. Heat coagulation test is commonly used to detect the presence of albumin in urine.
Flocculation: It is the process of protein precipitation at isoelectric pH. The precipitate is referred to as flocculum. Casein (milk protein) can be easily precipitated when adjusted to isoelectric pH (4.6) by dilute acetic acid.
Flocculation is reversible. On application of heat, flocculum can be converted into an irreversible mass, coagulum.

Proteins are classified in several ways. Three major types of classifying proteins based on their function, chemical nature and __ solubility properties and nutritional importance are discussed here.

Based on the functions they perform, proteins are classified into the following groups (with examples)
1. Structural proteins : Keratin of hair and
nails, collagen of bone.
2. Enzymes or catalytic proteins : Hexokinase,
pepsin.
3. Transport proteins : Hemoglobin, serum
albumin.
4. Hormonal
Insulin,
hormone.
proteins :
growth
5. Contractile proteins : Actin, myosin.
6. Storage proteins : Ovalbumin, glutelin.

7. Genetic proteins : Nucleoproteins.
8. Defense proteins : Snake venoms, Immun-
oglobulins.
9. Receptor proteins for hormones, viruses.

This is a more comprehensive and popular classification of proteins. It is based on the amino acid composition, structure, shape and solubility properties. Proteins are broadly classified into 3 major groups
1. Simple proteins: They are composed of
only amino acid residues.
2. Conjugated proteins : Besides the amino acids, these proteins contain a non-protein moiety known as_ prosthetic group or conjugating group.
3. Derived proteins : These are the denatured
or degraded products of simple and conjugated proteins.
The above three classes are further subdivided into different groups. The summary of protein classification is given in the Table 4.4.
+ Proteins are the most abundant organic molecules of life. They perform static (structural) and dynamic functions in the living cells.
+ The dynamic functions of proteins are highly diversified such as enzymes, hormones,
clotting factors, immunoglobulins, storage proteins and membrane receptors.
+ Half of the amino acids (about 10) that occur in proteins have to be consumed by humans in the diet, hence they are essential.
+ A protein is said to be complete (or first class) protein if all the essential amino acids are present in the required proportion by the human body e.g. egg albumin.
+ Cooking results in protein denaturation exposing more peptide bonds for easy digestion.
+ Monosodium glutamate (MSG) is used as a flavoring agent in foods to increase taste and flavour. In some individuals intolerant to MSG, Chinese restaurant syndrome (brief and reversible flu-like symptoms) is observed.
(vii)

(viii)
Lectins are
—
carbohydrate-binding
(a) Globular proteins : These are spherical or oval in shape, soluble in water or other solvents and digestible.
(i) Albumins: Soluble in water and dilute salt solutions and coagulated by heat. e.g. serum albumin, ovalbumin (egg), lactalbumin (milk).
(ii) Globulins: Soluble in neutral and dilute salt solutions e.g. serum globulins, vitelline (egg yolk).
(iii) Glutelins : Soluble in dilute acids and alkalies and mostly found in plants e.g. glutelin (wheat), oryzenin (rice).
(iv)
Prolamines
:
Soluble in
70%
alcohol e.g.
gliadin
(wheat), zein
(maize).
- (v) Histones: Strongly basic proteins, soluble in water and dilute acids but insoluble in dilute ammonium hydroxide e.g. thymus histones.
(vi)
Globins
:
These are
generally consi-
dered along
globins with
are not
not histones.
basic precipitated
Protamines
However, proteins
by
:
and are
NH,OH.
They and
resemble in
are strongly
histones size
and
Protamines association
basic but
smaller soluble
are in
also with
sperm proteins.
nucleic
NH,OH.
found in
acids e.g.
proteins, interaction
They help
structures.
and are
involved in
the between
cells to
and maintain
In tissue
the proteins.
and laboratory,
useful for
the carbohydrates
organ lectins
are purification
by of
affinity graphy
(b)
e.g.
chromato- concanavalin
Fibrous proteins
shape,
A,
:
These are
insoluble digestion.
in water
Albuminoids agglutinin.
fiber like
in and
or resistant
to scleroproteins
are predominant
group of
fibrous proteins.

(i) Collagens are connective _ tissue proteins lacking tryptophan. Collagens, on boiling with water or dilute acids, yield gelatin which is soluble and digestible (Chapter 22).
(ii) Elastins : These proteins are found in elastic tissues such as tendons and arteries.
(iii) Keratins: These are present in exoskeletal structures e.g. hair, nails, horns. Human hair keratin contains as much as 14% cysteine (Chapter 22).
2. Conjugated proteins
(a) Nucleoproteins : Nucleic acid (DNA or RNA) is the prosthetic group e.g. nucleohistones, nucleoprotamines.
(b) Glycoproteins : The prosthetic group is
carbohydrate, which
is less
than
4%
of
protein. The term mucoprotein is used if the carbohydrate content is more than 4%. e.g. mucin (saliva), ovomucoid (egg white).
(c) Lipoproteins: — Protein — found in combination with lipids as the prosthetic group e.g. serum lipoproteins.
(d) Phosphoproteins : Phosphoric acid is the prosthetic group e.g. casein (milk), vitelline (egg yolk).
(e) Chromoproteins : The prosthetic group is coloured in nature e.g. hemoglobins, cytochromes.
(f) Metalloproteins : These proteins contain metal ions such as Fe, Co, Zn, Cu, Mg etc., e.g. ceruloplasmin (Cu), carbonic anhydrase (Zn).
3. Derived proteins : The derived proteins are of two types. The primary derived are the denatured or coagulated or first hydrolysed products of proteins. The secondary derived are the degraded (due to breakdown of peptide bonds) products of proteins.

(i) Coagulated proteins: These are the denatured proteins produced by agents such as heat, acids, alkalies etc. e.g. cooked proteins, coagulated albumin (egg white).
Proteans: These are the earliest products of protein hydrolysis by enzymes, dilute acids, alkalies etc. which are insoluble in water. e.g. fibrin formed from fibrinogen. (ii
(iii Metaproteins : These are the second stage products of protein hydrolysis obtained by treatment with slightly stronger acids and alkalies e.g. acid and alkali metaproteins.
(b) Secondary derived proteins : These are the progressive hydrolytic products of protein hydrolysis. These include proteoses, peptones, polypeptides and peptides.

The nutritive value of proteins is determined
by the composition of essential amino acids
(described already). From the nutritional point of view, proteins are classified into 3 categories.
1. Complete proteins : These proteins have
all the ten essential amino acids in the required proportion by the human body to promote good growth. e.g. egg albumin, milk casein.
2. Partially incomplete proteins : These proteins partially lack one or more essential amino acids, and can promote moderate growth. e.g. wheat and rice proteins (limiting Lys, Thr).
3. Incomplete proteins: These proteins completely lack one or more essential amino acids. Hence they do not promote growth at all e.g. gelatin (lacks Trp), zein (lacks Trp, Lys).

Several peptides occur in the living organisms that display a wide spectrum of biological functions. Generally, the term 'peptide' is applied when the number of constituent amino acids is less than 10. Some examples of biologically active peptides and their functions are described here.
- 1. Glutathione : It is a tripeptide composed of 3 amino acids. Chemically, glutathione is yglutamyl-cysteinyl-glycine. It is widely distributed in nature and exists in reduced or oxidized states.
2G—SH
=
G=S-S-G
Reduced
Oxidized
Functions: In a_ steady state, the cells generally maintain a ratio of about 100/1 of GSH to G—S—S-—G. The reversible oxidationreduction of glutathione is important for many of its biological functions.
1 Glutathione serves as a coenzyme for certain enzymes e.g. prostaglandin PGE, synthetase, glyoxylase.
1 It prevents the oxidation of sulfhydryl (—SH) groups of several proteins to disulfide (-S—S—) groups. This is essential for the protein function, including that of enzymes.
1 It is believed that glutathione in association with glutathione reductase participates in the formation of correct disulfide bonds in several proteins.
1 Glutathione (reduced) performs specialized functions in erythrocytes
(i) It maintains RBC membrane structure and integrity.
(ii) It protects hemoglobin from getting oxidized by agents such as H,O,.
1 Glutathione is involved in the transport of amino acids in the intestine and kidney tubules via y-glutamyl cycle or Meister cycle (Refer Chapter 8).
1 Glutathione is involved in the detoxication process. The toxic substances (organo- phosphates, nitro compounds) are converted to mercapturic acids.
glutathione peroxidase (a selenium containing
enzyme).
2 GSH + H,0,
mS".
_g
-g-G+2H,0
2.
Thyrotropin
releasing
hormone
(TRH)
:
It
is
a_

tripeptide
secreted
by
hypothalamus.
TRH
stimulates
pituitary
gland
to
release
thyrotropic
hormone.
3. Oxytocin: It is a hormone secreted by posterior pituitary gland and contains 9 amino
acids (nonapeptide). Oxytocin causes contraction of uterus.
4.
(antidiuretic
hormone,
ADH)
:
ADH
is
also
a
nonapeptide
produced
by
posterior
pituitary
gland.
It
kidneys
to
retain
water
and
thus
increases
the
blood
pressure.
5.
Angiotensins
:
Angiotensin
|
is
a
decapep-
tide
(10
amino
acids)
which
is
converted
to
Il
(8
amino
acids).
The
later
has
1 Toxic amounts of peroxides and free radicals produced in the cells are scavanged by
more
hypertensive
the
adrenal
gland.

Collagen is the most abundant protein in mammals. It is rich in hydroxyproline and hydroxylysine.
Several biologically important peptides are known in the living organism. These include glutathione for the maintenance of RBC structure and integrity; oxytocin that causes uterus contraction; vasopressin that stimulates retention of water by kidneys; enkephalins that inhibit the sense of pain in the brain.
Antibiotics such as actinomycin, gramicidin, bacitracin and tyrocidin are peptide in nature.
y-Carboxyglutamic acid is an amino acid derivative found in certain plasma proteins involved in blood clotting.
Homocysteine has been implicated as a risk factor in the onset of coronary heart diseases.
Several non-protein amino acids of biological importance are known. These include ornithine, citrulline and arginosuccinic acid (intermediates of urea synthesis), thyroxine and triiodothyronine (hormones), and f-alanine (of coenzyme A).
The protein-free filtrate of blood, required for biochemical investigations (e.g. urea, sugar) can be obtained by using protein precipitating agents such as phosphotungstic acid and trichloroacetic acid.
Heat coagulation test is most commonly employed to detect the presence of albumin in urine.
effect.
II
also
release
of
aldosterone
from
6. Methionine enkephalin: It is a penta- peptide found in the brain and has opiate like function. It inhibits the sense of a pain.
7. Bradykinin and kallidin: They are nona-
and
decapeptides,
respectively.
Both
of
them
act
as
powerful
vasodilators.
They
are
produced
from
plasma
proteins
by
snake
venom
enzymes.
8. Peptide antibiotics: Antibiotics such as gramicidin, bacitracin, tyrocidin and actinomycin are peptide in nature.

9. Aspartame: It is a dipeptide (aspartylphenylalanine methyl — ester), — produced by a combination of aspartic acid and phenylalanine. Aspartame is about 200 times sweeter than sucrose, and is used as a low-calorie artificial sweetner in softdrink industry.
10. Gastrointestinal hormones: Gastrin, secretin etc. are the gastrointestinal peptides which serve as hormones.

1. Proteins are nitrogen containing, most abundant organic macromolecules widely distributed in animals and plants. They perform structural and dynamic functions in the organisms.
2. Proteins are polymers composed of L-o-amino acids. They are 20 in number and classified into different groups based on their structure, chemical nature, nutritional requirement and metabolic fate. Selenocysteine has been recently identified as the 21st amino acid, and is found in certain proteins.
3. Amino acids possess two functional groups namely carboxyl (~COOH) and amino (— NH). In the physiological system, they exist as dipolar ions commonly referred to as zwitterions.
4. Besides the 20 standard amino acids present in proteins, there are several non-standard amino acids. These include the amino acid derivatives found in proteins (e.g. hydroxy- proline, hydroxylysine) and, non-protein amino acids (e.g. ornithine, citrulline).
5. The structure of protein is divided into four levels of organization. The primary structure represents the linear sequence of amino acids. The twisting and spatial arrangement of polypeptide chain is the secondary structure. Tertiary structure constitutes the three dimensional structure of a functional protein. The assembly of similar or dissimilar polypeptide subunits comprises quaternary structure.
6. The determination of primary structure of a protein involves the knowledge of quality, quantity and the sequence of amino acids in the polypeptide. Chemical and enzymatic methods are employed for the determination of primary structure.
7. The secondary structure of protein mainly consists of a-helix and/or B-sheet. o-Helix is stabilized by extensive hydrogen bonding. B-Pleated sheet is composed of two or more segments of fully extended polypeptide chains.
8. The tertiary and quaternary structures of protein are stabilized by non-covalent bonds such as hydrogen bonds, hydrophobic interactions, ionic bonds etc.
9. Proteins are classified into three major groups. Simple proteins contain only amino acid residues (e.g. albumin). Conjugated proteins contain a non-protein moiety known as prosthetic group, besides the amino acids (e.g. glycoproteins). Derived proteins are obtained by degradation of simple or conjugated proteins.

10. In addition to proteins, several peptides perform biologically important functions. These include glutathione, oxytocin and vasopressin.

1. Describe the classification of amino acids along with their structures.
2. Discuss the organization of protein structure. Give an account of the determination of primary structure of protein.
3. Describe the classification of proteins with suitable examples.
4. Write an account of non-standard amino acids.
5. Discuss the important biologically active peptides.

(a) Essential amino acids, (b) Zwitterion, (c) Peptide bond, (d) Edman’s reagent, (e)
o-Helix,
(f) B-Pleated sheet, (g) Denaturation, (h) Isoelectric point, (i) Glutathione, (j) Quaternary structure of protein.

1. The average nitrogen content of proteins
. Proteins are the polymers of NY
. Name the sulfur containing essential amino acid WwW
. The charged molecule which is electrically neutral is known as KR
. The non —a amino acid present in coenzyme A DH
. The bonds forming the backbone of protein structure
. The amino acid that is completely destroyed by acid hydrolysis of protein oN . The number of peptide bonds present in a decapeptide
9. The chemical name of Sanger’s reagent
10. The phenomenon of disorganization of native protein structure is known as

11. The imino acid found in protein structure
(a) Arginine (b) Proline (c) Histidine (d) Lysine.
12. The following is a non-protein amino acid
(a) Ornithine (b) Homocysteine (c) Histamine (d) All of them.
13. The bonds in protein structure that are not broken on denaturation.
(a) Hydrogen bonds (b) Peptide bonds (c) lonic bond (d) Disulfide bonds.
14. Sequenator is an automatic machine to determine amino acid sequence in a polypeptide chain.
The reagent used in sequenator is
(a) Sanger’s reagent (b) CNBr (c) Trypsin (d) Edman’s reagent.
15. The reaction given by two or more peptide linkages is
(a) Biuret test (b) Ninhydrin test (c) Xanthoproteic reaction (d) Pauley’s test.
here are two types of nucleic acids, namely deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Primarily, nucleic acids serve as repositories and transmitters of genetic information.

DNA was discovered in 1869 by Johann Friedrich Miescher, a Swiss researcher. The demonstration that DNA contained genetic information was first made in 1944, by Avery, Macleod and MacCary.

DNA
is the
chemical basis
of may
be regarded
information.
as the
DNA
maintaining reserve
is heredity
and bank
exclusively the
organisms identity
over of
genetic responsible
of different
millions aspect
of cellular
DNA.
The of
function
DNA
fundamental for
species years.
Further, is
under is
organized units
of of
every the
control of
into genetic
genes, the
The genes control the protein synthesis through the
mediation of RNA, as shown below
(ova
The interrelationship of these three classes of biomolecules (DNA, RNA and proteins) constitutes the central dogma of molecular biology or more commonly the central dogma of life.

Nucleic acids are the polymers of nucleotides (polynucleotides) held by 3' and 5' phosphate bridges. In other words, nucleic acids are built up by the monomeric units—nucleotides (It may be recalled that protein is a polymer of amino acids).

Nucleotides are composed of a nitrogenous base, a pentose sugar and a phosphate. Nucleotides perform a wide variety of functions in the living cells, besides being the building blocks or
Fig. 5.1 : General structure of nitrogen bases
(A) Purine (B) Pyrimidine (The positions are numbered
according to the international system).
monomeric units in the nucleic acid (DNA and RNA) structure. These include their role as structural Components of some coenzymes of B-complex vitamins (e.g. FAD, NAD*), in the energy reactions of cells (ATP is the energy currency), and in the control of metabolic reactions.

As already stated, the nucleotide essentially consists of nucleobase, sugar and phosphate. The term nucleoside refers to base + sugar. Thus, nucleotide is nucleoside + phosphate.

The nitrogenous bases found in nucleotides (and, therefore, nucleic acids) are aromatic heterocyclic compounds. The bases are of two types—purines and pyrimidines. Their general structures are depicted in Fig.5.1. Purines are numbered in the anticlockwise direction while pyrimidines are numbered in the clockwise direction. And this is an internationally accepted system to represent the structure of bases.

The
structures
of
major
purines
and
pyrimidines
found
in
nucleic
acids
are
shown
in
Fig.5.2.
DNA
and
RNA
contain
the
same
purines
namely
adenine
(A)
and
guanine
(G).
Further,
the pyrimidine cytosine (C) is found in both DNA and RNA. However, the nucleic acids differ with respect to the second pyrimidine base. DNA contains thymine (T) whereas RNA contains uracil (U). As is observed in the Fig.5.2, thymine and uracil differ in structure by the presence (in T) or absence (in U) of a methyl group.

The existence of a molecule in a_ keto (lactam) and enol (lactim) form is known as tautomerism. The heterocyclic rings of purines and
Il
)
pyrimidines with
oxo
Li functional
groups exhibit tautomerism as simplified below.
Fig. 5.3 : The tautomeric forms of cytosine.
The purine—guanine and __ pyrimidines- cytosine, thymine and uracil exhibit tautomerism. The lactam and lactim forms of cytosine are represented in Fig.5.3.
At
physiological pH, the lactam (keto) tauto- forms predominantly present.
meric are

: the bases described above, several minor and unusual bases are often found in DNA and RNA. These include 5-methylcytosine, N¢4-acetylcytosine, N°-methyladenine, N®, N®-dimethyladenine, pseudouracil etc. It is believed that the unusual bases in nucleic acids will help in the recognition of specific enzymes.
Other biologically important bases : The bases such as hypoxanthine, xanthine and uric acid (Fig.5.4) are present in the free state in the cells. The former two are the intermediates in purine synthesis while uric acid is the end product of purine degradation.
Fig. 5.5 : Structures of sugars present in nucleic acids (ribose is found in RNA and deoxyribose in DNA; Note the structural difference at C2).

The five carbon monosaccharides (pentoses) are found in the nucleic acid structure. RNA contains D-ribose while DNA _ contains D-deoxyribose. Ribose and deoxyribose differ in structure at Cy. Deoxyribose has one oxygen less at C) compared to ribose (Fig.5.5).

The addition of a pentose sugar to base produces a nucleoside. If the sugar is ribose, ribonucleosides are formed. Adenosine, guanosine, cytidine and uridine are the ribonucleosides of A, G, C and U respectively. If the sugar is a deoxyribose, deoxyribonucleosides are produced.
Purine bases of plants : Plants contain certain methylated purines which are of pharmacological interest. These include caffeine (of coffee), theophylline (of tea) and theobromine (of cocoa).
The term mononucleotide is used when a single phosphate moiety is added to a nucleoside. Thus adenosine monophosphate (AMP) contains adenine + ribose + phosphate.
The principal bases, their respective nucleosides and nucleotides found in the structure of nucleic acids are given in Table 5.1. Note that the prefix 'd' is used to indicate if the sugar is deoxyribose (e.g. dAMP).

The atoms in the purine ring are numbered as 1 to 9 and_ for pyrimidine as 1 to 6 (See Fig.5.1). The carbons of sugars are represented with an associated prime = (')_ for differentiation. Thus the carbons are 1' to 5'. pentose

nucleotides.Ribonucleotide (5 c -monophosphate) = Deoxythymidine 5 c -monophosphate or deoxythymidylate. Thymine (T), T ABLE 5.1 Principal bases, nucleosides and nucleotides.Abbreviation = dTMP
The pentoses are bound to nitrogenous bases by B-N-glycosidic bonds. The N9 of a purine ring binds with Cj) of a pentose sugar to form a covalent bond in the purine nucleoside. In case of pyrimidine nucleosides, the glycosidic linkage is between N! of a pyrimidine and C'; of a pentose.
represents adenosine 5'-monophosphate. However, for adenosine 3'-monophosphate, the abbreviation 3'-AMP is used.
The structures of two selected nucleotides namely AMP and TMP are depicted in Fig.5.6.

The hydroxyl groups of adenosine are esterified with phosphates to produce 5'or 3'-monophosphates. 5'-Hydroxyl is the most commonly esterified, hence 5' is usually omitted while writing nucleotide names. Thus AMP
Nucleoside monophosphates possess only one phosphate moiety (AMP, TMP). The addition of second or third phosphates to the nucleoside results in nucleoside diphosphate (e.g. ADP) or triphosphate (e.g. ATP), respectively.
Fig. 5.6 : The structures of adenosine 5'-monophosphate (AMP) and thymidine 5'-monophosphate (TMP)
[k-Addition of second or third phosphate gives adenosine diphosphate
(ADP) and adenosine triphosphate (ATP)
respectively].
The anionic properties of nucleotides and nucleic acids are due to the negative charges contributed by phosphate groups.

It is possible to alter heterocyclic ring or sugar moiety, and produce synthetic analogs of purines, pyrimidines, nucleosides and nucleotides. Some of the synthetic analogs are highly useful in clinical medicine. The structures of selected purine and pyrimidine analogs are given in Fig.5.7.
The pharmacological applications of certain analogs are listed below
1. Allopurinol is used in the treatment of hyperuricemia and gout (For details, Refer Chapter 17).
2. 5-Fluorouracil, 6-mercaptopurine, 8-azaguanine, 3-deoxyuridine, 5or 6-azauridine, 5or 6-azacytidine and 5-idouracil are employed in the treatment of cancers. These compounds get incorporated into DNA and _ block cell proliferation.
3. Azathioprine (which gets degraded to 6-mercaptopurine) is used to — suppress immunological rejection during transplantation.
the viral used for disease, 4. Arabinosyladenine _ is treatment of neurological encephalitis.
5. Arabinosylcytosine is being used in cancer therapy as it interferes with DNA replication.
6. The drugs employed in the treatment of AIDS namely zidovudine or AZT (3-azido 2',3'-dideoxythymidine) and didanosine (dideoxyinosine) are sugar modified synthetic nucleotide analogs (For their structure and more details Refer Chapter 38).

DNA is polymer of deoxyribonucleotides a (or simply deoxynucleotides). It is composed of monomeric units namely deoxyadenylate (dAMP), deoxyguanylate (dGMP), deoxycytidylate ((CMP) and deoxythymidylate (dTMP) (It may be noted here that some authors prefer to use TMP for deoxythymidylate, since it is found only in DNA). The details of the nucleotide structure are given above.

The monomeric deoxynucleotides in DNA are held together by 3',5phosphodiester bridges (Fig.5.8). DNA (or RNA) structure is often represented in a short-hand form. The horizontal line indicates the carbon chain of sugar with base attached to Cy. Near the middle of the horizontal line is C3 phosphate linkage while at the other end of the line is Cs: phosphate linkage (Fig.5.8).

Erwin Chargaff in late 1940s quantitatively analysed the DNA hydrolysates from different species. He observed that in all the species he studied, DNA had equal numbers of adenine and thymine residues (A = T) and equal numbers of guanine and cytosine residues (G = C). This is known as Chargaff's rule of molar equivalence between the purines and pyrimidines in DNA structure. The significance of Chargaff's rule was not immediately realised. The double helical structure of DNA derives its strength from Chargaff's rule (discussed later).
Fig.
5.8
:
Structure of
a
polydeoxyribonucleotide segment
held part
is by
phosphodiester the
bonds.
On the
lower representation
of short
hand form
of oligonucleotides.
Single-stranded DNA, and RNAs which are usually single-stranded, do not obey Chargaff's rule. However, double-stranded RNA which is the genetic material in certain viruses satisfies Chargaff's rule.

The double helical structure of DNA was proposed
by
James
Watson and
Francis
Crick in
1953
(Nobel
Prize,
1962).
The elucidation
of
DNA structure is considered as a milestone in the era of modern biology. The structure of DNA double helix is comparable to a twisted ladder. The salient features of Watson-Crick model of DNA (now known as B-DNA) are described next (Fig.5.9).
Fig. 5.9 : (A) Watson—Crick model of DNA helix (B) Complementary base pairing in DNA helix.
Fig. 5.10 : Complementary base pairing in DNA (A) Thymine pairs with adenine by 2 hydrogen bonds (B) Cytosine pairs with guanine by 3 hydrogen bonds.
1. The DNA is a right handed double helix. It consists of two polydeoxyribonucleotide chains (strands) twisted around each other on a common axis.
2. The two strands are antiparallel, i.e., one strand runs in the 5' to 3' direction while the other in 3' to 5' direction. This is comparable to two parallel adjacent roads carrying traffic in opposite direction.

3. The width (or diameter) of a double helix 20 A° (2 nm).
4. Each turn (pitch) of the helix is 34 A° (3.4 nm) with 10 pairs of nucleotides, each pair placed at a distance of about 3.4 A°.
5. Each strand of DNA has a hydrophilic deoxyribose phosphate backbone (3'-5' phosphodiester bonds) on the outside (periphery) of the molecule while the hydrophobic bases are stacked inside (core).
6. The two polynucleotide chains are not identical but complementary to each other due to base pairing.
7. The two strands are held together by hydrogen bonds formed by complementary base pairs (Fig.5.10). The A-T pair has 2 hydrogen bonds while G-C_ pair has 3 hydrogen bonds. The G = C is stronger by about 50% than A=T.
8. The hydrogen bonds are formed between a purine and a pyrimidine only. If two purines face each other, they would not fit into the allowable space. And two pyrimidines would be too far to form hydrogen bonds. The only base arrangement possible in DNA structure, from spatial considerations is A-T, T-A, G-C and C-G.
9. The complementary base pairing in DNA helix proves Chargaff's rule. The content of adenine equals to that of thymine (A = T) and guanine equals to that of cytosine (G = C).
10. The genetic information resides on one of the two strands known as template strand or sense strand. The opposite strand is antisense strand. The double helix has (wide) major grooves and (narrow) minor grooves along the phosphodiester backbone. Proteins interact with DNA at these grooves, without disrupting the base pairs and double helix.

Variation in the conformation of the nucleotides of DNA_ is associated with conformational variants of DNA. The double helical structure of DNA exists in at least 6 different forms-A to E and Z. Among these, B, A and Z forms are important (Table 5.2). The B-form of DNA double helix, described by Watson and Crick (discussed above), is the most predominant form under __ physiological conditions. Each turn of the B-form has 10 base pairs spanning a distance of 3.4 nm. The width of the double helix is 2 nm.
The A-form is also a right-handed helix. It contains 11 base pairs per turn. There is a tilting of the base pairs by 20° away from the central axis.
The Z-form (Z-DNA) is a left-handed helix and contains 12 base pairs per turn. The
polynucleotide strands of DNA move in a somewhat 'zig zag' fashion, hence the name Z-DNA.
It is believed that transition between different
helical forms of DNA plays a significant role in
regulating gene expression.

It is now recognized that besides double helical structure, DNA also exists in certain unusual structures. It is believed that such structures are important for molecular recognition of DNA by proteins and enzymes. This is in fact needed for the DNA to discharge its functions in an appropriate manner. Some selected unusual structures of DNA are briefly described.

In general, adenine base containing DNA tracts are rigid and straight. Bent conformation of DNA occurs when A-tracts are replaced by other bases or a collapse of the helix into the minor groove of A-tract. Bending in DNA structure has also been reported due to photochemical damage or mispairing of bases.
Certain antitumor drugs (e.g. cisplatin) produce bent structure in DNA. Such changed structure can take up proteins that damage the DNA.

Triple-stranded DNA formation may occur due to additional hydrogen bonds between the bases. Thus, a thymine can selectively form two Hoogsteen hydrogen bonds to the adenine of A-T pair to form T-A-T. Likewise, a protonated cytosine can also form two hydrogen bonds with guanine of G-C pairs that results in C-G-C. An outline of Hoogsteen triple helix is depicted in Fig.5.11.
Triple-helical structure is less stable than double helix. This is due to the fact that the three negatively charged backbone strands in triple helix results in an increased electrostatic repulsion.

Polynucleotides with very high contents of guanine can form a novel tetrameric structure
Fig. 5.11 : An outline of Hoogsteen triple helical structure of DNA.
called G-quartets. These structures are planar and are connected by Hoogsteen hydrogen bonds (Fig.5.12A). Antiparallel four-stranded DNA structures, referred to as G-tetraplexes have also been reported (Fig.5.12B).
The ends of eukaryotic chromosomes namely telomeres are rich in guanine, and therefore form G-tetraplexes. In recent years, telomeres have become the targets for anticancer chemotherapies.
Fig. 5.12 : Four-stranded DNA structure (A) Parallel G-quartets (B) Antiparallel G-tetraplex.
G-tetraplexes have been implicated in the recombination of immunoglobulin genes, and in dimerization of double-stranded genomic RNA of the human immunodeficiency virus (HIV).

DNA molecules are huge in size. On an average, a pair of B-DNA with a_ thickness of 0.34 nm has a molecular weight of 660 daltons.
For the measurement of lengths, DNA doublestranded structure is considered, and expresssed in the form of base pairs (bp). A kilobase pair (kb) is 103 bp, and a megabase pair (Mb) is 10° bp and a gigabase pair (Gb) is 109 bp. The kb, Mb and Gb relations may be summarized as follows :
1
kb = 1000 bp
1
Mb = 1000 kb = 1,000,000 bp
1 Gb = 1000 Mb = 1,000,000,000 bp
It may be noted here that the lengths of RNA molecules (like DNA molecules) cannot be expressed in bp, since most of the RNAs are single-stranded.
The length of DNA varies from species to species, and is usually expressed in terms of base pair composition and contour length. Contour length represents the total length of the genomic DNA in a cell. Some examples of organisms with bp and contour lengths are listed.
1 & phage virus —4.8 x 104 bp — contour length 16.5 um.
1 E. coli—4.6 x 10° bp —contour length 1.5 um.
1
Diploid human
cell
(46
chromosomes)
—
6.0
x
10°
bp
—
contour length
2
meters.
It may be noted that the genomic DNA size is usually much larger the size of the cell or nucleus containing it. For instance, in humans, a 2-meter long DNA is packed compactly in a nucleus of about 10um diameter.
The genomic DNA may exist in linear or circular forms. Most DNAs in bacteria exist as closed circles. This includes the DNA of bacterial chromosomes and the extrachromosomal DNA of plasmids. Mitochondria and chloroplasts of eukaryotic cells also contain circular DNA.
Chromosomal DNAs in higher organisms are mostly linear. Individual human chromosomes contain a single DNA molecule with variable sizes compactly packed. Thus the smallest chromosome contains 34 Mb while the largest one has 263 Mb.

Fig. 5.13 : Diagrammatic representation of denaturation and renaturation of DNA.
following denaturation
is referred
The two strands of DNA helix are held together by hydrogen bonds. Disruption of hydrogen bonds (by change in pH or increase in temperature) results in the separation of polynucleotide strands. This phenomenon of loss of helical structure of DNA is known as denaturation (Fig.5.13)._ The phosphodiester bonds are not broken by denaturation. Loss of helical structure can be measured by increase in absorbance at 260 nm (in a_ spectrophotometer). The phenomenon of increase in the absorbance of purines and __ pyrimidines,
to
as
hyperchromicity.
Melting temperature (Tm) is defined as the temperature at which half of the helical structure of DNA is lost. Since G-C base pairs are more stable (due to 3 hydrogen bonds) than A-T base pairs (2 hydrogen bonds), the Tm is greater for DNAs with higher G-C content. Thus, the Tm is 65°C for 35% G-C content while it is 70°C for 50% G-C content. Formamide destabilizes hydrogen bonds of base pairs and, therefore, lowers Tm. This chemical compound is effectively used in recombinant DNA experiments.

DNA is the reserve bank of genetic information, ultimately responsible for the chemical basis of life and heredity.
DNA is organized into genes, the fundamental units of genetic information. Genes control protein biosynthesis through the mediation of RNA.
Nucleic acids are the polymers of nucleotides. Certain nucleotides serve as B-complex vitamin coenzymes (FAD, NAD*, CoA), carriers of high energy intermediates (UDP-glucose, S-adenosylmethionine) and second messengers of hormonal action (CAMP, cGMP).
Uric acid is a purine, and the end product of purine metabolism, that has been implicated in the disorder gout.
Certain purine bases from plants such as caffeine (of coffee), theophylline (of tea) and theobromine (of cocoa) are of pharmacological interest.
Synthetic analogs of bases (5-fluorouracil, 6-mercaptopurine, 6-azauridine) are used to inhibit the growth of cancer cells.
Certain antitumor drugs (e.g. cisplatin) can produce bent DNA structure and damage it.
Renaturation (or reannealing) is the process in which the separated complementary DNA strands can form a double helix.

As already stated, the double-stranded DNA helix in each chromosome has a length that is thousands times the diameter of the nucleus. For instance, in humans, a 2-meter long DNA is packed in a nucleus of about 10 um diameter! This is made possible by a compact and marvellous packaging, and organization of DNA inside in cell.

In prokaryotic cells, the DNA is organized as a single chromosome in the form of a doublestranded circle. These bacterial chromosomes are packed in the form of nucleoids, by interaction with proteins and certain cations (polyamines).

In the eukaryotic cells, the DNA is associated with various proteins to form chromatin which then gets organized into compact structures namely chromosomes (Fig.5.14).
The DNA double helix is wrapped around the core proteins namely histones which are basic in nature. The core is composed of two molecules of histones (H2A, H2B, H3 and H4). Each core with two turns of DNA wrapped round _ it (approximately with 150 bp) is termed as a nucleosome, the basic unit of chromatin. Nucleosomes are separated by spacer DNA to which histone H, is attached (Fig.5.15). This continuous string of nucleosomes, representing beads-on-a string form of chromatin is termed as 10 nm fiber. The length of the DNA is considerably reduced by the formation of 10 nm fiber. This 10-nm fiber is further coiled to produce 30-nm fiber which has a solenoid structure with six nucleosomes in every turn.
These 30-nm fibers are further organized into loops by anchoring the fiber at A/T-rich regions namely scaffold-associated regions (SARS) to a protein scafold. During the course of mitosis, the loops are further coiled, the chromosomes condense and become visible.

RNA is a polymer of ribonucleotides held together by 3'5"phosphodiester bridges. Although RNA has certain similarities with DNA structure, they have specific differences contrast
1. Pentose : The sugar in RNA is ribose in to deoxyribose in DNA.
uracil
2. Pyrimidine : RNA contains the pyrimidine in place of thymine (in DNA).
3. Single strand : RNA is usually a singlestranded polynucleotide. However, this strand may fold at certain places to give a doublestranded structure, if complementary base pairs are in close proximity.
4. Chargaff's rule—not obeyed : Due to the single-stranded nature, there is no specific relation between purine and__ pyrimidine contents. Thus the guanine content is not equal to cytosine (as is the case in DNA).
5. Susceptibility to alkali hydrolysis : Alkali can hydrolyse RNA to 2',3'-cyclic diesters. This is possible due to the presence of a hydroxyl group at 2' position. DNA cannot be subjected to alkali hydrolysis due to lack of this group.
6. Orcinol colour reaction : RNAs can be histologically identified by orcinol colour reaction due to the presence of ribose.

The three major types of RNAs with their respective cellular composition are given below
1. Messenger RNA (mRNA) : 5-10%
2. Transfer RNA (tRNA)
: 10-20%
3. Ribosomal RNA (rRNA):
50-80%

Messenger RNA, Abbreviation = mRNA. Messenger RNA, Function(s) = Transfers genetic information from genes to ribosomes to synthesize proteins.. Heterogeneous nuclear RNA, Abbreviation = hnRNA. Heterogeneous nuclear RNA, Function(s) = Serves as precursor for mRNA and other RNAs.. Transfer RNA, Abbreviation = tRNA. Transfer RNA, Function(s) = Transfers amino acid to mRNA for protein biosynthesis.. Ribosomal RNA, Abbreviation = rRNA. Ribosomal RNA, Function(s) = Provides structural framework for ribosomes.. Small nuclear RNA, Abbreviation = snRNA. Small nuclear RNA, Function(s) = Involved in mRNA processing.. Small nucleolar RNA, Abbreviation = snoRNA. Small nucleolar RNA, Function(s) = Plays a key role in the processing of rRNA molecules.. Small cytoplasmic RNA, Abbreviation = scRNA. Small cytoplasmic RNA, Function(s) = Involved in the selection of proteins for export.. Transfer-messenger RNA, Abbreviation = tmRNA. Transfer-messenger RNA, Function(s) = Mostly present in bacteria. Adds short peptide tags to proteins to facilitate the degradation of incorrectly synthesized proteins.
Besides the three RNAs referred above, other RNAs are also present in the cells. These include heterogeneous nuclear RNA (hnRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA) and small cytoplasmic RNA (scRNA). The major functions of these RNAs are given in Table 5.3.
The RNAs are synthesized from DNA, and are primarily involved in the process of protein biosynthesis (Chapter 25). The RNAs vary in their structure and function. A brief description on the major RNAs is given.

The mRNA is synthesized in the nucleus (in eukaryotes) as heterogeneous nuclear RNA (hnRNA). hnRNA, on processing, liberates the functional mRNA which enters the cytoplasm to participate in protein synthesis. mRNA has high molecular weight with a short half-life.
In general, MRNA of eukaryotes is more stable with longer half-life, compared to prokaryotic mRNA.
The eukaryotic mRNA _ is capped at the 5'-terminal end by 7-methylguanosine triphosphate. It is believed that this cap helps to prevent the hydrolysis of mRNA by _ 5'-exonucleases. Further, the cap may be also involved in the recognition of MRNA for protein synthesis.
The 3'-terminal end of mRNA _ contains a polymer of adenylate residues (20-250 nucleotides) which is known as poly (A) tail. This tail may provide stability to mRNA, besides preventing it from the attack of 3'-exonucleases. mRNA molecules often contain certain modified bases such as 6-methyladenylates in the internal structure.

Transfer RNA (soluble RNA) molecule contains 71-80 nucleotides (mostly 75) with a molecular weight of about 25,000. There are at least 20 species of tRNAs, corresponding to 20 amino acids present in protein structure. The structure of tRNA (for alanine) was _ first elucidated by Holley.
The structure of tRNA, depicted in Fig.5.16, resembles that of a clover leaf. tRNA contains mainly four arms, each arm with a base paired stem.
1. The acceptor arm : This arm is capped with a sequence CCA (5' to 3'). The amino acid is attached to the acceptor arm.
2. The anticodon arm : This arm, with the three specific nucleotide bases (anticodon), is responsible for the recognition of triplet codon of mRNA. The codon and anticodon are complementary to each other.
3. The D arm : It is so named due to the presence of dihydrouridine.
Fig. 5.16 : Structure of transfer RNA.
4.
The
T¥C
arm
:
This arm
contains sequence
of
T, pseudouridine
(represented
a
by psi,
W)
and
C.
5. The variable arm : This arm is the most variable in tRNA. Based on this variability, tRNAs are classified into 2 categories :
(a)
Class
|
tRNAs
:
The most
predominant
(about
75%)
form with
3-5
base pairs
length.
(b) Class Il tRNAs : They contain 13-20 base pair long arm.
Base pairs in tRNA : The structure of tRNA is maintained due to the complementary base pairing in the arms. The four arms with their respective base pairs are given below
The acceptor arm
—-7 bp
The T¥C arm
The anticodon arm
—5
bp
—
5
bp
The D arm
—4 bp

The ribosomes are the factories of protein synthesis. The eukaryotic ribosomes are composed of two major nucleoprotein complexes—60S subunit and 40S subunit. The 60S subunit contains 28S rRNA, 5S rRNA and 5.88 rRNA while the 40S subunit contains 18S rRNA. The function of rRNAs in ribosomes is not clearly known. It is believed that they play a significant role in the binding of mRNA to ribosomes and protein synthesis.

The various other RNAs and their functions are summarised in Table 5.3.

In certain instances, the RNA component of a ribonucleoprotein (RNA in association with protein) is catalytically active. Such RNAs are termed as ribozymes. A selected list of ribozymes along with their biochemical functions is given in Table 5.4.
Ribonuclease P (RNase P) is a_ ribozyme containing protein and RNA component. It cleaves tRNA precursors to generate mature tRNA molecules.
RNA molecules are known to adapt tertiary structure just like proteins (i.e. enzymes). The specific conformation of RNA may be responsible for its function as biocatalyst. It is believed that ribozymes (RNAs) were functioning as catalysts before the occurrence of protein enzymes, during the course of evolution.

It is now possible to design recombinant ribozymes that will cleave any RNA. These ribozymes are now being considered as therapeutic agents to cure diseases. Theoretically it is possible to selectively degrade faulty RNAs (mutated or inappropriately expressed RNAs in diseases) by rribozymes. This way specific RNAs can be eliminated from the cell that will help to inhibit the disease process.

Ribozyme(s)
Biochemical reaction(s)
rRNA
Peptide bond formation in
protein biosynthesis
RNase P
RNA clevage and ligation
Self-splicing RNAs
DNA cleavage
RNAs of splicesome
RNA splicing
In vitro selected RNAs
__
RNA polymerization,
RNA phosphorylation
RNA aminoacylation
Glycoside bond formation
Oxidation-reduction reactions
Disulfide exchange

. DNA is the chemical basis of heredity organized into genes, the basic units of genetic information.
. RNAs (mRNA, tRNA and rRNA) are produced by DNA which in turn carry out protein synthesis.
. Nucleic acids are the polymers of nucleotides (polynucleotides) held by 3' and 5' phosphodiester bridges. A nucleotide essentially consists of base + sugar (nucleoside) and phosphate.
. Besides being the constituents of nucleic acid structure, nucleotides perform a wide variety of cellular functions (e.g. energy carriers, metabolic regulators, second messengers etc.)
. Both DNA and RNA contain the purines-adenine (A) and guanine (G) and the pyrimidinecytosine (C). The second pyrimidine is thymine (T) in DNA while it is uracil (U) in RNA. The pentose sugar, D-deoxyribose is found in DNA while it is D-ribose in RNA.
. The structure of DNA is a double helix (Watson-Crick model) composed of two antiparallel strands of polydeoxynucleotides twisted around each other. The strands are held together by 2 or 3 hydrogen bonds formed between the bases i.e. A = T; G=C. DNA structure satisfies Chargaff's rule that the content of A is equal to T, and that of G equal to C.
. Besides the double helical structure, DNA also exists in certain unusual structures — bent DNA, triple-strand DNA, four-strand DNA.
. RNA is usually a single stranded polyribonucleotide. mRNA is capped at 5' terminal end by 7-methylIGTP while at the 3-terminal end, it contains a poly A tail. mRNA specifies the sequence of amino acids in protein synthesis.
. The structure of tRNA resembles that of a clover leaf with four arms (acceptor, anticodon, D-, and T¥C) held by complementary base pairs. tRNA delivers amino acids for protein synthesis.
10. Certain RNAs that can function as enzymes are termed as ribozymes. Ribozymes were probably functioning as catalysts before the occurrence of protein enzymes during evolution.

1. Describe the structure of DNA.
2. Name different RNAs and discuss their structure.
3. Write an account of structure, function and nomenclature of nucleotides.
4. Describe the structure of nitrogenous bases present in nucleic acids. Add a note on tautomerism.
5. “The backbone of nucleic acid structure is 3’-5’ phosphodiester bridge.”—justify.

(a) Chargaff’s rule, (b) Ribose and deoxyribose, (c) Hydrogen bonds in DNA, (d) Nucleoside,
(e) Different forms of DNA, (f) Transfer RNA, (g) Purine bases of plants, (h) Complementary base
pairs, (i) DNA denaturation, (j) hnRNA.

1. The fundamental unit of genetic information is known as
. DNA controls protein synthesis through the mediation of
. Nucleic acids are the polymers of
. The pyrimidine present in DNA but absent in RNA
. Ribose and deoxyribose differ in their structure around carbon atom
. Nucleotide is composed of
. The scientist who observed that there exists a relationship between the contents of purines and pyrimidines in DNA structure (A = T; G = C)
8. The base pair G-C is more stable and stronger than A-T due to
9. Under physiological condition, the DNA structure is predominantly in the form
10. The acceptor arm of tRNA contains a capped nucleotide sequence

11. The nitrogenous base not present in DNA structure
(a) Adenine (b) Guanine (c) Cytosine (d) Uracil.
12. The number of base pairs present in each turn (pitch) of B-form of DNA helix
(a) 9 (b) 10 (c) 11 (d) 12.
13. The backbone of nucleic acid structure is constructed by
(a) Peptide bonds (b) Glycosidic bonds (c) Phosphodiester bridges (d) All of them.
14. The following coenzyme is a nucleotide
(a) FAD (b) NAD* (c) CoASH (d) All of them.
15. The nucleotide that serves as an intermediate for biosynthetic reaction
(a) UDP-glucose (b) CDP-acylglycerol (c) S-Adenosylmethionine (d) All of them.
aes are biocatalysts — the catalysts of life. A catalyst is defined as a substance that increases the velocity or rate of a chemical reaction without itself undergoing any change in the overall process.
The student-teacher relationship may be a good example to understand how a catalyst works. The students often find it difficult to learn from a text-book on their own. The teacher explains the subject to the students and increases their understanding capability. It is no wonder that certain difficult things which the students take days together to understand, and sometimes do not understand at all — are easily learnt under the guidance of the teacher. Here, the teacher acts like a catalyst in enhancing the understanding ability of students. A good teacher is always a good catalyst in students' life!
Enzymes may be defined as_ biocatalysts synthesized by living cells. They are protein in nature (exception— RNA acting as ribozyme), colloidal and thermolabile in character, and specific in their action.
In the laboratory, hydrolysis of proteins by a strong acid at 100°C takes at least a couple of days. The same protein is fully digested by the enzymes in gastrointestinal tract at body temperature (37°C) within a couple of hours. This remarkable difference in the chemical reactions taking place in the living system is exclusively due to enzymes. The very existence of life is unimaginable without the presence of enzymes.

Berzelius in 1836 coined the term catalysis (Greek : to dissolve). In 1878, Kuhne used the word enzyme (Greek : in yeast) to indicate the catalysis taking place in the biological systems. Isolation of enzyme system from cell-free extract of yeast was achieved in 1883 by Buchner. He named the active principle as zymase (later found to contain a mixture of enzymes), which could convert sugar to alcohol. In 1926,
James Sumner first achieved the isolation and crystallization of the enzyme urease from jack bean and identified it as a protein.

enzyme reaction (type of reaction, cofactor requirement etc.)
Enzymes are sometimes considered under two broad categories : (a) Intracellular enzymes — They are functional within cells where they are synthesized. (b) Extracellular enzymes — These enzymes are active outside the cell; all the digestive enzymes belong to this group.
In the early days, the enzymes were given names by their discoverers in an arbitrary manner. For example, the names pepsin, trypsin and chymotrypsin convey no information about the function of the enzyme or the nature of the substrate on which they act. Sometimes, the suffix-ase was added to the substrate for naming the enzymes e.g. lipase acts on lipids; nuclease on nucleic acids; lactase on lactose. These are known as trivial names of the enzymes which, however, fail to give complete information of
The International Union of Biochemistry (IUB) appointed an Enzyme Commission in 1961. This committee made a thorough study of the existing enzymes and devised some basic principles for the classification and nomenclature of enzymes. Since 1964, the JUB system of enzyme classification has been in force. Enzymes are divided into six major classes (in that order). Each class on its own represents the general type of reaction brought about by the enzymes of that class (Table 6.1).

oglyph<c=3,font=/MDECLA+Symbol> A c. 6. Ligases Glutamine synthetase (L-glutamate ammonia ligase, E.C. 6.3.1.2), acetyl CoA carboxylase, succinate thiokinase, T ABLE 6.1 Classification of enzymes.Reaction catalysed = Condensation (usually dependent on ATP) A + B ATP ADP + Pi A - B
- 1. Oxidoreductases : Enzymes involved in
oxidation-reduction reactions.
2. Transferases : Enzymes that catalyse the transfer of functional groups.
3. Hydrolases : Enzymes that bring about hydrolysis of various compounds.
4. Lyases Enzymes specialised in the addition or removal of water, ammonia, CO, etc.
5. Isomerases : Enzymes involved in all the isomerization reactions.
6. Ligases : Enzymes catalysing the synthetic
reactions
(Greek
:
ligate—to bind)
where two
molecules are
[The joined
together word
may
OTHLIL
be memorised
and
ATP
(first letter
to remember
is in
each the
used.
class)
six classes
of enzymes
in the
correct order].
Each class in turn is subdivided into many sub-classes which are further divided. A four digit Enzyme Commission (E.C.) number is assigned to each enzyme representing the class (first digit), sub-class (second digit), sub-sub class (third digit) and the individual enzyme (fourth digit). Each enzyme is given a specific name indicating the substrate, coenzyme (if any) and the type of the reaction catalysed by the enzyme. Although the IUB names for the enzymes are specific and unambiguous, they have not been accepted for general use as they are complex and cumbersome to remember. Therefore, the trivial names, along with the E.C. numbers as and when needed, are commonly used and widely accepted.

All the enzymes are invariably proteins. In recent years, however, a few RNA molecules have been shown to function as enzymes. Each enzyme has its own tertiary structure and specific conformation which is very essential for its catalytic activity. The functional unit of the enzyme is known as holoenzyme which is often made up of apoenzyme (the protein part) and a coenzyme (non-protein organic part).
Holoenzyme
——>
Apoenzyme
+
Coenzyme
(active enzyme)
(protein part)
(non-protein part)
The term prosthetic group is used when the non-protein moiety tightly (covalently) binds with the apoenzyme. The coenzyme can be separated by dialysis from the enzyme while the prosthetic group cannot be.
The word monomeric enzyme is used if it is made up of a single polypeptide e.g. ribonuclease, trypsin. Some of the enzymes which possess more than one polypeptide (subunit) chain are known as oligomeric enzymes e.g. lactate dehydrogenase, aspartate —_—transcarbamoylase etc. There are certain multienzyme complexes possessing specific sites to catalyse different reactions in a sequence. Only the native intact multienzyme complex is functionally active and not the individual units, if they are separated e.g. pyruvate dehydrogenase, fatty acid synthase, prostaglandin synthase etc. The enzymes exhibit all the general properties of proteins (Chapter 4).

Recent advances in biotechnology have made it possible to modify the enzymes with desirable characters-improved catalytic abilities, activities under unusual conditions. This approach is required since enzymes possess enormous potential for their use in medicine and industry.
Hybrid enzymes : It is possible to rearrange genes and produce fusion proteins. e.g. a hybrid enzyme (of glucanase and cellulase) that can more efficiently hydrolyse barley B-glucans in beer manufacture.
Site-directed mutagenesis This is a technique used to produce a specified mutation at a predetermined position in a DNA molecule. The result is incorporation of a desired amino acid (of one's choice) in place of the specified amino acid in the enzyme. By this approach, it is possible to produce an enzyme with desirable characteristics. e.g. tissue plasminogen activator (used to lyse blood clots in myocardial

concentration on enzyme velociy.
infarction) with increased half-life. This is achieved by replacing asparagine (at position 120) by glutamine.
In recent years, it has also become possible to produce hybrid enzymes by rearrangement of genes. Another innovative approach is the production of abzymes or catalytic antibodies, the antibody enzymes.

The contact between the enzyme and substrate is the most essential pre-requisite for enzyme activity. The important factors that influence the velocity of the enzyme reaction are discussed hereunder

As the concentration of the enzyme _ is increased, the velocity of the reaction proportionately increases (Fig.6.1). In fact, this property of enzyme is made use in determining the serum enzymes for the diagnosis of diseases. By using a known volume of serum, and keeping all the other factors (substrate, pH, temperature etc.) at the optimum level, the enzyme could be assayed in the laboratory.

Increase in the substrate concentration gradually increases the velocity of enzyme reaction within the limited range of substrate levels. A rectangular hyperbola is obtained when velocity is plotted against the substrate concentration (Fig.6.2). Three distinct phases of the reaction are observed in the graph (A-linear; B-curve; C-almost unchanged).
Order of reaction : When the velocity of the reaction is almost proportional to the substrate concentration (i.e. [S] is less than K,,), the rate of the reaction is said to be first order with respect to substrate. When the [S] is much greater than Km, the rate of reaction is independent of substrate concentration, and the reaction is said to be zero order.

1
The enzyme (E) and substrate (S) combine with each other to form an unstable enzymesubstrate complex (ES) for the formation of product (P). k k,
E+s—
ES
—+E+P
Here k,, ky and k3 represent the velocity constants for the respective reactions, as indicated by arrows.
Ki, the Michaelis-Menten constant (or Brig's and
Haldane’s constant),
is given
by the
formula ky
_
tks m~
k, The following equation is obtained after suitable algebraic manipulation.
where
v
=
Measured velocity,
Vmax
S
= Maximum velocity,
= Substrate concentration,
K
=
Michaelis
—-
Menten constant.
Let us assume that the measured velocity (v) is equal to Vmax. Then the equation (1) may be substituted as follows
Fig. 6.2 : Effect of substrate concentration on enzyme velocity (A-linear; B-curve; C-almost unchanged).
K,, value is a constant and a characteristic feature of a given enzyme (comparable to a thumb impression or signature). It is a representative for measuring the strength of ES complex. A low K,, value indicates a strong affinity between enzyme and substrate, whereas a high K,, value reflects a weak affinity between them. For majority of enzymes, the K,, values are in the range of 10~> to 10-2 moles. It may however, be noted that K,, is not dependent on the concentration of enzyme.
Lineweaver-Burk double
reciprocal plot
:
For the
determination saturation
of
K,, curve
(Fig.6.2)
value, the
is not
very substrate
accurate since Vmax is approached asymptotically. By taking the reciprocals of the equation (1), a straight line graphic representation is obtained.

The above equation is similar to y = ax + b. Therefore, a plot of the reciprocal of the velocity (+} vs. the reciprocal of the substrate concentration (=) gives a straight line. Here the slope is Ky/Vmax and whose y intercept is 1/VmaxThe Lineweaver-Burk plot is shown in Fig.6.3. \t is much easier to calculate the K,, from the intercept on x-axis which is —(1/K,,). Further, the double reciprocal plot is useful in understanding the effect of various inhibitions (discussed later).
Enzyme reactions
with two
or more
:
The the
above presumption
reaction.
of discussion
a
In fact,
catalysed
a
reactions is
based on
single substrate-enzyme
majority involve
of the
enzyme- two
or more
substrates.
Even in
case of
multisubstrate
enzymes, despite the complex mathematical expressions, the fundamental principles conform to Michaelis-Menten Kinetics.

Velocity of an enzyme reaction increases with increase in temperature up to a maximum and then declines. A bell-shaped curve is usually observed (Fig.6.4).
Temperature coefficient or Qjg is defined as increase in enzyme velocity when the temperature is increased by 10°C. For a majority of enzymes, Qj9 is 2 between 0°C and 40°C. Increase in temperature results in higher activation energy of the molecules and more molecular (enzyme and substrate) collision and interaction for the reaction to proceed faster.
The optimum temperature for most of the enzymes is between 35°C—40°C. However, a few enzymes (e.g. Taq DNA polymerase, muscle adenylate kinase) are active even at 100°C. Some plant enzymes like urease have optimum activity around 60°C. This may be due to very stable structure and conformation of these enzymes.
In general, when the enzymes are exposed to a temperature above 50°C, denaturation leading to derangement in the native (tertiary) structure of the protein and active site are seen. Majority of the enzymes become inactive at higher temperature (above 70°C).
Fig. 6.4 : Effect of temperature on enzyme velocity.
Clinical significance : Foods can be preserved in refrigerators (at low temperatures) due to reduced bacterial enzyme activities. Certain surgeries are carried out by lowering the patient's body temperature (induced hyporthermia), and thus the matabolic rate.

Increase in the hydrogen ion concentration (pH) considerably influences the enzyme activity and a bell-shaped curve is normally obtained (Fig.6.5). Each enzyme has an optimum pH at which the velocity is maximum. Below and above this pH, the enzyme activity is much lower and at extreme pH, the enzyme becomes totally inactive.
Most of the enzymes of higher organisms show optimum activity around neutral pH (6-8). There are, however, many exceptions like pepsin (1-2), acid phosphatase (4-5) and alkaline phosphatase (10-11). Enzymes from fungi and plants are most active in acidic pH (4-6).
Hydrogen ions influence the enzyme activity by altering the ionic charges on the amino acids (particularly at the active site), substrate, ES complex etc.

reaction
enzyme
The
accumulation
of
generally
decreases
the
products
velocity.
For certain enzymes, the products combine with the active site of enzyme and form a loose complex and, thus, inhibit the enzyme activity. In the living system, this type of inhibition is generally prevented by a quick removal of products formed. The end product inhibition by feedback mechanism is discussed later.

Some of the enzymes require certain inorganic metallic cations like Mg2*+, Mn?*, Zn2*, Ca2+, Co2+, Cu2+, Nat, Kt etc. for their optimum activity. Rarely, anions are also needed for enzyme activity e.g. chloride ion (CI) for amylase. Metals function as activators of enzyme velocity through various mechanisms— combining with the substrate, formation of ES-metal complex, direct participation in the reaction and bringing a conformational change in the enzyme.
of metals
- for
Two categories enzymes requiring their activity are distinguished
- | Metal-activated enzymes : The metal is not tightly held by the enzyme and can be exchanged easily with other ions
e.g. ATPase (Mg2+ and Ca?+)
Enolase (Mg?*)
1 Metalloenzymes These enzymes hold the metals rather tightly which are not readily exchanged. e.g. alcohol dehydrogenase, carbonic anhydrase, alkaline phosphatase, carboxypeptidase and _ aldolase contain zinc.
Phenol oxidase (copper);
Pyruvate oxidase (manganese);
Xanthine oxidase (molybdenum);
Cytochrome oxidase (iron and copper).

Under ideal and optimal conditions (like pH, temperature etc.), the time required for an enzyme reaction is less. Variations in the time of the reaction are generally related to the alterations in pH and temperature.
Fig. 6.6 : A diagrammatic representation of an enzyme with active site.

Exposure of enzymes to ultraviolet, beta, gamma and X-rays inactivates certain enzymes due to the formation of peroxides. e.g. UV rays inhibit salivary amylase activity.

Enzymes are big in size compared to substrates which are relatively smaller. Evidently, a small portion of the huge enzyme molecule is directly involved in the substrate binding and catalysis (Fig.6.6).
The active site (or active centre) of an enzyme represents as the small region at which the substrate(s) binds and participates in the catalysis.

1. The existence of active site is due to the tertiary structure of protein resulting in threedimensional native conformation.
2. The active site is made up of amino acids (known as catalytic residues) which are far from each other in the linear sequence of amino acids (primary structure of protein). For instance, the enzyme lysozyme has 129 amino acids. The active site is formed by the contribution of amino acid residues numbered 35, 52, 62, 63 and 101.
3. Active sites are regarded as clefts or crevices or pockets occupying a small region in a big enzyme molecule.
4. The active site is not rigid in structure and shape. It is rather flexible to promote the specific substrate binding.
5. Generally, the active site possesses a substrate binding site and a catalytic site. The latter is for the catalysis of the specific reaction. 6. The coenzymes or cofactors on which some enzymes depend are present as a part of the catalytic site.
7. The substrate(s) binds at the active site by
weak noncovalent bonds.
8. Enzymes are specific in their function due
to the existence of active sites.
9. The commonly found amino acids at the active sites are serine, aspartate, histidine, cysteine, lysine, arginine, glutamate, tyrosine etc. Among these amino acids, serine is the most frequently found.
10. The substrate[S] binds the enzyme (E) at the active site to form enzyme-substrate complex (ES). The product (P) is released after the catalysis and the enzyme is available for reuse.

Enzyme inhibitor is defined as a substance which binds with the enzyme and brings about a decrease in catalytic activity of that enzyme. The inhibitor may be organic or inorganic in nature. There are three broad categories of enzyme inhibition
1. Reversible inhibition.
2. Irreversible inhibition.
3. Allosteric inhibition.

The inhibitor binds non-covalently with enzyme and the enzyme inhibition can be reversed if the inhibitor is removed. The reversible inhibition is further sub-divided into
|. Competitive inhibition (Fig.6.7A)
Il. Non-competitive inhibition (Fig.6.7B)
|. Competitive inhibition : The inhibitor (I) which closely resembles the real substrate (S) is regarded as a substrate analogue. The inhibitor competes with substrate and binds at the active site of the enzyme but does not undergo any catalysis. As long as the competitive inhibitor holds the active site, the enzyme is not available for the substrate to bind. During the reaction, ES and EI complexes are formed as shown below
The relative concentration of the substrate and inhibitor and their respective affinity with the enzyme determines the degree of competitive inhibition. The inhibition could be overcome by a high substrate concentration. In competitive inhibition, the K,, value increases whereas Vinay remains unchanged (Fig.6.8).
The enzyme succinate dehydrogenase (SDH) is a classical example of competitive inhibition with succinic acid as its substrate. The compounds, namely, malonic acid, glutaric acid and oxalic acid, have structural similarity with succinic acid and compete with the substrate for binding at the active site of SDH.
Some more examples of the enzymes with substrates and competitive inhibitors (of clinical and pharmacological significance) are given in Table 6.2.
Antimetabolites : These are the chemical compounds that block the metabolic reactions by their inhibitory action on enzymes. Antimetabolites are usually structural analogues of substrates and thus are competitive inhibitors (Table 6.2). They are in use for cancer therapy, gout etc. The term antivitamins is used for the antimetabolites which block the biochemical actions of vitamins causing deficiencies, e.g. sulphonilamide, dicumarol.
ll. Non-competitive inhibition : The inhibitor binds at a site other than the active site on the

Fig. 6.8 : Effect of competitive inhibitor (i) on enzyme velocity. (A) Velocity (v) versus substrate (S) plot. (B) Lineweaver-Burk plot (Red lines with inhibitor; competitive inhibitor increases K,,, unalters V,nay-)- enzyme surface. This binding impairs the enzyme function. The inhibitor has no structural resemblance with the substrate. However, there usually exists a strong affinity for the inhibitor to bind at the second site. In fact, the inhibitor does not interfere with the enzyme-substrate binding. But the catalysis is prevented, possibly due to a distortion in the enzyme conformation.
The inhibitor generally binds with the enzyme as well as the ES complex. The overall relation in non-competitive inhibition — is represented below
For non-competitive inhibition, the K,, value is unchanged while V,,ay is lowered (Fig.6.9).
Heavy metal ions (Ag*, Pb2+, Hg2* etc.) can non-competitively inhibit the enzymes by binding with cysteinyl sulfhydryl groups. The general reaction for Hg2+ is shown below.

The inhibitors
bind covalently
with the
irreversible.
These inhibitors
are usually
toxic substances
that poison
lodoacetate enzymes
like
3-phosphate enzymes.
is an
irreversible papain
inhibitor of
the and_—
dehydrogenase.
with sulfhydryl
glyceraldehyde lodoacetate
(—SH)
groups combines
at the
active site
of enzymes
and inactivate
them, which
_
is these
enzymes and
makes them
inactive.
Fig. 6.9 : Effect of non-competitive inhibitor (I) on enzyme velocity (A) Velocity (v) versus substrate (S) (B) LineweaverBurk plot (Red lines with inhibitor, non-competitive inhibitor does not change K,,, but decreases V,,,,.).
Diisopropy! fluorophosphate (DFP) is a nerve gas developed by the Germans during Second World War. DFP irreversibly binds with enzymes containing serine at the active site, e.g. serine proteases, acetylcholine esterase.
Many organophosphorus insecticides like melathion are toxic to animals (including man) as they block the activity of acetylcholine esterase (essential for nerve conduction), resulting in paralysis of vital body functions.
Disulfiram (Antabuse®) is a drug used in the treatment of alcoholism. \t irreversibly inhibits the enzyme aldehyde dehydrogenase. Alcohol addicts, when treated with disulfiram become sick due to the accumulation of acetaldehyde, leading to alcohol avoidance. (Note : Alcohol is metabolized by two enzymes. It is first acted upon by alcohol dehydrogenase to yield acetaldehyde. The enzyme aldehyde dehydrogenase converts acetaldehyde to acetic acid.)
The penicillin antibiotics act as irreversible inhibitors of serine — containing enzymes, and block the bacterial cell wall synthesis.
Cyanide inhibits cytochrome oxidase (binds to iron atom) of electron transport chain. Fluoride inhibits enolase (by removing manganese), and thus glycolysis.

Suicide inhibition is a specialized form of irreversible inhibition. In this case, the original inhibitor (the structural analogue/competitive inhibitor) is converted to a more potent form by the same enzyme that ought to be inhibited. The so formed inhibitor binds irreversibly with the enzyme. This is in contrast to the original inhibitor which binds reversibly.
A good example of suicide inhibition is allopurinol (used in the treatment of gout, Refer Chapter 17). Allopurinol, an inhibitor of xanthine oxidase, gets converted to alloxanthine, a more effective inhibitor of this enzyme.
The use of certain purine and pyrimidine analogues in cancer therapy is also explained on the basis suicide inhibition. For instance, 5-fluorouracil gets converted to fluorodeoxyuridylate which inhibits the enzyme thymidylate synthase, and thus nucleotide synthesis.

The details of this type of inhibition are given under allosteric regulation as a part of the regulation of enzyme activity in the living system.

Enzymes are the natural targets for development of pharmacologic agents. Many of the drugs used in the treatment of diseases act as enzyme inhibitors. For example :
1 Cholesterol loweing statin drugs (lovastatin) inhibit the enzyme HMG CoA reductase.
1 Drugs (tenofovir, emtricitabine) employed to block HIV replication inhibit the enzyme viral reverse transcriptase.
1 Hypertension is often treated by the drugs (captopril, enalapril )which inhibit angiotensin converting enzyme.

Enzymes are highly specific in their action when compared with the chemical catalysts. The occurrence of thousands of enzymes in the biological system might be due to the specific nature of enzymes. Three types of enzyme specificity are well-recognised-stereospecificity, reaction specificity, and substrate specificity.
Specificity is a characteristic property of the active site.
1. Stereospecificity or optical specificity : Stereoisomers are the compounds which have the same molecular formula, but differ in their structural configuration.
The enzymes act only on one isomer and, therefore, exhibit stereospecificity.
e.g. L-amino acid oxidase and D-amino acid oxidase act on Land D-amino acids respectively; hexokinase acts on D-hexoses; glucokinase on D-glucose; amylase acts on a-glycosidic linkages; cellulase cleaves B-glycosidic bonds.
Fig. 6.10 : Diagrammatic representation of stereospecificity (a', b', c')—three point attachment of substrate to the enzyme (a, b, Cc).
Stereospecificity is explained by considering three distinct regions of substrate molecule specifically binding with three complementary regions on the surface of the enzyme (Fig.6.10). The class of enzymes belonging to isomerases do not exhibit stereospecificity, since they are specialized in the interconversion of isomers.

2. Reaction specificity : The same substrate can undergo different types of reactions, each catalysed by a separate enzyme and _ this is referred to as reaction specificity. An amino acid can undergo transamination, oxidative deamination, decarboxylation, racemization etc. The enzymes however, are different for each of these reactions (For details, refer Chapter 15).
3. Substrate specificity The — substrate specificity varies from enzyme to enzyme. It may be either absolute, relative or broad.
| Absolute substrate specificity Certain enzymes act only on one substrate e.g. glucokinase acts on glucose to give glucose 6phosphate, urease cleaves urea to ammonia and carbon dioxide.
| Relative substrate specificity : Some enzymes act on structurally related substances. This, in turn, may be dependent on the specific group or a bond present. The action of trypsin is a good example for group specificity (Refer Fig.8.7). Trypsin hydrolyses peptide linkage involving arginine or lysine. Chymotrypsin
cleaves peptide bonds attached to aromatic amino acids (phenylalanine, tyrosine and tryptophan). Examples of bond specificityglycosidases acting on glycosidic bonds of carbohydrates, lipases cleaving ester bonds of lipids etc.
- | Broad specificity : Some enzymes act on closely related substrates which is commonly known as broad substrate specificity, e.g. hexokinase acts on glucose, fructose, mannose and glucosamine and not on galactose. It is possible that some structural similarity among the first four compounds makes them a common substrate for the enzyme hexokinase.

The protein part of the enzyme, on its own, is not always adequate to bring about the catalytic activity. Many enzymes require certain nonprotein small additional factors, collectively referred to as cofactors for catalysis. The cofactors may be organic or inorganic in nature.
The non-protein, organic, low molecular weight and dialysable substance associated with enzyme function is known as coenzyme.
The functional enzyme is referred to as holoenzyme which is made up of a protein part (apoenzyme) and a non-protein part (coenzyme). The term prosthetic group is used when a nonprotein moiety is tightly bound to the enzyme which is not easily separable by dialysis. The term activator is referred to the inorganic cofactor (like Ca2+, Mg2*, Mn2* etc.) necessary to enhance enzyme activity. It may, however, be noted that some authors make no distinction between the terms cofactor, coenzyme and prosthetic group and use them interchangeably.
Coenzymes are second _ substrates Coenzymes are often regarded as the second substrates or co-substrates, since they have affinity with the enzyme comparable with that of the substrates. Coenzymes undergo alterations during the enzymatic reactions, which are later regenerated. This is in contrast to the substrate which is converted to the product.

from vitamin = Biotin. Biocytin, T ABLE 6.3 Coenzymes of B-complex vitamins *.Atom or group transferred = CO 2. Biocytin, T ABLE 6.3 Coenzymes of B-complex vitamins *.Dependent enzyme (example) = Pyruvate carboxylase. Methylcobalamin; Deoxyadenosyl cobalamin, T ABLE 6.3 Coenzymes of B-complex vitamins *.Derived from vitamin = Cobalamin. Methylcobalamin; Deoxyadenosyl cobalamin, T ABLE 6.3 Coenzymes of B-complex vitamins *.Atom or group transferred = Methyl/isomerisation. Methylcobalamin; Deoxyadenosyl cobalamin, T ABLE 6.3 Coenzymes of B-complex vitamins *.Dependent enzyme (example) = Methylmalonyl CoA mutase. * Details for each coenzyme are given in Chapter 7 on vitamins, T ABLE 6.3 Coenzymes of B-complex vitamins *.Derived from vitamin = * Details for each coenzyme are given in Chapter 7 on vitamins. * Details for each coenzyme are given in Chapter 7 on vitamins, T ABLE 6.3 Coenzymes of B-complex vitamins *.Atom or group transferred = . * Details for each coenzyme are given in Chapter 7 on vitamins, T ABLE 6.3 Coenzymes of B-complex vitamins *.Dependent enzyme (example) = 
Coenzymes participate in various reactions involving transfer of atoms or groups like hydrogen, aldehyde, keto, amino, acyl, methyl, carbon dioxide etc. Coenzymes play a decisive role in enzyme function.
Coenzymes from
B-complex vitamins
:
Most of
the coenzymes
are soluble
B-complex biochemical
functions exerted
through chapter
their on
vitamins the
derivatives vitamins.
of
In of
B-complex respective
water fact,
the vitamins
coenzymes.
gives the
details are
The of
structure
Table.
6.3,
a
summary of
the vitamin
related coenzymes
along with
their
Non-vitamin are
coenzymes vitamin
derivatives.
organic functions
is
:

Not all
There substances,
vitamins which
but function
be considered
ATP, are
given.
coenzymes some
have no
other relation
as coenzymes.
as non-vitamin
CDP,
UDP
coenzymes etc.
along in
Table
6.4.
Nucleotide coenzymes
with
They coenzymes
The important
with their
may e.g.
non-vitamin functions
are given
Some of
the and
function of
(Chapter
7).
In coenzymes
possess nitrogenous
base, sugar
and

Adenosine triphosphate, Table 6.4 Coenzymes not related to B-complex vitamins.Abbreviation = ATP. Adenosine triphosphate, Table 6.4 Coenzymes not related to B-complex vitamins.Biochemical functions = Donates phosphate, adenosine and adenosine monophosphate (AMP) moieties.. Cytidine diphosphate, Table 6.4 Coenzymes not related to B-complex vitamins.Abbreviation = CDP. Cytidine diphosphate, Table 6.4 Coenzymes not related to B-complex vitamins.Biochemical functions = Required in phospholipid synthesis as carrier of choline and ethanolamine.. Uridine diphosphate, Table 6.4 Coenzymes not related to B-complex vitamins.Abbreviation = UDP. Uridine diphosphate, Table 6.4 Coenzymes not related to B-complex vitamins.Biochemical functions = Carrier of monosaccharides (glucose, galactose), required for glycogen synthesis.. S - Adenosylmethionine (active methionine), Table 6.4 Coenzymes not related to B-complex vitamins.Abbreviation = SAM. S - Adenosylmethionine (active methionine), Table 6.4 Coenzymes not related to B-complex vitamins.Biochemical functions = Donates methyl group in biosynthetic reactions.. Phosphoadenosine phosphosulfate (active sulfate), Table 6.4 Coenzymes not related to B-complex vitamins.Abbreviation = PAPS. Phosphoadenosine phosphosulfate (active sulfate), Table 6.4 Coenzymes not related to B-complex vitamins.Biochemical functions = Donates sulfate for the synthesis of mucopolysaccharides.

phosphate. Such coenzymes are, therefore, regarded as nucleotides e.g. NAD*t, NADP*, FMN, FAD, coenzyme A, UDPG etc. Protein coenzymes : Thioredoxin is a protein that serves as a coenzyme for the enzyme ribonucleotide reductase (Chapter 17). Coenzymes do not decide enzyme specificity : A particular. coenzyme may participate in catalytic reactions along with different enzymes. For instance, NAD* acts as a coenzyme for lactate dehydrogenase and alcohol dehydrogenase. In both the enzymatic reactions, NAD* is involved in hydrogen transfer. The specificity of the enzyme is mostly dependent on the apoenzyme and not on the coenzyme.

Catalysis is the prime function of enzymes. Enzymes are powerful catalysts. The nature of catalysis taking place in the biological system is similar to that of non-biological catalysis. For any chemical reaction to occur, the reactants have to be in an activated state or transition state.
Enzymes lower activation energy The energy required by the reactants to undergo the reaction is known as activation energy. The reactants when heated attain the activation energy. The catalyst (or the enzyme in the biological system) reduces the activation energy and this causes the reaction to proceed at a lower temperature. Enzymes do not alter the equilibrium constants, they only enhance the velocity of the reaction.
The role of catalyst or enzyme is comparable with a tunnel made in a mountain to reduce the barrier as illustrated in Fig.6.171. The enzyme lowers energy barrier of reactants, thereby making the reaction go faster. The enzymes reduce the activation energy of the reactants in such a way that all the biological systems occur at body temperature (below 40°C).

The prime requisite for enzyme catalysis is that the substrate (S) must combine with the enzyme (E) at the active site to form enzymesubstrate complex (ES) which ultimately results in the product formation (P).
Fig. 6.11 : Effect of enzyme on activation energy of a reaction (A is the substrate and B is the product. Enzyme decreases activation energy).
E+S—ES—+E+P
A few theories have been put forth to explain mechanism of enzyme-substrate complex formation.

This theory was proposed by a German biochemist, Emil Fischer. This is in fact the very first model proposed to explain an enzyme catalysed reaction.
According to this model, the structure or conformation of the enzyme is rigid. The substrate fits to the binding site (now active site) just as a key fits into the proper lock or a hand into the proper glove. Thus the active site of an enzyme is a rigid and pre-shaped template where only a specific substrate can bind. This model does not give any scope for the flexible nature of enzymes, hence the model totally fails to explain many facts of enzymatic reactions, the most important being the effect of allosteric modulators.

Koshland, in 1958, proposed a more acceptable and realistic model for enzymesubstrate complex formation. As per this model,
the active site is not rigid and pre-shaped. The essential features of the substrate binding site are present at the nascent active site. The interaction of the substrate with the enzyme induces a fit or a conformation change in the enzyme, resulting in the formation of a strong substrate binding site. Further, due to induced fit, the appropriate amino acids of the enzyme are repositioned to form the active site and bring about the catalysis (Fig.6.12).
Induced fit model has sufficient experimental evidence from the X-ray diffraction studies. Koshland's model also explains the action of allosteric modulators and competitive inhibition on enzymes.

In this model, the substrate is strained due to the induced conformation change in the enzyme. It is also possible that when a substrate binds to the preformed active site, the enzyme induces a strain to the substrate. The strained substrate leads to the formation of product.
In fact, a combination of the induced fit model with the substrate strain is considered to be operative in the enzymatic action.

The formation of an enzyme-substrate complex (ES) is very crucial for the catalysis to occur, and for the product formation. It is estimated that an enzyme catalysed reaction proceeds 10° to 10!2 times faster than a noncatalysed reaction. The enhancement in the rate of the reaction is mainly due to four processes :
1. Acid-base catalysis;
2. Substrate strain;
3. Covalent catalysis;
4. Entropy effects.
1. Acid-base catalysis : Role of acids and bases is quite important in enzymology. At the physiological pH, histidine is the most important amino acid, the protonated form of which functions as an acid and its corresponding conjugate as a base. The other acids are —OH group of tyrosine, —SH group of cysteine, and €-amino group of lysine. The conjugates of these acids and carboxyl ions (COO7) function as bases.
Ribonuclease which cleaves phosphodiester bonds in a pyrimidine loci in RNA is a classical example of the role of acid and base in the catalysis.
2. Substrate strain : Induction of a strain on the substrate for ES formation is discussed above. During the course of strain induction, the energy level of the substrate is raised, leading to a transition state.
The mechanism of lysozyme (an enzyme of tears, that cleaves B-1,4 glycosidic bonds) action is believed to be due to a combination of substrate strain and acid-base catalysis.
3. Covalent catalysis : In the covalent catalysis, the negatively charged (nucleophilic) or positively charged (electrophilic) group is present at the active site of the enzyme. This group attacks the substrate that results in the covalent binding of the substrate to the enzyme. In the serine proteases (so named due to the presence of serine at active site), covalent catalysis along with acid-base catalysis occur, e.g. chymotrypsin, trypsin, thrombin etc.

4. Proximity catalysis : The reactants should come in close proximity to the enzyme, for appropriate catalysis to occur. The higher the concentration of the substrate molecules, the greater will be the rate of reaction. As the enzyme binds with substrate molecules at the active site, the catalysis will increase several fold (at least a thousand fold)
In the actual catalysis of the enzymes, more than one of the processes — acid-base catalysis, substrate strain, covalent catalysis and proximity catalysis are simultaneously operative. This will help the substrate(s) to attain a transition state leading to the formation of products.

The enzyme catalysed reactions may be broadly grouped into three types based on thermodynamic (energy) considerations.
1. Isothermic
reactions
The
energy
exchange between reactants and products is
negligible e.g. glycogen phosphorylase
Glycogen + Pi
——>
Glucose 1-phosphate
2. Exothermic (exergonic) reactions : Energy
is liberated in these reactions e.g. urease
Urea
—+>
NH3
+
CO)
+
energy

3.
Endothermic
(endergonic)
reactions
Energy is consumed in these reactions e.g. glucokinase
Glucose + ATP —> Glucose 6-phosphate + ADP

In biological system, regulation of enzyme activities occurs at different stages in one or more of the following ways to achieve cellular economy.
1. Allosteric regulation
2. Activation of latent enzymes
3. Compartmentation of metabolic pathways
4. Control of enzyme synthesis
5. Enzyme degradation
6. Isoenzymes

Some of the enzymes possess additional sites,
known as allosteric sites (Greek : allo—other),
The existence of life is unimaginable without the presence of enzymes—the biocatalysts.
Majority of the coenzymes (TPP, NAD*, FAD, CoA) are derived from B-complex
vitamins in which form the latter exert their biochemical functions.
+ Competitive inhibitors of certain enzymes are of great biological significance. Allopurinol,
employed in the treatment of gout, inhibits xanthine oxidase to reduce the formation of uric acid. The other competitive inhibitors include aminopterin used in the treatment of cancers, sulfanilamide as antibactericidal agent and dicumarol as an anticoagulant.
+ The nerve gas (diisopropyl! fluorophosphate), first developed by Germans during Second
World
War,
inhibits
acetylcholine
esterase,
the
enzyme
essential
for
nerve
conduction
and
paralyses
the
vital
body
functions.
Many
organophosphorus
insecticides
(e.g.
melathion)
also
block
the
activity
of
esterase.
+ Penicillin antibiotics irreversibly inhibit serine containing enzymes of bacterial cell wall synthesis.
Fig. 6.13 : Diagrammatic representation of an allosteric enzyme (A) T-form; (B) R-form; (C) Effect of allosteric inhibitor; (D) Effect of allosteric activator.
besides the active site. Such enzymes are known as allosteric enzymes. The allosteric sites are unique places on the enzyme molecule.
Allosteric effectors Certain substances referred to as allosteric modulators (effectors or modifiers) bind at the allosteric site and regulate the enzyme activity. The enzyme activity is increased when a positive (+) allosteric effector binds at the allosteric site known as activator site. On the other hand, a negative (-) allosteric effector binds at the allosteric site called inhibitor site and inhibits the enzyme activity.

Classes of allosteric enzymes : Enzymes that are regulated by allosteric mechanism are referred to as allosteric enzymes. They are divided into two classes based on the influence of allosteric effector on Ky, and Vmax.
| K-class of allosteric enzymes, the effector changes the K,, and not the V,,,,. Double reciprocal plots, similar to competitive inhibition are obtained e.g. phosphofructokinase.
. V-class of allosteric enzymes, the effector alters the V,,,, and not the K,. Double reciprocal plots resemble that of non-competitive inhibition e.g. acetyl CoA carboxylase.
Conformational changes' in __ allosteric enzymes : Most of the allosteric enzymes are oligomeric in nature. The subunits may be identical or different. The non-covalent reversible binding of the effector molecule at the allosteric site brings about a conformational change in the active site of the enzyme, leading to the inhibition or activation of the catalytic activity (Fig.6.13). In the concerted model, allosteric enzymes exist in two conformational states — the T (tense or taut) and the R (relaxed). The T and R states are in equilibrium.
Allosteric activator
(or)
substrate
T
Allosteric inhibitor
Allosteric inhibitors favour T state whereas activators and substrates favour R state. The substrate can bind only with the R form of the enzyme. The concentration of enzyme molecule in the R state increases as more substrate is added, therefore the binding of the substrate to the allosteric enzyme is said to be cooperative. Allosteric enzymes give a sigmoidal curve (instead of hyperbola) when the velocity (v) versus substrate(S) concentration are plotted (Fig.6.14).
The term homotropic effect is used if the substrate influences the substrate binding through allosteric mechanism, their effect is always positive. Heterotropic effect is used when an allosteric modulator effects the binding of substrate to the enzyme. Heterotropic interactions are either positive or negative. Selected examples of allosteric enzymes responsible for rapid control of biochemical pathways are given in Table 6.5.

Fig. 6.14 : Effect of substrate concentration on allosteric enzyme (red line-sigmoidal curve) in comparison with normal enzyme (blue line-hyperbolic curve).

Hexokinase, Metabolic pathway = Glycolysis. Hexokinase, Allosteric.Inhibitor = Glucose 6-phosphate. Hexokinase, Allosteric.Activator = -. Phosphofructokinase, Metabolic pathway = Glycolysis. Phosphofructokinase, Allosteric.Inhibitor = ATP. Phosphofructokinase, Allosteric.Activator = AMP, ADP. Isocitrate dehydrogenase, Metabolic pathway = Krebs cycle. Isocitrate dehydrogenase, Allosteric.Inhibitor = ATP. Isocitrate dehydrogenase, Allosteric.Activator = ADP, NAD +. Pyruvate carboxylase, Metabolic pathway = Gluconeogenesis. Pyruvate carboxylase, Allosteric.Inhibitor = -. Pyruvate carboxylase, Allosteric.Activator = Acetyl CoA. Fructose 1, 6 - bisphosphatase, Metabolic pathway = Gluconeogenesis. Fructose 1, 6 - bisphosphatase, Allosteric.Inhibitor = AMP. Fructose 1, 6 - bisphosphatase, Allosteric.Activator = -. Carbamoyl phosphate synthetase I, Metabolic pathway = Urea cycle. Carbamoyl phosphate synthetase I, Allosteric.Inhibitor = -. Carbamoyl phosphate synthetase I, Allosteric.Activator = N - Acetylglutamate. Tryptophan oxygenase, Metabolic pathway = Tryptophan metabolism. Tryptophan oxygenase, Allosteric.Inhibitor = -. Tryptophan oxygenase, Allosteric.Activator = L - Tryptophan. Acetyl CoA carboxylase, Metabolic pathway = Fatty acid synthesis. Acetyl CoA carboxylase, Allosteric.Inhibitor = Palmitate. Acetyl CoA carboxylase, Allosteric.Activator = Isocitrate

The process of inhibiting the first step by the final product, in a series of enzyme catalysed reactions of a metabolic pathway is referred to as feedback regulation. Look at the series of reactions given below
A is the initial substrate, B, C, and D are the intermediates and E is the end product, in a pathway catalysed by four different enzymes (e1, €, €3, e4). The very first step (A > B by the enzyme ej) is the most effective for regulating the pathway, by the final end product E. This type of control is often called negative feedback regulation since increased levels of end product will result in its (e;) decreased synthesis. This is a real cellular economy to save the cell from the wasteful expenditure of synthesizing a compound which is already available within the cell.
Feedback inhibition or end product inhibition is a specialised type of allosteric inhibition necessary to control metabolic pathways for efficient cellular function.
Aspartate transcarbamoylase (ATCase) is a good example of an_ allosteric enzyme inhibited by a feedback mechanism. ATCase catalyses the very first reaction in pyrimidine biosynthesis.
Carbamoyl phosphate undergoes a sequence of reactions for synthesis of the end product, CTP. When CTP accumulates, it allosterically inhibits the enzyme aspartate transcarbamoylase by a feedback mechanism.

or Sometimes a distinction is made between these two usages. Feedback regulation represents a phenomenon while feedback inhibition involves the mechanism of regulation. Thus, in a true sense, they are not synonymous. For instance, dietary cholesterol decreases hepatic cholesterol biosynthesis through feedback regulation. This does not involve feedback inhibition, since dietary cholesterol does not directly inhibit the regulatory enzyme HMG CoA reductase. However, the activity of gene encoding this enzyme is reduced (repression) by cholesterol.

Latent enzymes, as such, are inactive. Some enzymes are synthesized as proenzymes or zymogens which undergo irreversible covalent
activation by the breakdown of one or more peptide bonds. For instance, proenzymes —namely chymotrypsinogen, pepsinogen and plasminogen, are respectively — converted to the active enzymes chymotrypsin, pepsin and plasmin.
Certain enzymes exist in the active and inactive forms which are interconvertible, depending on the needs of the body. The interconversion is brought about by the reversible covalent modifications, namely phosphorylation and dephosphorylation, and oxidation and reduction of disulfide bonds.
Glycogen phosphorylase is a muscle enzyme that breaks down glycogen to provide energy. This enzyme is a homodimer (two identical subunits) and exists in two interconvertible forms. Phosphorylase b (dephospho enzyme) is inactive which is converted by phosphorylation of serine residues to active form phosphorylase a. The inactive enzyme phosphorylase b is produced on dephosphorylation as illustrated below.
Other examples of phosphorylated active enzymes — citrate lyase, fructose 2,6-bisphosphatase.
There are some enzymes which are active in dephosphorylated state and become _ inactive when phosphorylated e.g. glycogen synthase, acetyl CoA carboxylase, HMG CoA reductase.
A few enzymes are active only with sulfhydryl (—SH) groups, e.g. succinate dehydrogenase, urease. Substances like glutathione bring about the stability of these enzymes.

There are certain substances in the body (e.g., fatty acids, glycogen) which are synthesized and also degraded. There is no point for simultaneous occurrence of both the pathways. Generally, the synthetic (anabolic) and breakdown (catabolic) pathways are operative in different cellular organelles to achieve maximum economy. For instance, enzymes for fatty acid synthesis are found in the cytosol whereas enzymes for fatty acid oxidation are present in the mitochondria.
The intracellular location of certain enzymes and
metabolic pathways
is given
in
Table
6.6.

Cytoplasm, Enzyme/metabolic pathway = Aminotransferases; peptidases; glycolysis; hexose monophosphate shunt; fatty acid synthesis; purine and pyrimidine catabolism.. Mitochondria, Enzyme/metabolic pathway = Fatty acid oxidation; amino acid oxidation; Krebs cycle; urea synthesis; electron transport chain and oxidative phosphorylation.. Nucleus, Enzyme/metabolic pathway = Biosynthesis of DNA and RNA.. Endoplasmic reticulum (microsomes), Enzyme/metabolic pathway = Protein biosynthesis; triacylglycerol and phospholipid synthesis; steroid synthesis and reduction; cytochrome P 450 ; esterase.. Lysosomes, Enzyme/metabolic pathway = Lysozyme; phosphatases; phospholipases; hydrolases; proteases; lipases; nucleases.. Golgi apparatus, Enzyme/metabolic pathway = Glucose 6-phosphatase; 5 c -nucleotidase; glucosyl- and galactosyl-transferases.. Peroxisomes, Enzyme/metabolic pathway = Catalase; urate oxidase; D-amino acid oxidase; long chain fatty acid oxidation.

Most of the enzymes, particularly the rate limiting ones, are present in very low concentration. Nevertheless, the amount of the enzyme directly controls the velocity of the reaction, catalysed by that enzyme. Many rate limiting enzymes have short half-lives. This helps in the efficient regulation of the enzyme levels.
There are two types of enzymes—(a) Constitutive enzymes (house-keeping enzymes)—the levels of which are not controlled and remain fairly constant. (b) Adaptive enzymes—their concentrations increase or decrease as per body needs and are well-regulated. The synthesis of enzymes (proteins) is regulated by the genes (Refer Chapter 26).
Induction and repression : The term induction is used to represent increased synthesis of enzyme while repression indicates its decreased synthesis. Induction or repression which ultimately determines the enzyme concentration at the gene level through the mediation of hormones or other substances.
Examples of enzyme induction : The hormone insulin induces the synthesis of glycogen synthetase, glucokinase, phosphofructokinase and pyruvate kinase. All these enzymes are involved in the utilization of glucose. The hormone cortisol induces the synthesis of many enzymes e.g. pyruvate carboxylase, tryptophan oxygenase and tyrosine aminotransferase.
Examples of repression : In many instances, substrate can repress the synthesis of enzyme. Pyruvate carboxylase is a key enzyme in the synthesis of glucose from non-carbohydrate sources like pyruvate and amino acids. If there is sufficient glucose available, there is no necessity for its synthesis. This is achieved through repression of pyruvate carboxylase by glucose.

Enzymes are not immortal, since it will create a series of problems. There is a lot of variability in the half-lives of individual enzymes. For some, it is in days while for others in hours or in minutes, e.g. LDH,— 5 to 6 days; LDH; — 8 to 12 hours; amylase — 3 to 5 hours.
In general, the key and regulatory enzymes are most rapidly degraded. If not needed, they immediately disappear and, as and when required, they are quickly sysnthesized. Though not always true, an enzyme with long half-life is usually sluggish in its catalytic activity.

Multiple forms of the same enzyme will also help in the regulation of enzyme activity, Many of the isoenzymes are tissue-specific. Although isoenzymes of a given enzyme catalyse the same reaction, they differ in Kn, Vmax or both. e.g. isoenzymes of LDH and CPK.

Enzymes are never expressed in terms of their concentration (as mg or wg etc.), but are expressed only as activities. Various methods have been introduced for the estimation of enzyme activities (particularly for the plasma enzymes). In fact, the activities have been expressed in many ways, like King-Armstrong units, Somogyi units, Reitman-Frankel units, spectrophotometric units etc.

In order to maintain uniformity in the expression of enzyme activities (as units) worldover, the Enzyme Commission of IUB has suggested radical changes. A new unit — namely katal (abbreviated as kat)—was_ introduced. One kat denotes the conversion of one mole substrate per second (mol/sec). Activity may also be expressed as millikatals (mkat), microkatals (ukat) and so on.

Some workers prefer to use standard units or SI units (System International). One SI unit or International Unit (IU) is defined as the amount of enzyme activity that catalyses the conversion of one micromol of substrate per minute. SI units and katal are interconvertible.
11U
=
16.67 nkat
(or)
1
kat
=
6x
107
1U

In the clinical laboratories, however, the units— namely katal or SI units—are yet to find a place. Many investigators still use the old units like King-Armstrong units, Somogyi units etc. while expressing the enzyme activities. It is therefore, essential that the units of enzyme activity, along with the normal values, be invariably stated while expressing the enzymes for comparison.

Ribozymes are a group of ribonucleic acids that function as biological catalysts, and they are regarded as non-protein enzymes.
Altman and his coworkers, in 1983, found that ribonuclease P— an enzyme till then known to cleave precursors of tRNAs to give tRNAs — was functional due to RNA component present in the enzyme and not the protein part of the enzyme (Refer Chapter 4).
The RNA part isolated from ribonuclease P exhibited a true enzyme activity and also obeyed Michaelis-Menten kinetics. Later studies have proved that RNA, in fact, can function as an enzyme and bring about the catalysis.
RNA molecules are known to adapt a tertiary structure just as in the case of proteins (i.e. enzymes). The specific conformation of RNA may be responsible for its function as biocatalyst. It is believed that ribozymes (RNAs) were functioning as catalysts before the occurrence of evolution.
protein enzymes during

Certain
enzymes
are
useful
as
therapeutic
agents,
analytical
reagents,
in
genetic
manipulations and for industrial
applications
(Table 6.7).

1. Streptokinase prepared from streptococcus is useful for clearing the blood clots. Streptokinase activates plasma plasminogen to plasmin which, in turn, attacks fibrin to convert into soluble products.
Plasminogen
|
Streptokinase
Plasmin
|
Fibrin
——+
Soluble products
(clot)
2. The enzyme asparaginase is used in the treatment of leukemias. Tumor cells are dependent on asparagine of the host's plasma for their multiplication. By administering asparaginase, the host's plasma levels of asparagine are drastically reduced. This leads to depression in the viability of tumor cells.

Some enzymes are useful in the clinical laboratory for the measurement of substrates, drugs, and even the activities of other enzymes. The biochemical compounds (e.g. glucose, urea, uric acid, cholesterol) can be more accurately and specifically estimated by enzymatic procedures compared to the conventional chemical methods. A good example is the estimation of plasma glucose by glucose oxidase and peroxidase method.

Enzymes can be used as catalytic agents in industrial and medical applications. Some of these enzymes are immobilized by binding them to a solid, insoluble matrix which will not affect the enzyme stability or its catalytic activity. Beaded gels and cyanogen bromide activated sepharose are commonly used for immobilization of enzymes. The bound enzymes can be preserved for long periods without loss of activity.
Glucose oxidase and peroxidase, immobilized and coated on a strip of paper, are used in the clinical laboratory for the detection of glucose in urine.
The intensity of the blue colour depends on the concentration of glucose. Hence, the strip method is useful for semi-quantitative estimation of glucose in urine.

Estimation of enzyme activities in biological fluids (particularly plasma/serum) is of great clinical importance. Enzymes in the circulation are divided into two groups — plasma functional and plasma non-functional.

Certain enzymes are normally present in the plasma and they have specific functions to perform. Generally, these enzyme activities are higher in plasma than in the tissues. They are mostly synthesized in the liver and enter the circulation e.g. lipoprotein lipase, plasmin, thrombin, choline esterase, ceruloplasmin etc.

These enzymes are either totally absent or present at a low concentration in plasma compared to their levels found in the tissues. The digestive enzymes of the gastrointestinal tract (e.g. amylase, pepsin, trypsin, lipase etc.) present in the plasma are known as secretory enzymes. All the other plasma enzymes associated with metabolism of the cell are collectively referred to as constitutive enzymes (e.g. lactate dehydrogenase, transaminases, acid and alkaline phosphatases, creatine phosphokinase).
Estimation of the activities of non-plasma specific enzymes is very important for the diagnosis and prognosis of several diseases. The normal serum level of an enzyme indicates the balance between its synthesis and release in the routine cell turnover. The raised enzyme levels could be due to cellular damage, increased rate of cell turnover, proliferation of cells, increased synthesis of enzymes etc. Serum enzymes are conveniently used as markers to detect the cellular damage which ultimately helps in the diagnosis of diseases.
(Note : Ther term biomarker refers to any laboratory analyte (enzyme, protein, antigen, antibody, metabolite etc.) that is useful for the

Amylase, Disease (most important) = Acute pancreatitis. Serum glutamate pyruvate transaminase (SGPT), Disease (most important) = Liver diseases (hepatitis). Serum glutamate oxaloacetate transaminase (SGOT), Disease (most important) = Heart attacks (myocardial infarction). Alkaline phosphatase, Disease (most important) = Rickets, obstructive jaundice. Acid phosphatase, Disease (most important) = Cancer of prostate gland. Lactate dehydrogenase (LDH), Disease (most important) = Heart attacks, liver diseases. Creatine phosphokinase (CPK), Disease (most important) = Myocardial infarction (early marker). Aldolase, Disease (most important) = Muscular dystrophy. 5 c -Nucleotidase, Disease (most important) = Hepatitis. J -Glutamyl transpeptidase (GGT), Disease (most important) = Alcoholism
diognosis/prognosis of any disease. Biomarker is a vague term, and less frequently used by biochemists.)
A summary of the important enzymes useful for the diagnosis of specific diseases is given in Table 6.8. Detailed information on the diagnostic enzymes including reference values is provided in Table 6.9. A brief account of selected diagnostic enzymes is discussed
It may be noted that SGPT is more specific for the diagnosis of liver diseases while SGOT is for heart diseases. This is mainly because of their cellular distribution—SGPT is a cytosomal enzyme while SGOT is found in cytosol and mitochondria.
Amylase : The activity of serum amylase is increased in acute pancreatitis (reference 80-180 Somogyi units/dl). The peak value is observed within 8-12 hours after the onset of disease which returns to normal by 3" or 4h day. Elevated activity of amylase is also found in urine of the patients of acute pancreatitis. Serum amylase is also important for the diagnosis of chronic pancreatitis, acute parotitis (mumps) and obstruction of pancreatic duct.

Alanine transaminase (ALT/SGPT) : SGPT is elevated in acute hepatitis of viral or toxic origin, jaundice and cirrhosis of liver (reference 3-40 IU/I).
Aspartate transaminase (AST/SGOT) : SGOT activity in serum is increased in myocardial infarction and also in liver diseases (reference 4-45 U/l).
Alkaline phosphatase (ALP) : \t is elevated in certain bone and liver diseases (reference 3-13 KA units/dl). ALP is useful for the diagnosis of rickets, hyperparathyroidism, carcinoma of bone, and obstructive jaundice.
Acid phosphatase (ACP) : It is increased in the cancer of prostate gland (reference 0.5-4 KA units/dl). The tartarate labile ACP (reference <1 KA units/dl) is useful for the diagnosis and prognosis of prostate cancers i.e. ACP is a good tumor marker.
Lactate dehydrogenase (LDH) : LDH is useful for the diagnosis of myocardial infarction, infective hepatitis, |eukemia and muscular dystrophy (serum LDH reference 50-200 IU/l). LDH has five isoenzymes, the details of which are described later.
Creatine kinase (CK) : It is elevated in myocardial infarction (early detection) and muscular dystrophy (reference 10-50 IU/I). CK has three isoenzymes (described later).

Amylase, T ABLE 6.10 Decrease in plasma (serum) enzymes in certain diseases.Reference values = 80-180 Somogyi units/dl. Amylase, T ABLE 6.10 Decrease in plasma (serum) enzymes in certain diseases.Disease(s) in which decreased = Liver diseases. Pseudocholinesterase (ChE II), T ABLE 6.10 Decrease in plasma (serum) enzymes in certain diseases.Reference values = 10-20 IU/dl. Pseudocholinesterase (ChE II), T ABLE 6.10 Decrease in plasma (serum) enzymes in certain diseases.Disease(s) in which decreased = Viral hepatitis, malnutrition, liver cancer, cirrhosis of liver. Ceruloplasmin, T ABLE 6.10 Decrease in plasma (serum) enzymes in certain diseases.Reference values = 20-50 mg/dl. Ceruloplasmin, T ABLE 6.10 Decrease in plasma (serum) enzymes in certain diseases.Disease(s) in which decreased = Wilson's disease (hepatolenticular degeneration). Glucose 6-phosphate dehydrogenase (G6PD) in RBC, T ABLE 6.10 Decrease in plasma (serum) enzymes in certain diseases.Reference values = 120-260 IU/10 12 RBC. Glucose 6-phosphate dehydrogenase (G6PD) in RBC, T ABLE 6.10 Decrease in plasma (serum) enzymes in certain diseases.Disease(s) in which decreased = Congenital deficiency with hemolytic anemia
y-Glutamyl transpeptidase (GGT) : It is a sensitive diagnostic marker for the detection of alcoholism. GGT is also increased in infective hepatitis and obstructive jaundice.

The multiple forms of an enzyme catalysing the same reaction are isoenzymes or isozymes. They, however, differ in their physical and chemical properties which include the structure, electrophoretic and immunological properties, Km and Vinax Values, pH optimum, relative susceptibility to inhibitors and degree of denaturation.

Many
possible
reasons
are
offered
to
explain
the presence of isoenzymes in the living systems.
1.
Isoenzymes
from
different
genes
e.g.
malate
of
cytosol
is
different from that found in mitochondria.
2.
Oligomeric
enzymes
consisting
of
more
than
one
type
of
subunits
e.g.
lactate
dehydro-
genase and creatine phosphokinase.
3. An enzyme may be active as monomer or oligomer e.g. glutamate dehydrogenase. 4. In glycoprotein enzymes, differences in carbohydrate content may be responsible for isoenzymes e.g. alkaline phosphatase.

most
Among the isoenzymes, LDH has been the thoroughly investigated.
LDH whose systematic name is L-lactateNAD+ oxidoreductase (E.C. 1.1.1.27) catalyses the interconversion of lactate and pyruvate as shown below
LDH
e
CH
3
COOH
OH
TN
CH3—C—COOH
NAD*
NADH+H*

LDH has five distinct isoenzymes LDH,, LDH), LDH3, LDHg and LDHs. They can be separated by electrophoresis (cellulose or starch gel or agarose gel). LDH, has more positive charge and fastest in electrophoretic mobility while LDHs is the slowest.
Structure of LDH isoenzymes : LDH is an oligomeric (tetrameric) enzyme made up of four polypeptide subunits. Two types of subunits namely M (for muscle) and H (for heart) are produced by different genes. M-subunit is basic while H subunit is acidic. The isoenzymes contain either one or both the subunits giving LDH, to LDHs. The characteristic features of LDH isoenzymes are given in Table 6.11.
CH
Significance of differential catalytic activity : LDH, (Hy) is predominantly found in heart muscle and is inhibited by pyruvate —the substrate. Hence, pyruvate is not converted to lactate in cardiac muscle but is converted to acetyl CoA which enters citric acid cycle. LDHs (M4) is mostly present in skeletal muscle and the inhibition of this enzyme by pyruvate is minimal, hence pyruvate is converted to lactate. Further, LDHs has low K,, (high affinity) while LDH, has high K,, (low affinity) for pyruvate. The differential catalytic activities of LDH, and LDHs in heart and skeletal muscle, respectively, are well suited for the aerobic (presence of oxygen) and anaerobic (absence of oxygen) conditions, prevailing in these tissues.
Diagnostic importance of LDH : Isoenzymes of LDH have immense value in the diagnosis of heart and liver related disorders (Fig.6.15). In healthy individuals, the activity of LDH, is higher than that of LDH, in serum. In the case of myocardial infarction, LDH, is much greater than LDH, and this happens within 12 to 24 hours after infarction. Increased activity of LDHs

in serum is an indicator of liver diseases. LDH activity in the RBC is 80-100 times more than that in the serum. Hence for estimation of LDH or its isoenzymes, serum should be totally free from hemolysis or else false positive results will be obtained.

Creatine kinase (CK) or creatine phosphokinase (CPK) catalyses the inter-conversion of phosphocreatine (or creatine phosphate) to creatine.
Phosphocreatine
Creatine
ADP
“>
ATP
Each two CPK exists as three isoenzymes. isoenzyme is a dimer composed of subunits—M (muscle) or B (brain) or both.
Isoenzyme
_—
Subunit
Tissue of origin
CPK,
CPK,
CPK,
BB
MB
MM
Brain
Heart
In healthy individuals, the isoenzyme CPK, (MB) is almost undetectable in serum with less than 2% of total CPK. After the myocardial infarction (MI), within the first 6-18 hours, CPK» increases in the serum to as high as 20% (against 2% normal). CPKz isoenzyme is not elevated in skeletal muscle disorders. Therefore, estimation of the enzyme CPKz (MB) is the earliest reliable indication of myocardial infarction.

As many as six isoenzymes of alkaline phosphatase (ALP) have been identified. ALP is a monomer, the isoenzymes are due to the difference in the carbohydrate content (sialic acid residues). The most important ALP isoenzymes are @1-ALP, )-heat labile ALP, @»-heat stable ALP, pre-B ALP, y-ALP etc.
Increase in @ -heat labile ALP suggests hepatitis whereas pre B-ALP indicates bone diseases.

+ In the living system, the regulation of enzyme activities occurs through allosteric inhibition, activation of latent enzymes, compartmentation of metabolic pathways, control of enzyme synthesis and degradation.
+ Feedback (or end product) inhibition is a specialized form of allosteric inhibition that controls several metabolic pathways e.g. CTP inhibits aspartate transcarbamoylase; Cholesterol inhibits HMG CoA reductase. The end product inhibition is utmost important to cellular economy since a compound is synthesized only when required.
+ Certain RNA molecules (ribozymes) function as non-protein enzymes. It is believed that ribozymes were functioning as biocatalysts before the occurrence of protein enzymes during evolution.
+ Certain enzymes are utilized as therapeutic agents. Streptokinase in used to dissolve blood clots in circulation while asparaginase is employed in the treatment of leukemias.
+ Determination of serum enzyme activities is of great importance for the diagnosis of several diseases (refer Table 6.8).
+ Lowered body temperature (hypothermia) is accompained by a decrease in enzyme activities. This principle is exploited to reduce metabolic demand during open heart surgery or transportation of organs for transplantation surgery.

Alcohol dehydrogenase (ADH) has two heterodimer isoenzymes. Among the white Americans and Europeans, aB; isoenzyme is predominant whereas in Japanese and Chinese (Orientals) &B is mostly present. The isomer a8» more rapidly converts alcohol to acetaldehyde. Accumulation of acetaldehyde is associated with tachycardia (increase in heart rate) and facial flushing among Orientals which is not commonly seen in whites. It is believed that Japanese and Chinese have increased sensitivity to alcohol due to the presence of a —isoenzyme of ADH.

For the right diagnosis of a particular disease, it is always better to estimate a few (three or more) serum enzymes, instead of a_ single enzyme. Examples of enzyme patterns in important diseases are given here.

The enzymes — namely creatine phosphokinase (CPK), aspartate transaminase (AST) and lactate dehydrogenase (LDH)—are important for the diagnosis of myocardial infarction (Ml). The elevation of these enzymes in serum in relation to hours/days of MI is given in the Fig.6.16.
Creatine phosphokinase (precisely isoenzyme MB) is the first enzyme to be released into circulation within 6-18 hours after the infarction. Therefore, CPK estimation is highly useful for the early diagnosis of MI. This enzyme reaches a peak value within 24-30 hours, and returns to normal level by the 2nd or 3rd day.
Aspartate transaminase (AST or SGOT) rises sharply
after
CPK, and
reaches
a
peak within
48
hours of
the myocardial
AST
takes
4-5
days to
return to
normal level.
Lactate dehydrogenase (LDHj) generally rises from the second day after infarction, attains a peak by the 3rd or 4th day and takes about 10-15 days to reach normal level. Thus, LDH is the last enzyme to rise and also the last enzyme to return to normal level in MI.
Cardiac troponins (CT) Although — not enzymes, the proteins cardiac troponins are highly useful for the early diagnosis of MI. Among these, troponin I (inhibitory element of actomysin ATPase) and troponin T (tropomysin binding element) are important. Cardiac troponin | (CTI) is released into circulation within four hours after the onset of MI, reaches a peak value by 12-24 hours, and remains elevated for about a week.
The protein myoglobin is also an early marker for the diagnosis of MI. However, it is not specific to cardiac diseases. High serum concentration of brain natriuretic peptide is a marker for congestive cardiac failure. In the Table 6.12, a summary of the diagnostic markers used in MI is given. Table 6.13 gives enzyme patterns in various diseases.

The following enzymes—when elevated in serum —are useful for the diagnosis of liver dysfunction due to viral hepatitis (jaundice), toxic hepatitis, cirrhosis and hepatic necrosis
1. Alanine transaminase
2. Aspartate transaminase
3. Lactate dehydrogenase.
The enzymes that markedly increase in intrahepatic and extrahepatic cholestasis are : (1) Alkaline phosphatase, (2) 5'-Nucleotidase Serum-y-glutamyl transpeptidase is useful in the diagnosis of alcoholic liver diseases.

In the muscular dystrophies, serum levels of certain muscle enzymes are increased. These include creatine phosphokinase, aldolase and aspartate transaminase.

Myoglobin, Time of peak elevation = 4-6 hrs. Myoglobin, Time of return to normal level = 20-25 hrs. Myoglobin, Diagnostic importance = Earliest marker, however not cardiac specific.. Cardiac troponin I, Time of peak elevation = 12-24 hrs. Cardiac troponin I, Time of return to normal level = 5-9 days. Cardiac troponin I, Diagnostic importance = Early marker and cardiac specific.. Cardiac troponin T, Time of peak elevation = 18-36 hrs. Cardiac troponin T, Time of return to normal level = 5-14 days. Cardiac troponin T, Diagnostic importance = Relatively early marker and cardiac specific. However, elevated in other degenerative diseases.. Creatine phosphokinase (MB), Time of peak elevation = 20-30 hrs. Creatine phosphokinase (MB), Time of return to normal level = 24-48 hrs. Creatine phosphokinase (MB), Diagnostic importance = Cardiac specific and early marker.. Lactate dehydrogenase (LDH I), Time of peak elevation = 48-72 hrs. Lactate dehydrogenase (LDH I), Time of return to normal level = 10-15 days. Lactate dehydrogenase (LDH I), Diagnostic importance = Relatively late marker and cardiac specific.. Aspartate transaminase, Time of peak elevation = 30-48 hrs. Aspartate transaminase, Time of return to normal level = 4-6 days. Aspartate transaminase, Diagnostic importance = Not cardiac specific.

Neuron-specific enolase serves
as
a
marker
Increase
in
the
serum
acid
phosphatase
(tartarate
labile)
is
specific
for
the
detection
of
prostatic carcinoma.
[Note
:
Prostate
specific
antigen
(PSA;
mol
wt.
32
KD),
though
not
an
enzyme,
is
a
more
reliable
marker
for
the
detection
of
prostate
cancer.
Normal
serum
of
PSA
is
1-4 ng/ml].
for lung
cancer, neuroblastoma,
pheochromo- cytoma etc.

Besides
serum/plasma
enzymes,
enzyme
estimations in other body fluids, and tissues also

, T ABLE 6.13 Serum enzyme profiles (patterns) in diseases.Disease/enzyme(s) = Acid phosphatase (ACP). , T ABLE 6.13 Serum enzyme profiles (patterns) in diseases.Significance = Marker enzyme for prostate cancer.. , T ABLE 6.13 Serum enzyme profiles (patterns) in diseases.Disease/enzyme(s) = Prostate specific antigen (PSA). , T ABLE 6.13 Serum enzyme profiles (patterns) in diseases.Significance = Significantly elvated in prostate cancer (not an enzyme).
have
some
diagnostic
A few
examples are lised.
importance.
Urine
:
Urinary
amylase
is
increased
in
acute
pencreatitis.
|
B-N-Acetylgalactosidase
in
urine
is
elevated
in
renal
graft
dysfunction.
is
increased
in
the
cancers
of
urinary bladder, pancreas etc.
Cerebrospinal fluid : Lactate dehydrogenase
is increased in CSF in meningitis.
Gastric
juice
:
$-Glucuronidase
activity
is
increased in gastric carcinoma.
Feces : Fetal trypsin levels are decreased in
cystic fibrosis.
Liver
:
Glucose
6-phosphatase
in
liver
is
significantly
lower
in
type
|
glycogen
storage
disease.
Muscle : Phosphorylase activity in muscle is
decreased in McArdle’s disease.
Erythrocytes
Glucose
_6-phosphate
delydrogenase_
in
RBC
causes
hemolytic
anemia.
Decreased
transketolase
activity in erythrocytes is used for diagnosis of thiamine deficiency.
In addition to the above, cultured fibroblasts,

and amniotic cells are frequently used for the
diagnosis
of
inborn
errors
of
e.g.
phenylalanine
hydroxylase
__in
phenylkonuria (in cultured amniotic cells).

1. Enzymes are the protein biocatalysts synthesized by the living cells. They are classified into six major classes—oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.
2. An enzyme is specific in its action, possessing active site, where the substrate binds to form enzyme-substrate complex, before the product is formed.
3.
Factors
like
of
enzyme,
substrate,
pH
etc.
influence
enzyme
activity.
The
substrate
to
produce
half-maximal
velocity
is
known
as
Michaelis
constant
(Km
value).
4. Enzyme activities are inhibited by reversible (competitive, and non-competitive), irreversible and allosteric manner.
5. Many enzymes require certain non-protein substances called cofactors (coenzymes) for their action. Most of the coenzymes are derivatives of B-complex vitamins (e.g. NAD*, FAD, TPP etc.)
6.
The
mechanism
of
enzyme
action
is
explained
by
lock
and
key
model
(of
Fischer),
more
recently
induced
fit
model
(of
Koshland)
and
substrate
strain
theory.
7. The enzymes enhance the rate of reaction through acid-base catalysis, covalent catalysis and/or proximity catalysis.
8. In the living system, there is a constant regulation of enzyme levels, brought about by allosteric mechanism, activation of proenzymes, synthesis and degradation of enzymes etc.
9. Estimation of serum enzymes is of great help in the diagnosis of several diseases. Serum amylase and lipase are increased in acute pancreatitis; alanine transaminase in hepatitis; aspartate transaminase, lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) in myocardial infarction; alkaline phosphatase in rickets and hyperparathyroidism; acid phosphatase in prostatic carcinoma; y-glutamyl transpep- tidase in alcoholism.

10. Isoenzymes are the multiple forms of an enzyme catalysing the same reaction which however, differ in their physical and chemical properties. LDH has five isoenzymes while CPK has three. LDH and CPKg2 are very important in the diagnosis of MI.

1. What are enzymes? Describe their classification and nomenclature.
a . Write an account of the importance of serum enzymes in the diagnosis of diseases.
& . What are coenzymes? Write briefly on the role of coenzymes in enzyme action.
WN . Write an account of the various factors affecting enzyme activity.
. Describe the mechanism of enzyme action.

(a) Enzyme specificity, (b) Competitive inhibition, (c) Coenzymes, (d) Allosteric enzymes, (e) Isoenzymes, (f) K,, value, (g) Serum enzymes in myocardial infarction, (h) Lactate dehydrogenase, (i) Role of metals in enzyme action, (j) Active site.

CoO . Phsophofructokinase is allosterically activated by
OWN . The enzyme glucose 6-phosphate dehydrogenase requires the coenzyme
DH . Examples of two enzymes containing zinc are
BW . The non-protein part of holoenzyme
KY . The class of enzymes involved in synthetic reactions are
= . The literal meaning of enzyme is
. Enzymes lose the catalytic activity at temperature above 70°C
due to
and
. The place at which substrate binds with the enzyme
. The E.C. number for alcohol dehydrogenase is
. The very first enzyme elevated in serum in myocardial infarction

11. Pepsin is an example for the class of enzymes namely
12. The coenzyme not involved in hydrogen transfer
13. In the feedback regulation, the end product binds at
14. y-Glutamyl transpeptidase activity in serum is elevated in
15. In recent years, a non-protein compound has been identified to bring about catalysis in biological system. The name of the compound is
(a) Oxidoreductases (b) Transferases (c) Hydrolases (d) Ligases.
(a) FMN (b) FAD (c) NADP* (d) FH.
(a) Active site (b) Allosteric site (c) E-S complex (d) of these.
(a) Pancreatitis (b) Muscular dystrophy (c) Myocardial infarction (d) Alcoholism.
(a) DNA (b) RNA (c) Lipids (d) Carbohydrates.

lt; is difficult to define vitamins precisely. Vitamins may be regarded as_ organic compounds required in the diet in small amounts to perform specific biological functions for normal maintenance of optimum growth and health of the organism. The bacterium E.coli does not require any vitamin, as it can synthesize all of them. It is believed that during the course of evolution, the ability to synthesize vitamins was lost. Hence, the higher organisms have to obtain them from diet. The vitamins are required in small amounts, since their degradation is relatively slow.
the natural foods. Funk (1913) isolated an active principle (an amine) from rice polishings and, later in yeast, which could cure beri-beri in pigeons. He coined the term vitamine (Greek : vita-life) to the accessory factors with a belief that all of them were amines. It was later realised that only few of them are amines. The term vitamin, however, is continued without the final letter 'e'.

In the beginning of 20th century, it was clearly understood that the diets containing purified carbohydrate, protein, fat and minerals were not adequate to maintain the growth and health of experimental rats, which the natural foods (such as milk) could do.
Hopkins coined the term accessory factors to the unknown and essential nutrients present in
The usage of A, B and C to vitamins was introduced in 1915 by McCollum and Davis. They first felt there were only two vitamins— fat soluble A and water soluble B (anti-beriberi factor). Soon another water soluble anti-scurvy factor named vitamin C was described. Vitamin A was later found to possess two componentsone that prevents night blindness (vitamin A) and another anti-ricket factor named as vitamin D. A fat soluble factor called vitamin E, in the absence of which rats failed to reproduce properly, was discovered. Yet another fat soluble vitamin concerned with coagulation was discovered in mid 1930s. It was named as vitamin K. In the
sequence of alphabets it should have been F, but K was preferred to reflect its function (koagulation).
As regards the water soluble factors, vitamin C was identified as a pure substance and named as ascorbic acid. Vitamin B was found to be a complex mixture and nomenclature also became complex. B, was clearly identified as anti-beriberi factor. Many investigators carried out intensive research between 1920 and 1930 and went on naming them as the water soluble vitamins B>, B3, By, Bs, Bg, Bz, Bg, Bo, Bio, B14 and By. Some of them were found to be mixtures of already known vitamins. And for this reason, a few members (numbers!) of the Bcomplex series disappeared from the scene. Except for B,, Bj, Bg and By; , names are more commonly used for other B-complex vitamins.

There are about 15 vitamins, essential for humans. They are classified as fat soluble (A, D, E and K) and water soluble (C and B-group) vitamins as shown in the Table 7.1. The B-complex vitamins may be sub-divided into energy-releasing (B,, B, Bg, biotin etc.) and hematopoietic (folic acid and B,). Most of the water soluble vitamins exert the functions through their respective coenzymes while only one fat soluble vitamin (K) has been identified to function as a
coenzyme.

As far as humans are concerned, it is believed that the normal intestinal bacterial synthesis, and absorption of vitamin K and biotin may be sufficient to meet the body requirements. For other B-complex vitamins, the synthesis and absorption are relatively less. Administration of anitibiotics often kills the vitamin synthesizing bacteria present in the gut, hence additional consumption of vitamins is recommended.

The four vitamins, namely vitamin A, D, E, and K are known as fat or lipid soluble. Their availability in the diet, absorption and transport are associated with fat. They are soluble in fats and oils and also the fat solvents (alcohol, acetone etc.). Fat soluble vitamins can be stored in liver and adipose tissue. They are not readily excreted in urine. Excess consumption of these vitamins (particularly A and D) leads to their accumulation and toxic effects.
All the fat soluble vitamins are isoprenoid compounds, since they are made up of one or more of five carbon units namely isoprene units (—CH=C.CH3—CH=CH-). Fat soluble vitamins perform diverse functions. Vitamin K has a specific coenzyme function.

The water soluble vitamins are a heterogenous group of compounds since they differ chemically from each other. The only common character shared by them is their solubility in water. Most of these vitamins are readily excreted in urine and they are not toxic to the body. Water soluble vitamins are not stored in the body in large quantities (except Bj2). For this reason, they must be continuously supplied in the diet. Generally, vitamin deficiencies are multiple rather than individual with overlapping symptoms. |t is often difficult to pinpoint the exact biochemical basis for the symptoms.
The water soluble vitamins form coenzymes (Refer Table 6.3) that participate in a variety of biochemical reactions, related to either energy generation or hematopoiesis. It may be due to this reason that the deficiency of vitamins results in a number of overlapping symptoms. The common symptoms of the deficiency of one or more vitamins involved in energy metabolism include dermatitis, glossitis (red and swollen tongue), cheilitis (rupture at the corners of lips), diarrhea, mental confusion, depression and malaise.
Deficiency of vitamins Bz, Bg and By is more closely associated with neurological manifestations.

The term vitamers represents the chemically similar substances that possess qualitatively similar vitamin activity. Some good examples of vitamers are given below
1 Retinol, retinal and retinoic acid are vitamers of vitamin A.
1 Pyridoxine, pyridoxal and pyridoxamine are vitamers of vitamin Be.

In the following pages, the individual members of the fat soluble and water soluble vitamins are discussed with regard to the chemistry, biochemical functions, recommended dietary/daily allowances (RDA), dietary sources, deficiency manifestations etc.

The fat soluble vitamin A, as such is present only in foods of animal origin. However, its provitamins carotenes are found in plants.
It is recorded in the history that Hippocrates (about 500 B.C.) cured night blindness. He prescribed to the patients ox liver (in honey), which is now known to contain high quantity of vitamin A.

In the recent years, the term vitamin A is collectively used to represent many structurally related and biologically active molecules (Fig.7.1). The term retinoids is often used to include the natural and synthetic forms of vitamin A. Retinol, retinal and retinoic acid are regarded as vitamers of vitamin A.
1. Retinol (vitamin A alcohol) : It is a primary alcohol containing B-ionone ring. The side chain has two isoprenoid units, four double bonds and one hydroxyl group. Retinol is present in animal tissues as retinyl ester with long chain fatty acids.
Fig. 7.1 : Structures of vitamin A and related compounds (Red colour represents the substituent groups in the respective compounds).
2. Retinal (vitamin A aldehyde) : This is an aldehyde form obtained by the oxidation of retinol. Retinal and retinol are interconvertible. Previously, the name retinine was used for retinal.
3. Retinoic acid (vitamin A acid) : This is produced by the oxidation of retinal. However, retinoic acid cannot give rise to the formation of retinal or retinol.
4. B-Carotene (provitamin A) : This is found in plant foods. \t is cleaved in the intestine to produce two moles of retinal. In humans, this conversion is inefficient, hence B-carotene possesses about one-sixth vitamin A activity compared to that of retinol.

Dietary retinyl esters are hydrolysed by pancreatic or intestinal brush border hydrolases in the intestine, releasing retinol and free fatty acids. Carotenes are hydrolysed by B-carotene 15-15'-dioxygenase of intestinal cells to release 2 moles of retinal which is reduced to retinol. In the intestinal mucosal cells, retinol is reesterified to long chain fatty acids, incorporated into chylomicrons and transferred to the lymph. The retinol esters of chylomicrons are taken up by the liver and stored (Fig.7.2).
As and when needed, vitamin A is released from the liver as free retinol. It is believed that zinc plays an important role in retinol mobilization. Retinol is transported in the circulation by the plasma retinol binding protein (RBP; mol. wt. 21,000) in association with pre-albumin. The retinol-RBP complex binds to specific receptors on the cell membrane of peripheral tissue and enters the cells. Many cells of target tissues contain a cellular retinolbinding protein that carries retinol to the nucleus and binds to the chromatin (DNA). It is here that retinol exerts its function in a manner analogous to that of a_ steroid hormone.

Vitamin A is necessary for a variety of functions such as vision, proper growth and differentiation, reproduction and maintenance of epithelial cells. In recent years, each form of vitamin A has been assigned specific functions
(Fig.7.3).

tion of vitamin A in the process of vision was first elucidated by George Wald (Nobel Prize 1968). The events occur in a cyclic process known as Rhodopsin cycle or Wald's visual cycle (Fig.7.4).
Fig. 7.2 : Summary of vitamin A absorption, transport and biochemical functions
(FFA-Free fatty acid; RBP-Retinol binding protein).

The retina of the eye possesses two types of cells—rods and cones. The human eye has about 10 million rods and 5 million cones. The rods are in the periphery while cones are at the centre of retina. Rods are involved in dim light vision whereas cones are responsible for bright light and colour vision. Animals—such as owls and cats for which night vision is more important— possess mostly rods.

Rhodopsin (mol. wt. 35,000) is a conjugated protein present in rods. It contains 11-cis retinal and the protein opsin. The aldehyde group (of retinal) is linked to €-amino group of lysine (of opsin).
The primary event in visual cycle, on exposure to light, is the isomerization of 11-cisretinal to all-trans retinal. This leads to a conformational change in opsin which _ is responsible for the generation of nerve impulse. The all-trans-retinal is immediately isomerized by retinal isomerase (of retinal epithelium) to 11-cis-retinal. This combines with opsin to regenerate rhodopsin and complete the visual cycle (Fig.7.4). However, the conversion of all trans-retinal to 11-cis retinal is incomplete. Therefore, most of the all-trans-retinal is transported to the liver and converted to all-trans retinol by alcohol dehydrogenase. The all-transretinol undergoes isomerization to 11-cis retinol which is then oxidized to 11-cis retinal to participate in the visual cycle.
Dark adaptation time : When a person shifts from a bright light to a dim light (e.g. entry into a dim cine theatre), rhodopsin stores are depleted and vision is impaired. However, within a few minutes, known as dark adaptation time, rhodopsin is resynthesized and vision is improved. Dark adaptation time is increased in vitamin A deficient individuals.
Bleaching of rhodopsin: When exposed to light, the colour of rhodopsin changes from red to yellow, by a process known as bleaching. Bleaching occurs in a few milliseconds and many unstable intermediates are formed during this process.

the retina, a number of biochemical changes leading to membrane hyperpolarization occur resulting in the genesis of nerve impulse. The hyperpolarization of the membrane is brought about by a visual cascade involving cyclic GMP. When a photon (from light) is absorbed by rhodopsin, metarhodopsin II is produced. The protein transducin is activated by metarhodopsin Il. This involves an exchange of GTP for GDP on inactive transducin. The activated transducin activates cyclic GMP phosphodiesterase. This
enzyme degrades cyclic GMP in the rod cells (Fig.7.5). A rapid decrease in cyclic GMP closes the Nat channels in the membranes of the rod cells. This results in hyperpolarization which is an excitatory response transmitted through the neuron network to the visual cortex of the brain.

Cones are specialized in bright and colour vision. Visual cycle comparable to that present in rods is also seen in cones. The colour vision is governed by colour sensitive pigments— porphyropsin (red), iodopsin (green) and cyanopsin (blue). All these pigments are retinalopsin complexes. When bright light strikes the retina, one or more of these pigments are bleached, depending on the particular colour of light. The pigments dissociate to all-trans-retinal and opsin, as in the case of rhodopsin. And_ this reaction passes on a nerve impulse to brain as a specific colour—red when porphyropsin splits, green when iodopsin splits or blue for cyanopsin. Splitting of these three pigments in different proportions results in the perception of different colours by the brain.

1. Retinol and retinoic acid function almost
like steroid hormones. They regulate the protein synthesis and thus are involved in the cell growth and differentiation.
2.
Vitamin
A
is essential
healthy epithelial
tissue.
retinol
This and
keratin retinoic
synthesis is
due acid
to the
are fact
required
(responsible
3.
Retinyl phosphate
is necessary
that to
for horny
synthesized for
prevent surface).
from retinol
the glycoproteins,
and are
4.
Retinol reproduction.
regulates synthesis
of mucopolysaccharides
growth certain
which and
mucus secretion.
It gene
for acts
like
a
expression.
5.
Vitamin the
A
is considered
maintenance normal
hormone to
and be
essential of
proper immune
for system
to fight
against various
infections.
6. Cholesterol synthesis requires vitamin A. Mevalonate, an intermediate in the cholesterol biosynthesis, is diverted for the synthesis of coenzyme Q in vitamin A deficiency. It is pertinent to note that the discovery of coenzyme Q was originally made in vitamin A deficient animals.
7. Carotenoids (most important B-carotene) function as antioxidants and reduce the risk of cancers initiated by free radicals and strong oxidants. B-Carotene is found to be beneficial to prevent heart attacks. This is also attributed to the antioxidant property.

The daily requirement of vitamin A_ is expressed as retinol equivalents (RE) rather than International Units (IU).
1
retinol equivalent
=1
pg retinol
=6
ug
=12
ug
B-carotene other
carotenoids
=3.33
IU
of activity
vitamin
A
=10
IU of vitamin A activity from B-carotene
The RDA of vitamin A for adults is around 1,000 retinol equivalents (3,500 IU) for man and 800 retinol equivalents (2,500 IU) for woman.
One International Unit (IU) equals to 0.3 mg of retinol. The requirement increases in pregnant women and lactating mothers.

Animal sources contain (preformed) vitamin A. The best sources are liver, kidney, egg yolk, milk, cheese, butter. Fish (cod or shark) liver oils are very rich in vitamin A.
Vegetable sources contain the provitamin A-carotenes. Yellow and dark green vegetables and fruits are good sources of carotenes e.g. carrots, spinach, pumpkins, mango, papaya etc.

The vitamin A deficiency may be due to inadequate dietary intake, impaired intestinal absorption, reduced storage in liver and chronic alcholism. The deficiency symptoms are not immediate, since the hepatic stores can meet the body requirements for quite sometime (2-4 months). The deficiency manifestations are related to the eyes, skin and growth.
Deficiency manifestations of the eyes : Night blindness (nyctalopia) is one of the earliest symptoms of vitamin A_ deficiency. The individuals have difficulty to see in dim light since the dark adaptation time is increased. Prolonged deficiency irreversibly damages a number of visual cells.
Severe deficiency of vitamin A leads to xerophthalmia. This is characterized by dryness in conjunctiva and cornea, and keratinization of epithelial cells. In certain areas of conjunctiva, white triangular plaques known as Bitot's spots are seen.
If xerophthalmia persisits for a long time, corneal ulceration and degeneration occur. This results in the destruction of cornea, a condition referred to as keratomalacia, causing total blindness. Therefore, adequate intake of vitamin A is necessary for the prevention of blindness.

Effect on growth: Vitamin A deficiency results in growth retardation due to impairment in skeletal formation.
Effect on reproduction: The reproductive system is adversely affected in vitamin A deficiency. Degeneration of germinal epithelium leads to sterility in males.
Effect on skin and epithelial cells : The skin becomes rough and dry. Keratinization of epithelial cells of gastrointestinal tract, urinary tract and respiratory tract is noticed. This leads to increased bacterial infection. Vitamin A deficiency is associated with formation of urinary stones.
The plasma level of retinol binding protein is decreased in vitamin A deficiency.

Excessive consumption of vitamin A leads to toxicity. The symptoms of hypervitaminosis A include dermatitis raised intracranial tension, enlargement of liver, skeletal decalcification, tenderness of long bones, loss of weight, irritability, loss of hair, joint pains etc. Elderly people are more susceptible to vitamin A toxicity, hence overdoses should be avoided.
Total serum vitamin A level (normal 20-50 ug/dl) is elevated in hypervitaminosis A. Free retinol or retinol bound to plasma lipoproteins is actually harmful to the body. It is now believed that the vitamin A toxicosis symptoms appear only after retinol binding capacity of retinol binding protein exceeds.
Higher concentration of retinol increases the synthesis of lysosomal hydrolases. The manifestations of hypervitaminosis A are attributed to the destructive action of hydrolases, particularly on the cell membranes.

Increased consumption of B-carotene is associated with decreased incidence of heart attacks, skin and lung cancers. This is attributed to the antioxidant role of B-carotene which is independent of its role as a precursor of vitamin A. Ingestion of high doses of B-carotene for long periods are not toxic like vitamin A.

Vitamin D is a fat soluble vitamin. It resembles sterols in structure and functions like a hormone.
Fig. 7.6 : Formation of ergocalciferol from ergosterol.
The symptoms of rickets and the benefical effects of sunlight to prevent rickets have been known for centuries. Hess (1924) reported that irradiation with ultraviolet light induced antirachitic activity in some foods. Vitamin D was isolated by Angus (1931) who named it calciferol.

Ergocalciferol (vitamin Dy) is formed from ergosterol and is present in plants (Fig.7.6). Cholecalciferol (vitamin D3) is found in animals. Both the sterols are similar in structure except that ergocalciferol has an additional methyl group and a double bond. Ergocalciferol and cholecalciferol are sources for vitamin D activity and are referred to as provitamins.
During the course of cholesterol biosynthesis (Chapter 14), 7-dehydrocholesterol is formed as an intermediate. On exposure to sunlight, 7-dehydrocholesterol is converted to cholecalciferol in the skin (Fig.2.7). Vitamin D_ is regarded as sun-shine vitamin.
The synthesis of vitamin D3 in the skin is proportional to the exposure to sunlight. Dark skin pigment (melanin) adversly influences the synthesis of cholecalciferol. (Note : The term vitamin D, is no more in use. It was originally used for fat soluble crystalline material, which later turned out to be a mixture).

Vitamin D is absorbed in the small intestine for which bile is essential. Through lymph, vitamin D enters the circulation bound to plasma Q-globulin and is distributed throughout the body. Liver and other tissues store small amounts of vitamin D.

Vitamins Dy and D3, as such, are not biologically active. They are metabolized identically in the body and converted to active forms. The metabolism and biochemical functions of vitamin D are depicted in Fig.7.8.

Cholecalciferol is first hydroxylated at 25th position to 25-hydroxycholecalciferol (25-OH D3) by a specific hydroxylase present in liver. 25-OH D3 is the major storage and circulatory form of vitamin D. Kidney possesses a specific enzyme, 25-hydroxycholecalciferol (calcidiol) 1-hydroxylase which hydroxylates 25-hydroxycholecalciferol at position 1 to produce 1,25dihydroxycholecalciferol (1,25-DHCC). 1,25 DHCC contains 3 hydroxyl groups (1,3 and 25 carbon) hence referred to as calcitriol. Both the hydroxylase enzymes (of liver and kidney) require cytochrome P4s59, NADPH and Op for the hydroxylation process. The synthesis of calcitriol is depicted in Figs.7.7 and 7.8.

The concentration of 1,25-DHCC is regulated by plasma levels of calcium and phosphate. They control hydroxylation reaction at position 1. Low plasma phosphate increases the activity of 25-hydroxycholecalciferol 1-hydroxylase. Low plasma calcium enhances the production of parathyroid hormone which in turn activates 1-hydroxylase. Thus the action of phosphate is direct while that of calcium is indirect on kidney 1-hydroxylase.
Chapter
7
:
VITAMINS

Calcitriol (1,25-DHCC) is the biologically active form of vitamin D. It regulates the plasma levels of calcium and phosphate. Calcitriol acts at 3 different levels (intestine, kidney and bone) to maintain plasma calcium (normal 9-11 mg/dl).
1. Action of calcitriol on the intestine : Calcitriol increases the intestinal absorption of calcium and phosphate. In the intestinal cells, calcitriol binds with a cytosolic receptor to form a calcitriol-receptor complex. This complex then approaches the nucleus and interacts with a specific DNA leading to the synthesis of a specific calcium binding protein. This protein increases the calcium uptake by the intestine. The mechanism of action of calcitriol on the target tissue (intestine) is similar to the action of a steroid hormone.
2. Action of calcitriol on the bone : In the osteoblasts of bone, calcitriol stimulates calcium uptake for deposition as calcium phosphate. Thus calcitriol is essential for bone formation. The bone is an important reservoir of calcium and phosphate. Calcitriol along with parathyroid hormone increases the mobilization of calcium and phosphate from the bone. This causes elevation in the plasma calcium and phosphate levels.
3. Action of calcitriol on the kidney Calcitriol is also involved in minimizing the excretion of calcium and phosphate through the kidney, by decreasing their excretion and enhancing reabsorption.
The sequence of events that take place in response to low plasma calcium concentration and the action of calcitriol on intestine, kidney and bone, ultimately leading to the increase in plasma calcium is given in Fig.7.9.
24,25-Dihydroxycholecalciferol (24,25-DHCC) is another metabolite of vitamin D. It is also synthesized in the kidney by 24-hydroxylase. The exact function of 24,25-DHCC is not known. It is believed that when calcitriol concentration is adequate, 24-hydroxylase acts leading to the synthesis of a less important compound 24, 25-DHCC. In this way, to maintain the homeostasis of calcium, synthesis of 24,25-DHCC
is also important.

Calcitriol (1,25-DHCC) is now considered as an important calciotropic hormone, while cholecalciferol is the prohormone. The following characteristic features of vitamin D (comparable with hormone) justify its status as a hormone.
1. Vitamin D3 (cholecalciferol) is synthesized in the skin by ultra-violet rays of sunlight.
2. The biologically active form of vitamin D,
calcitriol is produced in the kidney.
3.
Calcitriol has
target organs—intestine,
bone and
kidney, where
it specifically
acts.
4. Calcitriol action is similar to steroid hormones. |t binds to a receptor in the cytosol and the complex acts on DNA to stimulate the synthesis of calcium binding protein.
5. Actinomycin D_ inhibits the action of calcitriol. This supports the view that calcitriol exerts its effect on DNA leading to the synthesis of RNA (transcription).
6. Calcitriol synthesis is self-regulated by a feedback mechanism i.e., calcitriol decreases its own synthesis.

The daily requirement of vitamin D is 400 International Units or 10 mg of cholecalciferol.
In countries with good sunlight (like India), the RDA for vitamin D is 200 IU (or 5 mg cholecalciferol).

Good sources of vitamin D include fatty fish, fish liver oils, egg yolk etc. Milk is not a good source of vitamin D.
three
Vitamin D can be provided to the body in ways
- 1. Exposure of skin to sunlight for synthesis
of vitamin D;
- 2. Consumption of natural foods;
3. By irradiating foods (like yeast) that contain precursors of vitamin D and fortification of foods (milk, butter etc.).

Vitamin D = deficiency is relatively less common, since this vitamin can be synthesized in the body. However, insufficient exposure to sunlight and consumption of diet lacking vitamin D results in its deficiency.
Vitamin D_ deficiency occurs in strict vegetarians, chronic alcoholics, individuals with liver and kidney diseases or fat malabsorption syndromes. In some people, who cover the entire body (purdah) for religious customs, vitamin D deficiency is also observed, if the requirement is not met through diet.
Deficiency of vitamin D causes rickets in children and osteomalacia in adults. Rickets is derived from an old English word 'wrickken', meaning to twist. Osteomalacia is derived from Greek (osteon-bone; malakia-softness). Vitamin D is often called as antirachitic vitamin.
Rickets in children is characterized by bone deformities due to incomplete mineralization, resulting in soft and pliable bones and delay in teeth formation. The weight-bearing bones are bent to form bow-legs. In rickets, the plasma level of calcitriol is decreased and alkaline phosphatase activity is elevated. Alkaline phosphatase is concerned with the process of bone formation. There is an overproduction of
alkaline phosphatase related to more cellular activity of the bone. It is believed to be due to a vain attempt to result in bone formation. of osteomalacia (adult rickets)
In case demineralization of the bones occurs (bones become softer), increasing their susceptibility to fractures.

This is seen in patients with chronic renal failure. Renal rickets is mainly due to decreased synthesis of calcitriol in kidney. It can be treated by administration of calcitriol.

Vitamin D is stored mostly in liver and slowly metabolised. Among the vitamins, vitamin D is the most toxic in overdoses (10-100 times RDA). Toxic effects of hypervitaminosis D include demineralization of bone (resorption) and increased calcium absorption from the intestine, leading to elevated calcium in plasma (hypercalcemia). Prolonged hypercalcemia is associated with deposition of calcium in many soft tissues such as kidney and arteries. Hypervitaminosis D may lead to formation of stones in kidneys (renal calculi). High consumption of vitamin D is associated with loss of appetite, nausea, increased thirst, loss of weight etc.

Vitamin E (tocopherol) is a naturally occurring antioxidant. It is essential for normal reproduction in many animals, hence known as anti-sterility vitamin. Vitamin E is described as a 'vitamin in search of a disease.' This is due to the lack of any specific vitamin E deficiency disease in humans. Evans and his associates (1936) isolated the compounds of vitamin E activity and named them as tocopherols (Greek : tokos-child birth; pheros-to bear; ol-alcohol).

Vitamin E is the name given to a group of tocopherols and_ tocotrienols. About eight tocopherols (vitamin E vitamers) have been identified-a, B, y, 6& etc. Among _ these, a-tocopherol is the most active. The tocopherols are derivatives of 6-hydroxy chromane (tocol) ring with isoprenoid (3 units) side chain. The antioxidant property is due to the hydroxyl group of chromane ring.

Vitamin E is absorbed along with fat in the small intestine. Bile salts are necessary for the absorption. In the liver, it is incorporated into lipoproteins (VLDL and LDL) and transported. Vitamin E is stored in adipose tissue, liver and muscle. The normal plasma level of tocopherol is less than 1 mg/dl.

Most of the functions of vitamin E are related to its antioxidant property. It prevents the nonenzymatic oxidations of various cell components (e.g. unsaturated fatty acids) by molecular oxygen and free radicals such as superoxide (Oy) and hydrogen peroxide (H ,O,). The element selenium helps in these functions.
Vitamin E is lipophilic in character and is found in association with lipoproteins, fat deposits and cellular membranes. It protects the polyunsaturated fatty acids (PUFA) from peroxidation reactions. Vitamin E acts as a scavenger and gets itself oxidized (to quinone form) by free radicals (R) and spares PUFA, as shown below
The biochemical functions of vitamin E, related either directly or indirectly to its antioxidant property, are given hereunder
- 1. Vitamin E is essential for the membrane structure and integrity of the cell, hence it is regarded as a membrane antioxidant.
2. It prevents the peroxidation of polyunsaturated fatty acids in various tissues and membranes. It protects RBC from hemolysis by oxidizing agents (e.g. HO>).
3. It is closely associated with reproductive functions and prevents sterility. Vitamin E preserves and maintains germinal epithelium of gonads for proper reproductive function.
4. It increases the synthesis of heme by enhancing the activity of enzymes 6aminolevulinic acid (ALA) synthase and ALA dehydratase.
5. It is required for cellular respiration— through electron transport chain (believed to stabilize coenzyme Q).
6. Vitamin E prevents the oxidation of vitamin A and carotenes.
7. It is required for proper storage of creatine in skeletal muscle.
8. Vitamin E is needed for optimal absorption of amino acids from the intestine.
9. Itis involved in proper synthesis of nucleic acids.
10. Vitamin E protects liver from being damaged by toxic compounds such as carbon tetrachloride.
11. It works in association with vitamins A, C and B-carotene, to delay the onset of cataract.

12. Vitamin E has been recommended for the prevention of chronic diseases such as cancer and heart diseases. Clinical trials in this regard are rather disappointing, hence it is no more recommended. However, some __ clinicians continue to use it particularly in subjects susceptible to heart attacks. It is believed that vitamin E prevents the oxidation of LDL. (Note : The oxidized LDL have been implicated to heart promote diseases.)

The element selenium is found in the enzyme glutathione peroxidase that destroys free radicals. Thus, Se is also involved in antioxidant functions like vitamin E, and both of them act synergistically. To a certain extent, Se can spare the requirement vitamin E, and vice versa.

Intake of vitamin E is directly related to the consumption of polyunsaturated fatty acids (PUFA) i.e., requirement increases with increased intake of PUFA. A daily consumption of about 10 mg (15 IU) of «tocopherol for man and 8 mg (12 IU) for woman is recommended. One mg of a-tocopherol is equal to 1.5 IU. Vitamin E supplemented diet is advised for pregnant and lactating women.

Many vegetable oils are rich sources of vitamin E. Wheat germ oil, cotton seed oil, peanut oil, corn oil and sunflower oil are the good sources of this vitamin. It is also present in meat, milk, butter and eggs.

The symptoms of vitamin E deficiency vary from one animal species to another. In many animals, the deficiency is associated with sterility, degenerative changes in muscle, megaloblastic anaemia and changes in central nervous system. Severe symptoms of vitamin E deficiency are not seen in humans except increased fragility of erythrocytes and minor neurological symptoms.

Among the fat soluble vitamins (A, D, E, K), vitamin E is the least toxic. No toxic effect has been reported even after ingestion of 300 mg/ day for 23 years.

Vitamin K is the only fat soluble vitamin with a specific coenzyme function. It is required for

Vitamin K is taken in the diet or synthesized by the intestinal bacteria. Its absorption takes place along with fat (chylomicrons) and_ is dependent on bile salts. Vitamin K is transported along with LDL and is stored mainly in liver and, to a lesser extent, in other tissues.

The functions of vitamin K are concerned with blood clotting process. It brings about the posttranslational (after protein biosynthesis in the cell) modification of certain blood clotting factors. The clotting factors I (prothrombin), VII, IX and X are synthesized as inactive precursors (zymogens) in the liver. Vitamin K acts as a coenzyme for the carboxylation of glutamic acid residues present in the proteins and this reaction is catalysed by a carboxylase (microsomal). It involves the conversion of glutamate (Glu) to y-carboxyglutamate (Gla) and requires vitamin K, Oy, and CO, (Fig.7.12). The formation of y-carboxyglutamate is inhibited by dicumarol, an anticoagulant found in spoilt sweet clover. Warfarin is a synthetic analogue that can inhibit vitamin K action (Fig.7.13).
Vitamin K is also' required for the carboxylation of glutamic acid residues of osteocalcin, a calcium binding protein present in the bone.
The mechanism of carboxylation is not fully understood. It is known that a 2,3-epoxide derivative of vitamin K is formed as an intermediate during the course of the reaction. Dicumarol inhibits the enzyme (reductase) that converts epoxide to active vitamin K.
Role of Gla in clotting: The y-carboxyglutamic acid (Gla) residues of clotting factors are negatively charged (COO-) and_ they combine with positively charged calcium ions (Ca2*) to form a complex. The mechanism of action has been studied for prothrombin. The prothrombin —Ca complex binds to the phospholipids on the membrane surface of the platelets (Fig.7.14). This leads to the increased conversion of prothrombin to thrombin.

Strictly speaking, there is no RDA for vitamin K, since it can be adequately synthesized in the
Fig. 7.12 : Vitamin K dependent carboxylation of the precursors of clotting factors.
gut. It is however, recommended that half of the body requirement is provided in the diet, while the other half is met from the bacterial synthesis. Accordingly, the suggested RDA for an adult is 70-140 g/day.
diseases) and administration of antibiotics (killing of intestinal flora).

Cabbage, cauliflower, tomatoes, alfa alfa, spinach and other green vegetables are good sources. It is also present in egg yolk, meat, liver, cheese and dairy products.

The deficiency of vitamin K is uncommon, since it is present in the diet in sufficient quantity and/or is adequately synthesized by the intestinal bacteria. However, vitamin K deficiency may occur due to its faulty absorption (lack of bile salts), loss of vitamin into feces (diarrheal
Fig.
7.13
:
Summary of
vitamin
K
cycle in carboxylation reaction.
Deficiency of vitamin K leads to the lack of active prothrombin in the circulation. The result is that blood coagulation is adversely affected. The individual bleeds profusely even for minor injuries. The blood clotting time is increased.

Administration of large doses of vitamin K produces hemolytic anaemia and jaundice, particularly in infants. The toxic effect is due to increased breakdown of RBC.

The compounds—namely heparin, bishydroxycoumarin—act as anticoagulants and are antagonists to vitamin K. The salicylates and dicumarol are also antagonists to vitamin K.
Fig. 7.14 : Mechanism of action of y-carboxyglutamate of prothrombin in blood clotting.
dehydroascorbic acid ~—_are biologically active. However, D-ascorbic acid is inactive. The plasma and_ tissues _ predominantly contain ascorbic acid in the reduced form. The ratio of ascorbic acid to dehydroascorbic acid in many tissues is 15 : 1. On hydration, dehydroascorbic acid is irreversibly converted to 2,3diketogulonic acid which _ is inactive. Hydration reaction is
Dicumarol is structurally related to vitamin K and acts as a competitive inhibitor in the synthesis of active prothrombin.

Vitamin C is a water soluble versatile vitamin. It plays an important role in human health and disease. Vitamin C has become the most controversial vitamin in recent years. This is because of the claims and counter-claims on the use of vitamin C in megadoses to cure everything from common cold to cancer.
Scurvy has been known to man for centuries. It was the first disease found to be associated with diet. In the sixteenth century about 10,000 mariners died of a miraculous disease (scurvy) due to lack of fresh vegetables in their diet. James Lind, a surgeon of the English Navy, in 1753 published 'Treatise on Scurvy'. Based on Lind's observations, the Royal Navy since 1795 used to supply lime or lemon juice to all the crews. The English Navy used to carry crates of lemons, hence they were popularly known as
Limeys.

Ascorbic acid is a hexose (6 carbon) derivative and closely resembles monosaccharides in structure (Fig.7.15). The acidic property of vitamin C is due to the enolic hydroxyl groups. It is a strong reducing agent. L-Ascorbic acid undergoes oxidation to form dehydroascorbic acid and _ this reaction is reversible. Both ascorbic acid and almost spontaneous, in alkaline or neutral solution. It is for this reason that oxidation of vitamin C is regarded as biological inactivation (formation of diketogulonic acid). Oxidation of ascorbic acid is rapid in the presence of copper. Hence vitamin C becomes inactive if the foods are prepared in copper vessels.

Many animals can synthesize ascorbic acid from glucose via uronic acid pathway (Chapter 13). However, man, other primates, guinea pigs and bats cannot synthesize ascorbic acid due to the deficiency of a single enzyme namely L-gulonolactone oxidase.
Vitamin C is rapidly absorbed from the intestine. It is not stored in the body to a significant extent. Ascorbic acid is excreted in urine as such, or as_ its metabolites— diketogulonic acid and oxalic acid (Fig.7.15).

Most of the functions of vitamin C are related to its property to undergo reversible oxidation— reduction i.e., interconversion of ascorbic acid and dehydroascorbic acid.
1. Collagen formation : Vitamin C plays the role of a coenzyme in hydroxylation of proline and lysine while protocollagen is converted to collagen (i.e. post-translational modification). The hydroxylation reaction is catalysed by lysyl hydroxylase (for lysine) and prolyl hydroxylase (for proline). This reaction is dependent on vitamin C, molecular oxygen and a-ketoglutarate
(Fig.7.16).
Fig. 7.16 : Ascorbic acid dependent hydroxylation of proline of protocollagen.
Hydroxyproline and hydroxylysine are essential for the collagen cross-linking and the strength of the fiber. In this way, vitamin C is necessary for maintenance of normal connective tissue and wound healing process.
2. Bone formation : Bone tissues possess an organic matrix, collagen and the inorganic calcium, phosphate etc. Vitamin C is required for bone formation.
3. Iron and hemoglobin metabolism : Ascorbic acid enhances iron absorption by keeping it in the ferrous form. This is due to the reducing property of vitamin C. It helps in the formation of ferritin (storage form of iron) and mobilization of iron from ferritin.
Vitamin C is useful in the reconversion of methemoglobin to hemoglobin. The degradation of hemoglobin to bile pigments requires ascorbic acid.
4. Tryptophan metabolism: Vitamin C is essential for the hydroxylation of tryptophan (enzyme-hydroxylase) to hydroxytryptophan in the synthesis of serotonin.
5. Tyrosine metabolism: Ascorbic acid is required for the oxidation of p-hydroxy phenylpyruvate (enzyme hydroxylase) to homogentisic acid in tyrosine metabolism.

6. Folic acid metabolism : The active form of the vitamin folic acid is tetrahydrofolate (FH4). Vitamin C is needed for the formation of FH4 (enzyme-folic acid reductase). Further, in association with FH4, ascorbic acid is involved in the maturation of erythrocytes.
7. Peptide hormone synthesis : Many peptide hormones contain carboxyl terminal amide which is derived from terminal glycine. Hydroxylation of glycine is carried out by peptidylglycine hydroxylase which requires vitamin C.
8. Synthesis of corticosteroid hormones : Adrenal gland possesses high levels of ascorbic acid, particularly in periods of stress. It is believed that vitamin C is necessary for the hydroxylation reactions in the synthesis of corticosteroid hormones.
9. Sparing action of other vitamins: Ascorbic acid is a strong antioxidant. |t spares vitamin A, vitamin E, and some B-complex vitamins from oxidation.
10. Immunological function: Vitamin C enhances the synthesis of immunoglobulins (antibodies) and increases the phagocytic action of leucocytes.
11. Preventive action on cataract : Vitamin C reduces the risk of cataract formation.
12. Preventive action on chronic diseases : As an antioxidant, vitamin C reduces the risk of cancer, cataract, and coronary _ heart diseases.

About 60-70 mg vitamin C intake per day will meet the adult requirement. Additional intakes (20-40% increase) are recommended for women during pregnancy and lactation.

Citrus fruits, gooseberry (amla), guava, green vegetables (cabbage, spinach), tomatoes, potatoes (particularly skin) are rich in ascorbic acid. High content of vitamin C is found in adrenal gland and gonads. Milk is a poor source of ascorbic acid.

The deficiency of ascorbic acid results in scurvy. This disease is characterized by spongy and sore gums, loose teeth, anemia, swollen joints, fragile blood vessels, decreased immunocompetence, delayed wound _ healing, sluggish hormonal function of adrenal cortex and gonads, haemorrhage, osteoporosis etc. Most of these symptoms are related to impairment in the synthesis of collagen and/or the antioxidant property of vitamin C.

consumption of megadoses of ascorbic acid (even up to 18 g/day, 300 times the daily requirement!) to prevent and cure common cold. He is remembered as a scientist who suggested 'keep vitamin C in gunny bags and eat in grams.' This generated a lot of controversy worldover. It is now clear that megadose of vitamin C does not prevent common cold. But the duration of cold and the severity of symptoms are reduced. It is believed that ascorbic acid promotes leukocyte function.
Megadoses (1-4 g/day) of vitamin C are still continued
in common
cold, wound
_
healing, trauma
etc.
As an
antioxidant, ascorbic
acid certainly provides some health benefits.
Ascorbic acid, as such, has not been found to be toxic. But, dehydroascorbic acid (oxidized form of ascorbic acid) is toxic. Further, oxalate is a major metabolite of vitamin C. Oxalate has been implicated in the formation of kidney stones. However, there are controversial reports on the megadoses of vitamin C leading to urinary
Linus Pauling (1970) first advocated the stones.

+ It is believed that during the course of evolution, the ability to synthesize vitamins was lost by the higher organisms, hence they should be supplied through the diet.
+ For humans, the normal intestinal bacterial synthesis of vitamin K and biotin is almost sufficient to meet the body requirements.
+ Administration of antibiotics often destroys the vitamin synthesizing bacteria in the gut,
hence additional supplementation of vitamins is recommended during antibiotic therapy.
+ Vitamin A deficiency causes night blindness; vitamin D deficiency rickets (in children) or osteomalacia (in adults); vitamin E deficiency minor neurological symptoms; vitamin K deficiency bleeding.
+ Fat soluble vitamins are not readily excreted in urine, hence excess consumption leads to their accumulation and toxic effects.
+ Vitamin C deficiency causes scurvy. The manifestations of scurvy are related to the
impairment
in
the
synthesis
of
collagen
and/or
the
antioxidant
property
of
vitamin
C.
+ Megadoses of vitamin C are used in common cold, wound healing, trauma etc.
+ Carotene, vitamin E and ascorbic acid serve as antioxidants and reduce the risk of heart attacks and cancers.

Thiamine (anti-beri-beri_ or —antineuritic vitamin) is water soluble. It has a_ specific coenzyme, thiamine pyrophosphate (TPP) which is mostly associated with carbohydrate metabolism.

Thiamine contains a pyrimidine ring and a thiazole ring held by a methylene bridge (Fig.7.17). Thiamine is the only _ natural compound with thiazole ring.
The alcohol (OH) group of thiamine is esterfied with phosphate (2 moles) to form the coenzyme, thiamine pyrophosphate (TPP or cocarboxylase). The pyrophosphate moiety is donated by ATP and the reaction is catalysed by the enzyme thiamine pyrophosphate transferase.

The coenzyme, thiamine pyrophosphate or cocarboxylase is intimately connected with the energy releasing reactions in the carbohydrate metabolism (Fig.7.18).
1. The enzyme pyruvate dehydrogenase catalyses (oxidative decarboxylation) — the irreversible conversion of pyruvate to acetyl CoA. This reaction is dependent on TPP, besides the other coenzymes (details given in carbohydrate metabolism, Chapter 13).
respective
2. a-Ketoglutarate dehydrogenase is an enzyme of the citric acid cycle. This enzyme is comparable with pyruvate dehydrogenase and requires TPP.
3.
Transketolase is
dependent on
TPP.
This is
an enzyme
monophosphate of
the shunt
4.
hexose
(HMP
The branched
acid dehydrogenase
catalyses the
branched leucine
shunt).
chain a-keto
(decarboxylase)
decarboxylation chain
amino and
keto acids.
acids
(valine, isoleucine)
This enzyme
also to
the requires
TPP.
5. TPP plays an important role in the transmission of nerve impulse. |t is believed that TPP is required for acetylcholine synthesis and the ion translocation of neural tissue.

The daily requirement of thiamine depends on the intake of carbohydrate. A dietary supply of 1-1.5 mg/day is recommended for adults (about 0.5 mg/1,000 Cals of energy). For children RDA is 0.7-1.2 mg/day. The requirement marginally increases in pregnancy and lactation (2 mg/day), old age and alcoholism.

Cereals, pulses, oil seeds, nuts and yeast are good sources. Thiamine is mostly concentrated in the outer layer (bran) of cereals. Polishing of rice removes about 80% of thiamine. Vitamin B, is also present in animal foods like pork, liver, heart, kidney, milk etc. In the parboiled (boiling of paddy with husk) and milled rice, thiamine is not lost in polishing. Since thiamine is a water soluble vitamin, it is extracted into the water during cooking process. Such water should not be discarded.

The deficiency of vitamin B, results in a condition called beri-beri [Sinhalese : | cannot
Fig. 7.18 : Summary of the reactions dependent on thiamine pyrophosphate (TPP).
(said twice)]. Beri-beri is mostly seen in populations consuming exclusively polished rice as staple food. The early symptoms of thiamine deficiency are loss of appetite (anorexia), weakness, constipation, nausea, mental depression, peripheral neuropathy, irritability etc. Numbness in the legs complaints of 'pins and needles sensations' are reported.
3. Thiamine deficiency leads to impairment in nerve impulse transmission due to lack of TPP. 4. The transketolase activity in erythrocytes is decreased. Measurement of RBC transketolase activity is a reliable diagnostic test to assess thiamine deficiency.

1. Carbohydrate metabolism is impaired. Accumulation of pyruvate occurs in the tissues which is harmful. Pyruvate concentration in plasma is elevated and it is also excreted in urine.
2. Normally, pyruvate does not cross the blood-brain barrier and enter the — brain. However, in thiamine deficiency, an alteration occurs in the blood-brain barrier permitting the pyruvate to enter the brain directly. It is believed that pyruvate accumulation in brain results in disturbed metabolism that may be responsible for polyneuritis.
In adults, two types of beri-beri, namely wet beri-beri and dry beri-beri occur. Infantile beriberi that differs from adult beri-beri is also seen.
Wet beri-beri (cardiovascular beri-beri) : This is characterized by edema of legs, face, trunk and serous cavities. Breathlessness and palpitation are present. The calf muscles are slightly swollen. The systolic blood pressure is elevated while diastolic is decreased. Fast and bouncing pulse is observed. The heart becomes weak and death may occur due to heart failure.
Dry beri-beri (neurological beri-beri) : This is associated with neurological manifestations resulting in peripheral neuritis. Edema is not commonly seen. The muscles become
progressively weak and walking becomes difficult. The affected individuals depend on support to walk and become bedridden, and may even die, if not treated.
The symptoms of beri-beri are often mixed in which case it is referred to as mixed beri-beri.
Infantile beri-beri: This is seen in infants born to mothers suffering from thiamine deficiency. The breast milk of these mothers contains low thiamine content. Infantile beri-beri is characterized by sleeplessness, restlessness, vomiting, convulsions and bouts of screaming due to cardiac dilatation.

This disorder also known as cerebral beriberi, is mostly seen in chronic alcoholics. The body demands of thiamine increase in alcoholism. Insufficient intake or impaired intestinal absorption of thiamine will lead to this syndrome. It is characterized by loss of memory, apathy and a rhythmical to and fro motion of the eye balls.

The enzyme thiaminase is present in certain seafoods. Their inclusion in the diet will destroy thiamine by a cleavage action (pyrimidine and thiazole rings split) and lead to deficiency. Incidence of beri-beri in some parts of Japan is attributed to the consumption of fish (rich in thiaminase). Pyrithiamine, a structural analogue and an antimetabolite of thiamine is found in certain plants like ferns. Horses and cattle often develop thiamine deficiency (fern poisoning) due to the overconsumption of the plant fern.

are two
Pyrithiamine and oxythiamine the important antimetabolites of thiamine.

Riboflavin through its coenzymes takes part in a variety of cellular oxidation—-reduction reactions.

Riboflavin contains 6,7-dimethyl isoalloxazine (a heterocyclic 3 ring structure) attached to D-ribitol by a nitrogen atom. Ribitol is an open chain form of sugar ribose with the aldehyde group (CHO) reduced to alcohol (CH,OH).
Riboflavin is stable to heat but sensitive to light. When exposed to ultra-violet rays of sunlight, it is converted to lumiflavin which exhibits yellow fluorescence. The substances namely lactoflavin (from milk), hepatoflavin (from liver) and ovoflavin (from eggs) which were originally thought to be different are structurally identical to riboflavin.

Flavin mononucleotide (FMN) and _ flavin adenine dinucleotide (FAD) are the two coenzyme forms of riboflavin. The ribitol (5 carbon) is linked to a phosphate in FMN. FAD is formed from FMN by the transfer of an AMP moiety from ATP (Fig.7.19).

The flavin coenzymes (mostly FAD and to a lesser extent FMN) participate in many redox reactions responsible for energy production. The functional unit of both the coenzymes is isoalloxazine ring which serves as an acceptor of two hydrogen atoms (with electrons). FMN or FAD undergo identical reversible reactions accepting two hydrogen atoms forming FMNH) or FADH) (Fig.7.20).
Enzymes that use flavin coenzymes (FMN or FAD) are called flavoproteins. The coenzymes (prosthetic groups) often bind rather tightly, to the protein (apoenzyme) either by non-covalent bonds (mostly) or covalent bonds in the holoenzyme. Many flavoproteins contain metal atoms (iron, molybdenum etc.) which are known as

The coenzymes, FAD and FMN are associated with certain enzymes involved in carbohydrate, lipid, protein and purine metabolisms, besides the electron transport chain. A few examples are listed in Table 7.2. Further details are given in the respective chapters.

The daily
requirement riboflavin
for an
Higher intakes
are advised
—
adult is
1.2-1.7
(by of
mg.
0.2-0.5
for mg/day)
pregnant lactating
women.

Milk and
milk products,
eggs, liver,
Cereals, kidney
fruits, and
meat, are
rich sources.
vegetables and
are moderate
sources.

Riboflavin deficiency symptoms include cheilosis (fissures at the corners of the mouth), glossitis (tongue smooth and purplish) and dermatitis. Riboflavin deficiency as such is uncommon. It is mostly seen along with other vitamin deficiencies. Chronic alcoholics are susceptible to By deficiency. Assay of the enzyme glutathione reductase in erythrocytes will be useful in assessing riboflavin deficiency.
Antimetabolite
:
Galactoflavin is
an antimetabolite of riboflavin.
Niacin or
nicotinic acid
is known
as factor
of of
pellagra preventive
Goldberg.
niacin also
(P.P.)
The coenzymes
(NAD+
be and
synthesized amino
NADP?*)
by can
the acid,
essential tryptophan.
The disease
rough pellagra
skin)
has centuries.
been
However, the
(Italian known
its of
a
for relation
dietary first
identified
Goldberger to
factor was
by
Goldberger.
and conducted
an his
interesting associates
experiment fish
Fig. 7.19 : Structures and biosynthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD).
Chapter
7
:
VITAMINS

(FMN or FAD)
(FMNHp or FADH2)
Fig. 7.20 : Participation of FMN or FAD in oxidation-reduction reactions (R-represents the rest of the structure of FMN or FAD as depicted in Fig. 7.19).
for this purpose. Twelve convicts were promised pardon if they consumed diet of pellagrous families for one year. The diet consisted of corn meal, corn starch, rice, sweet potato and pork fat. More than half of the subjects showed symptoms of pellagra in less than an year, while no such symptoms were observed in other prisoners on a regular diet. Administration of dried meat or liver to the patients cured pellagra (Goldberger, 1928).
nicotine (present in tobacco leaves). The term 'niacin' was coined and more commonly used for nicotinic acid. This was done to emphasize the role of niacin as a_ vitamin and avoid the impression that nicotinic acid is an oxidized form of nicotine. However, most of the authors use niacin and nicotinic acid synonymously.

Much before it was recognized as a vitamin,
nicotinic
acid
was
well
known
as
a
chemical
compound,
produced
by
the
oxidation
of
Niacin is a pyridine derivative. Structurally, it is
pyridine
3-carboxylic acid.
The amide
form of
niacin is known as niacinamide or nicotinamide.

|. Carbohydrate metabolism
(a)
Pyruvate
complex”
(b)
a-Ketoglutarate
complex”
(c) Succinate dehydrogenase
Il. Lipid metabolism
(d) Acyl CoA dehydrogenase
Ill. Protein metabolism
(e)
Glycine
oxidase
(f) D-Amino acid oxidase
IV. Purine metabolism
(g) Xanthine oxidase

L-Amino acid oxidase
a
Dihydrolipoyl dehydrogenase component requires FAD
Reaction
Pyruvate
——>
Acetyl
CoA
—
>
Succinyl CoA
Succinate
——>
Fumarate
Acyl
CoA
—->
a,
B-Unsaturated
acyl
CoA
Glycine
——>
Glyoxylate
+
NH3
——>
o-Keto
acid
+
NHg
Xanthine
——
Uric acid
L-Amino acid
———>
o-Keto
acid
+
NH3
139
Dietary nicotinamide, niacin and tryptophan (an essential amino acid) contribute to the synthesis of the coenzymes—nicotinamide adenine dinucleotide (NAD*) and nicotinamide adenine dinucleotide phosphate (NADP*). The pathway for the biosynthesis of NAD* and NADP? is depicted in Fig.7.21. Nicotinamide is deaminated in the body to niacin. Niacin then undergoes a series of reactions to produce NAD* and NADP?*. Tryptophan produces quinolinate which then forms nicotinate mononucleotide and, ultimately, NAD* and NADPt. Sixty milligrams of tryptophan is equivalent to 1 mg of niacin for the synthesis of niacin coenzymes. Phosphoribosyl pyrophosphate and ATP, respectively, provide ribose phosphate and AMP. moieties for the synthesis of NAD*. Glutamine donates amide group. In the structure of the coenzymes, nitrogen atom of nicotinamide carries a positive charge (due to the formation of an extra bond, N is in quaternary state), hence the coenzymes are NAD* and = NADP?*. Nicotinamide, liberated on the degradation of NAD* and NADP? is mostly excreted in urine as N-methylnicotinamide.
(Note : Some authors use NAD/NADP to represent, in a general way, oxidized or reduced forms of niacin coenzymes.)

The coenzymes NAD*+ and NADP?* are involved in a variety of oxidation-reduction reactions. They accept hydride ion (hydrogen atom and one electron :H-) and undergo reduction in the pyridine ring. This results in the neutralization of positive charges. The nitrogen atom and the fourth carbon atom of nicotinamide ring participate in the reaction. While one atom of hydrogen (as hydride ion) from the substrate (AH) is accepted by the coenzyme, the other hydrogen ion (H*) is released into the surrounding medium.
This reaction is reversed when NADH is oxidized to NAD*. NADP* also functions like NAD+* in the oxidation-reduction reactions.
A large number of enzymes (about 40) belonging to the class oxidoreductases are
Fig. 7.21 : Outline of the biosynthesis of nicotinamide nucleotides, NAD+ and NADP* (NPRT-Nicotinate phosphoribosyltransferase; QPRT-Quinolinate phosphoribosyltransferase; PPi-Pyrophosphate; PRPP-Phosphoribosy! pyrophosphate).
Fig. 7.22 : Mechanism of oxidation and reduction of nicotinamide coenzyme—NAD* (Note : Similar mechanism operates for NADP* also).
dependent on NAD+ or NADP*. The coenzymes are loosely bound to the enzymes and can be separated easily by dialysis. NAD* and NADP* participate in almost all the metabolisms (carbohydrate, lipid, protein etc.). Some enzymes are exclusively dependent on NAD* whereas some require only NADPt. A few enzymes can use either NAD* or NADP*. Selected examples of enzymes and the reactions they catalyse are given in Table 7.3.
NADH produced is oxidized in the electron transport chain to generate ATP. NADPH is also important for many biosynthetic reactions as it donates reducing equivalents.

The daily requirement of niacin for an adult is 15-20 mg and for children, around 10-15 mg. Very often, the term niacin equivalents (NE) is used while expressing its RDA. One NE =1 mg niacin or 60 mg of tryptophan. Instead of mg, the daily requirements are known as_ niacin equivalents. Pregnancy and lactation in women impose an additional metabolic burden and increase the niacin requirement.

The rich natural sources of niacin include liver, yeast, whole grains, cereals, pulses like beans and peanuts. Milk, fish, eggs and vegetables are moderate sources. The essential amino acid tryptophan can serve as a precursor for the synthesis of nicotinamide coenzymes. On an average, 1 g of a good quality protein containing about 60 mg of tryptophan is equivalent to 1 mg of niacin (conversion ratio 60:1) for the synthesis of nicotinamide coenzymes. Tryptophan has many other essential and important functions in the body, hence dietary tryptophan cannot totally replace niacin. Increased conversion of tryptophan to niacin has been reported in low protein diet and starvation. Tryptophan can replace niacin to an extent of 10% for the synthesis of coenzymes. Therefore, both niacin and tryptophan have to be invariably provided in the diet.

Niacin deficiency results in a condition called pellagra (Italian: rough skin). This disease involves skin, gastrointestinal tract and central nervous system. The symptoms of pellagra are commonly referred to as three Ds. The disease also progresses in that order dermatitis, diarrhea, dementia, and if not treated may rarely lead to death (4th D). Pellagra is frequently observed in Hartnup's disease (See p-173).
Dermatitis (inflammation of skin) is usually found in the areas of the skin exposed to sunlight (neck, dorsal part of feet, ankle and parts of face). Diarrhea may be in the form of loose stools, often with blood and mucus. Prolonged diarrhea leads to weight loss. Dementia is associated with degeneration of nervous tissue. The symptoms of dementia include anxiety, irritability, poor memory, insomnia (sleeplessness) etc.
Pellagra is mostly seen among people whose staple diet is corn or maize. Niacin present in maize is unavailable to the body as it is in bound form, and tryptophan content is low in maize.

Administration of niacin in pharmacological doses (2-4 g/day, 200 times the RDA) results in a number of biochemical effects in the body, not related to its function as a vitamin. Most of the effects are believed to be due to the influence of niacin on cyclic AMP levels.

(a) Glyceraldehyde 3-phosphate dehydrogenase
(b) Lactate dehydrogenase
(c) Pyruvate dehydrogenase complex
(d) o-Ketoglutarate dehydrogenase complex

(e) B-Hydroxy acyl CoA dehydrogenase
(f) B-Hydroxybutyrate dehydrogenase
(g) Alcohol dehydrogenase
(h) Branched chain o-keto acid dehydrogenase
(i) Tyramine dehydrogenase

(a) 3-Ketoacyl reductase
(b) HMG CoA reductase
(c) Squalene epoxidase
(d) Cholesterol 7o-hydroxylase
(e) Phenylalanine hydroxylase
(f) Dihydrofolate reductase
Reaction
Glyceraldehyde 3-phosphate
——
>
1,
3-Bisphosphoglycerate
Pyruvate
——>
Lactate
Pyruvate
—
Acetyl
CoA
a.-Ketoglutarate ——> Succinyl CoA
B-Hydroxy
acyl
CoA
——>
B-Keto acyl CoA
B-Hydroxybutyrate
——>
Acetoacetate
Alcohol
——>
Acetaldehyde
a-Keto acids of branched chain amino acids
(Leu,
lle,
Val)
t—>
Corresponding acyl CoA thioesters
Tyramine
——>
p-Hydroxyphenyl acetate
Glutamate
——
o-Ketoglutarate
+
NH,
Isocitrate
——>
Oxalosuccinate
Glucose 6-phosphate
——>
6-Phosphogluconolactone
Malate
——>
Pyruvate
3-Ketoacyl enzyme
——>
3-Hydroxy acyl enzyme
HMG CoA
——>
Mevalonate
Squalene
——>
Squalene oxide
——>
7a-Hydroxy cholesterol
Phenylalanine
——>
Tyrosine
Folic acid
——>
Tetrahydrofolic acid.
1. Niacin inhibits lipolysis in
the
adipose
tissue and decreases the circulatory free fatty
acids.
2. Triacylglycerol synthesis in the liver is
3. The
serum
levels
of
low
density
lipoproteins
(LDL),
very
low
density
(VLDL),
and

are
lowered.
Hence
niacin
is
used
in
the
treatment
of hyperlipoproteinemia type II b (elevation of
LDL
and
VLDL).
Although megadoses of niacin are useful for
the treatment of hyperlipidemia, there are certain
harmful side effects also.
1. Glycogen and fat reserves of skeletal and
cardiac
muscle are depleted.
2. There is a tendency for the increased levels
of glucose and uric acid in the circulation.

Pyridoxal phosphate (PLP), the coenzyme of vitamin Bg is found attached to the ¢-amino group of lysine in the enzyme. PLP is closely associated with the metabolism of amino acids. The synthesis of certain specialized products such as_ serotonin, histamine, niacin coenzymes from the amino acids is dependent on pyridoxine. Pyridoxal phosphate participates in reactions like transamination, decarboxylation, deamination, transsulfuration, condensation etc.
1. Transamination : Pyridoxal phosphate is involved in the transamination reaction (by transaminase) converteing amino acids to keto acids. The keto acids enter the citric acid cycle and get oxidized to generate energy. Thus Bg is an energy releasing vitamin. It integrates carbohydrate and amino acid metabolisms (Fig.7.24).
During the course of transamination, PLP interacts with amino acid to form a Schiff base (Fig.7.25). The amino group is handed over to PLP to form pyridoxamine phosphate and the keto acid is liberated.
3. Pyridoxal phosphate is required for the synthesis of 5-amino levulinic acid, the precursor for heme synthesis.
4. The synthesis of niacin coenzymes (NAD* and NADP*) from tryptophan is dependent on PLP. The enzyme kynureninase requires PLP. |n Be deficiency, 3-hydroxy anthranilic acid is diverted to xanthurenic acid (Fig.7.26). Increased excretion of xanthurenate in urine is an indication of Bg deficiency.
5. PLP plays an important role in the metabolism of sulfur containing amino acids (Fig.7.27). Transsulfuration (transfer of sulfur) from homocysteine to serine occurs in the synthesis of cysteine. This is carried out by a PLP dependent cystathionine synthase. Taurine, a decarboxylated (PLP dependent) product of cysteine, is involved in the conjugation of bile acids.
6.
Deamination of
hydroxyl group
containing amino acids requires PLP.
Serine
Dehydratase
PLP
Pyruvate
+
NH3
Threonine
7.
Serine

PLP
is synthesized
a-Ketobutyrate
NH3
from glycine
by
a
PLP
dependent enzyme
hydroxymethyltransferase.
8. The enzyme glycogen phosphorylase (that cleaves glycogen to glucose 1-phosphate) contains PLP, covalently bound to lysine residue. Phosphorylase structure is stabilized by PLP, for effective enzymatic function.
9. PLP is needed for the absorption of amino acids from the intestine.
10. Adequate intake of Bg is useful to prevent hyperoxaluria and urinary stone formation.

The requirement of pyridoxine for an adult is 2-2.2 mg/day. During pregnancy, lactation and old age, an intake of 2.5 mg/day is recommended. As is observed from the coenzyme function, pyridoxine is closely associated with protein (amino acid) metabolism. The daily requirements of Bg are calculated on the assumption that the intake of protein is <100 g/day.

Animal sources such as egg yolk, fish, milk, meat are rich in Bg. Wheat, corn, cabbage, roots and tubers are good vegetable sources.
Fig. 7.27 : Role of pyridoxine in the metabolism of sulfur amino acids (PLP-Pyridoxal phosphate).
+

Pyridoxine deficiency is associated with neurological symptoms such as depression, irritability, nervousness and mental confusion. Convulsions and peripheral neuropathy are observed in severe deficiency. These symptoms are related to the decreased synthesis of biogenic amines (serotonin, GABA, norepinephrine and epinephrine). In children, Bg deficiency with a drastically reduced GABA production results in convulsions (epilepsy).
Decrease in hemoglobin levels, associated with hypochromic microcytic anaemia, is seen in Bg deficieny. This is due to a reduction in heme production.
The synthesis of niacin coenzymes (NADt and NADP*) from tryptophan is impaired. Xanthurenic acid, produced in high quantities is excreted in urine, which serves as a reliable index (particularly after tryptophan load test) for Bg deficiency.
Dietary deficiency of pyridoxine is rather rare and is mostly observed in women taking oral contraceptives, alcoholics and infants.

Isoniazid (isonicotinic acid hydrazide, INH) is a drug frequently used for the treatment of tuberculosis. \t combines with — pyridoxal phosphate to form inactive hydrazone derivatives which inhibit PLP dependent enzymes. Tuberculosis patients, on long term use of isoniazid, develop peripheral neuropathy which responds to Bg therapy.
The drug penicillamine (f-dimethy! cysteine) is used in the treatment of patients with rheumatoid arthritis, Wéilson's disease and cystinuria. This drug also reacts with PLP to form inactive thiazolidine derivative.
Administration of drugs namely isoniazid and penicillamine should be accompanied by pyridoxine supplementation to avoid Bg deficiency.

Isoniazid, deoxypyridoxine and methoxy pyridoxine are the antagonists of vitamin Bg.

Excess use of vitamin Bg (2.5 g/day) in the women of premenstrual syndrome is associated with sensory neuropathy. Some workers have suggested that vitamin Bg more than 200 mg/day may cause neurological damage.
Biotin (formerly known as anti-egg white injury factor, vitamin Bz or vitamin H) is a sulfur containing B-complex vitamin. It directly participates as a coenzyme in the carboxylation reactions.
Boas (1927) observed that rats fed huge quantity of raw egg white developed dermatitis and nervous manifestations, besides retardation in growth. She however, found that feeding cooked egg did not produce any of these symptoms. It was later shown that the egg white injury in rats and chicks was due to the presence of an anti-vitamin in egg white. The egg-white injury factor was identified as a glycoprotein— avidin and biotin was called as anti-egg white injury factor.

Biotin is a heterocyclic sulfur containing monocarboxylic acid. The structure is formed by fusion of imidazole and thiophene rings with a valeric acid side chain (Fig.7.28). Biotin is covalently bound to €-amino group of lysine to form biocytin in the enzymes. Biocytin may be regarded as the coenzyme of biotin.
Fig. 7.28 : Structure of biotin with binding sites.

Biotin serves as a carrier of CO ) in carboxylation reactions. The reaction catalysed by pyruvate carboxylase, converting pyruvate to oxaloacetate has been investigated in detail. This enzyme has biotin bound to the apoenzyme linked to the e-amino group of lysine, forming the active enzyme (holoenzyme). Biotin-enzyme reacts with CO, in presence of ATP (provides energy) to form a_ carboxybiotin-enzyme complex. This high energy complex hands over the CO, to pyruvate (carboxylation reaction) to produce oxaloacetate (Fig.7.29).
As a coenzyme, biotin is involved in various metabolic reactions.
1. Gluconeogenesis and citric acid cycle : The conversion of pyruvate to oxaloacetate by biotin dependent pyruvate carboxylase (described above) is essential for the synthesis of glucose from many non-carbohydrate sources. Oxaloacetate so formed is also required for the continuous operation of citric acid cycle.
2. Fatty acid synthesis : Acetyl CoA is the starting material for the synthesis of fatty acids. The very first step in fatty acid synthesis is a carboxylation reaction.
Acetyl CoA

Acetyl CoA carboxylase
Malonyl CoA
3. Propionyl CoA is produced in the metabolism of certain amino acids (valine, isoleucine, threonine etc.) and degradation of odd chain fatty acids. Its further metabolism is dependent on biotin.
Biotin
Propionyl! CoA
tJY—_—+
Propionyl CoA carboxylase
Methylmalonyl CoA
4.
In the
leucine, the
following reaction is dependent on biotin.
Biotin
B-Methylcrotonyl CoA
B-Methylcrotonyl
CoA
B-Methyl glutaconyl CoA
[Note : It was once believed that all the carboxylation reactions in the biological system are dependent on biotin. This was later proved to be wrong. There are a few carboxylation reactions which do not require biotin e.g. formation of carbamoyl phosphate in urea cycle, incorporation of CO, in purine synthesis.]

A daily intake of about 100-300 mg is recommended for adults. In fact, biotin is normally synthesized by the intestinal bacteria. However, to what extent the synthesized biotin contributes to the body requirements is not clearly known.

Biotin is widely distributed in both animal and plant foods. The rich sources are liver, kidney, egg yolk, milk, tomatoes, grains etc.

The symptoms of biotin deficiency include anemia, loss of appetite, nausea, dermatitis,
Fig. 7.29 : Role of biotin in the carboxylation reaction, catalysed by the enzyme pyruvate carboxylase (Enz-Enzyme).
glossitis etc. Biotin deficiency may also result. in depression, hallucinations, muscle pain and dermatitis.

it is
well distributed
in foods
and also
supplied by
deficiency the
intestinal may
bacteria.
however,
The be
the following
two causes.
1. Destruction of intestinal flora due to prolonged use of drugs such as sulfonamides.
2. High consumption of raw eggs. The raw egg white contains a glycoprotein— avidin, which tightly binds with biotin and blocks its absorption from the intestine. An intake of about 20 raw eggs per day is needed to produce biotin deficiency symptoms in humans. Consumption of an occasional raw egg will not result in deficiency.

Pantothenic acid (Greek pantos— everywhere), formerly known as chick anti-dermatitis factor (or filtrate factor) is widely distributed in nature. It's metabolic role as coenzyme A is also widespread.

Pantothenic acid
components, pantoic
held together
coenzyme consists
acid by
A
of two
and f-alanine,
a
peptide linkage.
from pantothenate
of reactions
(Fig.7.30).
phosphorylated
Synthesis occurs
Fig. 7.30 : (A) Summary of the synthesis of coenzyme A from pantothenic acid (B) Structure of coenzyme A.
of in
a
series
Pantothenate to
which
Decarboxylation, followed
moiety and
a
phosphate in
coenzyme cysteine
is by
addition
(each
A.
The structure
is first
added.
of from
AMP
ATP)
of results
coenzyme
A
consists of pantothenic acid, 1 = joined to. E -mercaptoethanolamine (thioethanolamine), 1 = at. one end. On the other side, pantothenic acid, 1 = is. held by a phosphate bridge to adenylic acid., 1 = The. adenylic acid is made up of adenine,, 1 = and a

The functions of pantothenic acid are exerted through coenzyme A or CoA (A for acetylation). Coenzyme A is a central molecule involved in all the metabolisms (carbohydrate, lipid and protein). It plays a unique role in integrating various metabolic pathways. More than 70 enzymes that depend on coenzyme A are known.
Coenzyme A has a terminal thiol or sulfhydryl group (—SH) which is the reactive site, hence CoA-SH is also used. Acyl groups (free fatty acids) are linked to coenzyme A by a thioester bond, to give acyl CoA. When bound to acetyl unit, it is called acetyl CoA. With succinate, succinyl CoA is formed. There are many other compounds bound to coenzyme A.
Coenzyme
A
may be
regarded as
a
coenzyme of metabolic integration, since acetyl CoA is a central molecule for a wide variety of biochemical reactions, as_ illustrated in Fig.7.32.
Succiny] CoA is also involved in many reactions, including the synthesis of porphyrins of heme.
Besides the various functions through coenzyme A, pantothenic acid itself is a component of fatty acid synthase complex and is involved in the formation of fatty acids.

The requirement of pantothenic acid for humans is not clearly known. A daily intake of about 5-10 mg is advised for adults.

Pantothenic acid is one of the most widely distributed vitamins found in plants and animals. The rich sources are egg, liver, meat, yeast, milk etc.

It is a surprise to biochemists that despite the involvement of pantothenic acid (as coenzyme A) in a great number of metabolic reactions,

Folic acid
pteridine glutamic
ring, consists
p-amino acid
of three
benzoic
(1
to
7
components—
acid residues).
has one
glutamic acid
residue
(PABA)
and
Folic and
acid mostly
is known
as

The active form of folic acid is tetrahydrofolate (THF or FH,). \t is synthesized from folic acid by the enzyme dihydrofolate reductase. The reducing equivalents are provided by 2 moles of NADPH. The hydrogen atoms are present at positions 5, 6, 7 and 8 of THF (Fig.7.33).

Most of the dietary folic acid found as polyglutamate with 3-7 glutamate residues (held by peptide bonds) is not absorbed in the intestine. The enzyme folate conjugase present in duodenum and jejunum splits the glutamate residues. Only the monoglutamate of folic acid is absorbed from the intestine. However, inside the cells, tetrahydrofolates are found as polyglutamates (with 5-6 amino acid residues) derivatives, which appear to be biologically most potent. As polyglutamate, folic acid is stored to some extent in the liver. The body can store 10-12 mg of folic acid that will usually last for 2-3. months. In the circulation, N®5-methyl tetrahydrofolate is abundantly present.

Tetrahydrofolate (THF or FH4), the coenzyme of folic acid, is actively involved in the one carbon metabolism. THF serves as an acceptor or donor of one carbon units (formyl, methyl etc.) in a variety of reactions involving amino acid and nucleotide metabolism.
The one carbon units bind with THF at position N° or N!° or on both N> and N!° of pteroyl structure. The attachment of formyl (—CHO) at position 5 of THF gives N5-formyl tetrahydrofolate which is commonly known as folinic acid or citrovorum factor. The other commonly found one carbon moieties and their binding with THF are given below.
THF-1 carbon derivative, 1 = R group (one carbon unit). N 5 -Formyl THF, 1 = CHO. N 10 -Formyl THF, 1 = CHO. N 5 -Formimino THF, 1 = CH NH. N 5 , N 10 -Methenyl THF, 1 = CH. N 5 , N 10 -Methylene THF, 1 = CH 2. N 5 - Methyl THF, 1 = CH 3
metabolism are summarized in Fig.7.34.
The interrelationship between the various 1-carbon THF derivatives along with their involvement in the synthesis of different compounds is given in Fig.15.32 (Chapter 15). Many important compounds are synthesized in one carbon metabolism.
- 1. Purines (carbon 2, 8) which are incorpora-
ted into DNA and RNA.
- 2. Pyrimidine — nucleotide-deoxythymidylic
acid (dTMP), involved in the synthesis of DNA.
- 3. Glycine, serine, ethanolamine and choline
are produced.
4.
N-Formylmethionine, the
initiator of
protein biosynthesis is formed.

Tetrahydrofolate is mostly trapped as N°-methyl THF in which form it is present in the circulation. Vitamin By, is needed for the conversion of N5-methyl THF to THF, in a reaction wherein homocysteine is converted to methionine. This step is essential for the liberation of free THF and for its repeated use in one carbon metabolism. In By, deficiency, conversion of N-methyl THF to THF is blocked (more details given under vitamin By»).

The daily requirement of folic acid is around 200 ug. In the women, higher intakes are recommended during pregnancy (400 ug/day) and lactation (300 g/day).

Folic acid is widely distributed in nature. The rich sources are green leafy vegetables, whole grains, cereals, liver, kidney, yeast and eggs. Milk is rather a poor source of folic acid.

Folic acid deficiency is probably the most common vitamin deficiency, observed primarily in the pregnant women, in both developed (including USA) and developing countries (including India). The pregnant women, lactating women, women on oral contraceptives, and alcoholics are also susceptible to folate
deficiency. The folic acid deficiency may be due to (one or more causes) inadequate dietary intake, defective _ absorption, use of anticonvulsant drugs (phenobarbitone, dilantin, phenyltoin), and increased demand.
In folic acid deficiency, decreased production of purines and dTMP is observed which impairs DNA synthesis. Due to a block in DNA synthesis, the maturation of erythrocytes is slowed down leading to macrocytic RBC. The rapidly dividing cells of bone marrow are seriously affected. The macrocytic anemia (abnormally large RBC) associated with megaloblastic changes in bone marrow is a characteristic feature of folate deficiency.
Folic acid deficiency in pregnant women may cause neural defects in the fetus. Hence high doses of folic acid are recommended in pregnancy to prevent birth defects.
Folic acid is associated with the metabolism of histidine. Formiminoglutamate (FIGLU), formed in histidine metabolism transfers the one carbon fragment, formimino group (—CH=NH) to tetrahydrofolate to produce N>-formimino THF. In case of folic acid deficiency, FIGLU accumulates and is excreted in urine. Histidine load test utilizing the excretion of FIGLU in urine is used to assess folic acid deficiency.

Elevated plasma levels of homocysteine are associated with increased risk of atherosclerosis, thrombosis and coronary heart disease. Hyperhomocysteinemia is mostly due to functional folate deficiency caused by impairment to form methyl-tetrahydrofolate by the enzyme methylene tetrahydrofolate reductase (See Fig.7.39). This results in a failure to convert homocysteine to methionine. Folic acid supplementation reduces hyperhomocysteinemia, and thereby the risk for various health complications.

Aminopterin and amethopterin (also called as methotrexate) are structural analogues of folic acid. They competitively inhibit dihydrofolate reductase and block the formation of THF. The biosynthesis of purines, thymine nucleotides and hence DNA is impaired. This results in the blockage of cell proliferation. Aminopterin and methotrexate are successfully used in the treatment of many cancers, including leukemia.
Trimethoprim (a component of the drug septran or bactrim) and pyrimethamine (antimalarial drug) are structurally related to folic acid. They inhibit dihydrofolate reductase, and the formation of THF. Trimethoprim is used to treat bacterial infections of sore throat, urinary tract, gastrointestinal tract etc.
Sulfonamides are structural analogues of PABA. They competitively inhibit the enzyme (dihydropteroate synthase) responsible for the incorporation of PABA into pteridine to produce folic acid. For this reason, sulfonamides are used as antibacterial drugs. Sulfonamides, have no effect on human body, since folic acid is not synthesized and supplied through the diet.

Vitamin Bj, is also known as anti-pernicious anemia vitamin. It is a unique vitamin, synthesized by only microorganisms and not by animals and plants. It was the last vitamin to be discovered.

Vitamin B,, is the only vitamin with a complex structure. The empirical formula of vitamin By (cyanocobalamin) is Cg3HgoN14 Oj,4PCo. The structure of vitamin B; consists of a corrin ring with a central cobalt atom. The corrin ring is almost similar to the tetrapyrrole ring structure found in other porphyrin compounds e.g. heme (with Fe) and chlorophyll (with Mg).
The corrin ring has four pyrrole units, just like a porphyrin. Two of the pyrrole units (A and D) are directly bound to each other whereas the other two (B and C) are held by methene bridges. The groups namely methyl, acetamide and
Fig. 7.35 : Structure of vitamin B,, (cyanocobalamin).
propionamide are the substituents on the pyrrole rings. Vitamin By) has cobalt atom in a coordination state of six. Cobalt present at the centre of the corrin ring is bonded to the four pyrrole nitrogens. Cobalt also holds (below the corrin plane) dimethylbenzimidazole (DMB) containing ribose 5-phosphate and aminoisopropanol. A nitrogen atom of dimethylbenzimidazole is linked to cobalt. The amide group of aminoisopropanol binds with D ring of corrin. The cobalt atom also possesses a sixth substituent group located above the plane of corrin ring (Fig.7.35). The substituent group may be one of the following
1. Cyanide (predominant) in cyanocobalamin (By 2a)
2. Hydroxyl in hydroxycobalamin (By 2p)
3. Nitrite in nitrocobalamin (Bj ,).
There are two coenzyme forms of vitamin By»
(Fig.7.36).
(a)
(b)
5’-Deoxyadenosyl cobalamin,
cyanide replaced
by
5’
deoxyadenosine an unusal carbon cobalt bond.
Methylcobalamin in
which cyanide
is forming
is replaced by methyl group.

The vitamin By is present in the diet in a bound form to proteins. By,» is liberated by the enzymes (acid hydrolases) in the stomach. The dietary source of B is known as extrinsic factor of Castle. The stomach secretes a special protein called intrinsic factor (IF). \t is a glycoprotein (8-15% carbohydrate) with a molecular weight
Fig. 7.36 : Coenzyme derivatives of vitamin B,. (Note : Corrin ring represented diagrammatically is identical in all; DMB-Dimethylbenzimidazole).

around 50,000. Intrinsic factor is resistant to proteolytic digestive enzymes. IF generally forms a dimer, binds strongly with 1 or 2 moles of vitamin By, . This binding protects vitamin By against its uptake and use by bacteria.
Methylcobalamin which is in excess is taken up by the liver, converted to deoxyadenosy! B, and stored in this form. It is believed that liver can store about 4-5 mg, an amount sufficient to meet the body requirements of B;, for 4-6 years.
The cobalamin-IF complex travels through the gut. The complex binds to specific receptors on the surface of the mucosal cells of the ileum. The binding of the complex and entry of By, into the mucosal cells is mediated by Ca2* ions. In the mucosal cells, By, is converted to methylcobalamin (Fig.7.37). It is then transported in the circulation in a bound form to proteins namely transcobalamins (TC-L, TC-Il). Methylcobalamin is mostly bound to TC-I (90%) and to a lesser degree to TC-II (10%). It is believed that TC-I acts as a repository of By, while TC-Il mediates the tissue uptake of By».

About ten enzymes requiring vitamin By have been identified. Most of them are found in bacteria (glutamate mutase, ribonucleotide reductase etc.). There are only two reactions in mammals that are dependent on vitamin By.
1. Synthesis of methionine from homocysteine : Vitamin B,, as methylcobalamin is used in this reaction. This is an important reaction involving N-methyl tetrahydrofolate from which tetrahydrofolate is liberated (enzyme-homocysteine methyltransferase or
2. Isomerization of methymalonyl CoA to succinyl CoA : The degradation of odd chain fatty acids, certain amino acids (valine, isoleucine etc.) and pyrimidines (thymine and uracil) produce directly or through the mediation of propionyl CoA, an important compound methylmalonyl CoA. This is converted by the enzyme methylmalonyl CoA mutase to succinyl] CoA in the presence of B,, coenzyme, deoxyadenosy! cobalamin (Fig.7.38). This reaction involves hydrogen transfer and intramolecular rearrangement. In B; deficiency, methylmalonyl CoA accumulates and is excreted in urine as methylmalonic acid.

A daily intake of about 3 ug of vitamin By is adequate to meet the adult requirements. For children, 0.5-1.5 pug/day is recommended. During pregnancy and lactation, the requirement is 4 ug/day.

Foods of animal origin are the only sources for vitamin By. The rich sources are liver, kidney, milk, curd, eggs, fish, pork and chicken. Curd is a better source than milk, due to the synthesis of By by Lactobacillus.
Vitamin By) is synthesized only by microorganisms (anaerobic bacteria). Plants cannot synthesize, hence Bj is never found in plant foods. Animals obtain By; either by eating foods, derived from other animals or from the intestinal bacterial synthesis.

The most important disease associated with vitamin Bj deficiency is pernicious anemia. It is
From the foregoing discussion, it is clear that pernicious anemia is more a disease of the stomach than due to the deficiency of vitamin By.
Bj deficiency is also associated with neuronal degeneration and demyelination of nervous system. The symptoms _ include paresthesia (numbness and tingling) of fingers and toes. In advanced stages, confusion, loss of memory and even psychosis may be observed. The neurological symptoms of pernicious anemia are believed to be due to the accumulation of methylmalony! CoA that interferes in myelin sheath formation in two possible ways.
1. The biosynthesis of fatty acids, required for myelin formation, is imparied. This is because, methylmalonyl CoA acts as a competitive inhibitor of malonyl CoA in fatty acid synthesis.
2. Methylmalonyl CoA can __ substitute malonyl CoA in fatty acid synthesis, resulting in a new type of branched chain fatty acids. These fatty acids will disrupt the normal membrane structure.
The excretion of methylmalonic acid (elevated) in urine and estimation of serum By) level are used to assess B,> deficiency.

Vitamin By» is administered in therapeutic doses (100-1000 ug) intramuscularly. Folic acid administration can also reverse hematological abnormalities observed in By, deficiency. However, the neurological symptoms. persist. Therefore, a combined supplementation of By and folate is employed to treat the patients with megaloblastic anemias.

The
of
either
folate
or
vitamin
By
results
in
a
similar
type
of
anemia.
This
suggests
a
probable
biochemical
interrelation
between
these
two
vitamins.
There
is
only
one
metabolic
reaction
known,
common
to
folate
and
vitamin
By (Fig.7.39).
In vitamin By, deficiency, increased folate levels are observed in plasma. The activity of the enzyme homocysteine _ methyltransferase (methionine synthase) is low in By deficiency. As a result, the only major pathway for the conversion of N-methyl THF to tetrahydrofolate is blocked and body THF pool is reduced. Essentially, almost the entire body folate becomes trapped as N>-methyl THF. This is known as folate trap or methyl trap. |n this manner, Bj deficiency results in decreased folate coenzymes that leads to reduced nucleotide and DNA synthesis.
Although the tissue folate levels are adequate or high, there is a functional folate deficiency due to the lack of THF pool. The outcome is the development of megaloblastic anemia. Administration of the amino acid methionine has been shown to partially correct the symptoms of By» deficiency. This is due to the fact that the formation of N°-methyl THF is inhibited by S-adenosylmethionine. A combined therapy of vitamin B;5 and folic acid is generally employed to treat the patients with megaloblastic anemia.

Besides the vitamins described above, there are many other compounds present in foods as accessory factors. Earlier workers have described these factors sometime or the other, as essential to higher animals. However, their essential nature and requirement in humans has not been established. Although not essential in the diet, they perform many important functions in the body. Selected examples of such substances which may be regarded as vitamin like compounds are described here.
Choline is trimethylhydroxy ethanolamine.
ors
HgC—N*—CHp—CH,OH
CH3
Fig. 7.39 : Interrelationship between folic acid and vitamin B,,.
It can be synthesized in the body (from serine). It is also available from many dietary
sources (e.g. milk, eggs, liver, cereals etc.).

1. Choline, as a component of phospholipids (lecithins), is involved in membrane structure and lipid transport.
2. Choline prevents the accumulation of fat in liver (as lipotropic factor). \t promotes the synthesis of phospholipids and lipoproteins and the disposal of triacylglycerols from liver.
3. Due to the presence of three methyl groups (one carbon fragments), choline is actively involved in one carbon metabolism.
4. Choline is a precursor for the synthesis of acetylcholine which is required for transmission of nerve impulse.

As such, choline can be synthesized and reutilized in humans. This may however, be insufficient to meet the body needs. Some workers label choline as an essential dietary nutrient with RDA in the range of 400-500 mg/day.

Inositol is hexahydroxy-cyclohexane. |t is also known as myo-inositol or meso-inositol.

1. Inositol is required for the synthesis of phosphatidylinositol (lipositol) which is a constituent of cell membrane.
2. It acts as a lipotropic factor (along with choline) and prevents the accumulation of fat in liver.
3. For some hormones, inositol] acts as a second messenger at the membrane level for the release of Ca2+ ions.
4. Inositol concentration in the heart muscle in high, the significance of which however, is
not known.
5. Phytin is hexaphosphate of inositol found
is plants. It prevents the absorption of iron and calcium from the intestine.

Lipoic containing
acid fatty
(thioctic acid)
is
a
acid
(6,8-dithiooctanoic sulfur
acid).
exists in
an oxidized
and reduced
form.
acid is fat as well as water soluble.
It
Lipoic
158
He0—CHe—GH~(CH,)
COOH
He0—CH—GH—(CH,)4—COOH
s
>
osH
SH
Lipoic acid (reduced)
Ss
Lipoic
acid
(oxidized)

Lipoic acid is involved in the decarboxylation reactions along with other vitamins (thiamine, niacin, riboflavin and pantothenic acid). The conversion of pyruvate to acetyl CoA (by pyruvate dehydrogenase) and o-ketoglutarate to succinyl CoA (by a-ketoglutarate dehydrogenase) requires lipoic acid.

lipoic acid) is gaining importance. Being fat and water soluble, it can comfortably reach various tissues. The therapeutic applications of lipoic acid are related to its antioxidant property (regarded as universal antioxidant), some of them are listed
1 Reduces the free radicals in brain that otherwise contribute to Alzheimer's disease and multiple sclerosis.
1
Lipoic acid
—
stimulates production
_
of glutathione
(GSH), besides
helping in
the recycle
of vitamins
E
and
C.
- 1 Reduces insulin resistance, and brings down plasma low density lipoproteins.
In recent years, administration of high doses mg/day) of lipoic acid (or dihydro-
- 1 May be useful in the prevention of stroke and myocardial infarction.

+ Distinct deficiency conditions of certain B-complex vitamins are known
Thiamine
— Beri-beri
Niacin
— Pellagra
Folic
acid
— Macrocytic anemia
Riboflavin — Cheilosis, glossitis
Pyridoxine — Peripheral neuropathy
Cobalamin — Pernicious anemia
+ B-complex vitamin deficiencies are usually multiple rather than individual with
overlapping symptoms.
+ A combined therapy of vitamin By9 and folic acid is commonly employed to treat the patients of megaloblastic anemias.
+ Megadoses of niacin are useful in the treatment of hyperlipidemia.
+ Long term use of isoniazid for the treatment of tuberculosis causes Bg deficiency.
+ Folic acid supplementation reduces elevated plasma homocysteine level which is associated with atherosclerosis and thrombosis.
+ Sulfonamides serve as antibacterial drugs by inhibiting the incorporation of PABA to produce folic acid.
+
Aminopterin
and
amethopterin,
the
structural
analogues
of
folic
acid,
are
employed
in
the treatment of cancers.
+ Lipoic acid is therapeutically useful as an antioxidant to prevent stroke, myocardial infarction, etc.

Para aminobenzoic acid (PABA) is a structural constituent of folic acid. PABA may be regarded as a vitamin in another vitamin (folic acid)
The deficiency of PABA was first found to be associated with failure of lactation and greying of black hair in rats. The specific functions of PABA in humans, except that it is a component of folic acid, have not been identified.
PABA is synthesized by the bacteria and is essential for their growth. The sulfa drug sulfonilamide (p-amino benzene sulfanilamide) is a structural analogue of PABA. Sulfonilamide competes with PABA and acts as a bacteriostatic agent. Ingestion of large doses of PABA will compete with the action of drugs and therefore should be avoided during sulfonilamide therapy (trade name—sulfonamides).

Szent-Gyorgi and his associates (1936) observed that flavonoids, isolated from lemon peel (known as citrin) were responsible for maintenance of normal capillary permeability. The term vitamin P (P for permeability) was used to this group of substances. However, they are commonly known as bioflavonoids.

Antivitamin/vitamin antgonists, 1 = Vitamin. Dicumarol Warfarin, 1 = Vitamin K. Thiaminase Pyrithiamine Oxythiamine, 1 = Thiamine. Galactoflavin, 1 = Riboflavin. Isoniazid Deoxypyridoxine, 1 = Niacin. Avidin Desthiobiotin, 1 = Biotin. Aminopterin Methotrexate Trimethoprim, 1 = Folic acid. Sulfonilamide, 1 = Para-aminobenzoic acid
Bioflavonoids act as antioxidants and protect ascorbic acid from being destroyed. It is suggested that this antioxidant property may be responsible for maintenance of capillary permeability. Bioflavonoids have been used to correct the vascular abnormality in humans.
Bioflavonoids are found in peel and pulp of citrus fruits, tobacco leaves and many vegetables. The requirement of these compounds in humans has not been established.

Antivitamins are
block)
the antagonistic
to action
structural of
similarities of
antivitamins vitamins.
They with
vitamins.
causes vitamin
(oppose and
usually have
Administration deficiencies.
The common
antivitamins are
discussed as
antagonists for each vitamin, and are given in Table 7.4.

in pernicious anemia.. 13., 1 = Vitamin C (ascorbic acid) is involved in the hydroxylation of proline and lysine in the formation of collagen. Scurvy is caused by ascorbic acid deficiency. Therapeutic use of megadoses of vitamin C, to cure everything from common cold to cancer, has become controversial.. 14., 1 = Certain vitamin like compounds (choline, inositol, PABA, lipoic acid) participate in many biochemical reactions.
Chapter
7
:
VITAMINS

1. Classify vitamins and briefly discuss their functions and deficiency disorders.
2. Describe the chemistry, biochemical functions, daily requirements, sources and deficiency manifestations of vitamin A.
3. Write an account of folic acid involvement in one carbon metabolism.
5. Write briefly about the coenzymes involved in oxidation-reduction reactions.
. Discuss the biochemical functions of vitamin C. Add a note on the therapeutic use of megadoses of this vitamin.

(a) Vitamin D is a hormone-justify, (b) Thiamine pyrophosphate, (c) Coenzymes of niacin,
(d) Pyridoxal phosphate in transamination, (e) Folate trap, (f) Tocopherol, (g) Vitamin K in
carboxylation, (h) Biocytin, (i) Choline, (j) Pernicious anemia.

1. The A in coenzyme A stands for.
BR . Anti-tuberculosis
WN . The vitamin containing isoalloxazine ring
CODON
DY . The egg injury factor present in raw egg white.
. The vitamin that is regarded as a vitamin in search of a disease.
drug, isonicotinic acid hydrazide (INH) leads to the deficiency of
vitamin
. The ‘burning feet syndrome’ in man is associated with the deficiency of.
. The vitamin that is synthesized by only microorganisms.
. The three Ds in pellagra stand for,
,
and
. The fat soluble vitamin required for carboxylation reaction
. FIGLU
(formimino
glutamic
acid) is
excreted
in urine
in
the
of
vitamin

11. Which one of the vitamin A functions as a steroid hormone (a) Retinal (b) Retinol (c) Provitamin A (d) B-Carotene.
12. The functionally active form of vitamin D is
13. The metabolite excreted in urine in thiamine deficiency
14. The coenzyme directly concerned with the synthesis of biogenic amines
15. Folic acid antagonist(s) used in the treatment of cancer
(a) Cholecalciferol (b) Ergocalciferol (c) Dehydrocholesterol (d) Calcitriol.
(a) Pyruvate (b) Glucose (c) Xanthurenic acid (d) FIGLU.
(a) TPP (b) NADP* (c) Biotin (d) Pyridoxal phosphate.
(a) Methotrexate (b) Trimethoprim (c) Sulfonamide (d) All the three.

Chapter
F ood is the basic and essential requirement of man for his very existence. The food we eat consists of carbohydrates, proteins, lipids, vitamins and minerals. The bulk of the food ingested is mostly in a complex macromolecular form which cannot, as such, be absorbed by the body.
Digestion is a process involving the hydrolysis of large and complex organic molecules of foodstuffs into smaller and preferably water-soluble molecules which can be easily absorbed by the gastrointestinal tract for utilization by the organism. Digestion of macromolecules also promotes the absorption of fat soluble vitamins and certain minerals.
Cooking of the food, and mastication (in the mouth) significantly improve the digestibility of foodstuffs by the enzymes.

Digestion as well as absorption are complicated processes that occur in the gastrointestinal tract (GIT) involving many organs. The

Organs of gastrointestinal tract with their ENaC eC MCC e cd
Mouth, Major function(s) = Production of saliva containing D -amylase; partial digestion of polysaccharides. Stomach, Major function(s) = Elaboration of gastric juice with HCl and proteases; partial digestion of proteins. Pancreas, Major function(s) = Release of NaHCO 3 and many enzymes required for intestinal digestion. Liver, Major function(s) = Synthesis of bile acids. Gall bladder, Major function(s) = Storage of bile. Small intestine, Major function(s) = Final digestion of foodstuffs; absorption of digested products. Large intestine, Major function(s) = Mostly absorption of electrolytes; bacterial utilization of certain non-digested and/or unabsorbed foods
diagrammatic representation of GIT is depicted in Fig.8.1, and the essential organs with their respective major functions are given in Table 8.1. The digestive organs possess a large
Fig. 8.1 : Diagrammatic representation of gastrointestinal tract.
reserve capacity. For instance, pancreas secretes enzymes 5-10 fold higher than required for digestion of foods normally ingested.
The digestion and absorption of individual foods, namely carbohydrates, proteins, lipids and nucleic acids, is described here. The gastrointestinal hormones are discussed under hormones (Chapter 19).

The principal
dietary carbohydrates
are polysaccharides (starch, glycogen), disaccharides
(lactose, sucrose) and, to a minor extent, mono- saccharides (glucose, fructose). The structures of
carbohydrates are described in Chapter 2.

The digestion of carbohydrates occurs briefly in mouth and largely in the intestine. The polysaccharides get hydrated during heating which is essential for their efficient digestion. The hydrolysis of glycosidic bonds is carried out by a group of enzymes called glycosidases (Fig.8.2). These enzymes are specific to the bond, structure and configuration of monosaccharide units.
Digestion in the mouth : Carbohydrates are the only nutrients for which the digestion begins in the mouth to a significant extent. During the process of mastication, salivary a-amylase (ptyalin) acts on starch randomly and cleaves o1,4-glycosidic bonds. The products formed include a-limit dextrins, (containing about 8 glucose units with one or more a-1,6-glycosidic bonds) maltotriose and maltose.
Carbohydrates not digested in the stomach : The enzyme salivary amylase is inactivated by high acidity (low pH) in the stomach. Consequently, the ongoing degradation of starch is stopped.

: dietary contents of the stomach, on reaching small intestine, are neutralized by bicarbonate produced by pancreas. The pancreatic a-amylase acts on starch and continues the digestion process. Amylase specifically acts on a-1,4-glycosidic bonds and not on a-1,6-bonds.
Fig. 8.2 : Hydrolysis of a glycosidic bond.
Fig. 8.3 : Degradation of amylopectin by salivary or pancreatic o-amylase.
contrast, lactase
(B-galactosidase)
limiting, and,
consequently, the
is the
rate- utilization
milk sugar
(lactose)
is limited
in of
humans.

The principal monosaccharides produced by the digestion of carbohydrates are glucose, fructose and galactose. Of these, glucose accounts. for nearly 80% of the _ total monosaccharides. The absorption of sugars mostly takes place in the duodenum and upper jejunum of small intestine.
Fig. 8.4 : Overview of digestion of carbohydrates.
Fig. 8.5 : Transport of glucose across intestinal epithelium (Note : Transport of amino acids also occurs by a similar mechanism; replace glucose in figure by amino acid).
It is observed that hexoses are more rapidly absorbed than pentoses. Further, among the monosaccharides, galactose is most efficiently absorbed followed by glucose and fructose. Insulin has no effect on the absorption of sugars.

Different sugars possess different mechanisms for their absorption. Glucose is transported into the intestinal mucosal cells by a carrier mediated and energy requiring process (Fig.8.5).
rehydration fluid contains glucose and sodium. Intestinal absorption of sodium is facilitated by the presence of glucose.
Glucose and Na* share the same transport system (symport) which is referred to as sodiumdependent glucose transporter. The concentration of Nat is higher in the intestinal lumen compared to mucosal cells. Nat, therefore, moves into the cells along its concentration gradient and simultaneously glucose _ is transported into the intestinal cells. This is mediated by the same carrier system. Thus, Nat diffuses into the cell and it drags glucose along with it. The intestinal Nat gradient is the immediate energy source for glucose transport. This energy is indirectly supplied by ATP since the reentry of Na* (against the concentration gradient) into the intestinal lumen is an energyrequiring active process. The enzyme Na+-Kt ATPase is involved in the transport of Nat in exchange of K+ against the concentration gradient (for details see Chapter 33).
Oral rehydration therapy (ORT) : ORT is the most common treatment of diarrhea. The oral
The mechanism of absorption of galactose is similar to that of glucose. The inhibitor phlorizin blocks the Nat dependent transport of glucose and galactose.
Absorption of fructose : Fructose absorption is relatively simple. It does not require energy and is independent of Na* transport. Fructose is transported by facilitated diffusion mediated by a carrier. Inside the epithelial cell, most of the fructose is converted to glucose. The latter then enters the circulation.
Pentoses are absorbed by a process of simple diffusion.

The plant foods are rich in fibrous material which cannot be digested either by the human enzymes or intestinal bacteria. The fibers are chemically complex carbohydrates — which include cellulose, hemicellulose, pectins, lignin and gums. Fiber in nutrition is of special importance which is described under nutrition
(Chapter 23).

In general, humans possess an efficient system of carbohydrate digestion and absorption. Since only the monosaccharides are absorbed, any defect in the activities of disaccharidases results in the passage of undigested disaccharides into the large intestine. The disaccharides draw water from intestinal mucosa by osmosis and cause diarrhea. Further, bacterial action of these undigested carbohydrates leads to flatulence.
Disaccharidases are the intestinal brush border enzymes. Any alteration in the mucosa of the small intestine caused by severe diarrhea, malnutrition, intestinal diseases or drug therapy will lead to a temporary acquired deficiency of disaccharidases. The patients with such disorders are advised to restrict the consumption of sucrose and lactose.
Hereditary disorders with deficiency of individual disaccharidases in infants and children cause intolerance of specific disaccharides.

Lactose intolerance is the most common disorder of carbohydrate digestion in humans. This is due to a defect in the enzyme lactase (B-galactosidase). It is estimated that more than half of the world's adult population is affected by lactose intolerance. It is more commonly found in Africans (blacks) and Asians compared to Europeans.
Continued consumption of lactose by lactose intolerant individuals causes typical symptoms of flatulence (described later).
Lactose intolerance may be _ primary (congenital) or secondary (acquired). Acquired lactose intolerance may occur due to a sudden and high intake of milk-based diets. Lactase is an inducible enzyme. Therefore in acquired intolerance, if milk is withdrawn temporarily, diarrhea will be limited. For lactose intolerant people, consumption of curd is benefical, since lactobacilli present in curd contain the enzyme lactase. Further, yeast rich in lactase, can also be used for treatment of lactose intolerance.
The best treatment for lactose intolerance is elimination of lactose from the diet (i.e. severe restriction of milk and dairy products).

The deficiency of the enzyme sucrase causes intolerance to dietary sucrose. It is estimated that about 10% of Eskimos of Greenland and 2% of North Americans are affected by this disorder. The treatment is to remove sucrose from the diet.

Flatulence is characterized by increased intestinal motility, cramps and irritation. This occurs after ingestion of certain carbohydrates and is explained as follows.
The carbohydrates (di-, oligo-, and polysaccharides) not hydrolysed by a-amylase and other intestinal enzymes cannot be absorbed. Lactose is not hydrolysed in some individuals due to the deficiency of lactase. The di-, and oligosaccharides can be degraded by the bacteria present in ileum (lower part of small intestine) to liberate monosaccharides. The latter can be metabolized by the bacteria.
As the monosaccharides are utilized by the intestinal bacteria, gases such as hydrogen, methane and carbon dioxide—besides lactate and short chain fatty acids—are released. These compounds cause flatulence.
The occurrence of flatulence after the ingestion of leguminous seeds (bengal gram, redgram, beans, peas, soya bean) is very common. They contain several nondigestible oligosaccharides by human intestinal enzymes. These compounds are degraded and utilised by intestinal bacteria causing flatulence. —Raffinose_—_ containing galactose, glucose and fructose is a predominant oligosaccharide found in leguminous seeds.

The proteins subjected to digestion and absorption are obtained from two sources— dietary and endogenous.
The intake of dietary protein is in the range of 50-100 g/day. About 30-100 g/day of endogenous protein is derived form the digestive enzymes and worn out cells of the digestive tract. The digestion and absorption of proteins is very efficient in healthy humans, hence very little protein (about 5-10 g/day) is lost through feces. Dietary proteins are denatured on cooking and therefore, easily digested.
Proteins are degraded by a class of enzymes— namely hydrolases—which specifically cleave the peptide bonds, hence known as peptidases. They are divided into two groups
1. Endopeptidases (proteases) which attack the internal peptide bonds and release peptide fragments, e.g. pepsin, trypsin.
2. Exopeptidases which act on the peptide bonds of terminal amino acids. Exopeptidases are subdivided into carboxypeptidases (act on C-terminal amino acid) and aminopeptidases (act on N-terminal amino acid).
The proteolytic enzymes responsible for the digestion of proteins are produced by the stomach, the pancreas and the small intestine. Proteins are not digested in the mouth due to the absence of proteases in saliva.

Protein digestion begins in the stomach. Gastric juice produced by stomach contains hydrochloric acid and a protease proenzyme namely pepsinogen.
Hydrochloric acid : The pH of the stomach is <2 due to the presence of HCI, secreted by parietal (oxyntic) cells of gastric gland. This acid performs two important functions-denaturation of proteins and killing of certain microorganisms. The denatured proteins are more susceptible to proteases for digestion.
Pepsin : Pepsin (Greek : pepsis—digestion) is produced by the serous cells of the stomach as pepsinogen, the inactive zymogen or proenzyme. Pepsinogen is converted to active pepsin either by autocatalysis, brought about by other pepsin molecules or by gastric HCI (pH < 2). Removal of a fragment of polypeptide chain (44 amino acids in case of pig enzyme) makes the inactive enzyme active after attaining a proper conformation.
Pepsin is an acid-stable endopeptidase optimally active at a very low pH (2.0). The active site of the enzyme contains 2 carboxyl groups, which are maintained at low pH. Pepsin A is the most predominant gastric protease which preferentially cleaves peptide bonds formed by amino groups of phenylalanine or tyrosine or leucine.
Pepsin digestion of proteins results in peptides and a few amino acids which act as stimulants for the release of the hormone cholecystokinin from the duodenum.
Rennin : This enzyme, also called chymosin, is found in the stomach of infants and children. Rennin is involved in the curdling of milk. It converts milk protein casein to calcium paracaseinate which can be effectively digested by pepsin. Rennin is absent in adults.

The proteases of pancreatic juice are secreted as zymogens (proenzymes) and then converted to active forms. These processes are initiated by the release of two polypeptide hormones, namely cholecystokinin and secretin from the intestine (Fig.8.6).
Fig. 8.7 : Digestion of proteins—Specificity of enzyme cleavage of peptide bonds. (R; can be from any amino acid)
Release and activation of zymogens : The key enzyme for activation of zymogen is enteropeptidase (formerly enterokinase) produced by intestinal (mostly duodenal) mucosal epithelial cells. Enteropeptidase cleaves off a hexapeptide (6 amino acid fragment) from the N-terminal end of trypsinogen to produce trypsin, the active enzyme. Trypsin, in turn, activates other trypsinogen molecules (autocatalysis). Further, trypsin is the common activator of all other pancreatic zymogens to produce the active proteases, namely chymotrypsin, elastase and carboxypeptidases (A and B).
Specificity and action of pancreatic proteases : Trypsin, chymotrypsin and elastase are endopeptidases active at neutral pH. Gastric HCl is neutralized by pancreatic NaHCO3 in the intestine and this creates favourable pH for the action of proteases.
The substrate specificity of pancreatic proteases is depicted in Fig.8.7. For instance, trypsin cleaves the peptide bonds, the carbonyl (-—CO-—) group of which is contributed by arginine or lysine.
The amino acid serine is essential at the active centre to bring about the catalysis of all the three pancreatic proteases, hence these enzymes are referred to as serine proteases.
Action of carboxypeptidases : The pancreatic carboxypeptidases (A and B) are metalloenzymes that are dependent on Zn2+ for their catalytic activity, hence they are sometimes called

Zn-proteases. They also possess certain degree of substrate specificity in their action. For example, carboxypeptidase B acts on peptide bonds of COOH-terminal amino acid, the amino group of which is contributed by arginine or lysine (Fig.8.7).
The combined action of pancreatic proteases results in the formation of free amino acids and small peptides (2-8 amino acids).

The luminal surface of intestinal epithelial cells contains aminopeptidases and dipeptidases. Aminopeptidase is a non-specific exopeptidase which repeatedly cleaves N-terminal amino acids one by one to produce free amino acids and smaller peptides. The dipeptidases act on different dipeptides to liberate amino acids (Fig.8.8).

The free amino acids, dipeptides and to some extent tripeptides are absorbed by intestinal epithelial cells.
The diand tripeptides, after being absorbed are hydrolysed into free amino acids in the cytosol of epithelial cells. The activities of dipeptidases are high in these cells. Therefore, after a protein meal, only the free amino acids are found in the portal vein.
Fig. 8.8 : Overview of digestion of proteins.
The small intestine possesses an efficient system to absorb free amino acids. L-Amino acids are more rapidly absorbed than D-amino acids. The transport of L-amino acids occurs by an active process (against a concentration gradient), in contrast to D-amino acids which takes place by a simple diffusion.
Three ATP are utilized for the transport of a single amino acid by this cycle. For this reason, Meister cycle is not a common. transport system for amino acid. However, this cycle is operative for rapid transport of cysteine and glutamine.

Amino acids are primarily absorbed by a similar mechanism, as described for the transport of D-glucose. |t is basically a Nat-dependent active process linked with the transport of Nat. As the Nat diffuses along the concentration gradient, the amino acid also enters the intestinal cell. Both Na+ and amino acids share a common carrier and are transported together. The energy is supplied indirectly by ATP (for details, see absorption of monosaccharides and Fig.8.5).
A Nat-independent system of amino acid transport across intestinal cells has also been identified. The compound cytochalasin B inhibits Nat-independent transport system.
Another transport system to explain the mechanism of amino acid transfer across membrane in the intestine and kidney has been put forth. This is known as y-glutamyl cycle or Meister cycle and involves a_ tripeptide namely glutathione (y-glutamylcysteinylglycine).
The y-glutamyl cycle appears to be important for the metabolism of glutathione, since this tripeptide undergoes rapid turnover in the cells. There may be more physiological significance of y-glutamyl cycle.

For a short period, immediately after birth, the small intestine of infants can absorb intact proteins and polypeptides. The uptake of proteins occurs by a process known as endocytosis or pinocytosis. The macromolecules are ingested by formation of small vesicles of plasma membrane followed by their internalization. The direct absorption of intact proteins is very important for the transfer of maternal immunoglobulins (y-globulins) to the offspring.
The intact proteins and polypeptides are not absorbed by the adult intestine. However, the macromolecular absorption in certain individuals appears to be responsible for antibody formation that often causes food allergy.

Any defect in the pancreatic secretion impairs protein and fat digestion. This causes the loss of undigested protein in the feces along with the abnormal appearance of lipids. Deficiency of pancreatic secretion may be due to pancreatitis, cystic fibrosis or surgical removal of pancreas.

Hartnup is the name of the family in whom this disease was first discovered. It is characterized by the inability of intestinal and renal epithelial cells to absorb neutral amino acids (tryptophan, alanine, serine, threonine, valine). Tryptophan absorption is most severely affected with a result that typical symptoms of pellagra are observed in the patients of Hartnup's disease. This is related to the impairment in the conversion of tryptophan to NAD+ and NADP*t, the coenzymes of niacin. The treatment includes high protein diet and nicotinamide supplementation.
There is considerable variation in the daily consumption of lipids which mostly depends on the economic status and dietary habits. The intake of lipids is much less (often < 60 g/day) in poorer sections of the society, particularly in the less developed countries. In the developed countries, an adult ingests about 60-150 g of lipids per day. Of this, more than 90% is fat (triacylglycerol). The rest of the dietary lipid is made up of phospholipids, cholesterol, cholesteryl esters and free fatty acids.
Lipids are insoluble or sparingly soluble in aqueous solution. The digestive enzymes, however, are present in aqueous medium. This poses certain problems for the digestion and absorption of lipids. Fortunately, the digestive tract possesses specialized machinery to
1. Increase the surface area of lipids for digestion;
2. Solubilize absorption. the digested products for

The digestion of lipids is initiated in the stomach, catalysed by acid-stable lipase. This enzyme (also called lingual lipase) is believed to originate from the glands at the back of tongue. Stomach contains a separate gastric lipase which can degrade fat containing short chain fatty acids at neutral pH. The digestion of lipids in the stomach of an adult is almost negligible, since lipids are not emulsified and made ready for lipase action. Further, the low pH in the stomach is unfavourable for the action of gastric lipase.
In case of infants, the milk fat (with short chain fatty acids) can be hydrolysed by gastric lipase to some extent. This is because the stomach pH of infants is close to neutrality, ideal for gastric lipase action.

Emulsification is the phenomenon — of dispersion of lipids into smaller droplets due to reduction in the surface tension. This is accompanied by increase in the surface area of lipid droplets. Emulsification is essential for effective digestion of lipids, since the enzymes can act only on the surface of lipid droplets. More correctly, lipases act at the interfacial area between the aqueous and lipid phase.
The process of emulsification occurs by three complementary mechanisms
Detergent action of bile salts; Surfactant action of degraded lipids; wn
Mechanical mixing due to peristalsis.
1. Bile salts : The terms bile salts and bile acids are often used interchangeably. At physiological pH, the bile acids are mostly present as anions. Bile salts are the biological detergents synthesized from cholesterol in the liver. They are secreted with bile into the duodenum. Bile salts possess steroid nucleus, the side chain of which is attached to either glycine (glycocholic acid) or taurine (taurocholic acid). For the synthesis and other details on bile acids, refer cholesterol metabolism (Chapter 14). Bile salts are the most effective biological emulsifying

agents. They interact with lipid particles and the aqueous duodenal contents and convert them into smaller particles (emulsified droplets). Further, bile salts stabilize the smaller particles by preventing them from coalescing.
(mol. wt. 12,000). It is also secreted by pancreas as procolipase and converted to active form by trypsin. Colipase binds at the lipid-aqueous interface and helps to anchor and stabilize lipase.
2. Surfactant action of degraded lipids : The initial digestive products of lipids (catalysed by lipase) namely free fatty acids, monoacylglycerols promote emulsification. These compounds along with phospholipids are known as surfactants. They are characterized by possessing polar and non-polar — groups. Surfactants get absorbed to the water-lipid interfaces and increase the interfacial area of lipid droplets. Thus the initial action of the enzyme lipase helps in further digestion of lipids.
3. Besides the action of bile salts and surfactants, the mechanical mixing due to peristalsis also helps in the emulsification of lipids. The smaller lipid emulsion droplets are good substrates for digestion.

The pancreatic
enzymes responsible
for the
are primarily
dietary triacyl-
glycerols, cholesteryl
esters and
phospholipids.

Pancreatic lipase is the major enzyme that digests dietary fats. This enzyme preferentially cleaves fatty acids (particularly long chain, above 10 carbons) at position 1 and 3 of triacylglycerols. The products are 2-monoacylglycerol (formerly 2-monoglyceride) and free fatty acids (Fig.8.9). The activity of pancreatic lipase is inhibited by bile acids which are present along with the enzyme in the small intestine. This problem is overcome by a small protein, colipase
Lipid esterase is a less specific enzyme present in pancreatic juice. It acts on monoacylglycerols, cholesteryl esters, vitamin esters etc. to liberate free fatty acids. The presence of bile acids is essential for the activity of lipid esterase.

A specific enzyme namely pancreatic cholesterol esterase (cholesteryl ester hydrolase) cleaves cholesteryl esters to produce cholesterol and free fatty acids (Fig.8.10).

Phospholipases are enzymes responsible for the hydrolysis of phospholipids. Pancreatic juice is rich in phospholipase Ay which cleaves the fatty acid at the 2nd position of phospholipids. The products are a free fatty acid and a lysophospholipid. Phospholipase Ay is secreted as a zymogen which is activated in the intestine by the action of trypsin.
An overview of the digestion of lipids is given in Fig.8.11.

The former and present theories to explain the absorption of lipids are briefly described hereunder
1. Lipolytic theory put forth by Verzar : According to this, fats are completely hydrolysed to glycerol and free fatty acids. The latter are absorbed either as soaps or in association with bile salts.
Fig. 8.10 : Enzymatic cleavage of cholesteryl ester.
2. Partition theory proposed by Frazer : This theory states that the digestion of triacylglycerols is partial and not complete. The partially digested triacylglycerols, in association with bile salts, form emulsions. The lipids are taken up by the intestinal mucosal cells. As per this theory, resynthesis of lipids is not necessary for their entry into the circulation.

3. Bergstrom theory : This is a more recent and comprehensive theory to explain lipid absorption. It has almost replaced the earlier theories, and is briefly described hereunder The primary products obtained from the lipid digestion are 2-monoacylglycerol, free fatty acids and free cholesterol.
Besides their participation in digestion, bile salts are essential for absorption of lipids. Bile salts form mixed micelles with lipids. These micelles are smaller in size than the lipid emulsion droplets (utilized for digestion, described above). The micelles have a disk like shape with lipids (monoacylglycerol, fatty acids, cholesterol and phospholipids) at the interior and bile salts at the periphery. The hydrophilic groups of the lipids are oriented to the outside (close to the aqueous environment) and the hydrophobic groups to the inside. In this fashion, the bile salt micelles exert a solubilizing effect on the lipids.
Fig. 8.11 : Overview of digestion of lipids.
Fig. 8.12 : Absorption of lipids by intestinal mucosal cell.

The mixed micelles serve as the major vehicles for the transport of lipids from the intestinal lumen to the membrane of the intestinal mucosal cells, the site of lipid absorption. The lipid components pass through the unstirred fluid layer and are absorbed through the plasma membrane by diffusion (Fig.8.12). Absorption is almost complete for monoacylglycerols and free fatty acids which are slightly water soluble. However, for water insoluble lipids, the absorption is incomplete. For instance, less than 40% of the dietary cholesterol is absorbed.
the intestinal cells, do not undergo any modification. They enter the portal circulation and are transported to the liver in a bound form to albumin.
The micelle formation is also essential for the absorption of fat soluble vitamins, particularly vitamins A and K.
The efficiency of lipid absorption — is dependent on the quantity of bile salts to solubilize digested lipids in the mixed micelles. It may, however, be noted that in the absence of bile salts, the lipid absorption occurs to a minor extent. This is mostly due to the slightly water soluble nature of monoacylglycerols and free fatty acids. Further, short and medium chain fatty acids are not dependent on micelle formation for the absorption.

The fatty acids of short and medium chain length (< 10 carbons), after their absorption into
The long chain fatty acids are activated by thiokinase (fatty acyl CoA synthetase) in the intestinal cells. The acyl CoA derivatives so formed combine with 2-monoacylglycerols to produce triacylglycerols. These reactions are catalysed by a group of enzymes, namely acyltransferases (Fig.8.173). Further, — within the intestinal cells, cholesterol is converted to cholesterylester, and phospholipids are regenerated from the absorbed lysophospholipids. The newly synthesized lipids are usually different from those consumed in the diet.

The lipids that are resynthesized (described above) in the intestinal cells are hydrophobic in nature. They are put together as lipid droplets and surrounded by a thin layer consisting of mostly apolipoproteins (A; and B-48) and phospholipids. This package of lipids enveloped in the layer stabilizes the droplets and increases their solubility. These particles are known as chylomicrons.
Chylomicrons migrate to the plasma membrane of intestinal mucosal cells. They are released into the lymphatic vessels by exocytosis.
Fig. 8.13 : Formation and secretion of chylomicrons by intestinal mucosal cells.
The presence of chylomicrons (Greek: chylosjuice) gives the lymph a milky appearance, which is observed after a lipid-rich meal. Chylomicrons enter the large body veins via the thoracic duct. Blood from here flows to the heart and then to the peripheral tissues (muscle, adipose tissue) and, finally, to the liver. Adipose tissue and muscle take up a large proportion of dietary lipids from chylomicrons for storage and transport. It is believed that this bypass arrangement (passage of chylomicrons through peripheral tissues) protects the liver from a lipid overload after a meal.

The gastrointestinal tract possesses an efficient system for digestion and absorption of lipids. It can comfortably handle as much as 4 times the normal daily intake of lipids.
Steatorrhea : It is a condition characterized by the loss of lipids in the feces. Steatorrhea may be due to
1. A defect in the secretion of bile or pancreatic juice into the intestine;
2. Impairment in the lipid absorption by the intestinal cells.
Steatorrhea is commonly seen in disorders associated with pancreas, biliary obstruction, severe liver dysfunction etc.

Cholesterol stone formation in gall-bladder (gall stones) is a frequent health complication. It is found more frequently in females than in males often in association with obesity. Cholesterol gall stones are formed when liver secretes bile (containing phospholipids, bile acids etc.), supersaturated with respect to cholesterol.

Obesity is a major problem in many parts of the
world as
the availability
of food
abundant and
overeating is
common.
Intake of
lipids largely
contributes to
obesity.
In recent
years, pharmacological interventions to prevent
Nucleic acids (DNA and RNA), and _ their bases purines and pyrimidines can be synthesized in the body, and thus they are dietarily non-essential.

+
Cooking of food significantly improves the digestibility by enzymes.
+ Lactose intolerance due to a defect in the enzyme lactase (B-galactosidase) is very common. The treatment advocated is severe restriction of lactose (milk and milk products) in the diet.
+ Flatulence, occurring after ingestion of certain non-digestible oligosaccharides, is
characterized by increased intestinal motility, cramps and irritation.
+ Direct intestinal absorption of proteins and polypeptides is observed in the infants, immediately after birth. This is important for the transfer of maternal immunoglobulins (via breast-feeding) to the offspring.
+ In some adults, macromolecular (protein) absorption by intestine is responsible for antibody formation, often causing food allergy.
+ Emulsification of lipids is essential for their effective digestion, since lipases can act only
on
the
surface
of
lipid
droplets.
Bile
salts
are
the
most
efficient
biological
emulsifying
agents.
+
Pharmacological
interventions
(e.g.
Orlistat,
Olestra)
to
block
fat
digestion
and/or
so
as
to
prevent
obesity
are
in
recent
use.
+ Steatorrhea, characterized by the loss of lipids in feces is commonly associated with impaired pancreatic function and biliary obstruction.
+
Gastric
ulcers
are
mainly
caused
by
the
bacterium
H.
pylori.
The
that
eliminate
this
bacterium
are
effective
in
the
+ Acute pancreatitis is caused by autodigestion of pancreas while chronic pancreatitis is associated with excessive consumption of alcohol.
fat digestion, absorption, and thus obesity are in use. Two approaches are given below
1. Pancreatic lipase degrades dietary triacylglycerol to fatty acids and glycerol which are absorbed. Orlistat is a non-hydrolysable analog of triacylglycerol, and is a powerful inhibitor of pancreatic lipase, hence prevents fat digestion, and absorption.

2. Olestra is a synthetic lipid, produced by esterification of natural fatty acids with sucrose (instead of glycerol). Olestra tastes like a natural lipid. However, it cannot be hydrolysed and therefore, gets excreted.
Fig. 8.14 : Overview of digestion of nucleic acids.
The digestion of dietary nucleic acids is carried out in the small intestine, primarily by the enzymes of pancreatic juice. Ribonucleases and deoxyribonucleases, respectively, hydrolyse RNA and DNA to oligonucleotides (Fig.8.14). The latter are degraded by phosphodiesterases to form mononucleotides. Nucleotidases act on nucleotides to liberate phosphate and nucleosides. The nucleosides may be either directly absorbed or degraded to free bases before absorption. Some of the unabsorbed purines are metabolized by the _ intestinal bacteria.

The dietary purines and pyrimidines are not of much utility for the synthesis of tissue nucleic acids. Further, the purines after their absorption are mostly converted to uric acid by the intestinal mucosal cells and excreted in the urine.

The following are the major abnormalities (of interest to biochemists) concerned with digestion and absorption of food in the gastrointestinal tract.
Lactose intolerance, deficiency of sucrase, Hartnup's disease and steatorrhea have already been described. Peptic ulcer, pancreatitis and celiac disease are other important disorders associated with digestive system.
Gastric and duodenal ulcers are collectively known as peptic ulcers. Ulceration occurs due to the autodigestion of mucosa by the gastric secretions (pepsin and HCl). In the patients of peptic ulcer, gastric HCI is always present in the pyloric regions of stomach and_ the duodenum. Gastic ulcers are mainly caused by the bacterium Helicobacter pylori which lives in the nutrient-rich gastric mucosa. H. pylori induces chronic inflammation in the stomach tissues, which gets exposed to acid damage. For this reason, the best mode of treatment for gastric ulcers is the use of antibiotics that eliminate H. pylori.
Achlorhydria is a less serious disorder involving the failure to secrete gastric HCl.

Inflammation of the pancreas is known as pancreatitis. Acute pancreatitis is caused by the autodigestion of pancreas due to the unusual conversion of zymogens into the active enzymes by trypsin. In normal circumstances, this is prevented by trypsin inhibitor.
Acute pancreatitis is a life-threatening disorder. Measurement of serum amylase (highly elevated) is used in the diagnosis of pancreatitis. Excessive consumption of alcohol over a long period is blamed as the prime cause of chronic pancreatitis.

Celiac disease is a disease of malabsorption caused
by immune-mediated
damage to
small intestine.
This occurs
in some
individuals

1. Digestion is a process that converts complex foodstuffs into simpler ones which can be readily absorbed by the gastrointestinal tract.
2. Stomach, duodenum and upper part of small intestine are the major sites of digestion. The small intestine is the prime site for the absorption of digested foods.
3. The digestion of carbohydrates is initiated in the mouth by salivary o-amylase and is completed in the small intestine by pancreatic a-amylase, oligosaccharidases and disaccharidases.
4. Monosaccharides are the final absorbable products of carbohydrate digestion. Glucose is transported into the intestinal mucosal cells by a carrier mediated, Na*-dependent energy requiring process.
5. Lactose intolerance due to a defect in the enzyme lactase (f-galactosidase) resulting in the inability to hydrolyse lactose (milk sugar) is the common abnormality of carbohydrate digestion.
6. Protein digestion begins in the stomach by pepsin, which is aided by gastric HCl. Pancreatic proteases (trypsin, chymotrypsin and elastase) and intestinal amino- peptidases and dipeptidases complete the degradation of proteins to amino acids and some dipeptides.
7. The intestinal absorption of amino acids occurs by different transport systems (at least six known). The uptake of amino acids is primarily a Nat-dependent energy requiring process.
8. Digestion of lipids occurs in the small intestine. Emulsification of lipids, brought about by bile salts, is a prerequisite for their digestion. Pancreatic lipase aided by a colipase degrades triacylglycerol to 2-monoacylglycerol and free fatty acids. Cholesterol esterase and phospholipases, respectively, hydrolyse cholesteryl esters and phospholipids.
9. Lipid absorption occurs through mixed micelles, formed by bile salts in association with products of lipid digestion (primarily 2-monoacylglycerol, cholesterol and free fatty acids). In the intestinal mucosal cells, lipids are resynthesized from the absorbed components and packed as chylomicrons which enter the lymphatic vessels and then the blood.

10. Dietary nucleic acids (DNA and RNA) are digested in the small intestine to nucleosides and/or bases (purines and pyrimidines) which are absorbed.
as a result of ingestion of gluten (or gliadin derived from gluten), a protein found in what, barley and rye.

aAbBWN
= . Write an account of the digestion and absorption of lipids.
. Describe briefly the digestion of carbohydrates and proteins.
. Give an account of the Nat dependent intestinal transport of glucose and amino acids.
. Describe the role of intestine in the digestion of foodstuffs.
. Write briefly on the enzymes of gastrointestinal tract involved in the digestion of foodstuffs.

(a) Mixed micelles, (b) Lactose intolerance, (c) Salivary amylase, (d) Disaccharidases, (e) y-
Glutamyl
cycle,
(f)
Zymogens,
(g)
Specificity
of
proteases,
(h)
Bile
salts,
(i)
Synthesis
of
chylomicrons
in the intestinal mucosal cells, (j) Pancreatic juice.

1. Cellulose is not digested in humans due to lack of the enzyme that hydrolyses
ND . Name of the peptide believed to be involved in the transport of amino acids
BW . Lactose intolerance is caused by the deficiency of the enzyme
10. The vehicles for the transport of lipids from the intestinal lumen to the membrane of mucosal cells
bonds.
. The most important carbohydrate associated with flatulence caused by ingestion of leguminous seeds
. The non-digested carbohydrates are collectively called
. Gastric HCI is secreted by
:
.
.
The
disease
characterized
by
in
the
of
neutral
amino
acids
. Trypsin hydrolyses peptide bonds, the carbonyl group of which is contributed by the amino acids or
.
The
of
the
enzyme
lipase
by
bile
salts
is
overcome
by
a
protein,
namely
.

11. Transport of glucose from the lumen to the intestinal mucosal cells is coupled with diffusion of (a) Na* (b) K* (c) CF (d) HCO3.
12. The key enzyme that converts trypsinogen to trypsin is
13. The products obtained by the action of pancreatic lipase on triacylglycerols are
14, The lipoproteins synthesized in the intestinal mucosal cells from the absorbed lipids are
15. Salivary «amylase becomes inactive in the stomach primarily due to
(a) Secretin (b) Chymotrypsin (c) Elastase (d) Enteropeptidase.
(a) Glycerol and free fatty acids (b) 1-Acylglycerol and free fatty acids (c) 2-Acylglycerol and free fatty acids (d) 3-Acylglycerol and free fatty acids.
(a) High density lipoproteins (b) Chylomicrons (c) Low density lipoproteins (d) Very low density lipoproteins.
(a) Inactivation by low pH (b) Degradation by gastric pepsin (c) Inhibition by Cl (d) Inhibition by peptides.
he plasma is the liquid medium of blood (55-60%), in which the cell components— namely erythrocytes, leukocytes, platelets—are suspended. If blood containing anticoagulants (e.g. heparin, potassium oxalate) is centrifuged, the plasma separates out as a supernatant while the cells remain at the bottom. The packed cell volume or hematocrit is about 45%.

The term serum is applied to the liquid medium which separates out after the blood clots (coagulates). Serum does not contain fibrinogen and other clotting factors. Thus, the main difference between plasma and serum is the presence or absence of fibrinogen.

The total volume of blood in an adult is around 4.5 to 5 liters. Blood performs several diversified functions. These include respiration, excretion, acid-base maintenance, water balance, transport of metabolites, hormones and drugs, body defense and coagulation.
The total concentration of plasma proteins is about 6-8 g/dl. The plasma is a complex mixture of proteins, and several techniques are employed to separate them. An age-old technique is based on the use of varying concentrations of ammonium sulfate or sodium sulfate. By this method, which is known as salting out process, the plasma proteins can be separated into three groups—namely albumin, globulins and fibrinogen.
Electrophoresis : This is the most commonly employed analytical technique for the separation of plasma (serum) proteins. The basic principles of electrophoresis are described in Chapter 43. Paper or agar gel electrophoresis with vernol buffer (pH-8.6) separates plasma proteins into 5 distinct bands namely albumin, 0c, Q, B and y globulins (Fig.9.1). The concentration of each one of these fractions can be estimated by a densitometer.
Fig. 9.1 : Electrophoresis of plasma proteins— (A) Separated bands, (B) Densitometer scannning.

Electrophoresis of
serum proteins
—
is conveniently used for the diagnosis of certain diseases
1.
Multiple myeloma
:
A
sharp and
distinct
M band appears in the y-globulin fraction.
2.
Acute infections
:
o4- and
a- globulins
are increased.
3.
Nephrotic with
sharp and
4.
Primary syndrome
:
prominent immune
Decreased
©
-globulin.
:
Diminished y-globulin
band.
- 5. ,-Antitrypsin deficiency : Diminished o1globulin band.
Albumin/globulin (A/G) ratio : The albumin concentration of plasma is 3.5 to 5.0 g/dl while that of total globulins is 2.5 to 3.5 g/dl. The normal A/G ratio is 1.2 to 1.5 : 1. The A/G ratio is lowered either due to decrease in albumin or increase in globulins, as found in the following conditions
1.
Decreased synthesis
of albumin
by liver—
usually found in liver diseases and severe protein malnutrition.
albumin
2.
Excretion damage.
3.
Increased associated
with albumin
into production
urine of
chronic infections,
in kidney
globulins multiple
myelomas etc.

The important plasma proteins along with their characteristics (based on electrophoretic pattern) and major functions are given in Table 9.1. Some selected plasma proteins are discussed hereunder.
Albumin is the major constituent (60%) of plasma proteins with a concentration of 3.5-5.0 g/dl. Human albumin has a molecular weight of 69,000, and consists of a single polypeptide chain of 585 amino acids with 17 disulfide bonds.

Albumin is exclusively synthesized by the liver. For this reason, measurement of serum albumin concentration is conveniently used to assess liver function (synthesis decreased in liver diseases). Liver produces about 12 g albumin per day which represents 25% of the total hepatic protein synthesis. Albumin has an half-life of 20 days.

Plasma albumin performs osmotic, transport and nutritive functions
1. Osmotic function Due to its high concentration and low molecular weight, albumin contributes to 75-80% of the total plasma osmotic pressure (25 mm Hg). Thus, albumin plays a predominant role in maintaining blood volume and body fluid distribution. Decrease in plasma albumin level results in a fall in osmotic pressure, leading to enhanced fluid retention in tissue spaces, causing edema. The edema observed in kwashiorkor, a disorder of protein-energy malnutrition, is attributed to a drastic reduction in plasma albumin level.
of

iron. Hemopexin, Plasma concentration = < 0.1 g/dl. Hemopexin, Molecular weight = 57,000. Hemopexin, Major function(s) = Transports heme. Plasminogen, Plasma concentration = < 0.05 g/dl. Plasminogen, Molecular weight = 140,000. Plasminogen, Major function(s) = Forms plasmin, involved in fibrinolysis. J -Globulins, Plasma concentration = 0.8-1.8 mg/dl. J -Globulins, Molecular weight = -. J -Globulins, Major function(s) = Antibody functions. (Immunoglobulins-IgG, IgA, IgM, IgD and IgE; refer Table 9.2 for details), Plasma concentration = (Immunoglobulins-IgG, IgA, IgM, IgD and IgE; refer Table 9.2 for details). (Immunoglobulins-IgG, IgA, IgM, IgD and IgE; refer Table 9.2 for details), Molecular weight = (Immunoglobulins-IgG, IgA, IgM, IgD and IgE; refer Table 9.2 for details). (Immunoglobulins-IgG, IgA, IgM, IgD and IgE; refer Table 9.2 for details), Major function(s) = (Immunoglobulins-IgG, IgA, IgM, IgD and IgE; refer Table 9.2 for details). Fibrinogen, Plasma concentration = 0.2-0.4 g/dl. Fibrinogen, Molecular weight = 340,000. Fibrinogen, Major function(s) = Participates in blood coagulation
2.
Transport functions
Plasma albumin
binds to
several compounds
circulation.
biochemically and_
These
3. Nutritive functions : Albumin serves as a source of amino acids for tissue protein synthesis to a limited extent, particularly in nutritional deprivation of amino acids.
bilirubin, steroid
[Note transports
them include
hormones,
:
Besides other

plasma prealbumin,
free important
in the
fatty calcium
albumin, transport
there proteins.
retinol binding
binding protein,
in the
and are
These protein,
transcortin functions
and acids,
copper.
several include
thyroxine others
of plasma
proteins as
stated in
Table
9.1].
4. Buffering function : Among the plasma proteins, albumin has the maximum buffering capacity. However, the buffering action of albumin in plasma is not significant compared to bicarbonate buffer system.

1. Albumin, binding to certain compounds in the plasma, prevents them from crossing the blood-brain barrier e.g. albumin-bilirubin complex, albumin-free fatty acid complex.
2. Hypoalbuminemia (lowered __ plasma albumin), observed in malnutrition, nephrotic syndrome and cirrhosis of liver is associated with edema.
The osmotic pressure of albumm significantly contributes to maintaining plasma volume, and fluid volume of interstitial fluids. In hypoalbuminemia (albumin <2g/dl), plasma osmotic pressure is decreased, leading to flow of water from plasma to interstitial compartment that results in edema of legs and other body parts.
3. Albumin is excreted into —_ urine (albuminuria) in nephrotic syndrome and_ in certain inflammatory conditions of urinary tract. Microalbuminuria (30-300 mg/day) is clinically important for predicting the future risk of renal diseases (Refer Chapter 36).
4. Albumin is therapeutically useful for the of burns and treatment hemorrhage.

Globulins constitute several proteins that are separated into four distinct bands (a,, , B and y-globulins) on electrophoresis (See Fig.9.1). Globulins, in general, are bigger in size than albumin. In Table 9.1, the important globulins are given, some of them are discussed hereunder.

a,-Antitrypsin, more recently called as @-antiproteinase, is a glycoprotein with 394 amino acids and a molecular weight of 54,000. It is a major constituent of o-globulin fraction of plasma proteins with a normal concentration of about 200 mg/dl. «1-Antitrypsin is a serine protease inhibitor. It combines with trypsin, elastase and other protease enzymes and inhibits their activity.

a,-Antitrypsin deficiency has been implicated in two diseases, namely, emphysema and a -AT deficiency liver disease.
Emphysema (Greek : emphusan—to inflate) is a term used to represent the abnormal distension of lungs by air. At least 5% of emphysema cases are due to the deficiency of a,-AT. This is associated with lung infections (e.g. pneumonia) and increase in the activity of macrophages to release elastase that damages lung tissues. In the normal circumstances, elastase activity is inhibited by a4-AT.
Effect of smoking on o-AT : The amino acid methionine at position 358 of @1-AT is involved in binding with proteases. Smoking causes oxidation of this methionine to methionine sulfoxide. As a result, «;-AT with methionine sulfoxide cannot bind and inactivate proteases. Emphysema is more commonly associated with heavy smoking and the situation becomes worse in persons with o-AT deficiency.
,-Antitrypsin deficiency and liver disease : This is due to the accumulation of a mutant a,-AT which aggregates to form polymers. These polymers, in turn cause liver damage (hepatitis) followed by accumulation of collagen resulting in fibrosis (cirrhosis).

@y-Macroglobulin concentration in plasma is elevated in nephrotic syndrome. This is due to the fact that majority of the low molecular weight proteins are lost in urine (proteinuria) in this disorder.

Haptoglobin (Hp), a glycoprotein, is an acute phase protein. Its plasma concentration is increased in several inflammatory conditions.

Haptoglobin binds with the free hemoglobin (known as extra-corpuscular hemoglobin) that spills into the plasma due to hemolysis. The haptoglobin-hemoglobin (Hp-Hb) complex
(mol. wt. 155,000) cannot pass through glomeruli of kidney while free Hb (mol. wt. 65,000) can. Haptoglobin, therefore, prevents the loss of free Hb into urine.
Clinical significance of Hp Plasma concentration of Hp is decreased in hemolytic anemia. This is explained as follows. The halflife of Hp is about 5 days while that of Hp-Hb complex is 90 min. In hemolytic anemia, free Hb in plasma is elevated leading to increased formation of Hp-Hb complex. This complex in turn, is rapidly cleared from the plasma resulting in decreased Hp levels.

Ceruloplasmin is a blue coloured, copper— containing 9-globulin with a molecular weight of 150,000. Its plasma concentration is about 30 mg/dl. Ceruloplasmin binds with almost 90% of plasma copper (6 atoms of Cu bind to a molecule). This binding is rather tight and, as a result, copper from ceruloplasmin is not readily released to the tissues. Albumin carrying only 10% of plasma copper is the major supplier of copper to the tissues. Ceruloplasmin possesses oxidase activity, and it is associated with Wilson's disease which is discussed under copper metabolism (Chapter 18).

Transferrin (Tf) is a glycoprotein with a molecular weight of 76,000. It is associated with B-globulin fraction. Transferrin is a transporter of iron in the circulation.

Acute phase response refers to a non-specific response to the stimulus of infection, injury, various inflammatory conditions (affecting tissue/organs), cancer etc. This phase is associated with a characteristic pattern of changes in certain plasma proteins, collectively referred to as acute phase proteins e.g. 0antitrypsin, ceruloplasmin, complement proteins, C-reactive protein. During the acute phase, synthesis of certain plasma proteins decreases, and they are regarded as negative acute phase reactants e.g. albumin, transferrin.

CRP is a major component of acute phase proteins. \t is produced in the liver and is present in the circulation in minute concentration (<1 mg/dl). C-reactive protein (C strands for carbohydrate to which it binds on the capsule of pneumococi) is involved in the promotion of immune system through the activation of complement cascade.
Estimation of CRP in serum is important for the evaluation of acute phase response. The response of CRP to surgery is depicted in Fig.9.2. In a normal surgery, serum CRP increases and returns to normal level within 7-10 days. If the recovery is complicated by any infection, it will be reflected by the continuous elevation of CRP which requires further treatment.
Increased levels of high sensitive CRP (hs-CRP) in th circulation (reference range 100-300 g/dl) are useful for predicting the risk of coronary heart disease.

The higher vertebrates, including man, have evolved
a
defense system
to protect
themselves against
virus,
a
the invasion
bacterium of
or
a
foreign substances—a
protein.
The defense
Fig. 9.3 : Diagrammatic representation of human IgG molecule (V—variable region, C—constant region; CHO—carbohyarate; Each heavy chain is composed of four units—V,,, C,,,, C, 'He? cm while light chain consists of two units—V,, C,).
strategies of the body are collectively referred to as immunity, and are briefly described under immunology (Chapter 42). Immunoglobulins (or antibodies) are described here.

Immunoglobulins, a specialised group of proteins are mostly associated with y-globulin fraction (on electrophoresis) of plasma proteins. Some immunoglobulins however, separate along with B and a-globulins. Therefore, it should be noted that y-globulin and immunoglobulin are not synonymous. Immunoglobulin is a functional term while y-globulin is a physical term.
chains (mol. wt. 53,000 to 75,000 each) and two identical light (L) chains (mol. wt. 23,000 each) held together by disulfide linkages and noncovalent interactions (Fig.9.3). Thus, immunoglobulin is a Y-shaped tetramer (HL). Each heavy chain contains approximately 450 amino acids while each light chain has 212 amino acids. The heavy chains of Ig are linked to carbohydrates, hence immunoglobulins are glycoproteins.

All the immunoglobulin (Ig) molecules basically consist of two identical heavy (H)
Constant and variable regions : Each chain (L or H) of Ig has two regions (domains), namely the constant and the variable. The amino terminal half of the light chain is the variable region (V,) while the carboxy terminal half is the constant region (C,). As regards heavy chain, approximately one-quarter of the amino terminal region is variable (V}) while the remaining threequarters is constant (Cy,, Cr, Cr3)The amino

mg/dl = 0.02-0.05. I gE, ABLE 9.2 Characteristics of human immunoglobulins.Major function(s) = Humoral sensitivity and histamine release.
acid sequence (with its tertiary structure) of variable regions of light and heavy chains is responsible for the specific binding of immunoglobulin (antibody) with antigen.

Proteolytic cleavage of Ig : An immunoglobulin can be split by the enzyme papain to their fragments. These are two identical antigen binding fragments (Fab) and one crystallizable fragment (Fc). Papain cleaves the immunoglobin molecule at the site between Cy and Cy regions which is referred to as hinge region.

Humans have five classes of immunoglobulins—namely IgG, IgA, IgM, IgD and IgE—containing the heavy chains y, a, p, 5 and €, respectively. The type of heavy chain ultimately determines the class and the function of a given lg.
Two types of light chains—namely kappa («) and lambda (A)—are found in immunoglobulins. They differ in their structure in C, regions. An immunoglobulin (of any class) contains two « or two A light chains and never a mixture. The occurrence of « chains is more common in human immunoglobulins than 4 chains.
The characteristics of the 5 classes of human immunoglobulins are given in Table 9.2.
IgG is the most abundant (75-80%) class of immunoglobulins. IgG is composed of a single Y-shaped unit (monomer). It can traverse blood vessels readily. IgG is the only immunoglobulin that can cross the placenta and transfer the mother's immunity to the developing fetus. IgG triggers foreign cell destruction mediated by complement system.

IgA occurs as a single (monomer) or double unit (dimer) held together by J chain. It is mostly found in the body secretions such as saliva, tears, sweat, milk and the walls of intestine. IgA is the most predominant antibody in the colostrum, the initial secretion from the mother's breast after a baby is born. The IgA molecules bind with bacterial antigens present on the body (outer epithelial) surfaces and remove them. In_ this way, IgA prevents the foreign substances from entering the body cells.

IgM is the largest immunoglobulin composed of 5 Y-shaped units (IgG type) held together by a J polypeptide chain. Thus IgM is a pentamer. Due to its large size, IgM cannot traverse blood vessels, hence it is restricted to the blood stream.
IgM is the first antibody to be produced in response to an antigen and is the most effective against invading microorganisms. |t may be noted that IgM can simultaneously combine with 5 antigenic sites due to its pentameric structure.

IgD is composed of a single Y-shaped unit and is present in a low concentration in the circulation. IgD molecules are present on the surface of B cells. Their function, however, is not known for certain. Some workers believe that IgD may function as B-cell receptor.

IgE is a single Y-shaped monomer. It is normally present in minute concentration in blood. IgE levels are elevated in individuals with allergies as it is associated with the body's allergic responses. The IgE molecules tightly bind with mast cells which release histamine and cause allergy.

As already discussed, immunoglobulins are composed of light and heavy chains. Each light chain is produced by 3 separate genes, namely a variable region (V,) gene, a constant region (C,) gene and a joining region (J) gene. Each heavy chain is produced by at least 4 different genes—a variable region (V4) gene, a constant region (Cy) gene, a joining region (J) gene and diversity region (D) gene. Thus multiple genes are responsible for the synthesis of any one of the immunoglobulins.
Antibody diversity : A person is capable of generating antibodies to almost an unlimited range of antigens (more than one billion!). It should, however, be remembered that humans do not contain millions of genes to separately code for individual immunoglobulin molecules. The antibody diversity is achieved by two special processes, namely combination of various structural genes and somatic mutations.

constitutes
Multiple myeloma, a plasma cell cancer, about 1% of all cancers affecting the population. Females are more susceptible than males for this disorder and it usually occurs in the age group 45-60 years.
Abnormal Ig production : Multiple myeloma is due to the malignancy of a single clone of plasma cells in the bone marrow. This results in the overproduction of abnormal immunoglobulins, mostly (75%) IgG and in some cases (25%) IgA or IgM. IgD type multiple myeloma found in younger adults is less common (<2%) but more severe. In patients of multiple myeloma, the synthesis of normal immunoglobulins — is diminished causing depressed immunity. Hence recurrent infections are common in these patients.

multiple myeloma patients shows a characteristic pattern of electrophoresis. There is a sharp and distinct band (M band, for myeloma globulin) between B-and y-globulins. Further, this M band almost replaces the y-globulin band due to the diminished synthesis of normal y-globulins.
Bence Jones proteins : Henry Bence Jones first described them in 1847. These are the light chains (« or A) of immunoglobulins that are synthesized in excess. Bence Jones proteins have a molecular weight of 20,000 or 40,000 (for dimer). In about 20% of the patients of multiple myeloma, Bence Jones proteins are excreted in urine which often damages the renal tubules.
Amyloidosis is characterized by the deposits of light chain fragments in the tissue (liver, kidney, intestine) of multiple myeloma patients. The presence of Bence Jones proteins in urine can be detected by specific tests.
1. Electrophoresis of a concentrated urine is the best test to detect Bence Jones proteins in urine.
2. The classical heat test involves the precipitation of Bence Jones proteins when slightly acidified urine is heated to 40-50°C. This precipitate redissolves on further heating of urine to boiling point. It reappears again on cooling urine to about 70°C.
3. Bradshaw's test involves layering of urine on concentrated HCI that forms a white ring of precipitate, if Bence Jones proteins are present.
190

The term hemostasis is applied to the sequence of physiological responses to stop bleeding (loss of blood after an injury). This is carried out by blood clotting.
Blood clotting or coagulation is the body's major defense mechanism against blood loss. A blood clot is formed as a result of a series of reactions involving nearly 20 different substances, most of them being glycoproteins, synthesized by the liver.
Blood _ clotting independent pathways process involves two
1. The extrinsic pathway is the initial process in clotting and involves the factors that are not present in the blood (hence the name).
2. The intrinsic pathway involves a series of reactions participated by the factors present in the blood.
Strictly speaking, the extrinsic and intrinsic pathways are not independent, since they are coupled together. Further, the final reactions are identical for both pathways that ultimately lead to the activation of prothrombin to thrombin and the conversion of fibrinogen to fibrin clot (Fig.9.4).
The blood coagulation factors in human plasma along with their common names and molecular weights are listed in Table 9.3. All but two of these factors are designated by a Roman numeral. It should, however, be noted that the numbers represent the order of their discovery and not the order of their action. The cascade of blood clotting process is depicted in Fig.9.5 and the salient features are discussed below. The active form of a factor is designated by a subscript a. The active clotting factors (with exception of fibrin) are serine proteases.

Fibrinogen (factor I) is a soluble glycoprotein that constitutes 2-3% of plasma proteins (plasma concentration 0.3 g/dl). Fibrinogen consists of 6 polypeptide chains-two A o, two B B and two y making the structure (A ©)» (B B)2 Y2.
Fibrinogen undergoes proteolytic cleavage catalysed by thrombin to release small fibrinopeptides (A and B). This results in the formation of fibrin monomers which can stick together to form hard clots (Fig.9.6). Clot formation is further stabilized by covalent crosslinking between glutamine and lysine residues. This reaction cross-links fibrin clots and_ is catalysed by fibrin stabilizing factor (XIII). The red colour of the clot is due to the presence of red cells entangled in the fibrin cross-links.

of
Prothrombin (Il) is the inactive zymogen form thrombin (lla). The activation of prothrombin
occurs on the platelets and requires the presence of factors Va and Xa, besides phospholipids and Ca2+.

The extrinsic pathway is very rapid and occurs in response to tissue injury. This pathway essentially involves the conversion of proconvertin (VII) to its active form (VIla) and the generation factor Xa. The tissue factor (Ill), found to be necessary to accelerate the action Vila on a factor X, is present in lung and brain.

The intrinsic pathway is rather slow. It involves the participation of a contact system (wounded surface) and a series of factors to generate factor Xa.
The Hageman factor (XII) is activated (XIla) on exposure to activating wound surface containing collagen or platelet membranes. The formation of Xlla is accelerated by kallikrein and HMK. The activated Hageman factor (Xlla) activates factor XI. The Xla activate the Christmas factor
(IX). The Christmas factor is also activated by active proconvertin (Vlla).
In the next step, the Staurt factor (X) is activated by Christmas factor (IXa) and_ this reaction requires the presence of antihemophilic factor (VIlla), Ca2+ and phospholipids.
The extrinsic and intrinsic pathways lead to the formation of factor Xa which then participates in the final common pathway to ultimately result in the formation of fibrin clot.

Several substances, known as anticoagulants, are in use to inhibit the blood clotting. Calcium is essentially required for certain reactions of blood coagulation. The substances which bind with Ca2+ are very effective as anticoagulants. These include oxalate, fluoride, EDTA and citrate.
Heparin is an anticoagulant used to maintain normal hemostasis. It is a heteropolysaccharide found in many tissues including mast cells in the endothelium of blood vessels. Heparin combines with antithrombin III which in turn, inhibits the

I, Common name(s) = Fibrinogen. I, Subunit molecular weight = 340,000. II, Common name(s) = Prothrombin. II, Subunit molecular weight = 720,000. III, Common name(s) = Tissue factor, thromboplastin. III, Subunit molecular weight = 370,000. IV, Common name(s) = Calcium (Ca 2+ ). IV, Subunit molecular weight = -. V, Common name(s) = Proaccelerin, labile factor. V, Subunit molecular weight = 330,000. VII, Common name(s) = Proconvertin, serum prothrombin conversion accelerator (SPCA). VII, Subunit molecular weight = 50,000. VIII, Common name(s) = Antihemophilic factor A, antihemophilic globulin (AHG). VIII, Subunit molecular weight = 330,000. IX, Common name(s) = Christmas factor, antihemophilic factor B,. IX, Subunit molecular weight = 56,000. , Common name(s) = Plasma thromboplastin component (PTC). , Subunit molecular weight = . X, Common name(s) = Staurt-Prower factor. X, Subunit molecular weight = 56,000. XI, Common name(s) = Plasma thromboplastin antecedent (PTA). XI, Subunit molecular weight = 160,000. XII, Common name(s) = Hageman factor. XII, Subunit molecular weight = 80,000. XIII, Common name(s) = Fibrin-stabilizing factor (FSF), fibrinoligase, Liki Lorand factor. XIII, Subunit molecular weight = 320,000. -, Common name(s) = Prekallikrein. -, Subunit molecular weight = 88,000. -, Common name(s) = High molecular weight kininogen (HMK). -, Subunit molecular weight = 150,000
Note : The numbers represent the order of their discovery and not the order of their action. Factor Va was once referred to as factor VI, hence there is no factor VI.

(the active forms of the factors are represented in red with subscript 'a').
clotting factors Il, IX, X, XI, XII and kallikrein.
Heparin can be administered to patients during and after surgery to retard blood clotting.
The blood contains another anticoagulant— namely protein C—which is activated by thrombin. Active protein C hydrolyses and inactivates clotting factors V and VIII.
Warfarin,
a
vitamin
K
antagonist may
be considered as an oral anticoagulant. This acts by reducing the synthesis of certain clotting factors
(Il, VII, IX and X).

The term fibrinolysis refers to the dissolution or lysis of blood clots. Plasmin is mostly responsible for the dissolution of fibrin clots. Plasminogen, synthesized in the kidney, is the inactive precursor of plasmin. Tissue plasminogen activator (TPA) and urokinase convert plasminogen to plasmin.

Several abnormalities associated with blood clotting are known. These are due to defects in clotting factors which may be inherited or acquired. Hemophilia, Von Willebrand's disease etc., are examples of inherited disorder while afibrinogenemia is an acquired disease.
and suffer from internal bleeding (particularly in joints and gastrointestinal tract). Hemophilia A has gained importance due to the fact that the Royal families of Britain are among the affected individuals.
Hemophilia A (classical hemophilia) : This is a sex-linked disorder transmitted by females affecting males. Hemophilia A is the most common clotting abnormality and is due to the deficiency of antihemophilic factor (VIII). The affected individuals have prolonged clotting time
Hemophilia B (Christmas disease) : This is due to the deficiency of Christmas factor (IX). The clinical symptoms are almost similar to that found in hemophilia A.
Von Willebrand's disease : This disorder is characterized by failure of platelets to aggregate and is due to a defect in the platelet adherence factor.

+ Albumin, the most abundant plasma protein, is involved in osmotic function, transport of several compounds (fatty acids, steroid hormones), besides the buffering action.
+ Hypoalbuminemia and albuminuria are observed in nephrotic syndrome.
+ a,-Antitrypsin deficiency has been implicated in emphysema (abnormal distension of lungs by air) which is more commonly associated with heavy smoking.
+ Haptoglobin prevents the possible loss of free hemoglobin from the plasma through the kidneys by forming haptoglobin-hemoglobin complex.
+ Immunoglobulins (antibodies), a specialized group of plasma globular proteins, are actively involved in immunity. IgG and IgM are primarily concerned with humoral immunity while IgE is associated with allergic reactions.
+ Multiple myeloma, a plasma cell cancer disease of bone marrow, is characterized by overproduction of abnormal immunoglobulins (mostly IgG). Laboratory diagnosis of multiple myeloma can be made by the presence of a distinct M band on plasma/serum electrophoresis.
+ Blood clotting or coagulation is the body’s major defense mechanism against blood loss. Defects in clotting factors cause coagulation abnormalities such as hemophilia A (deficiency of factor VIII) and Christmas disease (deficiency of factor IX).
+ Anticoagulants inhibit blood clotting. These include heparin, oxalate, fluoride, EDTA and citrate.

1. Describe the characteristics and major functions of plasma proteins.
2 . Give an account of different types of immunoglobulins along with their functions.
3. Discuss the cascade of blood clotting process.
4 . Describe the structure of different immunogloublins.
5. Discuss the role of acute phase proteins in health and disease.

(a) Electrophoresis of plasma proteins, (b) Functions of albumin, (c) a-Antitrypsin, (d) Haptoglobin,
(e)
Immunoglobulin
G,
(f)
Multiple
myeloma,
(g)
Bence-Jones
proteins,
(h)
Fibrinogen,
(i) Anticoagulants, (j) Hemophilia.

1. The difference between plasma and serum is the presence or absence of
2 . The most commonly employed technique for separation of plasma proteins
3 . Haptoglobin binds and prevents the excretion of the compound
4, The cells responsible for the production of immunoglobulins
5 . The immunoglobulin that can cross the placenta and transfer the mother’s immunity to the developing fetus
an . The immunoglobulins that can bind with mast cells and release histamine
7. Bence-Jones proteins are precipitated when urine is heated to
10. The factor mostly responsible for the lysis of blood clot
. The major component of acute phase proteins used for the evaluation of acute phase
response
.
The
extrinsic
and
intrinsic
pathways
result
in
the
formation
of
a
common
activated
factor

11. Hemophilia A is due to the deficiency of clotting factor
12. Plasma albumin performs the following functions
13. The immunoglobulin present in most abundant quantity
14. Name the immunoglobulin involved in body allergic reactions
15. The following anticoagulant binds with Ca2*+ and prevents blood clotting
(a) X (b) V (c) VII (d) IL
(a) Osmotic (b) Transport (c) Nutritive (d) All of them.
(a) IgG (b) IgA (©) IgM (d) IgE.
(a) IgA (b) IgE (c) IgD (d) IgM.
(a) Heparin (b) Oxalate (c) Protein C (d) All of them.
he structure, functions and abnormalities of hemoglobin, the synthesis and degradation of heme, the porphyrin containing compounds are discussed in this chapter.

Hemoglobin (Hb) is the red blood pigment, exclusively found in erythrocytes (Greek: erythrose—red; kytos—a hollow vessel). The normal concentration of Hb in blood in males is 14-16 g/dl, and in females 13-15 g/dl. Hemoglobin performs two important biological functions concerned with respiration
1. Delivery of Oy from the lungs to the tissues.
2. Transport of CO, and protons from tissues to lungs for excretion.

Hemoglobin (mol. wt. 64,450) is a conjugated protein,
containing globin—the apoprotein
Fig. 10.1 : Diagrammatic representation of hemoglobin with 2a and 2B chains (Red blocks—Heme).
part—and the heme—the non-protein part (prosthetic group). Hemoglobin is a tetrameric allosteric protein (Fig.10.1).
Structure of globin : Globin consists of four polypeptide chains of two different primary structures (monomeric units). The common form of adult hemoglobin (HbA) is made up of two a-chains and two B-chains (cf). Some authors consider hemoglobin consisting of two identical dimers—(a@), and (a). Each o-chain contains 141 amino acids while B-chain contains 146 amino acids. Thus HbA, has a total of 574
Fig. 10.2 : Structure of heme [M-Methyl (— CH,); V-Vinyl (— CH,— CH,—); P-Propionyl (— CH,—CH,— CH,).
amino acid residues. The four subunits of hemoglobin are held together by non-covalent interactions primarily hydrophobic, ionic and hydrogen bonds. Each subunit contains a heme group.
Structure of heme : The characteristic red colour of hemoglobin (ultimately blood) is due to heme. Heme contains a porphyrin molecule namely protoporphyrin IX, with iron at its center. Protoporphyrin IX consists of four pyrrole rings to which four methyl, two propionyl and two vinyl groups are attached (Fig.10.2).
Heme is common prosthetic group present in cytochromes, in certain enzymes such as catalase, tryptophan pyrolase, and chlorophyll (Mg?+). In case of cytochromes, oxidation and reduction of iron (Fe2+ == Fe3*) is essential for electron transport chain.

Besides the adult hemoglobin (HbA;) described above, other minor hemoglobins are also found in humans (Table 10.1). In adults a small fraction (< 5%) of hemoglobin, known as HbA, is present. HbA, is composed of two a and two 8 (delta) chains. Fetal hemoglobin (HbF) is synthesized during the fetal development and a little of it may be present even in adults.
Hereditary persistence of fetal hemoglobin (HPFH) is a condition in which fetal hemoglobin synthesis is not terminated at birth but continues into adulthood. Glycosylated hemoglobin (HbA,,), formed by covalent binding of glucose is also found in low concentration. It is increased in diabetes mellitus which is successfully utilized for the prognosis of these patients (Refer Chapter 36).

Myoglobin (Mb) is monomeric oxygen binding hemoprotein found in heart and skeletal muscle. It has a single polypeptide (153 amino acids) chain with heme moiety. Myoglobin (mol. wt. 17,000) structurally resembles the individual subunits of hemoglobin molecule. For this reason, the more complex properties of hemoglobin have been conveniently elucidated through the study of myoglobin.
Myoglobin functions as a_ reservoir for oxygen. It further serves as oxygen carrier that promotes the transport of oxygen to the rapidly respiring muscle cells.

Hemoglobin is largely responsible for the transport of O2 from lungs to tissues. It also helps to transport CO from the tissues to the lungs.

One molecule of hemoglobin (with four hemes) can bind with four molecules of O. This is in contrast to myoglobin (with one heme) which can bind with only one molecule of oxygen. In other words, each heme moiety can bind with one Op».
HbA 1, 10.1 Normal major types of hemoglobins.Composition and symbol = D 2 E 2. HbA 1, 10.1 Normal major types of hemoglobins.Percentage of total hemoglobin = 90%. HbA 2, 10.1 Normal major types of hemoglobins.Composition and symbol = D 2 G 2. HbA 2, 10.1 Normal major types of hemoglobins.Percentage of total hemoglobin = < 5%. HbF, 10.1 Normal major types of hemoglobins.Composition and symbol = D 2 J 2. HbF, 10.1 Normal major types of hemoglobins.Percentage of total hemoglobin = < 2%. HbA 1c, 10.1 Normal major types of hemoglobins.Composition and symbol = D 2 E 2 -glucose. HbA 1c, 10.1 Normal major types of hemoglobins.Percentage of total hemoglobin = < 5%
Fig. 10.3 : Oxygen dissociation curves of hemoglobin and myoglobin (pO,,— Partial pressure of oxygen).
Oxygen dissociation curve : The binding ability of hemoglobin with Op at different partial pressures of oxygen (pO) can be measured by a graphic representation known as Oy, dissociation curve. The curves obtained for hemoglobin and myoglobin are depicted in Fig.10.3.
It is evident from the graph that myoglobin has much higher affinity for Oy than hemoglobin. Hence Oy is bound more tightly with myoglobin than with hemoglobin. Further, pO, needed for half saturation (50% binding) of myoglobin is about 1 mm Hg compared to about 26 mm for
Hg hemoglobin.

The oxygen dissociation curve for hemoglobin is sigmoidal in shape (Fig.10.3). This indicates that the binding of oxygen to one heme increases the binding of oxygen to other hemes. Thus the affinity of Hb for the last Oy is about 100 times greater than the binding of the first Oj to Hb. This phenomenon is referred to as cooperative binding of Oj to Hb or simply heme-heme interaction (Fig.10.4). On the other hand, release of O, from one heme facilitates the release of Oz from others. In short, there is a communication among heme groups in the hemoglobin function.

In the lungs, where the concentration of Op is high (hence high pO.), the hemoglobin gets fully saturated (loaded) with O5. Conversely, at the tissue level, where the O, concentration is low (hence low pO,), the oxyhemoglobin releases (unloads) its Oy for cellular respiration. This is often mediated by binding Oy to myoglobin which serves as the immediate reservoir and supplier of O, to the tissues (Fig.10.5).
T

The four subunits (262) of hemoglobin are held together by weak forces. The relative position of these subunits is different in oxyhemoglobin compared to deoxyhemoglobin.
T-form of Hb : The deoxy form of hemoglobin exists in a T or taut (tense) form. The hydrogen and ionic bonds limit the movement of monomers. Therefore, the T-form of Hb has low oxygen affinity.
R-form of Hb : The binding of O» destabilizes some of the hydrogen and ionic bonds particularly between af dimers. This results in a relaxed form or R-form of Hb wherein the
Increasing affinity for Op
: T or taut form; © : R or relaxed form ). Fig. 10.4 : Cooperative binding of O, to hemoglobin (
subunits move
a
little freely.

The existence of hemoglobin in two forms (T and R) suitably explains the allosteric behaviour of hemoglobin (Fig. 10.4).

In aerobic metabolism, for every molecule of O» utilized, one molecule of CO, is liberated. Hemoglobin actively participates in the transport of CO from the tissues to the lungs. About 15% of CO} carried in blood directly binds with Hb. The rest of the tissue CO, is transported as bicarbonate (HCO3).
Carbon dioxide molecules are bound to the uncharged o-amino acids of hemoglobin to form carbamyl hemoglobin as shown below
Hb-NH)
+
CO
=
Hb-NH-COO™
+H*
The oxyHb can bind 0.15 moles CO,/ mole heme, whereas deoxyHb can bind 0.40 moles CO3/mole heme. The binding of CO, stabilizes the T (taut) form of hemoglobin — structure, resulting — in decreased Oy, affinity for Hb.
Hemoglobin also helps in the transport of CO, as bicarbonate, as explained below (Fig.10.6).
As the CO, enters the blood from tissues, the enzyme carbonic anhydrase present in erythrocytes catalyses the formation of carbonic acid (HyCO3). Bicarbonate (HCO3) and proton (H*) are released on dissociation of carbonic acid. Hemoglobin acts as a buffer and immediately binds with protons. It is estimated that for every 2 protons bound to Hb, 4 oxygen molecules are released to the tissues. In the lungs, binding Oy to Hb results in the release of protons. The bicarbonate and protons combine to form carbonic acid. The latter is acted upon by carbonic anhydrase to release CO), which is exhaled.
Fig. 10.5 : Diagrammatic representation of transport of O, and CO, by hemoglobin.

The binding of oxygen to hemoglobin decreases with increasing H* concentration (lower pH) or when the hemoglobin is exposed to increased partial pressure of CO, (pCO3). This phenomenon is known as Bohr effect. It is due to a change in the binding affinity of oxygen to hemoglobin. Bohr effect causes a shift in the oxygen dissociation curve to the right (Fig.10.7).
Bohr effect is primarily responsible for the release of Oy from the oxyhemoglobin to the tissue. This is because of increased pCO, and decreased pH in the actively metabolizing cells.
hemoglobin.

Any increase in protons and/or lower pO, shifts the equilibrium to the right to produce deoxyhemoglobin as happens in the tissues. On the other hand, any increase in pO, and / or a decrease in H* shifts the equilibrium to the left, which occurs in lungs.
When CO, binds to hemoglobin, carbamyl hemoglobin is produced (details described under transport of CO). This causes the removal of protons from the terminal NH» group and stabilizes the structure of Hb in the T form (deoxyhemoglobin). Therefore, the binding of CO, promotes the release of oxygen (in tissues). On the other hand, when hemoglobin is oxygenated in lungs, CO, is released as it binds loosely with R-form of Hb.

Chloride (CI) is bound more tightly to deoxyhemoglobin than to oxyhemoglobin. This facilitates the release of Oy which is explained as follows
Bicarbonate (HCO3) is freely permeable across the erythrocyte membrane. Once produced in the erythrocytes, HCO3 freely moves out and equilibrates with the surrounding
2,3-Bisphosphoglycerate (2,3-BPG; formerly, 2,3-diphosphoglycerate) is the most abundant organic phosphate in the erythrocytes. Its molar concentration is approximately equivalent to that of hemoglobin. 2,3-BPG is produced in the erythrocytes from an intermediate (1,3bisphosphoglycerate) of glycolysis. This short pathway, referred to as Rapaport-Leubering cycle, is described in carbohydrate metabolism (Chapter 13).
Fig. 10.7 : Effect of pH (Bohr effect) on oxygen dissociation curve (pO,-Partial pressure of O,).

2,3-BPG regulates the binding of Op, to hemoglobin. It specifically binds to deoxyhemoglobin (and not to oxyhemoglobin) and decreases the Oy affinity to Hb. The effect of 2,3-BPG on Hb may be summarized as follows
The reduced affinity of Oy to Hb facilitates the release of O» at the partial pressure found in the tissues. This 2,3-BPG shifts the oxygen dissociation curve to the right (Fig.10.8).

One molecule of 2,3-BPG binds with one molecule (tetramer) of deoxyhemoglobin in the central cavity of the four subunits. This central pocket has positively charged (e.g. histidine, lysine) two B-globin chains. lonic bonds (salt bridges) are formed between the positively charged amino acids (of 6 globins) with the negatively charged phosphate groups of 2,3-BPG (Fig.10.9). The binding of 2,3-BPG stabilizes the deoxygenated hemoglobin (T-form) by crosslinking the B-chains.
On oxygenation of hemoglobin, 2,3-BPG is expelled from the pocket and the oxyhemoglobin attains the R-form of structure.

1. In hypoxia : The concentration of 2,3-BPG in erythrocytes is elevated in chronic hypoxic conditions associated with difficulty in Oy supply. These include adaptation to high altitude, obstructive pulmonary emphysema (airflow in the bronchioles blocked) etc.
2. In anemia: 2,3-BPG levels are increased in severe anemia in order to cope up with the oxygen demands of the body. This is an adaptation to supply as much Oy as possible to the tissue, despite the low hemoglobin levels.
3. In blood transfusion : Storage of blood in acid citrate-dextrose medium results in the decreased concentration of 2,3-BPG. Such blood when transfused fails to supply O» to the tissues immediately.
Addition of inosine (hypoxanthine-ribose) to the stored blood prevents the decrease of 2,3BPG. The ribose moiety of inosine gets phosphorylated and = enters the hexose monophosphate pathway and _ finally gets converted to 2,3-BPG.
Fig. 10.9 : (A) Diagrammatic representation of binding of 2,3-BPG to deoxyhemoglobin; (B) Structure of 2,3-BPG.
4. Fetal hemoglobin (HbF) : The binding of 2,3-BPG to fetal hemoglobin is very weak. Therefore, HbF has higher affinity for Oy compared to adult hemoglobin (HbA). This may be needed for the transfer of oxygen from the maternal blood to the fetus.

Hemoglobin (specifically heme) combines with different ligands and forms hemoglobin derivatives. The normal blood contains oxyHb and deoxyHb. Besides these, methemoglobin (metHb) and carboxyhemoglobin are the other important Hb derivatives. The Hb derivatives have characteristic colour and they can be detected by absorption spectra.

For the biological function of hemoglobin—to carry oxygen—the iron should remain in the ferrous (Fe2*+) state. Hemoglobin (Fe2+) can be oxidized to methemoglobin (Fe?+). In normal circumstances, however, molecular oxygen does not oxidize Hb, it only loosely binds to form oxyhemoglobin.
The oxidation of — hemoglobin to methemoglobin (metHb) may be caused in the living system by HzO , free radicals and drugs. The methemoglobin (with Fe?*) is unable to bind to Op». Instead, a water molecule occupies the oxygen site in the heme of metHb.
In normal circumstances, the occasional oxidation of hemoglobin is corrected by the enzyme methemoglobin reductase present in erythrocytes (Fig.10.10).

Carbon monoxide (CO) is a toxic compound (an industrial pollutant) that can bind with Hb in the same manner as O, binds. However, CO has about 200 times more affinity than Oy for binding with Hb.
Clinical manifestations of CO toxicity are observed when the COHb concentration exceeds 20%. The symptoms include headache, nausea,
Fig. 10.10 : Conversion of hemoglobin to methemoglobin and vice versa.
breathlessness, vomiting and irritability. Administration of O) through oxygen masks will help to reverse the manifestations of CO toxicity.

Abnormal hemoglobins are the resultant of mutations in the genes that code for a or B chains of globin. As many as 400 mutant hemoglobins are known. About 95% of them are due to alteration in a single amino acid of globin.

For a better understanding of abnormal hemoglobins, it is worthwhile to have a basic knowledge of globin synthesis. The globin genes are organised into two gene families or clusters (Fig.10.11).
1. a-Gene family : There are two genes coding for a-globin chain present on each one of chromosome 16. The ¢-gene, other member of a-gene cluster is also found on chromosome 16 and is active during the embryonic development.
2. B-Gene family : The synthesis of B-globin occurs from a single gene located on each one of chromosome 11.
This chromosome also contains four other genes.
One e-gene expressed in the early stages of embryonic development.
Fig. 10.11 : Diagrammatic representation of globin genes with the synthesis of globin chains and hemoglobins (¢,£,-Hb Gower 1; c,Y,-HbF; 0,5,-HbA,; 0,8,-HbA,).
Two y-genes (Gy and Ay) synthesize y-globin chains of fetal hemoglobin (HbF).
One 5-gene producing 8-globin chain found
in adults to a minor extent (HbA>).

It is a term used to describe the disorders caused by the synthesis of abnormal hemoglobin molecule or the production of insufficient quantities of normal hemoglobin or rarely both.
Sickle-cell anemia (HbS) and hemoglobin C disease (HbC) are the classical examples of abnormal hemoglobins. Thalassemias, on the other hand, are caused by decreased synthesis of normal hemoglobin.

Sickle-cell anemia (HbS) is the most common form of abnormal hemoglobins. \t is so named because the erythrocytes of these patients adopt a sickle shape (crescent like) at low oxygen concentration
(Fig.10.12).

Sickle-cell anemia is largely confined to areas of the world. It occurs in
tropical primarily
the black population. It is estimated that 1 in
500
newborn
black
infants
in
the
USA
are
affected by sickle-cell anemia.

The structure of hemoglobin (as described already) contains two a-and two B-globin chains. In case of sickle-cell anemia, the hemoglobin (HbS) has two normal a-globin chains and two abnormal (mutant) B-globin chains. This is due to a difference in a single amino acid. In HbS, glutamate at sixth position of B-chain is replaced by valine (Glu Bb, —> Val).
Sickle-cell anemia is due to a change (missense mutation) in the single nucleotide (thymine > adenine) of B-globin gene. This error causes the formation of altered codon (GUG in
Fig. 10.13 : Formation of B-chain of hemoglobin in normal and sickle cell anemia (Note : Single base mutation in DNA (T >A) causes replacement of glutamate by valine at 6th position of B-chain).
place of GAG) which leads to the incorporation of valine instead of glutamate at the sixth position in B-chain (Fig.10.13).
4. Premature death : Homozygous individuals of sickle-cell anemia die before they reach adulthood (< 20 years).
Homozygous and heterozygous HbS : Sicklecell anemia is said to be homozygous, if caused by inheritance of two mutant genes (one from each parent) that code for B-chains. In case of heterozygous HbS, only one gene (of B-chain) is affected while the other is normal. The erythrocytes of heterozygotes contain both HbS and HbA and the disease is referred to as sicklecell trait which is more common in blacks (almost 1 in 10 are affected). The individuals of sickle-cell trait lead a normal life, and do not usually show clinical symptoms. This is in contrast to homozygous -sickle-cell anemia.

Sickle-cell anemia is characterized by the following abnormalities
1. Life-long hemolytic anemia : The sickled erythrocytes are fragile and their continuous breakdown leads to life-long anemia.
2. Tissue damage and pain : The sickled cells block the capillaries resulting in poor blood supply to tissues. This leads to extensive damage and inflammation of certain tissues causing pain.

Hemolysis and tissue damage are accompanied by increased susceptibility to infection and diseases.

Glutamate is a polar amino acid and it is replaced by a non-polar valine in sickle-cell hemoglobin. This causes a marked decrease in the solubility of HbS in deoxygenated form (T-form). However, solubility of oxygenated HbS is unaffected.

The substitution of valine for glutamate results in a sticky patch on the outer surface of B-chains. It is present on oxyand deoxyhemoglobin S$ but absent on HbA. There is a site or receptor complementary to sticky patch on deoxyHbS.
The sticky patch of one deoxyHbS binds with the receptor of another deoxyHbS and _ this process continuous resulting in the formation of long aggregate molecules of deoxyHbS (Fig.10.14). Thus, the polymerization of deoxyHbS molecules leads to long fibrous precipitates (Fig.10.15). These stiff fibres distort the erythrocytes into a sickle or crescent shape (Fig.10.12). The sickled erythrocytes are highly vulnerable to lysis.
In case of oxyHbS, the complementary receptor is masked, although the sticky patch is
bb tel
Fig. 10.14 : Diagrammatic representation of sticky patch (Blue) and sticky patch receptor (> ) in the formation of of
long aggregates deoxyhemoglobins.
present (Fig.10.14). Hence, the molecules of oxyHbS cannot bind among themselves or with the molecules of deoxyHbsS.
Normal deoxyHbA lacks sticky patches but contains receptors. Absence of sticky patches does not allow the deoxyHbA to participate in the formation of aggregates.
As explained above, sickling is due to polymerization of deoxyHbS. Therefore, if HbS is maintained in the oxygenated form (or with minimum deoxyHbS), sickling can be prevented.
2. More recent studies indicate that malarial parasite increases the acidity of erythrocytes (pH down by 0.4). The lowered pH increases the sickling of erythrocytes to about 40% from the normally occurring 2%. Therefore, the entry of malarial parasite promotes sickling leading to lysis of erythrocytes. Furthermore, — the concentration of K* is low in sickled cells which is unfavourable for the parasite to survive.

The incidence of sickle-cell disease coincides with the high incidence of malaria in tropical areas of the world (particularly among the black Africans).
Sickle-cell trait (heterozygous state with about 40% HbS) provides resistance to malaria which is a major cause of death in tropical areas. This is explained as follows
1. Malaria is a parasitic disease caused by Plasmodium falciparum in Africa. The malarial parasite spends a part of its life cycle in erythrocytes. Increased lysis of sickled cells (shorter life span of erythrocytes) interrupts the parasite cycle.
Sickle-cell trait appears to be an adaptation for the survival of the individuals in malariainfested regions. Unfortunately, homozygous individuals, the patients of sickle-cell anemia (much less frequent than the trait), cannot live beyond 20 years.
Fig. 10.15 : Diagrammatic representation a fibre of aggregated deoxyhemoglobin.

1. Sickling test : This is a simple microscopic examination of blood smear prepared by adding reducing agents such as sodium dithionite. Sickled erythrocytes can be detected under the microscope.
2. Electrophoresis When subjected to electrophoresis in alkaline medium (pH 8.6), sickle-cell hemoglobin (HbS) moves slowly towards anode (positive electrode) than does adult hemoglobin (HbA). The slow mobility of HbS is due to less negative charge, caused by the absence of glutamate residues that carry negative charge. In case of sickle-cell trait, the fast moving HbA and slow moving HbS are observed. The electrophoresis of hemoglobin obtained from lysed erythrocytes can be routinely used for the diagnosis of sicklecell anemia and sickle-cell trait (Fig.10.16).

Administration of sodium cyanate inhibits sickling of erythrocytes, Cyanate increases the affinity of Oy to HbS and lowers the formation of deoxyHbS. However, it causes certain sideeffects like peripheral nerve damage.
In patients with severe anemia, repeated blood transfusion is required. This may result in iron overload and cirrhosis of liver.
Replacement of HbS with other forms of hemoglobins has been tried. Fetal hemoglobin (HbF) reduces sickling. Sickle-cell disease awaits gene-replacement therapy!

Cooley's hemoglobinemia (HbC) is characterized by substitution of glutamate by lysine in the sixth position of B-chain. Due to the presence of lysine, HbC moves more slowly on electrophoresis compared to HbA and HbS. HbC disease occurs only in blacks. Both homozygous and heterozygous individuals of HbC disease are known. This disease is characterized by mild hemolytic anemia. No specific therapy is recommended.
Fig. 10.16 : Electrophoresis of hemoglobins at pH 8.6 (HbA—Normal adult hemoglobin; HbS-Sickle cell hemoglobin).

This is caused by the substitution of glutamine in place of glutamate in the 121st positioin of B-chain. Several variants of HbD are identified from different places indicated by the suffix. For instance, HbD (Punjab), HbD (Los Angeles). HbD, on electrophoresis moves along with HbsS.

This is the most common abnormal hemoglobin after HbS. It is estimated that about 10% of the population in South-East Asia (Bangladesh, Thailand, | Myanmar) suffer from HbE disease. In India, it is prevalent in West Bengal. HbE is characterized by replacement of glutamate by lysine at 26th position of B-chain. The individuals of HbE (either homozygous or heterozygous) have no clinical manifestations.

Thalassemias are a group of _ hereditary hemolytic disorders characterized by impairment/ imbalance in the synthesis of globin chains of Hb.
Thalassemias (Greek: thalassa-sea) mostly occur in the regions surrounding — the Mediterranean sea, hence the name. These diseases, however, are also prevalent in Central Africa, India and the Far East.

The basic concepts in the synthesis of globin chains have been described (See Fig.10.14). Hemoglobin contains 2a and 26 globin chains. The synthesis of individual chains is so coordinated that each a-chain has a B-chain partner and they combine to finally give hemoglobin (a5). Thalassemias are characterized by a defect in the production of a-or B-globin chain. There is however, no abnormality in the amino acids of the individual chains.
Thalassemias occur due to a variety of molecular defects
1. Gene deletion or substitution,
2. Underproduction or instability of mRNA,
3. Defect in the initiation of chain synthesis,
4. Premature chain termination.

a-Thalassemias are caused by a decreased synthesis or total absence of a-globin chain of Hb. There are four copies of a-globin gene, two on each one of the chromosome 16. Four types of a-thalassemias occur which depend on the
number of missing a-globin genes. The salient features of different a-thalassemias are given in Table 10.2.
1. Silent carrier state is due to loss of one of the four a-globin genes with no_ physical manifestations.
2. a-Thalassemia trait caused by loss of two
genes
(both
from
the
same
gene
pair
or
one
from
each
gene
pair).
Minor
anemia
is
observed.
3. Hemoglobin H disease, due to missing of
three
genes,
is
with
moderate
anemia.
4. Hydrops fetalis is the most severe form of a-thalassemias due to lack of all the four genes. The fetus usually survives until birth and then dies.

Decreased synthesis or total lack of the formation of f-globin§ chain' causes Bthalassemias. The production of o-globin chain continues to be normal, leading to the formation of a globin tetramer (a4) that precipitate. This causes premature death of erythrocytes. There are mainly two types of -thalassemias (Fig.10.17)

glyph<c=3,font=/MDECLA+Symbol>glyph<c=3,font=/MDECLA+Symbol>D 1 glyph<c=3,font=/MDECLA+Symbol>glyph<c=3,font=/MDECLA+Symbol>D 2 glyph<c=3,font=/MDECLA+Symbol>glyph<c=3,font=/MDECLA+Symbol>D 1 glyph<c=3,font=/MDECLA+Symbol>glyph<c=3,font=/MDECLA+Symbol>D 2. Hydrops fetalis, T ABLE 10.2 Summary of different types of D -thalassemias.Clinical symptoms = Fetal death usually occurs at birth.
Fig. 10.17: Diagrammatic representation of gene deletions in B-thalassemias (each one of the chromosome pair of 11 has one gene for f-globin).
1. B-Thalassemia minor : This is an heterozygous state with a defect in only one of the two B-globin gene pairs on chromosome 11. This disorder, also known as £-thalassemia trait, is usually asymptomatic, since the individuals can make some amount of f-globin from the affected gene.
Naturally = occurring = porphyrins —_ contain substituent groups replacing the 8 hydrogen atoms of the porphyrin nucleus.
2. B-Thalassemia major This is a homozygous state with a defect in both the genes responsible for B-globin synthesis. The infants born with B-thalassemia major are healthy at birth since B-globin is not synthesized during the fetal development. They become _ severely anemic and die within 1-2 years. Frequent blood transfusion is required for these children. This is associated with iron overload which in turn may lead to death within 15-20 years.

Porphyrins are cyclic compounds composed of 4 pyrrole rings held together by methenyl (=CH-—) bridges (Fig.10.18). Metal ions can bind with nitrogen atoms of pyrrole rings to form complexes. Heme is an_ iron-containing porphyrin (See Fig.10.2) while chlorophyll is a magnesium-containing porphyrin. Thus heme and chlorophyll are the classical examples of metalloporphyrins.

The structure of porphyrins (CygH)4N,) has four pyrrole rings namely |, Il, Ill and IV.
Hans Fischer, the father of porphyrin chemistry, proposed a shorthand model for presentation of porphyrin structures. Accordingly, each pyrrole ring is represented as a bracket. Thus porphyrin has 4 closed brackets with the 8 substituent positions numbered as shown in

Type | porphyrins : When the substituent groups on the 8 positions are symmetrically arranged they are known as type | porphyrins, e.g. uroporphyrin I.
Type III porphyrins : They contain asymmetric groups at the 8 positions and are more common in the biological system. Originally, Fischer placed them as IX series hence they are more popularly known as type IX porphyrins. It may be observed that the structure of uroporphyrin is asymmetric since on ring IV, the order of substituent groups is reversed (P, A instead of A, P).
The Fischer's shorthand models of important porphyrins (uroporphyrin | and Ill; coproporphyrin | and Ill; protoporphyrin IX and heme) are depicted in Fig.10.19.

The photodynamic properties of porphyrins can be used in the treatment of certain cancers. This is carried out by a technique called cancer phototherapy. Tumors are capable of taking up
8
7
H
Pyrrole
1
2
1
SN
oN
N
Il
6
5
Porphyrin
12
|
Il
6
HN
4
5
Fischer’s model
Fig. 10.18 : Structures of pyrrole and porphyrin [I-IV are pyrrole rings; 1-8 are substituent positions; at, B, y, 5 are methylene (— CH=) bridges.]
i
:
:
more porphyrins than normal tissues. The cancer phototherapy is carried out by administering : hematoporphyrin (or other related compounds) to the cancer patient. When the tumor is exposed to an argon laser, the porphyrins get excited and produce cytotoxic effects on tumor cells.
|v
|
|
NH
8
IV
7
a
ll
—3
II
—J4

Heme is the most important porphyrin containing compound. It is primarily synthesized in the liver and the erythrocyte-producing cells of bone marrow (erythroid cells). Heme synthesis also occurs to some extent in other tissues. However, mature erythrocytes lacking mitochondria are a notable exception. Biosynthesis of heme occurs in the following stages (Fig.10.20).
1. Formation of 5-aminolevulinate : Glycine, a non-essential amino acid and succinyl CoA, an intermediate in the citric acid cycle, are the starting materials for porphyrin synthesis. Glycine combines with succinyl CoA to form 5-aminolevulinate (ALA). This reaction catalysed by a pyridoxal phosphate dependent 5-aminolevulinate synthase occurs in the mitochondria. It is a rate-controlling step in porphyrin synthesis.
2. Synthesis of porphobilinogen : Two molecules of 5-aminolevulinate condense to form porphobilinogen (PBG) in the cytosol. This reaction is catalysed by a Zn-containing enzyme ALA dehydratase. It is sensitive to inhibition by heavy metals such as lead.
3. Formation of porphyrin ring Porphyrin synthesis occurs by condensation of four molecules of porphobilinogen. The four pyrrole rings in porphyrin are interconnected by methylene (—CHy) bridges derived from a-carbon of glycine.
The interaction of two enzymes—namely uroporphyrinogen | synthase and uroporphyrinogen III cosynthase—results in condensation of porphobilinogen followed by ring closure and isomerization to produce uroporphyrinogen III.
4.
Conversion of
uroporphyrinogen
III
to protoporphyrin
IX
of
:
This is
catalysed by
a
series reactions
(a) Uroporphyrinogen decarboxylase decarbo- xylates all the four acetate (A) side chains to form methyl groups (M), to produce coproporphyrinogen.

(b) Coproporphyrinogen oxidase converts (oxidative decarboxylation) two of the
propionate side chains (P) to vinyl groups (V) and results in the formation of protoporphyrinogen.
- (c) Protoporphyrinogen oxidase oxidizes methylene groups (—CH,—) interconnecting pyrrole rings to methenyl groups (=CH-—). This leads to the synthesis of protoporphyrin IX.
5. Synthesis of heme from protoporphyrin IX : The incorporation of ferrous iron (Fe2+) into protoporphyrin IX is catalysed by the enzyme ferrochelatase or heme synthetase. This enzyme can be inhibited by lead. It is found that the induction of Fe2+ into protoporphyrin IX can occur spontaneously but at a slow rate.

Heme production in the liver is required for the formation of hemoproteins (e.g. cytochrome P4sq involved in detoxification) while in the erythroid cells, it is necessary for the synthesis of hemoglobin. Two different mechanisms exist for the regulation of heme biosynthesis in the liver and the erythroid cells.
Regulation in the liver : The first committed step in heme biosynthesis, catalysed by 5-aminolevulinate (ALA) synthase, is regulatory. Heme or its oxidized product hemin (Fe3+) controls this enzyme activity by three mechanisms
1. Feedback inhibition
2. Repression of ALA synthatase
3. Inhibition of transport of ALA synthase
from cytosol
to mitochondria
(the site
of action).
Effect of drugs on ALA synthase activity : The activity of ALA synthase is markedly increased by the administration of a large number of drugs e.g. phenobarbital, insecticides, carcinogens etc. This is expected since these compounds are mostly metabolized by a heme containing protein, cytochrome P459. On administration of drugs, cellular levels of heme are depleted due to its increased incorporation into cytochrome P4590. The reduced heme concentration increases the synthesis (derepression) of ALA synthase to meet the cellular demands.
Fig. 10.21 : Summary of heme synthesis along with porphyrias (1-Acute intermittent porphyria; 2-Congenital erythropoietic porphyria; 3—Porphyria cutanea tarda; 4—Hereditary coproporphyria; 5—Variegate porphyria; 6—Protoporphyria).
Regulation in the erythroid cells : The enzyme ALA synthase does not appear to control the heme synthesis in the erythroid cells. Uroporphyrinogen synthase and ferrochelatse mostly regulate heme formation in these cells. Further, the cellular uptake of iron also influences heme synthesis. It is observed that heme stimulates globin synthesis. This ensures that heme and globin synthesis occur in the right proportion to finally form hemoglobin.

Porphyrias are the metabolic disorders of heme synthesis, characterized by the increased excretion of porphyrins or porphyrin precursors. Porphyrias are either inherited or acquired. They are broadly classified into two categories
1. Erythropoietic : Enzyme deficiency occurs in the erythrocytes.
- 2. Hepatic : Enzyme defect lies in the liver.
All the known porphyrias are inherited as autosomal dominant disorders. However, congenital erythropoietic porphyria is an exception, since it is autosomal recessive. The different types of porphyrias are described (Fig.10.21, Table 10.3)

This disorder occurs due to the deficiency of the enzyme uroporphyrinogen I synthase. Acute intermittent porphyria is characterized by increased excretion of porphobilinogen and 5-aminolevulinate. The urine gets darkened on exposure to air due to the conversion of porphobilinogen to porphobilin and porphyrin. The other characteristic features of acute intermittent porphyria are as follows
1
It is
usually expressed
after puberty
in humans.
- 1 The symptoms include abdominal pain, vomiting and cardiovascular abnormalities. The neuropsychiatric distrubances observed in these patients are believed to be due to reduced activity of tryptophan pyrrolase

Hepatic, Enzyme defect = . Hepatic, Characteristics = . Acute intermittent porphyria, Enzyme defect = Uroporphyrinogen I synthase. Acute intermittent porphyria, Characteristics = Abdominal pain, neuropsychiatric symptoms. Porphyria cutanea tarda, Enzyme defect = Uroporphyrinogen decarboxylase. Porphyria cutanea tarda, Characteristics = Photosensitivity. Hereditary coproporphyria, Enzyme defect = Corpoporphyrinogen oxidase. Hereditary coproporphyria, Characteristics = Abdominal pain, photosensitivity, neuropsychiatric symptoms. Variegate porphyria, Enzyme defect = Protoporphyrinogen oxidase. Variegate porphyria, Characteristics = Abdominal pain, photosensitivity, neuropsychiatric symptoms. Erythropoietic, Enzyme defect = . Erythropoietic, Characteristics = . Congenital erythropoietic porphyria, Enzyme defect = Uroporphyrinogen III cosynthase. Congenital erythropoietic porphyria, Characteristics = Photosensitivity, increased hemolysis. Protoporphyria, Enzyme defect = Ferrochelatase. Protoporphyria, Characteristics = Photosensitivity
(caused by depleted heme levels), resulting in the accumulation of tryptophan and 5-hydroxytryptamine.

1 The symptoms are more severe after administration of drugs (e.g. barbiturates) that induce the synthesis of cytochrome P,.9. This is due to the increased activity of ALA synthase causing accumulation of PBG and ALA.
1 These patients are not photosensitive since the enzyme defect occurs prior to the formation of uroporphyrinogen.
Acute intermittent porphyria is treated by administration of hematin which inhibits the enzyme ALA synthase and the accumulation of porphobilinogen.
[The disease—acute intermittent porphyria— has historical importance. King George III (17601820) ruled England during the period of American revolution. He was a victim of this disease and possessed the characteristic manifestations (such as red colour urine) and was considered mad. The decisions taken by the deranged King due to acute intermittent porphyria had led to a war followed by American Independence. It is widely believed that American history would have been different, had George Ill not inherited this metabolic disorder!]
This disorder is due to a defect in the enzyme uroporphyrinogen III cosynthase. Some workers, however, believe that congenital erythropoeitic porphyria is caused by an imbalance between the activities of uroporphyrinogen | synthase and uroporphyrinogen Ill cosynthase. This disease has certain characteristic features
1 It is a rare congenital disorder caused by autosomal recessive mode of inheritance, mostly confined to erythropoietic tissues.
| The individuals excrete uroporphyrinogen | and coproporphyrinogen | which oxidize respectively to uroporphyrin | and coproporphyrin | (red pigments).
1 The patients are photosensitive (itching and burning of skin when exposed to visible light) due to the abnormal prophyrins that accumulate.
1 Increased hemolysis is also observed in the individuals affected by this disorder.

This is also known as cutaneous hepatic porphyria and is the most common porphyria, usually associated with liver damage caused by
alcohol overconsumption or iron overload. The partial deficiency of the enzyme uroporphyrinogen decarboxylase appears to be responsible for the occurrence of porphyria cutanea tarda. The other characteristic features include
1 Increased excretion of uroporphyrins (I and III) and rarely porphobilinogen.
1 Cutaneous photosensitivity is the most important clinical manifestation of these patients.
1 Liver exhibits fluorescence due to high concentration of accumulated porphyrins.

This disorder is due to a defect in the enzyme coproporphyrinogen oxidase. As a result of this, coproporphyrinogen Ill and other intermediates (ALA and PBG) of heme synthesis prior to the blockade are excreted in urine and feces. The victims of hereditary coproporphyria are photosensitive. They exhibit the clinical manifestations observed in the patients of acute intermittent porphyria.
Infusion of hematin is used to control this disorder. Hematin inhibits ALA synthase and thus reduces the accumulation of various intermediates.

The enzyme protoporphyrinogen oxidase is defective in this disorder. Due to this blockade, protoporphyrin IX required for the ultimate synthesis of heme is not produced. Almost all the intermediates (porphobilinogen, coproporphyrin, uroporphyrin, protoporphyrin etc.) of heme synthesis accumulate in the body and are excreted in urine and feces. The urine of these patients is coloured and they exhibit photosensitivity.

This disorder, also known as erythropoietic protoporphyria, is caused by a deficiency of the enzyme ferrochelatase. Protoporphyrin IX accumulates in the tissues and is excreted into urine and feces. Reticulocytes (young RBC) and skin biopsy exhibit red flourescence.

The porphyrias, though not inherited, may be acquired due to the toxicity of several compounds. Exposure of the body to heavy metals (e.g. lead), toxic compounds (e.g. hexachlorobenzene) and drugs (e.g. griseofulvin) inhibits many enzymes in heme synthesis. These include ALA dehydratase, uroporphyrin | synthase and ferrochelatase.

Erythrocytes have a life span of 120 days. At the end of this period, they are removed from the circulation. Erythrocytes are taken up and degraded by the macrophages of the reticuloendothelial (RE) system in the spleen and liver. The hemoglobin is cleaved to the protein part globin and non-protein heme. About 6 g of hemoglobin per day is broken down, and resynthesized in an adult man (70 kg).
Fate of globin : The globin may be reutilized as such for the formation of hemoglobin or degraded to the individual amino acids. The latter undergo their own metabolism, including participation in fresh globin synthesis.
Sources of heme : It is estimated that about 80% of the heme that is subjected for degradation comes from the erythrocytes and the rest (20%) comes from immature RBC, myoglobin and cytochromes.
Heme oxygenase : A complex microsomal enzyme namely heme oxygenase _ utilizes NADPH and Oj and cleaves the methenyl bridges between the two pyrrole rings (A and B) to form biliverdin. Simultaneously, ferrous iron (Fe2+) is oxidized to ferric form (Fe?*) and released. The products of heme oxygenase reaction are biliverdin (a green pigment), Fe?* and carbon monoxide (CO). Heme promotes the activity of this enzyme.

(Note : Colours used in structures represent change in the specific reaction only).
Biliverdin is excreted in birds and amphibia while in mammals it is further degraded.
Biliverdin reductase : Biliverdin's methenyl bridges (between the pyrrole rings C and D) are reduced to methylene group to form bilirubin (yellow pigment). This reaction is catalysed by an NADPH dependent soluble enzyme, biliverdin reductase (Fig.10.22). One gram of hemoglobin on degradation finally yields about 35 mg bilirubin. Approximately 250-350 mg of bilirubin is daily produced in human adults. The term bile pigments is used to collectively represent bilirubin and its derivatives.
Transport of bilirubin to liver : Bilirubin is lipophilic and therefore insoluble in aqueous solution. Bilirubin is transported in the plasma in a bound (non-covalently) form to albumin. Albumin has two binding sites for bilirubin—a high affinity site and a low affinity site. Approximately 25 mg of bilirubin can bind
tightly to albumin (at high affinity sites) per 100 ml of plasma. The rest of the bilirubin binds loosely (at the low affinity sites) which can be easily detached from albumin to enter the tissues. Certain drugs and antibiotics (e.g sulfonamides, salicylates) can displace bilirubin from albumin. Due to this, bilirubin can enter the central nervous system and cause damage to neurons.
As the albumin-bilirubin complex enters the liver, bilirubin dissociates and is taken up by sinusoidal surface of the hepatocytes by a carrier mediated active transport. The transport system has a very high capacity and therefore is not a limitation for further metabolism of bilirubin. Inside the hepatocytes, bilirubin binds to a specific intracellular protein namely ligandin.

In the liver, bilirubin is conjugated with two molecules of glucuronate supplied by UDPglucuronate. This reaction, catalysed by bilirubin glucuronyltransferase (of smooth endoplasmic reticulum) results in the formation of a water soluble bilirubin diglucuronide (Figs.10.22 and 10.23). When bilirubin is in excess, bilirubin monoglucuronides also accumulate in the body. The enzyme bilirubin glucuronyltransferase can be induced by a number of drugs (e.g. phenobarbital),

Conjugated bilirubin is excreted into the bile canaliculi against a concentration gradient which then enters the bile. The transport of bilirubin diglucuronide is an active, energy-dependent and rate limiting process. This step is easily susceptible to any impairment in liver function. Normally, there is a good coordination between the bilirubin conjugation and its excretion into bile. Thus almost all the bilirubin (> 98%) that enters bile is in the conjugated form.

Bilirubin glucuronides are hydrolysed in the intestine by specific bacterial enzymes namely B-glucuronidases to liberate bilirubin. The latter is then converted to urobilinogen (colourless compound), a small part of which may be reabsorbed into the circulation. Urobilinogen can be converted to urobilin (an yellow colour compound) in the kidney and excreted. The characteristic colour of urine is due to urobilin.
A major part of urobilinogen is converted by bacteria to stercobilin which is excreted along with feces. The characteristic brown colour of feces is due to stercobilin.
The normal serum total bilirubin concentration is in the range of 0.2 to 1.0 mg/dl. Of this, about 0.2-0.6 mg/dl is unconjugated while 0.2 to 0.4 mg/dl is conjugated bilirubin.
Jaundice (French : Jaune-yellow) is a clinical condition characterized by yellow colour of the white of the eyes (sclerae) and skin. It is caused by the deposition of bilirubin due to its elevated levels in the serum. The term hyperbilirubinemia is often used to represent the increased concentration of serum bilirubin. (Note : For some more details on jaundice, refer Chapter 20)

Jaundice (also known as icterus) may be more appropriately considered as a symptom rather than a disease. It is rather difficult to classify jaundice, since it is frequently caused due to multiple factors. For the sake of convenience to understand, jaundice is classified into three major types—hemolytic, hepatic and obstructive.
1. Hemolytic jaundice : This condition is associated with increased hemolysis of erythrocytes (e.g. incompatible blood transfusion, malaria, sickle-cell anemia). This results in the overproduction of bilirubin beyond the ability of the liver to conjugate and excrete the same. It should, however be noted that liver possesses a large capacity to conjugate about 3.0 g of bilirubin per day against the normal bilirubin production of 0.3 g/day.
In hemolytic jaundice, more bilirubin is excreted into the bile leading to the increased
formation of urobilinogen and stercobilinogen. Hemolytic jaundice is characterized by
1 Elevation in the serum unconjugated bilirubin.
1 Increased excretion of urobilinogen in urine.
1 Dark brown colour of feces due to high content of stercobilinogen.
2. Hepatic (hepatocellular) jaundice : This type of jaundice is caused by dysfunction of the liver due to damage to the parenchymal cells. This may be attributed to viral infection (viral hepatitis), poisons and_ toxins (chloroform, carbon tetrachloride, phosphorus etc.) cirrhosis of liver, cardiac failure etc. Among these, viral hepatitis is the most common.
Damage to
the liver
adversely affects
the bilirubin
uptake and
its conjugation
by cells.
Hepatic jaundice
is characterized
by
- 1 Increased levels of conjugated unconjugated bilirubin in the serum.
and
- 1 Dark coloured urine due to the excessive excretion of bilirubin and urobilinogen.
liver

1 Increased activities of alanine transaminase (SGPT) and aspartate transaminase (SGOT) released into circulation due to damage to hepatocytes.
1 The patients pass pale, clay coloured stools
due to
the absence
of stercobilinogen.
The affected individuals experience
1 nausea and anorexia (loss of appetite).
3. Obstructive (regurgitation) jaundice : This
is due
to an
obstruction in
the bile
duct that
prevents the
passage
The of
obstruction tumors
bile into
may etc.
Due to
conjugated circulation.
the blockage
bilirubin be
caused the
by in
from
Obstructive the
jaundice intestine.
gall bile
liver stones,
duct, the
enters the
by
- 1 Increased concentration — of bilirubin in serum.
conjugated
1 Serum alkaline phosphatase is elevated as it is released from the cells of the damaged bile duct.
1 Dark coloured urine due to elevated excretion of bilirubin and clay coloured feces due to absence of stercobilinogen.
1 Feces contain excess fat indicating impairment in fat digestion and absorption in the absence of bile (specifically bile salts).
1 The patients experience nausea and gastrointestinal pain.

Physiological jaundice is not truly a genetic defect. It is caused by increased hemolysis coupled with immature hepatic system for the uptake, conjugation and secretion of bilirubin. The activity of the enzyme UDP-glucuronyltransferase is low in the newborn. Further, there is a limitation in the availability of the substrate UDP-glucuronic acid for conjugation. The net effect is that in some infants the serum
uncojugated bilirubin is highly elevated (may go beyond 25mg/dl), which can cross the bloodbrain barrier. This results in hyperbilirubinemic toxic encephalopathy or kernicterus that causes mental retardation. The drug phenobarbital is used in the treatment of neonatal jaundice, as it can induce bilirubin metabolising enzymes in liver. In some neonates, blood transfusion may be necessary to prevent brain damage.
bilirubin gets converted into a non-toxic isomer namely Jumirubin. Lumirubin can be easily excreted by the kidneys in the unconjugated form (in contrast to bilirubin which cannot be excreted). Serum bilirubin is monitored every 12-24 hours, and phototherapy is continuously carried out till the serum bilirubin becomes normal (<1 mg/dl).

Phototherapy : Bilirubin can absorb blue light (420-470 nm) maximally. Phototherapy deals with the exposure of the jaundiced neonates to blue light. By a process called photoisomerization, the toxic native unconjugated
This is also known as congenital nonhemolytic jaundice. It is a rare disorder and is due to a defect in the hepatic enzyme UDPglucuronyltransferase. Generally, the children die within first two of life.
years

+ Hemoglobin is primarily responsible for the delivery of Og from lungs to tissue and the transport of COg from tissues to lungs.
+ Increased erythrocyte 2,3-BPG levels in anemia and chronic hypoxia facilitate the release of more Og from the oxyhemoglobin to the tissues.
+ Storage of blood causes a decrease in the concentration of 2,3-BPG. This can be prevented by the addition of ionosine.
+ Hemoglobin (Fe2+) on oxidation by HOs, free radicals or drugs, forms methemoglobin (Fe3+) which cannot transport Oo.
+ Carboxyhemoglobin is produced when carbon monoxide, an industrial pollutant, binds to hemoglobin. The clinical manifestations of CO toxicity (> 20% COHb) include headache, nausea, breathlessness and vomiting.
+
Sickle
cell
hemoglobin
(HbS)
causes
hemolytic
anemia,
increased
susceptibility
to
infection
and
premature
death.
However,
HbS
offers
protection
against
malaria.
+ Thalassemias are hemolytic disorders caused by impairment/imbalance in the synthesis of globin chains of Hb. These include a-thalassemia trait, hydrops fetalis and B-thalassemias.
+ Administration of porphyrins can be used in the treatment certain cancers by phototherapy.
+ Abnormalities in heme synthesis cause porphyrias which may be erythropoietic (enzyme defect in RBC) or hepatic (enzyme defect in liver). Porphyrias are associated with elevated excretion of porphyrins, neuropsychiatric disturbances and cardiovascular abnormalities.
+ Jaundice is caused by elevated serum bilirubin (normal < 0.8 mg/dl) levels and is characterized by yellow coloration of white of the eyes, and skin.
+ Phototherapy (by exposure to blue light) is used in to control severe cases of neonatal physiologic jaundice.

Gilbert’s disease : This is not a single disease
but a combination of disorders. These include
This is again a rare hereditary disorder and is
due to a less severe defect in the bilirubin
conjugation.
It
is
believed
that
hepatic
UDP-
cells.
glucuronyltransferase that catalyses the addition
of
second
glucuronyl
group
is
defective.
The
serum
bilirubin
is
usually
less
than
1. A defect in the uptake of bilirubin by liver
2. An impairment in conjugation due to reduced activity of UDP-glucuronyltransferase.
20 mg/dl and this is less dangerous than type |.
3. Decreased hepatic clearance of bilirubin.

1. Hemoglobin (HbA,, mol. wt. 64,450) is a conjugated protein containing globin, the apoprotein and the heme, the nonprotein moiety (prosthetic group). It is a tetrameric, allosteric protein with 2a and 2B polypeptide chains held by non-covalent interactions. Each subunit contains a heme with iron in the ferrous state.
10. Jaundice is a clinical condition caused by elevated serum bilirubin concentration (normal <1.0 mg/dl). Jaundice is of three types-hemolytic (due to increased hemolysis), hepatic (due to impaired conjugation) and obstructive (due to obstruction in the bile duct).
Hemoglobin is responsible for the transport of Og from lungs to the tissues. Each heme (of Hb) can bind with one molecule of Og and this is facilitated by cooperative heme- heme interaction.
Hemoglobin actively participates in the transport of CO, from tissues to lungs. Increased partial pressure of COg (pCO) accompanied by elevated H* decreases the binding of Og to Hb, a phenomenon known as Bohr effect.
The four compounds namely 2,3-bisphosphoglycerate, COs, H* and CI” are collectively known as allosteric effectors. They interact with hemoglobin and facilitate the release of Og from oxyHb.
Sickle-cell anemia (HbS) is a classical example of abnormal hemoglobins. It is caused when glutamate at 6th position of B-chain is replaced by valine. HbS is characterized by hemolytic anemia, tissue damage, increased susceptibility to infection and premature death. Sickle-cell anemia, however offers resistance to malaria.
Thalassemias are a group of hereditary hemolytic disorders characterized by impairment/ imbalance in the synthesis of globin (a or B) chain of Hb. Hydrops fetalis, the most severe form of a-thalassemia is characterized by the death of infant at birth. B- Thalassemia major is another serious disorder with severe anemia and death of child within 1-2 years.

Heme is the most important porphyrin compound, primarily synthesized in the liver from the precursors-glycine and succinyl CoA. Heme productioin is regulated by d&-aminolevulinate synthase.
Porphyrias are the metabolic disorders of heme synthesis, characterized by the increased excretion of porphyrins or their precursors. Acute intermittent porphyria occurs due to the deficiency of the enzyme uroporphyrinogen I synthase and is characterized by increased excretion of porphobilinogen and Saminolevulinate. The clinical symptoms include neuropsychiatric disturbances and cardiovascular abnormalities.
Heme is degraded mainly to bilirubin, an yellow colour bile pigment. In the liver, it is conjugated to bilirubin diglucuronide, a more easily excretable form into bile.

1. Describe the structure of hemoglobin and discuss oxygen transport.
2. Write an account of hemoglobinopathies with special reference to sickle-cell anemia.
3. Discuss the biosynthesis of heme. Add a note on the regulation of heme synthesis.
4. What are porphyrias? Describe any three porphyrias in detail.
5. Write an account of the degradation of heme to bile pigments. Add a note on jaundice.

(a) Methemoglobin, (b) Heme—heme interaction, (c) Bohr effect, (d) 2,3—BPG, (e) Sickle cell anemia and malaria, (f) Thalassemias, (g) Acute intermittent porphyria, (h) Heme oxygenase, (i) Bilirubin diglucuronide, (j) Carboxyhemoglobin.

1. The total number of amino acids present in adult hemoglobin
2. The oxidation of ferrous (Fe2*) iron to ferric (Fe?*) iron in hemoglobin results in the formation of a compound namely
3. The enzyme that catalyses the formation of carbonic acid
4. Name the compound that is increased in RBC of anemic patients to facilitate the supply of O5 to the tissues
5. Sickling of RBC in sickle-cell anemia is due to polymerization of
6. The disorders characterized by decreased synthesis or total absence of globin chains of hemoglobin are collectively known as
7. The intermediate of citric acid cycle that is involved in heme synthesis
8. The enzyme defect in acute intermittent porphyria
9. The enzyme that is regulated by feedback inhibition in heme synthesis is
10. The product formed when heme oxygenase cleaves heme

11. The characteristic red colour of hemoglobin is due to
(a) Heme (b) a-Globin (c) B-Globin (d) All of them.
12. The number of heme groups present in myoglobin
13. The patients of sickle-cell anemia are resistant to
(a) Filaria (b) Malaria (c) Diabetes (d) Trypanosomiasis.
14. The compound that facilitates the release of O, from oxyhemoglobin
(a) 2, 3-BPG (b) H* (c) C1> (d) All of them.
15. Name the amino acid that directly participates in the synthesis of heme
(a) Methionine (b) Aspartate (c) Glycine (d) Tryptophan.
a a better understanding of biological oxidation, it is worthwhile to have a basic knowledge of bioenergetics and the role of highenergy compounds in biological processes.

Bioenergetics or biochemical thermodynamics deals with the study of energy changes (transfer and utilization) in biochemical reactions. The reactions are broadly classified as exergonic (energy releasing) and endergonic (energy consuming). Bioenergetics is concerned with the initial and final states of energy component of the reactants and not the mechanism of chemical reactions.

The energy actually available to do work (utilizable) is known as free energy. Changes in the free energy (AG) are valuable in predicting the feasibility of chemical reactions. The reactions can occur spontaneously if they are accompanied by decrease in free energy.
During a chemical reaction, heat may be released or absorbed. Enthalpy (AH) is a measure of the change in heat content of the reactants, compared to products.
Entropy (AS) represents a change in the randomness or disorder of reactants and products. Entropy attains a maximum as the reaction approaches equilibrium. The reactions of biological systems involve a temporary decrease in entropy.
The relation between the changes of free energy (AG), enthalpy (AH) and entropy (AS) is expressed as
T represents the absolute temperature in Kelvin (K = 273 + °C).
The term standard free energy represented by AG° (note the superscript?) is often used. It indicates the free energy change when the reactants or products are at a concentration of 1 mol/l at pH 7.0.

If free energy change (AG) is represented by a negative sign, there is a loss of free energy. The reaction is said to be exergonic, and proceeds spontaneously. On the other hand, a positive AG indicates that energy must be supplied to the reactants. The reaction cannot proceed spontaneously and is endergonic in character.
The hydrolysis of ATP is a classical example of exergonic reaction
ATP
+
HxO—>
ADP
+
Pi
(AG°
=
-7.3
Cal/mol)
The
reversal
of
the
reaction
(ADP
+
Pi
—
ATP)
is
endergonic
and
occurs
only
when
there
is
a
supply
of
energy
of
at
least
7.3
Cal/mol
(AG°
is
positive).
The free energy change becomes zero (AG = 0) when a reaction is at equilibrium.
At a constant temperature and pressure, AG is dependent on the actual concentration of reactants and products. For the conversion of reactant A to product B (A — B), the following mathematical relation can be derived
AG
=
AG®
+
RT
In
BL
[A]
where AG° = Standard free energy change
R = Gas constant (1.987 Cal/mol)
T = Absolute temperature (273 + °C)
In = Natural logarithm
[B] = Concentration of product
[A]
=
Concentration
of
reactant.

When a reaction AB is at equilibrium the free change The above
(eq), energy is zero. equation may be written as
AG
=
0
=
AG°
+
RT
In
——
Hence AG° = —RT In Keq.

Biochemical pathways often involve a series
of reactions. For such reactions, free energy
change is an additive value. The sum of AG is crucial in determining whether a_ particular pathway will proceed or not. As long as the sum of AGs of individual reactions is negative, the pathway can operate. This happens despite the fact that some of the individual reactions may have positive AG.

Certain compounds are encountered in the biological system which, on hydrolysis, yield energy. The term high-energy compounds or energy rich compounds is usually applied to substances which possess _ sufficient free energy to liberate at least 7 Cal/mol at pH 7.0 (Table 11.1). Certain other compounds which liberate less than 7.0 Cal/mol (lower than ATP hydrolysis to ADP + Pi) are referred to as lowenergy compounds.

High-energy phosphates, ' G° (Cal/mol) = . Phosphoenol pyruvate, ' G° (Cal/mol) = -14.8. Carbamoyl phosphate, ' G° (Cal/mol) = -12.3. Cyclic AMP, ' G° (Cal/mol) = -12.0. 1,3-Bisphosphoglycerate, ' G° (Cal/mol) = -11.8. Phosphocreatine, ' G° (Cal/mol) = -10.3. Acetyl phosphate, ' G° (Cal/mol) = -10.3. S-Adenosylmethionine *, ' G° (Cal/mol) = -10.0. Pyrophosphate, ' G° (Cal/mol) = -8.0. Acetyl CoA **, ' G° (Cal/mol) = -7.7. ATP o ADP + Pi, ' G° (Cal/mol) = -7.3. Low-energy phosphates, ' G° (Cal/mol) = . ADP o AMP + Pi, ' G° (Cal/mol) = -6.6. Glucose 1-phosphate, ' G° (Cal/mol) = -5.0. Fructose 6-phosphate, ' G° (Cal/mol) = -3.8. Glucose 6-phosphate, ' G° (Cal/mol) = -3.3. Glycerol 3-phosphate, ' G° (Cal/mol) = -2.2. * Sulfonium compound, ' G° (Cal/mol) = . ** Thioester, ' G° (Cal/mol) = 
All the high-energy compounds—when hydrolysed—liberate more energy than that of ATP. These include phosphoenol pyruvate, 1,3bisphosphoglycerate, phosphocreatine _ etc. Most of the high-energy compounds contain phosphate group (exception acetyl CoA) hence they are called high-energy phosphate compounds.

There are at least 5 groups of high-energy compounds.
1. Pyrophosphates e.g. ATP.
. Acyl phosphates e.g. 1,3-bisphosphoglycerate. Nh
. Enol phosphates e.g. phosphoenolpyruvate. WwW
. Thioesters e.g. acetyl CoA. &B
an . Phosphagens e.g. phosphocreatine.
Table 11.2 gives some more details on the high-energy compounds, including the highenergy bonds present in each category.
High-energy bonds The high-energy
: compounds possess acid anhydride bonds (mostly phosphoanhydride bonds) which are formed by the condensation of two acidic groups or related compounds. These bonds are referred to as high- energy bonds, since the free energy is liberated when these bonds are hydrolysed. Lipmann suggested use of the symbol ~ to represent highenergy bond. For instance, ATP is written as AMP~P~P.

Adenosine triphosphate (ATP) is a unique and the most important high-energy molecule in the living cells. It consists of an adenine, a ribose and a triphosphate moiety (Fig.11.1). ATP is a high-energy compound due to the presence of two phosphoanhydride bonds in the triphosphate unit. ATP serves as the energy currency of the cell as is evident from the ATP-ADP cycle.

The hydrolysis of ATP is associated with the release of large amount of energy.
ATP + H»O —> ADP + Pi + 7.3 Cal.
The energy liberated is utilized for various processes like muscle contraction, active transport etc. ATP can also act as a donor of high-energy phosphate to low-energy compounds, to make them energy rich. On the other hand, ADP can accept high-energy phosphate from the compounds possessing higher free energy content to form ATP.
ATP serves as an immediately available energy currency of the cell which is constantly
Fig. 11.2 : ATP-ADP cycle along with sources and utilization of ATP (Note that ~P does not exist in free form, but is only transferred).
being utilized and regenerated. This is represented by ATP-ADP cycle, the fundamental basis of energy exchange reactions in living system (Fig.11.2). The turnover of ATP is very high.
ATP acts as an energy link between the catabolism (degradation of molecules) and anabolism (synthesis) in the biological system.

ATP can be synthesized in two ways
1. Oxidative phosphorylation : This is the major source of ATP in aerobic organisms. It is linked with the mitochondrial electron transport chain (details described later).
2. Substrate level phosphorylation : ATP may be directly synthesized during substrate oxidation in the metabolism. The high-energy compounds such as__ phosphoenolpyruvate and 1,3-bisphosphoglycerate (intermediates of glycolysis) and succinyl CoA (of citric acid cycle) can transfer high-energy phosphate to ultimately produce ATP.

Phosphocreatine (creatine phosphate) stored in vertebrate muscle and brain is an energy-rich
compound.
In
invertebrates,
phosphoarginine
(arginine phosphate) replaces phosphocreatine.

Oxidation is defined as the loss of electrons and reduction as the gain of electrons. This may be illustrated by the interconversion of ferrous ion (Fe2*) to ferric ion (Fe3*).
The electron lost in the oxidation is accepted by an acceptor which is said to be reduced. Thus the oxidation-reduction is a_ tightly coupled process.
The general principle of oxidation-reduction is applicable to biological systems also. The oxidation of NADH to NAD* coupled with the reduction of FMN to FMNHy is illustrated
In the above illustration, there are two redox pairs NADH/NAD* and FMN/FMNH)b. The redox pairs differ in their tendency to lose or gain electrons.

The oxidation-reduction potential or, simply, redox potential, is a quantitative measure of the tendency of a redox pair to lose or gain electrons. The redox pairs are assigned specific standard redox potential (E, volts) at pH 7.0 and 25°C.
The redox potentials of some biologically important redox systems are given in Table 11.3. The more negative redox potential represents a greater tendency (of reductant) to lose electrons.

systems.E 0 Volts = + 0.25. Cytochrome a (Fe 3+ /Fe 2+ ), Table 11.3 Standard redox potential (E 0 ) of some oxidation-reduction systems.E 0 Volts = + 0.29. 1 2 O 2 /H 2 O, Table 11.3 Standard redox potential (E 0 ) of some oxidation-reduction systems.E 0 Volts = + 0.82
On the other hand, a more positive redox potential indicates a greater tendency (of oxidant) to accept electrons. The electrons flow from a redox pair with more negative Eg to another redox pair with more positive Eo.
The redox potential (Eg) is directly related to
the change in the free energy (AG°).

Fig.
11.3
:
Overview of
biological oxidation
(ETC-Electron transport
chain).
and FADH). The latter two reduced coenzymes pass through the electron transport chain (ETC) or respiratory chain and, finally, reduce oxygen to water. The passage of electrons through the ETC is associated with the loss of free energy. A part of this free energy is utilized to generate ATP from ADP and Pi (Fig.11.3).
An overview of the ETC is depicted in Fig.11.4.

The mitochondria are the centres for metabolic oxidative reactions to generate reduced coenzymes (NADH and FADH;,) which, in turn, are utilized in ETC to liberate energy in the form of ATP. For this reason, mitochondrion is appropriately regarded as the power house of the cell.

The mitochondrion consists of five distinct parts. These are the outer membrane, the inner membrane, the intermembrane space, the cristae and the matrix (Fig.11.5).
The energy-rich carbohydrates (particularly glucose), fatty acids and amino acids undergo a series of metabolic reactions and, finally, get oxidized to CO and H,O. The reducing equivlents from various metabolic intermediates are transferred to coenzymes NAD* and FAD to produce, respectively, NADH
Fig. 11.5 : Structure of mitochondrion depicting electron transport chain (ETC) (F,, F,—Protein subunits).
Inner mitochondrial membrane : The electron transport chain and ATP synthesizing system are located on the inner mitochondrial membrane which is a specialized structure, rich in proteins. It is impermeable to ions (H*, K*, Na*) and small molecules (ADP, ATP). This membrane is highly folded to form cristae. The surface area of inner mitochondrial membrane is greatly increased due to cristae. The inner surface of the inner mitochondrial membrane possesses specialized particles (that look like lollipops), the phosphorylating subunits which are the centres for ATP production.
Mitochondrial matrix : The interior ground substance forms the matrix of mitochondria. It is rich in the enzymes responsible for the citric acid cycle, B-oxidation of fatty acids and oxidation of amino acids.

The inner mitochondrial membrane can be disrupted into five distinct respiratory or enzyme complexes, denoted as complex I, II, III, 1V and V (Fig.11.6). The complexes I-IV are carriers of electrons while complex V is responsible for ATP synthesis. Besides these enzyme complexes, there are certain mobile electron carriers in the respiratory chain. These include NADH, coenzyme Q, cytochrome C and oxygen.
The enzyme complexes (I-IV) and the mobile carriers are collectively involved in the transport of electrons which, ultimately, combine with oxygen to produce water. The largest proportion of the oxygen supplied to the body is utilized by the mitochondria for the operation of electron transport chain.

There are five distinct carriers that participate in the electron transport chain (ETC). These carriers are sequentially arranged (Fig.11.7) and are responsible for the transfer of electrons from a given substrate to ultimately combine with proton and oxygen to form water.

Of the two coenzymes NAD+ and NADP* derived from the vitamin niacin, NAD* is more actively involved in the ETC. NAD* is reduced to NADH + Ht by dehydrogenases with the removal of two hydrogen atoms from the substrate (AH ). The substrates include
Fig. 11.6 : Multiprotein complexes in electron transport chain.
glyceraldehyde-3 phosphate, pyruvate, isocitrate, a-ketoglutarate and malate.
NADPH + H* produced by NADPt-dependent dehydrogenase is not usually a substrate for ETC. NADPH is more effectively utilized for anabolic reactions (e.g. fatty acid synthesis, cholesterol synthesis).

The enzyme NADH dehydrogenase (NADHcoenzyme Q reductase) is a flavoprotein with FMN as the prosthetic group. The coenzyme FMN accepts two electrons and a proton to form FMNH>. NADH dehydrogenase is a complex enzyme closely associated with non-heme iron proteins (NHI) or iron-sulfur proteins (FeS).
Succinate dehydrogenase (succinate-coenzyme Q reductase) is an enzyme found in the inner mitochondrial membrane. It is also a flavoprotein with FAD as the coenzyme.This can accept two hydrogen atoms (2H* + 2e-) from succinate.
Succinate
+
FAD
—-+>
Fumarate + FADH)

The iron-sulfur (FeS) proteins exist in the oxidized (Fe?+) or reduced (Fe*+) state. About half a dozen FeS proteins connected with respiratory chain have been identified. However, the mechanism of action of iron-sulfur proteins in the ETC is not clearly understood.
NADH
+
H*
+
FMN
—>
NAD*
+
FMNH)
One FeS participates in the transfer of electrons from FMN to coenzyme Q. Other FeS proteins associated with cytochrome b and cytochrome cy; participate in the transport of electrons.
Fig. 11.7 : Electron transport chain with sites of ATP synthesis and inhibitors (BAL—British antilewisite).

Coenzyme Q is also known as ubiquinone since it is ubiquitous in living system. It is a quinone derivative with a variable isoprenoid side chain. The mammalian tissues possess a quinone with 10 isoprenoid units which is known as coenzyme Qj, (CoQ)o).
Coenzyme Q is a lipophilic electron carrier. It can accept electrons from FMNH) produced in the ETC by NADH dehydrogenase or FADH) produced outside ETC (e.g. succinate dehydrogenase, acyl CoA dehydrogenase).
Coenzyme Q is not found in mycobacteria. Vitamin K performs similar function as coenzyme Q in these organisms. Coenzyme Q has no known vitamin precursor in animals. It is directly synthesized in the body. (Refer cholesterol biosynthesis, Chapter 14)

The cytochromes are conjugated proteins containing heme group. The latter consists of a porphyrin ring with iron atom. The heme group of cytochromes differ from that found in the structure of hemoglobin and myoglobin. The iron of heme in cytochromes is alternately oxidized (Fe?*) and reduced (Fe2*), which is essential for the transport of electrons in the ETC. This is in contrast to the heme iron of hemoglobin and myoglobin which remains in the ferrous (Fe2*) state.
Three cytochromes were initially discovered from the mammalian mitochondria. They were designated as cytochrome a, b and c depending on the type of heme present and the respective absorption spectrum. Additional cytochromes such as Cy, by, bo, a3 etc. were discovered later.
The electrons are transported from coenzyme Q to cytochromes (in the order) b, cy, c, a and a3. The property of reversible oxidation- reduction of heme iron Fe2+ = Fe3* present in cytochromes allows them to function as effective carriers of electrons in ETC.
Cytochrome c (mol. wt. 13,000) is a small protein containing 104 amino acids and a heme group. It is a central member of ETC with an intermediate redox potential. It is rather loosely bound to inner mitochondrial membrane and can be easily extracted.
Cytochrome a and a3 : The term cytochrome oxidase is frequently used to collectively represent cytochrome a and a3 which is the terminal component of ETC. Cytochrome oxidase is the only electron carrier, the heme iron of which can directly react with molecular oxygen. Besides heme (with iron), this oxidase also contains copper that undergoes oxidationreduction (Cu2+ = Cut) during the transport of electrons.
In the final stage of ETC, the transported electrons, the free protons and the molecular oxygen combine to produce water.

The transport of electrons through the ETC is linked with the release of free energy. The process of synthesizing ATP from ADP and Pi coupled with the electron transport chain is known as oxidative phosphorylation. The complex V (See Fig.11.6) of the inner mitochondrial membrane is the site of oxidative phosphorylation.

The P : O ratio refers to the number of inorganic phosphate molecules utilized for ATP generation for every atom of oxygen consumed. More appropriately, P : O ratio represents the number of molecules of ATP synthesized per pair of electrons carried through ETC.
The mitochondrial oxidation of NADH with a classical P : O ratio of 3 can be represented by the following equation :
NADH + Ht + +0, + 3ADP + 3Pi — NAD* + 3ATP + 4H ,O
Further, a P : O ratio of 2 has been assigned to the oxidation of FADH . There is a strong evidence now to suggest a P:O ratio of 2.5 for NADH, and 1.5 for FADH), since ten protons (for NADH) and six protons (for FADH)) are pumped across mitochondrial membrane. Synthesis of one ATP requires four protons.

There are three sites in the ETC that are exergonic to result in the synthesis of 3 ATP molecules (See Fig.11.7).
1. Oxidation of FMNH» by coenzyme Q.
2. Oxidation of cytochrome b by cytochrome cy.
3. Cytochrome oxidase reaction.
Each one of the above reactions represents a coupling site for ATP production. There are only two coupling sites for the oxidation of FADH) (P : O ratio 2), since the first site is bypassed.

The transport of electrons from redox pair NAD*/NADH (Ep, = — 0.32) to finally the redox pair +0,/H,0 (Ey = + 0.82) may be simplified and represented in the following equation
+0,
+
NADH
+
Ht
—>
H,O
+
NAD+
The redox potential difference between these two redox pairs is 1.14 V, which is equivalent to an energy 52 Cal/mol.
Three ATP are synthesized in the ETC when
NADH is oxidized which equals to 21.9 Cal (each ATP = 7.3 Cal).
The efficiency of energy conservation is calculated as
Therefore, when NADH is oxidized, about
42%
of energy
of
3
ATP
and the
is trapped
in the
form remaining
liberation is
lost is
allows not
a
ETC
to go
ATP.
Further, this
wasteful on
as heat.
process, continuously
heat to
to
The heat
since it
generate maintain

Several hypotheses have been put forth to explain the process of oxidative phosphorylation. The most important among them—namely, chemical coupling, and chemiosmotic—are discussed below.

This hypothesis was put forth by Edward Slater (1953). According to chemical coupling hypothesis, during the course of electron transfer in respiratory chain, a series of phosphorylated high-energy intermediates are first produced which are utilized for the synthesis of ATP. These reactions are believed to be analogous to the substrate level phosphorylation that occurs in glycolysis or citric acid cycle. However, this hypothesis lacks experimental evidence, since all attempts, so far, to isolate any one of the high-energy intermediates have not been successful.

This mechanism, originally proposed by Peter Mitchell (1961), is now widely accepted. It explains how the transport of electrons through the respiratory chain is effectively utilized to produce ATP from ADP + Pi. The concept of chemiosmotic hypothesis is comparable with energy stored in a battery separated by positive and negative charges.
Proton gradient : The inner mitochondrial membrane, as such, is impermeable to protons (H*) and hydroxyl ions (OH7). The transport of electrons through ETC is coupled with the translocation of protons (H*) across the inner mitochondrial membrane (coupling membrane) from the matrix to the intermembrane space. The pumping of protons results in an electrochemical or proton gradient. This is due to the accumulation of more H* ions (low pH) on the outer side of the inner mitochondrial membrane than the inner side (Fig.11.8). The proton gradient developed due to the electron flow in the respiratory chain is sufficient to result in the synthesis of ATP from ADP and Pi.
Fig. 11.8 : Outline of chemiosmotic hypothesis for oxidative phosphorylation.
The protons
that accumulate
intermembrane space
re-enter on
the the
mitochondrial matrix
leading to
the synthesis
of
ATP.

Paul
Boyer
in
1964
proposed
(Nobel
Prize,
1997)
that
a
conformational
change
in
the
Fig. 11.9 : Diagrammatic representation of chemiosmotic hypothesis for oxidative phosphorylation (1, Ill, IV and V-Respiratory chain complexes; F,, F,-Protein subunits for phosphorylation).
Fig. 11.10 : Structure of mitochondrial ATP synthase (FF) complex (C units-channel protein subunits; a, B, and y are the subunits of F,-ATP synthase).
mitochondrial membrane proteins leads to the synthesis of ATP. The original Boyer hypothesis, now considered as rotary motor/engine driving model or binding change model, is widely accepted for the generation ATP.
The enzyme ATP synthase is FoF; complex (of complex V). The Fy subcomplex is composed of channel protein 'C' subunits to which F,-ATP synthase is attached (Fig.11.10). F,-ATP synthase consists of a central y subunit surrounded by alternating a and B subunits (a3 and B3).
In response to the proton flux, the y subunit physically rotates. This induces conformational changes in the B3 subunits that finally lead to the release of ATP.
According to the binding change mechanism, the three B subunits of F;-ATP synthase adopt different conformations. One subunit has open (O) conformation, the second has loose (L) conformation while the third one has tight (T) conformation (Fig.11.11).
By an unknown mechanism, protons induce the rotation of y subunit, which in turn induces conformation changes in {6 subunits. The substrates ADP and Pi bind to B subunit in
L-conformation. The L_ site changes to T conformation, and this leads to the synthesis of ATP. The O site changes to L conformation which binds to ADP and Pi. The T site changes to O conformation, and releases ATP. This cycle of conformation changes of f subunits is repeated. And three ATP are generated for each revolution (Fig.11.11).
It may be noted that the ATP release in O conformation is energy dependent (and not ATP synthesis) and very crucial in rotary motor model for ATP generation.

The enzyme ATP synthase acts as a protondriving motor, and is an example of rotary catalysis. Thus, ATP synthase is the world's smallest molecular motor.

It is estimated that about 100 polypeptides are required for oxidative phosphorylation. Of these, 13 are coded by mitochondrial DNA (mtDNA) and synthesized in the mitochondria, while the rest are produced in the cytosol (coded by nuclear DNA) and transported. mtDNA is maternally inherited since mitochondria from the sperm do not enter the fertilized ovum.
Fig. 11.11 : The binding change model (rotary motor/engine driving model) for ATP synthesis by F,-ATP synthase.
Mitochondrial DNA is about 10 times more susceptible to mutations than nuclear DNA. mtDNA mutations are more commonly seen in tissues with high rate of oxidative phosphorylation (e.g. central nervous system, skeletal and heart muscle, liver).
Leber's hereditary optic neuropathy is an example for mutations in mtDNA. This disorder is characterized by loss of bilateral vision due to neuroretinal degeneration.

Many site-specific inhibitors of ETC have contributed to the present knowledge of mitochondrial respiration. Selected examples of these inhibitors haven been given in Fig.11.7. The inhibitors bind to one of the components of ETC and block the transport of electrons. This causes the accumulation of reduced components before the — inhibitor blockade step and oxidized components after that step.
The synthesis of ATP (phosphorylation)
is dependent on electron transport. Hence, all the site-specific inhibitors of ETC also inhibit ATP formation. Three possible sites of action for the inhibitors of ETC are identified
1. NADH and coenzyme Q : Fish poison rotenone, barbituate drug amytal and antibiotic piercidin A inhibit this site.
2. Between cytochrome b & c, : Antimycin A —an antibiotic, British antilewisite (BAL)— an antidote used against war-gas—are the two important inhibitors of the site between cytochrome b and cj.
3. Inhibitors of cytochrome oxidase : Carbon monoxide, cyanide, hydrogen sulphide and azide effectively inhibit cytochrome oxidase. Carbon monoxide reacts with reduced form of the cytochrome while cyanide and azide react with oxidized form.

+ The most important function of food is to supply energy to the living cells. This is finally achieved through biological oxidation.
+ The supply of Og is very essential for the survival of life (exception—anaerobic bacteria).
+ ATP, the energy currency of the cell, acts as a link between the catabolism and anabolism in the living system. The major production of body’s ATP occurs in the mitochondria through oxidative phosphorylation coupled with respiration.
+ Respiratory chain or electron transport chain (ETC) is blocked by site specific inhibitors such as rotenone, amytal, antimycin A, BAL, carbon monoxide and cyanide.
+ Uncoupling of respiration from oxidative phosphorylation under natural conditions assumes biological significance. The brown adipose tissue, rich in electron carriers, brings about oxidation uncoupled from phosphorylation. The presence of active brown adipose tissue in some individuals is believed to protect them from becoming obese. This is because the excess calories consumed by these people are burnt and liberated as heat instead of being stored as fat.
+ Inherited disorders of oxidative phosphorylation caused by the mutations in mitochondrial DNA have been identified e.g. Leber’s hereditary optic neuropathy.
Cyanide poisoning : Cyanide is probably the most potent inhibitor of ETC. It binds to Fe3*+ of cytochrome oxidase blocking mitochondrial respiration leading to cell death. Cyanide poisoning causes death due to tissue asphyxia (mostly of central nervous system).

The mitochondiral transport of electrons is tightly coupled with oxidative phosporylation (ATP synthesis). In other words, oxidation and phosphorylation proceed simultaneously. There are certain compounds that can uncouple (or delink) the electron transport from oxidative phosphorylation. Such compounds, known as uncouplers, increase the permeability of inner mitochondrial membrane to protons (H*). The result is that ATP synthesis does not occur. The energy linked with the transport of electrons is dissipated as heat. The uncouplers allow (often at accelerated rate) oxidation of substrates (via NADH or FADH)) without ATP formation.
The uncoupler, 2,4-dinitrophenol (DNP), has been extensively studied. It is a small lipophilic molecule. DNP is a proton-carrier and can easily diffuse through the inner mitochondrial membrane. In the people seeking to lose weight, DNP was used as a drug. However, this is now discontinued, as it produces hyperthermia and other side effects. In fact, Food and Drug Administration (USA) has banned the use of DNP.
The other uncouplers include dinitrocresol, pentachlorophenol, trifluorocarbonylcyanide phenylhydrazone (FCCP). The last compound (FCCP) is said to be 100 times more effective as an uncoupler than dinitrophenol. When administered in high doses, the drug aspirin acts as an uncoupler.
Physiological uncouplers : Certain physiological substances which act as uncouplers at higher concentration have been identified. These include thermogenin, thyroxine and long chain free fatty acids. The unconjugated bilirubin is also believed to act as an uncoupler. This is, be doubt.
however, yet to proved beyond

Uncoupling of respiration from oxidative phosphorylation under natural conditions assumes biological significance. The maintenance of body temperature is particularly important in hairless animals, hibernating animals and the animals adapted to cold. These animals possess a specialized tissue called brown adipose tissue in the upper back and neck portions. The mitochondria of brown adipose tissue are rich in electron carriers and are specialized to carry out an oxidation uncoupled from phosphorylation. This causes liberation of heat when fat is oxidized in the brown adipose tissue. Brown adipose tissue may be considered as a site of non-shivering thermogenesis. The presence of active brown adipose tissue in certain individuals is believed to protect them from becoming obese. The excess calories consumed by these people are burnt and liberated as heat, instead of being stored as fat.
Thermogenin (or uncoupling protein, UCPI) is a physiological uncoupler, located in the inner mitochondrial membrane of brown adipose tissue. It blocks the formation of ATP, and generates heat. This assumes significant in the newborn, and during hibernation in animals.
lonophores : The term 'ionophores' is used to collectively represent the lipophilic substances that promote the transport of ions across biological membranes.
All the uncouplers (described above) are, in fact, proton ionophores.
The antibiotics valinomycin, gramicidin A and nigercin act as ionophores for K* ions. Both these compounds are also capable of dissipating proton gradient across the inner mitochondrial membrane and inhibit oxidative phosphorylation.

Oligomycin : This antibiotic prevents the mitochondrial oxidation as well as phosphorylation. It binds with the enzyme ATP
Fig. 11.12 : Glycerol-phosphate shuttle (reducing equivalents transported are shown in Blue).
synthase and blocks the proton (H*) channels. It thus prevents the translocation (re-entry) of protons into the mitochondrial matrix. Due to this, protons get accumulated at higher concentration in the intermembrane space. Electron transport (respiration) ultimately stops, since protons cannot be pumped out against steep proton gradients.
Atractyloside : This is a plant toxin and inhibits oxidative phosphorylation by an indirect mechanism. Adenine nucleotide carrier system facilitates the transport of ATP and ADP. Atractyloside inhibits adenine nucleotide carrier and, thus, blocks the adequate supply of ADP, thereby preventing phosphorylation.

The inner mitochondrial membrane _ is impermeable to NADH. Therefore, the NADH produced in the cytosol cannot directly enter the mitochondria. Two pathways—namely glycerol- phosphate shuttle and malate-aspartate shuttle— are operative to do this job. They transport the reducing equivalents from cytosol to mitochondria and not vice versa.

Cytosolic glycerol 3-phosphate dehydrogenase oxidizes NADH to NAD+. The reducing equivalents are transported through glycerol 3-phosphate into the mitochondria. Glycerol 3-phosphate dehydrogenase—present on outer surface of inner mitochondrial membrane— reduces FAD to FADH). Dihydroxyacetone phosphate escapes into the cytosol and the shuttling continues as depicted in Fig.11.12. FADH, gets oxidized via ETC to generate 2 ATP.

In the cytosol, oxaloacetate accepts the reducing equivalents (NADH) and becomes malate. Malate then enters mitochondria where it is oxidized by mitochondrial malate dehydro-

genase. In this reaction, NADH and oxaloacetate are regenerated. NADH gets oxidized via electron transport chain and 3 ATP are produced. This is in contrast to glycerolphosphate shuttle where only 2 ATP are produced.
In the mitochondria, oxaloacetate participates in transamination reaction with glutamate to produce aspartate and a-ketoglutarate. The aspartate enters the cytosol and transaminates with a-ketoglutarate to give oxaloacetate and glutamate. The malate-aspartate shuttle is shown in
Fig.11.13.

Liver and heart utilize malate-aspartate shuttle, and yield 3 ATP per mole of NADH. Most of the other tissues, however, employ glycerol-phosphate shuttle and liberate 2 ATP from NADH.

All the enzymes participating in biological oxidation belong to the class oxidoreductases. These are further grouped into four categories
1. Oxidases
2. Dehydrogenases
3. Hydroperoxidases
4. Oxygenases.
1. Oxidases : These enzymes catalyse the elimination of hydrogen from the substrates which is accepted by oxygen to form mostly water, e.g. cytochrome oxidase, tyrosinase, monoamine oxidase (HO, formed instead of H,0).
the
___
terminal component of electron transport chain, transfers
electrons
(obtained from
the oxidation
of
substrate molecules by dehydrogenases) to final acceptor, oxygen.
the
Some flavoproteins containing FAD or FMN also belong to the category of oxidases. e.g., L-amino acid oxidase (FMN), xanthine oxidase (FAD).
2. Dehydrogenases : As the name indicates, these enzymes cannot utilize oxygen as hydrogen acceptor. They catalyse the reversible transfer of hydrogen from one substrate to another and, thus, bring about oxidationreduction reactions. There are a large number of enzymes belonging to this group
1 NAD* dependent dehydrogenases, e.g. alcohol dehydrogenase, glycerol 3-phosphate dehydrogenase.
1 NADP* dependent dehydrogenases, e.g. HMG CoA reductase, enoyl reductase.
1 FMN dependent dehydrogenases, e.g. NADH dehydrogenase.
1 FAD dependent dehydrogenases, e.g. succinate dehydrogenase, acyl CoA _ dehydrogenase.
1 The cytochromes : All the cytochromes of electron transport chain (b, c, and c) except the terminal cytochrome oxidase (a+a,) belong to this group.
3. Hydroperoxidases : Hydrogen peroxide is the substrate for these enzymes. There is a constant production of HjO, in the reactions catalysed by the aerobic dehydrogenases. The harmful effects of H,O j are prevented by hydroperoxidases, e.g. peroxidase and catalase.
2H,0)
—

>
2H20
+
Oy
(Note : The reader must refer Chapter 34 for details on free radicals and antioxidants)
4. Oxygenases This group of enzymes catalyses the direct incorporation of oxygen into the substrate molecules.
| Dioxygenases (true oxygenases) : They are responsible for the incorporation of both the atoms of oxygen (O,) into the substrate, e.g. homogentisate oxidase, L-tryptophan pyrrolase.
1 Monooxygenases (mixed function oxidases) : They catalyse the incorporation of one atom of oxygen (+0,) while the other oxygen atom is reduced to H,O. NADPH usually provides the reducing equivalents, e.g. cytochrome P,,, monooxygenase system of microsomes is responsible for the metabolism of many drugs (amino pyrine, morphine, aniline etc.) and biosynthesis of steroid hormones (from cholesterol). The action of Cyt P.sq is depicted here.

In contrast to eukaryotes, the prokaryotes lack mitochondria. However, prokaryotes possess a separate system for biological oxidation. A set of electron carriers (different from that found in mitochondria) and enzymes of oxidative phosphorylation are bound to the inner cell membrane in prokaryotes. This arrangement of oxidative machinery is one of the reasons to believe that mitochondria of higher organisms have descended from prokaryotic cells.

10. The enzymes participating in biological oxidation belong to the class oxidoreductases. There are five groups, namely oxidases, aerobic dehydrogenases, anaerobic dehydro- genases, hydroperoxidases and oxygenases.
. Bioenergetics deals with the study of energy changes in biochemical reactions. Change in free energy (AG) is valuable in predicting the feasibility of a reaction. A negative and a positive AG, respectively, represent an exergonic (energy-releasing) and endergonic (energy-consuming) reactions.
. High-energy compounds (AG > -7.0 Cal/mol) play a crucial role in the energy transfer of biochemical reactions (e.g. ATP, phosphocreatine, phosphoenolpyruvate).
. ATP is the energy currency of the cell. ATP-ADP cycle acts as a connecting energy link between catabolic and anabolic reactions.
. Respiratory chain or electron transport chain (ETC) located in the inner mitochondrial membrane represents the final stage of oxidizing the reducing equivalents (NADH and FADHs) derived from the metabolic intermediates to water.
. ETC is organized into five distinct complexes. The complexes I to IV are electron carriers while complex V is responsible for ATP production. The components of ETC are arranged in the sequence
NAD+
>
FMN
>
CoQ
>
Cyt
b
>
Cyt
cy
>
Cyt
c
>
Cyt
a
+
a3
—>
Op
. The process of synthesizing ATP from ADP and Pi coupled with ETC is known as oxidative phosphorylation. NADH oxidation with a P : O ratio 3 indicates that 3 ATP are synthesized while FADHg oxidation (P : O ratio 2) results in the production of 2 ATP.
. Among the hypotheses put forth to explain the mechanism of oxidative phosphorylation, the chemiosmotic hypothesis (of Mitchell) is widely accepted. The rotary motor model (of Boyer) involving the conformation changes in the B-subunits of ATP synthase explains the ATP generation.
. NADH produced in the cytosol cannot directly enter mitochondria. Glycerol-phosphate shuttle (generates 2 ATP) and malate-asparate shuttle (generates 3 ATP) operate to overcome the difficulty.

. There are many inhibitors of electron transport chain (rotenone, amytal, antimycin, CO,-CN, HS etc.) and oxidative phosphorylation (oligomycin, atractyloside). Uncouplers (e.g. dinitrophenol) are the substances that delink ETC from oxidative phosphorylation.

1. Write an account of the high-energy compounds in metabolism
2. Describe the components of electron transport chain and discuss the oxidation of NADH.
3. Define oxidative phosphorylation. Discuss chemiosmotic hypothesis in detail.
4. Give an account of the enzymes involved in biological oxidation.
5. Discuss about the inhibitors of ETC and oxidative phosphorylation.

(a) High-energy bonds, (b) Uncouplers, (c) P : O ratio, (d) Redox loops, (e) ATP synthase, (f) Cytochromes, (g) Sites of oxidative phosphorylation, (h) Coenzyme Q, (I) Redox potential, (j) ATP
as energy currency.

1.
The
relation
between
the
change
of
free
energy
(AG),
enthalpy
(AH)
and
entropy
(AS) is
expressed
by
the
equation
. A negative sign of free energy indicates that the reaction is NY
. The bonds responsible for a majority of high-energy compounds are BW
:
. The storage form of high-energy compound in invertebrates is
. A more negative redox potential represents a greater tendency to lose DH
.
. The electron transport chain is located in
. The prosthetic group present in cytochromes ON . The
component
of
electron
transport
chain
which
can
directly
react
with
molecular
oxygen
.
9. The site of ETC inhibited by cyanide
10. Superoxide is converted to H,O, by the enzyme

11. Name the compound with the greatest standard free energy.
(a) ATP (b) Phosphocreatine (c) Cyclic AMP (d) Phosphoenolpyruvate.
12. One of the following components of ETC possesses isoprenoid units
(a) Coenzyme Q (b) Cytochrome (c) Cytochrome b (d) Non-heme iron.
13. The P : O ratio for the oxidation of FADH, is
14. Inner mitochondrial membrane is impermeable
to
(a) H* (b) K* (c) OH” (d) All of them.
15. ATP synthase activity is associated with the mitochondrial enzyme complex
(a) V (b) HII (c) IV (d) I.

Hoss of reactions simultaneously take place in a living cell, in a well-organized and integrated manner. The entire spectrum of chemical reactions, occurring in the living system, are collectively referred to as metabolism.
A metabolic pathway (or metabolic map) constitutes a series of enzymatic reactions to produce specific products. The term metabolite is applied to a substrate or an intermediate or a product in the metabolic reactions.
Metabolism is broadly divided into two categories (Fig.12.1).
1. Catabolism : The degradative processes concerned with the breakdown of complex molecules to simpler ones, with a concomitant release of energy.
2. Anabolism : The biosynthetic reactions involving the formation of complex molecules from simple precursors.
A clear demarcation between catabolism and anabolism is rather difficult, since there are several intermediates common to both the processes. The term amphibolism is also in use for reactions which are both catabolic and anabolic in nature.

The very purpose of catabolism is to trap the energy of the biomolecules in the form of ATP and to generate the substances (precursors)
Fig. 12.2 : The three stages of catabolism (ETC-Electron transport chain).
required for the synthesis of complex molecules. Catabolism occurs in three stages (Fig.12.2).
1. Conversion of complex molecules into their building blocks : Polysaccharides are broken down to monosaccharides, lipids to free fatty acids and glycerol, proteins to amino acids.
few. These include pyruvate, acetyl CoA and the intermediates of citric acid cycle. Besides the availability of precursors, the anabolic reactions are dependent on the supply of energy (as ATP or GTP) and reducing equivalents (as NADPH + H?*).
2. Formation of simple intermediates : The building blocks produced in stage (1) are degraded to simple intermediates such as pyruvate and acetyl CoA. These intermediates are not readily identifiable as carbohydrates, lipids or proteins. A small quantity of energy (as ATP) is captured in stage 2.
3. Final oxidation of acetyl CoA : Acetyl CoA is completely oxidized to CO», liberating NADH and FADH), that finally get oxidized to release large quantity of energy (as ATP). Krebs cycle (or citric acid cycle) is the common metabolic pathway involved in the final oxidation of all energy-rich molecules. This pathway accepts the carbon compounds (pyruvate, succinate etc.) derived from carbohydrates, lipids or proteins.

For the synthesis of a large variety of complex molecules, the starting materials are relatively
The anabolic and catabolic pathways are not reversible and operate independently. As such, the metabolic pathways occur in specific cellular locations (mitochondria, microsomes etc.) and are controlled by different regulatory signals.
The terms—intermediary metabolism and energy metabolism—are also in use. Intermediary metabolism refers to the entire range of catabolic and anabolic reactions, not involving nucleic acids. Energy metabolism deals with the metabolic pathways concerned with the storage and liberation of energy.

types
The biochemical reactions are mainly of
four
1. Oxidation-reduction.
2. Group transfer.
3. Rearrangement and isomerization.
4. Make and break of carbon-carbon bonds.
These reactions are catalysed by specific
enzymes—more than 2,000 known so far.

The metabolic reactions do not occur in isolation. They are interdependent and integrated into specific series that constitute metabolic pathways. It is, therefore, not an easy task to study metabolisms. Fortunately, the basic metabolic pathways in most organisms are essentially identical. For this reason, many organisms can be used to understand metabolisms.
Several methods are employed to elucidate biochemical reactions and the metabolic pathways. These experimental approaches may be broadly divided into 3 categories
1. Use of whole organisms or its components.
2. Utility of metabolic probes.
3. Application of isotopes.
The actual methods employed may be either in vivo (in the living system) or in vitro (in the test tube) or, more frequently, both.
- 1. Use of whole organism or its components
:
- (a) Whole organisms : The ultimate aim of a biochemist is to know the metabolism in the organism as a whole. Glucose tolerance test (GTT),
employed to measure the response of man (or other animals) towards carbohydrate metabolism is a good example of the use of whole organism.
- S Isolated organs, tissue slices, whole cells, subcellular organelles, cell-free systems and recently purified components are frequently used to elucidate biochemical reactions and metabolic pathways.
2. Utility of metabolic probes : Two types of metabolic probes are commonly used to trace out biochemical pathways. These are metabolic inhibitors and mutations. |n both the cases, there is a specific blockade in a metabolic reaction which helps to understand the pathway. Inhibitors of electron transport chain have been largely responsible to elucidate the sequence of electron carriers (Chapter 11). The inborn errors of metabolism in higher organisms and the genetic manipulations in the microorganisms have also contributed a lot to the understanding of metabolisms.
3. Application of isotopes : Isotopes are the atoms with the same number of protons but different neutrons. By use of isotopes, the molecules of the living system can be labelled without altering their chemical properties. Application of isotopes in biochemistry has revolutionized the study of metabolisms. More details on the utility of isotopes in biochemistry are given elsewhere (Chapter 41).

1. The wide range of chemical reactions occurring in the living system are collectively known as metabolism. Catabolism is concerned with the degradation of complex molecules to simpler ones coupled with the liberation of energy (ATP). On the other hand, anabolism deals with the synthetic reactions converting simple precursors to complex molecules, coupled with the consumption of energy (ATP). A metabolic pathway constitutes a series of enzymatic reactions to produce specific products.
2. Several methods are employed to study metabolism. These include the use of the whole organism or its components (organ, tissue, cells, organelles etc.), utility of metabolic probes (inhibitors and mutations) and application of isotopes.
arbohydrates are the major source of energy for the living cells. As such, carbohydrates are the first cellular constituents, synthesized by green plants during photosynthesis from carbon dioxide and water, on absorption of light. Thus, light is the ultimate source of energy for all biological processes.
The monosaccharide glucose is the central molecule in carbohydrate metabolism since all the major pathways of carbohydrate metabolism are connected with it (Fig.13.1). Glucose is utilized as a source of energy, it is synthesized from non-carbohydrate precursors and stored as glycogen to release glucose as and when the need arises. The other monosaccharides important in carbohydrate metabolism are fructose, galactose and mannose.
The fasting blood glucose level in normal individuals is 70-100 mg/dl (4.5-5.5 mmol/l) and it is very efficiently maintained at this level (for details refer Chapter 36). Liver plays a key role in monitoring and_ stabilizing blood glucose levels. Thus liver may be appropriately considered as glucostat monitor.

The important pathways of carbohydrate metabolism are listed
1. Glycolysis (Embden-Meyerhof pathway) : The oxidation of glucose to pyruvate and lactate.
2. Citric acid cycle (Krebs cycle or tricarboxylic acid cycle) : The oxidation of acetyl CoA to CO,. Krebs cycle is the final common oxidative pathway for carbohydrates, fats or amino acids, through acetyl CoA.
3. Gluconeogenesis : The synthesis of glucose from non-carbohydrate precursors (e.g. amino acids, glycerol etc.).
4.
Glycogenesis
:
The formation
of glycogen
from glucose.
5. Glycogenolysis : The breakdown of glycogen to glucose.
6.
Hexose monophosphate
shunt
(pentose phosphate pathway or direct oxidative pathway) : This pathway is an alternative to glycolysis and
Fig. 13.1 : Overview of glucose metabolism. (Note : For majority of the pathways, glucose participates as glucose 6-phosphate).
TCA cycle for the oxidation of glucose (directly to carbon dioxide and water).
7. Uronic acid pathway Glucose is converted to glucuronic acid, pentoses and, in some animals, to ascorbic acid (not in man). This pathway is also an alternative oxidative pathway for glucose.
8. Galactose metabolism : The pathways concerned with the conversion of galactose to glucose and the synthesis of lactose.
9. Fructose metabolism : The oxidation of fructose to pyruvate and the relation between fructose and glucose metabolism.
10. Amino sugar and mucopolysaccharide metabolism : The synthesis of amino sugars and other sugars for the formation of mucopolysaccharides and glycoproteins.

Glucose concentration is very low in the cells compared to plasma (for humans < 100 mg/dl). However, glucose does not enter the cells by simple diffusion. Two specific transport systems are recognized for the entry of glucose into the cells
1. Insulin-independent transport system of glucose : This is a carrier mediated uptake of glucose which is not dependent on the hormone insulin. This is operative in hepatocytes, erythrocytes and brain.
2. Insulin-dependent transport system : This occurs in muscle and adipose tissue.
Glucose transporters : In recent years, at least six glucose transporters (GLUT-1 to GLUT-5 and GLUT-7) in the cell membranes have been identified. They exhibit tissue specificity. For instance, GLUT-1 is abundant in erythrocytes whereas GLUT-4 is abundant in skeletal muscle and adipose tissue.
Insulin increases the number and promotes the activity of GLUT-4 in skeletal muscle and adipose tissue. In type 2 diabetes mellitus, insulin resistance is observed in these tissues. This is due to the reduction in the quantity of GLUT-4 in insulin deficiency.

Glycolysis is derived from the Greek words (glycose—sweet or sugar; lysis—dissolution). It is a universal pathway in the living cells. The complete pathway of glycolysis was elucidated in 1940. This pathway is often referred to as Embden-Meyerhof pathway (E.M. pathway) in honour of the two biochemists who made a major contribution to the knowledge of glycolysis.
Glycolysis is defined as the sequence of reactions converting glucose (or glycogen) to pyruvate or lactate, with the production of ATP.

1. Glycolysis takes place in all cells of the body. The enzymes of this pathway are present in the cytosomal fraction of the cell.
2. Glycolysis occurs in the absence of oxygen (anaerobic) or in the presence of oxygen (aerobic). Lactate is the end product under anaerobic condition. In the aerobic condition, pyruvate is formed, which is then oxidized to CO, and H,O.
3. Glycolysis is a major pathway for ATP synthesis in tissues lacking mitochondria, e.g. erythrocytes, cornea, lens etc.
4. Glycolysis is very essential for brain which is dependent on glucose for energy. The glucose in brain has to undergo glycolysis before it is oxidized to COy and H,O.
5.
(anaerobic)
may
be
summarized
by the net reaction
Glucose + 2ADP + 2Pi —> 2Lactate + 2ATP
6. Glycolysis is a central metabolic pathway with many of its intermediates providing branch point to other pathways. Thus, the intermediates of glycolysis are useful for the synthesis of amino acids and fat.
7. Reversal of glycolysis along with the alternate arrangements at the _ irreversible steps, will result in the synthesis of glucose (gluconeogenesis).

The sequence of reactions of glycolysis is given in Fig.13.2. The pathway can be divided into three distinct phases
A. Energy investment phase or priming stage
B. Splitting phase
C. Energy generation phase.
The sequence of reactions are discussed
below.

- 1. Glucose is phosphorylated to glucose 6-phosphate by hexokinase — or glucokinase (both are isoenzymes). This is an irreversible reaction, dependent on ATP and Mg?*. The enzyme hexokinase is present in almost all the tissues. It catalyses the phosphorylation of various hexoses (fructose, mannose etc.), has low K,, for substrates (about 0.1 mM) and is inhibited by glucose 6-phosphate.
Glucokinase present
in liver,
phosphorylation has
not
Due of
high
K,, for
glucose inhibited
only glucose,
(10
by to
high glucose
mM)
and is
6-phosphate.
affinity is
utilized by
(low hexokinase
concentration, whereas
K,,), glucose
even at
low glucokinase
acts only at higher levels of glucose ie., after a meal when blood glucose concentration is above 100 mg/dl.
Glucose 6-phosphate is impermeable to the cell membrane. It is a central molecule with a variety of metabolic fates—glycolysis, glycogenesis, gluconeogenesis and pentose phosphate pathway.
. Glucose 6-phosphate undergoes isomerization to give fructose 6-phosphate in the presence of the enzyme phosphohexose isomerase and Mg2*.
. Fructose 6-phosphate is phosphorylated to fructose 1,6-bisphosphate by phosphofructokinase (PFK). This is an irreversible and a regulatory step in glycolysis.

4. The six carbon fructose —_1,6bisphosphate is split (hence the name glycolysis) to two three-carbon compounds, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate by the enzyme aldolase (fructose 1,6bisphosphate aldolase).
. The enzyme phosphotriose isomerase catalyses the reversible interconversion of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Thus, two molecules of glyceraldehyde 3-phosphate are obtained from one molecule of glucose.

- 6. Glyceraldehyde 3-phosphate dehydrogenase converts glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. This step is important as it is involved in the formation of NADH + Ht and a high energy compound _ 1,3-bisphosphoglycerate. lodoacetate and arsenate inhibit the enzyme — glyceraldehyde 3-phosphate dehydrogenase. In aerobic condition, NADH passes through the electron transport chain and 6 ATP (2 x 3 ATP) are synthesized by oxidative phosphorylation.
Fig
Fig. 13.2 : The reactions in the pathway of glycolysis (The three steps catalysed by hexokinase, phosphofructokinase and pyruvate kinase, shown in thick lines are irreversible).
- 8. 3-Phosphoglycerate is converted to 2-phosphoglycerate by phosphoglycerate mutase. This is an isomerization reaction.
phosphate to form a complex that binds with active site of enolase and blocks access of substrate. Thus, fluoride is an unusual competitive inhibitor).
10. The enzyme pyruvate kinase catalyses the transfer of high energy phosphate from phosphoenol pyruvate to ADP, leading to the formation of ATP. This step also is a substrate level phosphorylation. This reaction — is irreversible.

Under anaerobic conditions (lack of Oy), pyruvate is reduced by NADH to lactate in presence of the enzyme lactate dehydrogenase (competitive inhibitor-—oxamate). The NADH utilized in this step is obtained from the reaction catalysed by glyceraldehyde 3-phosphate dehydrogenase. The formation of lactate allows the regeneration of NAD*t which can be reused by glyceraldehyde 3-phosphate dehydrogenase so that glycolysis proceeds even in the absence of oxygen to supply ATP.
The occurrence of uninterrupted glycolysis is very essential in skeletal muscle during strenous exercise where oxygen supply is very limited. Glycolysis in the erythrocytes leads to lactate production, since mitochondria—the centres for aerobic oxidation—are absent. Brain, retina, skin, renal medulla and _ gastrointestinal tract derive most of their energy from glycolysis.

Lactic acid is a three carbon hydroxy acid. Elevation of lactic acid in the circulation (normal plasma 4-15 mg/dl) may occur due to _ its increased production or decreased utilization. Mild forms of lactic acidosis (not life-threatening) are associated with strenuous exercise, shock, respiratory diseases, cancers, low pyruvate dehydrogenase activity, von Gierke's disease etc.
Severe forms of lactic acidosis are observed due to impairment/collapse of circulatory system which is often encountered in myocardial infarction, pulmonary embolism, uncontrolled hemorrhage and severe shock. This type of lactic
acidosis is due to inadequate supply of Oy to the tissues with a drastic reduction in ATP synthesis (since the cells have to survive in anaerobic conditions) which may even lead to death. The term oxygen debt refers to the excess amount of O» required to recover. In clinical practice, measurement of plasma lactic acid is useful to know about the oxygen debt, and monitor the patient's recovery, and save the patient from morbidity and mortality.

The details of ATP generation in glycolysis (from glucose) are given in Table 13.1. Under anaerobic conditions, 2 ATP are synthesized while, under aerobic conditions, 8 or 6 ATP are synthesized—depending on the shuttle pathway that operates.
which forms glucose 6-phosphate). Thus, in anaerobic glycolysis, 3 ATP are produced from glycogen.

In the presence of mitochondria and oxygen, the NADH produced in glycolysis can participate in the shuttle pathways (Refer Chapter 11) for the synthesis of ATP. If the cytosolic NADH uses malate-aspartate shuttle, 3 ATP are generated from each molecule of NADH. This is in contrast to glycerolphosphate shuttle that produces only 2 ATP.

When the glycolysis occurs from glycogen, one more ATP is generated. This is because no ATP is consumed for the activation of glucose (glycogen directly produces glucose 1-phosphate
Cancer cells display increased uptake of glucose, and glycolysis. As the tumors grow rapidly, the blood vessels are unable to supply adequate oxygen, and thus a_ condition of hypoxia exists. Due to this, anaerobic glycolysis predominantly occurs to supply energy. The cancer cells get adapted to hypoxic

Glycolysis, Enzyme (method of ATP synthesis) = Glyceraldehyde 3-phosphate dehydrogenase (2 NADH, ETC, oxidative phosphorylation). Glycolysis, Number of ATP synthesized = 6(5). , Enzyme (method of ATP synthesis) = Phosphoglycerate kinase (substrate level phosphorylation). , Number of ATP synthesized = 2. , Enzyme (method of ATP synthesis) = Pyruvate kinase (substrate level phosphorylation). , Number of ATP synthesized = 2. , Enzyme (method of ATP synthesis) = Two ATP are consumed in the reactions catalysed by hexokinase and phosphofructokinase. , Number of ATP synthesized = -2. , Enzyme (method of ATP synthesis) = Net ATP synthesis in glycolysis in aerobic condition. , Number of ATP synthesized = 8(7). , Enzyme (method of ATP synthesis) = Pyruvate dehydrogenase (2 NADH, ETC, oxidative phosphorylation). , Number of ATP synthesized = 6(5). Citric acid cycle, Enzyme (method of ATP synthesis) = Isocitrate dehydrogenase (2 NADH, ETC, oxidative phosphorylation). Citric acid cycle, Number of ATP synthesized = 6(5). , Enzyme (method of ATP synthesis) = D -Ketoglutarate dehydrogenase. , Number of ATP synthesized = 6(5). , Enzyme (method of ATP synthesis) = Succinate thiokinase (substrate level phosphorylation). , Number of ATP synthesized = 2. , Enzyme (method of ATP synthesis) = Succinate dehydrogenase (2 FADH 2 , ETC, oxidative phosphorylation). , Number of ATP synthesized = 4(3). , Enzyme (method of ATP synthesis) = Malate dehydrogenase (2 NADH, ETC, oxidative phosphorylation). , Number of ATP synthesized = 6(5). , Enzyme (method of ATP synthesis) = Total ATP per mole of glucose under aerobic condition. , Number of ATP synthesized = 38(32). , Enzyme (method of ATP synthesis) = Total ATP per mole of glucose under anaerobic condition. , Number of ATP synthesized = 2

Most of the reactions of glycolysis are reversible. However, the three steps catalysed by the enzymes hexokinase (or glucokinase), phosphofructokinase and pyruvate kinase, are irreversible. These three stages mainly regulate glycolysis. The reversal of glycolysis, with alternate arrangements made at the three irreversible stages, leads to the synthesis of glucose from pyruvate (gluconeogenesis).

The three enzymes namely hexokinase (and glucokinase), phosphofructokinase and pyruvate kinase, catalysing the irreversible reactions regulate glycolysis.
Hexokinase_ is inhibited by — glucose 6-phosphate. This enzyme prevents the accumulation of glucose 6-phosphate due to product inhibition. | Glucokinase, — which specifically phosphorylates glucose, is an inducible enzyme. The substrate glucose, probably through the involvement of insulin, induces glucokinase.
Phosphofructokinase (PFK) is the most important regulatory enzyme in glycolysis. This enzyme catalyses the rate limiting committed step. PFK is an allosteric enzyme regulated by allosteric effectors. ATP, citrate and H* ions (low pH) are the most important allosteric inhibitors, whereas, fructose 2,6-bisphosphate, ADP, AMP and Pi are the allosteric activators.

Fructose 2,6-bisphosphate (F2,6-BP) is considered to be the most important regulatory factor
Fig. 13.3 : Regulation of fructose 2,6-bisphosphatase.
(activator) for controlling PFK and, ultimately, glycolysis in the liver. F2,6-BP is synthesized from fructose 6-phosphate by the enzyme phosphofructokinase called PFK-2 (PFK-1 is the glycolytic enzyme). F2,6-BP is hydrolysed by fructose 2,6-bisphosphatase. The function of synthesis and degradation of F2,6-BP is brought out by a single enzyme (same polypeptide with two active sites) which is referred to as bifunctional enzyme (Fig.13.3). \n fact, the combined name of phosphofructokinase-2/ fructose 2,6-bisphosphatase is used to refer to the enzyme that synthesizes and degrades F2,6-BP.
The activity of PFK-2 and fructose 2,6bisphosphatase is controlled by covalent modification which, in turn, is regulated by cyclic AMP (cAMP is the second messenger for certain hormones). Cyclic AMP brings about dephosphorylation of the bifunctional enzyme, resulting in inactivation of active site responsible for the synthesis of F2,6-BP but activation of the active site responsible for the hydrolysis of F2,6-BP.
Pyruvate kinase also regulates glycolysis. This enzyme is inhibited by ATP and activated by F1,6-BP. Pyruvate kinase is active (a) in dephosphorylated state and inactive (b) in phosphorylated state. Inactivation of pyruvate kinase by phosphorylation is brought about by cAMP-dependent protein kinase. The hormone— glucagon inhibits hepatic glycolysis by this mechanism (Fig.13.4).
Fig. 13.4 : Regulation of pyruvate kinase.

The inhibition of glycolysis by oxygen (aerobic condition) is known as Pasteur effect. It was discovered by Louis Pasteur, more than a century ago, while studying fermentation by yeast. He observed that when anaerobic yeast cultures were exposed to air, the utiliziation of glucose decreased by nearly seven fold.
In the aerobic condition, the levels of glycolytic intermediates from fructose 1,6bisphosphate onwards decrease while the earlier intermediates accumulate. This clearly indicates that Pasteur effect is due to the inhibition of the enzyme _ phosphofructokinase. The inhibitory effect of citrate and ATP (produced in the presence of oxygen) on phosphofructokinase explains the Pasteur effect.

The phenomenon of inhibition of oxygen consumption by the addition of glucose to tissues having high aerobic glycolysis is known as Crabtree effect. Basically, this is opposite to that of Pasteur effect. Crabtree effect is due to increased competition of glycolysis for inorganic phosphate (Pi) and NAD+ which limits — their availability for phosphorylation and oxidation.

Dental caries refers to the destruction or decalcification of hard teeth due to organic acids released by bacterial infections. The anaerobic bacteria (e.g. Streptococcus mutans, Lactobacillus sps) that colonize the oral cavily contribute to the development of dental caries. These bacteria grow optimally on refined and fermentable sugars (e.g. sucrose of chocolates, candies) by utilizing anaerobic glycolysis. Lactic acid and other acids (produced by bacteria) erode tooth enamel and dentin, and dissolve hydroxyapatite matrix of teeth that results in cavity formation. Low levels of fluoride, from tooth pastes or when applied topically can inhibit the enzyme enolase and reduce glycolysis and thus tooth decay. Further, fluoride integrates into tooth surface to form fluoroapatite which offers resistance to demineralization.

This is a supplementary pathway to glycolysis which is operative in the erythrocytes of man and other mammals. Rapaport-Leubering cycle is mainly concerned with the synthesis of 2,3-bisphosphoglycerate (2,3-BPG) in the RBC. 1,3-Bisphosphoglycerate (1,3-BPG) produced in glycolysis is converted to 2,3-BPG by the enzyme 2,3-bisphosphoglycerate mutase (Fig.13.5). 2,3-BPG is hydrolysed to 3-phosphoglycerate by bisphosphoglycerate phosphatase. It is now believed that bisphosphoglycerate mutase is a bifunctional enzyme with mutase and phosphatase activities catalysed by two different sites present on the same enzyme.
About 15-25% of the glucose that gets converted to lactate in erythrocytes goes via 2,3BPG synthesis.

1. Production of 2,3-BPG allows the glycolysis to proceed without the synthesis of ATP. Rapaport-Leubering cycle, therefore is a shunt pathway of glycolysis to dissipate or waste the energy not needed by erythrocytes.
2. 2,3-BPG, is not a waste molecule in RBC. It combines with hemoglobin (Hb) and reduces Hb. affinity with oxygen. Therefore, in the presence of 2,3-BPG, oxyhemoglobin unloads more oxygen to the tissues.
in
Increase in erythrocyte 2,3-BPG is observed hypoxic condition, high altitude, fetal tissues,

Pyruvate is converted to acetyl CoA by oxidative decarboxylation. This is an irreversible reaction, catalysed by a multienzyme complex, known as pyruvate dehydrogenase complex (PDH), which is found only in the mitochondria. High activities of PDH are found in cardiac muscle and kidney. The enzyme PDH requires five cofactors (coenzymes), namely—TPP, lipoamide, FAD, coenzyme A and NAD+ (lipoamide contains lipoic acid linked to €-amino group of lysine). The overall reaction of PDH is
Pyruvate
+
NADt
+
Coa
POH,
Acetyl
CoA
+
CO,
+
NADH
+
H*

The sequence of reactions brought about by different enzymes of PDH complex in association with the coenzymes is depicted in Fig.13.6. Pyruvate is decarboxylated to give hydroxyethyl TPP, catalysed by PDH (decarboxylase activity). Dihydrolipoyl transacetylase brings about the formation of acetyl lipoamide (from hydroxethyl-TPP) and then catalyses the transfer of acetyl group to coenzyme A to produce acetyl CoA. The cycle is complete when reduced lipoamide is converted to oxidized lipoamide by dihydrolipoyl dehydrogenase, transferring the reducing equivalents to FAD. FADHg, in turn, transfers the reducing equivalents to NAD*t to give NADH + Ht, which can pass through the respiratory chain to give 3 ATP (6 ATP from 2 moles of pyruvate formed from glucose) by oxidative phosphorylation.
The intermediates of PDH catalysed reaction are not free but bound with enzyme complex. In| mammals, the PDH complex has an approximate molecular weight of 9 x 10°. It contains 60 molecules of dihydrolipoyltransacetylase and about 20-30 molecules each of the other two enzymes (pyruvate dehydrogenase & dihydrolipoy! dehydrogenase). A comparable enzyme with PDH _ is a-ketoglutarate dehydrogenase complex of citric acid cycle which catalyses oxidative decarboxylation of a-ketoglutarate to succinyl CoA.
Fig. 13.6 : The mechanism of action of pyruvate dehydrogenase complex. (Note : The reaction involving the conversion of pyruvate to acetyl CoA requires five coenzymes—TPP, lipoamide, CoASH, FAD and NAD'*).
Arsenic poisoning : The enzymes PDH and a-ketoglutarate dehydrogenase are inhibited by arsenite. Arsenite binds to thiol (—SH) groups of lipoic acid and makes it unavailable to serve as cofactor.

Pyruvate dehydrogenase is a good example for end product (acetyl! CoA, NADH) inhibition. Besides this, PDH is also regulated by phosphorylation and dephosphorylation (Fig.13.7) PDH is active as a dephosphoenzyme while it is inactive as a phosphoenzyme. PDH phosphatase activity is promoted by Ca2+, Mg* and insulin (in adipose tissue). It is of interest to note that calcium released during muscle contraction stimulates PDH (by increasing phosphatase activity) for energy production.
PDH kinase (responsible to form inactive
PDH) is promoted by ATP, NADH and acetyl CoA, while it is inhibited by NAD*, CoA and pyruvate. The net result is that in the presence of high energy signals (ATP, NADH), the PDH is turned off.

1. Lack of TPP (due to deficiency of thiamine)
inhibits
PDH
activity resulting
in the
accumulation of pyruvate.
2. In the thiamine deficient alcoholics, pyruvate is rapidly converted to lactate, resulting in lactic acidosis.
3. In patients with inherited deficiency of PDH, lactic acidosis (usually after glucose load) is observed.
4. PDH activity can be inhibited by arsenic and mercuric ions. This is brought about by binding of these ions with —SH groups of lipoic acid.

Pyruvate is a key metabolite. Besides its conversion to acetyl CoA (utilized in a wide range of metabolic reactions-citric acid cycle, fatty acid synthesis etc.), pyruvate is a good substrate for gluconeogenesis.

The citric acid cycle (Krebs cycle or tricarboxylic acid—TCA cycle) is the most important metabolic pathway for the energy supply to the body. About 65-70% of the ATP is synthesized in Krebs cycle. Citric acid cycle essentially involves the oxidation of acetyl CoA to CO, and H,O. This cycle utilizes about twothirds of total oxygen consumed by the body. The name TCA cycle is used, since, at the outset of the cycle, tricarboxylic acids (citrate, cisaconitate and isocitrate) participate.

The citric acid cycle is the final common oxidative pathway for carbohydrates, fats and amino acids. This cycle not only supplies energy but also provides many intermediates required for the synthesis of amino acids, glucose, heme etc. Krebs cycle is the most important central pathway connecting almost all the individual metabolic pathways (either directly or indirectly).

The citric acid cycle was proposed by Hans Adolf Krebs in 1937, based on the studies of oxygen consumption in pigeon breast muscle. The cycle is named in his honour (Nobel Prize for Physiology and Medicine in 1953.)
[Note : It is of interest to note that the original manuscript on TCA cycle submitted by Krebs to the journal 'Nature' was not accepted. He published it in another journal Enzymoligia. Krebs used to carry the rejection letter (of Nature) with him, and advise the researches never to be discouraged by research paper rejection].

The enzymes of TCA cycle are located in mitochondrial matrix, in close proximity to the electron transport chain. This enables the synthesis of ATP by oxidative phosphorylation without hindrance.
any
Fig. 13.8 : An overview of Krebs cycle.

Krebs cycle _ basically involves the combination of a two carbon acetyl CoA with a four carbon oxaloacetate to produce a six carbon tricarboxylic acid, citrate. In the reactions that follow, the two carbons are oxidized to CO, and oxaloacetate is regenerated and_ recycled. Oxaloacetate is considered to play a catalytic role in citric acid cycle. An overview of Krebs cycle is depicted in Fig.13.8.

Krebs cycle is a cyclic process. However, it should not be viewed as a closed circle, since many compounds enter the cycle and leave. TCA cycle is comparable to a heavy traffic circle in a national highway with many connecting roads. Each intermediate of the cycle connecting another pathway is a road!

Oxidative decarboxylation of pyruvate to acetyl CoA by pyruvate dehydrogenase complex is discussed above. This step is a connecting link between glycolysis and TCA cycle. A few authors, however, describe the conversion of pyruvate to acetyl CoA along with citric acid cycle. The events of TCA cycle are described hereunder (Fig.13.9).
1. Formation of citrate : Krebs cycle proper starts with the condensation of acetyl CoA and oxaloacetate, catalysed by the enzyme citrate synthase.
2. and 3. Citrate is isomerized to isocitrate by the enzyme aconitase. This is achieved in a two stage reaction of dehydration followed by hydration through the formation of an intermediate—cis-aconitate.
4. and 5. Formation of o-ketoglutarate : The enzyme isocitrate dehydrogenase (ICD) catalyses the conversion (oxidative decarboxylation) of isocitrate to oxalosuccinate and then to a-ketoglutarate. The formation of NADH and the liberation of CO, occur at this stage.
6. Conversion of o-ketoglutarate to succinyl CoA occurs through oxidative decarboxylation, catalysed by o-ketoglutarate dehydrogenase complex. This enzyme is dependent on five cofactors—TPP, lipoamide, NAD*, FAD and CoA. The mechanism of the reaction is analogous to the conversion of pyruvate to acetyl

7. Formation of succinate : Succiny! CoA is converted to succinate by succinate thiokinase. This reaction is coupled with — the phosphorylation of GDP to GTP. This is a substrate level phosphorylation. GTP is converted to ATP by the enzyme nucleoside diphosphate kinase.
8. Conversion of succinate to fumarate : Succinate is oxidized by succinate dehydrogenase to fumarate. This reaction results in the production of FADH, and not NADH.
9. Formation of malate The enzyme fumarase catalyses the conversion of fumarate to malate with the addition of HO.
10. Conversion of malate to oxaloacetate : Malate is then oxidized to oxaloacetate by malate dehydrogenase. The third and _ final synthesis of NADH occurs at this stage. The oxaloacetate is regenerated which can combine with another molecule of acetyl CoA, and continue the cycle.

as
The events of Krebs cycle may be summarized given in the next column
Acetyl CoA + 3 NAD* + FAD + GDP + Pi
+
2H ,O —> 2CO, + 3NADH + 3Ht + FADH) +
GTP + CoA

There is no direct participation of oxygen in Krebs cycle. However, the cycle operates only under aerobic conditions. This is due to the fact that NAD* and FAD (from NADH and FADH), respectively) required for the operation of the cycle can be regenerated in the ETC only in the presence of O». Therefore, citric acid cycle is strictly aerobic in contrast to glycolysis which operates in both aerobic and anaerobic conditions.

During the process of oxidation of acetyl CoA via citric acid cycle, 4 reducing equivalents (3 as NADH and one as FADH)) are produced. Oxidation of 3 NADH by electron transport chain coupled with oxidative phosphorylation results in the synthesis of 9 ATP, whereas FADH) leads to the formation of 2 ATP. Besides, there is one substrate level phosphorylation. Thus, a total of twelve ATP (10 as per recent evidence) are produced from one acetyl CoA.

Four B-complex vitamins are essential for
Krebs cycle, and thus energy generation
1.
Thiamine
(as
TPP)
as
a
coenzyme for
a-ketoglutarate dehydrogenase.
2. Riboflavin (as FAD) as a coenzyme for succinate dehydrogenase.
3. Niacin (as NAD*) as electron acceptor for isocitrate dehydrogenase, a-ketoglutarate dehydrogenase and malate dehydrogenase.
4. Pantothenic acid (as coenzyme A) attached to active carboxylic acid residues i.e. acetyl CoA, succinyl CoA.

The important enzymes of TCA cycle inhibited by the respective inhibitors are listed
Enzyme
Inhibitor
Aconitase
Fluoroacetate
(non-competitive)
Arsenite
Succinate
Malonate
(competitive)
Fluoroacetate—a suicide substrate The inhibitor fluoroacetate is first activated to fluoroacety] CoA which then condenses with oxaloacetate to form fluorocitrate. TCA cycle (enzyme-aconitase) is inhibited by fluorocitrate. The compound fluoroacetate, as such, is a harmless substrate. But it is converted to a toxic compound (fluorocitrate) by cellular metabolism. This is a suicide reaction committed by the cell, and thus fluoroacetate is regarded as a suicide substrate

The cellular demands of ATP are crucial in controlling the rate of citric acid cycle. The regulation is brought about either by enzymes or the levels of ADP. Three enzymes—namely citrate synthase, isocitrate dehydrogenase and a-ketoglutarate dehydrogenase—regulate citric acid cycle.
1. Citrate synthase is inhibited by ATP, NADH, acetyl CoA and succinyl CoA.
- 2. Isocitrate dehydrogenase is activated by
ADP, and inhibited by ATP and NADH.
by
- 3. a-Ketoglutarate dehydrogenase is inhibited succinyl CoA and NADH.
4. Availability of ADP is very important for the citric acid cycle to proceed. This is due to the fact that unless sufficient levels of ADP are available, oxidation (coupled with phosphorylation of ADP to ATP) of NADH and FADH) through electron transport chain stops. The accumulation of NADH and FADH) will lead to inhibition of the enzymes (as stated above) and also limits the supply of NAD+ and FAD which are essential for TCA cycle to proceed.

The citric acid cycle provides various intermediates for the synthesis of many compounds needed by the body. Krebs cycle is both catabolic and anabolic in nature, hence regarded as amphibolic.
TCA cycle is actively involved in gluconeogenesis, transamination and deamination.
The most important synthetic (anabolic) reactions connected with TCA cycle are given (Fig.13.10)
- 1. Oxaloacetate and o-ketoglutarate,
respectively, serve as precursors for the synthesis of aspartate and glutamate which, in turn, are required for the synthesis of other non-essential amino acids, purines and pyrimidines.
2.
Succinyl
CoA
is used
for the
porphyrins and
heme.
3. Mitochondrial citrate is transported to the cytosol, where it is cleaved to provide acetyl CoA for the biosynthesis of fatty acids, sterols etc.

The synthetic reactions described above deplete the intermediates of citric acid cycle. The cycle will cease to operate unless the intermediates drawn out are replenished. The reactions concerned to replenish or to fill up the intermediates of citric acid cycle are called anaplerotic reactions or anaplerosis (Greek : fill up). In Fig.13.10, the important synthetic pathways that draw the intermediates of TCA cycle and the anaplerotic reactions to fill them up are given.
The salient features of important anaplerotic reactions are described
1. Pyruvate carboxylase catalyses the conversion of pyruvate to oxaloacetate. This is an ATP dependent carboxylation reaction.
Pyruvate
+
CO
+
ATP
—>
Oxaloacetate + ADP + Pi
The details of the above described under gluconeogenesis. reaction are
2. Pyruvate is converted to malate by NADP* dependent malate dehydrogenase (malic enzyme).
Pyruvate + CO, + NADPH + Ht ===
Malate + NADP* + H,O
3. Transamination is a process wherein an amino acid transfers its amino group to a keto acid and itself gets converted to a keto acid. The formation of a-ketoglutarate and oxaloacetate occurs by this mechanism.
Fig. 13.10 : Major synthetic and anaplerotic pathways of the intermediates of citric acid cycle.
4.
a-Ketoglutarate from
can also
be synthesized
glutamate by
action.
Glutamate
+ NAD(P)* + H,0 © o-Ketoglutarate + NAD(P)H + H* + NHZ
living system, energy is trapped leading to the synthesis of 38 ATP which is equivalent to 1,159 KJ (1 ATP has high energy bond equivalent to 30.5 KJ). That is, about 48% of the energy in glucose combustion is actually captured for ATP generation.

When a molecule of glucose (6 carbon) undergoes glycolysis, 2 molecules of pyruvate or lactate (3 carbon) are produced. Pyruvate is oxidatively decarboxylated to acetyl CoA (2 carbon) which enters the citric acid cycle and gets completely oxidized to CO, and HO. The overall process of glucose being completely oxidized to CO, and H,O via glycolysis and citric acid cycle is as follows
CH
1204
+
607
+
38ADP
+
38Pi
—>
6CO,
+
6H,O
+
38ATP
The enzymes of glucose metabolism responsible for generating ATP are given in Table 13.1.
When a molecule of glucose is burnt in a the calorimeter, 2,780 KJ of heat is liberated. In
glutamate dehydrogenase

The synthesis of glucose from noncarbohydrate compounds is known as gluconeogenesis. The major substrates/precursors for gluconeogenesis are lactate, pyruvate, glucogenic amino acids, propionate and glycerol.

Gluconeogenesis occurs mainly in the cytosol, although some precursors are produced in the mitochondria. Gluconeogenesis mostly takes place in liver (about 1 kg glucose synthesized everyday) and, to some extent, in kidney matrix (about one-tenth of liver capacity).

Glucose occupies a key position in the metabolism and its continuous supply is absolutely essential to the body for a variety of functions
1. Brain and central nervous — system, erythrocytes, testes and kidney medulla are dependent on glucose for continuous supply of energy. Human brain alone requires about 120 g of glucose per day, out of about 160 g needed by the entire body.
2. Glucose is the only source that supplies the skeletal muscle, under anaerobic energy to conditions.
3. In fasting even more than a day, gluconeogenesis must occur to meet the basal requirements of the body for glucose and to maintain the intermediates of citric acid cycle. This is essential for the survival of humans and other animals.
4. Certain metabolites produced in the tissues accumulate in the blood, e.g. lactate, glycerol, propionate etc. Gluconeogenesis effectively clears them from the blood.

Gluconeogenesis closely resembles the reversed pathway of glycolysis, although it is not the complete reversal of glycolysis. Essentially, 3 (out of 10) reactions of glycolysis are irreversible. The seven reactions are common for both glycolysis and gluconeogenesis (Fig.13.11). The three irreversible steps of glycolysis are catalysed by the enzymes, namely hexokinase, phosphofructokinase and pyruvate kinase. These three stages—bypassed by alternate enzymes specific to gluconeogenesis—are discussed
1. Conversion of pyruvate to phosphoenolpyruvate : This takes place in two steps (Fig.13.12). Pyruvate carboxylase is a biotin— dependent mitochondrial enzyme that converts pyruvate to oxaloacetate in presence of ATP and

+ Glycolysis is an important source of energy supply for brain, retina, skin and renal medulla.
+ The crucial significance of glycolysis is its ability to generate ATP in the absence of oxygen.
+ Skeletal muscle, during strenous exercise, requires the occurrence of uninterrupted glycolysis. This is due to the limited supply of oxygen.
+ The cardiac muscle cannot survive for long in the absence of oxygen since it is not well adapted for glycolysis under anaerobic conditions.
+ Glycolysis in erythrocytes is associated with 2, 3-bisphosphoglycerate (2,3-BPG) produc- tion. In the presence of 2, 3-BPG, oxyhemoglobin unloads more oxygen to the tissues.
+ The occurrence of glycolysis is very much elevated in rapidly growing cancer cells.
+ Lactic acidosis is also observed in patients with deficiency of the enzyme pyruvate dehydrogenase. It could also be due to collapse of circulatory system encountered in myocardial infarction and pulmonary embolism.
+ Citric acid cycle is the final common oxidative pathway for carbohydrates, fats and amino acids. It utilizes (indirectly) about 2/3 of the total oxygen consumed by the body and generates about 2/3 of the total energy (ATP).
+ Unlike the other metabolic pathways/cycles, very few genetic abnormalities of Krebs cycle are known. This may be due to the vital importance of this metabolic cycle for the survival of life.
Fig.
Fig. 13.11 : The pathway of gluconeogenesis. [The enzymes catalysing irreversible steps in glycolysis are shown in red. The important enzymes participating in gluconeogenesis are shown in shaded green. The substrates for gluconeogenesis are in blue. The numbers represent the entry of glucogenic amino acids : (1) Alanine, glycine, serine, cysteine, threonine and tryptophan; (2) Aspartate and asparagine; (3) Arginine, glutamate, glutamine, histidine, proline; (4) Isoleucine, methionine, valine; (5) Phenylalanine, tyrosine].

malate is catalysed by malate dehydrogenase, an enzyme present in both mitochondria and cytosol.
In the cytosol, phosphoenolpyruvate carboxykinase converts oxaloacetate to phosphoenolpyruvate. GTP or ITP (not ATP) is used in this reaction and the CO, (fixed by carboxylase) is liberated. For the conversion of pyruvate to phosphoenol pyruvate, 2 ATP equivalents are utilized. This is in contrast to only one ATP that is liberated in glycolysis for this reaction.
Fig. 13.12 : Conversion of pyruvate to phosphoenolpyruvate.
2. Conversion of fructose 1,6-bisphosphate to fructose 6-phosphate : Phosphoenolpyruvate undergoes the reversal of glycolysis until fructose 1,6-bisphosphate is produced. The enzyme fructose 1,6-bisphosphatase converts fructose 1,6-bisphosphate to fructose 6-phosphate. This enzyme requires Mg2* ions. Fructose 1,6bisphosphatase is absent in smooth muscle and heart muscle. This enzyme is also regulatory in gluconeogenesis.
3. Conversion of glucose 6-phosphate to glucose : Glucose 6-phosphatase catalyses the conversion of glucose 6-phosphate to glucose. The presence or absence of this enzyme in a tissue determines whether the tissue is capable of contributing glucose to the blood or not. It is mostly present in liver and kidney but absent in muscle, brain and adipose tissue.
The overall
summary of
gluconeogenesis for
the conversion of pyruvate to glucose is shown below
2 Pyruvate + 4ATP + 2GTP + 2NADH + 2Ht* + 6H,O —-> Glucose + 2NAD* + 4ADP + 2GDP + 6Pi + 6Ht

The carbon skeleton of glucogenic amino acids (all except leucine and lysine) results in the formation of pyruvate or the intermediates of citric acid cycle (Fig.13.11) which, ultimately, result in the synthesis of glucose.

Glycerol is liberated mostly in the adipose tissue by the hydrolysis of fats (triacylglycerols). The enzyme glycerokinase (found in liver and kidney, absent in adipose tissue) activates glycerol to glycerol 3-phosphate. The latter is converted to dihydroxyacetone phosphate by glycerol 3-phosphate dehydrogenase. Dihydroxyacetone phosphate is an intermediate in glycolysis which can be conveniently used for glucose production.

Oxidation of odd chain fatty acids and the breakdown of some amino acids (methionine, isoleucine) yields a three carbon propionyl CoA. Propionyl CoA carboxylase acts on this in presence of ATP and biotin and converts to methyl malonyl CoA which is then converted to succinyl CoA in presence of By, coenzyme (Refer Fig.7.38). Succinyl CoA formed from propionyl CoA enters gluconeogenesis via citric acid cycle.

Lactate produced by active skeletal muscle is a major precursor for gluconeogenesis. Under anaerobic conditions, pyruvate is reduced to lactate by lactate dehydrogenase (LDH)

Pyruvate + NADH + H* == Lactate + NAD*
Lactate is a dead end in glycolysis, since it must be reconverted to pyruvate for its further metabolism. The very purpose of lactate production is to regenerate NADH so _ that glycolysis proceeds uninterrupted in skeletal muscle. Lactate or pyruvate produced in the muscle cannot be utilized for the synthesis of

glucose due to the absence of the key enzymes of gluconeogenesis (glucose 6-phosphatase and fructose 1,6-bisphosphatase).

The plasma membrane is freely permeable to lactate. Lactate is carried from the skeletal muscle through blood and handed over to liver, where it is oxidized to pyruvate. Pyruvate, so produced, is converted to glucose' by gluconeogenesis, which is then transported to the skeletal muscle.
The cycle involving the synthesis of glucose in liver from the skeletal muscle lactate and the reuse of glucose thus synthesized by the muscle for energy purpose is known as Cori cycle
(Fig.13.13).

There is a continuous transport of amino acids from muscle to liver, which predominantly occurs during starvation. Alanine dominates among the transported amino acids. It is postulated that pyruvate in skeletal muscle undergoes transamination to produce alanine. Alanine is transported to liver and used for gluconeogenesis. This cycle is referred to as glucose-alanine cycle (Fig.13.13).
The hormone glucagon and the availability of substrates mainly regulate gluconeogenesis, as discussed hereunder.
Influence of glucagon : This is a hormone, secreted by a-cells of the pancreatic islets. Glucagon stimulates gluconeogenesis by two mechanisms
1. Active form of pyruvate kinase is converted to inactive form through the mediation of cyclic AMP, brought about by glucagon. Decreased pyruvate kinase results in the reduced conversion of phosphoenol pyruvate to pyruvate and the former is diverted for the synthesis of glucose.
2. Glucagon reduces the concentration of fructose 2,6-bisphosphate. This compound allosterically inhibits | phosphofructokinase and activates fructose 1,6-bisphosphatase, both favour increased gluconeogenesis.
Availability of substrates : Among the various substrates, glucogenic amino acids have stimulating influence on gluconeogenesis. This is particularly important in a condition like diabetes mellitus (decreased insulin level) where
amino acids are mobilized from muscle protein for the purpose of gluconeogenesis.
Acetyl CoA promotes gluconeogenesis During starvation—due to excessive lipolysis in adipose tissue—acetyl CoA accumulates in the liver. Acetyl CoA allosterically activates pyruvate carboxylase resulting in enhanced glucose production.

Ethanol oxidation in the liver to acetaldehyde by the enzyme alcohol dehydrogenase utilizes NAD*. The excess NADH produced in the liver interferes with gluconeogenesis as_ illustrated below.
Ethanol + NAD+ —> Acetaldehyde + NADH + H*
Pyruvate
+
NADH
+
H*
©
Lactate
+
NAD*
Oxaloacetate
+
NADH
+
H*<
Malate
+
NAD*
It is evident from the above reactions that pyruvate and oxaloacetate, the predominant substrates for gluconeogenesis, are made unavailable by alcohol intoxication. This happens due to overconsumption of NAD*t and excessive production of NADH by alcohol.
Alcohol consumption increases the risk of hypoglycemia (reduced plasma glucose) due to reduced gluconeogenesis. Hypoglycemia is frequently observed in diabetic patients (particularly on insulin treatment), and undernourished persons consuming alcohol.

It is often stated that glucose cannot be synthesized from fat. In a sense, it is certainly true, since the fatty acids (most of them being even chain), on oxidation, produce acetyl CoA which cannot be converted to pyruvate. Further, the two carbons of acetyl CoA disappear as 2 moles of CO, in TCA cycle. Therefore, even chain fatty acids cannot serve as precursors for glucose formation. The prime reason why animals cannot convert fat to glucose is the absence of glyoxylate cycle (described later).
However, the glycerol released from lipolysis and the propionate obtained from the oxidation of odd chain fatty acids are good substrates for gluconeogenesis, as discussed above.

Glycogen is the storage form of glucose in animals, as is starch in plants. It is stored mostly in liver (6-8%) and muscle (1-2%). Due to more muscle mass, the quantity of glycogen in muscle (250 g) is about three times higher than that in the liver (75 g). Glycogen is stored as granules in the cytosol, where most of the enzymes of glycogen synthesis and breakdown are present.

The prime function of liver glycogen is to maintain the blood glucose levels, particularly between meals. Liver glycogen stores increase in a well-fed state which are depleted during fasting. Muscle glycogen serves as a fuel reserve for the supply of ATP during muscle contraction.

As such, fat is the fuel reserve of the body. However, fat is not preferred, instead glycogen is chosen for a routine, and day to day use of energy for the following reasons
1 Glycogen can be rapidly mobilized
1 Glycogen can generate energy in the absence of oxygen
| Brain depends on continuous glucose supply (which mostly comes from glycogen.)
On the other hand, fat mobilization is slow, needs Oy for energy production and cannot produce glucose (to a significant extent). Thus, fat may be considered as a fixed deposit while glycogen is in the current/saving account in a bank!

The synthesis of glycogen from glucose is glycogenesis (Fig.13.14). Glycogenesis takes place in the cytosol and requires ATP and UTP, besides glucose.
1. Synthesis of UDP-glucose : The enzymes hexokinase (in muscle) and glucokinase (in liver) convert glucose to glucose 6-phosphate. Phosphoglucomutase catalyses the conversion of
glucose 6-phosphate to glucose 1-phosphate. Uridine diphosphate glucose (UDPG) is synthesized from glucose 1-phosphate and UTP by UDP-glucose pyrophosphorylase.
2. Requirement of primer to initiate glycogenesis A small fragment of pre-existing glycogen must act as a 'primer' to initiate glycogen synthesis. It is recently found that in the absence of glycogen primer, a_ specific protein—namely 'glycogenin'—can _ accept glucose from UDPG. The hydroxyl group of the amino acid tyrosine of glycogenin is the site at which the initial glucose unit is attached. The enzyme glycogen initiator synthase transfers the first molecule of glucose to glycogenin. Then glycogenin itself takes up a few glucose residues to form a fragment of primer which serves as an acceptor for the rest of the glucose molecules.
3. Glycogen synthesis by glycogen synthase : Glycogen synthase is responsible for the formation of 1,4-glycosidic linkages. This enzyme transfers the glucose from UDP-glucose to the non-reducing end of glycogen to form a1,4 linkages.

4. Formation of branches in glycogen Glycogen synthase can catalyse the synthesis of a linear unbranched molecule with 1,4 aglycosidic linkages. Glycogen, however, is a branched tree-like structure. The formation of branches is brought about by the action of a branching enzyme, namely glucosyl «-4-6 transferase. (amylo a 1,4 — 1,6 transglucosidase). This enzyme transfers a small fragment of five to eight glucose residues from the non-reducing end of glycogen chain (by breaking o-1,4 linkages) to another glucose residue where it is linked by o-1,6 bond. This leads to the formation of a new non-reducing end, besides the existing one. Glycogen is further elongated and branched, respectively, by the enzymes glycogen synthase and glucosyl 4-6 transferase.
The overall reaction of the glycogen synthesis for the addition of each glucose residue is
(Glucose),, + Glucose + 2ATP
—>
(Glucose),4; + 2 ADP + Pi
Fig. 13.14 : Glycogen synthesis from glucose (glycogenesis).
Of the two ATP utilized, one is required for the phosphorylation of glucose while the other is needed for conversion of UDP to UTP.

The degradation of stored glycogen in liver and muscle constitutes glycogenolysis. The pathways for the synthesis and degradation of glycogen are not reversible. An independent set of enzymes present in the cytosol carry out glycogenolysis. Glycogen is degraded by breaking a-1,4and «@-1,6-glycosidic bonds (Fig. 13.15).
1. Action of glycogen phosphorylase : The o1,4-glycosidic bonds (from the non-reducing ends) are cleaved sequentially by the enzyme glycogen phosphorylase to' yield glucose 1-phosphate. This process—called phosphorolysis—continues until four glucose residues remain on either side of branching point (-1,6glycosidic link). The glycogen so formed is known as limit dextrin which cannot be further degraded by — phosphorylase. Glycogen phosphorylase possesses a molecule of pyridoxal phosphate, covalently bound to the enzyme.
2. Action of debranching enzyme : The branches of glycogen are cleaved by two enzyme activities present on a single polypeptide called debranching enzyme, hence it is a bifunctional enzyme.
Glycosyl 4 : 4 transferase (oligo a-1,4 — 1,4 glucan transferase) activity removes a fragment of three or four glucose residues attached at a branch and transfers them to another chain. Here, one a-1,4-bond is cleaved and the same a-1,4 bond is made, but the places are different. Amylo a-1,6-glucosidase breaks the a-1,6 bond at the branch with a single glucose residue and releases a free glucose.
The remaining molecule of glycogen is again available for the action of phosphorylase and debranching enzyme to repeat the reactions stated in 1 and 2.
3. Formation of glucose 6-phosphate and glucose : Through the combined action of glycogen phosphorylase and debranching
Fig. 13.15 : Glycogen degradation to glucoseglycogenolysis. (The ratio of glucose 1-phosphate to glucose is 8 : 1).

enzyme, glucose 1-phosphate and free glucose in a ratio of 8 : 1 are produced. Glucose 1-phosphate is converted to glucose 6-phosphate by the enzyme phosphoglucomutase.
The fate of glucose 6-phosphate depends on the tissue. The liver, kidney and intestine contain the enzyme glucose 6-phosphatase that cleaves glucose 6-phosphate to glucose. This enzyme is absent in muscle and brain, hence free glucose cannot be produced from glucose 6-phosphate in these tissues. Therefore, liver is the major glycogen storage organ to provide glucose into the circulation to be utilised by various tissues.
In the peripheral tissues, glucose 6-phosphate produced by glycogenolysis will be used for glycolysis. It may be noted that though glucose 6-phosphatase is absent in muscle, some amount of free glucose (8-10% of glycogen) is produced in glycogenolysis due to the action of debranching enzyme (a-1,6-glucosidase activity).

Acid maltase or a-1,4-glucosidase is a lysosomal enzyme. This enzyme continuously degrades a small quantity of glycogen. The significance of this pathway is not very clear. However, it has been observed that the deficiency of lysosomal enzyme a-1,4 glucosidase results in glycogen accumulation, causing a serious glycogen storage disease type Il (ie. Pompe's disease).

A good coordination and glycogen synthesis and its degradation are essential to maintain the blood glucose levels. Glycogenesis and glycogenolysis are, respectively, controlled by the enzymes glycogen synthase and glycogen phosphorylase. Regulation of these enzymes is accomplished by three mechanisms regulation of
2. Hormonal regulation
3. Influence of calcium.
1. Allosteric regulation of glycogen metabolism : There are certain metabolites that allosterically regulate the activities of glycogen synthase and glycogen phosphorylase. The control is carried out in such a way that glycogen synthesis is increased when substrate availability and energy levels are high. On the other hand, glycogen breakdown is enhanced when glucose concentration and energy levels are low. The allosteric regulation of glycogen metabolism is depicted in Fig.13.16. In a well-fed state, the availability of glucose 6-phosphate is high which allosterically activates glycogen synthase for more glycogen synthesis. On the other hand, glucose 6-phosphate and ATP allosterically inhibit glycogen phosphorylase. Free glucose in liver also acts as an allosteric inhibitor of glycogen phosphorylase.
2. Hormonal regulation of glycogen metabolism : The hormones, through a complex series of reactions, bring about covalent modification, namely phosphorylation and dephosphorylation of enzyme proteins which, ultimately control glycogen synthesis or its degradation.

:
Hormones like epinephrine and norepinephrine, and glucagon (in liver) activate adenylate cyclase
to increase the production of cAMP. The enzyme phosphodiesterase breaks down cAMP. The hormone insulin increases the phosphodiesterase activity in liver and lowers the cAMP levels.
In the Fig.13.17, the inhibition of glycogen synthesis brought by epinephrine (also norepinephrine) and glucagon through cAMP by converting active glycogen synthase 'a' to inactive synthase 'b', is given.
Regulation of glycogen degradation by cAMP : The hormones like epinephrine and glucagon bring about glycogenolysis by their action on glycogen phosphorylase through cAMP as illustrated in Fig.13.18. Glycogen phosphorylase exists in two forms, an active 'a' form and inactive form 'b'.
The cAMP—formed due to hormonal stimulus—activates cAMP dependent _ protein kinase. This active protein kinase phosphorylates inactive form of glycogen phsophorylase kinase to active form. (The enzyme protein phosphatase removes phosphate and inactivates phosphorylase kinase). The active phosphorylase kinase phosphorylates inactive glycogen phosphorylase 'b' to active glycogen phospho-
Fig. 13.18 : Hormonal regulation of glycogen degradation (glycogenolysis).
rylase 'a' which degrades glycogen. The enzyme protein phosphatase | can dephosphorylate and convert active glycogen phosphorylase 'a' to inactive 'b' form.

3. Effect of Ca2+ ions on glycogenolysis : When the muscle contracts, Ca2* ions are released from the sarcoplasmic reticulum. Ca2*+ binds to calmodulin-calcium modulating protein and directly activates phosphorylase kinase without the involvement of cAMP-dependent protein kinase.
The overall effect of hormones on glycogen metabolism is that an elevated glucagon or epinephrine level increases glycogen degradation whereas an elevated insulin results in increased glycogen synthesis.
The synthesis and degradative pathways of metabolism (particularly reactions involving phosphorylation and dephosphorylation utilizing ATP) are well regulated and subjected to fine tuning to meet the body demands, with minimal wastage of energy and metabolites. Thus, glycolysis and gluconeogenesis (breakdown of glucose to pyruvate, and conversion of pyruvate to glucose), glycogenolysis and glycogenesis operate in a selective fashion to suit the cellular demands. If on the other hand, the synthesis and degradative metabolic pathways of a particular substance (say gluconeogenesis and glycolysis related to glucose) operate to the same extent
simultaneously, this would result in futile cycles. However, futile cycles, consuming energy (ATP) are wasteful metabolic exercises. They are minimally operative due to a well coordinated metabolic machinery.
Recent studies suggest that futile cycles may have some physiological (operative in arousal of hibernating animals) and pathological (malignant hyperthermia due to loss of control)significance.

The metabolic defects concerned with the glycogen synthesis and degradation are collectively referred to as glycogen storage diseases. These disorders are characterized by deposition of normal or abnormal type of glycogen in one or more tissues. A summary of glycogen metabolism along with the defective enzymes in the glycogen storage disorders is depicted in Fig.13.19. The biochemical lesions and the characteristic features of the disorders are given in Table 13.2.

The incidence of type | glycogen storage disease is 1 per 200,000 persons. It is transmitted by autosomal recessive trait. This disorder results in various biochemical manifestations.
1. Fasting hypoglycemia : Due to the defect in the enzyme glucose 6-phosphatase, enough free glucose is not released from the liver.
2. Lactic acidemia Glucose is not synthesized from lactate produced in muscle and liver. Lactate level in blood increases and the pH is lowered (acidosis).

features = Liver enlarged; liver glycogen cannot form glucose; mild hypoglycemia and ketosis seen.. VII, Name = Tarui's disease. VII, Enzyme defect = Phosphofructokinase. VII, Organ(s) involved = Skeletal muscle, erythrocytes. VII, Characteristic features = Muscle cramps due to exercise; blood lactate not elevated; hemolysis occurs.. Rare glycogen disorders VIII, IX, X and XI have been identified. They are due to defects in the enzymes concerned with activating and deactivating liver phosphorylase., Name = Rare glycogen disorders VIII, IX, X and XI have been identified. They are due to defects in the enzymes concerned with activating and deactivating liver phosphorylase.. Rare glycogen disorders VIII, IX, X and XI have been identified. They are due to defects in the enzymes concerned with activating and deactivating liver phosphorylase., Enzyme defect = Rare glycogen disorders VIII, IX, X and XI have been identified. They are due to defects in the enzymes concerned with activating and deactivating liver phosphorylase.. Rare glycogen disorders VIII, IX, X and XI have been identified. They are due to defects in the enzymes concerned with activating and deactivating liver phosphorylase., Organ(s) involved = Rare glycogen disorders VIII, IX, X and XI have been identified. They are due to defects in the enzymes concerned with activating and deactivating liver phosphorylase.. Rare glycogen disorders VIII, IX, X and XI have been identified. They are due to defects in the enzymes concerned with activating and deactivating liver phosphorylase., Characteristic features = Rare glycogen disorders VIII, IX, X and XI have been identified. They are due to defects in the enzymes concerned with activating and deactivating liver phosphorylase.
Fig. 13.19 : Summary of glycogen metabolism with glycogen storage diseases (Red blocks indicate storage disease, I-von Gierke's disease; I-Pompe's disease; I//—Cori's disease; 1V-Anderson's disease; V-Mc Ardle's disease; VI—Her's disease; VI/—Tarui's disease).
3.
:
There is
a
blockade in

The important features of the glycogen storage gluconeogenesis.
Hence more
meet energy
requirements results
fat of
plasma is
mobilized the
free to
body.
This fatty
acids and
ketone bodies.
4. Hyperuricemia : Glucose 6-phosphate that accumulates is diverted to pentose phosphate pathway, leading to increased synthesis of ribose phosphates which increase the cellular levels of phosphoribosyl pyrophosphate and enhance the metabolism of purine nucleotides to uric acid. Flevated plasma levels of uric acid (hyperuricemia) are often associated with gouty arthritis (painful joints).
diseases are given in Table 13.2.

Hexose monophosphate pathway or HMP shunt is also called pentose phosphate pathway or phosphogluconate pathway. This is an alternative pathway to glycolysis and TCA cycle for the oxidation of glucose. However, HMP shunt is more anabolic in nature, since it is concerned with the biosynthesis of NADPH and pentoses.

The pathway starts with glucose 6-phosphate. As such, no ATP is directly utilized or produced in HMP pathway. It is a unique multifunctional pathway, since there are several interconvertible substances produced which may proceed in different directions in the metabolic reactions.

The enzymes of HMP shunt are located in the cytosol. The tissues such as liver, adipose tissue, adrenal gland, erythrocytes, testes and lactating mammary gland, are highly active in HMP shunt. Most of these tissues are involved in the biosynthesis of fatty acids and steroids which are dependent on the supply of NADPH.

The sequence of reactions of HMP shunt (Fig.13.20) is divided into two phases—oxidative and non-oxidative.
1. Oxidative phase : Glucose 6-phosphate dehydrogenase (G6PD) is an NADP-dependent enzyme that converts glucose 6-phosphate to 6-phosphogluconolactone. The latter is then hydrolysed by the gluconolactone hydrolase to 6-phosphogluconate. The next reaction involving the synthesis of NADPH is catalysed by 6-phosphogluconate dehydrogenase to produce 3 keto 6-phosphogluconate which then undergoes decarboxylation to give ribulose 5-phosphate.
G6PD regulates HMP shunt : The first reaction catalysed by G6PD is most regulatory in HMP shunt. This enzyme catalyses an irreversible reaction. NADPH competitively inhibits G6PD. It is the ratio of NADPH/NADt that ultimately determines the flux of this cycle.
2. Non-oxidative phase : The non-oxidative reactions are concerned with the interconversion of three, four, five and seven carbon monosaccharides. Ribulose 5-phosphate is acted upon by an epimerase to produce xylulose 5-phosphate while ribose 5-phosphate ketoisomerase converts ribulose 5-phosphate to ribose 5-phosphate.
Fig. 13.20 contd. next page
Fig. 13.21 : Overview of hexose monophosphate shunt representing the number of molecules (prefix in red) and the number of carbon atoms (suffix in blue). Note that of the 6-molecules of glucose 6-phosphate that enter HMP shunt, one molecule is oxidized as 5-molecules are finally recovered.
6-phosphate to 6CO), we have to start with 6 molecules of glucose 6-phosphate. Of these 6, 5 moles are regenerated with the production of 12 NADPH.
The overall reaction may be represented as 6 Glucose 6-phosphate + 12 NADP* + 6H,O > 6CO, + 12 NADPH + 12H* + 5 Glucose

HMP._ shunt is unique in generating two important products—pentoses and NADPH— needed for the biosynthetic reactions and other functions.

In the HMP shunt, hexoses are converted into pentoses, the most important being ribose 5-phosphate. This pentose or its derivatives are useful for the synthesis of nucleic acids (RNA and DNA) and many nucleotides such as ATP, NAD+, FAD and CoA.
Skeletal muscle is capable of synthesizing pentoses, although only the first few enzymes of HMP shunt are active. It, therefore, appears that the complete pathway of HMP shunt may not be required for the synthesis of pentoses.

1. NADPH is required for the reductive biosynthesis of fatty acids and steroids, hence HMP shunt is more active in the tissues concerned with lipogenesis, e.g. adipose tissue, liver etc.
2. NADPH is used in the synthesis of certain amino acids involving the enzyme glutamate dehydrogenase.
3. There is a continuous production of H»O, in the living cells which can chemically damage unsaturated lipids, proteins and DNA. This is, however, prevented to a large extent through antioxidant (free radical scavenging) reactions involving NADPH. Gluta-thione mediated reduction of HjOz is given in the next column.
Glutathione (reduced, GSH) detoxifies H3O>, peroxidase catalyses this reaction. NADPH is responsible for the regeneration of reduced glutathione from the oxidized one.
4. Microsomal cytochrome P4s9 system (in liver) brings about the detoxification of drugs and foreign compounds by hydroxylation reactions involving NADPH.
5. Phagocytosis is the engulfment of foreign particles, including microorganisms, carried out by white blood cells. The process requires the supply of NADPH.
6. Special functions of NADPH in RBC : NADPH produced in erythrocytes has special functions to perform. It maintains the concentration of reduced glutathione (reaction explained in 3) which is essentially required to preserve the integrity of RBC membrane. NADPH is also necessary to keep the ferrous iron (Fe2*) of hemoglobin in the reduced state so that accumulation of methemoglobin (Fe?*) is prevented.
7. High concentration of NADPH in lens of eyes is necessary to preserve the transparency of the lenses.

G6PD deficiency is an inherited sex-linked trait. Although the deficiency occurs in all the cells of the affected individuals, it is more severe in RBC.
HMP shunt is the only means of providing NADPH in the erythrocytes. Decreased activity of G6PD impairs the synthesis of NADPH in RBC. This results in the accumulation of methemoglobin and peroxides in erythrocytes leading to hemolysis.
Most
Clinical manifestations in G6PD deficiency : of the patients with G6PD deficiency do not
usually exhibit clinical symptoms. Some of them, however, develop hemolytic anemia if they are administered oxidant drugs or exposed to a severe infection. The drugs such as primaquine (antimalarial), acetanilide (antipyretic), sulfamethoxazole (antibiotic) or ingestion of fava beans (favism) produce hemolytic jaundice in these patients. Severe infection results in the generation of free radicals (in macrophages) which can enter RBC and cause hemolysis (due to decreased NADPH and reduced GSH).

:
It is interesting to note that G6PD deficiency is associated with resistance to malaria (caused by Plasmodium falciparum). This is explained from the fact that the parasites that cause malaria are dependent on HMP_ shunt and reduced glutathione for their optimum growth in RBC. Therefore, G6PD_ deficiency—which is seen frequently in Africans—protects them from malaria, a common disease in this region. It is regarded as an adaptability of the people living in malaria-infected regions of the world.
Biochemical diagnosis can be done by detecting reduced activity of G6PD in RBC. The management of G6PD deficiency includes avoiding oxidative stress and symptomatic treatment of hemolysis.

This is a genetic disorder associated with HMP. shunt. An alteration in transketolase activity that reduces its affinity (by tenfold or so) with thiamine pyrophosphate is the biochemical lesion. The symptoms of Wernicke-Korsakoff syndrome include mental disorder, loss of memory and partial paralysis. The symptoms are manifested in vitamin-deficient alcoholics.

This is an alternative oxidative pathway for glucose and is also known as glucuronic acid pathway (Fig.13.22). It is concerned with the synthesis of glucuronic acid, pentoses and vitamin, ascorbic acid (except in primates and guinea pigs). Dietary xylulose enters uronic acid pathway through which it can participate in other metabolisms. In most of the pathways of carbohydrate metabolism, phosphate — esters participate, whereas, in uronic acid pathway, the free sugars or sugar acids are involved.
1. Formation and importance of UDPglucuronate Glucose 6-phosphate is first converted to glucose 1-phosphate. UDP-glucose is then synthesized by the enzyme UDP-glucose pyrophosphorylase. Till this step, the reactions are the same as described in glycogenesis (Fig.13.14). UDP-glucose dehydrogenase oxidizes UDP-glucose to UDP-glucuronate.
UDP-glucuronate is the metabolically active form of glucuronate which is utilized for conjugation with many substances like bilirubin, steroid hormones and certain drugs. Several insoluble compounds are converted to soluble ones through conjugation and, further, the drugs are detoxified. UDP-glucuronate is also required for the synthesis of glycosaminoglycans and proteoglycans.
2. Conversion of UDP-glucuronate to L-gulonate : UDP-glucuronate loses its UDP moiety in a hydrolytic reaction and releases Dglucuronate which is reduced to L-gulonate by an NADPH-dependent reaction.
3. Synthesis of ascorbic acid in some animals : L-Gulonate is the precursor for the synthesis of ascorbic acid (vitamin C) in many animals. The enzyme L-gulonolactone oxidase— which converts gulonate to ascorbic acid—is absent in man, other primates and guinea pigs. Therefore, vitamin C has to be supplemented in the diet for these animals.

4. Oxidation of L-gulonate : L-Gulonate is oxidized to 3-ketogulonate and then decarboxylated to a _ pentose, L-xylulose. L-Xylulose is converted to D-xylulose via xylitol by a reduction (NADPH-dependent) followed by an oxidation (NAD*-dependent) reaction. This is necessary since the D-xylulose (and not L-form)—after getting phosphorylated—can enter the hexose monophosphate shunt, for further metabolism.

Administration of drugs (barbital, chlorobutanol etc.) significantly increases the uronic
acid pathway to achieve more synthesis of glucuronate from glucose. Certain drugs (aminopyrine, antipyrine) were found to enhance the synthesis of ascorbic acid in rats.

This is a rare genetic disorder related to the deficiency of an NADPdependent enzyme xylitol dehydrogenase. Due to this enzyme defect, L-xylulose cannot be converted to xylitol. The affected individuals excrete large amounts of L-xylulose in urine. Essential pentosuria is asymptomatic and the individuals suffer from no ill-effects. It has been reported that the administration of drugs aminopyrine and antipyrine increases the excretion of L-xylulose in pentosuric patients.

The disaccharide lactose, present in milk and milk products, is the principal dietary source of galactose. Lactase (B-galactosidase) of intestinal mucosal cells hydrolyses lactose to galactose and glucose. Galactose is also produced within the cells from the lysosomal degradation of glycoproteins and glycolipids. As is the case for fructose, galactose entry into the cells is not dependent on insulin.
Fig. 13.22 : Uronic acid pathway [UDP—uridine diphosphate); Block in essential
(1) pentosuria; (2) Enzyme absent in primates (including man) and guinea pigs].
Galactose
Fig. 13.23 : Metabolism of galactose (defects in galactosemia are indicated by red blocks).
glycolipids. UDP-galactose can be converted to UDP-glucose by UDP hexose 4-epimerase. In this way, galactose can enter the metabolic pathways of glucose. It may be noted that galactose is not an essential nutrient since UDP-glucose can be converted to UDP-galactose by the enzyme UDPhexose 4-epimerase.
However, with increased levels of galactose, this pathway assumes significance. Galactitol (like sorbitol, discussed later) has been implicated in the development of cataract.

Galactosemia (incidence 1 : 30000) is due to the deficiency of the enzyme galactose 1phosphate uridyltrans-ferase. \t is a rare congenital disease in infants, inherited as an autosomal recessive disorder. The salient features of galactosemia are listed.
1. Galactose metabolism is impaired leading to increased galactose levels in circulation (galactosemia) and urine (galactosuria).
2. The accumulated galactose is diverted for the production of galactitol (dulcitol) by the enzyme aldose reductase (the same enzyme that converts glucose to sorbitol). Aldose reductase is present in lens, nervous tissue, seminal vesicles etc. The conversion of galactose to galactitol is insignificant in routine galactose metabolism.
3. The accumulation of galactose 1-phosphate and galactitol in various tissues like liver, nervous tissue, lens and kidney leads to impairment in their function.
4. High levels of galactose 1-phosphate in liver results in the depletion of inorganic phosphate (sequestering of phosphate) for other metabolic functions. Galactose 1-phosphate inhibits glycogen phosphorylase resulting in hypoglycemia.
5. The clinical symptoms of galactosemia are—loss of weight (in infants) hepatosplenomegaly, jaundice, mental retardation etc. In severe cases, cataract, amino aciduria and albuminuria are also observed.
Diagnosis : Early detection of galactosemia is possible (biochemical diagnosis) by measuring the activity of galactose 1-phosphate uridyltransferase in erythrocytes.
of
Treatment : The therapy includes the supply diet deprived of galactose and lactose.
: The defect in responsible _ for Galactokinase deficiency the enzyme galactokinase,

phosphorylation of galactose, will also result in galactosemia and galactosuria. Here again galactose is shunted to the formation of galactitol. Generally, galactokinase-deficient individuals do not develop hepatic and renal complications. Development of cataract occurs at a very early age, sometimes within an year after birth. The treatment is the removal of galactose and lactose from the diet.
found in free form in honey and many fruits. In the body, entry of fructose into the cells is not controlled by the hormone insulin. This is in contrast to glucose which is regulated for its entry into majority of the tissues.

The major dietary source of fructose is the disaccharide sucrose (cane sugar), and highfructose corn syrups (HFCS) used in the manufactured foods and beverages. It is also
Fructose is mostly phosphorylated by fructokinase to fructose 1-phosphate. Fructokinase has been identified in liver, kidney and intestine. Hexokinase, which phosphorylates various monosaccharides, can also act on fructose to produce fructose 6-phosphate. | However, hexokinase has low affinity (high K,,) for fructose, hence this is a minor pathway.
Fructose 1-phosphate is cleaved to glyceraldehyde and dihydroxyacetone — phosphate (DHAP) by aldolase B (Fig.13.24). This is in
contrast to fructose 6-phosphate which is converted to fructose 1, 6-bisphosphate and split by aldolase A (details in glycolysis—See Fig.13.2). Glyceraldehyde is phosphorylated by the enzyme triokinase to glyceraldehyde 3phosphate which, along with DHAP, enters glycolysis or gluconeogenesis.
Ingestion of large quantities of fructose or sucrose is linked with many _ health complications.

The fructose is more rapidly metabolized (via glycolysis) by the liver than glucose. This is due to the fact that the rate limiting reaction in glycolysis catalysed by phosphofructokinase is bypassed. Increased dietary intake of fructose significantly elevates the production of acetyl CoA and lipogenesis (fatty acid, triacylglycerol and very low density lipoprotein synthesis).
Polyol pathway (so termed since sorbitol is a polyhydroxy sugar) basically involves the conversion of glucose to fructose via sorbitol (Fig.13.24). This pathway is absent in liver. Sorbitol pathway is directly related to glucose concentration, and is higher in uncontrolled diabetes.
The enzyme aldose reductase reduces glucose to sorbitol (glucitol) in the presence of NADPH.

oF A continuous presence of glucose—supplied through diet or synthesized in the body (gluconeogenesis)—is essential for the survival of the organism. Alcohol intoxication reduces gluconeogenesis, particularly in diabetic patients and undernourished persons.
+ Human brain consumes about 120 g of glucose per day out of the 160 g needed by the body. Insufficient supply of glucose to brain may lead to coma and death.
+ Liver glycogen serves as an immediate source for maintaining blood glucose levels, particularly between the meals. The glycogen stores in the liver get depleted after 12- 18 hours of fasting.
+ Muscle glycogen is primarily concerned with the supply of hexoses that undergo glycolysis to provide energy during muscle contraction.
+ Glycogen storage diseases—characterized by deposition of normal or abnormal type of glycogen in one or more tissues—result in muscular weakness, or even death.
+ The occurrence of HMP shunt (NADPH production) in the RBC is necessary to maintain the methemoglobin. integrity of erythrocyte membrane and to prevent the accumulation of
+ Deficiency of glucose 6-phosphate dehydrogenase results in hemolysis of RBC, causing hemolytic anemia. The subjects of G6PD deficiency are, however, resistant to malaria.
+ Uronic acid pathway is concerned with the production of glucuronic acid (involved in detoxification), pentoses and vitamin C. Man is incapable of synthesizing vitamin C due to the absence of a single enzyme—L-gulonolactone oxidase.
+ The conversion of glucose to fructose is impaired in diabetes mellitus, causing accumulation of sorbitol. This compound has been implicated in the development of cataract, nephropathy, peripheral neuropathy etc.
+ Severe cases of galactosemia are associated with the development of cataract, believed to be due to the accumulation of galactitol.

Sorbitol is then oxidized to fructose by sorbitol dehydrogenase and NAD*. Aldose reductase is absent in liver but found in many tissues like lens and retina of the eye, kidney, placenta, Schwann cells of peripheral nerves, erythrocytes and seminal vesicles. The enzyme sorbitol dehydrogenase is present in seminal vesicle, spleen and ovaries. Fructose is a preferred carbohydrate for energy needs of sperm cells due to the presence of sorbitol pathway.

In uncontrolled diabetes (hyperglycemia), large amounts of glucose enter the cells which are not dependent on insulin. Significantly, the cells with increased intracellular glucose levels in diabetes (lens, retina, nerve cells, kidney etc.) possess high activity of aldose reductase and sufficient supply of NADPH. This results in a rapid and efficient conversion of glucose to sorbitol. The enzyme sorbitol dehydrogenase, however, is either low in activity or absent in these cells, hence sorbitol is not converted to fructose. Sorbitol cannot freely pass through the cell membrane, and accumulate in the cells where it is produced. Sorbitol—due to _ its hydrophilic nature—causes strong osmotic effects leading to swelling of the cells. Some of the pathological changes associated with diabetes (like cataract formation, peripheral neuropathy, nephropathy etc.) are believed to be due to the accumulation of sorbitol, as explained above.
It is clearly known that in diabetic animals sorbitol content of lens, nerve, and glomerulus is elevated. This causes damage to tissues. It thus appears that majority of the complications associated with diabetes share a common pathogenesis as a consequence of polyol pathway. Certain inhibitors of aldose reductase can prevent the accumulation of sorbitol, and thus the associated complications. However, this approach is still at the experimental stage.

1. Essential fructosuria Due to the deficiency of the enzyme hepatic fructokinase, fructose is not converted to fructose 1-phosphate. This is an asymptomatic condition with excretion of fructose in urine. Treatment involves the restriction of dietary fructose.
2. Hereditary fructose intolerance : This is due to the absence of the enzyme aldolase B. Hereditary fructose —_ intolerance —_ causes intracellular accumulation of fructose 1-phosphate, severe hypoglycemia, vomiting, hepatic ailure and jaundice. Fructose 1-phosphate allosterically inhibits liver phosphorylase and blocks glycogenolysis leading to hypoglycemia. Early detection and intake of diet free from ructose and sucrose, are advised to overcome ructose intolerance.
3. Consumption of high fructose : Fructose is rapidly converted to fructose 1-phosphate by ructokinase. The activity of the enzyme aldolase B is relatively less, and, due to this, fructose 1-phosphate accumulates in the cell. This leads o the depletion of intracellular inorganic phosphate (Pi) levels. The phenomenon of binding of Pi to the organic molecules (like ructose here)—that leads to the less availability of Pi for the essential metabolic functions—is nown as sequestering of phosphate. Due to the decreased availability of Pi, which happens in overconsumption of fructose, the _ liver metabolism is adversely affected. This includes he lowered synthesis of ATP from ADP and Pi. High consumption of fructose over a long period is associated with increased uric acid in blood leading to gout. This is due to the excessive breakdown of ADP and AMP (accumulated due to lack of Pi) to uric acid.

Athesosclerosis is characterized by thickening of arteries due to accumulation of lipids (Refer Chapter 14).
Fructose rapidly enters tissues and increases glycolysis, that ultimately results in lipogenesis. It is clearly established that in liver fructose increases fatty acid and triacylglycerol synthesis, and VLDL secretion. All these metabolic processes finally lead to elevated triacylglycerol
and LDL-cholesterol in circulation, increasing the risk of atherosclerosis. thereby of the amino sugars for the formation of glycosaminolgycans, glycoproteins and gangliosides is also indicated in this figure.

When a hydroxyl group of a sugar is replaced by an amino group, the resultant compound is an amino sugar.
The important amino sugars are glucosamine, galactosamine, mannosamine, sialic acid etc. They are essential components of glycosaminoglycans, glycolipids (gangliosides) and glycoproteins. They are also found in some oligosaccharides and certain antibiotics. It is estimated that about 20% of the glucose is utilized for the synthesis of amino sugars, which mostly occurs in the connective tissue.
The outline of the pathway for the synthesis of amino sugars is given in Fig.13.25. Fructose 6-phosphate is the major precursor for glucosamine, N-acetylgalactosamine and N-acetylneuraminic acid (NANA). The utilization

The lysosomal storage diseases caused by enzyme defects in the degradation of glycosaminoglycans (GAGs) are known as mucopolysaccharidoses (Table 13.3). Mucopolysaccharidoses are characterized by the accumulation of GAGs in various tissues that may result. in skeletal deformities, and mental retardation. Mucopolysaccharidoses are important for elucidating the role of lysosomes in health and disease.

The animals, including man, cannot carry out the net synthesis of carbohydrate from fat. However, the plants and many microorganisms are equipped with the metabolic machinery—

results in the diversion of galactose to produce galactitol which has been implicated in the development of cataract.. 9., 1 = Glucose can be converted to fructose via sorbitol pathway. In prolonged hyperglycemia (uncontrolled diabetes), sorbitol accumulates in the tissues, resulting in cataract, nephropathy, peripheral neuropathy etc.. 10., 1 = Amino sugars (glucosamine, galactosamine, mannosamine etc.), synthesized from fructose 6-phosphate are essential components of glycosaminoglycans, glycolipids and glycoproteins.
284

1. Describe briefly the metabolism of glucose 6-phosphate.
2. Give an account of glycogen metabolism.
3. Justify that citric acid cycle is the final common metabolic pathway for the oxidation of foodstuffs.
4. Discuss the synthesis of glucose from non-carbohydrate sources.
5. Describe the hexose monophosphate shunt and add a note on its significance.

(a) Glycogenolysis, (b) UDPG, (c) Galactosemia, (d) Cori cycle, (e) 2, 3- BPG, (f) Glycogen storage diseases, (g) Essential fructosuria, (h) Conversion of pyruvate to acetyl CoA, (i) Energetics of TCA TPP in metabolism.
cycle, (j) carbohydrate

1. Name the five vitamins required by pyruvate dehydrogenase or o-ketoglutarate dehydrogenase complex
SON Substrate level phosphorylation in citric acid cycle is catalysed by the enzyme
DMR Galactosemia is mostly due to the deficiency of the enzyme The two amino acids that are never glucogenic are
YH Ascorbic acid is not synthesized in man due to lack of the enzyme
10. The metabolite
Muscle glycogen does not directly contribute to blood glucose due to absence of the enyme
The compound implicated in the development of cataract in diabetic patients is
and
:
The
metabolic
pathway
concerned
with
the
of
L-xylulose
to
D-xylulose
is
The name of the protein that has been identified to serve as a primer for glycogen synthesis is
among
the
citric
acid
cycle
intermediates
performing
a
catalytic
role

11. One of the following enzymes in glycolysis catalyses an irreversible reaction.
12. Synthesis of 2, 3-bisphosphoglycerate occurs in the tissue namely.
13. The hormone that lowers cAMP concentration in liver cells is
14, The number of ATP produced when a molecule of acetyl CoA is oxidized through citric acid cycle
15. The connecting link between HMP shunt and lipid synthesis is
(a) Hexokinase (b) Phosphofructokinase (c) Pyruvate kinase (d) All of them.
(a) Liver (b) Kidney (c) Erythrocytes (d) Brain.
(a) Glucagon (b) Insulin (c) Epinephrine (d) Thyroxine.
(a) 12 (b) 24 (c) 38 (d) 15.
(a) Ribose (b) NADPH (c) Sedoheptulose 7-phosphate (d) NADH.
ipids are indispensable for cell structure and function. Due to their hydrophobic and nonpolar nature, lipids differ from rest of the body compounds and are unique in their action.
1. Triacylglycerols (TG) are highly concentrated form of energy, yielding 9 Cal/g, in contrast to carbohydrates and proteins that produce only 4 Cal/g. This is because fatty acids found in TG are in the reduced form.

Lipids constitute about 15-20% of the body weight in humans. Triacylglycerols (formerly triglycerides) are the most abundant lipids comprising 85-90% of body lipids. Most of the triacylglycerols (TG; also called neutral fat or depot fat) are stored in the adipose tissue and serve as energy reserve of the body. This is in contrast to carbohydrates and proteins which cannot be stored to a significant extent for energy purposes. Fat also acts as an insulating material for maintaining the body temperature of animals.

Triacylglycerols are the most predominant storage form of energy. There are two main reasons for fat being the fuel reserve of the body
2. The triacylglycerols are non-polar and hydrophobic in nature, hence stored in pure form without any association with water (anhydrous form). On the other hand, glycogen and proteins are polar. One gram of glycogen combines with 2 g of water for storage.
For the two reasons stated above, one gram of fat stored in the body yields nearly six times as much energy as one gram of (hydrated) glycogen. In a healthy adult individual (weighing 70 kg), about 10-11 kg of fat is stored (mostly in adipose tissue) which corresponds to a fuel reserve of 100,000 Cals. If this much of energy were to be stored as glycogen (instead of fat), then the weight of the person would increase by at least 55 kg! This explains why fat has been chosen as a fuel reserve during evolution.
Long chain fatty acids (of fat) are the ideal storage fuel reserves of the body. Fats can support the body's energy needs for long periods of food deprivation. In extreme cases, humans can fast and survive for 60-90 days, and the obese persons can survive even longer (6 months to one year!) without food.
Hibernating animals provide good example for utilizing fat reserve as fuel. For instance, bears go on hibernation for about 7 months and, during this entire period, the energy is derived from the degradation of fat stores. The rubythroated humming birds fly non-stop between New England and West Indies (2,400 km!) at a speed of 40 km/hr for 60 hours! This is possible only due to the stored fat.

Phospholipids, glycolipids and cholesterol are major components of cell membranes. Cholesterol is also a precursor for bile acids and steroid hormones. Arachidonic —acid—an unsaturated fatty acid—is the substrate for the synthesis of certain intercellular regulators— prostaglandins, thromboxanes, prostacyclins etc.

The insoluble lipids are solubilized in association with proteins to form lipoproteins in which form lipids are transported in the blood stream. Free lipids are undetectable in blood.
Chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL), high density lipoproteins (HDL) and albumin-free fatty acids are the different lipoprotein complexes that transport lipids in the blood stream. Details of plasma lipoproteins and their metabolism are discussed later.

The various fractions of lipids in the plasma can be estimated by different methods after extracting them with lipid solvents. The plasma levels of lipids (Table 14.1) are often useful for assessing the health of the individuals.

It was earlier thought that the lipids are inert storage compounds and are less significant

Total lipid, Reference values (mg/dl) = 400-600. Total cholesterol, Reference values (mg/dl) = 150-200. LDL-cholesterol, Reference values (mg/dl) = 80-150. HDL-cholesterol, Reference values (mg/dl) = 30-60. VLDL-cholesterol, Reference values (mg/dl) = 20-40. Triglycerides, Reference values (mg/dl) = 75-150. Phospholipids, Reference values (mg/dl) = 150-200. Free fatty acids, Reference values (mg/dl) = 5-15
metabolically. However, later experiments with isotope studies have proved that the body lipids are continuously being degraded and resynthesized. As already stated, fat stored in the adipose tissue is the fuel reserve of the body. This is in a dynamic state.
The triacylglycerols transported from intestine (as chylomicrons) and liver (as VLDL) are stored in the adipose tissue. Besides, they are also utilized by muscle, liver, heart etc., as per the needs of the body. An overview of fat metabolism is depicted in Fig.14.1.
Fig. 14.2 : Complete hydrolysis (lipolysis) of triacylglycerol.

Triacylglycerol (TG) is the stored fat in the adipose tissue. The enzyme, namely hormonesensitive triacylglycerol lipase, removes the fatty acid either from carbon 1 or 3 of the triacylglycerol to form diacylglycerol. The other two fatty acids of TG are cleaved by additional lipases specific for diacylglycerol and monoacylglycerol. The complete degradation of triacylglycerol to glycerol and free acids is known as lipolysis (Fig.14.2).

Hormone-sensitive TG-lipase is so named because its activity is mostly controlled by hormones. Lipase is present in an inactive form 'b' and is activated (phosphorylated) by a cAMP. dependent protein kinase to lipase 'a'. Several hormones—such as epinephrine (most effective), norepinephrine, glucagon, thyroxine, ACTH etc.— enhance the activity of adenylate cyclase and, thus, increase lipolysis. On the other hand, insulin decreases cAMP levels and thereby inactivates lipase. Caffeine promotes lipolysis by increasing CAMP levels through its inhibition on phosphodiesterase activity. The control of cAMP. mediated lipolysis is illustrated in Fig.14.3.
Fate of glycerol : The adipose tissue lacks the enzyme glycerol kinase, hence _ glycerol produced in lipolysis cannot be phosporylated here. It is transported to liver where it is activated to glycerol 3-phosphate. The latter may be used for the synthesis of triacylglycerols and phospholipids. Glycerol 3-phosphate may also enter glycolysis by getting converted to dihydroxyacetone phosphate (Fig.14.4).
Fate of free fatty acids : The fatty acids released in the adipocytes enter the circulation and are transported in a bound form to albumin. The free fatty acids enter various tissues and are utilized for the energy. About 95% of the energy obtained from fat comes from the oxidation of fatty acids. Certain tissues, however, cannot oxidize fatty acids, e.g. brain, erythrocytes.

During starvation, TG stored in adipose tissue is hydrolysed to free fatty acids (for oxidation) to provide energy to skeletal and cardiac muscle. However, about 65% of these FFA are converted to TG, and sent back to adipose tissue for deposition. This process of lipolysis of TG and resterification of FFA to TG is termed as triacylglycerol/fatty acid cycle.

The fatty acids in the body are mostly oxidized by B-oxidation. B-Oxidation may be defined as the oxidation of fatty acids on the B-carbon atom. This results in the sequential removal of a two carbon fragment, acetyl CoA.

The B-oxidation of fatty acids involves three stages
|. Activation of fatty acids occurring in the cytosol
Il. Transport of fatty acids into mitochondria
Ill.
B-Oxidation proper
in the
mitochondrial matrix.
Fatty acids are oxidized by most of the tissues in the body. However, brain, erythrocytes and adrenal medulla cannot utilize fatty acids for energy requirement.

Fatty acids are activated to acyl CoA by thiokinases or acyl CoA synthetases. The reaction occurs in two steps and requires ATP, coenzyme A and Mg?*. Fatty acid reacts with ATP to form acyladenylate which then combines with coenzyme A to produce acyl CoA (Fig.14.5). In the activation, two high energy phosphates are utilized, since ATP is converted to pyrophosphate (PPi). The enzyme inorganic pyrophosphatase hydrolyses PPi to phosphate (Pi). The immediate elimination of PPi makes this reaction totally irreversible.
Three different thiokinases, to activate long chain (10-20 carbon), medium chain (4-12 carbon) and short chain (<4 carbon) fatty acids have been identified.
The inner mitochondrial membrane _ is impermeable to fatty acids. A specialized carnitine carrier system (carnitine shuttle) operates to transport activated fatty acids from cytosol to the mitochondria. This occurs in four steps (Fig.14.6).
1. Acyl group of acyl CoA is transferred to carnitine (B-hydroxy y-trimethyl aminobutyrate), catalysed by carnitine acyltransferase | (present on the outer surface of inner mitochondrial membrane).
- 2. The acyl-carnitine is transported across the membrane to mitochondrial matrix by a specific carrier protein.
3. Carnitine acyl transferase II (found on the inner surface of inner mitochondrial membrane) converts acyl-carnitine to acyl CoA.
4. The carnitine released returns to cytosol for reuse.

It should be noted that the coenzyme A used for activation is different from the one that finally combines with fatty acid in the mitochondria to form acyl CoA. Thus, the cell has two separate pools (cytosolic and mitochondrial) of coenzyme A.
Inhibitor of carnitine shuttle : Carnitine acyl transferase | is inhibited by malonyl CoA, a key metabolite involved in fatty acid synthesis that occurs in cytosol (details given later). In other words, while the fatty acid synthesis is in progress (reflected by high concentration of malonyl CoA), their oxidation does not occur, since carnitine shuttle is impaired.

Each cycle
of
B-oxidation, carbon
unit-acetyl
CoA, occurs
liberating
a
two in
a
sequence of
four reactions
(Fig.14.7).
1. Oxidation Acyl CoA undergoes dehydrogenation by an FAD-dependent flavoenzyme, acyl CoA dehydrogenase. A double bond is formed between a and B carbons (i.e., 2 and 3 carbons).
2.
Hydration
:
Enoyl
CoA
hydratase brings
about the
hydration of
the double
bond to
form
B-hydroxyacyl
CoA.
3. Oxidation : B-Hydroxyacyl CoA dehydrogenase catalyses the second oxidation and generates NADH. The product formed is B-ketoacyl CoA.
4. Cleavage The final reaction in B-oxidation is the liberation of a 2 carbon fragment, acetyl CoA from acyl CoA. This occurs by a thiolytic cleavage catalysed by B-ketoacyl CoA thiolase (or simply thiolase).
The new acyl CoA, containing two carbons less than the original, reenters the B-oxidation cycle. The process continues till the fatty acid is completely oxidized.
Fig. 14.5 : Activation of fatty acid to acyl CoA by the enzyme thiokinase.

reaction for
each cycle
of
The standard free energy of palmitate = 2,340
C,
Acyl
CoASH
—>
CoA
+
FAD
Ciy_y)
Acyl
+
CoA
NAD*t
+
+
H,0
Acetyl
CoA
+
+
FADH,
+
NADH
+
Ht.
The scheme of fatty acid oxidation discussed above corresponds to saturated (no double bond) and even carbon fatty acids. This occurs most in predominantly biological system.

is
The summary of B-oxidation of palmitoyl CoA shown below
Palmitoy] CoA + 7 CoASH + 7 FAD + 7
NAD* + 7H »O —=> 8 Acetyl CoA + 7 FADH) +
7
NADH
+
7Ht
Palmitoyl CoA undergoes 7 cycles of B-oxidation to yield 8 acetyl CoA. Acety| CoA can enter citric acid cycle and get completely oxidized to CO, and H,O.

The ultimate aim of fatty acid oxidation is to generate energy. The energy obtained from the complete oxidation of palmitic acid (16 carbon) is given in Table 14.2 and Fig.14.8.
Cal.
The energy
yield by
its oxidation—129
ATP
(129 x 7.3 Cal) = 940 Cal.
The efficiency of energy conservation by fatty acid
oxidation
=
40
100
=
40%.
,
Fig. 14.8 : An overview of oxidation of palmitic acid.

The sudden infant death syndrome (SIDS) is an unexpected death of healthy infants, usually overnight. The real cause of SIDS is not known. It is now estimated that at least 10% of SIDS is due to deficiency of medium chain acyl CoA dehydrogenase. The enzyme defect has a frequency of 1 in 10,000 births and is, in fact, more prevalent than phenylketonuria. The occurrence of SIDS is explained as follows
Glucose is the principal source of energy, soon after eating or feeding babies. After a few hours, the glucose level and_ its utilization decrease and the rate of fatty acid oxidation must simultaneously increase to meet the energy needs. The sudden death in infants is due to a blockade in -oxidation caused by a deficiency in medium chain acyl CoA dehydrogenase (MCAD).

This disease is characterized by severe hypoglycemia, vomiting, convulsions, coma and death. It is caused by eating unripe ackee fruit which contains an unusual toxic amino acid, hypoglycin A. This inhibits the enzyme acyl
CoA dehydrogenase and thus B-oxidation of fatty acids is blocked, leading to various complications.

The f-oxidation of saturated fatty acids containing odd number of carbon atoms proceeds in the same manner, as described i above for even carbon fatty acids. The only difference is that in the last and final B-oxidation cycle, a three-carbon fragment is left behind (in place of 2 carbon unit for saturated fatty acids). This compound is . . . propionyl CoA which is converted to succinyl CoA as follows (Fig.14.9)
- 1. Propionyl CoA is carboxylated in the
presence of
ATP,
CO, and
vitamin biotin
to
D-methylmalonyl
CoA.
2. Methylmalonyl CoA racemase converts the methylmalonyl CoA to L-form. This reaction (D > L) is essential for the entry of this compound into the metabolic reactions of the body.
same extent as saturated fatty acids. Therefore, oxidation of unsaturated fatty acids, in general, provides Jess energy than that of saturated fatty acids.
3. The next enzyme, methylmalonyl CoA mutase, is dependent on vitamin By (deoxyadenosyl cobalamin). It catalyses the conversion of methylmalonyl CoA (a branched compound) to succinyl CoA (a straight chain compound), which can enter citric acid cycle.

1. Due to deficiency of vitamin B19;
2. Due to defect in the enzyme methylmalony| mutase.
CoA
In either case, there is an accumulation of methylmalonic acid in body, followed by its increased excretion in urine. This causes severe metabolic acidosis, damages the central nervous system and retards the growth. It is often fatal in the early years of life.

Due to the presence of double bonds, the unsaturated fatty acids are not reduced to the
Most of the reactions involved in the oxidation of unsaturated fatty acids are the same as found in the B-oxidation of saturated fatty acids. However, the presence of double bonds poses problem for B-oxidation to proceed. This is overcome by two additional enzymes—an isomerase and an epimerase.

in peroxisomes
Peroxisomes are organelles present in most eukaryotic cells. The f-oxidation occurs in a modified form in peroxisomes. Acyl CoA dehydrogenase (a flavoenzyme) leads to the formation of FADH , as in B-oxidation. The reducing equivalents from FADH, are not transferred to the electron transport chain, but handed over directly to Oy. This results in the formation of HO, which is cleaved by catalase.
Catalase
—
Fig. 14.9 : Conversion of propionyl CoA to succinyl CoA.
There is no ATP synthesized in peroxisomal B-oxidation of fatty acids, since the reducing equivalents do not pass through ETC. However, heat is liberated.
It is now believed that the peroxisomes carry out the initial oxidation of long chain (Cy9, Coo etc.) fatty acids which is followed by mitochondrial oxidation.
Peroxisomal oxidation is induced by high fat diet and administration of hypolipidemic drugs (e.g. clofibrate).
Zellweger syndrome : This is a rare disorder characterized by the absence of peroxisomes in almost all the tissues. As a result, the long chain fatty acids (Cy,—C3g) are not oxidized. They accumulate in tissues, particularly in brain, liver and kidney. Hence the disorder is also known as cerebrohepatorenal syndrome.

B-Oxidation is the most predominant pathway for fatty acid degradation. However, the removal of one carbon unit at a time by the oxidation of a-carbon atom of fatty acid is known. a-Oxidation does not involve the binding of fatty acid to coenzyme A and no energy is produced.
Refsum's disease is a rare but severe neurological disorder characterized by cerebral ataxia and peripheral neuropathy. The patients of this disease accumulate large quantities of an unusual fatty acid, phytanic acid. It is derived from phytol, a constituent of chlorophyll. Hence it is found mostly in plant foods. However, it is also present in milk lipids and animal fats. Phytanic acid cannot undergo B-oxidation due to the presence of a methyl group on carbon-3. This fatty acid undergoes initial o-oxidation (to remove a-carbon as carbon dioxide) and this is followed by B-oxidation.
Refsum's disease is caused by a defect in the a-oxidation due to the deficiency of the enzyme phytanic acid «-oxidase. The result is that phytanic acid cannot be converted to a compound that can be degraded by f-oxidation. The patients should not consume diets containing chlorophyll (i.e., green leafy vegetables).

This is a minor pathway. It involves hydroxylation followed by oxidation of w-carbon present as a methyl group at the other end (at one end carboxyl group is present) of fatty acid. This reaction requires cytochrome P4s59, NADPH and Op», besides the enzymes. The overall reaction may be represented as follows.

Fatty acid oxidation (even other forms of aerobic respiration) is accompanied by the production of water, referred to metabolic water. For instance, when one molecule of palmitic acid is oxidized, it releases 16 molecules of water. This metabolic water has _ great significance in some animals. Camel can store lipids in its hump which is good source of water, besides energy supply. For this reason, camel can travel in deserts for long periods even without food and water supply. Kangaroo rat is a small animal that is believed to live indefinitely without water. It consumes only oil rich seeds, and the metabolic water produced is adequate to meet its water needs. It may however, be noted that the use of metabolic water is an adaptation, and is accompanied by reduced output of urine.

The compounds namely acetone, acetoacetate and f-hydroxybutyrate (or 3-hydroxybutyrate) are known as ketone bodies (Fig.14.10). Only the first two are true ketones while Bhydroxybutyrate does not possess a keto (C=O) group. Ketone bodies are water-soluble and energy yielding. Acetone, however, is an exception, since it cannot be metabolized.

The synthesis of ketone bodies occurs in the liver. The enzymes for ketone body synthesis are located in the mitochondrial matrix. Acetyl CoA, formed by oxidation of fatty acids, pyruvate or some amino acids, is the precursor for ketone bodies. Ketogenesis occurs through the following reactions

1. Two moles of acetyl CoA condense to form acetoacetyl CoA. This reaction is catalysed by thiolase, an enzyme involved in the final step of B-oxidation. Hence, acetoacetate synthesis is appropriately regarded as the reversal of thiolase reaction of fatty acid oxidation.
2. Acetoacety] CoA combines with another molecule of acetyl CoA to produce B-hydroxy B-methy! glutaryl CoA (HMG CoA). HMG CoA synthase, catalysing this reaction, regulates the synthesis of ketone bodies.
3.
HMG
CoA
lyase cleaves
HMG
CoA
to produce acetoacetate and acetyl CoA.
4.
Acetoacetate can
undergo spontaneous
decarboxylation to
form acetone.
5. Acetoacetate can be reduced by a dehydrogenease to B-hydroxybutyrate.
The carbon skeleton of some amino acids (ketogenic) is degraded to acetoacetate or acetyl CoA and, therefore, to ketone bodies, e.g. leucine, lysine, phenylalanine etc.

The ketone bodies, being water-soluble, are easily transported from the liver to various tissues. The two ketone bodies—acetoacetate and -hydroxybutyrate serve as _ important
Fig. 14.11 : Synthesis of ketone bodies (ketogenesis).
sources of energy for the peripheral tissues such as skeletal muscle, cardiac muscle, renal cortex etc. The tissues which lack mitochondria (e.g. erythrocytes) however, cannot utilize ketone bodies. The production of ketone bodies and their utilization become more significant when glucose is in short supply to the tissues, as observed in starvation, and diabetes mellitus.
During prolonged starvation, ketone bodies are the major fuel source for the brain and other parts of central nervous system. It should be noted that the ability of the brain to utilize fatty acids for energy is very limited. The ketone bodies can meet 50-70% of the brain's energy needs. This is an adaptation for the survival of the organism during the periods of food deprivation.
intermediate in citric acid cycle. Thiophorase is absent in liver, hence ketone bodies are not utilized by the liver. Thiolase cleaves acetoacety| CoA to two moles of acetyl CoA (Fig.14.12).
The summary of ketone body synthesis, utilization and excretion is depicted in Fig.14.13.

Reactions of ketone bodies : B-Hydroxybutyrate is first converted to acetoacetate (reversal of synthesis) and metabolized. Acetoacetate is activated to acetoacetyl CoA by a mitochondrial enzyme thiophorase (succinyl CoA acetoacetate CoA transferase). The coenzyme A is donated by succinyl CoA, an
In normal individuals, there is a constant production of ketone bodies by liver and their utilization by extrahepatic tissues. The concentration of ketone bodies in blood is maintained around 1 mg/dl. Their excretion in urine is very low and undetectable by routine tests (Rothera's test).

+ An adult human body contains about 10-11 kg of fat reserve corresponding to about 100,000 Cal. This can meet the energy requirements for several weeks of food deprivation in man.
+ The sudden infant death syndrome (SIDS)—an unexpected overnight death of healthy infants—is attributed to a blockade in B-oxidation of fatty acids, caused by a deficiency of medium chain acyl CoA dehydrogenase (MCAD).
+ Jamaican vomiting sickness is due to consumption of unripe ackee fruit containing hypoglycin A which blocks f-oxidation.
+
acidemia
occurs
either
due
to
a
of
the
vitamin
B19
or
a
defect
in
an
enzyme
methyl
malonyl
CoA
mutase.
This
disorder
retards
growth
and
damages
central
nervous
system.
+ Zellweger syndrome is caused by the absence of peroxisomes in tissues; as a result, the long chain fatty acids cannot be oxidized.
+ Refsum’s disease is due to a defect in a-oxidation of fatty acids. The patients are advised not to consume diets containing chlorophyll.
+ Ketosis is commonly associated with uncontrolled diabetes mellitus and starvation. Diabetes ketoacidosis is dangerous—may result in coma or even death. Starvation, however, is not accompanied by ketoacidosis.
+ Insulin promotes fatty acid synthesis by stimulating the conversion of pyruvate to acetyl CoA.
+ The lack of the ability of the organisms to introduce double bonds in fatty acids beyond Cg and Cyg makes linoleic and linolenic acids essential to mammals.
Fig.
14.12
:
Metabolism
(utilization)
of ketone
bodies to
acetyl
CoA.

When the rate of synthesis of ketone bodies exceeds the rate of utilization, — their concentration in blood increases, this is known as ketonemia. Ketonemia is predominantly due to incresed production of ketone bodies rather than the deficiency in their utilization. The term ketonuria represents the excretion of ketone bodies in urine. The overall picture of ketonemia and ketonuria is commonly referred to as ketosis. Smell of acetone in breath is a common feature in ketosis. Ketosis is most commonly associated with starvation and severe uncontrolled diabetes mellitus.
Starvation : Starvation is accompanied by increased degradation of fatty acids (from the fuel reserve triacylglycerol) to meet the energy needs of the body. This causes an over- production of acetyl CoA which cannot be fully handled by citric acid cycle. Furthermore, TCA cycle is impaired due to deficiency of oxaloacetate, since most of it is diverted for glucose synthesis to meet the essential requirements (often unsuccessful) for tissues like brain. The result is an accumulation of acetyl CoA and its diversion for overproduction of ketone bodies.
Ketonuria and weight loss programs : The appearance of ketone bodies in urine is an indication of active fat metabolism. Some programs designed for body weight loss encourage reduction in carbohydrate and total calorie intake until ketone bodies appear in urine.
Diabetes mellitus Diabetes mellitus is associated with insulin deficiency. This results in impaired carbohydrate metabolism — and increased lipolysis, both of them ultimately leading to the accumulation of acetyl CoA and its conversion to ketone bodies. In severe diabetes, the ketone body concentration in blood plasma may reach 100 mg/dl and the urinary excretion may be as high as 500 mg/day.

The ketone body formation (particularly overproduction) occurs primarily due to nonavailability of carbohydrates to the tissues. This is an outcome of excessive utilization of fatty acids to meet the energy requirements of the cells. The hormone glucagon _ stimulates ketogenesis whereas insulin inhibits. The increased ratio of glucagon/insulin in diabetes mellitus promotes ketone body formation. This is due to disturbances caused in carbohydrate and lipid metabolisms in diabetes, as discussed elsewhere (Chapter 36).

The — ketogenic — substances —_— (promote ketogenesis) include fatty acids and certain amino acids (leucine, lysine, tyrosine etc.). The antiketogenic substances (inhibit ketogenesis) are glucose, glycerol and glucogenic amino acids (e.g. glycine, alanine, serine, glutamate etc.)
Fig. 14.13 : Summary of ketone body synthesis, utilization and excretion.

Both acetoacetate and B-hydroxybutyrate are strong acids. Increase in their concentration in blood would cause acidosis. The carboxyl group has a pk@ around 4. Therefore, the ketone bodies in the blood dissociate and release H* ions which lower the pH. Diabetic ketoacidosis is dangerous—may result in coma, and even death, if not treated. Ketosis due to starvation is not usually accompanied by ketoacidosis.
Treatment of ketoacidosis : Rapid treatment of diabetic ketoacidosis is required to correct the metabolic abnormalities and the associated water and electrolyte imbalance. Administration of insulin is necessary to stimulate uptake of glucose by tissues and inhibition of ketogenesis.

The dietary carbohydrates and amino acids, when consumed in excess, can be converted to fatty acids and stored as triacylglycerols. De novo (new) synthesis of fatty acids occurs predominantly in liver, kidney, adipose tissue and lactating mammary glands. The enzyme machinery for fatty acid production is located in the cytosomal fraction of the cell. Acetyl CoA is the source of carbon atoms while NADPH provides the reducing equivalents and ATP supplies energy for fatty acid formation. The fatty acid synthesis may be learnt in 3 stages
I. Production of acetyl CoA and NADPH
Il. Conversion of acetyl CoA to malonyl CoA
Ill. Reactions of fatty acid synthase complex.
I.
Production of
acetyl
CoA

Acetyl CoA and NADPH are the prerequisites for fatty acid synthesis. Acetyl CoA is produced in the mitochondria by the oxidation of pyruvate and fatty acids, degradation of carbon skeleton of certain amino acids, and from ketone bodies. Mitochondria, however, are not permeable to acetyl CoA. An _ alternate or a _ bypass arrangement is made for the transfer of acetyl CoA to cytosol. Acetyl CoA condenses with oxaloacetate in mitochondria to form citrate. Citrate is freely transported to cytosol where it is cleaved by citrate lyase to liberate acetyl CoA and oxaloacetate. Oxaloacetate in the cytosol is converted to malate
(Fig.14.14).
Fig. 14.14 : Transfer of acetyl CoA from mitochondria to cytosol
(HMP shunt—Hexose monophosphate shunt; **«—also known as malate dehydrogenase).
Malic enzyme converts malate to pyruvate. NADPH and CO, are generated in this reaction. Both of them are utilized for fatty acid synthesis.
Advantages of coupled transport of acetyl CoA and NADPH : The transport of acetyl CoA from mitochondria to cytosol is coupled with the cytosomal production of NADPH and CO, which is highly advantageous to the cell for optimum synthesis of fatty acids.

Acetyl CoA is carboxylated to malonyl CoA by the enzyme acetyl CoA carboxylase (Fig.14.15). This is an ATP-dependent reaction and requires biotin for CO, fixation. The mechanism of action of acetyl CoA carboxylase is similar to that of pyruvate carboxylase (Refer Chapter 7, Fig.7.29). Acetyl CoA carboxylase is a regulatory enzyme in fatty acid synthesis (details given later).

The remaining reactions of fatty acid synthesis are catalysed by a multifunctional enzyme known as fatty acid synthase (FAS) complex. |n eukaryotic cells, including man, the fatty acid synthase exists as a dimer with two identical units. Each monomer possesses the activities of seven different enzymes and an acyl carrier
Fig. 14.15 : Conversion of acetyl CoA to malonyl CoA.
Fig.
14.16
contd.
next column
Fig. 14.16 : Biosynthesis of long chain fatty acid-palmitate. (Cys—Cysteine; ACP-Acyl carrier protein; are
The pathway repeats 7 times to produce palmitate; the first two carbons at the methyl end directly from acetyl CoA, the rest of the carbons come from malonyl CoA).
(ACP)
protein
Fatty acid
bound to
4’-phosphopantetheine.
with a separate acyl carrier protein. This is in synthase
catalysing functions
all the
of the
as
a
single unit
seven synthase
reactions.
complex enzyme
activities.
In results
in the
Dissociation loss
of the
lower organisms
(prokaryotes), the
fatty acid
carried out by a multienzyme complex in association
contrast to
eukaryotes where
ACP
is
a
part of
fatty acid synthase.
The sequence of reactions of the extra—
mitochondrial synthesis of fatty acids (palmitate)
is depicted
in
Fig.14.16, and
described in
the next page.
1. The two carbon fragment of acetyl CoA is transferred to ACP of fatty acid synthase, catalysed by the enzyme, acetyl CoA-ACP transacylase. The acetyl unit is then transferred from ACP to cysteine residue of the enzyme. Thus ACP site falls vacant.
2. The — enzyme malonyl | CoA-ACP transacylase transfers malonate from malonyl CoA to bind to ACP.

3. The acetyl unit attached to cysteine is transferred to malonyl group (bound to ACP). The malonyl! moiety loses COg which was added by acetyl CoA carboxylase. Thus, CO, is never incorporated into fatty acid carbon chain. The decarboxylation is accompanied by loss of free energy which allows the reaction to proceed forward. This reaction is catalyzed by B-ketoacyl ACP synthase.
4. B-Ketoacyl ACP reductase reduces ketoacyl group to hydroxyacyl group. The reducing equivalents are supplied by NADPH.
5. B-Hydroxyacyl ACP undergoes dehydration. A molecule of water is eliminated and a double bond is introduced between @ and B carbons.
6. A second NADPH-dependent reduction, catalysed by enoyl-ACP reductase occurs to produce acyl-ACP. The four-carbon unit attached to ACP is butyryl group.
The carbon chain attached to ACP is transferred to cysteine residue and the reactions 2-6 are repeated 6 more times. Each time, the fatty acid chain is lengthened by a two-carbon unit (obtained from malonyl CoA). At the end of 7 cycles, the fatty acid synthesis is complete and a 16-carbon fully saturated fatty acid—namely palmitate—bound to ACP is produced.
7. The enzyme palmitoyl — thioesterase separates palmitate from fatty acid synthase. This completes the synthesis of palmitate.

Of the 16 carbons present in palmitate, only two come from acetyl CoA directly. The remaining 14 are from malonyl CoA which, in turn, is produced by acetyl CoA. The overall reaction of palmitate synthesis is summarized
8 Acetyl CoA + 7 ATP + 14 NADPH + 14 Ht*
—
Palmitate + 8 CoA + 7 ADP + 7 Pi + 6H,O

The diagrammatic representation of the model for fatty acid synthase (FAS) multienzyme complex is depicted in Fig.14.17. This model is tentative and is largely based on the work of Wakil.
Fatty acid synthase is a dimer composed of two identical subunits (monomers), each with a molecular weight of 240,000. Each subunit contains the activities of 7 enzymes of FAS and an ACP with 4'-phosphopantetheine —SH group. The two subunits lie in antiparallel (head-to-tail) orientation. The —SH_ group of phosphopantetheine of one subunit is in close proximity to the —SH of cysteine residue (of the enzyme ketoacyl synthase) of the other subunit.
Each monomer of FAS contains all the enzyme activities of fatty acid synthesis. But only the dimer is functionally active. This is because the functional unit consists of half of each subunit interacting with the complementary half of the other. Thus, the FAS structure has both functional division and subunit — division (Fig.14.17). The two functional subunits of FAS independently operate and synthesize two fatty acids simultaneously.

The organization of different enzymes of a metabolic pathway into a single multienzyme functional unit has distinct advantages for cellular function
1. The FAS complex offers great efficiency that is free from interference of other cellular reactions for the synthesis of fatty acids.
2. Since the entire process of the metabolic pathway is confined to the complex, there are no permeability barriers for the various intermediates.
3. The multienzyme polypeptide complex is coded by a single gene. Thus, there is a good coordination in the synthesis of all enzymes of the FAS complex.
Fig. 14.17 : Fatty acid synthase multienzyme complex (ACP-AcyI carrier protein; FAS has two identical subunits which organize into two functional subunits to simultaneously synthesize two fatty acids).

Fatty acid production is controlled by enzymes, metabolites, end products, hormones and dietary manipulations. Some of the important regulatory mechanisms are discussed hereunder.
Insulin stimulates tissue uptake of glucose, and conversion of pyruvate to acetyl CoA. This also facilitates fatty acid formation.
Acetyl CoA carboxylase This enzyme controls a committed step in fatty acid synthesis. Acetyl CoA carboxylase exists as an inactive protomer (monomer) or an active polymer. Citrate promotes polymer formation, hence increases fatty acid synthesis. On the other hand, palmitoy] CoA and malonyl CoA cause depolymerization of the enzyme and, therefore, inhibit fatty acid synthesis.
Hormonal influence : Hormones regulate acetyl CoA carboxylase by a separate mechanism—phosphorylation (inactive form) and dephosphorylation (active form) of the enzyme. Glucagon, epinephrine and norepinephrine inactivate the enzyme by cAMPdependent phosphorylation. Insulin, on the other hand, dephosphorylates and activates the enzyme. Thus, insulin promotes fatty acid synthesis while glucagon inhibits.
Dietary regulation : Consumption of high carbohydrate or fat-free diet increases the synthesis of acetyl CoA carboxylase and fatty acid synthase, which promote fatty acid formation. On the other hand, fasting or high fat diet decreases fatty acid production by reducing the synthesis of these two enzymes.
Availability of NADPH : The reducing equivalents for fatty acid synthesis are provided by NADPH which come either from citrate (acetyl CoA) transport or hexose monophosphate shunt. About 50-60% of required NADPH is obtained from HMP shunt, which significantly influences fatty acid synthesis.

A microsomal enzyme system called fatty acyl CoA desaturase is responsible for the formation of unsaturated fatty acids. This reaction also involves flavin-dependent cytochrome bs reductase, NADH and molecular Oy. The monounsaturated fatty acids—namely oleic

1.,  = Major tissues. 1., Fatty acid synthesis = Liver, adipose tissue. 1., E -Oxidation = Muscle, liver. 2.,  = Subcellular site. 2., Fatty acid synthesis = Cytosol. 2., E -Oxidation = Mitochondria. 3.,  = Precursor/substrate. 3., Fatty acid synthesis = Acetyl CoA. 3., E -Oxidation = Acyl CoA. 4.,  = End product. 4., Fatty acid synthesis = Palmitate. 4., E -Oxidation = Acetyl CoA. 5.,  = Intermediates are bound to. 5., Fatty acid synthesis = Acyl carrier protein. 5., E -Oxidation = Coenzyme A. 6.,  = Coenzyme requirement. 6., Fatty acid synthesis = NADPH (supplying reducing equivalents). 6., E -Oxidation = FAD and NAD + (get reduced). 7.,  = Carbon units added/degraded. 7., Fatty acid synthesis = Malonyl CoA. 7., E -Oxidation = Acetyl CoA. 8.,  = Transport system. 8., Fatty acid synthesis = Citrate (mitochondria oglyph<c=3,font=/MDECLA+Symbol> cytosol). 8., E -Oxidation = Carnitine (cytosol oglyph<c=3,font=/MDECLA+Symbol> mitochondria). 9.,  = Inhibitor. 9., Fatty acid synthesis = Long chain acyl CoA (inhibits acetyl CoA carboxylase). 9., E -Oxidation = Malonyl CoA (inhibits carnitine acyltransferase I). 10.,  = The pathway increased. 10., Fatty acid synthesis = After rich carbohydrate diet. 10., E -Oxidation = In starvation. 11.,  = Hormonal status that promotes. 11., Fatty acid synthesis = High ratio of insulin/glucagon. 11., E -Oxidation = Low ratio of insulin/glucagon. 12.,  = Status of enzyme(s). 12., Fatty acid synthesis = Multifunctional enzyme complex. 12., E -Oxidation = Individual enzymes

acid and palmitoleic acid—are, respectively, synthesized from stearate and palmitate.
Mammals lack the ability to introduce double bonds in fatty acids between carbon 10 and methyl terminal (@) end. Hence, linoleic acid (18 : 2; 9, 12) and linolenic acid (18 : 3; 9, 12, 15) are essential for man in the diet. However, arachidonic acid (20 : 4; 5, 8, 11, 14) can be synthesized from linoleic acid by desaturation and chain elongation. Arachidonic acid is the precursor for eicosanoids (prostaglan-dins and thromboxanes), a group of compounds with diversified functions, discussed elsewhere (Chapter 32).

Palmitate is the end product of the reactions of fatty acid synthase system that occurs in cytosol. Further, chain elongation can take place either in mitochondria or in endoplasmic reticulum (microsomes), by separate mechanisms. The microsomal chain elongation is more predominant and involves successive additions of malonyl CoA with the participation of NADPH.
These reactions are similar to that catalysed by fatty acid synthase. A specific group of enzymes, namely elongases, bring about fatty acid chain elongation.
The mitochondrial chain elongation is almost a reversal of B-oxidation of fatty acids. Acetyl CoA molecules are successively added to fatty acid to lengthen the chain. The reducing equivalents are derived from NADPH.

The synthesis of fatty acids and their oxidation are two distinct and independent pathways. A comparison between these two metabolic pathways in given in Table 14.3.

Triacylglycerol (TG) synthesis mostly occurs in liver and adipose tissue, and to a lesser extent in other tissues. Fatty acids and glycerol must be activated prior to the synthesis of triacylglycerols. Conversion of fatty acids to acyl CoA by thiokinase is already described (See Fig.14.5).

Two mechanisms are involved for the synthesis of glycerol 3-phosphate
1. In the liver, glycerol is activated by glycerol kinase. This enzyme is absent in adipose tissue. 2. In both liver and adipose tissue, glucose serves as a precursor for glycerol 3-phosphate. Dihydroxyacetone phosphate (DHAP) produced in glycolysis is reduced by glycerol 3-phosphate dehydrogenase to glycerol 3-phosphate.

Glycerol 3-phosphate acyltransferase catalyses the transfer of an acyl group to produce lysophosphatidic acid. DHAP can also accept acyl group, ultimately resulting in the formation of lysophosphatidic acid. Another acyl group is added to lysophosphatidic acid to form phosphatidic acid (1,2-diacylglycerol phosphate). The enzyme phosphatase cleaves off phosphate of phosphatidic acid to produce diacylglycerol. Incorporation of another acy! group finally results in synthesis of triacylglycerol (Fig.14.18).
The three fatty acids found in triacylglycerol are not of the same type. A saturated fatty acid is usually present on carbon 1, an unsaturated fatty acid is found on carbon 2, and carbon 3 may have either.
The intermediates of TG synthesis phosphatidic acid and diacylglycerol are also utilized for phospholipid synthesis (described later).

Phospholipids are a specialized group of lipids performing a variety of functions. These include the membrane structure and functions, involvement in blood clotting, and supply of arachidonic acid for the synthesis of prostaglandins (for details Refer Chapter 32).

Phospholipids are — synthesized ~— from phosphatidic acid and 1,2-diacylglycerol, intermediates in the production of triacylglycerols (Fig.14.18). Phospholipid synthesis occurs in the smooth endoplasmic reticulum.
1. Formation of lecithin and cephalin Choline and ethanolamine first get phosphorylated and then combine with CTP to form, respectively, CDP-choline and CDP-ethanolamine (Fig.14.19). Phosphatidylcholine (lecithin) is synthesized when CDP-choline combines with 1,2-diacylglycerol. Phosphatidyl ethanolamine (cephalin) is produced when CDP-ethanolamine reacts with 1,2-diacylglycerol. Phosphatidyl ethanolamine can be converted to phosphatidyl choline on methylation.
Choline and ethanolamine, used for phospholipid synthesis, are mostly derived from the preexisting phospholipids. Thus, the phospholipid synthesis starting with choline or ethanolamine is regarded as salvage pathway.
2. Synthesis of phosphatidylserine : Phosphatidy! ethanolamine can — exchange ___ its ethanolamine group with free serine to produce phosphatidylserine. The latter, on decarboxylation, gives the former.
3. Formation of phosphatidylinositol : CDPdiacylglycerol produced from phosphatidic acid combines with inositol to form phosphatidyl inositol (Pl). This phospholipid contains arachidonic acid on carbon 2 of glycerol which serves as a substrate for prostaglandin synthesis. Further, PI is important for signal transmission across membranes.

4. Synthesis of phosphatidyl glycerol and cardiolipin : CDP-diacylglycerol combines with glycerol 3-phosphate to form phosphatidyl glycerol 3-phosphate, which then forms phosphatidylglycerol. The latter combines with another molecule of phosphatidylglycerol to finally produce cardiolipin (Fig.14.19). Cardiolipin is the only phospholipid possessing antigenic properties.
5. Formation of plasmalogens : These are phospholipids with fatty acid at carbon 1 bound by an ether linkage instead of ester linkage. An important plasmalogen, 1-alkenyl 2-acetyl glycerol 3-phosphocholine, causes blood platelet aggregation and is referred to as_platelet-activating factor (PAF). The outline of the pathway for the synthesis of plasmalogens is depicted in Fig.14.20.

The enzyme sphingomyelinase of lysosomes hydrolyses sphingomyelins to ceramide and phosphorylcholine (Fig.14.23). Ceramide formed can be further degraded to sphingosine and free fatty acid.
Niemann-Pick disease : It is an inherited disorder due to a defect in the enzyme sphingomyelinase. This causes accumulation of
Fig. 14.21 : An outline of the synthesis of sphingomyelin.
sphingomyelins in liver and spleen, resulting in the enlargement of these organs. Victims of Niemann-Pick disease suffer from severe mental retardation, and death may occur in early childhood.
Farber's disease : A defect in the enzyme ceramidase results in Farber's disease. This disorder is characterized by skeletal deformation, subcutaneous nodules, dermatitis and mental retardation. It is fatal in early life.

The outline of the synthesis of cerebrosides
and sulfatide is given in Fig.14.24.
Fig. 14.25 : Degradation of cerebrosides and sphingomyelins with metabolic disorders.

The degradation of cerebrosides along with the associated inborn errors is depicted in Fig.14.25.
Gaucher's disease : This is due to a defect in the enzyme f-glucosidase. As a result, tissue glucocerebroside levels increase. This disorder is commonly associated with enlargement of liver and spleen, osteoporosis, pigmentation of skin, anemia, mental retardation etc. Sometimes, Gaucher's disease is fatal.
Krabbe's disease : Defect in the enzyme B-galactosidase results in the accumulation of galactocerebrosides. A total absence of myelin in the nervous tissue is a common feature. Severe mental retardation, convulsions, blindness, deafness etc. are seen. Krabbe's disease is fatal in early life.
Niemann-Pick disease and Farber's disease connected with sphingomylein metabolism are already described. They are also depicted in Fig.14.25.
Gangliosides are complex glycosphingolipids mostly found in ganglion cells. They contain one or more molecules of N-acetylneuraminic acid (NANA) bound ceramide oligosaccharides.
Defect in the degradation of gangliosides causes gangliosidosis, Tay-Sach's disease etc.

Lipid storage diseases, representing lysosomal storage defects, are inherited disorders. They are characterized by the accumulation of complex lipids.
The term sphingolipidoses is often used to collectively refer to the genetic disorders that lead to the accumulation of any one of the sphingolipids (glycosphingolipids and sphingomyelins). Some examples of sphiogolipidoses (lipid storage diseases) with important features are summarized in Table 14.4.

Cholesterol is found exclusively in animals, hence it is often called as animal sterol. The total body content of cholesterol in an adult man weighing 70 kg is about 140 g i.e., around 2 g/kg body weight. Cholesterol is amphipathic in nature, since it possesses both hydrophilic and hydrophobic regions in the structure.

Niemann-Pick disease, Missing/defective enzyme = Sphingomyelinase. Niemann-Pick disease, Major storage compound = Sphingomyelins. Niemann-Pick disease, Symptoms = Enlargement of liver, spleen, mental retardation.. Farber's disease, Missing/defective enzyme = Ceramidase. Farber's disease, Major storage compound = Ceramide. Farber's disease, Symptoms = Painful and deformed joints.. Gaucher's disease, Missing/defective enzyme = E -Glucosidase. Gaucher's disease, Major storage compound = Glucocerebroside. Gaucher's disease, Symptoms = Enlargement of liver and spleen, osteoporosis, mental retardation.. Krabbe's disease, Missing/defective enzyme = E -Galactosidase. Krabbe's disease, Major storage compound = Galactocerebrosides. Krabbe's disease, Symptoms = Absence of myelin formation, liver and spleen enlargement, mental retardation.. Tay-Sachs disease, Missing/defective enzyme = Hexosaminidase A. Tay-Sachs disease, Major storage compound = Ganglioside GM 2. Tay-Sachs disease, Symptoms = Blindness, mental retardation, death within 2-3 years.. Fabry's disease, Missing/defective enzyme = D -Galactosidase. Fabry's disease, Major storage compound = Ceramide trihexoside. Fabry's disease, Symptoms = Renal failure, skin rash, pain in lower extremities.

Cholesterol is essential to life, as it performs a number of functions
important
1. It is a structural component of cell membrane.
2. Cholesterol is the precursor for the synthesis of all other steroids in the body. These include steroid hormones, and bile
vitamin D acids.
3. It is an essential ingredient in the structure
of lipoproteins in which form the lipids in the body are transported.
4. Fatty acids are transported to liver as cholesteryl esters for oxidation.

About 1g of cholesterol is synthesized per day in adults. Almost all the tissues of the body participate in cholesterol biosynthesis. The largest contribution is made by liver (50%), intestine (15%), skin, adrenal cortex, reproductive tissue etc.
The enzymes involved in cholesterol synthesis are found in the cytosol and microsomal fractions of the cell. Acetate of acetyl CoA provides all the carbon atoms in cholesterol.
The reducing equivalents are supplied by NADPH while ATP provides energy. For the production of one mole of cholesterol, 18 moles of acetyl CoA, 36 moles of ATP and 16 moles of NADPH are required.
By administering acetate with '4C isotope label either on the methyl (—CH3) group or carboxyl (—~COO) group, the origin of carbon atoms in the entire molecule of cholesterol has been established. The sources of carbon atoms and the key intermediates of cholesterol formation are depicted in Fig.14.26, and the detailed reactions are given in Fig.14.27.
The synthesis of cholesterol may be learnt in
5
stages
. Synthesis of HMG CoA 1
. Formation of mevalonate (6C) 2
3. Production of isoprenoid units (5C)
4. Synthesis of squalene (30C)
5 . Conversion of squalene to cholesterol (27C).
1. Synthesis of B-hydroxy f-methylglutaryl CoA (HMG CoA) : Two moles of acetyl CoA condense to form acetoacetyl CoA. Another molecule of acetyl CoA is then added to produce HMG CoA. These reactions are similar to that of
Fig. 14.26 : Outline of cholesterol biosynthesis— (A) Derivation of carbon atoms from acetate, (B) Key intermediates with the carbon atoms.
ketone body synthesis. However, the two pathways are distinct, since ketone bodies are produced in mitochondria while cholesterol synthesis occurs in cytosol. Thus, there exist two pools of HMG CoA in the cell. Further, two isoenzymes of HMG CoA synthase are known. The cytosomal enzyme is involved in cholesterol synthesis whereas the mitochondrial HMG CoA synthase participates in ketone body formation.

2. Formation of mevalonate : HMG CoA reductase is the rate limiting enzyme in cholesterol biosynthesis. This enzyme is present in endoplasmic reticulum and catalyses the reduction of HMG CoA to mevalonate. The reducing equivalents are supplied by NADPH.

step reaction catalysed by kinases, mevalonate is converted to 3-phospho — 5-pyrophosphomevalonate which on decarboxylation forms isopentenyl pyrophosphate (IPP). The latter isomerizes to dimethylallyl pyrophosphate (DPP). Both IPP and DPP are 5-carbon isoprenoid units.
4. Synthesis of squalene : IPP and DPP condense to produce a 10-carbon geranyl pyrophosphate (GPP). Another molecule of IPP condenses with GPP to form a 15-carbon farnesyl pyrophosphate (FPP). Two units of farnesyl pyrophosphate unite and get reduced to produce a 30-carbon squalene.
5. Conversion of squalene to cholesterol : Squalene undergoes hydroxylation — and cyclization utilizing Oj, and NADPH and gets converted to lanosterol. The formation of cholesterol from lanosterol is a multistep process with a series of about 19 enzymatic reactions. The following are the most important reactions
1 Reducing the carbon atoms from 30 to 27.
1 Removal of two methyl groups from C, and one methyl group from C,,4.
| Shift of double bond from Cg to C;.
1 Reduction in the double bond present between C34 and Cys.
The enzymes (about 192) involved in the conversion of lanosterol to cholesterol are associated with endoplasmic reticulum. 14Desmethy! lanosterol, zymosterol, cholestadienol and desmosterol are among the intermediates in the cholesterol biosynthesis. The penultimate product is 7-dehydrocholesterol which, on reduction, finally yields cholesterol.
Cholesterol biosynthesis is now believed to be a part of a major metabolic pathway concerned with the synthesis of several other isoprenoid compounds. These _ include ubiquinone (coenzyme Q of electron transport chain) and dolichol (found in glycoprotein). Both of them are derived from farnesy! pyrophosphate.
Fig.
14.27
contd.
(15C)
Fig. 14.27 contd. next page

Cholesterol biosynthesis is controlled by the rate limiting enzyme HMG CoA reductase, at the beginning of the pathway (Fig.14.28). HMG CoA reductase is found in association with endoplasmic reticulum, and is subjected to different metabolic controls.
1. Feedback control The end product cholesterol controls its own synthesis by a feedback mechanism. Increase in the cellular concentration of cholesterol reduces the synthesis of the enzyme HMG CoA reductase. This is achieved by decreasing the transcription of the gene responsible for the production of HMG CoA reductase. Feedback regulation has been investigated with regard to LDL-cholesterol taken up by the cells, and the same mechanism is believed to operate whenever cellular cholesterol level is elevated.
2. Hormonal regulation : The enzyme HMG CoA reductase exists in two interconvertible forms. The dephosphorylated form of HMG CoA reductase is more active while the phosphorylated form is less active. The hormones exert their influence through cAMP by a series of reactions which are comparable with the control of the enzyme glycogen synthase. The net effect is that glucagon and glucocorticoids favour the formation of inactive HMG CoA _ reductase (phosphorylated form) and, thus, decrease cholesterol synthesis. On the other hand, insulin and thyroxine increase cholesterol production by enhancing the formation of active HMG CoA reductase (dephosphorylated form).
3. Inhibition by drugs : The drugs compactin and lovastatin (mevinolin) are fungal products. They are used to decrease the serum cholesterol level in patients with hypercholesterolemia. Compactin and lovastatin are competitive inhibitors of the enzyme HMG CoA reductase and, therefore, reduce cholesterol synthesis. About 50 to 60% decrease in serum cholesterol level has been reported by a combined use of these two drugs.
4. HMG CoA reductase activity is inhibited by bile acids. Fasting also reduces the activity of this enzyme.
Fig. 14.28 : Regulation of cholesterol biosynthesis by HMG CoA reductase (€)—Promoting effect; @-Inhibitory effect).

The steroid nucleus (ring structure) of the cholesterol cannot be metabolized in humans. Cholesterol (50%) is converted to bile acids, excreted in feces, serves as a precursor for the synthesis of steroid hormones, vitamin D, coprostanol and cholestanol. The latter two are the fecal sterols, besides cholesterol.
function as surfactants. In the bile, the conjugated bile acids exist as sodium and potassium salts which are known as bile salts.

The bile acids possess 24 carbon atoms, 2 or 3 hydroxyl groups in the steroid nucleus and a side chain ending in carboxyl group. The bile acids are amphipathic in nature since they possess both polar and non-polar groups. They serve as emulsifying agents in the intestine and actively participate in the digestion and absorption of lipids.
The synthesis of primary bile acids takes place in the liver and involves a series of reactions (Fig.14.29). The step catalysed by 7 a-hydroxylase is inhibited by bile acids and this is the rate limiting reaction. Cholic acid and chenodeoxycholic acid are the primary bile acids and the former is found in the largest amount in bile. On conjugation with glycine or taurine, conjugated bile acids (glycocholic acid, taurocholic acid etc.) are formed which are more efficient in their
In the intestine, a portion of primary bile acids undergoes deconjugation and dehydroxylation to form secondary bile acids (deoxycholic acid and lithocholic acid). These reactions are catalysed by bacterial enzymes in the intestine.

: bile salts synthesized in the liver accumulate in gall bladder. From there they are secreted into the small intestine where they serve as emulsifying agents for the digestion and absorption of fats and fat soluble vitamins. A large portion of the bile salts (primary and secondary) are reabsorbed and returned to the liver through portal vein. Thus the bile salts are recycled and reused several times in a day. This is known as enterohepatic circulation. About 1530 g of bile salts are secreted into the intestine each day and reabsorbed. However, a small portion of about 0.5 g/day is lost in the feces. An equal amount (0.5 g/day) is synthesized in liver to replace the lost bile salts. The fecal excretion of bile salts is the only route for the removal of cholesterol from the body.
Cholelithiasis : Bile salts and phospholipids are responsible for keeping the cholesterol in bile in a soluble state. Due to their deficiency (particularly bile salts), cholesterol crystals precipitate in the gall bladder often resulting in cholelithiasis—cholesterol gall stone disease. Cholelithiasis may be due to defective absorption of bile salts from the intestine, impairment in liver function, obstruction of biliary tract etc.
The patients of cholelithiasis respond to the administration of bile acid chenodeoxy cholic acid, commonly known as chenodiol. It is believed that a slow but gradual dissolution of gall stones occurs due to chenodiol. For severe cases of cholelithiasis, surgical removal of gall bladder is the only remedy.

hormones from cholesterol
Cholesterol is the precursor for the synthesis
of all the five classes of steroid hormones
(a) Glucocorticoids (e.g. cortisol)
(b) Mineralocorticoids (e.g. aldosterone)
(c) Progestins (e.g. progesterone)
Fig. 14.30 : Outline of steroid hormone synthesis from cholesterol (Numbers in the brackets represent the number of carbon atoms).
(d) Androgens (e.g. testosterone)
- (e) Estrogens (e.g. estradiol).
A brief outline of steroid hormonal synthesis is given in Fig.14.30 and more details are discussed under 'Hormones' (Chapter 19).

7-Dehydrocholesterol, an intermediate in the synthesis of cholesterol, is converted to cholecalciferol (vitamin D3) by ultraviolet rays in the skin.
A brief summary of prominent sources and the major pathways for utilization of cholesterol with the liver as the central metabolic organ is depicted in Fig.14.31.

Cholesterol is present in the lipoproteins in two forms plasma
1. About 70-75% of it is in an esterified form with long chain fatty acids.
2. About 25-30% as free cholesterol. This form of cholesterol readily exchanges between different lipoproteins and also with the cell membranes.
Role of LCAT : High density lipoproteins (HDL) and the enzyme lecithin-cholesterol acyltransferase (LCAT) are responsible for the transport and elimination of cholesterol from the
The cholesterol (cholesteryl) ester forms an integral part of HDL. In this manner, the cholesterol from the peripheral tissues is trapped in HDL, by a reaction catalysed by LCAT and then transported to liver for degradation and _ excretion. This mechanism is commonly known cholesterol transport.

In healthy individuals, the total plasma cholesterol is in the range of 150-200 mg/dl. In the new born, it is less than 100 mg/dl and rises to about 150 mg/dl within an year. The women have relatively lower plasma cholesterol which is attributed to the hormones-estrogens. Cholesterol level increases with increasing age (in women particularly after menopause), and also in pregnancy.
Plasma cholesterol is associated with different lipoprotein fractions (LDL, VLDL and HDL).
Total cholesterol can be estimated by many methods such as Libermann-Burchard reaction,
as_
reverse
Carr and Dructor method and, more recently, cholesterol oxidase method. HDLcholesterol can be determined after precipitating LDL and VLDL by polyethylene glycol (PEG). VLDL cholesterol is equivalent to 1/5thof plasma triacylglycerol (TG) in a fasting state. LDLcholesterol can be calculated from Friedewald formula given below.
LDL-cholesterol = Total cholesterol — (HDLcholesterol + TG/5).
The above formula is not valid if TG concentration is above 400 mg/dl.
In adults, the normal LDL-cholesterol is about 80-150 mg/dl while HDL-cholesterol is around 30-60 me/dl. Elevation in plasma HDLcholesterol is beneficial to the body, since it protects the body from atherosclerosis and coronary heart diseases (CHD). On the other hand, increase in LDL-cholesterol is harmful to the body as it may lead to various complications, including CHD.

Increase in plasma cholesterol (> 200 mg/dl) concentration is known as hypercholesterolemia and is observed in many disorders
1.
Diabetes mellitus
Due to
increased cholesterol
synthesis since
acetyl CoA is increased.
of
2. Hypothyroidism (myxoedema) : This is believed to be due to decrease in the HDL receptors on hepatocytes.
3. Obstructive jaundice Due to an obstruction in the excretion of cholesterol through bile.
4. Nephrotic syndrome : Increase in plasma globulin concentration is the characteristic feature of nephrotic syndrome. Cholesterol elevation is due to increase in plasma lipoprotein fractions in this disorder.
Hypercholesterolemia is associated with atherosclerosis and coronary heart disease (CHD). More specifically, LDL-cholesterol is positively correlated, whereas HDL-cholesterol is negatively correlated with CHD.
Bad cholesterol and good cholesterol Cholesterol is a natural metabolite performing a wide range of functions (membrane structure, precursor for steroid hormones, bile acids). The usages good and bad to cholesterol, although inappropriate, are still in use. The cholesterol in high concentration, present in LDL, is considered bad due to its involvement in altherosclerosis and related complications. Thus, LDL may be regarded as lethally dangerous lipoprotein. Small dense LDL (sdLDL) is considered to be the most dangerous fraction of LDL associated with CHD. On the other hand, HDL cholesterol is good since its high concentration counteracts atherogenesis. HDL may be considered as highly desirable lipoprotein.
Affects of lifestyles on serum cholesterol level : Individual lifestyles and habits certainly influence serum cholesterol, and thus play a significant role in the development coronary heart disease. The parametres such as high blood pressure, emotional stress, smoking, drinking of soft water (against hard water), coffee drinking, lack of exercise, obesity (particlarly of abdomen) elevate serum cholesterol level.

Several measures are advocated to lower the plasma cholesterol level
1. Consumption of PUFA : Dietary intake of polyunsaturated fatty acids (PUFA) reduces the plasma cholesterol level. PUFA will help in transport of cholesterol by LCAT mechanism (described earlier) and its excretion from the body. The oils with rich PUFA content include cottonseed oil, soya bean oil, sunflower oil, corn oil, fish oils etc. Ghee and coconut oil are poor sources of PUFA.
2. Dietary cholesterol : Dietary cholesterol influence on plasma cholesterol is minimal. However, avoidance of cholesterol-rich foods is advocated, and a dietary intake of <300 mg/day is advised. Certain drugs (e.g. ezetimide) inhibit intestinal cholesterol absorption.
3. Plant sterols : Certain plant sterols and their esters (e.g. sitostanol esters) reduce plasma cholesterol levels. They inhibit the intestinal absorption of dietary cholesterol.
4.
Dietary fiber
:
Fiber present
in vegetables
decreases the
cholesterol absorption
from the
intestine.
5. Avoiding high carbohydrate diet : Diets rich in carbohydrates (e.g. sucrose) should be avoided to control hypercholesterolemia.
6. Impact of lifestyles : Elevation in plasma cholesterol is obseved in people with smoking, abdominal obesity, lack of exercise, stress, high blood pressure, consumption of soft water etc. Therefore, adequate changes in the lifestyles will bring down plasma cholesterol.
7. Moderate alcohol cosumption The beneficial effects of moderate alcohol intake are masked by the ill effects of chronic alcoholism. Red wine is particularly beneficial due to its antioxidants, besides low alcohol content.

8. Use of drugs : Drugs such as lovastatin which inhibit HMG CoA reductase and decrease cholesterol synthesis are used. Statins currently in use include atorvastatin, simvastatin, fluvastatin and pravastatin. Statins are usually taken at night to ensure maximum effect (HMG CoA reductase activity at peak about 6 hours after dark). Certain drugs—cholestyramine and colestipol—bind with bile acids and decrease their intestinal reabsorption. This helps in the conversion of more cholesterol to bile acids and its excretion through feces. Clofibrate increases the activity of lipoprotein lipase and reduces the plasma cholesterol and triacylglycerols.
Fig. 14.33 : A general structure of lipoprotein complex. (Note : For the sake of clarity, only a part of the shell and core are filled with the constituents).

A decrease in the plasma cholesterol, although less common, is also observed. Hyperthyroidism, pernicious anemia, malabsorption syndrome, hemolytic jaundice etc., are some of the disorders associated with hypocholesterolemia.

Lipoproteins are molecular complexes that consist of lipids and proteins (conjugated proteins). They function as transport vehicles for lipids in blood plasma. Lipoproteins deliver the lipid components (cholesterol, triacylglycerol etc.) to various tissues for utilization.

A lipoprotein basically consists of a neutral lipid core (with triacylglycerol and/or cholesteryl ester) surrounded by a _ coat shell of phospholipids, apoproteins and cholesterol (Fig.14.33). The polar portions (amphiphilic) of phospholipids and cholesterol are exposed on the surface of lipoproteins so that lipoprotein is soluble in aqueous solution.

Five major classes of lipoproteins are identified in human plasma, based on_ their separation by electrophoresis (Fig.14.34).
1. Chylomicrons : They are synthesized in the intestine and transport exogenous (dietary) triacylglycerol to various tissues. They consist of highest (99%) quantity of lipid and lowest (1%) concentration of protein. The chylomicrons are the least in density and the largest in size, among the lipoproteins.
2. Very low density lipoproteins (VLDL) : They are produced in liver and intestine and are responsible for the transport of endogenously synthesized triacylglycerols.
3. Low density lipoproteins (LDL) : They are formed from VLDL in the blood circulation. They transport cholesterol from liver to other tissues.
4. High density lipoproteins (HDL) : They are mostly synthesized in liver. Three different fractions of HDL (1, 2 and 3) can be identified by ultracentrifugation. HDL particles transport cholesterol from peripheral tissues to liver (reverse cholesterol transport).
5. Free fatty acids—albumin : Free fatty acids in the circulation are in a bound form to albumin. Each molecule of albumin can hold about 20-30 molecules of free fatty acids. This lipoprotein cannot be separated by electrophoresis.
(-) Cathode
Fig. 14.34 : Electrophoresis of plasma
(serum)
lipoproteins.

The protein components of lipoproteins are known as apolipoproteins or, — simply, apoproteins. They perform the — following functions
1. Act as structural components of lipoproteins.
2. Recognize the cell membrane surface
3.
Activate enzymes
lipoprotein metabolism.
The comparative characteristic features of different lipoproteins with regard to electrophoretic patterns, size, composition etc. are given in Table 14.5.

A general picture of lipoprotein metabolism is depicted in Fig.14.35.
Chylomicrons (nascent) are synthesized in the small intestine during the course of fat absorption. They contain apoprotein Byg and mostly triacylglycerols. Apo Byg name is given since this apoprotein contains 48% of protein coded by apo B gene (apo Bygg is found in LDL and VLDL). Chylomicrons are produced when nascent particles combine with apo C Il and apo E, derived from HDL.
The liver synthesizes nascent VLDL containing apo Bygg which are rich in triacylglycerols and cholesterol. Circulating HDL donates apo C II and apo E to convert nascent VLDL to VLDL.
Role of lipoprotein lipase : The enzyme lipoprotein lipase is present in the capillary walls of adipose tissue, cardiac and skeletal muscle, besides other tissues. It hydrolyses a portion of triacylglycerols present in chylomicrons and VLDL to liberate free fatty acids and glycerol. Lipoprotein lipase is activated by apo C Il.
Uptake of chylomicron remnants by liver : As the triacylglycerols of chylomicrons and VLDL are degraded, they lose the apo C II which is returned to HDL. The chylomicron remnants are taken up by receptors present on the hepatocytes of liver.

40. Phospholipids, Chylomicrons = 8. Phospholipids, VLDL = 20. Phospholipids, LDL = 28. Phospholipids, HDL = 47. Free fatty acids, Chylomicrons = . Free fatty acids, VLDL = 1. Free fatty acids, LDL = 1. Free fatty acids, HDL = 1
Fig. 14.35 : Summary of metabolism of lipoproteins (Apoproteins—A, B,,, Bi, Cll and E; TG-Triacylglycerol; C—Cholesterol; P—Phospholipid; VLDL—Very low density lipoprotein;
/DL-Intermediate density lipoprotein; LDL—Low density lipoprotein; HDL—High density lipoprotein).

During the course of VLDL metabolism, intermediate density lipoprotein (IDL) is formed which lose apo-E and get converted to LDL. The apo E is returned to HDL. LDL contains high cholesterol (free and esterified) and less triacylglycerol.
:
CETP is synthesized in the liver, and it facilitates the exchange of components between different
lipoproteins. CETP can transfer cholesterol
©sters from HDL to VLDL or LDL, in exchange for
TG.

Fig. 14.36 : Metabolism of high density lipoproteins (P—Phospholipid; C-Cholesterol; CE—Cholesteryl ester; A, Cll, E-Apoproteins; LCAT-Lecithin cholesterol acyltransferase).
Deficiency of LDL receptors : A defect in LDL receptors results in the elevation of plasma LDL, hence plasma cholesterol. However, plasma triacylglycerol concentration remains normal. Deficiency of LDL receptors is observed in type Ha hyperbetalipoproteinemia. This disorder is associated with a very high risk of atherosclerosis (particularly of coronary artery).

High density lipoproteins are synthesized in the liver as discoidal particles — nascent HDL. They contain free cholesterol and phospholipids (mostly lecithin) and apoproteins (A, CII, E etc.).

plasma enzyme
_
lecithin-cholesterol
—acyl- transferase
(LCAT)
free cholesterol
in the
catalyses
(by the
esterification fatty
acid extrahepatic
HDL.
LCAT.
Apoprotein tissues
of and
A
promotes
HDL
also accepts
other lipoproteins
lecithin)
present transfers
to the
free in
of peripheral
cholesterol circulation
of from
and tissues
(Fig.14.36).
taken
LCAT-catalysed up
by esterification.
addition of
cholesterol, cell
Any
HDL
undergoes
Due
HDL
to the
particles become
spherical.
The HDL particles, with cholesteryl ester trapped inside, enter the hepatocytes by a receptor-mediated endocytosis. In the liver, the cholesteryl esters are degraded to cholesterol. The latter is utilized for the synthesis of bile acids and lipoproteins or excreted into bile (as cholesterol).

HDL is a good cholesterol and plays a cardioprotective role. It is attributed to the reverse cholesterol transport and removal of cholesterol from the peripheral tissue. Further, HDL plays an antioxidant role (due to the enzyme paroxanase activity) and protects LDL from getting oxidized. The result is that atherogenesis and related complications like heart attack are reduced.

Inherited disorders of lipoproteins are encountered in some individuals resulting in primary hyperor hypolipoproteinemias. These are due to genetic defects in lipoprotein metabolism and transport. The secondary acquired lipoprotein disorders are due to some other diseases (e.g. diabetes mellitus, nephrotic syndrome, atherosclerosis, hypothyrodism etc.), resulting in abnormal lipoprotein pattern which often resembles the primary inherited condition.

I, Increased plasma lipoprotein(s) = Chylomicrons. I, Increased plasma lipid (most) = Triacylglycerols. I, Probable metabolic defect = Deficiency of lipoprotein lipase. I, Risk of atherosclerosis = May increase. I, Suggested treatment = Low fat diet. IIa, Increased plasma lipoprotein(s) = LDL. IIa, Increased plasma lipid (most) = Cholesterol. IIa, Probable metabolic defect = Deficiency of LDL receptors. IIa, Risk of atherosclerosis = Very high (mostly in coronary artery). IIa, Suggested treatment = Low cholesterol fat diet; cholestyramine. IIb, Increased plasma lipoprotein(s) = LDL and VLDL. IIb, Increased plasma lipid (most) = Triacylglycerols and cholesterol. IIb, Probable metabolic defect = Overproduction of apo-B. IIb, Risk of atherosclerosis = do. IIb, Suggested treatment = do. III, Increased plasma lipoprotein(s) = IDL. III, Increased plasma lipid (most) = Triacylglycerols and cholesterol. III, Probable metabolic defect = Abnormality in apo-E. III, Risk of atherosclerosis = Very high (mostly in peripheral vessels). III, Suggested treatment = Low fat and low caloric diet; clofibrate. IV, Increased plasma lipoprotein(s) = VLDL. IV, Increased plasma lipid (most) = Triacylglycerols. IV, Probable metabolic defect = Overproduction of TG. IV, Risk of atherosclerosis = May or may not increase. IV, Suggested treatment = Low fat and low caloric diet; niacin. V, Increased plasma lipoprotein(s) = Chylomicrons and VLDL. V, Increased plasma lipid (most) = Triacylglycerols. V, Probable metabolic defect = . V, Risk of atherosclerosis = do. V, Suggested treatment = do

Elevation in one or more of the lipoprotein fractions constitutes —_ hyperlipoproteinemias. These disorders may be either primary or secondary. Some authors use hyperlipidemias or dyslipidemias instead of hyperlipoproteinemias. Frederickson's classification of hyperliporoteinemias—based on the electrophoretic patterns of plasma lipoproteins—is widely accepted to understand these disorders. It is given in Table 14.6 and briefly discussed hereunder.
1. Type I: This is due to familial lipoprotein lipase deficiency. The enzyme defect causes increase in plasma chylomicron and _triacyl-glycerol levels.
2. Type Ila : This is also known as hyperbetalipoproteinemia and is caused by a defect in LDL receptors. Secondary type Ila hyperlipoproteinemia is observed in association with diabetes mellitus, hypothyroidism, nephrotic syndrome etc. This disorder is characterized by hypercholesterolemia.
3. Type IIb : Both LDL and VLDL increase along with elevation in plasma cholesterol and triacylglycerol. This is believed to be due to overproduction of apo B.
4. Type III : This is commonly known as broad beta disease and characterized by the appearance of a broad 6-band corresponding to intermediate density lipoprotein (IDL) on electrophoresis.
5. Type IV : This is due to overproduction of endogenous triacylglycerols with a concomitant rise in VLDL. Type IV disorder is usually associated with obesity, alcoholism, diabetes mellitus etc.
6. Type V : Both chylomicrons and VLDL are elevated. This is mostly a secondary condition, due to disorders such as obesity, diabetes and excessive alcohol consumption etc.

Although low levels of plasma lipids (not HDL!) within the normal range may be beneficial to the body, very low lipid levels are undesirable. These are commonly associated with certain abnormalities
1. Familial hypobetalipoproteinemia : It is an inherited disorder probably due to an impairment in the synthesis of apoprotein B. The plasma LDL concentration in the affected individuals is between 10 to 50% of normal values. This disorder is harmless, and the individuals have healthy and long life.
2. Abetalipoproteinemia : This is a rare disorder due to a defect in the synthesis of apoprotein B. It is characterized by a total absence of f-lipoprotein (LDL) in plasma. Triacylglycerols are not found in plasma, but they accumulate in liver and intestine. Serum cholesterol level is low. Abetalipoproteinemia is associated with decreased absorption of fat
and fat-soluble vitamins. Impairment in physical growth and mental retardation are commonly observed.
Familial alpha-lipoprotein deficiency (Tangier disease) : The plasma HDL particles are almost absent. Due to this, the reverse transport of cholesterol is severely affected leading to the accumulation of cholesteryl esters in tissues. An absence of apoprotein C Il—which activates lipoprotein lipase—is also found. The plasma triacylglycerol levels are elevated. The affected individuals are at an increased risk for atheros- clerosis.

The normal concentration of lipid (mostly phospholipid) in liver is around 5%. Liver is not a storage organ for fat, unlike adipose tissue. However, in certain conditions, — lipids— especially the — triacylglycerols—accumulate excessively in liver, resulting in fatty liver
(Fig.14.37). In the normal liver, Kupffer cells contain lipids in the form of droplets. In fatty liver, droplets of triacylglycerols are found in the entire cytoplasm of hepatic cells. This causes impairment in metabolic functions of liver. Fatty liver is associated with fibrotic changes and cirrhosis, Fatty liver may occur due to two main causes.
1. Increased synthesis of triacylglycerols
2. Impairment in lipoprotein synthesis.
1. Increased triacylglycerol synthesis Mobilization of free fatty acids from adipose tissue and their influx into liver is much higher than their utilization. This leads to the overproduction of triacylglycerols and_ their accumulation in liver. Diabetes mellitus, starvation, alcoholism and high fat diet are associated with increased mobilization of fatty acids that often cause fatty liver. Alcohol also inhibits fatty acid oxidation and, thus, promotes fat and its synthesis deposition.

+ Niemann-Pick disease, caused by a defect in the enzyme sphingomyelinase, results in the accumulation of sphingomyelins in liver and spleen.
+ About a dozen glycolipid storage diseases are known. These include Gaucher’s disease and Krabbe’s disease.
+ Hypercholesterolemia is associated with atherosclerosis and coronary heart diseases. Consumption of polyunsaturated fatty acids and fiber decreases cholesterol in circulation. Drugs—such as lovastatin, cholestyramine, compactin and _ clofibrate— reduce plasma cholesterol.
+ Cholelithiasis, a cholesterol gall stone disease, is caused by a defect in the absorption of bile salts from the intestine or biliary tract obstruction.
+ High
density
lipoproteins—in
association
with
acyltrans-
ferase
(LCAT)—are
responsible
for
the
transport
and
elimination
of
from
the
body.
+ Hyperlipoproteinemias are a group of disorders caused by the elevation of one or more of plasma lipoprotein fractions.
+ Excessive accumulation of triacylglycerols causes fatty liver which can often be
prevented
by
the
of
lipotropic
factors
(choline,
betaine,
methionine).
Fig. 14.37 : Development of fatty liver along with responsible factors.
2. Impaired synthesis of lipoproteins : The synthesis of very low density lipoproteins (VLDL) actively takes place in liver. VLDL formation requires phospholipids and apoprotein B. Fatty liver caused by impaired lipoprotein synthesis may be due to :
1 a failure in the formation/secretion of lipoprotein.
1 a defect in phospholipid synthesis;
1 a block in apoprotein formation;
Among the three causes, fatty liver due to impairment in phospholipid synthesis has been studied in some detail. This is usually associated with the dietary deficiency of lipotropic factors such as choline, betaine, inositol etc. (more details given later). Deficiency of essential fatty

acids leads to a decreased formation — of phospholipids. Further, excessive consumption of cholesterol competes with essential fatty acids and impairs phospholipid synthesis.
Certain chemicals (e.g. puromycin, ethionine, carbon tetrachloride, chloroform, lead, phosphorus etc.) that inhibit protein synthesis cause fatty liver. This is due to a blockade in the synthesis of apoprotein B required for VLDL production.
Lipoprotein synthesis and their secretion require ATP. Decrease in the availability of ATP, sometimes found in pyridoxine and pantothenic acid deficiency, impairs lipoprotein formation. The action of ethionine in the development of fatty liver is believed to be due to a reduction in the availability of ATP. Ethionine competes with methionine and traps the available adenosine (as adenosylethionine)—thereby reducing ATP levels.
Deficiency of vitamin E is associated with fatty liver. Selenium acts as a protective agent in such a condition.
Endocrine factors : Certain hormones like ACTH, insulin, thyroid hormones, adrenocorticoids promote deposition of fat in liver.

These are the substances the deficiency of which causes fat (triacylglycerol) to accumulate in liver. This may happen despite the fatty acid synthesis and uptake by liver being normal.

These include choline, betaine, methionine and inositol. Folic acid, vitamin By», glycine and serine also serve as lipotropic factors to some extent.

+ Obesity is an abnormal increase in body weight due to excessive fat deposition (>25%). Overeating, lack of exercise and genetic predisposition play a significant role in the development of obesity.
+ Some individuals with active brown adipose tissue do not become obese despite overeating, since whatever they eat is liberated as heat due to uncoupling of oxidation and phosphorylation in the mitochondria.
+ A protein namely leptin, produced by the adipose tissue, has been identified in mice. Injection of leptin to obese mice caused reduction in body fat, increased metabolic rate and increased insulin concentration, besides reduced food intake. Leptin has also been detected in humans.
+ Anorexia nervosa is a psychiatric disorder associated with total loss of appetite—mostly found in females in the age group 10-30 years.
+ Atherosclerosis is characterized by hardening of arteries due to the accumulation of lipids and other compounds. The probable causes of atherosclerosis include hyperlipoproteinemias, diabetes mellitus, obesity, high consumption of saturated fat, lack of exercise and stress.
+ Atherosclerosis and coronary heart disease are directly correlated with plasma cholesterol and LDL, inversely with HDL. Elevation of plasma lipoprotein a suggests increased risk of CHD.
+ Alcoholism is associated with fatty liver, hyperlipidemia and atherosclerosis.

Choline and inositol are components of phospholipids and, hence, required for their synthesis. The other lipotropic factors are directly or indirectly concerned with transmethylation reactions and, ultimately, the synthesis of choline. Severe protein deficiency (e.g. kwashiorkor) causes fatty liver. This is due to a defect in the synthesis of choline as a result of insufficient amino acid (particularly methionine) supply. In other words the non-availability of methyl groups may lead to fatty liver (Fig.14.37).

Several explanations are offered to understand choline deficiency causing fatty liver : phospholipid
(1) Decreased synthesis (Fig.14.37); (2) Impaired formation of lipoprotein membrane; (3) Reduced synthesis of carnitine due to insufficient supply of methyl groups; (4) Impairment in fatty acid oxidation.

Obesity is an abnormal increase in the body weight due to excessive fat deposition.

Men and women are considered as obese if their weight due to fat (in adipose tissue), respectively, exceeds more than 20% and 25% of body weight. Obesity is basically a disorder of excess calorie intake, in simple language— overeating. It has to be remembered that every 7 calories of excess consumption leads to 1 g fat deposit and increase in body weight. Overeating—coupled with lack of physical exercise—contribute to obesity.
Obesity due to virus infection : It was found that around 15% of people weighing more than 120 kg had antibodies to adenovirus-36 in their blood, implying that this virus infection (causes cold, diarrhea etc.), by an unknown mechanism contributes to obesity. Surprisingly, adenovirus-36 infected individuals have normal serum cholesterol and other lipid parameters.

Clinical obesity is represented by body mass index. BMI is calculated as the weight (in kilograms) divided by the height (in meters").
Weight (kg)
BMI (kg/m?) =
——————
[height (m)?]
Healthy reference range for BMI is between
18.5-24.9 kg/m2.
1 Grade | obesity or overweight — BMI 25-30 kg/m?
1 Grade II or clinical obesity — BMI > 30 kg/m?
1 Grade III or morbid obesity — BMI > 40 kg/m?
Obesity complications hypertension, disease. is associated with many health e.g. type Il diabetes, CHD, stroke, arthritis, gall bladder
In recent years, the ratio between waist and hip sizes (for men <0.9 and for women < 0.85) is considered as more effective than BMI, particularly with regard to the risk of heart diseases. The lower is the waist to hip ratio, the lower the risk for health complications, and therefore better is the health.

There is strong evidence to suggest that obesity has genetic basis. Thus, a child born to two obese people has about 75% chances of being obese. One gene namely ob gene, expressed in adipocytes (of white adipose tissue) producing a protein called leptin (mol. wt. 16,000 daltons), is associated with obesity.
Leptin is regarded as a body weight regulatory hormone. |t binds to a_ specific receptor in the brain and functions as a lipostat. When the fat stores in the adipose tissue are adequate, leptin levels are high. This signals to restrict the feeding behaviour and limit fat deposition. Further, leptin stimulates lipolysis and inhibits lipogenesis. Any genetic defect in leptin or its receptor will lead to extreme overeating and obesity. Treatment of such obese individuals with leptin has been shown to reverse obesity. During starvation, leptin levels fall which promote feeding, and fat production and its deposition.

Obesity is due to an increase in both the number and size of adipocytes (of adipose tissue). There are two types of adipose tissues
- 1. White adipose tissue : The fat is mostly stored and this tissue is metabolically less active.
2.
Brown adipose
tissue
:
The stored
fat is
less but
the tissue
is metabolically
very active.
Brown adipose tissue possesses high proportion of mitochondria and cytochromes but low activity of ATP synthase. This is an active centre for the oxidation of fat and glucose and is responsible for the diet-induced thermogenesis.
The peculiarity of mitochondria of brown adipose tissue is that the oxidation and phosphorylation are not coupled. Mitochondrial oxidation produces more heat and less ATP. A specific protein—namely thermogenin—has been isolated in the inner membrane of these mitochondria. Thermogenin functions like an uncoupler and dissipates the energy in the form of heat, and thus blocks the formation of ATP.
Brown adipose tissue is mostly found in hibernating animals, and the animals exposed to cold, besides the newborn. In adult humans, though not a prominent tissue, it is located in the thoracic region. It is significant to note that brown adipose tissue is almost absent in obese persons. Some individuals are fortunate to have active brown adipose tissue. They eat and liberate it as heat, and therefore do not become obese.
Pharmacological treatment of obesity : In recent years, synthetic lipids such as Olestra and Orlistat are used to treat obesity. They taste like natural lipids but cannot be digested, and excreted unchanged.

Metabolic syndrome (MS) is a cluster of different conditions that adversely affect the health. The components contributing to MS include abdominal obesity, insulin resistance, dyslipidemia, elevated blood pressure, overnutrition, sedentary lifestyles etc. As per WHO criteria, metabolic syndrome has the following characteristics
1. Insulin resistance — identified either as type 2 diabetes or elevated fasting blood glucose (>100 mg/dl) or impaired glucose tolerance.
2. And any two of the following
(i) Hypertension (> = 140/90 mm Hg)
(ii) Dyslipidemia (serum TG > = 150 mg/dl or HDL cholesterol <35 mg/dl in men or <39 mg/dl in women.
(iii) BMI >30 kg/m? or waist : hip ratio of >0.9 in men or 0.85 in women.
Metabolic syndrome can be managed by healthy habits and change in lifestyles— restricted balanced diet, adequate intake of fiber, and antioxidants, exercise, avoiding smoking, stress-free life etc.

This is opposite of what is seen in obesity. Cachexia is characterized by a failure to maintain normal lipid stores in the body. It involves higher rate of fat mobilization than deposition.
Anorexia nervosa is a total loss of appetite. This is mostly seen in females in the age group 10-30 years. Surprisingly, majority of the affected individuals are from wealthy families where food is aplenty. And some members in these families may be even obese! Anorexia nervosa is more a psychiatric disease.

Atherosclerosis (Greek: athere—mush) is a complex disease characterized by thickening or hardening of arteries due to the accumulation of lipids (particularly — cholesterol, free, and esterified) collagen, fibrous tissue, proteoglycans, calcium deposits etc. in the inner arterial wall. Atherosclerosis is a progressive disorder that narrows and ultimately blocks the arteries. Infarction is the term used to indicate the stoppage of blood flow resulting in the death of affected tissue. Coronary arteries—the arteries supplying blood to heart—are the most commonly affected leading to myocardial infarction or heart attacks.
Causes of atherosclerosis and CHD : The development of atherosclerosis and the risk for
the coronary heart disease (CHD) is directly correlated with plasma cholesterol and LDL. On the other hand, plasma HDL is inversely correlated with CHD.

Certain diseases are associated with atherosclerosis. These include diabetes mellitus, hyperlipoproteinemias, nephrotic syndrome, hypothyroidism etc. Many other factors like obesity, high consumption of saturated fat, excessive smoking, lack of physical exercise, hypertension, stress etc., are the probable causes of atherosclerosis.

The increased levels of plasma HDL (good cholesterol) are correlated with a low incidence of cardiovascular disorders. Women have higher HDL and are less prone to heart diseases compared to men. This is attributed to estrogens in} women. Strenuous physical exercise, moderate alcohol intake, consumption of unsaturated fatty acids (vegetable and fish oils), reduction in body weight—all tend to increase HDL levels and reduce the risk CHD (see hypercholesterolemia, p-315).

Lipoprotein a (Lp-a) is almost identical in structure to LDL. Lp-a contains an additional apoprotein, apo-a. Lp-a_ inhibits fibrinolysis. Recent studies have shown that elevation of lipoprotein-a in the plasma (>30 mg/dl) suggests increased risk of CHD. \t is hypothesized that elevated Lp-a reduces the breakdown of blood clots by interfering with plasminogen activation. This results in intravascular thrombosis, and increased risk of heart attacks. Indians have higher levels of Lp-a compared to Western population.

Antioxidants, in
general, decrease
oxidation of
LDL.
There on
the is
some epidemiological
antioxidants
(vitamins studies
evidence, the
based that
taking
E
reduces the
and
C
of or
risk of
B-carotene)
atherosclerosis, and
CHD.

Walker has rightly said 'alcohol can be a food, a drug or a poison depending on the dose.' In small quantities, alcohol relieves tension and anxiety. Unfortunately, consumption of alcohol seldom ends with small doses, hence the beneficial effects are over-shadowed by the harmful effects.
Alcohol (ethanol or ethyl alcohol) is readily absorbed by the stomach and _ intestine. Consequently, less than 2% of the alcohol consumed is excreted through lungs, urine and sweat.
Alcohol gets oxidized in the liver by alcohol dehydrogenase to acetaldehyde.
Besides ADH, microsomal ethanol oxidizing system (MEOS) is also involved in the metabolism of alcohol. Aldehyde, produced by the action of either ADH or MEOS, is responsible for the manifestations of alcohol. The enzyme aldehyde dehydrogenase converts aldehyde to acetic acid which then enters Krebs cycle in the form of acetyl CoA.

CH3—CHO CH3COOH Acetaldehyde NAD* NADH+H* Acetic acid Since the activity of aldehyde dehydrogenase is less than that of alcohol dehydrogenase, acetaldehyde accumulates leading to various complications. Disulfiram, a drug used for the treatment of alcoholism, inhibits aldehyde dehydrogenase.

The metabolism
of alcohol
(by both
dehydrogenases)
involves
NAD*, and
consequently ratio.
This is
alterations are
listed.
1.
High conversion
the consumption
a
high mostly
responsible of
NADH/NAD*
for observed
in alcoholism.
concentration the
metabolic
Some of
NADH
to lactic
to acidosis.
lactate of
them favours
which the
may lead
2. Hypoglycemia due to reduced gluconeogenesis is observed. This happens as a result of decreased availability of pyruvate and oxaloacetate (the latter gets converted to malate by high NADH).
3. Citric acid cycle is impaired since the availability of oxaloacetate and NAD?* is reduced. As a result, acetyl CoA accumulates which gets diverted towards ketogenesis, cholesterologenesis, and fatty acid synthesis. Accumulation of fats leads to fatty liver and hyperlipidemia.
4. Increased concentration of serum uric acid
due
to
its
reduced
excretion
is
observed
in
alcoholism.
This
is
due
to
lactic
acidosis.
5. Acetaldehyde interferes with
the
functioning
of
neurotransmitters,
with
an
overall
effect
of
neurological
6. Acetaldehyde causes headache, nausea,
tachycardia,
reduced
blood
pressure
etc.

Chronic alcoholism is associated with cirrhosis of liver, neurodegenerative changes, cardiomyopathy, diuresis, impotence etc.

1. Triacylglycerols (TG) are the highly concentrated form of energy, stored in adipose tissue. Hormone-sensitive lipase hydrolyses TG to free fatty acids which are transported as albumin-FFA complexes.
2. Fatty acids are activated (acyl CoA) and transported by carnitine to mitchondria where they get oxidized (mostly by B-oxidation) to liberate energy. Complete oxidation of one mole palmitate liberates 129 ATP.
3. Excessive utilization of fatty acids occurs in uncontrolled diabetes mellitus and starvation. This results in the overproduction of ketone bodies (in liver), namely acetone, acetoacetic acid and f-hydroxy butyric acid. The last two ketone bodies serve as energy source for peripheral tissues.
4. Fatty acid biosynthesis occurs from acetyl CoA in the cytosol through the involvement of a multienzyme complex associated with acyl carrier protein (ACP). The reducing equivalents (NADPH + Ht*) are supplied mostly by HMP shunt.
5. Synthesis of triacylglycerols and phospholipids (PL) occurs from glycerol 3-phosphate and dihydroxyacetone phosphate with the addition of acyl CoA, and activated nitrogenous bases (for PL).
6. Cholesterol is synthesized from acetyl CoA in a series of reactions involving HMG CoA, mevalonate, isoprenoid units and squalene as the intermediates. Cholesterol serves as a precursor for bile acids, steroid hormones and vitamin D.
7.
Lipoproteins
are
the
transport
vehicles
for
lipids
in
the
plasma.
Lipoprotein
disorders
are
with
in
their
plasma
levels.
Elevation
in
LDL
and
VLDL—
in
with
and
TG—poses
a
serious
health
problem
with
increased
risk
of
atherosclerosis
and
CHD.
8.

Excessive accumulation of triacylglycerols in liver causes fatty liver, which may be due to increased production of TG or impairment in lipoprotein (VLDL) synthesis. The latter is mostly associated with the deficiency of certain substances called lipotropic factors (e.g. choline, betaine, methionine etc.)
9.
Obesity
is
an
abnormal
increase
in
body
weight
(with
more
than
25%
due
to
fat).
Among
the
many
causative
factors
of
obesity,
lack
of
active
brown
adipose
tissues
(which
burn
fat
and
liberate
heat)
in
these
is
gaining
10. Atherosclerosis is a complex disease characterized by thickening of arteries due to the accumulation of lipids. Atherosclerosis and CHD are directly correlated with LDL and inversely with HDL of plasma.

1. Describe the functions and metabolism of phospholipids.
5. Give an account of fatty acid oxidation.
. Give an account of cholesterol biosynthesis. Add a note on the significance of plasma cholesterol estimation.
. Describe in detail the extramitochondrial synthesis of fatty acids.
. Write about the types, characteristics and metabolism of lipoproteins. Add a note on lipoprotein disorders.

(a) Carnitine, (b) LCAT, (c) Fatty liver, (d) Ketone bodies, (e) Lipotropic factors, (f) Acyl carrier protein, (g) Degradation of cholesterol, (h) HDL, (i) Lipoprotein lipase, (j) Brown adipose tissue.

1. The most predominant lipid component of chylomicrons
2. Cholesterol synthesis is controlled by feedback inhibition of the enzyme
3. A compound possessing hydrophobic and hydrophilic groups in its structure is known
as
4. Niemann-Pick disease is due to a defect in the enzyme
9. Plasma lipoprotein that is inversely correlated with coronary heart disease is
10 . The fatty acid that is commonly found in the Cy of triacylglycerols is
. The lipoprotein involved in the reverse cholesterol transport is
.
. The total number of ATP produced by the oxidation of a molecule of palmitic acid is
. The long chain fatty acids (Cpg,—C35) are not oxidized due to the absence of peroxisomes. This disorder is known as
. Acetyl CoA from the mitochondria is transported into the cytosol after its conversion to

11. The following substance(s) is (are) ketogenic
12. The lipoprotein possessing the highest quantity of phospholipid
13. Hypercholesterolemia is observed in the disorder(s)
14. The two final products in the B-oxidation of odd chain fatty acids are
15. Hormone sensitive lipase activity is inhibited by the hormone
(a) Fatty acids (b) Leucine (c) Lysine (d) All of them.
(a) HDL (b) LDL (c) VLDL (d) Chylomicrons.
(a) Hypothyroidism (b) Diabetes mellitus (c) Nephrotic syndrome (d) All of them.
(a) Acetyl CoA and malonyl CoA (b) Acetyl CoA and acetyl CoA (c) Acetyl CoA and propionyl CoA (d) Acetyl CoA and succinyl CoA.
(a) Epinephrine (b) Insulin (c) Thyroxine (d) Glucocorticoids.
Pr: are the most abundant organic compounds and constitute a major part of the body dry weight (10-12 kg in adults). They perform a wide variety of static (structural) and dynamic (enzymes, hormones, clotting factors, receptors etc.) functions. About half of the body protein (predominantly collagen) is present in the supportive tissue (skeleton and connective) while the other half is intracellular.
Proteins are nitrogen-containing —macromolecules consisting of L-c-amino acids as the repeating units. Of the 20 amino acids found in proteins, half can be synthesized by the body (non-essential) while the rest have to be provided in the diet (essential amino acids).
The proteins on degradation (proteolysis) release individual amino acids. Amino acids are not just the structural components of proteins. Each one of the 20 naturally occurring amino acids undergoes its own metabolism and performs specific functions. Some of the amino acids also serve as precursors for the synthesis of many biologically important compounds (e.g.

melanin, serotonin, creatine etc.). Certain amino acids may directly act as neurotransmitters (e.g. glycine aspartate, glutamate). Protein metabolism is more appropriately learnt as metabolism of amino acids.

An adult has about 100 g of free amino acids which represent the amino acid pool of the body. The amino acid pool may be an oversimplification of the facts, since there is no single compartment—rather, several compartments exist.
Glutamate and glutamine together constitute about 50%, and essential amino acids about 10% of the body pool (100 g). The concentration of intracellular amino acids is always higher than the extracellular amino acids. Amino acids enter the cells against a concentration gradient by active transport.
represents body
protein turnover. There is a wide variation in the turnover of individual proteins. For instance, the plasma proteins and digestive enzymes are rapidly degraded, their half-lives being in hours or days. The structural proteins (e.g. collagen) have long half-lives, often in months and years.
Control of protein turnover : The turnover of the protein is influenced by many factors. A small polypeptide called ubiquitin (mol. wt. 8,500) tags with the proteins and_ facilitates degradation. Certain proteins with amino acid sequence proline, glutamine (one letter code E), serine and threonine (PEST sequence) are rapidly degraded.
(b) Dietary protein : There is a regular loss of nitrogen from the body due to degradation of amino acids. In healthy adults, it is estimated that about 30-50 g of protein is lost everyday from the body. This amount of protein (30-50 g/ day) must, therefore, be supplied daily in the diet to maintain nitrogen balance. The purpose of dietary protein is to supply amino acids (particularly the essential ones) for the synthesis of proteins and other nitrogen compounds.
There is no storage form of amino acids as is the case for carbohydrates (glycogen) and lipids (triacylglycerols). The excess intake of amino acids are metabolized—oxidized to provide energy, converted to glucose or fat. The amino groups are lost as urea and excreted. The protein consumption in developed countries is much higher than the recommended dietary allowance (i.e. 1g/kg body weight/day). The daily protein intake by an adult in most countries is 40-100 g. Protein is digested by proteolytic enzymes to amino acids which are absorbed in the intestine and enter the body pool of amino acids.

(c) Synthesis of non-essential amino acids : Ten out of the 20 naturally occurring amino acids can be synthesized by the body which contribute to the amino acid pool.

(a) Most of the body proteins (300-400 g/day) degraded are synthesized from the amino acid pool. These include enzymes, hormones, immunoproteins, contractile proteins etc.
(b) Many important nitrogenous compounds (porphyrins, purines, pyrimidines, etc.) are produced from the amino acids. About 30 g of protein is daily utilized for this purpose.
(c) Generally, about 10-15% of body energy requirements are met from the amino acids.
(d) The amino acids are converted to carbohydrates and _ fats. This becomes predominant when the protein consumption is in excess of the body requirements.

The amino acids undergo certain common reactions like transamination followed by deamination for the liberation of ammonia. The amino group of the amino acids is utilized for the formation of urea which is an excretory end product of protein metabolism. The carbon skeleton of the amino acids is first converted to keto acids (by transamination) which meet one or more of the following fates.
1. Utilized to generate energy.
2. Used for the synthesis of glucose.
3. Diverted for the formation of fat or ketone bodies.
4. Involved in the production of non-essential amino acids.
A general picture of amino acid metabolism is depicted in Fig.15.2.
The details of general and specific metabolic reactions of amino acids are described in the following pages.

The transfer of an amino (— NH) group from an amino acid to a keto acid is known as transamination. This process involves the interconversion of a pair of amino acids and a pair of keto acids, catalysed by a group of enzymes called transaminases (recently, aminotransferases).
Fig. 15.2 : An overview of amino acid metabolism.

1. All transaminases require pyridoxal phosphate (PLP), a coenzyme derived from vitamin Bg.
2. Specific transaminases exist for each pair of amino and keto acids. However, only two— namely, aspartate transaminase and alanine transaminase—make a significant contribution for transamination.
3. There is no free NH3 liberated, only the transfer of amino group occurs.
- 4. Transamination is reversible (Fig.15.3).
Fig. 15.3 : Transamination reaction.
4H

The removal of amino group from the amino acids as NH3 is deamination. Transamination (discussed above) involves only the shuffling of amino groups among the amino acids. On the other hand, deamination results in the liberation of ammonia for urea synthesis. Simultaneously, the carbon skeleton of amino acids is converted to keto acids. Deamination may be either oxidative or non-oxidative.
Although transamination and deamination are separately discussed, they occur simultaneously, often involving glutamate as the central molecule. For this reason, some authors use the term transdeamination while describing the reactions of transamination and deamination, particularly involving glutamate.

Oxidative deamination is the liberation of free ammonia from the amino group of amino acids coupled with oxidation. This takes place mostly in liver and kidney. The purpose of oxidative deamination is to provide NH3 for urea synthesis and o-keto acids for a variety of reactions, including energy generation.
Role of glutamate dehydrogenase : In the process of transamination, the amino groups of most amino acids are transferred to o-ketoglutarate to produce glutamate. Thus, glutamate serves as a 'collection centre' for amino groups in the biological system. Glutamate rapidly undergoes oxidative deamination, catalysed by glutamate dehydrogenase (GDH) to liberate ammonia. This enzyme is unique in that it can utilize either NAD* or NADPt as a coenzyme. Conversion of glutamate to a-ketoglutarate occurs through the formation of an intermediate, a-iminoglutarate (Fig.15.5).
Glutamate dehydrogenase catalysed reaction is important as it reversibly links up glutamate metabolism with TCA cycle through o-ketoglutarate. GDH is involved in both catabolic and anabolic reactions.
Regulation of GDH activity : Glutamate dehydrogenase is a zinc containing mitochondrial enzyme. It is a complex enzyme consisting of six identical units with a molecular weight of 56,000 each. GDH is controlled by allosteric regulation. GTP and ATP inhibit— whereas GDP and ADP activate—glutamate dehydrogenase. Steroid and thyroid hormones inhibit GDH.
After ingestion of a protein-rich meal, liver glutamate level is elevated. It is converted to a-ketoglutarate with liberation of NH3. Further, when the cellular energy levels are low, the degradation of glutamate is increased to provide a-ketoglutarate which enters TCA cycle to liberate energy.
Oxidative deamination by amino acid oxidases : L-Amino acid oxidase and D-amino acid oxidase are flavoproteins, possessing FMN and FAD, respectively. They act on_ the corresponding amino acids (L or D) to produce a-keto acids and NH3. In this reaction, oxygen is reduced to HO», which is later decomposed by catalase (Fig.15.6).

The activity of L-amino acid oxidase is much low while that of D-amino acid oxidase is high in tissues (mostly liver and kidney). L-Amino acid oxidase does not act on glycine and dicarboxylic
acids. This enzyme, due to its very low activity, does not appear to play any significant role in the amino acid metabolism.

Fate of D-amino acids : D-Amino acids are found in plants and microorganisms. They are, however, not present in the mammalian proteins. But D-amino acids are regularly taken in the diet and metabolized by the body. D-Amino acid oxidase converts them to the respective a-keto acids by oxidative deamination. The o-keto acids so produced undergo transamination to be converted to L-amino acids which participate in various metabolisms. Keto acids may be oxidized to generate energy or serve as precursors for glucose and fat synthesis. Thus, D-amino acid oxidase is important as it initiates the first step for the conversion of unnatural D-amino acids to L-amino acids in the body (Fig.15.7).

Ammonia is constantly being liberated in the metabolism of amino acids (mostly) and other nitrogenous compounds. At the physiological pH, ammonia exists as ammonium (NH) ion.

The production of NH3 occurs from the amino acids (transamination and deamination), biogenic amines, amino group of purines and pyrimidines and by the action of intestinal bacteria (urease) on urea.

Despite a regular and constant production of NH3 from various tissues, its concentration in
the circulation is surprisingly low (normal plasma 10-20 mg/dl). This is mostly because the body has an_ efficient mechanism for NH3 transport and _ its immediate utilization for urea synthesis. The transport of ammonia between various tissues and the liver mostly occurs in the form of glutamine or alanine and not as free ammonia. Alanine is important for NH3 transport from muscle to liver by glucose-alanine cycle (Refer Fig.13.13).
Role of glutamine : Glutamine is a storehouse of NH3. It is present at the highest concentration (8 mg/dl in adults)
in blood among the amino acids. Glutamine serves as a storage and transport form of NH3. Its synthesis mostly occurs in liver, brain and muscle. Ammonia is removed from the brain predominantly as glutamine. Glutamine is freely diffusible in tissues, hence easily transported.
Glutamine synthetase (a mitochondrial enzyme) is responsible for the synthesis of glutamine from glutamate and ammonia. This reaction is unidirectional and requires ATP and Mg2* ions.
Glutamine can be deaminated by hydrolysis to release ammonia by glutaminase (Fig.15.8) an enzyme mostly found in kidney and intestinal cells.

Ammonia is not just a waste product of nitrogen metabolism. It is involved (directly or via glutamine) for the synthesis of many compounds in the body. These include nonessential amino acids, purines, pyrimidines, amino sugars, asparagine etc. Ammonium ions (NH4*) are very important to maintain acid-base balance of the body.

The organisms, during the course of evolution, have developed different mechanisms for the disposal of ammonia from the body. The animals in this regard are of three different types
(a)
Ammoniotelic
The aquatic
animals dispose off NH3 into the surrounding water.
(b) Uricotelic : Ammonia is converted mostly to uric acid e.g. reptiles and birds.
(c) Ureotelic : The mammals including man convert NH3 to urea. Urea is a non-toxic and soluble compound, hence easily excreted.

Even a marginal elevation in the blood ammonia concentration is harmful to the brain. Ammonia, when it accumulates in the body, results in slurring of speech and blurring of the vision and causes tremors. It may lead to coma and, finally, death, if not corrected.
Hyperammonemiia : Elevation in blood NH3 level may be genetic or acquired. Impairment in urea synthesis due to a defect in any one of the five enzymes is described in urea synthesis. All these disorders lead to hyperammonemia and cause hepatic coma and mental retardation. The acquired hyperammonemia may be due to hepatitis, alcoholism etc. where the urea synthesis becomes defective, hence NH3 accumulates.
Explanation
for
NH3
toxicity
:
The
reaction
catalysed by glutamate dehydrogenase probably explains the toxic affects of NH3 in brain
NADPH
+
H*
NADP*
+
NH,
Glutamate
Accumulation of NH3 shifts the equilibrium to the right with more glutamate formation, hence more utilization of a-ketoglutarate. aKetoglutarate is a key intermediate in TCA cycle and its depleted levels impair the TCA cycle. The net result is that production of energy (ATP) by the brain is reduced. The toxic effects of NH3 on brain are, therefore, due to impairment in ATP formation.
Trapping and elimination of ammonia : When the plasma level of ammonia is highly elevated, intravenous administration of sodium benzoate and phenyllactate is done. These compounds can respectively condense with glycine and glutamate to form water soluble products that can be easily excreted. By this way, ammonia can be trapped and removed from the body. In some instances of toxic hyperammonemia, hemodialysis may become necessary.

Urea is the end product of protein metabolism (amino acid metabolism). The nitrogen of amino acids, converted to ammonia (as described above), is toxic to the body. It is converted to urea and detoxified. As such, urea accounts for 80-90% of the nitrogen containing substances excreted in urine.
Urea is synthesized in liver and transported to kidneys for excretion in urine. Urea cycle is the first metabolic cycle that was elucidated by Hans Krebs and Kurt Henseleit (1932), hence it is known as Krebs-Henseleit cycle. The individual reactions, however, were described in more detail later on by Ratner and Cohen.
Urea has two amino (—NH)) groups, one derived from NHz and the other from aspartate. Carbon atom is supplied by CO. Urea synthesis is a five-step cyclic process, with five distinct enzymes. The first two enzymes are present in mitochondria while the rest are localized in cytosol. The details of urea cycle are described (Figs.15.9 and 15.10).
1. Synthesis of carbamoyl phosphate Carbamoyl phosphate synthase | (CPS 1) of mitochondria catalyses the condensation of
Fig. 15.9 : Outline of urea cycle. (Note : In the synthesis of urea one amino group comes from ammonium ion while the other is from aspartate; carbon is derived from CO,, This is represented in colours.)
NHZ ions with CO, to form carbamoyl phosphate. This step consumes two ATP and is irreversible, and rate-limiting. CPS | requires Nacetylglutamate for its activity. Another enzyme, carbamoyl phosphate synthase Il (CPS Il)— involved in pyrimidine synthesis—is present in cytosol. It accepts amino group from glutamine and does not require N-acetylglutamate for its activity.

2. Formation of citrulline Citrulline is synthesized from carbamoyl phosphate and ornithine by ornithine — transcarbamoylase. Ornithine is regenerated and used in urea cycle. Therefore, its role is comparable to that of oxaloacetate in citric acid cycle. Ornithine and citrulline are basic amino acids. (They are never found in protein structure due to lack of codons). Citrulline produced in this reaction is transported to cytosol by a transporter system.
3. Synthesis of arginosuccinate : Arginosuccinate synthase condenses citrulline with aspartate to produce arginosuccinate. The second amino group of urea is incorporated in this reaction. This step requires ATP which is cleaved to AMP and pyrophosphate (PPi). The latter is immediately broken down to inorganic phosphate (Pi).
4. Cleavage of arginosuccinate : Arginosuccinase cleaves arginosuccinate to give arginine and fumarate. Arginine is the immediate precursor for urea. Fumarate liberated here provides a connecting link with TCA cycle, gluconeogenesis etc.
5. Formation of urea : Arginase is the fifth and final enzyme that cleaves arginine to yield urea and ornithine. Ornithine, so regenerated, enters mitochondria for its reuse in the urea cycle. Arginase is activated by Co2+ and Mn?*. Ornithine and lysine compete with arginine (competitive inhibition). Arginase is mostly found in the liver, while the rest of the enzymes (four) of urea cycle are also present in other tissues. For this reason, arginine synthesis may occur to varying degrees in many tissues. But only the liver can ultimately produce urea.

The urea cycle is irreversible and consumes 4 ATP. Two ATP are utilized for the synthesis of carbamoyl! phosphate. One ATP is converted to AMP and PPi to produce arginosuccinate which equals to 2 ATP. Hence 4 ATP are actually consumed.
NH4* + CO, + Aspartate + 3ATP
—>
Urea
+ Fumarate + 2 ADP + 2 Pi + AMP + PPi

The first reaction catalysed by carbamoyl phosphate synthase I (CPS |) is rate-limiting reaction or committed step in urea synthesis. CPS | is allosterically activated by N-acetylglutamate (NAG). It is synthesized from glutamate and acetyl CoA by synthase and degraded by a hydrolase (Fig.15.11).
The rate of urea synthesis in liver is correlated with the concentration of N-acetylglutamate. High concentrations of arginine increase NAG. The consumption of a protein-rich meal increases the level of NAG in liver, leading to enhanced urea synthesis.
Carbamoyl phosphate synthase | and glutamate dehydrogenase are localized in the mitochondria. They coordinate with each other in the formation of NH3, and its utilization for
the synthesis of carbamoyl phosphate. The remaining four enzymes of urea cycle are mostly controlled by the concentration of their respective substrates.

Urea produced in the liver freely diffuses and is transported in blood to kidneys, and excreted. A small amount of urea enters the intestine where it is broken down to CO) and NH3 by the bacterial enzyme urease. This ammonia is either lost in the feces or absorbed into the blood. In renal failure, the blood urea level is elevated (uremia), resulting in diffusion of more urea into intestine and its breakdown to NHs3. Hyperammonemia (increased blood NH3)_ is commonly seen in patients of kidney failure. For these patients, oral administration of antibiotics (neomycin) to kill intestinal bacteria is advised.

Urea cycle is linked with TCA cycle in three different ways (Fig.15.12). This is regarded as bicyclic integration between the two cycles.
1. The production of fumarate in urea cycle is the most important integrating point with TCA cycle. Fumarate is converted to malate and then to oxaloacetate in TCA cycle. Oxaloacetate undergoes transamination to produce aspartate which enters urea cycle. Here, it combines with citrulline to produce arginosuccinate. Oxaloacetate is an important metabolite which can combine with acetyl CoA to form citrate and get
Fig. 15.12 : Interrelation between urea and tricarboxylic acid (TCA) cycle (Depicted in blue colour).
finally oxidized. Oxaloacetate can also serve as a precursor for the synthesis of glucose (gluconeogenesis).
2. ATP (12) are generated in the TCA cycle while ATP (4) are utilized for urea synthesis.
intake marginally increases blood urea level, however this is well within normal range. About 15-30 g of urea (7-15 g nitrogen) is excreted in urine per day.
3.
Citric acid
cycle is
an important
metabolic pathway
for metabolites
the to
in
TCA
complete
CO)
cycle oxidation
and of
various
HO.
(in the
The
CO, liberated
mitochondria)
can be
urea cycle.

Metabolic defects associated with each of the five enzymes of urea cycle have been reported (Table 15.1). All the disorders invariably lead to a build-up in blood ammonia (hyperammonemia), leading to toxicity. Other metabolites of urea cycle also accumulate which, however, depends on_ the specific enzyme defect. The clinical symptoms associated with defect in urea cycle enzymes include vomiting, lethargy, irritability, ataxia and mental retardation.

In urea
healthy people, the normal blood mg/dl. Higher
concentration is 10-40 protein
Blood urea estimation is widely used as a screening test for the evaluation of kidney (renal) function. It is estimated in the laboratory either by urease method or diacetyl] monoxime (DAM) procedure. Elevation in blood urea may be broadly classified into three categories.
1. Pre-renal This is associated with increased protein breakdown, leading to a negative nitrogen balance, as observed after major surgery, prolonged fever, diabetic coma, thyrotoxicosis etc. In leukemia and bleeding disorders also, blood urea is elevated.
2. Renal : In renal disorders like acute glomerulonephritis, chronic nephritis, nephrosclerosis, polycystic kidney, blood urea is increased.
3. Post-renal : Whenever there is an obstruction in the urinary tract (e.g. tumors, stones, enlargement of prostate gland etc.), blood urea is elevated. This is due to increased reabsorption of urea from the renal tubules.
The term 'uremia' is used to indicate increased blood urea levels due to renal failure. Azotemia represents an elevation in blood urea/ or other nitrogen metabolites which may or may not be associated with renal diseases.

As is obvious from the name, the term NPN refers to all the nitrogen-containing substances other than proteins. These include urea (most abundant), creatinine, creatine, uric acid, peptides, amino acids etc. In healthy persons, NPN concentration in blood is 20-40 mg/dl.
The molecular weight of urea is 60 and about half of it (28) is contributed by the two nitrogen atoms. Thus, if blood urea concentration is 60 mg, then about half of it—28 mg—is blood urea nitrogen (BUN). Therefore,
In some countries, estimations of BUN or NPN are used rather than blood urea for assessing kidney function. The normal range for ratio of BUN to serum creatinine is 10:1 to 15:1.

In the preceding pages, the general aspects of amino acid metabolism have been discussed. A summary of the biologically important or specialized products obtained from or contributed by the amino acids is given in the Table 15.2. The metabolism of individual amino acids with special emphasis on the specialized products is described next.

Glycine, Specialized product(s) = Creatine, glutathione, heme, purines, conjugated bile acids.. Tyrosine, Specialized product(s) = Thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, melanin.. Tryptophan, Specialized product(s) = NAD + , NADP + (coenzymes of niacin), serotonin, melatonin.. Methionine, Specialized product(s) = Active methionine, creatine, epinephrine, polyamines.. Cysteine, Specialized product(s) = Glutathione, taurine, coenzyme A, active sulfate.. Histidine, Specialized product(s) = Histamine. Arginine, Specialized product(s) = Creatine, nitric oxide. Lysine, Specialized product(s) = Carnitine. Glutamate, Specialized product(s) = a -Amino butyric acid, glutathione, a -carboxyglutamate.. Glutamine, Specialized product(s) = Purines, pyrimidines, amino sugars.. Aspartate, Specialized product(s) = Purines, pyrimidines. Serine, Specialized product(s) = Phosphatidylserine, sphingomyelins, choline.. ` -Alanine, Specialized product(s) = Coenzyme A

Glycine (Gly, G) is a non-essential, optically inactive and glycogenic (precursor for glucose) amino acid. It is indispensable for chicks. The outline of glycine metabolism is depicted in Fig.15.13. Glycine is actively involved in the synthesis of many specialized products (heme, purines, creatine etc.) in the body, besides its incorporation into proteins, synthesis of serine and glucose and participation in one-carbon metabolism. Glycine is the most abundant amino acid normally excreted into urine (0.5-1.0 g/g creatinine).

Glycine is one among the commonest amino acids found in protein structure. Being small and non-polar, glycine is mostly present in the interior structure of protein. Collagen contains very high (about 30%) content of glycine.

Glycine is synthesized from serine by the enzyme serine hydroxymethyl transferase which is dependent on tetrahydrofolate (THF). Glycine can also be obtained from threonine, catalysed by threonine aldolase. Glycine synthase can convert a one-carbon unit (N5, N!9-methylene THF), COy and NH;3 to glycine.

Glycine undergoes oxidative deamination by glycine synthase to liberate NHZ, CO and onecarbon fragment as N°, N!9-methylene THF. This provides a major route for glycine breakdown in mammals. Glycine synthase is a multienzyme complex and requires PLP, NAD* and THF for its activity. This reaction is reversible and, therefore, glycine can be generated from one-carbon unit (methylene fragment of THF).
Glycine is reversibly converted to serine by THF dependent serine hydroxymethyl transferase. Pyruvate produced from serine by serine dehydratase, serves as a precursor for glucose. Serine is degraded to glyoxylate which undergoes transamination to give back glycine. Glyoxylate is also converted to oxalate, an excretory product and formate which enters onecarbon pool (Fig.15.14).
Fig.
15.14
:
Metabolism of
glycine
(THF—Tetrahydrofolate;
PLP-Pyridoxal phosphate;
==—Block reaction occurs in the kidney. It involves the transfer of guanidino group of arginine to glycine, catalysed by arginine-glycine transamidinase to produce guanidoacetate (glycocyamine). S-Adenosylmethionine (active methionine) donates methyl group to glycocyamine to produce creatine. This reaction occurs in liver. Creatine is reversibly phosphorylated to phosphocreatine (creatine phosphate) by creatine kinase. It is stored in muscle as high energy phosphate.
Creatinine is
an anhydride
of creatine.
It is
formed by
spontaneous creatine
phosphate.
urine.
cyclization
Creatinine of
creatine is
excreted or
in in
primary hyperoxaluria).
Fig. 15.15 : Outline of glutathione synthesis.

Estimation of serum creatinine (along with blood urea) is used as a diagnostic test to assess kidney function. Serum creatinine concentration is not influenced by endogenous and exogenous factors, as is the case with urea. Hence, some workers consider serum creatinine as a more reliable indicator of renal function.
The amount of creatinine excreted is proportional to total creatine phosphate content of the body and, in turn, the muscle mass. The daily excretion of creatinine is usually constant. Creatinine coefficient is defined as the mg of creatinine and creatine (put together) excreted per kg body weight per day. For a normal adult man, the value is 24-26 mg, while for a woman, it is 20-22 mg.
Increased output of creatine in urine is referred to as creatinuria. Creatinuria is observed in muscular dystrophy, diabetes mellitus, hyperthyroidism, starvation etc.

1. Glycinuria : This is a rare disorder. Serum glycine concentration is normal, but very high amount of it (normal 0.5-1 g/day) is excreted in urine. It is believed that glycinuria is due to a defective renal reabsorption. Glycinuria is characterized by increased tendency for the formation of oxalate renal stones. However, urinary oxalate level is normal in these patients.
2. Primary hyperoxaluria : This disorder is characterized by increased urinary oxalate resulting in oxalate stones. Deposition of oxalate (oxalosis) in various tissues is observed. The urinary oxalate is of endogenous origin and not due to dietary consumption of oxalate. Primary hyperoxaluria is due to a defect in glycine transaminase coupled with impairment in glyoxalate oxidation to formate.
It is now known that primary hyperoxaluria is mainly due to a defect in protein targeting (i.e. defect in transport of protein from one compartment to another). As a result, the enzyme glycine transaminase is found in mitochondria instead of its normal distribution in peroxisomes.
In vitamin Bg deficiency, urinary oxalate is elevated which can be corrected by Bg supplementation. However, Bg administration has no effect on endogenous hyperoxaluria.

Phenylalanine (Phe, F) and tyrosine (Tyr, Y) are structurally related aromatic amino acids. Phenylalanine is an essential amino acid while tyrosine is non-essential. Besides _ its incorporation into proteins, the only function of phenylalanine is its conversion to tyrosine. For this reason, ingestion of tyrosine can reduce the dietary requirement of phenylalanine. This phenomenon is referred to as 'sparing action' of tyrosine on phenylalanine.
compounds—epinephrine, norepinephrine, dopamine (catecholamines), thyroid hormones— and the pigment melanin (Fig.15.17). During the course of degradation, phenylalanine and tyrosine are converted to metabolites which can serve as precursors for the synthesis of glucose and fat. Hence, these amino acids are both glucogenic and ketogenic. Biochemists attach special significance to phenylalanine and tyrosine metabolism for two reasons—synthesis of biologically important compounds and the metabolic disorders due to enzyme defects.

The predominant metabolism of phenylalanine occurs through tyrosine. Tyrosine is incorporated into proteins and is involved in the synthesis of a variety of biologically important
Under normal circumstances, the degradation of phenylalanine mostly occurs through tyrosine. Phenylalanine is hydroxylated at para-position by phenylalanine hydroxylase to produce tyrosine (p-hydroxy phenylalanine). This is an irreversible reaction and requires the participation of a specific coenzyme biopterin

+ About 300-400 g of protein per day is constantly degraded and synthesized in the human body.
+ The amino acids are mainly utilized for protein biosynthesis, production of specialized products (creatine, porphyrin, amines, purines, pyrimidines) and generation of energy.
+ Glutamate is the collection centre for the amino groups in the biological system while glutamine is the storehouse of NH3. Free NHg3 can be liberated predominantly from glutamate.
+ Ammonia accumulation in blood is toxic to brain causing slurring of speech, blurring of vision, tremors and even death. Mammals convert NH3 to urea, a non-toxic excretory product. Metabolic defects in urea cycle enzymes result in hyperammonemia.
+ Dietary consumption of a protein rich meal increases the level of N-acetylglutamate in liver which enhances urea production.
+ Primary hyperoxaluria—a metabolic disorder due to a defect in the enzyme glycine transa- minase—is characterized by elevated urinary oxalate and the formation of oxalate stones.
+ Blood urea estimation is commonly used to assess renal function. Elevation of blood urea level (normal 10-40 mg/dl) is associated with several disorders which may be pre- renal (diabetic coma), renal (acute glomerulonephritis) and post-renal (tumors or stones in the urinary tract).
+ Estimation of serum creatinine (normal <1 mg/dl) is considered to be a more reliable indicator for the evaluation of kidney function.
Fig. 15.17 : Overview of phenylalanine and tyrosine metabolism (CNS—Central nervous system; T,-Triiodothyronine).
(containing pteridine ring) which is structurally related to folate. The active form of biopterin is tetrahydrobiopterin (H4-biopterin). In — the phenylalanine hydroxylase reaction, tetrahydrobiopterin is oxidized to dihydrobiopterin (H-biopterin). Tetrahydrobiopterin is then regenerated by an NADPH-dependent dihydrobiopterin reductase (Fig.15.18).

NADPH. Due to a defect in phenylalanine hydroxylase, the conversion of phenylalanine to tyrosine is blocked resulting in the disorder phenylketonuria (PKU).

The metabolism of phenylalanine and tyrosine is considered together. The sequence of the reactions in the degradation of these amino acids, depicted in Fig.15.19, is described hereunder
1. As phenylalanine is converted to tyrosine (details in Fig.15.18), a single pathway is responsible for the degradation of both these amino acids, which occurs mostly in liver.
2. Tyrosine first undergoes transamination to give p-hydroxyphenylpyruvate. This reaction is catalysed by tyrosine transaminase (PLP dependent).
The enzyme phenylalanine hydroxylase is present in the liver. In the conversion of phenylalanine to tyrosine, the reaction involves the incorporation of one atom of molecular oxygen (Oy) into the para position of phenylalanine while the other atom of Op» is reduced to form water. It is the tetrahydrobiopterin that supplies the reducing equivalents which, in turn, are provided by
3. p-Hydroxyphenylpyruvate hydroxylase (or dioxygenase) is a copper-containing enzyme. It catalyses oxidative decarboxylation as well as hydroxylation of the phenyl ring of p-hydroxyphenylpyruvate to produce homogentisate. This reaction involves a shift in hydroxyl group from para position to meta position, and incorporates a new hydroxyl group at para position. This step in tyrosine metabolism requires ascorbic acid.
4. Homogentisate oxidase (iron metalloprotein) cleaves the benzene ring of homogentisate to form 4-maleylacetoacetate.
Fig. 15.18 : Synthesis of tyrosine from phenylalanine (™_—Block in phenylketonuria).
different enzyme
systems exist
to convert
tyrosine to
DOPA.

DOPA undergoes PLP-dependent decarboxylation to give dopamine which, in turn, is hydroxylated to produce norepinephrine. Methylation of norepinephrine by S-adenosylmethionine gives epinephrine. The difference between epinephrine and norepinephrine is only a methyl group (remember that norepinephrine has no methyl group).
There exists tissue specificity in the formation of catecholamines. In adrenal medulla, synthesis of the hormones, norepinephrine — and epinephrine is prominent. Norepinephrine is produced in certain areas of the brain while dopamine is predominantly synthesized in substantia nigra and coeruleus of brain.
Functions of catecholamines Norepinephrine and epinephrine regulate carbohydrate and lipid metabolisms. They stimulate the degradation of triacylglycerol and glycogen. They cause an increase in the blood pressure. Dopamine and norepinephrine serve as neurotransmitters in the brain and autonomous nervous system.

Parkinson's disease is a common disorder in many elderly people, with about 1% of the population above 60 years being affected. It is characterized by muscular rigidity, tremors, expressionless face, lethargy, involuntary movements etc.
Biochemical basis : The exact biochemical cause of this disorder has not been identified. Parkinson's disease is, however, linked with a decreased production of dopamine. The disease is due to degeneration of certain parts of the brain (substantia nigra and locus coeruleus), leading to the impairment in the synthesis of dopamine.
Treatment : Dopamine cannot enter the brain, hence its administration is of no use. DOPA (levodopa or L-dopa) is used in the treatment of Parkinson's disease. In the brain, DOPA is decarboxylated to dopamine which alleviates the
symptoms of this disorder. Unfortunately, dopamine synthesis occurs in various other tissues and results in side-effects such as nausea, vomiting, hypretension etc. Administration of dopa analogs—that inhibit dopa decarboxylase (in various tissues) but not enter brain (due to blood-brain barrier)—are found to be effective. Carbidopa and y-methyl-dopa (dopa analogs) are administered along with dopa for the treatment of Parkinson's disease.

Several enzyme defects in phenylalanine/ tyrosine degradation leading to metabolic disorders are known. In Fig.15.19, the deficient enzymes and the respective inborn errors are depicted and they are discussed here under.

Phenylketonuria (PKU) is the most common metabolic disorder in amino acid metabolism. The incidence of PKU is 1 in 10,000 births. It is due to the deficiency of the hepatic enzyme, phenylalanine hydroxylase, caused by an autosomal recessive gene. In recent years, a variant of PKU—due to a_ defect in dihydrobiopterin reductase (relatively less)—has been reported. This enzyme deficiency impairs the synthesis of tetrahydrobiopterin required for the action of phenylalanine hydroxylase (See Fig.15.18). The net outcome in PKU is that phenylalanine is not converted to tyrosine.
Phenylalanine metabolism in PKU Phenylketonuria primarily causes the accumulation of phenylalanine in tissues and blood, and results in its increased excretion in urine. Due to disturbances in the routine metabolism, phenylalanine is diverted to alternate pathways (Fig.15.23), resulting in the excessive production of phenylpyruvate, phenylacetate, phenyllactate and phenylglutamine. All these metabolites are excreted in urine in high concentration in PKU. Phenylacetate gives the urine a mousey odour.
The name phenylketonuria is coined due to the fact that the metabolite phenylpyruvate is a keto acid (CgH5CH»—-CO-—COO) excreted in urine in high amounts.
Clinical/biochemical manifestations of PKU : The disturbed metabolism of phenylalanine— resulting in the increased concentration of phenylalanine and its metabolites in the body— causes many clinical and biochemical manisfestations.
1. Effects on central nervous system : Mental retardation, failure to walk or talk, failure of growth, seizures and tremor are the characteristic findings in PKU. If untreated, the patients show very low IQ (below 50). The biochemical basis of mental retardation in PKU is not well understood. There are, however, many explanations offered

1 Accumulation of phenylalanine in brain impairs the transport and metabolism of other aromatic amino acids (tryptophan and tyrosine).
1 The synthesis of serotonin (an excitatory neurotransmitter) from tryptophan is insufficient. This is due to the competition of phenylalanine and_ its metabolites with tryptophan that impairs the synthesis of serotonin.
| Defect in myelin formation is observed in PKU patients.
2. Effect on pigmentation : Melanin is the
pigment synthesized from tyrosine by tyrosinase.
Accumulation of phenylalanine competitively inhibits tyrosinase and = impairs melanin formation. The result is hypopigmentation that causes light skin colour, fair hair, blue eyes etc. Diagnosis of PKU : PKU is mostly detected by screening the newborn babies for the increased plasma levels of phenylalanine (PKU, 20-65 mg/ dl; normal 1-2me/dl). This is usually carried out by Guthrie test, which is a bacterial (Bacillus subtilis) bioassay for phenylalanine. The test is usually performed after the baby is fed with breast milk for a couple of days by testing elevated levels of phenylalanine. Phenylpyruvate in urine can be detected by ferric chloride test (a green colour is obtained). This test is not specific, since many other compounds give a false positive test. Prenatal diagnosis of PKU can also be done by using cultured amniotic cells.
Treatment of PKU : The maintenance of plasma phenylalanine concentration within the normal range is a challenging task in the treatment of PKU. This is done by selecting foods with low phenylalanine content and/or feeding synthetic amino acid preparations, low in phenylalanine. Dietary intake of phenylalanine should be adjusted by measuring plasma levels. Early diagnosis (in the first couple of months of baby's life) and treatment for 4-5 years can prevent the damage to brain. However, the restriction to protein diet should be continued for many more years in life. Since the amino acid tyrosine cannot be synthesized in PKU patients, it becomes essential and should be provided in the diet in sufficient quantity.

In some seriously affected PKU _ patients, treatment includes administration of 5-hydroxytryptophan and dopa to restore the synthesis of serotonin and catecholamines. PKU patients with tetrahydrobiopterin deficiency require tetrahydrobiopterin supplementation.

This disorder—also known as_ RichnerHanhart syndrome, is due to a defect in the enzyme tyrosine transaminase. The result is a blockade in the routine degradative pathway of tyrosine. Accumulation and excretion of tyrosine and its metabolites—namely p-hydroxyphenyl- pyruvate, p-hydroxyphenyllactate, phydroxyphenylacetate, N-acetyltyrosine—and tyramine are observed.
Tyrosinemia type Il is characterized by skin (dermatitis) and eye lesions and, rarely, mental retardation. A disturbed self-coordination is seen in these patients.

The absence of the enzyme p-hydroxyphenylpyruvate dioxygenase —_ causes __ neonatal tyrosinemia. This is mostly a temporary condition and usually responds to ascorbic acid. It is explained that the substrate inhibition of the enzyme is overcome by the presence of ascorbic acid.

Alkaptonuria has great historical importance. It was first described by Lusitanus in 1649 and characterized in 1859. Garrod conceived the idea of inborn errors of metabolism from his observation on alkaptonuria. The prevalance of this autosomal recessive disorder is 1 in 25,000. defect The defective in
Enzyme : enzyme alkaptonuria is homogentisate oxidase in tyrosine metabolism (See Fig.15.19). Homogentisate accumulates in tissues and blood, and is excreted into urine. Homogentisate, on standing, gets oxidized to the corresponding quinones, which polymerize to give black or brown colour. For this reason, the urine of alkaptonuric patients resembles coke in colour.
Biochemical manifestations : Homogentisate gets oxidized by polyphenol oxidase to benzoquinone acetate which undergoes _ polymerization to produce a pigment called alkapton (Fig.15.24). Alkapton deposition occurs in connective tissue, bones and various organs (nose, ear etc.) resulting in a condition known as ochronosis. Many alkaptonuric patients suffer from arthritis and this is believed to be due to the deposition of pigment alkapton (in the joints), produced from homogentisate.
Diagnosis : Change in colour of the urine on standing to brown or dark has been the simple traditional method to identify alkaptonuria.
Fig. 15.24 : Conversion of homogentisate to alkapton.
The urine gives a positive test with ferric chloride and silver nitrate. This is due to the strong reducing activity of homogentisate. Benedict's test—employed for the detection of glucose and other reducing sugars—is also positive with homogentisate.
Treatment : Alkaptonuria is not a dangerous disorder and, therefore, does not require any specific treatment. However, consumption of protein diet with relatively low phenylalanine content is recommended.

This is due to the deficiency of the enzymes fumarylacetoacetate hydroxylase and/or maleylacetoacetate isomerase. Tyrosinosis is a rare but serious disorder. It causes liver failure, rickets, renal tubular dysfunction and polyneuropathy. Tyrosine, its metabolites and many other amino acids are excreted in urine.
In acute tyrosinosis, the infant exhibits diarrhea, vomiting, and 'cabbage-like' odor. Death may even occur due to liver failure within one year. For the treatment, diets low in tyrosine, phenylalanine and methionine are recommended.

Albinism (Greek: albino—white) is an inborn error, due to the lack of synthesis of the pigment melanin. |t is an autosomal recessive disorder with a frequency of 1 in 20,000.
Biochemical basis : The colour of skin and hair is controlled by a large number of genes.
About 150 genes have been identified in mice. The melanin synthesis can be influenced by a variety of factors. Many possible causes (rather explanations) for albinism have been identified
1. Deficiency or lack of the enzyme tyrosinase.
. Decrease in melanosomes of melanocytes. 2
. Impairment in melanin polymerization. 3
4. Lack of protein matrix in melanosomes.
5. Limitation of substrate (tyrosine) availability.
6 . Presence of inhibitors of tyrosinase.
The most common cause of albinism is a defect in tyrosinase, the enzyme most responsible for the synthesis of melanin (See Fig.15.20).
Clinical manifestations : The most important function of melanin is the protection of the body from sun radiation. Lack of melanin in albinos makes them sensitive to sunlight. Increased susceptibility to skin cancer (carcinoma) is observed. Photophobia (intolerance to light) is associated with lack of pigment in the eyes. However, there is no impairment in the eyesight of albinos.

In some individuals, a reduced synthesis of melanin (instead of total lack) is often observed. Hypopigmentation disorders may be either diffuse or localized.
A good example of diffuse hypopigmentation is oculocutaneous albinism which is mostly due to mutations in the tyrosinase gene. The degree of hypopigmentation depends on the type and severity of mutated genes.
Vitiligo and leukoderma are the important among the localized hypopigmentation disorders. Vitiligo is an acquired progressive disease with loss of pigmentation around mouth, nose, eyes and nipples. Leukoderma is comparable with vitiligo, but lack of pigmentation usually begins with hands and then spreads.
Greying of hair is due to lack of melanin synthesis which usually occurs as a result of disappearance of melanocytes from the hair roots.

Tryptophan (Trp, W) was the first to be identified as an essential amino acid. It contains an indole ring and chemically it is a-amino B-indole propionic acid. Tryptophan is both glucogenic and ketogenic in nature. It is a precursor for the synthesis of important compounds, namely NADt and NADPt (coenzymes of niacin), serotonin and melatonin (Fig.15.25).
The metabolism of tryptophan is divided into
I. Kynurenine (kynurenine-anthranilate) pathway;
Il. Serotonin pathway.

This pathway mostly occurs in liver leading to oxidation of tryptophan and the synthesis of NAD* and NADP?

Tryptophan pyrrolase or oxygenase cleaves the five-membered ring of the indole nucleus to produce formylkynurenine. Tryptophan pyrrolase is a metalloprotein containing an iron porphyrin ring. It is a substrate inducible enzyme and is controlled by feedback regulation (by NADPH and other niacin derivatives). Tryptophan pyrrolase activity is also elevated by corticosteroids. _Formamidase hydrolyses formylkynurenine and liberates formate which enters the one carbon pool. Kynurenine formed in this reaction is a branch point with different fates. In the prominent pathway, kynurenine undergoes NADPH-dependent hydroxylation to give 3-hydroxykynurenine.
Kynureninase, a pyridoxal phosphate (PLP)dependent enzyme acts on the 3-hydroxykynurenine and splits off alanine. Tryptophan is glucogenic, since alanine is a good precursor for glucose. The enzyme kynureninase is very sensitive to vitamin Bg deficiency. Due to the lack of PLP, kynureninase reaction is blocked and 3-hydroxykynurenine is diverted to form xanthurenate. Elevated excretion of xanthurenate serves as an indication of Bg deficiency. Administration of isoniazid, an antituberculosis drug—induces Bg deficiency and results in xanthurenate excretion in urine. Defects in the activity of kynureninase (in Bg deficiency) cause reduced synthesis of NAD* and NADPt from tryptophan. The symptoms of pellagra —observed in Bg deficiency—are explained on this basis.
The enzyme kynurenine hydroxylase _ is inhibited by estrogen, hence women, in general, are more susceptible to pellagra.
3-Hydroxyanthranilate is cleaved by an oxidase (Fe2* dependent) to form an unstable intermediate, 2-amino 3-carboxy muconate semialdehyde. This compound has three fates.
1. It undergoes spontaneous cyclization quinolinate for NAD* synthesis.

form
2. Picolinate carboxylase decarboxylates the intermediate which cyclizes to produce picolinate. This enzyme competes with the formation of quinolinate. High activity of picolinate carboxylase in some animals (e.g. cat) deprives them of NAD* synthesis from tryptophan. In other words, cat is exclusively dependent on niacin for its coenzymes (NAD*, NADP*), since tryptophan cannot serve as a precursor.
3. The intermediate undergoes decarboxylation, catalysed by amino carboxysemialdehyde decarboxylase to produce 2-aminomuconate semialdehyde that enters glutarate pathway. The semialdehyde is converted to 2-aminomuconate by a dehydrogenase. The aminomuconate, in a series of reactions involving — reduction, deamination, decarboxylation etc., is converted to glutaryl CoA and finally to acety CoA. The latter is either completely oxidized via TCA cycle or converted to fat (hence tryptophan is ketogenic).
NADt Pathway Tryptophan is not a precursor for the synthesis of free niacin. Quinolinate undergoes decarboxylation and is converted to nicotinate mononucleotide by the enzyme quinolinate phosphoribosyl transferase (QPRT). The synthesis of NAD+ and NADPt from nicotinate mononucleotide is similar to that from niacin as described in vitamins (Refer Fig.7.21).
Conversion of tryptophan to indole acetate : Tryptophan undergoes deamination and decarboxylation to produce indolepyruvate and tryptamine, respectively. Both these compounds are converted to indoleacetate (Fig.15.27) and excreted in urine.

Serotonin or 5-hydroxytryptamine (5HT) is a neurotransmitter, synthesized from tryptophan. Normally, about 1% of the tryptophan _ is converted to serotonin. The production of 5HT occurs in the target tissues.
Synthesis of serotonin : In mammals, the largest amount of serotonin is synthesized in the intestinal cells. The formation of serotonin is comparable with _ the production — of catecholamines. Tryptophan is first hydroxylated at 5th carbon by tryptophan hydroxylase. This enzyme requires tetrahydrobiopterin as a cofactor. 5-Hydroxytryptophan is decarboxylated by aromatic amino acid decarboxylase (PLP- dependent)
to give
serotonin
(Fig.15.27).
Platelets contain high concentration of 5HT, the significance of which is not clear. As such, platelets cannot synthesize serotonin.
Degradation of serotonin Monoamine oxidase (MAO) degrades serotonin to 5hydroxyindoleacetate (SHIA) which is excreted in urine.
Functions of serotonin : Serotonin is a neurotransmitter and performs a variety of functions. 1. Serotonin is a powerful vasoconstrictor and results in smooth muscle contraction in bronchioles and arterioles.
2. It is closely involved in the regulation of cerebral activity (excitation).
controls the behavioural blood pressure and body 3. Serotonin patterns, sleep, temperature.
4. Serotonin evokes the release of peptide hormones from gastrointestinal tract.
5.
It is
also necessary
for the
motility of
GIT
(peristalsis).
Serotonin and_ brain The brain _ itself synthesizes 5HT which is in a bound form. The outside serotonin cannot enter the brain due to blood-brain barrier. Primarily, serotonin is a stimulator (excitation) of brain activity, hence its deficiency causes depression. Serotonin level is decreased in psychosis patients.
Defects in monoamine oxidase gene (lowered MAO activity) are linked to violent behaviour and slight mental retardation.

Effect of drugs on serotonin : The drug, iproniazid (isopropyl isonicotinyl hydrazine) inhibits MAO and elevates serotonin levels, therefore, this drug is a psychic stimulant. On the other hand, reserpine increases the degradation of serotonin, hence acts as a depressant drug. Lysergic acid diethylamide (LSD) competes with serotonin and, therefore, acts as a depressant.
Malignant carcinoid syndrome : Serotonin is produced by argentaffin cells of gastrointestinal tract. When these cells undergo uncontrolled growth, they develop into a tumor called malignant carcinoid or argentaffinomas. The patients exhibit symptoms like respiratory distress, sweating, hypertension etc.
Normally about 1% of the tryptophan is utilized for serotonin synthesis. In case of carcinoid syndrome, very high amount (up to 60%) of tryptophan is diverted for serotonin production. This disturbs the normal tryptophan metabolism and impairs the synthesis of NAD* and NADP*. Hence, the patients of carcinoid syndrome develop symptoms of pellagra (niacin deficiency). Further, negative nitrogen balance is also observed.
Diagnosis : The excretion of 5-hydroxy indole acetate in urine is tremendously elevated (upto 500mg/day against normal <5 mg/day) in carcinoid syndrome. The estimation of 5 HIA in urine is used for the diagnosis of this disorder. In general, urine concentration of 5 HIA above 25 mg/day should be viewed with caution as it may be suggestive of carcinoid syndrome. Sufficient precaution should, however, be taken for sample collection. During the course of urine collection, the patients should not ingest certain foods (banana, tomato etc.) that increase urine 5 HIA.

Melatonin is a hormone, mostly synthesized by the pineal gland. Serotonin—produced from tryptophan—is acted upon by _ serotonin N-acetylase (the rate limiting enzyme), to give N-acetylserotonin. The latter undergoes methylation, S-adenosylmethionine being the methyl group donor to produce melatonin or N-acetyl 5-methoxyserotonin (Fig.15.27). The synthesis and secretion of melatonin from pineal gland is controlled by light.

1. Melatonin is involved in circadian rhythms or diurnal variations (24 hr cyclic process) of the body. It plays a significant role in sleep and wake process.
2. Melatonin inhibits the production of melanocyte stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH).
3. It has some inhibitory effect on ovarian functions.
4. Melatonin also transmitter function. performs a neuro-

This disorder was first described in the family of Hartnup, hence the name—Hartnup's disease. It is a hereditary disorder of tryptophan metabolism. The clinical symptoms include dermatitis, ataxia, mental retardation etc. Hartnup's disease is characterized by low plasma levels of tryptophan and other neutral amino acids and their elevated urinary excretion. Increased urinary output of indoleacetic acid and indolepyruvic acid is also observed.
Pellagra-like symptoms are common in these patients. There is an impairment in the synthesis of NAD* and serotonin from tryptophan. Some authors (earlier) attributed Hartnup's disease to a defect in the enzyme tryptophan pyrrolase. This, however, does not appear to be true. Hartnup's disease is now believed to be due to an impairment in the absorption and/or transport of tryptophan and other neutral amino acids from the intestine, renal tubules and, probably brain. Some more details on Hartnup's disease are given under digestion and absorption (Chapter 8).

The sulfur-containing amino acids are methionine, cysteine and cystine. Among these, only methionine is essential. \t serves as a precursor for the synthesis of cysteine and cystine which are, therefore, non-essential. Cysteine can spare the requirement of methionine in the diet. Cysteine and cystine are interconvertible. Cystine is found exclusively in proteins. Methionine and cysteine, besides being present in proteins, are involved in many important metabolic reactions (Fig.15.28). Methionine is also required for the initiation of protein biosynthesis. The sulfurcontaining amino acids are almost an exclusive dietary source of sulfur to the body.

Methionine
(or
sulfur
amino
acids)
metabolism may be divided into three parts.
1. Utilization of methionine for transmethy-
lation reactions.
2. Conversion of methionine to cysteine and
cystine.
3. Degradation of cysteine and its conversion specialized products.
to

The transfer of methyl group (—CH3) from active methionine to an acceptor is known as transmethylation. Methionine has to be activated to S-adenosylmethionine (SAM) or active methionine to donate the methyl group.

The synthesis of S-adenosylmethionine occurs by the transfer of an adenosyl group from ATP to sulfur atom of methionine (Fig.15.29). This reaction is catalysed by methionine S-adenosyltransferase. The activation of methionine is unique as the sulfur becomes a sulfonium atom (SAM is a sulfonium compound) by the addition of a third carbon. This reaction is also unusual since all the three phosphates of ATP are eliminated as pyrophosphates (PPi) and inorganic phosphates (Pi). Three high energy phosphates (3 ATP) are consumed in the formation of SAM.

S-Adenosylmethionine is highly — reactive due to the presence of a positive charge.
The enzymes involved in the transfer of methyl group are collectively known as methyltransferases. S-Adenosy|lmethionine transfers the methyl group to an acceptor and gets itself converted to S-adenosylhomocysteine. The loss of free energy in this reaction makes the methyl transfer essentially irreversible. S-Adenosylhomocysteine is hydrolysed _ to homocysteine and adenosine. Homocysteine can be remethylated to methionine by N°-methyl tetrahydrofolate. In this manner, methionine can be regenerated for reuse. It should be noted that there is no net synthesis of methionine in the S-adenosyl-methionine cycle (homocysteine, the precussor for methionine has to be derived from methionine). Hence, methionine is an essential amino acid.
S-Adenosylmethionine (carbon fragment) is also involved in the synthesis of polyamines (spermidine, spermine). The most important transmethylation reactions are listed in Table 15.3.

Fig. 15.29 : S-Adenosylmethionine cycle—synthesis, utilization and regeneration.

. . . . 1. Transmethylation is of great biological significance since many compounds become functionally active only after methylation.
2. Protein (amino acid residues) methylation helps to control protein turnover. In general, methylation protects the proteins from immediate degradation.
3. In plants, S-adenosylmethionine is the precursor for the synthesis of a plant hormone, ethylene, which regulates plant growth and development and is involved in the ripening of fruits.

Homocysteine formed from methionine is a precursor for the synthesis of cysteine (Fig.15.30). Homocysteine condenses with serine to form cystathionine. This reaction is catalysed by a PLP-dependent cystathionine synthase. The enzyme cystathioninase (PLP-dependent) cleaves and deaminates cystathionine to cysteine and

Homocysteine is an intermediate in the synthesis of cysteine from methionine (Fig.15.30), Elevation in plasma homocysteine (normal <15 pmol/l) has been implicated in coronary artery diseases, although the mechanism is not known. It is believed that homocysteine reacts with collagen to produce reactive free radicals, besides interfering with collagen cross links. Homocysteine is also involved in the aggregation of LDL particles. All this leads to an increased tendency for atherogenesis, and consequently heart complications. Hyperhomocysteinemia in pregnancy increases the risk of neural tube defects in fetus.
Fig.
15.30
:
Synthesis of
cysteine from
methionine
(—For reactions
homocysteine of
see methionine
Fig.
only
15.29)
(*:::—In cysteine
synthesis, sulfur
is obtained
from homocysteine,
the rest
of the molecule is from serine).
Supplementation of diet with folic acid, vitamin B;» and vitamin Bg have some beneficial affects in lowering plasma homocysteine levels
(Refer Chapter 7).

Cystine and cysteine are interconvertible by an NAD*-dependent cystine reductase. Cysteine on decarboxylation produces _ mercaptoethanolamine' which is involved in the biosynthesis of coenzyme A from the vitamin pantothenic acid.
The enzyme cysteine dioxygenase oxidizes cysteine to cysteine sulfinate which, on further oxidation, is converted to cysteic acid. The latter undergoes decarboxylation to produce taurine which conjugates with bile acids. Cysteic acid can also be degraded to pyruvate, which is glycogenic (Fig.15.31).
Cysteine sulfinate cleaves off alanine to produce sulfite which is converted to sulfate and excreted in urine. Some amount of sulfate condenses with ATP to form active sulfate or 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Active sulfate (PAPS) is utilized for the synthesis of mucopolysaccharides (sulfation), besides being used in detoxification. Sulfate is also a structural component of some proteins, lipids etc.
Cysteine can be degraded by desulfhydrase to liberate sulfur (as HS), ammonia and pyruvate. Cysteine is a component of _ tripeptide glutathione (synthesis described in glycine metabolism).

Cystinuria (cystine-lysinuria) : Cystinuria is one of the most common inherited diseases with a frequency of 1 in 7,000. It is primarily characterized by increased excretion of cystine (25-40 times normal). Elevation in the urinary output of lysine, arginine and ornithine is also observed. A specific carrier system exists in kidney tubules for the reabsorption of amino acids, namely cysteine, ornithine, arginine and __ lysine (remember COAL to recall). In cystinuria, this carrier system becomes defective leading to the excretion of all these four amino acids in urine.
Cystine is relatively insoluble and increase in its concentration leads to precipitation and formation of cystine stones in kidney and urinary tract. Cystinuria is usually identified in the laboratory by cyanide nitroprusside test. The treatment includes restricted ingestion of dietary cystine and high intake of fluids.
Cystinosis (cystine storage disease) : Cystine crystals are deposited in many tissues and organs
Fig. 15.31 : Metabolism of cysteine and cystine.
of reticuloendothelial system throughout the body. These include spleen, lymph nodes, liver, kidney, bone marrow etc. A defect in the lysosomal function is said to be the primary cause of this disorder. In fact, cystine accumulates in the lysosomes of various tissues. Impairment in renal function is commonly seen in cystinosis. It is characterized by generalized amino aciduria. The affected patients die usually within 10 years, mostly due to renal failure. Although the underlying metabolic defect in cystinosis is not clearly known, some authors attribute this to the defect in the enzyme cystine reductase. This is, however, not accepted by others.
Homocystinurias : Homocystinurias are a group of metabolic disorders characterized by the accumulation and increased urinary excretion of homocysteine and S-adenosylmethionine. Plasma concentration of methionine is increased.

Homocystinuria type I has been more thoroughly investigated. It is due to a defect in the enzyme cystathionine synthase. Accumulation of homocysteine results in various complications—thrombosis, osteoporosis and, very often, mental retardation. Further, the deficiency of cystathionine is associated with damage to endothelial cells which might lead to atherosclerosis. Two forms of type | homocystinurias are known, one of them can be corrected with vitamin Bg supplementation (Bg responsive) while the other does not respond to By. The treatment includes consumption of diet low in methionine and high in cystine.
The patients of homocystinuria have high levels of homocysteine, and usually die of myocardial infarction, stroke, or pulmonary embolism.
The other homocystinurias are associated with enzyme defects (as stated below) in the conversion of homocysteine to methionine by remethylation.
Homocystinuria I: N°-N!°—Methylene THF reductase.
Homocystinuria III: N°-N!°—Methyl THFhomocysteine methyltransferase. This is mostly due to impairment in the synthesis of methylcobalamin.
Homocystinuria IV : N°—Methyl THF homocysteine methyl transferase. This is primarily due to a defect in the intestinal absorption of vitamin By5.

Amino acid metabolism is particularly important for the transfer or exchange of onecarbon units. The following one-carbon fragments are encountered in the biological reactions, which constitute one-carbon pool
Methyl
(—CH,)
Hydroxymethy]
(—CH,OH)
Methylene
(=CH,)
Methenyl
(—CH=)
Formyl
(—CH=O)
Formimino
(—CH=NH)
[Note : It may be stated here that CO, is also a one-carbon unit. Carbon dioxide is involved (carboxylation) in many biochemical reactions, which are dependent on biotin. For instance, conversion of pyruvate to oxaloacetate in gluconeogenesis. Most of the authors, however, ignore CO, as one-carbon unit and do not even consider it worth mentioning. This would be unfair to CO»!]
Tetrahydrofolate (THF) is a versatile coenzyme that actively participates in onecarbon metabolism. With regard to the transfer of methyl groups from S-adenosylmethionine, vitamin By,» is also involved besides THF.
The one-carbon unit covalently binds with THF at position N° or N!9 or on both N° and N!° of pteroyl structure of folate. The details of different one-carbon units binding with THF and the structures of THF derivatives are given under vitamin-folic acid (Chapter 7).
The one-carbon metabolism is rather complex, involving many reactions. For the sake of better understanding, it is divided into generation and utilization of one-carbon units, and the role of methionine and vitamin By».

Many compounds (particularly amino acids)
act as donors of one-carbon fragments
1. The formate released from glycine and
tryptophan
combines
with
THF
to
form N!0-formyl THF.
2. Histidine contributes formimino fragment
to produce N°-formimino THF.
3. When serine is converted to glycine, N°, N!°methylene THF is formed. This is the most predominant entry of one carbon units into one carbon pool.
4. Choline and betaine contribute to the formation of N°-methyl THF.
The different derivatives of THF carrying one- carbon units are interconvertible, and this is metabolically significant for the continuity of one- carbon pool (Fig.15.32).

One-carbon fragments from THF are used for the synthesis of a wide variety of compounds. These include purines, formylmethionine tRNA (required for initiation of protein synthesis), glycine, pyrimidine nucleotide (thymidylate) etc.

Methyl (—CH3) group is an important onecarbon unit. The role of active methionine as methyl donor in transmethylation reactions is already described. After the release of methyl group, methionine is converted to homocysteine. For the regeneration of methionine, free homocysteine and N°-methyl THF are required and this reaction is dependent = on methylcobalamin (vitamin B,2). The one-carbon pool, under the control of THF, is linked with methionine metabolism —_(transmethylation) through vitamin B,5. Hence vitamin B, is also involved in one-carbon metabolism.

Valine,
leucine
and
isoleucine
are
the
branched
chain
and
essential
amino
acids.
These
three
amino
acids
initially
undergo
a
common
pathway
and
then
diverge
to
result
in
different
end
products.
Based
on
the
products
obtained
from
the
carbon’
skeleton,
the
branched

coenzymes—TPP, lipoamide,
FAD, coenzyme
and
NADt—for dehydrogenase
its action.
catalyses lation
of the
keto
CoA
thioesters.
in the
acids to
This catabolism
of oa-Keto
oxidative
A
acid decarboxy-
the corresponding
is
a
acyl regulatory
enzyme branched
chain amino
acids.
3. Dehydrogenation : The dehydrogenation is similar to that in fatty acid oxidation. FAD is the coenzyme and there is an incorporation of a double bond. It is now believed that there are two enzymes responsible for dehydrogenation.
After the initial three common reactions, the metabolism of branched chain amino acids diverges and takes independent routes. In a series of reactions that follow, valine is converted

:
1 Accumulation of branched chain amino acids causes an impairment in transport and function of other amino acids.
1 Protein biosynthesis is reduced.
The treatment is to feed a diet with low (or no) content of branched amino acids. Mild variants of MSUD respond to high doses of thiamine. In severe cases of MSUD, liver transplantation is required.
1 Branched chain amino acids competitively inhibit glutamate dehydrogenase.
1 The disease results in acidosis, lethargy, convulsions, mental retardation, coma and, finally, death within one year after birth.
2. Intermittent branched chain ketonuria : This is a less severe variant form of maple syrup urine disease. The enzyme defect is the same— a-keto acid dehydrogenase. As such, there is an impairment and no total blockade in the conversion of a-keto acids to their respective
acyl CoA thioesters. The symptoms are not as severe as in maple syrup urine disease. Careful diet planning is adequate to ovecome this disorder.
3. Isovaleric acidemia : This is a specific inborn error of leucine metabolism. It is due to a defect in the enzyme isovaleryl CoA dehydrogenase. The conversion of isovaleryl CoA to methylcrotonyl CoA is impaired. The excretion of isovalerate is high in urine. The affected individuals exhibit a 'cheesy' odor in the breath and body fluids. The symptoms include acidosis and mild mental retardation.
4. Hypervalinemia : This inborn error is characterized by increased plasma concentration of valine while leucine and isoleucine levels remain normal. The transamination of valine alone is selectively impaired.

The metabolism of histidine, proline and arginine is considered together, as all the three are converted to glutamate and metabolized (Fig.15.34).

The metabolism of histidine is important for the generation of one-carbon unit, namely formimino group. The enzyme histidase acts on histidine to split off ammonia. Urocanate formed in this reaction is acted upon by urocanase to produce 4-imidazole 5-propionate. Imidazole ring of the product is cleaved by a hydrolase to give N-formiminoglutamate (FIGLU). Tetrahydrofolate (THF) takes up the formimino group to form N5-formimino THF, and glutamate is liberated. Deficiency of folate blocks this reaction and causes elevated excretion of FIGLU in urine. Histidine loading test is commonly employed to assess folate deficiency.
Histidine, on decarboxylation, gives the corresponding amine—histamine. Histamine regulates HCI secretion by gastric mucosa (Table 15.7). Excessive production of histamine causes asthma and allergic reactions.
Histidinemia : The frequency of histidinemia is 1 in 20,000. It is due to a defect in the enzyme histidase. Histidinemia is characterized by elevated plasma histidine levels and increased excretion of imidazole pyruvate and histidine in urine. Most of the patients of histidinemia are mentally retarded and have defect in speech. No treatment will improve the condition of the patients.

Proline is oxidized to pyrroline 5-carboxylate which undergoes a non-enzymatic conversion to glutamate 5-semialdehyde. The latter is converted to glutamate and then transaminated to a-ketoglutarate. The five carbons of proline are converted to a-ketoglutarate.
Hyperprolinemia type I : It is due to a defect in the enzyme proline oxidase (proline dehydrogenase).
Another metabolic disorder—hyperprolinemia type Il—associated with hydroxyproline metabolism is also reported.

Arginine is cleaved by arginase to liberate urea and produce ornithine. Ornithine undergoes transamination of §-amino group to form glutamate y-semialdehyde which is converted to glutamate. Hyperargininemia is an inborn error in arginine metabolism due to a defect in the enzyme arginase.
Nitric oxide (NO) : Arginine is the substrate for the production of nitric oxide (NO), a wonder molecule with a wide range of functions. The enzyme nitric oxide synthase (three isoenzymes known) cleaves the nitrogen from the guanidino group of arginine to form NO. This reaction requires NADPH, FMN, FAD, heme and tetrahydrobiopterin. NO has a very short half-life (about 5 seconds).
Fig.
15.34
:
histidine, proline,
arginine, glutamate
and glutamine
(THF-Tetrahydrofolate; a-KG—a-Ketoglutarate; Glu-Glutamate).
The occurrence of high concentrations of citrulline in human brain has been known for several years. Only recently it is realized that the citrulline is formed during the course of NO synthesis [Note : Nitric oxide (NO) should not be confused with nitrous oxide (NO»)—laughing gas— used as an anesthetic].
Functions of NO : The role of nitric oxide as a therapeutic drug (in the form of nitroglycerine and amyl nitrate) for the treatment of angina pectoris has been known since 1867. However, it is only recently that in vivo production and the biological importance of NO are recognized. In fact, the endothelium derived releasing factor
(EDRF) which causes smooth muscle relaxation is none other than a gas, NO.
Nitric oxide acts as a mediator for several biological functions.
- 1. NO functions as a vasodilator and causes
relaxation of smooth muscles.
2.
It is
a
key molecule
in blood
flow and
NO
synthesis the
the blood
pressure markedly
raise
3.
NO
acts as
an_
(inhibitors blood
inhibitor aggregation and adhesion.
4.

It functions
as
a
messenger the
nervous system
5.
NO
molecule
(neurotransmitter).
mediates the
bactericidal actions
macrophages.
- 6. It is involved in the erection of penis.
Mechanism of
action
:
Nitric oxide
promotes synthesis
the of
of the
actions cGMP.
of
It is
NO
believed are
that mediated
some through
cGMP
and protein
kinase
G.
Agmatine : It is a derivative of arginine produced in the brain. Agmatine possesses antihypertensive properties.
Lysine is an essential amino acid. Cereal proteins are deficient in lysine. It does not participate in transamination reactions. Some of the lysine residues in protein structure are present as hydroxylysine, methyllysine or acetyllysine. €-Amino group of lysine, forming salt bridges is necessary for the maintenance of structural conformation of protein.
Lysine is
a
ketogenic amino
acid.
The summary
of lysine
Fig.15.35.

Some of the lysine residues in proteins are found
in methylated
form.
The methyl
groups are
obtained proteins
from active
on release
the methionine
(SAM).
(by
Such proteolysis)
methyllysines.
The will
trimethyllysine is
depicted in
pressure).
of platelet
of of
of of
Fig. 15.35 : Summary of lysine metabolism.
serves as a precursor for the synthesis of carnitine, a compound involved in the transport of fatty acids to mitochondria for oxidation. It should be noted that free lysine is not methylated, hence it will not be a substrate for carnitine formation.
Synthesis of carnitine from trimethyllysine is a 4-step reaction involving oxidation, splitting off glycine residue, dehydrogenation and, finally, oxidation
(Fig.15.36).

Carnitine plays a key role in the fatty acid oxidation
(Chapter
15).
Human requirements of carnitine are usually met with the endogeneous biosynthesis and the dietary supply. Good sources of carnitine include meat, fish, poultry and dairy products.

Glutamate and glutamine are non-essential glycogenic amino acids. Both of them play a predominant role in the amino acid metabolism, and are directly involved in the final transfer of amino group for urea synthesis.
The amino acids—histidine, proline and arginine—are converted in their metabolism to glutamate (See Fig.15.34). o-Ketoglutarate—an intermediate in TCA cycle—serves as an immediate precursor for glutamate formation.
Glutamate—besides glutamine—is
involved specialized
products
1.
being converted
in the
_
to synthesis
(Fig.15.37).
Glutathione glutamate.
is
a
Its formation
glycine that
contains is
described metabolism.
2.
N-Acetylglutamate regulator
is of
carbamoyl under
an
__
allosteric phosphate
synthase
I, the
first enzyme
in urea
synthesis.
3. Glutamate is present in the clotting factors (I, VIL, IX, X) as y-carboxyglutamate and _ is involved in coagulation.
4. y-Aminobutyric acid (GABA) : Glutamate is decarboxylated to GABA—mostly in the brain. This is a sensitive and irreversible reaction, catalysed by a pyridoxal phosphate-dependent glutamate decarboxylase.
GABA undergoes transamination followed by oxidation to form succinate which enters TCA cycle (Fig.15.38). The reactions involving the fate of GABA constitute a bypass route for glutamate to enter TCA cycle, which is known as GABA shunt.
Fig. 15.37 : Overview of glutamate and glutamine metabolism.
of certain
Functions of GABA : It is one of the major inhibitory neurotransmitters in the brain. GABA regulates the activity of neurons by discouraging the transmission signals. It is believed that GABA opens chloride channels and increases the permeability of post-synaptic membranes. Thus GABA functions as an inhibitory neurotransmitter. Decreased GABA levels will cause convulsions.

Vitamin Bg deficiency and GABA : GABA synthesis requires pyridoxal phosphate, a coenzyme of vitamin Bg. In Bg deficiency, the production of GABA is reduced. The result is neuronal hyperexcitability, causing convulsions.

Glutamine
Ammonia is
a
versatile is
glutamine.
temporarily
Glutamine hence,
stored is
easily degradation
freely transported.
of
The glutamine
are amino
acid.
in the
form diffusible
of and,
synthesis described
and
(See

Glutamine is the donor of nitrogen atoms for purine and pyrimidine synthesis. It is the chief

Both these amino acids are non-essential and glycogenic. Aspartate is formed from oxaloacetate (an intermediate in TCA cycle) by transamination. Aspartate transaminase (AST) is an important enzyme for the interconversion of glutamate and aspartate.
The diagnostic importance of the enzyme AST has already been described (Chapter 6). Aspartate has certain important functions (Fig.15.39).
1.
It donates
one amino
group for
of urea
directly comes
2.
(the other
from
Aspartate forms
amino ammonia).
a
connecting group
in urea
link between
urea cycle
and
TCA
cycle
(via oxaloacetate).
Fig. 15.39 : Overview of aspartate and asparagine metabolism.
Fig. 15.40 : Synthesis of asparagine and its conversion to aspartate (Note : The reactions are independent and irreversible).
3. It is utilized for the synthesis of purines (N; and NH, at 6th position) and pyrimidines (N3, C4 Cs and Cg atoms).
4. Malate-aspartate shuttle is important for the transfer of reducing equivalents (NADH) from the cytosol to mitochondria (Refer Fig.11.13).
Asparagine is synthesized from aspartate by a synthetase in an_ irreversible ATP-dependent reaction. Asparaginase hydrolyses asparagine and liberates ammonia (Fig.15.40). These reactions are comparable to glutamine synthesis and its breakdown.
The non-essential amino acid alanine performs two important functions in the body— incorporation into proteins and participation in transamination and NH transport.

As already discussed, ammonia is toxic to the body, hence it cannot be transported in free form. Glutamate and glutamine shoulder the major burden of ammonia transport. Alanine is also important in this regard. In the peripheral tissues (most predominantly—muscle), pyruvate produced in glycolysis gets converted to alanine (by transamination) and is transported to liver. Pyruvate can be regenerated from alanine in liver and the pyruvate so produced serves as a precursor for glucose. Amino group is diverted for transamination or urea formation. This is an alanine-pyruvate shuttle for carrying nitrogen to be reutilized or converted to urea.
It is recently (2008) reported that people with higher levels of alanine in urine have increased risk for higher blood pressure.
The amino acid B-alanine is a constituent of the vitamin pantothenic acid, and thus the coenzyme A.
Serine is a non-essential glycogenic amino acid. As described in glycine metabolism, serine and glycine are interconvertible. Serine can be synthesized from the intermediates of glycolysis (3-phosphoglycerate). The metabolic reactions of serine are described hereunder (Fig.15.41)
1. Serine undergoes deamination to form pyruvate.
2. Serine is involved in one-carbon metabolism. It donates methylene (-CH») moiety to tetrahydrofolate (THF).
Fig. 15.41 : Overview of serine metabolism.

Threonine is an essential hydroxy amino acid. It is glycogenic and does not participate in transamination reactions. Threonine is often a carrier of phosphate group in the protein structure. The outline of threonine metabolism is depicted in Fig.15.42.
Threonine undergoes deamination (by threonine dehydratase) to «-ketobutyrate which is converted to propionyl CoA. Threonine can be cleaved to glycine and acetaldehyde by serine hydroxymethyltransferase. Dehydrogenation followed by decarboxylation of threonine results in aminoacetone which may be converted to pyruvate or lactate.

The metabolic reactions of individual amino acids are described above. After the removal of amino groups, the carbon skeleton of amino acids is converted to intermediates of TCA cycle or their precursors. The carbon skeleton finally has one or more of the following fates
1. Oxidation via TCA cycle to produce energy (about 10-15% of body needs).
- 2. Synthesis of glucose.
3.
Formation of
lipids—fatty acids
and ketone
bodies.
- 4. Synthesis of non-essential amino acids.
The carbon skeletons of the 20 standard (or more) amino acids (or the amino acids of proteins) are degraded to one of the following seven _products—pyruvate, a-ketoglutarate, succinyl CoA, fumarate, oxaloacetate, acetyl CoA and acetoacetate. Some authors use the term amphibolic (Greek: amphiboles—uncertain) intermediates to these compounds due to their multiple metabolic functions.
The amino acids are classified into two groups, based on the nature of the metabolic end products of carbon skeleton.
1. Glycogenic (glucogenic) amino acids : These are the amino acids whose carbon skeleton is finally degraded to pyruvate or one of the intermediates of TCA cycle (a-ketoglutarate, succinyl CoA, fumarate and oxaloacetate). These intermediates serve as good substrates for gluconeogenesis leading to the formation of glucose or glycogen.
Fig.
15.43
:
Summary of
the products
formed from
carbon skeleton
of amino
acids
(colour indication, Blue—glucogenic; Green shade—glucogenic and ketogenic; Red—-ketogenic).
2. Ketogenic amino acids : The amino acids whose carbon skeleton is metabolized to acetyl CoA or acetoacetate can be converted to fat (i.e., fatty acids or ketone bodies). Acetoacetate is a ketone body (besides acetone and 6-hydroxybutyrate).

Some of the amino acids are both glycogenic and ketogenic since they serve as precursors for glucose as well as fat.
The classification of amino acids (glycogenic, ketogenic, or both) is given in Table 15.4. The various products obtained from the carbon skeleton of amino acids and their connection with the citric acid cycle is depicted in Fig. 15.43.
The details on the formation of amphibolic intermediates by the degradation of amino acids are given in the metabolism of respective amino acids. They are summarized hereunder
- e Pyruvate Alanine, cysteine, — glycine, hydroxyproline, serine and threonine.
e
Glutamine, glutamate,
arginine, histidine
and proline.
- e Succinyl CoA Isoleucine, threonine and valine. methionine,
| Fumarate : Phenylalanine and tyrosine.
1 Oxaloacetate : Asparagine and aspartate.
1 Acetyl CoA and acetoacetate : Phenylalanine,
tyrosine, tryptophan, isoleucine, leucine and lysine.
Leucine and lysine are only ketogenic, since they produce acetoacetate or acetyl CoA.

Of the 20 amino acids, about half of them are non-essential in the diet, as they can be synthesized in human body. This is carried out by the biosynthesis of carbon skeleton, followed by the addition of amino group. via transamination. In the Table 15.5, the sources of carbon skeleton for the synthesis of non-essential amino acids are
given.

Several inherited disorders are associated with amino acid metabolism. The details of these metabolic disorders are described in the respective amino acids. Table 15.6 gives a summary of the inborn errors of amino acid metabolism.

+ Melanin—the pigment of skin, hair and eyes—is produced from tyrosine. Lack of melanin synthesis (mostly due to a deficiency of tyrosinase) causes albinism.
+ Parkinson’s disease—a common disorder of the elderly—is linked with decreased
synthesis
of
dopamine.
It
is
by
muscular
rigidity,
tremors,
lethargy
etc.
+ Phenylketonuria, due to a defect in the enzyme phenylalanine hydroxylase, is characterized by failure of growth, seizures and mental retardation (low IQ).
+ Alkaptonuria causes the accumulation of homogentisate which undergoes oxida- tion followed by polymerization to produce the pigment alkapton. Deposition of alkapton in connective tissue, causes ochronosis which is associated with arthritis.
+ Serotonin, an excitatory neurotransmitter, is synthesized from tryptophan. Psychic stimulant drugs (iproniazid) elevate serotonin levels while depressant drugs (LSD) decrease.
+ Malignant carcinoid syndrome, a tumor of argentaffin cells of gastrointestinal tract, is characterized by tremendously increased production of serotonin. This disorder can be diagnosed by the elevated levels of 5-hydroxyindoleacetate in urine.
+ Melatonin, produced from serotonin, is involved in circadian rhythms or diurnal variations, i.e., maintenance of body’s biological clock.
+ Homocysteine has been implicated as a risk factor in the onset of coronary heart diseases.
+ Histidine loading test, characterized by elevated excretion of N-formiminoglutamate (FIGLU) is commonly employed to assess the deficiency of the vitamin, folic acid.
+ Nitric oxide (NO), synthesized from arginine, is involved in several biological functions—
vasodilation,
platelet
aggregation,
neurotransmission
and
bactericidal
action.
NO
is
used
in
the
treatment
of
chronic
obstructive
pulmonary
disease
(COPD).

+ yAminobutyric acid (GABA), produced from glutamate, is an inhibitory neurotrans- mitter. Low levels of GABA result in convulsions.
+ The carbon skeleton of amino acids may be converted to glucose (glycogenic) or fat (ketogenic), besides being responsible for the synthesis of non-essential amino acids.
+ Polyamines (spermine, putrescine) are involved in the synthesis of DNA, RNA and proteins and, thus, they are essential for cell growth and differentiation.

The term amino aciduria is generally used to indicate the urinary excretion of amino acids. |t is frequently associated with defects in amino acid metabolism. Most of the amino acidurias manifest in mental retardation.

A neurotransmitter is an extracellular massenger that can transmit an extracellular message from a neuron to cells. Certain amino acids and or their derivatives can serve as neurotransmitters e.g. glycine, glutamate, serononin, GABA (Table 15.7).

In general, the decarboxylation of amino acids or their derivatives results in the formation of amines.
NH,
I
R-CH-coon
Amino acid
A
summary from
different functions
Pecatbowese
(PLP), co,
of the
amino are
biogenic
CH)—NHz
Amine amines
acids and
given in
Table derived
their
15.8.

Polyamines
(Greek:
poly—many)
possess
multiple
amino
groups.
Putrescine,
spermine
major and spermidine are the biologically important polyamines. Spermine and spermidine were originally detected in human semen (sperms), hence they are so named.

Ornithine and S-adenosylmethionine are the precursors for polyamine synthesis. It should, however, be noted that only the four-carbon moiety of SAM (not the methyl group) is involved in polyamine formation. Ornithine decarboxylase acts on ornithine to split off CO, and produce putrescine (Fig.15.44). The enzyme ornithine decarboxylase has the shortest halflife (about 10 minutes) among the known mammalian enzymes. It regulates polyamine synthesis. The activity of this enzyme is increased by hormones like corticosteroids, testosterone and growth hormone.
Putrescine is converted to spermidine and then spermine with the involvement of SAM. S-Adenosylmethionine is first decarboxylated to give decarboxylated SAM. SAM decarboxylase is a rare example of an enzyme that does not require pyridoxal phosphate as coenzyme. An amino acid residue bound to pyruvate is believed to function as a cofactor. The propylamino group of decarboxylated SAM is transferred to putrescine to give spermidine. Synthesis of spermine requires one more molecule of decarboxylated SAM _ and_ this reaction is catalysed by spermine synthase.

The enzyme polyamine oxidase (of liver peroxisomes) oxidizes spermine to spermidine and then to putrescine. Spermidine and putrescine are excreted in urine in a conjugated form, as acetylated derivatives. Some amount of putrescine is also oxidized to NH3 and CO).

1. Polyamines are basic in nature and possess multiple positive charges. Hence they are readily associated with nucleic acids (DNA and RNA).
RNA and
2. They are involved in the synthesis of DNA, proteins.

Disorder
Metabolic defect (enzyme/other)
Defects in urea synthesis
—Refer Table 15.1
Phenylalanine and tyrosine
1. Phenylketonuria
Phenylalanine hydroxylase
2. Tyrosinemia type II
Tyrosine transaminase
3. Neonatal tyrosinemia
p-Hydroxy phenylpyruvate dioxygenase
4.
Alkaptonuria
Homogentisate oxidase
5. Tyrosinosis (tyrosinemia type
|)
Maleyl acetoacetate isomerase or fumaryl acetoacetate hydrolase
6. Albinism
Tyrosinase

7. Cystinuria
Defect in renal reabsorption
8. Cystinosis
Impairment in cystine utilization (defect in lysosomal function)
9. Homocystinuria type
|
Cystathionine synthetase
10. Homocystinuria type
|I
N®, N'°-Methylene THF reductase
11. Homocystinuria type
III
N®-Methyl THF-homocysteine methyltransferase
12. Cystathionuria
Cystathioninase

13. Glycinuria
14. Primary hyperoxaluria
Glycine transaminase

15. Hartnup’s disease
Defective intestinal absorption
16. Maple syrup urine disease
Branched
chain
o-keto
acid
17. Intermittent branched chain ketonuria
Variant of the above enzyme (less severe)
18. Hypervalinemia
Valine transaminase
19. Isovaleric acidemia
Isovalery! CoA dehydrogenase
20. Histidinemia
Histidase
Proline
21. Hyperprolinemia type
|
Proline oxidase
Spermine
Fig. 15.44 : Biosynthesis of polyamines—putrescine, spermidine and spermine.
T ABLE 15.7 Amino acids and their derivatives as neurotransmitters, 1 = T ABLE 15.7 Amino acids and their derivatives as neurotransmitters. Amino acid/derivative, 1 = Major function(s). Glycine, 1 = Inhibitory neurotransmitter in spinal cord. Glutamate, 1 = Excitatory neurotransmitter. Dopamine, 1 = Increases blood pressure. Norepinephrine and epinephrine, 1 = Hormonal neurotransmitters, increase cardaic output and blood pressure. Serotonin, 1 = Regulates cerebral activity and behaviour. J -Aminobutyric acid (GABA), 1 = Inhibitory neurotransmitter in brain

The excretion of polyamines is found to be elevated in almost all types of cancers, e.g. leukemias; carcinoma of lungs, bladder, kidney etc. Diagnostically, putrescine is an ideal marker for cell proliferation whereas spermidine is suitable for the assessment of cell destruction.

Serine, Amine = Ethanolamine. Serine, Function(s) = Forms choline. Glutamate, Amine = J -Aminobutyric acid. Glutamate, Function(s) = Inhibitory neurotransmitter. Histidine, Amine = Histamine. Histidine, Function(s) = Vasodilator, promotes gastric HCl and pepsin synthesis. Phenylalanine, Amine = Dopamine. Phenylalanine, Function(s) = For the synthesis of nore- pinephrine and epinephrine. Tyrosine, Amine = Tyramine. Tyrosine, Function(s) = Vasoconstrictor (increases blood pressure). Tryptophan, Amine = Tryptamine Serotonin Melatonin. Tryptophan, Function(s) = Elevates blood pressure Stimulates cerebral activity Circadian rhythms. Cysteine, Amine = Taurine. Cysteine, Function(s) = Constituent of bile acid (taurocholic acid)

1., 1 = The body proteins are in a dynamic state (degradation and synthesis) and there is an active amino acid pool (100 g) maintained for this purpose.. 2., 1 = The amino acids undergo transamination and deamination to liberate ammonia for the synthesis of urea, the end product of protein metabolism.. 3., 1 = Besides being present as structural components of proteins, amino acids participate in the formation of several biologically important compounds.. 4., 1 = Glycine is involved in the synthesis of creatine, heme, purines, glutathione etc.. 5., 1 = Phenylalanine is hydroxylated to tyrosine, which is a precursor for the production of skin pigment (melanin), catecholamines (dopamine, epinephrine and norepinephrine) and thyroid hormones (T 3 and T 4 ).. 6., 1 = Tryptophan is converted to NAD + and NADP + , the coenzymes of niacin, serotonin (a neurotransmitter) and melatonin.. 7., 1 = The active methionine (SAM) is a donor of methyl group (transmethylation) for the synthesis of many biological compounds (epinephrine, choline, methylcytosine etc.).. 8., 1 = Many amino acids contribute to one-carbon fragments (formyl, formimino, methylene etc.) for participation in one-carbon metabolism-which is mostly under the control of tetrahydrofolate.. 9., 1 = The carbon skeleton of amino acids is involved either in the synthesis of glucose (glycogenic) or fat (ketogenic), or both-glucose and fat.. 10., 1 = Many inborn errors (mostly due to enzyme defects) in amino acid metabolism have been identified. These include phenylketonuria (defect-phenylalanine hydroxylase), albinism (defect-tyrosinase), maple syrup urine disease (defect glyph<c=16,font=/MDECLA+Symbol> D -keto acid dehydro- genase) etc.

1. Describe the reactions in the synthesis of urea.
2. Give an account of the formation of specialized products from glycine.
3. Discuss the metabolism of phenylalanine and tyrosine.
4. Describe the fate of carbon skeleton of amino acids.
5. Write briefly on various inborn errors of amino acid metabolism.

(a) Amino acid pool, (b) Transmethylation, (c) Transamination, (d) Deamination, (e) Ammonia toxicity, (f) One-carbon metabolism, (g) Albinism, (h) Serotonin, (i) Glutamate and glutamine,
(j) Polyamines.

1. The coenzyme that participates in transamination reactions is
CoN . The metabolite excreted (elevated) in alkaptonuria is
DH . The cofactor required for the conversion of phenylalanine to tyrosine is
FW . N-Acetylglutamate is required for the activation of the enzyme
NY . The most important enzyme involved in oxidative deamination is
9. The mammalian enzyme with the shortest half-life (about 10 minutes) is
10. The branched chain amino acid that is only ketogenic is
. Primary hyperoxaluria is due to a defect in the enzyme
. Parkinson's disease is linked with decreased synthesis of
. The disease in which very high amount of tryptophan (nearly 60%) is converted to serotonin
is

11. The synthesis of urea occurs in
12. The amino acid required for the formation of glutathione
13. In the synthesis of cysteine, the carbon skeleton is provided by
14. The amino acids are said to be ketogenic when the carbon skeleton is finally degraded to
15. The amino acid that does not participate in transamination
(a) Kidney (b) Liver (c) Muscle (d) Brain.
(a) Glycine (b) Cysteine (c) Glutamate (d) All of them.
(a) Serine (b) Methionine (c) Glutamate (d) Alanine.
(a) Succinyl CoA (b) Fumarate (c) Acetyl CoA (d) Pyruvate.
(a) Lysine (b) Glutamate (c) Alanine (d) Tryptophan.
Meeren is a continuous process, with thousands of reactions, simultaneously occurring in the living cell. However, biochemists prefer to present metabolism in the form of reactions and metabolic pathways. This is done for the sake of convenience in presentation and understanding.
In the preceeding three chapters (13-15), we have learnt the metabolism of carbohydrates, lipids and amino acids. We shall now consider the organism as a whole and _ integrate the metabolism with particular reference to energy demands of the body.

The organisms possess variable energy demands, hence the supply (input) is also equally variable. The consumed metabolic fuel may be burnt (oxidized to CO and HO) or stored to meet the energy requirements as per the body needs. ATP serves as the energy currency of the cell in this process (Fig. 16.1).
Fig. 16.1 : Asummary of body's energy supply and demands. (Note : ATP serves as the energy currency).
The humans possess enormous capacity for food consumption. It is estimated that one can consume as much as 100 times his/her basal requirements! Obesity, a disorder of overnutrition mostly prevalent in affluent societies, is primarily a consequence of overconsumption.

An overview of the interrelationship between the important metabolic pathways, concerned
Fig. 16.2 : An overview of integration of metabolic pathways of energy
(HMP
shunt-Hexose monophosphate
shunt).
with fuel metabolism depicted in Fig.16.2, is briefly described here. For detailed information on these metabolic pathways, the reader must refer the respective chapters.
1. Glycolysis : The degradation of glucose to pyruvate (lactate under anaerobic condition) generates 8 ATP. Pyruvate is converted to acetyl CoA.
different fuel sources (carbohydrates, lipids, amino acids). Acetyl! CoA enters citric acid (Krebs) cycle and gets oxidized to CO. Thus, citric acid cycle is the final common metabolic pathway for the oxidation of all foodstuffs. Most of the energy is trapped in the form of NADH and FADH).
2. Fatty acid oxidation : Fatty acids undergo sequential degradation with a _ release of 2-carbon fragment, namely acetyl CoA. The energy is trapped in the form of NADH and FADH).
3. Degradation of amino acids : Amino acids, particularly when consumed in excess than required for protein synthesis, are degraded and utilized to meet the fuel demands of the body. The glucogenic amino acids can serve as precursors for the synthesis of glucose via the formation of pyruvate or intermediates of citric acid cycle. The ketogenic amino acids are the precursors for acetyl CoA.
4. Citric acid cycle : Acetyl CoA is the key and common metabolite, produced from
5. Oxidative phosphorylation : The NADH and FADH,, produced in different metabolic pathways, are finally oxidized in the electron transport chain (ETC). The ETC is coupled with oxidative phosphorylation to generate ATP.
6. Hexose monophosphate shunt This pathway is primarily concerned with the liberation of NADPH and ribose sugar. NADPH is utilized for the biosynthesis of several compounds, including fatty acids. Ribose is an essential component of nucleotides and nucleic acids (Note : DNA contains deoxyribose).

7. Gluconeogenesis The synthesis of glucose from non-carbohydrate — sources constitutes gluconeogenesis. Several compounds (e.g. pyruvate, glycerol, amino acids) can serve as precursors for gluconeogenesis.
8. Glycogen metabolism : Glycogen is the storage form of glucose, mostly found in liver and muscle. It is degraded (glycogenolysis) and synthesized (glycogenesis) by independent pathways. Glycogen effectively serves as a fuel reserve to meet body needs, for a brief period (between meals).

The metabolic pathways, in general, are controlled by four different mechanisms
1. The availability of substrates
2. Covalent modification of enzymes
3. Allosteric regulation
4. Regulation of enzyme synthesis.
The details of these regulatory
processes are discussed under the individual metabolic pathways, in the respective chapters.

The various tissues and organs of the body work in a well coordinated manner to meet its metabolic demands. The major organs along with their most important metabolic functions, in a well-fed absorptive state (usually 2-4 hours after food consumption), are described.

The liver is specialized to serve as the body's central metabolic clearing house. \t processes and distributes the nutrients to different tissues for utilization. After a meal, the liver takes up the carbohydrates, lipids and most of the amino acids, processes them and routes to other tissues. The major metabolic functions of liver, in an absorptive state, are listed
1. Carbohydrate metabolism Increased glycolysis, glycogenesis and hexose monophosphate shunt and decreased gluconeogenesis.
fatty
2. Lipid metabolism : Increased synthesis of acids and triacylglycerols.
- 3. Protein metabolism : Increased
of amino acids and degradation protein synthesis.

Adipose tissue is regarded as the energy storage tissue. As much as 15 kg. of triacylglycerol (equivalent to 135,000 Cal) is stored in a normal adult man. The major metabolic functions of adipose tissue in an absorptive state are listed here.
1. Carbohydrate metabolism : The uptake of glucose is increased. This follows an increase in glycolysis and hexose monophosphate shunt.
2. Lipid metabolism : The synthesis of fatty acids and triacylglycerols is increased. The degradation of triacylglycerols is inhibited.

The metabolism of skeletal muscle is rather variable depending on its needs. For instance, the resting muscle of the body utilizes about 30% of body's oxygen consumption. However, during strenuous exercise, this may be as high as 90%. The important metabolic functions of skeletal muscle in an absorptive state are listed.
1.
Carbohydrate metabolism
:
The uptake
of glucose
is higher,
and glycogen
increased.
2. Lipid metabolism : Fatty acids taken up from the circulation are also important fuel sources for the skeletal muscle.
3. Protein metabolism Incorporation of acids into is amino proteins higher.
The human brain constitutes about 2% of the body's weight. But it utilizes as much as 20% of the oxygen consumed by the body. Being a vital organ, special priority is given to the metabolic needs of the brain.
1. Carbohydrate metabolism : In an absorptive state, glucose is the only fuel source to the brain. About 120 g of glucose is utilized per day by an adult brain. This constitutes about 60% of the glucose consumed by the body at

Liver, Energy compound(s) preferably utilized = Amino acids, glucose, fatty acids. Liver, Energy compound(s) exported = Glucose, fatty acids, ketone bodies. Adipose tissue, Energy compound(s) preferably utilized = Fatty acids. Adipose tissue, Energy compound(s) exported = Fatty acids, glycerol.. Skeletal muscle, Energy compound(s) preferably utilized = Fatty acids. Skeletal muscle, Energy compound(s) exported = None. , Energy compound(s) preferably utilized = Glucose. , Energy compound(s) exported = Lactate. Brain, Energy compound(s) preferably utilized = Glucose, ketone bodies (in starvation). Brain, Energy compound(s) exported = None
rest. It is estimated that about 50% of the energy consumed by brain is utilized by plasma membrane Na*-Kt-ATPase to maintain membrane potential required for nerve impulse transmission.
reserve of the body. The survival time of an individual on starvation is mostly dependent on his/her fat stores. And for this reason, obese individuals can survive longer than lean individuals without consuming food.
2. Lipid metabolism : The free fatty acids cannot cross the blood-brain barrier, hence their contribution for the supply of energy to the brain is insignificant. Further, in a fed state, ketone bodies are almost negligible as fuel source to the brain. However, brain predominantly depends on ketone bodies during prolonged starvation (details given later).
The metabolic interrelationship among the major tissues in an absorptive state are given in Fig.16.3. The fuel sources that are preferably utilized by the major organs and the compounds exported from them are listed in Table 16.1.

Starvation may be due to food scarcity or the desire to rapidly lose weight or certain clinical conditions (e.g. surgery, burns etc.). Starvation is a metabolic stress which imposes certain metabolic compulsions on the organism. The metabolism is reorganized to meet the new demands of starvation.
Glucose is the fuel of choice for brain and muscle. Unfortunately, the carbohydrate reserve of the body is so low that it cannot meet the energy requirements even for a day. The fuel stores (or energy reserves) of a 70 kg normal man are given in Table 16.2. Triacylglycerol (fat) of adipose tissue is the predominant energy
Protein is basically a structural constituent, mostly present in the muscle. However, during starvation, protein can also meet the fuel demands of the body. It is estimated that about '/srd of the body's protein can be utilized towards energy needs without compromising the vital functions.
Starvation is associated with a decrease in insulin level and an increase in glucagon. The metabolic changes during starvation are discussed with reference to the major organs/ tissues.

1. Carbohydrate metabolism : An important function of liver is to act as a blood glucose buffering organ. The action of liver is to suit the metabolic needs of the body. During starvation, increased gluconeogenesis and __ elevated glycogen degradation furnish glucose to the needy tissues (mostly brain).
384
Fig. 16.3 : Metabolic interrelationship among the major tissues in a well fed state
(HMP shunt—Hexose monophosphate shunt).
Fig. 16.4 : Metabolic interrelationship among major tissues during starvation.
2. Lipid metabolism : Fatty acid oxidation is increased with an elevated synthesis of ketone bodies. This is due to the fact that TCA (Krebs) cycle cannot cope up with the excess production of acetyl CoA, hence the latter is diverted for ketone body synthesis.
Ketone bodies
(primarily
B-hydroxybutyrate)
effectively serve
tissues.
as
The ketone
'/srd of
ketone brain
bodies.
slowly
Thus, the
brain’s for
the peripheral
adapts after
itself
a
3-day fuel
bodies, while,
they countribute
demands after
to about
to use
fast, about
are
40
days’
70%
met by
starvation, of
energy needs.

1.
Glucose uptake and its metabolism are lowered.
2. Lipid metabolism The degradation of triacylglycerol is elevated, leading to an increased release of fatty acids from the adipose tissue which serve as fuel source for various tissues (brain is an exception). The glycerol liberated in lipolysis serves as a precursor for glucose synthesis by liver. The synthesis of fatty acids and triacylglycerols is totally stopped in adipose tissue.

1. Carbohydrate metabolism Glucose uptake and its metabolism are very much depressed.
2. Lipid metabolism : Both fatty acids and ketone bodies are utilized by the muscle as fuel source. However, on prolonged starvation beyond 3 weeks, the muscle adapts to exclusively utilize fatty acids. This further increases the level of ketone bodies in the circulation.
3. Protein metabolism : During the early period of starvation, muscle proteins are degraded to liberate the amino acids which are effectively utilized by the liver for glucose synthesis (gluconeogenesis). On prolonged starvation, however, protein breakdown _ is reduced.

As already stated, glucose is the preferred fuel source by brain. During the first 2 weeks of starvation, the brain is mostly dependent on glucose, supplied by liver gluconeogenesis. This, in turn, is dependent on the amino acids released from the muscle protein degradation. Starvation beyond 3 weeks generally results in a marked increase in plasma ketone bodies. By this time, the brain adapts itself to depend on ketone bodies for the energy needs.
The metabolic interrelationship among the major organs in starvation are depicted in Fig.16.4. The biochemical changes that occur during starvation are such that an adequate supply of fuel molecules is maintained to various tissues to meet the energy demands. This is a natural adaptation for the survival of the organism.

1. The metabolism of carbohydrates, lipids and proteins is integrated to meet the energy and metabolic demands of the organism. The metabolic pathways—glycolysis, fatty acid oxidation, citric acid cycle and oxidative phosphorylation—are directly concerned with the generation of ATP. Gluconeogenesis, glycogen metabolism, hexose monophosphate shunt and amino acid degradation are also associated with energy metabolism.
2. The organs/tissues, with their respective specializations, coordinate with each other to meet the metabolic demands of the organism as a whole. Liver is specialized to serve as the body’s central metabolic clearing house. It processes and distributes the nutrients to different tissues for their utilization. Adipose tissue is primarily a storage organ of fat. The major bulk of the body protein is located in the muscle tissue.
3. Brain is a specialized organ which, in the normal situation, is exclusively dependent on the supply of glucose (120 g/day) for its fuel needs.
4. Starvation is a metabolic stress, as it imposes certain metabolic compulsions on the organism. The stored fat of adipose tissue and the muscle protein are degraded and utilized to meet the body’s fuel demands. Brain gradually adapts itself to use ketone bodies (instead of glucose) for its energy requirements. Starvation is, thus, associated with metabolic reorganization for the survival of the organism.
Niacceotses consist of a nitrogenous base, a pentose and a phosphate. The pentose sugar is D-ribose in ribonucleotides of RNA while in deoxyribonucleotides (deoxynucleotides) of DNA, the sugar is 2-deoxy D-ribose. Nucleotides participate in almost all the biochemical processes, either directly or indirectly. They are the structural components of nucleic acids (DNA, RNA), coenzymes, and are involved in the regulation of several metabolic reactions.

Many compounds contribute to the purine
ring of the nucleotides (Fig.17.1).
1. Ny of purine is derived from amino group
of aspartate.
2. Cy and Cg arise from formate of N!0-
formyl THF.
3. N3 and No are obtained from amide group
of glutamine.
Fig. 17.1 : The sources of individual atoms in purine ring. (Note : Same colours are used in the synthetic pathway Fig. 17.2).
- 4. C4, Cs and Nz are contributed by glycine.
5. Cg directly comes from CO).
It should be remembered that purine bases are not synthesized as such, but they are formed as ribonucleotides. The purines are built upon a pre-existing ribose 5-phosphate. Liver is the major site for purine nucleotide synthesis. Erythrocytes, polymorphonuclear leukocytes and brain cannot produce purines.
Fig. 17.2 contd. next column
Fig. 17.2 contd. next page
N'°-Formy!
Inosine monophosphate
Fig. 17.2 : The metabolic pathway for the synthesis of inosine monophosphate, the parent purine nucleotide (PRPP-Phosphoribosyl pyrophosphate; PPi-Pyrophosphate).
The pathway for the synthesis of inosine monophosphate (IMP or inosinic acid), the 'parent' purine nucleotide is given in Fig.17.2. The reactions are briefly described in the next column.
1. Ribose 5-phosphate, produced in the hexose monophosphate shunt of carbohydrate metabolism is the starting material for purine nucleotide synthesis. It reacts with ATP to form phosphoribosyl pyrophosphate (PRPP).
2. Glutamine transfers its amide nitrogen to PRPP to replace pyrophosphate and produce 5-phosphoribosylamine. The enzyme PRPP glutamyl amidotransferase is controlled by feedback inhibition of nucleotides (IMP, AMP and GMP). This reaction is the 'committed step' in purine nucleotide biosynthesis.

3. Phosphoribosylamine reacts with glycine in the presence of ATP to form glycinamide ribosyl 5-phosphate or glycinamide ribotide (GAR).
4. N!0Formyl tetrahydrofolate donates the formyl group and the product formed is formylglycinamide ribosyl 5-phosphate.
5. Glutamine transfers the second amido amino group to produce formylglycinamidine ribosyl 5-phosphate.
6. The imidazole ring of the purine is closed in an ATP dependent reaction to yield 5-aminoimidazole ribosyl 5-phosphate.
7. Incorporation of CO, (carboxylation) occurs to yield aminoimidazole carboxylate ribosyl 5-phosphate. This reaction does not require the vitamin biotin and/or ATP which is the case with most of the carboxylation reactions.
8. Aspartate condenses with the product in reaction 7 to form aminoimidazole 4-succinyl carboxamide ribosyl 5-phosphate.
9. Adenosuccinate lyase cleaves off fumarate and only the amino group of aspartate is retained to yield aminoimidazole 4-carboxamide ribosyl 5-phosphate.
10. N!9-Formyl tetrahydrofolate donates a one-carbon moiety to produce formaminoimidazole 4-carboxamide ribosyl 5-phosphate. With this reaction, all the carbon and nitrogen atoms of purine ring are contributed by the respective sources.
11. The final catalysed by cyclohydrolase leads to ring closure with an elimination of water molecule. The product obtained is inosine monophosphate (IMP), the parent purine nucleotide from which other purine nucleotides can be synthesized. reaction

Folic acid
(THF)
essential for
of is
the purine
(reactions
4
Sulfonamides synthesis
nucleotides and
10).
are structural
analogs aminobenzoic
(PABA).
These can
be used
the para-
acid sulfa
drugs to
inhibit synthesis
of folic
the acid
indirectly by
This reduces
synthesis therefore,
of purines
the nucleic
(DNA
and
Sulfonamides influence
the and,
acids
RNA).
have on
since folic
synthesized no
humans, acid
is not
and is
Fig. 17.3 : Synthesis of AMP and GMP from inosine monophosphate.
supplied through
diet.
The structural analogs of folic acid (e.g. methotrexate) are widely used to control cancer. They inhibit the synthesis of purine nucleotides (reaction 4 and 10) and, thus, nucleic acids. Both these reactions are concerned with the transfer of one-carbon moiety (formyl group). These inhibitors also affect the proliferation of normally growing cells. This causes many side-effects including anemia, baldness, scaly skin etc.

Inosine monophosphate is the immediate
precursor for the formation of AMP and GMP
(Fig.17.3). Aspartate condenses with IMP in the presence of GTP to produce adenylsuccinate which, on cleavage, forms AMP.
For the synthesis of GMP, IMP undergoes NADt dependent dehydrogenation to form xanthosine monophosphate (XMP). Glutamine then transfers amide nitrogen to XMP to produce GMP.
6-Mercaptopurine is an inhibitor of the synthesis of AMP and GMP. It acts on the enzyme adenylsuccinase (of | AMP pathway) and IMP dehydrogenase (of GMP. pathway).
of
Fig. 17.4 : Conversion of nucleoside monophosphates to diand triphosphates (NMP-Nucleoside monophosphate; NDP—Nucleoside diphosphate).

The nucleoside monophosphates (AMP and GMP) have to be converted to the corresponding diand triphosphates to participate in most of the metabolic reactions. This is achieved by the transfer of phosphate group from ATP, catalysed by nucleoside monophosphate (NMP) kinases and nucleoside diphosphate (NDP) kinases (Fig 17.4).

The free purines (adenine, guanine and hypoxanthine) are formed in the normal turnover of nucleic acids (particularly RNA), and also obtained from the dietary sources. The purines can be directly converted to the corresponding nucleotides, and this process is known as 'salvage pathway' (Fig.17.5).
Adenine phosphoribosyl transferase catalyses the formation of AMP from adenine. Hypoxanthine-guanine phosphoribosyl — transferase (HGPRT) converts guanine and hypoxanthine, respectively, to GMP and IMP. Phosphoribosyl pyrophosphate (PRPP) is the donor of ribose 5-phosphate in the salvage pathway.
AN
Fig. 17.5 : Salvage pathways of purine nucleotide synthesis (PRPP—Phosphoribosyl pyrophosphate; PPi-Inorganic pyrophosphate; AMP—Adenosine monophosphate; GMP-—Guanosine monophosphate; IMP-Inosine monophosphate; ** Deficiency of HGPRT causes Lesch-Nyhan syndrome).
Fig. 17.6 : Formation of deoxyribonucleotides from ribonucleotides.
The salvage pathway is particularly important in certain tissues such as erythrocytes and brain where de novo (a new) synthesis of purine nucleotides is not operative.
moiety (Fig.17.6). This reaction is catalysed by a multisubunit (two B,; and two B», subunits) enzyme, ribonucleotide reductase.
A defect in the enzyme HGPRT causes LeschNyhan syndrome (details given later).

The purine nucleotide synthesis is well coordinated to meet the cellular demands. The intracellular concentration of PRPP regulates purine synthesis to a large extent. This, in turn, is dependent on the availability of ribose 5-phosphate and the enzyme PRPP synthetase.
PRPP glutamy! amidotransferase is controlled by a feedback mechanism by purine nucleotides. That is, if AMP and GMP are available in adequate amounts to meet the cellular requirements, their synthesis is turned off at the amidotransferase reaction.
Another important stage of regulation is in the conversion of IMP to AMP and GMP. AMP inhibits adenylsuccinate synthetase while GMP inhibits IMP dehydrogenase. Thus, AMP and GMP control their respective synthesis from IMP by a feedback mechanism.

The synthesis of purine and pyrimidine deoxyribonucleotides occurs from __ ribonucleotides by a reduction at the Cy of ribose
Supply of reducing equivalents : The enzyme ribonucleotide reductase itself provides the hydrogen atoms needed for reduction from its sulfhydryl groups. The reducing equivalents, in turn, are supplied by thioredoxin, a monomeric protein with two cysteine residues.
NADPH-dependent thioredoxin reductase converts the oxidized thioredoxin to reduced form which can be recycled again and again. Thioredoxin thus serves as a protein cofactor in an enzymatic reaction.
Regulation of deoxyribonucleotide synthesis : Deoxyribonucleotides are mostly required for the synthesis of DNA. The activity of the enzyme ribonucleotide reductase maintains the adequate supply of deoxyribonucleotides.
The drug hydroxyurea inhibits ribonucleotide reductase by destroying free radicals required by this enzyme. Hydroxyurea is used in the treatment of cancers such as__ chronic myologenous leukemia.

The end product of purine metabolism in humans is uric acid. The sequence of reactions in purine nucleotide degradation is given in
Fig.17.7.
393

animals other than man.
1. The nucleotide monophosphates (AMP, IMP and GMP) are converted to their respective nucleoside forms (adenosine, inosine and guanosine) by the action of nucleotidase.
2. The amino group, either from AMP or adenosine, can be removed to produce IMP or inosine, respectively.
3. Inosine and guanosine are, respectively, converted to hypoxanthine and guanine (purine bases) by purine nucleoside phosphorylase. Adenosine is not degraded by this enzyme, hence it has to be converted to inosine.
4. Guanine undergoes deamination by guanase to form xanthine.
5. Xanthine oxidase is an important enzyme that converts hypoxanthine to xanthine, and xanthine to uric acid. This enzyme contains FAD, molybdenum and iron, and is exclusively found in liver and small intestine. Xanthine oxidase liberates HyOy which is harmful to the tissues. Catalase cleaves HO, to HjO and Op.
Uric acid (2,6,8-trioxypurine) is the final excretory product of purine metabolism in humans. Uric acid can serve as an important antioxidant by getting itself converted (nonenzymatically) to allantoin. It is believed that the antioxidant role of ascorbic acid in primates is replaced by uric acid, since these animals have lost the ability to synthesize ascorbic acid.
Most animals (other than primates) however, oxidize uric acid by the enzyme uricase to allantoin, where the purine ring is cleaved. Allantoin is then converted to allantoic acid and excreted in some fishes (Fig.17.8). Further degradation of allantoic acid may occur to produce urea (in amphibians, most fishes and some molluscs) and, later, to ammonia (in marine invertebrates).

Uric acid is the end product of purine metabolism in humans. The — normal concentration of uric acid in the serum of adults is in the range of 3-7 mg/dl. In women, it is slightly lower (by about 1 mg) than in men. The daily excretion of uric acid is about 500-700 mg.
Hyperuricemia refers to an elevation in the serum uric acid concentration. This is sometimes associated with increased uric acid excretion (uricosuria).
Gout is a metabolic disease associated with overproduction of uric acid. At the physiological pH, uric acid is found in a more soluble form as sodium urate. In severe hyperuricemia, crystals of sodium urate get deposited in the soft tissues, particularly in the joints. Such deposits are commonly known as_ tophi. This causes inflammation in the joints resulting in a painful gouty arthritis. Sodium urate and/or uric acid may also precipitate in kidneys and ureters that results in renal damage and stone formation.
Historically, gout was found to be often associated with high living, over-eating and alcohol consumption. |n the previous centuries, alcohol was contaminated with lead during its manufacture and storage. Lead poisoning leads to kidney damage and decreased uric acid excretion causing gout. In general, a diet rich is meat and seafoods is associated with increased risk of gout.
The prevalence of gout is about 3 per 1,000 persons, mostly affecting males. Post-menopausal

e PRPP synthetase : In normal circumstances, PRPP synthetase is under feedback control by purine nucleotides (ADP and GDP). However, variant forms of PRPP synthetase—which are not subjected to feedback regulation—have been detected. This leads to the increased production of purines.
e PRPP_ glutamylamidotransferase The lack of feedback control of this enzyme by purine nucleotides also leads to their elevated synthesis.
e HGPRT deficiency : This is an enzyme of purine salvage pathway, and its defect causes Lesch-Nyhan syndrome. This disorder is associated with increased — synthesis _—_ of purine nucleotides by a_ two-fold mechanism. Firstly, decreased utilization of purines (hypoxanthine and guanine) by salvage pathway, resulting in the accumulation and diversion of PRPP for purine nucleotides. Secondly, the defect in salvage pathway leads to decreased levels of IMP and GMP causing impairment in the tightly controlled feedback regulation of their production.
e Glucose 6-phosphatase deficiency : In type | glycogen storage disease (von Gierke's), glucose 6-phosphate cannot be converted to glucose due to the deficiency of glucose 6-phosphatase. This leads to the
Fig. 17.9 : Summary of possible enzyme alterations causing gout (t-Increased enzyme activity; |-Decreased enzyme activity; GSH-Reduced glutathione; G-S-S-G-—Oxidized glutathione; PRPP-Phosphoribosy! pyrophosphate; HGPRT—Hypoxanthineguanine phosphoribosyltransferase).
increased utilization of glucose 6-phosphate by hexose monophosphate shunt (HMP shunt), resulting in elevated levels of ribose 5-phosphate and PRPP and, ultimately, purine overproduction. von Gierke's disease is also associated with increased activity of glycolysis. Due to this, lactic acid accumulates in the body which interferes with the uric acid excretion through renal tubules.

- e Elevation of glutathione reductase : Increased glutathione reductase generates more NADPt which is utilized by HMP shunt. This causes increased ribose 5-phosphate and PRPP synthesis.
Among the five enzymes described, the first three are directly involved in purine synthesis. The remaining two indirectly regulate purine production. This is a good example to show how an abnormality in one metabolic pathway influences the other.
2. Secondary gout : Secondary hyperuricemia is due to various diseases causing increased synthesis or decreased excretion of uric acid. Increased degradation of nucleic acids (hence more uric acid formation) is observed in various cancers (leukemias, polycythemia, lymphomas, etc.) psoriasis and increased tissue breakdown (trauma, starvation etc.).
The disorders associated with impairment in renal function cause accumulation of uric acid which may lead to gout.

By administration of uric acid isotope (N!5), the miscible uric acid pool can be calculated. It is around 1,200 mg in normal subjects. Uric acid pool is tremendously increased to 3,000 mg. or even more, in patients suffering from gout.

The drug of choice for the treatment of primary gout is allopurinol. This is a structural analog of hypoxanthine that competitively inhibits the enzyme xanthine oxidase. Further, allopurinol is oxidized to alloxanthine by xanthine oxidase (Fig.17.10). Alloxanthine, in turn, is a more effective inhibitor of xanthine
Fig. 17.10 : Structures of hypoxanthine and its structural analogs.
oxidase. This type of inhibition is referred to as suicide inhibition (For more details, Refer Chapter 6).
Inhibition of xanthine oxidase by allopurinol leads to the accumulation of hypoxanthine and xanthine. These two compounds are more soluble than uric acid, hence easily excreted.
Besides the drug therapy, restriction in dietary intake of purines and alcohol is advised. Consumption of plenty of water will also be useful.
The anti-inflammatory drug colchicine is used for the treatment of gouty arthritis. Other antiinflammatory drugs—such as phenylbutazone, indomethacin, oxyphenbutazone, corticosteroids— are also useful.

The clinical manifestations of pseudogout are similar to gout. But this disorder is caused by the deposition of calcium pyrophosphate crystals in joints. Further, serum uric acid concentration is normal in pseudogout.

This disorder is due to the deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), an enzyme of purine salvage pathway (See Fig.17.5). It was first described in 1964 by Michael Lesch (a medical student) and William L. Nyhan (his teacher).
Lesch-Nyhan syndrome is a_ sex-linked metabolic disorder since the structural gene for HGPRT is located on the X-chromosome. It affects only the males and is characterized by excessive uric acid production (often gouty arthritis), and neurological abnormalities such as mental retardation, aggressive behavior, learning disability etc. The patients of this disorder have an irresistible urge to bite their fingers and lips, often causing self-mutilation.
The overproduction of uric acid in LeschNyhan syndrome' is explained. © HGPRT deficiency results in the accumulation of PRPP and decrease in GMP and IMP, ultimately leading to increased synthesis and degradation of purines (more details given under primary gout).
helps to decrease uric acid production, has no affect on the neurological manifestations in these patients.

Two different immunodeficiency disorders associated with the degradation of purine nucleosides are identified. The enzyme defects are adenosine deaminase and purine nucleoside phosphorylase, involved in uric acid synthesis (See Fig.17.7).
The biochemical basis for the neurological symptoms observed in Lesch-Nyhan syndrome is not clearly understood. This may be related to the dependence of brain on the salvage pathway for de novo synthesis of purine nucleotides. Uric acid is not toxic to the brain, since patients with severe hyperuricemia (not related to HGPRT deficiency) do not exhibit any neurological symptoms. Further, allopurinol treatment that
The deficiency of adenosine deaminase (ADA) causes severe combined immunodeficiency (SCID) involving T-cell and usually B-cell dysfunction. It is explained that ADA deficiency results in the accumulation of dATP which is an inhibitor of ribonucleotide reductase and, therefore, DNA synthesis and cell replication.
The deficiency of purine nucleotide phosphorylase is associated with impairment of T-cell function but has no effect on B-cell function. Uric acid synthesis is decreased and the tissue levels of purine nucleosides and nucleotides are higher. It is believed that dGTP inhibits the development of normal T-cells.

ts
Folic
acid
is
essential
for
the
synthesis
of
purine
nucleotides.
Folic
acid
analogs
(methotrexate)
are
employed
to
control
cancer.
ts
The
salvage
pathway,
involving
the
direct
of
purines
to
corresponding
nucleotides,
is
important
in
tissues—brain
and
erythrocytes.
t=
Gout
is
the
disorder
with
the
overproduction
of
uric
acid,
the
end
product
of
purine
Allopurinol
is
the
drug
of
choice
for
the
treatment
of
gout.
t=
syndrome
is
caused
by
a
defect
in
the
enzyme
hypoxanthine-guanine
phos-
phoribosyltransferase.
The
patients
have
an
irresistible
urge
to
bite
their
fingers
and
lips.
ts
A
defect
in
the
enzyme
adenosine
deaminase
(ADA)
results
in
severe
combined
immunodeficiency
(SCID)
involving
both
T-cell
and
B-cell
A
girl
suffering
from
SCID
was
cured
by
transferring
ADA
gene
(in
1990)
and
that
was
the
first
attempt
for
gene
therapy
in
modern
medicine.
ts
Orotic
aciduria,
a
metabolic
defect
in
is
by
anaemia
and
retarded
growth,
besides
the
excretion
of
orotic
acid
in
urine.

Fig. 17.11 : Sources of individual atoms in pyrimidine ring (Note : Same colours are used in the synthetic pathway, Fig. 17.12).

Decreased uric acid levels in the serum (<2 mg/dl) represent hypouricemia. This is mostly associated with a rare genetic defect in the enzyme xanthine oxidase. It leads to the increased excretion of xanthine and hypoxanthine. Xanthinuria frequently causes the formation of xanthine stones in the urinary tract.

The synthesis of pyrimidines is a much simpler process compared to that of purines. Aspartate, glutamine (amide group) and CO») contribute to atoms in the formation of pyrimidine ring (Fig.17.11). Pyrimidine ring is first synthesized and then attached to ribose 5-phosphate. This is in contrast to purine nucleotide synthesis wherein purine ring is built upon a pre-existing ribose 5-phosphate. The pathway of pyrimidine synthesis is depicted in Fig.17.12, and the salient features are described below.
Glutamine transfers its amido nitrogen to CO, to produce carbamoyl phosphate. This reaction is ATP-dependent and is catalysed by cytosomal enzyme carbamoyl phosphate synthetase II (CPS Il).
CPS Il is activated by ATP and PRPP and inhibited by UTP. Carbamoyl phosphate synthetase | (CPS I) is a mitochondrial enzyme which synthesizes carbamoyl phosphate from ammonia and CO, and, in turn urea (Refer protein metabolism, Chapter 15, for more details). Prokaryotes have only one carbamoyl phosphate synthetase which is responsible for the biosynthesis of arginine and pyrimidines.
Carbamoyl phosphate condenses with aspartate to form carbamoyl aspartate. This reaction is catalysed by aspartate transcarbamoylase. Dihydroorotase catalyses the pyrimidine ring closure with a loss of H,O.
The three enzymes—CPS II, aspartate transcarbamoylase and dihydroorotase are the domains (functional units) of the same protein. This is a good example of a multifunctional

The next step in pyrimidine synthesis is an NAD+ dependent dehydrogenation, leading to the formation of orotate.
Ribose 5-phosphate is now added to orotate to produce orotidine monophosphate (OMP). This reaction is catalysed by orotate phosphoribosyltransferase, an enzyme comparable with HGPRT in its function. OMP undergoes decarboxylation to uridine mono-phosphate (UMP).
Orotate phosphoribosyltransferase and OMP decarboxylase are domains of a single protein. A defect in this bifunctional enzyme causes orotic aciduria (details given later).
By an ATP-dependent kinase reaction, UMP is converted to UDP which serves as a precursor for the synthesis of (UMP, dTMP, UTP and CTP.
Ribonucleotide reductase converts UDP to dUDP by a thioredoxin-dependent reaction. Thymidylate synthetase catalyses the transfer of a methyl group from N°, N!9methylene tetrahydrofolate to produce deoxythymidine monophosphate (dTMP).
UDP undergoes an ATP-dependent kinase reaction to produce UTP. Cytidine triphosphate (CTP) is synthesized from UTP by amination. CTP synthetase is the enzyme and glutamine provides the nitrogen.

In bacteria, (ATCase) aspartate _transcarbamoylase catalyses a committed step in
Fig. 17.12 : Metabolic pathway for the synthesis of pyrimidine nucleotides.

the regulatory
enzyme of
pyrimidine synthesis
It is
PRPP
and
ATP
and inhibited by UDP and UTP. OMP decarboxylase,
inhibited by
UMP
and
CMP, also.
controls pyrimidine formation.

The pyrimidine nucleotides undergo similar reactions (dephosphorylation, deamination and cleavage of glycosidic bond) like that of purine nucleotides to liberate the nitrogenous bases— cytosine, uracil and thymine. The bases are then degraded to highly soluble products—f-alanine and f-aminoisobutyrate. These are the amino acids which undergo transamination and other reactions to finally produce acetyl CoA and succinyl CoA.

The pyrimidines (like purines) can also serve as precursors in the salvage pathway to be converted to the respective nucleotides. This reaction is catalysed by pyrimidine phosphoribosyltransferase which utilizes PRPP as the source of ribose 5-phosphate.

Orotic aciduria : This is a rare metabolic disorder characterized by the excretion of orotic acid in urine, severe anemia and_ retarded growth. It is due to the deficiency of the enzymes orotate phosphoribosyl — transferase — and OMP decarboxylase of pyrimidine synthesis (Fig.17.12). Both these enzyme activities are present on a_ single protein as domains (bifunctional enzyme).
Feeding diet rich in uridine and/or cytidine is an effective treatment for orotic aciduria. These compounds provide (through phosphorylation) pyrimidine nucleotides required for DNA and RNA synthesis. Besides this, UTP inhibits carbamoyl phosphate synthetase II and blocks synthesis of orotic acid.
Reye's syndrome : This is considered as a secondary orotic aciduria. It is believed that a defect in ornithine transcarbamoylase (of urea cycle) causes the accumulation of carbamoyl phosphate. This is then diverted for the increased synthesis and excretion of orotic acid.

The nucleotide coenzymes FMN, FAD, NAD* NADP? and coenzyme A are synthesized from the B-complex vitamins. Their formation is described under the section on vitamins (Chapter 7).

1., 1 = Nucleotides participate in a wide variety of reactions in the living cells-synthesis of DNA and RNA; as constituents of many coenzymes; in the regulation of metabolic reactions etc.. 2., 1 = Purine nucleotides are synthesized in a series of reactions starting from ribose 5-phosphate. Glycine, glutamine, aspartate, formate and CO 2 contribute to the synthesis of purine ring.. 3., 1 = Purine nucleotides can also be synthesized from free purines by a salvage pathway. The defect in the enzyme HGPRT causes Lesch-Nyhan syndrome.. 4., 1 = Deoxyribonucleotides are formed from ribonucleotides by a reduction process catalysed by ribonucleotide reductase. Thioredoxin is the protein cofactor required for this reaction.. 5., 1 = Purine nucleotides are degraded to uric acid, the excretory product in humans. Uric acid serves as a natural antioxidant in the living system.. 6., 1 = Uric acid in many animal species (other than primates) is converted to more soluble forms such as allantoin, allantoic acid etc., and excreted.. 7., 1 = Gout is a metabolic disease associated with overproduction of uric acid. This often leads to the accumulation of sodium urate crystals in the joints, causing painful gouty arthritis. Allopurinol, an inhibitor of xanthine oxidase, is the drug used for the treatment of gout.. 8., 1 = Pyrimidine nucleotides are synthesized from the precursors aspartate, glutamine and CO 2 , besides ribose 5-phosphate.. 9., 1 = Orotic aciduria is a defect in pyrimidine synthesis caused by the deficiency of orotate phosphoribosyltransferase and OMP decarboxylase. Diet rich in uridine and/or cytidine is an effective treatment for orotic aciduria.. 10., 1 = Pyrimidines are degraded to amino acids, namely ` -alanine and ` -aminoisobutyrate which are then metabolized.

1. Describe the catabolism of purine nucleotides and the associated metabolic disorders.
. Write an account of the biosynthesis of inosine monophosphate.
. Discuss the synthesis and degradation of pyrimidines. WN
. Describe the role of PRPP in purine and pyrimidine synthesis.
a . Write an account of salvage pathway in purine nucleotide synthesis. Add a note on Lesch- Nyhan syndrome.
(a) Gout, (b) PRPP, (c) Synthesis of deoxyribonucleotides, (d) Functions of nucleotides, (e) Immuno- deficiency diseases in purine metabolism, (f) Orotic aciduria, (g) Carbamoyl phosphate synthetase Il, (h) HGPRT, (1) Degradation of uric acid in different animals, (j) Regulation of purine synthesis
(k) Inhibitors of purine synthesis.

1. The amino acids required for the synthesis of purines and pyrimidines are
2. The enzyme xanthine oxidase is inhibited by
3. Tophi are mostly made up of
4. Hypouricemia is due to the deficiency of the enzyme
5.
The
disorder
in
which
the
patients
have
an
urge
to
bite
their
fingers
and
lips
is
6. The cofactor required by the enzyme ribonucleotide reductase is
7. The ‘parent’ nucleotide synthesized in the biosynthesis of purines is
8. Xanthine oxidase converts allopurinol to
9. The amino acid that contributes to the synthesis of more than half of the pyrimidine ring
10. The regulatory enzyme in the pyrimidine biosynthesis in animals is

11. Name the enzyme associated with hyperuricemia
(a) PRPP synthetase (b) HGPRT (c) Glucose 6-phosphatase (d) All of them.
12. An enzyme of purine metabolism associated with immunodeficiency disease
(a) Adenosine deaminase (b) Xanthine oxidase (c) PRPP synthetase (d) HGPRT.
13. Orotic aciduria can be treated by a diet rich in
(a) Adenine (b) Guanine (c) Uridine (d) Any one of them.
14. The end product of purine metabolism in humans is
(a) Xanthine (b) Uric acid (c) Urea (d) Allantoin.
15. The nitrogen atoms in the purine ring are obtained from
(a) Glycine (b) Glutamine (c) Aspartate (d) All of them.
he mineral (inorganic) elements constitute only a small proportion of the body weight. There is a wide variation in their body content. For instance, calcium constitutes about 2% of body weight while cobalt about 0.00004%.

Minerals perform several vital functions which are absolutely essential for the very existence of the organism. These include calcification of bone, blood coagulation, | neuromuscular irritability, acid-base equilibrium, fluid balance and osmotic regulation.
Certain minerals are integral components of biologically important compounds such as hemoglobin (Fe), thyroxine (I), insulin (Zn) and vitamin By) (Co). Sulfur is present in thiamine, biotin, lipoic acid and coenzyme A. Several minerals participate as cofactors for enzymes in metabolism (e.g. Mg, Mn, Cu, Zn, K). Some elements are essential constituents of certain enzymes (e.g. Co, Mo, Se).

The minerals are classified as _ principal elements and trace elements.
The seven principal elements (macrominerals) constitute 60-80% of the body's inorganic material. These are calcium, phosphorus, magnesium, sodium, potassium, chloride and sulfur.
The principal elements are required in amounts greater than 100 mg/day.
The (microminerals) are required in amounts less than 100 mg/day. They are subdivided into three categories
1. Essential trace elements : Iron, copper, iodine, manganese, zinc, molybdenum, cobalt, fluorine, selenium and chromium.
2.
Possibly essential
trace elements
:
Nickel, vanadium, cadmium and barium.
3.
Non-essential trace
elements
:
Aluminium, lead, mercury, boron, silver, bismuth etc.

Sulfur, T ABLE 18.1 A summary of the major characteristics of principal elements (macroelements).Recommended dietary allowance = -. Sulfur, T ABLE 18.1 A summary of the major characteristics of principal elements (macroelements).Major sources = Sulfur containing amino acids
A summary of the major characteristics of principal elements and trace elements is respectively given in Tables 18.1 and 18.2. The individual elements are described next.
the formation (of hydroxyapatite) and physical strength of skeletal tissue. Bone is regarded as a mineralized connective tissue. Bones which are in a dynamic state serve as reservoir of Ca. Osteoblasts are responsible for bone formation while osteoclasts result in demineralization.
Calcium is the most abundant among the minerals in the body. The total content of calcium in an adult man is about 1 to 1.5 kg. As much as 99% of it is present in the bones and teeth. A small fraction (1%) of the calcium, found outside the skeletal tissue, performs a wide variety of functions.

1. Development of bones and _ teeth Calcium, along with phosphate, is required for
2. Muscle contraction : Ca2+ interacts with troponin C to trigger muscle contraction. Calcium also activates ATPase, increases the interaction between actin and myosin.
3. Blood coagulation : Several reactions in the cascade of blood clotting process are dependent on Ca?*(factor IV).
4.
Nerve transmission
:
Ca2+
for the transmission of nerve impulse.
5.
Membrane integrity
and permeability
Ca2*
influences transport
of water
the membrane
and
:
structure several
and ions
across it.

erythrocytes. Cobalt, Deficiency disease/symptoms = Pernicious anemia (as in vitamin B 12 deficiency). Cobalt, Recommended dietary allowance = 5-8 + g/d. Cobalt, Major sources = Foods of animal origin. Fluorine, Major functions = Helps in the proper formation of bones and teeth. Fluorine, Deficiency disease/symptoms = Dental caries, osteoporosis. Fluorine, Recommended dietary allowance = 2-4 mg/d. Fluorine, Major sources = Drinking water. Selenium, Major functions = Involved in antioxidant function along with vitamin E; constituent of glutathione peroxidase and selenocysteine. Selenium, Deficiency disease/symptoms = Muscular degeneration, cardiomyopathy. Selenium, Recommended dietary allowance = 50-200 + g/d. Selenium, Major sources = Organ meats, sea foods. Chromium, Major functions = Promotes insulin function (as glucose tolerance factor). Chromium, Deficiency disease/symptoms = Impaired glucose tolerance. Chromium, Recommended dietary allowance = 10-100 + g/d. Chromium, Major sources = Brewer s yeast, meat, whole grains
Ca2*. hormonal glycogenolysis. messenger for some hormones e.g. antidiuretic hormone (ADH) acts through cAMP, and then mediation of Ca2+ (instead of CAMP). Calcium is regarded as a second messenger for such action e.g. Calcium liver third epinephrine in serves as a
9.
Release of
hormones
:
The release
of certain
hormones the
endocrine
(insulin,
PTH, glands
10.
Secretory microfilament
calcitonin)
is facilitated
processes and
from by
Ca2*.
Ca2*
microtubule regulates
mediated
processes such as endocytosis, exocytosis and
cell motility.
11. Contact inhibition : Calcium is believed to be involved in cell to cell contact and adhesion of cells in a tissue (Refer p. 692 also). The cell to cell communication may also require Ca?*.
12.
Action
on_
heart
Ca2*
acts
on

Adult men and women —
800 mg/day
Women during
pregnancy, lactation
and
post-menopause —
1.5 g/day
Children (1-18 yrs.)
— 0.8-1.2 g/day
Infants (<1 year)
— 300-500 mg/day

Best sources —
Milk and milk products
Good sources — _
Beans, leafy vegetables,
fish, cabbage, egg yolk.

The absorption of calcium mostly occurs in the duodenum by an energy dependent active
process. It is influenced by several factors.

1. Vitamin D (through its active form calcitriol) induces the synthesis of calcium binding protein in the intestinal epithelial cells and promotes Ca absorption.
2.
Parathyroid hormone
enhances
Ca absorption through the increased synthesis of calcitriol.
3. Acidity (low pH) is more favourable for Ca absorption.
tinal
4. Lactose promotes calcium uptake by intescells.
facilitate
5. The amino acids Ca absorption. lysine and arginine
Fig. 18.1 : Different forms of circulating calcium.

1. Phytates and oxalates form insoluble salts
and interfere with Ca absorption.
2. High content of dietary phosphate results
in
the
formation
of
insoluble
calcium
phosphate
and
prevents
Ca
uptake.
The
dietary
ratio
of
Ca
and
P—between
1
:
2
and
2
:
1—is
ideal
for
optimum Ca absorption by intestinal cells.
3. The free fatty acids react with Ca to form insoluble calcium soaps. This is particularly observed when the fat absorption is impaired.
4.
Alkaline
condition
(high
pH)
is
unfavourable for Ca absorption.
5. High content of dietary fiber interferes with absorption.
Ca

Most of the blood Ca is present in the plasma since the blood cells contain very little of it. The normal concentration of plasma or serum Ca is 9-11 mg/dl (4.5-5.5 mEq/l). About half of this (5 mg/dl) is in the ionized form which _ is functionally the most active (Fig.18.1). At least 1 mg/dl serum Ca is found in association with citrate and/or phosphate. About 40% of serum Ca (4-5mg/dl) is bound to proteins, mostly albumin and, to a lesser extent, globulin. lonized and citrate (or phosphate) bound Ca is diffusible from blood to the tissues while protein bound Ca is non-diffusible. In the usual laboratory determination of serum Ca, all the three fractions are measured together.
Fig. 18.2 : Overview of calcium homeostasis (PTH-Parathyroid hormone).

As already stated, calcium is almost exclusively present in blood plasma (or serum). The hormones—calcitriol, parathyroid hormone (PTH) and calcitonin are the major factors that regulate the plasma calcium (homeostasis of Ca; Fig.18.2) within a narrow range (9-11 mg/dl).

The physiologically active form of vitamin D is a hormone, namely calcitriol or 1,25-dihydroxycholecalciferol (1,25 DHCC). The synthesis of calcitriol and its wide range of biochemical actions are described under Vitamins (Chapter 7). Calcitriol induces the synthesis of specific a calcium binding protein in the intestinal cells. This protein increases the intestinal absorption of calcium as well as phosphate. Thus blood Ca level is increased by calcitriol (the active vitamin D). Furthermore, calcitriol stimulates calcium uptake by osteoblasts of bone and promotes calcification or mineralization (deposition of calcium phosphate) and remodelling.

Parathyroid hormone (PTH) is secreted by two pairs of parathyroid glands that are closely associated with thyroid glands. Parathyroid hormone (mol. wt. 95,000) is a single chain polypeptide, containing 84 amino acids. It is originally synthesized as preproPTH which is degraded to proPTH and, finally, to active PTH. The rate of formation (by degradation of proPTH) and_ the secretion of PTH are promoted by low Ca2+ concentration. Thus, the release of PTH from parathyroid glands is under the negative feedback regulation of serum Ca?*.
Mechanism of action of PTH : PTH binds to a membrane receptor protein on the target cell and activates adenylate cyclase to liberate cAMP. This, in turn, increases intracellular calcium that promotes the phosphorylation of proteins (by kinases) which, finally brings about the biological actions. PTH has 3 independent tissues—bone, kidneys and intestine—to exert its action. The prime function of PTH is to elevate serum calcium level.
Action on the' bone PTH causes decalcification or demineralization of bone, a process carried out by osteoclasts. This is brought out by PTH stimulated increased activity of the enzymes pyrophosphatase and collagenase. These enzymes result in bone resorption. Demineralization ultimately leads to an increase in the blood Ca level. The action of PTH on bone is quantitatively very significant to maintain Ca homeostasis. It must, however, be noted that this is being done at the expense of loss of Ca from bone, particularly in dietary Ca deficiency.
Action on the kidney : PTH increases the Ca reabsorption by kidney tubules. This is the most rapid action of PTH to elevate blood Ca levels. However, quantitatively, this is less important compared to the action of PTH on bone. PTH promotes the production of calcitriol (1,25 DHCC) in the kidney by stimulating 1-hydroxylation of 25-hydroxycholecalciferol.
Action on the intestine : The action of PTH on the intestine is indirect. It increases the intestinal absorption of Ca by promoting the synthesis of calcitriol.

Calcitonin is a peptide containing 32 amino acids. It is secreted by parafollicular cells of thyroid gland. The action of CT on calcium metabolism is antagonistic to that of PTH. Thus,
calcitonin promotes calcification by increasing the activity of osteoblasts. Further, calcitonin decreases bone reabsorption and increases the excretion of Ca into urine. CT, therefore, has a decreasing influence on blood calcium.

The ratio of plasma Ca : P is important for calcification of bones. The product of Ca x P (in mg/dl) in children is around 50 and in adults around 40. This product is less than 30 in rickets.

Calcium is excreted partly through the kidneys and mostly through the intestine. The renal threshold for serum Ca is 10 mg/dl. Calcium gets excreted into urine beyond this concentration.
Excretion of Ca into the feces is a continuous process and this is increased in vitamin D deficiency.

The teeth calcium is not subjected to regulation as observed for bone calcium. Thus the adult teeth, once formed, do not undergo decalcification to meet the body needs of calcium. However, proper calcification of teeth is important in the growing children.

The blood Ca level is maintained within a narrow range by the homeostatic control, most predominantly by PTH. Hence abnormalities in Ca metabolism are mainly associated with alterations in PTH.

Elevation in serum Ca level (normal 9-11 mg/dl) is hypercalcemia. Hypercalcemia is associated with hyperparathyroidism caused by increased activity of parathyroid — glands. Decrease in serum phosphate (due to increased renal losses) and increase in alkaline phosphatase activity are also found in hyperparathyroidism. Elevation in the urinary excretion of Ca and P, often resulting in the formation of urinary calculi, is also observed in these patients.
The determination of ionized serum calcium (elevated to 6-9mg/dl) is more useful for the diagnosis of hyperparathyroidism. It has been observed that some of the patients may have normal levels of total calcium in the serum but differ with regard to ionized calcium.
The symptoms of hypercalcemia include lethargy, muscle weakness, loss of appetite, constipation, nausea, increased myocardial contractility and susceptibility to fractures.

Hypocalcemia is a more serious and life threatening condition. It is characterized by a fall in the serum Ca to below 7 mg/dl, causing tetany. The symptoms of tetany include neuromuscular irritability, and convulsions.
Hypocalcemia is mostly due to hypoparathyroidism. This may happen after an accidental surgical removal of parathyroid glands or due to an autoimmune disease.
Treatment : Supplementation of oral calcium with vitamin D is commonly employed. In severe cases of hypocalcemia, calcium gluconate is intravenously administered.

Rickets is a disorder of defective calcification of bones. This may be due to a low levels of vitamin D in the body or due to a dietary deficiency of Ca and P—or both. The concentrations of serum Ca and P may be low or normal. An increase in the activity of alkaline phosphatase is a characteristic feature of rickets.

Renal rickets is associated with damage to renal tissue, causing impairment in the synthesis of calcitriol. It does not respond to vitamin D in ordinary doses, therefore, some workers regard this as vitamin D resistant rickets. Renal rickets can be treated by administration of calcitriol.

Osteoporosis is characterized by demineralization of bone resulting in the progressive loss of bone mass.
Occurrence : The elderly people (over 60 yr.) of both sexes are at risk for osteoporosis. However, it more predominantly occurs in the post-menopausal women. Osteoporosis results in frequent bone fractures which are a major cause of disability among the elderly. It is estimated that more than 50% of the fractures in USA are due to this disorder. Osteoporosis may be regarded as a silent thief.
Etiology : The etiology of osteoporosis is largely unknown, but it is believed that several causative factors may contribute to it. The ability to produce calcitriol from vitamin D_ is decreased with age, particularly in the postmenopausal women. Immobilized or sedentary individuals tend to decrease bone mass while those on regular exercise tend to increase bone mass. Deficiency of sex hormones (in| women) has been implicated in the development of osteoporosis.
Treatment : Estrogen administration along with calcium supplementation (in combination with vitamin D) to postmenopausal women reduces the risk of fractures. Higher dietary intake of Ca (about 1.5 g/day) is recommended for elderly people.

Osteopetrosis is characterized by increased bone density. This is primarily due to inability to resorb bone. This disorder is mostly observed in association with renal tubular acidosis (due to a defect in the enzyme carbonic anhydrase) and cerebral calcification.

An adult body contains about 1 kg phosphate and it is found in every cell of the body. Most of it (about 80%) occurs in combination with Ca in the bones and teeth. About 10% of body P is found in muscles and blood in association with proteins, carbohydrates and _ lipids. The remaining 10% is widely distributed in various chemical compounds.

1. Phosphorus is essential for the development of bones and teeth.
2. It plays a central role for the formation and utilization of high-energy phosphate compounds e.g. ATP, GTP, creatine phosphate etc.
3. Phosphorus is required for the formation of phospholipids, phosphoproteins and nucleic acids (DNA and RNA).
4. It is an essential component of several nucleotide coenzymes e.g. NAD*, NADPt, pyridoxal phosphate, ADP, AMP.
- 5. Several proteins and enzymes are activated by phosphorylation.
6. Phosphate buffer system is important for the maintenance of pH in the blood (around 7.4) as well as in the cells.

The recommended dietary allowance (RDA) of phosphate is based on the intake of calcium. The ratio of Ca : P of 1 : 1 is recommended (i.e. 800 mg/day) for an adult. For infants, however, the ratio is around 2 : 1, which is based on the ratio found in human milk. Calcium and phosphate are distributed in the majority of natural foods in 1 : 1 ratio. Therefore, adequate intake of Ca generally takes care of the P requirement also.

Phosphate absorption occurs from jejunum
1. Calcitriol promotes along with calcium. phosphate uptake
2. Absorption of phosphorus and calcium is optimum when the dietary Ca : P is between 1:2 and 2:1.
3. Acidity favours while phytate decreases phosphate uptake by intestinal cells.

The phosphate level of the whole blood is around 40 mg/dl while serum contains about 34 mg/dl. This is because the RBC and WBC have very high content of phosphate.
The serum phosphate may exist as free ions (40%) or in a complex form (50%) with cations such as Ca2+, Mg2+, Nat, Kt. About 10% of serum phosphate is bound to proteins. It is interesting to note that the fasting serum phosphate levels are higher than the postprandial. This is attributed to the fact that following the ingestion of carbohydrate (glucose), the phosphate from the serum is drawn by the cells for metabolism (phosphorylation reactions).

About 500 mg phosphate is excreted in urine per day. The renal threshold is 2 mg/dl. The reabsorption of phosphate by renal tubules is inhibited by PTH.

1. Serum phosphate level is increased in hypoparathyroidism and decreased in hyperpara- thyroidism.
2. In severe serum content is elevated causing acidosis.
renal diseases, phosphate
3. Vitamin D deficient rickets is characterized
by decreased serum phosphate (1-2 mg/dl).
4. Renal rickets is associated with low serum phosphate levels and increased alkaline phosphatase activity.
5. In diabetes mellitus, serum content of organic phosphate is lower while that of inorganic phosphate is higher.
The adult body contains about 20 g magnesium, 70% of which is found in bones in combination with calcium and phosphorus. The remaining 30% occurs in the soft tissues and fluids.
body

1. Magnesium is required for the formation of bones and teeth.
2. Mg2* serves as a cofactor for several enzymes requiring ATP e.g. hexokinase, glucokinase, phosphofructokinase, adenylate cyclase.
3. Mg2* is necessary for proper neuromuscular function. Low Mg?t levels lead to neuromuscular irritability.

Cereals, nuts, beans, vegetables (cabbage, cauliflower), meat, milk, fruits.

Magnesium is absorbed by the intestinal cells through a specific carrier system. About 50% of the dietary Mg is normally absorbed. Consumption of large amounts of calcium, phosphate and = alcohol diminishes Mg absorption. PTH increases Mg absorption.

Normal serum concentration of Mg is 2-3 mg/ dl. It is present in the ionized form (60%), in combination with other ions (10%) and bound to proteins (30%).

1. Magnesium deficiency causes neuro- muscular irritation, weakness and convulsions.
These symptoms are similar to that observed in tetany (Ca deficiency) which are relieved only by Mg. Malnutrition, alcoholism and cirrhosis of liver may lead to Mg deficiency.
2. Low levels of Mg may be observed in uremia, rickets and abnormal pregnancy.
Sodium is the chief cation of the extracellular fluid. About 50% of body sodium is present in the bones, 40% in the extracellular fluid and the the soft remaining (10%) in tissues.

1. In association with chloride and bicarbonate, sodium regulates the body's acidbase balance.
- 2. Sodium is required for the maintenance of and fluid balance.
osmotic pressure
3. It is necessary for the normal muscle irritability and cell permeability.
- 4. Sodium is involved in the intestinal absorption of glucose, galactose and amino acids.
5. It is necessary for maintaining heart beat. initiating and

For normal individuals, the requirement of sodium is about 5-10 g/day which is mainly consumed as NaCl. For persons with a family history of hypertension, the daily NaCl intake should be less than 5 g. For patients of hypertension, around 1 g/day is recommended. It may be noted that 10 g of NaCl contains 4 g of sodium. The daily consumption of Na _ is generally higher than required due to its flavour.

The common salt (NaCl) used in the cooking medium
is the
major source
of sodium.
The ingested
foods also
contribute to
sodium.
The good
sources of
sodium include
bread, whole
grains, leafy vegetables, nuts, eggs and milk.

Sodium is readily absorbed in the gastrointestinal tract and, therefore, very little of it (< 2%) is normally found in feces. However, in diarrhea, large quantities of sodium is lost in feces.

In the plasma (serum), the normal concentration of sodium is 135-145 mEgq/I. Sodium is an extracellular cation, therefore, the blood cells contain much less (35 mEq/l). The mineralocorticoids, secreted by adrenal cortex, influence sodium metabolism. A decrease in plasma sodium and an increase in its urinary excretion are observed in adrenocortical insufficiency.

Kidney is the major route of sodium excretion from the body. As much as 800 g Na/day is filtered by the glomeruli, 99% of this is reabsorbed by the renal tubules by an active process. This is controlled by aldosterone. Extreme sweating also causes considerable amount of sodium loss from the body. There is, however, individual variation in sodium loss through sweat.

1. Hyponatremia : This is a condition in which the serum sodium level falls below the normal. Hyponatremia may occur due to diarrhea, vomiting, chronic renal diseases, adrenocortical insufficiency (Addison's disease). Administration of salt free fluids to patients may also cause hyponatremia. This is due to overhydration. Decreased serum sodium concentration is also observed in edema which occurs in cirrhosis or congestive heart failure.
The manifestations of hyponatremia include reduced blood pressure and circulatory failure.
2. Hypernatremia This condition — is characterized by an elevation in the serum sodium level. The symptoms include increase in
blood volume and blood pressure. It may occur due to hyperactivity of adrenal cortex (Cushing's syndrome), prolonged ~— administration _ of cortisone, ACTH and/or sex hormones. Loss of water from the body causing dehydration, as it occurs in diabetes insipidus, results in hypernatremia. Rapid administration _ of sodium salts also increases serum sodium concentration. It may be noted that in pregnancy, steroid and placental hormones cause sodium and water retention in the body, leading to edema.
In edema, along with water, sodium concentration in the body is also elevated. Administration of diuretic drugs increases the urinary output of water along with sodium. In the patients of hypertension and congestive cardiac failure salt (Na*) restriction is advocated.

Potassium is the principal intracellular cation. It is equally important in the extracellular fluid for specific functions.

- 2. It is required for the regulation of acid-
base balance and water balance in the cells.
3. The enzyme pyruvate kinase (of glycolysis)
is dependent on Kt for optimal activity.
of
4. Potassium is required for the transmission nerve impulse.
5. Adequate intracellular concentration K* is necessary for proper biosynthesis of proteins by ribosomes.
6. Extracellular K* influences cardiac muscle activity.

Banana, orange, pineapple, potato, beans, chicken, and liver. Tender coconut water is a rich source of potassium.

The absorption of K* from the gastrointestinal tract is very efficient (90%) and very little is lost through feces. However, in subjects with diarrhea, a good proportion of K* is lost in the feces.

The plasma (serum) concentration — of potassium is 3.4-5.0 mEq/l. The whole blood contains much higher level of Kt (50 mEq/l), since it is predominantly an intracellular cation. Care should, therefore, be taken to avoid hemolysis of RBC for the estimation of serum K*.

Potassium is mainly excreted through urine. The maintenance of body acid-base balance influences K* excretion. Aldosterone increases excretion of potassium.

Serum potassium concentration is maintained within a narrow range. Either high or low concentrations are dangerous since potassium effects the contractility of heart muscle.
Hypokalemia : Decrease in the concentration of serum potassium is observed due to overactivity of adrenal cortex (Cushing's syndrome), prolonged cortisone __ therapy, intravenous administration of Kt-free fluids, treatment of diabetic coma with — insulin, prolonged diarrhea and vomiting.
The symptoms of hypokalemia include irritability, muscular weakness, tachycardia, cardiomegaly and cardiac arrest. Changes in the ECG are observed (flattened waves with inverted T wave).
Hyperkalemia : Increase in the concentration of serum potassium is observed in renal failure,
adrenocortical insufficiency (Addison's disease), diabetic coma, severe dehydration, intravenous administration of fluids with excessive potassium salts.
The manifestations of hyperkalemia include depression of central nervous system, mental confusion, numbness, bradycardia with reduced heart sounds and, finally, cardiac arrest. Changes in ECG are also observed (elevated T wave).
Chlorine is a constituent of sodium chloride. Hence, the metabolism of chlorine and sodium are intimately related.

1. Chloride is involved in the regulation of acid-base equilibrium, fluid balance and osmotic pressure. These functions are carried out by the interaction of chloride with Na* and Kt.
2.
Chloride is
necessary for
the formation
of
HCI
in the
gastric juice.
- 3. Chloride shift involves the active participation of CF.
4. The enzyme salivary amylase is activated by chloride.

The daily requirement of chloride as NaCl is 5-10 g. Adequate intake of sodium will satisfy the chloride requirement of the body.

Common salt as cooking medium, whole grains, leafy vegetables, eggs and milk.

In normal circumstances, chloride is almost totally absorbed in the gastrointestinal tract.

The normal plasma concentration of chloride
is
95-105
mEq/l.
Cerebrospinal
fluid
(CSF)
contains higher level of Cl(125 mEq/l). This is due to the fact that protein content is low in CSF and, therefore, Clis higher in order to maintain Donnan membrane equilibrium.

There exists a parallel relationship between excretion of chloride and sodium. The renal threshold for Clis about 110 mEq/l.

1. Hypochloremia : A reduction in the serum Cl level may occur due to vomiting, diarrhea, respiratory alkalosis, Addison's disease and excessive sweating.
2. Hyperchloremia : An increase in serum Clconcentration may be due to dehydration, respiratory acidosis and Cushing's syndrome.
Sulfur of the body is mostly present in the organic form. Methionine, cysteine and cystine are the three sulfur-containing amino acids present in the proteins. Generally, proteins contain about 1% sulfur by weight.

1. Sulfur-containing amino acids are very essential for the structural conformation and biological functions of proteins (enzymes, hormones, structural proteins etc.). The disulfide linkages (-S—S—) and sulfhydryl groups (—SH) are largely responsible for this.
2. The vitamins thiamine, biotin, lipoic acid, and coenzyme A of pantothenic acid contain sulfur.
3. Heparin, chondroitin sulfate, glutathione, taurocholic acid are some other important sulfurcontaining compounds.
4. Phosphoadenosine phosphosulfate (PAPS) is the active sulfate utilized for several reactions e.g. synthesis of glycosaminoglycans, detoxification mechanism.
5. The
—
sulfur-containing
amino
—
acid
methionine (as S-adenosylmethionine) is actively
involved in transmethylation reactions.

There is no specific dietary requirement for sulfur. Adequate intake of sulfur-containing essential amino acid methionine will meet the body needs. Food proteins rich in methionine and cysteine are the sources of sulfur.

The sulfur from different compounds is oxidized in the liver to sulfate and excreted in urine. The urine contains inorganic sulfate (80%), organic or conjugated or ethereal sulfate (10%) and unoxidized sulfur (10%). The unoxidized sulfur is in the form of sulfurcontaining amino acids, thiocyanates etc.
The total content of iron in an adult body is 3-5. g. About 70% of this occurs in the erythrocytes of blood as a constituent of hemoglobin. At least 5% of body iron is present in myoglobin of muscle. Heme is the most predominant iron-containing substance. It is a constituent of several — proteins/enzymes (hemoproteins)—hemoglobin, myoglobin, cytochromes, xanthine oxidase, catalase, tryptophan pyrrolase, peroxidase. Certain other proteins contain non-heme iron e.g. transferrin, ferritin, hemosiderin.

1.
Iron
mainly
exerts
its
functions
through
the
compounds
in
which
it
is
present.
and
myoglobin
are
required
for
the
transport
of
Oy and CO).
2. Cytochromes and certain non-heme proteins are necessary for electron transport chain and oxidative phosphorylation.
3. Peroxidase, the lysosomal enzyme, is required for phagocytosis and killing of bacteria
by neutrophils.
4. Iron is associated with effective immuno- competence of the body.

Adult man
—
10 mg/day
—
18 mg/day
Pregnant and lactating woman — 40 mg/day
Menstruating woman

Rich sources
—
Organ
meats
(liver,
heart,
kidney).
Leafy vegetables, pulses,
cereals, fish, apples, dried
fruits, molasses.
Milk,
rice.
Good sources
—
Poor sources
—
wheat,
—
polished

Iron is mainly absorbed in the stomach and duodenum. In normal people, about 10% of dietary iron is usually absorbed. However, in iron deficient (anemic) individuals and growing children, a much higher proportion of dietary iron is absorbed to meet the increased body demands.
Iron is mostly found in the foods in ferric form (Fe3+), bound to proteins or organic acids. In the acid medium provided by gastric HCI, the Fe?+ is released from foods. Reducing substances such as ascorbic acid (vitamin C) and cysteine convert ferric iron (Fe?+) to ferrous form (Fe2*). Iron in the ferrous form is soluble and readily absorbed.

1. Acidity, ascorbic acid, alcohol,
fructose
and cysteine promote iron absorption.
2. In iron deficiency anemia, Fe absorption is
increased to 2-10 times that of normal.
3. Small peptides and amino acids favour
iron uptake.
4. Phytate (found in cereals) and oxalate (found in leafy vegetables) interfere with Fe absorption.
Fig. 18.3 : Iron absorption and transport (GIT—Gastrointestinal tract; Hb-Hemoglobin; Mb-Myoglobin; Cyts—Cytochromes; NHI-Non-heme iron).
5. A diet with high phosphate content decreases Fe absorption while low phosphate promotes.
6. Impaired absorption of iron is observed in malabsorption syndromes such as steatorrhea.
7. In patients with partial or total surgical removal of stomach and/or intestine, iron absorption is severely impaired.
Storage of iron : Iron is stored in liver, spleen and bone marrow in the form of ferritin. In the mucosal cells, ferritin is the temporary storage form of iron. A molecule of apoferritin (mol. wt. 500,000) can combine with 4,000 atoms of iron. The maximum iron content of ferritin on weight basis is around 25%.
Iron in the mucosal cells : The iron (Fe?*) entering the mucosal cells by absorption is oxidized to ferric form (Fe?+) by the enzyme ferroxidase. Fe?+ then combines with apoferritin to form ferritin which is the temporary storage form of iron. From the mucosal cells, iron may enter the blood stream (which mainly depends on the body needs) or lost when the cells are desquamated.
Transport of Fe in the plasma : The iron liberated from the ferritin of mucosal cells enters the plasma in ferrous state. Here, it is oxidized to ferric form by a copper-containing protein, ceruloplasmin which possesses _ ferroxidase activity. Another cuproprotein ferroxidase II also helps for the conversion of Fe2+ to Fe3*.

Ferric iron then binds with a specific ironbinding protein, namely transferrin — or siderophilin (a glycoprotein with mol. wt. 90,000). Each transferrin molecule can bind with two atoms of ferric iron (Fe**+). The plasma transferrin (concentration 250 mg/dl) can bind with 400 mg of iron/dl plasma. This is known as total iron binding capacity (TIBC) of plasma.
Hemosiderin is another iron storage protein which can hold about 35% of iron by weight. Hemosiderin accumulates in the body (spleen, liver) when the supply of iron is in excess of body demands.

Iron metabolism is unique as it operates in a closed system. It is very efficiently utilized and reutilized by the body. Further, iron losses from the body are minimal (<1 mg/day) which may occur through bile, sweat, hair loss etc. Iron is not excreted into urine. Thus, iron differs from the vitamins or other organic and inorganic substances which are either inactivated or excreted during the course of metabolic function. Hence, iron is appropriately regarded as a one-way substance.
Iron entry into the body is controlled at the absorption level, depending on the body needs. Thus the periodical blood loss in menstruating women increases its requirements. Increased iron demands are also observed in pregnancy, lactation, and in growing children.
are
Strict vegetarians
more prone for iron deficieny anemia.
This is due to the presence of inhibitors of iron absorption in the vegetarian foods, besides the relatively low content of iron.
Iron deficiency anemia mostly occurs in growing children, adolescent girls, pregnant and lactating women. It is characterized by microcytic hypochromic anemia with reduced blood hemoglobin levels (<12 g/dl). The other manifestations include apathy (dull and inactive), sluggish metabolic activities, retarded growth and loss of appetite.
Treatment : Iron deficiency is treated by supplementation of iron along wih folic acid and vitamin C.
2. Hemosiderosis : This is a less common disorder and is due to excessive iron in the body. It is commonly observed in subjects receiving repeated blood transfusions over the years, e.g. patients of hemolytic anemia, hemophilia. As already stated, iron is a one-way compound, once it enters the body, it cannot escape. Excessive iron is deposited as ferritin and hemosiderin.
Hemosiderosis is commonly observed among the Bantu tribe in South Africa. This is attributed to a high intake of iron from their staple diet corn and their habit of cooking foods in iron
pots.

3. Hemochromatosis : This is a rare disease in which iron is directly deposited in the tissues (liver, spleen, pancreas and skin). Hemosiderosis is sometimes accompanied by hemochromatosis. Bronzed-pigmentation of the skin, cirrhosis of liver, pancreatic fibrosis are the manifestations of this disorder. Hemochromatosis causes a condition known as bronze diabetes.

The body contains about 100 mg copper distributed in different organs. It is involved in several important functions.

1. Copper is an essential constituent of several enzymes. These include cytochrome oxidase, catalase, tyrosinase, — superoxide dismutase, monoamine oxidase, ascorbic acid oxidase, ALA synthase, phenol oxidase and
uricase. Due to its presence in a wide variety of enzymes, copper is involved in many metabolic reactions.
2. Copper is necessary for the synthesis of hemoglobin (Cu is a constituent of ALA synthase, needed for heme synthesis).
3. Lysyl oxidase (a copper-containing enzyme) is required for the conversion of certain lysine residues of collagen and elastin to allysine which are necessary for cross-linking these structural proteins.
4. Ceruloplasmin serves as ferroxidase and is involved in the conversion of iron from Fe2* to Fe3+ in which form iron (transferrin) is transported in plasma.
5. Copper is necessary for the synthesis of melanin and phospholipids.
6. Development of bone and nervous system (myelin) requires Cu.
7. Certain copper-containing non-enzymatic proteins have been identified, although their functions are not clearly known. These include hepatocuprein (storage form in liver), cerebrocuprein (in brain) and hemocuprein (in RBC).
8. Hemocyanin, a copper protein complex in invertebrates, functions like hemoglobin for O4 transport.

Liver, kidney, meat, egg yolk, cereals, nuts and green leafy vegetables. Milk is a poor source.

About 10% of dietary copper is absorbed, mainly in the duodenum. Metallothionein is a transport protein that facilitates | copper absorption. Phytate, zinc and molybdenum decrease copper uptake.

The copper concentration of plasma is about 100-200 mg/dl. Most of this (95%) is tightly bound to ceruloplasmin while a small fraction (5%) is loosely held to albumin. Normal concentration of serum ceruloplasmin is 25-50 mg/dl. It contains about 0.34% copper (6-8 atoms of Cu per molecule, half in Cu2* state and the other half in Cut state).

1. Copper deficiency : Severe deficiency of copper causes demineralization of bones, demyelination of neural tissue, anemia, fragility of arteries, myocardial fibrosis, hypopigmentation of skin, greying of hair.
2. Menke's disease : This disorder is due to a defect in the intestinal absorption of copper. It is possible that copper may be trapped by metallothionein in the intestinal cells. The symptoms of Menke's disease include decreased copper in plasma and urine, anemia and depigmentation of hair.
3. Wilson's disease (hepatolenticular degeneration) : It is a rare disorder (1 : 50,000) of abnormal copper metabolism and _ is characterized by the following manifestations.
e Copper is deposited in abnormal amounts in liver and lenticular nucleus of brain. This may lead to hepatic cirrhosis and brain necrosis.
e Low levels of copper and ceruloplasmin in plasma (reference range 20-50 mg/dl) with increased excretion of copper in urine.
e Copper deposition in kidney causes renal damage. This leads to increased excretion of amino acids, glucose, peptides and hemoglobin in urine.
e Intestinal absorption of copper is very high, about 4-6 times higher than normal.
Probable causes of Wilson's disease : The
following explanations are offered to understand the etiology of this disease.
- 1. A failure to synthesize ceruloplasmin or an
impairment in the binding capacity of copper to
this protein or both. As a result of this, copper is free in the plasma which easily enters the tissues (liver, brain, kidney), binds with the proteins and gets deposited. The albumin bound copper is either normal or increased.
2. A mutation in the gene encoding copper binding ATPase is believed to be responsible for Wilson's disease. Defect in this ATPase reduces intestinal excretion of Cu through bile.
Treatment : Administration of penicillamine, a naturally occurring copper chelating agent, is used for the treatment of Wilson's disease.
The total body contains about 20 mg iodine, most of it (80%) being present in the thyroid gland. Muscle, salivary glands and ovaries also contain some amount of iodine.

The only known function of iodine is its requirement for the synthesis of thyroid hormones namely, thyroxine (T,) and triiodothyronine (T3). These hormones are involved in several biochemical functions (Chapter 19). Functionally, T3 is more active than Ty.

Seafoods, drinking water, vegetables, fruits (grown on seaboard). High altitudes are deficient in iodine content in water as well as soil. Plant and animal foods of these areas, therefore, contain lesser amount of iodine. In these regions, iodine is added to drinking water or to table salt.

lodine as iodide is mainly absorbed from the
small
Normally,
about
30%
of
dietary
iodine
is
taken
up
by
the
intestinal
cells.
lodine
absorption also occurs through skin and lungs.
About 80% of body's iodine is stored in the organic form as iodothyroglobulin (a glycoprotein) in the thyroid gland. This protein contains thyroxine, — diiodotyrosine — and triiodothyronine in different proportions.
Excretion of iodine mostly occurs through kidney. It is also excreted through saliva, bile, skin, and milk (in lactating women).

The normal concentration of plasma iodine is 4-10 mg/dl. Most of this is present as protein bound iodine (PBI) and represents the iodine contained in the circulating thyroid hormones. PBI level decreases in hypothyroidism and increases in hyperthyroidism. RBC do not contain iodine.

The disorders of iodine metabolism—simple goiter and toxic goiter—are discussed in detail under thyroid hormones (Chapter 19).
The total body content of manganese is about 15 mg. The liver and kidney are rich in Mn. Within the cells, Mn is mainly found in the nuclei in association with nucleic acids.

as a cofactor for several include arginase, pyruvate isocitrate dehydrogenase, dismutase (mitochondrial) and 1. Mn_ serves enzymes. These carboxylase, superoxide peptidase.
2. Mn is required for the formation of bone, proper reproduction and normal functioning of nervous system.
3.
Mn
is
necessary
for
the
synthesis
of
mucopolysaccharides and glycoproteins.
4. Hemoglobin synthesis involves Mn.
5. Mn inhibits lipid peroxidation.
6. Mn is necessary for cholesterol biosynthesis.

The exact requirement of Mn is not known. About 2-9 mg/day is recommended for an adult.

is
Cereals, nuts, leafy vegetables and fruits. Tea a rich source of Mn.

About 3-4% of dietary Mn is normally absorbed in the small intestine. Iron inhibits Mn absorption.

Manganese in the serum is bound to a specific carrier protein—transmagnanin (a -globulin). The normal blood contains about 5-20 mg/dl.

Mn deficiency in animals causes
- 1. Retarded growth, bone deformities and, in
severe deficiency, sterility.
2. Accumulation of fat in liver.
3. Increased alkaline phosphatase, and activity of serum
4. Diminished activity of B-cells of pancreas (low insulin).
The total content of zinc in an adult body is about 2 g. Prostate gland is very rich in Zn (100 mg/g). Zinc is mainly an intracellular element.

1. Zn is an essential component of several enzymes e.g. carbonic anhydrase, alcohol dehydrogenase, alkaline phosphatase, carboxypeptidase, superoxide dismutase (cytosolic).
- 2. Zinc may be regarded as an antioxidant
since the enzyme superoxide dismutase (Zn containing) protects the body against free radical damage.
- 3. The storage and secretion of insulin from
the B-cells of pancreas require Zn.
- 4. Zn is necessary to maintain the normal levels of vitamin A in serum. Zn promotes the
synthesis of retinol binding protein.
5. It is required for wound healing. Zn enhances cell growth and division, besides stabilizing biomembranes.
6.
Gusten,
a
zinc containing
protein of
the saliva, is important for taste sensation.
- 7. Zn is essential for proper reproduction.

Zinc requirement for an adult is 10-15 mg/ day. It is increased (by about 50%) in pregnancy and lactation.

Zinc is absorbed mainly in the duodenum. Zn from the animal sources is better absorbed than the vegetable sources. Zn absorption appears to be dependent on a transport protein—metallothionein. Phytate, calcium, copper and _ iron interfere while small peptides and amino acids promote Zn absorption.

The concentration of Zn in serum is about 100 mg/dl. Erythrocytes contain higher content of Zn (1.5 mg/dl) which is found in association with the enzyme carbonic anhydrase.

1. Zinc deficiency is associated with growth retardation, poor wound healing, anemia, loss of appetite, loss of taste sensation, impaired spermatogenesis etc. It is reported that Zn deficiency in pregnant animals causes congenital malformations of the fetus. Deficiency of Zn may result in depression, dementia and other psychiatric disorders. The neuropsychiatric manifestations of chronic alcoholism may be partly due to zinc deficiency.
Acrodermatitis enteropathica is a_ rare inherited metabolic disease of zinc deficiency caused by a defect in the absorption of Zn from the intestine.
2. Zinc toxicity is often observed in welders due to inhalation of zinc oxide fumes. The manifestations of Zn toxicity include nausea, gastric ulcer, pancreatitis, anemia and excessive salivation.

Molybdenum is a constituent of the enzymes xanthine oxidase, aldehyde oxidase and sulfite oxidase. Nitrite reductase (containing Mo) is a plant enzyme, required for nitrogen fixation.
The requirements of Mo are not clearly known. However, it is widely distributed in the natural foods. Dietary Mo is effectively (60%-70%) absorbed by the small intestine.
Some workers have reported that Mo decreases the mobilization and utilization of copper in the body.
Molybdenosis is a rare disorder caused by excessive consumption of Mo. Its manifestations include impairment in growth, diarrhea and anemia. Intestinal absorption of copper is diminished.

Cobalt is only important as a constituent of vitamin By. Cobalt content of vitamin By) is about 4% by weight. The functions of cobalt are the same as that of vitamin B,) (Chapter 7). Administration of cobalt stimulates the production of the hormone erythropoietin, which promotes erythropoiesis.
Prolonged administration of cobalt is toxic as it results in polycythemia (increased RBC in blood).

Fluoride is mostly found in bones and teeth. The beneficial effects of fluoride in trace amounts are overshadowed by its harmful effects caused by excess consumption.

1. Fluoride prevents the development of dental caries. |t forms a protective layer of acid resistant fluoroapatite with hydroxyapatite of the enamel and prevents the tooth decay by bacterial acids. Further, fluoride inhibits the bacterial enzymes and reduces the production of acids.
2. Fluoride is necessary for development of bones. the proper
3. It inhibits the activities of certain enzymes. Sodium fluoride inhibits enolase (of glycolysis) while fluoroacetate inhibits aconitase (of citric acid cycle).

An intake of less than 2 ppm of fluoride will meet the daily requirements. Drinking water is the main source.

1. Dental caries : It is clearly established that drinking water containing less than 0.5 ppm of fluoride is associated with the development of dental caries in children (Refer Chapter 13).
2. Fluorosis : Excessive intake of fluoride is harmful to the body. An intake above 2 ppm (particularly >5 ppm) in children causes mottling of enamel and discoloration of teeth. The teeth are weak and become rough with characteristic brown or yellow patches on their surface. These manifestations are collectively referred to as dental fluorosis.
An intake of fluoride above 20 ppm is toxic and causes pathological changes in the bones. Hypercalcification, increasing the density of the bones of limbs, pelvis and spine, is a characteristic feature. Even the ligaments of spine
and collagen of bones get calcified. Neurological disturbances are also commonly observed. The manifestations described here constitute skeletal fluorosis. In the advanced stages, the individuals are crippled and cannot perform their daily routine work due to stiff joints. This condition of advanced fluorosis is referred to as genu valgum.
The fluoride content of water in some parts of Andhra Pradesh, Punjab and Karnataka is quite high. Fluorosis is prevalent in these regions, causing concern to government and_ health officials.
with fluoride toothpastes. There is some rethinking on these aspects due to the toxic effects of excess fluoride.
Selenium was originally identified as an element that causes toxicity to animals (alkali disease) in some parts of USA, containing large amounts of Se in the soil. Later work, however, has shown that Se in smaller amounts is biologically important.
3. Fluoridation of water and use of fluoride tooth-pastes : In order to prevent the dental caries in children, some advanced countries like USA have started fluoridation of water. Further, the consumer markets till recently were flooded

1. Selenium, along with vitamin E, prevents and the development of hepatic necrosis muscular dystrophy.

t= Serum calcium level is increased (normal 9-11 mg/dl) in hyperparathyroidism. This condition is also associated with elevated urinary excretion of Ca and P, often leading to stone formation.
ts Tetany, caused by a drastic reduction in serum Ca, is characterized by neuromuscular irritability and convulsions.
ts Rickets is due to defective calcification of bones. This may be caused by deficiency of Ca and P or vitamin D or both.
ts Osteoporosis is the bone disorder of the elderly, characterized by demineralization resulting in a progressive loss of bone mass. It is the major cause of bone fractures and disability in the old people.
t= Decreased levels of serum Na (hyponatremia) is observed in diarrhea and vomiting, besides Addison’s disease, while increased serum Na (hypernatremia) is found in Cushing’s syndrome.
ts Jron deficiency anemia is the most prevalent nutritional disorder worldover. It is most commonly observed in pregnant and lactating women.
ts Wilson’s disease is due to an abnormal copper metabolism. It is characterized by abnormal deposition of copper in liver and brain, besides the low levels of plasma copper and ceruloplasmin.
ts Endemic goitre, due to dietary iodine deficiency, is very common. Consumption of iodized salt is advocated to overcome this problem.
ts Fluorosis is caused by an excessive intake of fluoride. The manifestations include mottling of enamel and discoloration of teeth. In the advanced stages, hypercalcification of limb bones and ligaments of spine get calcified, ultimately crippling the individual.
2. Se is involved in maintaining structural integrity of biological membranes.
3. Se as selenocysteine is an_ essential component of the enzyme _— glutathione peroxidase. This enzyme protects the cells against the damage caused by HjOp. It appears from recent studies that selenocysteine is directly incorporated during protein biosynthesis. Therefore, selenocysteine is considered as a separate (21st) amino acid.
4. Se prevents lipid peroxidation and protects the cells against the free radicals, including superoxide (O).

5. Se protects animals from carcinogenic chemicals. However, the precise role of Se in humans with regard to cancer prevention is not clearly identified.
6. Se binds with certain heavy metals (Hg, Cd) and protects the body from their toxic effects.
7. A selenium containing enzyme 5'-deio-dinase_ converts thyroxine (T4) to triiodothyronine in the thyroid gland.
8. Thioredoxin reductase, involved in purine
nucleotide metabolism, is also a selenoprotein.

A daily intake of 50-200 mg of Se has been recommended for adults. The good sources of Se are organ meats (liver, kidney) and sea foods.

Deficiency : Se deficiency in animals leads to muscular dystrophy, pancreatic fibrosis and reproductive disorders. In humans, Keshan disease, an endemic cardiomyopathy (in China) is attributed to the deficiency of Se. Epidemiological studies reveal that low serum Se levels are associated with increased risk of cardiovascular disease, and various cancers.

Selenosis is the toxicity due to very excessive intake of Se. The manifestations of selenosis include weight loss, emotional disturbances, diarrhea, hair loss and garlic odor in breath. The compound dimethyl selenide is responsible for the garlic odor.
The total human body contains about 6 mg chromium. The Cr content of blood is about 20 mg/dl. Cr performs several — biochemical functions.
1. In association with insulin, Cr promotes the utilization of glucose. Cr is a component of a protein namely chromodulin which facilitates the binding of insulin to cell receptor sites.
2. Cr lowers the total serum cholesterol level.
3. It is involved in lipoprotein metabolism. Cr decreases serum low density lipoproteins (LDL) and increases high density lipoproteins (HDL) and, thus, promotes health.
4. It is believed that Cr participates in the transport of amino acids into the cells (heart and liver).
The dietary requirement of Cr is not known.
It is estimated that an adult man consumes about 10 to 100 mg/day. The good sources of Cr include brewer's yeast, grains, cereals, cheese and meat.
Chromium deficiency causes disturbances in carbohydrate, lipid and protein metabolisms. Excessive intake of Cr results in toxicity, leading to liver and kidney damage.

enzymes (e.g catalase, cytochrome oxidase, tyrosinase). Ceruloplasmin is a copper containing protein required for the transport of iron (Fe 3+ ) in the plasma. Wilson's disease is an abnormality in copper metabolism, characterized by the deposition of copper in liver , brain and kidney.. 9., 1 = Iodine is important as a component of thyroid hormones (T 4 and T 3 ) while cobalt is a constituent of vitamin B 12 . Zinc is necessary for the storage and secretion of insulin and maintenance of normal vitamin A levels in serum, besides being a component of several enzymes (e.g. carbonic anhydrase, alcohol dehydrogenase).. 10., 1 = Fluorine in trace amounts (<2 ppm) prevents dental caries while its higher intake leads to fluorosis. Selenium is assigned an antioxidant role as it protects the cells from free radicals. Chromium promotes the utilization of glucose and reduces serum cholesterol.
424

1. Write briefly on the trace elements and their metabolism in the body.
. Discuss the biochemical functions, dietary requirements, sources and absorption of calcium.
. Write an essay on the iron metabolism in the body. WN
. Describe the metabolism of copper, zinc and manganese.
a . Write on the biochemical importance and disease states of fluorine and selenium.

(a) Homeostasis of calcium, (b) Osteoporosis, (c) Phosphorus, (d) Sodium and_ chlorine, (e) Potassium, (f) Factors affecting Fe absorption, (g) Hemosiderosis, (h) Wilson’s disease, (i) lodine,
(j) Magnesium.

1. The normal concentration of serum calcium
2. The vitamin derived hormone that regulates calcium homeostasis
w . The inorganic
element
found
in
the
structure
of
majority
of
high-energy
compounds
. Several kinase enzymes require the mineral cofactor
. The principal cation of extracellular fluid nn
. The normal concentration of serum potassium
. lron is transported in the plasma in a bound form to a protein rmn
. The copper containing protein involved for the conversion of ferrous iron (Fe2*) to ferric iron (Fe3+) in the plasma
wo . The zinc containing protein in the saliva involved in taste sensation
10. The element involved in the protection of cells against the damage of H)O, and other free
radicals

11. The following substance(s) is(are) involved in the regulation of plasma calcium level
(a) Calcitriol (b) Parathyroid hormone (c) Calcitonin (d) All of them.
12. The following is a sulfur containing essential amino acid
(a) Methionine (b) Cysteine (c) Cystine (d) All of them.
13. Iron in the mucosal cells binds with the protein
(a) Transferrin (b) Ferritin (c) Ceruloplasmin (d) Hemosiderin.
14. The following element is involved in wound healing
(a) Calcium (b) Sodium (c) Zinc (d) Magnesium.
15. Pick up element that prevents the development of dental caries
(a) Fluorine (b) Calcium (c) Phosphorus (d) Sodium.

he
living
body
possesses
a
remarkable
system
to
coordinate
its
This
is
achieved
by
two
distinctly organized functional systems.
1. The nervous system coordinates the body
functions through the transmission of electro-
chemical impulses.
2.
The
endocrine
system
acts
through
a
wide
range
of
chemical
messengers
known
as
hormones.
Hormones are conventionally defined as organic substances, produced in small amounts by specific tissues (endocrine glands), secreted into the blood stream to control the metabolic and biological activities in the target cells. Hormones may be regarded as the chemical messengers involved in the transmission of information from one tissue to another and from cell to cell. The major endocrine organs in human body are depicted in Fig.19.1).
ction;

Hormones may be classified in many ways
based on their characteristics and functions. Two
types of classification are discussed here
I.

The hormones can be categorized into three groups considering their chemical nature.
1. Protein or peptide hormones e.g. insulin,
glucagon, antidiuretic hormone, oxytocin.
2.
Steroid
hormones
e.g.
glucocorticoids,
mineralocorticoids, sex hormones.
3.
Amino
acid
derivatives
e.g.
epinephrine,
norepinephrine,
thyroxine
(T,),
triiodothyronine
(Ts).
Il.
Based
on
the
mechanism
of
action
Hormones
are
classified
into
two
broad
groups (I and Il) based on the location of the
Fig. 19.1 : Diagrammatic representation of major endocrine glands.
receptors to which they bind and the signals used to mediate their action.
1. Group I hormones : These hormones bind to intracellular receptors to form receptorhormone complexes (the intracellular messengers) through which their biochemical functions are mediated. Group | hormones are lipophilic in nature and are mostly derivatives of cholesterol (exception—T, and T,). e.g. estrogens, androgens, glucocorticoids, calcitriol.
2. Group II hormones : These hormones bind to cell surface (plasma membrane) receptors and

These hormones are lipophilic in nature and can easily pass across the plasma membrane. They act through the intracellular receptors located either in the cytosol or the nucleus. The hormone-receptor complex binds to specific regions on the DNA called hormone responsive element (HRE) and causes increased expression of specific genes (Fig.19.2). It is believed that the interaction of hormone receptor complex with HRE promotes initiation and, to a lesser extent, elongation and termination of RNA synthesis (transcription). The ultimate outcome is the production of specific proteins (translation) in response to hormonal action.

These hormones are considered as the first messengers. They exert their action through mediatory molecules, collectively called second messengers.

Origin
Major Function(s)
Group I. HORMONES THAT BIND TO INTRACELLULAR RECEPTORS, 1 = Group I. HORMONES THAT BIND TO INTRACELLULAR RECEPTORS. Group I. HORMONES THAT BIND TO INTRACELLULAR RECEPTORS, 2 = Group I. HORMONES THAT BIND TO INTRACELLULAR RECEPTORS. Estrogens, 1 = Ovaries and adrenal cortex Female sexual characteristics, menstrual cycle.. Estrogens, 2 = Ovaries and adrenal cortex Female sexual characteristics, menstrual cycle.. Progestins, 1 = Ovaries and placenta. Progestins, 2 = Involved in menstrual cycle and maintenance of pregnancy.. Androgens, 1 = Testes and adrenal cortex. Androgens, 2 = Male sexual characteristics, spermatogenesis.. Glucocorticoids, 1 = Adrenal cortex. Glucocorticoids, 2 = Affect metabolisms, suppress immune system.. Mineralocorticoids, 1 = Adrenal cortex. Mineralocorticoids, 2 = Maintenance of salt and water balance.. Calcitriol (1, 25-DHCC), 1 = Kidney (final form). Calcitriol (1, 25-DHCC), 2 = Promotes absorption of Ca 2+ from intestine, kidney and bone.. Thyroid hormones (T 3 , T 4 ), 1 = Thyroid. Thyroid hormones (T 3 , T 4 ), 2 = Promote general metabolic rate.

Adrenocorticotropic hormone (ACTH)
Follicle stimulating hormone (FSH)
_—_
Luteinizing hormone (LH)
Chorionic gonadotropin (hCG)
Thyroid stimulating hormone (TSH)
B-Endorphins and enkephalins
Antidiuretic hormone (ADH)
Glucagon
Parathyroid hormone (PTH)
Calcitonin
Epinephrine
Anterior pituitary
Posterior pituitary (stored)
Pancreas
Parathyroid
Thyroid
Adrenal medulla
Norepinephrine

Thyrotropin-releasing hormone (TRH)
Hypothalamus
Gonadotropin-releasing hormone (GnRH)
Gastrin
Stimulates the release of adrenocorticosteroids.
In females, stimulates ovulation and estrogen synthesis.
In males, promotes spermatogenesis.
Stimulates synthesis of estrogens and progesterone and causes ovulation. Promotes androgen synthesis by testes.
Stimulates progesterone release from placenta.
Promotes the release of thyroid hormones (T,, T,).
Natural endogenous analgesics (pain relievers).
Promotes water reabsorption by kidneys.
Increases blood glucose level, stimulates glycogenolysis and lipolysis.
Increases serum calcium, promotes Ca** release from bone.
Lowers serum calcium. Decreases Ca** uptake by bone and kidney.
Increases heart
rate and
blood pressure.
Promotes glycogen-
olysis in liver and muscle and lipolysis in adipose tissue.
Stimulates lipolysis in adipose tissue.
Promotes TSH release.
Stimulates release of FSH and LH.
Stimulates gastric HCI and pepsinogen secretion.
Stomach
Cholecystokinin (CCK)
Stimulates contraction of gall bladder and secretion of pancreatic enzymes.
Intestine

Growth hormone (GH)
Prolactin (PRL)
Oxytocin
Stimulates uterine contraction and milk ejection.
Promotes growth of the body (bones and organs).
Growth of mammary glands and lactation.
Insulin
Pancreas
Lowers blood glucose (hypoglycemic effect), promotes protein
synthesis and lipogenesis.
Growth related functions of GH are mediated.
Stimulates growth of cartilage.
Somatomedins (insulin-like
Liver
growth factors, IGF-I, IGF-II)
Once produced,
cAMP
performs messenger
chemical its
role as
a
in eliciting
responses cAMP
activates
(A
stands second
bio-
(Fig.19.4).
protein for
cAMP).
kinase
A
tramer consisting
of
2
regulatory is
a
heterote- subunits
(R)
and
Fig. 19.2 : Mechanism of action of steroid hormones (H-Hormone; R-Receptor; HR-Hormone-receptor complex).
protein that ultimately causes the biochemical response.
2 catalytic subunits (C).
cAMP binds to inactive protein kinase and causes the dissociation of R and C subunits.
AcAMP
+
RyCy
—>
R2(4
cAMP)
+
2C
(inactive)
The active
subunit
(C)
catalyses
(active)
phosphory- lation
of proteins
(transfer of
phosphate group
to serine and threonine residues). It is the phospho-
It should, however, be remembered that cAMP does not act on all protein kinases. For instance, on protein kinase C (the second messenger is diacylglycerol).

enzymes called
protein phosphatases
hydrolyse and
remove proteins.
the phosphate
group added
to
Fig. 19.3 : Synthesis and degradation of cAMP.
Degradation of cAMP : cAMP undergoes rapid hydrolysis, catalysed by the enzyme phosphodiesterase to 5' AMP which is inactive. Hence, the effect of cAMP will be shortlived if the hormone stimulating adenylate cyclase is removed. Caffeine and theophylline (methylxanthine — derivatives) = can inhibit phosphodiesterases and increase the intracellular levels of cAMP.

The pituitary gland or hypophysis (weighing about 1 g) is located below the hypothalamus of the brain. It consists of two distinct parts— the anterior pituitary (adenohypophysis) and the posterior pitutitary (neurohypophysis) connected by pars intermedia (Fig.19.5). The latter is almost absent in humans, although found in lower organisms.
Hypothalamus is a specialized center in the brain that functions as a master coordinator of hormonal action. |n response to the stimuli of central nervous system, hypothalamus liberates certain releasing factors or hormones. These factors stimulate or inhibit the release of corresponding tropic hormones from the anterior pituitary. Tropic hormones stimulate the target endocrine tissues to secrete the hormones they synthesize. The relationship between hypothalamus and_ pituitary with endocrine glands is illustrated in Fig.19.6. In general, the hormonal system is under feedback control. For instance, adrenocorticotropic hormone (ACTH) inhibits the release of corticotropin releasing hormone (CRH).

Hypothalamus produces at least six releasing factors or hormones.
1. Thyrotropin-releasing hormone (TRH) : It is a tripeptide consisting of glutamate derivative (pyroglutamate), histidine and proline. TRH stimulates anterior pituitary to release thyroidstimulating hormone (TSH or thyrotropin) which, in turn, stimulates the release of thyroid hormones (T3 and Ty).
2. Corticotropin-releasing hormone (CRH) : It stimulates anterior pituitary to release adrenocorticotropic hormone (ACTH) which in turn, acts on adrenal cortex to liberate adrenocorticosteroids. CRH contains 41 amino acids.
3. Gonadotropin-releasing hormone (GnRH) : It is a decapeptide. GnRH stimulates anterior pituitary to release gonadotropins, namely luteinizing hormone (LH) and follicle stimulating hormone (FSH).
4. Growth hormone-releasing hormone (GRH) with 44 amino acids stimulates the release of growth hormone (GH or somatotropin) which promotes growth.
5. Growth hormone _ release-inhibiting hormone (GRIH) : It contains 14 amino acids and is also known as somatostatin. GRIH inhibits the release of growth hormone from the anterior pituitary.
Fig. 19.5 : A diagrammatic view of pituitary gland.

The growth hormone (or somatotropin) is produced by somatotropes, a special group of acidophilic cells of anterior pituitary.
Regulation of GH release : Two hypothalamic factors play a prominent role in the release of growth hormones. These are the growth hormonereleasing hormone (GRH) that stimulates and the growth hormone release-inhibiting hormone (GRIH, somatostatin) that inhibits. This, in turn, is regulated by a feedback mechanism.
Growth hormone production is influenced by many factors such as sleep, stress (pain, cold, surgery), exercise, food intake etc. It is observed that the largest increase in the production of GH occurs after the onset of sleep. This supports the adage "If you don't sleep, you won't grow."
Growth hormone (GH), prolactin (PRL) and chorionic somatomammotropin (CS; placental lactogen) are protein hormones with many striking similarities in their structure.
Biochemical functions of GH Growth hormone promotes growth, and also influences the normal metabolisms (protein, carbohydrate, lipid and mineral) in the body.
1. Effects on growth : As is obvious from the name, GH is essential for the growth. The growth-related effects of GH are mediated through insulin like growth factor | (IGF-I) which is also known as somatomedin C (formerly sulfation factor), produced by liver.
2. Effects on protein metabolism : Growth hormone has an anabolic effect on protein metabolism. It promotes the uptake of amino acids into the tissues and increases the protein synthesis. The overall effect of GH is a positive nitrogen balance that leads to increase in body weight.
3. Effects on carbohydrate metabolism Growth hormone is antagonistic to insulin and causes hyperglycemia. GH increases gluconeogenesis, decreases glucose utilization, impairs glycolysis and reduces the tissue uptake of glucose.
4. Effects on lipid metabolism : Growth hormone promotes lipolysis in the adipose tissue and increases the circulatory levels of free fatty acids and their oxidation. It increases ketogenesis, particularly in diabetes.
5. Effects on mineral metabolism : Growth hormone promotes bone mineralization and its growth, as clearly observed in the growing children.

Deficiency of GH Impairment in the secretion of growth hormone in the growing age causes dwarfism. The other deficiency metabolic effects are not that serious in nature.
Overproduction of GH : Excessive production of GH causes gigantism in children and acromegaly in adults. This usually occurs in the acidophil tumor of pituitary gland. Gigantism is characterized by increased growth of long bones and this is observed before the epiphyseal plates close. Acromegaly occurs after epiphyseal closure and is characterized by increase in the size of hands, facial changes (enlarged nose, protruding jaw), excessive hair, thickening of skin etc.

Prolactin (PRL) is also called lactogenic hormone, luteotropic hormone, mammotropin or luteotropin.
Biochemical functions of PRL : Prolactin is primarily concerned with the initiation and maintenance of lactation in mammals. PRL increases the levels of several enzymes involved in carbohydrate and lipid metabolism. PRL promotes HMP — shunt, — increases _ lipid biosynthesis and stimulates lactose production in mammary glands.
Prolactin promotes the growth of corpus luteum (hence also known as luteotropic hormone) and stimulates the production of progesterone.

in
The
following
four
hormones
are
nature
and
possess
certain
—
similarities,
despite
their
functional
diversity.
1. Thyroid stimulating hormone (TSH)
2. Follicle stimulating hormone (FSH)
3. Luteinizing hormone (LH)
4. Human chorionic gonadotropin (hCG).
The last three hormones (2-4) are collectively referred to as gonadotropins due to_ their involvement in the function of gonads. The hormone hCG is produced by human placenta and not by pituitary. However, due to its structural resemblance with other hormones, it is also considered here.
1. Thyroid stimulating hormone (TSH) : TSH is a dimer (a@B) glycoprotein with a molecular weight of about 30,000.
Regulation of TSH production : The release of TSH from anterior pituitary is controlled by a feedback mechanism. This involves the hormones of thyroid gland (T3 and Ty) and thyrotropinreleasing hormone (TRH) of hypothalamus.
Functions of TSH : The biochemical effects of TSH on thyroid gland are briefly discussed here. TSH binds with plasma membrane receptors and stimulates adenylate cyclase with a consequent increase in cAMP level. TSH, through the mediation of cAMP, exerts the following effects.
1 Promotes the uptake of iodide (iodide pump) from the circulation by thyroid gland.
1 Enhances the conversion of iodide (I-) to active iodide (I*), a process known as organification.
1 Increases the proteolysis of thyroglobulin to release T; and T, into the circulation.
TSH increases the synthesis of proteins, nucleic acids and phospholipids in thyroid gland.
Gonadotropins The _ follicle-stimulating hormone (FSH), luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are commonly known as gonadotropins. All three are glycoproteins.
The release of FSH and LH from the anterior pituitary is controlled by gonadotropin-releasing hormone (GnRH) of hypothalamus.

2. Biochemical functions of FSH : In females, FSH stimulates follicular growth, increases the weight of the ovaries and enhances the production of estrogens.
In males, FSH — stimulates testosterone production, required for spermatogenesis. FSH also promotes growth of seminiferous tubules.
3. Biochemical functions of LH : Luteinizing hormone stimulates the production of progesterone from corpus luteum cells in females and testosterone from Leydig cells in males. LH and FSH are collectively responsible for the development and maintenance of secondary sexual characters in males.
4. Human chorionic gonadotropin (hCG) : hCG is a glycoprotein (mol. wt. 100,000), produced by syncytiotrophoblast cells of placenta. The structure of hCG closely resembles that of LH.
The levels of hCG in plasma and urine increase almost immediately after the implantation of fertilized ovum. The detection of ACG in urine is conveniently used for the early detection (within a week after missing the menstrual cycle) of pregnancy.

This family consists of the hormones— adrenocorticotropic hormone (ACTH), lipotropin (LPH) and melanocyte stimulating hormone (MSH) and_ several (about 24) neuromodulators such as endorphins and enkephalins.
The synthesis of POMC family. is very interesting. All the members of POMC are produced from a single gene of the anterior and intermediate lobes of pituitary. It is fascinating that a single polypeptide—proopiomelanocortin—is the precursor (approximately 285 amino acids) that contains multiple hormones. The name pro-opiomelano-cortin is derived since it is a prohormone to opioids, melanocyte-stimulating hormone and_ corticotropin.
Products of POMC The _ pituitary multihormone precursor is synthesized as preproopiomelanocortin from = which POMC is formed. The POMC consists of 3 peptide groups.
1. ACTH that can give rise to MSH and corticotropin like intermediate lobe peptide (CLIP).
2. B-Lipotropin (B-LPH) that can produce y-LPH, B-MSH and f-endorphin. The latter yields yand a-endorphins.
3. An N-terminal peptide that forms y-MSH.
The products obtained from POMC are depicted in Fig.20.7. These products undergo many modifications such as_ glycosylation, acetylation etc.
1. Adrenocorticotropic hormone (ACTH) : ACTH is a polypeptide with 39 amino acids and a molecular weight of 4,500. This hormone is primarily concerned with the growth and functions of adrenal cortex.
Regulation of ACTH production : The release of ACTH from the anterior pituitary is under the regulation of hypothalamic hormone, namely corticotropin releasing hormone (CRH).
Biochemical functions of ACTH

1 ACTH promotes the conversion of cholesterol to I in th | pregnenolone in the adrena cortex.
. 1 It enhances RNA and protein synthesis and thus promotes adrenocortical growth.
: : : 1 ACTH increases lipolysis by activating lipase of adipose tissue.
Overproduction of
ACTH
:
Cushing’s syndrome
by an
excessive
ACTH
to is
caused production
which of
may
a
tumor.
be due
This syndrome
by hyper-
pigmentation and increased production of adrenocorticosteroids. The associated symptoms include negative nitrogen balance, impaired glucose tolerance, hypertension, edema, muscle atrophy etc.
2. B-Lipotropin (B-LPH) : B-LPH is derived from POMC and contains 93 carboxy terminal amino acids. This polypeptide consists of y-LPH and f-endorphin from which B-MSH and y-endorphin are, respectively, formed. y-Endorphin can be converted to a-endorphin and then to enkephalins (Fig.19.7). B-LPH is found only in the pituitary and not in other tissues since it is rapidly degraded.
is derived from POMC (Fig.19.7). B-Lipotropin has 31 amino acids while its modified products a and y-endorphins have 15 and 14 amino acids, respectively. Methionine enkephalin (Tyr—Gly— Gly—Phe—Me?) and leucine enkephalin (Tyr-GlyGly-Phe-Leu) are the two important pentapeptide derivatives of B-endorphin.
The biochemical functions of B-LPH, as such, are limited. It promotes lipolysis and increases the mobilization of fatty acids. The most important function of B-LPH is its precursor role for the formation of f-endorphin and enkephalins.
Endorphins and enkephalins : These are the natural analgesics that control pain and emotions. They were discovered after an unexpected finding of opiate receptors in the human brain.

Synthesis Endorphins and enkephalins are produced from f-endorphin which, in turn,
Biochemical actions Endorphins and enkephalins are peptide neurotransmitters that produce opiate-like effects on the central nervous system, hence they are also known as opioid-peptides. They bind to the same receptors as the morphine opiates and are believed to control the endogenous pain perception. Endorphins and enkephalins are more potent (20-30 times) than morphine in their function as analgesics.
It is believed that the pain relief through acupuncture and placebos is mediated through opioid peptides.
3. Melanocyte-stimulating hormone (MSH) : Three types of MSH (a, B and y) are present in the precursor POMC molecule. In humans, y MSH is important while in some animals a and 6B are functional. The activity of y-MSH_ is contained in the molecule y-LPH or its precursor B-LPH (Fig.19.7).
Fig. 19.8 : Structures of (A) Human oxytocin and (B) Human antidiuretic hormone (ADH).
The functions of MSH has been clearly established in some animals. MSH promotes the synthesis of skin pigment melanin (melanogenesis) and disperses melanin granules that ultimately leads to darkening of the skin. In humans, MSH does not appear to play any role in melanin synthesis.

Two hormones namely oxytocin and antidiuretic hormone (ADH, vasopressin) are produced by the posterior pituitary gland (neurohypophysis). Both of them are nonapeptides (9 amino acids). Their structures are depicted in Fig.19.8.

The release of oxytocin from posterior pituitary gland is caused by the neural impulses of nipple stimulation. The other — stimuli responsible for oxytocin release include vaginal and uterine distention.

1. Effect on uterus : Oxytocin causes the contraction of pregnant uterus (smooth muscles) and induces labor.
2. Effect on milk ejection : In mammals, oxytocin causes contraction of myoepithelial cells (look like smooth muscle cells) of breast. This stimulates the squeezing effect, causing milk ejection from the breast.
3. Oxytocin synthesized in the ovary appears to inhibit the synthesis of steroids.

The release of ADH (also called vasopressin) is mostly controlled by osmoreceptors (of hypothalamus) and baroreceptors (of heart). Any increase in the osmolarity of plasma stimulates ADH secretion.
Biochemical functions : ADH is primarily concerned with the regulation of water balance in the body. It stimulates kidneys to retain water and, thus, increases the blood pressure.
In the absence of ADH, the urine output would be around 20 I/day. ADH acts on the distal convoluted tubules of kidneys and causes water reabsorption with a result that the urine output is around 0.5-1.5 I/day.
Mechanism of action ADH stimulates adenylate cyclase causing production of cAMP. Water reabsorption is promoted by cAMP. Inhibitors of adenylate cyclase (e.g. calcium) inhibit the activity of ADH. This supports the view that ADH action is mostly mediated through cAMP.
Diabetes insipidus : This disorder is characterized by the excretion of large volumes of dilute urine (polyuria). It may be due to insufficient levels of ADH or a defect in the receptors of target cells.

Thyroid gland (weighs about 30 g in adults) is located on either side of the trachea below the larynx. It produces two principal hormones (Fig.19.9)—thyroxine (Ty; 3,5,3',5'-tetraiodothyronine) and 3,5,3'-triiodothyronine (T3)— which regulate the metabolic rate of the body. Thyroid gland also secretes calcitonin, a hormone concerned with calcium homeostasis (discussed under calcium metabolism, Chapter 18).

lodine is essential for the synthesis of thyroid hormones.
More than
half of
the body’s
total iodine content is found in the thyroid gland.
Uptake of iodide : The uptake of iodide by the thyroid gland occurs against a concentration gradient (about 20 : 1). It is an energy requiring process and is linked to the ATPase dependent Na*-K* pump. lodide uptake is primarily controlled by TSH. Antithyroid agents such as thiocyanate and perchlorate inhibit iodide transport.
Formation of active iodine : The conversion of iodide (I>) to active iodine (I*) is an essential step for its incorporation into thyroid hormones. Thyroid is the only tissue that can oxidize I to a higher valence state I*. This reaction requires H,O, and is catalysed by the enzyme thyroperoxidase (mol. wt. 60,000). An NADPH dependent system supplies HO>.
TSH promotes the oxidation of iodide to active iodine while the antithyroid drugs (thiourea, thiouracil, methinazole) inhibit.
Thyroglobulin and synthesis of T; and Ty, : Thyroglobulin (mol. wt. 660,000) is a glycoprotein and precursor for the synthesis of T3 and Ty. Thyroglobulin contains about 140 tyrosine residues which can serve as substrates for iodine for the formation of thyroid hormones.
Tyrosine (of thyroglobulin) is first iodinated at position 3 to form monoiodotyrosine (MIT) and then at position 5 to form diiodotyrosine (DIT). Two molecules of DIT couple to form thyroxine (T4). One molecule of MIT, when coupled with one molecule of DIT, triiodothyronine (T3) is produced. The mechanism of coupling is not well understood. The details of synthesis of T3 and Ty are given under tyrosine metabolism (Chapter 15). A diagrammatic representation is depicted in Fig.19.10.
As the process of iodination is completed, each molecule of thyroglobulin contains about 6-8 molecules of thyroxine (T4). The ratio of T3 to T4 in thyroglobulin is usually around 1 : 10.

Thyroglobulin containing T, and T3 can be stored for several months in the thyroid gland. It is estimated that the stored thyroid hormones can meet the body requirement for 1-3 months.
Thyroglobulin is digested by lysosomal proteolytic enzymes in the thyroid gland. The free hormones thyroxine (90%) — and triiodothyronine (10%) are released into the blood, a process stimulated by TSH. MIT and DIT produced in the thyroid gland undergo deiodination by the enzyme deiodinase and the iodine thus liberated can be reutilized.

Two specific binding proteins—thyroxine binding globulin (TBG) and thyroxine binding prealbumin (TBPA)—are responsible for the transport of thyroid hormones. Both Ty and T3 are more predominantly bound to TBG. A small fraction of free hormones are biologically active. Ty has a half-life of 4-7 days while T3 has about one day.
Fig.
19.10
:
Biosynthesis of
thyroid hormones—
diagrammatic representation
synthesis
[Note
:
Refer
Fig.
with structures;
15.21
Tgb—Thyroglobulin;
iodine;
T-Triiodothyronine;
for
I*—Active
T,-Thyroxine;
MIT-Monoi- odotyrosine;
DIT—Diiodotyrosine;
A.
As—Amino acids].

Triiodothyronine (T3) is about four times more active in its biological functions than thyroxine (T4). The following are the biochemical functions attributed to thyroid hormones (T3 and T,).
1. Influence on the metabolic rate : Thyroid hormones stimulate the metabolic activities and increases the oxygen consumption in most of the tissues of the body (exception—brain, lungs, testes and retina).
Na*-K* ATP pump : This is an energy dependent process which consumes a major share of cellular ATP. Na*-K* ATPase activity is directly correlated to thyroid hormones and this, in turn, with ATP utilization. Obesity in some individuals is attributed to a decreased energy utilization and heat production due to diminished Nat-Kt ATPase activity.
2. Effect on protein synthesis : Thyroid hormones act like steroid hormones in promoting protein synthesis by acting at the transcriptional level (activate DNA to produce RNA). Thyroid hormones, thus, function as anabolic hormones and cause positive nitrogen balance and promote growth and development.
3. Influence on carbohydrate metabolism : Thyroid hormones promote intestinal absorption of glucose and its utilization. These hormones increase gluconeogenesis and_ glycogenolysis, with an overall effect of enhancing blood glucose level (hyperglycemia).
4. Effect on lipid metabolism : Lipid turnover and utilization are stimulated by thyroid hormones. Hypothyroidism is associated with elevated plasma cholesterol levels which can be reversed by thyroid hormone administration.

The synthesis of thyroid hormones _ is controlled by feedback regulation (Fig.19.11). T3 appears to be more actively involved than Ty in the regulation process. The production of thyroid stimulating hormone (TSH) by pituitary, and thyrotropin releasing hormone (TRH) by hypothalamus are inhibited by T3 and, to a lesser degree, by Ty. The increased synthesis of TSH and TRH occurs in response to decreased circulatory levels of T3 and Ty. As already discussed, the body has sufficient stores of hormones to last for several weeks. Hence it takes some months to observe thyroid functional deficiency.

Thyroid hormones undergo deiodination in the peripheral tissues. The iodine liberated may be reutilized by the thyroid. T; and Ty may get conjugated with glucuronic acid or sulfate in the liver and excreted through bile. Thyroid hormones are also subjected to deamination to
produce
(from
T4)
tetraiodothyroacetic and
triiodothyroacetic
(from
T3)
which acid
acid may
then undergo
conjugation and

Among the endocrine glands, thyroid is the most susceptible for hypoor hyperfunction.
Three abnormalities
thyroid
Goiter functions
are
:
in the
size known
as
Any known.
abnormal of
thyroid goiter.
thyroid gland
compensate gland
is
Enlargement is
mostly the
of thyroid
associated decreased
of to
synthesis hormones
and with
Goiter
_
is elevated
is primarily
due in
the to
a
autoregulation synthesis.
of
This may
deficiency or
excess
Goitrogenic
(goitrogens)
substances that
These
TSH.
failure
T3;
and
Ty caused
by iodide.
substances
These interfere
are the
with the
thyroid hormones.
include thiocyanates,
and
perchlorates nitrates
and the
drugs such
as thiourea,
thiouracil, thiocarbamide
etc. Certain plant foods—cabbage, cauliflower and turnip—contain goitrogenic factors (mostly thiocyanates).
Simple endemic goiter : This is due to iodine deficiency in the diet. It is mostly found in the geographical regions away from sea coast where the water and soil are low in iodine content. Consumption of iodized salt is advocated to overcome the problem of endemic goiter. In certain cases, administration of thyroid hormone is also employed.
Hyperthyroidism : This is also known as thyrotoxicosis and is associated —_ with overproduction of thyroid hormones. Hyperthyroidism is characterized by increased metabolic rate (higher BMR) nervousness, irritability, anxiety, rapid heart rate, loss of weight despite increased appetite, weakness, diarrhea, sweating, sensitivity to heat and often protrusion of eyeballs (exopthalmos).
Hyperthyroidism is caused by Grave's disease (particularly in the developed countries) or due to increased intake of thyroid hormones. Grave's disease is due to elevated thyroid stimulating IgG also known as long acting thyroid stimulator (LATS) which activates TSH and, thereby, increases thyroid hormonal production.

Thyrotoxicosis is diagnosed by scanning and/ or estimation of T3, T4 (both elevated) and TSH (decreased) in plasma. The treatment includes administration of antithyroid drugs. In severe cases, thyroid gland is surgically removed.
be of
Hypothyroidism This is due to an impairment in the function of thyroid gland that often causes decreased circulatory levels of T3 and Ty. Disorders of pituitary or hypothalamus also contribute to hypothyroidism. Women are more susceptible than men. Hypothyroidism is characterized by reduced BMR, slow heart rate, weight gain, sluggish behaviour, constipation, sensitivity to cold, dry skin etc.
Hypothyroidism in children is associated with physical and mental retardation, collectively known as cretinism. Early diagnosis and proper treatment are essential. Hypothyroidism in adult causes myxoedema, characterized by bagginess under the eyes, puffiness of face, slowness in physical and mental activities.
to
Thyroid hormonal administration is employed treat hypothyroidism.

Measurement of basal metabolic rate (BMR) was once used to reflect thyroid activity. The estimation of serum protein bound iodine (PBI), representing the circulating thyroid hormones, was employed for a long time to assess thyroid function. The normal serum PBI concentration is 3-8 ug/100 ml.
Hypothyroidism is associated with decreased
PBI and hyperthyroidism with increased PBI.
In recent years, more sensitive and reliable tests have been developed to assess thyroid activity. The concentration of free T3 and Ty, and TSH are measured (by RIA or ELISA) and their serum normal concentrations are
Free triiodothyronine
(T,;)
—
80-220
ng/dl
Free thyroxine (T4)
Total thyroxine (T,)
—
—
—
0.8-2.4
ng/dl
5-12
ug/dl
<10
pU/ml
Radioactive iodine
uptake
(RAIU)
and scanning of thyroid gland are also used for diagnosis.
Fig. 19.12 : Adrenal gland with zones (3) and medulla.

Serum cholesterol level is increased in hypothyroidism and decreased in hyperthyroidism. Unfortunately, cholesterol estimation will be of no value in the assessment of thyroid function. This is due to the fact that serum cholesterol level is elevated in many other disorders (diabetes, obstructive jaundice, nephrotic syndrome etc.). However, cholesterol estimation may be utilized for monitoring thyroid therapy.

The adrenal glands are two small organs (each weighing about 10 g), located above the kidneys. Each adrenal consists of two distinct tissues—an outer cortex (with 3 zones) and inner medulla (Fig.19.12).
As many as 50 steroid hormones (namely adrenocorticosteroids), produced by adrenal cortex, have been identified. However, only a few of them possess biological activity.
Adrenocorticosteroids are classified into three groups according to their dominant biological action. However, there is some overlap in their functions.
1. Glucocorticoids : These are 21-carbon steroids, produced mostly by zona fasciculata. They affect glucose (hence the name), amino acid and fat metabolism in a manner that is opposite to the action of insulin. Cortisol (also known as hydrocortisone) is the most important glucocorticoid in humans. Corticosterone is predominantly found in rats.
2. Mineralocorticoids : These are also 21- carbon containing steroids produced by zona glomerulosa. They regulate water and electrolyte balance. Aldosterone is the most prominent mineralocorticoid.
3. Androgens and estrogens : The innermost adrenal cortex zona reticularis produces small quantities of androgens (19-carbon) and estrogens (18-carbon). These hormones affecting sexual development and functions are mostly produced by gonads. Dehydroepiandrosterone— a precursor for androgens—is synthesized in adrenal cortex.

Cholesterol undergoes cleavage with an elimination of a 6-carbon fragment to form pregnenolone. Pregnenolone is the common precursor for the synthesis of all steroid hormones.
Conversion of cholesterol to pregnenolone is catalysed by cytochrome P4s59 side chain cleavage enzyme. This reaction is promoted by ACTH. The enzymes—hydroxylases, dehydrogenases/ isomerases and lyases associated with mitochondria or endoplasmic reticulum—are responsible for the synthesis of steroid hormones. The metabolic pathway for the formation of major adrenocorticosteroids is given in Fig.19.13.

1. Glucocorticoid hormones : The important glucocorticoids are—cortisol, cortisone and corticosterone. They bring about several biochemical functions in the body.
(a)
Effects
on
Glucocorticoids
promote
the
synthesis
of
glucose
(gluconeogenesis).
This
is
brought
about
by
increasing
the
amino
acids)
and
enhancing
the
synthesis
of
phosphoenolpyruvate
carboxykinase,
the
rate
limiting
enzyme
in
gluconeogenesis.
The overall influence of glucocorticoids on carbohydrate metabolism is to increase
blood glucose concentration. | The biological actions of glucocorticoids generally oppose that of insulin.
(b)
Effects
on
lipid
:
Glucocor-
ticoids
increase
the
free
fatty
acids.
This
is
caused
by
two
(i) Increased breakdown of storage triacyl- glycerol (lipolysis) in adipose tissue.
(ii)
Reduced
utilization
of
plasma
free
fatty
acids
for
the
synthesis
of
triacylglyce-
rols.
(c) Effects on protein and nucleic acid metabolism Glucocortiocoids exhibit both catabolic and anabolic effects on protein and nucleic acid metabolism. They promote transcription (RNA synthesis) and protein biosynthesis in liver. These anabolic effects of glucocorticoids are caused by the stimulation of specific genes.
Glucocorticoids (particularly at high concentration) cause catabolic effects in extrahepatic tissues (e.g. muscle, adipose tissue, bone etc.). This results in enhanced degradation of proteins.

Effects on water and electrolyte meta- bolism : The influence of glucocorticoids on water metabolism is mediated through antidiuretic hormone (ADH). Deficiency of glucocorticoids causes increased production of ADH. ADH decreases glomerular filtration rate causing water retention in the body.
Effects
on
the
immune
system
:
Gluco-
corticoids
cortisol),
in
high
doses,
suppress
the
host
immune
response.
The
steroid
hormones
act
at
different
levels—damaging
lymphocytes,
of
antibody
—
suppression
of
inflammatory
response
etc.
(f) Other physiological effects of glucocor- ticoids : Glucocorticoids are involved in several physiological functions.
(i) Stimulate the fight and flight response (to face sudden emergencies) of catecholamines.
- (ii) Increase the production of gastric HCI and pepsinogen.
(iii)
Inhibit the
bone formation,
hence the
subjects are
at
a
risk for
osteoporosis.

: Glucocorticoids bind to specific receptors on the target cells and bring about the action. These hormones mostly act at the transcription level and control the protein synthesis.
2. Mineralocorticoid hormones : The most active and potent mineralocorticoid _ is aldosterone. It promotes Na* reabsorption at the distal convoluted tubules of kidney. Nat retention is accompanied by corresponding excretion of Kt, H* and NH} ions.
Regulation of aldosterone synthesis : The production of aldosterone is regulated by different mechanisms. These include reninangiotensin, potassium, sodium and ACTH.
Mechanism of aldosterone action Aldosterone acts like other steroid hormones. It binds with specific receptors on the target tissue and promotes transcription and translation.

: steroid hormones are metabolized in the liver and excreted in urine as conjugates of glucuronides or sulfates.
The urine contains mainly two steroids— 17-hydroxysteroids and 17-ketosteroids—derived from the metabolism of glucocorticoids and mineralocorticoids. | Androgens — synthesized by gonads also contribute to the formation of 17-ketosteroids.
Urinary 17-ketosteroids estimated in the laboratory are expressed in terms of dehydroepiandrosterone and their normal excretion is in the range of 0.2-2.0 mg/day.

Addison's _ disease Impairment — in adrenocortical function results in Addison's disease. This disorder is characterized by decreased blood glucose level (hypoglycemia), loss of weight, loss of appetite (anorexia), muscle weakness, impaired cardiac function, low blood pressure, decreased Na* and increased K* level in serum, increased susceptibility to stress etc.
Cushing's syndrome Hyperfunction of adrenal cortex may be due to long term pharmacological use of steroids or tumor of adrenal cortex or tumor of pituitary. Cushing's syndrome is characterized by hyperglycemia (due to increased gluconeogenesis), fatigue, muscle wasting, edema, osteoporosis, negative nitrogen balance, hypertension, moon-face etc.

The adrenocortical function can be assessed by measuring plasma cortisol (5-15 g/dl at 9.00 AM), plasma ACTH, urinary 17-ketosteroids etc.

Adrenal medulla is an _ extension of sympathetic nervous system. It produces two important hormones—epinephrine (formerly adrenaline) and norepinephrine (formerly noradrenaline). Both these hormones are catecholamines since they are amine derivatives of catechol nucleus (dihydroxylated phenyl ring). Epinephrine is a methyl derivative of norepinephrine. Dopamine is another catecholamine, produced as an_ intermediate during the = synthesis of — epinephrine. Norepinephrine and dopamine are important neurotransmitters in the brain and autonomic nervous system. The structures of the three catecholamines are given in Fig.19.14.

The amino acid tyrosine is the precursor for the synthesis of catecholamines. The pathway is described under tyrosine metabolism (Chapter 15, Fig.15.22). Catecholamines are produced in response to fight, fright and flight. These include the emergencies like shock, cold, fatigue, emotional conditions like anger etc.

Catecholamines cause diversified biochemical effects on the body. The ultimate goal of their action is to mobilize energy resources and prepare the individuals to meet emergencies (e.g. shock, cold, low blood glucose etc.).
1.
Effects on
carbohydrate metabolism
:
Epinephrine norepinephrine
increase the
glycogen synthesis
and in
general degradation
of
(glycogenolysis), of
glucose
(gluconeogenesis)
and decrease
glycogen formation
(glycogenesis).
The overall effect of catecholamines is to elevate blood glucose levels and make_ it available for the brain and other tissues to meet the emergencies.
transferase (COMT) and monoamine oxidase (MAO), found in many tissues act on catecholamines. The metabolic products metanephrine and vanillylmandelic acid (VMA) are excreted in urine.
2. Effects on lipid metabolism Both epinephrine and norepinephrine enhance the breakdown of triacylglycerols (lipolysis) in adipose tissue. This causes increase in the free fatty acids in the circulation which are effectively utilized by the heart and muscle as fuel source.
The metabolic effects of catecholamines are mostly related to the increase in adenylate cyclase activity causing elevation in cyclic AMP levels (refer carbohydrate and lipid metabolisms for more details).
3. Effects on physiological functions : In general, catecholamines (most predominantly epinephrine) increase cardiac output, blood pressure and oxygen consumption. They cause smooth muscle relaxation in bronchi, gastrointestinal tract and the blood vessels supplying skeletal muscle. On the other hand, catecholamines stimulate smooth muscle contraction of the blood vessels supplying skin and kidney. Platelet aggregation is inhibited by catecholamines.

Catecholamines are rapidly inactivated and metabolized. The enzymes—catechol-O methyl-

Pheochromocytomas : These are the tumors of adrenal medulla. The diagnosis of pheochromocytoma is possible only when there is an excessive production of epinephrine and norepinephrine that causes severe hypertension. In the individuals affected by this disorder, the ratio of norepinephrine to epinephrine is increased. The measurement of urinary VMA (normal <8 mg/day) is helpful in the diagnosis of pheochromocytomas.

The gonads (testes in males, ovaries in females) perform closely related dual functions.
1. Synthesize sex hormones;
2. Produce germ cells.
The steroid sex hormones are responsible for growth, development, maintenance and regulation of reproductive system. Sex hormones are essentially required for the development of
germ cells.
- The sex hormones are categorized into three groups 1. Androgens or male sex hormones which are C-19 steroids. 2. Estrogens or female sex hormones which are C-18 steroids. Ring A of steroid nucleus is phenolic in nature and is devoid of C-19 methyl group. 3. Progesterone is a C-21 steroid produced during the luteal phase of menstrual cycle and also during pregnancy.

The male sex hormones or androgens are produced by the Leydig cells of the testes and to a minor extent by the adrenal glands in both the sexes. Ovaries also produce small amounts of androgens.

Cholesterol is the precursor for the synthesis of androgens. It is first converted to pregnenolone which then forms androstenedione by two pathways—either through progesterone or through 17-hydroxypregnenolone (Fig.19.15). Testosterone is produced from androstenedione. The production of androgens is under the control of LH and FSH.
Active form of androgen The primary product of testes is testosterone. However, the active hormone in many tissues is not testosterone but its metabolite dihydrotestosterone (DHT). Testosterone, on reduction by the enzyme 5 a-reductase, forms DHT. This conversion mostly occurs in the peripheral tissues. Some workers consider testosterone as a prohormone and dihydrotestosterone, the more potent form as the hormone.

1. Sex-related physiological functions : The androgens, primarily DHT and _ testosterone, influence :
1 Growth, development and maintenance of male reproductive organs. Sexual differentiation and secondary sexual
1 characteristics.
1 Spermatogenesis.
1 Male pattern of aggressive behavior.
2. Biochemical functions : Many specific biochemical effects of androgens that ultimately influence the physiological functions stated above are identified. Androgens are anabolic in nature.
| Effects on protein metabolism : Androgens promote RNA synthesis (transcription) and protein synthesis (translation). Androgens cause positive nitrogen balance and increase the muscle mass.
| Effects on carbohydrate and fat metabolisms : Androgens increase glycolysis fatty acid synthesis and citric acid cycle.
| Effects on mineral metabolism : Androgens promote mineral deposition and bone growth before the closure of epiphyseal cartilage.

Estrogens are predominantly — ovarian hormones, systhesized by the follicles and corpus luteum of ovary. These hormones are responsible for maintenance of menstrual cycle and reproductive process in women.

Estrogen synthesis occurs from the precursor cholesterol (Fig.19.15). Estrogens are produced by aromatization (formation of aromatic ring) of androgens. The ovary produces estradiol (E>) and estrone (E,) while the placenta synthesizes these two steroid hormones and estriol (E3). The synthesis of estrogens is under the control of LH and FSH.

- 1. Sex-related physiological functions : The
estrogens are primarily concerned with
1
Growth, development
and maintenance
female reproductive
organs.
Maintenance of menstrual cycles.
Development of female sexual characteristics.
of
1
1
19.15 : of steroid sex hormones from cholesterol
Fig. Biosynthesis
(Note : Male and female sex hormones are given together).
- 2. Biochemical functions : Estrogens are
involved in many metabolic functions.
. . . . Lipogenic effect Estrogens lipo-
| : increase genesis in adipose tissue and, for this reason, women have relatively more fat (about 5%) than men.
1 Hypocholesterolemic effect : Estrogens lower the plasma total cholesterol. The LDL fraction of lipoproteins is decreased while the HDL fraction is increased. This explains the low incidence of atherosclerosis and coronary heart diseases in the women during reproductive age.
1 Anabolic effect : Estrogens in general promote transcription and translation. The synthesis of many proteins in liver is elevated e.g. transferrin, ceruloplasmin.
| Effect on bone growth Estrogens like androgens promote calcification and bone growth. It is believed that decalcification of bone in the postmenopausal women leading to osteoporosis is due to lack of estrogens.
estrogens, the transhydrogenase activity is low. This results in the diversion of NADPH towards lipogenesis—causing obesity.

Progesterone is synthesized and secreted by corpus luteum and placenta. Progesterone, as such, is an intermediate in the formation of steroid hormones from cholesterol (See Fig.20.13). LH controls the production of progesterone.
- | Effect on transhydrogenase Transhydrogenase is an enzyme activated by estrogen. It is capable of transferring reducing equivalents from NADPH to NAD*. The NADH so formed can be oxidized. It is explained that in the women after menopause, due to deficiency of

1. Progesterone is essentially required for the
implantation of
fertilized ovum
of pregnancy.

+ Growth hormone deficiency causes dwarfism while its excessive production results in gigantism (in children) or acromegaly (in adults).
+ Identification of hCG in urine is employed for the early detection of pregnancy.
+ Cushing’s syndrome is due to overproduction of ACTH that results in the increased synthesis of adrenocorticosteroids. The symptoms of this syndrome include hypertension, edema and negative nitrogen balance.
+ Endorphins and enkephalins are the natural pain-killers in the brain. It is believed that the pain relief through acupuncture and placebos is mediated through these compounds.
+ Deficiency of ADH causes diabetes insipidus, a disorder characterized by excretion of large volumes of dilute urine (polyuria).
+ Thyroid hormones directly influence Nat — Kt ATP pump which consumes a major share of cellular ATP. Obesity in some individuals is attributed to decreased energy utilization (heat production) due to diminished Na* — K* ATPase activity.
+ Catecholamines are produced in response to fight, fright and flight. The ultimate goal of catecholamine function is to mobilize energy resources and prepare the individual to meet emergencies such as shock, cold, fatigue, anger etc.
+ Pheochromocytomas are the tumors of adrenal medulla, characterized by excessive production of epinephrine and norepinephrine, associated with severe hypertension.
+ Sex hormones are primarily responsible for growth, development, maintenance and regulation of reproductive system.
+ The low incidence of atherosclerosis and coronary heart disease in the women during reproductive age is due to estrogens.
Fig. 19.16 : Hormonal pattern in women during mestrual cycle (FSH-Follicle stimulating hormone; LH-Luteinizing hormone).
2. It promotes the growth of glandular tissue in uterus and mammary gland.
3. Progesterone increases the body temperature by 0.5-1.5 F°. The exact mechanism of this thermogenic effect is not clearly known. The measurement of temperature was used as an indicator for ovulation.

The occurrence of menstrual cycle is a good example of coordination among the hormonal functions. In humans, the menstrual cycle is under the control of FSH, LH, estrogens and progesterone. The cycle normally varies between 25 and 35 days in length, with a mean of 28 days. The menstrual cycle can be divided into two phases—follicular phase and luteal phase

1. Follicular phase : Follicular stimulating hormone (FSH) causes the development and maturation of ovarian follicles. As the follicle enlarges, estradiol progessively rises and reaches its peak value 24 hours before LH and FSH attain their respective maximum levels. LH surge or peak initiates ovulation-release of ovum from the ruptured follicles. The levels of progesterone are low during follicular phase
2. Luteal phase : After the ovulation occurs, the ruptured follicles form corpus luteum and start producing progesterone and estradiol. The predominant hormone of luteal phase is progesterone which prepares the endometrium of uterus for implantation of the fertilized ovum. LH maintains the corpus for a few days. In the absence of implantation, the corpus luteum regresses and sheds endometrium causing menstruation. And another new cycle begins.
The luteal phase is always fixed, with 14 + 2 days in length. The observed variations in the length of menstrual cycle are due to changes in the follicular phase. In case of implantation of the fertilized =ovum, human — chorionic gonadotropin (hCG) is produced by the cells of implanted early embryo. hCG stimulates corpus luteum to synthesize progesterone. This continues till the plancenta starts making high quantities of progesterone.

The menstrual cycles which begin in the women after puberty, continue till the age of 45-50 years. The cycles cease around this age which coincides with the loss of ovarian function. The progesterone and estrogen levels are very low in these women. However, the concentration of LH and FSH are elevated due to lack of feedback inhibition by estrogens.
Post-menopausal women are susceptible to two complications associated with insufficient levels of sex hormones.
1. Atrophy of secondary sex tissues : Mainly the epithelial tissue of vagina and lower urinary tract.
2. Osteoporosis : Decreased density of bones and increased susceptibility to fractures.

The digestion and absorption of nutrients (Chapter 8) is a complicated process which is regulated by the autonomic nervous system. This occurs in association with peptide hormones of gastrointestinal tract (GIT).
The specialized cells lining the GIT are responsible for the production of GIT hormones. Hence GIT may be considered as the largest mass of cells that secrete hormones. A large number of GIT hormones have been identified. However, only four GIT hormones have been well characterised.
1. Gastrin : This hormone contains 17 amino acids and is produced by gastric mucosa. It stimulates the secretion of gastric HCI and pepsinogen (proenzyme of pepsin). The release of gastrin is stimulated by vagus nerve of stomach and partially digested proteins. HCI and certain other hormones inhibit gastrin release.
2. Secretin : It is a 27-amino acid containing polypeptide and resembles glucagon in many ways. Secretin is synthesized by the mucosa of the upper small intestine. It is released in response to the presence of HCI in chyme in the duodenum which is passed on from the stomach. Secretin stimulates pancreatic cells to produce bicarbonate (HCO3) in order to neutralize HCI.
3. Cholecystokinin (CCK) : It contains 33 amino acids and is produced by the upper part of small intestine. The secretion of CCK is stimulated by the products of protein and lipid digestion, namely peptides, amino acids, monoor diacylglycerols, fatty acids and glycerol.
Cholecystokinin stimulates the contraction of gall bladder and increases the flow of bile into duodenum. It also promotes the secretion of digestive enzymes and HCO3 from pancreas.
4. Gastric inhibitory peptide (GIP) : It contains 43 amino acids and is produced by duodenal mucosa. The release of GIP is stimulated by the presence of glucose in the gut. The most important function of GIP is to stimulate the release of insulin from pancreas. This is evident from the fact that the plasma insulin level is elevated much before the increase in blood glucose. GIP also inhibits gastric HCI secretion, gastric motility and its emptying.

GIT hormones show certain — structural relations and may be considered under two families.
(i) Gastrin family : Some of the C-terminal amino acids are identical. This family includes gastrin and CCK.
(ii) Secretin family Secretin, GIP and glucagon are structurally related, hence may be considered under this family.
Besides the hormones described above, several other hormones (in hundreds!) from the GIT have been identified. These hormones are often known as candidate hormones, since their biological functions are yet to be precisely identified. The candidate hormones include vasoactive intestinal peptide (VIP), motilin, enteroglucagon, substance P, neurotensin, somatostatin and enkephalins.

Many of the GIT hormones have receptor sites specific for their action. At least two distinct mechanisms have been identified through which these hormones act.
1. Production of cAMP through the activation of adenylate cyclase e.g. secretin, VIP etc.
2. Stimulation of intracellular Ca2* usually mediated through the metabolism — of phosphatidylinositol e.g. gastrin , CCK.
Both these mechanisms ultimately influence the enzyme secretions/other biological effects.

Besides the hormones discussed above, there are a few other important hormones which are not referred to in this chapter. Insulin and glucagon are described under diabetes mellitus (Chapter 36) while parathyroid hormone and calcitonin' are discussed under calcium metabolism (Chapter 18) These hormones are not given here to avoid repetition.

1. Describe the role of second messengers in hormonal action.
. Write an account of the anterior pituitary hormones. wn
. Discuss in detail the synthesis and biochemical functions of thyroid hormones.
. Describe the hormones of adrenal cortex with special reference to glucocorticoids. BRB
a . Write briefly on the synthesis and biochemical functions of sex hormones.

(a) ‘G’-Proteins, (b) Inositol triphosphate, (c) Hypothalamic hormones, (d) ACTH, (e) Goiter,
(f) Epinephrine, (g) Cortisol, (h) Gastrin, (i) ADH, (j) Aldosterone.

1. The enzyme that catalyses the formation of cAMP from ATP is
. The inorganic ion that can act as a second messenger for certain hormones is . The endocrine organ responsible for the synthesis of trophic hormones is WN . The
compounds
that
produce
opiate-like
effects
on
the
central
nervous
system
are
. The enzyme that converts iodide (I>) to active iodine (I*) DU
. The most predominant mineralocorticoid synthesized by adrenal cortex
. The major urinary excretory product of catecholamines On . The male sex is converted
hormone,
testosterone,
to
a more active
form,
namely
. The precursor for the synthesis of steroid hormones wo
10. The gastrointestinal
hormone
that
increases
the
flow
of
bile
from
the
gall
bladder

11. Impairment in the synthesis of dopamine by the brain is a major causative factor for the disorder
(a) Parkinson’s disease (b) Addison’s disease (c) Cushing’s syndrome (d) Goiter.
12. One of the following hormones is an amino acid derivative
(a) Epinephrine (b) Norepinephrine (c) Thyroxine (d) All of them.
13. The most active mineralocorticoid hormone is
(a) Cortisol (b) Aldosterone (c) 11-Deoxycorticosterone (d) Corticosterone.
14. Name the hormone, predominantly produced in response to fight, fright and flight
(a) Thyroxine (b) Aldosterone (c) Epinephrine (d) ADH.
15. The hormone essentially required for the implantation of fertilized ovum and maintenance of
pregnancy
(a) Progesterone (b) Estrogen (c) Cortisol (d) Prolactin.
ach organ of the body has to perform its biochemical functions to keep the body, as a whole, in a healthy state. This is possible only when the cells of the organ are intact in structure and function. Any abnormality in the tissue, caused by exogenous or endogenous factors, will seriously impair the organ function which, in turn, influences the health of the organism.
Based on the functional capabilities of the organs, specific biochemical investigations have been developed in the laboratory, to assess their function. In this chapter, the biochemical investigations to assess the functioning of liver, kidney, stomach and pancreas are discussed. The tests to evaluate the function of endocrine organs are discussed elsewhere (Chapter 19).

Liver performs several diversified functions.
is the central organ of body's metabolism.
It

1. Metabolic functions : Liver actively participates in carbohydrate, lipid, protein, mineral and vitamin metabolisms.
2. Excretory functions : Bile pigments, bile salts and cholesterol are excreted in the bile into intestine.
3. Protective functions and detoxification : Kupffer cells of liver perform phagocytosis to eliminate foreign compounds. Ammonia _ is detoxified to urea. Liver is responsible for the metabolism of xenobiotics (detoxification).
4. Hematological _ functions Liver participates in the formation of blood (particularly in the embryo), synthesis of plasma proteins (including blood clotting factors) and destruction of erythrocytes.
5.
Storage functions
:
Glycogen, vitamins
A,
D
and liver.
By»
and trace
element iron
are stored
in

Hepatocellular damage may occur due to viruses (hepatitis A virus, hepatitis B virus), toxins (carbon — tetrachloride, aflatoxin), — alcohol, hepatocellular carcinoma, autoimmune hepatitis
etc.

The liver function tests (LFT) are the biochemical investigations to assess the capacity of the liver to carry out any of the functions it performs. LFT will help to detect the abnormalities and the extent of liver damage.
Two important facts should be borne in mind while carrying out LFT.
1. Liver is a large-size factory of safety. Therefore, it can perform many of its functions almost normally, despite the damage.
2. Selection of the right test is important in LFT. This is due to the fact that since liver participates in several functions, the function that is measured in LFT may not be the one that is adversely affected.
The major liver function tests may be classified as follows
1. Tests based on excretory function— Measurement of bile pigments, bile salts, bromosulphthalein.
2. Tests based on serum enzymes derived from liver—Determination of transaminases, alkaline phosphatase, 5’-nucleotidase, y-glutamyl- transpeptidase.
3.
Tests
based
on
metabolic
capacity—
Galactose
tolerance,
antipyrine
clearance.
4. Tests based on synthetic functions— Prothrombin time, serum albumin.
5. Tests based on detoxification—Hippuric acid synthesis.
This above list, contains the most important biochemical investigations to assess LFT. Among these, the commonly used tests are described in the following pages.

The important liver functions and the common plasma/serum markers for the impaired tas ew mmm ta mati FUE CRUE Cam ec Mm ire tC
Heme catabolism, Common plasma/serum marker(s) for impaired function = n Bilirubin. Enzymes, Common plasma/serum marker(s) for impaired function = n Alanine transaminase n Aspartate transaminase nJ -Glutamyltranspeptidase. Protein synthesis, Common plasma/serum marker(s) for impaired function = p Albumin n Prothrombin time. Protein catabolism, Common plasma/serum marker(s) for impaired function = n Urea n Ammonia. Lipid metabolism, Common plasma/serum marker(s) for impaired function = n Cholesterol n Triglycerides. Drug metabolism, Common plasma/serum marker(s) for impaired function = n Half-lives of drugs. Bile acid metabolism, Common plasma/serum marker(s) for impaired function = n Bile acids
functions are listed in Table 20.1. The most important markers namely, bilirubin, enzymes, albumin, prothrombin time and drug metabolism with special reference to jaundice and other liver diseases are described.

Bilirubin is a bile pigment, and is the excretory end product of heme degradation. It is conjugated in the liver to form bilirubin diglucuronide, and excreted in bile. The details of bilirubin metabolism are discussed elsewhere (Chapter 10).

The normal concentration of serum bilirubin is in the range of 0.2-1.0 mg/dl. Of this, the conjugated bilirubin (diglucuronide 75%; monoglucuronide 25%) is 0.2-0.4 mg/dl, while the unconjugated bilirubin is 0.2-0.6 mg/dl.

This is a simple test to measure the yellow colour of serum due to bilirubin. \t is rather crude and almost outdated. However, it is often useful for a rapid assessment of neonatal jaundice.

This is a specific reaction to identify the increase in serum bilirubin (above the reference level). Normal serum gives a negative van den Bergh reaction.
Mechanism of the reaction : van den Bergh reagent is a mixture of equal volumes of sulfanilic acid (in dilute HCI) and sodium nitrite. The principle of the reaction is that diazotised sulfanilic acid (in the above mixture) reacts with bilirubin to form a purple coloured azobilirubin.

: as such is insoluble in water while the conjugated bilirubin is soluble. van den Bergh reagent reacts with conjugated bilirubin and gives a purple colour immediately (normally within 30 seconds). This is referred to as a direct positive van den Bergh reaction. Addition of methanol (or alcohol) dissolves the unconjugated bilirubin which then gives the van den Bergh reaction (normally within 30 minutes) positive and this is referred to as indirect positive. If the serum contains both unconjugated and conjugated bilirubin in high concentration, the purple colour is produced immediately (direct positive) which is further intensified by the addition of alcohol (indirect positive). This type of reaction is known as van den Bergh reaction and jaundice : This reaction is highly useful in understanding the nature of jaundice. This is due to the fact that the type of jaundice is characterized by increased serum concentration of unconjugated bilirubin (hemolytic), conjugated bilirubin (obstructive) or both of them (hepatic). Therefore, the response of van den Bergh reaction can differentiate the jaundice as follows
Indirect positive
— Hemolytic jaundice
Direct positive
_—
Obstructive jaundice
Biphasic
—
Hepatic jaundice.

The conjugated bilirubin, being water soluble, is excreted in urine. This is in contrast to unconjugated bilirubin which is not excreted.
Bilirubin in urine can be detected by Fouchet's test or Gmelin's test.

Bromosulphthalein is a dye used to assess the excretory function of liver. \t is a non-toxic compound and almost exclusively excreted by the liver (through bile). BSP is administered intravenously (5 mg/kg body weight) and _ its serum concentration is measured at 45 min and at 2 hrs. In normal individuals, less than 5% of the dye is retained at the end of 45 min. Any impairment in liver function causes an increased retention of the dye. This test is quite sensitive to assess liver abnormality with particular reference to excretory function.

Liver cells contain several enzymes which may be released into the circulation in liver damage. Measurement of selected enzymes in serum is often used to assess the liver function. It must, however, be noted that there is no single enzyme that is absolutely specific to liver alone. Despite this fact, serum enzymes provide valuable information for LFT. Some of these enzymes are discussed hereunder.

The activities of two enzymes—namely serum glutamate pyruvate transaminase (SGPT; recently called as alanine transaminase—ALT) and serum glutamate oxaloacetate transaminase (SGOT; recently known as aspartate transaminase—AST) —are widely used to assess the liver function. ALT is a cytoplasmic enzyme while AST is found in both cytoplasm and mitochondria. The activity of these enzymes is low in normal serum (ALT 5-40 IU/l; AST 5-45 IU/l). Serum ALT and AST are increased in liver damage. However, alanine transaminase is more sensitive and reliable for the assessment of LFT.
The normal AST/ALT ratio is around 0.8. This ratio is increased (>2) in myocardial infarction, alcoholic hepatitis, and cirrhosis. AST/ALT ratio is decreased (i.e. ALT higher) in acute hepatocellular damage and cholestasis.

Alkaline phosphatase (ALP) is mainly derived from bone and liver (the cells lining the bile canaliculi). A rise in serum ALP (normal 3-13 KA units/dl), usually associated with elevated serum bilirubin is an indicator of biliary obstruction (obstructive/posthepatic jaundice). ALP is also elevated in cirrhosis of liver and hepatic tumors.
Liver is not the sole source of alkaline phosphatase. Therefore, its measurement has to be carefully viewed (along with others) before arriving at any conclusion. The liver and bone isoenzymes of ALP can be separated by electrophoresis.

This is a microsomal enzyme widely distributed in body tissues, including liver. Measurement of y-glutamy! transpeptidase (GGT) activity provides a sensitive index to asses liver abnormality. The activity of this enzyme almost parallels that of transaminases in hepatic damage. Serum GGT is highly elevated (normal 5-40 IU/I) in biliary obstruction and alcoholism. Further, several drugs (e.g. phenytoin) induce (liver synthesis) and increase this enzyme in circulation.

The serum activity of 5'-nucleotidase (normal 2-15 U/l) is elevated in hepatobiliary disease and this parallels ALP. The advantage with 5'-nucleotidase is that it is not altered in bone diseae (as is the case with ALP).

Serum
_
isocitrate isoenzymes
of lactate
dehydrogenase dehydrogenase
(LDH,
LDHs)
are also
useful in
LFT.

Very often, a combination of serum enzyme estimations (instead of a single one) is used for a better understanding of liver functions. For instance, a large increase in transaminases (particularly ALT) relative to a small increase in alkaline phosphatase indicates hepatocellular damage. On the other hand, a small increase in transaminases and a large increase of alkaline phosphatase shows biliary obstruction.
Jaundice (French : jaune—yellow) is characterized by yellow coloration of sclera (of eyes) and skin. This is due to the elevated serum bilirubin level, usually beyond 2 mg/dl (normal <1 mg/dl).
The metabolism of heme to produce bilirubin and its conjugated derivatives and the types of jaundice have already been described. The reader must refer this (Chapter 10) now. The biochemical changes and the related parameters for the differential diagnosis of the three types of jaundice (hemolytic, obstructive and hepatic) are given in Table 20.2.
In the Fig.20.1, the normal and abnormal bilirubin metabolism (along with the associated

Serum bilirubin, Hemolytic jaundice (preheptic jaundice) = Unconjugated bilirubin n. Serum bilirubin, Obstructive jaundice (posthepatic jaundice) = Conjugated bilirubin n. Serum bilirubin, Hepatic jaundice (Intrahepatic jaundice) = Both n. van den Bergh reaction, Hemolytic jaundice (preheptic jaundice) = Indirect positive. van den Bergh reaction, Obstructive jaundice (posthepatic jaundice) = Direct positive. van den Bergh reaction, Hepatic jaundice (Intrahepatic jaundice) = Biphasic. Serum enzymes, Hemolytic jaundice (preheptic jaundice) = ALT, AST and ALP o. Serum enzymes, Obstructive jaundice (posthepatic jaundice) = ALP nn , ALT and AST marginal n. Serum enzymes, Hepatic jaundice (Intrahepatic jaundice) = ALT and AST nn , ALP marginal n. Bilirubin in urine, Hemolytic jaundice (preheptic jaundice) = Not excreted. Bilirubin in urine, Obstructive jaundice (posthepatic jaundice) = Excreted. Bilirubin in urine, Hepatic jaundice (Intrahepatic jaundice) = Excreted. Urobilinogen in urine, Hemolytic jaundice (preheptic jaundice) = Excretion n. Urobilinogen in urine, Obstructive jaundice (posthepatic jaundice) = o or p. Urobilinogen in urine, Hepatic jaundice (Intrahepatic jaundice) = o or p
and and

Fig. 20.1 : Normal and abnormal bilirubin metabolism (A) Normal bilirubin metabolism (B) Alterations in bilirubin metabolism along with enzymes in three types of jaundice (Note : Colours indicate major changes; Red-changes in hemolytic jaundice; Green-changes in hepatic jaundice; Blue-changes in obstructive jaundice; Dotted lines indicate minor pathways; ALT—Alanine transaminase; AST—Aspartate transaminase; ALP—Alkaline phosphatase).
enzyme changes) are depicted. The major changes in the 3 types of jaundice are listed below
Fig.21.2. It may be noted that the transaminase activities (more predominantly ALT) are elevated much before the bilirubin starts increasing.
Hemolytic jaundice
Elevated serum
unconjugated bilirubin,
urinary excretion
of urobilinogen.
Obstructive jaundice Elevated serum conjugated bilirubin and increased activities of alkaline phosphatase (ALP), alanine transaminase (ALT) and aspartate transaminase (AST).
Hepatic jaundice Elevated serum unconjugated and conjugated bilirubin, and increased activities of ALT and AST.
The pattern
of rise
in the
serum alanine
transaminase, aspartate
bilirubin in
acute transaminase
viral hepatitis
and is

Galactose is a monosaccharide, almost exclusively metabolized by the liver. The liver function can be assessed by measuring the utilization of galactose. This is referred to galactose tolerance test. The subject is given intravenous administration of galactose (about 300 mg/kg body weight). Blood is drawn at 10 minute intervals for the next 2 hours and galactose estimated. In the normal individuals, the half-life of galactose is about 10-15 minutes. This is markedly elevated in hepatocellular damage (infective hepatitis, cirrhosis).
Fig. 20.2 : Pattern of rise in serum enzymes and bilirubin in viral hepatitis.

Albumin is solely synthesized by the liver. It has a half-life of about 20-25 days, therefore, it is a good marker to assess chronic (and not acute) liver damage. Low serum albumin is commonly observed in patients with severe liver damage. It must, however, be noted that the serum albumin concentration is also decreased due to other factors such as malnutrition.
Functional impairment of liver is frequently associated with increased synthesis of globulins. Cirrhosis of the liver causes a reversal of albumin/globulin ratio (A/G ratio). Serum electrophoresis of proteins reveals increased albumin and decreased y-globulin concentration. This, however, may not have much diagnostic importance since several diseases are associated with altered electrophoretic pattern of serum proteins.

The liver synthesizes all the factors concerned with blood clotting. A decrease in the concentration of plasma clotting factors is found in the impairment of liver function. This can be assessed in the laboratory by measuring prothrombin time which is prolonged in patients with liver damage, compared to normal. The half-lives of clotting factors are relatively short (5-72 hrs.), therefore, changes in prothrombin time occur quickly. Hence, this test is useful to assess acute as well as chronic liver damages; besides its help in the prognosis.
Vitamin K is required for the synthesis of blood clotting factors II, VII, IX and X. Therefore, vitamin K deficiency can also cause prolonged prothrombin time which must be ruled out, before drawing conclusions on the _ liver functions. This is done by measuring prothrombin time before and after administration of vitamin K.

The liver is the major site for the metabolism of xenobiotics (detoxification). Measurement of hippuric acid synthesis is an ideal test for assessing the detoxification function of liver. Hippuric acid is produced in the liver when benzoic acid combines with glycine.
About 6 g of sodium benzoate dissolved in (about 250 ml) water, is orally given to the subject, after a light breakfast (usually 2 hrs later) and after emptying the bladder. Urine collections are made for the next 4 hours and the amount of hippuric acid excreted is _ estimated. Theoretically, 6 g of sodium benzoate should yield 7.5 g of hippuric acid. In the healthy persons, about 60% of sodium benzoate (equivalent to 4.5 g hippuric acid) is excreted in urine. A reduction in hippuric acid excretion (particularly <3 g) indicates hepatic damage.

The choice of biochemical tests to measure liver functions mostly depends on the purpose of the investigation. The clinical history of the subject is often a guiding factor in this regard. A single test in isolation may have a little diagnostic value.
Frequently, a combination of laboratory investigations are employed in LFT. These include serum bilirubin (conjugated and

The kidneys
are the
vital organs
of the
body, performing
major functions.
Fig. 20.3 : Diagrammatic representation of a nephron.
2. Excretion of metabolic waste products : The end products of protein and nucleic acid metabolism are eliminated from the body. These include urea, creatinine, creatine, uric acid, sulfate and phosphate.

3. Retention of substances vital to body : The kidneys reabsorb and retain several substances of biochemical importance in the body e.g. glucose, amino acids etc.
4. Hormonal functions : The kidneys also function as endocrine organs by producing hormones.
. Erythropoietin, a peptide hormone, stimulates hemoglobin synthesis and formation of erythrocytes.
1 1,25-Dihydroxycholecalciferol (calcitriol) — the biochemically active form of vitamin D — is finally produced in the kidney. It regulates calcium absorption from the gut.
1 Renin, a proteolytic enzyme liberated by kidney, stimulates the formation of angiotensin Il which, in turn, leads to aldosterone production. Angiotensin II and aldosterone are the hormones involved in the regulation of electrolyte balance.
Nephron is the functional unit of kidney. Each kidney is composed of approximately one million nephrons. The structure of a nephron, as depicted in Fig.20.3, consists of a Bowman's capsule (with blood capillaries), proximal convoluted tubule (PCT), loop of Henle, distal convoluted tubule (DCT) and collecting tubule.
The blood supply to kidneys is relatively large. About 1200 ml of blood (650 ml plasma) passes through the kidneys, every minute. From this, about 120-125 ml is filtered per minute by the kidneys and this is referred to as glomerular filtration rate (GFR). With a normal GFR (120-125 ml/min), the glomerular filtrate formed in an adult is about 175-180 litres per day, out of which only 1.5 litres is excreted as urine. Thus, more than 99% of the glomerular filtrate is reabsorbed by the kidneys.
The process of urine formation basically involves two steps—glomerular filtration and tubular reabsorption.
1. Glomerular filtration : This is a passive process that results in the formation of ultrafiltrate of blood. All the (unbound) constituents of plasma, with a molecular weight
less than about 70,000, are passed into the filtrate. Therefore, the glomerular filtrate is almost similar in composition to plasma.

2. Tubular reabsorption : The renal tubules (PCT, DCT and collecting tubules) retain water and most of the soluble constituents of the glomerular filtrate by reabsorption. This may occur either by passive or active process. The excreted urine has an_ entirely different composition compared to glomerular filtrate from which it is derived. The normal composition of urine is given elsewhere (Refer inside backcover).

There are certain substances in the blood whose excretion in urine is dependent on their concentration. Such substances are referred to as renal threshold substances. At the normal concentration in the blood, they are completely reabsorbed by the kidneys, with a result that their excretion in urine is almost negligible.
The renal threshold of a substance is defined as its concentration in blood (or plasma) beyond which it is excreted into urine. The renal threshold for glucose is 180 mg/dl; for ketone bodies 3 mg/dl; for calcium 10 mg/dl and for bicarbonate 30 mEq/l. While calculating the renal threshold of a particular compound, it is assumed that both the kidneys are optimally functioning, without any abnormality. But this is not always true—in which case the renal threshold is altered. For instance, renal glycosuria is associated with reduced threshold for glucose due to_ its diminished tubular reabsorption.
The term tubular maximum (Tm) is used to indicate the maximum capacity of the kidneys to absorb a particular substance. For instance, tubular maximum for glucose (TmG) is 350 mg/min.

In view of the important and_ sensitive functions the kidney performs (described already), it is essential that the abnormalities (renal damages), if any, must be detected at the earliest. Several tests are employed in the laboratory to assess kidney (renal) function. It must, however, be remembered that about twothirds of the renal tissue must be functionally damaged to show any abnormality by these tests. The kidney function tests may be divided into four groups.
1. Glomerular function tests : All the clearance tests (inulin, creatinine, urea) are included in this group.
2. Tubular function tests : Urine concentration or dilution test, urine acidfication test.
3. Analysis of blood/serum : Estimation of blood urea, serum creatinine, protein and electrolyte are often useful to assess renal function.
4. Urine examination : Simple routine examination of urine for volume, pH, specific gravity, osmolality and presence of certain abnormal constituents (proteins, blood, ketone bodies, glucose etc.) also helps, of course to a limited degree, to assess kidney functioning.
Some of the important renal function tests are discussed in the following pages.

The clearance tests, measuring the glomerular filtration rate (GFR) are the most useful in assessing the renal function. The excretion of a substance can be expressed quantitatively by using the concept of clearance.
Clearance, in general, is defined as the volume of plasma that would be completely cleared of a substance per minute. |n other words, clearance of a substance refers to the milliliters of plasma which contains the amount of that substance excreted by kidney per minute. Clearance (C), expressed as ml/minute, can be calculated by using the formula
where U = Concentration of the substance in urine.
V =Volume of urine in ml excreted per minute.
P
=Concentration plasma.
of the
substance in
Care should be taken to express the concentrations of plasma and urine in the same units (mmol/I or mg/dl).
The clearance of a given substance is determined by its mode of excretion. The maximum rate at which the plasma can be cleared of any substance is equal to the GFR. This can be easily calculated by measuring the clearance of a plasma compound which is freely filtered by the glomerulus and is neither absorbed nor secreted in the tubule. Inulin (a plant carbohydrate, composed of fructose units) and 5!'Cr-EDTA satisfy this criteria. Inulin is intravenously administered to measure GFR.
In practice, however, measurement of clearance for the substances already present in the blood is preferred. The two compounds, namely creatinine and urea, are commonly employed for this purpose. Creatinine clearance (~145 ml/min) is marginally higher than the GFR as it is secreted by the tubules. On the other hand, urea clearance (~75 ml/min) is less than the GFR, since it is partially reabsorbed by the tubules.

Diodrast (diiodopyridone acetic acid) is used as a contrast medium to take urinary tract X-rays. Diodrast and para amino hippuric acid (PAH) are peculiar substances as they are entirely excreted by a single passage of blood through the kidneys. It is partly filtered by the glomerulus and mostly excreted by the tubules. PAH has a clearance of about 700 ml/min (or 1,200 ml, if expressed as blood). Thus clearance of PAH represents the renal plasma flow.

Creatinine is an excretory product derived from creatine phosphate (largely present in muscle). The excretion of creatinine is rather constant and is not influenced by body metabolism or dietary factors. As already stated, creatinine is filtered by the glomeruli and only marginally secreted by the tubules. The value of creatinine clearance is close to GFR, hence its measurement is a sensitive and good approach to assess the renal glomerular function. Creatinine clearance may be defined as the volume (ml) of plasma that would be completely cleared of creatinine per minute.
Procedure : In the traditional method, creatinine content of a 24 hr urine collection and the plasma concentration in this period are estimated. The creatinine clearance (C) can be calculated as follows :
where U = Urine concentration of creatinine
V
=
Urine output
in ml/min
(24
hr urine
volume divided
by
24
x
60)
- P = Plasma concentration of creatinine.
As already stated, creatinine concentration in urine and plasma should be expressed in the same units (mg/dl or mmol/l).
Modified procedure : Instead of a 24 hr urine collection, the procedure is modified to collect urine for 1 hr, after giving water. The volume of urine is recorded. Creatinine contents in plasma and urine are estimated. The creatinine clearance can be calculated by using the formula referred above.
Reference values : The normal range of creatinine clearance is around 120-145 ml/min. These values are slightly lower in women. In recent years, creatinine clearance is expressed in terms of body surface area.
Diagnostic importance A decrease in creatinine clearance value (<75% _ normal) serves as sensitive indicator of a decreased GFR, due to renal damage. This test is useful for an early detection of impairment in kidney function, often before the clinical manifestations are seen.

Urea is the end product of protein metabolism. After being filtered by the glomeruli, it is partially reabsorbed by the renal tubules. Hence, urea clearance is less than the GFR and, further, it is influenced by the protein content of the diet. For these reasons, urea clearance is not as sensitive as creatinine clearance for assessing renal function. Despite this fact, several laboratories traditionally use this test.
Diagnostic importance : A urea clearance value below 75% of the normal is viewed seriously, since it is an indicator of renal damage. Blood urea level as such is found to increase only when the clearance falls below 50% normal. As already stated, creatinine clearance is a better indicator of renal function.

This is a test to assess the renal tubular function. It is a simple test and involves the accurate measurement of specific gravity which depends on the concentration of solutes in urine. A specific gravity of 1.020 in the early morning urine sample is considered to be normal.
Several measures are employed to concentrate urine and measure the specific gravity. These include overnight water deprivation and administration of antidiuretic hormone. If the specific gravity of urine is above 1.020 for at least one of the samples collected, the tubular function is considered to be normal.
Fig. 20.4 : The relationship between glomerular filtration rate (GFR) and serum creatinine concentration.
Osmolality and specific gravity The osmolality of urine is variable. In normal individuals, it may range from 500-1,200 milliosmoles/kg. The plasma osmolality is around 300 milliosmoles/kg. The normal ratio of the osmolality between urine and plasma is around 2-4. It is found that the urine (without any protein or high molecular weight substance) with an osmolality of 800 mosm/kg has a specific gravity of 1.020. Therefore, measurement of urine osmolality will also help to assess tubular function.

Estimation of serum creatinine and blood urea are often used to assess the overall kidney function, although these tests are less sensitive than the clearance tests. Serum creatinine is a better indicator than urea in this regard. The diagnostic importance of urea and creatinine estimations are discussed elsewhere (Refer Chapter 15).
The relationship between GFR and serum creatinine levels is depicted in Fig.20.4. It is observed that the GFR must fall to about 50% of its normal value before a significant increase in serum creatinine occurs. Therefore, a normal
serum creatinine level does not necessarily mean that all is well with the kidney. It is estimated that a loss of 50% of the functions of nephrons leads to (approximate) doubling of serum creatinine concentration.
Cystatin C is a protein marker of kidney function (serum reference range 0.8-1.2 ug/dl), and is more sensitive than creatinine. Even minor changes in GFR in the early stages of chronic kidney diseases are associated with increased cystatin C.

The routine urine examination is undoubtedly a guiding factor for renal function. The volume of urine excreted, its pH, specific gravity, osmolality, the concentration of abnormal constituents (such as proteins, ketone bodies, glucose and blood) may help to have some preliminary knowledge of kidney function. More information on urine laboratory tests is given in the appendix.

In general, the assessment of kidney function starts with the routine urine examination, followed by serum creatinine and/or blood urea estimations and, finally, the specific tests to measure the tubular and glomerular functions (clearance tests).

Fig. 20.5 : Mechanism of HCI secretion
(O—represents K* activated ATPase).

The parietal (oxyntic) cells of gastric glands produce HCI. The pH in the gastric lumen is as low as 0.8 (against the blood pH 7.4). Therefore, the protons are transported against the concentration gradient by an active process.
A unique enzyme—namely K* activated ATPase—present in the parietal cells is connected with the mechanism of HCI secretion (Fig.20.5). The process involves an exchange of H?* ions (of the parietal cells) for Kt ions (of the lumen). This is coupled with the consumption of energy, supplied by ATP. The Ht are continuously generated in the parietal cells by the dissociation of carbonic acid which, in turn, is produced from CO). The bicarbonate ions (HCO3), liberated from the carbonic acid (H»CO3) dissociation, enter the blood in exchange for Clions. The latter diffuse into the gastric lumen to form HCI. Gastrin—a peptide hormone of gastrointestinal tract—stimulates HCI secretion.
Following a meal, there is a slight elevation in the plasma bicarbonate concentration which is linked to the gastric HCI secretion. This is referred to as alkaline tide.

There are several tests for gastric function evaluation, some of the important ones are briefly discussed.

This is rather old and not used these days. Fractional test meal involves the collection of stomach contents by Ryle's tube in fasting. This is followed by a gastric stimulation, giving a test meal (rice gruel, black coffee etc.) The stomach contents are aspirated by Ryle's tube at different time periods (usually every 15 min for 2 hrs.) The samples are analysed for free and _ total acidity in the laboratory. The results are normally represented by a graph.

Pentagastrin is a synthetic peptide which stimulates the gastric secretion in a manner similar to the natural gastrin. The test procedure adapted is as follows
The stomach contents are aspirated by Ryle's tube in a fasting condition. This is referred to as residual juice. The gastric juice elaborated for the next one hour is collected and pooled which represents the basal secretion. Pentagastrin (5 mg/kg body weight) is now given to stimulate gastric secretion. The gastric juice is collected at 15 minute intervals for one hour. This represents the maximum secretion.
Each sample of the gastric secretion collected is measured for acidity by titrating the samples with N/10 NaOH to pH 7.4. The end point may be detected by an indicator (phenol red) or a pH meter.
In this case, the test meal in the form of 100 ml of 7% alcohol is administered. The response to alcohol test meal is more rapid, and the test time can be reduced to 1'/) hour. Clear specimens can be collected by this test, and the free acidity levels are relatively higher compared to FTM.
Basal acid output (BAO) refers to the acid output (millimol per hour) under the basal conditions i.e. basal secretion.
Maximal acid output (MAO) represents the acid output (millimol per hour) after the gastric maximum stimulation secretion. by pentagastrin i.e.

+
The
in
the
functions
of
any
organ
in
the
body
will
adversely
influence
the
health of the organism. Organ function tests are the laboratory tools to biochemically evaluate the working of a given organ.
Acute viral hepatitis is associated with elevated alanine transaminase (predominantly), aspartate transaminase and bilirubin.
Increase in serum y-glutamyl transpeptidase is observed in biliary obstruction and alcoholism.
A combination of laboratory investigations—instead of a single one—are commonly
employed
in
assessing
organ
function.
Kidney
function
can
be
accurately
assessed
by
clearance
tests,
measuring
glomerular
filtration
rate.
A
reduction
in
clearance
reflects
renal
damage.
Zollinger-Ellison syndrome, a tumor of gastrin secreting cells of the pancreas, is associated with increased gastric HCI production.
In normal individuals, the BAO is 4-10 mmol/
hr while the MAO is 20-50 mmol/hr.

Histamine is a powerful stimulant of gastric secretion. The basal gastric secretion is collected for one hour. Histamine (0.04 mg/kg body weight) is administered subcutaneously and the gastric contents are aspirated for the next one hour (at 15 minute intervals). The acid content is measured in all these samples.

This is also known as Hollander's test. It is mainly done to assess the completeness of vagotomy (vagal resection). Insulin (0.1 unit/kg body weight) is administered intravenously which causes hypoglycemia (blood glucose about 40 mg/dl), usually within 30 minutes, in normal persons.
successful, cause any If the vagotomy operation is insulin administration does not increase in the acid output, compared to the basal level. This test has to be carefully perfomed, since hypoglycemia is dangerous.

In the traditional methods of gastric analysis, a tube is invariably passed into the stomach to collect the gastric juice. This causes inconvenience to the subject. Recently, some tests involving tubeless gastric analysis have been developed. Such tests, however, are mostly useful for preliminary screening.
The principle of tubeless gastric analysis involves administration of a cation exchange resin that gets quantitatively exchanged with the H* ions of the gastric juice. The resin is then excreted into urine which can be estimated for an indirect measure of gastric acidity (concentration of H* ions).
Diagnex blue containing azure-A-resin is employed in the tubeless gastric analysis.

Increased gastric HCI secretion is found in Zollinger-Ellison syndrome (a tumor of gastrin secreting cells of the pancreas), chronic duodenal ulcer, gastric cell hyperplasia, excessive histamine production etc.
A decrease in gastric HCI is observed in gastritis, gastric carcinoma, pernicious anemia etc.

The pancreas is a specialized organ with exocrine and endocrine functions. The endocrine functions are discussed under the topic diabetes mellitus (Chapter 36).
The exocrine functions involve the synthesis of pancreatic juice containing several enzymes (for the digestion of foodstuffs) and bicarbonate. The major enzymes of pancreatic juice are trypsin, | chymotrypsin, elastase, — carboxypeptidase, amylase and lipase.
Pancreatic enzymes in serum Serum amylase and lipase measurements are commonly employed to assess the pancreatic function. Both these enzyme activities are elevated in acute pancreatitis, obstruction in the intestine and/or pancreatic duct.

Thyroid gland produces two __ principal hormones—thyroxine (T4) and triiodothyronine which regulate the metabolic rate of the body. The laboratory tests employed for the diagnosis of thyroid function are described in the Chapter 19 on hormones.

5. Discuss the different laboratory investigations to evaluate gastric function.
1. Write briefly on the different laboratory investigations employed to assess liver function.
2. Discuss the biochemical parameters for the differential diagnosis of jaundice.
3. Give an account of the serum enzymes derived from liver and their importance in LFT.
4 . Describe the renal function tests.

(a) Serum bilirubin, (b) van den Bergh reaction, (c) Galactose tolerance test, (d) Prothrombin time as LFT, (e) Renal threshold substances, (f) Glomerular filtration rate, (g) Creatinine clearance, (h) Standard urea clearance, (i) Urine concentration test, (j) Gastric function tests.

1. Bilirubin is the excretory end product of
N . The laboratory reaction most commonly employed to detect the elevation of serum bilirubin is
CoO . Name the stomach tube used to aspirate gastric juice
ON
DOH . The excretory function of liver can be evaluated by using a dye
HR . Obstructive jaundice is characterized by an increase in the serum enzyme
WwW . The serum enzyme most predominantly elevated in viral hepatitis is
. The renal threshold for glucose is
. The exogenous substance used to measure glomerular filtration rate (GFR) is
. Standard urea clearance is calculated when the volume of urine output is less than
. Name the synthetic peptide used to stimulate gastric secretion for evaluation of gastric
function

11. In hemolytic jaundice, van den Bergh reaction is
12. The serum enzyme elevated in alcoholic cirrhosis of liver is
13. Bilirubin is not excreted in urine in
14. Urea clearance is less than GFR because it is
15. The serum enzyme used to evaluate pancreatic function is
(a) Indirect positive (b) Direct positive (c) Biphasis (d) of these.
(a) Alanine transaminase (b) Aspartate transaminase (c) Alcohol dehydrogenase (d) y-Glutamyl transpeptidase.
(a) Obstructive jaundice (b) Hepatic jaundice (c) Hemolytic jaundice (d) All three.
(a) Partially secreted by the renal tubules (b) Partially reabsorbed by the tubules (c) Only filtered by glomeruli (d) of these.
(a) Alkaline phosphatase (b) Amylase (c) Aspartate transaminase (d) Lactate dehydrogenase.
he organism possesses tremendous capacity to survive against odds and maintain homeostasis. This is particularly true with regard to water, electrolyte and acid-base status of the body. These three are interrelated, hence they are considered together for the discussion in this chapter. Kidney actively participates in the regulation of water, electrolyte and acid-base balance. The general functions of kidney have already been described (Chapter 20).

Water is the solvent of life. Undoubtedly, water is more important than any other single compound to life. It is involved in several body functions.

1. Water provides the aqueous medium to the organism which is essential for the various biochemical reactions to occur.
2. Water directly participates as a reactant in several metabolic reactions.
3. It serves as a vehicle for transport of solutes.
4. Water is closely associated with the regulation of body temperature.

Water is the major body constituent. An adult human contains about 60% water (men 55-70%, women 45-60%). The women and obese individuals have relatively less water which is due to the higher content of stored fat in an anhydrous form.
A 70 kg normal man contains about 42 litres of water. This is distributed in intracellular (inside the cells 28l) and extracellular (outside the cells 141) compartments, respectively known as intracellular fluid (ICF) and extracellular fluid (ECF). The ECF is further divided into interstitial fluid (10.51) and plasma (3.51). The distribution of water in man is given in Table 21.1.
Total, % Body weight = 60. Total, in.Volume (l) = 42. Intracellular fluid (ICF), % Body weight = 40. Intracellular fluid (ICF), in.Volume (l) = 28. Extracellular fluid (ECF), % Body weight = 20. Extracellular fluid (ECF), in.Volume (l) = 14. Interstitial fluid, % Body weight = 15. Interstitial fluid, in.Volume (l) = 10.5. Plasma, % Body weight = 5. Plasma, in.Volume (l) = 3.5

The body possesses tremendous capacity to regulate its water content. In a_ healthy individual, this is achieved by balancing the daily water intake and water output.

and
Water is supplied to the body by exogenous endogenous sources.
Exogenous water Ingested water and beverages, water content of solid foodsconstitute the exogenous source of water. Water intake is highly variable which may range from 0.5-5 litres. It largely depends on the social habits and climate. In general, people living in hot climate drink more water. Ingestion of water is mainly controlled by a thirst centre located in the hypothalamus. Increase in the osmolality of plasma causes increased water intake by stimulating thirst centre.

produced within
the body
is the
endogenous water.
This from
the water
(300-350
oxidation that
1
g
ml/day)
of foodstuffs.
each of
is derived
It is
carbohydrate, respectively,
yield water.
On an
estimated protein
0.6
ml,
0.4
ml average,
generated for
about
1,000
Cal and
fat, and
1.1
125
ml of
consumed by
ml of
water the
is body.

Water losses from the body are variable. There are four distinct routes for the elimination of water from the body—urine, skin, lungs and feces.
Urine : This is the major route for water loss from the body. In a healthy individual, the urine output is about 1-2 I/day. Water loss through kidneys although highly variable, is well regulated to meet the body demands—to get rid of water or to retain. It should, however, be remembered that man cannot completely shut down urine production, despite there being no water intake. This is due to the fact that some amount of water (about 500 ml/day) is essential as the medium to eliminate the waste products from the body.
Hormonal regulation of urine production : It is indeed surprising to know that about 180 litres of water is filtered by the glomeruli into the renal tubules everyday. However, most of this is reabsorbed and only 1-2 litres is excreted as urine. Water excretion by the kidney is tightly controlled by vasopressin also known as antidiuretic hormone (ADH) of the posterior pituitary gland. The secretion of ADH _ is regulated by the osmotic pressure of plasma. An increase in osmolality promotes ADH secretion that leads to an increased water reabsorption from the renal tubules (less urine output). On the other hand, a decrease in osmolality suppresses ADH secretion that results in reduced water reabsorption from the renal tubules (more urine output). Plasma osmolality is largely dependent on the sodium concentration, hence sodium indirectly controls the amount of water in the body.
Diabetes insipidus is a disorder characterized by the deficiency of ADH which results in an increased loss of water from the body.
Skin : Loss of water (450 ml/day) occurs through the body surface by perspiration. This is an unregulated process by the body which mostly depends on the atmospheric temperature and humidity. The loss is more in hot climate. Fever causes increased water loss through the skin. It is estimated that for every 1°C rise in body temperature, about 15% increase is observed in the loss of water (through skin).
Fig. 21.1 : Water balance in the body, represented by daily intake and output (values are variable).

Lungs : During respiration, some amount of water (about 400 ml/day) is lost through the expired air. The latter is saturated with water and expelled from the body. In hot climates and/or when the person is suffering from fever, the water loss through lungs is increased.
The loss of water by perspiration (via skin) and respiration (via lungs) is collectively referred to as insensible water loss.
Feces Most of the water entering the gastrointestinal tract is reabsorbed by the intestine. About 150 ml/day is lost through feces in a healthy individual. Fecal loss of water is tremendously increased in diarrhea.
A summary of the water intake and output in the body is depicted in Fig.21.1. It may be noted that water balance of the body is regulated predominantly by controlling the urine output. This happens after an obligatory water loss via skin, lungs and feces.
The abnormalities associated with water balance—dehydration and overhydration—will be described, following a discussion on electrolyte balance.

Electrolytes are the compounds which readily dissociate in solution and exist as ions i.e. positively and negatively charged particles. For instance, NaCl does not exist as such, but it exists as cation (Nat) and anion (Cl). The concentration of electrolytes are expressed as milliequivalents (mEq/l) rather than milligrams. A gram equivalent weight of a compound is defined as its weight in grams that can combine or displace 1 g of hydrogen. One gram equivalent weight is equivalent to 1,000 milliequivalents.
The following formula is employed to convert the concentration mg/l to mEq/l.
Eg/|
meq/
mg per
litre
x
Valency
=
—2——
Atomic weight

Electrolytes are well distributed in the body fluids in order to maintain the osmotic equilibrium and water balance. A comparison of electrolytes present in extracellular (plasma) and intracellular (muscle) fluids is given in Table 21.2. The total concentration of cations and anions in each body compartment (ECF or ICF) is equal to maintain electrical neutrality.
There is a marked difference in the concentration of electrolytes (cations and anions) between the extracellular and intracellular fluids. Na* is the principal extracellular cation while K* is the intracellular cation. This difference in the concentration is essential for the cell survival which is maintained by Na* — K* pump (for details, Refer Chapter 33). As regards anions, Cland HCO3_ predominantly occur in extracellular fluids, while HPO, proteins and organic acids are found in the intracellular fluids.

There are two ways of expressing the concentration of molecules with regard to the osmotic pressure.
1. Osmolarity : The number of moles (or millimoles) per liter of solution.
2. Osmolality : The number of moles (or millimoles) per kg of solvent.

mEq/l).Intracellular fluid.Cations = . , T ABLE 21.2 Composition of electrolytes in the body fluids (expressed as mEq/l).Intracellular fluid.Cations = . , T ABLE 21.2 Composition of electrolytes in the body fluids (expressed as mEq/l).Anions. = Organic acids. , T ABLE 21.2 Composition of electrolytes in the body fluids (expressed as mEq/l).Anions. = 5. , T ABLE 21.2 Composition of electrolytes in the body fluids (expressed as mEq/l).Extracellular fluid (plasma).Cations = 155. , T ABLE 21.2 Composition of electrolytes in the body fluids (expressed as mEq/l).Extracellular fluid (plasma).Anions = . , T ABLE 21.2 Composition of electrolytes in the body fluids (expressed as mEq/l).Extracellular fluid (plasma).Anions = 155. , T ABLE 21.2 Composition of electrolytes in the body fluids (expressed as mEq/l).Intracellular fluid.Cations = . , T ABLE 21.2 Composition of electrolytes in the body fluids (expressed as mEq/l).Intracellular fluid.Cations = 202. , T ABLE 21.2 Composition of electrolytes in the body fluids (expressed as mEq/l).Anions. = . , T ABLE 21.2 Composition of electrolytes in the body fluids (expressed as mEq/l).Anions. = 202
If the solvent is pure water, there is almost no difference between osmolarity and osmolality. However, for biological fluids (containing molecules such as proteins), the osmolality is more commonly used. This is about 6% greater than osmolarity.

Osmolality is a measure of the solute particles present in the fluid medium. The osmolality of plasma is in the range of 285-295 milliosmoles/ kg (Table 21.3). Sodium and its associated anions make the largest contribution (~90%) to plasma osmolality. Osmolality is generally measured by osmometer.
For practical purposes, plasma osmolality can be computed from the concentrations (mmol/l) of Nat, K*, urea and glucose as follows
2(Na*) + 2(Kt) + Urea + Glucose
The factor 2 is used for Na* and K* ions to account for the associated anion concentration (assuming complete ionization of the molecules). Since plasma Nat is the most predominant contributor to osmolality, the above calculation is further simplified as follows
Plasma
2
x
Plasma
Na*
osmolality
=
(mmol/kg)
(mmol/l)
The above calculation holds good only plasma
glucose and
urea are
the normal
range.
This calculation,
however, if
in will
not be
valid in
severe hyperproteinemia
and lipemia.

Movement of water across the biological membranes is dependent on the osmotic pressure differences between the intracellular fluid (ICF) and extracellular fluid (ECF). In a healthy state, the osmotic pressure of ECF, mainly due to Nat ions, is equal to the osmotic pressure of ICF which is predominantly due to
Constituent (solute), Distribution of plasma osmolality = Osmolality (mosm/kg). Sodium, Distribution of plasma osmolality = 135. Associated anions, Distribution of plasma osmolality = 135. Potassium, Distribution of plasma osmolality = 3.5. Associated anions, Distribution of plasma osmolality = 3.5. Calcium, Distribution of plasma osmolality = 1.5. Associated anions, Distribution of plasma osmolality = 1.5. Magnesium, Distribution of plasma osmolality = 1.0. Associated anions, Distribution of plasma osmolality = 1.0. Urea, Distribution of plasma osmolality = 5.0. Glucose, Distribution of plasma osmolality = 5.0. Protein, Distribution of plasma osmolality = 1.0. Total, Distribution of plasma osmolality = 293
Kt ions. As such, there is no net passage of water molecules in or out of the cells, due to this osmotic equilibrium.

Electrolyte and water balance are regulated together and the kidneys play a predominant role in this regard. The regulation is mostly achieved through the hormones aldosterone, ADH and renin-angiotensin.
Aldosterone : It is a mineralocorticoid produced by adrenal cortex. Aldosterone increases Nat reabsorption by the renal tubules at the expense of Kt and Ht ions. The net effect is the retention of Na* in the body.
Antidiuretic hormone (ADH) : An increase in the plasma osmolality (mostly due to Na*) stimulates hypothalamus to release ADH. ADH effectively increases water reabsorption by renal tubules.
Renin-angiotensin The secretion — of aldosterone is controlled by renin-angiotensin system. Decrease in the blood pressure (due to a fall in ECF volume) is sensed by juxtaglomerular apparatus of the nephron which secrete renin. Renin acts on angiotensinogen to produce angiotensin |. The latter is then converted to angiotensin II which stimulates the release of aldosterone.
The relation between renin, angiotensin and aldosterone in the regulation of Na* balance is depicted in Fig.21.2. Aldosterone and ADH coordinate with each other to maintain the normal fluid and electrolyte balance.
Atrial natriuretic factor (ANF) : ANF or atriopeptin is a 28-amino acids containing peptide. It is produced in the atrium of heart in response to increased blood volume, elevated blood pressure and high salt intake. ANF acts on kidneys to increase GFR, sodium excretion and urine output. Thus ANF opposes the actions of renin and aldosterone (which increase salt retention and blood pressure).

It is important to realise that Na* and _ its anions (mainly Cl) are confined to the extracellular fluid. And the retention of water in
Fig. 21.2 : Hormonal regulation of
Nat balance by the kidney.
the ECF is directly related to the osmotic effect of these ions (Nat and Cl-). Therefore, the amount of Na* in the ECF ultimately determines. its volume.

Generally, the consumption of a_ wellbalanced diet supplies the body requirement of electrolytes. Humans do not possess the ability to distinguish between the salt hunger and water hunger. Thirst, however, may regulate electrolyte intake also. In hot climates, the loss of electrolyte is usually higher. Sometimes it may be necessary to supplement drinking water with electrolytes.

Dehydration is a condition characterized by water depletion in the body. \t may be due to insufficient intake or excessive water loss or both. Dehydration is generally classified into two types.
1. Due to loss of water alone.
2.
Due
to
and
electrolytes.
deprivation
of
water
Causes of dehydration : Dehydration may occur as a result of diarrhea, vomiting, excessive sweating, fluid loss in burns, adrenocortical
dysfunction, kidney diseases (e.g. renal insufficiency), deficiency of ADH (diabetes insipidus) etc.
Characteristic features
of dehydration
:
There are three degrees of dehydration—mild, moderate and severe.
The salient features of dehydration are given hereunder
1. The volume of the extracellular fluid (e.g. plasma) is decreased with a concomitant rise in electrolyte concentration and osmotic pressure.
2. Water is drawn from the intracellular fluid that results in shrunken cells and disturbed metabolism e.g. increased protein breakdown.
3. ADH secretion is increased. This causes increased water retention in the body and consequently urine volume is very low.
4. Plasma protein and blood urea concentrations are increased.
5. Water depletion is often accompanied by a loss of electrolytes from the body (Nat, Kt etc.).
6. The principal clinical symptoms of severe dehydration include increased pulse rate, low blood pressure, sunken eyeballs, decreased skin turgor, lethargy, confusion and coma.
Treatment : The treatment of choice for dehydration is intake of plenty of water. In the subjects who cannot take orally, water should be administered intravenously in an isotonic solution (usually 5% glucose). If the dehydration is accompanied by loss of electrolytes, the same should be administered by oral or intravenous routes. This has to be done by carefully monitoring the water and electrolyte status of the body.

Cholera is transmitted through water and foods, contaminated by the bacterium Vibrio cholerae. This bacterium produces a toxin which stimulates the intestinal cells to secrete various ions (Cl, Na*, Kt, HCO3 etc.) into the intestinal
lumen. These ions collectively raise the osmotic pressure and suck the water into lumen. This results in diarrhea with a heavy loss of water (5-10 liters/day). If not treated in time, the victims of cholera will die due to dehydration and loss of dissolved salts. Thus, cholera and other forms of severe diarrhea are the major killers of young children in many developing countries.
Oral rehydration therapy (ORT) is commonly used to treat cholera and other diarrheal diseases.

Overhydration or water intoxication is caused by excessive retention of water in the body. This may occur due to excessive intake of large volumes of salt free fluids, renal failure, overproduction of ADH etc. Overhydration is observed after major trauma or operation, lung infections etc.
Water intoxication is associated with dilution of ECF and ICF with a decrease in osmolality. The clinical symptoms include headache, lethargy and convulsions. The treatment advocated is stoppage of water intake and administration of hypertonic saline.

The distribution of body water (in the ECF and ICF), dehydration and overhydration can be better understood by a water tank model (Fig.21.3). The tank has an inlet and outlet, respectively, representing the water intake (mostly oral) and water output (mainly urine) by the body.
Dehydration is caused when the water output exceeds the intake. On the other hand, overhydration is due to more water intake and less output.

The body distribution, dietary intake, intestinal absorption and biochemical functions — of individual electrolytes are discussed under the section mineral metabolism (Chapter 18). The
electrolyte disorders, particularly hypernatremia and hyponatremia (of sodium); hyperkalemia and hypokalemia (of potassium) must also be referred.

Diuretics are the drugs that stimulate water and sodium excretion, so that urine volume is increased. The commonly used diuretics are bendrofluazide, frusemide, spironolactone and mannitol. Diuretics are important in the treatment of edema, heart failure and hypertension.

The normal pH of the blood is maintained in the narrow range of 7.35-7.45, i.e. slightly alkaline. The pH of intracellular fluid is rather variable. Thus, for erythrocytes the pH is 7.2, while for skeletal muscle, it may be as low as 6.0.
Maintenance of blood pH is an important homeostatic mechanism of the body. In normal circumstances, the regulation is so effective that the blood pH varies very little. Changes in blood pH will alter the intracellular pH which, in turn, influence the metabolism e.g. distortion in protein structure, enzyme activity etc. It is estimated that the blood pH compatible to life is 6.8-7.8. (For a good understanding of acid-base balance, adequate knowledge on acids, bases, pH and buffers is essential (refer Chapter 40).
The metabolism of the body is accompanied by an overall production of acids. These include the volatile acids like carbonic acid (most predominent, about 20,000 mEq/day) or nonvolatile acids (about 80 mEq/day) such as lactic acid, sulfuric acid, phosphoric acid etc. Carbonic acid is formed from the metabolic product CO); lactic acid is produced in anaerobic metabolism; sulfuric acid is generated from proteins (sulfur containing amino acids); phosphoric acid is derived from organic phosphates (e.g. phospholipids). All these acids add up H* ions to the blood. A diet rich in animal proteins results in more acid production by the body that ultimately leads to the excretion of urine which is profoundly acidic.

The formation of basic compounds in the body, in the normal circumstances, is negligible. Some amount of bicarbonate is generated from the organic acids such as lactate and citrate. A vegetarian diet has a tendency for a net production of bases. This is due to the fact that vegetarian diet produces salts of organic acids such as sodium lactate which can utilize H* ions produced in the body. For this reason, a vegetarian diet has an alkalizing effect on the body. This is reflected by the excretion of neutral or slightly alkaline urine by these subjects.
It is evident that at a blood pH 7.4, the ratio of bicarbonate to carbonic acid is 20 : 1. Thus, the bicarbonate concentration is much higher (20 times) than carbonic acid in the blood. This is referred to as alkali reserve and is responsible for the effective buffering of H* ions, generated in the body. In normal circumstances, the concentration of bicarbonate and carbonic acid determines the pH of blood. Further, the bicarbonate buffer system serves as an index to understand the disturbances in the acid-base balance of the body.
2. Phosphate buffer system Sodium dihydrogen phosphate and disodium hydrogen phosphate (NaH »PO4 — Na»HPO,) constitute the phosphate buffer. It is mostly an intracellular buffer and is of less importance in plasma due to its low concentration. With a pK of 6.8 (close to blood pH 7.4), the phosphate buffer would have been more effective, had it been present in high concentration. It is estimated that the ratio of base to acid for phosphate buffer is 4 compared to 20 for bicarbonate buffer.
3. Protein buffer system The plasma proteins and hemoglobin together constitute the protein buffer system of the blood. The buffering capacity of proteins is dependent on the pK of ionizable groups of amino acids. The imidazole group of histidine (pK = 6.7) is the most effective contributor of protein buffers. The plasma proteins account for about 2% of the total buffering capacity of the plasma.
Hemoglobin of RBC is also an important buffer. It mainly buffers the fixed acids, besides being involved in the transport of gases (Oj and CO). More details on hemoglobin are given under respiratory mechanism for regulation of pH.

Respiratory system provides a_ rapid mechanism for the maintenance of acid-base balance. This is achieved by regulating the concentration of carbonic acid (HyCO3) in the blood i.e. the denominator in the bicarbonate buffer system. The details of CO, transport and the role of hemoglobin in this process are described elsewhere (Chapter 10, Refer Fig.10.6).
The large volumes of CO, produced by the cellular metabolic activity endanger the acidbase equilibrium of the body. But in normal circumstances, all of this CO, is eliminated from the body in the expired air via the lungs, as summarized below.

HCO,
CO,
+H,0-
The rate of respiration (or the rate of removal of CO ) is controlled by a respiratory centre, located in the medulla of the brain. This centre is highly sensitive to changes in the pH of blood. Any decrease in blood pH causes hyperventilation to blow off CO, thereby reducing the H»CO3 concentration. Simultaneously, the H* ions are eliminated as HjO.
Respiratory control of blood pH is rapid but only a short term regulatory process, since hyperventilation cannot proceed for long.
Hemoglobin as a buffer : Hemoglobin of erythrocytes is also important in the respiratory regulation of pH. At the tissue level, hemoglobin binds to H* ions and helps to transport CO, as HCO3 with a minimum change in pH (referred to as isohydric transport). In the lungs, as hemoglobin combines with Op», H* ions are removed which combine with HCO3 to form H»CO3. The latter dissociates to release CO, to be exhaled (Refer Fig.10.6).
Generation of HCO3 by RBC : Due to lack of aerobic metabolic pathways, RBC produce very little CO. The plasma CO, diffuses into the RBC along the concentration gradient where it combines with water to form H CO3. This reaction is catalysed by carbonic anhydrase (also called carbonate dehydratase). In the RBC, HCO 3 dissociates to produce H* and HCO; . The H* ions are trapped and buffered by hemoglobin. As the concentration of HCO; increases in the RBC, it diffuses into plasma along with the concentration gradient, in exchange for Clions, to maintain electrical neutrality. This phenomenon, referred to as chloride shift, helps to generate HCO3
(Fig.21.4).
Fig. 21.4 : Generation of bicarbonate by the erythrocyte (CA-Carbonic anhydrase; Hb—Hemoglobin).

The role of kidneys in the maintenance of acid-base balance of the body (blood pH) is highly significant. The renal mechanism tries to provide a permanent solution to the acid-base disturbances. This is in contrast to the temporary buffering system and a short term respiratory mechanism, described above.
The kidneys regulate the blood pH_ by maintaining the alkali reserve, besides excreting or reabsorbing the acidic or basic substances, as the situation demands.
Urine pH normally lower than blood pH : The pH of urine is normally acidic (~6.0). This clearly indicates that the kidneys have contributed to the acidification of urine, when it is formed from the blood plasma (pH 7.4). In other words, the H* ions generated in the body in the normal circumstances, are eliminated by acidified urine. Hence the pH of urine is normally acidic (~6.0), while that of blood is alkaline (7.4). Urine pH, however, is variable and may range between 4.5-9.5, depending on the concentration of H* ions.
Carbonic anhydrase and renal regulation of PH : The enzyme carbonic anhydrase (inhibited by acetazolamide) is of central importance in the renal regulation of pH which occurs by the following mechanisms.
1. Excretion of H* ions
2. Reabsorption of bicarbonate
3. Excretion of titratable acid
4. Excretion of ammonium ions.
1. Excretion of H* ions : Kidney is the only route through which the H* can be eliminated from the body. H* excretion occurs in the proximal convoluted tubules (renal tubular cells) and is coupled with the regeneration of HCO3. The process depicted in Fig.21.5, occurs as follows.

Carbonic anhydrase catalyses the production of carbonic acid (HyCO3) from CO, and HO in the renal tubular cell. H»CO3 then dissociates to H* and HCO3. The H* ions are secreted into the tubular lumen in exchange for Nat. The Na* in association with HCO3 is reabsorbed into the blood. This is an effective mechanism to eliminate acids (H*) from the body with a simultaneous generation of HCO3. The latter adds up to the alkali reserve of the body. The Ht combines with a non-carbonate base and is excreted in urine.
2. Reabsorption of bicarbonate : This mechanism is primarily responsible to conserve the blood HCO3, with a simultaneous excretion of H* ions. The normal urine is almost free from HCO3. This is explained as follows (Fig.21.6).
Fig. 21.5 : Renal regulation of blood pH-Excretion of H* ions (CA—Carbonic anhydrase).
Fig. 21.6 : Renal regulation of blood pH—Reabsorption of bicarbonate (CA—Carbonic anhydrase).
mechanism helps to maintain the steady state and will not be effective for the elimination of H* or generation of new HCO3.
3. Excretion of titratable acid : Titratable acidity is a measure of acid excreted into urine by the kidney. This can be estimated by titrating urine back to the normal pH of blood (7.4). In quantitative terms, titratable acidity refers to the number of milliliters of N/10 NaOH required to titrate 1 liter of urine to pH 7.4. Titratable acidity reflects the H+ ions excreted into urine which resulted in a fall of pH from 7.4 (that of blood). The excreted H* ions are actually buffered in the urine by phosphate buffer as depicted in Fig.21.7, and briefly described hereunder.

Bicarbonate freely diffuses from the plasma into the tubular lumen. Here HCO3 combines with Ht, secreted by tubular cells, to form H,CO3. H,CO3 is then cleaved by carbonic anhydrase (of tubular cell membrane) to form CO, and H,O. As the CO, concentration builds up in the lumen, it diffuses into the tubular cells along the concentration gradient. In the tubular cell, COy again combines with HO to form H CO; which then dissociates into H* and HCO3. The H* is secreted into the lumen in exchange for Na*. The HCO3 is reabsorbed into plasma in association with Na*. Reabsorption of HCO} is a cyclic process with the net excretion of H* or generation of new HCO3. This is because the H* is derived from water. This
As already discussed, Ht ion is secreted into the tubular lumen in exchange for Na* ion. This Nat is obtained from the base, disodium hydrogen phosphate (NayHPO,). The latter in turn combines with H* to produce the acid, sodium dihydrogen phosphate (NaH»PO,), in which form the major quantity of titratable acid in urine is present. As the tubular fluid moves down the renal tubules, more and more H* ions are added, resulting in the acidification of urine. This causes a fall in the pH of urine to as low as 4.5. Any further fall in the pH will cause depletion of Nat ions.
4. Excretion of ammonium ions : This is another mechanism to buffer H* ions secreted into the tubular fluid. The H* ion combines with
Fig. 21.7 : Renal regulation of blood pH—-Excretion of titratable acid by phosphate buffer mechanism (CA—Carbonic anhydrase).
Fig. 21.8 : Renal regulation of blood pH-Excretion of ammonium ions (CA—Carbonic anhydrase).

NH3 to form ammonium ion (NH). The renal tubular cells deamidate glutamine to glutamate and NH3. This reaction is catalysed by the enzyme glutaminase. The NH3, liberated in this reaction, diffuses into the tubular lumen where it combines with H* to form NHq_ (Fig.21.8). Ammonium ions cannot diffuse back into tubular cells and, therefore, are excreted into urine.
NHj@ is a major urine acid. It is estimated that about half to two-thirds of body acid load is eliminated in the form of NHj ions. For this reason, renal regulation via NHj excretion is very effective to eliminate large quantities of acids produced in the body. This mechanism becomes predominant particularly in acidosis.

As is observed from the foregoing discussion, CO, is of central importance in the acid-base balance of the body. It has the ability to combine with HO to from HyCO3 which can dissociate to HCO3 and H*. A summary of the interaction between the lungs, erythrocytes and kidneys in handling CO, to maintain pH of the blood is depicted in Fig.21.9. The CO, generated by aerobic metabolism may be exhaled via lungs, or converted to HCO3 by erythrocytes and kidneys to add up to the alkali reserve of the body.

The regulation of pH within the cells is as important as that discussed above for the extracellular fluid. The H* ions generated in the cells are exchanged for Na* and Kt ions. This is particularly observed in skeletal muscle which reduces the potential danger of H* accumulation in the cells.

The body has developed an efficient system for the maintenance of acid-base equilibrium with a result that the pH of blood is almost constant (7.4). The blood pH compatible to life is 6.8-7.8, beyond which life cannot exist.
For a better understanding of the disorders of acid-base balance, the Henderson-Hasselbalch equation must be frequently consulted.
Fig. 21.9 : Carbon dioxide—the central molecule of blood pH regulation.
It is evident from the above equation that the blood pH (H* ion concentration) is dependent on the relative concentration (ratio) of bicarbonate (HCO3) and carbonic acid (H3CO3).
The acid-base disorders are mainly classified as

(a)
Metabolic acidosis—due
to
a
in bicarbonate.
(b)
acidosis—due increase in carbonic acid.

(a)
Metabolic
—
alkalosis—due increase in bicarbonate.
(b)
alkalosis—due_
decrease to
an to
to an
a
decrease in carbonic acid.
The four acid-base disorders referred above are primarily due to alterations in either bicarbonate or carbonic acid. It may be observed that the metabolic acid-base balance disorders are caused by a direct alteration in bicarbonate concentration while the respiratory disturbances are due to a change in carbonic acid level (i.e. CO). This type of classification is more theoretical. In the actual clinical situations, mixed type of disorders are common.
The terms acidemia and _alkalemia, respectively, refer to an increase or a decrease in [H+] ion concentration in blood. They are, however, not commonly used.

The most important clinical causes/disease states that result in acid-base disorders are listed in Table 21.4. Metabolic acidosis could occur due to diabetes mellitus (ketoacidosis), lactic acidosis, renal failure etc. Respiratory acidosis is common in severe asthma and cardiac arrest. Vomiting and hypokalemia may result in metabolic alkalosis while hyperventilation and severe anemia may lead to respiratory alkalosis.
To counter the acid-base disturbances, the body gears up its homeostatic mechanism and

(ketoacidosis)
Pneumonia
Obstruction in airways
Chest deformities
Severe diarrhea
Depression of
respiratory center (by
drugs e.g. opiates)
Metabolic alkalosis
Severe vomiting
Anemia
Intravenous administration
of bicarbonate
Salicylate poisoning
makes every attempt to restore the pH to normal level (7.4). This is referred to as compensation which may be partial or full. Sometimes the acidbase disorders may remain uncompensated.
The principal acid-base disturbances, along with the blood concentration of HCO3 and H»CO3, in acute and compensated states are given in the Table. 21.5.
For the acute metabolic disorders (due to changes in HCO3), respiratory compensation sets in and regulates the HyCO3 (i.e. CO») by hyperor hypoventilation. As regards acute respiratory disorders (due to changes in H CO3), the renal compensation occurs to maintain the HCO3 level, by increasing or decreasing its excretion.
In the Table 21.6, a summary of the acid-base disorders with primary changes and compensatory mechanisms is given.

For a better understanding of acid-base disorders, adequate knowledge of anion gap is essential. The total concentration of cations and
anions (expressed as mEq/l) is equal in the body fluids. This is required to maintain electrical neutrality.
The commonly measured electrolytes in the plasma are Na*, Kt, Cland HCO3. Nat and Kt together constitute about 95% of the plasma cations. Cland HCO3 are the major anions, contributing to about 80% of the plasma anions. The remaining 20% of plasma anions (not normally measured in the laboratory) include proteins, phosphate, sulfate, urate and organic acids.
Anion gap is defined as the difference between the total concentration of measured cations (Na* and K*) and that of measured anion (CIand HCO3). The anion gap (A™) in fact represents the unmeasured anions in the plasma which may be calculated as follows, by substituting the normal concentration of electrolytes (mEq/l).
Na* +
KX
=
Ch
+
HCO;
+
A
136 +
4
=
100
+
25
+
A
A
=
15
mEq/l
The anion gap in a healthy individual is around 15 mEq/l (range 8-18 mEq/l). Acid-base disorders are often associated with alterations in the anion
gap.

The primary defect in metabolic acidosis is a reduction in bicarbonate concentration which leads to a fall in blood pH. The bicarbonate concentration may be decreased due to its utilization in buffering H* ions, loss in urine or gastrointestinal tract or failure to be regenerated.
The most important cause of metabolic acidosis is due to an excessive production of organic acids which combine with NaHCO3 and deplete the alkali reserve.
NaHCO3
+
Organic acids
—~>
Na salts
of organic
acids
+
CO,
Metabolic acidosis is commonly seen in severe uncontrolled diabetes mellitus which is associated with excessive production of acetoacetic acid and B-hydroxybutyric acid (both are organic acids).
Anion gap and metabolic acidosis : Increased production and accumulation of organic acids causes an elevation in the anion gap. This type of picture is seen in metabolic acidosis associated with diabetes (ketoacidosis).

Metabolic acidosis, T ABLE 21.6 Acid-base disorders with primary changes and compensatory mechanisms.Primary change = Decreased plasma bicarbonate. Metabolic acidosis, T ABLE 21.6 Acid-base disorders with primary changes and compensatory mechanisms.Compensatory mechanism = Hyperventilation (decrease in pCO 2 ). Metabolic acidosis, T ABLE 21.6 Acid-base disorders with primary changes and compensatory mechanisms.Timescale for compensation = Minutes to hours. Metabolic alkalosis, T ABLE 21.6 Acid-base disorders with primary changes and compensatory mechanisms.Primary change = Increased plasma bicarbonate. Metabolic alkalosis, T ABLE 21.6 Acid-base disorders with primary changes and compensatory mechanisms.Compensatory mechanism = Hypoventilation (increase in pCO 2 ). Metabolic alkalosis, T ABLE 21.6 Acid-base disorders with primary changes and compensatory mechanisms.Timescale for compensation = Minutes to hours. Respiratory acidosis, T ABLE 21.6 Acid-base disorders with primary changes and compensatory mechanisms.Primary change = Increased pCO 2. Respiratory acidosis, T ABLE 21.6 Acid-base disorders with primary changes and compensatory mechanisms.Compensatory mechanism = Elevation in plasma bicarbonate; increase in renal reabsorption of bicarbonate. Respiratory acidosis, T ABLE 21.6 Acid-base disorders with primary changes and compensatory mechanisms.Timescale for compensation = Days. Respiratory alkalosis, T ABLE 21.6 Acid-base disorders with primary changes and compensatory mechanisms.Primary change = Decreased pCO 2. Respiratory alkalosis, T ABLE 21.6 Acid-base disorders with primary changes and compensatory mechanisms.Compensatory mechanism = Reduction in plasma bicarbonate; decrease in renal reabsorption of bicarbonate. Respiratory alkalosis, T ABLE 21.6 Acid-base disorders with primary changes and compensatory mechanisms.Timescale for compensation = Days

Compensation of metabolic acidosis : The acute metabolic acidosis is usually compensated by hyperventilation of lungs. This leads to an increased elimination of CO, from the body (hence HyCO3J). but respiratory compensation is only short-lived. Renal compensation sets in within 3-4 days and the Ht ions are excreted as NHf@ ions.

The primary defect in respiratory acidosis is due to a retention of CO, (H»CO31). There may be several causes for respiratory acidosis which include depression of the respiratory centre (overdose of drugs), pulmonary disorders (bronchopneumonia) and breathing air with high content of CO).
The renal mechanism comes for the rescue to compensate respiratory acidosis. More HCO3 is generated and retained by the kidneys which adds up to the alkali reserve of the body. The excretion of titratable acidity and NHq_ is elevated in urine.

The primary abnormality in metabolic alkalosis is an increase in HCO3 concentration. This may occur due to excessive vomiting (resulting in loss of H*) or an excessive intake of sodium bicarbonate for therapeutic purposes (e.g. control of gastric acidity). Cushing's syndrome (hypersecretion of aldosterone) causes increased retention of Na* and loss of K* from the body. Metabolic alkalosis is commonly associated with low K* — concentration (hypokalemia). In severe Kt deficiency, H* ions are retained inside the cells to replace missing K* ions. In the renal tubular cells, H* ions are exchanged (instead of K*) with the reabsorbed Nat. Paradoxically, the patient excretes acid urine despite alkalosis.
The respiratory mechanism initiates the compensation by hypoventilation to retain CO (hence HyCO31). This is slowly taken over by renal mechanism which excretes more HCO3 and retains H*.

The primary abnormality in respiratory alkalosis is a decrease in HyCO3 concentration. This may occur due to _ prolonged hyperventilation resulting in increased exhalation of CO, by the lungs. Hyperventilation is observed in conditions such as_ hysteria, hypoxia, raised intracranial pressure, excessive artificial ventilation and the action of certain drugs (salicylate) that stimulate respiratory centre,
The renal mechanism tries to compensate by increasing the urinary excretion of HCO3.

Sometimes, the patient may have two or more acid-base disturbances occurring simultaneously. In such instances, both HCO3 and HzCO3 are altered. In general, if the biochemical data (of blood gas analysis) cannot be explained by a specific acid-base disorder, it is assumed that a mixed disturbance is occurring. Many a times, compensatory mechanisms may lead to mixed acid-base disorders.

Plasma potassium concentration
(normal
3.5-5.0
mEq/l)
is very
important as
it affects
the contractility of the heart. Hyperkalemia (high
plasma K*) or hypokalemia (low plasma Kt) can be life-threatening. The relevance of potassium balance in certain acid-base disorders _ is discussed briefly.
Potassium and diabetic ketoacidosis : The hormone insulin increases K* uptake by cells (particularly from skeletal muscle). The patient of severe uncontrolled diabetes (i.e. with metabolic acidosis) is usually with hypokalemia. When such a patient is given insulin, it stimulates Kt entry into cells. The result is that plasma K* level is further depleted. Hypokalemia affects heart functioning, and is life threatening. Therefore, in the treatment of diabetic ketoacidosis, potassium has to be given (unless the patients have high plasma K* concentration).
Potassium and_ alkalosis Low plasma concentration of K* (hypokalemia) leads to an increased excretion of hydrogen ions, and thus

+ Existence of life is unimaginable in the absence of water.
+ Kidneys play a predominant role in the regulation of water, electrolyte and acid-base balance.
+ Electrolyte and water balance regulation occurs through the involvement of hormones— aldosterone, ADH and renin-angiotensin.
+ Severe dehydration is characterized by low blood pressure, sunken eyeballs, lethargy, confusion and coma.
+ Sodium is the principal extracellular cation while K* is intracellular. The maintenance
of the differential concentration of these electrolytes is essential for the survival of life which is brought about by Na*-Kt+ pump.
+ The body metabolism is accompanied by the production of acids such as carbonic acid, sulfuric acid, phosphoric acid etc.
+ Vegetarian diet has an alkalizing effect on the body. This is attributed to the formation of organic acids such as sodium lactate which can deplete Ht ions by combining with them.
+ The blood pH is maintained by blood buffers, respiratory and renal mechanisms.
+ Carbon dioxide is the central molecule of acid-base regulation.
+ Disturbances in acid-base regulation result in acidosis (decreased blood pH) or alkalosis (raised blood pH).
+ Uncontrolled diabetes mellitus is associated with metabolic acidosis, commonly referred to as ketoacidosis (due to the overproduction of ketone bodies).
may cause metabolic alkalosis. Conversely, metabolic alkalosis is associated with increased renal excretion of K*.
In view of the importance discussed above, the measurement of plasma Kt concentration assumes significance in the acid-base disorders. In cases of these disorders associated with hypokalemia, potassium supplementation (with carefull monitoring of plasma K*) needs to be considered.

The measurement
of blood
gas is
investigation in
the laboratory
conditions associated
and/or service.
In with
acid-base certain
respiratory disorders,
O,)
measurement on
assumes the
results obtained
condition, failure
blood gas
(CO, significance.
and oxygen
and
Based the
severity treatment
of the
or
_
artificial ventilation
is carried
out.
For blood gas analysis, a sample of arterial blood collected from (most commonly) radial
artery in the forearm, or (less commonly) from the femoral artery in the leg is used. The biochemical profile measured include pOz, pCO, and pH (H* ion concentration). The concentration of bicarbonate is calculated by using Henderson-Hasselbalch equation. In fact, the blood gas analysers employed in the hospitals are designed to perform the various calculations automatically and give the final results. The reference ranges of blood gas analysis are given in Table 21.7.

1. Describe the role of kidney in the regulation of blood pH.
2. Give an account of the water distribution and its balance in the body.
3. Campare the composition of electrolytes in the extracellular and intracellular fluids. Discuss the regulation of electrolyte balance.
4. Describe the role of blood buffers in the acid-base balance.
5. Classify acid-base disorders and discuss them with compensatory mechanisms.

(a) Dehydration, (b) Vasopressin and water balance, (c) Osmolality of plasma, (d) Acids produced in the body, (e) Henderson-Hasselbalch equation, (f) Bicarbonate buffer, (g) Excretion of H* by kidney, (h) Titratable acidity, (i) Metabolic acidosis, (j) Anion gap.

. The hormone controlling water excretion via kidneys is
. The principal cation of extracellular fluid is . The normal osmolality of plasma is KRwWH
. Nat reabsorption by renal tubules is increased by the hormone
. The most predominant volatile acid generated in the body is
. The most important buffer system regulating blood pH is DUH
. Ata normal blood pH 7.4, the ratio of bicarbonate to carbonic acid is
. The body acid load is predominantly eliminated in the form of
OMAN
. The primary defect in metabolic acidosis is a reduction in the plasma concentration of
. The respiratory alkalosis is primarily associated with a decrease in the plasma concentration of Oo

11. The metabolic (endogenous) water is derived by the oxidation of
(a) Carbohydrate (b) Protein (c) Fats d) All of them.
12. The most predominant anion in the extracellular fluids
(a)
Cl
(b)
HCO3
(c)
HPOg
(d)
Protein.
13. The only route through which H+ ions are eliminated from the body
(a) Lungs (b) Stomach (c) Kidneys (d) of them.
14. Name the amino acid from which ammonia is derived in the renal tubular cells which is finally excreted as NH
(a) Asparagine (b) Glutamine (c) Glutamate (d) Aspartate.
15. The anion gap refers to the unmeasured plasma anion concentration (in the laboratory) and is represented by
(a) Proteins and organic acids (b) Phosphate and sulfate (c) Urate (d) All of them.
he body possesses a vast number of proteins designed with specific structures to perform specialized functions. A selected few of the most important proteins that are intimately connected with the tissue structure and functions are briefly described in this chapter. In addition, the body fluids are also discussed.

The connective tissue or extracellular matrix (ECM) refers to the complex material surrounding the mammalian cells in tissues. The major protein components of ECM include collagen, elastin, fibrillin, fibronectin, laminin and proteoglycans. Besides these proteins, the structural proteins namely keratins are also described.

Collagen is the most abundant protein in mammals, comprising approximately one-third
of the total body protein. Collagen is the predominant component of the connective tissue, although its distribution varies in different tissues. For instance, collagen forms 90% of the organic matrix of bones, 85% of tendons, 70% of skin, and 4% of liver.

1. Being a major component of the connective tissue, collagen gives strength, support and shape to the tissues. The tensile strength of collagen fiber is impressive. To break a collagen fiber of 1 mm in diameter, a load of 10-40 kg is needed! However, in diseased states with altered collagen structure, the tensile strength is reduced.
2. Collagen contributes to proper alignment of cells, which in turn helps in cell proliferation, and their differentiation to different tissues and organs.
3. Collagen (that is exposed in blood contributes to
vessels) thrombus formation.

Collagen is not a single homogeneous protein, but a group of structurally related and genetically distinct proteins. In humans, at least 19 different types of collagens, composed of 30 distinct polypeptide chains (encoded by separate genes), have been identified. The types of collagen are numbered (by Roman numerals) as I, Il...XIX. The different types of collagen are suited to perform specialized functions in tissues. For instance, collagens type | and type II are respectively found in skin and bone.
—_————[—
Collagen molecule

Fig. 22.1 : A diagrammatic representation of the structure of collagen and fibril
(X and Y represent amino acids other than glycine)
In principle, all types of collagen are triple helical structures. The triple helix may occur throughout the molecule, or only a part of the molecule.
Type | mature collagen, containing about 1000 amino acids (for each polypeptide chain) possesses triple helical structure throughout the molecule. It is composed of three similar polypeptide chains twisted around each other to form a rod like molecule of 1.4 nm diameter, and about 300 nm length (Fig.22.1). The amino acid composition of collagen is unique. Approximately '/3rd of the amino acids are contributed by glycine i.e. every third amino acid in collagen is glycine. Hence, the repetitive amino acid sequence of collagen is represented by (Gly-X-Y),, where X and Y represent other amino acids. Thus, collagen may be regarded as a polymer of glycine-led tripeptide. Among the other amino acids, proline and hydroxyproline are present in large quantities (about 100 residues each). These two amino acids confer rigidity to the collagen molecule.
The triple helical structure of collagen is stabilized by an extensive network of hydrogen bonds, covalent cross-links, electrostatic and hydrophobic interactions, and van der Waals forces.
The triple
helical molecules
of collagen
assemble and
like fibers
in form
elongated the
fibrils, tissues.
The and
fibril then
rod formation
occurs by a quarter staggered alignment i.e. each triple helix of collagen is displaced longitudinally from its neighbour by about one-quarter of its length (Fig.22.1).
The strength of the collagen fibers is contributed by the covalent cross links formed between lysine and hydroxylysine residues. The degree of collagen cross-linking increases with age. Thus, in older people, the collagen containing tissues (e.g. skin, blood vessels) become less elastic and more stiff, contributing to health complications.

Collagen synthesis occurs in fibroblasts, and the cells related to them e.g. osteoblasts in bones, chondroblasts in cartilage, odontoblasts in teeth.
Collagen is synthesized on the ribosomes in a precursor form namely preprocollagen. This contains a signal peptide which directs the protein to reach the endoplasmic reticulum (ER). In the ER, the signal peptide is cleaved to form procollagen. The latter undergoes extensive posttranslational modifications (hydroxylation and glycosylation) and disulfide bonds formation. The procollagen so formed is secreted into the extracellular medium, and subjected to the action of aminoproteinase and carboxyproteinase to remove the terminal amino acids.
This is followed by a spontaneous assembly of the polypeptide chains (with about 1000 amino acids in each) to form triple helical structures of collagen.

The biosynthesis of collagen is a complex process, involving at least 30 genes (in humans), and about 8 post-translational modifications. Expectedly, many inherited diseases due to gene mutations, linked with collagen formation have been identified. A few of them are listed below.
| Ehlers-Danlos syndrome-a group of inherited disorders characterized by hyperextensibility of skin, and abnormal tissue fragility.
1 Alport syndrome—-due to a defect in the formation of type IV collagen fibres found in the basement membrane of renal glomeruli. The patients exhibit hematuria and_ renal diseases.
1 Osteogenesis imperfecta—characterized by abnormal fragility of bones due to decreased formation of collagen.
| Epidermolysis bullosa—due to alteration in the structure of type VII collagen. The victims exhibit skin breaks and blisters formation even for a minor trauma.
Scurvy This is a disease due to the deficiency of vitamin C (ascorbic acid). Although not a genetic disease, scurvy is related to the improper formation of collagen, hence referred here (vitamin C is needed for the posttranslational modifications of collagen). Scurvy is characterized by bleeding of gums, poor wound healing and subcutaneous hemorrhages.
Lathyrism : It is a disease of bone deformities caused by the consumption of Kesari dal (Lathyrus sativa) in some parts of India. The toxic compound namely f-oxalyl aminoalanine (BOAA), found in kesari dal, interferes with the cross-linking of lysine amino acids in collagen. BOAA is found to inhibit enzyme lysyl oxidase.

Elastin
is
another
important
(besides
collagens) connective tissue protein. It is mainly
responsible for the extensibility and elasticity of tissues. Elastin is found in large quantities in lungs, arterial blood vessels, elastic ligments etc.
Elastin is synthesized as tropoelastin which undergoes __ post-translational = modifications (formation of hydroxyproline, and no hydroxylysine). Compared to collagen, elastin structure is simple—no triple helix, no repeat sequence of (Gly-X-Y),.

| Williams syndrome is a genetic disease due to impairment in elastin synthesis. The connective tissue and central nervous system are affected.
1 Decreased synthesis of elastin is found in aging of skin and pulmonary emphysema.

Fibrillin is a structural component of myofibrils found in various tissues.
Marfan syndrome is a genetic disorder due to a mutation in the gene for fibrillin. It is characterized by hyperextensibility of joints and skeletal system. Consequently, the patients of Marfan syndrome are tall, and have long digits. These patients may also have cardiovascular complications. Some researchers believe that Abraham Lincoln was a victim of Marfan syndrome.

Fibronectin, a glycoprotein, is closely involved in the interaction of cells with extracellular matrix. It actively participates in cell adhesion and cell migration. In general, tumor cells are deficient in fibronectin which results in the lack of adhesion among the tumor cells that may often lead to metastasis.

The basal lamina of glomerular membrane (of renal cells) contains laminin. In fact, laminin is one of the first extracellular proteins synthesized during embryogenesis. It is actively involved in
neuronal growth and nerve regeneration. In the patients of Alzheimer's disease, high concentrations of laminin are found.

Keratins are structural proteins found in hair, skin, nails, horns etc. The 3 polypeptides of keratin form a-helical structure and are held together by disulfide bonds. The toughness and strength of keratin are directly related to the number of disulfide bonds. Thus, the harder keratin possesses more disulfide bonds. The mechanical strength of the hair is attributed to disulfide bonds.

When the hair is exposed to moist heat, the a-helices of a-keratin can be stretched. This results in the formation of B-conformation from a-helices. On cooling, the hair structure is reverted back to a-conformation. This property of aand B-conformations of keratin is exploited in hair waving or curling.
The hair to be curled is first bent to appropriate shape. By applying a reducing agent, the disulfide bonds (of cystine) are converted to sulfhydyl groups (cysteine). This results in the uncoiling of o-helical structure. After some time, th reducing agent is removed, and an oxidizing agent is added. This allows the formation of some new disulfide bonds between cysteine residues (Fig.22.2). The hair is now washed and cooled. The desired curls are formed on the hair due to new disulfide bonds and altered a-helical structure of keratin. It may however, be noted that a permanent curling of hair is not possible. The new hair that grows will be the native original hair only (without curls).

Proteoglycans are conjugated proteins containing glycosaminoglycans (GAGs). Several proteoglycans with variations in core proteins and GAGs are known e.g. syndecan, betaglycan, aggrecan, fibromodulin. For more information on the structure and functions of proteoglycans Refer Chapter 2. GAGs, the components of proteoglycans, are affected in a group of
genetic disorders namely mucopolysaccharidoses (Chapter 13).

The proteins that are involved in the movement of body organs (e.g. muscle, heart, lung) are regarded as contractile proteins. It is worthwhile to understand the basic structure of muscle before learning the contractile proteins.

Muscle is the single largest tissue of the human
body.
Muscle constitutes
about
20%
of body
mass at
birth,
40%
in young
adults and
Fig. 22.4 : A diagrammatic representation of the thin filament of a sarcomere.
(anisotropic or A bands) and light bands (isotropic or I bands) are observed. The less dense central region of A band is referred to as H band (or H line). A narrow and dense Z line bisects the I band. The region of the muscle fibre between two Z lines is termed as sarcomere. Sarcomere is the functional unit of muscle.

In the electron microscopy, it is further observed that the myofibrils are composed of thick and thin longitudinal filaments. The thick filaments contain the protein myosin, and are confined to A band. The thin filaments lie in the I band, and can extend into A band (but not into H line). These thin filaments contain the proteins actin, tropomyosin and troponin.
During the course of muscle contraction, the thick and thin filaments slide over each other (sliding filament model of muscle contraction). Consequently, the H bands and I bands shorten. However, there is no change in the length of thick and thin filaments. The length of sarcomere which is around 2300 nm in an extended form of myofibril is reduced to 1500 nm in a contracted form (Fig.22.3B).
More than 20% of the muscle mass_ is composed of proteins. This is largely contributed by structural proteins namely actin, myosin, and the actin cross-linking proteins, tropomyosin and troponin. Muscle also contains other proteins — myoglobin, collagen, enzymes etc. The term sarcopenia is used to indicate the loss of skeletal muscle mass with age.

Actin is a major constituent of thin filaments of sarcomere. It exists in two forms — monomeric G-actin (i.e. globular actin) and polymeric F-actin (i.e. filament actin). G-actin constitutes about 25% of the muscle proteins by weight. In the presence of Mg2* ions, G-actin polymerizes to form an insoluble double helical F-actin with a thickness of 6-7 nm (Fig.22.4).
Tropomyosin and troponin : These two are cross-linking proteins found in association with actin. Although, minor in terms of mass, they are important in terms of their function. Tropomyosin, composed of two chains, attaches to
Fig. 22.5 : A diagrammatic representation of myosin along with its digested products by trypsin and papain (G-Globular head).
F-actin in the grooves (Fig.22.4). Troponin consists of three polypeptide chains — troponin T (TpT binding to tropomyosin), tropinin I (TpI that inhibits F-actin myosin interaction) and troponin C (TpC, calcium binding polypeptide). TpC is comparable to calmodulin.

Myosins are actually a family of proteins with about 15 members. The myosin that is predominantly present in muscle is myosin II.
In terms of quantity, myosin constitutes approximately 55% of muscle protein, and is found in thick filaments. Myosin is composed of six polypeptide chains (hexamer). It contains one pair of heavy (H) chains, and two pairs of light (L) chains.
Limited digestion of myosin with trypsin and papain has helped to understand its structure and function
(Fig.22.5).

When myosin is digested with trypsin, two fragments namely light meromyosin (LMM) and heavy meromyosin (HMM) are produced. Light meromyosin represents the a-helical fibres of the tail of myosin, and cannot bind to F-actin.

globular portions of myosin. HMM_ inhibits ATPase activity and binds to F-actin.
On digestion by papain, heavy meromyosin yields two sub-fragments S-1 and S-2 (HMM S-1, HMM S-2). HMM S-2 fragment is fiber-like, does not bind to F-actin and has no ATPase activity. On the other hand, HMM S-1 is globule-like, binds to L-chains, and possesses ATPase activity.

An outline of the reactions involving muscle contraction is depicted in Fig.22.6, and briefly described in the next page.
Fig. 22.6 : Major biochemical events occurring during a cycle of muscle contraction and relaxation (The numbers 1-5 represent the steps in muscle contraction; The zig zag rounds indicate high energy states).
1. During the relaxation phase of muscle contraction, the S-1 head of myosin hydrolyses ATP to ADP and Pi. This results in the formation high energy ADP-Pi myosin complex.
2. On contraction, the muscle gets stimulated (through the participation of actin, Ca2*, troponin, tropomyosin etc.) to finally form actinmyosin-ADP-Pi complex.
3. The next step is the power stroke which drives movement of actin filaments over myosin filaments. This is followed by the release of ADP and Pi, and a conformation change in myosin. The actin-myosin complex is in a low energy state.
4. A fresh molecule of ATP now binds to form actin-myosin ATP complex.
5. Actin is released, as myosin-ATP has low affinity for actin. This step is crucial for relaxation which is dependent on the binding of ATP to actin-myosin complex.
A fresh cycle of muscle contraction and relaxation now commences with the hydrolysis of ATP and the formation of ADP-Pi-myosin complex. It has to be noted that it is ultimately the ATP that is the immediate source of energy for muscle contraction.

ATP is a constant source of energy for muscle contraction and relaxation cycle. ATP can be generated from the following ways.
| By substrate level phosphorylation of glycolysis using glucose or glycogen.
| By oxidative phosphorylation.
1 From creatine phosphate.

There are a large number of other proteins that are involved in the structure and functions of muscle. These include titin, nebulin, dystrophin, calcineurin and desmin. Titin is the largest protein known. The gene coding for dystrophin is the largest gene (2,300 bp).

Muscular dystrophy is a hereditary disease in which muscles progressively deteriorate. This is caused by mutations in the gene (located on Xchromosome) coding for the protein dystrophin.

The process of protein folding is complex and has been briefly described in Chapter 25. Sometimes, improperly folded proteins may be formed (either spontaneous or by gene mutations). Such misfolded proteins usually get degraded within the cell. However, as the individuals age, the misfolded proteins accumulate and cause a number of diseases. Prion diseases and amyloidosis, two groups of diseases due to protein misfolding are briefly discussed.

The term prion represents proteinous infectious agents. Prion proteins (PrP) are the altered forms of normal proteins. However, no differences in the primary structure (i.e. amino acid sequence) and_ post-translational modifications are observed.
Prion proteins are implicated as causative agents in the following diseases.
1 Transmissible spongiform encephalopathies (TSEs) and Creutzfeldt Jacob disease in humans.
1 Scrapie in sheep
1 Bovine spongiform encephalopathy (popularly
known as
mad cow
disease)
in cattle.
Kuru is an interesting prion disease. It was first reported in Papau New Guinea in the tribal people who practice cannibalism (they eat the brains of the dead people).
As of now, there is no treatment for prion diseases. Transmissible spongiform encephalopathies are invariably fatal in humans.

The term amyloids is used to refer to the altered proteins (with B-sheets) that accumulate in the body, particularly in the nervous system. Amyloids are formed by protein misfolding or due to gene mutations. They are not infectious agents as prion proteins. However, as the age advances, amyloids accumulate, and they have been implicated in many degenerating diseases. A total of at least 15 different proteins are involved in amyloidosis.

disorder, affecting
about
5-10%
of the
people above
60
years of
memory personality
age.
It is
loss, characterized
confusion, changes
As the
disease
a
vegetative after
by
__
hallucinations, with
abnormal progresses,
state, the
onset behaviour.
the and
patient may
of the
disease may
enter die
after
10
years manifestations.
The
Fig. 22.7 : A model for the formation of infectious prions (Red thick lines represent B-sheets in protein).
accumulation of amyloids (in the form of amyloid plaque) has been clearly demonstrated in the patients of Alzheimer's disease.
A specific protein, namely B-amyloid which is prone for self aggregation is believed to be the causative agent of Alzheimer's disease,

Water, Human = 87.6. Water, Cow = 87.2. Water, Buffalo = 83.5. Water, Goat = 87.0. Total solids (g/dl), Human = 12.4. Total solids (g/dl), Cow = 12.8. Total solids (g/dl), Buffalo = 16.5. Total solids (g/dl), Goat = 13.0. Carbohydrates (g/dl), Human = 7.5. Carbohydrates (g/dl), Cow = 4.4. Carbohydrates (g/dl), Buffalo = 5.4. Carbohydrates (g/dl), Goat = 4.6. Lipids (g/dl), Human = 3.8. Lipids (g/dl), Cow = 3.8. Lipids (g/dl), Buffalo = 6.5. Lipids (g/dl), Goat = 3.5. Proteins (g/dl), Human = 1.1. Proteins (g/dl), Cow = 3.3. Proteins (g/dl), Buffalo = 4.3. Proteins (g/dl), Goat = 3.7. Calcium (mg/dl), Human = 35. Calcium (mg/dl), Cow = 150. Calcium (mg/dl), Buffalo = 160. Calcium (mg/dl), Goat = 175. Magnesium (mg/dl), Human = 2.2. Magnesium (mg/dl), Cow = 13. Magnesium (mg/dl), Buffalo = 10. Magnesium (mg/dl), Goat = 8. Phosphorus (mg/dl), Human = 16. Phosphorus (mg/dl), Cow = 100. Phosphorus (mg/dl), Buffalo = 100. Phosphorus (mg/dl), Goat = 70. Sodium (mg/dl), Human = 15. Sodium (mg/dl), Cow = 60. Sodium (mg/dl), Buffalo = 60. Sodium (mg/dl), Goat = 50. Potassium (mg/dl), Human = 55. Potassium (mg/dl), Cow = 140. Potassium (mg/dl), Buffalo = 130. Potassium (mg/dl), Goat = 85

B-Amyloid is formed from a conformational transformation of o-helix. Apolipoprotein E promotes the conformational change of a-amyloid to B-amyloid.
in Table 22.1. Water is the major constituent, with a concentration in the range of 83-87%, depending on the species. The remaining 13-17% is made up of solids—carbohydrates, lipids, proteins, minerals and vitamins.

The specialized fluids of the body are milk, cerebrospinal fluid, amniotic fluid, pleural fluid, aqueous humor, sweat and tears. In a broader perspective, blood and plasma also biological fluids. Their biochemical importance is discussed elsewhere (Hemoglobin, Chapter 10; Plasma proteins, Chapter 9; Diagnostic enzymes, Chapter 6; Acid-base balance, Chapter 21). Urine is an excretory biological fluid. (Note : Serum is prepared in the laboratory, hence in a strict sense, not a natural biological fluid)

Milk is secreted by mammary glands. It is almost a complete natural food. Colostrum refers to the mother's milk secreted during the first few days after delivery. Milk is the only food for the offsprings of mammals on their birth.

The major constituents of milk in different species—human, cow, buffalo and goat are given

Milk contains the disaccharide lactose which imparts sweetness. Human milk has a higher concentration of lactose (7.5%) compared to milk of other species. Thus, human milk is sweet enough for the babies to relish. Milk sugar (lactose) serves two major functions.
1. It provides galactose, a structural unit for the growing infant.
2. In the intestine, it gets metabolized to lactic acid which eliminates harmful bacteria.

The lipids in the milk are dispersed as small globules. Milk fat is mainly composed of triacylglycerols. Monoand diacylglycerols are also present in trace quantities. The fatty acids found in milk (i.e. in TG) are mostly medium or short chain, and saturated e.g. palmitic acid, myristic acid, stearic acid, lauric acid and butyric acid. Oleic acid, an unsaturated fatty acid, is also present.

The major milk proteins are casein (about 80%) and lactalbumin. Small concentrations of enzymes (proteases, lipase, lysozyme) and immunoglobulins are also found.
Milk casein (a phosphoprotein) is almost a complete protein (next to egg albumin), containing all the essential amino acids. It is present in milk in the form of aggregates called micelles. The white colour of milk is due to the dispersion of calcium caseinate micelles.
Whey proteins : If milk is acidified, casein gets precipitated at isoelectric point (pH 4.7). The supernatant fluid contains whey proteins (20% of milk proteins). These include lactalbumin, lactoglobulin and various enzymes.

Milk is rich in calcium, magnesium, phosphorus, sodium, potassium and_ chlorine. However, milk is a poor source of iron and copper.

Both fat soluble and water soluble vitamins are found in good concentration in milk. However milk is deficient in vitamin C.

Due to variability in the nutrient composition (carbohydrates, fats and proteins), the calorific value of milk from different species varies. Thus, human milk can provide about 70 Cal/100 ml, while for buffalo milk, it is around 95 Cal/100 ml.

Cerebrospinal fluid is a clear, colourless liquid formed within the cavities (ventricles) of brain and around the spinal cord. CSF originates in the choroid plexus (as an_ ultrafiltrate of plasma) and returns to blood through arachnoid villi. About 500 ml of CSF is formed everyday. However, at any given time, there is about 120-150 ml CSF in the system. Further, CSF is completely replaced about three times a day.

As the brain has no lymphatic system, CSF drains into the ventricular system and moves into spaces surrounding the brain and spinal cord. The major functions of CSF are listed.
1 CSF serves as a hydraulic shock absorber. It can diffuse the force from a hard blow to the skull that might otherwise cause severe injury.
1 It helps in the regulation of intracranial pressure.
1
It is
believed that
CSF
influences the
hunger sensation
and eating
behaviours.

Cerebrospinal fluid is usually collected by a spinal puncture for the purpose of biochemical analysis. The puncture is performed in the lumbar region, between the third and fourth, or between the fourth and fifth lumbar vertebrae.
The sterile lumbar puncture (spinal tap) is carried out in a side lying (lateral) position with head fixed into the chest and knees. This position helps to increase the space between the lumbar vertebrae so that the needle can be inserted with ease. A sitting position of the patient with head flexed to chest can also be used for lumbar puncture.

The normal composition of cerebrospinal fluid is given in the Table 22.2. From the diagnostic point of view, the total cell count of lymphocytes (Reference : 0-5 x 10°/1), protein concentration (15-45 mg/dl) and glucose concentration (45-85 mg/dl) are important.
In the Table 22.3, the major alterations in the CSF in the disease states are given. The total cell count and protein content are increased while glucose concentration is reduced in tuberculosis meningitis. In case of brain tumors, there is no change in total cell count while the protein concentration may be marginally increased.
The colour and appearance of CSF is sometimes a guiding factor in the disease diagnosis. For instance, CSF is opalescent and slightly yellow coloured in tuberculosis meningitis.

Volume, ABLE 22.2 Normal composition of cerebrospinal fluid.Description/concentration = 90-150 ml. Appearance, ABLE 22.2 Normal composition of cerebrospinal fluid.Description/concentration = Clear and colourless. Specific gravity, ABLE 22.2 Normal composition of cerebrospinal fluid.Description/concentration = 1.006-1.008. Osmolality, ABLE 22.2 Normal composition of cerebrospinal fluid.Description/concentration = 280-290 mOsm/kg. Total cell count (lymphocytes), ABLE 22.2 Normal composition of cerebrospinal fluid.Description/concentration = 0-5 u 10 6 /l. pH, ABLE 22.2 Normal composition of cerebrospinal fluid.Description/concentration = 7.3-7.4. Protein, ABLE 22.2 Normal composition of cerebrospinal fluid.Description/concentration = 15-45 mg/dl. A/G ratio (albumin/globulin), ABLE 22.2 Normal composition of cerebrospinal fluid.Description/concentration = 8 : 1. Glucose, ABLE 22.2 Normal composition of cerebrospinal fluid.Description/concentration = 45-85 mg/dl. Chloride, ABLE 22.2 Normal composition of cerebrospinal fluid.Description/concentration = 118-130 mEq/l. Calcium, ABLE 22.2 Normal composition of cerebrospinal fluid.Description/concentration = 2.1-2.7 mEq/l. Sodium, ABLE 22.2 Normal composition of cerebrospinal fluid.Description/concentration = 145-155 mEq/l. Potassium, ABLE 22.2 Normal composition of cerebrospinal fluid.Description/concentration = 2.0-3.5 mEq/l

Amniotic fluid
is
a
membranes throughout
liquid and
the fetus.
pregnancy.
fluid increases
produced
It surrounds
by the
the
The with
volume volume
the increases
gestation)
of gestational
from to
350
age.
30
ml
(at ml
to
500-1000
with
(at
20
weeks), ml.
Amniotic some
desquamated
2
and fluid
is fetal
fetus amniotic
Thus, the
weeks of
thereafter almost
cells clear
and
a
little

- 1 It provides physical protection to the fetus.
1
- Amniotic fluid is a medium for the exchange
of various chemicals.

amniotic fluid
The term amniocentesis is used for the process by which amniotic fluid is collected for analysis. The diagnostic importance of amniotic fluid is given below.
Assessment of fetal maturity : Fetal maturity can
be assessed
by cytological
staining of
fat cells,
and estimation
(>
1.6
mg/dl indicates
fetal maturity).
Lung maturity : The fetal lung maturity is evaluated by measuring lecithin-sphingomyelin (L/S) ratio. A L/S ratio of 2 : 1 or more indicates lung maturity. If L/S ratio is less than 1.2 : 1, it is better to delay the induced delivery until the lung has become more mature.
Diagnosis of congenital disorders : Amniotic fluid analysis is useful for the prenatal diagnosis of congenital disorders. Some of the important ones are listed.
1 Chromosomal disorders syndorme. such as Down's
Metabolic disorders e.g. cystic fibrosis.
Sex-linked disorders e.g. hemophilia.
lipid.
- 1 Enzyme defects e.g. Tay-Sachs disease.

Subarachnoid hemorrhage, T ABLE 22.3 Changes in cerebrospinal fluid in the disease states.Colour and appearance = Slightly blood colour. Subarachnoid hemorrhage, T ABLE 22.3 Changes in cerebrospinal fluid in the disease states.Total cell count = RBC and WBC present. Subarachnoid hemorrhage, T ABLE 22.3 Changes in cerebrospinal fluid in the disease states.Protein = Increased. Subarachnoid hemorrhage, T ABLE 22.3 Changes in cerebrospinal fluid in the disease states.Glucose = Almost normal
Assessment of hemolytic diseases : Estimation of bilirubin in amniotic fluid is useful to evaluate the severity of hemolytic diseases.
Measurement of o-fetoprotein : Increased levels of a-fetoprotein (normal 15-40 ug/ml during gestation; at 40 weeks <1.0 pg/ml) are associated with neural tube defects, fetal distress, Turner syndrome. Elevated o-fetoprotein may also indicate a possible death of the fetus.

Pleural fluid is the filtrate of plasma, and is present in a minimal quantity in pleural cavity. The amount of pleural fluid increases in disease states due to pleural effusion.
Pleural fluid may be transudate or exudate, based on the composition. If the ratio of protein content between pleural fluid and plasma is less than 0.5, it is transudate. If this ratio is more than 0.5, the pleural fluid is exudate.
(increased hydrostatic pressure) and obstruction of lymph flow (chylous effusion).
Exudate is viscous in nature with specific gravity >1.015. It may clot, and is associated with inflammation or malignancy. Exudate accumulates in various infections (tuberculosis, pneumonia, rheumatoid arthritis) and some cancers (e.g. lung cancer).
Biochemical analysis of exudate is useful in
certain disease states
1 Amylase activity increased in pancreatitis.
1 Rheumatoid factor elevated in rheumatoid arthritis.
1 Carcinoembryonic antigen (CEA) increased in malignancy.
1 Triacylglycerols elevated in chylothorax.

Transudate is fluid-like, with specific gravity <1.015. It will not clot, and is not associated with inflammation. Transudate is observed in nephrotic syndrome (due to low osmotic pressure of plasma), congestive cardiac failure
Aqueous humor is the fluid that fills the anterior chamber of the eye. This fluid is responsible for maintaining the intraocular tension. Aqueous humor, secreted by the ciliary body, enters the anterior chamber. Blockade in the flow of aqueous humor causes glaucoma due to increased intraocular pressure.

+ Improper formation of collagen is associated with certain genetic diseases e.g. Ehlers- Danlos syndrome (abnormal tissue fragility), osteogenesis imperfecta (abnormal fragility of bones).
+ Defective formation of collagen is observed in scurvy, caused by vitamin C deficiency. This results in bleeding of gums and poor wound healing.
+ Hair waving (curling) through artificial means is possible with suitable alterations in the structure of keratins.
+ Muscular dystrophy occurs due a mutation in the gene coding for the protein dystrophin.
+ Protein misfolding results in prion diseases (e.g. mad cow disease) and amyloidosis (Alzheimer’s disease).
+
Biochemical
analysis
of
CSF
is
useful
for
the
diagnosis
of
certain
diseases
—
tuberculosis
meningitis
(increased
protein
and
decreased
glucose
concentrations).
+ Amniotic fluid is analysed to assess fetal maturity (creatinine concentration > 1.6 mg/ dl), lung maturity (lecithin-sphingomyelin ratio > 2 : 1) and for the prenatal diagnosis of congenital disorders (e.g. hemophilia, Down’s syndrome).

1., 1 = The major proteins of connective tissue are collagen, elastin, fibrillin, laminin and proteoglycans. Among these, collagen is the most abundant, constituting one-third of the total body proteins.. 2., 1 = Type I mature collagen is a triple helical structure i.e. contains three polypeptide chains each with about 1000 amino acids. The repetitive amino acid sequence of collagen is (Gly-X-Y) n . Glycine constitutes about 1/3 rd of the amino acids while X and Y represent other amino acids.. 3., 1 = Keratins are structural proteins found in hair, skin, nails and horns. The strength of the keratins is directly related to the number of disulfide bonds.. 4., 1 = Muscle is the single largest tissue of the human body (30-40% of body weight). It is composed of fibre cells into which myofibrils are embedded. Each myofibril contains alternating A and I bands. Sarcomere is the functional unit of muscle.. 5., 1 = Actin, myosin, tropomyosin and troponin are the major contractile proteins found in muscles. The muscle contraction and relaxation occur due to the active involvement of these proteins. ATP is the immediate source of energy for muscle contraction.. 6., 1 = Proper folding of proteins is essential for their structure. Misfolding of proteins results in certain diseases e.g. mad cow disease, Alzheimer's disease.. 7., 1 = The specialized fluids of the body include milk, cerebrospinal fluid, amniotic fluid, aqueous humor, sweat and tears.. 8., 1 = Milk is almost a complete food with various nutrients-carbohydrates, lipids, proteins, vitamins and minerals. However, milk is deficient in vitamin C, iron and copper.. 9., 1 = Cerebrospinal fluid is an ultrafiltrate of plasma. In the disease, tuberculosis meningitis, the total cell count and protein concentration are increased, while glucose concentration is decreased in CSF.. 10., 1 = Amniotic fluid is a liquid produced by the fetus. Its biochemical analysis is important for the diagnostic purpose - assessment of fetal maturity, diagnosis of congenital diseases.

1 . Give an account of the structure and functions of collagen. Add a note on the abnormalities associated with collagen.
oP Give an account of the composition of milk.
WN Describe the muscle proteins, and muscle contraction.
Discuss the protein misfolding and various diseases related to it.
Describe the functions and composition of cerebrospinal fluid. Add a note on the alterations in CSF in diseased states.

(a) Biosynthesis of collagen, (b) Collagen and scurvy, (c) Elastin, (d) Light and heavy meromyosins, (e) Prion diseases, (f) Amyloidosis, (g) Hair waving, (h) Vitamins and minerals in milk, (i) Collection of Amniotic fluid.
CSF, (j)

1. The most abundant protein in mammals
2. The amino acid that contributes to one-third of the total number of amino acids in collagen
oO . Name the vitamin deficient in milk
ON . Name the major protein found in the structure of thin filaments of sarcomere . The white colour of milk is due to the dispersion of
DOH . Name the carbohydrates associated with the structure of proteoglycans
1 . The fetal lung maturity is evaluated by measuring
.
The toxic compound that interferes with the cross-linking of lysine in collagen, causing lathyrism
. Marfan syndrome is a genetic disorder due to a mutation of the gene coding for
. The region of the muscle fibre between two Z lines is termed as
ratio.

1 . The number of polypeptide chains present in collagen
12. The functional unit of muscle
13. The immediate source of energy for muscle contraction
14. One of the following minerals is lacking in milk
15. One of the following biochemical parameters is increased in tuberculosis meningitis
(a) 1 (b) 2 (c) 3d) 4.
(a) Fibre cell (b) Myofibril (c) H band d) Sarcomere.
(a) ATP (b) Creatine phosphate (c) GTP d) Phosphoenol pyruvate.
(a) Calcium (b) Sodium (c) Iron d) Potassium.
(a) Glucose (b) Protein (c) Sodium d) Chloride.
hether a man eats for living or lives for eating, food is his prime concern. Nutrition may be defined as the utilization of food by living organisms. Biochemists have largely
contributed to
the science
of nutrition.
areas—ideal nutrition, undernutrition and overnutrition. \deal nutrition is the concern of everyone. Undernutrition is the prime concern of developing countries while overnutrition is a serious concern of developed countries.
Nutrition significantly promotes man's development, his health and welfare. The subject nutrition, perhaps, is the most controversial. This is due to the fact that nutrition is concerned with food, and everyone feels competent enough to talk like an expert on nutrition. Further, high public awareness and the controversial reports by scientists also contribute to the controversy.
Methodology in nutrition : Most of the existing knowledge on nutrition is originally derived from animal experimentation. This is despite the fact that there may exist several differences in the biochemical composition between man and animals! For instance, some animals can synthesize ascorbic acid while man cannot do so.
Study of human nutrition : The study of nutrition may be logically divided into three
A sound knowledge of chemistry and metabolism of foodstuffs (carbohydrates, lipids, proteins, vitamins and minerals) is an essential prerequisite for a better understanding of nutrition. The reader must, therefore, first refer these chapters. The principles of nutrition with special reference to energy demands, carbohydrates, fats, proteins, recommended dietary/daily allowances (RDA), balanced diet and nutritional disorders are discussed in the following pages.

Food is the fuel source of the body. The ingested
food undergoes
metabolism to
liberate energy
body.
the vital
activities of
the

Carbohydrate, Energy value (Cal/g).In bomb calorimeter = 4.1. Carbohydrate, Energy value (Cal/g).In the body = 4. Fat, Energy value (Cal/g).In bomb calorimeter = 9.4. Fat, Energy value (Cal/g).In the body = 9. Protein, Energy value (Cal/g).In bomb calorimeter = 5.4. Protein, Energy value (Cal/g).In the body = 4. Alcohol, Energy value (Cal/g).In bomb calorimeter = 7.1. Alcohol, Energy value (Cal/g).In the body = 7

The calorific value (energy content) of a food is calculated from the heat released by the total combustion of food in a calorimeter.
Unit of heat : Calorie is the unit of heat. One calorie represents the amount of heat required to rise the temperature of one gram of water by 1°C (i.e. from 15° to 16°C). A calorie is too small a unit. Therefore, it is more conveniently expressed as kilocalories (1,000 times calorie) which is represented by kcal or simply Cal (with capital 'C').
The joule is also a unit of energy used in some countries. The relationship between calories and joules (J) is
1 Cal (1 kcal) = 4.128 KJ
The joule is less commonly used by nutritionists.
Calorie value of foods The values of

: energy the three principal foodstuffs—carbohydrate, fat and protein—measured in a bomb calorimeter and in the body are given in the Table 23.1. The carbohydrates and fats are completely oxidized (to CO and H,0) in the body; hence their fuel values, measured in the bomb calorimeter or in the body, are almost the same. Proteins, however, are not completely burnt in the body as they are converted to products such as urea, creatinine and ammonia, and excreted. Due to this reason, calorific value of protein in the body is less than that obtained in a bomb calorimeter. The energy values of carbohydrates, fats and proteins (when utilized in the body) respectively, are 9 and 4
Alcohol is a recent addition to the calorie
(7 Cal/g) contribution, as it is a significant dietary component for some people.
It must be noted that the nutrients, namely vitamins and minerals, have no calorific value, although they are involved in several important body functions, including the generation of energy from carbohydrates, fats and proteins.

The respiratory quotient (R. Q.) is the ratio of the
volume of
CO2
produced to
the volume
of
Oy utilized in the oxidation of foodstuffs.
Carbohydrates
The carbohydrates
are completely oxidized and their R. Q. is close
to 1, as represented below for glucose.
CoH1206 + 602 —~ 6CO?z + 6H2O
R. Q. for carbohydrate co. 2
=
Fats Fats have relatively lower R.Q. since
: they have a low oxygen content. For this reason, fats require more O, for oxidation. The R.Q. for the oxidation of the fat, tristearin is given below.
2 Cs57H119006 + 16 302 —> 114 CO2 + 110 H2O
Q.
for
CO.
R.
fat
2.
=
114
=
0.7.
O,
163
Proteins : The chemical nature of proteins is highly variable, and this cannot be represented by any specific formula. By — indirect measurements, the R.Q. of protein is found to be around 0.8.
Mixed diet : The R. Q. of the diet consumed is dependent of the relative composition of carbohydrates, fats and proteins. For a normally ingested diet, it is around 0.8.

Man consumes energy to meet the fuel demands _ of the three ongoing processess in the body.
1. Basal metabolic rate
2. Specific dynamic action
3. Physical activity.
Besides the above three, additional energy supply is needed during growth, pregnancy and lactation.

Basal metabolism or basal metabolic rate (BMR) is defined as the minimum amount of energy required by the body to maintain life at complete physical and mental rest in the postabsorptive state (i.e. 12 hours after the last meal). It may be noted that resting metabolic rate (RMR) is in recent use for BMR.
Under the basal conditions, although the body appears to be at total rest, several functions within the body continuously occur. These include working of heart and other organs, conduction of nerve impulse, reabsorption by renal tubules, gastrointestinal motility and ion transport across membranes (Nat-K+ pump consumes about 50% of basal energy).

Preparation of the subject For the measurement of BMR the subject should be awake, at complete physical and mental rest, in a post-absorptive state and in a comfortable surrounding (at 25°C).
Measurement : The BMR is determined either by the apparatus of Benedict and Roth (closed circuit device) or by the Douglas bag method (open circuit device). The former is more frequently used.
By Benedict-Roth method, the volume of Oy consumed (recorded on a graph paper) by the subject for a period of 2-6 minutes under basal conditions is determined. Let this be A liters for 6 minutes. The standard calorific value for one liter O2 consumed is 4.825 Cal.
Heat produced in 6 min = 4.825 x A
Heat produced in one hour = 4.825A x 10
Units of BMR : BMR is expressed as Calories per square meter of body surface area per hour ie. Cal/sq.m/hr.
For the calculation of body surface area, the simple formula devised by Du Bois and Du Bois is used.
A
=
H0-725
%
W425
x
71.84
where A = Surface area in cm*
H = Height in cm
W = Weight in kg.
To convert the surface area into square meters (m2), divide the above value (cm2) by 10,000. Nomograms of body surface area (directly in m2) from heights and weights are readily available in literature.
Normal values of BMR : For an adult man 35-38 Cal/sq. m/hr; for an adult woman 32-35 Cal/sq.m/hr. A BMR value between -15% and +20% is considered as normal.
Some authors continue to represent BMR as Cal/day. For an adult man BMR is around 1,600 Cal/day, while for an adult woman around 1,400 Cal/day. This is particularly important for easily calculating energy requirements per day.

1. Surface area : The BMR is directly proportional to the surface area. Surface area is related to weight and height.
2. Sex : Men have marginally higher (about 5%) BMR than women. This is due to the higher proportion of lean muscle mass in men.
3. Age : In infants and growing children, with lean muscle mass, the BMR is higher. In adults, BMR decreases at the rate of about 2% per decade of life.
4. Physical activity : BMR is increased in persons (notably athletes) with regular exercise. This is mostly due to increase in body surface area.
5. Hormones : Thyroid hormones (T3 and T4) have a stimulatory effect on the metabolism of the body and, therefore, BMR. Thus, BMR is raised in hyperthyroidism and reduced in hypothyroidism. |n fact, the measurement of BMR was earlier used to assess thyroid function.
The other hormones such as epinephrine, cortisol, growth hormone and sex hormones increase BMR.
6. Environment : In cold climates, the BMR is higher compared to warm climates.
7. Starvation During the periods of starvation, the energy intake has an inverse relation with BMR, a decrease up to 50% has been reported. This may be an adaptation by the body.
8. Fever : Fever causes an increase in BMR. An elevation by more than 10% in BMR is observed for every 1°C rise in body temperature.
9. Disease states : BMR is elevated in various infections, leukemias, polycythemia, cardiac failure, hypertension etc. In Addison's disease (adrenal insufficiency), BMR_ is marginally lowered.
10. Racial variations : The BMR of Eskimos is much higher. The BMR of Oriental women living in USA is about 10% less than the average BMR of American women.

BMR is important to calculate the calorie requirement of an individual and planning of diets. Determination of BMR is useful for the assessment of thyroid function. In hypothyroidism, BMR_ is lowered (by about -40%) while in hyperthyroidism it is elevated (by about + 70%). Starvation and certain disease conditions also influence BMR_ (described above).

The phenomenon of the extra heat production by the body, over and above the calculated caloric value, when a given food is metabolized by the body, is known as specific dynamic action (SDA). It is also known as calorigenic action or thermogenic action or thermic action (effect) of food.
SDA for different foods For a food containing 25 g of protein, the heat production from the caloric value is 100 Cal (25 x 4 Cal).
However, when 25 g protein is utilized by the body, 130 Cal of heat is liberated. The extra 30 Cal is the SDA of protein. Likewise, consumption of 100 Cal of fat results in 113 Cal and 100 Cal of carbohydrate in 105 Cal, when metabolized in the body. SDA _ for protein, fat and carbohydrate are 30%, 13% and 5%, respectively. Thus, proteins possess the highest SDA while carbohydrates have the lowest.
SDA for mixed diet : For a mixed diet, the SDA is not an additive value of different foods but it is much less. The presence of fats and carbohydrates reduces the SDA of proteins. Fats are most efficient in reducing SDA of foodstuffs. For a regularly consumed mixed diet, the SDA is around 10%.
Significance of SDA : For the utilization of foods by the body, certain amount of energy is consumed from the body stores. This is actually an expenditure by the body for the utilization of foodstuffs. It is the highest for proteins (30%) and lowest for carbohydrates (5%) and for a mixed diet around 10%. It is, therefore, essential that an additional 10% calories should be added to the total energy needs (of the body) towards SDA. And the diet should be planned, accordingly. (SDA is quite comparable to the handling charges levied by a bank for an outstation cheque).
The higher SDA for protein indicates that it is not a good source of energy. Fat is the best source of energy due to its lowering effect on SDA. However, excessive utilization of fat leads to ketosis.
Mechanism of SDA : The exact cause of SDA is not known. It is generally believed that SDA of foods is due to the energy required for digestion, absorption, transport, metabolism and storage of foods in the body.

Intravenous administration of amino acids or the oral ingestion of proteins gives the same SDA. This shows that the SDA of proteins is not due to their digestion and absorption. Hepatectomy abolishes SDA, thereby indicating that SDA is closely connected with the metabolic functions of liver. The SDA of proteins is primarily to meet the energy requirements for
Sitting (quietly), Type of physical energy expenditure BMR, about 65 Cal/hr). = 25. Standing (quietly), Type of physical energy expenditure BMR, about 65 Cal/hr). = 30. Writing/eating/reading, Type of physical energy expenditure BMR, about 65 Cal/hr). = 30. Car driving, Type of physical energy expenditure BMR, about 65 Cal/hr). = 60. Typing, Type of physical energy expenditure BMR, about 65 Cal/hr). = 75. Household work (dish washing), Type of physical energy expenditure BMR, about 65 Cal/hr). = 80. Walking (slow), Type of physical energy expenditure BMR, about 65 Cal/hr). = 130. Sexual intercourse, Type of physical energy expenditure BMR, about 65 Cal/hr). = 140. Cycling (slow), Type of physical energy expenditure BMR, about 65 Cal/hr). = 150. Running (moderate), Type of physical energy expenditure BMR, about 65 Cal/hr). = 500 600. Swimming Walking upstairs, Type of physical energy expenditure BMR, about 65 Cal/hr). = 800
deamination, synthesis of urea, biosynthesis of proteins, synthesis of triacylglycerol (from carbon skeleton of amino acids). It has been demonstrated that certain amino acids (phenylalanine, glycine and alanine) increase the SDA. It is a common. experience _ that consumption of a protein rich diet makes us feel warm and comfortable in cold weather. This is due to the high SDA of proteins.
The SDA of carbohydrates is attributed to the energy expenditure for the conversion of glucose to glycogen.
As regards fat, the SDA may be due to its storage, mobilization and oxidation.

The physical activity of the individual is highly variable. The amount of energy needed for this depends mainly on the duration and intensity of muscular activity. The expenditure of energy for the various physical activities has been calculated (Table 23.2).
For the sake of convenience, the individuals are grouped into four categories with regard to
their physical activity and the requirement of energy.
Light work
—
30-40% of BMR
(teachers, office workers, doctors)
Moderate work
—
40-50% of BMR
(housewives, students)
Heavy work
—
50-60% of BMR
(agricultural labourers, miners)
Very heavy work
—
60-100% of BMR
(construction workers, rickshaw pullers)

As already stated, the three factors—basal metabolic rate, specific dynamic action and physical activity—determine the energy needed by the body. In an individual with light work, about 60% of the calories are spent towards BMR, about 30% for physical activity and about 10% to take care of the SDA.
The daily requirement of energy is rather variable which depends on the BMR (in turn depends on age, sex, body size etc.) and physical activity. As per some rough calculation, caloric requirements of adults per day (Cal/day) are in the following ranges.
Light work
— 2,200-2,500
—
_
2,500-2,900
Heavy work
— 2,900-3,500
— 3,500-4,000

Dietary carbohydrates are the chief source of energy. They contribute to 60-70% of total caloric requirement of the body. Incidentally, carbohydrate rich foods cost less.
Carbohydrates are the most abundant dietary constituents, despite the fact that they are not essential nutrients to the body. From the nutritional point of view, carbohydrates are grouped into 2 categories.
1. Carbohydrates utilized by the body— starch, glycogen, sucrose, lactose, glucose, fructose etc.
2. Carbohydrates not utilized (not digested) by the body—cellulose, hemicellulose, pectin, gums etc.
Among the carbohydrates utilized by the body, starch is the most abundant. The consumption of starch has distinct advantages due to its bland taste, satiety value and slow digestion and absorption. Sucrose (the table sugar), due to its sweetness, can be consumed to a limited extent. Excessive intake of sucrose causes dental caries, and an_ increase in plasma lipid levels is associated with many health complications.

1. Major sources of energy : Carbohydrates are the principal source of energy, Yhplying 60-80% of the caloric requirements of the body.
2. Protein sparing action : Proteins perform a specialized function of body building and growth. The wasteful expenditure of proteins to meet the energy needs of the body should be curtailed. Carbohydrates come to the rescue and spare the proteins from being misused for caloric purpose.
3. Absolute requirement by brain : The brain and other parts of central nervous system are dependent on glucose for energy. Prolonged hypoglycemia may lead to irreversible brain damage.
4. Required for the oxidation of fat : Acetyl CoA is the product formed in fatty acid oxidation. For its further oxidation via citric acid cycle, acetyl CoA combines with oxaloacetate, the latter is predominantly derived from carbohydrates. It may therefore be stated 'Fat burns in a fuel of carbohydrate'.
5. Synthesis of pentoses : Pentoses (e.g. ribose) are the constituents of several compounds in the body e.g. nucleic acids (DNA, RNA), coenzymes (NADt, FAD). These pentoses are produced in carbohydrate metabolism.
6. Synthesis of fat : Excess consumption of carbohydrates leads to the formation of fat which is stored in the adipose tissue.
7. Importance of non-digestible carbohydrates : These are the carbohydrates not utilized by the body. Yet, they are important since they improve bowel motility, prevent constipation, lower cholesterol absorption and improve glucose tolerance (details discussed later).

HFCS are produced' from __ glucose by employing enzymatic processes — that convert glucose into fructose. HFCS contain approximately equal amount of glucose and fructose. They are commonly used as substitutes for sucrose in beverages, including soft drinks, and processed foods.
The composition and metabolism of HFCS and sucrose are similar except that HFCS is ingected as a mixture of monosaccharides. Further, most studies have shown that there is no significant difference between sucrose and HFCS with regard to post-prandial glucose and insulin response.

There are variations in the increase and fall of blood glucose levels after the ingestion of different carbohydrate containing foods. These quantitative differences are assayed by glycemic index which measures the time course of postprandial glucose concentrations from a graph. Glycemic index may be defined as the area under the blood glucose curve after the ingestion of a food compared with the area under the blood glucose curve after taking the same amount of carbohydrate as glucose. It is expressed as percentage.
Area under
the blood
glucose curve
after ingestion
of test
meal
Area under the curve after ingestion of glucose
A graphic representation of high and low glycemic
indices is
Fig.
23.1.
The glycemic index of a complex carbohydrate (i.e. starch) is lower than a refined carbohydrate (i.e. glucose). This is explained on the basis of slow digestion and absorption of
Glycemic index =
x
100
complex carbohydrates. Further, the glycemic index of carbohydrate is usually lower when it is combined with protein, fat or fiber. The glycemic index of some selected foods is given in Table 23.3.
The food item like ice cream has relatively lower glycemic index. This may be explained on the basis of high fat content which lowers the glucose absorption.
The nutritional importance of glycemic index is controversial. This is due to the fact that the foods with low glycemic index need not be good for health. However, low glycemic index foods usually have higher satiety value (creating a sense of stomachfulness), and thus may be helpful in limiting the caloric intake. Nutritionists are of the opinion that foods with high fiber content and low glycemic index (e.g. whole grains, fruits, vegetables) should be preferred for consumption.

Carbohydrates are
abundant naturally
occurring sugar
(99%), cereals
foods.
These
(60-80%), in
pulses table
(50-60%), roots
and tubers
(20-40%)
and bread
(50-60%).

In a well balanced diet, at least 40% of the caloric needs of the body should be met from carbohydrates.

The complex carbohydrates that are not digested by the human enzymes are collectively referred to as dietary fiber. Soluble fibers, mostly found in fruits and legumes, dissolve in water and form gels (e.g., pectins, gums, mucilages). Insoluble fibers, present in vegetables and grains, adsorb water and swell up (e.g. cellulose hemicellulose, lignin). Certain fibers (e.g. pectins, gums) are digestible by intestinal bacterial ezymes. It may be stated that once regarded as a nutritional waste, a lot of importance is now given to dietary fiber in human health.

1. Prevents constipation : Fiber can absorb 10-15 times its own weight of water, by drawing fluid into the lumen of the intestine. This increases bowel motility, and prevents constipation, besides decreasing the risk of hemorrhoids and diverticulosis.
2. Eliminates bacterial toxins : Fiber also adsorbs toxic compounds produced by intestinal bacteria and helps in their easier expulsion.
3. Decreases GIT cancers The lower incidence of cancers of gastrointestinal tract (e.g. colon and rectum) in vegetarians compared to non-vegetarians is attributed to dietary fiber.

Glucose, Glycemic index = 100. Carrots, Glycemic index = 90-95. Honey, Glycemic index = 80-90. Bread, rice, Glycemic index = 70-80. Banana, potato, Glycemic index = 60-70. Sweet potato, Glycemic index = 50-60. Oranges, apples, Glycemic index = 40-45. Ice cream, milk, Glycemic index = 35-40. Fructose, Glycemic index = 20-25. Soy beans, Glycemic index = 15-20
4. Improves glucose tolerance Fiber improves glucose tolerance by the body. This is mainly done by a diminished rate of glucose absorption from the intestine.
5. Reduces plasma cholesterol level : Fiber decreases the absorption of dietary cholesterol from the intestine. Further, fiber binds with the bile salts and reduces their enterohepatic circulation. Thus, degradation of cholesterol to bile salts and its disposal from the body is increased.
6. Satiety value : Dietary fiber adds to the weight of the foodstuff ingested and gives a sensation of stomachfullness, giving satiety without consumption of excess calories.

Some of the food fads went to the extent of ingesting huge quantities of rice bran to achieve all the benefits of fiber. This led to several complications. In general, the harmful effects are mostly observed in people consuming large quantities of dietary fiber.
1. Digestion and absorption of protein are adversely affected.
2. The intestinal absorption — of minerals (e.g. Ca, P, Mg) is decreased. certain
3. Intestinal bacteria ferment some fibers, causing flatulence and often discomfort.
Drinking plenty of water along with fiber is advocated
to reduce
adverse effects
of fiber.

Fruits, leafy vegetables, vegetables, whole wheat legumes, rice bran etc. are rich sources of fiber. The ideal way to increase fiber intake is to reduce intake of refined carbohydrates, besides eating vegetables, fresh fruits and whole grains. In general, vegetarians consume more fiber than non-vegetarians. An average daily intake of about 30 g fiber is recommended.

Triacylglycerols (fats and oils) are the concentrated dietary source of fuel, contributing
15-50% of the body energy requirements. Phospholipids and cholesterol (from animal sources) are also important in nutrition. The nutritional and biochemical functions of fat, phospholipids and cholesterol have already been discussed in detail and the reader must invariably refer them now (Chapters 3 and 14).

Dietary lipids have two major nutritive functions.
1. Supply triacylglycerols that normally constitute about 90% of dietary lipids which is a concentrated source of fuel to the body.
2. Provide essential fatty acids and fat soluble
vitamins (A, D, E and K).

The unsaturated fatty acids which the body cannot synthesize and, therefore, must be consumed in the diet are referred to as essential fatty acids (EFA).
The fatty acids—linoleic and linolenic acid— cannot be synthesized by humans. In a strict sense, only these two are essential fatty acids. Arachidonic acid can be synthesized from linoleic acid in some animal species, including man. However, the conversion efficiency of linoleic acid to arachidonic acid is not clearly known in man. And for this reason, some nutritionists recommend that it is better to include some amount of arachidonic acid also in the diet.

1. Essential fatty acids are the structural of biological membranes.
components
2. Participate in the transport and utilization
of
cholesterol.
3. Prevent fat accumulation in the liver.
4.
Required
for
the
—
synthesis
—
of
prostaglandins.
5. Maintain proper growth and reproduction
of the

The EFA deficiency in humans _ is characterized by a scaly dermatitis on the posterior and lateral parts of limbs and buttocks. This condition is referred to as phrynoderma or toad skin.

The essential fatty acids, more frequently called polyunsaturated fatty acids (PUFA), are predominantly present in vegetable oils and fish oils. The rich vegetable sources include sunflower oil, cotton seed oil, corn oil, soyabean oil etc. The fat of animal origin (exception—fish), contain less PUFA e.g. butter, fat of meat, pork.

Nutritionists recommend that at least 30% of the dietary fat should contain PUFA. Very high intake of PUFA (i.e. totally replacing saturated fatty acids) may not be advisable. This is due to the fact that excess PUFA, unless accompanied by antioxidants (vitamin E, carotenes), is believed to be injurious to the cells due to the overproduction of free radicals.

These are long chain PUFA with double bond beginning at 3rd (w-3) and 6th (@-6) position from the methyl end. o-3 fatty acids (e.g. linolenic acid, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and w-6 fatty acids (e.g. arachidonic acid) are known to reduce serum cholesterol and triacylglycerols, thereby decrease the tendency for thrombosis, lower blood presure and reduce the risk of CHD. In recent years, @-3 fatty acids are included in infant formulas to promote brain development.

TFA possess double bonds and are formed during partial hydrolysis of vegetable oils. TFA are widely used in food industry due to long shelf-life. They increase LDL and decrease HDL, and thus promote altherogenesis and heart diseases. Therefore, TFA should be avoided in the diet, as far as possible.

Animal foods are the only dietary source of cholesterol. However, the role of dietary cholesterol on plasma cholesterol is less important than the amount and types of fatty acids consumed.

Consumption of dietary fats and oils is considered in terms of their contribution towards the energy needs of the body. There is a wide variation in fat intake. It is much higher (up to 50% of daily calories) in affluent societies compared to the poorer sections of the people (about 15% of calories). The recommended fat intake is around 20-30% of the daily calorie requirement, containing about 50% of PUFA.

Proteins have been traditionally regarded as 'body-building foods'. However, 10-15% of the total body energy is derived from proteins. As far as possible, carbohydrates spare proteins and make the latter available for body-building process. The functions carried out by proteins in a living cell are innumerable, a few of them are listed hereunder.

1. Proteins are the fundamental basis of cell structure and its function.
2.
All the
enzymes, several
hormones, immunoglobulins,
etc., are
proteins.
3. Proteins are involved in the maintenance of osmotic pressure, clotting of blood, muscle contraction etc.
4. During starvation, proteins (amino acids) serve as the major suppliers of energy. It may be noted that the structural proteins themselves serve as 'storage proteins' to meet the emergency energy needs of the body. This is in contrast to lipids and carbohydrates which have storage forms.

The nutritional importance of proteins is based on the content of essential amino acids (Chapter 4). There are ten essential amino acids—arginine, valine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan and threonine (code to recall—AV HILL MP TT). Of these two—namely arginine and _histidine—are —semi-essential. | The requirement of 8 essential amino acids per kg body weight per day is given in Table 23.4. Cysteine and tyrosine can respectively spare the requirement of methionine and phenylalanine.

Dietary protein is almost an exclusive source of nitrogen to the body. Thus, nitrogen balance truly represents the protein (16% of which is nitrogen) utilization and its loss from the body. Nitrogen balance is determined by comparing the intake of nitrogen (chiefly by proteins) and the excretion of nitrogen (mostly undigested protein in feces; urea and ammonia in urine). A normal healthy adult is in a nitrogen equilibrium since the daily dietary intake (I) is equal to the loss through urine (U), feces (F) and sweat (S).

The term fudge factor (approximately 3g) is used to represent nitrogen lost in feces, sweat and nails etc.
Valine, Requirements of essential amino acids.Requirement (mg/kg body weight/day) = 14. Isoleucine, Requirements of essential amino acids.Requirement (mg/kg body weight/day) = 12. Leucine, Requirements of essential amino acids.Requirement (mg/kg body weight/day) = 16. Lysine, Requirements of essential amino acids.Requirement (mg/kg body weight/day) = 12. Methionine *, Requirements of essential amino acids.Requirement (mg/kg body weight/day) = 10. Phenylalanine *, Requirements of essential amino acids.Requirement (mg/kg body weight/day) = 16. Tryptophan, Requirements of essential amino acids.Requirement (mg/kg body weight/day) = 3. Threonine, Requirements of essential amino acids.Requirement (mg/kg body weight/day) = 8. * Cysteine and tyrosine can, respectively, requirement of methionine and phenylalanine., Requirements of essential amino acids.Requirement (mg/kg body weight/day) = spare (partly) the
Thus, an individual is said to be in a nitrogen balance if the intake and output of nitrogen are the same (Fig. 23.2). There are two other situations—a positive and a negative nitrogen balance.
Positive nitrogen balance : This is a state in which the nitrogen intake is higher than the output. Some amount of nitrogen is retained in the body causing a net increase in the body protein. Positive nitrogen balance is observed in growing children, pregnant women or during recovery after serious illness.
Negative nitrogen balance : This is a situation in which the nitrogen output is higher than the input. The result is that some amount of nitrogen is lost from the body depleting the body protein. Prolonged negative nitrogen balance may even lead to death. This is sometimes observed in children suffering from = kwashiorkor — or marasmus.
Negative nitrogen balance may occur due to inadequate dietary intake of protein (deficiency of even a single essential amino acid) or destruction of tissues or serious illness. In all these cases, the body adapts itself and increases the breakdown of tissue proteins causing loss of nitrogen from the body.

Besides the major factors discussed above (growth, pregnancy, protein deficiency, injury, illness) several other factors influence nitrogen balance.
Hormones : Growth hormone and insulin pro- mote positive nitrogen balance while corticoste- roids result in balance.
negative nitrogen
Disease states : Cancer and uncontrolled
diabetes cause negative nitrogen balance.

Knowledge on the quantity of dietary protein alone is not sufficient to evaluate the nutritional importance of proteins. This is in contrast to dietary carbohydrates and lipids. The quality of the proteins which depends on the composition of essential amino acids is more important. Several laboratory methods are in use to assess the nutritive value of proteins. Of these, four methods—protein efficiency ratio, biological value, net protein utilization and chemical score—are discussed briefly.

This test consists of feeding weaning (21 day old) albino rats with a 10% test protein diet and recording the gain in body weight for a period of 4 weeks. PER is represented by gain in the weight of rats per gram protein ingested.
Gain in
PER
body weight
(g)
=
Protein ingested (g)
3.0;
The PER for egg protein is 4.5; for milk protein for rice protein 2.2.

The biological value of protein is defined as the percentage of absorbed nitrogen retained by the body.
BV
Nitrogen retained
=
x
100
Nitrogen absorbed
For the measurement of BV, the experimental animals, namely weaning albino rats are chosen. They are first fed with a protein-free diet for 10 days. Then they are kept on a 10% protein diet to be tested for BV. Urine and feces are collected for both the periods i.e. protein-free diet and protein diet. Nitrogen is estimated in the diet,
The biological value provides a reasonably good index for the nutritive value of proteins. But unfortunately this method has an inherent drawback. It cannot take into account the nitrogen that might be lost during the digestion process. For instance, if the ingested nitrogen is 100 mg, absorbed is 10 mg and retained is 8 mg, the BV 8/10 x 100 = 80. This figure is erroneous, since the major part of the protein (90 mg) did not enter the body at all for utilization.

NPU is a_ better nutritional index than biological value, since it takes into account the digestibility factor. The experimental procedure for NPU is similar to that of BV. Net protein utilization can be calculated as
NPU
_
x100
Nitrogen ingested

This is based on the chemical analysis of the protein for the composition of essential amino acids which is then compared with a reference protein (usually egg protein). The chemical score is defined as the ratio between the quantitity of the most limiting essential amino acid in the test
protein to the quantity of the same amino acid in
the egg protein, expressed as percentage.
Chemical
score
mg
of
the
limiting
amino
acid
/g
test
protein
=
-
-
—_
x
100
mg
of
the
same
amino
acid
/g
egg
protein
The
chemical
score
of
egg
protein,
for
any
one
of
the
essential
amino
acids,
is
taken
as
100
and
the
rest
of
the
proteins
are
compared.
In the Table 23.5, the four methods employed (PER, BV, NPU and chemical score) for the assessment of nutritive value of proteins are compared with regard to the different sources of dietary proteins. Although there are certain variations, anyone of these methods provides sufficient information on the nutritive value of proteins.

As is observed from the Table 23.5, the animal proteins are superior in their nutritive value compared to the proteins of vegetable origin. Further, some of the essential amino acids are limiting in certain foods. For instance, rice and wheat proteins are limiting in lysine and threonine while the protein of Bengal gram is limited in sulfur-containing amino acids (methionine and cystine).
It is
fortunate that
humans
(worldover)
have the
habit of
consuming
a
mixed diet,
with different foods, simultaneously. This helps to overcome the deficiency of certain essential amino acids in one food by being supplemented from the others. This phenomenon is referred to as mutual supplementation. For instance, an Indian diet with cereals (wheat, rice) is taken along with pulses (dal). The limitation of lysine and threonine in cereal proteins is overcome by their supplementation from dal proteins. Simultaneously, the limitation of — sulfurcontaining amino acids in dal is also compensated by the cereals which are rich in them.
The nutritive value of protein of a particular food can be enhanced by appropriate combination with other foods. Due to the consumption of mixed diets, dietary deficiency of essential amino acids is most uncommon. Further, the principle of mixed diet takes care to supply adequate quantities of essential amino acids to the people subsisting on pure vegetarian diets. It has to be remembered that the effect of mutual supplementation in proteins is best observed with the same meal (or at least on the same day).

The requirement of protein is dependent on
its
nutritive
value,
caloric
intake
and
physiological
states
(growth,
—
pregnancy,

Egg, PER = 4.5. Egg, BV = 94. Egg, NPU = 90. Egg, Chemical score = 100. Egg, Limiting amino acid(s) = Nil. Milk, PER = 3.0. Milk, BV = 84. Milk, NPU = 75. Milk, Chemical score = 65. Milk, Limiting amino acid(s) = S-Containing amino acids. Fish, PER = 3.0. Fish, BV = 85. Fish, NPU = 70. Fish, Chemical score = 60. Fish, Limiting amino acid(s) = Tryptophan. Meat, PER = 2.7. Meat, BV = 75. Meat, NPU = 76. Meat, Chemical score = 70. Meat, Limiting amino acid(s) = S-Containing amino acids. Rice, PER = 2.2. Rice, BV = 68. Rice, NPU = 60. Rice, Chemical score = 60. Rice, Limiting amino acid(s) = Lysine, threonine. Wheat, PER = 1.5. Wheat, BV = 58. Wheat, NPU = 47. Wheat, Chemical score = 42. Wheat, Limiting amino acid(s) = Lysine, threonine. Bengal gram, PER = 1.7. Bengal gram, BV = 58. Bengal gram, NPU = 47. Bengal gram, Chemical score = 45. Bengal gram, Limiting amino acid(s) = S-Containing amino acids. Red gram, PER = 1.5. Red gram, BV = 57. Red gram, NPU = 46. Red gram, Chemical score = 45. Red gram, Limiting amino acid(s) = S-Containing amino acids. Groundnut, PER = 1.7. Groundnut, BV = 55. Groundnut, NPU = 45. Groundnut, Chemical score = 44. Groundnut, Limiting amino acid(s) = Lysine, threonine, S-amino acids. Soyabean, PER = 2.1. Soyabean, BV = 65. Soyabean, NPU = 55. Soyabean, Chemical score = 55. Soyabean, Limiting amino acid(s) = S-Containing amino acids

lactation) of the individual. For an adult, 0.8-1.0 g protein/kg body weight/day is adequate. The requirement should be nearly double for growing children, pregnant and lactating women.

The protein content of foods is variable, cereals have 6-12%; pulses 18-22%; meat 1825%, egg 10-14%; milk 3-4% and_ leafy vegetables 1-2%. In general, the animal proteins are superior than vegetable proteins as the dietary source.

The nutritional aspects including metabolism, biochemical functions, dietary sources, requirements and associated disorders for vitamins (Chapter 7) and for minerals (Chapter 18) have already been discussed in much detail.

The
(RDA)
to be
good recommended
dietary/daily allowances
represents the
quantities provided
of the
in health
the diet
daily and
It must
be physical
for efficiency
remembered that
RDA
nutrients maintaining
of the
body.
is not
minimum amount
to just
meet the
body the
needs, but allowance is given for a safe margin.

1. Sex : The RDA for men is about 20% higher than that for women. Iron is an exception as the requirement is greater in menstruating women. Additional requirements (20-30% above normal) are needed for pregnant and lactating women.
2.
Age
:
the nutrient
requirement is
much higher
in the
growing age.
For instance,
the protein
requirement for
a
growing child
is about 2 g/kg body wt/day compared to 1 g/kg
body wt/day for adults.

The details of RDA for each of the nutrients in relation to age, sex and physiological status is described in the respective chapters. For a quick recapitulation, the RDA of macronutrients (carbohydrate, fat and protein) and selected micronutrients (vitamins and minerals) for an adult man weighing 70 kg are given in Table 23.6.

After discussing the nutritional aspects of dietary ingredients and their RDA, it is worthwhile to formulate a diet for man. A
Vay eR Ce cara UV mee me Cg PUM ME Ue
Nutrient(s)
Carbohydrates, RDA = 400. Carbohydrates, RDA = g. Fats, RDA = 70. Fats, RDA = g. Proteins, RDA = 56. Proteins, RDA = g *. Essential fatty acids, RDA = 4. Essential fatty acids, RDA = g. Vitamin A, RDA = 1,000. Vitamin A, RDA = P g **. Vitamin D, RDA = 5. Vitamin D, RDA = P g ***. Vitamin E, RDA = 10. Vitamin E, RDA = P g. Vitamin K, RDA = 70. Vitamin K, RDA = P g. Ascorbic acid, RDA = 60. Ascorbic acid, RDA = mg. Thiamine, RDA = 1.5 mg. Thiamine, RDA = 1.5 mg. Riboflavin, RDA = 2. Riboflavin, RDA = mg. Niacin, RDA = 20. Niacin, RDA = mg. Pyridoxine, RDA = 2. Pyridoxine, RDA = mg. Folic acid, RDA = 150. Folic acid, RDA = P g. Cobalamin, RDA = 2. Cobalamin, RDA = P g. Calcium, RDA = 800. Calcium, RDA = mg. Phosphorus, RDA = 800. Phosphorus, RDA = mg. Iron, RDA = 10. Iron, RDA = mg. * 0.8 g/kg body weight/day ; ** Retinol equivalents ; *** As cholecalciferol, RDA = * 0.8 g/kg body weight/day ; ** Retinol equivalents ; *** As cholecalciferol. * 0.8 g/kg body weight/day ; ** Retinol equivalents ; *** As cholecalciferol, RDA = * 0.8 g/kg body weight/day ; ** Retinol equivalents ; *** As cholecalciferol

balanced diet or prudent diet is defined as the diet which contains different types of foods, possessing the nutrients—carbohydrates, fats, proteins, vitamins and minerals—in a proportion to meet the requirements of the body. A balanced diet invariably supplies a little more of each nutrient than the minimum requirement to withstand the short duration of leanness and keep the body in a state of good health.
The basic composition of balanced diet is highly variable, as it differs from country to country, depending on the availability of foods. Social and cultural habits, besides the economic status, age, sex and physical activity of the individual largely influence the intake of diet.
The Nutrition Expert Group, constituted by the Indian Council of Medical Research has recommended the composition of balanced diets for Indians. This is done taking into account the commonly available foods in India. The composition of balanced diet (vegetarian and non-vegetarian), for an adult man is given Table 23.7.
fats and oils, sugar and groundnuts. Meat, fish and eggs are present in the non-vegetarian diets. In case of vegetarians, an additional intake of milk and pulses is recommended. The nutritional composition of the most commonly consumed Indian foods given in the Appendix VII. The nutritional aspects of milk are given in the Chapter 22.

Some people in developed countries (e.g. U.S.A) consume excessive quantities of certain nutrients. It is recommended that such people have to reduce the intake of total calories, total fat, saturated fatty acids, cholesterol, refined sugars and salt. The U.S. Government recommends a daily intake of less than 30% fat against the present 40-50% towards calories.

The Indian balanced diet is composed of cereals (rice, wheat, jowar), pulses, vegetables, roots and tubers, fruits, milk and milk products,
While the people of developing countries suffer from undernutrition, overnutrition is the major concern of the developed countries. Some of the important nutritional diseases are discussed hereunder.

work.Non-vegetarian (g) = 30. Sugar and jaggery, Sedentary work.Vegetarian (g) = 30. Sugar and jaggery, Sedentary work.Non-vegetarian (g) = 30. Sugar and jaggery, Moderate work.Vegetarian (g) = 40. Sugar and jaggery, Moderate work.Non-vegetarian (g) = 40. Sugar and jaggery, Heavy work.Vegetarian (g) = 55. Sugar and jaggery, Heavy work.Non-vegetarian (g) = 55. Groundnuts, Sedentary work.Vegetarian (g) = . Groundnuts, Sedentary work.Non-vegetarian (g) = . Groundnuts, Moderate work.Vegetarian (g) = . Groundnuts, Moderate work.Non-vegetarian (g) = . Groundnuts, Heavy work.Vegetarian (g) = 50. Groundnuts, Heavy work.Non-vegetarian (g) = 50
** Formulations based on the recommended dietary (daily) allowances (RDA) of the Indian Council of Medical Research (1989)

Protein-energy malnutrition (PEM)—sometimes called protein-calorie —_ malnutrition (PCM)—is the most common nutritional disorder of the developing countries. PEM is widely prevalent in the infants and pre-school children. Kwashiorkor and marasmus are the two extreme forms of protein-energy malnutrition.

The term kwashiorkor was introduced by Cicely Williams (1933) to a nutritional disease affecting the people of Gold Coast (modern Ghane) in Africa. Kwashiorkor literally means sickness of the deposed child i.e. a disease the child gets when the next baby is born.
Occurrence and causes Kwashiorkor is predominantly found in children between 1-5 years of age. This is primarily due to insufficient intake of proteins, as the diet of a weaning child mainly consists of carbohydrates.
Clinical symptoms The major clinical manifestations of kwashiorkor include stunted growth, edema (particularly on legs and hands), diarrhea, discoloration of hair and skin, anemia, apathy and moonface.
Biochemical manifestations : Kwashiorkor is associated with a decreased plasma albumin concentration (<2 g/dl against normal 3-4.5 g/dl), fatty liver, deficiency of K* due to diarrhea. Edema occurs due to lack of adequate plasma proteins to maintain water distribution between blood and_ tissues. Disturbances in the metabolism of carbohydrate, protein and fat are also observed. Several vitamin deficiencies occur. Plasma retinol binding protein (RBP) is reduced. The immunological response of the child to infection is very low.
Treatment : Ingestion of protein-rich foods or the dietary combinations to provide about 3-4 g of protein/kg body weight/day will control kwashiorkor. The treatment can be monitored by measuring plasma albumin concentration, disappearance of edema and gain in body weight.

Marasmus literally means 'to waste'. It mainly occurs in children under one year age. Marasmus is predominantly due to the deficiency of calories. This is usually observed in children given watery gruels (of cereals) to supplement the mother's breast milk.
The symptoms of marasmus include growth retardation, muscle wasting (emaciation), anemia and weakness. A marasmic child does not show edema or decreased concentration of plasma albumin. This is a major difference to distinguish marasmus from kwashiorkor. In the Table 23.8, a comparison between kwashiorkor and marasmus is given.

The patients of certain chronic diseases like cancer and AIDS are fequently undernourished, resulting in a codition called cachexia. This is mainly due to the loss of body proteins as a result of hypermetabolism, particularly increased basal metabolic rate. Further, increased metabolisms leading to thermogenesis is also observed in cancer and AIDS.

Anemia is characterized by lower concentration of hemoglobin (reference 14-16 g/dl) with a reduced ability to transport oxygen. Nutritional anemias are classified based on the size of erythrocytes.
| Microcytic anemia—most common, with reduced RBC size. Occurs due to the deficiency of iron, copper and pyridoxine.
1 Macrocytic anemia—RBC are large and immature. Mostly due to the deficiency of folic acid and vitamin By).
1 Normocytic anemia—Size of the RBC is normal, but their quantity in blood is low. Mostly found in _ protein-energy malnutrition.

Age of onset, Kwashiorkor = Pre-school children (1-5 yr). Age of onset, Marasmus = Weaned infants (< 1 yr). Main nutritional cause, Kwashiorkor = Low protein intake. Main nutritional cause, Marasmus = Low calorie intake. Body weight, Kwashiorkor = 60-80% of normal. Body weight, Marasmus = Less than 60% of normal. Growth, Kwashiorkor = Mild retardation. Growth, Marasmus = Severe retardation. Oedema, Kwashiorkor = Present. Oedema, Marasmus = Absent. Facial appearance, Kwashiorkor = Moon face. Facial appearance, Marasmus = Like old man's face. Abdomen, Kwashiorkor = Protruding. Abdomen, Marasmus = Shrunken. Skin, Kwashiorkor = Dermatitis. Skin, Marasmus = Dry and atrophic. Muscles, Kwashiorkor = Undergo wasting. Muscles, Marasmus = Weak and atrophic. Subcutaneous fat, Kwashiorkor = Present. Subcutaneous fat, Marasmus = Absent. Vitamin deficiencies, Kwashiorkor = Present. Vitamin deficiencies, Marasmus = Present. Serum albumin, Kwashiorkor = 0.5-2 g/dl. Serum albumin, Marasmus = 2-3 g/dl. Serum cortisol, Kwashiorkor = Normal or decreased. Serum cortisol, Marasmus = Increased. Fasting blood glucose, Kwashiorkor = Decreased. Fasting blood glucose, Marasmus = Decreased. Serum K +, Kwashiorkor = Decreased. Serum K +, Marasmus = Normal

demands as per the situation. Selected examples of therapeutic diets are listed
There are several other nutritional disorders which have been discussed elesewhere. These include obesity, body mass index and atherosclerosis (Chapter 14); vitamin deficiency disorders—xerophthalmia, _ rickets, _ beri-beri, pellagra, scurvy and pernicious anemia (Chapter 7); goiter and other disorders of minerals (Chapter 18). The biochemical ramifications of starvation are discussed along with the integration of metabolism (Chapter 16).

Diet therapy in disease states is a part of nutrition. Therapeutic diets are usually not palatable. However, they possess high or low amounts of specific nutrients to meet the body
1 Liquid diets — for post-operative patients
1 Low sodium diets — for hypertensive people
1 Low fat diet —for patients of malabsorption syndrome
1 Low protein diet—for patients of hepatic encephalopathy, renal failure
| High fiber diet — for patients of constipation, CHD, diabetes mellits

In Atkins diet, fat and protein are high, and carbohydrate very low (<50g/day; <10% of a 2000 Cal/day). Atkins diet is advocated for weight loss programmes of obese people. High fat diet reduces appetite, and thus food intake. However, long term human consumption of Atkins diet is controversial.

The nutritional status of an individual is important in clinical practice. The dietary requirements of nutrients are variable, and are mostly related to age and sex, and physiological status. Some examples are listed
1 Infants and young children have increased needs of protein, iron and calcium.
1 During teenage, high calcium and magnesium are recommended.
1 In pregnancy, and lactation, the requirements of iron, calcium, magnesium, folic acid and vitamin B, and B,, are increased.
1 Elderly people have to take more of vitaminsB, and Bj), folic acid and vitamin D, and minerals chromium, zinc etc. However, vitamin A intake should be restricted in the elderly to avoid toxicity.

Drug
Risk of nutrient deficiencies
Diuretics
Anticonvulsants
Isoniazid
Corticosteroids
Vitamin
B,, vitamin
B,,, folic
acid
Potassium, zinc
Folic acid, vitamin D, vitamin K
Vitamin B,
Vitamin D, calcium, potassium, zinc
Alcohol
Thiamine, vitamin B,, folic acid liver and kidney diseases reduce the formation of active vitamin D (calcitriol), and storage and utilization of vitamins—folic acid, vitamin By and vitamin D.

In general, illness and metabolic stress increase the nutritional demands. For instances,
Many drugs are known to lead to potential nutrient deficiencies (Table 23.9). For instance, oral contraceptives may result in deficiency manifestations of vitamin Bg, By and folic acid.

+ Most of the information on human nutrition is based on the research carried out in experimental animals.
+ The body at total rest (physical and mental) requires energy to meet the basal require-
ments
such
as
working
of
heart,
of
nerve
impulse,
membrane
transport
etc.
+ Carbohydrates are the most abundant dietary constituents despite the fact that they are not essential nutrients to the body.
+ Adequate intake of dietary fiber prevents constipation, eliminates bacterial toxins, reduces GIT cancers, improves glucose tolerance and reduces plasma cholesterol.
+ In general, vegetable oils are good sources for essential fatty acids while animal proteins are superior for the supply of essential amino acids.
+ The biological value (BV) of protein represents the percentage of absorbed nitrogen
retained
in
the
body.
The
BV
for
egg
protein
is
94
while
that
for
rice
is
68.
+ The recommended dietary allowance (RDA) of nutrients depends on the sex and age, besides pregnancy and lactation in the women.
+ The habit of consuming mixed diet by man is largely responsible to enhance the nutritive value of foods, besides preventing several nutritional deficiencies (e.g. amino acids).
+ Kwashiorkor and marasmus, the two extreme forms of protein-energy malnutrition in infants and pre-school children, are highly prevalent in developing countries.

Nutrigenomics, a new field of nutrition, relates to 3 distinct areas of nutrient-gene interactions
1.
Nutritional genetics
involves
__
the individual
genetic on
the differences
nutrient intake.
2.
Nutritional nutrient
epigenetics induced
and their
influence describes
changes in
DNA, the
such as
DNA
- methylation, histone post-translational changes etc.
3. Nutritional transcriptomics involves the effects of nutrients on gene expression. The role vitamins A and D on nuclear receptors, and in turn on gene expression is well known.
Nutrigenomics may soon revolutionize the clinical and nutritional practice, and result in individualized RDA for disease prevention and treatment.

1., 1 = The calorific values of carbohydrates, fats and proteins respectively are 4, 9 and 4 Cal/g. These three nutrients (macronutrients) supply energy to the body to meet the requirements of basal metabolic rate, specific dynamic action and physical activity.. 2., 1 = Basal metabolic rate (BMR) represents the minimum amount of energy required by the body to maintain life at complete physical and mental rest, in the post-absorptive state. The normal BMR for an adult man is 35-38 Cal/m 2 body surface/hr.. 3., 1 = Specific dynamic action (SDA) is the extra heat produced by the body over and above the calculated calorific value of foodstuff. It is higher for proteins (30%), lower for carbohydrates (5%), and for a mixed diet, it is around 10%.. 4., 1 = Carbohydrates are the major source of body fuel supplying about 40-70% of body calories. The non-digested carbohydrates (cellulose, pectin), referred to as fiber, prevent constipation, improve glucose tolerance and reduce plasma cholesterol.. 5., 1 = Lipids are the concentrated source of energy. They also provide essential fatty acids (linoleic and linolenic acids) and fat-soluble vitamins (A, D, E and K).. 6., 1 = Proteins are the body building foods that supply essential amino acids, besides meeting the body energy requirement partly (10-15%).. 7., 1 = Several methods are employed to assess the nutritive value of proteins. These include protein efficiency ratio, biological value, net protein utilization and chemical score.. 8., 1 = The recommended dietary allowance (RDA) represents the quantities of nutrients to be provided daily in the diet for maintaining good health and physical efficiency. The RDA for protein is 1g/kg body weight/day.. 9., 1 = A balanced diet is the diet which contains different types of foods with the nutrients, namely carbohydrates, fats, proteins, vitamins and minerals, in a proportion to meet the body requirements.. 10., 1 = Protein-energy malnutrition (PEM) is the most common nutritional disorder in the developing countries. Kwashiorkor is primarily due to inadequate protein intake while marasmus is mainly caused by calorie deficiency.

. Define BMR. Discuss the factors affecting BMR.
. Describe the different methods employed for the nutritional evaluation of proteins.
. Define a balanced diet. Formulate a diet for a medical student.
. Discuss the protein-energy malnutrition with special reference to kwashiorkor.
aA . Give an account of the recommended dietary allowance (RDA) for macro- and micronutrients.
bBwWN
(a) Essential amino acids, (b) Mutual supplementation of proteins, (c) Caloric value of foods,
(d)
Specific
dynamic
action,
(e)
Energy
requirements
of
man,
(f)
Fiber
in
nutrition,
(g)
Kwashiorkor,
(h) Limiting amino acids, (i) Nitrogen balance, (j) Biological value of proteins.
1. One calorie of energy is equivalent to
Joules (KJ).
2. The endocrine organ most predominantly associated with BMR is
3. The non-digestible carbohydrates are collectively known as
4. The major source of energy to the body is supplied by
5. The nutritional assessment method used to know the most limiting essential amino acid in relation to a standard is
protein
. The daily normal requirement of protein in an adult is DO
. The percentage of absorbed nitrogen retained in the body represents ON
. The proteins of Bengal gram are limiting in the amino acids
. The nutrient required in greater amounts in menstruating women compared to men is oD
. The biochemical parameter often used as an index for monitoring the recovery from kwashiorkor
is
11. The specific dynamic action (SDA) is the greatest for the following foodstuff
(a) Protein (b) Carbohydrate (c) Fat (d) Vitamins.
12. The reference protein for the calculation of chemical score
(a) Meat protein (b) Fish protein (c) Milk protein (d) Egg protein.
13. The essential amino acid limiting in rice
(a) Methionine (b) Tryptophan (c) Lysine (d) Histidine.
14. A continuous supply of energy to the body is necessary to meet the requirements of
(a) Basal metabolic rate (b) Specific dynamic action (c) Physical activity (d) All of them.

15. One of the following is the most important essential fatty acid in the diet
(a) Linoleic acid (b) Arachidonic acid (c) Oleic acid (d) Palmitic acid.

A single mammalian fetal cell contains only a few picograms (10-!2 g) of DNA. It is surprising that this little quantity of DNA stores information that will determine the differentiation and every function of an adult animal.

RNA molecules, in principle, can perform the cellular functions that are carried out by DNA. In fact, many viruses contain RNA as the genetic material. Chemically, DNA is more stable than RNA. Hence, during the course of evolution, DNA is preferred as a more suitable molecule for long-term repository of genetic information.

The biological information flows from DNA to RNA, and from there to proteins. This is the central dogma of life (Fig.24.1). It is ultimately the DNA that controls every function of the cell through protein synthesis.
As the carrier of genetic information, DNA in a cell must be duplicated (replicated), maintained and passed down accurately to the daughter cells. Three distinct processes are designed for this purpose. The 'three Rs' of DNA-replication, recombination, and repair, are dealt with in this chapter. There are certain common features between the three Rs.
1 They act on the same substrate (DNA).
1 They are primarily concerned with the making and breaking of phosphodiester bonds (the backbone of DNA structure).
1 Enzymes used in the three processes are mostly similar/comparable.

DNA is the genetic material. When the cell divides, the daughter cells receive an identical copy of genetic information from the parent cell.
Replication is a process in which DNA copies itself to produce identical daughter molecules of DNA. Replication is carried out with high fidelity which is essential for the survival of the species. Synthesis of a new DNA molecule is a complex process involving a series of steps.
The salient features of replication in prokaryotes are described first. This is followed by some recent information on the eukaryotic replication.

The parent DNA has two strands complementary to each other. Both the strands undergo simultaneous replication to produce two daughter molecules. Each one of the newly synthesized DNA has one-half of the parental DNA (one strand from original) and one-half of new DNA (Fig.24.2). This type of replication is known as semiconservative since half of the original DNA is conserved in the daughter DNA. The first experimental evidence for the semiconservative DNA replication was provided by Meselson and Stahl (1958).

The initiation of DNA synthesis occurs at a site called origin of replication. In case of prokaryotes, there is a single site whereas in eukaryotes, there are multiple sites of origin.
Fig. 24.2 : DNA replication—semiconservative.
These sites mostly consist of a short sequence of A-T base pairs. A specific protein called dna A (20-50 monomers) binds with the site of origin for replication. This causes the double-stranded DNA to separate.

The two complementary strands of DNA separate at the site of replication to form a bubble. Multiple replication bubbles are formed in eukaryotic DNA molecules, which is essential for a rapid replication process (Fig.24.3).

For the synthesis of new DNA, a short fragment of RNA (about 5-50 nucleotides, variable with species) is required as a primer. The enzyme primase (a_ specific RNA polymerase) in association with single-stranded binding proteins forms a complex called primosome, and produces RNA primers. A constant synthesis and supply of RNA primers should occur on the lagging strand of DNA. This is in contrast to the leading strand which has almost a single RNA primer.
Fig. 24.3 : Schematic representation of multiple replication bubbles in DNA replication.

The replication of DNA occurs in 5' to 3' direction, simultaneously, on both the strands of DNA. On one strand, the leading (continuous or forward) strand—the DNA _ synthesis _ is continuous. On the other strand, the lagging (discontinuous or retrograde) strand—the synthesis of DNA is discontinuous. Short pieces of DNA (15-250 nucleotides) are produced on the lagging strand.
In the replication bubble, the DNA synthesis occurs in both the directions (bidirectional) from the point of origin.

The separation of the two strands of parent DNA results in the formation of a replication fork. The active synthesis of DNA occurs in this region. The replication fork moves along the parent DNA as the daughter DNA molecules are synthesized.
DNA helicases : These enzymes bind to both the DNA strands at the replication fork. Helicases move along the DNA helix and separate the strands. Their function is comparable with a zip opener. Helicases are dependent on ATP for energy supply.
These
Single-stranded DNA binding (SSB) proteins : are also known as DNA helix-destabilizing proteins. They possess no enzyme activity. SSB proteins bind only to single-stranded DNA (separated by helicases), keep the two strands separate and provide the template for new DNA synthesis. It is believed that SSB proteins also protect the single-stranded DNA degradation by
nucleases.

The synthesis of a new DNA strand, catalysed by DNA _ polymerase Ill, occurs in 5'—3' direction. This is antiparallel to the parent template DNA strand. The presence of all the four deoxyribonucleoside triphosphates (dATP, dGTP, dCTP and dTTP) is an_ essential prerequisite for replication to take place.
The synthesis of two new DNA strands, simultaneously, takes place in the opposite direction—one is in a direction (5'3') towards the replication fork which is continuous, the other in a direction (5'—3') away from the replication fork which is discontinuous (Fig.24.4).
The incoming deoxyribonucleotides are added one after another, to 3' end of the growing DNA chain (Fig.24.5). A molecule of pyrophosphate (PPi) is removed with the addition of each nucleotide. The template DNA strand (the parent) determines the base sequence of the newly synthesized complementary DNA.

The DNA strand (leading strand) with _ its 3'-end (3'-OH) oriented towards the fork can be elongated by sequential addition of new nucleotides. The other DNA strand (lagging strand) with 5'-end presents some problem, as there is no DNA polymerase enzyme (in any organism) that can catalyse the addition of nucleotides to the 5' end (i.e. 3'-45' direction) of the growing chain. This problem however is solved by synthesizing this strand as a series of small fragments. These pieces are made in the normal 5'-3' direction, and later joined together.
Okazaki pieces : The small fragments of the discontinuously synthesized DNA are called
Fig. 24.5 : DNA replication with a growing complementary strand.
Another enzyme—DNA
II]—has been isolated. It participates in the DNA repair
process.
at least five distinct DNA polymerases are known in eukaryotes. Greek letters are used to number these enzymes.

As the double helix of DNA separates from one side and replication proceeds, supercoils are formed at the other side. The formation of supercoils can be better understood by comparing DNA helix with two twisted ropes tied at one end. Hold the ropes at the tied end in a fixed position. And let your friend pull the ropes apart from the other side. The formation of supercoils is clearly observed.
Type | DNA topoisomerase cuts the single DNA strand (nuclease activity) to overcome the problem of supercoils and then reseals the strand (ligase activity). Type Il DNA topoisomerase (also known as DNA gyrase) cuts both strands and reseals them to overcome the problem of supercoils. DNA topoisomerases are targeted by drugs (campthoterin for topoisomerase |, and amsacrime and etoposide for topoisomerase I, and amsacrime and etoposide for topoisomerase Il) in the treatment of cancers.

Replication of DNA in eukaryotes closely resembles that of prokaryotes. Certain differences, however, exist. Multiple origins of replication is a characteristic feature of eukaryotic cell. Further,
1. DNA polymerase @ is responsible for the synthesis of RNA primer for both the leading and lagging strands of DNA.
2.
DNA
f
is involved
in the
repair of DNA. Its function is comparable with DNA polymerase | found in prokaryotes.
3.
DNA
y
participates in
the replication of mitochondrial DNA.
4.
DNA
6
is responsible
replication on
the leading
strand of
DNA.
for
It the
also possesses
proof-reading activity.
5. DNA polymerase é is involved in DNA synthesis on the lagging strand and proof-reading function.
The differences in the DNA replication between bacteria and human cells, attributed to the enzymes, are successfully used — in antibacterial therapy to target pathogen (bacterial) replication and spare the host (human) cells.

The replication on the leading (continuous) strand of DNA is rather simple, involving DNA polymerase 5 and a sliding clamp called proliferating cell nuclear antigen (PCNA). PCNA is so named as it was first detected as an antigen in the nuclei of replicating cells. PCNA forms a ring around DNA to which DNA polymerase 5 binds. Formation of this ring also requires another factor namely replication factor C (RFC).
The replication on the lagging (discontinuous) strand in eukaryotes is more complex when compared to prokaryotes or even the leading strand of eukaryotes. This is depicted in Fig.24.7, and briefly described hereunder.
The parental strands of DNA are separated by the enzyme helicase. A single-stranded DNA binding protein called replication protein A (RPA) binds to the exposed single-stranded template. This strand has been opened up by the replication fork (a previously formed Okazaki fragment with an RNA primer is also shown in Fig.24.4).
The enzyme primase forms a complex with DNA polymerase « which initiates the synthesis of Okazaki fragments. The primase activity of pol a-primase complex is capable of producing 10-bp RNA primer. The enzyme activity is then switched from primase to DNA polymerase which elongates the primer by the addition of 20-30 deoxyribonucleotides. Thus, by the action of pol a-primase complex, a short stretch of DNA attached to RNA is formed. And now the complex dissociates from the DNA.
The next step is the binding of replication factor C (RFC) to the elongated primer (short RNA-DNA). RFC serves as a clamp loader, and catalyses the assembly of proliferating cell nuclear antigen (PCNA) molecules. The DNA polymerase 5 binds to the sliding clamp and elongates the Okazaki fragment to a final length of about 150-200 bp. By this elongation, the replication complex approaches the RNA primer of the previous Okazaki fragment.
The RNA primer removal is carried out by a pair of enzymes namely RNase H and _ flap endonuclease | (FENI). This gap created by RNA removal is filled by continued elongation of the new Okazaki fragment (carried out by polymerase 8, described above). The small nick that remains is finally sealed by DNA ligase.

Eukaryotic DNA is tightly bound to histones (basic proteins) to form nucleosomes which, in turn, organize into chromosomes. During the course of replication, the chromosomes are relaxed and the nucleosomes get loosened. The DNA strands separate for replication, and the parental histones associate with one of the parental strands. As the synthesis of new DNA strand proceeds, histones are also produced simultaneously, on the parent strand. At the end of replication, of the two daughter chromosomal DNAs formed, one contains the parental histones while the other has the newly synthesized histones.

Bacteria. contain a_ specific type Il topoisomerase namely gyrase. This enzyme cuts and reseals the circular DNA (of bacteria), and thus overcomes the problem of supercoils. Bacterial gyrase is inhibited by the antibiotics ciprofloxacin, novobiocin and nalidixic acid. These are widely used as antibacterial agents since they can effectively block the replication of DNA and multiplication of cells. These antibacterial agents have almost no effect on human enzymes.
Certain compounds that inhibit human topoisomerases are used as anticancer agents e.g. adriamycin, etoposide, doxorubicin. The nucleotide analogs that inhibit DNA replication are also used as anticancer drugs e.g. 6-mercaptopurine, 5-fluorouracil.
Fig. 24.7 : An outline of DNA replication on the lagging strand in eukaryotes (RPA-—Replication protein A; PCNA-Proliferating cell nuclear antigen; RFC-—Replication factor C; RNase H—-Ribonuclease H; FENI-Flap endonuclease |; Note : Leading strand not shown).

The cell cycle consists of four distinct phases in higher organisms—mitotic, G;, S and G) phases (Fig.24.8). When the cell is not growing, it exists in a dormant or undividing phase (Go). G, phase is characterized by active protein synthesis.
Replication of DNA occurs only once in S-phase and the chromosomes get doubled i.e. diploid genome gets converted into tetraploid. The entire process of new DNA synthesis takes place in about 8-10 hours, and a large number of DNA polymerases (500-1,000) are simultaneously involved in this process. It is believed that methylation of DNA serves as a marker to inhibit replication.
The G» phase is characterized by enlargement of cytoplasm and this is followed by the actual cell division that occurs in the mitotic phase.

Cyclins are a group of proteins that are closely associated with the transition of one phase of cell cycle to another, hence they are so named. The most important cyclins are cyclin A, B, D and E. The concentrations of cyclins increase or decrease during the course of cell cycle. These cyclins act on cyclin-dependent kinases (CDKs) that phosphorylate certain substances essential for the transition of one cycle to another.

As depicted in Fig.24.8, there occurs a continuous monitoring of the cell cycle with respect to DNA replication, chromosome segregation and integrity. If any damage to DNA is detected either in G, or Gy phase of the cycle, or if there is a formation of defective spindle (i.e. incomplete chromosomal segregation), the cell cycle will not progress until appropriately corrected. If it is not possible to repair the damage done, the cells undergo apoptosis (programmed cell death).
Fig. 24.8 : The cell cycle of a mammalian cell (M-Mitotic phase; G;-Gap1 phase; Gy—Dormant phase; S phase — Period of replication; G.-Gap 2 phase).

Cancer represents an excessive division of cells. In cancer, a large quantity of cells are in mitosis and most of them in S-phase.
Majority of the drugs used for cancer therapy are designed to block DNA replication or inhibit the enzymes that participate in replication (directly or indirectly). Methotrexate (inhibits dihydrofolate reductase) and 5-fluorouracil (inhibits thymidylate synthase) block nucleotide synthesis.
In recent years, topoisomerase inhibitors are being used. They block the unwinding of parental DNA strands and prevent replication.

There are certain difficulties in the replication of linear DNAs (or chromosomes) of eukaryotic cells. The leading strand of DNA can be completely synthesized to the very end of its template. This is not possible wih the lagging strand, since the removal of the primer RNA leaves a small gap which cannot be filled (Fig.24.9A). Consequently, the daughter chromosomes will have shortened DNA molecules. This becomes significant after several cell cycles involving — replication of chromosomes. The result is that over a period of time, the chromosomes may lose certain essential genes and the cell dies. This is however, avoided to a large extent.

Telomeres are maintained by the enzyme telomerase, also called as telomere terminal transferase. Telomerase is an unusual enzyme as it is composed of both protein and RNA. In case of humans, the RNA component is 450 nucleotides in length, and at the 5'-terminal and it contains the sequence 5'-CUAACCCUAAC-3'. It may be noted that the central region of this sequence is complementary to the telomere repeat sequence 5'-TTAGGG-3'. The telomerase RNA sequence can be used as a template for extension of telomeres (Fig.24.9B).
The telomerase RNA base pairs to the end of the DNA molecule with telomeres and extends to a small distance. Then translocation of telomerase occurs and a fresh extension of DNA takes place. This process of DNA synthesis and translocation is repeated several times until the chromosome gets sufficiently extended. The extension process gets completed through the participation of DNA polymerase and primase complex and sealing of the new DNA formed.
SRS
It may be noted here that as such the telomeres do not encode proteins. Hence, when extended by telomerase, they need not have to remain the same length, and some shortening will not pose any problem. During the course of repeated cell cycles, there occurs progressive shortening of telomeres, and this has to be prevented, which is appropriately carried out by telomerase.

Telomerase is highly active in the early embryo, and after birth it is active in the reproductive and stem cells. Stem cells divide continuously throughout the lifetime of an organism to produce new cells. These cells in turn are responsible to tissues and organs in the functional state e.g. hematopoietic stem cells of bone marrow.
Many biologists link the process of telomere shortening with cell senescence (i.e. cell death). This is mainly based on the observations made in the jin vitro mammalian cell cultures. However, some researchers question this relation between telomere shortening and senescence.
Cancerous cells are able to divide continuously. There is a strong evidence to suggest that the absence of senescence in cancer cells is linked to the activation of the enzyme telomerase. Thus, telomere length is maintained throughout multiple cell divisions. It is however, not clear whether telomerase activation is a cause or an effect of cancer. There is however, evidence to suggest that telomerase activation is in fact the cause of certain cancers e.g. dyskeratosis congenita due to a mutation in the gene responsible for the RNA component of telomerase.
The enzyme telomerase is an attractive target for cancer chemotherapy. The drugs have been designed to inactivate telomerase, and consequently induce senescence in the cancer cells. This in turn prevents the rapid cell proliferation.

Recombination basically involves _ the exchange of genetic information. There are mainly two types of recombinations.
1. Homologous recombination : This is also called as general recombination, and occurs between identical or nearly __ identical chromosomes (DNA sequences). The best example is the recombination between the paternal and = maternal chromosomal _ pairs (Fig.24.10).

homologous recombination.
2. Non-homologous recombination : This is regarded as illegitimate recombination and does not require any special homologous sequences. Transposition is a good example of nonhomologous recombination. Random integration of outside genes into mammalian chromosomes is another example.

It is a known fact that the chromosomes are not passed on intact from generation to generation. Instead, they are inherited from both the parents. This is possible due to homologous recombination. Three models have been put forth to explain homologous recombinations.
1
Holliday model
1
Meselson-Radding model
: Double-strand break model.

Holliday model (proposed by Holliday in 1964) is the simplest among the homologous recombination models. It is depicted in Fig.24.11, and briefly explained in the next page.
The two homologous chromosomes come closer, get properly aligned, and form single-
strand breaks. This results in two aligned DNA duplexes. Now the strands of each duplex partly unwind and invade in the opposite direction to form a two strands cross between the DNA molecules.
There occurs simultaneous unwinding and rewinding of the duplexes in such a way that there is no net change in the amount of base pairing, but the position of crossover moves. This phenomenon referred to as branch migration, results in the formation of heteroduplex DNA. The enzyme DNA ligase seals the nick. The two DNA duplexes (4 strands of DNA), joined by a single crossover point can rotate to create a fourstanded Holliday junction. Now the DNA molecules are subjected to symmetrical cuts in either of the two directions, and the cut ends are resealed by ligase.
The DNA exchange is determined by the direction of the cuts, which could be horizontal or vertical. If the corss strands are cut horizontally (cut 1), the flanking genes (or markers, i.e. AB/ab) remain intact, and no recombination occurs. On the other hand, if the parental strands are cut vertically (cut 2), the flanking genes get exchanged (i.e. Ab/aB) due to recombination.

The recombination process without any special homologous sequences of DNA is regarded as non-homologous recombination.

Transposition primarily involves __ the movement of specific pieces of DNA in the genome. The mobile segments of DNA are called transposons or transposable elements. They were first discovered by Barbara McClintock (in 1950) in maize, and their significance was ignored for about two decades by other workers.
Transposons are mobile and can move almost to any place in the target chromosome. There are two modes of transposition. One that involves an RNA intermediate, and the other which does not involve RNA intermediate.
Fig. 24.12 : A diagrammatic representation of retrotransposition.
Retrotransposition : Transposition involving RNA intermediate represents retrotransposition (Fig.24.12). By the normal process of transcription, a copy of RNA formed from a transposon (also called as retrotransposon). Then by the enzyme reverse transcriptase, DNA is copied from the RNA. The newly formed DNA which is a copy of the transposon gets integrated into the genome. This integration may occur randomly on the same chromosome or, on a different chromosome. As a result of the retrotransposition, there are now two copies of the transposon, at different points on the genome.
DNA transposition : Some transposons are capable of direct transposition of DNA to DNA. This may occur either by replicative transposition or conservative transposition (Fig.24.13). Both the mechanisms require enzymes that are mostly coded by the genes within the transposons.
DNA transposition is less common than retrotransposition in case of eukaryotes. However, in case of prokaryotes, DNA transposons are more important than RNA transposons.

It is now widely accepted that a large fraction of the human genome has resulted due to the accumulation of transposons. Short interspersed elements (SINEs) are repeats of DNA sequences which are present in about 500,000 copies per haploid human genome e.g. Alu sequences.

repeated DNA sequences and are present in about 50,000 copies in the human genome e.g. L1 elements.
Some of the diseases caused by mutations are due to insertion of transposons into genes.

Being the carrier of genetic information, the cellular DNA must be replicated (duplicated), maintained, and passed down to the daughter cells accurately. In general, the accuracy of replication is extremely high. However, there do occur replication errors. It is estimated that approximately one error is introduced per billion base pairs during each cycle of replication. The cells do posses the capability to repair damages done to DNA to a extent.
large

Despite an efficient repair system for the damaged DNA, replication errors do accumulate that ultimately result in mutations. The human body possesses 10!'4 nucleated cells, each with 3 x 109 base pairs of DNA. It is estimated that about 10!° cell divisions occur in a lifetime. If 10-!9 mutations per base pair per cell generation escape repair, this results in about one mutation per 10° base pairs in genome.
Fig. 24.13 : A diagrammatic representation of DNA transposition (coloured blocks represent transposons).
Besides the possible errors in replication, the DNA is constantly subjected to attack by both physical and chemical agents. These include radiation, free radicals, chemicals etc., which also result in mutations.
It is fortunate that a great majority of the mutations probably occur in the DNA that does not encode proteins, and consequently will not have any serious impact on the organism. This is not, however, all the time true, since mutations do occur in the coding regions of DNA also. There are situations in which the change in a single base pair in the human genome can cause a serious disease e.g. sicklecell anemia.

The damages done to DNA by physical, chemical and environmental agents may be broadly classified into four categories with different types (Table 24.1).
The DNA damage may occur due to singlebase alterations (e.g. depurination, deamination), two-base alterations (e.g. pyrimidine dimer) chain breaks (e.g. ionizing radiation) and crosslinkages (e.g. between bases). Some selected DNA damages are briefly described.
The occurrence of spontaneous deamination bases in aqueous solution at 37°C is well known. Cytosine gets deaminated to form uracil while adenine forms hypoxanthine.
Spontaneous depurination, due to cleavage of glycosyl bonds (that connect purines to the backbone) also occurs. It is estimated that 2000-10,000 purines may be lost per mammalian cell in 24 hours. The depurinated sites are called as abasic sites. Originally, they were detected in purines, and called apurinic sites (AP sites) which represent lack of purine. Now, the term AP sites is generally used to base in DNA.

The production of reactive oxygen species is often associated with alteration of bases e.g. formation of 8-hydroxy guanine. Free radical formation and oxidative damage to DNA increases with advancement of age.

Single-base alteration, Types = Deamination (C o U; A o hypoxanthine). Two-base alteration, Types = UV light induced pyrimidine dimer alteration (T-T). Chain breaks, Types = Oxidative free radical formation Ionizing radiation. Cross-linkage, Types = Between bases in the same or opposite strands Between the DNA and protein molecules
Ultraviolet radiations result in the formation of covalent links between adjacent pyrimidines along the DNA strand to form pyrimidine dimers. DNA chain breaks can be caused by ionizing radiations (e.g. X-rays).

The genetic macromolecule DNA is highly stable with regard to its base composition and sequence. However, DNA is not totally exempt from gradual change.
Mutation refers to a change in the DNA structure of a gene. The substances (chemicals) which can induce mutations are collectively known as mutagens.
The changes that occur in DNA on mutation are reflected in replication, transcription and translation.

Mutations are mainly of two major types— point mutations, frameshift mutations (Fig.24.14). 1. Point mutations : The replacement of one base pair by another results in point mutation. They are of two sub-types.
(a) Transitions : In this case, a purine (or a pyrimidine) is replaced by another.
(b)
Transversions
:
These
are
by
replacement
of
a
purine
by
a
or
vice
versa.
2.
Frameshift
mutations
:
These
occur
when
one
or
more
base
pairs
are
inserted
in
or
deleted
from
the
DNA,
respectively,
causing
insertion
or
deletion
mutations.

The change in a single base sequence in point mutation may cause one of the following (Fig.24.15).
1. Silent mutation : The codon (of mRNA) containing the changed base may code for the same amino acid. For instance, UCA codes for serine and change in the third base (UCU) still codes for serine. This is due to degeneracy of the genetic code. Therefore, there are no detectable effects in silent mutation.
2.
Missense
mutation
In
this
case,
the
changed
base
may
code
for
a
different
amino
acid.
For
example,
UCA
codes
for
serine
while
ACA
codes
for
threonine.
The
mistaken
(or

(A)
—C—G—A—G—
Transition
|
—C—G—G—G—
I}
|
|
———
+
11
i
|
—G—C—T—C—
—G—C—C—C—
—C—G—A—G—
Transversion
—C—G—T—G—
Fig. 24.14 : An illustration of mutations (A)-Point mutations; (B)-Frameshift mutations.
missense) amino acid may be acceptable, partially acceptable or unacceptable with regard to the function of protein molecule. Sickle-cell anemia is a classical example of missense mutation.
A few micrograms (10-12 g) of DNA in a fetal cell stores the genetic information that will determine the differentiation and every function of an adult animal. This is the marvel of molecular biology.
Topoisomerase inhibitors (e.g. adriamycin, etoposide) are useful to prevent DNA replication, and thus uncontrolled cell proliferation in cancer. These compounds block the unwinding of DNA strands.
The
progressive
shortening
of
telomeres
(DNA
sequences
at
chromosomal
ends)
is
prevented
by
telomerase.
This
enzyme
is
an
attractive
target
for
cancer
therapy.
Mutations may sometimes result in serious diseases. e.g. sickle-cell anemia, cancer.
Xeroderma pigmentosum is a rare disease characterized by photosensitivity and risk for
skin
cancer.
This
is
due
to
a
defect
in
the
nucleotide
excision
repair
of
the
damaged
DNA
(caused
by
UV
rays).
Hereditary nonpolyposis colon cancer is a common inherited cancer, and is due to a faulty mismatch repair of defective DNA.
3.
Nonsense mutation
Sometimes, the
codon with
the termination
change in
(UCA)
acts as
may
(or altered
base nonsense)
the may
become codon.
second base
result in
a
For of
UAA.
a
stop signal
The and

causes instance,
serine codon
altered codon
termination protein
synthesis, at
that point.

in the reading of codons, translation continues. The result is that the protein synthesized will have several altered amino acids and/or prematurely terminated protein.

Mutations are permanent alterations in DNA structure, which have been implicated in the etiopathogenesis of cancer.

As already stated, damage to DNA caused by replication errors or mutations may have serious consequences. The cell possesses an_ inbuilt system to repair the damaged DNA. This may be achieved by four distinct mechanisms (Table 24.2).
1. Base excision-repair
2. Nucleotide excision-repair
3. Mismatch repair
4 . Double-strand break repair.

The insertion or deletion of a base in a gene results in an altered reading frame of the mRNA (hence the name _ frameshift). The machinery of mRNA (containing codons) does not recognize that a base was missing or a new base was added. Since there are no punctuations
The bases cytosine, adenine and guanine can undergo spontaneous depurination to respectively form uracil, hypoxanthine and xanthine. These altered bases do not exist in the normal DNA, and therefore need to be removed. This is carried out by base excision repair (Fig.24.16).

Base excision-repair, Damage to DNA = Damage to a single base due to spontaneous alteration or by chemical or radiation means.. Base excision-repair, DNA repair = Removal of the base by N-glycosylase; abasic sugar removal, replacement.. Nucleotide excision-repair, Damage to DNA = Damage to a segment of DNA by spontaneous, chemical or radiation means.. Nucleotide excision-repair, DNA repair = Removal of the DNA fragment ( ï 30 nt length) and replacement.. Mismatch repair, Damage to DNA = Damage due to copying errors (1-5 base unpaired loops).. Mismatch repair, DNA repair = Removal of the strand (by exonuclease digestion) and replacement.. Double-strand break repair, Damage to DNA = Damage caused by ionizing radiations, free radicals, chemotherapy etc.. Double-strand break repair, DNA repair = Unwinding, alignment and ligation.
of
Fig. 24.16 : A diagrammatic representation of base excision-repair of DNA.
A defective DNA in which cytosine is deaminated to uracil is acted upon by the enzyme uracil DNA glycosylase. This results in the removal of the defective base uracil. An endonuclease cuts the backbone of DNA strand near the defect and removes a few bases. The gap so created is filled up by the action of repair DNA polymerase and DNA ligase.

The DNA damage due to ultraviolet light, ionizing radiation and other environmental factors often results in the modification of certain bases, strand breaks, cross-linkages etc. Nucleotide excision-repair is ideally suited for such large-scale defects in DNA. After the identification of the defective piece of the DNA, the DNA double helix is unwound to expose the damaged part. An excision nuclease (exinuclease) cuts the DNA on either side (upstream and downstream) of the damaged DNA. This defective piece is degraded. The gap created by the nucleotide excision is filled up by DNA polymerase which gets ligated by DNA ligase

Xeroderma pigmentosum (XP) is a_ rare autosomal recessive disease. The affected patients are photosensitive and susceptible to skin cancers. It is now recognized that XP is due to a defect in the nucleotide excision repair of the damaged DNA.

Despite high accuracy in replication, defects do occur when the DNA is copied. For instance, cytosine (instead of thymine) could be incorporated opposite to adenine. Mismatch
repair corrects a single mismatch base pair e.g. C to A, instead of T to A.
The template strand of the DNA exists in a methylated form, while the newly synthesized strand is not methylated. This difference allows the recognition of the new strands. The enzyme GATC endonuclease cuts the strand at an adjacent methylated GATC sequence (Fig.24.18). This is followed by an exonuclease digestion of the defective strand, and thus its removal. A new DNA strand is now synthesized to replace the damaged one.
Hereditary nonpolyposis colon cancer (HNPCC) is one of the most common inherited cancers. This cancer is now linked with faulty mismatch repair of defective DNA.

Double-strand breaks (DSBs) in DNA are dangerous. They result in genetic recombination which may lead to chromosomal translocation, broken chromosomes, and finally cell death. DSBs can be repaired by homologous recombination or non-homologous end joining. Homologous recombination occurs in yeasts while in mammals, non-homologous and joining dominates.

Cancer
develops
when
certain
genes
that regulate normal cell division fail or are
altered. Defects in the genes encoding proteins involved in nucleotide-excision repair, mismatch repair and recombinational repair are linked to human cancers. For instance, as already referred above, HNPCC is due to a defect in mismatch repair.

1., 1 = The central dogma of life revolves around the flow of information from DNA to RNA, and from there to proteins.. 2., 1 = Replication is a process in which DNA copies itself to produce identical daughter molecules of DNA. DNA replication is semiconservative, bidirectional and occurs by the formation of bubbles and forks.. 3., 1 = Prokaryotic DNA syntheis is catalysed by the enzyme DNA polymerase III. This enzyme possesses proof-reading activity and edits the mistakes that might occur during nucleotide incorporation.. 4., 1 = Replication in eukaryotes (particularly on the lagging strand) is more complex and involves several factors e.g. replication protein A, replication factor C, flap endonuclease.. 5., 1 = Telomeres (repeat TTAGGG sequences) are the special structures that prevent the continuous loss of DNA at the end of the chromosome during the course of replication.. 6., 1 = Recombination involves the exchange of genetic information through the exchange of DNA. Transposition refers to the movement of specific pieces of DNA (called transposons) in the genome.. 7., 1 = Damage to DNA may be due to single base alteration, two-base alteration, chain breaks and cross linkages. The cells possess an inbuilt system to repair the damaged DNA

bBwWN
. Describe the replication of DNA.
. Give an account of recombination of DNA.
. Discuss different types of DNA damages, and the repair mechanisms.
. What are mutations? Describe different types, and consequences of mutations.
aA . Give an account of telomeres and their role in senescence and cancer.

(a) Replication fork, (b) Okazaki pieces, (c) RNA primer, (d) DNA topoisomerases, (e) Inhibitors of
DNA replication, (f) Telomerase, (g) Holliday model of DNA recombination, (h) Transposition,
(i) Frameshift mutations, (j) Missense mutation, (k) Mismatch repair, (I) Xeroderma pigmentosum.

1. DNA strands for replication process are separated by the enzyme
2. The small fragments of DNA produced during replication are called
3. During the course of DNA replication, the proof-reading function is carried out by the enzyme

. The problem of supercoils in DNA replication is overcome by a group of enzymes, namely
.
The
proteins
that
are
with
the
transition
of
one
phase
of
cell
cycle
to
another
.
Name
the
DNA
sequence
that
prevents
the
continuous
loss
of
DNA
at
the
end
of
the
chromosome during the course of replication
. The mobile segments of DNA are called
. Any change in the DNA sequence of a gene is commonly referred to as
.
. Sickle-cell anemia is a good example of
mutation.
10. One common example of inherited cancer with faulty mismatch repair of defective DNA

11. The chemical nature of the primer required for the synthesis of DNA
12. The enzyme responsible for the synthesis of RNA primer in eukaryotes
13. The repeat sequence of nucleotides in telomeres
14. The DNA damage caused by deamination is an example of
15. The mutation involving the replacement of one purine by another
(a) DNA (b) Histone (c) RNA (d) hnRNA.
(a) DNA polymerase « (b) DNA polymerase
B (c) DNA polymerase y (d) Topisomerases.
(a) TTGGGA (b) TTAGGG (c) GGGATT (d) TTGAGG.
(a) Single-base alteration (b) Two-base alteration (c) Chain breaks (d) Cross linkage.
(a) Frameshift mutation (b) Transition (c) Transversion (d) of the above.
te, in character;
he conventional concept of central dogma of life which in essence is "DNA makes RNA makes protein' is an oversimplification of molecular biology. With the advances in cell biology and rapid developments in bioinformatics, the terms genome, transcriptome and proteome are in current use to represent the central dogma of life (Fig.25.1).

The total DNA _ (genetic information) contained in an organism or a cell is regarded as the genome. Thus, the genome is the storehouse of biological information. It includes the chromosomes in the nucleus and the DNA in mitochondria, and chloroplasts.
Genomics : The study of the structure and function of genome is genomics. The term functional genomics is used to represent the gene expression and relationship of genes with gene products. Structural genomics refers to the structural motifs and complete protein structures. Comparative genomics involves the study of

The RNA copies of the active protein coding genes represent transcriptome. Thus, transcriptome is the initial product of gene
expression which directs the synthesis of proteins.
Transcriptomics : The study of transcriptome that involves all the RNA molecules made by a cell, tissue or an organism is transcriptomics.

The cell's repertoire (repository/storehouse) of proteins with their nature and _ biological functions is regarded as proteome. Thus, proteome represents the entire range of proteins and their biological functions in a cell.

Metabolomics : The use of genome sequence analysis for determining the capability of a cell, tissue or an organism to synthesize small molecules (metabolites) is metabolomics.
Whether the central dogma of life is represented in the conventional or more recent form, replication, transcription and translation are the key or core processes that ultimately control life. Replication of DNA has been described in Chapter 24, while transcription and translation are discussed in this chapter.

Transcription is a process in which ribonucleic acid (RNA) is synthesized from DNA. The word gene refers to the functional unit of the DNA that can be transcribed. Thus, the genetic information stored in DNA is expressed through RNA. For this purpose, one of the two strands of DNA serves as a template (non-coding strand or antisense strand) and_ produces working copies of RNA molecules. The other DNA strand which does not participate in transcription is referred to as coding strand or sense strand or non-template strand. (Coding strand commonly used since with the exception of T for U, primary mRNA contains codons with the same base sequence).

The entire molecule of DNA is not expressed in transcription. RNAs are synthesized only for
Fig. 25.2 : RNA polymerase of E. coli.
some selected regions of DNA. For certain other regions of DNA, there may not be any transcription at all. The exact reason for the selective transcription is not known. This may be due to some inbuilt signals in the DNA molecule.
The product formed in transcription is referred to as primary transcript. Most often, the primary RNA transcripts are inactive. They undergo certain alterations (splicing, terminal additions, base modifications etc.) commonly known as post-transcriptional modifications, to produce functionally active RNA molecules.
There exist certain differences in the transcription between prokaryotes and eukaryotes. The RNA synthesis in prokaryotes is given in some detail. This is followed by a brief discussion on eukaryotic transcription.

A single enzyme—DNA dependent RNA polymerase or simply RNA _ polymerase— synthesizes all the RNAs in prokaryotes. RNA polymerase of E. coli is a complex holoenzyme (mol wt. 465 kDa) with five polypeptide subunits—2a, 1B and 1B' and one sigma(s) factor (Fig.25.2). The enzyme without sigma factor is referred to as core enzyme (Bf).
An overview of RNA synthesis is depicted in Fig.25.3. Transcription involves three different stages—initiation, elongation and_ termination (Fig.25.4).
Fig. 25.3 : An overview of transcription.

The binding of the enzyme RNA polymerase to DNA is the prerequisite for the transcription to start. The specific region on the DNA where the enzyme binds is known as promoter region. There are two base sequences on the coding DNA strand which the sigma factor of RNA polymerase can recognize for initiation of transcription (Fig.25.5).
for RNA polymerase and, further, this enzyme does not possess endoor exonuclease activity. Due to lack of the latter function (proof-reading activity), RNA polymerase has no ability to repair the mistakes in the RNA synthesized. This is in contrast to DNA replication which is carried out with high fidelity. It is, however, fortunate that mistakes in RNA synthesis are less dangerous, since they are not transmitted to the daughter cells.
1. Pribnow box (TATA box) : This consists of 6 nucleotide bases (TATAAT), located on the left side about 10 bases away (upstream) from the starting point of transcription.
2. The '-35' sequence : This is the second recognition site in the promoter region of DNA. It contains a base sequence TTGACA, which is located about 35 bases (upstream, hence —35) away on the left side from the site of transcription start.

As the holoenzyme, RNA _ polymerase recognizes the promoter region, the sigma factor is released and transcription proceeds. RNA is synthesized from 5' end to 3' end (5'-3') antiparallel to the DNA_ template. RNA polymerase utilizes ribonucleotide triphosphates (ATP, GTP, CTP and UTP) for the formation of RNA. For the addition of each nucleotide to the growing chain, a pyrophosphate moiety is released.
The sequence of nucleotide bases in the mRNA is complementary to the template DNA strand. It is however, identical to that of coding strand except that RNA contains U in place of T in DNA (Fig.25.6).
RNA polymerase differs from DNA polymerase in two aspects. No primer is required
The double helical structure of DNA unwinds as the transcription goes on, resulting in supercoils. The problem of supercoils is overcome by topoisomerases (more details in Chapter 24).

The process of transcription stops by termination signals. Two types of termination are identified.
1. Rho (p) dependent termination : A specific protein, named p factor, binds to the growing RNA (and not to RNA polymerase) or weakly to DNA, and in the bound state it acts as ATPase and terminates transcription and releases RNA. The p factor is also responsible for the dissociation of RNA polymerase from DNA.
2. Rho (p) independent termination : The termination in this case is brought about by the formation of hairpins of newly synthesized RNA. This occurs due to the presence of palindromes. A palindrome is a word that reads alike forward and backward e.g. madam, rotor. The presence of palindromes in the base sequence of DNA template (same when read in opposite direction) in the termination region is known. As a result of this, the newly synthesized RNA folds to form hairpins (due to complementary base pairing) that cause termination of transcription.
:G¢
NOLLdIdOSNVYL
GNV
NOILVISNVHL

RNA synthesis in eukaryotes is a much more complicated process than the transcription described above for prokaryotes. As such, all the details of eukaryotic transcription (particularly about termination) are not clearly known. The salient features of available information are given hereunder.

2. RNA polymerase II synthesizes the precursors for mRNAs and small nuclear RNAs. 3. RNA polymerase III participates in the formation of tRNAs and small ribosomal RNAs. Besides the three RNA polymerases found in the nucleus, there also exists a mitochondrial RNA polymerase in eukaryotes. The latter resembles prokaryotic RNA _ polymerase _ in structure and function.
The nuclei of eukaryotic cells possess three distinct RNA polymerases (Fig.25.7).
1. RNA polymerase | is responsible for the synthesis
of precursors
for the
large ribosomal
RNAs.

In eukaryotes, a sequence of DNA bases— which is almost identical to pribnow box of prokaryotes—is identified (Fig.25.8). This sequence, known as Hogness box (or TATA box),
Fig. 25.7 : An overview of transcription in eukaryotes.
Fig. 25.8 : Promoter regions of DNA in eukaryotes.
is located on the left about 25 nucleotides away (upstream) from the starting site of mRNA synthesis. There also exists another site of recognition between 70 and 80 nucleotides upstream from the start of transcription. This second site is referred to as CAAT box. One of these two sites (or sometimes both) helps RNA polymerase Il to recognize the requisite sequence on DNA for transcription.

The molecular events required for the initiation of transcription in eukaryotes are complex, and broadly involve three stages.
1. Chromatin containing the promoter sequence made accessible to the transcription machinery.
- 2. Binding of transcription factors (TFs) to
DNA sequences in the promoter region.
- 3. Stimulation of transcription by enhancers.
A large number of transcription factors interact with eukaryotic promoter regions. In humans, about six transcription factors have been identified (TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH). It is postulated that the TFs bind to each other, and in turn to the enzyme RNA polymerase.
Enhancer can increase gene expression by about 100 fold. This is made possible by binding of enhancers to transcription factors to form activators. |t is believed that the chromatin forms a loop that allows the promoter and enhancer to be close together in space to facilitate transcription.

The primary mRNA transcript produced by RNA polymerase II in eukaryotes is often referred to as heterogeneous nuclear RNA (hnRNA). This is then processed to produce mRNA needed for protein synthesis.

The RNAs produced during transcription are called primary transcripts. They undergo many alterations—terminal base additions, base modifications, splicing etc., which are collectively referred to as post-transcriptional modifications. This process is required to convert the RNAs into the active forms. A group of enzymes, namely ribonucleases, are responsible for the processing of tRNAs and rRNAs of both prokaryotes and eukaryotes.
The prokaryotic mRNA synthesized in transcription is almost similar to the functional MRNA. In contrast, eukaryotic mRNA (i.e. hnRNA) undergoes extensive post-transcriptional changes. An outline of the — post-transcriptional modifications is given in Fig.25.9, and some highlights are described.

The primary transcript of mRNA is the hnRNA in eukaryotes, which is subjected to many changes before functional mRNA is produced.
1. The 5' capping : The 5' end of mRNA is capped with 7-methylguanosine by an unusual
Fig.
25.9
:
An outline
of post-transcriptional
modifications of
RNA
(hnRNA-Heterogeneous nuclear
RNA).
5'5' triphosphate linkage. | S-Adenosylmethionine is the donor of methyl group. This cap is required for translation, besides stabilizing the structure of mRNA.
2. Poly-A tail : A large number of eukaryotic mRNAs possess an adenine nucleotide chain at the 3'-end. This poly-A tail, as such, is not produced during transcription. It is later added to stabilize mRNA. However, poly-A chain gets reduced as the mRNA enters cytosol.
3. Introns and their removal : Introns are the intervening nucleotide sequences in mRNA which do not code for proteins. On the other hand, exons of mRNA possess genetic code and are responsible for protein synthesis. The splicing and excision of introns is illustrated in Fig.25.10. The removal of introns is promoted by small nuclear — ribonucleoprotein particles (snRNPs). snRNPs (pronounced as snurps) in turn, are formed by the association of small nuclear RNA (snRNA) with proteins.
The term spliceosome is used to represent the snRNP association with hnRNA at the exon-intron junction.
Post-transcriptional modifications of mRNA occur in the nucleus. The mature RNA then enters the cytosol to perform its function (translation).

Alternate patterns of hnRNA splicing result in different mRNA molecules which can produce
Fig. 25.10 : Formation of mature RNA from eukaryotic mRNA (SnRNPs-Small nuclear ribonucleoprotein particles).
Fig. 25.11 : A diagrammatic representation of the relationship between eukaryotic chromosomal DNA and MRNA (bp-Base pair; nt-Nucleotides).
different proteins. Alternate splicing results in mRNA heterogeneity. In fact, the processing of hnRNA molecules becomes a site for the regulation of gene expression.
Faulty splicing can cause diseases : Splicing of hnRNA has to be performed with precision to produce functional mRNA. Faulty splicing may result in diseases. A good example is one type of B-thalassemia in humans. This is due to a mutation that results in a nucleotide change at an exon-intron junction. The result is a diminished or lack of synthesis of B-chain of hemoglobin, and consequently the disease B-thalassemia.

The sequence in the DNA determines the coding sequence in mRNA, and finally the amino acid sequence in the protein. However, in recent years, changes in the coding information by editing of mRNA have been reported. It is estimated that about 0.01% of the mRNAs undergoes editing. One example is the conversion of CAA codon in mRNA (of apoprotein B gene) to UAA by the enzyme cytidine deaminase. As a result, originating from the same gene, the liver synthesizes a 100-kDa protein (apoB 100) while the intestinal cells synthesize 48-kDa protein (apoB 48). This happens due to formation of a termination codon (UAA) from CAA in RNA editing.

All the tRNAs of prokaryotes and eukaryotes undergo post-transcriptional modification. These include trimming, converting the existing bases into unusual ones, and addition of CCA nucleotides to 3' terminal end of tRNAs.

The preribosomal RNAs originally synthesized are converted to ribosomal RNAs by a series of post-transcriptional changes.

The synthesis of RNA is inhibited by certain antibiotics and toxins.
Actinomycin D : This is also known as dactinomycin. It is synthesized by Streptomyces. Actinomycin D binds with DNA template strand and blocks the movement of RNA polymerase. This was the very first antibiotic used for the treatment of tumors.
Rifampin : It is an antibiotic widely used for the treatment of tuberculosis and_ leprosy. Rifampin binds with the B-subunit of prokaryotic RNA polymerase and inhibits its activity.
a-Amanitin It is a toxin produced by mushroom, Amanita phalloides. This mushroom is delicious in taste but poisonous due to the toxin a@-amanitin which tightly binds with RNA polymerase Il of eukaryotes and _ inhibits transcription.

A typical, 1 = bacterium normally contains. 0.05-0.10, 1 = pg of RNA which contributes to about. 6% of the, 1 = total weight. A mammalian cell, being. larger in, 1 = size, contains 20-30 pg RNA, and this
Fig. 25.12 : A diagrammatic representation of RNA content of a cell (Note : RNAs represented in black are found in all organisms; RNAs in colour and exclusively present in eukaryotes only; hnRNA-Heterogeneous nuclear RNA; rRNA—Ribosomal RNA : tRNA-Transfer RNA; snRNA-Small nuclear RNA; snoRNA-Small nucleolar RNA; scRNA-Small cytoplasmic RNA).
represents only 1% of the cell weight. Transcriptome, representing the RNA derived from protein coding genes actually constitutes only 4%, while the remaining 96% is the non-coding RNA (Fig.25.12). The different non-coding RNAs are ribosomal RNA, transfer RNA, small nuclear RNA, small nucleolar RNA and small cytoplasmic RNA. The functions of different RNAs are described in Chapter 5 (Refer Table 5.3).

Some of the viruses—known as retroviruses— possess RNA as the genetic material. These viruses cause cancers in animals, hence known as oncogenic. They are actually found in the transformed cells of the tumors.
The enzyme RNA dependent DNA polymerase —or simply reverse transcriptase— is responsible for the formation of DNA from RNA (Fig.25.13). This DNA is complementary (cDNA) to viral RNA and can be transmitted into host DNA.
Synthesis of cDNA from mRNA : As already described, the DNA expresses the genetic information in the form of RNA. And the mRNA determines the amino acid sequence in a protein. The mRNA can serve as a template for synthesis of double-stranded complementary
DNA (cDNA) by using the enzyme reverse transcriptase. This CDNA can be used as a probe to identify the sequence of DNA in genes.

The genetic information stored in DNA is passed on to RNA (through transcription), and ultimately expressed in the language of proteins. The biosynthesis of a protein or a polypeptide in a living cell is referred to as translation. The term translation is used to represent the biochemical translation of four-letter language information from nucleic acids (DNA and then RNA) to 20 letter language of proteins. The sequence of amino acids in the protein synthesized is determined by the nucleotide base sequence of mRNA.

There are wide variations in the cells with respect to the quality and quantity of proteins synthesized. This largely depends on the need and ability of the cells. Erythrocytes (red blood cells) lack the machinery for translation, and therefore cannot synthesize proteins.
In general, the growing and dividing cells produce larger quantities of proteins. Some of the cells continuously synthesize proteins for export. For instance, liver cells produce albumin and blood clotting factors for export into the blood for circulation. The normal liver cells are very rich in the protein biosynthetic machinery, and thus the liver may be regarded as the protein factory in the human body.

The three nucleotide (triplet) base sequences in mRNA that act as code words for amino acids in protein constitute the genetic code or simply codons. The genetic code is regarded as a dictionary of nucleotide bases (A, G, C and U ) that determines the sequence of amino acids in proteins.
The codons consist of the four nucleotide bases, the purines—adenine (A) and guanine (G), and the pyrimidines—cytosine (C) and uracil (U). These four bases produce 64 _ different combinations (43) of three base codons, as depicted in Table 25.1. The nucleotide sequence of the codon on mRNA is written from the 5'end to 3' end. Sixty one codons code for the 20 amino acids found in protein.
The three codons UAA, UAG and UGA do not code for amino acids. They act as stop signals in protein synthesis. These three codons are collectively known as termination codons or non-sense codons. The codons UAG, UAA and UGA are often referred to, respectively, as amber, ochre and opal codons.
are
The codons AUG—and, sometimes, GUG— the chain initiating codons.

The genetic code is universal, specific, nonoverlapping and degenerate.
1. Universality : The same codons are used to code for the same amino acids in all the living organisms. Thus, the genetic code has been conserved during the course of evolution. Hence genetic code is appropriately regarded as universal. There are, however, a few exceptions. For instance, AUA is the codon for methionine in mitochondria. The same codon (AUA) codes for isoleucine in cytoplasm. With some exceptions noted, the genetic code is universal.
2. Specificity : A particular codon always codes for the same amino acid, hence the genetic code is highly specific or unambiguous e.g. UGG is the codon for tryptophan.
3. Non-overlapping : The genetic code is read from a fixed point as a continuous base sequence. It is non-overlapping, commaless and without any punctuations. For instance, UUUCUUAGAGGG is read as UUU/CUU/AGA/GGG. Addition or deletion of one or two bases will radically change the message sequence in mRNA. And the protein synthesized from such mRNA will be totally different. This is encountered in frameshift mutations which cause an alteration in the reading frame of mRNA.
4. Degenerate : Most of the amino acids have more than one codon. The codon is degenerate or redundant, since there are 61 codons available to code for only 20 amino acids. For instance, glycine has four codons. The codons that designate the same amino acid are called synonyms. Most of the synonyms differ only in the third (3' end) base of the codon.
The Wobble hypothesis codon degeneracy (described later). explains

The codon of the mRNA is recognized by the anticodon of tRNA (Fig.25.14). They pair with each other in antiparallel direction (5'— 3' of mRNA with 3' 5' of tRNA). The usual conventional complementary base _ pairing (A=U, C=G) occurs between the first two bases of codon and the last two bases of anticodon. The third base of the codon is rather lenient or flexible with regard to
the complementary
region base.
of tRNA
consists and
it recognizes
The of
seven the
three mRNA.

Wobble hypothesis,
the phenomenon
can due
in recognize
more to
the fact
in the
that put
forth which
letter anticodon
nucleotides codon
in by
a
than one
the codon
often fails
specific complementary
(5’-base).
third to
base
Wobbling difference
5’-end of
of
5’-end
Crick, single
is tRNA
codon.
This is
base
(3’-base)
recognize in
the the
is in
anticodon attributed
the spatial
the anticodon.
base to
the arrangement
The of
possible anticodon
of the
pairing
(of tRNA)
with the
3’-end base
of codon
(mRNA)
is given
C, 1 = ¼ CUU CUC CUA CUG. C, 2 = Leu. C, 3 = CCU CCC CCA CCG. C, 4 = Pro. C, 5 = CAU CAC º ¼ » His CAA CAG º ¼ » Gln. C, 6 = CGU CGC CGA CGG. C, 7 = Arg. C, 8 = U C A G. C, 1 = AUU AUC Ile AUA º ¼ » AUG *. C, 2 = Met. C, 3 = ACU ACC ACA ACG. C, 4 = Thr. C, 5 = º ¼ » Asn º ¼ » Lys º. C, 6 = AGU AGC AGA AGG. C, 7 = º ¼ » Ser º ¼ » Arg. C, 8 = U C A G. C, 1 = GUU GUC GUA GUG. C, 2 = Val. C, 3 = GCU GCC GCA GCG. C, 4 = Ala. C, 5 = GAU GAC ¼ » Asp GAA GAG º ¼ » Glu. C, 6 = GGU GGC GGA GGG. C, 7 = Gly. C, 8 = U C A G
Fig. 25.14 : Complementary binding of codon (of mRNA) and anticodon (of tRNA).

Mutations result in the change of nucleotide sequences in the DNA, and consequently in the RNA. The different types of mutations are described in Chapter 24. The ultimate effect of mutations is on the translation through the alterations in codons. Some of the mutations are harmful.
The occurrence of the disease sickle-cell anemia due to a_ single base alteration (CTC > CAC in DNA, and GAG GUG in RNA) is a classical example of the seriousness of mutations. The result is that glutamate at the 6th position of B-chain of hemoglobin is replaced by valine. This happens since the altered codon GUG of mRNA codes for valine instead of glutamate (coded by GAG in normal people).
Frameshift mutations are caused by deletion or insertion of nucleotides in the DNA that generate altered mRNAs. As the reading frame of mRNA is continuous, the codons are read in continuation, and amino acids are added. This results in proteins that may contain several altered amino acids, or sometimes the protein synthesis may be terminated prematurely.

The protein synthesis which involves the translation of nucleotide base sequence of mRNA into the language of amino acid sequence may be divided into the following stages for the convenience of understanding.
I. Requirement of the components
Il. Activation of amino acids
Ill. Protein synthesis proper
IV. Chaperones and protein folding
V. Post-translational modifications.

The protein synthesis may be considered as a biochemical factory operating on the ribosomes. As a factory is dependent on the supply of raw materials to give a final product, the protein synthesis also requires many components.
1. Amino acids : Proteins are polymers of amino acids. Of the 20 amino acids found in protein structure, half of them (10) can be synthesized by man. About 10 essential amino acids have to be provided through the diet. Protein synthesis can occur only when all the amino acids needed for a particular protein are available. If there is a deficiency in the dietary supply of any one of the essential amino acids, the translation stops. It is, therefore, necessary that a regular dietary supply of essential amino acids, in sufficient quantities, is maintained, as it is a prerequisite for protein synthesis.
As regards prokaryotes, there is no requirement of amino acids, since all the 20 are synthesized from the inorganic components.
2. Ribosomes : The functionally active ribosomes are the centres or factories for protein synthesis. Ribosomes may also be considered as workbenches of translation. Ribosomes are huge complex structures (70S for prokaryotes and 80S for eukaryotes) of proteins and ribosomal RNAs. Each ribosome consists of two subunits—one big and one small. The functional ribosome has two
Fig. 25.15 : A polyribosome in protein synthesis.
sites—A site and P site. Each site covers both the subunits. A site is for binding of aminoacyl tRNA and P site is for binding peptidyl tRNA, during the course of translation. Some authors consider A site as acceptor site, and P site as donor site. In case of eukaryotes, there is another site called exit site or E site. Thus, eukaryotes contain three sites (A, P and E) on the ribosomes.
5. Energy sources : Both ATP and GTP are required for the supply of energy in protein synthesis. Some of the reactions involve the breakdown of ATP or GTP, respectively, to AMP and GMP with the liberation of pyrophosphate. Each one of these reactions consumes two high energy phosphates (equivalent to 2 ATP).

The ribosomes are located in the cytosomal fraction of the cell. They are found in association with rough endoplasmic reticulum (RER) to form clusters RER—ribosomes, where the protein synthesis occurs. The term polyribosome (polysome) is used when several ribosomes simultaneously translate on a single mRNA (Fig.25.15).
3. Messenger RNA (mRNA) : The specific information required for the synthesis of a given protein is present on the MRNA. The DNA has passed on the genetic information in the form of codons to mRNA to translate into a protein sequence.
4. Transfer RNAs (tRNAs) : They carry the amino acids, and hand them over to the growing peptide chain. The amino acid is covalently bound to tRNA at the 3'-end. Each tRNA has a three nucleotide base sequence—the anticodon, which is responsible to recognize the codon (complementary bases) of mRNA for protein synthesis.
In man, there are about 50 different tRNAs whereas in bacteria around 40 tRNAs are found. Some amino acids (particularly those with multiple codons) have more than one tRNA.
6. Protein factors : The process of translation involves a number of protein factors. These are needed for initiation, elongation and termination of protein synthesis. The protein factors are more complex in eukaryotes compared to prokaryotes.

Amino acids are activated and attached to tRNAs in a two step reaction. A group of enzymes—namely aminoacyl tRNA synthetases— are required for this process. These enzymes are highly specific for the amino acid and the corresponding tRNA.
The amino acid is first attached to the enzyme utilizing ATP to form enzyme-AMP-amino acid complex. The amino acid is then transferred to the 3' end of the tRNA to form aminoacyl tRNA (Fig.25.16).

The protein or polypeptide synthesis occurs on the ribosomes (rather polyribosomes). The mRNA is read in the 53' direction and the polypeptide synthesis proceeds from N-terminal end to C-terminal end. Translation is directional and collinear with mRNA.
Fig. 25.16 : Formation of aminoacyl tRNA (AA-Amino acid; E-Enzyme).
The prokaryotic mRNAs are polycistronic, since a single mRNA has many coding regions that code for different polypeptides. In contrast, eukaryotic mRNA is monocistronic, since it codes for a single polypeptide.
protein biosynthesis in eukaryotes is better understood now.
In case of prokaryotes, translation commences before the transcription of the gene is completed. Thus, simultaneous transcription and translation are possible. This is not so in case of eukaryotic organisms since transcription occurs in the nucleus whereas translation takes place in the cytosol. Further, the primary transcript (hnRNA) formed from DNA has to undergo. several modifications to generate functional mRNA.
Protein synthesis is comparatively simple in case of prokaryotes compared to eukaryotes. Further, many steps in eukaryotic translation were not understood for quite sometime. For these reasons, majority of the textbooks earlier used to describe translation in prokaryotes in detail, and give most important and relevant information for eukaryotic translation. With the advances in molecular biology, the process of
Translation in eukaryotes is briefly described here, along with some relevant features of prokaryotic protein biosynthesis. Translation proper is divided into three stages—initiation, elongation and termination (as it is done for transcription).

The initiation of translation in eukaryotes is complex, involving at least ten eukaryotic initiation factors (elFs). Some of the elFs contain multiple (3-8) subunits. The process of translation initiation can be divided into four steps (Fig.25.17).
1. Ribosomal dissociation.
2. Formation of 43S preinitiation complex.
. Formation of 48S initiation complex.
. Formation of 80S initiation complex.
Fig. 25.17 : A diagrammatic representation of initiation of protein biosynthesis (translation) in eukaryotic cells (The eukaryotic initiation factors are represented by symbols A, and ©.. By pretixing with elF,
the full names of the factors are obtained e.g. elF-3).
(7), [8] represents

The 80S ribosome dissociates to form 40S and 60S subunits. Two initiating factors namely elF3 and elF-1A bind to the newly formed 40S subunit, and thereby block its reassociation with 60S subunit. For this reason, some workers name elF-3. as anti-association factor.

A ternary complex containing met-tRNA' and elF-2 bound to GTP attaches to 40S ribosomal subunit to form 43S preinitiation complex. The presence of elF-3 and elF-1A stabilizes this complex (Note Met-tRNA is specifically involved in binding to the initiation condon AUGs; hence the superscript' is used in mettRNA').

The binding of mRNA to 43S preinitiation complex results in the formation of 48S initiation complex through the intermediate 43S initiation complex. This, however, involves certain interactions between some of the elFs and activation of mRNA.
elF-4F complex is formed by the association of elF-4G, elF-4A with elF-4E. The so formed elF-4F (referred to as cap binding protein) binds to the cap of mRNA. Then elF-4A and elF-4B bind to mRNA and reduce its complex structure. This mRNA is then transferred to 43S complex. For the appropriate association of 43S preinitiation complex with mRNA, energy has to be supplied by ATP.
Recognition of initiation codon The ribosomal initiation complex scans the mRNA for the identification of appropriate initiation codon. 5'-AUG is the initiation codon and its recognition is facilitated by a specific sequence of nucleotides surrounding it. This marker sequence for the identification of AUG is called as Kozak consensus sequences. |n case of prokaryotes the recognition sequence of initiation codon is referred to as ShineDalgarno sequence.

48S initiation complex binds to 60S ribosomal subunit to form 80S initiation complex. The binding involves the hydrolysis of GTP (bound to elF-2). This step is facilitated by the involvement of elF-5.
As the 80S complex is formed, the initiation factors bound to 48S initiation complex are released, and recycled. The activation of elF-2 requires elF-2B (also called as guanine nucleotide exchange factor) and GTP. The activated elF-2 (i.e. bound to GTP) requires elF2C to form the ternary complex.

The elF-4F, a complex formed by the assembly of three initiation factors controls initiation, and thus the translation process. elF4E, a component of elF-4F is primarily responsible for the recognition of mRNA cap. And this step is the rate-limiting in translation.
elF-2 which is involved in the formation of 43S preinitiation complex also controls protein biosynthesis to some extent.

The formation of translation initiation complex in prokaryotes is less complicated compared to eukaryotes. The 30S ribosomal subunit is bound to initiation factor 3 (IF-3) and attached to ternary complex of IF-2, formyl mettRNA and GTP. Another initiation factor namely IF-l also participates in the formation of preinitiation complex. The recognition of initiation codon AUG is done through ShineDalgarno sequence. A 50S ribosome unit is now bound with the 30S unit to produce 70S initiation complex in prokaryotes.

Ribosomes elongate the polypeptide chain by a sequential addition of amino acids. The amino acid sequence is determined by the order of the codons in the specific mRNA. Elongation, a cyclic process involving certain elongation
factors (EFs), may be divided into three steps (Fig.25.18).
1. Binding of aminoacyl t-RNA to A-site.
2. Peptide bond formation.
3. Translocation.

The 80S initiation complex contains mettRNAi in the P-site, and the A-site is free. Another aminoacyl-tRNA is placed in the A-site. This requires proper codon recognition on the mRNA and the involvement of elongation factor 1a (EF-la) and supply of energy by GTP. As the aminoacyl-tRNA is placed in the A-site, EF-10 and GDP are recycled to bring another aminoacyl-tRNA.

The enzyme peptidyltransferase catalyses the formation of peptide bond (Fig.25.19). The activity of this enzyme lies on 28S RNA of 60S ribosomal subunit. It is therefore the rRNA (and not protein) referred to as ribozyme that catalyses the peptide bond formation. As the amino acid in the aminoacyl-tRNA is already activated, no additional energy is required for peptide bond formation.
The net result of peptide bond formation is the attachment of the growing peptide chain to the tRNA in the A-site.

As the peptide bond formation occurs, the ribosome moves to the next codon of the mRNA (towards 3'-end). This process called translocation, basically involves the movement of growing peptide chain from A-site to P-site. Translocation requires EF-2 and GTP. GTP gets hydrolysed and supplies energy to move mRNA. EF-2. and GTP complex recycles for translocation.
In recent years, another site namely exit site (E-site) has been identified in eukaryotes. The deacylated tRNA moves into the E-site, from where it leaves the ribosome.
In case of prokaryotes, the elongation factors are different, and they are EF-Tu, EF-Ts (in place of of EF-1a) and EF-G (instead of EF-2).

It is estimated that about six amino acids per second are incorporated during the course of elongation of translation in eukaryotes. In case of prokaryotes, as many as 20 amino acids can be incorporated per second. Thus the process of protein/polypeptide synthesis in translation occurs with great speed and accuracy.

Termination is a simple process when compared to initiation and elongation. After several cycles of elongation, incorporating amino acids and the formation of the specific protein/ polypeptide molecule, one of the stop or termination signals (UAA, UAG and UCA) terminates the growing polypeptide. The termination codons which act as stop signals do not have specific tRNAs to bind. As the termination codon occupies the ribosomal A-site, the release factor namely eRF recognizes the stop signal. eRF-GTP complex, in association with the enzyme peptidyltransferase, cleaves the peptide bond between the polypeptide and the tRNA occupying P-site. In this reaction, a water molecule, instead of an amino acid is added. This hydrolysis releases the protein and tRNA from the P-site. The 80S ribosome dissociates to form 40S and 60S subunits which are recycled. The mRNA is also released.

Translation is a complex process and it has become a favourite target for inhibition by antibiotics. Antibiotics are the substances produced by bacteria or fungi which inhibit the growth of other organisms. Majority of the antibiotics interfere with the bacterial protein synthesis and are harmless to higher organisms. This is due to the fact that the process of translation sufficiently differs between prokaryotes and eukaryotes. The action of a few important antibiotics on translation is described next.
:
i
i
i
|
Fig. 25.18 : Protein biosynthesis — Elongation and termination (for initiation See Fig. 25.17). Met-Methionine; P-site — Peptidyl tRNA binding site; A-site — Aminoacyl tRNA binding site. AA-Amino acid; EF-Elongation factor; RF-Releasing factor.
Fig. 25.19 : Formation of peptide bond in translation (P-site — Peptidyl tRNA site; A-site — Aminoacyl tRNA site).
Streptomycin : Initiation of protein synthesis is inhibited by streptomycin. It causes misreading of mRNA and interferes with the normal pairing between codons and anticodons.

Tetracycline It inhibits the binding of aminoacyl tRNA to the ribosomal complex. In fact, tetracycline can also block eukaryotic protein synthesis. This, however, does not happen since eukaryotic cell membrane is not permeable to this drug.
Puromycin : This has a structural resemblance to aminoacyl tRNA. Puromycin enters the A site and gets incorporated into the growing peptide chain and causes its release. This antibiotic prevents protein synthesis in both prokaryotes and eukaryotes.
Chloramphenicol : It acts as a competitive inhibitor of the enzyme peptidyltransferase and thus interferes with elongation of peptide chain.
Erythromycin : It inhibits translocation by binding with 50S subunit of bacterial ribosome.
Diphtheria toxin : It prevents translocation in eukaryotic protein synthesis by inactivating elongation factor eEF.
The three dimensional conformation of proteins is important for their biological functions. Some of the proteins can spontaneously generate the correct functionally active conformation e.g. denatured pancreatic ribonuclease. However, a _ vast majority of proteins can attain correct conformation, only through the assistance of certain proteins referred to as chaperones. Chaperones are heat shock proteins (originally discovered in response to heat shock). They facilitate and favour the interactions on the polypeptide surfaces to finally give the specific conformation of a __ protein. Chaperones can reversibly bind to hydrophobic regions of unfolded proteins and folding intermediates. They can _ stabilize intermediates, prevent formation of incorrect intermediates, and also prevent undesirable interactions with other proteins. All these activities of chaperones help the protein to attain compact and_ biologically active conformation.

Chaperones are categorized into two major
groups
1. Hsp70 system : This mainly consists of Hsp70 (70-kDa heat shock protein) and Hsp40 (40-kDa_ Hsp). These proteins can bind individually to the substrate (protein) and help in the correct formation of protein folding.
2. Chaperonin system This is a large oligomeric assembly which forms a structure into which the folded proteins are inserted. The chaperonin system mainly has Hsp60 and Hsp10 i.e. 60 kDa Hsp and 10 kDa Hsp. Chaperonins are required at a later part of the protein folding process, and often work in association with
Hsp70 system.

The failure of a protein to fold properly generally leads to its rapid degradation. Cystic fibrosis (CF) is a common autosomal recessive disease. Some cases of CF with mutations that result in altered protein (cystic _ fibrosis transmembrane conductance regulator or in short CFTR) have been reported. Mutated CFTR cannot fold properly, besides not being able to get glycosylated or transported. Therefore, CFTR gets degraded.
Certain neurological diseases which are due to cellular accumulation of aggregates of misfolded proteins or their partially degraded products have been identified. The term prions (proteinous infectious agents) is used to collectively represent them.
Prions exhibit the characteristics of viral or microbial pathogens and have been implicated in many diseases. e.g. mad cow disease, Creutzfeldt-Jacob disease, Alzheimer's disease, Huntington's disease (Refer Chapter 22).

The proteins synthesized in translation are, as such, not functional. Many changes take place in the polypeptides after the initiation of their synthesis or, most frequently, after the protein synthesis is completed. These modifications include protein folding (described already), trimming by proteolytic degradation, intein splicing and covalent changes which are collectively known as __ post-translational modifications (Fig.25.20).

Faulty splicing of hnRNA may result in certain diseaes e.g. B-thalassemia.
Inhibitors of transcription are used as therapeutic agents. Thus, actinomycin D was the first antibiotic used in the treatment of tumors. Rifampin is employed to treat tuberculosis and leprosy.
Retroviruses (RNA is the genetic material) are oncogenic i.e. cause cancers.
Several antibiotics selectively block bacterial translation, and thus inhibit their growth e.g. streptomycin, tetracycline, puromycin.
+ Protein misfolding often results in the formation of prions (proteinous infectious agents) which have been implicated in many diseases e.g. mad cow disese, Alzheimer’s disease.
+ Lebers’ hereditary optic neuropathy is caused by mutation in mtDNA in males. The victims become blind due to loss of central vision as a result of neuroretinal degeneration.
Fig. 25.20 : An outline of post-translational modifications of proteins.

Many proteins are synthesized as the precursors which are much bigger in size than the functional proteins. Some portions of precursor molecules are removed by proteolysis to liberate active proteins. This process is commonly referred to as trimming. The formation of insulin from — preproinsulin, conversion of zymogens (inactive digestive enzymes e.g. trypsinogen) to the active enzymes are some examples of trimming.

Inteins are intervening sequences in certain proteins. These are comparable to introns in mRNAs. Inteins have to be removed, and exteins ligated in the appropriate order for the protein to become active.

The proteins synthesized in translation are subjected to many covalent changes. By these modifications in the amino acids, the proteins may be converted to active form or inactive form. Selected examples of — covalent modifications are described below.
1. Phosphorylation The hydroxyl group containing amino acids of proteins, namely serine, threonine and tyrosine are subjected to phosphorylation. The phosphorylation may either increase or decrease the activity of the proteins. A
group of enzymes called protein kinases catalyse phosphorylation while protein phosphatases are responsible for dephosphorylation (removal of phosphate group). Many enzymes that undergo phosphorylation or dephosphorylation are known in metabolisms (e.g. glycogen synthase).
2. Hydroxylation : During the formation of collagen, the amino acids proline and lysine are respectively converted to hydroxyproline and hydroxylysine. This hydroxylation occurs in the endoplasmic reticulum and requires vitamin C.
3. Glycosylation : The attachment of carbohydrate moiety is essential for some proteins to perform their functions. The complex carbohydrate moiety is attached to the amino acids, serine and threonine (O-linked) or to asparagine (N-linked), leading to the synthesis of glycoproteins.
Vitamin K dependent carboxylation of glutamic acid residues in certain clotting factors is also a post-translational modification.
In the Table 25.2, selected examples of posttranslational modification of proteins through their amino acids are given.

The eukaryotic proteins (tens of thousands) are distributed between the cytosol, plasma membrane and a number of cellular organelles

Amino acid, 1 = Post-translational modification(s). Amino-terminal amino acid, 1 = Glycosylation, acetylation, myristoylation, formylation.. Carboxy terminal amino acid, 1 = Methylation, ADP-ribosylation. Arginine, 1 = Methylation. Aspartic acid, 1 = Phosphorylation, hydroxylation. Cysteine ( -SH), 1 = Cystine ( -S-S-) formation, selenocysteine formation, glycosylation.. Glutamic acid, 1 = Methylation, J -carboxylation.. Histidine, 1 = Methylation, phosphorylation.. Lysine, 1 = Acetylation, methylation, hydroxylation, biotinylation.. Methionine, 1 = Sulfoxide formation.. Phenylalanine, 1 = Glycosylation, hydroxylation.. Proline, 1 = Hydroxylation, glycosylation.. Serine, 1 = Phosphorylation, glycosylation.. Threonine, 1 = Phosphorylation, methylation glycosylation.. Tryptophan, 1 = Hydroxylation.. Tyrosine, 1 = Hydroxylation, phosphorylation, sulfonylation, iodination.
(nucleus, mitochondria, endoplasmic reticulum etc.). At the appropriate places, they perform their functions.
The proteins, synthesized in translation, have to reach their destination to exhibit their biological activities. This is carried out by a process called protein targeting or protein sorting or protein localization. The proteins move from one compartment to another by multiple mechanisms.
The protein transport from the endoplasmic reticulum through the Golgi apparatus, and beyond uses carrier vesicles. It may be, however, noted that only the correctly folded proteins are recognized as the cargo for transport. Protein targeting and_ post-translational modifications occur in a well coordinated manner.
Certain glycoproteins are targeted to reach lysosomes, as the lysosomal proteins can recognize the glycosidic compounds e.g. N-acetylglucosamine phosphate.

For the transport of secretory proteins, a special mechanism is operative. A signal peptide containing 15-35 amino acids, located at the amino terminal end of the secretory proteins facilitates the transport.

Most of the proteins of mitochondria are synthesized in the cytosol, and their transport to mitochondria is a complex process. Majority of the proteins are synthesized as larger preproteins with N-terminal presequences for the entry of these proteins into mitochondria. The transport of unfolded proteins is often facilitated by chaperones.
One protein namely mitochondrial matrix targeting signal, involved in protein targeting has been identified. This protein can recognize mitochondrial receptor and transport certain proteins from cytosol to mitochondria. This is an energy-dependent process.

Specific signals for the transport of proteins to organelles such as nuclei and peroxisomes have been identified.
The smaller proteins can easily pass through nuclear pores. However, for larger proteins, nuclear localization signals are needed to facilitate their entry into nucleus.

The mitochondrial DNA (mtDNA) _ has structural and functional resemblances with prokaryotic DNA. This fact supports the view that mitochondria are derivatives of prokaryotes. mtDNA is circular in nature and contains about 16,000 nucleotide bases.
mtDNA is inherited from the mother. Mitochondrial DNA is subjected to high rate of mutations (about 10 times more than nuclear DNA) that causes inherited defects in oxidative phosphorylation. The best known among them are certain mitochondrial myopathies and Leber's hereditary optic neuropathy. The latter is mostly found in males and is characterized by blindness due to loss of central vision as a result of neuroretinal degeneration. Leber's hereditary optic neuropathy is a consequence of single base mutation in mtDNA. Due to this, the amino acid histidine, in place of arginine, is incorporated into the enzyme NADH coenzyme Q reductase.

1., 1 = Transcription is the process in which RNA is synthesized from DNA, which is carried out in 3 stages-initiation, elongation and termination.. 2., 1 = In case of prokaryotes, a single enzyme synthesizes all the RNAs. In eukaryotes, RNA polymerase I, II and III respectively catalyse the formation of rRNAs, mRNAs and tRNAs.. 3., 1 = The primary mRNA transcript (i.e. hnRNA) undergoes post-transcriptional modifications e.g. base modifications, splicing etc.. 4., 1 = Reverse transcription is the process of synthesizing DNA from RNA by the enzyme reverse transcriptase.. 5., 1 = Biosynthesis of a protein or a polypeptide is known as translation. The amino acid sequence of a protein is determined by the triplet nucleoside base sequences of mRNA, arranged as codons.. 6., 1 = The genetic code (codons)-composed of A, G, C and U-is universal, specific, non- overlapping and degenerate. Of the 64 codons, three (UAA, UAG, UGA) are termination codons while the rest code for amino acids.. 7., 1 = Ribosomes are the factories of protein biosynthesis. Translation involves activation of amino acids, protein synthesis proper (initiation, elongation and termination), protein folding and post-translational modifications.. 8., 1 = The post-translational modifications include proteolytic degradation, intein splicing and covalent modifications (phosphorylation, hydroxylation, glycosylation etc.). These modifications are required to make the proteins biologically active.. 9., 1 = The proteins synthesized in translation reach the destination to exhibit their biological activity. This is carried out by a process called protein targeting or protein sorting.. 10., 1 = The mitochondria possess independent DNA with the machinery for transcription and translation. However, only a few proteins (around 13) are actually synthesized in the mitochondria.

1
a kB wWN . Describe protein biosynthesis (translation). . Discuss the inhibitors of transcription and translation. . Give an account of post-transcriptional and post-translational modifications.
.
Give
an
account
of
transcription.
Compare
the
RNA
synthesis
between
and
eukaryotes.
. What is genetic code? Describe the characteristics of genetic code. Add a note on the effects of mutations on genetic code.

(a) Genome, (b) Heterogeneous nuclear RNA (hnRNA), (c) Eukaryotic RNA polymerases, (d) Introns and exons, (e) Reverse transcription, (f) Wobble hypothesis, (g) Anticodon, (h) Shine-Dalgarno sequence, (i) Peptidyltransferase, (j) Chaperones, (k) Protein targeting.

1. The total DNA (genetic information) contained in an organism (or a cell) is referred to
as
2. The primary transcript produced by RNA polymerase II is eukaryotes
9. The common term used for the diseases due to misfolding of proteins
10. The process of delivery of proteins in a cell to the site their biological activity is
. The intervening nucleotide sequences in mRNA that do not code for proteins
:
. The synthesis of complementary DNA (cDNA) from mRNA is catalysed by the enzyme
. Asingle tRNA is capable of recognizing more than one codon, and this phenomenon is referred
to
as
. The factories for protein biosynthesis are
:
. The enzyme peptidyltransferase calalyses the formation of peptide bond during translation. The
chemical nature of this enzyme is
.
The
proteins
that
the
formation
of
specific
conformation
of
proteins
are
.

11. The codon(s) that terminate(s) protein biosynthesis
12. The nitrogenous base that is never found in the genetic code
13. The total DNA (genetic information) contained in a living cell (or organism) is regarded as
14. The enzyme responsible for the synthesis of mRNAs in eukaryotic cells
15. Mitochondrial DNA is inherited from
(a) UAA (b) UAG (c) UGA (d) All of them.
(a) Adenine (b) Guanine (c) Thymine (d) Cytosine.
(a) Genome (b) Transciptome (c) Proteome (d) Gene.
(a) RNA polymerase | (b) RNA polymerase II (c) RNA polymerase III (d) RNA polymerase o.
(a) Mother only (b) Father only (c) Both of them (d) Either mother or father.
D™ the chemical vehicle of heredity, is composed of functional units, namely genes. The term genome refers to the total genetic information contained in a cell. The bacterium Escherichia coli contains about 4,400 genes present on a single chromosome. The genome of humans is more complex, with 23 pairs of (diploid) chromosomes containing 6 billion (6 x 109) base pairs of DNA, with an estimated 30,000-40,000 genes. At any given time, only a fraction of the genome is expressed.
The living cells possess a remarkable property to adapt to changes in the environment by regulating the gene expression. For instance, insulin is synthesized by specialized cells of pancreas and not by cells of other organs (say kidney, liver), although the nuclei of all the cells of the body contain the insulin genes. Molecular regulatory mechanisms facilitate the expression of insulin gene in pancreas, while preventing its expression in other cells.

The regulation of the expression of genes is absolutely essential for the growth, development, differentiation and the very existence of an organism. There are two types of gene regulation-positive and negative.
1. Positive regulation : The gene regulation is said to be positive when its expression is increased by a regulatory element (positive regulator).
2. Negative regulation : A decrease in the gene expression due to the presence of a regulatory element (negative regulator) is referred to as negative regulation.
It may be noted here that double negative effect on gene regulation results in a positive phenomenon.

The genes are generally considered under two categories.
1. Constitutive genes: The — products (proteins) of these genes are required all the time in a cell. Therefore, the constitutive genes (or housekeeping genes) are expressed at more or less constant rate in almost all the cells and, further, they are not subjected to regulation e.g. the enzymes of citric acid cycle.
2. Inducible genes : The concentration of the proteins synthesized by inducible genes is regulated by various molecular signals. An inducer increases the expression of these genes while a repressor decreases, e.g. tryptophan pyrrolase of liver is induced by tryptophan.
The term pseudogenes is used to represent DNA sequences that have significant homology to a functional gene, but they cannot express due to mutations. Thus pseudogenes are nonfunctional. However, they significantly increase the size of the eukaryotic genome without any contribution to the expression of genes.

The chemical product of a gene expression is a protein which may be an enzyme. It was originally believed that each gene codes for a specific enzyme, leading to the popular concept, one gene-one enzyme. This however, is not necessarily valid due to the fact that several enzymes (or proteins) are composed of two or more nonidentical subunits (polypeptide chains).
The cistron is the smallest unit of genetic expression. It is the fragment of DNA coding for the subunit of a protein molecule. The original concept of one gene-one enzyme is replaced by one cistron-one subunit.

Elucidation of the regulation of gene expression in prokaryotes has largely helped to understand the principles of the flow of information from genes to mRNA to synthesize specific proteins. Some important features of prokaryotic gene expression are described first. This is followed by a brief account of eukaryotic gene expression.

The operon is the coordinated unit of genetic expression in bacteria. The concept of operon was introduced by Jacob and Monod in 1961 (Nobel Prize 1965), based on their observations on the regulation of lactose metabolism in E. coli. This is popularly known as lac operon.

The lac operon (Fig.26.1) consists of a regulatory gene (I; | for inhibition), operator gene (O) and three structural genes (Z, Y, A). Besides these genes, there is a promoter site (P), next to the operator gene, where the enzyme RNA polymerase binds. The structural genes Z, Y and A respectively, code for the enzymes B-galactosidase, galactoside permease and galactoside acetylase. B-Galactosidase hydrolyses lactose (B-galactoside) to galactose and glucose while permease is responsible for the transport of lactose into the cell. The function of acetylase (coded by A gene) remains a mystery.
The structural genes Z, Y and A transcribe into a single large mRNA with 3 independent translation units for the synthesis of 3 distinct enzymes. An mRNA coding for more than one protein is known as_ polycistronic mRNA. Prokaryotic organisms contain a large number of polycistronic mRNAs.

The regulatory gene (I) is constitutive. It is expressed at a constant rate leading to the synthesis of lac repressor. Lac repressor is a tetrameric (4 subunits) regulatory protein (total mol. wt. 150,000) which specifically binds to the operator gene (O). This prevents the binding of the enzyme RNA polymerase to the promoter site (P), thereby blocking the transcription of structural genes (Z, Y and A). This is what happens in the absence of lactose in EF. coli. The repressor molecule acts as a negative regulator of gene expression.
galactoside permease and galactoside acetylase. Lactose acts by inactivating the repressor molecules, hence this process is known as derepression of lac operon.
Gratuitous inducers There are certain structural analogs of lactose which can induce the lac operon but are not the substrates for the enzyme f-galactosidase. Such substances are known as_ gratuitous inducers. lsopropylthiogalactoside (IPTG) is a gratuitous inducer, extensively used for the study of lac operon.

gene : cells of E. coli utilize glucose in preference to lactose; when both of them are present in the medium. After the depletion of glucose in the medium, utilization of lactose starts. This indicates that glucose somehow interferes with the induction of lac operon. This is explained as follows.
The attachment of RNA polymerase to the promoter site requires the presence of a catabolite gene activator protein (CAP) bound to cyclic AMP (Fig.26.2). The presence of glucose lowers the intracellular concentration of cAMP by inactivating the enzyme adenylyl cyclase responsible for the synthesis of CAMP. Due to the diminished levels of cAMP, the formation of CAPcAMP is low. Therefore, the binding of RNA polymerase to DNA (due to the absence of CAPcAMP) and the transcription are almost negligible in the presence of glucose. Thus, glucose interferes with the expression of lac operon by depleting cAMP levels. Addition of exogenous cAMP is found to initiate the transcription of many inducible operons, including lac operon.
It is now clear that the presence of CAP-cAMP is essential for the transcription of structural genes of lac operon. Thus, CAP-cAMP acts as a positive regulator for the gene expression. It is, therefore, evident that lac operon is subjected to both positive (by repressor, described above) and negative regulation.

Tryptophan is an aromatic amino acid, and is required for the synthesis of all proteins that
contain tryptophan. If tryptophan is not present in the medium in adequate quantity, the bacterial cell has to make it, as it is required for the growth of the bacteria.
The tryptophan operon of E. coli is depicted in Fig.26.3. This operon contains five structural genes (trpE, trpD, trpC, trpB, trpA), and the regulatory elements—primary promoter (trpP), operator (frpO), attenuator (trpa), secondary internal promoter (TrpP ), and terminator (trpt). The five structural of tryptophan genes operon code for three enzymes (two enzymes contain two different subunits) required for the synthesis of tryptophan from chorismate.
The tryptophan repressor is always turned on, unless it is repressed by a specific molecule called corepressor. Thus lactose operon (described already) is inducible, whereas tryptophan operon is repressible. The tryptophan operon is said to be derepressed when it is actively transcribed.

Tryptophan acts as a corepressor to shut down the synthesis of enzymes from tryptophan operon. This is brought out in association with a specific protein, namely tryptophan repressor.
Fig. 26.3 : Tryptophan operon in E.coli [regulatory elements are promoter (trpP), operator (trpO), attenuator (trpa), secondary internal promoter (trpP,) and terminator (trpt); Col, Coll-Component | and component II; PRPP — 5-Phosphoribosyl 1-pyrophosphate; CdRP-Carboxyl-phenylamino 1 deoxyribulose 5-phosphate; InGP-Indole 3-glycerol phosphate].
Tryptophan repressor, a homodimer (contains two identical subunits) binds with two molecules of tryptophan, and then binds to the trp operator to turn off the transcription. It is of interest to note that tryptophan repressor also regulates the transcription of the gene (trpR) responsible for its own synthesis.
Two polycistronic mRNAs are produced from tryptophan operon—one derived from all the five structural genes, and the other obtained from the last three genes.
Besides acting as a corepressor to regulate tryptophan operon, tryptophan can inhibit the activity of the enzyme anthranilate synthetase. This is referred to as feedback inhibition, and is brought out by binding of tryptophan at an allosteric site on anthranilate synthetase.

Attenuator gene (trpa) of tryptophan operon lies upstream of trpE gene. Attenuation is the second level of regulation of tryptophan operon. The attenuator region provides RNA polymerase which regulates transcription. In the presence of tryptophan, transcription is prematurely terminated at the end of attenuator region. However, in the absence of tryptophan, the attenuator region has no effect on transcription. Therefore, the polycistronic mRNA of the five structural genes can be synthesized.

Each cell of the higher organism contains the entire genome. As _ in_ prokaryotes, gene expression in eukaryotes is regulated to provide the appropriate response to biological needs. This may occur in the following ways
1
Expression of
certain genes
(housekeeping genes)
in most
of the
cells.
' Activation of selected genes upon demand.
| Permanent inactivation of several genes in all but a few types.
In case of prokaryotic cells, most of the DNA is organized into genes which can be transcribed. In contrast, in mammals, very little of the total DNA is organized into genes and their associated regulatory sequences. The function of the bulk of the extra DNA is not known.
Eukaryotic gene expression and its regulation are highly complex. Some of the important aspects are briefly described.

The DNA in higher organisms is extensively folded and packed to form protein-DNA complex called chromatin. The — structural organization of DNA in the form of chromatin plays an important role in eukaryotic gene expression. In fact, chromatin — structure provides an additional level of control of gene expression.
A selected list of genes (represented by the products) along with the respective chromosomes on which they are located is given in Table 26.1. In general, the genes that are transcribed within a particular cell are less condensed and more open in structure. This is in contrast to genes that are not transcribed which form highly condensed chromatin.

DNA
segments are
wrapped around
histone proteins
to form
nucleosome.

Alkaline phosphatase, Chromosome number = 1. Apolipoprotein B, Chromosome number = 2. Transferrin, Chromosome number = 3. Alcohol dehydrogenase, Chromosome number = 4. HMGCoA reductase, Chromosome number = 5. Steroid 21-hydroxylase, Chromosome number = 6. Arginase, Chromosome number = 7. Carbonic anhydrase, Chromosome number = 8. Interferon, Chromosome number = 9. Parathyroid hormone, Chromosome number = 11. Glyceraldehyde 3-phosphate dehydrogenase, Chromosome number = 12. Adenosine deaminase, Chromosome number = 13. D 1 -Antitrypsin, Chromosome number = 14. Cytochrome P 450, Chromosome number = 15. Hemoglobin D -chain, Chromosome number = 16. Growth hormone, Chromosome number = 17. Prealbumin, Chromosome number = 18. Creatine phosphokinase (M chain), Chromosome number = 19. Adenosine deaminase, Chromosome number = 20. Superoxide dismutase, Chromosome number = 21. Immunoglobulin ( O chain), Chromosome number = 22. Glucose 6-phosphate dehydrogenase, Chromosome number = X. Steroid sulfatase, Chromosome number = Y
Acetylation or deacetylation of histones is an important factor in determining the gene expression. In general, acetylation of histones leads to activation of gene expression while deacetylation reverses the effect.
Acetylation predominantly occurs on the lysine residues in the amino terminal ends of histones. This modification in histones reduces the positive charges of terminal ends (tails), and decreases their binding affinity to negatively charged DNA. Consequently, nucleosome structure is disrupted to allow transcription.

Cytosine in the sequence CG of DNA gets methylated to form 5'-methylcytosine. A major portion of CG sequences (about 20%) in human DNA exists in methylated form. In general, methylation leads to loss of transcriptional activity, and thus inactivation of genes. This occurs due to binding of methylcytosine binding proteins to methylated DNA. As a_ result, methylated DNA is not exposed and bound to transcription factors. It is interesting to note that methylation of DNA correlates with deacetylation of histones. This provides a double means for repression of genes.
The activation and normal expression of genes, and gene inactivation by DNA methylation are depicted in Fig.26.4.

Enhancers (or activators) are DNA elements that facilitate or enhance gene expression. The enhancers provide binding sites for specific proteins that regulate transcription. They facilitate binding of the transcription complex to promoter regions.
Some of the enhancers possess the ability to promote transcription in a tissue-specific manner. For instance, gene expression in lymphoid cells for the production immunoglobulins (Ig) is promoted by the enhancer associated with lg genes between J and C regions.
Transgenic animals are frequently used for the study of tissue-specific expression. The available evidence from various studies indicates that the tissue-specific gene expression is largely mediated through the involvement of enhancers.

in mammals
is
a
complicated process
with stimuli
on
a
of the
gene brought
single gene.
which out
by and
several environmental
The ultimate
may be
response positive
association or
negative of
DNA
is elements
In the illustration given in the Fig.26.5, gene | is activated by a combination of activators 1, 2 and 3. Gene II is more effectively activated by the combined action of 1, 3 and 4. Activator 4 is not in direct contact with DNA, but it forms a bridge between activators 1 and 3, and activates gene II. As regards gene III, it gets inactivated by a combination of 1, 5 and 3. In this case, protein 5 interferes with the binding of protein 2 with the DNA, and inactivates the gene.

A motif literally means a dominant element. Certain motifs in proteins mediate the binding of regulatory proteins (transcription factors) to
(A)
Fig. 26.5 : A diagrammatic representation of the association of DNA elements and proteins in gene regulation. A, B andC represent genes |, II and III J (1...5 represent proteins).
A great majority of specific protein-DNA interactions are brought out by four unique motifs—helix-turn-helix (HTH), zinc finger, leucine zipper, helix-loop-helix (HLH).
These amino acid motifs bind with high affinity to the specific site and low affinity to other parts of DNA. The motif-DNA interactions are maintained by hydrogen bonds and van der Waals forces.

The helix-turn-helix (HTH) motif is about 20 amino acids which represents a small part of a large protein. HTH is the domain part of the protein which specifically interacts with the DNA (Fig.26.6A). Examples of helix-turn-helix motif proteins include lactose repressor, and cyclic AMP catabolite activator protein (CAP) of E. coli, and several developmentally important transcription factors in mammals.
DNA. The specific control of transcription occurs by the binding of regulatory proteins with high affinity to the correct regions of DNA.

Sometime ago, it was recognized that the
transcription factor TFIIIA requires zinc for its

+ Regulation of gene expression to adapt to the changes in the environment is a remarkable property of living cells e.g. synthesis of insulin by P-cells of pancreas and nowhere else.
+ The growth, development and differentiation of an organism involves complex mechanisms which ultimately depend on gene regulation.
+ The house-keeping genes or constitutive genes are expressed at almost a constant rate in the cells, and they are not usually subjected to regulations e.g. enzymes of Krebs cycle.
+ The malignant cells develop drug resistance to long term administration of methotrexate. This occurs by amplification of the genes coding for dihydrofolate reductase.
+ The human body has the capability to produce around 10 billion antigen-specific immunoglobulins. This is achieved by a process called gene rearrangement.
+ Knowledge on the gene expression and its regulation helps in the understanding and control of several diseases, including cancer.

Fig. 26.6 : A diagrammatic representation of common motifs in proteins interacting with DNA.
activity. On analysis, it was revealed that each TFIIIA contains zinc ions as a_ repeating coordinated complex. This complex is formed by the closely spaced amino acids cysteine and cysteine, followed by a histidine—histidine pair. In some instances, His-His is replaced by a second
Cys-Cys pair (Fig.26.6B).
The zinc fingers bind to the major groove of DNA, and lie on the face of the DNA. This binding makes a contact with 5 bp of DNA. The steroid hormone receptor transcription factors use zinc finger motifs to bind to DNA.

The basic regions of leucine zipper (bZIP) proteins are rich is the amino acid leucine. There occurs a periodic repeat of leucine residues at every seventh position. This type of repeat structure allows two identical monomers or heterodimers to zip together and form a dimeric complex. This protein-protein complex associates and interacts with DNA (Fig.26.6C€). Good examples of leucine zipper proteins are the enhancer binding proteins (EBP)—fos and jun.

Two amphipathic (literally means a feeling of closeness) at-helical segments of proteins can form helix-loop-helix motif and bind to DNA. The dimeric form of the protein actually binds to DNA (Fig.26.6D).

The important features of eukaryotic gene expression along with the regulatory aspects are described in the preceeding pages. Besides transcription, eukaryotic cells also employ variety of other mechanisms to regulate gene expression. The most important ones are listed below, and briefly described next.
. Gene amplification 1
. Gene rearrangement 2
. Processing of RNA 3
4. Alternate mRNA splicing
5. Transport of mRNA from nucleus to cytoplasm
6. Degradation of mRNA.

In this mechanism, the expression of a gene is increased several fold. This is commonly observed during the developmental stages of eukaryotic organisms. For instance, in fruit fly
Fig. 26.7 : A diagrammatic representation of gene amplification (the genes are depicted in blue and red colours).
(Drosophila), the amplification of genes coding for egg shell proteins is observed during the course of oogenesis. The amplification of the gene (DNA) can be observed under electron microscope (Fig.26.7).
The occurrence of gene amplification has also been reported in humans. Methotrexate is an anticancer drug which inhibits the enzyme dihydrofolate reductase. The malignant cells develop drug resistance to long term administration of methotrexate by amplifying the genes coding for dihydrofolate reductase.

The body possesses an enormous capacity to synthesize
estimated that the human body can produce about 10 billion (1019) antibodies in response to antigen stimulations. The molecular mechanism of this antibody diversity was not understood for long. It is now explained on the basis of gene rearrangement or transposition of genes or somatic recombination of DNA.
The structure of a typical immunoglobulin molecule consists of two light (L) and two heavy (H) chains. Each one of these chains (L or H) contains an N-terminal variable (V) and C-terminal constant (C) regions (Refer Fig.9.3). The V_ regions of immunoglobulins are responsible for the recognition of antigens. The phenomenon of gene rearrangement can be understood from the mechanism of the synthesis of light chains of immunoglobulins (Fig.26.8).
Each light chain can be synthesized by three distinct DNA segments, namely the variable (V,), the joining (,) and the constant (C,). The mammalian genome contains about 500 V, segments, 6 J, segments and 20 C, segments. During the course of differentiation of B-lymphocytes, one V, segment (out of the 500) is brought closer to J, and C, segments. This occurs on the same chromosome. For the sake of illustration, 100% Vi 3rd J, and qoth C, segments are rearranged in Fig.26.8. The rearranged DNA (with V,, J, and C, fragments) is then transcribed to produce a single mRNA for the synthesis of a specific light chain of the antibody. By
a
wide range
of antibodies.
It is
Fig. 26.8 : A diagrammatic representation of gene rearrangement for the synthesis of light chain of immunoglobulin.
innumerable combinations of V,, J, and C, segments, the body's immune system can generate millions of antigen specific immunoglobulin molecules.
The formation of heavy (H) chains of immunoglobulins also occurs by rearrangement of 4 distinct genes—variable (V4), diversity (D), joining (Jy) and constant (Cy).

The RNA synthesized in transcription undergoes modifications resulting in a functional RNA. The changes include intron-exon splicing, polyadenylation etc. (Chapter 25).

Eukaryotic cells are capable of carrying out alternate mRNA _ processing to control gene expression. Different mRNAs can be produced by alternate splicing which code for different proteins (for more details, Refer Chapter 25).

The expression of genes is indirectly influenced by the stability of mRNA. Certain hormones regulate the synthesis and degradation of some mRNAs. For instance, estradiol prolongs the half-life of vitellogenin mRNA from a few hours to about 200 hours.
It appears that the ends of mRNA molecules determine the stability of mRNA. A_ typical eukaryotic mRNA has 5'-non-coding sequences (5'-NCS), a coding region and a 3'-NCS. All the mRNAs are capped at the 5' end, and most of them have a polyadenylate sequence at the 3' end (Fig.26.9). The 5' cap and poly (A) tail protect the mRNA _ against the attack by exonuclease. Further, stem-loop structures in NCS regions, and AU rich regions in the 3' NCS also provide stability to mRNA.

The term epigenetics is used to describe the changes in the characteristics of a cell or an organism that are not due to changes in the nucleotide sequence of the DNA.
Epigenetics regulates gene expression by modulating chromatin structure via histone modification or modification of DNA _ via methylation. For instance, acetylation of histones leads to activation of gene expression, while DNA methylation is associated with a reduction in gene expression.
The pattern of covalent modifications in histones that provide a tunable means of regulating gene expression is referred to as histone code.
Although there is no change in DNA sequence, epigenetics is heritable due to coordination between histone modification and methylation of DNA.

Hypermethylation of DNA in some parts of tumor suppressor genes is found in certain cancers. The enzyme DNA methyltransferase (DNMT) responsible for DNA methylation is targeted for cancer therapy. Many inhibitors of DNMT (e.g. 5-azacytidine) have been approved by FDA, and are in use for the treatment of leukemia.
Likewise, inhibitors of histone deacetylase (HDAC) are also employed in the epigenetic therapy cancers. HDAC _ inhibitors stimulate tumor suppressor gene expression by allowing acetylation of histones in chromatin structure. FDA has aproved the use of vorinostat for the treatment of T-cell lymphoma.
Epigenetic therapy of cancer by employing various inhibitors (individually or in combination) holds a great promise.

1. DNA, the chemical vehicle of heredity, is composed of genes. The regulation of gene expression is absolutely essential for the growth, development and differentiation of an organism. A positive regulation increases gene expression while a negative regulation decreases.
2. The operon is the coordinated unit of gene expression. The lac operon of E. coli consists of regulatory genes and structural genes. The lac repressor binds to the DNA and halts the process of transcription of structural genes. However, the presence of lactose inactivates the repressor (derepression) leading to the expression of structural genes.
3. Tryptophan operon is regulated by a repressor. Tryptophan repressor binds to tryptophan, and then to trp operator gene to turn off the transcription.
4. Eukaryotic gene expression and its regulation are highly complex. Acetylation of histones leads to gene expression while deacetylation reverses the effect. In general, methylation of DNA results in the inactivation of genes.
5.
The
protein-DNA
interactions,
brought
out
by
motifs
(helix-turn-helix,
zinc
finger,
leucine
zipper,
helix-loop-helix),
are
involved
in
the
control
of
gene
6. Eukaryotic cells have developed several mechanisms to regulate gene expression. These include gene amplification, gene rearrangement, and processing, transport and degradation of DNA.

1. Describe lactose (lac) operon.
2. Write briefly on the gene expression and its regulation in eukaryotes.

(a) One cistron-one subunit concept, (b) Catabolite gene activator protein, (c) Gene inactivation by DNA methylation, (d) Zinc finger motif, (e) Gene amplification.

1. The number of genes found in human genome
2. The earlier concept of one gene-one enzyme is replaced by
3. The chromatin in higher organisms is chemically composed of

4. The structural ‘Z’ gene of lactose (lac) operon is responsible for the synthesis of the enzyme(s)
(a) B-Galactosidase (b) Permease (c) Acetylase (d) All of them.
5. Methylation of DNA results in
(a) Activation of genes (b) Inactivation of genes (c) No effect on genes (d) Inactivation of protein motifs.
6. The specific control of transcription involves the following motif(s)
(a) Helix-turn-helix (b) Zinc finger (c) Leucine zipper (d) All of them.
he term biotechnology represents a fusion or an alliance between biology and technology. Frankly speaking, biotechnology is a newly discovered discipline for age-old practices e.g. preparation of wine, beer, curd, bread. These natural processes are regarded as old or traditional biotechnology.
The new or modern biotechnology embraces all the genetic manipulations, cell fusion techniques, and improvements made in the old biotechnological processes. The biotechnology with particular reference to recombinant DNA in human health and disease is briefly described in this chapter.
Genetic engineering primarily involves the manipulation of genetic material (DNA) to achieve the desired goal in a pre-determined way. Some other terms are also in common use to describe
genetic engineering.
1 Gene manipulation
| Recombinant DNA (rDNA) technology
| Gene cloning (molecular cloning)
| Genetic modifications
| New genetics.

The present day DNA technology has its roots in the experiments performed by Boyer and Cohen in 1973. In their experiments, they successfully recombined two plasmids (pSC 101 and pSC 102) and cloned the new plasmid in E.coli. In the later experiments the genes of a frog could be successfully transplanted, and expressed in E.coli. This made the real beginning of modern rDNA technology and laid foundations for the molecular

Some biotechnologists who admire BoyerCohen experiments divide the subject into two chronological categories.
1. BBC-biotechnology Before Boyer and Cohen.
2. ABC-biotechnology After Boyer and
Cohen.
Fig. 27.1 : The basic principle of recombinant DNA technology.
Recombinant DNA technology is a vast field. The basic principles and techniques of rDNA technology along with the most important applications are briefly described in this chapter.

There are many diverse and complex techniques involved in gene manipulation. However, the basic principles of recombinant DNA technology are reasonably simple, and broadly involve the following stages (Fig.27.1).
1.
Generation selection
of the
human gene).
2.
Insertion vector
(e.g.
of desired
DNA
_
piece of
the selected
a
DNA
plasmid)
or
Greek chimeric
fragments of
DNA
DNA
to create
DNA
mythology body
a
(Chimera that
has and
a
a
lion’s serpent’s
tail.
and
(e.g.
a
into
a
cloning recombinant
is
a
monster in
head,
This
a
goat’s may
be comparable
to
Narasimha in
Indian mythology).
- 3. Introduction of the recombinant vectors into host cells (e.g. bacteria).
4.
Multiplication and
selection of
clones containing
the recombinant
5.
Expression molecules.
of the
gene desired
product.
Recombinant to
DNA
produce technology
the with
special reference
to the
following aspects
is described
1 Molecular tools of genetic engineering.
| Host cells-the factories of cloning.
1 Vectors-the cloning vehicles.
1 Methods of gene transfer.
1 Gene cloning strategies.

The term genetic engineer may be appropriate for an individual who is involved in genetic manipulations. The genetic engineer's toolkit or molecular tools namely the enzymes most commonly used in recombinant DNA experiments are briefly described.

Restriction endonucleases are one of the most important groups of enzymes for the manipulation of DNA. These are the bacterial enzymes that can cut/split DNA (from any source) at specific sites. They were first discovered in E.coli restricting the replication of bacteriophages, by cutting the viral DNA (The host E.coli DNA is protected from cleavage by addition of methyl groups). Thus, the enzymes that restrict the viral replication are known as restriction enzymes or restriction endonucleases.
Nomenclature : Restriction endonucleases are named by a standard procedure, with particular reference to the bacteria from which they are isolated. The first letter (in italics) of the enzymes indicates the genus name, followed by the first two letters (also in italics) of the species, then comes the strain of the organism and finally a Roman numeral indicating the order of discovery. A couple of examples are given below.
EcoRI is from Escherichia (BE) coli (co), strain Ry13 (R), and first endonuclease (I) to be discovered. HindIII is from Haemophilus (H) influenzae (in), strain Rd (d) and, the third endonucleases (III) to be discovered.
Recognition sequences : Recognition sequence is the site where the DNA is cut by a restriction endonuclease. Restriction endonucleases can specifically recognize DNA with a_ particular sequence of 4-8 nucleotides and cleave.
Cleavage patterns : Majority of restriction endonucleases (particularly type II) cut DNA at defined sites within recognition sequence. A selected list of enzymes, recognition sequences, and their products formed is given in Table 27.1.
The cut DNA _ fragments by _ restriction endonucleases may have mostly sticky ends (cohesive ends) or blunt ends, as given in Table 27.1. DNA fragments with sticky ends are particularly useful for recombinant DNA experiments. This is because the single-stranded sticky DNA ends can easily pair with any other DNA fragment having complementary sticky ends.
Fig. 27.2 : Action of DNA ligase in the formation of phosphodiester bond (B-base).

The cut DNA fragments are covalently joined together by DNA ligases. These enzymes were originally isolated from viruses. They also occur in E.coli and eukaryotic cells. DNA ligases actively participate in cellular DNA repair process.
Many enzymes are used in the recombinant DNA technology/genetic engineering. A selected list of these enzymes and the reactions catalysed by them is given in Table 27.2.

The hosts are the living systems or cells in which the carrier of recombinant DNA molecule or vector can be propagated. There are different types of host cells-prokaryotic (bacteria) and eukaryotic (fungi, animals and plants). Some examples of host cells used in genetic engineering are given in Table 27.3.
The action of DNA ligases is absolutely required to permanently hold DNA pieces. This is so since the hydrogen bonds formed between the complementary bases (of DNA strands) are not strong enough to hold the strands together. DNA ligase joins (seals) the DNA fragments by forming a phosphodiester bond between the phosphate group of 5'-carbon of one deoxyribose with the hydroxyl group of 3'-carbon of another deoxyribose (Fig.27.2).
Host cells, besides effectively incorporating the vector's genetic material, must be conveniently cultivated in the laboratory to collect the products. In general, microorganisms are preferred as host cells, since they multiply faster compared to cells of higher organisms (plants or animals).

Escherichia coli : The bacterium, Escherichia coli was the first organism used in the DNA technology experiments and continues to be the host of choice by many workers.

Alkaline phosphatase, Use/reaction = Removes phosphate groups from 5 c -ends of double/single-stranded DNA (or RNA).. Bal 31 nuclease, Use/reaction = For the progressive shortening of DNA.. DNA ligase, Use/reaction = Joins DNA molecules by forming phosphodiester linkages between DNA segments.. DNA polymerase I, Use/reaction = Synthesizes DNA complementary to a DNA template.. DNase I, Use/reaction = Produces single-stranded nicks in DNA.. Exonuclease III, Use/reaction = Removes nucleotides from 3 c -end of DNA.. O exonuclease, Use/reaction = Removes nucleotides from 5 c -end of DNA.. Polynucleotide kinase, Use/reaction = Transfers phosphate from ATP to 5 c -OH ends of DNA or RNA.. Restriction enzymes, Use/reaction = Cut double-stranded DNA with a specific recognition site.. Reverse transcriptase, Use/reaction = Synthesizes DNA from RNA.. RNase A, Use/reaction = Cleaves and digests RNA (and not DNA).. RNase H, Use/reaction = Cleaves and digests the RNA strand of RNA-DNA heteroduplex.. Taq DNA polymerase, Use/reaction = Used in polymerase chain reaction. SI nuclease, Use/reaction = Degrades single-stranded DNA and RNA.. Terminal transferase, Use/reaction = Adds nucleotides to the 3 c -ends of DNA or RNA. Useful in homopolymer tailing.
T ABLE, 1 = 27.3 Some examples of host cells used in genetic engineering. Group, 1 = Examples. Prokaryotic, 1 = . Bacteria, 1 = Escherichia coli Bacillus subtilis Streptomyces sp. Eukaryotic, 1 = . Fungi, 1 = Saccharomyces cerevisiae Aspergillus nidulans. Animals, 1 = Insect cells Oocytes Mammalian cells Whole organisms Protoplasts. Plants, 1 = Intact cells Whole plants
The major drawback however, is that E.coli (or even other prokaryotic organisms) cannot perform post-translational modifications.

Bacillus subtilis : Bacillus subtilis is a rod shaped non-pathogenic bacterium. It has been used as a host in industry for the production of enzymes, antibiotics, insecticides etc. Some workers consider B.subtilis as an alternative to E.coli.

Eukaryotic organisms are preferred to produce human proteins since these hosts with complex structure (with distinct organelles) are more suitable to synthesize complex proteins. The most commonly used eukaryotic organism is the

Mammalian cells : Despite the practical difficulties to work with and high cost factor, mammalian cells (such as mouse cells) are also employed as hosts. The advantage is that certain complex proteins which cannot be synthesized by bacteria can be produced by mammalian cells e.g. tissue plasminogen activator. This is mainly because the mammalian cells possess the machinery to modify the protein to the active form (post-translational modifications).

Vectors are the DNA molecules, which can carry a foreign DNA fragment to be cloned. They are self-replicating in an appropriate host cell. The most important vectors are plasmids, bacteriophages, cosmids and artificial chromosome vectors.

Plasmids are extrachromosomal, doublestranded, circular, self-replicating DNA molecules. Almost all the bacteria have plasmids containing a low copy number (1-4 per cell) or a high copy number (10-100 per cell). The size of the plasmids varies from 1 to 500 kb. Usually, plasmids contribute to about 0.5 to 5.0% of the total DNA of bacteria (Note : A few bacteria contain linear plasmids e.g. Streptomyces sp, Borella burgdorferi).
Nomenclature of plasmids : It is a common practice to designate plasmid by a lower case p, followed by the first letter(s) of researcher(s) names and the numerical number given by the workers. Thus, pBR322 is a plasmid discovered by Bolivar and Rodriguez who designated it as 322. Some plasmids are given names of the places where they are discovered e.g. pUC is plasmid from University of California.
pBR322 — the most common plasmid vector : pBR322 of E.coli is the most popular and widely used plasmid vector, and is appropriately regarded as the parent or grand parent of several other vectors.
pBR322 has a DNA sequence of 4,361 bp. It carries genes resistance for ampicillin (Amp") and tetracycline (Tet') that serve as markers for the identification of clones carrying plasmids. The plasmid has unique recognition sites for the action of restriction endonucleases such as EcoRI, HindlIlIl, BamHI, Sall and Pstll (Fig.27.3).
Other plasmid cloning vectors : The other plasmids employed as cloning vectors include pUC19 (2,686 bp, with ampicillin resistance gene), and derivatives of pBR322-pBR325, pBR328 and pBR329.
Fig. 27.3 : Genetic map of plasmid cloning
vector pBR322.

Bacteriophages or simply phages are the viruses that replicate within the bacteria. In case of certain phages, their DNA gets incorporated into the bacterial chromosome and remains there permanently. Phage vectors can accept short fragments of foreign DNA into their genomes. The advantage with phages is that they can take up larger DNA segments than plasmids. Hence phage vectors are preferred for working with genomes of human cells. The most commonly used phages are bacteriophage A (phage A) and bacteriophage (phage Mq3).

Cosmids are the vectors possessing the characteristics of both plasmid and_bacterio-phage A. Cosmids can be constructed by adding a fragment of phage A DNA including cos site, to plasmids. A foreign DNA (about 40 kb) can be inserted into cosmid DNA. The recombinant DNA so formed can be packed as phages and injected into E.coli. Once inside the host cell, cosmids behave just like plasmids and_ replicate. The advantage with cosmids is that they can carry larger fragments of foreign DNA compared to plasmids.

Human artificial chromosome (HAC) Developed in 1997 (by H. Willard), human artificial chromosome is a__ synthetically produced' vector DNA, _ possessing the characteristics of human chromosome. HAC may be considered as a_ self-replicating microchromosome with a size ranging from 1/ 10th to '/;th of a human chromosome. The advantage with HAC is that it can carry human genes that are too long. Further, HAC can carry genes to be introduced into the cells in gene therapy.
Yeast artificial chromosomes (YACs) Introduced in 1987 (by M. Olson), yeast artificial chromosome (YAC) is a synthetic DNA that can accept large fragments of foreign DNA (particularly human DNA). It is thus possible to clone large DNA pieces by using YAC.
Bacterial artificial chromosomes (BACs) : The construction of BACs is based on one F-plasmid which is larger than the other plasmids used as cloning vectors. BACs can accept DNA inserts of around 300 kb.

Among the several factors, the size of the foreign DNA is very important in the choice of vectors. The efficiency of this process is often crucial for determining the success of cloning. The sizes of DNA insert that can be accepted by different vectors is shown in Table 27.4.

Introducing a foreign DNA (i.e. the gene) into the cells is an important task in biotechnology. The efficiency of this process is often crucial for determining the success of cloning. The most commonly employed gene transfer methods, namely transformation, conjugation, electroporation and lipofection, and direct transfer of DNA are briefly described.

Transformation is the method of introducing foreign DNA into bacterial cells (e.g. E.coli). The
uptake of plasmid DNA by E.coli is carried out in ice-cold CaCly (0-5°C), and a subsequent heat shock (37-45°C for about 90 sec). By this technique, the transformation frequency, which refers to the fraction of cell population that can be transferred, is reasonably good e.g. approximately one cell per 1000 (10-3) cells.

Conjugation is a natural microbial recombination process. During conjugation, two live bacteria (a donor and a recipient) come together, join by cytoplasmic bridges and transfer singlestranded DNA (from donor to recipient). Inside the recipient cell, the new DNA may integrate with the chromosome (rather rare) or may remain free (as is the case with plasmids).
The natural phenomenon of conjugation is exploited for gene transfer. This is achieved by transferring plasmid-insert DNA from one cell to another. In general, the plasmids lack conjugative functions and therefore, they are not as such capable of transferring DNA to the recipient cells. However, some plasmids with conjugative properties can be prepared and used.

Electroporation is based on the principle that high voltage electric pulses can induce cell plasma membranes to fuse. Thus, electroporation is a technique involving electric field-mediated membrane permeabilization. Electric shocks can also induce cellular uptake of exogenous DNA (believed to be via the pores formed by electric pulses) from the suspending solution. Electroporation is a simple and rapid technique for introducing genes into the cells from various organisms (microorganisms, plants and animals).

Liposomes are circular lipid molecules, which have an aqueous interior that can carry nucleic acids. Several techniques have been developed to encapsulate DNA in liposomes. The liposomemediated gene transfer is referred to as lipofection.
On treatment of DNA _ fragment with liposomes, the DNA pieces get encapsulated inside liposomes. These liposomes can adhere to cell membranes and fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus. The positively charged liposomes very efficiently complex with DNA, bind to cells and transfer DNA rapidly.

It is possible to directly transfer the DNA into the cell nucleus. Microinjection and particle bombardment are the two techniques commonly used for this purpose.

A clone refers to a group of organisms, cells, molecules or other objects, arising from a single individual. Clone and colony are almost synonymous.
Gene cloning strategies in relation to recombinant DNA technology broadly involve the following aspects (Fig.27.4).
1 Generation of desired DNA fragments.
1 Insertion of these fragments into a cloning vector.
Introduction of the vectors into host cells.
' Selection or screening of the recipient cells for the recombinant DNA molecules.
GENERATION OF DNA FRAGMENTS.
|
(Restriction endonuclease digestion,
cDNA synthesis, PCR, chemical synthesis)
INSERTION INTO A CLONING VECTOR
|
(Ligation of blunt ends or cohesive ends, homopolymer tailing, linker molecules)
INTRODUCTION INTO HOST CELLS
|
(Transformation, transfection, transduction)
SELECTION OR SCREENING
(Hybridization, PCR, immunochemical methods, protein-protein interactions, functional complementation)
Fig. 27.4 : An overview of cloning strategies in recombinant DNA technology.

DNA represents the complete genetic material of an organism which is referred to as genome. Theoretically speaking, cloning from genomic DNA is supposed to be ideal. But the DNA contains non-coding sequences (introns), control regions and repetitive sequences. This complicates the cloning strategies, hence DNA as a source material is not preferred, by many workers. However, if the objective of cloning is to elucidate the control of gene expression, then genomic DNA has to be invariably used in cloning.
The use of mRNA in cloning is preferred for
the following reasons.
1 mRNA - represents the information being expressed. actual genetic
1 Selection and isolation mRNA are easy.
1 As introns are removed during processing, mRNA reflects the coding sequence of the gene.
1 The synthesis of recombinant protein is much easier with MRNA cloning.
Besides the direct use of genomic DNA or mRNA, it is possible to synthesize DNA in the laboratory (by polymerase chain reaction), and use it in cloning experiments. This approach is
useful if the gene sequence is short and the complete sequence of amino acids is known.

There are several techniques used in recombinant DNA _ technology or gene manipulation. The most frequently used methods are listed.
1 Isolation and purification of nucleic acids.
1 Nucleic acid blotting techniques.
1 DNA sequencing.
1 Methods of gene transfer (described already).
1 Polymerase chain reaction.
1
Production of monoclonal — antibodies (Chapter 41).
1 Construction of gene library.
1 Site-directed engineering.
mutagenesis
and __
protein

Almost all the experiments dealing with gene manipulations require pure forms of either DNA or RNA, or sometimes even both. Hence there is a need for the reliable isolation of nucleic acids from the cells. The purification of nucleic acids broadly involves three stages.
1. Breaking or opening of the cells to expose
nucleic acids.
2. Separation of nucleic acids from other cellular
components.
3. Recovery of nucleic acids in a pure form.
The
basic
principles
and
procedures
for
nucleic acid purification are briefly described.

The first step for DNA purification is to open the cells and release DNA. The method should be gentle to preserve the native DNA. Due to variability in cell structure, the approaches to break the cells are also different.

Bacterial cells : The bacterial cells (e.g. E. coli) can be lysed by a combination of enzymatic and chemical treatments. The enzyme lysozyme and the chemical ethylenediamine tetraacetate (EDTA) are used for this purpose. This is followed by the addition of detergents such as sodium dodecyl sulfate (SDS).
Animal cells Animal cells, particularly cultured animal cells, can be easily opened by direct treatment of cells with detergents (SDS).
Plant cells : Plant cells with strong cell walls require harsh treatment to break open. The cells are frozen and then ground in a morter and pestle. This is an effective way of breaking the cellulose walls.

There are two different approaches to purify DNA from the cellular extracts.
1. Purification of DNA by removing cellular components : This involves the degradation or complete removal of all the cellular components other than DNA. This approach is suitable if the cells do not contain large quantities of lipids and carbohydrates.
The cellular extract is centrifuged at a low speed to remove the debris (e.g. pieces of cell wall) that forms a pellet at the bottom of the tube. The supernatant is collected and treated with phenol to precipitate proteins at the interface between the organic and aqueous layers. The aqueous layer, containing the dissolved nucleic acids, is collected and treated with the enzyme ribonuclease (RNase). The RNA is degraded while the DNA remains intact. This DNA can be precipitated by adding ethanol and isolated after centrifugation, and suspended in an appropriate buffer.
2. Direct purification of DNA In_ this approach, the DNA itself is selectively removed from the cellular extract and isolated. There are two ways for direct purification of DNA.
In one method, the addition of a detergent cetyltrimethyl ammonium (CTAB) results in the formation of an insoluble complex with nucleic acids. This complex, in the form of a precipitate is collected after centrifugation and suspended in a high-salt solution to release nucleic acids. By treatment with RNase, RNA is degraded. Pure DNA can be isolated by ethanol precipitation.
The second technique is based on_ the principle of tight binding between DNA and silica particles in the presence of a denaturing agent such as guanidinium thiocyanate. The isolation of DNA can be achieved by the direct addition of silica particles and guanidinium thiocyanate to the cellular extract, followed by centrifugation. Alternately, a column chromatography containing silica can be used, and through this the extract and guanidinium thiocyanate are passed. The DNA binds to the silica particles in the column which can be recovered.

Among the RNAs, mRNA _ is frequently required in a pure form for genetic experiments.
After the cells are disrupted on lysis by different techniques (described above), the cellular extract is deproteinised by treatment with phenol or phenol/chloroform mixtures. On centrifugation, the nucleic acids get concentrated in the upper aqueous phase which may then be precipitated by using isopropanol or ethanol.
The purification of mRNA can be achieved by affinity chromatography using oligo (dT)cellulose (Fig.27.5). This is based on the principle that oligo (dT)-cellulose can specifically bind to the poly (A) tails of eukaryotic mRNA. Thus, by this approach, it is possible to isolate mRNA from DNA, rRNA and tRNA.
As the nucleic acid solution is passed through an affinity chromotographic column, the oligo(dT) binds to poly(A) tails of mRNA. By washing the column with high-salt buffer, DNA, tRNA and tRNA can be eluted, while the mRNA is tightly bound. This mRNA can be then eluted by washing with low-salt buffer. The mRNA is precipitated with ethanol and collected by centrifugation (Fig.27.5).

Blotting techniques are very widely used analytical tools for the specific identification of desired DNA or RNA fragments from thousands of molecules. Blotting refers to the process of immobilization of sample nucleic acids on solid support (nitrocellulose or nylon membranes). The blotted nucleic acids are then used as targets in the hybridization experiments for their specific detection. An outline of the nucleic acid blotting technique is depicted in Fig.27.6.

The most comonly used blotting techniques listed below are
» Southern blotting (for DNA)
» Northern blotting (for RNA)
1 Dot blotting (DNA/RNA)
The Southern blotting is named after the scientist Ed Southern (1975) who developed it. The other names Northern blotting and Western blotting are laboratory jargons which are now accepted. Western blotting involves the transfer of protein blots and their identification by using specific antibodies.
A diagrammatic representation of a typical blotting apparatus is depicted in Fig.27.7.

Northern blotting is the technique for the specific identification of RNA molecules. The procedure adopted is almost similar to that described for Southern blotting and is depicted in Fig.27.9. RNA molecules are subjected to electrophoresis, followed by blot transfer, hybridization and autoradiography.
RNA molecules do not easily bind to nitrocellulose paper or nylon membranes. Blottransfer of RNA molecules is carried out by using a chemically reactive paper prepared by diazotization of aminobenzyloxymethyl to create diazobenzyloxymethy!l (DBM) paper. The RNA can covalently bind to DBM paper.
Northern blotting is theoretically, a good technique for determining the number of genes (through mRNA) present on a given DNA. But this is not really practicable since each gene may give rise to two or more RNA transcripts. Another drawback is the presence of exons and introns.

Dot-blotting is a modification of Southern and Northern blotting techniques described above. In
Fig. 27.9 : An outline of Northern blotting.
this approach, the nucleic acids (DNA or RNA) are directly spotted onto the filters, and not subjected to electrophoresis. The hybridization procedure is the same as in original blotting techniques.
Dot-blotting technique is particularly useful in obtaining quantitative data for the evaluation of gene expression.

Western blotting involves the identification of proteins. It is very useful to understand the nucleic acid functions, particularly during the course of gene manipulations.
The technique of Western blotting involves the transfer of electrophoresed protein bands from polyacrylamide gel to nylon' or _ nitrocellulose membrane. These proteins can be detected by specific protein-ligand interactions. Antibodies or lectins are commonly used for this purpose.

Autoradiography is the process of localization and recording of a radiolabel within a solid specimen, with the production of an image in a photographic emulsion. These emulsions are composed of silver halide crystals suspended in gelatin.
When a f-particle or a y-ray from a radiolabel passes through the emulsions, silver ions are converted to metallic silver atoms. This results in the development of a visible image which can be easily detected.

As already described, autoradiography is closely associated with blotting techniques for the detection of DNA, RNA and proteins.

Determination of nucleotide sequence in a DNA molecule is the basic and fundamental
requirement in biotechnology. DNA sequencing is important to understand the functions of genes, and basis of inherited disorders. Further, DNA cloning and gene manipulation invariably require knowledge of accurate nucleotide sequence.

The first DNA sequencing technique, using chemical reagents, was developed by Maxam and Gilbert (1977). This method is briefly described below (Fig.27.10).
A strand of source DNA is labeled at one end with 32P. The two strands of DNA are then separated. The labeled DNA is distributed into four samples (in separate tubes). Each sample is subjected to treatment with a chemical that specifically destroys one (G, C) or two bases (A + G, T + ©) in the DNA. Thus, the DNA strands are partially digested in four samples at sites G, A +G, T + Cand C. This results in the formation of a series of labeled fragments of varying lengths. The actual length of the fragment depends on the site at which the base is destroyed from the labeled end. Thus for instance, if there are C residues at positions 4, 7, and 10 away from the labeled end, then the treatment of DNA that specifically destroys C will give labeled pieces of length 3, 6 and 9 bases. The labeled DNA fragments obtained in the four tubes are subjected to electrophoresis side by side and they are detected by autoradiograph. The sequence of the bases in the DNA can be constructed from the bands on the electrophoresis.

Currently, the preferred technique — for determining nucleotide sequence in DNA is the one developed by Sanger (1980). This is an enzymatic procedure commonly referred to as the dideoxynucleotide method or chain termination method (Note : Fredrick Sanger won Nobel prize twice, once for determining the structure of protein, insulin; the second time for sequencing the nucleotides in an RNA virus).
A dideoxynucleotide is a laboratory-made chemical molecule that lacks a hydroxyl group
0
Bands on autoradiograph
ATACTGCGACT
Sequenced strand
TATGACGCTGA
Complementary strand
Fig. 27.10 : Maxam and Gilbert method for DNA sequencing.
at both the 2' and 3' carbons of the sugar (Fig.27.11). This is in contrast to the natural deoxyribonucleotide that possesses at 3' hydroxyl group on the sugar.
Termination role of dideoxynucleotide
:
In the
normal process
of
DNA
replication, an
(A)
incoming nucleoside triphosphate is attached by its 5'-phosphate group to the 3'-hydroxyl group of the last nucleotide of the growing chain (Refer Chapter 24) when a dideoxynucleotide is incorporated to the growing chain, no further replication occurs. This is because dideoxynucleotide, lacking a 3'-hydroxyl group, cannot form a phosphodiester bond and thus the DNA synthesis terminates.
Sequencing method The process of sequencing DNA by dideoxynucleotide method is briefly described. A single-stranded DNA to be sequenced is chosen as a template. It is attached to a primer (a short length of DNA oligonucleotide) complementary to a_ small section of the template. The 3'-hydroxyl group of the primer initiates the new DNA synthesis.
DNA synthesis is carried out in four reaction tubes. Each tube contains the primed DNA, Klenow subunit (the larger fragment of DNA polymerase of EF. coli), four dideoxyribonucleotides (ddATP, ddCTP, ddGTP or ddTTP). It is necessary to radiolabel (with 32P) the primer or one of the deoxyribonucleotides.

As the new DNA synthesis is completed, each one of the tubes contains fragments of DNA of varying length bound to primer. Let us consider the first reaction tube with dideoxyadenosine (ddATP). In this tube, DNA synthesis terminates whenever the growing chain incorporates ddA (complementary to dT on the template strand). Therefore, this tube will contain a series of different length DNA fragments, each ending with ddA. In a similar fashion, for the other 3 reaction tubes, DNA synthesis stops as the respective dideoxynucleotides are incorporated.
The synthesis of new DNA fragments in the four tubes is depicted in Fig.27.12.
The DNA pieces are denatured to yield free strands with radiolabel. The samples from each tube are separated by polyacrylamide gel electrophoresis. This separation technique resolves DNA pieces, different in size even by a single nucleotide. The shortest DNA will be the fastest moving on the electrophoresis.
The sequence of bases in a DNA fragment is determined by identifying the electrophoretic (radiolabeled) bands by autoradiography. In the Fig.27.13, the sequence of the newly synthesized DNA fragment that is complementary to the original DNA piece is shown. It is conventional to read the bands from bottom to top in 5' to 3' direction. By noting the order of the bands first C, second G, third T and so on, the sequence of the DNA can be determined accurately. As many as 350 base sequences of a DNA fragment can be clearly identified by using autoradiographs.
Modifications of dideoxynucleotide method : Replacement of 32P-radiolabel by 33P or 35S improves the sharpness of autoradiographic images. DNA polymerase of the thermophilic bacterium, Thermus aquaticus (in place of Klenow fragment of E. coli DNA polymerase 1) or a modified form of phage T7 DNA polymerase (sequenase) improves the technique.

DNA sequencing in the recent years is carried out by an automated DNA sequencer. |n this technique, flourescent tags are attached to chainterminating nucleotides (dideoxynucleotides). This tag gets incorporated into the DNA molecules, while terminating new — strand synthesis. Four different fluorescent dyes are used to identify chain-terminating reactions in a sequencing gel. The DNA bands are separated by electrophoresis and detected by their fluorescence. Recently, four dyes that exhibit strong absorption in laser are in use for automated sequencing.
Reaction
tube
Primer
with
Primer
with
sequence
of
with
dideoxynucleotide
extended
nucleotides
extended
ddATP
Primer
+
4
Primer-CGTddA
Primer + 9
Primer-CGTAGCTTddA
ddCTP
Primer +1
Primer—ddC
Primer
+
6
Primer-CGTAGddC
ddGTP
Primer
+
2
Primer-—CddG
Primer
+
5
Primer-CGTAddG
ddTTP
Primer + 3
Primer-CGddT
Primer + 7
Primer-CGTAGCddT
Primer
+
8
Primer-CGTAGCTddT
Fig.
27.12
:
Synthesis
of
new
DNA
fragments
in
the
presence
of
dideoxynucleotides
(**the
size
of
the
new
DNA
is
variable,
depending
on
the
chain
termination).
ddATP
ddCTP
ddGTP
ddTTP
Sequence
3
Largest
a
A
+
5
Smallest
Advantages of automated sequencing : It is a rapid and accurate technique. Automated DNA sequencer can accurately sequence up to 100,000 nucleotides per day. The cost works out to be not more than $0.2 per nucleotide. Automated DNA _ sequencing has been successfully used in the human genome project.

DNA chips or DNA microarrays are recent developments for DNA sequencing as result of advances made in automation and miniarization. A large number of DNA probes, each one with different sequence, are immobilized at defined positions on the solid surface, made up of either nylon or glass. The probes can be short DNA molecules such as cDNAs or synthetic oligonucleotides.
For the preparation of high density arrays, oligonucleotides are synthesized in situ on the surface of glass or silicon. This results in an oligonucleotide chip rather than a DNA chip.

A DNA chip carrying an array of different oligonucleotides can be used for DNA sequencing. For this purpose, a fluorescently labeled DNA test molecule, whose sequence is to be determined, is applied to the chip. Hybridization occurs between the complementary sequences of the test DNA molecule and oligonucleotides of the chip. The positions of these hybridizing oligonucleotides can be determined by confocal microscopy. Each hybridizing oligonucleotide represents an 8nucleotide sequence that is present in the DNA probe. The sequence of the test DNA molecule can be deduced from the overlaps between the sequences of the hybridizing oligonucleotides

There have been many successes with this relatively new technology of DNA chips. Some of them are listed.
- 1 Identification of genes responsible for the development of nervous systems.

The major limitation of DNA chips at present is the unavailability of complete genome arrays for higher eukaryotes, including humans. It is expected that within the next few years such DNA chips will be available. This will help the biotechnologists to capture the functional snapshots of the genome in action for higher organisms.

The polymerase chain reaction (PCR) is a laboratory (in vitro) technique for generating large quantities of a specified DNA. Obviously, PCR is a cell-free amplification technique for synthesizing multiple identical copies (billions) of any DNA of interest. Developed in 1984 by Karry Mullis (Nobel Prize, 1993), PCR is now considered as a basic tool for the molecular biologist. As is a photocopier a_ basic requirement in an office, so is the PCR machine in a molecular biology laboratory!

The double-stranded DNA of interest is denatured to separate into two_ individual strands. Each strand is then allowed to hybridize with a primer (renaturation). The primer-template duplex is used for DNA synthesis (the enzymeDNA — polymerase). These three steps— denaturation, renaturation and synthesis are repeated again and again to generate multiple forms of target DNA.

The essential for listed
requirements PCR are below
1. A target DNA (100-35,000 bp in length).
2. Two primers (synthetic oligonucleotides of
17-30
length)
that
are
complementary
to
regions
flanking
the
target
DNA.
3. Four deoxyribonucleotides (dATP, dCTP, dGTP, dTTP).
4. A DNA polymerase that can withstand at a
temperature
up
to
95°C
(i.e.,
thermostable).
The actual technique of PCR_ involves repeated cycles for amplification of target DNA. Each cycle has three stages.
1. Denaturation : On raising the temperature to about 95°C for about one minute, the DNA gets denatured and the two strands separate.
2. Renaturation or annealing : As_ the temperature of the mixutre is slowly cooled to about 55°C, the primers base pair with the complementary regions flanking target DNA strands. This process is called renaturation or annealing. High concentration of primer ensures annealing between each DNA strand and the primer rather than the two strands of DNA.
3. Synthesis : The initiation of DNA synthesis occurs at 3'-hydroxyl end of each primer. The primers are extended by joining the bases complementary to DNA strands. The synthetic process in PCR is quite comparable to the DNA replication of the leading strand (Refer Chapter 24). However, the temperature has to be kept optimal as required by the enzyme DNA polymerase. For Taq DNA _ polymerase, the optimum temperature is around 75°C (for E. coli DNA polymerase, it is around 37°C). The reaction can be stopped by raising the temperature (to about 95°C).
The 3 stages of PCR in relation to temperature and time are depicted in Fig.27.15. Each cycle of PCR takes about 3-5 minutes. In the normal practice, the PCR is carried out in an automated machine.
As is evident from the Fig.27.16 (cycle |), the new DNA strand joined to each primer is beyond the sequence that is complementary to the second primer. These new strands are referred to as long templates, and they will be used in the second cycle.

For the second cycle of PCR, the DNA strands (original + newly synthesized long template) are denatured, annealed with primers and subjected to DNA synthesis. At the end of second round, long templates, and short templates (DNA
1 Nested PCR
1 Inverse PCR
1 Anchored PCR
1 Reverse transcription PCR (RT-PCR)
1 Asymmetric PCR
1 Real-time quantitative PCR
1 Random amplified polymorphic DNA (RAPD)
1 Amplified (AFLP) fragment length polymorphism
1 Rapid amplification of cDNA ends (RACE).

The advent of PCR had, and continues to have tremendous impact on molecular biology. The applications of PCR are too many to be listed here. Some of them are selectively and very briefly described. Other applications of PCR are discussed at appropriate places.

The specificity and sensitivity of PCR is highly useful for the diagnosis of various diseases in humans. These include diagnosis of inherited disorders (genetic diseases), viral diseases, bacterial diseases etc.
Prenatal diagnosis of inherited diseases : PCR is employed in the prenatal diagnosis of inherited diseases by using chorionic villus samples or cells from amniocentesis. Thus, diseases like sickle-cell anemia, B-thalassemia and phenylketonuria can be detected by PCR in these samples.
Diagnosis of retroviral infections : PCR from cDNA is a valuable tool for diagnosis and monitoring of retroviral infections, e.g., HIV infection.
Diagnosis of bacterial infections : PCR is used for the detection of bacterial infections e.g., tuberculosis by Mycobacterium tuberculosis.
Diagnosis of cancers : Several virally-induced cancers (e.g., cervical cancer caused by human papilloma virus) can be detected by PCR. Further, some cancers which occur due to chromosomal translocation (chromosome 14 and
18 in follicular lymphoma) involving known genes are identified by PCR.

human and live stock embryos fertilized in vitro, can be determined by PCR, by using primers and DNA probes specific for sex chromosomes. Further, this technique is also useful to detect sex — linked disorders in fertilized embryos of embryos.

As the PCR technique is much simpler and quicker to amplify the DNA, it is conveniently used for sequencing. For this purpose, singlestrands of DNA are required.

The differences in the genomes of two organisms can be measured by PCR with random primers. The products are separated by electrophoresis for comparative identification. Two genomes from closely related organisms are expected to yield more similar bands.
PCR is very important in the study of evolutionary biology, more specifically referred to as phylogenetics. As a technique which can amplify even minute quantities of DNA from any source (hair, mummified tissues, bone, or any fossilized material), PCR has revolutionized the studies in palaentology and archaelogy. The movie 'Jurassic Park', has created public awareness of the potential applications of PCR!

A single molecule of DNA from any source (blood strains, hair, semen etc.) of an individual is adequate for amplification by PCR. Thus, PCR is very important for identification of criminals.
The reader may refer DNA finger printing technique described later in this chapter.

The collection of DNA fragments (specifically genes) from a particular species represents gene libraries. The creation or construction of gene libraries (broadly genomic libraries) is
accomplished by isolating the complete genome (entire DNA from a cell) which is cut into fragments, and cloned in suitable vectors. Then the specific clone carrying the desired (target) DNA can_ be identified, isolated and characterized. In this manner, a library of genes or clones (appropriately considered as gene bank) for the entire genome of a species can be constructed.

The human cellular DNA (the entire genome) may be subjected to digestion by restriction endonucleases (e.g., EcoRI). The fragments formed on an average are of about 4 kb size. (i.e., 4000 nitrogenes bases). Each human chromosome, containing approximately 100,000 kb can be cut into about 25,000 DNA fragments. As the humans have 23 different chromosomes (24 in man), there are a total of 575,000 fragments of 4 kb length formed. Among these 575,000 DNA fragments is the DNA or gene of interest (say insulin gene).
Now is the selection of a vector and cloning process. E.coli, a harmless bacterium to humans is most commonly used. The plasmids from E. coli are isolated. They are digested by the same restriction enzyme as was used for cutting human genome to form open plasmids. The human chromosomal DNA fragments and open plasmids are joined to produce recombined plasmids. These plasmids contain different DNA fragments of humans. The recombined plasmids are inserted into F. coli and the cells multiply (Fig.27.17). The E. coli cells possess all the human DNA in fragments. It must, however be remembered that each E. coli cell contains different DNA fragments. All the E. coli cells put together collectively represent genomic library (containing about 575,000 DNA fragments).

Once a DNA library is created, the clones (i.e., the cell lines) must be screened for identification of specific clones. The screening techniques are mostly based on the sequence of the clone or the structure/function of its product.

sequence in a DNA can be determined with a DNA probe (Fig.27.18). To start with, the double-stranded DNA of interest is converted into single strands by heat or alkali (denaturation). The two DNA strands are kept apart by binding to solid matrix such as nitrocellulose or nylon membrane. Now, the single strands of DNA probe (100-1,000 bp) labeled with radioisotope are added. Hybridization (i.e., base pairing) occurs between the complementary nucleotide sequences of the target DNA and the probe. For a stable base pairing, at least 80% of the bases in the two strands (target DNA and the probe) should be matching. The hybridized DNA can be detected by autoradiography.
(Note : DNA probe or gene probe represents a segment of DNA that is tagged with a label (i.e. isotope) so as to detect a complementary base sequence with sample DNA after hybridization)

Modifications in the DNA sequence of a gene are ideal to create a protein with desired properties. Site-directed mutagenesis is the technique for generating amino acid coding changes in the DNA (gene). By this approach specific (site-directed) change (mutagenesis) can be made in the base (or bases) of the gene to produce a desired enzyme. The net result in site-directed mutagenesis is incorporation of a desired amino acid (of one's choice) in place of a specific amino acid in a protein or a polypeptide. By employing this technique, enzymes that are more efficient and more suitable than the naturally occurring counterparts can be created for industrial applications. But it must be remembered that site-directed mutagenesis is a trial and error method that may or may not result in a better protein.
A couple of proteins developed by sitedirected mutagenesis and protein engineering are given next.
Fig. 27.17 : Creation of a genomic library for humans. (Note : Double-stranded DNA is represented by single lines or circles for clarity; human DNA fragments are coloured).

Tissue plasminogen activator is therapeutically used to lyse the blood clots that cause myocardial infarction. Due to its shorter half-life (around 5 minutes), tPA has to be repeatedly administered. By replacing asparagine residue (at position 120) with glutamine, the half-life of tPA can be substantially increased. This is due to the fact that glutamine is less glycosylated than asparagine and this makes a difference in the half-life of tPA.
Hirudin is a protein secreted by leech salivary gland, and is a strong thrombin inhibitor (i.e., acts as an anticoagulant). By replacing asparagine (at 47 position) with lysine, the potency of hirudin can be increased severalfold.

DNA, being the genetic material of the living
organisms,
contains
the
information
—
that
contributes
to
various
characteristic
features
of
the specific organism. Thus, the presence of a disease-causing pathogen can be detected by identifing a gene or a set of genes of the organism. Likewise an inherited genetic defect can be diagnosed by identifying the alterations in the gene. In the modern laboratory diagnostics, DNA analysis is a very useful and a sensitive tool.
The basic principles underlying the DNA diagnostic systems, and their use in the diagnosis of certain pathogenic and genetic diseases are described. Besides these, the various approaches for DNA fingerprinting (or DNA profiling) are also discussed.

The specific identification of the DNA sequence is absolutely essential in the laboratory diagnostics. This can be achieved by employing the following principles/tools.

Hybridization of nucleic acids (particularly DNA) is the basis for reliable DNA analysis. Hybridization is based on the principle that a single-stranded DNA molecule recognizes and specifically binds to a complementary DNA strand amid a mixture of other DNA strands. This is comparable to a specific key and lock relationship. The general procedure adopted for nucleic acid hybridization has been described (See p. 597 and Fig.27.18). Some more information is given below (Fig.27.19).
The single-stranded target DNA is bound to a membrane support. Now the DNA probe (single-
stranded and labeled with a detector substance) is added. Under appropriate conditions (temperature, ionic strength), the DNA probe pairs with the complementary target DNA. The unbound DNA probe is removed. Sequence of nucleotides in the target DNA can be identified from the known sequence of DNA probe.
There are two types of DNA hybridizationradioactive and non-radioactive respectively using DNA probes labeled with isotopes and non-isotopes as detectors.

The DNA chip or Genechip contains thousands of DNA probes (4000,000 or even more) arranged on a small glass slide of the size of a postage stamp. By this recent and advanced approach, thousands of target DNA molecules can be scanned simultaneously.

The unknown DNA molecules are cut into fragments by _ restriction endonucleases. Fluorescent markers are attached to these DNA fragments. They are allowed to react with the probes of the DNA chip. Target DNA fragments with complementary sequences bind to DNA probes. The remaining DNA fragments are washed away. The target DNA pieces can be identified by their fluorescence emission by passing a laser beam. A computer is used to record the pattern of fluoresence emission and DNA identification.
The technique of employing DNA chips is very rapid, besides being sensitive and specific for the identification of several DNA fragments simultaneously. Scientists are trying to develop Genechips for the entire genome of an organism.

The presence of mutations in a DNA sequence can be conveniently identified. In fact, Genechip probe array has been successfully used for the detection of mutations in the p53 and BRCA I genes. Both these genes are involved in cancer (See p. 593 also).

The use of DNA analysis (by employing DNA probes) is a novel and revolutionary approach for specifically identifying the disease-causing pathogenic organisms. This is in contrast to the traditional methods of disease diagnosis by detection of enzymes, antibodies etc., besides the microscopic examination of pathogens. Although at present not in widespread use, DNA analysis may soon take over the traditional diagnostic tests in the years to come. Diagnosis of selected diseases by genetically engineered techniques or DNA probes or direct DNA analysis is briefly described.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis. The commonly used diagnostic tests for this disease are very slow, and sometimes may take several weeks. This is because M. tuberculosis multiplies very slowly (takes about 24 hrs. to double; E. coli takes just 20 minutes to double).
A novel diagnostic test for tuberculosis was developed by genetic engineering, and_ is illustrated in Fig.27.20. A gene from firefly, encoding the enzyme luciferase is introduced into the bacteriophage specific for M. tuberculosis. The bacteriophage is a bacterial virus, frequently referred to as luciferase reporter phage or mycophage. The genetically engineered phage is added to the culture of M. tuberculosis. The phage attaches to the bacterial cell wall, penetrates inside, and inserts its gene (along with luciferase gene) into the M. tuberculosis chromosome. The enzyme luciferase is produced by the bacterium. When luciferin and ATP are added to the culture medium, luciferase cleaves luciferin. This reaction is accompanied by a flash of light which can be detected by a luminometer. This diagnostic test is quite sensitive for the confirmation of tuberculosis.
The flash of light is specific for the identification of M. tuberculosis in the culture. For other bacteria, the genetically engineered phage cannot attach and enter in, hence no flash of light would be detected.
Fig. 27.20 : Diagnosis of tuberculosis by using a genetically engineered bacteriophage (phage).
Malaria, mainly caused by Plasmodium falciparum, and P. vivax, affects about one-third of the world's population. The commonly used laboratory tests for the diagnosis of malaria include microscopic examination of blood smears, and detection of antibodies in the circulation. While the former is time consuming and frequently gives false-negative tests, the latter cannot distinguish between the past and present infections.
A specific DNA diagnostic test for identification of the current infection of P. falciparum has been developed. This is carried out by using a DNA probe that can bind and hybridize with a DNA fragment of P. falciparum genome, and not with other species of Plasmodium. It is reported that this DNA probe can detect as little as 1 ng of P. falciparum in blood or 10 pg of its purified DNA.

DNA probes, with radioisotope label, for HIV DNA are now available. By using PCR and DNA probes, AIDS can be specifically diagnosed in the laboratory.

Traditional laboratory tests for the diagnosis of genetic diseases are mostly based on the estimation of metabolites and/or enzymes. This is usually done after the onset of symptoms.
The laboratory tests based on DNA analysis can specifically diagnose the inherited diseases at the genetic level. DNA-based tests are useful to discover, well in advance, whether the individuals or their offsprings are at risk for any genetic disease. Further, such tests can also be employed for the prenatal diagnosis of hereditary disorders, besides identifying the carriers of genetic diseases.
Although not in routine use in the laboratory service, methods have been developed or being developed for the analysis of DNA in the diagnosis of several genetic diseases. These include sickle-cell anemia, cystic _ fibrosis, Duchenne's muscular dystrophy, Huntington's disease, fragile X syndrome, Alzheimer's disease, certain cancers (e.g. breast cancer, colon cancer), type II diabetes, obesity, Parkinson's disease and baldness.

Sickle-cell anemia is a genetic disease characterized by the irregular sickle (crescent like) shape of the erythrocytes. Biochemically, this disease results in severe anemia and progressive damage to major organs in the body (heart, brain, lungs, joints).
Sickle-cell anemia occurs due to a single amino acid change in the f-chain of hemoglobin. Specifically, the amino acid glutamate at the 6th position of B-chain is replaced by valine. At the molecular level, sickle-cell anemia is due to a single-nucleotide change (A= T) in the B-globin gene of coding (or antisense) strand. In the normal B-globin gene the DNA sequence is CCTGAGGAG, while in sickle-cell anemia, the sequence is CCTGTGGAG. This single-base mutation can be detected by using restriction enzyme Mstll to cut
Fig. 27.21 : Single-base change resulting in sickle-cell anemia. (Note : A small and relevant DNA fragment of B-globin gene magnified and shown with encoded amino acids).
DNA fragments in and around B-globin gene, followed by the electrophoretic pattern of the DNA fragments formed. The change in the base from A to T in the B-globin gene destroys the recognition site (CCTGAGG) for Mstll (Fig.27.21). Consequently, the DNA fragments formed from a sickle-cell anemia patient for B-globin gene differ from that of a normal person. Thus, sickle-cell anemia can be detected by digesting mutant and normal f-globin genes by restriction enzyme and_ performing a hybridization with a cloned B-globin DNA probe.

As the search continues by scientists for the identification of more and more genes responsible for various diseases, the enlightened public (particularly in the developed countries), is very keen to enjoy the fruits of this research outcome. As of now, DNA probes are available for the detection a limited number of diseases. Researchers continue to develop DNA probes for a large number of genetically predisposed disorders.
Gene banks are the centres for the storage of individual's DNAs for future use to diagnose diseases. For this purpose, the DNA isolated from a person's cells (usually white blood cells) is stored. As and when a DNA probe for the detection of a specific disease is available, the stored DNA can be used for the diagnosis or risk assessment of the said genetic disease.
In fact, some institutions have established gene banks. They store the DNA samples of the interested customers at a fee (one firm was charging $200) for a specified period (say around 20-25 years). For the risk assessment of any disease, it is advisable to have the DNAs from close relatives of at least 2-3 generations.

DNA fingerprinting is the present day genetic detective in the practice of modern medical forensics. The underlying principles of DNA fingerprinting are briefly described.
The structure of each person's genome is unique. The only exception being monozygotic identical twins (twins developed from a single fertilized ovum). The unique nature of genome structure provides a good opportunity for the specific identification of an individual.
It may be remembered here that in the traditional fingerprint technique, the individual is identified by preparing an ink impression of the skin folds at the tip of the person's finger. This is based on the fact that the nature of these skin folds is genetically determined, and thus the fingerprint is unique for an individual. In contrast, the DNA fingerprint is an analysis of the nitrogenous base sequence in the DNA of an individual.

The original DNA fingerprinting technique was developed by Alec Jaffreys in 1985. Although the DNA fingerprinting is commonly used, a more general term DNA profiling is preferred. This is due to the fact that a wide range of tests can be carried out by DNA sequencing with improved technology.

The amount of DNA required for DNA fingerprint is remarkably small. The minute quantities of DNA from blood strains, body fluids, hair fiber or skin fragments are enough. Polymerase chain reaction is used to amplify this DNA for use in fingerprinting. DNA profiling has wide range of applications—most of them related to medical forensics. Some important ones are listed below.
1 Identification of criminals, rapists, thieves etc.
1 Settlement of paternity disputes.
1 Use in immigration test cases and disputes.
In general, the fingerprinting technique is carried out by collecting the DNA from a suspect (or a person in a paternity or immigration dispute) and matching it with that of a reference sample (from the victim of a crime, or a close relative in a civil case).

The DNA markers are highly useful for genetic mapping of genomes. There are four types of DNA sequences which can be used as markers.
1. Restriction fragment length polymorphisms (RFLPs, pronounced as rif-lips).
2. Minisatellites or variable number tandem repeats (VNTRs, pronounced as vinters).
3. Microsatellites or simple tandem repeats (STRs).
4. Single nucleotide polymorphisms (SNPs, pronounced as snips).
The general aspects of the above DNA markers are described along with their utility in disease diagnosis and DNA fingerprinting.

A RFLP represents a stretch of DNA that serves as a marker for mapping a specified gene. RFLPs are located randomly throughout a
—$
3 fragments
Fig. 27.22 : An outline of the restriction fragment length polymorphism (RFLP) (R,, R,, R, represent the sites for the action of restriction endonucleases).
person's chromosomes and have no apparent function.
A DNA molecule can be cut into different fragments by a group of enzymes called restriction endonucleases (See Table 27.1). These fragments are called polymorphisms (literally means many forms).
An outline of RFLP is depicted in Fig.27.22. The DNA molecule 1 has three restriction sites (Ry, Ry, R3), and when cleaved by restriction endonucleases forms 4 fragments. Let us now consider DNA 2 with an inherited mutation (or a genetic change) that has altered some base pairs. As a result, the site (R>) for the recognition by restriction endonuclease is lost. This DNA molecule 2 when cut by restriction endonuclease forms only 3 fragments (instead of 4 in DNA 1).
As is evident from the above description, a stretch of DNA exists in fragments of various lengths (polymorphisms), derived by the action of restriction enzymes, hence the name restriction fragment length polymorphisms.

If the RFLP lies within or even close to the locus of a gene that causes a particular disease, it is possible to trace the defective gene by the
analysis of RFLP in DNA. The person's cellular DNA is isolated and treated with restriction enzymes. The DNA fragments so obtained are separated by electrophoresis. The RFLP patterns of the disease suspected individuals can be compared with that of normal people (preferably with the relatives in the same family). By this approach, it is possible to determine whether the individual has the marker RFLP and the disease gene. With 95% certainity, RFLPs can detect single gene-based diseases.
2. Polymerase chain reaction : RFLPs can also be scored by PCR. For this purpose, PCR primers that can anneal on either side of the suspected restriction site are used. After amplification by PCR, the DNA molecules are treated with restriction enzyme and_ then analysed by agarose gel electrophoresis. If the restriction site is absent only one band is seen, while two bands are found if the site is found (Fig.27.23B).
Methods of RFLP scoring : Two methods are in common use for the detection of RFLPs (Fig.27.23).
1. Southern hybridization : The DNA is digested with appropriate restriction enzyme, and separated by agarose gel electrophoresis. The so obtained DNA fragments are transferred to a nylon membrane. A DNA probe that spans the suspected restriction site is now added, and the hybridized bands are detected by autoradiograph. If the restriction site is absent, then only a single restriction fragment is detected. If the site is present, then two fragments are detected (Fig.27.23A).
Applications of RFLPs : The approach by RFLP is very powerful and has helped many genes to be mapped on the chromosomes. e.g. sickle-cell anemia (chromosome 11), cystic fibrosis (chromosome 7), Huntington's desease (chromosome 4), retinoblastoma (chromosome 13), Alzheimer's disease (chromosome 21).

VNTRs, also known as minisatellites, like RFLPs, are DNA fragments of different length. The main difference is that RFLPs develop from random mutations at the site of restriction enzyme activity while VNTRs are formed due to
different number of base sequences between two points of a DNA molecule. In general, VNTRs are made up of tandem repeats of short base sequences (10-100 base pairs). The number of elements in a given region may vary, hence they are known as variable number tandem repeats.
An individual's genome has many different VNTRs and RFLPs which are unique to the individual. The pattern of VNTRs and RFLPs forms the basis of DNA fingerprinting or DNA profiling.
In the Fig.27.24, two different DNA molecules with different number of copies (bands) of VNTRs are shown. When these molecules are subjected to restriction endonuclease action (at two sites R; and Ry), the VNTR sequences are released, and they can be detected due to variability in repeat sequence copies. These can be used in mapping of genomes, besides their utility in DNA fingerprinting.
VNTRs are useful for the detection of certain genetic diseases associated with alterations in the degree of repetition of microsatellites e.g. Huntington's chorea is a disorder which is found when the VNTRs exceed 40 repeat units.
Limitations of VNTRs : The major drawback of VNTRs is that they are not evenly distributed throughout the genome. VNTRs tend to be localized in the telomeric regions, at the ends of the chromosomes.

RFLPs caused by variations in the number of VNTRs between two restriction sites can be detected (Fig.27.25). The DNAs from three individuals with different VNTRs are cut by the specific restriction endonuclease. The DNA fragments are separated by electrophoresis, and identified after hybridization with a probe complementary to a specific sequence on the fragments.
Fig. 27.25 : Use of restriction fragment length polymorphisms (RFLPs) caused by variable number tandem repeats (VNTRs) in genetic fingerprinting (A) An illustration of DNA structure from three individuals (B) Hybridized pattern of DNA fragment with a probe complementary to the sequence shown in green circles (1, 2 and 3 represent the individuals; R, and R, indicate restriction sites; coloured Squares are the number of VNTRs)

Microsatellites are short repeat units (10-30 copies) usually composed of dinucleotide or tetranucleotide units. These simple tandem repeats (STRs) are more popular than minisatellites (VNTRs) as DNA markers for two reasons.
1.
Microsatellites are
evenly
—
distributed throughout the genome.
2. PCR can be effectively and conveniently used to identify the length of polymorphism.
Two variants (alleles) of DNA molecules with
5 and 10 repeating units of a dimer nucleotides
(GA) are depicted in Fig.27.26.
By use of PCR, the region surrounding the microsatellites is amplified, separated by agarose gel electrophoresis and identified.

SNPs represent the positions in the genome where some individuals have one nucleotide (e.g. G) while others have a different nucleotide (e.g. C). There are large numbers of SNPs in genomes. It is estimated that the human genome contains at least 3 million SNPs. Some of these SNPs may give rise to RFLPs.
SNPs are highly useful as DNA markers since there is no need for gel electrophoresis and this saves a lot of time and labour. The detection of SNPs is based on the oligonucleotide hybridization analysis (Fig.27.27).
An oligonucleotide is a short single-stranded DNA molecule, synthesized in the laboratory with a length not usually exceeding 50 nucleotides. Under appropriate conditions, this nucleotide sequence will hybridize with a target DNA strand if both have completely base paired structure. Even a single mismatch in base pair will not allow the hybridization to occur.
DNA chip technology is most commonly used to screen SNPs hybridization with oligonucleotide (See p. 593).

In the forensic analysis of DNA, the original techniques based on RFLPs and VNTRs are now largely replaced by microsatellites (short tandem repeats). The basic principle involves the amplification of microsatellites by polymerase chain reaction followed by their detection.
It is now possible to generate a DNA profile by automated DNA detection system (comparable to the DNA sequencing equipment).
(A)
CAGCTGTCGAT
CAGCTCTCGAT
>
SNP
SPT
GTCGACAGCTA
FTS
RAT
‘
—
Matched base
Complete and stable hybridization of base pairs
-—
SNP
GTCGAGAGCTA *
———
fate
Oligonucleotide
Target DNA
—
Mismatched base
Hybridization not formed due to mismatch base pair
Fig. 27.27 : (A) An illustration of single nucleotide polymorphism (SNP) (B) Oligonucleotide hybridization to detect SNP.
(B)

The advent of recombinant DNA technology heralded a new chapter for the production of a wide range of therapeutic agents in sufficient quantities for human use. The commercial exploitation of recombinant DNA _ (rDNA) technology began in late 1970s by a few biotechnological companies to produce proteins. There are at least 400 different proteins being produced (by DNA technology) which may serve as therapeutic agents for humans. A selected list of some important human proteins produced by recombinant DNA technology potential for the treatment of human disorders is given in Table 27.5. As of now, only a selected few of them (around 30) have been approved for human use, and the most important among these are given in Table 27.6.

Diabetes mellitus is characterized by increased blood glucose concentration (hyperglycemia) which occurs due to insufficient or inefficient insulin. In the early years, insulin isolated and purified from the pancreases of pigs and cows was used for the treatment of severe diabetics. This often resulted in allergies. Recombinant DNA technology has become a boon to diabetic patients.

Attemps to produce insulin by recombinant DNA technology started in late 1970s. The basic technique consisted of inserting human insulin gene and the promoter gene of lac operon on to the plasmids of E. coli. By this method human insulin was produced. It was in July 1980, seventeen human volunteers were, for the first time, administered recombinant insulin for treatment of diabetes at Guy's Hospital, London. And in fact, insulin was the first ever pharmaceutical product of recombinant DNA technology administered to humans. Recombinant insulin worked well, and this gave hope to scientists that DNA technology could be successfully employed to produce substances of
Taste 27.5 A selected list of human proteins eri Mee a Wry ey Va Cg MMU a)

Insulin, Trade name(s) = Humulin. Insulin, Applications/uses = Diabetes. Growth hormone, Trade name(s) = Protropin/Humatrope. Growth hormone, Applications/uses = Pituitary dwarfism. D -Interferon, Trade name(s) = Intron A. D -Interferon, Applications/uses = Hairy cell leukemia. Hepatitis B vaccine, Trade name(s) = Recombinax HB/Engerix B. Hepatitis B vaccine, Applications/uses = Hepatitis B. Tissue plasminogen activator, Trade name(s) = Activase. Tissue plasminogen activator, Applications/uses = Myocardial infarction. Factor VIII, Trade name(s) = Kogenate/Recombinate. Factor VIII, Applications/uses = Hemophilia. DNase, Trade name(s) = Pulmozyme. DNase, Applications/uses = Cystic fibrosis. Erythropoietin, Trade name(s) = Epogen/Procrit. Erythropoietin, Applications/uses = Severe anemia with kidney damage
medical and commercial importance. An a : approval, by the concerned authorities, for using recombinant insulin for the treatment of diabetes mellitus was given in 1982. And in 1986, Eli Lilly company received approval to market human insulin under the trade name Hamulin.
Technique for production of recombinant insulin : The orginal technique (described briefly above) of insulin synthesis in EF. coli has undergone several changes, for improving the yield. e.g. addition of signal peptide, synthesis of A and B chains separately etc.
SS

The procedure employed for the synthesis of two insulin chains A and B is illustrated in Fig.27.28. The genes for insulin A chain and B chain are separately inserted to the plasmids of two different £. coli cultures. The lac operon system (consisting of inducer gene, promoter gene, operator gene and structural gene Z for B-galactosidase) is used to express both the genes. The presence of lactose in the culture medium induces the synthesis of insulin A and B chains in separate cultures. The so formed insulin chains can be isolated, purified and joined together to give a full-fledged human insulin.

DNA
_
technology
in
recent
years,
has
become
a
boon
to
produce
new
generation
vaccines.
By
this
approach,
some
of
Fig. 27.28 : The production of recombinant insulin in E. coli (l-Inducer gene, P-Promoter gene, O-Operator gene, Z-B-Galactosidase gene; all these genes are of lac operon system).
the limitations (low yield, high cost, side effects) of traditional vaccine production could be overcome.
The list of diseases for which recombinant vaccines are developed or being developed is given in Table 22.7. It may be stated here that due to very stringent regulatory requirements to use in humans, the new generation vaccines are first tried in animals, and it may take some more years before most of them are approved for use in humans.

In 1987, the recombinant vaccine for hepatitis B (i.e. HBsAg) became the first synthetic vaccine for public use. It was marketed by trade names Recombivax and Engerix-B. Hepatitis B vaccine is safe to use, very effective and produces no allergic reactions. For these reasons, this recombinant vaccine has been in use since 1987. The individuals must be administered three doses over a period of six months. Immunization against hepatitis B is strongly recommended to anyone coming in contact with blood or body secretions. All the health professionals—physicians, surgeons, medical laboratory technicians, nurses, dentists, besides police officers, firefighters etc., must get vaccinated against hepatitis B.

India is the fourth country (after USA, France and Belgium) in the world to develop an indigenous hepatitis B vaccine. It was launched in 1997, and is now being used.

Genetic immunization
by using
vaccines is
being in
a
novel approach
1990.
The immune
that came
response
DNA
into of
the body
is stimulated
by
a
DNA
molecule sequence
of pathogen's genome. This DNA is basically a
Taste 27.7 A selected list of diseases along Um UC UC CI CMLL] CUE Meee eC aval or being developed
Disease
Pathogenic organism

Acute infantile
Rotavirus
gastroenteritis
Acute respiratory
Influenza A and B viruses
diseases
AIDS
Chicken pox
Varicella-zoster virus
Japanese encephalitis virus
Genital ulcers
Herpes simplex virus type-2
Hemorrhagic fever
Liver damage
Dengue virus
Hepatitis A virus
Upper and lower
respiratory tract lesions
Hepatitis B virus
Yellow fever virus

Cholera
Vibrio cholerae
Diarrhea
E.
coli
Dysentery
Shigella strain
Gonorrhea
Niesseria gonorroheae
Leprosy
Mycobacterium leprae
Neisseria meningitidis
Pneumonia
Streptococcus pneumoniae
Rheumatic fever
Streptococcus group A
Tetanus
Clostridium tetani
Tuberculosis
Mycobacterium tuberculosis
Typhoid
Salmonella typhi
Urogenital tract
Streptococcus group B
infection

Filariasis
Wuchereria bancrofti
Malaria
Plasmodium sp
River blindness
Onchocerca volvulus
Schistosomiasis
Schistosoma mansoni
Sleeping sickness
Trypanosoma sp
bacterial plasmid engineered to include the sequence of an antigenic protein from the pathogen. After its entry into different cell types, this DNA can be expressed there using cellular transcription and translation machinary. Thus, DNA vaccines behave like viruses. They cannot, however become infectious due to limited amout of genetic information they contain.
DNA vaccine—plasmids can be administered
1
Nasal spray
1 Intramuscular injection
1 Intravenous injection
1 Intradermal injection
1 Gene gun or biolistic delivery (involves pressure delivery of DNA-coated gold beads).

to the animals by one of the following delivery methods.
An_ illustration of a DNA _ vaccine and the mechanism of its action in developing

+ Biotechnology is a newly discovered discipline for age-old practices (e.g. preparation of curd, wine, beer), with special emphasis on genetic manipulations.
+ Human artificial chromosome (HAC) is a synthetic vector, possessing the characteristics of human chromosome. HAC is capable of carrying large-sized human genes that may be useful in gene therapy.
+ Southern blotting technique (that specifically detects DNA) is employed for the identification of thieves, rapists, and settlement of parenthood.
+ Polymerase chain reaction is useful for the diagnosis of inherited diseases, in DNA sequencing, and in forensic medicine.
+ By employing site-directed mutagenesis, it is possible to produce more efficient and more suitable enzymes for therapeutic and industrial purposes.
+ The analysis of genetic material DNA (gene/genes) is employed for the diagnosis of certain diseases, and in medical forensics e.g. AIDS, sickle-cell anemia, certain cancers, DNA fingerprinting.
+
The
pharmaceutical
products
of
rDNA
have
revolutionized
the
treatment
of
certain
diseases
e.g.
diabetes,
asthma,
atherosclerosis,
heart
attacks,
hemophilia.
+ Recombinant vaccine for hepatitis B is the first synthetic vaccine. It is effective, safe and produces no allergic reactions.
+ Genetic immunization by using DNA vaccines is a novel concept. It has been shown that the immune response (humoral and cellular) of the body can be stimulated by a DNA molecule.
+ Transgenic mice that serve as animal models for human diseases have been developed. These include human mouse (model for immune system), Alzheimer’s mouse, oncomouse (model for cancer), transplantation etc.). prostate mouse, knockout mice (for allergy,
+ Transgenic animals serve as bioreactors for the production of therapeutically important proteins e.g. interferon, lactoferrin, urokinase.
+ Certain pet animals (cats, dogs) are being cloned by some companies.

immunity is given in Fig.27.29. The plasmid vaccine carrying the DNA (gene) for antigenic protein enters the nucleus of the inoculated target cell of the host. This DNA produces RNA, and in turn the specific antigenic protein. The antigen can act directly for developing humoral immunity or as fragments in association with major histocompatability class (MHC) molecules for developing — cellular immunity. DNA vaccines may hold some promise for vaccination against cancer, HIV, malaria, tuberculosis etc.

As the antigens bind to B-lymphocytes, they trigger the production of antibodies which can destroy the pathogens. Some of the B-lymphocytes become memory cells that can protect the host against future infections.

The protein fragments of the antigen bound to MHC molecules can activate the cytotoxic T-lymphocytes. They are capable of destroying the infected pathogenic cells. Some of the activated T-lymphocytes become memory cells which can_ kill the future infecting pathogens.

With the advent of modern biotechnology, it is now possible to carry out manipulations at the genetic level to get the desired characteristics in animals. Transgenesis refers to the phenomenon of introduction of exogeneous DNA into the genome to create and maintain a stable heritable character. The foreign DNA that is introduced is called transgene. And the animal whose genome is altered by adding one or more transgenes is said to be transgenic. The transgenes behave like other genes present in the animals' genome, and are passed on to the offsprings. Thus, transgenic animals are genetically engineered or genetically modified organisms (GMOs) with a new heritable character.

Transgenesis has now become a powerful tool for studying the gene expression and developmental processes in higher organisms, besides the improvement in their genetic characteristics. Transgenic animals serve as good models for understanding the human diseases. Further, several proteins produced by transgenic animals are important for medical and pharmaceutical applications. | Thus, the transgenic farm animals are a part of the lucrative world-wide biotechnology industry, with great benefits to mankind.

Mouse, although not close to humans in its biology, has been and continues to be the most exploited animal model in __ transgenesis experiments. The common feature between man and mouse is that both are mammals. Transgenic mice are extensively used as animal models for understanding human diseases, and for the production of therapeutic agents. Adequate care, however, must be exercised before extrapolating data of transgenic mice to humans.
Mouse models for several human diseases (cancers, muscular — dystrophy, arthritis, Alzheimer's disease, hypertension, allergy, coronary heart disease, endocrine diseases, neurodegenerative disorders _— etc.) ~— have been developed. A selected few of them are listed.
| The human mouse, the transgenic mouse that displays human immune system.
| The Alzheimer's mouse to understand the pathological basis of Alzheimer's disease.
| The oncomouse, the animal model for cancer.
| The prostate mouse, the transgenic mouse to understand prostate cancer.
| The knockout mice, (developed by eliminating specific genes) for certain diseases e.g. SCID mouse, knockout mouse for transplantation.

Transgenesis is wonderfully utilized for production of proteins for pharmaceutical and medical use. In fact, any protein synthesized in the human body can be made in the transgenic animals, provided that the genes are correctly programmed. The advantage with transgenic animals is to produce scarce human proteins in huge quantities. Thus, the animals serving as factories for production of biologically important products are referred to as animal bioreactors or sometimes pharm animals. Some transgenic animals that serve as bioreactors are listed
1 Transgenic cow for the production lactoferrin and interferons.
of
1 Transgenic goat to synthesize tissue plasminogen activator, and antithrombin III.
1
Transgenic mouse
for the
immunoglobulins, and
urokinase.
- 1 Transgenic pig to produce hemoglobin.

Dolly, the first ever mammal clone was developed by Wilmut and Campbell in 1997. It is a sheep (female lamb) with a mother and no father.
The technique primarily involves nuclear transfer and the phenomenon of totipotency. The character of a cell to develop into different cells, tissues, organs, and finally an organism is referred to as totipotency or pluripotency. Totipotency is the basic character of embryonic cells. As the embryo develops, the cells specialize to finally give the whole organism. As such, the cells of an adult lack totipotency. Totipotency was induced into the adult cells for developing Dolly.
The cloning of sheep for producing Dolly, illustrated in Fig.27.30, is briefly described here. The mammary gland cells from a donor ewe were isolated. They were subjected to total nutrient deprivation (starvation) for five days. By this process, the mammary cells abandon their
normal growth cycle, enter a dormant stage and regain totipotency character. An ovum (egg cell) was taken from another ewe, and its nucleus was removed to form an enucleated ovum. The dormant mammary gland cell and the enucleated ovum were fused by pulse electricity. The mammary cell outer membrane was broken, allowing the ovum to envelope the nucleus. The fused cell, as it had gained totipotency, can multiply and develop into an embryo. This embryo was then implanted into another ewe which served as a surrogate/foster mother. Five months later, Dolly was born.
As reported by Wilmut and Campbell, they fused 277 ovum cells, achieved 13 pregnancies, and of these only one pregnancy resulted in live birth of the offspring-Dolly.

Some of the companies involved in transgenic experiments have started cloning pet animals like cats and dogs. Little Nicky was the first pet cat that was cloned at a cost of $50,00 by an American company (in Dec. 2004). More cloned cats and dogs will be made available to interested parties (who can afford) in due course. Some people who own pet animals are interested to continue the same pets which is possible through cloning. There is some opposition to this approach as the cloned animals are less healthy, and have shorter life span, besides the high cost factor.

Advances in biotechnology, and __ their applications are most frequently associated with controversies. Based on their perception to biotechnology, the people may be grouped into three broad categories.
1. Strong opponents who oppose the new technology, as it will give rise to problems, issues and concerns humans have never faced before. They consider biotechnology as an unnatural manipulative technology.
2. Strong proponents who consider that the biotechnology will provide untold benefits to society. They argue that for centuries the society has safely used the products and processes of biotechnology.
3. A neutral group of people who have a balanced approach to biotechnology. This group believes that research on biotechnology (with regulatory systems), and extending its fruits to the society should be pursued with a cautious approach.

The fruits of biotechnology are beneficial to the fields of healthcare, agriculture, food production, manufacture of industrial enzymes and appropriate environmental management.
It is a fact that modern technology in various forms is woven tightly into the fabric of our lives. Our day-to-day life is inseparable from technology. Imagine life about 1-2 centuries ago where there was no electricity, no running water, sewage in the streets, unpredictable food supply and an expected life span of less than 40 years. Undoubtedly, technology has largely contributed to the present day world we live in. Many pepople consider biotechnology as a technology that will improve the quality of life in every country, besides maintaining living standards at a reasonably higher level.

Why so much uproar and negativity to biotechnology? This is mainly because the major part of the modern biotechnology deals with genetic manipulations. These unnatural genetic manipulations, as many people fear, may lead to unknown consequences.
ELSI is the short form to represent the ethical, legal and social implications of biotechnology. ELSI broadly covers the relationship between biotechnology and society with particular reference to ethical and legal aspects.

The modern biotechnology deals with genetic manipulations
of viruses,
bacteria, plants,
animals, fish and birds. Introduction of foreign genes into various organisms raises concerns about the safety, ethics and unforeseen consequences. Some of the popular phrases used in the media while referring to experiments on recombinant DNA technology are listed.
1 Manipulation of life
1 Playing God
1 Man-made evolution
The major apprehension of — genetic engineering is that through recombinant DNA experiments, unique microorganisms or viruses (either inadvertently, or sometimes deliberately for the purpose of war) may be developed that would cause epidemics and environmental catastrophes. Due to these fears, the regulatory guidelines for research dealing with DNA manipulation were very stringent in the earlier years.
So far, risk assessment studies have failed to demonstrate any hazardous properties acquired by host cells/organisms due to transfer of DNA. Thus, the fears of genetic manipulations may be unfounded to a_ large extent. Consequently, there has been some relaxation in the regulatory guidelines for recombinant DNA research.
It is now widely accepted that biotechnology is certainly beneficial to humans. But it should not cause problems of safety to people and environment, and create unacceptable social, moral and ethical issues.

1., 1 = Recombinant DNA (rDNA) technology is primarily concerned with the manipulation of genetic material (DNA) to achieve the desired goal in a pre-determined way.. 2., 1 = The procedure for rDNA technology involves molecular tools (enzymes e.g. restriction endonucleases), host cells ( E. coli, S. cerevisiae) , vectors (plasmids, bacteriophages), gene transfer (transformation, electroporation) and the strategies of gene cloning.. 3., 1 = Blotting techniques are employed for the identification of desired DNA (Southern blot), RNA (Northern blot), and protein (Western blot).. 4., 1 = Polymerase chain reaction is an in vitro technique for generating large quantities of a specified DNA i.e. cell-free amplification.. 5., 1 = Gene libraries or genomic libraries represents the collection of DNA fragments (i.e. genes) from a genome of a particular species.. 6., 1 = Site-directed mutagenesis is the technique for generating amino acid coding changes in the DNA (gene) to produce a desired protein/enzyme.. 7., 1 = Analysis of DNA (i.e. detection of gene/genes) can be used as a diagnostic system for the detection of many pathogenic and genetic diseases e.g. tuberculosis, malaria, AIDS, sickle-cell anemia, certain cancers.. 8., 1 = DNA fingerprinting or DNA profiling is the present day genetic detective in the practice of modern medical forensics. Four types of DNA markers are used in DNA fingerprinting-RFLFs, VNTRs, STRs, and SNPs.. 9., 1 = Many pharmaceutical compounds of health importance (for disease treatment) are being produced by rDNA technology e.g. insulin, growth hormone, interferons, erythropoietin, hepatitis B vaccine.. 10., 1 = Transgenic animals can be developed by introducing a foreign DNA (transgene). These animals are genetically modified or engineered with new heritable characters e.g. oncomouse, knockout mouse, prostate mouse.

. Describe the basic principles underlying the recombinant DNA technology. =
. Give an account of the nucleic acid blotting techniques. Add a note on their importance.
. Describe the polymerase chain reaction along with its applications. kWNnN
. Write briefly on the utility of DNA in disease diagnosis and medical forensics.
. Give an account of the pharmaceutical products of DNA technology. a

(a) Restriction endonucleases, (b) Plasmids, (c) Methods of gene transfer, (d) Purification of nucleic acids, (e) Western blotting, (f) DNA sequencing, (g) DNA chips (h) Gene libraries, (i) Restriction fragment length polymorphisms, (j) Recombinant vaccines.

1. The most commonly used prokaryotic host in rDNA technology is
2. Northern blotting technique is used for the detection of
3. Name the blotting technique in which nucleic acids (DNA or RNA) are directly blotted onto the filters without electrophoresis.
4. The bacterial source of the enzyme Taq DNA polymerase, that is widely used in polymerase
chain
reaction
5. The collection of DNA fragments from the genome of a particular species represents
6. The technique for generating amino acid coding changes in the DNA (gene) is regarded as
7. The trade name for insulin produced by rDNA technology
8. The first synthetic vaccine developed by rDNA technology
9. The most commonly used animal model in transgenesis to represent humans
10. Name the first ever mammmal that has been cloned

11. One of the following enzyme produces single-stranded nicks in DNA
(a) DNA ligase (b) DNA polymerase (c) DNase | (d) SI nuclease.
12. Western blotting is the technique for the identification of
(a) DNA (b) RNA (c) Carbohydrates (d) Proteins.
13. The DNA markers used in the diagnosis of diseases and DNA fingerprinting
(a) Restriction fragment length polymorphisms, (b) Minisatellites and microsatellites, (c) Single nucleotide polymorphisms, (d) Any one of the above.
14. The first pharmaceutical product of recombinant DNA technology approved for human use (a) Insulin (b) Growth hormone (c) Interferon (d) Hypatitis B vaccine.
15. Genetic immunization involves the administration of
(a) Antigens (b) Antibodies (c) DNA (d) RNA.
he most important features of a DNA molecule are the nucleotide sequences, and the identification of genes and their activities. Since 1920, scientists have been working to determine the sequences of pieces of DNA.

The human genome project (HGP) was conceived in 1984, and officially begun in earnest in October 1990. The primary objective of HGP was to determine the nucleotide sequence of the entire human nuclear genome. In addition, HGP was also entrusted to elucidate the genomes of several other model organisms e.g. Escherichia coli, Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (roundworm), Mus musculus (mouse). James Watson (who elucidated DNA structure) was the first Director of HGP.
In 1997, United States established the National Human Genome Research Institute (NHGRI). The HGP was an international venture involving research groups from six countries — USA, UK, France, Germany, Japan and China, and several individual laboratories and a large number of scientists and technicians from various disciplines. This collaborative venture was named as International Human Genome Sequencing Consortium (IHGSC) and was headed by Francis Collins. A total expenditure of $3 billion, and a time period of 10-15 years for the completion of HGP was expected. A second human genome project was set up by a private company — Celera Genomics, of Maryland USA in 1998. This team was led by Craig Venter.

The date 26th June 2000 will be remembered as one of the most important dates in the history of science or even mankind. It was on this day, Francis Collins and Craig Venter, the leaders of the two human genome projects, in the presence of the President of U.S., jointly announced the working drafts of human genome sequence. The detailed results of the teams were later published
Fig. 28.1 : Different types of genome maps.
in February 2001 in scientific journals Nature (IHGSC) and Science (Celera Genomics).
The human genome project results attracted worldwide attention. This achievement was hailed with many descriptions in the media.
1 The mystery of life unravelled.
| The library of life.
1 The periodic table of life.
1 The Holy grail of human genetics.

The most important objective of human genome project was to construct a series of maps for each chromosome. In Fig.28.1, an outline of the different of is types maps given.
1. Cytogenetic map : This is a map of the chromosome in which the active genes respond to a chemical dye and display themselves as bands on the chromosome.
2. Gene linkage map : A chromosome map in which the active genes are identified by locating closely associated marker genes. The most commonly used DNA markers are restriction fragment length polymorphism (RFLP), variable number tandem repeats (VNTRs) and short tandem repeats (STRs). VNTRs are also called as minisatellites while STRs are microsatellites.
3. Restriction fragment map : This consists of the random DNA fragments that have been sequenced.
4. Physical map : This is the ultimate map of the chromosome with highest resolution base sequence. Physical map depicts the location of the active genes and the number of bases between the active genes.

A list of different methods used for mapping of human genomes is given in Table 28.1. These techniques are also useful for the detection of normal and disease genes in humans.
For elucidating human genome, different approaches were used by the two HGP groups. IHGSC predominantly employed map first and sequence later approach. The principal method was_ heirarchical shotgun sequencing. This technique involves fragmentation of the genome into small fragments (100-200 kb), inserting them into vectors (mostly bacterial artificial chromosomes, BACs) and cloning. The cloned fragments could be sequenced.
Celera Genomics used whole genome shotgun approach. This bypasses the mapping step and saves time. Further, Celera group was lucky to have high-throughput sequenators and powerful computer programmes that helped for the early completion of human genome sequence.

One of the intriguing questions of human genome project is whose genome is being sequenced and how will it relate to the 6 billion or so population with variations in world? There is no simple answer to this question. However, looking from the positive side, it does not matter whose genome is sequenced, since the phenotypic differences between individuals are due to variations in just 0.1% of the total genome sequences. Therefore many individual genomes can be used as source material for sequencing.
Much of the human work genome was performed on the material supplied by the Centre for Human Polymorphism in Paris, France. This institute had collected cell lines from sixty different French families, each spanning three generations. Thus, the material supplied from Paris was used for human genome sequencing.

DNA sequencing, Comments = Physical map of DNA can be identified with highest resolution.. Use of probes, Comments = To identify RFLPs, STS and SNPs.. Radiation hybrid mapping, Comments = Fragment genome into large pieces and locate markers and genes. Requires somatic cell hybrids.. Fluorescence in situ hybridization (FISH), Comments = To localize a gene on chromosome.. Sequence tagged site (STS) mapping, Comments = Applicable to any part of DNA sequence if some sequence information is available.. Expressed sequence tag (EST) mapping, Comments = A variant of STS mapping; expressed genes are actually mapped and located.. Pulsed-field gel electrophoresis (PFGE), Comments = For the separation and isolation of large DNA fragments.. Cloning in vectors (plasmids, phages, cosmids, YACs, BACs), Comments = To isolate DNA fragments of variable lengths.. Polymerase chain reaction (PCR), Comments = To amplify gene fragments. Chromosome walking, Comments = Useful for cloning of overlapping DNA fragments (restricted to about 200 kb).. Chromosome jumping, Comments = DNA can be cut into large fragments and circularized for use in chromosome walking.. Detection of cytogenetic abnormalities, Comments = Certain genetic diseases can be identified by cloning the affected genes e.g. Duchenne muscular dystrophy.. Databases, Comments = Existing databases facilitate gene identification by comparison of DNA and protein sequences.. ( RFLP-Restriction fragment length polymorphism; STS-Sequence tagged site; SNP-Single nucleotide polymorphism; YAC-Yeast, Comments = ( RFLP-Restriction fragment length polymorphism; STS-Sequence tagged site; SNP-Single nucleotide polymorphism; YAC-Yeast
chromosome)

The information on the human genome projects is too vast, and only some highlights can be given (Table 28.2). Some of them are briefly described.

(SNPs) have been identified.. ●, 28.2 Major highlights of human genome = Human DNA is about 98% identical to that of chimpanzees.
Fig. 28.2 : An overview of the organization of human genome (LINEs-Long interspersed nuclear elements; SINEs-Short interspersed nuclear elements; LTR-Long terminal repeats).

About 90% of the human genome has been sequenced. It is composed of 3.2 billion base pairs (3200 Mb or 3.2 Gb). If written in the format of a telephone book, the base sequence of human genome would fill about 200 telephone books of 1000 pages each. Some other interesting analogs/sidelights of genome are given in Table 28.3.
Individual differences in genomes : It has to be remembered that every individual, except identical twins, have their own versions of genome sequences. The differences between individuals are largely due to single nucleotide polymorphisms (SNPs). SNPs represent positions in the genome where some individuals have one nucleotide (i.e. an A), and others have a different nucleotide (i.e. a G). The frequency of occurrence of SNPs is estimated to be one per 1000 base pairs. About 3 million SNPs are believed to be present and at least half of them have been identified.

An outline of the organization of the human genome is given in Fig.28.2. Of the 3200 Mb,
The base sequence in human genome would fill about 200 telephone books of 1000 pages each.
If the genome is recited at the rate of one base per second for 24 hours a day, it would take a century to recite the book of life.
If a typist types at the rate of 60 words per minute (ie. 360 letters) for 8 hours a day, he/she would take around 50 years to type human genome.
If the DNA sequence is typed in lines 10 cm containing 60 nucleotide bases and printed, the human genome sequence (from a single cell) would stretch a distance of 5000 km.
If the DNA in the entire human body is put end to end, it would reach to the sun and back over 600 times (Note : The human body contains 100 trillion cells; the length of DNA in a cell is 6 feet; the distance between the sun and earth is 93 million miles).
The total expenditure for human genome project was $3 billion. The magnitude of this huge amount has to be appreciated. If one starts counting at a non-stop rate of a dollar per second, it would take about 90 years to complete.
only a small fraction (48 Mb) represents the actual genes, while the rest is due to gene-related sequences (introns, pseudogenes) and intergenic DNA (long interspersed nuclear elements, short interspread nuclear elements, microsatellites, DNA transposons etc.). Intergenic DNA represents the parts of the genome that lie between the genes which have no known function. This is appropriately regarded as junk DNA.

The two genome projects differ in their estimates of the total number of genes in humans. Their figures are in the range of 30,000— 40,000 genes. The main reason for this variation is that it is rather difficult to specifically recognize the DNA sequences which are genes and which are not.
Before the results of the HGP were announced, the best guess of human genes was in the range of 80,000-100,000. This estimate was based on the fact that the number of proteins in human cells is 80,000-100,000, and thus so many genes expected. The fact that the number of genes is much lower than the proteins suggests that the RNA _ editing (RNA processing) is widespread, so that a single mRNA may code for more than one protein.
A diagrammatic representation of a typical structure of an average human gene is given in Fig.28.3. |\t has exons and introns.
A broad categorization of human gene catalog in the form of a pie chart is depicted in Fig.28.4. About 17.5% of the genes participate in the general biochemical functions of the cells, 23% in the maintenance of genome, 21% in signal transduction while the remaining 38% are involved in the production of structural proteins, transport proteins, immunoglobins etc.

It is now clear that only 1.1-1.5% of the human genome codes for proteins. Thus, this figure 1.1-1.5% represents exons of genome. As already described, a huge portion of the genome is composed of introns, and intergenic sequences (junk DNA).
The major categories of the proteins encoded by human genes are listed in Table 28.4. The functions of at least 40% of these proteins are not known.

It is expected that the sequencing of human genome, and the genomes of other organisms will dramatically change our understanding and perceptions of biology and medicine. Some of the benefits of human genome project are given.
- 1 Identification of human genes and_ their functions.
Fig. 28.4 : A pie chart showing a broad categorization of the human gene catalog (About 13000 genes whose functions are not known are not included).
Unknown functions, Percentage = 41.0%. Unknown functions, Actual number of genes = 12,809. Nucleic acid enzymes, Percentage = 7.5%. Nucleic acid enzymes, Actual number of genes = 2,308. Transcription factors, Percentage = 6.0%. Transcription factors, Actual number of genes = 1,850. Receptors, Percentage = 5.0%. Receptors, Actual number of genes = 1,543. Hydrolases, Percentage = 4.0%. Hydrolases, Actual number of genes = 1,227. Regulatory proteins (G-proteins, cell cycle regulators etc.), Percentage = 3.2%. Regulatory proteins (G-proteins, cell cycle regulators etc.), Actual number of genes = 988. Protooncogenes, Percentage = 2.9%. Protooncogenes, Actual number of genes = 902. Structural proteins of cytoskeleton, Percentage = 2.8%. Structural proteins of cytoskeleton, Actual number of genes = 876. Kinases, Percentage = 2.8%. Kinases, Actual number of genes = 868. ( Note : This table is based on the reported by Celera Genomics. The a total of 26,383 genes), Percentage = rough percentages. ( Note : This table is based on the reported by Celera Genomics. The a total of 26,383 genes), Actual number of genes = draft of human genome are derived from
|
Improved diagnosis of diseases
1 Development of pharmacogenomics
1
Genetic basis of psychiatric disorders
1
Understanding of complex social trait
1
Improved knowledge on mutations
| Better understanding of developmental biology
1 Comparative genomics
1 Development of biotechnology

The research on human genomes will make very sensitive data available that will affect the personal and private lives of individuals. For instance, once it is known that a person carries genes for an incurable disease, what would be the strategy of an insurance company? How will the society treat him/her? There is a possibility that individuals with substandard genome sequences may be discriminated. Human genome results may also promote racial discrimination categorizing the people with good and bad genome sequences. Considering the gravity of ethics related to a human genome, about 3% of the HGP budget was earmarked for ethical research.

1. Human Genome Project is an international venture involving several laboratories, and
a large number of scientists and technicians from various disciplines.
2. About 90% of the human genome has been sequenced. It is composed of 3.2 billion base pairs.
3. The total number of genes in the humans is in the range of 30,000-40,000.
4. About 1.1-1.5% of the human genome codes for proteins while the remaining portion
is regarded as junk DNA (composed of introns and intergenic sequences).
5.
Human
genome
sequencing
has
wide
range
of
applications—better
understanding
of
genetic
diseases,
improvements
in
gene
therapy,
development
of
pharmacogenomics,
and
advancement
of
biotechnology.
dvances in biochemistry and molecular biology have helped to understand the genetic basis of inherited diseases. It was a dream of the researchers to replace the defective genes with good ones, and cure the genetic disorders.
Gene therapy is the process of inserting genes into cells to treat diseases. The newly introduced genes will encode proteins and correct the deficiencies that occur in genetic diseases. Thus, gene therapy primarily involves genetic manipulations in animals or humans to correct a disease, and keep the organism in good health. The initial experiments on gene therapy are carried out in animals, and then in humans. Obviously, the goal of the researchers is to benefit the mankind and improve their health.
An overview of gene therapy strategies is depicted in Fig.29.1. In gene augmentation therapy, a DNA is inserted into the genome to replace the missing gene product. In case of gene inhibition therapy, the antisense gene inhibits the expression of the dominant gene.

There are two approaches to achieve gene therapy.
1. Somatic cell gene therapy : The nonreproductive (non-sex) cells of an organism are referred to as somatic cells. These are the cells of an organism other than sperm or egg cells, e.g., bone marrow cells, blood cells, skin cells, intestinal cells. At present, all the research on gene therapy is directed to correct the genetic defects in somatic cells. In essence, somatic cell gene therapy involves the insertion of a fully functional and expressible gene into a target somatic cell to correct a genetic disease permanently.
2. Germ cell gene therapy : The reproductive (sex) cells of an organism constitute germ cell line. Gene therapy involving the introduction of DNA into germ cells is passed on to the successive generations. For safety, ethical and technical reasons, germ cell gene therapy is not being attempted at present.
The genetic alterations in somatic cells are not carried to the next generations. Therefore, somatic cell gene therapy is preferred and extensively studied with an ultimate objective of correcting human diseases.
A large number of genetic disorders and other diseases are currently at various stages of gene therapy trials. A selected list of some important ones is given in Table 29.1.
There are two types of gene therapies.
|. Ex vivo gene therapy : This involves the transfer of genes in cultured cells (e.g., bone marrow cells) which are then reintroduced into the patient.

Severe combined immunodeficiency (SCID)
Familial hypercholesterolemia
Emphysema
Hemophilia B
Thalassemia
Sickle-cell anemia
Lesch-Nyhan syndrome
Gaucher's disease
Peripheral artery disease
Fanconi anemia
Melanoma
Melanoma, renal cancer
Glioblastoma (brain tumor), AIDS, ovarian cancer
Head and neck cancer
Breast cancer
AIDS
Colorectal cancer, melanoma, renal cancer
Duchenne muscular dystrophy
Short
stature**
Diabetes**
Phenylketonuria**
Citrullinemia**
Mostly confined to animal experiments
Adenosine deaminase (ADA).
Cystic fibrosis transmembrane regulator (CFTR).
Low density lipoprotein (LDL) receptor.
o4,Antitrypsin
Factor IX
a- or B-Globin
B-Globin
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT).
Glucocerebrosidase
Vascular endothelial growth factor (VEGF)
Fanconi anemia C
Tumor necrosis factor (TNF)
Interleukin-2 (IL-2)
Thymidine kinase (herpes simplex virus)
ps3
Multidrug resistance |
rev and env
Histocompatability locus antigen-B7 (HLA-Bz)
Dystrophin
Growth hormone
Glucose transporter-2, (GLUT-2), glucokinase
Arginosuccinate synthetase
Il. In vivo gene therapy : The direct delivery of genes into the cells of a particular tissue is referred to as in vivo gene therapy.

The ex vivo gene therapy can be applied to only selected tissues (e.g., bone marrow) whose cells can be cultured in the laboratory.
The technique of ex vivo gene therapy involves the following steps (Fig.29.2).
- 1. Isolate cells with genetic defect
from a patient.
2. Grow the cells in culture.
3. Introduce the therapeutic gene to
gene
correct defect.
4. Select the genetically corrected cells (stable transformants) and grow.
5. Transplant the modified cells to the patient.
The procedure basically involves the use of the patient's own cells for culture and genetic correction, and then their return back to the patient. This technique is therefore, not associated with adverse immunological responses after transplanting the cells. Ex vivo gene therapy is efficient only if the therapeutic gene (remedial gene) is stably incorporated and continuously expressed. This can be achieved by use of vectors.

The carrier particles or molecules used to deliver genes to somatic cells are referred to as vectors. The important vectors employed in ex vivo gene therapy are listed below and briefly described next.
1 Viruses
» Human artificial chromosome
1 Bone marrow cells.

The vectors frequently used in gene therapy are viruses, particularly retroviruses. RNA is the genetic material in retroviruses. As the retrovirus enters the host cell, it synthesizes DNA from RNA (by reverse transcription). The so formed viral DNA (referred to as provirus) gets incorporated into the DNA of the host cell. The proviruses are normally harmless. However, there is a tremendous risk, since some of the retroviruses can convert normal cells into cancerous ones. Therefore, it is absolutely essential to ensure that such a thing does not happen.

The human. artificial chromosome (HAC) is a synthetic chromosome that can replicate with other chromosomes, besides encoding a human protein. As already discussed above, use of retroviruses as vectors in gene therapy is associated with a heavy risk. This problem can be overcome if HAC is used. Some success has been achieved in_ this direction.

Bone marrow contains totipotent embryonic stem (ES) cells. These cells are capable of dividing and differentiating into various cell types (e.g., red blood cells, platelets, macro-
phages, osteoclasts, Band T-lymphocytes). For this reason, bone marrow transplantation is the most widely used technique for several genetic diseases. And there is every reason to believe that the genetic disorders that respond to bone marrow transplantation are likely to respond to ex vivo gene therapy also e.g. sicklecell anemia, SCID, thalassemia.

The first and the most publicised human gene therapy was carried out to correct the deficiency of the enzyme adenosine deaminase (ADA). This was done on September 14, 1990 by a team of workers led by Blaese and Anderson at the National Institute of Health, USA (The girl's name is Ashanti, 4 years old then).

SCID is rare inherited immune disorder associated with T-lymphocytes, and (to a lesser extent) B-lymphocytes dysfunction. About 50% of SCID patients have a defect in the gene (located on chromosome 20, and has 32,000 base pairs and 12 exons) that encodes for adenosine deaminase. In the deficiency of ADA, deoxyadenosine and its metabolites (primarily deoxyadenosine 5'-triphosphate) accumulate and destroy T-lymphocytes. T-Lymphocytes are essential for body's immunity. Besides participating directly in body's defense, they promote the function of B-lymphocytes to produce antibodies. Thus, the patients of SCID (lacking ADA) suffer from infectious diseases and die at an young age. Previously, the children suffering from SCID were treated with conjugated bovine ADA, or by bone marrow transplantation.

The general scheme of gene therapy adopted for introducing a defective gene in the patient has been depicted in Fig.29.2. The same procedure with suitable modifications can also be applied for other gene therapies.
A
plasmid vector
bearing
a
proviral
DNA
is selected. A part of the proviral DNA is replaced by the ADA gene and a gene (G 418) coding for antibiotic resistance, and then cloned. The antibiotic resistance gene will help to select the desired clones with ADA gene.
A diagrammatic representation of the treatment of ADA deficient patient is depicted in Fig.29.3.
Circulating lymphocytes are removed from a patient suffering from ADA deficiency. These cells are transfected with ADA gene by exposing to billions of retroviruses carrying the said gene. The genetically-modified lymphocytes are grown in cultures to confirm the expression of ADA gene and returned to the patient. These lymphocytes persist in the circulation and synthesize ADA. Consequently, the ability of the patient to produce antibodies is increased. However, there is a limitation. The lymphocytes have a short life span (just live for a few months), hence the transfusions have to be carried out frequently.

In 1995, ADA gene was transferred into the stem cells, obtained from the umbilical cord blood, at the time of baby's delivery. Four days after birth, the infant received the modified cells back. By this way, a permanent population of ADA gene producing cells was established.

The direct delivery of the therapeutic gene (DNA) into the target cells of a particular tissue of a patient constitutes in vivo gene therapy (Fig.29.4). Many tissues are the potential candidates for this approach. These include liver, muscle, skin, spleen, lung, brain and blood cells. Gene delivery can be carried out by viral or nonviral vector systems. The success of in vivo gene
Fig. 29.3 : Treatment of adenosine deaminase (ADA) deficient patient by somatic ex vivo gene therapy (SCID-Severe combined immunodeficiency).
therapy mostly depends on parameters the following
retroviruses,
adenoviruses,
adeno-associated
viruses and herpes simplex virus.
- | The efficiency of the uptake of the remedial
(therapeutic) gene by the target cells.
- 1 Intracellular degradation of the gene and its
uptake by nucleus.
- 1 The expression capability of the gene.

Many viral vector systems have been developed for gene delivery. These include

There are certain limitations in using viral vectors in gene therapy. In addition to the prohibitive cost of maintaining the viruses, the viral proteins often induce — inflammatory responses in the host. Therefore, there is a continuous search by researchers to find alternatives to viral vector systems.
Fig. 29.4 : Diagrammatic representation of in vivo gene therapy. (p-Promoter gene specific for therapeutic gene)
The non-viral gene delivery systems are listed
1 Pure DNA constructs that can be directly introduced into target tissues.
| Lipoplexes, lipid-DNA complexes that have
DNA surrounded by lipid layers.
- 1 Human artificial chromosome which can carry large DNA (one or more genes).

Cancer is the leading cause of death throughout the world, despite the intensive treatment strategies (surgery, chemotherapy, radiation therapy). Gene therapy is the latest and a new approach for cancer treatment. Some of the developments are briefly described hereunder.

Tumor necrosis factor (TNF) is a protein produced by human macrophages. TNF provides defense against cancer cells. This is brought out by enhancing the cancer-fighting ability of tumor-infiltrating lymphocytes (TILs), a special type of immune cells.
The tumor-infiltrating lymphocytes were transformed with a TNF gene (along with a neomycin resistant gene) and used for the treatment of malignant melanoma (a cancer of melanin producing cells, usually occurs in skin). TNF as such is highly toxic, and fortunately no toxic side effects were detected in the melanoma patients injected with genetically altered TILs with TNF gene. Some improvement in the cancer patients was observed.

The gene encoding the enzyme thymidine kinase is often referred to as suicide gene, and is used for the treatment of certain cancers.
Thymidine kinase (TK) — phosphorylates nucleosides to form nucleotides which are used for the synthesis of DNA during cell division. The drug ganciclovir (GCV) bears a close structural resemblance to certain nucleosides

+ Theoretically, gene therapy is the permanent solution for genetic diseases.
+ A large number of genetic disorders and other diseases are at various stages of gene therapy trials e.g. sickle-cell anemia, cystic fibrosis, AIDS, cancer.
+ Ganciclovir (a drug with structural resemblance to thymidine) has been used (suicide
gene
therapy)
for
the
treatment
of
brain
tumors,
although
with
limited
success.
+ Despite extensive research and trials, as of now, no disease has been permanently cured by gene therapy. However, a breakthrough may come at anytime.
Fig. 29.5 : The action of ganciclovir mediated by thymidine kinase to inhibit the growth of cancer cells.
(thymidine). By mistake, TK phosphorylates ganciclovir to form triphosphate-GCV, a false and unsuitable nucleotide for DNA synthesis. Triphosphate-GCV inhibits DNA polymerase (Fig.29.5). The result is that the elongation of the DNA molecule abruptly stops at a point containing the false nucleotide (of ganciclovir). Further, the triphospate-GCV can enter and kill the neighbouring cancer cells, a phenomenon referred to as bystander effect. The ultimate result is that the cancer cells cannot multiply, and therefore die. Thus, the drug ganciclovir can be used to kill the cancer cells.
Ganciclovir is frequently referred to as a prodrug and this type of approach is called prodrug activation gene therapy. Ganciclovir has been used for treatment of brain tumors (e.g., glioblastoma, a cancer of glial cells in brain), although with a limited success.

A gene named p®? codes for a protein with a molecular weight of 53 kilodaltons (hence p>). p?? is considered to be a tumor-suppressor gene, since the protein it encodes binds with DNA and inhibits replication. The tumor cells of several tissues (breast, brain, lung, skin, bladder, colon, bone) were found to have altered genes of p°? (mutated p°), synthesizing different proteins from the original. These altered proteins cannot inhibit DNA replication. It is believed that the damaged p®* gene may be a causative factor in tumor development.
Some workers have tried to replace the damaged p°? gene by a normal gene by employing adenovirus vector systems. There are some encouraging results in the patients with liver cancer.
The antisense therapy for cancer is discussed as a part of antigene and antisense therapy.

In general, gene therapy is carried out by introducing a therapeutic gene to produce the defective or the lacking protein. But there are certain disorders (cancer, viral and_ parasitic infections, inflammatory diseases) which result in an overproduction of certain normal proteins. It is possible to treat these diseases by blocking transcription using a single-stranded nucleotide sequence (antigene oligonucleotide) that hybridizes with the specific gene, and this is called antigene therapy. Antisense therapy refers to the inhibition of translation by using a singlestranded nucleotide (antisense oligonucleotide). Further, it is also possible to inhibit both transcription and translation by blocking (with oligonucleotides) the transcription factor responsbile for the specific gene expression.
Nucleic acid therapy refers to the use of DNA or RNA molecules for therapeutic purposes, as stated above. The naturally occurring sequences of DNA and RNA (with suitable modifications)
nucleic acid bases attached to a polypeptide in place of sugar-phosphate backbone. Antisense PNAs have been developed to inhibit translation of HIV viral transcript, gag-pol, and to block translation of cancer genes e.g. Has-ras and bcl-2.

Theoretically, gene therapy is the permanent solution for genetic diseases. But it is not as simple as it appears since gene therapy has several inbuilt complexicities. Gene therapy broadly involves isolation of a specific gene, making its copies, inserting them into target tissue cells to make the desired protein. The story does not end here. It is absolutely essential to ensure that the gene is harmless to the patient and it is appropriately expressed (too much or too little will be no good). Another concern in gene therapy is the body's immune system which reacts to the foreign proteins produced by the new genes.
The public, in general, have exaggerated expectations on gene therapy. The researchers, at least for the present, are unable to satisfy them. As per the records, by 1999 about 1000 Americans had undergone clinical _ trails involving various gene therapies. Unfortunately, the gene therapists are unable to categorically claim that gene therapy has permanently cured any one of these patients! Some people in the media (leading news papers and magazines) have openly questioned whether it is worth to continue research on gene therapy!!
It may be true that as of now, gene therapy due to several limitations, has not progressed the way it should, despite intensive research. But a breakthrough may come anytime, and of course, this is only possible with persistent research. And a day may come (it might take some years) when almost every disease will have a gene therapy, as one of the treatment modalities. And gene therapy will revolutionize the practice of medicine!

1. Gene therapy is the process of inserting genes into cells to treat diseases. Somatic cell gene therapy, involving the insertion of an expressible gene into somatic cells, is the preferred approach.
2. Ex vivo gene therapy involves the transfer of genes in cultured cells which are then reintroduced into the patient. The direct delivery of genes into the cells of a particular tissue is regarded as in vivo gene therapy.
3. Gene therapy was successfully carried out in a patient of severe combined immunodeficiency (caused by the deficiency of the enzyme adenosine deaminase).
4. Antigene therapy involves blocking of transcription (by antigene oligonucleotide) while in antisense therapy, translation is inhibited (by antisense oligonucleotide). These approaches are in the experimental stages for the therapy of cancer and AIDS.
5. Although as of now, gene therapy has not offered any permanent cure to any human patients, a breakthrough may come anytime. And gene therapy may revolutionize the practice of medicine.
ioinformatics is the combination (or marriage!) of biology and information technology. Basically, bioinformatics is a recently developed science using information to understand biological phenomenon. It broadly involves the computational tools and methods used to manage, analyse and manipulate volumes and volumes of biological data.
management and analysis of the data pertaining to DNA, RNA and protein sequences. As the biological data is being produced at an unprecedented rate, their management and interpretation invariably requires bioinformatics. Thus, bioinformatics now includes many other types of biological data. Some of the most important ones are listed below
Bioinformatics may also be regarded as a part of the computational biology. The latter is concerned with the application of quantitative analytical techniques in modeling and solving problems in the — biological — systems. Bioinformatics is an interdisciplinary approach requiring advanced knowledge of computer science, mathematics and statistical methods for the understanding of biological phenomena at the molecular level.

The term bioinformatics was first introduced
- in' 1990s.
Originally, it
dealt with
the
| Gene expression profiles
| Protein structure
1 Protein interactions
1 Microarrays (DNA chips)
' Functional analysis of biomolecules
1 Drug designing.
Bioinformatics is largely (not exclusively) a computer-based discipline. Computers are in fact very essential to handle large volumes of biological data, their storage and retrieval.
We have to accept the fact that there is no computer on earth (however advanced) which
can store information, and perform the functions like a living cell. Thus a highly complex information technology lies right within the cells of an organism. This primarily includes the organism's genes and their dictates for the organisms biological processes and behaviour.

Bioinformatics covers many specialized and advanced areas of biology.
Functional genomics : Identification of genes and their respective functions.
functions of
Structural genomics : Predictions related to proteins.
Comparative genomics : For understanding the genomes of different species of organisms.
DNA microarrays : These are designed to measure the levels of gene expression in different tissues, various stages of development and in different diseases.
Medical informatics This involves the management of biomedical data with special reference to biomolecules, in vitro assays and clinical trials.

1. Creation of databases : This involves the organizing, storage and management of the biological data sets. The databases are accessible to researchers to know the existing information and submit new entries. e.g. protein sequence data bank for molecular structure. Databases will be of no use until analysed.
2. Development of algorithms and statistics : This involves the development of tools and resources to determine the relationship among the members of large data sets e.g. comparison of protein sequence data with the already existing protein sequences.
3. Analysis of data and interpretation : The appropriate use of components 1 and 2 (given above) to analyse the data and interpret the results in a biologically meaningful manner. This includes DNA, RNA and _ protein sequences, protein structure, gene expression profiles, and biochemical pathways.

The internet is an international computer network. A computer network involves a group of computers that can communicate (usually over a telephone system) and exchange data between users.
It is the internet protocol (IP) that determines
Bioinformatics comprises three components how the packets of information are addressed

+ Bioinformatics has largely benefited biological and medical sciences, particularly related to molecular biology and biotechnology. Some applications are listed :
1 Sequencing of macromolecules (proteins, DNA, RNA)
1 Human genome sequencing
1 Molecular modelling of biomolecules
1 Handling of vast biological data
| Designing of drugs for the treatment of diseases
1 Development of models for the functioning of cells, tissues and organs
+ As such, there is no field of biological science that is not benefited by bioinformatics.
and routed over the network. To access the internet, a computer must have the correct hardware (modem/network card), appropriate software and permission for access to network. For this purpose, one has to subscribe to an internet service provider (ISP).
World wide web (www) : www involves the exchange of information over the internet using a programme called browser. The most widely used browsers are Internet explorer and Netscape navigator.
www works
on the
basis of
Uniform resource
locator
(URL)
which is
a
document with
a
unique address.
URLs takes
the format
http.//
(hypertext transfer protocol) that can identify the protocol for communication over www.

The collection of the biological data on a computer which can be manipulated to appear in varying arrangements and subsets is regarded as a database. The biological information can be stored in different databases. Each database has its own website with unique navigation tools.
The biological databases are, in general, publicly accessible. Selected examples of biological databases are briefly described (Table 30.1).

Primary nucleotide sequence databases, Salient features = . GenBank (www.ncbi.nih.gov/GeneBank/), Salient features = Provides nucleotide sequence databases maintained by the National Center for Biotechnology Information (NCBI), USA.. Other nucleotide sequence databases, Salient features = . UniGene (www.ncbi.nih.gov/UniGene/), Salient features = The nucleotide sequences of GenBank in the form of clusters, representing genes are available.. Genome Biology (www.ncbi.nlm.nih.gov/Genomes/), Salient features = The information about the completed genomes is available.. Protein sequence database, Salient features = . SWISS-PROT (www.expasy.ch/sport), Salient features = Provides the description of the structure of a protein, its domains structure, post-translational modifications, variants etc. It has high level of integration with other databases and minimal level of redundancy.. Protein sequence motif databases, Salient features = . PROSITE (www.expasy.ch/prosite/), Salient features = Provides information on protein families and domains. It also has patterns and profiles for sequences and biological functions.. Macromolecular databases, Salient features = . PDB (www.rcsb.org/pdb), Salient features = This is the primary database for 3-dimensional (3-D) structures of biological macromolecules (determined by X-ray and NMR studies).. Other databases, Salient features = . KEGG (www.genome.ad.jp/kegg/), Salient features = The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database with latest computerised information on biomolecules and cell biology. KEGG provides details on information pathways, interacting molecules and the connecting links with genes.

The nucleotide sequence data submitted by the scientists and genome sequencing groups is at the databases namely GenBank, EMBL (European Molecular Biology Laboratory) and DDB} (DNA Data Bank of Japan). There is a good coordination between these three databases as they are synchronized on daily basis.
Besides the primary nucleotide databases (referred above), there are some other databases also to provide information on genes, genomes and ongoing research projects.

Protein sequence databases are usually prepared from the existing literature and/or in consultation with the experts. In fact, these databases represent the translated DNA databases.

The three dimensional (3-D) structures of macromolecules are determined by X-ray crystallography and nuclear magnetic resonance (NMR). PDB and SCOP are the primary databases of 3-D_ structures of biological molecules.

KEGG database is an important one that provides information on the current knowledge of molecular biology and cell biology with special reference to information on metabolic pathways, interacting molecules and genes.

The advent of bioinformatics has revolutionized the advancements in biological science. And biotechnology is largely benefited by bioinformatics. The best example is the sequencing of human genome in a record time which would not have been possible without bioinformatics. A selected list of applications of bioinformatics is given below.
- 1 Sequence mapping of biomolecules (DNA, RNA, proteins).
1
Identification of
of functional
genes.
| Finding of sites that can be cut by restriction enzymes.
| Prediction of functional gene products.
| To trace the evolutionary trees of genes.
| For the prediction of 3-dimensional structure of proteins.
1 Molecular modelling of biomolecules.
| Designing of drugs for medical treatment.
| Handling of vast biological data which otherwise is not possible.
1 Development of models for the functioning various cells, tissues and organs.
The above list of applications however, may be treated as incomplete, since at present there is no field in biological sciences that does not involve bioinformatics.

1. Bioinformatics (a computer-based discipline) represents an alliance between biology and information technology.
2. The storage, management and interpretation of vast biological data invariably requires bioinformatics.
3.
comprises
three
components-creation
of
data
base,
of
algorithms
and
statistics,
and
analysis
of
data
and
interpretation.
4. Biological databases, containing the biological information, are publicly accessible e.g.
GenBank
(www.ncbi.nih.gov/GeneBank).
5. Bioinformatics has revolutionized the advancements of biological and medical sciences e.g. sequencing of human genome.
an is continuously exposed to several foreign compounds such as_ drugs, pollutants, food additives, cosmetics, pesticides etc. Certain unwanted compounds are produced in the large intestine by the bacteria which enter the circulation. These include indole from tryptophan, cadaverine from lysine, tyramine from tyrosine, phenol from phenylalanine etc. In the normal metabolism of the body, certain waste compounds (e.g. bilirubin) are formed. A vast majority of the foreign compounds or the unwanted substances, produced in the body, are toxic and, therefore, they should be quickly than the original substance (e.g. procarcinogens to carcinogens). It appears that the body tries to get rid of a foreign substance by converting it into a more soluble (often polar), and easily excretable compound, and this may be sometimes associated with increased toxicity (e.g. conversion of methanol to formaldehyde).
eliminated from the body.
The term detoxication or detoxification refers to the series of biochemical reactions occurring in the body to convert the foreign (often toxic) compounds to non-toxic or less toxic, and more easily excretable forms.

Detoxification is rather misleading, since sometimes a detoxified product is more toxic
In recent years, the term detoxification is replaced by biotransformation or metabolism of xenobiotics (Greek xenos—strange, foreign) or simply metabolism of foreign compounds.

The detoxification reactions are carried out mainly in the liver which is equipped with the enzyme machinery. Kidney and other organs may sometimes be involved. The products formed by detoxification are mostly excreted by the kidneys, less frequently excreted via feces or expired air.
Fig. 31.1 : Phase | and phase II reactions in the metabolism of xenobiotics.

The metabolism of xenobiotics may be divided into two phases which may occur together or separately (Fig.31.1).
Phase I : The reactions of phase | are oxidation, reduction and hydrolysis.
Phase II: These are the conjugation reactions, involving compounds such as glucuronic acid, amino acids (glycine), glutathione, sulfate, acetate and methyl group.
Generally, detoxification of a compound involves phase | as well as phase II reactions. For instance, oxidation followed by conjugation is the most frequent process in the metabolism of xenobiotics.

A large number of foreign substances are detoxified by oxidation. These include alcohols, aldehydes, amines, aromatic hydrocarbons and sulfur compounds. In general, aliphatic compounds are more easily oxidized than aromatic ones.
Alcohols : Aliphatic and aromatic alcohols undergo
oxidation to
form the
corresponding acids.
CH30H
-——>
HCOOH
Methanol
Formic
acid
mixed function oxidase, is associated with the microsomes. The usage P4sg refers to the absorption peak (at 450 nm), exhibited by the enzyme when exposed to carbon monoxide.
Most of the reactions of cytochrome P4509 involve the addition of a hydroxyl group to aliphatic or aromatic compounds which may be represented as
RH
+
O,
+
NADPH
——>
ROH
+
H,O
+
NADP*

1. Multiple forms of cytochrome Pysq are believed to exist, ranging from 20 to 200. At least 6 species have been isolated and worked in detail.
- 2. They are all hemoproteins,
heme as group.
containing the prosthetic
3. Cytochrome P45 species are found in the highest concentration in the microsomes of liver. In the adrenal gland, they occur in mitochondria.
4. The mechanism of action of cytochrome
P4s59 is complex and is dependent on NADPH.
- 5. The phospholipid-phosphatidylcholine is a
constituent of
P459
system which
for the
action of
this enzyme.
- 6. Cytochrome P4s9 is an inducible enzyme. Its synthesis is increased by the administration of drugs such as phenobarbitol.
7. A distinct species namely cytochrome Py4g (with absorption peak at 448 nm) has been studied. It is specific for the metabolism of polycyclic aromatic hydrocarbons, hence it is also known as_ aromatic hydrocarbon hydroxylase.

The hydrolysis of the bonds such as ester, glycoside and amide is important in the metabolism of xenobiotics. Several compounds undergo hydrolysis during the course of their detoxification. These include aspirin, acetanilide, diisopropylfluorophosphate, atropine and procaine.

Several xenobiotics undergo detoxification by conjugation to produce less toxic and/or more easily excretable compounds. Conjugation is the process in which a foreign compound combines with a substance produced in the body. The process of conjugation may occur either directly or after the phase | reactions. At least 8 different conjugating agents have been identified in the body. These are glucuronic acid, glycine, cysteine (of glutathione), glutamine, methyl group, sulfate, acetic acid and thiosulfate.
Glucuronic acid Conjugation — with glucuronic acid is the most common. The active form of glucuronic acid is UDP-glucuronic acid produced in the uronic acid pathway (Chapter 13). The microsomal enzymes UDPglucuronyl transferases participate in glucuronide formation. A general reaction of glucuronide conjugation is shown below (X-OH represents xenobiotic).

+ Knowledge of the metabolism of xenobiotics is essential for the understanding of toxicology, pharmacology and drug addiction.
+ The body possesses the capability to get rid of the foreign substances by converting them into more easily excretable forms.
+ Detoxification is not necessarily associated with the conversion of toxic into compounds. For instance, methanol is metabolized to a more toxic formaldehyde.
non-toxic
+ Detoxification primarily occurs in the liver through one or more of the reactions, namely oxidation, reduction, hydrolysis and conjugation.
+ British antilewisite (BAL), a compound developed during Second World War, was used to detoxify certain war poisons.

1. Detoxification deals with the series of biochemical reactions occurring in the body to convert the foreign (often toxic) compounds to non-toxic or less toxic and more easily excretable forms. Liver is the major site of detoxification. In recent years, the term detoxification is replaced by biotransformation or metabolism of xenobiotics.
2. Detoxification may be divided into phase I (oxidation, reduction, hydrolysis) and phase II reactions (conjugation). Oxidation is a major process of detoxification, involving the microsomal enzyme cytochrome P59 which is an inducible, NADPH dependent hemoprotein.
3. Conjugation is a process in which a foreign compound combines with a substance produced in the body. The process of conjugation may occur either directly or after phase I reactions. At least 8 different conjugating agents have been identified in the body—glucuronic acid, glycine, cysteine, glutamine, methyl group, sulfate, acetic acid and thiosulfate.
Sec
[Proszetencins and their related compounds— prostacyclins (PGI), thromboxanes (TXA), leukotrienes (LT) and lipoxins are collectively known as_ eicosaniods, since they all contain 20 carbons (Greek : eikosi-twenty). Eicosanoids are considered as locally acting hormones with a wide range of biochemical functions.
History : Prostaglandins (PGs) were first discovered in human semen by UIf von Euler (of Sweden) in 1930. These compounds were found to stimulate uterine contraction and reduce blood pressure. von Euler presumed that they were synthesized by prostate gland and hence named them as prostaglandins. It was_ later realized that PGs and other eicosanoids are synthesized in almost all the tissues (exception— erythrocytes). By then, however, the name prostaglandins was accepted worldwide, and hence continued.
The prostaglandins E and F were first isolated from the biological fluids. They were so named due to their solubility in ether (PGE) and phosphate buffer (PGF, F for fosfat, in Swedish). All other prostaglandins discovered later were denoted by a letter—PGA, PGH etc.

Prostaglandins are derivatives of a hypothetical 20-carbon fatty acid namely prostanoic acid, hence known as prostanoids. This has a cyclopentane ring (formed by carbon atoms 8 to 12) and two side chains, with carboxyl group on one side. Prostaglandins differ in their structure due to substituent group and double bond on cyclopentane ring. The different prostaglandins are given in Fig.32.1.
The structures of the most important prostaglandins (PGF and PGF a), prostacyclins (PGly), thromboxanes (TXA) and leukotrienes (LTA,) along with arachidonic acid are depicted in Fig.32.2. A subscript numeral indicates the number of double bonds in the two side chains. A subscript o-denotes that the hydroxyl group at Cg of the ring and the carboxyl group are on the same side of the ring.

10 11 12, 7 6 5 4 3 2 = 13 14 15 16 17 18 Prostanoic acid. 10 11 12, COOH 1 = 19 20 PGE. 10 11 12, Substituent group Prostaglandin = Keto group at C 9 OH group at C 11. 10 11 12, Structure = R 1 R 2 O. Prostaglandin PGA, 7 6 5 4 3 2 = Substituent group Double bond C 10 - C 11 Keto group C 9. Prostaglandin PGA, COOH 1 = Structure O R 1 R 2 PGF. Prostaglandin PGA, Substituent group Prostaglandin = OH group at C 9 and at C 11. Prostaglandin PGA, Structure = R 1 R 2 OH. PGB, 7 6 5 4 3 2 = Double bond C 8 - C 12 Keto group C 9. PGB, COOH 1 = R 1 R 2 PGG. PGB, Substituent group Prostaglandin = Two oxygen atoms present at C 9 and C 11 are bonded; hydroper- oxide group at C 15. PGB, Structure = R 1 O. PGC, 7 6 5 4 3 2 = Double bond C 11 - C 12 Keto group C 9. PGC, COOH 1 = O R 1 PGH. PGC, Substituent group Prostaglandin = Ring the same as in PGG; OH group at C 15. PGC, Structure = R 2 O. PGD, 7 6 5 4 3 2 = OH group at C 9 Keto group at C 11. PGD, COOH 1 = R 1 R 2 O PGI. PGD, Substituent group Prostaglandin = Attachment of oxygen between C 6 and C 9 forming another 5-carbon ring. PGD, Structure = R 2 OH

Fig. 32.1 : Prostaglandins with substituent groups and structures (Note : Prostanoic acid is the parent nucleus for all the PGs; R, represents C, to C, of prostanoic acid, except in PGI where R, is C, to C,; R, represents C,, to C,, of prostanoic acid).

2. Oxidation and cyclization of arachidonic
Arachidonic acid (5,8,11,14-eicosatetraenoic acid) is the precursor for most of the prostaglandins in humans. The biosynthesis of PGs was described by scene Bergstrom and Bengt Samuelsson (1960). It occurs in the endoplasmic reticulum in the following stages, as depicted in Fig.32.3.
1. Release of arachidonic acid from membrane bound phospholipids by phospholipase Ay—this reaction occurs due to a specific stimuli by hormones such as epinephrine or bradykinin.
acid to PGG which is then converted to PGH)
by a reduced glutathione dependent peroxidase.
3.
PGHy serves
as the
immediate precursor
for the synthesis of a number of prostaglandins, including prostacyclins and thromboxanes.
The above pathway is known as_ cyclic pathway of arachidonic acid. |n the linear pathway of arachidonic acid, leukotrienes and lipoxins are synthesized (details given later).
Cyclooxygenase-a suicide
enzyme
:
It is
interesting to
note that
prostaglandin synthesis
can be partly controlled by suicidal activity of
cortisol) prevent the formation of arachidonic acid the by inhibiting enzyme phospholipase A>. Many non-steroidal anti-inflammatory drugs inhibit the synthesis of prostaglandins, prostacyclins and thromboxanes. They do so by blocking the enzyme cyclooxygenase.

Aspirin inhibits PG synthesis : Aspirin (acetyl salicylic acid) has been used since nineteenth century as an antipyretic (fever-reducing) and analgesic (pain relieving). The mechanism of action of aspirin however, was not known for a long period. It was only in 1971, John Vane discovered that aspirin inhibits the synthesis of PG from arachidonic acid. Aspirin acetylates serine at the active site of cyclooxygenase and irreversibly inhibits. Other antiinflammatory drugs, such as indomethacin and phenylbutazone act as reversible inhibitors of the enzyme cyclooxygenase. Paracetamol is also a reversible inhibitor.
Degradation of prostaglandins : Almost all the eicosanoids are metabolized rapidly. The lung and liver are the major sites of PG degradation. Two enzymes, namely 15-ahydroxy PG dehydrogenase and 13-PG reductase, convert hydroxyl group at C;5 to keto group and then to C,3 and Cy,4 dihydroderivative.

Prostaglandins act as local hormones in their function. They, however, differ from the true hormones in many ways. Prostaglandins are produced in almost all the tissues in contrast to hormonal synthesis which occurs in specialized glands. PGs are not stored and they are degraded to inactive products at the site of their production. Further, PGs are produced in very small amounts and have low half-lives.
Prostaglandins are involved in a variety of biological functions. The actions of PGs differ in different tissues. Sometimes, PGs bring about opposing actions in the same tissue.
Overproduction of PGs results in many symptoms which include pain, fever, nausea, vomiting and inflammation.
and lipolysis, increases glycogen formation promotes calcium mobilization from the bone.
10. Platelet aggregation and thrombosis : The prostaglandins, namely prostacyclins (PGI), inhibit platelet aggregation. On the other hand, thromboxanes (TXA») and prostaglandin E promote platelet aggregation and blood clotting that might lead to thrombosis. PGlj, produced by endothelial cells lining the blood vessels, prevents the adherence of platelets to the blood vessels. TXAg is released by the platelets and is responsible for their spontaneous aggregation. Thus, prostacyclins and thromboxanes are antagonists in their action. In the overall effect PGI, acts as a vasodilator, while TXA is a vasoconstrictor. The balance between PGI and TXA, is important in the regulation of hemostasis and thrombosis.
The mechanism of action of prostaglandins is not known for certain. It is believed that PGs may act through the mediation of cyclic nucleotides. PGE increases CAMP levels whereas PGF elevates cGMP.

aggregation and thrombus formation. This helps to prevent heart attacks to some extent.

Prostaglandins perform diversified functions. And for this reason, PGs (or other derivatives) are the most exploited in therapeutic applications. They are used in the treatment of gastric ulcers, hypertension, thrombosis, asthma etc. Prostaglandins are also employed in the medical termination of pregnancy, prevention of conception, induction of labor etc.
Inhibitors of prostaglandin synthesis (e.g. aspirin, ibuprofen) are utilized in controlling fever, pain, migraine, inflammation etc.

Leukotrienes are synthesized by leucocytes, mast cells, lung, heart, spleen etc., by lipoxygenase pathway of arachidonic acid. The synthesis of different leukotrienes (Ay, By, Cy, D4 and E4) through the intermediate, 5—hydroperoxyeicosatetraenoic acid (5—-HPETE) is depicted in Fig.32.4.
At low doses (80-325 mg/day), aspirin inhibits platelet cyclooxygenase, thereby reduces thromboxane (TXA,) formation, and thus platelet
Anaphylaxis is a violent and fatal allergic reaction.
It is
now known
that leukotrienes
(C4,
D, and E,) are the components of slow-reacting

+ Prostaglandins, synthesized in almost all the tissues (exception-erythrocytes) of the
body, act as local hormones.
+ PGs perform diversified biochemical functions. These include lowering of blood pressure, inhibition of gastric HCI secretion, decrease in immunological response and induction of labor.
+ Overproduction of PGs causes symptoms such as pain, fever, vomiting, nausea, inflammation etc. Aspirin/ibuprofen/corticosteroid administration inhibits PG synthesis and relieves these symptoms.
+ Platelet aggregation that may lead to thrombosis is promoted by thromboxanes and
prostaglandins E and inhibited by prostacyclins.
+ Leukotrienes are implicated in hypersensitivity (allergy) and asthma.
+ Consumption of fish foods containing the unsaturated fatty acid namely eicosapentaenoic acid is advocated to prevent heart attacks.
Fig. 32.4 : Synthesis of leukotrienes from arachidonic acid (5-HPETE—5-Hydroperoxycicosatetraenoic acid).
substances of anaphylaxis (SRS-A), released after immunological challenge. SRS-A is 100-1,000 times more potent than histamine — or prostaglandins in its action as a stimulant of allergic reactions. Leukotrienes are implicated in asthma, inflammatory reactions, hypersensitivity (allergy) and heart attacks.
Leukotrienes cause contraction of smooth muscles, bronchoconstriction, vasoconstriction, adhesion of white blood cells and release of lysosomal enzymes.
Lipoxins are involved in vasoactive, and immunoregulatory functions. There is a strong evidence to support that lipoxins act as counterregulatory compounds of immune response.

Eskimos of
Greenland have
a
low incidence
coronary heart
they consume
terol.
lipids diseases,
high
This is
despite quantities
due containing
The the
of fact
that of
fat to
the high
unsaturated most
predominant consumed
UFA
by
Eskimos sapentaenoic
leukotrienes-5
is acid
(EPA).
series in
their activity
5, and
intake choles-
of fatty
acids in
the
8,
EPA
which
11, is
fish marine
(UFA).
foods
14, the
17-eico- precursor
for are
much than
produced from
eicosapentaenoic the
lower leukotriene-4
arachidonic acid.
acid thromboxanes
inhibits
(TXA9).
promotes platelet
The diet
decreases
As already
aggregation rich
plasma
These factors,
TXA)
series,
Further, the
formation described,
TXA>
and in
marine cholesterol
along are
thrombosis.
lipids
(with
EPA)
and triacylglycerols.
with reduced
believed incidence
to of
heart be
responsible attacks
for in
the low
Eskimos.

1. Prostaglandins (PGs) and related compounds prostacyclins (PGI), thromboxanes (TXA) and leukotrienes (LT) are collectively known as eicosanoids. They are the derivatives of a hypothetical 20 carbon fatty acid, namely prostanoic acid. Prostaglandins are synthesized from arachidonic acid, released from the membrane bound phospholipids. Corticosteroids and aspirin inhibit PG synthesis.
2. Prostaglandins act as local hormones and are involved in a wide range of biochemical functions. In general, PGs are involved in the lowering of blood pressure, induction of inflammation, medical termination of pregnancy, induction of labor, inhibition of gastric HCI secretion, decrease in immunological response and increase in glomerular filtration rate. Thromboxanes (TXAg) and prostaglandin E, promote while prostacyclins (PGI) inhibit platelet aggregation.
he plasma membrane is an_ envelope surrounding the cell (Refer Fig.1.1). It separates and protects the cell from the external hostile environment. Besides being a protective barrier, plasma membrane provides a connecting system between the cell and its environment. The subcellular organelles such as nucleus, mitochondria, lysosomes are also surrounded by membranes.

The membranes are composed of lipids, proteins and carbohydrates. The actual composition differs from tissue to tissue. Among the lipids, amphipathic lipids (containing hydrophobic and hydrophilic groups) namely phospholipids, glycolipids and cholesterol, are found in the animal membranes.
Many animal cell membranes have thick coating of complex polysaccharides referred to as glycocalyx. The oligosaccharides _ of glycocalyx interact with collagen of intercellular matrix in the tissues.

A lipid bilayer model originally proposed for membrane structure in 1935 by Davson and Danielle has been modified.
Fluid mosaic model, proposed by Singer and Nicolson, is a more recent and acceptable model for membrane — structure. The _ biological membranes usually have a thickness of 5-8 nm. A membrane is essentially composed of a lipid bilayer. The hydrophobic (nonpolar) regions of the lipids face each other at the core of the bilayer while the hydrophilic (polar) regions face outward. Globular proteins are irregularly embedded in the lipid bilayer (Fig.33.1). Membrane proteins are categorized into two groups.
1. Extrinsic (peripheral) membrane proteins are loosely held to the surface of the membrane and they can be easily separated e.g. cytochrome c of mitochondria.
2. Intrinsic (integral) membrane proteins are tightly bound to the lipid bilayer and they can be separated only by the use of detergents or
organic receptors,
solvents e.g.
The membrane
due to
hormone
P45.
is the
irregular proteins.
The subunits
asymmetric distribution
of lipid
of the
appearance tile.
Unlike membrane
of and
protein membrane
mosaic or
a
a
fixed give
an ceramic
ceramic freely
the structure
tile, the
changes, of
the hence
membrane is
considered as
fluid mosaic.
Fig. 33.1 : The fluid mosaic model of membrane structure.

The biological membranes are relatively impermeable. The membrane, therefore, forms a barrier for the free passage of compounds across it. At least three distinct mechanisms have been identified for the transport of — solutes (metabolites) through the membrane (Fig.33.2).
1. Passive diffusion
2. Facilitated diffusion
3. Active transport.
2. Facilitated diffusion : This is somewhat comparable with diffusion since the solute moves along the concentration gradient (from higher to lower concentration) and no energy is needed. But the most important distinguishing feature is that facilitated diffusion occurs through the mediation of carrier or transport proteins. Specific carrier proteins for the transport of glucose, galactose, leucine, phenylalanine etc. have been isolated and characterized.
1. Passive diffusion : This is a simple process which depends on the concentration gradient of a particular substance across the membrane. Passage of water and gases through membrane occurs by passive diffusion. This process does not require energy.
Mechanism of facilitated diffusion : A pingpong model is put forth to explain the occurrence of facilitated diffusion (Fig.33.3). According to this mechanism, a transport (carrier) protein exists in two conformations. In the pong conformation, it is exposed to the side with high solute
Fig. 33.2 : Mechanism of transport across biological membrane
(Note : Transport molecule are represented in blue; the carrier proteins in red).
Fig. 33.3 : A diagrammatic representation of 'ping-pong' model for facilitated diffusion.
concentration. This allows the binding of solute to specific sites on the carrier protein. The protein then undergoes a conformational change (ping state) to expose to the side with low solute concentration where the solute molecule is released. Hormones regulate facilitated diffusion. For instance, insulin increases glucose transport in muscle and adipose tissue; amino acid transport in liver and other tissues.
3 Na' (in) + 2K* (out) + ATP —> 3Na* (out) + 2K* (in) + ADP + Pi
3. Active transport : Active transport occurs against a concentration gradient and this is dependent on the supply of metabolic energy (ATP). Active transport is also a carrier mediated process like facilitated diffusion. The most important primary active transport systems are ion-pumps (through the involvement of pump ATPases or ion transporting ATPases).

Nat-K* pump : The cells have a high intracellular Kt concentration and a low Na* concentration. This is essentially needed for the survival of the cells. High cellular K* is required for the optimal glycolysis (pyruvate kinase is dependent on K*) and for protein biosynthesis. Further, Na* and K* gradients across plasma membranes are needed for the transmission of nerve impulse.
The Na*-K+ pump is responsible for the maintenance of high K*t and low Na* concentrations in the cells. This is brought about by an integral plasma membrane protein, namely the enzyme Nat-Kt ATPase (mol. wt. 250,000). It consists of two a and two B subunits which may be represented as (a). Na*-K* ATPase pumps 3Nat ions from inside the cell to outside and brings 2K+ ions from the outside to the inside with a concomitant hydrolysis of intracellular ATP. The Nat-Kt+ pump, depicted in Fig.33.4, is summarized.
A major portion of the cellular ATP (up to 70% in nerve cells) is in fact utilized by Na*-Kt pump to maintain the requisite cytosolic Nat and K* levels. Ouabain (pronounced as Wahbain) inhibits Na*-K+ ATPase. Ouabain is a steroid derivative extracted from the seeds of an African shrub. It is a poison used to tip the hunting arrows by the tribals in Africa. Digoxin a steroid glycoside and an inhibitor of Nat-Kt ATPase, is used in the treatment of congestive cardiac failure (digoxin improves cardiac contractility).
Nat-cotransport system : The amino acids and sugars are transported into the cells by a Nat*-cotransport system. This process essentially consists of the passage of glucose (or amino acid) into the cell with a simultaneous movement of Nat. ATP is required to pump out the intracellular Nat through the mediation of Nat-K*+ ATPase. More details on the cotransport system are given under digestion and absorption (Chapter 8).
Fig. 33.5 : Diagrammatic representation of transport systems.

The transport systems may be divided into 3 categories (Fig.33.5).
1. Uniport system This involves the movement of a single molecule through the membrane e.g. transport of glucose to the erythrocytes.
Cotransport system In cotransport, the transport of a substance through the membrane is coupled to the spontaneous movement of another substance. The symport and antiport systems referred to above are good examples of cotransport system.
2. Symport system The simultaneous transport of two different molecules in the same direction e.g. transport of Na* and glucose to the intestinal mucosal cells from the gut.
3. Antiport system : The simultaneous transport of two different molecules in the opposite direction e.g exchange of Cland HCO3 in the erythrocytes. Uniport, symport and antiport systems are considered as secondary active transport systems.
Proton pump in the stomach : This is an antiport transport system of gastric parietal cells. It is brought out by the enzyme Ht-Kt ATPase to maintain highly acidic (pH=1) conditions in the lumen of the stomach. Proton pump antiports two cytoplasmic protons (2H*) and two extracellular potassium (2K*) ions for a molecule of ATP hydrolysed. The chloride ions secreted by Clchannels combine with protons to form gastric HCl. Omeprazole is a drug used in the treatment of peptic ulcer. It inhibits H*-K* ATPase and results in reduced secretion of HCl.

+
Biological
membranes
are
impermeable
protective
barriers
that
provide
a
connecting
link
between
the
cell
(or
its
organelle)
and
its
environment.
+ The cells must contain high K* and low Na* concentrations for their survival. Na*-Kt pump, which consumes a major portion of the cellular metabolic energy (ATP), is responsible for this.
+ QOuabain inhibits Na*-K+ ATPase (Nat-K*+ pump). It is extracted from the seeds of an African shrub and used as poison to tip the hunting arrows by the tribals.
+ Disturbances is osmosis are associated with diarrhea, edema, inflammation. Changes in membrane fluidity have been suggested to be involved in LCAT deficiency, hypertension and Alzheimer’s disease.

Osmosis is the phenomenon _ of movement of water from low osmotic pressure (dilute solution) to high osmotic pressure (concentrated solution) across biological membranes. The movement of water in the body occurs through osmosis, and this process does not require energy (ATP). Certain medical and __ health complications are due to disturbances in osmosis. e.g. edema, diarrhea, cholera, inflammation of tissues. The reader may refer Chapter 40 for more information on osmosis, water and electrolyte imbalance in cholera/diarrhea.

The transport of macromolecules such as proteins, polysaccharides and polynucleotides across the membranes is equally important. This is brought about by two_ independent mechanisms namely endocytosis—intake of macromolecules by the cells (e.g. uptake of LDL by cells) and exocytosis—release of macromolecules from the cells to the outside (e.g. secretion of hormones-insulin, PTH).

Alterations in transport systems result in a number of pathological conditions, selected examples are listed.
Fig. 33.6 : Diagrammatic representation of endocytosis and exocytosis (Note : The red coloured particles indicate the transport material).
Hartnup's disease due to a decrease in the transport of neutral amino acids in the intestinal cells and renal tubules.
Cystinuria characterized by _ increased excretion of cystine, lysine, arginine and ornithine. This results in the formation of renal cystine stones.
Decreased glucose uptake in some individuals due to lack of the specific sodium-glucose transporter.
Renal reabsorption of phosphate is decreased in vitamin D resistant rickets.

1. The biological membranes are the barriers that protect the cell and the subcellular organelles from the hostile environment. The membranes are primarily composed of a lipid bilayer onto which the globular proteins are irregularly embedded to form a fluid mosaic model.
2. Transport of molecules through membranes occurs either by passive diffusion, facilitated diffusion or active transport. Active transport occurs against a concentration gradient which is dependent on the supply of metabolic energy (ATP). Nat-K* pump is responsible for the maintenance of high Kt and low Na* concentrations inside the cells, an essential requisite for the survival of cells.
3. The transport systems are divided into 3 categories—uniport, symport and antiport.
4. The transport of macromolecules takes place by endocytosis (ingestion by the cells) and exocytosis (release from the cells).
he supply of oxygen is absolutely essential for the existence of higher organisms. As the saying goes too much of even the best is bad. Very high concentrations of Oy are found to be toxic, and can damage tissues. The present day concept of oxygen toxicity is due to the involvement of oxygen free radicals or reactive oxygen species (ROS). |n fact, the generation of reactive metabolites of Oy is an integral part of our daily life.
A free radical is defined as a molecule or a molecular species that contains one or more unpaired electrons, and is capable of independent existence.

Oxygen is required in many metabolic reactions, particularly for the release of energy. During these processes, molecular Oy is completely reduced, and converted to water. However, if the reduction of O, is incomplete, a series of reactive radicals are formed, as shown in the next column.
Besides the above (03, H»O», OH), the other free radicals and reactive oxygen species of biological importance include singlet oxygen ('O5), hydroperoxy radical (HOO), lipid peroxide radical (ROO-), nitric oxide (NO-) and peroxynitrite (ONOO-).
The common characteristic features of free radicals are listed
| Highly reactive
1 Very short half-life
1 Can generate new radicals by chain reaction
1 Cause damage to biomolecules, cells and tissues
Free radicals and reactive oxygen species (ROS)—not synonymous : By definition, a free radical contains one or more unpaired electrons. e.g. O7, OH, ROO-. There are certain nonradical derivatives of O2 which do not contain unpaired electrons e.g. H»O7, 'O7. The term reactive oxygen species is used in a broad sense to collectively represent free radicals, and nonfree radicals (which are extremely reactive) of the biological systems. However, most authors do not make a clear distinction between free radicals and ROS, and use them interchangebly.

The major sources responsible for the generation of free radicals may be considered under two categories
1. Due to processes (or
normal biological cellular metabolism).
Il. Due to environmental effects.
It is estimated that about 1-4% of the O, taken up by the body is converted to free radicals. A summary of the sources for generation of free radicals is given in the Table 34.1, and a couple of the processes are briefly described.

Free radical-induced peroxidation __ of membrane lipids occurs in three stages-initiation, propagation and termination
Initiation phase : This step involves the removal of hydrogen atom (H) from polyunsaturated fatty acids (LH), caused by hydroxyl radical
LH
+
OH>
—>
L>
+
H)O
Propagation
—_
phase
Under
—
aerobic conditions,
the fatty
acid radical
(L~)
takes up
oxygen to form peroxy radical (LOO-). The

I Cellular metabolism
1 Leakage of electrons from the respiratory chain (ETC).
1 Production of H,O, or O; by oxidase enzymes
(e.g.
xanthine oxidase,
NADPH
oxidase).
1 Due to chain reactions of membrane lipid peroxidation.
1 Peroxisomal generation of O, and H,O,.
1 During the synthesis of prostaglandins.
1 Production of nitric oxide from arginine.
: During the course of phagocytosis (as a part of bactericidal action).
1 In the oxidation of heme to bile pigments.
1 As a result of auto-oxidation e.g. metal ions [Fe2*, Cu?*]; ascorbic acid, glutathione, flavin coenzymes.

1 As a result of drug metabolism e.g. paracetamol, halothane, cytochrome P,,, related reactions.
1 Due to damages caused by ionizing radiations (e.g. X-rays) on tissues.
1 Photolysis of O, by light.
1 Photoexcitation of organic molecules
1 Cigarette smoke contains free radicals, and trace
metals that generate OH.
- 1 Alcohol, promoting lipid peroxidation.
latter, in turn, can remove H-atom from another PUFA (LH) to form lipid hydroperoxide (LOOH).
L>
+
O)7
—>
LOO-
LOO- + LH — > LOOH + L~
The hydroperoxides are capable of further
stimulating
lipid
peroxidation
as
they
can
form
alkoxy (LO-) and peroxyl (LOO”) radicals.
Fe, 2S"
_
2LOOH
+
LO-
+
LOZ
+
H,O
LOOH —> LO™ + LOO + aldehydes
Termination phase Lipid peroxidation
proceeds
as
a
chain
reaction
until
the
available
PUFA gets oxidized.

Most of the products of lipid peroxidation are unstable e.g. —_ carbonyls, esters, alkanes, alkenes, 2alkenal, 2,4-alkadienal, MDA. Of these, malondialdehyde (—CHO—CH,—CHO-) is the most extensively studied, and is used as a_ biochemical marker for the assessment of lipid peroxidation. MDA and
other aldehydes react with thiobarbituric acid and produce red-coloured products namely thiobarbituric acid reactive substances (TBARS) which can be measured colorimetrically. The estimation of serum MDA is often used to assess oxidative stress, and free radical damage to the body.

During the course of phagocytosis, macrophages produce superoxide (O}), by a reaction catalysed by NADPH oxidase (Fig.34.1). This O3 radical gets converted to H,O , and then to hypochlorous acid (HCIO). The superoxide radical along with hypochlorous ions brings about bactericidal action. This truly represents the beneficial affects of the free radicals generated by the body. A large amount of Oy is consumed by macrophages during _ their bactericidal function, a phenomenon referred to as respiratory burst. |t is estimated that about 10% of the Oy taken up by macrophages is utilized for the generation of free radicals.

1. Radiation therapy uring cobalt-60 or y-rays to destroy tumor tissue involves ROS (hydroxyl radicals, organic radicals). Thus, the biochemical basis of radiation therapy is localized oxidative stress, causing damage to all biomolecules. Most important is the damage to DNA that prevents tumor cell replication and tumor growth.
2. Sterilization of foods by _ irradiation destroying viral or bacterial contaminants also involves ROS.
3. Nuclear accidents explosions result in ionizing radiations. This causes oxidative damage to DNA and mutations which may lead to cancers.

Free radicals are highly reactive, and are capable of damaging almost all types of biomolecules (proteins, lipids, carbohydrates, nucleic acids). Free radicals beget free radicals i.e. generate free radicals from normal compounds which continues as a chain reaction.
Proteins : Free radicals cause oxidation of sulfhydryl groups, and modification of certain amino acids (e.g. methionine, cysteine, histidine, tryptophan, tyrosine). ROS may damage proteins by fragmentation, cross-linking and aggregation. The net result is that free radicals may often result in the loss of biological activity of proteins.
Lipids : Polyunsaturated fatty acids (PUFA) are highly susceptible to damage by free radicals. (see lipid peroxidation)
Carbohydrates At physiological pH, oxidation of monosaccharides (e.g. glucose) can produce HyO, and oxoaldehydes. It appears that the linkage of carbohydrates to proteins (glycation) increases the susceptibility of proteins to the attack by free radicals. This character assumes significance in diabetes mellitus where
protein glycation is associated with many health complications e.g. diabetic microangiopathy, diabetic nephropathy.
Nucleic acids : Free radicals may cause DNA strand breaks, fragmentation of bases and deoxyribose. Such damages may be associated with cytotoxicity and mutations.

As discussed above, free radicals are harmful to biomolecules, and in turn cells and tissues. Free radicals have been implicated in the causation and progress of several diseases.
Cardiovascular diseases (CHD) : Oxidized low density lipoproteins (LDL), formed by the action of free radicals, promote atherosclerosis and CHD.
Cancer : Free radicals can damage DNA, and cause mutagenicity and cytotoxicity, and thus play a key role in carcinogenesis. It is believed that ROS can induce mutations, and inhibit DNA repair process, that results in the inactivation of certain tumor suppressor genes leading to cancer. Further, free radicals promote biochemical and molecular changes for rapid growth of tumor cells.
Inflammatory diseases : Rheumatoid arthritis is a chronic inflammatory disease. The free radicals produced by neutrophils are the predominant causative agents. The occurrence of other inflammatory —disorders—chronic glomerulonephritis and ulcerative colitis is also due to the damages caused by ROS on the extracellular components (e.g. collagen, hyaluronic acid).
Respiratory diseases : Direct exposure of lungs to 100% oxygen for a long period (more than 24 hrs) is known to destroy endothelium and cause lung edema. This is mediated by free radicals. ROS are also responsible for adult respiratory distress syndrome (ARDS), a disorder characterized by pulmonary edema.
Cigarette smoke, as such, contains free radicals, and further it promotes the generation of more free radicals. The damages caused to lungs in the smokers are due to ROS.
Diabetes : Destruction of islets of pancreas due to the accumulation of free radicals is one of the causes for the pathogenesis of insulindependent diabetes mellitus.
Cataract : Increased exposure to oxidative stress contributes to cataract formation, which is mostly related to aging.
Male infertility : Free radicals are known to reduce sperm motility and viability, and thus may contribute to male infertility.
Aging process : Free radicals are closely associated with the various biochemical and morphological changes that occur during normal aging.
Other diseases : Free radicals play a key role in' Parkinson's disease, Alzheimer's disease, multiple sclerosis, liver cirrhosis, muscular dystrophy, toxemia of pregnancy etc.

To mitigate the harmful/damaging effects of free radicals, the aerobic cells have developed antioxidant defense mechanisms. A biological antioxidant may be defined as a substance (present in low concentrations compared to an oxidizable substrate) that significantly delays or inhibits oxidation of a substrate. Antioxidants may be considered as the scavengers of free radicals.
The production of free radicals and their neutralization by antioxidants is a normal bodily process. There are different ways of classifying antioxidants.

1. Preventive antioxidants that will block the initial production of free radicals e.g. catalase, glutathione peroxidase.
2. Chain breaking antioxidants that inhibit the propagative phase of lipid peroxidation e.g. superoxide dismutase, vitamin E, uric acid.

1. Plasma antioxidants e.g. B-carotene, ascorbic acid, bilirubin, uric acid, ceruloplasmin, transferrin.
2. Cell membrane antioxidants e.g. o-tocopherol.
3. Intracellular antioxidants e.g. superoxide
dismutase, catalase, glutathione peroxidase.

1. Enzymatic antioxidants e.g. superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase.
2. Non-enzymatic antioxidants
(a) Nutrient antioxidants e.g. carotenoids (B-carotene), a-tocopherol, ascorbic acid, selenium.
(b) Metabolic antioxidants e.g. glutathione, ceruloplasmin, albumin, bilirubin, transferrin, ferritin, uric acid

The antioxidant enzymes are truly the scavangers of free radicals. The major reactions of these enzymes are depicted in Fig.34.2, some highlights are given below.
Superoxide dismutase : It converts superoxide (O3) to hydrogen peroxide and O» (Fig.34.2A). This is the first line of defense to protect cells from the injurious effects of superoxide.
Catalase : Hydrogen peroxide, produced by superoxide dismutase, is metabolised by catalase (Fig.34.2B).
Glutathione peroxidase : It detoxifies HO to H,O, while reduced glutathione (G—SH) is converted to oxidized glutathione (GS—SG). The reduced glutathione can be regenerated by the enzyme glutathione reductase utilizing NADPH (Fig.34.2C). The hexose monophosphate shunt is the major source of NADPH.

Tocopherols (vitamin E) : Vitamin E is fat soluble, and among the tocopherols, o-tocopherol is biologically the most active. It is an antioxidant present in all cellular membranes, and protects against lipid peroxidation.
dismutase
a-Tocopherol can directly act on oxyradicals (e.g. O03, OH", singlet oxygen), and thus serves as an important chain breaking antioxidant.
Ascorbic acid (vitamin C) : It is a vitamin that participates in many normal metabolic reactions of the body. Ascorbic acid is an important watersoluble antioxidant in biological fluids. Vitamin C efficiently scavanges free radicals, and inhibits lipid peroxidation. It also promotes the regeneration of a-tocopherol (from atocopheroxy| radical produced during scavenging of ROS).
Carotenoids These are the natural compounds with lipophilic properties. About 500 different carotenoids have been identified, among them f-carotene is the most important. It can act as an antioxidant under low partial pressure of Oy. B-Carotene usually functions in association with vitamins C and E. Lycopene, a fat soluble pigment is a carotenoid. It is responsible for colour of certain fruits and vegetables (e.g. tomato). Lycopene possesses antioxidant property. Lutein and zeaxanthin are also carotenoid pigments that impart yellow or green colour to fruits and vegetables. These pigments can also serve as antioxidants.
Selenium : It is an essential trace element, and is proved to be a significant antioxidant. Selenium works with vitamin E in fighting free radicals. It is also required for the function of an important antioxidant enzyme, namely glutathione peroxidase.
a-Lipoic acid : It is vitamin-like compound, produced in the body, besides the supply from plant and animal sources. o-Lipoic acid plays a key role in recycling other important antioxidants such as ascorbic acid, a-tocopherol and glutathione.
Caffeine : Coffee contains flavonoids which are antioxidant in nature. Recent studies indicate that caffeine can directly act as an antioxidant.
Besides the above, there are many other important nutrient antioxidants, some of them are listed below

1 Coenzyme Qi of ubiquinone family
1 Proanthocyanidins of grape seeds
1 Catechins of green tea
1 Curcuminoids of turmeric
1 Quercetin of onions
In the Table 34.2, some important nutrient antioxidants and their dietary sources are given. Consumption of a variety of nutrient antioxidants is important, since each antioxidant targets certain types of damaging free radicals.

Glutathione : Reduced glutathione (GSH) plays a key role in the biological antioxidant enzyme system (See Fig.34.2C). GSH and HO) are the twin substrates for glutathione peroxidase. The reduced glutathione (GSH) gets regenerated from the oxidized glutathione

Vitamin E (tocopherols), Dietary Source = Unprocessed vegetable oils (cotton seed oil, peanut oil, sunflower oil) whole grains, leafy vegetables, legumes. Vitamin C (ascorbic acid), Dietary Source = Citrus fruits (oranges, grapes) gooseberry (amla), guava, green vegetables (cabbage, spinach), cauliflower, melons. E -Carotene (provitamin A), Dietary Source = Carrots, green fruits and vege- tables, spinach, turnip, apricots.. Lycopene, Dietary Source = Tomatoes, and their products (tomato sauce), papaya, pink guava, watermelon.. Leutein and zeaxanthin, Dietary Source = Egg yolk, fruits, green leafy vegetables, corn, green peas.. Selenium, Dietary Source = Sea foods, meats, organ meats, whole grains. D -Lipoic acid, Dietary Source = Red meat, liver, yeast. Coenzyme Q 10, Dietary Source = Organ meats (best heart), beef, chicken.. N-Acetylcysteine, Dietary Source = Available as supplement or drug.. Proanthocyanidins, Dietary Source = Grape seeds. Catechins, Dietary Source = Green tea. Curcuminoids, Dietary Source = Turmeric. Quercetin, Dietary Source = Onions, red wine, green tea. Ellagic acid, Dietary Source = Berries, walnuts, pomegranates. Hesperidin, Dietary Source = Citrus fruits (oranges), lemon.

+ Free radicals have been implicated in the causation and progress of several diseases e.g. atherosclerosis and CHD, cancer, respiratory diseases, aging.
+ The estimation of serum malondialdehyde is often used to assess oxidative stress and free radical damage to the body.
+ The respiratory burst of macrophages, accompanied by the generation of ROS (H202 and HCIO), brings about bactericidal action, and is beneficial to the body.
+ Dietary consumption of a variety of nutrient antioxidants (vitamins C and E, pB- carotene, lycopenes, Se, Plipoic acid) is desirable since each antioxidant targets certain types of damaging free radicals.
(GS—SG) through the participation of glutathione reductase and NADPH. It is sugested that the ability to synthesize GSH decreases as age advances, and this has been implicated in certain diseases e.g. cataract.
There are many more metabolic antioxidants of biological importance. A selected few of them are listed below
1 Uric acid, a powerful scavenger of singlet oxygen ('O,) and OH™ radicals.
1 Ceruloplasmin inhibits iron and copper dependent lipid peroxidation.
| Transferrin binds to iron and prevents ironcatalysed free radical formation.
1 Albumin can scavange the free radicals formed on its surface.
| Bilirubin protects the albumin bound free fatty acids from peroxidation.
1 Haptoglobin binds to free hemoglobin and prevents the acceleration of lipid peroxidation.

Free radicals damage biomolecules (proteins, nucleic acids, lipids), and are implicated in the causation and progress of several diseases (CHD, cancer, autoimmune diseases). To counter the action of free radicals, many protective antioxidant nutrients (vitamins E and C, B-carotene, selenium) are in use as dietary supplements.
Some recent studies show that antioxidant supplements may be beneficial to the people who are on deficient states, but not to all. On the other hand, some clinical trials indicate that supplementation of B-carotene and vitamin E is associated with increased mortality. However, this is controversial, and needs to be proved beyond doubt.
In any case, caution should be exercised in the supplementation of antioxidants, and their overuse should be avoided.

. Free radicals are the molecules or molecular species containing one or more unpaired electrons with independent existence. e.g. O05, HzOz, OH-, 10s.
.
ROS
are
constantly
formed
during
the
normal
cellular
(e.g.
lipid
peroxidation)
and
due
to
various
environmental
influences
(e.g.
ionizing
radiations).
. Free radicals are highly reactive and are capable of damaging almost all types of biomolecules (proteins, lipids, carbohydrates, nucleic acids), and have been implicated in the causation of many diseases e.g. cardiovascular diseases, cancer, inflammatory diseases.
. To mitigate the harmful effects of free radicals, the aerobic cells have developed antioxidant defense mechanisms-enzymatic antioxidants (superoxide dismutase, catalase) and non-enzymatic antioxidants (glutathione, Se, a-tocopherol, B-carotene).
nvironment constitutes the non-living (air, water, land, energy etc.) as well as the living (biological and social) systems surrounding man. Environmental biochemistry primarily deals with the metabolic (biochemical) responses and adaptations in man (or other organisms) due to the environmental factors.
A healthy environment is required for a healthy life which is however, not really possible or practicable. This is mainly because of the atmospheric (climatic) changes and environmental pollution.
Environmental biochemistry is a very vast subject. The basic concepts regarding the atmospheric changes and _— environmental pollution on humans are dealt with here.

The climatic changes include cold, heat etc.
The body makes every effort to maintain its normal temperature (despite cold and heat surroundings) for optimal physiological and biochemical functions.

Short-term exposure to cold causes shivering (mainly due to skeletal muscle) to produce extra heat. Heat is generated by the hydrolysis of ATP.

Chronic exposure to cold results in nonshivering phase which is characterized by several metabolic adaptations.
- | Energy metabolism : Heat generation by a process called chemical thermogenesis occurs in non-shivering phase. The foodstuffs undergo oxidation to generate heat at the expense of growth and other anabolic _ processes. Elevation in BMR, and increased intake of foods are observed.
|
Lipid metabolism
:
Stored fat
(triacylglycerol)
in the adipose tissue is mobilized to supply
free fatty acids for oxidation and production of energy. Brown edipose tissue, particularly in neonatal life, significantly contributes to thermogenesis.
- 1 Hormonal changes : Thyroxine, a hormone closely associated with energy metabolism, is elevated. Further, corticosteroids are increased on exposure to cold.

There is a continuous generation of heat by the body due to the ongoing biochemical processes, referred to as metabolic heat. This heat has to be exchanged with the environment to maintain a constant body temperature. On exposure to heat in surroundings, as happens in summer, the body is subjected to an uncomfortable situation (since temperature of the surroundings is much higher than that of the body). However, heat is still lost from the body through sweating and evaporation. Normally, the body (thermoregulation) gets acclimatized to higher temperature within 3-5 days.
Heat stroke : It is characterized by the failure of the heat regulatory system (thermoregulation) of the body. The manifestations of heat stroke include high body temperature, convulsions, partial (some times total) loss of consciousness. In extreme cases, heat stroke may cause irreversible damage to brain. The treatment for the heat stroke involves rapid cooling of the body.
The milder form of heat stroke is referred to as heat syncope. Although the body temperature is not raised much in this condition, the blood pressure falls and the person may collapse suddenly. Heat syncope is easily reversible.

Environmental pollution may be regarded as the addition of extraneous (foreign) materials to air, water or land which adversely affects the quality of life. Pollution may be caused by physical, chemical or biological processes.
The term pollutant refers to a substance which increases in quantity due to human activity and adversely affects the environment (e.g. carbon monoxide, sulfur dioxide, lead). A substance which is not present in nature but released during human activity is the contaminant (e.g. methyl isocyanate, DDT, malathion). A contaminant however, is regarded as a pollutant when it exerts detrimental effects. Environmental pollution may be considered in different ways—industrial pollution; agricultural pollution; pollution due to gaseous wastes, liquid wastes and solid wastes. Environmental pollution with reference to air, water and foodstuffs is briefly discussed.

The major components of air include nitrogen (78.1%), oxygen (20.93%) and carbon dioxide (0.03%), along with water vapour and suspended particles. The rapid growth of industries coupled with changing lifestyles of man (urbanization, smoking, use of motor vehicles etc.) largely contribute to air pollution. The major chemical constituents of air pollution are sulfur dioxide, oxides of carbon (CO, and CO), oxides of nitrogen, hydrocarbons and _ particulates. The biochemical affects of air pollution are described.

Sulfur dioxide (SO) is the most dangerous pollutant gas to man. Industrial activities such as burning of coal and oil emit large quantities of SO>.
Sulfur dioxide pollution primarily affects respiratory system in man. Irritation of the respiratory tract and increasing airway resistance (breathing difficulty) are observed. Lung tissue may get damaged due to acidic pH. Further, dipalmity! lecithin, the phospholipid acting as the lung surfactant, gets affected. Continuous exposure to SO) (> 1 ppm) for several days causes bronchitis and in some individuals lung cancer. Atmospheric SOy when dissolved in rain water becomes very acidic (acid rain) damaging soil, plants and vegetables. Exposure of plants to SO» destroys leaves.

Carbon monoxide (CO) is mostly produced by incomplete combustion of fuel or carboncontaining compounds. Automobiles, aircrafts, rail engines and burning of coal in factories contribute to CO pollution.
Carbon monoxide combines with hemoglobin to form carboxyhemoglobin (Refer Chapter 10). This causes a drastic reduction in the supply of Oy to tissues. Ata CO concentration around 1 ppm, impairment in mental performance and visual perception take place. With a further increase in CO level, headache, dizziness and loss of consciousness occur. Death may be inevitable in persons exposed to above 750 ppm of CO.

Carbon dioxide (CO ), constituting only a fraction (0.03%) of the atmospheric gases, plays a significant role in controlling the climate. This is done by trapping the heat radiation from the earth's surface. Without the presence of CO, the earth would be as cold as moon!
Carbon dioxide is often referred to as greenhouse gas. The term greenhouse effect refers to an elevation in CO near earth's surface that traps sunlight and increases atmospheric _ temperature. Deforestation, burning of coal, oils etc., elevate atmospheric CO), resulting in greenhouse effect. Hence the global propaganda for increased plantation of trees!
Fortunately, marginal variations in atmospheric CO, are tolerated by the cells. The body gets adapted to prolonged exposure to higher concentrations of CO, (even upto 1%) with minor alterations in electrolyte balance.

Nitrogen dioxide (NO») like carbon monoxide (CO), combines with hemoglobin and reduces the supply of Oy to the tissues. NO» is more harmful to human health than CO. It is fortunate that the atmospheric concentration of NO, is relatively lower.
with
Nitrogen dioxide (in the form of HNO3) along SO, (as H»SO4) contributes to acid rain.

Many hydrocarbons polluting the environment affect human life. The aromatic hydrocarbons cause irritation to injuries.

The solid dust particles suspended in the atmosphere constitute particulates. The sources of particulates are grinding, spraying, erosion, smoking etc.
The particulates have ill-affects on humans. These include interference in respiratory function (coughing, sneezing) and toxicity caused by the absorption particulate chemicals. Further, the dust particles carry microorganisms and other infective agents to spread diseases.

Ozone is formed from atmospheric oxygen during high energy radiations of electrical discharges. This ozone forms a layer above the earth's surface (15-35 km). It absorbs harmful ultraviolet radiations of sun which would otherwise cause skin diseases and mutations, besides increasing the temperature of earth.
In recent years, a decrease in the ozone layer is observed due to chemical pollution in the air. Nitrogen oxides (released from engines of aeroplanes) and chlorofluoro carbons (used in refrigerators and air conditioners) deplete the ozone layer.

Water is the most predominant constituent of living matter. The very existence of life is unimaginable without water.
As such, pure water does not exist in nature. The available water contains dissolved gases, minerals and some suspended particles. Pollution of water occurs due to waste disposal from industries, agriculture and municipalities. The pollutants may be organic, inorganic, sediments, radioactive, thermal etc., in nature.

The organic pollutants include agents carrying water borne diseases, oxygen demanding wastes and organic chemicals.

Several
pathogenic
organisms
find
their
entry
into
water
and
cause
diseases.
The
water
borne
disease
include
typhoid,
paratyphoid,
cholera,
amoebiasis,
giardiasis
and
infectious
hepatitis.
These
diseases
can
be
prevented
by
disinfection
techniques
employed
for
the
treatment
of
water.

Sewage, and wastes from industries and agriculture provide good nutrients for algae. As the algae grow utilizing the wastes, oxygen depletion occurs. This phenomenon of water deoxygenation is technically referred to as eutrophication. As a consequence _ of eutrophication, fish and other acquatic animals die (due to lack of O), causing foul smell.

The organic chemical pollutants of water include pesticides and several synthetic compounds (detergents, paints, _ plastics, pharmaceuticals, food additives etc.)

Pesticides
is
a
broad
term
used
for
insecticides,
herbicides,
_
fungicides
—_
and

rodenticides. Based on their structure, pesticides
are classified as follows.
(a) Chlorinated hydrocarbons : e.g.
aldrin,
dieldrin, endrin, dichlorodiphenyl trichloro- ethane (DDT).
(b) Organophosphates
diazinon.
e.g.
malathion,
(c) Carbamates e.g. baygon, carbaryl (sevin)
(d)
Chlorophenoxy
e.g.
2,4-dichlorophenoxy
acetic acid.
The use of pesticides has helped in controlling certain diseases (malaria, typhus), besides boosting food production. However, pesticides pollute water and cause several health complications to humans, besides damaging acquatic life.
Dichloro-diphenyl trichloroethane (DDT) is a widely used pesticide to control cotton and peanut pests, besides malaria. However, continuous use of DDT leads to its accumulation in foods causing ill effects (hence banned in some countries like USA).
DDT, being fat soluble, accumulates in the adipose tissue and is not excreted. Thus, its concentration in the body goes on increasing. DDT causes nervous irritability, muscle twitching and convulsions.
Aldrin and dialdrin are also fat soluble and
their effects on humans are comparable with that
of
DDT.
+ The body makes every effort to maintain its normal temperature, despite cold and heat surroundings, for optimal physiological and biochemical functions.
+ Failure of heat regulatory system (thermoregulation) leads to heat stroke characterized by high body temperature, convulsions etc.
+ Sulfur dioxide (SOg) is the most dangerous industrial pollutant gas to man. It primarily affects the respiratory system, and may result in bronchitis, and even lung cancer.
+ Carbon monoxide (CO) combines with hemoglobin to form carboxyHb. This reduces O2 supply to tissues.
+ Pollution of water with pathogenic organisms causes many diseases e.g. typhoid, cholera, amoebiasis.
+ Lead toxicity affects central nervous system—learning disabilities, mental retardation etc.
+ Radioactive pollution may lead to gene mutations, cancer etc.

Organophosphates and carbamates are powerful neurotoxic agents. They prevent the transmission of nerve impulse by inhibiting the enzyme cholinesterase.

Heavy metals (lead, mercury, cadmium, aluminium, arsenic etc.) are the most dangerous among the inorganic pollutants.

Lead is the most common inorganic pollutant found in water, air, foods and soils. The sources of lead pollution include petrol, gasoline, paints, cigarettes, news papers, lead pipes and xerox copies. The plasma concentration of > 25 ug/dl in adults and > 10 ug/dl in children results in toxic manifestations.
The principal target of lead toxicity is central nervous system. In the growing children, Pb causes learning disabilities, behavioural changes (hyperexcitability) and mental retardation. In adults, confusion, irritability, abdominal colic and severe anemia are associated with lead toxicity.
Lead inhibits several enzymes, particularly, 5-aminolevulinate (ALA) — synthase, © ALA dehydratase and ferrochelatase of heme synthesis (Refer Chapter 10 also). This results in severe anemia. There has been an increasing awareness worldover on the toxic manifestations of lead. This has lead to the supply of unleaded petrol in most countries.

Mercury is a common industrial (plastic, paints, electrical apparatus, fungicides) pollutant. Acute mercuric poisoning causes — gastritis, vomiting and pulmonary edema. Chronic manifestations of Hg include emotional changes, loss of memory and other neuropsychiatric disturbances. In addition, deposition of mercuric salts may cause renal failure.
Organic mercuric poisoning is commonly referred to as minamata disease (as it first occurred in Minamata, Japan in 1953-60 by consuming fish containing methyl mercury, as industrial pollutant).

The outbreak of cadmium toxicity was reported in Japan in the form of itai itai or ouch disease. Cadmium poisoning causes fragile bones, anemia, bone marrow disorders and kidney damage. Biochemically, cadmium replaces zinc and adversely influences several metabolic reactions.

The sources of aluminium include cooking vessels, building materials, food additives and cosmetics. Aluminium toxicity is associated with Alzheimer's disease, anemia and osteomalacia.

Arsenic, commonly found in = many insecticides and fungicides, is toxic to the body. Arsenic binds with-SH groups of several enzymes and inhibits biochemical reactions e.g. pyruvate dehydrogenase. Further, arsenic causes coagulation of proteins and blockage of ATP generation (functions as an uncoupler).

The unwanted sound is noise, which is a major urban environmental pollutant. Man can tolerate noise upto 100 decibels (speaking — 60 decibels; telephone bell 70 decibels; motor cycle 110 decibels; rockets 170 decibels). A noise above 150 decibels is uncomfortable.
The affects of noise pollution include headache, increased blood pressure, irritability, neuromuscular tension, confusion, disturbed vision and digestion, depression and loss of hearing.

The pollution due to radioactive substances is the most dangerous to human life. The health hazards of radioactive pollution include gene mutations, cancer, destruction of living cells etc.

The foodstuffs consumed by humans contain several toxic compounds which may be either normally present or enter foodstuffs during the course of cultivation, processing or storage.

Neurotoxins : Kesari dal (Lathyrus sativus) is a pulse grown in some parts of Madhya Pradesh, Bihar and Uttar Prodesh. Excessive consumption of kesari dal causes paralysis of lower limbs referred to as lathyrism. This is due to a neurotoxin namely — B-oxalylaminoalanine (BOAA). BOAA damages upper motor neurons, and inhibits the enzyme lysyl oxidase (reduces collagen cross-linking). Cooking of kesari dal 2-3 times and removal of the supernatant water will eliminate the toxin.
Protease inhibitors : Certain legumes (soya bean, peanut) contain inhibitors of protease enzymes particularly trypsin. Normally, protease inhibitors are destroyed by cooking. However, partial cooking does not totally destroy them. In such a case, protease inhibitors can inhibit digestion and proteins.
Goitrogens These compounds prevent uptake and utilization of iodine by thyroid gland. Goitrogens are found in cabbage and turnips (thioglycosides), mustard and rape seed oils (thiocyanates), ground nuts and almonds (polyphenolic glycosides).
Biogenic amines Bananas and cheese contain biogenic amines namely histamine, tryptamine, tyramine serotonin and epinephrine. In normal metabolism, they are degraded by monoamine oxidase (MAO). However, in persons taking MAO—inhibitors, the foodstuffs with amines may cause hypertension.
Anti-vitamins : Avidin of raw egg is a good example of anti-vitamin of biotin.

The foodstuffs may get polluted with several toxic chemicals which might occur during cultivation, processing or storage.
Cultivation
:
Pesticides and
other unnatural
chemicals used during cultivation do find an entry into the foodstuffs. It is fortunate that most of these chemicals can be removed by peeling the outer layers of vegetables and fruits, besides repeated washings.
Processing : Defects in freezing, and packing provide a suitable environment for the growth of several organisms which release toxic products e.g. milk contamination by Salmonella.
Several food additives are in use for preservation and enchancing flavour. Not all of them are safe e.g. aniline dyes used as colouring agents are carcinogenic; sweetening agent cyclamate may cause bladder cancer.
Storage : Contamination of stored foods occurs mostly due to fungal infections. Aflatoxins are produced by Aspergillus favus when ground nuts or coconuts are stored in moist conditions. Aflatoxins are hepatotoxic and carcinogenic.
The group of chemicals that cause cancer in man and animals are collectively referred to as carcinogens (Refer Chapter 37). Environmental pollution is undoubtedly associated with increased risk of cancer. The topic 'cancer may be considered as a part of environmental biochemistry for learning purpose.

1., 1 = Environmental biochemistry deals with the biochemical responses and adaptations in man (and other organisms) due to environmental factors.. 2., 1 = The atmospheric (climatic) changes like cold and heat influence the body. Several metabolic adaptations occur to overcome the adverse affects.. 3., 1 = The major chemical constituents of air pollution include SO 2 , CO, CO 2 and oxides of nitrogen. Among these, sulfur dioxide is the most dangerous.. 4., 1 = Water pollution occurs mainly due to waste disposal from industries, agriculture and municipalities. The pollutants may be organic (pathogenic organisms, pesticides), or inorganic (lead, mercury).. 5., 1 = The foodstuffs consumed by humans may contain several toxic compounds. These may be normally present (e.g. BOAA causing lathyrism) or enter the foodstuffs during the course of cultivation (e.g. pesticides), or storage (e.g. aflatoxins).
De mellitus is the third leading cause of death (after heart disease and cancer) in many developed countries. It affects about 6 to 8% of the general population. The complications of diabetes affect the eye, kidney and nervous system. Diabetes is a major cause of blindness, renal failure, amputation, heart attacks and stroke. (The term diabetes, whenever used, refers to diabetes mellitus. It should, however, be noted that diabetes insipidus is another disorder characterized by large volumes of urine excretion due to antidiuretic hormone deficiency).
Diabetes mellitus is a clinical condition characterized by increased blood glucose level (hyperglycemia) due to insufficient or inefficient (incompetent) insulin. In other words, insulin is either not produced in sufficient quantity or inefficient in its action on the target tissues. As a consequence, the blood glucose level is elevated which spills over into urine in diabetes mellitus (Greek : diabetes—a siphon or running through; mellitus—sweet).
An important feature of diabetes is that the body cells are starved of glucose despite its very high concentration around i.e. scarcity in plenty. For a comprehensive understanding of diabetes, the relevant hormones, namely insulin and glucagon, homeostasis of blood glucose, besides the biochemical aspects of diabetes, are discussed in this chapter.

Insulin is a polypeptide hormone produced by the f-cells of islets of Langerhans of pancreas. |t has profound influence on the metabolism of carbohydrate, fat and protein. Insulin is considered as anabolic hormone, as it promotes the — synthesis of — glycogen, triacylglycerols and proteins. This hormone has been implicated in the development of diabetes mellitus.
Insulin occupies a special place in the history of biochemistry as well as medicine. Insulin was the first hormone to be isolated, purified and
synthesized; first hormone to be sequenced; first hormone to be produced by recombinant DNA technology.

Human insulin (mol. wt. 5,734) contains 57 amino acids, arranged in two polypeptide chains. The chain A has 21 amino acids while B has 30 amino acids. Both are held together by two interchain disulfide bridges, connecting Az to Bz and Ajg to Byg. In addition, there is an intrachain disulfide link in chain A between the amino acids 6 and 11.

Insulin is produced by the B-cells of the islets of Langerhans of pancreas. The gene for this protein synthesis is located on chromosome 11. The synthesis of insulin involves two precursors, namely preproinsulin with 108 amino acids (mol. wt. 11,500) and proinsulin with 86 amino acids (mol. wt. 9,000). They are sequentially degraded (Fig.36.1) to form the active hormone insulin and a connecting peptide (C-peptide). Insulin and C-peptide are produced in equimolar concentration. C-peptide has no_ biological activity, however its estimation in the plasma serves as a useful index for the endogenous production of insulin.
In the B-cells, insulin (and also proinsulin) combines with zinc to form complexes. In this form, insulin is stored in the granules of the cytosol which is released in response to various stimuli (discussed below) by exocytosis.

About 40-50 units of insulin is secreted daily by human pancreas. The normal insulin concentration in plasma is 20-30 wU/ml. The important factors that influence the release of insulin from the B-cells of pancreas are discussed hereunder.
1. Factors stimulating insulin secretion These include glucose, amino acids and gastrointestinal hormones.
- 1 Glucose is the most important stimulus for insulin release. The effect is more predominant when glucose is administered orally (either direct or through a carbohydrate-rich
Fig. 36.1 : Formation of insulin from preproinsulin.
meal). A rise in blood glucose level is a signal for insulin secretion.
Amino acids induce the secretion of insulin. This is particularly observed after the ingestion of protein-rich meal that causes transient rise in plasma amino acid concentration. Among the amino acids, arginine and leucine are potent stimulators of insulin release.

- 1 Gastrointestinal hormones (secretin, gastrin, pancreozymin) enhance the secretion of insulin. The GIT hormones are released after the ingestion of food.

2. Factors inhibiting insulin secretion : Epinephrine is the most potent inhibitor of insulin release. In emergency situations like stress, extreme exercise and trauma, the nervous system stimulates adrenal medulla to release epinephrine. Epinephrine suppresses — insulin release and promotes energy metabolism by mobilizing energy-yielding compounds—glucose from liver and fatty acids from adipose tissue.

In the plasma, insulin has a normal half-life of 4-5 minutes. This short half-life permits rapid metabolic changes in accordance to the alterations in the circulating levels of insulin. This is advantageous for the therapeutic purposes. A protease enzyme, namely insulinase (mainly found in liver and kidney), degrades insulin.
Insulin plays a key role in the regulation of carbohydrate, lipid and protein metabolisms (Table 36.1). Insulin exerts anabolic and anticatabolic influences on the body metabolism. 1. Effects on carbohydrate metabolism : In a normal individual, about half of the ingested glucose is utilized to meet the energy demands of the body (mainly through glycolysis). The other half is converted to fat (~40%) and glycogen (~ 10%). This relation is severely impaired in insulin deficiency. Insulin influences glucose metabolism in many ways. The net effect is that insulin lowers blood glucose level (hypoglycemic effect) by promoting _ its utilization and storage and by inhibiting its production.
- . Effect on glucose uptake by tissues : Insulin is required for the uptake of glucose by muscle (skeletal, cardiac and smooth), adipose tissue, leukocytes and mammary glands. Surprisingly, about 80% of glucose uptake in the body is
not dependent on insulin. Tissues into which glucose can freely enter include brain, kidney, erythrocytes, retina, nerve, blood vessels and intestinal mucosa. As regards liver, glucose entry into hepatocytes does not require insulin. However, insulin stimulates glucose utilization in liver and, thus, indirectly promotes its uptake.
. Effect on glucose utilization : Insulin increases glycolysis in muscle and liver. The activation as well as the quantities of certain key enzymes of glycolysis, namely glucokinase (not hexokinase) phosphofructokinase and pyruvate kinase are increased by insulin. Glycogen production is enhanced by insulin by increasing the activity of glycogen synthetase.
| Effect on glucose production Insulin decreases gluconeogenesis by suppressing the enzymes pyruvate carboxylase, phosphoenol pyruvate carboxykinase and glucose 6phosphatase. Insulin also inhibits glycogenolysis by inactivating the enzyme glycogen phosphorylase.

2. Effects on lipid metabolism : The net effect of insulin on lipid metabolism is to reduce the release of fatty acids from the stored fat and decrease the production of ketone bodies. Among the tissues, adipose tissue is the most sensitive to the action of insulin.
| Effect on lipogenesis : Insulin favours the synthesis of triacylglycerols from glucose by providing more glycerol 3-phosphate (from glycolysis) and NADPH (from HMP._ shunt). Insulin increases the activity of acetyl CoA carboxylase, a key enzyme in fatty acid synthesis.
| Effect on lipolysis : Insulin decreases the activity of hormone-sensitive lipase and thus reduces the release of fatty acids from stored fat in adipose tissue. The mobilization of fatty acids from liver is also decreased by insulin. In this way, insulin keeps the circulating free fatty acids under a constant check.
1 Effect on ketogenesis Insulin reduces ketogenesis by decreasing the activity of HMG CoA synthetase. Further, insulin promotes the
utilization of acetyl CoA for oxidation (Krebs cycle) and lipogenesis. Therefore, the availability of acetyl CoA for ketogenesis, in the normal circumstances, is very low.
- 3. Effects on protein metabolism : Insulin is an anabolic hormone. It stimulates the entry of amino acids into the cells, enhances protein synthesis and reduces protein degradation.
Besides the metabolic effects described above, insulin promotes cell growth and replication. This is mediated through certain factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF) and prostaglandins.

It is now recognized that insulin binds to specific plasma membrane receptors present on the target tissues, such as muscle and adipose. This results in a series of reactions ultimately leading to the biological action. Three distinct mechanisms of insulin action are known. One concerned with the induction of transmembrane signals (signal transduction), second with the glucose transport across the membrane and the third with induction of enzyme synthesis.
1. Insulin receptor mediated signal transduction
Insulin receptor : It is a tetramer consisting of 4 subunits of two types and is designated as QB». The subunits are in the glycosylated form. They are held together by disulfide linkages. The a-subunit (mol. wt. 135,000) is extracellular and it contains insulin binding site. The B-subunit (mol. wt. 95,000) is a transmembrane protein which is activated by insulin. The cytoplasmic domain of B-subunit has tyrosine kinase activity.
The insulin receptor is synthesized as a single polypeptide and cleaved to a and f subunits which are then assembled. The insulin receptor has a half-life of 6-12 hours. There are about 20,000 receptors per cell in mammals.
Signal transduction : As the hormone insulin binds to the receptor, a conformational change is induced in the a-subunits of insulin receptor. This results in the generation of signals which
Fig.
36.2
:
Insulin receptor
mediated signal
transduction
(IRS—Insulin receptor
substrate).
are transduced to f-subunits. The net effect is that insulin binding activates tyrosine kinase activity of intracellular B-subunit of insulin receptor. This causes the autophosphorylation of tyrosine residues on B-subunit. It is believed that receptor tyrosine kinase also phosphorylates insulin receptor substrate (IRS). The phosphorylated IRS, in turn, promotes activation of other protein kinases and phosphatases, finally leading to biological action (Fig.36.2).

2. Insulin-mediated glucose transport : The binding of insulin to insulin receptors signals the translocation of vesicles containing glucose transporters from intracellular pool to the plasma membrane. The vesicles fuse with the membrane recruiting the glucose transporters. The glucose transporters are responsible for the insulin—mediated uptake of glucose by the cells. As the insulin level falls, the glucose transporters move away from the membrane to the intracellular pool for storage and_ recycle (Fig.36.3).
3. Insulin mediated enzyme synthesis Insulin promotes the synthesis of enzymes such as glucokinase, | phosphofructokinase — and pyruvate kinase. This is brought about by increased transcription (mRNA _ synthesis), followed by translation (protein synthesis).
Fig. 36.3 : Insulin mediated glucose transport.

Glucagon, secreted by a-cells of the pancreas, opposes the actions of insulin. It is a polypeptide hormone composed of 29 amino acids (mol. wt. 3,500) in a single chain. Glucagon is actually synthesized as proglucagon (mol. wt. 9,000) which on sequential degradation releases active glucagon. Unlike insulin, the amino acid sequence of glucagon is the same in all mammalian species (so far studied). Glucagon has a short half-life in plasma i.e. about 5 minutes.

The secretion of glucagon is stimulated by low blood glucose concentration, amino acids derived from dietary protein and low levels of epinephrine. Increased blood glucose level markedly inhibits glucagon secretion.

Glucagon influences carbohydrate, lipid and protein metabolisms. In general, the effects of this hormone oppose that of insulin.
1. Effects on carbohydrate metabolism Glucagon is the most potent hormone that enhances the blood glucose level (hyperglycemic). Primarily, glucagon acts on liver to cause increased synthesis of glucose (gluconeogenesis) and enhanced degradation of glycogen (glycogenolysis). The actions of glucagon are mediated through cyclic AMP (Chapter 13).
2. Effects on lipid metabolism : Glucagon promotes fatty acid oxidation resulting in energy production and ketone body synthesis (ketogenesis).
3. Effects on protein metabolism : Glucagon increases the amino acid uptake by liver which, in turn, promotes gluconeogenesis. Thus, glucagon lowers plasma amino acids.

Glucagon binds to the specific receptors on the plasma membrane and acts through the mediation of cyclic AMP, the second messenger. The details are given elsewhere (Chapter 19).

Glucose is carbohydrate currency of the body. An adult human body contains about 18 g free glucose. This amount is just sufficient to meet the basal energy requirements of the body for one hour! The liver has about 100 g stored glycogen. Besides this, it is capable of producing about 125-150 mg glucose/minute or 180-220 9/24 hrs.
Expression of glucose concentration : In most developed countries, plasma glucose (instead of blood glucose) is estimated and expressed as SI units (mmol/l). This is not however so, in developing countries for practical reasons. It may be noted that the plasma concentration of glucose is slightly higher (about 15%) than blood glucose. Further, a glucose concentration of 180 mg/dl (plasma or blood) corresponds to 10 mmol/l. In this book, expression of blood glucose as mg/dl is more frequently used.
A healthy individual is capable of maintaining the blood glucose concentration within a narrow range. The fasting blood glucose level in a postabsorptive state is 70-100 mg/dl (plasma glucose 80-120 mg/dl). Following the ingestion of a carbohydrate meal, blood glucose may rise to 120-140 mg/dl. The fasting blood glucose value is comparatively lower in ruminant animals (sheep 30-40 mg/dl; cattle 50-60 mg/dl), while it is higher in birds (250-300 mg/dl).
The term hyperglycemia refers to an increase in the blood glucose above the normal level. Hypoglycemia represents a decreased blood glucose concentration. Excretion of glucose in urine is known as glycosuria. The concentration of blood glucose is dependent on the quantity of glucose that enters the circulation from various sources (dietary carbohydrates, glycogenolysis, gluconeogenesis etc.) and the amount that is utilized for different metabolic purposes (glycolysis, glycogenesis, fat synthesis etc.) as illustrated in
Fig.36.4.
Fig. 36.4 : Overview of blood glucose homeostasis.

1. Dietary sources The dietary carbohydrates are digested and absorbed as monosaccharides (glucose, fructose, galactose etc.). The liver is capable of converting fructose and _ galactose into glucose, which can readily enter blood.
2. Gluconeogenesis : The degradation of glycogen in muscle results in the formation of lactate. Breakdown of fat in adipose tissue will produce free glycerol and propionate. Lactate, glycerol, propionate and some amino acids are good precursors for glucose synthesis (gluconeogenesis) that actively occurs in liver and kidney. Gluconeogenesis continuously adds glucose to the blood. Cori cycle is responsible for the conversion of
muscle lactate
to glucose
in liver.
3. Glycogenolysis : Degradation of glycogen in liver produces free glucose. This is in contrast to muscle glycogenolysis where glucose is not formed in sufficient amount due to lack of the enzyme glucose 6-phosphatase. However, the contribution of liver glycogenolysis to blood glucose is rather limited and can meet only the short intervals of emergency. This is due to the limited presence of glycogen in liver. An adult liver (weighing about 1.5 kg) can provide only 40-50 g of blood glucose from glycogen, that can last only for a few hours to meet the body requirements.
In the Fig.36.5, the sources of blood glucose during a normal day (24 hours) are given. Glucose is primarily derived from glycogenolysis (of hepatic glycogen) between the meals. Gluconeogenesis becomes a predominant source of glucose in late night (after depletion of hepatic

Fig. 36.6 : Hormonal regulation of blood glucose.
glycogen). During day time, gluconeogenesis may be more or less active, depending on the frequency of consumption of snacks, coffee, tea, fruit juices etc.

Certain tissues like brain, erythrocytes, renal medulla and bone marrow are_ exclusively dependent on glucose for their energy needs. When the body is at total rest, about two-thirds of the blood glucose is utilized by the brain. The remaining one-third by RBC and skeletal muscle. A regular supply of glucose, by whatever means it may be, is absolutely required to keep the brain functionally intact.
The different metabolic pathways (glycolysis, glycogenesis, HMP shunt etc.) responsible for the utilization of blood glucose are already discussed (Chapter 13). The synthesis of fat from acetyl CoA and_ glycerol is described in lipid metabolism (Chapter 14).
Kidney plays a special role in the homeostasis of blood glucose. Glucose is continuously filtered by the glomeruli, reabsorbed and returned to the blood. If the level of glucose in blood is above 160-180 me/dl, glucose is excreted in urine (glycosuria). This value (160-180 mg/dl) is referred to as_ renal threshold for glucose. The maximum ability of the renal tubules to reabsorb glucose per minute is known as tubular maximum for glucose (TmG). The value for TmG is 350 mg/minute.

Hormones play a significant role in the regulation of blood glucose concentration (Figs.36.6 and 36.7). Primarily, insulin lowers blood glucose level (hypoglycemic) while the rest of the hormones oppose the actions of insulin (hyperglycemia).
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Insulin : Insulin is produced by B-cells of the islets of Langerhans in response to hyperglycemia (elevated blood glucose level). Some amino acids, free fatty acids, ketone bodies, drugs such as tolbutamide also cause the secretion of insulin.
Insulin is basically a hypoglycemic hormone that lowers in blood glucose level through various means. It is an anti-diabetogenic hormone. For details of insulin action on glucose homeostasis refer metabolic effects of insulin (carbohydrate metabolism) in this chapter.
Glucagon : Glucagon is synthesized by a-cells of the islets of Langerhans of the pancreas. Hypoglycemia (low blood glucose _ level) stimulates its production. Glucagon is basically involved in elevating blood — glucose concentration. It enhances gluconeogenesis and glycogenolysis.
Epinephrine : This hormone is secreted by adrenal medulla. It acts both on muscle and liver to bring about glycogenolysis by increasing phosphorylase activity. The end product is glucose in liver and lactate in muscle. The net outcome is that epinephrine increases blood glucose level.
Thyroxine : It is a hormone of thyroid gland. It elevates blood glucose level by stimulating hepatic glycogenolysis and gluconeogenesis.
Glucocorticoids These hormones are produced by adrenal cortex. Glucocorticoids stimulate protein metabolism and_ increase gluconeogenesis (increase the activities of enzymes—glucose 6-phosphatase and fructose 1,6-bisphosphatase). The glucose utilization by extrahepatic tissues is inhibited by glucocorticoids. The overall effect of glucocorticoids is to elevate blood glucose concentration.

Growth hormone and adrenocorticotropic hormone (ACTH) : The anterior pituitary gland secretes growth hormone and ACTH. The uptake of glucose by certain tissues (muscle, adipose tissue etc.) is decreased by growth hormone. ACTH decreases glucose utilization. The net effect of both these hormones is hyperglycemic.
[In Fig.36.7, regulation of blood glucose level by hormones is depicted as a game of tug of war with elephant (representing insulin) on one side and the other animals (as rest of the hormones) on the opposite side. This is just an illustration (a cartoon) for a quick understanding of glucose homeostasis].

When the blood glucose concentration falls to less than 45 mg/dl, the symptoms of hypoglycemia appear. The manifestations include headache, anxiety, confusion, sweating, slurred speech, seizures and coma, and, if not corrected, death. All these symptoms are directly and indirectly related to the deprivation of glucose supply to the central nervous system (particularly the brain) due to a fall in blood glucose level.
The mammalian body has developed a well regulated system for an efficient maintenance of blood glucose concentration (details already described). Hypoglycemia, therefore, is not commonly observed. The following three types of hypoglycemia are encountered by physicians.
1. Post-prandial hypoglycemia : This is also called reactive hypoglycemia and is observed in subjects with an elevated insulin secretion following a meal. This causes transient hypoglycemia and is associated with mild symptoms. The patient is advised to eat frequently rather than the 3 usual meals.
2. Fasting hypoglycemia : Low blood glucose concentration in fasting is not very common. However, fasting hypoglycemia is observed in patients with pancreatic B-cell tumor and hepatocellular damage.
3. Hypoglycemia due to alcohol intake : In some individuals who are starved or engaged in prolonged exercise, alcohol consumption may cause hypoglycemia. This is due to the accumulation of NADH (during the course of alcohol metabolism by alcohol dehydrogenase) which diverts the pyruvate and oxaloacetate (substrates of gluconeogenesis) to form,
respectively, lactate and malate. The net effect is that gluconeogenesis is reduced due to alcohol consumption.
4. Hypoglycemia due to insulin overdose : The most common complication of insulin therapy in diabetic patients is hypoglycemia. This is particularly observed in patients who are on intensive treatment regime.
5. Hypoglycemia in premature infants Premature and underweight infants have smaller stores of liver glycogen, and are susceptible to hypoglycemia.

Diabetes mellitus is a metabolic disease, more appropriately a disorder of fuel metabolism. It is mainly characterized by hyperglycemia that leads to several long term complications.
Diabetes mellitus is broadly divided into 2 groups, namely insulin-dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM). This classification is mainly based on the requirement of insulin for treatment.

IDDM, also known as type I diabetes or (less frequently) juvenile onset diabetes, mainly occurs in childhood (particularly between 12-15 yrs age). IDDM accounts for about 10 to 20% of the known diabetics. This disease is characterized by almost total deficiency of insulin due to destruction of B-cells of pancreas. The B-cell destruction may be caused by drugs, viruses or autoimmunity. Due to certain genetic variation, the B-cells are recognized as non-self and they are destroyed by immune mediated injury. Usually, the symptoms of diabetes appear when 80-90% of the B-cells have been destroyed. The pancreas ultimately fails to secrete insulin in response to glucose ingestion. The patients of IDDM require insulin therapy.

Recent research findings on NIDDM suggest that increased levels of tumor necrosis factor-a (TNF-a) and resistin, and reduced seretion of adiponectin by adipocytes of obese people cause insulin resistance (by impairing insulin receptor function).
The comparison between IDDM and NIDDM is given in Table 36.2. (For metabolic syndrome refer p-326)

The diagnosis of diabetes can be made on the basis of individual's response to oral glucose load, the oral glucose tolerance test (OGTT).

The person should have been _ taking carbohydrate-rich diet for at least 3 days prior to the test. All drugs known to influence carbohydrate metabolism should be discontinued (for at least 2 days). The subject should avoid strenuous exercise on the previous day of the test. He/she should be in an overnight (at least 10 hr) fasting state. During the course of GTT, the person should be comfortably seated and should refrain from smoking and exercise.

Glucose tolerance test should be conducted preferably in the morning (ideal 9 to 11 AM). A fasting blood sample is drawn and_ urine collected. The subject is given 75 g glucose orally, dissolved in about 300 ml of water, to be drunk in about 5 minutes. Blood and urine samples are collected at 30 minute intervals for at least 2 hours. All blood samples are subjected to glucose estimation while urine samples are qualitatively tested for glucose.

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Fig.36.8.
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Fasting, Plasma glucose concentration as mmol/l (mg/dl).Normal = <6.1. Fasting, Plasma glucose concentration as mmol/l (mg/dl).Impaired glucose tolerance = <7.0. Fasting, Plasma glucose concentration as mmol/l (mg/dl).Diabetes = >7.0. , Plasma glucose concentration as mmol/l (mg/dl).Normal = (<110). , Plasma glucose concentration as mmol/l (mg/dl).Impaired glucose tolerance = (<126). , Plasma glucose concentration as mmol/l (mg/dl).Diabetes = (>126). 2 hours after, Plasma glucose concentration as mmol/l (mg/dl).Normal = <7.8. 2 hours after, Plasma glucose concentration as mmol/l (mg/dl).Impaired glucose tolerance = <11.1. 2 hours after, Plasma glucose concentration as mmol/l (mg/dl).Diabetes = >11.1. glucose, Plasma glucose concentration as mmol/l (mg/dl).Normal = (<140). glucose, Plasma glucose concentration as mmol/l (mg/dl).Impaired glucose tolerance = (<200). glucose, Plasma glucose concentration as mmol/l (mg/dl).Diabetes = (>200)
In individuals with impaired glucose tolerance, the fasting (110-126 mg/dl) as well as 2 hour (140-200 mg/dl) plasma glucose levels are elevated. These subjects slowly develop frank diabetes at an estimated rate of 2% per year. Dietary restriction and exercise are advocated for the treatment of impaired glucose tolerance.
The WHO criteria for the diagnosis of diabetes by OGTT is presented in Table 36.3. A person is said to be suffering from diabetes mellitus if his/her fasting plasma glucose exceeds 7.0 mmol/l (126 mg/dl) and, at 2 hrs. 11.1 mmol/| (200 mg/dl).

1. For conducting GTT in children, oral glucose is given on the basis of weight (1.5 to 1.75 g/kg).
2. In case of pregnant women, 100 g oral glucose is recommended. Further, the diagnostic criteria for diabetes in pregnancy should be more stringent than WHO recommendations.
3. In the mini GTT carried out in some laboratories, fasting and 2 hrs. sample (instead of 1/y hr. intervals) of blood and urine are collected.
4. The GTT is rather unphysiological. To evaluate the glucose handling of the body under physiological conditions, fasting blood sample is drawn, the subject is allowed to take heavy breakfast, blood samples are collected at 1 hour and 2 hrs (post-prandiaI—meaning after food). Urine samples are also collected. This type of test is commonly employed in established diabetic patients for monitoring the control.
5. For individuals with suspected malabsorption, intravenous GTT is carried out. 6. Corticosteroid stressed GTT is employed to detect latent diabetes.

The commonest cause of glucose excretion in urine (glycosuria) is diabetes mellitus. Therefore, glycosuria is the first line screening test for diabetes. Normally, glucose does not appear in urine until the plasma glucose concentration exceeds renal threshold (180 mg/dl). As age advances, renal threshold for glucose increases marginally.
Renal glycosuria : Renal glycosuria is a benign condition due to a reduced renal threshold for glucose. |t is unrelated to diabetes and, therefore, should not be mistaken as diabetes. Further, it is not accompanied by the classical symptoms of diabetes.

blood glucose level rises rapidly after meals resulting in its spill over into urine. This condition is referred to as alimentary (lag storage) glycosuria. |t is observed in some normal people, and in patients of hepatic diseases, hyperthyroidism and peptic ulcer.

Diabetes mellitus is associated with several metabolic alterations. Most important among them are hyperglycemia, ketoacidosis and hypertriglyceridemia (Fig.36.9).
1. Hyperglycemia : Elevation of blood glucose concentration is the hallmark of uncontrolled diabetes. Hyperglycemia is primarily due to
Fig. 36.9 : Major metabolic alterations in diabetes mellitus.
reduced glucose uptake by tissues and_ its increased production via gluconeogenesis and glycogenolysis. When the blood glucose level goes beyond the renal threshold, glucose is excreted into urine (glycosuria).
Glucose toxicity : High concentrations of glucose can be harmful causing osmotic effects/ hypertonic effects (water drawn from cells into extracellular fluid and excreted into urine, resulting in dehydration), B-cell damage by free radicals (due to enhanced oxidative phosphorylation, oxidative stress, and increased free radicals) and glycation of proteins (associated with diabetic complicationsneuropathy, nephropathy, retinopathy etc.).
2. Ketoacidosis : Increased mobilization of fatty acids results in overproduction of ketone bodies which often leads to ketoacidosis.
3. Hypertriglyceridemia : Conversion of fatty acids to triacylglycerols and the secretion of VLDL and chylomicrons is comparatively higher in diabetics. Further, the activity of the enzyme lipoprotein lipase is low in diabetic patients. Consequently, the plasma levels of VLDL, chylomicrons and_ triacylglycerols are increased. Hypercholesterolemia is also frequently seen in diabetics.

Hyperglycemia is directly or indirectly associated with several complications. These include atherosclerosis, retinopathy, nephropathy and neuropathy. The biochemical basis of these complications is not clearly understood. It is believed that at least some of them are related to microvascular changes caused by glycation of proteins.

Diet, exercise, drug and, finally, insulin are the management options in diabetics. Approximately, 50% of the new cases of diabetes can be adequately controlled by diet alone, 2030% need oral hypoglycemic drugs while the remaining 20-30% require insulin.
Dietary management : A diabetic patient is advised to consume low calories (i.e. low carbohydrate and fat), high protein and fiber rich diet. Carbohydrates should be taken in the form of starches and complex sugars. As far as possible, refined sugars (sucrose, glucose) should be avoided. Fat intake should be drastically reduced so as to meet the nutritional requirements of unsaturated fatty acids. Diet control and exercise will help to a large extent obese NIDDM patients.
Hypoglycemic drugs : The oral hypoglycemic drugs are broadly of two categories-sulfonylureas and biguanides. The latter are less commonly used these days due to side effects.
mide
Sulfonylureas such as acetohexamide, tolbutaand glibenclamide are frequently used.
They promote the secretion of | endogenous insulin and thus help in reducing blood glucose level.
Management with insulin Two_ types of insulin preparations are commercially available—short acting and long acting. The short acting insulins are unmodified and their action lasts for about 6 hours. The long acting insulins are modified ones (such as adsorption to protamine) and act for several hours, which depends on the type of preparation.
The advent of genetic engineering is a boon to diabetic patients since bulk quantities of insulin can be produced in the laboratory.

For a diabetic patient who is on treatment (drug or insulin therapy), periodical assessment of the efficacy of the treatment is essential. Urine glucose detection and blood glucose estimations are traditionally followed in several laboratories. In recent years, more reliable and long-term biochemical indices of diabetic control are in use.
HbA, is produced by the condensation of glucose with N-terminal valine of each B-chain of HbA.
Diagnostic importance of HbA,, : The rate of synthesis of HbAj. is directly related to the exposure of RBC to glucose. Thus, the concentration of HbA;,. serves as an indication of the blood glucose concentration over a period, approximating to the half-life of RBC (hemoglobin) i.e. 6-8 weeks. A close correlation between blood glucose and HbA, concentrations has been observed when simultaneously monitored for several months.
Normally, HbA;. concentration is about 3-5% of the total hemoglobin. In diabetic patients, HbA;, is elevated (to as high as 15%). Determination of HbA;, is used for monitoring of diabetes control. HbA, reflects the mean blood glucose level over 2 months period prior to its measurement.
In the routine clinical practice, if the HbAj, concentration is less than 7%, the diabetic patient is considered to be in good control.
Glycated hemoglobin Glycated or glycosylated hemoglobin refers to the glucose derived products of normal adult hemoglobin (HbA). Glycation is a post-translational, non- enzymatic addition of sugar residue to amino acids of proteins. Among the _ glycated hemoglobins, the most abundant form is HbA1¢.
Estimated average glucose(eAG) : eAG is a new term (introduced by American Diabetic Association) used in diabetic management. It is a laboratory tool to understand the approximate relationship between HbA;, and glucose concentrations, and is given by the following formula eAG (mg/dl) = (28.7 x HbAj,) — 46.7

+ Diabetes affects about 2-3% of the population and is a major cause of blindness, renal failure, heart attack and stroke.
The hormone insulin has been implicated in the development of diabetes.
Diabetic ketoacidosis is frequently encountered in severe uncontrolled diabetics. The
management includes administration of insulin, fluids and potassium.
+ The hypoglycemic drugs commonly used in diabetic patients include tolbutamide, glibenclamide and acetohexamide.
+ Measurement of glycated hemoglobin (HbA,,) serves as a marker for diabetic control.
(e.g. for HBA;, values of 6%, 8% and 10%, the eAG values respectively are 126 mg/dl, 183 mg/ dl and 240 mg/dl)
Fructosamine : Besides HbAj,, several other proteins in the blood are glycated. Glycated serum proteins (fructosamine) can also be measured in diabetics. As albumin is the most abundant plasma protein, glycated albumin largely contributes to plasma _ fructosamine measurements. Albumin has shorter half-life than Hb. Thus, glycated albumin represents glucose status over 3 weeks prior to_ its determination.
Microalbuminuria
Microalbuminuria —
is defined as the excretion of 30-300 mg of albumin in urine per day. |t may be noted that microalbuminuria represents an intermediary stage between normal albumin excretion (2.5-30 mg/d) and macroalbuminuria (> 300 mg/d). The small increase in albumin excretion predicts impairment in renal function in diabetic patients. Microalbuminuria serves as a signal of early reversible renal damage.
Serum lipids
:
serum lipids
(total cholesterol, HDL, triglycerides) serves as an index for overall metabolic control in diabetic patients. Hence, serum lipids should be frequently measured.

1. Diabetes mellitus is a common metabolic disorder, characterized by insufficient or inefficient insulin.
2. Insulin is a polypeptide hormone, secreted by the f-cells of pancreas. It has a profound influence on carbohydrate, fat and protein metabolisms. Insulin lowers blood glucose concentration (hypoglycemic effect).
3. Glucagon, secreted by the orcells of pancreas, in general opposes the actions of insulin. The net effect of glucagon is to increase blood glucose concentration (hyperglycemic effect).
4. Ina healthy person, the blood glucose level (fasting 70-100 mg/dl) is maintained by a well coordinated hormonal action regulating the sources that contribute to glucose (gluconeogenesis, glycogenolysis), and the utilization pathways (glycolysis, glycogenesis, lipogenesis). Insulin is hypoglycemic while other hormones (glucagon, epinephrine, thyroxine, glucocorticoids) are hyperglycemic.
5.
In
(blood
glucose
<45
mg/dl),
there
is
of
glucose
supply
to
brain
resulting
in
symptoms
such
as
headache,
confusion,
anxiety
and
seizures.
6. Diabetes mellitus is broadly classified into 2 categories—insulin dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM).
7. The diagnosis of diabetes is frequently carried out by oral glucose tolerance test (GTT). As per WHO criteria, a person is said to be suffering from diabetes if his/her fasting blood glucose exceeds 126 mg/dl, and 2 hrs. after OGTT goes beyond 200 mg/dl.
8. Diabetes is associated with several metabolic derangements such as ketoacidosis and hypertriglyceridemia, besides hyperglycemia. The chronic complications of diabetes include atherosclerosis, retinopathy, nephropathy and neuropathy.
9. Diet, exercise, drug and insulin are the options for diabetic control. It is estimated that about half of the new diabetic patients can be controlled by diet and exercise.

10.Estimation of glycated hemoglobin (HbA,,), plasma fructosamine, microalbumin in urine, and serum lipids serve as biochemical indices to monitor diabetic control.
I the normal circumstances, the proliferation of body cells is under strict control. The cells differentiate, divide and die in a sequential manner in a_ healthy organism. Cancer is characterized by loss of control of cellular growth and development leading to excessive proliferation and spread of cells. Cancer is derived from a Latin word meaning crab. It is presumed that the word cancer originated from the character of cancerous cells which can migrate and adhere and cause pain (like a crab) to any part of the body.
Neoplasia literally means new growth. Uncontrolled growth of cells results in tumors (a word originally used to represent swelling). Oncology (Greek : oncos—tumor) deals with the study of tumors.

1. Benign tumors : They usually grow by expansion and remain encapsulated in a layer of connective tissue. Normally benign tumors are not life-threatening e.g. moles, warts. These types of benign tumors are not considered as cancers.
2. Malignant tumors or cancers : They are characterized by uncontrolled proliferation and spread of cells to various parts of the body, a process referred to as metastasis. Malignant tumors are invariably life-threatening e.g. lung cancer, leukemia.
About 100 different types of human cancers have been recognized. Cancers arising from epithelial cells are referred to as carcinomas while that from connective tissues are known as sarcomas. Methods for the early detection and treatment of cancers have been developed. However, little is known about the biochemical basis of cancer.

Cancer is the second largest killer disease (the first being coronary heart disease) in the developed countries. It is estimated that cancer accounts for more than 20% of the deaths in United States. Based on the current rate of incidence, it is believed that one in every 3 persons will develop cancer at sometime during his life.
Although humans of all ages develop cancer, the incidence increases with advancement of age. More than 70% of the new cancer cases occur in persons over 60 years. Surprisingly, cancer is a leading cause of death in children in the age group 3-13 years, half of them die due to leukemia.

In general, cancers are multifactorial in origin. The causative agents include physical, chemical, genetic and environmental factors. A survey in USA has shown that about 90% of all cancer deaths are due to avoidable factors such as tobacco, pollution, occupation, alcohol and diet. Most of the cancers are caused by chemical carcinogens, radiation energy and viruses. These agents may damage DNA or interfere with its replication or repair.

It is estimated that almost 80% of the human cancers are caused by chemical carcinogens in nature. The chemicals may be organic (e.g. dimethylbenzanthracene, benzo (a) pyrene, dimethyl nitrosamine) or inorganic (arsenic, cadmium) in nature. Entry of the chemicals into the body may occur by one of the following mechanisms.
1. Occupation e.g. asbestos, benzene.
2. Diet e.g. aflatoxin B produced by fungus (Aspergillus flavus) contamination of foodstuffs, particularly peanuts.
3. Drugs—certain therapeutic drugs can be carcinogenic e.g. diethylstibesterol.
4. Life style e.g. cigarette smoking.
Mechanism of action : Although a few of the chemicals are directly carcinogenic, majority of them require prior metabolism to become carcinogenic. The enzymes such as cytochrome P4sq responsible for the metabolism of xenobiotics (Chapter 31) are involved in dealing with the chemical carcinogens. In general, a chemically non-reactive — procarcinogen __ is converted to an ultimate carcinogen by a series of reactions.
The carcinogens can covalently bind to purines, pyrimidines and phosphodiester bonds of DNA, often causing unrepairable damage. The chemical carcinogens frequently = cause mutations (a change in the nucleotide sequence of DNA) which may finally lead to the development of cancer, hence they are regarded as mutagens.
Ames assay : This is a laboratory test to check the carcinogenecity of chemicals. Ames assay employs the use of a special mutant strain of bacterium, namely Salmonella typhimurium (His-). This organism cannot synthesize histidine; hence the same should be supplied in the medium for its growth. Addition of chemical carcinogens Causes mutations (reverse mutation) restoring the ability of the bacteria to synthesize histidine (Hist). By detecting the strain of Salmonella (His*) in the colonies of agar plates, the chemical mutagens can be identified. The Ames assay can detect about 90% of the chemical carcinogens. This test is regarded as a preliminary screening procedure. Animal experiments are conducted for the final assessment of carcinogenecity.

Promoters of carcinogenesis : Some of the chemicals on their own are not carcinogenic. Certain substances known as promoting agents make them carcinogenic. The application of benzo(a)pyrene to the skin, as such, does not cause tumor development. However, if this is followed by the application of croton oil, tumors will develop. In this case, benzo(a)pyrene is the initiating agent while croton oil acts as a promoting agent or promoter. Several compounds that act as promoting agents in various organs of the body have been identified. These include saccharin and phenobarbital.

Ultraviolet rays, X-rays and y-rays have been proved to be mutagenic in nature causing cancers. These rays damage DNA which is the basic mechanism to explain the carcinogenicity
of radiation energy. For instance, exposure to UV rays results in the formation of pyrimidine dimers in DNA _ while X-rays cause the production of free radicals. This type of molecular damages are responsible for the carcinogenic effects of radiations.

The involvement of viruses in the etiology of cancer was first reported by Rous in 1911. He demonstrated that the cell-free filtrates from certain chicken sarcomas (tumors of connective tissues) promote new sarcomas in chickens. Unfortunately, this epoch-making discovery of Rous was ignored for several years. This is evident from the fact that Rous was awarded the Nobel Prize in 1966 at the age of 85 for his discovery in 1911!
The presence of viral particles and the enzyme reverse transcriptase, besides the occurrence of base sequence in the DNA of malignant cells, complementary to tumor viruses indicate the involvement of viruses in cancer. The viruses involved in the development of cancer, commonly known as oncogenic viruses, may contain either DNA or RNA. A selected list of tumor viruses is given in Table 37.1.

DNA is the ultimate critical macromolecule in carcinogenesis. This fact is supported by several evidences.
1. Cancers are transmitted from mother to daughter cells. In other words, cancer cells beget cancer cells.
2. Chromosomal abnormalities are observed
in many tumor cells.
3. Damage to DNA caused by mutations
often results in carcinogenesis.
4. Laboratory experiments have proved that purified oncogenes can transform normal cells into cancer cells.

Cancer is caused by a genetic change in a single cell resulting in its uncontrolled multiplication. Thus, tumors are monoclonal. Two types of regulatory genes—oncogenes and antioncogenes are involved in the development of cancer (carcinogenesis). In recent years, a third category of genes that control the cell death or apoptosis are also believed to be involved in carcinogenesis.

The genes capable of causing cancer are known as oncogenes (Greek : oncos—tumor or mass). Oncogenes were originally discovered in tumor causing viruses. These viral oncogenes were found to be closely similar to certain genes present in the normal host cells which are referred to as protooncogenes. Now, about 40 viral and cellular protooncogenes have been identified. Protooncogenes encode for growthregulating proteins. The activation of protooncogenes to oncogenes is an important step in the causation of cancer.
selected list of and the In the Table 37.2, a oncoproteins, — protooncogenes associated human cancers is given.

There are several mechanisms for converting the protooncogenes to oncogenes, some of the
important ones are described next.
Growth factors, T ABLE 37.2 Selected oncoproteins, protooncogenes and associated cancers.Protooncogene = . Growth factors, T ABLE 37.2 Selected oncoproteins, protooncogenes and associated cancers.Associated human cancer(s) = . Platelet derived growth factor (PDGF) Epidermal growth factor (EGF), T ABLE 37.2 Selected oncoproteins, protooncogenes and associated cancers.Protooncogene = sis hst -1. Platelet derived growth factor (PDGF) Epidermal growth factor (EGF), T ABLE 37.2 Selected oncoproteins, protooncogenes and associated cancers.Associated human cancer(s) = Osteosarcoma Cancers of stomach, breast and bladder. Growth factor receptors, T ABLE 37.2 Selected oncoproteins, protooncogenes and associated cancers.Protooncogene = erb -B. Growth factor receptors, T ABLE 37.2 Selected oncoproteins, protooncogenes and associated cancers.Associated human cancer(s) = Lung cancer Stomach cancer Breast cancer. , T ABLE 37.2 Selected oncoproteins, protooncogenes and associated cancers.Protooncogene = 1 erb -B 2 erb -B 3. , T ABLE 37.2 Selected oncoproteins, protooncogenes and associated cancers.Associated human cancer(s) = . Signal-transducing proteins GTP- binding proteins Non-receptor tyrosine kinase, T ABLE 37.2 Selected oncoproteins, protooncogenes and associated cancers.Protooncogene = ras abl. Signal-transducing proteins GTP- binding proteins Non-receptor tyrosine kinase, T ABLE 37.2 Selected oncoproteins, protooncogenes and associated cancers.Associated human cancer(s) = Leukemias, cancers of lung, pancreas and colon Leukemia

1. Viral insertion into chromosome : When certain retroviruses (genetic material RNA) infect cells, a complementary DNA (cDNA) is made from their RNA by the enzyme reverse transcriptase. The cDNA so produced gets inserted into the host genome (Fig.37.1). The integrated double-stranded cDNA is referred to as provirus. This pro-viral DNA takes over the control of the transcription of cellular chromosomal DNA and transforms the cells. Activation of protooncogene myc to oncogene by viral insertion —_ ultimately — causing carcinogenesis is well known (e.g. avian leukemia).
Some DNA viruses also get inserted into the host chromosome and _ activate the protooncogenes.
2. Chromosomal translocation : Some of the tumors exhibit chromosomal abnormalities. This is due to the rearrangement of genetic material (DNA) by chromosomal translocation i.e. splitting off a small fragment of chromosome which is joined to another chromosome. Chromosomal translocation usually results in overexpression of protooncogenes.
Burkitt's lymphoma, a cancer of human B-lymphocytes, is a good example of chromosomal translocation. In this case, a fragment from chromosome 8 is split off and joined to chromosome 14 containing myc gene
(Fig.37.2). This results in the activation of inactive myc gene leading to the increased synthesis of certain proteins which make the cell malignant.
3. Gene amplification : Severalfold amplifications of certain DNA sequences are observed in some cancers. Administration of anticancer drugs methotrexate (an inhibitor of the enzyme dihydrofolate reductase) is associated with gene amplification. The drug becomes inactive due to gene amplification resulting in a severalfold (about 400) increase in the activity of dihydrofolate reductase.
4. Point mutation : The ras protooncogene is the best example of activation by point mutation (change in a single base in the DNA). The mutated ras oncogene produces a_ protein
Fig. 37.1 : Integration of viral DNA into host DNA.
Fig. 37.2 : Diagrammatic representation of reciprocal translocation occurring in Burkitt's lymphoma.

(GTPase) which differs in structure by a single amino acid. This alteration diminishes the activity of GTPase, a key enzyme involved in the control of cell growth (details described later).
The presence of ras mutations is detected in several human tumors—90% of pancreatic, 50% of colon and 30% of lung. However, ras mutations have not been detected in the breast cancer.

Oncogenes encode for certain proteins, namely oncoproteins. These proteins are the altered versions of their normal counterparts and are involved in the transformation and multiplication of cells. Some of the products of oncogenes are discussed below.
Growth factors Several growth factors stimulating the proliferation of normal cells are known. They regulate cell division by transmitting the message across the plasma membrane to the interior of the cell (transmembrane _ signal transduction). It is believed that growth factors play a key role in carcinogenesis.
A selected list of polypeptide growth factors, their sources and major functions is given in Table 37.3.
The cell proliferation is stimulated by growth factors. In general, a growth factor binds to a protein receptor on the plasma membrane. This binding activates cytoplasmic protein kinases leading to the phosphorylation of intracellular target proteins. The phosphorylated proteins, in turn, act as intracellular messengers to stimulate cell division, the mechanism of which is not clearly known.
Transforming growth factor (TGF-a) is a protein synthesized and required for the growth of epithelial cells. TGF-a is produced in high concentration in individuals suffering from psoriasis, a disease characterized by excessive proliferation of epidermal cells.
Growth factor receptors : Some oncogenes encoding growth factor receptors have been identified. Overexpression and/or — structural alterations in growth factor receptors are associated with carcinogenesis. For instance, the overexpression of gene erb-B, encoding EGFreceptor is observed in lung cancer.
GTP-binding proteins : These are a group of signal transducing __ proteins. | Guanosine triphosphate (GTP)-binding proteins are found in about 30% of human cancers. The mutation of ras protooncogene is the single-most dominant cause of many human tumors.

The involvement of ras protein (product of ras gene) with a molecular weight 21,000 (P 1) in cell multiplication is illustrated in Fig.37.3. The inactive ras is in a bound state with GDP. When the cells are stimulated by growth factors, ras P51 gets activated by exchanging GDP for GTP. This exchange process is catalysed by guanine nucleotide releasing factor (GRF). The active ras P51, stimulates regulators such as cytoplasmic kinases, ultimately causing DNA replication and cell division. In normal cells, the activity of ras P51 is shortlived. The GTPase activity, which is an integral part (intrinsic) of ras Py;, hydrolyses GTP to GDP, reverting ras 21 to the original state. There are certain proteins, namely GTPase activating proteins (GAP), which accelerate the

Stimulates growth and maturation of T-cells
Fig. 37.3 : Model for the mechanism of action of ras P,, protein (GRF—Guanine nucleotide releasing factor; GAP—GTPase activating proteins).
hydrolysis of f hus, i ydrolysis of GTP of ras Pj;. Thus, in norma cells, the activity of ras Py; is well regulated.
Point mutations
in ras
gene result
in the
altered activity.
This leads
to in
a
permanently ras
P»1, lacking
the occurrence
activated of
state,
GTPase ras
Py, causing
uncontrolled multiplication
of cells.
N ine ki Th on-receptor tyrosine inases : ese proteins are found on the interior of the inner plasma membrane. They phosphorylate the A . : Biot cellular target proteins (involved in cell division) in response to external growth stimuli. Mutations in the protooncogenes (e.g. abl) encoding nonreceptor tyrosine kinases increase the kinase activity and, in turn, phosphorylation of target proteins causing unlimited cell multiplication.

A special category of genes, namely cancer suppressor genes (e.g. p>? gene) or, more commonly, antioncogenes, have been identified. The products of these genes apply breaks and regulate cell proliferation. The loss of these
Fig.
37.4
:
A
simplified hypothesis
for the
cancer.
suppressor genes removes the growth control of cells and is believed to be a key factor in the development of several tumors, e.g. retinoblastoma, one type of breast cancer, carcinoma of lung, Wilms' kidney tumor.
With the rapid advances in the field of genetic engineering, introducing antioncogenes to a normal chromosome to correct the altered growth rate of cells may soon become a reality.

A new category of genes that regulate programmed cell death (apoptosis) have been discovered. These genes are also important in the development of tumors.
The gene, namely bcl-2, causes B-cell lymphoma by preventing programmed cell death. It is believed that overexpression of bcl-2 allows other mutations of protooncogenes that, ultimately, leads to cancer.

The multifactorial origin of cancer can be suitably explained by oncogenes. The physical and chemical agents, viruses and mutations all lead to the activation of oncogenes causing carcinogenesis. The antioncogenes and the genes regulating apoptosis are intimately involved in development of cancer. A simplification of a unified hypothesis of carcinogenesis is depicted in Fig.37.4.

The biochemical indicators employed to detect the presence of cancers are collectively referred to as tumor markers. These are the abnormally produced molecules of tumor cells such as surface antigens, cytoplasmic proteins, enzymes and hormones. Tumor markers can be measured in serum (or plasma). In theory, the tumor markers must ideally be useful for screening the population to detect cancers. In practice, however, this has not been totally true. As such, the tumor markers support the diagnosis of cancers, besides being useful for monitoring the response to therapy and for the early detection of recurrence.
A host of tumor markers have been described and the list is evergrowing. However, only a few of them have proved to be clinically useful. A selected list of tumor markers and the associated cancers are given in Table 37.4.
A couple of the most commonly used tumor markers are discussed hereunder.
1. Carcinoembryonic antigen (CEA) : This is a complex glycoprotein, normally produced by the embryonic tissue of liver, gut and pancreas. The presence of CEA in serum is detected in several cancers (colon, pancreas, stomach, lung). In about 67% of the patients with colorectal cancer, CEA can be identified. Unfortunately, serum CEA is also detected in several other disorders such as alcoholic cirrhosis (70%), emphysema (57%) and diabetes mellitus (38%). Due to this, CEA lacks specificity for cancer detection. However, in established cancer patients (particularly of colon and breast), the serum level of CEA is a useful indicator to detect the burden of tumor mass, besides monitoring the treatment.
2. Alpha-fetoprotein (AFP) : It is chemically a glycoprotein, normally synthesized by yolk sac in early fetal life. Elevation in serum levels of AFP mainly indicates the cancers of liver and germ cells of testis and, to some _ extent, carcinomas of lung, pancreas and colon. As is the case with CEA, alpha-fetoprotein is not specific for the detection of cancers. Elevated

Oncofetal antigens, Associated cancer(s) = . Carcinoembryonic antigen (CEA) Alpha fetoprotein (AFP) Cancer antigen-125 (CA-125), Associated cancer(s) = Cancers of colon, stomach, lung, pancreas and breast Cancer of liver and germ cells of testis Ovarian cancer. Hormones, Associated cancer(s) = . Human chorionic gonadotropin (hCG) Calcitonin Catecholamines and their metabolites (mainly vanillyl mandelic acid), Associated cancer(s) = Choriocarcinoma Carcinoma of medullary thyroid Pheochromocytoma and neuroblastoma. Enzymes, Associated cancer(s) = . Prostatic acid phosphatase Neuron specific enolase Alkaline phosphatase, Associated cancer(s) = Prostate cancer Neuroblastoma Bone secondaries. Specific proteins, Associated cancer(s) = . Prostate specific antigen (PSA) Immunoglobulins Bence-Jones proteins, Associated cancer(s) = Prostate cancer Multiple myeloma Multiple myeloma
levels of AFP are observed in cirrhosis, hepatitis and pregnancy. However, measurement of serum AFP provides a sensitive index for tumor therapy and detection of recurrence.
they form monolayers and cannot move away from each other. The cancer cells form multilayers due to loss of contact inhibition (Fig.37.5). As a result, the cancer cells freely move and get deposited in any part of the body, a property referred to as metastasis.

The morphological and biochemical changes in the growing tumor cells are briefly described here. These observations are mostly based on the in vitro culture studies. Knowledge on the biochemical profile of tumor cells guides in the selection of of cancers.
chemotherapy
1. General and morphological changes
1 Shape of cells : The tumor cells are much rounder in shape compared to normal cells.
| Alterations in cell structures : The cytoskeletal structure of the tumor cells with regard to actin filaments is different.
1 Loss of contact inhibition : The normal cells are characterized by contact inhibition i.e.
Loss of anchorage dependence : The cancer cells can grow without attachment to the surface. This is in contrast to the normal cells which firmly adhere to the surface.
Alteration in permeability properties : The tumor cells have altered permeability and transport.
Fig. 37.5 : Growth cells in culture (A) Normal cells forming monolayer (exhibiting contact inhibition); (B) Cancer cells forming multilayers (loss of contact inhibition).
2. Biochemical changes
| Increased replication and transcription : The synthesis of DNA and RNA is increased in cancer cells.
: Increased glycolysis : The fast growing tumor cells are characterized by elevation in aerobic and anaerobic glycolysis due to increased energy demands of multiplying cells.
1 Reduced requirement of growth factors : The tumor cells require much less quantities of growth factors. Despite this fact, there is an increased production of growth factors by these cells.
. Synthesis of fetal proteins : During fetal life, certain genes are active, leading to the synthesis of specific proteins. These genes are suppressed in adult cells. However, the tumor cells synthesize the fetal proteins e.g. carcinoembryonic antigen, alfa fetoprotein.
| Alterations in the structure of molecules : Changes in the structure of glycoproteins and glycolipids are observed.

anchorage dependence and alterations in the structure of certain macromolecules are among the important factors responsible for metastasis.

Chemotherapy, employing certain anticancer drugs, is widely used in the treatment of cancer. In the Table 37.5, a selected list of the most commonly used drugs, and their mode of action is given. The effectiveness of anticancer drugs is inversely proportional to the size of the tumor i.e. the number of cancer cells. The major limitation of cancer chemotherapy is that the rapidly dividing normal cells (of hematopoietic system, gastrointestinal tract, hair follicles) are also affected. Thus, the use of anticancer drugs is associated with toxic manifestations.
Cisplatin is used in the treatment of testicular, ovarian and several other cancers (bone, lung). The side effects of cisplatin include bone marrow depletion, loss of hearing and impairment in kidney function. About 80% of testicular cancer patients survive with a new combination therapy of cisplatin, etoposide, and bleomycin.
Metastasis refers to the spread of cancer cells from the primary site of origin to other tissues of the body where they get deposited and grow as secondary tumors. Metastasis is the major cause of cancer related morbidity and mortality. It is believed that the morphological changes in tumor cells, loss of contact inhibition, loss of
The term tumor lysis syndrome (TLS) represents all the metabolic consequences that occur during cancer treatment. These include increased uric acid levels in serum and urine, acute renal failure, hyperkalemia, hyperphosphatemia etc. Recombinant urate oxidase (converts uric acid to soluble allantion) is successfully used in the treatment of TLS.

+ About 80% of the human cancers are caused by chemical carcinogens.
+ The products of oncogenes (e.g. growth factors) have been implicated in the development of cancer. Antioncogenes apply breaks and regulate the cell proliferation.
+ The physical and chemical agents, viruses and mutations result in the activation of oncogenes causing carcinogenesis.
+ The abnormal products of tumor cells, referred to as tumor markers (CEA, AFP, PSA) are useful for the diagnosis and prognosis of cancer.
+ Anticancer drugs (e.g. methotrexate, cisplatin) are commonly used in the treatment of cancer. Antioxidants (vitamins E and C, fcarotene, Se) decrease the risk of carcinogenesis and hence their increased consumption is advocated.

Methotrexate, Chemical nature = Folic acid analogue. Methotrexate, Mode of action = Blocks the formatin of tetrahydrofolate (inhibits the enzyme dihydrofolate reductase). THF is required for nucleotide synthesis.. 6-Mercaptopurine, Chemical nature = Purine analogue. 6-Mercaptopurine, Mode of action = Inhibits the formation of AMP from IMP.. 6-Thioguanine, Chemical nature = Purine analogue. 6-Thioguanine, Mode of action = Blocks thymidylate synthase reaction.. Mitomycin C, Chemical nature = Antibiotic. Mitomycin C, Mode of action = Results in cross bridges between DNA base pairs.. Actinomycin D, Chemical nature = Antibiotic. Actinomycin D, Mode of action = Blocks transcription. Vinblastine and vincristine, Chemical nature = Alkaloids. Vinblastine and vincristine, Mode of action = Inhibit cell division and cytoskeleton formation. Cisplatin, Chemical nature = Platinum compound. Cisplatin, Mode of action = Results in the formation of intrastrand DNA adducts.. Cyclophosphamide, Chemical nature = Alkylating agent. Cyclophosphamide, Mode of action = Cross-links bases and inhibits DNA strand separation. Imatinib, Chemical nature = Monoclonal antibody. Imatinib, Mode of action = Tyrosine kinase inhibitor

In recent years, certain precautionary measures are advocated to prevent or reduce the occurrence of cancer. The most important among them, from the biochemical perspective, are the antioxidants namely vitamin E, B-carotene, vitamin C and selenium. They prevent the formation or detoxify the existing free radicals (free radicals are known to promote carcinogenesis). In addition, antioxidants stimulate body's immune system, and promote detoxification of various carcinogens.
In general, most of the vegetables and fruits are rich in antioxidants. Their increased consumption is advocated to prevent cancer. (For more details on free radicals and antioxidants, Refer Chapter 34).

1., 1 = Cancer is characterized by uncontrolled cellular growth and development, leading to excessive proliferation and spread of cells. Cancer is the second largest killer disease (next to heart disease) in the developed world.. 2., 1 = Regulatory genes-namely oncogenes, antioncogenes and genes controlling cell death- are involved in the development of cancer. Activation of oncogenes is a fundamental step in carcinogenesis. This may occur by insertion of viral DNA into host chromosome, translocation of chromosomes, gene amplification and point mutation.. 3., 1 = The products of activated oncogenes such as growth factors, growth factor receptors, GTP-binding proteins, non-receptor tyrosine kinases have all been implicated in the development of cancer.. 4., 1 = Tumor markers of cancers include carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), cancer antigen-125 and prostate specific antigen (PSA). They are mainly useful to support diagnosis, monitor therapy and detect recurrence.. 5., 1 = There are several morphological and biochemical changes in the tumor cells which distinguish them from the normal cells. The cancer cells are characterized by loss of contact inhibition, altered membrane transport, increased DNA and RNA synthesis, increased glycolysis, alteration in the structure of certain molecules etc.
cquired immunodeficiency syndrome (AIDS) was first reported in 1981 in homosexual men. AIDS is a retroviral disease caused by human immunodeficiency virus (HIV). The disease is characterized by immunosuppression, secondary neoplasma_ and___ neurological manifestations. AIDS is invariably fatal, since there is no cure. In the USA, it is the fourth leading cause of death in men between the ages 15 to 55 years.
No other disease has attracted as much attention as AIDS by the governments, public and scientists. AIDS has stimulated an unprecedented amount of biomedical research which led to a major understanding of this deadly disease within a short period of time. So rapid is the research on AIDS (particularly relating to molecular biology), any review is destined to be out of date by the time it is published!
The isolation of human immunodeficiency virus (HIV) from lymphocytes of AIDS patients was independently achieved by Gallo (USA) and Montagnier (France) in 1984.

AIDS was first described in USA and this country has the majority of reported cases. The prevalence of AIDS has been reported from almost every country. The number of people living with HIV worldwide is estimated to be around 40 million by the end of the year 2005. (India alone has about 5 million persons). At least 5 million deaths occurred in 2005, due to AIDS. AIDS is truely a global disease with an alarming increase in almost every country.

essentially requires the exchange of body fluids (semen, vaginal secretions, blood, milk) containing the virus or virus-infected cells. There are three major routes of HIV transmission— sexual contact, parenteral inoculation, and from infected mothers to their newborns.
The distribution of risk factors for AIDS trans- mission are as follows.
Sex between men (homosexuals)
Sex between men and women
—
—
60%
15%
It should, however, be noted that HIV cannot be transmitted by casual personal contact in the household or work place. Further, the transmission of AIDS from an infected individual to health personnel attending on him is extremely rare.

AIDS is caused by a retrovirus, namely human immunodeficiency virus (HIV), belonging to lentivirus family. Retroviruses contain RNA as the genetic material. On entry into the host cell, they transcribe DNA which is a complementary copy of RNA. The DNA, in turn is used, as a template to produce new viral RNA copies.
Two different forms of HIV, namely HIV-1 and HIV-2 have been isolated from AIDS patients. HIV-1 is more common, being found in AIDS patients of USA, Canada, Europe and Central Africa while HIV-2 is mainly found in West Africa. Both the viruses are almost similar except they differ in certain immunological properties.
HIV-1 is described in some detail.
Structure of HIV : The virus is spherical with a diameter of about 110 nm. It contains a core, surrounded by a lipid envelop derived from the host plasma membrane (Fig.38.1). The core of the HIV has two strands of genomic RNA and four core proteins, p24, P1g, reverse transcriptase (Pe6/P51) and endonuclease (p33). Note that the naming of the proteins is based on the molecular weight. For instance, a protein with a molecular weight of 24,000 is designated as po4.
Fig. 38.1 : Diagrammatic representation of HIV (p represents protein with molecular weight €.9. Py, is with molecular weight 18,000; gp represents glycoprotein).
The lipid membrane of the virus is studded with two glycoproteins gp;j) and gp4,;The surface antigen gp; 9 is very important for the viral infection and the detection of AIDS.
Genome and gene products of HIV : The HIV genome contains 3 structural genes—gag, pol and env that, respectively, code for core proteins, reverse transcriptase and envelop proteins. On either side of the HIV genome are long terminal repeat (LTR) genes which control transcription. Besides the structural genes, HIV contains several regulatory genes including vif, vpr, tat, rev, vpu and nef (Fig.38.2). These genes control the synthesis and assembly of infectious viral proteins. In fact, the regulatory genes of HIV play a key role in the development of AIDS.

As is evident from the name_ AIDS, immunodeficiency (or immunosuppression) is the hallmark of this disease. AIDS primarily affects the cell-mediated immune system which protects the body from intracellular parasites such as viruses, protozoa and mycobacteria. This is caused by a reduction in CD, (cluster determinant antigen 4) cells of T-lymphocytes, besides impairment in the functions of surviving CD, cells.
CD, cells may be regarded as master cells of cell mediated immunity. They produce cytokines, macrophage chemotactic factors, hemopoietic growth factors, and others involved in the body immunity.
Entry of HIV and lysis of CD, cells : The virus enters the CD, T-lymphocytes. HIV binds to the specific receptors on CD, cells by using its surface membrane — glycoprotein — (gpj 9). Following the entry into the host cells, RNA of HIV is transcribed into DNA by the viral enzyme reverse transcriptase. The viral DNA _ gets incorporated into the host genomic DNA. The virus may remain locked in the host genome for months or years and this is considered as the latent period. The viral DNA may undergo replication and translation, — respectively, producing viral RNA and viral proteins. The latter two, on assembly, result in new viruses. The newly synthesized viruses leave the host cells by forming buds on plasma membrane. Extensive viral budding is associated with lysis and death of CD, cells (Fig.38.3). The new viral particles infect other host cells and repeat the whole process, ultimately resulting in a profound loss of CD4 cells from the blood. Most of the immunodeficiency symptoms of AIDS are associated with the reduction in CD, cells.

The viral membrane protein gpj;29_ binds with normal T-helper cells and kills them. AIDS patients also display abnormalities in antibody production by B-lymphocytes (humoral immunity).
Abnormalities of central nervous system : HIV also infects the cells of central nervous system. It is believed that HIV _ infected monocytes enter the brain and cause damage, the mechanism of which remains obscure.
Consequences of immunodeficiency : The various clinical symptoms (fever, diarrhea, weight loss, neurological complications, multiple opportunistic infections, generalized lymphadenopathy, secondary neoplasma etc.) of AIDS
Fig. 38.3 : Immunological abnormalities in CD4 cells on HIV infection.
Fig. 38.4 : Graphic representation of a typical course of HIV infection.
are directly or indirectly related to the immunosuppression caused by HIV. Due to the deficiency in the immune system, the body of AIDS patient is freely exposed to all sorts of infections (viral, bacterial, fungal).
hence this phase is also referred to as seropositive period.

Three distinct phases of HIV interaction with the immune system of infected body have been identified. These are the early, acute phase; the intermediate, chronic phase; the final, crisis phase (Fig.38.4).
1. Acute phase : This represents the initial body response to HIV infection. It is characterized by high rate of production of viruses which are lodged in the lymphoid tissues and the antiviral immune response of the body. This period may last for about 8-12 weeks.
2. Chronic phase : During this period that may last for 5 to 10 years or even more, the body tries to contain the virus. The immune system is largely intact. The person obviously appears normal, although he/she is the carrier of HIV. which can be transmitted to others. Antibodies to HIV are found in the circulation,
3. Crisis phase : A failure in the defense system of the body, caused by immunosuppression by HIV, represents the crisis phase. The plasma level of virus is tremendously increased. CD, T-lymphocyte concentration drastically falls. A patient with lower than 200 CD, T-lymphocytes/ul blood is considered to have developed AIDS. Crisis phase _ is characterized by opportunistic infections and the related clinical manifestations. In Western countries, a cancer—Kaposis sarcoma—is associated with AIDS.
In general, AIDS patients die between 5-10 years after HIV infection. Treatment may, however, prolong the life.

to
The following laboratory tests are employed diagnose the HIV infection.
1.
The detection
of antibodies
in the
circulation by ELISA (enzyme-linked immunosorbant assay).
Fig. 38.5 : Structure of anti-AIDS drugs.
2. Western blot technique, a more specific test for the HIV antibodies, is employed for confirmation of ELISA positive cases. 3. A more recent and sophisticated PCR can be used to detect the presence of the HIV genome in the peripheral blood lymphocytes.

Although there is no cure for AIDS, use of certain drugs can prolong the life of AIDS patients. Zidovudine or AZT (3'-azido 2', 3'-dideoxy thymidine), a structural analog of deoxythymidine was the first drug used and continues to be the drug of choice for the treatment of AIDS. Didanosine (dideoxyionosine, DDI) is another drug employed to treat AIDS. The structures of AZT and DDI are shown in Fig.38.5.
Mechanism of action : AZT is taken up by the lymphocytes and converted to AZT triphosphate which inhibits the enzyme HIV reverse transcriptase. AZT triphosphate competes with dTTP for the synthesis of DNA from viral RNA. Further, AZT is added to the growing DNA chain and the synthesis is halted. This drug is not toxic to the T-lymphocytes since cellular DNA polymerase has low affinity for AZT. However, AZT is found to be toxic to the bone marrow cells, therefore, the patients develop anemia.
The mechanism of action of dideoxyinosine is almost similar to that of AZT.

HIV exhibits genetic heterogenecity with a result that several species of virus may be found in the same AIDS patient. The principal cause for the genetic variation is the lack of proof-reading activity by the enzyme reverse transcriptase. This leads to very frequent alterations in the DNA base sequence synthesized from viral RNA which, in turn, influences the amino acid sequence of proteins. Thus, the protein products of HIV are highly variable in the amino acid composition and, therefore, the antigenic properties. For this reason, it has not been possible to develop a vaccine against AIDS. However, there have been some encouraging animal and in vitro experiments which raise fresh hopes for a vaccine in the near future.

+ AIDS is a global disease with an alarming increase in the incidence of occurrence. By the year 2005, more than 40 million people were globally affected by AIDS.
+ Homosexuality (predominantly in men) and intravenous drug abuse are the major factors in the risk of AIDS transmission.
+ The patients of AIDS are destined to die (within 5-10 years after infection), since there is no cure. However, administration of certain drugs (AZT, DDI) prolongs life.
+ The clinical manifestations of AIDS are directly or indirectly related to immunosuppression (mostly due to reduced CDy cells). AIDS patients are freely exposed to all sorts of infections (viral, bacterial, fungal).

1. AIDS is a retorviral disease caused by human immunodeficiency virus (HIV). It is characterized by immunosuppression, secondary neoplasms and_ neurological manifestations. Transmission of HIV occurs by sexual contact (more in male homosexuals), parental inoculation (intravenous drug abusers) and from infected mothers to their newborns.
2. HIV enters CDg T-lymphocytes where its genetic material RNA is transcribed into DNA by the enzyme reverse transcriptase. The viral DNA gets incorporated into the host genome ultimately leading to the multiplication of the virus and the destruction of CD4 cells. This is the root cause of immunosuppression leading to opportunistic infections in AIDS.
3. The natural course of AIDS has 3 distinct phases—acute, chronic and crisis. A patient with lower than 200 CD4 T-lymphocytes/ul is considered to have developed AIDS. The sensitive laboratory tests for AIDS detection are—ELISA, Western blot technique and, recently PCR.
4. There is no cure for AIDS. The patients generally die within 5-10 years after HIV infection. Administration of drugs (zidovudine and didanosine), however, prolongs the life of AIDS patients. These drugs inhibit the viral enzyme reverse transcriptase and halt the multiplication of the virus.
5. The attempts to produce vaccine for AIDS have been unsuccessful due to the variations in the genome (and, therefore, the protein products) of the HIV.

s life comes from the existing life, it was believed for a long that the carbon compounds of organisms (hence the name organic) arose from life only. This is referred to as vital force theory. Friedrich Wohler (1825) first discovered that urea (NH3—CO—NHy), the organic compound, could be prepared by heating ammonium cyanate (NH4NCO), in the laboratory. Thereafter, thousands and thousands of organic compounds have been synthesized outside the living system.
Organic chemistry broadly deals with the chemistry of carbon compounds, regardless of their origin. Biochemistry, however, _ is concerned with the carbon chemistry of life only. The general principles of organic chemistry provide strong foundations for understanding biochemistry. However, biochemistry exclusively deals with the reactions that occur in the living system in aqueous medium.

The organic compounds, namely carbohydrates, lipids, proteins, nucleic acids and vitamins are the most common organic compounds of life. Their chemistry has been
discussed in Section | (Chapters 1-7).

Most of the physical and chemical properties of organic compounds are determined by their functional groups. Biomolecules possess certain functional groups which are their reactive centres. The common functional groups of importance in biomolecules are presented in
Table 39.1.
There are many homocyclic and heterocyclic rings,
commonly encountered
in biomolecules.
A selected list of them is given in Fig.39.1.
Homocyclic rings : Phenyl ring derived from benzene is found in several biomolecules (phenylalanine, tyrosine, | catecholamines). Phenanthrene and cyclopentane form the backbone of steroids (cholesterol, aldosterone).

formula.General structural formula = R 1 O R 2. Ether, T ABLE 39.1 Common functional groups of importance in biomolecules.Type of compound.Type of compound = Ether. Ether, T ABLE 39.1 Common functional groups of importance in biomolecules.Examples of biomolecule(s).Examples of biomolecule(s) = Thromboxane A 2. Ester, T ABLE 39.1 Common functional groups of importance in biomolecules.Functional group.Group = C O R 1 O. Ester, T ABLE 39.1 Common functional groups of importance in biomolecules.General structural formula.General structural formula = R 2 C O R 1 O. Ester, T ABLE 39.1 Common functional groups of importance in biomolecules.Type of compound.Type of compound = Ester. Ester, T ABLE 39.1 Common functional groups of importance in biomolecules.Examples of biomolecule(s).Examples of biomolecule(s) = Cholesterol ester. Amido, T ABLE 39.1 Common functional groups of importance in biomolecules.Functional group.Group = C N O R 1 R 2. Amido, T ABLE 39.1 Common functional groups of importance in biomolecules.General structural formula.General structural formula = R 3 C N R 1 R 2 O. Amido, T ABLE 39.1 Common functional groups of importance in biomolecules.Type of compound.Type of compound = Amide. Amido, T ABLE 39.1 Common functional groups of importance in biomolecules.Examples of biomolecule(s).Examples of biomolecule(s) = N-Acetylglucosamine
Coenzyme Q is an example of benzoquinone while vitamin K is a naphthoquinone.
and indole are examples of fused heterocyclic rings.
Heterocyclic rings : Furan is the ring structure found in pentoses. Pyrrole is the basic unit of porphyrins found in many biomolecules (heme) while pyrrolidine is the ring present in the amino acid, proline. Thiophene ring is a part of the vitamin biotin. The amino acid histidine contains imidazole.
Pyran structure
is found
in hexoses.
Pyridine nucleus
is
a

part pyridoxine.
of the
Pyrimidines purines
(adenine, of
vitamins-niacin
(cytosine, guanine)
nucleotides is
and found
in the
and thymine)
are the
nucleic amino
acid and
constituents acids.
Indole ring
tryptophan.
Purine
The compounds possessing __ identical molecular formulae but different structures are referred to as isomers. The phenomenon of existence of isomers is called isomerism (Greek : isos—equal; meros—parts). Isomers differ from each other in physical and chemical properties. Isomerism is partly responsible for the existence of a large number of organic molecules.
705

C H H H H H Isomerism gories-structural, CHEMISTRY = isomerism ) or difference in the position functional groups ( position isomerism ) difference in both molecular chains functional groups ( functional isomerism ). Structural isomerism, as such, is common in general organic Tautomerism, a type of structural occurs due to the migration of an atom or from one position to the other e.g. purines pyrimidines (Chapter 5).. Consider the molecular formula-C 2 H 6 O. There are two important isomers of this-ethyl alcohol (C 2 H 5 OH) and diethyl ether (CH 3 OCH 3 ) depicted next. H C C H H H O H H Ethyl alcohol Diethyl ether H C O C H H H H H Isomerism gories-structural, 705 = of or and more molecules. isomerism, group and. Structural isomerism The difference in the arrangement of the atoms in the molecule (i.e. molecular framework) is responsible for structural isomerism. This may be due to variation in carbon chains ( chain, INTRODUCTION TO BIOORGANIC = Structural isomerism The difference in the arrangement of the atoms in the molecule (i.e. molecular framework) is responsible for structural isomerism. This may be due to variation in carbon chains ( chain. Structural isomerism The difference in the arrangement of the atoms in the molecule (i.e. molecular framework) is responsible for structural isomerism. This may be due to variation in carbon chains ( chain, CHEMISTRY = Stereoisomerism Stereoisomerism ( Greek : stereos-space pying) is, perhaps, more relevant and to biomolecules. The differential arrangement of atoms or groups in molecules. Structural isomerism The difference in the arrangement of the atoms in the molecule (i.e. molecular framework) is responsible for structural isomerism. This may be due to variation in carbon chains ( chain, 705 = occu- important space
gives rise to stereoisomerism. Thus, stereoisomers have the same structural formula but differ in their spatial arrangement.
Stereoisomerism is
of two
types—geometric

Geometrical isomerism : This is also called cis-trans isomerism and is exhibited by certain molecules possessing double bonds. Geometrical isomerism is due to restriction of freedom of rotation of groups around a carbon-carbon double bond (C=C). Maleic acid and fumaric acid are classical examples of cis-trans isomerism.
H-G—COOH
H-C—COOH
H-—C—COOH
HOOC—C—H
Maleic
acid
(cis)
Fumaric
acid
(trans)
When similar groups lie on the same side, it is called cis isomer (Latin : cis—on the same side). On the other hand, when similar groups lie on the opposite sides, it is referred to as trans isomer (Latin : trans—across). As is observed from the above structure, maleic acid is a cis form while fumaric acid is a trans form.
Geometric isomerism is also observed in sterols and porphyrins. cis-trans isomers differ in physical and chemical properties.
Optical isomerism Optical isomers or enantiomers occur due to the presence of an asymmetric carbon (a chiral carbon). Optical isomers differ from each other in their optical activity to rotate the plane of polarized light.

An object is said to be symmetrical if it can be divided into equal halves e.g. a ball. Objects which cannot be divided into equal halves are asymmetric, e.g. hand (Fig.39.2). An asymmetric object cannot coincide with its mirror image. For instance, left hand is the mirror image of right hand and these two can never be superimposed. In contrast, a symmetrical object like a ball superimposes its image.
A carbon is said to be chiral (Greek : hand) or asymmetric when it is attached to four different groups. Their mirror images do not superimpose with each other.
Fig. 39.2 : Asymmetric and symmetric objects.
The number of possible optical isomers of a molecule depends upon the specific number of chiral carbon (n). It is given by 2".

The ordinary light propagates in all directions. However, on passing ordinary light through a Nicol prism, the plane of polarized light vibrates in one direction only (Fig.39.3).
Certain organic compounds (optical isomers) which are said to exhibit optical activity rotate the plane of polarized light either to the left or to the right.
The term levorotatory (indicated by 1 or (-) sign) is used for the substances which rotate the plane of polarized light to the left. On the other hand, the term dextrorotatory (indicated by d or (+) sign) is used for substances rotating the plane of polarized light to right (Fig.39.3).
The term racemic mixture represents equal concentration of d and | forms which cannot rotate the plane of polarized light.

While representing the configuration of chiral molecules, the configuration of glyceraldehyde is taken as a reference standard.
he general laws and principles of chemistry and physics are applicable to biochemistry as well. It is, therefore, worthwhile to have a brief understanding of some of the basic chemical and physical principles that have direct relevance to life.
It must, however, be remembered that this chapter deals with quite unrelated topics to each other.
Water is the most abundant fluid on earth. It is justifiably regarded as the solvent of life. As much as 70% of a typical cell is composed of water. The unique physical and chemical properties of water have profound biological importance. The structures of biomolecules (proteins, nucleic acids, lipids and carbohydrates) are maintained due to their interaction with water, which forms an aqueous environment. This is essential for sustaining life.

The H,O molecule exists in a bent geometry. The bond angle of H—O-H is 104.5° and the O-H bond has a distance of 0.958A°. There exists electrical polarity in H)O due to electronegativity (the power of an atom in a molecule to attract electrons) difference between H and O. This results in the polarization of a positive charge on H and a negative charge on O. Thus HO molecule, although carrying no net charge, possesses an electrical dipole. The polar character of water has tremendous biological significance.
Hydrogen bonds between HO molecules : The presence of electrical dipoles on H,O molecules is responsible for their attraction. Hydrogen bonds are formed due to polarity between two atoms with different electronegativities. Thus, in HO, the transient negative charge on the O atom of one H,O molecule and the transient positive charge on the H atom of another H,O molecule attract each other to form a hydrogen bond. The water
Fig. 40.1 : Diagrammatic representation of water structure along with hydrogen bonds.
molecules are interlinked with each other by profuse hydrogen bonding. The energy of each hydrogen bond is very small compared to that of a covalent bond. But the collective strength of H-bonds is due to their large numbers. Hydrogen bonds are important for the three-dimensional structures of biomolecules.
Water expands on freezing : Water is one of the very few substances that expands on freezing. Thus, ice has a density of 0.92 g/ml, while water at 0°C has density of 1.0 g/ml. For this reason, ice floats on water. And this property is essential to maintain water equilibrium in the environment, and to sustain life.
(Imagine that water contracted on cooling and becomes denser. In such a case, ice would sink to the bottom of seas and lakes and would never get exposed to sun rays. Thus, frozen water would permanently remain as ice. If this were to happen, earth would have a permanent ice age!)

According to Lowry and Bronsted, an acid is defined as a substance that gives off protons while base is a substance that accepts protons. Thus, an acid is a proton (H*) donor and a base is a proton acceptor. A few examples of acids and their corresponding bases are given in the next column.
HCl, Base = H + + Cl -. H 2 CO 3, Base = H + + HCO - 3. H 2 PO 4, Base = H + + HPO - 4. NH + 4, Base = H + + NH 3. H 2 O, Base = H + + OH -. CH 3 COOH, Base = H + + CH 3 COO -. HA (general), Base = H + + A - (general)
It is evident that an acid dissociates to form proton and base. On the other hand, the base combines with proton to form acid. The difference between an acid and its corresponding base (more commonly referred to as conjugate base) is the presence or absence of a proton. In general, a strong acid has a weak base while a weak acid has a strong base. For instance, strong acid HCI has weak base Cl-, weak acid HCN has a strong base CN-.
Alkalies : The metallic hydroxides such as NaOH and KOH are commonly referred to as alkalies. These compounds do not directly satisfy the criteria of bases. However, they dissociate to form metallic ion and OH" ion. The latter, being a base, accepts H* ions.
Ampholytes : The substances which can function both as acids and bases are referred to as ampholytes. Water is the best example for ampholytes.

Water is a weak electrolyte and dissociates as follows.
The proton reacts with another molecule of water to form hydronium ion (H30%*).
For the sake of convenience, the presence of proton as H3O* is ignored.
By applying the law of mass action for the dissociation of water.
Here K is a constant; the concentrations are expressed in molarity. Since the degree of dissociation is very small, the concentration of undissociated [H,O] may be taken as constant.

Kw is the dissociation constant for water. Its value is 10-'4 at 25°C.
[H+] [OH-] = 10-14
In a neutral solution
[H*]
=
[OH]
=
10-7

The acidic or basic nature of a solution is measured by H* ion concentration. The strength of H?* ions in the biological fluids is exceedingly low. For this reason, the conventional units such as moles/I or g/l are not commonly used to express H* ion concentration.
Sorenson (1909) introduced the term pH to express H* ion concentration. pH is defined as the negative logarithm of H* ion concentration.

The pH (may be considered as potential of H* ions) is a narrow scale, ranging from 0 to 14 which corresponds to 1 M solution to 10-'4 M solution of [H*] concentration.
As explained under dissociation of water, pure water has an equal concentration of H* and OH ions i.e. 10-7 M each. Thus, pure water has a pH 7 which is neutral. Solutions with pH less than 7 are said to be acidic while those with pH greater than 7 are alkaline. It must be remembered that the term acidic or alkaline are not absolute but only relative. Thus, a solution with pH 3.0 is more acidic when compared with a solution of pH 4.5.
A rise in H* concentration decreases pH while a fall in H* concentration increases pH. The reverse is true for OH~ concentration. The pH of a solution containing 1N [H*] is 0 while that containing 1N [OH] is 14.
The pH of important biological fluids is presented in Table 40.1.

Pancreatic juice, pH = 7.5. Pancreatic juice, pH = -. Pancreatic juice, pH = 8.0. Blood plasma (or whole blood), pH = 7.35. Blood plasma (or whole blood), pH = -. Blood plasma (or whole blood), pH = 7.45. Cerebrospinal fluid, pH = 7.2. Cerebrospinal fluid, pH = -. Cerebrospinal fluid, pH = 7.4. Tears, pH = 7.2. Tears, pH = -. Tears, pH = 7.4. Interstitial fluid, pH = 7.2. Interstitial fluid, pH = -. Interstitial fluid, pH = 7.4. Human milk, pH = 7.2. Human milk, pH = -. Human milk, pH = 7.4. Saliva, pH = 6.4. Saliva, pH = -. Saliva, pH = 7.0. Intracellular fluid (cytosol), pH = 6.5. Intracellular fluid (cytosol), pH = -. Intracellular fluid (cytosol), pH = 6.9. Gastric juice, pH = 1.5. Gastric juice, pH = -. Gastric juice, pH = 3.0. Urine, pH = 5.0. Urine, pH = -. Urine, pH = 7.5
The pH of a given solution can be easily altered by the addition of acids or bases. Buffers are defined as the solutions which resist change in pH by the addition of small amounts of acids or bases. A buffer usually consists of a weak acid and its salt (e.g. acetic acid and sodium acetate) or a weak base and its salt (e.g. ammonium hydroxide and ammonium chloride). Several buffers can be prepared in the laboratory. Nature has provided many buffers in the living system.

Let us consider the buffer pair of acetic acid and sodium acetate. Acetic acid, being a weak acid, feebly ionizes. On the other hand, sodium acetate ionizes to a large extent.
CH3;COOH = CH;COO-+ Ht
CH3COONa == CH;COO-+ Nat
When an acid (say HCl) is added, the acetate ions of the buffer bind with H* ions (of HCl) to form acetic acid which is weakly ionizing. Therefore, the pH change due to acid is resisted by the buffer.
H+
+
CH;COO-
—>
CH,COOH
When a base (say NaOH) is added the Ht ions of the buffer (acetic acid) combine with OH ions to form water, which is weakly
dissociated. Thus, the pH change due to base addition is also prevented by the buffer.
Buffering capacity : The efficiency of a buffer in maintaining a constant pH on the addition of acid or base is referred to as buffering capacity. It mostly depends on the concentration of the buffer components. The maximum buffering capacity is usually achieved by keeping the same concentration of the salt as well as the acid.
For a comprehensive discussion on blood buffers, refer Chapter 21.

Solutions may be regarded as mixtures of substances. In general, a solution is composed of two parts—solute and solvent. The substance that is dissolved is solute and the medium that dissolves the solute is referred to as solvent. The particle size of a solute in solution is < 1 nm.
solution
The relative concentrations of substances in a can be measured by several ways.
Per cent concentration : This represents parts per 100. The most frequently used is weight per volume (w/v) e.g. 9% saline (9 9/100 ml solution). For expressing smaller concentration, mg (10-3g), wg (10-° g), ng (10-9 g) and pg (10-!2 g) are used.
Parts per million (ppm) : This refers to the number of parts of a substance in one million parts of the solution. Thus 10 ppm chlorine means 10 ug of chlorine in 1 g of water.
Molarity (M) : It is defined as the number of moles of solute per liter solution. NaCl has a molecular weight of 58.5. To get one molar (1 M) or one mole solution of NaCl, one gram molecular weight (58.5 g) of it should be dissolved in the solvent (HjO) to make to a final total volume of 1 liter. For smaller concentrations, millimole and micromole are used.
Molality : It represents the number of moles of solute per 1,000 g of solvent. One molal solution can be prepared by dissolving 1 mole of solute in 1,000 g of solvent.
Normality : Molarity is based on molecular weight while normality is based on equivalent weight. One gram equivalent weight of an element or compound represents its capacity to combine or replace 1 mole of hydrogen. In general, the gram equivalent weight of an element or a compound is equal to its molecular weight divided by the total positive valence of the constituent ions. Thus, for NaOH and KOH, the molecular and equivalent weights are the same, while, for HSO4, equivalent weight is half of the molecular weight. The term milliequivalent per liter (mEq/l) is used for smaller concentrations.

Thomas Graham (1861), regarded as the 'father of colloidal chemistry', divided substances into two classes—crystalloids and colloids.
Crystalloids are the substances which in solution can freely pass (diffuse) through parchment membrane e.g. sugar, urea, NaCl. Colloids (Greek : glue-like), on other hand, are the substances that are retained by parchment membrane e.g. gum, gelatin, albumin. The above classification of Graham is no longer tenable, since any substance can be converted into a colloid by suitable means. For instance, sodium chloride in benzene forms a colloid.
Colloidal state : As such, there are no group of substances as colloids, rather, substances can exist in the form of colloidal state or colloidal system. Colloidal state is characterized by the particle size of 1 to 100 nm. When the particle size is <1 nm, it is in true solution. For the particle sizes >100 nm, the matter exists as a visible precipitate. Thus, the colloidal state is an intermediate between true solution and precipitate.
Phases of colloids : Colloidal state is heterowith two phases.
geneous
1. Dispersed phase (internal phase) which constitutes the colloidal particles.
2. Dispersion medium (external phase) which refers to the medium in which the colloidal particles are suspended.

Based on the affinity of dispersion medium with dispersed phase, colloids are classified as lyophobic_ and lyophilic colloids.
1. Lyophobic (Greek : solvent-hating) : These colloids do not have any attraction towards dispersion medium. When water is used as dispersion medium, the colloids are referred to as hydrophobic.
2. Lyophilic (Greek : solvent-loving) : These colloids have distinct affinity towards dispersion medium. The term hydrophilic is used for the colloids when water is the dispersion medium.
The terms gel and sol are, respectively, used to jelly-like and solution-like colloids. Emulsions are the colloids formed by two immiscible liquids (e.g. oil + water). Frequently, emulsions can be stabilized by using agents known as emulsifiers. For instance, the protein casein acts as an emulsifier for milk.
Micelles are the aggregates of colloidal particles. Soap (sodium palmitate) in water is the classical example for the micelles formation.

1. Brownian movement : The continuous and haphazard motion of the colloidal particles is known as Brownian movement.
2. Optical properties : When light is passed through a colloidal solution, it gets scattered. This phenomenon is referred to as Tyndal effect. 3. Electrical properties The colloidal particles carry electrical charges, either positive or negative. The electrical charge may be due to ionization of the colloidal particles or adsorption of the ions from the medium, or both. The stability and precipitation of colloids is determined by the ionic charges they carry. The separation of charged colloids can be achieved by the analytical technique—electrophoresis (Refer Chapter 41).
4. Osmotic pressure : Since the colloidal particles are larger in size, their contribution to osmotic pressure is relatively less.
5. Non-dialysable nature : The colloidal particles, being larger in size, cannot pass through a membrane (cellophane or parchment). The membrane, however, allows dispersion medium and smaller particles to escape through the pores. This process is referred to as dialysis and is useful for the separation of colloids.
6. Donnan membrane equilibrium : The presence of non-diffusible colloidal particles (e.g. protein) in the biological systems influences the concentration of diffusible ions across the membrane. This is an important phenomenon, the details of which are given on page 714.

1. Biological fluids as colloids These include blood, milk and cerebrospinal fluid.
2. Biological compounds as _ colloidal particles : The complex molecules of life, the high molecular weight proteins, complex lipids and polysaccharides exist in colloidal state.
3. Blood coagulation : When blood clotting occurs, the sol is converted finally into the gel.
4. Fat digestion and absorption The formation of emulsions, facilitated by the emulsifying agents bile salts, promotes fat digestion and absorption in the intestinal tract.
is on
5. Formation of urine : The filtration of urine based the principle of dialysis.
The molecules in liquids or gases are in continuous motion. Diffusion may be regarded as the movement of solute molecules from a higher concentration to a lower concentration. Diffusion is more rapid in gases than in liquids.
The smaller particles diffuse faster than the larger ones. The greater the temperature, the higher is the rate of diffusion.
Diffusion occurs in true solutions as well as in colloidal solutions.

1. Exchange of Oy and COg in lungs and in tissues occurs through diffusion.
2. Certain nutrients are absorbed by diffusion in the gastrointestinal tract e.g. pentoses, minerals, water soluble vitamins.
3. Passage of the waste products namely ammonia, in the renal tubules occurs due to diffusion.
Osmosis (Greek push) refers to the movement of solvent (most frequently water) through a semipermeable membrane.
The flow of solvent occurs from a solution of low concentration to a solution of high concentration, when both are separated by a semipermeable membrane. In a strict sense, the semipermeable membrane is expected to be permeable to the solvent and not to the solute.

Osmotic pressure may be defined as the excess pressure that must be applied to a solution to prevent the passage of solvent into the solution, when both are separated by a semipermeable membrane.
Osmosis is a colligative property i.e. a character which depends on the number of solute particles and not their nature. Osmotic pressure is directly proportional to the concentration (number) of the solute molecules or ions. Low molecular weight substances (e.g. NaCl, glucose) will have more number of molecules compared to high molecular weight substances (albumin, globulin) for unit mass. Therefore, the substances with low molecular weight, in general, exhibit greater osmotic pressure. Further, for ionizable compounds, the total osmotic pressure is equivalent to the sum of the individual pressures exerted by each ion. For instance, one molar solution of NaCl will exert double the osmotic pressure of one molar solution of glucose. This is because NaCl ionizes to Nat and CF while glucose is non-ionizable.
The solutions that exert the same osmotic pressure are said to be isoosmotic. The term isotonic is used when a cell is in direct contact with an isoosmotic solution (0.9% NaCl) which does not change the cell volume and, thus, the cell tone is maintained. A solution with relatively greater osmotic pressure is referred to as hypertonic. On the other hand, a solution with relatively lower pressure is hypotonic.
The term oncotic pressure is commonly used to represent the osmotic pressure of colloidal substances (e.g. albumin, globulin).
Units of osmotic pressure : Osmole is the unit of osmotic pressure. One osmole is the number of molecules in gram molecular weight of undissociated solute. One gram molecular weight of glucose (180 g) is one osmole. However, one gram molecular weight of NaCl (58.5 g) is equivalent to 2 osmoles, since NaCl ionizes to give two particles (Nat, CI).
Osmotic pressure of biological fluids is frequently expressed as milliosmoles. The osmotic pressure of plasma is 280-300 milliosmoles/I.

1. Fluid balance and blood volume : The fluid balance of the different compartments of the body is maintained due to osmosis. Further, osmosis significantly contributes to the regulation of blood volume and urine excretion.
2. Red blood cells and fragility : When RBC are suspended in an isotonic (0.9% NaCl) solution, the cell volume remains unchanged and they are intact. In hypertonic solution (say 1.5% NaCl), water flows out of RBC and the cytoplasm shrinks, a phenomenon referred to as crenation.
On the other hand, when the RBC are kept in hypotonic solution (say 0.4% NaCl), the cells bulge due to entry of water which often causes of membrane of RBC
rupture plasma (hemolysis).
Osmotic fragility test for RBC is employed in laboratories for diagnostic purposes. For a normal human blood, RBC begin to hemolyse in 0.45% NaCl and the hemolysis is almost complete in 0.33% NaCl. Increased fragility of RBC is observed in hemolytic jaundice while it is decreased in certain anemias.
3. Transfusion : Isotonic solutions of NaCl (0.9%) or glucose (5%) or a suitable combination of these two are commonly used in transfusion in hospitals for the treatment of dehydration, burns etc.
4. Action of purgatives : The mechanism of action of purgatives is mainly due to osmotic phenomenon. For instance, epson (MgSO4 7H,O) or Glauber's (Na,SO4 10H O) salts withdraw water from the body, besides preventing the intestinal water absorption.
5. Osmotic diuresis : The high blood glucose concentration causes osmotic diuresis resulting in the loss of water, electrolytes and glucose in the urine. This is the basis of polyuria observed in diabetes mellitus. Diuresis can be produced by administering compounds (e.g. mannitol) which are filtered but not reabsorbed by renal tubules.

6. Edema due to _ hypoalbuminemia Disorders such as kwashiorkor and glomerulonephritis are associated with lowered plasma albumin concentration and edema. Edema is caused by reduced oncotic pressure of plasma, leading to the accumulation of excess fluid in tissue spaces.
7. Cerebral edema : Hypertonic solutions of salts (NaCI, MgSO,4) are in use to reduce the volume of the brain or the pressure of cerebrospinal fluid.
8. Irrigation of wounds : Isotonic solutions are used for washing wounds. The pain experienced by the direct addition of salt or sugar to wounds is due to osmotic removal of water.
Fig. 40.2 : Diagrammatic representation of Donnan membrane equilibrium.

When membrane is freely permeable to ions (say Nat, Cl) and if the concentration of ions on both the sides is different, the ions freely diffuse to attain equal concentration. Gibbs-Donnan observed that the presence of a non-diffusible ion on one side of the membrane alters the diffusion of diffusible ions.
In the molecule sodium proteinate (NatPr-), the protein (Pr~) ion is non-diffusable through the membrane. Let us consider two sides of a compartment separated by a membrane. Initially, sodium proteinate is on side | while sodium chloride is on side II (Fig.40.2). Diffusible ions (Na*, Cl) can freely pass through the membrane. On side |, Na* ions will balance the incoming Cl ions besides Pr~ ions, while on side II Nat ions have to balance only Clions. Therefore, the concentration of Na* on side | is greater than on side II. However, from the thermodynamical point of view, at equilibrium, the concentration of Nat CF on both the sides should be the same.
Thus
Nat*ChF
(I)
=
Nat
Ch
(Il)
Since
Na?
(I)
>
Nat
(Il)
Cr
()
<Ch (Il)
Consequently, the concentration of Cl" ions should be greater on side II. Further, the total concentration of ions on side | is higher than on side Il.
The salient features of Donnan membrane equilibrium are listed next.
1. The presence of a non-diffusible ion influences the concentration of diffusible ions across the membrane.
2. The concentration of oppositely charged ions (Nat), is greater on the side of membrane containing non-diffusible ions (Pr-).
3. The concentration of similarly charged ions (CI) is higher on the side of the membrane not containing non-diffusible ions (Pr-).
4. The net concentration of total ions will be greater on the side of the membrane containing non-diffusible ions. This leads to a difference in the osmotic pressure on either side of the membrane.

1. Difference in the ionic concentrations of biological fluids : The lymph and _ interstitial fluids have lower concentration of inorganic cations (Nat, K*) and higher concentration of anions (CI) compared to plasma. This is due to the higher protein (Pr) content in plasma.
2. Membrane _ hydrolysis The relative strength of Ht and OH™ ions and, therefore, the acidic or alkaline nature on either side of a membrane, is influenced by the presence of non-diffusible ions. This phenomenon is referred to as membrane hydrolysis. Donnan membrane equilibrium explains the greater concentration of H* ions in the gastric juice.
3. Lower pH in RBC : Hemoglobin of RBC is negatively charged and, this causes accumulation of positively charged ions including H*. Therefore, the pH of RBC is slightly lower (7.25) than that of plasma (7.4).
4. Osmotic imbalance : Donnan membrane equilibrium—which results in the differential distribution of ions in different compartments of the body—partly explains the osmotic pressure differences.
5. Dialysis in renal failure Donnan membrane equilibrium is the basic principle involved in the artificial means of purifying blood by dialysis in the patients of renal failure.

Liquid or fluid has a tendency to flow which is referred to as fluidity. The term viscosity may be defined as the internal resistance offered by a liquid or a gas to flow. The property of viscosity is due to frictional forces between the layers while their movement occurs. Viscosity may be appropriately regarded as the internal friction of a liquid.
Liquids vary widely as regards their viscosity. For instance, ether has very low viscosity while honey and blood are highly viscous. Among the several factors that contribute to viscosity, density of the liquid, concentration of dissolved substances and their molecular weight and the molecular interactions are important. Increase in temperature decreases viscosity while increase in pressure increases viscosity to some extent.
Viscosity of
colloidal solutions,
particularly lyophilic colloids, is generally higher than true solutions.
Units of viscosity : The unit of viscosity is poise, after the scientist Poiseuille, who first systematically studied the flow of liquids. A poise represents dynes/cm?.

1. Viscosity of blood : Blood is about 4 times more viscous than water. The viscosity of blood is mainly attributed to suspended blood cells and colloidal plasma proteins. As the blood flows through capillaries the viscosity decreases to facilitate free flow of blood. Blood viscosity is increased in polycythemia (elevation of RBC), while it is reduced in anemia and nephritis. A more viscous blood increases cardiac work load. When dehydration occurs, the viscosity of the blood increases.
2. Viscosity change in muscle : Excitation of the muscle is associated with increase in the viscosity of the muscle fibres. This delays the change in the tension of the contracting muscle.
3. Vitreous body : This is an amorphous viscous body located in the posterior chamber of the eye. It is rich in albumin and hyaluronic acid.

4. Synovial fluid : It contains hyaluronic acid which imparts viscosity and helps in the lubricating function of joints.

A molecule in the interior of a liquid is attracted by other molecules in all directions. In contrast, a molecule on the surface is attracted only downwards and sideways and not upwards (Fig.40.3). Due to this, the surface layer behaves like a stretched film. Surface tension is the force with which the molecules on the surface are held together. \t is expressed as dynes/cm. Surface tension decreases with increase in temperature.
Due to the phenomenon of surface tension, any liquid occupies the minimum possible volume.
According to the principle of Gibbs-Thomson, the compounds which lower the surface tension get concentrated at the surface (or interface) layer while those compounds which increase surface tension get distributed in the interior portion of the liquid. In general, organic substances (proteins, lipids) decrease whereas inorganic substances (NaCl, KCl) increase surface tension.

1. Digestion and absorption of fat : Bile salts reduce the surface tension. They act as detergents and cause emulsification of fat, thereby allowing the formation of minute particles for effective digestion and absorption.
2. Hay's sulfur test : This is a common laboratory test employed for the detection of bile salts in urine of jaundice patients. Sulfur powder, when sprinkled on the surface of urine possessing bile salts, sinks. This is in contrast to a normal urine where sulfur powder floats. Hay's test is based on the principle that bile salts in urine lower surface tension which is responsible for sulfur to sink.
3. Surfactants and lung function : The low surface tension of the alveoli keeps them apart and allows an efficient exchange of gases in lungs. In fact, certain surfactants, predominantly dipalmitoyl phosphatidyl choline (dipalmitoyl lecithin) are responsible for maintaining low surface tension in the alveoli. Surfactant deficiency causes respiratory distress syndrome in the infants.
4. Surface tension and adsorption : Adsorption, being a surface phenomenon, is closely related to surface tension. Due to the coupled action of these two processes, the formation of complexes of proteins and lipids occurs in the biological systems.
5. Lipoprotein complex membranes : The structure of plasma membrane is composed of surface tension reducing substances, namely lipids and proteins. This facilitates absorption of these compounds.

Adsorption is a surface phenomenon. It is the process of accumulation of a substance (adsorbate) on the surface of another substance (adsorbent). Adsorption differs from absorption, as the latter involves the diffusion into the interior of the material.
The capacity of an adsorbent depends on the surface area. Therefore, porous substances serve as better adsorbents e.g. charcoal, alumina, silica gel. Adsorption is a dynamic and reversible process which decreases with rise in temperature.

1. Formation of enzyme-substrate complex : For the catalysis to occur in biological system, formation of enzyme-substrate complex is a
prerequisite. This happens by adsorption of substrate on the enzyme.
2. Action of drugs and poisons : On adsorption at the cell surface, drugs and poisons exert their action.

:
The principle of adsorption is widely employed in the chromatography technique for the separation and_ purification of compounds (enzymes, immunoglobulins).

Isotopes have revolutionized biochemistry when they became available to investigators soon after Second World War. Isotopes are defined as the elements with same atomic number but different atomic weights. They possess the same number of protons but differ in the neutrons in their nuclei. Therefore, isotopes (Greek : iso—equal; tope—place) occupy the same place in the periodic table. The chemical properties of different isotopes of a particular element are identical.
Isotopes are of two types—stable and unstable. The latter are more commonly referred to as radioactive isotopes and they are of particular interest to biochemists. Conventionally while representing isotopes, the atomic weight is written on upper left side of the element symbol.

They are naturally occurring and do not emit radiations (non-radioactive) e.g. deuterium (heavy hydrogen) 2H; !3C; '5N; '80. Stable isotopes can be identified and quantitated by mass spectrometry or nuclear magnetic resonance (NMR). They are less frequently used in biochemical investigations.

The atomic nucleus of radioactive isotopes is unstable and, therefore, undergoes decay. The radioactive decay gives rise to one of the following 3 ionizing radiations.
- 1. a-Rays—an a particle possessing 2 protons
ie. helium nuclei.
2. B-Rays—due to the emission of electrons.
3. y-Rays—due to emission of high energy
photons.
The radiations emmitted by radioactive nuclei are characteristic of the isotope. For instance, 3H, '4C, and 32P all emit B-particles in the respective energies of 0.018, 0.155 and 1.71 MeV.
The 6 and y emitting radioisotopes are employed in biochemical research. These isotopes are produced in nuclear reactors. The simple chemicals so produced are then converted to radiolabelled biochemicals by chemical or enzymatic synthesis.

unit of radioactive decay. It is defined as the amount of radioactivity equivalent to 1 g of radium i.e. 2.22 x 10!2 disintegrations per minute (dpm). Millicurie (mCi) and microcurie (uCi), respectively, corresponding to 2.2 x 109 and 2.2 x 10° dpm, are more commonly used.
Half-lives of isotopes The unstable radioisotopes undergo decay. The radioactivity gets reduced to half of the original within a fixed time. This represents the half-life which is characteristic for a given isotope.
Some of the commonly used radioactive isotopes in biochemical research with their characteristics are given in Table 40.2.

Several techniques are in use for the detection of radioactivity of the isotopes. The most commonly employed in biochemical research are—Geiger counters, liquid _ scintillation counter and autoradiography. Geiger counters are almost outdated. Liquid scintillation counters are now widely used.
In the liquid scintillation counter, the sample is dissolved or suspended in a_ solution containing one or two fluorescent organic compounds (fluors). The fluors emit a pulse of

3 H, Radiation = E. 3 H, Half-life = 12.2 years. 14 C, Radiation = E. 14 C, Half-life = 5,700 years. 22 Na, Radiation = J. 22 Na, Half-life = 2.5 years. 32 P, Radiation = E. 32 P, Half-life = 14.5 days. 35 S, Radiation = E. 35 S, Half-life = 87 days. 45 Ca, Radiation = E. 45 Ca, Half-life = 164 days. 59 Fe, Radiation = E , J. 59 Fe, Half-life = 45 days. 60 Co, Radiation = J. 60 Co, Half-life = 5.25 years. 125 I, Radiation = J. 125 I, Half-life = 60 days. 131 I, Radiation = E , J. 131 I, Half-life = 8.1 days
light when struck by radiation. The light, proportional to the radiation energy, can be detected. The advantage with liquid scintillation counter is that it can discriminate the particles of different energies. Thus, two or more isotopes can be simultaneously detected.
In autoradiography, the radiations are detected by its blackening of photographic film. This technique is commonly used for the detection of radioactive substances separated in polyacrylamide gel electrophoresis (PAGE).

Radioactive isotopes have become indispensable tools of biochemistry. They can be conveniently used as tracers in biochemical research since the chemical properties of different isotopes of a particular element are identical. Therefore, the living cells cannot distinguish the radioactive isotope from a normal atom.
Radioisotopes are widely used in establishing the precursor-product relationships in metabolisms and understanding of the complex metabolic pathways.
A few important application of radioisotopes are
1. By the use of isotope tracers, the metabolic origin of complex molecules such as heme, cholesterol, purines and phospholipids can be determined. As early as 1945, it was established that nitrogen atom of heme was derived from glycine. This was done by feeding rats with (!5N) glycine and detecting ('5N) heme.
2. The precursor-product relationship in several metabolic pathways has been investigated by radioisotopes. e.g. Krebs cycle, B-oxidation of fatty acids, urea cycle, fatty acid synthesis.
3. Radioisotopes are conveniently used in the study of metabolic pools (e.g. amino acid pool) and metabolic turnovers (e.g. protein turnover).
4. Certain endocrine and immunological studies also depend on the use of radioisotopes e.g. radioimmunoassay.
5. Radioisotopes are employed in elucidating drug metabolism.

Certain radioisotopes are used in the scanning of organs—thyroid gland ('3'l), bone °Sr) and kidney (1311 hippuran).
Radioactivity has been employed in the treatment of cancers. This is based on the principle that radiations produce ionizations which damage nucleic acids. Thus, the uncontrolled proliferation of cells is restricted.
Bhivctersy is
an experimental
rather than
a
- 1 Methods of DNA assay
theoretical development
science.
The understanding
of concepts
result of
continuous evidence
obtained to
state that
the in
and biochemistry
are experimentation
therein.
the
a
and
It is
no exaggeration
foundations future,
are of
course!)
based on
for for
the knowledge
present
(and of
biochemistry the
biochemical development
laboratory tools
experimentation.
of analytical
contributed sensitive
and
_
techniques has
to our
A
understanding detailed
biochemistry discussion
employed
Thus, the
sophisticated tremendously
of biochemistry.
on is
beyond
The basic
the the
principles employed
scope of
some tools
reader are
described must,
however, following
tools of
of of
the this
book.
commonly in
this chapter.
refer
Chapter techniques
related
The
27, for
the to
molecular biology,and
recombinant
DNA
1 Isolation and purification of nucleic acids
1 Nucleic acid blotting techniques
1 DNA sequencing
1 Polymerase chain reaction
1 DNA fingerprinting or DNA profiling.

Chromatography is one of the most useful and popular tools of biochemistry. It is an analytical technique dealing with the separation of closely related compounds from a mixture. These include proteins, peptides, amino acids, lipids, carbohydrates, vitamins and drugs.

as physicochemical
principles partition,
ion-exchange, such
molecular adsorption,
sieving and
affinity.
The interaction between stationary phase and mobile phase is often employed in the classification chromatography e.g. partition, adsorption, ion-exchange. Further, the classification of chromatography is also based either on the nature of the stationary phase (paper, thin layer, column), or on the nature of both mobile and stationary phases (gas-liquid chromatography). A summary of the different methods (classes) of chromatography is given in Fig.41.1.
1. Partition chromatography : The molecules of a mixture get partitioned between the stationary phase and mobile phase depending on their relative affinity to each one of the phases.
(a) Paper chromatography This technique is commonly used for the separation of amino acids, sugars, sugar derivatives and peptides. In paper chromatography, a few drops of solution containing a mixture of the compounds to be separated is applied
(spotted) at one end, usually ~2 cm above, a strip of filter paper (Whatman No. 1 or 3). The paper is dried and dipped into a_ solvent mixture consisting of butanol, acetic acid and water in 4: 1:5 ratio (for the separation of amino acids). The aqueous component of the solvent system binds to the paper and forms a stationary phase. The organic component that migrates on the paper is the mobile phase. When the' migration of the solvent is upwards, it is referred to as ascending chromatography. In descending chromatography, _ the solvent moves downwards (Fig.41.2). As the solvent flows, it takes along with it the unknown substances. The rate of migration of the molecules depends on the relative solubilities in the stationary phase (aqueous) and mobile phase (organic).
After a sufficient migration of the solvent front, the paper (chromatogram) is removed, dried and developed for the identification of the specific spots.
(b)
(TLC)
Fig. 41.2 : Paper chromatography—ascending and descending types.
Ninhydrin, complex
frequently which
forms
_
purple with
a-amino used
The chemical
spots as
acids,
a
colouring nature
is reagent.
of the
can be
known individual
identified standards
mixture.

The migration
frequently substance, such as cellulose, is employed as supporting material. Cellulose is spread as a thin layer on glass or plastic plates. The chromatographic separation is comparatively rapid in TLC.
by with
running the
of
a
expressed of
fronts)
Distance travelled
Distance as
unknown substance
is
R;
by travelled
The
R;
value characteristic
value
(ratio the
substance by
solvent of
each of
and paper,
identification
Sometimes, separate
a
given often
front substance,
solvent helps
of system
for unknown.
it
a
is rather
complex by
a
solvent single
system.
second the
difficult mixture
run
In run
is such
carried solvent
system, perpendicular
to referred
to as
chromatography separation
out by
in the
first two
_
a
to of
with
a
one case,
a
a
different direction
run.
This is
dimensional which
enhances of
Thin layer
principle described
(partition).
a
the mixture
into components.
chromatography of
TLC
is
:
the
The the
for
In paper
place of
same as
chromatography
a
paper, an
inert
In case of adsorption thin layer chromatography, adsorbents such as activated silica gel, alumina, kieselguhr are used.
Gas-liquid chromatography (GLC) This is the method of choice for the separation of volatile substances or the volatile derivatives of certain nonvolatile substances. In GLC, the stationary phase is an inert solid material (diatomaceous earth or powdered firebrick), impregnated with a non-volatile liquid (silicon or polyethylene glycol). This is packed in a narrow column and maintained at high temperature (around 200°C). A mixture of volatile material is injected into the column along with the mobile phase, which is an inert gas (argon, helium or nitrogen). The separation of the volatile mixture is based on the partition of the components between the mobile phase (gas) and stationary phase (liquid), hence the name gasliquid chromatography. The separated compounds can be _ identified and

Fig. 41.3 : Diagrammatic representation of gas-liquid chromatography (GLC).
quantitated by a detector (Fig.41.3). The detector works on the principles of ionization or thermal conductivity.
Gas-liquid chromatography
is sensitive,
rapid and
for the
biological reliable.
It is
frequently quantitative
materials used
estimation of
such as_
lipids, drugs
and vitamins.
2. Adsorption column chromatography : The adsorbents such as silica gel, alumina, charcoal powder and calcium hydroxyapatite are packed into a column in a glass tube. This serves as the stationary phase. The sample mixture in a solvent is loaded on this column. The individual components get differentially adsorbed on to the adsorbent. The elution is carried out by a buffer system (mobile phase). The — individual compounds come out of the column at different rates which may be separately collected and identified (Fig.41.4). For instance, amino acids can be identified by ninhydrin calorimetric
Fig. 41.4 : Diagrammatic representation of adsorption column chromatography.
Fig. 41.5 : Diagrammatic representation of amino acid analyser.
method. An automated column chromatography apparatus—fraction collector—is frequently used nowadays.
3. lon-exchange chromatography lonexchange chromatography involves __ the separation of molecules on the basis of their electrical charges. lon-exchange resins—cation exchangers and anion exchangers—are used for this purpose. An anion exchanger (RtA>) exchanges its anion (A~) with another anion (B-) in solution.
Similarly,
a
cation exchanger
exchanges its
cation
(C*)
in
(H+)
with another
solution.
Thus, in ion-exchange chromatography, ions in solution are reversibly replaced by ionexchange resins. The binding abilities of ions bearing positive or negative charges are highly pH dependent, since the net charge varies with
(H*R>)
cation pH. This principle is exploited in the separation
of molecules in ion-exchange chromatography.
A mixture of amino acids (protein hydrolysate)

or proteins can be conveniently separated by ion-exchange chromatography. The amino acid mixture (at pH around 3.0) is passed through a cation exchange and the individual amino acids can be eluted by using buffers of different pH. The various fractions eluted, containing individual amino acids, are allowed to react with ninhydrin reagent to form coloured complex. This is continuously monitored for qualitative and quantitative identification of amino acids. The amino acid analyser, first developed by Moore and Stein, is based on this principle (Fig.41.5).
Several types of ion exchangers are commercially available. These include polystyrene resins (anion exchange resin, Dowex 1; cation exchange resin, Dowex 50), DEAE (diethyl! aminoethyl) cellulose, CM (carboxy methyl) cellulose, DEAE-sephadex and CM-sephadex.
4. Gel filtration chromatography : In gel filtration chromatography, the separation of molecules is based on their size, shape and molecular weight. This technique is also referred to as molecular sieve or molecular exclusion chromatography. The apparatus consists of a column packed with spongelike gel beads (usually cross-linked polysaccharides) containing pores. The gels serve as molecular sieves for the separation of smaller and bigger molecules (Fig.41.6).
The solution mixture containing molecules of different sizes (say proteins) is applied to column and eluted with a buffer. The larger molecules cannot pass through the pores of gel and, therefore, move faster. On the other hand, the smaller molecules enter the gel beads and are left behind which come out slowly. By selecting the gel beads of different porosity, the molecules can be separated. The commercially available gels include Sephadex (G-10, G-25, G-100), Biogel (P-10, P-30, P-100) and sepharose (6B, 4B, 2B).
The gel-filtration chromatography be used can for an approximate determination of molecular weights. This is done by using a calibrated column with substances of known molecular weight.

5. Affinity chromatography : The principle of affinity chromatography is based on the property of specific and non-covalent binding of proteins to other molecules, referred to as ligands. For instance, enzymes bind specifically to ligands such as substrates or cofactors.
The technique involves the use of ligands covalently attached to an inert and porous matrix in a column. The immobilized ligands act as
molecular fishhooks to selectively pick up the desired protein while the remaining proteins pass through the column. The desired protein, captured by the ligand, can be eluted by using free ligand molecules. Alternately, some reagents that can break protein-ligand interaction can also be employed for the separation.
Affinity chromatography is useful for the purification of enzymes, vitamins, nucleic acids, drugs, hormone receptors, antibodies etc.
6. High performance liquid chromatography (HPLC) In general, the chromatographic techniques are slow and time consuming. The separation can be greatly improved by applying high pressure in the range of 5,000-10,000 psi (pounds per square inch), hence this technique is also referred (less frequently) to as high pressure liquid chromatography. HPLC requires the use of non-compressible resin materials and strong metal columns. The eluants of the column are detected by methods such as UV absorption and fluorescence.

The movement of charged particles (ions) in an electric field resulting in their migration towards the oppositely charged electrode is
known as electrophoresis. Molecules with a net positive charge (cations) move towards the negative cathode while those with net negative charge (anions) migrate towards positive anode. Electrophoresis is a widely used analytical technique for the separation of biological molecules such as plasma proteins, lipoproteins and immunoglobulins.
The rate of migration of ions in an electric field depends on several factors that include shape, size, net charge and solvation of the ions, viscosity of the solution and magnitude of the current employed.

Among the electrophoretic techniques, zone electrophoresis (paper, gel), isoelectric focussing and immunoelectrophoresis are important and commonly employed in the laboratory. The original moving boundary — electrophoresis, developed by Tiselius (1933), is less frequently used these days. In this technique, the U-tube is filled with protein solution overlaid by a buffer solution. As the proteins move in solution during electrophoresis, they form boundaries which can be identified by refractive index.
1. Zone electrophoresis : A simple and modified method of moving boundary electrophoresis is the zone electrophoresis. An inert supporting material such as paper or gel are used.
- (a) Paper electrophoresis : In this technique, the sample is applied on a strip of filter paper wetted with desired buffer solution. The ends of the strip are dipped into the buffer reservoirs in which the electrodes are placed. The electric current is applied allowing the molecules to migrate for sufficient time. The paper is removed, dried and stained with a dye that specifically colours the substances to be detected. The coloured spots can be identified by comparing with a set of standards run simultaneously.
For the separation of serum proteins, Whatman No. 1 filter paper, veronal or tris buffer at pH 8.6 and the stains amido black or bromophenol blue are employed. The serum proteins are separated into five distinct bands—albumin, ©4-, O -, Band y-globulins (Refer Fig.9.1). For the electrophoretic pattern of serum lipoproteins, refer Fig.14.34.
- Ss Gel electrophoresis : This technique involves the separation of molecules based on their size, in addition to the electrical charge. The movement of large molecules is slow in_ gel electrophoresis (this is in contrast to gel filtration). The resolution is much higher in this technique. Thus, serum proteins can be separated to about 15 bands, instead of 5 bands on paper electrophoresis.
The gels commonly used in gel electrophoresis are agarose and_poly-acrylamide, sodium dodecyl sulfate (SDS). Polyacrylamide is employed for the determination of molecular weights of proteins in a popularly known electrophoresis technique known as SDS-PAGE.

2. Isoelectric focussing : This technique is primarily based on the immobilization of the molecules at __ isoelectric pH during electrophoresis. Stable pH gradients are set up (usually in a gel) covering the pH range to include the isoelectric points of the components in a mixture. As the electrophoresis occurs, the molecules (say proteins) migrate to positions corresponding to their isoelectric points, get immobilized and form sharp stationary bonds. The gel blocks can be stained and identified. By isoelectric focussing, serum proteins can be separated to as many as 40 bands. Isoelectric focussing can be conveniently used for the purification of proteins.
3. Immunoelectrophoresis : This technique involves combination of the principles of electrophoresis and immunological reactions. Immunoelectrophoresis is useful for the analysis of complex mixtures of antigens and antibodies.
Fig. 41.8 : Diagrammatic representation of immunoelectrophoresis.
The complex proteins of biological samples (say human serum) are subjected to electrophoresis. The antibody (antihuman immune serum from rabbit or horse) is then applied in a trough parallel to the electrophoretic separation. The antibodies diffuse and, when they come in contact with antigens, precipitation occurs, resulting in the formation of precipitin bands which can be identified (Fig.41.8).
depends on the concentration of the absorbing molecules). And according to Lambert's law, the transmitted light decreases exponentially with increase in the thickness of the absorbing molecules (i.e. the amount of light absorbed is dependent on the thickness of the medium).
By combining the two laws (Beer-Lambert law), the following mathematical derivation can be obtained
where I = Intensity of the transmitted light
Ig
=
Intensity of
the incident
light e¢
c
=
=
ll
Molar extinction
(characteristic of
the investigated)
Concentration of
substance
(moles/I
Thickness coefficient
substance being
the absorbing
or g/dl)
of medium
through which
light passes.

When the thickness of the absorbing medium is kept constant (i.e. Lambert's law), the intensity of the transmitted light depends only on concentration of the absorbing material. In other words, the Beer's law is operative.
The ratio of transmitted light (I) to that of incident light (Ig) is referred to as transmittance (T).
Absorbance (A) or optical density (OD) is very commonly used in laboratories. The relation between absorbance and_ transmittance — is expressed by the following equation.
A
=
2
-
logig
T
=
2
-
log%
T

Photometry broadly deals with the study of the phenomenon of light absorption by molecules in solution. The specificity of a compound to absorb light at a_ particular wavelength (monochromatic light) is exploited in the laboratory for quantitative measurements. From the biochemist's perspective, photometry forms an important laboratory tool for accurate estimation of a wide variety of compounds in biological samples. Colorimeter and spectrophotometer are the laboratory instruments used for this purpose. They work on the principles discussed below.
When a light at a particular wavelength is passed through a solution (incident light), some amount of it is absorbed and, therefore, the light that comes out (transmitted light) is diminished. The nature of light absorption in a solution is governed by Beer-Lambert law.
Beer's law states that the amount of transmitted light decreases exponentially with an increase in the concentration of absorbing material (i.e. the amount of light absorbed
727
Fig. 41.9 : Diagrammatic representation of the components in a colorimeter.

Colorimeter (or photoelectric colorimeter) is the instrument used for the measurement of coloured substances. This instrument is operative in the visible range (400-800 nm) of the electromagnetic spectrum of light. The working of colorimeter is based on the principle of Beer-Lambert law (discussed above).
The colorimeter, in general consists of light source, filter sample holder and detector with display (meter or digital). A filament lamp usually serves as a light source.The filters allow the passage of a small range of wave length as incident light. The sample holder is a special glass cuvette with a_ fixed thickness. The photoelectric selenium cells are the most common detectors used in colorimeter. The diagrammatic representation of a colorimeter is depicted in

The spectrophotometer primarily differs from colorimeter by covering the ultraviolet region (200-400 nm) of the electromagnetic spectrum. Further, the spectrophotometer is more sophisticated with several additional devices that ultimately increase the sensitivity of its operation severalfold when compared to a colorimeter. A precisely selected wavelength (say 234 nm or 610 nm) in both ultraviolet and visible range can be used for measurements. In place of glass cuvettes (in colorimeter), quartz cells are used in a spectrophotometer.
The spectrophotometer has similar basic components as described for a colorimeter (Fig.41.9), and its operation is also based on the Beer-Lambert law (already discussed).
When certain compounds are subjected to light of a particular wavelength, some of the molecules get excited. These molecules, while they return to ground state, emit light in the form of fluorescence which is proportional to the concentration of the compound. This is the principle in the operation of the instrument fluorometer.

Flame photometry primarily deals with the quantitative measurement of electrolytes such as sodium, potassium and lithium. The instrument, namely flame photometer, works on the following principle. As a solution in air is finally sprayed over a burner, it dissociates to give neutral atoms. Some of these atoms get excited and move to a higher energy state. When the excited atoms fall back to the ground state, they emit light of a characteristic wavelength which can be measured. The intensity of emission light is proportional to the concentration of the electrolyte being estimated.

The ultracentrifuge was developed by a Swedish biochemist Svedberg (1923). The principle is based on the generation of centrifugal force to as high as 600,000 g (earth's gravity g = 9.81 m/s?) that allows the sedimentation of particles or macromolecules. Ultracentrifugation is an indispensable tool for the isolation of subcellular organelles, proteins and nucleic acids. In addition, this technique is also employed in the determination of molecular of macromolecules.
weights
Fig. 41.10 : Separation of subcellular fractions by differential centrifugation.
The rate at which the sedimentation occurs in ultracentrifugation primarily depends on the size and shape of the particles or macromolecules (i.e. on the molecular weight). It is expressed in terms of sedimentation coefficient(s) and is given by the formula.
where
v
@
=
=
Migration molecule
Rotation of
radians/sec
Distance in
(sedimentation)
the centrifuge
cm of
the rotor
from the
in centre
of rotor
The sedimentation coefficient has the units of seconds. It was usually expressed in units of 10~ 135 (since several biological macromolecules occur in this range), which is designated as one Svedberg unit. For instance, the sedimentation coefficient of hemoglobin is 4 x 10-!3 s or 45; ribonuclease is 2 x 10-!3 s or 2S. Conventionally, the subcellular organelles are often referred to by their S value e.g. 70S ribosome.

The cells are subjected to disruption by sonication or osmotic shock or by use of homogenizer. This is usually carried out in an isotonic (0.25 M) sucrose. The advantage with sucrose medium is that it does not cause the organelles to swell. The subcellular particles can be separated by differential centrifugation. The most commonly employed laboratory method separates subcellular organelles into 3 major fractions—nuclear, mitochondrial and microsomal (Fig.41.10).
When the homogenate is centrifuged at 700 g for about 10 min, the nuclear fraction (includes plasma membrane) gets sedimented. On centrifuging the supernatant (I) at 15,000 g for about 5 min mitochondrial fraction (that includes lysosomes, peroxisomes) is pelleted. Further centrifugation of the supernatant (II) at 100,000 g for about 60 min separates microsomal fraction (that includes ribosomes and endoplasmic reticulum). The supernatant (III) then obtained corresponds to the cytosol.
The purity (or contamination) of the subcellular fractionation can be checked by the use of marker enzymes. DNA polymerase is the marker enzyme for nucleus, while glutamate dehydrogenase and glucose 6-phosphatase are the markers for mitochondria and ribosomes, respectively. Hexokinase is the marker enzyme for cytosol.

Radioimmunoassay (RIA) was developed in 1959 by Solomon, Benson and Rosalyn Yalow for the estimation of insulin in human serum. This technique has revolutionized the estimation of several compounds in biological fluids that are found in exceedingly low concentrations (nanogram or picogram). RIA is a highly sensitive and specific analytical tool.

Radioimmunoassay combines the principles of radioactivity of isotopes and immunological reactions of antigen and antibody, hence the name.
The principle of RIA is primarily based on the competition between the labelled and unlabelled antigens to bind with antibody to form antigenantibody complexes (either labelled — or unlabelled). The unlabelled antigen is the substance (say insulin) to be determined. The antibody to it is produced by injecting the antigen to a goat or a rabbit. The specific antibody (Ab) is then subjected to react with unlabelled antigen in the presence of excess amounts of isotopically labelled ('3"l) antigen (Ag*) with known radioactivity. There occurs a competition between the antigens (Ag* and Ag) to bind the antibody. Certainly, the labelled Agt will have an upper hand due to its excess presence.
As the concentration of unlabelled antigen (Ag) increases the amount of labelled antigenantibody complex (Agt-Ab) decreases. Thus, the concentration of Agt-Ab is inversely related to the concentration of unlabelled Ag i.e. the substance to be determined. This relation is almost linear. A standard curve can be drawn by using different concentrations of unlabelled antigen and the same quantities of antibody and labelled antigen.
The labelled antigen-antibody (Ag*-Ab) complex is separated by precipitation. The radioactivity of '3'l present is Agt-Ab_ is determined.

RIA is no more limited to estimating of hormones and proteins that exhibit antigenic properties. By the use of haptens (small molecules such as dinitrophenol, which, by themselves, are not antigenic), several substances can be made antigenic to elicit specific antibody responses. In this way, a wide variety of compounds have been brought under the net of RIA estimation. These include peptides, steroid hormones, vitamins, drugs, antibiotics, nucleic acids, structural proteins and hormone receptor proteins.
Radioimmunoassay has tremendous application in the diagnosis of hormonal disorders, cancers and therapeutic monitoring of drugs, besides being useful in biomedical research.

Enzyme-linked immunosorbant assay (ELISA) is a non-isotopic immunoassay. An enzyme is used as a label in ELISA in place of radioactive isotope employed in RIA. ELISA is as sensitive as or even more sensitive than RIA. In addition, there is no risk of radiation hazards (as is the case with RIA) in ELISA.

ELISA is based on the immunochemical principles of antigen-antibody reaction. The stages of ELISA, depicted in Fig.41.11, are summarized.
1. The antibody against the protein to be determined is fixed on an inert solid such as polystyrene.
2. The biological sample containing the protein to be estimated is applied on the antibody coated surface.
3. The protein antibody complex is then reacted with a second protein specific antibody to which an enzyme is covalently linked. These enzymes must be easily assayable and produce preferably coloured products. Peroxidase, amylase and alkaline phosphatase are commonly used.
4. After washing the unbound antibody linked enzyme, the enzyme bound to the second antibody complex is assayed.
5. The enzyme activity is determined by its action on a substrate to form a product (usually coloured). This is related to the concentration of the protein being estimated.
The principle for the use of the enzyme peroxidase in ELISA is illustrated next.
Fig. 41.11 : Diagrammatic representation of enzyme-linked immunosorbant assay (ELISA).

ELISA is widely used for the determination of small quantities of proteins (hormones, antigens, antibodies) and other biological substances. The most commonly used pregnancy test for the detection of human chorionic gonadotropin (hCG) in urine is based on ELISA. By this test, pregnancy can be detected within few days after conception. ELISA is also been used for the diagnosis of AIDS.

Conventional methods
adopted in
laboratory for
the antigens
lead antibodies.
production to
the of
antisera formation
Among the
against of
heterogeneous these
antibodies
a
few may
have the desired properties but are found with many other antibodies which undoubtedly are not required. A simple, convenient and desirable method for the large scale production of specific antibodies remained a dream for immunologists for a long period. In 1975, George Kohler and Cesar Milstein (Nobel Prize 1984) made this dream a reality. They created hybrid cells that will make unlimited quantities of antibodies with defined specificities, which are termed as monoclonal antibodies (McAb). This discovery, often referred to as hybridoma technology, has revolutionized methods for antibody production.

This is based on the fusion between myeloma cells (malignant plasma cells) and spleen cells from a suitably immunized animal. Spleen cells die in a short period under ordinary tissue culture conditions while myeloma cells are adopted to grow permanently in culture. Mutants of
[rereretee' deals with the study of immunity and immune systems of vertebrates. Immunity (immunis literally means exempt/free from burden) broadly involves the resistance shown, and protection offered by the host organism against the infectious diseases. The immune system consists of a complex network of cells and molecules, and their interactions. It is specifically designed to eliminate infectious organisms from the body. This is possible since the organism is capable of distinguishing the self from non-self, and eliminate non-self.
Immunity is broadly divided into two types
—
innate (non-specific) immunity and adaptive or acquired (specific) immunity.

Innate immunity
is non-specific,
and represents the inherent capability of the organism
to offer resistance against diseases. It consists of defensive barriers.

The skin is the largest organ in the human body, constituting about 15% of the adult body weight. The skin provides mechanical barrier to prevent the entry of microorganisms and viruses. The acidic (pH 3-5) environment on the skin surface inhibits the growth of certain microorganisms. Further, the sweat contains an enzyme lysozyme that can destroy bacterial cell wall.

Despite the
physical barriers,
organisms do
enter the
itself and
body.
eliminates non-specific
the
The invading
mechanisms secretions
of also
tries
(involving the
mucus.
such the
micro- body
defends organisms
as by
sneezing
In and
addition, kill
the pathogens
macrophages the
body by
phagocytosis and
complement system).
The inflammatory
response and
fever response of the body also form a part of innate immunity.
to

The immune system represents the third and most potent defense mechanism of the body. Acquired (adaptive or specific) immunity is capable of specifically recognizing and eliminating the invading microorganisms and foreign molecules (antigens). In contrast to innate immunity, the acquired immunity displays four distinct characteristics
1 Antigen specificity
1 Recognition diversity
1» Immunological memory
1 Discrimination between self and non-self.
The body possess tremendous capability to specifically identify various antigens (antigen is a foreign substance, usually a protein or a carbohydrate that elicits immune response). Exposure to an antigen leads to the development of immunological memory. As a result, a second encounter of the body to the same antigen results in a heightened state of immune response. The immune system recognizes and responds to foreign antigens as it is capable of distinguishing self and non-self. Autoimmune diseases are caused due to a failure to discriminate self and non-self antigens.

The immune system consists of several organs distributed throughout the body (Fig. 42.1). These lymphoid organs are categorized as primary and secondary.

These organs provide appropriate microenvironment for the development and maturation of antigen-sensitive lymphocytes (a type of white blood cells). The thymus (situated above the heart) and bone marrow are the central or primary lymphoid organs. T-lymphocyte maturation occurs in the thymus while B-lymphocyte maturation takes place in the bone marrow.
Fig. 42.1 : A diagrammatic representation of human lymphatic system.

These are the sites for the initiation of immune response. e.g. spleen, tonsils, lymph nodes, appendix, Peyers patches in the gut. Secondary lymphoid organs provide the microenvironment for interaction between antigens and mature lymphocytes.

Two types of lymphocytes namely B-cells and T-cells are critical for the immune system. In addition, several accessory cells and effector cells also participate.

The site of development and maturation of B-cells occurs in bursa fabricius in birds, and bone marrow in mammals. During the course of immune response. B-cells mature into plasma cells and secrete antibodies (immunoglobulins). The B-cells possess the capability to specifically recognize each antigen and produce antibodies (i.e. immunoglobulins) against it. B-lymphocytes are intimately associated with humoral immunity. Immunoglobulins are described in Chapter 9.

The maturation of T-cells occurs in the thymus, hence the name. The T-cells can identify viruses and microorganisms from the antigens displayed on their surfaces. There are at least four different types of T-cells.
| Inducer T-cells that mediate the development of T-cells in the thymus.
1 Cytotoxic T-cells (T,), capable of recognizing and killing the infected or abnormal cells.
| Helper T-cells (Tj) that initiate immune responses.
1 Suppressor T-cells mediate the suppression of immune response.
T-lymphocytes are responsible for the cellmediated immunity.

The major histocompatibility complex (MHO), represents a special group of proteins, present on the cell surfaces of T-lymphocytes. MHC is involved in the recognition of antigens on T-cells. It may be noted here that the B-cell receptors (antibodies) can recognize antigens on their own, while T-cells can do so through the mediation of MHC.
In humans, the MHC proteins are encoded by a cluster of genes located on chromosome 6 (it is on chromosome 17 for mice). The major histocompatibility complex in humans is referred to as human leukocyte antigen (HLA). Three classes of MHC molecules (chemically glycoproteins) are known in human. Class | molecules are found on almost all the nucleated cells of the body. Class Il molecules are associated only with leukocytes involved in cell-mediated immune response. Class Ill molecules are the secreted proteins possessing immune functions e.g. complement components (C5, C4), tumor necrosis factor.

The complement system is composed of about 20 plasma proteins that 'complement' the function of antibodies in defending the body from the invading antigens. The complementary factors are heat labile and get inactivated if heated at 56°C for about 30 minutes. The complement system helps the body immunity in 4 ways
1. Complement fixation : The complement system binds to the foreign invading cells and causes lysis of the cell membranes.
2. Opsonization : The process of promoting the phagocytosis of foreign cells is referred to as opsonization.
3. Inflammatory reaction : The complement stimulates local inflammatory reaction attracts cells.
system and phagocytic
4. Clearance of antigen-antibody complexes : The complement system promotes the clearance of antigen-antibody complexes from the body.
Nomenclature of complement system : The complement proteins are designated by the letter 'C', followed by a component number—C,, Co, C; ete.
Types of
reaction
:
The complement
system brings about two sets of reactions :
1. Antibody dependent classical pathway.
2. Antibody independent alternative pathway.
Each one
of the
pathways consists
of
a
series of
reactions converting
active products
inactive by
serine precursors
proteases to
which resembles
blood coagulation.

The immune response refers to the series of reactions carried out by the immune system in the body against the foreign invader. When an infection takes place or when an antigen enters the body, it is trapped by the macrophages in lymphoid organs. The phagocytic cells which are guarding the body by constant patrolling engulf and digest the foreign substance. However, the partially digested antigen (i.e. processed antigen) with antigenic epitopes attaches to lymphocytes.
T-helper cells (T,,) play a key role the immune response (Fig. 42.2). This is brought out through
Fig. 42.2 : A diagrammatic representation of the central role of helper T cells (T,, cells) in immune response (APC—Antigen presenting cell; T, cell—Cytotoxic T-cell).
the participation of antigen presenting cell (APC), usually a macrophage. Receptors of T,, cell bind to class Il MHC-antigen complex displayed on the surface of APC. APC secretes interleukin-I, which activates the T,, cell. This activated T,, cell actively grows and divides to produce clones of T,, cells. All the T,, cells possess receptors that are specific for the MHC-antigen complex. This facilitates triggering of immune response in an exponential manner. The T,, cells secrete interleukin-2 which promotes the prolifiration of cytotoxic T cells (Tc cells) to attack the infected cells through cell-mediated immunity. Further, interleukin-2 also activates B-cells to produce immunoglobulins which — perform humoral immunity.

Cytokines are a group of proteins that bring about communication between different cell types involved in immunity. They are low molecular weight glycoproteins and are produced by lymphoid and non-lymphoid cells during the course of immune response. Cytokines may be regarded as soluble messenger molecules of immune system. They can act as short messengers between the cells or long range messengers by circulating in the blood and affecting cells at far off sites. The latter function is comparable to that of hormones.
The term interleukin (IL) is frequently used to represent cytokines. There are more than a dozen interleukins (IL-l...... IL12), produced by different cells with wide range of functions. The main function (directly or indirectly) of cytokines is to amplify immune responses and_ inflammatory responses.

It is now possible to produce cytokines in vitro. Some of the cytokines have potential applications in the practice of medicine. For instance, IL-2 is used in cancer immunotherapy, and in the treatment of immunodeficiency diseases. IL-2 induces the proliferation and differentiation of T-and_ B-cells, besides increasing the cytotoxic capacity of natural killer cells.
A group of cytokines namely interferons can combat viral infection by inhibiting — their replication.

The prime function of immune system is to protect the host against the invading pathogens. The body tries its best to overcome various strategies of infectious agents (bacteria, viruses), and provides immunity.
Some of the important immunological aspects in human health and disease are briefly described.

In general, the immune system is self-tolerant i.e. not responsive to cells or proteins of self. Sometimes, for various reasons, the immune system fails to discriminate between self and non-self. As a consequence, the cells or tissues of the body are attacked. This phenomenon is referred to as autoimmunity and the diseases are regarded as autoimmune diseases. The antibodies produced to self molecules are regarded as autoantibodies. Some examples of autoimmune diseases are listed.
1 Insulin-dependent diabetes (pancreatic P-cell autoreactive T-cells and antibodies).
1 Rheumatoid arthritis (antibodies against proteins present in joints).
1 Myasthenia gravis (acetylcholine receptor autoantibodies).
1 Autoimmune hemolytic anemia (erythrocyte autoantibodies).
Mechanism of autoimmunity : It is widely accepted that autoimmunity generally occurs as a consequence of body's response against bacterial, viral or any foreign antigen. Some of the epitopes of foreign antigens are similar (homologous) to epitopes present on certain host proteins. This results in cross reaction of antigens and antibodies which may lead to autoimmune diseases.

The phenomenon of transfer of cells, tissues or organs from one site to another (in the same organism, autograft or from another organism allograft) is regarded as organ transplantation.
In case of humans, majority of organ transplantations are allografts (between two individuals). The term xenograft is used if tissues/organs are transferred from one species to another e.g. from pig to man.
Organ transplantation is associated with immunological complications, and __ tissue rejection. This is because the host body responds to the transplanted tissue in a similar way as if it were an invading foreign organism. Major histocompatibility complex (MHC) is primarily involved in allograft rejection. This is due to the fact that MHC proteins are unique to each individual, and the immune system responds promptly to foreign MHCs.
Organ transplantation between closely related family members is preferred, since their MHCs are also likely to be closely related. And major immunological complications can be averted.

Growth of tumors is often associated with the formation of novel antigens. These tumor antigens (also referred to as oncofetal antigens e.g. a-fetoprotein) are recognized as non-self by the immune systems. However, tumors have developed several mechanisms to evade immune responses.

Acquired immunodeficiency — syndrome (AIDS), caused by human immunodeficiency virus, is characterized by immunosuppression, secondary neoplasma and __ neurological manifestations. AIDS primarily affects the cellmediated immune system which protects the body from intracellular parasites such as viruses, and bacteria. Most of the immunodeficiency symptoms of AIDS are associated with a reduction in CD, (cluster determinant antigen 4) cells.
(Ca is the study of heredity. It is appropriately regarded as the science that explains the similarities and differences among the related organisms.

For many centuries, it was customary to explain inheritance in humans through blood theory. People used to believe that the children received blood from their parents, and it was the union of blood that led to the blending of characteristics. That is how the terms 'blood relations', 'blood will tell', and 'blood is thicker than water' came into existence. They are still used, despite the fact that blood is no more involved in inheritance. With the advances in genetics, the more appropriate terms should be as follows
1 Gene relations in place of blood relations.
1 Genes will tell instead of blood will tell.

Genetics is relatively young, not even 150 years. The blood theory of inheritance was questioned in 1850s, based on the fact that the semen contained no blood. Thus, blood was not being transferred to the offspring. Then the big question was what was the hereditary substance.

: Austrian monk named Gregor Johann Mendel, for the first time reported the fundamental laws of inheritance. He conducted several experiments on the breeding patterns of pea plants. Mendel put forth the theory of transmissible factors which _ states that inheritance is controlled by certain factors passed from parents to offsprings. His results were published in 1866 in an obscure journal Proceedings of the Society of Natural Sciences.
For about 35 years, the observations made by
Mendel went
unnoticed, and
were almost
forgotten. Two European botanists (Correns and Hugo de Vries) in 1900, independently and simultaneously rediscovered the theories of Mendel. The year 1900 is important as it marks the beginning the modern era of genetics.
The origin of the word gene : In the early years of twentieth century, it was believed that the Mendel's inheritance factors are very closely related to chromosomes (literally coloured bodies) of the cells. It was in 1920s, the term gene (derived from a Greek word gennan meaning to produce) was introduced by Willard Johannsen. Thus, gene replaced the earlier terms inheritance factor or inheritance unit.
Chemical basis of heredity : There was a controversy for quite sometime on the chemical basis of inheritance. There were two groups— the protein supporters and DNA supporters. It was in 1944, Avery and his associates presented convincing evidence that the chemical basis of heredity lies in DNA, and not in protein. Thus, DNA was finally identified as the genetic material. Its structure was elucidated in 1952 by Watson and Crick.

Monozygotic or identical twins contain the same genetic material — DNA or genes. Studies conducted on identical twins make. startling revelations with regard to inheritance. One such study is described here.
Oskar Stohr and Jack Yufe were identical twins separated at birth. Oskar was taken to Germany where he was brought up by _ his grandmother as a Christian. Jack was raised by his father in Israel as a Jew. The two brothers were reunited at the age of 47. Despite the different environmental influences, _ their behavioural patterns and personalities were remarkably similar
- : Both men had moustaches, wore two pocket shirts, and wire-rimmed glasses.
1
Both loved
spicy foods
and tended
to fall
asleep in
front of
television.
- 1 Both flushed the toilet before using.
1
- Both read maganizes from back to front.
:
Both stored rubber bands on their wrists.
: Both liked to sneeze in a room of strangers.
Besides Oskar and Jack, many other studies conducted on identical twins point out the importance of genes on the inherited characters related to personality and mannerisms.

The understanding of how _ genetic characteristics are passed on from one generation to the next is based on the principles developed by Mendel.
As we know now, the human genome is organized into a diploid (2n) set of 46 chromosomes. They exist as 22 pairs of autosomes and one pair of sex chromosomes (XX/XY). During the course of meiosis, the chromosome number becomes haploid (n). Thus, haploid male and female gametes — sperm and oocyte respectively, are formed. On fertilization of the oocyte by the sperm, the diploid status is restored. This becomes possible as the zygote receives one member of each chromosome pair from the father, and the other from the mother. As regards the sex chromosomes, the males have X and Y, while the females have XX. The sex of the child is determined by the father.

The genetic traits or characters are controlled by single genes or multiple genes. The changes in genes are associated with genetic diseases.
Monogenic disorders : These are the single gene disease traits due to alterations in the corresponding gene e.g. sickle-cell anemia, phenylketonuria. Inheritance of monogenic disorders usually follows the Mendelian pattern of inheritance.
Polygenic disorders The genetic traits conferred by more than on gene (i.e multiple genes), and the disorders associated with them are very important e.g. height, weight, skin colours, academic performance, blood pressure, aggressiveness, length of life.

The pattern of inheritance of monogenic traits
The heredity is transmitted from parent to offspring as individual characters controlled by
genes.
The genes
are linearly
distributed on
- chromosomes at fixed positions called loci.
A gene may have different forms referred to as alleles. Usually one allele is transferred from the father, and the other from the mother. The allele is regarded as dominant if the trait is exhibited due to its presence. On the other hand, the allele is said to be recessive if its effect is masked by a dominant allele. The individuals are said to be homozygous if both the alleles are the same. When the alleles are different they are said to be heterozygous.
may occur in the following ways (Fig. 43.1).
1. Autosomal dominant
2. Autosomal recessive
3. Sex-linked.
1. Autosomal dominant inheritance : A normal allele may be designated as a while an autosomal dominant disease allele as A (Fig. 43.1A). The male with Aa genotype is an affected one while the female with aa is normal. Half of the genes from the affected male will carry the disease allele. On mating, the male and female gametes are mixed in different combinations. The result is that half of the

examples of genetic disorders (monogenic traits) in humans.Salient features = . Colour blindness, T ABLE 43.1 Selected examples of genetic disorders (monogenic traits) in humans.Estimated incidence = 1 : 50 males. Colour blindness, T ABLE 43.1 Selected examples of genetic disorders (monogenic traits) in humans.Salient features = Unable to distinguish colours. Hemophilia (A/B), T ABLE 43.1 Selected examples of genetic disorders (monogenic traits) in humans.Estimated incidence = 1 : 10,000 males. Hemophilia (A/B), T ABLE 43.1 Selected examples of genetic disorders (monogenic traits) in humans.Salient features = Defective blood clotting. Duchenne muscular dystrophy, T ABLE 43.1 Selected examples of genetic disorders (monogenic traits) in humans.Estimated incidence = 1 : 7000 males. Duchenne muscular dystrophy, T ABLE 43.1 Selected examples of genetic disorders (monogenic traits) in humans.Salient features = Muscle wastage. Mitochondrial, T ABLE 43.1 Selected examples of genetic disorders (monogenic traits) in humans.Estimated incidence = . Mitochondrial, T ABLE 43.1 Selected examples of genetic disorders (monogenic traits) in humans.Salient features = . Leber hereditary optic neuropathy, T ABLE 43.1 Selected examples of genetic disorders (monogenic traits) in humans.Estimated incidence = Not known. Leber hereditary optic neuropathy, T ABLE 43.1 Selected examples of genetic disorders (monogenic traits) in humans.Salient features = Damage to optic nerves, may lead to blindness
3.
Sex
(X)-linked inheritance
:
In the
Fig.
43.1C, depicted.
sex-linked
A
female
(XY)
of the
male child
male normal
pattern of
inheritance male
will
(XY)
produce children
is and
children are
affected
This children
and
a
carrier wherein,
while is
due to
possess there
(as is
no only
dominant is
the case
and one
allele with
half no
female the
fact that
the
X
chromosome, to
females).

hemophilia are
mark its
effects
Colour good
blindness
X-linked diseases.
A
selected
list
of
genetic
disorders
(monogenic
traits)
due
to
autosomal
and
sex-
linked
inheritance
in
humans
is
given
in
Table 43.1.

The pattern of inheritance and monogenic traits along with some of the associated disorders are described above (Table 43.1). Besides gene mutations, chromosomal abnormalities (aberrations) also result in genetic diseases.
Aneuploidy : The presence of abnormal number of chromosomes within the cells is referred to as aneuploidy. The most common aneuploid condition is trisomy in which three copies of a particular chromosome are present in a cell instead of the normal two e.g. trisomy-21 causing Down's syndrome; trisomy-18 that results in Edward's syndrome. These are the examples of autosomal aneuploidy.
In case of sex-linked aneuploidy, the sex chromosomes occur as three copies. e.g. phenotypically male causing Klinefelter's syndrome has XXY; trisomy-X is phenotypically a female with XXX.

Eugenics is a science of improving human race based on genetics. Improving the traits of plants and = animals through _ breeding programmes has been in practice for centuries.
Eugenics is a highly controversial subject due to social, ethical, and political reasons. The proponents of eugenics argue that people with desirable and good traits (good blood) should reproduce while those with undersirable characters (bad blood) should not. The advocates of eugenics, however, do not force any policy, but they try to convince the people to perform their duty voluntarily. The object of eugenics is to limit the production of people who are unfit to live in the society.

Germany developed its own _ eugenic programme during 1930s. A law on eugenic sterilization was passed in 1933. In a span of three years, compulsory sterilization was done on about 250,000 people, who allegedly suffered from hereditary disabilities, feeble mindedness, epilepsy, schizophrenia, blindness, physical deformaties, and drug or alcohol addiction.
The German Government committed many atrocities in the name of racial purity. Other countries, however do not support this kind of eugenics.

1. Sucrose,
2. Glyceraldehyde,
3. Epimers,
4. Anomers,
5. Aglycone,
6. Streptomycin,
7. &-1,6-Glycosidic bond,
8. Inulin,
9. Hyaluronic acid,
10. N-Acetylneuraminic acid,
11. b,
12. d,
13. a,
14. d,
15. a.
N . Dipalmitoyl lecithin,
DD . Saponification number,
VW . Stereospecific number,
RB . Triacylglycerols,
WN . Geometric isomerism (cis-trans isomerism),
. Triacylglycerolds,
. Chaulmoogric acid,
8. Phosphatidylinositol,
9. Gangliosides,
10. Cyclopentanoperhydrophenanthrene,
11. a,
12. d,
13. d,
14. c,
15. b.
1. 16%,
. L-a-Amino acids, NY
. Methionine, . Zwitterion, FW
. B-Alanine, DH
. Peptide bonds,
. Tryptophan, - 9, ON
. 1-Fluro 2,4-dinitrobenzene (FDNB), Oo
10. Denaturation,
11. b,
12. d,
13. b,
14. d,
15. a.
746
1 . Gene,
2. RNA,
3. Nucleotides,
4. Thymine,
5. 2,
6. Base + sugar + phosphate,
7 . Erwin Chargaff,
8 . 3 Hydrogen bonds (in place of 2 in A-T),
9 . B-Form,
10. CCA(5' to 3'),
11. d,
12. b,
13. ¢,
14. d,
15. d.
1. Acetylation,
WN . Riboflavin,
. Vitamin E (tocopherol),
KF . Pyridoxine (B,),

DH . Avidin,
. Pantothenic acid,
ON . Cobalamin (B, 5),
. Dermatitis, diarrhea and dementia,
Oo . Vitamin K,
10. Folic acid,
11. b,
12. d,
13. a,
14. d,
15. a.
1. In yeast,
. Ligases, 2
. Coenzyme, 3
. Denaturation, 4
. Alcohol dehydrogenase, carbonic anhydrase, 5
6. Active site,
7 . NADP+,
8 / EC. 1.1.1.1,
9 . AMP/ADP,
10. Creatine phosphokinase (CPK),
11. c,
12. d,
13. b,
14. d,
15. b.
1. B-Glycosidic bonds,
Oo . Colipase
ON . Hartnup’s disease,
DH . Parietal (oxyntic) cells, . Glutathione,
FW . Lactase (B-galactosidase), . Fiber,
NY . Raffinose,
. Arginine, lysine,
10. Mixed micelles,
11.
a,
12. d,
13. ¢,
14. b,
15. a.

1. Fibrinogen,, 1 = 1. Δ G = Δ H - T Δ S (T = Absolute temperature),. 2. Electrophoresis,, 1 = 2. Exergonic or spontaneous,. 3. Hemoglobin,, 1 = 3. Phosphoanhydride bonds,. 4. B-Lymphocytes,, 1 = 4. Phosphoarginine,. 5. IgG,, 1 = 5. Electrons,. 6. IgE,, 1 = 6. Inner mitochondrial membrane,. 7. 40-50°C,, 1 = 7. Heme (porphyrin with iron),. 8. C-reactive protein,, 1 = 8. Cytochrome oxidase (cyt a + a 3 ),. 9. Staurt factor (Xa),, 1 = 9. Cytochrome a + a 3 ,. 10. Plasmin,, 1 = 10. Superoxide dismutase,. 11. c,, 1 = 11. d,. 12. d,, 1 = 12. a,. 13. a,, 1 = 13. b,. 14. b,, 1 = 14. d,. 15. b., 1 = 15. a.
1. 574,
. Methemoglobin, NY
. Carbonic anhydrase, WwW
. 2,3-Bisphosphoglycerate, &F
. Deoxyhemoglobin, . Thalassemias, DoH
. Succinyl CoA, OmN
. Uroporphyrinogen synthase
I,
Oo
.
5
-Aminolevulinate synthase,
10. Biliverdin,
11. a,
12. a,
13. b,
14. d,
15. c.
1. Thiamine, riboflavin, lipoic acid, niacin,
pantothenic acid,
2. Absence of glucose 6-phosphatase,
. L-Gulonolactone oxidase, 3
. Sorbitol, 4
. Galactose 1-phosphate uridyltransferase, 5
6. Leucine and lysine,
7. Succinate thiokinase,
8. Uronic acid pathway,
9. Glycogenin,
10. Oxaloacetate,
11.
d,

1. Triacylglycerols,
2. HMG CoA reductase,
. . 3. Ampipathic,
: 4. Sphingomyelinase,
5. HDL,
6. 129 ATP,
7. Zellweger syndrome,
8. Citrate,
9. HDL,
‘i 1 10. Unsaturated fatty acid,
11. d,
12. a,
13. d,
14 .C,
1s b a

1. Pyridoxal phosphate,
2. Glutamate dehydrogenase,
3. Carbamoyl phosphate synthase |,
4. Glycine transaminase,
5. Tetrahydrobiopterin,
6. Dopamine,
7H . . Homogentisate,
8. Malignant carcinoid syndrome,
9. Ornithine decarboxylase,
10. Leucine,
11. b,
12. d,
13. a,
14. c,
15. a.
1. Glutamine and aspartate,
2. Allopurinol,
3.
Sodium urate,
4. Xanthine oxidase,
5. Lesch-Nyhan syndrome,
6. Thioredoxin,
. 7. Inosine monophosphate,
8. Alloxanthine,
9. Aspartate,
10. Carbamoyl phosphate synthetase II,
11. d,
12. a,
13.c,
14. b,
15.d.

1. 9-11 mg/dl. (4.5-5.5 mEq,/.),
2. Calcitriol,
. Phosph 3. Phosphorus,
4. Magnesium,
5. Sodium,
6. 3.5-5.0 mEq/l,
7. Transferrin,
8. Ceruloplasmin,
9. Gusten,
10. Selenium,
11. d,
12. a,
i. b,
15. .a.
o
1. Adenylate cyclase,
. Ca?*, WN
N . Vanillyl mandelic acid (VMA),
DW
BB . Endorphins and enkephalins,
. Anterior pituitary,
. Thyroperoxidase,
. Aldosterone,

8. Dihydrotestosterone (DHT),
9. Cholesterol,
10. Cholecystokinin (CCK),
11. a,
12. d,
13. b,
14. ¢,
15. a.

1. Heme,
2. van den Bergh reaction,
3. Alanine transaminase,
4. Alkaline phosphatase,
5. Bromosulphthalein (BSP),
6. 180 mg/dl,
7. Inulin,
8. 2ml/min,
9. Ryle's tube,
10. Pentagastrin,
11. a,
12. d,
13. ¢,
14. b,
15. b.

1. Antidiuretic hormone (ADH),
Na*,
. 285-295 milliosmoles /kg,
. Carbonic acid (H,CO,),
. Bicarbonate buffer,
20:1,
. Ammonium ion (NH}),
9. Bicarbonate (HCO3),
10. Carbonic acid (H,CO,) or CO,,
11. d,
1 2. a,
1 3. ¢,
1 4. b,
1 5. d.
1 . Collagen,
- o . Lecithin/Sphingomyelin,
ODO . Vitamin C,
ON . Actin,
DH . Glycosaminoglycans, . Sarcomere,
FW . B-oxalyl aminoalamine, . Fibrillin,
N . Glycine,
RWN £
1 5. b.
. Calcium caseinate,
©

= -4.128,
. Thyroid gland,
. Fiber,
. Carbohydrates,
KBWN
DU . Chemical score,
. 1g/kg body weight/day,
AOAN . Biological value (BV) of protein, . Sulfur containing amino acids,
OD . Iron,
-o . Plasma albumin,
2
awn 2
uw »
750

1. DNA helicase,
. Okazaki pieces, 2
. DNA polymerase III, 3
. DNA topoisomerases, 4
5. Cyclins,
6. Telomere,
7. Transposons or transposable elements,
8. Mutation,
9. Missense,
10. Hereditary nonpolyposis colon cancer,
. Genome,
. hnRNA,
Introns,
. Reverse transcriptase,
. Wobble hypothesis,
. Ribosomes, rRNA,
. Chaperones,
9. Prion diseases,
10. Protein targeting,
11. d,
12. ¢,
13. a,
14. b,
15. a.

1.
30,000-40,000,
. One cistron-one subunit concept,
. Protein-DNA complex, Rw
a,
7 d. au
. Escherichia coli,
RNA,
. Dot-blotting,
. Thermus aquaticus,
. Genomic library/DNA library,
. Site-directed mutagenesis,
. Humulin,
. Hepatitis B vaccine,
. Mouse,
. Sheep (Dolly), - oO Cc
.d, .d, BW Nn
.a,
15.c¢.

AMP, 1 = adenosine monophosphate. AMP, 2 = CEA. AMP, 3 = carcinoembryonic antigen. ANF, 1 = atrial natriuretic factor. ANF, 2 = CF. ANF, 3 = cystic fibrosis. APC, 1 = antigen presenting cell apoprotein-A. APC, 2 = CFTR. APC, 3 = cystic fibrosis transmembrane regulator. Apo-A AP sites AST, 1 = apurinic sites aspartate transaminase. Apo-A AP sites AST, 2 = cGMP. Apo-A AP sites AST, 3 = 3',5'-cyclic guanosine monophos- phate. AT, 1 = D 1 -antitrypsin aspartate. AT, 2 = C H. AT, 3 = constant heavy chain. ATCase ATP, 1 = transcarbamoylase adenosine triphosphate. ATCase ATP, 2 = CHD. ATCase ATP, 3 = coronary heart disease. BAL, 1 = British antilewisite. BAL, 2 = ChE. BAL, 3 = cholinesterase. BAO, 1 = basal acid output. BAO, 2 = Chl. BAO, 3 = chlorophyll. BHA, 1 = butylated hydroxyanisole. BHA, 2 = C L. BHA, 3 = constant light chain. BHT BMR, 1 = butylated hydroxy toluene basal metabolic rate. BHT BMR, 2 = CLIP. BHT BMR, 3 = corticotropin like intermediate lobe peptide. BNP, 1 = brain natriuretic peptide. BNP, 2 = CMP. BNP, 3 = cytidine monophosphate. BOAA, 1 = E -oxalylaminoalanine base pair. BOAA, 2 = CNS CoAorCoASH. BOAA, 3 = central nervous system coenzyme A. bp, 1 = . bp, 2 = . bp, 3 = carboxyhemoglobin. , 1 = blood. , 2 = . , 3 = . BP, 1 = pressure. BP, 2 = COHb. BP, 3 = 
752

DHA, 1 = docosahexaenoic acid. DHA, 2 = FH 4. DHAP, 1 = dihydroxyacetone phosphate. DHAP, 2 = FIGLU. , 1 = dihydroxycholecalciferol (1, 25-. , 2 = fMet. DHCC, 1 = DHCC; 24, 25-DHCC). DHCC, 2 = FMN. DHEA DHF, 1 = dehydroepiandrosterone dihydrofolate. DHEA DHF, 2 = FMNH 2 F 1-P. DHT, 1 = dihydrotestosterone. DHT, 2 = F 6-P. DIT, 1 = diiodotyrosine. DIT, 2 = Fp. dl, 1 = deciliter. dl, 2 = FSH. DMB, 1 = dimethyl benzimidazole. DMB, 2 = FTM. DMS, 1 = dimethyl sulfate. DMS, 2 = G. DNA, 1 = deoxyribonucleic acid. DNA, 2 = g. DNase, 1 = deoxyribonuclease. DNase, 2 = ' G. DNMT, 1 = DNA methyltransferase. DNMT, 2 = GABA. DNP, 1 = 2, 4-dinitrophenol. DNP, 2 = GAG. , 1 = . , 2 = Gal-Cer. DOPA, 1 = dihydroxy phenylalanine. DOPA, 2 = GAR. DPG DPP, 1 = diphosphoglycerate dimethyl allyl pyrophosphate. DPG DPP, 2 = GDH. , 1 = . , 2 = GDP. dTMP, 1 = deoxythymidine monophosphate. dTMP, 2 = GFR. E O, 1 = redox potential. E O, 2 = GGT (GT). eAG, 1 = estimated average glucose. eAG, 2 = GH. EC ECF, 1 = enzyme commission extracellular fluid. EC ECF, 2 = GHRH GIP. EDRF, 1 = endothelium-derived releasing. EDRF, 2 = GIT. , 1 = factor. , 2 = 
ethylene diamine
elongation factor essential

eukaryotic initiation factors epidermal growth factor
enzyme-linked immunosorbent assay
Embden-Meyerhof endoplasmic
reticulum enzyme-substrate complex
embryonic stem cells exist
site electron
transport fatty
acid antigen
binding flavin
adenine reduced
chain fragment
dinucleotide
FAD
fatty acid
synthase fructose 1, 6-bisphosphate
fructose
2,
6-bisphosphate crystalline
fragment food and drug administration
1-fluoro
2,
4-dinitrobenzene free
fatty acid
fibroblast growth factor tetrahydrofolate
formiminoglutamic acid
N-formylmethionine flavin
mononucleotide reduced
FMN
fructose 1-phosphate fructose
6-phosphate flavoprotein
follicle stimulating
fractional hormone
test meal
guanine, gram
change y-aminobutyric
acid glycosaminoglycans
galactocerebroside glycinamide
ribotide glutamate dehydrogenase
guanosine diphosphate
glomerular filtration rate y-glutamy!
transpeptidase growth hormone
growth hormone releasing hormone gastric inhibitory peptide
gastrointestinal tract guanosine
tetraacetate
Gla
GLC
Glu-Cer
Gly
GLUT
GN
GMP
GnRH
GRH
GRIH
G
6-P
G
6-PD
GPP
GSH
GSSG
GTP
GTT
AH
HAC
Hb
HbA,
HbA,
HbF
¢
HbO,
HBsAg
HbS
hCG
HDAC
HDL
HFCS
HGPRT
HGPS
HIAA
HIF
HIV
HLA
HLH
HMG
CoA
HMP
HNPCC
hnRNA
Hp
HPFH
HPFH
HPFC
y-carboxy glutamate gas
liquid chromatography

glucocerebroside glycine
glucose transporters glucose-nitrogen
(ratio)
guanosine monophosphate gonadotropin releasing hormone
growth hormone releasing hormone growth
hormone release-inhibiting
hormone glucose
6-phosphate glucose 6-phosphate dehydrogenase
gerany!
pyrophosphate glutathione (reduced form)
glutathione
(oxidized form)
guanosine triphosphate
glucose tolerance
test change in enthalpy
human artificial chromosome hemoglobin
adult hemoglobin
glycosylated hemoglobin etal
hemoglobin oxyhemoglobin
hepatitis B surface antigen sickle-cell
hemoglobin human chorionic gonadotropin
histone deacetylase
high density
lipoproteins high fructose-corn syrups
hypoxanthine guanine
phosphoribosyltransferase
Hutchinson-Gilford progeria
syndrome hydroxy
indole hypoxia
factor human
immunodeficiency human
acetic acid
inducible transcription
virus leukocyte
antigen helix-loop-helix
B-hydroxy
B-methylglutaryl hexose
monophosphate hereditary
nonpolyposis cancer
heterogeneous nuclear
RNA
haptoglobin hereditary
persistence hemoglobin
hereditary perristence
of fetal
of fetal
hemoglobin hereditary persistence of fetal
hemoglobin colon
CoA
HPLC
HRE
hs-CRP
Hsp
5HT
HTH
ICD
IDDM
IDL
IDP
IF
Ig
IgG
IGF
IL
IMP
INH
InsP,
InsP,
IPP
IR
ITP
IU
IV
K
MAO
MAO.
Mb
(IP)
(IP3)
high performance liquid chromatography
hormone responsive element high sensitive C-reactive protein
heat shock
protein
5-hydroxytryptamine helix-turn-helix
isocitrate dehydrogenase insulin dependent diabetes mellitus
intermediate density lipoproteins inosine diphosphate

initiation factor immunoglobulin
immunoglobulin G
insulin-like growth factor interleukins
inosine monophosphate isonicotinic acid hydrazide
(isoniazid)
inositol 1, 4-bisphosphate inositol 1, 4, 5-triphosphate
isopentenyl pyrophosphate infrared
inosine triphosphate international unit
intravenous dissociation constant
King Armstrong dissociation constant of acid
kilo base
pair kilodalton
equilibrium constant a-ketoglutarate
inhibition constant
Michaelis constant kilojoule
long acting thyroid stimulator lecithin cholesterol acyltransferase
lactate dehydrogenase low
density lipoproteins
liver function
tests luteinizing
hormone
Long interspesed elements lipotrophic hormone (lipotropin)
leukotrienes lipoprotein-a
lysergic acid diethylamide molar
maximal acid
output monoamine oxidase
myoglobin
754

2 = POMC. ng, oxymyoglobin = nanogram (10 -9 g) nerve growth. ng, pCO 2 = PPi. NGF, oxymyoglobin = factor. NGF, pCO 2 = ppm. NIDDM, oxymyoglobin = non-insulin dependent diabetes mellitus. NIDDM, pCO 2 = PRIH. NMR, oxymyoglobin = nuclear magnetic resonance. NMR, pCO 2 = PRPP. NPN, oxymyoglobin = non-protein nitrogen. NPN, pCO 2 = PT. NPU, oxymyoglobin = net protein utilization. NPU, pCO 2 = PTH. , oxymyoglobin = obese. , pCO 2 = . Ob, oxymyoglobin = . Ob, pCO 2 = PUFA. OD, oxymyoglobin = optical density. OD, pCO 2 = QPRT. OMP Osm, oxymyoglobin = orotidine monophosphate osmoles. OMP Osm, pCO 2 = RACE. PABA, oxymyoglobin = para amino benzoic acid. PABA, pCO 2 = RAIU. , oxymyoglobin = platelet-activating factor. , pCO 2 = RAPD. PAF, oxymyoglobin = . PAF, pCO 2 = ras. PAGE PAH, oxymyoglobin = polyacrylamide gel electrophoresis para amino hippurate. PAGE PAH, pCO 2 = RBC. PAPS, oxymyoglobin = phosphoadenosine phosphosulfate. PAPS, pCO 2 = RBP. , oxymyoglobin = porphobilinogen. , pCO 2 = RDA. PBG PBI, oxymyoglobin = protein bound iodine. PBG PBI, pCO 2 = rDNA. PCM, oxymyoglobin = protein-calorie malnutrition. PCM, pCO 2 = RE. PCNA, oxymyoglobin = proliferating cell nuclear antigen. PCNA, pCO 2 = 
partial presence
polymerase of
CO, chain
reaction proximal
convoluted platelet
derived tubule
polyethylene glycol
protein-energy malnutrition
phosphoenol pyruvate
protein efficiency
ratio proline,

glutamine, serine,
phosphofructokinase prostaglandins
pteroyl glutamic
negative acid
log of
(H*)
phosphatidyl inositol
inorganic phosphate
isoelectric pH
prolactin inhibitory
inositol
4, factor
5-bisphosphate negative
log of
Ka phenylketonuria
phospholipid pyridoxal
phosphate peptide
nucleic acid
partial pressure
of
O, pro-opiomelanocortin
inorganic pyrophosphate
parts per
million prolactin
release-inhibiting hormone
prolactin
5-phosphoribosyl prothrombin
1-pyrophosphate time
parathyroid hormone
phenyl thiohydantoin
polyunsaturated fatty
acids quinolinate
phosphoribosyltransferase rapid
amplification of
cDNA
radioactive iodine
random amplified
rat sarcoma
red blood
cells retinol
binding recommended
ends uptake
polymorphic
DNA
protein dietary
allowance recombinant
DNA
retinol equivalents
rough endoplasmic
(daily)
threonine reticulum

Acid (Latin) acidus—sour
Acidosis (Latin) acidus—sour; osis—condition
Albinism (Greek) albino—white
Alkali (Arabic) al-qite-ashes of saltwort
Allergy (Greek) allos—other; ergon-work
Alloseric (Greek) allo-the other
Amentia
(Latin)
amentis—-mental
Amnesia (Greek) a—not; mnesis-memory
Amphipathic (Greek) amphi-both; pathos—feeling
Amphiphilic (Greek) amphi-both; philic-love
Anaerobe (Greek) a—not; aer—air; bios-—life
Anaplerotic (Greek) ana-up; plerotikos—to fill
Androgen (Greek) aner—man; genesis—production
Anemia (Greek) a—not; haima—blood
Anorexia (Greek) a—not; orexis—appetite
Anticoagulant
anti
(Greek)-against;
coagulare
(Latin)-to curdle
(Greek)
anti-against;
metabole-
change
Arteriosclerosis
arteria_
(Latin)-artery;
sclerosis
(Greek)
hardening.
Arthritis (Greek) arthron-joint; itis—inflammation
Atherosclerosis
(Greek)
athere—porridge;
sclerosis—
hardening
Beri-beri (Singhalese)-| cannot (said twice)
Biochemistry (Greek) bios—life; chymos-juice
Biology
(Greek) bios—life; logos—discourse
Bovine
(Latin) bovinus—pertaining to cow or ox
Calorie
(Latin)
calor—heat
Cancer (Latin) crab
Carbohydrate carbo (Latin/-coal; hydor (Greek)- water
Caries
(Latin)-decay
Casein (Latin) caseus—cheese
Catabolism
(Greek)
kata-down;
ballein—-to
throw

Catalysis (Greek) kata~down; lysis-degradation
Cathepsin
(Greek)
to
digest
Cephalins (Greek) kephale-head
Cheilitis (Greek) cheilos-lip; itis—inflammation
Cheilosis (Greek) cheilos—lip; osis-condition
Chirality
(Greek)
cheir-hand
Chlorophyll
(Greek)
chloros—pale
green;
phyllon—
leaf
Cholelithiasis
(Greek)
chole—bile;
lithos—stone;
asis—
condition
Cholesterol (Greek) chole—bile; sterol-solid alcohol
Chromatography (Greek) chroma-colour; graphein -to write
Chromosome
(Greek)
chroma-—colour;
soma—body
Chyle
(Greek)
chylos—juice
Chyluria
(Greek)
chylos—juice;
auron-urine
Chyme
(Greek) chymos-juice
Appendix
II
ORIGINS
OF
IMPORTANT
BIOCHEMICAL
WORDS
:
Cirrhosis
(Greek)
kirrhos-orange-tawny;
osis—
condition
Cis
(Latin)
same
side
Coagulation
(Greek)
coagulare-to
curdle
Collagen
(Greek)
kolla—glue;
produced
genesthai-to
be
Colloid
(Greek)
kolla-glue;
eidos-form
Consanguinity
(Latin)
con-with;
sanguis—blood
Creatine (Greek) kreas—flesh
Cristae
(Latin)
crests
Cutaneous (Latin) cutis—skin
Cytology
(Greek)
kytos—cell;
logos—discourse
Cytoplasm (Greek) kytos—cell; plassein-to mould
Dermatitis
(Greek)
derma-skin;
itis—inflammation
Diabetes
mellitus
(Greek)

diabetes—running
through
(or a siphon); mellitus-sweet
Dialysis
(Greek)
dia—through;
lysis-loosening
Doctor
(Latin) docere—to teach
Eicosanoids (Greek) eikosi-twenty
Embolism
(Greek)
embolos-to
plug
Emphysema (Greek) emphysan-to inflate
Enkephalin
(Greek) in the brain
Enthalpy
(Greek)
to
warm
within
Entropy
(Greek) in turning
Enzyme
(Greek) in yeast
Erythrocyte (Greek) erythros-red; kytos—cell
Eukaryotes
(Greek)
eu-true;
karyon—nucleus
Ferrous
(Latin)
ferrum-iron
Folate
(Latin) folium-—leaf
Galactose
(Greek)
gala—milk
Gastritis
(Greek)
gaster—belly;
Gene
(Greek) genesis—descent
Genome
(Greek) genos—birth
Globin
(Latin)
globus—ball
Globulin
(Latin)
globulus—tittle
ball
Glossitis
(Greek)
glossa—tongue;
(Greek)
glycos—sweet;
lysis—dissolution
Goitre
(Latin) gultur—throat
Gonadotrophin
(Greek)
gona-generation;
trophe—
nourishment
haima
(Greek)-blood;
globus
(Latin)/-
ball
Hepatitis (Greek) hepar-liver; itis—inflammation
Hippos
(Greek) horse
Hormone
(Greek) hormain-to excite
Hydrophilic
(Greek)
hydro-water;
philic—living
(Greek) hydro-water; phobic-hating
(Greek)
hyper—above;
glycos-sweet;

haima—blood
Hypertonic (Greek) hyper-above; tonos-tension
(Greek)
hypo-below;
haima-blood
Hypotonic (Greek) hypo—below; tonos-tension
Icterus (Greek) ikteros-jaundice
Immunity
(Latin)
immunis-exempt
from
public
burden
(Latin)
inflammare-to
set
on
fire
In situ (Latin) in the correct position
Insulin
(Latin)
insula—island
In
vitro
(Latin)
in
a
test
tube
In vivo (Latin) in the living tissue
Isomerism
(Greek)
iso-equal;
mesos—part
Isotonic
(Greek)
iso—equal;
tonos—tension
Isotope
(Greek) iso—equal; topos—place
Jaundice
(French)
jaune-yellow
Keratin
(Greek)
keras—horn
Kwashiorkor (Ga-African) sickness of the deposed
child
Lactalbumin
(Greek)
lac-milk;
albumin—white
Lecithin
(Greek) lekithos-egg yolk
Lipids (Greek) lipos—fat
Lactosuria
lac
(Latin)-milk;
ovron
(Greek)-urine
Leukocytes (Geek) leukos—white; kytos—cell
Leukoderma (Greek) leukos—white; derma-skin
Ligase
(Greek)
ligate-to
bind
Malaria
(Italian)
bad
air
Malnutrition
(Latin)
malus—bad;
nutrire—
Marasmus
(Greek)
to
waste
Melanin (Greek) melan-black
758

Pellagra (Italian) -rough skin, 1 = Pellagra (Italian) -rough skin. Phagocytosis (Greek) phagein-to eat; osis-condition, 1 = kytos-cell;. Phobia (Greek) phobos-fear, 1 = Phobia (Greek) phobos-fear. Polysaccharide (Greek) poly-many;, 1 = saccharin-
sugar
Porphyrin (Greek) porphyra—purple colour
Post-prandial (Latin)-after food
Prokaryotes (Greek) pro—before; karyon-nucleus
Proteins (Greek) proteios—holding first place
Rickets (Old English) wrickken-to twist
Serum
(Latin)-whey
Sphingosine (Greek) sphingein-to bind tight
Steatorrhea
(Greek) stear—fat; rheein-to flow
Stereoisomerism (Greek) stero-space
Sterol (Greek) steros-solid; ol- alcohol
Syndrome (Greek) syn-together; dramein-to run
Thalassemia (Greek) thalassa—sea
Thermodynamics (Greek) therme-heat; dynamics—
power
Thermogenesis
(Greek)
therme-heat;
production
genesis—
Thrombosis (Greek) thrombos-—clot; osis—condition
Thylakoid (Greek) thylakos—a sac or pouch
Tocopherol (Greek) tokos—child birth; pheros—to
bear; ol-alcohol
Trans (Latin) across
Tumor
(Latin) swelling
Vitamin (coined inappropriately in 1906) (Latin)
vita—life;
amine
Xanthoma (Greek) xanthos—yellow
Xenobiotics (Greek) xenos-strange
Zwitterion (German) zwitter-hybrid.

Acetone;
acetate
—
Acetone is a ketone; acetate is
a carboxylic acid.
Acetyl CoA; acyl CoA — Acetyl CoA is a specific compound containing acetate bound to coenzyme A; acyl CoA is a general term used to refer to any fatty acid (acyl group) bound to coenzyme A.
Albumin; albinism — Albumin is a serum protein; albinism is a genetic disease in tysosine metabolism.
Amino; imino — Amino group (—NH,) is found in majority of amino acids; imino group (=NH) is present in a few amino acids like proline and hydroxyproline.
Anabolism;
catabolism
—
Anabolism
refers
to
the
biosynthetic
reactions
involving
the
formation
of
complex
molecules
from
simpler
ones;
is
concerned
with
the
of
complex
molecules
to
simpler
ones
with
a
concomitant
release
of
energy.
Anomers; epimers — Anomers refer to two stereoisomers of a sugar that differ in configuration around a single carbonyl atom; epimers are two stereoisomers that differ in configuration around one asymmetric carbon of a sugar possessing two or more asymmetric carbon atoms.
Apoenzyme;
coenzyme
—
Apoenzyme
is
the
protein
part
of
the
enzyme
(holoenzyme);
coenzyme
is
the
non-protein
organic
part
with
enzyme
activity.
Bile
pigments;
bile
salts
—
Bile
pigments
(biliverdin,
bilirubin)
are
the
breakdown
products
of
heme; bile salts are the sodium and potassium salts of bile acids (glycocholate, taurocholate) produced by cholesterol.

Biliverdin; bilirubin — Both are bile pigments. Biliverdin is produced from heme in the reticuloendothelial cells; bilirubin is formed by reduction of biliverdin.
Biotin;
biocytin
—
Biotin is
a
B-complex vitamin;
biocytin refers
to enzymes
to the
covalently bound
(through
B-Lymphocytes;
€-amino biotin
group
T-lymphocytes produce
of lysine).
—
immunoglobulins involved
B-lymphocytes
(antibodies)
in humoral
immunity;
and are
T-lymphocytes are
responsible for
cellular immunity.
Bisphosphate; diphosphate — Bisphosphate has two phosphates held separately e.g. 2,3-BPG; diphosphate has two phosphates linked together e.g. ADP.
Calcitriol;
calcitonin
—
Calcitriol
(1,25-DHCC)
is the
physiologically calcitonin
active is
a
form peptide
by thyroid
Calorimetry;
of vitamin
hormone, gland.
colorimetry the
measurement
—
of organism;
colorimetry measurement
of colour
Carboxyl;
carbonyl groups
found in
group
—COOH;
Calorimetry
D;
synthesized deals
heat with
production is
concerned with
compounds.
—
These two
are functional
organic substances;
carboxyl
Il carbonyl
—C—.
Carnitine;
creatine;
creatinine
-— Carnitine
transports activated fatty acids (acyl CoA) from by
the
cytosol to mitochondria; creatine is mostly found in the muscle as creatine phosphate, a high energy compound; creatinine is the anhydride of creatine.
Choline; cholic acid — Choline is a trimethyl quaternary base and is a constituent of acetylcholine; cholic acid is an important bile acid.

Chyle; chyme — Chyle refers to lymph with milky appearance due to chylomicrons; chyme is the partially digested food in the stomach that passes to deodenum.
Configuration; conformation — Configuration is the geometric relationship between a given set of atoms (e.g. Land D-amino acids). Conformation is the special relationship of every atom in a molecule (e.g. secondary structure of protein).
Cysteine; cystine — Both are sulfur containing nonessential amino acids. Cysteine contains sulfhydryl (—SH) group; cystine is formed by condensation of two cysteine residues and contains a disulfide (-~S—S—) group.
Dextrins; dextrans; dextrose — The first two are polysaccharides composed of glucose. Dextrins are the breakdown products of starch; dextrans are gels produced by bacteria from glucose. Dextrose is glucose in solution (dextrorotatory) used in medical practice.
Diabetes mellitus; diabetes insipidus — Diabetes mellitus is primarily an impairment in glucose metabolism due to the deficiency of, or inefficient insulin; diabetes insipidus — is characterized by excretion of large volumes of urine (polyuria), caused by the deficiency of antidiuretic hormone (ADH).
Endocytosis;
exocytosis
—
Endocytosis is
the intake
of macromolecules by the cells; exocytosis refers to the release of macromolecules from the cells to the outside.
Epinephrine;
norepinephrine
—
Both are
catecho- lamines
Epinephrine phrine
does
Exons;
introns coding
is methylated
not contain
from while
a
—
Exons for
methyl are
proteins;
the introns
DNA
are the
tyrosine.
norepine- group.
sequences intervening
DNA
sequences that
do not
code for
proteins.
GABA; PABA — y-Aminobutyric acid (GABA) is a neurotransmitter; p-aminobenzoic acid (PABA) is a vitamin.
Gene;
genome
—

A
gene
refers
to
the
DNA
fragment
of a chromosome that codes for a single polypeptide; all the genes of a cell or an organism are collectively known as genome.
Glu;
Gla
—
Glu
is
the
code
for
glutamic
acid;
Gla
is
the
code
for
y-carboxy
glutamic
acid.
Glucuronic
acid;
gluconic
acid
—
Both
are
derived
from
glucose;
oxidation
of
C,
results
in
glucuronic
acid
while
oxidation
of
C,
yields
gluconic
acid.
acid
is
produced
in
uronic
acid
pathway;
gluconic
acid
is
formed
in
hexose
shunt.
Glutaric
acid;
glutamic
acid
—
Glutaric
acid
is
a
dicarboxylic
acid;
glutamic
acid
(a-amino
glutaric
acid)
is
an
amino
acid.
Glycogen; glycogenin — Glycogen is a storage form of carbohydrate (polysaccharide) in the animal body; glycogenin is a protein which serves as a primer for the initiation of glycogen synthesis.
Glycoproteins;
mucoproteins
-
Both
are
proteins
containing
as
the
prosthetic
group.
The
term
is
used
if
the
content
is
<4%;
mucoprotein
contains
>4%
carbohydrate.
Hydrophilic;
—
refers
to
affinity
to
water;
hydrphobic
means
hatred
towards
water.
Hypercalcemia;
hyperkalemia
—
Elevation
in
serum
calcium
is
hypercalcemia;
increase

in
serum
potassium
is
hyperkalemia.
Hypocalcemia;
hypokalemia
—
refers
to
a
fall
in
serum
calcium;
is
a
decrease
in
serum
potassium.
Insulin;
inulin
—
Insulin
is
a
peptide
hormone;
inulin
is
a
polysaccharide
composed
of
fructose.
In
vivo;
in
vitro
—
In
vivo
refers
to
within
the
cell
or
organism;
in
vitro
means
in
the
test
tube.
Isoniazid;
iproniazid
—
Isoniazid
is
an
anti-
drug;
is
an
anti-
depressant
drug.
Lactam; lactim — These terms are used to represent tautomerism. Lactam indicates the existence of a molecule in keto form; lactim represents a molecule in enol form.
Lactose; lactase; lactulose - Lactose is a disaccharide; lactase is an enzyme that cleaves lactose to glucose and _ gulactose; lactulose is a disaccharide containing galactose and fructose.
Linoleic acid; linolenic acid — Both are 18 carbon unsaturated fatty acids. Linoleic acid has two double bonds; linolenic acid has three double bonds.
Lipoproteins; lipotropic factors — Lipoproteins are molecular complexes composed of lipids and proteins; lipotropic factors are the substances (e.g. choline, betaine), the deficiency of which causes accumulation of fat in liver.
6-Lipoprotein; B-lipotropin — B-Lipoprotein refers to the low density lipoproteins; B-lipotropin is a peptide hormone derived from proopiomelanocortin (POMC) peptide.

lyases; ligases — Lyases are the enzymes that catalyse the addition or removal of water, ammonia, CO, etc.; ligases catalyse the synthetic reactions where two molecules are joined together.
Malate; malonate; mevalonate — Malate is an intermediate in the citric acid cycle; malonate is a competitive inhibitor of the enzyme succinate dehydrogenase; mevalonate is an intermediate in cholesterol biosynthesis.
Melanin;
melatonin
—
Melanin is
the pigment
of skin
and hair;
synthesized melatonin
by
Maltose;
pineal maltase
maltase gland.
—
Maltose is
an enzyme
Methyl, is
that of
glucose.
methenyl;
one-carbon methylene
fragments methyl
as
(—CH,);
(—CH,-).
Molarity;
molality number
methenyl
—
Molarity of
moles molality
a
disaccharide;
cleaves maltose
to
—
All the
shown three
are in
(—CH=);
brackets, methylene
is of
a
defined solute
as the
per liter
solution;
number of
moles of
a
solute per
1,000
g
of solvent.
Nicotinic acid; nicotine — Nicotinic acid is a B-complex vitamin; nicotine is an alkaloid present in tobacco leaves.
Nucleoside; nucleotide — A nucleoside is composed of a nitrogen base and a sugar; nucleotide contains one or more phosphate groups bound to nucleoside.
Oncogens; oncogenes -— Oncogens are the chemicals that cause cancer; oncogenes are the genes causing cancer.
Osmolarity; osmolality — Osmolarity represents osmotic pressure exerted by the number of moles (milli moles) per liter solution; osmolality refers to the osmotic pressure exerted by the number of moles (milli moles) per kg solvent.

Oxidase; Oxygenase — Oxidase accepts O, but oxygen atoms are not incorporated into substrate; oxygenase incorporates one or both oxygen atoms into substrate.
is
a
hormone
Palmitate; palmitoleate — Both are even chain (16-carbon) fatty acids. Palmitate is a saturated fatty acid; palmitoleate is a monounsaturated fatty acid.
Phosphatase; phosphorylase — Phosphatase uses water to remove phosphoryl group; phosphorylase utilizes Pi to break a bond and produce a phosphorylated compound.
Phosphatidyl ethanolamine; phosphatidal ethanolamine -— Both are phospholipids. In phosphatidyl ethanolamine, the fatty acid is bound by an ester linkage. The fatty acid is held by an ether linkage in phosphatidal ethanolamine.
Phytic acid; phytanic acid — Phytic acid is formed by the addition of six phosphate molecules to inositol, it is an inhibitor of the intestinal absorption of calcium and iron; phytanic acid is an unusal fatty acid derived from phytol, a constituent of chlorophyll.
Prokaryotes; eukaryotes — Prokaryotes are the cells that lack a well defined nucleus; eukaryotes possess a well-defined nucleus.
Prolamines; protamines — Both are simple proteins. Prolamines are soluble in alcohol; protamines are basic protein soluble in NH,OH.
Pyridine; pyrimidine; pteridine — All the three are heterocyclic rings containing nitrogen, as depicted below.
Pyridine ring is found in niacin and pyridoxine; pyrimidine is present in thiamine (vitamin B,), thymine, cytosine and uracil; folic acid contains pteridine ring.
Pyridoxine; pyridoxal — Pyridoxine is the primary alcohol form of vitamin B,; pyridoxal is the aldehyde form of Bg.

RDA; SDA — RDA (recommended dietary/daily allowance) represents the quantities of nutrients to be provided in the diet daily for maintenance of good health and_ physical efficiency; specific dynamic action (SDA) is the extra heat produced by the body over and above the caloric value of foodstuffs.
Renin;
Rennin
—
Renin and
hypertension;
in by
the vasoconstriction
rennin gastric
juice is
an enzyme
responsible kidneys
causing found
for in
coagulation of
milk.
Ribosomes; ribozymes — Ribosomes are the sites of protein biosynthesis; ribozymes refer to the RNA molecules which function as enzymes.
Retinol;
retinal
—
Retinol is
the alcohol
form of
vitamin
A;
obtained by
Scleroproteins;
retinal is
the the
oxidation selenoproteins
a
group contain
of aldehyde
form retinol.
—
Scleroproteins of
fibrous proteins;
the amino
Serotonin;
acid melatonin
transmitter are
selenoproteins selenocysteine.
—
Serotonin synthesized
melatonin serotonin
in is
a
from is
a
hormone the
pineal neuro-
tryptophan;
derived gland.
Somatotropin;
somatostatin;
Somatotropin somatomedin
is the
other hormone
(GH);
growth inhibiting
hormone somatostatin;
from
—- name
for growth
hormone
(GRIH)
is also
somatomedin insulin-like
growth by
factor liver
in
-I
response
Sucrose;
sucrase to
GH
—
Sucrose sucrase
is an
enzyme refers
(IGF-I), action.
is that
glucose and fructose.
a
cleaves sucrose
to release
called to
the produced

Synthase; synthetase — Both the enzymes are concerned with biosynthetic _ reactions. Synthase does not require ATP; synthetase is dependent on ATP for energy supply. (Note : This distinction between synthase and synthetase however, is not maintained strictly by most authors).
Thiamine;
thymine
—
Thiamine is
a
vitamin thymine
(B,);
is
a
pyrimidine base
found in
DNA
Thiokinase; thiolase — Thiokinase activates fatty acids to acyl CoA; Thiolase catalyses the final reaction in B-oxidation to liberate acetyl CoA from acyl CoA.
Transcription;
translation
—
Transcription refers
to the
synthesis involves
of
RNA
the
RNA.
acid;
uronic end
product acids
protein from
DNA;
translation synthesis
acid of
are
—
purine from
the
Uric acid
is metabolism;
formed group
acid).
Ureotelic;
uricotelic mammals)
by the
the uronic
(e.g.
—
Ureotelic convert
organisms
NH,
(e.g.
glucuronic organisms
to reptiles)
acid.
Vitamin
A;
coenzyme vitamin;
A
(e.g.
urea;
uricotelic convert
—
Vitamin coenzyme
soluble
NH,
A
A
is derived
vitamin, pantothenic
acid.
to uric
is fat
soluble from
water
Uric

Several laboratory
qualitative experiments
performed to
importance non-protein
reduction is carried out in mild acidic medium. Since acidic medium is not favourable for reduction, only strong reducing sugars (monosaccharides) give this test positive. Thus, Barfoed's test serves as a key reaction to distinguish monosaccharides form disaccharides.
the indentify
the compounds
(carbohydrates, are
of biochemical
proteins/amino nitrogenous
substances)
abnormal constituents
acids, and
to of
urine.
detect
The principles
the reactions
pertaining to
the most
widely of
employed qualitative tests are described here.

The carbohydrates used in the laboratory for the qualitative tests include glucose and fructose (monosaccharides), sucrose, lactose and maltose (disaccharides) and starch (polysaccharide). The principles of the reactions of carbohydrates are given :
1. Molisch test : It is a general test for the detection of carbohydrates. The strong H2SO4 hydrolyses carbohydrates (polyand disaccharides) to liberate monosaccharides. The monosaccharides get dehydrated to form furfural (from pentoses) or hydroxy methylfurfural (from hexoses) which condense with -naphthol to form a violet coloured complex.
2. lodine test : Polysaccharides combine with iodine to form a coloured complex. Thus, starch gives blue colour while dextrins give red colour with iodine.
3. Benedict's test : This is a test for the identification of reducing sugars, which form enediols (predominantly under alkaline conditions). The enediol forms of sugars reduce cupric ions (Cu2+) of copper sulfate to cuprous ions (Cut) which form a yellow precipitate of cuprous hydroxide or a red precipitate of cuprous oxide.
4.
Barfoed’s test
:
The principle
of this
test is
the same
as that
of
Benedict’s test
except that
the
5. Seliwanoff's test : This is a specific test for ketohexoses. Concentrated hydrochloric acid dehydrates ketohexoses to form furfural derivatives which condense with resorcinol to give a cherry red complex.
6. Foulger's test : This is also a test for ketohexoses. The furfural derivatives formed from ketohexoses condense with urea in the presence of stannous chloride to give a blue colour.
7.
Rapid furfural
test
:
Ketohexoses are
converted to
furfural derivatives
by
HCI
which form
a
purple colour
complex with
a-naphthol.

8. Osazone test : Phenylhydrazine in acetic acid, when boiled with reducing sugars forms osazones. The first two carbons (C; and C9) are involved in this reaction. The sugars that differ in their configuration on these two carbons give the same type of osazones, since the difference is marked by binding with phenylhydrazine. Thus, glucose, fructose and mannose give the same type (needle shaped) of osazones. However, the osazones of reducing disaccharides differ — maltose gives sunflower-shaped while lactose powder-puff shaped.
9. Sucrose hydrolysis test : Sucrose is a nonreducing sugar, hence it does not give Benedict's and Barfoed's tests. Sucrose can be hydrolysed by concentrated HCl, to be converted to glucose and fructose (reducing monosaccharides) which answer the reducing reactions. However, after sucrose hydrolysis, the medium has to be made alkaline (by adding NajCO3) for effective reduction process.

The
proteins
employed
in
the
laboratory
for
the
qualitative
tests
include
albumin,
globulins,
casein,
gelatin
and
peptones.
The
principle
of
the
most
common
reactions
of
proteins/amino
acids
performed in the laboratory are given hereunder.

Proteins exist in colloidal solution due to hydration of polar groups (-COO~, —NH%3, —OH). They can be precipitated by dehydration or neutralization of polar groups. Several methods are in use to achieve protein precipitation.
1. Precipitation by neutral salts : The process of protein precipitation by the addition of neutral salts such as ammonium sulfate or sodium sulfate is referred to as salting out. This phenomenon is explained on the basis of dehydration of protein molecules by salts. This causes increased proteinprotein interaction, resulting in molecular aggregation and precipitation.
The amount of salt required for protein precipitation depends on the size (molecular weight) of the protein molecule. In general, the higher is the protein molecular weight, the lower is the salt required for precipitation. Thus, serum globulins are precipitated by half saturation with ammonium sulfate while albumin is precipitated by full saturation.
2. Precipitation by salts of heavy metals : Heavy metal ions like Pb2+, Hg2+, Fe2+, Zn2+, Cd2+ cause precipitation of proteins. These metals being positively charged, when added to protein solution (negatively charged) in alkaline medium result in precipitate formation.
3. Precipitation by anionic or alkaloid reagents : Proteins can be precipitated by trichloroacetic acid, sulphosalicylic acid, phosphotungstic acid, picric acid, tannic acid, phosphomolybdic acid etc. By the addition of these acids, the proteins existing as cations are precipitated by the anionic form of acids to produce protein-sulphosalicylate, protein-tungstate, protein-picrate etc.
The anionic
reagents such
as phosphotungstic
acid and
trichloroacetic protein-free
filtrate of
acid are
blood used
to prepare
needed for
several estimations
(e.g., urea,
sugar)
in the
laboratory.
- 4. Precipitation by organic solvents : Organic
solvents such
as alcohol
are good
protein precipitating agents. They dehydrate the protein molecule by removing that water envelope and cause precipitation.

The proteins give several colour reactions which are often useful to identify the nature of the amino acids present in them as shown in the table.
Colour reactions of proteins/amino acids
1.
Biuret reactions
:
Biuret is
a
compound formed
by heating
with dilute
purple urea
to copper
colour test
used
180°C.
When sulfate
is obtained.
for biuret
is in
alkaline
This identification
treated medium,
is the
of basis
a
of biuret
proteins and
peptides.
Biuret test
is answered
by compounds
containing two or more CO—NH groups i.e., peptide bonds. All proteins and peptides possessing atleast two peptide linkages i.e., tripeptides (with 3 amino acids) give positive biuret test. The principle of biuret test is conveniently used to detect the presence of proteins in biological fluids. The mechanism of biuret test is not clearly known. It is believed that the colour is due to the formation of a copper co-ordinated complex.
2. Ninhydrin reaction : The o-amino acids react with ninhydrin to form a purple, blue or pink colour complex (Ruhemann's purple).
Amino acid + Ninhydrin
——>
Keto acid + NH3 + CO» + Hydrindantin
Hydrindantin + NH3 + Ninhydrin
——>
Ruhemann's purple
3. Xanthoproteic reaction : Xanthoproteic reaction is due to nitration of aromatic amino acids (tryptophan, tyrosine and phenylalanine) on treatment with strong nitric acid at high temperature.
4. Millon's test : This test is given by the amino acid tyrosine, or any other compound containing hydroxyphenyl ring. A red colour or precipitate is obtained in this reaction due to the formation of mercury complex of nitrophenol derivative.
5. Hopkins-Cole reaction : This reaction is specific for the indole ring of tryptophan. It combines with formaldehyde in the presence of the oxidizing agent (sulfuric acid with mercuric sulfate) to form a violet or purple coloured compound.

6. Sakaguchi reaction : Arginine, containing guanidino group, reacts with a-naphthol and alkaline hypobromite to form a red colour complex.
7. Sulfur test : This is a test specific for sulfur containing amino acids namely cysteine and cystine, but not methionine. When cysteine and cystine are boiled with sodium hydroxide, organic sulfur is converted to inorganic sodium sulfide. This reacts with lead acetate to form a black precipitate of lead sulfide. Methionine does not give this test, since sulfur of methionine is not split by alkali.
8. Pauly's test : This reaction is specific for histidine (imidazole ring). Diazotised sulfanilic acid reacts with imidazole ring in alkaline medium to form a red coloured complex.
9. Molisch test : This is a specific test for the detection of carbohydrates. The proteins containing carbohydrates (e.g., glycoproteins) give this test positive. Albumin contains carbohydrate bound to it, hence answers Molisch test.

The non-protein nitrogenous (NPN) substances of biochemical importance include urea, uric acid and creatinine.
1. Sodium hypobromite test : This is a test for the detection of urea. Sodium hypobromite decomposes urea to liberate nitrogen. The latter can be identified by brisk effervescence.
2.
Specific
urease
test
:
The
enzyme
urease
(source-
horse
gram)
specifically
acts
on
urea
to
liberate
ammonium
carbonate
(alkali).
The
latter
can
be
identified by a colour change in phenophthalein indicator (pink colour in alkaline medium).
3.
Benedict’s uric
acid test
:
Uric acid,
being
a
strong reducing agent, reduces phosphotungstate to tungsten blue in alkaline medium.
- 4. Murexide test : Uric acid is oxidized by nitric acid to give purpuric acid (reddish yellow). This in turn combines with ammonia to form purple red colour ammonium purpurate (murexide).
5.
Jaffe’s test
:
Creatinine reacts
with picric
acid in
alkaline medium
to form
orange red
colour complex.

Urine is the most important excretory fluid from the body. Some of the diseases are associated with an excretion of abnormal constituents in urine. The identification of such compounds in urine is of great diagnostic importance.

Associated disorder(s)
constituent
Albumin
Glucose
Bile salts
Bile pigments
Kidney damage (glomerulonephritis)
Damage to
kidneys or
urinary tract.
renal glycosuria.
Diabetes mellitus, starvation.
Obstructive jaundice and hepatic jaundice.
1.
Sulfosalicylic acid
test
:
Proteins get
precipitated by sulfosalicylic acid by forming proteinsulfosalicylate.
- 2. Heat coagulation test : This is a test for the detection of albumin and/or globulins in urine. Heat coagulation test is based on the principle of denaturation of proteins, followed by coagulation.
(Note
:
Small amounts
of dilute
are added
to dissolve
the phosphates
and sulfates
that get
precipitated on
heating.)
3. Benzidine test : This test detects the presence of blood. Hemoglobin (acts like peroxidase) decomposes hydrogen peroxide to liberate nascent oxygen (O-) which oxidises benzidine to a green or blue coloured complex.
(Note : Pus cells of urine possess peroxidase activity
which interferes
in benzidine
test.
This can
be eliminated
by boiling
inactivate the enzyme).
the urine
prior to
the test
to
4. Benedict's test : This is a semiquantitative test for the detection of urine reducing sugars (primarily glucose). Benedict's test is based on the principle of reducing property of sugars (described in detail under reactions of carbohydrates). Colour of the precipitate formed indicates the approximate amount of glucose present in urine. Thus, green turbidity = traces; green precipitate = 0.5%; yellow precipitate = 1%; orange precipitate = 1.5% brick red precipitate = 2%. (Note Benedict's test is not specific to glucose, since it can be answered by any reducing substance).

5. Glucose oxidase test : This is a strip test for the specific detection of glucose. The enzyme glucose oxidase oxidizes glucose to liberate hydrogen peroxide which in turn is converted to nascent oxygen (O-) by peroxidase enzyme. The compound O-diansidine combines with nascent oxygen to form a coloured (yellow to red) complex.
- 6. Rothera's test : Nitroprusside in alkaline medium reacts with keto group of ketone bodies (acetone and acetoacetate) to form a purple ring. This test is not given by B-hydroxybutyrate.
7. Hay's test : This test is based on the surface tension lowering property of bile salts (sodium glycocholate and sodium taurocholate). Sulfur powder sprinkled on the surface of urine containing bile salts sinks to the bottom.
8. Petternkofer's test : This test is employed for the detection of bile salts. The furfural derivatives (by reacting sugar with concentrated H2SO,4) condense with bile salts to form a purple ring.
9. Gmelin's test : Nitric acid oxidizes the bile pigment bilirubin to biliverdin (green) or bilicyanin (blue). Gmelin's test gives a play of colours and is used for the identification of bile pigments.
10. Fouchet's test : This test is also employed for the detection of bile pigments. Bile pigments are adsorbed on barium sulfate. Fouchet's reagents (containing ferric chloride in trichloroacetic acid) oxidizes bilirubin to biliverdin (green) and bilicyanin (blue).

Quantitative experiments, dealing with the determination of concentrations of several biologically important compounds and the assay of many enzymes, are of great significance in the laboratory practice. Very often, the ultimate diagnosis and prognosis of a large number of diseases are guided by the quantitative biochemical investigations.
The principles involved in some of the quantitative experiments, commonly employed in the biochemistry laboratory by an undergraduate student, are briefly described here.

The quantitative determination of blood (plasma/ serum) glucose is of great importance in the diagnosis and monitoring of diabetes mellitus.

Folin Wu method Alkaline copper (cupric ions) is reduced by glucose when boiled with protein free blood filtrate to cuprous oxide. The cuprous oxide in turn reacts with phosphomolybdic acid to form blue coloured oxides of molybdenum. The intensity of the colour can be measured in a colorimeter at a wavelength 680 nm. {Folin Wu method is rather old and is not specific for glucose determination, since other substances (e.g., fructose, lactose, glutathione) also bring about reduction. Consequently the blood glucose level when estimated by Folin Wu method is higher i.e., normal fasting is 80-120 mg/dl against true glucose 60-100 mg/dl]
- (ii) O-Toluidine method : Glucose combines with O-toluidine when boiled in acid medium to form a green coloured complex which can be measured in a colorimeter at a wavelength 630 nm. (This method determines glucose alone).
(iii)
Glucose oxidase-peroxidase
(GOD—POD)
method
This is
an determination
gets of
blood oxidized
by enzymatic
glucose.
glucose
Glucose oxidase
to
gluconic acid and hydrogen peroxide. The enzyme peroxidase converts hydrogen peroxide to water and oxygen. The oxygen in turn reacts with 4-aminophenzone in the presence of phenol to form a pink coloured complex, the intensity of which can be measured at 530 nm.

Determination of blood urea (reference range 10-40 mg/dl) is important for the evaluation of kidney (renal) function. Elevation of blood urea is associated with pre-renal (diabetic coma, thyrotoxicosis), renal (acute glomerulonephritis, polycystic kidney) and post-renal (obstruction in the urinary tract, due to tumors, stones) conditions.
Diacetyl monoxime (DAM) method : Urea when heated with diacetyl] monoxime forms a yellow coloured complex of dioxime derivatives which can be measured at 520 nm.

Estimation of serum creatinine (reference range 0.5-1.5 mg/dl) is used as a diagnostic test to assess kidney function. Serum creatinine is not influenced by endogenous and exogenous factors, as is the case with urea. Hence, some workers consider serum creatinine as a more reliable indicator of renal function.
Alkaline picrate method : This method is based on Jaffe's reaction. Creatinine reacts with alkaline picrate to form creatinine picrate, an orange red coloured complex, which can be measured in a colorimeter at 530 nm.
(Note : Urinary creatinine can also be determined by employing the same principle given above).

The normal concentration of total serum proteins is in the range 6-8 g/dl (albumin 3.5-5.0 g/dl; globulins 2.5-3.5 g/dl; A/G ratio is 1.2 to 1.5 : 1). The A/G ratio is lowered either due to a decrease in albumin or an increase in globulins.
Serum albumin concentration is decreased in liver diseases, severe protein malnutrition, and excretion of albumin in urine (due to renal damage). Serum globulin concentration is elevated in chronic infections and multiple myeloma.
Biuret method : Peptide bonds (~CO—NH) of proteins react with cupric ions in alkaline medium to form a violet colour complex which is measured at a wavelength 530 nm. This method is suitable for total serum proteins with estimation.

technique is employed for the estimation of serum albumin. BCG dye reacts with albumin to form an intense blue-green coloured complex which can be measured at 628 nm.

The total bilirubin concentration in serum is 0.2-1 mg/dl (conjugated ~ 0.6 mg/dl; unconjugated ~0.4 mg/dl). Elevation in serum bilirubin concentration is observed in jaundice. Unconjugated bilirubin is increased in hemolytic jaundice, conjugated bilirubin in obstructive jaundice, while both of them are increased in hepatic jaundice.
van den Bergh reaction : Serum bilirubin estimation is based on van den Bergh reaction. The principle of the reaction is that diazotised sulfanilic acid (formed by mixing equal volumes of sulfanilic acid in HCI and sodium nitrite) reacts with bilirubin to form a purple coloured azobilirubin which can be measured at 540 nm.

Serum cholesterol concentration (reference range 150-225 mg/dl) is elevated in atherosclerosis, diabetes mellitus, obstructive jaundice and hypothyroidism. Decreased levels are observed in hyperthyroidism.
Acetic anhydride method : Serum cholesterol reacts with acetic anhydride in the presence of glacial acetic acid and concentrated H2SO, to form a green coloured complex. Intensity of this colour is measured at 560 nm.

Uric acid is the end product of purine metabolism. Its concentration in serum is increased (reference range -men 4-8 mg/dl; women 3-6 mg/ dl) in gout.
Henry-Caraway's method : Uric acid in the protein-free filtrate when treated with phosphotungstic acid in the presence of sodium carbonate (alkaline solution) gives a blue coloured complex which can be measured at 660 nm.

Serum calcium level is elevated (reference range
9-11 mg/dl) in hyperparathyroidism and decreased in hypothyroidism.
O-Cresolphthalein complexone method : Calcium reacts with the dye, O-cresolphthalein complexone (CPC) in alkaline solution to form a complex which can be measured at a wavelength 660 nm.

Serum phosphate (reference range 3-4.5 mg/dl) is increased in hypoparathyroidism, and decreased in hyperparathyroidism and renal rickets.
For the determination of serum phosphate, serum proteins are precipitated by trichloroacetic acid. The protein-free filtrate containing inorganic phosphate is reacted with molybdic acid reagent to form phosphomolybdate. The latter in turn is reduced to molybdenum blue by treatment with 1-amino 2naphthol-4 sulfonic acid (ANSA). The intensity of the blue colour is measured at 689 nm.

Serum glutamate pyruvate transaminase (SGPT; alanine transaminase) and serum glutamate oxaloacetate transaminase (SGOT; aspartate transaminase) are two important diagnostic enzymes. SGPT activity (reference range 5-40 IU/L) is more specifically increased in liver diseases (hepatic jaundice). SGOT activity is elevated (reference range 5-45 IU/L) in heart diseases (myocardial infarction).

reaction
L-Alanine +
a-ketoglutarate
—>
L-glutamate + pyruvate
SGOT brings about the following reaction
L-Aspartic acid +
—>
L-glutamate + oxaloacetate
The keto acid (pyruvate or oxaloacetate), formed in the above reaction, when treated with 2, 4-dinitropheny! hydrazine forms dinitropheny| hydroazone (brown colour) in alkaline medium which can be measured at 505 nm.

The activity of the enzyme serum alkaline phosphatase (normal range 3-13 KA Units/dl) is elevated in rickets and obstructive jaundice.

hydrolyses disodium phenylphosphate liberating phenol. On treatment with 4-amino antipyrine in alkaline medium, phenol gives ferricyanide (reddish colour) which can be measured at 520 nm.

Serum amylase activity is increased (reference range 80-180 Somogyi Units/dl) in acute pancreatitis.
Principle of assay : Amylase acts on starch and hydrolyses to dextrins and maltose. Starch forms blue coloured complex with iodine, a decrease in the colour (measured at 670 nm) is proportional to the activity of amylase.

Cerebrospinal fluid (CSF) is the aqueous medium surrounding the brain and spinal cord. From the biochemical perspective, estimation of proteins and glucose in CSF is important. Increase in protein (reference range 15-40 mg/dl) and decrease in glucose (reference range 50-75 mg/dl) in the cerebrospinal fluid are observed in tuberculosis meningitis.
CSF protein estimation : Sulfosalicylic acid (in sodium sulfate solution) precipitates CSF proteins and the turbidity is measured at 680 nm.
CSF glucose estimation : Any one of the standard methods employed for the determination of blood glucose (already described) can be used for CSF glucose estimation.

The ultimate application of the biochemistry subject is for the health and welfare of mankind. Clinical biochemistry (also known as clinical chemistry or chemical pathology) is the laboratory service absolutely essential for medical practice. The results of the biochemical investigations carried out in a clinical chemistry laboratory will help the clinicians to determine the diseases (diagnosis) and for follow-up of the treatment/recovery from the illness (prognosis). Biochemical investigations hold the key for the diagnosis and prognosis of diabetes mellitus, jaundice, myocardial infarction, gout, pancreatitis, rickets, cancers, acid-base imbalance etc. Successful medical practice is unimaginable without the service of clinical biochemistry laboratory.

The biological fluids employed in the clinical biochemistry laboratory include blood, urine, cerebrospinal fluid and pleural fluid. Among these, blood (directly or in the form of plasma or serum) is frequently used for the investigations in the clinical biochemistry laboratory.

Venous blood is most commonly used for a majority of biochemical investigations. It can be drawn from any prominent vein (usually from a vein on the front of the elbow). Capillary blood (<0.2 ml) obtained from a finger or thumb, is less frequently employed. Arterial blood (usually drawn under local anesthesia) is used for blood gas determinations.
Precautions for blood collection : Use of sterile (preferably disposable) needles and syringes, cleaning of patients skin, blood collection in clean and dry vials/tubes are some of the important precautions.
Potasium oxalate and sodium fluoride : These anticoagulants are employed for collecting blood to estimate glucose. Further sodium fluoride inhibits glycolysis and preserves blood glucose concentration. Ammonium oxalate and potassium oxalate : A mixture of these two compounds in the ratio 3 : 2 is used for blood collection to carry out certain hematological tests.
Enthylene diaminetetracetic acid (EDTA) : It chelates with calcium and blocks coagulation. EDTA is employed to collect blood for hematological examinations.

The rupture or lysis of RBC, releasing the cellular constituents interferes with the laboratory investigations. Therefore, utmost care should be taken to avoid hemolysis when plasma or serum are used for biochemical tests. Use of dry syringes, needles and containers, allowing slow flow of blood into syringe are among the important precautions to avoid hemolysis.

Plasma or serum should be separated within 2 hours after blood collection. It is ideal and advisable to analyse blood, plasma or serum, immediately after the specimen collection. This however, may not be always possible. In such a case, the samples (usually plasma/serum) can be stored at 4°C until analysed. For enzyme analysis, the sample are preserved at — 20°C.

The biochemical investigations (on blood/ plasma/serum) carried out in the clinical biochemistry laboratory may be grouped into different types.
1. Discretionary or on-off tests : Most common clinical biochemistry tests that are designed to answer specific questions. e.g., does the patient have increased blood urea/glucose concentration? Normally, these tests are useful to support the diagnosis.
2. Biochemical profiles : These tests are based on the fact that more useful information on the patients disease status can be obtained by analysing more
constituents
rather
than
one
e.g.,
plasma
electrolytes
(Nat,
K+,
Cl-,
urea);
liven
function
tests
(serum bilirubin, ALT, AST).
3. Dynamic function tests : These tests are designed to measure the body’s response to external stimulus e.g., oral glucose tolerance test (to assess glucose homeostasis) : bromosulphthalein test (to assess liver function).
4. Screening tests : These tests are commonly employed to identify the inborn errors of metabolism, and to check the entry of toxic agents (pesticides, lead, mercury) into the body.
5.
Metabolic
work-up
tests
:
The
intensive
investigations
carried
out
to
identify
the
endocrinological
disorders
come
under
this
category.
The term emergency tests is frequently used in the clinical laboratory. It refers to the tests to be performed immediately to help the clinician for proper treatment of the patient e.g., blood glucose, urea, serum electrolytes.

Urine, containing the metabolic waste products of the body in water is the most important excretory fluid. For biochemical investigations, urine can be collected as a single specimen or for 24 hours. Single specimens of urine, normally collected in the morning, are useful for qualitative tests e.g., sugar, proteins. Twenty four hour urine collections (done between 8 AM to 8 AM) are employed for quantitative estimation of certain urinary constituents e.g., proteins, hormones, metabolites.
Preservatives for urine : For the collection of 24 hr urine samples, preservatives have to be used or else urine undergoes changes due to bacterial action. Hydrochloric acid, toluene, light petroleum, thymol, formalin etc., are among the common preservatives used.

CSF is a fluid of the nervous system. It is formed by a process of selective dialysis of plasma by the choroid plexuses of the ventricles of the brain. The total volume CSF is 100-200 ml.
Collection of CSF : CSF is collected by
puncturing
the
interspace
between
the
3rd
and
the
5th
lumbar
vertebrae,
under
aseptic
and
local anesthesia.
Biochemical investigations on CSF : Protein, glucose and_ chloride estimations are usually performed in the clinical biochemistry laboratory.

Quality control in clinical biochemistry laboratory refers to the reliability of investigative service. Any error in the laboratory will jeopardize the lives of patients. It is therefore utmost important that the laboratory errors are identified and rectified. Quality control comprises of four interrelated factors namely precision, accuracy, specificity and sensitivity.
Precision refers to the reproducibility of the result when the same sample is analysed on different occasions (replicate measurements) by the same person. For instance, the precision is good, if the blood glucose level is 78, 80 and 82 mg/dl on replicates.
Accuracy means the closeness of the estimated result
to the
true value
e.g., if
true blood
urea level
is
50
mg/dl, the
laboratory reporting
45
mg/dl is
more accurate than the one reporting 35 mg/dl.
Specificity refers
to the
ability of
the analytical
method to specifically determine a particular parameter e.g., glucose can be specifically estimated by enzymatic glucose oxidase method.
Sensitivity deals with the ability of a particular method to detect small amounts of the measured constituent.

Internal quality control refers to the analysis of the same pooled sample on different days in a
laboratory, the results should vary within a narrow range.
External quality control deals with the analysis of a sample received from outside, usually from a national or regional quality control centre. The results obtained are then compared.

The heavy work load in the clinical biochemistry laboratory has lead to the discovery of autoanalysers. These modern equipment are useful to analyse hundreds of samples in a short time. Single channel and multi-channel machines (autoanalysers) based on the principles of either continuous or discrete analysis are available on the market.

As already stated, clinical biochemistry laboratory is a service-oriented establishment for the benefit of patient health care. The reader may refer tools of biochemistry (Chapter 41) and principles of practical biochemistry (Appendix-V) for a brief knowledge on the principles of some of the equipment used and the laboratory investigations employed.
The details on the biochemistry of health and disease states in relation to the normal and abnormal biochemical data are described in the text of this book. For ready reference, the most common reference biochemical values are given on the inside of back cover.

giving the child rice gruel, along with breast milk. The laboratory data of the child are given hereunder.

A 3-year old female child had stunted growth, edema (particularly on legs and hands) discoloration of skin and hair, apathy and moon-face. She also had frequent respiratory infections and diarrhea. On enquiring, the mother informed the physician that the child was mostly breast-fed until 2 years of age, and for the past one year she was being given dilute buffalo milk and a small quantities of rice with ghee and dhal. The following are the laboratory data of the child
Hemoglobin, Subject = 7 g/dl. Hemoglobin, Reference range = 13-15 g/dl. Serum proteins (total), Subject = 4 g/dl. Serum proteins (total), Reference range = 6-8 g/dl. Serum albumin, Subject = 2 g/dl. Serum albumin, Reference range = 3-4.5 g/dl. Serum potassium, Subject = 2.8 mEq/1. Serum potassium, Reference range = 3.5-4.5 mEq/1

The clinical manifestations and the nutritional history, supported by the laboratory data clearly indicate that the child was suffering from kwashiorkor, a predominant nutritional disorder, in the developing countries. Edema occurred due to lack of adequate serum proteins to maintain water distribution between blood and tissues. The immunological response of the child to infections was very low. Deficiency of serum K+ was observed due to diarrhea.

A
one
year
old
male
baby
had
growth
retardation,
reduced
physical
activity,
muscle
wasting
(emaciation),
loose
folds
of
skin
wrapped
over
bones.
The
mother
of
the
child,
now
in
third
month
of
gestation,
informed
the
physician
that
she
had
been
Hemoglobin, Subject = 7 g/dl. Hemoglobin, Reference range = 13-15 g/dl. Serum proteins (total), Subject = 6 g/dl. Serum proteins (total), Reference range = 6-8 g/dl. Serum albumin, Subject = 3 g/dl. Serum albumin, Reference range = 3-4.5 g/dl. Serum potassium, Subject = 3.5 mEq/1. Serum potassium, Reference range = 3.5-5 mEq/1

A 6-year old boy had bone deformities such as bow legs and pigeon chest. He had a history of delayed eruption of teeth. On enquiring, the mother informed the physician that the boy had been on a strict vegetarian diet with low intake of milk as well as fats and oils. The following are the laboratory findings of this boy.
Calcium, Subject = 8.1 mg/dl. Calcium, Reference range = 9-11 mg/dl. Phosphate, Subject = 2.5 mg/dl. Phosphate, Reference range = 3-4.5 mg/dl. Alkaline phosphatase, Subject = 40 KAU/dl. Alkaline phosphatase, Reference range = 3-13 KAU/dl

A 25-year old strict vegetarian woman, with two children aged 5 years and 3 years, complained of tiredness and appeared pale. In the recent few months, the woman had heavy and prolonged menstrual flow. Enquiries revealed that her consumption of milk and milk products was reasonably good, but leafy vegetables was low. The laboratory investigations showed that the woman's hematocrit was 28% (reference range 40% to 50%), while her hemoglobin concentration was 8 g/dl (reference range 13-15 g/dl).

The woman with a low hemoglobin concentration and reduced hematocrit depicts a very common nutritional disorder — iron deficiency anemia. A strict vegetarian diet coupled with low consumption of leafy vegetables led her to iron deficiency.

A boy, aged 12 years was given treatment for prolonged diarrhea. After improvement, he complained abdominal discomfort and diarrhea with a feeling of being bloated, after consumption of milk. He was taken to a physician who advised him to stop his intake of milk. He felt better in 3 days.

The boy was suffering from acquired lactose intolerance. This was precipitated by diarrhea where the intestinal mucosal cells were denuded faster. The brush border of the intestine houses the enzyme lactase which is lost due to diarrhea, and hence typical symptoms of flatulence.

A normal one month old baby had a history of vomiting and diarrhea that frequently occurred after breast feeding. The urine gave a positive test for reducing sugars (Benedict's test) while the test was negative by Glucostix (specific for glucose). The RBC were found to be totally deprived of activity of the enzyme galactose 1-phosphate uridyltransferase (Reference range 4-30 units/g of hemoglobin).

A 25-year old man was on treatment with antimalarial drug primaquine. As the treatment was in progress, he developed complications. This subject's laboratory investigative data are given hereunder.
Hematocrit, Subject = 30 %. Hematocrit, Reference range = 45-50 %. Hemoglobin, Subject = . Hemoglobin, Reference range = . before treatment, Subject = 12 g/dl. before treatment, Reference range = 13-15 g/dl. after treatment, Subject = 8 g/dl. after treatment, Reference range = . Serum bilirubin, Subject = 3.5 mg/dl. Serum bilirubin, Reference range = < 1.2 mg/dl
The laboratory tests also indicated increased fragility of erythrocytes.

This subject obviously had the deficiency of the glucose 6-phosphate dehydrogenase (G6PD). It was severe in erythrocytes resulting in an impairment in the production of NADPH, a coenzyme required by glutathione peroxidase for the inactivation of free radicals (superoxide and H2O2). The drugs such as primaquine cause increased generation of free radicals which cannot be effectively inactivated due to G6PD deficiency. This leads to hemolysis of RBC and consequently jaundice, as is evident from the laboratory data-reduced hemoglobin and increased serum bilirubin.

A 30-year old man was admitted in a cardiology ward after he complained chest pain. His clinical and biochemical investigations (ECG changes, isoenzymes of creatine phosphokinase etc.) indicated that he suffered a mild myocardial infarction. Timely intervention and appropriate treatment saved him from death. The lipid profile data of this patient are given in the next page.
Serum total cholesterol, Subject (mg/dl) = Serum total cholesterol. Serum total cholesterol, Reference = 450. Serum total cholesterol, range = 150-225. LDL-cholesterol, Subject (mg/dl) = LDL-cholesterol. LDL-cholesterol, Reference = 380. LDL-cholesterol, range = 80-150. HDL-cholesterol, Subject (mg/dl) = HDL-cholesterol. HDL-cholesterol, Reference = 40. HDL-cholesterol, range = 30-60. VLDL-cholesterol, Subject (mg/dl) = VLDL-cholesterol. VLDL-cholesterol, Reference = 30. VLDL-cholesterol, range = 20-40. Serum triglycerides, Subject (mg/dl) = Serum triglycerides. Serum triglycerides, Reference = 150. Serum triglycerides, range = 75-150

This man had highly elevated LDL-cholesterol while other lipid profile parameters are within the normal limits. This is a case of familial hypercholesterolemia (hyperlipoproteinemia type Ila). The victims of this disorder have decreased number of LDL receptors. They have very high risk of coronary heart diseases in the 3rd and 4th decades of life. Lipid-lowering drugs, besides low cholesterol, low fat diet and regular exercise are useful for these people.

A 5-year old boy had delayed developments and was unable to speak or walk properly. He was found to be mentally retarded with characteristic seizures and tremors. The boy had a plasma phenylalanine level of 30 mg/dl (reference range of 1-2 mg/dl). His urine gave a positive ferric chloride test, indicating an elevated excretion of phenylpyruvate.

A 55-year old man complained severe pain in the joints. He was a non-vegetarian and consumed alcohol occasionally. His laboratory findings are as follows.
Serum uric acid, Subject = 12 mg/dl. Serum uric acid, Reference range = 3-7 mg/dl. Blood urea, Subject = 25 mg/dl. Blood urea, Reference range = 15-40 mg/dl. Urinary uric acid, Subject = 2.5 g/day. Urinary uric acid, Reference range = 0.5-0.7 g/day. Urinary pH, Subject = 4.5. Urinary pH, Reference range = 5.5-7.0
The subject responded to the treatment by the drug-allopurinol.

Elevation of uric acid in serum and urine along with case history, further supported by the response of the subject to the drug allopurinol, clearly indicate that the patient was suffering from gout. Overproduction of uric acid, the excretory end product of purine metabolism, causes gout. Crystals of sodium urate get deposited in the joints causing gouty arthritis. Allopurinol competitively inhibits the enzyme xanthine oxidase and lowers the formation of uric acid, hence the relief by administering this drug.

A 4-year old boy showed signs of learning disability and aggressive behaviour, besides pain in the joints. It was observed that he had an irresistible urge to bite his fingers and lips. The laboratory investigations revealed that the boy had serum uric acid concentration of 10 mg/dl (reference range 4-6 mg/dl).

An African negro boy (aged 12 years), studying in an Indian High School, was admitted in a hospital with complaints of fever and severe pain in the arms and legs. He was found to have hepatosplenomegaly. The boy's laboratory investigative data are given in the next page.

The laboratory data of the body with sickleshaped erythrocytes and altered hemoglobin electrophoretic pattern with a distinct Hbs band supported by anemia and reduced hematocrit, along with the clinical manifestations support the diagnosis of sickle cell anemia. This disorder primarily occurs in black population and the patients may die before they reach adulthood (< 20 years). Sickle cell anemia is caused by an abnormality in hemoglobin. It occurs due to the replacement of glutamate by valine at the 6th position of B-chain of hemoglobin.

A school boy aged 12 years complained of abdominal pain and was admitted in a hospital. He was weak and tired, and had a history of behavioural disturbance and epileptic form of seizures. Clinical examination revealed that the boy had an enlarged liver. His cornea showed the presence of Kayser Fleishw ring. The laboratory data of the boy are given below.
Serum copper, Subject = 40 P g/dl. Serum copper, Reference range = 100-200 P g/dl. Serum ceruloplasmin, Subject = 5 mg/dl. Serum ceruloplasmin, Reference range = 25-50 mg/dl. Urinary copper, Subject = 200 P g/dl. Urinary copper, Reference range = < 25 P g/dl

This boy
had low
serum level
ceruloplasmin urine.
The biochemical
with an
clinical of
copper and
elevated copper
excretion manifestations
data from
indicate
Wilson’s with
disease.
the brain.
in and
the that
the boy
was
This deposition
of disorder
copper in
suffering is
liver, associatd
kidney and

An 18-year old male medical student had the complaints of loss of appetite (anorexia), nausea, headache and malaise. On clinical examination, his liver was found to be slightly enlarged. He was passing pale coloured stools but dark coloured urine. The data of his laboratory investigations are as follows.
Bilirubin (total) (van den Bergh reaction, Subject = 8 mg/dl Biphasic. Bilirubin (total) (van den Bergh reaction, Reference range = 0.2-1 mg/dl Negative). Conjugated bilirubin, Subject = 4.5 mg/dl. Conjugated bilirubin, Reference range = <0.4 mg/dl. Unconjugated bilirubin, Subject = 3.5 mg/dl. Unconjugated bilirubin, Reference range = <0.6 mg/dl. Alanine transaminase (ALT), Subject = 170 IU/l. Alanine transaminase (ALT), Reference range = 5-40 IU/l. Aspartate transaminase (AST), Subject = 80 IU/l. Aspartate transaminase (AST), Reference range = 5-45 IU/l. Alkaline phosphatase (ALP), Subject = 20 KAU/dl. Alkaline phosphatase (ALP), Reference range = 3-13 KAU/dl

A biphasic van den Bergh positive reaction (elevated serum conjugated and unconjugated bilirubin), along with increased activity of ALT and AST clearly indicates that this is a case of hepatic jaundice. This is due to impairment in liver cell function caused by viral infection (viral hepatitis), poisons and toxins or cirrhosis of liver. Damage to the liver cells adversely affects the bilirubin uptake and its conjugation (hence conjugated and unconjugated bilirubins are elevated). The pale coloured stools are due to the absence of stercobilinogen while the dark coloured urine is due to urobilinogen.

A 20-year old man was treated for malaria a month ago. For the past one week, he was excreting slightly dark coloured urine and dark brown coloured stools. His laboratory findings are given hereunder.

A
30-year
old
man
had
fever
and
abdominal
pain.
His
stools
were
pale
in
colour
and
contained
fat
(steatorrhea).
The
man’s
urine
was
found
to
be
dark
in
colour.
His
findings
are
given
below.
Bilirubin (total) (van den Bergh reaction, Subject = 12 mg/dl Direct positive. Bilirubin (total) (van den Bergh reaction, Reference range = 0.2-1 mg/dl Negative). Conjugated bilirubin, Subject = 11.5 mg/dl. Conjugated bilirubin, Reference range = <0.4 mg/dl. Unconjugated bilirubin, Subject = 0.5 mg/dl. Unconjugated bilirubin, Reference range = <0.6 mg/dl. Alanine transaminase (ALT), Subject = 60 IU/l. Alanine transaminase (ALT), Reference range = 5-40 IU/l. Aspartate transminase (AST), Subject = 65 IU/l. Aspartate transminase (AST), Reference range = 5-45 IU/l. Alkaline phosphatase (ALP), Subject = 180 KAU/dl. Alkaline phosphatase (ALP), Reference range = 3-13 KAU/dl
In addition, the urine gave a positive test for bile salts (Hay's test) and bile pigments (Fouchet's test).

A
direct van
den
Bergh reaction
elevation in
that the
man
(cholestasis).
conjugated bilirubin,
serum alkaline
was suffering
A
ALT
and reflecting
along an
with phosphatase
from marginal
AST
Cholestasis obstructive
increase is
is also
an indicates
jaundice in
observed due
stones, tumors
into the
to the
to an
etc.)
that
The excretion
stools were
pale stercobilinogen,
Feces contained
fat digestion
and bile sats.
in obstruction
prevents of
this disorder.
(caused the
urine was
of conjugated
in colour
by gall
passage dark
of in
colour bilirubin
due
a
pigment bile
due while
the to
the deficiency
derived excess
fat of
from indicating
absorption bilirubin.
impairment due
to the
in absence
of

A 55-year old man was brought to the hospital with severe chest pain, breathlessness and vomiting. He could be rushed to the city hospital 5 hours after the onset of chest pain. His blood was immediately drawn, and the laboratory data are given below.
Creatine phosphokinase (CPK) 410 IU/l, Reference range = 10-50 IU/l. Aspartate transaminase (AST) 67 IU/l, Reference range = 5-45 IU/l. Lactate dehydrogenase (LDH) 315 IU/l, Reference range = 50-200 IU/l
Further laboratory evaluation revealed that the isoenzymes
CPK2(MB)
and
LDH;
were highly
elevated.

The man obviously, 1 = had an attack of myocardial. -, 1 = elevation in CPK (MB), LDH (isoenzyme LDH 1 ). and, 1 = aspartate transaminase. It may be noted that. CPK, 1 = starts to rise at 4-6 hours and reaches a peak. value, 1 = within 24-30 hours. As regards LDH and AST,. they, 1 = rise from the second day onwards. Thus, CPK is. the, 1 = earliest marker enzyme for the diagnosis of

A 50-year old man, an occasional alcoholic,
had the complaint of severe abdominal pain following
a
large
meal
or
alcohol
intake.
He
also
had
the
symptoms
of
nausea
and
vomiting.
His
data are given hereunder.
Serum amylase, Subject = 500. Serum amylase, Subject = Somogyi U/dl. Serum amylase, Reference range = 80-180. Serum amylase, Reference range = Somogyi U/dl. Urine amylase, Subject = 1320. Urine amylase, Subject = Somogyi U/24 hr. Urine amylase, Reference range = 250-850. Urine amylase, Reference range = Somogyi U/24hr

Elevation in the amylase activity is an indication of
pancreatitis.
It may
be noted
that serum
amylase activity
falls rapidly
on the
due to
renal first
day of
the urinary
the disease
Very is
more the diagnosis of pancreatitis.

A 20-year old man had generalized edema of the body with puffiness of the face in the mornings. His laboratory findings are given below.
Serum total proteins, Subject = 4.5 g/dl. Serum total proteins, Reference range = 6-8 g/dl. albumin, Subject = 1.5 g/dl. albumin, Reference range = 3.5-5 g/dl. globulins, Subject = 3.0 g/dl. globulins, Reference range = 2.5-3.5 g/dl. Serum cholesterol, Subject = 350 mg/dl. Serum cholesterol, Reference range = 150-225 mg/dl. Blood urea, Subject = 30 mg/dl. Blood urea, Reference range = 15-40 mg/dl. Serum creatinine, Subject = 1.2 mg/dl. Serum creatinine, Reference range = 0.5-1.5 mg/dl. Urinary proteins, Subject = 15 g/day. Urinary proteins, Reference range = < 100 mg/day
The serum electrophoresis of the subject showed a sharp and prominent
2-globulin band.

This is a, 1 = case of nephrotic syndrome ,. characterized, 1 = by generalized edema heavy. proteinuria and, 1 = hypoproteinemia (particularly low. albumin). Increased serum cholesterol level in this, 1 = albumin). Increased serum cholesterol level in this. disorder is quite common. The serum electrophoretic, 1 = disorder is quite common. The serum electrophoretic. pattern (a prominent D 2 -globulin band) supports the, 1 = pattern (a prominent D 2 -globulin band) supports the. diagnosis of nephrotic syndrome. Generalized edema, 1 = diagnosis of nephrotic syndrome. Generalized edema. is due to low plasma colloidal osmotic pressure, as, 1 = is due to low plasma colloidal osmotic pressure, as
a result of reduced albumin concentration.
and frequently,
important for

A 30-year old woman, married five years ago, had no children. She complained of tiredness, weight gain, cold intolerance, neuromuscular pains and constipation. She was found to be anemic. Her laboratory data are given hereunder.
Triiodothyronine (T 3 ), Subject = 100 ng/dl. Triiodothyronine (T 3 ), Reference range = 70-200 ng/dl. Thyroxine, total (T 4 ), Subject = 4 P g/dl. Thyroxine, total (T 4 ), Reference range = 4.2-12 P g/dl. Thyroid stimulating hormone (TSH), Subject = 20 P U/ml. Thyroid stimulating hormone (TSH), Reference range = 0.5-4 P U/ml. Cholesterol, Subject = 250 mg/dl. Cholesterol, Reference range = 150-225 mg/dl

The woman with the described clinical manifestations, elevated serum TSH and marginally lower T4 and T3 was a case of hypothyroidism. Serum cholesterol level is also increased in hypothyroidism. (Note : This has no diagnositic importance, since serum cholesterol gets elevated in several other diseases e.g. diabetes, nephrotic syndrome).

given
Blood glucose (mg/dl), Fasting = 150. Blood glucose (mg/dl), 2 hr = 240. (Normal, Fasting = < 110. (Normal, 2 hr = < 140). Urine glucose, Fasting = -ve. Urine glucose, 2 hr = ++. Ketone bodies, Fasting = +. Ketone bodies, 2 hr = ++

The laboratory data of the boy-increase in blood glucose coupled with urinary excretion of glucose and ketone bodies, along with the results obtained in the OGTT point out that this is a good example of juvenile onset diabetes mellitus (type | diabetes or insulin dependent diabetes mellitus, IDDM). IDDM, which mainly occurs in childhood, is characterized
by almost total deficiency of insulin due to the destruction of B-cells of pancreas. The boy has to be treated by insulin administration.

A 45-year old woman visited her physician with complaints of increased appetite and thirst with high frequency of urination. She also had the symptoms of diminished or impalpable pulses in the feet, besides gangrene of the feet. Her fasting blood glucose level was 160 mg/dl, with no presence of glucose or ketone bodies in urine. Her laboratory findings on the oral glucose tolerance test are as follows.
Blood glucose (mg/dl), Fasting = 155. Blood glucose (mg/dl), 2 hr = 205. (Normal Reference, Fasting = <110. (Normal Reference, 2 hr = <140). Urine glucose, Fasting = -ve. Urine glucose, 2 hr = ++. Ketone bodies, Fasting = -ve. Ketone bodies, 2 hr = -ve

The woman's fasting (elevated) blood glucose and the data obtained on OGTT indicate that she was suffering from non-insulin dependent diabetes mellitus (NIDDM or type II diabetes). NIDDM mainly occurs in adults (>30 yrs) with a strong genetic predisposition. The patients of NIDDM can be usually treated with oral hypoglycemic drugs. Before prescribing the drugs, diet control and exercise are tried.

A 55-year old man was brought to the hospital in a confused and semiconscious state. He had low BP and feeble pulse. His breath had fruity odour. The data of his laboratory investigations are given below.

The blood pH was reduced due to a decrease in plasma bicarbonate (HCO-3) concentration. This is case of metabolic acidosis. The blood glucose level was highly increased along with urinary excretion of glucose and ketone bodies. The breath of the subject was fruity due to exhalation of acetone. Obviously, this diabetic patient was not under control. The acidosis was due to the accumulation of ketone bodies. This may be appropriately regarded as diabetic ketoacidosis.

One patient with severe vomiting and hyokalemia was admitted in an intensive care unit. His laboratory data and acid-base status are given below.

This patient with increased blood pH due to an elevation in the plasma bicarbonate concentration is a typical case of metabolic alkalosis.

A 6-year old boy had high temperature with sweats during sleep, marked tachycardia and loss of weight. He also had the complaints of headache, neck stiffness and vomiting. The liver was found to be slightly enlarged. The following are the laboratory findings of this boy.
Cerebrospinal fluid (CSF), Subject = . Cerebrospinal fluid (CSF), Reference range = . Glucose, Subject = 30 mg/dl. Glucose, Reference range = 50-75 mg/dl. Proteins, Subject = 100 mg/dl. Proteins, Reference range = 15-40 mg/dl. Chloride, Subject = 105 mEq/l. Chloride, Reference range = 120-130 mEq/l. Blood glucose, Subject = 100 mg/dl. Blood glucose, Reference range = 70-100 mg/dl

A decrease in the CSF glucose and chloride concentrations along with an increase in protein level indicates that the boy was a victim of tuberculosis meningitis. This is supported by the clinical manifestations.
A
A-site,
558
Abasic sites,
535
Abetalipoproteinemia,
521
Abnormal hemoglobins,
Abzymes,
202
88
327
Acetanilide,
640
Acetohexamide,
Acetazolamide,
682
477
Acetic acid,
640,
Acetoacetate,
642
293
Acetone,
293
Acetylcholine,
Acetylcholine
149
esterase,
Acetyl
CoA,
149,
297,
309,
242,
94
252,
381,
642
Acetyl
CoA
N-Acetylcysteine,
147,
51
N-Acetylglucosamine,
24,
N-Acetylglutamate,
337,
281
Achlorhydria,
Achrodextrin,
Acid(s),
474,
Acid-base
179
21
709
balance,
Acid-base
Acid-base
Acidemia, catalysis,
disorders,
480
Acidic amino
Acid acids,
maltase,
Acid number,
Acidosis,
480
266
34
287,
298
281
369
acid,
474
99
474
47
6,
18,
780
210
Androgens,
446
Anemia,
201,
Aneuploidy,
416
741
Angiotensin,
66
Angiotensinogen,
Aniline,
639
Animal starch,
Anion
21
gap,
480
Annealing,
Anomers,
Anorexia
594
14
nervosa,
Anterior pituitary,
Antibodies,
472
326
432
187,
729
monoclonal,
Antibody enzymes,
730

88
Anticoagulants,
191
Anticodon,
552
Antidiuretic hormone,
472,
669
Anti egg-white
Antifreeze injury
factor, glycoproteins,
Antigen,
729,
437,
469,
146
25
733
Antigen therapy,
631
Anti-inflammatory drugs,
Antimetabolites,
92
Antimycin
A,
227,
Antioncogenes,
232
690
394,
510,
Antioxidant
33,
128,
658
enzymes,
659
system,
659
Antioxidant vitamins,
Antipernicious
659
anemia
152
Antiport system,
Antiproteinase, vitamin,
653
185
Antisense therapy,
Antisterility
631
vitamin,
Antithyroid
128
drugs,
438
a,-Antitrypsin,
183,
Antivitamins,
93,
185
159
87
415
Apolipoproteins
(apoproteins),
Apoptosis,
8,
691
Apurinic sites,
535
Aqueous humor,
Arabinosyl
499
adenine,
Arabinosyl cytosine,
Arachidonic
73
73
acid,
31,
Argentaffinomas,
509,
356
396
132,
274,
318
645
Biological databases,
oxidation,
636
221
value of
proteins,
Biomaker,
106
Biomembranes,
Biopterin,
345,
650
356
Biotin,
146,
259,
292,
298
Biotransformation,
638
1,3-Bisphosphoglycerate,
247
2,3-Bisphosphoglycerate,
200,
Bisphosphoglycerate mutase,
Biuret reaction,
61
Bitot’s
Blood
Blood
Blood
Blood
Blood spots,
123

buffers,
475
clotting,
190
gas analysis,
group
484
antigens, pH,
26
474
Blood urea
nitrogen,
Blotting techniques,
B-Lymphocytes,
341
587
733
Body fluids,
496
BMR
Body
Bohr
(see basal
mass metabolic
index, effect,
325
199
Bone marrow
cells
Bovine spongiform
encephalopathy,
495
Bowman’s capsule,
Bradshaws
Bradykinin, test,
459
189
67
Branched chain
363
o-keto dehydrogenase,
rate),
45, acid
135,
365
ketonuria,
Brain natriuretic
Brig’s-Haldane’s
365
peptide,
112
constant,
British antilewisite,
88
227,
642
Broad beta
disease,
Bromosulphthalein
321
test,
Bronze diabetes,
416
tissue,
Brownian movement,
Buffering capacity,
Buffers,
455
233,
326
712
711
475,
710
Buffer systems
Burkitt’s of
blood, lymphoma,
Burning
Butyric
475
688
feet syndrome,
acid,
30
251
251
150
512
319
293

639
Down’s syndrome,
Du
Bois
741
and
Du
Bois
Duchenne muscular
626
Dulcitol,
16, formula,
dystrophy,
277
Duodenal ulcer,
179
Dyslipidemias,
321
Dystrophin,
494
ECoRI,
580
Edman’s reagent,
56
Edward’s syndrome,
741
syndrome,
32,
489
644
Eicosapentanoic
Elaidic acid,
31
Elastins,
64,
Electrolyte
689
balance,
Electron
470
components,
226
inhibitors,
232
in prokaryotes,
organization,
236
225
oxiditive phosphorylation,
sites of
ATP
228
227
Electrophoresis,
724
of hemoglobins,
of
206
plasma proteins,
of serum
182
lipoproteins, types,
725
Electroporation,
584
Electrostatic bonds,
59
ELISA
(enzyme-linked immunosorbant
assay),
Elongation factors,
Embden-Meyerhof
317
729
557
pathway,
185
Emulsification of
Emulsions,
40,
Enantiomers, lipids,
712
13
Endocytosis,
172,
Endopeptidases,
654
170
Endoplasmic reticulum,
Endorphins,
Endothelium
6
436
derived factor,
367
End product
inhibition, releasing
102
acid,
510,
649
504
245
173
785
786
Gabapentin,
G-quartets,
G-tetraplex,
51
430
77
77
GABA
shunt,
370
Galactitol,
277
Galactoflavin,
138
277

Galactosamine,
Galactose,
12,
Galactosemia,
281
276
277
Galactose
1-phosphate uridyltransferase,
277
Galactose tolerance
B-Galactosidase, test,
167,
568
Galactosuria,
Gall stones,
277
178,
Ganciclovir,
630
37,
308
Gas-liquid chromatography,
Gastric function
Gastric
Gastric
Gastric
Gastric
Gastric tests,
463
HCl,
170,
463
inhibitory juice,
polypeptide,
170
lipase, ulcers,
173
179
Gastrin,
450,
463
Gastrointestinal hormones,
450,
672
Gastrointestinal tract,
Gaucher's
165
disease,
Geiger counters,
Gelatin,
308
717
65
Gel electrophoresis,
Gel-filtration
725
chromatography,
Gene(s)
amplification,
574,
688
chip,
600
constitutive,
567
450
67,
314
457
276,
308,
307
721
724
expression, inducible,
571
567
library,
597
535
rearrangement,
575
expression, therapy,
625
Gene augmentation,
Gene
Gene
Gene
Gene
Gene
Gene
Gene
625
augmentation cloning,
578
delivery by
viruses, family,
202
inhibition therapy,
596
linkage map,
Gene therapy,
Genetic
625
code,
Genetic
551
engineering,
578,
Genetic immunization,
Genetics,
585
609
737
Genome,
Genomic
542,
619
library,
Genu valgum,
Geometric
596
421
isomerism,
31,
Geranyl pyrophosphate,
Germ cell
706
311
gene therapy,
Gibbs-Thomson

Gigantism,
434
Gilbert’s disease,
Glibenclamide,
219
682
Globin(s),
64,
196,
Globular protein,
Globulins,
202
64
64,
185
Glomerular filtration
Glomerulus,
459
Glossitis,
138
Glucagon,
262,
674,
678
aaa aa
ucan,
20
ucocorticoids,
312, ucogenic
amino
372
ucokinase,
246
uconeogenesis,
675
Gluconic acid,
Glucosamine,
Glucosan,
20
Glucose absorption,
168
alanine cycle,
262
16
281
267, rate,
459
288,
312,
441, acids,
678
48,
147,
258,
261,
381,
625
principle,
716
620
therapy,
625
629
625
566
Guanine,
70,
391
Guanosine,
72,
393
diphosphate,
(GDP), monophosphate
391
(GMP), triphosphate
(GTP),
L-Gulonate,
275
L-Gulonolactone,
L-Gulonolactone
Gusten,
419
Guthrie test,
Gyrase,
528
Hagemen factor,
Hair waving,
390
391
276
oxidase,
132,
191
490
Half-maximal
Hapten,
729
Haptoglobin,
185
Haptoglobin-hemoglobin complex,
185
Hartnup’s disease,
HAT
medium,
Haworth
173,
731
projections,
Hay’s test,
Heat
Heat
15
716
shock protein,
stroke,
663
Heat syncope,
663
Heavy meromyosin,
Helicobacter
560
493
pylori, a-Helix,
56
Helix-loop-helix motif,
Helix-turn-helix
Hematocrit,

179
574
motif,
182
Heme,
197,
Heme
210,
214, oxygenase,
Heme
214
synthase,
210
Hemicellulose,
508
Hemin,
210
Hemochromatosis,
Hemocuprein,
417
Hemocyanin,
abnormal,
202
as buffer,
476
lemoglobin(s)
contd.
biochemical functions,
CO, transport,
derivatives,
199
202
glycated,
683
O, transport,
417
573
342, velocity,
352
89
416
198
358,
275
654
414
197
High-sensitive
CRP,
Hippuric acid,
Hirudin,
599
144,
Histamine
366,
624
186
342,
458
377, stimulation
test,
335,
366
46,
366
366
Histone acetylation,
Histone code,
576
Histone deacetylase,
Histones,
64,
571
576
79
HIV
(human immunodeficiency
virus),
695
HMG
CoA,
HMG
294,
CoA
310
Hollander’s
546
test,
Holliday
465
model,
Holoenzyme,
87
Homeostasis

Homeostasis of
plasma of
102,
312
calcium, blood
Homocyclic glucose,
rings,
703
154,
Homocysteine
360
methyltransferase,
156
Homocystinuria(s),
362
Homogentisate,
346,
Homogentisate
352
oxidase,
352
Homologous recombination,
Homopolysaccharides,
532
10,
20
Homoserine,
52,
145
Hoogsteen hydrogen
Hopkins-Cole test,
bonds,
61
Hormone(s),
427
adrenal adrenal
cortex,
441
medulla, anterior
444
pituitary, classification,
427
gastrointestinal,
449
gonads,
445
hypothalamic, mechanism
of ovarian,
446
670,
674
posterior pituitary,
receptors,
437
428
second messengers,
thyroid, vitamin
430
437
D,
127
Hormone sensitive
Host cells
House lipase,
in cloning,
keeping
Human
287
581
genes, artificial
567
chromosome,
431
action,
432
674
532
464
464
76
428
583
INDEX
Human
Human
435
Human body
composition, chorionic
4
gonadotropin, genome
project,
Human
619
leukocyte
Humoral antigen,
immunity,
Humulin,
Hunter’s
734
733
608
syndrome,
Hurler’s syndrome,
Hutchinson-Gilford syndrome,
6
Hyaluronidase,
23
24
=
brid enzymes,
87
Hybridoma technology,
170
Hydrogen bonds,
Hydrogen
58,
75
ion concentration,
Hydrogen peroxide,
710
236,
394,
7,
87,
170
Hydrolysis,
640
Hydroperoxidases,
236

Hydroperoxyeicosatetraenoic acid,
648
ydrophobic bonds,
Hydrops fetalis,
207
Hydroxyapatite,
404,
392
B-Hydroxy butyrate,
294
25-Hydroxycholecalciferol,
-Hydroxychromane,
128
Hydroxycobalamin,
153
5-Hydroxy indole
acetate,
Hydroxylysine,
132
B-Hydroxy
B-methyglutaryl
(see
HMG
CoA,
CoA)
Hydroxyproline,
132
17-Hydroxysteroids,
5-Hydroxytryptamine
444
(see
Hyperammonemia,
336
Hyperargininemia,
366
Hyperbetalipoproteinemia,
Hyperbilirubinemic toxic
218
Hypercalcemia,
408
315
Hyperchloremia,
413
Hyperchromicity,
78
Hyperglycemia,
669,
674,
Hyperhomocysteinemia,
681
152
Hyperkalemia,
412
Hyperlipidemia,
269,
321
Hyperlipoproteinemias,
142,
Hypermetabolism,
516
Hypernatremia,
411
Hyperoxaluria,
344
58
420
655
281
281
progeria
730
125
358
serotonin)
320
321
Indole ring,
Induced
47,
354
fit theory,
98
Induced hyperthermia,
Inducible
90
enzymes,
104
Inducible genes,
567
Infantile beri-beri,
Initiation
137
codons, factor(s)
of
DNA
of synthesis,
524
of transcription,
Inophores,
555
544
233
Inorganic pollutants,
Inosine,
666
201,
Inosine
393
monophosphate,
Inositol,
19,
35,
389
157
Insecticides,
210
Insulin blood
glucose deficiency,
679

glucose transport,
history,
673
669
action, metabolic
effects, receptors,
671
672
recombinant,
607
resistance,
679
structure, synthesis,
59,
670
670
Insulinase,
671
Insulin like
Insulin test
Interferon,
Intergenic growth
factor, meal,
465
609
DNA,
623
Interleukin,
735
Intermediary metabolism, 242
Union of
Biochemistry, 86
Human
Genome
Sequencing Consortium, 619
Intermediate density
lipoproteins,
321
Internet,
635
Intracellular fluid, 468
Intracellular enzymes,
Intrinsic factor,
153
Intrinsic pathway, 190
Introns,
548
Inulin,
21,
461
Inulin clearance, 461
Invertase,
20
Invert sugar,
lodide, 438
20
551
86
672
434
676
In vivo
lodine, gene
therapy,
418,
628
438
lodine number,
33
lodoacetate,
246
lodothyroglobulin,
418
lon-exchange chromatography,
lonophores,
233
proniazid,
Iron,
133, lron-sulfur
356
414
proteins, islets
of
Langerhans,
Isoalloxazine,
137
Isocitrate,
256
Isocitrate isoelectric
dehydrogenase, focussing,
725
Isoelectric pH,
49,
60,
Isoenzymes,
109,
725
112
sohydric transport,
isoleucine,
476
45,
363
Isomaltose,
Isomerases, isomerism,
19
87
704
isomers,
704
Isoniazid
(see hydrazide)
Isonicotinic acid
Isopentenyl hydrazide,
isonicotinic acid
146
pyrophosphate,
Isoprene,
118
Isoprenoid(s),
118,

Isotonic solution,
Isotopes,
228,
713
243,
717
Isovaleric
Isozymes acidemia,
366
(see isoenzymes)
J
J-polypeptide chain,
Jacob and
188
Monod
567
Jamaican
Jaundice,
Joule, operon
vomiting
216, concept,
sickness,
456
503
Junk
DNA,
Juvenile
623
onset diabetes,
291
679
311
227
669
256
310
723
L
Loop of
Henle,
459
Long interspersed
elements, nuclear
622
312,
316
Low-energy phosphates,
Lumirhodopsin,
222
121
Lumiflavin,
137
Lumirubin,
218
Lung maturity,
Lung
498
Luteal
716
phase,
449
Lutein,
659
Luteinizing
Lyases,
87
Lycopene,
660
733
Lymphoma,
Burkitt's,
Lyophilic,
712
Lyophobic,
712
Lysine,
46,
368
Lysolecithin,
36,
306
Lysosomal storage
Lysosomes,
Lysozyme, diseases,
7
91
Lysyl hydroxylase,
Macroglobulins,
105
403
Macrophages,
214
Mad cow
disease,
495
410
Major histocompatibility
734
Malaria,
205,
275
Malate,
256,
260
Malate-aspartate shuttle,
Malate dehydrogenase,
Maleic complex,
234
260,
298
acid,
706
Malic enzyme,
Malignant
298
carcinoid
356
Malnutrition,
458,
516
Malonate,
256
Malondialdehyde, syndrome,
132
hormone,
435
688
281

152
657
Metabolic probes,
243
791
590
Metabolic water,
293, definition,
469
241
in starvation,
Metabolomics,
383
543
Metagenomics,
542
Metalloenzymes,
91
Metalloflavoproteins,
Metalloproteins,
65
Metanephrine,
445
Metallothionein,
417,
Metaproteins,
65
Metarhodopsin,
121
Metastasis,
685,
Methanol,
639
Methemoglobin,
Methionine,
693
202,
137
419
274
46,
156,
358
enkephalin,
67
sulfoxide,
185
Methotrexate,
152,
390,
575
Methylation
(see transmethylation)
Methylcobalamin,
153
Methylcytosine,
71
Methyldopa,
351
Methylfolate trap,
Methyl malonic
156
acid,
Methyl malonyl
CoA,
N-Methylnicotinamide,
155
155,
140
N®-Methyl
THF,
151,
364
Methyltransferases,
359
Mevalonate,
310
Micelles,
39,
175
Microalbuminuria,
600
Microbodies,
7
Microcytic hypochromic

Microminerals,
403
Microsatellites,
Microsomes,
6
Microtubules,
8
Migraine,
647
Milliequivalents,
711
Millons reaction,
61
Milk composition,
496
Mineralocorticosteroids,
442
Mineral metabolism,
403
Minisatellites,
620
Mismatch repair,
Missense
538
536
620
constant,
88,
92
684
292
anemia,
252,
Myxoedema,
NAD+,
140,
NADP+,
30
316,
441
253,
140,
NADPH,
290,
271,
142,
Nat-K+
354
354
274,
ATPase,
297,
168,
Na*-K+
pump,
470,
Naphthoquinones,
334
653
504,
652
130
National
Human
Genome
Research
Institute,
619
Neonatal-physiologic
217
Neonatal
Nephron,
Nephrotic
316
Nerve gas,
Nerve
95
growth factor,
690
Net protein
utilization,
Neurodegenerative
512
diseases,
Neurohypophysis,
495
431
Neuroretinal degeneration,
Neurological
Neurotensin, beri-beri,
136
450
Neurotoxins,
667
Neurotransmitter,
Niacin,
356,
138,
354
Niacin equivalents,
Nicotine,
141
139
Nicotinamide,
Nicotinamide
140
adenine
(see
NAD*)
Nicotinamide adenine
phosphate
226
Nicotinic acid
(see niacin)
NIDDM
(non-insulin dependent
diabetes mellitus),
679
disease,
Nigercin,
233
Night blindness,
Ninhydrin
123
reagent,
Nitric oxide,
51
366

synthase,
Nitrocellulose,
589
Nitrocobalamin,
153
307
366
dinucleotide dinucleotide
(see
NADP*)
140,
232
tyrosinemia,
459
syndrome, jaundice,
352
183,
185,
370
Nitrogen balance,
511
Nitrogen dioxide, 604
Nitrogen equilibrium,
511
Noise pollution,
666
Non-competitive inhibition, 93
Non-essential amino
acids,
48
Nonheme iron,
414
Non-homologous recombination,
Non-oxidative deamination,
Non-protein nitrogen, 341
Nonsense mutations,
537
Noradrenaline (see norepinephrine)
Norepinephrine,
349,
445
Normality,
711
Northern blot
(see
DNA

Nucleoplasm,
6
Nucleoproteins,
Nucleosidases,
64
179
Nucleosides,
72
Nucleosome(s),
6,
Nucleotidase,
79
107,
Nucleotide
179, excision
Nucleotides,
393, repair,
69,
274,
Nucleus,
456
538
387
5
Nutrient antioxidants,
Nutrigenomics,
519
Nutrition,
502
659
517
515
epigenetics,
519
genetics,
519
Nutritional importance
carbohydrates, of
506
lipids,
509
proteins,
510
Nutritional transcriptomics, 519
Nyctalopia
(night blindness),123
Obesity,
Obesity
Obesity
325
and and
fat absorption,
178
leptin,
Ob gene,
325
Ochronosis,
325
217,
352
technique,
589
69
and
RNA)
335
316,
Osteoblasts,
407
Osteoclasts,
407
Osteogenesis imperfecta, 489
Osteomalacia,
127
Osteopetrosis,
Osteosarcoma,
409
688
Osteoporosis,
409,
Ouabain,
18
Overhydration,
473
Oxalic acid
(oxalate),
Oxaloacetate,
256
Oxalosis,
344
Oxalosuccinate,
256
B-Oxalylamino alanine, 667
Oxidases,
235
Oxidation,
224, a-Oxidation,
87
639
293
B-Oxidation,
@-Oxidation,
287
293
Oxidative deamination,
Oxidative
Oxi ative
381
Oxidative stress,
334
decarboxylation, phosphorylation,
657
Oxygenases, 236
Oxygen debt,
249

Oxygen dissociation
198
Oxyntic cells,
170
Oxythiamine,
Oxytocin,
66,
Ozone layer,
P
P-site,
554
PABA (see p-amino benzoic acid)
Paget's disease,
108
Palindromes, 544
Palmitic acid
(palmitate),
Palmitoleic acid,
30
Palmitoyl
CoA,
290
Pancreatic function tests, 465
137
437
664
87,
140, curve,
93,
449
132,
252,
228,
235
198
30,
290,
Pancreatitis,
179
Pantoic acid,
Pantothenic
149
acid,
Papain,
174
149
187
Paper chromatography,
Paper electrophoresis,
720
725
PAPS
(see phosphoadenosyl
phosphosulfate)
Parathyroid hormones,
Parkinson’s
124,
120
disease,
350
Partition chromatography,
Passive
Pasteur
Paulys
720
651
effect,
251
test,
PCR
61
(see polymerase
reaction)
Pectins,
25,
Pellagra,
168, chain
508
138,
141
Pellagra preventive
Penicillamine,
(PP)
51,
hexose factor,
146
pathway
(see monophosphate
Pentoses,
11,
Pentosuria,
274
essential,
Pentagastrin
Pepsin,
170
170
Peptic ulcers,
Peptide(s),
179
53,
65
Peptide antibiotics,
Peptide
Peptide bond,
53
nucleic acids,
632
Peptidyl site
(see
P-site)
Peptidyltransferase,
558
Peptones,
64
Peripheral

Pernicious neuropathy,
174
anemia,
659
Peroxidation,
656
Proxisome biogenesis
Peroxisomes, disorders,
7
Pesticides,
665
pH,
90,
710
Phenanthrene
Phenobarbitol,
Phenol,
641
Phenylalanine,
351
155
276
stimulation test,
shunt)
464
274
ring,
210,
37
640
47,
345
hydroxylase,
67
407
136
138
8
346,
475
Pregnancy detection,
Racemic
mixture,
12
Radioactivity, 717
Radioactive isotopes, 717
252
Radioactive pollution,
Radiation therapy,
657
Radioimmunoassay,
729
Raffinose,
169
Rancidity,
33
Rapaport-Leubering cycle,
251
Reactive oxygen
Recombinant species,
200,
656
DNA
578
Recombinant technology,
insulin,
607
ribozymes,
82
vaccines,
688
Recommended dietary
allowance,
514
Redox potential,
Reducing
224
equivalents,
16
Reduction,
224
Refsum’s disease,
Regulation
293
of, citric
acid fatty
cycle,
257
gene expression,
301
566
gluconeogenesis, glycogenesis,
266
glycogenolysis,
266
heme synthesis,
ketogenesis,
210
296
purine synthesis,
392
synthesis, urea
cycle,
Release factors,
Reichert-Meiss|
Renal
339
558
number, function
Renal
Renal
Renal
Renal tests,
459
glycosuria,
460, plasma
flow, regulation
681
459
of rickets,
128,
Renal threshold
Renaturation pH,
477
408

substances, of
Renaturation
Renin,
459, of
472
Rennin,
170
Renin-angiotensin,
472
Replication, in
eukaryotes, in
prokaryotes,
Replication
527
524
bubbles,
Repression,
524
factor
C, fork,
526
protein
A,
566
528
528
DNA,
79
protein,
62
262
(daily)
666
234
398
34
460
37
476
RNA
RNA
RNA
polymerase, primer,
524
viruses,
Rods,
121
Rotary motor
Rotenone, model,
227
Rothera’s test,
Rous sarcoma
550,
688
230
295
virus purple,
Ryle’s tube,
Sakagauchi
646
reaction,
Salicylic acid,
640
Salivary amylase,166
Salkowski’s test,
38
Salting in,
Salting
60
out,
60,
182
Salvage pathways,
Sanfilippo
303, syndrome,
Sanger’s reagent,
687
51
61
391,
281
56
Saponification,
33
Saponification number,
Sarcopenia,
492
Schiff base,
143,
Scleroproteins,
64
Scurvy,
134
Secondary messengers,
Secondary structure
of
Secretin,
170,
33
428,
430
protein,
450
Sedimentation
Sedoheptulose,
Sedoheptulose
Selenium,
49,
Selenocysteine,
Selenoproteins,
Selenosis,
422
Sequenator,
56
Serine,
45,
361,371
Serine proteases,
Serotonin,
95,
144,
356
Serotonin pathway,
coefficient,
728
14
7-phosphate,
273
128,
421,
659
48,
48

422
171
354
Severe combined
immunodeficiency,
Sex chromosomes,
738
Sex hormones,
445
Sex-linked inheritance,
SGOT
(see
740
aspartate transaminase)
SGPT
(see alanine
transaminase)
Shine-Dalgarno sequence,
557
56
543,
546
333
397
400

Ubiquinone
(CoQ),
331
UDP,
398
276
263
UDP-glucuronate,
275
UDP-glucuronyltransferase,
215
Ultracentrifugation,
727
UMP,
72
Uncouplers,
233
Uncoupling protein,
Undernutrition,
515
Uniport
Uracil,
Urea,
Urea locator,
systems,
653
70
337,
340
clearance
337
Urease,
90
Uremia,
341
Ureotelic,
336
Uric acid,
Uric acid
71, pool,
Uricase,
394
Uricotelic,
336
Uridine,
72,
Uridylic
Urinary
398
acid
(see
UMP)
stones,
134
Urine formation,
459
Urine production,
hormonal regulation,
Urobilin,
216
215
Urocanate,
335
pathway,
275
Uroporphyrin, 209
Uroporphyrinogen, 211
Vmax,
88
Valeric
acid,
30
Valine,
45,
363
Valinomycin,
233
van den Bergh reaction, 455
Van der Waals forces, 59
test,
394
396
228
233
461
636
469
Xanthine,
71,
393
Xanthine
oxidase,
393
Xanthine
stone,
398
Xanthinuria,
398
Xanthoproteic reaction,
Xanthurenate,
145,
354
X-chromosome,
397,
Xeaxanthin,
659
638
Xenograft,
736
Xeroderma pigmentosum,
538
123
Xylitol,
273
Xylitol dehydrogenase,
Xylose,
11

Xylulose,
z
Zak's test,
Zein,
65
Zellweger syndrome,
Zidovudine,
11
38
8
699
Zinc,
419
Zinc finger
motif,
Zn-proteases,
171
Zona fasciculata,
Zona
441
glomerulosa,
Zona
441
reticularis,
441
Zollinger-Ellison syndrome,
Zone electrophoresis,
Zoo
725
blot,
588
Zymogens,
102,
Zymosterol,
310
49,
60
170,
740
573
61
273
562
799
465

Clinical Associate Professor Weill Cornell Medical College New York, New York
NUTT life) tm oa tld lellCrys

10987654321
To
Daniel Joseph Marino, my 26-year-old son, who has become the best friend
I hoped he would be.
I would especially commend the physician who, in acute diseases, by which the bulk of mankind are cut off,
conducts the treatment better than others.
HIPPOCRATES

The fourth edition of The ICU Book marks its 23rd year as a fundamental sourcebook for the care of critically ill patients. This edition continues the original intent to provide a "generic textbook" that presents fundamental concepts and patient care practices that can be used in any adult intensive care unit, regardless of the specialty focus of the unit. Highly specialized topics, such as obstetrical emergencies, burn care, and traumatic injuries, are left to more qualified specialty textbooks.
This edition has been reorganized and completely rewritten, with updated references and clinical practice guidelines included at the end of each chapter. The text is supplemented by 246 original illustrations and 199 original tables, and five new chapters have been added: Vascular Catheters (Chapter 1), Occupational Exposures (Chapter 4), Alternate Modes of Ventilation (Chapter 27), Pancreatitis and Liver Failure (Chapter 39), and Nonpharmaceutical Toxidromes (Chapter 55). Each chapter ends with a brief section entitled "A Final Word," which highlights an insight or emphasizes the salient information presented in the chapter.
The ICU Book is unique in that it represents the voice of a single author, which provides a uniformity in style and conceptual framework. While some bias is inevitable in such an endeavor, the opinions expressed in this book are rooted in experimental observations rather than anecdotal experiences, and the hope is that any remaining bias is tolerable.

Acknowledgements are few but well deserved. First to Patricia Gast, who is responsible for all the illustrations and page layouts in this book. Her talent, patience, and counsel have been an invaluable aid to this author and this work. Also to Brian Brown and Nicole Dernoski, my longtime editors, for their trust and enduring support.
1 Units and Conversions
2 Selected Reference Ranges
3 Additional Formulas
Index

He who works with his hands is a laborer.
He who works with his head and his hands is a craftsman.
Louis Nizer
Between You and Me
1948

Charles Babbage (1791 — 1871)
One of the most dramatic events in medical self-experimentation took place in a small German hospital during the summer of 1929 when a 25 year old surgical resident named Werner Forssman inserted a plastic urethral catheter into the basilic vein in his right arm and then advanced the catheter into the right atrium of his heart (1). This was the first documented instance of central venous cannulation using a flexible plastic catheter. Although a success, the procedure had only one adverse consequence; i.e., Dr. Forssman was immediately dismissed from his residency because he had acted without the consent of his superiors, and his actions were perceived as reckless and even suicidal. Upon dismissal, he was told that "such methods are good for a circus but not for a respected hospital"(1). Forssman went on to become a country doctor, but his achievement in vascular cannulation was finally recognized in 1956 when he was awarded the Nobel Prize in Medicine for performing the first right-heart catheterization in a human subject.
Werner Forssman's self-catheterization was a departure from the standard use of needles and rigid metal cannulas for vascular access, and it marked the beginning of the modern era of vascular cannulation, which is characterized by the use of flexible plastic catheters like the ones described in this chapter.

Vascular catheters are made of synthetic polymers that are chemically inert, biocompatible, and resistant to chemical and thermal degradation. The most widely used polymers are polyurethane and silicone.

Polyurethane is a versatile polymer that can act as a solid (e.g., the solid tires on lawn mowers are made of polyurethane) and can be modified to exhibit elasticity (e.g., Spandex fibers used in stretchable clothing are made of modified polyurethane). The polyurethane in vascular catheters provides enough tensile strength to allow catheters to pass through the skin and subcutaneous tissues without kinking. Because this rigidity can also promote vascular injury, polyurethane catheters are used for short-term vascular cannulation. Most of the vascular catheters you will use in the ICU are made of polyurethane, including peripheral vascular catheters (arterial and venous), central venous catheters, and pulmonary artery catheters.

Silicone is a polymer that contains the chemical element silicon together with hydrogen, oxygen, and carbon. Silicone is more pliable than poly-urethane (e.g., the nipple on baby bottles is made of silicone), and this reduces the risk of catheter-induced vascular injury. Silicone catheters are used for long-term vascular access (weeks to months), such as that required for prolonged administration of chemotherapy, antibiotics, and parenteral nutrition solutions in outpatients. The only silicone-based catheters inserted in the ICU setting are peripherally-inserted central venous catheters (PICCs). Because of their pliability, silicone catheters cannot be inserted percutaneously without the aid of a guidewire or introducer sheath.

The size of vascular catheters is determined by the outside diameter of the catheter. There are two measures of catheter size: the gauge size and the "French" size.

The gauge system was introduced (in England) as a sizing system for iron wires, and was later adopted for hollow needles and catheters. Gauge size varies inversely with outside diameter (i.e., the higher the gauge size, the smaller the outside diameter); however, there is no fixed relationship between gauge size and outside diameter. The International Organization for Standardization (ISO) has proposed the relationships shown in Table 1.1 for gauge sizes and corresponding outside diameters in peripheral catheters (2). Note that each gauge size is associated with a range of outside diameters (actual OD), and further that there is no fixed relationship between the actual (measured) and nominal outside diameters. Thus, the only way to determine the actual outside diameter of a catheter is to consult the manufacturer. Gauge sizes are typically used for peripheral catheters, and for the infusion channels of multilumen catheters.

The French system of sizing vascular catheters (named after the country of origin) is superior to the gauge system because of its simplicity and uniformity. The French scale begins at zero, and each increment of one French unit represents an increase of 1/3 (0.33) millimeter in outer diameter (3): i.e., French size x 0.33 = outside diameter (mm). Thus, a catheter that is 5 French units in size will have an outer diameter of 5 x 0.33 = 1.65 mm. (A table of French sizes and corresponding outside diameters is included in Appendix 2 in the rear of the book.) French sizes can increase indefinitely, but most vascular catheters are between 4 French and 10 French in size. French sizes are typically used for multilumen catheters and for large-bore single lumen catheters (like introducer sheaths, de-scribed later in the chapter).

24, Range of ActualOD (mm) = 0.65 — 0.749. 24, Nominal OD (mm) = 0.7. 22, Range of ActualOD (mm) = 0.75 -— 0.949. 22, Nominal OD (mm) = 0.8, 0.9. 20, Range of ActualOD (mm) = 0.95 — 1.149. 20, Nominal OD (mm) = 1G AA. 18, Range of ActualOD (mm) = 1.15 — 1.349. 18, Nominal OD (mm) = ANA dee. 16, Range of ActualOD (mm) = 1.55 — 1.849. 16, Nominal OD (mm) = 16,1 18. 14, Range of ActualOD (mm) = 1.85 — 2.249. 14, Nominal OD (mm) = 1.9, 2.0, 2.1, 2.2
+From tha Intarnational Organization for Standardization; ISO 10555-5; 1996 (available at wwaw.iso.org). OD = outside diameter.

Steady flow (Q) through a hollow, rigid tube is proportional to the pressure gradient along the length of the tube (Pi, — Poy, or 2P), and the constant of proportionality is the resistance to flow (R):
Q=AP*x 1/R
(1.1)
The properties of flow through rigid tubes was first described by a Ger-man physiologist (Gotthif Hagen) and a French physician (Jean Louis Marie Poiseuille) working independently in the mid-19th century. They both observed that flow (Q) through rigid tubes is a function of the inner radius of the tube (r), the length of the tube (L) and the viscosity of the fluid (u). Their observations are expressed in the equation shown below, which is known as the Hagen-Poiseuille equation (4).
Q= AP x (mrt / 8uL)
(1.2)
This equation states that the steady flow rate (Q) in a rigid tube is directly related to the fourth power of the inner radius of the tube (r+), and is inversely related to the length of the tube (L) and the viscosity of the fluid (u). The term enclosed in parentheses (#r4/8uL) is equivalent to the reciprocal of resistance (1/R, as in equation 1.1), so the resistance to flow can be expressed as R = 8uL/#r4.
Since the Hagen-Poiseuille equation applies to flow through rigid tubes, it can be used to describe flow through vascular catheters, and how the dimensions of a catheter can influence the flow rate (see next).

According to the Hagen-Poiseuille equation, the inner radius of a catheter has a profound influence on flow through the catheter (because flow is directly related to the fourth power of the inner radius). This is illustrated in Figure 1.1, which shows the gravity-driven flow of blood through catheters of similar length but varying outer diameters (5). (In studies such as this, changes in inner and outer diameter are considered to be equivalent.) Note that the relative change in flow rate is three times greater than the relative change in catheter diameter (2 flow/2 diameter=3). Although the magnitude of change in flow in this case is less than predicted by the Hagen-Poiseuille equation (a common observation, with possible explanations that are beyond the scope of this text), the slope of the graph in Figure 1.1 clearly shows that changes in catheter diameter have a marked influence on flow rate.

The Hagen-Poiseuille equation indicates that flow through a catheter will decrease as the length of the catheter increases, and this is shown in Figure 1.2. (6) Note that flow in the longest (30 cm) catheter is less than half the flow rate in the shortest (5 cm) catheter; in this case, a 600% increase in catheter length is associated with a 60% reduction in catheter flow (2flow/2length = 0.1). Thus, the influence of catheter length on flow rate is

Figure 1.1 Relationship between flow rate and outside diameter of a vascular catheter. From Reference 5.
The comparative influence of catheter diameter and catheter length, as indicated by the Hagen-Poiseuille equation and the data in Figures 1.1 and 1.2, indicates that when rapid volume infusion is necessary, a large-bore catheter is the desired choice, and the shortest available large-bore catheter is the optimal choice. (See Chapter 11 for more on this subject.) The flow rates associated with a variety of vascular catheters are presented in the re-maining sections of this chapter.
FIGURE 1.2 The influence of catheter length on flow rate. From Reference 6.

There are three basic types of vascular catheters: peripheral vascular catheters (arterial and venous), central venous catheters, and peripherally inserted central catheters.

The catheters used to cannulate peripheral blood vessels in adults are typically 16-20 gauge catheters that are 1-2 inches in length. Peripher-al catheters are inserted using a catheter-over-needle device like the one shown in Figure 1.3. The catheter fits snugly over the needle and has a tapered end to prevent fraying of the catheter tip during insertion. The needle has a clear hub to visualize the "flashback" of blood that occurs when the tip of the needle enters the lumen of a blood vessel. Once flashback is evident, the catheter is advanced over the needle and into the lumen of the blood vessel.
FIGURE 1.3 A catheter-over-needle device for the cannulation of peripheral blood vessek.
The characteristics of flow through peripheral catheters are demonstrated in Table 1.2 (7,8). Note the marked (almost 4-fold) increase in flow in the larger-bore 16 gauge catheter when compared to the 20 gauge catheter and also note the significant (43%) decrease in flow rate that occurs when the length of the 18 gauge catheter is increased by less than one inch. These observations are consistent with the relationships in the Hagen-Poiseuille equation, and they demonstrate the power of catheter diameter in determining the flow capacity of vascular catheters.

16, 1 = 30 mm (1.2 in). 16, 2 = 220. 16, 3 = 13.2. 18, 1 = 30 mmm (1.2 in). 18, 2 = 105. 18, 3 = 6.0. , 1 = 50 mm (2 in}. , 2 = 60. , 3 = . 20, 1 = 30 mm (1.2 in). 20, 2 = 60. 20, 3 = 3.6
From Referances 6 and 7. All flow rates are for gravity-drivan flow of watar.

Cannulation of larger, more centrally placed veins (i.e., subclavian, internal jugular, and femoral veins) is often necessary for reliable vascular access in critically ill patients. The catheters used for this purpose, commonly known as central venous catheters, are typically 15 to 30 cm (6 to 12 inches) in length, and have single or multiple (2—4) infusion channels. Multilumen catheters are favored in the ICU because the typical ICU patient recquires a multitude of parenteral therapies (e.g., fluids, drugs, and nutrient mixtures), and multilumen catheters make it possible to deliver these therapies using a single venipuncture. The use of multiple infusion channels does not increase the incidence of catheter-related infections (9), but the larger diameter of multilumen catheters creates an increased risk of catheter-induced thrombosis (10).
Triple-lumen catheters like the one shown in Figure 1.4 are the consensus favorite for central venous access. These catheters are available in diameterss of 4 French to 9 French, and the 7 French size (outside diameter = 2.3 mm) is a popular choice in adults. Size 7 French triple lumen catheters typically have one 16 gauge channel and two smaller 18 gauge channels. To prevent mixing of infusate solutions, the three outflow ports are separated as depicted in Figure 1.4.

The features of triple lumen catheters (7 French size) from one manufacturer are shown in Table 1.3. Note the much slower flow rates in the 16 gauge and 18 gauge channels when compared to the 16 and 18 gauge peripheral catheters in Table 1.2. This, of course, is due to the much longer length of central venous catheters, as predicted by the HagenPoiseuille equation. There are 3 available lengths for the triple lumen catheter: the shortest (16 cm) catheters are intended for right-sided catheter insertions, while the longer (20 cm and 30 cm) catheters are used in left-sided cannulations (because of the longer path to the superior vena cava). The 20 cm catheter is long enough for most leftsided cannulations so (to limit catheter length tand thereby preserve flow), it seems wise to avoid central venous catheters that are longer than 20 cm, if possible.

? Fr, Length = 16cm (6 In). ? Fr, Lumens = Distal Medial Proximal. ? Fr, = Lumen Size = 16 ga 18 ga 18 ga. ? Fr, Flow Rate (L/hr)t = 3.4 1.8 1.9. 7 Fr, Length = 20 om 6h). 7 Fr, Lumens = Distal Mediial Proximal. 7 Fr, = Lumen Size = 16 ga 18 ga 18 ga. 7 Fr, Flow Rate (L/hr)t = 3.1 1.5 1.6. Cir, Length = 30 cm (12 in). Cir, Lumens = Distal Meciial Proximal. Cir, = Lumen Size = 16 ga 18 ga 18 ga. Cir, Flow Rate (L/hr)t = 2.3 1.0 1.1
- +All flow rates are for gravity-drivan flow of isotonic saline from a height of 40 inches above tha catheters. Fr= French size; ga = gauge siza.
From Arrow Intemational www.arrowintlcom); accessed 84/2011.

Central venous catheters are inserted by threading the catheter over a guidewire (a technique introduced in the early 1950s and called the Seldinger technique after its founder). This technique is illustrated in Figure 1.5. A small bore needle (usually 20 gauge) is used to probe for the target vessel. When the tip of the needle enters the vessel, a long, thin wire with a flexible tip is passed through the needle and into the vessel lumen. The needle is then removed, and a catheter is advanced over the guidewire and into the blood vessel. When cannulating deep vessels, a larger and more rigid "dilator catheter" is first threaded over the guide-wire to create a tract that facilitates insertion of the vascular catheter.

Central venous catheters are available with two types of antimicrobial coating: one uses a combination of chlorhexidine and silver sulfadiazine (available from Arrow International, Reading PA), and the other uses a combination of minocycline and rifampin (available from Cook Critical Care, Bloomington, IN). Each of these antimicrobial catheters has proven effective in reducing the incidence of catheter-related septicemia (11,12).
A single multicenter study comparing both types of antimicrobial coating showed superior results with the minocycline-rifampin catheters (13). A design flaw in the chlorhexidinesilver sulfadiazine catheter (i.e., no antimicrobial activity on the luminal surface of the catheter) has since been corrected, but a repeat comparison study has not been performed. Therefore, the evidence at the present time favors the minocycline rifampin catheters as the most effective antimicrobial catheters in clinical use (12). This situation could (and probably will) change in the future.
FIGURE 1.5 The steps involved in guidewire-assisted cannulation of blood vessels (the Seldinger technique).
What are the indications for antimicrobial catheters? According to the most recent guidelines on preventing catheter-related infections (14), antimicrobial catheters should be used if the expected duration of central venous catheterization is >5 days and if the rate of catheter-related infections in your ICU is unacceptably high despite other infection control efforts.
5 Fr, 1 = 50 cm (195 in). 5 Fr, 2 = Single. 5 Fr, 3 = 16 ga. 5 Fr, 4 = 1.26. & Fr, 1 = 7oOcm (27.5 In). & Fr, 2 = Single. & Fr, 3 = 16 ga. & Fr, 4 = 1.30. 5 Fr, 1 = 50 cm (19.5 In). 5 Fr, 2 = Distal Proximal. 5 Fr, 3 = 18 ga 20 ga. 5 Fr, 4 = 0.58 0.16. 5 Fr, 1 = 7oOcm (27.5 in). 5 Fr, 2 = Distal Proximal. 5 Fr, 3 = 18 ga 20 ga. 5 Fr, 4 = 0.44 0.12

- +All flow rates are for gravity-drivan flow of isotonic saline from a height of 40 inches above the catheters. Fr= French size; ga = gauge siza.
From Arrow Intemational Wwwaww.arrowintlcom); accessed 81/2011.

Concern for the adverse consequences of central venous cannulation (e.g., pneumothorax arterial puncture, poor patient acceptance) prompted the introduction of peripherally inserted central catheters (PICCs), which are inserted in the basilic or cephalic vein in the arm (just above the antecubital fossa) and advanced into the superior vena cava (15). (Insertion of PICCs is described in the next chapter). In the ICU, PICCs are used primarily when traditional central venous access sites are considered risky (e.g., severe thrombocytopenia) or are difficult to obtain (e.g., morbid obesity).
The characteristics of PICC devices from one manufacturer are shown in Table 1.4. These catheters are smaller in diameter than central venous catheters because they are introduced into smaller veins. However, the major distinction between PICCs and central venous catheters is their length; i.e., the length of the catheters in Table 1.4 (50 cm and 70 cm) is at least double the length of the triple lumen catheters in Table 1.3. The tradeoff for this added length is a reduction in flow capacity, which is evident when comparing the flow rates in Table 1.4 and Table 1.3. Flow is particularly sluggish in the double lumen PICCs because of the smaller diameter of the infusion channels. The flow limitation of PICCs (especially the double lumen catheters) makes them ill-suited for aggressive volume therapy.

The catheters described in this section are designed to perform specific tasks, and are otherwise not used for patient care. These specialty devices include hemodialysis catheters, introducer sheaths, and pulmonary art-ery catheters.

One of the recognized benefits of intensive care units is the ability to provide emergent hemodialysis for patients with acute renal failure, and this is made possible by a specially designed catheter like the one shown in Figure 1.6. The features of this catheter are shown in Table 1.5.

12 Fr, Length = 16cm (6 In). 12 Fr, Lumens = Proximal. 12 Fr, LumenSize = 12 ga. 12 Fr, Flow Rate (L/hrjt = 23.7. 12 Fr, Length = 16cm (6 In). 12 Fr, Lumens = Distal. 12 Fr, LumenSize = 12 ga. 12 Fr, Flow Rate (L/hrjt = 17.4. 12 Fr, Length = 20 cm (8 in). 12 Fr, Lumens = Proximal. 12 Fr, LumenSize = 16 ga. 12 Fr, Flow Rate (L/hrjt = 13.8. 12 Fr, Length = 20 cm (8 in). 12 Fr, Lumens = Distal. 12 Fr, LumenSize = 12 ga. 12 Fr, Flow Rate (L/hrjt = 15.5
- *All flow ratas are for gravity-driven flow of isotonic saline from a height of 40 inches above the catheters. Fr= French size; ga = gauge size.
From Arrow Intemational Wwww.arrowintlcom); accessed 8/1/2011.
Hemodialysis catheters are the wide-body catheters of critical care, with diameters up to 16 French (5.3 mm), and they are equipped with dual 12 gauge infusion channels that can accommodate the high flow rates (200-300 mL/min) needed for effective hemodialysis. One channel carries blood from the patient to the dialysis membranes, and the other channel returns the blood to the patient.
Hemodialysis catheters are usually placed in the internal jugular vein and are left in place until alternate access is available for dialysis. Can-nulation of the subclavian vein is forbidden because of the propensity for subclavian vein stenosis (16), which hinders venous outflow from the ipsilateral arm and thereby prevents the use of that arm for chronic hemodialysis access with an arteriovenous shunt.

Introducer sheaths are large-bore (8-9 French) catheters that serve as conduits for the insertion and removal of temporary vascular devices. In the ICU, they are used primarily to facilitate the placement of pulmonary artery (PA) catheters (see Figure 8.1 for an illustration of an introducer sheath and its companion PA catheter). The introducer sheath is first placed in a large, central vein, and the PA catheter is then threaded through the sheath and advanced into the pulmonary artery. The placement of PA catheters often requires repeated trials of advancing and retracting the catheter to achieve the proper position in the pulmonary artery, and the introducer sheath facilitates these movements. When the PA catheter is no longer needed, the introducer sheath allows the catheter to be removed and replaced with a central venous catheter, if needed, without a new venipuncture.

Introducer sheaths can also serve as stand-alone infusion devices by vir-tue of a side infusion port on the hub of the catheter. The large diameter of introducer sheaths has made them popular as rapid infusion devices for the management of acute blood loss. When introducer sheaths are used with pressurized infusion systems, flow rates of 850 mL/min have been reported (17). The use of introducer sheaths for rapid volume infusion is revisited in Chapter 11.

Pulmonary artery balloon-flotation catheters are highly specialized de-vices capable of providing as many as 16 measures of cardiovascular function and systemic oxygenation. These catheters have their own chapter (Chapter 8), so proceed there for more information.

The performance of vascular catheters as infusion devices is rooted in the HagenPoiseuille equation, which describes the influence of catheter dimensions on flow rate. The following statements from this equation are part of the "essential knowledge base" for vascular catheters.
1. Flow rate is directly related to the inner radius of a catheter (i.e., both vary in the same direction), and is inversely related to the length of the catheter (i.e., vary in opposite directions).
2. The inner radius (lumen size) of a catheter has a much greater influence on flow rate than the length of the catheter.
3. For rapid infusion, a large bore catheter is essential, and a short, large bore catheter is optimal.
As for the performance of individual catheters, each ICU has its own stock of vascular catheters, and you should become familiar with the sizes and flow capabilities of the catheters that are available.

1. Mueller RL, Sanborn TA. The history of interventional cardiology: Cardiac catheterization, angioplasty, and related interventions. Am Heart J 1995; 129:146- 172.

16. Hernandez D, Diaz F, Rufino M, et al. Subclavian vascular access stenosis in dialysis patients: Natural history and risk factors. J Am Soc Nephrol 1998; 9:1507—-1510.
17. Barcelona SL, Vilich F Cote CJ. A comparison of flow rates and warming capabilities of the Level 1 and Rapid Infusion System with various-size intravenous catheters. Anesth Analg 2003; 97:358-363.

J. Wilis Hurst, MD
Vascular access in critically ill patients often involves the insertion of long, flexible catheters (like those described in the last chapter) into large veins entering the thorax or abdomen; this type of central venous access is the focus of the current chapter. The purpose of this chapter is not to teach you the technique of central venous cannulation (which must be mastered at the bedside), but to describe the process involved in establishing central venous access and the adverse consequences that can arise.

Catheters placed in small, peripheral veins have a limited life expectancy because they promote localized inflammation and thrombosis. The inflammation is prompted by mechanical injury to the blood vessel and by chemical injury to the vessel from caustic drug infusions. The thrombosis is incited by the inflammation, and is propagated by the sluggish flow in small, cannulated veins. (The viscosity of blood varies inversely with the rate of blood flow, and thus the low flow in small, cannulated veins is associated with an increase in blood viscosity, and this increases the propensity for thrombus formation. )
Large veins offer the advantages of a larger diameter and higher flow rates. The larger diameter allows the insertion of larger bore, multilumen catheters, which increases the efficiency of vascular access (i.e., more infusions per venipuncture). The higher flow rates reduce the damaging effects of infused fluids and thereby reduce the propensity for local thrombosis. The diameters and flow rates of some representative large and small veins are shown in Table 2.1. Note that the increase in flow rate is far greater than the increase in vessel diameter; e.g., the diameter of the subclavian vein is about three times greater than the diameter of the metacarpal veins, but the flow rates in the subclavian vein are as much as 100 times higher than flow rates in the metacarpal veins. This relationship between flow rate and vessel diameter is an expression of the HagenPoiseuille equation described in Chapter 1 (see Equation 1.2).

Upper Body:, Vein = . Upper Body:, Diameter = . Upper Body:, Flow Ratet = . , Vein = Superior Vena Cava Internal Jugular Vein Subclavian Vein. , Diameter = 18-22 mm. , Flow Ratet = 1800 — 2000 mL/min. , Vein = . , Diameter = 10-22 mm. , Flow Ratet = 500 — 1400 mL/min. , Vein = . , Diameter = 7-12 mm. , Flow Ratet = 350 — 800 mL/min. , Vein = Metacarpal Veins. , Diameter = 2-65mm. , Flow Ratet = 8 —10 mL/min. Lower Body:, Vein = . Lower Body:, Diameter = . Lower Body:, Flow Ratet = . , Vein = Inferior Vena Cava. , Diameter = 27 -36 mm. , Flow Ratet = 1200 — 2000 mL‘min. , Vein = Femoral Vein. , Diameter = 8-16 mm. , Flow Ratet = 700 — 1100 mL‘min
+Hlow rates ara for healthy adults.

The major indications for central venous access are summarized as follows (1):
1. When peripheral venous access is difficult to obtain (e.g., in obese patients or intravenous drug abusers) or difficult to maintain (e.g., in agitated patients).
2. For the delivery of vasoconstrictor drugs (e.g., dopamine, norepinephrine), hypertonic solutions (e.g., parenteral nutrition formulas), or multiple parenteral medications (taking advantage of the multilumen catheters described in Chapter 1).
3. For prolonged parenteral drug therapy (i.e., more than a few days).
4. For specialized tasks such as hemodialysis, transvenous cardiac pacing, or hemodynamic monitoring (e.g., with pulmonary artery catheters).

There are no absolute contraindications to central venous cannulation (1), including the presence or severity of a coagulation disorder (2,3). However, there are risks associated with cannulation at a specific site, and these are described later in the chapter.

Infection control is an essential part of vascular cannulation, and the pre-ventive measures recommended for central venous cannulation are shown in Table 2.2 (4,5). When used together (as a "bundle"), these five measures have been effective in reducing the incidence of catheter-related bloodstream infections (6,7). The following is a brief description of these preventive measures.

Hand Hygeine, ‘Recommendations = Use an alcohol-based handrub or a scap and water hancwash before and after inserting or manipulatng catheters.. Barrier Precautions, ‘Recommendations = Use maximal barrier precautions, including cap, mask, sterile gloves, sterile gown, and sterile full body drape, for catheter insertion or guidewire exchange.. Skin Antisepsis, ‘Recommendations = Apply a chlorhexadine-based solution to the catheter insertion site and allow 2 minutes to air-dry.. Cannulation Site, ‘Recommendations = When possible, avoid femoral vein cannulation, and cannulate the subclavian vein rather than the internal jugular vein.. Catheter Removal, ‘Recommendations = Remove catheter promptly when itis no longer needed.
From the Instituta for Healthcare Improvement (5). Adherence to all recommendations in this bundk has been shown to reduce tha incidence of catheterralated bloodstraam infections (6,7).

Proper hand hygiene is considered one of the most important, and most neglected, methods of infection control. Alcohol-based hand rubs are preferred if available (4,8); otherwise, handwashing with soap (plain or antimicrobial soap) and water is acceptable (4). Hand hygiene should be performed before and after palpating catheter insertion sites, and before and after glove use (4).
The skin around the catheter insertion site should be decontaminated just prior to cannulation, and the preferred antiseptic agent is chlorhexidine (4—7). This preference is based on clinical studies showing that chlorhexidine is superior to other antiseptic agents for limiting the risk of catheter-associated infections (9). The enhanced efficacy of chlorhexidine is attributed to its prolonged (residual) antimicrobial activity on the skin, which lasts for at least 6 hours after a single application (10). Anti-microbial activity is maximized if chlorhexidine is allowed to air-dry on the skin for at least two minutes (4).

All vascular cannulation procedures, except those involving small peripheral veins, should be performed using full sterile barrier precautions, which includes caps, masks, sterile gloves, sterile gowns, and a sterile drape from head to foot (4). The only barrier precaution advised for peripheral vein cannulation is the use of gloves, and nonsterile gloves are acceptable as long as the gloved hands do not touch the catheter (4).

According to the current guidelines for preventing catheter-related infections (4), femoral vein cannulation should be avoided, and cannulating the subclavian vein is preferred to cannulating the internal jugular vein. These recommendations are based on the perceived risk of catheter-related infections at each site (i.e., the highest risk from the femoral vein and the lowest risk from the subclavian vein). However, there are other considerations that can influence the preferred site of catheter insertion; e.g., the subclavian vein is the least desirable site for insertion of hemodialysis catheters (for reasons explained later). Hence the qualifying term "when possible" is added to the recommendation for catheter insertion site in the central line bundle. The special considerations for each central venous access site are presented later in the chapter.

Since its introduction in the early 1990s, the use of real-time ultrasound imaging to locate and cannulate blood vessels has added considerably to the success rate and safety of vascular cannulation (11,12). The following is a brief description of ultrasound-guided vascular cannulation.

Ultrasound imaging is made possible by specialized transducers (gray scale adapters) that convert the amplitude of reflected ultrasound waves (echoes) into colors representing shades of gray in the black-white continuum. Higher amplitude echoes produce brighter or whiter images, while lower amplitude echoes produce darker or blacker images. This methodology is knows as B-mode (brightness-mode) ultrasound, and it produces two-dimensional, gray-scale images. The frequency of the ultrasound waves is directly related to the resolution of the ultrasound image, and is inversely related to the depth of tissue penetration; i.e., higher frequency waves produce higher resolution images, but the area visualized is smaller.
Ultrasound waves pass readily through fluids, so fluid-filled structures like blood vessels have a dark gray or black interior on the ultrasound image.

Vascular ultrasound uses probes that emit high-frequency waves to produce highresolution images, but visualization is limited to only a few centimeters from the skin. Ultrasound images are used in real time to locate the target vessel and assist in guiding the probe needle into the target vessel. This process in influenced by the orientation of the ultrasound beam, as depicted in Figure 2.1.
FIGURE 2.2 Ultrasound image showing a long-axis view of the internal jugular vein, with a visible probe needk advancing towards the vein. From Reference 12. (Image digitally enhanced. )

Tilting the body so the head is below the horizontal plane (the Trendelenburg position) will distend the large veins entering the thorax from above to facilitate cannulation of the subclavian vein and internal jugular vein. In healthy subjects, head-down body tilt to 15° below horizontal is associated with a diameter increment of 20-25% in the internal jugular vein (14), and 8—10% in the subclavian vein (15). Further increases in the degree of body tilt beyond 15° produces little or no incremental effect (14). Thus, the full benefit of the head-down position is achieved with small degrees of body tilt, which is advantageous because it limits the undesirable effects of the head-down position (e.g., increased intracranial pressure and increased risk of aspiration). The head-down body tilt is not necessary in patients with venous congestion (e.g., from left or right heart failure), and is not advised in patients with increased intracranial pressure.

The following is a brief description of central venous cannulation at four different access sites: i.e., the internal jugular vein, the subclavian vein, the femoral vein, and the veins emerging from the antecubital fossa. The focus here is the location and penetration of the target vessel; once this is accomplished, cannulation proceeds using the Seldinger technique, which is described in Chapter 1 (see Figure 1.5).

The internal jugular vein is located under the sternocleidomastoid muscle on either side of the neck, and it runs obliquely down the neck along a line drawn from the pinna of the ear to the sternoclavicular joint. In the lower neck region, the vein is often located just anterior and lateral to the carotid artery, but anatomic relationships can vary (16). At the base ofthe neck, the internal jugular vein joins the subclavian vein to form the innominate vein, and the convergence of the right and left innominate veins forms the superior vena cava. The supine diameter of the internal jugular vein varies widely (from 10 mm to 22 mm) in healthy subjects (14).
The right side of the neck is preferred for cannulation of the internal jugular vein because the vessels run a straight course to the right atrium. The right side is particularly well suited for the placement of temporary pacer wires, hemodialysis catheters, and pulmonary artery catheters.

A head-down body tilt of 15° will distend the internal jugular vein and facilitate cannulation, as described earlier. The head should be turned slightly in the opposite direction to straighten the course of the vein, but turning the head beyond 30° from midline is counterproductive because it stretches the vein and reduces the diameter (16).

The internal jugular vein is well suited for ultrasound imaging because it is close to the skin surface and there are no intervening structures to interfere with transmission of the ultrasound waves. A short-axis view of the internal jugular vein and carotid artery on the right side of the neck is shown in Figure 2.3. (This image was obtained by placing the ultrasound probe across the triangle created by the two heads of the sternocleidomastoid muscle, which is shown in Figure 2.4.) The image on the left shows the large jugular vein situated anterior and lateral to the smaller carotid artery. The image on the right shows the vein collapsing when a compressive force is applied to the overlying skin; this is a popular maneuver for determining if a blood vessel is an artery or vein.
When ultrasound guidance is used for internal jugular vein cannulation, there is an increased success rate, fewer cannulation attempts, a shorter time to cannulation, and a reduced risk of carotid artery puncture (16-18). As a result of these benefits, ultrasound guidance has been recommended as a standard practice for cannulation of the internal jugular vein (16).
FIGURE 2.3 Ultrasound images (short-axis view) of the author's internal jugular vein (IJV) and carotid artery (CA) on the right side of the neck. The image on the right shows collapse of the vein when downward pressure is applied to the overlying skin. The green dots mark the lateral side of each image. (Images courtesy of Cynthia Sullivan, R.N. and Shawn Newvine, R.N.).

When ultrasound imaging is not available, cannulation of the internal jugular vein is guided by surface landmarks. There are two approaches to the internal jugular vein using surface landmarks, as described next.
FIGURE 2.4 Anatomic relationships of the internal jugular vein and subclavian vein.
ANTERIOR APPROACH: For the anterior approach, the operator first identifies the triangular area at the base of the neck created by the separation of the two heads of the sternocleidomastoid muscle (see Figure 2.4). The internal jugular vein and carotid artery run through this triangle. The operator first locates the carotid artery pulse in this triangle; once the artery is located by palpation, it is gently retracted toward the midline and away from the internal jugular vein. The probe needle is then inserted at the apex of the triangle (with bevel facing up) and the needle is advanced toward the ipsilateral nipple at a 45° angle from the skin. If the vein is not entered by a depth of 5 cm, the needle should be drawn back and advanced again in a more lateral direction.
POSTERIOR APPROACH: For the posterior approach, the insertion point for the probe needle is 1 cm above the point where the external jugular vein crosses over the lateral edge of the sternocleidomastoid muscle (see Figure 2.4). The probe needle is inserted at this point (with the bevel at 3 o'clock) and then advanced along the underbelly of the muscle in a direction pointing to the suprasternal notch. The internal jugular vein should be encountered 5 to 6 cm from the insertion point.

Accidental puncture of the carotid artery is the most feared complication of jugular vein cannulation, and has a reported prevalence of 0.5-11% when anatomic landmarks are used (17,19,20), and 1% when ultrasound imaging is employed (17). If the artery is punctured by the small-bore probe needle, it is usually safe to remove the needle and compress the site for at least 5 minutes (double the compression time for patients with a coagulopathy). Insertion of a catheter into the carotid artery is more of a problem because removing the catheter can be fatal (20,21). If confronted with a catheterized carotid artery, leave the catheter in place and consult a vascular surgeon pronto (21).
OTHERS: Accidental puncture of the pleural space (resulting in hemothorax and/or pneumothorax) is not expected at the internal jugular vein site because it is located in the neck. However, this complication is reported in 1.3% of internal jugular vein cannulations using the landmark approach (19). The principal complication of indwelling jugular vein catheters is septicemia, which has a reported incidence that varies from zero to 2.3 cases per 1000 catheter days (22,23). Catheters in the internal jugular vein are considered a greater infectious risk than catheters in the subclavian vein (4,5), but this is not supported by some clinical surveys (22).

The internal jugular vein should be the favored site for central venous access when ultrasound imaging is available (16), and the right internal jugular vein is the preferred site for insertion of transvenous pacemaker wires, pulmonary artery catheters, and hemodialysis catheters. Awake patients often complain of discomfort and limited neck mobility from indwelling jugular vein catheters, so other sites should be considered for central venous access in conscious patients. (Peripherally inserted central catheters, which are described later, may be a better choice for central venous access in conscious patients.)

The subclavian vein is a continuation of the axillary vein as it passes over the first rib (see Figure 2.4). It runs most of its course along the underside of the clavicle (sandwiched between the clavicle and first rib), and at some points is only 5 mm above the apical pleura of the lungs. The underside of the vein sits on the anterior scalene muscle along with the phrenic nerve, which comes in contact with the vein along its posteroinferior side. Situated just deep to the vein, on the underside of the anterior scalene muscle, is the subclavian artery and brachial plexus. At the thoracic inlet, the subclavian vein meets the internal jugular vein to form the innominate vein. The subclavian vein is 3-4 cm in length, and the diameter is 7-12 mm in the supine position (24). The diameter of the subclavian vein does not vary with respiration (unlike the internal jugular vein), which is attributed to strong fascial attachments that fix the vein to surrounding structures and hold it open (24). This is also the basis for the claim that volume depletion does not collapse the subclavian vein (25), which is an unproven claim.

The head-down body tilt distends the subclavian vein (24) and can facilitate cannulation. However, other maneuvers used to facilitate cannulation, such as arching the shoulders or placing a rolled towel under the shoulder, actually cause a paradoxical decrease in the cross-sectional area of the vein (24,26).

Ultrasound imaging can improve the success rate and reduce the adverse consequences of subclavian vein cannulation (25). However, the subclavian vein is not easily visualized because the overlying clavicle blocks transmission of ultrasound waves. Because of this technical difficulty, ultrasound guidance is not currently popular for subclavian vein cannulation.

The subclavian vein can be located by identifying the portion of the sternocleidomastoid muscle that inserts on the clavicle (see Figure 2.4). The subclavian vein lies just underneath the clavicle at this point, and the vein can be entered from above or below the clavicle. This portion of the clavicle can be marked with a small rectangle, as shown in Figure 2.4, to guide insertion of the probe needle.
INFRACLAVICULAR APPROACH: The subclavian vein is typically entered from below the clavicle. The probe needle is inserted at the lateral border of the rectangle marked on the clavicle, and the needle is advanced (with the bevel at 12 o'clock) along the underside of the clavicle in a direction that would bisect the rectangle into two triangles. The needle should enter the subclavian vein within a few centimeters from the surface. It is important to keep the needle on the underside of the clavicle to avoid puncturing of the subclavian artery, which lies deep to the subclavian vein. When the needle enters the subclavian vein, the bevel of the needle should be rotated to 3 o'clock so the guidewire will advance in the direction of the superior vena cava.
SUPRACLAVICULAR APPROACH: Identify the angle formed by the lateral margin of the sternocleidomastoid muscle and the clavicle. The probe needle is inserted so that it bisects this angle. Keep the bevel of the needle at 12 o'clock and advance the needle along the underside of the clavicle in the direction of the opposite nipple. The vein should be entered at a distance of 1 to 2 cm from the skin surface (the subclavian vein is more superficial in the supraclavicular approach). When the vein is entered, turn the bevel of the needle to 9 o'clock so the guidewire will advance in the direction of the superior vena cava.

The immediate complications of subclavian vein cannulation include puncture of the subclavian artery ("5%), pneumothorax ("5%), brachial plexus injury ("3%), and phrenic nerve injury (1.5%) (19,25). All are less frequent when ultrasound guidance is used (25).
Complications associated with indwelling catheters include septicemia and subclavian vein stenosis. The incidence of septicemia in one survey was less than one case per 1000 catheter days (22). Stenosis of the subclavian vein appears days or months after catheter removal, and has a reported incidence of 15-50% (27). The risk of stenosis is the principal reason to avoid cannulation of the subclavian vein in patients who might require a hemodialysis access site (e.g., arteriovenous fistula) in the ipsilateral arm (27).

The major advantage of the subclavian vein site is patient comfort after catheters are placed. The claim that infections are less frequent with subclavian vein catheters (4,5) is not supported by some clinical studies (22).

The femoral vein is a continuation of the long saphenous vein in the groin, and is the main conduit for venous drainage of the legs. It is located in the femoral triangle along with the femoral artery and nerve, as shown in Figure 2.5. The superior border of the femoral triangle is formed by the inguinal ligament, which runs from the anterior superior iliac spine to the pubic symphysis, just beneath the inguinal crease on the skin. At the level of the inguinal ligament (crease), the femoral vein lies just medial to the femoral artery, and is only a few centimeters from the skin. The vein is easier to locate and cannulate when the leg is placed in abduction.

Ultrasound visualization of the femoral artery and vein is possible by placing the ultrasound probe over the femoral artery pulse, which is typically located just below and medial to the midpoint of the inguinal crease. A cross-sectional (short-axis) view of the femoral artery and femoral vein in this location is shown in Figure 2.6. In the image on the left, the femoral artery and vein are identified by their lateral and medial positions, respectively. In the image on the right, the color Doppler mode of ultrasound is used to distinguish between the femoral artery (red color) and femoral vein (blue color). (The red and blue colors do not identify arterial vs. venous flow, but indicate the direction of flow in relation to the ultrasound probe. The red color indicates movement towards the probe, and the blue color indicates movement away from the probe, as indicated by the color legend to the left of the color Doppler image.)
FIGURE 2.6 Ultrasound images (short-axis view) of the femoral vein (FV) and femoral artery (FA) in the left groin. The image on the right identifies the femoral vein (blue color) and femoral artery (red color) using the color Doppler mode of ultrasound. The color legend indicates the directional color assignment for the color Doppler image. The green dots mark the lateral side of each image.

To cannulate the femoral vein when ultrasound imaging is not available, begin by locating the femoral artery pulse (as described in the prior section) and insert the probe needle (with the bevel at 12 o'clock) 1 to 2 cm medial to the pulse; the vein should be entered at a depth of 2 to 4 cm from the skin. If the femoral artery pulse is not palpable, draw an imaginary line from the anterior superior iliac crest to the pubic tubercle, and divide the line into three equal segments. The femoral artery should be just underneath the junction between the middle and medial segments, and the femoral vein should be 1 to 2 cm medial to this point. This method of locating the femoral vein results in successful cannulation in over 90% of cases (28).

The major concerns with femoral vein cannulation include puncture of the femoral artery, femoral vein thrombosis, and septicemia. Thrombus formation from indwelling catheters is more common than suspected, but is clinically silent in most cases. In one study of indwelling femoral vein catheters, thrombosis was detected by ultrasound in 10% of patients, but clinically evident thrombosis occurred in less than 1% of patients (23). The incidence of septicemia from femoral vein catheters is 2 to 3 infections per 1000 catheter days, which is no different than the incidence of septicemia from indwelling catheters in the subclavian vein or internal jugular vein (22,23). This is contrary to the claim that femoral vein catheters have the highest risk of infection amongst central venous catheters (4), and it does not support the recommendation in the "central line bundle" (see Table 2.2) to avoid femoral vein cannulation as an infection control measure.

The femoral vein is generally regarded as the least desirable site for central venous access, but the observations just presented indicate that the negative publicity directed at femoral vein catheters do not seem justified. The femoral vein is a favored site for temporary hemodialysis catheters (23), and for central venous access during cardiopulmonary resuscitation (because it does not disrupt resuscitation efforts in the chest) (29). However, the use of leg veins for vascular access is not advised during cardiac arrest because drug delivery may be delayed (30). Avoiding femoral vein cannulation is mandatory in patients with deep vein thrombosis of the legs, and in patients with penetrating abdominal trauma (because of the risk of vena cava disruption) (1).

Catheters can be advanced into the superior vena cava from peripheral veins located just above the antecubital fossa in the arm. These peripherally inserted central catheters (PICCs) are described in Chapter 1 (see Table 1.4). There are two veins that emerge from the antecubital fossa, as shown in Figure 2.7. The basilic vein runs up the medial aspect of the arm, and the cephalic vein runs up the lateral aspect of the arm. The basilic vein is preferred for PICC placement because it has a larger diameter than the cephalic vein, and it runs a straighter course up the arm.

PICC insertion is performed with ultrasound guidance. Once the basilic vein is located and cannulated, the catheters are advanced a predetermined distance to place the catheter tip in the lower third of the superior vena cava, just above the right atrium. The distance the catheter must be advanced is estimated by measuring the distance from the antecubital fossa to the shoulder, then from the shoulder to the right sternoclavicular joint, then down to the right 3rd intercostal space. In an average sized adult, the distance from the right antecubital fossa to the right atrium is 52-54 cm, and the distance from the left antecubital fossa to the right atrium is 56-58 cm. When the catheter has been advanced the desired distance, a portable chest x-ray is obtained to locate the catheter tip. Malposition of the catheter tip is reported in 6—7% of PICC insertions (31).
FIGURE 2.7 Anatomy of the major veins in the region of the antecubital fossa in the right arm.

The most common complication of PICC insertion is catheter-induced thrombosis, which most often involves the axillary and subclavian veins (32). Occlusive thrombosis with swelling of the upper arm has been reported in 2-11% of patients with indwelling PICC devices (32,33); the highest incidence occurs in patients who have a history of venous thrombosis (32) and in cancer patients (33). Septicemia from PICCs occurs at a rate of one infection per 1000 catheter days (31), which is similar to the rate of infection from

PICCs are very appealing for central venous access for the following reasons. First, they eliminate many of the risks associated with cannulation of the subclavian vein and internal jugular vein (e.g., puncture of a major artery, pneumothorax). Second, PICC insertion is relatively easy (thanks to ultrasound) and causes less discomfort than cannulation at other central venous access sites. Third, PICCs can be left in place for prolonged periods of time (several weeks) with only a minimal risk of infection. These features make PICC insertion a desirable choice for central venous access in the ICU.

Air entry into the venous circulation is an uncommon but potentially lethal complication of central venous cannulation. The following is a brief description of this feared complication.

Pressure gradients that favor the movement of air into the venous circulation are created by the negative intrathoracic pressure generated during spontaneous breathing, and by gravitational gradients between the site of air entry and the right atrium (i.e., when the site of air entry is vertically higher than the right atrium). A pressure gradient of only 5 mm Hg across a 14 gauge catheter (internal diameter=1.8 mm) can entrain air at a rate of 100 mL per second, and this is enough to produce a fatal venous air embolism (35). Both the volume of air and the rate of entry determine the consequences of venous air embolism.
The consequences can be fatal when air entry reaches 200-300 mL (3-5 mL/kg) over a few seconds (35). The adverse consequences of venous air embolism include acute right heart failure (from an air lock in the right ventricle) that can progress to cardiogenic shock, leaky-capillary pulmonary edema, and acute embolic stroke from air bubbles that pass through a patent foramen ovale (35).

Prevention is the most effective approach to venous air embolism. Positive-pressure mechanical ventilation reduces the risk of air entry through central venous catheters by creating a positive pressure gradient from the central veins to the atmosphere. Other preventive measures include the Trendelenburg position (head-down body tilt) for insertion and removal of internal jugular vein and subclavian vein catheters, and a supine or semirecumbent position for insertion and removal of femoral vein catheters. These measures will reduce, but not eliminate, the risk of venous air embolism. In one study employing appropriate body positions for 11,500 central venous cannulation procedures (34), 15 cases of venous air embolism were observed (incidence=0.13%).

Venous air embolism can be clinically silent (34). In symptomatic cases, the earliest manifestation is sudden onset of dyspnea, which may be accompanied by a distressing cough. In severe cases, there is rapid progression to hypotension, oliguria, and depressed consciousness (from cardiogenic shock). In the most advanced cases, the mixing of air and blood in the right ventricle can produce a drum-like, mill wheel murmur just prior to cardiovascular collapse (35).
Venous air embolism is usually a clinical diagnosis, but there are some diagnostic aids. Transesophageal echocardiography is the most sensitive method of detecting air in the right heart chambers, and precordial Doppler ultrasound is the most sensitive noninvasive method of detecting air in the heart (35). (Doppler ultrasound converts flow velocities into sounds, and air in the cardiac chambers produces a characteristic high-pitched sound.) The drawback of these diagnostic modalities is limited availability in emergency situations.

The management of venous air embolism includes measures to prevent air entrainment, and general cardiorespiratory support. The first step is to make sure that there is no disruption in the catheter or intravenous tubing that could introduce air into the circulation. If air entrainment is suspected through an indwelling catheter, you can attach a syringe to the hub of the catheter and attempt to aspirate air from the bloodstream. Placing the patient in the left lateral decubitus position is a traditional recommendation aimed at relieving an air lock blocking outflow from the right ventricle, but the value of this maneuver has been questioned (35). Chest compressions can help to force air out of the pulmonary outflow tract and into the pulmonary circulation, but the clinical benefits of this maneuver are unproven (35). Pure oxygen breathing is used to reduce the volume of air in the bloodstream by promoting the movement of nitrogen out of the air bubbles in the blood. However, the efficacy of this maneuver is also unproven.

Pneumothorax is an infrequent event during central venous cannulation, and most cases are associated with subclavian vein cannulation. When pneumothorax is suspected, the chest x-ray should be obtained in the upright position and after a forced exhalation (if possible). The forced exhalation will decrease lung volume but will not decrease the volume of air in a pneumothorax; the result is an increase in the relative size of the pneumothorax on the chest x-ray, which can facilitate detection. Unfortunately, few patients in an ICU may be capable of performing a forced exhalation.

Critically ill patients are often unable to sit upright, so chest x-rays are frequently taken in the supine position. This creates a problem for the detection of a pneumothorax. The problem is the distribution of pleural air in the supine position (36); i.e., pleural air does not collect at the apex of the lungs in the supine position, but instead collects anteriorly (because the anterior thorax is the nondependent region in the supine position). Pleural air in this location will be in front of the lungs on the supine chest x-ray, and it can go undetected because of the lung markings behind the pneumothorax. Clinical studies have shown that portable chest x-rays fail to detect 25 to 50% of pneumothoraces when patients are in the supine position (37-39). B-mode ultrasound is superior to portable chest x-rays for detecting supine pneumothoraces (38,39). (An example of a supine pneumothorax that is not apparent on a portable chest x-ray is shown in Chapter 27.)

Pneumothoraces from central venous cannulation may not be radiographically evident for 24 to 48 hours (40), and these will be missed on chest x-rays obtained immediately after catheter insertion. However, serial chest x-rays over the first 48 hours post-insertion are not necessary if patients remain asymptomatic.

Post-insertion chest x-rays are also used to identify the location of the catheter tip, which should be positioned in the distal one-third of the superior vena cava, 1-2 cm above the junction of the right atrium. The appropriate position for a central venous catheter is shown in Figure 2.8. The cannulation site in this case is the right internal jugular vein, and the catheter follows a straight course down the mediastinum, within the long axis of the superior vena cava shadow. The tip of the catheter is just above the carina, which is the bifurcation of the trachea to form the right and left mainstem bronchi. The carina is located just above the junction between the superior vena cava and the right atrium, so a catheter tip that is at the level of the carina, or slightly above it, is appropriately positioned in the distal superior vena cava. The carina is thus a useful landmark for evaluating catheter tip location (41).
FIGURE 2.8 Portable chest x-ray showing the proper placement of an internal jugular vein catheter with the tip of the catheter located at the level of the carina, where the trachea bifurcates to form the right and left mainstem bronchi. The dotted lines are used to highlight the region of the tracheal bifurcation. (Catheter image digitally enhanced.)
FIGURE 2.9 Malposition of a left subclavian vein catheter with the tip abutting the lateral wall of the superior vena cava (SVC). (Catheter image digitally enhanced.)
Misplaced catheters are found in 5% to 25% of cannulations involving central venous catheters and PICC devices (19,31,39). The following are some aberrant catheter tip positions that can prove harmful.

Catheters inserted from the left side must make an acute turn downward when they enter the superior vena cava from the left innominate vein. Catheters that do not make this turn can end up in a position like the one shown in Figure 2.9. The tip of the catheter is at the lateral edge of the superior vena cava shadow, suggesting that the catheter tip is in contact with the lateral wall of the superior vena cava. In this position, any forward movement of the catheter (e.g., from shrugging the left shoulder) could puncture the vessel wall and produce a hemothorax (see Figure 3.1). Catheters in this position should be withdrawn into the innominate vein.

As mentioned earlier, the tip of a central venous catheter will be in the right atrium if it is located below the level of the carina on a chest x-ray. This is a common occurrence; eé.g., in one study, one of every four central venous catheters was positioned with the tip in the right atrium (39). This malposition creates a risk of right atrial perforation and cardiac tamponade, which is fatal in over 50% of cases (42). Fortunately, this complication occurs only rarely (42), and the risk of cardiac perforation can be eliminated entirely by repositioning catheters when the tip is below the level of the carina on the chest x-ray.

Levitov A, Mayo P, Slonim A, eds. Critical Care Ultrasonography. New York: McGraw-Hill, 2009.
Noble VE, Nelson BP. Manual of Emergency and Critical Care Ultrasound. 2nd ed., New York: Cambridge University Press, 2011.

al. Real-time ultrasound-guided subclavian vein cannulation versus the landmark method in critical care patients: A prospective randomized study. Crit Care Med 2011; 39:1607-1612.. 26., 1 = Rodriguez CJ, Bolanowski A, Patel K, et al. Classic positioning decreases cross- sectional area of the subclavian vein. Am J Surg 2006; 192:135-137.. 27., 1 = Hernandez D, Diaz F, Rufino M, et al. Subclavian vascular access stenosis in dialysis patients: Natural history and risk factors. J Am Soc Nephrol 1998; 9:1507-1510.. 28., 1 = Getzen LC, Pollack EW. Short-term femoral vein catheterization. Am J Surg 1979; 138:875-877.. 29., 1 = Hilty WM, Hudson PA, Levitt MA, Hall JB. Real-time ultrasound-guided femoral vein catheterization during cardiopulmonary resuscitation. Ann Emerg Med 1997; 29:311- 316.. 30., 1 = Cummins RO (ed). ACLS Provider Manual. Dallas, TX; American Heart Association, 2001: pp. 38-39.
31., 1 = Ng P, Ault M, Ellrodt AG, Maldonado L. Peripherally inserted central catheters in general medicine. Mayo Clin Proc 1997; 72:225-233.. 32., 1 = Evans RS, Sharp JH, Linford LH, et al. Risk of symptomatic DVT associated with peripherally inserted central catheters. Chest 2010; 138:803-810.. 33., 1 = Hughes ME. PICC-related thrombosis: pathophysiology, incidence, morbidity, and the effect of ultrasound guided placement technique on occurrence in cancer patients. JAVA 2011; 16:8-18.

Immediate, 1 = Concerns. 34., 1 = Vesely TM. Air embolism during insertion of central venous catheters. J Vasc Interv Radiol 2001; 12:1291-1295.. 35., 1 = Mirski MA, Lele AV, Fitzsimmons L, Toung TJK. Diagnosis and treatment of vascular air embolism. Anesthesiology 2007; 106:164-177.. 36., 1 = Tocino IM, Miller MH, Fairfax WR. Distribution of pneumothorax in the supine and semirecumbent critically ill adult. Am J Radiol 1985;144:901-905.. 37., 1 = Blaivas M, Lyon M, Duggal S. A prospective comparison of supine chest radiography and bedside ultrasound for the diagnosis of traumatic pneumothorax. Acad Emerg Med 2005; 12:844-849.. 38., 1 = Ball CG, Kirkpatrick AW, Laupland KB, et al. Factors related to the failure of radiographic recognition of occult posttraumatic pneumothoraces. Am J Surg 2005; 189:541-546.. 39., 1 = Vezzani A, Brusasco C, Palermo S, et al. Ultrasound localization of central vein catheter and detection of postprocedural pneumothorax: an alternative to chest radiography. Crit Care Med 2010; 38:533-538.. 40., 1 = Collin GR, Clarke LE. Delayed pneumothorax: a complication of central venous catheterization. Surg Rounds 1994;17:589-594.. 41., 1 = Stonelake PA, Bodenham AR. The carina as a radiological landmark for central venous catheter tip position. Br J Anesthesia 2006; 96:335-340.. 42., 1 = Booth SA, Norton B, Mulvey DA. Central venous catheterization and fatal cardiac tamponade. Br J Anesth 2001; 87:298-302.

My dear Watson, you see but you do not observe.
Sir Arthur Conan Doyle, Scandal in Bohemia, 1891
Every patient in the ICU is equipped with at least one indwelling vascular catheter, and attention to the maintenance and adverse consequences of these devices is part of everyday patient care. This chapter describes the routine care and troublesome complications of indwelling vascular catheters. Many of the recommendations in this chapter are taken from the clinical practice guidelines listed at the end of the chapter (13).

The recommendations for routine catheter care are summarized in Table 3.1.

Catheter insertion sites should be covered with a sterile dressing for the life of the catheter. The sterile dressing can be a covering of sterile gauze pads, or an adhesive, transparent plastic membrane (called occlusive dressings). The transparent membrane in occlusive dressings is semipermeable, and allows the loss of water vapor, but not liquid secretions, from the underlying skin. This prevents excessive drying of the underlying skin to promote wound healing. Occlusive dressings are favored because the transparent membrane allows daily inspection of the catheter insertion site. Sterile gauze dressings are preferred when the catheter insertion site is difficult to keep dry (1).

Sterile Dressings, Recommendations: = Adhesive transparent dressings are favored because they allow inspection of the catheter insertion site. Sterile gauze dressings are used for skin areas that are difficult to keep dry. Adhesive transparent dressings and sterile gauze dressings provide equivalent protection against catheter colonization.. Antimicrobial Gels, Recommendations: = Do not apply antimicrobial gels to catheter insertion sites, except for hemodialysis catheters.. Replacing Catheters, Recommendations: = Regular replacement of central venous catheters is not recommended.. Flushing Catheters, Recommendations: = Avold using heparin in catheter flush solutions.
From the dinical practice quidelines in Reference 1.
Sterile gauze dressings and occlusive dressings are roughly equivalent in their ability to limit catheter colonization and infection (1,4-6). However, occlusive dressings can promote colonization and infection when moisture accumulates under the sealed dressing (4,6), so occlusive dressings should be changed when fluid accumulates under the transparent membrane.

The application of antimicrobial gels to the insertion site of central venous catheters does not reduce the incidence of catheter-related infections (1), with the possible exception of hemodialysis catheters (7). As a result, topical antimicrobial gels are recommended only for hemodialysis catheters (1), and are applied after each dialysis.

The major concern with peripheral vein catheters is phlebitis (from the catheter or infusate), which typically begins to appear after 3-4 days (1,8). Catheter replacement is thus recommended every 3—4 days (1), but peripheral catheters are usually left in place as long as there is no evidence of localized phlebitis ( i.e., pain, erythema and swelling around the insertion site).

Replacing central venous catheters at regular intervals, using either guidewire exchange or a new venipuncture site, does not reduce the incidence of catheter-related infections (9), and can actually promote complications (both mechanical and infectious) (10). One study showed a 7% complication rate associated with replacement of central venous catheters (11). The combination of no benefit and added risk is the reason that routine replacement of indwelling central venous catheters is not recommended (1). This recommendation also applies to peripherally inserted central catheters (PICCs), hemodialysis catheters, and pulmonary artery catheters (1). Catheter replacement is also not necessary when there is erythema around the catheter insertion site, since erythema alone is not evidence of infection (12).

Vascular catheters are flushed at regular intervals to prevent thrombotic obstruction, although this may not be necessary for peripheral catheters that are used intermittently (13). The standard flush solution is hep-arinized saline (with heparin concentrations ranging from 10 to 1,000 units/mL) (14). Catheters that are used only intermittently are filled with heparinized saline and capped when not in use; this is known as a heparin lock. Arterial catheters are flushed continuously at a rate of 3 mL/hour using a pressurized bag to drive the flush solution through the catheter (15).

The use of heparin in catheter flush solutions has two disadvantages: i.e., cost (considering all the catheter flushes performed in the hospital each day) and the risk of heparin-induced thrombocytopenia (see Chapter 19). These disadvantages can be eliminated by using heparin-free flush solutions. Saline alone is as effective as heparinized saline for flushing venous catheters (14), but this is not the case for arterial catheters (15), where 1.4% sodium citrate is a suitable alternative to heparinized saline for maintaining catheter patency (16).

The noninfectious complications of indwelling central venous catheters include catheter occlusion, thrombotic occlusion of the cannulated central vein, and perforation of the superior vena cava or right atrium.

Occlusion of central venous catheters can be the result of sharp angles or kinks in the catheter (usually created during insertion), thrombosis (from backwash of blood into the catheter), insoluble precipitates in the infusates (from medications or inorganic salts), and lipid residues (from propofol or total parenteral nutrition). Thrombosis is the most common cause of catheter obstruction, and is reported in up to 25% of central venous catheters (17). Occlusion from insoluble precipitates can be the result of water-insoluble drugs (e.g., diazepam, digoxin, phenytoin, trimethoprim-sulfa) or anion—cation complexes (e.g., calcium phosphate) that precipitate in an acid or alkaline solution (18).

Every effort should be made to restore patency and avoid replacing the catheter. Advancing a guidewire to dislodge an obstructing mass is not advised because of the risk of embolization. Chemical dissolution of the obstructing mass (described next) is the preferred intervention.
THROMBOTIC OCCLUSION: Since thrombosis is the most common cause of catheter obstruction, the initial attempt to restore patency should involve the local instillation of a thrombolytic agent. Alteplase (recombinant tissue plasminogen activator) is currently the favored thrombolytic agent for restoring catheter patency, and the regimen shown in Table 3.2 can restore patency in 80-90% of occluded catheters (19,20). There are no reports of abnormal bleeding associated with this regimen (19).

Preparation, Alteplase (recombinant tissue plasminogen activator) = Cathflo Activase (Genentech Inc.) available as a powder in 2 mq vials of alteplase Add 2 mL sterile water to each vial for a drug concentration of 1 mg/mL.. Regimen, Alteplase (recombinant tissue plasminogen activator) = 1. Instill 2 mL (2 mg) of drug solution into the occluded catheter and cap the hub of the catheter. 2. Wait 30 minutes and attempt to withdraw blood from the catheter. . If the occlusion persists, wait another 90 minutes (total dwell time=120 min) and attempt to withdraw blood from the catheter. . If the occlusion persists, prepare a second dose of alteplase (2 mg) and repeat steps 1-3. . ff patency Is restored, withdraw 5 mL of blood through the catheter to remove the drug solution and any residual clot. . If alteplase instillation does not restore patency, consider instilling 0.1N HGL for drug or calcium phosphate precipi- tates, or 70% ethanol ff lipid residues are suspected. co & on D
NON-THROMBOTIC OCCLUSION: Dilute acid will promote the dissolution of occlusive precipitates (e.g., calcium phosphate precipitates), and catheter occlusion that is refractory to thrombolysis is occasionally relieved after instillation of 0.1N hydrochloric acid (21). If lipid residues are suspected as a cause of catheter occlusion (e.g., from propofol infusions or lipid emulsions used for parenteral nutrition), instillation of 70% ethanol can restore catheter patency (18).
is described in Chapter 6.

Catheter-induced perforation of the superior vena cava or right atrium is uncommon but has potentially life-threatening complications of central venous cannulation, as described at the end of Chapter 2. These perforations are avoidable with vigilance and prompt correction of misplaced catheters.

Perforation of the superior vena cava is most often caused by left-sided central venous catheters that enter the superior vena cava but do not make the acute turn downward toward the right atrium. The tip of the catheter then abuts the lateral wall of the superior vena cava, as shown in Figure 2.9 in the last chapter. Most perforations occur within 7 days of catheter placement (28). The clinical symptoms (substernal chest pain, cough, and dyspnea) are nonspecific, and suspicion of perforation is often prompted by the sudden appearance of mediastinal widening or a pleural effusion on a chest x-ray, like the one in Figure 3.1. The unexpected appearance of a pleural effusion in a patient with a left-sided central venous catheter should always raise suspicion of superior vena cava perforation.
DIAGNOSIS: The pleural effusions associated with catheter-induced perforation of the superior vena cava are the result of intravenous fluids flowing into the pleural space. Thoracentesis will thus support the diagnosis of vena cava perforation if the pleural fluid is similar in composition to the intravenous infusion fluid. Pleural fluid glucose levels can be useful if a parenteral nutrition formula was infusing through the catheter. The perforation can be confirmed by injecting radiocontrast dye through a catheter in the superior vena cava and noting the presence of dye in the mediastinum.
MANAGEMENT: When vena cava perforation is first suspected, the infusion should be stopped immediately. If the diagnosis is confirmed, the catheter should be removed immediately (this does not provoke mediastinal bleeding) (28). Antibiotic therapy is not necessary unless there is evidence of infection in the pleural fluid (28).

The most life-threatening complication of central venous catheterization is cardiac tamponade from catheter-induced perforation of the right atrium. Although considered rare, the actual incidence of this complication is not known (29). The first sign of tamponade is usually the abrupt onset of dyspnea, which can progress to cardiovascular collapse within an hour. The diagnosis requires ultrasound evidence of a pericardial effusion with diastolic collapse of the right heart, and immediate pericardiocentesis is necessary to relieve the tamponade. Emergency thoracotomy may also be necessary if there is a large tear in the wall of the heart.
FIGURE 3.1 Chest x-ray of a patient with a perforated superior vena cava caused by a left-sided subclavian catheter (which is positioned like the catheter in Figure 2.9). Image courtesy of John E. Heffner, MD (from Reference 27).
Catheter-associated pericardial tamponade is often overlooked, and the mortality rate varies from 40% to 100% in published reports (29). The most effective approach to this condition is prevention, which requires proper positioning of central venous catheters so the tip is at, or slightly above, the tracheal carina. The proper position of a central venous catheter is shown in Figure 2.8 in the last chapter.

Pathogenic organisms can colonize the intravascular portion of central venous catheters, and dissemination of these organisms in the bloodstream (i.e., catheter-related bloodstream infections) can be fatal in up to 25% of cases (30). Fortunately, the incidence of these infections has declined by almost 60% over the past decade (31), presumably as a result of preventive measures like those in Table 2.3 in the last chapter. The following is a description of the etiology and management of these infections.

The sources of catheter-related bloodstream infections are indicated in Figure 3.2. Each is described below using the corresponding numbers in the illustration.
1. Microbes can gain access to the bloodstream via contaminated infusates (e.g., blood products), but this occurs rarely.
2. Contamination of the internal lumen of vascular catheters can occur through break points in the infusion system, such as catheter hubs. This may be a prominent route of infection for catheters inserted through subcutaneous tunnels.
3. Microbes on the skin can migrate along the subcutaneous tract of an indwelling catheter and eventually reach (and colonize) the intravascular portion of the catheter. This is considered the principal route of infection for percutaneous (non-tunneled) catheters, which includes most of the catheters inserted in the ICU .
4. Microorganisms in circulating blood can attach to the intravascular portion of an indwelling catheter. This is considered a secondary seeding of the catheter from a source of septicemia elsewhere, but proliferation of the microbes on the catheter tip could reach the point where the catheter becomes a source of septicemia.
FIGURE 3.2 Sources of microbial colonization at the distal end of vascular catheters. See text for explanation.
(For a contrary view of the importance of skin microbes in catheter-related infections, see the very last section of the chapter: A Final Word.)

Microbes are not freely moving organisms, and have a tendency to congregate on inert surfaces. When a microbe comes in contact with a surface, it releases adhesive molecules (called adhesins, of course) that firmly attach it to the surface. The microbe then begins to proliferate, and the newly formed cells release polysaccharides that coalesce to form a matrix known as slime (because of its physical properties), which then encases the proliferating microbes. The encasement formed by the polysaccharide matrix is called a biofilm. Biofilms are protective barriers that shield microbes from the surrounding environment, and this protected environment allows microbes to thrive and proliferate (32).
Biofilms are ubiquitous in nature, and predominate on surfaces that are exposed to moisture (the slippery film that covers rocks in a stream is a familiar example of a biofilm). They also form on indwelling medical devices such as vascular catheters (33). In fact, the organism that is most frequently involved in catheter-related infections, Staphylococcus epidermidis, shows a propensity for adherence to polymer surfaces and slime production (34). A biofilm of S. epidermidis is shown in .
FIGURE 3.3 Electron micrograph of Staphylococcus epidermidis encased in a biofilm. Image courtesy of Jeanne VanBriesen, Ph.D., Carnegie Mellon University. Image colorized digitally.
BIOFILM RESISTANCE: Biofilms on medical devices are problematic because they show a resistance to host defenses and antibiotic therapy. Phagocytic cells are unable to ingest organisms that are embedded in a biofilm, and antibiotic concentrations that eradicate free-living bacteria must be 100 to 1,000 times higher to eradicate bacteria in biofilms (35). Chemical substances that disrupt biofilms, such as tetrasodium EDTA, may have a prominent role in the eradication of biofilms on medical devices (36).

Each day that a catheter remains in place carries a risk of infection, so the frequency of catheter-related infections is expressed in terms of the total number of catheter-days. The incidence of catheter-associated infections in Table 3.3 is expressed as the number of infections per 1,000 catheter-days. The information in this table, which is organized by type of specialty ICU, is from the National Healthcare Safety Network Report of 2010, which includes data from about 2,500 hospitals in the United States (37). The most striking feature of this data is the remarkably low incidence of catheter-associated infections in all the ICUs, regardless of specialty Furthermore, this data overestimates the actual incidence of infection, as described next.

Bum Units, 1 = 3.5. Bum Units, 2 = 0-8.0. Trauma Units, 1 = 1.9. Trauma Units, 2 = 0-4.0. Medical ICUs, 1 = 1.8. Medical ICUs, 2 = 0-35. Surgical ICUs, 1 = 14. Surgical ICUs, 2 = 0-3.2. Med/Surg ICUs, 1 = 1.4. Med/Surg ICUs, 2 = 0-34. Coronary Gare Units, 1 = 1.3. Coronary Gare Units, 2 = 0-27. Neurosurgical ICUs, 1 = 1.3. Neurosurgical ICUs, 2 = 0-27. Cardiothoracic ICUs, 1 = 0.9. Cardiothoracic ICUs, 2 = O0-2.0
From the Nationa Healthcare Safety Network Raport (37). Includes only ICUs in major teaching hospitals.

The following two definitions are used to identify infections attributed to central venous catheters:
Catheter-Associated Bloodstream Infections (CABI) are bloodstream infections that have no apparent source other than a vascular catheter in patients who either have an indwelling catheter or have had one within 48 hours of the positive blood culture. This is the definition used in epidemiological surveys (like the one in Table 3.3), and it requires no evidence of microbial growth on the suspected catheter.
Catheter-Related Bloodstream Infections (CRBI) are bloodstream infections where the organism identified in peripheral blood is also present in significant quantities on the tip of the catheter or in a blood sample drawn through the catheter (the criteria for a significant quantity is presented later). This is the definition used in clinical practice, and it requires evidence of catheter involvement with the same organism present in peripheral blood.
The diagnostic criteria for CABI (which are used in clinical surveys) are far less rigorous than the diagnostic criteria for CRBI (which are used in clinical practice), so the incidence of CABI (like the one in Table 3.3) can overestimate the incidence of CRBI (the actual incidence in clinical practice). In one comparison study, the incidence of CABI exceeded that of CRBI by one infection per 1,000 catheter-days (38). If this difference is applied to the data in Table 3.3 (i.e., subtract one from the incidences in the table), the mean incidence of catheter-related infections falls to less than one per 1,000 catheter days in most of the ICUs.

Catheter-related infections do not appear in the first 48 hours after catheter insertion (which presumably is the time required for colonization of the catheter tip). When they do appear, the clinical manifestations are typically non-specific signs of systemic inflammation (e.g., fever, leukocytosis). Inflammation at the catheter insertion site has no predictive value the presence of septicemia (12), and purulent drainage from the catheter insertion site is uncommon, and can be a manifestation of an exit-site infection without bloodstream invasion (2). The diagnosis of CRBI is thus not possible on clinical grounds, and one of the culture methods described next is required to conform or exclude the diagnosis.

There are three culture-based approaches to the diagnosis of CRBI, and these are included in Table 3.4. The culture method you select in each case will be determined by the decision to retain or replace the suspect catheter.

The evaluation of suspected CRBI requires one of three possible decisions for the suspect catheter:
1. Remove the catheter and insert a new catheter at a new venipuncture site.
2. Replace the catheter over a guidewire using the same venipuncture site.
3. Leave the catheter in place.
The first option (remove the catheter and insert a new one at a new site) is recommended for patients with neutropenia, a prosthetic valve, indwelling pacemaker wires, evidence of severe sepsis or septic shock, or purulent drainage from the catheter insertion site (2). Otherwise, catheters can be left in place or replaced over a guidewire. Option #3 (leave the catheter alone) is desirable because most evaluations for CRBI do not confirm the diagnosis (so replacing the catheter is not necessary), and because guidewire exchanges can have adverse effects (10,11).

Semiquantitative Culture of Catheter Tip, Diagnostic Criteria for CRBI = Same organism on catheter tip and In peripheral blood, and growth from the catheter tip >15 colony-forming units (cfu) in 24 hours.. Differential Quantitative Blood Cultures, Diagnostic Criteria for CRBI = Same organism in peripheral blood & catheter blood, and colony count from catheter blood 23 times greater than colony count from peripheral blood.. Differential Time to Positive Culture, Diagnostic Criteria for CRBI = Same organism in peripheral blood & catheter blood, and onset of growth in catheter blood at least two hours before onset of growth in peripheral blood.
From the clinical practice guidelines in Reference 2.

The standard approach to suspected CRBI is to remove the catheter and culture the tip, as outlined below.
1. Before the catheter is removed, swab the skin around the catheter insertion site with an antiseptic solution.
2. Remove the catheter using sterile technique and sever the distal 5 cm (2 inches) of the catheter. Place the severed segment in a sterile culturette tube for transport to the microbiology laboratory, and request a semiquantitative or roll-plate culture (the tip of
This method is also designed for catheters that remain in place, and is based on the expectation that when a catheter is the source of a bloodstream infection, the blood withdrawn through the catheter will show microbial growth earlier than blood obtained from a peripheral vein. This method uses routine (qualitative) blood cultures; and requires 10 mL of blood drawn through the catheter, and 10 mL of blood from a peripheral vein. The diagnosis of CRBI is confirmed if the same organism is isolated from the catheter blood and peripheral blood, and growth is first detected at least 2 hours earlier in the catheter blood. This approach is technically easier and less costly than comparing quantitative blood cultures, but the diagnostic accuracy is lower (39).
FIGURE 3.4 Culture plates showing colonies of bacterial growth from blood drawn from a central venous catheter (Catheter Blood) and a peripheral vein (Peripheral Blood). The denser growth from catheter blood is evidence of catheterrelated septicemia. (From Curtas S, Tramposch K. Culture methods to evaluate central venous catheter sepsis. Nutr Clin Pract 1991;6:43). Image colorized digitally.

The organisms involved in CRBI are (in order of prevalence) coagulase-negative staphylococci, Gram-negative aerobic bacilli (Pseudomonas aeruginosa, Klebsiella pneumoniae, E. coli, etc), enterococci, Staphylococcus aureus and Candida species (40). Coagulase-negative staphylococci (mostly Staphylococcus epidermidis) are responsible for about one-third of infections, while Gram-negative bacilli and other organisms that inhabit the bowel (enterococci and Candida species) are involved in about half the infections. This microbial spectrum is important to consider when selecting empiric antimicrobial therapy.

Empiric antibiotic therapy is recommended for all ICU patients with suspected CRBI, and should be started immediately after cultures are obtained. The recommendations for empiric antibiotic coverage from published guidelines (2) are shown in Table 3.5.

Organism
Antibiotic
Staphylococel,  = Vancomycin. Staphylococel, Comment = If MRSA isolates with MICG>2 mg/mL are prevalent, use daptomycin.. Enterococci,  = Vancomycin. Enterococci, Comment = If vancomycin resistance Is a concern, use daptomycin.. Gram-Negative Bacill,  = Carbepenen? or Cefepime or Pipericillin- Tazobactam. Gram-Negative Bacill, Comment = Add aminoglycoside for neutropenia or concern for multidrug-resistant organisms.. Candida species,  = Echinocandin®. Candida species, Comment = Indications: Femoral catheter, TPN hematologic malignancy, pro- longed antibiotic Rx, recent transplant, or Candida sp. elsewhere.. Cardiothoracic ICUs,  = 0.9. Cardiothoracic ICUs, Comment = 0-2.0
From the cinical practice guidelines in Reference 2. See Chapter 52 for antibiotic dosing.
aCarbapenems include inmipenem,merapenam, and doripenem.
bEchinocandins include caspefungin, micafungin, and anidulafungin.

Vancomycin is the backbone of the empiric antibiotic regimen because it is the most active agent against staphylococci (including coagulase-negative and methicillin-resistant strains), and enterococci, which together are responsible for about 50% of catheterrelated infections (40). Daptomycin can substitute for vancomycin if there is a risk of infection with vancomycin-resistant enterococci. Empiric coverage for enteric Gramnegative bacilli is advised because these organisms are the second most common isolates in ICU patients with CRBI (40). The antibiotics best-suited for empiric Gram-negative coverage include the carbepenems (e.g., meropenem), the fourth-generation cephalosporins (e.g., cefepime), and the B-lactam/B-lactamase inhibitor combinations (e.g., pipericillin/tazobactam ). Additional Gram-negative coverage (with an aminoglycoside) is recommended for patients with neutropenia, and when multidrugresistant Gram-negative bacilli are possible offenders.
Empiric coverage for candidemia is recommended when the conditions listed in Table 3.5 are present. The echinocandins (e.g., caspofungin) are favored over the azoles (e.g., fluconazole) for empiric coverage because some Candida species (i.e., Candida krusei and Candida glabrata) are resistant to azoles. The dosing of antifungal agents is described in

If the culture results confirm the diagnosis of CRBI, further antibiotic therapy is dictated by the identified organisms and antibiotic susceptibilities. The pathogen-specific antibiotic recommendations from the most recent guidelines on CRBI (2) are shown in Table 3.6.

Antibiotics = . Iv. Canalda sp.u, Alternative Antibiotics = . Candida albicans, Prefered Antibiotics = Fluconazole. Candida albicans, Alternative Antibiotics = Echinocandin®. Candida krusel and Candida glabrata, Prefered Antibiotics = Echinocandin®. Candida krusel and Candida glabrata, Alternative Antibiotics = Amphotericin B
From the clinical practice guidslines in Reference 2. See Chapter 52 for antibiotic dosing.
oCarbapenems include imipsnem,merapenem, and doripenam.
'Echinocandins include caspofungin, micafungin, and aniduafungin.
CATHETER MANAGEMENT: When the diagnosis of CRBI is confirmed, catheters that were left in place or changed over a guidewire should be removed and reinserted at a new venipuncture site, unless the offending organism is a coagulase-negative staphylococcus (e.g.,S. epidermidis) or an enterococcus, and the patient shows a favorable response to empiric antimicrobial therapy (2).
Decontamination of catheters that are left in place can be difficult with systemic antibiotic therapy (probably because of biofilm resistance), and recurrent infections are common (41). Instillation of concentrated antibiotic solutions into indwelling catheters (antibiotic lock therapy) enhances the ability to disrupt biofilms and eradicate persistent organisms (see next).

Antibiotic lock therapy is recommended for all catheters that are left in place during systemic antibiotic therapy (2). The antibiotic lock solution contains the same antibiotic used systemically, in a concentration of 2-5 mg/mL in heparinized saline. This solution is injected into each lumen of the indwelling catheter and allowed to dwell for 24 hours, and the solution is then replaced every 24 hours for the duration of systemic antibiotic therapy. If the catheter is never idle and antibiotic lock therapy is not possible, then the systemic antibiotic(s) should be delivered through the suspect lumen. (For a list of pathogen-specific antibiotic lock solutions, see the clinical practice guidelines in Reference 2.)

The duration of antibiotic therapy is determined by the offending pathogen, the status of the catheter (i.e., replaced or retained), and the clinical response. For patients who show a favorable response in the first 72 hours of systemic antibiotic therapy, the recommended duration of treatment is as follows (2):
1. If coagulase-negative staphylococci are involved, antibiotic therapy is continued for 5— 7 days if the catheter is removed, and for 10-14 days if the catheter is left in place.
2. If S. aureus is the culprit, antibiotic therapy can be limited to 14 days if the catheter is removed and the following conditions are satisfied: the patient is not diabetic or immunosuppressed, there are no intravascular prosthetic devices in place, and there is no evidence of endocarditis on transesophageal ultrasound (2). (Some recommend that all cases of S. aureus bacteremia include an evaluation for endocarditis with transesophageal ultrasound, which should be performed 5-7 days after the onset of bacteremia.) If any of these conditions are present, 4-6 weeks of antibiotic therapy is recommended (2).
3. For infections caused by enterococci or Gram-negative bacilli, 7-14 days of antibiotic therapy is recommended, regardless of whether the catheter is replaced or retained (2).
4. For uncomplicated Candida infections, antifungal therapy should be continued for 14 days after the first negative blood culture (2).

Continued signs of sepsis or persistent septicemia after 72 hours of antimicrobial therapy should prompt an evaluation for the following conditions.

As mentioned earlier, thrombus formation on indwelling catheters is common, and these thrombi can trap microbes from a colonized catheter. Proliferation of these microbes can then transform the thrombus into an intravascular abscess. This condition is known as suppurative thrombophlebitis, and the most common offending organism is Staphylococcus aureus (2). Clinical manifestations are often absent, but can include purulent drainage from the catheter insertion site, limb swelling from thrombotic venous occlusion, multiple cavitary lesions in the lungs from septic emboli, and embolic lesions of the hand if arterial catheters are involved.
The diagnosis of septic thrombophlebitis requires evidence of thrombosis in the cannulated blood vessel (e.g., by ultrasound) and persistent septicemia with no other apparent source. Treatment includes catheter removal and systemic antibiotic therapy for 4-6 weeks (2). Surgical excision of the infected thrombus is usually not necessary, and is reserved for cases of refractory septicemia. There is no consensus on the use of heparin anticoagulation in suppurative thrombophlebitis; according to the most recent guidelines on catheter-related infections (2), heparin therapy is a consideration (not a requirement) for this condition.

Nosocomial endocarditis is uncommon; the reported incidence in university teaching hospitals is 2-3 cases annually (42,43). Vascular catheters are implicated in 30 to 50% of cases, and staphylococci (mostly S. aureus) are the offending organisms in up to 75% of cases (42,43). Methicillin-resistant strains of S. aureus (MRSA) predominate in some reports (44).
Typical manifestations of endocarditis (e.g., new or changing cardiac murmur) can be absent in as many as two-thirds of patients with nosocomial endocarditis involving Staphylococcus aureus (44). As a result, endocarditis should be considered in all cases of S. aureus bacteremia, including patients who appear to respond to antimicrobial therapy (2). The diagnostic procedure of choice for endocarditis is transesophageal (not transthoracic) ultrasound. Diagnostic findings include valvular vegetations, new-onset mitral regurgitation, and perivalvular abscess.
Antimicrobial therapy for 4-6 weeks is standard recommendation for endocarditis. Unfortunately, despite our best efforts for antibiotic therapy, about 30% of patients do not survive the illness (42-44).

1. O'Grady NP, Alexander M, Burns LA, et. al. and the Healthcare Infection Control Practices Advisory Committee (HICPAC). Guidelines for the Prevention of Intravascular Catheter-related Infections. Clin Infect Dis 2011; 52:e1—e32. (Available at www.cdc.gov/hicpac/pdf/guidelines/bsi-guidelines-2011.pdf)
2. Mermel LA, Allon M, Bouza E, et al. Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis 2009; 49:1—45.
3. Debourdeau P, Chahmi DK, Le Gal G, et al. 2008 guidelines for the prevention and treatment of thrombosis associated with central venous catheters in patients with cancer: report from the working group. Ann Oncol 2009; 20:1459-1471.

intravascular lines. Heart Lung 1991; 20:631-642.. 15., 1 = American Association of Critical Care Nurses. Evaluation of the effects of heparinized and nonheparinized flush solutions on the patency of arterial pressure monitoring lines: the AACN Thunder Project. Am J Crit Care 1993; 2:3-15.. 16., 1 = Branson PK, McCoy RA, Phillips BA, Clifton GD. Efficacy of 1.4% sodium citrate in maintaining arterial catheter patency in patients in a medical ICU. Chest 1993; 103:882-885.
, 1 = therapy. Ann Surg 1963; 157:847-858.. 47., 1 = Marshall JC, Christou NV, Horn R, Meakins JL. The microbiology of multiple organ failure. Arch Surg 1988; 123:309-315.. 48., 1 = Mrozek N, Lautrette A, Aumeran C, et al. Bloodstream infection after positive catheter cultures: what are the risks in the intensive care unit when catheters are routinely cultured on removal. Crit Care Med 2011; 39:1301-1305.. 49., 1 = Atela I, Coll P, Rello J, et al. Serial surveillance cultures of skin and catheter hub specimens from critically ill patients with central venous catheters: Molecular epidemiology of infection and implications for clinical management and research. J Clin Microbiol 1997; 35:1784-1790.. 50., 1 = Sing R, Marino PL. Bacterial trasnslocation: an occult cause of catheter-related sepsis. Infect Med 1993; 10:54-57.

The only thing necessary for the triumph of evil is for good men to do nothing.
Edmund Burke 1770

The risk of nosocomial (hospital-acquired) infections is not limited to the patient population; i.e., hospital workers are also at risk of acquiring infections from occupational exposure to bloodborne and airborne pathogens. The bloodborne pathogens include the human immunodeficiency virus (HIV), and the hepatitis B and C viruses, while the airborne pathogens include Mycobacterium tuberculosis, and the respiratory viruses (e.g., the influenza virus). This chapter describes the modes and risks of disease transmission, and the recommended protective measures, for these potentially harmful occupational exposures. Most of the recommendations in this chapter are from the clinical practice guidelines listed at the end of the chapter (1-5).

The transmission of bloodborne pathogens occurs primarily by accidental puncture wounds from contaminated needles, and less frequently by exposure of mucous membranes and nonintact skin to splashes of infected blood. The risk of transmission for each of the bloodborne pathogens is summarized in Table 4.1.

Neediestick Injury, 1 = HBY (+) Blood. Neediestick Injury, 2 = 22 — 31%. Neediestick Injury, 3 = 3-45. Needilestick Injury, 1 = HCV (+) Blood. Needilestick Injury, 2 = 1.8%. Needilestick Injury, 3 = 56. Needlestick Injury, 1 = HIV (+) Blood. Needlestick Injury, 2 = 0.3%. Needlestick Injury, 3 = 333. Mucous Membrane, 1 = HIV (+) Blood. Mucous Membrane, 2 = 0.09%. Mucous Membrane, 3 = Tan
From the cinical practice guidelines in References 2 and 3.
H8V = hepatitis B virus; HCV = hepatitis C virus; A/V = human immunedeficiancy virus.

Each year, about 10% of hospital workers experience an accidental puncture wound from a hollow-bore needle or suture needle; i.e., a needlestick injury (5,6). High-risk activities include the manipulation of suture needles, and the recapping and disposal of used hollow-bore needles. The incidence of needlestick injuries is highest in staff surgeons and surgical trainees; e.g., in one survey of 17 surgical training programs, 99% of the residents claimed at least one needlestick injury by the last year of training, and 53% of the injuries involved high-risk patients (7). Over half of the needlestick injuries in this survey were not reported, which is consistent with other studies showing that needlestick injuries are often dismissed as insignificant events (8).

The emergence of HIV in the 1980s created concern for needlestick in-juries and, in the year 2000, the United States Congress passed the Needlestick Safety and Prevention Act that mandates the use of "safety-engineered" needles in all American healthcare facilities. An example of a safety-engineered needle is shown in Figure 4.1. The needle is equipped with a rigid, plastic housing that is attached by a hinge joint to the hub of the needle. The protective housing is normally positioned away from the needle so it does not interfere with normal use. After the needle is used, it is locked into the protective housing as shown in the illustration. The needle and attached syringe are then placed in a puncture-proof "sharps container" for eventual disposal. (Sharps containers are found in every room in the ICU.) This procedure avoids any contact between the hands and the needle, thereby eliminating the risk of needlestick injury.

Once the needle is locked in its protective housing, it is not possible to remove it for further use. In situations where a needle may need to be reused (e.g., for repeated lidocaine injections during a prolonged procedure), the needle can be rendered harmless when idle by recapping it with the one-handed "scoop technique" shown in Figure 4.2. With the syringe still attached, the needle is advanced into the needle cap and then rotated vertically until it is perpendicular to the horizontal surface. The needle is then pushed into the cap until it locks in place. The hands never touch the needle while it is recapped , thereby eliminating the risk of a needlestick injury.

Occupational transmission of HIV is a universally feared but infrequent occurrence. From 1981 through December 2002, there were 57 documented cases of HIV transmission to healthcare workers (9). Of these 57 cases, 19 cases (33.3%) involved laboratory personnel and 2 cases (3.5%) in-volved housekeeping and maintenance workers, leaving only 36 cases involving hospital personnel that work at the bedside. These 36 cases represent an average of only 1.6 cases annually over the 22-year survey period. If all these cases occurred in the 6,000 ICUs in this country, the average yearly risk of HIV transmission in an ICU setting would be about one case per 3,750 ICUs. Not much of a risk,
FIGURE 4.1 A safety-engineered needle that allows the needk to be locked into a rigid plastic housing after it is used. The hands never touch the needle, thereby eliminating the risk of a needlestick injury.

A needlestick puncture from a hollow needle will transfer an average of one microliter (10-6 L) of blood (10). During the viremic stages of HIV infection, there are as many as 5 infectious particles per microliter of blood (11). Therefore, puncture of the skin with a hollow needle that contains HIV-infected blood is expected to transfer at least a few infectious particles. Fortunately, this is not enough to transmit the disease in most cases. As shown in Table 4.1, the average risk for HIV transmission from a single needlestick injury with HIV-infected blood is 0.3% (2,3), which translates to one infection for every 333 needlestick injuries involving HIV-infected blood. The likelihood of HIV transmission is greater in the following circumstances: when the source patient has advanced HIV disease, when the skin puncture is deep, when there is visible blood on the needle, and in cases where the needle entered an artery or vein in the source patient (12).
FIGURE 4.2 The one-handed "scoop technique" for safely recapping needles that may need to be reused.

Exposure of mucous membranes to HIV-infected blood (e.g., a blood splash to the face) is much less likely to result in HIV transmission than a needlestick injury. As shown in Table 4.1, the average risk for HIV transmission from a single mucous membrane exposure to HIV-infected blood is 0.09% (2,3), which translates to one infection for every 1,111 mucous membrane exposures to HIV-infected blood (i.e., a one-in-a-thousand chance of disease transmission).

The postexposure management of needlestick injuries or mucous membrane exposures is determined by the HIV status of the source patient. If the HIV status is unknown, this can be quickly resolved in the hospital setting by performing a rapid HIV-antibody test on a blood sample from the source patient. This is an enzyme-linked immunoabsorbent assay (ELISA) that yields results in only 10 to 15 minutes. A negative test does not eliminate the possibility of HIV infection (because it takes 4-6 weeks for antibodies to appear in plasma after the onset of infection), but it does obviate the need for postexposure drug prophylaxis. A positive ELISA test in the source patient is an indication to begin postexposure drug prophylaxis, but the result must be confirmed by another test; i.e., a Western Blot or immunofluorescent antibody assay. The recommendations for postexposure prophylaxis are shown in Table 4.2 (3). When indicated, prophylactic drugs should be started within 36 hours of the exposure (12).

Neediestick Injuries, __HlV status of Source Patient __.HIV(+): Class 17 = . Neediestick Injuries, __HlV status of Source Patient __.HIV(+): Class 27 = . Neediestick Injuries, __HlV status of Source Patient __.HIV(-)_ = . Less Severe!, __HlV status of Source Patient __.HIV(+): Class 17 = 2 drugs. Less Severe!, __HlV status of Source Patient __.HIV(+): Class 27 = 23 drugs. Less Severe!, __HlV status of Source Patient __.HIV(-)_ = No drugs. More Severe1, __HlV status of Source Patient __.HIV(+): Class 17 = 3 drugs. More Severe1, __HlV status of Source Patient __.HIV(+): Class 27 = 23 drugs. More Severe1, __HlV status of Source Patient __.HIV(-)_ = No drugs. Mucous Membrane Exposures, __HlV status of Source Patient __.HIV(+): Class 17 = . Mucous Membrane Exposures, __HlV status of Source Patient __.HIV(+): Class 27 = . Mucous Membrane Exposures, __HlV status of Source Patient __.HIV(-)_ = . Small Volume?, __HlV status of Source Patient __.HIV(+): Class 17 = 2 drugs (275. Small Volume?, __HlV status of Source Patient __.HIV(+): Class 27 = 2 drugs. Small Volume?, __HlV status of Source Patient __.HIV(-)_ = No drugs. Large Volume?, __HlV status of Source Patient __.HIV(+): Class 17 = 2 drugs. Large Volume?, __HlV status of Source Patient __.HIV(+): Class 27 = =3 drugs. Large Volume?, __HlV status of Source Patient __.HIV(-)_ = No drugs
From the clinical practice guidelines in Reference 3. See text for the recommended crug combinations.
tHIV[+}: Class 1 -— asymptomatic HIV infaction or viral load <1,500 copies'mL

- +HIVi+): Class 2 — symptomatic HIV infection or vird lead =1,500 copies/mL, or acute seroconversion
*Drugs ara optiond,
- iLeass Severe: solid needle or superficial injury. More Severe: deep puncture, visible blood on the needie, or aneedia that entered an artery or vain of the source patient.
2Small Volume: a few crops. Large Volume: a major blood splash.

The standard two-drug regimen is a combination of two nucleoside reverse transcriptase inhibitors: zidovudine (300 mg BID) and lamivudine (150 mg BID). These two drugs are available in a combination tablet (Combivir, containing 300 mg zidovudine and 150 mg lamivudine per tablet), which is taken twice daily. If additional drugs are indicated, the preferred regimen is a combination of two protease inhibitors: lopinavir/ ritinovir (400 mg/100 mg), available as a single tablet (Kaletra) taken three times daily (3). In highrisk exposures, 28 days of drug therapy is recommended. However, as many as 50% of hospital workers who receive antiretroviral drugs following HIV exposure are unable to complete the four-week treatment period because of adverse drug effects (3).
ADVERSE DRUG EFFECTS: Side Effects Are Common With Antiretroviral Drug Therapy, And The Frequency Of Side Effects Is Higher When The Drugs Are Taken For Postexposure Prophylaxis. The Most Frequent Side Effects In-Clude Nausea, Malaise, Fatigue, And Diarrhea (3). More Serious Drug Toxicities Include Pancreatitis And Lactic Acidosis From Nucleoside Reverse' Transcriptase Inhibitors, And Severe Hypertriglyceridemia From Protease Inhibitors (3)
DRUG INTERACTIONS: Protease Inhibitors Have A Number Of Serious Drug Interactions. Drugs That Are Contraindicated During Therapy With Protease Inhibitors
Since most healthy adults achieve immunity after completion of the first vaccination series, postvaccination anti-HBs levels are not measured routinely. The principal indications for postvaccination anti-HBs levels are occupational exposure to HBV-infected blood, and high-risk occupations (e.g., hemodialysis technicians).

The management strategies following possible exposure to HBV are outlined in Table 4.3. Management decisions are dictated by the immune status of the exposed individual, and the HBV status of the source patient (as determined by the presence or absence of hepatitis B surface antigen in the blood).
Not vaccinated, oo. = HBIG (0.06 mL/kg IM) and start HBV vaccination. Not vaccinated, oo.“HBSAGE i = Start HBV vaccination. Vaccinated and Immunet, oo. = No treatment. Vaccinated and Immunet, oo.“HBSAGE i = No treatment. Vaccinated and Not Immunet, oo. = HBIG (0.06 mL/kg IM) and repeat HBV vaccination* or HBIG x25. Vaccinated and Not Immunet, oo.“HBSAGE i = Repeat HBV vaccination*
From the clinical practice quidelines in Reference 2.
HBsAg
=
hepatitis
B
surface antigen;
HBIG
=
hepatitis
B
immune dobuin.
tmmunity requires the prasence in blood of the antibody to hapatitis B surface antigen {antiHEs) at a concentration 210 miLimL.
"if immunity is not achiaved after 2 coursas of HBV vaccination, no further immunization is warranted. SHBIG x2 = Hepatitis B immune globulin in two intramuscular doses of 0.06 miLl'mL each. This ragimen is reserved for subjects whe doen't achiave immunity after 2 courses of HEV vaccination.

Following exposure to HBV-infected blood (i.e., the blood of the source patient is positive for hepatitis B surface antigen), exposed individuals who have not achieved immunity to HBV (either because they did not receive the vaccination series or because the postvaccination anti-HBs levels are <10 mIU/mL) should receive hepatitis B immune globulin (HBIG) by deep intramuscular injection at a dose of 0.06 mL/kg, along with the first dose of HBV vaccine. The HBV vaccination series is recommended for all nonimmune individuals who are exposed to HBV-positive blood, except for individuals who are nonresponders after two completed immunizations. This latter group of subjects (i.e., nonresponders) should receive two injections of HBIG (0.06mL/kg for each injection).

Transmission of the hepatitis C virus (HCV) is considered a rare event in the hospital setting, with the possible exception of the hemodialysis suite. As shown in Table 4.1, the average risk for disease transmission from a single needlestick exposure to HCV-infected blood is 1.8% (2), which translates to one infection per 56 exposures. Transmission from mucous membrane exposure is rare, and there are no documented cases of HCV transmission through nonintact skin.
The antibody produced in response to HCV infection (anti-HCV) is not protective (2), which means there is no vaccine for HCV and no effective antibody prophylaxis following exposure to HCV-infected blood. When a hospital worker sustains a needlestick injury, the HCV status of the source patient can be determined by the presence or absence of antiHCV in blood. If the source patient has evidence of HCV infection (i.e., has a positive assay for anti-HCV), serial determinations of anti-HCV antibody in the exposed subject is recommended for 6 months following the exposure (2). A positive assay for anti-HCV in the exposed individual is evidence of HCV transmission.

Pathogens that are transmitted through the air are generated by coughing or sneezing (one cough or sneeze can produce 3,000 airborne particles), and by procedures such as airway suctioning, endotracheal intubation, and cardiopulmonary resuscitation. The transmission of these path-ogens is classified according to the size of the dispersed particles and the mode of transmission. Figure 4.3 shows a summary of the types of transmission, the pathogens involved, and the infection control measures recommended to prevent transmission.
FIGURE 4.3 Respiratory precautions for pathogens that are dispersed in the air.

Droplets are relatively large particles (>5 microns in diameter) that do not travel far from the source (typically <3 feet). These particles are transmitted by impact on the nasal mucosa and oral mucosa, and are not inhaled into the lungs. The principal pathogens that are transmitted via the droplet route are respiratory viruses, including the influenza virus and the corona virus responsible for Severe Acute Respiratory Syndrome (SARS), along with Mycoplasma pneumoniae, Nisseria meningiditis, and Bordatella pertussis (the causative agent for diphtheria).

The recommendations for preventing droplet transmission include: a private room for the patient if possible, masks for hospital workers and visitors while in the patient's room, and a mask for the patient whenever outside the room (1). Either surgical masks or procedure masks (i.e., the ones with elastic loops for the ears) are sufficient for this purpose. If a private room is not available, the patient's bed should be separated from other beds by a curtain and a distance of at least 3 feet (1).

Airborne transmission is the term used to describe the transmission of small infectious particles (<5 microns in diameter) that are suspended in the air and can reach distances that extend well beyond the patient's room. These particles are also small enough to be inhaled into the lungs. The principal pathogen transmitted via the airborne route in adults is Mycobacterium tuberculosis.

The features that distinguish airborne precautions from droplet precautions include the use of negative-pressure isolation rooms to prevent the movement of airborne pathogens out of the room, and the use of particulate respirators to prevent inhalation of airborne pathogens (1). For cases of active pulmonary tuberculosis, these precautions are continued until there are 3 consecutive sputum samples that are devoid of acid-fast bacilli on microscopic exam (1).

Protective face masks (surgical masks and procedure masks) are de-signed to prevent large infectious droplets from impacting on the nasal and oral mucosa, and do not block inhalation of small airborne particles into the lungs. Respirators, on the other hand, are designed to block in-halation of pathogenic material into the lungs; particulate respirators block inhalation of small (<5y in diameter) infectious particles, while gas mask respirators block the inhalation of toxic gases. In the hospital setting, particulate respirators are recommended primarily for protection against Mycobacterium tuberculosis (1). The Centers for Disease Control (CDC) currently recommends the "N95" respirator for this use (15); the "N" indicates that the mask will block non-oil based or aqueous aerosols (the type that transmits the tubercle bacillus), and the "95" indicates the mask will block 95% of airborne infectious particles. Respirators must create a tight seal around the nose and mouth to be effective, and they are usually fit-tested before use.

It is important to distinguish infections caused by Mycobacterium tuberculosis from those caused by atypical mycobacteria (e.g., Mycobacterium avium complex) because there is no evidence for person-to-person transmission of atypical mycobacteria. This obviates the need for respiratory precautions (i.e., droplet precautions and airborne precautions) when caring for patients with atypical pulmonary tuberculosis (1).

There are two take-home messages in this chapter.
1. Vaccination against hepatitis B virus (HBV) virtually eliminates the risk of HBV transmission in the typical hospital worker (i.e. not elderly and _ not immunocompromised), so avoiding HBV immunization is both foolish and dangerous.
2. HIV is rarely transmitted in the hospital setting, and over 99% of healthcare workers who are exposed to HIV-infected blood are not expected to acquire the illness, even in the absence of postexposure prophylaxis with antiretroviral drugs (12). This should help to relieve the trepidation that often accompanies a needlestick injury with a blood-stained needle from an HIV-infected patient.

1. Siegel JD, Rhinehart E, Jackson M, Chiarello L, and the Healthcare Infection Control Practices Advisory Committee. 2007 Guideline for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings. Available at http://www.cdc.gov/ncidod/dhqp/pdf/isolation2007.pdf. Accessed 1/31/12.
. Centers for Disease Control and Prevention. Updated U.S. Public Health Service Guidelines for the management of occupational exposures to HBV, HCV, and HIV and recommendations for postexposure prophylaxis. MMWR 2001; 50 (No. RR-11):1-52.
. Centers for Disease Control and Prevention. Updated U.S. Public Health Service guidelines for the management of occupational exposures to HIV and recommendations for postexposure prophylaxis. MMWR 2005; 54 (No. RR-9):1-17.
. Centers for Disease Control and Prevention. Immunization of health-care workers: Recommendations of the Advisory Committee on Immunization Practices (ACIP) and the Hospital Infection Control Practices Advisory Committee (HICPAC). MMWR 1997, 46(RR-18): 1-42.
. National Institute for Occupational Safety and Health. Preventing needlestick injuries in health care settings. DHHS (NIOSH) Publication Number 2000-108; November, 1999. http://www.cdc.gov/niosh/docs/2000-108.pdf. Accessed 1/31/12.

We are told the most fantastic biological tales. For exampk, that it is dangerous to have acid in your stomach.
JBS Haldane (1939)
Standard antiseptic practices are designed to prevent microbial invasion from the skin, but (as mentioned at the end of Chapter 3) the skin is not the only body surface that can be breached by microbes. The alimentary tract, which extends from the mouth to the rectum, is outside the body (like the hole in a donut), and the mucosal lining of the alimentary tract is the largest body surface area in contact with the outside world (about 300 m2, or about the size of a tennis court). This mucosa serves as a barrier to microbial invasion, just like the skin. However, unlike the skin, which is multilayered and covered with a keratinized surface, the mucosa of the alimentary tract is a single layer of columnar epithelial cells that is only 0.1 mm thick. Considering this paper-thin "inner skin," and the unfathomable number of infectious organisms in the alimentary tract ( i.e., up to one trillion microbes in each gram of stool), it seems that the real threat of microbial invasion comes from the alimentary tract, not the skin. This chapter will introduce you to the importance of the alimentary tract as a source of infection in critically ill patients, and what can be done to reduce the risk of infection from the mouth all the way down to the rectum. Included is a section on stress-related mucosal injury in the stomach, and the measures used to prevent troublesome bleeding from this condition.

Microbial organisms are aquatic creatures that require moisture to thrive, and the moisture-rich environment in the alimentary tract is ideal for microbial proliferation. The alimentary tract in adults is home to 400-500 species of bacteria and fungi (1,2), with a total mass of about 2 kg (4.4 pounds) (3). The distribution of this mass of microbes is not uniform, as depicted in Figure 5.1 (1). The rectum is the most populated region of the alimentary tract (with up to one trillion microbes per gram of stool), and the stomach is the least populated region (with less than 1,000 organisms per mL of gastric contents). The reason for this uneven distribution will be explained shortly.
FIGURE 5.1 The population density of microorganisms in different regions of the alimentary tract. Numbers indicate colony forming units per gram or mL of luminal contents. (From Reference 1)

There are three levels of infection control in the alimentary tract.
1. The first level takes place in the stomach, where the antimicrobial actions of gastric acid eradicates microorganisms swallowed in food and saliva and maintains a relatively sterile environment in the upper GI tract.
2. The second level of protection occurs at the bowel wall, where the mucosal lining of the bowel acts as a physical barrier that blocks the movement of enteric pathogens and proinflammatory substances (e.g., endotoxin) into the systemic circulation.
3. The third level of protection takes place on the extraluminal side of the bowel wall, where the reticuloendothelial system traps and destroys microbes that breach the mucosal barrier. About two-thirds of the reticuloendothelial system in the body is located in the ab-domen (4), which suggests that microbial invasion across the bowel wall may be a frequent occurrence.
Failure of any of these protective mechanisms can lead to the systemic spread of enteric pathogens, as depicted in Figure 5.2. The movement of enteric microbes across the bowel wall is known as translocation (5), and it plays an important role in the pathogenesis of ICU-acquired bloodstream infections (described later) and progressive multiorgan failure (described next).

Multiorgan failure is a life-threatening (and often lethal) condition characterized by persistent systemic inflammation and progressive dysfunction in two or more major organs (6). Septicemia may or may not be present. The unrelenting systemic inflammation in this condition is the source of the multiorgan injury, and the driving force for this inflammation is the translocation of enteric pathogens and proinflammatory substances (e.g., endotoxin) across a disrupted mucosal barrier in the GI tract (6,7).
FIGURE 5.2 Illustration of an intestinal villus depicting three conditions that promote the systemic spread of enteric pathogens.
THE GI TRACT AS SITE OF ORIGIN: According to the "gut hypothesis" of multiorgan failure, the problem begins with a period of splanchnic hypoperfusion (e.g., from hypovolemia or hypotension) that leads to ischemic injury of the GI mucosa. The damaged mucosa then permits enteric pathogens and/or proinflammatory triggers to gain access to the systemic circulation. This initiates a systemic inflammatory response (e.g., fever, leukocytosis), which is accompanied by hemodynamic changes (i.e., sympathetic nervous system activation with splanchnic vasoconstriction) that promotes further splanchnic hypoperfusion and mucosal injury. The result is a self-sustaining process that drives systemic inflammation to the point of widespread inflammatory injury and progressive multiorgan failure. According to this scenario (and to borrow a popular phrase), the GI tract is the 'motor' of multiorgan failure (7).

Gastric acid is often misperceived as a digestive aid. An acid environment in the stomach facilitates the absorption of iron and calcium, and triggers the production of pepsin; however, patients with achlorhydria (inability to acidify gastric secretions) are not troubled by malabsorption (8). The principal function of gastric acid is not to facilitate digestion, but to serve as an antimicrobial defense mechanism, as described next.

The benefits of antisepsis were first recognized in the mid-nineteenth century by a British surgeon named Joseph Lister, who treated penetrating skin wounds with a chemical agent that was used to treat sewage, and observed a marked decline in suppurative wound infections. Lister's observations were published in 1867 in a treatise entitled On the Antiseptic Principle in the Practice of Surgery (9). In the following excerpt from this treatise, Lister describes the chemical agent that he used:
The material which I have employed is carbolic acid, a volatile compound which appears to exercise a peculiarly destructive influence upon low forms of life, and hence is the most powerful antiseptic with which we are at present acquainted.
As indicated, the very first antiseptic agent used in clinical medicine was an acid. Thus, Joseph Lister not only discovered the benefits of antisepsis in preventing infections, he also discovered the benefits of acids in eradicating infectious microbes. (In recognition of Lister's discoveries, his name is now immortalized by a mouthwash, Listerine!)

The influence of gastric pH on the growth of a pathogenic organism is shown in Figure 5.3 (10). The pathogen in this case is Salmonella typhimurium, a common cause of infectious enteritis in humans. The graph in Figure 5.3 shows the survival of S. typhimurium in gastric juice at three different pH levels. The organism thrives at a pH of 4. However, survival begins to decline at a pH of 3, and the organism is almost completely eradicated at a pH of 2. These survival curves indicate that gastric secretions are bactericidal when the pH falls below 4. The normal pH of gastric secretions is well within this range.
FIGURE 5.3 The influence of gastric pH on the survival of Salmonella typhimurium, a common cause of infectious enteritis. (From Reference 10)
Considering the bactericidal effects of gastric acidity, it is likely that gastric acid serves as an antimicrobial guardian that protects the GI tract from unwanted pathogens. The benefits of gastric acid as an infection control device are summarized below.

1. Gastric acid eradicates pathogens that are ingested in contaminated food products. This is demonstrated in studies showing that reduced gastric acidity is associated with an increased incidence of infectious gastroenteritis from Salmonella and Campylobacter species (8,10—13). Food processing techniques do not always remove infectious organisms, and gastric acid serves as a built-in backup mechanism for disinfecting the food we eat.
. Gastric acid can block the transmission of illnesses via the fecal-oral route. This is demonstrated by the association of gastric-acid suppressing drugs with Clostridium difficile enterocolitis (14). This is an important and often-overlooked complication of drugs that suppress gastric acidity, and will be described in more detail later in the chapter.
. Gastric acid eradicates microorganisms that are swallowed in saliva, which explains the marked drop in microbial density between the mouth and stomach in Figure 5.1. Loss of gastric acidity leads to colonization of the stomach with microbes that are swallowed in saliva. This may have little consequence in healthy subjects, who have harmless saprophytes in their saliva. However, critically ill patients often have pathogenic Gram-negative bacilli in their saliva (described later), and colonization of the stomach with these pathogens can promote Gram-negative septicemia (from translocation across the gastric mucosa) (15), as well as lung infections (from aspiration of infected gastric secretions into the airways) (16).
The antiseptic benefits of gastric acid will resurface in the next section.

Stress-related mucosal injury is a term used to describe erosions in the gastric mucosa that occur in almost all patients with acute, life-threatening illness (18,19). These erosions can be superficial and confined to the mucosa, or they can bore deeper and extend into the submucosa. The deeper lesions are called stress ulcers, and are more likely to cause troublesome bleeding. For the remainder of this chapter, the term "stress ulcer" will indicate both types of gastric erosions.

The gastric mucosa must protect itself against injury from the acid environment in the stomach, and gastric mucosal blood flow is considered to play an important part in this protection (by supplying nutrients to maintain the functional integrity of the mucosa). The importance of mucosal blood flow is demonstrated by the fact that 70 to 90% of the blood supply to the stomach is delivered to the gastric mucosa (20). Splanchnic vasoconstriction and hypoperfusion is common in critically ill patients, and the resultant decrease in gastric mucosal blood flow is considered the principal cause of gastric erosions (18-20). Once the gastric mucosa is disrupted, the acidity in the gastric lumen can aggravate the surface lesions.

1. Mechanical Ventilation 48 h) 2. Coagulopathy a. platelets <50,000 or DINE 4-6 OF c. PTT > 2x control 3. Bums involving > 30% of the body surface, Other High-Risk Conditions = 1. Circulatory Shock 2. Severe Sepsis 3. Multisystem Trauma 4. Traumatic Brain & Spinal Cord Injury 5. Renal Failure 6. Steroid Therapy

Erosions are visible on luminal surface of the stomach in 75% to 100% of patients within 24 hours of admission to the ICU (19). These lesions often ooze blood from eroding into superficial capillaries, but clinically significant bleeding (i.e., results in a significant drop in blood pressure or a drop in hemoglobin >2 g/dL) is observed in less than 5% of ICU patients (18,19,21).

The conditions that predispose to stress ulcer bleeding are listed in Table 5.1 (18,21). The independent risk factors (i.e., require no other risk factors to promote bleeding) include mechanical ventilation for longer than 48 hours, and a significant coagulopathy (i.e., platelets <50,000, INR>1.5, or activated PTT>twice control) (21). However, all of the conditions in Table 5.1 are indications for prophylaxis to prevent stress ulcer bleeding.

The goal of prophylaxis for stress ulcers is not to prevent their appearance (since they appear almost immediately after ICU admission), but to prevent clinically significant bleeding from these lesions. Surveys indicate that about 90% of ICU patients receive some form of prophylaxis for stress ulcer bleeding (22), but this is excessive. Prophylaxis is primarily indicated for the conditions listed in Table 5.1, and is especially important for patients who are ventilator-dependent for longer than 48 hours, or have a significant coagulopathy.

Famotidine, Type. = = H» Blocker. Famotidine, Usual Route = IV. Famotidine, Usual Dose. = 20 mg every 12 hr2. Raniticinet, Type. = = He Blocker. Raniticinet, Usual Route = Vv. Raniticinet, Usual Dose. = 50 mg every 8 hr2. Lansoprazole, Type. = = PPI. Lansoprazole, Usual Route = NG. Lansoprazole, Usual Dose. = 30 mg once daily. Omeprazole, Type. = = PPI. Omeprazole, Usual Route = NG. Omeprazole, Usual Dose. = 20 mg once daily. Pantoprazole, Type. = = PPI. Pantoprazole, Usual Route = IV. Pantoprazole, Usual Dose. = 40 mg once daily. Sucratfate, Type. = = Protectant. Sucratfate, Usual Route = NG. Sucratfate, Usual Dose. = 1 gram every 6 hr.
The dose of gastric acid-suppressing drugs may need to be adjusted to maintain pH = 4 in gastric aspiratas.
"Dose reduction is necessary in renal failure.
Abbreviations: PA = proton pump inhibitor; NG = nasogastric instillation.

The principal method of prophylaxis for stress ulcer bleeding is to block the production of gastric acid using histamine type-2 receptor antagonists or proton pump inhibitors, and maintain a pH=4 in gastric aspirates. The other method of prophylaxis involves the use of a cytoprotective agent (sucralfate) that protects damaged areas of the gastric mucosa without altering gastric acidity. The individual drugs that are used for prophylaxis of stress ulcer bleeding are shown in Table 5.2.

Inhibition of gastric acid secretion with histamine Hz-receptor antagonists (Hz blockers) is the most popular method of stress ulcer prophylaxis (22). The drugs most frequently used for this purpose are ranitidine and famotidine; both drugs are typically given as an intravenous bolus in the doses shown in Table 5.2. Ranitidine is the most studied gastric acid—suppressing drug for stress ulcer prophylaxis. A single 50 mg dose of ranitidine given as an IV bolus will reduce gastric acidity (pH>4) for 6-8 hrs. (24), so the typical ranitidine dosing regimen is 50 mg IV every 8 hours. Famotidine has a longer duration of action; i.e., a single 20 mg dose of famotidine given as an IV bolus will reduce gastric acidity (pH>4) for 10-15 hours (23), so the typical famotidine dosing regimen is 20 mg IV every 12 hours.

sucralfate. However if the desired result is fewer adverse events (bleeding and pneumonia), then sucralfate is superior to ranitidine. It seems that the goal of any preventive strategy is fewer adverse events, in which case the results in Figure 5.4 would favor sucralfate over ranitidine (i.e., gastric acid suppression) for prophylaxis of stress ulcer bleeding.
PHYSICIAN PREFERENCE: The most recent survey of critical care physicians showed that 64% used H> blockers, 23% used proton pump inhibitors, and only 12% used sucralfate for stress ulcer prophylaxis (22). However, only 30% of the physicians based their preferences on the efficacy and side effects of the drugs (!).

One of the most compelling reasons for avoiding gastric acid—suppressing drugs is the increased risk of Clostridium difficile enterocolitis associated with these drugs. This has been reported in both outpatients (14,33) and inpatients (34,35), and is a greater risk with proton pump inhibitors than with Hz blockers (14,34). In fact, the increase in C. difficile enterocolitis that has been observed in recent years coincides with the increased use of proton pump inhibitors in both outpatients and inpatients. It is very possible that the increasing incidence of C. difficile enterocolitis in hospitalized patients is not the result of increased antibiotic usage (since antibiotics have always been overused), but instead is a consequence of the escalating use of gastric acid suppression for stress ulcer prophylaxis.
FIGURE 5.4 Effects of stress ulcer prophylaxis with ranitidine and sucralfate on the incidence of clinically significant upper GI bleeding and pneumonia in ventilator-dependent patients. The height of the columns in each graph are significantly different at the p <0.01 level. (From Reference 17)

Enteral tube feedings exert a trophic effect on the GI mucosa that helps to maintain the structural and functional integrity of the mucosal surface (see Chapter 48). Enteral feeding solutions also raise the pH in the lumen of the stomach. Both of these effects should protect against stress ulcer bleeding. The benefit of enteral tube feedings is revealed in the combined results of three clinical studies, which showed that the ability of H> blockers to reduce stress ulcer bleeding was lost when patients received a full regimen of enteral tube feedings (36). These results suggest that patients who receive enteral tube feedings do not require any further prophylactic measures for stress ulcer bleeding. Unfortunately, more studies are required before the experts will commit to tube feedings as a Suitable method of stress ulcer prophylaxis.

Monitoring occult blood in gastric aspirates has no predictive value for identifying patients who will develop clinically significant bleeding from stress ulcers because gastric aspirates almost always contain occult blood in the presence of stress ulcers (37). For cases where testing for occult blood in gastric aspirates is performed, guaiac and Hemoccult tests are not reliable because of false-positive and false-negative results when the test fluid has a pH <4 (38). The Gastroccult test (Smith, Kline Laborato-ries) is not influenced by pH (38), and is a more reliable test for occult blood in gastric aspirates.

The microbes that normally inhabit the oral cavity and GI tract seem to live in peaceful coexistence with us. However, in the presence of severe or chronic illness, the alimentary tract becomes populated by more pathogenic organisms capable of causing invasive infections. This section de-scribes two methods for combating this pathogenic colonization. Both methods haven proven effective in reducing the incidence hospitalacquired infections.

The aspiration of mouth secretions into the upper airways is believed to be the inciting event in most cases of hospital-acquired pneumonia. An average of 1 billion (109) microorganisms are present in each milliliter of saliva (39), so aspiration of one microliter (10-3 mL) of saliva will introduce about one million (106) microbes into the airways. Fortunately, the microbes that normally inhabit the mouth are harmless saprophytes (e.g., lactobacillus and a-hemolytic streptococci) that show little tendency to produce invasive infection. Critically ill patients are not as fortunate, as described next.

The oral cavity in hospitalized patients is often colonized with patho-genic organisms, most notably aerobic Gram-negative bacilli like Pseudomonas aeruginosa (40,41). The change in microflora is not environmentally driven, but is directly related to the severity of illness in each patient. This is demonstrated in Figure 5.5 (41). Note that healthy subjects were not colonized with aerobic Gram-negative bacilli, regardless of the environment. This highlights the importance of host-specific factors in the colonization of body surfaces.
BACTERIAL ADHERENCE: The specific organisms that colonize body surfaces are determined by specialized receptor proteins on epithelial cells that bind to adhesion proteins (called adhesins) on the surface of bacteria. These receptors are specific for certain groups or species of microorganisms, and this determines the microbes that can attach to the body surface. In healthy subjects, the epithelial cells in the mouth have receptors that bind harmless saprophytes (e.g., lactobacillus), while in seriously ill patients, the same epithelial cells have receptors that bind pathogenic organisms (e.g., Pseudomonas aeruginosa). The presence of a serious illness somehow induces the epithelial cells to express a different receptor for bacterial adherence; identifying the mechanism for this change is the first step towards the goal of manipulating epithelial cell receptors to prevent pathogenic colonization and infection in seriously ill patients.
that SOD was associated with a 33% (relative) decrease in the incidence of Gramnegative bacteremias.
The influence of oral decontamination on Gram-negative bacteremia is an unanticipated observation, and indicates that the mouth is a source of bacteremia in ICU patients. Since brushing your teeth can result in bacteremia, it is possible that movements of endotracheal tubes and nasogastric tubes (e.g., with head movements) can damage the oropharyngeal mucosa and promote bacterial translocation in the mouth.
FIGURE 5.6 The effects of selective oral decontamination (SOD) on the incidence of tracheal colonization and pneumonia in ventilator-dependent patients. The height of the columns in each graph are significantly different at the p = 0.01 level. (From Reference 45)

RESISTANCE: Despite dramatic effects like those in Figure 5.6, oral decontamination with nonabsorbable antibiotics is largely ignored in the United States (not so in Europe) because of concerns about the possible emergence of antibiotic-resistant organisms. However, there is no evidence of antibiotic resistance in any of the reports of antibioticbased oral decontamination.

Selective digestive decontamination (SDD) is essentially an extension of SOD to include the entire alimentary canal. One popular SDD regimen is shown below (49).
Oral cavity: A paste containing 2% polymyxin, 2% tobramycin, and 2% amphotericin is applied to the inside of the mouth with a gloved finger every 6 hours.
GI
tract:
A
10-mL
solution containing
100
mg polymyxin
E,
80
mg tobramycin,
and
500
mg amphotericin is given via a nasogastric tube every 6 hours.
Systemic: Intravenous cefuroxime, 1.5 grams every 8 hours, for the first four days.
Note that the same drug combination used for SOD is also used to decontaminate the oral cavity and the GI tract. This nonabsorbable antibiotic regimen is designed to eradicate staphylococci, Gram-negative aerobic bacilli, and fungal species, while sparing the normal inhabitants of the bowel (mostly anaerobes) to prevent colonization with opportunistic pathogens like Clostridium difficile. The intravenous antibiotic provides systemic protection while the bowel regimen begins to take effect (decontamination of the bowel takes about one week). The SDD regimen is intended for all patients who will stay in the ICU longer than 72 hours, and is administered from admission until the patient is well enough to be discharged from the ICU.

Several studies have shown a significant reduction in ICU-acquired infections associated with SDD (47-49). The results of one of these studies is shown in Figure 5.7 (47). This was a large, randomized trial that evaluated the influence of SOD and SDD on ICUacquired bacteremias involving Gram-negative bacilli. Both bar graphs show that SDD patients had 69% drop in the incidence of Gram-negative bacteremias when compared with control patients. Although not shown in the figure, the SDD effect was accompanied by a decrease in mortality. Improved survival in patients receiving SDD has also been observed in other studies (50).
FIGURE 5.7 The incidence of ICU-acquired, Gram-negative bacteremia in patients randomized to receive sekctive oral decontamination (SOD), selective digestive decontamination (SDD), or standard care (control). The graph on the left shows the number of bacteremias per 1,000 days (incidence density), and the graph on the right shows the percentage of patients with bacteremia (cumulative incidence). N is the number of patients in each study group. (From Reference 47)

The principal fear with SDD is possible emergence of antibiotic-resistant organisms. However, this fear is not supported by clinical observations. (50,51).

Numerous clinical studies over the past 25 years have shown that SDD is an effective method for preventing ICU-acquired infections, yet SDD is rarely used in the United States. Some of the resistance to SDD is based on an unfounded fear of antibiotic resistance, as just described. Another concern is a lack of consistent survival benefit with SDD. Early studies of SDD typically showed no survival benefit, but more recent and larger clinical trials have shown a significant improvement in survival associated with SDD (48,50). Thus, there is little justification for the continued disregard of SDD.

One of the recurring themes in this book is the importance of the alimentary tract as a source of infection in critically ill patients. The summary statements and recommendations listed below are designed to assist you in limiting the risk of infection from this important site., 1 = One of the recurring themes in this book is the importance of the alimentary tract as a source of infection in critically ill patients. The summary statements and recommendations listed below are designed to assist you in limiting the risk of infection from this important site.. 1., 1 = Prophylaxis for stress ulcer bleeding is overused in the ICU (22), and should be limited to the conditions in Table 5.1.. 2., 1 = Gastric acid suppression is overused as a method of stress ulcer prophylaxis, and this has probably contributed to the surge in C. difficile infections in recent years (34,35).. 3., 1 = Proton pump inhibitors should be avoided for stress ulcer prophylaxis because they are not more effective than H 2 blockers (27), and they are more likely to promote unwanted infections (14,28).. 4., 1 = Recent evidence suggests that total enteral nutrition is an effective method of stress ulcer prophylaxis (36).. 5., 1 = Oral decontamination is advised for all ventilator-dependent patients, and the use of non-absorbable antibiotics provides better antimicrobial coverage than chlorhexidine (44), and has more proven efficacy for preventing ventilator-associated pneumonia (45,46).. 6., 1 = Selective digestive decontamination (SDD) is a proven method for reducing the incidence of ICU-acquired infections (47-50), and there is no evidence that it promotes the emergence of antibiotic-resistant organisms (50,51). As a result, SDD deserves much more consideration for the prevention of ICU-acquired infections.

Marston A, Bulkley GB, Fiddian-Green RG, Haglund UH, eds. Splanchnic Ischemia and Multiple Organ Failure. St. Louis: C.V. Mosby, 1989.

= Dial S, Delaney JAC, Barkun AN, Suissa S. Use of gastric acid-suppressing agents and the risk of community-acquired Clostridium difficile-associated disease. JAMA 2005; 294:2989-2994.. 15., 1 = MacFie J, Reddy BS, Gatt M, et al. Bacterial translocation studied in 927 patients over 13 years. Br J Surg 2006; 93:87-93.. 16., 1 = Gulmez SE, Holm A, Frederiksen H, et al. Use of proton pump inhibitors and the risk of community-acquired pneumonia. Arch Intern Med 2007; 167:950-955.. 17., 1 = Huang J, Cao Y, Liao C, et al. Effect of histamine-2-receptor antagonists versus sucralfate on stress ulcer prophylaxis in mechanically ventilated patients: A meta- analysis of 10 randomized controlled trials. Crit Care 2010; 14:R194-R204.
, 1 = hospitalized patients prescribed proton pump inhibitors: cohort and case-control studies. Canad Med Assoc J 2004; 171:33-38.. 35., 1 = Aseri M, Schroeder T, Kramer J, Kackula R. Gastric acid suppression by proton pump inhibitors as a risk factor for Clostridium difficile-associated diarrhea in hospitalized patients. Am J Gastroenterol 2008; 103:2308-2313.. 36., 1 = Marik PE, Vasu T, Hirani A, Pachinburavan M. Stress ulcer prophylaxis in the new millenium: a systematic review and meta-analysis. Crit Care Med 2010; 38:2222- 2228.. 37., 1 = Maier RV, Mitchell D, Gentiello L. Optimal therapy for stress gastritis. Ann Surg 1994; 220:353-363.. 38., 1 = Rosenthal P, Thompson J, Singh M. Detection of occult blood in gastric juice. J Clin Gastroenterol 1984; 6:119.

Two words best characterize the mortality and morbidity due to venous thromboembolism in the United States: substantial and unacceptable.
Kenneth M. Moser, MD
The threat of pulmonary embolism is a daily concern in ICU patients, who typically have one or more risk factors for venous thrombosis (the precursor of pulmonary embolism). Thrombus formation occurs most frequently in proximal leg veins, and becomes apparent only when a portion of the thrombus breaks loose and travels to the lungs to become a pulmonary embolus. This progression from silent leg thrombosis to symptomatic pulmonary embolism is a significant, and preventable, problem in hospitalized patients. In fact, pulmonary embolism is considered the most common preventable cause of death in hospitalized patients, and the prevention of venous thrombosis is considered the single most important measure for ensuring patient safety during hospitalization (see Federal Reports in bibliography).
This chapter presents the current practices for the prevention, diagnosis, and treatment of venous thrombosis and pulmonary embolism (i.e., venous thromboembolism) in the ICU patient population. Several clinical practice guidelines and reviews of this subject are included in the bibliography at the end of the chapter (1-8).

Several conditions promote venous thromboembolism (VTE) in hospitalized patients, and these are listed in Table 6.1 (1). One or more of these conditions is present in almost all ICU patients, and thus VTE is considered a universal risk in critically ill patients (1,5). The incidence of VTE in different groups of patients, which is shown in Figure 6.1 (1), can be used to estimate of the likelihood of VTE in individual ICU patients. The presence of additional risk factors (e.g., prior VTE) will add further to the risk of VTE in any of the clinical groups shown in Figure 6.1.

Surgery
Major surgery, especially cancer-related surgery, hip and knee surgery
Trauma
Multisystem trauma, especially spinal cord injury and fractures of the spine
Any malignancy, active or occult Chemotherapy and radiotherapy
Acute Medical Illness
Stroke, right-sided heart fallure, sepsis, inflammatory bowel disease, nephrotic syndrome, myeloproliferative disorders
Drugs
Erythropoiesis-stimulating drugs, estrogen-containing compounds
Patient-Specific Factors
Prior thromboembolism, obesity, increasing age, pregnancy
|CU-Related Factors
Prolonged mechanical ventilation, neuromuscular paralysis, severe sepsis, vasopressors, platelet transfusions, immobility
From Referanceas 1 and 5.

Major surgery (i.e., surgery performed under general or spinal anesthesia that lasts longer than 30 minutes) is the most recognized cause of VTE in hospitalized patients, and autopsy studies have shown that VTE is responsible for about 10% of postoperative deaths (9). The propensity for VTE after major surgery is primarily due to thromboplastin release during the surgical procedure, which produces a generalized hypercoagulable state. The risk of VTE is particularly high following cancer-related surgery (1,5).

The highest incidence of postoperative VTE occurs after major orthopedic procedures involving the hip and knee (1,5). Vascular injury in the lower extremities contributes to the enhanced risk of VTE following hip and knee surgery.
FIGURE 6.1 The prevalence of venous thromboembolism in different groups of hospitalized patients. From Reference 4.

The risk of VTE is highest following major or multisystem trauma. Victims of major trauma have a greater than 50% chance of developing VTE, and pulmonary embolism is the third leading cause of death in those who survive the first day (1). The trauma conditions with the highest risk of VTE are brain and spinal cord injuries, spinal fractures, and fractures of the hip and pelvis (1,5). Several factors contribute to the propensity for VTE after major trauma, including thromboplastin release from injured tissues, vascular injury, and prolonged immobility.

Hospitalization for acute medical illness is associated with an eightfold increase in the risk of VTE (10). Although VTE is less common in medical patients than in postoperative patients or trauma victims (see Figure 6.1), the majority (70% to 80%) of deaths from VTE occur in medical patients (1). Medical conditions with a particularly high risk of VTE include cancer, acute stroke with lower extremity weakness, and right-sided heart failure. In a Medical ICU, additional risk factors for VTE can include mechanical ventilation, severe sepsis, and prolonged immobility.

Autopsy studies in critically ill patients reveal pulmonary emboli in as many as 27% of patients (5), and in the majority of these cases there is no clinical suspicion of venous thrombosis or pulmonary embolism prior to death (5). Because of the insidious nature of VTE, prevention is the best means of limiting the adverse consequences of this condition. In fact, preventive strategies for VTE should have the greatest impact on patient safety in the ICU setting because of the ubiquitous presence of risk factors for VTE in critically ill patients.
This section describes the methods that are used to prevent thrombus formation in critically ill patients. The preventive measures that are recommended in a variety of conditions are included in Table 6.2, and the prophylactic anticoagulant dosing regimens are shown in Table 6.3.

Acute Medical Ilness, High-Risk Regimens' = LDUH or LMWH. Major Abdominal Surgery, High-Risk Regimens' = LDUH or LMWH + GCS or IPG. Thoracic Surgery, High-Risk Regimens' = LDUH or LMWH + GCS or IPC. Cardiac Surgery with Complications, High-Risk Regimens' = LDUH or LMWH + IPC. Craniotomy, High-Risk Regimens' = IPG. Hip or Knee Surgery, High-Risk Regimens' = LMWH. Major Trauma, High-Risk Regimens' = LDUH or LMWH or IPG. Head or Spinal Cord Injury, High-Risk Regimens' = LDUH or LMWH + IPG. Any of the Above + Active Bleeding or High Risk of Bleeding, High-Risk Regimens' = IPG
From Reference 1. Abbreviations: LDUH=low-dess unfractionated heparin; LMWH = low-melacular weight haparin; GCS = graded compression stockings; IPC= intermittant pneumatic compression.
1Sritically ill patients ara considerad to be in the high-risk category for each of the conditions listed.
dosing regimens for LDUH (every 8 hrs. and every 12 hrs.) differ in preventing VTE (4,13), so the twice-daily LDUH regimen is the more prudent choice.

Unfractionated Heparin
Usual Dose:
5,000 Units SC every 12 hrs. or
5,000 Units SC every 8 hrs.
Obesity:
5,000 Units SC every 8 hrs. (BMI< 50)
7,000 Units SC every 8 hrs. (BMI 50)
Enoxaparin (LMWH)
40 mg SC once daily or 80 mg SC twice dally
Obesity:
0.6 mg/kg SC once dally (BMI>40)
Renal Fallure:
30 mg once dally (for CrCL < 30 ml/min)
Dalteparin (LMWH)
Usual Dose;
2,500 Units SC once dally or
5,000 Units SC once dally
Renal Failure:
No recommended dose adjustment
From References 4, 14, 17-19.

Anticoagulant prophylaxis can promote hematoma formation during the insertion and removal of intrathecal and epidural catheters. This is a feared complication because spinal and epidural hematomas can compress the spinal cord and produce paralysis. To limit the risk of this complication, the insertion and removal of intrathecal and epidural catheters should be performed at a time when anticoagulant effects are minimal; e.g., if a twice daily dosing regimen is used, these procedure should be delayed at least 12 hours after the prior anticoagulant dose, and for a once-daily dosing regimen, the delay should be at least 24 hours after the prior anticoagulant dose (4). In addition, at least 2 hours should elapse after these procedures before an anticoagulant dose is administered (4).

As mentioned earlier, thrombosis in the deep veins of the legs is often clinically silent, and VTE is suspected only when a symptomatic pulmonary embolus appears. The diagnostic approach to pulmonary em-bolism is accurately described by the following quote from a recent review of the topic (6):
The one certainty surrounding the issue of thromboembolism diagnosis in critically ill patients is that considerable uncertainty remains.
In patients who are evaluated for possible pulmonary embolism, the di-agnosis is confirmed in as few as 10% of cases (23), and when physicians have a highest degree of certainty that a patient has a pulmonary em-bolism, they are correct in only 17-25% of the patients (23). What this means is that when you suspect a pulmonary embolus, it usually isn't there. It also means that the diagnostic evaluation of pulmonary em-bolism serves prlimarily to exclude the diagnosis rather than confirm it.

The clinical presentation of acute pulmonary embolism (PE) includes nonspecific manifestations such as dyspnea, tachypnea, tachycardia, and hypoxemia. The predictive value of clinical and laboratory findings in suspected cases of PE is shown in Table 6.4 (24). Note that none of the findings provides more than a 50% chance of identifying pulmonary embolism (i.e., the positive predictive value), and further that the ab-sence of these findings does not exclude PE (i.e., the negative predictive value should be =98% to exclude the presence of a condition). Of particular interest is the negative predictive value of 70% for hypoxemia, which means that 30% of patients with a pulmonary embolus have a normal arterial PO. Although not included in Table 6.4, the alveolar— arterial PO» (A-a PO) gradient can also be normal in patients with pulmonary emboli (25).

Active thrombosis is accompanied by some degree of clot lysis, and this produces crosslinked fibrin monomers, also called fibrin D-dimers or D-dimers. Plasma D-dimer levels are elevated in patients with VTE, and the D-dimer assay has become a_ popular screening test for suspected VTE in non-ICU patients. However, D-dimer levels have little predictive value in ICU patients. The problem is the multitude of other conditions that can elevate plasma D-dimer levels, including sepsis, malignancy, pregnancy, heart failure, renal failure, and even advanced age (26). As a result, a majority (up to 80%) of ICU patients have elevated plasma D-dimer levels in the absence of VTE (27). This is reflected in the poor positive predictive value of the D-dimer assay in Table 6.4. Because of the high prevalence of elevated D-dimer levels in critically ill patients, the D-dimer assay is not considered a useful test for VTE in the ICU setting.
able 6.4 Predictive Value of Clinical and Laboratory Findings in Suspected
Dyspnea, 1 = S7%. Dyspnea, 2 = 75%. Tachycardia, 1 = 47%. Tachycardia, 2 = 86%. Tachypnea, 1 = AB %. Tachypnea, 2 = 15%. Pleuritic Chest Pain, 1 = 39%. Pleuritic Chest Pain, 2 = 11%. Hemoptysis, 1 = 82%. Hemoptysis, 2 = 67%. Pulmonary Infiltrate, 1 = 33%. Pulmonary Infiltrate, 2 = 11%. Pleural Effusion, 1 = 40%. Pleural Effusion, 2 = 69%. Hypoxemia, 1 = 34%. Hypoxemia, 2 = TO%. Elevated D-Dimert, 1 = 27%. Elevated D-Dimert, 2 = 92%
+Positiva pradictive value is the percentage of patients with the finding who have a pulmonary embolus.
*Negative pradictive value is the percentage of patients without the finding who do not have a pulmonary embolus.
1From Raferance 26. Other data from Refarance 24.

The information just presented shows that there are no clinical or laboratory findings that will confirm or exclude the presence of a pulmonary embolus. As a result, the diagnostic approach to PE requires specialized tests, which are included in the flow diagram shown in Figure 6.2.

Pulmonary emboli originate primarily from thrombi in proximal leg veins (28), so the evaluation of suspected PE can begin with a search for thrombosis in the proximal leg veins using ultrasound imaging at the bedside. The basics of vascular ultrasound are described in Chapter 2.
FIGURE 6.2 Flow diagram for the evaluation of suspected pulmonary embolism.

There are two ultrasound methods for identifying venous thrombosis. The principal method is compression ultrasound, where a compressive force is applied to the skin overlying the target vein. This will normally compress the underlying vein and obliterate its lumen (see Figure 2.3). However, when a vein is filled with blood clots (which are often not visualized by ultrasound), external compression does not compress the vein. Therefore, an incompressible vein is used as evidence of a clot-filled vein (29).
Thecolor Doppler mode of ultrasound can be used as an adjunct to compression ultrasonography. This method converts flow velocities into color images, and the flow in arteries and veins can be identified by the direction of flow in relation to the ultrasound

In patients with lung disease (i.e., most patients in the ICU), the next step in the evaluation of suspected PE is to visualize the pulmonary arteries with computed tomographic (CT) angiography. This is a specialized method of CT that uses a spiral or helical scanner that rotates around the patient to produce a "volumetric" two-dimensional view of the lungs (33). When spiral CT is combined with the peripheral injection of a contrast agent, the central pulmonary arteries can be visualized. Pulmonary emboli appear as filling defects, as shown in
FIGURE 6.3 CT angiogram showing a pulmonary embolus (filing defect) in the left main pulmonary artery (PA). AO = aorta. Image digitally retouched.

In studies using newer, multidetector CT scans, CT angiography (CTA) has a sensitivity of 83%, a specificity of 96%, a positive predictive value of 86%, and a negative predictive value of 95% for the diagnosis of PE (34). Sensitivity is highest (>90%) for detecting clots in the main pulmonary arteries, while, emboli in smaller, subsegmental vessels can be missed. However, the importance of detecting smaller, subsegmental emboli is questionable because withholding anticoagulant therapy based on a negative CT scan does not seem to adversely affect clinical outcomes (35).
One of the shortcomings of CTA is the risk of nephrotoxicity from the radiocontrast dye. This is particularly relevant in ICU patients, who often have one or more risk factors for dye-induced nephropathy (e.g., renal insufficiency, diabetes, volume depletion, etc.). In one study that included non-ICU patients, 18% of the patients with suspected pulmonary embolism were not eligible for CTA because of elevated serum creatinine levels (34). If CTA is necessary in a patient with mild renal compromise (a creatinine clearance >60 mL/min) volume expansion and N-acetylcysteine can help to limit the risk of dye-induced nephrotoxicity (if you can convince the radiologist to perform the procedure). See
34 for more information on dye-induced renal injury.

Ventilation-perfusion lung scans are widely used in the evaluation of suspected PE, but they secure the diagnosis in only about 25% to 30% of cases (36). The problem is the presence of lung disease (particularly infiltrative disease), which produces an abnormal scan in about 90% of cases (36). Lung scans are most helpful in patients with no underlying lung disease (which, unfortunately, excludes most ICU patients). If the decision is made to proceed with a lung scan, the results can be used as follows (36):
1. A normal lung scan excludes the presence of a pulmonary embolus, whereas a highprobability lung scan carries a 90% probability that a pulmonary embolus is present.
2. A low-probability lung scan does not reliably exclude the presence of a PE. However, when combined with a negative ultrasound evaluation of the legs, a low-probability scan is sufficient reason to stop the diagnostic workup and observe the patient.
3. An intermediate-probability or indeterminate lung scan has no value in predicting the presence or absence of a pulmonary embolus. In this situation, the options include spiral CT angiography (see Figure 6.2) or conventional pulmonary angiography (see next).

Conventional pulmonary angiography is considered the most accurate method for detecting pulmonary emboli, and is reserved for cases where the other diagnostic tests are unable to confirm or exclude a pulmonary embolism that is highly suspected and associated with significant morbidity. Conventional angiography is rarely performed, in part because the other diagnostic tests are considered sufficient in most cases, and in part because the procedure is time-consuming and can be risky (i.e., in patients with right heart failure or renal insufficiency).

The initial treatment of VTE that is not life-threatening is anticoagulation with one of the heparin preparations. The pharmacology and comparative features of unfractionated heparin and low-molecular-weight heparin (LMWH) have been described earlier in the chapter.

For full anticoagulation, unfractionated heparin is administered as an intravenous bolus followed by a continuous infusion. Dosing based on body weight, like the regimen in Table 6.5, achieves more rapid anticoagulation than fixed-dosing regimens (37). The anticoagulant effect is monitored with the activated partial thromboplastin time (PTT); the target PTT is 46-70 seconds, or a PTT ratio (test/control) between 1.5 and 2.5 (3).

1. Give initial bolus dose of 80 IU/kg and follow with continuous Infusion of 18 IU/kg/nr. (Use actual body weight.)
2, Check PTT 6 hrs, after start of infusion, and adjust heparin dose as indicated below.
3. Check PTT 6 hrs. after each dose adjustment. When in the desired range (46-70 sec), monitor daily.
<35, PTTRatio = <1.2. <35, Bolus Dose = 80 IU/kg. <35, —_ Continuous Infusion. = Increase by 4 IU/kg/hr. 35-45, PTTRatio = 1.3-1.5. 35-45, Bolus Dose = 40 |LUkg. 35-45, —_ Continuous Infusion. = Increase by 2 IU/ka/hr. 46-70, PTTRatio = 1.5-2.3. 46-70, Bolus Dose = —. 46-70, —_ Continuous Infusion. = —. 71-90, PTTRatio = 2.3 -3.0. 71-90, Bolus Dose = _—. 71-90, —_ Continuous Infusion. = Decrease by 2 ILU/kq/hr. >90, PTTRatio = =3. >90, Bolus Dose = —. >90, —_ Continuous Infusion. = Stop infusion for 1 hr. then decrease by 3 IU/kg/hr
Fram Referanca 37.

The weight-based dosing regimen in Table 6.5 was derived in patients weighing less than 130 kg (286 Ibs.). For body weights in excess of 130 kg, this regimen can result in excessive anticoagulation (38). To avoid this problem, the adjusted body weight shown here is recommended for patients with morbid obesity (i.e., a body mass index or BMI>40 kg/m2) (14).
Adjusted weight (ke) = IBW + 0.4 x (actual weight - IBW)
Men: IBW (kg) = 50 + 2.3 x (height > 60 in)
Women:
IBW (kg) = 45 + 2.3 x (height > 60 in)
The adjusted body weight is almost halfway between the ideal body weight (IBW) and the actual body weight.

An antibody-mediated thrombocytopenia can appear 5-10 days after in-itiation of heparin therapy (or sooner with prior heparin exposure), and the resultant platelet clumping can lead to symptomatic venous and arterial thrombosis. This condition is described in detail in Chapter 19.

The anticoagulation effects of heparin can be protein (from fish sperm) that binds to intravenous protamine dose of 1 mg will minutes (3). The following are some protamine:, 1 = rapidly reversed with protamine sulfate, a heparin and forms an inactive compound. An neutralize about 100 units of heparin within 5 recommendations for heparin reversal with. 1. For IV-bolus heparin: if only a few minutes have elapsed, give 1 mg of protamine IV per 100 units of heparin administered; if 30 minutes have elapsed, give 0.5 mg of protamine IV per 100 units of heparin; if ≥2 hours have elapsed, give 0.25-0.375 mg of protamine IV per 100 units heparin administered (39)., 1 = 1. For IV-bolus heparin: if only a few minutes have elapsed, give 1 mg of protamine IV per 100 units of heparin administered; if 30 minutes have elapsed, give 0.5 mg of protamine IV per 100 units of heparin; if ≥2 hours have elapsed, give 0.25-0.375 mg of protamine IV per 100 units heparin administered (39).

Oral anticoagulation with warfarin should be started as soon as possible after the start of heparin anticoagulation. The initial dose is 5-10 mg daily for the first 2 days, with subsequent dosing tailored to the international normalized ratio (INR). The target INR is 2-3. When the INR reaches the therapeutic range, anticoagulation with unfractionated heparin or LMWH can be discontinued. Warfarin anticoagulation is primarily an outpatient management issue, and will not be described here. The management of warfarinassociated hemorrhage is described in Chapter 19. (For more information on warfarin anticoagulation, see Reference 40.)

1. Pulmonary embolism with obstructive shock
2, Pulmonary embolism with right ventricular dysfunction (7)
3. No contraindication to thrombolytic therapy

1. Continuous infusion heparin is used in conjunction with lytic therapy. Infusion can be continued during lytic drug delivery.
2, Standard thrombolytic regimen: Alteplase: 100 mg infused over 2
hrs..
3. Regimens aimed at accelerated clot lysis:
Alteplase:
0.6 mg/kg infused over 15 min.
Reteplase:
10 by IV bolus and repeat in 30 min.
U
4, Systemic drug administration is preferred to pulmonary artery instillation

1. Major hemorrhage: 9-125
2. Intracranial hemorrhage: 1-29
From Referances 7, 8, 42, and 43.

If you're lucky enough to work in a medical center with an experienced team that is readily available around-the-clock, embolectomy (either surgical or catheter-based) is an option for life-threatening PE, particularly when there is a contraindication to thrombolytic therapy (2). Survival rates of 83% have been reported with emergency embole ctomy
FIGURE 6.4 The Greenfield Filter. The struts have hooked ends to anchor the fitter to the wall of the vena cava, and the elongated, conical shape allows the filter to trap blood clots without obstructing blood flow.
IVC filters are inserted percutaneously, usually through the internal jugular vein or femoral vein, and are placed below the renal veins, if possible. Removable filters can be retrieved when the condition responsible for the insertion (e.g., bleeding from anticoagulation) is no longer present.

Although not devoid of risk, IVC filters are remarkably safe and effective. The incidence of symptomatic PE after filter placement is about 5% (48), and troublesome complications (e.g., migration of the filter) are reported in less than 1% of cases (48). One of the intriguing features of IVC filters (which receives little attention) is the fact that they never seem to get infected, even when exposed to bacteremia.

The experience with venous thromboembolism, which is far from satisfying, can be summarized as follows:
1. When you think it's there, it usually isn't (i.e, when a pulmonary embolism is suspected, the diagnosis is confirmed in as few as 10% of cases).
2. When you don't think it's there, it can be (i.e., in most cases of pulmonary embolism, the venous thrombosis is clinically silent prior to the embolic event).
3. When you finally find it, it may be too late (i.e., in massive pulmonary embolism, there is no treatment that consistently improves survival).
4. Because of the conditions stated above, the best approach to venous thromboembolism is to prevent it.

Shojania KG, Duncan BW, McDonald KM, et al, eds. Making healthcare safer: a critical analysis of patient safety practices. Evidence report/technology assessment No. 43. AHRQ Publication No. 01-E058. Rockville, MD: Agency for Healthcare Research and Quality, July, 2001.

1. Guyatt GH, Aki EA, Crowther M, et al. Executive summary: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012; 141(Suppl):7S—47S.
2. Kearon C, Akl EA, Comerota AJ, et al. Antithrombotic therapy for VTE disease. Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: Am-erican College of Chest Physicians Evidence-Based Clinical Practice Guide-lines. Chest 2012; 141(Suppl):e419S—e494s.
3. Garcia DA, Baglin TP, Weitz JI, Samama MM. Parenteral anticoagulants. Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012; 141(Suppl):e24S—e43S.
4. Geerts WH, Bergqvist D, Pineo GF, et. al. Prevention of venous thromboembolism. American College of Chest Physicians Evidence-Based Clinical Prac-tice Guidelines (8th edition). Chest 2008; 133(Suppl):381S—453S.

Not everything that counts can be counted.
And not everything that can be counted counts.
Albert Einstein

It should be clearly recognized that arterial pressure cannot be measured with precision by means of sphygmomanometers.
Committee for Arterial Pressure Recording,
American Heart Association, 1951
About 60 years have elapsed since the warning in the introductory quote, yet the same imprecise method continues to be the accepted standard for the measurement of arterial blood pressure (1). (This is not good news for the 75 million people in the United States with hypertension, who have a diagnosis that is based on a flawed measurement.) The accuracy of the standard (noninvasive) blood pressure measurement is even more of a problem in patients who are hemodynamically unstable (for reasons described later), and blood pressure monitoring in these patients often requires direct intra-arterial pressure recordings. This chapter describes the indirect and direct methods of blood pressure monitoring, and the problems associated with each method.

Although the blood pressure measurement is one of the most frequently performed measurements in clinical practice, observational studies have shown that almost no one measures the blood pressure properly (i.e., according to the guidelines of the American Heart Association). In surveys that included primary care physicians, nurses, medical specialists, and surgeons (3), none of the examinees measured the blood pressure correctly (2,3). In one of these studies (3), only 3% of the general practitioners and 2% of the nurses obtained blood pressure measurements that were reliable (3).
The American Heart Association publishes guidelines for the indirect blood pressure measurement, and these are included in the bibliography at the end of this chapter (1). The principal recommendations from these guidelines are included in this section.

The indirect blood pressure is measured with a device called a sphygmomanometer (sphygmos is the Greek term for pulse, and manometer is a pressure measuring device) which consists of an inflatable bladder covered by a cloth sleeve, and a gauge or column of mercury to measure pressure. The cloth sleeve is wrapped around the upper arm or thigh in an area that overlies a major artery (usually the brachial artery), and the bladder in the sleeve is inflated to compress the underlying artery.
The effects of arterial compression are illustrated in Figure 7.1. As the cuff pressure increases and the underlying artery is compressed, the pulsations in the artery gradually increase and then decrease until the artery is occluded. These "counterpulsations" produce oscillations in cuff pressure (as shown in the figure), and measuring these pressure oscillations is the basis for the oscillometric method of blood pressure recording. Counterpulsations can also be converted into sound waves, which is the basis for the auscultation method of measuring blood pressure.

Counterpulsations are more reproducible, and blood pressure measurements are more reliable, when an artery is compressed uniformly. The tendency for cuff inflation to produce uniform compression of an underlying artery is determined by the dimensions of the inflatable bladder in the cuff and the circumference of the upper arm or thigh. The optimal relationships between the dimensions of the cuff bladder and the circumference of the upper arm are shown in Figure 7.2. To produce uniform arterial occlusion, the length of the bladder should be at least 80% of the circumference of the upper arm (measured midway between the shoulder and elbow), and the width of the bladder should be at least 40% of the upper arm circumference (1). If the cuff bladder is too small for the size of the upper arm, the pressure measurements will be falsely elevated (1). Errors in measurement are much less pronounced when the cuff bladder is inappropriately large relative to arm circumference.
FIGURE 7.1 Comparison of the auscultation method and oscilometric method of blood pressure measurement. See text for explanation.
FIGURE 7.2 Optimal relationships between the width (W) and length (L) of the cuff bladder and the circumference (C) of the

A mismatch between cuff size and limb size (called miscuffing) is the most common source of error in indirect blood pressure measurements (1-4). To indicate the magnitude of this problem, one study showed that 97% of primary care physicians routinely used blood pressure cuffs of inappropriate size (2). Most cases (80-90%) of miscuffing involve cuffs that are too small for the size of the upper arm (1,2). As an aid in selecting the appropriate cuff size, Table 7.1 shows the recommended cuff sizes for upper arm circumferences ranging from 22 cm (about 9 inches) to 52 cm (about 21 inches). An easier way to determine the appropriate cuff size is de-scribed next.

Align the cuff so that the long axis runs along the long axis of the arm. Then turn the cuff over so the bladder on the underside is facing you, and wrap the cuff around the upper arm. The bladder (width) should encircle close to half (40%) of the upper arm (circumference). If the bladder encircles less than half of the upper arm, the cuff is too small, and the blood pressure measurement will be spuriously elevated. No change in cuff size is needed if the cuff bladder is too big (i.e., if it encircles more than half of the upper arm) because the errors in measurement in this situation are small or nonexistent (1).

22 to 26 cm, _Blood Pressure Cuff_.‘Size = Small Adult. 22 to 26 cm, _Blood Pressure Cuff_.: Dimensions = 12x24 0m. 27 to 34 cm, _Blood Pressure Cuff_.‘Size = Adult. 27 to 34 cm, _Blood Pressure Cuff_.: Dimensions = 16x 30 cm. 35 to 44 cm, _Blood Pressure Cuff_.‘Size = Large Adult. 35 to 44 cm, _Blood Pressure Cuff_.: Dimensions = 16x 36cm. 45 to 52 cm, _Blood Pressure Cuff_.‘Size = Adult Thigh. 45 to 52 cm, _Blood Pressure Cuff_.: Dimensions = 16 x 42 cm
From Referance 1.

The limited accuracy of indirect blood pressure measurements is magnified in critically ill patients, and this is demonstrated in Figure 7.3. The data in this figure is from two studies comparing direct intraarterial pressures with pressures obtained with the auscultation method (in patients with circulatory shock) and with the oscillometric method (in unselected ICU patients) (4,9). All auscultatory pressures differed from the intraarterial pressures by more than 10 mm Hg (which is the threshold for an unacceptable difference), and the discrepancy was more than 20 mm Hg in almost threequarters of the measurements (auscultatory pressures were always lower than intraarterial pressures). The discrepancy for oscillometric pressures was unacceptable in 61% of measurements.
The particularly poor performance of the auscultatory method in pa-tients with circulatory shock is the result of reduced systemic flow (from hypotension and vasopressors), which curtails the arterial counterpulsations and reduces the intensity of the Korotkoff sounds. Since reliable blood pressure measurements are essential in the management of circulatory shock (e.g., to guide fluid resuscitation), direct measurements of intraarterial pressure is the consensus recommendation.
Discrepancy Between Indirect and Direct Measurements
FIGURE 7.3 Discrepancy between indirect and direct blood pressure measurements in critically ill patients. From References 4 and 9.

Intraarterial pressure is typically measured from the radial, brachial, axillary, or femoral arteries. The arterial cannulation technique is not de-scribed here, but many of the practices related to arterial cannulation are similar to those described in Chapter 2 for central venous cannulation.

The contour of the arterial pressure waveform changes as it moves away from the proximal aorta. This is shown in Figure 7.4. Note that as the pressure wave moves toward the periphery, the systolic pressure gradually increases and the systolic portion of the waveform narrows. The systolic pressure can increase as much as 20 mm Hg from the proximal aorta to the radial or femoral arteries (10). This increase in peak systolic pressure is offset by the narrowing of the systolic pressure wave, so the mean arterial pressure (described later) remains unchanged.

The increase in systolic pressure in peripheral arteries is the result of pressure waves that are reflected back from vascular bifurcations and narrowed blood vessels (11). Reflected waves move faster when the arteries are stiff, and they reach the arterial pressure waveform before it has time to decrement; the convergence of antegrade and retrograde pressure waves serves to heighten the peak of the antegrade pressure waveform. (You can see this effect when ocean waves meet from opposing directions, and this effect has been implicated in the formation of "monster waves".) Amplification of the systolic pressure by reflected waves is the mechanism for systolic hypertension in the elderly (11). Because systolic amplification is the result of retrograde pressure waves, it does not promote systemic blood flow.
FIGURE 7.4 Arterial pressure waveforms at specific points along the arterial circulation.

Fluid-filled recording systems can produce artifacts that further distort the arterial pressure waveform. Failure to recognize these artifacts can lead to errors in blood pressure management.

Vascular pressures are transmitted through fluid-filled plastic tubes that connect the arterial catheter to the pressure transducer. This fluid-filled system can oscillate spontaneously, and the oscillations can distort the arterial pressure waveform (13,14). The performance of a resonant system is defined by two factors: the resonant frequency and the damping factor. The resonant frequency is the frequency of oscillations that occur when the system is disturbed. When the frequency of an incoming signal approaches the resonant frequency of the system, the resident oscillations add to the incoming signal and amplify it. This type of system is called an underdamped system. The damping factor is a measure of the tendency for the system to attenuate the incoming signal. A resonant system with a high damping factor is called an overdamped system.

Three waveforms from different recording systems are shown inFigure 7.5. The waveform in panel A, with the rounded peak and the dicrotic notch, is the normal waveform expected from a recording system with no distortion. The waveform in panel B, with the sharp systolic peak, is from an underdamped recording system. Underdamped systems are popular for pressure recording because of their rapid response characteristics, but these systems can amplify the systolic pressure by as much as 25 mm Hg (15). The final waveform in panel C has an attenuated peak and a narrow pulse pressure. This waveform is from an overdamped system. Overdamping reduces the gain of the system and attenuates the pressure waveform. Overdamping can be the result of partial obstruction of the catheter with a thrombus, or air bubbles in the recording circuit.
FIGURE 7.5 The rapid flush test. Panel A: Normal response; Panel B: Underdamped response; Panel C: Overdamped response. See text for explanation.

A pressurized flush of the catheter-tubing system can also help to identify a recording circuit that is distorting the pressure waveform (14,15). Most commercially available transducer systems are equipped with a one-way valve that can be used to deliver a flush from a pressurized source. Figure 7.5 shows the results of a flush test in three different situations; the response when the flush is released will help characterize the system In panel A, the flush release is followed by a high-frequency burst. This is the normal behavior of a fluid-filled system. In panel B, the flush release produces a more sluggish frequency response. This is characteristic of an underdamped system, which will produce some degree of systolic amplification (as suggested by the narrowed peak on the pressure waveforms). The flush release in panel C does not produce oscillations. This is a sign of an overdamped system, which will attenuate the arterial pressure waveform and produce a spuriously low systolic pressure.

Most ignorance is vincible ignorance; we don't know because we don't want to know.
Aldous Huxley
The most disturbing feature of indirect blood pressure (BP) measurements is not their limited accuracy, but the universal lack of competence in the methodology. In two studies presented earlier in the chapter (2,3), no one measured the blood pressure correctly! This is perplexing, unacceptable, and dangerous. Because BP measurements are performed so frequently, the impact of false readings can be enormous. For example, about 85% of adults in the United States (about 180 million) have a BP measurement at least once yearly—if 3% of the measurements are falsely elevated (a common error), then as many as 5.4 million new cases of hypertension will be created each year. This would explain why there are currently 74.6 million adults in the United States with hypertension (17) (more than the entire population of France!). The American Heart Association publishes guidelines for indirect blood pressure measurements (1), and you are urged to read these guidelines. And then read them again.

- 1. Pickering TG, Hall JE, Appel LJ, et al. Recommendations for blood pressure measurement in humans and experimental animals: Part 1: Blood pressure measurement in humans: a statement for professionals from the Sub-committee of Professional and Public Education of the American Heart Assoc-iation Council on High Blood Pressure Research. Circulation 2005; 111:697-716.

A searchlight cannot be used effectively without a fairly thorough knowedge of the territory to be searched.
Fergus Macartney, FRCP
The pulmonary artery catheter is a versatile monitoring device that provides a wealth of information on cardiac performance and systemic oxygen transport. Introduced in 1970 (1), the catheter rapidly gained in popularity and became a staple in critical care management in the latter part of the twentieth century. Unfortunately, the benefits of the pulmonary artery catheter as a monitoring device have not translated into a survival benefit in most patients (2-4). As a result, the popularity of the catheter has declined precipitously over the past decade, and use of the catheter is currently reserved for cases of refractory heart failure or life-threatening hemodynamic instability of uncertain etiology (5,6).
This chapter presents the spectrum of hemodynamic parameters that can be monitored with pulmonary artery catheters. The physiologic relationships and clinical applications of these parameters are described in Chapters 9 and 10.

The pulmonary artery (PA) catheter was conceived by a cardiologist named Jeremy Swan (1), who designed a catheter that is equipped with a small inflatable balloon. When inflated, the balloon allows the flow of venous blood to carry the catheter through the right side of the heart and into one of the pulmonary arteries (like floating down a river on an inflatable rubber raft). This balloon flotation principle allows a right heart catheterization to be performed at the bedside, without fluoroscopic guidance.

The basic features of a PA catheter are shown in Figure 8.1. The catheter is 110 cm long and has an outside diameter of 2.3 mm (about 7 French). There are two internal channels: one channel emerges at the tip of the catheter (the distal or PA lumen), and the other channel emerges 30 cm proximal to the catheter tip, which should be situated in the right atrium (the proximal or RA lumen). The tip of the catheter has a small inflatable balloon (1.5 mL capacity) that helps to carry the catheter to its final destination (as just described). When the balloon is fully inflated, it creates a recess for the tip of the catheter that prevents the tip from damaging the vessel wall as the catheter is advanced. A small thermistor (a temperature-sensing transducer) is placed near the tip of the catheter. This device is involved in the measurement of cardiac output, as described later in the chapter.

The PA catheter is inserted through a large-bore (8-9 French) introducer sheath that has been placed in the subclavian vein or internal jugular vein (see Figure 8.1). The distal lumen of the catheter is attached to a pressure transducer to monitor vascular pressures as the catheter is ad-vanced. When the catheter is passed through the introducer sheath and enters the superior vena cava, a venous pressure waveform appears. When this occurs, the balloon is inflated with 1.5 mL of air, and the cath-eter is advanced with the balloon inflated. The location of the catheter tip is determined by the pressure tracings recorded from the distal lumen, as shown in Figure 8.2.
FIGURE 8.1 The basic features of a pulmonary artery (PA) catheter. Note that the PA catheter has been threaded through a large-bore introducer catheter that has a side-arm infusion port.
1. The superior vena cava pressure is identified by a venous pressure waveform, which appears as small amplitude oscillations. This pressure remains unchanged after the catheter tip is advanced into the right atrium.
. When the catheter tip is advanced across the tricuspid valve and into the right ventricle, a pulsatile waveform appears. The peak (systolic) pressure is a function of the strength of right ventricular contraction, and the lowest (diastolic) pressure is equivalent to the right-atrial pressure.
. When the catheter moves across the pulmonic valve and into a main pulmonary artery, the pressure waveform shows a sudden rise in diastolic pressure with no change in the systolic pressure. The rise in diastolic pressure is caused by resistance to flow in the pulmonary circulation.
4. As the catheter is advanced along the pulmonary artery, the pulsatile waveform

The wedge pressure is obtained by slowly inflating the balloon at the tip of the PA catheter until the pulsatile pressure disappears, as shown in Figure 8.3. Note that the wedge pressure is at the same level as the diastolic pressure in the pulmonary artery. This relationship is altered in pulmonary hypertension, where the wedge pressure is lower than the pulmonary artery diastolic pressure.
FIGURE 8.2 The pressure waveforms at different points along the normal course of a pulmonary artery catheter. These waveforms are used to identify the location of the catheter tip as it is advanced.

The wedge pressure represents the venous pressure on the left side of the heart, and the magnified section of the wedge pressure in Figure 8.3 shows a typical venous contour that is similar to the venous pressure on the right side of the heart. The a wave is produced by left atrial contraction, the c wave is produced by closure of the mitral valve (during isometric contraction of the left ventricle), and the v wave is produced by systolic contraction of the left ventricle against a closed mitral valve. These components are often difficult to distinguish, but prominent v waves are readily apparent in patients with mitral regurgitation.

The principle of the wedge pressure is illustrated in Figure 8.4. When the balloon on the PA catheter is inflated to obstruct flow (Q=0), there is a static column of blood between the tip of the catheter and the left atrium, and the wedge pressure at the tip of the catheter (Py) is equivalent to the pulmonary capillary pressure (P,) and the pressure in the left atrium (Pi,). To summarize: if Q=0, then Pw = P, = Pia. If the mitral valve is behaving normally, the left atrial pressure (wedge pressure) will be equivalent to the end-diastolic pressure (the filling pressure) of the left ventricle. Therefore, in the absence of mitral valve disease, the wedge pressure is a measure of left ventricular filling pressure,
FIGURE 8.3 Pressure tracing showing the transition from a pulsatile pulmonary artery pressure to a balloon occlusion (wedge) pressure. The magnified area shows the components of the wedge pressure: a wave (atrial contraction), c wave (mitral valve closure), and v wave (ventricular contraction).

The wedge pressure will reflect left atrial pressure only if the pulmonary capillary pressure is greater than the alveolar pressure (P.>P, in Figure 8.4); otherwise the wedge pressure will reflect the alveolar pressure. Capillary pressure exceeds alveolar pressure when the tip of the PA catheter is below the level of the left atrium, or posterior to the left atrium in the supine position. Most PA catheters enter dependent lung regions naturally (because the blood flow is highest in these regions), and lateral chest x-rays are rarely obtained to verify catheter tip position.
FIGURE 8.4 The principle of the wedge pressure measurement. When flow ceases because of balloon inflation (Q=0), the wedge pressure (Pyy) is equivalent to the pulmonary capillary pressure (P-) and the pressure in the left atrium (Pla). This occurs only in the most dependent lung region, where the pulmonary capilary pressure (Pc) is greater than the alveolar pressure (Pa).
Respiratory variations in the wedge pressure suggest that the catheter tip is in a region where alveolar pressure exceeds capillary pressure (7). In this situation, the wedge pressure should be measured at the end of expiration, when the alveolar pressure is closest to atmospheric (zero) pressure. The influence of intrathoracic pressure on cardiac filling pressures is described in more detail in Chapter 9.

In addition to respiratory variations, the CVP and wedge pressures can vary spontaneously, independent of any change in the factors that influence these pressures. The spontaneous variation in wedge pressure is "4 mm Hg in 60% of patients, but it can be as high as 7 mm Hg (8). In general, a change in the wedge pressure should exceed 4 mm Hg to be considered a clinically significant change.

The wedge pressure is often mistaken as the hydrostatic pressure in the pulmonary capillaries, but this is not the case (9,10). The wedge pressure is measured in the absence of blood flow. When the balloon is deflated and flow resumes, the pressure in the pulmonary capillaries (P.) will be higher than the pressure in the left atrium (P,,), and the difference in pressures will be dependent on the flow rate (Q) and the resistance to flow in the pulmonary veins (Ry); i.é.,
(8.1)
Since the wedge pressure is equivalent to left atrial pressure, Equation 8.1 can be restated using the wedge pressure (Py) as a substitute for left atrial pressure (P),).
(8.2)
Therefore the wedge pressure and capillary hydrostatic pressure must be different to create a pressure gradient for venous flow to the left side of the heart. The magnitude of this difference is unclear because it is not possible to determine Ry. However, the discrepancy between wedge and capillary hydrostatic pressures may be magnified in ICU patients because conditions that promote pulmonary venoconstriction (i.e., increase Ry), such as hypoxemia, endotoxemia, and the acute respiratory distress syndrome (11,12), are common in these patients.

The wedge pressure is used to differentiate hydrostatic pulmonary edema from the acute respiratory distress syndrome (ARDS); a normal wedge pressure is considered evidence of ARDS (13). However, since the capillary hydrostatic pressure is higher than the wedge pressure, a normal wedge pressure measurement will not rule out the diagnosis of hydrostatic pulmonary edema. Therefore, the use of a normal wedge pressure as a diagnostic criterion for ARDS should be abandoned.

The ability to measure cardiac output increases the monitoring capacity of the PA catheter from 2 parameters (i.e., central venous pressure and wedge pressure) to at least 10 parameters (see Tables 8.1 and 8.2), and allows a physiologic evaluation of cardiac performance and systemic oxygen transport.
FIGURE 8.5 The thermodilution method of measuring cardiac output. See text for explanation.
The indicator-dilution method of measuring blood flow is based on the premise that, when an indicator substance is added to circulating blood, the rate of blood flow is inversely proportional to the change in concentration of the indicator over time. If the indicator is a temperature, the method is known as thermodilution.
The thermodilution method is illustrated in Figure 8.5. A dextrose or saline solution that is colder than blood is injected through the proximal port of the catheter in the right atrium. The cold fluid mixes with blood in the right heart chambers, and the cooled blood is ejected into the pulmonary artery and flows past the thermistor on the distal end of the catheter. The thermistor records the change in blood temperature with time; the area under this curve is inversely proportional to the flow rate in the pulmonary artery, which is equivalent to the cardiac output in the absence of intracardiac shunts. Electronic monitors integrate the area under the temperature-time curves and provide a digital display of the calculated cardiac output.

Examples of thermodilution curves are shown in Figure 8.6. The low cardiac output curve (upper panel) has a gradual rise and fall, whereas the high output curve (middle panel) has a rapid rise, an abbreviated peak, and a steep downslope. Note that the area under the low cardiac output curve is greater than the area under the high output curve (i.e., the area under the curves is inversely related to the flow rate).

Serial measurements are recommended for each cardiac output determination. Three measurements are sufficient if they differ by 10% or less, and the cardiac output is taken as the average of all measurements. Serial measurements that differ by more than 10% are considered unreliable (14).

Thermodilution cardiac output can vary by as much as 10% without any apparent change in the clinical condition of the patient (15). Therefore, a change in thermodilution cardiac output should exceed 10% to be considered clinically significant.

Regurgitant flow across the tricuspid valve can be common during positive-pressure mechanical ventilation. The regurgitant flow causes the indicator fluid to be recycled, producing a prolonged, low-amplitude thermodilution curve similar to the one in the bottom frame of Figure 8.6. This results in a falsely low cardiac output measurement (16).

Intracardiac shunts produce falsely high thermodilution cardiac output measurements. In right-to-left shunts, a portion of the cold indicator fluid passes through the shunt, thereby creating an abbreviated thermodilution curve similar to the high-output curve in the middle panel of Figure 8.6. In left-to-right shunts, the thermodilution curve is abbreviated be-cause the shunted blood increases the blood volume in the right heart chambers, and this dilutes the indicator solution that is injected.
FIGURE 8.6 Thermodilution curves for a low cardiac output (upper panel), a high cardiac output (middle panel), and tricuspid insufficiency (lower panel). The sharp inflection in each curve marks the end of the measurement period. CO = cardiac output.

The PA catheter provides a wealth of information on cardiovascular function and systemic oxygen transport. This section provides a brief de-scription of the hemodynamic parameters that can be measured or de-rived with the PA catheter. These parameters are included in Table 8.1.

Hemodynamic parameters are often expressed in relation to body size, and the popular measure of body size for hemodynamic measurements is the body surface area (BSA), which can be determined with the following simple equation (17).
BSA (m2?) = Ht (cm) + Wt (ke) — 60/100
(8.3)
Why not use body weight to adjust for body size? BSA was chosen for hemodynamic measurements because cardiac output is linked to metabolic rate, and the basal metabolic rate is expressed in terms of body surface area. The average-sized adult has a body surface area of 1.7 m2.
Central Venous Pressure, 1 = CVP. Central Venous Pressure, 2 = 0-5 mm Hg. Pulmonary Artery Wedge Pressure, 1 = PAWP. Pulmonary Artery Wedge Pressure, 2 = 6-12 mm Hg. Cardiac Index, 1 = Cl. Cardiac Index, 2 = 2.4—4.0 Lémin/m?. Stroke Index, 1 = Sl. Stroke Index, 2 = 20-40 mL/m2. Systemic Vascular Resistance Index, 1 = SVRI. Systemic Vascular Resistance Index, 2 = 25-30 Woed Unitst. Pulmonary Vascular Rasistance Index, 1 = PVRI. Pulmonary Vascular Rasistance Index, 2 = 1-2 Wood Unitst. Oxygen Delivery (Index), 1 = DO;. Oxygen Delivery (Index), 2 = 520-570 mL/min/m2. Oxygen Uptake (Index), 1 = VOo. Oxygen Uptake (Index), 2 = 110-160 mL/min/m?. Oxygen Extraction Ratio, 1 = OER. Oxygen Extraction Ratio, 2 = 0.2-0.3
tmm HaLiminém2
(8.7)
The stroke index is a measure of the systolic performance of the heart during one cardiac cycle. The normal range in adults is 20—40 mL/m2.

The hydraulic resistance in the systemic circulation is not a measurable quantity for a variety of reasons (e.g., resistance is flow-dependent and varies in different regions). Instead, the systemic vascular resistance (SVR) is a global measure of the relationship between systemic pressure and flow. The SVR is directly related to the pressure drop from the aorta to the right atrium (MAP — CVP), and inversely related to the cardiac output (CI).
SVRI = (MAPCVP) /CI
(8.8)
The SVRI is expressed in Wood units (mm Hg/L/min/m2), which can be multiplied by 80 to obtain more conventional units of resistance (dynesesectecm-5/m2), but this conversion offers no advantage (18).

The pulmonary vascular resistance (PVR) has the same limitations as mentioned for the systemic vascular resistance. The PVR is a global measure of the relationship between pressure and flow in the lungs, and is derived as the pressure drop from the pulmonary artery to the left atrium, divided by the cardiac output. Because the pulmonary artery wedge pressure (PAWP) is equivalent to the left atrial pressure, the pressure gradient across the lungs can be expressed as the difference between the mean pulmonary artery pressure and the wedge pressure (PAP — PAWP).
PVRI= (PAP -PAWP)/CI
(8.9)
Like the SVRI, the PVRI is expressed in Wood units (mm Hg/L/min/m2), which can be multiplied by 80 to obtain more conventional units of resistance (dynesesec-tecm-5/m2).
(8.14)
The OzER is a measure of the balance between Op) delivery and O> uptake. It is normally about 25%, which means that 25% of the oxygen delivered to the systemic capillaries is taken up into the tissues.

Most hemodynamic problems can be identified by noting the pattern of changes in three hemodynamic parameters: cardiac filling pressure (CVP or PAWP), cardiac output, and systemic or pulmonary vascular resistance. This is demonstrated in Table 8.2 using the three classic forms of shock: hypovolemic, cardiogenic, and vasogenic. Each of these conditions produces a distinct pattern of changes in the three parameters. Since there are 3 parameters and 3 possible conditions (low, normal, or high), there are 33 or 27 possible hemodynamic patterns, each representing a distinct hemodynamic condition.

CVP or PAWP, 1 = Low. CVP or PAWP, 2 = High. CVP or PAWP, 3 = Low. Cardiac Output, 1 = Low. Cardiac Output, 2 = Low. Cardiac Output, 3 = High. Systemic Vascular Resistance, 1 = High. Systemic Vascular Resistance, 2 = High. Systemic Vascular Resistance, 3 = Low

The hemodynamic patterns just described can identify a hemodynamic problem, but they provide no information about the impact of the problem on tissue oxygenation. The addition of the oxygen uptake (VO>) will correct this shortcoming, and can help identify a state of clinical shock. Clinical shock can be defined as a condition where tissue oxygenation is inadequate for the needs of aerobic metabolism. Since a VO> that is below normal can be used as indirect evidence of oxygen-limited aerobic metabolism, a subnormal VO, can be used as indirect evidence of clinical shock. The following example shows how the VO; can add to the evaluation of a patient with cardiac pump failure.

High CVP, Cardiogenic Shock = High CVP. ore!, Cardiogenic Shock = Low Cl. High Sve, Cardiogenic Shock = High SVRI. Normal VO>, Cardiogenic Shock = row Oe
Without the VO measurement in Table 8.3, it is impossible to differentiate compensated heart failure from cardiogenic shock. This illustrates how oxygen transport monitoring can be used to determine the consequences of hemodynamic abnormalities on systemic oxygenation. Oxygen transport monitoring is described in more detail in Chapter 10.

Despite the wealth of physiologically relevant information provided by the PA catheter, the catheter has been vilified and almost abandoned in recent years because clinical studies have shown added risk with little or no survival benefit, associated with use of the catheter (2—4). The following points are made in support of the PA catheter.
1. First and foremost, the PA catheter is a monitoring device, not a therapy. If a PA catheter is placed to evaluate a problem and it uncovers a disorder that is untreatable (e.g., cardiogenic shock), the problem is not the catheter, but a lack of effective therapy. Clinical outcomes should be used to evaluate therapies, not measurements.
2. In addition, surveys indicate that physicians often don't understand the measurements provided by PA catheters (19,20). Any tool can be a weapon in the wrong hands.
3. Finally, the incessant use of mortality rates to evaluate critical care interventions is problematic because the presumption that every intervention has to save lives to be of value is flawed. Interventions should (and do) have more specific and immediate goals other than life or death. In the case of a monitoring device, the goal is to provide clinical information, and the PA catheter achieves this goal with distinction.

When is a piece of matter said to be alive?
When it goes on "doing something", moving, exchanging material with its environment.
Erwin Schrodinger What is Life? (1944)
The human organism has an estimated 100,000,000,000,000 cells that must go on exchanging material with the external environment to stay alive. This exchange is made possible by the human circulatory system: a closed hydraulic circuit with an automatic stroke pump that averages 100,000 strokes daily, a volumetric flow that averages 8,000 liters/day, and a network of conducting vessels that, if placed end-to-end, would stretch more than 60,000 miles (more than twice the circumference of the Earth!) (1). The design and performance of this circulatory system is a reminder of the following quote by Aristotle: In all things of nature, there is something of the marvelous (2).
This chapter describes the forces that govern blood flow through the circulatory system, both pulsatile flow (cardiac stroke output) and steady flow (peripheral blood flow), and the available methods for monitoring these forces in the clinical setting. Many of the concepts in this chapter are old friends from the physiology classroom.

If one end of a muscle fiber is suspended from a rigid strut and a weight is attached to the free end of the muscle, the muscle will be stretched to a new length. The added weight in this situation represents a force called the preload. (The prefix pre indicates that the load is imposed prior to the onset of muscle contraction.) Preload is thus defined as the force imposed on resting muscle that stretches the muscle to a new length. According to the length-tension relationship of muscle, an increase in the length of a resting muscle will increase the strength of muscle contraction (because more crossbridges are formed between contractile elements in the muscle) (3). Therefore, the preload force acts to augment the strength of muscle contraction.

In the intact heart, the volume in the ventricles at the end of diastole is the force that stretches the resting muscle to a new length. Therefore, the end-diastolic volume of the ventricles is the preload force of the intact heart (3).
The influence of end-diastolic volume (preload) on cardiac performance is demonstrated in Figure 9.1. The lower curve shows the changes in end-diastolic pressure, which is a reflection of the distensibility of the ventricle , and the upper curve shows the peak pressure developed during systole. At any given end-diastolic volume, the increment from end-diastolic pressure to peak systolic pressure is a reflection of the strength of ventricular contraction. This increment in pressure increases as the end-diastolic volume increases, indicating that the preload force augments the strength of ventricular contraction. This relationship between preload and the strength of ventricular contraction was discovered independently by Otto Frank (a German engineer) and Ernest Starling (a British physiologist), and their discovery is commonly referred to as the Frank-Starling relationship of the heart (3). This relationship can be stated as follows: In the normal heart, diastolic volume is the principal force that governs the strength of ventricular contraction (3).
End-Diastolic Volume
FIGURE 9.1 The influence of ventricular end-diastolic volume (preload) on end-diastolic pressure and peak systolic pressure. At any given end-diastolic volume, the increment from end-diastolic pressure to peak systolic pressure is a reflection of the strength of ventricular contraction during systok.

Ventricular end-diastolic volume is not easily measured at the bedside, and end-diastolic pressure is used as the clinical measure of ventricular preload. The end-diastolic pressure in the right and left ventricles is measured as follows:
- 1. The pressure in the superior vena cava, also called the central venous pressure (CVP), is equivalent to the right atrial pressure (RAP). In the absence of tricuspid valve dysfunction, the RAP is equivalent to the right ventricular end-diastolic pressure (RVEDP); i.e.,

Therefore, the CVP can be used as the filling pressure of the right ventricle when tricuspid valve function is normal.
- 2. The pulmonary artery wedge pressure (PAWP), which is described in the last chapter, is equivalent to the left atrial pressure (LAP). In the absence of mitral valve dysfunction, the LAP is equivalent to the left ventricular end-diastolic pressure (LVEDP); i.e.,

Therefore, the wedge pressure can be used as the left ventricular filling pressure when mitral valve function is normal.
The reference ranges for the CVP and wedge pressures are shown in Table 9.1 (4,5). Note the very low pressure range for the CVP, which helps to promote venous return to the heart. Note also that the wedge pressure is slightly higher than the CVP; the higher pressure in the left atrium closes the flap over the foramen ovale and prevents right-toleft shunting in patients with a patent foramen ovale (about 30% of adults).

Right Ventricle, 1 = . Right Ventricle, 2 = . End-Diastolic Pressure, 1 = RVEDP. End-Diastolic Pressure, 2 = 6-5 mmHg. End-Diastolic Volume, 1 = RVEDV. End-Diastolic Volume, 2 = 45—90 mLém?. Stroke Volume, 1 = SV. Stroke Volume, 2 = 20-40 mL/m2. Ejection Fraction, 1 = EF. Ejection Fraction, 2 = = 44%. Left Ventricle, 1 = . Left Ventricle, 2 = . End-Diastolic Pressure, 1 = LVEDP. End-Diastolic Pressure, 2 = 6-12 mm Hg. End-Diastolic Volume, 1 = LVEDV. End-Diastolic Volume, 2 = 35-75 mLim?. Stroke Volume, 1 = sv. Stroke Volume, 2 = 20-40 mL/m2. Ejection Fraction, 1 = EF. Ejection Fraction, 2 = 255%
From Referance 4 and 5. End-diastolic volumes and stroke volumes ara expressed relative to body surface area.

The relationship between ventricular end-diastolic pressure and cardiac stroke output is described with ventricular function curves like the ones in Figure 9.2 (6). The principal feature of the normal curve is the steep slope, which results in a 2.5-fold increase in cardiac output over the normal range of right atrial pressure (0-5 mm Hg). This demonstrates the profound effect of ventricular filling on the strength of ventricular contraction, as predicted by the Frank-Starling relationship of the heart. The ventricular function curve is displaced downward in patients with heart failure, indicating that the strength of ventricular contraction is reduced at any given ventricular filling pressure in patients with heart failure.
FIGURE 9.2 Ventricular function curves describing the relationship between right atrial pressure and cardiac output. Normal curve is redrawn from Reference 6.

Although the end-diastolic pressure is the clinical measure of preload, clinical studies have shown a poor correlation between end-diastolic pressure and end-diastolic volume (preload) (7-9). This is demonstrated in Figure 9.3, which shows the results of a study that compared measurements of right ventricular end-diastolic pressure (i.e., CVP) and right ventricular end-diastolic volume (RVEDV) before and after a volume challenge with isotonic saline (9). The graph on the left shows the corresponding measures of CVP and RVEDV prior to volume infusion, and the graph on the right shows the corresponding changes in CVP and RVEDV in response to the volume infusion. The distribution of data points in both graphs shows no relationship between CVP and RVEDV, or between changes in CVP and RVEDV. This is confirmed by the correlation coefficients (r) in the upper left corner of each graph. Similar results have been reported for the left ventricle (8,9). These studies indicate that ventricular filling pressures (i.e. CVP and wedge pressures) are unreliable as surrogate measures of ventricular filling.
FIGURE 9.3 Graphs showing the relationships between right ventricular end-diastolic pressure (CVP) and right ventricular end-diastolic volume (RVEDV) in healthy adults who received a volume infusion with isotonic saline (3 liters over 3 hours). The graph on the left shows the baseline measurements of CVP and RVEDV, and the graph on the right shows the changes in CVP and RVEDV in response to the volume infusion. The correlation coefficients (r) are shown in the upper left corner of each graph. Graphs redrawn from Reference 9.
The poor correlation between end-diastolic pressures and volume in Figure 9.3 is particularly noteworthy because the subjects were healthy adults with normal cardiac function. When ventricular distensibility is impaired (i.e., diastolic dysfunction), which is common in critically ill patients (10), the discrepancy between end-diastolic pressures and volumes will be greater than usual. The influence of ventricular distensibility on diastolic pressure-volume relationships is described next.

Ventricular filling is influenced by the tendency of the ventricular walls to stretch during diastole (i.e., distensibility). The more popular term for distensibility is compliance. Ventricular compliance is derived as the ratio of associated changes in end-diastolic volume (ZEDV) and end-diastolic pressure (SEDP):
A decrease in ventricular compliance will result in a greater change in EDP for a given change in EDV, or a smaller change in EDV for a given change in EDP.
The influence of compliance on diastolic pressure-volume relationships is shown in Figure 9.4 (11). The lower curve in this figure is from a control subject with no cardiac disease, and the upper curve is from a patient with hypertrophic cardiomyopathy. Note the increased slope of the curve for the hypertrophic cardiomyopathy, indicating a decrease in ventricular compliance. Comparing the position of the two curves shows that, at any given end-diastolic volume, the end-diastolic pressure is higher in the noncompliant ventricle. Therefore, when ventricular compliance is reduced, the end-diastolic pressure will overestimate the end-diastolic volume.
FIGURE 9.4 Diastolic pressure-volume curves for the left ventricle in a control subject and a patient with hypertrophic cardiomyopathy. Data from Reference 11.

In the early stages of cardiac disease where ventricular distensibility is impaired (i.e., diastolic dysfunction), ventricular end-diastolic volume is preserved but end-diastolic pressure rises. As the condition progresses, the progressive increase in end-diastolic pressure eventually results in a decrease in venous return, and this is accompanied by a decrease in ventricular filling and a subsequent decrease in cardiac output. When impaired ventricular distensibility compromises ventricular filling, the condition is known as diastolic heart failure (11,12).
Diastolic heart failure can be difficult to distinguish from heart failure due to contractile dysfunction (systolic heart failure) because both conditions are associated with increased end-diastolic pressures and a downward shift in the ventricular function curve. (In Figure 9.2, the lower "heart failure" curve could represent diastolic or systolic heart failure.) The changes in end-diastolic pressure (EDP), end-diastolic volume (EDV), and ventricular ejection fraction (EF) in the two types of heart failure are shown below (12):
Systolic Failure: High EDP / High EDV / Low EF
Diastolic Failure: High EDP / Low EDV / Normal EF
The EDV and EF can distinguish diastolic from systolic heart failure. (The EF, which is the ratio of stroke volume to end-diastolic volume, is the standard distinguishing feature. Table 9.1 includes the normal EF for the right and left ventricles). The fact that the EDP and EDV change in opposite directions in diastolic heart failure highlights the discrepancy be-tween EDP and ventricular preload (EDV) when ventricular compliance is reduced. (Note: The terms "diastolic heart failure" and "systolic heart failure" have recently been abandoned. Diastolic failure is now called "heart failure with normal ejection fraction," and systolic failure is called "heart failure with a reduced ejection fraction." These two conditions will be described in more detail in Chapter 13.)

Despite the shortcomings of EDP as a measure of ventricular filling, CVP monitoring continues to be a popular practice in ICUs. However, errors in the CVP measurement are common (13), and this section highlights the potential sources of error.

The catheters used for CVP monitoring are multilumen central venous catheters (15 to 20 cm in length) that are inserted in the subclavian or internal jugular veins and advanced into the superior vena cava. Peri-pherally inserted central catheters (PICCs) are not used for CVP monitoring because of concerns that the length of the catheters (up to 70 cm) will attenuate the pressure signal. However, there is a study showing that PICCs can provide accurate CVP measurements when a continuous infusion of saline (at rates comparable to those used for arterial catheters) is used to maintain catheter patency (14). Monitoring through PICCs is appealing because it eliminates the risks (i.e., arterial puncture and pneumothorax) associated with cannulation of the subclavian and internal jugular veins.

The CVP is a hydrostatic pressure, so it is important that the fluid-filled transducer is at the same level as the right atrium. The traditional reference point for the right atrium is the intersection of the mid-axillary line (midway between the anterior and posterior axillary folds) and the fourth intercostal space, with the patient in the supine position. An alternative reference point that can be used in the semirecumbent position (up to 60°) is located 5 cm directly below the sternal angle (the angle of Louis), where the sternum meets the second rib (15).

The CVP and wedge pressure measurements can be misleading because the recorded pressure differs from the physiologically relevant pressure. This is demonstrated in the illustration in Figure 9.5. The pressure in the superior vena cava (the CVP) is recorded as an intravascular pressure; i.e., the pressure in the blood vessel relative to atmospheric (zero) pressure. However, the pressure that distends the ventricles to allow ventricular filling is the transmural pressure, which is the difference between the intravascular pressure and the surrounding intrathoracic pressure. Therefore, the recorded (intravascular) pressure will reflect the relevant (transmural) pressure only when the intrathoracic pressure is equivalent to atmospheric pressure. This normally occurs at the end of expiration. Therefore, the CVP and wedge pressure should be measured at the end of expiration.

When intrathoracic pressure changes (i.e., during spontaneous breathing or positive pressure ventilation), the pressure change can be transmitted into the lumen of the veins within the thorax, resulting in a change in the measured (intravascular) pressure without a change in the relevant (transmural) pressure. An example of this phenomenon is shown in the CVP tracing in Figure 9.6. The undulations in this tracing are the result of respiratory changes in intrathoracic pressure that are transmitted into the superior vena cava. Although the recorded (intravascular) pressure is changing, the relevant (transmural) pressure is unchanged. Therefore, respiratory variations in the CVP (and wedge pressure) do not represent changes in ventricular filling pressure. When respiratory variations are evident, the cardiac filling pressure should be measured at the end of expiration, when intrathoracic pressure is normally at atmospheric (zero) level. For the CVP tracing in Figure 9.6, which was recorded during positive-pressure ventilation, the end-expiratory pressure is the lowest pressure on the tracing, so the CVP is 0-3 mm Hg. When respiratory variations occur during spontaneous (negative pressure) breathing, the end-expiratory pressure will be the highest pressure in the tracing.
FIGURE 9.5 Illustration showing the difference between the intravascular pressure (Py) recorded electronically and the transmural pressure (PT) that is the responsible for distending the ventricles during diastole. Pat = atmospheric pressure; PTHORAX = intrathoracic pressure; SVC = superior vena cava.
FIGURE 9.6 Respiratory variations in the central venous pressure (CVP).

Positive end-expiratory pressure (PEEP) can falsely elevate the cardiac filling pressures at end-expiration because the intrathoracic pressure is higher than atmospheric pressure. When PEEP is applied during mech-anical ventilation (which is a routine practice), the patient can be briefly disconnected from the ventilator to measure the CVP (16). For patients with "intrinsic PEEP" (caused by incomplete emptying of the lungs), accurate measurement of the cardiac filling pressures can be difficult (17). Chapter 28 includes a description of intrinsic PEEP, and a method for correcting the effect of intrinsic PEEP on the recorded filling pressures.

The CVP and wedge pressure can vary spontaneously by as much as 4 mm Hg (18), so changes in these pressures must exceed 4 mm Hg to be considered clinically significant.

When a weight is attached to one end of a contracting muscle, the force of muscle contraction must be enough to lift the weight before the muscle begins to shorten. The weight in this situation represents a force called the afterload, which is the load imposed on a muscle after the onset of muscle contraction. Unlike the preload force, which facilitates muscle contraction, the afterload force opposes muscle contraction. In the intact heart, the afterload force is equivalent to the peak tension developed across the wall of the ventricles during systole (3). Afterload is thus the wall stress associated with ejection of the stroke volume.

The determinants of ventricular wall tension are derived from observations on soap bubbles made by the Marquis de Laplace in 1820. His observations are expressed in the Law of Laplace, which states that the wall tension in a thin-walled sphere is directly related to the chamber pressure and the radius of the sphere: A modified version of the Laplace Law is shown below.
Wall Tension = (pressure * radius) / (2 = wall thickness)
(9.2)
When the Laplace relationship is applied to the heart, the relevant pressure is the peak transmural pressure across the ventricle during systole, and the relevant radius is the end-diastolic radius of the ventricular chamber. The relationships in equation 9.2 allow the following statements:
1. The greater the peak transmural pressure during systole, the greater the wall stress.
2. The larger the ventricular chamber size, the greater the wall stress.
3. The greater the ventricular hypertrophy, the less the wall stress.

The forces that contribute to ventricular afterload can be identified by their relationship to the variables in the Laplace equation. This is demonstrated by the flow diagram in Figure 9.7. The component forces of ventricular afterload include end-diastolic volume (preload), pleural pressure, vascular impedance, and peripheral vascular resistance. Each of these forces is briefly described in this section.

Since afterload is a transmural wall tension, it will be influenced by the pleural pressure surrounding the heart.
NEGATIVE PLEURAL PRESSURE: Negative pressure surrounding the heart will impede ventricular emptying by opposing the inward movement of the ventricular wall during systole (19,20). This effect is responsible for the transient decrease in systolic blood pressure that occurs during the inspiratory phase of spontaneous breathing. When the inspiratory drop in systolic pressure is greater than 15 mm Hg, the condition is called "pulsus paradoxus" (which is a misnomer, since the response is not paradoxical, but is an exaggeration of the normal response).
POSITIVE PLEURAL PRESSURE: Positive pressures surrounding the heart will promote ventricular emptying by facilitating the inward movement of the ventricular wall during systole (19,21). This effect is responsible for the phenomenon shown in Figure 9.8. The tracings in this figure show the effect of a positive-pressure lung inflation on the arterial blood pressure. Note that when intrathoracic pressure rises during a positive-pressure breath, there is a transient rise in systolic blood pressure (reflecting an increase in the stroke output of the heart). The inspiratory rise in blood pressure during mechanical ventilation is known as "reverse pulsus paradoxus."
The "unloading" effect of positive intrathoracic pressure is the basis for the use of positive-pressure breathing as a "ventricular assist" maneuver for patients with advanced heart failure (21,22). The cardiovascular ef-fects of mechanical ventilation are described in more detail in Chapter 25.
FIGURE 9.8 Changes in arterial blood pressure in response to a positive-pressure breath.

The stroke output of the left ventricle produces pulsatile pressure and flow patterns in the aorta and major arteries, but the phasic changes in pressure and flow are progressively dampened as the blood moves peri-pherally, and by the time the blood reaches the small peripheral arterioles, pressure and flow are steady and non-pulsatile. The force that opposes pulsatile flow is known as impedance, and the force that opposes steady flow is resistance.
IMPEDANCE: Vascular impedance is the force that opposes the rate of change in pressure and flow, and it is expressed primarily in the large, proximal arteries, where pulsatile flow is predominant. Impedance in the ascending aorta is considered the principal afterload force for the left ventricle, and impedance in the main pulmonary arteries is considered the principal afterload force for the right ventricle (23). Vascular impedance is a dynamic force that changes frequently during a single cardiac cycle, and it is not easily measured in the clinical setting.
RESISTANCE: Vascular resistance is the force that opposes non-pulsatile or steady flow, and is expressed primarily in small, terminal blood vessels, where non-pulsatile flow is predominant. About 75% of the vascular resistance is in arterioles and capillaries (24). Vascular resistance is calculated as described next, but the relevance of these calculations is questioned.

As mentioned earlier, the design of the heart as an intermittent stroke pump results in a phasic or pulsatile pattern of pressure and flow in the large, proximal arteries. As the blood moves away from the heart, the arterial circuit acts to progressively dampen the pulsatile pattern of pressure and flow, culminating in a non-pulsatile or steady flow rate by the time the blood reaches the microcirculation. (Steady flow permits more efficient exchange in the microcirculation.) The pulsations in the proximal arteries represent wasted cardiac work (i.e., not involved in promoting capillary flow and exchange), whereas the maintenance of steady flow in peripheral blood vessels represents the energy-efficient portion of cardiac work.

Because flow in the peripheral circulation is predominantly non-pulsatile, it can be described with the Hagen-Poiseuille equation, which identifies the determinants of steady flow through small rigid tubes (27). This equation is shown below, and is also included in Chapter 1 to describe flow through vascular catheters.
Q=AP x (nw rt/8uL)
(9.6)
According to this equation, steady flow (Q) through a rigid tube is directly related to the pressure gradient along the tube (2P) and the fourth power of the radius (r) of the tube, and is inversely related to the length (L) of the tube and the viscosity (u) of the fluid. The final term in the equation is the reciprocal of resistance (1/R), so resistance to flow can be described as:
R= 8uL/rrt
(9.7)
This equation identifies the radius of blood vessels as the single most important factor in determining the resistance to steady flow in the peripheral circulation; i.e., a two fold increase in vessel radius will result in a 16-fold increase in flow rate (r4 x r4 = r16), This highlights the importance of vasodilator therapy for promoting cardiac output in patients with heart failure.

According to equations 9.6 and 9.7, steady flow will vary inversely with changes in the viscosity (u) of blood. Viscosity is defined as the resistance of a fluid to changes in flow rate (28), and has also been called the "gooiness" of a fluid (29). The viscosity of whole blood is the result of cross-linking of erythrocytes by plasma fibrinogen, and the principal determinant of whole blood viscosity is the concentration of erythrocytes (the hematocrit). The influence of hematocrit on blood viscosity is shown in Table 9.2. Note that blood viscosity can be expressed in absolute or relative terms (relative to water). The viscosity of plasma (zero hematocrit) is only slightly higher than that of water, while the viscosity of whole blood at a normal hematocrit (45%) is about 3 times greater than plasma and about 4 times greater than water. The influence of hematocrit on blood viscosity is the single most important factor that determines the hemodynamic effects of anemia and blood transfusions (see later).

0, Relative Viscosity (water = 1) = 1.4. 0, Absolute Viscosity (centipoise) = a. 10, Relative Viscosity (water = 1) = 1.8. 10, Absolute Viscosity (centipoise) = 1.2. 20, Relative Viscosity (water = 1) = 21. 20, Absolute Viscosity (centipoise) = 15. 30, Relative Viscosity (water = 1) = 2.8. 30, Absolute Viscosity (centipoise) = 1.8. 40, Relative Viscosity (water = 1) = 3.7. 40, Absolute Viscosity (centipoise) = 2.3. 50, Relative Viscosity (water = 1) = 48. 50, Absolute Viscosity (centipoise) = 2.9. 60, Relative Viscosity (water = 1) = 5.8. 60, Absolute Viscosity (centipoise) = 3.8
From Documenta Geigy Scientific Tables. 7th Ed. Basel: Documenta Geigy, 1966:557-8.

The viscosity of some fluids varies inversely with changes in the velocity of flow (28). Blood is one of these fluids. (Another is ketchup, which is thick and difficult to get out of the bottle, but once it starts to flow, it thins out and flows more easily.) The velocity of blood flow increases as the blood vessels narrow (like the nozzle on a garden hose works), and the velocity of plasma increases more than the velocity of erythrocytes. This results in a relative increase in plasma volume (and a decrease in blood viscosity) in the small, peripheral blood vessels. This process is called shear thinning (shear is a tangential force that influences flow rate), and it facilitates flow through small vessels.

The influence of blood viscosity on cardiac output is shown in Figure 9.9. The data in this graph is from a patient with polycythemia who was treated with phlebotomy to achieve a therapeutic reduction in hematocrit and blood viscosity (30). The progressive decrease in hematocrit is associated with a steady rise in cardiac output, and the change in cardiac output is proportionally greater than the change in hematocrit. The disproportionate increase in cardiac output can be explained by the inverse relationship between blood viscosity and flow velocity; i.e. as cardiac output is increased in response to hemodilution, the increase in flow velocity will result in a further reduction in viscosity, which will then lead to a further increase in cardiac output, and so one. This process magnifies the influence of blood viscosity on cardiac output.

Viscosity is rarely measured in the clinical setting because of the concern that in vitro measurements of viscosity do not take into account in vivo conditions like shear thinning that influence viscosity and blood flow. Despite the lack of meaningful measurements, viscosity is an important consideration for understanding the hemodynamic effects of conditions like anemia, blood transfusions, and dehydration.
FIGURE 9.9 The influence of progressive hemodilution on cardiac output in a patient with polycythemia. CO = cardiac output. Data from Reference 30.

One of the distinguishing features of critical care medicine is the opportunity to apply basic principles of cardiovascular and respiratory physiology to bedside patient care. This, of course, requires a working knowledge of the concepts in this chapter (and the ones in Chapters 10, 20, and 25). Some of the salient points in this chapter are summarized below.
1. The heart is a volume-regulated stroke pump, and the ventricular end-diastolic volume (preload) is the major determinant of the strength of ventricular contraction.
2. Ventricular filling pressures (i.e., the CVP and wedge pressure) are used as a surrogate measure of ventricular filling volumes, but are unreliable.
3. When ventricular compliance is reduced, which is common in critically ill patients, ventricular filling pressures will overestimate ventricular filling volumes.
4. Pleural pressure can have a significant effect on ventricular emptying. Negative pleural pressure impedes ventricular emptying, and positive pleural pressure promotes ventricular emptying.
5. Ventricular afterload has several component forces, and is not measurable.

1. Vogel S. Vital circuits. New York: Oxford University Press, 1992:1-17.
2. Aristotle. De Partibus Animalum. circa 350 B.C.: p 645, first column, line 16.

19., 1 = Pinsky MR. Cardiopulmonary interactions: the effects of negative and positive changes in pleural pressures on cardiac output. In Dantzger DR (ed). Cardio- pulmonary critical care. 2nd ed. Philadelphia: WB Saunders, 1991:87-120.. 20., 1 = Hausnecht N, Brin K, Weisfeldt M, Permutt s, Yin F. Effects of left ventricular loading by negative intrathoracic pressure in dogs. Circ Res 1988; 62:620-631.. 21., 1 = Yan AT, Bradley TD, Liu PP. The role of continuous positive airway pressure in the treatment of congestive heart failure. Chest 2001; 120:1675-1685.. 22., 1 = Boehmer JP, Popjes E. Cardiac failure: Mechanical support strategies. Crit Care Med 2006; 34(Suppl):S268-S277.. 23., 1 = Nichols WW, O'Rourke MF. Input impedance as ventricular load. In: McDonald's Blood Flow in Arteries, 3rd ed. Philadelphia: Lea & Febiger, 1990:330-342.. 24., 1 = Nichols WW, O'Rourke MF. The nature of flow of a fluid. In: McDonald's Blood Flow in Arteries, 3rd ed. Philadelphia: Lea & Febiger, 1990:27.. 25., 1 = Pinsky MR. Hemodynamic monitoring in the intensive care unit. Clin Chest Med 2003; 24:549-560.. 26., 1 = Lang RM, Borrow KM, Neumann A, et al. Systemic vascular resistance: an unreliable index of left ventricular afterload. Circulation 1986; 74:1114-1123.

27., 1 = Chien S, Usami S, Skalak R. Blood flow in small tubes. In Renkin EM, Michel CC (eds). Handbook of Physiology. Section 2: The cardiovascular system. Volume IV. The microcirculation. Bethesda: American Physiological Society, 1984:217-249.. 28., 1 = Merrill EW. Rheology of blood. Physiol Rev 1969; 49:863-888.. 29., 1 = Vogel S. Life in Moving Fluids. Princeton: Princeton University Press, 1981:11-24.. 30., 1 = LeVeen HH, Ahmed N, Mascardo T, et al. Lowering blood viscosity to overcome vascular resistance. Surg Gynecol Obstet 1980; 150:139-149.. 31., 1 = Lowe GOD. Blood rheology in vitro and in vivo. Bailleres Clin Hematol 1987; 1:597.. 32., 1 = Reggiori G, Occhipinti G, de Gasperi A, et al. Early alterations of red blood cell rheology in critically ill patients. Crit Care Med 2009; 37:3041-3046.

Oxygen may be necessary for life, but it doesn't prevent death.
P.L.M.
Critical care management is dominated by interventions that promote tissue oxygenation, yet there are no direct measurements of the oxygen tension in tissues. Instead, a variety of global, indirect measures of tissue oxygenation are used to guide aerobic support. This chapter describes these indirect measures, and how they are obtained. Because of the global nature of these measurements, the term systemic oxygenation seems more suitable for what is measured.

The oxygenation of arterial and venous blood is frequently involved in the evaluation of systemic oxygenation. The relevant measures of oxygen (O03) in blood include the partial pressure of O» (PO), the O, saturation of hemoglobin (SO>), the concentrations of hemoglobin-bound O> and dissolved O>, and the total O5 concentration (also called O, content). The normal values of these measures in arterial and venous blood are shown in Table 10.1.

The oxygenation of hemoglobin is evaluated by the fraction of the hemoglobin in blood that is fully saturated with O>. This is called the O> saturation (SO), and is the ratio of fully oxygenated hemoglobin to the total hemoglobin in blood.
SO, = Oxygenated Hb/Total Hb
(10.1)
This ratio is typically reported as a percentage (the percent saturation of hemoglobin). The SO, can be measured using spectrophotometry (which is called oximetry, and is described in Chapter 21), or it can be estimated using the PO> of blood, as described next.

Partial Pressure of Os, Arterial Blood = 90 mm Hg. Partial Pressure of Os, “Venous Blood. = 40 mm Hg. OQ, Saturation of Hb, Arterial Blood = 98%. OQ, Saturation of Hb, “Venous Blood. = 13%. Hb-Bound OQ,, Arterial Blood = 19.7 mL/de. Hb-Bound OQ,, “Venous Blood. = 14.7 mde. Dissolved C5, Arterial Blood = 0.3 mLU/dL. Dissolved C5, “Venous Blood. = 0.1 mL/dL. Total Oz Content, Arterial Blood = 20 mL/dL. Total Oz Content, “Venous Blood. = 14.8 mL‘dl. Blood Volumet, Arterial Blood = 1.25L. Blood Volumet, “Venous Blood. = 3.75 .L. Total Volume of O,, Arterial Blood = 250 mL. Total Volume of O,, “Venous Blood. = 555 mL
Values shown are for a body temperatura of 37°C and a hamedoebin concantration of 15 afd.
Tolume estimates basad on a total blood volume (TBV) of 6 Liters, artarial blood volume = 25% of TEV, and venous blood volume = 75% of TRV.
Abbreviations: Hb = hamoglebin; dl = decilitar (400 mL}.

The SO, is determined by the PO; in blood and the tendency of the iron moieties in hemoglobin to bind O>. The relationship between SO, and POQ> is described by the oxyhemoglobin dissociation curve like the one shown in Figure 10.1. The "S" shape of the curve offers two advantages. First, the arterial PO> (PaO) is normally on the upper, flat part of the curve, which means that a large drop in PaOz (down to 60 mm Hg) results in only minor changes in the arterial O2 saturation (SaO2). Secondly, the capillary PO2 (which is equivalent to the venous PO) or PvO> after equilibration with the tissues) is on the steep portion of the curve, which facilitates the exchange of O> in both the pulmonary and systemic capillaries.
SHIFTS IN THE CURVE: A number of conditions can alter the affinity of hemoglobin for O, and shift the position of the oxyhemoglobin dissociation curve. These are listed in the boxes in Figure 10.1. A shift of the curve to the right facilitates oxygen release in the systemic capillaries, while a shift to the left facilitates oxygen uptake in the pulmonary capillaries. The position of the curve is indicated by the Psp, which is the PO, that corresponds to an O> saturation of 50%. The Psp is normally about 27 mm Hg (1), and it increases when the curve shifts to the right, and de-creases when the curve shifts to the left. A decrease in the Psp to 15 mm Hg has been reported in blood that is stored in acidcitrate-dextrose (ACD) preservative for 3 weeks, due to a leftward shift in the oxyhemoglobin dissociation curve from depletion of 2,3 diphosphoglycerate (2,3-DPG) in the red blood cells (2).
FIGURE 10.1 Oxyhemoglobin dissociation curve showing the normal relationship between the PO? in blood and the O2 saturation of hemoglobin. The Psg is the PO? that corresponds to 50% saturation of hemoglobin with O3. Abbreviations: PaO? = arterial PO2; PvO2 = venous PO; SaO2 = arterial SO7; SvO7 = venous SO3.

Shifts in the oxyhemoglobin dissociation curve have opposing effects in the pulmonary and systemic capillaries that seem to cancel each other. For example, a rightward shift of the curve caused by acidemia (the Bohr effect) will facilitate O2 release in the systemic capillaries but will hinder O5 uptake in the pulmonary capillaries. So what is the net effect of acid-emia on tissue oxygenation? The answer is based on the influence of shifts in the oxyhemoglobin dissociation curve on different portions of the curve; i.e., shifts in the curve cause less of a change in the flat portion of the curve (where the arterial POz and SO, reside) than in the steep portion of the curve (where the capillary PO2 and SO, reside). Therefore, a rightward shift of the curve from acidemia will facilitate O release
in the systemic capillaries more than it hinders Oz uptake in the pulmonary capillaries, and the overall effect benefits tissue oxygenation.
equation 10.4, but the SOz and PO> are for mixed venous blood (SvO> and PvO;).
CvO, = (1.34 x [Hb] «x S5vO,) + (0.003 x PvOs)
(10.5)
As shown in Table 10.1, the normal mixed venous O> content is about 15 mL/dL, and less than 1% (0.1 mL/dL) represents dissolved O3. Note also that the difference between the arterial and venous O> content (CaO — CvO3) is 5 ml/dL, or 50 mL/L, which means that 50 mL of Op is extracted from each liter of blood flowing through the capillaries. At a normal cardiac output of 5 L/min, the O> extracted from capillary blood would be 5 x 50 = 250 mL/min, which is the normal Oz consumption in an adult at rest. This demonstrates how the oxygenation of blood can provide information about tissue oxygenation.

The dissolved O> is such a small fraction of the total O> content that it is usually eliminated from the equation describing Oz content, as shown below.
©, Content = 1.34 x [Hb] x SO,
(10.6)
The O> content of blood is thus equivalent to the Hb-bound QO, as de-scribed in Equation 10.2.
FIGURE 10.2 The effects of equivalent (50%) reductions in hemoglobin concentration [Hb] and arterial PO? (PaO?) on the oxygen content in arterial blood (CaO2).

There is a tendency to use the arterial PO (PaOz) as an indication of how much QO; is in the blood. However, the O > content of blood is determined primarily by [Hb], as shown in equation 10.6. The influence of proportional decreases in [Hb] and PaO; on the arterial O> content is shown in Figure 10.2. A 50% reduction in [Hb] (from 15 to 7.5 g/dL) results in an equivalent 50% reduction in CaO» (from 20 to 10 mL/dL), while a 50% reduction in the PaO> (from 90 to 45 mm Hg, which corresponds to a decrease in SaOz from 98% to 78%) results in only a 20% decrease in CaO> (from 20 to 16 mL/dL). This demonstrates that anemia has a much greater influence on arterial oxygenation than hypoxemia. The PaO» measurement is useful for evaluating gas exchange in the lungs (as described in Chapter 20), not for evaluating the oxygenation of blood.

The business of nutrient metabolism is to extract the energy stored in nutrient fuels (which is accomplished by disrupting high-energy carbon bonds) and transfer the energy to storage molecules like adenosine triphosphate (ATP). The energy yield from this process is determined by the balance between rate of O> transport to metabolizing tissues and the rate of metabolism. This balance is illustrated in Figure 10.3. Oxygen transport has two components: the rate of O2 delivery to the microcirculation (DO2), and the rate of O» uptake into the tissues (VO>). When the VO matches the metabolic rate (MR), glucose is completely oxidized to yield 36 ATP molecules (673 kcal) per mole. When VO, is less than the metabolic rate (i.e., when VO2<MR), some of the glucose is diverted to form lactate, and the energy yield falls to 2 ATP molecules (47 kcal) per mole.

The condition where the energy yield of nutrient metabolism is limited by the availability of oxygen is called dysoxia (5), and the clinical expression of this condition is multiorgan dysfunction progressing to multiorgan failure. Dysoxia can be the result of an inadequate supply of O2, which results in tissue hypoxia, or it can be caused by a defect in oxygen utilization in the mitochondria, which is called cytopathic hypoxia (6,7). Tissue hypoxia is the mechanism of organ injury in hypovolemic and cardiogenic shock (6), whereas cytopathic hypoxia is operative in severe sepsis and septic shock (7). As demonstrated in Figure 10.3, the DO) and VO> play an important role in determining the energy yield from nutrient metabolism. The remainder of this section will describe how the DO; and VO> are derived, and how the relationship between DO> and VO> can be used to evaluate the state of tissue oxygenation. These parameters require a measurement of cardiac output, which can be obtained using the thermodilution technique (described in Chapter 8) or a variety of noninvasive techniques (de-scribed in Reference 8). The normal range of values for the O> transport parameters are shown in Table 10.2.
FIGURE 10.3 Illustration of the factors that determine the energy yield from glucose metabolism. When the rate of oxygen uptake (VO2) into the tissues is unable to match the metabolic rate (MR), glucose metabolism is diverted to lactate production, and the energy yield drops dramatically. Abbreviations: DO? = rate of O2 delivery; HbO2 = oxygenated hemoglobin; ATP = adenosine triphosphate.

Cardiac Output, 1 = 5-6 L/min. Cardiac Output, 2 = 2.4— 4.0 Limin/m?. Oy Delivery, 1 = 900-1100 mL/min. Oy Delivery, 2 = 520-600 mL/min/m2. Oy Uptake, 1 = 200-270 mL'min. Oy Uptake, 2 = 110-160 mL/min’m2. ©, Extraction Ratio, 1 = 0,20-0.30. ©, Extraction Ratio, 2 = 
tSize-adjusted values ara the absolute values divided by the patient's body surface araa in square moaters {m2}.

The rate of O> transport from the heart to the systemic capillaries is called the oxygen delivery (DO3), and is a function of the cardiac output (CO) and the O> content of arterial blood (CaO) (9).
DO, =CO x CaQ, x 10 (mL/min)
(10.7)
(The multiplier of 10 is used to convert the CaO, from mL/dL to mL/L.) If the CaO> is broken down into its components (1.34 x [Hb] x SaQ>), equation 10.7 can be rewritten as:
DO, = CO x (1.34 x [Hb] x $aQ,) x 10
(10.8)
Three measurements are needed to calculate the DO >: cardiac output, hemoglobin concentration, and arterial O> saturation. The DO, in healthy adults at rest is 900-1100 mL/min, or 500-600 mL/min/m2 when adjusted for body size (see Table 10.2).

The rate of O> transport from the systemic capillaries into the tissues is called the oxygen uptake (VO). Since oxygen is not stored in tissues, the VO> is also a global measure of the oxygen consumption of metabolizing tissues. The VOz can be described as the product of the cardiac output (CO) and the difference between arterial and venous O> content (CaO> — CvO>).
VO, = CO x (CaO, -CvO,) x 10 (mL/min)
(10.9)
(The multiplier of 10 is included for the same reason as explained for the DO>.) This equation is a modified version of the Fick equation for cardiac output (CO = VO2/CaQO> — CvO>); using this equation to calculate the VO, is called the reverse Fick method (10). The CaO, and CvO, in equation 10.9 share a common term (1.34 x [Hb]), so the equation can be restated as:
VO, = CO x 1.34 x [Hb] x (SaQ, -SvO,) x 10
(10.10)
Four measurements are required to calculate the VO»: the 3 measurements used for the DO> calculation, plus the O> saturation in "mixed" venous blood (SvQ>) in the pulmonary artery, which requires a pulmon-ary artery catheter. The VO > in healthy adults at rest is 200-300 mL/min, or 110-160 mL/min/m2 when adjusted for body size (see Table 10.2).

Each of the 4 measurements used to derive the VO> has an inherent variability, and these are shown in Table 10.3 (10-12). The variability of the calculated VO> is +18%, which is the summed variability of the component measurements. Therefore, the VO, that is calculated from the modified Fick equation must change by at least 18% for the change to be considered significant.

The calculated VO> from the modified Fick equation is not the whole body VO because it does not include the O» consumption of the lungs (10,13,14). Normally, the VOz of the lungs accounts for less than 5% of the whole body VO, (13), but it can make up 20% of the whole body VO> when there is inflammation in the lungs (which is common in ICU patients) (14).
WHOLE BODY VO>: The whole body VO, is measured by monitoring the QO, concentration in inhaled and exhaled gas. This requires a specialized in-strument equipped with an oxygen analyzer (such as the metabolic carts used by nutrition support services). The instrument is connected to the proximal airway (usually in intubated patients), and it records the VO as the product of minute ventilation (VE) and the fractional concentration of O2 in inhaled and exhaled gas (FiO2 and FeO).
The measured (whole body) VO, has a variability of 5% (10,12), which is much less than the variability of the calculated VO2, as shown in Table 10.3. The major drawback of the measured VO> is the need for specialized equipment and trained personnel, which is costly and limits the availability of the measurement.

Thermodilution, Variability = +10%. Hemoglobin Concentration, Variability = +2%. Qo Saturation of Hemoglobin, Variability = +2%. O, Content of Blood, Variability = +4%. CaQ, — CvO5, Variability = +8%. Calculated VO,, Variability = +18%. Measured VO>, Variability = £59
Fram Referances 10-12.

The two conditions associated with a low VO) are a decreased metabolic rate (hypometabolism) and inadequate tissue oxygenation resulting in anaerobic metabolism. Since hypometabolism is uncommon in ICU pa-tients, an abnormally low VO, (<200 mL/min or <110 mL/min/m2) can be used as evidence of inadequate tissue oxygenation. An example of this is shown in Figure 10.4, which shows serial measurements of cardiac index (CI), systemic O, uptake (VO), and serum lactate levels during the first postoperative day in a patient who underwent an abdominal aortic aneurysm repair. Note that the VO, is abnormally low throughout the study period, while the serum lactate began to rise above normal (>4 mM/L) at the 8th postoperative hour. The abnormally low VO> represents inadequate tissue oxygenation, as confirmed by the eventual rise in blood lactate levels. However, there is a lag time of 6 hours from the first evidence of a low VO; to the first evidence of an elevated lactate level. This indicates that the VO) may be a more sensitive marker of inadequate tissue oxygenation than the serum lactate level. Note that the cardiac index remains in the normal range despite the evidence of impaired tissue oxygenation, demonstrating the nonvalue of cardiac output monitoring for evaluating tissue oxygenation.
FIGURE 10.4 Serial measurements of cardiac index, systemic O2 uptake (VO2), and blood lactate levels in the postoperative period following an abdominal aortic aneurysm repair. The dotted lines indicate the upper or lower limits of normal for each measurement. The shaded area represents the oxygen debt.
OXYGEN DEBT: The shaded area in the VO> curve in Figure 10.4 shows the magnitude of the VO> deficit over time. The cumulative deficit in tissue oxygenation is called the oxygen debt, and clinical studies have shown a direct relationship between the size of the oxygen debt and the risk of multiorgan failure (15,16).

resulting in an increase in (SaOQz — SvO>) to almost 50%. The point where the VOz begins to decrease is the point where O> extraction is maximal (about 50%) and is unable to increase further. Beyond this point, decreases in DO) are accompanied by similar decreases in VO), indicating the onset of tissue hypoxia. Thus, the point where O> extraction is maximal is the anaerobic threshold.
FIGURE 10.5 Graph showing the relationship between O2 delivery (DO?) and O2 up-take (VO2). O2 extraction is represented by (SaO? — SvO2). See text for explanation.

The O> extraction can be monitored as the (SaO» — SvO>) as long as the SaQO> is above 90%. The SaQ> is monitored by pulse oximetry (described in Chapter 21) and the SvQ> is monitored with pulmonary artery catheters (or central venous catheters, as described later). The following general rules can be applied to the interpretation of (SaOz — SvO>). These interpretations are based on the assumption that the metabolic rate is normal or unchanging.
1. The normal (SaO> — SvO>) is 20% to 30%.
2. An increase in (SaQz — SvO5) above 30% indicates a decrease in O> delivery (i.e., usually anemia or a low cardiac output).
3. An increase in (SaQz — SvO>) that approaches 50% indicates either threatened or inadequate tissue oxygenation.
4.
A
decrease in
(SaOz
—
SvO>)
below
20%
indicates
a
defect in
O>
utilization in
tissues, which
is usually
When the
the result
SaQ>
as of
approaches described
next.
100%, cell
injury
O>
extraction in
can severe
be sepsis
monitored or
septic using
shock.
only the
SvO;,

1, VO <200 mL/min or <110 mL/min/m?
2. (Sa, — SvOz) 250%
3. SVO>5 <50%

1. Serum Lactate >2 mM/L (or =4 mM/L)
2. Arterial Base Deficit > 2 mM/L

The serum lactate level and arterial base deficit are readily available measurements that have both diagnostic and prognostic value. The lactate level is the superior measurement, as will be shown.
patients with elevated lactate levels (29). The lowest mortality rate occurred when lactate levels normalized within 24 hours, and the mortality rate increased dramatically when lactate levels did not normalize within 48 hours . This relationship between the rate of lactate clearance and the mortality rate has been observed in several studies (25,27,29,30), and occurs predominantly in patients with severe sepsis and septic shock. The rate of lactate clearance in these patients has greater prognostic value than the initial lactate level (25, 30). Lactate clearance can be incorporated into the early goals of management in patients with severe sepsis and septic shock (which are described in Chapter 14) because of the observation that a lactate clearance >10% in the first 6 hours after diagnosis is associated with improved survival (30).
FIGURE 10.6 Graphs showing the prognostic value of monitoring serum lactate levels. Graph on the left (from Reference 28) shows the association between initial lactate levels and in-hospital mortality rate, including the mortality rate in the first 3 days after the initial lactate. Graph on the right (from Reference 29) shows the association between the time for lactate levels to normalize (lactate clearance) and in-hospital mortality rate.

Lactate accumulation in sepsis is not the result of an inadequate supply of Oz, but instead appears to be related to the accumulation of pyruvate as a result of inhibition of pyruvate dehydrogenase (the enzyme that converts pyruvate to acetyl coenzyme A and moves glycolysis from the cytoplasm to the Krebs cycle in mitochondria) (31). Endotoxin and other components of bacterial cell walls have been implicated in the inhibition of this enzyme (31). This mechanism of lactate accumulation is consistent with the notion that a defect in O> utilization in mitochondria (i.e., cytopathic hypoxia) is the cause of cell injury in severe sepsis and septic shock (7), as mentioned earlier. The notion that tissue O> levels are not deficient in patients with severe sepsis and septic shock has important implications for the management of these patients (which is described in Chapter 14).

The association of elevated serum lactate levels with poor outcomes has created a perception that the lactate molecule has deleterious effects. This is unproven, and it is possible that lactate may have a beneficial role as an "adaptive fuel" in critically ill patients (32). The energy yield from the oxidative metabolism of lactate is equivalent to that of glucose, as shown in Table 10.5. The caloric density (kcal/g) of lactate and glucose are equivalent and, since one glucose molecule produces 2 lactate molecules, the energy yield from the complete oxidation (kcal/mole) of lactate and glucose are equivalent. There is evidence that lactate oxidation in the heart is increased in patients with septic shock (33), and evidence that lactate oxidation is an important source of energy in neuronal tissue subjected to hypoxia and ischemia (34). Therefore, it is possible that lactate is more friend than foe in critically ill patients.

Glucose, 1 = 180. Glucose, 2 = 673 kcal/mole. Glucose, 3 = 3.74 keal/g. Lactate, 1 = 30. Lactate, 2 = 326 kcal/mole. Lactate, 3 = 3.62 kcal/g. Lactate 2, 1 = 180. Lactate 2, 2 = 673 kcal/mole. Lactate 2, 3 = 3.62 keal/g

The "base deficit" is considered a more specific marker of metabolic acidosis than the serum bicarbonate (35), and is defined as the amount (in millimoles) of base that must be added to one liter of blood to raise the pH to 7.40 (at a PCO> of 40 mm Hg). Most blood gas analyzers determine the base deficit routinely using a PCO2/HCO3 nomogram, and the re-sults are included in the blood gas report. The normal arterial base deficit is <2 mmol/L; increases above 2 mmol/L are classified as mild (2 to 5 mmol/L), moderate (6 to 14 mmol/L), and severe (215 mmol/L).
Arterial base deficit has been a popular marker of impaired tissue oxygenation in acute surgical emergencies, especially trauma. Studies in trauma victims show a correlation between the magnitude of acute blood loss and the magnitude of elevation in arterial base deficit (36), and acute trauma resuscitation that normalizes the arterial base deficit within hours is associated with a favorable outcome (36). Based on these observations, normalization of the arterial base deficit is one of the end-points of trauma resuscitation (37).

When used as a marker of tissue oxygenation, the arterial base deficit is a surrogate measure of serum lactate levels. However, base deficit is not specific for lactate because it is influenced by other causes of metabolic acidosis (e.g., ketosis, renal insufficiency). In one study comparing base deficit and lactate levels in patients admitted to a surgical ICU (38), both were similar in predictive value on admission, but lactate was superior to base deficit for predicting outcome when serial measurements of both were followed after ICU admission. This observation, combined with the lack of specificity of arterial base deficit as a measure of lactate, indicates that the arterial base deficit offers no advantages over blood lactate levels in the evaluation of tissue oxygenation.

Near infrared spectroscopy (NIRS) is a noninvasive method of measuring the venous O> saturation in tissues using the optical properties of hemoglobin in the oxygenated (HbO>) and dexoxygenated (Hb) state. This is described in Chapter 21 in relation to pulse oximetry. NIRS is essentially tissue oximetry without the "pulse" component. A light source is placed on the skin that emits light with wavelengths specific for HbO2 (990 nm) and Hb (660 nm), and the light of each wavelength that is reflected back from the underlying tissues (subcutaneous tissue and muscle) is picked up by a photodetector and processed to display the tissue O> saturation (StO>):
StO, = HbO,/HbO, + Hb

The StO> includes the O> saturation in arteries, capillaries, and veins within the tissue, but most of the blood in tissues (70-75%) is in veins, so the StO, is assumed to be a measure of the venous O> saturation in the underlying tissue, which is then used as a measure of the balance between O> delivery and O> consumption in that tissue (i.e., equation 10.18 for tissues instead of the whole body). It has been used mostly in the brain and skeletal muscle (39), and is not without problems; e.g., several other factors can influence StOz, such as skin color, tissue thickness and composition, and myoglobin (for muscle), and a few studies indicate 50% to 100% of the NIRS signal from skeletal muscle is from myoglobin (40). This is one of the problems with NIRS; i.e., you are not sure what is being measured.

The importance of monitoring systemic or tissue oxygenation is based on the premise that inadequate tissue oxygenation is responsible for cell in-jury, multiorgan failure, and fatal outcomes in critically ill patients. This premise is difficult to evaluate because of the inability to directly measure tissue O> levels. Increased serum lactate levels have been used as evidence of inadequate tissue oxygenation in critically ill patients, but the elevated lactate levels in septic shock are not the result of limited O> availability in tissues (31), as described earlier in the chapter. In fact, the consensus opinion is that inflammatory cell injury is the culprit responsible for multiorgan failure and fatal outcomes in septic shock (see Chapter 14). Since septic shock is the leading cause of death in ICUs, it seems that inadequate tissue oxygenation is not as important as we think it is in critically ill patients. This, of course, has obvious implications for the current emphasis on promoting tissue oxygenation in critical care management.

1. Nunn JF Oxygen. In Nunn's Applied Respiratory Physiology. 4th ed. London: Butterworth-Heinemann Ltd, 1993:247-305.
2. McConn R, Derrick JB. The respiratory function of blood: transfusion and blood storage. Anesthesiology 1972; 36:119-127.
3. Zander R. Calculation of oxygen concentration. In: Zander R, Mertzlufft F, eds. The oxygen status of arterial blood. Basel: S. Karger, 1991:203-209.
4. Christoforides C, Laasberg L, Hedley-Whyte J. Effect of temperature on solubility of O> in plasma. J. Appl Physiol 1969; 26:56-60.

19:231-243.. 16., 1 = Shoemaker WC, Appel PL, Krom HB. Role of oxygen debt in the development of organ failure, sepsis, and death in high-risk surgical patients. Chest 1992; 102:208- 215.. 17., 1 = Leach RM, Treacher DF. The relationship between oxygen delivery and consumption. Disease-a-Month 1994; 30:301-368.. 18., 1 = Maddirala S, Khan A. Optimizing hemodynamic support in septic shock using central venous and mixed venous oxygen saturation. Crit Care Clin 2010; 26:323-333.. 19., 1 = Noll ML, Fountain RL, Duncan CA, et al. Fluctuations in mixed venous oxygen saturation in critically ill medical patients: a pilot study. Am J Crit Care 1992; 3:102- 106.. 20., 1 = Krafft P, Stelzer H, Heismay M, et al. Mixed venous oxygen saturation in critically ill septic shock patients. Chest 1993; 103:900-906.. 21., 1 = Reinhart K, Kuhn H-J, Hartog C, Bredle DL. Continuous central venous and pulmonary artery oxygen saturation monitoring in the critically ill. Intensive Care Med 2004; 30:1572-1578.
22., 1 = Dueck MH, Kilmek M, Appenrodt S, et al. Trends but not individual values of central venous oxygen saturation agree with mixed venous oxygen saturation during varying hemodynamic conditions. Anesthesiology 2005; 103:249-257.. 23., 1 = Pope JV, Jones AE, Gaieski DF, et al. Multicenter study of central venous oxygen saturation (ScvO 2 ) as a predictor of mortality in patients with sepsis. Ann Emerg Med 2010; 55:40-46.. 24., 1 = Dellinger RP, Levy MM, Carlet JM, et al. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2008. Crit Care Med 2008; 36:296-327.

39. Boushel R, Piantadosi CA. Near-infrared spectroscopy for monitoring muscle oxygenation. Acta Physiol Scandinav 2000; 168:615-622.
40. Ward KR, Ivatury RR, Barbee RW, et al. Near infrared spectroscopy for evaluation of the trauma patient: a technological review. Resuscitation 2006; 27-64.
41. Cooper CE, Springett R. Measurement of cytochrome oxidase and mitochondrial energetics by near-infrared spectroscopy. Phil Trans R Soc Lond 1997; 352:669-676.

Variability is the law of life...no two individuals react alike or behave alike under the abnormal conditions which we know as disease.
Sir William Osler
On the Educational Value of the Medical
Society
1903

Hippocrates
The human circulatory system operates with a small volume and a volume-responsive stroke pump. This is an energy-efficient design that limits the workload of the heart, but the system quickly falters when volume is removed. While most internal organs like the lungs, liver, and kidneys can lose as much as 75% of their functional mass without lifethreatening organ failure, loss of only 35% to 40% of the blood volume can be fatal. This intolerance of the circulatory system to loss of blood volume is the dominant concern in the bleeding patient.
This chapter describes the evaluation and management of hypovolemia, with emphasis on acute blood loss, and includes a section on damage-control resuscitation for the severely injured patient.

The volume of selected body fluids in adults is shown in Table 11.1. Total body fluid accounts for about 60% of the lean body weight in males (600 mL/kg) and 50% of the lean body weight in females (500 mL/kg). An average sized adult male who weighs 75 kg (165 Ibs) will thus have 0.6 x 75=45 liters of total body fluid, and an average sized adult female who weighs 60 kg (132 Ibs) will have 0.5 x 60=30 liters of total body fluid. The volume of blood accounts for 6% to 7% of body weight (66 mL/kg in males, and 60 mL/kg in females) (1). As shown in Table 11.1, the blood volume is 5 liters in an average sized male, and only 3.6 liters in an average sized female. Comparing the volumes of blood and total body fluid indicates that blood represents only 11-12% of total body fluid. The meager distribution of total body fluid in the vascular compartment is an important factor in the intolerance to blood loss.

Total Body Fluid, 1 = 600. Total Body Fluid, 2 = 45L. Total Body Fluid, 3 = 500. Total Body Fluid, 4 = 30 L. Interstitial Fluid, 1 = 150. Interstitial Fluid, 2 = 1163L. Interstitial Fluid, 3 = 125. Interstitial Fluid, 4 = 1.5L. Blood, 1 = 66. Blood, 2 = SL. Blood, 3 = 60. Blood, 4 = 3.6L. Red Cell, 1 = 26. Red Cell, 2 = 2L. Red Cell, 3 = 24. Red Cell, 4 = TAL. Plasma, 1 = 40. Plasma, 2 = oL. Plasma, 3 = 36. Plasma, 4 = 2.2L
tLesn bedy weight for an average sized adut male and fama.
Volume of blood, rad cells, and plasma {in mL'kg) are from Reference 1.

Extracellular fluid accounts for about 40% of the total body fluid, and is composed of extravascular (interstitial) and intravascular (plasma) fluid compartments. Comparison of interstitial fluid and plasma volumes in Table 11.1 shows that plasma volume is about 25% of interstitial fluid volume. This relationship is important for understanding the volume effects of sodium-based (saline) fluids; i.e., since sodium equilibrates throughout the extracellular fluid, 75% of infused saline solutions will distribute in the interstitial fluid, and 25% will distribute in the plasma. Thus, the principal effect of saline solutions is to enhance the interstitial fluid volume, not the plasma volume (2).

The importance of an accurate assessment of the intravascular volume is matched by the difficulties encountered, and the clinical evaluation of intravascular volume is so flawed that it has been called a "comedy of errors" (7).

The changes in pulse rate and blood pressure that occur in acute hypovolemia are listed in Table 11.2, along with the reported sensitivities and specificities at two levels of blood loss (8,9). Supine tachycardia and hypotension are absent in a large majority of patients with blood volume deficits up to 1.1 liters (up to a 25% loss of blood volume in average sized males). The absence of tachycardia is contrary to traditional beliefs, yet bradycardia may be more prevalent in patients with acute blood loss (8).
Supine Tachycardia!, Sensitivity/Specificity._ _ ‘Moderate Blood Loss (450-630 mL)jt = 0/ 96%. Supine Tachycardia!, Sensitivity/Specificity.- Severe Blood Loss. (630-1150 mL)}5 = 12% / 96%. Supine Hypotension?, Sensitivity/Specificity._ _ ‘Moderate Blood Loss (450-630 mL)jt = 13% / 97%. Supine Hypotension?, Sensitivity/Specificity.- Severe Blood Loss. (630-1150 mL)}5 = 33% / 97%. Postural Pulse Increment?, Sensitivity/Specificity._ _ ‘Moderate Blood Loss (450-630 mL)jt = 22% / 98%. Postural Pulse Increment?, Sensitivity/Specificity.- Severe Blood Loss. (630-1150 mL)}5 = 97% / 98%. Postural Hypotension*, Sensitivity/Specificity._ _ ‘Moderate Blood Loss (450-630 mL)jt = O76 / 94% /. Postural Hypotension*, Sensitivity/Specificity.- Severe Blood Loss. (630-1150 mL)}5 = Not studied Not studied. Postural Hypotension*, Sensitivity/Specificity._ _ ‘Moderate Blood Loss (450-630 mL)jt = 27% 86%. Postural Hypotension*, Sensitivity/Specificity.- Severe Blood Loss. (630-1150 mL)}5 = 

1Pulse rate >100 beats/min; Systolic pressure <95 mm Ha; increase in pulse rate = 30 beats/min:
4Decreasa in systolic pressure > 20 mm Hg.
tEquivalent to a 10-12.5% lees of blood volume in.an average sized adut mala.
SEquivalent to a 12.5-25% loss of blood in an averaga sized adult maa.
From Raferancas 8 and 9.

Moving from the supine to the standing position causes a shift of 7 to 8 mL/kg of blood to the lower extremities (8). In healthy subjects, this change in body position is associated with a small increase in heart rate (about 10 beats/min) and a small decrease in systolic blood pressure (about 3 to 4 mm Hg). These changes can be exaggerated in hypovolemia. The expected postural changes in hypovolemia include an increment in pulse rate of at least 30 beats/minute, and a decrease in systolic blood pressure that exceeds 20 mm Hg. As shown in Table 11.2, these postural changes are uncommon when blood loss is less than 630 mL (<12% decrease in blood volume), but above this level, the postural pulse increment is a sensitive and specific marker of acute blood loss (as indicated by the boxed numbers in Table 11.2).
In summary, vital signs provide little benefit in the evaluation of hypovolemia, particularly in excluding the diagnosis. Supine hypotension may suggest the presence of profound hypovolemia, but this condition should be accompanied by other more reliable markers of severe volume loss (e.g., diminished urine output, elevated serum lactate levels).

The use of the hematocrit (and hemoglobin concentration) to evaluate the presence and severity of acute blood loss is both common and inappropriate. Changes in hematocrit show a poor correlation with blood volume deficits and erythrocyte deficits in acute hemorrhage (10), and the reason for this discrepancy is demonstrated in Figure 11.1. Acute blood loss involves the loss of whole blood, which results in proportional decreases in the volume of plasma and erythrocytes. As a result, acute blood loss results in a decrease in blood volume, but not a decrease in hematocrit. (There is a small dilutional effect from transcapillary refill in acute blood loss, but this is usually not enough to cause a significant decrease in hematocrit.) In the absence of volume resuscitation, the hematocrit will eventually decrease because hypovolemia activates the renin-angiotensinaldosterone system, and the renal retention of sodium and water that follows will have a dilutional effect on the hematocrit. This process begins 8 to 12 hours after acute blood loss, and can take a few days to become fully established.

The influence of fluid resuscitation on the hematocrit is demonstrated in Figure 11.1. Infusion of isotonic saline augments the plasma volume but not the red cell volume, resulting in a dilutional decrease in the hematocrit. All asanguinous fluids (i.e., colloid and crystalloid fluids) have a similar dilutional effect on the hematocrit (11), and the volume of fluid infused will determine the magnitude of the decrease in hematocrit. Resuscitation with erythrocyte-containing fluids will have a different effect. This is demonstrated in Figure 11.1 using whole blood as the resuscitation fluid. In this situation, erythrocyte and plasma volumes are increased proportionately, so there is no change in hematocrit. This demonstrates how, in the early hours after acute blood loss, the hematocrit is a reflection of the resuscitation effort (the type and volume of fluids infused), and not the extent of blood loss.
FIGURE 11.1 Influence of acute hemorrhage and fluid resuscitation on blood volume and hematocrit. See text for explanation

Cardiac filling pressures (i.e., central venous pressure and pulmonary artery occlusion pressure) have traditionally played a prominent role in the evaluation of ventricular volume and circulating blood volume. However, neither role is justified because experimental studies have shown a poor correlation between cardiac filling pressures and ventricular end-diastolic volume (see Figure 9.3 in Chapter 9) (12), and even less of a correlation between cardiac filling pressures and circulating blood volume (13-15). The latter observation is demonstrated in Figure 11.2, which shows the relationship between paired measurements of central venous pressure (CVP) and circulating blood volume in a group of postoperative patients. The scattered distribution of the data points illustrates the lack of a significant relationship between the two measurements, which is confirmed by the correlation coefficient (r) and p value in the upper left corner of the graph. Similar results have been reported in other clinical studies (13,15). The consistent lack of correlation between CVP and blood volume measurements has prompted the recommendation that the CVP should never be used to make decisions regarding fluid management (13).
FIGURE 11.2 Scatter plot showing 112 paired measurements of circulating blood volume (CBV) and central venous pressure (CVP) in a group of postoperative patients. Correlation coefficient (R) and p value indicate no significant relationship between CVP and blood volume. Redrawn from Reference 14.

The systemic O> transport parameters are described in detail in Chapter 10. The typical pattern with hemorrhage or hypovolemia is a decrease in systemic O> delivery (DO2) with an increase in O> extraction (SaOz — SvO>). Systemic O> consumption (VO) is normal in cases of compensated hypovolemia (when the increase in QO) extraction fully compensates for the decrease in DO, as shown inFigure 10.5), and the VO, is abnormally low in cases of hypovolemic shock. It is usually not possible to monitor these parameters in cases of acute hemorrhage, and chemical markers of tissue dysoxia are used to determine if acute blood loss results in hemorrhagic shock.

Active hemorrhage can reduce systemic O> delivery to levels that are un-able to sustain aerobic energy metabolism. The resulting oxygen-limited energy metabolism, also known as dysoxia, is accompanied by enhanced production of lactic acid via anaerobic glycolysis. The clinical expression of this condition is hemorrhagic shock, which is characterized by an elevated lactate concentration in blood. Since this condition can appear after loss of only 30% of the blood volume (1.5 liters in an average sized male), cases of active hemorrhage are monitored routinely for evidence of hemorrhagic shock using serum lactate concentrations or arterial base deficit. These two markers of impaired tissue oxygenation are described in detail in Chapter 10, and are mentioned only briefly here.

As just mentioned, an elevated serum lactate level in the setting of acute blood loss is presumptive evidence of hemorrhagic shock. The possibility that lactate accumulation in low flow states is the result of reduced lactate clearance is not supported by clinical studies showing equivalent rates of lactate removal in healthy adults and patients with cardiogenic shock (16). Although the threshold for an elevated serum lactate level is 2 mM/L, lactate levels >4 mM/L are more predictive of increased mortality (17), soa threshold of 4 mM/L is often used to identify life-threatening elevations of serum lactate.
LACTATE CLEARANCE: According to the bar graphs in Figure 10.6 (Chapter 10), the mortality rate in critically ill patients is not only related to the initial lactate level, but is also a function of the rate of decline in lactate levels after treatment is initiated (lactate clearance). The panel on the right in Figure 10.6 indicates that mortality is lowest when lactate levels return to normal within 24 hours. In one study of trauma victims with hemorrhagic shock, there were no deaths when lactate levels returned to normal within 24 hours, while 86% of the patients died when lactate levels remained elevated after 48 hours (18). Therefore, normalization of lactate levels within 24 hours can be used as an end-point of resuscitation for hemorrhagic shock (see later).

Arterial base deficit is a non-specific marker of metabolic acidosis that was adopted as a surrogate measure of lactic acidosis because of its availability in blood gas reports. However, lactate-specific analyzers are now routinely available that provide lactate measurements within a few minutes, and this obviates the need for arterial base deficit in the evaluation and management of hemorrhagic shock.

Concern about the liberal use of fluids in critical care management ( which creates risks without apparent rewards) led to the practice of evaluating patients for fluid responsiveness before infusing fluids empirically. This practice is not aimed at uncovering occult hypovolemia, but is an attempt to limit volume therapy to those who are likely to respond. It is primarily intended for patients who have an uncertain intravascular volume and are hemodynamically unstable. Mechanical methods of modulating cardiac preload have been proposed for evaluating fluid responsiveness (19), but these methods can be problematic, and fluid challenges remain the recommended method for evaluating fluid responsiveness (20).

There is no standard protocol for fluid challenges. The principal concern is to ensure that the fluid challenge will increase ventricular preload (i.e., end-diastolic volume), and the rate of infusion is more important than the volume infused for achieving this goal (21). The fluid challenge favored in clinical studies is 500 mL of isotonic saline infused over 1015 minutes (22). Fluid responsiveness is evaluated by the response of the cardiac output (which can be measured noninvasively using Doppler ultrasound techniques). An increase in cardiac output of at least 12-15% after a fluid challenge is used as evidence of fluid responsiveness (23). About 50% of critically ill patients are fluid responsive when tested in this manner (21,23). This percentage is much lower than expected, and may indicate that fluid challenges often fall short of augmenting ventricular preload.
PASSIVE LEG RAISING: Elevating the legs to 45° above the horizontal plane while in the supine position will move 150 mL to 750 mL of blood out of the legs and towards the heart (19), thereby serving as a "built-in" fluid challenge. This maneuver augments aortic blood flow within 30 seconds (22), and an increase in flow rate of 10-15% predicts fluid responsiveness with a sensitivity and specificity of 90% (23). Passive leg raising is recommended as an alternative to fluid challenges when volume restriction is desirable. It is not advised in patients with increased intra-abdominal pressure because the hemodynamic effects are attenuated or lost (24).

Blood volume measurements have traditionally required too much time to perform to be clinically useful in an ICU setting, but this has changed with the introduction of a semiautomated blood volume analyzer (Daxor Corporation, New York, NY) that provides blood volume measurements is less than an hour. The information in Figure 11.3 was provided by measurements obtained with this device in a surgical ICU (25). In this case, blinded measurements of blood, red cell, and plasma volumes were performed in patients with circulatory shock who were managed with pulmonary artery catheters, and the results show that blood and plasma volumes were considerably higher than normal. When blood volume measurements were made available for patient care, 53% of the measurements led to a change in fluid management, and this was associated with a significant decrease in mortality rate (from 24% to 8%) (25). These results will require corroboration, but they highlight the limitations of the clinical assessment of blood volume, and the potential for improved outcomes when blood volume measurements are utilized for fluid management.
FIGURE 11.3 Deviations from normal for blood, plasma, and red cell volumes in patients with circulatory shock being managed with pulmonary artery catheters. Data from Reference 25.

The steady flow of fluids through small, rigid tubes is described by the Hagen-Poiseuille equation shown below (26).
Q= AP (nrt/ BuL)
(11.1)
This equation states that steady flow (Q) through a rigid tube is directly related to the driving pressure (2P) for flow and the fourth power of the inner radius (r) of the tube, and is inversely related to the length (L) of the tube and the viscosity (°<) of the infusate. These relationships also de-scribe fluid flow through vascular catheters, as presented next.

There is a tendency to cannulate the large central veins for volume resuscitation because of the perception that larger veins allow more rapid infusion of fluids. However, infusion rates are determined by the dimensions of the catheter, not the size of the vein. The influence of catheter dimensions on flow rates is described in detail in Chapter 1. According to the Hagen Poiseuille equation, infusion rates will be higher in shorter or larger bore catheters. This is demonstrated in Figure 11.4, which shows the gravity-driven flow of water through short, peripheral catheters and a longer, triple-lumen central venous catheter. Flow in the peripheral catheters is at least 4 times greater than flow in the lumen of equivalent diameter in the central venous catheter. This demonstrates why short, large-bore peripheral catheters are preferred to central venous catheters for aggressive volume resuscitation.

The resuscitation of trauma victims sometimes requires infusion of more than 5 liters in the first hour (>83 mL/min) (27), and resuscitation using more than 50 liters in one hour has been reported (28). Because flow increases with the fourth power of the radius of a catheter, very rapid flow rates are best achieved with large-bore introducer sheaths used as conduits for pulmonary artery catheters (see Figure 8.1). These sheaths can be used as stand-alone infusion catheters, and are available in sizes of 8.5 French (2.7 mm outside diameter) or 9 French (3 mm outside diameter). Flow through introducer sheaths can reach 15 mL/sec (900 mL/min or 54 L/hr), which is only slightly less than the maximum flow (18 mL/sec) through standard (3 mm diameter) intravenous tubing (29). Some introducer sheaths have an additional side infusion port on the hub (see Figure 8.1), but the flow capacity of this port is only 25% of the flow capacity of the introducer sheath (29), so it should be bypassed for rapid infusion rates. Introducer sheaths are available without side infusion ports (e.g., Cook Access Plus, Cook Critical care), and these are preferred for rapid infusion rates.
FIGURE 11.4 The influence of catheter dimensions on the gravity-driven infusion rate of water. The triple-lumen central venous catheter (CVC) is a popular size (7 French, 20 cm in length). Flow rates from Table 1.2 and Table 1.3 in Chapter 1,

Whole blood is not available for replacement of blood loss, and erythrocyte losses are replaced with stored units of concentrated erythrocytes called packed red blood cells. Each unit of packed RBCs has a hematocrit of 55% to 60%, which imparts a high viscosity (see Table 9.2 for the relationship between hematocrit and blood viscosity). As a result, packed RBCs can flow sluggishly unless diluted with saline (as predicted in the HagenPoiseuille equation).
The influence of dilution with isotonic saline on the infusion rate of packed RBCs is shown in Figure 11.5 (30). When infused alone, the flow rate of packed RBCs through average sized (18-gauge or 20-gauge) peri-pheral catheters is 3-5 mL/min, which means that one unit of undiluted packed RBCs (which has a volume of about 350 mL) can be infused over 70-117 minutes (about 1-2 hours). This is sufficient for replacing erythrocyte losses in hemodynamically stable patients, but more rapid flow rates may be needed for patients with active bleeding. Figure 11.5 shows that dilution of packed RBCs with 100 mL of isotonic saline results in 7-fold to 8-fold increases in infusion rates, while dilution with 250 mL saline increases flow rates over 10-fold. At the highest infusion rate of 96 mL/min in the 16gauge catheter, one unit of packed RBCs (350 mL plus 250 mL saline) can be infused in 6-7 minutes. More rapid rates require pressurized infusions, which can increase infusion rates to 120 mL/min using a 16-gauge catheter (30).

The immediate goal of resuscitation for acute blood loss is to support oxygen delivery (DO) to vital organs. The determinants of DO, are identified in the following equation (the derivation of this equation is described in Chapter 10):
DO, = CO x (1.34 x [Hb] x SaQ,) x 10
(11.2)
Acute blood loss affects two components of this equation: cardiac output (CO) and hemoglobin concentration in blood, [Hb]. Therefore, the immediate goals of resuscitation are to promote cardiac output and maintain an adequate [Hb]. (Other goals will emerge as we proceed.)
we
FIGURE 11.5 Influence of dilution with isotonic saline on the gravity-driven infusion rate of erythrocyte concentrates (packed red blood cells) through peripheral catheters. Data from Reference 26.

The consequences of a low cardiac output are far more threatening than the consequences of anemia, so the first priority in the bleeding patient is to support cardiac output.

The different types of resuscitation fluids are shown in Table 11.3. The fluids used to promote cardiac output are crystalloid fluids and colloid fluids. Plasma is used to provide clotting factors, and is not used as a volume expander. The distinction between crystalloid and colloid fluids is briefly described below.
1. Crystalloid fluids are sodium-rich electrolyte solutions that distribute throughout the extracellular space, and these fluids expand the extracellular volume.
2. Colloid fluids are sodium-rich electrolyte solutions that contain large molecules that do not pass readily out of the bloodstream. The retained molecules hold water in the intravascular compartment; as a result, colloid fluids primarily expand the intravascular (plasma) volume.

Colloid Fluid, Products - = Albumin (5%, 2596) Hetastarch (695) extrans. Colloid Fluid, Principal Use or Result = Expands the plasma volume. Crystalloid Fluid, Products - = lsotonic Saline Ringer’s lactate Normosol. Crystalloid Fluid, Principal Use or Result = Expands the extracellular volume. RBC Concentrate, Products - = Packed RBC's. RBC Concentrate, Principal Use or Result = Increases O, content of blood. Stored Plasma, Products - = Fresh Frozen Plasma. Stored Plasma, Principal Use or Result = —s- Provides coagulation factors. Procoagulant Mixture, Products - = Cryoprecipitate. Procoagulant Mixture, Principal Use or Result = Low-volumeé source of fibrinogen. Platelet Concentrate, Products - = Pooled platelets Apharesis platelets. Platelet Concentrate, Principal Use or Result = Restores circulating platelet pool
The influence of different types of resuscitation fluids on cardiac output is shown in Figure 11.6 (31). The infusion volume of each fluid (except Ringer's lactate) is roughly the same. The colloid fluid (dextran-40) is clearly the most effective fluid for augmenting cardiac output, while the crystalloid fluid (Ringer's lactate) is only about 25% as effective, despite having twice the infusion volume. Packed RBCs are the least effective in promoting cardiac output, and have actually been shown to decrease cardiac output (32). This is due to the viscosity effect of the concentrated erythrocytes in packed RBCs, and this viscosity effect also explains why whole blood is less effective than the colloid fluid for augmenting cardiac output.
Figure 11.6 thus demonstrates that colloid fluids are much more effective than crystalloid fluids for promoting cardiac output.

The superiority of colloid fluids over crystalloid fluids for augmenting cardiac output is explained by the distribution of each fluid. Crystalloid fluids are primarily sodium chloride solutions, and sodium is distributed uniformly in the extracellular fluid. Because plasma represents only 25% of the extracellular fluid, only 25% of the infused volume of crystalloid fluids will remain in the vascular space and add to the plasma volume, while the remaining 75% will add to the interstitial fluid volume (33). Colloid fluids, on the other hand, have large molecules that do not readily escape from the bloodstream, and these retained molecules hold water in the intravascular compartment. As a result, as much as 100% of the infused volume of colloid fluids will remain in the vascular space and add to the plasma volume (33), at least in the first few hours after infusion. The increase in plasma volume augments cardiac output not only by increasing ventricular preload (volume effect) but also by decreasing ventricular afterload (dilutional effect on blood viscosity).
FIGURE 11.6 Change in cardiac index after a one-hour infusion of different resuscitation fluids. The infusion volumes are roughly equivalent (500 mL), except for Ringers lactate (1 liter). From Reference 31.

Despite the superiority of colloid fluids for increasing plasma volume and promoting cardiac output, crystalloid fluids have been the preferred resuscitation fluid for hemorrhagic shock for the past 50 years. The origins of this preference will be described in the next chapter; the principal reasons for the crystalloid preference are the low cost of crystalloid fluids and the lack of a documented survival benefit with colloid resuscitation (34). The favored crystalloid fluid is Ringer's lactate, which does not produce the metabolic acidosis that accompanies high-volume resuscitation with isotonic saline (see Figure 12.3).
Colloid fluids remain a reasonable choice in hypovolemia that is not associated with acute blood loss, particularly since ICU patients often have a low plasma oncotic pressure, which will be lowered further by crystalloid resuscitation. There is also much concern about the deleterious effects of high-volume crystalloid resuscitation, including edema formation in the lungs, heart, and intestinal tract, and the abdominal compartment syndrome (34). Chapter 12 has a more detailed comparison of colloid and crystalloid fluid resuscitation.

The standard practice for managing a trauma victim who presents with active bleeding or hypotension is to infuse 2 liters of crystalloid fluid over 15 minutes (35). If hypotension or bleeding continue, packed RBCs are infused along with crystalloid fluids to maintain a mean blood pressure >65 mm Hg. The volume of crystalloid resuscitation will be about 3 times the estimated volume of plasma loss, as shown in Table 11.4 (assume a >30% loss of blood volume in hemorrhagic shock). When bleeding is controlled and the patient is stable hemodynamically, the threshold for further RBC transfusions is a hemoglobin of 7 g/dL, or =9 g/dL in patients with active coronary artery disease (36).

1. Estimate normal blood volume (BV), ‘Methods = BV = 66 mL/kg (M) = 60 mL/kg (F). 2. Estimate 9% loss of blood volume, ‘Methods = Class I: <15% Class Il: 15-30% Class Ill: 90-45% Class IV: > 45%. 3. Calculate blood volume defect (BYD), ‘Methods = BVD = BV x % loss BY. 4. Calculate plasma volume deficit (PVD), ‘Methods = PVD = 0.6 x BVD. 6. Estimate resuscitation volume (RV), ‘Methods = RV = PVD x 1 (colloids) = PVD x 3 (crystalloids)

Because uncontrolled exsanguinating hemorrhage is the leading cause of death in hemorrhagic shock, the following practices are being adopted to limit the extent of bleeding in cases of massive blood loss (defined as the loss of one blood volume in 24 hours). These practices are part of an overall approach known as damage control resuscitation (37).

Observations with combat injuries and penetrating trauma have shown that aggressive volume replacement can exacerbate bleeding before the hemorrhage is controlled (34,37). This has led to an emphasis on permitting low blood pressures (i.e., systolic BP = 90 mm Hg or mean BP = 50 mm Hg) in trauma patients with hemorrhagic shock until the bleeding is controlled. This strategy has been shown to reduce resuscitation volumes (38,39), and increase survival rates (38). Low blood pressures are allowed only if there is evidence of adequate organ perfusion (e.g., patient is awake and follows commands).

FRESH FROZEN PLASMA: For the resuscitation of massive blood loss, the traditional practice has been to give one unit of fresh frozen plasma (FFP) for every 6 units of packed RBCs (34). However, the discovery that severely injured trauma victims often have a coagulopathy on presentation (40) has led to the practice of giving one unit of FFP for every one or two units of packed RBCs, and several studies have shown improved survival rates with this practice (34,37,41). Transfusion of FFP is aimed at maintaining an INR <1.5 and an activated PTT <1.5 times normal (42).
CRYOPRECIPITATE: Although FFP is a good source of fibrinogen (2-5 g/L), cryoprecipitate provides equivalent amounts of fibrinogen in a much smaller volume (3.24 grams in 150-200 mL, which is 2 "pools" of cryoprecipitate) (42). Therefore, cryoprecipitate can be used to maintain serum fibrinogen levels (>1 g/L) if volume control is desirable.
PLATELETS: The standard practice of giving one unit of platelets for every 10 units of packed RBCs has also been questioned, and improved survival rates have been recorded when one unit of platelets is given for every 2 to 5 units of packed RBCs (34). The optimal ratio of platelets to packed RBCs has yet to be determined, and platelet transfusions can be guided by the platelet count. The standard goal is to maintain a platelet count >50,000/°e<L when bleeding is active, but some advocate a platelet count >75,000/e¢L until bleeding is controlled (42).

Severe trauma is accompanied by loss of thermoregulation, and trauma-related hypothermia (body temp <32° C) is associated with increased mortality, possibly from reduced activity of coagulation factors and platelets (37). Since hypothermia is a risk with infusion of roomtemperature fluids and cold blood products (stored at 4° C), in-line fluid warmers are used routinely for the resuscitation of massive blood loss (28). The use of warming blankets and in-line fluid warmers has reduced the incidence of hypothermia to <1% in combat support hospitals (37).

A summary of the general goals and associated end-points of resuscitation for hemorrhagic shock are shown in Figure 11.7. (Some of these end-points, such as the cardiac output and the O> transport parameters, are not routinely available.) Limiting blood loss and prompt reversal of tissue ischemia are the most important considerations, and normalization of serum lactate levels within 24 hours is one of the most important end-points.

The restoration of blood pressure and hemoglobin levels in hemorrhagic shock does not ensure a Satisfactory outcome because it can be followed in 48-72 hours by progressive multiorgan failure (43). The earliest manifestation of postresuscitation injury is progressive respiratory dysfunction from the acute respiratory distress syndrome (described in Chapter 23), and this can be followed within 5-6 days by progressive dysfunction involving the kidneys, liver, heart, and central nervous system. The mortality rate is determined by the number of organs involved, and averages 50-60% (43).

Postresuscitation injury is a form of reperfusion injury (44) that is believed to originate in the splanchnic circulation, where reperfusion of ischemic bowel releases proinflammatory cytokines that gain access to the systemic circulation and activate circulating neutrophils, which adhere to the walls of capillaries and migrate into the parenchyma of vital organs to incite inflammatory injury (43). (This is the underlying mechanism for the multiorgan failure associated with sepsis, as described in Chapter 14.)
FIGURE 11.7 The general goals and associated end-points of resuscitation for hemorrhagic shock. CI = cardiac index; MABP = mean arterial blood pressure; DO? = systemic O2 delivery; [Hb] = hemoglobin concentration in blood; VO? = systemic O2 consumption; SvO7 = mixed venous O9 saturation; INR = international normalized ratio; aPTT = activated partial thromboplastin time.
Several factors predispose to postresuscitation injury, including the time required to reverse tissue ischemia (e.g., lactate clearance >24 hours), the volume of crystalloid resuscitation (i.e., predisposes to the abdominal compartment syndrome), the number of units of RBCs transfused (>6 units in 12 hrs), and the age of the transfused blood (stored >3 weeks) (43). Infection may be involved if the onset of multiorgan failure is more than 3 days after the resuscitation effort (43).

There is no specific therapy for postresuscitation injury, and preventive measures such as rapid reversal of ischemia, limiting the resuscitation volume (both crystalloid fluids and RBC products) and avoiding the transfusion of old blood, if possible, are advised. In lateonset multiorgan failure (onset >72 hrs after resuscitation), recognition and prompt treatment of underlying sepsis is crucial.

The following points in this chapter deserve emphasis:
1. The clinical evaluation of intravascular volume, including the use of central venous pressure (CVP) measurements, is so flawed it has been called a "comedy of errors" (7).
. Direct measurements of blood volume are clinically feasible, but are underutilized .
. Colloid fluids are much more effective than crystalloid fluids for expanding the plasma volume and promoting cardiac output, yet crystalloid fluids are preferred for the resuscitation of hemorrhagic shock because of cost considerations and the lack of a survival benefit with colloid resuscitation.
. In the severely injured patient, damage control resuscitation incorporates alternative strategies like hypotensive resuscitation (maintaining a lower-than-usual blood pressure until bleeding is controlled) and hemostatic resuscitation (giving fresh frozen plasma and platelets more frequently than usual).
. Return of serum lactate levels to normal within 24 hours is the end-point of resuscitation that is the most predictive of a satisfactory outcome.
. Multiorgan failure can appear 48—72 hours after resuscitation of hemorrhagic shock as a result of reperfusion-induced systemic inflammation.

1. Walker RH (ed). Technical Manual of the American Association of Blood Banks. 10th ed., Arlington, VA: American Association of Blood Banks, 1990:650.
2. Moore FD, Dagher FJ, Boyden CM, et al. Hemorrhage in normal man: I. Distribution and dispersal of saline infusions following acute blood loss. Ann Surg 2966; 163:485504.
. Moore FD. Effects of hemorrhage on body composition. New Engl J Med 1965; 273:567-577.
. American College of Surgeons. Advanced Trauma Life Support Manual, 7th ed. Chicago, IL: American College of Surgeons, 2004.
. Schadt JC, Ludbrook J. Hemodynamic and neurohumoral responses to acute hypovolemia in conscious animals. Am J Physiol 1991; 260:H305—-H318.
. Fiddian-Green RG. Studies in splanchnic ischemia and multiple organ failure. In Marston A, Bulkley GB, Fiddian-Green RG, Haglund UH, eds. Splanchnic ischemia and multiple organ failure. St. Louis, CV Mosby, 1989:349-364.
24. Mahjoub Y, Touzeau J, Airapetian N, et al. The passive leg-raising maneuver cannot accurately predict fluid responsiveness in patients with intra-abdominal hypertension. Crit Care Med 2010; 36:1824—1829.
25. Yu M, Pei K, Moran S, et al. A prospective randomized trial using blood volume analysis in addition to pulmonary artery catheter, compared with pulmonary artery catheter alone to guide shock resuscitation in critically ill surgical patients. Shock 2011; 35:220-228.

26., 1 = Chien S, Usami S, Skalak R. Blood flow in small tubes. In Renkin EM, Michel CC (eds). Handbook of Physiology. Section 2: The cardiovascular system. Volume IV. The microcirculation. Bethesda: American Physiological Society, 1984:217-249.. 27., 1 = Buchman TG, Menker JB, Lipsett PA. Strategies for trauma resuscitation. Surg Gynecol Obstet 1991; 172:8-12.. 28. Barcelona SL, Vilich F, Cote CJ. A comparison of flow rates and warming capabilities of the Level 1 and Rapid Infusion Systems with various-size intravenous catheters. Aneth Analg 2003; 97:358-363., 1 = 28. Barcelona SL, Vilich F, Cote CJ. A comparison of flow rates and warming capabilities of the Level 1 and Rapid Infusion Systems with various-size intravenous catheters. Aneth Analg 2003; 97:358-363.. 29. Hyman SA, Smith DW, England R, et al. Pulmonary artery catheter introducers: Do the component parts affect flow rate? Anesth Analg 1991; 73:573-575., 1 = 29. Hyman SA, Smith DW, England R, et al. Pulmonary artery catheter introducers: Do the component parts affect flow rate? Anesth Analg 1991; 73:573-575.. 30. de la Roche MRP, Gauthier L. Rapid transfusion of packed red blood cells: effects of dilution, pressure, and catheter size. Ann Emerg Med 1993; 22:1551-1555., 1 = 30. de la Roche MRP, Gauthier L. Rapid transfusion of packed red blood cells: effects of dilution, pressure, and catheter size. Ann Emerg Med 1993; 22:1551-1555.

- 43. Dewar D, Moore FA, Moore EE, Balogh Z. Postinjury multiorgan failure. Injury 2009; 40:912-918.
44. Eltzschig HK, Collard CD. Vascular ischaemia and reperfusion injury. Br Med Bull 2004; 70:71-86.

The secret of science is to ask the right question.
Sir Henry Tizard
In 1861, Thomas Graham's investigations on diffusion led him to classify substances as crystalloids or colloids based on their ability to diffuse through a parchment membrane. Crystalloids passed readily through the membrane, whereas colloids (from the Greek word for glue) did not. Intravenous fluids are similarly classified based on their ability to pass through capillary walls that separate the intravascular and interstitial fluid compartments (see Figure 12.1). This chapter presents the variety of crystalloid and colloid fluids available for use, and describes the salient features of these fluids, both individually and as a group.

Crystalloid fluids are electrolyte solutions with small molecules that can diffuse freely from intravascular to interstitial fluid compartments. The principal component of crystalloid fluids is sodium chloride. Sodium is the principal determinant of extracellular volume, and is distributed uniformly in the extracellular fluid. The sodium in crystalloid fluids also distributes uniformly in the extracellular fluid. Because the plasma volume is only 25% of the interstitial fluid volume (see Table 11.1), only 25% of an infused crystalloid fluid will expand the plasma volume, while 75% of the infused volume will expand the interstitial fluid. Thus, the predominant effect of crystalloid fluids is to expand the interstitial volume, not the plasma volume.
FIGURE 12.1 Illustration depicting the different tendencies of colloid and crystalloid fluids to expand the plasma volume and the interstitial fluid volume. See text for further explanation.

One of the most widely used crystalloid fluids is 0.9% sodium chloride (1), with annual sales of 200 million liters in the United States (data from Baxter Healthcare). This solution has a variety of names, including normal saline, physiologic saline, and isotonic saline, none of which is appropriate (see next). The term used for 0.9% NaCL in this text is isotonic saline, to distinguish it from hypertonic saline (described later).

The most popular term for 0.9% NaCL is normal saline, but this solution is neither chemically nor physiologically normal. It is not normal chemically because the concentration of a one-normal (1 N) NaCL solution is 58 grams per liter (the combined molecular weights of sodium and chloride), while 0.9% NaCL contains only 9 grams of NaCL per liter It is not normal physiologically because the composition of 0.9% NaCL differs from the composition of extracellular fluid. This is shown in Table 12.1. When compared to plasma (extracellular fluid), 0.9% NaCL has a higher sodium concentration (154 vs. 140 mEq/L), a much higher chloride concentration (154 vs. 103 mEq/L), a higher osmolality (308 vs. 290 mOsm/L), and a lower pH (5.7 vs. 7.4). These differences can have deleterious effects on fluid and acid-base balance, as described next.

Ringer's Lactate, nee.Mg = -—. Ringer's Lactate, nee.Buffers = Lactate (28). Ringer's Lactate, pH = 6.5. Ringer's Lactate, es.(mOsm/L) = 273. Ringer's Acetate, nee.Na = 131. Ringer's Acetate, nee.CL = 109. Ringer's Acetate, nee.K = 4. Ringer's Acetate, nee.Gai = 3. Ringer's Acetate, nee.Mg = -. Ringer's Acetate, nee.Buffers = Acetate (28). Ringer's Acetate, pH = 6.7. Ringer's Acetate, es.(mOsm/L) = 275. seater A I, nee.Na = re. seater A I, nee.CL = ms. seater A I, nee.K = . seater A I, nee.Gai = . seater A I, nee.Mg = Ss. seater A I, nee.Buffers = oes. seater A I, pH = . seater A I, es.(mOsm/L) = ae
SConcentration of ionized calcium in mod.
tNot commercially available.

The effects of 0.9% NaCL on expanding the plasma volume and interstitial fluid volume are shown in Figure 12.2. Infusion of one liter of 0.9% NaCL adds 275 mL to the plasma volume and 825 mL to the interstitial volume (2). This is the distribution expected of a crystalloid fluid. However, there is one unexpected finding; i.e., the total increase in extracellular volume (1,100 mL) is slightly greater than the infused volume. This is the result of a fluid shift from the intracellular to extracellular fluid, which occurs because 0.9% NaCL is slightly hypertonic in relation to extracellular fluid, as shown in Table 12.1.
INTERSTITIAL EDEMA: Infusions of 0.9% NaCL promote interstitial edema more than crystalloid fluids with a lower sodium content (e.g., Ringer's lactate, Plasma-Lyte) (3). This is related to the increased sodium load from 0.9% NaCL, which increases the "tonicity" of the interstitial fluid (as just described) and promotes sodium retention by suppressing the renin-angiotensin-aldosterone axis (4). Decreases in renal perfusion have also been observed after infusion of 0.9% NaCL (3), presumably as a result of chloridemediated renal vasoconstriction. The increase in interstitial edema with 0.9% NaCL can have a negative influence on clinical outcomes (5).
FIGURE 12.2 The effects of selected colloid and crystalloid fluids on the plasma volume and interstitial fluid volume. The infusion volume of each fluid is shown in parentheses. Data from Reference 2.

Large-volume_ infusions of 0.9% NaCL produce ametabolic acidosis (6,7), as demonstrated in Figure 12.3. In this clinical study (6), infusion of isotonic saline (0.9% NaCL) at a rate of 30 mL/kg/h was accompanied by a progressive decline in the pH of blood (from 7.41 to 7.28) over two hours, while the pH was unchanged when Ringer's lactate solution was infused at a similar rate. The saline-induced metabolic acidosis is a hyperchloremic acidosis, and is caused by the high concentration of chloride in 0.9% saline relative to plasma (154 versus 103 mEq/L). The close match between the chloride concentration in Ringer's lactate solution and plasma (see Table 12.1) explains the lack of a pH effect associated with large-volume infusion of Ringer's lactate solution.
STRONG ION DIFFERENCE: The influence of crystalloid fluids on acid-base balance can also be explained using the strong ion difference (SID), which is the difference between readily dissociated (strong) cations and anions in extracellular fluid (8). (The SID of plasma is roughly equivalent to the plasma [Na] — plasma [CL] since these are the most prevalent strong ions in extracellular fluid. Plasma HCO3 is not included in the SID because HCO3 is not a strong ion.) The principle of electrical neutrality requires an equal concentration of cations and anions in the extracellular fluid, so the relationship between SID and the ions that dissociate from water (H+ and OH-) can be described as follows:
FIGURE 12.3 The effects of isotonic saline (0.9% NaCL) versus lactated Ringer's solution on the pH of blood in patients undergoing elective surgery. Total volume infused after 2 hours was 5 to 6 liters for each fluid. Data from Reference 6.
SID + [H*] -[OH-]=0
(12.1)
Since the [OH-] is negligible in the physiologic pH range, equation 12.1 can be rewritten as:
SID + [H*]=0
(12.2)

According to this relationship, a change in SID must be accompanied by a reciprocal change in [H+] (or a proportional change in pH) to maintain electrical neutrality. The relationship between the SID and pH of plasma is shown in Figure 12.4 (9). Note that the SID and pH change in the same direction (because pH is used instead of [H+]). The normal SID of plasma is 40 mEq/L (as indicated by the dotted line), which is roughly equivalent to the normal plasma [Nat] — [CL-] difference (140 — 103 mEq/L).
The SID of intravenous fluids determines their ability to influence the pH of plasma. The SID of 0.9% NaCL is zero (Na — CL=154 — 154=0) , so infusions of 0.9% NaCL will reduce the SID of plasma and thereby reduce the plasma pH. The SID of Ringer's lactate fluid is 28 mEq/L (Na + K + Ca — CL=130 + 4 + 3 -— 109=28) if all the infused lactate is metabolized. This SID is not far removed from the normal SID of plasma, so Ringer's

FIGURE 12.4 The relationship between the strong ion difference (SID) and the pH of extracellular fluid (plasma). The normal SID of plasma is about 40 mEq/L. The SID of a crystalbid fluid relative to plasma determines the tendency of the fluid to influence acid-base status. See text for further explanation. Graph redrawn from Reference 9.

Sydney Ringer, a British physician who studied the contraction of isolated frog hearts, introduced a sodium chloride solution in 1880 that contained calcium and potassium to promote cardiac contraction and cell viability (10). This solution is shown as Ringer's injection in Table 12.1, and is essentially 0.9% NaCL with potassium and ionized calcium added.

In the early 1930's, an American pediatrician named Alexis Hartmann added sodium lactate to Ringer's solution to provide a buffer for the treatment of metabolic acidosis (10). This solution was originally called Hartmann's solution, and is now known as Ringer's lactate solution. The composition of this solution is shown in Table 12.1. The sodium concentration in Ringer's lactate is reduced to compensate for the sodium released from sodium lactate, and the chloride concentration is reduced to compensate for the negatively-charged lactate molecule; both changes result in an electrically neutral salt solution.

Because of concerns that large-volume infusions of Ringer's lactate solution could increase plasma lactate levels in patients with impaired lactate clearance (e.g., from liver disease), the lactate buffer was replaced by acetate to create Ringer's acetate solution. Acetate is metabolized in muscle rather than liver (10), which makes Ringer's acetate a reasonable alternative to Ringer's lactate in patients with liver failure. (The influence of Ringer's lactate on serum lactate levels is described below). As shown in Table 12.1, the composition of Ringer's acetate and Ringer's lactate solutions is almost identical with the exception of the added buffer.

The principal advantage of Ringer's lactate and Ringer's acetate over isotonic saline (0.9% NaCL) is the lack of a significant effect on acid-base balance. The principal disadvantage of Ringer's solutions is the calcium content; i.e., the ionized calcium in Ringer's solutions can bind to the citrated anticoagulant in stored RBCs and promote clot formation. For this reason, Ringer's solutions are contraindicated as diluent fluids for the transfusion of erythrocyte concentrates (packed red blood cells) (11). However, clot formation does not occur if the volume of Ringer's solution does not exceed 50% of the volume of packed RBCs (12).
LACTATE CONSIDERATIONS: As mentioned above, the lactate content in Ringer's lactate solution (28 mM/L) creates concern about the risk of spurious hyperlactatemia with large-volume infusions of the fluid. In healthy subjects, infusion of one liter of lactated Ringer's over one hour does not raise serum lactate levels (8). In critically ill patients, who may have impaired lactate clearance from circulatory shock or hepatic insufficiency, the impact of lactated Ringer's infusions on serum lactate levels is not known. However, if lactate clearance is zero, the addition of one liter of lactated Ringer's to a blood volume of 5 liters (which would require infusion of 3—4 liters of fluid) would raise the serum lactate level by 4.6 mM/L (13). Therefore, lactated Ringer's infusions are unlikely to have a considerable impact on serum lactate levels unless large volumes are infused in patients with virtually no capacity for clearing lactate from the bloodstream. Blood samples obtained from intravenous catheters that are being used for lactated Ringer's infusions can yield spuriously high serum lactate determinations (14). Therefore, in patients receiving lactated Ringer's infusions, blood samples for lactate measurements should be obtained from sites other than the infusion catheter.

Two of the crystalloid fluids in Table 12.1 (i.e., Normosol and Plasma-Lyte) contain magnesium instead of calcium, and contain both acetate and gluconate buffers to achieve a pH of 7.4 These fluids are not as popular as isotonic saline or Ringer's lactate, but the absence of calcium makes them suitable as diluents for RBC transfusions, and PlasmaLyte has shown less of a tendency to promote interstitial edema when compared with isotonic saline (3,5).

Hypertonic saline solutions like 7.5% NaCL (which has an osmolality 8-9 times greater than plasma) are much more effective at expanding the extracellular volume than isotonic crystalloid fluids. This is demonstrated in Figure 12.2, which shows that infusion of 250 mL of 7.5% NaCL results in a 1,235 mL increase in extracellular fluid, which is about 5 times greater than the infusion volume. (The added volume comes from the intracellular fluid.) Animal studies have shown that hypertonic saline is effective for limited-volume resuscitation of hemorrhagic shock. This is demonstrated in Figure 12.5, which shows that hypertonic saline can restore and maintain cardiac output with 1/5 the volume required with isotonic saline (15).
FIGURE 12.5 A comparison of the cumulative volume of three intravenous fluids needed to maintain a normal rate of aortic blood flow in an animal model of hemorrhagic shock. Data from Reference 15.
Observations like the one in Figure 12.5 suggested that hypertonic saline would be well suited for situations where small volumes of resuscitation fluid are advantageous; e.g., the prehospital resuscitation of trauma victims, particularly those with traumatic brain injury (16). Unfortunately, the accumulated evidence shows no apparent survival benefit from hypertonic saline compared to isotonic crystalloids for the management of traumatic shock (17) or traumatic brain injury (18). The addition of 6% dextran-70 to hypertonic saline to make a hyperoncotic-hypertonic fluid has not improved the results (16.18,19). As a result, hypertonic resuscitation is currently in the graveyard of resuscitation strategies.

The once-popular use of 5% dextrose solutions (Ds solutions) has fallen out of favor, as explained in this section.

Prior to the standard use of enteral tube feedings and total parenteral nutrition (TPN), 5% dextrose solutions were used to provide calories in patients who were unable to eat. Dextrose provides 3.4 kilocalories (kcal) per gram when fully metabolized, so a 5% dextrose solution (50 grams dextrose per liter) provides 170 kcal per liter. Infusion of 3 liters of a Ds solution daily (125 mL/min) provides 3 x 170=510 kcal/day, which is enough nonprotein calories to limit the breakdown of endogenous proteins to provide calories (i.e., protein-sparing effect). This is no longer necessary, as most patients can tolerate enteral tube feedings, and those who cannot will receive TPN.

The addition of dextrose to intravenous fluids increases osmolality (50 g of dextrose adds 278 mOsm/L to an intravenous fluid). For a 5% dextrose-in-water solution (D;W), the added dextrose brings the osmolality close to that of plasma. However, since the dextrose is taken up by cells and metabolized, this osmolality effect rapidly wanes, and the added water then moves into cells. This is shown in Figure 12.2. The infusion of one liter of D5|W results in an increase in extracellular fluid (plasma plus interstitial fluid) of about 350 mL, which means the remaining 650 mL (two-thirds of the infused volume) has moved intracellularly. Therefore, the predominant effect of D5W is cellular swelling. An effect opposite to that of D;W can occur when dextrose is added to 0.9% NaCL. As described previously, the osmolality of 0.9% NaCL is slightly higher than extracellular fluid (308 vs. 290 mOsm/L), and this results in some movement of water out of cells. When 50 grams of dextrose is added to make Ds-normal saline, the osmolality of the fluid increases to 560 mOsm/L, which is almost twice the normal osmolality of the extracellular fluid. If glucose utilization is impaired (as is common in critically ill patients), largevolume infusions of D>W can result in cellular dehydration.

In healthy subjects, only 5% of an infused glucose load will result in lactate formation, but in critically ill patients with tissue hypoperfusion, as much as 85% of glucose metabolism is diverted to lactate production (20). This latter effect is demonstrated in Figure 12.6. In this case, tissue hypoperfusion was induced by aortic clamping during abdominal aortic aneurysm surgery (21). Patients received intraoperative fluids to maintain normal cardiac filling pressures using either a Ringer's solution or a 5% dextrose solution. When the dextrose-containing fluid was infused, the serum lactate levels began to rise after the aorta was cross-clamped, and the increase in circulating lactate levels persisted throughout the remainder of the surgery. These results indicate that, when circulatory flow is compromised, infusion of 5% dextrose solutions can result in lactic acid production and significant elevations of serum lactate.

About 20% of patients admitted to ICUs are diabetic (22), and as many as 90% of patients will develop hyperglycemia at some time during their ICU stay (23). Hyperglycemia has several deleterious effects in critically ill patients, including immune suppression (22), increased risk of infection (23), aggravation of ischemic brain injury (24), and increased mortality, particularly following cardiac surgery (23). Because of the association between hyperglycemia and increased morbidity and mortality, blood glucose levels are typically not allowed to remain above 180 mg/dL in ICU patients (25).
FIGURE 12.6 The effect of intravenous fluid therapy with and without dextrose on blood lactate kevels in patients undergoing abdominal aortic aneurysm repair. Each point represents the mean lactate level in 10 study patients. The average infusion volume for each fluid is indicated in parentheses. Data from Reference 21.
Considering the high risk of hyperglycemia in ICU patients, and the numerous adverse consequences of hyperglycemia, infusion of dextrose-containing fluids should be avoided whenever possible. In fact, considering the potential for harm, it seems that the routine use of 5% dextrose solutions should be abandoned in critically ill patients.

In chemical terms, a colloidal solution is a particulate solution with particles that do not dissolve completely. (These solutions are also called suspensions). In clinical terms, a colloid fluid is a saline solution with large solute molecules that do not pass readily from plasma to interstitial fluid. The retained molecules in a colloid fluid create an osmotic force called the colloid osmotic pressure or oncotic pressure that holds water in the vascular compartment, as reviewed next.

The direction and rate of fluid exchange (Q) between capillary blood and interstitial fluid is determined, in part, by the balance between the hydrostatic pressure in the capillaries (P.), which promotes the movement of fluid out of capillaries, and the colloid osmotic pressure of plasma (COP), which favors the movement of fluid into capillaries.
Q=P.-COP
(12.3)
In the supine position, the normal Pc averages about 20 mm Hg (30 mm Hg at the arterial end of the capillaries and 10 mm Hg at the venous end of the capillaries); the normal COP of plasma is about 28 mm Hg (26), so the net forces normally favor the movement of fluid into capillaries (which preserves the plasma volume). About 80% of the plasma COP is due to the albumin fraction of plasma proteins (26), so a reduction in the plasma albumin concentration (hypoalbuminemia) favors the movement of fluid out of the capillaries and promotes interstitial edema.

The volume distribution of colloid and crystalloid fluids can be explained by their influence on the plasma COP. Crystalloid fluids re-duce the plasma COP (dilutional effect), which favors the movement of these fluids out of the bloodstream. Colloid fluids can preserve the normal COP (iso-oncotic fluids), which holds these fluids in the bloodstream, or they can increase the plasma COP (hyperoncotic colloid fluids), which pulls interstitial fluid into the bloodstream.

The volume distribution of a (nearly iso-oncotic) colloid fluid is demonstrated in Figure 12.2. The colloid fluid in this case is a 5% albumin solution, which has a COP of 20 mm Hg. Infusion of one liter of this solution results in a 700 mL increment in the plasma volume and a 300 mL increment in the interstitial fluid volume. When compared with the increment in plasma volume after one liter of 0.9% NaCL (275 mL), the colloid fluid is about 3 times more effective in expanding the plasma volume than the crystalloid fluid. Most observations indicate that, in equivalent volumes, colloid fluids are at least three times more effective than crystalloid fluids for expanding the plasma volume (2, 27-29). As expected from Figure 12.2, colloid fluids promote cardiac output at much lower infusate volumes than required with crystalloid fluids. This is demonstrated in Figure 12.5 (described earlier), which shows that restoring the cardiac output in hemorrhagic shock requires five times more volume when a crystalloid fluid (isotonic saline) is infused instead of a colloid fluid (dextran-70).

As mentioned earlier, the ability of a colloid fluid to augment the plasma volume is determined by the COP of the fluid relative to the plasma COP. This is demonstrated in Table 12.2, which includes the commonly used colloid fluids in the United States, along with the oncotic pressure (COP) of each fluid and the increment in plasma volume produced by a given infusate volume. Note that the higher the COP of the fluid, the greater the increment in plasma volume relative to the infusate volume. Fluids with a COP of 20-30 mm Hg are considered to be iso-oncotic fluids (i.e., fluid COP equivalent to plasma COP); these fluids produce increments in plasma volume that are roughly equivalent to the infusate volume (range = 70—-130% of infusate volume). Colloid fluids with a COP >30 mm Hg are hyperoncotic fluids (fluid COP >plasma COP); these fluids produce increments in plasma volume that are usually greater than the infusate volume. This is most apparent with 25% albumin, which has a COP of 70 mm Hg, and produces an increment in plasma volume that is 3 to 4 times greater than the infusate volume.

25% Albumin, Average os = 69. 25% Albumin, —Oncotic. fae = 70. 25% Albumin, Anioar wus ‘Infusate Volume = 3.0-4.0. 25% Albumin, Duration of | Effect = 12h. 10% Dextran-40, Average os = 26. 10% Dextran-40, —Oncotic. fae = 40. 10% Dextran-40, Anioar wus ‘Infusate Volume = 1.0-1.5. 10% Dextran-40, Duration of | Effect = 6h. 6% Hetastarch, Average os = 450. 6% Hetastarch, —Oncotic. fae = 30. 6% Hetastarch, Anioar wus ‘Infusate Volume = 1.0-1.3. 6% Hetastarch, Duration of | Effect = 24h. 5% Albumin, Average os = 69. 5% Albumin, —Oncotic. fae = 20. 5% Albumin, Anioar wus ‘Infusate Volume = 0.7 -1.3. 5% Albumin, Duration of | Effect = 12h
Data from Refarances 2, 27-29, 38.

Albumin is a versatile plasma protein with several functions. It is the principal determinant of plasma COP (26), the principal transport protein in blood (see Table 12.3), has significant antioxidant activity (30), and helps maintain the fluidity of blood by inhibiting platelet aggregation (31). As much as 2/3 of the albumin in the body is located outside blood vessels (32); the role of the extravascular albumin pool is unclear.

Albumin solutions are heat-treated preparations of human serum albumin that are available as a 5% solution (50 g/L) and a 25% solution (250 g/L) in 0.9% NaCL. The 5% albumin solution is usually given in aliquots of 250 mL; the colloid osmotic pressure is 20 mm Hg, and the plasma volume increment averages 100% of the infused volume. The volume effect begins to dissipate at 6 hours, and can be lost after 12 hours (2,27).

Benzodiazepines Cephalosporine Furosemide NSAIDS Phenytoin Quinidine Salloylates Sulfonamides Vallproic Acid Warfarin, Others = Bilirubin Copper pai awe Fatty Acids Progesterone Prostaglandins Testosterone Zine
The 25% albumin solution is a hyperoncotic fluid with a colloid osmotic pressure of 70 mm Hg (more than twice that of plasma). It is given in aliquots of 5 0-100 mL, and the plasma volume increment is 3 to 4 times the infusate volume. The effect is produced by fluid shifts from the interstitial space, so interstitial fluid volume decreases as plasma volume increases. Because it does not replace lost volume, but instead shifts fluid from one compartment to another, 25% albumin should not be used for volume resuscitation in patients with blood loss. This fluid should be reserved for instances where hypovolemia is the result of hypoalbuminemia, which promotes fluid shifts from plasma to interstitial fluid.

Albumin's reputation was sullied in 1998 when a clinical review suggested that one of every 17 patients who received albumin infusions died as a result of the fluid (33). This has been refuted in subsequent studies showing that albumin poses no greater risk of death than other volume expanders (34,35). The consensus opinion at the present time is that 5% albumin is safe to use as a resuscitation fluid (32), except possibly in traumatic head injury, where one large study has shown a higher mortality rate in patients who received albumin instead of isotonic saline (36). Hyperoncotic (25%) albumin has been associated with an increased risk of renal injury and death in patients with circulatory shock (37), which is similar to the renal injury reported with other hyperoncotic colloid fluids (see next).

Hydroxyethyl starch (HES) is a chemically modified polysaccharide composed of long chains of branched glucose polymers substituted periodically by hydroxyl radicals (OH), which resist enzymatic degradation. HES elimination involves hydrolysis by amylase enzymes in the bloodstream, which cleave the parent molecule until it is small enough to be cleared by the kidneys. The following is a summary of the important features of HES preparations (32,38).

MOLECULAR WEIGHT: HES preparations have different molecular weights, and are classified as high MW (450 kilodaltons or kD), medium MW (200 kD), and low MW (70 kD). High MW preparations have a prolonged duration of action because amylase cleavage results in progressively smaller molecules that are osmotically active. When the cleavage products reach a molecular weight of 50 kD, they can be cleared by the kidneys (32).
MOLAR SUBSTITUTION RATIO: HES preparations are also classified by the ratio of hydroxyl radical substitutions per glucose polymer (OH/glucose), which is called the molar substitution ratio and ranges from zero to one (32). Since hydroxyl radicals resist enzymatic degradation, higher OH/glucose ratios are associated with prolonged activity. Higher molar substitution ratios increase the risk of HES-associated coagulopathy (see later).
INDIVIDUAL PREPARATIONS: Individual HES preparations are described by their concentration, MW, and molar substitution ratio, as shown in Table 12.4. Most preparations are available as 6% solutions in 0.9% NaCL. The prefix of the HES preparation indicates the molar substitution ratio (e.g., pentastarch = 0.5, tetrastarch = 0.4). Hetastarch is the most commonly used HES preparation in the United States, and has a high MW(450 kD) and a high molar substitution ratio (0.7). Tetrastarch is the most recent HES preparation introduced for use in the United States, and has the lowest MW (130 kD) and the lowest molar substitution ratio (0.4). Tetrastarch is available as Voluven (Hospira).

The performance of 6% HES solutions as plasma volume expanders is very similar to 5% albumin. The oncotic pressure is higher than 5% albumin, and the increment in plasma volume can be higher as well (see Table 12.2). The effect on plasma volume can last up to 24 hours with high MW preparations such as hetastarch (38). The duration of action of the lower MW preparations is at least 6 hours, but effects begin to dissipate within one hour (4).
Hetastarch, Concentration: = 6%. Hetastarch, MW = 450 kD. Hetastarch, _ (OH/Glucoss) = 0.7. Hexastarch, Concentration: = 6%. Hexastarch, MW = 200 kD. Hexastarch, _ (OH/Glucoss) = 0.6. Pentastarch, Concentration: = 6%, 109. Pentastarch, MW = 200 kD. Pentastarch, _ (OH/Glucoss) = 0.5. Tetrastarch, Concentration: = 6%. Tetrastarch, MW = 130 kD. Tetrastarch, _ (OH/Glucoss) = 0.4

HES can impair hemostasis by inhibition of Factor VII and von Wille-brand factor, and impaired platelet adhesiveness (32,39). This effect was originally attributed to high MW preparations, but high (OH/glucose) ratios are now considered more important in determining the risk of altered hemostasis (32). Clinically significant coagulopathies are uncommon unless large volumes of HES are infused (e.g., >50 mL/kg for tetra-starch) (28).

Several studies have shown an association between HES infusions and an increased risk of renal injury and death; this association has been reported with hetastarch (40), pentastarch (41), and tetrastarch (42). The colloid osmotic pressure of HES preparations (30 mm Hg for hetastarch and 36 mm Hg for tetrastarch) has been implicated in renal injury, although the precise mechanism is not clear. HES-associated renal injury has been reported mostly in patients with life-threatening conditions such as severe sepsis and circulatory shock (32,41,42). In patients who are less severely ill, there is no association between HES and renal injury (32), and some studies show a favorable responses to HES in such patients (43).

The amylase enzymes involved in the hydrolysis of HES attach to the HES molecules, and this reduces amylase clearance by the kidneys. This can result is an increase in serum amylase levels to 2—3 times above normal (38,44). Levels usually return to normal within one week after HES is discontinued. Serum lipase levels are unaffected by HES infusions (44).

There is a longstanding debate concerning the type of fluid that is most appropriate for volume resuscitation, and each type of fluid has its loyalists who passionately defend the merits of their chosen fluid. The following is a brief description of the issues involved in this debate, and a suggested compromise.

Early studies of acute blood loss in the 1960s showed that hemorrhagic shock was associated with an interstitial fluid deficit, partly as a result of a fluid shift from the interstitial fluid into the bloodstream (46). In an animal model of hemorrhagic shock, replacement of the shed blood was almost universally fatal, while survival improved significantly if Ringer's lactate fluid was added to the replacement of shed blood (47). These results were interpreted as indicating that replacement of the interstitial fluid deficit (with Ringer's lactate) was the critical factor in the successful resuscitation of hemorrhagic shock. This led to the popularity of crystalloid fluids for the resuscitation of blood loss. Therefore, crystalloid fluids were popularized for volume resuscitation because of their ability to resuscitate the interstitial volume, not the plasma volume.

Since those early studies, the importance of promoting cardiac output and systemic O> delivery have emerged as the primary focus of volume resuscitation. To this end, colloid fluids have proven superior to crystalloid fluids, as demonstrated in Figure 11.6 (Chapter 11). Despite this superiority, crystalloid fluids remain the popular choice for volume resuscitation (at least in the United States). The principal argument in favor of crystalloid resuscitation is the lack of proven survival benefit with colloid resuscitation (48,49), and the lower cost of crystalloid fluids (see Table 12.5). The problem with crystalloid resuscitation is the relatively large volumes needed to expand the plasma volume (at least 3 times greater than the volume of colloid fluids), which promotes edema formation and a positive fluid balance, both of which are associated with increased morbidity and mortality in critically ill patients (5,50).

Crystalloid Fluids, ‘Manufacturer = . Crystalloid Fluids, Unit = . Crystalloid Fluids, Size_=——_Unit Cost = . 0.9% NaCl, ‘Manufacturer = Baxter. 0.9% NaCl, Unit = 7,000 mL. 0.9% NaCl, Size_=——_Unit Cost = $1.95. Ringer's Lactate, ‘Manufacturer = Baxter. Ringer's Lactate, Unit = 1,000 mL. Ringer's Lactate, Size_=——_Unit Cost = $2.06. Colloid Fluids, ‘Manufacturer = . Colloid Fluids, Unit = . Colloid Fluids, Size_=——_Unit Cost = . 5% Albumin, ‘Manufacturer = Grifols. 5% Albumin, Unit = 250 mL. 5% Albumin, Size_=——_Unit Cost = $43.92. 25% Albumin, ‘Manufacturer = Grifols. 25% Albumin, Unit = 50 mL. 25% Albumin, Size_=——_Unit Cost = $43.92. 6% Hetastarch, ‘Manufacturer = Hospira. 6% Hetastarch, Unit = 500 mL. 6% Hetastarch, Size_=——_Unit Cost = $41.72. 6% TetrastarchS, ‘Manufacturer = Hospira. 6% TetrastarchS, Unit = 500 mL. 6% TetrastarchS, Size_=——_Unit Cost = $60.27
tHaspital cost in 2012. SAvailable as Voluven.

, 1 = 5%Dextrose Solutions. 20., 1 = Gunther B, Jauch W, Hartl W, et al. Low-dose glucose infusion in patients who have undergone surgery. Arch Surg 1987; 122:765-771.. 21., 1 = DeGoute CS, Ray MJ, Manchon M, et al. Intraoperative glucose infusion and blood lactate: endocrine and metabolic relationships during abdominal aortic surgery. Anesthesiology 1989; 71;355-361.. 22., 1 = Turina M, Fry D, Polk HC, Jr . Acute hyperglycemia and the innate immune system: Clinical, cellular, and molecular aspects. Crit Care Med 2005; 33:1624-1633.. 23., 1 = Van Den Berghe G, Wouters P, Weekers F, et al. Intensive insulin therapy in critically ill patients. New Engl J Med 2001;345:1359-1367.. 24., 1 = Sieber FE, Traystman RJ. Special issues: glucose and the brain. Crit Care Med 1992; 20:104-114.. 25., 1 = Kavanagh BP, McCowen KC. Glycemic control in the ICU. N Engl J Med 2010; 363:25402546.

Intensive Care Med 1999; 25:258-268.. 39., 1 = de Jonge E, Levi M. Effects of different plasma substitutes on blood coagulation: A comparative review. Crit Care Med 2001; 29:1261-1267.. 40., 1 = Lissauer ME, Chi A, Kramer ME. et al. Association of 6% hetastarch resuscitation with adverse outcomes in critically ill trauma patients. Am J Surg 2011; 202:53-58.. 41., 1 = Brunkhorst FM, Engel C, Bloos F, et al. Intensive insulin therapy and pentastarch resuscitation in severe sepsis. N Engl J Med 2008; 358:125-139.. 42., 1 = Perner A, Haase N, Guttormsen AB, et al. Hydroxyethyl starch 130/0.4 versus Ringer's acetate in severe sepsis. N Engl J Med 2012; 367:124-134.. 43., 1 = Magder S, Potter BJ, De Varennes B, et al. Fluids after cardiac surgery: A pilot study of the use of colloids versus crystalloids. Crit Care Med 2010; 38:2117-2124.. 44., 1 = Nearman HS, Herman ML. Toxic effects of colloids in the intensive care unit. Crit Care Clin 1991; 7:713-723.
45. Drumi W, Polzleitner D, Laggner AN, et al. Dextran-40, acute renal failure, and elevated plasma oncotic pressure. N Engl J Med 1988; 318:252-254.

Leonardo da Vinci Notebooks, Vol. I
Acute heart failure is responsible for about one million hospital admissions each year in the United States (1), and about 80% of the admissions involve the elderly (age 265 yrs) (2). The first appearance of acute, decompensated heart failure often marks the beginning of a progressive decline in clinical status. Although most patients (>95%) survive the initial hospitalization for heart failure (5), 50% of patients are readmitted within 6 months (2), and 25-35% of patients die within 12 months after hospital discharge (2).
Heart failure is not a single entity, but is classified according to the portion of the cardiac cycle that is affected (systolic or diastolic dysfunction) and the side of the heart that is involved (right-sided or left-sided heart failure). This chapter describes each of these heart failure syndromes, and focuses on the advanced stages of heart failure that require management in an intensive care unit. Many of the recommendations in this chapter are derived from the clinical practice guidelines listed in the bibliography at the end of the chapter (2-5).

Heart failure can originate from pathologic disorders involving the pericardium, myocardium, endocardium, or great vessels, as indicated in Figure 13.1. Most cases of heart failure originate in the myocardium, and are the result of ischemic injury or hypertrophy (2).
FIGURE 13.1 Possible causes of acute heart failure, indicated by the anatomic region involved. RV = right ventricle, LV = left ventricle.

The changes in cardiac performance that occur in progressive stages of heart failure are shown in Figure 13.2. Three distinct stages are identified, and each is summarized below (using the corresponding numbers in Figure 13.2).
1. The earliest sign of ventricular dysfunction is an increase in cardiac filling pressure (i.e., the pulmonary artery wedge pressure). The stroke volume is maintained, but at the expense of the elevated filling pressure, which produces venous congestion in the lungs and the resulting sensation of dyspnea.
2. The next stage is marked by a decrease in stroke volume and an increase in heart rate. The tachycardia offsets the reduction in stroke volume, so the minute output of the heart (the cardiac output) is preserved.
3. The final stage is characterized by a decrease in cardiac output. and a further increase in the filling pressure. The point at which the cardiac output begins to fall marks the transition from compensated to decompensated heart failure.
FIGURE 13.2 Changes in cardiac performance during progressive stages of left-sided heart failure in a postoperative patient. See text for further explanation.

Heart failure triggers a multitude of endogenous responses, some beneficial and some counterproductive. The responses described here have the most clinical relevance (6).

Increases in atrial and ventricular wall tension are accompanied by the release of four structurally similar natriuretic peptides from cardiac myocytes. These peptides "unload" the ventricles by promoting sodium excretion in the urine (which reduces ventricular preload) and dilating systemic blood vessels (which reduces ventricular preload and afterload). Natriuretic peptides also stimulate lipolysis in adipose tissue (7), but the relevance of this action is unclear. Natriuretic peptides play an important role in the evaluation of suspected heart failure, as described later in this section.

Decreases in stroke volume are sensed by baroreceptors in the carotid and pulmonary arteries, and activation of these receptors (through complex mechanisms) results in brainstem activation of the sympathetic nervous system. This occurs in the early stages of heart failure, and the principal results are positive inotropic and chronotropic effects in the heart, peripheral vasoconstriction, and activation of the renin-aldosterone-angiotensin system.

Specialized cells in the renal arterioles release renin in response to renal hypoperfusion and adrenergic B-Receptor stimulation. Renin release has three consequences: the formation of angiotensin II, the production of aldosterone in the adrenal cortex, and the (angiotensin-triggered) release of arginine vasopressin from the posterior pituitary. Angiotensin produces systemic vasoconstriction, while aldosterone promotes renal sodium and water retention, and vasopressin promotes both vasoconstriction and renal water retention.
Activation of the renin-angiotensin-aldosterone (RAA) system is not fully developed until the advanced stages of heart failure (8), when the principal effects (i.e., vasoconstriction and renal retention of sodium and water) are counterproductive. One beneficial effect of RAA activation is the angiotensin-mediated constriction of arterioles on the efferent side of the glomerulus, which promotes glomerular filtration by increasing the filtration pressure across the glomerulus. The deleterious effects of the RAA system are confirmed by the beneficial effects of angiotensin converting enzyme (ACE) inhibitors in the treatment of heart failure (2).

One of the natriuretic peptides described earlier, brain-type or B-type natriuretic peptide (BNP), is released as a precursor or prohormone (proBNP) from both ventricles in response to increased wall tension. The prohormone is cleaved to form BNP (the active hormone) and N-terminal (NT)-proBNP, which is metabolically inactive. The clearance of BNP and NT-proBNP is primarily via the kidneys. Peptide receptors in adipose tissue also contribute to BNP clearance (7), which might explain why plasma BNP levels are inversely related to body mass index (BMI) (8) .NT-proBNP has a longer half-life than BNP, resulting in plasma levels that are 3-5 times higher than BNP levels.

Plasma levels of both BNP and NT-proBNP are used as biomarkers for evaluating the presence and severity of heart failure (4). The predictive value of BNP and NT-proBNP levels for detecting heart failure is shown in Table 13.1 (9-11). As indicated, advancing age and renal insufficiency can also elevate natriuretic peptide levels. Severe sepsis also elevates natriuretic peptide levels, and the magnitude of the elevation can be as great as in heart failure (12). Because elevated peptide levels lack specificity, natriuretic peptide levels are better suited for excluding the presence of heart failure (4).

BNP (pq/mL):, Likelihood of Acute Heart Failure.Unlikely = . BNP (pq/mL):, Likelihood of Acute Heart Failure.‘Uncertain: = . BNP (pq/mL):, Likelihood of Acute Heart Failure.: Likely = . Age 218 yrs, Likelihood of Acute Heart Failure.Unlikely = <100. Age 218 yrs, Likelihood of Acute Heart Failure.‘Uncertain: = 100-500. Age 218 yrs, Likelihood of Acute Heart Failure.: Likely = > 500. GFR <60 mL/min, Likelihood of Acute Heart Failure.Unlikely = < 200. GFR <60 mL/min, Likelihood of Acute Heart Failure.‘Uncertain: = 200-500. GFR <60 mL/min, Likelihood of Acute Heart Failure.: Likely = > 500. NT-proBNP (pg/mL):, Likelihood of Acute Heart Failure.Unlikely = . NT-proBNP (pg/mL):, Likelihood of Acute Heart Failure.‘Uncertain: = . NT-proBNP (pg/mL):, Likelihood of Acute Heart Failure.: Likely = . Age 18-49 yrs, Likelihood of Acute Heart Failure.Unlikely = <300. Age 18-49 yrs, Likelihood of Acute Heart Failure.‘Uncertain: = 300-450. Age 18-49 yrs, Likelihood of Acute Heart Failure.: Likely = >450. Age 50-75 yrs, Likelihood of Acute Heart Failure.Unlikely = < 300. Age 50-75 yrs, Likelihood of Acute Heart Failure.‘Uncertain: = 300-900. Age 50-75 yrs, Likelihood of Acute Heart Failure.: Likely = > 900. Age >75 yrs, Likelihood of Acute Heart Failure.Unlikely = < 300. Age >75 yrs, Likelihood of Acute Heart Failure.‘Uncertain: = 300-1800. Age >75 yrs, Likelihood of Acute Heart Failure.: Likely = > 1800
Fram Referances 9-11.

Natriuretic peptide levels are most useful in the emergency department, for evaluating patients with suspected heart failure. For patients with heart failure that are admitted to the ICU, the use of serial measurements of natriuretic peptide levels to evaluate the response to therapy has not been studied. However, spurious elevations in natriuretic peptide levels from renal insufficiency and severe sepsis are likely to be commonplace in critically ill patients, and thus it seems unlikely that natriuretic peptide levels will have a clinical role in the ICU setting.

As mentioned earlier, heart failure can be classified by the portion of the cardiac cycle that is affected (systolic and diastolic heart failure) and the side of the heart that is involved (left-sided and right-sided heart failure). These distinctions are the focus of this section of the chapter.

Early descriptions of heart failure attributed most cases to contractile failure during systole (i.e., systolic heart failure). However, observations over the past 30 years indicate that diastolic dysfunction is responsible for up to 60% of cases of heart failure (2). The hallmark of diastolic heart failure is a decrease in ventricular distensibility with impaired ventricular filling during diastole (13). Common causes of diastolic heart failure include ventricular hypertrophy, myocardial ischemia (stunned myocardium), restrictive or fibrotic cardiomyopathy, and pericardial tamponade. Additional sources of impaired diastolic filling in ICU patients are positive-pressure ventilation and positive end-expiratory pressure (PEEP).

The graphs in Figure 13.3 show the influence of systolic and diastolic dysfunction on measures of cardiac performance in decompensated heart failure. The upper graph shows the relationship between end-diastolic pressure and stroke volume (similar to the graph in Figure 9.2) The curve representing heart failure has a decreased slope, and the point on the curve indicates that heart failure is associated with an increase in end-diastolic pressure and a decrease in stroke volume (similar to stage 2 and stage 3 in Figure 13.2). The lower graph shows the relationship between end-diastolic pressure and end-diastolic volume. The curve representing diastolic dysfunction has a decreased slope, which reflects a decrease in ventricular compliance (distensibility) according to the following relationship:
Compliance = A EDV /A EDP
(13.1)
The points on the ventricular compliance curves indicate that the in-crease in enddiastolic pressure in heart failure is associated with different end-diastolic volumes with systolic and diastolic dysfunction; i.e., systolic dysfunction results in an increase in enddiastolic volume, and diastolic dysfunction results in a decrease in end-diastolic volume. Therefore, the end-diastolic volume (not the end-diastolic pressure) can distinguish between systolic and diastolic dysfunction in patients with heart failure. This is evident in the diagnostic criteria shown in Table 13.2, where a left ventricular end-diastolic volume of 97 mL/m2 (measured relative to body surface area in m2) is the threshold value for identifying systolic vs. diastolic dysfunction as the cause of heart failure (14).
End-Diastolic Pressure
FIGURE 13.3 Graphs showing the influence of systolic and diastolic dysfunction on measures of cardiac performance in decompensated heart failure. Lower panel shows diastolic pressure-volume curves, and upper panel shows ventricular function curves. See text for further explanation

End-Diastolic Pressure, 1 = >16 mm Hg. End-Diastolic Pressure, 2 = >16 mm Hg. End-Diastolic Volume, 1 = > 97 mL/m?. End-Diastolic Volume, 2 = <97 mL/m?. Ejection Fraction, 1 = < 45%. Ejection Fraction, 2 = > 50%
From Referanceas 14 and 16.

The fraction of the end-diastolic volume that is ejected during systole, known as the ejection fraction (EF), is equivalent to the ratio of stroke volume to end-diastolic volume (EDV):
EF = SV/EDV
(13.2)
The EF is directly related to the strength of ventricular contraction, and is used a measure of systolic function. The normal EF of the left ventricle is >55% (15,16), but lower values of 45-50% are used as normal in the evaluation of heart failure because increases in

Right-sided heart failure is more prevalent than suspected in ICU pa-tients (17), and can be difficult to detect in the early stages. Most cases are the result of pulmonary hypertension (e.g., from pulmonary emboli or chronic lung disease) and inferior wall myocardial infarction.

Acute right heart failure is a contractile (systolic) failure that results in an increase in right ventricular end-diastolic volume (RVEDV). However, the central venous pressure (CVP) does not rise until the increase in RVEDV is restricted by the pericardium (pericardial constraint) (17). The delayed rise in the CVP is one of the reasons that the early stages of right heart failure are often undetected. The following hemodynamic criteria have been proposed for the diagnosis of right heart failure (18): CVP >10 mm Hg and CVP = PAWP or CVP within 5 mm Hg of PAWP. (PAWP is the pulmonary artery wedge pressure.) Equalization of right and left ventricular filling pressures is also characteristic of cardiac tamponade, and this similarity shows the importance of pericardial constraint in right heart failure.
INTERVENTRICULAR INTERDEPENDENCE: Because of pericardial constraint, progressive distension of the right ventricle pushes the interventricular septum towards the left ventricle and reduces the size of the left ventricular chamber, as shown in Figure 13.4. This septal displacement impairs left ventricular filling and increases the left ventricular end-diastolic pressure. In this situation, the filling pressures of both ventricles "equilibrate" to produce equalization of pressures, as indicated by the diastolic pressures in the right and left ventricle in Figure 13.4. This mechanism whereby right-sided heart failure can produce diastolic dysfunction in the left ventricle is Known as interventricular interdependence.

Cardiac ultrasound is an invaluable tool for detecting right heart failure in the ICU. The variety of measurements used to evaluate the right heart is beyond the scope of this chapter, and the most recent guidelines on this subject are included in the bibliography at the end of the chapter (Ref-erence 19). The diameter of the right ventricular chamber is a popular measurement to identify right heart dilatation. Measurements of right ventricular end-diastolic volume and ejection fraction require 3-dimensional echocardiography, and validation studies are currently ongoing to determine reliable reference ranges (19). The lower limit of normal for right ventricular ejection fraction is currently set at 44% (19).

The management of acute heart failure described here is limited to the advanced stages of heart failure, where cardiac output is impaired and the perfusion of vital organs is threatened. The management is described using measures of cardiac performance rather than symptomatology, and most of the drugs are administered by continuous intravenous infusion.
FIGURE 13.4 Interventricular interdependence: ie., the mechanism whereby right heart failure can impair left ventricular filing and produce diastolic left-heart failure. The numbers in each chamber represent the peak systolic and end-diastolic pressures. RV = right ventricle; LV = left ventricle.

The following management pertains to non-valvular heart failure resulting from systolic or diastolic dysfunction, where the hemodynamic changes are characterized by an increased pulmonary artery wedge pressure (PAWP), a decreased cardiac output (CO), and an increased systemic vascular resistance (SVR). Three approaches are described, based on the blood pressure ( i.e., high, normal, and low).
Profile: High PAWP /Low CO /High SVR /High BP.
Management: Vasodilator therapy with nitroglycerin, nitroprusside, or nesiritide, followed by diuretic therapy with furosemide if there is evidence of volume overload, or if the PAWP remains above 20 mm Hg despite vasodilator therapy.
The dosing regimens for continuous-infusion vasodilator therapy are shown in Table 13.3 (21). The vasodilators in this table are capable of dilating both arteries and veins, and will decrease both ventricular preload and afterload. The decrease in preload reduces venous congestion in the lungs, and the decrease in afterload promotes cardiac output. The overall effect is a decrease in arterial blood pressure, an increase in cardiac output, and a decrease in hydrostatic pressure in the pulmonary capillaries.

Nitroglycerin, Dosing Regimens and Precautions = 1. Do NOT infuse through polyvinylchloride (PVC) tubing (drug binds to PVC). 2. Start infusion at 5 ua/min, and increase by 5 ug/min every 5 min to achieve the desired effect. The effective dose is 5-100 g/min in most cases, and doses above 200 ug/min are not advised.. Nitroprusside, Dosing Regimens and Precautions = 1. Start infusion at 0.2 j1g/kg/min and titrate upward every 5 min to achieve the desired effect. The effective dose Is 2-5 pg/kg/min in most cases, and the maximum dose allowed is 10 pq/kg/min. 2. To reduce the risk of cyanide toxicity, avoid prolonged infu- sions >3 pg/kg/min, and avold the drug in patients with renal failure. Thiosulfate (500 mg) can be added to the infusate to bind the cyanide released from nitroprusside.. Nesiritide, Dosing Regimens and Precautions = 1. Do NOT infuse through heparin-bonded catheters (drug binds to heparin). 2, Start with a bolus dose of 2 g/kg, and infuse at 0.01 ng/kg/min. If necessary, a sacond bolus dose of 1 ug/kg can be given, followed by an increase in infusion rate of 0.005 wg/kg/min, and this can be repeated every 3 hrs to a maximum rate of 0.03 pq/kg/min.
NITROPRUSSIDE: The vasodilating effects of nitroprusside are the result of nitric oxide release from the nitroprusside molecule. Unfortunately, cyanide ions are also released (5 atoms per molecule) and accumulation of these ions can produce life-threatening cyanide intoxication (22,23). Both the liver and kidneys participate in cyanide clearance, and thus nitroprusside is not recommended in patients with renal or hepatic insufficiency. Thiosulfate binds cyanide and reduces the risk of cyanide toxicity (23), and sodium

Dobutamine, Dosing Regimens and Precautions = 4. Do NOT infuse with alkaline solutions. 2. Start at infusion rate of 5 .g/kg/min, and increase in increments of 3-5 ug/kg/min, If necessary. Usual dose range is 5-20 ug/kg/min.. Levosimendan, Dosing Regimens and Precautions = 1. Initial dose Is 12 ug/kg (over 10 min), followed by an Infusion rate of 0.1 uwg/kg/min. Rate can be increased to 0.2 .g/kg/min, if necessary. 2. Infusions are usually limited to 24 hrs, but long-acting active metabolites produce salutary effects for at least 7 days.. Milrinone, Dosing Regimens and Precautions = 1. Initial dose is 50 g/kg (over 10 min), followed by an infusion rate of 0.375-0.75 p.g/kg/min. Dally dose should not exceed 1.13 ma/kg.. , Dosing Regimens and Precautions = 2. The following dose adjustments are recommended for patients with renal insufficiency:. , Dosing Regimens and Precautions = . , Dosing Regimens and Precautions = . , Dosing Regimens and Precautions = . , Dosing Regimens and Precautions = . , Dosing Regimens and Precautions = . , Dosing Regimens and Precautions = . , Dosing Regimens and Precautions = 
WHICH INODILATOR IS PREFERRED?: Levosimendan is emerging as the preferred inodilator, particularly in the setting of myocardial ischemia or infarction, and is the only inodilator that is associated with improved survival (34). The benefit of levosimendan over dobutamine in reducing plasma BNP levels is demonstrated in Figure 13.5 (36). Dobutamine is the least favored inodilator because of the deleterious effects of adrenergic stimulation in the failing heart.
DIURETICS: The indications for diuretic therapy with furosemide are the same as those described for heart failure with high blood pressure.

Acute heart failure accompanied by hypotension is a life-threatening condition that often represents cardiogenic shock (when accompanied by an elevated serum lactate level). This condition is most often the result of acute myocardial infarction.
Profile: High PAWP /Low CO/High SVR /Low BP.
Treatment: Dobutamine or vasoconstrictor therapy with dopamine, combined with mechanical cardiac support.
Dobutamine can sometimes increase blood pressure (when the increase in stroke volume is greater than the decrease in systemic vascular resistance); otherwise a vasoconstrictor drug is required to raise the blood pressure. Since systemic vasoconstriction is a prominent feature of cardiogenic shock, drug-induced vasoconstriction can further aggravate tissue hypoperfusion. To limit this risk, a vasoconstrictor drug that also promotes cardiac output is the favored choice in cardiogenic shock. Dopamine is such a

DOPAMINE: Dopamine stimulates both cardiac B-receptors (which promotes cardiac output) and peripheral B-receptors (which promotes systemic vasoconstriction). In moderate doses (3-10 g/kg/min), the B-receptor effect predominates, while in higher doses (>10 eg/kg/min), a-receptor stimulation predominates. At dose rates of 5-15 ecg/kg/min, dopamine can promote cardiac output and produce systemic vasoconstriction (29). Therefore dopamine infusion at a rate of 5-15 e<g/kg/min is a reasonable choice in the management of cardiogenic shock. (See Chapter 53 for a more detailed description of dopamine. )
The mortality rate in cardiogenic shock remains high (about 80%) with the use of hemodynamic drugs alone, and other measures, such as mechanical cardiac support and coronary revascularization, are required to improve outcomes. Mechanical cardiac support using intra-aortic balloon counterpulsation is described later in the chapter.

Diuretic therapy is a cornerstone of management for chronic heart failure. However, the following observations indicate that diuretic therapy with intravenous furosemide should be used cautiously in patients with acute, decompensated heart failure.
FIGURE 13.5 Changes in plasma BNP kvels associated with short-term (24 hr) infusions of dobutamine and levosimendan in patients with acute decompensated heart failure. Graph redrawn from Reference 35.
Pulmonary Artery Wedge Pressure(mm Hg)
FIGURE 13.6 Ventricular function curves for the normal and failing left ventricle with arrows indicating the expected changes associated with each type of drug therapy. The shaded area indicates the high-risk region for pulmonary edema.
1. Intravenous furosemide causes a decrease in cardiac output in acute heart failure (3638), as indicated in Figure 13.6. This effect is the result of a decrease in venous return and an increase in left ventricular afterload; the latter effect is due to the acute vasoconstrictor response to furosemide mentioned earlier (31).
2. The presence of pulmonary edema in acute heart failure is NOT evidence of excess
CONTINUOUS INFUSION FUROSEMIDE: Because the diuretic effect of furosemide is a function of the urinary excretion rate and not the plasma concentration (43), continuous infusions of the drug often (but not always) produce a more vigorous diuresis than bolus injection. The dosing regimen for continuous-infusion furosemide is influenced by renal function, as shown below (41,42):
>75 mL/min, Loading Dose = 100 mg. >75 mL/min, Initial Infusion Rate = 10 mg /hr. 25-75 mL/min, Loading Dose = 100-200 mg. 25-75 mL/min, Initial Infusion Rate = 10-20 mg/hr. <25 mL/min, Loading Dose = 200 mg. <25 mL/min, Initial Infusion Rate = 20-40 mg /hr
The infusion rate can be increased as needed to achieve the desired urine output (e.g., 2100 mL/hr). The maximum recommended infusion rate is 240-360 mg/hr (42), or 170 mg/hr in elderly patients (44).

Intra-aortic balloon counterpulsation is used for temporary cardiac support in cases of unstable angina or cardiogenic shock where cardiac pump function is expected to improve as a result of some intervention; i.e., coronary angioplasty or coronary artery bypass surgery (49). This technique is contraindicated in patients with aortic valve insufficiency and aortic dissection.

The intra-aortic balloon is an elongated polyurethane balloon that is inserted percutaneously into the femoral artery and advanced up the aorta until the tip lies just below the origin of the left subclavian artery (see Figure 13.7). A pump attached to the balloon uses helium, a low density gas, to rapidly inflate and deflate the balloon (inflation volume is generally 35 to 40 mL). Inflation begins at the onset of diastole, just after the aortic valve closes (the R wave on the ECG is a common trigger). The balloon is then deflated at the onset of ventricular systole, just before the aortic valve opens (during isovolumic contraction). This pattern of balloon inflation and deflation produces two changes in the aortic pressure waveform, which are illustrated in Figure 13.7.

As described in Chapter 9, positive intrathoracic pressure reduces left ventricular afterload by decreasing the transmural wall pressure developed by the ventricle during systole. This promotes ventricular emptying by facilitating the inward movement of the ventricular wall during systole. As a result, positive pressure breathing can augment the stroke output of the left ventricle (see Figure 9.8).
Clinical studies have demonstrated that breathing with continuous positive airway pressure (CPAP) reduces left ventricular transmural pressure (54) and, increases cardiac output (55) in patients with left-sided heart failure. Furthermore, in patients with cardiogenic pulmonary edema, CPAP hastens clinical improvement when added to conventional therapy for acute heart failure (56,57). As a result of these observations, CPAP (along with non-invasive pressure support ventilation) has emerged as a treatment modality for acute heart failure associated with pulmonary edema.

The management of acute heart failure suffers from the following shortcomings:, 1 = The management of acute heart failure suffers from the following shortcomings:. 1., 1 = Despite the increasing prevalence and poor prognosis associated with acute, decompensated heart failure, the management of this condition has changed very little in the last 10 to 15 years.. 2., 1 = The treatment of heart failure is actually treating the consequences of heart failure (e.g., pulmonary venous congestion) and has little impact on the functional derangements in the myocytes. (Coronary revascularization is an exception to this rule.). 3., 1 = Many of the drug therapies for acute heart failure produce effects that are counterproductive (e.g., diuretics reduce cardiac output, which promotes sodium retention, vasodilators stimulate renin release, which results in vasoconstriction).. While these shortcomings are not unique to heart failure, they are more evident because of the prominence of cardiovascular disease as the leading cause of death in this country., 1 = While these shortcomings are not unique to heart failure, they are more evident because of the prominence of cardiovascular disease as the leading cause of death in this country.

1. Roger V, Go AS, Lloyd-Jones D, et al. Heart disease and stroke statistics — 2012 update: a report from the American Heart Association. Circulation 2012; 125:e2— e220.
2. Hunt SA, Abraham WT, Chin MH, Feldman AM, et al. 2009 focused update incorporated into the ACC/AHA 2005 guidelines for the diagnosis and management of heart failure in adults: a report of the American College of Cardiology Foundation/ American Heart Association Task Force on Practice Guidelines. Circulation 2009; 119:e391-e479.
3. Gheorghiade M, Pang PS. Acute heart failure syndromes. J Am Coll Cardiol 2009; 53:557-573.
4. Weintraub NL, Collins SP, Pang PS, et al. Acute heart failure syndromes: emergency department presentation, treatment, and disposition: current approaches and future aims: a scientific statement from the American Heart Association. Circulation 2010; 122:1975-1996.
5. Task Force for the Diagnosis and Treatment of Acute and Chronic Heart Failure 2012 of the European Society of Cardiology. ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2012. Eur Heart J 2012; 33:1787-1847.

Types of, 1 = Heart Failure. 13., 1 = Zile MR, Baicu CF, Gaasch WH. Diastolic heart failure - Abnormalities in active relaxation and passive stiffness of the left ventricle. New Engl J Med 2004; 350:1953-1959.. 14., 1 = Paulus WJ, Tschope C, Sanderson JE, et al. How to diagnose diastolic heart failure: a consensus statement on the diagnosis of heart failure with normal left ventricular ejection fraction by the Heart Failure and Echocardiography Associations of the European Society of Cardiology. Europ Heart J 2007; 28:2539-2550.. 15., 1 = Lang RM, Bierig M, Devereux RB, et al. Recommendations for chamber quantification: a report from the American Society of Echocardiography and the European Association of Echocardiography. J Am Soc Echocardiogr 2005; 18:1440- 1463.. 16., 1 = Hess OM, Carroll JD. Clinical assessment of heart failure. In: Libby P, Bonow RO, Mann DL, Zipes DP, eds. Braunwald's Heart Disease. 8th ed. Philadelphia: Saunders Elsevier, 2008:561-581.. 17., 1 = Hurford WE, Zapol WM. The right ventricle and critical illness: a review of anatomy, physiology, and clinical evaluation of its function. Intensive Care Med 1988; 14:448- 457.. 18., 1 = Lopez-Sendon J, Coma-Canella I, Gamello C. Sensitivity and specificity of hemodynamic criteria in the diagnosis of right ventricular infarction. Circulation 1981; 64:515-525.. 19., 1 = Rudski LG, Lai WW, Afilalo J, et al. Guidelines for the echocardiographic assessment of the right heart in adults: A report from the American Society of Echocardiography. J Am Soc Echocardiogr 2010; 23:685-713.

45., 1 = Isner JM. Right ventricular myocardial infarction. JAMA 1988; 259:712-718.. 46., 1 = Reuse C, Vincent JL, Pinsky MR. Measurement of right ventricular volumes during fluid challenge. Chest 1990; 98:1450-1454.. 47., 1 = Dell'Italia LJ, Starling MR, Blumhardt R, et al. Comparative effects of volume loading, dobutamine and nitroprusside in patients with predominant right ventricular infarction. Circulation 1986; 72:1327-1335.. 48., 1 = Russ MA, Prondzinsky R, Carter JM, et al. Right ventricular function in myocardial infarction complicated by cardiogenic shock: improvement with levosimendan. Crit Care Med 2009; 37:3017-3023.

Inflammation is not itself considered a disease but a salutary operation . . . but when it cannot accomplish that salutary purpose . . . it does mischief.
John Hunter, MD (1728-1793)
The introductory quote is from a distinguished 18th century Scottish surgeon who is most remembered for a reckless self-experiment where he intentionally injected himself with a purulent discharge from a patient with venereal disease, and subsequently developed both gonorrhea and syphilis (I). On the brighter side, John Hunter was a skilled observer, and his observations on inflammation revealed a tendency to produce harm, as indicated in his statement. A quarter of a millennium later, the harmful effects of inflammation are recognized as a leading source of morbidity and mortality in critically ill patients. This chapter describes the features of inflammatory injury, and presents the manifestations and management of two inflammatory shock syndromes: i.e., septic shock and anaphylactic shock. These conditions will demonstrate the widespread damage that occurs when inflammation "does mischief."

The inflammatory response is a complex process that is triggered by conditions that threaten the functional integrity of the host (e.g., physical injury or microbial invasion). Once activated, the inflammatory response generates a variety of noxious substances that are designed to control or eliminate the threat, while the host organism is not adversely affected. However, persistent or widespread inflammation can produce tissue damage in any or all of the vital organs. Inflammatory injury is problematic because it tends to become a self-sustaining process; i.e., the inflammatory tissue damage triggers more inflammation, which produces more tissue damage, and so on. This condition of self-sustaining and progressive inflammatory injury is known as malignant inflammation, and it is characterized by progressive multiorgan dysfunction and multiorgan failure (1,2).

One of the principal sources of inflammatory injury is the release of toxic oxygen metabolites from activated neutrophils (3,4). The purpose of neutrophil activation is to generate these metabolites, as described next.

Activation of neutrophils, which occurs in the early stages of the inflammatory response, is associated with a 20to 50-fold increase in O2 consumption. This is called the respiratory burst (4), which is a misleading term because it is not associated with increased energy production, but is designed to generate toxic metabolites of oxygen (6). This is illustrated in Figure 14.1. When neutrophils are activated, a specialized oxidase en-zyme on the inner surface of the cell membrane is activated; this triggers the metabolic reduction of oxygen to water, which generates a series of highly-reactive metabolites that include the superoxide radical, hydrogen peroxide, and the hydroxyl radical. Neutrophils also have a myelo-peroxidase enzyme that converts hydrogen peroxide to hypochlorite, a powerful germicidal agent that is the active ingredient in household bleach (5). The oxygen metabolites generated during the respiratory burst are stored in cytoplasmic granules, and are released during neutrophil degranulation.

Oxygen metabolites are powerful oxidizing agents or oxidants that can disrupt cell membranes, denature proteins, and fracture DNA molecules. Once released, these metabolites are capable or producing lethal damage in invading microorganisms, while the cells of the host are normally protected by endogenous antioxidants. However, when oxidant activity exceeds antioxidant protection (a condition known as oxidant stress), the cells of the host are also damaged by the oxygen metabolites. This oxidant cell injury is the major source of damage produced by the inflammatory response, and the spectrum of organ damage than can occur is shown in Table 14.1.

Free radicals such as the superoxide radical and the hydroxyl radical are highly reactive because they have an unpaired electron in their outer orbitals. When a free radical reacts with a non-radical, the non-radical loses an electron and is transformed into a free radical. Such radical-regenerating reactions become repetitive, creating a series of selfsustaining reactions known as a chain reaction (6). These self-sustaining reactions are troublesome because they continue after the inciting event is eliminated, and tend to produce widespread damage. Fires are a familiar example of an oxidative chain reaction. The oxidation of membrane lipids, which is a major component of oxidant cell injury, also proceeds as a chain reaction (7).
FIGURE 14.1 The sequence of chemical reactions involved in neutrophil activation, which generates a series of highly reactive oxygen metabolites that are stored in cytoplasmic granules. SOD = superoxide dismutase. See text for further explanation.
Clinical Conditions Attributed to Inflammatory Injur
Brain, Condition | = Septic encephalopathy. Bone Marrow, Condition | = Anemia of critical Illness. Cardiovascular, Condition | = Septic shock. Kidneys, Condition | = Acute kidney injury. Lungs, Condition | = Acute respiratory distress syndrome. Peripheral Nerves, Condition | = Critical iliness polyneuropathy. Skeletal Muscle, Condition | = Critical ilIness myopathy

The following definitions have been adopted for the clinical syndromes associated with systemic inflammation (8,9):
1. The condition that is characterized by signs of systemic inflammation (e.g., fever, leukocytosis) is called the systemic inflammatory response syndrome (SIRS).
2. When SIRS is the result of an infection, the condition is called sepsis.
3. When sepsis is accompanied by dysfunction in one or more vital organs, or an elevated blood lactate level (>4 mM/L), the condition is called severe sepsis.
4. When severe sepsis is accompanied by hypotension that is refractory to volume infusion, the condition is called septic shock.
5. Inflammatory injury involving more than one vital organ is called multiorgan dysfunction syndrome (MODS), and the subsequent failure of more than one organ system is called (surprise!) multiorgan failure (MOF).

The diagnostic criteria for the SIRS are shown in Table 14.2. SIRS is a common condition; i.e., in one survey of patients in a surgical ICU, SIRS was identified in 93% of the patients (10). The presence of SIRS does not imply the presence of infection. Infection is identified in only 25 to 50% of patients with SIRS (10,11). The distinction between inflammation and infection is an essential ingredient in the rational approach to patients with fever and leukocytosis.

ee
The diagnosis of SIRS requires at least 2 of the following:
1. Temperature > 38°C or < 36°C
2. Heart rate > 90 beats/min
3. Respiratory rate > 20 breaths/min, or arterial PCO. <32 mm Hg
4. WBC count >12,000/mm? or <4000/mm?, or >10% immature neutrophils (band forms)
From Referance 8.
The organs most often damaged by systemic inflammation are the lungs, kidneys, cardiovascular system, and central nervous system (see Table 14.1). The most common manifestation of inflammatory organ injury is the acute respiratory distress syndrome (ARDS), which has been reported in 40% of patients with severe sepsis (12), and is one of the leading causes of acute respiratory failure in critically ill patients (see Chapter 23). The number of organs that are damaged by inflammatory injury has important prognostic implications. This is shown in Figure 14.2, which includes surveys from the United States (12) and Europe (13) showing a direct relationship between the mortality rate and the number of organ failures related to inflammation. This demonstrates the lethal potential of uncontrolled systemic inflammation.
FIGURE 14.2 The relationship between mortality rate and the number of inflammation-related organ failures. Data from References 12 and 13.

Severe sepsis and septic shock (which are essentially the same condition with different blood pressures) have been implicated in one of every four deaths worldwide (9), and the incidence of these conditions is steadily rising. The mortality rate averages about 3050% (12,14), and varies with age and the number of associated organ failures (as just described). The mortality rate is not related to the site of infection or the causative organism, including multidrug-resistant organisms (14). This observation is evidence that inflammation, not infection, is the principal determinant of outcome in severe sepsis and septic shock.

As mentioned in Chapter 10, the impaired energy metabolism in septic shock is not the result of inadequate tissue oxygenation, but is caused by a defect in oxygen utilization in mitochondria (17,18). This condition is known as cytopathic hypoxia (17), and the culprit is oxidant-induced inhibition of cytochrome oxidase and other proteins in the electron transport chain (19). A decrease in oxygen utilization would explain the observation shown in Figure 14.3, where the PO> in skeletal muscle is increased in patients with severe sepsis (19).
The proposed decrease in oxygen utilization in sepsis is not consistent with the increase in whole-body Oz consumption that is often observed in sepsis. This discrepancy can be resolved by proposing that the in-creased Oz consumption in sepsis is not a reflection of aerobic metabolism, but is a manifestation of the increased O> consumption that occurs during neutrophil activation (i.e., the respiratory burst) (21).

The discovery that tissue oxygenation is (more than) adequate in severe sepsis and septic shock has important implications because it means that efforts to improve tissue oxygenation in these conditions (e.g., with blood transfusions) are not justified.
FIGURE 14.3 Direct measurements of tissue PO? in the forearm muscles of healthy volunteers and patients with severe sepsis. The height of the columns represents the mean value for each group, and the crossbars represent the standard error of the mean. Data from Reference 20.

As mentioned in Chapter 10, the increase in serum lactate levels in severe sepsis and septic shock is not the result of inadequate tissue oxygenation, but instead appears to be the result of enhanced production of pyruvate and inhibition of pyruvate dehydrogenase (22,23), the enzyme that converts pyruvate to acetyl coenzyme A in mitochondria.
Endotoxin and other bacterial cell wall components have been implicated in the inhibition of this enzyme (22). This mechanism of lactate accumulation is consistent with the notion that tissue oxygenation is not impaired in severe sepsis and septic shock.

Acute Sepsis Bundle: Complete within 6 hrs of diagnosis, On ent is = fF wo N . Obtain appropriate cultures. . Obtain plasma lactate level. . Administer appropriate antibiotics. . Achieve the following goals: a. CVP= 8-12 mmHg b. MAP 265 mm Hg c. Urine output 20.5 mL/kg/hr d. SvO; >65% or SevO, > 70%. Sepsis Management Bundle: Complete within 24 hrs of diagnosis, On ent is = 1. 2. Administer low-close steroid if indicated. Maintain blood glucose levels at 120-150 mg/dL. . Maintain plateau airway pressure at <30 om H,0 In ventilator-dependent patients (see Chapter 23).
From Referancas 9 and 24. CVP=cantral venous pressure; MAP'= maan arterial prassura; SvO> = mixed venous O> saturation; ScvO = centra venous O» saturation.
BLOOD CULTURES: One dose of an intravenous antibiotic can sterilize blood cultures within a few hours, so blood cultures should be obtained prior to administering antibiotics. At least 2 sets of blood cultures are recommended (9). Two sets of blood cultures will detect about 90% of bloodstream infections, while 3 sets of blood cultures will detect close to 98% of bloodstream infections (32). The yield from blood cultures is influenced by the volume of blood that is cultured, and a volume of at least 20 mL is recommended for each set of blood cultures (33).

Urticaria, 1 = 85-90%. Subcutaneous angioedema, 1 = 85-90%. Upper airway angioedema, 1 = 50-60%. Bronchospasm and wheezing, 1 = 45-50%. Hypotension, 1 = 30-3595. Abdominal cramping, diarrhea, 1 = 25-30%. Substernal chest pain, 1 = 4-6%. Pruritis without rash, 1 = 2-5%
From Referance 35.

Anaphylaxis is an acute multiorgan dysfunction syndrome produced by the immunogenic release of inflammatory mediators from basophils and mast cells. The characteristic feature is an exaggerated immunoglobulin E (IgE) response to an external antigen; i.e., a hypersensitivity reaction. The manifestations of anaphylaxis typically involve the skin, lungs, gastrointestinal tract, and cardiovascular system (35). Identical manifestations can occur without the involvement of IgE; these are called anaphylactoid reactions, and are not immunogenic in origin (36). Common triggers for anaphylactic reactions include food, antimicrobial agents, and insect bites, while common triggers for anaphylactoid reactions include opiates and radiocontrast dyes. Anaphylaxis can also appear without an identifiable external trigger.

Anaphylactic reactions are typically abrupt in onset, and appear within minutes of exposure to the external trigger. Some reactions are delayed, and can appear as late as 72 hours after exposure (35). A characteristic feature of anaphylactic reactions is edema and swelling in the involved organ, caused by increased vascular permeability with extravasation of fluid. As much as 35% of the intravascular volume can be lost within 10 minutes in severe anaphylactic reactions (35).
The clinical manifestations of anaphylaxis are shown in Table 14.4, and are listed by their frequency of occurrence. The most common manifestations are urticaria and subcutaneous angioedema (typically involving the face), and the most concerning manifestations are angioedema of the upper airway (e.g., laryngeal edema), bronchospasm, and hypotension. The most feared manifestation of anaphylaxis is profound hypotension with evidence of systemic hypoperfusion, which represents anaphylactic shock.

The management of anaphylaxis includes drugs that halt the progress of anaphylactic reactions (i.e., epinephrine), and drugs that alleviate signs and symptoms (e.g., bronchodilators).

Epinephrine is the most effective drug available for treating anaphylaxis, and is capable of blocking the release of inflammatory mediators from sensitized basophils and mast cells. The drug is available in a (confusing) variety of aqueous solutions, and these are shown in Table 14.5. The usual treatment for anaphylactic reactions is 0.3-0.5 mg of epinephrine (0.3-0.5 mL of 1:1000 epinephrine solution) administered by deep intramuscular (IM) injection in the lateral thigh, and repeated every 5 minutes if necessary (35). Drug absorption is slower with subcutaneous injection (36), and with drug injection in the deltoid muscle instead of the thigh (35). Epinephrine can be nebulized for patients with laryngeal edema using the dosing regimen shown in Table 14.5; however, the efficacy of nebulized epinephrine is unclear.
GLUCAGON: The actions of epinephrine to inhibit degranulation of mast cells and basophils is mediated by B-adrenergic receptors, and ongoing therapy with B-receptor antagonists can attenuate or eliminate the response to epinephrine. When anaphylactic reactions are refractory to epinephrine in patients receiving B-blocker drugs, glucagon can be effective (for reasons described in Chapter 54). The dose of glucagon is 1-5 mg by slow intravenous injection (over 5 min), followed by a continuous infusion at 5-15 g/min, titrated to the desired response (35). Glucagon can trigger vomiting, and patients with depressed consciousness should be placed on their side to limit the risk of aspiration when glucagon is administered.

The following drugs can be given after epinephrine is administered, and should never be used as a replacement for epinephrine.
ANTIHISTAMINES: Histamine receptor antagonists are often used for cutaneous anaphylactic reactions, and can help in alleviating pruritis. The histamine H, blocker diphenhydramine (25-50 mg PO, IM, or IV) and the histamine-H2 blocker ranitidine (50 mg IV or 150 mg PO) should be given together because they are more effective in combination.
hrine Solutions and Their Clinical Uses
From Referance 35. PEA = Pulseless Electrical Activity.

1:100 (19 mg/mL), == Condition = = Laryngeal Edema. 1:100 (19 mg/mL), Dosing Regimen = 0.25 mL (2.5 mg) in 2 mL saline, administered by nebulizer.. 1:1,000 (1 mg/mL), == Condition = = Anaphylaxis. 1:1,000 (1 mg/mL), Dosing Regimen = 0.3-0.5 mL (mq) by deep IM injection in the thigh avery 5 min as needed.. 1:10,000 (0.1 mg/mL), == Condition = = Asystole or PEA. 1:10,000 (0.1 mg/mL), Dosing Regimen = 10 mL (1 mg) IV every 3-5 min as needed. 1:100,000 (1Oug/mL), == Condition = = Anaphylactic Shock. 1:100,000 (1Oug/mL), Dosing Regimen = Add 1 mL of 1:1,000 solution to 100 mL of saline (1 mg/100 mL or 10 wg/mL) and infuse at 30-100 mL/hr (5-15 pg/min).

BRONCHODILATORS: Inhaled B-receptor agonists like albuterol are used to relieve bronchospasm, and are administered by nebulizer (2.5 mL or a 0.5% solution) or by metered-dose inhaler.
CORTICOSTEROIDS: Despite the popularity of steroids for treating hypersensitivity reactions, there is no evidence that steroids are effective in reversing, slowing, or preventing the recurrence of anaphylactic reactions (35). As a result, the most recent practice guideline on treating anaphylaxis does not include a recommendation for steroid therapy (35).

Anaphylactic shock is an immediate threat to life, with profound hypotension from systemic vasodilatation and massive fluid loss through leaky capillaries (35). The hemodynamic alterations in anaphylactic shock are similar to those in septic shock, but are often more pronounced. Because of the potential for rapid deterioration, anaphylactic shock requires prompt and aggressive management using the measures described next.

There is no standardized dosing regimen for epinephrine in anaphylactic shock, but the epinephrine infusion regimen in Table 14.5, which uses dose rates of 5-15 yg/min, has been cited for its efficacy (35). An intravenous bolus dose of epinephrine (5-10 wg) can precede the continuous drug infusion (37).

Aggressive volume resuscitation is essential in anaphylactic shock be-cause at least 35% of the intravascular volume can be lost through leaky capillaries (35), which is enough to produce hypovolemic shock (see Chapter 11). Volume resuscitation can begin by infusing 1-2 liters of crystalloid fluid (or 20 mL/kg), or 500 mL of iso-oncotic colloid fluid (e.g., 5% albumin), over the first 5 minutes (35). Thereafter, the infusion rate of fluids should be tailored to the clinical condition of the patient.

Persistent hypotension despite epinephrine infusion and volume resuscitation can be managed by adding glucagon or another vasopressor such as norepinephrine or dopamine (the dosing regimens for these drugs have been described earlier).

The discovery that inflammation is the source of multiorgan failure and fatal outcomes in septic shock has created interest in therapies aimed at inhibiting the inflammatory response in septic shock. So far, these therapies have failed to produce the anticipated benefits. This is not unexpected, because the problem with inflammatory injury is not the inflammation, but the inability of the host to protect itself from inflammatory injury. Since the damage inflicted by inflammation is largely due to oxidation (i.e., oxidant cell injury), inflammatory injury is a manifestation of oxidant stress, where the production of oxidants (like the reactive oxygen metabolites in Figure 14.1) overwhelms the body's endogenous antioxidant defenses. Therefore, inflammatory injury can be the result of inadequate antioxidant protection.
Conditions like severe sepsis and septic shock create an oxidation-rich environment in tissues, which requires an antioxidant-rich defense system. However, antioxidant support is never provided for critically ill patients. It seems likely that persistent oxidation will eventually deplete endogenous antioxidants like glutathione (the major intracellular antioxidant) and vitamin E (which protects cell membranes from oxidant injury), ensuring the progressive march of inflammatory injury and multiorgan failure. There is some evidence that daily administration of endogenous antioxidants improves outcomes in septic shock (38), and the promise of this approach deserves much more attention.
Moore W. The Knife Man: Blood, Body Snatching, and the Birth of Modern Surgery. New York: Broadway Books, 2005.

2012.. 26., 1 = Boyd JH, Forbes J, Nakada T-a, et al. Fluid resuscitation in septic shock: a positive fluid balance and elevated central venous pressure are associated with increased mortality. Crit Care Med 2011; 39:259-265.. 27., 1 = Hollenberg SM. Inotropes and vasopressor therapy of septic shock. Crit Care Clin 2009; 25:781-802.. 28., 1 = Polito A, Parisini E, Ricci Z, et al. Vasopressin for treatment of vasodilatory shock: an ESICM systematic review and meta-analysis. Intensive Care Med 2012; 38:9-19.. 29., 1 = Sprung CL, Annane D, Keh D, et al. Hydrocortisone therapy for patients with septic shock. N Engl J Med 2008; 358:111-124.. 30., 1 = Sherwin RL, Garcia AJ, Bilkovski R. Do low-dose corticosteroids improve mortality or shock reversal in patients with septic shock? J Emerg Med 2012; 43:7-12.. 31., 1 = Gaieski DF, Mikkelsen ME, Band RA, et al. Impact of time to antibiotics on survival in patients with severe sepsis or septic shock in whom early goal-directed therapy was initiated in the emergency department. Crit Care Med 2010; 38:1045-1053.
32. Lee A, Mirrett S, Reller B, Weinstein MP. Detection of bloodstream infections in adults: how many blood cultures are needed? J Clin Microbiol 2007; 45:3546—-3548.
33. Cockerill FR III, Wilson JW, Vetter EA, et al. Optimal testing parameters for blood cultures. Clin Infect Dis 2004; 38:1724—1730.
34. Marik PE, Preiser J-C. Toward understanding tight glycemic control in the ICU. Chest 2010; 137:544-551.

35. Lieberman P, Nicklas RA, Oppenheimer J, et al. The diagnosis and management of anaphylaxis practice parameter: 2010 update. J Allergy Clin Immunol 2010; 126:480.e1-—480.e42.
36. Simons FER, Gu X, Simons KJ. Epinephrine absorption in adults: intramuscular versus subcutaneous injection. J Allergy Clin Immunol 2001; 108(5):871-873.
37. Sampson HA, Munoz-Furlong A, Campbell RL, et al. Second symposium on the definition and management of anaphylaxis: summary report — second National Institute of Allergy and Infectious Disease/Food Allergy and Anaphylaxis Network symposium. Ann Emerg Med 2006; 47:373-380.

- 38. Angstwurm MWA, Engelmann L, Zimmermann T, et al. Selenium in intensive care (SIC): results of a prospective randomized placebo-controlled study in patients with severe systemic inflammatory response syndrome, sepsis, and septic shock. Crit Care Med 2007; 35:118-126.

Nothing is so firmly believed as that which is least known.
Francis Jeffrey (1773-1850)

A rapid heart rate or tachycardia while at rest is usually evidence of a problem, but the tachycardia may not be the problem. This chapter de-scribes tachycardias that are a problem (i.e., tachyarrhythmias), and require prompt evaluation and management. Most of the recommendations in this chapter are borrowed from the clinical practice guidelines listed at the end of the chapter (1-4).

The evaluation of tachycardias (heart rate >100 beats/min) is based on 3 ECG findings: i.e., the duration of the QRS complex, the uniformity of the R-R intervals, and the characteristics of the atrial activity. The results of this evaluation are demonstrated in Figure 15.1. The duration of the QRS complex is used to distinguish narrow-QRS-complex tachycardias (QRS duration "0.12 sec) from wide-QRS-complex tachycardias (QRS duration >0.12 sec). This helps to identify the point of origin of the tachycardia, as described next.

Tachycardias with a narrow QRS complex (0.12 sec) originate from a site above the AV conduction system. These supraventricular tachycardias include sinus tachycardia, atrial tachycardia, AV nodal re-entrant tachycardia (also called paroxysmal supraventricular tachycardia), atrial flutter, and atrial fibrillation. The specific arrhythmia can be identified using the uniformity of the R-R interval (i.e., the regularity of the rhythm), and the characteristics of the atrial activity, as described next.

If the R-R intervals are uniform in length (indicating a regular rhythm), the possible arrhythmias include sinus tachycardia, AV nodal re-entrant tachycardia, or atrial flutter with a fixed (2:1, 3:1) AV block. The atrial activity on the ECG can identify each of these rhythms using the following criteria:
1. Uniform P waves and P-R intervals indicate a sinus tachycardia.
2. The absence of P waves suggests an AV nodal re-entrant tachycardia (see Figure 15.2).

If the R-R intervals are not uniform in length (indicating an irregular rhythm), the most likely arrhythmias are multifocal atrial tachycardia and atrial fibrillation. Once again, the atrial activity on the ECG helps to identify each of these rhythms; i.e.,
1. Multiple P wave morphologies with variable P-R intervals is evidence of multifocal atrial tachycardia (see Panel A, Figure 15.3).
2. The absence of P waves with highly disorganized atrial activity (fibrillation waves) is evidence of atrial fibrillation (see Panel B, Figure 15.3).
FIGURE 15.2 Narrow-QRS-complex tachycardia with a regular rhythm. Note the absence of P waves, which are hidden in the QRS complexes. This is an AV nodal re-entrant tachycardia.

Tachycardias with a wide QRS complex (>0.12 sec) can originate from a site below the AV conduction system (i.e., ventricular tachycardia), or they can represent a supraventricular tachycardia (SVT) with prolonged AV conduction (e.g., from a bundle branch block). These two arrhythmias can be difficult to distinguish. An irregular rhythm is evidence of an SVT with aberrant AV conduction, while certain ECG abnormalities (e.g., AV dissociation) provide evidence of VT. The distinction between VT and SVT with aberrant conduction is described in more detail later in the chapter.
FIGURE 15.3 Narrow-QRS-compkx tachycardias with an irregular rhythm. Panel A shows a multifocal atrial tachycardia (MAT), identified by multiple P wave morphologies and variable PR interval. Panel B is atrial fibrillation, identified by the absence of P waves and the highly disorganized atrial activity (fibrillation waves).

Atrial fibrillation (AF) is the most common cardiac rhythm disturbance in clinical practice, and can be paroxysmal (resolves spontaneously), recurrent (2 or more episodes), persistent (present for at least 7 days), or permanent (present for at least one year) (1,2). Most patients with AF are elderly (median age 75 years) and have underlying cardiac disease. About 25% of patients are less than 60 years of age and have no underlying cardiac disease (1): a condition known as lone atrial fibrillation.

Postoperative AF is reported in up to 45% of cardiac surgeries, up to 30% of non-cardiac thoracic surgeries, and up to 8% of other major surgeries (5). It usually appears in the first 5 postoperative days (6), and is associated with longer hospital stays and increased mortality (5,6). Several predisposing factors have been implicated, including heightened adrenergic activity, magnesium depletion, and oxidant stress. Prophylaxis with B-blockers and magnesium is currently popular (5,7), and there is evidence that the antioxidant Nacetylcysteine (a glutathione surrogate) provides effective prophylaxis following cardiac surgery (7). Most cases of postoperative AF resolve within a few months.

The adverse consequences of AF include impaired cardiac performance and thromboembolism.

Atrial contraction is responsible for 25% of the ventricular end-diastolic volume in the normal heart (8). Loss of the atrial contribution to ventricular filling in AF has little noticeable effect when cardiac function is normal, but it can result in a significant decrease in stroke output when diastolic filling is impaired by mitral stenosis or reduced ventricular compliance (1). This effect is more pronounced at rapid heart rates (because of the reduced time for ventricular filling).

Atrial fibrillation predisposes to thrombus formation in the left atrium, and these thrombi can dislodge and embolize in the cerebral circulation, thereby producing an acute ischemic stroke. The average yearly incidence of ischemic stroke is 3 to 5 times higher in patients with AF, but only when the AF is accompanied by certain risk factors (e.g., heart failure, mitral stenosis, advanced age) (1,2). Recommendations for antithrombotic therapy are presented later.

The acute management of AF can be divided into 3 components: i.e., control of the heart rate, cardioversion (electrical and pharmacological), and thromboprophylaxis.

The typical strategy for uncomplicated AF is to slow the ventricular response with drugs that prolong AV conduction. A variety of drugs are available for this purpose, and the popular ones are included in Table 15.1. The following is a brief description of these drugs.

titration. Effective In AF associated with hyperadrenergic states.. Digoxin, Dosing Regimen and Comments: = Dosing:. Digoxin, Dosing Regimen and Comments: = 0.25 mg IV every 2 hr to a total dose of 1.5 mg, then 0.125-0.375 mg IV dally.. Digoxin, Dosing Regimen and Comments: = Comment:. Digoxin, Dosing Regimen and Comments: = Slow-acting drug that should not be used alone for acute rate control. Preferred for AF with heart failure.
From the cinical practice guidelines in References 1 and 4.
DILTIAZEM: Diltiazem is a calcium channel blocker that achieves satisfactory rate reduction in up to 90% of cases of uncomplicated AF (9). The acute response to diltiazem is shown in Figure 15.4: note the superiority of diltiazem over amiodarone and digoxin after the first hour of therapy. Adverse effects of diltiazem include hypotension and cardiac depression. Although diltiazem has negative inotropic effects, it has been used safely in patients with moderate to severe heart failure (10).
B-RECEPTOR ANTAGONISTS: £-blockers achieve successful rate control in 70% of cases of acute AF (11), and they are the preferred agents for rate control when AF is associated with hyperadrenergic states (such as acute MI and post-cardiac surgery) (1,5). Two B-blockers with proven efficacy in AF are esmolol (Brevibloc) and metoprolol (Lopressor). Both are cardioselective agents that preferentially block B-1 receptors in the heart. Esmolol is more attractive than metoprolol because it is an ultra short-acting drug (with a serum half-life of 9 minutes), which allows rapid dose titration to the desired effect (12).

AMIODARONE: Amiodarone prolongs conduction in the AV node, but is less effective for acute rate control than diltiazem, as shown in Figure 15.4. However, amiodarone produces less cardiac depression than diltiazem (13), and it is favored by some for AF with heart failure (1). Amiodarone is also an antiarrhythmic agent (Class III), and is capable of converting AF to a sinus rhythm. The success rate for converting recent-onset AF is 55% to 95% when a loading dose and continuous infusion are used, and the daily dose exceeds 1500 mg (1,14). However, unanticipated cardioversion with amiodarone can be a problem when patients are not adequately anticoagulated (see later).
FIGURE 15.4 Comparison of acute rate control with intravenous diltiazem, amiodarone, and digoxin in patients with uncomplicated atrial fibrilation. Data points marked with a star indicate a significant difference with diltiazem compared to the other two drugs. Data from Reference 10.
The adverse effects of short-term intravenous amiodarone include hypo-tension (15%), infusion phlebitis (15%), bradycardia (5%), and elevated liver enzymes (3%) (15,16). Hypotension is the most common side effect, and is related to the combined vasodilator actions of amiodarone and the solvent (polysorbate 80 surfactant) used to enhance water solubility of injectable amiodarone (17). Amiodarone also has several drug interactions by virtue of its metabolism by the cytochrome P450 enzyme system in the liver (16). The most relevant interactions in the ICU setting are inhibition of digoxin and warfarin metabolism, which requires attention if amiodarone is continued orally for long-term management.

DIGOXIN: Digoxin prolongs conduction in the AV node, and is a popular drug for longterm rate control in AF. However, the response to intravenous digoxin is slow to develop; i.e., it is usually not apparent for at least one hour, and the peak response can take longer than 6 hours to develop (1). In comparison, the response to an intravenous dose of diltiazem is apparent in 3-5 minutes, and the peak response occurs at 5-7 minutes (17). The superiority of diltiazem over digoxin for acute rate control in AF is demonstrated in Figure 15.4. Note that the heart rate remains above 100 beats/min (the threshold for tachycardia) at 6 hours after the start of rate-control therapy with digoxin. Digoxin may have a role in treating AF associated with heart failure, but it should not be used alone for acute rate control in AF (1,4).

More than 50% of episodes of recent-onset AF will spontaneously revert to sinus rhythm within the first 72 hours (18). For the remaining cases of AF that are complicated by hypotension, pulmonary edema, or myocardial ischemia, direct-current cardioversion is the appropriate intervention. Biphasic shocks have replaced monophasic shocks as the standard of care for cardioversion because they require less energy for a successful result. An energy of 100 J is usually enough for successful cardioversion using biphasic shocks, but 200 J is recommended for the initial cardioversion attempt in the most recent guidelines on AF (1). If additional shocks are needed, the energy level is increased in increments of 100 J to a maximum energy level of 400 J. Success may be fleeting when AF has persisted for more than a year (1).

Drug-induced cardioversion is used in cases of uncomplicated AF that are refractory to rate control, or for first episodes of uncomplicated AF that are less than 48 hours in duration, to eliminate the need for anticoagulation (see later). Several antiarrhythmic agents can be effective in terminating AF, including amiodarone and ibutilide. The success rate of amiodarone in recent-onset AF has been mentioned previously. Ibutilide (at a dose of 1 mg IV over 10 minutes, repeated once if necessary) has a reported success rate of about 50% in recent-onset AF (15). Ibutilide prolongs the QT interval, and is one of the high-risk drugs for promoting polymorphic ventricular tachycardia (torsade de pointes) (15), as shown later in Table 15.4

Recommendations for antithrombotic therapy in AF are shown in Table 15.2. To summarize, anticoagulation is recommended for any patient with AF and one or more of the following risk factors: mitral stenosis, coronary artery disease, congestive heart failure, hypertension, age =75 yrs, diabetes mellitus, and prior stroke or TIA (1,2). This, of course, excludes patients who have a contraindication to anticoagulation.

tCHADS, Score = 0, _ Recommendations: = No anticoagulation. tCHADS, Score 21, _ Recommendations: = Long-term R,, with dabigatran, 150 mg BID". Mitral Stenosis, Stable Coronary Disease, _ Recommendations: = Long-term R,, with warfarin to INR of 2-3. Elective Cardioversion: AF > 48 hr or unknown, _ Recommendations: = Therapeutic anticoagulation from 3 weeks before to 4 weeks after the procedure. Urgent Cardioversion, _ Recommendations: = Therapeutic anticoagulation during, and for 4 weeks after the procedure
TCHADS-: Congestive heart failure 1 point), history of Hypertension (1 point), Ags =75 yrs, (1 point), Diabstes mellitus (1 point), prior Stroke or TIA (2 points).
eDecraase dose ta 75 mg BID for creatinine charance of 15-30 mL'min, and do NOT use if creatinine daarance <15 mL'min.
Recommendations from tha ACCP guidelines in Refarance 2.
DABIGATRAN: The ACCP guidelines (2) recommend the use of dabigatran (150 mg BID), a direct thrombin inhibitor, for patients with AF and one or more risk factors in the CHADS; score. This is based on one study showing that dabigatran in a dose of 150 mg BID (but not lower) was associated with fewer strokes than warfarin (19). This study excluded patients with renal insufficiency because dabigatran is cleared by the kidneys. In fact, it is important to note that a dose reduction of 50% (to 75 mg BID) is required for patients with renal impairment (i.e., creatinine clearance of15-30 mL/min), while the drug is contraindicated in patients with renal failure (i.e., creatinine clearance <15 mL/min) (2).

Since the degree of anticoagulation is not routinely monitored when using thrombin inhibitors, and there is no antidote to reverse dabigatran in cases of drug-associated bleeding, it is probably wise to avoid this drug in patients with any degree of renal impairment.
CARDIOVERSION: Thromboembolic events following cardioversion have been reported in 1% to 7% of patients who were not anticoagulated at the time of the procedure (1,2). This is the basis for the recommendation to begin anticoagulation 3 weeks prior to an elective cardioversion, and continue anticoagulation for 4 weeks after the procedure (2). For emergent cardioversion, anticoagulation with heparin should be initiated as soon as possible prior to the procedure, followed by four weeks of anticoagulation after the procedure. When AF is less than 48 hours in duration, cardioversion has a low risk of thromboembolism (<1%), and peri-procedural anticoagulation is not necessary (1).

The Wolff-Parkinson-White (WPW) syndrome (short P-R interval and delta waves before the QRS) is characterized by recurrent supraventricular tachycardias that originate from an accessory pathway in the AV node. (The mechanism for these tachycardias is explained in the section on re-entrant tachycardias.) When atrial fibrillation occurs in a patient with an accessory pathway, drugs that block conduction in the AV node (e.g., calcium channel blockers, B-blockers, digoxin) are unlikely to slow the ventricular rate because the accessory pathway is not blocked (1,4). Furthermore, selective block of the AV node can precipitate ventricular fibrillation (4). Therefore, drugs that block the AV node (e.g., calcium channel blockers, B-blockers, and digoxin) should NOT be used when AF is associated with the WPW syndrome (1,4). The preferred management in this situation is electrical cardioversion or antiarrhythmic drugs such as amiodarone or procainamide.

Multifocal atrial tachycardia or MAT (see Panel A in Figure 15.3) is a disorder of the elderly (average age = 70), and over half of the cases occur in patients with chronic lung disease (20). Other associated conditions include magnesium and potassium depletion, and coronary artery disease (21).

Paroxysmal supraventricular tachycardias (PSVT) are narrow-QRS-complex tachycardias that are second only to atrial fibrillation as the most prevalent rhythm disturbances in the general populace.

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There are 5 different types of PSVT, based on the location of the re-entrant pathway. The most common PSVT is AV nodal re-entrant tachycardia, where the re-entrant pathway is located in the AV node.

AV nodal re-entrant tachycardia (AVNRT) accounts for 50 to 60% of cases of PSVT (23). It typically appears in patients who have no history of heart disease, and is more common in women than men. The onset is abrupt, and the predominant complaints are palpitations and lightheadedness. There is no evidence of heart failure or myocardial ischemia, and significant hemodynamic compromise is uncommon. The ECG shows a narrow-QRS-complex tachycardia with a regular rhythm and a heart rate between 140 and 250 bpm (23). There are often no visible P waves on the ECG, as shown in Figure 15.2.
AVNRT is frequently mistaken for sinus tachycardia, but the onset is different (abrupt onset vs. gradual onset for sinus tachycardia), the heart rate is often different (usually higher than 140 beats/min in AVNRT and rarely higher than 150 beats/min in sinus tachycardia), and the ECG appearance is different (no visible P waves in AVNRT and P waves before each QRS complex in sinus tachycardia).

Maneuvers that increase vagal tone are recommended as an initial attempt to terminate AVNRT. A variety of maneuvers have been identified, including carotid sinus massage and the Valsalva maneuver (maximal expiratory effort with a closed glottis). The success of these maneuvers has not been adequately studied; one study of 148 patients with paroxysmal SVT showed a success rate of 18% with the Valsalva maneuver and 12% with carotid sinus massage (24).

When vagal maneuvers are ineffective, adenosine is the drug of choice for terminating reentrant tachycardias involving the AV node (25,26). Adenosine is an endogenous nucleotide (the backbone of the ATP molecule) that relaxes vascular smooth muscle and slows conduction in the AV node. When given by rapid intravenous injection, adenosine has a rapid onset of action (<30 sec) and produces a transient AV block that can terminate AV nodal re-entrant tachycardias. Adenosine is quickly cleared from the bloodstream (by receptors on RBCs and endothelial cells), and the effects last only 1-2 minutes.

The dosing regimen for adenosine is shown in Table 15.3. The initial dose is 6 mg, which is injected rapidly into a peripheral vein and followed by a saline flush. Optimal results are obtained if the drug is injected at the hub of the catheter. If conversion to sinus rhythm does not occur after 2 minutes, a second 12 mg dose is given, and this can be repeated once if necessary. This regimen terminates re-entrant tachycardias in over 90% of cases (24-26). The effective dose of adenosine was determined using drug injection into peripheral veins, and ventricular asystole has been reported when standard doses of adenosine are injected through central venous catheters (27). As a result,a dose reduction of 50% has been recommended by some (including the manufacturer) when adenosine is injected through a central venous catheter (27).
able 15.3 Intravenous Adenosine for Paroxysmal SV
From Referancas 4, 25, 26.

Dosing Regimen, 1 = 1. Deliver through a peripheral vein. 2, Give 6 mg by rapid IV injection and flush catheter with saline. 3. If response inadequate after 2 min, give 12 mg by rapid IV injection and flush catheter with saline. 4. If response still inadequate after 2 min, another 12 mg can be given by rapid IV injection.. Dose Adjustments, 1 = Decrease dose by 50% for: * Drug injection into the superior vena cava « Patient receiving calcium antagonist, B-blocker, or dipyridamole. Drug Interactions, 1 = ¢ Dipyridamole (blocks adenosine uptake) * Theophylline (blocks adenosine receptors). Contraindications, 1 = e Asthma e 2nd or Sid degree AV block « Sick sinus syndrome. Adverse Effects, 1 = e Bradycardia, AV block (50%) e Facial flushing (20%) e Dyspnea (1295) « Chest pressure (7%)

The adverse effects of adenosine are short-lived because of the drug's ultra-short duration of action. The most frequent adverse effect is post-conversion bradycardia, including various degrees of AV block. The AV block is refractory to atropine, but resolves spontaneously within 60 seconds (26). Dipyridamole enhances the AV block produced by adenosine (26). Adenosine is contraindicated in patients with asthma because of reports of adenosine-induced bronchospasm (28), but more recent studies indicate that adenosine produces a sense of dyspnea, but not bronchospasm, in asthmatic subjects (29).

Methylxanthines like theophylline block adenosine receptors and reduce the effectiveness of adenosine in terminating re-entrant tachycardias (26). This interaction has been minimized by the diminished use of theophylline as a bronchodilator. When PSVT does not respond to adenosine, the calcium channel blockers diltiazem or verapamil can be effective. (The dosing regimen for diltiazem is shown in Table 15.1, and the dosing regimen for verapamil was presented in the section on paroxysmal SVTs).

Ventricular tachycardia (VT) is a wide-QRS-complex tachycardia that has an abrupt onset, a regular rhythm, and a rate above 100 bpm (usually 140-200 bpm). The appearance can be monomorphic (uniform QRS complexes) or polymorphic (multiple QRS morphologies). VT rarely occurs in the absence of structural heart disease (30), and when it is sustained (i.e., lasts longer than 30 seconds), it can be an immediate threat to life.

Monomorphic VT can be difficult to distinguish from an SVT with prolonged AV conduction. This is demonstrated in Figure 15.5. The tracing in the upper panel shows a wide-QRScomplex tachycardia that looks like monomorphic VT. The tracing in the lower panel shows spontaneous conversion to sinus rhythm. Note that the QRS complex remains unchanged after the arrhythmia is terminated, revealing an underlying bundle branch block. Thus, the apparent VT in the upper panel is actually an SVT with a preexisting bundle branch block.
FIGURE 15.5 Upper panel shows a wide-QRS-compkex tachycardia that looks like monomorphic VT. However, the lower panel shows spontaneous conversion to a sinus rhythm, which reveals an underlying bundle branch block, indicating that the rhythm in the upper panel is an SVT with a preexisting bundle branch block. Tracings courtesy of Richard M. Greenberg, M.D.

There are two abnormalities on an ECG that will identify VT as the cause of a wide-QRScomplex tachycardia.
1. The atria and ventricles beat independently in VT, and this results in AV dissociation on the ECG, where there is no fixed relationship between P waves and QRS complexes. This may not be evident on a single-lead tracing, and is more likely to be discovered on a 12-lead ECG. (P waves are most visible in the inferior limb leads and the anterior precordial leads. )
. The presence of fusion beats like the one in Figure 15.6 is indirect evidence of VT. A fusion beat is produced by the retrograde transmission of a ventricular ectopic impulse that collides with a supra-ventricular (e.g., sinus node) impulse. The result is a hybrid QRS complex that is a mixture of the normal QRS complex and the ventricular ectopic impulse. The presence of a fusion beat (which should be evident on a single-lead ECG tracing) is indirect evidence of ventricular ectopic activity.
If there is no definitive evidence of VT on the ECG, the presence or ab-sence of heart disease can be useful; i.e., VT is the cause of 95% of wide-QRS-complex tachycardias in patients with underlying heart disease (31). Therefore, a wide-QRS-complex tachycardia should be treated as probable VT in any patient with underlying heart disease.
FIGURE 15.6 An exampk of a fusion beat (circled in red), which is a hybrid QRS complex produced by the collision of a ventricular ectopic impube and a supraventricular (e.g., sinus node) impule. The presence of fusion beats is evidence of ventricular ectopic activity.

The management of patients with a wide-QRS-complex tachycardia can proceed as follows. This approach is organized in a flow diagram in Figure 15.7.
1. If there is evidence of hemodynamic compromise, electrical cardioversion is the appropriate intervention, regardless of whether the rhythm is VT or SVT with aberrant conduction. The shocks should be synchronized (timed with the QRS complex), with an initial shock of 100 J (biphasic or monophasic shocks) (30). This should terminate most cases of monomorphic VT, but shocks of 200 J (biphasic shocks) and up to 360 J (monophasic shocks) may be necessary.
2. If there is no hemodynamic compromise and the diagnosis of VT is certain, intravenous amiodarone should be used to terminate the arrhythmia. Amiodarone is the favored drug for suppression of monomorphic VT (4).
3. If there is no hemodynamic compromise and the diagnosis of VT is uncertain, the response to adenosine can be helpful because adenosine will abruptly terminate most cases of paroxysmal SVT, but will not terminate VT. If a wide-QRS-complex tachycardia is refractory to adenosine, the likely diagnosis is VT, and intravenous amiodarone is indicated for arrhythmia suppression.

Torsade de pointes ("twisting around the points") is a polymorphic VT with QRS complexes that appear to be twisting around the isoelectric line of the ECG, as shown in Figure 15.8. This arrhythmia is associated with a prolonged QT interval, and it can be congenital or acquired. The acquired form is much more prevalent, and is caused by a variety of drugs and electrolyte abnormalities that prolong the QT interval (32,33). There is also a polymorphic VT that is associated with a normal QT interval, and the predisposing condition in this arrhythmia is myocardial ischemia (4).
FIGURE 15.7 Flow diagram for the acute management of patients with wide-QRS-compkex tachycardia. Based on recommendations in Reference 4.

The drugs most frequently implicated in torsade de pointes are listed in Table 15.4 (33). The prominent offenders are antiarrhythmic agents (Class IA and III), macrolide antibiotics, neuroleptic agents, cisapride (a pro-motility drug), and methadone. The electrolyte disorders that prolong the QT interval include hypokalemia, hypocalcemia, and hypomagnesemia.
FIGURE 15.8 Torsade de pointes, a polymorphic ventricular tachycardia described as "twisting around the (isoelectric) points." Tracing courtesy of Richard M. Greenberg, M.D.
IA « ; ,, { |, Antiarrhythmics = Quinidine Disopyramide Procainamide (bute Sotalol. IA « ; ,, { |, Antimicrobials = Clarithromycin Erythromycin Pentamidine. IA « ; ,, { |, Neuroleptics = Chlorpromazine Thioridazine Droperidol Haloperidol :. IA « ; ,, { |, Others: = Clsapride Methadone
From Reference 33. For a complete list of drugs, go to ww.torsades.org.

The QT interval is the ECG manifestation of ventricular depolarization and repolarization, and is measured from the onset of the QRS complex to the end of the T wave. The longest QT intervals are usually in precordial leads V3 and V4, and these leads are most reliable for assessing QT prolongation. The QT interval varies inversely with heart rate (e.g., an increase in heart rate shortens the QT interval), and therefore a rate-corrected QT interval (QTc) provides a more accurate assessment of QT prolongation. The accepted method for determining the QTc is to divide the QT interval by the square root of the R-R interval (33-35); i.e.,

A normal QT interval corresponds to a QTc "0.44 seconds, and a QTc >0.5 seconds represents an increased risk of torsade de pointes (35). The risk of torsade de points when the QTc = 0.45-0.5 seconds is not clear (33,35). There is no consensus on how to measure the QTc in highly irregular rhythms like atrial fibrillation.

The management of polymorphic VT is summarized as follows:
1. Sustained polymorphic VT requires nonsynchronized electric cardio-version (i.e., defibrillation) (4).
2. For torsade de pointes, intravenous magnesium is used for pharmacological management (and can be combined with electrical cardioversion). There is no universal dosing regimen for magnesium in this setting. The ACLS guidelines

The tachyarrhythmia that will dominate your time more than all the others combined is atrial fibrillation (AF). This is rarely a primary problem (in a non-coronary ICU), but it can require more attention than the primary problem. This arrhythmia is like an annoying person you meet who demands a lot of attention but is not threatening (not lifethreatening, in the case of the arrhythmia).
The tachyarrhythmias that are an immediate threat to life (i.e., ventricular tachycardia) are not prevalent in non-coronary ICUs, even in patients with circulatory shock and multiorgan failure. In fact, in patients who are near death and are not resuscitated, the progression to ventricular asystole is usually populated by bradyarrhythmias (e.g. AV blocks). This paucity of troublesome ventricular tachycardia outside the coronary care unit may be explained by the trigger for ventricular tachycardia (i.e., focal, rather than global, myocardial ischemia), which is not a common occurrence in critical illness when coronary artery disease is not the dominant problem.

1. Fuster V, Ryden LE, Cannom DS, et al. 2011 ACCF/AHA/ESC focused updates incorporated into the ACC/AHA/ESC 2006 guidelines for the management of patients with atrial fibrillation. J Am Coll Cardiol 2011; 57:e101-e198.
2. You JJ, Singer DE, Howard PA, et al. Antithrombotic therapy for atrial fibrillation. Antithrombotic Therapy and Prevention of Thrombosis, 9th ed. American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012; 141(Suppl):e531S—e575S.
3. The Task Force for the Management of Atrial Fibrillation of the European Society of Cardiology (ESC). Guidelines for the management of atrial fibrillation. Europace 2010; 12:1360-1420.
4. Neumar RW, Otto CW, Link MS, et al. Part 8: adult advanced cardiovascular life support. 2010 American Heart Association Guidelines for Cardiopul-monary Resuscitation and Emergency' Cardiovascular Care. Circulation 2010; 122(Suppl):S729-S767.

20. Kastor J. Multifocal atrial tachycardia. N Engl J Med 1990; 322:1713-1720.
21. Iseri LT, Fairshter RD, Hardeman JL, Brodsky MA. Magnesium and potassium therapy in multifocal atrial tachycardia. Am Heart J 1985; 312:21-26.
22. McLean RM. Magnesium and its therapeutic uses: a review. Am J Med 1994; 96:63-— 76.
Paroxysmal Supraventricular Tachycardias, 1 = Paroxysmal Supraventricular Tachycardias. 23., 1 = Trohman RG. Supraventricular tachycardia: implications for the internist. Crit Care Med 2000; 28(Suppl):N129-N135.. 24., 1 = Lim SH, Anantharaman V, Teo WS, et al. Comparison of treatment of supraventricular tachycardia by Valsalva maneuver and carotid sinus massage. Ann Emerg Med 1998; 31:30-35.. 25., 1 = Rankin AC, Brooks R, Ruskin JM, McGovern BA. Adenosine and the treatment of supraventricular tachycardia. Am J Med 1992; 92:655-664.. 26., 1 = Chronister C. Clinical management of supraventricular tachycardia with adenosine. Am J Crit Care 1993; 2:41-47.. 27., 1 = McCollam PL, Uber W, Van Bakel AB. Adenosine-related ventricular asystole. Ann Intern Med 1993; 118:315-316.. 28., 1 = Cushley MJ, Tattersfield AE, Holgate ST. Adenosine-induced bronchoconstriction in asthma. Am Rev Respir Dis 1984; 129:380-384.. 29., 1 = Burki NK, Alam M, Lee L-Y. The pulmonary effects of intravenous adenosine in asthmatic subjects. Respir Res 2006; 7:139-146.

The study of the causes of things must be preceded by the study of things caused.
John Hughlings Jackson (1835-1911)
The dominance of coronary artery disease in the Western world is highlighted by a recent claim that fatal coronary events occur once every minute in the United States (1). Despite this dismal estimate, appropriate early intervention in patients with acute myocardial infarction can save lives. This survival benefit, however, is time-dependent, and steadily declines in the hours that follow the first signs of ischemic injury. This is the famous time is muscle axiom that drives the early management of acute myocardial infarction.
This chapter describes the early interventions that provide a clinical benefit in acute myocardial infarction, using the recommendations in the clinical practice guidelines listed at the end of the chapter (2-8).

Ischemic myocardial injury is the result of an occlusive thrombus in one or more coronary arteries. The trigger for thrombus formation is rupture of an atherosclerotic plaque (9), which releases thrombogenic lipids and activates platelets and clotting factors (see Figure 16.1). Plaque disruption may be the result of liquefaction caused by local inflammation (10). Hydraulic stress may also play a role, because ruptured plaques are typically located at branch points in the coronary circulation (11).

Coronary artery thrombosis is responsible for three clinical conditions: ST-segment elevation myocardial infarction (STEMI), non-ST-segment elevation myocardial infarction (NSTEMI), and unstable angina (UA). The first condition (STEMI) is the result of complete thrombotic occlusion of the involved artery, while the other two conditions (NSTEMI and UA) are the result of either partial thrombotic occlusion or transient complete occlusion with spontaneous revascularization (2-6). These 3 clinical conditions are called acute coronary syndromes (ACS).
FIGURE 16.1 Illustration showing the pathogenesis of acute coronary syndromes. Rupture of an atherosclerotic plaque leads to activation of platelets and clotting factors (upper panel), and results in the formation of an occlusive thrombus (lower panel).

The following measures are used when ACS is first suspected, often before the diagnostic evaluation is completed. Some of these interventions, which are summarized in Table 16.1, are used in the pre-hospital setting.

, 1 = Dosing:. , 2 = ©, by nasal prongs or face mask to maintain $aO, >94%. Nitroglycerin, 1 = Dosing: Comment:. Nitroglycerin, 2 = For chest pain, 0.4 mg SL or by mouth spray, and repeat every 5 min x 2 if needed. For re- current pain, CHF, or high BP, Infuse at 5 g/min and Increase by 5-10 ug/min every 5 min to desired effect or to dose rate of 200 pg/min. Avoid in patients with RV infarction and for 24 hr. Morphine, 1 = Dosing: Comment:. Morphine, 2 = 2-4 mg with increments of 2-8 mg IV every 5-15 min as needed. Morphine-induced respiratory depression Is uncommon In acute coronary syndromes.. Aspirin, 1 = Dosing: Comment:. Aspirin, 2 = 162-325 mg (chewable form) Initially, then 75-162 mg (enteric-coated tablets) daily. Initial dose should be chewed to enhance buccal absorption.. B-Blockers, 1 = Dosing:. B-Blockers, 2 = Atenolol: 10 mg IV, then 100 mg PO daily, or Metoprolol: 5 mg lV avery 5 min for 3 doses, then 50 mg PO q 6h for 48 hr, then 100 mg PO BID.
Dosing regimens from the clinical practica guidelines in Referances 2, 3, and 5.

Providing supplemental O> has been a standard practice in patients with ACS (3,5), even when arterial oxygenation is normal. This practice has been questioned in recent years (12) because oxygen promotes coronary vasoconstriction (oxygen is a vasoconstrictor in all organs except the lungs, where it acts as a vasodilator), and because oxygen is the source of toxic oxygen metabolites (see Figure 14.1), which have been implicated in reperfusion injury (13). The deleterious effects of oxygen are emphasized in a recent study showing an increased risk of unfavorable outcomes associated with O, therapy in acute MI (14).
Concern about the potential for harm from unregulated O> breathing is evident in the most recent guidelines on acute coronary syndromes from the American Heart Association (2), which recommends supplemental O> only when the arterial O> saturation (SaQ>) falls below 94% (2). This is higher than the standard threshold for O» breathing (SaOz <90%), but it is still a significant step in the realization that oxygen can be a toxic gas.

The benefit of B-Receptor antagonists in ACS is based on their ability to reduce cardiac work and decrease myocardial energy requirements. Early institution of beta-blocker therapy is recommended for all patients with ACS who do not have a contraindication to B-Receptor blockade (2,3,5,7,8). These contraindications include high-grade AV block, systolic heart failure, hypotension, and reactive airway disease. In addition, B-Receptor antagonists should not be used for cocaine-induced chest pain or myocardial infarction because there is a risk of aggravated coronary vasospasm from unopposed a-adrenergic receptor activity (3).
Oral B-blocker therapy is suitable for most cases of ACS, while intravenous therapy is reserved for patients with persistent chest pain, tachycardia, and hypertension (2,3,7). The B-blockers used most often in clinical trials of ACS are atenolol (Tenormin) and metoprolol (Lopressor), and the dosing regimens for these drugs is shown in Table 16.1.

The principal determinant of outcomes in acute coronary syndromes is the ability to restore patency in occluded coronary arteries using thrombolytic therapy or percutaneous coronary intervention (PCI), which includes coronary angioplasty and stent placement, when indicated.
Hours from Onset of Chest Pain to Thrombolytic Therapy
FIGURE 16.2 The survival benefit of thrombolytic therapy in relation to the time elapsed from the onset of chest pain to the start of therapy. STEMI = ST-segment elevation myocardial infarction, LBB = left bundle branch block. Data from Reference 19.

Active bleeding other than menses Malignant Intracranial neoplasm (primary or metastatic) Cardiovascular anomaly (8.g. AV malformation) Suspected aortic dissection Ischemic stroke within 3 months (but not within 3 hrs) Prior history of intracranial hemonhage Significant closed-head or facial trauma in past 3 months, ‘Relative Contraindications = Systolic BP >180 mm Hg or diastolic BP >110 mm Hg Active bleeding In past 4 weeks Noncompressible vascular punctures Major surgery in past 3 weeks Traumatic or prolonged (=10 min) CFR Ischemic stroke over 3 months ago Dementia Active peptic ulcer disease Pregnancy Ongoing therapy with warfarin
Fram Referance 2.

Thrombolytic agents act by converting plasminogen to plasmin, which then breaks fibrin strands into smaller subunits. The fibrinolytic drugs used in ACS are shown in Table 16.3 These drugs act primarily on the plasminogen that is bound to fibrin (clot-specific fibrinolysis), and this limits the extent of systemic fibrinolysis and thereby limits the risk of unwanted hemorrhage.
ALTEPLASE (Activase) is a recombinant tissue plasminogen activator (tPA) that was popularized in 1993 by the GUSTO trial (21), which showed superior clinical outcomes with alteplase compared to streptokinase (the original fibrinolytic agent used in ACS). Alteplase is given as a fixed IV bolus dose followed by a 90-minute weight-based drug infusion (see Table 16.3). It has largely been replaced by the more rapid-acting fibrinolyitc agents, but there is no evidence of inferior clinical outcomes with alteplase compared to the more rapidly-acting fibrinolyitc agents (see next).
RETEPLASE (Retavase) is a recombinant variant of tPA that is given as an IV bolus (10 Units), which is repeated in 30 minutes. It produces more rapid clot lysis than alteplase (22), but clinical trials have shown no survival advantage with reteplase when compared to alteplase (23). Rete-plase is the only fibrinolytic agent that is given in fixed (not weight-based) doses, and this has created some popularity for the drug.
TENECTEPLASE (TNkK-ase) is another variant of tPA that is given as a single IV bolus using a weight-based dosing regimen (see Table 16.3). It produces more rapid clot lysis than reteplase (24), but clinical trials have shown no survival advantage with tenecteplase compared to alteplase (25).

Alteplase, Comments ng Regimen and = Dosing:. Alteplase, Comments ng Regimen and = 15 mg IV bolus, then 0.75 ma/kg (not to exceed 50 mg) over 30 min, then 0.5 ma/kg (not to exceed 35 mq) over 60 min. Maximum dose Is 100 mg over 90 min.. , Comments ng Regimen and = Comment:. , Comments ng Regimen and = The original (and slowest-acting) clot-specific thrombolytic agent.. Reteplase, Comments ng Regimen and = Dosing: Comment:. Reteplase, Comments ng Regimen and = 10 Units as lV bolus and repeat in 30 min. Produces more rapid clot lysis than alteplase, but clinical outcomes are no different.. Tenecteplase, Comments ng Regimen and = Dosing:. Tenecteplase, Comments ng Regimen and = Given as a single IV bolus using weight-based dosing: 30 mg for <60 kg, 35 mg for 60-69 kg, 45 mg for 80-89 kg, and 50 mg for = 90 kg. Produces the most rapid clot lysis, but clinical outcomes are not superior to other thrombolytic. , Comments ng Regimen and = Comment:. , Comments ng Regimen and = agents.
Dosing regimens ara manufacturer's racommendations.

Clot-specific fibrinolytic agents produce some degree of systemic fibrinolysis, which can deplete circulating fibrinogen and create an increased risk of bleeding. The risk of major bleeding such as intracerebral hemorrhage (0.5-1%) and extracranial bleeding that requires blood transfusions (5-15%) is equivalent with alteplase, reteplase, and tenecteplase (24,26). Major bleeding from thrombolysis can be treated with cryoprecipitate (10 to 15 bags) followed by fresh frozen plasma (up to 6 units) if necessary (the goal is a serum fibrinogen =1 mg/ml). The use of antifibrinolytic agents such as epsilon-aminocaproic acid (5 grams IV over 15-30 min) is discouraged because of the risk for thrombosis (26).

Despite differences in pharmacokinetic properties, the fibrinolytic agents in Table 16.3 are equivalent in terms of survival benefit and risk of bleeding. The clinical experience with thrombolytic therapy then leads to the following summation: the important issue in thrombolytic therapy is not which agent to use, but how quickly to use it.

In 1977, a Swiss cardiologist named Andreas Gruntzig used a homemade balloon-tipped catheter to reopen an occluded left main coronary artery. This "Gruntzig procedure" (coronary angioplasty) was promptly sanctioned by the American Heart Association, and was introduced in the 1980s as an alternative to thrombolytic therapy. (Unfortunately, Dr. Gruntzig died in a plane crash in 1985, just as his procedure was gaining widespread use.) In the late 1990s, the placement of stents was introduced to maintain vascular patency after coronary angioplasty. The combination of coronary arteriography, angioplasty, and stent placement is known as percutaneous coronary intervention (PCI).

Several clinical trials have shown that PCI is superior to thrombolytic therapy for restoring flow in occluded arteries and reducing the incidence of unfavorable outcomes (1-3, 27— 29). This is shown in Figure 16.3. The bar graphs on the left (depicting vascular events) show superior results with PCI for restoring normal flow in infarct-related arteries and preventing reocclusion. The bar graphs on the right (depicting clinical outcomes) show superior results with PCI for reducing the mortality rate and the reinfarction rate.
TIMING: The survival benefit of PCI is time-dependent, similar to thrombolytic therapy (see Figure 16.2). This is demonstrated in Figure 16.4, which shows the mortality rate (at 30 days) in relation to the time elapsed from hospital arrival to coronary angioplasty (the "door-to-balloon" time) (30). The mortality rate rises steadily with increasing delays to angioplasty, and the increased mortality becomes significant when the delay exceeds 2 hours. This is the basis for the recommendation that PCI should be performed within 90 minutes after hospital arrival (2-4).
FIGURE 16.3 Comparative effects of coronary angioplasty and thrombolytic therapy on vascular events (graph on the left) and clinical outcomes (graph on the right) in patients with ST-elevation myocardial infarction. Data from references
FIGURE 16.4 Mortality rate in relation to the time elapsed from hospital arrival to coronary angioplasty (door-to-balloon time). Asterisk indicates a significant difference compared to the initial time period (0-60 min). Adapted from data in Reference 30.

INTERHOSPITAL TRANSFER: The major limitation of PCI is availability; i.e., less than 25% of hospitals in the United States can provide PCI on a timely basis. One solution for this problem is a facilitated transfer from "have-not" hospitals to hospitals that can provide emergent PCI. Clinical studies have shown that interhospital transfer for PCI can result in a survival benefit if the transfer can be completed within 1-2 hours of the patient's arrival at the "have not" hospital (31). Therefore, for patients who are candidates for reperfusion therapy, interhospital transfer for PCI is encouraged if the total door-to-balloon time, including the transfer time, does not exceed 90 minutes (2-4). Strict adherence to this time requirement is not inviolate because delayed PCI may still be a better option than no PCI (e.g., in patients with heart failure or hemodynamic instability).

Decisions regarding immediate reperfusion therapy in suspected cases of STEMI can be organized as a series of four questions, as shown in Table 16.4. The first two questions determine if the patient is a candidate for early reperfusion therapy, and the last two questions determine the type of reperfusion therapy that is most appropriate (i.e., PCI or thrombolytic therapy).

PCI is also advantageous in the following situations:
- 1. For patients with non-ST elevation MI (NSTEMI) who have risk factors for a poor outcome (e.g., unrelenting chest pain, heart failure, or hemodynamic instability) (4-6). 2. When thrombolytic therapy fails.
When an acute coronary syndrome ts first suspected, ask yourself the following questions immediately:
- 1. Does the ECG show ST elevation (2 0.1 mV) in at least 2 contiguous leads, or a new left bundle branch block?
If the answer is YES, go to the next question
- 2. Did the chest pain begin less than 12 hours ago?
- 3. Gan PCI be performed in a timely manner (here or elsewhere?)
If the answer is YES, proceed to PCI If the answer is NO, go to the next question
- 4. Does the patient have any reason to withhold lytic therapy?
If the answer is NO, begin thrombolytic therapy

Antithrombotic therapy with heparin and antiplatelet agents other than aspirin have proven benefits in ACS when used with or without reperfusion therapy.

When platelets are activated, specialized glycoprotein receptors on the platelet surface (named IIb and IIIa) change their configuration and begin to bind fibrinogen. This allows fibrinogen molecules to form bridges between adjacent platelets, which promotes platelet aggregation. Glycoprotein receptor antagonists (also called IIb/IIIa inhibitors) block fibrinogen binding to activated platelets and inhibit platelet aggregation. These drugs are the most potent antiplatelet agents available, and are sometimes referred to as the superaspirins.

Drug
Dosing
From the cinical practice guidelines in Reference 6.

Abciximab, Regimen = Dosing: Load with 0.25 mg as IV bolus, then infuse at 0.125 pg/kg/min (maximum rate is 10 ug/min) for up to 12 hr.. Eptifibatide, Regimen = Dosing: Load with 180 ug/kg as IV bolus, then infuse at 2 ug/kg/min for 12-18 hr. For PCI in STEMI, repeat bolus cose in 10 min if renal function is normal. Reduce infusion rate by 50% when creatinine clearance is <50 mL/min.. Tirofiban, Regimen = Dosing: Load with 25 ug/kg as IV bolus, then infuse at 0.1 wg/kg/min for 12-24 hr. Reduce infusion rate by 50% when creatinine clearance Is <30 mL/min.

The IIb/IIIa inhibitors available for clinical use include abciximab (ReoPro), eptifibatide (Integrilin), and tirofiban (Aggrastat). All three are given by in-travenous infusion using the dosing regimens in Table 16.5. These drugs are used in high-risk patients who receive emergent PCI, and are given just before or at the start of the procedure (6,32). They are managed primarily by invasive cardiologists, and are described only briefly here. Abciximab is (an almost unpronounceable name for) a monoclonal antibody that is the most potent, most expensive, and longest-acting IIb/IIa inhibitor. After discontinuing abciximab, bleeding times can take 12 hours to normalize (32). Eptifibatide (a synthetic peptide) and tirofiban (a tyrosine derivative) are short-acting agents that are cleared by the kidneys. After discontinuing these drugs, bleeding times return to normal in 15 minutes for eptifibatide and 4 hours for tirofiban (32). Dose adjustments are recommended for both drugs in renal insufficiency, as indicated in Table 16.5.

The appearance of decompensated heart failure and cardiogenic shock in the first few days after an acute MI is an ominous sign, and usually indicates a catastrophic structural defect like acute mitral regurgitation, or extensive muscle injury resulting in cardiac pump failure. Fatal outcomes are common in these conditions despite timely interventions.

The following defects are usually the result of transmural (ST-elevation) infarctions.

Acute mitral regurgitation is the result of papillary muscle rupture, and presents with the sudden onset of pulmonary edema and the characteristic holosystolic murmur radiating to the axilla. The pulmonary artery occlusion pressure should show prominent V waves, but this can be a non-specific finding. Diagnosis is by echocardiography, and arterial vasodilators (e.g., hydralazine) are used to relieve pulmonary edema pending surgery. Mortality is 70% without surgery and 40% with surgery (34).

Rupture of the septum separating the two ventricles can occur anytime in the first 5 days after an acute MI, and the diagnosis can be elusive without cardiac ultrasound. There is a step-up in O> saturation from right atrial to pulmonary artery blood, but this is rarely measured. Initial management involves vasodilator (e.g., nitroglycerin) infusions and the intra-aortic balloon pump if needed. Mortality is 90% without surgery and 20% to 50% with surgery (3).

Ventricular free wall rupture occurs in up to 6% of cases of STEMI, and is more common with anterior MI, fibrinolytic or steroid therapy, and advanced age (3). The first signs of trouble are usually return of chest pains and new ST-segment abnormalities on the ECG. Accumulation of blood in the pericardium often leads to rapid deterioration and cardiovascular collapse from pericardial tamponade. Diagnosis is made by cardiac ultrasound (if time permits), and prompt pericardiocentesis combined with aggressive volume resuscitation is required for hemodynamic support. Immediate surgery is the only course of action, but fewer than half of the patients survive despite surgery (3).

About 10% of cases of ST-elevation MI (STEMI) result in enough muscle damage to produce decompensated heart failure and cardiogenic shock (35). Management involves hemodynamic support followed by coronary angioplasty or coronary bypass surgery. Unfortunately, the mortality rate in cardiogenic shock can be as high as 80% (36), but hospitals that provide timely PCI can expect 10% fewer deaths (36).

Hemodynamic support for acute heart failure and cardiogenic shock is described in Chapter 13. When these conditions are the result of coronary insufficiency, hemodynamic support should be designed to augment cardiac output without increasing cardiac work and myocardial oxygen consumption. Table 16.6 shows the effects of hemodynamic support measures on the determinants of myocardial Oz consumption (preload, contractility, afterload, and heart rate) for acute heart failure and cardiogenic shock. As judged by the net effect on myocardial O2 consumption, vasodilator therapy is the best choice for acute heart failure, and the intra-aortic balloon pump is the best choice for cardiogenic shock.

Hemodynamic support is, of course, a bridge to interventions that re-establish flow in the infarct-related blood vessels. The ACC/AHA guidelines (3) recommend immediate PCI when cardiogenic shock appears within 36 hours of STEMI and when the angioplasty can be performed within 18 hours of the onset of shock. Coronary artery bypass surgery is considered if the cardiac catheterization reveals multivessel disease that is not amenable to angioplasty.
Preload, 1 = ¥. Preload, 2 = ¥. Preload, 3 = ¥. Preload, 4 = -. Contractility, 1 = —. Contractility, 2 = tt. Contractility, 3 = a. Contractility, 4 = ”. Afterload, 1 = v4. Afterload, 2 = ¥. Afterload, 3 = ¥. Afterload, 4 = :. Heart Rate, 1 = =. Heart Rate, 2 = tt. Heart Rate, 3 = a. Heart Rate, 4 = =”. Net effect on myocardial VO,, 1 = bey. Net effect on myocardial VO,, 2 = ht. Net effect on myocardial VO,, 3 = ve. Net effect on myocardial VO,, 4 = TPT EP
IABP = intra-aortic balloon pump; VO = oxygen consumption

Aortic dissection involving the ascending aorta can be mistaken as ACS, and can also be a cause of ACS. However, unlike ACS, aortic dissection is a surgical emergency that is often fatal if not managed appropriately.

Aortic dissection occurs when a tear in the aortic intima allows blood to dissect between the intimal and medial layers of the aorta, creating a false lumen. This process can be the result of atherosclerotic damage in the aorta from hypertension, or accelerated degradation of the aortic wall from a genetic disorder (e.g., Marfan's syndrome). The dissection can originate in the ascending or descending aorta, and can propagate in antegrade and retrograde directions. When a dissection involves the ascending aorta, retrograde propagation can result in coronary insufficiency, aortic insufficiency, and pericardial tamponade, while antegrade propagation can result in neurologic deficits (from involvement of the aortic arch vessels) (37).

The most common complaint is the abrupt onset of chest pain. The pain is often sharp, and can be substernal (ascending aortic dissection) or in the back (descending aortic dissection). Most importantly, the chest pain can subside spontaneously for hours to days (39.40), and this can be a source of missed diagnoses. The return of the pain after a pain-free interval is often a sign of impending aortic rupture. About 5% of patients with acute aortic dissection are devoid of pain (37).

The most common clinical findings are hypertension (50% of patients) and aortic insufficiency (50% of patients) (38,39). Unequal pulses in the upper extremities (from obstruction of the left subclavian artery in the aortic arch) are found in as few as 15% of patients (39). The chest x-ray can show mediastinal widening (60% of cases) (39), but normal chest x-rays are reported in up to 20% of cases (37). The ECG can show ischemic changes (15% of cases) or evidence of MI (5% of cases), but the ECG is normal in 30% of cases (37). Because of the limited sensitivity of clinical findings, additional imaging studies are required for the diagnosis.

The diagnosis of aortic dissection requires one of four imaging modalities (40): magnetic resonance imaging (MRI) (sensitivity and_ specificity 98%), transesophageal echocardiography (sensitivity 98%, specificity 77%), contrast-enhanced computed tomography (sensitivity 94%, specificity 87%), and aortography (sensitivity 88%, specificity 94%). As indicated MRI is the most sensitive and specific imaging modality for the diagnosis aortic dissection. However, the immediate availability of MRI is limited in some hospitals, and CT angio-graphy is often the diagnostic test when aortic dissection is suspected.

There are two essential management goals in aortic dissection: i.e., control of hypertension (to prevent aortic rupture) and prompt surgical correction.

There is one major caveat for blood pressure control in aortic dissection: the reduction in blood pressure should not be accompanied by an increase in cardiac stroke output because increased flow in the aorta can promote further dissection. As a result, ?-receptor antagonists are preferred for blood pressure control in aortic dissection because of their ability to reduce the force of ventricular contraction (negative inotropic effect). The drug regimens used for blood pressure control in aortic dissection are presented in Table 16.7, and outlined below.
1. The B-blocker that is most favored is esmolol (Brevibloc), which has a short duration of action (9 minutes) and can be rapidly titrated to achieve the desired end-point (i.e., systolic blood pressure of 120 mm Hg and a heart rate of 60 bpm).
2. If the B-blocker does not achieve the desired effect, a vasodilator like nitroprusside can be added. Vasodilators should, however, never be used as monotherapy because they will increase cardiac output and increase the risk of progressive aortic dissection.
3. An alternative to dual therapy with a B-blocker and a vasodilator is the drug labetalol, which is a combined a-, B-Receptor antagonist that can be used as monotherapy for blood pressure control in aortic dissection.
With medical management alone, the mortality in acute aortic dissection increases 1-2% per hour after the onset of symptoms (37). Surgical repair reduces the mortality rate to 10% at 24 hours and 12% at 48 hours (37).

Esmolol, Dosing Regimen and Comments = Dosing:. Esmolol, Dosing Regimen and Comments = 500 «kg IV bolus, then infuse at 50 ug/kg/min. Increase rate by 25 ug’kg/min every & min until systolic BP 120 mm Hg or heart rate 60 bpm. Maximum dose is 200 pg/kg/min.. Esmolol, Dosing Regimen and Comments = Comment:. Esmolol, Dosing Regimen and Comments = An ultra rapic-acting B-blocker, which is the pre- ferred drug for BP control in aortic dissaction.. Nitroprusside, Dosing Regimen and Comments = Dosing:. Nitroprusside, Dosing Regimen and Comments = Start infusion at 0.2 j.g/kg/min and titrate to achieve a systolic BP of 120 mm Hg. (See Table 13.3 for further dosing recommendations.). Nitroprusside, Dosing Regimen and Comments = Comment:. Nitroprusside, Dosing Regimen and Comments = Use only in combination with B-blockers. Do not use in renal failure (risk of thiocyanate toxicity).. Labetalol, Dosing Regimen and Comments = Dosing:. Labetalol, Dosing Regimen and Comments = 20mg IV over 2 min, then give 20-40 mg IV every 10 min if needed or infuse at 1-2 mg/min to the same endpoints as esmolol. Maximum cumulative dose Is 300 mg.. Labetalol, Dosing Regimen and Comments = Comment:. Labetalol, Dosing Regimen and Comments = A combined «- and B-blocker that can be used as monotherapy in aortic clissection.

The discovery that acute myocardial infarctions are caused by blood clots that obstruct a coronary artery is at odds with the traditional teaching that global decreases in myocardial oxygen delivery (e.g., from anemia and hypoxemia) can promote ischemic injury in patients with coronary artery disease. This teaching is responsible for the current emphasis on blood transfusions and oxygen breathing to prevent myocardial ischemia in ICU patients with coronary artery disease. However, since blood clots cause heart attacks, not anemia or hypoxemia, these practices are unfounded.
Ever wonder why myocardial infarction is an uncommon occurrence during progressive circulatory shock and multiorgan failure (where myocardial O> delivery is progressively threatened)? Now you have the answer.

- 1. Roger V, Go AS, Lloyd-Jones D, et al. Heart disease and stroke statistics—2012 update: a report from the American Heart Association. Circulation 2012; 125:e2— e220.

7. Trost JC, Lange RA. Treatment of acute coronary syndrome: Part 1: non STsegment acute coronary syndrome. Crit Care Med 2011; 39:2346-2353.
8. Trost JC, Lange RA. Treatment of acute coronary syndrome: Part 2: ST-segment elevation myocardial infarction. Crit Care Med 2012; 40:1939-1945.

9. Davies MJ, Thomas AC. Plaque fissuring: the cause of acute myocardial in-farction. Br Heart J 1985; 53:363-373.
10. Van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation 1994; 89:36—-44.
11. Malek AM, Alper SL, Izumo S. Hemodynamic shear stress and its role in atherosclerosis. JAMA 1999; 282:2035-2042.

Thrombolytic, 1 = Therapy. 18., 1 = Gruppo Italiano per lo Studio della Streptochinasi nell'Infarto Miocardico (GISSI). Effectiveness of intravenous thrombolytic treatment in acute myocardial infarction. Lancet 1986; 1:397-401.. 19., 1 = Fibrinolytic Therapy Trialists Collaborative Group. Indications for fibrinolytic therapy in suspected acute myocardial infarction: collaborative overview of early mortality and major morbidity results from all randomized trials of more than 1000 patients. Lancet 1994; 343:311-322.. 20., 1 = Boden WE, Kleiger RE, Gibson RS, et al. Electrocardiographic evolution of posterior acute myocardial infarction: importance of early precordial ST-segment depression. Am J Cardiol 1987; 59:782-787.. 21., 1 = GUSTO Investigators. An international randomized trial comparing four thrombolytic strategies for acute myocardial infarction. N Engl J Med 1993; 329:673-682.. 22., 1 = Smalling RW, Bode C, Kalbfleisch J, et al. More rapid, complete, and stable coronary thrombolysis with bolus administration of reteplase compared with alteplase infusion in acute myocardial infarction. Circulation 1995; 91:2725-2732.. 23., 1 = GUSTO-III Investigators. An international, multicenter , randomized comparison of reteplase with alteplase for acute myocardial infarction. N Engl J Med 1997; 337:1118-1123.. 24., 1 = Llevadot J, Giugliano RP, Antman EM. Bolus fibrinolytic therapy in acute myocardial infarction. JAMA 2001; 286:442-449.. 25., 1 = Assessment of the Safety and Efficacy of a New Thrombolytic (ASSENT-2) Investigators. Single-bolus tenecteplase compared with front-loaded alteplase in acute myocardial infarction. Lancet 1999; 354:716-722.. 26., 1 = Young GP, Hoffman JR. Thrombolytic therapy. Emerg Med Clin 1995; 13:735-759.

Percutaneous Coronary Intervention, 1 = Percutaneous Coronary Intervention. 27., 1 = The GUSTO IIb Angioplasty Substudy Investigators. A clinical trial comparing primary coronary angioplasty with tissue plasminogen activator for acute myocardial infarction. New Engl J Med 1997; 336:1621-1628.. 28., 1 = Keeley EC, Boura JA, Grines CL. Primary angioplasty versus intravenous thrombolytic therapy for acute myocardial infarction: a quantitative review of 23 randomized trials. Lancet 2003; 361:13-20.. 29., 1 = Stone GW, Cox D, Garcia E, et al. Normal flow (TIMI-3) before mechanical reperfusion therapy is an independent determinant of survival in acute myo-cardial infarction. Circulation 2001; 104:636-641.. 30., 1 = Cannon CP, Gibson CM, Lambrew CT, et al. Relationship of symptom onset to balloon time and door-to-balloon time with mortality in patients undergoing angioplasty for acute myocardial infarction. JAMA 2000; 283:2941-2947.. 31., 1 = Andersen HR, Nielsen TT, Rasmussen K, et al. for the DANAMI-2 Investi-gators. A comparison of coronary angioplasty with fibrinolytic therapy in acute myocardial infarction. New Engl J Med 2003; 349:733-742.

32. Patrono C, C, Coller B, Fitzgerald G, et al. Platelet-active drugs: the relationship among dose, effectiveness, and side effects. Chest 2004; 126:234S—264S.
33. COMMIT (Clopidogrel and Metoprolol in Myocardial Infarction Trial) collaborative group. Addition of clopidogrel to aspirin in 45,852 patients with acute myocardial infarction: randomized placebo-controlled trial. Lancet 2005; 366:1607—1621.
Complications, 1 = Complications. 34., 1 = Thompson CR, Buller CE, Sleeper LA, et al. Cardiogenic shock due to acute severe mitral regurgitation complicating acute myocardial infarction: a re-port from the SHOCK trial registry. J Am Coll Cardiol 2000; 36:1104-1109.. 35., 1 = Samuels LF , Darze ES. Management of acute cardiogenic shock. Cardiol Clin 2003; 21:43-49.. 36., 1 = Babaev A, Frederick PD, Pasta D, et al. Trends in the management and outcomes of patients with acute myocardial infarction complicated by cardiogenic shock. JAMA 2005; 294:448-454.
Aortic Dissection, 1 = Aortic Dissection. 37., 1 = Tsai TT, Nienaber CA, Eagle KA. Acute aortic syndromes. Circulation 2005; 112:3802-3813.. 38., 1 = Khan IA, Nair CK. Clinical, diagnostic, and management perspectives of aortic dissection. Chest 2002; 122:311-328.. 39., 1 = Knaut AL, Cleveland JC. Aortic emergencies. Emerg Med Clin N Am 2003; 21:817- 845.. 40., 1 = Zegel HG, Chmielewski S, Freiman DB. The imaging evaluation of thoracic aortic dissection. Appl Radiol 1995; (June):15-25.

Wakter Lippmann (1889-1974)
In 1960, an article was published in the Journal of the American Medical Association that would eventually change the way we approach the dying process. The article, titled "Closed-Chest Cardiac Massage" (1), was a report of 5 cases of cardiorespiratory arrest that were successfully managed with chest compressions, defibrillation shocks, and assisted ventilation. This report marks the birth of what is known today as cardiopulmonary resuscitation (CPR). In the more than 50 years since its inception, CPR has become a universally mandated practice that requires certification, and is withheld only on request. All this for an intervention that doesn't work in most cases, as demonstrated in Figure 17.1 (2,3).
This chapter describes the essential elements of resuscitation for cardiac arrest, including induced hypothermia for patients who remain comatose after successful resuscitation. The material in this chapter is drawn heavily from the most recent guidelines on CPR from the American Heart Association (4,5).

The essential elements of basic life support are chest compressions, establishing a patent airway in the oropharynx, and periodic lung inflations. The original mnemonic for these elements, ABC (Airway, Breathing, and Circulation), has been rearranged to CAB (Circulation, Airway, Breath-ing), reflecting a recent shift in emphasis from ventilation to chest compressions in the resuscitation effort.

One of the limiting factors in the success of CPR is the narrow time window between the cessation of blood flow and irreversible cell death. This time period can be estimated using the determinants of systemic oxygenation described in Chapter 10. The volume of QO, in circulating blood is normally about 800 mL (see Table 10.1), which is equivalent to the totalbody Oz content (because oxygen is not stored in tissues). The whole-body O> consumption in adults at rest is about 250 mL/min (see Table 10.2), which will consume the total-body O> content of 1 liter in just 4 minutes. Therefore, following the cessation of blood flow from a cardiac arrest, oxygen depletion and anoxic cell death can be expected after 4-5 minutes. Therefore, CPR must be initiated within this limited time period to have any chance of success.
FIGURE 17.1 Rates of survival and satisfactory neurologic recovery in cardiac arrests that occur inside the hospital (graph on the left) and outside the hospital (graph on the right), grouped according to the responsible rhythm disturbance. N=number of cases included in each survey, VF=ventricular fibrillation, VT =ventricular tachycardia, PEA=pulsekess electrical activity. Data from References 2 and 3.

The most recent recommendations for chest compressions are summarized in Table 17.1 (6). The principal feature is the emphasis on early and uninterrupted chest compressions.

For the initial resuscitation, first responders should begin with a series of 30 chest compressions, followed by 2 rescue breaths. Chest compressions should be delivered at a rate of at least 100/min. The initial compression:ventilation ratio of 30:2 should be repeated until an advanced airway (e.g., endotracheal tube) is placed. Thereafter, lung inflations are provided at regular intervals (see later), while the chest compressions continue without interruption.
RATIONALE: The emphasis on early chest compressions is based on the observation that delays to initiating CPR have an adverse effect on survival (see later) (6). The importance of chest compressions over ventilation is demonstrated by the practice of hands-only bystander CPR (chest compressions only), which has the same survival benefit as standard CPR (chest compressions plus rescue breaths) in the initial resuscitation period (7). Chest compressions can produce cardiac outputs that are 25% or 30% of normal (6), but this effect diminishes rapidly with delays to initiating CPR.

ay . Chest compressions should be delivered at a rate of at least 100/min.
2, Each chest compression should depress the mid-sternum at least 2 inches (5 cm), and the chest should be allowed to recoil completely to allow the heart to fill before the next compression.
co . First responders should begin CPR with a series of 30 chest compressions, followed by 2 rescue breaths. This compression:ventilation ratio (30:2) Is continued until an advanced airway Is placed (e.g., endotracheal tube).
4. Chast compressions should not be interrupted unless absolutely necessary (e.g., to deliver electric countershocks).
5. When possible, those performing chest compressions should be replaced every 1-2 minutes to prevent shallow compressions from fatigue.
Fram Referance 6.

Avoiding unnecessary interruptions in chest compressions is also emphasized. Observational studies have shown that interruptions in chest compressions are common, and the accumulated time of these interruptions can be as much as 50% of the total resuscitation time (8). Prolonging the time without chest compressions is considered to have a negative effect on outcomes (6), although there is evidence that is contrary to this view (9).

Prior to endotracheal intubation, rescue breathing is provided by a face mask that is connected to a self-inflating ventilation bag (e.g., Ambu Respirator) that fills with oxygen. The bag is compressed by hand to deliver the breath, and 2 breaths are provided for every 30 chest compressions, as mentioned earlier. After an endotracheal tube is in place, lung inflations are delivered at 6to 8-second intervals (8 to 10 breaths/min) using the same type of self-inflating ventilation bag that is used for the bag-mask device. The recommended volume for each lung inflation is 6-7 mg/kg (6), or about 500 mL in a normal sized adult.

The volume of lung inflations is not monitored during "bagged ventilation," and large inflation volumes are considered to be common during CPR (6), resulting in troublesome hyperinflation of the lungs (10). Adherence to the recommended inflation volumes (6—7 mL/kg) is possible if the volume capacity of the inflation bag is known. For example, if the inflation bag has a volume capacity of 1 liter, compressing the bag until it is about half full will deliver close to 500 mL for lung inflation. (The volume capacity of most adult ventilation bags is 1 to 2 liters.) An alternative method is to use one hand to compress the ventilation bag; this generates a volume of 600 to 800 mL (personal observation), and is unlikely to produce short-term hyperinflation.

Rapid lung inflation rates are common during CPR (10,11), and average rates of 30 inflations/min (3 times the recommended rate) have been reported (11). Rapid breathing is problematic because there is insufficient time for the lungs to empty, and this results in progressive hyperinflation and positive end-expiratory pressure (PEEP). This is called dynamic hyperinflation, and is described in more detail Chapter 27. The increased intrathoracic pressure associated with PEEP has two adverse effects. First, it reduces venous return to the heart, which limits the ability of chest compressions to augment cardiac output. Secondly, it reduces coronary perfusion pressure (10), which is an important determinant of outcome in cardiac arrest. These adverse effects provide a reason to avoid rapid rates of "bagged ventilation."

The practices listed in Table 17.2 (which have all been described in this section) are considered essential for providing high-quality CPR (6). This table is included to provide you with a checklist for achieving the highest level of performance in CPR.

1. Rate of chest compressions at least 100 per minute
2. Depth of chest compressions at least 2 inches (5 crm)
3. Allow chest to recoil completely after each compression.
4. Minimize Interruptions In chest compressions.
5. Avoid excessive ventilation.
From Referance 6.

Advanced cardiovascular life support, or ACLS, includes a variety of interventions such as airway intubation, mechanical ventilation, defibrillation, and the administration of lifesupporting drugs (12). This section will focus on defibrillation and life-supporting drugs, and will describe these interventions using a rhythm-based approach. This approach divides the management of cardiac arrest into 2 pathways: one for the management of ventricular fibrillation (VF) and pulseless ventricular tachycardia (VT), and the other for the management of pulseless electrical activity (PEA) and asystole.

The outcomes in cardiac arrest are most favorable when the initial rhythm is VF or pulseless VT, as demonstrated in Figure 17.1. This is the result of prompt electrical cardioversion, as described next.

Electrical cardioversion using asynchronous shocks (i.e., not timed to the QRS complex), which is called defibrillation, is the most effective resuscitation measure for cardiac arrest associated with VF and pulseless VT. The survival benefit from defibrillation is timedependent; i.e., the time elapsed from the cardiac arrest to the first electric shock is the most important factor in determining the likelihood of survival (12-14). This is demonstrated in Figure 17.2 (14). Note that 40% of patients survived when the first shock was delivered 5 minutes after the arrest, while only 10% of patients survived if defibrillation was delayed until 20 minutes after the arrest. These results emphasize the importance of prompt defibrillation when VF or pulseless VT is first apparent in a cardiac arrest victim.
IMPULSE ENERGY: Modern defibrillators deliver current based on stored energy, and the strength of the impulses is expressed in joules (J), which is a unit of thermal energy (see Appendix 1). The appropriate strength for effective cardioversion is determined by the waveform of the energy impulse. Biphasic waveforms (used by newer defibrillators) are effective at lower energy levels than monophasic waveforms (used by older defibrillators). For termination of VF and pulseless VT, the effective impulse strengths are 120 to 200 J for biphasic shocks, and 360 J for monophasic shocks (12,13). Manual defibrillators used in hospitals require the operator to select the desired energy level of the impulse, while automated external defibrillators (AEDs) use a preselected energy level.
FIGURE 17.2 Survival in out-of-hospital cardiac arrests with "shockable" arrhythmias (VF/pulseless VT) in relation to the time elapsed from the onset of the cardiac arrest to the initial defibrilation attempt. N=number of cases studied. Data

The management of cardiac arrest associated with pulseless electrical activity (PEA) or ventricular asystole is notoriously unsuccessful, as indicated in Figure 17.1. The principal elements of the management are shown in the right half of Figure 17.3. The major intervention is vasopressor therapy with epinephrine using the same dosing regimen used for VF and pulseless VT. Defibrillation is not attempted unless the cardiac rhythm changes to VF or VT.

PEA offers some hope because there are reversible causes, and the prominent ones are easily remembered with the letter T: i.e., Tension pneumothorax, pericardial Tamponade, pulmonary Thromboembolism, and Throm-botic occlusion of the coronary arteries. Unfortunately, there is little time for a diagnostic workup during a cardiac arrest, but pericardial tamponade and tension pneumothorax can be rapidly identified at the bedside using ultrasound imaging, which is readily available in many ICUs.

The use of drugs is considered a second-line treatment in cardiac arrest because there is no documented survival benefit (12). This may not be surprising, considering that the drugs are administered at a time of cardiovascular collapse, when drug delivery to target sites is sluggish and may not occur at all. The cardiac arrest drugs are either vasopressors or antiarrhythmic agents, and the recommended dosing regimen for each drug is shown in Table 17.3.

EPINEPHRINE produces systemic vasoconstriction, which is accompanied by an increase in coronary perfusion pressure (the difference between aortic and right atrial relaxation pressures during the time between chest compressions). This is demonstrated in Figure 17.4 (15). In this case, there is a 30% increase in coronary perfusion pressure following IV epinephrine, and the effect lasts at least 3 minutes (the recommended time interval between epinephrine doses). The disadvantage with epinephrine is the B-Receptormediated cardiac stimulation, which can erase the benefit of increased coronary perfusion, and has also been implicated in postresuscitation heart failure (15). Epinephrine use is associated with an increased rate of return to spontaneous circulation (ROSC), but the mortality rate is unchanged (12,16).
VASOPRESSIN is a non-adrenergic vasoconstrictor that is recommended only as a single dose (40 Units IV bolus) that can be used to replace the first or second dose of epinephrine (12). The advantage of vasopressin is the absence of cardiac stimulation, but vasopressin also causes coronary vasoconstriction, which is counterproductive. Clinical trials have shown no apparent advantage of vasopressin over epinephrine (17).

AMIODARONE is the preferred antiarrhythmic agent for VF/VT-associated cardiac arrest that is refractory to defibrillation and vasopressor therapy (12). This preference is based on clinical studies that show superior results with amiodarone compared to placebo (18) or lidocaine (19). However, the superior results with amiodarone are limited to increased survival to hospital admission, but not to hospital discharge.
LIDOCAINE is the original antiarrhythmic agent used for shock-resistant VF and pulseless VT, but it is less effective than amiodarone, and should be used only when amiodarone is not available.
MAGNESIUM is used for polymorphic VT, but only when the arrhythmia is associated with QT prolongation (torsade de pointes). The recognition and treatment of polymorphic VT are described in Chapter 15.
In the rare instance when intravenous or intraosseous access is not available, some ACLS drugs (i.e., epinephrine, vasopressin, and lidocaine) can be administered by injection into the upper airway through an endotracheal tube. The drug dose for endotracheal injection should be 2—2.5 times the intravenous dose (e.g., 2—2.5 mg for epinephrine) (12).

Vasoconstrictor Drugs, 1 = Vasoconstrictor Drugs. Vasoconstrictor Drugs, 2 = Vasoconstrictor Drugs. Epinephrine, 1 = Dosing: Comment:. Epinephrine, 2 = 1 mg Iv/lO every 3-5 minutes. Vasopressor effect can Increase coronary perfusion pressure, but cardiac stimulation Is counterproduc- tive.. Vasopressin, 1 = Dosing: Comment:. Vasopressin, 2 = 40 Units I'V/lO as a single dose. Can be used to replace the 1st or 2nd dose of api- nephrine, to reduce cardiac stimulation. No proven advantage.. Antiarrhythmic Agents, 1 = Antiarrhythmic Agents. Antiarrhythmic Agents, 2 = Antiarrhythmic Agents. Amiodarone, 1 = Dosing: Comment:. Amiodarone, 2 = 300 mg IV/lO, then another 150 mg If needed. Antiarrhythmic drug of choice for VF/VT that Is refractory to defibrillation and vasopressors.. Lidecaine, 1 = Dosing: Comment:. Lidecaine, 2 = 1-5 mg/kg Iv/lO, then 0.5-0.75 mg/kg every 5-10 min, as needed, to a total of 3 mg/kg. Can use a 1-4 mg/min for maintenance. Alternative to amiodarone, but much less effec- tive.. Magnesium, 1 = Dosing: Comment:. Magnesium, 2 = 1-2 grams IV/lO over 5 minutes. Used for polymorphic VT associated with pro- longed QT interval (torsade de pointes).
From the ACLS guidelinas in Refarance 12.

Monitoring for the return of spontaneous circulation (ROSC) is typically limited to palpation for carotid artery pulsations, but this is discouraged as a sole practice (12) because the search for pulses often requires interruption of chest compressions for extended periods of time, and there are often uncertainties about the presence or absence of pulses (20a). The end-tidal PCOz and central venous O> saturation provide a more reliable evaluation of the circulation, and can be used to predict the likelihood of ROSC.
FIGURE 17.4 The effect of intravenous epinephrine on coronary perfusion pressure (CPP) during resuscitation of cardiac arrest with VF/pulseless VT. Data from Reference 15.

The end-tidal PCO; measurement is described in detail in Chapter 21. The PCO) in exhaled gas at the end of expiration (end-tidal PCO) is a measure of the balance between ventilation and perfusion in the lungs (V/Q balance). The end-tidal PCO> varies directly (in the same direction) with changes in cardiac output relative to ventilation, and when alveolar ventilation is constant, changes in end-tidal PCO, reflect proportional changes in cardiac output (e.g., a 30% decrease in end-tidal PCO, indicates a 30% decrease in the cardiac output). The end-tidal PCO> is normally equivalent to the arterial PCO) (i.e., about 40 mm Hg), but it can be lower than the arterial PCO> in pulmonary conditions associated with increased physiologic dead space (i.e., V/Q ratio >1).

End-tidal CO) monitoring provides a measure of exhaled PCO, for each breath, and serial measurements during CPR can be used to identify when ROSC occurs, or is unlikely to occur. The graph in Figure 17.5 shows the serial changes in end-tidal PCO) during 20 minutes of CPR in patients who achieved ROSC and in patients who did not achieve ROSC (20b). Note that patients who achieved ROSC showed a progressive increase in end-tidal PCO> during the resuscitation period, whereas patients who did not achieve ROSC showed a progressive decline in end-tidal PCO». The end-tidal PCO, that separated responders from nonresponders in this study was 15 mm Hg after 20 minutes of CPR. Other studies have shown a discriminant value of 10 mm Hg separating survivors and nonsurvivors (21,22).
The available studies indicate that a successful outcome is unlikely if the end-tidal PCO, is not higher than 10-15 mm Hg after 20 minutes of CPR. When the end-tidal PCO, remains above this level, continued resuscitation for as long as 11/> hours has been associated with a favorable outcome (23).

As described in Chapter 10, the O, saturation of hemoglobin in central venous (superior vena cava) blood (ScvOz) is a measure of the balance between systemic O, delivery (DO) and systemic O, uptake (VO ). When VO; is constant, a decrease in DO> (e.g., from a low cardiac output) is accompanied by a decrease in ScvO>. The normal ScvO> is about 70-80% (see Chapter 10), and there is evidence that failure to achieve an ScvO> 230% during CPR is associated with failure to achieve ROSC (24). The value of ScvO> during CPR is limited by the need for a central venous catheter.
FIGURE 17.5 Serial changes in end-tidal PCO? in relation to return of spontaneous circulation (ROSC) during the resuscitation of cardiac arrest with VF/pulseless VT. Data points represent mean values for the patients in each group. From Reference 20b.

The immediate goal of CPR is a return of spontaneous circulation (ROSC), but this does not ensure a satisfactory outcome. In fact, surveys of cardiac arrest victims who survive the initial resuscitation indicate that about 70% do not survive to leave the hospital (25). This section describes the common problems encountered in the days following ROSC, and a few treatment modalities that can alleviate some of these problems.

Induced hypothermia, previously known as human refrigeration (29), was introduced for the management of post-cardiac arrest victims in the mid-twentieth century (30), but was abandoned because of uncertain benefit and the risks of severe hypothermia (<30°C) used at the time. About 40 years later (in 2002), two studies were published showing that comatose survivors of cardiac arrest who were treated with 12 to 24 hours of mild hypothermia (32—34°C) had more favorable neurologic outcomes and fewer deaths (31,32). The results of one of these studies (the larger one) is shown in Figure 17.6 (31). According to these results, mild hypothermia prevented one unfavorable neurologic outcome for every 6 patients treated, and prevented one death for every 7 patients treated. Since these results were published, mild induced hypothermia, now called targeted temperature management or TTM (33), has been adopted with enthusiasm for patients who do not awaken after ROSC. The general features of TTM are presented in Table 17.4 (26,27,30-33).

The original reports of benefit with mild hypothermia included cases of out-of-hospital cardiac arrest associated with VF/pulseless VT (31,32), and this condition was initially considered the only indication for mild hypothermia. However, because of the great promise with TTM, it is now a consideration for any patient who does not awaken following ROSC (26), regardless of the associated rhythm or location of the cardiac arrest. The only absolute contraindications to TTM are pre-existing hypothermia (<34°C), major bleeding, or cryoglobulinemia. Hemodynamic instability and cardiogenic shock are not considered absolute contraindications to TTM (26).
FIGURE 17.6 Clinical outcomes with mild hypothermia (32-34°C) and conventional management (normothermia) in comatose survivors of out-of-hospital cardiac arrest with VF/pulseless VT. Data from Reference 31.

Surface cooling has been the most popular method of TTM, but endovascular cooling is gaining favor because it creates less risk of shivering, and avoids the problem of erratic surface cooling caused by cold-induced vasoconstriction in the skin. The endovascular cooling method requires insertion of a specialized central venous catheter, but this is easily accomplished during the induction phase of cooling (see next). Both types of cooling are optimal when automated, and a number of automated systems are commercially available.
Patients who receive TIM are intubated and mechanically ventilated, and have thermistor-equipped bladder catheters to monitor body temperature. Although the patients are comatose, infusions of sedative agents (e.g., midazolam, propofol, or fentanyl) are often used to alleviate anticipated shivering. The cooling process in TTM is divided into 3 phases: induction, maintenance, and rewarming.

Patients who do not awaken after ROSC
Contraindications
Body temp <34°C, major bleeding, cryoglobulinemia
Treatment Plan
Reduce body temp to 32-34°C, for 24 hrs. Begin as soon as possible after ROSC.
Cooling Methods
Surface cooling and endovascular cooling
Protocol
Infuse ice-cold (4°C) saline until body temp at 34°C, then maintain temp at 32-34°C for 24 hrs, and rewarm at 0.25-0.5°C/hr.
Complications
Shivering, bradycardia, hypotension, diuresis, hypokalemia, infection
Benefit
Prevents one unfavorable neurologic outcome for every 6 patients treated, and prevents one fatal outcome for every 7 patients treated.t
tRaported for out-of-hospital cardiac amests with VF/pulseless VT (31).
From Raferencas 25, 26, 30-33. ROSS = retum of spontaneous circuation.
INDUCTION: Rapid cooling is advised with ice-cold (4°C) saline or lactated Ringers fluid infused in 500 mL aliquots every 10 minutes until the body temperature falls to 34°C or the infusion volume reaches 30 mL/kg. Shivering is common during the induction phase, and is counterproductive because it increases the body temperature. Shivering can be controlled with propofol (0.1-0.2 mg/kg/min IV), midazolam (0.02—0.1 mg/kg/hr IV), or fentanyl (25-100 e<g/hr IV), while magnesium (5 grams IV over 5 hours) can also be effective (25). Refractory shivering is managed with neuromuscular blockade (e.g., cisatracurium, 0.15—0.2 mg/kg IV bolus, then 1-2 e<g/kg/min, if needed).

MAINTENANCE: Following induction, automated surface cooling or endo-vascular cooling systems maintain the body temperature at 32-34°C for the next 24 hours. Bradycardia is common during hypothermia, but usually requires no intervention. Hypotension can occur as a result of cold-induced diuresis and cardiac depression, and is managed with volume infusions initially, followed by a vasopressor (e.g., norepinephrine)

As expected from the beneficial effects of hypothermia, fever following a cardiac arrest is associated with unfavorable neurologic outcomes (35). Therefore, prompt antipyretic therapy is advised for patients with fever who do not receive TTM, or for rebound fever that can follow TTM. Acetaminophen is the standard antipyretic agent, and is given in doses of 650 mg or 1000 mg IV or via feeding tube every 6 hours, with a maximum daily dose of 4 grams. Patients with hepatic insufficiency should not receive acetaminophen.

Hyperglycemia following cardiac arrest is associated with a poor neurologic outcome (36), although there is no evidence that glycemic control after cardiac arrest improves neurologic outcome. Strict glycemic control in ICU patients is associated with frequent episodes of hypoglycemia (37), so a higher-than-normal range of 144-180 mg/dL for blood glucose is considered a reasonable target (26). As an adjunct to this practice, it is wise to avoid dextrose-containing intravenous solutions whenever possible.

In patients who do not regain consciousness after CPR or induced hypo-thermia, the single most important determination is the likelihood of neurologic recovery. The traditional signs that predict a poor neurologic outcome are based on observations performed before hypothermia was adopted as a treatment modality (38,39), and these signs may not apply to patients who are treated with hypothermia (40-42). The following sections describe the signs that predict a poor neurologic outcome after CPR, and whether or not these signs also apply to patients treated with hypothermia. This information is summarized in Table 17.5

Poor
No pupillary light reflex on day 3, ‘After CPR = v. No pupillary light reflex on day 3, Hypothermia = wv. No corneal reflexes on day 3, ‘After CPR = v. No corneal reflexes on day 3, Hypothermia = v. GCS motor score <= 2 on day 3t, ‘After CPR = v. GCS motor score <= 2 on day 3t, Hypothermia = . Generalized status epilepticus, ‘After CPR = v. Generalized status epilepticus, Hypothermia = . Myoconic status epilepticus on day 1, ‘After CPR = v. Myoconic status epilepticus on day 1, Hypothermia = a
Signs
After
—
TA motor score of £2 on the Glasgow Coma Scare indicates no motor response or an abnormal extensor moter rasponse (dacarebrate posturing) to a painful stimulus.
Fram Raferencas 38-43.

Following CPR, most (80-95%) of the patients who regain consciousness are awake after 72 hours (38,40), but it can take 7 days or even longer for all the patients to awaken (38). There is a perception that hypothermia prolongs the time to awaken, but this remains unproven. In one retrospective study (40), the median time to awaken was the same (2 days) after CPR and after hypothermia, and a larger percentage of the patients who received hypothermia were awake at 72 hours (91%) compared to patients who did not receive hypothermia (79%). In general, the time to awaken can be a poor predictor of neurologic outcome in the first week following CPR, with or without hypothermia. Residual sedation may play a role in prolonging the time to awaken, particularly in patients who are treated with hypothermia (40).

Absence of brainstem reflexes is highly predictive of a poor outcome in patients who remain comatose after CPR and hypothermia. When papillary light reflexes or corneal reflexes are absent 3 days after CPR or hypothermia, none of the patients have a satisfactory neurologic recovery (39,41,42).

Patients who remain comatose for 72 hours after CPR have no chance of a satisfactory neurologic recovery if they show no motor response to pain, or an abnormal extensor response (i.e., decerebrate posturing) (38,39). However, when hypothermia is used, as many as 25% of patients who show no motor response at 72 hours will have a satisfactory recovery (43). Therefore, poor motor responses at 72 hours do not predict a poor outcome in patients treated with hypothermia.

Myoclonic status epilepticus (repetitive, irregular movements of the face, trunk, and extremities) often appears in the first 24 hours after a cardiac arrest (41), and is a poor prognostic sign for recovery in all patients, including those who receive hypothermia (39,41,42). Generalized status epilepticus (repetitive, tonic-clonic movements of the face, trunk, and extremities), including nonconvulsive status epilepticus (no seizure-like movements), is a poor prognostic sign without hypothermia, but does not always indicate a poor outcome following hypothermia (41).

The prognostic evaluation following hypothermia requires further study; however, one issue that is emerging is the possible interference of unsuspected sedation in the neurologic evaluation following hypothermia. The administration of opiates and other sedating drugs is commonplace during hypothermia and rewarming (which can last for 30 hours or longer), and slowed drug metabolism from hypothermia could result in prolonged sedation after the procedure. This could prolong the time to awaken, and create misleading signs of a poor neurologic recovery. Attention to limiting the use of sedating drugs, if possible, during hypo-thermia and rewarming will limit the risk of errors in interpreting persistent unresponsiveness, and can avoid the embarrassing situation where a patient wakes up after you have assured the family that there is little or no chance of awakening.

Cardiopulmonary resuscitation has always enjoyed a popularity far greater than deserved. This is evident in surveys of the general public, where 95% of respondents have unrealistic expectations about CPR (44), including the perception that more than half of cardiac arrest victims not only survive, but return to daily life with no residual effects (45). Television shows mirror this perception, where CPR is portrayed as a success in 67 to 75% of cases (46).

- 1. Kouwenhoven WB, Ing, Jude JR, Knickerbocker GG. Closed-chest cardiac massage. JAMA 1960; 173:1064—-1067.

2. Nadkarni VM, Laarkin GL, Peberdy MA, et al, for the National Registry of Cardiopulmonary Resuscitation Investigators. First documented rhythm and clinical outcome from in-hospital cardiac arrest among children and adults. JAMA 2006; 295:50-57.
3. Yasanuga H, Horiguchi H, Tanabe S, et al. Collaborative effects of bystander-initiated cardiopulmonary resuscitation and prehospital advanced cardiac life support by physicians on survival of out-of-hospital cardiac arrest: a nationwide populationbased observational study. Crit Care 2010; 14:R199-R210.

4. 2010 American Heart Association Guidelines for Cardiopulmonary Resusci-tation and Emergency Cardiovascular Care Science with Treatment Recom-mendations. Circulation, volume 122, issue 16, supplement 2, October 16, 2010. (Available online @ http://circ.ahajournals.org/content/122/16_suppl_2.toc)
5. Advanced Cardiovascular Life Support Provider Manual. Dallas, TX: Amer-ican Heart Association, 2011.

Basic Life Support, 1 = Basic Life Support. 6., 1 = Berg RA, Hemphill R, Abella BS, et al. Part 5: Adult basic life support: 2010 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Circulation 2010; 122 (suppl 3):S685-S705.. 7., 1 = Hupfl M, Selig H, Nagele P. Chest compression-only CPR: a meta-analysis. Lancet 2010; 376:1552-1557.. 8., 1 = Wit L, Kramer-Johansen J, Mykelbust H, et al. Quality of cardiopulmonary resuscitation during out-of-hospital cardiac arrest. JAMA 2005; 293:299-304.. 9. Jost D, Degrance H, Verret C, et al. DEFI 2005: a randomized controlled trial of the effect of automated external defibrillator cardiopulmonary resuscitation protocol on outcome from out-of-hospital cardiac arrest. Circulation 2010; 121:1614-1622., 1 = 9. Jost D, Degrance H, Verret C, et al. DEFI 2005: a randomized controlled trial of the effect of automated external defibrillator cardiopulmonary resuscitation protocol on outcome from out-of-hospital cardiac arrest. Circulation 2010; 121:1614-1622.. 10. Aufderheide TP, Lurie KG. Death by hyperventilation: A common and life-threatening problem during cardiopulmonary resuscitation. Crit Care Med 2004; 32(Suppl):S345- S351., 1 = 10. Aufderheide TP, Lurie KG. Death by hyperventilation: A common and life-threatening problem during cardiopulmonary resuscitation. Crit Care Med 2004; 32(Suppl):S345- S351.. 11. Abella BS, Alvarado JP, Mykelbust H, et al. Quality of cardiopulmonary resuscitation during in-hospital cardiac arrest. JAMA 2005; 293:305-310., 1 = 11. Abella BS, Alvarado JP, Mykelbust H, et al. Quality of cardiopulmonary resuscitation during in-hospital cardiac arrest. JAMA 2005; 293:305-310.

12., 1 = Neumar RW, Otto CW, Link MS, et al. Part 8: adult advanced cardiovascular life support: 2010 American Heart Association Guidelines for Cardiopul-monary Resuscitation and Emergency Cardiovascular Care. Circulation 2010; 122 (suppl 3):S729-S767.. 13., 1 = Link MS, Atkins DL, Passman RS, et al. Part 6: electrical therapies: automated external defibrillators, defibrillation, cardioversion, and pacing: 2010 American Heart Association Guidelines for Cardiopulmonary Resuscitation and Emerg-ency Cardiovascular Care. Circulation 2010; 122 (suppl 3):S706-S719.. 14., 1 = Larsen MP, Eisenberg M, Cummins RO, Hallstrom AP. Predicting survival from out of hospital cardiac arrest: a graphic model. Ann Emerg Med 1993; 22:1652-1658.. 15., 1 = Sun S, Tang W, Song F, et al. The effects of epinephrine on outcomes of mormothermic and therapeutic hypothermic cardiopulmonary resuscitation. Crit Care Med 2010; 38:2175-2180.. 16., 1 = Herlitz J, Ekstrom L, Wennerblom B, et al. Adrenaline in out-of-hospital ventricular fibrillation. Does it make any difference? Resuscitation 1995; 29:195-201.. 17., 1 = Aung K, Htay T. Vasopressin for cardiac arrest: a systematic review and meta- analysis. Arch Intern Med 2005; 165:17-24.. 18., 1 = Kudenchuk PJ, Cobb LA, Copass MK, et al. Amiodarone for out-of-hospital cardiac arrest due to ventricular fibrillation. New Engl J Med 1999; 341:871-878.. 19., 1 = Dorian P, Cass D, Schwartz B, et al. Amiodarone as compared to lidocaine for shock- resistant ventricular fibrillation. New Engl J Med 2002; 346:884-890.

44., 1 = Jones GK, Brewer KL, Garrison HG. Public expectations of survival following cardiopulmonary resuscitation. Acad Emerg Med 2000; 7:48-53.. 45., 1 = Marco CA, Larkin GL. Cardiopulmonary resuscitation: knowledge and opinions among the U.S. general public. State of the science-fiction. Resuscitation 2008; 79:490-498.. 46., 1 = Diem SJ, Lantos JD, Tulsky JA. Cardiopulmonary resuscitation on television. Miracles and misinformation. N Engl J Med 1996; 334:1578-1582.. 47., 1 = Bohm K, Rosenqvist M, Herlitz J, et al. Survival is similar after standard treatment and chest compressions only in out-of-hospital bystander cardiopulmonary resuscitation. Circulation 2007; 116:2908-2912.

The golden rule is there are no golden rules.
George Bernard Shaw

Fundamental progress requires the reinterpretation of basic ideas.
Alfred North Whitehead (1861-1947)
Anemia is almost universal in patients who spend a few days in the ICU (1), and about 50% of ICU patients receive erythrocyte transfusions to alleviate anemia (1,2). Unfortunately, few ICUs employ guidelines to standardize transfusion therapy (2), and erythrocyte transfusions are typically guided by personal preference and tradition rather than evidence of need or benefit. The discovery in recent years that erythrocyte transfusions often create risks instead of benefits has prompted a mandate for change in this fickle and arbitrary practice.
This chapter describes the effects (physiological and clinical) of anemia and erythrocyte transfusions, and presents the indications, methods, and risks of erythrocyte transfusions in critically ill patients (3,4). Transfusion practices that lack a scientific basis or justification are highlighted to comply with Whitehead's edict in the introductory quote.

The variety of erythrocyte-related measurements and their reference ranges are shown in Table 18.1. The use of hematocrit and hemoglobin concentration as measures of anemia is problematic in ICU patients, as described next.

Anemia is defined as a decrease in the oxygen (O2) carrying capacity of blood. The most accurate measure of the O» carrying capacity of blood is the red cell mass, which is measured using chromium-tagged erythrocytes. This measurement is not readily available for clinical use, so the hematocrit and hemoglobin (Hb) concentration are used as clinical measures of O> carrying capacity.

Red Cell Count
Mean Cellular Volume (MCV)
Males:
4.6 10'/L
Males:
80-100 « 1075/L
Females:
4.2 x 101/L
Females:
same
Reticulocyte Count
Hematocrit
Males:
25-75 x 10°/L
Males: 40-54%
Females:
same
Females:
38-479

Hemaglobin*®
Males:
26 mL/kg
Males:
14-18 g/dL
Females:
24 mL/kg
Females:
12-16 g/dL
tNormal values are 10% lower in the elderly (265 years of age).
aNormal values are 0.5 a/dL lower in blacks
Sources: (1) Walker RH fad.) Techrica Manual of the American Association of Blood Banks, 10th ad., VA: American Association of Blood Banks, 1990:649-650; (2) Billman Rs, Finch GCA Red cell manual. 6th ad. Philadalphia, PA: Davis, 1994:46.

The problem with the hematocrit and Hb concentration as measures of O> carrying capacity is the influence of plasma volume on these variables. This is demonstrated in Figure 18.1, which shows the postural changes in hematocrit and plasma volume in a group of healthy adults (5). When changing from the standing to supine positions, there is a decrease in the hydrostatic pressure in the veins and capillaries in the legs (due to loss of the gravitational effect), and interstitial fluid moves into the bloodstream and increases the plasma volume (by 420 mL in this study). The hematocrit then decreases by dilution, but there is no change in the Op, carrying capacity of blood. The postural change in hematocrit (4.1%) is equivalent to one unit of erythrocytes (packed red blood cells), so a dilutional drop in Hct similar to the one in Figure 18.1 could be misinterpreted as indicating a decrease in O> carrying capacity equivalent to one unit of blood.
Increased plasma volume is common in critically ill patients (see Figure 11.3 in Chapter 11), which means the hematocrit and Hb concentration will overestimate the occurrence and severity of anemia. Clinical studies have confirmed that the Hct and Hb concentration are unreliable measures of anemia in critically ill patients (6,7). Unfortunately, these are the measures used in all the clinical studies evaluating anemia and erythrocyte transfusions in critically ill patients.
FIGURE 18.1 Postural changes in hematocrit (Hct) and plasma volume (PV) in a group of healthy adults. The numbers above the columns are mean values for each measurement. From Reference 5.

Two conditions have been implicated in the anemia that appears during the ICU stay (8): systemic inflammation, and repeated phlebotomy for laboratory studies.

Inflammation is responsible for the anemia of chronic disease, which is now called the anemia of inflammation (8). The hematologic effects of inflammation include inhibition of erythropoietin release from the kidneys, re-duced marrow responsiveness to erythropoietin, iron sequestration in macrophages, and increased destruction of RBCs (12,13). The changes in plasma include a decrease in plasma iron, total iron binding capacity, and transferrin levels, and an increase in plasma ferritin levels.

An average of 40-70 mL of blood is withdrawn daily from each ICU patient to perform laboratory tests (9,10), which is at least 4 times more than the daily phlebotomy volume in non-ICU patients (9). The cumulative phlebotomy volume can reach 500 mL (one unit of whole blood) after one week, and this volume loss can result in an iron-deficiency anemia if allowed to continue.
The daily phlebotomy volume can be reduced by ordering fewer laboratory tests, and by reducing the volume of blood discarded for each laboratory blood draw. When blood is withdrawn through vascular catheters for laboratory testing, the initial aspirate is discarded to eliminate interference from intravenous fluid in the lumen of the catheter. The volume of discarded blood is typically about 5 mL for each laboratory blood draw, and returning this blood to the patient can reduce the daily phlebotomy volume by 50% (11).

Anemia elicits two responses that help to preserve tissue oxygenation: (a) an increase in cardiac output, and (b) an increase in O> extraction from capillary blood.

The graph in Figure 9.9 (page 166) shows the increase in cardiac output that occurs in response to a progressive decrease in hematocrit. This response is explained by the influence of anemia on blood viscosity. The hematocrit is the principal determinant of blood viscosity, and decreases in hematocrit are accompanied by similar decreases in blood viscosity. The relationship between hematocrit and blood viscosity is shown in Table 9.2 (page 165). The final section of Chapter 9 contains a detailed description of blood viscosity and its influence on circulatory blood flow.

The influence of progressive anemia on measures of systemic oxygenation are shown in Figure 18.2 (12). The principal findings are explained using the following relationships between O> uptake (VO>), O2 delivery (DO>), and O> extraction:
VO, = DO, x O, Extraction

1. The progressive decrease in hematocrit is associated with a steady decrease in O> delivery (DO2). However, there is also an equivalent increase in O> extraction, and the reciprocal changes in DO and O> extraction results in an unchanged O> uptake (VO>).
2. When the hematocrit falls below 10%, the increase in O> extraction is no longer able to match the decrease in DO, and the VO, begins to fall. The decrease in VO> represents a decrease in O> availability in tissues, and is accompanied by lactate accumulation in the blood.
3. The maximum O, extraction is about 50%, and it marks the threshold for impaired tissue oxygenation. Therefore, an OQ, extraction of 50% could be used as a trigger point for transfusion of erythrocytes. This point will be revisited later in the chapter.

Animal studies have shown that, when the intravascular volume is maintained, hematocrits as low as 5 to 10% (Hb=1.5 to 3 g/dL) do not adversely affect tissue oxygenation (12-14), even in awake animals breathing room air (14). The lowest tolerable hematocrit or hemoglobin has not been determined in humans, but in one study of progressive hemodilution in healthy adults, Hb levels of 5 g/dL showed no apparent harm (15). The important message from studies of severe anemia is not the lowest tolerable hematocrit, but the realization that severe anemia is tolerated when the intravascular volume is maintained.
FIGURE 18.2 The influence of progressive isovolemic anemia on measures of systemic oxygenation. DO? = systemic O2 delivery, VO = systemic O2 uptake. Data from Reference 12.

The graph in Figure 18.3 reveals an effect of anemia that few would predict; i.e., enhanced tissue oxygenation! The data in this graph is from a study that used direct measurements of the PO, in subcutaneous tissue to evaluate normovolemic hemodilution in isolated skin flaps (16). As indicated in the graph, progressive decreases in hematocrit were accompanied by increases in the subcutaneous PO; in both normal and ischemic skin regions, and this relationship continued until the hematocrit fell to 15%. Similar results in other studies has led to the use of normovolemic anemia to promote the viability of skin flaps.
FIGURE 18.3 The effect of progressive isovolemic anemia on the subcutaneous PO? in normal and ischemic regions of the skin. From Reference 16.
So how can anemia improve tissue oxygenation? Only if the flow augmentation in response to anemia is greater than the decrease in hematocrit. Exaggerated flow responses to anemia have been reported in the coronary and cerebral circulations (17), which may be a protective mechanism for the heart and brain, but the effect on tissue oxygenation is not known. Regional variations in the flow response to anemia add further to the problem of selecting an appropriate transfusion trigger.

Surveys indicate that 90% of the erythrocyte transfusions in ICU patients are given to alleviate anemia (18), and are guided by the hemoglobin concentration in blood. This is a flawed practice, as described in this section.

As described earlier (and shown in Figure 18.2) anemia elicits a compensatory increase in O, extraction from capillary blood, which serves to maintain a constant rate of O2 uptake into tissues. However, the O> extraction cannot increase much further than 50%, and, when the QO, extraction is maximum at 50%, further decreases in Hb are accompanied by proportional decreases in O> uptake into tissues (indicating tissue dysoxia). Therefore, an O, extraction of 50% can be used as a transfusion trigger because it identifies the threshold for impaired tissue oxygenation. The O> extraction is roughly equivalent to the (SaO>—-ScvO>), and can be monitored continuously using pulse oximetry (for the SaQ>) and a central venous oximetry catheter (PreSep Catheter, Edwards Life sciences) to monitor the central venous O> saturation (ScvO2). The (SaOz—ScvO3) is very appealing as a transfusion trigger (23) because it provides information about the adequacy of tissue oxygenation.

When the SaQ3 is close to 100%, the (SaOQ>—ScvO>) is equivalent to (1—-ScvO>), and the ScvO, can be used as a transfusion trigger. An ScvO> <70% has been proposed as a transfusion trigger (24), although it seems a lower ScvQ) (i.e., closer to 50%) would be more appropriate for identifying the threshold for impaired tissue oxygenation.

Whole blood is stored only on request, and is otherwise separated into its component parts; i.e., red blood cells, platelets, plasma, and cryoprecipitate. This practice allows each unit of donated blood to serve multiple transfusion needs. The erythrocyte preparations that are available for transfusion are shown in Table 18.2.

Packed RBCs, 1 = 1. Each unit has a volume of 350 mL and hematocrit of about 6096. 2. Contains leukocytes and residual plasma (15-30 mL per unit}. 3. Can be stored for 42 days with appropriate additives.. Leukocyte-Poor RBCs, 1 = 1. Donor RBCs aré passed through specialized filters to remove most of the leukocytes. This reduces the risk of febrile reactions to RBC and platelet transfusions. 2. Indicated for patients with a history of febrile trans- fusion reactions.. Washed RBCs, 1 = 1. Saline washing of packed RBCs removes residual plasma, which reduces the risk of hypersensitivity reactions. 2. Used in patients with a history of transfusion- related allergic reactions, and in patients with IgA deficiency, who are at risk for transfusion- related anaphylaxis.
Fram Referance 25.

The erythrocyte fraction of donated blood is placed in a preservative fluid and stored at 1 to 6°C. Newer preservative solutions contain adenine, which helps to maintain ATP levels in stored erythrocytes, and allows storage of donor erythrocytes for up to 42 days (25). Each unit of donor erythrocytes, known as packed red blood cells (packed RBCs), has a hematocrit of about 60% and a volume of about 350 mL. Packed RBCs also contain 30-50 mL of residual plasma, and a considerable numbers of leukocytes (1-3 billion leukocytes per unit of packed RBCs) (25).

The leukocytes in packed RBCs can trigger an antibody response in the recipient after repeated transfusions, and this is responsible for febrile nonhemolytic transfusion reactions (see later). To reduce the occurrence of this reaction, donor RBCs are passed through specialized filters to remove most of the leukocytes. This is performed routinely in many blood banks, but universal leukocyte reduction has yet to be adopted in the United States. Leukocyte-reduced RBCs are recommended for pa-tients with prior febrile nonhemolytic transfusion reactions (25).

Donor RBCs can be washed with isotonic saline to remove residual plasma. This reduces the risk of hypersensitivity reactions caused by prior sensitization to plasma proteins in donor blood. Washed RBC preparations are recommended for patients with a history of hypersensitivity reactions to blood transfusions, and for patients with immunoglobulin A deficiency, who have an increased risk of transfusion-related anaphylaxis (25). Saline washing does not effectively remove leukocytes.

The infusion of packed RBCs is described in Chapter 11 (see page 205). Rapid infusion rates are not necessary when packed RBCs are used to alleviate anemia, and this eliminates the need for infusion pumps or dilution with saline to increase infusion rates. The gravity-driven flow rate of packed RBCs through an 18-gauge peripheral catheter is 5 mL/min (see Figure 11.5 on page 206), which corresponds to 70 minutes for the transfusion of one unit (350 mL) of packed RBCs. This is well within the recommended transfusion time of 2 hrs per unit of packed RBCs for hemodynamically stable patients (26).

Standard blood filters (pore size 170 to 260 microns) are required for the transfusion of all blood products (26). These filters trap blood clots and other debris, but they do not trap leukocytes, and are not effective for leukocyte reduction (25). These filters can become an impediment to flow as they collect trapped debris, and sluggish infusion rates should prompt replacement of the blood filter.

In an average sized adult, one unit of packed RBCs is expected to raise the hemoglobin concentration and hematocrit by 1 g/dL and 3%, respectively (25). The effects of RBC transfusions on measures of systemic oxygenation are shown in Figure 18.4. The data in this figure is from a group of postoperative patients with severe normovolemic anemia (Hb <7 g/dL) who were transfused with 1-2 units of packed RBCs to raise the Hb above 7 g/dL. The RBC transfusions increased the mean Hb concentration from 6.4 to 8 g/dL (25% increase), and there was a similar increase in O> delivery (DO>). However, the systemic O» uptake (VO2) was unchanged. The constant VO, in the face of an increased DO, indicates that Oz extraction was reduced by the RBC transfusions, as predicted by equation 18.1. These changes in DO) and Op» extraction are the reverse of the changes produced by anemia, as shown in Figure 18.2.

The lack of an effect on VO, indicates that RBC transfusions do not en-hance tissue oxygenation. This has been confirmed in several clinical studies (27-30), and prolonged storage of RBCs can actually impair tissue oxygenation after transfusion (31). These studies have prompted the following statement in the most recent clinical practice guidelines for red blood cell transfusions (3): "RBC transfusion should not be considered an absolute method to improve tissue oxygenation in critically ill patients." If RBC transfusions do not provide a benefit for tissue oxygenation, then why do we give RBC transfusions? Unfortunately, there is no satisfying answer to this question.
FIGURE 18.4 Effects of erythrocyte transfusions (1-2 units packed RBCs) on hemoglobin concentration (Hb), systemic oxygen delivery (DO2), and systemic oxygen uptake (VO7) in 11 postoperative patients with severe anemia (Hb <7 g/dL). Data points represent mean values for each parameter. Numbers in parentheses are the mean hemoglobin concentrations before and after transfusion. Data from personal observations.
The inability of RBC transfusions to enhance tissue oxygenation raises serious questions about the practice of transfusing RBCs simply to raise the blood Hb level. Adding to the concern are studies indicating that RBC transfusions are a source of morbidity and even mortality in critically ill patients (32). The risks associated with RBC transfusions are described next.

The spectrum of adverse events associated with blood transfusions are shown in Table 18.3, along with the incidence of each event expressed in relation to the number of units transfused (33-37). (Only acute events are included.) Note that transfusion errors are much more frequent than the feared transmission of HIV or the hepatitis B virus. The following is a brief description of the principal transfusion reactions (37-41).

Nonhemolytic fever (1 In 200) Hypersensitivity Reactions: Urticaria (1 in 100) Anaphylaxis (1 in 1,000) Anaphylactic shock (1 in 50,000) TAcute lung injury (1 in 12,000) * Nos lal Infecti 2 losocomial Infections (7) Acute hemolytic reaction (1 In 35,000) Fatal hemolytic reaction (1 in 1 million), 1 = Transmitted Infections: Bacterial (1 500,000) Hepatitis B vrus (1 in 220,000) Hepatitis C virus (1 In 1.6 million) HIV (1 In 1.6 milion) Transfusion Errors: Wrong person transfused (1 in 15,000) Incompatible transfusion (1 in 33,000)
Includes only acute complications. From Raferances 33-49.
tThe leading cause of transfusion-olated deaths.
"May be the most common adverse consequence of RBC transfusions.

A febrile nonhemolytic transfusion reaction is defined as a temperature elevation >1°C (1.8°F) that occurs during transfusion or up to 6 hours after transfusion, and is not attributed to another cause (e.g., acute hemolytic reaction) (35). The culprit is the presence of antileukocyte antibodies in recipient blood that react with antigens on donor leukocytes. This triggers the release of endogenous pyrogens from phagocytes, which is the source of the fever. This reaction is reported in 0.5% of RBC transfusions (once per 200 transfusions), and occurs in patients who have received prior transfusions, and in multiparous women. Transfusion of leukocyte-reduced RBCs reduces, but does not eliminate, the risk of this reaction (35).

The fever typically does not appear in the first hour after the start of transfusion (unlike the fever associated with acute hemolytic reactions), and it can be accompanied by rigors and chills.

1. The initial approach to transfusion-related fever is the same as described for hemolytic transfusion reactions, even though the fever may not appear until after the transfusion is completed. The diagnosis is confirmed by excluding the presence of hemolysis with the tests described previously.
2. The blood bank will perform a Gram stain on the donor blood, and may request blood cultures on the recipient. This is usually unrewarding because microbial contamination in stored blood is rare (1 per 5,000,000 units). The organism most frequently isolated in stored RBCs is Yersinia enterocolitica (34).
3. More than 75% of patients with a nonhemolytic fever will not experience a similar reaction to subsequent transfusions (34). Therefore, no special precautions are needed for future transfusions. If a second febrile reaction occurs, leukocyte-reduced RBCs are advised for all subsequent transfusions.

Hypersensitivity reactions are the result of sensitization to plasma proteins in donor blood from prior transfusions. Patients with IgA deficiency are prone to hypersensitivity transfusion reactions, and prior exposure to plasma products is not required. The most common hypersensitivity reaction is urticaria, which is reported in one of every 100 units transfused (36). More severe anaphylactic reactions (e.g., bronchospasm) are much less common, and anaphylactic shock is rare.

The usual manifestation is mild urticaria that appears during the transfusion and is not accompanied by fever. The abrupt onset of dyspnea during a transfusion could represent laryngeal edema or bronchospasm, and hypotension from anaphylactic shock can be mistaken for an acute hemolytic reaction.

1. Mild urticaria without fever does not require interruption of the transfusion. However, the popular practice is to stop the transfusion temporarily and administer an antihistamine for symptom relief (e.g., diphenhydramine, 25-50 mg PO, IM, or IV).
2. Severe anaphylactic reactions should be managed as described in Chapter 14. The transfusion should be stopped immediately if severe anaphylaxis is suspected.
3. Washed RBCs should be used for all future transfusions in patients with hypersensitivity reactions. However, in patients with severe anaphylactic reactions, future transfusions are risky, even with washed RBCs, and should be avoided unless absolutely necessary.
4. Patients who develop hypersensitivity reactions should be tested for an underlying IgA deficiency.

Transfusion-related acute lung injury (TRALI) is an inflammatory lung injury associated with RBC and platelet transfusion (38), and resembles the acute respiratory distress syndrome (ARDS), which is described in Chapter 23. Recent surveys show an incidence of 1 per 12,000 transfusions (38), and a mortality rate of 6% (37). TRALI is considered the leading cause of transfusion-related deaths (37).

The prevailing theory is that TRALI is the result of antileukocyte antibodies in donor blood that bind to antigens on circulating neutrophils in the recipient. This triggers neutrophil activation, and the activated neutrophils become sequestered in pulmonary capillaries and migrate into the lungs to produce the inflammatory injury. The risk of TRALI is higher when donor blood contains high levels of antileukocyte antibodies, and in donor blood from females (37). The link between female blood and TRALI is not well understood.

Signs of respiratory compromise (dyspnea, tachypnea, hypoxemia, etc.) can appear for up to 6 hours after the start of a transfusion, but they usually appear within the first hour after the transfusion begins (37). Fever is common, and the chest x-ray eventually looks like the one in Figure 18.5, with diffuse, homogeneous infiltrates in both lungs that are indistinguishable from ARDS. The diagnosis of TRALI is based on the clinical setting (i.e., ARDS that appears within 6 hours after the start of a blood transfusion). The chest x-ray can be mistaken for acute hydrostatic pulmonary edema, but this can be excluded based on the clinical setting. TRALI can be severe at the outset, and often requires mechanical ventilation, but the condition typically resolves within a week (37).

The immunosuppressive effects of blood transfusions became evident with the discovery (in the early 1970s) that pre-transplant blood transfusions improved the survival rate of renal allografts (39). Since then, a multitude of clinical studies have shown that patients who receive blood transfusions have a higher incidence of nosocomial infections (32,39— 41). The risk of infection increases with the volume of blood transfused (see Figure 18.6), and with the storage time of donor blood (42). There has been some concern that the association between blood transfusions and infection is not a causal relationship, but instead is a reflection of severity of illness (i.e., sicker patients develop more infections and they also require more blood transfusions). However, at least 22 studies have shown that blood transfusion is an independent risk factor for nosocomial infections (32). Transfusion-related immunosuppression is poorly understood, but the emerging opinion is that nosocomial infections are a major source of transfusion-related morbidity and mortality in critically ill patients (32).

A review of 45 clinical studies evaluating RBC transfusions in critically ill patients, which included 272,596 patients, revealed the following findings (32):
1. In 42 of the 45 studies, the adverse effects of RBC transfusions outweighed any benefits.
2. Only 1 of 45 studies showed that the benefits of RBC transfusions outweighed the adverse effects.
3. Eighteen studies evaluated the relationship between RBC transfusions and survival, and 17 of the 18 studies showed that RBC transfusions were an independent risk factor for death. The likelihood of a fatal outcome was, on average, 70% higher in patients who re-ceived an RBC transfusion.
Not a very good report card, is it? These observations, combined with those showing that RBC transfusions do not improve tissue oxygenation, suggest that the current practice of transfusing RBCs to increase the Hb concentration in blood may simply be bad medicine.
FIGURE 18.6 Results of a large multicenter study showing the relationship between the incidence of nosocomial infections and the volume of blood transfused in critically ill patients. N = number of patients in the study. From Reference 40.

The practice of transfusing RBCs to raise the Hb level in blood is rooted in the belief that anemia is a threat to tissue oxygenation. However, as described earlier in the chapter, the severest anemias do not threaten tissue oxygenation as long as the intravascular volume (and hence cardiac output) is maintained. The supremacy of blood volume over RBCs in supporting tissue oxygenation is evident when you consider that hypovolemia is a recognized cause of impaired tissue oxygenation (i.e., hypovolemic shock), but anemia is not (i.e., "anemic shock" is not a clinical entity). The importance of blood volume is often overlooked, even by the American Red Cross, whose popular slogan, blood saves lives, deserves a more accurate update, as shown in Figure 18.7. Awareness of the attributes of blood volume would help to curb the undeserved emphasis on the transfusion of RBCs to support tissue oxygenation.
FIGURE 18.7 A popular slogan of the American Red Cross on the left, and an amended version on the right that recognizes the contribution of intravascular volume in the survival benefit of blood transfusions.

King KE (ed). Blood Transfusion Therapy: A Physician's Handbook. 9th ed. Bethesda, MD: American Association of Blood Banks, 2008.

1. Hebert PC, Tinmouth A, Corwin HL. Controversies in RBC transfusions in the critically ill. Chest 2007; 131:1583-1590.
2. Bennet-Guerrero E, Zhao Y, O'Brien SM, et al. Variation in the use of blood transfusion in coronary artery bypass graft surgery. JAMA 2010; 304:1568—-1575.

3. Napolitano LM, Kurek S, Luchette FA, et al. Clinical practice guideline: Red blood cell transfusion in adult trauma and critical care. Crit Care Med 2009; 37:3124—3157.
4. Ferraris VA, Ferraris SP, Saha SP, et al. Perioperative blood transfusion and blood conservation in cardiac surgery; the Society of Thoracic Surgeons and the Society of Cardiovascular Anesthesiologists Clinical Practice Guideline. Ann Thorac Surg 2007; 83(suppl):S27—S86.
Transfusion Triggers, 1 = Transfusion Triggers. 18., 1 = Corwin HL, Gettinger A, Pearl R, et al. The CRIT study: anemia and blood transfusion in the critically ill - Current clinical practice in the United States. Crit Care Med 2004; 32:39-52.. 19., 1 = Adam RC, Lundy JS. Anesthesia in cases of poor risk: Some suggestions for decreasing the risk. Surg Gynecol Obstet 1942; 74:1011-1101.. 20., 1 = Hebert PC, Wells G, Blajchman MA, et al. A multicenter , randomized, controlled clinical trial of transfusion requirements in critical care. N Engl J Med 1999; 340:409- 417.. 21., 1 = Hebert PC, Yetisir E, Martin C, et al. Is a low transfusion threshold safe in critically ill patients with cardiovascular disease. Crit Care Med 2001; 29:227-234.. 22., 1 = Consensus Conference on Perioperative Red Blood Cell Transfusion. JAMA 1988; 260:2700-2702.. 23., 1 = Levy PS, Chavez RP, Crystal GJ, et al. Oxygen extraction ratio: a valid indicator of transfusion need in limited coronary vascular reserve? J Trauma 1992; 32:769-774.. 24., 1 = Vallet B, Robin E, Lebuffe G. Venous oxygen saturation as a physiologic transfusion trigger. Crit Care 2010; 14:213-217.

Erythrocyte Transfusions, 1 = Erythrocyte Transfusions. 25., 1 = King KE (ed). Blood Transfusion Therapy: A Physician's Handbook. 9th ed. Bethesda, MD: American Association of Blood Banks, 2008:1-18.. 26., 1 = Ibid, pp. 91-95.. 27., 1 = Conrad SA, Dietrich KA, Hebert CA, Romero MD. Effects of red cell transfusion on oxygen consumption following fluid resuscitation in septic shock. Circ Shock 1990; 31:419-429.. 28., 1 = Dietrich KA, Conrad SA, Hebert CA, et al. Cardiovascular and metabolic response to red blood cell transfusion in critically ill volume-resuscitated nonsurgical patients. Crit Care Med 1990; 18:940-944.. 29., 1 = Marik PE, Sibbald W. Effect of stored-blood transfusion on oxygen delivery in patients with sepsis. JAMA 1993; 269:3024-3029.. 30., 1 = Fuller BM, Gajera M, Schorr C, et al. Transfusion of packed red blood cells is not associated with improved central venous oxygen saturation or organ function in patients with septic shock. J Emerg Med 2012; 43:593-598.. 31., 1 = Kiraly LN, Underwood S, Differding JA, Schreiber MA. Transfusion of aged packed red blood cells results in decreased tissue oxygenation in critically ill trauma patients. J Trauma 2009; 67:29-32.. 32., 1 = Marik PE, Corwin HL. Efficacy of red blood cell transfusion in the critically ill: A systematic review of the literature. Crit Care Med 2008; 36:2667-2674.

Hemorrhage upon a strong pulsation in wounds is bad.
Hippocrates Aphorisms (400 B.C.)
The last chapter was devoted to the red blood cell and its relationship with tissue oxygenation. The spotlight now shifts to the platelet and plasma components of blood, and their relationship with the tendency to bleed.

The vascular endothelium is a thromboresistant surface that helps to maintain the "fluidity" of blood in three ways (1). First, endothelial cells secrete substances like nitric oxide and prostacyclin that inhibit platelet adhesiveness. Secondly, a glycoprotein on the surface of endothelial cells, known as thrombomodulin, serves as an endogenous anticoagulant by activating protein C (which inactivates clotting factors V and VII). Finally, the endothelium serves as a barrier that separates the blood from tissue elements that can trigger thrombosis.

When the endothelium is disrupted, platelets adhere to exposed collagen in the subendothelium and begin to form a platelet plug. The platelets release calcium, which activates the glycoprotein receptor IIb/IIIa complex on the platelet surface. This receptor complex binds irreversibly to von Willebrand factor on the surrounding endothelial cells, which helps to anchor the platelet plug to the vessel wall. The IIb/IIIa receptor complex also binds fibrinogen, and the subsequent formation of fibrin bridges between adjacent platelets allows the platelet plug to grow and mature into a fibrin-platelet thrombus. (Drugs that inhibit the IIb/IIIa receptor complex are used in the management of acute coronary syndromes—see page 315).
Endothelial and tissue injury also promote the formation of fibrin, which is essential for the growth and stability of the developing thrombus. There are two pathways to fibrin formation (2). The major pathway is called the Tissue Factor Pathway (formerly called the Extrinsic Pathway), and is activated by the release of thromboplastin (tissue factor) from the subendothelium. The second pathway is the Contact Activation Pathway (formerly called the Intrinsic Pathway), and is activated by endogenous peptides known as kininogens, which are precursors of bradykinin. Both pathways involve the activation of specific procoagulant proteins known as clotting factors, and they both lead to the activation of prothrombin (factor IT) and the subsequent conversion of fibrinogen (factor I) to fibrin monomers.
The end-product of the response to injury is the thrombus, which is essentially a clump of platelets embedded in a meshwork of fibrin strands and anchored to vessel wall in the area of injury.

Thrombocytopenia is the most common hemostatic disorder in critically ill patients, with a reported incidence as high as 60% (3,4). The traditional definition of thrombocytopenia is a platelet count below 150,000/c<L, but the ability to form a hemostatic plug is retained until the platelet count falls below 100,000/°<L (4), so a platelet count <100,000/ccL is more appropriate for identifying clinically significant thrombocytopenia. However, the risk of major bleeding is not determined by the platelet count alone, but also requires a structural lesion that is prone to bleeding. In the absence of such a lesion, platelet counts as low as 5,000/e<L can be tolerated without evidence of major hemorrhage (5). The major risk with a platelet count <10,000/e<L is spontaneous intracerebral hemorrhage, which is uncommon (4).

Pseudothrombocytopenia is a condition where antibodies to EDTA (the anticoagulant in blood collection tubes) produce clumping of platelets in vitro. The clumped platelets are misread as leukocytes by automated machines that perform cell counts, and this results in a spuriously low platelet count (4,6). This phenomenon has been reported in 2% of platelet counts performed in hospitalized patients (6), and is most often seen in patients with severe sepsis or autoimmune, neoplastic, or liver disease (4). Suspicion of pseudothrombocytopenia is usually prompted by a platelet count that is lower than expected, or by the presence of clumped platelets on the peripheral blood smear. If suspected, blood collection tubes that use citrate or heparin as an anticoagulant should be used for subsequent platelet counts.

The most likely causes of thrombocytopenia in the ICU setting are listed in Table 19.1. Systemic sepsis is the most common cause of thrombocytopenia in ICU patients (7), and is the result of increased platelet destruction by macrophages (8). Other less common but more life-threatening causes of thrombocytopenia include heparin and the thrombotic micro-angiopathies; i.e., disseminated intravascular coagulation (DIC), thrombotic thrombocytopenia purpura (TTP), and the pregnancy-related HELLP syndrome.

Cardiopulmonary Bypass Disseminated Intravascular Coagulation (DIC 9 (OK) HELLP Syndrome Hemolytic-Uremic Syndrome HIV Infection Liver Disease/Hypersplenism Intra-Aortic Balloon Pump Massive Transfusion Renal Replacement Therapy Systemic Sepsist Thrombotic Thrombocytopenia Purpura (TTP), Pharmacological = Anticonvulsants: Phenytoin Valproic Acid Antimicrobial Agents: B-Lactams Linezolid TMP/SMX Vancomycin Antineoplastic Agents Antithrombotic Agents Heparin IIbvilla Inhibitors Histamine H; Blockers Miscellaneous Drugs Amiodarone Furosemide Thiazides Morphine
+The most common cause of thrombocytopenia in the ICU satting.
From Referances 3, 4, 8, 9.
Drugs like antineoplastic agents can produce thrombocytopenia by suppressing platelet production in the bone marrow, but the most common mechanism for drug-induced thrombocytopenia is the production of antibodies that cross-react with platelets (8). This immune-mediated thrombocytopenia is most frequently observed with heparin, and less frequently with platelet glycoprotein receptor (IIb/IIIa) antagonists and selected antibiotics (particularly linezolid, B-lactams, and vancomycin).

There are two types of thrombocytopenia associated with heparin. The first is a nonimmune response that results in mild thrombocytopenia (platelet counts may not fall below 100,000/c<L) in the first few days after starting heparin. This reaction is reported in 10 to 30% of patients receiving heparin (10), and it resolves spontaneously without interruption of heparin, and without adverse consequences. The second type of thrombocytopenia is an immune response that typically appears 5 to 10 days after starting heparin (10,11). This reaction is much less common (incidence = 1—3%) but is much more serious; i.e., it can produce life-threatening thrombosis (not hemorrhage), and has a mortality rate as high as 30% if left unnoticed (10). The term heparin-induced thrombocytopenia (HIT) is reserved for the immune-mediated thrombocytopenia, and this condition is the focus of the current presentation.

Heparin is not immunogenic itself, but it binds to a protein (platelet factor 4) on platelets to form an antigenic complex that can trigger the formation of IgG antibodies. These antibodies bind to platelets and induce a strong platelet activation response to promote thrombosis. These antibodies can also bind to endothelial cells and promote the release of tissue factor from the endothelium; this promotes fibrin formation and further accelerates the thrombotic process. The reticuloendothelial system can clear antibodycoated platelets, and this helps to limit the incidence of thrombosis. Heparin-associated antibodies usually disappear within 3 months after discontinuing heparin (10).

One of the most important features of HIT is the fact that it is not a dose-dependent reaction, and can occur as a result of heparin exposure from heparin-based flushes of intravascular catheters, or even heparin-coated pulmonary artery catheters (12). The type of heparin preparation does, however, influence the risk of HIT; i.e., the risk of HIT is ten times greater with unfractionated heparin (UFH) than with low-molecular-weight heparin (LMWH) (11). The risk of HIT also varies by patient population; i.e., it is highest in patients undergoing orthopedic and cardiac surgical procedures, and lowest in medical patients (10,11). The reported incidence of HIT with UFH is 1-—5% following orthopedic or cardiac surgery, and 0.1—-1% in medical patients (11).

HIT typically appears 5 to 10 days after the first exposure to heparin, but can appear within 24 hours in patients with HIT antibodies due to heparin exposure within the past 3 months (11). Platelet counts are usually between 50,000/ecL and 150,000/e<L. Severe thrombocytopenia (<20,000/mL) is uncommon in HIT (10,11). In up to 25% of cases of HIT, the thrombosis precedes the thrombocytopenia (11).
THROMBOSIS: Venous thrombosis is more common than arterial thrombosis. Reports indicate that 17% to 55% of patients with untreated HIT develop deep vein thrombosis in the legs and/or pulmonary embolism, whereas only 1% to 3% of patients develop arterial thromboses resulting in limb ischemia, thrombotic stroke, or acute myocardial infarction (11). Limb gangrene from thrombotic veno-occlusion has been reported in 5% to 10% of patients with HIT who are treated with a vitamin K antagonist (e.g., coumadin).

About 8 different assays are currently used to detect HIT antibodies. The most popular of these is an enzyme-linked immunosorbent assay (ELISA) for antibodies to the platelet factor 4-heparin complex. A negative assays helps to exclude the diagnosis of HIT, but a positive assay does not confirm the diagnosis because HIT antibodies do not always promote thrombocytopenia or thrombosis (11). The diagnosis of HIT requires a positive antibody assay in combination with a high index of clinical suspicion.

, Dosing Regimen and Comments = 1.6-2 mg/dl. , Dosing Regimen and Comments = 50%. , Dosing Regimen and Comments = 21-3. , Dosing Regimen and Comments = 21-3. , Dosing Regimen and Comments = 70%. , Dosing Regimen and Comments = 3.1-6. , Dosing Regimen and Comments = 3.1-6. , Dosing Regimen and Comments = 85%. , Dosing Regimen and Comments = >6. , Dosing Regimen and Comments = >6. , Dosing Regimen and Comments = Avoid. , Dosing Regimen and Comments = Comment:. , Dosing Regimen and Comments = Severe anaphylactic reactions can occur with re-exposure to lepirudin, so only a single course of therapy is advised.. , Dosing Regimen and Comments = Severe anaphylactic reactions can occur with re-exposure to lepirudin, so only a single course of therapy is advised.
From Referances 10, 11, 14.

Heparin must be discontinued immediately (don't forget to discontinue heparin flushes and remove heparin-coated catheters). Therapeutic anticoagulation with one of the direct thrombin inhibitors shown in Table 19.2 should be started immediately, even in cases where HIT is not accompanied by thrombosis (11). The recommendation for full anticoagulation without evidence of thrombosis is based on studies showing a 10-fold higher incidence of thrombosis following the appearance of HIT when anticoagulation is delayed (13).
ARGATROBAN: Argatroban is a synthetic analogue of L-arginine that reversibly binds to the active site on thrombin. It has a rapid onset of action, and is given by continuous infusion using the dosing regimen in Table 19.2. The therapeutic goal is an activated partial thromboplastin time (aPTT) of 1.5 to 3 times control values. The drug is cleared primarily by the liver, and a dose adjustment is necessary in hepatic insufficiency. Argatroban is recommended in patients with renal insufficiency (11) because a dose adjustment is not necessary.
LEPIRUDIN: Lepirudin is a recombinant form of hirudin, an anticoagulant found in leech saliva (!) that binds irreversibly to thrombin. Lepirudin is also given by continuous infusion, which can be preceded by a bolus injection in cases of life-threatening thrombosis. The therapeutic goal is the same as with argatroban (aPTT=1.5-3 x control). Lepirudin is cleared by the kidneys, and a dose adjustment is necessary when renal function is even mildly impaired (i.e., when the serum creatinine is above 1.5 mg/dL), as shown in Table 19.2 (14). Using argatroban in patients with renal impairment will avoid dosing adjustments. Finally, re-exposure to lepirudin can produce lifethreatening anaphylactic reactions (11), so the treatment of HIT with lepirudin is usually a one-time affair.

DURATION OF TREATMENT: Full anticoagulation with argatroban or lepirudin is recommended until the platelet count rises above 150,000/ecL (11). Thereafter, coumadin can be used for long-term anticoagulation if HIT is associated with thrombosis, but there are 2 caveats: (a) coumadin should NOT be started until the platelet count increases beyond 150,000/e<¢L, and (b) the initial coumadin dose should not exceed 5 mg (11). These precautions are intended to reduce the risk of limb gangrene associated with coumadin therapy during the active phase of HIT (as mentioned earlier). The antithrombin agents should be continued until coumadin achieves full anticoagulation.
Schistocytes, 1 = Present. Schistocytes, 2 = Present. Schistocytes, 3 = Present. Platelets, 1 = Low. Platelets, 2 = Low. Platelets, 3 = Low. INR, 1 = Elevated. INR, 2 = Normal. INR, 3 = Normal. aPTT, 1 = Prolonged. aPTT, 2 = Normal. aPTT, 3 = Normal. Fibrinogen, 1 = Low. Fibrinogen, 2 = Normal. Fibrinogen, 3 = Normal. Plasma D-dimer, 1 = Elevated. Plasma D-dimer, 2 = Normal. Plasma D-dimer, 3 = Normal. Liver Enzymes, 1 = Variable. Liver Enzymes, 2 = Normal. Liver Enzymes, 3 = Elevated
Fram Referance 4.

Disseminated intravascular coagulation (DIC) is a secondary disorder that is triggered by conditions that produce widespread tissue injury such as multisystem trauma, severe sepsis and septic shock, and obstetric emergencies (amniotic fluid embolism, abruptio placentae, eclampsia, and retained fetus syndrome). The inciting event is release of tissue factor, which (as described earlier) activates a series of clotting factors in the bloodstream that culminates in the formation of fibrin. This leads to widespread microvascular thrombosis and secondary depletion of platelets and clotting factors, resulting in a consumptive coagulopathy (15).

The microvascular thrombosis in DIC can lead to multiorgan failure, most often involving the lungs, kidneys, and central nervous system, while depletion of platelets and coagulation factors can promote bleeding, particularly from pre-existing lesions in the GI tract such as stress ulcers. DIC can also be accompanied by symmetrical necrosis and ecchymosis involving the limbs, a condition known as purpura fulminans that is usually seen with overwhelming systemic sepsis, most notably with meningococcemia (7).
HEMATOLOGIC ABNORMALITIES: In addition to thrombocytopenia, DIC is usually (but not always) associated with elevation of the INR (i.e., prolongation of the prothrombin time) and prolongation of the activated partial thromboplastin time (aPTT), both abnormalities being the result of consumption and subsequent depletion of clotting factors in blood. The enhanced thrombosis is also accompanied by enhanced fibrinolysis, which elevates the fibrin degradation products in plasma (i.e., plasma D-dimers). Finally, the microangiopathic hemolytic anemia is identified by the presence of damaged or fragmented erythrocytes in a peripheral blood smear, like the ones in Figure 19.1. The fragmented erythrocytes are known as schistocytes, and they are the hallmark of the thrombotic microangiopathies.
hemolytic anemia.

There is no specific treatment for DIC other than supportive care. Uncontrolled bleeding often prompts consideration of replacement therapy with platelets and coagulation factors (plasma products), but this rarely helps and can be deleterious by "adding fuel" to the microvascular thrombosis. In severe cases of DIC associated with multiorgan failure, the mortality rate is 80% or higher (7,15).

Thrombotic thrombocytopenia purpura (TTP) is thrombotic microangiopathy that is caused by platelet binding to abnormal von Willebrand factor on microvascular endothelium (4). This can be a devastating condition that is fatal within 24 hours of onset. There is often no predisposing condition, although it seems to follow a nonspecific viral illness in some cases.

TTP presents with a characteristic pentad of clinical manifestations that includes fever, altered mental status, acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia. The presence of all 5 conditions is not necessary for the diagnosis of TTP, but the diagnosis does require thrombocytopenia and evidence of a microangiopathic hemolytic anemia (e.g., schistocytes in the peripheral blood smear). TTP can be distinguished from DIC because clotting factors are not depleted in TTP, so the INR, aPTT, and fibrinogen levels are normal in TTP.

Platelet transfusions are contraindicated in TTP because they can aggravate the underlying thrombosis. The treatment of choice for TTP is plasma exchange (16,17), where blood from the patient is diverted to a device that separates and discards the patient's plasma and reinfuses plasma from a healthy donor. This is continued until 1.5 times the normal plasma volume is exchanged, and this process is repeated daily for 3-7 days. Acute fulminant TTP is almost always fatal if untreated, but if plasma exchange is started early (with 48 hours of symptom onset), as many as 90% of patients can survive the illness (16,17).

HELLP (Hemolysis, Elevated Liver enzymes, Lo w Platelets) syndrome is a thrombotic microangiopathy that occurs late in pregnancy or in the early postpartum period (18). About 20% of cases are associated with severe pre-eclampsia, and there is also an association with the antiphospholipid syndrome (19). The culprit in the HELLP syndrome is unexplained activation of clotting factors and platelets leading to microvascular thrombosis. There is also an unexplained elevation in liver enzymes, principally the transaminases (18).

As the name indicates, HELLP is identified by the characteristic triad of hemolysis, thrombocytopenia, and elevated liver enzymes. HELLP can be confused with DIC (which can occur in the same clinical settings), but the INR and aPTT are usually normal in HELLP because there is no depletion of clotting factors, and this feature should distinguish HELLP from DIC (see Table 19.3).
The HELLP syndrome is an obstetric emergency, and a detailed description of this condition is beyond the scope of this text. For more information on HELLP, some recent reviews are included in the bibliography at the end of the chapter (18,19).

Platelets are obtained either by pooling the platelets from multiple donors, or by extracting platelets from a single donor using apheresis techniques.

Platelets are separated from fresh whole blood by differential centrifugation, and the resulting platelet concentrates from 5 units of whole blood (from 5 individual donors) are pooled together prior to storage. The pooled platelet concentrate contains about 38 x 1010 platelets in 260 mL plasma, which is equivalent to a platelet count of about 130 x 109/ecL. This is six orders of magnitude higher than the normal platelet count in blood (150-400 x 103/cecL). Platelets are stored at 20—24°C, and can be stored for up to 5 days.

Apheresis platelets are collected from a single donor and have a platelet count and volume that is equivalent to the pooled platelets from 5 donors. The presumed benefit of single-donor platelet transfusions is a lower risk of transmitted infections and a lower incidence of platelet alloimmunization (i.e., developing antibodies to donor platelets). How-ever, neither of these proposed benefits has been documented in clinical trials (20), and when leukocytes are removed from platelet products, there is no difference in the risk of platelet alloimmunization with single-donor and multiple-donor platelet transfusions (22).

Leukocytes in donor blood have been implicated in several adverse reactions, and leukocyte removal using specialized filters is now a routine practice for erythrocyte transfusions (see last chapter). Platelet concentrates are not free of leukocytes, and leukocyte reduction for platelet transfusions has the following advantages (20,22): a lower incidence of cytomegalovirus transmission (because this organism is transmitted in leukocytes), fewer febrile reactions, and a lower incidence of platelet alloimmunization. Because of these advantages, leukocyte reduction is becoming a routine practice for platelet transfusions.

In an average sized adult with no ongoing blood loss, a platelet concentrate from one unit of whole blood should raise the circulating platelet count by 7,000 to 10,000/°<L at one hour post-transfusion (20). Since an average of 5 platelet concentrates are pooled together for each platelet transfusion, the expected (or ideal) increase in platelet count is 35,000 to 50,000/e«L at one hour post-transfusion. The increment is about 40% lower after 24 hours, as shown in Figure 19.2. Note: The number of platelet concentrates that are pooled together can differ slightly (by +1 unit) in individual platelet transfusions, so if you want to be precise about estimating post-transfusion platelet increments (which is usually not necessary), you need to enquire about the number of units of platelets included in the transfusion pack.

The increment in platelet count declines with multiple transfusions. This is shown in Figure 19.2, where the platelet increment is about 25% lower after 5 platelet transfusions (23). As mentioned earlier, this phenomenon of "platelet refractoriness" is the result of antiplatelet antibodies in the recipient directed at ABO antigens on donor platelets. This effect can be mitigated by transfusing ABO-matched platelets.

In the presence of active bleeding other than ecchymoses or petechiae, platelet transfusions are recommended to maintain a platelet count >50,000/ecL (21). For intracranial hemorrhage, higher platelet counts (>100,000/e¢L) should be maintained (21).
Platelet
FIGURE 19.2 Post-transfusion increment in platelet counts in relation to the time elapsed after the transfusion (one hour versus 24 hours) and the number of transfusions given. Data from Reference 23.

Despite evidence that spontaneous bleeding through an intact vascular system is uncommon with platelet counts down to 5,000/e<L (20), most experts are reluctant to adopt a platelet transfusion trigger as low as 5,000/e<L. In the absence of bleeding (other than ecchymoses or petechiae), prophylactic platelet transfusions are usually recommended when the platelet count reaches 10,000/e¢L (21).

In the absence of associated coagulation abnormalities:
1. Platelet counts >40,000/ecL are sufficient to perform laparotomy, craniotomy, tracheotomy, percutaneous liver biopsy, and bronchoscopic or endoscopic biopsy (20).
2. Platelet counts >20,000/e<L are sufficient to perform lumbar punctures (20).
3. Platelet counts >10,000/°<¢L are sufficient to perform central venous cannulation safely (24,25).

Bacteria are much more likely to flourish in platelet concentrates than in RBC concentrates (packed cells) because platelets are stored at room temperature (22°C), while RBCs are refrigerated at about 4°C. It is estimated that one in every 2,000 to 3,000 platelet concentrates harbors bacteria, and that one in 5,000 concentrates will produce sepsis in the recipient (16). Cultures of all platelet concentrates are now required (16) but, since platelets can be stored for only 5 days, the platelets can be transfused before the culture results are available.

Febrile nonhemolytic reactions have been reported in as many as 30% of platelet transfusions (26), which is much greater than the 0.5% rate of similar reactions reported with RBC transfusions (see Table 18.3 on page 359). Part of this difference may be related to the multiple donors used for platelet transfusions. Since antibodies to donor leukocytes are implicated in this reaction, leukoreduction of platelet products will help to alleviate this problem.

Hypersensitivity reactions (urticaria, anaphylaxis, anaphylactic shock) are also more common with platelet transfusions that with erythrocyte transfusions (20). Since this is a reaction to proteins in donor plasma, removing the plasma from platelet concentrates will reduce the risk of hypersensitivity transfusion reactions.

Transfusion-related acute lung injury (TRALI) is described in Chapter 18 (see pages 361363). This condition, which is an inflammatory lung injury similar to the acute respiratory distress syndrome (ARDS), is most often associated with erythrocyte transfusions, but has also been reported in association with platelet transfusions (27). The culprit is believed to be antileukocyte antibodies in donor blood that activate neutrophils in the recipient. TRALI usually appears within 6 hours after the start of a transfusion, and management is supportive.
Plasma products are used as a source of coagulation factors, but surveys indicate that about 50% of plasma transfusions are inappropriate (28).

Plasma is separated from donor blood and frozen at —18°C within 8 hours of blood collection. This fresh frozen plasma (FFP) has a volume of about 230 mL, and can be stored for one year. Once thawed, FFP can be stored at 1 to 6°C for up to 5 days. The principal uses of FFP include the resuscitation of massive blood loss, and the reversal of excess anticoagulation with coumadin.

As described in Chapter 11 (see pages 209-210), the use of FFP in massive blood loss (blood loss equivalent to one blood volume within 24 hours) has become more aggressive in recent years, mostly as a result of experiences in combat injuries. Whereas the traditional practice was to transfuse one unit of FFP for every 6 units of packed RBCs to prevent a dilutional coagulopathy, there is now evidence that severe trauma is accompanied by a coagulopathy (30), and survival rates have improved with FFP:RBC ratios of 1:2 to 1:3 during massive transfusion (31,32). This approach is called hemostatic resuscitation, and the goal is an INR that remains below 1.5. (Note: the international normalized ratio or INR is the ratio of the patient's prothrombin time and an international standard for the normal or control prothrombin time; i.e., INR = Patient's PT/standardized control PT.)

The annual incidence of major bleeding during warfarin anticoagulation is 3 to 12%, and the incidence of fatal hemorrhage is 1 to 3% (33), with intracerebral hemorrhage causing most of the fatalities. The management of major or life-threatening hemorrhage related to excessive warfarin anticoagulation is shown in Table 19.4 (34,35). Since warfarin acts by inhibiting vitamin K-dependent clotting factors (i.e., factors II, VII, IX, X), vitamin K is administered to block ongoing anticoagulant activity. Clotting factors are then replenished, and this has traditionally involved the infusion of FFP at a volume of 15 mL/kg. There are two shortcomings with the use of FFP in this setting: the time to normalize the INR can be prolonged, and the volume of fluid required can aggravate the bleeding. These problems can be mitigated by using the plasma product described next.
PROTHROMBIN COMPLEX CONCENTRATE: Normalizing the INR quickly with a limited infusion volume is possible with prothrombin complex concentrates (PCC) using the dosing recommendations in Table 19.4. There are 3-factor and 4-factor PCC preparations (the name indicating the number of vitamin K-dependent clotting factors in the preparation), but only the 3-factor PCC is approved for clinical use. PCC is a lyophilized powder that is quickly reconstituted, thereby avoiding the time delays involved in thawing FFP. Whereas FFP can take hours to normalize the INR, PCC can accomplish this task in less than 30 minutes (34,35). The rapid response and limited volume associated with PCC make it well suited for the treatment of warfarin-induced bleeding, particularly intracerebral hemorrhage (35).

1, Give 10 mg vitamin K intravenously over 10 minutes. (Do not exceed 1 mg/min to avoid hypersensitivity-like reactions.), 1 = 1, Give 10 mg vitamin K intravenously over 10 minutes. (Do not exceed 1 mg/min to avoid hypersensitivity-like reactions.). 2. If available, give prothrombin complex concentrate (PCC) using the dosing recommendations shown below. Check the INR 30 min after each dose of PCC., 1 = 2. If available, give prothrombin complex concentrate (PCC) using the dosing recommendations shown below. Check the INR 30 min after each dose of PCC.. INR, 1 = PGG Dose. 2-3.9, 1 = 35-39 IU/kg. 4-6, 1 = 40-45 IU/kg. >6, 1 = 46-50 IU/kg. 8. If PCC Is not available, give fresh frozen plasma, 15 mL/kg., 1 = 8. If PCC Is not available, give fresh frozen plasma, 15 mL/kg.. 4, The management goal is an INR <1.5., 1 = 4, The management goal is an INR <1.5.
From Referancas 34, 35.

The risks associated with transfusion of plasma products are essentially the same risks associated with transfusion of erythrocytes and/or platelets. The exception is nonhemolytic febrile transfusion reactions, which are caused by donor leukocytes and thus should not occur with plasma transfusions.

Acute hemolytic reactions are caused by anti-A and anti-B antibodies in the transfused plasma that react with A and B antigens on recipient RBCs. Since cross-matching of plasma transfusions is not a universal practice, acute hemolytic reactions continue to be reported with plasma transfusions. The evaluation of suspected acute hemolytic transfusion reactions is described in Chapter 18 (see pages 359-360).

Plasma transfusions carry a minimal risk of transmitting infections. The risk of hepatitis B transmission is 1 per 900,000 transfusions, the risk of hepatitis C transmission is 1 per 30 million transfusions, and the risk of HIV transmission is one per 8 million transfusions (39). The risk of bacterial transmission is reported as "rare" and CMV transmission, which occurs in transfused leukocytes, has not been reported with plasma transfusions (39).

Hypersensitivity reactions (urticaria, anaphylaxis, anaphylactic shock), which are caused by sensitization to proteins in donor plasma, are more common with plasma transfusions than with erythrocyte or platelet transfusions. These reactions are, however, uncommon; e.g., the reported incidence of allergic reactions in the United Kingdom is approximately 1 case per 17,000 plasma transfusions (39).

Transfusion-related acute lung injury (TRALI) is attributed to antileukocyte antibodies in donor blood, and is a complication of erythrocyte, platelet, and plasma transfusions. The reported incidence of TRALI after platelet transfusions is 1 per 60,000 units (39), which is much less than the reported incidence following RBC transfusions (1 per 12,000 units). The clinical features of TRALI are described in Chapter 18 (see pages 361-363).

1. King KE (ed). Overview of hemostasis. In: Blood transfusion therapy: A physician's handbook. 9th ed. Bethesda, MD: American Association of Blood Banks, 2008.
2. Wheeler AP, Rice TW. Coagulopathy in critically ill patients. Part 2 — Soluble clotting factors and hemostatic testing. Chest 2010; 137:185—-194.

P, Lubenow N, et al. Heparin-induced thrombocytopenia with thromboembolic complications: a meta-analysis of 2 prospective trials to assess the value of parenteral treatment with lepirudin and its therapeutic aPTT range. Blood 2000; 96:846-851.. 14., 1 = Lepirudin drug monograph. In McEvoy GK, ed. AHFS Drug Information, 2012. Bethesda, MD: American Society of Health System Pharmacists, 2012:1476-1478.. 15., 1 = Senno SL, Pechet L, Bick RL. Disseminated intravascular coagulation (DIC). Pathophysiology, laboratory diagnosis, and management. J Intensive Care Med 2000; 15:144-158.. 16., 1 = Rock GA, Shumack KH, Buskard NA, et al. Comparison of plasma exchange with plasma infusion in the treatment of thrombotic thrombocytopenia purpura. N Engl J Med 1991; 325:393-397.. 17., 1 = Hayward CP, Sutton DMC, Carter WH Jr , et al. Treatment outcomes in patients with adult thrombotic thrombocytopenic purpura-hemolytic uremic syndrome. Arch Intern Med 1994; 154:982-987.. 18. Kirkpatrick CA. The HELLP syndrome. Acta Clin Belg 2010; 65:91-97., 1 = 18. Kirkpatrick CA. The HELLP syndrome. Acta Clin Belg 2010; 65:91-97.. 19., 1 = Di Prima FAF, Valenti O, Hyseni E, et al. Antiphospholipid syndrome during
pregnancy: the state of the art. J Prenat Med 2011; 5:41-53.

Platelet Transfusions, 1 = Platelet Transfusions. 20., 1 = Slichter SJ. Platelet transfusion therapy. Hematol Oncol Clin N Am 2007; 21:697- 729.. 21., 1 = Slichter SJ. Evidence-based platelet transfusion guidelines. Hematol 2007; 2007:172-178.. 22., 1 = The Trial to Reduce Alloimmunization to Patients Study Group. Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. N Engl J Med 1997; 337:1861-1869.. 23., 1 = Slichter SJ, Davis K, Enright H, et al. Factors affecting post-transfusion platelet increments, platelet refractoriness, and platelet transfusion intervals in thrombocytopenic patients. Blood 2005; 105:4106-4114.. 24., 1 = Doerfler ME, Kaufman B, Goldenberg AS. Central venous catheter placement in patients with disorders of hemostasis. Chest 1996; 110:185-188.. 25., 1 = DeLoughery TG, Liebler JM, Simonds V, et al. Invasive line placement in critically ill patients: Do hemostatic defects matter? Transfusion 1996; 36:827-831.. 26., 1 = Gelinas J-P , Stoddart LV, Snyder EL. Thrombocytopenia and critical care medicine. J Intensive Care Med 2001; 16:1-21.. 27., 1 = Sayah DM. Looney MR, Toy P. Transfusion reactions. Newer concepts on the pathophysiology, incidence, treatment, and prevention of transfusion-related acute lung injury. Crit Care Clin 2012; 28:363-372.

I breathe for my own necessity, for my survival.
Ayn Rand
The Fountainhead
1943

Respiration is thus a process of combustion, in truth very slow, but otherwise exactly like that of charcoal.
Antoine Lavoisier
Antoine Lavoisier was an 18th-century French scientist who was the first to identify oxygen as the essential element for metabolism, and the first to discover that aerobic metabolism is essentially a combustion reaction, where oxygen reacts with an organic fuel and produces carbon dioxide as a by-product. (One of the many tragedies of the French Revolution was the senseless beheading of Antoine Lavoisier in 1794.) Providing the oxygen and removing the carbon dioxide is the responsibility of the lungs, and this chapter describes how the lungs perform this task, and how abnormalities in lung function can lead to deficits in arterial oxygenation (hypoxemia) and accumulation of carbon dioxide (hypercapnia). The last part of the chapter presents a physiological approach to the evaluation of hypoxemia and hypercapnia in individual patients.

The efficiency of gas exchange in the lungs is determined by the balance between alveolar ventilation and pulmonary capillary blood flow (1-4). This balance is commonly expressed as the ventilation—perfusion (V/Q) ratio. The influence of V/Q ratios on pulmonary gas exchange can be described using an alveolar—capillary unit, as shown in Figure 20.1. The upper panel shows a perfect match between ventilation and perfusion (V/Q = 1). This is the reference point for defining the abnormal patterns of gas exchange.

A V/Q ratio above 1.0 (Fig. 20.1, middle panel) describes the condition where ventilation is excessive relative to pulmonary capillary blood flow. The excess ventilation, Known as dead space ventilation, does not participate in gas exchange with the blood. Dead space ventilation includes anatomic dead space, which is the gas in the large conducting airways that does not come in contact with capillary blood (about half of the anatomic dead space is in the pharynx), and physiologic dead space, which is alveolar gas that does not equilibrate fully with capillary blood. In normal subjects, dead space ventilation (Vp) accounts for 20% to 30% of the total ventilation (V+); i.e., Vp/Vr = 0.2 to 0.3 (1,3).
, Wa vo) = . , TERM = V-Q Match. , - CONSEQUENCES = Normal PaO». , Wa vo) = >1. , TERM = Dead Space Ventilation. , - CONSEQUENCES = 1PaO. tPacos. , Wa vo) = . , TERM = Venous Admixture. , - CONSEQUENCES = $Pad, Normal or }PaCO.
FIGURE 20.1 Ventilation—perfusion (V/Q) relationships and associated blood gas abnormalities.

Dead space ventilation increases in the following situations:
1. When the alveolar—capillary interface is destroyed; e.g., emphysema
2. When blood flow is reduced; i.e., low cardiac output
3. When alveoli are overdistended; e.g., during positive-pressure ventilation
ARTERIAL BLOOD GASES: An increase in Vp/V7 above 0.3 results in both hypoxemia (decreased arterial PO) and hypercapnia (increased arterial PCO), which is analogous to what would happen if you held your breath. The hypercapnia usually appears when the Vp/V+z is above 0.5 (5).

A V/Q ratio below 1.0 (Fig. 20.1, lower panel) occurs when pulmonary capillary blood flow is excessive relative to ventilation. The excess blood flow, known as intrapulmonary shunt, does not participate in pulmonary gas exchange. There are two types of intrapulmonary shunt. True shunt indicates the total absence of gas exchange between capillary blood and alveolar gas (V/Q = 0), and is equivalent to an anatomic shunt between the right and left sides of the heart. Venous admixture represents the capillary flow that does not equilibrate completely with alveolar gas (0<V/Q <1). As the venous admixture increases, the V/Q ratio decreases until it becomes a true shunt (V/Q = 0).
The fraction of the cardiac output that represents intrapulmonary shunt is known as the shunt fraction. In normal subjects, intrapulmonary shunt flow (Qs) represents less than 10% of the total cardiac output (Qt), so the shunt fraction (Qs/Qt) is less than 10% (1,2,4).

Intrapulmonary shunt fraction is increased in the following situations:
1. When the small airways are occluded; e.g., asthma
2. When the alveoli are filled with fluid; e.g., pulmonary edema, pneumonia
3. When the alveoli collapse; e.g., atelectasis
4. When capillary flow is excessive; e.g., in nonembolized regions of the lung in pulmonary embolism
ARTERIAL BLOOD GASES: The influence of shunt fraction on arterial O27 and CO, tensions (PaO>z, PaCO>, respectively) is shown in Figure 20.2. The PaO, falls progressively as shunt fraction increases, but the PaCO» remains constant until the shunt fraction exceeds 50% (4). The PaCO, is often below normal in patients with increased intrapulmonary shunt as a result of hyperventilation triggered by the disease process or by the accompanying hypoxemia.

The shunt fraction also determines the influence of inhaled oxygen on the arterial PO>. This is shown in Figure 20.3 (4). As intrapulmonary shunt increases from 10 to 50%, an increase in fractional concentration of inspired oxygen (FIO) produces less of an increment in the arterial PO2. When the shunt fraction exceeds 50%, the arterial PO> is independent of changes in FIOz, and the condition behaves like a true (anatomic) shunt. This means that, in conditions associated with a high shunt fraction (e.g., acute respiratory distress syndrome), the FIO2 can often be lowered to non-toxic levels (FIO below 60%) without further compromising arterial oxygenation. This can be a valuable maneuver for preventing pulmonary oxygen toxicity.
FIGURE 20.2 The influence of shunt fraction on arterial PO? (PaO?) and arterial PCO? (PaCO2). From Reference 4.
FIGURE 20.3 The influence of shunt fraction on the relationship between the inspired oxygen (FIO2) and the arterial PO? (PaO2). From Reference 4.

The calculation of dead space ventilation (Vp/V+) is based on the difference between the PCO, in exhaled gas and end-capillary (arterial) blood. In the normal lung, the capillary blood equilibrates fully with alveolar gas, and the exhaled PCO, (PECO>) is equivalent to the arterial PCO (PaCO>). As dead space ventilation (Vp/Vr) increases, the PECO, decreases relative to the PaCO>. The Bohr equation shown below (derived by Christian Bohr, father of Neils Bohr, one of the founders of quantum mechanics) is based on this principle.
Thus, when the PECO> decreases relative to the PaCO>, the calculated Vp/V+y rises. The PECO> is measured in a random sample of expired gas (mean ex-haled PCO3), and is not measured at the end of expiration (end-tidal PCO3).

The intrapulmonary shunt fraction (Qs/Qt) is derived by the relationship between the O> content in arterial blood (CaO), mixed venous blood (CvO2), and pulmonary capillary blood (CcO3).
The problem with this formula is the inability to measure the pulmonary capillary O> content (CcO>) directly. As a result, pure oxygen breathing (to produce 100% oxyhemoglobin saturation in pulmonary capillary blood) is recommended for the shunt calculation. However in this situation, Qs/Qt measures only true shunt.
able 20.1 Normal Arterial Blood Gase
20, PaO, (mmHg) = 84-95. 20, PaCO, (mm Hg) = 33-47. 20, A-a PO, (mm Hg) = 4-17. 30, PaO, (mmHg) = 81-92. 30, PaCO, (mm Hg) = 34-47. 30, A-a PO, (mm Hg) = 7-21. 40, PaO, (mmHg) = 78-90. 40, PaCO, (mm Hg) = 34-47. 40, A-a PO, (mm Hg) = 10-24. 50, PaO, (mmHg) = 75-87. 50, PaCO, (mm Hg) = 34-47. 50, A-a PO, (mm Hg) = 14-27. 60, PaO, (mmHg) = 72-84. 60, PaCO, (mm Hg) = 34-47. 60, A-a PO, (mm Hg) = 17-31. 70, PaO, (mmHg) = 70-81. 70, PaCO, (mm Hg) = 34-47. 70, A-a PO, (mm Hg) = 21-34. 80, PaO, (mmHg) = 67-79. 80, PaCO, (mm Hg) = 34-47. 80, A-a PO, (mm Hg) = 25-38
All valuas partain ta room air braathing at saa level,

From the Intermountain Thoracic Society Manual of Uniform Laboratory Procedures. Sat Laka City, 41984:44-45,

The influence of inspired oxygen on the A-a PO> gradient is shown in Figure 20.4 (7). The A-a PO> gradient increases from 15 to 60 mm Hg as the FIO; increases from 21% (room air) to 100%. According to this relationship, the normal A-a PO> gradient increases 5 to 7 mm Hg for every 10% increase in FIO>. This effect is presumably caused by the loss of regional hypoxic vasoconstriction in the lungs. Hypoxic vasoconstriction in poorly ventilated lung regions diverts blood to more adequately ventilated regions, and this helps to preserve the normal V/Q balance. Loss of regional hypoxic vasoconstriction during supplemental O> breathing maintains blood flow in poorly ventilated lung regions, and this increases intrapulmonary shunt fraction and increases the A-a PO> gradient. The FIQ; is difficult to estimate accurately when supplemental O, is delivered via nasal prongs or "open" face masks (see Chapter 22), and this limits the accuracy of the A-a PO» gradient in these situations.

Positive-pressure mechanical ventilation elevates the pressure in the airways above the ambient barometric pressure. Therefore, when determining the A-a PO» gradient in a ventilator-dependent patient, the mean airway pressure should be added to the barometric pressure (8). In the example presented in equation 20.6, a mean airway pressure of 30 cm H20 would increase the A-a PO» gradient from 10 to 16 mm Hg (a 60% increase). Thus, neglecting the contribution of positive airway pressure during mechanical ventilation will underestimate the degree of abnormal gas exchange.

Unlike the A-a PO> gradient, the a/A PO ratio is relatively unaffected by the FIO>. This is demonstrated in Figure 20.4. The independence of the a/A PQ> ratio in relation to the FIO, is explained by the equation below.
a/A PO, =1-(A-a PO;)/ PaO,
(20.7)
FIGURE 20.4 The influence of FIO2 on the alveolar-arterial PO? gradient (A-a PO2) and the arteriakalveolar PO? ratio (a/A PO2) in normal subjects. From Reference 7.
Because the alveolar PO; is in both the numerator and denominator of the equation, the influence of FIO, on the PAO; is eliminated. Thus, the a/A PO, ratio is a mathematical manipulation that eliminates the influence of FIO2 on the A-a PO> gradient. The normal a/A PQ; ratio is 0.74 to 0.77 when breathing room air, and 0.80 to 0.82 when breathing 100% oxygen (7).

The PaO>/FIO, ratio is used as an indirect estimate of shunt fraction. The following correlations have been reported (9).
PaO, / FiO,, 1 = Qs/Qt. <200, 1 = >20%. >200, 1 = <20%
The major limitation of the PaO2/FIO> ratio is the inability to estimate the FIOz accurately when supplemental O, is delivered through nasal prongs or "open" face masks (see Chapter 22). (This limitation has also been described for the A-a PO> gradient.)

The arterial PO) and PCO, can vary spontaneously without a change in the clinical condition of the patient. This is demonstrated in Table 20.2, which shows the spontaneous variation in arterial PO2 and PCO, over a one-hour period in a group of clinically stable trauma victims (10). Note that the arterial PO varied by as much as 36 mm Hg, while the arterial PCO2 varied by as much as 12 mm Hg. This variability has also been observed in patients in a medical ICU (11). Because of this degree of spontaneous variation, routine monitoring of arterial blood gases can be misleading.

Mean,  = 13mm Hg. Mean, = = 2.5 mm Hg. 95th Percentile,  = +18mm Hg. 95th Percentile, = = +4 mm Hg. Range,  = 2-37 mmHg. Range, = = 0-12 mm Hg
Represents variations over a 1-hour pariod in 28 ventilator-dependent trauma victins who were clinically stable.
From Referance 10.

Hypoxemia can be defined as an arterial PO below what is expected for a patient's age, as defined in Table 20.1. However, hypoxemia usually doesn't raise red flags until the arterial PO falls below 60 mm Hg (or the arterial O> saturation falls below 90%). The causes of hypoxemia can be separated into 3 categories based on the physiological process involved (12,13). Each group of disorders can be distinguished by the A-a PO» gradient and/or the mixed venous PO>, as shown in Table 20.3.

Hypoventilation, 1 = Normal. Hypoventilation, 2 = Normal. V/Q mismatch, 1 = Increased. V/Q mismatch, 2 = Normal. DO, /VO; Imbalance, 1 = Increased. DO, /VO; Imbalance, 2 = Decreased

Alveolar hypoventilation causes both hypoxemia and hypercapnia, similar to breathholding. There is no V/Q imbalance in the lungs, so the A-a PO> gradient is not elevated. The common causes of alveolar hypoventilation are listed in Table 20.4. Most cases of hypoventilation in the ICU are the result of drug-induced respiratory depression or neuromuscular weakness. Obesity-related hypoventilation (Pickwickian syndrome) is also a consideration, as this condition is present in up to one-third of morbidly obese patients (body mass index >35 kg/m2) (14).

Brainstem Respiratory Depression
1. Drugs (@.g., opiates)
2. Obesity-hypoventilation syndrome

1. Critical lliness myopathy
2. Hypophosphatemia
3. Myasthenia gravis

Most cases of respiratory muscle weakness in the ICU are the result of an idiopathic polyneuropathy and myopathy that is specific to ICU patients, particularly those with sepsis, prolonged mechanical ventilation, and prolonged neuromuscular paralysis (15). The standard method of evaluating respiratory muscle strength is to measure the maximum inspiratory pressure (Plma,x), which is the maximum pressure recorded during a maximum inspiratory effort against a closed valve. The normal Pla, varies with age and gender, but most healthy adults can generate a negative Pla, of at least 80 cm H20 (16). A PIma, that does not exceed -25 cm H20 is considered evidence of respiratory muscle failure (17). (See Chapter 45 for more information on neuromuscular weakness syndromes in the ICU.)

Most cases of hypoxemia are the result of a V/Q mismatch in the lungs. Virtually any lung disease can be included in this category, but the common ones encountered in the ICU are pneumonia, inflammatory lung injury (acute respiratory distress syndrome), obstructive lung disease, hydrostatic pulmonary edema, and pulmonary embolism. The Aa PO, gradient is almost always elevated in these conditions, but the elevation can be minimal in patients with severe airways obstruction (which behaves like hypoventilation).

As explained in Chapter 10, a decrease in systemic O> delivery (DO>) is usually accompanied by an increase in O, extraction from capillary blood, and this serves to maintain a constant rate of O> uptake (VO>) into the tissues. The increased O, extraction from capillary blood results in a decrease in the PO> of venous blood, and this can have an deleterious effect on arterial oxygenation, as explained below.

The QO; in arterial blood represents the sum of the O> in mixed venous (pulmonary artery) blood and the O> added from alveolar gas. When gas exchange is normal, the PO, in alveolar gas is the major determinant of the arterial PO. However, when gas exchange is impaired, the contribution of the alveolar PO declines and the contribution of the mixed venous PO) rises (18). The greater the impairment in gas exchange, the greater the contribution of the mixed venous PO; to the arterial PO>. (If there is no gas exchange in the lungs, the mixed venous PO> would be the sole determinant of the arterial PO.) The diagram in Figure 20.5 demonstrates the influence of mixed venous PO, on the arterial PO2 when gas exchange is impaired. The curves in the graph represent the transition from mixed venous PO; to arterial PO> as blood flows through the lungs. The slope of each curve reflects the efficiency of gas exchange in the lungs. Note that the curve representing the V/Q abnormality results in a lower arterial PO) because the slope is decreased (indicating impaired oxygen exchange in the lungs). If this curve begins at a lower mixed venous PO;, as indicated, the curve shifts downward, resulting in a further decrease in arterial PO>. This illustrates how a decrease in mixed venous PO, can aggravate the hypoxemia caused by a V/Q abnormality. It also indicates that, in the presence of a V/Q abnormality, the mixed venous PO, is an important consideration in the evaluation of hypoxemia.
The relationship between O> delivery (DO2), O2 uptake (VO2), and the mixed venous PO> (PvO>) can be stated as follows:
PvO, = k x (DO) /'VO,)
(20.8)
(k is a proportionality constant.) Thus, any condition that reduces DO> (e.g., low cardiac output, anemia) or increases VO> (e.g., hypermetabolism) can decrease the PvO> and aggravate the hypoxemia caused by abnormal gas exchange in the lungs.

FIGURE 20.5 The influence of a V/Q abnormality on the transition from venous to arterial PO, and the added effect of low mixed venous a PO? (PvO2).

The evaluation of hypoxemia can proceed according to the flow diagram in Figure 20.6. This approach uses three measures: A-a PO> gradient, mixed venous POz, and maximum inspiratory pressure. The PO» in superior vena cava blood (central venous PO) can be used as the mixed venous PO> when there is no indwelling pulmonary artery catheter.
The first step in the approach involves a determination of the A-a PO» gradient. After correcting for age and FIO, the A-a PO> gradient can be interpreted as follows:
1. Normal A-a PO, gradient indicates hypoventilation rather than a cardiopulmonary disorder. In this situation, the most likely problems are drug-induced respiratory depression and neuromuscular weakness. The latter condition can be uncovered by measuring the maximum inspiratory pressure (Plmax), which is described earlier.
2. Increased A-a PO> gradient indicates a V/Q abnormality (cardiopulmonary disorder) and a possible superimposed DO2/VO> imbalance (e.g., a decrease in cardiac output). The mixed venous (or central venous) PO) will help to identify a DO2/VO> imbalance.
a. If the venous PO, is 40 mm Hg or higher, the problem is solely a V/Q mismatch in the lungs.
b. If the venous PO> is below 40 mm Hg, there is a DO3/VO> imbalance adding to the hypoxemia created by a V/Q mismatch in the lungs. The source of this imbalance is either a decreased DO> (from anemia or a low cardiac output) or an increased VO> (from hypermetabolism).
FIGURE 20.6 Flow diagram for the evaluation of hypoxemia.

Spurious hypoxemia is a rarely reported phenomenon that is characterized by hypoxemia in an arterial blood sample without corresponding hypoxemia in circulating blood (as measured by pulse oximetry) (19). This phenomenon seems to occur only in patients with hematologic malignancies who have marked leukocytosis (WBC >100,000) or thrombocytosis (platelet count >1,000,000). The reduced PO, in the blood sample has been attributed to O2 consumption by activated leukocytes in the sample, a phenomenon that has been called leukocyte larceny (20). This does not explain why marked thrombocytosis can also produce spurious hypoxemia because platelets are not oxygenguzzlers like activated leukocytes. Regardless of the mechanism, there is no accepted method of preventing spurious hypoxemia (rapid cooling of blood samples has had inconsistent results), so you should be aware of the phenomenon and the value of pulse oximetry for validating in vitro PO2 measurements (pulse oximetry is described in the next chapter).

Hypercapnia is defined as an arterial PCO, (PaCO) above 46 mm Hg that does not represent compensation for a metabolic alkalosis (21). The causes of hypercapnia can be identified by considering the determinants of PaCO> in the following relationship, where VCO is the rate of CO> production in the body, Va is the rate of alveolar ventilation, and k is a proportionality constant (1).
PaCO, =k x (VCO; /'V,)
(20.9)
Alveolar ventilation is the portion of the total ventilation (Ve) that is not dead space ventilation (Vp/Vr); that is, Va = Ve (1 — Vp/Vr). Therefore, equation 20.9 can be restated as follows:
PaCQ, = k x [VCO,/Vz (1Vp/Vy)]
(20.10)
This equation identifies three major sources of hypercapnia: (a) increased CO, production (VCO>), (b) hypoventilation (1/V_), and (c) increased dead space ventilation (Vp/Vr).

Hypoventilation was discussed briefly in the last section on hypoxemia, and Table 20.4 shows the common causes of hypoventilation. Because hypoxemia is so common in ICU patients, hypercapnia may be the first sign of hypoventilation from neuromuscular weakness or drug-induced respiratory depression. This is also the case in obesityhypoventilation syndrome, where hypercapnia while awake is often the first evidence of hypoventilation. On the other hand, hypercapnia is a relatively late sign in neuromuscular disorders, and does not appear until the maximum inspiratory pressure or Plax (described earlier) is below 50% of normal (17).

As mentioned earlier, hypercapnia is not a feature of increased intrapulmonary shunt until late in the process (which is why hypercapnia is not a feature of pulmonary edema or other infiltrative lung processes until they are far advanced). Hypercapnia is more a feature of increased dead space ventilation (Such as occurs in advanced emphysema, where there is destruction of the alveolar-capillary interface), and the PaCO> usually begins to rise when dead space ventilation accounts for more than 50% of total ventilation (Vp/V7 >0.5).

An increase in COQ, production is usually related to oxidative metabolism, but nonmetabolic CO, production is possible when extracellular acids generate hydrogen ions that combine with bicarbonate ions and generate CO>. Whatever the source, increased CO, production is normally accompanied by an increase in minute ventilation, which eliminates the excess CO> and maintains a constant arterial PCO. Therefore, excess CO> production does not normally cause hypercapnia. However, when CO, excretion is impaired, an increase in CO, production can lead to an increase in PaCO>. Thus, increased CO, production is an important factor in promoting hypercapnia only when the ability to eliminate CO is impaired.

Overfeeding, or the provision of calories in excess of daily needs, is a recognized cause of hypercapnia in patients with severe lung disease and acute respiratory failure (22). Nutrition-associated hypercapnia occurs predominantly in ventilator-dependent patients, and can delay weaning from mechanical ventilation. Overfeeding with carbohydrates is particularly problematic because the oxidative metabolism of carbohydrates generates more carbon dioxide than the other nutrient substrates (lipids and proteins). This is described in more detail in Chapter 47.

The bedside evaluation of hypercapnia is shown in Figure 20.7. The evaluation of hypercapnia, like hypoxemia, begins with the A-a PO» gradient (23). A normal or unchanged A-a PO> gradient indicates that the problem is alveolar hypoventilation (the same as described for the evaluation of hypoxemia). An increased A-a PO> gradient indicates a V/Q abnormality (an increase in dead space ventilation) that may or may not be accompanied by an increase in CO production.

The rate of CO) production (VCO2) can be measured at the bedside with specialized metabolic carts that are normally used to perform nutritional assessments. These carts are equipped with infrared devices that can measure the CO) in expired gas (much like the end-tidal CO) monitors described in Chapter 21), and can determine the volume of CO, excreted per minute. In steady-state conditions, the rate of CO, excretion is equivalent to the VCO>. The normal VCO; is 90 to 130 L/minute/m2, which is roughly 80% of the VO>. As mentioned earlier, an increased VCO > is evidence for one of the following conditions: generalized hypermetabolism, overfeeding (excess calories), or metabolic acidoses.

It is important to remember that the arterial PO> is not a useful measure for determining the amount of oxygen in the blood (this requires the hemoglobin concentration in blood and the percent saturation of hemoglobin with oxygen, as shown in Equation 10.6 in Chapter 10). Instead, the PaO» (along with the PaCO>) is used to evaluate gas exchange in the lungs, and can be useful in identifying the source of the problem with gas exchange.
An approach to Oz and CO balance that is superior in many ways to the measurement of arterial blood gases is described in the next chapter.
Bell SM. Lavoisier in the Year One. New York: W.H. Norton & Co., 2005.

1. Dantzger DR. Pulmonary gas exchange. In: Dantzger DR, ed. Cardiopul-monary critical care. 2nd ed. Philadelphia: WB Saunders, 1991; 25-43.
2. Lanken PN. Ventilation-perfusion relationships. In: Grippi MA, ed. Pul-monary Pathophysiology. Philadelphia: JB Lippincott, 1995; 195-210.
3. Buohuys A. Respiratory dead space. In: Fenn WO, Rahn H, eds. Handbook of physiology: respiration. Bethesda: American Physiological Society, 1964; 699-714.
4. D'Alonzo GE, Dantzger DR. Mechanisms of abnormal gas exchange. Med Clin North Am 1983; 67:557-571.

Measures of Gas Exchange, 1 = Measures of Gas Exchange. 5., 1 = Gammon RB, Jefferson LS. Interpretation of arterial oxygen tension. UpToDate Web Site, 2006. (Accessed 3/11/2006). 6., 1 = Harris EA, Kenyon AM, Nisbet HD, et al. The normal alveolar-arterial oxygen tension gradient in man. Clin Sci 1974; 46:89-104.. 7., 1 = Gilbert R, Kreighley JF . The arterial/alveolar oxygen tension ratio. An index of gas exchange applicable to varying inspired oxygen concentrations. Am Rev Resp Dis 1974; 109:142-145.. 8. Carroll GC. Misapplication of the alveolar gas equation. N Engl J Med 1985; 312:586., 1 = 8. Carroll GC. Misapplication of the alveolar gas equation. N Engl J Med 1985; 312:586.. 9. Covelli HD, Nessan VJ, Tuttle WK. Oxygen derived variables in acute respiratory failure. Crit Care Med 1983; 11:646-649., 1 = 9. Covelli HD, Nessan VJ, Tuttle WK. Oxygen derived variables in acute respiratory failure. Crit Care Med 1983; 11:646-649.. 10. Hess D, Agarwal NN. Variability of blood gases, pulse oximeter saturation, and end- tidal carbon dioxide pressure in stable, mechanically ventilated trauma patients. Clin Monit 1992; 8:111-115., 1 = 10. Hess D, Agarwal NN. Variability of blood gases, pulse oximeter saturation, and end- tidal carbon dioxide pressure in stable, mechanically ventilated trauma patients. Clin Monit 1992; 8:111-115.. 11., 1 = Sasse SA, Chen P, Mahutte CK. Variability of arterial blood gas values over time in stable medical ICU patients. Chest 1994; 106:187-193.

Hypercapnia, 1 = Hypercapnia. 21., 1 = Weinberger SE, Schwartzstein RM, Weiss JW. Hypercapnia. N Engl J Med 1989; 321:1223-1230.. 22., 1 = Talpers SS, Romberger DJ, Bunce SB, Pingleton SK. Nutritionally associated increased carbon dioxide production. Chest 1992; 102:551-555.. 23., 1 = Gray BA, Blalock JM. Interpretation of the alveolar-arterial oxygen difference in patients with hypercapnia. Am Rev Respir Dis 1991; 143:4-8.

The kiling vice of the young doctor is intellectual laziness.
Sir Wiliam Osler
On the Educational Value of the Medical Society, in
Aequanimitas, 1904
The introduction of optical techniques for the continuous and noninvasive measurement of oxyhemoglobin saturation in blood (oximetry) and carbon dioxide in exhaled gas (capnometry) has provided the most useful advances in critical care monitoring in the last 30 years. Oximetry has had an enormous impact on patient care throughout the hospital, and the arterial O> saturation has been called the fifth vital sign (1,2), while infrared capnometry has emerged as an_ indispensable component of cardiopulmonary resuscitation (see pp. 334-335).
Despite the prominent role of oximetry and capnography in critical care management, surveys have shown that 97% of house staff and ICU nurses have little or no understanding of these techniques or the parameters that are monitored (3). The material in this chapter should help to correct this situation.

All atoms and molecules absorb specific wavelengths of light, and this property is the basis for the technique known as spectrophotometry, where light waves of specific wavelengths are transmitted through a medium to determine the molecular composition of the medium. The absorption of specific wavelengths of light as they pass through a medium is proportional to the concentration of the substance that absorbs the light waves, and the length of the path that the waves travel (as defined by the Lambert-Beer Law). The application of this principle to the detection of hemoglobin in its different forms is known as oximetry.

Hemoglobin (like all proteins) changes its structural configuration when it participates in a chemical reaction, and each of the configurations has a distinct pattern of light absorption. The patterns of light absorption for the different forms of hemoglobin are shown in Figure 21.1 (4). Four different forms of hemoglobin are represented in the figure: oxygenated hemoglobin (HbO>), deoxygenated hemoglobin (Hb), methemoglobin (metHb), and carboxyhemoglobin (COHb). In the red region of the light spectrum (represented by the wavelength of 660 nm), oxygenated hemoglobin (HbO), does not absorb light as well as deoxygenated hemoglobin (Hb), which is why oxygenated blood is more intensely red than deoxygenated blood. The opposite is true in the infrared region of the spectrum (represented by the wavelength of 940 nm), where HbO> absorbs light more effectively than Hb. Based on these absorption patterns, two wavelengths of light (660 nm and 940 nm) can be used to identify oxygenated and deoxygenated hemoglobin.
FIGURE 21.1 The absorption spectrum for the different forms of hemoglobin: oxygenated hemoglobin (HbO2), deoxygenated hemoglobin (Hb), carboxyhemoglobin (COHb), and methemoglobin (metHb) The vertical lines represent the two wavekngths of light (660 nm and 940 nm) used by puke oximeters. Adapted from Reference 4.

Oximetry was introduced in the 1940s to detect hypoxemia in fighter pilots. Early oximetry, which measured the transmission of red and infra-red light waves through the earlobe, had two major shortcomings: (a) the transmission of light was influenced by factors other than hemoglobin (e.g., skin pigments, and the thickness of the earlobe), and (b) it was not possible to differentiate between hemoglobin in arteries and veins. As a result of these problems, oximetry failed to gain acceptance as a monitoring tool, but this situation changed in the 1970s with the introduction of pulsatile oximetry.
FIGURE 21.2 Fingertip pulse oximetry. The fingertip probe has light-emitting diodes (LEDs) on one side that emit light in the red (660 nm) and infrared (940 nm) spectrum, and a photodetector on the opposite side that processes light waves of alternating intensity (using an AC amplifier). The lower panel shows the pulsatile oximetry signal (SpO2) that is displayed on the bedside monitor.

, 1 = Spo,". , 2 = ‘$v0,"". , 3 = PerCO,*. Time period, 1 = 60 min. Time period, 2 = 120 min. Time period, 3 = 60 min. Mean variation, 1 = 1%. Mean variation, 2 = 69. Mean variation, 3 = 2mm Hg. Range of variation, 1 = 0-5%. Range of variation, 2 = 1-199. Range of variation, 3 = o-7 mm Hg
Cliricaly stable patients. 95% of measurements obtained during mechanical ventiation.
*From Reference 8.
"From Reference 23.

Standard pulse oximeters do not detect carboxyhemoglobin (COHb) or methemoglobin (metHb) in blood. Normally, these hemoglobin variants account for less than 5% of the total hemoglobin pool in blood (7,9). When metHb or COHb levels are abnormally elevated, the arterial O) saturation (SaOz) decreases because HbO; is a lower fraction of the total hemoglobin pool. However, the SpO> from pulse oximetry is not influenced by COHb or metHb levels (10,11). Therefore, in cases of methemoglobinemia and carbon monoxide poisoning, the SpO> overestimates the true SaQ;, and is not a reliable marker

Hospital laboratories have large oximeters that use 8 wavelengths of light to measure all the hemoglobin variants in blood. Therefore, when abnormal elevations of metHb or COHb are suspected, an arterial blood sample should be sent to the hospital laboratory for a complete oximetry assessment. Newer blood gas analyzers are also equipped with 8 wavelengths of light, and can measure the levels of metHb and COHb in blood. (Carbon monoxide poisoning and methemoglobinemia are de-scribed in Chapter 55.)
Note: A pulse oximeter is now available that uses multiple wavelengths of light to detect all forms of hemoglobin (Rainbow Pulse CO-oximeter, Masimo Corp, Irvine, CA) (12). These devices are used by firefighters and emergency first responders for rapid, on-site detection of carbon monoxide exposure, and are not meant for routine SpOz monitoring in a hospital setting.

Although pulse oximetry is based on the presence of pulsatile blood flow, SpO> is an accurate reflection of SaO, at blood pressures as low as 30 mm Hg (13). Damped pulsations also do not affect the accuracy of fingertip SpO> recordings distal to a cannulated radial artery (14). When fingertip SpO> recordings are unreliable as a result of hypotension or peripheral vasoconstriction, the forehead is an alternate site for SpO> monitoring (see later).

In the absence of hypoxemia, pulse oximetry is accurate down to hemoglobin levels as low as 2 to 3 g/dL (15). At hemoglobin levels between 2.5 and 9 g/dL, SpO3 is within 1% of the SaQ> (15).

The influence of skin and fingernail color on the accuracy of SpO>) measurements has been severely curtailed by the introduction of pulse oximetry. Dark skin can produce a up to a 10% discrepancy between SpO> and SaQ3, but this occurs at Oz saturations between 70% and 80% (16), which are much lower than allowed in ICU patients. Dark fingernail polish produces a very small (2%) discrepancy between SpO, and SaQ,> (17), but the clinical significance of this effect is in doubt.

The forehead is an appealing site for pulse oximetry because the arterial circulation in the forehead (which originates from the internal carotid artery) is less prone to vasoconstriction than the digital arteries in the fingers (18). Clinical studies have shown that pulse oximetry in the forehead can provide suitable SpO) measurements when fingertip SpO> recordings are compromised by hypotension or peripheral vasoconstriction (19). The basic features of forehead pulse oximetry are shown in Figure 21.3. Forehead SpO> sensors are placed just above the eyebrows, where vascular density is greatest. The forehead sensors have light-emitting diodes and photodetectors positioned next to each other, and they record the light intensity reflected back from the underlying arteries to derive the SpO>. This method of reflectance oximetry differs from the method of transmission oximetry used by the fingertip SpO, sensors.

The major limitation of forehead pulse oximetry is the risk of spuriously low SpO, readings when there is localized venous congestion (e.g., from positive-pressure mechanical ventilation). This effect is attributed to en-hanced venous pulsations, which are misread as arterial pulsations, resulting in SpO> readings that include reflections from HbO> and Hb in venous blood. This effect can be minimized with an elastic headband, as shown in Figure 21.3, which helps to dispel venous blood from the forehead (20). These headbands are often provided with the forehead SpO> sensors.
FIGURE 21.3 Forehead puke oximetry. The forehead probe (circled in red) is placed just above the eyebrows, and an elastic headband is used to reduce venous pulsations. The probe has light-emitting diodes (LEDs) and photodetectors placed next to each other, and the intensity of pulsatile light waves that are reflected back from the underlying arteries is used to determine the SpO3.

In theory, pulse oximetry is indicated in any situation where arterial oxygenation is a concern. In reality, pulse oximetry is considered a requirement for patient safety in certain areas of the hospital (e.g., ICUs and operating suites), and pulse oximetry is mandatory for all patients in these areas. Therefore, the important issue concerning the use of pulse oximetry in the ICU is not when to use it, but how to use it. The following information is relevant in this regard.

As a surrogate measure of the O, saturation of hemoglobin in arterial blood (SaO>), the SpO> is one of the determinants of the O> concentration in arterial blood (CaQ>), as described in the following equation.
CaQ, = 1.34 x [Hb] x 5aQ, (mL/dL)

(1.34 is the oxygen binding capacity of hemoglobin in mL/g, and [Hb] is the hemoglobin concentration in blood in g/dL.) In an idealized situation where [Hb] = 15 g/dL and SpO> = 0.98, the CaO, is 19.7 mL/dL (or 197 mL/L). A 10% decrease in SpO, (from 0.98 to 0.88), which is considered a clinically relevant change, results in a 10% decrease in CaO> (from 19.7 to 17.7 mL/dL). Based on this information, the following statements about the SpO> are valid:
1. Since the SaQ> is not the sole determinant of the arterial O2 content, monitoring the SpO> provides only partial information about arterial oxygenation.
2. Changes in SpO> that are considered clinically relevant are associated with only minor changes in the O> content of arterial blood.

A standard practice in the management of respiratory failure is to maintain the SpO> above a certain level by adjusting the O3 concentration in inspired gas. However, opinions vary about the lowest tolerable SpO>. In one survey of 25 ICU directors, the lowest acceptable SpO> varied from 85% to 95% (21). Studies in ventilator-dependent patients have shown that the threshold for hypoxemia (PaOz = 60 mm Hg) occurs at SpO> levels of 92% to 95% (21). However, it is important to emphasize that the lowest SpO> needed to support aerobic metabolism has never been identified, so the choice of a minimally acceptable SpO; is largely empirical.

Specialized oximetry catheters are available that can monitor the O, saturation of hemoglobin in the superior vena cava or pulmonary artery, and the operation of these catheters is illustrated in Figure 21.4. Oximetry catheters contain fiberoptic bundles that transmit two wavelengths of light (red and infrared) from an external light source to the tip of the catheter. Another channel of the catheter is connected to a photodetector that records the intensity of light that is reflected back from the hemoglobin in circulating erythrocytes. This technique (reflectance ox-imetry) is similar to the one used in forehead pulse oximetry. Oximetry catheters process and display the venous O> saturation every 5 seconds.
FIGURE 21.4 Venous oximetry using a specialized catheter that records the venous O2 saturation (SvO2) using reflectance oximetry.

The O> saturation in mixed venous (pulmonary artery) blood and central venous (superior vena cava) blood (SvO, and ScvQ;, respectively) are described in Chapter 10 (see pp. 183-184). Both measurements are influenced by the balance between systemic O> delivery (DO2) and O consumption (VO>), as described below:
SvO, or SevO, = 1 -VO,/DO,

(For the derivation of this equation, see Equations 10.17 and 10.18 on page 183.) A decrease in venous O> saturation below the normal range (SvOz <65% or ScvOz <70%) identifies a condition where O> delivery is low relative to O> consumption. This condition can be the result of a decrease in DO, (from low cardiac output, anemia, or arterial O> desaturation) or an increase in VO> (from hypermetabolism).

Measurements of SvO, with pulmonary artery oximetry catheters are typically within 1—-
2% of in vitro measurements (22). The spontaneous variations in SvOz can be considerable, as demonstrated in Table 21.1 (23). As a general rule, a greater than 5% change in SvO> that persists for longer than 10 minutes is considered a significant change (24).

Measurements of ScvO, with central venous oximetry catheters (PreSep Catheter, Edwards Life Sciences) are slightly lower than the SvO>, and this difference is magnified in the presence of circulatory shock (25). Single measurements of ScvO> can differ from SvO>z by as much as 10%, but the difference is reduced (to within 5%) when multiple measurements are obtained (26).

The predictive value of SvO» or ScvO> can be increased by adding the SpO> from pulse oximetry. The difference (SpO2—-SvO>) or (SpO2-ScvO>) is roughly equivalent to the O, extraction from capillary blood (27). Therefore, using the equation for the O, extraction ratio (see Equation 10.12 on page 181), the following relationships can be defined (using SvO> instead of ScvO>):
SpO, -SvO, = (VO,/DO,) «100
(21.4)
An increase in (SpOz—-SvO>2) above the normal range (above 30%) can be the result of an increasing VO» (hypermetabolism), or a decreasing DO> (from progressive anemia or a declining cardiac output). An (SpO -SvO>) that reaches 50% can also be used as a marker of tissue hypoxia, as shown in Figure 10.5 (see page 182).

Capnometry is the measurement of CO, in exhaled gas using colorimetric techniques or infrared spectrophotometry.

The colorimetric detection of CO in exhaled gas is a quick and simple method of determining if an endotracheal tube has been placed in the lungs (28). This is a standard of care following placement of an endotracheal tube because auscultation for breath sounds is an unreliable method of determining if an endotracheal tube is in the esophagus or trachea (29).
A popular device for the colorimetric detection of COz in exhaled gas is shown in Figure 21.5. The central area of the device contains filter paper that is impregnated with a pHsensitive indicator that changes color as a function of pH. When exhaled gas passes over the filter paper, the CO> in the gas is hydrated by a liquid film on the filter paper, and the resulting pH is detected by a color change. The outer perimeter of the device contains color-coded sections indicating the concentrations of exhaled CO, associated with each color change.

The accuracy of this colorimetric device for predicting the success of endotracheal intubation is shown in Table 21.2 (28). A color change from purple to tan or yellow almost always indicates successful intubation of the trachea. The absence of a color change from purple indicates that the endotracheal tube is not in the trachea, except during a cardiac arrest, when successful intubation of the trachea does not always produce a color change from purple. This latter observation is explained by the fact that exhaled CO> decreases when cardiac output is reduced, and the absence of a functional cardiac output during a cardiac arrest will result in very low levels of exhaled CO). Therefore, the lack of a color change from purple on the colorimetric CO, detector is not evidence of failure to intubate the lungs during a cardiac arrest.

No cardiac arrest (n=83), 1 = Tube In esophagus In 100% of cases. No cardiac arrest (n=83), 2 = Tube in trachea in 99% of cases. Cardiac arrest (n=144), 1 = Tube in trachea In 779 of cases and tube in esophagus In 23% of cases. Cardiac arrest (n=144), 2 = Tube in trachea in 100% of cases
tFrom Reference 28.

Carbon dioxide absorbs light in the infrared spectrum, which is the basis for the use of infrared light absorption to measure the PCO) in exhaled gas (30). This provides a more quantitative measure of exhaled CO, than the colorimetric method. Figure 21.6 shows an infrared CO> probe that has an airway attachment (which is placed in series with the expiratory tubing during mechanical ventilation) and a fitted transducer. When in place, the probe emits a continuous infrared light beam that travels through the exhaled gas. The photodetector has a rapid response, and can measure changes in PCO> during a single exhalation. This is recorded as an expiratory capnogram like the one in Figure 21.6.

The shape of the normal capnogram has been described as "the outline of a snake that has swallowed an elephant" (31). The PCO, at the onset of expiration is negligible because the gas in the upper airways is first to leave the lungs. As exhalation proceeds, gas from the alveoli begins to contribute to the exhaled gas, and the PCO, begins to rise steadily. The rate of rise eventually declines, and the exhaled PCO, reaches a plateau. When gas exchange is normal, the PCO, at the end of expiration (called the end-tidal PCO3) is equivalent to the PCO; in end-capillary (arterial) blood.

When pulmonary gas exchange is normal, the end-tidal PCO, is only 2 to 3 mm Hg lower than the arterial PCOz (30). However, when gas exchange in the lungs is impaired, and specifically when there is increased dead space ventilation, the end-tidal PCO, decreases relative to the arterial PCO. In this situation, the PaCO,—PETCO, difference is greater than 3 mm Hg. The conditions associated with an increased PaCO,—PETCO, difference are listed in Table 21.3.
FIGURE 21.6 Infrared capnography. The airway adapter and fitted transducer allow for a steady beam of infrared light to pass through exhaled gas. The photodetector records the PCO? continuously during each exhalation, which is displayed in the expired capnogram.

Increased Anatomic Dead Space, Conditions = « Open ventilator circuit « Shallow breathing. Increased Physiologic Dead Space, Conditions = « Obstructive lung disease « Excessive lung Inflation « Low cardiac output « Pulmonary embolism
The end-tidal PCO can be higher than arterial PCO> in the following situations (32): (a) when CO; production is high (from hypermetabolism or a metabolic acidosis) and there is a low inflation volume or a high cardiac output, or (b) when the concentration of inhaled O, is very high (the O, displaces CO from Hb).

End-tidal PCO) can be monitored in nonintubated patients using a modified nasal cannula. These are commercially available (Salter divided nasal cannula, DRE Medical, Louisville, KY), or a nasal cannula can be modified as shown in Figure 21.7 (33). The tubing between the two nasal prongs must be occluded (either with a cotton ball inserted through one of the nasal prongs or with a small screw clamp). This allows one nasal prong to be used for oxygen inhalation, while the other nasal prong is used to transmit exhaled gas. A 14-gauge intravascular catheter (2 inches long) is inserted into the exhalation side of the nasal cannula to transmit gas to a CO> detector. A sidestream CO > detector (i.e., one that applies suction to draw gas from the tubing) is best suited for this application. If one of these is not available, a mainstream infrared CO) detector (such as the one shown in Fig. 20.5) can be used with a suction pump to draw gas samples from the cannula (at 150 mL/minute). The respiratory therapy department can help with this modification.

The following are some useful applications of end-tidal CO monitoring.

The end-tidal PCO, can be used as a noninvasive method of monitoring the arterial PCO>. The arterial PCOz should be measured simultaneously with the end-tidal PCO, to establish the baseline PaCO,—PETCO> gradient. This gradient should remain the same as long as no other process intervenes to disturb pulmonary gas exchange. Changing ventilator settings will influence the PaCO2-PETCO, gradient (34), so the arterial PCO> should be measured after each change in ventilator settings to determine the new relationship between arterial and end-tidal PCO>.
FIGURE 21.7 A modified nasal cannula to monitor end-tidal PCO? during spontaneous breathing.

The most promising application of end-tidal PCO2 monitoring is the noninvasive detection of changes in cardiac output. There is a close correlation between changes in end-tidal PCO, and changes in cardiac output, as demonstrated in Figure 21.8 (35). This can be useful for detecting acute changes in cardiac output (e.g., in response to volume loading), and is proving very useful for monitoring changes in cardiac output during cardiopulmonary resuscitation (see pages 334-335).

A sudden decrease in end-tidal PCO with an increase in the PaCOz—PETCO, gradient can
be an early warning sign for any of the following conditions:
1. Overdistension of alveoli from high tidal volumes or PEEP.
2. Migration of an endotracheal tube into a mainstem bronchus (36).
3. Acute pulmonary embolism (37).

During weaning from mechanical ventilation, end-tidal PCO2 monitoring can serve several purposes (38). In uneventful weaning (e.g., following surgery), it serves as a noninvasive measure of PaCO>. In difficult or complicated weaning, it can help determine the success or failure of the wean attempt. For example, a progressive rise in end-tidal PCO can be a sign of an increase in the work of breathing (a sign of wean failure), while a decrease in end-tidal PCOz can be a sign of respiratory muscle weakness with shallow breathing (another sign of failure to wean).
FIGURE 21.8 Relationship between changes in end-tidal PCO? and changes in cardiac output in a group of postoperative patients. r = correlation coefficient. Data from Reference 35.

The fall from grace of the pulmonary artery catheter has created a void in the ability to monitor the cardiac output, but the techniques described in this chapter can help to fill this void. Both dual oximetry (i.e., combining pulse oximetry and central venous oximetry) and end-tidal PCO, monitoring can be useful for evaluating the adequacy of cardiac output, and for detecting changes in cardiac output (e.g., in response to volume loading). End-tidal PCO) monitoring has the advantage of being totally noninvasive, while dual oximetry has the advantage of monitoring the balance between systemic O> delivery (DO2) and O, uptake (VO>). Of course, nothing can replace the pulmonary artery catheter (at least at the present time) for monitoring cardiac output and systemic oxygenation.

1. Neff TA. Routine oximetry. A fifth vital sign? Chest 1988; 94:227.
2. Mower WR, Myers G, Nicklin EL, et. al. Pulse oximetry as a fifth vital sign in emergency geriatric assessment. Acad Emerg Med 1998; 5:858-869.
3. Stoneham MD, Saville GM, Wilson IH. Knowledge about pulse oximetry among medical and nursing staff. Lancet 1994; 344:1339-1342.

22., 1 = Armaganidis A, Dhinaut JF , Billard JL, et al. Accuracy assessment for three fiberoptic pulmonary artery catheters for SvO 2 monitoring. Intensive Care Med 1994; 20:484- 488.. 23., 1 = Noll ML, Fountain RL, Duncan CA, et al. Fluctuation in mixed venous oxygen saturation in critically ill medical patients: a pilot study. Am J Crit Care 1992; 3:102- 106.. 24., 1 = Krafft P, Steltzer H, Heismay M, et al. Mixed venous oxygen saturation in critically ill septic shock patients. Chest 1993; 103:900-906.. 25., 1 = Rivers EP, Ander DS, Powell D. Central venous oxygen saturation monitoring in the critically ill patient. Curr Opin Crit Care 2001; 7:204-211.. 26., 1 = Dueck MH, Kilmek M, Appenrodt S, et al. Trends but not individual values of central venous oxygen saturation agree with mixed venous oxygen saturation during varying hemodynamic conditions. Anesthesiology 2005; 103:249-257.. 27., 1 = Bongard FS, Leighton TA. Continuous dual oximetry in surgical critical care. Ann Surg 1992; 216:60-68.

28. Ornato JP, Shipley JB, Racht EM, et al. Multicenter study of a portable, hand-size, colorimetric end-tidal carbon dioxide detection device. Ann Emerg Med 1992; 21:518-523.
29. Mizutani AR, Ozake G, Benumoff JL, et al. Auscultation cannot distinguish esophageal from tracheal passage of tube. J Clin Monit 1991; 7:232-236.
Infrared Capnography, 1 = Infrared Capnography. 30. 31., 1 = Stock MC. Capnography for adults. Crit Care Clin 1995; 11:219-232. . Gravenstein JS, Paulus DA, Hayes TJ. Capnography in clinical practice. Boston ButterworthHeinemann, 1989; 11.. 32., 1 = Moorthy SS, Losasso AM, Wilcox J. End-tidal PCO2 greater than PaCO 2 . Crit Care Med 1984; 12:534-535.. 33., 1 = Roy J, McNulty SE, Torjman MC. An improved nasal prong apparatus for carbon. , 1 = end-tidal dioxide monitoring in awake, sedated patients. J Clin Monit 1991; 7:249-252.. 34., 1 = Hoffman RA, Kreiger PB, Kramer MR, et al. End-tidal carbon dioxide in critically ill patients during changes in mechanical ventilation. Am Rev Respir Dis 1989; 140:1265-1268.. 35., 1 = Shibutani K, Shirasaki S, Braatz T, et al. Changes in cardiac output affect PETCO2, CO2 transport, and O 2 uptake during unsteady state in humans. J Clin Monit 1992; 8:175-176.. 37., 1 = endobronchial migration of an endotracheal tube. J Clin Monit 1991; 7:35-38. Rodger MA, Gwynne J, Rasuli P. Steady-state end-tidal alveolar dead space fraction and D-dimer. Bedside tests to exclude pulmonary embolism. Chest 2001; 120:115-. 38., 1 = 119. Healey CJ, Fedullo AJ, Swinburne AJ, Wahl GW. Comparison of noninvasive measurements of carbon dioxide tension during weaning from mechanical

Carbon structures life. Oxygen ignites it.
Eric Roston
The Carbon Age
2008
One of the rare sights in any ICU is a patient who is NOT receiving supplemental oxygen to breathe. Oxygen is used liberally in ICU patients, and oxygen therapy is guided by measures (i.e., the arterial PO> and O> saturation) that have no proven relationship with tissue oxygenation. As emphasized in this chapter, the excessive use of oxygen without evidence of impaired tissue oxygenation is problematic because it promotes the formation of toxic oxygen metabolites, which are capable of lethal cell injury.
This chapter begins with some insights on the design of the human body in relation to oxygen and oxygen therapy. This is followed by a description of the different oxygen delivery systems. The final section is devoted to the dark side of oxygen; i.e., the propensity for oxygen to damage aerobic organisms.

Despite our dependence on oxygen for metabolic energy production, aerobic metabolism is carried out in an oxygen-restricted environment. Oxygen does not readily dissolve in water, which is why we need hemoglobin to transport oxygen, and this limits the volume of dissolved oxygen in the tissues of the body. The estimated volume of oxygen in the interstitial fluid and cells of the human body is shown in Table 22.1. According to these estimates, there is only about 13 mL of Q> in all the tissues of the human body!
0, Contentt, 1 = 0.45 mU/L. 0, Contentt, 2 = 0.15 mL/L. Fluid Volume®, 1 = 16L. Fluid Volume®, 2 = 23L. Volume of Oz, 1 = 9.6 mL. Volume of Oz, 2 = 3.5 mL
- tDissoWed O2 content =o x PO: where ce (solubility coafficient) = 0.03 mL/Limm Hg for Os in water at 37s.
SVolume estimatas are basad on total body water (TBW) of 42 liters, an intracallular volume that is 55% of TBW, and an interstitial volume that is 38% of TBW.
The estimates in Table 22.1 are based on the determinants of dissolved O 2 in the equation shown below.
Dissolved O, (mL,'L) = 0.03 x PO;
(22.1)

where 0.03 is the solubility coefficient of O> in water (expressed as mL Op per liter of body water per mm Hg of PO2) at a body temperature of 37°C. Experimental studies show that the intracellular PO is about 5 mm Hg (1) and interstitial PO2 is about 15 mm Hg (2). Using these PO> values in equation 22.1, the concentration of dissolved O> will be 0.03x5= 0.15 mL/L inside cells and 0.03x15=0.45 mL/L in the interstitial fluid. An average sized adult has an intracellular volume of about 23 liters and an interstitial fluid volume of about 16 liters, so the total volume of dissolved O> will be 0.15x23=3.5 mL in cells and 0.45x16=9.6 mL in the interstitial fluid.

Humans are described as obligate aerobic organisms; i.e., organisms that require oxygen to survive. However, according to the estimates in Table 22.1, humans are more accurately described as microaerophilic organisms; i.e., organisms that require only low concentrations of oxygen to survive.
Implications
The oxygen-restricted environment in tissues can be viewed as a safeguard against the damaging effects of oxygen metabolites (described later in the chapter). This safeguard will be threatened or lost if oxygen therapy exposes the tissues to more O> than is needed to sustain aerobic metabolism. The remaining observations in this section are relevant to this issue.

Oxygen therapy is used to prevent hypoxemia (i.e., an arterial PO7 <60 mm Hg or an arterial Oz saturation <90%) (3). However, there is no evidence that hypoxemia impairs tissue oxygenation, regardless of severity (4-6). The data points in Figure 22.1 show the blood lactate levels in patients with severe hypoxemia (i.e., arterial PO2 <40 mm Hg) from an acute exacerbation of chronic obstructive lung disease (4). Note that the blood lactate levels fail to rise above 2 mmol/L (the threshold for hyperlactatemia) despite arterial PO levels as low as 22 mm Hg. This provides evidence that severe hypoxemia did not impair aerobic metabolism in these patients. Similar observations have been reported in patients with acute respiratory distress syndrome (5), indicating that tolerance to severe hypoxemia is not an adaptation that develops over time. (Note: Tolerance to hypoxemia is the reason that "hypoxemic shock" is not a clinical entity. )
FIGURE 22.1 The relationship between arterial PO? and blood lactate kvels in seven patients with severe hypoxemia (arterial PO? <40 mm Hg) from acute exacerbation of chronic obstructive lung disease. All lactate levels are within the normal range (<2 mmol/L), suggesting that severe hypoxemia did not impair tissue oxygenation in these patients. Data from Reference 4.

Since hypoxemia does not impair aerobic metabolism, then oxygen therapy (which is aimed at relieving hypoxemia) should not be necessary for preserving aerobic metabolism. This is supported by observations like those in Figure 22.2. The graphs in this figure show the effects of breathing 24% and 28% oxygen on the arterial PO) (PaO>2) and systemic oxygen consumption (VO>) in patients with acute exacerbation of chronic ob-structive lung disease. There is a significant increase in the PaO» with each increment in inhaled Oz, but the VOz remains unchanged. Similar results have been reported in other clinical studies (8,9), and since the VO> represents the rate of aerobic metabolism, these studies indicate that oxygen therapy does not promote aerobic metabolism.
FIGURE 22.2 Acute effects of oxygen therapy on the arterial PO2 (PaO2) and systemic oxygen consumption (VO72) in ICU patients with acute exacerbation of chronic obstructive lung disease. Numbers in parentheses are the mean values for each measurement. FIO? = fraction of O2 in inhaled gas. Data from Reference 7.
Pure oxygen breathing (a condition known as normobaric hyperoxia) is accompanied by a 10% to 20% decrease in VO, (10,11), indicating that hyperoxia can inhibit aerobic metabolism(!). This effect has been attributed to oxygen-induced changes in microvascular flow (described next), and it is blocked by the antioxidant N-acetylcysteine (12), indicating that toxic oxygen metabolites are involved.

Oxygen therapy produces systemic vasoconstriction (not pulmonary vasoconstriction) that is most pronounced in the small arterioles that control capillary blood flow (12-14). At high concentrations of inhaled O,, the arteriolar constriction can be accompanied by a decrease in functional capillary density (15), and this could result in a decrease in O> availability in the capillaries. This has been implicated in the reduction of VO that occurs during normobaric hyperoxia (described above), and it may also be a mechanism for protecting the tissues from oxygen-induced injury (oxidant injury) during periods of hyperoxia.

The information just presented indicates that aerobic metabolism is normally conducted in an oxygen-restricted environment, and this condition persists in the face of severe hypoxemia. Furthermore, attempts to provide more oxygen are met with resistance, as arteriolar vasoconstriction reduces the capillary surface area available for oxygen entry into tissues. Thus, the tissues are normally oxygen-restricted, and it seems they want to remain that way. This is understandable in light of the propensity for oxygen to produce lethal cell damage, as described in the last section of the chapter. If the scenario just presented is accurate, then oxygen therapy, which does not promote aerobic metabolism, is merely creating an added risk of oxygen-induced (oxidant) damage in the vital organs. The observation that arterial hyperoxia following cardiac arrest is associated with an increased mortality rate (16) is consistent with the notion that oxygen therapy can be dangerous. Considering the prominent role played by oxidant injury in the damaging effects of inflammation (which is a leading cause of morbidity and mortality in the ICU), the current liberal use of oxygen in critically ill patients deserves scrutiny.

Oxygen delivery systems are classified as low-flow systems, reservoir systems, and highflow systems (17). Low-flow systems use standard nasal prongs, while reservoir systems use standard face masks and face masks with reservoir bags, and high-flow systems use air-entrainment masks or heated, humidified Oz delivered through nasal prongs. Each system is characterized by the following features: (a) the manner in which the fractional concentration of inspired oxygen (FIO2) is determined, (b) the achievable FIO, range, (c) the variability of the FIO2, and (d) the type of patient that is most appropriate for the system. The features of different oxygen delivery systems are summarized in Table 22.2

Low-Flow Nasal O,, 1 = 1-6 L/min. Low-Flow Nasal O,, 2 = —. Low-Flow Nasal O,, 3 = 24-40%. Low-Flow Nasal O,, 4 = Variable. Standard Face Mask, 1 = 5-10L/min. Standard Face Mask, 2 = 100-200mL. Standard Face Mask, 3 = 35-50%. Standard Face Mask, 4 = Variable. Partial Rebreather Mask, 1 = >10L/min. Partial Rebreather Mask, 2 = 600-1000 mL. Partial Rebreather Mask, 3 = 40-70%. Partial Rebreather Mask, 4 = Variable. Nonrebreather Mask, 1 = >10L/min. Nonrebreather Mask, 2 = 600-1000 mL. Nonrebreather Mask, 3 = 60-80%. Nonrebreather Mask, 4 = Variable. Ait-Entrainment Mask, 1 = >60L/min. Ait-Entrainment Mask, 2 = 100-200 mL. Ait-Entrainment Mask, 3 = 24-50%. Ait-Entrainment Mask, 4 = Constant. High-Flow Nasal O5, 1 = =40 L/min. High-Flow Nasal O5, 2 = _. High-Flow Nasal O5, 3 = 21-100%. High-Flow Nasal O5, 4 = Variable

The standard device for low-flow O> therapy is the nasal cannula or nasal prongs, which deliver oxygen into the nasopharynx at flow rates of 1 to 6 L/min. (The normal inspiratory flow rate during quiet breathing is about 15 L/min (0.25 L/sec), so low-flow nasal O2 represents only a fraction of the inspiratory flow generated by the patient.) A large fraction of the inspired volume is drawn from room air, which means that low-flow nasal O, does not achieve high concentrations of inhaled oxygen. The FIO) range during quiet breathing is 24% Op, (at 1 L/min) up to 40% Op, (at 6 L/min), as shown in Table 22.2. In patients with acute respiratory failure, peak inspiratory flow rates can increase to 30— 120 L/min (18). In this situation, low-flow nasal O, provides an even smaller fraction of the inspiratory flow needs of the patient. For this reason, low-flow nasal O> is often not adequate for O> therapy in patients with high ventilatory demands.

The major advantages of nasal prongs are simplicity of use and patient acceptance, including the ability for patients to eat and converse. The major disadvantage is the inability to achieve high concentrations of inhaled O >, particularly in patients with increased ventilatory demands.

Face masks are considered a reservoir system because the mask encloses a volume of 100 to 200 mL. Standard face masks deliver oxygen at flow rates between 5 and 10 L/min; a minimum flow rate of 5 L/min is needed to clear exhaled gas from the mask. Exhalation ports on the side of the face mask also allow room air to be inhaled. This system can achieve a maximum FIO, of about 60% during quiet breathing.

Standard face masks can provide a slightly higher maximum FIO, than low-flow nasal prongs, but like nasal prongs, the FIO, varies with the ventilatory demands of the patient. Face masks are more confining than nasal prongs, and they do not permit oral feeding.

The addition of a reservoir bag to a standard face mask increases the capacity of the oxygen reservoir by 600 to 1000 mL (depending on the size of the bag). If the reservoir bag is kept inflated, the patient will draw primarily from the gas in the bag. There are two types of reservoir bag devices: partial rebreathers and nonrebreathers.

The device shown in Figure 22.3 is a partial rebreather. This device allows the gas exhaled in the initial phase of expiration to return to the reservoir bag. As exhalation proceeds, the expiratory flow rate declines, and when the expiratory flow rate falls below the oxygen flow rate, exhaled gas can no longer return to the reservoir bag. The initial part of expiration contains gas from the upper airways (anatomic dead space), so the gas that is rebreathed is rich in oxygen and largely devoid of CO . The patient can inhale room air through the exhalation ports on the mask, but the gas in the reservoir bag is under positive pressure, and inhalation will draw primarily from the gas in the bag. Partial rebreather devices can achieve a maximum FIO> of about 70%.

The device shown in Figure 22.4 is a nonrebreather system. The expiratory ports on the mask are covered with flaps that allow exhaled gas to escape but prevent inhalation of room air gas. There is also a one-way valve between the reservoir bag and the mask that allows inhalation of gas from the bag but prevents exhaled gas from entering the bag (to prevent rebreathing of exhaled gas). Nonrebreather devices can theoretically achieve an FIO> of 100%, but in reality the maximum FIO) is closer to 80% (because of leaks around the mask).
FIGURE 22.3 Partial rebreather system. The initial 100 to 150 mL of exhaled gas (anatomic dead space) is returned to the reservoir bag for rebreathing. Exhaled gas stops entering the reservoir bag when the expiratory flow rate falls below the oxygen flow rate.
FIGURE 22.4 Nonrebreather system. Flaps on the exhalation ports of the mask prevent inhalation of room air, and a one-way valve between the mask and reservoir bag prevents exhaled gas from entering the bag for rebreathing.

The principal advantage of the reservoir bags is the ability to deliver higher concentrations of inhaled oxygen. The disadvantages are the same as described for face masks. In addition, aerosolized bronchodilator therapy is not possible with reservoir bag devices.

The newest technique of O> delivery (in adults) is high-flow nasal Oz using heated and humidified gas. Using oxygen that is heated to body temperature and supersaturated with water (to a relative humidity of 99%), O, flow rates up to 40-60 L/min can be delivered through wide nasal prongs without discomfort and mucosal injury. A commercially available product for high-flow nasal O2 (Vapotherm, Stevensville, MD) allows adjustments for flow rate (1-40 L/min), FIO) (21-100%), and temperature (usually at 37°C).
FIGURE 22.5 Function of an air-entrainment device. A narrowing at the oxygen inlet creates a high-velocity stream of gas that creates viscous drag, which puls in room air (RA). This "jet mixing" keeps the concentration of inhaled oxygen constant, regardless of changes in the flow rate of oxygen. See text for further explanation.

The initial experience with high-flow nasal O» has been very encouraging. Preliminary studies have shown that, in patients who require a high FIO) using mask devices, switching to humidified, high-flow nasal Oz was associated with a significant improvement in measures of respiratory distress (e.g., reduced respiratory rate, less dyspnea), along with a decrease in the PaO>/FIO> ratio (indicating improved gas exchange) (20,21). The beneficial effect on gas exchange may be explained by the observation that high-flow nasal O> creates positive pressure in the nasopharynx (22), which could act like positive end-expiratory pressure (PEEP) to prevent the collapse of alveoli at the end-of expiration (PEEP is described in Chapter 26). The most exciting observation regarding high-flow nasal O» comes from a study where the improvements resulting from high-flow nasal O> made it possible to avoid intubation and mechanical ventilation in 75% of the patients studied (21).

The balance sheet for high-flow nasal O» shows all advantages and no disadvantages (so far). The advantages include improved oxygenation and gas exchange, and a possible role in avoiding intubation and mechanical ventilation in patients with refractory
hypoxemia. You will certainly hear more about this promising method of O> therapy in the near future.

Ever wonder why food is stored in vacuum-sealed containers, or why we wrap food in cellophane to keep it fresh? These measures are designed to protect food from exposure to oxygen, which oxidizes and disrupts all organic molecules (including the carbohydrates, proteins, and lipids in food), and is responsible for the decomposition of food. The metabolites of oxygen are even more damaging than the parent molecule, and are capable of inflicting lethal cell injury (23). In fact, contrary to the popular perception that oxygen protects cells from injury in critically ill patients, the accumulating evidence indicates that oxygen (via the production to toxic metabolites) is a source of cell injury in critically ill patients (23-26). The following is a brief description of the toxic nature of oxygen.

The metabolism of oxygen takes place at the end of the electron transport chain in mitochondria (within the cytochrome oxidase complex), where the electrons that accumulate as a result of ATP production are cleared by the chemical reduction of oxygen to water. The reaction sequence for this process is shown in Figure 22.6. Oxygen has two unpaired electrons in its outer orbitals with the same directional spin. According to Pauli's Exclusion Principle (from the Austrian physicist Wolfgang Pauli), no two electrons can occupy the same orbital if they have the same directional spin. This means it is not possible to add an electron pair to oxygen and reduce it to water as a one-step process because one orbital would have two electrons with the same directional spin, which is a quantum impossibility. Because of this spin restriction, oxygen is metabolized in a series of single-electron reduction reactions, and this produces some highly reactive intermediates.
The intermediates in oxygen metabolism include the superoxide radical, hydrogen peroxide, and the hydroxyl radical. The behavior of these metabolites is summarized in the following statements (23):
Ground-State Oxygen, Outer Orbital Electrons = . Superoxide = Radical 02, Outer Orbital Electrons = . Hyd drogen H,0. Peroxide, Outer Orbital Electrons = . Hydroxyl Radical & lon "OH + OF, Outer Orbital Electrons = . 2 H20, Outer Orbital Electrons = 
FIGURE 22.6 The metabolism of molecular oxygen to water involves a series of four single-electron reduction reactions. Orbital diagrams on the right side of the figure show the directional spin of electrons in the outer orbitals of each reactant. Free radical metabolites are indicated by a superscripted dot. See text for explanation.

Oxidative (oxygen-related) injury is kept in check by a vast array of endogenous antioxidants (i.e., atoms or molecules that prevent or block the actions of oxidizing agents.) The following is a brief description of the major antioxidants and their possible role in critical illness.

Superoxide dismutase (SOD in Figure 22.7) is an enzyme that facilitates the conversion of superoxide radicals to hydrogen peroxide. Although SOD is considered essential for aerobic life (27), its role in critical illness is unclear, and it can act as a pro-oxidant as well as an antioxidant (27,29). The pro-oxidant effect may be the result of hydrogen peroxide production.

Glutathione is a sulfur-containing tripeptide that is considered the major intracellular antioxidant in the human body (29,30). In its reduced form, glutathione (GSH in Figure 22.7) donates electrons to reduce hydrogen peroxide to water in a reaction that is catalyzed by a selenium-dependent enzyme, glutathione peroxidase (Se-GPx in Figure 22.7):
FIGURE 22.7 Actions of endogenous and exogenous antioxidants (highlighted in red). SOD = superoxide dismutase, SeGPx = selenium-glutathione peroxidase complex, GSH = reduced glutathione, GSSG = oxidized glutathione (a dipeptide connected by a disulfide bridge). See text for explanation.
powerful reducing agent that can serve as a scavenger (i.e., donate electrons) for the superoxide and hydroxyl radicals (30). High-dose vitamin C has shown some promise in reducing pulmonary complications in trauma patients (see later).

Ceruloplasmin and transferrin account for most of the antioxidant activity in plasma (41). The antioxidant activity of both proteins is related to their actions in limiting free iron in the reduced form, Fe (II), which will limit the production of the hydroxyl radical. Caeruloplasmin oxidizes iron from the Fe (II) to the Fe (III) state, and transferrin binds iron in the oxidized or Fe (III) state. The role of free iron as a pro-oxidant (42) may explain why most of the iron in the body is bound to proteins or sequestered (e.g., in the bone marrow).

The risk of oxidant injury is determined by the balance between oxidant and antioxidant activities. When oxidant activity exceeds the neutralizing capacity of the antioxidants, the excess or unopposed oxidation can promote tissue injury. This condition of unopposed biological oxidation is known as oxidant stress. Unfortunately, there is no clinical measure of oxidant stress, so the presence of this condition must be inferred.

Pulmonary oxygen toxicity is described as an inflammatory lung injury (similar to the acute respiratory distress syndrome described in the next chapter) that occurs in patients who have inhaled gas with an FIO, >60% for longer than 48 hours. This description, however, does not take into account the information that follows.

The tendency to develop pulmonary oxygen toxicity varies in different species. For example, laboratory rats will die of respiratory failure after 5 to 7 days of breathing 100% O,, while sea turtles can breathe pure O> indefinitely without harm (43). This speciesspecific effect is important because experimental studies of pulmonary oxygen toxicity have been conducted almost solely in laboratory rats. There is limited information about the tendency of humans to develop pulmonary oxygen toxicity.
HUMAN STUDIES: In healthy volunteers, inhalation of 100% O, for 6 to 12 hours results in a tracheobronchitis and a decrease in vital capacity attributed to absorption atelectasis (44). Prolonged exposure to 100% O, has been reported in only 6 humans: 5 with irreversible coma who received 100% O> for 3 to 4 days (45), and one healthy volunteer who inhaled pure O, for 4.5 days (46). In all these cases, the subjects developed a pulmonary condition that was consistent with inflammatory lung injury.

Based on the observation of a decreased vital capacity when the FIO) exceeds 60% (44), the threshold FIO, for pulmonary O> toxicity was set at 60%. However, adopting a single threshold FIO; for all patients neglects the contribution of endogenous antioxidants to the risk of oxygen toxicity. If the antioxidant stores in the lungs are depleted, oxygen toxicity can be expected at FIO> levels below 60%. Since antioxidant depletion seems to be common in ICU patients (33,38,39), it is reasonable to assume that any FIOz above 21% (room air) can represent a toxic exposure to oxygen in critically ill patients. Therefore, the best practice is to reduce the FIO> to the lowest tolerable level; e.g., the lowest FIO> needed to maintain an arterial O> saturation >90%.

There is no clinical measure of pulmonary O toxicity. However, considering the evidence of antioxidant depletion in critically ill patients (33,38,39), antioxidant supplementation is a reasonable consideration for reducing the risk of pulmonary oxygen toxicity. Evidence of benefit from such an approach is provided by a study in trauma patients (47), which showed that a high-dose antioxidant cocktail of vitamin C (1000 mg every 8 hrs), vitamin E (1000 units every 8 hrs), and selenium (200 eg daily) for 7 days was associated with a significant decline in cases of respiratory failure and ventilator dependence (47).
Antioxidant supplementation could be guided by available measures of antioxidant protection (e.g., serum levels of selenium), or it could be used routinely in patients considered at risk for pulmonary O> toxicity.

For those who like teleological explanations for biological design, the observation that oxygen is an arteriolar vasoconstrictor merits some attention. As shown in the first section of the chapter, aerobic metabolism is normally conducted in an oxygen—restricted environment; i.e., most of the O> in the body is attached to hemoglobin, while very little is present in tissues because Oz does not readily dissolve in water. Further-more, attempts to enhance tissue oxygenation with O, therapy are thwarted because of oxygen's actions as an arteriolar vasoconstrictor, which reduces functional capillary flow. Thus, the body resists attempts to increase tissue oxygenation, and the likely purpose of this design is to limit the risk of oxygen-induced tissue injury. In summary, it seems the human body is designed to maintain an oxygen-restricted environment in the tissues to limit the risk of oxygen-induced cell injury. If this is the case, then the liberal use of oxygen therapy in critically ill patients deserves re-evaluation.

Lane N. Oxygen: The Molecule that Made the World. New York: Oxford University Press, 2002.
Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. 4th ed, New York: Oxford University Press, 2007.
Banerjee R (ed). Redox Biochemistry. Hoboken, NJ: Wiley & Sons, 2008.

17., 1 = Heuer AJ, Scanlan CL. Medical gas therapy. In Wilkins RL, Stoller JK, Kacmarek RM, eds. Egan's Fundamentals of Respiratory Care, 9th ed, St. Louis: Mosby Elsevier , 2009.. 18., 1 = L'Her E, Deye N, Lellouche F, et al. Physiologic effects of noninvasive ventilation during acute lung injury. Am J Respir Crit Care Med 2005; 172:1112-1118.. 19., 1 = Scacci R. Air entrainment masks: jet mixing is how they work. The Bernoulli and Venturi principles is how they don't. Respir Care 1979; 24:928-931.. 20., 1 = Roca O, Riera J, Torres F, Masclans JR. High-flow oxygen therapy in acute respiratory failure. Respir Care 2010; 55:408-413.. 21., 1 = Sztrymf B, Messika J, Bertrand F, et al. Beneficial effects of humidified high flow nasal oxygen in critical care patients: a prospective pilot study. Inten-sive Care Med 2011; 37:1780-1786.. 22., 1 = Parke R, MacGuinness S, Eccleston M. Nasal high-flow therapy delivers low-level positive airway pressure. Br J Anesth 2009; 103:886-890.

JL, Cotgreave IA, et al. Skeletal muscle glutathione is depleted in critically ill patients. Crit Care Med 1997; 25:78-84.. 34., 1 = Robinson M, Ahn MS, Rounds JD, et al. Parenteral glutathione monoester enhances tissue antioxidant stores. J Parent Ent Nutrit 1992; 16:413-418.. 35., 1 = Suter PM, Domenighetti G, Schaller MD, et al. N-acetylcysteine enhances recovery from acute lung injury in man: a randomized, double-blind, placebo-controlled clinical study. Chest 1994; 105:190-194.. 36., 1 = Institute of Medicine, Food and Nutrition Board. Dietary reference intakes: vitamin C, vitamin E, selenium, and carotenoids. Washington, DC: National Academy Press, 2000.. 37., 1 = Sando K, Hoki M, Nezu R, et al. Platelet glutathione peroxidase activity in long-term total parenteral nutrition with and without selenium supplementation. J Parent Ent Nutrit 1992; 16:54-58.. 38., 1 = Angstwurm MWA, Engelmann L, Zimmerman T, et al. Selenium in Intensive Care
, 1 = (SIC): results of a prospective randomized, placebo-controlled, multiple-center study in patients with severe systemic inflammatory response syndrome, sepsis, and septic shock. Crit Care Med 2007; 35:118-126.. 39., 1 = Pincemail J, Bertrand Y, Hanique G, et al. Evaluation of vitamin E deficiency in patients with adult respiratory distress syndrome. Ann NY Acad Sci 1989; 570:498- 500.. 40., 1 = Meydani M. Vitamin E. Lancet 1995; 345:170-176.. 41., 1 = Halliwell B, Gutteridge JMC. Role of free radicals and catalytic metal ions in human disease. Methods Enzymol 1990; 186:1-85.. 42., 1 = Herbert V, Shaw S, Jayatilleke E, Stopler-Kasdan T. Most free-radical injury is iron- related: it is promoted by iron, hemin, haloferritin and vitamin C, and inhibited by desferrioxamine and apoferritin. Stem Cells 1994; 12:289-303.

Pulmonary Oxygen Toxicity, 1 = Pulmonary Oxygen Toxicity. 43., 1 = Fanburg BL. Oxygen toxicity: why can't a human be more like a turtle? Intensive Care Med 1988; 3:134-136.. 44., 1 = Lodato RF. Oxygen toxicity. Crit Care Clin 1990; 6:749-765.. 45., 1 = Barber RE, Hamilton WK. Oxygen toxicity in man. N Engl J Med 1970; 283:1478- 1483.. 46., 1 = Winter PM, Smith G. The toxicity of oxygen. Anesthesiology 1972; 37:210-212.. 47., 1 = Dossett GAM, Fleming SB, Abumrad NN, Cotton BA. High-dose antioxidant administration is associated with a reduction in post-injury complications in critically ill trauma patients. Injury 2011; 42:78-82.

Physicians think they do a lot for a patient when they give his disease a name.
Immanuel Kant
The condition described in this chapter has had several names over the years, including shock lung, Da Nang lung (from the Vietnam war), stiff-lung syndrome, leaky capillary pulmonary edema, noncardiogenic pulmonary edema, acute lung injury, adult respiratory distress syndrome, and most recently, acute respiratory distress syndrome, or ARDS. of these names provides any useful information about this disease entity, which is a diffuse inflammatory injury of the lungs, and one of the leading causes of acute respiratory failure in modern times (1).

The first clinical report of ARDS appeared in 1967 (2), and included 12 patients with refractory hypoxemia and diffuse infiltrates on chest x-ray. Seven patients died, and autopsy findings revealed dense infiltration of the lungs with an inflammatory exudate. There was no evidence of infection, which indicated that ARDS is an acute inflammatory lung injury.

The lung consolidation in ARDS is believed to originate with the activation of circulating neutrophils (3). This leads to neutrophil sequestration in the pulmonary microcirculation, where the neutrophils attach to the vascular endothelium and move between endothelial cells (by diapedesis) and into the lung parenchyma. The neutrophils then degranulate to release the contents of their cytoplasmic granules (i.e., proteolytic en-zymes and toxic oxygen metabolites). Subsequent damage of the capillary walls then leads to exudation of protein-rich fluid, erythrocytes, and platelets into the lungs. The cellular and proteinaceous exudation eventually fills and obliterates the distal airspaces, as shown in Figure 23.1. The inflammatory exudate contains fibrin, and progressive inflammation results in fibrin accumulation, which can lead to structural remodeling and pulmonary fibrosis (similar to the process that occurs in wound healing). The source of fibrin is a procoagulant state triggered by release of tissue factor from the lungs (4).
FIGURE 23.1 Microscopic images of a normal lung and a lung in the advanced stages of ARDS, which shows a dense infiltration of kukocytes and proteinaceous material that fills and obliterates the normal architecture of the lungs.

ARDS is not a primary disorder, but is a consequence of a variety of infectious and noninfectious conditions. The common conditions that predispose to ARDS are listed in Table 23.1. The most frequent offenders are pneumonia and the "sepsis syndromes" (i.e., septicemia, severe sepsis, and septic shock) (1,5). One feature shared by many of these conditions is the ability to trigger a systemic inflammatory response, which involves neutrophil activation, the principal inciting event in ARDS.

Pneumonia, 1 = Gastric Aspiration. Septicemia, 1 = Blood Transfusions. See Spas, 1 = Pancreatitis. Septic Shock, 1 = Drug Overdose
Other sources of ARDS include bums, cardiopulmonary bypass, pulmonary contusion, fat embolism syndrome, inhalation injury, and pulmonary oxygen toxicity.

The clinical features of ARDS are listed in Table 23.2. The principal features include an acute onset, severe hypoxemia, and bilateral pulmonary infiltrates without evidence of left heart failure or volume overload. The earliest signs of ARDS are the sudden appearance of hypoxemia and signs of respiratory distress (e.g., dyspnea, tachypnea). The chest x-ray can be unrevealing in the first few hours after symptom onset, but bilateral pulmonary infiltrates begin to appear within 24 hours. Progressive hypoxemia requiring mechanical ventilation often occurs in the first 48 hours of the illness.

ee
1. Acute onsat
2. Bilateral infiltrates on frontal chest x-ray
3. PaO,/FIO, = 300 mm Hgt
4. No evidence of left heart failure or fluid overload
5. The presence of a predisposing condition
TFrom Referanca 7. This differs from earlier definitions of ARDS, which requirad a PaQ./FiO2 = 200 mm Hg for tha diagnosis of ARDS (6). See text for further explanation.

Despite over 40 years of clinical experience with ARDS, there is still some uncertainty about the defining features of this condition. In 1994, a consensus conference of experts published a set of diagnostic criteria for ARDS and a clinical entity known as acute lung injury (ALI) (6). These criteria included: (a) PaO2/FIO2 "200 mm Hg for ARDS, (b) PaO>/FIOz "300 mm Hg for ALI, and (c) a pulmonary artery wedge pressure (PAWP) "18 mm Hg (to rule out left heart failure). In 2012, a European task force published a set of revised criteria for the diagnosis of ARDS (7) that included the following changes: (a) ALI was eliminated as a clinical entity, and the PaO 2/FIO2 for ARDS was set at "300 mm Hg, (b) a requirement was added that the PaO>/FIO, determination should be conducted at a positive end-expiratory pressure (PEEP) of 5 cm H,O, and (c) the wedge pressure measurement was eliminated (because of the diminished use of pulmonary artery catheters). These revised criteria are known as the Berlin Criteria, and they are combined with the original diagnostic criteria for ARDS in Table 23.2. Not included is the requirement for a standard level of PEEP during the PaO2/FIO2 determination because PEEP requires mechanical ventilation, and the diagnosis of ARDS can occur during spontaneous breathing.

Many of the diagnostic criteria for ARDS are non-specific, and are shared by other common causes of acute respiratory failure. This creates a tendency for misdiagnosis, which is demonstrated in Table 23.3 (8). The information in this table is from an autopsy study of patients who died with a premortem diagnosis of ARDS, and the postmortem diagnoses are listed in the table along with the prevalence of each diagnosis. Only half of the patients with the premortem diagnosis of ARDS had evidence of ARDS on postmortem examination, and the conditions most commonly mistaken for ARDS were pneumonia and hydrostatic pulmonary edema. The likelihood of identifying ARDS was 50% in this study, which is no better than the likelihood of heads or tails with a coin flip!

Inflammatory Injury (ARDS), % of Autopsies = 5096. Acute Pneumonia, % of Autopsies = 25%. Pulmonary Congestion, % of Autopsies = 11%. Invasive Aspergillosis, % of Autopsies = 6%. Pulmonary Embolism, % of Autopsies = 3%. Other Diagnoses, % of Autopsies = 5%
One source of error in the diagnosis of ARDS is the appearance of the chest x-ray. The classic radiographic appearance of ARDS is shown in Figure 23.2. The infiltrate has a finely granular or ground-glass appearance, and is evenly distributed in all lung fields, with no evidence of a pleural effusion. Unfortunately, these characteristic features are not always present. This is demonstrated by the chest x-ray in Figure 23.3. In this case, the infiltration shows a hilar prominence and is confined to the lower lung fields, with obliteration of the left hemidiaphragm suggesting a possible pleural effusion. These features could be mistaken for cardiogenic pulmonary edema. Because of such variability in the radiographic ap-pearance of ARDS, it is not possible to identify ARDS reliably using the chest x-ray alone (9).
FIGURE 23.2 Portable chest x-ray showing the classic radiographic appearance of ARDS. The infiltrate has a finely granular or "ground glass" appearance, and is evenly distributed throughout both lungs, with a relative sparing of the lung bases. There is no evidence of a pleural effusion.

When the chest-x-ray shows overlapping features of ARDS and cardiogenic pulmonary edema, like the one in Figure 23.3, the pulmonary artery wedge pressure (PAWP) has been used to distinguish between these two conditions (i.e., a PAWP "18 mm Hg is considered evidence of ARDS) (6). This is problematic because the wedge pressure is not a measure of capillary hydrostatic pressure, as explained in Chapter 8 (see pages 140141). The PAWP is measured in the absence of blood flow, when the static column of blood between the catheter tip and left atrium results in an equalization of pressures between the wedge pressure and left atrial pressure. However, when flow resumes, the pressure in the pulmonary capillaries must be higher than the left atrial pressure to provide a pressure gradient for flow in the pulmonary veins. Therefore, the left atrial (wedge) pressure is lower than the capillary hydrostatic pressure, and this will lead to an overdiagnosis of ARDS.
FIGURE 23.3 Portable chest x-ray of ARDS in a patient with urosepsis and Gram-negative septicemia. The infiltrates appear to emanate from the hilar areas and are confined to the lower lung fields. There is also obliteration of the left hemidiaphragm suggesting the presence of a pleural effusion. These radiographic features could be mistaken for hydrostatic pulmonary edema.

Although rarely used, bronchoalveolar lavage is a reliable method for distinguishing ARDS from cardiogenic pulmonary edema (10). This procedure is performed at the bedside using a flexible fiberoptic bronchoscope that is advanced into one of the involved lung segments. Once in place, the lung segment is lavaged with isotonic saline, and the lavage fluid is analyzed for the presence of neutrophils and protein.
1. In normal subjects, neutrophils make up less than 5% of the cells recovered in lung lavage fluid, whereas in patients with ARDS, as many as 80% of the recovered cells are neutrophils (10). A low neutrophil count in lung lavage fluid can be used to exclude the diagnosis of ARDS, while a high neutrophil count is evidence of ARDS.
2. Because inflammatory exudates are rich in proteinaceous material, lung lavage fluid that is rich in protein is used as evidence of ARDS. When the protein concentration in lung lavage fluid is expressed as a fraction of the protein concentration in plasma, the following criteria can be applied (11):
Hydrostatic Edema:Lavage fluid [protein] / plasma [protein] <0.5
ARDS:Lavage fluid [protein] / plasma [protein] >0.7

One of the most important discoveries in critical care medicine in the last quarter-century is the role of mechanical ventilation as a source of lung injury, particularly in patients with ARDS. This has led to a management strategy known as lung protective ventilation (12), which is described here.

Since the introduction of positive-pressure mechanical ventilation, the use of large inflation volumes (tidal volumes) has been a standard practice to reduce the tendency for atelectasis during mechanical ventilation. The conventional tidal volumes are 12 to 15 mL/kg (13), which are twice the size of tidal volumes achieved during quiet breathing (6 to 7 mL/kg). In patients with ARDS, these large inflation volumes are delivered into lungs that have only a fraction of the normal functional lung volume, as described next.

Although portable chest x-rays show an apparent homogeneous pattern of lung infiltration in ARDS, CT images reveal that the lung infiltration in ARDS is confined to dependent lung regions (13). This is shown in the CT images in Figure 23.4. Note the dense consolidation in the posterior lung regions (which are the dependent lung regions in the supine position), and the normal or uninvolved lung is restricted to the anterior half of the thorax. The uninvolved areas represent the functional portion of the lungs, and the portion that receives the inflation volumes. Therefore, the high inflation volumes used during conventional mechanical ventilation are being delivered to a markedly reduced volume of available lung, and this results in overdistension and rupture of the distal airspaces (15).
FIGURE 23.4 Computed tomographic image of lung slices in the region of the hilum from a patient with ARDS. The lung consolidation is confined to the posterior lung regions, which are the dependent regions in the supine position. The uninvolved lung in the anterior one-third of the thorax represents the functional portion of the lung. CT image is from Reference 14, and is digitally retouched.

Excessive inflation of the distal airspaces produce stress fractures in the alveolar capillary interface, and this leads to infiltration of the lung parenchyma and distal airspaces with an inflammatory exudate. This condition is known as ventilator-induced lung injury (VILI), and is strikingly similar to ARDS (15). The lung injury is volume-related rather than pressure-related (16), and is called volutrauma. (Pressure-related lung injury is called barotrauma, and is associated with the escape of air from the lungs.)
BIOTRAUMA: During conventional, high-volume mechanical ventilation, proinflammatory cytokines can appear in the lungs and systemic circulation, even though there is no structural damage in the lungs (17). This proinflammatory condition is known as biotrauma, and it can lead to neutrophil activation and inflammatory infiltration in the lungs (16). The systemic inflammatory response associated with biotrauma can promote inflammatory injury in other organs, which means that mechanical ventilation can be as source of inflammatory-mediated multiorgan failure (18)!
ATELECTRAUMA: The decrease in lung distensibility in ARDS can result in the collapse of small airways at the end of expiration. When this occurs, mechanical ventilation can be associated with cyclic opening and closing of small airways, and this process can be a source of lung injury (19). This type of lung injury is called atelectrauma (16), and it may be the result high-velocity shear forces created by the opening of collapsed airways, which can damage the airway epithelium.

Lung protective ventilation employs low tidal volumes (6 mL/kg) to limit the risk of volutrauma and biotrauma, and uses positive end-expiratory pressure (PEEP) to limit the risk of atelectrauma. A protocol for lung protective ventilation that has a proven survival benefit in ARDS (20) is shown in Table 23.4. This protocol was developed by the ARDS Clinical Network (a network created by governmental health agencies to evaluate potential therapies for ARDS), and is available at www.ardsnet.org. The tidal volume in this protocol is 6 mL/kg, based on predicted body weight, which is the body weight associated with normal lung volumes. Note that one of the stated goals is an endinspiratory "plateau" pressure (Ppl) "30 cm H20. This pressure is described in detail in Chapter 25.

|. 1st Stage, 1 = 1. Galculate patient's predicted body weight (PBW)t. Males: PBW = 50 + [2.3 x (height in inches — 60)] Females: PBW = 45.5 + [2.3 x (height In inches — 60)] . Set initial tidal volume (V7) at 8 mL/kg PBW. . Add positive end-expiratory pressure (PEEP) of 5 cm H,0. . Select the lowest FiO. that achieves an SpO, of 88-95%. . Reduce V; by 1 mL/kg every 2 hours until V7 =6 mL/kg. on -& co. Il. 2nd Stage, 1 = — . When V, = 6 mL/kg, measure plateau pressure (Ppl). . If Ppl > 80 cm H,0, decrease V; in 1 mL/kg Increments until Ppl < 80 cm H,O or V;= 4 mLikg. ho. lll. Sra Stage, 1 = —_ . Monitor arterial blood gases for respiratory acidosis. 2. If pH = 7.15-7.30, increase respiratory rate (RR) until pH > 7.30 or RR = 35 bpm. 3. If PH < 7.15, increase RR to 35 bom. If pH Is still < 7.15, increase V; In 1 mL/kg Increments until pH > 7.15.. lV. Optimal Goals, 1 = V7 = 6 mL/kg, Ppl = 30 cm HO, SpO, = 88-95%, pH = 7.30-7.45
Adapted fram the protocol devaloped by ARDS Network, available at weve.
the ardsnet.org.
tPradicted body weight is tha weight associated with normal lung volumes.

(For a detailed description of this pressure, see Chapter 26.) Lung protective ventilation employs a positive end-expiratory pressure (PEEP) of at least 5 cm H>0 to prevent the collapse of small airways at the end of expiration. The goal is to prevent the cyclic opening and closing of small airways and reduce the risk of atelectrauma. Higher PEEP levels (e.g., 15 cm H30) have been associated with a shorter duration of mechanical ventilation and a borderline increase in survival in ARDS, but only when the PaQ2/FIO> ratio is "200 mm Hg (21). However, PEEP levels above 10 cm H>0 are not typically used unless there is a problem maintaining arterial oxygenation (see next).
In situations where a toxic level of inhaled oxygen (FIO2 >50%) is needed to maintain the target SpO> of 88-95%, PEEP levels above 5 cm H;0 can be used to improve arterial oxygenation and reduce the FIO, to safer levels. However, it is important to emphasize that increases in PEEP can reduce the cardiac output, and if the goal of increasing PEEP is to maintain the same SpO> at a lower FIO,, the reduced cardiac output will reduce the systemic O> delivery.

One of the consequences of low tidal volume ventilation is a decrease in CO, elimination in the lungs, which can result in hypercapnia and respiratory acidosis. Because of the benefits of low volume ventilation, hypercapnia is allowed to persist as long as there is no evidence of harm. This practice is known as permissive hypercapnia (22). The limits of tolerance to hypercapnia and respiratory acidosis are unclear, but data from clinical trials of permissive hypercapnia show that arterial PCO, levels of 60-70 mm Hg and arterial pH levels of 7.2-7.25 are safe for most patients (23). The target pH is 7.30—7.45 in the protocol for lung protective ventilation in Table 23.4.

Lung protective ventilation is one of the few measures that has been shown to improve survival in ARDS. The largest and most successful trial of lung protective ventilation was conducted by the ARDS Network (20), and enrolled over 800 ventilator-dependent patients with ARDS who were randomly assigned to receive tidal volumes of 6 mL/kg or 12 mL/kg (using predicted body weight). Ventilation with the lower tidal volume (6 mL/kg), and an end-inspiratory plateau pressure (Ppl) "30 cm H20, was associated with a shorter duration of mechanical ventilation and a 9% absolute reduction in mortality rate (40% to 31%, P=0.007).
A total of 5 clinical trials have compared tidal volumes of 6 mL/kg and 12 mL/kg during mechanical ventilation in patients with ARDS. In two of the trials, low tidal volumes were associated with fewer deaths, while in three of the trials, there was no survival benefit associated with low tidal volumes (24). Despite the lack of a consistent survival benefit, lung protective ventilation using tidal volumes of 6 mL/kg has become a standard practice in patients with ARDS. A recent multicenter survey of lung protective ventilation in ARDS showed an in-hospital mortality rate of 48% (5), which is no better than mortality rates reported before the introduction of lung protective ventilation. An observation that is relevant in this context is that mortality rates for ARDS tend to be lower in controlled clinical trials than in clinical practice surveys (25).
(Note: A possible explanation for the lack of a consistent survival benefit with low tidal volumes is presented in Chapter 25.)

There is convincing evidence that mechanical ventilation can damage the lungs in ARDS as a result of overdistension of functional alveoli (volutrauma) and collapse of small airways (atelectrauma). Lung protective ventilation is designed to mitigate the mechanical forces that create ventilator-induced lung injury, and it has been adopted as a standard method of mechanical ventilation in ARDS.

The treatment of ARDS begins by treating the inciting condition (e.g., septicemia), if possible. Therapies directed at the ARDS have been marked by failure more than success. The failed therapies in ARDS in-clude surfactant (in adults), inhaled nitric oxide, pentoxyphylline, ibuprofen, prostaglandin E1, and antifungal agents (to inhibit thromboxane) (26). Clinical benefits have been reported with fluid management that avoids fluid accumulation in the lungs, and with high-dose corticosteroids in severe or unresolving ARDS. The management described in this section is limited to the measures that have documented benefits.

The lung consolidation in ARDS is an inflammatory exudate, and should not be influenced by fluid balance (for the same reason that diuresis will not clear an infiltrate caused by pneumonia). However, avoiding a positive fluid balance will prevent unwanted fluid accumulation in the lungs, which could aggravate the respiratory insufficiency in ARDS. Clinical studies have shown that avoiding a positive fluid balance in patients with ARDS can reduce the time on mechanical ventilation (27), and can even reduce mortality (28).
However, it is also important to avoid fluid deficits and maintain intra-vascular volume because the positive intrathoracic pressures during mechanical ventilation will magnify the tendency for the cardiac output to decrease in response to deficits in intravascular volume.

There is a long history of clinical trials evaluating steroid therapy in ARDS, and the aggregated results of these studies show no consistent survival benefit associated with steroid therapy (29). However, there is evidence of other benefits provided by steroid therapy in ARDS, and these include a reduction in markers of inflammation (both pulmonary and systemic inflammation), improved gas exchange, shorter duration of mechanical ventilation, and shorter length of stay in the ICU (29). Steroid therapy is currently recommended only in cases of early severe ARDS and unresolving ARDS (28).

In early severe ARDS, defined as a PaOQ2/FIO2 <200 mm Hg with PEEP of 10 cm H,0, the following steroid regimen in recommended (29):
Methylprednisolone: Start with an IV loading dose of 1 mg/kg (ideal body weight) over 30 minutes, then infuse at 1 mg/kg/day for 14 days, then gradually taper the dose over the next 14 days and discontinue therapy. Five days after the patient is able to ingest oral medications, the dose can be given orally (as prednisone or prednisolone) as a single daily dose.

ARDS has a fibrinoproliferative phase that begins 7-14 days after the onset of illness (30), and eventually results in irreversible pulmonary fibrosis. High-dose steroid therapy started in the developing phase of fibrinoproliferation can help to halt the progression to pulmonary fibrosis. In cases where ARDS does not begin to resolve after 7 days, highdose steroid therapy is recommended, but should begin no later than 14 days after the onset of illness. The following steroid regimen is recommended (29):
Methylprednisolone: Start with an IV loading dose of 2 mg/kg (ideal body weight) over 30 minutes, then infuse at 2 mg/kg/day for 14 days, and 1 mg/kg/day for the next 7 days. After this, gradually taper the dose and discontinue therapy at 2 weeks after extubation. Five days after the patient is able to ingest oral medications, the dose can be given orally (as prednisone or prednisolone) as a single daily dose.
The risks of high-dose steroid therapy include worsening glycemic control and prolonged neuromuscular weakness when combined with neuromuscular blocking agents. There is no evidence of an increased risk of nosocomial infections with the steroid regimens described here (28).

Although the therapeutic focus for ARDS has been the lungs, the principal cause of death in ARDS is multiorgan failure, not respiratory failure (5,31). As many as 70% of deaths in ARDS are the result of multiorgan failure (31), and the mortality rate is directly related to the number of organs that fail. The relationship between mortality rate and extrapulmonary organ failure is shown in Figure 23.5. This relationship indicates that mortality is a function of a progressive systemic condition, which most likely represents progressive systemic inflammation. The similarities between Figure 23.5 and Figure 14.2 (see page 267) supports the notion of a relationship between progressive systemic inflammation and mortality in ARDS. If this is the case, then limiting the therapeutic focus to the lungs in ARDS is a prescription for failure.

A minority (10 to 15%) of patients with ARDS develop severe hypoxemia that is refractory to oxygen therapy and mechanical ventilation (32). This condition is an immediate threat to life, and the following "rescue therapies" can produce an immediate improvement in arterial oxygenation. Unfortunately, these rescue measures often provide little or no survival benefit.

High frequency oscillatory ventilation (HFOV) delivers small tidal volumes (1-2 mL/kg) using rapid pressure oscillations (300 cycles/min). The small tidal volumes limit the risk of volutrauma, and the rapid pressure oscillations create a mean airway pressure that prevents small airway collapse and limits the risk of atelectrauma. When used in patients with severe ARDS, HFOV can improve arterial oxygenation, but there is no documented survival benefit (33). HFOV requires a specialized ventilator (Sensormedics 3100B, Viasys Healthcare, Yorba Linda, CA), and may not be available in all hospitals. This mode of ventilation is de-scribed in more detail in Chapter 27.

Inhaled nitric oxide (5-10 ppm) is a selective pulmonary vasodilator that can improve arterial oxygenation in ARDS by increasing flow to areas of high dead space ventilation (34). However, the increase in arterial oxygenation is temporary (1-4 days), and there is no associated survival benefit (35). Adverse effects of inhaled nitric oxide include methemoglobinemia (usually mild) and renal dysfunction (35). An added risk is the potential for nitric oxide to form peroxinitrite, a potent toxin capable of oxidant cell injury.
FIGURE 23.5 The relationship between extrapulmonary organ failure and mortality in ARDS. Data from the electronic supplementary material for Reference 5.

Switching from the supine to prone position can improve pulmonary gas exchange by diverting blood away from poorly aerated lung regions in the posterior thorax and increasing blood flow in aerated lung regions in the anterior thorax (see Figure 23.4). Prone positioning has had little impact on mortality in ARDS, but a recent study combining lung protective ventilation with prone positioning showed a lower than expected mortality rate in patients with severe ARDS (PaO>/FIO, <100 mm Hg) (36). Prone positioning is labor intensive and creates problems with nursing care (e.g., airway care and skin care), but it may be the only measure available for refractory hypoxemia in hospitals with limited resources.

Extracorporeal membrane oxygenation (ECMO) has had variable success in patients with refractory hypoxemia, and is a consideration only in medical centers with established ECMO programs, and only when other rescue therapies have failed (37).

One of the important messages in this book is the prominent role of inflammation as a source of organ damage and multiorgan failure in critically ill patients, and the lack of an effective treatment for this destructive force. This message appears in Chapter 14, which describes inflammatory shock and multiorgan failure, and it appears again in this chapter. Although a survival benefit has been documented with lung protective ventilation in ARDS, this is not a treatment for ARDS, but rather is a lessening of the damage produced by mechanical ventilation. Until a remedy is available for the destructive effects of inflammation, conditions like ARDS will continue to be a major source of morbidity and mortality in the ICU.

1. Matthay MA, Ware LB, Zimmerman GA. The acute respiratory distress syndrome. J Clin Invest 2012; 122:2731-2740.

Tzouanakis AE, Cardenas VJ, Zwischenberger JB. Permissive hypercapnia in acute respiratory failure. JAMA 1994; 272:957-962.. 23., 1 = Hickling KG, Walsh J, Henderson S, et al. Low mortality rate in adult respiratory distress syndrome using low-volume, pressure-limited ventilation with permissive hypercapnia: A prospective study. Crit Care Med 1994; 22:1568-1578.. 24., 1 = Fan E, Needham DM, Stewart TE. Ventilator management of acute lung injury and acute respiratory distress syndrome. JAMA 2005; 294:2889-2896.. 25., 1 = Phua J, Badia JR, Adhikari NKJ, et al. Has mortality from acute respiratory distress syndrome decreased over time? Am J Respir Crit Care Med 2009; 179:220-227.
Nonventilatory Management, 1 = Nonventilatory Management. 26., 1 = Calfee CS, Matthay MA. Nonventilatory treatment for acute lung injury and ARDS. Chest 2007; 131:913-920.. 27., 1 = The Acute Respiratory Distress Syndrome Network. Comparison of two fluid management strategies in acute lung injury. N Engl J Med 2006; 354:2564-2575.. 28., 1 = Murphy CV, Schramm GE, Doherty JA, et al. The importance of fluid management in acute lung injury secondary to septic shock. Chest 2009; 136:102-109.. 29., 1 = Marik PE, Meduri GU, Rocco PRM, Annane D. Glucocorticoid treatment in acute lung injury and acute respiratory distress syndrome. Crit Care Clin 2011; 27:589-607.. 30., 1 = Meduri GU, Chinn A. Fibrinoproliferation in late adult respiratory distress syndrome. Chest 1994; 105(Suppl):127S-129S.. 31., 1 = Estenssoro E, Dubin A, Laffaire E, et al. Incidence, clinical course, and outcome in 217 patients with acute respiratory distress syndrome. Crit Care Med 2002; 30:2450- 2456.

Asthmatic distress cals for procedures which stimulate the sympathetic and dilate the bronchi.
Lawrason Brown, MD The Practical Medicine Series 1931.
This chapter describes the management of severe exacerbations of asthma and chronic obstructive pulmonary disease (COPD), and includes strategies for ventilatory assistance in these conditions. The non-ventilator management of these conditions has experienced little change in recent years. In fact, Dr. Brown's comment in the introductory quote, which appeared 80 years ago, is remarkably similar to the current emphasis on adrenergic bronchodilators in the management of "asthmatic distress."

The management of obstructive airways disease is guided by the severity of obstruction in the airways (1,2). During acute exacerbations, the clinical examination is often unreliable for determining the severity of airway obstruction (3,4). As a result, more objective measures of airway obstruction like the ones described here have been recommended for guiding the management of obstructive airways diseases like asthma and COPD (1,2).

For spontaneously breathing patients, the recommended measures of airway obstruction are the forced expiratory volume in 1 second (FEV;), and the peak expiratory flow rate (PEFR) (1). Hand-held devices that are easy to operate are available for both measurements. The FEV, and PEFR vary with age, gender, and height (5), and measurements are typically ex-pressed as "% predicted" (observed result/predicted resultx 100), with the predicted values derived from standard reference equations. (The PEFR is also expressed in relation to the highest flow achieved by each patient; i.e., the "personal best" PEFR.) The correlations between the % predicted values of FEV, and PEFR and the severity of airway obstruction are shown below (1).
270%, Severity of Obstruction = Mild. 40-69%, Severity of Obstruction = Moderate. < 40%, Severity of Obstruction = Severe
Both measurements are volume-dependent and effort-dependent, so maximum patient effort is required for reliable recordings. The FEV, is the preferred measurement because it is less variable and more likely to detect obstruction in the smaller airways (1,5). Comparison of both mea-surements in the acute care setting have shown that the PEFR underestimates the severity of airway obstruction (4).
SEVERE EXACERBATIONS: The measurement of FEV; and PEFR requires a maximum inspiratory effort (to total lung capacity) followed by a maximum expiratory effort (to residual lung volume), and three consecutive measurements are recommended for optimal results. Patients with se-vere exacerbations of asthma and COPD are often unable to perform these maneuvers because of respiratory distress, and thus the FEV, and PEFR have a limited role in the management of severe exacerbations of asthma and COPD (which includes most cases admitted to the ICU).

For patients with asthma and COPD who require mechanical ventilation, the severity of airflow obstruction can be evaluated by monitoring a pressure known as intrinsic PEEP.
This pressure is described in the final section of the chapter.

Drug aerosols play a major role in the management of obstructive airways diseases. There are two basic designs for aerosol generators in clinical medicine: the jet nebulizer and the metered-dose inhaler. The operation of these devices is illustrated in Figure 24.1

The pneumatic or jet nebulizer uses the same principle as the air-entrainment device in Figure 22.5 (see page 435). A high-pressure gas source (e.g., 50 psi from a wall outlet) is passed through a narrow opening in the nebulizer, creating a high-velocity (jet) stream of gas that is passed over the opening of a narrow tube submerged in a drug solution. The gas jet draws the drug solution up the tube (by creating viscous drag) and then pulverizes the solution to create an aerosol spray that is inhaled by the patient. Standard jet nebulizers have a reservoir volume of 3-6 mL, and can completely aerosolize the reservoir volume in less than 10 minutes (6). A larger version (with a reservoir volume >200 mL) is available for continuous aerosol therapy (see later).
FIGURE 24.1 Devices used for aerosol drug therapy. See text for explanation.
LUNG DEPOSITION: Although small-volume nebulizers can completely aerosolize a drug solution, only a fraction of the drug aerosol reaches the lungs. This is demonstrated in Table 24.1, which shows the distribution of aerosolized albuterol with different aerosol generator systems (7). When the nebulizer is used, most of the aerosol impacts on the delivery apparatus or is exhaled, and only 12% of the intended dose reaches the lungs. Inefficient drug delivery is a characteristic feature of aerosol drug therapy, and is not specific for the jet nebulizer.

A metered-dose inhaler (MDI) operates like a canister of hair spray. The MDI has a pressurized canister that contains a drug solution with a boiling point below room temperature. When the canister is squeezed between the thumb and fingers, a valve opens that releases a fixed volume of the drug solution. The liquid immediately vaporizes when it emerges from the canister, and a liquid propellant in the solution creates a highvelocity spray.
LUNG DEPOSITION: The spray emerging from an MDI can have a velocity in excess of 30 meters per second (over 60 miles per hour) (8), and when this high-velocity spray is delivered directly into the mouth, most of the spray impacts on the posterior wall of the oropharynx and is not inhaled. This inertial impaction is reduced by using a spacer device or holding chamber to reduce the velocity of aerosol delivery. The influence of a holding chamber on drug delivery is shown in Table 24.1. When the MDI is used alone, 80% of the drug aerosol is deposited in the oropharynx, but when a holding chamber is used with the MDI, drug deposition in the mouth is almost completely eliminated, and the drug dose reaching the lungs is doubled. Results like this are the reason that holding chambers are recommended for all bronchodilator treatments with MDIs (6).

of
Exhaled, ‘Nebulizer (2.5 mg) = 20%. Exhaled, “MDI (180 pg) = 1%. Exhaled, MDI+ HC. (180 po) = 1%. Apparatus, ‘Nebulizer (2.5 mg) = 66%. Apparatus, “MDI (180 pg) = 10%. Apparatus, MDI+ HC. (180 po) = 789. Oropharynx, ‘Nebulizer (2.5 mg) = 2%. Oropharynx, “MDI (180 pg) = 80%. Oropharynx, MDI+ HC. (180 po) = 19%. Lungs, ‘Nebulizer (2.5 mg) = 12%. Lungs, “MDI (180 pg) = 9%. Lungs, MDI+ HC. (180 po) = 20%
From Referance 7. MDI= metered dose inhaler; HC = holding chamber. The MDI dose of 180 ug raprasents 2 puffs.

One of the notable features of aerosol drug therapy is the equivalent bronchodilator responses produced by nebulizers and MDIs despite a large difference in drug dosage. This is demonstrated in Figure 24.2, which compares the bronchodilator response to albuterol delivered by nebulizer and MDI (with a holding chamber) in patients with acute exacerbation of asthma (9). There is no difference in the response in each of the 3 treatments. Using the distribution patterns in Table 24.1, the dose of albuterol deposited in the lungs in Figure 24.2 would be 12% of 2.5 mg or 250 wg for the nebulizer and 20% of 360 wg or 72 yg for the MDI with holding chamber. Thus, there is a 3.5-fold difference in drug dose in the airways, yet the bronchodilator responses are equivalent.

Equivalent responses like those in Figure 24.2 have also been observed in ventilatordependent patients (10,11). Drug deposition in the lungs is reduced further during mechanical ventilation (11) due to condensation on the endotracheal tube and ventilator tubing. However, the impact of this loss on bronchodilator responses is not clear (12). The response to MDIs is optimal when a holding chamber is used (11): the chamber is connected to the inspiratory limb of the ventilator tubing, and 5-8 puffs from the MDI are delivered into the chamber for inhalation during the ensuing lung inflations. Regardless of the aerosol device used, drug delivery into the airways can be enhanced by decreasing the inspiratory flow rate and increasing the duration of inspiration (13).

One thing is clear about aerosol therapy; i.e., it is an inefficient method of drug administration, regardless of the type of aerosol generator that is used. Yet, despite this inefficiency, drug aerosols are favored for administering bronchodilators in acute exacerbations of asthma and COPD, as described next.
FIGURE 24.2 Equivalent responses to a bronchodilator (albuterol) given by nebulzer or metered-dose inhaler (MDI) with a holding chamber in patients with acute exacerbation of asthma. Treatments were given every 30 minutes using the doses indicated in the upper right portion of the graph (the MDI dose of 0.36 mg represents 4 puffs). FEV; = forced expiratory volume in one second. Data from Reference 9.

The flow diagram in Figure 24.3 summarizes the early management of adults with acute exacerbation of asthma (1). This protocol is based on objective measures of airway obstruction (i.e., FEV; and peak expiratory flow rate), but clinical measures of disease severity (e.g., respiratory rate, use of accessory muscles) are also suitable (14,15). The recommended drugs and dosing regimens for acute asthma are summarized in Table 24.2.

The favored bronchodilators are drugs that stimulate a-adrenergic receptors in bronchial smooth muscle (2 subtype). Aerosol delivery of these B-agonists is preferred because it is more effective than oral (16) or intravenous (17) drug therapy, and has fewer side effects. Short-acting B:-agonists are preferred for the acute management of asthma because they can be given in rapid succession with less risk of drug accumulation (1).
FIGURE 24.3 Flow diagram showing the early management of acute exacerbations of asthma, as recommended by the National Asthma Education Program (1). FEV, = forced expiratory volume in one second, PEFR = peak expiratory flow

prednisone (IV) dally in 1 or 2 divided doses for 7-10 days. Oral therapy as effective as intravenous therapy.
Fram Referance 1.
Albuterol is the most widely used short-acting B-agonist for the acute management of asthma (1,14,15). Aerosolized albuterol has a rapid onset of action (less than 5 minutes), and a bronchodilator effect that lasts 2-5 hours (18). Levalbuterol is the R-enantiomer of albuterol (a more active form of the drug) that is equally effective at half the dose. However, clinical studies have not shown an advantage with levalbuterol over albuterol (1,14).

The following regimens of aerosolized albuterol are recommended for acute exacerbations of asthma (1,14,15):
1. The initial treatment involves up to three 20-minute treatments using 2.5 to 5 mg albuterol by nebulizer or 4-8 puffs (90 mg al-buterol per puff) by MDI with a holding chamber. Nebulizer delivery is preferred for patients with severe airflow obstruction
(1), even though there is no evidence that nebulizers produce better bronchodilator responses than MDIs in acute asthma (19).
2. If further therapy is needed, albuterol can be given hourly for up to 3 hours, or can be given by continuous nebulization using doses of 5-15 mg/hr. Continuous aerosol therapy is popular, and may be more effective than intermittent aerosol therapy in patients with severe airflow obstruction (20).
3. For patients who are admitted to the hospital, albuterol (2.5—5 mg by nebulizer or 4-8 puffs by MDI) is given every 4-6 hours for the duration of the hospital stay.

For the rare asthmatic patient who does not tolerate bronchodilator aerosols (usually because of excessive coughing) parenteral therapy can be given using subcutaneous epinephrine (0.3 to 0.5 mg every 20 minutes for 3 doses) or subcutaneous terbutaline (0.25 mg every 20 minutes for 3 doses) (1). However, it is important to emphasize that parenteral B-agonist therapy is not more effective than aerosol therapy, and is more likely to produce unwanted side effects (21).

High-dose aerosol therapy with B>-agonists can produce a number of side effects, including tachycardia, tremors, hyperglycemia, and a decrease in serum potassium, magnesium, and phosphate levels (22,23). Cardiac ischemia has been reported, but is rare (22). The decrease in serum potassium is the result of a B-Receptor-mediated shift of potassium into cells. This effect is notable because large doses of inhaled Bj-agonists (e.g., 20 mg albuterol) have been used for the acute management of hyperkalemia (24). (Note: This is not a favored treatment for hyperkalemia because of the side effects of high-dose B>-agonists. )

Anticholinergic agents offer only marginal benefits in acute asthma, and their use is restricted to combination therapy with short-acting Bz-agonists for severe exacerbations, and only for the first 3-4 hours of management (1,25). The only anticholinergic agent approved for use in the United States is ipatropium bromide, a derivative of atropine that blocks muscarinic receptors in the airways. The dose in acute asthma is 0.5 mg (which can be mixed with albuterol in the nebulizer) every 20 minutes for 3 doses, then as needed, or 8 puffs (18 eg per puff) by MDI every 20 min as needed for up to 3 hours (1). Systemic absorption is minimal, and there is little risk of anticholinergic side effects (e.g., tachycardia, dry mouth, blurred vision, urinary retention). Ipatropium has no proven benefit beyond the first few hours of management, and it should be discontinued in patients who are admitted for continued asthma management (1).
able 24.3 Comparison of Therapeutic Corticosteroid
Hydrocortisone, 1 = 20. Hydrocortisone, 2 = 1. Hydrocortisone, 3 = 20. Prednisone, 1 = 6. Prednisone, 2 = 3.5. Prednisone, 3 = 1. Methylprednisone, 1 = 4. Methylprednisone, 2 = 6. Methylprednisone, 3 = 0.5. Dexamethasone, 1 = 0.75. Dexamethasone, 2 = 30-40. Dexamethasone, 3 = 0
From Zsiss CR. Intense pharmacotherapy. Chest 1992; 104 (Suppl):4075.

An acute myopathy has been reported in ventilator-dependent asthmatic patients treated with high-dose steroids and neuromuscular blocking agents (31). Unlike the traditional steroid myopathy, which is characterized by proximal muscle weakness, this condition involves both proximal and distal muscles, and is often associated with rhabdomyolysis. The muscle weakness can be prolonged (although it usually resolves) and can hamper weaning from mechanical ventilation. Because of the risk of this myopathy, it seems wise to avoid neuromuscular paralysis whenever possible in steroid-treated ventilatordependent asthmatic patients.
Additional Considerations, 1 = Additional Considerations. The following are some additional considerations for the management of acute asthma:, 1 = The following are some additional considerations for the management of acute asthma:. 1. Asthma 2., 1 = exacerbations are often triggered by viral infections, and empiric antibiotic therapy is not advised the absence of a treatable infection (1). Intravenous magnesium (2 grams over 20 min) has mild bronchodilator effects (possibly as a result of calcium channel blockade), and can be used as an adjunctive measure for severe exacerbations of asthma (1). However, magnesium administration has no impact on the clinical course of acute asthma (32).. 3. An arterial blood gas is advised for patients who do not show a satisfactory response to bronchodilators in the emergency department. A normal PCO 2 in acute asthma indicates severe airflow obstruction, and warrants admission to the ICU., 1 = 3. An arterial blood gas is advised for patients who do not show a satisfactory response to bronchodilators in the emergency department. A normal PCO 2 in acute asthma indicates severe airflow obstruction, and warrants admission to the ICU.. 4., 1 = Intubation and mechanical ventilation can be problematic in acute exacerbations of asthma; this aspect of management is described in the final section of the chapter.

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States (33) and, despite a marked reduction in the prevalence of cigarette smoking in adults, hospital admissions for acute exacerbations of COPD increased by 60% over a recent 10-year period (34). About half of these admissions require care in an ICU (35).
An acute exacerbation of COPD is defined as "a change in the patient's baseline dyspnea, cough, or sputum production that is beyond the normal day-to-day variation" (2). Most of these exacerbations are the result of infection (usually confined to the airways), but as many one of every four or five cases may be the result of an acute pulmonary embolism (36). In about one-third of patients, a precipitating event is never identified (2).

Although a distinguishing feature of COPD is lack of responsiveness to bronchodilators (in contrast to asthma), bronchodilator therapy is used routinely in patients with COPD (2). The same bronchodilator drugs used in acute ex-acerbations of asthma are also recommended for acute exacerbations of COPD, but the dosing regimens differ, as shown in Table 24.4. Ipatropium is used as combination therapy when the response to shortacting B2-agonists is less than satisfactory, although at least three clinical studies do not support this practice (37).

A short course (7-10 days) of corticosteroid therapy is associated with fewer treatment failures and a shorter duration of mechanical ventilation in acute exacerbations of COPD (38,39). However, at least 10 patients must be treated with corticosteroids to produce one favorable response (38), so the impact of steroid therapy is limited. The recommended steroid regimen for acute exacerbations of COPD is shown in Table 24.4 (2). Note that the dose range is slightly lower than the dose range for treating acute asthma. Intravenous steroids offer no advantage over oral steroids in acute exacerbations of COPD (40), similar to the observations in acute asthma (26,29).

Airways infections (viral and bacterial) are responsible for at least 50% of acute exacerbations of COPD (2), but many are nontreatable viral infections, and this limits the benefit of antimicrobial therapy.

Antibiotic therapy is not determined by sputum cultures in acute exacerbations of COPD because the same organisms are often isolated during clinically stable periods and during acute exacerbations (41). Instead, the clinical severity of disease is used to determine the need for antibiotic therapy. Clinical studies have shown that antibiotic therapy is most likely to improve clinical outcomes when exacerbations of COPD are severe enough to require hospitalization (34,42), and particularly when mechanical ventilation is required (2,43). This means that all ICU patients with acute exacerbations of COPD are candidates for antibiotic therapy.

Albuterol, ‘Dosing Regimen and Comments = Dosing:. Albuterol, ‘Dosing Regimen and Comments = 2.5-5 mg by nebulizer or 2-8 puffs (90 pg/purh) by MDI every 4-6 hrs.. Albuterol, ‘Dosing Regimen and Comments = Comment:. Albuterol, ‘Dosing Regimen and Comments = First-line broncho-dilator that is equally effective (or ineffective) when given by nebulizer or MDI with holding chamber.. Levalbuterol, ‘Dosing Regimen and Comments = Dosing:. Levalbuterol, ‘Dosing Regimen and Comments = 1.25-2.5 mg by nebulzer or 2-8 puts (45 ug/pufh by MDI every 4-6 hrs.. Levalbuterol, ‘Dosing Regimen and Comments = Comment:. Levalbuterol, ‘Dosing Regimen and Comments = More potent form of albuterol that has no proven clinical superiority over albuterol.. lpatropium, ‘Dosing Regimen and Comments = Dosing:. lpatropium, ‘Dosing Regimen and Comments = 0.5 mg by nebulizer or 2-8 puffs (18 pg/putt) by MDI every 4-6 hrs... lpatropium, ‘Dosing Regimen and Comments = Comment:. lpatropium, ‘Dosing Regimen and Comments = Used as combination therapy when response to short-acting B2-agonists is less than satisfactory.. Corticosteroids, ‘Dosing Regimen and Comments = Dosing:. Corticosteroids, ‘Dosing Regimen and Comments = 30-40 mg of prednisone (PO) or methyl prednisone (IV) daily in 1 or 2 divided doses for 7-10 days.. Corticosteroids, ‘Dosing Regimen and Comments = Comment:. Corticosteroids, ‘Dosing Regimen and Comments = Oral therapy Is equivalent to intravenous therapy.
From Referance 2.

The pathogens most often isolated from the lower respiratory tract in acute exacerbations of COPD are Hemophilus influenzae and Streptococcus pneumoniae (2,41). Pseudomonas aeruginosa is also a prominent isolate in advanced cases of COPD and in ventilator-dependent patients (44). Antibiotics with activity against all these pathogens are advised for ICU patients with acute exacerbations of COPD. These antibiotics include levofloxacin, pipericillin-tazobactam, and imipenem or meropenem. Duration of antibiotic therapy is typically 5-7 days.

Over 50% of patients admitted to the ICU because of asthma or COPD are placed on mechanical ventilation (47,48), and the following are some of the major considerations related to positive pressure ventilation in these patients.

During spontaneous breathing in normal subjects, inhaled gas is completely exhaled before the end of expiration. In this situation, there is no expiratory airflow at the end of expiration, so the pressure in the distal airspaces is equivalent to atmospheric (zero reference) pressure. This is illustrated in the lower pressure-volume loop in Figure 24.4. In patients with airways obstruction from asthma or COPD, exhalation is prolonged, and when the airways obstruction is severe, exhalation is not completed before the next inhalation. This results in hyperinflation, called dynamic hyperinflation, and the trapped gas in the distal airspaces creates a positive end-expiratory pressure (PEEP), which is called intrinsic PEEP or auto-PEEP (49). This is illustrated by the upper hysteresis loop in Figure 24.4. Note that, in the presence of hyperinflation and intrinsic PEEP, breathing occurs on a flatter portion of the pressure-volume curve, which means that the respiratory muscles must generate a higher transpulmonary pressure to draw the normal volume of air into the lungs. This creates an increased work of breathing in patients with severe airflow obstruction.

The shift in the pressure volume curves for breathing caused by dynamic hyperinflation means that positive-pressure mechanical ventilation will create positive intrathoracic pressures that are higher than normal in patients with dynamic hyperinflation. Furthermore, mechanical ventilation can add to the hyperinflation (e.g., by delivering high inflation volumes) (50). Dynamc hyperinflation can have two adverse consequences: (a) overdistention of alveoli, which can lead to ventilator-induced lunginjury, and (b) an increase in intrathoracic pressure, which can impede venous return to the heart. The following measures can help to reduce the risk of these adverse consequences.
FIGURE 24.4 Pressure-volume curves showing the effects of severe airways obstruction on lung volumes and transpulmonary pressures. The hysteresis loops show the pressure and volume changes during inspiration (I) and expiration (E) for a single breath. See text for further explanation.

Dynamic hyperinflation can be detected by monitoring the flow waveforms displayed by mechanical ventilators. This is illustrated in Figure 24.5. The normal flow waveforms in the upper panel show that the expiratory flow ceases before the next lung inflation, while the flow waveforms in the lower panel show that expiratory flow is continuing when the next lung inflation is delivered. The presence of expiratory flow at the end of expiration is evidence of dynamic hyperinflation.
INTRINSIC PEEP: When there is evidence of dynamic hyperinflation on the flow waveforms, the severity of the problem can be evaluated by monitoring the level of intrinsic PEEP. (The measurement of intrinsic PEEP is described in Chapters 25 and 27). The intrinsic PEEP level provides a measure of the severity of airways obstruction in patients with asthma and COPD who require mechanical ventilation.

The following measures are designed to limit dynamic hyperinflation:
1. Ventilate with low tidal volumes (6 mL/kg) using the lung protective ventilation protocol in Table 23.4 (see page 455).
2. Maximize the time for expiration by: (a) preventing rapid respiratory rates (with sedation, if possible, or neuromuscular paralysis, if necessary) and (b) maintaining inspiratory:expiratory ratio of 1:2 or higher.

Positive pressure ventilation can be delivered with tight-fitting face masks instead of endotracheal tubes. This type of noninvasive mechanical ventilation avoids the adverse effects of endotracheal intubation (e.g., patient discomfort, increased risk of nosocomial pneumonia), but is not appropriate for all patients. Patients are not candidates for noninvasive ventilation if they are unresponsive, have facial trauma, have severe circulatory compromise, have impending cardiac or respiratory arrest, or have copious respiratory secretions and are unable to clear secretions effectively.
Noninvasive ventilation has been used most successfully in acute exacerbations of COPD associated with progressive hypercapnia (51). There is far less experience in acute asthma, but the few available studies show that noninvasive ventilation can hasten the resolution of the acute episode and reduce the number of patients who require intubation (52).
FIGURE 24.5 Flow waveforms during positive pressure mechanical ventilation. The waveforms in the lower panel show continuing expiratory flow at the end of expiration (indicated by the arrows), which indicates the presence of dynamic hyperinflation. I = inspiration, E = expiration.

Noninvasive ventilation is a consideration in patients with acute exacerbation of asthma or COPD who do not respond adequately to initial medical management, and it is most likely to be successful when instituted early, before progression to life-threatening CO> retention and impending respiratory arrest. This is particularly important in patients with severe asthma, who do not develop hypercapnia until late in the progression to lifethreatening ventilatory failure (52).
(Note: Noninvasive ventilation is presented in detail in Chapter 27.)

The management of patients admitted to the ICU because of severe exacerbations of asthma or COPD can be summarized in the following two statements:
1. Give bronchodilators and corticosteroids to all patients, plus antibiotics for patients with severe COPD.
2. If the condition progresses despite the above measures, use noninvasive ventilation if possible, or conventional mechanical ventilation if necessary.
That's about it.

1. National Asthma Education and Prevention Program Expert Panel Report 3: Guidelines for the diagnosis and management of asthma. Full Report 2007. NIH Publication No. 07-4051; August, 2007. (Available at www.nhlbi.nih.gov/ guidelines/asthma)
2. Rabe KF, Hurd S, Anzueto A, et al. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. The GOLD executive summary. Am J Respir Crit Care Med 2007; 176:532-555.

Mechanical Ventilation, 1 = Mechanical Ventilation. 47., 1 = Peters JI, Stupka JE, Singh H, et al. Status asthmaticus in the medical intensive care unit: a 30-year experience. Respir Med 2012; 106:344-348.. 48., 1 = Soo Hoo GW, Hakimian N, Santiago SM. Hypercapnic respiratory failure in COPD patients' response to therapy. Chest 2000; 117:169-177.. 49., 1 = Blanch L, Bernabe F, Lucangelo U. Measurement of air trapping, intrinsic positive end-expiratory pressure, and dynamic hyperinflation in mechanically ventilated patients. Respir Care 2005; 50:110-123.. 50., 1 = Pepe P, Marini JJ. Occult positive end-expiratory pressure in mechanically ventilated patients with airflow obstruction. The auto-PEEP effect. Am Rev Respir Dis 1982; 126:166-170.. 51., 1 = Boldrini R, Fasano L, Nava S. Noninvasive mechanical ventilation. Curr Opin Crit Care 2012; 18:48-53.. 52., 1 = Scala R. Noninvasive ventilation in severe acute asthma? Still far from the truth. Respir Care 2010; 55:630-637.

All who drink of this remedy will recover . . . except those in whom it does not help, who will die. Therefore, it is obvious that it fails only in incurable diseases.
Galen

", . . an opening must be attempted in the trunk of the trachea, into which a tube of reed or cane should be put; you wil then blow into this, so that the lung may rise again. . . and the heart becomes strong... "
Andreas Vesalius 1555
Vesalius is credited with the first description of positive pressure ventilation, but it took 400 years to apply his concept to patient care. The occasion was the polio epidemic of 1955, when the demand for assisted ventilation outgrew the supply of negative-pressure tank ventilators (known as iron lungs). In Denmark, all medical schools shut down and medical students worked in 8-hour shifts as human ventilators, manually inflating the lungs of afflicted patients. In Boston, the nearby Emerson Company made available a prototype positive-pressure lung inflation device, which was put to use at the Massachusetts General Hospital, and became an instant success. Thus began the era of positive pressure mechanical ventilation (and the era of intensive care medicine).

The fundamental operation of positive pressure ventilation is to create a pressure that moves a volume of gas into the lungs. There are two general methods of positive pressure ventilation, which are summarized below and illustrated in Figure 25.1.
1. Volume-controlled ventilation, where the inflation volume (tidal volume) is preselected, and the ventilator automatically adjusts the inflation pressure to deliver the desired volume. The rate of lung inflation can be constant (as in Figure 25.1) or decelerating.
2. Pressure-controlled ventilation, where the inflation pressure is preselected, and the duration of inflation is adjusted (by the operator) to deliver the desired tidal volume. The rate of lung inflation is high at the onset of lung inflation (to achieve the desired inflation pressure), then rapidly decelerates (to maintain a constant inflation pressure).
The uses, advantages, and disadvantages of these modes of ventilation are described in the next chapter. This section will describe the measurements that are used to evaluate the mechanical properties of the lungs and chest wall (i.e., airflow resistance and distensibility) with each method of ventilation.
During positive pressure ventilation, pressures in the thorax are monitored at the level of the endotracheal tube or ventilator These proximal airway pressures can differ from pressures at the level of the alveoli (as described next).

The proximal airway pressure at the end of a lung inflation has different interpretations for volume-cycled and pressure-cycled ventilation.

During volume-controlled ventilation, the pressure in the airways rises steadily until the preselected volume is delivered. The airway pressure (Paw) at the end of each lung inflation, the peak airway pressure (Ppeak), is the pressure needed to overcome both resistive and elastic forces in the lungs and chest wall (Pres and Pel, respectively).
Ppeak = Pres + Pel
(25.1)
FIGURE 25.1 Waveforms for inflation (tidal) volume, airway pressure, and flow rate for volume-controlled and pressurecontroled ventilation. See text for explanation.
Pres is a function of the resistance to flow in the airways (R), and the inspiratory flow rate [Pres = RxV (insp)], while Pel is a function of the elastic recoil force of the lungs and chest wall, and the lung volume (Pel = elastancexV). The resistive component of the peak airway pressure can be eliminated by eliminating airflow. This is accomplished by occluding the expiratory circuit at the very end of lung inflation, to prevent the patient from exhaling. During this "inflationhold" maneuver (which typically lasts for one second), the pressure in the airways decreases initially and then remains constant until the occlusion is released and the patient is allowed to exhale. This is illustrated in Figure 25.2. The steady occlusion pressure, which is called the plateau pressure, is the peak pressure in the alveoli (Palv) at the end of inspiration.
The difference between the plateau pressure and the level of positive end-expiratory pressure (PEEP) is the pressure needed to overcome the elastic recoil forces of the lungs and chest wall (Pel).
Pplateau —-PEEP = Pel
(25.3)
FIGURE 25.2 Airway pressure profile for a constant-flow, volume-controlled lung inflation with a brief end-inspiratory occlusion (inflation-hold). Ppeak is the peak airway pressure, Pplateau is the end-inspiratory occlusion pressure, Palv (peak) is the peak alveolar pressure at end-inspiration, Pres is the pressure attributed to airway resistance, and Pel is the pressure attributed to the elastic recoil force of the lungs and chest wall. See text for explanation.

The difference between the peak and plateau pressures represents the pressure needed to overcome the resistance to airflow at any given inspiratory flow rate.
Ppeak -Pplateau = Pres
(25.4)

During pressure-controlled ventilation, there should be no airflow at the end of inspiration, and the end-inspiratory airway pressure (Paw) will be equivalent to the peak alveolar pressure at the end of inspiration.
Paw (end-insp) = Palv (peak)
(25.5)
The change in pressure attributed to the elastic forces of the lungs and chest wall is the pressure difference between the end-inspiratory Paw and PEEP:
Paw (end-insp) — PEEP = Pel
(25.6)
(Note: These relationships are valid only when there is no airflow at the end of inspiration. Inspiratory flow may not return to zero at end-inspiration in patients who are breathing rapidly; in this situation, the end-inspiratory airway pressure will be higher than alveolar pressure. )
Since inspiratory flow is not constant during pressure-controlled ventilation, it is not possible to evaluate airway resistance during lung inflation. Resistance to expiratory flow
(described later) can be used to evaluate airways resistance during pressure-controlled ventilation.

The end-expiratory pressure is the minimum pressure in the alveoli (not airways) during a ventilatory cycle. The different forms of end-expiratory pressure are illustrated in Figure 25.3.
FIGURE 25.3 End-expiratory airway pressures. See text for explanation.

During appropriate ventilation in the normal lung, there is no airflow at the end of expiration, and the pressure in the alveoli is equivalent to atmospheric pressure. Since atmospheric pressure is a zero reference point for breathing, this condition is called zero end-expiratory pressure, or ZEEP.

Positive end-expiratory pressure (PEEP) can be added through the ventilatory circuit (via a pressure-sensitive valve in the expiratory limb of the circuit) so that exhalation will cease when the airway pressure falls to the preselected PEEP level. Applied PEEP is used routinely during mechanical ventilation to prevent the collapse of distal airspaces at the end of expiration, and to open collapsed alveoli (recruitment). This is described in the next chapter.

When there is continued airflow at the end of expiration, the lungs do not completely empty, and the alveolar pressure remains positive even though the proximal airway pressure falls to atmospheric (zero) pressure. This pressure is sometimes called intrinsic PEEP or auto-PEEP, but occult PEEP seems more appropriate because the PEEP is not apparent in proximal airway pressure recordings (2). Occult PEEP can be the result of dynamic hyperinflation in patients with asthma and COPD, as described in Chap-ter 24 (see pages 477-480), or it can be the result of ventilator settings that predispose to endexpiratory airflow (e.g., high inflation volumes, decreased time for exhalation).
Occult PEEP can be detected on the flow tracing by noting the presence of airflow at the end of expiration, as illustrated in Figure 24.5 (see page 479). If occult PEEP is present, it can be quantified by occluding the expiratory circuit at the end of expiration. During an end-expiratory occlusion, alveolar pressure will equilibrate with proximal airway pressure, and the occult PEEP becomes apparent as a abrupt increase in airway pressure, as shown in Figure 25.3. Occult PEEP is described in more detail in Chapter 28.

The mean airway pressure is the average pressure in the airway during the ventilatory cycle, and is influenced by several variables, including the peak airway pressure, the contour of the pressure waveform, the PEEP level, the respiratory rate, and the inflation time relative to the total time of the ventilatory cycle (T;/Tiot). The mean airway pressure that is displayed by ventilators is obtained by integrating the area under the airway pressure waveform.
The mean airway pressure is linked to the hemodynamic effects of positive pressure ventilation. (The intrapleural pressure is the important influence on cardiac function, but this pressure is measured with an intraesophageal balloon, and is not monitored routinely.) Typical values for mean airway pressure during positive pressure ventilation are 5-10 cm H20 for normal lungs, 10-20 cm H20 for airflow obstruction, and 20-30 cm H>0 for noncompliant (stiff) lungs (3).

Compliance (Avolume/Apressure) is the reciprocal of elastance, and is the traditional term used to express the elastic properties of structures with chambers (like the heart and lungs). Compliance expresses distensibility, or the tendency of a chamber to increase in volume when exposed to a given distending pressure. The compliance that is measured during mechanical ventilation is the thoracic compliance, and includes both the lungs and the chest wall.

During volume-controlled ventilation, the static compliance of the thorax (Cstat) is expressed as the preselected tidal volume (V+) divided by the difference between the plateau pressure and the total PEEP level (applied plus occult PEEP):
Cstat = V_ / [Pplateau -PEEP(tot)]
(25.7)
This is a "static" compliance because the pressures involved are measured in the absence of airflow. In patients with normal lungs, Cstat is 50-80 mL/cm H>20 (4), and in patients with infiltrative lung diseases (e.g., pulmonary edema or acute respiratory distress syndrome), Cstat is typically <25 mL/cm H20 (1).

Compliance measurements are difficult during pressure-controlled ventilation because: (a) there must be no inspiratory flow at the end of inspiration (which is not always the case), and (b) the tidal volume varies with changes in airway resistance and thoracic compliance during pressure-controlled ventilation. Under ideal conditions (i.e., no airflow at end-inspiration and stable lung mechanics) Cstat is equivalent to the exhaled tidal volume (exhaled Vy) divided by the difference between the end-inspiratory airway pressure (Paw) and the total PEEP level:
Cstat = Exhaled Vz /[Paw(end-insp) — PEEP(tot)]
(25.8)

1. During passive ventilation, the chest wall can account for 35% of the total thoracic compliance (5,6), and this contribution increases when the chest wall muscles contract. Therefore, to avoid interference from respiratory muscle contraction, static compliance measurements should be performed only in patients who are not actively breathing.
2. Thoracic compliance is volume-dependent; i.e., it decreases as lung volume increases. Absolute lung volumes cannot be measured during mechanical ventilation. However, serial measurements of Cstat should be performed at the same tidal volume(s).
3. The tidal volume used for the compliance measurement should be adjusted for the compliance of the ventilator tubing, which is typically
3 mL/cm H2O (1). For example, if the preselected tidal volume during volume-controlled ventilation is 500 mL and the peak airway pressure is 40 cm H>0, then 3x40 = 120 mL
of the delivered volume will be lost to expansion of the ventilator tubing, and the actual tidal volume reaching the patient will be 500 — 120 = 380 mL. When using exhaled tidal volumes, the peak alveolar pressure at end-inspiration should be used for the volume adjustments.

Normal breathing is accomplished by pulling air into the lungs, and as much as 5 liters of air can be drawn in during a single breath (from residual volume to total lung capacity) without noticeable injury to the lungs. On the other hand, positive pressure ventilation pushes air into the lungs, and this push creates abnormal stresses and strains that can damage the architecture of the lungs, particularly diseased lungs, with single-breath volumes of less than one-half liter (7,8). An example of the structural damage produced by mechanical ventilation is shown in Figure 25.4 (9).
There are several types of ventilator-induced lung injury (VILI), as described in this section. Since VILI has been studied primarily in patients with acute respiratory distress syndrome (ARDS), a brief description of VILI is also included in the chapter describing ARDS (see pages 454-455).

In the early days of positive pressure ventilation, large tidal volumes were adopted to prevent atelectasis (10). Tidal volumes of 10 to 15 mL/kg (ideal body weight) became standard during mechanical ventilation, compared to normal tidal volumes of 5 to 7 mL/kg during spontaneous breathing. From the outset, it was clear that mechanical ventilation could rupture alveoli and produce air leaks (barotrauma), but in the 1970s, a study was published showing that high inflation pressures could produce diffuse pulmonary infiltration that resembles pulmonary edema (11). This was followed by a landmark study in the 1980s showing that high inflation volumes, rather than high inflation pressures, were responsible for the lung injury produced by mechanical ventilation (12). The results of this study are shown in Figure 25.5. Note that marked increases in extravascular lung water occurred only when inflation volumes were high, regardless of the inflation pressure. As a result of this study, the term volutrauma was adopted to describe the underlying mechanism for the pulmonary infiltration produced by mechanical ventilation.
The lung injury in VILI is not watery pulmonary edema, but is the result of overdistension of alveoli and disruption of the alveolar-capillary interface, leading to inflammatory infiltration of the lungs and a clinical condition that resembles ARDS (13).

The effects of high inflation volumes are more marked in infiltrative lung diseases like pneumonia and ARDS because the inflation volumes are distributed preferentially to regions of normal lung function. In this situation, the high inflation volumes are being delivered to lungs with a decrease in functional lung volume (see Figure 23.4 on page 454), and this predisposes to volutrauma from overdistension of alveoli in normal lung regions.
FIGURE 25.4 Electron micrographs showing a tear at the alveolar-capilary interface attributed to alveolar overdistension during mechanical ventilation. Post mortem specimen from a patient with ARDS. Scales shown in lower right corner of each image. Images from Reference 7, and digitally retouched.
FIGURE 25.5 Effects of high pressures and high volumes on extravascular lung water (EVLW) during positive pressure ventilation. Height of the bars represents mean values, and hatched bars represent standard deviations of the mean. Dotted line represents the upper 95% limit of EVLW measurements obtained during ventilation at normal pressures and volumes. Data from Reference 12.

Concern for the risk of lung injury during conventional, high-volume ventilation prompted clinical studies evaluating lower tidal volumes during mechanical ventilation, mostly in patients with ARDS. The largest study to date included over 800 patients with ARDS (14), and compared ventilation with tidal volumes of 6 mL/kg and 12 mL/kg using predicted body weight (which is the weight at which lung volumes are normal). Ventilation with low tidal volumes was associated with a shorter duration of mechanical ventilation and 9% (absolute) reduction in mortality. As a result of this study (and other corroborating studies), a low-volume ventilation protocol known as lung protective ventilation is now recommended for all patients with ARDS. This protocol is presented later in the section.

During mechanical ventilation, small airways tend to collapse by the end of expiration, particularly in pulmonary conditions associated with reduced lung compliance (e.g., pulmonary edema, ARDS). Repetitive opening and closing of small airways during positive pressure ventilation can damage the airway epithelium, possibly by generating excessive shear forces (15). This type of lung injury is called atelectrauma (8), and it can be mitigated by the use of positive end-expiratory pressure (PEEP), which provides a pressurized "stent" to keep the small airways open during expiration.

The most intriguing form of VILI is the ability of positive pressure ventilation to promote proinflammatory cytokine release from the lungs at inflation volumes that do not produce structural damage in the lungs (16). This is called biotrauma, and it can trigger the systemic inflammatory response syndrome, and can lead to inflammatory injury in the lung as well as other organs. This means that mechanical ventilation can be a source of inflammatory-mediated multiorgan failure (17), like that seen in severe sepsis and septic shock. This is not good.

Positive pressure ventilation can also produce air leaks from a rupture in the airways and distal airspaces. The air that escapes can enter the pleural space (pneumothorax), or it can move along' the bronchovascular bundles' into the mediastinum (pneumomediastinum) and up into the subcutaneous — tissues (subcutaneous emphysema), or it can track down the mediastinum and enter the peritoneal cavity (pneumoperitoneum). This form of VILI is called barotrauma, even though the source of injury can be high alveolar volume (i.e., volutrauma).

1. Calculate patient's predicted body weight (PBW):
Males: PBW=50 + [2.3 x (height in inches — 60] Females: PBW = 45.5 + [2.3 x (height in inches — 60]
2, Use volume-controlled ventilation and set initial tidal volume (V4) to 8 mL/kg (PBW).
3, Set respiratory rate (RR) to match baseline minute ventilation, but not > 35 bpm.
4, Set positive end-expiratory pressure (PEEP) at 5 cm H,0.
5. Reduce V7 by 1 mL/kg every 1 to 2 hours until V7 = 6 mL/kg (PBW)
6. Adjust PEEP and FiQ; to maintain SpO, of 88-95%.

- 1. If Ppl >30 cm H,O and V; at 6 mL/kg, decrease Vj In 1 mL/kg increments until Ppl falls to =<30 cm H,0 or Vy reaches a minimum of 4 mL/kg.

1. If PH = 7.15-7.30, increase RR until pH =7.30, PaCO, <25 mm Hg, or AR = 85 bpm.
2. If pH <7.15, increase RR to 35 bpm. If pH remains <7.15, increase in V7 In 4 mL/kg increments until pH >7.15 (Ppl target may be exceeded).
3. If pH >7.45, decrease AR, if possible.
Adapted fram the protocol devaloped by the ARDS Clirica Network, available at wavw.ardsnet.org.
THE PLATEAU PRESSURE: As mentioned in Chapter 23, clinical studies have not shown a consistent survival benefit with lung protective ventilation in ARDS, and this discrepancy appears to be related to the plateau pressure; i.e., a survival benefit with low-volume, lung protective ventilation is demonstrated only when the plateau pressure is above 30 cm HO during conventional, high-volume (10-15 mL/kg) ventilation (19). Therefore, a plateau pressure <30 cm H 0 seems more important than a low tidal volume for protecting the lungs from ventilator-induced injury.
A STANDARD PRACTICE?: Although lung protective ventilation is recommended only for patients with ARDS, there are reports that VILI can occur during conventional ventilation in patients who do not have ARDS (20), and that lung protective ventilation is associated with improved outcomes in non-ARDS patients (21). These observations, combined with the strong physiological basis for lung protective ventilation, should lead to the adoption of lung protective ventilation for all patients who require mechanical ventilation.

The influence of positive pressure ventilation on cardiac performance is complex, and involves the preload and afterload forces for the right and left sides of the heart (22). These forces are described in Chapter 9.

Positive pressure ventilation can reduce ventricular filling (preload) in several ways, and these are indicated in Figure 25.6. First and foremost, positive intrathoracic pressure decreases the pressure gradient for venous inflow into the thorax (although positivepressure ventilation also increases intra-abdominal pressure, which tends to maintain the pressure gradient for venous inflow into the thorax). Second, positive pressure on the outer surface of the heart decreases the transmural pressure during diastole, and this will reduce ventricular filling. Finally, positive pressure ventilation increases pulmonary vascular resistance, and this can impede right ventricular stroke output and hence impede left ventricular filling. In this situation, the right ventricle can dilate and push the interventricular septum toward the left ventricle, and this reduces left ventricular chamber size and further reduces left ventricular filling. This phenomenon, known as ventricular interdependence, is one of the mechanisms whereby right heart failure can impair the performance of the left side of the heart (see Fig. 13.4 on page 247).
FIGURE 25.6 The mechanisms for a decrease in ventricular filing (preload) during positive pressure ventilation.

Left ventricular afterload is a function of the peak transmural wall pressure during systole (see Figure 9.7 on page 161), which is the pressure that ventricular contraction must overcome to eject the stroke volume. Positive intrapleural pressure decreases this transmural pressure, and thereby decreases left ventricular afterload. (Conceptually, positive intrapleural pressure acts like a hand that squeezes the ventricles during systole.) Therefore, positive pressure ventilation reduces ventricular afterload, and this effect can augment cardiac output in certain situations (see next).

The overall effect of positive pressure ventilation on cardiac output is determined by the balance between the decrease in ventricular preload (which reduces cardiac output) and the decrease in ventricular afterload (which increases cardiac output) during positive pressure ventilation. This balance is determined by cardiac function, intravascular volume, and intrathoracic pressure.

The curves in Figure 25.7 show the superimposed influence of preload and afterload on cardiac output in the normal and failing heart. The normal heart operates on the steep portion of the preload curve and the flat portion of the afterload curve. In this situation, a decrease in preload has a greater influence on cardiac output than a decrease in afterload (as indicated by the arrows), so cardiac output is expected to decrease during positive pressure ventilation. However, as mentioned earlier, positive pressure ventilation also increases intra-abdominal pressure (by pushing the diaphragm down into the abdomen), and this serves to maintain the venous inflow into the thorax. As a result, the decrease in venous return (preload) can be minimal during positive pressure ventilation, and the cardiac output can decrease slightly, show no change, or even increase slightly (from the afterload reducing effect).
In contrast, the failing heart operates on the flat portion of the preload curve and the steep portion of the afterload curve. In this situation, the decrease in afterload during positive pressure ventilation has a greater influence on cardiac output than the decrease in preload, and cardiac output will increase during positive pressure ventilation. This is the basis for the use of positive pressure breathing as a "ventricular assist" maneuver in patients with advanced heart failure (23,24).

When the intravascular volume is reduced, the preload-reducing effect of positive pressure ventilation predominates in both the normal and failing heart, and cardiac output will decline (22). This is particularly the case when intrathoracic pressures are high during mechanical ventilation. Maintaining a normal or adequate intravascular volume is essential during positive pressure ventilation to avoid significant deficits in cardiac output.
Afterload
FIGURE 25.7 Superimposed curves for the relationship between preload, afterload, and cardiac output in the normal and failing heart. Arrows indicate the effects of positive pressure ventilation.

For the first 30 years after its introduction, positive pressure ventilation was dominated by large inflation volumes and high pressures, but this has changed since the discovery that pushing air into the lungs creates stresses and strains that damage the lungs, and can lead to damage in other organs as well. The emphasis now is on limiting inflation volumes and preventing overdistension of alveoli. This "less is best" strategy is currently advised only for patients with ARDS, but it seems likely that it will (and should) be adopted for all patients who require positive pressure ventilation.

Papadakos PJ, Lachmann B, eds. Mechanical ventilation: clinical applications and pathophysiology. Philadelphia: Saunders, Elsevier, 2008.

1. Bekos V, Marini JJ. Monitoring the mechanically ventilated patient. Crit Care Clin 2007; 23:575-611.
2. Pepe P, Marini JJ. Occult positive end-expiratory pressure in mechanically ventilated patients with airflow obstruction. The auto-PEEP effect. Am Rev Respir Dis 1982; 126:166-170.
3. Hess DR, Kacmarek RM. Basic pulmonary mechanics during mechanical ventilation. In: Essentials of Mechanical Ventilation. New York, McGraw-Hill, 1996:171-176.
4. Tobin MJ. Respiratory monitoring. JAMA 1990; 264:244—-251.
5. Marini JJ. Lung mechanics determinations at the bedside: instrumentation and clinical application. Respir Care 1990; 35:669-696.
6. Katz JA, Zinn SE, Ozanne GM, Fairley BB. Pulmonary, chest wall, and lung— thorax elastances in acute respiratory failure. Chest 1981; 80:304—311.

22. Singh I, Pinsky MR. Heart-lung interactions. In Papadakos PJ, Lachmann B, eds. Mechanical ventilation: clinical applications and pathophysiology. Philadelphia: Saunders Elsevier, 2008:173-184.
23. Yan AT, Bradley TD, Liu PP. The role of continuous positive airway pressure in the treatment of congestive heart failure. Chest 2001; 120:1675—-1685.
24. Boehmer JP, Popjes E. Cardiac failure: Mechanical support strategies. Crit Care Med 2006; 34(Suppl):S268-S277.

Development in most fields of medicine appears to occur according to sound scientific principles. However, exceptions can be found, and the development of mechanical ventilatory support is one of them.
J. Rasdanen, MD
A recently published textbook on respiratory care equipment lists 174 specific methods of positive pressure ventilation (1), yet in the 50-plus years since positive pressure ventilation was introduced, only one method has improved clinical outcomes (2 ), and only because it reduces the lung damage produced by positive intrathoracic pressures. What this means is that positive pressure ventilation is much more complicated than it needs to be (3).
This chapter describes six basic methods of positive pressure ventilation (volume control, pressure control, assist-control, pressure support, intermittent mandatory ventilation, and positive end-expiratory pressure). Although this is far less than the bloated number of available methods, these six methods should be all that is needed to provide effective ventilatory support in a majority of patients.

As introduced in the last chapter, there are two basic methods of positive-pressure lung inflation: (a) volume control, where the inflation volume is constant, and (b) pressure control, where the inflation pressure is constant (1). The changes in pressure and flow that occur with each type of ventilator breath are shown in Figure 26.1.
FIGURE 26.1 Pressure and flow changes during a single ventilator breath using volume control and pressure control methods of lung inflation at equivalent inflation (tidal) volumes. Changes in airway pressure (Paw) are indicated by the solid lines, and changes in alveolar pressure (Palv) are indicated by the dashed lines. I = inspiration, E = expiration.

With volume control ventilation (VCV), the inflation (tidal) volume is preselected, and the lungs are inflated at a constant flow rate until the desired volume is delivered. Because there is airflow at the end of inspiration, the peak pressure in the proximal airways (peak Paw) is greater than the peak pressure in the alveoli (peak Palv); and the difference (peak Paw — peak Palv) is the pressure dissipated by the resistance to flow in the airways. The peak alveolar pressure is a reflection of the alveolar volume at the end of lung inflation.

CONSTANT TIDAL VOLUME: The major advantage of VCV is the ability to deliver a constant tidal volume despite changes in the mechanical properties of the lungs. When airways resistance increases or lung compliance decreases, the ventilator generates a higher pressure to deliver the preselected volume. This maintains the desired minute ventilation in the face of abrupt or undetected changes in airway resistance or lung compliance.

AIRWAY PRESSURE: At any given tidal volume, the airway pressures at the end of inspiration are higher during VCV than during pressure control ventilation (PCV), as shown in Figure 26.1, and this is mistakenly perceived as an increased risk of ventilator-induced lung injury. However, the risk of ventilator-induced lung injury is related to the peak alveolar pressure (2,4), and this pressure is the same during VCV and PCV (at equivalent tidal volumes). Therefore, the higher peak airway pressures during volume-controlled ventilation do not increase the risk of alveolar overdistension and ventilator-induced lung injury.
INSPIRATORY FLOW: There are disadvantages related to the constant inspiratory flow rate during VCV. First, the duration of inspiration is relatively short, and this can lead to uneven alveolar filling. In addition, the maximum inspiratory flow is limited when flow is constant, and the inspiratory flow rate can be inadequate in patients with high flow demands, resulting in patient distress. A decelerating flow pattern is available for VCV, and it has been shown to improve patient comfort VCV (5).

With pressure control ventilation (PCV), the desired inflation pressure is preselected, and a decelerating inspiratory flow rate provides high flows at the onset of the lung inflation, to attain the desired inflation pressure quickly. The inspiratory time is adjusted to allow enough time for the inspiratory flow rate to fall to zero at the end of inspiration. Since there is no airflow at the end of the inspiration, the end-inspiratory airway pressure is equivalent to the peak alveolar pressure (see Figure 26.1).

ALVEOLAR PRESSURE: The major benefit of PCV is the ability to control the peak alveolar pressure, which is the pressure most closely related to the risk of alveolar overdistension and ventilator-induced lung injury. Clinical studies indicate that the risk of ventilator-induced lung injury is negligible if the peak alveolar pressure is <30 cm H20 (2,4 ). (Note: Maintaining a peak alveolar pressure <30 cm H30 is also possible during VCV by monitoring the end-inspiratory occlusion pressure or "plateau pressure," which is described on pages 489-490.)
PATIENT COMFORT: PCV is more likely to promote patient comfort than VCV (6), and this has been attributed to the high initial flow rates and the longer duration of inspiration with PCV.

ALVEOLAR VOLUME: The major disadvantage of PCV is the decrease in alveolar volume that occurs when there is an increase in airway resistance or a decrease in lung compliance. This is a particular concern in acute respiratory failure, where steady-state conditions of airway resistance and lung compliance are unlikely.

Pressure-regulated, volume control ventilation (PRVC) is a hybrid mode of ventilation that provides a constant tidal volume (like volume control) but limits the end-inspiratory airway pressures (like pressure control). PRVC operates like an intelligent form of volume control; i.e., the ventilator monitors lung compliance, and uses these measurements to select the lowest airway pressure needed to deliver the desired tidal volume. Hybrid modes like PRVC are gaining in popularity, although there is no documented clinical advantage with PRVC when compared to more conventional modes of ventilation.

Assist-control ventilation (ACV) allows the patient to initiate a ventilator breath (assisted or patient-triggered ventilation), but if this is not possible, ventilator breaths are delivered at a preselected rate (controlled or time-triggered ventilation). The ventilator breaths during ACV can be volume-controlled or pressure-controlled.
FIGURE 26.2 Airway pressure patterns in assist-control ventilation (ACV) and synchronized intermittent mandatory ventilation (SIMV),. See text for further explanation.

A general rule of thumb during mechanical ventilation is to allow at least twice the amount of time for expiration as allowed for inspiration. This is equivalent to an inspiration:expiration time ratio (I:E ratio) of at least 1:2. The goal is to allow enough time for complete exhalation to prevent dynamic hyperinflation and intrinsic or occult PEEP (see page 491). If the duration of exhalation is too short, the I:E ratio can be increased by: (a) increasing the inspiratory flow rate, (b) reducing the tidal volume, or (c) decreasing the inspiratory time (for pressure control).

When each breath is a patient-triggered ventilator breath, rapid breathing like that shown in Figure 26.2 can severely curtail the time for exhalation and increase the risk of incomplete alveolar emptying and intrinsic PEEP. When rapid breathing is the result of a condition other than discomfort or anxiety, attempts to reduce the respiratory rate with sedation or inspiratory flow adjustments are often unsuccessful. In this situation, the appropriate mode of ventilation is described next.

Difficulties with rapid breathing during ACV in neonates with respiratory distress syndrome, who typically have respiratory rates above 40 breaths/minute, led to the introduction of intermittent mandatory ventilation (IMV). IMV is designed to allow spontaneous breathing between ventilator breaths. (Spontaneous breathing is the condition where the patient initiates and terminates each lung inflation.) This is accomplished by placing a spontaneous breathing circuit in parallel with the ventilator circuit with a unidirectional valve that opens the spontaneous breathing circuit when a ventilator breath is not being delivered. The ventilatory pattern for IMV is illustrated in the lower panel of Figure 26.2. Note that the ventilator breath is delivered in synchrony with the spontaneous breath; this is called synchronized IMV (SIMV).
The ventilator breaths during SIMV can be volume-controlled or pressure-controlled. The rate of ventilator breaths is adjusted as needed to match the total minute ventilation (spontaneous plus assisted breaths) to the patient's baseline level.

The principal adverse effects of IMV are: (a) an increased work of breathing, and (b) a decrease in cardiac output, primarily in patients with left ventricular dysfunction. Both effects are the result of the spontaneous breathing period.

The heightened work of breathing during the spontaneous breathing period in IMV is attributed to resistance in the ventilator circuit, which can have an exaggerated effect on ventilatory work when breathing is rapid (the condition that usually prompts the use of IMV). Pressure-support ventilation (described in the next section) overcomes the added resistance of the ventilator circuit, and thereby reduces the work of breathing (10). As a result, pressure-support ventilation (at 10 cm HO) is now used routinely during the spontaneous breathing periods in IMV.

As described at the end of the last chapter, positive pressure ventilation reduces left ventricular afterload and increases cardiac output, primarily in patients with left ventricular dysfunction (see Figure 25.7 on page 501) (11). IMV has the opposite effect and increases left ventricular afterload (during the spontaneous breathing periods), which results in a decrease in cardiac output in patients with left ventricular dysfunction (12).

The major indication for IMV is rapid breathing with incomplete exhalation during assistcontrol ventilation. The spontaneous breathing periods during IMV promote alveolar emptying and reduce the risk of air trapping and intrinsic PEEP. IMV can increase the work of breathing and impair cardiac output in patients with left ventricular dysfunction; as a result, IMV is not advised for patients with respiratory muscle weakness or left heart failure.

Pressure support ventilation (PSV) is pressure-augmented spontaneous breathing (13). It differs from patient-triggered PCV because it allows the patient to terminate the lung inflation, whereas the ventilator terminates the lung inflation during patient-triggered PCV. Therefore, PSV is a more interactive form of ventilation than PCV because it allows the patient to control the inspiratory time and tidal volume.

The changes in pressure, and flow during a lung inflation with PSV are shown in Figure 26.3. PSV uses a decelerating inspiratory flow rate, with high flow rates early in inspiration to achieve the desired pressure level. The pressure-augmented breath is terminated when the inspiratory flow rate falls to 25% of the peak level. This allows the patient to determine the duration of lung inflation and the tidal volume.
FIGURE 26.3 Changes in pressure and flow during a singk lung inflation with pressure support ventilation. The lung inflation is terminated when the inspiratory flow rate falls to 25% of the peak flow rate, which allows the patient to determine the inspiratory time and tidal volume.

Low levels of PSV (5-10 cm H,0) can be used during weaning from mechanical ventilation, to overcome the resistance to flow in the artificial airways and ventilator tubing. The goal of PSV in this setting is to reduce the work of breathing without augmenting the tidal volume (14). Higher levels of PSV (15-30 cm H20) can be used to augment the tidal volume; in this case, PSV is used as a form of noninvasive ventilation (which is described in the next chapter) (15).

Progressive narrowing of the airways during expiration can culminate airspaces (small airways and alveoli) at the end of dependent lung regions, where transpulmonary pressures effect). The transpulmonary pressure where the distal called the closing pressure, and is normally about 3 cm H 2 higher in the presence of small airway obstruction (e.g., compliance (e.g., in ARDS), and this results in more expiration. This has two adverse consequences: (a) atelectasis, and (b) atelectrauma from repetetive, 1 = in collapse of distal expiration. This normally occurs in are more positive (gravitational airspaces begin to collapse, is O (16). The closing pressure is in COPD) and reduced lung extensive airspace collapse at end- impaired gas exchange from closing and opening of the distal. The risk of alveolar collapse at pressure from falling below the closing by creating a positive pressure in the to the closing pressure. This positive pressure-relief valve in the expiratory to proceed until the pressure falls to maintained for the duration of expiration. pressure in the clinical setting, a PEEP collapse in all patients., 1 = end-expiration can be eliminated by preventing the airway pressure at the end of expiration. This is achieved airways at the end of expiration that is equivalent end-expiratory pressure (PEEP) is created by a limb of the ventilator circuit that allows exhalation the preselected level, and this pressure is then Since it is not possible to identify the closing level of 5-7 cm H 2 O is used to prevent alveolar

The influence of PEEP on inflation pressures is illustrated in Figure 26.4. Note that the addition of PEEP moves the inflation pressure waveform upward, and results in a higher peak alveolar pressure and a higher mean airway pressure. The following statements are relevant in this regard.
1. The effects of PEEP are not related to the PEEP level, but are determined by the influence of PEEP on the peak alveolar pressure and the mean airway pressure.
2. The change in peak alveolar pressure determines the influence of PEEP on alveolar ventilation (and hence arterial oxygenation), and also determines the risk of alveolar overdistension and volutrauma (see page 494).
3. The change in mean airway pressure determines the influence of PEEP on cardiac output.
FIGURE 26.4 Airway pressure waveforms during pressure control ventilation showing the effects of positive endexpiratory pressure (PEEP) on the peak alveolar pressure (Palv) and the mean airway pressure (Paw).

While low levels of PEEP (5-10 cm H>0) help to prevent the collapse of distal airspaces, high levels of PEEP (20-30 cm H20) help to reopen distal airspaces that are persistently collapsed. This effect is demonstrated in Figure 26.5 (19). The thoracic CT image on the left shows consolidation in the posterior (dependent) regions of both lungs, and this disappears after the addition of PEEP (at 19 cm H 0). This effect is known as alveolar recruitment, and it increases the available surface area in the lungs for gas exchange (19,20).

The addition of PEEP may not result in alveolar recruitment, but instead can overdistend alveoli in normal lung regions, which increases the risk of ventilator-induced lung injury. The volume of "recruitable lung" (i.e., areas of atelectasis that can be aerated) determines if PEEP will promote alveolar recruitment or alveolar overdistension; i.e., if there is a significant volume of recruitable lung, then PEEP will promote alveolar recruitment and improve pulmonary gas exchange, but if there is a negligible volume of recruitable lung, then PEEP will promote alveolar overdistension and increase the risk of ventilator-induced lung injury.
The volume of recruitable lung varies widely in individual patients (from 2% to 25% of total lung volume) (20). Although it is not possible to measure recruitable lung volume reliably, the following measures can be used to determine if PEEP is promoting alveolar recruitment (favorable response) or promoting alveolar overdistension (unfavorable response).
LUNG COMPLIANCE: When PEEP is promoting alveolar recruitment, the compliance (distensibility) of the lungs will increase, but when PEEP is overdistending alveoli in normal lung regions, lung compliance will decrease. The measurement of lung compliance during mechanical ventilation is described in Chapter 25 (see pages 492-493).
PAO2/FIO2 RATIO: The relationship between the arterial PO) and the fractional concentration of inspired oxygen, as expressed by the PaO>/FIO> ratio, is a measure of the efficiency of gas exchange in the lungs. When PEEP promotes alveolar recruitment, the PaO>/FIO> ratio will increase, and when PEEP does not promote alveolar recruitment, the PaO>/FIO> ratio will remain unchanged or will decrease. An example of a beneficial response in the PaO>/FIO> ratio (indicating alveolar recruitment) is shown in Figure 26.6.

The beneficial effects of PEEP on arterial oxygenation may not be accompanied by a similar benefit in systemic oxygen transport, as described next.

The influence of positive pressure ventilation on cardiac performance is described at the end of Chapter 25 (see pages 499-501). PEEP magnifies these effects, particularly the tendency for positive pressure ventilation to decrease cardiac output. There are several mechanisms whereby PEEP can decrease cardiac output, including (a) impaired venous return, (b) decreased ventricular compliance, (c) increased right ventricular afterload, and (d) external constraint of the ventricles (21,22). These effects are more prominent in the presence of hypovolemia.

PEEP-induced decreases in cardiac output can erase the beneficial effects of PEEP on alveolar recruitment. This is illustrated by the following equation for systemic oxygen delivery (DO2). (see page 178 for the derivation of this equation. )
DO, = CO « (1.34 x [Hb] x 5a0,) x 10 (mL/min)
(26.1)
Thus, PEEP can promote alveolar recruitment and increase arterial oxygenation (SaO>), but systemic O> delivery may not improve if PEEP also decreases the cardiac output (CO). The opposing effects of PEEP on arterial oxygenation and cardiac output are demonstrated in Figure 26.6 (23).
BEST PEEP: The optimal benefit from PEEP occurs when an increase in arterial oxygenation is accompanied by an increase in systemic O> delivery. Therefore, the optimal or best PEEP for an individual patient is the level of PEEP that results in the greatest increase in systemic O> delivery (24). Best PEEP can be determined by measuring the systemic O, delivery at increasing levels of PEEP. Unfortunately, the decline in popularity of the pulmonary artery catheter (which provides the most reliable measurements of cardiac output) has also decreased the popularity of best PEEP determinations.
FIGURE 26.6 The opposing effects of positive end-expiratory pressure (PEEP) on arterial oxygenation (PaO7/FIO2) and cardiac index in patients with ARDS. Data from Reference 23.

When mechanical ventilation is initiated, the respiratory therapist will ask you for the following parameters: (a) mode of ventilation, (b) tidal volume, (c) respiratory rate, (d) PEEP level, and (e) inspired Oz concentration. The following is a list of suggestions for setting up mechanical ventilation.

1. Select assist-control as the initial mode of ventilation.
2. It may be necessary to switch to synchronized IMV in patients who are breathing too rapidly in the assist-control mode (see later).

personal
1. The use of volume control or pressure control is largely a matter of preference, although some patients breathe more comfortably with pressure control.
2. A suitable compromise is the pressure-regulated volume control mode of ventilation (PRVC), which allows for control of the tidal volume, but limits the airway pressures.

1. Select an inspiratory flow rate of 60 L/min if the patient is breathing quietly or has no spontaneous respirations.
2. Use higher inspiratory flow rates (e.g., =80 L/min) for patients with respiratory distress or a high minute ventilation (=10 L/min).

1. The I:E ratio should be =1:2.
2. If the I:E ratio is <1:2, the options for increasing the I:E ratio include: (a) increasing the inspiratory flow rate, (b) decreasing the tidal volume, or (c) decreasing the respiratory rate, if possible.

1. Set the initial PEEP to 5 cm H,0 to prevent the collapse of distal airspaces at endexpiration.
2. Further increases in PEEP may be required if either of the following conditions is present: (a) a toxic level of inhaled oxygen (>60%) is required to maintain adequate oxygenation (SaOz =90%), or (b) hypoxemia is refractory to oxygen therapy.

Considering that the human capacity to store and process information is limited to 4 variables (25), the 174 documented modes of ventilatory assistance mentioned in the introduction represents an incomprehensible load of information. What seems to be lost in this maze of technology is the simple fact that mechanical ventilation is a temporary support measure for acute respiratory failure, and is not a treatment modality for lung disease. Therefore, if we want to improve outcomes in ventilator-dependent patients, less attention should be given to the knobs on ventilators, and more attention should be directed at the diseases that create ventilator dependency.

1. Cairo JM, Pilbean SP. Mosby's Respiratory Care Equipment. 8th ed. St. Louis: Mosby Elsevier; 2010.
2. The Acute Respiratory Distress Syndrome Network. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med 2000; 342(18):1301-1308.
3. Mireles-Cabodevila E, Hatipoglu U, Chatburn RL. A rational framework for selecting modes of ventilation. Respir Care 2013; 58:348-366.

7. Hess DR, Kacmarek RM. Physiologic effects of mechanical ventilation. In: Essentials of Mechanical Ventilation. New York, McGraw-Hill, 1996:1—10.
8. Leung P, Jubran A, Tobin MJ. Comparison of assisted ventilator modes on triggering, patients' effort, and dyspnea. Am J Respir Crit Care Med 1997; 155:1940-1948.
9. Laureen H, Pearl R. Flow triggering, pressure triggering, and autotriggering during mechanical ventilation. Crit Care Med 2000; 28:579-581.

10. Shelledy DC, Rau JL, Thomas-Goodfellow L. A comparison of the effects of assistcontrol, SIMV, and SIMV with pressure-support on ventilation, oxygen consumption, and ventilatory equivalent. Heart Lung 1995; 24:67-75.
11. Singh I, Pinsky MR. Heart-lung interactions. In Papadakos PJ, Lachmann B, eds. Mechanical ventilation: clinical applications and pathophysiology. Philadelphia: Saunders Elsevier, 2008:173-184.
12. Mathru M et al. Hemodynamic responses to changes in ventilatory patterns in patients with normal and poor left ventricular reserve. Crit Care Med 1982; 10:423426.
25. Cowan N. The magical number 4 in short-term memory: a reconsideration of mental storage capacity. Behav Brain Sci 2001; 24:87-114.

The reasonable man adapts himself to the world; the unreasonabk one persists in trying to adapt the world to himseff. Therefore, all progress depends on the unreasonable man.
George Bernard Shaw 1903
The conventional modes of ventilation described in the last chapter are appropriate for most patients with acute respiratory failure. However, there are occasions when conventional mechanical ventilation is either unable to support gas exchange in the lungs, or is not necessary as a means of ventilatory support. This chapter describes alternate modes of ventilation that are available when conventional mechanical ventilation is either not sufficient, or not necessary, for patients with acute respiratory failure. Included are rescue modes of ventilation (i.e., high frequency oscillatory ventilation and airway pressure release ventilation) and noninvasive modes of ventilation (i.e., continuous positive airway pressure, bilevel positive airway pressure, and pressure support ventilation).

A small percentage (10-15%) of patients with acute respiratory distress syndrome (ARDS) develop hypoxemia that is refractory to oxygen therapy and conventional mechanical ventilation (1). In this situation, the appropriate strategy for ventilatory support is defined by the concept described next.

Alveolar collapse (atelectasis) in ARDS not only impairs alveolar ventilation, it also promotes ventilator-induced lung injury. There are two mechanisms for the lung injury produced by atelectasis. First, if the alveolar collapse is extensive, like that shown in Figure 23.4 (on page 454), the tidal volumes delivered by the ventilator will distribute in the uninvolved (normal) areas of the lungs, and this will overdistend normal alveoli and promote volutrauma. Secondly, if alveolar collapse occurs only at end-expiration, repetitive opening and closing of alveoli promotes atelectrauma by creating excessive shear forces that damage the airway epithelium. (See pages 494-497 for more information on ventilator-induced lung injury.)
The adverse consequences of alveolar collapse can be mitigated by a mode of ventilation that both prevents alveolar collapse and opens collapsed alveoli. This is the open lung concept of mechanical ventilation, where the goal is to: "open up the lung and keep the lung open" (2). The modes of ventilation described next are designed to achieve this goal.

High frequency oscillatory ventilation (HFOV) uses high-frequency, low volume oscillations like the ones shown in Figure 27.1. These oscillations create a high mean airway pressure, which improves gas exchange in the lungs by opening collapsed alveoli (alveolar recruitment) and preventing further alveolar collapse. The small tidal volumes (typically 1-2 mL) limit the risk of alveolar overdistension and volutrauma. For optimal results, an alveolar recruitment maneuver (e.g., with high PEEP levels, as shown in Figure 26.5, page 514) is recommended just prior to switching from conventional mechanical ventilation (CMV) to HFOV (3).
FIGURE 27.1 Airway pressure oscillations during high frequency oscillatory ventilation (HFOV), with a superimposed lung inflation during conventional mechanical ventilation (CMV). Dotted line represents mean airway pressure. From Reference 3.

HFOV requires a specialized ventilator (Sensormedics 3100B, Viasys Healthcare, Yorba Linda, CA) that allows the following adjustments: (a) the frequency and amplitude of the oscillations, (b) the mean airway pressure, (c) the bias flow rate (similar to an inspiratory flow rate), and (d) the inspiratory time (time of the bias flow). Some recommendations for these settings are listed in Table 27.1.
OSCILLATIONS: The frequency range for the oscillations is 4-7 Hz (1 Hz is one oscillation per second or 60 oscillations/min, so a range of 4-7 Hz is 240-420 oscillations/min), and the specific frequency selected is determined by the arterial pH (which represents the CO> burden). The pulse amplitude (tidal volume) determines CO, removal, and pulse amplitude is inversely related to the frequency of oscillations; i.e., lower oscillation frequencies result in higher tidal volumes and more effective CO» removal. The initial pulse amplitude is set at 70-90 cm H>0.
MEAN AIRWAY PRESSURE: The end-inspiratory alveolar pressure (e.g., the plateau pressure during volume control ventilation) should be measured just prior to switching from CMV to HFOV. This pressure is a reflection of: (a) alveolar volume, and (b) the risk of alveolar overdistension and volutrauma (see Chapter 25). The mean airway pressure is usually set 5 cm H20 higher than the end-inspiratory alveolar pressure recorded during CMV (3), but should not exceed 30 cm H20 (to prevent volutrauma).

Frequency: 4 Hz: (pH <7.1) 5-6 Hz: (pH 7.1—7.35) EES Bit aE a8) Amplitude: 70-90 cm H,O Mean Paw: 5 cm H,0 >P On CAA to nok oF 30 cm H,O Bias Flow: 40 L/min Inspiratory Time: 33% FiO,: 100%, APRV- = Pressure: High: Same a5 Postean On CMY, to max of 30 em H,O Low: Atmospheric (zero) pressure Time: High Pressure: 4-6 sec Low Pressure: 0.6-0.8 sec FiO: 100%
From Referance 3. HFOV=high frequency oscilatory ventilation; APRV=arway pressure relaasa ventiation; GMV= conventional mechanical ventilation; Paw=<airway pressure; Poiatea, =Pplateau pressure.

Clinical trials comparing HFOV with CMV, primarily conducted in patients with ARDS, have shown a 16-24% increase in the PaO>/FIO> ratio associated with HFOV (4). Early studies of HFOV showed no impact on mortality rate (3), but a very recent meta-analysis of all available studies shows a significant survival benefit with HFOV (4). However, HFOV has not been compared with lung protective ventilation (which also shows a survival benefit) so it is not known if HFOV represents an advance over lung protective ventilation in patients with ARDS.

The disadvantages of HFOV are listed below (3,4):
1. A special ventilator is needed, along with trained personnel to operate the device.
2. Cardiac output is often decreased during HFOV because of the high mean airway pressures. This effect requires augmentation of the intravascular volume during HFOV.
3. Aerosolized bronchodilators are ineffective during HFOV.

FIGURE 27.2 Related modes of pressure-regulated spontaneous ventilation. CPAP=continuous positive airway pressure, APRV=airway pressure release ventilation, BIPAP=bikvel positive airway pressure, IPAP=inspiratory positive airway pressure, EPAP=expiratory positive airway pressure, Prnean=mean airway pressure, I=inspiration, E=expiration. See text for explanation.

The benefits of APRV are lost if the patient has no spontaneous breathing efforts. Additional disadvantages of APRV are listed below.
1. Severe asthma and COPD are relative contraindications to APRV because of the inability to empty the lungs rapidly during the pressure release phase of APRV (3).
2. Cardiac output is often decreased during APRV because of the high mean airway pressures, but this effect is less pronounced than the cardiac depression during HFOV (3).

Noninvasive ventilation (NIV) is intended for patients with acute respiratory failure who will benefit from ventilatory support, but may not need conventional mechanical ventilation (which requires endotracheal intubation). NIV is delivered via tight-fitting face masks, which obviates the need (and the complications) of endotracheal intubation. Although NIV has applications in the outpatient and inpatient setting, the following description is limited to the use of NIV in acute respiratory failure (7,8).

Patient selection is the single most important factor in determining the success or failure of NIV (7,8). The patient selection criteria for NIV are presented as a checklist in Table 27.2.
1. The first step is to identify patients who might need ventilatory support. These patients have signs of respiratory distress (e.g., tachypnea, use of accessory muscles of respiration, abdominal paradox) plus either severe hypoxemia (PaO2/FIOz <200) or hypercapnia (PaCOz >45 mm Hg).
2. The next step is to identify patients who are candidates for NIV. While some causes of acute respiratory failure are more successfully managed with NIV than others (see later), all patients should be considered candidates for NIV if all of the following conditions are present: (a) the acute respiratory failure is not an immediate threat to life, (b) there is no life-threatening circulatory disorder (e.g., circulatory shock), (c) the patient is awake or arousable and cooperative, (d) airway protective mechanisms are intact (e.g., gag and cough reflexes), (e) there is no hematemesis or recurrent vomiting, (f) there is no facial anomaly that will prevent the use of a tight-fitting face mask (e.g., recent facial trauma), and (g) there is no obstruction that will prevent effective ventilatory support through a face mask (e.g., laryngeal edema).
3. Progression of the respiratory failure can limit the success of NIV (7,8), so there should be no delays in initiating NIV for appropriate candidates.

A. Does the patient have:
1. Signs of respiratory distress?
2. PaOz/FiO, <200 and/or PaCO, > 45 mm Hg?
=< Isis 7
LUws
B. If the answer is YES to both, answer the following questions.
~< 5 ow
C. Does the patient have:
1. Respiratory failure that is an immediate threat to life?
2. A life-threatening circulatory disorder (e.g., shock)?
3. Coma, severe agitation, or uncontrolled seizures?
4. Inability to protect the always?
5, Hematemesis or recurrent vomiting?
6. Laryngeal edema, facial trauma, or recent head and neck surgery?
UU LSi
Ls|
Ls|
Ls
Ls.
3
isi
D. If the answer Is NO to all of the above questions, the patient is a candidate for noninvasive ventilation.

The success of NIV in avoiding endotracheal intubation varies in different clinical disorders, which can be organized according to the major blood gas abnormality associated with the respiratory failure; i.e., hypercapnia or hypoxemia.

ACUTE EXACERBATION OF COPD: The greatest benefit with NIV in acute respiratory failure is in patients with acute exacerbation of COPD and CO) retention (9,10). The benefits of NIV in patients with acute exacerbation of COPD are demonstrated in Figure 27.3 (9). The use of NIV is associated with a marked decline in both the rate of tracheal intubation and the mortality rate. These results are corroborated in 14 clinical studies (10); as a result, noninvasive ventilation is considered a first-line therapy for acute exacerbations of COPD associated with hypercapnia (7,8). The preferred mode of ventilation in this condition is PSV with CPAP.
FIGURE 27.3 Impact of noninvasive ventilation (NIV) on rate of intubation and in-hospital mortality rate in patients with hypercapnic respiratory failure secondary to acute exacerbation of COPD. Patients assigned to NIV received pressure support ventilation for at least 6 hrs each day until intubation or recovery. N = number of patients in each study group. Data from Reference 9.
OBESITY HYPOVENTILATION SYNDROME: NIV reduces the severity of hypercapnia in outpatients with obesity hypoventilation syndrome (11) and, despite few studies in ICU patients, NIV is recommended as a routine measure for patients with obesity hypoventilation syndrome who are admitted with acute respiratory failure (12). Either CPAP or BiPAP can be used in this condition.
ASTHMA: NIV has not been adequately evaluated in status asthmaticus, but the available evidence shows that NIV hastens the resolution of the acute illness and decreases the length of stay in both the ICU and the hospital (13).
FIGURE 27.4 Failure rate of noninvasive ventilation for preventing endotracheal intubation in relation to the etiology of hypoxemic respiratory failure. CPE=cardiogenic pulmonary edema, Pulm Cont=pulmonary contusion, NP=nosocomial pneumonia, CAP=community-acquired pneumonia, ARDS=acute respiratory distress syndrome. Data from reference 14.

The failure rate of NIV for avoiding endotracheal intubation in disease states associated with hypoxemic respiratory failure is summarized in Figure 27.4 (14). The failure rate is lowest in patients with cardiogenic pulmonary edema, and highest in patients with community-acquired pneumonia and acute respiratory distress syndrome (ARDS).
CARDIOGENIC PULMONARY EDEMA: NIV is successful in reducing the need for intubation, and reducing the mortality rate, in a large majority of patients with cardiogenic pulmonary edema (15,16). Most of the experience in this condition has been with CPAP (at 10 cm H>O), but BiPAP produces equivalent results (17). The improved outcomes may be related to improved cardiac performance because NIV can increase cardiac output in patients with systolic heart failure (17). This effect is attributed to the afterload-reducing effects of positive intrathoracic pressure (See page 500).
ARDS: NIV has had limited success in patients with ARDS. The success rate is greater with PSV plus CPAP (17) than with CPAP alone (18), and success is more likely when ARDS has an extrapulmonary source (e.g., septicemia) (14). If NIV is attempted in patients with ARDS, the preferred mode is PSV with CPAP, while CPAP alone should be avoided (8).

The success or failure of NIV in any individual patient should not be predetermined by the etiology of the respiratory failure, but rather by the response of the patient in the first hour after the onset of NIV. This is illustrated in Figure 27.5 for patients with hypercapnic and hypoxemic respiratory failure (14, 19). Failure to improve gas exchange significantly after one hour of NIV is evidence that NIV is failing as a support modality; at this point, the appropriate course of action is immediate endotracheal intubation. Delaying intubation only invites problems (see A FINAL WORD).
FIGURE 27.5 Responses to the first hour of noninvasive ventilation for predicting success or failure in patients with hypercapnic and hypoxemic respiratory failure. Data from References 14 and 19.

Adverse events during NIV include gastric distension, pressure ulcers on the bridge of the nose from tight-fitting masks, and nosocomial pneumonia.

The principal concern during NIV is gastric distension from insufflated gas. However, this does not seem to be a common problem, and studies measuring upper esophageal opening pressures indicate that pressures less than 30 cm H20 should not cause gastric insufflation (20). Although nasogastric tubes are commonly placed for gastric decompression during NIV, withholding nasogastric tubes is a safe practice in patients who do not develop abdominal distension during NIV (21).

The application of positive pressure to the airways can retard mucociliary clearance and predispose to nosocomial pneumonia during NIV. In studies comparing NIV to endotracheal intubation, the incidence of nosocomial pneumonia during NIV was 8 — 10%, but this was less than half the incidence of nosocomial pneumonia during endotracheal intubation (19-22%) (22,23).

The rising popularity of noninvasive ventilation tends to overshadow the value of endotracheal intubation. The following simple rules about endotracheal intubation deserve mention.
Rule 1: Hesitation invites trouble. There is a tendency to rely on noninvasive ventilation and delay intubation as long as possible in the hopes that it will be unnecessary. However, delays in intubation create unnecessary dangers for the patient because emergency intubations in patients who are in extremis can be troublesome as well as dangerous. As soon as intubation becomes a serious consideration, you should intubate the patient and get control of the airway without delay.
Rule 2: Endotracheal intubation is not the 'kiss of death'. The perception that "once on a ventilator, always on a ventilator" is a fallacy that should never influence the decision to intubate a patient for full ventilatory support. Being on a ventilator does not create ventilator dependence, having a severe cardiopulmonary or neuromuscular disease does.

1., 1 = Pipeling MR, Fan E. Therapies for refractory hypoxemia in acute respiratory distress syndrome. JAMA 2010; 304:2521-2527.. 2., 1 = Lachmann B. Open up the lung and keep the lung open. Intensive Care Med 1992; 18:319-321.. 3., 1 = Stawicki SP, Goyal M, Sarini B. High-frequency oscillatory ventilation (HFOV) and airway pressure release ventilation (APRV): a practical guide. J Intens Care Med 2009; 24:215-229.. 4., 1 = Sud S, Sud M, Freiedrich JO, et al. High frequency ventilation versus conventional ventilation for treatment of acute lung injury and acute respiratory distress syndrome. Cochrane Database Syst Rev 2013; Feb 28:CD004085.. 5., 1 = Kallet RH. Patient-ventilator interaction during acute lung injury, and the role of spontaneous breathing: Part 2: airway pressure release ventilation. Respir Care 2011; 56:190-206.. 6., 1 = Sydow M, Burchardi H, Ephraim E, et al. Long-term effects of two different ventilatory modes on oxygenation in acute lung injury. Comparison of airway pressure release ventilation and volume-controlled inverse ratio ventilation. Crit Care Med 1994; 149:1550-1556.
21., 1 = Meduri GU, Fox RC, Abou-shala N, et al. Noninvasive mechanical ventilation via face mask in patients with acute respiratory failure who refused endotracheal intubation. Crit Care Med 1994; 22:1584-1590.. 22., 1 = Girou E, Schotgen F, Delclaux C, et al. Association of noninvasive ventilation with nosocomial infections and survival in critically ill patients. JAMA 200; 284:2361- 2367.. 23., 1 = Carlucci A, Richard J-C, Wysocki M, et al. Noninvasive versus conventional mechanical ventilation: an epidemiological study. Am J Respir Crit Care Med 2001; 163:874-880.

Eyes and ears are bad witnesses to men if they have souk that understand not their language.
Heraclitus 6th Century BC
This chapter describes the practices and common concerns involved in the care of ventilator-dependent patients. The focus in this chapter is on artificial airways (endotracheal and tracheostomy tubes) and mechanical complications of positive pressure ventilation (e.g., pneumothorax). The infectious complications of mechanical ventilation (i.e., tracheobronchitis and pneumonia) are described in the next chapter.

Positive pressure ventilation is delivered through a variety of plastic tubes that are passed into the trachea through the vocal cords (endotracheal tubes) or are inserted directly into the trachea (tracheostomy tubes). These tubes are equipped with an inflatable balloon at the distal end (called a cuff) that is used to seal the trachea and prevent inflation volumes from escaping out through the larynx.

Endotracheal tubes vary in length from 25 to 35 cm, and are sized according to their internal diameter, which varies from 5 to 10 mm (e.g., a "size 7" endotracheal tube will have an internal diameter of 7 mm). For adults, the diameter of endotracheal tubes should be at least 7 mm, and preferably 8 mm. Smaller tubes impede the clearance of secretions, and create increased airflow resistance when weaning from the ventilator.

Evaluation of endotracheal tube position is mandatory after intubation. The portable chest film in Figure 28.1 shows the proper position of an endotracheal tube. When the head is in a neutral position, the tip of the endotracheal tube should be 3 to 5 cm above the carina, or midway between the carina and vocal cords. (If not visible, the main carina is usually over the T4-T5 interspace on a portable chest x-ray). The head is in the neutral position when the inferior border of the mandible projects over the lower cervical spine (C5-C6). Flexion or extension of the head and neck causes a 2 cm displacement of the tip of the endotracheal tube (2).
FIGURE 28.1 Portabk chest x-ray showing proper position of an endotracheal tube, with the tip located midway between the thoracic inlet and the carina.

Endotracheal tubes can migrate distally, and enter the right mainstem bronchus (which runs a straight course down from the trachea). What can happen next is shown in Figure 28.2; i.e., selective ventilation of one lung results in progressive atelectasis in the nonventilated lung.
There are two measures that can reduce the incidence of endotracheal tube migration.
First, for orotracheal intubations, don't allow the tip of endotracheal tube to advance further than 21 cm from the teeth in women and 23 cm from the teeth in men (3). Second, monitor the position of the endotracheal tube with periodic chest x-rays, and keep the tip of the tube at least 3 cm above the carina.
FIGURE 28.2 Portabk chest x-ray showing the tip of an endotracheal tube in the right mainstem bronchus. Opacification of the left hemothorax represents atelectasis of the left lung.

The risk for laryngeal injury with endotracheal tubes is a major concern, and is one of the reasons for performing tracheostomies when prolonged intubation is anticipated. The spectrum of laryngeal damage includes ulceration, granulomas, vocal cord paresis, and laryngeal edema. Some type of laryngeal damage is usually evident after 72 hours of translaryngeal intubation (4), and laryngeal edema is reported in 5% of cases. Fortunately, most cases of laryngeal injury do not result in significant airways obstruction or permanent injury, and the problem resolves within weeks after extubation (5). The problem of laryngeal edema after extubation is described in Chapter 30.

The prominent role played by aspiration of mouth secretions in the pathogenesis of ventilator-associated pneumonia has led to the introduction of specially designed endotracheal tubes capable of draining mouth secretions that accumulate just above the inflated cuff. These tubes can reduce the incidence of ventilator-associated pneumonia (6), and they are described in more detail in the next chapter.
insertion, the patient should be reintubated before attempting to reinsert the tracheostomy tube.
TRACHEAL STENOSIS: The most feared complication of tracheostomy is tracheal stenosis, which is a late complication that appears in the first 6 months after the tracheostomy tube is removed. Most cases of tracheal stenosis occur at the site of the tracheal incision, and are the result of tracheal narrowing after the stoma closes. The incidence of tracheal stenosis ranges from zero to 15% in individual reports (12), but most cases are asymptomatic. The risk of tracheal stenosis is the same with surgical and percutaneous tracheostomies (9).

Positive pressure ventilation requires a seal in the trachea that prevents gas from escaping out through the larynx. This is created by inflating the balloon (called a cuff) that surrounds the distal portion of the tracheal tube. A tracheostomy tube with an inflated cuff is shown in Figure 28.3. The cuff is attached to a pilot balloon that has a oneway valve. A syringe is attached to the pilot balloon and air is injected into the cuff until there is no audible leak around the cuff. (The pilot balloon will inflate as the cuff inflates.) The pressure in the cuff (measured with a pressure gauge attached to the pilot balloon) should not exceed 25 mm Hg (13). This pressure limit is based on the assumption that the capillary hydrostatic pressure in the wall of the trachea is 25 mm Hg, and thus external (cuff) pressures above 25 mm Hg can compress the underlying capillaries and produce ischemic injury and tracheal necrosis. Fortunately, the elongated design of cuffs allows for greater dispersion of pressure, and also allows for a tracheal seal at relatively low pressures.

Cuff leaks are usually detected by audible sounds during lung inflation (created by gas flowing through the vocal cords). Cuff leaks are rarely caused by disruption of the cuff (14), and are usually the result of nonuniform contact between the cuff and the wall of the trachea, or dysfunction of the valve on the pilot balloon.
TROUBLESHOOTING: If a cuff leak is audible, the patient should be separated from the ventilator and the lungs should be manually inflated with an anesthesia bag. If the cuff leak involves an endotracheal tube, check the position of the tube to determine if the tube is coming out of the trachea. If this is a possibility, you can deflate the cuff and advance the tube. If this is unsuccessful, the tube should be replaced (or a fiber-optic instrument can be advanced through the tube to make sure it is still in the lungs). Air should never be added to the cuff blindly because the tube may be coming out of the trachea, and cuff inflation will damage the vocal cords. If the cuff leak involves a tracheostomy tube, air can be added to the cuff in an attempt to produce a seal. If this eliminates the leak, the cuff pressure should be measured, and should be <25 mm Hg. If the cuff pressure is above 25 mm Hg, or the leak persists despite adding volume, the tracheostomy tube should be replaced (with a larger diameter tube, if possible).
FIGURE 28.3 A tracheostomy tube with an inflated cuff.

Routine suctioning to clear secretions has been a standard practice in the care of ventilator-dependent patients. However, the inner surface of artificial airways become colonized with biofilms containing pathogenic organisms (see Figure 28.4), and passing a suction catheter through the tubes can dislodge these biofilms and inoculate the lungs with pathogenic organisms (15,16). As a result, endotracheal suctioning is no longer recommended as a routine procedure, but should be performed only when respiratory secretions are present (17).
FIGURE 28.4 Electron micrograph showing a biofilm on the inner surface of an endotracheal tube. Image from Reference 16.

Saline is often instilled into the trachea to facilitate the clearing of secretions, but this practice is no longer advised as a routine procedure (17) for two reasons: (a) saline will not liquefy or reduce the viscosity of respiratory secretions (as described next), and (b) saline can dislodge pathogenic organisms colonizing the inner surface of tracheal tubes. One study has shown that injection of 5 mL of saline can dislodge up to 300,000 colonies of viable bacteria from the inner surface of endotracheal tubes (18).

The respiratory secretions create a blanket that covers the mucosal surface of the airways. This blanket has a hydrophilic (water soluble) layer, and a hydrophobic (water insoluble) layer. The hydrophilic layer faces inward, and keeps the mucosal surface moist. The hydrophobic layer faces outward, towards the lumen of the airways. This outer layer is composed of a meshwork of mucoprotein strands (called mucus threads) held together by disulfide bridges. This meshwork traps particles and debris in the airways, and the combination of the mucoprotein meshwork and the trapped debris is what determines the viscoelastic behavior of the respiratory secretions. Since the layer that contributes to the viscosity of respiratory secretions is not water soluble, saline will not reduce the viscosity of respiratory secretions. (Adding saline to thick, respiratory secretions is like pouring water over grease.)
The accumulation of viscous secretions can result in a condition like the one shown in Figure 28.5, where a tenacious "plug" is completely ob-structing a major airway. In this situation, a mucolytic agent like N-Acetylcysteine (Mucomyst) can help to disrupt the plug and relieve the obstruction. N-acetylcysteine (NAC) is a sulfhydryl-containing tripeptide that is better known as the antidote for acetaminophen overdose, but it is also a mucolytic agent that acts by disrupting the disulfide bridges between mucoprotein strands in sputum (19). The drug is available in a liquid preparation (10 or 20% solution) that can be given as an aerosol spray, or injected directly into the airways (Table 28.1). Aerosolized NAC should be avoided when possible because it is irritating to the airways and can provoke coughing and bronchospasm (particularly in asthmatics). Direct instillation of NAC into the tracheal tube is preferred, especially when there is an obstruction.

Aerosol Therapy:, 1 = « Use 10% NAC solution. © Mix 2.5 mL NAC with 2.5 mL saline and place mixture (5 mL) in a small volume ebullzer for aerosol delivery. « Warning: this can provoke bronchospasm, and Is hot recommended in asthmatics. Tracheal Injection:, 1 = « Use 20% NAC solution. « Mix 2 mL NAG with 2 mL saline and inject 2 mL aliquots into the trachea. « Warning: Excessive volumes can produce bronchorrhea.
If intratracheal injection of NAC does not relieve an obstruction, bronchoscopy should be performed (the NAC is then applied directly to the mucous plug). Following relief of the obstruction, NAC can be instilled two or three times a day for the next day or two. Daily use of NAC is not advised because the drug solution is hypertonic (even with the saline additive) and can provoke bronchorrhea.

One of the manifestations of ventilator-induced lung injury is alveolar distension and volutrauma, and one of the clinical consequences of volutrauma is alveolar rupture with the escape of gas from the distal airspaces. This form of injury, which is mistakenly called pulmonary barotrauma, occurs in up to 25% of patients receiving mechanical ventilation (20).

Escape of gas from the alveoli can produce a variety of clinical manifestations. The alveolar gas can dissect along tissue planes and _ produce pulmonary interstitial emphysema, and air can move into the mediastinum and produce pneumomediastinum. Mediastinal gas can move into the neck to produce subcutaneous emphysema, or can pass below the diaphragm to produce pneumoperitoneum. Finally, if the rupture involves the visceral pleura, gas will collect in the pleural space and produce a pneumothorax. Each of these entities can occur alone or in combination (20,21).
FIGURE 28.5 Portabk chest x-ray of an intubated patient showing atelectasis of the left lung, which is usually caused by a mucous plug in the left mainstem bronchus.

Radiographic evidence of pneumothorax occurs in 5 to 15% of ventilator-dependent patients (20,21).

Clinical manifestations are either absent, minimal, or nonspecific. The most valuable clinical sign is subcutaneous emphysema in the neck and upper thorax, which is pathognomonic of alveolar rupture. Breath sounds are unreliable in ventilator-dependent patients because sounds transmitted from the ventilator tubing can be mistaken for airway sounds.

The radiographic detection of pleural air can be difficult in the supine position, because pleural air does not collect at the lung apex when patients are supine (22). Figure 28.6 illustrates this difficulty. In this case of a traumatic pneumothorax, the chest x-ray is unrevealing, but the CT scan reveals an anterior pneumothorax on the left. Pleural air will collect in the most superior region of the hemithorax and, in the supine position, this region is just anterior to both lung bases. Therefore, basilar and subpulmonic collections of air are characteristic of pneumothorax in the supine position (22).
REDUNDANT SKIN FOLDS: When the film cartridge used for portable chest x-rays is placed under the patient, the skin on the back can fold over on itself, and the edge of this redundant skin fold creates a radiographic shadow that can be mistaken for a pneumothorax. The radiographic appearance of a redundant skin fold is shown in Figure 28.7. Note that there is a gradual increase in radiodensity that produces a wavy line. The gradual increase in density is produced by the skin that is folded back on itself. A pneumothorax would appear as a sharp white line with dark shadows (air) on either side.
FIGURE 28.6 A portable chest x-ray and CT image of the thorax in a young male with blunt trauma to the chest. An anterior pneumothorax is evident on the CT image (indicated by the asterisk) but is not apparent on the portable chest xray.
FIGURE 28.7 Portable chest x-ray showing a wavy line in the left hemithorax. This line is the edge of a redundant skin fold, and not the edge of the lung.

Evacuation of pleural air is accomplished by inserting a chest tube through the fourth or fifth intercostal space along the mid-axillary line. The tube should be advanced in an anterior and superior direction (because this is where pleural air collects in the supine position). The pleural space is drained of fluid and air using a three-chamber system like the one shown in Figure 28.8 (23).

The first bottle in the system collects fluid from the pleural space and allows air to pass through to the next bottle in the series. Because the inlet of this chamber is not in direct contact with the fluid, the pleural fluid that is collected does not impose a back pressure on the pleural space.

The second bottle acts as a one-way valve that allows air to escape from the pleural space but prevents air from entering the pleural space. This one-way valve is created by submerging the inlet tube under water. This imposes a back-pressure on the pleural space that is equal to the depth that the tube is submerged. The positive pressure in the pleural space then prevents atmospheric air (at zero pressure) from entering the pleural space. The water thus "seals" the pleural space from the surrounding atmosphere. This water-seal pressure is usually 2 cm H20.
Air that is evacuated from the pleural space passes through the water in the second bottle and creates bubbles. Thus, the presence of bubbles in the water-seal chamber is evidence of a continuing bronchopleural air leak.

The third bottle in the system is used to set a maximum limit on the negative suction pressure that is imposed on the pleural space. This maximum pressure is determined by the height of the water column in the air inlet tube. Negative pressure (from wall suction) draws the water down the air inlet tube, and when the negative pressure exceeds the height of the water column, air is entrained from the atmosphere. Therefore, the pressure in the bottle can never become more negative than the height of the water column in the air inlet tube.
Water is added to the suction-control chamber to achieve a water level of 20 cm. The wall suction is then activated and slowly increased until bubbles appear in the water. This bubbling indicates that atmospheric air is being entrained, and thus the maximum negative pressure has been achieved. The continuous bubbling causes water evaporation, so it is imperative that the height of the water in this chamber is checked periodically, and more water is added when necessary.
FIGURE 28.8 A standard pleural drainage system for evacuating air and fluid from the pleural space.

The practice of using suction to evacuate pleural air is often unnecessary and potentially harmful. Although there is a perception that suction will help the lungs reinflate, the lungs will reinflate without the use of suction. Furthermore, creating a negative pressure in the pleural space also creates a higher transpulmonary pressure (the pressure difference between alveoli and the pleural space), and this increases the rate of the air flowing through a bronchopleural fistula. Thus applying suction to the pleural space increases bronchopleural air leaks, and this can prevent bronchopleural fistulas from closing. If a persistent air leak is present when suction is applied to the pleural space, the suction should be discontinued. Any air that collects in the pleural space will continue to be evacuated when the pleural pressure becomes more positive than the water-seal pressure,

Intrinsic, occult PEEP (also called auto-PEEP) is described in Chapter 24 (see pages 477— 479) and inChapter 25 (see pages 490-491). The features of intrinsic PEEP are illustrated in Figure 28.9. This pressure is the result of incomplete exhalation, and can occur in patients with severe airflow obstruction, or during mechanical ventilation that is too rapid to allow complete exhalation (24). During conventional mechanical ventilation, intrinsic PEEP is probably universal in patients with severe asthma and COPD (25,26), while it is also common in patients with ARDS (27), although at low levels (<3 cm H)0). It is important to emphasize that intrinsic PEEP is not apparent when monitoring airway pressures (hence the term "occult PEEP").

Intrinsic PEEP has several adverse effects, which are summarized below (24). As is the case with applied PEEP, many of these adverse effects are not the result of the PEEP level, but rather are due to the influence of PEEP on the end-inspiratory alveolar pressure and the mean intrathoracic pressure.
1. Intrinsic PEEP increases mean intrathoracic pressure, which impairs venous return and can decrease cardiac output.
2. The hyperinflation that produces intrinsic PEEP in patients with severe airflow obstruction can increase the work of breathing when the region of tidal breathing moves to the upper flat portion of the pressure-volume curve (see Figure 24.4 on page 478). (To appreciate this effect, take a deep breath, and then try to breathe in further.)
. Intrinsic PEEP increases end-inspiratory alveolar pressure, which increases the risk of volutrauma and alveolar rupture.
. When intrinsic PEEP goes undetected, the increase in end-inspiratory alveolar pressure (plateau pressure) is misinterpreted as a decrease in the compliance of the lungs and chest wall. When thoracic compliance is calculated (see page 492), the total PEEP level (applied PEEP plus intrinsic PEEP) must be subtracted from the end-inspiratory alveolar (plateau) pressure.
FIGURE 28.9 The features of intrinsic PEEP resulting from incomplete exhalation. The presence of airflow (V.) at endexpiration indicates a pressure drop from the alveolus (Pa; y) to the proximal airways (Pppox). As shown at the top of the figure, the proximal airway pressure returns to zero at end-expiration whik the alveolar pressure remains positive, hence the term occult PEEP. The upper left panel illustrates the end-expiratory occlusion method for measuring occult PEEP.
- 5. Intrinsic PEEP can be transmitted into the superior vena cava, resulting in the mistaken impression that end-diastolic (transmural) pressure has increased.

Intrinsic PEEP is easy to detect but difficult to quantify. The easiest method of detection is inspecting the expiratory flow waveform for the presence of airflow at the end of expiration (see Figure 24.5 on page 479). If intrinsic PEEP is evident on the expiratory flow waveform, the level of intrinsic PEEP can be measured with the end-expiratory occlusion method.

Occlusion of the expiratory circuit at the end of expiration will "unmask" intrinsic PEEP, as shown in Figure 28.9. Accuracy requires that the occlusion occur at the very end of expiration, and this cannot be timed properly if patients are breathing spontaneously. Therefore, the end-expiratory occlusion method can only be performed in the patients who are not triggering ventilator breaths.

The maneuvers used to prevent or reduce hyperinflation and intrinsic PEEP are all directed at promoting alveolar emptying during expiration. These maneuvers include reducing the tidal volume, increasing the inspiratory flow rate, reducing the inspiratory time (in pressure control ventilation), and reducing the respiratory rate, if possible. Some of these maneuvers will reduce the minute ventilation, which may not be desirable.

The addition of external PEEP can reduce hyperinflation (and intrinsic PEEP) by holding the small airways open at end-expiration. The level of applied PEEP must be enough to counterbalance the pressure causing small airways collapse (the closing pressure) but should not exceed the level of intrinsic PEEP (so that it does not impair expiratory flow) (28). To accomplish this, the level of applied PEEP should match the level of intrinsic PEEP. Since intrinsic PEEP will be difficult to quantify in spontaneously breathing patients, an alternative method in these patients is to monitor the response of the end-expiratory airflow to applied PEEP; i.e., if the applied PEEP reduces or eliminates end-expiratory flow, then it is reducing the level of intrinsic PEEP. Although the end result is still PEEP (applied PEEP instead of intrinsic PEEP), the applied PEEP will help to reduce the risk of atelectrauma from repetitive opening and closing of small airways at end-expiration.

The patient's clinical course in the first few days of mechanical ventilation will give you a fairly accurate indication of what is coming. If the patient is not improving, proceed to a tracheostomy as soon as it can be done safely. Most of the day-to-day management of the ventilator-dependent patient involves vigilance for adverse events (e.g., pneumothorax). You will learn that, in many cases, you are not the one controlling the course of the patient's illness.

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7., Tracheostomy = Terragni PP, Antonelli M, Fumagalli R, et al. Early vs. late tracheotomy for prevention of pneumonia in mechanically ventilated adult ICU patients. JAMA 2010; 303:1483- 1489.. 8., Tracheostomy = Trouillet JL, Luyt CE, Guiguet M, et al. Early percutaneous tracheotomy versus prolonged intubation of mechanically ventilated patients after cardiac surgery: A randomized trial. Ann Intern Med 2011; 154:373-383.. 9., Tracheostomy = Freeman BD, Morris PE. Tracheostomy practice in adults with acute respiratory failure. Crit Care Med 2012; 40:2890-2896.. 10., Tracheostomy = Ciagla P. Technique, complications, and improvements in percutaneous dilatational tracheostomy. Chest 1999; 115:1229-1230.. 11., Tracheostomy = Freeman BD, Isabella K, Lin N, Buchman TG. A meta-analysis of prospective trials comparing percutaneous and surgical tracheostomy in critically ill patients. Chest 2000; 118:1412-1418.. 12., Tracheostomy = Tracheotomy: application and timing. Clin Chest Med 2003; 24:389-398.. 13., Tracheostomy = Heffner JE, Hess D. Tracheostomy management in the chronically ventilated patient. Clin Chest Med 2001; 22:5; 10:561-568.. 14., Tracheostomy = Kearl RA, Hooper RG. Massive airway leaks: an analysis of the role of endotracheal tubes. Crit Care Med 1993; 21:518-521.

Airway Care, 1 = Airway Care. 15., 1 = Adair CC, Gorman SP, Feron BM, et al. Implications of endotracheal tube biofilm for ventilator-associated pneumonia. Intensive Care Med 1999; 25:1072-1076.. 16., 1 = Gil-Perontin S, Ramirez P, Marti V, et al. Implications of endotracheal tube biofilm in ventilator associated pneumonia response: a state of concept. Crit Care 2012; 16:R93 (available at ccforum.com/content/16/3/R93).. 17., 1 = AARC Clinical Practice Guideline. Endotracheal suctioning of mechanically ventilated patients with artificial airways 2010. Respir Care 2010; 55:758-764.. 18., 1 = Hagler DA, Traver GA. Endotracheal saline and suction catheters: sources of lower airways contamination. Am J Crit Care 1994; 3:444-447.. 19., 1 = Holdiness MR. Clinical pharmacokinetics of N-acetylcysteine. Clin Pharma-cokinet 1991; 20:123-134.
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Everything hinges on the matter of evidence.
Carl Sagan
The approach to pneumonias that develop during mechanical ventilation can be characterized by one word: problematic. The problems extend from the lack of a "gold standard" method for identifying parenchymal lung infections in ventilator-dependent patients, to the lack of standardized methods for collecting and culturing respiratory secretions and, finally, to the difficulties in eradicating multidrug-resistant organisms that are becoming prevalent in ventilator-associated pneumonias.
This chapter presents the current state of knowledge regarding ventilator-associated pneumonias, and includes recommendations from the most recent clinical practice guidelines and reviews of this topic (1-4).

The, 1 = following statements include some basic information about ventilator-associated pneumonia (VAP).. 1., 1 = Infections involving the lungs are the most common nosocomial infections in ICU patients, accounting for 65% of all nosocomial infections in this patient population (5). However, this prevalence is overstated because many cases of presumed ICU-acquired pneumonia are not corroborated on postmortem examination (see later).. 2., 1 = Over 90% of ICU-acquired pneumonias occur during mechanical ventilation, and 50% of these ventilator-associated pneumonias (VAPs) begin in the first 4 days after intubation (2).. 3., 1 = Unlike community-acquired pneumonias, where the predominant pathogens are pneumococci, atypical organisms, and viruses, three-quarters of the isolates in VAP are Gram-negative aerobic bacilli (most frequently Pseudomonas aeruginosa) and Staphylococcus aureus (see Table 29.1).. 4., 1 = The crude mortality rate associated with VAP is 5% to 65% (3). However, there is a paucity of strong evidence directly linking VAP with fatal outcomes (4,6), and several studies show no direct relationship between VAP and mortality rate (7-9). As a result, the growing consensus is that VAP is not a life-threatening illness, but it does prolong the duration of mechanical ventilation and increases the length of stay in the ICU and hospital (4).

Gram-Negative Bacilll, Frequency = . Pseudomonas aeruginosa, Frequency = 18.9%. Escherichia coll, Frequency = 9.2%. Hemophilus spp, Frequency = 1.1%. Enterobacter spp, Frequency = 3.8%. Proteus, Frequency = 3.8%. Klebsiella pneumoniae, Frequency = 3.2%. Others, Frequency = 10.5%. Gram-Positive Cocel, Frequency = . Staphylococcus aureus, Frequency = 18.9%. Streptococcus pneumoniae, Frequency = 13.2%. Haemophilus spp, Frequency = 1.4%. Others, Frequency = 8.6%. Fungal Isolates, Frequency = 1.3%
From Ghastra J. et a. JAMA 2008; 200:2558.

Aspiration of pathogenic organisms from the oropharynx is believed to be the inciting event in most cases of VAP (10). The pathogens that most often colonize the oropharynx in ICU patients are Gram-negative aerobic bacilli (see Figure 5.5 on page 89), which explains the predominance of these pathogens in VAP. The instances of VAP that appear within 4 days of intubation are most likely caused by pathogens that have been dragged into the airways during the intubation procedure.

The realization that VAP begins with pathogenic colonization of the oropharynx prompted the introduction of measures to decontaminate the oropharynx as a preventive measure for VAP. The methods of oral decontamination (i.e., with antiseptic agents and nonabsorbable antibiotics) are described in Chapter 5 (see pages 88-90), and the benefits of oral decontamination in reducing tracheal colonization and VAP are shown in Figure 5.6 (page 90). Oral decontamination (usually with the antiseptic agent chlorhexidine) is now a standard measure in all ventilator-dependent patients.

(Note: This topic is described in more detail on pages 540-542 in the last chapter.) Tracheal tubes serve as a nidus for pathogenic colonization and biofilm formation, and these biofilms protect the colonized pathogens and allow them to proliferate (see Figure 28.4 on page 541). Endotracheal suctioning can disrupt the biofilms and dislodge microbes colonizing the inner surface of tracheal tubes, which introduces pathogens into the lower airways. For this reason, endotracheal suctioning is not recommended as a routine procedure (11), but only when necessary to clear secretions from the airways.

The traditional clinical criteria for the diagnosis of VAP include: (a) fever or hypothermia, (b) leukocytosis or leukopenia, (c), an increase in volume of respiratory secretions or a change in character of the secretions, and (d) a new or progressive infiltrate on the chest x-ray (4). Unfortu-nately, in patients who are suspected of having VAP based on these clinical criteria, the incidence of pneumonia on postmortem exam is only 30% to 40% (14). The diagnostic accuracy for VAP based on traditional clinical criteria is demonstrated in Table 29.2. This table shows the results of two studies that used autopsy evidence of pneumonia to evaluate the premortem diagnosis of VAP based on clinical findings (15,16). In both studies, the clinical criteria for identifying VAP were just as likely to occur in the presence or absence of pneumonia. These studies demonstrate that the diagnosis of VAP is not possible using clinical criteria alone.
FIGURE 29.1 Endotracheal tube with a suction port placed just above the cuff to clear secretions that accumulate in the subglottic region.
Table 29.2 Predictive Value of Clinical Criteria in the Diagnosis of VentilatorAssociated Pneumonia
Fagon et al. (15), 1 = Radiographic infiltrate + purulent sputum + fever or leukocytosis. Fagon et al. (15), 2 = 1.03. Timset et al. (16), 1 = = Radiographic infiltrate + 2 of the following: fever, leukocytosis, or purulent sputum.. Timset et al. (16), 2 = 0.96
TAs reported in Reference 14. Tha likalihood ratio is the likelihood that patients with pneumonia will have the cinical findings compared to the likelihood that patients without pneumonia will have the same cinica findings. A likelinood ratio of 1 indicates that a pneumonia is just as Ikely to be prasent or absent based on the clinical findings.

The limited diagnostic accuracy in VAP is primarily due to the nonspecific nature of pulmonary infiltrates. Pneumonia accounts for only one-third of all pulmonary infiltrates in ICU patients (17,18), which means that conditions other than pneumonia are the most frequent cause of pulmonary infiltrates in ICU patients. The noninfectious causes of pulmonary infiltrates in the ICU include pulmonary edema, acute respiratory distress syndrome, and atelectasis.
An example of how a decrease in lung volume can produce spurious changes in a portable chest x-ray is illustrated in Figure 29.2. Both images in this figure were obtained within minutes of each other in the same patient. The image on the left, which was taken during expiration in the supine position (a common body position in ICU patients) shows a marked reduction in lung volume accompanied by a crowding of lung markings at the base of the right lung (dotted triangle). In a patient with fever, this radiographic change could be mistaken for a basilar pneumonia.
FIGURE 29.2 The effect of a decrease in lung volume on the appearance of a portable chest x-ray. Both images were obtained within minutes of each other in the same patient. The dotted triangle outlines an area of crowded lung markings that could be mistaken for a basilar pneumonia in a febrile patient. Image on the left digitally enhanced.
ARDS: The most common noninfectious cause of pulmonary infiltrates in ICU patients is the acute respiratory distress syndrome (ARDS) (18). This condition, which is described in
Chapter 23, is an inflammatory disorder of the lungs that produces bilateral infiltrates on chest x-ray (see Figure 23.2 on page 451). Because it is often accompanied by fever, ARDS can be difficult to distinguish from a multilobar pneumonia.

The other limitation of chest radiography is a limited sensitivity for the detection of pulmonary infiltrates. This is demonstrated in Figure 29.3, where the portable chest x-ray of a patient with cough and fever shows no apparent infiltrates, while the CT image of the same patient shows a fine pattern of infiltration in the posterior region of both lungs. Since CT scans are not obtained routinely in patients with suspected pneumonia and an unrevealing chest x-ray, it is not clear how often the example in Figure 29.3 actually occurs. However, this example clearly demonstrates that the diagnosis of pneumonia cannot be ruled out with a portable chest x-ray.
FIGURE 29.3 Demonstration of the limited sensitivity of portable chest radiography in the detection of pulmonary infiltrates. A portable chest x-ray of a patient with fever shows no apparent pulmonary infiltrates, while the CT image from the same patient reveals infiltrates in the posterior region of both lungs (indicated by the arrows).

The National Healthcare Safety Network (NHSN) has recently published an algorithm for the diagnosis of VAP that does not include findings on a chest x-ray (1). This algorithm is shown in Figure 29.4. The clinical suspicion of VAP in this algorithm is triggered by a deterioration in arterial oxygenation (ventilator-associated complication) combined with a change in body temperature or leukocyte count in the blood (infection-related ventilatorassociated complication). Although this algorithm has not been validated, it does recognize the limitations of chest radiography in the diagnosis of VAP.

The diagnosis of VAP rests heavily on identifying a responsible patho-gen, but there is little agreement on the optimal method of either collecting or culturing respiratory secretions. Blood cultures have limited value in the diagnosis of VAP because organisms isolated from blood in cases of suspected VAP are often from extrapulmonary sites of origin (14). The NHSN algorithm in Figure 29.4 relies on quantitative cultures of respiratory secretions or lung biopsies for the diagnosis of VAP, but quantitative cultures are not a standard practice in the diagnostic evaluation of suspected VAP. The following is a brief review of the methods used to collect and culture respiratory secretions in the diagnostic approach to VAP.

The traditional approach to suspected VAP involves aspiration of respiratory secretions through an endotracheal or tracheostomy tube, and these specimens can be contaminated with mouth secretions that are aspirated into the upper airway. A screening method for identifying contaminated and uncontaminated specimens is described next. (This method should be performed routinely in the microbiology laboratory.)

The cells identified in Figure 29.5 can help to determine if secretions collected by aspiration through tracheal tubes are contaminated by mouth secretions, and if there is evidence of infection. Each type of cell can be identified and interpreted as follows.
- 1. The squamous epithelial cells that line the oral cavity are large and flattened with abundant cytoplasm and a small nucleus. The presence of more than 10 squamous epithelial cells per low-power field (x 100) indicates that the specimen is contaminated with mouth secretions, and is not an appropriate specimen for culture (1).

After 2 days of stability or improvement on the ventilation, the patient has at least one of the following indications of worsening oxygenation:
1. Increase in daily minimum FiQz 20% for at least 2 days.
2. Increase in daily minimum PEEP 3 cm H30 for at least 2 days.

After at least 3 days of mechanical ventilation, and within 2 days of worsening oxygenation, the patient has:
1. Body temperature 38C or <36 OR
2. WBC count 12,000/mm? or 4,000/mm3.

After at least 3 days of mechanical ventilation, and within 2 days of worsening oxygenation, the patient has one of the following:
1. Purulent secretions (25 neutrophils and 10 squamous cells per low power field) AND one of the following:
a. Positive culture of endotracheal aspirate at 105 CFU/mL.t
b. Positive culture of broncoalveolar lavage at 104 CFU/mL.t
c. Positive culture of lung tissue at 104 CFU/mL.
d. Positive culture of protected specimen brush at 104 CFU/mL.t
2. One of the following (with or without purulent secretions):
a. Positive pleural fluid culture.
b. Positive lung histopathology.
c. Positive diagnostic test for Legionella spp.
d. Positive diagnostic test on respiratory secretions for influenza virus, adenovirus, respiratory syncytial virus, rhinovirus, human metapneumovirus, or coronavirus.
texcludes the following: (a) normal respiratory flora, (b) Candida species or yeast not otherwise specified, (c) coagulase-negative Staphylococcus spp., and (d) Enterococcus species.

2. Lung macrophages are large, oval-shaped cells with a granular cytoplasm and a small, eccentric nucleus. The size of the nucleus in a macrophage is roughly the same size as a neutrophil. Although macrophages can inhabit the airways (20), the predominant home of the macrophage is the distal airspaces. Therefore, the presence of macrophages, regardless of the number, is evidence that the specimen is from the lower respiratory tract.
3. The presence of neutrophils in respiratory secretions is not evidence of infection because neutrophils can make up 20% of the cells recovered from a routine mouthwash (20). The neutrophils should be present in abundance to indicate infection. More than 25 neutrophils per low-power field (x100) can be used as evidence of
FIGURE 29.5 Microscopic appearance (magnification 400) of bronchial brushings from a ventilator-dependent patient. The paucity of squamous epithelial cells and the presence of alveolar macrophages is evidence that the specimen is from the distal airways (and thus would be an appropriate specimen for culture in a case of suspected VAP).

The standard practice is to perform qualitative cultures on endotracheal aspirates (where the growth of organisms is reported, but there is no assessment of growth density). These cultures have a high sensitivity (usually >90%) but a very low specificity (1540%) for the diagnosis of VAP (22). This means that for qualitative cultures of tracheal aspirates, a negative culture can be used to exclude the diagnosis of VAP, but a positive culture cannot be used to confirm the presence of VAP. The poor predictive value of positive cultures is due to contamination of tracheal aspirates with secretions from the mouth and upper airways.

For quantitative cultures of tracheal aspirates (where growth density on the culture plate is reported), the threshold growth for the diagnosis of VAP is 105 colony-forming units per mL (CFU/mL). This threshold has a sensitivity and specificity of 76% and 75%, respectively, for the diagnosis of VAP (see Table 29.3) (2,22). Comparing these results to the sensitivity and specificity of qualitative cultures (i.e., sensitivity >90% and specificity <40%) shows that, for cultures of tracheal aspirates, quantitative cultures are less sensitive but more specific than qualitative cultures for the diagnosis of VAP.
Quantitative Cultures for the Diagnosis of Pneumonia in Ventilator-
Diagnostic Threshhold (CFU/mL), 1 = 108. Diagnostic Threshhold (CFU/mL), 2 = 103. Diagnostic Threshhold (CFU/mL), 3 = 104. Sensitivity (mean), 1 = 76%. Sensitivity (mean), 2 = 66%. Sensitivity (mean), 3 = 13%. Specificity (mean), 1 = 75%. Specificity (mean), 2 = 90%. Specificity (mean), 3 = 82%. ane ae, 1 = Most Sensitive. ane ae, 2 = Most Specific. ane ae, 3 = Most Accurate
Abbraviations: TA = trachadl aspirates; PSB = protected spacimen brushings; BAL = broncoalveolar lavage.
From Referancas 2, 22, 24.

Bronchoalveolar lavage (BAL) is performed by wedging the bronchoscope in a distal airway and performing a lavage with sterile isotonic saline. A minimum lavage volume of 120 mL is recommended for adequate sampling of the lavaged lung segment (23), and this is achieved by performing a series of 6 lavages using 20 mL for each lavage. The same syringe is used to introduce the fluid and aspirate the lavage specimen (only 25% or less of the volume instilled will be returned via aspiration). The first lavage is usually discarded, and the remainder of the lavage fluid is pooled and sent to the microbiology lab for microscopic analysis and quantitative culture.

The threshold for a positive BAL culture is 104 CFU/mL (1). The reported sensitivity and specificity of BAL cultures are shown in Table 29.3 (2,24). BAL does not have the highest sensitivity or specificity when compared to the other diagnostic methods in Table 29.3, but when sensitivity and specificity are considered together, BAL cultures have the highest overall accuracy for the diagnosis of pneumonia.

Inspection of BAL specimens for intracellular organisms can help in guiding initial antibiotic therapy until culture results are available. When intracellular organisms are present in more than 3% of the cells in the lavage fluid, the likelihood of pneumonia is over 90% (25). This is not done on a routine Gram's stain, but requires special processing and staining, and will require a specific request of the microbiology lab.

BAL can also be performed without the aid of bronchoscopy using a sheathed catheter like the one illustrated in Figure 29.6. This catheter (COMBICATH,, KOL Bio-Medical, Chantilly, VA) is inserted through a tracheal tube and advanced "blindly" until it wedges in a distal airway. An absorbable polyethylene plug at the tip of the catheter prevents contamination while the catheter is advanced. Once wedged, an inner cannula is advanced for the BAL, which is performed with 20 mL of sterile saline. Only 1 mL of BAL aspirate is required for culture and microscopic analysis.
Nonbronchoscopic BAL (also called mini-BAL because of the lower lavage volume) is a safe procedure that can be performed by respiratory therapists (26). Despite the uncertainty regarding the location of the catheter tip in relation to the region of suspected infection, the yield from quantitative cultures with mini-BAL is equivalent to the yield with bronchoscopic BAL (2,27).
FIGURE 29.6 Protected catheter for performing bronchoalveolar lavage without the aid of bronchoscopy. See text for explanation.

Aspiration of secretions through a bronchoscope produces false-positive cultures because of contamination as the bronchoscope is advanced through the tracheal tubes and upper respiratory tract (23). To eliminate this problem, a specialized brush called a protected specimen brush (PSB) was developed to collect uncontaminated secretions from the distal airways during bronchoscopy. The catheter design for the PSB is similar to the catheter design in Figure 29.6, and the brush is advanced from the inner cannula to collect samples from the distal airways.

The threshold for the diagnosis of VAP with PSB cultures is 103 CFU/mL (1,2), and the yield from PSB cultures is shown in Table 29.3 (2). A positive culture result has a low sensitivity (66%) but a high specificity (90%) for the diagnosis of VAP. Therefore, a negative PSB culture does not exclude VAP, but a positive PSB culture is considered evidence of VAP.

Pleural effusions are present in up to 50% of bacterial pneumonias (29), and these parapneumonic effusions typically do not require an intervention unless the conditions described next are indentified.

Antimicrobial therapy for pneumonia accounts for half of all antibiotic use in the ICU, and 60% of this antibiotic use is for suspected pneumonias that are not confirmed by bacteriologic studies (30). The aggressive use of antibiotics in cases of suspected VAP is fueled by studies showing that the mortality rate in VAP is increased by delays in initiating appropriate antibiotic therapy (31). However, there is a paucity of strong evidence directly linking VAP with fatal outcomes (4,6), and several studies show no direct relationship between VAP and mortality rate (7-9). (See the last section of this chapter for a comment on this situation.)

Empiric antimicrobial therapy for VAP should include coverage for Gram-negative aerobic bacilli and Staphylococcus aureus (i.e., the principal pathogens listed in Table 29.1), unless the results of a sputum Gram stain or BAL specimen indicates one prominent type of organism. Accepted regimens include pipericillin/tazobactam or a carbepenem (imipenem or meropenem) or antipseudomonal cephalosporin (ceftazidime or cefepime) plus vancomycin or linezolid (2). (See Chapter 52 for recommended dosing regimens for these antibiotics).
There is a tendency to continue empiric antibiotics despite negative culture results if patients are improving, but this is not justified unless there is some other evidence of a treatable infection (e.g., a sputum Gram stain showing a dense population of organisms) (32).

Antibiotic therapy of documented VAP will be dictated by the responsible pathogen(s) and by the antibiotic susceptibilities of pathogenic organisms in your hospital (i.e., the antibiogram).

The traditional duration of antibiotic therapy for VAP has been 14 to 21 days (2). However, there is evidence that 8 days of antibiotic therapy for VAP is as effective as 15 days of therapy (33), and the popular opinion now is that one week of antibiotic therapy is adequate for most patients with VAP.

1. Centers for Disease Control, National Healthcare Safety Network. Device-associated Module: Ventilator-Associated Event Protocol. January 2013. Available on the National Healthcare Safety Network website (www.cdc.gov/nhsn).
2. American Thoracic Society and Infectious Disease Society of America. Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. Am J Respir Crit Care Med 2005; 171:388-416.
3. Muscedere J, Dodek P, Keenan S, et al. for the VAP Guidelines Committee and the Canadian Critical Care Trials Group. Comprehensive evidence-based clinical practice guidelines for ventilator-associated pneumonia: Prevention. J Crit Care 2008; 23:126-137.

General Information, 1 = General Information. 4., 1 = Kollef MH. Ventilator-associated complications, including infection-related complications: The way forward. Crit Care Clin 2013; 29:33-50.. 5., 1 = Vincent J-L, Rello J, Marshall J, et al. International study of the prevalence and outcomes of infection in intensive care units. JAMA 2009; 302:2323-2329.. 6., 1 = Nguile-Makao M, Zahar JR, Francais A, et al. Attributable mortality of ventilator- associated pneumonia: respective impact of main characteristics at ICU admission and VAP onset using conditional logistic regression and multi-state models. Intensive Care Med 2010; 36:781-789.. 7., 1 = Rello J, Quintana E, Ausina A, et al. Incidence, etiology, and outcome of nosocomial pneumonia in mechanically ventilated patients. Chest 1991; 100:439-444.. 8., 1 = Papazian L, Bregeon F, Thirion X, et al. Effect of ventilator-associated pneumonia on mortality and morbidity. Am J Respir Crit Care 1996; 154:91-97.. 9. Bregeon F, Cias V, Carret V, et al. Is ventilator-associated pneumonia an independent risk factor for death? Anesthesiology 2001; 94:554-560., 1 = 9. Bregeon F, Cias V, Carret V, et al. Is ventilator-associated pneumonia an independent risk factor for death? Anesthesiology 2001; 94:554-560.

14., 1 = Wunderink RG. Clinical criteria in the diagnosis of ventilator-associated pneumonia. Chest 2000; 117:191S-194S.. 15., 1 = Fagon JY, Chastre J, Hance AJ, et al. Detection of nosocomial lung infection in ventilated patients: use of a protected specimen brush and quantitative culture techniques in 147 patients. Am Rev Respir Dis 1988; 138:110-116.. 16., 1 = Timsit JF . Misset B, Goldstein FW, et al. Reappraisal of distal diagnostic testing in the diagnosis of ICU-acquired pneumonia. Chest 1995; 108:1632-1639.. 17., 1 = Louthan FB, Meduri GU. Differential diagnosis of fever and pulmonary densities in mechanically ventilated patients. Semin Resp Infect 1996; 11:77-95.. 18., 1 = Singh N, Falestiny MN, Rogers P, et al. Pulmonary infiltrates in the surgical ICU. Chest 1998; 114:1129-1136.

, 1 = Microbiological Evaluation. 19., 1 = Luna CM, Videla A, Mattera J, et al. Blood cultures have limited value in predicting severity of illness and as a diagnostic tool in ventilator-associated pneumonia. Chest 1999; 116:1075-1084.. 20., 1 = Rankin JA, Marcy T, Rochester CL, et al. Human airway macrophages. Am Rev Respir Dis 1992; 145:928-933.. 21., 1 = Wong LK, Barry AL, Horgan S. Comparison of six different criteria for judging the acceptability of sputum specimens. J Clin Microbiol 1982; 16:627-631.. 22., 1 = Cook D, Mandell L. Endotracheal aspiration in the diagnosis of ventilator-associated pneumonia. Chest 2000; 117:195S-197S.. 23., 1 = Meduri GU, Chastre J. The standardization of bronchoscopic techniques for ventilator-associated pneumonia. Chest 1992; 102:557S-564S.. 24., 1 = Torres A, El-Ebiary M. Bronchoscopic BAL in the diagnosis of ventilator-associated pneumonia. Chest 2000; 117:198S-202S.. 25., 1 = Veber B, Souweine B, Gachot B, et al. Comparison of direct examination of three types of bronchoscopy specimens used to diagnose nosocomial pneumonia. Crit Care Med 2000; 28:962-968.. 26., 1 = Kollef MH, Bock KR, Richards RD, Hearns ML. The safety and diagnostic accuracy of minibronchoalveolar lavage in patients with suspected ventilator-associated pneumonia. Ann Intern Med 1995; 122:743-748.. 27., 1 = Campbell CD, Jr . Blinded invasive diagnostic procedures in ventilator-associated pneumonia. Chest 2000; 117:207S-211S.. 28., 1 = Shorr AF, Sherner JH, Jackson WL, Kollef MH. Invasive approaches to the diagnosis of ventilator-associated pneumonia: a meta-analysis. Crit Care Med 2005; 33:46-53.. 29., 1 = Light RW, Meyer RD, Sahn SA, et al. Parapneumonic effusions and empyema. Clin Chest Med 1985; 6:55-62.

30., 1 = Bergmanns DCJJ, Bonten MJM, Gaillard CA, et al. Indications for antibiotic use in ICU patients: a one-year prospective surveillance. J Antimicrob Chemother 1997; 111:676-685.. 31., 1 = Iregui M, Ward S, Sherman G, et al. Clinical importance of delays in the initiation of appropriate antibiotic treatment for ventilator-associated pneumonia. Chest 2002; 122:262-268.. 32., 1 = Singh N, Rogers P, Atwood CW, et al. Short-course empiric antibiotic therapy for patients with pulmonary infiltrates in the intensive care unit. Am J Respir Crit Care Med 2000; 162:505-511.. 33., 1 = Chastre J, Wolff M, Fagon J-Y, et al. Comparison of 8 vs. 15 days of antibiotic therapy for ventilator-associated pneumonia in adults. JAMA 2003; 290:2588-2598.

See, and then reason and compare and control. But see first.
Discontinuing mechanical ventilation (also known as weaning from mechanical ventilation) is a rapid and uneventful affair for most patients, but for one of every four or five patients, the transition from mechanical ventilation to unassisted breathing is a prolonged process that can consume almost half of the time spent on a ventilator. This chapter describes the process of removing patients from mechanical ventilation, and the difficulties that can occur in the transition to unassisted breathing (1-4).

The duration of mechanical ventilation is primarily determined by the severity of the cardiopulmonary disorder that is responsible for the ventilatory support. The measures described in this section can also contribute to shorter stays on the ventilator by facilitating the attempts to discontinue mechanical ventilation when the time arises.

Although the diaphragm is an involuntary muscle that contracts automatically with each lung inflation, mechanical ventilation "unloads" the diaphragm and can promote diaphragm weakness (5). This ventilator-induced diaphragm dysfunction is particularly prominent when contractions of the diaphragm are suppressed (e.g., during controlled mechanical ventilation), and is attenuated when the diaphragm is allowed to contract and initiate a ventilator breath (i.e., during patient-triggered ventilation). This is demonstrated inFigure 30.1, which shows that controlled ventilation (where diaphragmatic contractions are suppressed) is associated with a significant (~40%) reduction in the power output of the diaphragm, while assisted mechanical ventilation ( where the diaphragm is allowed to initiate the ventilator breath) is associated with a much smaller and nonsignificant (~20%) decrement in diaphragmatic power (6).
FIGURE 30.1 The power of diaphragmatic contractions (equivalent to the product of contractile force and velocity) during spontaneous breathing (control) and after 3 days of assisted (i.e., patient-triggered) mechanical ventilation (AMV) or controled mechanical ventilation (CMV). Note that CMV (but not AMV) was associated with a significant reduction in the power output of the diaphragm. Data from Reference 5.
Observations like those in Figure 30.1 indicate that allowing patients to trigger ventilator breaths (e.g., by avoiding controlled ventilation and neuromuscular paralysis) will help to preserve the strength of the diaphragm, and this should facilitate the transition from ventilatory support to spontaneous breathing. (The role of diaphragm weakness in weaning from the ventilator is described later in the chapter.)

Prolonged bed rest and physical inactivity during mechanical ventilation often leads to deconditioning and generalized muscle weakness, and this is considered a contributing factor in ventilator-dependent patients who have difficulty in the transition to unassisted breathing. Supporting this contention are studies showing that early physical rehabilitation, including ambulation, is associated with a shorter duration of mechanical ventilation (7). Therefore, early and regular physical rehabilitation (including ambulation in patients who are awake and hemodynamically stable) is encouraged in selected patients to facilitate the transition to spontaneous breathing.

Several studies have shown that both deep sedation (where the patient is not arousable) and sustained use of benzodiazepines (midazolam and lorazepam) for sedation are associated with delays in discontinuing mechanical ventilation (8). As a result of these studies, the most recent guidelines on sedation in ventilator-dependent patients (8) include the following recommendations:
1. Maintain a light level of sedation, where patients are easily aroused.
2. Avoid or minimize the use of benzodiazepines for sedation. Non-benzodiazepine sedatives include propofol and dexmedetomidine, which are described in Chapter 51.

y PaQ,/FiQ; =150-200 mm Hg with FlO, =50% and PEEP <8 cm H,0.
y PaCO, normal or at baseline levels.
Patient is able to initiate an inspiratory effort.

y No evidence of myocardial ischemia.
Heart rate <140 beats/minute.
WY Blood presure adequate with minimal or no vasopressors.

The management of ventilator-dependent patients requires constant vigilance for signs that ventilatory support may no longer be necessary. These signs are listed in Table 30.1. Candidates for possible removal of mechanical ventilation should have adequate gas exchange in the lungs (i.e., PaO>2/FIO2 >150-200 mm Hg and a normal or baseline arterial PCO2) while breathing non-toxic concentrations of oxygen (FIO2 <50%) and at low levels of PEEP (<8 cm H,0). In addition, there should be no evidence of myocardial ischemia, severe tachycardia (>140 beats/min), circulatory shock, or ongoing sepsis (e.g., fever).
When the criteria in Table 30.1 have been satisfied, the patient should be removed from the ventilator briefly to obtain the measurements listed in Table 30.2. These measurements (which are called "weaning parameters") are used to predict the likelihood of success or failure during a trial of spontaneous or unassisted breathing. However, as indicated by the range of likelihood ratios, each of these parameters can be a poor predictor of success or failure in individual patients. (Monitoring serial changes in these parameters during a trial of spontaneous breathing may have a greater predictive value than measurements obtained immediately after removing ventilatory support) (9). Because of the variable predictive value of the weaning parameters in Table 30.2, the emerging consensus is that trials of spontaneous breathing can begin when the readiness criteria in Table 30.1 are satisfied.

Tidal Volume (\V7), preaeecer na “Threshold for ‘Success = 4-6 mL/kg. Tidal Volume (\V7), Range of Likelihood Ratios® = 0.7 -3.8. Respiratory Rate (RR), preaeecer na “Threshold for ‘Success = 30-38 bpm. Respiratory Rate (RR), Range of Likelihood Ratios® = 1.0-3.8. RRV; Ratio, preaeecer na “Threshold for ‘Success = 60-105 bpmv/L. RRV; Ratio, Range of Likelihood Ratios® = 08-47. Maximum Inspiratory Pressure (Plax), preaeecer na “Threshold for ‘Success = -15 to -30 cm H,0. Maximum Inspiratory Pressure (Plax), Range of Likelihood Ratios® = 1.0 -3.0
TAIl measuraments should ba obtained during the first 1-2 minutes of spontaneous breathing.
SThe likelihood ratio is tha likelihood that the measurement will pradict success, divided by the likelihood that tha measurement will preciict failure. From Reference 2.

The traditional approach to discontinuing mechanical ventilation emphasized a gradual reduction in ventilatory support (over hours to days), and this created unnecessary delays in removing ventilatory support for patients who were capable of unassisted breathing. (This delayed approach is still evident in the practice of placing patients back on a ventilator at night to "rest them".) In contrast, spontaneous breathing trials (SBTs) are conducted with no ventilatory support, so that patients capable of unassisted breathing can be identified quickly. There are two methods of conducting an SBT, as described next.

SBTs are often conducted while the patient breathes through the ventilator circuit. The advantage of this method is the ability to monitor the tidal volume (V7) and respiratory rate (RR), since rapid, shallow breathing (indicated by an increase in the RR/V;+ ratio) is a common breathing pattern in patients who fail the SBT (9). The drawback of this method is the resistance to breathing through the ventilator circuit, which can increase the work of breathing (particularly in patients who are breathing rapidly).

To counteract the resistance to breathing through the ventilator circuit, low levels of pressure support (5 cm HO) are routinely used when SBTs are conducted through the ventilator circuit. (For a description of pressure support, see pages 511-512.) However, as demonstrated in Figure 30.2, the use of pressure support results in only a small and insignificant decrease in the work of breathing (10). These results suggest that the benefit of low-level pressure support is minimal, and clinically irrelevant.

SBTs can also be conducted when the patient is disconnected from the ventilator, using the simple circuit design illustrated in Figure 30.3. A source of O> (usually from a wall outlet) is delivered to the patient at a high flow rate (higher than the patient's inspiratory flow rate), and this not only facilitates the inhalation of O5, it also carries exhaled CO> out to the atmosphere, to prevent CO> rebreathing. Because this circuit employs a T-shaped adapter, it is popularly known as a T-piece circuit.
FIGURE 30.2 The work of breathing (in joules per liter) during spontaneous breathing trials conducted with and without the aid of pressure support ventilation (PSV) at 5 cm H20, and one hour after extubation. The asterisk indicates a significant difference at the p=0.05 level. Data from Reference 10.
The work of breathing is considered to be lower when breathing through a T-piece circuit compared to a ventilator circuit (although this is unproven). The major disadvantage of the T-piece circuit is the inability to monitor the respiratory rate and tidal volume.
FIGURE 30.3 Simpk breathing circuit for spontaneous breathing trials that are independent of the ventilator. The Tshaped adapter in the circuit is responsible for the popular term T-piece that is used for this circuit.

There is no clinically proven advantage with either method of SBT (3). However, the Tpiece method has the following theoretical advantages: (a) it is better suited for patients with increased ventilatory demands (which is a common cause of difficulty during an SBT), and (b) it is a closer approximation of the normal conditions for breathing than breathing through a ventilator circuit with low-level pressure support as an adjunct.

Rapid breathing during SBTs may be the result of breathlessness (dyspnea) provoked by anxiety rather than ventilatory failure (12). Monitoring the tidal volume can be useful in distinguishing anxiety from ventilatory failure; i.e., anxiety produces hyperventilation, where the respiratory rate and tidal volume are both increased, whereas ventilatory failure usually produces rapid and shallow breathing, where the respiratory rate is increased but the tidal volume is decreased. Therefore, for the patient who develops rapid respirations during a trial of unassisted breathing, an increased tidal volume suggests anxiety as the underlying problem, while a decreased tidal volume suggests ventilatory failure. Worsening of gas exchange may not distinguish between anxiety and ventilatory failure for the reasons described next.

Regardless of the cause, rapid breathing during SBTs can be detrimental in several ways, as summarized below.
1. In patients with asthma and COPD, rapid breathing promotes hyperinflation and intrinsic PEEP, which can: (a) decrease the cardiac output, (b) increase dead space ventilation, (c) decrease lung compliance, and (d) produce diaphragm dysfunction by flattening the diaphragm.
2. For patients with infiltrative lung disease (e.g., ARDS), rapid breathing reduces ventilation in diseased lung regions (where time constants for alveolar ventilation are prolonged), and this promotes alveolar collapse and hypoxemia.
3. For all patients with acute respiratory failure, rapid breathing can increase whole-body O> consumption, which places an added burden on systemic O> transport.

If ventilatory failure is suspected as the cause of rapid breathing, the patient should be placed back on the ventilator. If anxiety is suspected as the culprit, administration of a sedative drug should be considered. Opiates may be preferred in this situation because they are particularly effective in curbing the sensation of dyspnea (13). Despite the fear of opiate use in patients with COPD, these drugs have been used safely for relief of dyspnea in patients with severe or end-stage COPD (13).
A failed trial of spontaneous breathing is usually a sign that the pathologic condition requiring ventilatory support needs further improvement. However, there are other conditions that create difficulties in discontinuing mechanical ventilation, and the principal ones are described next.
FIGURE 30.4 Mixed venous O92 saturation (SvO2) during successful and failed trials of spontaneous breathing. Data
There is surprisingly little information on methods for correcting the cardiac dysfunction that develops during spontaneous breathing trials. Patients who develop systolic dysfunction should benefit from continuous positive airway pressure (CPAP), which promotes cardiac output by cancelling the afterload-increasing effect of negative intrathoracic pressure (20,21). Since CPAP is delivered noninvasively, it will not prevent the removal of mechanical ventilation, including extubation.
values for predicting successful trials of spontaneous breathing (see Table 30.2). Unfortunately, patients with acute respiratory failure have difficulty performing the maneuvers involved in the measurement of PIpj3,. AS a result, the Pl max is not measured regularly in patients who require ventilatory support.
ULTRASOUND: Ultrasound has recently emerged as a potential method of assessing diaphragm strength. The ultrasound measures of diaphragm strength include the thickness of the diaphragm, and the length of excursion of the diaphragm during inspiration (29). In a preliminary study that used ultrasound assessments of diaphragm strength in patients who were weaned from mechanical ventilation, there was a significant correlation between failed SBTs and diaphragm weakness identified by ultrasound (29).
The reliability of ultrasound measurements for detecting diaphragm weakness is uncertain at the present time because the criteria for diaphragm weakness are arbitrary, and have not been validated. (This will require ultrasound measurements in large numbers of normal subjects to determine the range of normal measurements.)

When respiratory muscle weakness is strongly suspected, trials of spontaneous breathing should continue, but should be terminated before patients show evidence of respiratory distress (to avoid aggravating the weakness). Strategies designed to promote muscle strength, such as patient-triggered ventilation and physical rehabilitation (described earlier), are considered particularly important in patients with documented muscle weakness.

Once there is evidence that mechanical ventilation is no longer necessary, the next step is to remove the artificial airway. This section focuses on removing endotracheal tubes, although some of the principles also apply to removing tracheostomy tubes. (The removal of tracheostomy tubes is a more gradual process, and often occurs after patients leave the ICU.)
Extubation should never be performed to reduce the work of breathing, because the work of breathing can actually increase after extubation, as demonstrated in Figure 30.2. (The increased work of breathing can be the result of an increased respiratory rate or breathing through a narrowed glottis, but it occurs in patients who tolerate extubation, so it is not always a cause for concern.) The considerations that must be addressed prior to extubation include: (a) the patient's ability to clear secretions from the airways, and (b) the risk of symptomatic laryngeal edema following extubation.

The ability to protect the airway from aspirated secretions is determined by the strength of the gag and cough reflexes. Cough strength can be assessed by holding a piece of paper 1-2 cm from the end of the endotracheal tube and asking the patient to cough. If wetness appears on the paper, the cough strength is considered adequate (30). Diminished strength or even absence of cough or gag reflexes will not necessarily prevent extubation, but will identify patients who need special precautions to prevent aspiration.

Upper airway obstruction from laryngeal edema is the major cause of failed extubations, and is reported in 5-22% of patients who have been intubated for longer than 36 hours (3,31,32). Contributing factors include difficult and prolonged intubation, endotracheal tube diameter, and self-extubation.

The cuff-leak test measures the volume of inhaled gas that escapes through the larynx when the cuff on the endotracheal tube is deflated. This test is designed to determine the risk of symptomatic upper airway obstruction from laryngeal edema after the endotracheal tube is removed. According to a recent analysis of the cuff leak test (33), the absence of an air leak indicates a high risk of upper airway obstruction following extubation, but the presence of an air leak does not indicate a low risk of upper airway obstruction following extubation, regardless of the volume of the leak. The value of the cuff leak test has been debated for years, and the test is not universally accepted. Since the results of a cuff leak test do not alter patient management, including the decision to extubate, the clinical relevance of the test is unproven.

Two clinical studies have shown that pretreatment with intravenous corticosteroids ( methylprednisolone, 20-40 mg every 4-6 hrs) for 12 to 24 hours prior to extubation results in fewer cases of laryngeal edema and upper airway obstruction following extubation, and fewer reintubations (33,34). The results of one of these studies is shown in Figure 30.5. Steroid pretreatment in this study consisted of three doses of intravenous methylprednisolone (20 mg every 4 hours), with the first dose given 12 hours prior to a planned extubation. Note that this pretreatment was associated with about a 7-fold decrease in the incidence of symptomatic laryngeal edema following extubation, and a 50% drop in reintubation rate.
Although the use of corticosteroids for "anything that swells" is questionable (35), the results of the study shown in Figure 30.5 are compelling enough to consider a brief period (12 to 24 hrs) of corticosteroid therapy prior to planned extubations, particularly in patients who have a high risk of post-extubation laryngeal edema (e.g., from prior selfextubations). A single dose of methylprednisolone (40 mg IV) given one hour prior to extubation did not reduce the incidence of post-extubation laryngeal edema in one study (36), so there is no reason to administer steroids only at the time of extubation.

The first sign of a significant laryngeal obstruction may be stridorous breathing (noisy breathing), also called stridor. The sounds may be high-pitched and wheezy, or lowpitched and harsh, but they are always audible without a stethoscope, and they are always most apparent during inspiration. This inspiratory prominence is due to the extrathoracic location of laryngeal obstruction because negative intrathoracic pressures generated during inspiration are transmitted to the upper airways outside the thorax, and this results in a narrowing of the extrathoracic airways during inspiration. Therefore, extrathoracic obstructions are always magnified during inspiration.
FIGURE 30.5 Results of a large, multicenter study showing the effects of pretreatment with a corticosteroid (methylprednisolone, 20 mg IV every 4 hrs for the 12 hours prior to extubation) on the incidence of post-extubation laryngeal edema and the rate of reintubation. Data from Reference 30.
Post-extubation stridor is apparent within 30 minutes of extubation in a large majority (~80%) of cases (30), but delays in appearance of up to 2 hours can occur (personal observation). Reintubation is not always re-quired, but close scrutiny is required because there is no proven method for reducing laryngeal edema after extubation.

Inhalation of an epinephrine aerosol (2.5 mL of 1% epinephrine) is a popular practice for post-extubation stridor However, while effective in children (36), this practice is unproven in adults. Aerosol therapy with a racemic mixture of epinephrine (which has equal amounts of the levoand dextroisomers) is also popular for post-extubation stridor, but clinical studies in children have shown no advantage with racemic epinephrine over standard (I-isomer) epinephrine (37).

Noninvasive ventilation (which is described on pages 526-531) is effective in reducing the rate of reintubation when used immediately after extubation in patients with a high risk of laryngeal edema (38), but similar success has not been demonstrated in patients who develop post-extubation respiratory failure (39). Thus, the benefit of noninvasive ventilation occurs when it is used as a preventive measure early after extubation.

The ultimate goal of mechanical ventilation is to no longer need it, and the introductory quote by Sir William Osler is intended to emphasize that vigilance is required to identify when you have reached this goal in a timely manner. Vigilance involves early recognition of candidates for trials of unassisted breathing (with daily assessments using the readiness criteria in Table 30.1), and early recognition that the candidates can sustain spontaneous ventilation (with trials of spontaneous breathing). This approach will free patients from mechanical ventilation without delay, and end the misfortune of being tethered to a machine.

- 1. MacIntyre NR, Cook DJ, Ely EW Jr, et al. Evidence-based guidelines for weaning and discontinuing ventilatory support: a collective task force facilitated by the American College of Chest Physicians, the American Associa-tion for Respiratory Care, and the American College of Critical Care Med-icine. Chest 2001; 120(Suppl):375S—395S.
Spontaneous Breathing Trials, 1 = Spontaneous Breathing Trials. 10., 1 = Mehta S, Nelson DL, Klinger JR, et al. Prediction of post-extubation work of breathing. Crit Care Med 2000; 28:1341-1346.. 11., 1 = Ely W, Baker AM, Dunagen DP, et al. Effect of duration of mechanical ventilation of identifying patients capable of breathing spontaneously. N Engl J Med 1996; 335:1864-1869.. 12., 1 = Bouley GH, Froman R, Shah H. The experience of dyspnea during weaning. Heart Lung 1992; 21:471-476.. 13., 1 = Raghavan N, Webb K, Amornputtisathaporn N, O'Donnell DE. Recent advances in pharmacotherapy for dyspnea in COPD. Curr Opin Pharmacol 2011; 11:204-210.

isolated diaphragm weakness. Am Rev Respir Dis 1988; 138:862-866.. 24., 1 = Hudson LD, Lee CM. Neuromuscular sequelae of critical illness. N Engl J Med 2003; 348:745-747.. 25., 1 = Benotti PN, Bistrian B. Metabolic and nutritional aspects of weaning from mechanical ventilation. Crit Care Med 1989; 17:181-185.. 26., 1 = Malloy DW, Dhingra S, Solren F, et al. Hypomagnesemia and respiratory muscle power. Am Rev Respir Dis 1984; 129:427-431.. 27., 1 = Mier-Jedrzejowicz A, Brophy C, Moxham J, Geen M. Assessment of diaphragm weakness. Am Rev Respir Dis 1988; 137:877-883.. 28., 1 = Bruschi C, Cerveri I, Zoia MC. et al. Reference values for maximum respiratory mouth pressures: A population-based study. Am Rev respir Dis 1992; 146:790-793.. 29., 1 = Kim WY, Suh HJ, Hong S-S, et al. Diaphragm dysfunction assessed by
ultrasonography: Influence on weaning from mechanical ventilation. Crit Care Med 2011; 39:2627-2630.

Life is a struggle, not against sin, not against money power... but against hydrogen ions.
H.L. Mencken

Alfred North Whitehead
Managing ICU patients without a working knowledge of acid-base disorders is like trying to clap your hands when you have none; i.e., it simply can't be done. This chapter presents a structured approach to the identification of acid-base disorders based on the traditional relationships between the pH, PCO , and bicarbonate (HCO3) concentration in plasma. Also included is a section on the evaluation of metabolic acidosis using the anion gap and a measurement known as the "gap-gap." Alternative approaches to acid-base analysis, such as the "Stewart method," are not included here because it is unlikely, at the present time, that these methods will replace the traditional approach to acid-base analysis.

The hydrogen ion concentration [H+] in aqueous solutions is traditionally expressed by the pH, which apparently means the power of hydrogen, and is a logarithmic function of the [H+]; i.e., pH = log (1/[H*]) = -log [H*]
(31.1)
The physiological range of pH and corresponding [H+] is shown in Table 31.1. The normal pH of plasma is indicated as 7.40, which corresponds to a [H+] of 40 nEq/L.

The relationships in Table 31.1 illustrate 3 unfortunate features of the pH: (a) it is a dimensionless number, which has no relevance in chemical or physiological events, (b) it varies in the opposite direction to changes in [H+], and (c) changes in pH are not linearly related to changes in [H+]. Note that as the pH decreases, the changes in [H+] become gradually larger with each change in pH. This means that changes in pH will have different implications for acid-base balance at different points along the pH spectrum. Although it is unlikely that the pH will be abandoned, it is not a representative measure of the acid-base events in the body.

6.9, [HA] (nEq/t) = 1.26. 6.9, PH = 7.4. 6.9, [HA] (nEq/L) = 40. 7.0, [HA] (nEq/t) = 100. 7.0, PH = 7.5. 7.0, [HA] (nEq/L) = 32. 74, [HA] (nEq/t) = 80. 74, PH = 7.6. 74, [HA] (nEq/L) = 25. 7.2, [HA] (nEq/t) = 64. 7.2, PH = 7.7. 7.2, [HA] (nEq/L) = 20. 7.3, [HA] (nEq/t) = 50. 7.3, PH = 7.8. 7.3, [HA] (nEq/L) = 16

Also evident in Table 31.1 is the fact that [H+] is expressed as nanoequivalents per liter (nEq/L). One nanoequivalent is one-millionth of a milliequivalent (1 nEq = 1x10-6 mEq), so hydrogen ions are about a million times less dense than the principal ions in extracellular fluid (sodium and chloride), whose concentration is expressed in mEq/L. This gives hydrogen ions the status of a trace element. How can such a small quantity of an ion have all the effects attributed to acidosis and alkalosis? Other trace elements certainly have important biological effects, but it is also possible that changes in the [H+] are just one of several physicochemical changes that are taking place in the extracellular fluid. This would explain why the same degree of acidosis is more life-threatening in lactic acidosis than in ketoacidosis (as described in the next chapter); i.e., the acidosis is not the problem.

According to traditional concepts of acid-base physiology, the [H+] in extracellular fluid is determined by the balance between the partial pressure of carbon dioxide (PCO) and the concentration of bicarbonate (HCO3) in the fluid. This relationship is expressed as follows (1):
[H*] = 24 x (PCO,/HCO;)
(31.2)
The PCO2/HCO3 ratio identifies the primary acid-base disorders and secondary responses, which are shown in Table 31.2.

According to equation 31.2, a change in either the PCO> or the HCO3 will cause a change in the [H+] of extracellular fluid. When a change in PCO) is responsible for a change in [H+], the condition is called a respiratory acid-base disorder: an increase in PCO) is a respiratory acidosis, and a decrease in PCO; is a respiratory alkalosis. When a change in HCO3 is responsible for a change in [H+], the condition is called a metabolic acid-base disorder: a decrease in HCO3 is a metabolic acidosis, and an increase in HCO3 is a metabolic alkalosis.

tSecondary rasponses are always in the sama diraction as tha primary change.

Respiratory Acidosis, ATH#] = APCO,/AHCO,.Primary Change = TPCO,. Respiratory Acidosis, ATH#] = APCO,/AHCO,.Secondary Response? = THGO,. Respiratory Alkalosis, ATH#] = APCO,/AHCO,.Primary Change = IPCO,. Respiratory Alkalosis, ATH#] = APCO,/AHCO,.Secondary Response? = LHCO,. Metabolic Acidosis, ATH#] = APCO,/AHCO,.Primary Change = LHCO,. Metabolic Acidosis, ATH#] = APCO,/AHCO,.Secondary Response? = PCO;. Metabolic Alkalosis, ATH#] = APCO,/AHCO,.Primary Change = THCO,. Metabolic Alkalosis, ATH#] = APCO,/AHCO,.Secondary Response? = TPCO,

Secondary responses are designed to limit the change in [H+] produced by the primary acid-base disorder, and this is accomplished by changing the other component of the PaCO>/HCO3 ratio in the same direction. For example, if the primary problem is an increase in PaCO> (respiratory acidosis), the secondary response will involve an increase in HCO3, and this will limit the change in [H+] produced by the increase in PaCO>. Secondary responses should not be called "compensatory responses" because they do not completely correct the change in [H+] produced by the primary acid-base disorder (2). The specific features of secondary responses are described next. The equations described in the next section are included in Figure 31.1.

Responses to Respiratory Acid-Base Disorders, 1 = Responses to Respiratory Acid-Base Disorders. The secondary response to changes in PaCO 2 occurs in the kidneys, where HCO 3 absorption in the proximal tubes is adjusted to produce the appropriate change in plasma HCO 3 . This renal response is relatively slow, and can take 2 or 3 days to reach completion. Because of the delay in the secondary response, respiratory acid-base disorders are separated into acute and chronic disorders. Acute Respiratory Disorders, 1 = The secondary response to changes in PaCO 2 occurs in the kidneys, where HCO 3 absorption in the proximal tubes is adjusted to produce the appropriate change in plasma HCO 3 . This renal response is relatively slow, and can take 2 or 3 days to reach completion. Because of the delay in the secondary response, respiratory acid-base disorders are separated into acute and chronic disorders. Acute Respiratory Disorders. Acute changes in PaCO 2 have a small effect on the plasma HCO 3 , as indicated in the following two equations (2)., 1 = Acute changes in PaCO 2 have a small effect on the plasma HCO 3 , as indicated in the following two equations (2).. For acute respiratory acidosis:, 1 = (31.7). For acute respiratory alkalosis:, 1 = (31.8). EXAMPLE : For an acute increase in PaCO 2 to 60 mm Hg, the ∅ HCO 3 is 0.1×20 = 2 mEq/L for an acute respiratory acidosis, and 0.2×20 = 4 mEq/L for an acute respiratory alkalosis. Neither of these changes would be recognized as significant., 1 = 
FIGURE 31.1 Predictive equations for evaluating secondary responses to primary acid-base disorders. All equations are from Reference 2.

The renal response to an increase in PaCO> is an increase in HCO3 reabsorption in the proximal renal tubules, which raises the plasma HCO3 concentration. The response to a decrease in PaCO, is a decrease in renal HCO3 reabsorption, which lowers the plasma HCO3 concentration. The magnitude of this response is similar, regardless of the directional change in PaCO, so the equation below applies to both chronic respiratory acidosis and alkalosis.
AHCO, = 0.4 x APaCO,
(31.9)
Using a normal PaCO> of 40 mm Hg and a normal HCO3 of 24 mEq/L, the above equation can be rewritten as follows:
For chronic respiratory acidosis:
Expected HCO, = 24 + [0.4 x (current PaCO, — 40)]
For chronic respiratory alkalosis:
Expected HCO, = 24 -[0.4 x (40 -current PaCO,)]
(31.11)
EXAMPLE: For an increase in PaCO> to 60 mm Hg that persists for at least a few days, the 2PaCO, is 60 — 40 = 20 mm Hg, the 2HCO3 is 0.4x20 = 8 mEq/L, and the expected HCO3 is 24 + 8 = 32 mEq/L.
(31.10)

The following is a structured, rule-based approach to the diagnosis of primary, secondary, and mixed acid-base disorders using the relationships between the [H+], PCOz, and HCO3 concentration described previously. Several examples are included as instructional aids. The reference ranges for arterial pH, PCO2, and HCO3 are shown below.
pH = 7.36-7.44
PCO, = 36-44 mm Hg
HCO3 = 22-26 mEq/L

In the first stage of the approach, the PaCO, and pH are used to identify the primary acid-base disorder.
Rule
If the
PaCO, and/or
the pH
is outside
the normal
range, there
is an
acid-base
1:
Rule disorder.
If the
PaCO>
and pH
are both
abnormal, compare
the directional
change.
2a
If the
PaCO>
and pH
change in
the same
direction, there
is
a
primary metabolic
“acid-base disorder.
- ab If the PaCO> and pH change in opposite directions, there is a primary respiratory acid-base disorder.
EXAMPLE:
Consider
a
case where
the arterial
pH
=
7.23
and the
PaCO>
=
23
mm
Hg.
The pH
and
PaCO;
are both
reduced
(indicating
a
disorder)
and is
low
(indicating an
acidosis), so
the diagnosis
is
a
acidosis.
Rule If only the pH or PaCQ> is abnormal, the condition is a mixed metabolic and
- 3: respiratory disorder (i.e., equal and opposite disorders).
If the PaCO> is abnormal, the directional change in PaCO> identifies the type of
3a.
3b.
respiratory disorder
(e.g., high
PaCO>
indicates
a
respiratory acidosis),
and the
opposing metabolic
If the
pH
disorder disorder.
is abnormal,
(e.g., the
directional low
pH
indicates change
a
pH
identifies acidosis)
the and
type of
the opposing
metabolic respiratory
disorder.

EXAMPLE: Consider a case where the arterial pH = 7.38 and the PaCO> = 55 mm Hg. Only the PaCO, is abnormal, so there is a mixed metabolic and respiratory disorder. The PaCOQ> is elevated, indicating a respiratory acidosis, so the metabolic disorder must be a metabolic alkalosis. Therefore, this condition is amixed respiratory acidosis and metabolic alkalosis. Both disorders are equivalent in severity because the pH is normal.
the pH

The final stage of this approach is for patients with a metabolic acidosis, where the use of measurements called gaps can help to uncover the underlying cause of the acidosis. These are described in the next section.

There are numerous potential sources of a metabolic acidosis in critically ill patients, and the measurements described in this section are designed to help in the search for the culprit.

The anion gap is a rough estimate of the relative abundance of unmeasured anions, and is used to determine if a metabolic acidosis is due to an accumulation of non-volatile acids (e.g., lactic acid) or a primary loss of bicarbonate (e.g., diarrhea) (6,7).

To achieve electrochemical balance, the concentration of negatively charged anions must equal the concentration of positively charged cations. This electrochemical balance is expressed in the equation shown below using electrolytes that are routinely measured, including sodium (Na), chloride (CL), and bicarbonate (HCO3), as well as the unmeasured cations (UC) and unmeasured anions (UA).
Na+ UC = (CL+ HCO.) +UA
(31.12)
Rearranging the terms in this equation yields the following relationships:
Na -(CL + HCO;) =UA-UC
(31.13)
The difference (UA — UC) is a measure of the relative abundance of unmeasured anions, and is called the anion gap (AG).
AG = Na(CL + HCO,)
(31.14)
Reference Range: The original reference range for the AG was 12+4 mEq/L (range = 8 to 16 mEq/L) (7). With subsequent improvements in automated systems that measure serum electrolytes, the reference range for the AG has decreased to 7+4 mEq/L (range = 3 to 11 mEq/L) (8).

The unmeasured anions and cations that normally contribute to the anion gap are shown in Table 31.3. Note that albumin is the principal unmeasured anion, and the principal determinant of the anion gap. Albumin is a weak (i.e., poorly dissociated) acid that contributes about 3 mEq/L to the AG for each 1 g/dL of albumin in plasma (at a normal pH) (3). A low albumin level in plasma will lower the AG, and this could mask the presence of an unmeasured anion (e.g., lactate) that is contributing to a metabolic acidosis. Since hypoalbuminemia is present in as many as 90% of ICU patients (9), the following formula for the "corrected AG" (AGc) has been proposed to include the contribution of albumin:
AGc = AG + 2.5 x (4.5 — [albumin in g/dL))
(31.15)
(4.5 represents the normal concentration of albumin in plasma). For a patient with an AG of 10 mEq/L and a plasma albumin of 2 g/dL, the AGc is 10 + (2.5x2.5) = 16 mEq/L, which represents a 60% increase in the AG.

Albumin (15 mEq/L), ‘Unmeasured Cations = Calcium (5 mEq/L). Organic Acids (5 mEq/L) Phosphate (2 mEq/L), ‘Unmeasured Cations = Potassium (4.5 mEq/L). Sulfate (1 mEq/L), ‘Unmeasured Cations = Magnesium (1.5 mEq/L). Total UA: (23 mEq/L), ‘Unmeasured Cations = Total UC: (11 mEq/L). Anion Gap = UA - UC = 12 mEq/L, ‘Unmeasured Cations = Anion Gap = UA - UC = 12 mEq/L

The AG can be used to identify the underlying mechanism of a metabolic acidosis, which then helps to identify the underlying clinical condition. An elevated AG occurs when there is an accumulation of fixed or non-volatile acids (e.g., lactic acidosis), while a normal AG occurs when there is a primary loss of bicarbonate (e.g., diarrhea) (7). Table 31.4 shows the causes of metabolic acidosis grouped according to the AG.
HIGH AG: Common causes of high AG metabolic acidosis are lactic acidosis, diabetic ketoacidosis, and advanced renal failure (where there is loss of H+ secretion in the distal tubules of the kidneys). Also included are toxic ingestions of methanol (which produces formic acid), ethylene glycol (which produces oxalic acid), and salicylates (which produce Salicylic acid) (10).

Lactic Acidosis, Normal AG = Diarrhea. Ketoacidosis, Normal AG = Isotonic Saline Infusion. End-Stage Renal Failure, Normal AG = Early Renal Insufficiency. Methanol Ingestion, Normal AG = Renal Tubular Acidosis. Ethylene Glycol Ingestion, Normal AG = Acetazolamide. Salicylate Toxicity, Normal AG = Uretercenterostomy
NORMAL AG: Common causes of a normal AG metabolic acidosis are diarrhea, saline infusion (see Figure 12.3), and early renal failure (where there is loss of bicarbonate reabsorption in the proximal tubules). The loss of HCO3 is counterbalanced by a gain of chloride ions to maintain electrical charge neutrality; hence the term hyperchloremic metabolic acidosis is used for normal AG metabolic acidoses. (In high AG metabolic acidoses, the remaining anions from the dissociated acids balance the loss of HCO3, so there is no associated hyperchloremia.)
RELIABILITY The AG has shown a limited ability to detect non-volatile acids, and there are several reports where the AG was normal in patients with lactic acidosis (11,12). The poor performance of the AG may be due to the confounding influence of albumin, which was not considered in early studies of the AG. A recent study shows that the albumincorrected AG (AGc) provides a more accurate assessment of metabolic acidosis than the AG (13).

In the presence of a high AG metabolic acidosis, it is possible to detect another metabolic acid-base disorder (a normal AG metabolic acidosis or a metabolic alkalosis) by comparing the AG excess (the difference between the measured and normal AG) to the HCO3 deficit (the difference between the measured and normal HCO3 in plasma). This is accomplished with the equation shown below, which includes 12 mEq/L for the normal AG and 24 mEq/L for the normal HCO3 in plasma.
AG Excess/ HCO, Deficit = (AG — 12) /24 -HCO,)
(31.16)
This ratio is sometimes called the gap-gap ratio because it involves two gaps (the AG excess and the HCO3 deficit). Some applications of the gap-gap ratio are described next.

In metabolic acidoses caused by non-volatile acids (high AG metabolic acidosis), the decrease in serum HCO3 is equivalent to the increase in AG, and the gap-gap (AG Excess/HCO3 deficit) ratio is unity or 1. However, if there is a second acidosis that has a normal AG, the decrease in HCO3 is greater than the increase in AG, and the gap-gap ratio falls below unity (<1). Therefore, in the presence of a high AG metabolic acidosis, a gap-gap ratio <1 indicates the co-existence of a normal AG (hyperchloremic) metabolic acidosis (6,14).
DIABETIC KETOACIDOSIS: A popular question on the medicine boards is a case where the management of a patient with diabetic ketoacidosis (DKA) is associated with improvement in the blood glucose and the clinical condition of the patient, but the acidosis persists, and you are asked what to do (more insulin, more fluids, etc). The answer lies in the gap-gap ratio; i.e., DKA presents with a high AG metabolic acidosis, but the aggressive infusion of isotonic saline during the initial management creates a hyperchloremic (normal AG) metabolic acidosis, which replaces the high AG acidosis as the ketoacids are cleared. In this situation, the serum bicarbonate remains low, but the gap-gap ratio falls below 1 as the acidosis switches from a high AG to a normal AG acidosis (15). Therefore, monitoring the serum HCO3 alone will create a false impression that the DKA is not resolving, while the gap-gap ratio provides an accurate measure of the acid-base status of the patient.

When alkali is added in the presence of a high AG acidosis, the decrease in serum bicarbonate is less than the increase in AG, and the gap-gap is greater than unity (>1). Therefore, in the presence of a high AG metabolic acidosis, a gap-gap >1 indicates the co-existence of a metabolic alkalosis. This is an important consideration because metabolic alkalosis is common in ICU patients (from the frequent use of nasogastric suction and diuretics).

For more than 100 years, the evaluation of acid-base balance has been based on a single reaction sequence (which is shown below), and a single determinant of plasma pH (the PCO /HCO3 ratio).
CO, + H;,0 = H,CO; = Ht+HCo;
This approach is appealing because of its simplicity, but as Whitehead advises in the introductory quote, the simplicity of this approach is also a reason for "distrust." The following are a few reasons to distrust the traditional view of acid-base balance.
1. The use of the PCO2-HCO3 relationship to identify acid-base disorders has two flaws:
a. The PCO, and HCO3 are both dependent variables, and thus it is not possible detect acid-base conditions that operate independent of these variables.
b. Since the CO, in plasma is present primarily as HCO3, it is difficult to establish an independent identity for HCO3.
2. Bicarbonate does not act as a buffer in the physiological pH range (which is demonstrated in the next chapter). The plasma [H+] is a function of the anionic charge equivalence of plasma proteins (which act as the buffers in plasma), and is not directly related to the plasma [HCO3] (16).
A novel view of acid-base balance that challenges traditional concepts was introduced by Peter Stewart (a Canadian physiologist working at Brown) about 30 years ago, and I have included a textbook, and one of Stewart's original papers on the subject (17), for your interest.

Rose BD, Post T, Stokes J. Clinical Physiology of Acid-Base and Electrolyte Disorders. 6th ed. New York:McGraw-Hill, 2013.
Kellum JA, Elbers WG, ed. Stewart's Textbook of Acid-Base. 2nd ed. Amsterdam: AcidBase.org, 2009.
Gennari FJ, Adrogue HJ, Galla JH, Maddias N, eds. Acid-Base Disorders and Their Treatment. Boca Raton: CRC Press, 2005.

It is incident to physicians, I am afraid, beyond all other men, to mistake subsequence for consequence.
Samuel Johnson
This chapter describes the clinical conditions associated with the accumulation of organic acids; i.e., lactic acid and ketoacids (diabetic and alcoholic ketoacidosis). Also included is a presentation of the issues regarding alkali therapy for metabolic acidosis. One of the goals of this chapter is to introduce the notion that the problem with these metabolic acidoses is not the acidosis, but the metabolic derangement that produces the acidosis.

Lactate is the end-product of glucose metabolism in the cytoplasm (glycolysis), and is formed by the reduction of pyruvate in a reaction catalyzed by lactate dehydrogenase (LDH) (see Figure 32.1). About 1,500 mmoles of lactate are produced daily under aerobic conditions (1,2), and the principal sites of production are skeletal muscle (25%), skin (25%), red blood cells (20%), brain (20%), and intestine (10%). Activated neutrophils are an additional source of lactate in inflammatory conditions like acute respiratory distress syndrome (ARDS) (3,4). The concentration of lactate in plasma is usually "2 mmol/L, with a lactate:pyruvate ratio of 10:1 (1,2). Lactate is cleared from plasma by the liver (60%), kidneys (30%), and heart (10%).

The diagram in Figure 32.1 shows the energy yield from the metabolism of glucose and lactate. Anaerobic glycolysis generates 32 kilocalories (kcal) per mole of glucose, which is only 5% of the energy yield from the oxidative metabolism of glucose (673 kcal/mole) (3). The energy deficit from anaerobic lactate production can be corrected by the oxidative metabolism of lactate, which generates 326 kcal/mole (5), or 326 x 2 = 652 kcal per mole of glucose (since 1 mole of glucose produces 2 moles of lactate). In fact, lactate is classified as an organic fuel, and has a caloric density (3.62 kcal/g) equivalent to glucose (3.74 kcal/g) (5).
FIGURE 32.1 The energy yield from the oxidative metabolism of glucose and lactate, and the actions of selected factors that promote lactic acidosis. PDH = pyruvate dehydrogenase; LDH = lactate dehydrogenase.
Lactate Shuttle: Lactate is used as a fuel source during exercise (the lactate shuttle (6), and it is possible that lactate serves a similar function in critically ill patients (7). This is consistent with the observation that lactate uptake by the myocardium is increased in patients with septic shock (8). The possibility of a lactate shuttle in critically ill patients suggests that the enhanced production of lactate in these patients could be an adaptive response that helps to maintain energy metabolism in vital organs like the heart. This differs radically from the traditional perception of lactate as a source of fatal outcomes in critically ill patients.

LINEZOLID: Lactic acidosis has been reported during therapy with linezolid (22,23). Most cases are mild and without adverse consequences, but lactate levels as high as 10 mmol/L have been reported (23). The mechanism is unknown, and lactate levels normalize after discontinuing the drug.

Lactic acidosis can be the result of intoxications with cyanide, carbon monoxide, and propylene glycol. The latter agent is used as a solvent for intravenous drugs, and can be overlooked as a cause of lactic acidosis.
PROPYLENE GLYCOL: The intravenous drugs that use propylene glycol as a solvent include lorazepam, diazepam, esmolol, nitroglycerin, and phenytoin. About 55-75% of propylene glycol is metabolized by the liver and the primary metabolites are lactate and pyruvate (24). Propylene glycol toxicity (i.e., agitation, coma, seizures, hypotension, and lactic acidosis) has been reported in 19% to 66% of patients receiving high-dose intravenous lorazepam for more than 2 days (24,25). If suspected, the drug infusion should be stopped and another sedative agent selected (midazolam and propofol do not use propylene glycol as a solvent). An assay for propylene glycol in blood is available, but the acceptable range has not been determined.

Severe alkalosis (respiratory or metabolic) can raise blood lactate levels as a result of increased activity of pH-dependent enzymes in the glycolytic pathway (26). When liver function is normal, the liver clears the extra lactate generated during alkalosis, and lactic alkalosis becomes evident only when the blood pH is 7.6 or higher. However, in patients with impaired liver function, hyperlactatemia can be seen with less severe degrees of alkalemia.

Other possible causes of hyperlactatemia in ICU patients include generalized seizures (from hypermetabolism) (27), hepatic insufficiency (from reduced lactate clearance) (28), acute asthma (from enhanced lactate production by the respiratory muscles) (29), and hematologic malignancies (rare) (30). Hyperlactatemia associated with hepatic insufficiency is often mild (28). Hyperlactatemia that accompanies generalized seizures can be severe (with lactate levels of 15 mmol/L) but is transient (27).

The normal lactate concentration in blood is "2 mmol/L, but increases in lactate concentration do not have prognostic value until they reach 4 mmol/L (1), so a higher threshold of 4 mmol/L is used to define'clinically significant" hyperlactatemia. Measurements can be obtained on arterial or venous blood samples. If immediate measurements are unavailable, the blood should be placed on ice to retard lactate production by red blood cells.
The anion gap (described on pages 594-596) should be elevated in lactic acidosis, but there are numerous reports of a normal anion gap in patients with lactic acidosis (31). As a result, the anion gap should not be used as a screening test for lactic acidosis.

The lactate produced by mammalian tissues is a levo-isomer (I-lactate), whereas a dextro-isomer of lactate (d-lactate) is produced by certain strains of bacteria that can populate the bowel (32). D-lactate generated by bacterial fermentation in the bowel can gain access to the systemic circulation and produce a metabolic acidosis, often combined with a metabolic encephalopathy (33). Most cases of d-lactic acidosis have been reported after extensive small bowel resection or after jejunoileal bypass for morbid obesity (32— 34).
DIAGNOSIS: D-lactic acidosis can produce an elevated anion gap, but the standard laboratory assay for blood lactate measures only I-lactate. If d-lactic acidosis is suspected, you must request the laboratory to perform a d-lactate assay.

The primary goal of therapy in lactic acidosis is to correct the underlying metabolic abnormality. Alkali therapy aimed at correcting the pH is of questionable value (35). The following is a brief summary of the pertinent issues regarding alkali therapy for lactic acidosis.

The principal fear from acidosis is the risk of impaired myocardial contractility (36). However, in the intact organism, acidemia is often accompanied by an increase in cardiac output (37). This is explained by the ability of acidosis to stimulate catecholamine release from the adrenals and to produce vasodilation. Therefore, impaired contractility from acidosis is less of a concern in the intact organism. In addition, acidosis may have a protective role in the setting of clinical shock. For example, extracellular acidosis has been shown to protect energy-depleted cells from cell death (38).

Sodium bicarbonate is the standard buffer used for lactic acidosis, but has limited success in raising the serum pH (39). This can be explained by the titration curve for the carbonic acid-bicarbonate buffer system, which is shown in Figure 32.2. The HCO3 buffer pool is generated by the dissociation of carbonic acid (H2CO3):
CO, + H,O = H,CO, = H+ + HCO-
(32.4)
The dissociation constant (pK) for carbonic acid (i.e., the pH at which the acid is 50% dissociated) is 6.1, as indicated on the titration curve. Buffers are most effective within 1 pH unit on either side of the pK (40), so the effective range of the bicarbonate buffer system should be an extracellular pH between 5.1 and 7.1 pH units (indicated by the shaded area on the titration curve). Therefore, bicarbonate is not expected to be an effective buffer in the usual pH range of extracellular fluid. Bicarbonate is not really a buffer; rather, it is a transport form of carbon dioxide in blood.

A number of undesirable effects are associated with sodium bicarbonate therapy. One of these is the abilityof bicarbonate to generate CO;, which can actually lower the intracellular pH and cerebrospinal fluid pH (41,42). In fact, considering that the PCO; is 200 mm Hg in standard bicarbonate solutions (see Table 32.1), bicarbonate is really a CO, burden (an acid load!) that must be removed by the lungs.
Finally, bicarbonate infusions can increase blood lactate levels (42). Although this effect is attributed to alkalosis-induced augmentation of lactate production, it is not a desirable effect for a therapy of lactic acidosis.
FIGURE 32.2 The titration curve for the carbonic acid-bicarbonate buffer system. The large, shaded area indicates the effective pH range for the bicarbonate buffer system, which does not coincide with the physiological pH range of extracellular fluid. Adapted from Reference 40.

Carbicarb is a commercially available buffer solution that is a 1:1 mixture of sodium bicarbonate and disodium carbonate. As shown in Table 32.1, carbicarb has less bicarbonate and a much lower PCO, than the standard 7.5% sodium bicarbonate solution. As a result, carbicarb does not produce the increase in PCO, seen with sodium bicarbonate infusions (41).

Sodium,  = 0.9 mEq/mL. Sodium, Carbicarb = 0.9 mEq/mL. Bicarbonate,  = 0.9 mEq/mL. Bicarbonate, Carbicarb = 0.3 mMEq/mL. Dicarbonate,  = —. Dicarbonate, Carbicarb = 0.3 mMEq/mL. PCO.,  = > 200 mm Hg. PCO., Carbicarb = 3mm Hg. Osmolality,  = 1461 mOsm/kg. Osmolality, Carbicarb = 1667 mOsm/kg. pH (25°C),  = 8.0. pH (25°C), Carbicarb = 9.6

Alkali therapy has no role in the routine management of metabolic acidosis. However, when patients are deteriorating rapidly in the setting of a severe acidemia (pH </7.0), a trial infusion of bicarbonate can be attempted as a desperation measure by administering one-half of the estimated HCO3 deficit (42).
HCO, deficit (mEq) = 0.6 x wt(kg) x (15 -measured HCOS)
(32.5)
(where 15 mEq/L is the desired plasma [HCO3]). If cardiovascular im-provement occurs, bicarbonate therapy can be continued to maintain the plasma HCO3 at 15 mEq/L. If no improvement or further deterioration occurs, further bicarbonate administration is not warranted.

When carbohydrates are not available for metabolic energy production, there is a breakdown of triglycerides in adipose tissue (lipolysis) to generate fatty acids, which are transported to the liver and metabolized to form 3 ketone bodies; i.e., acetoacetate, Bhydroxybutyrate, and acetone. This is illustrated in Figure 32.3. These ketones are released from the liver and can be used as oxidative fuels by vital organs such as the heart and central nervous system. The oxidative metabolism of ketones yields 4 kcal/g, which is slightly in excess of the energy yield from the oxidative metabolism of glucose (3.7 kcal/g).
FIGURE 32.3 Ketogenesis in the liver, which occurs in response to diminished availability of glucose. Acetone is a ketone, but is not a ketoacid.

The normal concentration of ketones in the blood is negligible (0.1 mmol/L), but blood ketone levels increase tenfold (to 1 mmol/L) after just 3 days of starvation. Acetone is not a ketoacid, but is responsible for the "fruity" odor of breath in patients with ketoacidosis. Acetoacetate (AcAc) and B-hydroxybutyrate (B-OHB) are strong acids (i.e., readily dissociate), and they promote a decrease in plasma pH when their plasma concentrations reach 3 mmol/L (43). The balance of AcAc and B-OHB in blood is determined by the following redox reaction (see Figure 32.3):
AcAc+ NADH © B-OHB + NAD

The balance of this reaction favors the formation of B-OHB. In conditions of enhanced ketone production, the B-OHB:AcAc ratio ranges from 3:1 in diabetic ketoacidosis, to as high as 8:1 in alcoholic ketoacidosis. The concentration of ketoacids in the blood in diabetic and alcoholic ketoacidosis is shown in Figure 32.4. Note the preponderance of BOHB in both conditions. Because of this preponderance, ketoacidosis is more accurately called B-hydroxybutyric acidosis.

The nitroprusside reaction is a colorimetric method for detecting AcAc and acetone in blood and urine. The test can be performed with tablets (Acetest) or reagent strips (Ketostix, Labstix, Multistix). A detectable reaction requires a minimum AcAc concentration of 3 mmol/L. Because this reaction does not detect the predominant ketoacid, B-hydroxybutyrate (43), it is an insensitive method for monitoring the severity of ketoacidosis. This is illustrated in Figure 32.4. In alcoholic ketoacidosis, the total concentration of ketoacids in blood is 13 mmol/L, which represents more than a hundredfold increase over the normal concentration of blood ketones, yet the nitroprusside reaction will be negative because the AcAc concentration is below 3 mmol/L.

There are portable "ketone meters" that can provide reliable measurements of B-OHB concentrations in fingerstick (capillary) blood, and results are available in about 10 seconds (44). (Available devices include Precision Xtra meter from Abbot Laboratories and Nova Max PLUS from Nova Biomedical.) The American Diabetes Association considers measurements of plasma B-OHB with these meters as the preferred method for monitoring patients with diabetic ketoacidosis (45).

Diabetic ketoacidosis (DKA) is usually seen in insulin-dependent diabetic patients, but there is no previous history of diabetes mellitus in 27-37% of cases (46). The most common precipitating factors in DKA are inappropriate insulin dosing and concurrent illness (e.g., infection). The mortality rate of DKA is 1-5% (46).
FIGURE 32.4 The concentrations of acetoacetate and 8-hydroxybutyrate in the blood in diabetic ketoacidosis (DKA) and alcoholic ketoacidosis (AKA). The horizontal hatched line represents the minimum concentration of acetoacetate required to produce a positive nitroprusside reaction.

The management of DKA is summarized in Table 32.2. The following are some of the details.

Volume deficits in DKA average 50-100 mL/kg (4-8 L for an 80 kg adult) (45). Volume resuscitation begins with isotonic (0.9%) saline infused at a rate of 1 liter per hour (or 15-20 mL/kg/hr) (45). When the patient stabilizes (e.g., blood pressure returns to normal), the infusion rate can be decreased to 250-500 mL/hr (4-14 mL/kg/hr). When the blood glucose falls to 250 mg/dL, change the intravenous fluid to 5% dextrose in 0.45% saline and decrease the infusion rate to 150-250 mL/hr.

Insulin therapy is started with regular insulin given intravenously, starting with a bolus dose of 0.15 units per kilogram body weight and followed with a continuous infusion at 0.1 units/kg/hr. Because insulin adsorbs to intravenous tubing, the initial 50 mL of infusate should be run through the IV setup before the insulin drip is started. The blood glucose levels should decrease by 50 to 75 mg/dL per hour (46), and the insulin infusion should be adjusted to achieve this goal. When the blood glucose level falls to 200 mg/dL, the insulin infusion should be decreased to 0.05 to 0.1 units/kg/hr, and dextrose should be added to the intravenous fluids. Thereafter, the blood glucose levels should be maintained between 150-200 mg/dL (45,46). Achieving euglycemia is not recommended because of the risk of hypoglycemia.
able 32.2 Management of Diabetic Ketoacidosi

1. Start with isotonic saline at 1 L/hr (15-20 mL/kg/hn. When BP normalizes, decrease rate to 250-500 mL/hr.
2. When blood glucose falls to 250 mg/dL, change to 5% dextrose in 0.45% saline and infuse at 150-250 mL/hr.
3. Volume deficit in DKA Is usually 50-100 mL/kg (4-8 Lin 80 kg adult).

1. Regular insulin: 0.15 units'kg as an IV bolus, then infuse at 0.1 units'kg/nr.
2. Adjust infusion so that blood glucose clecreases by 50-75 ma/dL per hour.
3. When blood glucose reaches 250 mg/dL, decrease infusion rate to 0.1 -0.5 units'ka/hr, and maintain blood glucose at 150-200 mg/dL.
4. Begin SC insulin when DKA resolves (pH 7.3) and oral fluids are tolerated, but continue IV insulin for a few hours after starting SC insulin.

1. If initial serum [K+] is <3.3 mEq/L, hold the insulin infusion and give 40 mEq K+ per hour until the [K+] ls 23.3 mEq/L.
2. If initial serum [K+] ls 3.3-4.9 mEq/L, give 20-30 mEq K* in each liter of IV fluid to keep serum [K+] at 4—5 mEq/L.
3. If initial serum [K*] Is =5 mEq/L, do not give K+, but check serum [K*] every 2 hours.
4, Average Kt deficit in DKA is 3-5 mEq/kg.
From the American Diabetes Association guidelines in Raferencea 45.
AFTER DKA RESOLVES: When DKA has resolved (i.e., glucose <200 mg/dL, plasma [HCO3] >18 mEq/L, plasma pH >7.3) and the patient can tolerate oral fluids, subcutaneous insulin can be started. Patients who were insulin-dependent prior to admission can be placed on their usual outpatient regimen. Patients who are new to insulin should receive 0.5—0.8 Units/kg per day in divided doses (46).
Potassium depletion is universal in DKA, and the average deficit is 3 to 5 mEq/kg (47). However, the initial serum K+ is often normal (74% of patients) or even elevated (22% of patients) (47). The serum K+ can fall dramatically during insulin therapy (transcellular shift), so if the initial serum K+ is low (<3.3 mEq/L), the insulin should be withheld until the serum K+ can be normalized (by giving 40 mEq KCL hourly). If the serum K+ is high (25 mEq/L), no potassium is given initially, and when the serum K+ is normal, 20-30 mEq of KCL should be added to each liter of IV fluid (45). Regardless of the initial potassium regimen, the serum K+ should be checked every 1-2 hours for the first 4-6 hours.

Phosphate depletion is also common in DKA, and serum phosphate levels are typically 11.5 mmol/kg (47). However, phosphate replacement has no documented benefit in DKA, and is not recommended unless the hypophosphatemia is severe (45-47). If the serum
PO, is <1 mg/dL, give 20-30 mEq potassium phosphate with each liter of IV fluid (45).

Bicarbonate therapy does not improve the outcome in DKA, and is not recommended as a routine measure (45-47). However, in patients who are in extremis with a pH <7.0, the bicarbonate regimen presented earlier can be used as a desperation measure.

The serum [HCO3] is not a reliable measurement for following the acid-base status during the management of DKA because the isotonic saline produces a hyperchloremic acidosis that prevents the bicarbonate from rising despite a resolving ketoacidosis. In this situation, monitoring the gap-gap ratio is more informative. The gap-gap ratio is the ratio of the anion gap excess to the bicarbonate deficit, and is described at the end of the last chapter (see pages 596-597). This ratio is unity (1.0) in pure ketoacidosis, and it decreases as the ketoacidosis resolves and is replaced by the hyperchloremic acidosis. When the ketones have been cleared from the bloodstream, the ratio approaches zero.

Alcoholic ketoacidosis (AKA) is a complex acid-base disorder that occurs in chronic alcoholics and usually appears 1 to 3 days after a period of heavy binge drinking (53,54). Several mechanisms are involved, including reduced nutrient intake (which initiates enhanced ketone production), hepatic oxidation of ethanol (which generates NADH and enhances B-hydroxybutyrate formation), and dehydration (which impairs ketone excretion in the urine).

Patients with AKA tend to be chronically ill and have several comorbid conditions. The presentation usually includes nausea, vomiting, and abdominal pain (53). Electrolyte abnormalities are common, particularly the hypos (e.g., hyponatremia, hypokalemia, hypophosphatemia, hypomagnesemia, hypoglycemia). Mixed acid-base disorders are also common in AKA. More than half the patients can have lactic acidosis (caused by other conditions), and metabolic alkalosis occurs in patients with protracted vomiting.

The diagnosis of AKA is suggested by the clinical setting (i.e., after a period of binge drinking), an elevated anion gap, and the presence of ketones in the blood or urine. However, the nitroprusside reaction for detecting ketones can be negative in AKA. This is shown in Figure 32.4. The oxidation of ethanol in the liver generates NADH, and this favors the conversion of acetoacetate to B-hydroxybutyrate, and results in a low concentration of acetoacetate blood and urine. Even though most cases of AKA have a positive nitroprusside reaction for ketones (53), the severity of the ketoacidosis is significantly underestimated.

The management of AKA is notable for its simplicity; i.e., infusion of dextrose-containing saline solutions is usually all that is required. The glucose infusion slows hepatic ketone production, while the infused volume promotes the renal clearance of ketones. The ketoacidosis usually re-solves within 24 hours. Other electrolyte deficiencies are corrected as needed, and thiamine supplementation is recommended because glucose infusions can deplete marginal thiamine reserves.

One of the important messages in this chapter is the fact that the clinical problem created by lactic acidosis or diabetic ketoacidosis is not because of the acidosis, but because of the underlying metabolic derangement. The same degree of acidosis can be associated with diabetic ketoacidosis and lactic acidosis, but the survival is much better with ketoacidosis because the underlying problem is an exaggeration of an adaptive response to intracellular glucose deprivation, while the problem with lactic acidosis is a generalized derangement in cellular energy metabolism.
The other point that deserves emphasis is the fact that bicarbonate is not much of a buffer in the physiological pH range. In fact, bicarbonate can be viewed as a consequence of the accumulation of CO>, the principal volatile acid in the human body.

1. Okorie ON, Dellinger P. Lactate: biomarker and potential therapeutic target. Crit Care Clin 2011; 27:299-326.
2. Vernon C, LeTourneau JL. Lactic acidosis: Recognition, kinetics, and associated prognosis. Crit Care Clin 2010; 26:255-283.

43. Cartwright MM, Hajja W, Al-Khatib S, et al. Toxigenic and metabolic causes of ketosis and ketoacidotic syndromes. Crit Care Clin 2012; 601-631.
44. Pludderman A, Hemeghan C, Price C, et al. Point-of-care blood test for ketones in patients with diabetes: primary care diagnostic technology update. Br J Clin Pract 2011; 61:530-531.
45. American Diabetes Association. Hyperglycemic crisis in diabetes. Diabetes Care 2004; 27(Suppl):S94—S102.

53. Wrenn KD, Slovis CM, Minion GE, Rutkowsli R. The syndrome of alcoholic ketoacidosis. Am J Med 1991; 91:119-128.
54. McGuire LC, Cruickshank AM, Munro PT. Alcoholic ketoacidosis. Emerg Med J 2006; 23:417-420.

To do common things perfectly is far better worth our endeavor, than to do uncommon things respectably.
Harriet Beecher Stowe 1864
Although the spotlight usually falls on metabolic acidosis, the most common acid-base disturbance in hospitalized patients is metabolic alkalosis (1-3). The prevalence of metabolic alkalosis can be attributed to three factors: (a) common predisposing conditions (e.g., diuretic therapy), (b) the ability of the alkalosis to sustain itself (thanks to chloride), and (c) the tendency for the condition to go unnoticed and untreated. The latter factor reveals that metabolic alkalosis is not just a common disorder, it is a common and often untreated disorder.

Metabolic alkalosis is defined as an increase in the bicarbonate (HCO3) concentration in extracellular fluid (>26 mEq/L) that is not an adaptive response to hypercapnia. This condition can be the result of any of the following: (a) loss of hydrogen ions (H+) from extracellular fluid, (b) a gain in bicarbonate ions in extracellular fluid, or (c) a decrease in extracellular volume. Once developed, metabolic alkalosis is sustained by a decrease in HCO3 excretion in the urine, which is the result of both an increase in HCO3 reabsorption and a decrease in HCO3 secretion in the distal nephron. These renal adjustments are promoted by chloride depletion, hypokalemia, and aldosterone, as explained next.

The kidneys play a major role in the maintenance of metabolic alkalosis by decreasing HCO3 excretion in the urine, as just mentioned. The following is a description of the mechanisms involved.

Bicarbonate is readily filtered at the glomerulus, and is almost entirely reabsorbed in the renal tubules. Most (90%) of the filtered HCO3 is reabsorbed in the proximal tubules, and the remainder is reabsorbed by specialized cells in the collecting ducts. The distal site is the principal site of increased HCO3 reabsorption in metabolic alkalosis (4). The mechanism of HCO3 reabsorption in the collecting ducts is shown in Figure 33.1 A membrane ATPase pump moves H+ into the tubular lumen, where it reacts with HCO3 to form carbonic acid, which dissociates to form CO) and HO. The CO> moves into the tubular cell and is hydrated to regenerate HCO3 and H+. The HCO3 moves into the bloodstream and the H+ is transported back into the tubular lumen for another go-round. Chloride (CL-) moves out of the peritubular capillaries in exchange for HCO3.
anhydrase, Pn = pendrin (the chloride-bicarbonate exchanger). See text for explanation.

Bicarbonate can also be secreted from specialized cells in the collecting ducts. This does not occur normally, but appears when there is excess HCO3 in plasma (i.e., metabolic alkalosis). The mechanism for HCO3 secretion is the reverse of HCO3 reabsorption, with the H+ pump on the capillary side of the tubular cells (see Figure 33.1). The principal difference is the presence of a chloride-bicarbonate exchange protein, called pendrin, on the luminal surface of the cells. This anion exchange protein has an important role in HCO3 secretion (5-8). The pendrin gene is up-regulated in metabolic alkalosis (6), and pendrin activity is directly related to the luminal chloride concentration (7).

Chloride depletion plays a major role in promoting metabolic alkalosis by increasing HCO3 reabsorption and inhibiting HCO3 secretion. Both effects are mediated by a decrease in luminal chloride concentration in the distal nephron, which: (a) increases HCO3 reabsorption by promoting the movement of chloride and H+ into the tubular lumen, and (b) impairs HCO3 secretion by reducing the activity of the anion exchange protein, pendrin. The effects of luminal chloride on HCO3 secretion are considered the principal mechanism for the ability of chloride depletion to promote metabolic alkalosis, and for the ability of chloride replacement to correct metabolic alkalosis (5).

Like chloride depletion, hypokalemia promotes metabolic alkalosis by increasing HCO3 reabsorption and decreasing HCO3 secretion in the distal nephron. The increase in HCO3 reabsorption is caused by the simultaneous movement of K+ out of cells and H+ into cells, and the resulting decrease in intracellular pH promotes HCO3 reabsorption in the distal nephron (4). The decrease in HCO3 secretion in hypokalemia is attributed to a decrease in pendrin activity (8).

Aldosterone (a mineralocorticoid produced in the adrenal cortex) promotes HCO3 reabsorption in the distal nephron by stimulating the membrane H+ pump on the luminal surface of acid-secreting renal tubular cells (9).

Metabolic alkalosis has no apparent deleterious effects in most patients. In one case report, an elderly patient with protracted vomiting and a plasma [HCO3] of 151 mEq/L showed no signs of an immediate threat to life, and recovered fully after correction of the alkalosis (11). The lack of clinical consequences raises questions about the value of recognition and management of metabolic alkalosis.

The neurologic manifestations attributed to alkalosis include depressed consciousness, generalized seizures, paresthesias, and carpopedal spasms. However, these manifestations are usually associated with respiratory alkalosis, not metabolic alkalosis. This is traditionally explained by the greater tendency for respiratory alkalosis to influence intracellular and central nervous system pH.
Neurologic manifestations seem to be more prominent in cases of metabolic alkalosis due to baking soda (sodium bicarbonate) ingestion (12), but this condition is accompanied by other electrolyte abnormalities (particularly hypercalcemia) that could be the responsible factor.

The ventilatory response to metabolic alkalosis is hypoventilation, with a subsequent rise in arterial PCO». However, this is not a vigorous response, and a considerable rise in plasma HCO3 may be necessary before significant hypoventilation is evident (13). The ventilatory response to metabolic alkalosis is described by the following equation (14).
APaCOQ, = 0.7 x AHCO,
(33.1)
(PaCO, is the arterial PCO2 and HCO3 is the plasma bicarbonate concentration.) This equation was used to construct the curve in Figure 33.2 that shows the relationship between PaCO, and plasma HCO3 in progressive metabolic alkalosis. (A normal PaCO> of 40 mm Hg and a normal plasma HCO3 of 24 mEq/L were used in constructing this curve.) Note that hypercapnia (i.e., PaACO> >46 mm Hg ) does not occur until the plasma HCO3 increases from 24 to 32 mEq/L, which represents a 40% increase in plasma HCO3. In patients who have an increased ventilatory drive from an acute pulmonary process (é.g., pneumonia), even greater increases in plasma HCO3 will be necessary to produce significant hypoventilation.

Alkalosis shifts the oxyhemoglobin dissociation curve to the left (Bohr effect), which results in a decreased tendency for hemoglobin to release oxygen into the tissues. When the O» extraction from capillary blood is constant, a leftward shift of the oxyhemoglobin dissociation curve results in a decrease in venous PO, (15), which typically indicates a decrease in tissue PO2. However, there is no evidence of inadequate tissue oxygenation (e.g., lactate accumulation) from this effect.
FIGURE 33.2 The relationship between plasma bicarbonate (HCO3) and arterial PCO? (PaCO72) in metabolic alkalosis, as predicted by the equation shown at the top of the graph (using a normal PaCOz of 40 mm Hg and a normal plasma [HCO3] of 24 mEq/L).

The likely source(s) of metabolic alkalosis in ICU patients are loss of gastric secretions, diuretics, volume depletion, and hypokalemia, and these are readily apparent. In the rare case of uncertainty, the urinary chloride concentration can be informative, as described next.

The urinary chloride concentration is used to classify metabolic alkalosis as chlorideresponsive or chloride-resistant, and the conditions associated with each category are listed in Table 33.1.

A chloride-responsive metabolic alkalosis is characterized by a low urinary chloride concentration (i.e., <15 mEq/L), indicating chloride depletion. The principal conditions that produce this type of metabolic alkalosis include loss of gastric secretions, therapy with chloruretic diuretics (i.e., diuretics that promote urinary chloride excretion), and volume depletion. In cases of metabolic alkalosis due to chloruretic diuretics (e.g., thiazides or furosemide), the urinary chloride may be inappropriately high when the drug is active, but this effect is lost when the drug effect dissipates.

Chloride-Responsive, Criteria = Urine [CL-] <15 mEq/L. Chloride-Responsive, “Conditions = = Vomiting Nasogastric suction Choluretic diuretics Volume depletion Laxative abuse. Chloride-Resistant, Criteria = Urine [CL] >25 mEq/L. Chloride-Resistant, “Conditions = Primary aldosteronism Licorice ingestion Severe hypokalemia (plasma Kt <2 mEq/L)
Chloride-responsive metabolic alkalosis is typically accompanied by volume depletion, and improves with infusion of isotonic saline. Almost all cases of metabolic alkalosis in ICU patients are chloride-responsive.
LAXATIVE ABUSE: Diarrhea usually produces a hyperchloremic metabolic acidosis from bicarbonate losses in stool, but the diarrhea associated with chronic laxative abuse is rich in potassium and chloride (70-90 mEq/L), and it typically results in hypokalemia and a saline (chloride)-responsive metabolic alkalosis (16). The diagnosis can be elusive because patients often deny both laxative abuse and diarrhea.

A chloride-resistant metabolic alkalosis is characterized by an elevated urinary chloride concentration (i.e., >25 mEq/L). Most cases of chloride-resistant alkalosis are caused by primary mineralocorticoid excess (e.g., primary aldosteronism). Whereas volume depletion is common in chloride-responsive metabolic alkalosis, volume expansion is common in chloride-resistant metabolic alkalosis. As a result, this type of metabolic alkalosis does not improve with infusions of isotonic saline. This type of metabolic

SEVERE K+ DEPLETION: Mineralocorticoid excess promotes urinary potassium loss, so hypokalemia is common in chloride-resistant metabolic alkalosis. However, a chlorideresistant metabolic alkalosis has been reported in patients with hypokalemia that is not caused by mineralocorticoid excess (17). The hypokalemia is typically severe (plasma Kt <2 mEq/L), and the proposed mechanism is diminished chloride reabsorption is the distal renal tubules. This condition is saline-resistant, but it can be corrected with potassium repletion (17).

Since maintenance of a metabolic alkalosis is primarily the result of chloride and potassium depletion, these ions must be replenished to correct the alkalosis. This is accomplished with isotonic saline (0.9% NaCL) and potassium chloride.

Because volume depletion is common in chloride-responsive metabolic alkalosis, infusion of isotonic saline will help to correct the alkalosis. The volume of isotonic saline needed to correct the alkalosis can be estimated by first estimating the chloride (CL-) deficit, as shown below (2,18):
CL" deficit (mEq) =0.2 x wt (kg) x (100 -plasma [CL7])
(33.2)
(wt is lean body weight in kg, and 100 is the desired plasma chloride concentration). The required volume of isotonic saline (in liters) is then estimated as follows:
Volume of isotonic saline (L) = CL™ deficit /154
(33.3)
(154 is the chloride concentration in mEq/L in isotonic saline). This method is summarized in Table 33.2. If the patient is not hemodynamically compromised, rapid saline infusion is not necessary, and a rate of 100-125 mL/hr above total hourly fluid losses (including insensible loss) is appropriate.

From Referances 2 and 16.
EXAMPLE: A 70 kg adult with protracted vomiting has a metabolic alkalosis with a plasma chloride of 80 mEq/L. The chloride deficit in this case (using a normal plasma [CL-] of 100 mEq/L) is 0.2x70x(100 — 80) = 280 mEq. The volume of isotonic saline needed to correct this deficit is 280/154 = 1.8 liters.

Isotonic saline infusion is contraindicated in edematous states because crystalloid fluids are distributed primarily in the interstitial fluid compartment. For every liter of isotonic saline infused, 825 mL will be added to the interstitial edema fluid (See Figure 12.2 on page 220) (19). Metabolic alkalosis in edematous states is often accompanied by hypokalemia, so potassium chloride replacement therapy can be used to treat the alkalosis.

Potassium replacement is used to correct hypokalemia, and is always given as KCL, to take advantage of chloride replacement for correcting metabolic alkalosis. It is important to emphasize that diuretic-induced hypokalemia can be resistant to potassium replacement if there is concurrent magnesium depletion (8). Since magnesium depletion is also common during diuretic therapy, the plasma magnesium level should be checked at the outset of K+ replacement. (The evaluation of magnesium depletion is described in Chapter 37.)

Alkalosis from mineralocorticoid excess (primary or secondary) is associated with an increased extracellular volume (which may be apparent as peripheral edema), and saline infusions will be counterproductive. Hypokalemia is common in this type of metabolic alkalosis, so K+ replacement (as KCL) helps in correcting the alkalosis. However, corrective therapy may require one of the following measures.

Acetazolamide (Diamox) inhibits carbonic anhydrase, the enzyme involved in HCO3 reabsorption and secretion (see Figure 33.1). The major effect of acetazolamide is inhibition of HCO3 reabsorption (proximal and distal sites) and an increase in urinary HCO3 excretion (the urine pH should rise to 7 or higher during acetazolamide therapy). The in-crease in HCO3 excretion is accompanied by an increase in sodium excretion, so acetazolamide provides the dual benefit of diuresis and correction of the metabolic alkalosis. The recommended dose is 5 to 10 mg/kg IV (or PO), and the maximum effect occurs after an average of 15 hours (21).

Alkalosis that is not corrected with K+ replacement and acetazolamide can be treated with an infusion of dilute hydrochloric acid (HCL), but this can be risky, and is reserved only for severe alkalemia (plasma pH >7.5).
METHOD: The "dose" of HCL is determined by estimating the hydrogen ion (H+) deficit with the equation below (see also Table 33.3).
H* deficit (mEq) = 0.5 x wt (kg) x (plasma [HCO,] -30)
(33.4)
(wt is the lean body weight in kg, and 30 is the desired plasma [HCO3], which is higher than normal because the goal is to curb the alkalosis and not correct it). The preferred HCL solution for intravenous use is 0.1N HCL, which contains 100 mEq H+ and 100 mEq

The final word in metabolic alkalosis is chloride, the principal element in both the maintenance and correction of metabolic alkalosis in ICU patients. Chloride plays an important role in metabolic acidosis as well. In addition to its role in acid-base balance, chloride is the second most abundant electrolyte in the extracellular fluid, and is a major factor in determining the osmolality and volume of extracellular fluid. In light of chloride's participation in numerous physiological processes, a recent review of chloride suggested that it is the "queen of electrolytes" (24). Although a more appropriate term might be warranted, it highlights an increasing awareness that chloride is much more than sodium's passive partner in the extracellular fluid.

1. Laski ME, Sabitini S. Metabolic alkalosis, bedside and bench. Semin Nephrol 2006; 26:404—421.
2. Khanna A, Kurtzman NA. Metabolic alkalosis. Respir Care 2001; 46:354—365.
3. Galla JH. Metabolic alkalosis. J Am Soc Nephrol 2000; 11:360—375.

16. Roerig JL, Steffen KJ, Mitchell JE, Zunker C. Laxative abuse. Epidemiology, diagnosis, and management. Drugs 2010; 70:1487-1503.
17. Garella S, Chazan JA, Cohen JJ. Saline-resistant metabolic alkalosis or "chloridewasting nephropathy". Report of four patients with severe potassium depletion. Ann Intern Med 1970; 73:31-38.
18., Management = Androgue HJ, Madias N. Management of life-threatening acid-base disorders. Part 2. N Engl J Med 1998; 338:107-111.. 19., Management = Imm A, Carlson RW. Fluid resuscitation in circulatory shock. Crit Care Clin 1993; 9:313-333.. 20., Management = Whang R, Flink EB, Dyckner T, et al. Mg depletion as a cause of refractory potassium depletion. Arch Intern Med 1985; 145:1686-1689.. 21., Management = Marik PE, Kussman BD, Lipman J, Kraus P. Acetazolamide in the treatment of metabolic alkalosis in critically ill patients. Heart Lung 1991; 20:455-458.. 22., Management = Brimioulle S, Vincent JL, Dufaye P, et al. Hydrochloric acid infusion for treatment of metabolic alkalosis: effects on acid-base balance and oxygenation. Crit Care Med 1985; 13:738-742.. 23., Management = Buchanan IB, Campbell BT, Peck MD, Cairns BA. Chest wall necrosis and death secondary to hydrochloric acid infusion for metabolic alkalosis. South Med J 2005; 98:822.
24. Berend K, van Hulsteijn LH, Gans RO. Chloride: the queen of electrolytes? Eur J Intern Med 2012; 23:203-211.

Paul A.M. Dirac (1903-1984)
As many as 70% of ICU patients have some degree of renal dysfunction, and about 5% of ICU patients require renal replacement therapy (1-3). The renal dysfunction that occurs in critically ill patients is now called acute kidney injury. This condition is similar to the acute respiratory distress syndrome (ARDS) because it typically occurs as part of multiorgan failure in patients with progressive systemic inflammation (1). Patients with acute kidney injury who require hemodialysis have a mortality rate of 50-70% (3), which has not changed over the last 30 years (4). The inability of hemodialysis to curb the mortality rate in acute renal failure has apparently escaped the notice of the "evidencebased medicine" junkies, who preach that an intervention should be discarded if it does not improve mortality.

The term "acute kidney injury" (AKI) was introduced over a decade ago to include the spectrum of renal dysfunction that occurs in critically ill patients. Also introduced was a classification system for severity of illness and outcome. The intent is to standardize the description of renal dysfunction in critically ill patients, but the reality (as will be presented) is a system of competing criteria that seems to complicate rather than simplify the approach to renal dysfunction in critically ill patients.

In 2002, a group of experts known as the Acute Dialysis Quality Initiative (ADQI) proposed a classification system to define the progressive states of acute kidney injury (AKI). This system included 5 categories, and was given the name RIFLE, which is an acronym for Risk, Injury, Failure, Loss, and End-stage renal disease. The RIFLE criteria are shown in Table 34.1. There are 3 severity categories and 2 clinical outcome categories. The severity categories are defined by the serum creatinine and the urine output. The first (Risk) category identifies the minimum requirements for the diagnosis of AKI: i.e., a 50% increase in the serum creatinine concentration and a decrease in urine output to 0.5 mL/kg/hr (the definition of oliguria) for at least a 6-hour time period. If the creatinine and urine output criteria don't match, the "worst" measurement is used to determine the category).
The RIFLE criteria have 2 limitations: (a) there is no defined time period for the change in serum creatinine, and (b) the minimum change in serum creatinine required for the diagnosis of AKI is considered too large.

Serum
RIFLE:, Categories Creatinine = . RIFLE:, Output Criteriat = . Risk, Categories Creatinine = f in Scr to 1.5-<2 baseline. Risk, Output Criteriat = UO: <0.5 mL’kg/hr for 6 hrs. Injury, Categories Creatinine = tT in SCr to 2- <3 x baseline. Injury, Output Criteriat = UO: <0.5 mL/ka/hr for 12 hrs. Failure, Categories Creatinine = ? in SCr to =3 x baseline. Failure, Output Criteriat = Uo: <0.3 mL’kg/hr for 24 hrs or anuria for 12 hrs. Loss, Categories Creatinine = Loss of Kidney function for >4 wks. Loss, Output Criteriat = . ESRD, Categories Creatinine = Loss of Kidney function for >3 mos. ESRD, Output Criteriat = . AKIN":, Categories Creatinine = . AKIN":, Output Criteriat = . Stage 1, Categories Creatinine = t in SCr to 20.3 mg/dL or to 1.5-2 x baseline. Stage 1, Output Criteriat = UO: <0.5 mL/ka/hr for =8 hrs. Stage 2, Categories Creatinine = in Scrto>2-3 x baseline. Stage 2, Output Criteriat = Uo: <0.5 mL’ka/hr for > 12 hrs. Stage 3, Categories Creatinine = f in SCr to>& x baseline or SCr = 4 mg/dL with an acute increase of 20.5 mg/dL. Stage 3, Output Criteriat = UO: <0.5 mL/ka/hr for 24 hrs or anuria for 12 hrs
Criteria
_Urine
"The AKIN critaria require the increase in serum creatirine to occur within 4 hrs.
Tideal bodly weight is recommended for urine output determinations.
From Aeferances 1 and 2. ESRD=end-stage renal disease; SCr=sarum creatining; UO=urine output.

Because of the limitations of the RIFLE criteria just mentioned, revised criteria were introduced by the Acute Kidney Injury Network (AKIN), and these criteria are shown at the bottom of Table 34.1. The AKIN criteria require a smaller change in creatinine (20.3 mg/dL) for the diagnosis of AKI, and a time limit of 48 hours is imposed on the change in serum creatinine. Unfortunately, the RIFLE criteria were not abandoned after introduction of the AKIN criteria, so there are two competing systems for the diagnosis and classification of AKI at the present time.

So, which criteria should be used to diagnose and stage AKI? The AKIN criteria seem to be favored in published reviews, but comparison studies have not demonstrated a difference between the RIFLE and AKIN criteria for predicting outcomes. This is demonstrated in Figure 34.1, which shows that the two sets of criteria are equivalent for predicting mortality rates (5).

Despite the intent of simplifying the approach to renal failure in critically ill patients, the newly minted condition known as acute kidney injury has created the following sources of confusion:
1. The diagnosis of AKI includes prerenal conditions (e.g., hypovolemia) where there is no "injury" in the kidneys.
2. Oliguria (i.e., urine output <0.5 mL/kg/hr) is required for the diagnosis of AKI, which neglects cases of nonoliguric acute renal failure (e.g., interstitialnephritis, myoglobinuric renal failure).
3. There is a lack of agreement about the minimum increase in serum creatinine required for the diagnosis of AKI.
FIGURE 34.1 Comparison of in-hospital mortality rates for corresponding RIFLE and AKIN criteria in 291 patients with AKI. There is no difference between the classification systems for predicting mortality. Data from Reference 5.
(3,9). AKI has been reported in up to 40% of postoperative patients following major surgery, particularly cardiopulmonary bypass surgery (3). AKI has also been reported in 30% of major trauma victims (3), and rhabdomyolysis is responsible for 30% of these cases (3). Nephrotoxic drugs and radiocontrast dye are implicated in about 20% of cases of AKI (9). Increased abdominal pressure is emerging as a common, and often overlooked, source of AKI. This condition is described later in the chapter.
FIGURE 34.2 Photomicrograph of acute tubular necrosis (ATN) showing a proximal tubule (outlined by the dotted line) filled with exfoliated renal tubular cells.

Sepsis* Major Surgery Hypovolemia Low Cardiac Output Nephrotoxic Agents, 1 = Increased Abdominal Pressure Cardiopulmonary Bypass Trauma Rhabdomyolysis
tFrom Raferance 9.
"The leading cause of acuta kidnay injury

The evaluation of AKI begins with a bedside ultrasound evaluation of the kidneys for evidence of postrenal obstruction. If there is no obstruction, the remaining evaluation is aimed at determining if the problem is a prerenal disorder (e.g., hypovolemia or a reduced cardiac output) or an intrinsic renal disorder (e.g. ATN or AIN). The measurements in Table 34.3 can help to distinguish prerenal from renal disorders, but only in patients with oliguria.

In prerenal disorders, the renal hypoperfusion is accompanied by an increase in sodium reabsorption in the renal tubules and a subsequent decrease in urine sodium concentration. In contrast, renal "tubulo-pathies" like ATN are characterized by impaired sodium reabsorption and increased urinary sodium losses. Therefore, when a random urine sample (spot urine) is obtained in a patient with AKI, a urine sodium <20 mEq/L is used as evidence of a prerenal disorder, while a urine sodium >40 mEq/L is used as evidence of an intrinsic renal disorder (10).
EXCEPTIONS: A prerenal disorder can be associated with a high urine sodium (>40 mEq/L) if there is ongoing diuretic therapy, or the patient has chronic renal disease (where there is "obligatory" sodium loss in the urine).

Spot Urine Sodium, Prerenal Disorder = <20 mEq/L. Spot Urine Sodium, Renal Disorder = >40 mEq/L. Fractional Excretion of Na, Prerenal Disorder = <1%. Fractional Excretion of Na, Renal Disorder = > 2%. Fractional Excretion of Urea, Prerenal Disorder = <35%. Fractional Excretion of Urea, Renal Disorder = > 50%. Urine Osmolality, Prerenal Disorder = > 500 mOsm/kg. Urine Osmolality, Renal Disorder = 300-400 mOsm/kg. U/P Osmolality, Prerenal Disorder = 21.5. U/P Osmolality, Renal Disorder = 1-1.3

The fractional excretion of sodium (FENa) is considered a more accurate measure of renal tubular function than the spot urine sodium concentration. The FENa is equivalent to the fractional sodium clearance divided by the fractional creatinine clearance, as expressed by the following equation:
(34.1)
(U/P is the urine-to-plasma ratio for sodium and creatinine concentrations.) In euvolemic patients with normal renal function, the FENa is 1% (i.e., only 1% of the filtered sodium is excreted in the urine). In prerenal disorders like hypovolemia, the FENa is <1% (reflecting sodium conservation), and in intrinsic renal disorders like ATN, the FENa is typically >2% (reflecting an increase in urinary sodium excretion) (11).
EXCEPTIONS: Like the spot urine sodium, the FENa can be falsely elevated (>1%) by diuretic therapy and chronic renal insufficiency (11). In addition, the FENa can be falsely low (<1%) in patients with renal failure due to sepsis (12), radiocontrast dyes (13), and hemoglobinuria or myoglobinuria (14).

The fractional excretion of urea (FEU) is conceptually similar to the FENa, and is equivalent to the fractional urea clearance divided by the fractional creatinine clearance, as expressed by the following equation:
(34.2)
(U/P is the urine-to-plasma ratio for urea and creatinine concentrations.) The FEU is low (<35%) in prerenal disorders like hypovolemia, and high (>50%) in renal disorders like ATN. However, the FEU is not influenced by diuretics (15), which is the major advantage of the FEU over FENa.

Distinguishing between prerenal and intrarenal causes of AKI can be difficult, and often a fluid challenge is necessary to distinguish between these two conditions (see next section).

The early management of AKI should include the following: (a) volume infusion to promote renal blood flow, (b) discontinuing any nephrotoxic drugs, and (c) treating any conditions that predispose to AKI (e.g., sepsis).

If a prerenal source of AKI has not been ruled out, prompt volume infusion is warranted. Delays in correcting renal hypoperfusion can lead to intrarenal damage, so prompt attention to volume resuscitation is mandatory. Fluid challenges can be given in 500 ml to 1,000 mL aliquots for crystalloid fluids and 300 mL to 500 mL aliquots for colloid fluids, infused over 30 minutes (16). The fluid challenges are continued until there is a response (i.e., an increase in urine output), or until you are concerned about volume overload. (Remember that only 20-25% of infused crystalloid fluids remain in the intravascular space, so 500 mL of infused crystalloid fluid will increase the plasma volume by only 100— 125 mL. Therefore, fluid challenges with crystalloid fluids should not stop if a volume of 500 ml does not produce a favorable response.) Diuretics should never be used to increase urine output until the possibility of a prerenal condition has been eliminated.

Several studies have shown an association between hydroxyethyl starch solutions and AKI (see page 231). Therefore, it is wise to avoid starch solutions for fluid challenges in AKI.

The following considerations are relevant in patients with AKI due to intrarenal disorders (i.e., ATN and AIN). Unfortunately, the only early option available for halting or reversing the course of AKI is to discontinue possible offending drugs.

Despite its popularity in AKI, intravenous furosemide does not improve renal function in AKI, and does not convert oliguric to non-oliguric renal failure (3,17). Furosemide can increase urine output during the recovery phase of AKI (18), and a trial of furosemide is reasonable during this period to relieve fluid accumulation.

Low-dose dopamine (2 e<g/kg/min) can act as a renal vasodilator, but it does not improve renal function in patients with AKI (19,20). Furthe-rmore, low-dose dopamine can have deleterious effects on hemodynamics (decreased splanchnic blood flow) immune function (inhibition of T-cell lymphocyte function) and endocrine function (inhibition of thyroid-stimulating hormone release from the pituitary) (20). Because of the lack of benefit combined with the risk of harm, the use of low-dose dopamine in patients with AKI is considered bad medicine (borrowing the title from Reference 20).

As mentioned, discontinuing possible offending drugs is the most effective early measure for halting or reversing the course of AKI. A variety of drugs can be responsible for AKI, as indicated in Table 34.4.

Iodinated contrast agents can damage the kidneys in several ways, including direct renal tubular injury, renal vasoconstriction, and the generation of toxic oxygen metabolites (21). Using the AKIN criteria for the diagnosis of AKI, the incidence of AKI after contrast studies is 8-9% (22). The AKI usually appears within 72 hours after the contrast study. The incidence is greater in patients with multiorgan failure, chronic renal insufficiency, or during therapy with other nephrotoxic agents (23). Most cases resolve within two weeks, and few require renal replacement therapy (24).

INTRAVENOUS HYDRATION: The most effective preventive measure for contrastinduced nephropathy in high-risk patients is intravenous hydration (if permitted). The recommended regimen is isotonic saline at 100-150 mL/hr started 3 to 12 hours before the procedure and continued for 6-24 hours after the procedure (23). For emergency procedures, at least 300-500 mL isotonic saline should be infused just prior to the procedure.

Intrarenal Hemodynamics, Offending Drugs = Most Frequent: Others:. Intrarenal Hemodynamics, Offending Drugs = Nonsteroidal anti-inflammatory agents (NSAIDs) ACE inhibitors, angiotensin receptor-blocking drugs, cyclosporine, tacrolimus. Osmotic Nephropathy, Offending Drugs = Most Frequent: Others:. Osmotic Nephropathy, Offending Drugs = Hydroxyethyl starches Mannitol, intravenous immunoglobulins. Renal Tubular Injury, Offending Drugs = Most Frequent: Others:. Renal Tubular Injury, Offending Drugs = Aminoglycosides Amphotericin B, antiretrovirals, cisplatin. Interstitial Nephritis, Offending Drugs = Most Frequent: Others:. Interstitial Nephritis, Offending Drugs = Antimicrobials (penicillins, cephalosporins, sulfonamides, vancomycin, macrolides, tetracyclines, rifampin) Anticonvulsants (phenytoin, valproic acid), H; blockers, NSAIDs, proton pump inhibitors
Adapted from Refarance 21.
N-ACETYLCYSTEINE: N-acetylcysteine (NAC) is a glutathione surrogate with antioxidant actions that has had mixed results as a protective agent for contrast-induced nephropathy (3). However, an analysis of 16 studies using high-dose NAC (exceeding 1,200 mg daily) showed a 50% risk-reduction for contrast-induced nephropathy (24). The high-dose NAC regimen is 1,200 mg orally twice daily for 48 hours, beginning the night before the contrast procedure. For emergency procedures, the first 1,200 mg dose should be given
just prior to the procedure. Despite continuing debate, NAC is a popular preventive agent because of its low cost and safety.

AIN is an inflammatory condition that involves the renal interstitium and presents as acute renal failure. However, oliguria is not always a feature of AIN (25), which means that AIN does not always qualify for the diagnosis of AKI. Most cases of AIN are the result of a hypersensitivity drug reaction, but infections (usually viral or atypical pathogens) can also be involved. The drugs most often implicated in AIN are listed in Table 34.4 (26). Antibiotics are the most common offenders, particularly the penicillins. Drug-induced AIN is often (but not always) accompanied by signs of a hypersensitivity reaction; i.e., fever, rash, and eosinophilia. The onset of renal injury usually appears several weeks after the first exposure (26), but can appear within a few days after a second exposure. Sterile pyuria and eosinophiluria are common manifestations (26). A renal biopsy can secure the diagnosis, but these are rarely performed. AIN usually resolves spontaneously after the offending agent is discontinued, but recovery can take months

Acute renal failure develops in about one-third of patients with diffuse muscle injury (rhabdomyolysis) (27,28). The culprit is myoglobin, which is released by the injured muscle and is capable of damaging the renal tubular epithelial cells. The source of cell injury may be the iron moiety in heme (29), which is capable of oxidative cell injury via the production of hydroxyl radicals (see Figure 22.6 on page 437). This would explain why hemoglobin is also capable of producing renal tubular injury.
The diagnosis of AKI can be difficult in the setting of rhabdomyolysis because the injured muscle releases creatine, which is measured as creatinine, and falsely elevates the serum creatinine concentration (29).

Myoglobin can be detected in urine with the orthotoluidine dipstick reaction (Hemastix), which is used to detect occult blood in urine. If the test is positive, the urine should be centrifuged (to separate erythrocytes) and the supernatant should be passed through a micropore filter (to remove hemoglobin). A persistently positive test after these measures is evidence of myoglobin in urine. An alternative approach is to inspect the urine sediment for red blood cells; i.e., a positive dipstick test for blood without red blood cells in the urine sediment can be used as evidence of myoglobinuria.
The presence of myoglobin in urine does not ensure the diagnosis of AKI, but the absence of myoglobin in urine can be used to exclude the diagnosis of myoglobinuric renal injury (28).

Aggressive volume resuscitation to promote renal blood flow is the most effective measure for preventing or limiting the renal injury in rhabdomyolysis. Alkalinizing the urine can also help to limit the renal injury, but this is difficult to accomplish, and is often not necessary. The potassium and phosphate levels in plasma must be monitored closely in rhabdomyolysis because these electrolytes are released by injured skeletal muscle, and their concentrations in plasma can increase dramatically, especially when renal function is impaired. About 30% of patients who develop myoglobinuric renal injury will require dialysis (28).

Abdominal compartment syndrome (ACS) is the condition where an increase in abdominal pressure leads to dysfunction in one or more vital organs (30,31). This organ dysfunction usually involves the bowel (splanchnic ischemia), the kidneys (AKI), and cardiovascular system (reduced cardiac output).

The pertinent definitions related to ACS are shown inTable 34.5 (30). The intraabdominal pressure (IAP) is normally 5-7 mm Hg in the supine position (the IAP measurement is described later), and intraabdominal hypertension (IAH) is defined as a sustained increase in IAP to >12 mm Hg. ACS occurs when the IAP rises above 20 mm Hg and there is evidence of a newly-developed organ dysfunction.

The pressure in the abdominal cavity, which Is normally 5-7 mm Hg in the supine position.

A sustained increase In IAP to =12 mm Hg in the supine position.

A sustained increase in IAP to 220 mm Hg In the supine position that is accompanied by newly-developed organ dysfunction.

A measure of visceral perfusion pressure, equivalent to the difference between the mean arterlal pressure and the intraabdominal pressure: APP = MAP -IAP The desired APP is =60 mm Hg.

The mechanical force across the glomerulus, equivalent to the difference between the glomerular filtration pressure (or MAP — IAP) and the proximal tubular pressure (or IAP): FG = MAP -(IAP x 2).
Fram Referance 30.

ACS is traditionally associated with abdominal trauma, but several conditions can raise the IAP and predispose to ACS, including gastric distension, bowel obstruction, ileus, peritoneal hemorrhage, ascites, bowel wall edema, hepatomegaly, positive-pressure breathing, upright body position, and obesity (31). Several of these factors can co-exist in critically ill patients, which explains why IAH is discovered in as many as 60% of patients in medical and surgical ICUs (32).
LARGE VOLUME RESUSCITATION: One of the more common and unrecognized causes of IAH is large volume resuscitation, which can raise the IAP by promoting edema in the abdominal organs (particularly the bowel). In one report of ICU patients with a net
Covington, GA). The following conditions are required for each measurement (30): (a) the patient must be in the supine position, with the pressure transducer zeroed along the mid-axillary line, (b) a small volume (25 mL) of isotonic saline in injected into the bladder 30-60 seconds prior to each measurement, and (c) the IAP is measured only at the end of expiration, and only when there is no evidence of abdominal muscle contractions. The IAP is measured in mm Hg, not cm H20 (1 mm Hg = 1.36 cm H20).

General measures for reducing IAP include sedation (to reduce abdominal muscle contractions), avoiding elevation of the head more than 20° above the horizontal plane (35), and avoiding a positive fluid balance. Specific measures are dictated by the source of the elevated IAP, and can include decompression of the stomach, small bowel, or colon, percutaneous drainage of peritoneal fluid, or surgery (e.g., for abdominal injuries or bowel obstruction). As mentioned earlier, efforts to maintain an APP >60 mm Hg (with vasopressors to increase MAP, if necessary) is associated with improved outcomes in ACS. Surgical decompression is recommended for patients with ACS when the IAP cannot be reduced by conventional measures (35). However, this procedure has considerable risks (e.g., the abdomen is often left open for continued drainage), and these risks must be weighed against the risks of not performing the procedure.

About 70% of patients with acute renal failure will require some form of renal replacement therapy (RRT). The usual indications for RRT in acute renal failure include (a) volume overload, (b) life-threatening hyperkalemia or metabolic acidosis that is refractory to conventional measures, and (c) removal of toxins (e.g., ethylene glycol). Otherwise, the optimal timing for RRT in acute renal failure is unclear (36).
There is a growing body of RRT techniques, which includes not only hemodialysis and hemofiltration, but also hemodiafiltration, high flux dialysis, and plasmafiltration. The descriptions that follow are limited to hemodialysis and hemofiltration. The mechanisms of fluid and solute removal by each of these techniques is shown in Figure 34.3.

Hemodialysis removes solutes by diffusion, which is driven by the concentration gradient of solutes across a semipermeable membrane. To maintain this concentration gradient, a technique called countercurrent exchange is used, where blood and dialysis fluid are driven in opposite directions across the dialysis membrane. A blood pump is used to move blood in one direction across the dialysis membrane at a rate of 200-300 mL/min. The dialysis fluid on the other side of the membrane moves about twice as fast, at a rate of 500 to 800 mL/min (37). Large-bore, double lumen catheters are required for acute hemodialysis, and these catheters are described in Chapter 1 (see Table 1.5 on page 13, and Figure 1.6 on page 14).

The principal benefit of hemodialysis is rapid clearance of small solutes. Only a few hours of hemodialysis is needed to clear life-threatening accumulations of potassium or organic acids, or to remove a day's worth of accumulated nitrogenous waste. The disadvantages of hemodialysis include (a) limited removal of large molecules (e.g., inflammatory cytokines), and (b) the need to maintain a blood flow of 200-300 mL/min through the dialysis chamber. This latter requirement creates a risk of hypotension, which occurs in about one-third of hemodialysis treatments (37).

Hemofiltration removes solutes by convection, where a hydrostatic pressure gradient is used to move a solute-containing fluid across a semipermeable membrane. Since the bulk movement of fluid "drags" the solute across the membrane, this method of solute removal is also known as solvent drag (37).
Hemofiltration can remove large volumes of fluid (up to 3 liters per hour), but the rate of solute clearance is much slower than during hemodialysis. Therefore, hemofiltration must be performed continuously to provide effective solute clearance. Because solutes are cleared with water, the plasma concentration of these solutes (e.g., urea) does not decrease during hemofiltration unless a solute-free intravenous fluid is infused to replace some of the ultrafiltrate that is lost (this is often necessary because of the large volumes removed during hemofiltration).

Hemofiltration was originally performed by cannulating an artery (radial, brachial, or femoral) and a large vein (internal jugular or femoral). This method of continuous arteriovenous hemofiltration (CAVH) uses the mean arterial pressure as the filtration pressure, and does not require a pump in the circuit. However, it is not well suited for patients with labile blood pressures.
FIGURE 34.3 Mechanisms of solute clearance by hemodialysis and hemofitration. The smaller particles represent small solutes (e.g., urea), which can be cleared by both techniques, whi the larger particles represent larger molkecuks (e.g., inflammatory cyto-kines) that can be cleared by hemofiltration, but not by hemodialysis.
The more popular method at present is continuous venovenous hemofiltration (CVVH), where venous blood is removed and returned through large-bore, double-lumen catheters like the ones used for hemodialysis. This method does not require cannulation of an artery, but a pump in the circuit is required to create an effective filtration pressure.

There are two major advantages with hemofiltration. First, hemofiltration allows more gradual fluid removal than hemodialysis, and thus is less likely to produce hemodynamic compromise. Secondly, hemofiltration removes larger molecules than hemodialysis, which makes it the preferred method for removing toxins such as ethylene glycol. This feature also allows for the removal of inflammatory mediators, which can provide a therapeutic advantage in patients with systemic inflammation and multiorgan failure (38). The major disadvantage of hemofiltration is the slow solute removal, which is not well suited when rapid removal of solutes is necessary (e.g., for life-threatening hyperkalemia or acidosis). A newer method of RRT known as hemodiafiltration (which combines the features of dialysis and hemofiltration) is more suitable than hemofiltration for patients who require rapid solute removal as well as volume removal.

The author of the introductory quote, Paul Dirac, was a prominent (and eccentric) theoretical physicist who introduced the concept of antimatter (39). His quote refers to his equation (the Dirac equation) for describing the behavior of electrons, which was later shown to have some limitations. Like the Dirac equation, the concept of acute kidney injury has not achieved its intent to describe the spectrum of renal failure experienced by critically ill patients. The sources of confusion created by the concept of acute kidney injury are described on page 635.
The concept of acute kidney injury has one feature that is unlike the Dirac equation; i.e., the Dirac equation added considerably to the understanding of how electrons behave, while the concept of acute kidney injury adds little to the understanding of how or why renal failure occurs in critically ill patients.

1. Fliser D, Laville M, Covic A, et al. A European Renal Best Practice (ERBP) Position Statement on Kidney Disease Improving Global Outcomes (KDIGO) clinical practice guidelines on acute kidney injury. Nephrol Dial Transplant 2012; 27:4263-4272.
2. Brochard L, Abroug F, Brenner M, et al. An official ATS/ERS/ESICM/SCCM/ SRLF statement: Prevention and management of acute renal failure in the ICU patient. Am J Respir Crit Care Med 2010; 1126-1155.

3. Dennen P, Douglas IS, Anderson R. Acute kidney injury in the intensive care unit: an update and primer for the intensivist. Crit Care Med 2010; 38:261-275.
4. Ympa YP, Sakr Y, Reinhart K, et al. Has mortality from acute renal failure decreased? A systematic review of the literature. Am J Med 2005; 118:827—-832.
5. Chang CH, Lin C-Y, Tian Y-C, et al. Acute kidney injury classification: comparison of AKIN and RIFLE criteria. Shock 2010; 33:247-252.
Contrast-Induced Renal Injury, 1 = Contrast-Induced Renal Injury. 21., 1 = Pierson PB, Hansell P, Lias P. Pathophysiology of contrast medium-induced nephropathy. Kidney Int 2005; 68:14-22.. 22., 1 = Ehrmann S, Badin J, Savath L, et al. Acute kidney injury in the critically ill: Is iodinated contrast medium really harmful? Crit Care Med 2013; 41:1017-1025.. 23., 1 = McCullough PA, Soman S. Acute kidney injury with iodinated contrast. Crit Care Med 2008; 36(Suppl):S204-S211.. 24., 1 = Triverdi H, daram S, Szabo A, et al. High-dose N-acetylcysteine for the prevention of contrast-induced nephropathy. Am J Med 2009; 122:874.e9-15.

25. Ten RM, Torres VE, Millner DS, et al. Acute interstitial nephritis. Mayo Clin Proc 1988; 3:921-930.
26. Bentley ML, Corwin HL, Dasta J. Drug-induced acute kidney injury in the critically ill adult: Recognition and prevention strategies. Crit Care Med 2010; 38(Suppl): S169S174.
Myoglobinuric Renal Injury, 1 = Myoglobinuric Renal Injury. 27., 1 = Beetham R. Biochemical investigation of suspected rhabdomyolysis. Ann Clin Biochem 2000; 2000:37:581-587.. 28., 1 = Sharp LS, Rozycki GS, Feliciano DV. Rhabdomyolysis and secondary renal failure in critically ill surgical patients. Am J Surg 2004; 188:801-806.. 29., 1 = Visweswaran P, Guntupalli J. Rhabdomyolysis. Crit Care Clin 1999; 15:415-428.

30., 1 = Malbrain ML, Cheatham ML, Kirkpatrick A, et al. Results from the International Conference of Experts on Intra-abdominal Hypertension and Abdominal Compartment Syndrome. I. Definitions. Intensive Care Med 2006; 32:1722-1723.. 31., 1 = Al-Mufarrej F, Abell LM, Chawla LS. Understanding intra-abdominal hypertension: from bench to bedside. J Intensive Care Med 2012; 27:145-160.. 32., 1 = Malbrain ML, Chiumello D, Pelosi P, et al. Prevalence of intra-abdominal hypertension in critically ill patients: A multicenter epidemiological study. Intensive Care Med 2004; 30:822-829.. 33., 1 = Daugherty EL, Hongyan L, Taichman D, et al. Abdominal compartment syndrome is common in medical ICU patients receiving large-volume resuscitation. J Intensive Care Med 2007; 22:294-299.. 34., 1 = Sugrue M, Bauman A, Jones F, et al. Clinical examination is an inaccurate predictor of intraabdominal pressure. World J Surg 2002; 26:1428-1431.. 35., 1 = Cheatham ML, Malbrain ML, Kirkpatrick A, et al. Results from the International Conference of Experts on Intra-abdominal Hypertension and Abdominal Compartment Syndrome. II. Recommendations. Intensive Care Med 2007; 33:951- 962.
Renal Replacement Therapy, 1 = Renal Replacement Therapy. 36., 1 = Pannu N, Klarenbach S, Wiebe N, et al. Renal replacement therapy in patients with acute renal failure. A systematic review. JAMA 2008; 299:793-805.. 37., 1 = O'Reilly P, Tolwani A. Renal replacement therapy III. IHD, CRRT, SLED. Crit Care Clin 2005; 21:367-378.. 38., 1 = Morgera S, Haase M, Kuss T, et al. Pilot study on the effects of high cutoff hemofiltration on the need for norepinephrine in septic patients with acute renal failure. Crit Care Med 2006; 34:2099-2104.

39. Farmelo G. The Strangest Man. The Hidden Life of Paul Dirac, Mystic of the Atom. New York: Basic Books, 2009.

In dealing with any subject matter, find out what entities are undeniably involved, and state everything in terms of
these entities.
Bertrand Russell 1914
As many as 40% of ICU patients have a disorder involving the osmotic balance between intracellular and extracellular fluids, and in many cases, the problem is acquired after admission to the ICU (1). The presenting feature of these disorders is a change in the plasma sodium concentration (i.e., hypernatremia or hyponatremia), but the pathological problem is a change in cell volume, which is most apparent in the central nervous system.
This chapter presents a simple approach to osmotic disorders based on a single variable: the extracellular volume. The first part of the chapter is a brief review of osmotic forces and how they influence the distribution of total body water.

The movement of water between fluid compartments is determined by a property of fluids known as osmotic activity, which is a reflection of the number of solute particles per unit volume of solvent. Osmotic activity is a colligative property, and depends only on the number of solute particles in a fluid, and not on the electrical charge, size, or chemical behavior of the solutes. The total osmotic activity of a solution is the summed osmotic activity of all the solute particles in the solution.

When two fluid compartments are separated by a membrane that is permeable to solutes and water, the solutes in each fluid compartment will equilibrate across the membrane, and the osmotic activity will be equivalent in both fluid compartments. Since water movement follows solute movement, the volume in both fluid compartments will be equivalent. This is illustrated in the panel on the left in Figure 35.1. In this situation, the two fluids are described as isotonic (i.e., the term "tonicity" refers to the relative osmotic activity of two fluids).

When two fluid compartments are separated by a membrane that is permeable to water but is not freely permeable to solutes, the solutes will not distribute evenly in the fluid compartments, and the osmotic activity will be different in each fluid. In this situation, water moves from the fluid with the lower osmotic activity to the fluid with the higher osmotic activity, as demonstrated in the panel on the right in Figure 35.1. The difference in osmotic activity between the fluid compartments is called the effective osmotic activity, and it is the force that drives the movement of water between fluids with different osmotic activities. This force is also called the osmotic pressure. The fluid with the higher osmotic activity is described as hypertonic, and the fluid with the lower osmotic activity is described as hypotonic.
FIGURE 35.1 Illustration of the relationship between osmotic activity and the movement of water between fluid compartments. See text for explanation.

The following statements summarize the relationship between osmotic activity and the transcellular shift of water.
1. A change in the relative osmotic activity of extracellular fluid produces a transcellular water shift.
2. When the extracellular fluid is hypertonic, water will move out of cells.
3. When the extracellular fluid is hypotonic, water will move into cells.

The unit of measurement for osmotic activity is the osmole (osm), which is defined as one gram molecular weight (one mole) of a nondissociable substance, and is equivalent to 6x 1023 particles (Avogadro's Number). Osmotic activity can be expressed in relation to the volume of water in a solution, or the total volume of the solution (3,4).
1. Osmotic activity per volume of solution is called osmolarity, and is expressed as milliosmoles per liter (mosm/L).
2. Osmotic activity per volume of water is called osmolality, and is expressed as milliosmoles per kilogram of H2O (mosm/kg H20, or mosm/kg).
Plasma is mostly (93%) water, so the osmotic activity of plasma solutes is typically expressed as osmolality (mosm/kg HO). However, there is little difference between the osmolality and osmolarity of extracellular fluid, and the two terms are often used interchangeably (4).

The following formulas (where n is the number of nondissociable particles) can be used to convert plasma solute concentrations to units of osmolality:
- 1. For solute concentrations expressed in mEq/L:
(35.1)
Thus, for univalent ions like Nat, the plasma concentration in mEq/L is equivalent to the osmotic activity in mosm/kg H20.
- 2. For solute concentrations expressed in mg/dL:
```
mg/dL x 10 xn=mosm/kg H,O
```
(35.2)
mol wt where mol wt is molecular weight, and the factor 10 is used to convert deciliters (dL) to liters. For example, glucose has a molecular weight of 180, so a plasma glucose concentration of 90 mg/dL is equivalent to (90x 10/180)x1 = 5 mosm/kg H20.

The standard method for measuring plasma osmolality is the freezing point depression method. Solute-free water freezes at 0°C, and this freezing point decreases by 1.86°C for each osmole of solute that is added to one kilogram of water. Therefore, depression of the freezing point of plasma is proportional to the osmotic activity in plasma.

Plasma osmolality can also be calculated using the concentrations of the principal solutes in plasma (sodium, chloride, glucose, and urea) (3); i.e.,
$$Posm = (2 x [Nat]) + aor a BUN 2.8$$
$$(35.3)$$
where Posm is the plasma osmolality in mosm/kg H 20, [Na] is the plasma sodium concentration in mEq/L, [glucose] and BUN are the glucose and urea concentrations in plasma in mg/dL, and the factors 18 and 2.8 are the molecular weights of glucose and urea divided by 10, respectively, to express their concentrations in mosm/kg H 20 (analogous to Equation 35.2). The [Nat] is doubled to include the osmotic activity of chloride.
EXAMPLE: Using normal plasma concentrations of Na+ (140 mEq/L), glucose (90 mg/dL), and BUN (14 mg/dL), the plasma osmolality is: (2x140) + 90/18 + 14/2.8 = 290 mosm/kg H20.
EFFECTIVE PLASMA OSMOLALITY: Urea readily crosses cell membranes, so an increase in blood urea nitrogen (BUN) will not increase the relative osmotic activity of plasma (i.e., azotemia is a hyperosmotic, but not a hypertonic, condition). Therefore, the calculation of effective plasma osmolality does not include the BUN; i.e.,
$$[glucose] Effective Posm = (2 x [Nat]} + ; &$$
(35.4)
Using normal plasma concentrations of Na+ (140 mEq/L) and glucose (90 mg/dL) yields an effective plasma osmolarity of (2x140) + 90/18 = 285 mosm/kg H20. Note the following:
1. There is only a minor difference (5 mosm/kg HO) between the total and effective plasma osmolarity.

2. Plasma sodium accounts for 98% (280 of 285 mosm/kg H20) of the effective osmotic activity of the extracellular fluid. This highlights the fact that the sodium concentration in extracellular fluid is the principal determinant of the distribution of total body water in the intracellular and extracellular fluid compartments.

Because solutes other than sodium, glucose, and urea are present in the extracellular fluid, the measured plasma osmolality will be greater than the calculated osmolality. This osmolal gap (i.e., the difference between measured and calculated plasma osmolality) is normally " 10 mosm/kg H20 (3,5). Accumulation of a nondiffusable solute (like an exogenous toxin) will increase the osmolal gap, and this has led to the use of the osmolal gap to detect the presence of ingested toxins that are not easily measured (6).

The normal plasma [Nat] is 135-145 mEq/L, and thus hypernatremia is defined as a plasma [Na+] >145 mEq/L. This condition is reported in as many as 25% of ICU patients (1), and is acquired in the ICU in most cases (1,7).

Hypernatremia can be the result of 3 conditions: (a) Loss of sodium and water, with water loss >sodium loss (i.e., hypotonic fluid loss), (b) free water loss, and (c) gain of sodium and free water, with sodium gain >free water gain (i.e., gain of hypertonic fluid) (8).

Each of the conditions responsible for hypernatremia is associated with a different extracellular volume (ECV); i.e., hypotonic fluid loss is associated with a low ECV, free water loss is associated with a normal ECV, and hypertonic fluid gain is associated with a high ECV. Therefore, assessment of the extracellular volume (ECV) can be used to identify the condition responsible for hypernatremia. The approach to hypernatremia based on the ECV is shown in Figure 35.2. (Assessment of the extracellular volume is not presented here. See Chapter 11 for the evaluation of hypovolemia.)
FIGURE 35.2 Flow diagram for the approach to hypernatremia based on the extracellular volume (ECV).

The principal consequence of hypernatremia is an increase in the effective osmolality (tonicity) of the extracellular fluid, which draws water out of cells. This is most evident in the central nervous system.

The clinical manifestations of hypernatremic encephalopathy range from agitation and lethargy to coma and generalized or focal seizures (1). Encephalopathy is more likely to occur with a rapid rise in plasma Nat, and possible mechanisms include shrinkage of neuronal cell bodies (9) and osmotic demyelination (10). The presence of encephalopathy is a poor prognostic sign in hypernatremia, and carries a mortality rate as high as 50% (9).

Hypernatremia associated with a low extracellular volume (ECV) is the result of loss of hypotonic fluids (i.e., fluids with a [Na+] <135 mEq/L). This is the most common cause of community-acquired (but not ICU-acquired) hypernatremia (1).

The average sodium concentration in fluids that can be lost is shown in Table 35.1 (11-
13). Note the following:
1. All fluid losses involve the loss of sodium, which promotes a de-crease in ECV.
2. All lost fluids are hypotonic to plasma (i.e., have a sodium concentration <135 mEq/L), and will eventually result in hypernatremia if not replaced.
Common sources of hypotonic fluid loss include (a) excessive diuresis (osmotic or druginduced), (b) excessive sweat loss in heat-related illnesses, and (c) normal or accentuated fluid losses in elderly, debilitated patients, particularly those with sepsis.

The normal response to hypotonic fluid loss is an increase in the sensation of thirst, which promotes water ingestion to limit the rise in plasma osmolality. Patients who develop symptomatic hypernatremia often have an impaired sensation of thirst from a debilitating or chronic illness, or advanced age. For this reason, symptomatic hypernatremia from hypotonic fluid loss can be a sign of a more serious condition; i.e., "failure to thrive."
Normal Stool, ‘[Nat] (mEq/L) = 25. Normal Stool, Other Fluids = Normal Urine Diuretic. Normal Stool, [Na+] (mEq/L) = <10*. Vomitus/NG Drainage, ‘[Nat] (mEq/L) = 60. Vomitus/NG Drainage, Other Fluids = Urine. Vomitus/NG Drainage, [Na+] (mEq/L) = 80. lleostomy Drainage, ‘[Nat] (mEq/L) = 125. lleostomy Drainage, Other Fluids = Paul. lleostomy Drainage, [Na+] (mEq/L) = S. Secretory Diarrhea, ‘[Nat] (mEq/L) = 90. Secretory Diarrhea, Other Fluids = Paul. Secretory Diarrhea, [Na+] (mEq/L) = S. , ‘[Nat] (mEq/L) = . , Other Fluids = Paul. , [Na+] (mEq/L) = S
From Referancas 141-13.
"Dependent on sodium intake.

Hypotonic fluid loss has less of an effect on plasma volume than ECV because the accentuated loss of free water increases the colloid osmotic pressure in plasma (i.e., the osmotic pressure generated by plasma proteins, principally albumin, that do not move readily into the interstitial fluid), and this draws fluid from the interstitial fluid and helps to maintain the plasma volume. The influence of hypotonic fluid loss on plasma volume is determined by the severity of sodium losses; i.e., the greater the sodium loss, the greater the likelihood of a significant decrease in plasma volume (and a subsequent decrease in cardiac output).

Hypernatremia with a normal ECV is the result of free water loss with no net sodium loss. This condition is common in ICU patients with hypernatremia (1), and usually occurs when sodium losses are replaced, leaving a net free water deficit. The condition described next is the best example of a free water deficit.

Hypernatremia with a high ECV is uncommon, and is usually the result of sodium bicarbonate infusions for metabolic acidosis, or aggressive use of hypertonic saline to treat increased intracranial pressure. Excessive ingestion of table salt (often in females with a psychiatric disorder) should also be considered in patients admitted with hypervolemic hypernatremia (21).

In patients with normal renal function, excess sodium and water are excreted rapidly. When renal sodium excretion is impaired, it might be necessary to increase renal sodium excretion with a diuretic (e.g., furo-semide). Because the urinary sodium concentration during furosemide diuresis (~80 mEq/L) is less than the plasma sodium concentration, diuresis can aggravate the hypernatremia. If this occurs, urine losses should be partially replaced with a fluid that is hypotonic to urine (e.g., quarter-normal saline).

Although normoglycemia has little osmotic impact, severe hyperglycemia has a considerable influence on plasma osmolality (e.g., a plasma glucose level of 600 mg/dL is equivalent to 600/18 = 40 mosm/kg H20).
hypovolemia. Therefore, in patients who are hypo-volemic, insulin should be withheld until the vascular volume is restored. Once this is accomplished, insulin is started using the same regimen recommended for diabetic ketoacidosis (see pages 610-613). However, the insulin requirement will diminish as the hypertonic condition is corrected (because hypertonicity promotes insulin resistance), so plasma glucose levels should be monitored frequently during insulin infusions to prevent hypoglycemia.

Hyponatremia (plasma [Na] <135 mEq/L) (26), is reported in 40-50% of ICU patients (26,27), and is particularly prevalent in neurosurgical patients (27). Although hyponatremia is typically a hypotonic condition, there are instances of isotonic hyponatremia (e.g., pseudohyponatremia) and hypertonic hyponatremia (i.e., non-ketotic hyperglycemia, de-scribed in the last section).

The traditional method of measuring the plasma [Na] (flame photometry) includes both aqueous and nonaqueous phases of plasma, while sodium is restricted to the aqueous phase of plasma. Therefore, the measured plasma [Na]can be lower than the actual (aqueous phase) plasma [Na]. Plasma is about 93% water, so the difference between measured and actual plasma [Na] is small (about 7%).
Marked increases in lipid or protein levels in plasma will add to the nonaqueous phase of plasma, and this can significantly lower the measured plasma [Na] without affecting the actual (aqueous phase) plasma [Na]. This condition is called pseudohyponatremia (28). It usually does not occur until the plasma lipid levels rise above 1,500 mg/dL or the plasma protein levels rise above 12-15 g/dL (28). The influence of hypertriglyceridemia on the measured plasma [Na] is described by the following equation (28):
% decrease in [Na]= 2.1 x triglycerides (g/dL) -0.6
(35.9)

The diagnosis of pseudohyponatremia can be confirmed or eliminated by measuring the plasma osmolality, which will be normal in pseudohyponatremia, and reduced in "true" or hypotonic hyponatremia. An alternative method involves measuring the plasma [Na] with an ion-specific electrode. These electrodes measure the [Na] in the aqueous phase of plasma, and the plasma [Na] will be normal in cases of pseudohyponatremia.

Hypotonic hyponatremia is the result of excess free water relative to sodium in the extracellular fluid. Most cases involve loss of the normal control mechanisms for antidiuretic hormone (ADH) release.

Antidiuretic hormone (ADH) is released by the posterior pituitary in response to an increase in the osmolality of extracellular fluid, and it helps to curb the hyperosmolar condition by promoting water reabsorption in the distal renal tubules. ADH release is normally suppressed at a plasma [Na] " 135 mEq/L (1). ADH is also released in response to nonosmotic factors like a decrease in blood pressure (via baroreceptors) or "physiological stress" (i.e., the same stimulus for ACTH release from the anterior pituitary).
When nonosmotic stimuli for ADH release are active, ADH release persists despite a plasma [Na] "135 mEq/L, and the resulting water reabsorption in the kidneys aggravates the hyponatremia. Therefore, nonosmotic or "inappropriate" release of ADH is an important factor in the development of severe and sustained hyponatremia (29).

ENCEPHALOPATHY: The principal consequence of hypotonic hyponatremia is a lifethreatening encephalopathy characterized by cerebral edema, increased intracranial pressure, and the risk of brain herniation (29,30). Symptoms range from headache, nausea and vomiting to seizures, coma, and brain death. The risk and severity of encephalopathy are greater with acute (<48 hrs) hyponatremia (29,30).
EXTRACELLULAR VOLUME: Like hypernatremia, the extracellular volume can be low, normal, and high in hyponatremia, and the approach can be organized according to the extracellular volume (ECV). This is shown in Figure 35.3.

Hyponatremia with a low ECV is the result of sodium loss with excess free water retention. The sodium loss decreases the ECV, and the excess free water retention decreases the extracellular [Na]. The excess free water retention is due to baroreceptormediated (nonosmotic) ADH release (29) combined with free water intake (oral intake or infusion of a fluid that is hypotonic to the fluid lost).

The principal conditions associated with hypovolemic hyponatremia are shown in Table 35.2. Thiazide diuretics are common offenders, probably because they impair renal diluting ability. Other conditions in critically ill patients are primary adrenal insufficiency and cerebral salt wasting.
PRIMARY ADRENAL INSUFFICIENCY: Primary adrenal insufficiency is accompanied by mineralocorticoid deficiency, which results in renal sodium wasting. In contrast, secondary (hypothalamic) adrenal insufficiency is primarily a glucocorticoid deficiency, and does not promote renal sodium loss.
CEREBRAL SALT WASTING: Cerebral salt wasting is a syndrome that occurs with traumatic brain injury, subarachnoid hemorrhage, and neurosurgery (27). The mechanism for the renal sodium loss is unclear (29).

The source of Nat loss is usually apparent. If this is unclear, the spot urine [Na] can help to distinguish renal from extrarenal losses; i.e., a high urine [Na] (>20 mEq/L) suggests a renal source of sodium loss, while a low urine [Na] (<20 mEq/L) suggests an extrarenal source (see Figure 35.3).
Renal NAt Loss Diuretics Cerebral Salt Wasting Primary Adrenal Insufficiency Extrarenal NA* Loss Gl Losses, NormalECV = = ADH-Related SADH Physiological Stress Hypothyroidism Not ADH-Related Primary Polydipsia. Renal NAt Loss Diuretics Cerebral Salt Wasting Primary Adrenal Insufficiency Extrarenal NA* Loss Gl Losses, High ECV. = Cimhosis Heart Failure Renal Failure
"Must bs combined with water intake to produce hyponatremia.

Hypervolemic hyponatremia is the result of Na+ and HO retention, with H2O retention exceeding Nat retention. This condition occurs in advanced heart failure, cirrhosis, and renal failure. Renal failure is associated with a high urine [Na+] (>20 mEq/L), while the urine [Na+] is low (<20 mEq/L) in heart failure and cirrhosis, except during diuretic therapy when the drug is active.
FIGURE 35.3 Flow diagram for the approach to hyponatremia based on the extracellular volume (ECV).
furosemide diuresis (29). For asymptomatic patients, fluid restriction and furosemide diuresis are the standard measures.

Demeclocycline is a tetracycline derivative that blocks the effects of ADH in the renal tubules. It is used primarily in patients with SIADH and chronic hyponatremia who do not tolerate fluid restriction. The drug is given orally, and the dose is 600-1200 mg daily in divided doses (29). Maximum effect takes several days, and success is variable. Demeclocycline can be nephrotoxic, so monitoring renal function is advised while using the drug.

Since 2005, two drugs have been introduced that block receptors for arginine vasopressin (the other term for antidiuretic hormone). These drugs, conivaptan and tolvaptan (the "vaptans") can be used as an alternative to fluid restriction in patients with euvolemic or hypervolemic hyponatremia (except renal failure). At the present time, their principal use is in patients with SIADH-related hyponatremia. These drugs are contraindicated in hypovolemic hyponatremia because of their aquaretic effects.
CONIVAPTAN: Conivaptan is the original vaptan, and blocks vasopressin effects in the kidneys and elsewhere. It is given intravenously with a loading dose of 20 mg, followed by a continuous infusion of 40 mg/day for 96 hours. The effect of this dosing regimen on the plasma [Na] is shown in Figure 35.4 (32). Note the 6-7 mEq/L increase in plasma [Na] in the first 24 hours of drug infusion, which is maintained for the 96-hour infusion period.
TOLVAPTAN: Tolvaptan is more selective than conivaptan, and blocks only vasopressin receptors in the kidneys. The drug is given orally, starting at a dose of 15 mg once daily, and increasing the dose, if necessary, to a maximum of 60 mg daily. Increases in plasma [Na] of 6-7 mEq/L have been observed in the first 4 days of tolvaptan therapy (31), which is the time to maximal drug effect.
sodium concentration in patients with euvolemic and hypervolemic hyponatremia. N is the number of study patients. Data from Reference 32.
COMMENT: The vaptans have not gained widespread acceptance, although they may have a role as an alternative to fluid restriction in SIADH-related hyponatremia. However, these agents offer no apparent advantages for the acute management of symptomatic hyponatremia in critically ill patients.

The following points from this chapter deserve emphasis:
1. Sodium is the principal determinant of the distribution of total body water in the intracellular and extracellular fluids.
2. An abnormal plasma sodium concentration is not about sodium, it's about cell volume.
3. A single variable (the extracellular volume), can be used to understand, identify, and correct the osmotic impact of hypernatremia and hyponatremia. (Bertrand Russell would be proud.)

1. Pokaharel M, Block CA. Dysnatremia in the ICU. Curr Opin Crit Care 2011; 17:581- 593.
, 1 = Osmotic Activity. 2., 1 = Rose BD, Post TW. The total body water and the plasma sodium concentration. In: Clinical physiology of acid-base and electrolyte disorders. 5th ed. New York, NY: McGraw-Hill, 2001; 241-257.. 3., 1 = Gennari FJ. Current concepts. Serum osmolality. Uses and limitations. N Engl J Med 1984; 310:102-105.. 4., 1 = Erstad BL. Osmolality and osmolarity: narrowing the terminology gap. Pharmacother 2003; 23:1085-1086.. 5., 1 = Turchin A, Seifter JL, Seely EW. Clinical problem-solving. Mind the gap. N Engl J Med 2003; 349:1465-1469.. 6., 1 = Purssell RA, Lynd LD, Koga Y. The use of the osmole gap as a screening test for the presence of exogenous substances. Toxicol Rev 2004; 23:189-202.

26., 1 = Hoorn EJ, Lindemans J, Zietse R. Development of severe hyponatremia in hospitalized patients: treatment-related risk factors and inadequate management. Nephrol Dial Transplant 2006; 21:70-76.. 27., 1 = Upadhyay UM, Gormley WB. Etiology and management of hyponatremia in neurosurgical patients. J Intensive Care Med 2012; 27:139-144.. 28. Aw TC, Kiechle FL. Am J Emerg Med 1985; 3:236-239., 1 = 28. Aw TC, Kiechle FL. Am J Emerg Med 1985; 3:236-239.. 29., 1 = Verbalis JG, Goldsmith SR, Greenberg A, et al. Hyponatremia treatment guidelines 2007: Expert panel recommendations. Am J Med 2007; 120(Suppl): S1-S21.. 30. Arieff AI, Ayus JC. Pathogenesis of hyponatremic encephalopathy. Current concepts. Chest 1993; 103:607-610., 1 = 30. Arieff AI, Ayus JC. Pathogenesis of hyponatremic encephalopathy. Current concepts. Chest 1993; 103:607-610.. 31., 1 = Lehrich RW, Greenberg A. Hyponatremia and the use of vasopressin receptor antagonists in critically ill patients. J Intensive Care Med 2012; 27:207-218.. 32., 1 = Zeltser D, Rosansky S, van Rensburg H, et al. Assessment of efficacy and safety of intravenous conivaptan in euvolemic and hypervolemic hyponatremia. Am J Nephrol 2007; 27:447-457.

No stream can rise higher than its source.
Frank Lloyd Wright
1875
The earliest sea-living organisms showed a preference for intracellular potassium and a disdain for intracellular sodium, and this behavior eventually changed the composition of the oceans from a potassium salt solution to a sodium salt solution. The same preferences are found in the human organism, where 98% of the total body potassium is located inside cells, and only 2% remains in the extracellular fluid (1-3). As a result, monitoring the plasma (extracellular) potassium level as an index of total body potassium is like evaluating the size of an iceberg by its tip. With this limitation in mind, this chapter describes the causes and consequences of abnormalities in the plasma potassium concentration.

The intracellular preponderance of potassium is the result of a sodium-potassium (Na+K+) exchange pump on cell membranes that moves Na+ out of cells and moves K+ into cells in a 3:2 ratio (1). One of the major roles of this pump is to create a voltage gradient across cell membranes in "excitable" tissues (i.e., nerves and muscle), which allows for the transmission of electrical impulses in these tissues.
The small fraction of total body K+ that is present outside cells is illustrated in Figure 36.1. The total body potassium in healthy adults is about 50-55 mEq per kg body weight (1). Using the conservative estimate of 50 mEq/kg in a 70 kg adult yields a total body potassium of 3,500 mEq, with 70 mEq (2%) in extracellular fluid. Because the plasma accounts for about 20% of the extracellular fluid, the potassium content of plasma will be about 15 mEq, whichis only 0.4% of the total body potassium. This emphasizes the limited size of the K+ pool that is available for evaluating total body potassium.
FIGURE 36.1 Illustration of the small fraction of total body K+ that is present outside cells. This example pertains to a 70 kg adult with an estimated total body Kt of 50 mEq/kg body weight. Each gold bar represents 100 mEq of Kt.

The relationship between total body K+ and serum (plasma) K+ is shown in Figure 36.2 (4,5). Note the curvilinear shape of the curve, with the flat portion of the curve in the region of potassium deficiency. In an averaged-size adult with a normal serum K+ of 4 mEq/L, a total body K+ deficit of 200-400 mEq is required to produce a decrease in plasma K+ of 1 mEq/L, while a total body K+ excess of 100-200 mEq is required to produce a similar (1 mEq/L) increase in plasma K (5). Therefore, for a given change in serum K+, the change in total body K+ is twofold greater with K+ depletion (hypokalemia) than with K+ excess (hyperkalemia). The larger deficit associated with hypokalemia is due to the large pool of intracellular K+ that can replenish extracellular K+ (and help to maintain serum K+) when K+ is lost.

Small amounts of K+ are lost in stool (5-10 mEq/day) and sweat (0-10 mEq/day), but the majority of K+ loss is in urine (40-120 mEq/day, depending on K+ intake) (1).

Most of the K+ that is filtered at the glomerulus is passively reabsorbed in the proximal tubules (along with sodium and water), and K+ is then secreted in the distal tubules and collecting ducts (1). Potassium excretion in urine is primarily a function of K+ secretion in the distal nephron, which is controlled by plasma K+ and (primarily by ) aldosterone. When renal function is normal, the capacity for renal K+ excretion is great enough to prevent a sustained rise in serum K+ in response to an increased K+ load (1).
FIGURE 36.2 Relationship between the serum potassium concentration and total body potassium. Redrawn from Reference 4.
ALDOSTERONE: Aldosterone is a mineralocorticoid that is released by the adrenal cortex in response to an increase in plasma K+ (and angiotensin II), and it increases Kt excretion in the urine by stimulating K+ secretion in the distal nephron. Potassium secretion is linked to sodium reabsorption, so aldosterone also promotes sodium and water retention. The diuretic spironolactone acts by blocking the actions of aldosterone in the kidneys. As a result, spironolactone is a potassium-sparing diuretic.

Hypokalemia (serum K+ <3.5 mEq/L) can be the result of K+ movement into cells (transcellular shift), or a decrease in total body K+ ( K+ depletion) (3-6).

The movement of K+ into cells is facilitated by stimulation of B2 adrenergic receptors on cell membranes in muscle, and this explains the decrease in serum K+ associated with inhaled B-agonist bronchodilators (e.g., albuterol) (7). This effect is mild ("0.5 mEq/L) in the usual therapeutic doses (7), but is more significant when inhaled B>-agonists are used in combination with diuretics (8). Other conditions that promote K+ movement into cells include alkalosis (respiratory or metabolic), hypothermia (accidental or induced), and insulin. Alkalosis has a variable and unpredictable effect on serum K+ (9). Hypothermia causes a transient drop in serum K+ that resolves on rewarming (10).

Potassium depletion can be the result of K+ loss via the kidneys or gastrointestinal tract. The site of K+ loss (renal or extrarenal) is usually obvious, but can be identified by measuring the urinary K+ and chloride concentrations, as shown in Figure 36.3.
FIGURE 36.3 Diagnostic approach to hypokalemia.

The leading cause of renal K+ loss is diuretic therapy. Other causes likely to be seen in the ICU include nasogastric drainage, alkalosis, and magnesium depletion. Nasogastric drainage has a low concentration of K+ (10-15 mEq/L), but the resulting loss of volume and H+ promotes K+ loss in the urine. The urine chloride is low (<15 mEq/L) with nasogastric drainage and alkalosis, and it is high (>25 mEq/L) with diuretic therapy and magnesium depletion. Magnesium depletion impairs K+ reabsorption in the renal tubules and may play a very important role in promoting K+ depletion in critically ill patients, particularly those receiving diuretics (12).

The major cause of extrarenal K+ loss is diarrhea. Normal K+ loss in stool is only 5-10 mEq daily. In secretory and inflammatory diarrhea, the concentration of K+ in stool is 15—40 mEq/L, and the daily stool volume can reach 10 liters in severe cases. Therefore, K+ losses can reach 400 mEq daily in severe cases of inflammatory or secretory diarrhea (11).

Severe hypokalemia (serum K+ <2.5 mEq/L) can be associated with diffuse muscle weakness (3), but in most cases, hypokalemia is asymptomatic. Abnormalities in the ECG are the major manifestation of hypokalemia, and can be present in 50% of cases (13). The ECG abnormalities include prominent U waves (more than 1 mm in height), flattening and inversion of T waves, and prolongation of the QT interval. However, these changes are not specific for hypokalemia; i.e., the T wave changes and U waves can be seen with digitalis or left ventricular hypertrophy, and QT prolongation can be the result of drugs, hypocalcemia, or hypomagnesemia.

Contrary to popular belief, hypokalemia alone is not a risk for serious arrhythmias (3,13). However, hypokalemia can add to the risk of serious arrhythmias from other conditions (e.g., myocardial ischemia) (3).

The first concern in hypokalemia is to eliminate or treat any condition that promotes transcellular potassium shifts (e.g., alkalosis) (3). If the hypokalemia is due to Kt depletion, proceed as described next.

About 10% of total body K+ is lost for every 1 mEq/L decrease in serum K+ (14). For a 70 kg adult with a normal total body K+ of 50 mEq/kg, the estimated K+ deficits associated with progressive hypokalemia are shown in Table 36.1. Note that even mild hypokalemia (serum K+ = 3 mEq/L) is associated with a considerable K+ deficit (175 mEq).

3.0, ee = 175. 3.0, ee = 5. 2.5, ee = 350. 2.5, ee = 10. 2.0, ee = 470. 2.0, ee = 15. 1.5, ee = 700. 1.5, ee = 20. 1.0, ee = 875. 1.0, ee = 25. , ee = . , ee = 
"Estimated deficits ara for a 70 kg adut with a total body potassium of 50 mEq/kg.

FLUIDS: The usual replacement fluid is potassium chloride, which is available as a concentrated solution (1-2 mEq/mL) in ampules containing 10, 20, 30, and 40 mEq of potassium. These solutions are extremely hyperosmotic (the 2 mEq/mL solution has an osmolality of 4,000 mosm/kg H20) and must be diluted (15). A potassium phosphate solution is also available that contains 4.5 mEq potassium and 3 mmol phosphate per mL, and this solution is preferred by some for potassium replacement in diabetic ketoacidosis (because of the phosphate depletion in diabetic ketoacidosis).
RATE OF REPLACEMENT: The standard method of intravenous K+ replacement is to add 20 mEq of K+ to 100 mL of isotonic saline and infuse this mixture over 1 hour (16). The maximum rate of intravenous potassium replacement is usually set at 20 mEq/hr (16), but dose rates of 40 mEq/hour may be necessary (e.g., serum K+ <1.5 mEq/L or serious arrhythmias), and dose rates as high as 100 mEq/hour have been used safely (17). Infusion through a large, central vein is preferred, if possible, because of the irritating properties of the hyperosmotic KCL solutions. However, delivery into the superior vena cava is not recommended if the desired rate of replacement exceeds 20 mEq/hr because there is a (poorly documented) risk of an abrupt rise in plasma K+ in the right heart chambers severe enough to produce asystole.
RESPONSE: The serum K+ may be slow to rise initially, as predicted by the flat portion of the curve inFigure 36.2. If the hypokalemia is resistant or refractory to Kt replacement, magnesium depletion should be considered. Magnesium depletion promotes urinary K+ loss (as described earlier), andin patients who are magnesium deficient, hypokalemia is often refractory to K+ replacement until the magnesium is repleted (18). Magnesium deficiency may play an important role in diuretic-induced hypokalemia, as described in the next chapter.

While hypokalemia is often well tolerated, hyperkalemia (serum K+ >5.5 mEq/L) can be a life-threatening condition (2,3,19,20).

Hyperkalemia can be the result of potassium release from cells (transcellular shift), or impaired renal excretion of potassium. If the source of the hyperkalemia is unclear, a spot urine K+ can be useful. A high urine K+ (>30 mEq/L) suggests a transcellular shift, and a low urine K+ (<30 mEq/L) indicates impaired renal excretion. If the hyperkalemia is unexpected, the condition described next should be considered.

Hyperkalemia that is present ex vivo (in the blood sample), but not in vivo, is known as pseudohyperkalemia. The leading cause of this condition is potassium release from traumatic hemolysis during the venipuncture. This is more common than suspected, and has been reported in 20% of blood samples with hyperkalemia (21). Other sources of pseudohyperkalemia in-clude: (a) K+ release from muscles during fist clenching (22), and (b) potassium release from clot formation in the blood collection tube in patients with severe leukocytosis (>50,000/mm3) or severe thrombocytosis (platelet count >1 million/mm3). When pseudohyperkalemia is suspected, a repeat blood sample should be obtained using precautions to mitigate the suspected problem (e.g., minimize suction when withdrawing blood).

The conditions associated with K+ movement out of cells include acidosis, rhabdomyolysis, tumor lysis syndrome, drugs, and blood transfusions.
ACIDOSIS: The presumed mechanism for the relationship between acidosis and hyperkalemia is competition between H+ and K+ for the same site on the membrane pump that moves K+ into cells. However, a causal link between acidosis and hyperkalemia is being questioned because the organic acidoses (lactic acidosis and ketoacidosis) are not associated with hyperkalemia (9), and respiratory acidosis has an inconsistent and unpredictable association with hyperkalemia (9).
TUMOR LYSIS SYNDROME: Tumor lysis syndrome is an acute, life-threatening condition that appears within 7 days after the initiation of cytotoxic therapy for selected malignancies (e.g., non-Hodgkins lymphoma). Characteris-tic features include the combination of hyperkalemia, hyperphosphatemia, hypocalcemia, and hyperuricemia, often accompanied by acute kidney injury (23). Hyperkalemia is the most immediate threat to life.
DRUGS: The drugs that promote K+ movement out of cells are listed in Table 36.2. Digitalis inhibits the membrane NatK+ exchange pump, but hyperkalemia occurs only with acute (not chronic) digitalis toxicity (24). Succinylcholine is an ultra short-acting neuromuscular blocking agent that also inhibits the membrane Nat+K+ exchange pump (a depolarizing effect), and this effect is associated with a minor increase in serum K+ (<1 mEq/L) that lasts only 5—10 minutes (25). Life-threatening increases in serum K+
have been reported when succinylcholine is used in patients with "denervation injury"of skeletal muscle (e.g., spinal cord injury). This is attributed to a an exaggerated response to depolarizing signals following denervation (denervation hypsersensitivity).

B-blockers Digitalis Succinyleholine, Impair Renal K+ Excretion: = AGE Inhibitors Angiotensin-receptor blockers K*- sparing diuretics NSAIDS Heparin Trimethoprim-sulfamethoxazole

As mentioned earlier in the chapter, when renal function is normal, the capacity for urinary K+ excretion is great enough to prevent a sustained increase in serum K+ in response to a K+ load (1). As a result, hyperkalemia always involves a defect in renal K+ excretion. The common causes of impaired renal K+ excretion include renal failure, adrenal insufficiency, and drugs. In renal failure, hyperkalemia usually doesn't occur until the GFR drops below 10 mL/min, but it can appear earlier when renal failure is the result of interstitial nephritis. Adrenal insufficiency impairs renal potassium excretion, but hyperkalemia is seen only in chronic adrenal insufficiency.
DRUGS: Drugs that impair renal potassium excretion represent a common source of hyperkalemia. A list of common offenders is shown in Table 36.2 (25-29). The drugs most often implicated are angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, potassium sparing diuretics, and nonsteroidal antiinflammatory drugs. All of these agents promote hyperkalemia by inhibiting the renin—angiotensin—aldosterone system. The hyperkalemia from these drugs often occurs in combination with Kt supplements or renal insufficiency.

Hyperkalemia is a recognized (but inconsistent) complication of massive blood transfusion (i.e., blood replacement equivalent to the blood volume). The temperature used to store red blood cells (4°C) shuts off the Na+K+ exchange pump in the erythrocyte cell membrane, and this results in a steady leakage of K+ from the cells (30). The K+ concentration in the supernatant increases steadily as the storage time increases. After 18 days of storage (the average time that blood is stored), the potassium load in one unit of packed red blood cells is 2 to 3 mEq (30), so massive transfusion (usually at least 6 units of red blood cells) represents a K+ load of at least 12-18 mEq. This is a considerable load, considering that plasma contains about 9-10 mEq of K+ in an averagesized adult.
The K+ load in transfused blood is normally cleared by the kidneys, but when systemic blood flow is compromised (which applies to most patients who need massive blood transfusions), renal K+ excretion is impaired, and the K+ infused in blood transfusions will accumulate. The transfusion volume needed to produce hyperkalemia will vary, but one study has shown that hyperkalemia begins to appear after transfusion of 7 units of red blood cells (31).

In 1985, a case report was published describing a patient with severe hyperkalemia who was discovered to be ingesting 1,500 burnt match heads daily (a pica) (32). It turns out that burnt match heads are rich in potassium chlorate, and the habit of eating burnt match heads is known as cautopyreiophagia. This case is mentioned to demonstrate that anything is possible in medicine, and that there is a name for everything.

The principal threat of hyperkalemia is slowed impulse transmission in the heart (from depolarization of cardiac muscle), which can progress to heart block and bradycardic cardiac arrest.

The ECG changes in progressive hyperkalemia are shown in Figure 36.4. The earliest change is the appearance of a tall, tapering (tented) T wave that is most evident in precordial leads V> and V3. As the hyperkalemia progresses, the P wave amplitude decreases and the PR interval lengthens. The P waves eventually disappear and the QRS complex widens. The final event is ventricular fibrillation or asystole.
ECG changes usually begin to appear when the serum K+ reaches 7 mEq/L (33), but the threshold for ECG changes can vary widely. This is demonstrated by one case report that showed no ECG abnormalities at a serum K+ of 14 mEq/L (!) (34). Because of the variable relationship between the serum K+ and the ECG, both are used for management decisions in hyperkalemia.

Severe hyperkalemia is defined as a serum K+ >6.5 mEq/L, or any serum K+ associated with ECG changes (33). The management of this condition has 3 goals: (a) antagonism of the cardiac effects of hyperkalemia, (b) transcellular shift of K+ into cells, and (c) removal of excess K+ from the body. The methods used to achieve these goals are described next, and are summarized in Table 36.3.
FIGURE 36.4 ECG abnormalties in progressive hyperkalemia.

Calcium increases the electrical charge difference across myocardial cell membranes and opposes the depolarization produced by hyperkalemia (35). The preferred calcium preparation is calcium gluconate, which is given in the dosing regimen shown in Table 36.3. The response to calcium is short-lived (20-30 minutes) and it does not reduce the serum K+, so other measures (e.g., insulin-glucose) should be initiated to reduce serum K+ levels. Calcium must be used cautiously in patients receiving digitalis because hypercalcemia aggravates digitalis cardiotoxicity. For patients receiving digitalis, calcium gluconate can be added to 100 mL of isotonic saline and infused over 20 to 30 minutes.

Antagonize Cardiac Effects, Treatment Regimen = e 10% calcium gluconate; 10 mLs IV over 3 min, and repeat after 5 min, if necessary e Use calcium chloride for circulatory shock. « Effect lasts SO-60 min. * Do NOT use calcium for digitalis toxicity.. Transcellular Shift, Treatment Regimen = « Regular insulin: 10 units as IV bolus + 50% dextrose: 50 mLs as IV bolus « Start dextrose Infusion « Peak effect at 30-60 min. Potassium Removal, Treatment Regimen = * Kayexalate (oral): 30 g In 20% sorbitol (50 mL) (rectal): 50 g in 20% sorbitol (200 mL) * Slow-acting (onset at 2 hrs, peak at 6 hrs) « Small risk of bowel necrosis
When hyperkalemia is associated with circulatory shock or cardiac arrest, calcium chloride is preferred to calcium gluconate. One ampule (10 mL) of 10% calcium chloride has three times the elemental calcium in one ampule of 10% calcium gluconate (270 mg vs. 90 mg, respectively), and the extra calcium has a potential advantage by promoting cardiac output and preserving peripheral vascular tone. The osmolality of calcium chloride is 2,000 mosm/kg H20, so administration through a free-flowing central venous catheter is strongly advised.

INSULIN-DEXTROSE: Insulin drives K+ into skeletal muscle cells by activating the membrane NatK+ exchange pump (36), and the insulin-dextrose regimen in Table 36.3 will decrease the serum K+ by at least 0.6 mEq/L (33). A dextrose infusion is advised after insulin-dextrose (unless the patient is hyperglycemic) because there is a risk of hypoglycemia at one hour (33). In the setting of hyperglycemia, insulin should be used without dextrose (33). The insulin effect is temporary (peak effect at 30-60 min), so measures that promote K+ removal should be started.
82-AGONISTS: The dose of inhaled B2agonists (e.g., albuterol) needed to produce a significant (0.5—-1 mEq/L) drop in serum K+ is at least 4 times the therapeutic dose (33), and this can produce unwanted side effects (e.g., tachycardia). Therefore, these agents are not advised for severe hyperkalemia.
BICARBONATE: There are two reasons to avoid bicarbonate for the management of severe hyperkalemia: (a) short-term infusions of bicarbonate (up to four hours) have no effect on serum K+ levels (33), and (b) bicarbonate can form complexes with calcium,

1. Rose BD, Post TW. Potassium homeostasis. In: Clinical physiology of acid-base and electrolyte disorders. 5th ed. New York, NY: McGraw-Hill, 2001; 372-402.
2. Alfonzo AVM, Isles C, Geddes C, Deighan C. Potassium disorders—clinical spectrum and emergency management. Resusc 2006; 70:10-25.
3. Schaefer TJ, Wolford RW. Disorders of potassium. Emerg Med Clin North Am 2005; 23:723-747, viii-ix.
4. Brown RS. Extrarenal potassium homeostasis. Kidney Int 1986; 30:116—-127.
5. Sterns RH, Cox M, Feig PU, et al. Internal potassium balance and the control of the plasma potassium concentration. Medicine 1981; 60:339-354.

You can know the name of a bird in all the languages of the world, but you'l know absolutely nothing about the bird. So let's look at the bird and see what it's doing — that's what counts.
Richard Feynman The Physics Teacher 1969
Magnesium is an essential element for the utilization of energy in the organic world. In green plants, magnesium is the heart of the chlorophyll molecule that captures the energy in sunlight in order to produce oxygen and carbohydrates (i.e., photosynthesis). Aerobic organisms then use the oxygen to release the energy stored in organic nutrients (including carbohydrates), and this energy is stored as adenosine triphosphate (ATP). The release of energy from ATP requires magnesium, which is an essential cofactor for the ATPase enzymes that hydrolyze ATP. Therefore, magnesium is essential for providing us with energy, and for allowing us to utilize this energy to sustain life. Now that's an element.
More specific roles of magnesium include the proper functioning of the Na+-K+ exchange pump (which is a magnesium-dependent ATPase) that generates the electrical gradient across cell membranes. As a result, magnesium plays an important role in the activity of electrically excitable tissues (1-4). Magnesium also regulates the movement of calcium into smooth muscle cells, which gives it a pivotal role in the maintenance of cardiac contractile strength and peripheral vascular tone (4).

The distribution of magnesium (Mg) in the human body is shown in Table 37.1 (5). The average-sized adult contains approximately 24 g (1 mole, or 2,000 mEq) of magnesium; a little over half is located in bone, whereas less than 1% is located in plasma. This lack of representation in the plasma limits the value of the plasma Mg as an index of total body magnesium (similar to plasma potassium). This is particularly true in patients with magnesium deficiency, in whom plasma Mg levels can be normal in the face of total body magnesium depletion (5,6).

Bone, 1 = 12. Bone, 2 = 1060. Bone, 3 = 53. Muscle, 1 = 30. Muscle, 2 = 540. Muscle, 3 = ar. Soft Tissue, 1 = 23. Soft Tissue, 2 = 384. Soft Tissue, 3 = 19. RBC, 1 = 2. RBC, 2 = 10. RBC, 3 = 0.7. Plasma, 1 = 3. Plasma, 2 = 6. Plasma, 3 = . Total, 1 = f0 kg. Total, 2 = 2,000 mEq. Total, 3 = 100%
Fram Referance 5.

Serum is favored over plasma for magnesium assays because the anticoagulant used for plasma samples can be contaminated with citrate or other anions that bind magnesium (5). The reference range for serum Mg depends on the daily magnesium intake, which varies according to geographic region. The normal range of serum Mg for healthy adults in the United States is shown in Table 37.2 (7).
Note: The clinical laboratory typically reports the serum Mg concentration in mg/dL (because Mg is partially bound to plasma proteins), while the medical literature typically uses mEq/L for the serum Mg concentration. The conversion is as follows:
(37.1)
where mol wt is the molecular weight (atomic weight in the case of magnesium) and valence is the number of charges on the atom or molecule. Magnesium has an atomic weight of 24 and a valence of 2, so a serum Mg concentration of 1.7 mg/dL is equivalent to (1.7x10)/24x2 = 1.4 mEq/L.

About 67% of the magnesium in plasma is in the ionized (active) form, and the remaining 33% is either bound to plasma proteins (19% of the total) or chelated with divalent anions such as phosphate and sulfate (14% of the total) (8). The standard assay for magnesium (i.e., spectrophotometry) measures all three fractions. Therefore, when the serum Mg is abnormally low, it is not possible to determine if the problem is a decrease in the ionized (active) fraction, or a decrease in the bound fractions (e.g., hypoproteinemia) (9). The level of ionized Mg can be measured with an ion-specific electrode (10), but this is not routinely available. However, because only a small amount of magnesium resides in plasma, the difference between the ionized and bound magnesium content may not be large enough to be clinically relevant.

Serum Magnesium:, Traditional Units = . Serum Magnesium:, SI Units = . Total, Traditional Units = 1.7-2.4 mg/dl 1.4-2.0 mEq/L. Total, SI Units = 0.7-1.0 mmoVvL. lonized, Traditional Units = 0.8-1.1 mEq/L. lonized, SI Units = 0.4-0.6 mmol/L. Urinary Magnesium:, Traditional Units = 5-15 mEq/24 hr. Urinary Magnesium:, SI Units = 2.5-7.5 mmol/24 hr
Pertains to healthy adults residing in the United States. From Reference 7.
Conversions: mEq/L = [imag/dL x 10)/24] x 2; MEq/L = mmol/L x 2.

The normal range for urinary Mg excretion is shown in Table 37.2. Under normal circumstances, only small quantities of magnesium are excreted in the urine (3). When magnesium intake is deficient, the kidneys conserve magnesium, and urinary magnesium excretion falls to negligible levels. This is shown in Figure 37.1. Note that the serum Mg remains in the normal range one week after starting a Mg-3 diet, while the urinary Mg excretion has dropped to negligible levels. This illustrates the relative value of urinary magnesium excretion in the detection of magnesium deficiency.
FIGURE 37.1 Urinary magnesium excretion and plasma magnesium kvels in a healthy volunteer placed on a magnesium-free diet. Solid bars on the vertical axes indicate the normal range for each variable. Adapted from Shils ME. Experimental human magnesium deficiency. Medicine 1969;48:61-82.

Hypomagnesemia is reported in as many as 65% of patients in ICU's (1-3). Because magnesium depletion may not be accompanied by hypomagnesemia, the incidence of magnesium depletion is probably higher. In fact, magnesium depletion has been described as "the most underdiagnosed electrolyte abnormality in current medical practice" (11).

Because serum Mg levels have a limited ability to detect magnesium depletion, recognizing the conditions that predispose to magnesium depletion may be the only clue of an underlying electrolyte imbalance. The common predisposing conditions for magnesium depletion are listed in Table 37.3.

Drug Therapy:* Furosemide (50%) Aminoglycosides (309s) Amphotericin, pentamidine Digitalis (20%) Cisplatin, cyclosporine Diarrhea (secretory) Alcohol abuse (chronic) Diabetes mellitus Acute Ml, Clinical Findings = Electrolyte abnormalities: Hypokalemla (40%) Hypophosphatemla (30%) Hyponatremia (27%) Hypocalcemla (22%) Cardiac manifestations: Ischemia Arhythmias Digitalis toxicity Hyperactive CNS Syndrome
"Numbers in paranthesas indicata incidenca of associated hypomagnasemia.

Diuretics are the leading cause of magnesium deficiency. Diuretic-induced inhibition of sodium reabsorption also interferes with magnesium reabsorption, and the resultant urinary magnesium losses can parallel urinary sodium losses. Urinary magnesium excretion is most pronounced with the loop diuretics (furosemide and ethacrynic acid). Magnesium deficiency has been reported in 50% of patients receiving chronic therapy with furosemide (12). The thiazide diuretics show a similar tendency for magnesium depletion, but only in elderly patients (13). Magnesium depletion does not occur with "potassium-sparing" diuretics (14).

The antibiotics that promote magnesium depletion are the aminoglycosides, amphotericin and pentamidine (15,16). The aminoglycosides block magnesium reabsorption in the ascending loop of Henley, and hypomagnesemia has been reported in 30% of patients receiving aminoglycoside therapy (16).

There are several case reports indicating that prolonged use of proton pump inhibitors (14 days to 13 years) can be associated with severe hypomagnesemia (17), possibly due to diminished magnesium absorption in the GI tract. Other drugs associated with magnesium depletion include digitalis, epinephrine, and the chemotherapeutic agents cisplatin and cyclosporine (15,18). The first two agents shift magnesium into cells, whereas the latter two promote renal magnesium excretion.

Hypomagnesemia is reported in 30% of hospital admissions for alcohol abuse, and in 85% of admissions for delirium tremens (19,20). The magnesium depletion is due to a number of factors, including generalized malnutrition and chronic diarrhea. In addition, there is an association between magnesium deficiency and thiamine deficiency (21). Magnes-ium is required for the transformation of thiamine into thiamine pyrophosphate, SO magnesium deficiency can promote thiamine deficiency in the face of adequate thiamine intake. For this reason, the magnesium status should be monitored periodically in patients receiving daily thiamine supplements.

Secretions from the lower GI tract are rich in magnesium (10-14 mEq/L) (22), and secretory diarrhea can be accompanied by profound magnesium depletion (20). Upper GI tract secretions are not rich in magnesium (1-2 mEq/L), so vomiting does not pose a risk for magnesium depletion.

Magnesium depletion is common in insulin-dependent diabetic patients, probably as a result of urinary Mg losses that accompany glycosuria (23). Hypomagnesemia is reported in only 7% of admissions for diabetic ketoacidosis, but the incidence increases to 50% over the first 12 hours after admission (24), probably as a result of insulin-induced movement of magnesium into cells.

Hypomagnesemia is reported in as many as 80% of patients with acute myocardial infarction (25). The mechanism is unclear, but may be due to an intracellular shift of Mg from excess catecholamines.
metabolic acidosis, excessive salivation, diffuse muscle spasms, generalized seizures, and progressive obtundation (33). The clinical features are often brought out by loud noises or bodily contact, and thus the term reactive central nervous system magnesium deficiency has been used to describe this disorder. This syndrome is associated with reduced magnesium levels in cerebrospinal fluid, and it resolves with magnesium infusion. The prevalence of this disorder is unknown at present.

As mentioned several times, the serum Mg level is an insensitive marker of magnesium depletion. When magnesium depletion is due to nonrenal factors (e.g., diarrhea), the urinary magnesium excretion is a more sensitive test for magnesium depletion (34). However, most cases of magnesium depletion are due to enhanced renal magnesium excretion, so the diagnostic value of urinary magnesium excretion is limited.

In the absence of renal magnesium wasting, the urinary excretion of magnesium in response to a magnesium load may be the most sensitive index of total body magnesium stores (35,36). This method is outlined in Table 37.4. The normal rate of magnesium reabsorption is close to the maximum rate (Tmax), SO most of an infused magnesium load will be excreted in the urine when magnesium stores are normal. However, when magnesium stores are deficient, the magnesium reabsorption rate is much lower than the Tmax, SO more of the infused magnesium will be reabsorbed and less will be excreted in the urine. Magnesium deficiency is likely when less than 50% of the infused Mg is recovered in the urine, and is unlikely when more than 80% of the infused Mg is excreted in the urine. This test can be also be valuable for identifying the end-point of magnesium replacement therapy (see later). It is important to emphasize that this test will be unreliable in patients with impaired renal function or when there is ongoing renal magnesium wasting.

1. For suspected Mg deficiency when serum Mg is normal
2. For identifying the end-point of Mg replacement therapy

1. Add 24 mmol of magnesium (6g of MgSQ,) to 250 mL of isotonic saline and infuse over 1 hour.
2. Collect urine for 24 hrs, beginning at the onset of the magnesium infusion.

1. Urinary Mg excretion <12 mmol (24 mEq) In 24 hrs (i.¢., less than 50% of the infused Mg) is evidence of Mg depletion.
2. Urinary Mg excretion >19 mmol (38 mEq) in 24 hrs (L¢., more than 8096 of the infused Mg) Is evidence against Mg depletion.

The magnesium preparations available for oral and parenteral use are listed in Table 37.5 (37,38). The oral preparations can be used for daily maintenance therapy (5 mg/kg in normal subjects). However, because intestinal absorption of oral magnesium is erratic, parenteral magnesium is advised for managing hypomagnesemia.
The standard intravenous magnesium preparation is magnesium sulfate (MgSO,). Each gram of MgSO, has 8 mEq (4 mmol) of elemental magnesium (4). A 50% magnesium sulfate solution (500 mg/mL) has an osmolarity of 4,000 mosm/L (43), so it must be diluted to a 10% (100 mg/mL) or 20% (200 mg/mL) solution for intravenous use. Ringer's solutions should not be used as the diluent for MgSO, because the calcium in Ringer's solutions will counteract the actions of the infused magnesium.

Oral Preparations:, Elemental Mg = . Magnesium chloride enteric coated tablets, Elemental Mg = 64 mg (5.3 mEq). Magnesium oxide tablets (400 mg), Elemental Mg = 241 mg (19.8 mEq). Magnesium oxide tablets (140 mg), Elemental Mg = 85 mg (6.9 mEq). Magnesium gloconate tablets (500 mg), Elemental Mg = 27 mg (2.3 mEq). Parenteral solutions, Elemental Mg = . Magnesium sulfate (5095)*, Elemental Mg = 500 mg/dL (4 mEq/L). Magnesium sulfate (12.5%), Elemental Mg = 120 mg/dL (1 mEq/L)
"Should be diluted to a 20% solution for intravenous irjaction.

Magnesium accumulation occurs much less frequently than magnesium depletion. In one survey, hypermagnesemia (i.e., serum Mg >2 mEq/L) was observed in 5% of hospitalized patients (40).

>4mEq/L, Manifestation = Hyporeflexia. >5 mEq/L, Manifestation = Ist? AV Block. >10 mEq/L, Manifestation = Complete Heart Block. >13 mEq/L, Manifestation = Cardiac Arrest
Magnesium has been described as nature's physiologic calcium blocker (43), and most of the serious consequences of hypermagnesemia are due to calcium antagonism in the cardiovascular system. Most of the cardiovascular depression is the result of cardiac conduction delays. Depressed contractility and vasodilation are not prominent.

Hemodialysis is the treatment of choice for severe hypermagnesemia. Intravenous calcium gluconate (1 g IV over 2 to 3 minutes) can be used to antagonize the cardiovascular effects of hypermagnesemia temporarily, until dialysis is started (44). If fluids are permissible and some renal function is preserved, aggressive volume infusion combined with furosemide may be effective in reducing the serum magnesium levels in less advanced cases of hypermagnesemia.

Magnesium often takes a back seat to sodium and potassium, but as mentioned in the introduction to this chapter, magnesium is required for the release of energy stored in ATP, and is also required for the proper functioning of the membrane Na-K exchange pump that allows the transmission of electrical impulses in excitable tissues. Therefore, magnesium de-serves more attention that it receives.
The following are some specific points about magnesium that warrant emphasis:
1 . The serum Mg can be normal in patients who are magnesium depleted.
2. Hypomagnesemia is reported in over 50% of ICU patients, and the frequency of magnesium depletion is likely to be even higher. Diuretic therapy with furosemide is the leading cause of magnesium depletion in ICUs.
. Magnesium depletion should be suspected in any patient with diuretic-induced hypokalemia, and especially when hypokalemia is refractory to potassium replacement.
. Magnesium replacement will correct the serum Mg before total body stores of magnesium are replenished. The best indicator of magnesium repletion is the urinary retention test (see Table 37.4)

1. Noronha JL, Matuschak GM. Magnesium in critical illness: metabolism, assessment, and treatment. Intensive Care Med 2002; 28:667-679.
2. Tong GM, Rude RK. Magnesium deficiency in critical illness. J Intensive Care Med 2005; 20:3-17.
3. Martin KJ, Gonzalez EA, Slatpolsky E. Clinical consequences and management of hypomagnesemia. J Am Soc Nephrol 2009; 20:2291-2295.
4. White RE, Hartzell HC. Magnesium ions in cardiac function. Regulator of ion channels and second messengers. Biochem Pharmacol 1989; 38:859-867.
Magnesium Balance, 1 = Magnesium Balance. 6., 1 = Reinhart RA. Magnesium metabolism. A review with special reference to the relationship between intracellular content and serum levels. Arch Intern Med 1988; 148:2415-2420.. 7., 1 = Lowenstein FW, Stanton MF. Serum magnesium levels in the United States, 1971- 1974. J Am Coll Nutr 1986; 5:399-414.. 8. Altura BT, Altura BM. A method for distinguishing ionized, complexed and protein- bound Mg in normal and diseased subjects. Scand J Clin Lab Invest 1994; 217:83-87., 1 = 8. Altura BT, Altura BM. A method for distinguishing ionized, complexed and protein- bound Mg in normal and diseased subjects. Scand J Clin Lab Invest 1994; 217:83-87.. 9., 1 = Kroll MH, Elin RJ. Relationships between magnesium and protein concentrations in serum. Clin Chem 1985; 31:244-246.. 10., 1 = Alvarez-Leefmans FJ, Giraldez F, Gamino SM. Intracellular free magnesium in excitable cells: its measurement and its biologic significance. Can J Physiol Pharmacol 1987; 65:915-925.

25:749- 755.
Magnesium Replacement Therapy, 1 = Magnesium Replacement Therapy. 37., 1 = DiPalma JR. Magnesium replacement therapy. Am Fam Physician 1990; 42:173-176.. 38., 1 = Trissel LA. Handbook on injectable drugs. 13th ed. Bethesda, MD: Amer Soc Health System Pharmcists, 2005.. 39., 1 = Oster JR, Epstein M. Management of magnesium depletion. Am J Nephrol 1988; 8:349-354.

Nature never deceives us, it is always we who deceive ourselves.
Jean Jacques Rousseau
1754
Calcium and phosphorus are responsible for much of the structural integrity of the bony skeleton. Although neither is found in abundance in the soft tissues, both have an important role in vital cell functions. Phosphorus participates in aerobic energy production, whereas calcium participates in blood coagulation, neuromuscular transmission, and smooth muscle contraction. Considering the important functions of these electrolytes, it is surprising that abnormalities in calcium and phosphorus balance are so well tolerated.

Calcium is the most abundant electrolyte in the human body (the average adult has more than half a kilogram of calcium), but 99% is in bone (1,2). In the soft tissues, calcium is 10,000 times more concentrated than in the extracellular fluids (2,3).

The calcium in plasma is present in three forms, as depicted in Figure 38.1. Approximately half of the calcium is ionized (biologically active) and the remainder is complexed (biologically inactive) (1). About 80% of the bound calcium is bound to albumin, while 20% is complexed to plasma anions such as proteins and sulfates. The concentration of total and ionized calcium in plasma is shown in Table 38.1. These values may vary slightly in different clinical laboratories.
FIGURE 38.1 The three fractions of calcium in plasma and the contribution of each to the total calcium concentration. The column on the right shows how a decrease in plasma albumin can reduce the total plasma calcium without affecting the ionized calcium.

The calcium assay used by most clinical laboratories measures all three fractions of calcium, which can be misleading. This is demonstrated in the column on the right in Figure 38.1, which shows that a decrease in the plasma albumin concentration will decrease the total calcium concentration in plasma without affecting the ionized (biologically active) calcium concentration.
A variety of correction factors have been proposed for adjusting the plasma calcium concentration in patients with hypoalbuminemia. However, none of these correction factors are reliable (4,5), and the only reliable method of determining the concentration of ionized calcium is to measure it with ion-specific electrodes.

Total Calcium,  = 9.0-10.0. Total Calcium,  = 0.25. Total Calcium, (n = 2.25-2.50. lonzed Calcium,  = 4,6-5.0. lonzed Calcium,  = 0.25. lonzed Calcium, (n = 1.15-1.25. Phosphorus,  = 2.5-5.0. Phosphorus,  = 0.32. Phosphorus, (n = 0.8-1.6
"Multiply traditional urits by conversion factor to deriva SI Units or divide SI units by coriversion factor to derive traditional units.

Ionized calcium can be measured in whole blood, plasma, or serum with ion-specific electrodes that are now available in most clinical laboratories.
BLOOD COLLECTION Some precautions are necessary when collecting blood samples for an ionized calcium measurement (7). Loss of carbon dioxide from a blood sample could falsely lower the ionized calcium (by increasing calcium binding to albumin), so it is important to avoid gas bubbles in the blood sample. Anticoagulants (e.g., heparin, citrate, and EDTA) can bind calcium, so blood samples should not be placed in collection tubes that contain these anticoagulants. Tubes with red stoppers ("red top" tubes) contain silicone, and are adequate for measuring ionized calcium in serum samples.

In a large, multicenter survey that included over 7,000 ICU patients, 88% of the patients had at least one episode of mild ionized hypocalcemia (0.9-1.14 mmol/L), and 3.3% of patients had at least one episode of severe ionized hypocalcemia (<0.8 mmol/L) (7). The common disorders associated with ionized hypocalcemia in ICU patients are listed in Table 38.2. Hypoparathyroidism is a leading cause of hypocalcemia in outpatients, but is not a consideration in the ICU unless a patient has had recent neck surgery.

Alkalosis
Fat Embolism
Blood Transfusions (15%)
Mag Sni Gp ea)
5 rugs:
Aminoglycosides (40%) Heparin (1096)
Renal Insufficiency (50%)
Sepsis (3095)
Numbers in parentheses show the fraquency of iorizad hypocalcemia raportad in each condition.
causal relationship between hypocalcemia and fatal outcomes has not been proven.

The major consequences of hypocalcemia are enhanced cardiac and neuromuscular excitability, and reduced contractile force in cardiac muscle and vascular smooth muscle. However, most cases of ionized hypocalcemia in the ICU have no apparent adverse consequences (7).

Hypocalcemia can be accompanied by tetany (of peripheral or laryngeal muscles), hyperreflexia, paresthesias, and seizures (11). Chvostek's and Trousseau's signs are often listed as manifestations of hypocalcemia, but Chvostek's sign is nonspecific (it is present in 25% of normal adults), and Trousseau's sign is insensitive (it can be absent in at least 30% of patients with hypocalcemia) (12).

The cardiovascular complications of hypocalcemia include hypotension, decreased cardiac output, and ventricular ectopic activity. However, these complications are reported only in cases of extreme ionized hypocalcemia (<0.65 mmol/L) (8).

The treatment of ionized hypocalcemia should be directed at the underlying cause of the problem. If calcium replacement is needed, the intravenous calcium solutions and a recommended dosing regimen are shown in Table 38.3.

The calcium solutions available for intravenous use are 10% calcium chloride and 10% calcium gluconate. Calcium chloride contains three times more elemental calcium than calcium gluconate, but calcium gluconate is usually preferred because it has a lower osmolarity, and is less irritating when injected. However, both calcium solutions are hyperosmolar, and should be given via a large central vein, if possible.

A bolus dose of 200 mg elemental calcium (diluted in 100 mL isotonic saline and given over 5-10 minutes) should raise the total serum calcium by 0.5 mg/dL, but levels will begin to fall after 30 minutes (8). Therefore, the bolus dose of calcium should be followed by a continuous infusion at a dose rate of 1-2 mg/kg/hr (elemental calcium) for at least 6 hours. Individual responses will vary, so calcium dosing should be guided by the level of ionized calcium in blood (8).
CAUTION: Intravenous calcium can be risky in select patient populations. Calcium infusions can promote vasoconstriction and ischemia in any of the vital organs (13). The risk of calcium-induced ischemia should be particularly high in patients with low cardiac output who are already vasoconstricted. In addition, aggressive calcium replacement can promote intracellular calcium overload, which can produce lethal cell injury (14), particularly in patients with circulatory shock. Because of these risks, it is wise to avoid correcting ionized hypocalcemia with intravenous calcium unless there is evidence of a serious complication linked to the hypocalcemia.

The daily maintenance dose of calcium is 2—4 g in adults. This can be administered orally using calcium carbonate (e.g., Oscal) or calcium gluconate tablets (500 mg calcium per tablet).

In the large survey of ionized calcium levels mentioned previously (7), 23% of ICU patients had at least one episode of mild ionized hypercalcemia (1.26—1.35 mmol/L), and 17% of patients had at least one episode of ionized hypercalcemia and ionized hypocalcemia. The source of ionized hypercalcemia in ICU patients has not been adequately studied, but common causes of hypercalcemia outside the ICU are hyperparathyroidism and malignancy (15-17).

Treatment is indicated when the hypercalcemia is associated with adverse effects, or when the serum calcium is greater than 14 mg/dL (or ionized calcium >3.5 mmol/L). Most cases of severe, symptomatic hypercalcemia (hypercalcemic crisis) are cancerrelated, and the treatment is summarized in Table 38.4 (1,15-17).

Hypercalcemia usually is accompanied by hypercalciuria, which produces an osmotic diuresis. This eventually leads to hypovolemia, which reduces calcium excretion in the urine and precipitates a rapid rise in the serum calcium. Therefore, volume infusion to correct hypovolemia and promote renal calcium excretion is the first goal of management for hypercalcemia. Isotonic saline (200-500 mL/hr) is recommended for the volume infusion (16) because natriuresis promotes renal calcium excretion. The goal is a urine output of 100-150 mL/hr (15-17).

Saline infusion does not correct hypercalcemia in over 70% of cases (15), and furosemide is often recommended (40 to 80 mg IV every 2 hours) to further promote urinary calcium excretion. However, this can be counterproductive because it promotes hypovolemia, so furosemide is recommended only in cases of volume overload (15,17).

Calcitonin is a naturally occurring hormone that inhibits bone resorption. It is available as salmon calcitonin, which is given subcutaneously or intramuscularly in a dose of 4 U/kg every 12 hours. The response is rapid (onset within a few hours), but the effect is mild (the maximum drop in serum calcium is 0.5 mmol/L) and tachyphylaxis is common (15). As a result, calcitonin has largely been abandoned for the treatment of severe hypercalcemia (15).

Glucocorticoids have several effects that can decrease the serum calcium, including increased renal excretion of calcium, decreased osteoclast activity in bone, and decreased extrarenal production of calcitriol in lymphoma and myeloma (15). Glucocorticoid regimens include oral prednisone (40-100 mg daily) or intravenous hydrocortisone (200— 400 mg daily) for 3-5 days. On the negative side, glucocorticoid effects may not be evident for 4 days, and they can precipitate tumor lysis syndrome (15).

The biphosphonates are potent inhibitors of osteoclast activity. Two drugs in this class, zoledronate (4 or 8 mg IV over 15 min) and pamidronate (90 mg IV over 2 hours) are considered first line agents in the treatment of severe hypercalcemia (15). Zoledronate is the more effective agent, but the higher dose carries a risk of renal injury. Both drugs have a delayed onset of action (2-4 days). The peak effect is at 4-7 days, and the effect lasts 1-4 weeks (15).

Dialysis (hemodialysis or peritoneal dialysis) is effective in removing calcium in patients with renal failure.

Unlike calcium, inorganic phosphate (PO,) is predominantly intracellular in location, where it participates in glycolysis and high energy phosphate (ATP) production. The normal concentration of PO, in plasma is shown in Table 38.1 (18).
Hypophosphatemia (serum PO, <2.5 mg/dL or <0.8 mmol/L) is reported in 17-28% of critically ill patients (19,20), and can be the result of an intracellular shift of phosphorus, an increase in the renal excretion of phosphorus, or a decrease in phosphorus absorption from the GI tract. Most cases of hypophosphatemia are due to movement of PO, into cells.

The movement of glucose into cells is accompanied by a similar movement of PO, into cells, and this can lead to hypophosphatemia if extracellular PO, levels are marginal. Glucose loading is the most common cause of hypophosphatemia in hospitalized patients (19,21,22), and typically occurs during refeeding in alcoholic, malnourished, or debilitated patients. The influence of parenteral nutrition on serum PO, levels is shown in Figure 38.2. Note the gradual decline in the serum PO, and the severe degree of hypophosphatemia (serum PO, <1 mg/dL) after 7 days of intravenous nutrition. The risk of hypophosphatemia is one of the reasons why parenteral nutrition regimens are advanced gradually for the first few days.
FIGURE 38.2 The cumulative effect of total parenteral nutrition (TPN) on the serum phosphate level. Data from Reference 21.

A phenomenon similar to glucose loading is seen in patients with prolonged hyperglycemia who receive insulin. The hyperglycemia produces an osmotic diuresis (from glycosuria), which promotes urinary PO, losses, but the hypophosphatemia appears only when insulin is given (which drives PO, into cells). An example of this phenomenon is the hypophosphatemia that appears during the treatment of diabetic ketoacidosis (see page 613).

Respiratory alkalosis can increase intracellular pH, and this accelerates glycolysis. The increase in glucose utilization is accompanied by an increase in glucose and phosphorus movement into cells (23). This may be an important source of hypophosphatemia in ventilator-dependent patients because overventilation and respiratory alkalosis are common in these patients.

Stimulation of a-adrenergic receptors can move PQ, into cells and promote hypophosphatemia. This effect is evident in patients treated with fB-agonist bronchodilators. In one study of patients with acute asthma who were treated aggressively with nebulized albuterol (2.5 mg every 30 minutes), the serum PQ, decreased by 1.25 mg/dL (0.4 mmol/L) 3 hours after the onset of therapy (24).

There is an inverse relationship between serum PO, and circulating levels of inflammatory cytokines (25). Possible explanations include in-creased PO, utilization by activated neutrophils, and a transcellular shift of PO, caused by elevated levels of endogenous catecholamines.

Aluminum can form insoluble complexes with inorganic phosphates. As a_ result, aluminum-containing compounds such as sucralfate (Carafate) can impede the absorption of phosphate in the upper GI tract and promote phosphate depletion (26).

Intravenous phosphate replacement is recommended for all patients with severe hypophosphatemia (i.e., serum PO, <1.0 mg/dL or 0.3 mmol/L), and for patients with hypophosphatemia of any degree who also have cardiac dysfunction, respiratory failure, muscle weakness, or impaired tissue oxygenation. The phosphate solutions and dosing regimen are shown in Table 38.5 (31).

The normal daily maintenance dose of phosphorus is 1,200 mg if given orally (32). The IV maintenance dose of PO, is lower, at 800 mg/day, because only 70% of orally administered phosphate is absorbed from the GI tract.
FIGURE 38.3 The effects of phosphate depletion that can impair energy utilzation in cells. See text for explanation.
able 38.5 Phosp lacement Therap
Sodium Phosphate, 1 = 93 mg (3 mmol/mL. Sodium Phosphate, 2 = Nat: 4.0 mEq. Potassium Phosphate, 1 = 93 mg (3 mmol)/mL. Potassium Phosphate, 2 = Kt: 4.3 mEq. , 1 = 40-60 ka 61-80 kg. , 2 = 81-120 kg. rd, 1 = 30 mmol 40 mmol. rd, 2 = 50 mmol. 1-1.7, 1 = 20 mmol 30 mmol. 1-1.7, 2 = 40 mmol. 1.8-2.5, 1 = 10 mmol 15 mmol. 1.8-2.5, 2 = 20 mmol
Tif tha plasma K+ is 24 mEq/L, use sodium phosphata, and if the plasma K+ is <4 mEq/L, usa potassium phosphate. Infusion time is 6 hrs. From Referance 31.

Most cases of hyperphosphatemia in the ICU are the result of impaired PO, excretion from renal insufficiency, or PO, release from disrupted cells (e.g., rhabdomyolysis or tumor lysis).

The clinical manifestations of hyperphosphatemia include: (a) the formation of insoluble calcium—phosphate complexes (with deposition into soft tissues), and (b) acute hypocalcemia (with tetany) (11). There is little information about the prevalence or significance of these manifestations in ICU patients.

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1. Bushinsky DA, Monk RD. Electrolyte quintet: Calcium. Lancet 1998; 352:306—311.
2. Baker SB, Worthley LI. The essentials of calcium, magnesium and phosphate metabolism: part I. Physiology. Crit Care Resusc 2002; 4:301-306.
3. Weaver CM, Heaney RP. Calcium. In: Shils ME, et al., eds. Modern nutrition in health and disease. 10th ed. Philadelphia, PA: Lippincott, Williams & Wilkins, 2006; 194— 210.
Plasma Calcium, 1 = Plasma Calcium. 4., 1 = Slomp J, van der Voort PH, Gerritsen RT, et al. Albumin-adjusted calcium is not suitable for diagnosis of hyper- and hypocalcemia in the critically ill. Crit Care Med 2003; 31:1389-1393.. 5., 1 = Byrnes MC, Huynh K, Helmer SD, et al. A comparison of corrected serum calcium levels to ionized calcium levels among critically ill surgical patients. Am J Surg 2005; 189:310-314.. 6., 1 = Forman DT, Lorenzo L. Ionized calcium: its significance and clinical usefulness. Ann Clin Lab Sci 1991; 21:297-304.

15. McCurdy MT, Shanholtz CB. Oncologic emergencies. Crit Care Med 2012; 40:22122222.
16. Stewart AF. Clinical practice. Hypercalcemia associated with cancer. N Engl J Med 2005; 352:373-379.
17. Body JJ. Hypercalcemia of malignancy. Semin Nephrol 2004; 24:48—-54.

33. Kraft MD, Btaiche IF, Sacks GS, et al. Treatment of electrolyte disorders in adult patients in the intensive care unit. Am J Health Syst Pharm 2005; 62:1663-1682.
34. Lorenzo Sellares V, Torres Ramirez A. Management of hyperphosphataemia in dialysis patients: role of phosphate binders in the elderly. Drugs Aging 2004; 21:153-165.

Obscurity is painful to the mind, as well as the eye.
David Hume

Medicine is a science that hath been more labored than advanced. I find much iteration, but small addition.
Sir Francis Bacon 1605
The conditions described in this chapter (i.e., necrotizing pancreatitis and liver failure) share the following features: (a) both are associated with injury in multiple organs, (b) both are plagued by infections from pathogens that reside in the bowel, (c) the management of both conditions is mostly supportive care, and (d) mortality rates are high, and have not changed in recent memory. The management of patients with liver failure is a particular challenge because of the enormous number of life-supporting functions (including the production of over 20,000 proteins) that are lost when the liver fails.

Acute pancreatitis is an inflammatory condition of the pancreas that is characterized by abdominal pain and elevated levels of pancreatic enzymes (amylase, lipase) in blood. Two types of pancreatitis are identified (1):
1. Edematous Pancreatitis is the most common form of pancreatitis, and is characterized by inflammatory infiltration of the pancreas without involvement of other organs. The clinical presentation is usually a self-limited period of abdominal pain, nausea, and vomiting. The mortality rate is low (<2%) (2), and management rarely requires ICUlevel care.
2. Necrotizing Pancreatitis occurs in 10-15% of cases (1), and is characterized by areas of necrotic destruction in the pancreas, usually accompanied by progressive systemic inflammation and inflammatory injury in one or more extraabdominal organs (e.g., lungs, kidneys, and circulatory system). The mortality rate can be as high as 40% (2), and management usually requires ICU-level care.

Pancreatitis can have a variety of etiologies, as shown in Table 39.1. About 90% of cases are related to gallstones (40% of cases), alcohol abuse (30% of cases), or are idiopathic (20% of cases) (2,4,5). Less common causes include abdominal trauma, severe hypertriglyceridemia (serum levels >1,000 mg/dL), drugs (e.g., acetaminophen, pentamidine, tri-methoprim-sulfamethoxazole, omeprazole, furosemide), infections (e.g., HIV, cytomegalovirus, mycoplasma, Legionella), and vasculitides (lupus and polyarteritis nodosa).

Gallstones (4096) Alcohol (30%) Idiopathic (20%), 1 = Abdominal trauma Hypertrighyceridemia Drugst Infections’ Vasculitis
Reported prevalence indicated in paranthasas.
Tincludes acetaminophen, omeprazole, metronidazole, trimethoprim-sulfamethoxazole, furosemide and valproic acid.
includes HIM cytomegabvirus, mycoplasma, and Lagionaiiz.

The diagnosis of acute pancreatitis requires: (a) an increase in serum levels of pancreatic enzymes (amylase and lipase) to at least 3 times the upper limit of normal, and (b) evidence of pancreatitis on contrast-enhanced computed tomography (1).

AMYLASE: Amylase is an enzyme that cleaves starch into smaller polysaccharides. The principal sources of amylase are the pancreas, salivary glands, and fallopian tubes. Serum amylase levels begin to rise 6-12 hours after the onset of acute pancreatitis, and they return to normal in 3-5 days. An increase in serum amylase levels to 3 times the upper limit of normal (the threshold for the diagnosis of acute pancreatitis) has a high sensitivity (>90%) but a low specificity (as low as 70%) for the diagnosis of acute pancreatitis (6).
The low specificity of serum amylase is a reflection of the numerous conditions that can elevate serum amylase levels. These are listed in Table 39.2 (7). About 25% of the nonpancreatic conditions in this table can result in serum amylase levels that overlap those seen in acute pancreatitis (8). The ones that deserve mention include parotitis, ruptured ectopic pregnancy, and acute alcohol intoxication. Of particular note, hyperamylasemia of salivary origin is reported in 40% of cases of acute alcohol intoxication (6). (Note: The reference range for serum amylase is not mentioned because it often varies in different clinical laboratories. )

Pancreatitis Cholecystitis Renal Failure Parotitis (amylase) Peptic Ulcer Disease Bowel Obstruction or Infarction Liver Disease Ruptured Ectopic Pregnancy (amylase) Diabetic Ketoacidosis, Drugs and Other Agentst = Amylase: Ethanol intoxication Hydroxyethy! starch Histamine Hs blockers Metaclopramide Opiates Lipase: Lipid infusions: Methylprednisone Opiates
tincludes orly substances that ara likaly to be encountered in ICU patients. For a more complete list, sae Raferance 7.
LIPASE: Lipase is an enzyme that hydrolyses triglycerides to form glycerol and free fatty acids. The principal sources of lipase are the tongue, pancreas, liver, intestine, and circulating lipoproteins. In acute pancreatitis, serum lipase levels begin to rise earlier than serum amylase (at 4 to 8 hours), and the serum levels remain elevated longer than serum amylase (for 8 to 14 days).
Like amylase, there are several nonpancreatic conditions that can elevate serum lipase levels, as shown in Table 39.2. However, unlike amylase, nonpancreatic conditions rarely raise serum lipase levels high enough to overlap with the levels seen in acute pancreatitis (8). Therefore, serum lipase is considered more specific than serum amylase for the diagnosis of acute pancreatitis. An increase in serum lipase to three times the upper limit of normal has a sensitivity and specificity of 80-100% for acute pancreatitis (6).
RECOMMENDATION: The serum lipase can be used alone for the diagnostic evaluation of pancreatitis. Adding the serum amylase assay does not increase diagnostic accuracy (6). However, pancreatic enzyme assays cannot be used to evaluate the severity of illness (6).

Contrast-enhanced computed tomography (CT) is the most reliable diagnostic test for acute pancreatitis, and can identify the type of pancreatitis (edematous vs. necrotizing) as well as localized complications (e.g., infection). Figure 39.1 shows a contrast-enhanced CT image of edematous pancreatitis. The pancreas is thickened and enhances completely, and the border of the pancreas is blurred, which is characteristic of pancreatic edema. Compare this with the image in Figure 39.2, which shows a large area that is not contrast-enhanced in the region of the neck and body of the pancreas. This represents pancreatic necrosis, and identifies the condition as necrotizing pancreatitis. The full extent of pancreatic necrosis may not be evident on CT imaging for the first week after the onset of symptoms (1), so repeat imaging is advised in patients with persistent symptoms or severe pancreatitis.
FIGURE 39.1 Contrast-enhanced CT image showing edematous pancreatitis. The pancreas (outlined by the dotted line) is enlarged and enhances compktely. There is also blurring of the pancreatic border, which is characteristic of edema formation. Image digitally enhanced.
When IV contrast cannot be administered (because of a dye allergy or a serum creatinine above 1.5 mg/dL) CT imaging is less likely to distinguish between edematous and necrotizing pancreatitis.

Since gallstones are the leading cause of acute pancreatitis in the United States (4), an evaluation of the gall bladder and biliary tree is advised in all cases of confirmed acute pancreatitis. Contrast-enhanced CT images may suffice for this evaluation, but in cases where a CT scan is inconclusive, or has not been performed, ultrasonography is recommended.
FIGURE 39.2 Contrast-enhanced CT image showing necrotizing pancreatitis. The area that is not contrast-enhanced (indicated by the arrows) represents necrosis in the neck and body of the pancreas. Image from Reference 1.

Severe pancreatitis is defined as acute (usually necrotizing) pancreatitis that is associated with persistent (>48 hrs) injury in at least one other organ system (1). The cause of the extrapancreatic organ injury is progressive systemic inflammation (similar to severe sepsis and septic shock), and the organs that are typically involved include the lungs (acute respiratory distress syndrome, or ARDS), the kidneys (acute kidney injury), and the circulatory system (hypotension and circulatory shock). Pancreatic enzymes and CT images have a poor correlation with the clinical severity of this condition
The management of severe pancreatitis is best conducted in an ICU setting, and includes the following measures: (a) removing the precipitating condition (e.g., obstructing gallstones), (b) providing supportive care for the extrapancreatic organ injuries (e.g., mechanical ventilation for ARDS), (c) early nutritional support with enteral tube feedings, and (d) managing intraabdominal complications (e.g., infection).

Circulatory support includes volume resuscitation and vasopressor drugs, if necessary.

Severe pancreatitis is associated with loss of intravascular fluid through leaky systemic capillaries, and the resulting hypovolemia can produce additional pancreatic necrosis. For this reason, aggressive fluid therapy has been recommended early in the course of severe pancreatitis (9). There is no agreement on which type of fluid (colloid or crystalloid) is best, but crystalloid fluids are currently the popular choice. The initial regimen for volume resuscitation is summarized as follows:
1. For crystalloid fluids, infuse 20 mL/kg (about 1.5 liters) over 60 to 90 minutes.
2. Follow with an infusion rate up to 250 mL/hr for the next 24—48 hours, to maintain a mean arterial pressure >65 mm Hg, and a urine output 20.5 mL/kg/hr.
CAUTION: Aggressive volume infusion has not been shown to improve outcomes in severe pancreatitis (10), and this practice can be deleterious by promoting edema formation, which can aggravate conditions like ARDS, and increases the risk of abdominal compartment syndrome. Therefore, after the initial volume infusion of 20 mL/kg, the infusion rate should be titrated to the desired blood pressure and urine output, but should not exceed 250 mL/hr. If volume infusion does not achieve the desired hemodynamic goals, vasopressor therapy should be initiated.

There are no official recommendations regarding vasopressor therapy in severe pancreatitis, but norepinephrine is an appropriate choice. The initial infusion rate is 0.1 Ug/kg/hr, which is then titrated to maintain a mean arterial pressure >65 mm Hg. All vasoconstrictor drugs can reduce splanchnic blood flow (especially phenylephrine), and could aggravate pancreatic necrosis, so careful titration of infusion rates (and avoiding phenylephrine) is advised.

About one-third of patients with necrotizing pancreatitis develop infections in the necrotic areas of the pancreas (11). The pathogens are almost always Gram-negative enteric organisms, and the infections typically appear 7-10 days after the onset of illness. These infections are difficult to eradicate, and they are associated with increased mortality rates (11). Unfortunately, antibiotic prophylaxis does not reduce the incidence of pancreatic infections, and does not influence the mortality rate in severe pancreatitis (12). As a result, prophylactic antibiotics are not recommended in necrotizing pancreatitis (11).

Nutrition support should be started early (within 48 hours after the onset of illness) using enteral tube feedings, if possible (13).

The preference for enteral nutrition is based on the ability of tube feedings to exert a trophic effect on the bowel mucosa. This helps to maintain the structural and functional integrity of the bowel mucosa, and thereby reduce the risk of bacterial translocation across the bowel wall (which is considered the major route leading to pancreatic infections). Clinical studies have shown that enteral nutrition is associated with fewer infections, less multiorgan failure, and a lower mortality rate than total parenteral nutrition in patients with severe pancreatitis (14). (The effect of enteral feedings on the bowel mucosa is described in more detail in Chapter 48.)
FEEDING SITE: Tube feedings should be infused into the jejunum using long feeding tubes that can be placed with fluoroscopic or endoscopic guidance. Alternately, a feeding jejunostomy can be created in patients who require laparotomy for pancreatic debridement. Nasogastric feedings are not currently recommended, although one small study has shown no apparent harm from nasogastric feedings in severe pancreatitis (15).
FEEDING REGIMEN: The jejunum does not have the reservoir capacity of the stomach, so jejunal feedings should be advanced more slowly than gastric feedings. The diluting effect of gastric secretions is also lost in the jejunum, so isotonic feeding formulas are preferred to hypertonic formulas. Standard (polymeric) tube feedings can be used for jejunal feedings (13), but in patients with diarrhea, elemental feeding formulas may be preferred. (Elemental formulas are low in fat, and the protein is available as individual amino acids, which are presumably easier to digest.)

The appearance of infection in necrotizing pancreatitis is often heralded by reappearance, persistence, or progression of systemic inflammation and multiorgan failure at 7-10 days after the onset of illness. A contrast-enhanced CT scan may show gas bubbles in the necrotic areas of the pancreas, as shown in Figure 39.3. If infection is suspected but gas bubbles are not evident on CT imaging, cultures must be obtained from the necrotic areas of the pancreas (using CT-guided needle aspiration). The treatment of choice for infected pancreatic necrosis is surgical debridement (necrosectomy) (11).

There are several sources of increased intraabdominal pressure in severe pancreatitis, including peripancreatic fluid collections, ascites, and edema of the bowel wall (which is exaggerated by aggressive volume infusion). Abdominal compartment syndrome (ACS) has been reported in as many as 55% of patients with severe pancreatitis (16), but this may be an exaggeration because it is based on studies that used a relatively low abdominal pressures (15-20 mm Hg) for the diagnosis of ACS. Nevertheless, ACS is more common than suspected in severe pancreatitis, and measurements of abdominal pressure are warranted in any patient with acute pancreatitis who develops acute oliguric renal failure. (See Chapter 34 for more information on ACS.)
FIGURE 39.3 Contrast-enhanced CT image showing extensive necrosis of the pancreas with numerous gas bubbks, indicating infection.

When acute pancreatitis is associated with gallstones, early endoscopic retrograde cholangio-pancreatography (ERCP) is indicated for biliary obstruction or evidence of cholangitis (i.e., fever and increasing liver enzymes) (17).

There are two types of liver failure that can appear in the ICU: (a) acute liver failure, and (b) acute-on-chronic liver failure.

Acute liver failure is an abrupt and rapid deterioration in liver function that occurs de novo, without prior liver disease. This is an uncommon condition with an annual incidence of 1 to 6 cases per million people in developed countries (18). Most cases are the result of viral hepatitis or drug-induced liver injury, and the principal clinical manifestation is severe hepatic encephalopathy. The leading cause of acute liver failure in the United States is acetaminophen overdose. (See Chapter 54 for a description of acetaminophen hepatotoxicity. )

Most cases of liver failure involve patients with chronic liver disease (cirrhosis) who develop an abrupt deterioration in liver function as a result of a precipitating condition, usually an infection or variceal hemorrhage (19). The clinical manifestations often include signs of systemic inflammation (fever, leukocytosis, etc.), worsening ascites, mental status changes (hepatic encephalopathy), and a deterioration in renal function. The evaluation and management of this group of patients is described in the following text. The mortality rate in these patients is considerable, and ranges from 35% to 70% (19,20).

In patients with acute-on-chronic liver failure and ascites, 10% to 27% have evidence of infection in the ascitic fluid without an apparent primary site of infection (21). This condition is called spontaneous bacterial peritonitis (SBP), and the presumed mechanism is translocation of enteric pathogens across the bowel mucosa and into the peritoneal fluid. Cirrhosis predisposes to SBP because it impairs the normal function of the liver in eradicating enteric pathogens that translocate across the bowel wall. A single organism is isolated in most cases of SBP, and the isolates are Gram-negative aerobic bacilli (especially Escherichia coli) in 75% of cases, and Gram-positive aerobic cocci (especially streptococcal species) in 25% of cases (21).

Fever, abdominal pain, and rebound tenderness are present in at least 50% of cases of SBP, but the condition can be asymptomatic in one-third of cases (21).

A diagnostic paracentesis should be performed on all patients with cirrhosis and ascites who are admitted for acute-on-chronic liver failure. An absolute neutrophil count =250 cells/mm3 in the ascitic fluid is presumptive evidence of infection, and is an indication to begin empiric antimicrobial therapy. Culture samples of ascitic fluid should be injected directly into blood culture bottles at the bedside because standard culture methods have a diagnostic yield of only 50% in cases of probable SBP (22).

The preferred antibiotic for suspected SBP is cefotaxime (2 grams IV every 8 hours), or another third-generation cephalosporin (21-23). Unfortunately, the mortality rate in SBP is 30 to 40% despite adequate antibiotic coverage (22); this may be explained by the fact that 30% of patients with SBP develop hepatorenal syndrome (23), which has a mortality rate in excess of 50% (see later for a description of this syndrome).
ALBUMIN INFUSIONS: Because renal hypoperfusion plays an important role in the pathogenesis of hepatorenal syndrome (see later), clinical studies have evaluated the role of albumin infusions in SBP. The results of these studies indicate that infusions of albumin can reduce the incidence of hepatorenal syndrome in SBP, but only in high-risk patients; i.e., those with a BUN >30 mg/dL, creatinine >1 mg/dL and bilirubin >4 mg/dL (24). The recommended albumin infusion regimen is as follows (24):
Day 1: 1.5 g /kg body weight, infused within 6 hours of the diagnosis of SBP. Day 3: 1.0 g/kg body weight.
It is unclear at this time if the benefit from albumin is a volume effect, or is related to some other effect (e.g., albumin has antioxidant activity, and can also bind cytokines).

Hepatorenal syndrome (HRS) is a functional renal failure (i.e., occurs without intrinsic renal disease) that occurs in patients with advanced cirrhosis, especially those with spontaneous bacterial peritonitis or sepsis from another source (26).

HRS is the result of hemodynamic alterations in the splanchnic and renal circulations. Cirrhosis is associated with splanchnic vasodilation, and the neurohumoral (renin system) response to this vasodilation results in vasoconstriction in other organs, including the kidneys (26). The renal vasoconstriction creates a situation where the glomerular filtration rate is vulnerable to small decrements in cardiac output. Sepsis is also associated with splanchnic vasodilation, which could explain the association between sepsis and HRS.

The diagnostic criteria for HRS are shown in Table 39.3. The criteria include renal impairment (serum creatinine >1.5 mg/dL) that does not respond to albumin infusions, and no other likely source of renal dysfunction (i.e., nephrotoxic drugs, shock, or parenchymal kidney disease).

1. Cirrhosis with ascites 2, Serum creatinine >1.5 mg/dL 3. No improvement in renal function after 2 days of albumin infusions and no diuretics 4. No evidence of shock 5. No nephrotoxic drugs 6. No evidence of parenchymal Kidney disease, "Management = 1. Terlipressin: 1-2 mg IV every ete Albumin: 1 g/kg (to 100 mg) on day 1, then 20-40 mg daily. 2. TIPPS: If response to #1 Is suboptimal. 3. Liver transplant: Optimal
TDiscontinus if sarum albumin +4.5 gvdL.
Fram Referance 26.

The management of HRS is designed to correct the hemodynamic changes that are responsible for HRS. First-line therapy includes a splanchnic vasoconstrictor (terlipressin, a vasopressin analogue) and a volume expander (albumin). An effective dosing regimen is shown in Table 39.3. Over 50% of patients with HRS will show improvement in renal function with this regimen (26,27). However, HRS often relapses after drug therapy is discontinued, and long-term survival requires liver transplantation (26). Transjugular intrahepatic portosystemic stent-shunt (TIPPS) can improve renal function in HRS (26), but this procedure promotes hepatic encephalopathy, so it is reserved for transplant
candidates that are unresponsive to pharmacotherapy.

Liver failure produces an encephalopathy that is characterized by cerebral edema, disordered thinking, and altered consciousness. Hepatic encephalopathy is the dominant manifestation in acute liver failure, whereas in acute-on-chronic liver failure, the encephalopathy is usually preceded by an acute insult (e.g., variceal hemorrhage). Ammonia has been identified as a key factor in the pathogenesis of hepatic encephalopathy (28).

Ammonia (NH3) is a byproduct of protein degradation, and is produced primarily in the bowel (and to a lesser degree in skeletal muscle and kidneys). The liver plays a major role in clearing ammonia by converting it to urea in the urea cycle. This clearance mechanism is impaired or lost in liver failure, resulting in a progressive rise in ammonia levels in blood. Am-monia eventually crosses the blood-brain barrier and is taken up by astrocytes, which use the ammonia to convert glutamate to glutamine; i.e.,
glutamate + NH; + ATP — glutamine + ADP
(Astrocytes normally provide glutamine for neurons, which use the glutamine as a substrate for the production of glutamate, a neurotransmitter.) The ammonia load in astrocytes leads to accumulation of glutamine, and this creates an osmotic force that draws water into the astrocytes. The result is cerebral edema, astrocyte damage, and impaired synaptic transmission in the brain.

The principal features of progressive hepatic encephalopathy are shown in Table 39.4 (29). Agitation and disorientation are prominent in the early stages, while depressed consciousness is the dominant feature in the late stages. The cranial nerves are not affected, but dysarthria can be present (30). Involuntary movements like tremors or asterixis (clonic movements during wrist extension) can appear, while sensation is intact. Focal neurological deficits are considered evidence of an alternative diagnosis (30).
Stage 0, 1 = « No encephalopathy. Stage 1, 1 = ¢ Short attention span « Euphoria or depression « Asterixis may be present. Stage 2, 1 = « Lethargy or apathy ¢ Disorientation « Asterixis usually present. Stage 3, 1 = « Somnolent, but responsive to verbal commands * Savere disorientation « Asterixis absent. Stage 4, 1 = « Coma
The “Wast Haven Criteria," from Refarence 22.

The diagnosis of hepatic encephalopathy is usually made by excluding other causes of altered mentation. Other conditions that should be considered in patients with cirrhotic liver failure include drug overdose, subdural hematoma, and Wernicke's encephalopathy (from thiamine deficiency). Neuroimaging studies are performed to eliminate other diagnoses. The only diagnostic test that can help to identify hepatic enceph-alopathy is the serum ammonia level.

Considering the role played by ammonia in the pathogenesis of hepatic encephalopathy, it is no surprise that serum ammonia levels are typically elevated in patients with hepatic encephalopathy. This is demonstrated in Figure 39.4, which shows the relationship between ammonia levels in blood (arterial and venous) and the presence and severity of hepatic encephalopathy (31). Although the ammonia levels are mildly elevated in the absence of hepatic encephalopathy (stage 0), the levels are higher in the presence of hepatic encephalopathy, and the degree of elevation corresponds to the severity of the condition. Note that the ammonia levels are higher in arterial blood. Although the difference between arterial and venous ammonia levels was not statistically significant in this study (31), arterial blood seems optimal for identifying hepatic encephalopathy in the early stages of the condition.
FIGURE 39.4 Relationship between arterial and venous ammonia (NH3) and the presence and severity of hepatic encephalopathy. The stages of encephalopathy correspond to those in Table 39.3. The horizontal dotted line represents the upper limit of the normal range for serum ammonia (47 pmol/L) at the study hospital. N indicates number of patients studied. Data from Reference 31.

Protein restriction (which would reduce the ammonia burden from the bowel) is not recommended for patients with hepatic encephalopathy because these patients have increased rates of protein catabolism, and restricting protein intake would promote a negative nitrogen balance (34). The recommended protein intake in critically ill patients is 1.2 to 1.5 g/kg/day (see Chapter 47), so staying at the low end of this range (1.2 g/kg/day) might be the best choice in patients with hepatic encephalopathy.

One of the recurring themes in this book is the importance of the bowel as a source of infection in critically ill patients (see Chapters 5 and 40). Two observations in this chapter demonstrate the normal defense mechanisms that prevent infections of bowel origin.
The first observation is the ability of enteral tube feedings to reduce the incidence of sepsis and multiorgan failure in patients with severe pancreatitis. This highlights the trophic effect of bulk nutrients on the structural and functional integrity of the mucosal barrier in the gut; i.e., the "non-nutritive" function of enteral feeding. (See Chapter 48 for more information on this topic.)
The second observation is the occurrence of spontaneous bacterial peritonitis in patients with cirrhosis and ascites. This is a classic example of an infection that is caused by the translocation of enteric pathogens across the bowel mucosa, and it highlights the importance of the reticuloendothelial system in the bowel (mostly represented by the liver) in defending against the spread of enteric pathogens.

1., 1 = Banks PA, Bollen TL, Dervenis C, et al. Classification of acute pancreatitis - 2012: revision of the Atlanta classification and definitions by international consensus. Gut 2012; 62:102-111.. 2., 1 = Cavallini G, Frulloni L, Bassi C, et al. Prospective multicentre survey on acute pancreatitis in Italy (Proinf-AISP). Dig Liver Dis 2004; 36:205-211.. 3., 1 = Greer SE, Burchard KW. Acute pancreatitis and critical illness. A pancreatic tale of hypoperfusion and inflammation. Crit Care Med 2009; 136:1413-1419.. 4., 1 = Forsmark CE, Baille J. AGA Institute technical review on acute pancreatitis. Gastroenterol 2007; 132:2022-2044.. 5., 1 = Yang AL, Vadhavkar S, Singh G, Omary MB. Epidemiology of alcohol-related liver and pancreatic disease in the United States. Arch Intern Med 2008; 168:649-656.. 6., 1 = Yadav D, Agarwal N, Pitchumoni CS. A critical evaluation of laboratory tests in acute pancreatitis. Am J Gastroenterol 2002; 97:1309-1318.. 7., 1 = Gelrud D, Gress FG. Elevated serum amylase and lipase. UpToDate (accessed on May 30, 2013).. 8., 1 = Gumaste VV, Roditis N, Mehta D, Dave PB. Serum lipase levels in nonpancreatic abdominal pain versus acute pancreatitis. Am J Gastroenterol 1993; 88:2051-2055.

Ascites, 1 = Ascites. 21., 1 = Gilbert JA, Kamath PS. Spontaneous bacterial peritonitis: an update. Mayo Clin Proc 1995; 70:365-370.. 22., 1 = Runyon BA. Management of adult patients with ascites caused by cirrhosis. Hepatology 1998; 27:264-272.. 23., 1 = Moore CM, van Thiel DH. Cirrhotic ascites review: pathophysiology, diagnosis, and management. World J Hepatol 2013; 5:251-263.. 24., 1 = Narula N, Tsoi K, Marshall JK. Should albumin be used in all patients with spontaneous bacterial peritonitis? Can J Gastroenterol 2011; 25:373-376.. 25., 1 = Peltekian KM, Wong F, Liu PP, et al. Cardiovascular , renal, and neurohumoral responses to single large-volume paracentesis in cirrhotic patients with diuretic resistant ascites. Am J Gastroenterol 1997; 92:394-399.
Hepatorenal Syndrome, 1 = Hepatorenal Syndrome. 26., 1 = Dalerno F, Gerbes A, Gines P, et al. Diagnosis, prevention and treatment of hepatorenal syndrome in cirrhosis. Gut 2007l 56:131-1318.. 27., 1 = Rajekar H, Chawla Y. Terlipressin in hepatorenal syndrome: evidence for present indications. J Gastroenterol Hepatol 2011; 26(Suppl):109-114.

Hepatic Encephalopathy, 1 = Hepatic Encephalopathy. 28. 29., 1 = Clay AS, Hainline BE. Hyperammonemia in the ICU. Chest 2007; 132: 1368-1378. Blei AT, Cordoba J, and the Practice Parameters Committee of the American College of Gastroenterology. Hepatic encephalopathy. Am J Gastroenterol 2001; 96:1968- 1976.. 30., 1 = Ferenci P, Lockwood A, Mullen K, et al. Hepatic encephalopathy - definition,nomenclature, diagnosis and quantification: Final report of the Working Party at the 11th World Congress of Gastroenterology, Vienna, 1998. Hepatol 2002; 55:716-721.. 31., 1 = Ong JP, Aggarwal A, Krieger D, et al. Correlation between ammonia levels and the severity of hepatic encephalopathy. Am J Med 2003; 114:188-193.. 32., 1 = Salminen S, Salminen E. Lactulose, lactic acid bacterial, intestinal microecology, and mucosal protection. Scand J Gastroenterol 1997; 222(Suppl):45-48.. 33., 1 = Lawrence KR, Klee JA. Rifaximin for the treatment of hepatic encephalopathy. Pharmacotherapy 2008; 28:1019-1032.. 34., 1 = Nutrition in end-stage liver disease: principles and practice. Gastroenterol-ogy 2008; 134:1729-1740.

If you know your enemies and know yourself, you will not be imperiled in a hundred battles.
Sun Tzu
The Art of War
The concept of the bowel as noxious reservoir first appeared in the early years of the twentieth century, when a Scottish surgeon William Arbuthnot-Lane began performing total colectomies in patients with chronic constipation, to prevent "autointoxication" from toxic bowel contents (1). This practice was abandoned (along with the surgeon), but the concept of autointoxication has been revived, and the bowel is now recognized as a leading source of morbidity and mortality in critically ill patients.
This chapter describes abdominal infections that occur in the ICU, including infections of the biliary tree (acalculous cholecystitis), bowel (Clostridium diffcile enterocolitis), and peritoneal cavity (postoperative infections) (2,3).

Acalculous cholecystitis accounts for only 5-15% of cases of acute cholecystitis (4), but it is more common in critically ill patients, and has a mortality rate (about 45%) that rivals that of septic shock (4,5).

Most cases of acalculous cholecystitis are not discovered until complications arise (é.g., gangrenous cholecystitis or gallbladder perforation), so the clinical manifestations reported for acalculous cholecystitis are often those of advanced, complicated cholecystitis. The diagnosis of acalculous cholecystitis is often delayed because pain and tenderness in the right upper quadrant can be absent in one-third of patients with acalculous cholecystitis (2). Fever (100%), elevated bilirubin (90%), hypotension (90%), and multiorgan failure (65-80%) are common but nonspecific findings (4,5). Blood cultures are positive in 90% of cases (2) and Gram-negative aerobic bacilli are isolated in almost all cases.

Ultrasound is the favored diagnostic test for acalculous cholecystitis because it can be performed at the bedside. Gallbladder distension and sludge are suggestive findings, but are nonspecific. The ultrasound image in Figure 40.1 shows more specific findings; i.e., marked thickness of the gallbladder wall and sloughed mucosa in the lumen of the gallbladder. The diagnostic yield from ultrasound varies widely in different reports (4,8), and may be operator-specific. If ultrasound is not helpful, the next step is a hepatobiliary scan, which is the "gold standard" method for the diagnosis of acute cholecystitis (but requires a functional liver to move the tracer into the bile ducts).

Prompt intervention is mandatory. Cholecystectomy is the procedure of choice, but for patients who are too unstable for surgery, percutaneous drainage of the gallbladder is a suitable alternative. Empiric antibiotic therapy should be started as soon as the diagnosis is confirmed. The recommended antibiotics are piperacillin-tazobactam, or a carbapenem (imipenem or meropenem) (2).

The microbial landscape in the GI tract is altered in critically ill patients, and the infections that can appear as a result of this change are described in this section.
FIGURE 40.1 Transverse sonogram of the gallbladder showing marked thickening of the gallbladder wall and an echogenic mass projecting into the lumen of the gallbladder. This mass represents slughed mucosa, and is characteristic of gangrenous chokcystitis.

Clostridium difficile is a spore-forming, Gram-positive anaerobic bacillus that does not inhabit the bowel in healthy subjects, but is able to colonize and proliferate in the bowel when the normal microflora has been altered by antibiotic therapy (12). The typical host for C. difficile colonization is an elderly or debilitated patient or nursing home resident who has received antibiotics at some time in the last 2 weeks. Colonization is uncommon in healthy subjects who live in the community (although this may change).

C. difficile is transmitted from patient to patient via the fecal-oral route. There is a dormant (spore) form that can survive on environmental surfaces for months, but patientto-patient transmission usually occurs via the hands of hospital personnel (13). As a result, strict adherence to the use of disposable gloves can significantly reduce transmission (14).
FIGURE 40.2 Correlation between the most frequent isolates in the stomach and the most frequent isolates in nosocomial infections in critically ill patients. Data from Reference 4.
C. difficile is not an invasive organism, but releases cytotoxins that damage the bowel mucosa. This leads to inflammatory infiltration of the bowel wall and symptomatic disease. Severe inflammation is accompanied by raised, plaque-like lesions on the mucosal surface known as "pseudomembranes." The presence of these lesions (pseudomembranous colitis) is evidence of severe disease.
GASTRIC ACID SUPPRESSION: There are several reports showing that use of acidsuppressing drugs, particularly proton pump inhibitors, is associated with an increased
FIGURE 40.3 Radiographic appearance of toxic megacolon in a patient with C. difficile enterocolitis.

Mild CDI «Temp 2101°F «WBC £15,000, Category = Preferred Alternate (PO) Alternate (IV). Mild CDI «Temp 2101°F «WBC £15,000, Category = Metronidazole: 500 mg PO every 8 hrs Vancomycin: 125 mg PO every 6 hrs or Fidaxomicin: 200 mg PO every 12 hrs Metronidazole: 500 mqg IV every 8 hrs. Mild CDI «Temp 2101°F «WBC £15,000, Drug Regiment = Metronidazole: 500 mg PO every 8 hrs Vancomycin: 125 mg PO every 6 hrs or Fidaxomicin: 200 mg PO every 12 hrs Metronidazole: 500 mqg IV every 8 hrs. Severe CDI, Category = Preferred Alternate (IV). Severe CDI, Category = Vancomycin: 125 mg PO every 6 hrs Metronidazole: 500 mg IV every 8 hrs. Severe CDI, Drug Regiment = Vancomycin: 125 mg PO every 6 hrs Metronidazole: 500 mg IV every 8 hrs. Life-Threatening CDI, Category = Preferred. Life-Threatening CDI, Category = Vancomycin: 500 mg every 6 hrs via NG tube or by enema, plus Metronidazole: 500 mg IV every 8 hrs. Life-Threatening CDI, Drug Regiment = Vancomycin: 500 mg every 6 hrs via NG tube or by enema, plus Metronidazole: 500 mg IV every 8 hrs. Relapsed CDI, Category = Preferred Alternate (PO). Relapsed CDI, Category = Same regimens as in first episode Fidaxomicin: 200 mg PO every 12 hrs. Relapsed CDI, Drug Regiment = Same regimens as in first episode Fidaxomicin: 200 mg PO every 12 hrs
tDuration of tharapy is 10-44 days.
Fram Referancas 19, 23-25.
including donor screening, see Reference 28.)

Postoperative abdominal infections are located in the peritoneal cavity, and are the result of peritoneal seeding during the procedure, or leakage of bowel contents from an anastomotic site or undetected injury to the bowel wall. These infections can present as diffuse peritonitis or an abdominal abscess.
FIGURE 40.4 Abdominal x-ray in the upright position showing free air under both hemidiaphragms. In the absence of a recent laparoscopy, this finding is evidence of a perforated hollow viscus.
ANTIBIOTICS: Antibiotic therapy should be started as soon as possible using antibiotics that are active against the frequent isolates as presented in Table 40.2. Single-agent antibiotic coverage using piperacillin-tazobactam, or a carbapenem (imipenem-cilastatin, meropenem, or doripenem) is recommended (2). In patients who may be colonized by Candida species (e.g., patients who have received antibiotics recently), additional empiric coverage with an antifungal agent (e.g., fluconazole) seems wise.
Gram-negative aerobic bacilli, 1 = . Eschernchia coll, 1 = 71. Klebsiella spp, 1 = 14. Pseudomonas aeruginosa, 1 = 14. Proteus mirabilis, 1 = 5. Enterobacter spp, 1 = 5. Anaerobic organisms, 1 = . Bacterojdes fragilis, 1 = 35. Other Bacterojdes spp, 1 = 7A. Closindium sop, 1 = 29. Other anaerobes, 1 = 55. Gram-positive aerobic coccl, 1 = . Streptococcus spo, 1 = 38. Enterococcus spp, 1 = 23. Staphylococcus aureus, 1 = 4

Abdominal abscesses typically serve as an occult source of sepsis, and are difficult to detect with routine clinical evaluations.

Fever is almost always present (30), but localized abdominal tenderness can be absent in 60% of cases, and a palpable abdominal mass is evident in less than 10% of cases (30,31). Abdominal x-rays can show extraluminal air, but this occurs in less than 15% of cases (31).

Computed tomography (CT) of the abdomen is the most reliable diagnostic method of detection for abdominal abscesses, with a sensitivity and specificity of 90% or higher (31). However, CT imaging in the early postoperative period can be misleading because collections of blood or irrigant solutions in the peritoneal cavity can be misread as an abscess. CT scans are most reliable when performed after the first postoperative week (when peritoneal fluid collections have resorbed) (31). The CT appearance of an abdominal abscess is shown in Figure 40.5.
FIGURE 40.5 Abdominal CT scan showing a multiloculated abscess in the left upper quadrant in a post-splenectomy patient.

Immediate drainage is advised for postoperative abdominal abscesses. Precise localization with CT imaging allows many abscesses to be drained percutaneously with CT-guided drainage catheters (30). Empiric antibiotic therapy should be started while awaiting the results of abscess fluid cultures. The empiric antibiotic regimen is the same as described for peritonitis.

1. Arbuthnot-Lane W. Remarks on the operative treatment of chronic constipation. Reprinted in Dis Colon & Rectum 1985; 28:750—-757.

2. Solomkin JS, Mazuski JE, Bradley JS, et al. Diagnosis and management of complicated intra-abdominal infection in adults and children: guidelines by the Surgical Infection Society and the Infectious Disease Society of America. Clin Infect Dis 2010; 50:133-164.
3. Sarteli M, Viale P, Catena F, et al. 2013 WSES guidelines for the management of intra-abdominal infections. World J Emerg Surg 2013; 8:3.

4., 1 = McChesney JA, Northrup PG, Bickston SJ. Acute acalculous cholecystitis associated with systemic sepsis and visceral arterial hypoperfusion. A case series and review of pathophysiology. Dig Dis Sci 2003; 48:1960-1967.. 5., 1 = Laurila J, Syrjälä H, Laurila PA, et al. Acute acalculous cholecystitis in critically ill patients. Acta Anesthesiol Scand 2004; 48:986-991.. 6., 1 = Messing B, Bories C, Kuntslinger C. Does parenteral nutrition induce gallbladder sludge formation and lithiasis? Gastroenterology 2983; 84:1012-1019.. 7., 1 = Jüngst C, Killack-Ublick GA, Jüngst D. Gallstone disease: microlithiasis and sludge. Best Prect Res Clin Gastroenterol 2006; 20:1053-1062.. 8., 1 = Puc MM, Tran HS, Wry PW, Ross SE. Ultrasound is not a useful screening tool for acalculous cholecystitis in critically ill trauma patients. Am Surg 2002; 68:65-69.

9., 1 = Huang J, Cao Y, Liao C, et al. Effect of histamine-2-receptor antagonists versus sucralfate on stress ulcer prophylaxis in mechanically ventilated patients: A meta- analysis of 10 randomized controlled trials. Crit Care 2010; 14:R194-R204.. 10., 1 = MacFie J, Reddy BS, Gatt M, et al. Bacterial translocation studied in 927 patients over 13 years. Br J Surg 2006; 93:87-93.. 11., 1 = Marshall JC, Christou NV, Meakins JL. The gastrointestinal tract: the 'un-drained abscess' of multiple organ failure. Ann Surg 1993; 218:111-119.

SF, Young-Fadok TM, Zein NN, Pardi DS. Clostridium difficile- associated diarrhea and colitis. Mayo Clin Proc 2001; 76:725-730.. 23., 1 = van Nispen tot Pannerden CMF, Verbon A, Kuipers E. Recurrent Clostridium difficile infection. What are the treatment options. Drugs 2011; 71:853-868.. 24., 1 = Zar FA, Bakkanagari SR, Moorthi KM, Davis MB. A comparison of vancomycin and metronidazole for the treatment of Clostridium difficile-associated diarrhea, stratified by disease severity. Clin Infect Dis 2007; 45:302-307.. 25., 1 = Louie TJ, Miller MA, Mullane KM, et al. Fidaxomicin versus vancomycin for Clostridium difficile infection. N Engl J Med 2011; 364:422-431.. 26., 1 = Lipsett PA, Samantaray DK, Tam ML, et al. Pseudomembranous colitis: a surgical disease? Surgery 1994; 116:491-496.. 27., 1 = Aslam S, Hamill RJ, Musher DM. Treatment of Clostridium difficile-associated disease: old therapies and new strategies. Lancet Infect Dis 2005; 5:549-557.. 28., 1 = Aas J, Gessert CE, Bakken JS. Recurrent Clostridium difficile colitis: case series involving 18 patients treated with donor stool administered via nasogastric tube. Clin
Infect Dis 2003; 36:580—-585.

Complicated Abdominal Infections, 1 = Complicated Abdominal Infections. 29., 1 = Miller RE, Nelson SW. The roentgenologic demonstration of tiny amounts of free intraperitoneal gas: experimental and clinical studies. AJR Am J Roentgenol 1971; 112:574-585.. 30., 1 = Khurrum Baig M, Hua Zao R, Batista O, et al. Percutaneous postoperative intra- abdominal abscess drainage after elective colorectal surgery. Tech Coloproctol 2002; 6:159-164.. 31. Fry DE. Noninvasive imaging tests in the diagnosis and treatment of intra-abdominal abscesses in the postoperative patient. Surg Clin North Am 1994; 74:693-709., 1 = 31. Fry DE. Noninvasive imaging tests in the diagnosis and treatment of intra-abdominal abscesses in the postoperative patient. Surg Clin North Am 1994; 74:693-709.

32. Lin P-C, Chang C-H, Hsu P-I, et al. The efficacy and safety of proton pump inhibitors vs. histamine-2 receptor antagonists for stress ulcer bleeding prophylaxis among critical care patients: A meta-analysis. Crit Care Med 2010; 38:1197-1205.
33. Marik PE, Vasu T, Hirani A, Pachinburavan M. Stress ulcer prophylaxis in the new millennium: a systematic review and meta-analysis. Crit Care Med 2010; 38:2222— 2228.

Throughout nature, infection without disease is the rule rather than the exception.
René Dubois
Man Adapting
1966
Urethral catheters are commonplace in critically ill patients, and surveys indicate that catheter-associated urinary tract infections account for 40% of all hospital-acquired infections in the United States (1). However, these surveys are misleading, because a majority of the catheter-associated infections represent asymptomatic bacteriuria (infection without disease, according to Dubois), and do not require antimicrobial therapy. This chapter describes the current recommendations for the diagnosis and treatment of catheter-associated symptomatic urinary tract infection.

The presence of a urethral catheter is associated with a 3-8% incidence of bacteriuria (2105 colony forming units/mL) per day (1). This is assumed to be the result of bacterial migration along the outer surface of the catheter and into the bladder. Bacteria also form biofilms on the inner and outer surface of urethral catheters (2), and these biofilms can serve as a source of continued microbial colonization in the bladder. However, this is not the full story, because direct injection of pathogens into the bladder of healthy subjects does not result in a urinary tract infection (3). Furthermore, the continuous flow of urine that is allowed by bladder drainage catheters should wash away microbes that migrate up the urethra.
FIGURE 41.1 Photomicrograph showing non-pathogenic Lactobacillus organisms adhering to a bladder epithelial cell. From Reference 3. Image has been digitally enhanced.

The missing piece of the puzzle is the ability of pathogenic organisms to adhere to the bladder epithelium. The epithelial cells of the bladder are normally coated with nonpathogenic organisms, as shown in Figure 41.1 (4); this prevents the attachment of pathogenic organisms, which is the precipitating event that leads to infections of the lower urinary tract (5). This is the same phenomenon that occurs in colonization of the oral mucosa with pathogenic Gram-negative aerobic bacilli (see Figure 5.5), which serves as a prelude to nosocomial pneumonia. The link between bladder catheters and the change in bacterial adherence is unclear. However, it is possible that an increase in severity of illness is responsible for both the change in bacterial adherence and the need for a bladder catheter. (For more on the role of bacterial adherence in nosocomial infections, see "A Final Word" at the end of the chapter.)

The pathogens isolated in catheter-associated bacteriuria are shown in Table 41.1 (6). The predominant organisms are Gram-negative aerobic bacilli (especially Escherichia coli), enterococci, and Candida species, while staphylococci are infrequent isolates. A single organism predominates in bacteriuria associated with short-term catheterization (<30 days), whereas bacteriuria is often polymicrobial in long-term catheterization (=30 days).

Escherichia coll, 1 = 21.4. Escherichia coll, 2 = 22.3. Enterococci, 1 = 15.5. Enterococci, 2 = 15.8. Candida albicans, 1 = 14.5. Candida albicans, 2 = 15.3. Other Candida species, 1 = 6.5. Other Candida species, 2 = 9.5. Pseudomonas aeruginosa, 1 = 10.0. Pseudomonas aeruginosa, 2 = 13.3. Klebsiella peararia, 1 = Tot. Klebsiella peararia, 2 = f5. Enterobacter species, 1 = 44. Enterobacter species, 2 = 6.5. Gram-neg. staphylococci, 1 = 2.5. Gram-neg. staphylococci, 2 = 4.6. Staphylococcus aureus, 1 = 2.2. Staphylococcus aureus, 2 = 25. Acinetobacter baumannil, 1 = 1.2. Acinetobacter baumannil, 2 = 1.5
Adapted fram Refarance 6. Some of tha percentagas reprasent median values.

The recommendations in this section are taken from the most recent guidelines developed by the Infectious Disease Society of America (1).

1., 1 = Screening for , or antibiotic treatment of, asymptomatic bacteriuria is NOT advised unless the patient is scheduled for a urologic procedure that is associated with mucosal bleeding (e.g., transurethral resection of the prostate) (9). These recommendations are based on the following observations: (a) few cases of asymptomatic bacteriuria progress to CA-UTI, (b) antibiotic therapy does not reduce the incidence of CA-UTI, and (3) antibiotic therapy promotes the emergence of resistant organisms.. 2., 1 = Empiric antibiotics are recommended for patients with suspected CA-UTI. Single agent therapy with piperacillin-tazobactam or a carbapenem (imipenem or meropenem) is recommended, while levofloxacin (750 mg IV once daily) is a second-line agent (10).. 3., 1 = If the diagnosis of CA-UTI is confirmed by urine culture, antibiotic therapy should be adjusted according to the organism isolated and the reported sensitivities. Catheters that have been in place for >2 weeks should be replaced.. 4., 1 = The duration of antibiotic therapy for CA-UTI should be 7 days for patients who respond promptly, and 10-14 days for patients with a delayed response.

The presence of Candida species in urine usually represents colonization in patients with indwelling urethral catheters, but candiduria can also be a sign of disseminated candidiasis (i.e., the candiduria being the result, not the cause, of the disseminated candidiasis). Disseminated candidiasis can be an elusive diagnosis because blood cultures are unrevealing in more than 50% of cases (11), and candiduria may be the only evidence of disseminated disease. The clinical condition of the patient becomes an important factor in the approach to candiduria in the ICU.

In cases of candiduria, the colony count has no predictive value for identifying renal or disseminated candidiasis (11). The most frequent isolate is Candida albicans (about 50% of cases), followed by Candida glabrata (about 15% of cases) (11). The latter organism is notable for resistance to the antifungal agent fluconazole.

Asymptomatic candiduria does not require treatment unless the patient is neutropenic (12,13). Removal of the catheter is always advised, when possible, because this can eradicate candiduria in 40% of cases (13). Repeat urine cultures are recommended, and persistent candiduria in high-risk (immunosuppressed) patients should be investigated with blood cultures and imaging studies of the kidneys.
In neutropenic patients with asymptomatic candiduria, the recommended prophylaxis includes caspofungin: 70 mg IV as a loading dose, followed by 50 mg IV daily (12).

The unifying feature in nosocomial infections that involve the gastrointestinal, respiratory, and urinary tracts is a change in the character of microbes that adhere to epithelial surfaces. In healthy subjects, the epithelial surfaces in the mouth, GI tract, and urinary tract are covered by harmless, commensal organisms, but in patients who develop severe or chronic illness, these surfaces are covered with pathogenic organisms, and this serves as a prelude to nosocomial infections. Of interest in this regard is a study conducted in patients with spinal cord injuries and long-term urinary catheters, where injection of nonpathogenic E. coli into the bladder was associated with 50% fewer urinary tract infections (15).
However, the population of epithelial surfaces is not just a matter of "territorial imperative" (where one population takes over, or defends, a territory) but is the result of receptors on epithelial cells that bind to specific groups of microorganisms. A change in the configuration of these receptors allows pathogens to bind to epithelial surfaces, and this is the precipitating event that leads to nosocomial infections. As such, we need to study how microbes bind to epithelial surfaces if we want to eliminate the threat of nosocomial infections.

There is no possibility of escaping the entropic doom imposed on all natural phenomena.
Aharon
Katchalsky
1965

Heat not a furnace for your foe so hot that you singe yourself.
William Shakespeare
Henry
VIII
The human body is a metabolic furnace that generates enough heat to raise the body temperature by 1°C every hour, even at rest (1). For-tunately, the external surface of the body acts like a radiator, and discharges excess heat into the surrounding environment. The behavior of this radiator is guided by a thermostat (the thermoregulatory system) that limits the daily variation in body temperature to +0.6°C (2). This chapter describes what happens when this thermostat fails, and allows the body temperature to rise or fall to life-threatening levels.

The distinction between hyperthermia and fever deserves mention at the outset. Both conditions are characterized by an elevated body temperature, but hyperthermia is the result of a defect in temperature regulation, while fever is the result of a normal thermoregulatory system operating at a higher set point. The elevations in body temperature in this chapter represent hyperthermia, not fever. Because the underlying mechanisms involved in the production of hyperthermia and fever are different, the antipyretic agents used to treat fever (e.g., acetaminophen) are _ ineffective in hyperthermia.

The maintenance of body temperature in conditions of thermal stress (e.g., hot weather, strenuous exercise) is primarily achieved by enhanced blood flow to the skin (convective heat loss) and the loss of sweat (evaporative heat loss).

When heat is lost from the skin, it warms the air just above the skin surface, and the increase in surface temperature limits the further loss of body heat by conduction. However, when an air current (e.g., from a fan or gust of wind) is passed across the skin, it displaces the warm layer of air above the skin and replaces it with cooler air, and this process facilitates the continued loss of body heat by conduction. The same effect is produced by increases in blood flow just underneath the skin. The action of currents (air and blood) that promotes heat loss is known as convection.

The transformation of water from a liquid to a gas requires heat (called the 'latent heat of vaporization'), and the heat required for the evaporation of sweat from the skin is provided by body heat. The evaporation of one liter of sweat from the skin is accompanied by the loss of 580 kilocalories (kcal) of heat from the body (3). This is about one-quarter of the daily heat production by an average-sized adult at rest. Thermal sweating (as opposed to "nervous sweating") can achieve rates of 1-2 liters per hour (3), which means that over 1,000 kcal of heat can be lost in one hour during profuse sweating. It is important to emphasize that sweat must evaporate to ensure loss of body heat. Wiping sweat off the skin will not result in heat loss, so this practice should be discouraged during strenuous exercise.

Heat-related illnesses are conditions where the thermoregulatory system is no longer able to maintain a constant body temperature in response to thermal stress. There are a number of minor heat-related illnesses, such as heat cramps and heat rash (prickly heat), but the following descriptions are limited to the major heat-related illnesses: heat exhaustion and heat stroke. The comparative features of these conditions are shown in Table 42.1

Body Temperature, 1 = <39°C. Body Temperature, 2 = 241°C. CNS Dysfunction, 1 = Mild. CNS Dysfunction, 2 = Severe. Sweat Production, 1 = Yes. Sweat Production, 2 = Minimal. Dehydration, 1 = Yes. Dehydration, 2 = Yes. Multiorgan Involvement, 1 = No. Multiorgan Involvement, 2 = Yes

Heat exhaustion is the most common form of heat-related illness. Patients with heat exhaustion experience flu-like symptoms that include hyperthermia (usually <39°C or 102°F), muscle cramps, nausea, and malaise. The hallmark of this condition is volume depletion without signs of hemodynamic compromise. The volume loss can be accompanied by hypernatremia (from sweat loss) or hyponatremia (when sweat loss is partly replaced with water intake). There is no evidence of significant neurologic impairment.
The management of heat exhaustion includes volume repletion and other general supportive measures. Cooling measures to reduce body temperature are not necessary.

Skeletal muscle injury (rhabdomyolysis) is a common complication of hyperthermia syndromes, including heat stroke (particularly the exertional type) and drug-induced hyperthermia (described later in the chapter). Disruption of myocytes in skeletal muscle leads to the release of creatine kinase (CK) into the bloodstream, and the measurement of CK levels in plasma is used to determine the presence and severity of rhabdomyolysis. There is no standard CK level for the diagnosis of rhabdomyolysis, but CK levels that are five times higher than normal (or about 1,000 Units/liter) have been used to identify rhabdomyolysis in clinical studies (7). Plasma CK levels above 15,000 Units/L indicate severe rhabdomyolysis and an increased risk of acute renal failure from myoglobin released by disrupted myocytes (7).

Renal tubular injury from myoglobin results in acute renal failure in about one-third of patients with rhabdomyolysis (8). This condition is described in Chapter 34.

The heat-related illnesses just described are triggered by thermal stress in the environment. The source of the thermal stress in the following conditions is drug-induced metabolic heat production.

Malignant hyperthermia (MH) is an uncommon disorder that occurs once every 15,000 exposures to inhalational anesthesia, and affects approximately 1 in 50,000 adults (9). It is an inherited disorder with an autosomal dominant pattern, and is characterized by excessive release of calcium from the sarcoplasmic reticulum in skeletal muscle in response to halogenated inhalational anesthetic agents (e.g., halothane, isoflurane, servoflurane, and desflurane) and depolarizing neuromuscular blockers (e.g., succinylcholine) (9). The calcium release leads to uncoupling of oxidative phosphorylation and a marked rise in metabolic rate.

The clinical manifestations of MH include muscle rigidity, hyperthermia, depressed consciousness, and autonomic instability. The first sign of MH may be a sudden and unexpected rise in end-tidal PCO, (reflecting the underlying hypermetabolism) in the operating room (9,10). This is followed (within minutes to a few hours) by generalized muscle rigidity, which can progress rapidly to widespread myonecrosis (rhabdomyolysis) and subsequent myoglobinuric renal failure. The heat generated by the muscle rigidity is responsible for the marked rise in body temperature (often above 40°C or 104°F) in MH. The altered mental status in MH can range from agitation to coma. Autonomic instability can lead to cardiac arrhythmias, fluctuating blood pressure, or persistent hypotension.

The first suspicion of MH should prompt immediate discontinuation of the offending anesthetic agent.
DANTROLENE: Specific treatment for the muscle rigidity is available with dantrolene sodium, a muscle relaxant that blocks the release of calcium from the sarcoplasmic reticulum. When given early in the course of MH, dantrolene can reduce the mortality rate from 70% or higher (in untreated cases) to 10% or less (9,10). The dosing regimen for dantrolene in MH is as follows:
Regimen: 1-2 mg/kg as IV bolus, and repeat every 15 minutes if needed to a total dose of 10 mg/kg. Follow the initial dosing regimen with a dose of 1 mg/kg IV or 2 mg/kg orally four times daily for 3 days.
Treatment is extended to 3 days to prevent recurrences. The most common side effect of dantrolene is muscle weakness, particularly grip strength, which usually resolves in 2—4 days after the drug is discontinued (11). The most troublesome side effect of dantrolene is hepatocellular injury, which is more common when the daily dose exceeds 10 mg/kg (9). Active hepatitis and cirrhosis are contraindications to dantrolene therapy (11) but in light of the high mortality in MH if left untreated, these contraindications should not be absolute.

All patients who survive an episode of MH should be given a medical bracelet that identifies their susceptibility to MH. In addition, because MH is a genetic disorder with an known inheritance pattern (autosomal dominant), immediate family members should be informed of their possible susceptibility to MH. A test is available to identify the responsible gene for MH in family members (10).

The neuroleptic malignant syndrome (NMS) is strikingly similar to malignant hyperthermia in that it is a drug-induced disorder characterized by hyperthermia, muscle rigidity, altered mental status, and autonomic instability (12).

NMS is associated with drugs that influence dopamine-mediated synaptic transmission in the brain. A decrease in dopaminergic transmission in the basal ganglia and hypothalamic-pituitary axis may be responsible for many of the clinical manifestations of NMS (12). As indicated in Table 42.2, NMS can be the result of therapy with drugs that inhibit dopaminergic transmission (most cases), or can be triggered by discontinuing drugs that facilitate dopaminergic transmission. (Note that not all drugs associated with NMS are neuroleptic drugs.) The drugs most frequently implicated in NMS are haloperidol and fluphenazine (12). The incidence of NMS during therapy with neuroleptic agents is reported at 0.2—1.9% (13).

Antipsychotic agents
Butyrophenones (6.g., haloperidol, phenothiazines, clozapine, olanzapine, respiracone
Antiemetic agents
Metaclopramide, droperidol, prochlorperazine
CNS stimulants
Amphetamines, cocaine
Other
Lithium, tricyclic antidepressants (overdose)

Dopaminergic drugs
Amantidine, bromocriptine, levodopa
tDiscontinuing these drugs can trigger the neuroleptic malignant syndrome.
There is no relationship between the intensity or duration of drug therapy and the risk of NMS (12), so NMS is an idiosyncratic drug reaction and not a manifestation of drug toxicity. There is some evidence of a familial tendency, but a genetic pattern of transmission has not been identified (14).

Most cases of NMS begin to appear 24—72 hours after the onset of drug therapy, and almost all cases are apparent in the first 2 weeks of drug therapy. The onset is usually gradual, and can take days to fully develop. In 80% of cases, the initial manifestation is muscle rigidity or altered mental status (12). The muscle rigidity has been described as lead-pipe rigidity to distinguish it from the rigidity associated with tremulousness (cogwheel rigidity). The change in mental status can range from agitation to coma. Hyperthermia (body temperature can exceed 41°C) is required for the diagnosis of NMS (12), but the increase in body temperature can be delayed for 8-10 hours after the

Overstimulation of serotonin receptors in the central nervous system produces a combination of mental status changes, autonomic hyperactivity, and neuromuscular abnormalities that is known as the serotonin syndrome (SS) (17). The recent growth in popularity of seritonergic drugs such as selective serotonin reuptake inhibitors (SSRIs) has led to a marked increase in the prevalence of SS in recent years. The severity of illness can vary widely, and the most severe cases can be confused with the other druginduced hyperthermia syndromes.

Serotonin is a neurotransmitter that participates in sleep—wakefulness cycles, mood, and thermoregulation. A variety of drugs can enhance serotonin neurotransmission and produce SS, and a list of these drugs is shown in Table 42.3. Many of these drugs work in combination to produce SS, although single-drug therapy can also result in SS. Many of the drugs involved in SS are mood enhancers, including illegal substances like "ecstasy," an amphetamine derivative implicated in life-threatening cases of SS (18).

Increased serotonin synthesis, Drugs = L-tryptophan. Decreased serotonin breakdown, Drugs = MAOIs (including linezolic), ritonavir. Increased serotonin release, Drugs = Amphetamines, MDMA (ecstasy), cocaine, fanfluramine. Decreased serotonin reuptake, Drugs = SSRIs, TCAs, dextromethorphan, meperidine, fentanyl, tramadol. Serotonin receptor agonists, Drugs = Lithium, sumitriptan, buspirone, LSD
tSea Raferance 17 for a comprahansive list of drugs. Abbreviations: MACIs = monoamine oxidase inhibitors; MDMA = methyenadioxy-methamphetamine; SSRIs = selective serotonin rauptake inhibitors; TCAs = tricyclic antidepressanits .

The onset of SS is usually abrupt (in contrast to NMS, where the full syndrome can take days to develop), and over half of the cases are evident within 6 hours after ingestion of the responsible drug(s) (17). The clinical findings include mental status changes (e.g., confusion, delirium, coma), hyperthermia, autonomic hyperactivity (e.g., mydriasis, tachycardia, hypertension), and neuromuscular abnormalities (e.g., hyperkinesis, hyperactive deep tendon reflexes, clonus, and muscle rigidity). The clinical presentation can vary markedly (17). Mild cases may include only hyperkinesis, hyperreflexia, tachycardia, diaphoresis, and mydriasis. Moderate cases often have additional findings of hyperthermia (temperature >38°C) and clonus. The clonus is most obvious in the patellar deep-tendon reflexes, and horizontal ocular clonus may also be present. Severe cases of SS often present with delirium, hyperpyrexia (temperature >40°C), widespread muscle rigidity, and spontaneous clonus. Life-threatening cases are marked by rhabdomyolysis, renal failure, metabolic acidosis, and hypotension.
A useful worksheet for the diagnosis of SS is shown in Table 42.4. The first step in the diagnostic evaluation is to establish recent ingestion of seritonergic drugs. Although the worksheet in Table 42.2 indicates drug ingestion in the past five weeks, most cases of SS follow within hours of drug ingestion (17). Hyperthermia and muscle rigidity can be absent in mild cases of the illness. The features that most distinguish SS from other druginduced hyperthermia syndromes are hyperkinesis, hyperreflexia, and clonus. However, muscle rigidity can mask these clinical findings in severe cases of SS.

As in other drug-induced hyperthermia syndromes, removal of the precipitating drug(s) is the single most important task in the management of SS. The remainder of the management includes measures to control agitation and hyperthermia, and the use of serotonin antagonists. Many cases of SS will resolve within 24 hours after initiation of therapy, but seritonergic drugs with long elimination half-lives can produce more prolonged symptomatology.
Sedation with benzodiazepines is important for controlling agitation in SS. Physical restraints should be avoided because they encourage isometric muscle contraction, which can aggravate skeletal muscle injury and promote lactic acidosis (19).
CYPROHEPTADINE: Cyproheptadine is a serotonin antagonist that can be given in severe cases of SS (20). This drug is available for oral administration only, but tablets can be broken up and administered through a nasogastric tube.
Regimen: The initial dose is 12 mg, followed by 2 mg every 2 hrs for persistent symptoms. The maintenance dose is 8 mg every 6 hours.
Cyproheptadine can be sedating, but this should aid in the control of agitation in SS. Neuromuscular paralysis may be required in severe cases of SS to control muscle rigidity and extreme elevations of body temperature (>41°C). Nondepolarizing agents (e.g., vecuronium) should be used for muscle paralysis because succinylcholine can aggravate the hyperkalemia that accompanies rhabdomyolysis. Dantrolene does not reduce muscle rigidity or hyperthermia in SS (17).

Hypothermia is defined as a decrease in body temperature below 35°C (95°F), and can be the result of environmental forces (accidental hypo-thermia), a metabolic disorder (secondary hypothermia), or a therapeutic intervention (induced hypothermia). This section will focus primarily on environmental (accidental) hypothermia.

Physiologically, the human body is better equipped to survive in hot rather than cold environments. The physiological response to cold includes cutaneous vasoconstriction (to reduce convective heat loss) and shivering (which can double metabolic heat production). These physiological adaptations are protective only in mild hypothermia (see later in the text); otherwise, protection from the cold is dependent on behavioral responses (e.g., wearing warm clothing and seeking shelter from the cold). Because of the importance of behavioral responses, hypothermia is particularly pronounced when these responses are impaired (e.g., in the intoxicated or confused patient).

Environmental hypothermia is most likely to occur in the following situations: (a) prolonged submersion in cold water (the transfer of heat to cold water occurs much more readily than the transfer of heat to cold air), (b) exposure to cold wind (wind promotes heat loss by convection, as described earlier in the chapter), (c) when the physiological response to cold is impaired (e.g., the vasoconstrictive response to cold is impaired by alcohol consumption), and (d) when the behavioral responses to cold are impaired (as mentioned in the previous paragraph).

Most standard thermometers measure temperatures down to 34°C (94°F). For more accurate recordings in hypothermia, electronic temperature probes are available that can record temperatures down to 25°C (77°F), and can be placed in the bladder, rectum, or esophagus.

The consequences of progressive hypothermia are summarized in Table 42.5.

Mild, Body Temp = 32-35°C 90-95°F. Mild, Clinical Manifestations = Confusion, cold and pale skin, shivering, tachycardia. Moderate, Body Temp = 28-31.9°C 82-89.9°F. Moderate, Clinical Manifestations = Lethargy, reduced or absent shivering, bradycardia, bradypnea. Severe, Body Temp = <28°C <82°F. Severe, Clinical Manifestations = Obtundation er cama, no shivering, edema, dilated and fixed pupils, bradycardia, hypotension, oliguria. Fatal, Body Temp = <25°C <1 fF. Fatal, Clinical Manifestations = Apnea, asystole
MILD HYPOTHERMIA: In mild hypothermia (32—35°C or 90-95°F), pa-tients are usually confused, and show signs of adaptation to cold; i.e., cold, pale skin from cutaneous vasoconstriction. There is brisk shivering, and a rapid heart rate.
MODERATE HYPOTHERMIA: In moderate hypothermia (28-31.8°C or 82-89°F), shivering may be absent, and patients are lethargic. Bradycardia and a decreased respiratory rate (bradypnea) become evident, and pupillary light reflexes can be absent.
SEVERE HYPOTHERMIA: In severe hypothermia (<28°C or <82°F), patients are usually obtunded or comatose with dilated, fixed pupils (which are not a sign of brain death in this situation). Additional findings include hypo-tension, severe bradycardia, oliguria, and generalized edema. Apnea and asystole are expected at body temperatures below 25°C (77°F).

The laboratory tests of interest in hypothermia include arterial blood gases, serum electrolytes (particularly potassium), tests of coagulation, and tests of renal function. A generalized coagulopathy (with elevation of the INR and prolonged partial thromboplastin times) is common in hypothermia (21), but may not be evident if the coagulation profile is run at normal body temperatures. Arterial blood gases (which should be run at normal body temperatures) can reveal a respiratory acidosis or a metabolic acidosis (21). Serum electrolytes can reveal hyperkalemia, which is presumably due to potassium release by skeletal muscle from shivering or rhabdomyolysis. Serum creatinine levels can be elevated as a result of rhabdomyolysis, acute renal failure, or cold diuresis (caused by diminished renal tubular responsiveness to antidiuretic hormone).

About 80% of patients with hypothermia will have prominent J waves at the QRS-ST junction on the electrocardiogram (see Figure 42.1). These waves, which are called Osborn waves, are not specific for hypothermia, and can occur with hypercalcemia, subarachnoid hemorrhage, cerebral injuries, and myocardial ischemia (22). Despite the attention these waves have received, they are merely a curiosity, and have little or no diagnostic or prognostic value in hypothermia (21-13).
ARRHYTHMIAS: A multitude of rhythm disturbances can occur in hypo-thermia, including first, second, and third-degree heart block, sinus and junctional bradycardia, idioventricular rhythm, premature atrial and ventricular beats, and atrial and ventricular fibrillation (22).
FIGURE 42.1 The (overhyped) Osborn wave.

EXTERNAL REWARMING: External rewarming (removing wet clothes, covering the patient in blankets, etc.) can increase body temperature at a rate of 1-2°C per hour (21), and is adequate for most cases of hypothermia (23). There is a risk of a further decrease in body temperature during external rewarming (called afterdrop), which can trigger ventricular fibrillation (24). This phenomenon is attributed to central displacement of cold

Intentional cooling to a body temperature of 32—34°C (89.6—-93.2°F) is now a popular treatment modality for patients who remain comatose after resuscitation from cardiac arrest. This topic is presented in Chapter 17 (see pages 336-339).

The number of deaths from heat exposure is estimated at only 400 per year in the United States (27), and in a 20-year survey of a large urban hospital in France, severe hypothermia accounted for only 0.4% of admissions to the ICU (24). These small numbers are a testament to the human ability to adapt (both physiologically and behaviorally) to environmental extremes.

1. Keel CAm Neil E, Joels N. Regulation of body temperature in man. In: Samson Wright's Applied Physiology, 13th ed. New York: Oxford University Press, 1982:346.
2. Guyton AC, Hall JE. Body temperature, temperature regulation, and fever. In: Medical Physiology, 10th ed. Philadelphia, WB Saunders, 2000:822-833.
Malignant Hyperthermia, 1 = Malignant Hyperthermia. 9., 1 = Rusyniakn DE, Sprague JE. Toxin-induced hyperthermic syndromes. Med Clin N Am 2005;89:1277-1296.. 10., 1 = Litman RS, Rosenberg H. Malignant hyperthermia. J Am Med Assoc 2005; 293:2918- 2924.. 11., 1 = McEvoy GK, ed. AHFS Drug Information, 2001. Bethesda, MD: American Society of Health-System Pharmacists, 2001, pp. 1328-1331.
Serotonin Syndrome, 1 = Serotonin Syndrome. 17., 1 = Boyer EH, Shannon M. The serotonin syndrome. N Engl J Med 2005; 352:1112-1120.. 18., 1 = Demirkiran M, Jankivic J, Dean JM. Ecstacy intoxication: an overlap between serotonin syndrome and neuroleptic malignant syndrome. Clin Neurophar-macol 1996; 19:157-164.. 19., 1 = Hick JL, Smith SW, Lynch MT. Metabolic acidosis in restraint-associated cardiac arrest. Acad Emerg Med 1999; 6:239-245.. 20., 1 = Graudins A, Stearman A, Chan B. Treatment of serotonin syndrome with cyproheptadine. J Emerg Med 1998; 16:615-619.

Hypothermia, 1 = Hypothermia. 21., 1 = Hanania NA, Zimmerman NA. Accidental hypothermia. Crit Care Clin 1999; 15:235- 249.. 22., 1 = Aslam AF, Aslam AK, Vasavada BC, Khan IA. Hypothermia: evaluation, electrocardiographic manifestations, and management. Am J Med 2006; 119:297- 301.. 23., 1 = Cornell HM. Hot topics in cold medicine: controversies in accidental hypothermia. Clin Ped Emerg Med 2001; 2:179-191.. 24., 1 = Vassal T, Bernoit-Gonin B, Carrat F, et al. Severe accidental hypothermia treated in an ICU. Chest 2001; 120:1998-2003.. 25., 1 = Ireland AJ, Pathi VL, Crawford R, et al. Back from the dead: Extracorporeal rewarming of severe accidental hypothermia victims in accidental emergency. J Accid Emerg Med 1997; 14:255-303.. 26., 1 = Handrigen MT, Wright RO, Becker BM, et al. Factors and methodology in achieving ideal delivery temperatures for intravenous and lavage fluid in hypothermia. Am J Emerg Med 1997; 15:350-359.

27. Morbidity and Mortality Weekly Report, 2002; 51:567—570.
Give me the power to produce fever, and I wil cure all diseases.
Parmenides ca 500 B.C
The appearance of a new fever is always a matter of concern in a hospitalized patient. This chapter presents a practical approach to the ICU patient with a new-onset fever, and includes: (a) the definition of fever in ICU patients, (b) the appropriate sites for measuring body temperature, (c) the optimal method for obtaining blood cultures, and (d) the potential sources of fever in the ICU (1,2). The final section focuses on the practice of suppressing fever, and why Parmenides might disapprove of this practice.

Two scales (Celsius and Fahrenheit) are used to record body temperature, and the conversion from one scale to the other is shown in Table 43.1. Although readings on the Celsius scale are often called "degrees centigrade," this unit is intended for the degrees on a compass, not for temperatures (3). The appropriate term for temperatures on the Celsius scale is degrees Celsius.

The definition of a normal body temperature is not straightforward, as demonstrated by the following observations.
1. The traditional norm of 37°C (98.6°F) is a mean value derived from a study of axillary temperatures in 25,000 healthy adults, conducted in the late 19th century (4). However, axillary temperatures can vary by as much as 1.0°C (1.8°F) from core body temperatures (5), and axillary temperatures are not advised in ICU patients (1).
2. Core body temperature can be 0.5°C (0.9°F) higher than oral temperatures (6), and 0.2—0.3°C lower than rectal temperatures (1).
3. Elderly subjects have a mean body temperature about 0.5°C (0.9°F) lower than younger adults (4,7).
4. Body temperature has a diurnal variation, with the nadir in the early morning (between 4 and 8 a.m.) and the peak in the late afternoon (between 4 and 6 p.m.). The range of diurnal variation varies, but can be as high as 1.3°C (2.4°F) (8).
These observations indicate that the normal body temperature is influenced by age, measurement site, and time of day. As such, the best definition of a normal body temperature is the usual range of temperatures for an individual patient, measured at the same site, over a 24-hour period.

Answer the following questions:,  = YES. Answer the following questions:, NO = . Has the patient received a serotinergic drug in the past 5 wks?,  = Mf. Has the patient received a serotinergic drug in the past 5 wks?, NO = L!. If the answer is YES, proceed to the questions below.,  = . If the answer is YES, proceed to the questions below., NO = . Does the patient have any of the following?,  = . Does the patient have any of the following?, NO = . Tremor + hyperreflexia,  = . Tremor + hyperreflexia, NO = . Spontaneous clonus,  = ISLS. Spontaneous clonus, NO = . Rigidity + Temp >38°C + ocular or inducible clonus,  = ISLS. Rigidity + Temp >38°C + ocular or inducible clonus, NO = UUs. Ocular clonus + agitation or dlaphoresis,  = . Ocular clonus + agitation or dlaphoresis, NO = . Inducible clonus + agitation or diaphoresis,  = iS. Inducible clonus + agitation or diaphoresis, NO = L|. lf the answer Is YES to ANY of the above conditions, the patient has Serotonin Syndrome.,  = lf the answer Is YES to ANY of the above conditions, the patient has Serotonin Syndrome.. lf the answer Is YES to ANY of the above conditions, the patient has Serotonin Syndrome., NO = lf the answer Is YES to ANY of the above conditions, the patient has Serotonin Syndrome.
"Adapted from Refarence 17.

The most recent guidelines on fever in the ICU (1) contain the following recommendations for measuring body temperature.
1. The most accurate measurements are obtained with thermistor-equipped catheters placed in the pulmonary artery, esophagus, or urinary bladder.
2. Less accurate measurements are obtained with rectal, oral, and tympanic membrane

Any condition capable of triggering an inflammatory response is capable of causing a fever. The notable sources of nosocomial fever in the ICU are shown in Figure 43.1.
FIGURE 43.1 Potential sources of nosocomial fever in the ICU.

As mentioned earlier, infection is responsible for only half of ICU-acquired fevers (9,10). The conditions described in this section are responsible for most of the remaining 50% of ICU-acquired fevers. The ones that deserve mention are included in Table 43.2.

SIRS, Less Common Causes = Drug Fever. Early Postop Fever, Less Common Causes = Adrenal Failure. Pulmonary Embolism, Less Common Causes = Acalculous Cholecystitis. Platelet Transfusions, Less Common Causes = latrogenic Fever

The clinical entity known as the systemic inflammatory response syndrome (SIRS) is characterized by signs of systemic inflammation (see Table 14.2), and may not be associated with infection. Noninfectious sources of SIRS include tissue injury (e.g., from ischemia or major surgery), and translocation of endotoxin and inflammatory cytokines from the GI tract. SIRS can be accompanied by inflammatory injury in one or more vital organs (e.g., acute respiratory distress syndrome), and can have a fatal outcome. This condition is described in more detail in Chapter 14.

Major surgery is itself a source of tissue injury. (In the words of Dr. John Millili, a surgeon and close friend, major surgery is like being hit with a baseball bat!) Because inflammation and fever are the normal response to tissue injury, it is no surprise that fever in the first postoperative day is reported in 15-40% of cases of major surgery (1315) and, in most cases, there is no apparent infection (13,14). These fevers are shortlived, and usually resolve within 24—48 hours.

There is a long-standing misconception that atelectasis is a common cause of fever in the early postoperative period. One possible source of this misconception is the high incidence of atelectasis in patients who develop a postoperative fever This is demonstrated in Figure 43.2 (see the graph on the left), which is from a study involving patients who underwent open heart surgery (15). Close to 90% of the patients with a fever on the first postoperative day had radiographic evidence of atelectasis. This, however, is not evidence that the atelectasis is the source of fever. In fact, the graph on the right in Figure 43.2 shows that most (75%) of the patients with atelectasis did not have a fever. The inability of atelectasis to produce fever was demonstrated over 50 years ago in an animal study where lobar atelectasis produced by ligation of a mainstem bronchus was not accompanied by fever (16).
To summarize, atelectasis is a common complication of major surgery, and occurs in over 90% of cases of general anesthesia (17). However, it is not a common cause of postoperative fever. Most fevers that appear in the first 24 hours after surgery are the result of the tissue injury sustained during the procedure.
FIGURE 43.2 The relationships between fever and atelectasis in the first postoperative day in 100 consecutive patients who had open heart surgery. The graph on the left shows that most patients with fever had atelectasis, but the graph on the right shows that most patients with atelectasis did not have a fever. Data from Reference 15.

An uncommon but treatable cause of elevated body temperatures in the immediate postoperative period is malignant hyperthermia, an inherited disorder characterized by muscle rigidity, hyperpyrexia (temperature >40°C or 104°F), and rhabdomyolysis in response to halogenated inhalational anesthetics. This disorder is described in Chapter 42.

Several groups of patients are at risk for venous thromboembolism (see Table 6.1 and Figure 6.1), but the risk is highest in trauma victims and postoperative patients. Most cases of hospital-acquired deep vein thrombosis are asymptomatic, but acute pulmonary embolism can produce a fever that lasts up to 1 week (18). The diagnostic approach to acute pulmonary embolism is outlined in Figure 6.2.

Febrile non-hemolytic transfusion reactions occur in 0.5% of erythrocyte transfusions. These reactions are the result of antileukocyte antibodies in the recipient that react with donor leukocytes, and they are more likely to occur in patients who have received multiple transfusions. The fever usually appears during, or up to 6 hours after, the transfusion. For more information on these reactions, see pages 360-361.

Fever is much more common with platelet transfusions; i.e., the reported incidence is as high as 30% (see page 380). These reactions are also caused by antibodies to donor leukocytes, and the frequent appearance of these reactions with platelet transfusions is probably due to the multiple donors used for routine platelet transfusions.

Drug-induced fever can be the result of a hypersensitivity reaction or an idiosyncratic reaction. Any drug can trigger a hypersensitivity reaction, but the drugs most often implicated in drug fever are listed in Table 43.3.
Drug fever is poorly understood. The onset of the fever varies from a few hours to more than three weeks after the onset of drug therapy (1). The fever can appear as an isolated finding, or can be accompanied by the other manifestations listed in Table 43.3 (19). Note that about half of patients have rigors, and about 20% develop hypotension, indicating that patients with a drug fever can appear seriously ill. Evidence of a hypersensitivity reaction (i.e., eosinophilia and rash) is absent in over 75% of cases of drug fever (19).
Suspicion of drug fever usually occurs when there are no other likely sources of fever. When suspected, possible offending drugs should be discontinued, if possible. The fever should disappear in 2-3 days, but it can persist for up to 7 days (20).

Amphotericin, Occasional Offenders = Cimetidine. Amphotericin, —_—_Clinical Findings* = Rigors (53%). Cephalosporins, Occasional Offenders = Carbamazepine. Cephalosporins, —_—_Clinical Findings* = Myalgias (2596). Penicillins, Occasional Offenders = Hydralazine. Penicillins, —_—_Clinical Findings* = Leukocytosis (229). Phenytoin, Occasional Offenders = Rifampin. Phenytoin, —_—_Clinical Findings* = Eosinophilia (22°). Procainamide, Occasional Offenders = Streptokinase. Procainamide, —_—_Clinical Findings* = Rash (18%). Quinidine, Occasional Offenders = Vancomycin. Quinidine, —_—_Clinical Findings* = Hypotension (18%)
Fram Referance 19.

The drug-induced hyperthermia syndromes include malignant hyperthermia (mentioned earlier), neuroleptic malignant syndrome, and serotonin syndrome. These disorders are characterized by muscle rigidity, hyperpyrexia (temperature >40°C or 104°F), and rhabdomyolysis, and are described in detail in Chapter 42. The neuroleptic malignant syndrome is a particular concern in patients receiving haloperidol for sedation.

There are several other potential causes of noninfectious fever, and the most notable of these are included in the following text.

Acalculous cholecystitis is an uncommon but serious disorder reported in 1.5% of critically ill patients (21). It is believed to be the result of ischemia and stasis within the gallbladder, resulting in edema of the cystic duct that blocks drainage of the gallbladder. The diagnosis and management of this condition is described in Chapter 40.

The endocrine disorders known to produce fever are thyrotoxicosis and adrenal crisis. Thyrotoxicosis is unlikely to appear de novo in the ICU, but adrenal crisis due to spontaneous adrenal hemorrhage is a recognized complication of anticoagulant therapy and disseminated intravascular coagulation (DIC). These endocrine disorders are described in Chapter 50.

Faulty thermal regulators in water mattresses and aerosol humidifiers can cause fever by transference (22). It takes only a minute to check the temperature settings on heated mattresses and ventilators, but it can take far longer to explain why such a simple cause of fever was overlooked.

The incidence of ICU-acquired infections in medical and surgical ICU patients is shown in Table 43.4 (23). Four infections account for over three-quarters of ICU-acquired infections (pneumonia, urinary tract infections, bloodstream infections, and surgical site infections), and three of these infections involve indwelling plastic devices: i.e., 83% of the pneumonias occur in intubated patients, 97% of the urinary tract infections occur in catheterized patients, and 87% of bloodstream infections originate from intravascular catheters (23).

Pneumonia, 1 = 30%. Pneumonia, 2 = . Pneumonia, 3 = 33%. Pneumonia, 4 = 78%. Urinary Tract Infection, 1 = 30%. Urinary Tract Infection, 2 = ) 76%. Urinary Tract Infection, 3 = 18%. Urinary Tract Infection, 4 = 78%. Bloodstream Infection, 1 = 16%. Bloodstream Infection, 2 = . Bloodstream Infection, 3 = 13%. Bloodstream Infection, 4 = 78%. Surgical Site Infection, 1 = —. Surgical Site Infection, 2 = . Surgical Site Infection, 3 = 14%. Surgical Site Infection, 4 = 78%. Cardiovascular Infection, 1 = 5%. Cardiovascular Infection, 2 = . Cardiovascular Infection, 3 = 4%. Cardiovascular Infection, 4 = . GI Tract Infection, 1 = 5%. GI Tract Infection, 2 = . GI Tract Infection, 3 = 4%. GI Tract Infection, 4 = . Ear, Nose & Throat Infection, 1 = 4%. Ear, Nose & Throat Infection, 2 = . Ear, Nose & Throat Infection, 3 = 4%. Ear, Nose & Throat Infection, 4 = . Skin & Soft Tissue Infection, 1 = 3%. Skin & Soft Tissue Infection, 2 = . Skin & Soft Tissue Infection, 3 = 3%. Skin & Soft Tissue Infection, 4 = . Others, 1 = 7%. Others, 2 = . Others, 3 = 7%. Others, 4 = 
From Referance 23.

The three most common ICU-acquired infections in Table 43.4 are described elsewhere in this book. The diagnosis and management of these conditions can be found in Chapter 3 (for vascular catheter-related infections), Chapter 29 (for ventilator-associated pneumonia), and Chapter 41 (for urinary tract infections). The following are some other nosocomial infections that should be considered in a patient with ICU-acquired fever.

Surgical site infections (SSIs) continue to be a considerable source of postoperative morbidity despite attention to preventive measures (24). These infections typically appear 5-7 days after surgery. Superficial infections are less likely to produce fever than infections with deep tissue involvement. Sternal wound infections following open heart surgery show a particular propensity for deep tissue involvement (i.e., mediastinitis) (25). For patients with fever following open heart surgery, sternal instability can be an early sign of sternal wound infection.
The pathogens involved in SSIs are determined by the surgical procedure. SSIs from clean surgical procedures (where the chest or abdomen has not been opened) usually involve Staphylococcus aureus, while SSIs from contaminated procedures (where the chest or abdomen has been opened) often involve organisms that are part of the indigenous flora of the organ that was surgically repaired (e.g., infections following bowel surgery typically involve Gram-negative aerobic bacilli and anaerobes) (1).

Superficial infections can usually be managed with debridement only. The management of deep-seated infections is dependent on the character of the infection. Localized collections (abscesses) can often be managed with drainage only, while more diffuse involvement of deep tissues should prompt antimicrobial therapy.

Necrotizing wound infections are produced by Clostridium species or a-hemolytic streptococci (1). Unlike other wound infections (which typically appear 5—7 days after surgery), necrotizing infections are evident in the first few postoperative days. There is often marked edema, and fluid-filled bullae, around the incision, and crepitance may be present. Spread to deeper structures is rapid, and progressive disease is often accompanied by rhabdomyolysis and myoglobinuric renal failure. Treatment involves extensive debridement and intravenous penicillin. The mortality rate is high (>60%) when treatment is delayed.

Indwelling nasogastric and nasotracheal tubes can block the ostia that drain the paranasal sinuses, leading to accumulation of infected secretions in the sinuses. The maxillary sinuses are almost always involved, and the resulting acute sinusitis can be an occult source of fever. Paranasal sinusitis is reported in 15-20% of patients with indwelling nasal tubes (26,27), and can be a source of fever and bacteremia. However, the clinical significance of this condition, in many cases, is unclear (see later).

The pathogens involved in ICU-acquired sinusitis are the same ones that colonize the oropharynx in critically ill patients. The most frequent isolates are Gram-negative aerobic bacilli (in 60% of cases), followed by Gram-positive aerobic cocci (particularly Staph. aureus and coagulase-negative staphylococci) in 30% of cases, and yeasts (mostly Candida albicans) in 5-10% of cases (1).

Purulent drainage from the nares is absent in about 75% of cases (1), and the diagnosis is suggested by radiographic features of sinusitis (i.e., opacification or air—fluid levels in the involved sinuses). Although CT scans are recommended for the diagnosis of nosocomial sinusitis (26,27), portable sinus films obtained at the bedside can also be revealing, as shown in Figure 43.3. The maxillary sinuses can be viewed with a single occipitomental view, also called a "Waters view," which can be obtained at the bedside (28). (Avoiding a CT scan also avoids the risks and manpower involved in transporting a patient out of the ICU.)
Radiographic or CT evidence of sinusitis is not sufficient for the diagnosis of sinusitis because 30-40% of patients with evidence of sinusitis on imaging studies do not have an infection documented by aspiration of the involved sinus (26,27). The diagnosis requires aspiration of the involved sinus with a quantitative culture that grows 2103 colony forming units per mL (26,27).

A trial of empiric antibiotic therapy is warranted when there is radiographic evidence of sinusitis in a febrile patient with no other apparent source of fever. The antibiotic regimen should provide coverage for Gram-negative aerobic bacilli and staphylococci. Single-agent therapy with imipenem or meropenem should be adequate if MRSA has not been isolated on routine nasal swabs. If MRSA has been isolated on a nasal swab, or MRSA is a frequent isolate in your ICU, then vancomycin should be added to the Gram-negative coverage. In addition, nasal tubes should be removed and replaced with oral tubes. If there is no improvement with empiric antibiotics, sinus puncture for Gram stain and quantitative culture is warranted (1).

Despite the fact that nosocomial sinusitis is documented in 15-20% of patients with indwelling nasal tubes (26,27), sinusitis is often overlooked in the evaluation of ICUacquired fever, without apparent harm. This creates uncertainty about the clinical significance of ICU-acquired sinusitis.

ICU-acquired fever that is associated with new-onset diarrhea should always prompt suspicion of Clostridium difficile enterocolitis. The diagnosis and management of this condition are described in Chapter 40 (see pages 738-742).

Infections that should be considered in specific patient populations include: (a) abdominal abscesses in patients who have had a laparotomy or laparoscopy (see pages 744-745), (b) endocarditis in patients with prosthetic or damaged valves, (c) meningitis in neurosurgery patients, and (d) spontaneous bacterial peritonitis in patients with cirrhosis and ascites (see pages 725-726).
FIGURE 43.3 Portabk sinus film (Waters view) showing opacification of the left maxillary and frontal sinuses in a patient with indwelling nasotracheal and nasogastric tubes. The diagnosis of paranasal sinusitis was subsequently confirmed by a maxilary sinus puncture at the bedside, with the aspirate growing Staph. epidermidis at 103 cfu/mL.

The appearance of an ICU-acquired fever is not a license to perform an extensive evaluation and start empiric antimicrobial therapy. Instead, an evaluation should be made to determine the likelihood of a noninfectious or infectious source of fever. If a noninfectious source of fever is unlikely, the following measures are relevant.

Blood cultures are recommended for all cases of ICU-related fever where a noninfectious source is unlikely (1). The yield from blood cultures is dependent on the volume of blood withdrawn during a venipuncture, and the number of venipuncture sites.

The yield from blood cultures is optimal when 20-30 mL of blood is withdrawn from each venipuncture site (1). The standard practice is to withdraw 20 mL of blood from a venipuncture site: one-half (10 mL) is then injected into each of the two bottles of broth (one aerobic and one anaerobic) provided in a blood culture set. Increasing from 20 mL to 30 mL of blood increases the yield from blood cultures by about 10% (29). When using 30 mL per venipuncture, the extra 10 mL aliquot of blood should be injected into an aerobic broth bottle (29).

In the terminology of blood cultures, one blood culture refers to a single venipuncture site. (For example, culture of blood specimens from each lumen of a multilumen catheter still represents one blood culture.) The relationship between the number of blood cultures and the detection of bacteremia is shown in Figure 43.4 (30). This is from a study of patients with bacteremia documented by four or more blood cultures drawn over a 24 hour period. The two curves in the graph represent patients with endocarditis and patients with other infections. The majority of the bacteremias (94%) were detected with two blood cultures in the patients with endocarditis, while three blood cultures were required to detect over 90% of the bacteremias in patients with other infections. The enhanced detection rate in endocarditis is due to the continuous bacteremia associated with endocarditis.
Based on the data in Figure 43.4, three blood cultures drawn over a 24-hour period will detect a majority (>90%) of bacteremias (1). However, two blood cultures will detect a majority of bacteremias in patients with endocarditis.
FIGURE 43.4 Relationship between the number of blood cultures drawn over a 24-hour period (20 mL per blood culture) and the detection rate for bacteremia. See text for explanation. Data from Reference 30.

The popular perception of fever as a malady that must be corrected is rooted in hearsay. In fact, fever is a normal adaptive response that enhances the ability to eradicate infection (12). This section contains some observations about fever that are intended to make you think twice about starting antipyretic therapy in critically ill patients.

An increase in body temperature can enhance immune function by increasing the production of antibodies and cytokines, activating T-lymphocytes, facilitating neutrophil chemotaxis, and enhancing phagocytosis by neutrophils and macrophages (32,33). In addition, high temperatures inhibit bacterial and viral replication. The effect of body temperature on the growth of bacteria in blood is demonstrated in Figure 43.5 (34). Note that an increase in temperature of 4°C completely suppresses growth. Similar results have been demonstrated in an animal model of pneumococcal meningitis (35).

The benefits of fever as a host defense against infection is supported in clinical studies showing that septic patients who develop hypothermia have at least twice the mortality rate of septic patients who develop a fever (36,37). The results of these studies are shown in Figure 43.6. Although these studies cannot establish a causal relationship between body temperature and outcomes, they do show that higher body temperatures are associated with improved outcomes. A more recent observational study showed that antipyretic therapy was associated with higher mortality rates in septic patients (38).
FIGURE 43.5 The influence of body temperature on the growth of Pasteurella multocida in the blood of infected laboratory animals. The range of temperatures in the figure is the usual range of febrile temperatures for the study animal (rabbits). Data from Reference 34.
ina
FIGURE 43.6 The relationship between body temperature and survival in two clinical studies of patients with severe sepsis. Data from References +36 and #37.

One of the claims in support of suppressing fever is the assumed effect of fever in promoting tachycardia, which can be harmful in patients with heart disease. However, the association between fever and tachycardia was established in animal models of sepsis, and it is likely that the inflammatory response to sepsis is the source of tachycardia, and not an elevation in body temperature.

There is convincing evidence that increased body temperatures aggravate ischemic brain injury following cardiac arrest (see Chapter 17) and ischemic stroke (see Chapter 46). However, the effects of increased body temperatures in the non-ischemic brain have not been adequately studied. The popular claim that hyperpyrexia (temperature =40°C or 2104°F) promotes injury in the non-ischemic brain can neither be supported nor refuted because hyperpyrexia is rarely left untreated in clinical practice.

Prostaglandin E mediates the febrile response to endogenous pyrogens, and drugs that interfere with prostaglandin E synthesis are effective in reducing fever (39). These drugs include aspirin, acetaminophen, and nonsteroidal anti-inflammatory agents (NSAIDS). Only the latter two are used for fever suppression in the ICU.

Acetaminophen is the favored drug for antipyresis, despite the fact that it is the leading cause of acute liver failure in the United States (see Chapter 54). Acetaminophen is contraindicated in patients with hepatic insufficiency.
DOSING REGIMENS: Acetaminophen is usually given orally or by rectal suppository in a dose of 650 mg every 4-6 hrs, with a maximum daily dose of 4 grams. An intravenous preparation is now available in the United States (OFIRMEV™), and the recommended dose for adults =>50 kg is 650 mg every 4 hrs, or 1,000 mg every 6 hrs, with a maximum dose of 4 grams daily (40). This dosing regimen is equivalent to oral acetaminophen for suppressing fever in adults (41). Intravenous acetaminophen is costly, and is recommended only for patients who cannot tolerate oral or rectal drug administration.

Ibuprofen is apopular over-the-counter NSAID that provides safe and effective antipyresis at an intravenous dose of 400-800 mg every six hours (42) . Ketorolac is another intravenous NSAID that has been effective in suppressing fever (in a single dose of 0.5 mg/kg) (43). See Chapter 51 for more information on these drugs.

Cooling blankets are inappropriate for the treatment of fever. The febrile response raises the body temperature by promoting cutaneous vasoconstriction and increasing skeletal muscle activity (via rigors and shivering). This is what the body normally does in response to a cold environment, so the febrile response mimics the physiological response to cold. Stated another way, the febrile response makes the body behave like it is wrapped in a cooling blanket. Adding a cooling blanket will only aggravate the cutaneous vasoconstriction and increased muscle activity involved in the febrile response. This explains why cooling blankets are notoriously ineffective in reducing fever. Cooling blankets are more appropriate for hyperthermia syndromes, when normal thermoregulation is faulty (see Chapter 42).

There's a wrong way and a right way to approach a new-onset fever in the ICU. The wrong way is to culture everything available, order a barrage of laboratory tests and imaging studies, and start antibiotics without hesitation. The right way is to make sure the fever is real (and not the result of an iatrogenic problem), and then evaluate the patient for the likelihood of an infectious or noninfectious source of the fever. Remem-ber that there is a 50-50 chance of finding an underlying infection, so don't start antibiotics unless an_ infection is apparent or highly suspected, or the patient is immunocompromised. And finally, please think twice about suppressing fever, and stay away from cooling blankets.

1. O'Grady NP, Barie PS, Bartlett J, et al. Guidelines for the evaluation of new fever in critically ill adult patients: 2008 update from the American College of Critical Care Medicine and the Infectious Disease Society of America. Crit Care Med 2008; 36:1330-1349.
2. Laupland KB. Fever in the critically ill medical patient. Crit Care Med 2009; 37(Suppl):S273-S278.

23., 1 = Richards MJ, Edwards JR, Culver DH, Gaynes RP. The National Nosocomial Infections Surveillance System. Nosocomial infections in combined medical-surgical intensive care units in the United States. Infect Control Hosp Epidemiol 2000; 21:510-515.. 24., 1 = Alexander JW, Solomkin JS, Edwards MJ. Updated recommendations for control of surgical site infections. Ann Surg 2011; 253:1082-1093.. 25., 1 = Loopp FD, Lytle BW, Cosgrove DM, et al. Sternal wound complications after isolated coronary artery bypass grafting: early and late mortality, morbidity, and cost of care. Ann Thorac Surg 1990; 49:179-187.. 26., 1 = Holzapfel L, Chevret S, Madinier G, et al. Influence of long-term oro- or nasotracheal intubation on nosocomial maxillary sinusitis and pneumonia: results of a prospective, randomized, clinical trial. Crit Care Med 1993; 21:1132-1138.. 27., 1 = Rouby J-J, Laurent P, Gosnach M, et al. Risk factors and clinical relevance of nosocomial maxillary sinusitis in the critically ill. Am Rev Respir Dis 1994; 150:776- 783.. 28., 1 = Diagnosing sinusitis by x-ray: is a single Waters view adequate? J Gen Intern Med 1992; 7:481-485.

29. Patel R, Vetter EA, Harmsen WS, et al. Optimized pathogen detection with 30compared to 20-milliliter blood culture draws. J Clin Microbiol 2011; 49:4047-4051.
30. Cockerill FR, Wilson JW, Vetter EA, et al. Optimal testing parameters for blood cultures. Clin Infect Dis 2004; 38:1724—1730.
31. Hughes WH, Armstrong D, Bodey GP, et al. 2002 guidelines for the use of antimicrobial agents in neutropenic patients with cancer. Clin Infect Dis 2002; 34(6):730-751.

There is no delusion more damaging than to get the idea in your head that you understand the functioning of your brain.
Lewis Thomas
1983

I think, therefore I am.
René
Descartes
1644
The ability to recognize and interact with the surroundings (i.e., consciousness) is the sina qua non of the life experience, and loss of this ability is one of the dominant (and most prevalent) signs of a life-threatening illness. This chapter describes the principal disorders of consciousness encountered in the ICU, including delirium, coma, and the ultimate disorder of consciousness, brain death.

Consciousness has two components: arousal and awareness.
1. Arousal is the ability to experience your surroundings, and is also known as wakefulness.
2. Awareness is the ability to understand your relationship to your surroundings, and is also known as responsiveness.
These two components are used to identify the altered states of consciousness in Table 44.1.

Anxiety, 1 = Delirium. Anxiety, 2 = Coma. Lethargy, 1 = Dementia. Lethargy, 2 = Brain Death. Locked-In State, 1 = Psychosis. Locked-In State, 2 = . , 1 = Vegetative State. , 2 = 

The nontraumatic causes of altered consciousness are indicated in Figure 44.1. In a prospective survey of neurologic complications in a medical ICU (3), ischemic stroke was the most frequent cause of altered consciousness on admission to the ICU, and septic encephalopathy was the most common cause of altered consciousness that developed after admission to the ICU. Nonconvulsive status epilepticus should always be considered when the source of altered consciousness is not clear (see Chapter 45).

Septic encephalopathy is a global brain disorder associated with infections that originate outside the central nervous system. This condition is reported in 50-70% of ICU patients with sepsis, and can be an early sign of infection, especially in elderly patients (3,4). Septic encephalopathy is similar to hepatic encephalopathy (described in Chapter 39) in that both conditions are characterized by cerebral edema, and involve the accumulation of ammonia and aromatic amino acids (e.g., tryptophan) in the central nervous system (4,5). The origins of septic encephalopathy may be the actions of inflammatory mediators to increase the permeability of the blood-brain barrier, which then allows ammonia and other toxic substances to gain entry into the central nervous system. This is similar to the capillary leak that promotes peripheral edema in septic and anaphylactic shock.
FIGURE 44.1 Sources of altered consciousness in ICU patients.

Delirium is reported in 16-89% of ICU patients (6), and is particularly prevalent in ventilator-dependent patients (7), and elderly postoperative patients (8). The delirium that accompanies alcohol withdrawal is a different entity than hospital-acquired delirium, and is described in a separate section.

The clinical features of delirium are summarized in Figure 44.2 (9). Delirium is an acute confusional state with attention deficits, disordered thinking, and a fluctuating course (the fluctuations in behavior occur over a 24-hour period). Over 40% of hospitalized patients with delirium have psychotic symptoms (e.g., visual hallucinations) (10); as a result, delirium is often inappropriately referred to as "ICU psychosis" (11).

The following subtypes of delirium are recognized:
1. Hyperactive delirium is characterized by restless agitation. While this form of delirium is common in alcohol withdrawal, it is rare in hospital-acquired delirium, accounting for "2% of cases (6).
2.Hypoactive delirium is characterized by lethargy and somnolence. This is the most common form of hospital-acquired delirium, and is responsible for 45-64% of cases (6).
3 .. Mixed delirium is characterized by episodes of delirium that alternate between hyperactive and hypoactive forms of the illness. This type of delirium is reported in 6— 55% of patients with hospital-acquired delirium (6).
As indicated, the popular perception of delirium as a state of agitated confusion does not
apply to hospital-acquired delirium, where the most common presentation of delirium is lethargy and somnolence. Failure to recognize the hypoactive form of delirium may explain why the diagnosis of delirium is missed in as many as 75% of patients (12).

Delirium and dementia are distinct mental disorders that are often confused because they have overlapping clinical features (i.e., attention deficits and disordered thinking). Furthermore, as many as two-thirds of hospitalized patients with dementia can have a superimposed delirium (8,13), which further blurs the distinction between these two conditions. The principal features of delirium that distinguish it from dementia are the acute onset and fluctuating course.

Several conditions promote delirium in hospitalized patients, including (a) advanced age, (b) sleep deprivation, (c) unrelieved pain, (d) prolonged bed rest, (e) major surgery, (f) encephalopathy, (g) systemic inflammation, and (h) deliriogenic drugs (6,8,11).

Several types of drugs can promote delirium, including (a) anticholinergic drugs, (b) dopaminergic drugs, (c) seritonergic drugs, and (d) drugs that promote gamma-aminobutyric-acid (GABA)-mediated neurotransmission, such as benzodiazepines and propofol (6).

Validated screening tools are recommended for the detection of delirium because (as mentioned earlier) the diagnosis of delirium is frequently missed (12). The Confusion Assessment Method for the ICU (CAM-ICU) is the most reliable tool for the detection of delirium (6,9), and it is available (along with an_ instructional video) at www. icudelirium.org.

Alcohol withdrawal delirium, also Known as delirium tremens or DTs, is characterized by increased motor activity and increased activity on the electroencephalogram (EEG). In contrast, hospital-acquired delirium is characterized by decreased motor activity and slowing of the EEG activity (6).

The central nervous system depressant effects of ethanol are the result of stimulation of GABA receptors (the major inhibitory pathway in the brain) and inhibition of N-methyl-Daspartate (NMDA) receptors (the major excitatory pathway in the brain). When ethanol is withdrawn, the resulting effects on both receptors results in central nervous system excitation, which leads to the agitation, delirium, and seizures that are characteristic features of alcohol withdrawal.

The clinical features of alcohol withdrawal are shown in Table 44.2. About 5% of patients who experience alcohol withdrawal symptoms will develop DTs (17). Risk factors include a prolonged drinking history, prior episodes of DTs, comorbid illness, and time since last drink. Signs of DTs usually appear 2—3 days after the last drink, and include agitated delirium, low-grade fever, tachycardia, hypertension, diaphoresis, nausea, and vomiting. Associated conditions include dehydration, hypo-kalemia, hypomagnesemia, and generalized seizures. The condition typically lasts for 3-5 days (17), but severe cases can last for up to 2 weeks (personal observation). The reported mortality is 5-15% (17).
WERNICKE'S ENCEPHALOPATHY: Alcoholic patients who are admitted with borderline thiamine stores and receive an intravenous glucose load can develop acute Wernicke's encephalopathy from thiamine deficiency (because thiamine is a cofactor for enzymes involved in glucose metabolism) (18). In this situation, the acute changes in mental status occur 2—3 days after admission, and can be confused with alcohol withdrawal delirium. The presence of nystagmus or oculomotor palsies (e.g., lateral gaze paralysis) will help to identify Wernicke's encephalopathy. (For more information on thiamine deficiency, see Chapter 47.)
able 44.2 Clinical Features of Alcohol Withdrawal
Early Withdrawal Anxiety Tremulousness Nausea, Onset after Last Drink = 6-8 hours. Early Withdrawal Anxiety Tremulousness Nausea, Duration = 1-2 days. Generalized Seizures, Onset after Last Drink = 6-48 hours. Generalized Seizures, Duration = 2-3 days. Hallucinations Visual Auditory Tactile Delirium Tremens, Onset after Last Drink = 12-48 hours 48-96 hours. Hallucinations Visual Auditory Tactile Delirium Tremens, Duration = 1-2 days 1-5 days
Adapted from Refarance 17.

The drugs of choice for treating alcohol withdrawal delirium are the benzodiazepines (19), which mimic the CNS depressant effects of alcohol by stimulating GABA receptors in the brain. An added benefit of benzodiazepines is protection against generalized seizures.
ICU REGIMEN: For patients who require care in the ICU, intravenous lorazepam is an appropriate choice for the management of DTs (19). For initial control, give 2-4 mg IV every 5-10 minutes until the patient is calm. Thereafter, administer IV lorazepam every 1-2 hours in a dose that maintains calm (a dose of 2—4 mg should be sufficient in most cases). After at least 24 hours of calm, the dose can be tapered to determine if the delirium persists. It is important to taper benzodiazepines as soon as possible because they accumulate and can produce prolonged sedation and a prolonged ICU stay. An additional concern with prolonged administration of IV lorazepam is propylene glycol toxicity (see page 605). For more information on benzodiazepines, see Chapter 51.
THIAMINE: The clinical manifestations of DTs can mask an acute Wernicke's encephalopathy that is precipitated by glucose infusions in IV fluids, as described earlier. Therefore, thiamine supplementation is a standard practice during the treatment of DTs. The popular dose is 100 mg daily, which can be given intravenously without harm.

The patient who is comatose (i.e., unarousable and unaware) is one of the most challenging problems in critical care practice, and the management includes not only the patient, but the patient's family and other intimates as well.
able 44.3 Conditions That Affect Pupillary Size and Reactivi
, a _ Associated Conditions = Atropine, anticholinergic toxicity, adrenergic agonists (2.g., dopamine), stimulant drugs (é.g., amphetamines), or nonconvulsive seizures. , a _ Associated Conditions = Diffuse brain injury, hypothermia (< 28°C), or brainstem compression from an expanding intracranial mass or intracranial hypertension. , a _ Associated Conditions = Expanding Intracranial mass (e.g., uncal hermia- tion), ocular trauma or surgery, or focal seizure. , a _ Associated Conditions = Toxic/metabolic encephalopathy, sedative over- dose, or neuromuscular blockade. @ @ Oo ©, a _ Associated Conditions = Acute liver failure, postanoxic encephalopathy, or brain death. HG), a _ Associated Conditions = Horner's Syndrome. © © 4) 44), a _ Associated Conditions = Opiate overdose, toxic/metabolic encephalopathy, hypercapnia or pontine injury
44) and (-indicate a reactive and nonreactive pupil, respectively. From Referencas 21, 22, and 24.

1. Dilated, reactive pupils can be the result of drugs (anticholinergics, CNS stimulants, or adrenergic agonists) or nonconvulsive seizures, while dilated, unreactive pupils are a sign of diffuse brain injury or brainstem compression (e.g., from an expanding intracranial mass).
. A unilateral, dilated and fixed pupil can be the result of ocular trauma or recent ocular surgery, or can be evidence of third cranial nerve dysfunction from an expanding intracranial mass.
Midposition, reactive pupils can be the result of a metabolic enceph-alopathy, a sedative overdose, or neuromuscular blocking drugs, while midposition, unreactive pupils can be seen with acute liver failure, postanoxic encephalopathy, or brain death.
. Small, reactive pupils can be the result of a metabolic encephalopathy, while pinpoint pupils can be the result of opiate overdose (pupils reactive) or pontine injury (pupils unreactive).

Spontaneous eye movements (conjugate or dysconjugate) are a nonspecific sign of toxic or metabolic encephalopathies (22). However, a fixed gaze preference involving one or both eyes is highly suggestive of a mass lesion or seizure activity.

The ocular reflexes are used to evaluate the functional integrity of the lower brainstem (22). These reflexes are illustrated in Figure 44.3.
OCULOCEPHALIC REFLEX: The oculocephalic reflex is assessed by briskly rotating the head from side-to-side. When the cerebral hemispheres are impaired but the lower brainstem is intact, the eyes will deviate away from the direction of rotation and maintain a forward field of view. When the lower brainstem is damaged (or the patient is awake), the eyes will follow the direction of head rotation. The oculocephalic reflex should not be attempted in patients with an unstable cervical spine.
OCULOVESTIBULAR REFLEX: The oculovestibular reflex is performed by injecting 50 mL of cold saline in the external auditory canal of each ear (using a 50 mL syringe and a 2-inch soft plastic angiocatheter). Before the test is performed, check to make sure that the tympanic membrane is intact and that nothing is obstructing the ear canal. When brainstem function is intact, both eyes will deviate slowly toward the irrigated ear. This conjugate eye movement is lost when the lower brainstem is damaged. After the test is performed on one side, wait for 5 minutes before testing the opposite side.

The Glasgow Coma Scale, which is shown in Table 44.4, was introduced to evaluate the severity of traumatic brain injuries (25,26), but has been adopted for use in patients with nontraumatic brain injuries. The Scale consists of three components: 1) eye opening, 2) verbal communication, and 3) motor response to verbal or noxious stimulation. The Glasgow Coma Score (GCS) is the sum of the three components. A minimum score of 3 indicates total absence of awareness and responsiveness, while a maximum score of 15 is normal.

The GCS is not reliable in patients who are paralyzed, heavily sedated, or hypotensive. Otherwise, the GCS (best score) can be used as follows: 1. To define coma (GCS 8).

2. To stratify the severity of head injury (mild: GCS = 13-15, moderate: GCS = 9-12, severe: GCS "8 ) (25,26).
3. To identify candidates for intubation; i.e., airway protective reflexes are typically defective at a GCS "8, which is used as an indication for endotracheal intubation.
4. AS a prognostic marker; e.g., in the initial evaluation of nontraumatic coma, patients with a GCS =6 are seven-times more likely to awaken within two weeks than patients with a GCS "5 (27).
5. The influence of induced hypothermia on the prognostic value of the GCS is described in Chapter 17 (see Table 17.5).
INTUBATED PATIENTS: One of the major shortcomings of the Glasgow Coma Scale is the inability to evaluate verbal responses in intubated patients. These patients are assigned a verbal pseudoscore of 1 (for a maximum GCS of 11).

Eye Opening:, 1 = . Eye Opening:, 2 = . Spontaneous, 1 = . Spontaneous, 2 = . To speech, 1 = Oo. To speech, 2 = . To pain, 1 = ony. To pain, 2 = . , 1 = . , 2 = (J Points. Verbal Communication:, 1 = . Verbal Communication:, 2 = . Oriented, 1 = Om. Oriented, 2 = . Confused conversation, 1 = wo fe. Confused conversation, 2 = . Inappropriate but recognizable words, 1 = . Inappropriate but recognizable words, 2 = . Incomprehensible sounds, 1 = . Incomprehensible sounds, 2 = . , 1 = -+-. , 2 = (J Points. Motor Response:, 1 = . Motor Response:, 2 = . Obeys commands, 1 = OD. Obeys commands, 2 = . Localizes to pain, 1 = Oo. Localizes to pain, 2 = . Withdraws to pain, 1 = NO fF. Withdraws to pain, 2 = . Abnormal flexion (decorticate response), 1 = . Abnormal flexion (decorticate response), 2 = . Abnormal extension (decerebrate response), 1 = . Abnormal extension (decerebrate response), 2 = . No movement, 1 = +. No movement, 2 = (J Points. Glasgow Coma Score (Total of3 scales)", 1 = . Glasgow Coma Score (Total of3 scales)", 2 = LJ Points
"Worst score is 3 points, and bast score is 15 points. With endotrached intubation, the highast score is 17.

The Uniform Determination of Death Act states the following: "An individual who has sustained either 1) irreversible cessation of circulatory and respiratory functions, or 2) irreversible cessation of all functions of the entire brain, including the brainstem, is dead." (28). The second requirement in this statement is the purpose of the brain death determination described here.
Brain death is not a common consequence of the conditions listed in Figure 44.1, and is most often the result of traumatic brain injury and intracerebral hemorrhage, where increased intracranial pressure results in cessation of blood flow to all areas of the brain (29).

A checklist for the diagnosis of brain death in adults is shown in Table 44.5 (30-32). There is a lack of consensus about minor aspects of the brain death determination, but the consensus goal is to establish: (a) irreversible coma, (b) the absence of brainstem reflexes, and (c) the absence of spontaneous respirations. Prior to performing a brain death examination, other confounding conditions (e.g., hypothermia) should be corrected. If the etiology of the coma is unclear, an electroencephalogram should be performed to search for nonconvulsive status epilepticus. If the clinical evaluation for brain death is equivocal, then confirmatory testing may be required. (See the bottom of Table 44.5 for a list of the accepted confirmatory tests).
able 44.5 Checklist for Brain Death Determination in Adults
Step 1: Prerequisite to Exam: All of the following conditions should be corrected before beginning the brain death evaluation. * Hypotension (mean arterial pressure <65 mm Hg) * Hypothermia (core temp <32°C or <90°F) ¢ Hypothyroidism © Hypoglycemia e Effects of CNS depressant drugs, Check (“) Item if Confirmed = J. Step 2: Establish the Cause of Coma:, Check (“) Item if Confirmed = . The cause of coma Is known, and Is sufficient to account for irreversible brain death., Check (“) Item if Confirmed = _J. A. The patient is comatose (unaware and unresponsive)., Check (“) Item if Confirmed = UL. B. The following brainstem reflexes are absent: * Absent pupillary response to bright light * Absent comeal reflex « Absent gag and cough reflexes * Absent oculocephalic reflex * Absent deviation of eyes with cold water stimulation, Check (“) Item if Confirmed = _]. Step 4: Absence of Spontaneous Breathing Efforts: There are no spontaneous breathing efforts when the arterial PCO; Is 20 mm Hg above the patient's baseline level (positive apnea test)., Check (“) Item if Confirmed = LU. Step 5: Consider Confirmatory Tests:t Confirmatory tests may be necessary when Steps 1-4 cannot be completed or unequivocally interpreted., Check (“) Item if Confirmed = 

tConfirmatory testing can include: electroancephalography, transcranial Doppler flow study somatosensory evoked potentials, brain scan with taechnetum-99m, or cerebral angiography.
From Referancas 30-22.

The most convincing evidence of brain death is the absence of spontaneous respiratory efforts in the face of an acute increase in arterial PCOz (which is normally a potent respiratory stimulus). This is evaluated with the apnea test, which involves removing the patient from ventilatory support and observing for spontaneous breathing efforts as the arterial PCO rises. Because the apnea test can cause hypotension, hypoxemia and cardiac arrhythmias, it is typically the last test performed in the brain death determination. The following steps are involved in the apnea test: 1. Prior to the test, the patient is preoxygenated with 100% Os, and an arterial blood gas
is obtained to establish the baseline arterial PCOz (PaCO>).
2. The patient is then separated from the ventilator and oxygen is insufflated into the endotracheal tube (apneic oxygenation) to help prevent O, desaturation during the apneic period. (A pulse oximeter should be used to monitor the arterial O> saturation. )
3. The goal of the apnea test is to allow the PaCO> to rise 20 mm Hg above baseline. The PaCO, rises about 3 mm Hg per minute during apnea at normal body temperatures (33), So an apnea period of 6-7 minutes should be sufficient for achieving the target PaCO>. A repeat arterial blood gas is obtained at the end of the apnea period, and the patient is placed back on the ventilator.
4. If apnea persists despite a rise in PaCO, =20 mm Hg, the test confirms the diagnosis of brain death.
5. The apnea test is risky, and often cannot be completed because of severe O> desaturation, hypotension, or serious cardiac arrhythmias (34). If the apnea test cannot be completed, confirmatory testing may be required to establish the diagnosis of brain death.

Brain-dead patients can exhibit brief, spontaneous movements of the head, torso, or upper extremities (Lazarus' Sign), especially after they are removed from the ventilator (35). These movements are the result of neuronal discharges in the cervical spinal cord, possibly in response to hypoxemia, and they can be a source of angst when they appear after the patient has been pronounced brain dead and is removed from the ventilator.

For the potential organ donor, the following measures can be used to en-hance organ viability (36).

Potential organ donors should have a mean arterial pressure 265 mm Hg and a urine output =1 mL/kg per hour, and fluids and vasopressors should be used, if necessary, to achieve these goals. Circulatory support for the potential organ donor should follow the same principles of circulatory support used for other critically ill patients (see pages 270— 272).

More than half of patients with brain death will develop pituitary failure with diabetes insipidus and secondary adrenal insufficiency (37). Both conditions can lead to profound hypovolemia (with reduced organ perfusion) and hypertonic hypernatremia (with cell dehydration). If there is evidence of central diabetes insipidus (i.e., spontaneous diuresis with a urine osmolality below 200 mOsm/L), treatment with desmopressin, a vasopressin analog that does not cause vasoconstriction, is advised (38). The usual dose of desmopressin is 0.5-2.0 yg IV every 2-3 hours, with dose titration to maintain a urine output of 100-200 mL/hr.

In the care of the patient with persistent coma or a persistent vegetative state, spending time with the patient's family (or other intimates) is as important as patient care. These people will look to you for guidance, and avoiding the conspiracy of silence (39) is one of the greatest services you can perform as a physician.

1. Leén-Carridn J, van Eeckhout P, Dominguez-Morales Mdel R. The locked-in syndrome: a syndrome looking for a therapy. Brain Inj 2002; 16:555—569.
2. The Multi-Society Task Force on PVS. Medical aspects of the persistent vegetative state (Part 1). N Engl J Med 1994; 330:1499-1508.
3. Bleck TP, Smith MC, Pierre-Louis SJ, et al. Neurologic complications of critical medical illnesses. Crit Care Med 1993; 21:98-103.
4. Papadopoulos M, Davies D, Moss R, et al. Pathophysiology of septic encephalopathy: a review. Crit Care Med 2000; 28:3019-3024.
5. Sprung CL, Cerra FB, Freund HR, et al. Amino acid alterations and encephalopathy in the sepsis syndrome. Crit Care Med 1991; 19:753-757.
Delirium Tremens, 1 = Delirium Tremens. 17., 1 = Tetrault JM, O'Connor PG. Substance abuse and withdrawal in the critical care setting. Crit Care Clin 2008; 24:767-788.. 18., 1 = Attard O, Dietermann JL, Diemunsch P, et al. Wernicke encephalopathy: a complication of parenteral nutrition diagnosed by magnetic resonance imaging. Anesthesiology 2006; 105:847-848.. 19., 1 = Mayo-Smith MF, Beecher LH, Fischer TL, et al. Management of alcohol withdrawal delirium: an evidence-based practice guideline. Arch Intern Med 2004; 164:1405- 1412.

28., Brain Death = Uniform Determination of Death Act, 12 uniform laws annotated 589 (West 1993 and West suppl 1997).. 29., Brain Death = Smith AJ, Walker AE. Cerebral blood flow and brain metabolism as indicators of cerebral death. A review. Johns Hopkins Med J 1973; 133:107-119.. 30., Brain Death = The Quality Standards Subcommittee of the American Academy of Neur-ology. Practice parameters for determining brain death in adults (summary statement). Neurology 1995; 45:1012-1014.. 31., Brain Death = Wijdicks EFM, Varelas PNV, Gronseth GS, Greer DM. Evidence-based guideline update: determining brain-death in adults. Report of the Quality Standards Sub- committee of the American Academy of Neurology. Neurology 2010; 74:1911-1918.. 32. Wijdicks EF. The diagnosis of brain death. N Engl J Med 2001; 344:1215-1221., Brain Death = 32. Wijdicks EF. The diagnosis of brain death. N Engl J Med 2001; 344:1215-1221.. 33., Brain Death = Dominguez-Roldan JM, Barrera-Chacon JM, Murillo-Cabezas F, et al. Clinical factors influencing the increment of blood carbon dioxide during the apnea test for the diagnosis of brain death. Transplant Proc 1999; 31:2599-2600.. 34., Brain Death = Goudreau JL, Wijdicks EF, Emery SF. Complications during apnea testing in the determination of brain death: predisposing factors. Neurology 2000; 55:1045-1048.. 35., Brain Death = Ropper AH. Unusual spontaneous movements in brain-dead patients. Neurology 1984; 34:1089-1092.

The, 1 = Potential Organ Donor. 36., 1 = Wood KE, Becker BN, McCartney JG, et al. Care of the potential organ donor. N Engl J Med 2004; 351:2730-2739.. 37., 1 = Detterbeck FC, Mill MR. Organ donation and the management of the multiple organ donor. Contemp Surg 1993; 42:281-285.. 38., 1 = Guesde R, Barrou B, Leblanc I, et al. Administration of desmopressin in brain-dead donors and renal function in kidney recipients. Lancet 1998; 352:1178-1181.

39. Fallowfield LJ, Jenkins VA, Beveridge HA. Truth may hurt but deceit hurts more: communication in palliative care. Palliative Med 2002; 16:297-303.

When we contemplate this life, we see motion as its principal characteristic, and when we take a farther view, we see that this motion must necessarily waste the machine in which it resides.
John Young
Senior Dissertation
Univ. PA School of Medicine
1803
This chapter describes three types of movement disorder that you are likely to encounter in the ICU: (a) involuntary movements (i.e., seizures), (b) weak or ineffective movements (i.e., Neuromuscular weakness), and (c) no movements (i.e., drug-induced paralysis). These disorders share one common trait, which is the ability to "waste" the human machine.

Seizures are second only to metabolic encephalopathy as the most common neurological complication in critically ill patients (1). The incidence of new-onset seizures in ICU patients is 0.8-3.5% (1,2).
without an intervening period of consciousness (7). Status epilepticus can involve any type of seizure, and can be "convulsive" (i.e., associated with abnormal body movements) or "nonconvulsive" (i.e., not associated with abnormal body movements).
NONCONVULSIVE STATUS EPILEPTICUS: Most cases of nonconvulsive status epilepticus involve partial complex seizures (which are not common in ICU patients), but as many as 25% of generalized seizures can be nonconvulsive (8). Generalized nonconvulsive status epilepticus is also known as subtle status epilepticus, and typically occurs when generalized convulsive seizures have not been adequately treated (4). These seizures are associated with loss of consciousness, and they are a source of unexplained coma in the ICU. In one study, generalized nonconvulsive seizures were responsible for 8% of cases of coma in ICU patients (9). The diagnosis requires evidence of epileptiform discharges on the EEG.

A variety of conditions can produce seizures in critically ill patients, as indicated in Table 45.1. In one survey of new-onset seizures in ICUs, the most common predisposing conditions were drug intoxication, drug withdrawal, and metabolic abnormalities (e.g., hypoglycemia) (2).

Drug Toxicity Amphetamines Cocaine Tricyclics Drug Withdrawal Barbiturates Benzodiazepines Ethanol Opiates Metabolic Hypoglycemia Hypoxia Uremia Liver failure, 1 = Ischemic Stroke Cardiac arrest Traumatic Intracranial hemorrhage Intracranial hypertension Infectious Abscess Meningoencephalitis Septic Emboll Hematologic DIC TTP
tFrom Raferenca 2, DIC = disseminated intravascular coaguopathy, TTP = thrombotic thrombocytopenia purpura.

Ten percent of patients with GSE are refractory to stage 1 and 2 drugs (8). The recommended treatment at this point is anesthetic doses of one of the drugs in Table 45.3. Pentobarbital may be the favored drug in this situation (16). At this stage, a neurology consult is the best option.

than valproic acid, but the clinical experience in status epilepticus ts limited.
Dosing regimens from References 6 and 7.

The in-hospital mortality rates are as high as 21% for convulsive GSE, as high as 52% for nonconvulsive GSE, and as high as 61% for refractory GSE (7).

Pentobarbital, Dosing Regimens = Load with 5-15 mg/kg IV over one hr, then Infuse at 0.5-1 mg/kg/h. If necessary, Increase Infusion rate up to 3 mg/Kg/hr (maximum rate).. Thiopental, Dosing Regimens = Start with an IV bolus of 3-5 mg/kg, and follow with 1-2 mg/kg every 2-3 min until seizures subside. Then infuse 3-7 mg/kg/hr for the next 24 hrs.. Midazolam, Dosing Regimens = Load with 0.2 mg/kg IV, then infuse at 4-10 mg/kg/h.. Propofol, Dosing Regimens = Start with IV bolus of 2-3 mg/kg, and use further boluses of 1-2 mg/kg, if needed, until seizure activity subsides. Then infuse at 4-10 mg/kg/hr for 24 hrs.
Dosing regimens from Reference 6.

The following is a description of the acute neuromuscular weakness syndromes that you may encounter in an ICU.

Ocular weakness, 1 = Yes. Ocular weakness, 2 = No. Fluctuating weakness, 1 = Yes. Fluctuating weakness, 2 = No. Bulbar weakness, 1 = Yes. Bulbar weakness, 2 = No. Deep tendon reflexes, 1 = Intact. Deep tendon reflexes, 2 = Depressed. Autonomic instability, 1 = No. Autonomic instability, 2 = Yes. Nerve conduction, 1 = Normal. Nerve conduction, 2 = Slowed

The Guillain-Barré syndrome is a subacute inflammatory demyelinating polyneuropathy that often follows an acute infectious illness (by 1-3 weeks) (22,23). An immune etiology is suspected.

The presenting features of the Guillain-Barré syndrome include paresthesias and symmetric limb weakness that evolves over a period of a few days to a few weeks. Progression to respiratory failure occurs in 25% of cases (22), and autonomic instability can be a feature in advanced cases (24). The condition resolves spontaneously in about 80% of cases, but residual neurological deficits are common (22).

The diagnosis of Guillain-Barré syndrome is based on the clinical presentation (paresthesias and symmetric limb weakness), nerve conduction studies (slowed conduction), and cerebrospinal fluid analysis (elevated protein in 80% of cases) (22). The features that distinguish the Guillain-Barré syndrome from myasthenia gravis are shown in Table 45.4.

Treatment is mostly supportive, but in advanced cases with respiratory failure, plasmapharesis or intravenous immunoglobulin G (0.4 g/kg/day for 5 days) are equally effective in producing short-term improvement (23). Immunoglobulin G is often preferred because it is easier to carry out.

Drug-induced paralysis is used in the following situations: (a) to facilitate endotracheal intubation, (b) to prevent shivering during induced hypothermia (in comatose survivors of cardiac arrest), and (c) to facilitate mechanical ventilation in severely agitated patients (30). The latter practice is frowned upon for reasons stated later.
Neuromuscular blocking agents act by binding to acetylcholine receptors on the postsynaptic side of the neuromuscular junction. Once bound, there are two different modes of action: (a) depolarizing agents act like acetylcholine and produce a sustained depolarization of the post-synaptic membrane, and (b) non-depolarizing agents act by inhibiting depolarization of the post-synaptic membrane.

The comparative features of three commonly used neuromuscular blocking agents are shown in Table 45.5 (31).

IV Bolus Dose, ‘Succinylcholine = 1 mg/kg. IV Bolus Dose, Rocuronium = 0.6 mg/kg. IV Bolus Dose, —_Cisatracurium = 0.1 mg/kg. Onset Time, ‘Succinylcholine = 1-1.5 min. Onset Time, Rocuronium = 1.5-3 min. Onset Time, —_Cisatracurium = 5-7 min. Recovery Time, ‘Succinylcholine = 10-12 min. Recovery Time, Rocuronium = 380-40 min. Recovery Time, —_Cisatracurium = 40-5 min. Infusion Rate, ‘Succinylcholine = —. Infusion Rate, Rocuronium = 5-10 pg/kg/min. Infusion Rate, —_Cisatracurium = §=2-5 pg/kg/min. Cardiovascular Effects, ‘Succinylcholine = Bradycarcdia. Cardiovascular Effects, Rocuronium = . Cardiovascular Effects, —_Cisatracurium = . Effects of Renal or Liver Failure, ‘Succinylcholine = . Effects of Renal or Liver Failure, Rocuronium = Prolonged effect in liver failure. Effects of Renal or Liver Failure, —_Cisatracurium = 
From Reaferance 2.

Succinylcholine is a depolarizing agent with a rapid onset of action (60-90 seconds) and a rapid recovery time (10-12 minutes). Because of these features, succinylcholine is used to facilitate endotracheal intubation.
SIDE EFFECTS: Succinylcholine-induced depolarization of skeletal muscle promotes the efflux of potassium from muscle cells. This can be associated with a 0.5 mEq/L rise in the serum potassium (32), but this effect is transient and without consequence. However, life-threatening hyperkalemia can occur when succinylcholine is given to patients with skeletal muscle "denervation injury" (e.g., from head or spinal cord injury), or patients with rhabdomyolysis, burns, or chronic immobility. As a result, succinylcholine is not advised for patients with these conditions.

Rocuronium is a non-depolarizing neuromuscular blocker with a rapid onset of action (1.5-3 minutes) and an "intermediate" recovery time (30-40 min). Because of the rapid onset of action, rocuronium can be used for endotracheal intubation when succinylcholine is not advised. However, larger doses (1 mg/kg) are required for intubation, and this will prolong the recovery time (31). Rocuronium can be infused at a rate of 5-10 eg/kg/min for prolonged neuromuscular paralysis. The drug is well tolerated, and has no cardiovascular side effects. Rocuronium has gradually replaced a related neuromuscular blocker, vecuronium, be-cause of its rapid onset of action.

Cisatracurium is a non-depolarizing agent with a prolonged onset of action (5-7 min) and an "intermediate" recovery time. It is an isomer of atracurium (another neuromuscular blocker), and was developed to eliminate the histamine release associated with atracurium. Cisatracur-ium can be infused at a rate of 2-5 eg/kg/min for prolonged neuromuscular paralysis, and the drug is well suited for ICU patients because blood levels are not influenced by renal or liver dysfunction (31).

The standard method of monitoring drug-induced paralysis is to apply a series of four low-frequency (2 Hz) electrical pulses to the ulnar nerve at the forearm, and observe for adduction of the thumb. Total absence of thumb adduction is evidence of excessive block. The desired goal is 1 or 2 perceptible twitches, and the drug infusion is adjusted to achieve that end-point (30).

The experience of being awake while paralyzed is both horrifying and painful (33), and it is imperative to keep patients heavily sedated while they are paralyzed. However, it is not possible to evaluate the adequacy of sedation or pain control while a patient is paralyzed. The inability to insure adequate sedation and pain control is the major reason
to avoid drug-induced paralysis whenever possible. Avoiding prolonged periods of neuromuscular paralysis will also reduce the risk of the following complications:
1. Critical illness myopathy (explained earlier).
2. "Hypostatic" pneumonia (from pooling of respiratory secretions in dependent lung regions).
3. Venous thromboembolism (from prolonged immobilization).
4. Pressure ulcers on the skin (also from prolonged immobilization).

One of the central themes in this book is the widespread harm inflicted by progressive systemic inflammation in critically ill patients. Inflam-matory injury is responsible for the acute respiratory distress syndrome (see Chapter 23) and acute kidney injury (see Chapter 34), as well as the multiorgan failure associated with septic shock (see Chapter 14). In this chapter, we learn that inflammation can also damage peripheral nerves (i.e., critical illness polyneuropathy) and skeletal muscle (i.e., critical illness myopathy). These injuries support the notion that inflammation is the most lethal force that you will face in the ICU.

22. Hughes RA, Cornblath DR. Guillain-Barré syndrome. Lancet 2005; 366:1653-1666.
23. Hund EF, Borel CO, Cornblath DR, et al. Intensive management and treatment of severe Guillain-Barré syndrome. Crit Care Med 1993; 21:433-446.
24. Pfeiffer G, Schiller B, Kruse J, et al. Indicators of dysautonomia in severe GuillainBarré syndrome. J Neurol 1999; 246:1015-1022.
Drug-Induced Paralysis, 1 = Drug-Induced Paralysis. 30., 1 = Murray MJ, Cowen J, DeBlock H, et al. Clinical practice guidelines for sustained neuromuscular blockade in the adult critically ill patient. Crit Care Med 2002; 30:142-156.. 31., 1 = Donati F, Bevan DR. Neuromuscular blocking agents. In: Barash PG, Cullen BF, Stoelting RK, et al, eds. Clinical Anesthesia. 6th ed. Philadelphia: Lippincott Williams & Wilkins, 2009:498-530.. 32., 1 = Koide M, Waud BE. Serum potassium concentrations after succinylcholine in patients with renal failure. Anesthesiology 1972; 36:142-145.. 33., 1 = Parker MM, Schubert W, Shelhamer JH, et al. Perceptions of a critically ill patient experiencing therapeutic paralysis in an ICU. Crit Care Med 1984; 12:69-71.

Disease makes men more physical, it leaves them nothing but body.
Thomas Mann
The focus of this chapter is a cerebrovascular disorder that was first described over 2,400 years ago, and since then has suffered through a variety of inappropriate names like apoplexy, cerebrovascular accident (what accident?), stroke, and the most recent embarrassment, brain attack. Considering that this disorder is responsible for one death every 4 seconds in the United States (1), it deserves a better name.
This chapter describes the initial evaluation and management of acute stroke, with an emphasis on the recommendations for thrombolytic therapy in acute stroke from the American Heart Association (2).

Stroke is defined as "an acute brain disorder of vascular origin accompanied by neurological dysfunction that persists for longer than 24 hours" (3). The neurological dysfunction is usually localized or focal (which is typical of vascular occlusion); however, global dysfunction can occur when vascular rupture leads to hemorrhage and mass effect.

Stroke is classified according to the responsible mechanism.
1. Ischemic stroke accounts for 87% of all strokes (1): 80% of ischemic strokes are thrombotic strokes, and 20% are embolic strokes. Most emboli originate from thrombi in the left atrium (from atrial fibrillation) or left ventricle (from acute MI), but some originate from venous thrombi in the legs that reach the brain through a patent foramen ovale (4).
2.Hemorrhagic stroke accounts for 13% of all strokes: 97% of hemorrhagic strokes involve intracerebral hemorrhage, and 3% are the result of subarachnoid hemorrhage (1).

A transient ischemic attack (TIA) is an acute episode of ischemia with focal loss of brain function that lasts less than 24 hours (3). The feature that distinguishes TIA from stroke is the reversibility of clinical symptoms in TIAs. This does not apply to reversibility of cerebral injury, because one-third of TIAs are associated with cerebral infarction (5,6).

The evaluation of a patient with suspected acute stroke must proceed quickly. Each minute of cerebral infarction results in the destruction of 1.9 million neurons and 7.5 miles of myelinated nerves (7), and continued tissue destruction eventually leads to a point where reperfusion of occluded arteries with thrombolytic therapy will not promote neurological recovery. This point occurs 4—5 hours after stroke onset, where the benefit from thrombolytic therapy is lost.

Stroke has traditionally been a clinical diagnosis (although diffusion-weighted MRI will change this, as described later), and the area of brain that is injured determines the clinical presentation. Figure 46.1 shows the clinical features of stroke in relation to the area of brain that is damaged. Some of the notable manifestations of acute stroke are described next.

Most cerebral infarctions are unilateral, and do not result in loss of consciousness (8). When focal neurological deficits are accompanied by coma, the most likely conditions are intracerebral hemorrhage, brainstem infarction, or nonconvulsive seizures.
APHASIA: Injury in the left cerebral hemisphere (which is the dominant hemisphere for speech in 90% of patients) produces aphasia, which is a disturbance in the comprehension and/or formulation of language. Pa-tients with aphasia can have difficulty with verbal comprehension (receptive aphasia), or difficulty with verbal expression (expressive aphasia), or both (global aphasia).

The hallmark of injury involving one cerebral hemisphere is weakness on the opposite or contralateral side of the face and body (i.e., hemiparesis). The presence of hemiparesis or isolated limb weakness creates a high index of suspicion for stroke (or TIA), but focal limb weakness can be the result of nonconvulsive status epilepticus, and hemiparesis has been reported in patients with hepatic and septic encephalopathy (9,10).
FIGURE 46.1 Areas of brain injury and corresponding neurological abnormalities. Indicates deficits involving the same side of the face and the opposite side of the body.

For patients admitted to the hospital with a suspected stroke based on clinical findings, as many as 30% of the patients will have another condition that mimics an acute stroke (11). The most common stroke mimics are seizures, sepsis, metabolic encephalopathies, and space-occupying lesions (in that order) (11). Since stroke is primarily a clinical diagnosis, at least in the first 24-48 hours, stroke mimics are a source of excessive hospital admissions (and thrombolytic therapy) for suspected stroke.

The use of a clinical scoring system is recommended to standardize the evaluation of acute stroke (2), and the most validated scoring system is the NIH Stroke Scale (NIHSS). The NIHSS evaluates 11 different aspects of performance, and rates each performance with a number from zero to 3 or 4. The total score is a measure of the severity of illness, and ranges from zero (best performance) to 41 (worst performance). A score of 22 or higher generally indicates a poor prognosis. Trained personnel can complete the NIHSS in less than 5 minutes, and the score can be used to assess the likelihood of an acute stroke (i.e., a stroke is unlikely if the score is 10 or lower) and to follow the clinical course of the
illness. (The NIHSS can be downloaded from http://stroke.nih.gov/documents.)

The imaging techniques described next have become an integral part of the stroke evaluation, and each technique has a specific role in the evaluation.

Noncontrast computed tomography (NCCT) is a reliable method for visualizing intracranial hemorrhage, as shown in Figure 46.2. This reliability is particularly important in the decision to administer thrombolytic therapy, which is contraindicated if NCCT reveals intracranial bleeding. The sensitivity of NCCT for intracranial hemorrhage is close to 100% (5).
NCCT is not a reliable method for visualizing ischemic changes. One-half of ischemic strokes are not apparent on NCCT (12), and the diagnostic yield is even less in the first 24 hours after an acute stroke (when infarct size is the smallest) (13). The nonvalue of CT imaging in the early post-infarct period is demonstrated in Figure 46.3 (13). The CT image on day 3 shows a large area of infarction with mass effect, which is not apparent in the CT image on day 1 (the day of the stroke). These images demonstrate why, in the initial evaluation of suspected stroke, a negative CT scan does not eliminate the possibility of an ischemic stroke.
FIGURE 46.2 Noncontrast CT scan showing a high-density mass with adjacent low-density areas in the left hemisphere representing hematoma with adjacent areas of edema. CT is a reliable method for visualizing intracranial hemorrhage.
FIGURE 46.3 Noncontrast CT scans from the first and third day following an ischemic stroke. The CT scan on day 1 is unrevealing, while the CT scan on day 3 shows a large hypodense area (outlined by the dotted line) with mass effect, representing extensive tissue destruction with intracerebal edema. Images from Reference 13.

MRI with diffusion-weighted imaging (DWI) is the most sensitive and specific technique for the detection of ischemic stroke (2). This technique, which is based on water movement through tissues, can detect ischemic changes within 5-10 minutes after onset (14), and it has a sensitivity of 90% for the detection of ischemic stroke in the early period after stroke onset (5). The appearance of diffusion-weighted MRI in ischemic stroke is shown in Figure 46.4 (15). The image on the left shows a large, hyperdense area representing ischemic changes. (This differs from CT scans, which show ischemic changes as hyopodense areas.) The image on the right is a time-delay technique that detects regions of hypoperfusion using the adjacent color palette. If the ischemic areas on the DWI image are digitally subtracted from the areas of hypoperfusion in the timedelay image, the remaining colored areas on the time-delay map would represent areas of threatened infarction. This digital subtraction technique allows an assessment of continued risk in patients with acute ischemic stroke.

When the initial evaluation identifies a patient with suspected acute stroke, the next step is to determine if the patient is a candidate for thrombolytic therapy.

The selection criteria for thrombolytic therapy in ischemic stroke are presented as a checklist in Table 46.1. Some comments about the criteria are presented next.

The use of thrombolytic therapy in ischemic stroke was prompted by a single study (16), which showed that a 60-minute infusion of recombinant tissue plasminogen activator (tPA) was associated with improved neurologic recovery (not survival), but only when the drug infusion started within 3 hours after the onset of symptoms. The FDA subsequently approved the use of tPA in ischemic stroke (in 1996), but with the restriction that drug administration must be started within 3 hours of symptom onset. This 3-hour time restriction has limited the use of thrombolytic therapy in ischemic stroke; i.e., surveys indicate that only 2% of patients with ischemic stroke receive thrombolytic therapy (17).
able 46.1 Checklist for Thrombolytic Therapy in Ischemic Stroke

yw Time of symptom onset can be identified accurately.
y Thrombolytic therapy can be started within 4.5 hrs of symptom onset. lf both boxes are checked, proceed to Step 2.

|_J Evidence of active bleeding
|_J Systolic pressure = 185 mm Hg or diastolic pressure = 110 mm Hg
|_J History of previous intracranial hemomhage
|_J Intracranial neoplasm, aneurysm, or AV malformation
|_J intracranialntraspinal surgery, serious head trauma, or stroke in past 3 months
|_J Thrombin inhibitor or factor Xa Inhibitor in past 2 days
|_J Laboratory evidence of a coagulopathy (e.g., platelets <100,000/uL)
|_J Blood glucose <50 mg/dL (2.7 mmol/L)
|_J CT scan shows multilobar infarction (hypodense area >1/s cerebral hemispheres)
If no boxes are checked, proceed to Step 3.

|_J Major surgery or serious trauma in past 14 days
‘_J GI or urinary tract bleeding within past 21 days
|_J Acute MI within past 3 months
|_J Seizure since onset with continued postictal state
Additional criteria for thrombolytic therapy at S—4.5 hrs after symptom onset:
|_J Age >80 years
|_] Oral anticoagulant therapy, regardless of INR
|_] Severe stroke (NIHSS +25)
|_] Diabetes plus a previous stroke
Proceed to Step 4 when no boxes are checked, or when 1 or more boxes are checked, but a risk-benefit analysis favors thrombolytic therapy.
Step 4: Thrombolytic Therapy (begin immediately)
From Referanca 2.
EXPANDED TIME LIMIT: A more recent clinical study has shown that thrombolytic therapy started between 3 and 4.5 hours after the onset of ischemic stroke also improves neurologic recovery (18). Based on these results, the time for initiating thrombolytic therapy has recently been expanded to 4.5 hours after the onset of ischemic stroke (2). There are additional exclusion criteria for the expanded time limit, and these are included in Table 46.1.
Why adhere to these time restrictions? Because about 6% of patients who receive thrombolytic therapy for ischemic stroke will suffer from an intracerebral hemorrhage, so evidence of benefit is necessary to justify this therapy.

The time restriction for thrombolytic therapy makes it imperative to pinpoint the time when the stroke began (i.e., became symptomatic). This can be difficult, because patients are unable to provide a reliable history and, in many cases, the onset of

One of the exclusion criteria for thrombolytic therapy is an elevated blood pressure; i.e., a systolic pressure >185 mm Hg, or a diastolic pressure >110 mm Hg (see Table 46.1). If the patient is otherwise a candidate for thrombolytic therapy, the blood pressure can be lowered to qualify for thrombolytic therapy using the drug regimens shown in Table 46.2 (1). The methods and concerns with blood pressure reduction in acute stroke are described in the last section of the chapter. If the blood pressure reduction is successful and the patient receives thrombolytic therapy, the blood pressure should be maintained at <180/105 for the next few days to limit the risk of intracranial hemorrhage.

tSBP >185 mm Hg or DBP >=110 mm Hg, Drugs & Dosing Regimens — = Labetalol: 10-20 mg IV over 1-2 min. Can repeat once after 10 min. Nicardipine: Infuse at 5 mogvhr, and increase by 2.5 mg/hr every 5-15 min, if necassary, up to 15 mg/hr.. SBP >220 mm Hg or DBP >120 mm Hg, Drugs & Dosing Regimens — = Labetalol: 10mg IV bolus, then infuse at 2-5 ma/min. Nicardipine: Infuse at 5 mgvhr, and increase by 2.5 mg/hr every 5-15 min, if necessary, up to 15 mg/hr.. DBP>140 mmHg _, Drugs & Dosing Regimens — = Nitroprusside: Infuse at 0.2 ug/kg/min and titrate to desired effect.
tBlood pressure reduction to allow thrombolytic therapy.
Adapted fram Reference 2.

Several studies have failed to show a beneficial effect of heparin anticoagulation in ischemic stroke (2). This lack of benefit, combined with the risks associated with heparin (i.e., bleeding and thrombocytopenia) is the reason that heparin anticoagulation is not recommended in ischemic stroke (2). The only recommended use of heparin in acute stroke is for prevention of thromboembolism (2).

Despite the apparent lack of benefit, aspirin therapy is recommended as a routine measure in ischemic stroke (2). The initial dose is 325 mg (oral), which is given 24—48 hours after stroke onset (or after thrombolytic therapy), and the daily maintenance dose is 75-150 mg (2). Additional antiplatelet agents are not recommended.

The measures described in this section are designed to protect the brain from further injury following an acute stroke.

Hypertension is reported in 60-65% of patients with acute stroke (21), and is attributed to several factors, including activation of the sympathetic nervous system, cerebral edema, and a prior history of hypertension. Blood pressures usually return to baseline levels in 2-3 days. Patients with stroke-associated hypertension have more extensive neurologic def-icits, but blood pressure reduction is not routinely recommended (2). The indications for blood pressure reduction include a systolic pressure >220 mm Hg, a diastolic blood pressure >120 mm Hg, or a complication of hypertension (e.g., acute MI).

Table 46.2 shows the recommended drugs and dosing regimens for blood pressure reduction in patients with acute stroke. Labetalol (a combined 8, a-adrenergic receptor antagonist) and nicardipine (a calcium channel blocker) share the ability to decrease blood pressure while preserving cardiac output (and cerebral blood flow). Labetalol is probably the preferred drug because it does not cause tachycardia, but there are no studies comparing these drugs for blood pressure control in acute stroke. Sodium nitroprusside is recommended for severe hypertension (diastolic BP >140 mm Hg) (2), but nitroprusside infusions are accompanied by an increase in intracranial pressure (22), which is not a desirable condition in the patient with ischemic brain injury.

Fever develops within 48 hours in 30% of patients with acute stroke (2), and the presence of fever is associated with worse clinical outcomes (23).

Fever typically appears within 48 hours after stroke onset (24), which suggests a noninfectious origin (e.g., from tissue necrosis or intracerebral blood). However, some studies have found infections in a majority of patients with stroke-related fever (25). Therefore, stroke-related fever should be evaluated as potentially infectious in origin.

There is convincing evidence from animal studies that fever is harmful for ischemic brain tissue (27), and thus antipyretic therapy is justified for stroke-related fever. Antipyretic therapy is described in Chapter 43.

The success of thrombolytic therapy in coronary occlusive syndromes expectations for thrombolytic therapy in acute, ischemic stroke, and these prompted a massive effort to create 'stroke centers' in major hospitals, 'stroke team' to direct the management of acute stroke. The following is an of what this effort has accomplished., 1 = created high expectations each with a accounting. Number of strokes each year in the United States, 1 = 700,000. Number of ischemic strokes (88% ), 1 = 616,000. Number of stroke patients who receive lytic therapy (2%), 1 = 12,320. Number of patients who benefit from lytic therapy (1 in 9), 1 = 1,369. Percent of strokes that benefit from lytic therapy (1369/700,000) 0.2%, 1 = Percent of strokes that benefit from lytic therapy (1369/700,000) 0.2%. Enough said., 1 = 

- 1. Go AS, Mozaffarian D, Roger VL, et al. Heart disease and stroke statistics — 2013 update: A report from the American Heart Association. Circulation 2013; 127:e6— e245.

2. Jauch EC, Saver JL, Adams HP, et al. Guidelines for the early management of patients with acute ischemic stroke. A guideline for healthcare professionals from The American Heart Association/American Stroke Association. Stroke 2013; 44:1-78.
Thrombolytic Therapy, 1 = Thrombolytic Therapy. 16., 1 = Tissue plasminogen activator for acute ischemic stroke. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. N Engl J Med 1995; 333:1581-1587.. 17., 1 = Caplan LR. Thrombolysis 2004: the good, the bad, and the ugly. Rev Neurol Dis 2004; 1:16-26.. 18. Hacke W, Kaste M, Bluhmki E, et al. Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke. N Engl J Med 2008; 359:1317-1329., 1 = 18. Hacke W, Kaste M, Bluhmki E, et al. Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke. N Engl J Med 2008; 359:1317-1329.

, 1 = Protective Measures. 19., 1 = Ronning OM, Guldvog B. Should stroke victims routinely receive supplemental oxygen. A quasi-randomized controlled trial. Stroke 1999; 30:2033-2037.. 20., 1 = Kety SS, Schmidt CF. The effects of altered tensions of carbon dioxide and oxygen on cerebral blood flow and cerebral oxygen consumption of normal young men. J Clin Invest 1984; 27:484-492.. 21., 1 = Qureshi AI, Ezzeddine MA, Nasar A, et al. Prevalence of elevated blood pressure in 563,704 adult patients with stroke presenting to the ED in the United States. Am J Emerg Med 2007; 25:32-38.. 22., 1 = Candia GJ, Heros RC, Lavyne MH, et al. Effect of intravenous sodium nitroprusside on cerebral blood flow and intracranial pressure. Neurosurgery 1978; 3:50-53.. 23., 1 = Reith J, Jorgensen HS, Pedersen PM, et al. Body temperature in acute stroke: relation to stroke severity, infarct size, mortality, and outcome. Lancet 1996; 347:422-425.. 24., 1 = Wrotek SE, Kozak WE, Hess DC, Fagan SC. Treatment of fever after stroke: conflicting evidence. Pharmacotherapy 2011; 31:1085-1091.. 25., 1 = Grau AJ, Buggle F, Schnitzler P, et al. Fever and infection early after ischemic stroke. J Neurol Sci 1999; 171:115-120.. 26., 1 = Baena RC, Busto R, Dietrich WD, et al. Hyperthermia delayed by 24 hours aggravates neuronal damage in rat hippocampus following global ischemia. Neurology 1997; 48:768-773.. 27., 1 = Sulter G, Elting JW, Mauritis N, et al. Acetylsalicylic acid and acetaminophen to combat elevated temperature in acute ischemic stroke. Cerebrovasc Dis 2004; 17:118-122.

The more impure bodies are fed, the more diseased they will become.
Hippocrates Aphorisms

What is food to one man, may be fierce poison to others.
Lucretius
(99-55 BC)
The fundamental goal of nutritional support is to provide the daily nutrient and energy needs of each patient. This chapter will describe how to evaluate those needs in critically ill patients (1), and will try to do so without pretending that anyone knows how to support metabolism in patients who are critically ill.

Oxidative metabolism captures the energy stored in nutrient fuels (carbohydrates, lipids, and proteins) and uses this energy to sustain life. This process consumes oxygen, and generates carbon dioxide, water, and heat. The quantities involved in the oxidation of each type of nutrient fuel are shown in Table 47.1. The following points deserve mention.
1. The heat generated by the complete oxidation of a nutrient fuel is equivalent to the energy yield (in kcal/g) of that fuel.
2. Lipids have the highest energy yield (9.1 kcal/gram), while glucose has the lowest energy yield (3.7 kcal/gram).
The summed metabolism of all three nutrient fuels determines the whole-body O> consumption (VO), COs production (VCO>), and heat production for any given time period. The 24-hour heat production is equivalent to the daily energy expenditure (in kcal) for each patient. The daily energy expenditure determines how many calories to provide each day in nutritional support, and it can be calculated or measured.

Glucose, ©, Consumption = 0.74 Lig. Glucose, CO, Production = 0.74 Lig. Glucose, Heat Productiont = 3.7 keal/g. Lipid, ©, Consumption = 2.00 L/g. Lipid, CO, Production = 1.40 Lig. Lipid, Heat Productiont = 9.1 keal/g. Protein, ©, Consumption = 0.96 L/g. Protein, CO, Production = 0.78 Lig. Protein, Heat Productiont = 4.0 keal/g
tTha anergy yield from each nutrient fusl.

It is not possible to measure metabolic heat production in hospitalized patients, but if the whole-body Oz consumption (VO) and CO, production (VCO ) are known, the relationships in Table 47.1 can be used to determine the metabolic heat production. This is the principle of indirect calorimetry, which measures the resting energy expenditure (REE) using the following relationships (2):
REE (keal/'min) = (3.6 x VO.) + (1.1 x VCO,) -61
(47.1)

Indirect calorimetry is performed with "metabolic carts" that measure whole-body VO> and VCO, at the bedside by measuring the concentrations of O2 and CO) in inhaled and exhaled gas (usually in intubated pa-tients). Steady-state measurements are obtained for 15-30 minutes to determine the REE (kcal/min), which is then multiplied by 1,440 (the number of minutes in 24 hours) to derive the daily energy expenditure (kcal/24 hr) (3). Clinical studies have shown that REE measurements obtained over 30 minutes and extrapolated to 24 hours are equivalent to REE measurements performed for 24 hours (4). The oxygen sensor in metabolic carts is not reliable at O2 concentrations above 60% (3), so indirect calorimetry can be unreliable when inhaled O> concentrations are >60%.
Although indirect calorimetry is the most accurate method for determining daily energy requirements, it requires expensive equipment along with trained personnel, and is not universally available. As a result, daily energy requirements are usually estimated, as described next.

There are more than 200 cumbersome equations available for estimating daily energy requirements (1), but none is considered more predictive than the following relationship:
REE (keal/day) = 25 x body weight (kg)
(47.2)
This simple predictive relationship is remarkably accurate in most ICU patients (5) and is considered suitable for estimating daily energy requirements in the ICU (1). Actual body weight is used unless it is 25% higher than ideal body weight. When actual weight is more than 125% of ideal weight, the adjusted weight (wt) can be used, as determined by the following equation (6):
Adjusted wt (kg) = [(actual -ideal) wt x 0.25] + ideal wt
(47.3)

The daily energy requirement is provided by nonprotein calories from carbohydrates and lipids, while protein intake is used to maintain essential enzymatic and _ structural proteins.

Standard nutrition regimens use carbohydrates to provide about 70% of the nonprotein calories. The human body has limited carbohydrate stores (Table 47.2), and daily intake of carbohydrates is necessary to ensure proper functioning of the central nervous system, which relies heavily on glucose as a nutritive fuel. However, excessive carbohydrate intake promotes hyperglycemia, which has several deleterious effects, including impaired immune responsiveness in leukocytes (7).

Adipose Tissue Fat, 1 = 15.0. Adipose Tissue Fat, 2 = 141,000. Muscle Protein, 1 = 6.0. Muscle Protein, 2 = 24,000. Total Glycogen, 1 = 0.08. Total Glycogen, 2 = 900. , 1 = . , 2 = Total: 165,900
Data from Cahill GF. Jr N Eng J Mad 1970; 282:668-875.

Standard nutrition regimens use lipids to provide approximately 30% of the daily energy needs. Dietary lipids have the highest energy yield of the three nutrient fuels (Table 47.1), and lipid stores in adipose tissues represent the major endogenous fuel source in healthy adults (Table 47.2).

Dietary lipids are triglycerides, which are composed of a glycerol molecule linked to three fatty acids. The only dietary fatty acid that is considered essential (i.e., must be provided in the diet) is linoleic acid, a long chain, polyunsaturated fatty acid with 18 carbon atoms (8). A deficient intake of this essential fatty acid produces a clinical disorder characterized by a scaly dermopathy, cardiac dysfunction, and increased susceptibility to infections (8). This disorder is prevented by providing 0.5% of the dietary fatty acids as linoleic acid. Safflower oil is used as the source of linoleic acid in most nutritional support regimens.

Propofol, an intravenous anesthetic agent that is popular for short-term sedation in the ICU, is mixed in a 10% lipid emulsion very similar to 10% Intralipid (Baxter Healthcare) that provides 1.1 kcal/mL. As a result, the calories provided by propofol infusions must be considered when calculating the nonprotein calories in a nutrition support regimen (1).

The daily protein requirement is dependent on the rate of protein catabolism. The normal daily protein intake is 0.8-1 grams/kg, but in ICU patients, the daily protein intake is higher at 1.2-1.6 grams/kg because of hypercatabolism (9).

The adequacy of protein intake can be evaluated with the nitrogen balance; i.e., the difference between intake and excretion of protein-derived nitrogen.
- 1. Nitrogen Excretion: Two-thirds of the nitrogen derived from protein breakdown is excreted in the urine (8), and about 85% of this nitrogen is contained in urea (the remainder is in ammonia and creatinine). Thus, the urinary urea nitrogen (UUN), measured in grams excreted in 24 hours, represents the bulk of nitrogen derived from protein breakdown. The remainder of the protein-derived nitrogen (usually about 4-6 grams/day) is excreted in the stool. Therefore, protein-derived nitrogen excretion can be expressed as follows:
Nitrogen Excretion (2 /24h) = UUN + (4-6)
(47.4)
If the UUN is greater than 30 g/24 h, 6 grams is more appropriate for the estimated nonurinary nitrogen losses (10). In the presence of diarrhea, non-urinary nitrogen losses cannot be estimated accurately, and nitrogen balance determinations are unreliable. Intake: each of 1/6.25
- 2. Nitrogen Protein is 16% nitrogen, so gram protein contains grams of nitrogen. Therefore, protein-derived nitrogen intake is derived as follows:
Nitrogen Intake (¢/24 h) = Protein Intake (g/24h) /6.25
(47.5)
- 3. Nitrogen Balance: The equations for nitrogen intake and nitrogen excretion are combined to determine the daily nitrogen balance.
Nitrogen Balance (g//24h) = Protein Intake 6.25 — [UUN + (4-6)]
(47.6)
The goal of nutritional support is a positive nitrogen balance of 4—6 grams.
FIGURE 47.1 Relationship between nitrogen balance and the daily intake of nonprotein calories relative to daily calorie requirements. Protein intake is constant. REE = resting energy expenditure.

The first step in achieving a positive nitrogen balance is to provide enough nonprotein calories to spare proteins from being degraded to provide energy. This is demonstrated in Figure 47.1. When the daily protein intake is constant, the nitrogen balance becomes positive only when the intake of nonprotein calories is sufficient to meet the daily energy needs (i.e., the REE). Therefore, increasing protein intake will not achieve a positive nitrogen balance unless the intake of nonprotein calories is adequate.

Thirteen vitamins are considered an essential part of the daily diet, and Table 47.3 shows the recommended daily dose and the maximum tolerable daily dose of these vitamins. The daily vitamin requirements have not been identified in critically ill patients (and probably vary with each patient) but they are likely to be higher than the recommended daily doses in Table 47.3. This is supported by reports of vitamin deficiencies in hospitalized patients who were receiving daily vitamin supplementation (11,12). Two vitamin deficiencies that deserve mention are described next.

Thiamine (vitamin B,) plays an essential role in carbohydrate metabolism, where it serves aS a coenzyme (thiamine pyrophosphate) for pyruvate dehydrogenase, the enzyme that allows pyruvate to enter mitochondria and undergo oxidative metabolism to generate high-energy ATP molecules (13). Thiamine deficiency can thus have an adverse effect on cellular energy production, particularly in the brain, which relies heavily on glucose metabolism.

Vitamin A, “pally intake = 900 yg. Vitamin A, ally Intake = 3,000 jug. Vitamin By», “pally intake = 2 ug. Vitamin By», ally Intake = 5 ug. Vitamin C, “pally intake = 90 mg. Vitamin C, ally Intake = 2,000 mg. Vitamin DB, “pally intake = 15 yg. Vitamin DB, ally Intake = 100 pg. Vitamin E, “pally intake = 15 mg. Vitamin E, ally Intake = 1,000 mg. Vitamin K, “pally intake = 120 ug. Vitamin K, ally Intake = ND. Thiamine (By), “pally intake = 1 mg. Thiamine (By), ally Intake = ND. Riboflavin (B.), “pally intake = 1 mg. Riboflavin (B.), ally Intake = ND. Niacin (Ba), “pally intake = 16 mq. Niacin (Ba), ally Intake = 35 mg. Pyridoxine (Bg), “pally intake = 2mg. Pyridoxine (Bg), ally Intake = 100 mg. Pantothenic acid (B,), “pally intake = 5 mg. Pantothenic acid (B,), ally Intake = ND. Biotin, “pally intake = 30 ag. Biotin, ally Intake = ND. Folate, “pally intake = 400 .g. Folate, ally Intake = 1,000 jug
Intakes for adult males, age 51-70 yrs. From the Food & Nutrition Board, Institute of Medicine. Available at the Food & Nutrition Information Centar (htto."inic.nal.usda.gov), Accessed July 2013. Doses rounded off to the nearest whole number. ND = not determined.

The prevalence of thiamine deficiency in ICU patients is not known, but the are several conditions in ICU patients that promote thiamine deficiency, including alcoholism, hypermetabolic states like trauma (14), increased urinary thiamine excretion by furosemide (15), and magnesium depletion (16). Furthermore, thiamine is degraded by sulfites (used as preservatives) in parenteral nutrition solutions (17), so thiaminecontaining multivitamin preparations should not be mixed with parenteral nutrition solutions.

The consequences of thiamine deficiency include cardiomyopathy (wet beriberi), Wernicke's encephalopathy (18), lactic acidosis (19), and a peripheral neuropathy (dry beriberi). Cardiomyopathies, encephalo-pathies, and lactic acidosis are common in ICU patients, and the possible contribution of thiamine deficiency to these conditions should not be overlooked.

The laboratory evaluation of thiamine status is shown in Table 47.4. Plasma levels of thiamine can be useful in detecting thiamine depletion, but the most reliable measure of functional thiamine stores is the erythrocyte transketolase assay (21). This assay measures the activity of a thiamine pyrophosphate-dependent (transketolase) enzyme in the patient's red blood cells in response to the addition of thiamine pyrophosphate (TPP). An increase in enzyme activity of greater than 25% after the addition of TPP indicates functional thiamine deficiency.

, Thiamine Fraction = Total. , Normal Range = 3.4-4.8 pg/dl. , Thiamine Fraction = Free. , Normal Range = 0.8-1.1 pg/dL. , Thiamine Fraction = Phosphorylated. , Normal Range = 2.6-3.7 wg/db. Erythrocyte Transketolase Activityt, Thiamine Fraction = Erythrocyte Transketolase Activityt. Erythrocyte Transketolase Activityt, Normal Range = Erythrocyte Transketolase Activityt. Enzyme activity measured in response to thiamine pyrophosphate (TPP) 1. <20% increase in activity after TPP indicates normal thiamine levels., Thiamine Fraction = Enzyme activity measured in response to thiamine pyrophosphate (TPP) 1. <20% increase in activity after TPP indicates normal thiamine levels.. Enzyme activity measured in response to thiamine pyrophosphate (TPP) 1. <20% increase in activity after TPP indicates normal thiamine levels., Normal Range = Enzyme activity measured in response to thiamine pyrophosphate (TPP) 1. <20% increase in activity after TPP indicates normal thiamine levels.
tFrom Raferenca 24.

Vitamin E is the major lipid-soluble antioxidant in the body, and plays a major role in preventing damage from lipid peroxidation in cell membranes (22). The incidence of vitamin E deficiency in ICU patients is not known, but vitamin E deficiency is common during parenteral nutrition (23). The reperfusion injury that follows aortic cross-clamping is associated with reduced blood levels of vitamin E, and pre-treatment with vitamin E ameliorates this reperfusion injury (24). Considering that oxidant stress plays an important role in the pathogenesis of inflammatory-mediated organ injury (25), attention to the status of vitamin E in critically ill patients seems warranted. The normal plasma concentration of vitamin E is 11.6-30.8 pmol/L (0.5-1.6 mg/dL) (26).

A trace element is a substance that is present in the body in amounts less than 50 ug per gram of body tissue (27). Seven trace elements are considered essential in humans (i.e., are associated with deficiency syndromes), and these are listed in Table 47.5 , along with the recommended and maximum daily intake for each element. As mentioned for vitamins, the essential trace element requirements are not known in critically ill patients, and are probably higher than normal. The following trace elements deserve mention because of their relevance to oxidant cell injury.

Chromium, 1 = 30 Hg. Chromium, 2 = ND. Copper, 1 = 900 jug. Copper, 2 = 10,000 jg. ledine, 1 = 150 pg. ledine, 2 = 1,100 pg. lron, 1 = 8 mg. lron, 2 = 45 mg. Manganese, 1 = 2.3 mg. Manganese, 2 = 11 mg. Selenium, 1 = 55 wg. Selenium, 2 = 400 pg. Zinc, 1 = 414mg. Zinc, 2 = 40 mg
Intakes for adult males, 51-70 yrs. From the Food & Nutrition Board, Institute of Medicine. Available at the Food & Nutrition Information Center (http:/Ainic.nal.usda.gov). Accessed July 2013. ND = not detarmined.

One of the interesting features of iron in the human body is how little is allowed to remain as free, unbound iron. The normal adult has approximately 4.5 grams of iron, yet there is virtually no free iron in plasma (28). Most of the iron is bound to hemoglobin, and the remainder is bound to ferritin in tissues and transferrin in plasma. Furthermore, the transferrin in plasma is only approximately 30% saturated with iron, so any increase in plasma iron will be quickly bound by transferrin, thus preventing any rise in plasma free iron.

One reason for the paucity of free iron is the ability of free iron to promote oxidantinduced cell injury (28,29). Iron in the reduced state (Fe-II) promotes the formation of hydroxyl radicals (see Figure 22.6), and hydroxyl radicals are considered the most reactive oxidants known in biochemistry. In this context, the ability to bind and sequester iron has been called the major antioxidant function of blood (29). This might explain why hypoferremia is a common occurrence in patients who have conditions associated with hypermetabolism (30) (because this would limit the destructive effects of hypermetabolism).
In light of this description of iron, a reduced serum iron level in a critically ill patient should not prompt iron replacement therapy unless there is evidence of total-body iron deficiency. This latter condition can be detected with a plasma ferritin level; i.e., iron deficiency is likely if the plasma ferritin is below 18 ug/L, and is unlikely if the plasma ferritin is above 100 yg/L (31).

Selenium is an endogenous antioxidant by virtue of its role as a co-factor for glutathione peroxidase (see Figure 22.7). The recommended daily re-quirement for selenium is 55 yg in healthy adults (32), but selenium utilization is increased in acute illness (33), so daily requirements are likely to be higher in critically ill patients. A recent review of studies evaluating selenium in patients with severe sepsis has shown that low plasma levels of selenium are common in severe sepsis, and selenium supplementation in severe sepsis is associated with a lower mortality rate (34). In light of this study, attention to monitoring serum selenium levels in severe sepsis, as well as other conditions associated with systemic inflammation, seems warranted. The normal plasma selenium concentration is 89-113 yg/L (35).

Before leaving this chapter, it is important to point out a fundamental problem with promoting nutrient intake in critically ill patients (36). The problem is the difference in mechanisms for the malnutrition associated with critical illness and the malnutrition associated with starvation; i.e., the malnutrition from starvation is due to depletion of essential nutrients, while the malnutrition associated with critical illnesses is the result of abnormal nutrient processing. Because malnutrition in critically ill patients is caused by metabolic derangements, providing nutrients will not correct the malnutrition until the metabolic derangements resolve.
FIGURE 47.2 Influence of dextrose infusion on arterial lactate levels during abdominal aortic surgery. Each point represents the mean lactate level for 10 patients receiving Ringer's solution (closed squares) and 10 patients receiving 5% dextrose solution (open squares). Total volume infused is equivalent with both fluids. Data from Reference 38.
An example of abnormal nutrient processing in acute illness is illustrated by the fate of a glucose load; i.e., less than 5% of glucose is metabolized to lactate in healthy subjects, while as much as 85% of a glucose load can be recovered as lactate in acutely ill patients (37). This is demonstrated in Figure 47.2 (38). In this case, patients undergoing abdominal aneurysm surgery were given intraoperative fluid therapy with either Ringer's solutions or 5% dextrose solutions. In the patients who received dextrose (an average of 200 grams), the blood lactate level increased by more than 3 mmol/L, whereas the blood lactate level increased <1 mmol/L in the patients who received dextrose-free fluids. This demonstrates that nutrient intake can have very different consequences in critically ill patients, and the consequences can be toxic (e.g., the accumulation of an organic acid). It seems Lucretius had the right idea, over 2,000 years ago.

1. McClave SA, Martindale RG, Vanek VW, et al. Guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient: Society of Critical Care Medicine and American Society for Parenteral and Enteral Nutrition. J Parent Ent Nutr 2009; 33:277-316.

Forced feeding . . . goes on because the wort still believes it can eat itself well.
Herbert Shelton
1978
For patients who are unable to eat, the preferred method of nutrition support is the infusion of liquid feeding formulas into the stomach or small bowel (1,2). This mimics the normal process of nutrition support, and also serves as an infection control measure, as will be described.
This chapter presents the fundamentals of nutrition support with enteral tube feedings, and will show you how to create a tube feeding regimen for each patient in the ICU.

The preference for enteral over parenteral nutrition is based on numerous studies showing that enteral nutrition is associated with fewer infections (1-4), particularly pneumonias. This is attributed to the trophic effects of nutritional bulk that maintain the barrier and immunological functions of the bowel, as described next.

The role of enteral tube feedings in protecting against infections is summarized in the following statements.
1. The presence of food or tube feedings in the lumen of the bowel has a trophic influence on the bowel mucosa that preserves the structural integrity of the mucosa (5,6). This maintains the barrier function of the bowel mucosa, which protects against invasion from enteric pathogens, a phenomenon known as translocation (7).
2. The trophic influence of luminal nutrition also extends to the im-mune defenses in the bowel, such as the production of immunoglobulin A (IgA) by monocytes in the bowel wall, which blocks the attachment of pathogens to the bowel mucosa (8).
3. These effects are triggered by the presence of nutritional bulk in the lumen of the bowel (9), and are mediated, in part, by the release of gastrin and cholecystokinin in response to gastric distension (1). Specific nutrients in the bowel lumen also participate in these ef-fects. One of these nutrients is glutamine, which is a principal fuel for enterocytes in the bowel mucosa (10).
4. The trophic effects of nutritional bulk are lost during periods of bowel rest, and this results in progressive atrophy of the bowel mucosa (6), and can lead to translocation and the systemic spread of enteric pathogens (11). Parenteral nutrition does not prevent the deleterious effects of prolonged bowel rest (1,11).
The sum of these observations indicates that the normal antimicrobial defenses in the bowel are sustained by the presence of nutritional bulk in the bowel lumen. This is how nutrition support with enteral tube feedings serves as an infection control measure, as stated in the introduction to the chapter.

Patients who are unable to eat and do not have the absolute contraindications described next are candidates for enteral tube feeding. The presence of bowel sounds is not required to initiate enteral tube feedings (1). Tube feedings should be started within 24— 48 hours of admission to the ICU (1) to take advantage of the protective effects of tube feedings. There is evidence that early institution of enteral nutrition is associated with fewer septic complications and a shorter hospital stay (12).

Absolute contraindications to enteral tube feedings include complete bowel obstruction, bowel ischemia, ileus, and circulatory shock with high-dose vasopressor requirements (1,2). Gastric feedings can be attempted in stable patients on low doses of vasopressors (1), but any signs of intolerance should prompt immediate cessation of feedings.

There are at least 200 enteral feeding formulas that are commercially available, and many of the formulas used at individual hospitals are from the same manufacturer (because of contractual obligations). The following is a brief description of some characteristic features of feeding formulas. Examples are provided in Tables 48.1 and 48.2

Feeding formulas are available with caloric densities of 1 kcal/mL, 1.5 kcal/mL, and 2 kcal/mL. Most tube feeding regimens use formulas with 1 kcal/mL. The high-calorie formulas (2 kcal/mL) are intended for patients with severe physiological stress (e.g., multisystem trauma and burns), but they are frequently used when volume restriction is a priority.

Osmolite, Caloric geamy = 1. Osmolite, — Nonprotein “ay = 86. Osmolite,  = 37. Osmolite, eeu = 300. Osmolite HN, Caloric geamy = 1. Osmolite HN, — Nonprotein “ay = 83. Osmolite HN,  = 44. Osmolite HN, eeu = 300. Isocal, Caloric geamy = 1. Isocal, — Nonprotein “ay = or. Isocal,  = 34. Isocal, eeu = 300. Isocal HN, Caloric geamy = 1. Isocal HN, — Nonprotein “ay = 83. Isocal HN,  = 44. Isocal HN, eeu = 300. Isocal HCN, Caloric geamy = 2. Isocal HCN, — Nonprotein “ay = 85. Isocal HCN,  = “5. Isocal HCN, eeu = 690. Twocal HN, Caloric geamy = 2. Twocal HN, — Nonprotein “ay = 83. Twocal HN,  = 84. Twocal HN, eeu = 690

The caloric density of feeding formulas includes both protein and nonprotein calories, but daily caloric requirements should be provided by nonprotein calories (as mentioned in Chapter 47). In standard feeding formulas, nonprotein calories account for about 85% of the total calories (see Table 48.1).

The osmolality of feeding formulas is determined primarily by the caloric density. Feeding formulas with 1 kcal/mL have an osmolality similar to plasma (280-300 mosm/kg H20), and feeding formulas with 2 kcal/mL have an osmolality about twice that of plasma. Hypertonic feedings create little risk of diarrhea when they are delivered into the stomach, where the large volume of gastric secretions attenuates the osmolality.

Standard feeding formulas provide 35-40 grams of protein per liter. High-protein formulas, often designated by the suffix HN (for "high nitrogen"), provide about 20% more protein than the standard formulas (compare Isocal and Isocal HN in Table 48.1). Most enteral formulas contain intact proteins that are broken down into amino acids in the upper GI tract. These are called polymeric formulas. Feeding formulas are also available that contain small peptides (called semi-elemental formulas) and individual amino acids (called elemental formulas) that are absorbed more readily than intact protein. Semi-elemental and elemental formulas promote water reabsorption from the bowel, and could benefit patients with troublesome diarrhea. However, the clinical
benefit of these formulas is unproven (14). Examples of semi-elemental and elemental formulas include Optimental, Peptamen, Perative, Vital HN, and Vivonex T.E.N.

Carbohydrates (usually polysaccharides) are the major source of calories in feeding formulas, and provide 40-70% of the total calories. Low-carbohydrate formulas, in which carbohydrates provide 30-40% of the calories, are available for diabetics. One example of a low-carbohydrate formula is Glucerna.

The term "fiber" refers to polysaccharides from plants that are not digested by humans. Fiber is fermented by bacteria in the colon, and is broken down into short-chain fatty acids, which are an important energy source for the mucosal cells in the large bowel (13). The uptake of these fatty acids into the bowel mucosa also promotes sodium and water absorption. This "fermentable" fiber promotes the growth and viability of the surface mucosa in the large bowel, and can also reduce the water content of stool. There is also "nonfermentable" fiber that is not broken down by gut bacteria. This type of fiber draws water into the bowel and increases the water content of stool.
Fiber is added to some feeding formulas to promote the viability of the mucosa in the large bowel, and some examples of fiber-containing formulas are shown in Table 48.2. The fiber in most feeding formulas is a mixture of fermentable and nonfermentable varieties.

Jevity - 1 Cal, 1 = 1. Jevity - 1 Cal, 2 = ary. Jevity - 1 Cal, 3 = 14. Jevity - 1 Cal, 4 = 300. Jevity - 1.5 Cal, 1 = 4.5. Jevity - 1.5 Cal, 2 = 64. Jevity - 1.5 Cal, 3 = 22. Jevity - 1.5 Cal, 4 = 525. Promote with Fiber, 1 = 1. Promote with Fiber, 2 = 63. Promote with Fiber, 3 = 14. Promote with Fiber, 4 = 380

Standard feeding formulas contain polyunsaturated fatty acids from vegetable oils. The lipid content is adjusted to provide about 30% of the caloric density of the formula.

Polyunsaturated fatty acids from vegetable oils (which make up the lipid content of standard feeding formulas) can serve as precursors for inflammatory mediators (eicosanoids) that are capable of promoting inflammatory cell injury. This concern has prompted the introduction of feeding solutions that contain polyunsaturated fatty acids from fish oils (omega-3 fatty acids), which do not promote the production of inflammatory mediators. Some of these feeding formulas are shown in Table 48.3. The use of feeding formulas that influence the inflammatory response is known as immunonutrition (14).

Impact, Calories (kcal/mL) = 1. Impact, Q-3FAs — (g/L) = 57.. Impact, Arginine oe = 13. Impact, | aces = Selenium, B-carotene. Impact 1.5, Calories (kcal/mL) = T.8. Impact 1.5, Q-3FAs — (g/L) = 2.5. Impact 1.5, Arginine oe = 19. Impact 1.5, | aces = Selenium, B-carotene. Optimental, Calories (kcal/mL) = 1. Optimental, Q-3FAs — (g/L) = 2.3. Optimental, Arginine oe = 6. Optimental, | aces = Vitamins G & E, B-carotene. Oxepa, Calories (kcal/mL) = 1.5. Oxepa, Q-3FAs — (g/L) = 4.6. Oxepa, Arginine oe = 0. Oxepa, | aces = Vitamins G & E, B-carotene
42-3 FAs = omega-3 fatty acids.
Clinical studies have shown that patients with acute respiratory distress syndrome (ARDS) derive some benefit (fewer days on the ventilator) from feeding formulas enriched with omega-3 fatty acids and antioxidants (15). However, the benefits are marginal, and there is a general reluctance to adopt these feeding formulas for patients with ARDS.

Despite the confusing array of liquid feeding formulas, including the "designer formulas," there is very little consistent or convincing evidence that one feeding formula, or one type of formula, is superior to the others. In other words a single feeding formula could be used for all ICU patients (with an occasional exception), as long as it is used appropriately.

This section describes a simple four-step method for creating an enteral feeding regimen. This method is summarized in Table 48.4.

The very first consideration is the patient's daily requirement for calories and protein, and both requirements can be estimated with the simple predictive formulas in Table 48.2. (See Chapter 47 for more information on these formulas.) You can use actual body weight in the formulas as long as actual body weight is not above 125% of the ideal weight. If actual body weight exceeds 125% of ideal body weight, you should use an adjusted body weight, which is derived in Equation 47.3. If available, indirect calorimetry should be used to measure the resting energy expenditure (see Chapter 47).

A standard formula, with 1—1.5 kcal/mL, should be sufficient for most patients. Use formulas with higher caloric densities if volume restriction is a priority.

The final step in the process is to determine if the feeding regimen will provide enough protein to satisfy the daily protein requirement (from step 1). The projected protein intake is simply the daily feeding volume multiplied by the protein concentration in the feeding formula. If the projected protein intake is less than the desired protein intake, powdered protein is added to the tube feedings to correct the discrepancy.

Step 1: Estimate daily calorie and protein requirements.
Calories (kcal/day) = 25 x wt (kg) Protein (a/day) = (1.2-1.6) x wt (kg)
Step 2: Select feeding formula.
Step 3: Calculate desired infusion rate.
Feeding volume (mL) =
keal/day required
keal/mL in feading formula
Infusion rate (mL/hn) =
Feeding volume im
Feeding time (hrs)

a. Calculate the projected protein intake (g/day) as:
Feeding volume (L‘day) x protein (g/L) In feedings
b. If the projected intake Is less than desired intake, add protein to the feeding regimen to correct the discrepancy.

Feeding tubes are inserted through the nares and advanced blindly into the stomach or duodenum. The distance required to reach the stomach can be estimated by measuring the distance from the tip of the nose to the earlobe and then to the xiphoid process (typically 50-60 cm) (20). Once the tube is advanced the desired length, a portable chest x-ray is required to verify proper tube position before the feeding formula is infused. The common practice of evaluating tube placement by pushing air through the tube and listening for bowel sounds is not reliable, because sounds emanating from a misplaced tube in the distal airways or pleural space can be transmitted into the upper abdomen (21,22).

Feeding tubes end up in the trachea during 1% of insertions (23). In-tubated patients often do not cough when feeding tubes enter the trachea (unlike healthy subjects); as a result, feeding tubes can be advanced deep into the lungs without any warning signs, and can puncture the visceral pleura and create a pneumothorax (21,22). The portable chest x-ray in Figure 48.1 shows a feeding tube that has been advanced almost to the edge of the right lung in a patient with a tracheostomy. This was a routine post-insertion chest xray, and there was no evidence that the tube was in the airways other than the chest xray. This illustrates the value of a portable chest x-ray immediately after a feeding tube is inserted, and before the feeding formula is infused. (The pleural effusion on the right in Figure 48.1 was present prior to insertion of the feeding tube.)

Advancing the tip of the feeding tube into the duodenum, to reduce the risk of aspiration, is not necessary (1) because most studies show that there is no difference in the risk of aspiration with gastric versus duodenal feedings (24,25). However, duodenal placement may be necessary in patients who regurgitate intragastric feedings.

The traditional practice is to begin tube feedings at a low infusion rate (10-20 ml/hr), and then gradually advance to the target infusion rate over the next 6-8 hours. However, gastric feedings can begin at the desired (target) rate in most patients without the risk of vomiting or aspiration (26,27). Starter regimens are more appropriate for small bowel feedings (particularly in the jejunum) because of the limited reservoir capacity of the small bowel.

The complications associated with enteral tube feedings include occlusion of the feeding tube, regurgitation of the feeding formula into the mouth and airways, and diarrhea.
FIGURE 48.1 Routine chest x-ray following the insertion of a feeding tube. See text for explanation. Image digitally enhanced.

Narrow-bore feeding tubes can become occluded by protein precipitates that form when acidic gastric secretions reflux into the feeding tubes (28). Standard preventive measures include flushing the feeding tubes with 30 mL of water every 4 hours, and using a 10-mL water flush after medications are instilled.

If flow through the feeding tube is sluggish, flushing the tube with warm water can restore flow in 30% of cases (29). If this is ineffective, pancreatic enzyme (Viokase) can be used as follows (17):
Regimen: Dissolve 1 tablet of Viokase and 1 tablet of sodium carbonate (324 mg) in 5 mL of water. Inject this mixture into the feeding tube and clamp for 5 minutes.
Follow with a warm water flush. This should relieve the obstruction in about 75% of cases (17).
If the tube is completely occluded, advance a flexible wire or a drum cartridge catheter through the feeding tube in an attempt to clear the obstruction. If this is unsuccessful, replace the feeding tube without delay.
Metoclopramide, Dosing Regimens and Comments = Dosing: Comment:. Metoclopramide, Dosing Regimens and Comments = 10 mg every 6 hrs. A dopamine antagonist in the GI tract. Can decrease gastric residual volume by 30%, but efficacy diminishes over a few days. IV may be more effective than PO therapy.. Erythromycin, Dosing Regimens and Comments = Dosing: Comment:. Erythromycin, Dosing Regimens and Comments = 200 mg IV every 12 hrs. Stimulates motilin receptors in the Gl tract. May be more effective than metoclopramide, but efficacy wanes over a few days.. Enteral Naloxone, Dosing Regimens and Comments = Dosing: Comment:. Enteral Naloxone, Dosing Regimens and Comments = 8 mg via nasogastric tube every 6 hrs. Blocks opiate receptors in the bowel without antagonizing the analgesic effects of opiates. Used only for gastric dysmotility associated with opiates.
Fram Referancas 34-37.
METOCLOPRAMIDE: Metoclopramide promotes gastric emptying by antagonizing the actions of dopamine in the GI tract. At a dose of 10 mg IV every 6 hours, metoclopramide can decrease gastric residual volumes by 30% after 24 hours, but the effect wanes rapidly (35). Metoclopramide is more effective when given in combination with erythromycin (36).

ENTERAL NALOXONE: In critically patients who have gastric dysmotility associated with opiates, direct intragastric administration of the opioid antagonist naloxone (8 mg via nasogastric tube every 6 hours) can selectively block opioid receptors in the bowel and promote gastric emptying without antagonizing the analgesic effects of the opiate (37). The opiate antagonist methylnaltrexone, has also been shown to promote postoperative recovery of bowel function in opiate users (38).
RECOMMENDATION: For a trial of prokinetic therapy, either start with erythromycin, and add metoclopramide after 24 hours if needed, or start with both drugs. And don't expect much.

For patients who continue to be intolerant of tube feedings (e.g., repeatedly regurgitate feedings or develop abdominal distention), a switch to parenteral nutrition may be necessary. However, the infusion of tube feedings should be continued at a lower, tolerable rate, whenever possible, to provide some support for the antimicrobial defenses in the bowel.

Diarrhea occurs in approximately 30% of patients receiving enteral tube feedings (26). The feeding formulas were originally implicated, but the consensus opinion now is that other factors are involved (39). The principal offender in tube-feeding diarrhea may be liquid drug preparations.

Liquid preparations are favored for drug delivery through narrow-bore feeding tubes because there is less risk of obstruction. However, liquid preparations have two features that create a risk for diarrhea: (a) they can be extremely hyperosmolar (23,000 mosm/kg H,O), and (b) they can contain sorbitol (to improve palatability), a well-known laxative that draws water into the bowel lumen (40). Table 48.6 includes a list of diarrhea-prone liquid preparations that might be used in ICU patients. These preparations should be discontinued, if possible, in any patient who develops diarrhea of uncertain etiology during enteral nutrition.

Acetaminophen elixir, _ Contain Sorbitol = Acetaminophen liquid. Dexamethasone solution, _ Contain Sorbitol = Cimetidine solution. Ferrous sulfate liquid, _ Contain Sorbitol = Isoniazid syrup. Hydroxyzine syrup, _ Contain Sorbitol = latrogenic Fever. Metoclopramide syrup, _ Contain Sorbitol = Lithium syrup. Multivitamin liquid, _ Contain Sorbitol = Metoclopramide syrup. Potassium chloride liquid, _ Contain Sorbitol = Theophylline solution. Promethazine syrup, _ Contain Sorbitol = Tetracycline suspension. Sodium phosphate liquid, _ Contain Sorbitol = 
Fram Referance 40.

The mucosal surface of the bowel is constantly changing, with new cells to replace the old ones every few days, and the force behind this dynamic process is the presence of food in the lumen of the bowel. Removing the nutritional bulk from the bowel lumen disrupts the normal process of renewal in the bowel mucosa, and makes us vulnerable to invasion by the hordes of enteric pathogens that inhabit the bowel. This is one of the major advantages of enteral tube feedings over total parenteral nutrition, and it also means that eating is a defense against infection.

2. Kreymann KG, Berger MM, Deutz NEP, et al. ESPEN guidelines on enteral nutrition: intensive care. Clin Nutr 2006; 25:210-223.

Initiating Tube Feedings, 1 = Initiating Tube Feedings. 20., 1 = Stroud M, Duncan H, Nightingale J. Guidelines for enteral feeding in adult hospital patients. Gut 2003; 52 Suppl 7:vii1-vii12.. Kolbitsch C, Pomaroli A, Lorenz I, et al. Pneumothorax following nasogastric feeding tube insertion in a tracheostomized patient after bilateral lung transplantation. Intensive Care Med 1997; 23:440-442., 1 = Kolbitsch C, Pomaroli A, Lorenz I, et al. Pneumothorax following nasogastric feeding tube insertion in a tracheostomized patient after bilateral lung transplantation. Intensive Care Med 1997; 23:440-442.. 22., 1 = Fisman DN, Ward ME. Intrapleural placement of a nasogastric tube: an unusual complication of nasotracheal intubation. Can J Anaesth 1996; 43:1252-1256.. 23., 1 = Baskin WN. Acute complications associated with bedside placement of feeding tubes. Nutr Clin Pract 2006; 21:40-55.. 24., 1 = Neumann DA, DeLegge MH. Gastric vsersus small bowel tube feeding in the intensive care unit: a prospective comparison of efficacy. Crit Care Med 2002; 30:1436-1438.. 25. Marik PE, Zaloga GP. Gastric versus post-pyloric feeding: a systematic review. Crit Care 2003; 7:R46-R51., 1 = 25. Marik PE, Zaloga GP. Gastric versus post-pyloric feeding: a systematic review. Crit Care 2003; 7:R46-R51.. 26., 1 = Rees RG, Keohane PP, Grimble GK, et al. Elemental diet administered nasogastrically without starter regimens to patients with inflammatory bowel disease. J Parenter Enteral Nutr 1986; 10:258-262.. 27., 1 = Mizock BA. Avoiding common errors in nutritional management. J Crit Illness 1993; 10:1116-1127.

To lengthen thy life, lessen thy meak.
Benjamin Franklin
When full nutritional support is not possible in the alimentary canal, the intravenous route is available for nutrient delivery. This chapter describes the basic features of intravenous nutritional support, and demonstrates how to create a parenteral nutrition regimen to meet the needs of individual patients.

Standard nutrition support regimens use carbohydrates to supply approximately 70% of the daily (nonprotein) caloric requirements. The carbohydrate source for total parenteral nutrition (TPN) is dextrose (glucose), which is available in the solutions shown in Table 49.1. Because the energy yield from dextrose is relatively low (see Table 47.1), the dextrose solutions must be concentrated to provide enough calories to satisfy daily requirements. (The standard solution is 50% dextrose, or Dso.) These solutions are hyperosmolar, and must be infused through large central veins.

5%, 1 = 50. 5%, 2 = 170. 5%, 3 = 253. 10%, 1 = 100. 10%, 2 = 340. 10%, 3 = 505. 20%, 1 = 200. 20%, 2 = 680. 20%, 3 = 1,080. 50%, 1 = 500. 50%, 2 = 4,700. 50%, 3 = 2,525. 70%, 1 = 700. 70%, 2 = 2,380. 70%, 3 = 3,530
"Based on an oxidative energy yield of 3.4 keal'g for dextrose.

Protein is provided as amino acid solutions that contain varying mixtures of essential (N=9), semi-essential (N=4), and nonessential (N=10) amino acids. These solutions are mixed with dextrose solutions in a 1:1 volume ratio. Examples of standard and "specialty" amino acid solutions are shown in Table 49.2.

EAA = essential arrino acids; BCAA = branched chain amino acids.
Strengths, Aminosyn_ = 3.5%, 5%, 7%, 8.5%, 10%. Strengths, Aminosyn-HBC = 1%. Strengths, Aminosyn RF = 5.2%. Indications, Aminosyn_ = Standard TPN. Indications, Aminosyn-HBC = Hypercatabolism. Indications, Aminosyn RF = Renal Failure. % EAA, Aminosyn_ = . % EAA, Aminosyn-HBC = 68%. % EAA, Aminosyn RF = . % BCAA, Aminosyn_ = 25%. % BCAA, Aminosyn-HBC = 46%. % BCAA, Aminosyn RF = 33%

Standard amino acid solutions (e.g., Aminosyn in Table 49.2) are balanced mixtures of 50% essential amino acids and 50% nonessential and semi-essential amino acids. Available concentrations range from 3.5 % up to 10%, but 7% solutions (70 g/L) are used most often.

Specially designed amino acid solutions are available for patients with severe metabolic stress (e.g., from multisystem trauma or burns), and for patients with renal or liver failure.
1. Solutions designed for metabolic stress (e.g., Aminosyn-HBC in Table 49.2) are enriched with branched chain amino acids (iso-leucine, leucine, and valine), which are preferred fuels in skeletal muscle when metabolic demands are high.
2. Renal failure solutions (e.g., Aminosyn RF in Table 49.2) are rich in essential amino acids, because the nitrogen in essential amino acids is partially recycled to produce nonessential amino acids, which results in smaller increments in blood urea nitrogen (BUN) when compared with breakdown of nonessential amino acids.
3. Solutions designed for hepatic failure (e.g., HepaticAid) are en-riched with branched chain amino acids, which block the transport of aromatic amino acids across the bloodbrain barrier (which is implicated in hepatic encephalopathy).
It is important to emphasize that none of these specialized formulas have improved the outcomes in the disorders for which they are designed (3).

Glutamine is the principal metabolic fuel for rapidly dividing cells like intestinal epithelial cells and vascular endothelial cells (4). Because of studies showing that glutamine is important for maintaining the integrity of the bowel mucosa (5), and studies showing a glutamine-associated decrease in infectious complications in ICU patients (6,7), glutamine has been recommended as a daily nutrional supplement in ICU patients (0.2— 0.4 g/kg/day) (1). However, a recent multicenter study has shown a glutamineassociated increase in mortality rate in ICU patients with multiorgan failure (8), and until this issue is resolved, the recommendation for daily glutamine administration in ICU patients must be re-evaluated. (Note: Glutamine is not included in any of the commercially available amino acid solutions, so it must be added to the solutions by the pharmacy. This alone will limit the popularity of daily glutamine supplementation. )

Calories (kcal/mL), 1 = 1.1. Calories (kcal/mL), 2 = 2. Calories (kcal/mL), 3 = 1.1. Calories (kcal/mL), 4 = 2. %6 calories as EFA (Linoleic acid), 1 = 50%. %6 calories as EFA (Linoleic acid), 2 = 50%. %6 calories as EFA (Linoleic acid), 3 = 66%. %6 calories as EFA (Linoleic acid), 4 = 66%. Cholesterol (mg/dL), 1 = 250-300. Cholesterol (mg/dL), 2 = 250-300. Cholesterol (mg/dL), 3 = 13-22. Cholesterol (mg/dL), 4 = 13-22. Osmolarity (mosm/L), 1 = 260. Osmolarity (mosm/L), 2 = 260. Osmolarity (mosm/L), 3 = 276. Osmolarity (mosm/L), 4 = 258. Unit Volumes (mL), 1 = 50. Unit Volumes (mL), 2 = 50. Unit Volumes (mL), 3 = 100. Unit Volumes (mL), 4 = 200. , 1 = 100. , 2 = 100. , 3 = 200. , 4 = 500. , 1 = 250. , 2 = 250. , 3 = 500. , 4 = . , 1 = 500. , 2 = 500. , 3 = . , 4 = 
EFA = essential fatty acid.

Lipids are provided as emulsions composed of submicron droplets of cholesterol, phospholipids, and triglycerides (9). The triglycerides are derived from vegetable oils (safflower or soybean oils) and are rich in linoleic acid, an essential fatty acid (10). Lipids are used to provide 30% of daily caloric requirements, and 4% of the daily calories should be provided as linoleic acid to prevent essential fatty acid deficiency (11). As shown in Table 49.3, lipid emulsions are available in 10% and 20% strengths (the percentage refers to grams of triglyceride per 100 mL of solution). The 10% emulsions provide approximately 1 kcal/mL, and the 20% emulsions provide 2 kcal/mL. Unlike the hypertonic dextrose solutions, lipid emulsions are roughly isotonic to plasma and can be infused through peripheral veins. The lipid emulsions are available in unit volumes of 50 to 500 mL, and can be infused separately (at a maximum rate of 50 mL/hour) or added to the dextrose—amino acid mixtures. The triglycerides introduced into the bloodstream are not cleared for 8 to 10 hours, and lipid infusions often produce a transient, lipemicappearing plasma.
Commercially available mixtures of electrolytes, vitamins, and trace elements are added directly to the dextrose—amino acid mixtures.

There are more than 15 electrolyte mixtures available. Most have a volume of 20 mL, and contain sodium, chloride, potassium, and magnesium. You must check the mixture used at your hospital to determine if additional electrolytes must be added. Additional requirements for po-tassium or other electrolytes can be specified in the TPN orders.

Aqueous multivitamin preparations are added to the dextrose—amino acid mixtures. One unit vial of a standard multivitamin preparation will provide the normal daily requirements for most vitamins (see Table 47.3 ) (16). The daily vitamin requirements in ICU patients are not known (and probably varies with each patient). However, vitamin deficiencies are rcommon in ICU patients despite the provision of normal daily requirements, suggesting that critically ill patients have increased daily vitamin requirements.

A variety of trace element additives are available, and one of the commercial preparations is shown in Table 49.4, along with the recommended daily requirement for trace elements. Note the poor correlation between the daily requirements and the trace element content of the commercial mixture. Trace element mixtures don't contain iron and iodine, and some don't contain selenium. Iron is not advised in critically ill patients because of its pro-oxidant effects (see Chapter 47 for more information on iron and oxidant injury). However, selenium should be given daily to ICU patients, particularly those with severe sepsis.

The most important consideration regarding trace elements is selenium, which is a cofactor for glutathione peroxidase, an enzyme that participates in endogenous antioxidant protection (see Figure 22.7). Plasma levels of selenium are reduced in severe sepsis, and selenium replacement is associated with improved survival (12). The proposed daily requirement for selenium is 55 yg, but this is probably inadequate for critically ill patients. A daily dose of 200 ug is used in many studies, and doses of 400 ug daily are considered safe (see Table 47.3).

Chromium, Daily — -Requirementt = 30 pg. Chromium, _ Multitrace-5. -Goncentrateds = 10 yg. Copper, Daily — -Requirementt = 900 kg. Copper, _ Multitrace-5. -Goncentrateds = img. lodine, Daily — -Requirementt = 150 pug. lodine, _ Multitrace-5. -Goncentrateds = a. lron, Daily — -Requirementt = 8 mg. lron, _ Multitrace-5. -Goncentrateds = _. Manganese, Daily — -Requirementt = 2.3 mg. Manganese, _ Multitrace-5. -Goncentrateds = 0.5 mg. Selenium, Daily — -Requirementt = 55 yg. Selenium, _ Multitrace-5. -Goncentrateds = 60 pg. Zinc, Daily — -Requirementt = 14 mg. Zinc, _ Multitrace-5. -Goncentrateds = 5 mg
tFroam the Food and Nutrition Information Centar (http./Anic.nal.usda.gov). Accessed July, 2013.
§Product description, America Raagant, Inc.

The following is a stepwise approach to creating a standard TPN regimen for an individual patient. The patient in this example will be a 70-kg adult who is not malnourished and has no volume restrictions.

The first step is to determine the daily requirement for calories and protein. There are two very simple approximations that can be used: i.e., the daily requirement for calories is 25 kcal/kg, and the daily protein requirement is 1.2-1.6 g/kg. (See Chapter 47 for more information on these estimates.) You can use actual body weight in these estimates as long as actual body weight is within 125% of the ideal weight. If actual body weight exceeds 125% of ideal body weight, you can use an adjusted body weight (see equation 47.3). If available, indirect calorimetry should be used to measure the resting energy expenditure (see Chapter 47).
For the 70 kg patient, we'll use actual body weight, and a daily protein requirement of 1.4 g/kg. Therefore, the daily requirement for calories and protein will be:
Calories: 25 x 70 = 1,750 keal/day
Protein: 1.4 x 70 = 98 grams/ day
(49.1)
Note: If propofol is being infused, you should determine the calories provided by propofol and subtract those calories from the daily caloric requirement. Propofol is infused in a 10% lipid emulsion, which has a caloric density equivalent to 10% Intralipid (1 kcal/mL). Therefore, the hourly infusion rate of propofol (mL/hr) is equivalent to the hourly yield of calories (kcal/hr).

The next step is to take a standard mixture of 10% amino acids (500 mL) and 50% dextrose (500 mL) and determine the volume of this mixture that is needed to deliver the estimated daily protein requirement. Although the dextrose—amino acid mixture is referred to as Ajg-Ds0, the final mixture actually represents 5% amino acids (50 grams of protein per liter) and 25% dextrose (250 grams of dextrose per liter). The volume of the Ayo-Ds9 mixture that will provide the daily protein requirement is equivalent to the daily protein requirement (98 g/day), divided by the protein concentration in the amino acid mixture (50 g/L); i.e.,
Volume of AygDe9 = 98 50 = 1.9 Liters
(49.2)
If this mixture is infused over 24 hours, the infusion rate will be:
Infusion rate = 1,900 mL/24 hr = 81 mL/hr
(49.3)
Step 3
Now, determine how many nonprotein calories will be provided by 1.9 liters of Aj9-Dso. (Only nonprotein calories are used to provide the daily energy needs.) First, determine how much dextrose is in 1.9 liters of Ayg-Dso:
250 2 /'L = 1.9 L= 475 grams of dextrose
(49.4)
Now, using an energy yield of 3.4 kcal/g for dextrose, calculate the calories provided by 475 grams of dextrose:
Dextrose calories = 475 x 3.4 = 1,615 keal/day
(49.5)

The next step is to use lipid calories to make up the difference between the calories supplied by dextrose and the daily requirement for calories, as shown below:
Daily Requirement:
1,750 kcal
Daily Dextrose Calories: 1,615 kcal
Deficit: 135 keal
(49.6)
The remaining 135 calories will be provided by lipids. If a 10% lipid emulsion (1 kcal/mL) is used, the volume will be 135 mL/day. (Lipid emulsions are available in unit volumes of 50 mL, so the volume can be adjusted to 150 mL to avoid wastage). The maximum infusion rate is 50 mL/hr.

The TPN orders for this example can be written as follows:
1. Aj9-D59 to run at 80 mL/hour.
2. 10% Intralipid, 150 mL, to infuse over 3 hours.
3. Add standard electrolytes, multivitamins, and trace elements.
TPN orders are rewritten each day. Specific electrolytes, vitamins, and trace elements are added to the daily orders when needed.

As mentioned earlier, the hyperosmolarity of the dextrose and amino acid solutions requires infusion through large veins, so central venous cannulation, or a peripherally inserted central catheter (PICC), is required. The complications associated with these catheters are described in Chapters 2 and 3.

Insertion of subclavian vein catheters and peripherally inserted central catheters (PICCs) can occasionally result in advancement of a catheter into the internal jugular vein, like the one shown in Figure 49.1. In one survey (13), 10% of subclavian vein cannulations (mostly on the right) resulted in misplacement of the catheter in the internal jugular vein. The standard recommendation is to reposition such catheters because of the risk of thrombosis (13), but there is no evidence to support this claim.
FIGURE 49,1 X-ray showing a central venous catheter misdirected into the neck.

Hyperglycemia is common during TPN; e.g., in one study, blood glucose levels above 300 mg/dL were recorded in 20% of postoperative patients receiving TPN (vs. 1.5% of control patients) (14). This is attributed to the glucose load in TPN. (A standard TPN regimen with 1,800 nonprotein calories has about 350 grams of glucose, compared to 230 grams in a standard tube feeding regimen.) Tight glycemic control is not recommended in critically ill patients because of the risk of hypoglycemia, which has more serious consequences than hyperglycemia (15). The current recommendation for hospitalized patients is a target range of 140-180 mg/dL for blood glucose (1,16).
INSULIN If insulin therapy is required, a variety of biosynthetic insulins are available, and the ones used most frequently are shown in Table 49.5 (17). A continuous infusion of regular insulin is preferred for critically ill patients who are unstable or have type 1 diabetes (2), to prevent wide swings in glucose levels. This can be accomplished by adding insulin to the TPN solutions. One shortcoming of intravenous insulin infusions is the propensity for insulin to adsorb to the plastic tubing in IV infusion sets. This has a variable effect on the bioavailability of insulin, but this variability can be reduced by priming the IV infusion set with an insulin solution (e.g., 20 mL of a saline containing 1 unit/mL of regular insulin). This stabilizes the bioavailability of infused insulin (at about 30-40%) for several days, but the priming procedure must be repeated each time the IV infusion set is changed (2).

Rapid-Acting, Name — = Aspart. Rapid-Acting, Onset = 10-20 min. Rapid-Acting, Peak = 1-3 hr. Rapid-Acting, Duration = 3-5 hr. Rapid-Acting, Name — = Glulisine. Rapid-Acting, Onset = 25 min. Rapid-Acting, Peak = 45-50 min. Rapid-Acting, Duration = 4-5 hr. Rapid-Acting, Name — = Lispro. Rapid-Acting, Onset = 15-30 min. Rapid-Acting, Peak = 0.5-2.5 hr. Rapid-Acting, Duration = 3-6 hr. Short-Acting, Name — = Regular. Short-Acting, Onset = 30-60 min. Short-Acting, Peak = 1-5 hr. Short-Acting, Duration = 6-10 hr. Intermediate, Name — = NPH. Intermediate, Onset = 1-2 hr. Intermediate, Peak = 6-14 hr. Intermediate, Duration = 16-24 hr. Long-Acting, Name — = Glargine. Long-Acting, Onset = 1 hr. Long-Acting, Peak = 2-20 hr. Long-Acting, Duration = 24 hr
Fram Referance 17.
Subcutaneous insulin can be used for patients who are stable. Regimens will vary in each patient, but the combination of an intermediate or long-acting insulin with a rapid-acting insulin, when needed, is a popular for hospitalized patients.

The movement of glucose into cells is associated with a similar movement of phosphate into cells, and this provides phosphate for co-factors (e.g., thiamine pyrophosphate) that participate in glucose metabolism. This intracellular shift of phosphate can result in hypophosphatemia if extracellular phosphate levels are marginal. This is the most common cause of hypo-phosphatemia in hospitalized patients (18), and plasma phosphate levels typically show a steady decline after TPN is initiated (see Table 38.2).

Glucose movement into cells is also accompanied by an intracellular shift of potassium (which is the basis for the use of glucose and insulin to treat severe hyperkalemia). This effect is usually transient, but continued glucose loading during TPN can lead to persistent hypokalemia.

Excess carbohydrate intake promotes CQO, retention in patients with respiratory insufficiency. This was originally attributed to the high respiratory quotient (VCO>/VO>) associated with carbohydrate metabolism. However, CO> retention is a consequence of overfeeding, and not overfeeding with carbohydrates (19).

Overfeeding with lipids may contribute to hepatic steatosis. However, a more serious concern with lipid infusions is the potential to promote inflammation. The lipid emulsions used in TPN regimens are rich in oxidizable lipids (20), and the oxidation of infused lipids will trigger an inflammatory response. In fact, infusions of oleic acid, one of the lipids in TPN, is a standard method for producing the acute respiratory distress syndrome (ARDS) in animals (21), and this might explain why lipid infusions are associated with impaired oxygenation (22,23). The possible role of lipid infusions in promoting oxidant-induced injury deserves more attention.

Fat accumulation in the liver (hepatic steatosis) is common in patients receiving longterm TPN, and is believed to be the result of chronic overfeeding with carbohydrates and lipids. Although this condition is associated with elevated liver enzymes in blood (24), it may not be a pathological entity.

The absence of lipids in the proximal small bowel prevents cholecystokinin-mediated contraction of the gallbladder. This results in bile stasis and the accumulation of sludge in the gallbladder, and can lead to acalculous cholecystitis (25), which is described in Chapter 40.

The absence of nutritional bulk in the GI tract leads to atrophic changes in the bowel mucosa, and impairs bowel-associated immunity, and these changes can lead to the systemic spread of enteric pathogens. This topic is described in Chapter 48.

Peripheral parenteral nutrition (PPN) is a truncated form of TPN that can be used to provide nonprotein calories in amounts that will spare the breakdown of proteins to provide energy (i.e., protein-sparing nutrition support). PPN can be used as a supplement to enteral feeding, or as a source of calories during brief periods of inadequate nutrition. It is not intended for hypercatabolic or malnourished patients, who need full nutritional support,
The osmolarity of peripheral vein infusates should be kept below 900 mosm/L, with a pH between 7.2 and 7.4, to slow the rate of osmotic damage to vessels (26,27). This requires dilute amino acid and dextrose solutions, which limits nutrient intake. However, lipid emulsions are isotonic to plasma, and lipids can be used to provide a considerable portion of the nonprotein calories in PPN.

A popular solution in PPN is a mixture of 3% amino acids and 20% dextrose (final concentration of 1.5% amino acids and 10% dextrose), which has an osmolarity of 500 mosm/L. The dextrose will provide 340 kcal/L, so 2.5 L of the mixture will provide 850 kcal. If 250 mL of 20% Intralipid is added to the regimen (adding 500 kcal), the total nonprotein calories will increase to 1,350 kcal/day. This should be close to the nonprotein caloric requirement of an average-size, unstressed adult (20 kcal/kg/day).

The final word on parenteral nutrition is . . . avoid . . . whenever possible. For an explanation, see the first section of Chapter 48.

1. Singer P, Berger MM, Van den Berghe G, et al. ESPEN guidelines on parenteral nutrition: Intensive care. Clin Nutr 2009; 387-400.
2. Jacobi J, Bircher N, Krinsley J, et al. Guidelines for the use of insulin infusion for the management of hyperglycemia in critically ill patients. Crit Care Med 2012; 40:32513276.

Substrate 3., 1 = Solutions Andris DA, Krzywda EA. Nutrition support in specific diseases: back to basics. Nutr Clin Pract 1994; 9:28-32.. 4., 1 = Souba WW, Klimberg VS, Plumley DA, et al. The role of glutamine in maintaining a healthy gut and supporting the metabolic response to injury and infection. J Surg Res 1990; 48:383-391.. 5., 1 = De-Souza DA, Greene LJ. Intestinal permeability and systemic infections in critically ill patients: effect of glutamine. Crit Care Med 2005; 33:1125-1135.. , 1 = Dechelotte P, Hasselmann M, Cynober L, et al. L-alanyl-L-glutamine dipeptide- supplemented total parenteral nutrition reduces infectious complications and glucose intolerance in critically ill patients: the French controlled, randomized, double-blind,. 6. 7., 1 = multicenter study. Crit Care Med 2006; 34:598-604. Fuentes-Orozco C, Anaya-Prado R, Gonzalez-Ojeda A, et al. L-alanyl-L-glutamine-. , 1 = peritonitis. Clin Nutr 2004; 23:13-21. Heyland D, Muscedere J, Wischmeyer PE, et al. A randomized trial of glutamine antioxidants in critically ill patients. N Engl J Med 2013; 368:1489-1497.. 8., 1 = and. 9., 1 = Driscoll DF. Compounding TPN admixtures: then and now. J Parenter Enteral Nutr 2003; 27:433-438.. 10., 1 = Warshawsky KY. Intravenous fat emulsions in clinical practice. Nutr Clin Pract 1992; 7:187-196.. 11., 1 = Barr LH, Dunn GD, Brennan MF. Essential fatty acid deficiency during total parenteral nutrition. Ann Surg 1981; 193:304-311.. 12., 1 = Alhazzani W, Jacobi J, Sindi A, et al. The effect of selenium therapy on mortality in patients with sepsis syndrome. Crit Care Med 2013; 41:1555-1564.

Peripheral Parenteral Nutrition, 1 = Peripheral Parenteral Nutrition. 26., 1 = Culebras JM, Martin-Pena G, Garcia-de-Lorenzo A, et al. Practical aspects of peripheral parenteral nutrition. Curr Opin Clin Nutr Metab Care 2004; 7:303-307.. 27., 1 = Anderson AD, Palmer D, MacFie J. Peripheral parenteral nutrition. Br J Surg 2003; 90:1048-1054.

Judge a tree from its fruit, not from the eaves.
Euripedes
484-406 BC
Adrenal and thyroid disorders are rarely the primary reason for admission to an ICU. However, critical illness can influence adrenal and thyroid function, and there is concern that this influence can have a negative impact on outcomes. This chapter describes the spectrum of adrenal and thyroid disorders that occur in critically ill patients, and how to detect and manage each of these disorders.

The adrenal gland plays a major role in the adaptive response to stress. The adrenal cortex releases glucocorticoids and mineralocorticoids that promote glucose availability and maintain extracellular volume, while the adrenal medulla releases catecholamines that support the circulation. Attenuation or loss of this adrenal response leads to hemodynamic instability, volume depletion, and defective energy metabolism (1,2). Adrenal insufficiency can remain silent until the adrenal gland is called on to respond to a physiologic stress. When this occurs, adrenal insufficiency becomes an occult catalyst that speeds the progression of acute, life-threatening conditions.
The activity of the adrenal glands is governed by the release of adrenocorticotrophic hormone (ACTH) from the anterior pituitary gland, which, in turn, is governed by the production of corticotrophin-releasing hormone (CRH) in the hypothalamus (see Figure 50.1). Adrenal insufficiency can be the result of suppression at the hypothalamic-pituitary level, or primary suppression of adrenal gland activity.

Cortisol (hydrocortisone) is the major glucocorticoid released by the adrenal cortex. The daily production of cortisol in the normal (un-stressed) adult is 15-25 mg/day, and this can increase up to 350 mg/day during periods of maximum physiological stress (2).
PLASMA CORTISOL: About 90% of the cortisol in plasma is bound to corticosteroidbinding globulin (CBG) and albumin, while the remaining 10% is the free or biologically active form (1,2). The commercial assay for plasma cortisol measures both bound and unbound fractions; i.e., total cortisol. This assay can be misleading in acutely ill patients because plasma levels of CBG fall by as much as 50% during an acute illness (2). In one study of ICU patients with sepsis, total cortisol levels were diminished in 40% of the patients, while free cortisol levels were consistently elevated (4).

Adrenal insufficiency is common in critically ill patients. The overall prevalence is 10-20% (1), but rates as high as 60% have been reported in patients with severe sepsis and septic shock (3). The adrenal suppression in critically ill patients is often reversible, and is called critical illness-related corticosteroid insufficiency (CIRCI) (1). The mechanisms involved in CIRCI are complex, and are shown in Figure 50.1 (1-3,5). The systemic inflammatory response plays a major role in CIRCI. Suppression at the hypothalamicpituitary level is particularly prominent, and is responsible for as many as 75% of the cases of adrenal suppression in patients with severe sepsis and septic shock (3).

As mentioned, severe sepsis and septic shock are the leading causes of adrenal suppression in critically ill patients. Other infection-related causes of adrenal suppression include HIV infection, systemic fungal infections, and meningococcemia (which can precipitate adrenal hemorrhage) (2,5).
Noninfectious sources of adrenal suppression in ICU patients include: (a) abrupt discontinuation of chronic steroid therapy, (b) adrenal hemorrhage from disseminated intravascular coagulation (DIC) or anticoagulant therapy, and (c) drugs that inhibit the synthesis of cortisol (e.g., etomidate and ketoconazole) or accelerate the metabolism of cortisol (e.g., phenytoin or rifampin) (2,5).

The principal manifestation of adrenal suppression in critically ill patients is hypotension that is refractory to volume resuscitation (1-3). The typical electrolyte abnormalities that accompany adrenal insufficiency (i.e., hyponatremia and hyperkalemia) are uncommon in the adrenal suppression associated with critical illness.

Adrenal suppression should be suspected in any ICU patient with hypotension that does not respond to volume resuscitation. Unfortunately, the diagnosis of adrenal suppression in critically ill patients is steeped in uncertainty. (Note: The use of total cortisol levels to evaluate adrenal function is a major problem in critically ill patients because of the confounding influence of plasma proteins on total cortisol measurements, as mentioned previously.)
FIGURE 50.1 Mechanisms of adrenal suppression in ICU patients. CRF = cortico-trophin-releasing hormone; ACTH = adrenocorticotrophic hormone.

A popular (but often unnecessary) test of adrenal function in ICU patients is the rapid
ACTH stimulation test, which can be performed at any time of the day or night. A blood sample is obtained for a baseline (random) plasma cortisol level, and the patient is given synthetic ACTH (Cosyntropin) intravenously in a dose of 250 eg. One hour after the ACTH injection, a second blood sample is obtained for a repeat plasma cortisol level. The interpretation of the test results is as follows (1,2):
The best predictor of adrenal suppression in critically ill patients is a random plasma cortisol level <10 e<g/dL, or an increment in plasma cortisol of <9 ecg/dL after the intravenous injection of synthetic ACTH (250 <g).
The favorite approach to evaluating adrenal function in the ICU is to rely on the random plasma cortisol level. A level that is 235 e<g/dL is evidence of normal or adequate adrenal function, while a baseline cortisol level that is below 10 e<g/dL is evidence of adrenal suppression. A rapid ACTH stimulation test can be performed when the random serum cortisol level is 10-34 o<g/dL. However, a normal response to ACTH (i.e., an increment in serum cortisol of =>9 e<g/dL) does not eliminate the possibility of secondary adrenal suppression due to hypothalamic-pituitary dysfunction (which may be more common than suspected in ICU patients).

In patients with septic shock, plasma cortisol levels are not necessary for identifying patients who might benefit from corticosteroid therapy. In these patients, a trial of intravenous hydrocortisone is recommended when hypotension is refractory to volume resuscitation (and requires a vasopressor drug).

The treatment of critical illness-related adrenal suppression is intravenous hydrocortisone in a dose of 200-300 mg daily (i.e., 50 mg every 6 hours, or 100 mg every 8 hours) (1). The addition of a mineralocorticoid (i.e., fludrocortisone, 50 eg orally once daily) is considered optional (1), because hydrocortisone has excellent mineralocorticoid activity (see Table 50.1).
Hydrocortisone can be discontinued after satisfactory resolution of the underlying condition. In septic shock, hydrocortisone can be discontinued when vasopressor therapy is no longer necessary, and serum lactate levels have normalized. A gradual taper of the hydrocortisone dose (over at least a few days) is recommended to prevent a rebound increase in proinflammatory mediators (1).

Corticosteroid, 1 = . Corticosteroid, 2 = . Corticosteroid, 3 = . Hydrocortisone, 1 = 20 mg. Hydrocortisone, 2 = 4. Hydrocortisone, 3 = 1. Prednisone, 1 = 5 mg. Prednisone, 2 = 3. Prednisone, 3 = 2. Methylprednisone, 1 = 4mg. Methylprednisone, 2 = 2. Methylprednisone, 3 = 2. Dexamethasone, 1 = 0.75 mg. Dexamethasone, 2 = 1. Dexamethasone, 3 = 4
tAnti-inflammatory activity (1 = best, 4 = worst)
4Minerdocorticoid activity (1 = bast, 4 = worst)
From Referanca 4.

Laboratory tests of thyroid function can be abnormal in up to 90% of critically ill patients (6). In most cases, the abnormality is a consequence of non-thyroidal illness, and is not a sign of pathologic thyroid disease (6,7). This section describes the laboratory evaluation of thyroid function, and explains how to identify non-thyroidal illness as a cause of abnormal thyroid function tests.

Thyroxine (T.4) is the principal hormone secreted by the thyroid gland, but the active form is triiodothyronine (T3), which is formed by deiodination of thyroxine in extrathyroidal tissues. Both T3 and T,4 are extensively (>99%) bound to plasma proteins (especially thyroxine-binding globulin), and less than 1% of either hormone is present in the free, or biologically active, form (8). Because of the potential for alterations in plasma proteins and protein binding in acute illness, free T4 and T3 levels are more reliable for assessing thyroid function in ICU patients. Free T3 levels are not routinely available, so free T, levels are used to evaluate thyroid function in acutely ill patients.

The plasma level of thyroid-stimulating hormone (TSH) is considered the most reliable test of thyroid function, and is useful for identifying non-thyroidal illness, and for distinguishing between primary and secondary thyroid disorders. Plasma TSH levels have a diurnal variation, with the lowest values in late afternoon, and the highest values around the hour of sleep. TSH levels can vary by as much as 40% over a 24-hour period (9), and this diurnal variation must be considered when interpreting changes in plasma TSH levels. The reference range for serum TSH is 0.3-4.5 mU/dL (10).

Because of the negative feedback exerted by the thyroid hormones on TSH secretion, plasma TSH levels can distinguish primary from secondary thyroid disorders. For example, in patients with hypothyroidism, an elevated plasma TSH level is evidence of primary hypothyroidism, while a reduced TSH level is evidence of secondary hypothyroidism due to hypothalamic—pituitary dysfunction.

Plasma TSH levels are normal in a majority of instances where abnormal thyroid function tests are the result non-thyroidal illness (6). However, plasma TSH levels can be depressed in 30%, and elevated in 10%, of these patients (6). TSH secretion can be depressed by sepsis, corticosteroids, and dopamine infusions (11), and these factors must be considered when interpreting plasma TSH levels.

The interpretation of free T4 and TSH levels is shown in Table 50.2.

Normal Range, Free T, = 0.8-1.8 ng/L. Normal Range, ‘TSH = 0.35-4.5 mU/mL. Non-Thyroidal lliness (severe), Free T, = 4. Non-Thyroidal lliness (severe), ‘TSH = NL or +. Primary Hypothyroidism, Free T, = 4. Primary Hypothyroidism, ‘TSH = t. Secondary Hypothyroidism, Free T, = +. Secondary Hypothyroidism, ‘TSH = +. Primary Hyperthyroidism, Free T, = t. Primary Hyperthyroidism, ‘TSH = +

Acute, non-thyroidal illness is associated with low plasma levels of free T3, as a result of impaired conversion of T, to T3 in non-thyroidal tissue (6). With increasing severity of illness, both free T3 and free T, levels are depressed, which is the pattern reported in 30— 50% of ICU patients (6,7). As mentioned earlier, plasma TSH levels are normal in a majority of patients with non-thyroidal illness, but TSH levels can be depressed in selected conditions (e.g., sepsis, corticosteroid therapy, or dopamine infusions).

Primary thyroid disorders are characterized by reciprocal changes in free T4 and TSH levels, while in secondary thyroid disorders (due to hypothalamic-pituitary dysfunction), free T, and TSH levels change in the same direction.

Thyrotoxicosis is almost always the result of primary hyperthyroidism. Notable causes include autoimmune thyroiditis, and chronic therapy with amiodarone (12).

The principal manifestations of thyrotoxicosis are agitation, tachycardias (including atrial fibrillation), and fine tremors. Elderly patients with hyperthyroidism can be lethargic rather than agitated; this condition is called apathetic thyrotoxicosis. The combination of lethargy and atrial fibrillation is a frequently cited presentation for apathetic thyrotoxicosis in the elderly (13).

An uncommon but severe form of hyperthyroidism known as thyroid storm can be precipitated by acute illness or surgery. This condition is characterized by hyperpyrexia (body temperatures can exceed 104°F), severe agitation or delirium, and severe tachycardia with high-output heart failure. Advanced cases are associated with obtundation or coma, generalized seizures, and hemodynamic instability. If overlooked and left untreated, the outcome is uniformly fatal (12).

The plasma TSH assay is the most sensitive and specific diagnostic test for hyperthyroidism, and is recommended as the initial screening test for suspected hyperthyroidism (12). TSH levels are <0.01 mU/dL in mild cases of hyperthyroidism, and TSH levels are undetectable in most cases of overt thyrotoxicosis (12). A normal TSH level (0.3—0.45 mmU/dL) excludes the diagnosis of hyperthyroidism (12).

Table 50.3 includes the drugs and dosing regimens used for the treatment of thyrotoxicosis and thyroid storm.

Treatment with B-Receptor antagonists relieves the tachycardia, agitation, and fine tremors in thyrotoxicosis. Propranolol has been the most widely used B-Receptor antagonist in hyperthyroidism (see Table 50.3 for the dosing recommendations), but it is a non-selective B-Receptor antagonist, which is not optimal for patients with asthma. More selective B-Receptor antagonists like metoprolol (25-50 mg PO every 4 hours) or esmalol (see Table 15.1 for dosing recommendations) can be used for thyrotoxicosis. However, propranolol remains the drug of choice for treatment of thyroid storm (12).

Two drugs are used to suppress thyroxine production: methimazole and propylthiouracil (PTU). Both are given orally. Methimazole is preferred for the treatment of thyrotoxicosis, while PTU is favored for the treatment of thyroid storm (12). Uncommon but serious side effects include cholestatic jaundice for methimazole, and fulminant hepatic necrosis, plus agranulocytosis, for PTU (12). (See Table 50.3 for the dosing regimens for each drug).
for Thyrotoxicosis and Thyroid Storm

Propanolol, 1 = Desing:. Propanolol, 2 = 10-40 mg PO TID or GID for thyrotoxicosis, and 60-80 mg IV or PO every 4 hrs for thyroid storm.. Propanolol, 1 = Comment:. Propanolol, 2 = Blocks conversion of T, to T, In high doses. Use cautiously in patients with systolic heart failure. Use selective B-blocker In asthmatics.. Methimazole, 1 = Dosing:. Methimazole, 2 = 10-20 mg PO once dally for thyrotoxicosis, and 60-80 mg PO once dally for thyroid storm.. Methimazole, 1 = Comment:. Methimazole, 2 = Blocks synthesis of T,. Preferred to PTU for thyrotoxicosis, but not for thyroid storm.. Propylthiouracil, 1 = Dosing:. Propylthiouracil, 2 = 50-150 mg PO TID for thyrotoxicosis, and 500-—1,000 mg PO loading dose, then 250 mg PO every 4 hrs for thyroid storm.. Propylthiouracil, 1 = Comment:. Propylthiouracil, 2 = Blocks both T, synthesis and the conversion of T, to T,. Preferred to methimazole for thyroid storm.. lodine, 1 = Dosing:. lodine, 2 = 50 drops of saturated potassium iodide (250 mg iodine) PO every 6 hrs, for severe thyrotoxicosis or thyroid storm.. lodine, 1 = Comment:. lodine, 2 = Blocks synthesis and secretion of Ty. Used in combination with antithyroid drugs.. Hydrocortisone, 1 = Dosing:. Hydrocortisone, 2 = 300 mg IV as a loading dose, then 100 mg avery 8 hrs, for thyroid storm only.. Hydrocortisone, 1 = Comment:. Hydrocortisone, 2 = Used for prophylaxis of the relative adrenal insufficiency in thyroid storm.
Dosing regimens from Refarence 12.

In severe cases of hyperthyroidism, iodine (which blocks the synthesis and release of T4) can be added to antithyroid drug therapy. The iodine is given orally as a saturated potassium iodide solution (Lugol's solution). In patients with an iodine allergy, lithium (300 mg orally every 8 hours) can be used as a substitute (14).

In addition to the above measures, the management of thyroid storm often requires aggressive volume resuscitation to replace fluid losses from vomiting, diarrhea, and heightened insensible fluid loss. Thyroid storm can accelerate glucocorticoid metabolism and create a relative adrenal insufficiency, and prophylactic therapy with intravenous hydrocortisone (300 mg IV as a loading dose, followed by 100 mg IV every 8 hours) is recommended for thyroid storm (12).

Symptomatic hypothyroidism is uncommon, with a prevalence of only 0.3% in the general population (15). Most cases are the result of chronic autoimmune thyroiditis (Hashimoto's thyroiditis). Other causes include radioiodine, thyroidectomy, or hypothalamic-pituitary dysfunction from tumors and hemorrhagic necrosis (Sheehan's syndrome), and drugs (lithium, amiodarone).

The clinical manifestations of hypothyroidism are often subtle, and include dry skin, fatigue, muscle cramps, and constipation. Contrary to popular perception, obesity is not a consequence of hypothyroidism (15). More advanced cases of hypothyroidism can be accompanied by hypo-natremia and a skeletal muscle myopathy, with elevations in muscle enzymes (creatine phosphokinase and aldolase), and an increase in the serum creatinine (from creatine released by skeletal muscle) that is not caused by renal dysfunction (16).

The most common cardiovascular manifestation of hypothyroidism is pericardial effusion, which is the most common cause of an enlarged cardiac silhouette in patients with hypothyroidism (17). These effusions usually accumulate slowly and do not cause cardiac compromise. Pleural effusions are also common in hypothyroidism. The pleural and pericardial effusions in hypothyroidism are due to an increase in capillary permeability, and are exudative in quality.

Advanced cases of hypothyroidism are accompanied by an edematous appearance known as myxedema. This condition is mistaken for edema, but is caused by the intradermal accumulation of proteins (18). Myx-edema is also associated with hypothermia and depressed consciousness. The latter condition is called myxedema coma, even though unresponsiveness is uncommon (18).

Serum T3 levels can be normal in hypothyroidism, but free T, levels are always reduced (15). Serum TSH levels are elevated (often above 10 mU/dL) in primary hypothyroidism, and are depressed in hypothyroidism due to hypothalamic-pituitary dysfunction.

The treatment for mild to moderate hypothyroidism is levothyroxine (T4), which is given orally in a single daily dose of 50 to 200 eg (19). The initial dose is usually 50 g/day, and this is increased in 50 eg/day increments every 3 to 4 weeks. The optimal replacement dose of levothyroxine is determined by monitoring the serum TSH level. Intravenous thyroxine is recommended for severe hypothyroidism (at least initially), because of the risk of impaired gastrointestinal motility in severe hypothyroidism. One recommended regimen includes an initial intravenous dose of 250 eg, followed on the next day by a dose of 100 g, and followed thereafter by a daily dose of 50 eg (19).

Because the conversion of T4 to T3 (the active form of thyroid hormone) can be depressed in critically ill patients (18), oral therapy with T3 can be used to supplement thyroxine (T4) replacement therapy. In patients with depressed consciousness, T3 can be given (via NG tube) in a dose of 25 eg every 12 hours until the patient awakens (20). Studies evaluating the benefits of T3 supplementation have shown mixed results (15).

1. Marik PE, Pastores SM, Annane D, et al. Recommendations for the diagnosis and management of corticosteroid insufficiency in critically ill adult patients: consensus statement from an international task force by the American College of Critical Care Medicine. Crit Care Med 2008; 36L1937-1949.
. Marik PE. Critical illness-related corticosteroid insufficiency. Chest 2009; 135:181193.
. Annane D, Maxime V, Ibrahim F, et al. Diagnosis of adrenal insufficiency in severe sepsis and septic shock. Am J Respir Crit Care Med 2006; 174:1319-1326.
. Hamrahian AH, Oseni TS, Arafah BM. Measurements of serum free cortisol in critically ill patients. N Engl J Med 2004; 350:1629-1638.
. Bornstein SR. Predisposing factors for adrenal insufficiency. N Engl J Med 2009; 360:2328-2339.
12., Thyrotoxicosis = Bahn RS, Burch HB, Cooper DS, et al. Hyperthyroidism and other causes of thyrotoxicosis: Management guidelines of the American Thyroid Association and the American Association of Clinical Endocrinologists. Thyroid 2011; 21:593-646.. 13., Thyrotoxicosis = Klein I. Thyroid hormone and the cardiovascular system. Am J Med 1990; 88:631- 637.. 14., Thyrotoxicosis = Migneco A, Ojetti V, Testa A, et al. Management of thyrotoxic crisis. Eur Rev Med Pharmacol Sci 2005; 9:69-74.

The desire to take medicine is, perhaps, the great feature that distinguishes man from other animals.
Sir William Ostler

"Pain is perfect misery, the worst of evils...
John Milton Paradise Lost
Contrary to popular perception, our principal function in patient care is not to save lives (since this is impossible on a consistent basis), but to relieve pain and suffering. And the patients that experience the greatest pain and suffering are the ones in the ICU. If you want an idea of how prepared we are to relieve pain and suffering in the ICU, take a look at Figure 51.1 (1). This might explain why most patients who are discharged from the ICU remember discomfort and unrelieved pain as a dominant experience during their ICU stay (2).
This chapter describes the use of intravenous analgesics and sedatives to achieve patient comfort in the ICU. Pertinent reviews, along with the most recent clinical practice guidelines on analgesia and sedation in the ICU, are included in the bibliography at the end of the chapter (3-5).

In patients who have been discharged from an ICU, surveys conducted at 6 months to 4 years after discharge show that 20-40% of the patients have no recollection of what happened during their ICU stay (2,6,7). This may be a reflection of the amnestic effects of benzodiazepines, because patients who have not been heavily sedated while in the ICU are more likely to recall experiences during the ICU stay (7). Regardless of the mechanism, amnesia for the ICU stay seems beneficial because it eliminates memories of a stressful experience.

It seems axiomatic that the ICU experience is stressful for a majority of patients. Several stressors have been identified by patients after discharge from the ICU, and the major ones are (a) unrelieved pain, (b) inadequate sedation, (c) inability to communicate (in intubated patients), (d) difficulty sleeping, and (e) hallucinations and nightmares (2,6,7). The most frequently cited source of stress is painful procedures (8). Stressful experiences during the ICU stay can have prolonged neuropsychiatric effects. One study has reported that 25% of patients with stressful experiences in the ICU showed symptoms of posttraumatic stress disorder 4 years later (9).
FIGURE 51.1 Percentage of housestaff physicians and ICU nurses who answered incorrectly when asked if diazepam (Valium) relieved pain.

Surveys indicate that 50-80% of ICU patients experience pain and discomfort while in the ICU (2,6,7). Surprisingly, the prevalence of pain is the same in surgical ICUs and medical ICUs (10).

Critically ill patients experience pain more readily than healthy subjects (hypernociception). For example, the most painful experiences for ICU patients are endotracheal suctioning, and being turned in bed (8). In addition, 30-50% of critically ill patients experience pain while at rest, without a noxious stimulus (8,10). This type of pain typically involves the back and lower extremities.
The heightened pain experience in critically ill patients is attributed to immobility and systemic inflammation. (The rest pain experienced by ICU patients is similar to the myalgias experienced during a systemic infection.) Failure to recognize this heightened nociceptive state is a major source of inadequate pain relief in the ICU. Frequent pain assessments using pain intensity scales (described next) can help to correct problems with inadequate pain control in the ICU (3).

Pain is defined as "an unpleasant sensory or emotional experience" (11), which highlights the subjective nature of pain. The pain sensation can be described in terms of intensity, duration, location, and quality, but pain intensity is the parameter most often monitored because it reflects the "unpleasantness" of pain.

Facial Expression, Description = Relaxed. Facial Expression, Description = Relaxed. Facial Expression, ‘Score = +. , Description = Partially tightened. , Description = Partially tightened. , ‘Score = Oh. , Description = Fully tightened. , Description = Fully tightened. , ‘Score = . , Description = Grimacing. , Description = Grimacing. , ‘Score = &. Upper Limbs, Description = No movement. Upper Limbs, Description = No movement. Upper Limbs, ‘Score = —. , Description = Partially bent. , Description = Partially bent. , ‘Score = OM. , Description = Fully bent, fingers flexed. , Description = Fully bent, fingers flexed. , ‘Score = . , Description = Permanently retracted. , Description = Permanently retracted. , ‘Score = &. Compliance with, Description = Tolerating ventilator. Compliance with, Description = Tolerating ventilator. Compliance with, ‘Score = =. Ventilation, Description = Coughing, but tolerating ventilator. Ventilation, Description = Coughing, but tolerating ventilator. Ventilation, ‘Score = . , Description = Fighting ventilator. , Description = Fighting ventilator. , ‘Score = ow. , Description = Unable to control ventilation. , Description = Unable to control ventilation. , ‘Score = |~. , Description = Total Score. , Description = Total Score. , ‘Score = Total Score. , Description = Score. , Description = Interpretation. , ‘Score = . , Description = 1. , Description = No pain. , ‘Score = . , Description = 1-5. , Description = Acceptable pain control. , ‘Score = . , Description = 12. , Description = Maximal pain. , ‘Score = 
Fram Referance 10.

Pain intensity scales are used to determine the need for, and evaluate the effectiveness of, analgesic therapy. There are 6 different pain intensity scales, but only a few are needed to assess pain intensity in most ICU patients.
1. When patients can reliably self-report pain, the Numerical Ranking Scale can be used for pain assessment (12). This is a ruler with 10 equally spaced divider markings, numbered 1 (no pain) to 10 (maximum pain). The patient points to one of the numbered markings to indicate the severity of pain. A score of 3 or less is considered adequate pain control.
2. When patients are sedated and on a ventilator, the Behavioral Pain Scale (BPS) shown in Table 51.1 is recommended for pain assessments (3). This scale evaluates pain intensity by elicited behaviors (i.e., facial expression, arm flexion, and tolerance of mechanical ventilation) (11). Scores can range from 3 (no pain) to 12 (maximum pain). A score of 5 or less represents adequate pain control (3).

Vital signs (e.g., heart rate) show a poor correlation with patients' reports of pain intensity (the gold standard), and they can remain unchanged in the presence of pain (13). As a result, vital signs are not recommended for pain assessments (3).

The natural chemical derivatives of opium are called opiates. Opiates and other substances that produce their effects by stimulating discrete opioid receptors in the central nervous system are called opioids. Stimulation of opioid receptors produces a variety of effects, including analgesia, sedation, euphoria, pupillary constriction, respiratory depression, bradycardia, constipation, nausea, vomiting, urinary retention, and pruritis (14). The term narcotic refers to a general class of drugs that blunt sensation and depress consciousness (i.e., narcotize).
Opioids are the most frequently used drugs for pain relief in the ICU (3,8), and are given intravenously as intermittent bolus doses or continuous infusions. Opioids also produce mild sedation but, unlike benzodiazepines, they have no amnestic effects (15).

The opioids used most often in the ICU are morphine, fentanyl, and hydromorphone. The recommended intravenous doses for each drug are shown in Table 51.2. It is important to emphasize that opioid dose requirements can vary widely in individual patients, and that the effective dose of an opioid is determined by each patient's response, not by the recommended dose range of the drug.

Fentanyl has replaced morphine as the most popular opioid analgesic in ICUs (8). The advantages of fentanyl over morphine include a more rapid onset of action (because fentanyl is 600 times more lipid soluble than morphine), less risk of hypotension (because fentanyl does not promote histamine release) (16), and the absence of active metabolites. The relative lack of adverse hemodynamic effects is a major source of fentanyl's appeal in critically ill patients.

Opioids are metabolized primarily in the liver, and the metabolites are excreted in the urine. Morphine has several active metabolites that can accumulate in renal failure. One metabolite (morphine 3-glucuronide) can produce central nervous system excitation with myoclonus and seizures (17), while another metabolite (morphine-6-glucuronide) has more potent analgesic effects than the parent drug (14). To avoid accumulation of these metabolites, the maintenance dose of morphine should be reduced by 50% in patients with renal failure (18). Fentanyl does not have active metabolites, and dose not require dose adjustments in renal failure.
Morphine also promotes the release of histamine, and this can produce systemic vasodilation and a decrease in blood pressure (14). Hypotension can occur, and is typically seen in patients with a hyperadrenergic state and increased peripheral vascular tone (4). Morphine-induced histamine release does not promote bronchoconstriction, and morphine doses of 1.5 mg/kg have been given to asthma patients without adverse consequences (19).

Hydromorphone is a morphine derivative that may produce more effective analgesia (according to a recent meta-analysis) (20). However, other than the lack of a need for dose alterations in renal failure, hydromorphone has no apparent clinical advantage over morphine in critically ill patients.
able 51.2 Commonly Used Intravenous Opioid
Onset, Morphine = 5-10 min. Onset, Hydrocodone = 5-15 min. Onset, —_—— Fentanyl = 1-2 min. Bolus Dosing, Morphine = 2-4 mg q 1-2 hr. Bolus Dosing, Hydrocodone = 0.2-0.6 mg q 1-2 hr. Bolus Dosing, —_—— Fentanyl = 0.35-0.5 pg/kg q 0.5-1 hr. Infusion rate, Morphine = 2-30 mg/hr. Infusion rate, Hydrocodone = 0.5-3 mg/hr. Infusion rate, —_—— Fentanyl = 0.7-10 uwgq/kg/nr. PCA, Morphine = . PCA, Hydrocodone = 1. PCA, —_—— Fentanyl = 4. Demand (bolus) Lockout Interval, Morphine = 0.5-3 mg 10-20 min. Demand (bolus) Lockout Interval, Hydrocodone = 0.1-0.5 mg 5-15 min. Demand (bolus) Lockout Interval, —_—— Fentanyl = 15-75 ug 3-10 min. Lipid Solubility, Morphine = x. Lipid Solubility, Hydrocodone = 0.2x. Lipid Solubility, —_—— Fentanyl = 600x. Active Metabolites, Morphine = yes. Active Metabolites, Hydrocodone = yes. Active Metabolites, —_—— Fentanyl = No. Histamine Release, Morphine = yes. Histamine Release, Hydrocodone = No. Histamine Release, —_—— Fentanyl = No. Dose Adjustment for Renal Failure, Morphine = 450%. Dose Adjustment for Renal Failure, Hydrocodone = . Dose Adjustment for Renal Failure, —_—— Fentanyl = 

Remifentanil is an ultra-short acting opioid that is given by continuous intravenous infusion, using the dosing regimen shown below.
Dosing Regimen: 1.5 ug/kg as a loading dose, followed by a continuous infusion at 0.5— 15 yg/kg/hr (3).
Analgesic effects are lost within 10 minutes after stopping the drug infusion. The short duration of action is a reflection of drug metabolism; i.e., remifentanil is broken down by nonspecific esterases in plasma. Because drug metabolism does not take place in the liver or kidneys, dose adjustments are not necessary in renal or hepatic failure.
Remifentanil's short duration of action is most advantageous in conditions that require frequent evaluations of cerebral function (e.g., traumatic head injuries). The abrupt cessation of opioid activity can precipitate acute opioid withdrawal (5), which can be prevented by combining remifentanil with a longer-acting opioid.

Meperidine (Demerol, Pethidine) is an opioid analgesic that is no longer favored for pain control in the ICU because of the potential for neurotoxicity. Meperidine is metabolized in the liver to normeperidine, a metabolite that is slowly excreted by the kidneys (elimination half-life is 15-40 hours) (21). Accumulation of normeperidine can produce central nervous system excitation, with agitation, myoclonus, delirium, and generalized seizures (21). Since renal dysfunction is prevalent in ICU patients, there is a high risk for accumulation of the neurotoxic metabolite of meperidine in ICU patients.

For patients who are awake and capable of drug self-administration, patient-controlled analgesia (PCA) can be an effective method of pain control, and may be superior to intermittent opioid dosing. The PCA method uses an electronic infusion pump that can be activated by the patient. When pain is sensed, the patient presses a button connected to the pump to receive a small intravenous bolus of drug. After each bolus, the pump is disabled for a mandatory time period called the "lockout interval," to prevent overdosing. The opioid dosing regimens for PCA are shown in Table 51.2. The minimum lockout interval is determined by the time required to achieve peak drug effect (22). When writing orders for PCA, you must specify the initial loading dose (if any), the lockout interval, and the repeat bolus dose. PCA can be used alone or in conjunction with a lowdose opioid infusion.

Opioids produce a centrally mediated, dose-dependent decrease in respiratory rate and tidal volume (23,24), but respiratory depression and hypoxemia are uncommon when opioids are given in the usual doses (25). Opioid doses that impair arousal also impair ventilation and produce hypercapnia (23). Patients with sleep apnea syndrome or chronic hypercapnia are particularly prone to respiratory depression from opioids.

Opioid analgesia is often accompanied by decreases in blood pressure and heart rate, which are the result of decreased sympathetic activity and in-creased parasympathetic activity. These effects are usually mild and well-tolerated, at least in the supine position (26). Decreases in blood pressure can be pronounced in patients with hypovolemia or heart failure (where there is an increased baseline sympathetic tone), or when opioids are given in combination with benzodiazepines (27). Opioid-induced hypo-tension is rarely a threat to tissue perfusion, and the blood pressure re-sponds to intravenous fluids or small bolus doses of vasopressors.

Opioids are well known for their ability to depress bowel motility via activation of opioid receptors in the GI tract. This is a source of troublesome constipation in cancer patients. In critically ill patients, impaired GI motility can promote reflux of enteral tube feedings into the oropharynx, creating a risk for aspiration pneumonia. Opioid-induced bowel hypomotility can be partially reversed with enteral naloxone (8 mg every 6 hours) without affecting opioid analgesia. In one small study (28), this approach resulted in fewer cases of aspiration pneumonia.

Opioids can promote vomiting via stimulation of the chemoreceptor trigger zone in the lower brainstem (23). All opioids are equivalent in their ability to promote vomiting, but substituting one opioid for another sometimes resolves the problem.

The list of intravenous non-opioid analgesics is a very short one, and includes only three drugs: acetaminophen, ketorolac, and ibuprofen. These drugs are used primarily for pain control in the early postoperative period. They can be used alone for mild pain, but are used in combination with an opioid analgesic for moderate-to-severe pain. The goal of combination analgesic therapy is to reduce the opioid dose ("opioid sparing" effect) and hence reduce the risk of an opioid-related adverse effect. The intravenous dosing regimens for the non-opioid analgesics are presented in Table 51.3.

Ketorolac is a nonsteroidal anti-inflammatory drug (NSAID) that was introduced in 1990 as the first parenteral analgesic agent for postoperative pain that does not produce respiratory depression (29). Ketorolac has a proven opioid sparing effect, and the dose of opioid analgesics can often be reduced by 25-50% (30).

Ketorolac can be given by intravenous (IV) or intramuscular (IM) injection. The recommended dosing regimen for moderate-to-severe pain in adults is 30 mg IV or IM every 6 hrs, for up to 5 days (31). A dose reduction of 50% is recommended for elderly patients (age 265 yrs), and for patients with a body weight <50 kg. IM injection of ketorolac can produce hematomas (32), so IV bolus injection may be preferred.

The beneficial actions of ketorolac and other NSAIDS are attributed to inhibition of prostaglandin production, but this also creates a risk for adverse effects, particularly gastric mucosal injury, upper GI hemorrhage, and impairment of renal function (31). These adverse effects are typically associated with excessive dosing or prolonged exposure to NSAIDS, and they are uncommon when ketorolac is given in the recommended doses, and the treatment period is limited to 5 days (32-34).

Keterolac, Dosing Regimens and Comments = Dosing: Comment:. Keterolac, Dosing Regimens and Comments = 30 mg IV or IM every 6 hrs, for up to 5 days. Reduce dose by 50% for age 265 yrs or body weight <50 kg. Keterolac is an NSAID, and has anti-inflam- matory and antipyretic effects. Serious compli- cations are uncommon when treatment is limited to 5 days.. Ibuprofen, Dosing Regimens and Comments = Dosing: Comment.. Ibuprofen, Dosing Regimens and Comments = 400-800 mg IV every 6 hrs. Ibuprofen is also an NSAID, with actions simi- lar to kerotolac. Serious complications are uncommon when used for short-term pain relief.. Acetaminophen, Dosing Regimens and Comments = Dosing:. Acetaminophen, Dosing Regimens and Comments = 1 gram IV every 6 hrs. Daily dose should not exceed 4 grams.

Ibuprofen is very similar to ketorolac because (a) it is an NSAID that can be given intravenously, (b) it has an opioid sparing effect, and (c) it is safe when used for shortterm pain control (35). The intravenous dose of ibuprofen is 400-800 mg IV every 6 hrs, with a maximum daily dose of 3.2 grams (35). Unlike ketorolac, the treatment period for ibuprofen has no recommended time limit. Clinical trials of IV ibuprofen typically employ a treatment period of 24—48 hrs, and serious complications are uncommon over that time period.

Acetaminophen was approved for intravenous use in 2010, and is intended for the shortterm treatment of pain and fever in postoperative patients who are unable to receive acetaminophen via the oral or rectal routes (36). The recommended dose for IV acetaminophen is 1 gram every 6 hrs, with a maximum allowable dose of 4 grams daily (to prevent acetaminophen hepatotoxicity) (36). This dosing regimen has a documented opiate sparing effect in postoperative patients (37).

Acetaminophen has no anti-inflammatory activity, which is a major disadvantage in critically ill patients. Furthermore, although the daily dose limitation of 4 grams is intended to avoid acetaminophen hepatotoxicity, the toxic dose of acetaminophen has not been evaluated in critically ill patients. For these reasons, IV acetaminophen is not the optimal choice for opiate-sparing analgesia in critically ill patients. (For a description of acetaminophen hepatotoxicity, see Chapter 54.)

Non-opioid analgesia is usually required for neuropathic pain (e.g., from diabetic neuropathy), and the recommended drugs for this type of pain are gabapentin and carbamazepine (3). Both drugs must be given enterally. Effective drug doses vary in individual patients, but typical doses are 600 mg every 8 hrs for gabapentin, and 100 mg every 6 hours for carbamazepine (oral suspension).

Anxiety and related disorders (agitation and delirium) are observed in as many as 85% of patients in the ICU (38). These disorders can be defined as follows.
1. Anxiety is characterized by exaggerated feelings of fear or apprehension that are sustained by internal mechanisms more than external events.
2. Agitation is a state of anxiety that is accompanied by increased motor activity.
3. Delirium is an acute confusional state that may, or may not, have agitation as a component. Although delirium is often equated with agitation, there is a hypoactive form of delirium that is characterized by lethargy. (Delirium is described in more detail in Chapter 44.)
The common denominator in these disorders is the absence of a sense of well-being.

Sedation is the process of relieving anxiety and establishing a state of calm. This process includes general supportive measures (like frequent communication with patients and families), and drug therapy. The drugs used most often for sedation in ICUs are midazolam and propofol (3).

+4, 1 = Combative. +4, 2 = Overly combative or violent; immediate danger to staff. +3, 1 = Very agitated. +3, 2 = Pulls on or removes tube(s}/catheter(s), or aggressive behavior. +2, 1 = Agitated. +2, 2 = Frequent non-purposeful movement or patient- ventilator asynchrony. +1, 1 = Restless. +1, 2 = Anxious or apprehensive but movements not aggressive or vigorous. 0, 1 = Alert & calm. 0, 2 = . +1, 1 = Drowsy. +1, 2 = Not fully alert, but awakens for >10 sec, with eye contact, to voice. -2, 1 = Light sedation. -2, 2 = Briefly awakens (<10 sec), with eye contact, to voice. 3, 1 = Moclerate sedation. 3, 2 = Any movernent (but no eye contact) to voice. -4, 1 = Deep sedation. -4, 2 = No respomse to voice, but movernent to physical stimulation. -5, 1 = Unarousable. -5, 2 = No response to voice or physical stimulation
To determine the RASS, proceed as follows:
Step 1 Observation: Observe the patient without interaction. If patient is alert, assign the appropriate score (0 to +4). If patient Is not alert, go to Step 2.
Step 2 Verbal Stimulation: Address patient by name in a loud voice and ask the patient to look at you. Can repeat once if necessary. If patient responds to voice, assign the appropriate score (-1 to -3). If there is no response, go to Step 3.
Step 3 Physical Stimulation: Shake the patient's shoulder. If there is no response, rub the sternum vigorously. Assign the appropriate score (4 to -5).
From Referance 39.

The routine use of sedation scales is instrumental in achieving effective sedation (3). The sedation scales that are most reliable in ICU patients are the Sedation-Agitation Scale (SAS) and the Richmond Agitation-Sedation Scale (RASS) (3), and the latter scale is shown in Table 51.4 (39). The RASS includes 4 possible scores for progressive agitation (+1 to +4) and 5 possible scores for progressive sedation (-1 to -5). The optimal RASS score is zero (alert and calm). The added advantage of RASS is the ability to monitor serial changes in a patient's mental state (40). This latter feature allows the RASS score to be used as the end-point of sedative drug therapy. (Sedative drug infusions can be titrated to achieve a RASS score of —1 to —2, which represents light sedation. )

Benzodiazepines are currently the most popular drugs for sedation in ICUs (3,8), but they are gradually losing ground to other sedatives because of problems with drug accumulation and excessive sedation.

Two benzodiazepines are used for sedation in ICUs: midazolam and lorazepam. (Diazepam is no longer used because of excessive sedation with prolonged use.) Both drugs are given intravenously, and a brief profile of each drug is presented in Table 51.5.

Midazolam (Versed) is a rapid-acting drug by virtue of its high lipid solubility. Sedative effects are apparent within 1-2 minutes after an intravenous (IV) injection of midazolam, and this makes midazolam the preferred benzodiazepine for rapid sedation (e.g., for a severely agitated or combative patient). The avid uptake of midazolam into tissues also results in rapid clearance from the bloodstream, resulting in a short duration of action (41). Because of the short-lived effect (1-2 hrs), midazolam is given as a continuous IV infusion, preceded by a bolus loading dose. However, because the brief drug effect is due to avid drug uptake into tissues, rather than drug elimination from the body, a continuous infusion of midazolam will result in progressive drug accumulation in tissues. To avoid excessive sedation from drug accumulation, midazolam infusions should be limited to <48 hrs (4).

Lorazepam (Ativan) is a longer-acting drug than midazolam, with effects lasting up to 6 hours after a single intravenous dose (3). Lorazepam can given by intermittent IV injections, or by continuous IV infusion. The intravenous preparation of lorazepam contains propylene glycol, a solvent used to increase drug solubility in plasma. This solvent has adverse effects (see later), which is why the lorazepam dosing recommendations in Table 51.5 have a maximum allowable dose (2 mg for bolus doses of lorazepam, and 10 mg/hr for continuous infusions).

Benzodiazepines are metabolized in the liver. Midazolam is metabolized by the cytochrome P450 enzyme system, and drugs that interfere with this enzyme system (e.g., diltiazem, erythromycin) can inhibit midazolam metabolism and potentiate it effects. Midazolam has one active metabolite, 1-hydroxymidazolam, that is cleared by the kidneys, so alterations in renal function can also influence midazolam sedation. Lorazepam is metabolized by glucuronidation, and has no active metabolites.
able 51.5 Sedation with Intravenous Benzodiazepines
Loading Dose, 1 = 0.01-0.05 mg/kg. Loading Dose, 2 = 0.02-0.04 mg/kg (s2 mg). Onset of Action, 1 = 2-5 min. Onset of Action, 2 = 15-20 min. Duration (after bolus), 1 = 1-2 hr. Duration (after bolus), 2 = 2-6 hr. Continuous Infusion, 1 = 0.02-0.1 mg/kg/nr. Continuous Infusion, 2 = 0.01-0.1 mg/kg/hr (£10 mg/h. Intermittent Bolus Dosing, 1 = . Intermittent Bolus Dosing, 2 = 0.02-0.06 mg/kg q 2-6 hr, pm. Lipophilicity, 1 = +++. Lipophilicity, 2 = ++. Specific Concems, 1 = Active Metabolitest. Specific Concems, 2 = Propylene Glycol ToxicityS
TActive Metabolites prolong sedation, aspecially in ranal failura.
SLorazepam, 2 mg/mL, contains propylene glycol, 830 mg/mL. as a solvent.
Dosing recommendations from Refarence 3.

Advantages, 1 = Advantages. The 1., 1 = advantages of sedation with benzodiazepines include the following. Benzodiazepines have a dose-dependent amnestic effect that is distinct from the sedative effect. The amnesia extends beyond the sedation period (antegrade amnesia), and this may be responsible for the surprising percentage (up to 40%) of patients who, after discharge from the ICU, have no recollection of events during their ICU stay (2,6,7). As mentioned earlier , this amnesia should be beneficial because it eliminates memories of stressful experiences.. 2., 1 = Benzodiazepines have anticonvulsant effects (see Chapter 45), which is always a benefit in critically ill patients.. 3., 1 = Benzodiazepines are the sedatives of choice for drug withdrawal syndromes, including alcohol, opiate, and benzodiazepine withdrawal.

The major disadvantages of sedation with benzodiazepines are (a) drug accumulation with prolonged sedation, and (b) the apparent tendency for benzodiazepines to promote delirium.

Maintaining sedation in ICU patients is often a long-term affair, especially in ventilatordependent patients, and both midazolam and lorazepam accumulate in tissues with prolonged use. This produces deeper levels of sedation, and prolongs the time for awakening when the drugs are discontinued. This can result in delays in weaning patients from mechanical ventilation, and longer ICU stays (3,4). Prolonged sedation is more of a problem with midazolam, because of its greater lipid solubility, and the accumulation of its active metabolite. In one study of sedation in ICU patients, the time to emerge from sedation was 1,815 min (30.2 hours) for midazolam, versus 261 min (4.4 hr) for lorazepam (42).

1. Daily interruption of benzodiazepine infusions (until the patient awakens) curtails drug accumulation, and has been shown to shorten the duration of mechanical ventilation, and reduce the length of stay in the ICU (43). This is the accepted practice for limiting excessive sedation from benzodiazepines.
2. Titration of benzodiazepine infusions to maintain light levels of sedation, using routine monitoring with a sedation scale (SAS or RASS), has been proposed in the most recent guidelines on sedation in ICUs (3). This is a more logical solution to the problem than daily interruption of benzodiazepine infusions, and should be a standard practice.
3. The final solution to the problem is to avoid benzodiazepines for sedation, which is a current trend.

The prevailing opinion is that the frequent appearance of delirium in ICU patients (described in Chapter 44) is at least partly due to the frequent use of benzodiazepines in ICU patients (3,4). Benzodiazepines produce their effects by binding to receptors for gamma-amino-butyric-acid (GABA), which are involved in the development of delirium (44), and sedation with drugs that do not involve GABA receptors is associated with fewer cases of delirium in ICU patients (see later). The role of benzodiazepines in promoting delirium will be a severe blow to their popularity if suitable alternatives are available.

Intravenous preparations of lorazepam contain propylene glycol (415 mg/mg lorazepam) to enhance drug solubility in plasma. Propylene glycol is converted to lactic acid in the liver, and excessive intake of propylene glycol can produce a toxidrome characterized by a metabolic (lactic) acidosis, delirium (with hallucination), hypotension, and (in severe

Concerns about the delayed emergence from sedation with benzodiazepines has led to the increasing popularity of two sedatives that allow rapid arousal: propofol and dexmedetomidine.

Propofol (Deprivan) is a powerful sedative that exerts its effects by binding to GABA receptors (similar to benzodiazepines, but different receptors). It is used for induction of general anesthesia, and has become popular in ICUs because arousal is rapid when the drug infusion is stopped. A profile of this drug is presented in Table 51.6.

Propofol has sedative and amnestic effects, but no analgesic effects (49). A single intravenous bolus of propofol produces sedation within 1-2 minutes, and the drug effect lasts 5-8 minutes (49). Because of the short duration of action, propofol is given as a continuous infusion. When the infusion is stopped, awakening occurs within 10-15 minutes, even with prolonged infusions (49). Loading doses of propofol can be used in pa-tients who are hemodynamically stable.
Propofol was originally intended for short-term sedation when rapid awakening is desired (e.g., during brief procedures), but it is being used for longer periods of time in ventilatordependent patients, to avoid delays in weaning from ventilatory support. Propofol can be useful in neurosurgical patients and patients with head injuries because it reduces intracranial pressure (49), and the rapid arousal allows for frequent evaluations of mental status.

Propofol is highly lipophilic, and is suspended in a 10% lipid emulsion to enhance solubility in plasma. This lipid emulsion is almost identical to 10% Intralipid used in parenteral nutrition formulas, and it has a caloric density of 1 kcal/mL, which should be included as part of the daily caloric intake. Propofol dosing is based on ideal rather than actual body weight, and no dose adjustment is required for renal failure or moderate hepatic insufficiency (49). Loading doses are not advised in patients who are hemodynamically unstable (because of the risk of hypotension) (3).

Loading Dose, Propofol = 5 ug/kg/min over 5 mint. Loading Dose, Dexmedetomidine = 1 pg/kg over 10 mint. Onset of Action, Propofol = 1-2 min. Onset of Action, Dexmedetomidine = 5-10 min. Maintenance Infusion, Propofol = 5-50 pg/kg/min. Maintenance Infusion, Dexmedetomidine = 0.2-0.7 ua/ka/hr. Time to Arousal, Propofol = 10-15 min. Time to Arousal, Dexmedetomidine = 6-10 min. Respiratory Depression, Propofol = Yes. Respiratory Depression, Dexmedetomidine = No. Adverse Effects, Propofol = Hypotension Hyperlipidemia Propofol Infusion Syndrome. Adverse Effects, Dexmedetomidine = Hypotension Bradycardia Sympathetic
tUse loading dosa only in hemodynamically stable patients.
Dosing recommendations from Reference 3.

Dexmedetomidine is an alpha-2 receptor agonist that has sedative, amnestic, and mild analgesic effects, and does not depress ventilation. A brief profile of the drug is presented in Table 51.6. The most distinguishing feature of dexmedetomidine is the type of the sedation it produces, which is described next.

The sedation produced by dexmedetomidine is unique because arousal is maintained, despite deep levels of sedation. Patients can be aroused from sedation without discontinuing the drug infusion, and when awake, patients are able to communicate and follow commands. When arousal is no longer required, the patient is allowed to return to the prior state of sedation. This has been called cooperative sedation (5), and is similar to temporary awakening from sleep. In fact, the EEG changes in this type of sedation are similar to the EEG changes in natural sleep (5).
Cooperative sedation with dexmedetomidine is very different than the sedation produced by GABAergic drugs (benzodiazepines and propofol), where arousal occurs only after the drug is discontinued and the sedative effects abate. In fact, daily interruption of benzodiazepine sedation is aimed at achieving the type of sedation produced by dexmedetomidine; i.e., arousable and cooperative. Dexmedetomidine should be well suited for ventilator-dependent patients, because sedation can be continued during the transition period from mechanical ventilation to spontaneous breathing.

Clinical studies have shown a lower prevalence of delirium in patients who are sedated with dexmedetomidine instead of midazolam (55), and based on these studies, dexmedetomidine is recommended over benzodiazepines for the sedation of patients with ICU-acquired delirium (3).

Dexmedetomidine produces dose-dependent decreases in heart rate, blood pressure, and circulating norepinephrine levels (sympatholytic effect) (5). Patients with heart failure and cardiac conduction defects are particularly susceptible to the sympatholytic actions of dexmedetomidine. Life-threatening bradycardia has been reported, primarily in patients treated with high infusion rates of dexmedetomidine (>0.7 ug/kg/min) together with a loading dose (56 ). Patients with cardiac conduction defects should not receive dexmedetomidine, and patients with heart failure or hemodynamic instability should not receive a loading dose of the drug.

Haloperidol (Haldol) is a first-generation antipsychotic agent that has a long history of treating agitation and delirium (57-58).

Haloperidol produces its sedative and antipsychotic effects by blocking dopamine receptors in the central nervous system. Following an intravenous bolus dose of haloperidol, sedation is evident in 10-20 minutes, and the effect lasts 3-4 hours. There is no respiratory depression, and hypotension is unusual in the absence of hypovolemia.
DOSING: The recommended dosing for intravenous haloperidol is shown in Table 51.7 (59,60). Because haloperidol has a delayed onset of action, midazolam can be given with the first dose of haloperidol to achieve more rapid sedation. Individual patients show a wide variation in serum drug levels after a given dose of haloperidol (61). If there is no evidence of a sedative response after 10 minutes, the dose should be doubled. If there is a partial response at 10-20 minutes, a second dose can be given along with 1 mg lorazepam (preferred to midazolam because of the longer duration of action) (61). Lack of response to a second dose of haloperidol should prompt a switch to another agent.

1. Administer dose by IV push. 2. Allow 10-20 min for response: a. Ifno response, double the drug dose, or b. Add lorazepam (1 ma) 3. If still no response, switch to another sedative. 4, Give 1/4 of the loading dase 6 hr for maintenance of sedation., Dose.0.5-2 mg 5-10 mg 10-20 mg = 1. Administer dose by IV push. 2. Allow 10-20 min for response: a. Ifno response, double the drug dose, or b. Add lorazepam (1 ma) 3. If still no response, switch to another sedative. 4, Give 1/4 of the loading dase 6 hr for maintenance of sedation.
Adapted fram References 59 and 6D.

The adverse effects of haloperidol include: (a) extrapyramidal reactions, (b) neuroleptic malignant syndrome, and (c) ventricular tachycardia.
1. Extrapyramidal reactions (e.g., rigidity, spasmodic movements) are dose-related side effects of oral haloperidol therapy, but these reactions are uncommon when haloperidol is given intravenously (for unclear reasons) (61).
2. The neuroleptic malignant syndrome (described on pages 763-765) is an idiosyncratic reaction to neuroleptic agents that consists of hyperpyrexia, severe muscle rigidity, and rhabdomyolysis. This condition has been reported with intravenous haloperidol (62), but is rare.
3. The most publicized risk of therapy with haloperidol is prolongation of the QT interval
on the electrocardiogram, which can trigger a polymorphic ventricular tachycardia (torsade de pointes, shown in Figure 15.8). This arrhythmia is reported in up to 3.5% of patients receiving intravenous haloperidol (63), which provides a reason to avoid haloperidol in patients with a prolonged QT interval.

It is not possible to completely eliminate the stress experienced by ICU patients because being critically ill is inherently stressful. However, the following considerations might help to reduce the "unpleasantness" of the ICU experience.
1. Critically ill patients experience pain in situations that are not normally painful. For example, being turned in bed is one of the most painful experiences reported by ICU patients, and up to 50% of patients experience pain at rest, without any noxious stimulus (8,10). This indicates that pain control is a full-time endeavor in ICU patients.
. Unrelieved pain can be a source of agitation, so make sure that pain is relieved before considering a sedative drug for agitation.
. When a benzodiazepine is used for prolonged (>48 hrs) sedation, attention to preventing drug accumulation and prolonged sedation can result in a shorter time in the ICU. The preventive measures include daily interruption of drug infusions, or maintaining light sedation using a reliable sedation scale to guide adjustments in drug dosage.
. Consider using dexmedetomidine for sedation, because this drug allows the patient to be aroused while still sedated (e.g., to help with repositioning or speak with family members). When arousal is no longer needed, the patient will resume the prior level of sedation. This is more like sleep than a drug-induced stupor.
. Finally, communicate with patients (e.g., tell them what you are going to do before doing it), and allow some "down time" for patients to sleep.

1. Loper KA, Butler S, Nessly M, Wild L. Paralysed with pain: the need for education. Pain 1989; 37:315-316.
2. Rotondi AJ, Chelluri L, Sirio C, et al. Patients' recollections of stressful experiences while receiving prolonged mechanical ventilation in an intensive care unit. Crit Care Med 2002; 30:746-752.

3. Barr J, Fraser GL, Puntillo K, et al. Clinical practice guidelines for the management of pain. agitation, and delirium in adult patients in the intensive care unit. Crit Care Med 2013; 41:263-306.

4. Devlin JW, Roberts RJ. Pharmacology of commonly used analgesics and sedatives in the ICU: benzodiazepines, propofol, and opioids. Crit Care Clin 2009; 25:431—449.
5. Panzer O, Moitra V, Sladen RN. Pharmacology of sedative-analgesic agents: dexmedetomidine, remifentanil, ketamine, volatile anesthetics, and the role of peripheral mu antagonists. Crit Care Clin 2009; 25:451—469.

, 1 = Non-Opioid Analgesics. 29., 1 = Buckley MM, Brogden RN. Ketorolac. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential. Drugs 1990; 39:86-109.. 30., 1 = Gillis JC, Brogden RN. Ketorolac. A reappraisal of its pharmacodynamic and pharmacokinetic properties and therapeutic use in pain management. Drugs 1997; 53:139-188.. 31., 1 = Ketorolac Tromethamine. In: McEvoy GK, ed. AHFS Drug Information, 2012. Bethesda: American Society of Health System Pharmacists, 2012:2139-2148.. 32., 1 = Ready LB, Brown CR, Stahlgren LH, et al. Evaluation of intravenous ketorolac administered by bolus or infusion for treatment of postoperative pain. A double- blind, placebo-controlled, multicenter study. Anesthesiology 1994; 80:1277-1286.. 33., 1 = Strom BL, Berlin JA, Kinman JL, et al. Parenteral ketorolac and risk of gastrointestinal and operative site bleeding. A postmarketing surveillance study. JAMA 1996; 275:376-382.. 34., 1 = Reinhart DI. Minimising the adverse effects of ketorolac. Drug Safety 2000; 22:487- 497.. 35. Scott LJ. Intravenous ibuprofen. Drugs 2012; 72:1099-1109., 1 = 35. Scott LJ. Intravenous ibuprofen. Drugs 2012; 72:1099-1109.. 36., 1 = Yeh YC, Reddy P. Clinical and economic evidence for intravenous acetaminophen. Pharmacother 2012; 32:559-579.. 37., 1 = Sinatra RS, Jahr JS, Reynolds LW, et al. Efficacy and safety of single and repeated administration of 1 gram intravenous acetaminophen injection (paracetamol) for pain management after major orthopedic surgery. Anesthesiology 2005; 102:822- 831.

38. Ely EW, Inouye SK, Bernard GR, et al. Delirium in mechanically ventilated patients: validity and reliability of the confusion assessment method for the intensive care unit (CAM-ICU). JAMA 2001;286:2703-2710.
39. Sessler CN Gosnell MS, Grap MJ, et al. The Richmond Agitation-Sedation Scale: validity and reliability in adult intensive care units. Am J Resp Crit Care Med 2002; 166:1338-1344.
40. Ely EW, Truman B, Shintani A, et al. Monitoring sedation status over time in ICU patients: reliability and validity of the Richmond Agitation-Sedation Scale (RASS). JAMA 2003; 289:2983-2991.
Propofol, 1 = Propofol. 49., 1 = McKeage K, Perry CM. Propofol: a review of its use in intensive care sedation of adults. CNS drugs 2003; 17:235-272.. 50., 1 = Riker RR, Fraser GL. Adverse events associated with sedatives, analgesics, and other drugs that provide patient comfort in the intensive care unit. Pharmacotherapy 2005; 25:8S-18S.. 51., 1 = Devaud JC, Berger MM, Pannatier A. Hypertriglyceridemia: a potential side effect of propofol sedation in critical illness. Intensive Care Med 2012; 38:1990-1998.. 52., 1 = Fong JT, Sylvia L, Ruthazer R, et al. Predictors of mortality in patients with suspected propofol infusion syndrome. Crit Care Med 2008; 36:2281-2287.. 53., 1 = Fodale V, LaMonaca E. Propofol infusion syndrome: an overview of a perplexing disease. Drug Saf 2008; 31:293-303.

54. Bhana N, Goa KL, McClellan KJ. Dexmedetomidine. Drugs 2000; 59:263-268.
55. Riker RR, Shehabi Y, Bokesch PM, et al. SEDCOM (Safety and Efficacy of Dexmedetomidine Compared With Midazolam) Study Group: Dexmedetomi-dine vs. midazolam for sedation of critically ill patients. JAMA 2009; 301:489-499.
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The danger with germ-killing drugs is that they may kill the patient as well as the germ.
J.B.S. Haldane
Antibiotic therapy is inescapable in the ICU, and the antibiotics used most often are included in the list shown below. Each of these will be presented in alphabetical order, as listed.
. Aminoglycosides HE
. Antifungal agents
. Cephalosporins
. Carbapenems BRWN
. Fluoroquinolones . Penicillins NOU
. Vancomycin & Alternatives

The aminoglycosides are a group of antibiotics derived from cultures of Streptomyces (hence the name _ streptomycin for the first aminoglycoside). There are three aminoglycosides available for intravenous use in the United States: gentamicin, tobramycin, and amikacin (introduced in 1966, 1975, and 1981, respectively).

The aminoglycosides are bactericidal, and are among the most active antibiotics against Gram-negative aerobic bacilli (See Figure 52.1), including Pseudomonas aeruginosa (see Figure 52.2) (1,2). Amikacin is the most active of the aminoglycosides, probably because it has been in clinical use for a shorter period of time (giving microbes less time to develop resistance). Because of the risk of nephrotoxicity (see later), aminoglycosides are usually reserved for infections involving Pseudomonas aeruginosa. However, there is some evidence that, in cases of Gram-negative septicemia associated with neutropenia or septic shock, empiric antibiotic coverage is more effective if an aminoglycoside is added to another drug with activity against Gram-negative aerobic bacilli (e.g., a carbapenem, cefepime, or piperacillin/tazobactam) (3).

Aminoglycosides are given in one daily dose, which is based on body weight and renal function, as shown in Table 52.1.
, 1 = 7 mg/kg. , 2 = 20 makg. , 3 = . 60-79, 1 = 5 mg/kg. 60-79, 2 = 15 mg/kg. 60-79, 3 = . 40-59, 1 = 4 mg/kg. 40-59, 2 = 12 mg/kg. 40-59, 3 = 24 hrs. 20-39, 1 = 4 mg/kg. 20-39, 2 = 12 mg/kg. 20-39, 3 = 48 hrs. 10-19, 1 = 3 mg/kg. 10-19, 2 = 10 mg/kg. 10-19, 3 = 48 hrs. <10, 1 = 2.5 mg/kg. <10, 2 = 7.5 mg/kg. <10, 3 = 48 hrs
From Referance 1. Or GL = creatinine dearance. Gant = gentamicin; Tobra = tobramycin.

Aminoglycoside dosing is based on ideal body weight (see Appendix 2 for charts of ideal body weights). For obese patients, dosing should be based on an adjusted body weight (ABW), which is equivalent to the ideal body weight (IBW) plus 45% of the difference between the total body weight (TBW) and ideal body weight (1); i.e.,
ABW = IBW +0.45 (TBW -IBW)
(52.1)

The aminoglycosides are cleared by filtration in the kidneys, and dose adjustments are necessary when renal clearance is impaired. Note in Table 52.1 that the strength of the dose is progressively decreased with decreasing creatinine clearance, and at a creatinine clearance below 40 mL/min, the dosing interval increases to 48 hours. (Formulas for estimating the creatinine clearance are in the Appendix at the end of the book.) Hemodialysis for 6 hours will eliminate about 40-50% of the accumulated aminoglycoside, so 50% of a full dose should be given after each dialysis session (1).

Serum aminoglycoside levels should be monitored to determine if dosing is appropriate in individual patients. Target peak levels are 4-8 mg/L for gentamicin and tobramycin, and 15-20 mg/L for amikacin (4). Target trough levels (obtained just before a dose) are 1-2 mg/L for gentamicin and tobramycin, and 5-10 mg/L for amikacin (4).
FIGURE 52.1 Antibiotic susceptibilties of Gram-negative aerobic bacili isolated from abdominal infections in 37 hospitals in North America from 2005 to 2010. Data from Reference 2.

Nephrotoxicity is the principal adverse effect of aminoglycosides.

Aminoglycosides have been called obligate nephrotoxins because renal impairment will eventually develop in all patients if treatment is continued (5). The site of renal injury is the proximal tubules, and the risk of renal injury is the same with each of the aminoglycosides. The earliest signs of injury include cylindrical casts in the urine, proteinuria, and inability to concentrate urine (3). The urinary changes appear in the first week of drug treatment, and the serum creatinine begins to rise 5-7 days after the start of therapy. The renal impairment can progress to acute renal failure, which is usually reversible. Nephrotoxic effects are enhanced by hypovolemia and pre-existing renal disease (5,6).

Other adverse effects, which include ototoxicity and neuromuscular blockade, are rarely a problem. The ototoxicity can produce irreversible hearing loss and vestibular damage, but these changes are usually asymptomatic (5). Aminoglycosides can block acetylcholine release from presynaptic nerve terminals, but this is never clinically apparent with therapeutic dosing (4). There is a small risk that aminoglycosides will aggravate the neuromuscular blockade associated with myasthenia gravis and nondepolarizing muscle relaxants (7,8), and it is wise to avoid aminoglycosides in these conditions.
FIGURE 52.2 Susceptibility of Pseudomonas aeruginosa to selected antibiotics. Data from a survey of abdominal infections in 37 hospitals in North America from 2005 to 2010. From Reference 2.

Aminoglycosides were once the darlings of the infectious disease community because they were the first antibiotics capable of treating serious Gram-negative infections. However, because of their nephrotoxicity, and the availability of less harmful drugs capable of treating serious Gram-negative infections, aminoglycosides should be reserved for Pseudomonas bacteremias in patients with neutropenia or septic shock.

Antifungal therapy in the critical care setting is primarily directed against Candida species, and this is the focus of the following presentation.

Amphotericin B (AmB) is a naturally occurring antibiotic that is fungicidal for most of the pathogenic fungi in humans (9). It is one of the most effective antifungal agents available, but is plagued by toxic reactions; i.e., infusion-related inflammatory responses, and nephrotoxicity. As a result, AmB is used mostly as a backup, for patients who are intolerant of, or refractory to, less toxic antifungal drugs (see Table 52.2) (10).

AmB is available for intravenous use only, and contains a vehicle (sodium deoxycholate) to enhance solubility in plasma. It is given once daily in a dose of 0.5—1 mg/kg. The dose is initially delivered over a 4-hour time period, but can be delivered in one hour, if tolerated. Daily infusions are continued until a specified cumulative dose is achieved. The total AmB dose is determined by the type and severity of the fungal infection: it can be as little as 500 mg (for catheter-related candidemia) or as much as 4 grams (for lifethreatening invasive aspergillosis).

Infusions of AmB are accompanied by fever, chills, nausea, vomiting, and rigors in about 70% of instances (11). This reaction is most pronounced with the initial infusion, and often diminishes in intensity with repeated infusions. The following measures are used to reduce the intensity of this reaction (11):
1. Thirty minutes before the infusion, give acetaminophen (10-15 mg/kg orally) and diphenhydramine (25 mg orally or IV). If rigors are a problem, premedicate with meperidine (25 mg IV).
2. If the premedication regimen does not give full relief, add hydrocortisone to the AmB infusate (0.1 mg/mL).
Central venous cannulation is preferred for AmB infusions to reduce the risk of infusionrelated phlebitis, which is common when AmB is infused through peripheral veins (9).

AmB binds to cholesterol on the surface of renal epithelial cells and produces an injury that clinically resembles a renal tubular acidosis (distal type), with increased urinary excretion of potassium and magnesium (12). Azotemia is reported in 30-40% of patients during daily infusions of AmB (13), and can occasionally progress to acute renal failure that requires hemodialysis (14). The renal impairment from AmB usually stabilizes with continued infusions, and improvement is expected if AmB is discontinued. Hypovolemia aggravates the renal injury, and maintaining intravascular volume is important for mitigating the injury. An increase in the serum creatinine above 3.0 mg/dL should prompt cessation of AmB infusions for a few days (11).
ELECTROLYTE ABNORMALITIES: Hypokalemia and hypomagnesemia are common during AmB therapy, and the hypokalemia can be difficult to correct until the magnesium deficits are replaced (as described in Chapter 37). Oral magnesium (300-600 mg elemental magnesium daily) is recommended during AmB therapy, except for patients with progressive azotemia.

Specialized lipid preparations of AmB have been developed to enhance AmB binding to fungal cell membranes and reduce binding to mammalian cells (thereby reducing the risk of renal injury). There are 2 lipid preparations, liposomal amphotericin, and amphotericin B lipid complex, and the recommended dose is 3-5 mg/kg daily (10). Both reduce the incidence of nephrotoxic reactions, and the decrease is greater with the liposomal preparation (15). Both preparations are costly.

The triazoles are synthetic antifungal agents that are less toxic alternatives to AmB for selected fungal infections. There are 3 drugs in this class, fluconazole, itraconazole, and voriconazole, but fluconazole is the one used for Candida infections.

Fluconazole is the drug of choice for infections involving Candida albicans, C. tropicalis, and C. parapsilosis, but not for infections involving C. glabrata or C. krusei (see Table 52.2) (10).

Candida albicans, “First-Line Drug = Fluconazole. Candida albicans, ‘Alternate Drug = Echinocandin. Candida tropicals, “First-Line Drug = Fluconazole. Candida tropicals, ‘Alternate Drug = Echinecandin. Candida parapsilosis, “First-Line Drug = Fluconazole. Candida parapsilosis, ‘Alternate Drug = Amphotericin B. Candida glabrata, “First-Line Drug = Echinocandin. Candida glabrata, ‘Alternate Drug = Amphotericin B. Candida krusel, “First-Line Drug = Echinocandin. Candida krusel, ‘Alternate Drug = Amphotericin B
Adapted fram Reference 10.

Fluconazole can be given orally or intravenously. The usual dose is 400 mg daily, given as a single dose. Doses of 800 mg daily are recommended for clinically unstable patients. The time to reach steady state levels after the start of therapy is 4-5 days, and this can be shortened by doubling the initial dose. Adjustments are necessary for renal impairment: if the creatinine clearance is <50 mL/min, the dose should be reduced by 50% (9).

The triazoles inhibit the cytochrome P450 enzyme system in the liver, and they can potentiate the activity of several drugs. For fluconazole, the significant interactions include phenytoin, cisapride, and the statins (lovastatin, atorvastatin) (9).

Fluconazole is largely devoid of serious toxicity. Asymptomatic elevation of liver enzymes has been reported (9), and there are rare reports of severe and even fatal hepatic necrosis during fluconazole therapy in HIV patients (16).

The echinocandins are antifungal agents that are active against a broader spectrum of Candida species than fluconazole (except C. parapsilosis) (10). The drugs in this class include caspofungin, micafungin, and anidulafungin. These agents can be used as alternatives to fluconazole for treating invasive candidiasis involving C. albicans and C. tropicalis, and are the preferred agents for infections involving C. glabrata and C. krusei (10). They are also preferred by some for prophylaxis of invasive candidiasis in unstable or immunocompromised patients (10).

Caspofungin is the flagship drug in this class, and is equivalent to amphotericin for treating invasive candidiasis (17). The drug is given intravenously, and the usual IV dose is 70 mg initially, then 50 mg daily thereafter. Like all echinocandins, no dose adjustment is needed for renal insufficiency (18).

Anidulafungin (200 mg IV on day 1, then 100 mg IV daily) and micafungin (100 mg IV daily) are equivalent to caspofungin (10), but there is much less clinical experience with these drugs.

The echinocandins are relatively non-toxic. Transient elevations in liver enzymes can occur, and there are occasional reports of hepatic dysfunction associated with these drugs (18).

The carbapenems have the broadest spectrum of antibacterial activity of any class of antibiotics currently available. There are 4 carbapenems available for clinical use: imipenem, meropenem, doripenem, and ertapenem. Some notable features of these drugs are included in Table 52.3. The following description is limited to imipenem and meropenem, which are currently the most popular carbapenems. Doripenem is largely indistinguishable from meropenem, and ertapenem is not active against Pseudomonas aeruginosa, which makes it the least desirable carbapenem for critically ill patients.

Usual Dose, 1 = O5gq6hr. Usual Dose, 2 = 2gqq6hr. Usual Dose, 3 = O.5gq8hr. Usual Dose, 4 = 1gq24hr. Seizure Risk, 1 = Yes. Seizure Risk, 2 = Not. Seizure Risk, 3 = No. Seizure Risk, 4 = Possibly. Pseudomonas Activity, 1 = Was. Pseudomonas Activity, 2 = Yes. Pseudomonas Activity, 3 = Yes. Pseudomonas Activity, 4 = No. Renal Dose Adjustment, 1 = Yes. Renal Dose Adjustment, 2 = ‘Yes. Renal Dose Adjustment, 3 = Yes. Renal Dose Adjustment, 4 = Yes
TMeropenam does not cause seizures, but it can raduce serum levels of valproic acid, and will increase the risk of seizures in patients being treated with this anticorwulsant.

Imipenem and meropenem are active against all common bacterial pathogens except methicillin-resistant staphylococci (MRSA) and vancomycin-resistant enterococci (19). As demonstrated in Figures 52.1 and 52.2, imipenem is one of the most active agents for aerobic Gram-negative bacilli, and is also active against Pseudomonas aeruginosa (although less so). These agents also provide adequate coverage for pneumococci, methicillin-sensitive staphylococci, coagulase-negative staphylococci, and anaerobes, including Bacteroides fragilis and Enterococcus faecalis. Furthermore, acquired resistance to the carbapenems has increased much less than with other antimicrobials used in critical care (19).

Because of their broad spectrum of activity, imipenem and meropenem are well suited for empiric antibiotic coverage of suspected Gram-negative infections (e.g., abdominal infections), or mixed aerobic/anaerobic infections (e.g., pelvic infections). Imipenem has also been effective as single-agent empiric coverage for neutropenic patients with fever (20). Meropenem readily crosses the blood-brain barrier (21), and can be used for empiric antibiotic coverage of suspected Gram-negative meningitis.

The carbapenems can only be given intravenously. Imipenem is inactivated by enzymes on the luminal surface the proximal renal tubules, so it is impossible to achieve high levels of the drug in urine. To overcome this problem, the commercial preparation of imipenem contains an enzyme inhibitor, cilastatin (22). The combination imipenem— cilastatin preparation is available as Primaxin. The usual dose of imipenem-—cilastatin is 500 mg IV every 6 hours. In suspected Pseudomonas infections, the dose is doubled to 1 g every 6 hours. In renal failure, the dose should be reduced by 50-75% (23). Meropenem does not require the cilastatin addition, and the usual dose if 1 gram IV every 8 hours, which can be increased to 2 grams every 8 hours for serious infections. A 50% dose reduction is recommended in patients with renal failure (23).

The major risk associated with imipenem is generalized seizures, which has been reported in 1-3% of patients receiving the drug (22). Most of the seizure cases are in patients with a history of a seizure disorder, or an intracranial mass, who did not receive an adjusted dose of imipenem for renal insufficiency (22).
There is little risk of seizures with meropenem (19,21). However, mero-penem can reduce serum levels of valproic acid, and this can increase the risk of seizures in patients being treated with this anticonvulsant (19).

Patients with hypersensitivity reactions to penicillin can occasionally have a hypersensitivity reaction to carbapenems. The incidence of this cross-reactivity is not known, but the allergic reactions usually include a rash or urticaria, and are almost never life-threatening (24).

The ideal antibiotic would be effective against all pathogens and produce no adverse reactions. Meropenem comes closer to this ideal than any antibiotic currently available, with imipenem as a close second. These agents have been my personal favorites for several years because they cover almost everything, which simplifies the selection of empiric antibiotic coverage. Meropenem plus vancomycin (for MRSA) provides adequate empiric coverage for most patients in the ICU (unless there is a problem with vancomycin-resistant enterococci in your ICU). Add fluconazole for empiric coverage if invasive candidiasis is a possibility, and that should be it. The risk of seizures with imipenem is also overstated, and should not be a problem if you adjust the dose for renal insufficiency.

The first cephalosporin (cephalothin) was introduced in 1964, and this was followed by a small army of other cephalosporins. There are currently more than 20 cephalosporins available for clinical use (24,25). They are divided into generations, and some of the parenteral agents in the first four generations are shown in Table 52.4.

Cefazolin (Ancef), 1 = 1. Cefazolin (Ancef), 2 = Stats. Cefazolin (Ancef), 3 = cies. Cefazolin (Ancef), 4 = . Cefazolin (Ancef), 5 = . Cefazolin (Ancef), 6 = ++. Cefoxatin (Mefoxin), 1 = 2. Cefoxatin (Mefoxin), 2 = +4. Cefoxatin (Mefoxin), 3 = HE. Cefoxatin (Mefoxin), 4 = . Cefoxatin (Mefoxin), 5 = ++. Cefoxatin (Mefoxin), 6 = ++. Ceftriaxone (Rocephin), 1 = co. Ceftriaxone (Rocephin), 2 = 4. Ceftriaxone (Rocephin), 3 = H+. Ceftriaxone (Rocephin), 4 = . Ceftriaxone (Rocephin), 5 = . Ceftriaxone (Rocephin), 6 = t+t+. Ceftazidime (Fortaz), 1 = 3. Ceftazidime (Fortaz), 2 = _—. Ceftazidime (Fortaz), 3 = H+. Ceftazidime (Fortaz), 4 = ++. Ceftazidime (Fortaz), 5 = . Ceftazidime (Fortaz), 6 = . Cefepime (Maxipime), 1 = 4. Cefepime (Maxipime), 2 = ++. Cefepime (Maxipime), 3 = H+. Cefepime (Maxipime), 4 = +++. Cefepime (Maxipime), 5 = . Cefepime (Maxipime), 6 = 
SDoes not include coagulase-negative or methicillin-resistant staphylococci, or anterococci.
Relative antibacterial activity is indicated by number of plus signs.
Adapted from Refarance 24.

The doses for the more popular parenteral cephalosporins are shown in Table 52.5, along with dose adjustments for renal failure. Note that the dose in renal failure is adjusted by extending the dosing interval rather than decreasing the amount of drug given with each dose (22). This is done to preserve concentration-dependent bacterial killing. Note also that ceftriaxone requires no dose adjustment in renal failure.

Cefazolin, Serious Infections = 1g every 6 hr. Cefazolin, Renal Failure* = 1 gevery 24 hr. Ceftriaxone, Serious Infections = 2g every 12 hr. Ceftriaxone, Renal Failure* = 2g every 12 hr. Ceftazidime, Serious Infections = 2 g every Shr. Ceftazidime, Renal Failure* = 2 g every 48 hr. Cefepime, Serious Infections = 2 g every 8 hr. Cefepime, Renal Failure* = 2g every 24 hr
"From Refarance 22.

Adverse reactions to cephalosporins are uncommon and nonspecific (e€.g., nausea, rash, and diarrhea). There is a 5-15% incidence of cross-antigenicity with penicillin (24), and cephalosporins should be avoided in patients with a history of serious anaphylactic reactions to penicillin.

The only cephalosporins that continue to have some value in the ICU are ceftriaxone (for empiric therapy of serious community-acquired pneumonias) and cefepime (for empiric coverage of Gram-negative enteric pathogens).

The fluoroquinolone era began in the mid-1980s with the introduction of norfloxacin. Since then, several fluoroquinolones have been introduced, but only three survive: ciprofloxacin, levofloxacin, and moxifloxacin.

The fluoroquinolones are active against methicillin-sensitive staphylococci, and the newer agents (levofloxacin and moxifloxacin) are active against streptococci (including penicillin-resistant pneumococci), and "atypical" organisms like Mycoplasma pneumoniae and Hemophilus influenza (26). When first introduced, the fluoroquinolones were very active against Gram-negative aerobic bacilli, including Pseudomonas aeruginosa, but rapidly emerging resistance has reduced their activity against Gram-negative organisms, as shown in Figures 52.1 and 52.2.
The development of resistance in Gram-negative organisms has severely curtailed the use of fluoroquinolones in the ICU. Levofloxacin and moxifloxacin are used primarily for community-acquired pneumonia, exacerbations of chronic obstructive lung disease, and for uncomplicated urinary tract infections.

Table 52.6 shows the intravenous dosing regimens for the quinolones. The newer quinolones have longer half-lives than ciprofloxacin, and require only one once-daily dosing. Dose adjustments are required for renal failure for all but moxifloxacin, which is metabolized in the liver (26).
Ciprofloxacin,  = 400 g every 8 hr. Ciprofloxacin,  = 400 g every 18 hr. Levofloxacin,  = 500 g every 24 hr. Levofloxacin,  = 250 g every 48 hr. Moxtfloxacin,  = 400 g every 24 hr. Moxtfloxacin,  = 400 g every 24 hr

Ciprofloxacin interferes with the hepatic metabolism of theophylline and warfarin, and can potentiate the actions of both of these drugs (27,28). Ciprofloxacin causes a 25% increase in serum theophylline levels, and combined therapy has resulted in symptomatic theophylline toxicity (29). Although no dose adjustments are necessary, serum theophylline levels and prothrombin times should be monitored carefully when ciprofloxacin is given in combination with theophylline or warfarin.

The fluoroquinolones are relatively safe. Neurotoxic reactions (confusion, hallucinations, seizures) can develop days after starting quinolone therapy in 1-2% of patients (30). Prolongation of the QT interval and polymorphic ventricular tachycardia (torsade de pointes) have been reported with all quinolones except moxifloxacin, but is a rare occurrence (31).

The emerging resistance of Gram-negative pathogens to fluoroquin-olones has just about eliminated fluoroquinolones from the ICU formulary. Levofloxacin is a popular antibiotic for community-acquired pneumonias, and is also used for exacerbations of COPD, but these conditions are often managed outside the ICU.

The penicillin discovered by Alexander Fleming in 1929 is benzylpenicillin, or penicillin G, which is (was) active against aerobic streptococci (S. pneumoniae, S. pyogenes) and anaerobic mouth flora. The emergence of penicillin-resistant pneumococci in recent years, along with broader spectrum drugs for treating anaerobic infections, has eliminated penicillin G from the ICU formulary.

The penicillins in this category have an extended antibacterial spectrum that covers aerobic Gram-negative bacilli. This category includes the aminopenicillins (ampicillin and amoxicillin), the carboxypenicillins (carbenicillin and ticarcillin), and the ureidopenicillins (azlocillin, mezlocillin, and piperacillin). All groups are active against Gram-negative pathogens, but the latter two groups are active against Pseudomonas aeruginosa (32). These agents are also known as antipseudomonal penicillins. The most popular drug in this class is piperacillin, which is available in a special combination product (see next).

When used for serious Gram-negative infections, piperacillin is given in combination with tazobactam, a B-lactamase inhibitor that has synergistic activity when combined with piperacillin. The commercial product (Zosyn) contains piperacillin in an 8:1 ratio with tazobactam. The recommended dose of the combination product is 3.375 grams (3 grams piperacillin and 0.375 mg tazobactam) IV every 4-6 hours. In the presence of renal insufficiency, the dose should be changed to 2.25 grams every 8 hours (33).

Piperacillin-tazobactam is a favorite drug for empiric coverage of suspected Gramnegative infections in the ICU. However, Figures 52.1 and 52.2 show that there are more effective drugs than piperacillin/tazobactam for empiric coverage, and treatment, of Gram-negative infections in the ICU.

Vancomycin is the bedrock of antibiotic therapy in the ICU, and has been for several years.

Vancomycin is active against all Gram-positive cocci, including all strains of Staphylococcus aureus (coagulase-positive, coagulase-negative, moethicillin-sensitive, methicillin-resistant) as well as aerobic and anaerobic streptococci (including pneumococcus and enterococcus) (34). It is the drug of choice for penicillin-resistant pneumococci, and is one of the most active agents against Clostridium difficile. Enterococci can be resistant to vancomycin. The prevalence of vancomycin-resistant enterococci (VRE) varies from 2% to 60%, depending on the species involved (34).

Vancomycin is the drug of choice for infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis. However, as much as 2/3 of the vancomycin use in ICUs is not directed at a specific pathogen, but is used for empiric antibiotic coverage in patients with suspected infections (35). The popularity of vancomycin in empiric antibiotic regimens is a reflection of the prominent role played by MRSA and S. epidermidis in ICU-related infections.

Standard dosing recommendations for vancomycin (1 gram IV every 12 hrs for normal renal function) frequently results in subtherapeutic vancomycin levels in blood. As a result, weight-based dosing is now recommended for vancomycin. The loading dose for most patients is 15-20 mg/kg, and a largerloading dose of 25-30 mg/kg is recommended for critically ill patients (36). Actual body weight can be used unless the weight is more than 20% above the upper limit of normal for ideal body weight. For overweight patients, an adjusted body weight should be calculated using Equation 52.1 (37).
After the loading dose, subsequent dosing is determined by renal function and the target vancomycin level in blood. An example of a vancomycin dosing nomogram based on body weight, renal function, and a target vancomycin blood level, is shown in Table 52.7. Most hospital pharmacies have nomograms, and will determine the appropriate vancomycin dose for you. After the dosing begins, serum vancomycin levels are used to adjust the dosing regimen.
DRUG LEVELS: Monitoring vancomycin blood levels is recommended when the drug is used to treat serious infections. Steady-state drug levels are usually reached after the fourth intravenous dose (36). Trough blood levels should be >10 mg/L to prevent the development of resistance. For serious infections, trough blood levels of 15-20 mg/L are recommended (36).

Rapid administration of vancomycin can be accompanied by vasodilation, flushing, and hypotension (red man syndrome) as a result of histamine release from mast cells (34). The trigger for this release is unknown, but slowing the infusion rate (to less than 10 mg/min) usually corrects the problem.
Original reports of vancomycin-induced nephrotoxicity were likely due to impurities in the vancomycin preparation, or other nephrotoxic drugs, because recent reports are unable to confirm nephrotoxicity with vancomycin monotherapy (34). There is evidence of an immune-mediated thrombocytopenia in 20% of patients receiving vancomycin (38), and vancomycin-induced neutropenia has been reported in 2—12% of patients who receive the drug for more than 7 days (39).

1,250 mg. 10-19, 4 = 1,250 mg. , 1 = q 48 hr. , 2 = q 48 hr. , 3 = q 48 hr. , 4 = q 48 hr. <10, 1 = Repeat dose when spot serum vancomycin <20 mg/L. <10, 2 = Repeat dose when spot serum vancomycin <20 mg/L. <10, 3 = Repeat dose when spot serum vancomycin <20 mg/L. <10, 4 = Repeat dose when spot serum vancomycin <20 mg/L
From UptoDate twww.uptodate.com), Accessed 6/2013.
Based on a target vancomycin level (trough) of 145-29 mL.

Vancomycin continues to be a solid performer in the ICU, but alternative drugs are needed for infections with vancomycin-resistant enterococci, and for patients who are unable to receive vancomycin (e.g., because of toxic reactions). There is also a need for a vancomycin replacement, to curb the current use of vancomycin, and slow the rate of microbial resistance.

Linezolid, Dosing Regimens and Comments = Desing: Comment:. Linezolid, Dosing Regimens and Comments = 400-600 mg IV every 12 hrs. Currently the closest to a replacement for vancomycin.. Daptomycin, Dosing Regimens and Comments = Dosing: Comment:. Daptomycin, Dosing Regimens and Comments = 4-6 mg/kg IV every 24 hrs. Ineffective for pneumonia (is inactivated by surfactants in the lung).. Quinupristin- cameaeh), Dosing Regimens and Comments = Dosing: Comment:. Quinupristin- cameaeh), Dosing Regimens and Comments = 7.5 mg/kg IV every 8-12 hrs. Use has been limited by side effects (e.g., painful arthralgias and myalgias).

Linezolid is a synthetic antibiotic that has the same spectrum of activity as vancomycin (including MRSA), but is also active against vancomycin-resistant enterococci (VRE) (34). The intravenous dose is 600 mg twice daily. Linezolid has much better penetration into lung secretions than vancomycin, but original studies suggesting improved outcomes with linezolid in MRSA pneumonia have not been confirmed in a review of all available studies (40).
Linezolid could be used as a replacement for vancomycin, although resistance is already beginning to appear (34). Toxicity associated with linezolid includes thrombocytopenia (with prolonged use) (34), a partially-reversible optic neuropathy (41), and serotonin syndrome (see Table 42.3).

Daptomycin is a naturally occurring antibiotic that is active against Gram-positive organisms, including MRSA and VRE (34). The recommended IV dose is 4-6 mg/kg given once daily. Dose reductions are recommended for creatinine clearance <30 mL/min (34). Daptomycin can be used to treat soft tissue infections or bacteremias involving MRSA and VRE (34). However, it cannot be used to treat pneumonias (42) because it is inactivated by surfactants in the lung. The major toxicity of daptomycin is a skeletal muscle myopathy, and monitoring of serum CPK levels is recommended during therapy with daptomycin (34).

Quinupristin-dalfopristin is a combination of naturally occurring compounds that was the first antibiotic introduced for the treatment of VRE infections. The recommended dose is 7.5 mg/kg IV every 8 hours (34). The principal use of this drug is for infections involving VRE, and use has been curtailed because of troublesome side effects, including painful myalgias and arthralgias (34).

The first rule of antibiotics is try not to use them, and the second rule is try not to use too many of them for too long. If empiric antibiotic coverage is necessary, pending culture results, the combination of vancomycin and meropenem will provide adequate coverage for most infections. If invasive candidiasis is a concern, add fluconazole or caspofungin. You can then tailor antibiotic therapy according to the culture results, or discontinue antibiotics if the cultures are sterile (unless you suspect invasive candidiasis, when antifungal therapy should be continued until you have an answer). Remember that fever and leukocytosis are signs of systemic inflammation, not infection, and that only 25-50% of ICU patients with signs of systemic inflammation will have a documented infection (see page 266).

1. Craig WA. Optimizing aminoglycoside use. Crit Care Clin 2011; 27:107-111.
2. Babinchak T, Badal R, Hoban D, Hackel M, et al. Trends in susceptibility of selected gram-negative bacilli isolated from intra-abdominal infections in North America: SMART 2005-2010. Diag Micro Infect Dis 2013; 76:379-381.
. Martinez JA, Cobos-Triqueros N, Soriano A, et al. Influence of empiric therapy with a beta-lactam alone or combined with an aminoglycoside on prognosis of bacteremia due to gram-negatice organisms. Antimicrob Agents Chemother 2010; 54:35903596.
. Wallach J. Interpretation of diagnostic tests. 8th ed. Philadelphia: Lippincott, Williams & Wikins, 2007:1095.
. Turnidge J. Pharmacodynamics and dosing of aminoglycosides. Infect Dis Clin N Am 2003; 17:503-528.
. Wilson SE. Aminoglycosides: assessing the potential for nephrotoxicity. Surg Gynecol Obstet 1986; 171(Suppl):24-30.
. Lippmann M, Yang E, Au E, Lee C. Neuromuscular blocking effects of tobramycin, gentamicin, and cefazolin. Anesth Analg 1982; 61:767—770.
. Drachman DB. Myasthenia gravis. N Engl J Med 1994; 330:179-1810.

Carbapenems, 1 = Carbapenems. 19., 1 = Baughman RP. The use of carbapenems in the treatment of serious infections. J Intensive Care Med 2009; 24:230-241.. 20., 1 = Freifield A, Walsh T, Marshall D, et al. Monotherapy for fever and neutropenia in cancer patients: a randomized comparison of ceftazidime versus imi-penem. J Clin Oncol 1995; 13:165-176.. 21., 1 = Cunha B. Meropenem for clinicians. Antibiotics for Clinicans 2000; 4:59-66.. 22., 1 = Hellinger WC, Brewer NS. Imipenem. Mayo Clin Proc 1991; 66:1074-1081.. 22., 1 = Bennett WM, Aronoff GR, Golper TA, et al. eds. Drug prescribing in renal failure. 3rd ed. Philadelphia: American College of Physicians, 1994.. 23., 1 = Carbapenems. In: McEvoy GK, ed. AHFS Drug Information, 2012. Bethesda: American Society of Health-System Pharmacists, 2012:166-182.

24. Asbel LE, Levison ME. Cephalosporins, carbapenems, and monobactams. Infect Dis Clin N Am 2000; 14:1-10.
25. Cephalosporins: General statement. In: McEvoy GK, ed. AHFS Drug Infor-mation, 2012. Bethesda: American Society of Health-System Pharmacists, 2012:68-83.

32. Wright AJ. The penicillins. Mayo Clin Proc 1999; 74:290-307.
33. Piperacillin and Tazobactam. In: McEvoy GK, ed. AHFS drug information, 2012. Bethesda: American Society of Hospital Pharmacists, 2012:340-344.
34., Vancomycin = Nailor MD, Sobel JD. Antibiotics for gram-positive bacterial infections: vancomycin, teicoplanin, quinupristin/dalfopristin, oxazolidinones, daptomycin, dalbavancin, and telavancin. Infect Dis Clin N Am 2009; 23:965-982.. 35., Vancomycin = Ena J, Dick RW, Jones RN. The epidemiology of intravenous vancomycin usage in a university hospital. JAMA 1993; 269:598-605.. 36., Vancomycin = Rybak M, Lomaestro B, Rotschafer JC, et al. Therapeutic monitoring of vancomycin in adult patients: A consensus review of the American Society of Health System Pharmacists, the Infectious Disease Society of America, and the Society of Infectious Diseases Pharmacists. Am J Heath-Syst Pharm 2009; 66:82-98.. 37., Vancomycin = Leong JVB, Boro MS, Winter ME. Determining vancomycin clearance in an overweight and obese population. Am J Heath-Syst Pharm 2011; 68:599-603.. 38., Vancomycin = Von Drygalski A, Curtis B, Bougie DW, et al. Vancomycin-induced immune thrombocytopenia. N Engl J Med 2007; 356:904-910.. 39., Vancomycin = Black E, Lau TT, Ensom MHH. Vancomycin-induced neutropenia. Is it dose- or duration-related? Ann Pharmacother 2011; 45:629-638.

Alternatives to Vancomycin, 1 = Alternatives to Vancomycin. 40., 1 = Kali AC, Murthy MH, Hermsen ED, et al. Linezolid versus vancomycin or teicoplanin for nosocomial pneumonia: A systematic review and meta-analysis. Crit Care Med 2010; 38:1802-1808.. 41., 1 = Rucker JC, Hamilton SR, Bardenstein D, et al. Linezolid-associated toxic optic neuropathy. Neurology 2006; 66:595-598.. 42., 1 = Daptomycin. In: McEvoy GK, ed. AHFS drug information, 2012. Bethesda: American Society of Hospital Pharmacists, 2012:454-457.

In the successful resuscitation of the shocked patient, the physician achieves his or her greatest victory.
Evan Geler 1993
Pharmacological support of blood pressure and blood flow is one of the fundamental practices in the care of critically ill patients. This chapter describes the principal drugs that are used for circulatory support in the ICU, and includes only drugs that are administered by continuous intravenous infusion. The very last section of the chapter includes a brief comment on the shortcomings of circulatory support drugs in the critically ill.

Catecholamines are drugs that promote blood flow and blood pressure by stimulating adrenergic receptors. The different types of adrenergic receptors are summarized in Table 53.1, and the effects of catecholamine drugs on adrenergic receptors is summarized in Table 53.2. Despite differences in adrenergic receptor activation and physiological responses, no catecholamine drug has proven superior to the others for improving clinical outcomes (1,2).

Vasoconstriction Iris Dilatation Piloerection,  = Cardioacceleration Increased Cardiac omy Lipolysis. Vasoconstriction Iris Dilatation Piloerection,  = Vasodilatation Bronchedilatation Increased Glycolysis Uterine Relaxation
Dobutamine,  = _. Dobutamine,  = ++. Dobutamine,  = +. Dopamine (Moderate Dose),  = =. Dopamine (Moderate Dose),  = ote. Dopamine (Moderate Dose),  = IRAE. Dopamine (High Dosa),  = ++. Dopamine (High Dosa),  = +++. Dopamine (High Dosa),  = +++. Epinephrine,  = +++. Epinephrine,  = HE. Epinephrine,  = +H. Norepinephrine,  = ++H+. Norepinephrine,  = +. Norepinephrine,  = —_—. Phenylephrine,  = +H. Phenylephrine,  = _—. Phenylephrine,  = _

Dobutamine is a synthetic catecholamine that is classified as an inodilator because it has positive inotropic and vasodilator effects.

Dobutamine is primarily a B,-receptor agonist, but also has weak B>-receptor agonist activity. The B j-receptor stimulation produces an in-crease in heart rate and stroke volume, while the B2-receptor stimulation produces peripheral vasodilatation (3,4). The stroke volume augmentation produced by dobutamine is shown in Figure 53.1 (4). Because the increase in stroke volume is accompanied by a decrease in systemic vascular resistance, the blood pressure is usually unchanged or slightly increased (3). The response to dobutamine, however, can vary widely in critically ill patients (5). The cardiac stimulation produced by dobutamine is often accompanied by an increase in cardiac work and myocardial O) consumption (3). These effects can be deleterious in heart failure because cardiac work and myocardial energy needs are already heightened in the failing myocardium.

Dobutamine has been used to augment cardiac output in patients with decompensated heart failure due to systolic dysfunction. However, the unfavorable effects of dobutamine on myocardial energetics has created a preference for other inodilators in decompensated heart failure (see pages 250-251). Dobutamine remains the preferred inotropic agent for the treatment of myocardial depression associated with septic shock (1), but it usually must be combined with a vasoconstrictor agent (e.g., norepinephrine) to raise the blood pressure,

Dobutamine is started at an infusion rate of 3-5 ug/kg/min (without a loading dose), and this can be increased in increments of 3—5 yg/kg/min, if necessary, to achieve the desired effect. (A pulmonary artery catheter is usually needed to guide dobutamine dosing.) The usual dose range is 5-20 wg/kg/min (3), but doses as high as 200 ug/kg/min have been used safely (5). Therapy should be driven by hemodynamic end-points, and not by preselected dose rates.

Dobutamine produces only mild increases in heart rate (5-15 beats/min) in most patients, but it occasionally causes significant tachycardia (rate increases > 30 beats/min) (3), which can be deleterious in patients with coronary artery disease. Like all positive inotropic agents, dobutamine is contraindicated in patients with hypertrophic cardiomyopathy.
FIGURE 53.1 Stroke volume augmentation produced by equivalent doses of dobutamine and dopamine in postcardiopulmonary bypass patients. Data from Reference 4.
EXTRAVASATION OF VASOPRESSORS: The risk of tissue necrosis from extra-vasation of dopamine is a concern with all vasopressor (vasoconstrictor) drug infusions, and eliminating this risk is the reason that large, central veins are recommended for all vasopressor drug infusions. If dopamine or any other vasopressor drug escapes from a peripheral vein into the surrounding tissues, the tendency for ischemic tissue necrosis can be reduced by injecting phentolamine (an a-receptor antagonist) into the involved area. The recommended injectate is a solution containing 5-10 mg phentolamine in 15 mL of isotonic saline (6).

Epinephrine is an endogenous catecholamine that is released by the adrenal medulla in response to physiological stress. It is the most potent natural B-agonist.

Epinephrine stimulates both a-adrenergic and a-adrenergic receptors (B; and Bp» subtypes), and produces dose-dependent increases in heart rate, stroke volume, and blood pressure (11). Epinephrine is a more potent B,-receptor agonist than dopamine, and produces a greater increase in stroke volume and heart rate than comparable doses of dopamine (12). This is demonstrated in Figure 53.2. The B-Receptor stimulation produces a non-uniform peripheral vasoconstriction, with the most prominent effects in the subcutaneous, renal, and splanchnic circulations. Epinephrine also has several metabolic effects, including lipolysis, increased glycolysis, and increased lactate production (from B-Receptor activation), and hyperglycemia from B-Receptor-mediated inhibition of insulin secretion (11,13).

Epinephrine plays an important role in the resuscitation of cardiac arrest (see pages 330-— 332), and it is the drug of choice for hemodynamic support in anaphylactic shock (see pages 274-276). Epinephrine is also used for hemodynamic support in the early postoperative period following cardiopulmonary bypass surgery (4). Although epinephrine is as effective as other catecholamines in septic shock (12,13), concerns about side effects have limited its popularity in septic shock.
FIGURE 53.2 Cardiac effects of epinephrine (Epi), norepinephrine (Norepi), and dopamine (Dopa) at infusion rates needed to maintain a mean arterial pressure of 75 mm Hg in patients with septic shock. Data from Reference 12.

The dosing regimens for epinephrine in cardiac arrest and anaphylactic shock are presented in Table 14.5 (see page 275). Epinephrine infusions are not preceded by a loading dose. The initial infusion rate is usually 1-2 g/min (or 0.02 ~g/kg/min), and the rate is then increased in increments of 1-2 g/min to achieve the desired effect (11). The usual dose range for augmenting cardiac output or correcting hypotension is 5-15 g/min.

Epinephrine creates a greater risk of unwanted cardiac stimulation (which can be deleterious in patients with coronary artery disease) than the other catecholamine drugs (11,12). Other adverse effects include hyperglycemia, increased metabolic rate, and splanchnic hypoperfusion (which can damage the mucosal barrier in the bowel) (11-13). Epinephrine infusions are accompanied by an increase in serum lactate levels (11), but this is not an adverse effect because it reflects an increased rate of glycolysis (not tissue hypoxia), and the lactate can be used as an alternative fuel source (See page 187).

Norepinephrine is an endogenous catecholamine that normally functions as an excitatory neurotransmitter. When used as an exogenous drug, norepinephrine functions as a vasopressor.

The principal action of norepinephrine is B-Receptor-mediated peripheral vasoconstriction. However, the adrenergic response to norepinephrine is altered in patients with septic shock (15). For example, norepinephrine infusions are usually accompanied by a decrease in renal blood flow (15), but in patients with septic shock, renal blood flow is increased by norepinephrine infusions (15,16). Similar alterations may also occur with splanchnic blood flow (i.e., normally reduced, but not in septic shock) (15). Norepinephrine is also a weak B,-receptor agonist, but the effects of norepinephrine on stroke volume and heart rate can be comparable to dopamine (a more potent B,;-receptor agonist) in patients with septic shock (see Figure 53.2).

Norepinephrine is the preferred catecholamine for circulatory support in patients with septic shock. This preference is not based on improved outcomes, because the mortality rate in septic shock is the same regardless of the catecholamine used for circulatory support (1,2,12). Instead, norepinephrine is favored in septic shock because it has fewer adverse effects than dopamine or epinephrine (8,12).

Norepinephrine infusions are usually started at a rate of 8-10 ~g/min, and the dose rate is then titrated upward or downward to maintain a mean blood pressure of at least 65 mm Hg. The effective dose rate in septic shock varies widely in individual patients, but is usually below 40 wg/min. Hypotension that is refractory to norepinephrine usually prompts the addition of dopamine or vasopressin, but there is no evidence that this practice improves outcomes.

Adverse effects of norepinephrine include local tissue necrosis from drug extravasation, and intense systemic vasoconstriction with organ dysfunction when high dose rates are required. However, whenever high doses of a vasoconstrictor drug are required to correct hypotension, it is difficult to distinguish between adverse drug effects and adverse effects of the circulatory shock.

Phenylephrine is a potent vasoconstrictor that has very few applications in the ICU.

Phenylephrine in a pure B-Receptor agonist that produces widespread vasoconstriction. The consequences of this vasoconstriction can include bradycardia, a decrease in cardiac stroke output (usually in patients with cardiac dysfunction), and hypoperfusion of the kidneys and bowel.

The principal use of phenylephrine is for the reversal of severe hypotension produced by spinal anesthesia. However, pure B-Receptor agonists are not universally favored in this situation because they can aggravate the decrease in cardiac stroke output that occurs in spinal shock (17). Phenylephrine is not recommended for hemodynamic support in septic shock, although a clinical study comparing phenylephrine and norepinephrine for the early management of septic shock showed no differences in hemodynamic effects or clinical outcomes with the use of either drug (18).

Phenylephrine can be given as intermittent IV doses. The initial IV dose is 0.2 mg, which can be repeated in increments of 0.1 mg to a maximum dose of 0.5 mg (17). Phenylephrine can be infused at an initial dose rate 0.1-0.2 mg/min, which is progressively decreased after the blood pressure is stabilized (17).

The principal adverse effects of phenylephrine are bradycardia, low cardiac output, and renal hypoperfusion. These effects are magnified in hypovolemic patients.

The following drugs can be added to vasopressor therapy with catecholamines in selected situations.

Terlipressin is a vasopressin analogue that has two advantages over vasopressin. First, it is a selective V; receptor agonist, and does not produce the side effects associated with stimulation of the other vasopressin receptors. Secondly, terlipressin has a much longer duration of action than vasopressin, and a single IV dose of 1—2 mg can raise the blood pressure for 5 hours (19). The long duration of action allows terlipressin to be given by intermittent IV dosing. Terlipressin is a potent splanchnic vasoconstrictor, and may prove valuable in the management of variceal bleeding. However, there is an increased risk of splanchnic ischemia with terlipressin, and ischemic effects cannot be reversed for 5 hours after the drug is administered. Like vasopressin, there is no survival advantage associated with the addition of terlipressin in patients with septic shock (20).

Drugs that produce vasodilatation via nitric oxide-—mediated relaxation of vascular smooth muscle are known as nitrovasodilators (21). There are two drugs that act in this manner: nitroglycerin and nitroprusside.

Nitroglycerin is an organic nitrate that produces a dose-dependent dilation of arteries and veins (22,23).

The biochemical basis for the vasodilator actions of nitroglycerin is illustrated in Figure 53.3 Nitroglycerin (glyceryl trinitrate) binds to the surface of endothelial cells and releases inorganic nitrite (NO2), which is converted to nitric oxide (NO) in the endothelial cells. The nitric oxide then moves out of the endothelial cells and into adjacent smooth muscle cells, where it produces muscle relaxation by promoting the formation of cyclic guanosine monophosphate (cGMP).
Venodilatation predominates at lower infusion rates of nitroglycerin (<50 g/min), while higher infusion rates produce arterial vasodilatation as well. Both of these effects are advantageous in patients with heart failure; i.e., the venodilatation reduces cardiac filling pressures (which reduces edema formation), and the arterial vasodilatation reduces ventricular afterload (which increases cardiac stroke output).
FIGURE 53.3 The biochemical basis for the vasodilator actions of nitroglycerin (NTG) and nitroprusside (NTP). Chemical symbok: nitroglycerin [C3H503-(NO2)3], nitroprusside (FeCNs5-NO), inorganic nitrite (NO), nitric oxide (NO), guanosine triphosphate (GTP), cyclic guanosine monophosphate (cGMP).

Nitrates inhibit platelet aggregation, and nitric oxide is believed to mediate this effect as well (24). Because platelet thrombi play an important role in the pathogenesis of acute coronary syndromes, the antiplatelet actions of nitroglycerin have been proposed as the mechanism for the antianginal effects of the drug (24). This would explain why
because this solvent makes up 30-50% of some nitroglycerin preparations (25). (For a description of propylene glycol toxicity, see page 911-912).
NITRATE TOLERANCE: Tolerance to the vasodilator and antiplatelet actions of nitroglycerin is a well-described phenomenon, and can appear after only 24—48 hours of continuous drug administration (25). The underlying mechanism may be oxidative stressinduced endothelial dysfunction (29). The most effective measure for preventing or reversing nitrate tolerance is a daily drug-free interval of at least 6 hours (25).

Nitroprusside is a rapidly-acting vasodilator that is favored for the treatment of hypertensive emergencies. The popularity of this drug is limited by the risk of cyanide intoxication.

The vasodilator actions of nitroprusside, like those of nitroglycerin, are mediated by nitric oxide (21). The nitroprusside molecule contains one nitrosyl group (NO), which is released as nitric oxide when nitroprusside enters the bloodstream. The nitric oxide somehow ends up in endothelial cells, where it proceeds as shown in Figure 53.3.
Like nitroglycerin, nitroprusside dilates both arteries and veins, but it is less potent than nitroglycerin as a venodilator, and more potent as an arterial vasodilator. Nitroprusside has variable effects on cardiac output in subjects with normal cardiac function (30), but it consistently improves cardiac output in patients with decompensated heart failure (30,31).

The principal uses of nitroprusside are the treatment of hypertensive emergencies, where rapid blood pressure reduction is desirable, and management of acute, decompensated heart failure, as described on page 248.

Nitroprusside infusions are started at 0.2 yg/kg/min, and then titrated upward every 5 minutes to the desired result. Control of hypertension usually requires infusion rates of 2— 5 yg/kg/min, but infusion rates should be kept below 3 yg/kg/min, if possible, to limit the risk of cyanide intoxication (30). In renal failure, the infusion rate should be kept below 1 Ug/kg/min to limit thiocyanate accumulation (described later) (30).

Nitroprusside infusions carry a considerable risk of cyanide intoxication. In fact, cyanide accumulation is common during therapeutic infusions of nitroprusside (25,32,33). The origin of the cyanide is the nitroprusside molecule, which is a ferricyanide complex with 5 cyanide molecules bound to an oxidized iron core (see Figure 53.4). This cyanide is released in the bloodstream when nitroprusside breaks up to release nitric oxide and exert its vasodilator actions. The clearance mechanisms for the released cyanide are shown. in Figure 53.4. Two chemical reactions help to remove cyanide from the bloodstream. One involves the binding of cyanide to the oxidized iron moiety in methemoglobin. The other reaction involves the transfer of sulfur from a donor molecule (thiosulfate) to cyanide to form a thiocyanate compound, which is then cleared by the kidneys. The latter (transulfuration) reaction is the principal mechanism for removing cyanide from the human body.
FIGURE 53.4 The fate of cyanide (CN) that is released from nitroprusside molecules. See text for explanation.
Healthy adults have enough methemoglobin to bind the cyanide in 18 mg of nitroprusside, and enough thiosulfate to bind the cyanide in 50 mg of nitroprusside (25). This means that healthy adults can detoxify 68 mg of nitroprusside. At a nitroprusside infusion rate of 2 ug/kg/minute (therapeutic dose) in an 80-kg adult, the 68 mg limit of detoxification is reached in 500 minutes (8.3 hours) after the start of the infusion. Thereafter, the cyanide released by nitroprusside will combine with the oxidized iron in cytochrome oxidase and block the utilization of oxygen in the mitochondria.
The capacity for cyanide removal is reduced by thiosulfate depletion, which is common in smokers and postoperative patients (25,32). To eliminate the risk of thiosulfate depletion, thiosulfate can be routinely added to the nitroprusside infusate. About 500 mg of thiosulfate should be added for every 50 mg of nitroprusside (24).

CLINICAL MANIFESTATIONS: One of the early signs of cyanide accumulation is nitroprusside tachyphylaxis (25); i.e., progressively increasing requirements for nitroprusside to maintain the desired blood pressure. Signs of impaired oxygen utilization (i.e., an increase in central venous O> saturation, and an increase in plasma lactate levels) often do not appear until the late stages of cyanide intoxication (34). As a result, the absence of lactic acidosis during nitroprusside infusion does not exclude the possibility of cyanide accumulation (25,32).
Evidence of possible cyanide intoxication should prompt immediate discontinuation of nitroprusside. Whole blood cyanide levels can be used to confirm the diagnosis of cyanide intoxication, but results are not immediately available, and clinical suspicion is the impetus to begin detoxification measures. These measures are described in Chapter 55.

The most important mechanism for cyanide removal is the formation of thiocyanate, which is slowly excreted in the urine. When renal function is impaired, thiocyanate can accumulate and produce a toxic syndrome that is distinct from cyanide intoxication. The clinical features of thiocyanate intoxication include anxiety, confusion, pupillary constriction, tinnitus, hallucinations, and generalized seizures (25,32). Thiocyanate can also promote hypothyroidism by blocking thyroidal uptake of iodine (32).
The diagnosis of thiocyanate toxicity is established by the serum thiocyanate level. Normal levels are below 10 mg/L, and clinical toxicity is usually accompanied by levels above 100 mg/L (32). Thiocyanate intoxication can be treated by hemodialysis or peritoneal dialysis.

One of the frustrating aspects of critical care practice is the continuing high mortality rates in circulatory shock, particularly septic shock, de-spite correction of the blood pressure with vasopressor drugs. The likely explanation for this is the probability that low blood pressure plays little or no role in the pathogenesis of circulatory shock, or in the clinical outcomes. This is consistent with observations in septic shock indicating that the pathological injury is a defect in oxygen utilization in mitochondria, and the culprit is uncontrolled inflammation, not a low blood pressure. In light of this explanation, the decrease in blood pressure that occurs in shock is more likely to be the result of the pathological cell injury (i.e., shock of the blood vessels) rather than a cause of the injury. Hypotension then becomes one of several consequences of cellular shock, and correcting the hypotension is not expected to correct the primary pathological process. After at least 50 years of focusing on vasopressor therapy in shock, it's time for a do-over.

1. Holmes CL, Walley KR. Vasoactive drugs for vasodilatory shock in ICU. Curr Opin Crit Care 2009; 15:398—402.
2. Beale RJ, Hollenberg SM, Vincent J-L, Parrillo JE. Vasopressor and inotropic support in septic shock: An evidence-based review. Crit Care Med 2004; 32(Suppl): S455—S465.
Dobutamine, 1 = Dobutamine. 3., 1 = Dobutamine hydrochloride. In McEvoy GK, ed. AHFS Drug Information, 2012. Bethesda: American Society of Health-System Pharmacists, 2012:1314-1316.. 4., 1 = Steen PA, Tinker JH, Pluth JR, et al. Efficacy of dopamine, dobutamine, and epinephrine during emergence from cardiopulmonary bypass in man. Circulation 1978; 57:378-384.. 5., 1 = Hayes MA, Yau EHS, Timmins AC, et al. Response of critically ill patients to treatment aimed at achieving supranormal oxygen delivery and consumption. Relationship to outcome. Chest 1993; 103:886-895.
Norepinephrine, 1 = Norepinephrine. 14., 1 = Norepinephrine bitartrate. In: McEvoy GK, ed. AHFS Drug Information, 2012. Bethesda: American Society of Health System Pharmacists, 2012:1371-1374.. 15., 1 = Bellomo R, Wan L, May C. Vasoactive drugs and acute kidney injury. Crit Care Med 2008; 36(Suppl):S179-S186.. 16., 1 = Desairs P, Pinaud M, Bugnon D, Tasseau F. Norepinephrine therapy has no deleterious renal effects in human septic shock. Crit Care Med 1989; 17:426-429.

17. Phenylephrine hydrochloride. In: McEvoy GK, ed. AHFS Drug Information, 2012. Bethesda,: American Society of Health System Pharmacists, 2012:1306—-1311.
18. Morelli A, Ertmer C, Rehberg S, et al. Phenylephrine versus norepinephrine for initial hemodynamic support of patients with septic shock: a randomized, controlled trial. Crit Care 2008; 12:R143.

19. Treschan TA, Peters J. The vasopressin system: physiology and clinical strategies. Anesthesiology 2006; 105:599-612.
20. Polito A, Parisini E, Ricci Z, et al. Vasopressin for treatment of vasodilatory shock: an ESICM systematic review and meta-analysis. Intensive Care Med 2012; 38:9-19.
Nitroprusside, 1 = Nitroprusside. 30., 1 = Sodium nitroprusside. In: McEvoy GK, ed. AHFS Drug Information, 2012. Bethesda: American Society of Health System Pharmacists, 2012:1811-1814.. 31., 1 = Guiha NH, Cohn JN, Mikulic E, et al. Treatment of refractory heart failure with infusion of nitroprusside. New Engl J Med 1974; 291:587-592.. 32., 1 = Hall VA, Guest JM. Sodium nitroprusside-induced cyanide intoxication and prevention with sodium thiosulfate prophylaxis. Am J Crit Care 1992; 2:19-27.. 33., 1 = Robin ED, McCauley R. Nitroprusside-related cyanide poisoning. Time (long past due) for urgent, effective interventions. Chest 1992; 102:1842-1845.. 34., 1 = Arieff AI. Is measurement of venous oxygen saturation useful in the diagnosis of cyanide poisoning? Am J Med 1992; 93:582-583.

There are two things which Man cannot look at directly without flinching: the sun and death.
Francois De La Rochefoucauld 1630-1680

Poisons and medicine are oftentimes the same substance given with different intents.
Peter Latham 1865
Prescription drug use in the United States is astronomical (see Figure 54.1), and among the massively consumed prescription drugs are "drugs of abuse" like opioid analgesics. The magnitude of the opioid prescribing frenzy is demonstrated by a recent estimate that the number of prescribed opioid analgesics in 2010 was enough to medicate every single adult in the United States for a period of one month (using typical doses) (1). As a result of the liberal prescribing practices for potentially dangerous drugs, prescription drugs have replaced "street drugs" as the principal offenders in toxic drug ingestions (2), and there has been an increase in the number of drug overdoses, as well as an increase in the number of fatal drug overdoses, in recent years (2a).
This chapter describes overdoses involving 5 pharmaceutical drugs: 3 prescription drugs (benzodiazepines, B-Receptor antagonists, and opioids) and 2 over-the-counter drugs (acetaminophen and salicylates).

Acetaminophen is a ubiquitous analgesic-antipyretic agent that is included in over 600 commercial drug preparations. It is also a hepatotoxin, and is the most common cause of acute liver failure in the United States (3). Acetaminophen overdoses are responsible for half of the cases of acute liver failure in the United States, and half of the overdoses are unintentional (4). Because of the hepatotoxic risk, the Food and Drug Administration issued a mandate in 2009 for more prominent labeling of the risks associated with acetaminophen, and the recommended maximum dose of acetaminophen was reduced from 4 to 3.25 grams daily (5). The impact of these changes is unclear at the present time.
FIGURE 54.1 Prescription drug use in the United States in 2007-2010, by age group. Data from Health United States, 2012. DHHS Publication No. 2013-1232, May, 2013.

The toxicity of acetaminophen is related to its metabolism in the liver, and the metabolic pathways are shown Figure 54.2. The bulk of acetaminophen metabolism involves the formation of nontoxic sulfated conjugates, which are excreted in the urine. About 10% of the metabolism involves the oxidation of acetaminophen to form a toxic metabolite that is capable of producing oxidant cell injury. When the daily dose of acetaminophen is not excessive, the toxic metabolite is removed by conjugation with glutathione, an intracellular antioxidant. When the daily dose of acetaminophen exceeds 4 grams, the sulfated conjugation pathways become saturated, which diverts acetaminophen metabolism to the pathway with the toxic metabolite. The increased traffic in this pathway eventually depletes glutathione reserves, and when glutathione stores fall to 30% of normal, the toxic acetaminophen metabolite can accumulate and promote hepatocellular damage (3).

The toxic dose of acetaminophen can vary widely in individual patients, but is somewhere between 7.5 and 15 grams in most adults (6,7). However, several conditions can increase the susceptibility to acetaminophen hepatotoxicity, such as malnutrition, chronic illness, and chronic ethanol ingestion, and the toxic dose of acetaminophen may be lower in these patients. A general rule adopted by poison control centers is to recommend evaluation for acute ingestions of 10 grams of acetaminophen (20 extra-strength Tylenol tablets) (3). For patients who may have an increased susceptibility to acetaminophen hepatotoxicity, an acute ingestion of 4 grams of acetaminophen warrants an evaluation (3). The maximum recommended daily dose of acetaminophen is 3.25 grams (5).

In the first 24 hours following a toxic ingestion, symptoms are either absent or nonspecific (e.g., nausea, vomiting, malaise), and liver enzymes do not begin to rise until 24-36 hours post-ingestion (6). Elevated aspartate aminotranferase (AST) is the most sensitive marker of acetaminophen toxicity; the rise in AST precedes the hepatic dysfunction, and peak levels are reached at 72—96 hours. Evidence of hepatic injury becomes apparent 24—48 hours after ingestion, with steadily rising liver enzymes, and the appearance of jaundice and a coagulopathy. Peak hepatic injury occurs at 3-5 days after the toxic ingestion, and hepatic encephalopathy may be evident at this time, along with acute, oliguric renal failure (mechanism unclear), and lactic acidosis (from reduced hepatic clearance) (3). Death from hepatic injury usually occurs 3-5 days post-ingestion. Patients who survive often recover completely, although recovery can be prolonged.
FIGURE 54.2 The hepatic metabolism of acetaminophen. See text for explanation.

In many cases of acetaminophen overdose, the initial encounter with the patient occurs within 24 hours after drug ingestion, when there are no manifestations of hepatic injury. The principal task at this time is to determine the risk of hepatocellular injury, and this involves two considerations: the time elapsed from the toxic ingestion, and the plasma level of acetaminophen. The ingested dose is not used to determine hepatotoxic risk because it is not possible to determine if the patient's recollection of the ingested dose is accurate. In addition, the toxic dose of acetaminophen can vary in individual patients.

Plasma acetaminophen levels obtained from 4-24 hours after drug ingestion can be used to predict the risk of hepatotoxicity using the nomogram in Figure 54.3 (7). If the plasma level is in the high-risk region of the nomogram, the risk of developing hepatotoxicity is 60% or higher, and antidote therapy is warranted. The risk of hepatotoxicity is only 13% in the low-risk region of the nomogram, and this does not warrant antidote therapy (7). This nomogram is useful only when the time of drug ingestion can be identified, and when the plasma drug level can be measured between 4 and 24 hours post-ingestion.
FIGURE 54.3 Nomogram for predicting the risk of hepatotoxicity according to the plasma acetaminophen level between 4 and 24 hours after ingestion. A plasma level that falls on, or above, the treatment line is an indication to begin antidotal therapy with N-acetykysteine. Nomogram redrawn from Reference 7.

Glutathione does not readily cross cell membranes, so exogenous glutathione is not a viable treatment option for acetaminophen hepatotoxicity. N-acetylcysteine (NAC) is a glutathione analogue that readily crosses cross cell membranes, and can serve as an intracellular glutathione surrogate (see Figure 54.2) (8). The cysteine residue in NAC contains a sulfhydryl group (SH), which is capable of reducing (and inactivating) the toxic acetaminophen metabolite.

NAC is most effective when therapy is started within 8 hours after ingestion; in this situation, NAC reduces the risk of hepatotoxicity to <5% (3). NAC is less protective after 10 hours have elapsed, but some protection is provided for 24 hours after drug ingestion (6,9). NAC can also have beneficial effects after the onset of acetaminophen hepatotoxicity (3,10). Therefore, evidence of hepatotoxicity after 24 hours post-ingestion is an indication to begin NAC therapy (3).

NAC can be given intravenously or orally using the dosing regimens shown in Table 54.1. Despite significant differences in total NAC dose and duration of therapy, the two dosing regimens are considered equally effective (13,14). The intravenous route is preferred because it is the most reliable mode of drug delivery, and is not as disagreeable as oral ingestion of NAC (see "Adverse Reactions").
EXTENDED TREATMENT: The standard duration of treatment is 21 hours for the IV regimen, and 72 hours for the oral regimen. However, because NAC can hasten the resolution of acetaminophen-induced liver injury (3) , treatment with NAC should be continued beyond the normal course of therapy if there is evidence of continued liver injury (3). For the IV regimen, therapy is continued using the recommendations for the last 16 hours of the regimen. NAC can be discontinued when ALT levels have peaked and are improving, and when the INR is <1.3 (3).

Intravenous NAC can cause anaphylactoid reactions, and fatal reactions have been reported in asthmatics (15). Oral NAC has a very disagreeable taste (because of the sulfur content), and often causes nausea and vomiting. The oral NAC regimen produces diarrhea in about 50% of patients, but this usually resolves with continued therapy (16).

Acetaminophen is rapidly absorbed from the GI tract, and activated charcoal (1 g/kg body weight) is recommended only if given within the first 4 hours after drug ingestion (17). However, following massive drug ingestions, charcoal can provide a benefit when given as late as 16 hours after drug ingestion (3). Activated charcoal does not curb the efficacy of oral NAC (3).

Use 20% NAC (200 mg/mL) for each of the doses below and infuse in sequence.
1. 150 mg/kg In 200 mL DsW over 60 min
2, 50 mg/kg in 500 mL D.W over 4 hrs
3, 100 mg/kg in 1,000 mL DW over 16 hrs
Total
dose:
300 mg/kg over 21 hrs

Use 10% NAC (100 mg/mL) and dilute 2:1 In water or juice to make a 5% solution (50 mg/mL).
Initial dose:
140 mg/kg
Maintenance dosage: 70 mg/kg every 4 hrs for 17 doses
Total dose:
1,330 mg/kg over 72 hrs
From Referances 11 and 12.

Benzodiazepines are second only to opiates as the drugs most frequently involved in medication-related deaths (2). However, benzodiazepines are rarely fatal when ingested alone (18), and other respiratory depressant drugs (e.g., opiates) are almost always involved in benzodiazepine-related fatalities (2).

Because overdoses involving benzodiazepines also involve other drugs, the clinical presentation can vary (according to the drugs ingested). Pure benzodiazepine overdoses produce deep sedation, but rarely result in coma (18). Respiratory depression (2-12% of cases), bradycardia (1-2% of cases) and hypotension (5-7% of cases) are also uncommon (18). Benzodiazepine intoxication can also produce an agitated confusional state (with hallucinations) that could be mistaken for alcohol withdrawal (18). Involvement of benzodiazepines in an apparent overdose can be difficult to establish because there are no serum assays for benzodiazepines, and qualitative tests for benzodiazepines in urine are unreliable because of a limited spectrum of detection (19). Benzodiazepine involvement is usually based on the clinical history.

Intentional B-blocker overdoses are uncommon, but can be life-threatening. An effective antidote is available, if needed.

The typical manifestations of B-blocker overdose are bradycardia and hypotension (26). The bradycardia is usually sinus in origin, and is well tolerated. The hypotension can be due to peripheral vasodilatation (renin blockade), or a decrease in cardiac output, (B,receptor blockade). Hypotension that is sudden in onset is usually a reflection of a decrease in cardiac output and is an ominous sign.

Excessive doses of B-blockers can exert a membrane-stabilizing effect that is independent of the B-Receptor blockade. The principal consequence of this membrane-stabilizing effect is prolonged atrioventricular (AV) conduction, which can progress to complete heart block (27).

Most B-blockers are lipophilic, and have a tendency to accumulate in lipid-rich tissues like the central nervous system. As a result, B-blocker overdose is often accompanied by lethargy, depressed consciousness, and generalized seizures. The latter manifestation is more prevalent than suspected, and has been reported in 60% of overdoses with propranolol (28). Like the prolonged AV conduction, the neurological manifestations are not the result of B-Receptor blockade, and may be related to the membrane-stabilizing effect.

Glucagon is a regulatory hormone that acts in opposition to insulin by stimulating glycogen breakdown to raise blood glucose levels. In a seemingly unrelated role, glucagon antagonizes the cardiac depression produced by B-Receptor antagonists.

The illustration in Figure 54.4 shows how glucagon can correct the cardiac depression caused by B-blockers and calcium channel blockers. The glucagon receptor and BReceptor are linked to the adenyl cyclase en-zyme on the inner surface of the cell membrane. Activation of each receptor-enzyme complex results in the hydrolysis of adenosine triphosphate (ATP) and the formation of cyclic adenosine monophosphate (cyclic AMP). The cyclic AMP then activates a protein kinase that promotes the inward movement of calcium through the cell membrane. The influx of calcium promotes interactions between contractile proteins to enhance the strength of cardiac contraction. This sequence of reactions just described is responsible for the positive inotropic and chronotropic effects of B-Receptor stimulation. Since the same reactions occur with activation of the glucagon receptor, glucagon has positive inotropic and chronotropic effects that are equivalent to B-Receptor agonists. More importantly, the parallel orientation of the gluca-gon receptor and B-Receptor allows glucagon to retain its cardiostimulatory effects when B-Receptors are quiescent. This is the basis for gluca-gon's role as an antidote for B-blocker toxicity.

Glucagon is indicated for the treatment of hypotension and symptomatic bradycardia associated with toxic exposure to B-blockers. When used in the appropriate doses, glucagon will elicit a favorable response in 90% of patients (29). Glucagon is not indicated for reversing the prolonged AV conduction or neurological abnormalities in Bblocker overdoses because these effects are not mediated by B-Receptor blockade.
CALCIUM ANTAGONIST TOXICITY: Glucagon is also capable of antagonizing the effects of calcium channel blockers (30), as illustrated in Figure 54.4. However, glucagon is less effective in reversing the cardiac depression precipitated by calcium channel blocker overdoses. This is not unexpected, because glucagon and calcium blockers act at different sites in the pathways that modulate cardiac contractile strength.
FIGURE 54.4 Mechanisms of drug-induced alterations in cardiac contractile strength. See text for explanation. Abbreviations: ATP=adenosine triphosphate; cAMP=cyclic adenosine monophosphate; PDE=phosphodiesterase; AMP=adenosine monophosphate.

Dosing recommendations for glucagon are presented in Table 54.2. The effective dose of glucagon can vary in individual patients, but a bolus dose of 3-5 mg IV should be effective in most adults (28,29). The initial dose is 3 mg (or 0.05 mg/kg) as an IV bolus. The response to glucagon is usually evident within 3 minutes (30). If the response is not satisfactory, a second IV bolus dose of 5 mg (or 0.07 mg/kg) can be administered. The effects of glucagon can be short-lived (5 minutes), and so a favorable response should be followed by a continuous infusion (5 mg/hr). The chronotropic response to glucagon is optimal when the plasma ionized calcium is normal (31).

When the actions of B-receptor antagonists or calcium channel blockers result in: 1. Hypotension, or 2. Symptomatic Bradycardia, Dosing Regimen = 1. Start with 50 pg/kg (or 3 mg) as IV bolus. 2. If response Is not satisfactory, give a second IV bolus of 70 ng/kg (or 5 mg). 3. Follow a satisfactory response with 4 continuous infusion at: 70 uwa/kg/hr (or 5 ma/hr}.

Nausea and vomiting are common at glucagon doses above 5 mg/hr. Mild hyperglycemia is common, and is the result of glucagon's actions to stimulate glycogenolysis. The insulin response to the hyperglycemia can drive potassium into cells and promote hypokalemia.
Finally, glucagon stimulates catecholamine release from the adrenal medulla, and this can cause unwanted increases in blood pressure in patients with hypertension.

Phosphodiesterase inhibitors (e.g., inamrinone, milrinone) augment cardiac contractile strength by inhibiting the breakdown of cyclic AMP, as illustrated in Figure 54.4. These agents can increase cardiac output in the setting of B-blockade (32), and they should add to the increase in cyclic AMP produced by glucagon. However, it is unclear if phosphodiesterase inhibitors add to the effectiveness of glucagon in B-blocker toxicity. Because these drugs are vasodilators, and can produce unwanted decreases in blood pressure, they are generally reserved for cases of B-blocker toxicity that are resistant, or refractory, to glucagon.

Opiates are implicated in 75% of fatal drug overdoses in the United States (2a), and the problem is likely to grow rather than shrink. The adverse effects of opioids are described in Chapter 51; the following description focuses on the use of the narcotic antagonist, naloxone.

The classic description of an opiate overdose is a patient who presents with stupor, pinpoint pupils, and slow breathing (bradypnea). However, these clinical findings are either absent or nonspecific, and it is not possible to identify an opiate overdose based on the clinical presentation or physical examination (33). The response to the narcotic antagonist, naloxone, is probably the most reliable method of identifying an opioid overdose.
Table 54.3 Naloxone Dosing
1. Start with 0.4 mg IV bolus. 2. If no response In 2-3 min, give another 0.4 mg IV bolus. 3. Ifno response in 2-3 min, give 2 mg IV bolus. 4. lf no response in 2-3 min, STOP and reassess., Respiratory Depression = 1. Start with 2 mg IV bolus. 2. If no response in 2-3 min, give 4 mg IV bolus. 3. If no response in 2-3 min, give 10 mg IV bolus. 4. If no response in 2-3 min, give 15 mg IV bolus. 5. If no response in 2-3 min, STOP and reassess,

Empiric therapy with naloxone (0.2-8 mg IV bolus) has been used in patients with altered mentation to identify occult cases of opioid overdose. However, this practice is effective in fewer than 5% of patients with altered mental status of unknown etiology (38). An alternative approach has been proposed where empiric naloxone is indicated only for patients with pinpoint pupils and circumstantial evidence of opioid abuse (e.g., needle tracks) (24,38). When naloxone is used in this manner, a favorable response (indicating an opioid overdose) is expected in about 90% of patients (38).

Naloxone has few undesirable effects. The most common adverse reaction is the opioid withdrawal syndrome (anxiety, abdominal cramps, vomiting, and piloerection). There are case reports of acute pulmonary edema (most in the early postoperative period) and generalized seizures following naloxone administration (24), but these are rare occurrences.
Despite a steady decline in prevalence, salicylate intoxication is the 14th leading cause of drug-induced deaths in the United States (39).

Ingestion of 10-30 grams of aspirin (150 mg/kg) can have fatal consequences. Once ingested, acetylsalicylic acid (aspirin) is promptly converted to salicylic acid, which is the active form of the drug. Salicylic acid is readily absorbed from the upper GI tract, and metabolism takes place in the liver. The hallmark of salicylate intoxication is the combination of a respiratory alkalosis and a metabolic acidosis.

Within hours after a toxic ingestion of aspirin, there is an increase in respiratory rate and tidal volume. This is the result of direct stimulation of brainstem respiratory neurons by Salicylic acid, and the subsequent increase in minute ventilation results in a decrease in arterial PCO; (i.e., acute respiratory alkalosis).

Salicylic acid is a weak acid that does not readily dissociate, and thus does not produce a metabolic acidosis. However, salicylic acid activates proteins in the mitochondria that uncouple oxidative phosphorylation, and this results in a marked increase in the anaerobic production of lactic acid, and this is the principal source of the metabolic acidosis in salicylate intoxication. The mixed metabolic acidosis-respiratory alkalosis produces an arterial blood gas with a low PCO3, a low bicarbonate, and a normal pH (see Rule 3 on page 593). As the lactic acidosis progresses, the serum pH will eventually fall, which is a poor prognostic sign (40).

Clinical features in the early stages of salicylate toxicity include nausea, vomiting, tinnitus, and agitation. Progression of the toxidrome is associated with neurologic changes (delirium, seizures, and progression to coma), fever (from the uncoupled oxidative phosphorylation), and acute respiratory distress syndrome (ARDS).

The plasma salicylate level is used to confirm or exclude the diagnosis of salicylate toxicity. The therapeutic range of salicylates in plasma is 10-30 mg/L (0.7—2.2 mmol/L), and levels above 40 mg/L (2.9 mmol/L) are considered toxic (40). Plasma salicylate levels are usually elevated within 4-6 hours after a toxic ingestion.

Management of salicylate toxicity includes general supportive care (i.e., fluids, vasopressors, and mechanical ventilation), if necessary. Multiple-dose activated charcoal is recommended, if it can be started within 2-3 hours of drug ingestion. The dosing regimen is 25 grams orally every 2 hours for 3 doses.

Alkalinization of the urine to enhance salicylate excretion is the cornerstone of management for salicylate intoxication. Salicylic acid has a pK of 3 (the pK is the pH at which 50% of the acid is dissociated), which means that the acid will dissociate more readily as the pH rises. An alkaline pH in the urine will promote dissociation of salicylic acid in the renal tubules, and this essentially "traps" the salicylic acid in the renal tubular lumen, where it can be excreted in the urine. Bicarbonate infusions are used to raise the urine pH using the following regimen, which is summarized in Table 54.4.

1. Greate a bicarbonate solution by adding 3 amps NaHCO, (44 mEq Nat/amp).
2. Start with a bicarbonate loading dose of 1-2 mEq/kg.
3. Follow with infusion of the bicarbonate solution at 2-3 mL'kg/nr.
4, Maintain a urine output of 1-2 mL/kg/hr, and a urine pH of =7.5.
Fram Referances 40 and 44.
REGIMEN: Start IV bicarbonate therapy with 1-2 mEq/kg as an IV bolus, and follow with a continuous bicarbonate infusion. To create the bicarbonate infusate, add 3 ampules of 40% sodium bicarbonate (43 mEq/amp) to 1 liter of D>7jW (129 mEq/L), and infuse this solution at 2-3 mL/kg/hr (40). The urine pH should be maintained at 27.5 (41). Treatment continues until the plasma salicylate levels fall below the toxic range.
HYPOKALEMIA: Bicarbonate infusions will lower the serum potassium (intracellular shift), and hypokalemia hampers the ability to alkalinize the urine. This is explained by the K+ H+ exchanger in the distal renal tubules, which releases H+ into the tubular fluid as K+ is reabsorbed. Since K+ reabsorption is increased in the setting of hypokalemia, the extra H+ released into the tubular fluid will hamper attempts to alkalinize the urine. Potassium should be added to the bicarbonate solution (40 mEq/L) to reduce the risk of hypokalemia.

Hemodialysis is the most effective method of clearing salicylates from the body (42). The indications for hemodialysis include a serum salicylate level >100 mg/L, the presence of renal failure or ARDS, or progression of the toxidrome despite alkalinization therapy (39).

If any over-the-counter drug deserves to be regulated, it's acetaminophen. Not because it is the leading cause of acute liver failure in the United States (which should be enough of a reason), but because half of the overdoses are unintentional, indicating a general lack of awareness of the toxic potential of acetaminophen (which can be fatal). The FDA has called for stronger warnings on drug packaging, but who reads that small print? Requiring a prescription that specifies how the drug should be used is the best way to ensure that acetaminophen is used safely in the general population.
Acetaminophen gained its popularity in the 1970s because of concerns about the toxicity of aspirin, and it looks like we moved from the frying pan into the fire.

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2. Centers for Disease Control and Prevention. National Vital Statistics System. 2010 Multiple Cause of Death File. Hyattsville, MD: US Department of Health and Human Services, Centers for Disease Control and Prevention; 2012.
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18. Gaudreault P, Guay J, Thivierge RL, Verdy I. Benzodiazepine poisoning. Drug Saf 1991; 6:247-265.
19. Wu AH, McCay C, Broussard LA, et al. National Academy of Clinical Biochemistry laboratory medicine practice guidelines: Recommendations for the use of laboratory tests to support poisoned patients who present to the emergency department. Clin Chem 2003; 49:357-379.
, 1 = Flumazenil. 20., 1 = Howland MA. Flumazenil. In: Flomenbaum NE, et al., eds. Goldfrank's Toxicologic Emergencies. 8th ed. New York: McGraw-Hill, 2006; 1112-1117.. 21., 1 = Shalansky SJ, Naumann TL, Englander FA. Effect of flumazenil on benzodiazepine- induced respiratory depression. Clin Pharm 1993; 12:483-487.. 22., 1 = Roche Laboratories. Romazicon (flumazenil) package insert. 2004.. 23., 1 = Bodenham A, Park GR. Reversal of prolonged sedation using flumazenil in critically ill patients. Anaesthesia 1989; 44:603-605.. 24., 1 = Doyon S, Roberts JR. Reappraisal of the 'coma cocktail'. Dextrose, flumazenil, naloxone, and thiamine. Emerg Med Clin North Am 1994; 12:301-316.. 25., 1 = Haverkos GP, DiSalvo RP, Imhoff TE. Fatal seizures after flumazenil administration in a patient with mixed overdose. Ann Pharmacother 1994; 28:1347-1349.

ß-Receptor Antagonists, 1 = ß-Receptor Antagonists. 26., 1 = Newton CR, Delgado JH, Gomez HF. Calcium and beta receptor antagonist overdose: a review and update of pharmacological principles and management. Semin Respir Crit Care Med 2002; 23:19-25.. 27., 1 = Henry JA, Cassidy SL. Membrane stabilising activity: a major cause of fatal poisoning. Lancet 1986; 1:1414-1417.. 28., 1 = Weinstein RS. Recognition and management of poisoning with beta-adrenergic blocking agents. Ann Emerg Med 1984; 13:1123-1131.. 29., 1 = Kerns W, 2nd, Kline J, Ford MD. Beta-blocker and calcium channel blocker toxicity. Emerg Med Clin North Am 1994; 12:365-390.

30. Howland MA. Glucagon. In: Flomenbaum NE, et al., eds. Goldfrank's Toxicologic Emergencies. 8th ed. New York: McGraw-Hill, 2006; 942-945.
31. Chernow B, Zaloga GP, Malcolm D, et al. Glucagon's chronotropic action is calcium dependent. J Pharmacol Exp Ther 1987; 241:833-837.
32. Travill CM, Pugh S, Noblr MI. The inotropic and hemodynamic effects of intravenous milrinone when reflex adrenergic stimulation is suppressed by beta adrenergic blockade. Clin Ther 1994; 16:783-792.

In clinical matters, ignorance can be dangerous, but ignorance of ignorance can be fatal.
P.L.M.
This chapter describes toxic syndromes that are not the result of medications, and includes sections on carbon monoxide, cyanide, and the toxic alcohols (methanol and ethylene glycol). While these toxidromes will not be encountered often, they can be lethal if not recognized.

Carbon monoxide (CO) is a gaseous product of incomplete combustion involving organic (carbon-based) matter, and is one oxidation reaction shy of carbon dioxide: i.e., 2CO + O> B 2 CO. The principal source of CO poisoning is smoke inhalation during structural fires. The exhaust from automobile engines was once a major source of CO, but the catalytic converters that are mandated for all automobiles (and convert CO to CO>) have reduced CO emissions by more than 95%.

Carbon monoxide binds to the heme moieties in hemoglobin (at the same site that binds oxygen) to produce carboxyhemoglobin (COHb). The affinity of CO for binding to hemoglobin is 200-300 times greater than the affinity of O2, and CO pressures of only 0.4 mm Hg can fully saturate hemoglobin (2). The effects of COHb on systemic oxygenation are demonstrated in Figure 55.1. The curves in this graph show the relationship between oxygen tension (PO2) and oxygen content when hemoglobin is normal (upper curve), and when COHb makes up 50% of the hemoglobin molecules (lower curve) (3). The arterial O> content (point A) decreases in proportion to the increase in COHb, reflecting the ability of CO to block O» binding to hemoglobin. The venous point (point V) on both curves was identified by assuming a normal arteriovenous O, content difference (CaOz — CvO> = 5 mL/dL) for both curves. The venous PO> (PvQ>) is a close approximation of tissue PO, and it is much lower when COHb is present. This provides indirect evidence that CO poisoning can impair tissue oxygenation.
FIGURE 55.1 Influence of 50% carboxyhemoglobin on the relationship between oxygen tension (PO2) and oxygen content.

Carbon monoxide has toxic effects that are unrelated to COHb (4). These effects include: (a) inhibition of cytochrome oxidase, which impairs the ability to generate ATP from oxidative metabolism, (b) release of nitric oxide from platelets, which promotes the formation of peroxynitrite, a powerful oxidant capable of widespread cell injury, and (c)
activation of neutrophils, which is an additional source of oxidant injury (see Figure 14.1). These effects may play an important role in CO poisoning, because there is a poor correlation between COHb levels and the severity of carbon monoxide poisoning (1,4).

The treatment for CO poisoning is inhalation of 100% oxygen. The elimination half-life of COHb is 320 minutes while breathing room air, and 74 minutes while breathing 100% oxygen (1), so only a few hours of breathing pure oxygen is needed to return COHb levels to normal.

Hyperbaric oxygen has been advocated as a means of reducing the delayed neurologic sequelae in CO poisoning, but evidence of benefit has not been convincing (7).

House fires generate cyanide as well as carbon monoxide, and the possibility of cyanide poisoning must be considered in all patients with CO poisoning from a house fire. When smoke inhalation from a house fire is accompanied by a severe metabolic acidosis (pH <7.2) or a markedly elevated serum lactate level (210 mmol/L), empiric treatment for cyanide poisoning is recommended (1). See the next section for the treatment of cyanide poisoning.

Cyanide is a lethal toxin with a nefarious history. The Nazis used hydrogen cyanide gas (Zyklon-B) for mass murder in the 1940s, and in 1978, cyanide-laced fruit drinks were used for mass murder/suicide by Jim Jones and his followers. The principal source of cyanide poisoning is inhalation of hydrogen cyanide gas during domestic fires (8,9). Infusion of the vasodilator, sodium nitroprusside, is an additional source of cyanide toxicity in ICU patients (see Figure 53.4).

Cyanide ions have a high affinity for metalloproteins, including the oxidized iron (Fe3+) in cytochrome oxidase and methemoglobin, and the cobalt in hydroxocobalamin (a precursor of vitamin B,2). Cytochrome oxidase is situated at the end of the electrontransport chain in mitochondria, and is the site where the electrons collected during ATP production are used to reduce oxygen to water (thereby clearing the way for continued ATP production). Cyanide-induced inhibition of cytochrome oxidase halts the process of oxidative metabolism in mitochondria. This halts the uptake of pyruvate into mitochondria, and results in excess production of lactic acid and a progressive metabolic (lactic) acidosis. The defect in cellular energy production is fatal if not corrected.

There are two endogenous mechanisms for clearing cyanide from the body, and these are shown in Figure 53.4 (see page 953). The principal mechanism is the transfer of sulfur from thiosulfate (S203) to cyanide, which forms thiocyanate (SCN). This is called a transulfuration reaction.
$,0, + CN > SCN + SO;
(55.1)
Thiocyanate is then cleared by the kidneys. Thiocyanate can accumulate in patients with renal failure, and can cause an acute psychosis (10).
The second (minor) mechanism for cyanide clearance is the reaction of cyanide with methemoglobin to form cyanomethemoglobin; i.e.,
Hb-Fe** + CN — Hb-Fe**— CN
(55.2)
The hemoglobin-bound cyanide is eventually cleared via transulfuration. These two clearance mechanisms are easily overwhelmed, especially in the setting of thiosulfate deficiency (e.g., in smokers).

Early signs of cyanide poisoning include agitation, tachycardia, and tachypnea, representing the compensatory stage of metabolic acidosis. Progressive cyanide accumulation eventually results in loss of consciousness, bradycardia, hypotension, and cardiac arrest. Plasma lactate levels are typically very high (>10 mmol/L), and venous blood can look "arterialized" because of the marked decrease in tissue O> utilization. Progression is rapid after smoke inhalation, and the time from onset of symptoms to cardiac arrest can be less than 5 minutes (8).

Cyanide poisoning is a clinical diagnosis. Whole blood cyanide levels can be used for documentation, but results are not immediately available, and cyanide antidotes must be given quickly for optimal results. The clinical diagnosis of cyanide poisoning is particularly challenging in victims of smoke inhalation, because many of the clinical features of cyanide poisoning are indistinguishable from carbon monoxide (CO) poisoning. As a general rule of thumb, severe metabolic (lactic) acidosis and hemodynamic instability are the clinical features that distinguish cyanide poisoning from CO poisoning in victims of smoke inhalation (8,9).

Antidote therapy should begin immediately after cyanide poisoning is first suspected. The antidotes for cyanide poisoning are presented in Table 55.1.

The antidote of choice for cyanide poisoning is hydroxocobalamin, a cobalt-containing precursor of vitamin By that combines with cyanide to form cyanocobalamin, which is then excreted in the urine. The recommended dose is 5 grams, as an IV bolus. A second dose of 5 grams is recommended for patients with cardiac arrest (8). Hydroxocobalamin is relatively safe to use, but urine and other body fluids can have a reddish color for a few days.

Hydroxocobalamin, Dosing Regimens and Comments. = Dosing: Comment:. Hydroxocobalamin, Dosing Regimens and Comments. = 5 grams as IV bolus. Give 10 g for cardiac arrest. The antidote of choice for cyanide poisoning. May cause reddish color in urine for a few days.. Sodium Thiosulfate (25%), Dosing Regimens and Comments. = Dosing: Comment:. Sodium Thiosulfate (25%), Dosing Regimens and Comments. = 50 mL (12.5 g) as IV bolus. Used in combination with hydroxocobalamin. Avold, if possible, in patients with renal failure.. Amy! Nitrate Inhalant, Dosing Regimens and Comments. = Dosing: Comment:. Amy! Nitrate Inhalant, Dosing Regimens and Comments. = Inhale for 30 sec. each min, for up to 5 min. Used only for temporary relief when IV access Is not available. Contraindicated in smoke inhalation.
From Referances 8, 9.

Sodium thiosulfate converts cyanide to thiocyanate (see Equation 55.1), and is used in combination with hydroxocobalamin. The recommended dose is 12.5 grams as an IV bolus. Since thiocyanate can accumulate in renal failure and cause an acute psychosis (10), thiosulfate should not be used in patients with renal failure. If thiosulfate is given before evidence of renal failure is available, watch for signs of thiocyanate toxicity (which is treated with hemodialysis).

Nitrates promote cyanide clearance by promoting the formation of methemoglobin. However, nitrates have undesirable side effects, and are contraindicated in smoke inhalation (because they cause a leftward shift in the oxyhemoglobin dissociation curve, and can aggravate the similar effect of carbon monoxide). The only role for nitrates in cyanide poisoning is the use of inhaled amyl nitrate as a temporary measure when IV access is not available.

There are specialized antidote kits for cyanide poisoning (e.g., Akorn Cyanide Antidote Kit) that contain amyl nitrate for inhalation, sodium nitrite (300 mg in 10 mL) for IV injection, and sodium thiosulfate (12.5 g in 50 mL) for IV injection. These kits can be used as a source of thiosulfate, but they do not contain hydroxocobalamin (at least at the present time).

Ethylene glycol and methanol are common components of household, automotive, and industrial products, and they both produce toxidromes characterized by a metabolic acidosis. They are known as toxic alcohols (10), which is a misnomer, because it implies that ethanol is non-toxic. Toxic alcohols are the 12th leading source of toxic exposures in the United States (11).

Ethylene glycol is the main ingredient in many automotive antifreeze products. It has a sweet, agreeable taste, which makes it a popular method of attempted suicide.

Ethylene glycol is readily absorbed from the GI tract, and 80% of the ingested dose is metabolized in the liver. As shown in Figure 55.2, the metabolism of ethylene glycol involves the formation of a series of acids, with the participation of alcohol dehydrogenase and lactate dehydrogenase enzymes, and ends with the formation of oxalic acid (12). Each of the intermediate reactions involves the conversion of NAD to NADH, which promotes the conversion of pyruvate to lactate. As a result, serum lactate levels are also elevated in ethylene glycol poisoning (12). Each of the acid intermediates in ethylene glycol metabolism is a strong acid that readily dissociates, and can contribute to a metabolic acidosis. The oxalic acid also combines with calcium to form insoluble calcium oxalate crystals that precipitate in several tissues, and are particularly prominent in the renal tubules. These crystals can be a source of renal tubular injury.

Early signs of ethylene glycol intoxication include nausea, vomiting, and apparent inebriation (altered mental status, slurred speech, and ataxia). Because ethylene glycol is odorless, there is no odor of alcohol on the breath. Severe cases are accompanied by depressed consciousness, coma, generalized seizures, renal failure, pulmonary edema, and cardiovascular collapse (12). Renal failure can be a late finding (24 hours after ingestion).
Laboratory studies show a metabolic acidosis with an elevated anion gap and an elevated osmolal gap (see page 656 for a description of the osmolal gap). Serum lactate levels can be elevated (usually 5-6 mEq/L). A plasma assay for ethylene glycol is available, and a level >20 mg/dL is considered toxic, but the results are not immediately available, and are not used in the decision to initiate therapy (12). (Plasma levels can be used to guide decisions about discontinuing therapy, as described later.)
FIGURE 55.2 The metabolism of ethylene glycol and methanol in the liver. AD = alcohol dehydrogenase; LDH = lactate dehydrogenase; FMP = fomepizok.
CRYSTALLURIA: Calcium Oxalate Crystals Can Be Visualized In The Urine In About 50% Of Cases Of Ethylene Glycol Poisoning (14). The Presence Of Calcium Oxalate Crystals Is Not Specific For Ethylene Glycol Poisoning, But The Shape Of The Crystals Is More Specific; I.E., Thin, Monohydrate Crystals, Like The Ones Shown In Figure 55.3, Are More Characteristic Of Ethylene Glycol Poisoning Than The Box-Shaped Dihydrate Crystals (12). Most Hospital Laboratories Do Not Routinely Inspect Urine For Crystals, So Make Sure To Request A Search For Crystalluria When Ethylene Glycol Toxicity Is Supected.

The management of ethylene glycol poisoning involves measures to alter the metabolism of ethylene glycol, along with hemodialysis, if necessary.
FOMEPIZOLE: Fomepizole Inhibits Alcohol Dehydrogenase, The Enzyme Involved In The Initial Step Of Ethylene Glycol Metabolism (See Figure 55.2). The Recommended Dosing Regimen For Both Ethylene Glycol And Methanol Poisoning Is Shown In Table 55.2. The Best Results Are Obtained If Therapy Begins Within 4 Hours Of Ingestion. Fomepizole Should Be Continued Until The Metabolic Acidosis Has Resolved, And The Plasma Level Of Ethylene Glycol Is In The Non-Toxic Range (12).
FIGURE 55.3 Microscopic appearance of calcium oxalate monohydrate crystals. The presence of these thin, needleshaped crystals in the urine is highly suggestive of ethylene glycol poisoning.
1. Start with an IV loading dose of 15 mg/kg.
2. Follow with a dose of 10 mq/kg IV every 12 hrs for 4 doses.
3. Then increase the dose to 15 mg/kg IV every 12 hrst, and continue until the following end-points are reached:
(a) the plasma toxin level is <20 mg/dL,
(b) the plasma pH is normal,
(c) the patient Is asymptomatic.
4. lf more than one hemodialysis session is required, change the dose to 15 mg/kg IV every 4 hrs, until dialysis Is no longer necessary.
The increased dose represents compensation for 4 sal-inducad incraasa in metabolism. Fram Referance 12.
HEMODIALYSIS: The clearance of ethylene glycol and all its metabolites is enhanced by hemodialysis. The indications for immediate hemodialysis include severe acidemia (pH <7.1), and evidence of significant end-organ damage (e.g., coma, seizures, and renal insufficiency) (12). Multiple courses of hemodialysis may be necessary, and fomepizole dosing should be adjusted, if hemodialysis is continued, as indicated in Table 55.2.

ADJUNCTS: THIAMINE (100 MG IV DAILY) AND PYRIDOXINE (100 MG IV DAILY) ARE RECOMMENDED TO DIVERT GLYOXYLIC ACID TO THE FORMATION OF NON-TOXIC METABOLITES (SEE Figure 55.2).

Methanol (also Known as wood alcohol because it was first distilled from wood) is a common ingredient in shellac, varnish, windshield washer fluid, and solid cooking fuel (Sterno) (12).

Like ethylene glycol, methanol is readily absorbed from the upper GI tract, and is metabolized by alcohol dehydrogenase in the liver. The principal metabolite is formic acid, a strong acid that readily dissociates and produces a metabolic acidosis with a high anion gap. Formic acid is also a mitochondrial toxin that inhibits cytochrome oxidase and blocks oxidative energy production. Tissues that are particularly susceptible to damage are the retina, optic nerve, and basal ganglia (12). Methanol metabolism promotes the conversion of pyruvate to lactate in the same manner described for ethylene glycol metabolism, and lactate production is increased further by the actions of formic acid on cytochrome oxidase activity.

Early manifestations (within 6 hours of ingestion) include signs of apparent inebriation without the odor of alcohol on the breath (as in ethylene glycol intoxication). Later signs (6-24 hours after ingestion) include visual disturbances (e.g., scotoma, blurred vision, complete blindness), depressed consciousness, coma, and generalized seizures (12). Examination of the retina can reveal papilledema and generalized retinal edema. Visual disturbances are characteristic of methanol poisoning, and are not a feature of ethylene glycol poisoning.
Laboratory studies show a high-anion-gap metabolic acidosis and an elevated osmolal gap, similar to ethylene glycol poisoning. However, there is no crystalluria in methanol poisoning. A plasma assay for methanol is available, and a level above 20 mg/dL is considered toxic. However, the re-sults of the plasma assay are not immediately available, and are not used in the decision to initiate therapy.

Treatment for methanol poisoning is the same as described for ethylene glycol poisoning, except for the following: (a) visual impairment is an indication for dialysis in methanol poisoning, and (b) folinic acid is used as adjunctive therapy in methanol poisoning, instead of thiamine and pyridoxine.
OLINIC ACID: Polinic Acid (Leucovorin) Can Convert Formic Acid To Non-Toxic Metabolites. The Recommended Dose Is 1 Mg/Kg Iv, Up To 50 Mg, At 4-Hour Intervals (12). Folic Acid Should Be Used If Folinic Acid Is Unavailable.

1. Carbon monoxide poisoning can be missed if you rely on pulse oximetry readings to detect O> desaturation. Detection of elevated carboxyhemoglobin levels requires an 8wavelength CO-oximeter, which is available in most clinical laboratories.
. A victim of smoke inhalation who has a severe metabolic acidosis should be treated empirically for cyanide poisoning.
. Methanol and ethylene glycol poisoning should be considered in any patient who presents with a high-anion-gap metabolic acidosis of unclear etiology.
. If ethylene glycol poisoning is a consideration, inspection of the urine for calcium oxalate monohydrate crystals can provide useful information.

. Hampson NB, Piantadosi CA, Thom SR, Weaver LK. Practice recommendations in the diagnosis, management, and prevention of carbon monoxide poisoning. Am J Resp Crit Care Med 2012; 186:1095-1101.
. Guyton AC, Hall JE. Medical Physiology, 10th ed. Philadelphia: W.B. Saunders, Co, 2000:470.
. Nunn JF Nunn's Applied Respiratory Physiology. 4th ed. Oxford: Butterworth Heinemann, 1993:279-280.
. Guzman JA. Carbon monoxide poisoning. Crit Care Clin 2012; 28:537-548.
. Kalay N, Ozdogru I, Cetinkaya Y, et al. Cardiovascular effects of carbon monoxide poisoning. Am J Cardiol 2007; 99:322-324.
. Choi IS. Delayed neurologic sequelae in carbon monoxide intoxication. Arch Neurol 1983; 40:433-435.
. Buckley NA, Juurlick DN, Isbister G, et al. Hyperbaric oxygen for carbon monoxide poisoning. Cochrana Database Syst Rev 2011; 4:CD002041.

8. Anseeuw K, Delvau N, Burill-Putze G, et al. Cyanide poisoning by fire smoke inhalation: a European expert consensus. Eur J Emerg Med 2013; 20:29.
9. Baud FJ. Cyanide: critical issues in diagnosis and treatment. Hum Exp Toxicol 2007; 26:191-201.
Toxic Alcohols, 1 = Toxic Alcohols. 10., 1 = Weiner SW. Toxic alcohols. In Nelson LS, Lewin NA, Howland MA, et al., eds. Goldfrank's Toxicologic Emergencies. 9th ed. New York: McGraw-Hill, 2006:1400- 1410.. 11., 1 = Bronstein AC, Spyker DA, Cantilena LR, Jr , et al. 2011 Annual Report of the American Association of Poison Control Centers' National Poison Data System (NPDS): 29th Annual Report. Clin Toxicol 2012; 50:911-1164.. 12., 1 = Kruse PA. Methanol and ethylene glycol intoxication. Crit Care Clin 2012; 28:661- 711.
When you're through learning, you're through.
Vernon Law

The units of measurements in the medical sciences are taken from the metric system (centimeter, gram, second) and the Anglo-Saxon system (foot, pound, second). The metric units were introduced during the French Revolution and were revised in 1960. The revised units are called Systeme Internationale (SI) units and are currently the worldwide standard.

Length, Dimensions = L. Length, Basic SI Unit (Symbol) = Meter (m). Length, _ Equivalencies = 1 inch = 2.54 cm. Area, Dimensions = L2. Area, Basic SI Unit (Symbol) = Square meter (m2). Area, _ Equivalencies = 1 square centimeter (cm?) = 104 m?. Volume, Dimensions = i. Volume, Basic SI Unit (Symbol) = Cubic meter (m4). Volume, _ Equivalencies = 1 liter (LK) = 0.001 m3 { milliliter (mL) = 1 cubic centimeter (cm?). Mass, Dimensions = M. Mass, Basic SI Unit (Symbol) = Kilogram (kg). Mass, _ Equivalencies = 1 pound (lb) = 453.5 g 1 kg = 2.2 Ibs. Density, Dimensions = MLS. Density, Basic SI Unit (Symbol) = Kilogram per cuble meter (kg/m?). Density, _ Equivalencies = 1 kg/m?= 0.001 kg/d? Density of water = 1.0 kg/dms Density of mercury = 13.6 kg/dm. Velocity, Dimensions = UT. Velocity, Basic SI Unit (Symbol) = Meters per second (msec). Velocity, _ Equivalencies = 1 mile per hour (mph) = 0.4 m/sec. Acceleration, Dimensions = LT?. Acceleration, Basic SI Unit (Symbol) = Meters persecond squared (m/sec?). Acceleration, _ Equivalencies = 1 ft/sec®= 0.03 m/sec?. Force, Dimensions = M x (LT?). Force, Basic SI Unit (Symbol) = Newton (Nj = kg x (m/sec?). Force, _ Equivalencies = 1 dyne = 105 N
Units of Measurement in the Systeme Internationale (SI

Pressure, 1 = Mx (LT?) L2. Pressure, 2 = Pascal (Pa)=N/m?. Pressure, 3 = 1kPa=7.5mmHg = 10.2 cm H,0 1mm Hg =1 x 10-9 torr (See conversion table for kPa and cm H20). Heat, 1 = Mx(U/T3)xL. Heat, 2 = Joule ()=Nxm. Heat, 3 = 1 kilocalorie (kcal) = 4184 J. Temperature, 1 = . Temperature, 2 = Kelvin (K). Temperature, 3 = O° C =-273 K (Se conversion table for °C and °F). Viscosity, 1 = M, 1/L, 1/T. Viscosity, 2 = ~=Newton x second per square meter (N « sec/m?). Viscosity, 3 = Centipoise (cP) = 10-3 N « sec/m?. Amount of a substance, 1 = N. Amount of a substance, 2 = Mole (mol = molecular weight in grams. Amount of a substance, 3 = Equivalent (Eq) = mol x valence
- 1. For ions that exist freety in an aqueous solution, the concentration is expressed as milliequivalents per liter (mEq/L). To convert to millimoles per liter (mmol/L):
a. For a unlvalent ion like potassium (K*), the concentration In mmol/L is the same as the concentration in mEq/L.
b. For a divalent ion like magnesium (Mg**), the concentration in mmol/L is one-half the concentration in mEq/L.
2. For ions that are partially bound or complexed to other molecules (2.g., plasma Ca'), the concentration is usually expressed as milligrams per deciliter (mg/dL). To convert to mEq/L:
mol wt where mol wt is molecular weight, and the factor 10 is used to convert deciliters (100 mL) to liters.

EXAMPLE: Ca** has a molecular weight of 40 and a valence of 2, so a plasma Gat concentration of 8 mg/dL Is equivalent to: (8x 10/40) x 2= 4 mEq.
Part 2 Converting Units of Solute Concentration
- 3. The concentration of uncharged molecules (@.g., glucose) is also expressed as milligrams per deciliter (mg/dL). To convert to (mmol/L):
EXAMPLE: Glucose has a molecular weight of 180, so a plasma glucose concentration of 80 mg/dL Is equivalent to: (90 x 10/180) = 5 mmolL.
- 4, The concentration of solutes can also be expressed in terms of osmotic pressure, which determines the distribution of water in different fluid compartments. Osmotic activity in aqueous solutions (called osmolality) is expressed as millosmoles per kg water (mosmkg H,O or mosm/kq). The following formulas can be used to express the osmolality of solute concentrations (n Is the number of nondissociable particles per molecule).
EXAMPLE:
- a. Aplasma Nat concentration of 140 mEq/L has the following osmolality:
- b. Aplasma glucose concentration of 90 mg/dL has the following osmotality:
The sodium in plasma has 4 much greater osmotic activity than the glucose in plasma because osmotic activity Is determined by the number of particles in solution, and Is independent of the size of the particles (1.¢., one sodium ion has the same osmotic activity as one glucose molecule).

1 grain = 60mg 1 ounce = 30mg 1 fluid ounce = 30 mL 1 pint = 500 mL 1 quart = 947 mL, Household = 1 teaspoonful = 5 mL 1 tablespoonful = 15 mL 1 wineglassful = 60 mL 1 teacupful = 120 mL
emperature Conversion
A, 1 = 105.8. A, 2 = 35. A, 3 = 95. 40, 1 = 104. 40, 2 = 34. 40, 3 = 93.2. 39, 1 = 102.2. 39, 2 = 33. 39, 3 = 91.4. 38, 1 = 100.4. 38, 2 = 32. 38, 3 = 89.6. 37, 1 = 98.6. 37, 2 = 31. 37, 3 = 87.8. 36, 1 = 96.8. 36, 2 = 30. 36, 3 = 86. °F = (9/5 °C) + 82, 1 = °F = (9/5 °C) + 82. °F = (9/5 °C) + 82, 2 = °C = 5/9 (°F — 32). °F = (9/5 °C) + 82, 3 = °C = 5/9 (°F — 32)

= 7.58. , 2 = v7. , 3 = 10.24. , 4 = . , 5 = 12.90. , 1 = eek. , 2 = 18. , 3 = 10.37. , 4 = 98. , 5 = 13.03. , 1 = 7.85. , 2 = 79. , 3 = 10.51. , 4 = . , 5 = 13.17. , 1 = 7.98. , 2 = 80. , 3 = 10.64. , 4 = 100. , 5 = 13.90
Kilopascal (kPa) = 0.133 x mmHg; mmHg =7.5 x kPa
1, 1 = 0.01. 1, 2 = 0.3. 4, 1 = 0.05. 4, 2 = 1.3. 8, 1 = 0.10. 8, 2 = 2.6. 10, 1 = 013. 10, 2 = 3.3. 12, 1 = 0.16. 12, 2 = 4.0. 14, 1 = 0.18. 14, 2 = 46. 16, 1 = 0.21. 16, 2 = 5.3. 18, 1 = 0.23. 18, 2 = 6.0. 20, 1 = 0.26. 20, 2 = 6.6. 22, 1 = 0.28. 22, 2 = 7.3. 24, 1 = 0.31. 24, 2 = 8.0. 26, 1 = 0.34. 26, 2 = 8.6. 28, 1 = 0.36. 28, 2 = 3.3. 30, 1 = 0.39. 30, 2 = 10.0. 32, 1 = 0.41. 32, 2 = 10.6. 34, 1 = 0.44. 34, 2 = 11.3. 36, 1 = 0.47. 36, 2 = 12.0. 38, 1 = 0.50. 38, 2 = 12.6
"Diameters can vary with manufacturers. However a useful ruia of thumb is OD (mm) x 3 = French size.

26, 1 = 0.018. 26, 2 = 0.45. 25, 1 = 0.020. 25, 2 = 0.50. 24, 1 = 0.022. 24, 2 = 0.56. 23, 1 = 0.024. 23, 2 = 0,61. 22, 1 = 0.028. 22, 2 = 0.71. 21, 1 = 0.032. 21, 2 = 0.81. 20, 1 = 0.036. 20, 2 = 0.91. 19, 1 = 0.040. 19, 2 = 1.02. 18, 1 = 0.048. 18, 2 = 1.22. 16, 1 = 0.040. 16, 2 = 1.62. 14, 1 = 0.080. 14, 2 = 2.03. 12, 1 = 0.104. 12, 2 = 2.64
"Diameters can vary with manufacturers.

(SGOT), SIUnits = 0-0.58 ukat/L. B-Hydroxybutyrate, ree = S$. B-Hydroxybutyrate, Traditional Units = <1.0 mg/dL. B-Hydroxybutyrate, xk = = 96.05. B-Hydroxybutyrate, SIUnits = <100 umol/L. Bicarbonate, ree = s. Bicarbonate, Traditional Units = 22-26 mEq/L. Bicarbonate, xk = = 1. Bicarbonate, SIUnits = 22-26 mmol/L. Bilirubin: Total Conjugated, ree = s §. Bilirubin: Total Conjugated, Traditional Units = 0.1-1.0 mg/dL =0.2 mg/dL. Bilirubin: Total Conjugated, xk = = 74. Bilirubin: Total Conjugated, SIUnits = 2-18 pmol/L <4 umol/L. Blood urea nitrogen (BUN), ree = RS. Blood urea nitrogen (BUN), Traditional Units = 818 mg/dl. Blood urea nitrogen (BUN), xk = = 0.367. Blood urea nitrogen (BUN), SIUnits = 3.0-6.5 mmol/L. Calcium: Total lonized, ree = s e. Calcium: Total lonized, Traditional Units = 8.5-10.5 mg/dl 2.2-2.3 mEq/L. Calcium: Total lonized, xk = = 0.26 0.49. Calcium: Total lonized, SIUnits = 2.2-2.6 mmol/L 4.41-1.15 mmol/L
Reference Ranges for Selected Clinical Laboratory Test

5, 1 = 2. 5, 2 = 128-134. 5, 3 = 131-141. 5, 4 = 138-150. 5, 1 = 3. 5, 2 = 130-136. 5, 3 = 183-143. 5, 4 = 140-153. 5, 1 = 4. 5, 2 = 132-138. 5, 3 = 135-145. 5, 4 = 142-156. 5, 1 = 5. 5, 2 = 134-140. 5, 3 = 137-148. 5, 4 = 144-160. 5, 1 = 6. 5, 2 = 136-142. 5, 3 = 139-151. 5, 4 = 146-164. 5, 1 = if. 5, 2 = 138-145. 5, 3 = 142-154. 5, 4 = 149-168. 5, 1 = 8. 5, 2 = 140-148. 5, 3 = 145-157. 5, 4 = 152-172. 5, 1 = 9. 5, 2 = 142-151. 5, 3 = 148-160. 5, 4 = 155-176. 5, 1 = 10. 5, 2 = 144-154. 5, 3 = 151-163. 5, 4 = 158-180. 5, 1 = 11. 5, 2 = 146-157. 5, 3 = 154-166. 5, 4 = 161-184. 6, 1 = 0. 6, 2 = 149-160. 6, 3 = 157-170. 6, 4 = 164-188. 6, 1 = 1. 6, 2 = 152-164. 6, 3 = 160-174. 6, 4 = 168-192. 6, 1 = 2. 6, 2 = 155-163. 6, 3 = 164-178. 6, 4 = 172-197. 6, 1 = 3. 6, 2 = 158-172. 6, 3 = 167-182. 6, 4 = 172-202. 6, 1 = 4. 6, 2 = 162-176. 6, 3 = 171-187. 6, 4 = 181-207

4, 1 = 10. 4, 2 = 102-111. 4, 3 = 109-121. 4, 4 = 112-131. 4, 1 = 11. 4, 2 = 103-113. 4, 3 = 111-123. 4, 4 = 120-134. 5, 1 = 0. 5, 2 = 104-115. 5, 3 = 113-126. 5, 4 = 122-137. 5, 1 = 1. 5, 2 = 106-118. 5, 3 = 115-129. 5, 4 = 125-140. 5, 1 = 2. 5, 2 = 108-121. 5, 3 = 118-132. 5, 4 = 128-143. 5, 1 = 3. 5, 2 = 111-124. 5, 3 = 121-135. 5, 4 = 131-147. 5, 1 = 4. 5, 2 = 114-127. 5, 3 = 124-138. 5, 4 = 134-151. 5, 1 = 5. 5, 2 = 117-130. 5, 3 = 127-141. 5, 4 = 137-155. 5, 1 = 6. 5, 2 = 120-133. 5, 3 = 130-144. 5, 4 = 140-159. 5, 1 = ze. 5, 2 = 123-136. 5, 3 = 133-147. 5, 4 = 143-163. 5, 1 = 8. 5, 2 = 126-139. 5, 3 = 186-150. 5, 4 = 146-167. 5, 1 = 9. 5, 2 = 129-142. 5, 3 = 139-153. 5, 4 = 149-170. 5, 1 = 10. 5, 2 = 132-145. 5, 3 = 142-156. 5, 4 = 152-173. 5, 1 = 11. 5, 2 = 135-148. 5, 3 = 145-159. 5, 4 = 155-176. 6, 1 = 1. 6, 2 = 138-151. 6, 3 = 148-162. 6, 4 = 158-179
"Unclothad weights associated with the longest lita expectancies. From the statistics bureau of the Metropolitan Life Insurance Company, 1983.
Body Mass Inde

20, 1 = 602. 20, 2 = 649. 20, 3 = 693. 20, 4 = 740. 25, 1 = 580. 25, 2 = 636. 25, 3 = 679. 25, 4 = 725. 30, 1 = Sf?. 30, 2 = 622. 30, 3 = 664. 30, 4 = 710. 35, 1 = 565. 35, 2 = 609. 35, 3 = 651. 35, 4 = 695. 40, 1 = 552. 40, 2 = 596. 40, 3 = 636. 40, 4 = 680. 45, 1 = 540. 45, 2 = 583. 45, 3 = 622. 45, 4 = 665. 50, 1 = 527. 50, 2 = 569. 50, 3 = 607. 50, 4 = 649. 55, 1 = 515. 55, 2 = 556. 55, 3 = 593. 55, 4 = 634. 60, 1 = 502. 60, 2 = 542. 60, 3 = 578. 60, 4 = 618. 65, 1 = 490. 65, 2 = 529. 65, 3 = 564. 65, 4 = 603. 70, 1 = Ar?. 70, 2 = 515. 70, 3 = 550. 70, 4 = 587
Peak How (Lémin) = [3.95 — (0.0151 x Agel] x Ht (em)
Ragression equation from Leiner GC et al. Arm Rev Raspir Dis 1983; 88:646.
Peak Expiratory Flow Rates for Healthy Females

20, 1 = 308. 20, 2 = 423. 20, 3 = 460. 20, 4 = 496. 25, 1 = 385. 25, 2 = 418. 25, 3 = 454. 25, 4 = 490. 30, 1 = 380. 30, 2 = AIS. 30, 3 = 448. 30, 4 = 483. 35, 1 = 375. 35, 2 = 408. 35, 3 = 442. 35, 4 = 476. 40, 1 = 370. 40, 2 = 402. 40, 3 = 436. 40, 4 = 470. 45, 1 = 365. 45, 2 = 3g7. 45, 3 = 430. 45, 4 = 464. 50, 1 = 360. 50, 2 = 391. 50, 3 = 424. 50, 4 = AS?. 55, 1 = 355. 55, 2 = 386. 55, 3 = 418. 55, 4 = 451. 60, 1 = 350. 60, 2 = 380. 60, 3 = 412. 60, 4 = 445. 65, 1 = 345. 65, 2 = 375. 65, 3 = 406. 65, 4 = 439. 70, 1 = 340. 70, 2 = 369. 70, 3 = 400. 70, 4 = 432
Peak Flow (Limin) = [2.93 - (0.0072 x Agal] x Ht (cm)
Ragression equation from Leiner GC et d. Am Rev Respir Dis 1963; 88:645,

Males: IBW (kg) = 50 + 2.3 (Ht in inches — 60)
Females:
IBW (kg) = 45.5 + 2.3 (Ht in inches — 60)
Body Mass Indext
BMI =

Dubois Formulat
BSA (m2) = Ht (in cm) + Wt (in kg)P-425 x 0.007184
Jacobson Formula®
BSA (m2) =
Ht (in cm) 2 (in kg) — 60
"Devine BJ. Drug Intell Clin Pharm 1974; 8:650.
TMatz R. Ann Intam Med 1993; 118:232.
+ Dubois EF, Basal metabolism in health and disease, Philadelphia: Lea & Febigar, 19365.
QJacobson B. Medicine and clinical engineering. Englewood Cliffs, NJ: Prentice-Hall, 1977.

cr cp = 140=Age) x Welght (KG) tnt sminy
SCr (mg/dL) * 72
x 0.85 (for females)

Gr CL= 170 x SCr (mg/dL) x Age®.176 (mL/min/1.73 m2)
* 0.762 (for females)
* 1.180 (for African Americans)
SCr = sarum creatinine; MDMR = Modification of Diet in Renal Disease; 1.73 m? is the body surfaca area Of an average-sized adult.
(1) Cockcroft DW, Gault MH. Nephron. 1976; 16:34-44.
(2) Lavey AS, Bosch JP Lewis JB, et al. Ann Intern Med 1999; 120:461-470.
Wt (in Ibs)
Ht (in inches}? * 703

Page numbers followed by f indicate figures and those followed by t indicate tables.

A-a PO; gradient, 395-397, 402. See also Hypoxemia and hypercapnia
age and, 396, 396t
inspired oxygen on, influence of, 396-397
positive-pressure ventilation and, 397
a/A PO; ratio, 397-398, 397f
Abciximab (ReoPro), in acute coronary syndromes, 315
Abdominal abscesses, 746-747
clinical features, 746
computed tomography in, 746-747
management of, 747
Abdominal compartment syndrome (ACS), 643-645, 725-726
definitions, 643, 643t
intraabdominal pressure and, 645
management of, 645
predisposing conditions, 644
renal dysfunction and, 644-645
surgical decompression for, 645
Abdominal infections, in ICU, 737-750
acalculous cholecystitis, 737-738
clinical features, 738
diagnostic test for, 738
management of, 738
pathogenesis, 737—738
microbial colonization in GI tract, 738-744
Clostridium difficile infections, 740-744
gastric colonization, 739-740
organisms isolated in, 746t
postoperative infections, 744-747
abdominal abscesses, 746-747
peritonitis, 744-746
Abdominal perfusion pressure (APP), 644
defined, 643t
renal dysfunction and, 644
Abnormal thyroid function test, 892, 892t
Acalculous cholecystitis, 737-738, 783
Accidental decannulation, 538-539
Accidental hypothermia, 770-773
clinical features, 770-771
electrocardiogram of, 771-772
laboratory tests of, 771
rewarming, 772-773
temperature recordings in, 770
for fever, 339, 791-792
hepatic metabolism of, 965f
in ICU, 909

nomogram for, 966f
overdoses of, 963-968
activated charcoal for, 967
clinical presentation, 965
N-acetylcysteine for, 967, 968t
Acetaminophen (Continued)
risk assessment, 966
toxic mechanism, 964—965
toxic dose of, 964-965
Acetazolamide (Diamox), for saline-resistant alkalosis, 627
Acetoacetate (AcAc), 609
in AKA and DKA, 610f
Acid-base analysis, 587-599
basic concept of, 587-592
acid-base disorders, classification of, 588-589
hydrogen ion concentration and pH, 587-588, 588t
metabolic acid-base disorder and, 589-590
respiratory acid-base disorders and, 590-592
features of pH, 587-588
gaps and, 594-597
anion gap, 594-596
gap-gap ratio, 596-597
hydrogen ions as trace element, 588
overview, 587
stepwise approach to, 592-594
evaluate secondary responses, 593-594
gaps to evaluate metabolic acidosis, 594
identification of primary acid-base disorder, 592-593
Acid-base disorders, 588-589
classification of, 588-589
primary, 588-589, 589t
respiratory, 589
secondary responses, 589, 589t
Acid-bicarbonate buffer system, 607f
Acidosis
D-lactic, 606
and hyperkalemia, 679
lactic (See Lactic acidosis)
metabolic (See Metabolic acidosis)
ACLS. See Advanced cardiovascular life support (ACLS)
ACS. See Abdominal compartment syndrome (ACS)
ACTH. See Adrenocorticotrophic hormone (ACTH)
Activase. See Alteplase
Activated charcoal, 967
Acute coronary syndromes, 303-321
and acute aortic dissection, 318-320
adjunctive antithrombotic therapy, 313-316
glycoprotein receptor antagonists, 315-316

heparin, 313-314
thienopyridines, 314-315
complications, 316-317
cardiac pump failure, 317
structural defects, 316
coronary thrombosis, 303-305
and clinical conditions, 303-304
thrombocentric management, 304-305
pathogenesis of, 304f
reperfusion therapy, 307-313
percutaneous coronary intervention, 311-313
thrombolytic therapy, 308-310
routine measures in, 305-307, 305t
aspirin, 305t, 307
beta-receptor antagonists, 305t, 307
morphine, 305t, 306-307
nitroglycerin, 305t, 306
oxygen therapy, 305t, 306
relieving chest pain in, 306-307
Acute Dialysis Quality Initiative (ADQI), 633
Acute heart failure. See Heart failure
Acute hemolytic reactions, 359
clinical features, 360
management, 360
Acute interstitial nephritis (AIN), 636, 642
Acute kidney injury (AKI), 633-651
common causes of, 636-637, 637t
diagnostic considerations, 636-639
categories, 636-637
diagnostic evaluation, 638-639
diagnostic criteria, 633-635
AKIN criteria, 634-635, 634t
RIFLE criteria, 633-634, 634t
Dirac equation and, 648
early management of, 639-640
fluid challenges, 639-640
intrarenal disorders, 640
in-hospital mortality rates for, 635f
overview, 633
renal replacement therapy, 645-648
hemodialysis, 646, 647f
hemofiltration, 646-648, 647f
sources of confusion in, 635
specific conditions in, 640-645
abdominal compartment syndrome, 643-645
acute interstitial nephritis, 642
contrast-induced renal injury, 640-641
myoglobinuric renal failure, 642-643
urinary measurements of, 638t
Acute Kidney Injury Network (AKIN) criteria, 634-635, 634t

Acute liver failure, 727
Acute lung injury (ALI), 450
Acute mitral regurgitation, 316
Acute myocardial infarction, hypomagnesemia and, 691
Acute-on-chronic liver failure, 727
Acute pancreatitis, 719-722
biliary evaluation, 722
contrast-enhanced CT for, 721-722, 722f
diagnosis of, 720-721
etiology of, 720, 720t
Acute respiratory disorders, 590-591
Acute respiratory distress syndrome (ARDS), 447—460
alveolar collapse in, 521-522
clinical features of, 449-453, 449t
bronchoalveolar lavage, 452—453
diagnostic problems, 449-450
radiographic appearance, 451, 451f
wedge pressure, and ARDS diagnosis, 451-452
inflammation and, 267
lung protective ventilation in, 497-498, 498t
NIV in, 530
nonventilatory management, 457-458
corticosteroid therapy, 457-458
early severe ARDS, 458
fluid management, 457
unresolving ARDS, 458
omega-3 fatty acids for, 863
pathogenesis, 447-449
inflammatory injury, 447-448
predisposing conditions, 448-449
and refractory hypoxemia, 459
extracorporeal membrane oxygenation, 460
high frequency oscillatory ventilation, 459
inhaled nitric oxide, 459-460
prone position, 460
sources of, 449t
ventilator management in, 453-457
lung protective ventilation, 455-457, 455t
mechanical ventilation, 453-455
Acute stroke, 831-843
antithrombotic therapy, 839
initial evaluation, 832-836
bedside evaluation, 832-834
imaging techniques, 834-836
protective measures in, 839-840
oxygen therapy, 839-840
treatment of fever, 840
treatment of hypertension, 840
thrombolytic therapy, 836-839
checklist for, 837t
dosage regimen, 839

selection criteria for, 836-838
Acute tubular necrosis (ATN), 636
photomicrograph of, 637f
ACV. See Assist-control ventilation (ACV)
Adenosine, for paroxysmal SVT, 293-295, 294t
adverse effects of, 294
dosing regimen, 293, 294t
ADH. See Antidiuretic hormone (ADH)
Adjunctive vasopressors, 950-951
terlipressin, 951
vasopressin, 950-951
actions of, 950
adverse effects, 951
clinical uses, 950
dosing regimen for, 950-951
ADQI. See Acute Dialysis Quality Initiative (ADQT)
Adrenal cortex/gland
in adrenal suppression, 887-888
cortisol released by, 888
glucocorticoids and mineralocorticoids, 887
Adrenal suppression, in ICU, 887-890
clinical manifestation of, 888
critically ill patients, 888
diagnosis of, 888-890
mechanisms of, 889f
predisposing conditions in, 888
rapid ACTH stimulation test for, 890
treatment of critical illness-related, 890
Adrenocorticotrophic hormone (ACTH), 887, 889f
and adrenal suppression, 890
Advanced cardiovascular life support (ACLS), 328-329
pulseless electrical activity or asystole, 330, 332
VF and pulseless VT, 329
defibrillation, 329-330
management protocol, 330
Aerosol therapy, for post-extubation stridor, 581
AF. See Atrial fibrillation (AF)
Afterdrop, 772
Aggrastat. See Trofiban
Aggressive volume infusion, in severe pancreatitis, 724
Agitation, 909
AIN. See Acute interstitial nephritis (AIN)
Airborne pathogens, 73
airborne transmission, 74-75
droplet transmission, 74
respiratory precautions for, 73f
Air entrainment devices, 434-435
advantages and disadvantages of, 435
function of, 435, 435f
Airway care, 540-542

pleural evacuation, 545-547
collection bottle, 545, 546f
reasons for, 547
suction-control bottle, 546, 546f
ethylene glycol, 987-990
clinical features, 987-988
pathophysiology, 987
treatment for, 988-990
methanol, 990-991
clinical features, 990
pathophysiology, 990
treatment for, 990-991
Alcohol-related illness, and magnesium deficiency, 691
Alcohol withdrawal delirium, 804-805
clinical features of, 804, 805t
NMDA receptors in, 804
pathogenesis of, 804
thiamine in, 805
treatment of, 805

and bicarbonate reabsorption, 621
and plasma potassium (K+), 675
Alimentary tract, 77, 92-93. See also Stress ulcer bleeding
distribution of microbes in, 77-78, 78f
gastric acid, function of, 80-81
infection control in, levels of, 78-79
microbial invasion from, 77-82
and multiorgan failure, 79-80
oral decontamination of, 88
chlorhexidine for, 89
colonization of oral cavity and, 88-89
nonabsorbable antibiotics for, 89-91, 90f
selective digestive decontamination of, 91-92, 92f
spread of enteric pathogens across, 79, 79f
stress-related mucosal injury, 82-88 (see also Stress ulcer bleeding)

ionized hypocalcemia and, 704
respiratory, 709
Alteplase, 309, 310t
for restoring catheter patency, 44, 44t
Altered consciousness, in ICU, 799-801
principal states of, 799t, 800
sources of, 800-801, 801f
Alveolar collapse
in ARDS, 521-522
in intubated patient, 543f
Alveolar hypoventilation. See also Hypoxemia and hypercapnia
causes, 399, 399t
and hypoxemia, 399
Alveolar pressure, PCV and, 507
Alveolar recruitment, 513-514
Alveolar rupture, 542-547
clinical presentation, 542-543
pneumothorax, 543-545
clinical presentation, 543
radiographic detection, 543-545, 544f
Ambu Respirator, 327
American Heart Association, 124
Amino acid solutions
glutamine, 877
specialty, 876, 876t
standard, 876, 876t
Aminoglycosides, 923-926
activity against microbes, 923-924
adverse effects, 925-926
dosing regimens, 924-925
Aminosyn-HBC, 876, 876t
Aminosyn RF, 876, 876t
Amiodarone
in atrial fibrillation, 287t, 288-289
in cardiac arrest, 332, 333t
in torsade de pointes, 299
in ventricular tachycardia, 296
Ammonia, 730-731
Ammonia burden, in hepatic encephalopathy, 733
Amphotericin B (AmB), 927-928
dosing regimen, 927
infusion-related inflammatory response, 927
lipid preparations of, 928
nephrotoxicity, 927-928
Anaphylactic shock, 274, 275
epinephrine in, 276
glucagon in, 276
volume resuscitation in, 276
Anaphylaxis, 273. See also Anaphylactic shock
clinical features, 273-274

corticosteroids in, 275
management of, 274-275
antihistamines, 274
bronchodilators, 275
epinephrine, 274, 275t
Anatomic dead space, 392
Anemia, 349
definition of, 349-350
erythrocyte transfusions, 356-359, 356t
packed red blood cells, 356-357
preparations, 356t
systemic oxygenation, 357-359
ICU-related anemia
anemia of inflammation, 351
phlebotomy, 351-352
physiological effects of, 352
cardiac output, 352
lowest tolerable hematocrit, 352-353
paradoxical effect, 353-354, 354f
systemic oxygenation, 352, 353f
plasma volume, influence of, 350t
reference ranges for red cell parameters, 350t
transfusion risks, 359-364, 359t
acute hemolytic reactions, 359-360
acute lung injury, 361-363
clinical outcomes, 363-364
febrile nonhemolytic reactions, 360-361
hypersensitivity reactions, 361
nosocomial infections, 363
transfusion triggers, 354-356
Anemia of inflammation, 351
Anion gap (AG), 594-596
determinants of, 594-595, 595t
in DKA, 611
gap-gap ratio and, 596-597
influence of albumin, 595
in lactic acidosis, 606
metabolic acidosis, classification, 596t
reference range for, 595
Antiarrhythmic agents, in cardiac arrest, 332-333, 333t
Antibiotic therapy
for CDI, 742-744, 743t
for COPD, 475-476
of documented pneumonia, 566
for magnesium deficiency, 690-691
for peritonitis, 746
for septic shock, 272-273
Anticholinergic agents, in asthma, 472—473
in diabetes insipidus, 661
in hyponatremia, 664-665
Antifungal agents, 926-929
amphotericin B, 927-928

echinocandins, 929
for ICU-acquired fever, 789
for invasive candidiasis, 928t
triazoles, 928-929
clinical uses, 928
dosing regimens, 928
drug interactions, 929
toxicity, 929
Antihypertensive therapy, in aortic dissection, 319-320, 320t
Antimicrobial gels, at catheter insertion site, 42, 42t
Antimicrobial-impregnated urinary catheters, 753
Antimicrobial therapy, 923-941
aminoglycosides, 923-926
antifungal agents, 926-929
amphotericin
B, 927-928
carbapenems, 929-931, 930t
activity against microbes, 930
adverse effects, 931
clinical uses, 930
dosing regimens, 930-931
cephalosporins, 931-933
dosing regimens, 933, 933t
generations of, 932-933, 932t
toxicity, 933
fluoroquinolones, 933-935
activity against microbes, 933-934
dosing regimens, 934, 934t
drug interactions, 934
toxicity, 934
penicillin, 935
vancomycin, 935-937
alternatives for, 937-938, 938t
antibacterial spectrum of, 935-936
clinical use of, 936
dosing nomogram, 937t
dosing regimens of, 936
toxicity, 936-937
for VAP, 565-566
Antineoplastic agents, and thrombocytopenia, 371
Antioxidants, 439-441
Antipyretic therapy
for fever during stroke, 840

for ICU-acquired fever, 791-792
acetaminophen, 791-792
NSAIDs, 792
Antiretroviral agents, and hyperlactatemia, 605
Antithrombotic therapy
for acute stroke, 839
in atrial fibrillation, 290-291
Antithyroid drugs, for thyrotoxicosis, 893-894
Anxiety
arousal and awareness in, 800
in ICU, 909-911
Aortic dissection, 318
clinical manifestations, 318
clinical findings, 318
diagnostic tests, 318-319, 319f
management, 319-320, 320t
pathophysiology, 318
Aphasia, 832
Apheresis platelets, 378
Apnea test, for brain death, 812
APP. See Abdominal perfusion pressure (APP)
Applied PEEP, 549
APRV. See Airway pressure release ventilation (APRV)
ARDS. See Acute respiratory distress syndrome (ARDS)
ARDS Clinical Network, 455
Argatroban, 373t, 374
Arginine, in enteral tube feeding, 863-864
Arousal, 799
Arrhythmias, 772
hypokalemia and risk for, 677
and magnesium deficiency, 692
Arterial base deficit, 187-188
Arterial blood pressure, measurement of, 123-132. See also Blood pressure monitoring
Arterial PCOz (PaCO>), 622-623, 623f
Artificial airways, 535-540
cuff management, 539-540, 540f
endotracheal tubes, 535-537
migration of, 536
position of, 535-536, 536f
risk of laryngeal damage, 537
subglottic drainage tubes, 537
tracheostomy, 538-539
complications in, 538-539
techniques for, 538
timing for, 538

management of, 728-729
furosemide for, 729
large-volume paracentesis, 729
sodium restriction, 728
spironolactone for, 728
sodium restriction in, 728
Aspartate aminotranferase (AST), 965

in acute coronary syndromes, 305t, 307
in acute stroke, 839
Assist-control ventilation (ACV), 508-509
airway pressure patterns in, 509f
rapid breathing in, 509
respiratory cycle in, 509
triggers for, 508
ventilator settings, 516
AST. See Aspartate aminotranferase (AST)
Asthma, 465. See also Mechanical ventilation
acute exacerbation of, 469-474, 470f, 471t
additional considerations in, 474
anticholinergic agents in, 472-473
B>-receptor agonists in, 469, 471-472
and chronic obstructive pulmonary disease, 465-480
corticosteroids in, 473-474, 474t
Asthma, NIV in, 529-530
Astrocytes, in hepatic encephalopathy, 731
Asymptomatic candiduria, 755
Atelectasis, fever and, 781, 781f
Atelectrauma, 455, 496-497, 522
ATN. See Acute tubular necrosis (ATN)
Atrial fibrillation (AF), 286
adverse consequences of, 286
cardiac performance, 286
thromboembolism, 286
lone, 286
management of, 286
electrical cardioversion, 289
heart rate control, 287-289, 287t, 288f
pharmacological cardioversion, 289-290
thromboprophylaxis, 290-291, 290t
postoperative, 286
Auscultation method, 126
“Auto-intoxication,” 737
Automatisms, 818
Autonomic instability, malignant hyperthermia and, 764-765
AV nodal re-entrant tachycardia (AVNRT), 292-293
adenosine for, 293-295, 294t
vagal maneuvers for, 293
Awareness, 799
Azathioprine, for myasthenia gravis, 823

Bacterial adherence, 752, 756
Bacteroides fragilis, 930
B-adrenergic receptors, in hypophosphatemia, 709-710
Bagged ventilation, 327-328
B.—agonists, in hyperkalemia, 684
BAL. See Bronchoalveolar lavage (BAL)
Barotrauma, 454, 497
Bedside evaluation, of acute stroke, 832-834
mental status, 832
NIH Stroke Scale, 833-834
sensorimotor loss, 832
stroke mimics, 833
Behavioral Pain Scale (BPS), 903t

in alcohol withdrawal delirium, 805
in ICU, 911-914, 912t
advantages, 912
disadvantages, 912-914
delirium, 913
prolonged sedation, 912-913
propylene glycol toxicity, 913-914
withdrawal syndrome, 914
drug profiles, 911-912
lorazepam, 911
metabolism, 911-912
midazolam, 911
overdoses of, 968-969
clinical features, 968
management, 968-969
Berlin Criteria, ARDS and, 450
Beta-blockers
in atrial fibrillation, 287t, 288
B-hydroxybutyrate (B-OHB), 609
concentration in AKA and DKA, 610f
testing for ketogenesis, 610
B-hydroxybutyric acidosis, 609
for diabetic ketoacidosis, 613
in hyperkalemia, 684
for lactic acidosis, 606-607
Bicarbonate-containing buffer solutions, 608t
Bicarbonate reabsorption, 620-621, 620f
Bicarbonate secretion, 620f, 621
Bilevel positive airway pressure (BiPAP), 525f
mode for NIV, 527
Biliary evaluation, in acute pancreatitis, 722
Biofilms, 49-50, 49f
Biotrauma, 454, 497
BiPAP. See Bilevel positive airway pressure (BiPAP)
Biphosphonates, for ionized hypercalcemia, 708
Bloodborne pathogens, transmission of, 65, 65t
hepatitis B virus, 70-72
hepatitis C virus, 72-73
human immunodeficiency virus, 66—70
needlestick injuries and, 66

before antibiotics in septic shock, 272-273
for ICU-related fever, 787-788, 788f
influence of volume, 787—788
number of, 788, 788f
Blood filters, 357
Blood-gas variability, 398
Blood pressure control, in acute stroke, 838t
Blood pressure monitoring, 123
direct methods, 128-132
artifacts and, 130-132
mean arterial pressure, 129-130
systolic amplification, 128-129
indirect methods, 123-128
accuracy of, 127-128
auscultation method, 126
oscillometric method, 126-127
sphygmomanometry, 124-126

fever, 782
and hyperkalemia, 680-681
and ionized hypocalcemia, 704
Blood urea nitrogen (BUN), 656
Blood viscosity, 164-165
Blunt trauma, to chest, 544f
BNP. See B-type natriuretic peptide (BNP)
Body mass index (BMI), 1002, 1005
Body surface area (BSA), 144, 1005
Body temperature, 777-779. See also Fever
and growth of Pasteurella multocida, 790f
febrile response, 779
malignant hyperthermia and rise in, 782
normal, 777-779
temperature conversion, 778f
Body weight, aminoglycoside dosing and, 924
Bowel mucosa
K+ clearance across, 684
trophic influence on, 859
visualization of, 741-742
Bowel sepsis, parenteral nutrition and, 884
BPS. See Behavioral Pain Scale (BPS)
Brain death, 810-813
diagnosis of, 810-812
apnea test, 812
checklist for, 811t
Lazarus’ sign, 812
hemodynamics and, 813
pituitary failure and, 813
in potential organ donor, 813
Uniform Determination of Death Act and, 810

areas of, 833f
post-cardiac arrest syndrome and, 336
B-receptor agonists, in asthma, 469, 471-472
B—-receptor antagonists
membrane-stabilizing effect of, 969
overdoses of, 969-972
glucagon and, 970-972
phosphodiesterase inhibitors and, 972
toxic manifestations of, 969-970
for thyrotoxicosis, 893
Brevibloc. See Esmolol
Bromocriptine mesylate, 767
Bronchial brushings, 561f
Bronchoalveolar lavage (BAL), 452-453, 562-564
intracellular organisms, 562-563
protected catheter for, 563f
quantitative cultures, 562
without bronchoscopy, 563-564
in anaphylaxis, 275
in COPD, 475
B-type natriuretic peptide (BNP), 242, 243t
in cardiac dysfunction, 576
clinical use, 242
role in ICU, 243t
BUN. See Blood urea nitrogen (BUN)
Cc
CAB (Circulation, Airway, Breathing), in basic life support, 325
CABI. See Catheter-associated bloodstream infections (CABI)
Caeruloplasmin, 441
Calcitonin, for ionized hypercalcemia, 708
Calcium, in hyperkalemia, 682-683
Calcium antagonist toxicity, glucagon and, 970
Calcium chloride, 683
Calcium gluconate, 682-683
Calcium in plasma, 701-703, 713
fractions of, 702f
ionized calcium
measurement, 702-703
vs. total calcium, 702f
normal ranges for, 702t
Calcium oxalate monohydrate crystals, 989f
Calcium replacement therapy, for ionized hypocalcemia, 705-706
calcium salt solutions, 705
dosing regimen in, 705-706
intravenous, 705t
maintenance therapy, 706
Calcium salt solutions, 705

Caloric density, in enteral tube feeding, 860-862
nonprotein calories, 861
osmolality, 861
protein content, 861-862
Cancer patients, thrombosis risk in, 45
Cancer-related surgery, and venous thromboembolism, 98
Candida albicans, 755, 785
Candida glabrata, 755
Candiduria, 755-756
asymptomatic, 755
microbiology of, 755
symptomatic, 755-756
Capnometry, 417-422
clinical applications, 420
arterial PCOz, 420-421
cardiac output, 421, 422f
nosocomial complications, 421
ventilator weaning, 421-422
colorimetric, 417-418, 418f, 418t
infrared, 418-420
Carbamazepine, in ICU, 909
Carbapenems, 755, 929-931, 930t
Carbicarb, for lactic acidosis, 608
Carbohydrates, 849
in enteral tube feeding, 862
Carbon monoxide poisoning, 981-984
carboxyhemoglobin and, 981-982, 982f
clinical features, 983
cyanide poisoning and, 984
diagnosis of, 983-984
neurologic sequelaea and, 983
pathophysiology, 981-982
treatment for, 984
Carboxyhemoglobin (COHb), 410, 981-982, 982f
Cardiac arrest, 325-341, 326f
advanced life support, 328-333
algorithm for, 331f
resuscitation drugs, 332-333, 333t
ventricular asystole or PEA, 330-332
VF and pulseless VT, 329-330
basic life support, 325-328
chest compressions, 326-327, 327t
high-quality CPR, 328, 328t
time factor, 325-326
ventilation, 327-328
post-resuscitation period, 335-341

fever, 339
glycemic control, 339
neurologic recovery, predictive signs of, 339-341
post-cardiac arrest syndrome, 336
targeted temperature management, 336-339, 338t
resuscitation monitoring, 333-335
central venous O> saturation, 335
end-tidal PCO2, 334-335, 335f
Cardiac contractile strength, drug-induced alterations in, 971f
Cardiac dysfunction
in septic shock, 268
Cardiac index (CI), 145
Cardiac output
anemia and, 352
in hypophosphatemia, 710
in IMV, 510
positive pressure ventilation on, effect of, 500, 50if
systemic O> transport, 514
Cardiac performance
effect of atrial fibrillation on, 286
positive pressure ventilation, influence of, 499-501
Cardiac pump failure, hemodynamic support in, 317
Cardiac tamponade, catheter-associated, 46-47
Cardiogenic pulmonary edema, NIV in, 530
Cardiopulmonary resuscitation (CPR), 325. See also Cardiac arrest
Cardiovascular parameters, 144, 144t
cardiac index, 145
central venous pressure, 144-145
pulmonary artery wedge pressure, 145
pulmonary vascular resistance index, 146
stroke index, 145
systemic vascular resistance index, 145-146
Carnitine, in enteral tube feeding, 864
Caspofungin, 929
for asymptomatic candiduria, 755
Catecholamines, 943-950
and adrenal medulla, 887
adrenergic receptors and, 943t, 944t
dobutamine, 944-945
actions of, 944
adverse effects, 945
clinical uses of, 944
dosing regimen for, 945
stroke volume augmentation by, 945f
dopamine, 945-947
actions of, 945-946
adverse effects, 946-947
cardiac effects of, 947f
clinical uses of, 946
dosing regimen for, 946
epinephrine, 947-948
actions of, 947

adverse effects, 948
clinical uses of, 947-948
dosing regimens for, 948
norepinephrine, 948-949
actions of, 948
adverse effects of, 949
clinical uses of, 948
dosing regimen for, 949

catheter tip positions, 34-36
pneumothorax, 33-34
venous air embolism, 32-33
contraindications to, 18
indications for, 18
and infection control measures, 18-19, 19t
barrier precautions, 19-20
site selection, 20
skin antisepsis, 19

femoral vein, 28-30, 29f
internal jugular vein, 23-26, 24f, 25f
peripherally inserted central catheters, 30-31, 31f
subclavian vein, 26-28
small vs. large veins, 17-18, 18t
ultrasound-guided, 20
body tilt, 23
long-axis view, 21, 22f
short-axis view, 21-22, 24f
vascular ultrasound, 20-21, 21f
Central venous catheters, 9
antimicrobial catheters, 10-12
insertion technique, 10, 11f
replacement of, 42-43
triple-lumen catheters, 9-10, 9f, 10t
Central venous oximetry catheters, 416-417
Central venous oxygen saturation, 183-184, 356, 576
Central venous pressure (CVP), 144-145, 153, 157
catheter-transducer circuit and, 157
respiratory variations in, 159, 159f
venous pressures in thorax, 158-159
volume resuscitation in septic shock and, 271
Cephalosporins, 931-933
Cerebral salt wasting, 665
cGMP. See Cyclic guanosine monophosphate (cGMP)
Chain reactions, 264-266, 438
Chest compressions, 326
early, 326-327
recommendations for, 327t

in, 475-476 bronchodilator therapy in, 475, 3 = . proton pump inhibitors and, 85, 1 = . proton pump inhibitors and, 85, 2 = . proton pump inhibitors and, 85, 3 = . difficile, 740, 936. See also Clostridium (CDI), 1 = . difficile, 740, 936. See also Clostridium (CDI), 2 = . difficile, 740, 936. See also Clostridium (CDI), 3 = . difficile infections cytotoxins released by, 741 enterocolitis, gastric acid-suppressing drugs and, 86-87, 1 = . difficile infections cytotoxins released by, 741 enterocolitis, gastric acid-suppressing drugs and, 86-87, 2 = . difficile infections cytotoxins released by, 741 enterocolitis, gastric acid-suppressing drugs and, 86-87, 3 = . difficile infections (CDI), 740-744, 786, 1 = . difficile infections (CDI), 740-744, 786, 2 = . difficile infections (CDI), 740-744, 786, 3 = . Clostridium, 1 = . Clostridium, 2 = . Clostridium, 3 = . , 1 = Clostridium. , 2 = antibiotic treatment, 742-744, 743t. , 3 = clinical features, 741
Continuous venovenous hemofiltration (CVVH), 647
“Contraction alkalosis,” 622
Contrast-induced renal injury, 640-641
intravenous hydration, 641
prevention of, 641
Convective heat loss, 762
Cooling blankets, for fever in ICU, 792
Cooperative sedation, with dexmedetomidine, 916
COPD. See Chronic obstructive pulmonary disease (COPD)
Coronary artery bypass surgery, 317
Corticosteroid-binding globulin (CBG), 888
in ARDS, 457-458
in asthma, 473-474, 474t

comparison of, 891t
in COPD, 475, 476t
in laryngeal edema, 580, 581f
in septic shock, 272
Corticotrophin-releasing hormone (CRH), 887, 889f
Cortisol (hydrocortisone), and adrenal cortex, 888
Countercurrent exchange, 646
CPAP. See Continuous positive airway pressure (CPAP)
CPR. See Cardiopulmonary resuscitation (CPR)
CRBI. See Catheter-related bloodstream infections (CRBI)
Creatine kinase (CK), 764
CRH. See Corticotrophin-releasing hormone (CRH)
Critical illness myopathy (CIM), 824-825
Critical illness neuromyopathy, 824-825
clinical features, 824-825
pathogenesis, 824
and respiratory muscle weakness, 578
Critical illness polyneuropathy (CIP), 824-825
Critical illness-related corticosteroid insufficiency (CIRCI), 888
Cryoprecipitate, 210
hypofibrinogenemia and, 383
uremic bleeding and, 383
Crystalloid fluids, 206, 207t, 217-225
hypertonic saline, 224-225
isotonic saline, 218-222
other balanced salt solutions, 223-224
Ringer's fluids, 222—223
and volume distribution, 217, 218f
CT angiography (CTA), for pulmonary embolism, 109
Cuff leaks, 539-540
Cuff-leak test, for laryngeal edema, 580
Cuff management, 539-540, 540f
CVP. See Central venous pressure (CVP)
CVVH. See Continuous venovenous hemofiltration (CVVH)
Cyanide intoxication, 954-956
Cyanide poisoning, 984-987
and carbon monoxide poisoning, 984
clinical features, 985
diagnosis of, 985
pathophysiology, 984-985
treatment for, 986-987
antidotes, 986t, 987
hydroxocobalamin, 986
nitrates, 986-987
sodium thiosulfate, 986

Cyclic guanosine monophosphate (cGMP), 951
in magnesium deficiency, 691
in myasthenia gravis, 823
Cyproheptadine, in serotonin syndrome, 769-770
Cytocrome oxidase, 188-189
Cytopathic hypoxia, 176, 269
Cytoprotective agent, 85. See also Sucralfate
Cytotoxin-positive diarrhea, 743
Cytotoxins, and Clostridium difficile, 741

Dabigatran, in atrial fibrillation, 290-291
Daily energy expenditure, 847-849
equations to measure, 848-849
indirect calorimetry for, 848
oxidation of nutrient fuels, 847-848, 848t
Dalteparin, 102t, 103-104
Damage control resuscitation, 209-210
for malignant hyperthermia, 765
for neuroleptic malignant syndrome, 767
Daptomycin, 938
in catheter-related bloodstream infections, 55
D-dimer assay, 105-106
Dead space ventilation, 391-392, 392f
Deep vein thrombosis (DVT)
of lower extremities, 46
of upper extremities, 45
Defibrillation, 329-330
Dehydration, in DKA, 611
Deliriogenic drugs, 803
Delirium, 801-805, 909
alcohol withdrawal, 804-805
pathogenesis, 804
benzodiazepines and, 913
clinical features of, 801-803, 802f
dexmedetomidine and, 916
diagnosis of, 803
hyperactive, 802
hypoactive, 802
management of, 803-804
drug therapy, 804
preventive measures, 803
mixed, 802
predisposing conditions, 803
subtypes of, 802-803
vs. dementia, 803
Delirium tremens (DTs). See Alcohol withdrawal delirium
Demeclocycline, for hyponatremia, 668-669
Dementia
vs. delirium, 803
Denervation injury, succinylcholine for, 680
Deoxygenated hemoglobin (Hb), 410
Depressed sensorium, naloxone and, 973
Desmopressin, 383, 813
Dexmedetomidine
in ICU, 916
for sedation in delirium, 803, 804
Dextrans, 231-232
disadvantages, 232
features, 232

Dextrose infusion, 683
and arterial lactate levels, 855f
Dextrose solutions, 875

5% dextrose solutions, 225
and enhanced lactate production, 225-226
hyperglycemia and, 226-227
protein-sparing effect, 225
volume effects, 225
Diabetes insipidus (DI), 661-662
antidiuretic hormone and, 661
central, 661
diagnosis of, 661
management of, 662
nephrogenic, 661
vasopressin in, 662
Diabetes mellitus, and magnesium deficiency, 691
Diabetic ketoacidosis (DKA), 610-613
acid-base status monitoring, 613
clinical features of, 611
gap-gap ratio and, 597
management of, 611-613, 612t
bicarbonate therapy, 613
insulin therapy, 611-612
intravenous fluids, 611
phosphate therapy, 613
potassium therapy, 612-613
Diaphragmatic contractions, power of, 570f

cytotoxin-positive, 743
enteral tube feeding and, 870
and extrarenal K+ loss, 677
lactulose and, 733
liquid drug preparations, 870, 870t
magnesium deficiency and secretory, 691
Diastolic heart failure, 156-157, 243-245
Diazepam (Valium), for pain, 902f
DIC. See Disseminated intravascular coagulation (DIC)
Diffusion-weighted MRI, in acute stroke, 835, 836f
Digitalis toxicity, and hyperkalemia, 679-680
Digoxin, in atrial fibrillation, 287t, 289
Diltiazem, in atrial fibrillation, 287-288, 287t
Diphenhydramine, in anaphylaxis, 274
Dirac equation, and AKI, 648
Discontinuing mechanical ventilation, 569-584
extubation, 579-581
airway protective reflexes, 579
laryngeal edema and, 579-580
post-extubation stridor, 580-581
overview, 569
patient-triggered ventilation, 569-570
physical rehabilitation, 570-571
preliminary concerns, 569-572
readiness criteria, 571-572
ventilatory support strategies, 569-571
sedation practices in, 571
spontaneous breathing trial, 572-579
cardiac dysfunction during, 576-577
checklist for identifying candidates for, 571t
by disconnecting ventilator, 573-574
measurements used to predict, 572t
method preference for, 574
rapid breathing during, 575-576
respiratory muscle weakness, 577-579
simple breathing circuit for, 574f
success vs. failure, 574-575, 577f
using ventilator circuit, 573
work of breathing during, 573f
Disseminated intravascular coagulation (DIC), 375
clinical features, 375-376, 376f
management, 376
Diuretic therapy
with furosemide
for ascites, 729
in heart failure, 249-250, 252-255

in magnesium deficiency, 690
in metabolic alkalosis, 622
and renal K+ loss, 677
DKA. See Diabetic ketoacidosis (DKA)
D-lactic acidosis, 606
Dobutamine, 944-945
in heart failure, 250, 251t, 252
Documented pneumonia, antibiotic therapy of, 566
Dopamine, 945-947
in anaphylactic shock, 276
cardiac effects of, 947f clinical uses of, 946
in heart failure, 252
in septic shock, 271
Doppler ultrasound, for venous thrombosis, 107-108
Droplet transmission, 74
Drug-induced cardioversion, in atrial fibrillation, 289-290
Drug-induced hyperthermia, 764-770
malignant hyperthermia, 764—765
clinical manifestations of, 764—765
management of, 765
prevention of, 765
neuroleptic malignant syndrome, 765-767
clinical features, 766
drugs implicated in, 766t dystonic reactions in, 766-767
laboratory studies for, 766-767
management of, 767
pathogenesis of, 765-766
serotonin syndrome, 767-770
clinical manifestations, 768-769
diagnostic worksheet for, 769t drugs can produce, 768t
management of, 769-770
pathogenesis of, 768
Drug-induced hyperthermia syndromes, 783
Drug-induced paralysis, in ICU, 825-827
avoiding, 826-827
monitoring of, 826
neuromuscular blocking agents in, 825-826, 825t
Drug-induced thrombocytopenia, 371
Drug therapy, for delirium, 804
Duplex ultrasound, for venous thrombosis, 108

DVT. See Deep vein thrombosis (DVT)
Dynamic hyperinflation, 327, 477-478
Dysoxia, 176
Dystonic reactions, in NMS, 766-767
End-tidal CO2 monitoring, 334-335
Energy availability, hypophosphatemia and, 710
Energy metabolism, in hypophosphatemia, 710
Enoxaparin, 102t, 103
Enteral feedings, stress ulcer bleeding and, 87
Enteral nutrition, in severe pancreatitis, 725
Enteral tube feeding, 859-873
complications associated with, 866-870
diarrhea, 870
regurgitation, 868-870
tube occlusion, 867-868
contraindications to, 860
creating regimen for, 864-865, 865t
daily energy and protein requirements, 864
desired infusion rate, 864-865
protein intake, 865
selecting feeding formula, 864
criteria for, 860
feeding formulas, 860-864, 861t
caloric density, 860-862
carbohydrate content, 862
conditionally essential nutrients, 863-864
enriched with fiber, 862, 862t
immune-modulating, 863t
lipid content, 862-863
single feeding formula, 864
infectious risk, 859-860
initiating, 866
placement of tubes, 866
starter regimens, 866
Enterococcus faecalis, 930
Enzyme-linked immunosorbent assay (ELISA), 373
EPAP. See Expiratory positive airway pressure (EPAP)
Epilepsia partialis continua, 818
Epinephrine, 947-948
in anaphylaxis, 274, 275t
Epinephrine aerosol, for post-extubation stridor, 581
Eptifibatide, in acute coronary syndromes, 315

Erythrocytes, in hypophosphatemia, 710
Erythrocyte transfusions, 356-359, 356t
in critically ill patients, 354-355
and fever, 782
and blood filters, 357
infusion of, 357
leukocytes in, 357
washed red blood cells, 357
and systemic oxygenation, 357-359, 358f
tissue oxygenation, 358-359
Erythrocyte transketolase assay, 853
Erythromycin, for regurgitation, 868-869, 869t
Escherichia coli, 727, 752
Esmolol
in aortic dissection, 319, 320t
Essential trace elements, 853-855
daily requirements, 853-854
dietary allowances for, 854t, 879t
iron, 854
selenium, 855, 878-879
Ethylene glycol poisoning, 987-990
fomepizole, 988-989
hemodialysis, 989-990
pyridoxine, 990
thiamine, 990
Euvolemic hyponatremia, 666
Evaporative heat loss, 762
Exertional heat stroke, 763
Expiratory positive airway pressure (EPAP), 525f, 527
Expressive aphasia, 832
External rewarming, 772
Extracellular volume (ECV)
associated with hypernatremia, 657-658, 657f
hyponatremia and, 665, 667f, 668
metabolic alkalosis and, 621-622
Extracorporeal membrane oxygenation (ECMO), 460
Extrapyramidal reactions, haloperidol and, 917
Extrarenal potassium loss, 677
Extravasation, of vasopressors, 946-947
Extubation, 579-581

work of breathing and, 579
F
Face masks, 74, 432
with reservoir bags, 432-434
Famotidine, for stress ulcer bleeding, 83t, 84
Fast flush test, 131f, 132
Febrile nonhemolytic reactions, 360
definition, 360
management, 360-361
platelet transfusions and, 380
Fecal transplantation, in CDI, 744
Feeding formulas, for enteral tube feeding, 860-864, 861t caloric density, 860-862
enriched with fiber, 862, 862t immune-modulating, 863t
Feeding tube misplacement, 866
Feeding tubes insertion of, 867f
placement of, 866
gastric vs. duodenal placement, 866
Femoral vein cannulation, 28, 28f, 30
anatomy, 28, 28f complications, 30
landmark method, 29
ultrasound imaging, 29, 29f
Fentanyl, in ICU, 904
Fever
during acute stroke, 840
as adaptive response, 779
after cardiac arrest, 339
definition of, 779
drug-induced, 782-783, 783t
early postoperative, 781-782
febrile response to, 779
iatrogenic, 783
ICU-acquired, 777-795
antipyretic therapy, 791-792
cooling blankets for, 792
as host defense mechanism, 789-791
initial approach in, 787-789
neurologic injury and, 791
noninfectious sources of, 780-783, 780t
tachycardia and, 791
potential sources of nosocomial, 780f
relationships with atelectasis, 781, 781f
sources of, 779

vs. hyperthermia, 761
FFP. See Fresh frozen plasma (FFP)
FG. See Filtration gradient (FG)
Fiber, in enteral tube feeding, 862, 862t
Fiberoptic catheters, 184
Fibrin, formation of, 370
Fidaxomicin, for CDI, 743
Filtration gradient (FG), 643t, 644
Fluconazole, 928
for Candida cystitis, 756
for pyelonephritis, 756
Flucytosine, for Candida UTIs, 756
in ARDS, 457
in diabetic ketoacidosis, 611
in hyponatremia, 667—668
in NKH, 663
in severe pancreatitis, 724
Flumazenil, overdoses of benzodiazepines and, 969
Fluoroquinolones, 933-935
Flush solution, 43
Folinic acid, for methanol poisoning, 991
Fomepizole
dosing regimen for, 989t
for ethylene glycol poisoning, 988-989
Forced expiratory measurements, 465-466
Forehead pulse oximetry, 414, 414f
Fosphenytoin, in seizures, 820
Fractional excretion of sodium (FENa), 638-639
Fractional excretion of urea (FEU), 639
Fragmin. See Dalteparin
Frank-Starling relationship of heart, 152
Free water deficits, 660
Freezing point depression method, 655
Fresh frozen plasma (FFP), 209-210, 381
massive blood loss and, 381
warfarin-induced hemorrhage and, 381-382, 382t
in AKI, 640
hypercalcemia, 707, 707t

GABA. See Gamma-amino-butyric-acid (GABA)
Gabapentin, in ICU, 909
Gallbladder, transverse sonogram of, 739f
Gallbladder distension, 738
Gallstone pancreatitis, 726
Gamma-amino-butyric-acid (GABA), 913
Gap-gap ratio, 596-597, 613
metabolic acidosis and alkalosis, 597
in mixed metabolic acidoses, 596-597
Gaps, 594-597
metabolic acidosis, evaluation of, 594
Gastric acid, 80
antiseptic effects of, 80-82, 81f loss, in metabolic alkalosis, 622
suppression, 741
Gastric colonization, 739-740
Gastric insufflation, during NIV, 531
Gastric residual volume, in regurgitation, 868
Gastroccult test, 88
GCS. See Glasgow Coma Score (GCS)
Generalized seizures, 818
imipenem and, 931
Generalized status epilepticus
acute management, 819-820
drug regimens for, 821t
outcomes, 821
GFP. See Glomerular filtration pressure (GFP)
Glasgow Coma Score (GCS), 808-809, 810t
interpretation of, 808-809
intubated patients and, 809
Global aphasia, 832
Glomerular filtration pressure (GFP), 644
Glucagon, 970-972
adverse effects of, 970
in anaphylaxis, 274
as antidote, 971t
clinical uses of, 970
dosing regimen for, 971
mechanism of action, 970
Glucocorticoids, for ionized hypercalcemia, 708
Glutamine, 877
Glutathione, 439-440
Glycoprotein receptor antagonists, in acute coronary syndromes, 315-316, 315t
Graded compression stockings, 104
Gram-negative aerobic bacilli, 727, 752

antibiotic susceptibility of, 925f
Gram-positive aerobic cocci, 727
Greenfield Filter, 115-116, 115f
Gruntzig procedure, 311
Guillain-Barré syndrome, 823-824
clinical features, 824
diagnosis of, 824
treatment of, 824
vs. myasthenia gravis, 823t
H
Hagen-Poiseuille equation, 5-7, 15, 164
Haloperidol (Haldol), in ICU, 917-918
adverse effects of, 917-918
for agitated patient, 917t
features of, 917
Heart failure, 239-258
management strategies, 246-255
left heart failure, 247-255
right heart failure, 255
mechanical cardiac support, 255-258
intra-aortic balloon counterpulsation, 255-257
positive pressure breathing, 257
pathophysiology, 239-243, 240f
B-type natriuretic peptide, 242-243
neurohumoral responses, 241-242
progressive heart failure, 240, 241f
types of, 243-246
right heart failure, 246
systolic and diastolic heart failure, 243-245
Heat exhaustion, 762-763
management of, 763
volume depletion and, 763
vs. heat stroke, 762t

convective, 762
evaporative, 762
Heat-related illness, 761-764
heat exhaustion, 762-763, 762t
heat stroke, 762t, 763
hyperthermia vs. fever, 761
response to thermal stress, 761-762
syndromes, 762
Heat stroke, 763
types of, 763
vs. heat exhaustion, 762t
HELLP syndrome, 377
Hemidiaphragms, 745f
Hemithorax, 545f
Hemodiafiltration, 648
in AKI, 646
advantages and disadvantages of, 646
mechanisms of solute clearance by, 647f
for clearing salicylates, 976
ethylene glycol poisoning and, 989-990
for hypermagnesemia, 696
for potassium removal, 684
Hemodialysis catheters, 13, 13t, 14f
Hemodynamic drugs, 943-959
adjunctive vasopressors, 950-951
catecholamines, 943-950
phenylephrine, 949-950
nitrovasodilators, 951-956
nitroglycerin, 951-953
nitroprusside, 954-956
Hemofiltration, in AKI, 646-648
advantages and disadvantages of, 648
CAVH, 646
CVVH, 647
methods of, 646-647
Hemoglobin, absorption patterns, 410, 410f
Hemolysis, hypermagnesemia and, 696
Hemophilus influenza, 476, 934
Hemorrhage and hypovolemia, 195-212
blood volume, assessment of, 197-203
blood volume measurements, 202-203, 203f

Hemorrhage and hypovolemia (Continued)
dysoxia, chemical markers of, 201-202
fluid responsiveness, 202
hematocrit, 198-200, 199f
invasive measures, 200-201
vital signs, 197-198, 198t
body fluids & blood loss, 195-197
blood loss and compensatory responses, 196
classification system for blood loss, 196-197
plasma vs. interstitial fluid, 196
volume of body fluids in adults, 195, 196t
fluids infusion, 203-205
central vs. peripheral catheters, 204—205
packed red blood cells, 205
and nephrotoxicity, 231
volume effects, 230-231
Hyperactive delirium, 802
Hyperbaric oxygen, for carbon monoxide poisoning, 984
Hypercapnia, excess carbohydrate and, 883
Hypercapnic respiratory failure, during NIV, 529-530
Hyperchloremic metabolic acidosis, 596
Hyperdynamic shock, 268. See also Septic shock
in critically ill patients, 226
5% dextrose solutions and, 226-227
hypertonic, 662-663
and hypophosphatemia, 709
and plasma, 663
during TPN, 882
Hyperkalemia, 679-684
blood transfusion and, 680-681
cautopyreiophagia, 681
clinical consequences of, 681
drugs promote, 680t
ECG abnormalities in progressive, 681, 682f
etiology of, 679-680
impaired renal excretion, 680
pseudohyperkalemia, 679
transcellular shift, 679-680
management of severe, 681-684, 683t
membrane antagonism, 682-683
potassium removal, 684
transcellular shift, 683-684
tumor lysis syndrome and, 679
Hyperlactatemia, causes of, 604-606
clinical shock syndromes, 604
lactic alkalosis, 605
non-pharmaceutical toxidromes, 605
pharmaceutical agents, 604-605
systemic inflammatory response syndrome, 604

thiamine deficiency, 604
Hypermagnesemia. See also Magnesium accumulation
hypovolemic, 658-661
without hypovolemia, 661-662
Hypernatremic encephalopathy, 658
Hypernociception, 902
Hyperphosphatemia, in ICU, 712
Hypersensitivity reactions, 273, 361
clinical features, 361
management, 361
plasma transfusions and, 384
acute stroke and, 838, 840
sodium nitroprusside for, 840
Hyperthermia
malignant, 782
vs. fever, 761
Hypertonic hyperglycemia, 662-663
Hypertonicity, 658
Hypertonic saline, 224-225
hyponatremia and, 667f
infusion rate for, 668
Hypertriglyceridemia, propofol infusions and, 915
Hyperventilation, during CPR, 328
Hypervolemic hypernatremia, 662
Hypervolemic hyponatremia, 666
Hypoactive delirium, 802
Hypocalcemia, and magnesium deficiency, 692
Hypochlorite, 438
Hypodense area, 835
Hypokalemia, 675-678
carbohydrate causes, 883
clinical manifestations of, 677
diagnostic approach to, 676f
extrarenal loss, 677
management of, 677-678
potassium deficits, 677, 678t
potassium replacement, 678
potassium depletion, 676
promotes metabolic alkalosis, 621
renal loss, 677
transcellular shift in, 676
Hypomagnesemia, 691. See also Magnesium deficiency
life-threatening, 695
mild, asymptomatic, 694
moderate, 694-695
and renal insufficiency, 695
Hyponatremia, 664-670
encephalopathy and, 665
euvolemic, 666
flow diagram for approach to, 667f
fluid management, 667-668
hypervolemic, 666
hypotonic, 664

hypovolemic, 665-666
nonosmotic ADH in, 664-665
pharmacotherapy, 668-670
predisposing conditions for, 666t
pseudohyponatremia, 664
Hypophosphatemia, 708-711
carbohydrate and, 883
clinical manifestations, 710-711
energy metabolism, 710
muscle weakness, 711
phosphate replacement therapy, 711, 712t
predisposing conditions, 708-710
B-adrenergic receptors, 709-710
glucose loading, 708-709
hyperglycemia, 709
phosphate binding agents, 710
respiratory alkalosis, 709
systemic inflammation, 710
Hypotensive resuscitation, 209
Hypothermia, 770-773
accidental, 770-773
adaptation to cold, 770
induced, 773
manifestations of progressive, 771t
mild, 771
moderate, 771
reducing incidence of, 210
severe, 771
Hypothyroidism, 895-896
clinical manifestations of, 895
diagnosis of, 895
thyroid replacement therapy in, 896
Hypotonic fluid loss, 658-659
impaired thirst, 658-659
plasma volume, 659
sodium concentration in, 659T
Hypotonic hyponatremia, 664
Hypoventilation
and hypercapnia, 403-404
in metabolic alkalosis, 622-623
Hypovolemic hypernatremia, 658-661
hypotonic fluid loss, 658-659
management of, 659-661
free water replacement, 659-661
volume resuscitation, 659
Hypovolemic hyponatremia, 665-666
diagnosis of, 665-666
etiology of, 665

Hypoxemia and hypercapnia, 391—406
hypercapnia, 403-405
causes, 403
CO, production in, increase in, 404
definition, 403
diagnostic evaluation, 404—405, 405f
hypoventilation and, 403-404
overfeeding and, 404
V/Q abnormality and, 404
hypoxemia, 398-403
causes, 398-399, 399t
definition, 398
diagnostic evaluation, 401-403, 402f
DO>/VO> imbalance and, 400-401
hypoventilation and, 399-400, 399t
oxygen therapy and, 428-429, 429f
spurious, 403
V/Q mismatch and, 400
measures of gas exchange and, 395-398
A-a PO gradient, 395-397
a/A PQ; ratio, 397-398, 397f
blood-gas variability, 398
intrapulmonary shunt fraction, 395
PaO,/FIO> ratio, 398
pulmonary gas exchange and, 391-394
dead space ventilation, 391-392, 392f intrapulmonary shunt, 393, 394f
Hypoxemic respiratory failure, during NIV, 531
Hypoxia, 176

IAH. See Intraabdominal hypertension (IAH)
IAP. See Intraabdominal pressure (IAP)
Iatrogenic fever, 783
Ibuprofen for fever, 792
in ICU, 908
Ibutilide, in atrial fibrillation, 290
ICU-acquired fever, 777-795
acalculous cholecystitis and, 783
endocrine disorders and, 783
noninfectious sources of, 780-783
blood transfusions, 782
drug-induced fever, 782-783, 783t early postoperative fever, 781-782
SIRS, 781
venous thromboembolism, 782
Ideal body weight, 1005
Idiogenic osmoles, 660
I:E ratio, ventilator settings and, 517
Imipenem, 930-931
Immune-modulating feeding formulas, 863t
Immunoglobulin G, for Guillain-Barré syndrome, 824
Immunonutrition, 863
Impaired renal excretion, and hyperkalemia, 680
Impaired thirst, 658-659
Impedance, 162-163
IMV. See Intermittent mandatory ventilation (IMV)
Indirect calorimetry, 848
Induced hypothermia, 773
Infection control measures, central venous cannulation and, 18-20, 19t
Inferior vena cava (IVC) filters, 115-116
Inflammation, malignant, 264
Inflammatory injury, 263-268
clinical conditions attributed to, 266t
and clinical syndromes, 266-268
oxidant injury, 264-266
chain reaction, 264—266
neutrophil activation and, 264, 265f
oxidant stress, 264, 266t
Inflammatory organ failure, 267, 267f
Inflammatory response, 263
Inflammatory shock syndromes, 263-276

anaphylaxis, 273-276
inflammatory injury, 263-268
septic shock, 268-273
Infrared capnometry, 418-420, 419f
Infused crystalloid fluid, in AKI, 639-640
Inhaled nitric oxide, in ARDS, 459-460
Inorganic iodine, for thyrotoxicosis, 894
Inspiratory flow rate, ventilator settings and, 516
Inspiratory positive airway pressure (IPAP), 525f, 527
Insulin-dextrose, in hyperkalemia, 683
for diabetic ketoacidosis, 611-612
for NKH, 663
Intensive care unit (ICU)
anxiety in, 909-911
benzodiazepines for sedation, 911-914, 912t
dexmedetomidine in, 916
haloperidol in, 917-918
non-opioid analgesia in, 907-909
acetaminophen, 909
carbamazepine, 909
gabapentin, 909
ibuprofen, 908
ketorolac, 907-908
opioid analgesia in, 904-907, 905t
adverse effects of, 906-907
dosage, 904-906
patient-controlled analgesia, 906
patient experience in, 901-904
monitoring pain, 903-904
pain, 902
stressful experience, 901-902
propofol in, 914-916
Intermittent mandatory ventilation (IMV), 510-511
Intermittent pneumatic compression (IPC), 104-105
Internal jugular vein, for central venous access, 23-26
anatomy, 23
complications, 25-26
positioning, 23-24
surface landmarks, 24-25, 25f
ultrasound guidance, 24, 24f
Internal rewarming, 772
International Organization for Standardization (ISO), 4
Interventricular interdependence, 246
Intestinal motility, opioids and, 907

Intraabdominal hypertension (IAH)
Intraabdominal pressure (IAP)
ACS and measurement of, 645
Intra-aortic balloon counterpulsation, 255-257, 256f
complications, 257
contraindications to, 255
methodology, 255-257, 256f
Intracellular preponderance, of potassium, 673
Intralipid, 850
Intrapulmonary shunt, 393
and arterial blood gases, 393, 394f
inhaled oxygen on arterial PO;, influence of, 393, 394f
pathophysiology, 393
shunt fraction, 393
true shunt, 393
venous admixture, 393
Intrapulmonary shunt fraction, 395
Intrarenal disorders, management of, 640
Intrathoracic pressure, influence of, 158-159
Intravascular pressure, 158
Intrinsic PEEP. See Occult PEEP
Introducer sheaths, 14
Ionized calcium
measurement, 702—703
Ionized hypercalcemia, 706-708
clinical manifestations of, 706
management of, 706-707, 706t
biphosphonates for, 708
calcitonin for, 708
dialysis, 708
furosemide for, 707
glucocorticoid for, 708
isotonic saline infusion for, 707
Ionized hypocalcemia, in ICU, 703-706
blood transfusions and, 704
calcium replacement therapy, 705-706, 705t
causes of, 703t
clinical manifestations of, 704—705
cardiovascular complications, 705
neuromuscular, 705-706
drugs promote, 704
predisposing conditions in, 703-704
renal failure and, 704
Ionized magnesium, 688-689

IPAP. See Inspiratory positive airway pressure (IPAP)
Ipatropium
in asthma, 471t, 472-473
in COPD, 476t
Iron, 854
and oxidant injury, 854
Iron lungs, 487
Ischemic stroke, 286, 831
Isotonic saline, 218-219
acid-base effect, 220-222
and interstitial edema, 219-220
and plasma, 219t
volume effects, 219-220, 220f
Isotonic saline infusion, for ionized hypercalcemia, 707
J
Jet mixing, 435
Jet nebulizer, 466-467, 467f

Kayexalate, 684
Ketoacids, 608-610
Ketogenesis, 608
in blood, 609-610
B-hydroxybutyrate testing, 610
nitroprusside reaction, 609-610
in liver, 609f
Ketorolac in fever, 792
in ICU, 907-908
Kininogens, 370

in aortic dissection, 320, 320t to control blood pressure, 840
Laboratory tests, reference ranges for, 999-1000
Lactate, 184-185
and acidosis, 603-604
as adaptive fuel, 187, 187t as biomarker, 602-603
in blood, 185
as fuel, 601-602
oxidative metabolism of, 602f prognostic value, 185-186, 186f
in sepsis, 186-187
in septic shock, 270
Lactate dehydrogenase (LDH), 601, 602f
Lactate metabolism, 601-602
Lactate shuttle, 602
Lactic acidosis, 601-606
alkali therapy for, 606-608
bicarbonate therapy, 606-607
carbicarb, 608
recommendation, 608
anion gap in, 606
diagnostic considerations, 606
hyperlactatemia, causes of, 604-606
Lactic acidosis (Continued)
lactate as biomarker, 602-603
lactate metabolism, 601-602
Lactic alkalosis, 605
Lactobacillus acidophilus, 733
Lactobacillus organisms, non-pathogenic, 752f
Lactulose, for hepatic encephalopathy, 733
Large-volume paracentesis, for ascites, 729
Laryngeal damage, endotracheal tubes and, 537
Laryngeal edema
cuff-leak test, 580
and extubation, 579-580
Law of Laplace, 160
Laxative abuse, 624-625
LDH. See Lactate dehydrogenase (LDH)
LDUH. See Low-dose unfractionated heparin (LDUH)
Lead-pipe rigidity, 766
Left atrial pressure (LAP), 153
Left heart failure, management of, 247-248

diuretic therapy with furosemide, 249-250, 252-255
inodilators, 250-251, 251t
dobutamine, 250
levosimendan, 250
milrinone, 250
vasodilator therapy, 248, 248t
nesiritide, 249
nitroglycerin, 249
nitroprusside, 248-249
Left ventricular end-diastolic pressure (LVEDP), 153
Lepirudin, 373t, 374
Lethargy, 800
Leukocyte larceny, 403
Leukocyte reduction, for platelet transfusions, 378
Leukocytosis, and DKA, 611
Levalbuterol
in asthma, 471, 471t
Levetiracetam, in seizures, 820
Levofloxacin, 934
Levothyroxine (T4), 896
Lidocaine, in cardiac arrest, 332-333, 333t
Light reactivity, condition affects, 807t
Limb weakness, in Guillain-Barré syndrome, 824
Linezolid, 938
and hyperlactatemia, 605
Linoleic acid, 849-850
Lipase, in acute pancreatitis, 721
sources of elevated, 721t
Lipid complications, parenteral nutrition and, 883
Lipid emulsions, 877-878
intravenous, 877t
propofol preparations and, 915
Lipid preparations, of AmB, 928
Lipids, 849-850
for enteral tube feeding, 862-863
linoleic acid, 849-850
propofol, 850
Liquid drug preparations, diarrhea and, 870, 870t
Liver
metabolism of toxic alcohols in, 988f
role in clearing ammonia, 731
Liver failure, 726-730
acute, 727
acute-on-chronic, 727
hepatorenal syndrome, 729-730
clinical approach to, 730t
diagnosis of, 729-730
management of, 730
pathogenesis of, 729

management of ascites, 728-729
spontaneous bacterial peritonitis, 727-728
albumin infusions in, 728
clinical features, 727
diagnosis of, 727
management of, 728
types of, 726-727
LMWH. See Low-molecular-weight heparin (LMWH)
Locked-in state, arousal and awareness in, 800
Lone atrial fibrillation, 286
Lopressor. See Metoprolol
Lorazepam
in ICU, 911
in seizures, 820
Lovenox. See Eptifibatide
Low airway pressure, in APRV, 524
Low-dose dopamine, in AKI, 640
Low-dose unfractionated heparin (LDUH), 101
indications for, 101
in obesity, 101
Low-flow nasal oxygen, 432
Low-molecular-weight heparin (LMWH), 102
in acute coronary syndromes, 314
advantages of use of, 102
dosing regimens for, 102t, 103
dalteparin, 102t, 103-104
enoxaparin, 102t, 103
drawback with, 103
thromboprophylaxis with, 102-104
for venous thromboembolism, 112-113
vs. low-dose unfractionated heparin, 103
Lung compliance, PEEP, 514
Lung macrophages, 560-561
Lung protective ventilation, 455-456, 497-498, 498t
impact on survival, 456-457
and permissive hypercapnia, 456
positive end-expiratory pressure in, 456
protocol for, 455, 455t
Lung scans, for pulmonary embolism, 110
Lung volume, 557f

Magnesium (Mg), 687-699. See also Potassium (K+)
accumulation of, 695-696
clinical features, 696
management, 696
predisposing conditions, 695-696
in asthma, 474
in cardiac arrest, 333, 333t
deficiency of, 690-695
clinical manifestations, 691-692
diagnosis, 693
magnesium replacement in, 694-695
predisposing conditions, 690-691
distribution of, 687-688
in adults, 688t
ionized, 688-689
in multifocal atrial tachycardia, 291-292
overview, 687
preparations, 694, 694t
reference ranges for, 689t
serum, 688-689
urinary, 689-690
Magnesium accumulation, 695-696. See also Hypermagnesemia
Magnesium deficiency, 690-695
monitoring of, 694-695
magnesium retention test for, 693, 693t
markers of, 690t
acute myocardial infarction, 691
alcohol-related illness, 691
antibiotic therapy, 690-691
diabetes mellitus, 691
diuretic therapy, 690
secretory diarrhea, 691
Magnesium depletion
and ionized hypocalcemia, 703
Magnesium replacement therapy
in magnesium deficiency, 694-695
monitoring of, 695
Magnesium retention test, 693, 693t
Malignant hyperthermia (MH), 764-765

. defined,, 2 = . evaluation of, 624-625, 1 = . evaluation of, 624-625, 2 = . , 1 = clinical. , 2 = Metabolic
hypokalemia promotes, 621
management of, 625-628
potassium chloride, 626
saline infusion for, 625-626, 626t
saline-resistant alkalosis, 626-628
overview, 619
pathogenesis of, 619-622
predisposing conditions, 621-622
renal mechanisms, 619-621
secondary response to, 590
Metabolic stress, solutions for, 876
Metered-dose inhaler (MDI), 467-468, 468t
Metformin, and hyperlactatemia, 604
Methanol poisoning, 990-991
Methemoglobin (metHb), 410
Methemoglobinemia, 953
Methicillin-resistant Staphylococcus aureus (MRSA), 786, 936
Methimazole, for thyrotoxicosis, 893-894
Methylprednisolone
in asthma, 473, 474t
in laryngeal edema, 580
Metoclopramide, for regurgitation, 869, 869t
Metoprolol
in thyrotoxicosis, 893
Metronidazole, for CDI, 743
MG. See Myasthenia gravis (MG)
Microaerophilic organisms, man as, 428
Microbial therapy, for CDI, 744
Microbiological evaluation, in VAP, 559-564
bronchoalveolar lavage, 562-564
preference for method, 564
protected specimen brush, 564
tracheal aspirate, 559-562
microscopic analysis, 559-561
qualitative cultures, 561
quantitative cultures, 562, 562t
“Microliths,” 738
Midazolam
in ICU, 911
Mild hypothermia, 771

Miscuffing, 125
Mixed delirium, 802
Moderate hypothermia, 771
MOF. See Multiorgan failure (MOF)

in acute coronary syndromes, 305t, 306-307
in ICU, 904-905
Motor response, in coma, 806
Movement disorder, in ICU, 817-829
drug-induced paralysis, 825-827
neuromuscular blocking agents, 825-826, 825t
neuromuscular weakness syndromes, 822-825
critical illness neuromyopathy, 824-825
myasthenia gravis, 822-823
seizures, 817-822
predisposing conditions, 819, 819t
types of, 817-819
Moxifloxacin, 934
MRSA. See Methicillin-resistant Staphylococcus aureus (MRSA)
Mucolytic therapy, with N-acetylcysteine, 542t
Multifocal atrial tachycardia (MAT), 291
in elderly, 291
management of, 291-292
Multiloculated abscess, in post-splenectomy patient, 747f
Multiorgan dysfunction syndrome (MODS), 266
Multiorgan failure (MOF), 79, 266
GI tract and, 80
Muscle weakness
in hypophosphatemia, 711
in MG, 822
Myasthenia gravis (MG), 822-823
clinical features of, 822
diagnosis of, 823
predisposing conditions, 822
treatment of, 823
vs. Guillain-Barré syndrome, 823t
Mycobacterium tuberculosis, 74
Mycoplasma pneumoniae, 934
Myoclonic status epilepticus, after cardiac arrest, 341
Myoclonus, 818
Myoglobin, in urine, 642
Myoglobinuric renal failure, 642-643, 764
management of, 642-643
myoglobin in urine, 642
Myxedema, 895
Myxedema coma, 895

N-acetylcysteine (NAC), 286, 440
for contrast-induced renal injury, 641
mucolytic therapy with, 542t
overdoses of acetaminophen and, 967, 968t
Naloxone, 972-974
adverse reactions of, 974
dosing regimen for, 972-973, 973t
empiric, 973-974
enteral, for regurgitation, 869, 869t
Narrow-QRS-complex tachycardias, 283-284, 285f
irregular rhythm, 284, 285f
regular rhythm, 283, 285f
Nasal cannula, 420, 421f
Nasogastric feedings, and severe pancreatitis, 725
National Asthma Education Program, 473
National Healthcare Safety Network (NHSN), 559
Natriuretic peptides, 241
Nausea, opioids and, 907
NCCT. See Noncontrast computed tomography (NCCT)
Near infrared spectroscopy (NIRS), 188-189
Necrosis, of pancreas, 726f
Necrotizing pancreatitis, 723f
Necrotizing wound infections, 785
Needlestick injuries, 66
incidence of, 66
prevention of
one-handed recapping technique, 66, 68f
safety-engineered needle, 66, 67f
Needlestick Safety and Prevention Act, 66
Neomycin, for hepatic encephalopathy, 733
Nephrotoxic agents, in AKI, 640
Nephrotoxicity
aminoglycosides and, 925
amphotericin B and, 927-928
Nesiritide (Natrecor), in heart failure, 248t, 249
Neuraxial analgesia, 104
Neurohumoral responses, heart failure and, 241
natriuretic peptides, 241
renin-angiotensin-aldosterone system, 242
sympathetic nervous system, 241-242
Neuroleptic malignant syndrome (NMS), 765-767
drugs implicated in, 766t
dystonic reactions in, 766-767
haloperidol and, 917-918

Neurological abnormalities, areas of, 833f
Neurologic injury, fever and, 791
Neuromuscular blocking agents, in drug-induced paralysis, 825-826, 825t
Neuromuscular weakness syndromes, in ICU, 822-825
Neuronal cells, 660
Neurotoxicity, B-receptor antagonists and, 969-970
Neutrophils, 561
activation, inflammatory response and, 264, 265f
oxygen metabolism in, 438
NHSN. See National Healthcare Safety Network (NHSN)
Nicardipine, for blood pressure, 840
NIHSS. See NIH Stroke Scale (NIHSS)
NIH Stroke Scale (NIHSS), 833-834
NIRS. See Near infrared spectroscopy (NIRS)
Nitrates, for cyanide poisoning, 986-987
Nitrate tolerance, nitroglycerin and, 953
Nitrogen balance, 850
nonprotein calories and, 851, 851f
Nitrogen excretion, 850
Nitrogen intake, 850
Nitroglycerin, 951-953
in acute coronary syndromes, 305t, 306
adverse effects, 953
antiplatelet effects of, 952
clinical uses, 952
dosing regimen for, 952-953
in heart failure, 248t, 249
vasodilator actions of, 951-952, 952f
Nitroprusside, 954-956
in aortic dissection, 320, 320t
clinical uses of, 954
cyanide intoxication, 954-956, 955f
dosing regimen for, 954

in heart failure, 248-249, 248t
thiocyanate intoxication, 956
vasodilator actions of, 954
Nitroprusside reaction, in ketogenesis, 609-610
Nitroprusside tachyphylaxis, 956
Nitrovasodilators, 951-956
NIV. See Noninvasive ventilation (NIV)
NKH. See Non-ketotic hyperglycemia (NKH)
N-methyl-D-aspartate (NMDA) receptors, 804
NMS. See Neuroleptic malignant syndrome (NMS)
Nonabsorbable antibiotics, for hepatic encephalopathy, 733
Nonabsorbable antibiotics, for oral decontamination, 89-91, 90f
Noncontrast computed tomography (NCCT)
in acute stroke, 834-835, 834f
hypodense area, 835f
Nonconvulsive status epilepticus, 818-819
Noninvasive ventilation (NIV), 479-480, 526-532
adverse events during, 531
checklist for, 528t
efficacy, 529-530
failure rate of, 530f
impact of, 529f
modes of ventilation, 526-527
BiPAP, 527
CPAP, 526
PSV, 527
monitoring, 531
nosocomial pneumonia during, 531-532
patient selection criteria, 528
for post-extubation stridor, 581
responses to first hour of, 531f
Non-ketotic hyperglycemia (NKH), 662-663
clinical manifestations, 663
fluid management, 663
insulin therapy for, 663

Non-opioid analgesia, in ICU, 907-909
Nonprotein calories, 849
intake of, 851f
and nitrogen balance, 851
Nonrebreather system, 433-434, 434f
Nonsteroidal anti-inflammatory agents (NSAIDs), for fever, 792
Non-ST-segment elevation myocardial infarction (NSTEMI), 303
Non-thyroidal illness, plasma TSH levels and, 892
Norepinephrine, 948-949
Normobaric hyperoxia, 430
Normosol, 219t
Nosocomial endocarditis, 58
Nosocomial infections, in ICU, 363, 784-786, 784t
Clostridium difficile infection, 786
common, 784
isolates, 740f
paranasal sinusitis, 785-786
clinical significance of, 786
diagnosis of, 785-786
management of, 786
pathogenesis of, 785
patient-specific infections, 786
surgical site infections, 784-785
Nosocomial pneumonia, during NIV, 531-532
Numerical Ranking Scale, 903
Nutrient fuels, metabolism of, 847-848, 848t
Nutritional bulk, trophic effects of, 860
Nutritional requirements, 847-858
daily energy expenditure, 847-849
essential trace elements, 853-855
dietary allowances for, 854t
iron, 854
selenium, 855
substrate requirements, 849-851
carbohydrates, 849

lipids, 849-850
nonprotein calories, 849
protein requirements, 850-851
vitamin requirements, 851-853
thiamine deficiency, 851-853
vitamin E deficiency, 853
Nutrition-associated hypercapnia, 404
Nutrition support
in hepatic encephalopathy, 734
problem with, 855-856
in severe pancreatitis, 725

Obesity hypoventilation syndrome, NIV in, 529
Obesity-related hypoventilation, 399
Obligate nephrotoxins, 925
Occlusion, of central venous catheters, 43
causes of, 43-44
restoring patency in, 44, 44t
non-thrombotic occlusion, 44-45
thrombotic occlusion, 44, 44t
Occult blood testing, 87-88
Occult PEEP, 466, 477, 478, 491, 517, 547-549
adverse effects of, 547-548
features of, 548f
management of, 549
monitoring of, 548-549
Occupational exposures, 65, 75
from airborne pathogens, 73-75
from bloodborne pathogens, 65-73
Ocular motility, coma, 808
Ocular reflexes
in evaluation of coma, 809f
bedside evaluation, 808
oculocephalic reflex, 808
oculovestibular reflex, 808
Oculocephalic reflex, 808
Oculovestibular reflex, 808
Oliguria, 635
Omega-3 fatty acids, for enteral tube feeding, 862-863
One handed “scoop technique,” 66, 68f
Open lung concept, 521-522
Opiates, 904
Opioid analgesia, in ICU, 904-907, 905t
cardiovascular effects, 907
intestinal motility, 907
nausea and vomiting, 907
respiratory depression, 906
fentanyl, 904
hydromorphone, 905
meperidine, 906
morphine, 904-905
remifentanil, 905-906
Opioids, overdoses of, 972-974
clinical features, 972
naloxone and, 972-974
Oral cavity, colonization of, 88-89
Oral decontamination, in VAP, 554-555
Organic acidoses, 601-618

alcoholic ketoacidosis, 613-614
diabetic ketoacidosis, 610-613
ketoacids, 608-610
lactic acidosis, 601-606
Orthopedic surgery, and venous thromboembolism, 98
Osborn waves, 772, 772f
Oscillometric method, 126-127
Osmolal gap, 656
Osmolality, 655
of feeding formulas, 861
plasma, 655-656
Osmolarity, 655
of peripheral vein, 884
Osmotic activity, 653-656
defined, 653
effective, 654
osmolal gap, 656
of plasma, 655-656
relationship with water movement, 654f
relative, 653-654
units of, 655
Osmotic disorders, in ICU, 653-672
hypernatremia, 657-658
approach to, 657-658, 657f
extracellular volume associated with, 657-658, 657f
hypertonicity, 658
hypertonic hyperglycemia, 662—663
hyponatremia, 664-670
nonosmotic ADH release in, 664-665
osmotic activity, 653-656
Osmotic pressure, 654
Overfeeding, and hypercapnia, 404
Oxidant injury, iron and, 854
Oxidant stress, 264, 266t, 441
Oxidative chain reaction, 264-266
Oximetry, 409-417
early, 410-411
forehead pulse, 414
light absorption by hemoglobin, 410, 410f

pulse, 411-413, 412f, 415 (see also Pulse oximetry)
venous, 415-417
Oxygenated hemoglobin (HbO>), 410
Oxygen debt, 180
Oxygen delivery, 146, 178
Oxygen extraction, 181-182, 355
monitoring of, 182
Oxygen extraction ratio, 147
Oxygen in blood, 171-176, 172t
anemia vs. hypoxemia, 176
oxygenation of hemoglobin, 171-173
oxygen content, 174-175
arterial oxygen content, 174
dissolved oxygen, 174
hemoglobin-bound oxygen, 174
oxygen content equation, 175, 175f
venous oxygen content, 174-175
Oxygen metabolism, 436-438, 437f
and chain reactions, 438
and neutrophil activation, 438
Oxygen saturation, 171
Oxygen therapy, 427-443
in acute stroke, 839-840
and aerobic metabolism, 429-431, 430f
in COPD, 476-477
delivery systems, 431-436, 431t
air entrainment devices, 434-435, 435f
high-flow systems, 435-436
low-flow systems, 432
masks with reservoir bags, 432—434
standard face masks, 432
hypoxemia and, 428-429, 429f
and microcirculation, 430
paucity of oxygen in tissues and, 427-428, 428t
toxic nature of oxygen and, 436-442
antioxidant protection, 439-441
oxidant stress, 441-442
oxygen metabolism, 436-438
Oxygen transport, 176
and energy metabolism, 176-177, 177f
parameters, 146
oxygen delivery, 146
oxygen extraction ratio, 147
oxygen uptake, 146-147
parameters and normal range of values, 177t
Oxygen uptake, 146-147, 178-180
control of, 181-182
deficit over time, 180
low, conditions associated with, 179-180
from modified Fick equation, 179
variability in, 178, 179t
whole body, 179

Oxyhemoglobin dissociation, hypophosphatemia and, 710
Oxyhemoglobin dissociation curve, 172, 173f, 623-624
shifts in, 172-173

in critically ill patients, 902
definition, 903
monitoring, 903-904
pain intensity scales, 903-904
vital signs, 904
Pain intensity scales, 903-904
Pamidronate, for ionized hypercalcemia, 708
Pancreas, necrosis of, 726f
Pancreatic enzyme
for acute pancreatitis diagnosis, 720-721
Viokase, 867-868
Pancreatic infection, 725
acute, 719-722
contrast-enhanced computed tomography for, 721-722, 722f
edematous, CT image of, 722f
gallstone, 726
necrotizing, CT image of, 723f
severe, 723-726
abdominal complications in, 725-726
circulatory support in, 724
gallstone pancreatitis, 726
nutrition support in, 725
prophylactic antibiotics for, 724
PaO>/FIO> ratio, 398, 514
Paranasal sinusitis, 785-786
Clinical significance of, 786
Parapneumonic effusions, 565
Parenteral nutrition, 875-886
additives, 878-879
and complications, 881-884
bowel sepsis, 884
carbohydrate complications, 882-883
catheter-related complications, 881
hepatobiliary complications, 883-884
lipid complications, 883
creating regimen for total, 879-881
peripheral, 884
substrate solutions, 875-878
amino acid solutions, 876-877
dextrose solutions, 875
lipid emulsions, 877-878, 877t
Paresthesias, in Guillain-Barré syndrome, 824
antibiotic therapy for, 746
clinical features, 744-745

fluids loss and, 745
management of, 745-746
Permissive hypercapnia, 456, 497
Peroxynitrite, 982
Persistent vegetative state, 800
family care in, 813
Phenylephrine, 949-950
Phenytoin, in seizures, 820
Phlebitis, peripheral vein catheters and, 42
Phlebotomy, 351-352
Phosphate, for diabetic ketoacidosis, 613
Phosphate binding agents
Phosphate depletion
effects of, 711f
energy metabolism and, 710
Phosphate in blood, 713
Phosphate replacement therapy, in hypophosphatemia, 711, 712t
Phosphodiesterase inhibitors, 972
Phosphorous depletion, and respiratory muscle weakness, 578
Physiologic dead space, 392
PICC. See Peripherally inserted central catheter (PICC)
Pickwickian syndrome, 399
Piperacillin-tazobactam, 755, 935
Pituitary failure, and brain death, 813
Plasma acetaminophen levels, 966, 966f
Plasma bicarbonate (HCO3), 623, 623f
Plasma cortisol, in adrenal suppression, 888
Plasma exchange, in thrombotic thrombocytopenia purpura, 377
Plasma-Lyte A, 219t
Plasma magnesium levels, 689f
Plasma osmolality, 655-656
calculated, 656
effective, 656
measured, 655
Plasma products, 381
adverse effects, 383
acute hemolytic reactions, 383-384
acute lung injury, 384
hypersensitivity reactions, 384
transmitted infections, 384
cryoprecipitate, 382-383
fresh frozen plasma, 381-382
Plasma sodium concentration (PNa), 659-660
Plateau pressure, 498
Platelet plug, 369
Platelet refractoriness, 379
Platelets, 210
Platelet transfusions, 378-381

acute lung injury, 381
bacterial transmission, 380
fever, 380
hypersensitivity reactions, 380
indications for
active bleeding, 379
no active bleeding, 380
procedures, 380
platelet products, 378
apheresis platelets, 378
leukoreduction, 378
pooled platelets, 378
response to, 378-379
Plavix. See Clopidogrel
Pleural drainage system, standard, 546
in bacterial pneumonias, 565
in hypothyroidism, 895
superior vena cava perforation and, 46
Pleural evacuation, 545-547
water-seal bottle, 545-546, 546f
Pleural pressure, 160
negative, 160-161
positive, 161-162, 162f
Pneumothorax, 543-545
central venous cannulation and, 33-34
redundant skin folds and, 544-545

Positive end-expiratory pressure (PEEP), 159, 328, 456, 512-516
airway pressure waveforms and, 513f
alveolar recruitment, 513-514
influence on inflation pressures, 512-513, 513f
influence on lung aeration, 514f
occult, 517
opposing effects of, 515f
rationale, 512
recruitable lung volume, 513-514
systemic O> transport, 514-516
cardiac output, 514
oxygen delivery, 515-516
ventilator settings and, 517
Positive pressure breathing, 257
Positive-pressure lung inflation
pressure control ventilation, 507
pressure-regulated, volume control ventilation, 508
volume control ventilation, 506-507
Positive pressure ventilation, 487-501
basics of
airway resistance, 493
end-expiratory pressure, 490-491, 490f
end-inspiratory pressures, 488-490
mean airway pressure, 491-492
thoracic compliance, 492—493
and cardiac performance, 499-501
afterload, 500
cardiac output, 500, 501f
preload, 499, 499f
end-expiratory pressure
applied PEEP, 491
occult PEEP, 491
zero end-expiratory pressure, 491
end-inspiratory pressures
pressure-controlled ventilation, 490
volume-controlled ventilation, 488-490
lung injury, 494-498, 495f
atelectrauma, 496—497
barotrauma, 497
biotrauma, 497
lung protective ventilation, 497-498, 498t
volutrauma, 494, 496
Post-cardiac arrest syndrome, 336
Post-extubation stridor, 580-581
Postrenal obstruction, AKI and, 636
Postresuscitation injury, 210-211
management of, 212
pathophysiology of, 211-212
predisposing factors, 212
Potassium (K+), 673-686. See also Magnesium (Mg)
for diabetic ketoacidosis, 612-613
distribution of, 673

excretion, 674-675
fraction of total body, 673, 674f
hyperkalemia, 679-684
management of severe of, 681-684, 683t
hypokalemia, 675-678
intracellular preponderance of, 673
overview, 673
serum (plasma), 674
and total body potassium, 675f
spironolactone and, 675
Potassium chloride
for metabolic alkalosis, 626
in potassium replacement, 678
Potassium deficits, in hypokalemia, 677, 678t
Potassium depletion, 676
chloride-resistant metabolic alkalosis and, 625
Potassium excretion, 674-675
renal, 674-675, 680
Potassium removal, 684
Potassium replacement
in hypokalemia, 678

PPIs. See Proton pump inhibitors (PPIs)
PPN. See Peripheral parenteral nutrition (PPN)
Prednisone

and cardiac performance, 152
definition of, 151-152
Prerenal disorders, AKI and, 636
Prescription drug, use in the United States, 964f
Pressure control ventilation (PCV), 507
Pressure-regulated, volume control ventilation (PRVC), 508
Pressure-regulated spontaneous ventilation, 525f
Pressure-supported breath, 511
Pressure support ventilation (PSV), 511-512
changes in pressure and flow, 511f
clinical uses, 512
pressure-supported breath, 511
Primary acid-base disorders, 588-589, 589t
identification of, 592-593
predictive equations for, 591f
Primary adrenal insufficiency, 665
Probiotics, for CDI, 744
Prokinetic therapy, 869t
for regurgitation, 868-869
Prophylactic antibiotics, for severe pancreatitis, 724
Propofol, in ICU, 850, 914-916
actions and uses of, 914
adverse effects, 915
dosage of, 914-915
Propofol infusion syndrome, 915-916
Propranolol, for thyrotoxicosis, 893
Propylene glycol, and hyperlactatemia, 605
Propylene glycol toxicity, 805
benzodiazepines and, 913-914
Propylthiouracil (PTU), 893-894
Prostaglandin E, in fever, 791
Protamine sulfate, 111-112
Protected specimen brush (PSB), 564
Protein
in enteral tube feeding, 861-862
requirements, 850-851
Prothrombin complex concentrates (PCC), 382
Proton pump inhibitors (PPIs)
and clopidogrel, 85
for stress ulcer bleeding, 84-85
Proximal tubular pressure (PTP), 644
PRVC. See Pressure-regulated, volume control ventilation (PRVC)
PSB. See Protected specimen brush (PSB)
Pseudohyperkalemia, 679
Pseudohyponatremia, 664

“Pseudomembranes,” 741
Pseudomonas aeruginosa, 88, 476, 554, 930, 934, 935
susceptibility of, 926f
Pseudothrombocytopenia, 370
PSV. See Pressure support ventilation (PSV)
PSVT. See Paroxysmal supraventricular tachycardias (PSVT)
PTP. See Proximal tubular pressure (PTP)
Pulmonary angiography, for pulmonary embolism, 110
Pulmonary artery catheter, 15, 135-148
applications, 147-148
hemodynamic patterns, 147, 147t
tissue oxygenation, 148, 148t
balloon flotation principle and, 135
design of, 135
features of, 136, 136f
and hemodynamic parameters, 143-147, 144t
body size and, 143-144
placement of, 136-137
thermodilution cardiac output, 141-143, 141f, 143f
intracardiac shunts and, 142-143
thermodilution curves, 142, 143f
tricuspid regurgitation and, 142
variability in, 142
wedge pressure and, 137-141
Pulmonary artery wedge pressure (PAWP), 137, 138f, 145, 153
in ARDS, 140-141
principle of, 138-140, 139f
vs. hydrostatic pressure, 140
wedge pressure tracing, 138
Pulmonary barotrauma, 542
Pulmonary embolism, 97. See also Venous thromboembolism (VTE)
Pulmonary oxygen toxicity, 441-442
Pulmonary vascular resistance (PVR), 163
Pulmonary vascular resistance index (PVRI), 146
Pulse oximetry, 411
and anemia, 413
dyshemoglobinemias and, 413
features, 411, 412f

hypotension and, 413
pigments and, 413
reliability, 411, 412t
use of, 415
Pulsus paradoxus, 161
Pupillary size, condition affects, 807t
Purpura fulminans, 375
Pyridostigmine (Mestinon), for myasthenia gravis, 823
Pyridoxine, for ethylene glycol poisoning, 990

Rainbow Pulse CO-oximeter, 413
Rancidity, 440
in stress ulcer bleeding, 83t, 84
Rapid breathing
ACV and, 509
during SBT, 575-576
adverse effects, 575
management of, 575-576
RASS. See Richmond Agitation Sedation Scale (RASS)
Receptive aphasia, 832
Recruitable lung volume, 513-514
Red man syndrome, 936
Refractory hypoxemia, 459-460
advantages of, 525-526
disadvantages of, 526
ventilator settings, 524-525
high frequency oscillatory ventilation, 522-524
advantages of, 523
disadvantages of, 524
ventilator settings, 522-523
open lung concept, 521-522
Reservoir bag devices, 432
advantages and disadvantages, 434
nonrebreathers, 433-434, 434f
partial rebreathers, 432-433, 433f
Reservoir system, for oxygen therapy
Resonant systems, 130
damping factor and, 131
overdamped system, 131, 132
resonant frequency and, 131
underdamped system, 131
Respirators, 74-75
Respiratory acid-base disorders, 589
response to, 590-592
acute respiratory disorders, 590-591
chronic respiratory disorders, 591-592
Respiratory acidosis, salicylates and, 974
Respiratory alkalosis, 589, 709
Respiratory burst, 264, 438
Respiratory depression
naloxone and, 973
opioids and, 906
Respiratory disorders
acute, 590-591
chronic, 591-592
Respiratory muscle strength, evaluation of, 399-400
Respiratory muscle weakness
magnesium depletion and, 578
maximum inspiratory pressure and, 578
mechanical ventilation and, 577-578
spontaneous breathing trial and, 577-579
ultrasound for, 578-579

Respiratory rate, ventilator settings and, 517
Respiratory secretions, viscosity of, 541-542
Resting energy expenditure (REE), 848
Resuscitation fluids, 206-207, 207t
distribution of, 207-208
influence of, on cardiac output, 207, 208f
preferred fluid, 208
Retention enema, of lactulose, 733
Reteplase (Retavase), 309-310, 310t
Return of spontaneous circulation (ROSC), monitoring for, 333-334
Reverse Fick method, 178
Reverse pulsus paradoxus, 161
Rewarming, during hypothermia, 772-773
afterdrop and, 772
external, 772
internal, 772
rewarming shock, 772—773
Rewarming shock, 772-773
Rhabdomyolysis, 764
Richmond Agitation Sedation Scale (RASS), 910-911, 910t
Rifamaxin, for hepatic encephalopathy, 733
RIFLE criteria. See Risk, Injury, Failure, Loss, and End-stage renal disease (RIFLE) criteria
Right atrial pressure (RAP), 144, 153
Right heart failure, 246
management of, 255
Right-ventricular end-diastolic pressure (RVEDP), 144, 153
Right ventricular end-diastolic volume (RVEDV), 246
Ringer's acetate, 223
Ringer's fluids, 222
advantages & disadvantages, 223
lactate considerations, 223
Ringer's lactate, 222-223
Ringer's lactate, 207, 208, 222-223
Risk, Injury, Failure, Loss, and End-stage renal disease (RIFLE) criteria, 633-634, 634t

Saccharomyces boulardii, 744
Salicylates, overdoses of, 974-976
clinical features, 974-975
diagnosis, 975
hemodialysis, 976
management, 975-976
Salicylic acid, 974

in edematous states, 626
for metabolic alkalosis, 625-626, 626t
Saline instillation, 541-542
Saline-resistant alkalosis, 626-628
acetazolamide for, 627
hydrochloric acid infusion for, 627-628, 627t
adverse effects of, 628
method, 627
Salmonella typhimurium, 80
SBP. See Spontaneous bacterial peritonitis (SBP)
SBT. See Spontaneous breathing trial (SBT)
Schistocytes, 376
Secretory diarrhea, magnesium deficiency and, 691
Sedation, in ICU, 909-911
benzodiazepines for, 911-914, 912t
dexmedetomidine for cooperative, 916
monitoring, 910-911
with rapid-arousal drugs, 915t
Seizures, in ICU, 817-822
stage 1 drugs, 819-820
stage 2 drugs, 820
stage 3 drugs, 820
carbapenems and, 931
levetiracetam in, 820
abnormal movements, 817-818
generalized seizures, 818
myoclonus, 818
partial seizures, 818
status epilepticus, 818-819
Seldinger technique, 10, 11f, 23
Selective digestive decontamination (SDD), 91-92
Selective oral decontamination (SOD), 90-91, 90f
Selenium, 440, 855, 878-879
Semiquantitative catheter tip cultures, for CRBI diagnosis, 52-53
Sensorimotor loss, 832
Sepsis, 266
AKI and, 637
body temperature and, 790f
bowel, 884
source of lactate levels in, 603
Septic encephalopathy, and altered consciousness, 800-801

Septic shock, 266, 268
adrenal suppression and, 890
hemodynamic alterations in, 268
management of, 270-273, 270t
antibiotic therapy, 272-273
in bundles, 270, 270t
corticosteroids, 272
vasopressors, 271-272
volume resuscitation, 271
mortality rate, 268
serum lactate levels in, 270
tissue oxygenation in, 269, 269f
Serotonin syndrome (SS), 767-770
diagnostic worksheet for, 769t
drugs can produce, 768t
Serum ammonia, in hepatic encephalopathy, 732-733
Serum amylase, in acute pancreatitis, 720-721
Serum magnesium, 688-689
Serum phosphate level, TPN effects on, 709f
Serum potassium, 674
Severe hypothermia, 771
Severe pancreatitis, 723-726
abdominal compartment syndrome, 725-726
pancreatic infection, 725
Severe sepsis, 266. See also Septic shock
Shear thinning, 165
Shivering, in targeted temperature management, 338
SIADH. See Syndrome of inappropriate ADH (SIADH)
SID. See Strong ion difference (SID)
Silicone catheters, 4
SIMV. See Synchronized IMV (SIMV)
Single ventilator breath, 506f
SIRS. See Systemic inflammatory response syndrome (SIRS)
Skeletal muscle injury, 764
"Sludge," 738
SOD. See Selective oral decontamination (SOD)
Sodium bicarbonate, for lactic acidosis, 607
Sodium nitroprusside, for severe hypertension, 840
Sodium polystyrene sulfonate, 684

Sodium restriction, in ascites, 728
Sodium thiosulfate, and cyanide poisoning, 986
Solvent drag, 646
Solvent toxicity, nitroglycerin and, 953
Spectrophotometry, 409
Sphygmomanometer, 124
Spironolactone, for ascites, 728
Spontaneous bacterial peritonitis (SBP), 727-728
Spontaneous breathing trial (SBT), 572-579
management of, 577
monitoring of, 576
checklist for, 571t by disconnecting ventilator, 573-574
measurements for, 572t method preference for, 574
management of, 579
monitoring of, 578-579
potential sources, 577-578
simple breathing circuit for, 574f success vs. failure, 574-575
Spontaneous eye movements, in coma, 808
Spontaneous eye opening, in coma, 806-807
Spot urine sodium measurements, for AKI, 638, 638t
Spurious hypoxemia, 403
Squamous epithelial cells, 559-560
SSI. See Surgical site infections (SSI)
Staphylococcus aureus, 58, 554, 566, 785, 935
Staphylococcus epidermidis, 49, 49f, 59, 936
Startle myoclonus, 818
Status epilepticus, 818-819
drug regimens for generalized, 821t
drug regimens for refractory, 822t
nonconvulsive, 818-819
subtle, 819
Steady flow, resistance to, 164
Sterile dressing, at catheter insertion site, 41-42, 42t
Sterile pyuria, AIN and, 642
Steroid myopathy, 474

Streptococcus pneumoniae, 476
Stress hyperlactatemia, 604
Stress ulcer bleeding, 82
drugs used for prophylaxis of, 83-84, 83t
and Clostridium difficile enterocolitis, 86-87
histamine H2-receptor antagonists, 84
proton pump inhibitors, 84-85
sucralfate, 85-86
enteral tube feedings and, 87
occult blood testing and, 87-88
preventive measures, 83-88
risk factors for, 82t, 83
Stridorous breathing, 580

classification, 831-832
definition, 831
“Stroke centers,” 841
Stroke index (SI), 145
Stroke mimics, 833
“Stroke team,” 841
Strong ion difference (SID), 220-222, 222f, 603-604
ST segment elevation myocardial infarction (STEMI), 303
Subclavian vein cannulation, 26-28
anatomy, 26
complications, 27-28
landmark method, 27
positioning, 26
ultrasound guidance, 27
Subglottic secretions, in VAP, 555
Substrate requirements, 849-851
Substrate solutions, 875-878
intravenous, 875t
lipid emulsions, 877-878
Subtle status epilepticus, 819
Succinylcholine, in hyperkalemia, 680
Sucralfate, for stress ulcer bleeding, 85-86
Suction-control bottle, 546, 546f
Suctioning, to clear secretions, 540
Superaspirins, 315
Superior vena cava
catheter-induced perforation of, 46, 47f
syndrome, 45
Superoxide dismutase (SOD), 439, 439f
Suppurative thrombophlebitis, 57-58
Supraventricular tachycardias, 283. See also Tachyarrhythmias
Surgical decompression, for patients with ACS, 645
Surgical site infections (SSI), 784-785
Surviving Sepsis Campaign, 270
Suspensions, 227
Sympathetic nervous system, activation of, 241-242
Symptomatic candiduria, 755-756

Synchronized IMV (SIMV), 510
Syndrome of inappropriate ADH (SIADH), 666
Systemic inflammatory response syndrome (SIRS), 266, 604, 780
after cardiac arrest, 336
diagnostic criteria for, 267t
Systemic O> transport, PEEP and, 514-516
Systemic oxygenation, 171-189
anemia on, influence of, 352, 353f
chemical markers, 184-188
arterial base deficit, 187-188
serum lactate level, 184-187
near infrared spectroscopy and, 188-189
oxygen in blood, 171-176
systemic oxygen balance, 176-184
oxygen delivery, 178
oxygen extraction, 181-182
oxygen transport & energy metabolism, 176-177, 177t
oxygen uptake, 178-180
venous oxygen saturation, 183-184
Systemic vascular resistance (SVR), 163
Systemic vascular resistance index (SVRI), 145-146
Systolic heart failure, 243-245
T
Tachyarrhythmias, 283
atrial fibrillation, 286-291
evaluation of, 283, 284f
narrow-QRS-complex tachycardias, 283-284, 285f
wide-QRS-complex tachycardias, 285
multifocal atrial tachycardia, 291-292
paroxysmal supraventricular tachycardias, 292-295
ventricular tachycardia, 295-299
Tachycardia, 283. See also Tachyarrhythmias
fever and, 791
Tachyphylaxis, nitroglycerin infusions and, 249
Targeted temperature management (TTM), 336-339, 338t
contraindications to, 337, 338t
indications for, 337, 338t
method of, 337-338
induction, 338
contraindications to, 309t thrombolytic agents, 309-310, 310t

alteplase, 309, 310t reteplase, 309-310, 310t
and risk of major bleeding, 310
tenecteplase, 310, 310t for venous thromboembolism, 113-114
Thrombomodulin, 369
Thrombosis, catheter-associated, 45
in cancer patients, 45
lower extremity thrombosis, 46
upper extremity thrombosis, 45
Thrombotic microangiopathy, 374-375, 375t
Thrombotic thrombocytopenia purpura (TTP), 376-377
clinical features, 377
management, 377
Thrombus, 370
Thyroid disorders, 892
primary vs. secondary, 891-892
Thyroid function, evaluation of, 891-892
Thyroid replacement therapy, in hypothyroidism, 896
Thyroid-stimulating hormone (TSH), 891-892
levels, 892t non-thyroidal illness and plasma, 892
thyroid disorders, 892
Thyroid storm, 893
drug therapy for, 894t propylthiouracil for, 893-894
special concerns in, 894-895
Thyrotoxicosis, 783, 892-895
clinical manifestations of, 893
diagnosis of, 893
drug therapy for, 894t thyroid storm and, 893, 894-895
treatment of, 893-895
antithyroid drugs for, 893-894
B-receptor antagonists for, 893
inorganic iodine, 894
Thyroxine (T4), 891
patterns of free, 892t
TIA. See Transient ischemic attack (TIA)

TIPPS. See Transjugular intrahepatic portosystemic stent-shunt (TIPPS) Tissue factor, 375 factor pathway, 370 Tissue hypoxia, 176 oxygenation, 171-189. See also Systemic oxygenation impaired, markers of, 184t in septic shock, 269, 269f Tissue oxygen saturation, 188, 1 = . Tissue, 1 = . Tissue, 1 = . Tolvaptan, for hyponatremia, 669-670 'Tonicity,' 654, 1 = . Torsade de pointes, 297-299, 298f management of, 299 predisposing factors, 298, 298t QT intervals, measuring of, 298, 1 = . Total body water (TBW), 659-660 Total parenteral nutrition (TPN) creating regimen for, 879-881 effects on serum phosphate level, 709f, 1 = . hyperglycemia during, 882, 1 = . insulin and, 882, 882t Toxic megacolon, 742f, 1 = . methanol, 990-991 carbon monoxide poisoning, 981-984 clinical features, 983, 1 = . ethylene glycol, 987-990, 1 = . diagnosis of, 983-984, 1 = . pathophysiology, 981-982, 1 = . treatment for, 984, 1 = . poisoning, 984-987, 1 = . cyanide antidotes for, 986t, 1 = . clinical features, 985 diagnosis of, 985, 1 = . pathophysiology,, 1 = . treatment for, 986-987, 1 = . and, 605 elements, 878-879, 1 = . hyperlactatemia aspirate, in VAP,, 1 = . microscopic analysis, 559-561, 1 = . 559-562 lung macrophages, 560-561, 1 = . neutrophils, 561, 1 = . squamous epithelial cells, 559-560, 1 = . Trace Tracheal, 1 = . , 1 = 984-985

= . 3 hypothyroidism,, 3 = . Triazoles, 928-929 clinical uses, 928, 1 = . Triazoles, 928-929 clinical uses, 928, 2 = . Triazoles, 928-929 clinical uses, 928, 3 = . dosing regimens, 928, 1 = . dosing regimens, 928, 2 = . dosing regimens, 928, 3 = . drug interactions, 929 toxicity, 929, 1 = . drug interactions, 929 toxicity, 929, 2 = . drug interactions, 929 toxicity, 929, 3 = . (T 3 ), 891, 1 = . (T 3 ), 891, 2 = . (T 3 ), 891, 3 = . Triiodothyronine in acute coronary, 1 = . Triiodothyronine in acute coronary, 2 = . Triiodothyronine in acute coronary, 3 = . Trofiban,, 1 = . Trofiban,, 2 = . Trofiban,, 3 = . Troponin I levels, in DKA, 611, 1 = . Troponin I levels, in DKA, 611, 2 = . Troponin I levels, in DKA, 611, 3 = . syndromes,, 1 = . syndromes,, 2 = . syndromes,, 3 = . 315, 1 = . 315, 2 = . 315, 3 = . TSH. See Thyroid-stimulating hormone, 1 = . TSH. See Thyroid-stimulating hormone, 2 = . TSH. See Thyroid-stimulating hormone, 3 = . (TSH), 1 = . (TSH), 2 = . (TSH), 3 = . , 1 = TTM. See Targeted temperature management. , 2 = (TTM). , 3 = TTP. See Thrombotic thrombocytopenia purpura (TTP)
Tube occlusion, enteral tube feeding and, 867-868
Tubuloglomerular feedback, 636
Tumor lysis syndrome, 679

in acalculous cholecystitis, 738
for cardiac dysfunction, 576
for respiratory muscle weakness, 578-579
Ultrasound-guided vascular cannulation, 20-23, 21f, 22f
Unfractionated heparin (UFH), 100-101
in acute coronary syndromes, 313-314
low-dose, 101
thromboprophylaxis with, 100-101
for venous thromboembolism, 110-112, 11it
Uniform Determination of Death Act, 810
Units and conversions, 993-997
apothecary and household conversions, 995
converting units of solute concentration, 994-995
French sizes, 997
gauge sizes, 997
pressure conversions, 996
temperature conversions, 996
units of measurements in SI system, 993-994
Unstable angina (UA), 303
Uremic bleeding, 383
cryoprecipitate in, 383
desmopressin in, 383
Urinary magnesium, 689-690
Urinary magnesium excretion, 689f
Urinary tract infections, in ICU, 751-757
candiduria, 755-756
common symptoms of, 754
diagnosis of, 754
pathogenesis of, 751-753
bacterial adherence, 752
microbiology, 752, 753t prevention of, 753
treatment of, 754-755
Urinary urea nitrogen (UUN), 850
UUN. See Urinary urea nitrogen (UUN)

Valproic acid, in seizures, 820
Vancomycin, 935-937
alternatives for, 937-938, 938t antibacterial spectrum of, 935-936
for CDI, 743
dosing nomogram, 937t dosing regimens of, 936
Vancomycin, in catheter-related bloodstream infections, 55, 55t
VAP. See Ventilator-associated pneumonia (VAP)
Vascular catheters, 3
flow through, 5—7
material for, 3
polyurethane, 4
silicone, 4
size of, 4
French size, 4—5
gauge size, 4, 5t specialty devices, 13
hemodialysis catheters, 13, 13t, 14f introducer sheaths, 14
pulmonary artery catheters, 15
types of, 7
central venous catheters, 9-12
peripherally inserted central catheters, 12-13
peripheral vascular catheters, 8, 8f, 8t
Vascular catheters, indwelling, 41
catheter-related infections, 48-58
noninfectious complications, 43
catheter occlusion, 43-45
vascular perforation, 46-47
venous thrombosis, 45-46
routine care, 41, 42t catheter replacement, 42-43
flushing catheters, 43
site dressing, 41-42
Vascular endothelium, 369-370
Vascular impedance, 162-163
Vascular perforation, 46-47
Vascular resistance, 163
Vasopressin, 950-951
in diabetes insipidus, 662
for hyponatremia, 669-670
in hyponatremia, 669-670
Vasopressors. See also Adjunctive vasopressors
in cardiac arrest, 332
in septic shock, 271-272

VCV. See Volume control ventilation (VCV)
Vegetative state, arousal and awareness in, 800
Vena cava interruption, 114-116
Venous air embolism, central venous cannulation and, 32-33
Venous oximetry, 415-416, 416f
central venous O> saturation, 416-417
dual oximetry, 417
mixed venous O> saturation, 416
venous O> saturation, 416
Venous oxygen saturation, 183
central, 183-184
monitoring of, 183
Venous thromboembolism (VTE), 97-116
diagnostic evaluation, 105-110
clinical evaluation, 105-106, 106t
CT angiography, 108-109, 109f
pulmonary angiography, 110
radionuclide lung scan, 110
venous ultrasound, 106-108
prevalence of, 99f
risk factors for, 97-98, 98t
acute medical illness, 99
orthopedic procedures, 98
surgery, 98
trauma, 99
thromboprophylaxis, 100-105, 100t, 102t
low-molecular-weight heparin, 102-104
mechanical, 104-105
neuraxial analgesia, 104
unfractionated heparin, 100-101
treatment of, 110-116
embolectomy, 114
low-molecular-weight heparin, 112-113
thrombolytic therapy, 113-114, 113t
unfractionated heparin, 110-112, 111t
vena cava interruption, 114-116
warfarin, 113
Venous thrombosis, 97. See also Venous thromboembolism (VTE)
Venous ultrasound, 106-108
Ventilation, during CPR, 327-328
Ventilation-perfusion lung scans, for pulmonary embolism, 110
Ventilation—perfusion (V/Q) ratio, 391, 392f
Ventilator-associated pneumonia (VAP), 553-538
antimicrobial therapy for, 565-566
empiric, 566
basic information on, 553-554

chest radiography for, 556-559
sensitivity of, 557-559
specificity of, 556-557
clinical criteria, 556t, 559
clinical features, 555-559
diagnostic accuracy, 555-559
microbiological evaluation, 559-564
mortality rate associated with, 554
NHSN algorithm for diagnosis of, 560f
overview, 553
parapneumonic effusions, 565
pathogenic isolates in, 554t
preventive measures, 554-555
clearance of subglottic secretions, 555
oral decontamination, 554—555
routine airway care, 555
Ventilator breath, 505-508
pressure and flow changes during, 506f
pressure control, 507
volume control, 506—507
Ventilator-dependent patients, oral decontamination for, 88-91
Ventilator-induced lung injury (VILI), 454-455, 494
atelectrauma, 496-497
and lung protective ventilation, 497-498, 498t
volutrauma, 494, 496f
infiltrative lung diseases, 494
low-volume ventilation, 496
Ventilator settings, 516-517
assist-control ventilation, 516
I:E ratio, 517
inspiratory flow rate, 516
PEEP and, 517
respiratory rate, 517
tidal volume, 516
volume vs. pressure controlon, 516
Ventilator weaning, 421-422
Ventricular afterload, 159-166
components of, 160
peripheral vascular resistance, 163
pleural pressure, 160-162
vascular impedance, 162-163
definition of, 159-160
forces contributing to, 161f
Ventricular compliance, 155-156
Ventricular end-diastolic volume, 153
Ventricular function curves, 154, 154f

Ventricular septum, rupture of, 316
Ventricular tachycardia (VT), 295
clues for, 295-296
management in, 296-297, 297f
monomorphic, 295
polymorphic, 295, 297-299
versus SVT, 295, 295f
torsade de pointes, 297-299
Ventricular wall, rupture of, 316
Venturi masks, 435
Verapamil, in multifocal atrial tachycardia, 292
VILI. See Ventilator-induced lung injury (VILI)
Viscosity, 164-165
Viscous drag, 435
Vitamin C (ascorbic acid), 441
Vitamin E (alpha-tocopherol), 440-441
Vitamin E deficiency, 853
Vitamin requirements, 851-853
dietary allowances, 852t
clinical manifestations, 852
diagnosis, 853
laboratory evaluation, 853t
predisposing factors, 852
Vitamins, 878
Volume control ventilation (VCV), 506-507
advantages of, 506
disadvantages of, 506-507
inspiratory flow and, 507
Volume infusion, in NKH, 663
Volume resuscitation
in ACS, 644
in hvolemic hyprnatremia, 659
in myoglobinuric renal failure, 642-643
Volutrauma, 454, 494, 496, 522
Vomiting, opioids and, 907
VT. See Ventricular tachycardia (VT)
VTE. See Venous thromboembolism (VTE)

Warfarin, anticoagulation with, 113
Warfarin-induced hemorrhage and, 381-382, 382t
Warm shock, 268. See also Septic shock
Water replacement, in hvolemic hyprnatremia, 659-661
Water-seal bottle, 545-546, 546f
Wedge pressure, 137—141. See also Pulmonary artery wedge pressure
Weights, desirable, for adults, 1001
Wernicke's encephalopathy, in alcohol withdrawal, 804
Wide-QRS-complex tachycardias, 285
Wolff-Parkinson-White (WPW) syndrome, 291
Work of breathing and extubation, 579
in IMV, 510
Withdrawal syndrome, benzodiazepines and, 914
Y
Yersinia enterocolitica, 361
Z
Zero end-expiratory pressure (ZEEP), 491 Zoledronate, for ionized hypercalcemia, 708

MBBS, MS, PhD, FAMS
Department of Anatomy
G.R. Medical College
Gwalior, India
Edited by

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MBBS, MS, PhD, FIMSA, FIAMS, FAMS & Chikitsa Ratan
Ex. Prof. & Head, Deptt. of Anatomy,
Lady Hardinge Medical College, New Delhi
CBS PUBLISHERS & DISTRIBUTORS PVT. LTD.
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Reprint: 2010, 2011, 2012, 2013
First Edition: 1978
Second Edition: 1983
Third Edition: 1996
All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system without permission, in writing, from the author and the publisher.
Published by Satish Kumar Jain for

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| feel a sense of pride and enthusiasm in presenting to you the fourth edition of this popular book. Now, simple coloured diagrams extensively illustrate each chapter. Once initial interest to read text supplemented by diagrams is developed, learning general anatomy is hardly problematic.
Clinical anatomy has been illustrated with coloured diagrams. Students have always been encouraging me in improving both text and diagrams.
The help of Ms. Priya, MBBS student of Lady Hardinge Medical College during 1990-91, is being acknowledged for improving the "Anatomical word meanings and historical names."
Mr. Ajit Kumar, first year student of Banarasidas Chandiwala Institute of Physiotherapy (BCIP) 2004-05, gave constructive suggestions for its betterment. Ms. Stuti Malhotra, first year student of BCIP (2007-08), provided me with a number of tables in various chapters. I feel highly obliged to them.
The editor is obliged to Mr. Y.N. Arjuna, Publishing Director, CBS for timely and much needed guidance. Page layout and four colour diagrams work have been diligently done by Ms. Nishi Verma and Mr. Chand Singh Naagar of M/s. Limited Colors.
Mr. Vinod Jain, Production Director, and Mr. Satish Jain, Chairman, CBS Publishers and Distributors, have been helping me from time to time. Comments from the students are welcome.

This handbook of general anatomy has been written to meet the requirements of students who are newly admitted to medical colleges. It thoroughly introduces the greater part of medical terminology, as well as the various structures which constitute the human body. On account of the late admissions and the shorter time now available for teaching anatomy, the coverage of general anatomy seems to suffer maximum. Since it lays down the foundation of the entire subject of medicine, it was felt necessary to produce a short, simple and comprehensive handbook on this neglected, though important, aspect of the subject. It has been written in a simple language, with the text classified in small parts to make it easier for the students to follow and remember. It is hoped that this will prove quite useful to the medical students.
Gwalior
November 1978
B D CHAURASIA

Human anatomy is the science which deals with the structure of the human body. The term, 'anatomy', is derived from a Greek word, "anatome", meaning cutting up. The term 'dissection' is a Latin equivalent of the Greek anatome. However, the two words, anatomy and dissection, are not synonymous. Dissection is a mere technique, whereas anatomy is a wide field of study.
Anatomy forms firm foundation of the whole art of medicine and introduces the student to the greater part of medical terminology. "Anatomy is to physiology as geography is to history, i.e. it describes the theatre in which the action takes place."

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Initially, anatomy was studied mainly by dissection. But the scope of modern anatomy has become very wide because it is now studied by all possible techniques which can enlarge the boundaries of the anatomical knowledge.
The main subdivisions of anatomy are:
1. Cadaveric anatomy is studied on dead embalmed (preserved) bodies usually with the naked eye (macroscopic or gross anatomy). This can be done by one of the two approaches: (a) In 'regional anatomy' the body is studied in parts, like the upper limb, lower limb, thorax, abdomen, head and neck, and brain; (b) in 'systemic anatomy' the body is studied in systems, like the skeletal system (osteology) (Fig. 1.1), muscular system (myology), articulatory system (arthrology or syndesmology), vascular system (angiology), nervous system (neurology), and respiratory, digestive, urogenital and endocrine systems (splanchnology). The locomotor system inslidac ctan no: arhen newanec uvalacy
ll
Fig. 1.1: Skeletal system
2. Living Anatomy is studied by inspection, palpation (Fig. 1.2), percussion, auscultation, endoscopy (bronchoscopy, gastroscopy), radiography, electromyography, etc.
Fig. 1.2: Contracted muscles for palpation
3. Embryology (developmental anatomy) is the study of the prenatal developmental changes in an individual. The developmental history is called 'ontogeny'. The evolutionary history on the other hand, is called 'phylogeny'.
4. Histology (microscopic anatomy) is the study of structures with the aid of a microscope.
5. Surface anatomy (topographic anatomy) is the study of deeper parts of the body in relation to the skin surface. It is helpful in clinical practice and surgical operations (Fig. 1.3).
6. Radiographic and imaging anatomy is the study of the bones and deeper organs by plain and contrast radiography by ultrasound and computerised tomographic (CT) scans (Fig. 1.4).
Fig. 1.3: Palpating the dorsalis pedis artery
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Fig. 1.4: X-ray of chest (plain radiograph)

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7. Comparative anatomy is the study of anatomy of the other animals to explain the changes in form, structure and function (morphology) of different parts of the human body.
8. Physical anthropology deals with the external features and measurements of different races and groups of people, and with the study of the prehistoric remains.
9. Applied anatomy (clinical anatomy) deals with application of the anatomical knowledge to the medical and surgical practice (Fig. 1.6).
10. Experimental anatomy is the study of the factors which influence and determine the form, structure and function of different parts of the body.
. Genetics deals with the study of information present in the chromosomes. —_ —
Fig. 1.5: Physical anthropology
Fig. 1.6: The relation of nerves to the humerus and likelihood of their injury in case of fracture

Hippocrates of Cos (circa 400 B.C.), the 'Father of Medicine', is regarded as one of the founders of anatomy. Parts of Hippocratic collection are the earliest anatomical descriptions.
Herophilus of Chalcedon (circa 300 B.C.) is called the "father of anatomy". He was a Greek physician, and was one of the first to dissect the human body. He distinguished cerebrum from cerebellum, nerves from tendons, arteries from veins, and the motor from sensory nerves. He described and named the parts of eye, meninges, torcular Herophili, fourth ventricle with calamus scriptorius, hyoid bone, duodenum, prostate gland, etc. We owe to him the first description of the lacteals. Herophilus was a very successful teacher, and wrote a book on anatomy, A special treatise of the eyes, and a popular handbook for midwives.
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5

Galen of Pergamum, Asia Minor (circa 130-200 A.D.), the "prince of physicians", practised medicine at Rome. He was the foremost' practitioner of his days and the first experimental physiologist. He wrote voluminously and theorized and dogmatized on many medical subjects like anatomy, physiology, pathology, symptomatology and treatment. He demonstrated and wrote on anatomy De anatomicisadministrationibus. His teachings were followed and considered as the infallible authority on the subject for nearly 15 centuries.

Mundinus or Mondino d'Luzzi (1276-1326), the 'restorer of anatomy', was an Italian anatomist and professor of anatomy at Bologna. He wrote a book Anathomia which was the standard anatomical text for over a century. He taught anatomy by dissection for which his text was used as a guide. He was the most renowned anatomist before Vesalius.

Leonardo da Vinci of Italy (1452-1519), the originator of cross-sectional anatomy, was one of the greatest geniuses the world has known. He was a master of arts and contributed substantially in mathematics, science and engineering. He was the first to describe the moderator band of the right ventricle. The most admirable of his works are the drawings of the things he observed with perfection and fidelity. His 60 notebooks containing 500 diagrams were published in 1898.

Vesalius (1514-1564), the 'reformer of anatomy', was German in origin, Belgian (Brussels) by birth, and found an Italian (Padua) university favourable for his work. He was professor of anatomy at Padua. He is regarded as the founder of modern anatomy because he taught that anatomy could be learnt only through dissections. He opposed and corrected the erroneous concepts of Galen and fought against his authority, thus reviving anatomy after a deadlock of about 15 centuries. His great anatomical treatise De Febricia Humani Corporis, written
7
in seven volumes, revolutionized the teaching of anatomy and remained as authoritative text for two centuries.
Vesalius studied first at Louvain and then at Paris under Gunther and Sylvius. Eustachius was the rival of Vesalius. The followers of Vesalius included Servetus, Columbus, Fallopius, Varolio, Vidius, etc.; all of them lived during 16th century.

William Harvey (1578-1657) was an English physician who discovered the circulation of blood, and published it as Anatomical Exercise on the Motion of the Heart and Blood in Animals. He also published a book on embryology.
The other events of this century included: (a) the first recorded human dissection in 1638 in Massachusetts; (b) foundation of microscopic anatomy by Malpighi; and (c) introduction of alcohol as a preservative.

William Hunter (1718-1783) was a London anatomist and obstetrician. He introduced the present day embalming with the help of Harvey's discovery, and founded with his younger brother (John Hunter) the famous Hunterian museum.

Dissection by medical students was made compulsory in Edinburgh (1826) and Maryland (1833). Burke and Hare scandal of 16 murders took place in Edinburgh in 1828. Warburton Anatomy Act (1932) was passed in England under which the unclaimed bodies were made available for dissection. The 'Act' was passed in America (Massachusetts) in 1831. Formalin was used as a fixative in 1890s.
X-rays were discovered by Roentgen in 1895. Various endoscopes were devised between 1819 and 1899. The anatomical societies were founded in Germany (1886), Britain (1887) and America (1888).
The noted anatomists of this century include Ashley Cooper (1768-1841; British surgeon), Cuvier (1769-1832; French naturalist), Meckel (1724-1774; German anatomist), and Henry Gray (1827-1861; the author of Gray's Anatomy).

The electron microscope was invented in 20th century. It was applied in clinical practice, which made startling changes in the study of normal and diseased conditions. Various modifications of electron microscope, transmission EM and SEM, etc. were devised. These helped in better understanding of the body tissues.
Besides plain X-rays, in this century, ultrasonography and echocardiography were discovered. This was the non-invasive safe-procedure.
Also computer-axial tomography or CT scan, a non-invasive procedure and magnetic resonance imaging were devised. These were extremely useful, sensitive means of understanding the dynamics of body structure in health and disease.
Tissue culture was developed which was new and exciting field of research.
New advances in cases of infertility were discovered, which gave hopes to some infertile couples. GIFT: Gamete Intrafallopian Transfer got started

Foetal medicine is emerging as a newer subject. Even treatment
'in-utero' is being practised in some cases.
Human genome is being prepared.
New research in drugs for many diseases, especially AIDS, is being done very enthusiastically. There is also a strong possibility of gene therapy.

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Dr. Inderjit Dewan worked chiefly on osteology and anthropology.
Dr. D.S. Choudhry did notable work on carotid body.
Dr. H. Chaterjee and Dr. H. Verma researched on embryology.
Dr. S.S. Dayal did good work in cancer biology.
Dr. Shamer Singh and his team did pioneering work on teratology.
Chaturvedi and Dr. C.D. work was on
Dr. Gupta's prominent corrosion cast.

Galen (2nd century) wrote his book in Greek and Vesalius (16th century) did it in Latin. Most of the anatomical terms, therefore, are either in Greek or Latin. By 19th century about 30,000 anatomical terms were in use in the books and journals. In 1895, the German Anatomical Society held a meeting in Basle, and approved a list of about 5000 terms known as Basle Nomina Anatomica (BNA). The following six rules were laid down to be followed strictly: (1) Each part shall have only one name; (2) each term shall be in Latin; (3) each term shall be as short and simple as possible; (4) the terms shall be merely memory signs; (5) the related terms shall be similar, e.g. femoral artery, femoral vein, and femoral nerve; and (6) the adjectives shall be arranged as opposites, e.g. major and minor, superior and inferior.
BNA was revised in 1933 by a committee of the Anatomical Society of Great Britain and Ireland in a meeting held at Birmingham. The revised BNA was named as Birmingham Revision (BR). An independent revision of the BNA was also done by German anatomists in 1935, and was known as Jena Nomina Anatomica (JNA or INA). However, the BR and JNA found only local and restricted acceptance.
In 1950, it was agreed at an International Congress of Anatomists held at Oxford that a further attempt should be made to establish a generally acceptable international nomenclature. In the Sixth International Congress of Anatomists held at Paris (1955), a somewhat conservative revision of BNA with many terms from BR and INA was approved. Minor revisions and corrections were made at the International Congresses held in New York (1960), and Wiesbaden, Germany (1965), and the 3rd edition of Nomina Anatomica (Ed. GA.G. Mitchell, 1968) was published by the Excerpta Medica Foundation.

The drafts on Nomina Histologica and Nomina Embryologica prepared by the subcommittee of the International Anatomical Nomenclature Committee (IANC) were approved in a plenary session of the Eleventh International Congress of Anatomists held in Leningrad in 1970. After a critical revision, the 4th edition of Nomina'Anatomica (Ed. Roger Warwick, 1977) containing Nomina Histologica and Nomina Embryologica was published by the same publisher.

Various positions, planes, terms in relation to various regions and movements are described.

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» Anatomical position: When a person is standing straight with eyes looking forwards, both arms by the side of body, palms facing forwards, both feet together, the position is anatomical position (Fig. 1.7).
» Supine position: When a person is lying on her/his back, arms by the side, palms facing upwards and feet put together, the position is supine position (Fig. 1.8).
* Prone position: Person lying on his/her face, chest and abdomen is said to be in prone position (Fig. 1.9).
Fig. 1.7: Anatomical position
Fig. 1.9: Prone position
+ Lithotomy position: Person lying on her back with legs up and feet supported in straps. This position is mostly used during delivery of the baby (Fig. 1.10).
Fig. 1.10: Lithotomy position

« A plane passing through the centre of the body dividing it into two equal right and left halves, is the median or midsagittal plane (Fig.1.11). Plane parallel to median or midsagittal plane is the sagittal plane.
Fig. 1.11: Planes of the body
-A plane at right angles to sagittal or median plane which divides the body into anterior and posterior halves is called a coronal plane (Fig. 1.12).
-A plane at right angles to both sagittal and coronal planes which divides the body into upper and lower parts is called a transverse plane (Fig. 1.12).
Fig. 1.12: Planes of the body in a child

° Ventral or Anterior is the front of trunk.
° Dorsal or Posterior is the back of trunk (Fig. 1.13).
° Medial is a plane close to the median plane (Fig. 1.13).
° Lateral is plane away from the median plane.
° Proximal/Cranial/Superior is close to the head end of trunk

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° Distal/Caudal/Inferior is close to the lower end of the trunk.
Superficial is close to skin/towards surface of body (Fig. 1.15).
* Deep away from skin/away from surface of body.
Ipsilateral on the same side of the body as another structure (Fig. 1.13).
* Contralateral on opposite side of body from another structure.
» Invagination is projection inside.
» Evagination is projection outside (Fig. 1.16).
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Fig. 1.14: Language of anatomy
Fig. 1.15: Language of anatomy
Fig. 1.16: Language of anatomy

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° Ventral or Anterior is the front aspect (Fig. 1.13).
* Dorsal or Posterior is the back aspect.
* Medial border lies along the little finger, medial border of forearm and arm.
° Lateral border follows the thumb, lateral border of forearm and arm (Fig. 1.14).
* Proximal is close to root of limb, while distal is away from the root.
* Palmar aspect is the front of the palm (Fig. 1.14).
* Dorsal aspect of hand is on the back of palm.
* Flexor aspect is front of upper limb.
° Extensor aspect is back of upper limb.

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* Posterior aspect is the back of lower limb.
» Anterior aspect is front of lower limb.
» Medial border lies along the big toe or hallux, medial border of leg and thigh (Fig. 1.13).
16 | Handbook of General Anatomy
« Lateral border lies along the little toe, lateral border of leg and thigh.
¢ Flexor aspect is back of lower limb.
* Extensor aspect is front of lower limb (Fig. 1.13).
+ Proximal is close to the root of limb, while distal is away from it.

(a) Ventral — Towards the belly (like anterior).
(b) Dorsal — Towards.the back (like posterior).
(c) Cranial or Rostral — Towards the head (like superior).
(d) Caudal — Towards the tail.

Movements in general at synovial joints are divided into four main categories.
1. Gliding movement: Relatively flat surfaces move back-andforth and from side-to-side with respect to one another. The angle between articulating bones does not change significantly.
2. Angular movements: Angle between articulating bones decreases or increases. In flexion there is decrease in angle between articulating bones and in extension there is increase in angle between articulating bones. Lateral flexion is movement of trunk sideways to the right or left at the waist. Adduction is movement of bone toward midline whereas abduction is movement of bone away from midline.
3. Rotation: A bone revolves around its own longitudinal axis. In medial rotation anterior surface of a bone of limb is turned towards the midline. In lateral rotation anterior surface of bone of limb is turned away from midline.
4. Special movements: These occur only at certain joints, e.g. pronation, supination at radioulnar joints, protraction and retraction at temporo-mandibular joint.

* Flexion: When two flexor surfaces are brought close to each other, e.g. in elbow joint when front of arm and forearm are opposed to each other [Fig. 1.17 (i-v)].
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»
Extension of thigh: When person stands erect.
» Abduction: When thigh is taken away from the median plane.
» Adduction: When thigh is brought close to median plane.
» Medial rotation: When thigh is turned medially.
» Lateral rotation: When thigh is turned laterally (Fig. 1.18).
*
Flexion of
knee:
When back
of thigh
and back
of leg
come opposition.
contact with
front in
Fig. 1.17: Terms related to movements of upper limb

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° Flexion: When face comes closer to chest.
» Extension: When face is brought away from the chest.
Fig. 1.18: Terms related to movements of lower limb
»
Lateral flexion: When ear is brought close to shoulder.
» Rotation: When neck rotates so that chin goes to opposite side.
»
Opening the mouth: When lower jaw is lowered to open the mouth.
* Closure of the mouth: When lower jaw is opposed jaw, closing the mouth. to the upper
* Protraction: When lower jaw slides forwards in its socket in the
temporal bone of skull.
»
Retraction:
When lower
jaw slides
temporal bone of skull (Fig. 1.19).
backwards in
its socket
in the
ry
Fig. 1.19: Retraction and protraction at temporo-mandibular joints

» Backward bending is called extension.
» Forward bending is flexion (Fig. 1.20).
° Sideward movement is lateral flexion.
Sideward rotation is lateral rotation.
Fig. 1.20: Movements of the trunk: (a) extension, (b) flexion, (c) lateral flexion

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(a) Origin: The end of a muscle which is relatively fixed during its contraction (Fig. 1.21).
(b) Insertion: The end of a muscle which moves during its contraction.
Fig. 1.21: Terms used for describing muscles
The two terms, origin and insertion, are sometimes interchangeable, when the origin moves and the insertion is fixed.
(c) Belly: The fleshy and contractile part of a muscle.
(d) Tendon: The fibrous noncontractile and cord-like part of a muscle.
(e) Aponeurosis: The flattened tendon.
(f) Raphe: A fibrous band made up of interdigitating fibres of the tendons or aponeuroses. Unlike a ligament, it is stretchable. Ligaments are fibrous, inelastic bands which connect two segments of a joint.

(a) Arteries carry oxygenated blood away from the heart, with the exception of the pulmonary and umbilical arteries which carry deoxygenated blood. Arteries resemble trees because they have branches (arterioles) (Fig. 1.22).
Fig. 1.22: Artery, capillaries and vein
(b) Veins carry deoxygenated blood towards the heart, with the exception of the pulmonary and umbilical veins which carry oxygenated blood. Veins resemble rivers because they have tributaries (venules). Veins have valves to allow unidirectional flow of blood.
(c) Capillaries are networks of microscopic vessels connecting arterioles to venules.
(d) Anastomosis is a precapillary or postcapillary communication between the neighbouring vessels (Fig. 1.23).

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1. Linear elevation may be a line, lip, ridge, or crest.
2. Sharp elevation may be a spine, styloid process, cornu (horn), or hamulus (Fig. 1.24).
3. Rounded or irregular elevation may be a tubercle, tuberosity, epicondyle, malleolus, or trochanter. A ramus is a broad arm or process projecting from the main part or body of the bone (Figs 1.25—1.27).
(b) Depressions may be a pit, impression, fovea, fossa, groove (sulcus), or notch (incisura).
(c) Openings may be a foramen, canal, hiatus, or aqueduct.
Fig. 1.23: Anastomoses around elbow joint
Fig. 1.24: Norma basalis
Fig. 1.25: Norma frontalis
Fig. 1.26: Right scapula and humerus (anterior view)
Fig. 1.27 Femur—anterior surface (a) and posterior surface (b)
(d) Cavities: A large cavity within a bone is called sinus, cell or antrum.
(e) Smooth articular areas may be a facet, condyle, head, capitulum, or trochlea.

1. The suffix, '-itis', means inflammation, e.g. appendicitis, tonsillitis, arthritis, neuritis, dermatitis, etc.
2. The suffix, '-ectomy', means removal from the body, e.g. appendicectomy, tonsillectomy, gastrectomy, nephrectomy, etc.
3. The suffix, '-otomy', means to open and then close a hollow organ, e.g. laparotomy, hysterotomy, cystotomy, cystolithotomy, etc.

end in necrosis.
, 1 = Introduction ! 27. 23., 1 = Gangrene: A form of necrosis (death) combined with putrefaction.. 24., 1 = Infarction: Death (necrosis) of a tissue due to sudden obstruction of its artery of supply (often an end-artery).. 25., 1 = Atrophy: Diminution in the size of cells, tissue, organ, or a part due to loss of its nutrition.. 26., 1 = Dystrophy: Diminution in the size due to defective nutrition.. 27., 1 = Hypertrophy: Increase in the size without any increase in the number of cells.. 28., 1 = Hyperplasia: Increase in the size due to increase in the number of cells.. 29., 1 = Hypoplasia: Incomplete development.. 30., 1 = Aplasia: Failure of development.. 31 ., 1 = Syndrome: Agroup of diverse symptoms and signs constituting together the picture of a disease.. 32., 1 = Paralysis: Loss of motor power (movement) of a part of body due to denervation or primary disease of the muscles.. 33., 1 = Hemiplegia: Paralysis of one-half of the body.. 34., 1 = Paraplegia: Paralysis of both the lower limbs.. 35. 36., 1 = Monoplegia: Paralysis of any one limb. Quadriplegia: Paralysis of all the four limbs.. 37., 1 = Anaesthesia: Loss of the touch sensibility.. 38., 1 = Analgesia: Loss of the pain sensibility.. 39., 1 = Thermanaesthesia: Loss of the temperature sensibility.. 40., 1 = Hyperaesthesia: Abnormally increased sensibility.. 41. 42., 1 = Paraesthesia: Perverted feeling of sensations.. , 1 = Coma: Deep unconsciousness. Tumour (neoplasm): A circumscribed,. 43., 1 = noninflammatory, abnormal growth arising from the body tissues.. , 1 = Benign: Mild illness or growth which does not endanger life.. 44., 1 = Severe form of illness or growth, which is. 45., 1 = Malignant: resistant to treatment and ends in death.. 47., 1 = Sarcoma: Malignant growth arising from connective tissue (mesoderm).

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48. Cancer: A general term used to indicate any malignant neoplasm which shows invasiveness and results in death of the patient.
49. Metastasis: Spread of a local disease (like the cancer cells) to distant parts of the body.
50. Convalescence: The recovery period between the end of a disease and restoration to complete health.
51. Therapy: Medical treatment.

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1. Bones form the supporting framework of the body.
2. Muscles are attached to bones.
3. Blood vessels, nerves and lymphatics form neurovascular bundles which course in between the muscles, along the fascial planes.
4. The thoracic and abdominal cavities contain several internal organs called viscera.
5. The whole body has three general coverings, namely (a) skin; (b) superficial fascia; and (c) deep fascia.
Skeleton includes bones and cartilages. It forms the main supporting framework of the body, and is primarily designed for a more effective production of movements by the attached muscles.

1. Os (L),
2. Osteon (G).
Compare with the terms, osteology, ossification, osteomyelitis, osteomalacia, osteoma, osteotomy, etc.

Bone is one-third connective tissue. It is impregnated with calcium salts which constitute two-thirds part. The inorganic calcium salts (mainly calcium phosphate, partly calcium carbonate, and traces of other salts) make it hard and rigid, which can afford resistance to compressive forces of weight-bearing and impact forces of jumping. The organic connective tissue (collagen fibres) makes it tough and resilient (flexible), which can afford resistance to tensile forces. In strength, bone is comparable to iron and steel.
Despite its hardness and high calcium content the bone is very much a living tissue. It is highly vascular, with a constant turn-over of its calcium content. It shows a characteristic pattern of growth. It is subjected to disease and heals after a fracture. It has greater regenerative power than any other tissue of the body, except blood. It can mould itself according to changes in stress and strain it bears. It shows disuse atrophy and overuse hypertrophy.

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1. Bones give shape and support to the body, and resist any forms of stress (Fig. 2.1).
2. These provide surface for the attachment of muscles, tendons, ligaments, etc.
3. These serve as levers for muscular actions.
4. The skull, vertebral column and thoracic cage protect brain, spinal cord and thoracic viscera, respectively.
5. Bone marrow manufactures blood cells.
6. Bones store 97% of the body calcium and phosphorus.
7. Bone marrow contains reticulo-endothelial cells which are phagocytic in nature and take part in immune responses of the body.
8. The larger paranasal air sinuses affect the timber of the voice.
Fig. 2.1: Human skeleton: (a) Anterior view, (b) Posterior view

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1. Long bones: Each long bone has an elongated shaft (diaphysis) and two expanded ends (epiphyses) which are smooth and articular. The shaft typically has 3 surfaces separated by 3 borders, acentral medullary cavity, and a nutrient foramen directed away from the growing end. Examples:
(a) typical long bones like humerus, radius, ulna, femur, tibia and fibula;
(b) miniature long bones have only one epiphysis like metacarpals, metatarsals and phalanges; and
(c) modified long bones have no medullary cavity like clavicle (Fig. 2.2).
2. Short bones: Their shape is usually cuboid, cuneiform, trapezoid, or scaphoid. Examples: tarsal and carpal bones (Fig. 2.3).
3. Flat bones resemble shallow plates and form boundaries of certain body cavities. Examples: bones in the vault of the skull, ribs, sternum and scapula (Fig. 2.4).
4. Irregular bones: Examples: vertebra, hip bone, and bones in the base of the skull (Fig. 2.5).
5. Pneumatic bones: Certain irregular bones contain large air spaces lined by epithelium Examples: maxilla, sphenoid, ethmoid, etc. They make the skull light in weight, help in resonance of voice, and act as air conditioning chambers for the inspired air (Fig. 2.6).
6. Sesamoid bones: These are bony nodules found embedded in the tendons or joint capsules. They have no periosteum and ossify after birth. They are related to an articular or nonarticular bony surface, and the surfaces of contact are covered with hyaline cartilage and lubricated by a bursa or synovial membrane. Examples: patella, pisiform, fabella, etc. (Fig. 2.7).
Functions of the sesamoid bones are:
(a) to resist pressure;
(b) to minimise friction;
(c) to alter the direction of pull of the muscle; and
(d) to maintain the local circulation.
lT

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Fig. 2.2: Long bones: (a) Humerus, (b) Metacarpals, (c) Clavicle
Fig. 2.4: Flat bones: (a) Rib, (b) Scapula
Fig. 2.6: Pneumatic bone: Ethmoid
Fig. 2.7: Sesamoid bone: Patella
7. Accessory (supernumerary) bones are not always present. These may occur as ununited epiphyses developed from extra centres of ossification. Examples: sutural bones, os trigonum (lateral tubercle of talus), os vesalianum (tuberosity of Sth metatarsal), etc. In medicolegal practice, accessory bones may be mistaken for fractures. However, these are often bilateral, and have smooth surfaces without any callus.
8. Heterotopic bones: Bones sometimes develop in soft tissues. Horse riders develop bones in adductor muscles (rider's bones).

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1.° Membrane (dermal) bones ossify in membrane (intramembranous or mesenchymal ossification), and are thus derived from mesenchymal condensations. Examples: bones of the vault of skull and facial bones.
° Cartilaginous bones ossify in cartilage (intracartilaginous or endochondral ossification), and are thus derived from preformed cartilaginous models. Examples: bones of limbs, vertebral column and thoracic cage.
¢ Membrano-cartilaginous bones ossify partly in membrane and partly in cartilage. Examples: clavicle, mandible, occipital, temporal, sphenoid.
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2. ° Somatic bones: Most of the bones are somatic.
e Visceral bones: These develop from pharyngeal arches. Examples are hyoid bones, part of mandible and ear ossicles.

1. Axial skeleton includes skull, vertebral column, and thoracic cage.
2. Appendicular skeleton includes bones of the limbs.

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I. Macroscopically, the architecture of bone may be compact or cancellous (Fig. 2.8).
1. Compact bone is dense in texture like ivory, but is extremely porous. It is best developed in the cortex of the long bones. This is an adaptation to bending and twisting forces (a combination of compression, tension and shear).
2. Cancellous or spongy, or trabecular bone is open in texture, and is made up of a meshwork of trabeculae (rods and plates) between which are marrow containing spaces. The trabecular meshworks are of three primary types, namely:
(a) meshwork of rods,
Fig. 2.8: Structural components of a bone

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(b) meshwork of rods and plates, and
(c) meshwork of plates (Singh, 1978).
Cancellous bone is an adaptation to compressive forces.
Bones are marvellously constructed to combine strength, elasticity and lightness in weight. Though the architecture of bone may be modified by mechanical forces, the form of the bone is primarily determined by heredity.
According to Wolff's law (Trajectory Theory of Wolff, 1892), the bone formation is directly proportional to stress and strain. There are two forces, tensile force and compressive force. Both the tensile and compressive forces can stimulate bone formation in proper conditions.
The architecture of cancellous bone is often interpreted in terms of the trajectorial theory. Thus the arrangement of bony trabeculae (lamellae) is governed by the lines of maximal internal stress in the bone. Pressure lamellae are arranged parallel to the line of weight transmission, whereas tension lamellae are arranged at right angles to pressure lamellae. The compact arrangement of pressure lamellae forms bony buttress, for additional support, like calcar femorale (Fig. 2.9).
Fig. 2.9: Diagrammatic representation of the compression (continuous lines) and tension (interrupted lines) lamellae in a sagittal section of the upper end femur.
oe
.
.
II. Microscopically, the bone is of five types, namely lamellar (including both compact and cancellous), woven, fibrous, dentine and cement.
is Lamellar bone: Most of the mature human bones, whether compact or cancellous, are composed of thin plates of bony tissue called lamellae. These are arranged in piles in a cancellous bone, but in concentric cylinders (Haversian system or secondary osteon) in a compact bone.
. Woven Bone: seen in fetal bone, fracture repair and in cancer of bone
. Fibrous bone is found in young foetal bones, but are common in reptiles and amphibia.
4. Dentine and
. Cement occur in teeth.
Compact bone ar SN
Cancellous (spongy) bone a ee
La
eg
See
ined
Location
In shaft (diaphysis) of long

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_In the epiphyses of long bone
bone
Lamellae
Arranged to form Haversian system
Arranged
in
a
meshwork,
so
Haversion
systems
are
not
present
Yellow
which
stores
fat
after
Red, produce RBCs, granular series of WBC and platelets
puberty.
It
is
red
before
puberty
Nature
Hard and ivory like
Spongy
Table 2.1: Comparison of compact and cancellous bones

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Naked eye examination of the longitudinal and transverse sections of a long bone shows the following features.
1. Shaft: From without inwards, it is composed of periosteum, cortex and medullary cavity (Fig. 2.10).
(a) Periosteum is a thick fibrous membrane covering the surface of the bone. It is made up of an outer fibrous layer, and an inner cellular layer which is osteogenic in nature. Periosteum is united to the underlying bone by Sharpey's fibres, and the
union is particularly strong over the attachments of tendons, and ligaments. At the articular margin the periosteum is continuous with the capsule of the joint. The abundant periosteal arteries nourish the outer part of the underlying cortex also. Periosteum has a rich nerve supply which makes it the most sensitive part of the bone.
(b) Cortex is made up of a compact bone which gives it the desired strength to withstand all possible mechanical strains.
(c) Medullary cavity is filled with red or yellow bone marrow. At birth the marrow is red everywhere with widespread active haemopoiesis. As the age advances, the red marrow at many places atrophies and is replaced by yellow, fatty marrow, with no power of haemopoiesis. Red marrow persists in the cancellous ends of long bones. In the sternum ribs, iliac crest, vertebrae and skull bones the red marrow is found throughout life.
2. The two ends of a long bone are made up of cancellous bone covered with hyaline (articular) cartilage (Fig. 2.10).
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A typical long bone ossifies in three parts, the two ends from secondary centres, and the intervening shaft from a primary centre (Fig. 2.11). Before ossification is complete the following parts of the bone can be defined.
Fig. 2.11: Parts of a young long bone.

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The ends and tips of a bone which ossify from secondary centres are called epiphyses. These are of the following types.
(a) Pressure epiphysis is articular and takes part in transmission of the weight. Examples: head of femur; lower end of radius, etc. (Fig. 2.12)
(b) Traction epiphysis is nonarticular and does not take part in the transmission of the weight. It always provides attachment to one or more tendons which exert a traction on the epiphysis. The traction epiphyses ossify later than the pressure epiphyses. Examples: trochanters of femur and tubercles of humerus (Figs 1.26 and 1.27).
(c) Atavistic epiphysis is phylogenetically an independent bone which in man becomes fused to another bone.
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Fig. 2.12: Types of epiphyses
Examples: coracoid process of scapula and os trigonum or lateral tubercle of talus.
(d) Aberrant epiphysis is not always present. Examples: epiphysis at the head of the first metacarpal and at the base of other metacarpal bones.

It is the elongated shaft of a long bone which ossifies from a primary centre (Fig. 2.11).

The epiphysial ends of a diaphysis are called metaphyses.
Each metaphysis is the zone of active growth. Before epiphysial fusion, the metaphysis is richly supplied with blood through end arteries forming 'hair-pin' bends.
This is the common site of osteomyelitis in children because the bacteria or emboli are easily trapped in the hair-pin bends, causing infarction.
After the epiphysial fusion, vascular communications are established between the metaphysial and epiphysial arteries. Now the metaphysis contains no more end-arteries and is no longer subjected to osteomyelitis.

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It separates epiphysis from metaphysis.
Proliferation of cells in this is responsible for cartilaginous plate lengthwise growth of a long bone.
After the epiphysial fusion, the bone can no longer grow in length.
The growth cartilage is nourished by both the epiphysial and arteries.
metaphysial

blood supply of a long bone is derived from the following sources
The (Fig. 2.13).

* Itenters the shaft through the nutrient foramen, runs obliquely through the cortex, and divides into ascending and descending branches in the medullary cavity.

Each branch divides into a number of small parallel channels which terminate in the adult metaphysis by anastomosing with the epiphysial, metaphysial and periosteal arteries.
- » The nutrient artery supplies medullary cavity, inner 2/3 of cortex and metaphysis.
°
The nutrient foramen is directed away from the growing end of the bone; their directions are indicated by a jingle, 'To the elbow I go, from the knee I flee' (Fig. 2.14).

The details and variations in the diaphysial nutrient foramina are described by a number of authors (Ujwal, 1962; Mysorekar, 1967; Chhatrapati and Misra, 1967; Kate, 1971; Patake and Mysorekar, 1977; Mysorekar and Nandedkar, 1979; Longia et al, 1980).
* The growing ends of bones in upper limb are upper end of humerus and lower ends of radius and ulna. In lower limb, the lower end of femur and upper end of tibia are the growing ends.

*
These are especially numerous beneath the muscular and ligamentous attachments.
* They ramify beneath the periosteum and enter the Volkmann's canals to supply the outer 1/3 of the cortex.

° These are derived from periarticular vascular arcades (circulus vasculosus) found on the nonarticular bony surface.
* Out of the numerous vascular foramina in this region, only a few admit the arteries (epiphysial and metaphysial), and the rest are venous exits.
* The number and size of these foramina may give an idea of the relative vascularity of the two ends of a long bone (Tandon, 1964).

* These are derived from the neighbouring systemic vessels.
¢ They pass directly into the metaphysis and reinforce the metaphysial branches from the primary nutrient artery.
In miniature long bones, the infection begins in the middle of the shaft rather than at the metaphysis because, the nutrient artery breaks up into a plexus immediately upon reaching the medullary cavity. In the adults, however, the chances of infection are minimized because the nutrient artery is mostly replaced by the periosteal vessels.

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Short bones are supplied by numerous periosteal vessels which enter their nonarticular surfaces.
Ina vertebra, the body is supplied by anterior and posterior vessels; and the vertebral arch by large vessels entering the bases of transverse processes. Its marrow is drained by two large basivertebral veins.
A rib is supplied by : (a) the nutrient artery which enters it just beyond the tubercle; and (b) the periosteal arteries.
Veins are numerous and large in the cancellous, red marrow containing bones (e.g., basivertebral veins). In the compact bone, they accompany arteries in the Volkmann's canals.
Lymphatics have not been demonstrated within the bone, although some of them do accompany the periosteal blood vessels, which drain to the regional lymph nodes.

Nerves accompany the blood vessels. Most of them are sympathetic and vasomotor in function.

A few of them are sensory which are distributed to the articular ends and periosteum of the long bones, to the vertebra, and to large flat bones.

Bones are first laid down as mesodermal (connective tissue) condensations. Conversion of mesodermal models into bone is called intramembranous or mesenchymal ossification, and the bones are called membrane (dermal) bones.
However, mesodermal stage may pass through cartilaginous stage by chondrification during 2nd month of intrauterine life. Conversion of cartilaginous model into bone is called intracartilaginous or endochondral ossification, and such bones are called cartilaginous bones (Fig. 2.15).
Ossification takes place by centres of ossification, each one of which is a point where laying down of lamellae (bone formation) starts by the osteoblasts situated on the newly formed capillary loops. The centres of ossification may be primary or secondary. The primary centres appear before birth, usually during 8th week of intrauterine life; the secondary centres appear after birth, with a few exceptions of lower end of femur and upper end of tibia. Many secondary centres appear during puberty.
A primary centre forms diaphysis, and the secondary centres form epiphyses. Fusion of epiphyses with the diaphysis starts at puberty and is complete by the age of 25 years, after which no more bone growth can take place. The Jaw of ossification states that secondary centres of ossification which appear first are last to unite. The end ofa long bone where epiphysial fusion is delayed is called the growing end of the bone.

1. Bone grows in length by multiplication of cells in the epiphysial plate of cartilage (Fig. 2.15).
2. Bone grows in thickness by multiplication of cells in the deeper layer of periosteum.
3. Bones grow by deposition of new bone on the surface and at the ends. This process of bone deposition by osteoblasts is called appositional growth or surface accretion. However, in order to maintain the shape the unwanted bone must be removed. This

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When a skeleton or isolated bones are received for medicolegal examination, one should be able to determine:
(a) whether the bones are human or not;
(b) whether they belong to one or more persons;
(c) the age of the individual;
(d) the sex;
(e) the stature; and
(f) the time and cause of death.
For excellent details of all these points consult Modi (1977).

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Up to the age of 25 years, the skeletal age can be estimated to within
1-2 years of correct age by the states of dentition and ossification, provided the whole skeleton is available.
From 25 years onwards, the skeletal age can be estimated to within
+ 5 years of the correct age by the state of cranial sutures and of the bony surfaces of symphysis pubis.
In general, the appearance of secondary centres and fusion of epiphyses occur about one year earlier in females than in males.
These events are also believed to occur 1-2 years (Bajaj et al, 1967) or 2-3 years (Pillai, 1936) earlier in India than in Western countries.
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However, Jit and Singh (1971) did not find any difference between the eastern and western races.

Sex can be determined after the age of puberty.
Sexual differences are best marked in the pelvis and skull, and accurate determination of sex can be done in over 90% cases with either pelvis or skull alone.
However, sexual dimorphism has been worked out in a number of other bones, like sternum (Jit et al, 1980), atlas (Halim and Siddiqui, 1976), and most of the limb bones.

It is a common experience that trunk and limbs show characteristic ratios among themselves and in comparison with total height.
Thus a number of regression formulae have been worked out to determine height from the length of the individual limb bones (Siddiqui and Shah, 1944; Singh and Sohal, 1952; Jit and Singh, 1956; Athawale, 1963; Kolte and Bansal, 1974; Kate and Majumdar, 1976).
Height can also be determined from parts of certain long bones (Mysorekar et al), from head length (Saxena et al, 1981), and from foot measurements (Charnalia, 1961; Qamra et al, 1980).
CR length has been correlated with diaphysial length of foetal bones (Vare and Bansal, 1977) and with the neonatal and placental parameters (Jeyasingh et al, 1980; Saxena et al, 1981).

It is of interest to anthropologists. A number of metrical (like cranial and facial indices) and non metrical features of the skull, pelvis, and certain other bones are of racial significance (Krogman, 1962; Berry, 1975).

1. Chondros (G); 2. Gristle. Compare with the terms chondrification, chondrodystrophy, synchondrosis, etc.

Cartilage is a connective tissue composed of cells (chondrocytes) and fibres (collagen or yellow elastic) embedded in a firm, gel-like matrix which is rich in a mucopolysaccharide. It is much more elastic than bone.

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1. Cartilage has no blood vessels or lymphatics. The nutrition of cells diffuses through the matrix.
2. Cartilage has no nerves. It is, therefore, insensitive.
3. Cartilage is surrounded by a fibrous membrane, called perichondrium, which is similar to periosteum in structure and function. The articular cartilage has no perichondrium, so that its regeneration after injury is inadequate.
4. When cartilage calcifies, the chondrocytes die and the cartilage
is replaced by bone like tissue.
Table 2.2 shows the comparison between bone and cartilage.
1. Bone is hard
Cartilage is firm
2. Matrix has inflexible material
It has chondroitin providing flexibility
called ossein
3. Matrix possesses calcium salt
Calcium salts not present
4. Bone has rich nerve supply.
It does not have nerve supply.
It is vascular in nature
It is avascular in nature
5. Bone marrow is present
Bone marrow is absent
6. Growth is only by apposition
Growth is appositional and interstitial
(by surface deposition)
(from within)

There are three types of cartilages:
1. Hyaline cartilage (Fig. 2.16)
2. Fibrocartilage (Fig. 2.17)
3. Elastic cartilage (Fig. 2.16)

Table 2.3 reveals the comparison between three types of cartilages.
Fig. 2.16: Hyaline and elastic cartilages
Fig. 2.17: Fibrocartilage: Intervertebral disc
+ A defect in membranous ossification causes a rare syndrome called cleidocranial dysostosis. \t is characterized by three cardinal features: (a) Varying degrees of aplasia of the clavicles; (b) increase

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in the transverse diameter of cranium, and (c) retardation in fontanelle ossification (Srivastava et al, 1971). It may be hereditary or environmental in origin.
°
A defect in endochondral ossification causes a common type of dwarfism called achondroplasia, in which the limbs are short, but the trunk is normal. It is transmitted as a Mendelian dominant character.
» Periosteum is particularly sensitive to tearing or tension. Drilling into the compact bone without anaesthesia causes only mild pain or an aching sensation; drilling into spongy bone is much more painful. Fractures, tumours and infections of the bone are very painful.
*
Blood supply of bone is so rich that it is very difficult to interrupt it sufficiently to kill the bone. Passing a metal pin into the medullary cavity hardly interferes with the blood supply of the bone.
°
Fracture is a break in the continuity of a bone. The fracture which is not connected with the skin wound is known as simple (closed) fracture. The fracture line may be (a) oblique or (b) horizontal. The fracture which communicates with the skin wound is known as (c) compound (open) fracture. A fracture requires "reduction" by which the alignment of the broken ends is restored (Fig. 2.18).
Healing (repair) of a fracture takes place in three stages:
(a) Repair by granulation tissue;
(b) union by callus; and
(c) consolidation by mature bone.
»
- Axis or 2nd cervical vertebra may get fractured. If dens of axis gets separated from the body, it hits the vital centres in the medulla oblongata causing instantaneous death (Fig. 2.19). Even fracture of laminae may cause death.
Fig. 2.19: "Hanging till death" occurs due to fracture of dens of axis vertebra

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°In rickets (deficiency of vitamin D), calcification of cartilage fails and ossification of the growth zone is disturbed. Rickets affects the growing bones and, therefore, the disease develops during the period of most rapid growth of skeleton, i.e. 3 months to 3 years. Osteoid tissue is formed normally and the cartilage cells proliferate freely, but mineralization does not take place. This results in craniotabes, rachitic rosary at the costochondral junctions, Harrison's sulcus at the diaphragmatic attachments, enlarged epiphyses in limb bones (Fig. 2.20) and the spinal and pelvic deformities.
* For proper development of bones, a child requires adequate amounts of proteins, calcium, vitamin D, etc. Deficiency of calcium and vitamin D in growing children leads to widening of ends of bones with inadequate ossification. This condition is called as rickets (Fig. 2.20).
aa
Fig. 2.20: (a) Rickets, (b) Normal
»
Inscurvy (deficiency of vitamin C), formation of collagenous fibres and matrix is impaired. Defective formation of the intercellular cementing substances and lack of collagen cause rupture of capillaries and defective formation of new capillaries. Haematoma in the muscles and bones (subperiosteal) cause severe pain and tenderness. The normal architecture at the growing ends of the bones is lost.
*
Many skeletal defects are caused by genetic factors, or by a combination of genetic, hormonal, nutritional and pathological factors.
* The vertebral arch or laminae of the vertebral column may remain deficient, the spinal cord may be covered by skin, i.e. (a) spina bifida occulta. There may be protrusion of the meninges surrounding the spinal cord placed in the vertebral canal, i.e. (b) meningocele or there may be protrusion of the spinal cord as well as meninges, i.e. (c) meningo-myelocele (Fig. 2.21).
Fig. 2.21: Types of spina bifida: (a) Spina bifida occulta, (b) Meningocele,
ee
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- «If deficiency of calcium, vitamin D occurs in adult life, it leads osteomalacia. The bones on X-rays examination do not reveal enough trabeculae.
Deficiency of calcium in bones in old age leads to osteoporosis, seen both in females and males. Due to osteoporosis, there is forward bending of the vertebral column, leading to kyphosis (Fig. 2.22).
- * Nerves are closely related to bones in some areas. Fracture of the bones of those areas may lead to injury to the nerve, leading to paralysis of muscles supplied including the sensory loss (Fig. 2.23).
Fig. 2.22: Kyphosis due to osteoporosis
Fig. posterior
2.23: Nerves in contact with surface of humerus
Osteoporosis, Table 2.4: Comparison of osteoporosis and osteomalacia.Calcium & phosphate = Normal. Osteoporosis, Table 2.4: Comparison of osteoporosis and osteomalacia.Alkaline phosphatase = Normal. Osteoporosis, Table 2.4: Comparison of osteoporosis and osteomalacia.Osteo- blast = Normal. Osteoporosis, Table 2.4: Comparison of osteoporosis and osteomalacia.Trabeculae = Thin and small. Osteomalacia, Table 2.4: Comparison of osteoporosis and osteomalacia.Calcium & phosphate = May be low. Osteomalacia, Table 2.4: Comparison of osteoporosis and osteomalacia.Alkaline phosphatase = Raised. Osteomalacia, Table 2.4: Comparison of osteoporosis and osteomalacia.Osteo- blast = Increased. Osteomalacia, Table 2.4: Comparison of osteoporosis and osteomalacia.Trabeculae = Thick uncalcified osteoid
'Bone marrow biopsy: Bone marrow can be taken either from manubrium sterni or iliac crest in various clinical conditions (Fig. 2.24).
»
Bone tumour: Benign or malignant tumours can occur in the bone (Fig. 2.25).
Fig. 2.25: Malignant tumour of the bone

1. Arthron (G. a joint). Compare with the terms arthrology, synarthrosis, diarthrosis, arthritis, arthrodesis, etc.
2. Articulatio (L a joint); articulation (NA).
3. Junctura (L a joint).
4. Syndesmology (G. syndesmosis = ligament) is the study of ligaments and related joints.

Joint is a junction between two or more bones or cartilages. It is a device to permit movements.
However, immovable joints are primarily meant for growth, and may permit moulding during childbirth.
There are more joints in a child than in an adult because as growth proceeds some of the bones fuse together, e.g. the ilium, ischium and pubis to form the pelvic bone; the two halves of the infant frontal bone, and of the infant mandible; the five sacral vertebrae and the four coccygeal vertebrae.

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2. Cartilaginous joints
(a) Primary cartilaginous joints or synchondrosis
(b) Secondary cartilaginous joints or symphysis
3. Synovial joints
(a) Ball-and-socket or spheroidal joints
(b) Sellar or saddle joints
(c) Condylar or bicondylar joints
(d) Ellipsoid joints
(e) Hinge joints
(f) Pivot or trochoid joints
(g) Plane joints

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1. Synarthrosis (immovable), like fibrous joints (Fig.3.1).
2. Amphiarthrosis (slightly movable), like cartilaginous joints (Fig. 3.2).
3. Diarthrosis (freely movable), like synovial joints (Fig. 3.3).
Synarthroses are fixed joints at which there is no movement. The articular surfaces are joined by tough fibrous tissue. Often the edges of the bones are dovetailed into one another as in the sutures of the skull.
Amphiarthroses are joints at which slight movement is possible. A pad of cartilage lies between the bone surfaces, and there are fibrous ligaments to hold the bones and cartilage in place. The cartilages of such joints also act as shock absorbers, e.g. the intervertebral discs between the bodies of the vertebrae, where the cartilage is strengthened by extra collagen fibres.
Fig. 3.1: Synarthrosis: Fibrous joint
Fig. 3.3: Diarthrosis: Simple synovial joint
Diarthroses or synovial joints are known as freely movable joints, though at some of them the movement is restricted by the shape of the articulating surfaces and by the ligaments which hold the bones together. These ligaments are of elastic connective tissue.
A synovial joint has a fluid-filled cavity between articular surfaces which are covered by articular cartilage. The fluid, known as synovial fluid, produced by the synovial membrane which lines the cavity except for the actual articular surfaces and covers any ligaments or tendons which pass through the joint. Synovial fluid acts as a lubricant.
The form of the articulating surfaces controls the type of movement which takes place at any joint.
The movements possible at synovial joints are:
Angular flexion
:
decreasing the angle between two bones;

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f
(i
extension
:
increasing the angle between two bones;
abduction
:
moving the part away from the mid-line;
adduction
:
bringing the part towards the mid-line.
Rotary
rotation
:
turning upon an axis;
circumduction: moving the extremity of the part round
in a circle so that the whole part
inscribes a cone.
Gliding
one part slides on another.

1. Skull type: immovable.
2. Vertebral type: slightly movable.
3. Limb type: freely movable.

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1. Simple joint: When two bones articulate, e.g. interphalangeal joints (Fig. 3.3).
2. Compound joint: More than two bones articulate within one capsule, e.g. elbow joint, wrist joint (Fig. 3.4).
3. Complex joint: When joint cavity is divided by an intra-articular disc, e.g., temporomandibular joint (Fig. 3.5) and sternoclavicular joint.
Fig. 3.4: Compound joint: Wrist joint
Fig. 3.5: Complex joint: Temporomandibular joint
The structural classification is most commonly followed, and will be considered in detail in the following paragraphs.

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In fibrous joints the bones are joined by fibrous tissue. These joints are either immovable or permit a slight degree of movement. These can be grouped in the following three subtypes.
1. Sutures: These are peculiar to skull, and are immovable. According to the shape of bony margins, the sutures can be:
(i) Plane, e.g. internasal suture
(ii) Serrate, e.g. interparietal suture
(iii) Squamous, e.g. temporo-parietal suture
(iv) Denticulate, e.g. lambdoid suture
(v) Schindylesis type (Fig. 3.6), e.g. between rostrum of sphenoid and upper border of vomer.
Neonatal skull reveals fontanelles which are temporary in nature. At six specific points on the sutures in new born skull are membrane filled gaps called "fontanelles". These allow the underlying brain to increase in size. Anterior fontanelle is used to judge the hydration of the infant. All these fontanelles become bone by 18 months (Fig. 3.7).
2. Syndesmosis: The bones are connected by the interosseous ligament. Example: inferior tibiofibular joint (Fig. 3.8).
3. Gomphosis (peg and socket joint). Example: root of the tooth in its bony socket (Fig. 3.9).
Fig. 3.6: Types of sutures
Fig. 3.7: Fontanelles
Fig. 3.8: Inferior tibiofibular joint
Fig. 3.9: Gomphosis

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In this type of joints the bones are joined by cartilage. These are of the following two types:
1,
Primary cartilaginous
joints
(synchondrosis, or
hyaline cartilage
joints):
The that
the
These bones
are joint
united is
immovable joints
are the
by
a
plate of
hyaline and
temporary cartilaginous
strong.
in nature
plate is
because replaced
by cartilage
after
a
so certain
bone age
(synostosis).
Examples:
- (a) Joint between epiphysis and diaphysis of a growing long bone (Fig. 3.10)
Fig. 3.10: Primary cartilaginous joint

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(b) Spheno-occipital joint
(c) First chondrosternal joint
(d) Costochondral joints.
. Secondary cartilaginous joints (symphyses or fibrocartilaginous joints): The articular surfaces are covered by a thin layer of hyaline cartilage, and united by .a disc of fibrocartilage.
These joints are permanent and persist throughout life. In this respect symphysis menti is a misnomer as it is a synostosis. Typically the secondary cartilaginous joints occur in the median plane of the body, and permit limited movements due to compressible pad of fibro-cartilage such as in the pubic symphysis and manubriosternal joints.
The thickness of fibrocartilage is directly related to the range of movement. Secondary cartilaginous joints may represent an intermediate stage in the evolution of synovial joints.
Examples:
(a) Symphysis pubis
(b) Manubriosternal joint
(c) Intervertebral joints between the vertebral bodies (Fig. 3.2).

Synovial joints are most evolved, and, therefore, most mobile type of joints.

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1, The articular surfaces are covered with hyaline (articular) cartilage (fibrocartilage in certain membrane bones).
Articular cartilage is avascular, non-nervous and elastic. Lubricated with synovial fluid, the cartilage provides slippery surfaces for free movements, like 'ice on ice'.
The surface of the cartilage shows fine undulations filled with synovial fluid.
2. Between the articular surfaces there is a joint cavity filled with synovial fluid. The cavity may be partially or completely subdivided by an articular disc or meniscus (Fig. 3.5).
3. The joint is surrounded by an articular capsule which is made up of a fibrous capsule lined by synovial membrane.
Because of its rich nerve supply, the fibrous capsule is sensitive to stretches imposed by movements. This sets up appropriate reflexes to protect the joint from any sprain. This is called the 'watch-dog' action of the capsule.
The fibrous capsule is often reinforced by :
(a) Capsular or true ligaments representing thickenings of the fibrous capsule
(b) The accessory ligaments (distinct from fibrous capsule) which may be intra or extracapsular.
The synovial membrane lines whole of the interior of the joint, except for the articular surfaces covered by hyaline cartilage.
The membrane secretes a slimy viscous fluid called the synovia or synovial fluid which lubricates the joint and nourishes the articular cartilage. The viscosity of fluid is due to hyaluronic acid secreted by cells of the synovial membrane.
4. Varying degrees of movements are always permitted by the synovial joints.

Articular surfaces are more or less flat (plane). They permit gliding movements (translations) in various directions.

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Examples:
(a) Intercarpal joints (Fig. 3.4)
(b) Intertarsal joints
(c) Joints between articular processes ofi vertebrae
(d) Cricothyroid joint
(e) Cricoarytenoid joint
(f) Superior tibiofibular
(g) Interchondral joint (5—9 ribs)
(h) Costovertebral
(i) Costotransverse
(j) Acromioclavicular with intra-articular disc
(k) Carpometacarpal (except first)
(1) Tarsometatarsal
(m) Intermetacarpal
(n) Intermetatarsal
(0) Chondrosternal (except first)
(p) Sacroiliac

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Articular surfaces are pulley-shaped. There are strong collateral ligaments. Movements are permitted in one plane around a transverse axis.
Examples:
(a) Elbow joint (Fig. 3.11)
(b) Ankle joint
(c) Interphalangeal joints.

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Articular surfaces comprise a central bony pivot (peg) surrounded by an osteoligamentous ring. Movements are permitted in one plane around a vertical axis.
Examples:
(a) Superior and inferior radio-ulnar joints (Fig. 3.12)
(b) Median atlanto-axial joint (Fig. 3.13).
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Fig. 3.12: Radioulnar joints
Fig. 3.13: Median atlantoaxial joint

Articular surfaces include two distinct condyles (convex male surfaces) fitting into reciprocally concave female surfaces (which are also, sometimes, known as condyles, such as in tibia). These joints permit movements mainly in one plane around a transverse axis, but partly in another plane (rotation) around a vertical axis.

(a) Knee joint (Fig. 3.14)
(b) Right and left jaw joints or temporomandibular joint (Fig. 3.15).
Fig. 3.14: Knee joint
Fig. 3.15: Temporomandibular joint

Articular surfaces include an oval, convex, male surface fitting into an elliptical, concave female surface. Free movements are permitted around both the axes, flexion and extension around the transverse axis, and abduction and adduction around the anteroposterior axis. Combination of movements produces circumduction. Typical rotation around a third (vertical) axis does not occur.

(a) Wrist joint (Fig. 3.4)
(b) Metacarpophalangeal joints
(c) Atlanto-occipital joints.

Articular surfaces are reciprocally concavoconvex. Movements are similar to those permitted by an ellipsoid joint, with addition of some rotation (conjunct rotation) around a third axis which, however, cannot occur independently.

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(a) First carpometacarpal joint (Fig. 3.16)
(b) Sternoclavicular joint (Fig. 3.17)
(c) Calcaneocuboid joint (Fig. 3.18)
(d) Incudomalleolar joint
(e) Between femur and patella.
Fig. 3.16: First carpometacarpal joint: Saddle variety
Fig. 3.17: Sternoclavicular joint: Saddle variety
Fig. 3.18: Calcaneocuboid joint: Saddle variety

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Articular surfaces include a globular head (male surface) fitting into a cup-shaped socket (female surface). Movements occur around an indefinite number of axes which have one common centre. Flexion, extension, abduction, adduction, medial rotation, lateral rotation, and circumduction, all occur quite freely.
Examples:
(a) Shoulder joint
(b) Hip joint (Fig. 3.19)
(c) Talo-calcaneonavicular joint (Fig. 3.20)
(d) Incudostapedial joint

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Human Kinesiology: Study of geometry of surfaces and their associated movements.
Male surface: An articulating surface which is larger in surface area and always convex in all directions (Fig. 3.3).
Female surface: An articulating surface which is smaller and concave in all directions (Fig. 3.3).
Simple joints: Joints with only two articulating surfaces, i.e. male and female.
Compound joints: Joint possessing more than one pair of articulating surfaces.
Degrees of freedom: Number of axes at which the bone in a joint can move.
Uniaxial: Movement of bone at a joint is limited to one axis, i.e.
with one degree of freedom, e.g. interphalangeal joints.
Biaxial: With two degrees of freedom, e.g. wrist joint.
Multi-axial: Three axes along with intermediate positions also, e.g. shoulder joint
Translation: Sliding movements of one articulating surface over the other.

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A. Angular movement: Movement leading to diminution or increase in angle between two adjoining bones. They are of two types:
(a) Flexion and extension: Bending and straightening respectively.
(b) Abduction and adduction: Movement away and towards the median plane respectively.
Circumduction: When a long bone circumscribes a conical space.
B. Rotation: Bone moves around a longitudinal axis.
(a) Adjunct rotation: independent rotations, e.g. locking of knee joint.
(b) Conjunct rotation: rotations which accompany other movements as in Ist carpometacarpal joint.

The common shapes of the articular surface are:
(a) Ovoid: When concave—female ovoids. When convex—male ovoids.
(b) Sellar/saddle-shaped: These are convex in one plane, concave in the perpendicular plane.

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It is a reference point around which joint mechanics can be studied and around which the most habitual conjunct rotation occurs.
Spin:
Simple rotation
around the
bone’s stationary
mechanical axis.
Swing:
Any other
displacement of
the bone
and its
mechanical apart from spin is termed a swing.
Swing may be pure or impure (swing + element of spin).
Ovoid of motion: This represents the imaginary surface which would include all possible paths of a point on the mechanical axis at some distance from its related joint.
Cardinal swing: When the mechanical axis moves in the shortest pathway when bone moves.
Arcuate swing: When the mechanical axis moves in the longest pathway along with the bony movement.
Co-spin: When the effect of adjunct rotation is additive to the rotation.
Anti-spin: Adjunct rotation which has a nullifying effect on rotation.
Basic components
of movements
of the
synovial joints
are:
(1)
Spin,
(2)
Sliding, and
(3)
Rolling.
1. Spin: It occurs around a fixed mechanical axis.
axis

fe
Slide: During sliding movement, the mechanical axis of the joint and both ends of a moving bone move in the same direction. The transverse axis of movement is not fixed and it undergoes gliding or translation or linear movement. pS
3. Rolling: In rolling movement, one end of the mechanical axis moves ina particular direction and the other end moves in opposite direction. The transverse axis of movement is almost fixed. The resultant movement is rolling along
an arc. Rolling and sliding occur together in knee joint.

Close packed position: When the joint surfaces become completely congruent, their area of contact is maximal and they are tightly compressed.
In this position fibrous capsule and ligaments are maximally spiralized and tense; no further movement is possible; surfaces cannot be separated by disruptive forces; articular surfaces are liable to trauma (Table 3.1).

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Joint
Close packed position
Temporomandibular
Clenched teeth
Spine
Extension
Shoulder
Abduction and lateral rotation
Elbow
Extension
Wrist
Extension with radial deviation
Trapeziometacarpal
Metacarpophalangeal
»
Metacarpophalangeal flexion (finger)
and
*
Opposition (thumb)
Interphalangeal
»
Interphalangeal extension
Hip
Extension and medial rotation
Knee
Extension with locking
Ankle
Dorsiflexion
|
Subtalar and mid-tarsal
Inversion
|
Metatarsophalangea! and
Metatarsophalangeal extension
interphalangeal
Loose packed: All other positions of incongruency.
Examples: Least packed position.
Shoulder — semiabduction
Hip — semiflexion
Knee — semiflexion
Ankle — plantar flexion.

¢ Reflex contraction of antagonistic muscles.
¢ Due to stimulations of mechanoreceptors in articular tissue.
Ligaments tension;
e Approximation of soft parts.

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1. Synovial fluid, secreted by synovial membrane, is sticky and viscous due to hyaluronic acid (a mucopolysaccharide). It serves the main function of lubrication of the joint, but also nourishes the articular cartilage.
2. Hyaline cartilage covering the articular surfaces possesses inherent slipperiness, like that of the ice.
3. Intra-articular fibrocartilages, articular discs or menisci, complete or incomplete, help in spreading the synovial fluid throughout the joint cavity, but particularly between the articular surfaces, e.g. temporomandibular joint (Fig. 3.15).
4. Haversian fatty pads (Haversian glands) occupy extra spaces in the joint cavity between the incongruous bony surfaces. All of them are covered with synovial membrane, and perhaps function as swabs to spread the synovial fluid.
5. Bursa is a synovial fluid filled bag in relation to joints and bones, to prevent friction. The inflammation of bursa is called bursitis.

The articular and epiphysial branches given off by the neighbouring arteries form a periarticular arterial plexus. Numerous vessels from

re
——i(ststéiéisz this plexus pierce the fibrous capsule and form a rich vascular plexus in the deeper parts of synovial membrane. The blood vessels of the synovial membrane terminate around the articular margins in a fringe of looped anastomoses termed the circulus vasculosus (circulus articularis vasculosus). It supplies capsule, synovial membrane, and the epiphysis. The articular cartilage is avascular.
After epiphysial fusion, communications between circulus vasculosus and the end arteries of metaphysis are established, thus minimizing the chances of osteomyelitis in the metaphysis.

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- 1. The capsule and ligaments possess a rich nerve supply, which makes them acutely sensitive to pain. The synovial membrane has a poor nerve supply and is relatively insensitive to pain. The articular cartilage is non-nervous and totally insensitive.
Articular nerves contain sensory and autonomic fibres.
Some of the sensory fibres are proprioceptive in nature; these are sensitive to position and movement, and are concerned with the reflex control of posture and locomotion. Other sensory fibres are sensitive to pain.
Autonomic fibres are vasomotor or vasosensory.
The joint pain is often diffuse, and may be associated with nausea, vomiting, slowing of pulse, and fall in blood pressure.
The pain commonly causes reflex contraction of muscles which fix the joint in a position of maximum comfort. Like visceral pain, the joint pain is also referred to uninvolved joints.
- 2. The principles of distribution of nerves to joints were first described by Hilton (1891). Hilton's law states that a motor nerve to the muscle acting on joint tends to give a branch to that joint (capsule) and another branch to the skin covering the joint.
The concept of innervation of a joint was further elucidated by Gardner (1948) who observed that each nerve innervates a specific region of the capsule, and that the part of the capsule which is rendered taut by a given muscle is innervated by the nerve supplying its antagonists. Thus the pattern of innervation is concerned with the maintenance of an efficient stability at the joint.

Lymphatics form a plexus in the subintima of the synovial membrane, and drain along the blood vessels to the regional deep nodes.

The various factors maintaining stability at a joint are described here in order of their importance.
1. Muscles: The tone of different groups of muscles acting on the joint is the most important and indispensable factor in maintaining the stability. Without muscles, the knee and shoulder would be unstable, and arches of the foot would collapse.
2. Ligaments: Are important in preventing any over-movement, and in guarding against sudden accidental stresses. However, they do not help against a continuous strain, because once stretched, they tend to remain elongated. In this respect the elastic ligaments (ligamenta flava and ligaments of the joints of auditory ossicles) are superior to the common type of white fibrous ligaments.
3. Bones: Help in maintaining stability only in firm type of joints, like the hip and ankle. Otherwise in most of the joints (shoulder, knee, sacroiliac, etc.) their role is negligible.

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* Intervertebral disc forms secondary cartilaginous joint between the bodies of the vertebrae. If the nucleus pulposus part of the disc gets protruded backwards, it may press on the spinal nerve leaving out from the intervertebral foramina. The condition is known as herniation of the disc or disc prolapse. If disc prolapse occurs in lumbar or sacral nerves, there is radiating pain in the lower limb, then the condition is called sciatica (Fig. 3.21).
* The joints may get dislocated, i.e. the end of one of the bones gets out of its socket. In subluxation, the end of the bone partially leaves its socket (Fig. 3.22).
* Rheumatic fever causes fleeting pain in the joints, accompanied by streptococcal pharyngitis. It is mostly temporary pain in the joints. The toxins of the bacteria may affect the mitral valve of the heart or the kidneys.
Fig. 3.21: Disc prolapse leading to sciatica
Fig. 3.22: (a) Subluxation, (b) Dislocation
* Rheumatoid arthritis is an inflammatory systemic disease involving the synovial membranes of small joints of the hands. Due to chronic inflammatory process there is deformity of the fingers (Fig. 3.23).
* Osteoarthritis is a degenerative condition of the large weightbearing joints. The articular cartilage wears out, degenerates and there is formation of peripheral osteophytes. The patients feels lots of pain due to rubbing of the bones together during movements of the joints (Fig. 3.24).
* The degenerative changes or spondylitis may occur in the cervical spine, leading to narrowed intervertebral foramen, causing pressure on the spinal nerve (Fig. 3.25).
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Fig. 3.23: Changes in rheumatoid arthritis with clinical features
Fig. 3.25: Spondylitis
- * There may be injury to various structures in the joints. At times the medial meniscus of the knee joint may get injured. In that case it needs to be removed (Fig. 3.26).
Fig. 3.26: Injury to medial meniscus

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The metaphysis, the end of diaphysis or shaft is the actively growing end of the bone. In some joints, the capsule encloses the metaphysis as well. In such joints, infection from metaphysis would reach the joint cavity and cause septic arthritis (Fig. 3.27).
There may be fracture into the joint space leading to collection of blood and broken pieces of ends of the bones in the joint cavity (Fig. 3.28).
If joints have been diseased for a very long time with no hope of recovery, these can be replaced. The X-rays shows replaced hip joints and knee joint (Figs 3.29 and 3.30).
»
»
*
Fig. 3.28: Injury to a joint
Fig. 3.29: Right hip joint replaced
Fig. 3.30: Both knee joints replaced
Courtesy: Dr. Naresh Chand, Senior Consultant, Department of Orthopaedics, Narendra Mohan Hospital, Mohan Nagar, Ghaziabad.
°
*
Stiffness of joints related to weather. The viscosity of synovial fluid increases with fall in temperature. This accounts for stiffness of the joints in cold weather. Mobility of joint in itself is an important factor in promoting lubrication. Thus the stiffness of joints experienced in the morning gradually passes off as the movements are resumed.
Neuropathic joint is the result of its complete denervation, so that all reflexes are eliminated and the joint is left unprotected and liable to mechanical damage. A neuropathic joint shows painless swelling, excessive mobility and bony destruction. It is commonly caused by leprosy, tabes dorsalis and syringomyelia.

Muscles (L. Mus = mouse) are so named because, many of them resemble a mouse, with their tendons representing the tail.

Muscle is a contractile tissue which brings about movements. Muscles can be regarded as motors of the body.

The muscles are of three types, skeletal, smooth and cardiac. The characters of each type are summarized in Table 4.1.

1. Striped muscles
2. Striated muscles
3. Somatic muscles
4. Voluntary muscles

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1. Origin is one end of the muscle which remains fixed during its contraction.
2. Insertion is the other end which moves during its contraction.
In the limb muscles, the origin is usually proximal to insertion.
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Fig. 4.3: Cardiac muscle
However, the terms, origin and insertion, are at times interchangeable (e.g. climbing action of latissimus dorsi), and at other times difficult to define, as in the intercostal muscles.

1. Fleshy part is contractile, and is called the 'belly'.
2. Fibrous part is noncontractile and inelastic. When cord-like or rope-like, it is called tendon (Fig. 4.4); when flattened, it is called aponeurosis.

Each muscle is composed of numerous muscle fibres. Each muscle fibre is a multinucleated, cross-striated cylindrical cell (myocyte) 1-300 mm long. It is made up of sarcolemma (cell membrane) enclosing sarcoplasm (cytoplasm).
Embedded in the sarcoplasm there are (a) several hundred of nuclei arranged at the periphery beneath the sarcolemma and (b) a number of evenly distributed longitudinal threads called myofibrils. Each myofibril shows alternate dark and light bands. Dark bands are known as A bands (anistropic) and the light bands as I bands (isotropic). The bands of adjacent fibrils are aligned transversely so that the muscle fibre appears cross-striated. In the middle of dark band there is a light H band with M band (dark), in its middle. In the middle of I band there is a dark Z disc or Krause's membrane. the segment of myofibril between two Z discs is called sarcomere (Fig. 4.5).
Fig. 4.5: A sarcomere in skeletal muscle between two Z lines

It helps in organization of the muscle. Endomysium surrounds each muscle fibre separately. Perimysium surrounds bundles (fasciculi or myonemes) of muscle fibres of various sizes. Epimysium surrounds the entire muscle. The connective tissue of the muscle becomes continuous with the tendon (Fig. 4.6).
Slow and Fast Muscle Fibres (Dubowitz, 1969; Gauthier and Schaeffer, 1974)
Fig. 4.6: Supporting tissue

Show a slow 'tonic' contraction characteristic of postural muscles. These are red in colour because of large amounts of myoglobin. The fibres are rich in mitochondria and oxidative enzymes, but poor in phosphorylases.
Because of a well-developed aerobic metabolism, slow fibres are highly resistant to fatigue.

Show a fast 'phasic' contraction required for large-scale movements of body segments.
These are paler (white) in colour because of small amounts of myoglobin. The fibres are rich in glycogen and phosphorylases, but poor in mitochondria and oxidative enzymes.
Because of a glycolytic respiration, the fast fibres are quite easily fatigued.

Represent a variant of type II (fast) fibres which are relatively resistant to fatigue, although less than type I (slow) fibres (Burke et al, 1973).
In man, most of the skeletal muscles show a mixture of fibre types, but any one type may predominate.

The arrangement of muscle fibres varies according to the direction, force and range of habitual movement at a particular joint. The force of movement is directly proportional to the number and size of muscle fibres, and the range of movement is proportional to the length of fibres. The muscles can be classified according to the arrangement of their fasciculi into the following groups (Fig. 4.7).

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When the fasciculi are parallel to the line of pull, the muscle may be :
1. Quadrilateral (thyrohyoid),
2. Strap-like (sternohyoid and sartorius).
3. Strap-like with tendinous intersections (rectus abdominis).
4. Fusiform (biceps brachii, digastric, etc.). The range of movement in such muscles is maximum (Fig. 4.7).
Fig. 4.7: Fascicular architecture of muscles

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When the fasciculi are oblique to the line of pull, the muscle may be triangular, or pennate (feather-like) in the construction. This arrangement makes the muscle more powerful, although the range of movement is reduced. Oblique arrangements are of the following types:
- 90 | Handbook of General Anatomy
a
ao
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1. Triangular, e.g. temporalis, adductor longus.
2. Unipennate, e.g. flexor pollicis longus, extensor digitorum longus, peroneus tertius, palmar interossei (Fig. 4.8).
3. Bipennate, e.g. rectus femoris, dorsal interossei (Fig. 4.7), peroneus longus, flexor hallucis longus.
4. Multipennate, e.g. subscapularis, deltoid (acromial fibres) (Fig. 4.8).
5. Circumpennate, e.g. tibialis anterior (Fig. 4.7).

Spiral or twisted fibres are found in trapezius, pectoralis major, latissimus dorsi, supinator, etc. (Fig. 4.9). In certain muscles the fasciculi are crossed. These are called cruciate muscles, e.g. sternocleido-mastoid, masseter, and adductor magnus.
Fig. 4.9: Spiral fasciculi: Latissimus dorsi

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The muscles have been named in a number of ways.
I. According to their shape, e.g. trapezius, rhomboideus, serratus anterior (Fig. 4.10), latissimus dorsi, etc.
. According to the number of heads of origin, e.g. biceps, triceps (Fig. 4.11), quadriceps (Fig. 4.7), digastric, etc.
. According to their gross structure, e.g. semitendinosus, semimembranosus, etc. (Fig. 4.12).
. According to their location, e.g. temporalis (Fig. 4.13), supraspinatus, intercostales.
. According to their attachments, e.g. stylohyoid, cricothyroid (Fig. 4.14), ete.
. According to their action, e.g. adductor longus, flexor carpi ulnaris (Fig. 4.15), abductor pollicis longus, etc. orbicularis oculi (Fig. 4.7).
. According to direction of their fibres, e.g. rectus abdominis, transversus abdominis, orbicularis oculi (Fig. 4.7).
. A muscle with two bellies with an intervening tendon is called digastric muscle (Fig. 4.7). Muscle with number of intervening tendons or intersections is the rectus abdominis.
Fig. 4.10: Serratus anterior muscle
Fig. 4.11: Triceps brachii
Muscles
!
93
Fig. 4.12: According to gross structure: Semitendinosus
Fig. 4.13: According to shape: Temporalis
Fig. 4.14: According to attachment: Cricothyroid

The muscles that extend over two or more joints are called diarthric or polyarthric muscles, e.g. flexor carpi radialis (Fig. 4.15) and flexor digitorum profundus (Fig. 4.4).
Fig. 4.15: Muscles named according to action

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The nerve supplying a muscle is called motor nerve. In fact it is a mixed nerve and consists of the following types of fibres.
1. Motor fibres (60%) comprise:
(a) Large myelinated alpha efferents which supply extrafusal muscle fibres (Fig. 4.16).
(b) Smaller myelinated gamma efferents which supply intrafusal fibres of the muscle spindles which refine and control muscle contraction.
(c) The fine non-myelinated autonomic efferents which supply smooth muscle fibres of the blood vessels.
2. Sensory fibres (40%) comprise: Myelinated fibres distributed to muscle spindles for proprioception, also to tendons.
Muscle spindles are spindle-shaped sensory end organs of the skeletal muscle. Each spindle contains 6-14 intrafusal muscle fibres
which are to two types, the larger nuclear bag fibres, and the smaller nuclear chain fibres (Fig. 4.16). The spindle is innervated by both the sensory and motor nerves. The sensory endings are of two types, the primary sensory endings (annulospiral endings) around the central nuclear region of the intrafusal fibres, and the secondary sensory endings (flower spray endings) beyond the nuclear region on either side of these fibres.

The motor nerve supply of the spindle is derived from gamma motor neurons of the spinal cord. Muscles spindles act as stretch receptors. They record and help regulate the degree and rate of contraction of the extrafusal fibres by influencing the alpha neurons.
Motor point is the site where the motor nerve enters the muscle. It may be one or more than one. Electrical stimulation at the motor point is more effective.
Motor unit (myone) is defined as a single alpha motor neuron together with the muscle fibres supplied by it. The size of motor unit depends upon the precision of muscle control. Small motor units (5-10 muscle fibres) are found in muscles of fine movements (extraocular muscles). Large motor units (100-2000 muscle fibres) are found in muscles of gross movements (proximal limb muscles).
Composite/hybrid muscle: Muscle supplied by two different motor nerves with different root values is called a composite or hybrid muscle, e.g. adductor magnus, flexor digitorum profundus and pectoralis major.

According to nerve supply the smooth muscles are classified into:
Single-unit type: Seen in intestines. The nerve impulse reaches one muscle cell, is transmitted to other cells by the mechanical pull through the fused cell membrane. The nerve supply is sparse.
Multi-unit type: Seen in the muscles of the ductus deferens. Each muscle cell receives a separate nerve fibre. The contraction is simultaneous. The nerve supply is rich (Fig. 4.17).
Fig. 4.17: Nerve supply of smooth muscle

Heart is supplied by sympathetic and parasympathetic nerve fibres. Sympathetic nerves stimulate both the heart rate and blood pressure and dilate the coronary arteries. The sensory fibres convey painful impulses from heart.
Parasympathetic fibres decrease the heart rate. Their sensory fibres are involved with visceral reflexes.

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1. Broadly, when a muscle contracts, it shortens by one-third (30%) of its belly-length, and brings about a movement. The range of
movement depends on the length of fleshy fibres, and the power or force of movement on the number of fibres.
However, the actual behaviour of muscle contraction is more complex.
During contraction the length of the muscle may decrease (isotonic contraction).
May remain unchanged (isometric contraction).
May increase, according to the functional demands of the body. In each circumstance the tension generated at the ends may either increase, persist, or decrease, depending upon the number and state of its active motor units and the external conditions like loading.
- . Each movement at a joint is brought about by a coordinated activity of different groups of muscles. These muscle groups are classified and named according to their function.
(a)
Prime movers
(agonists)
bring about
the desired
movement.
When a prime mover helps opposite action by active controlled lengthening against gravity, it is known as action of paradox. For example, putting a glass back on the table is assisted by gravity but controlled by a gradual active lengthening of biceps (paradoxical or eccentric action).
(b) Antagonists (opponents) oppose the prime movers. They help the prime movers by active controlled relaxation, so that the desired movement is smooth and precise. Thus, the antagonists cooperate rather than oppose the prime movers. This is due to reciprocal innervation of the opposite groups of muscles, regulated by the spinal cord through stretch reflex (Fig. 4.18).
(c) Fixators are the groups of muscles which stabilize the proximal joints ofa limb, so that the desired movement at the distal joint may occur on a fixed base. Muscles acting on shoulder joint fix it for better movement of fingers.
(d) Synergists: When the prime movers cross more than one joint, the undesired actions at the proximal joints are prevented by certain muscles known as synergists. For example, during making a tight fist by long digital flexors the wrist is kept fixed in extension by the synergists (extensors of wrist). Thus, the synergists are special fixators and partial antagonists to the prime movers.
Two or more muscles causing one movement are synergist (Fig. 4.15).
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Loss of motor power (power of movement) is called paralysis. This is due to inability of the muscles to contract, caused either by damage to the motor neural pathways (upper or lower motor neuron), or by the inherent disease of muscles (myopathy). Damage to the upper motor neuron causes spastic paralysis with exaggerated tendon jerks. Damage to the lower motor neuron causes flaccid paralysis with loss of tendon jerks.
¢

These are quite painful. Localized muscle spasm is commonly caused by a 'muscle pull'. In order to relieve its pain the muscle should be relaxed by appropriate treatment. Generalized muscle spasms occur in tetanus and epilepsy.

The muscles which are not used for long times become thin and weak. This is called disuse atrophy. Conversely, adequate or excessive use of particular muscles causes their better development, or even hypertrophy (Fig. 1.2). Muscular 'wasting' (reduction in size) is a feature of lower motor neuron paralysis and generalized debility.

Skeletal muscle is capable of limited regeneration. If large regions are damaged, regeneration does not occur and the missing muscle is replaced by connective tissue.

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Increase in number of smooth muscle fibres. Usually occurs in uterus during pregnancy.
Myasthenia gravis is an autoimmune disease of muscle of unknown origin. Antibodies are produced that bind to acetylcholine receptor and block it. The nerve impulse transmission to muscle fibres is therefore blocked. This leads to extensive and progressive muscle weakness although the muscles are normal. Extraocular and eyelid muscles are affected first, followed by those of the neck and limbs. It affects more women that men and usually those between age of 20 and 40 years.
Polymyositis is a disease of muscle characterized by inflammation of the muscle fibres. It starts when white blood cells (immune cells of inflammation) spontaneously invade the muscle. Muscles close to trunk or torso are mostly affected by polymyositis that results in severe weakness. Polymyositis associated with skin rash is referred to as "dermamyositis".
Fibrillation is the abnormal contraction of cardiac muscle. The cardiac chambers do not contract as a whole resulting in the disruption of pumping action. In atrial fibrillation, there is rapid and uncoordinated contraction of atria, ineffective pumping and abnormal contraction of the AV node. Ventricular fibrillation is characterized by very rapid and disorganized contraction of ventricle. This leads to disruption of ventricular function.
Angina pectoris is episode of chest pain due to temporary ischaemia of cardiac muscle. It is usually relieved by rest and nitrites.

Persistent ischaemia due to blockage of more than one arteries results in necrosis (death) of the cardiac muscle (Fig. 4.19). Pain, not relieved by rest, gets referred to left arm, chest, and neigbhouring areas.
Fig. 4.19: Myocardial ischaemia

Cardiovascular system is the transport system of the body, through which the nutrients are conveyed to places where these are utilized, and the metabolites (waste products) are conveyed to appropriate places from where these are expelled.
The conveying medium is a liquid tissue, the blood, which flows in tubular channels called blood vessels. The circulation is maintained by the central pumping organ called the heart.

Cardiovascular system is a closed system of tubes made up of the following parts based on their structural and topographical characteristics (Fig. 5.1).
Fig.
5.1:
Heart

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Pan)
we
2
1. Heart: It is a four-chambered muscular organ which pumps blood to various parts of the body. Each half of the heart has a receiving chamber called atrium, and a pumping chamber called ventricle.
2. Arteries: These are distributing channels which carry blood away from the heart.
(a) They branch like trees on their way to different parts of the body.
(b) The large arteries are rich in elastic tissue, but as branching progresses there is an ever-increasing amount of smooth muscle in their walls.
(c) The minute branches which are just visible to naked eye are called arterioles.
(d) Angeion is a Greek word, meaning a vessel (blood vessel or lymph vessel). Its word derivatives are angiology, angiography, haemangioma, and thromboangitis obliterans.
3. Veins: These are draining channels which carry blood from different parts of the body back to the heart.
(a) Like rivers, the veins are formed by tributaries.
(b) The small veins (venules) join together to form larger veins, which in turn unite to form great veins called venae cavae (Fig. 5.2).
Capillaries: These are networks of microscopic vessels which connect arterioles with the venules.
* These come in intimate contact with the tissues for a free exchange of nutrients and metabolites across their walls between the blood and the tissue fluid.
* The metabolites are partly drained by the capillaries and partly by lymphatics.
» Capillaries are replaced by sinusoids in certain organs, like liver and spleen.
Functionally, the blood vessels can be classified into the following groups.

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(a) Distributing vessels, including arteries;
(b) Resistance vessels, including arterioles and precapillary sphincters;
(c) Exchange vessels, including capillaries, sinusoids, and postcapillary venules;
(d) Reservoir (capacitance) vessels, including larger venules and veins; and
(e) Shunts, including various types of anastomoses.

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Systemic (greater) circulation: The blood flows from the left ventricle, through various parts of the body, to the right atrium, i.e. from the left to the right side of the heart (Fig. 5.3).
Pulmonary (lesser) circulation: The blood flows from the right ventricle, through the lungs, to the left atrium, i.e. from the right to the left side of the heart.
Portal circulation: It is a part of systemic circulation, which has the following characteristics.
(a)
The
blood
passes
through
two
sets
of
capillaries
before
draining
into
a
systemic
vein.
(b) The vein draining the first capillary network is known as portal vein which branches like an artery to form the second set of capillaries or sinusoids. Examples: hepatic portal circulation hypothalamo hypophyseal portal circulation and renal portal circulation (Fig. 5.3).

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1. Arteries are thick-walled, being uniformly thicker than the accompanying veins, except for the arteries within the cranium and vertebral canal where these are thin.
2. Their /umen is smaller than that of the accompanying veins.
3. Arteries have no valves.
4. An artery is usually accompanied by vein(s) and nerve(s), and the three of them together form the neurovascular bundle which is surrounded and supported by a fibroareolar sheath.
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1. Large arteries of elastic type, e.g. aorta and its main branches (brachiocephalic, common carotid, subclavian and common iliac) and the pulmonary arteries.
2. Medium and small arteries of muscular type, e.g. temporal, occipital, radial, popliteal, etc.
WwW . Smallest arteries of muscular type are called arterioles. They measure 50-100 micron in diameter. Arterioles divide into terminal arterioles with a diameter of 15—20 micron, and having one or two layers of smooth muscle in their walls. The side branches from terminal arterioles are called metarterioles which measure 10—15 micron at their origin and about 5 micron at their termination (Fig. 5.4).
The terminal narrow end of metarteriole is surrounded by a precapillary sphincter which regulates blood flow into the capillary bed.
Fig. 5.4: Capillary bed between arteriole and venule

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It is important to know that the muscular arterioles are responsible for generating peripheral resistance, and thereby for regulating the diastolic blood pressure.
Microscopically, all arteries are made up of three coats.
(a) The inner coat is called tunica intima (Fig. 5.5).
(b) The middle coat is called tunica media.
(c) The outer coat is called tunica adventitia. It is strongest of all coats and merges with the perivascular sheath.
The relative thickness of the coats and the relative proportion of the muscular, elastic and fibrous tissues vary in different types of arteries.
Fig. 5.5: Microscopic structure of (a) artery, (b) vein, and (c) lymph vessel

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The large arteries (of more than | mm diameter) are supplied with blood vessels.
The nutrient vessels, called vasa vasorum, form a dense capillary network in the tunica adventitia, and supply the adventitia and the outer part of tunica media.
The rest of the vessel wall (intima + inner part of media) is nourished directly by diffusion from the luminal blood.
Minute veins accompanying the arteries drain the blood from the outer part of arterial wall.
Lymphatics are also present in the adventitia.

Some arteries can be palpated through the skin. These are: common carotid, facial, brachial, radial, abdominal aorta, femoral, posterior tibial and dorsalis pedis (Fig. 5.6).

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The nerves supplying an artery are called nervi vascularis.
The nerves are mostly non-myelinated sympathetic fibres which are vasoconstrictor in function. A few fibres are myelinated, and are believed to be sensory to the outer and inner coats of the arteries.
Vasodilator innervation is restricted to the following sites.
(a) The skeletal muscle vessels are dilated by cholinergic sympathetic nerves.
(b) The exocrine gland vessels are dilated on parasympathetic stimulation.
(c) The cutaneous vessels are dilated locally to produce the flare (redness) after an injury. The vasodilatation is produced by the afferent impulses in the cutaneous nerves which pass antidromically in their collaterals to the blood vessels (axon reflex).

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1. Veins are thin-walled, being thinner than the arteries.
2. Their /umen is larger than that of the accompanying arteries.
3. Veins have valves which maintain the unidirectional flow of blood, even against gravity.
Since the venous pressure is low (7 mm Hg), the valves are of utmost value in the venous return. However, the valves are absent:
(a) In the veins of less than 2 mm diameter.
(b) In the venae cavae.
(c) Inthe hepatic, renal, uterine, ovarian (not testicular), cerebral, spinal, pulmonary, and umbilical veins.
4. The muscular and elastic tissue content of the venous walls is much less than that of the arteries. This is directly related to the low venous pressure.
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Handbook of
General
Anatomy
r
Fig. 5.6: Palpable arteries
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- 5. Large veins have dead. space around them for their dilatation during increased venous return. The dead space commonly contains the regional lymph nodes (Fig. 5.7).
Fig. 5.7: Dead space with large veins

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Veins are made up of usual three coats which are found in the arteries. But the coats are ill-defined, and the muscle and elastic tissue content is poor.
In poorly developed tunica media, the amount of collagen fibres is more than the elastic and muscle fibres. The adventitia is thickest and best developed. The smooth muscle is altogether absent:
(a) in the veins of maternal part of placenta;
(b) in the cranial venous sinuses and pial veins;
(c) in the retinal veins;
(d) in the veins of cancellous bone; and
(e) in the venous spaces of the corpora cavernosa and corpus spongiosum.

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Table
5.1.
Comparison
of
arteries
and
veins
Arteries
Veins
1.
Arteries
carry
oxygenated
blood,
Veins carry deoxygenated
|
away from the heart except
blood, towards the heart except
pulmonary artery
pulmonary veins
2.
These
are
mostly
deeply
situated
These
are
superficial
and
deep
in
in
the
body
location
3.
These
are
thick-walled,
highly
These are thin-walled
muscular
except
arteries
of
cranium and vertebral column
These posses narrow lumen
These posses wide lumen
5. Valves are absent
Valves are present which provide
unidirectional flow of blood
6. These are reddish in colour
These are bluish in colour
These show spurty movement of blood giving pulse
These show sluggish movement
of blood
8.
Blood
in
arteries
moves
with
Blood in veins moves under very
pressure
low pressure
9. Arteries empty up at the time of
Veins get filled up at time of death
death
.
If
arterial
wall
is
injured,
the
blood
If venous wall is injured, blood
comes out like a ‘fountain’ in a
comes out, collects in a pool ina
large area all around the artery
small area around vein
1
oO

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The larger veins, like the arteries, are supplied with nutrient vessels called vasa vasorum. But in the veins, the vessels may penetrate up to the intima, probably because of the low venous pressure and the low oxygen tension.
Nerves also are distributed to the veins in the same manner as to the
but are fewer in number.
pushed
from
behind
arteries
,
2. Negative intrathoracic pressure sucks the blood into the heart from all over the body.
3. Gravity helps venous return in the upper part of the body.
4. Arterial pulsations press on the venae comitantes intermittently and drive the venous blood towards the heart.
5. Venous valves prevent any regurgitation (back flow) of the luminal blood (Fig. 5.8).

- 6. Muscular contractions press on the veins and form a very effective mechanism of venous return. This becomes still more effective. within the tight sleeve of the deep fascia, as is seen in the lower limbs. The calf muscles (soleus) for this reason are known as the peripheral heart. Thus the muscle pumps are important factors in the venous return.

Capillaries (capillus = hair) are networks of microscopic endothelial tubes interposed between the metarterioles and venules (Fig. 5.4). The true capillaries (without any smooth muscle cell) begin after a transition zone of 50-100 micron beyond the precapillary sphincters.
The capillaries are replaced by cavernous (dilated) spaces in the sex organs, splenic pulp and placenta.

The average diameter of a capillary is 6-8 micron, just sufficient to permit the red blood cells to pass through in 'single file'. But the size varies from organ to organ. It is smallest in the brain and intestines, and is largest (20 micron) in the skin and bone marrow.

The capillaries are classified as continuous and fenestrated according to the type of junctions between the endothelial cells.
1. Continuous capillaries are found in the skin, connective tissue, skeletal and smooth muscles, lung and brain. These allow passage across their walls of small molecules (up to 10 micron size).
2. Fenestrated capillaries are found in the renal glomeruli/intestinal mucosa, endocrine glands and pancreas. These allow passage across their walls of larger molecules (up to 20-100 micron size).
The capillary bed and postcapillary venules form an enormous area for the exchange of nutrients, gases, metabolites and water, between the blood and interstitial fluid. Capillaries also allow migration of leucocytes out of the vessels.

Sinusoids, replace capillaries in certain organs, like liver, spleen, bone marrow, suprarena! glands, parathyroid glands, carotid body, etc.

Sinusoids are large, irregular, vascular spaces which are closely surrounded by the parenchyma of the organ. These differ from capillaries in the following respects;
1. Their lumen is wider (upto 30 micron) and irregular
2. Their walls are thinner and may be incomplete. They are lined by endothelium in which the phagocytic cells (macrophages) are often distributed. The adventitial support is absent.
3. These may connect arteriole with venule (spleen, bone marrow), or venule with venule (liver).

A precapillary or postcapillary communication between the neighbouring vessels is called anastomoses. Circulation through the anastomosis is called collateral circulation.

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A. Arterial anastomoses is the communication between the arteries, or branches of arteries. It may be actual or potential.
1. In actual arterial anastomosis the arteries meet end to end. For example, palmar arches (Fig. 5.9), plantar arch, circle of Willis, intestinal arcades, labial branches of facial arteries.
2. In potential arterial anastomoses the communication takes place between the terminal arterioles. Such communications can dilate only gradually for collateral circulation. Therefore on sudden occlusion of a main artery, the anastomoses may fail to compensate the loss. The examples are seen in the coronary arteries (Fig. 5.10) and the cortical branches of cerebral arteries, etc.
B. Venous anastomoses is the communication between the veins or tributaries of veins. For example, the dorsal venous arches of the hand and foot.
C. Arteriovenous anastomosis (shunt) is the communication between an artery and a vein. It serves the function of phasic activity of the organ. When the organ is active these shunts are closed and the blood circulates through the capillaries.
1
)
ia
Fig. 5.10: Potential arterial anastomosis between coronary arteries
‘ie
Pe!
However, when the organ is at rest, the blood bypasses the capillary bed and is shunted back through the arteriovenous anastomosis. The shunt vessel may be straight or coiled, possesses a thick muscular coat, and is under the influence of sympathetic system (Fig. 5.11).
Fig. 5.11: Arteriovenous shunt bypassing the capillaries
Shunts of simple structure are found in the skin of nose, lips and external ear; in the mucous membrane of nose and alimentary canal; the coccygeal body; the erectile tissue of sexual organs; the tongue; the thyroid gland and sympathetic ganglia.
Specialized arteriovenous anastomoses are found in the skin of digital pads and nail beds. They form a number of small units called glomera.
Preferential 'thoroughfare channels are also a kind of shunts. They Course through the capillary network. Many true capillaries arise as their side branches.

4
One thoroughfare channel with its associated capillaries forms a microcirculatory unit. The size of the unit is variable from 1-2 to 20-30 true capillaries. The number of active units varies from time to time.

Arteries which do not anastomose with their neighbours are called end arteries (Fig. 5.12).

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1. Central artery of retina and labyrinthine artery of internal ear are the best examples of an absolute end arteries.
2. Central branches of cerebral arteries and vasa recta of mesenteric arteries.
3. Arteries of spleen, kidney, lungs and metaphyses of long bones.
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Occlusion of an end-artery causes serious nutritional disturbances resulting in death of the tissue supplied by it. For example, occlusion of central artery of retina results in blindness.

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The blood pressure is the arterial pressure exerted by the blood on the arterial walls. The maximum pressure during ventricular systole is called systolic pressure; the minimum pressure during ventricular diastole is called diastolic pressure. The systolic pressure is generated by the force of contraction of the heart; the diastolic pressure is chiefly due to arteriolar tone (peripheral resistance). The heart has to pump the blood against the diastolic pressure which is a direct load on the heart. Normally, the blood pressure is roughly 120/80 mm Hg, the systolic pressure ranging from 110-130, and the diastolic pressure from 70-80. The difference between systolic and diastolic pressure is called 'pulse pressure'.
» Haemorrhage (bleeding) is the obvious result of rupture of the blood vessels. Venous haemorrhage causes oozing of blood; arterial haemorrhage causes spurting of blood (Fig. 5.13).
Fig. 5.13: (a) Spurting of blood in arterial haemorrhage, (b) Pooling of blood in venous
haemorrhage
Vascular catastrophies are of three types:
(a) Thrombosis
(b)
Embolism

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j
- (c) Haemorrhage. All of them result in a loss of blood supply to the area of distribution of the vessel involved, unless it is compensated by collateral circulation.
° Arteriosclerosis: In old age the arteries become stiff. This phenomenon it called arteriosclerosis. This causes a variable reduction in the blood supply to the tissues and a rise in systolic pressure.
» Arteritis and Phlebitis: Inflammation of an artery is known as arteritis, and inflammation of a vein as phlebitis.
» Atheroma are patchy changes developed in the tunica intima of arteries due to accumulation of cholesterol and other lipid compounds. Arteries most commonly narrowed are those in the heart, brain, small intestine, kidneys and lower limbs. The changes are called thrombi (Fig. 5.14).
Fig. 5.14: Thrombi formation due to atheroma
» Coronary arteries blockage: These may be opened up by stents (Fig. 5.15). Blocked coronary artery may be replaced by a graft (Fig. 5.16).
»
Aneurysm is the swelling or dilation of blood vessels where part of the wall of artery inflates like a balloon. The wall of the blood vessel at the site of aneurysm is weaker and thinner than the rest of the blood vessels. Due to its likelihood to burst it poses a serious risk to health (Fig. 5.17).
Fig. 5.16: Graft for bypassing the blocked coronary artery
' Buerger's disease (thromboangitis obliterans): This is a very painful condition. There is inflammation of small peripheral arteries of the legs. The victim is a young person and a heavy smoker.
»
- Raynaud's phenomenon: In this condition there is spasmodic attack of pallor of the fingers due to constriction of small arteries and arterioles in response to cold.
Fig. 5.17: Aneurysms in circle of Willis
¢ Acute phlebothrombosis: The veins of the lower limbs are affected. Due to lack of movement of the legs there is thrombus formation with mild inflammation. This thrombus may get dislodged and flow in the blood and may block any other artery. This condition is called as embolism.
¢

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Varicose veins: When the vein wall is subjected to increased pressure over long time there is atrophy of muscle and elastic tissue with fibrous replacement. This leads to stretching of the vein with tortuosity and localized bulging. Venous congestion of the feet is relieved by putting feet on the stool, that is higher than the trunk, helping in venous return and relief in tiredness (Fig. 5.18). Varicose veins may occur at the lower end of oesophagus or in the anal canal.
Fig. 5.18: Varicose veins lower
limb in
ee
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» At times parenteral nutrition can be given through the right subclavian vein (Fig. 5.19).
- Blood vessels can be examined in the retina by ophthalmoscope, especially in cases of diabetes and hypertension.
Fig. 5.19: Parenteral nutrition through right subclavian vein
Lymphatic system is essentially a drainage system which is accessory to the venous system (Fig. 6.1).
Most of the tissue fluid formed at the arterial end of capillaries is absorbed back into the blood by the venous ends of the capillaries and the postcapillary venules. The rest of the tissue fluid (10-20%) is absorbed by the lymphatics which begin blindly in the tissue spaces.
Fig. 6.1: Lymphatic system
It is important to know that the larger particles (proteins and particulate matter) can be removed from the tissue fluid only by the lymphatics. Therefore, the lymphatic system may be regarded as 'drainage system of coarse type' and the venous system as 'drainage system of fine type'.

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eH
Certain parts of the lymphatic system (lympho-reticular organs), however, are chiefly involved in phagocytosis, raising immune responses, and contributing to cell populations of the blood and lymph.
The tissue fluid flowing in the lymphatics is called lymph. It passes through filters (lymph nodes) placed in the course of lymphatics, and finally drains into the venous blood.
Lymph from most of the tissues is clear and colourless, but the lymph from small intestine is milky-white due to absorption of fat. The intestinal milky lymph is called chyle, and lymph vessels, the Jacteals.

The lymphatic system comprises: (1) lymph vessels; (2) central lymphoid tissues; (3) peripheral lymphoid organs; and (4) circulating lymphocytes.

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The lymph capillaries begin blindly in the tissue spaces (Fig. 6.2) and form intricate networks. Their calibre is greater and less regular than that of blood capillaries, and their endothelial wall is permeable to substances of much greater molecular size (Fig. 5.5).
Lymph capillaries are absent from the cellular structures like brain, spinal cord, splenic pulp, and bone marrow.
Lymph capillaries have been compared to blood capillaries in Table 6.1.
Table
6.1.
of
lymph
and
blood
Lymph capillaries
Blood capillaries
1. Colourless, difficult to observe.
Reddish, easy to observe.
2. Blind (closed at the tip).
Joined to arterioles at one end and
to venules at another end.
3. Wider than blood capillaries.
Narrower than lymph capillaries
4. Wall consist of thin endothelium
Wall consist of normal endothelium
and poorly developed basement membrane.
and basement membrane.
5. Contain colourless lymph.
Contain red blood.
6. Have relatively low pressure.
Have relatively high pressure.
7. Absorb tissue fluid from inter- cellular spaces.
Add tissue fluid to intercellular
spaces.
es:
|
Fig. 6.2: Relation of lymphatic and circulatory system
The lymph capillaries join to form lymphatics, which are superficial and deep lymphatics. The superficial lymphatics accompany veins, while the deep lymphatics accompany arteries.
The lymph passes through filters or barriers of the regional lymph nodes which trap the particulate matter.
The filtered lymph passes through larger lymphatics and is eventually collected into two large trunks, the thoracic duct and right lymphatic duct, which pour their lymph into the brachiocephalic veins (Fig. 6.3). Thoracic duct drains both lower limbs, abdomen, left halves of thorax, head and neck and left upper limb. Right lymphatic duct drains right halves of thorax, head and neck and right upper limb.

The lymphatics anastomose freely with their neighbours of the same side as well as of the opposite side. Larger lymphatics are supplied with their vasa vasorum and are accompanied by a plexus of fine blood vessels which form red streaks seen in lymphangitis.

Central lymphoid tissues comprise bone marrow and thymus.
All 'pluripotent' lymphoid stem cells are initially produced by bone marrow, except during early fetal life when these are produced by liver

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4
and spleen. The stem cells undergo differentiation in the central lymphoid tissues, so that the lymphocytes become competent defensive elements of the immune system.
Bone marrow helps differentiation of the (committed B-lymphocytes which are capable of synthesizing antibodies after gettin; transformed into plasma cells.
In birds, B-cells are differentiated in the wall of the bursa of Fabricius a hindgut diverticulum.
Thymus helps differentiation of immunologically competent but uncommitted T-lymphocytes (10% of thymic population) which are longlived, join the circulating pool of lymphocytes, and populate the thymusdependent areas of lymph nodes and other peripheral lymphoid organs. T-cells being uncommitted can react to a wide range of foreign antigenic stimuli.
These respond by cytotoxic cell killing (killing virus-infected cells, neoplastic cells, fungi, tissue grafts, etc.), by 'arming' macrophages, and by triggering the large mononuclear cells (killer cells) and the 'helper' activity of B-lymphocytes.

Peripheral lymphoid organs comprise lymph nodes, spleen, and epithelio-lymphoid tissues (lymphoid nodules developed in the alimentary and respiratory tracts). Any part of this may become overactive on appropriate stimulation.
The progenies of Band T-lymphocytes reach these organs where the cells may proliferate and mature into competent cells. The mature lymphocytes join the circulating pool of lymphocytes.

The pool contains mature progenies of Band T-lymphocytes which may be called upon during antigenic emergencies (Roitt, 1977).

Collections of lymphocytes occur at many places in the body. Everywhere there is a basic pattern, the /ymphatic follicle. The follicle is a spherical collection of lymphocytes with a pale centre known as germinal centre, where the lymphocytes are more loosely packed.
The central cells are larger in size, stain less deeply, and divide more Tapidly, than the peripheral cells.

Lymph nodes are small nodules of lymphoid tissue found in the course of smaller lymphatics.
The lymph passes through one or more lymph nodes before reaching the larger lymph trunks.

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a
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CC
The nodes are oval or reniform in shape, 1-25 mm long, and light brown, black (pulmonary), or creamy white (intestinal) in colour.
Usually they occur in groups (cervical, axillary, inguinal, mesenteric, mediastinal, etc.), but at times there may be a solitary lymph node.
Superficial nodes are arranged along the veins, and the deep nodes along the arteries.
Cervical lymph nodes form a ring at the junction of head and neck and vertical chains in the neck (Fig. 6.4). These drain whole of head and neck. On right side lymph vessels drain into right lymphatic duct, while on left side these drain into thoracic duct. Lymph vessels of abdominal wall above a line passing horizontally through umbilicus drain into respective sides of axillary lymph nodes. Lymph vessels below this line drain into inguinal group of lymph nodes. This line is called "watershed" (Fig. 6.5).
Fig. 6.4: Lymph nodes in the neck
Each lymph node has a slight depression on one side, called hilum: The artery enters the node, and the vein with efferent lymphatic comes out of it, at the hilum.
The afferent lymphatics enter the node at different parts of its periphery.
Structurally, a lymph node is made up of the following parts (Fig. 6.6).
- 1. Fibrous and reticular framework: The lymph node is covered by a capsule. From the deep surface of the capsule a number of
trabeculae extend radially into the interior of the node, where they are continuous with the fine reticulum which forms the supporting framework for the lymphoid tissue.
Fig. 6.6. Structure of lymph node (diagrammatic)
a
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and the postcapillary venules are more abundant in the paracortical zones for lymphatic migration.
Fig. 6.7. Zones in lymph node. AL = afferent lymphatics; EL = efferent lymphatic

These are small lymphatic bodies resembling lymph nodes in their Structure, which are found in the course of blood vessels.
The afferent and efferent lymphatics are absent. Their sinuses are filled with blood rather than lymph.
These are found in some animals in relation to their abdominal and thoracic viscera.
Haemal nodes may represent an intermediate stage between a lymph node and the spleen. In man, the spleen is a large haemal node.

This system is closely related to lymphatic system because the two are independent structurally and functionally. The macrophage system is

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eke
a
made up of highly phagocytic cells which are widely distributed in the body. These cells include:
(a) Macrophages of connective tissue, reticular tissue and lungs
(b) Monocytes of blood
(c) Kupffer's cells of liver
(d) Meningocytes of meninges
(e) Microglial cells of nervous tissue
(f) Foreign body giant cells.
The endothelial cells, fibroblasts, and most leucocytes are not included in this system because of their poor power of phagocytosis.

1. The system forms first line of defence of the body against microorganisms, because of the amoeboid and phagocytic properties of its cells.
2. The macrophages of lymphoid tissue are now considered to be intimately concerned with mounting specific immune responses by the neighbouring cells.
3. Many of the prominent sites of RES are also important sites of haemopoiesis.

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Lymphoid tissue of the body is prominent at birth, and grows rapidly during childhood.
The growth ceases at about the time of puberty, and is followed by partial atrophy in the later years.
This growth pattern is shared by lymph nodes, thymus, tonsils, lymphoid tissue of the intestines, and the follicles of spleen.
However, the lymph nodes may enlarge again in response to inflammation (lymphadenitis) or tumour formation (Hodgkin's disease, lymphosarcoma, etc.).
Lymph nodes are commonly enlarged by metastasis (spread) of the malignant growths (carcinoma).

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- 1. Lymph capillaries absorb and remove the large protein molecules and other particulate matter from the tissue spaces. Thus the
SARE
MES.
cellular debris and foreign particles (dust particles inhaled into the lungs, bacteria and other microorganisms) are conveyed to the regional lymph nodes. Lymphatics (lacteals) help in transportation of fat from the gut.
2. Lymph nodes serve a number of functions.
(a) They act as filters for the lymph which percolates slowly
through the intricate network of its spaces. Thus the foreign particles are prevented from entering the bloodstream.
(b) The foreign particles are engulfed by the macrophages in the sinuses.
(c) Antigens are also trapped by the phagocytes.
(d) The mature B-lymphocytes (plasma cells capable of producing antibodies) and mature T-lymphocytes are produced in the node.
(e) Both the cellular and humoral immune responses are mounted against the antigen-laden phagocytes.
(t) The circulating lymphocytes can pass back into the lymphatic channels within the node.
(g) Humoral antibodies are freely produced by the lymph nodes.
3. Production (proliferation) and maturation of Band T-lymphocytes is the main function of lymphoid tissue.

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Lymphatics are primarily meant for coarse drainage, including cell debris and microorganisms, from the tissue spaces to the regional lymph nodes, where the foreign and noxious material is filtered off by the phagocytic activity of the macrophage cells for its final disposal by the appropriate immune responses within the node. Thus the lymphatic system forms the first line of defence of the body. While draining from an infected area, the lymphatics and lymph nodes carrying infected debris may become inflammed, resulting in lymphangitis (Fig. 6.8) and lymphadenitis. In acute cases the lymphatics are marked on the skin as painful red lines leading to the painful and tender swollen lymph nodes which may suppurate. Chronic infections (tuberculosis, syphilis, etc.) cause chronic lymphadenitis.
:
ag
Fig. 6.8: Lymphangitis and lymphadenitis
* The filarial parasite lives in the lymphatics, which may become blocked, giving rise to solid oedema (elephantiasis) in the peripheral area of drainage. Elephantiasis is characterized by enormous enlargement of the limb or scrotum (Fig. 6.9) due to the thickened skin. The microfilariae enter the blood stream only during night and, therefore, the blood for examination must be collected during night.
Fig. 6.9: Elephantiasis involving the scrotum
»
The lymphatics provide the most convenient route of spread of the cancer cells (Fig. 6.10). Therefore, the lymphatic drainage of those organs which are commonly involved in cancer should be studied in greater details and with special interest for many reasons: (a) It is helpful in the diagnosis of the primary site of the cancer.
Eee
(b) It helps in predicting the prognosis and in classifying the stage of cancer.
(c) Ithelps the surgeon in doing the block dissections during operative removal of the cancer.
The spread of cancer causes enlargement of the regional lymph nodes, which become fixed and stony hard. Many atime the primary site of cancer is quite insignificant or even difficult to define, and the enormous enlargement of the draining lymph nodes due to secondary malignant deposits forms the most prominent part of the disease. A retrograde spread of cancer cells, after the blockage of lymphatics, may occur by a reversed flow of the lymph.

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Fig. 6.10: Spread of cancer cells via the lymphatics
°
Splenomegaly is the enlargement of spleen mainly due to infections, circulatory disorders, blood diseases and malignant neoplasms. It causes excessive and premature haemolysis of red cells or phagocytosis of normal white cells and platelets leading to anaemia, leukopenia and thrombocytopenia. Spleen also may enlarge due to congestion of blood in portal venous congestion, in right-sided heart failure and in fibrosis caused due to cirrhosis of liver. Splenomegaly also occurs to meet the extra workload for removing damaged and abnormal blood cells. Commonest cause of splenomegaly is malaria.
¢Enlargement of thymus may cause myasthenia gravis, which produces extreme weakness of the skeletal muscles. It may be treated by removal of enlarged thymus, or by drug treatment.
Nervous system is the chief controlling and coordinating system of the body. It controls and regulates all activities of the body, whether voluntary or involuntary, and adjusts the individual (organism) to the given surroundings.
This is based on the special properties of sensitivity, conductivity and responsiveness of the nervous system.
The protoplasmic extensions of the nerve cells form the neural pathways called nerves. The nerves resemble the electricity wires. Like the electric current flowing through the wires, the impulses (sensory and motor) are conducted through the nerves.
The sensory impulses are transmitted by the sensory (afferent) nerves from the periphery (skin, mucous membranes, muscles, tendons, joints, and special sense organs) to the central nervous system (CNS).
The motor impulses are transmitted by the motor (efferent) nerves from the central nervous system to the periphery (muscles and glands) (Fig. 7.1).
Thus the CNS is kept continuously informed about the surroundings (environment) through various sensory impulses, both general and special.
The CNS in turn brings about necessary adjustment of the body by issuing appropriate orders which are passed on as motor impulses to the muscles, vessels, viscera and glands. The adjustment of the organism to the given surroundings is the most important function of the nervous system, without which it will not be possible for the organism to survive.

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The nervous system is broadly divided into central and peripheral parts which are continuous with each other. Further subdivisions of each part are given below.
A. Central nervous system (CNS) includes:
1. Brain or encephalon, which occupies cranial cavity, and contains the higher governing centres (Fig. 7.2).
2. Spinal cord or spinal medulla, which occupies upper twothirds of the vertebral canal, and contains many reflex centres.
Fig. 7.1: Afferent and efferent pathways through the spinal cord
Fig. 7.2: Brain and spinal cord
A
- B. Peripheral nervous system (PNS) is subdivided into the following two components.
1.
Cerebrospinal nervous system is the somatic component of the peripheral nervous system, which includes 12 pairs of cranial nerves (Fig. 7.3) and 31 pairs of spinal nerves. It innervates the somatic structures of the head and neck, limbs and body wall, and mediates somatic sensory and motor functions.
2. Peripheral autonomic nervous system is the visceral component of the peripheral nervous system, which includes the visceral or splanchnic nerves that are connected to the CNS through the somatic nerves. It innervates the viscera, glands, blood vessels and nonstriated muscles, and mediates the visceral functions.
The cerebrospinal and autonomic nervous systems differ from each other in their efferent pathways. Table 7.1 shows comparison of the two systems.
The somatic efferent pathway is made up of one neuron which passes directly to the effector organ (skeletal muscles) Neuron I axon Skeletal muscle, Table 7.1. Comparison of cerebrospinal and peripheral autonomic nervous systems.Peripheral autonomic nervous system = The autonomic efferent pathway is made up of two neurons (preganglionic and postganglionic) with an intervening ganglion for the relay of the preganglionic fibre. The effector organ (viscera) are supplied by the postganglionic fibre

The nervous tissue is composed of two distinct types of cells:
(a) The excitable cells are the nerve cells or neurons; and
(b) The non-excitable cells constitute neuroglia and ependyma in the CNS, and Schwann cells in the PNS.

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Each nerve cell or neuron has:
(a) A cell body or perikaryon, having a central nucleus and Niss] granules in its cytoplasm (Fig. 7.4).
Fig. 7.3: Central Nervous System and 31 pairs of spinal nerves
Fig. 7.4: Components of a neuron with a peripheral nerve
(b)
Cell processes
called neurites,
which are
of two
types.
short afferent
processes, which
are freely
Many branching
and varicose,
are called
dendrites.
A single long efferent process called axon, which may give off occasional branches (collaterals) and is of uniform diameter.
The terminal
branches of
the axon
are called
axon terminals
or telodendria.
The cell
bodies nuclei
in
(somata)
the
CNS, of
the and
neurons ganglia
form in
the
PNS.
grey
The matter
and cell
processes
(axons)
form tracts
in the
CNS, and
nerves in
the
PNS.
Table 7.2 shows the differences between axon and dendrite.
1. Only one axon is present in a neuron., Table 7.2. Comparison of axon and dendrite.Dendrite = Usually multiple in a neuron.

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Types of neurons: Neurons can be classified in several ways.
I. According to the number of their processes (neurites) they may be:
(a) Unipolar, e.g. mesencephalic nucleus;
(b) Pseudo-unipolar, e.g. sensory ganglia or spinal ganglia (Fig. 7.5);
(c) Bipolar, e.g. spiral and vestibular ganglia and bipolar neurons of retina.
(d) Multipolar, neurons in cerebrum and cerebellum.
II. According to the length of axon, the neurons are classified as
(a) Golgi type I neurons, with a long axon; and
(b) Golgi type II neurons (microneurons), with a short or no axon.
Fig. 7.5: Types of neurons: (a) Pseudounipolar, (b) bipolar, (¢) multipolar
Dynamic polarity: The neurons show dynamic polarity in their processes. The impulse flows towards the soma in the dendrites, and away from the soma in the axon (Fig. 7.6a). However, in certain microneurons, where the axon is absent, the impulse can flow in either direction through their dendrites.
Synapse: The neurons form long chains along which the impulses are conducted in different directions. Each junction between the neurons is called a synapse (Fig. 7.6b). It is important to know that the contact between the neurons is by contiguity and not by continuity. This is neuron theory of Waldeyer (1891). The impulse is transmitted across a synapse by specific neurotransmitters, like acetylcholine, catecholamines (noradrenalin and dopamine), serotonin, histamine, glycine, GABA and certain polypeptides.

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The most common types of the synapse are axo-dendritic, somatosomatic, somato-dendritic. In synaptic glomeruli, groups of axons make contact with the dendrites of one or more neurons for complex interactions.
Functionally, a synapse may either be inhibitory or excitatory.

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The non-excitable supporting cells of the nervous system form a major component of the nervous tissue. These cells include the following.
1. Neuroglial cells, found in the parenchyma of brain and spinal cord.
2. Ependymal cells lining the internal cavities or ventricles.
3. Capsular or satellite cells, surrounding neurons of the sensory and autonomic ganglia.
4. Schwann cells, forming sheaths for axons of peripheral nerves.
5. Several types of supporting cells, ensheathing the motor and sensory nerve terminals, and supporting the sensory epithelia.
The neuroglial cells, found in the parenchyma of brain and spinal cord, are broadly classified as :
A. Macroglia, of ectodermal (neural) origin, comprising astrocytes, oligodendrocytes, and glioblasts.
B. Microglia, of mesodermal origin.
All glial cells are much smaller but far more numerous than the nerve cells.

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Astrocytes:
As the
name suggests,
these cells
are star-shaped
because of
their numerous
processes radiating
in all
directions.
Astrocytes are
of two
types.
- Protoplasmic astrocytes, with thick and symmetrical processes are found in the grey matter.
Fibrous astrocytes, with thin and asymmetrical processes, are found in the white matter.
The processes of astrocytes often end in plate-like expansions on the blood vessels, ependyma, and pial surface of the CNS (Fig. 7.7).
The functions of various glial cells are enumerated below.

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(b)
Oligodendrocytes:
As the
name suggests
fewer cell
processes.
According to
their these
cells have
distribution, the
oligodendrocytes may
be intrafascicular,
or perineuronal.
The intrafascicular cells are found in the myelinated tracts. The perineuronal cells are seen on the surface of the somata of neurons.
- Glioblast: These are stem cells which can differentiate into macroglial cells. They are particularly numerous beneath the ependyma. (c YS
(d
Microglia:
These are
the smallest
of the
glial cells
which have
~
a
flattened
They cell
are body
often in
nature.
Microglial monocytes
with related
cells
a
few short,
to capillaries,
are which
possibly fine
and processes.
are said
derived migrate
into to
be phagocytic
from the
CNS
and early
postnatal life.

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1. They provide mechanical support to neurons.
2. Because of their non-conducting nature, the glial cells act as
the during
circulating the
late foetal

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insulators between the neurons and prevent neuronal impulses from spreading in unwanted directions.
3. They can remove the foreign material and cell debris by phagocytosis.
4. They can repair the damaged areas of nervous tissue. By proliferation (gliosis) they form glial scar tissue, and fill the gaps left by degenerated neurons.
5. Glial cells can take up and store neurotransmitters released by the neighbouring synapses. These.can either be metabolized or released again from the glial cells.
6. They help in neuronal functions by maintaining a suitable metabolic and ionic environment for the neurons.
7. Oligodendrocytes myelinate tracts.
8. Ependymal cells are concerned with exchanges of materials between brain and CSF.

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Certain dyes, when injected intravenously, fail to stain the parenchyma of brain and spinal cord, although they pass easily into the non-nervous tissues. However, the same dyes, when injected into the ventricles, enter the. brain substances easily. This indicates that a barrier exists at the capillary level between the blood and nerve cells. The possible structures constituting the blood-brain barrier are as follows.
(a) Capillary endothelium without fenestrations.
(b) Basement membrane of the endothelium.
(c) The end feet of astrocytes covering the capillary walls.
The barrier permits a selective passage of blood contents to the nervous tissue, and thus the toxic and harmful substances are ordinarily prevented from reaching the brain.

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A reflex arc is the basic functional unit of the nervous system which can perform an integrated neural activity. In its simplest form, i.e. mono-synaptic reflex arc, is made up of:
(a) A receptor, e.g. skin;
(b) A sensory or afferent neuron;
(c) A motor or efferent neuron; and
(d) An effector, e.g. muscle.
The complex forms of reflex arc are polysynaptic due to addition of one or more internuncial neurons (interneurons) in between the afferent and efferent neurons (Fig. 7.8).
An involuntary motor response of the body is called a reflex action. The stretch reflexes (tendon jerks) are the examples of monosynaptic reflexes (Fig. 7.9) whereas the withdrawal reflex (response to a painful stimulus) is a polysynaptic reflex.
Fig. 7.8: Polysynaptic reflex

The nerves are solid white cords composed of bundles (fasciculi) of nerve fibres.
Each nerve fibre is an axon with its coverings.
The nerve fibres are supported and bound together by connective tissue sheaths at different levels of organization of the nerve. The whole Nerve trunk is ensheathed by epineurium, each fasciculus by berineurium, and each nerve fibre by a delicate endoneurium. The toughness of a nerve is due to its fibrous sheaths, otherwise the nerve tissue itself is very delicate and friable (Fig. 7.10).

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There are 31 pairs of spinal nerves, including 8 cervical, 12 thoracic, 5 lumbar, 5 sacral, and 1 coccygeal.
Fig. 7.9: Reflex arc of the stretch reflex
Fig. 7.10: Fibrous support of the nerve fibres
Area of skin supplied by a single segment of spinal cord is called a dermatome (Fig. 7.11). Each spinal nerve is connected with the spinal cord by two roots, a ventral root which is motor, and a dorsal root which is sensory (Fig. 7.12).
The dorsal root is characterized by the presence of a spinal ganglion at its distal end. In the majority of nerves the ganglion lies in the intervertebral foramen.
The ventral and dorsal nerve roots unite together within the intervertebral foramen to form the spinal nerve.
The nerve emerges through the intervertebral foramen, gives off recurrent meningeal branches, and then divides immediately into a dorsal and a ventral ramus.
Fig. 7.11: Dermatomes: (a) Anterior aspect, (b) Posterior aspect
Fig. 7.12: Course of typical thoracic spinal nerve
The dorsal ramus passes backwards and supplies the intrinsic muscles of the back, and the skin covering them.
The ventral ramus is connected with the sympathetic ganglion, and is distributed to the limb or the anterolateral body wall.
In case of a typical (thoracic) spinal nerve, the ventral ramus does not mix with neighbouring rami, and gives off several muscular branches, a lateral cutaneous branch, and an anterior cutaneous branch. However, the ventral rami of other spinal nerves are plaited to form the nerve plexuses for the limbs, like the brachial plexus, lumbar plexus, etc.

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All nerve plexuses are formed only by the ventral rami, and never by the dorsal rami.
These supply the limbs.
Against each plexus the spinal cord is enlarged, e.g. 'cervical enlargement' for the brachial and 'lumbar for the plexus, enlargement' lumbosacral plexus. Plexus formation resembles a tree (Fig. 7.13).
Fig. 7.13: Nerve plexus likened to a tree
Each nerve root of the plexus (ventral ramus) divides into a ventral, a dorsal division.
The ventral division supplies the flexor compartment, and the dorsal division, the extensor compartment, of the limb.
The flexor compartment has a richer nerve supply than the extensor compartment. The flexor skin is more sensitive than the extensor skin, and the flexor muscles (antigravity, bulkier muscles) are more efficient and are under a more precise control than the coarse extensor muscles.
The plexus formation (Fig. 7.14) is a physiological or functional adaptation, and is perhaps the result of the following special features in the limbs.
. Overlapping of dermatomes . Overlapping of myotomes WN
. Composite nature of muscles
. Possible migration of muscles from the trunk to the limbs; and bk
. Linkage of the opposite groups of muscles in the spinal cord for reciprocal innervation. vA

The peripheral nerves are supplied by vessels, called vasa nervorum, which form longitudinal anastomoses on the surface ofthe nerves. The nerves distributed to the sheaths ofthe nerve trunks are called nervi nervorum.

Larger axons are covered by a myelin sheath and are termed myelinated or medullated fibres.
The fatty nature of myelin is responsible for the glistening whiteness of the peripheral nerve trunks and white matter of the CNS.
Thinner axons, of less than one micron diameter, do not have the myelin sheath and are therefore termed non-myelinated or nonmedullated (Fig. 7.15).

However, all the fibres whether myelinated or non-myelinated have a neurolemmal sheath, which is uniformly absent in the tracts. In peripheral nerves, both the myelin and neurolemmal sheaths are derived from Schwann cells.

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Myelinated fibres form the bulk of the somatic nerves. Structurally, they are made up of following parts from within outwards.
1. Axis cylinder forms the central core of the fibre. It consists of axoplasm covered by axolemma (Fig. 7.16).
2. Myelin sheath, derived from Schwann cells, surrounds the axis cylinder. It is made up of alternate concentric layers of lipids and proteins formed by spiralization of the mesaxon; the lipids include cholesterol, glycolipids and phospholipids.
Myelin sheath is interrupted at regular intervals called the nodes of Ranvier where the adjacent Schwann cells meet.
Collateral branches
of the
axon arise
at the
nodes of
Ranvier.
Thicker axons possess a thicker coat of myelin and longer internodes.
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Each internode is myelinated by one Schwann cell. Oblique clefts in the myelin, called incisures of Schmidt Lantermann, provide conduction channels for metabolites into the depth of the myelin and to the subjacent axon.
Myelin sheath acts as an insulator for the nerve fibres.
- 3. Neurolemmal sheath (sheath of Schwann) surrounds the myelin sheath.
It represents the plasma membrane (basal lamina) of the Schwann cell.
Beneath the membrane there lies a thin layer of cytoplasm with the nucleus of the Schwann cell.
The sheaths of two cells interdigitate at the nodes of Ranvier.
Neurolemmal sheath is necessary for regeneration of a damaged nerve.
Tracts do not regenerate because of absence of neurolemmal sheath.
- 4. Endoneurium is a delicate connective tissue sheath which
surrounds the neurolemmal sheath.

Non-myelinated fibres comprise the smaller axons of the CNS, in addition to peripheral postganglionic autonomic fibres, several types of fine sensory fibres (C fibres of skin, muscle and viscera), olfactory nerves, etc. Structurally, a 'non-myelinated fibre' consists of a group of small axons (0.12—2 microns diameter) that have invaginated separately 4 single Schwann cell (in series) without any spiralling of the mesaxon
SS.
(Fig.7 .15). The endoneurium, instead of ensheathing individual axons, surrounds all the neurolemmal sheath by virtue of which the non-myelinated fibres, like the myelinated fibres, can regenerate after damage.

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A.According to their function, the cranial nerves have following nuclear columns:
1 . General somatic efferent, to supply striated muscles of somatic origin, e.g. III, IV, VI, XII.
2. Special visceral efferent (branchial efferent) to supply striated muscles of branchial origin, e.g. V, VII, IX, X, XI.
3. General visceral efferent to supply smooth muscles and glands, e.g. Ill, VI, IX, X.
4. General visceral afferent, to carry visceroceptive impulses (like pain) from the viscera, e.g. X.
5. Special visceral afferent, to carry the sensation of taste, e.g. VII, IX, X.
6. General somatic afferent, to carry exteroceptive impulses from the skin of face and proprioceptive impulses from the muscles, tendons and joints (Fig. 7.17), e.g. V.
7. Special somatic afferent to carry the sensations of smell vision, hearing and equilibrium, e.g. VIII.
Fig. 7.17: Functional nuclear columns of cranial nerves
B.According to their size and speed of conduction, the nerve fibres are divided into three categories, namely A, B and C. These have been compared in Table 7.3.

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Autonomic nervous system controls involuntary activities of the body, like sweating, salivation, peristalsis, etc. It differs fundamentally from the somatic nervous system in having:
(a) The preganglionic fibres arising from the CNS;
(b) The ganglia for relay of the preganglionic fibres; and
(c) The postganglionic fibres arising from the ganglia which supply the effectors (smooth muscles and glands).
In contrast, the somatic nerves after arising from the CNS reach their destination without any interruption (Fig. 7.1).
Autonomic nervous
system is
divided into
two more
or less
complementary parts,
The the
sympathetic sympathetic
activities and
parasympathetic are
widespread systems.
and diffuse,
and combat
the acute
emergencies.
The parasympathetic activities are usually discrete and isolated, and provide a comfortable environment.
Both systems function in absolute coordination and adjust the body involuntarily to the given surroundings.

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- 1. It is also known as 'thoracolumbar' outflow because it arises from lateral horn of T1 to L2 segments of the spinal cord (Fig7.18 and © of Fig. 7.20a).
Fig. 7.18. Plan of sympathetic nervous system. Green line: afferent from viscera. Red line: preganglionic fibres. Red dotted lines: postganglionic fibres
2. The medullated preganglionic fibres (white rami communicantes) arise from the lateral column of the spinal cord, emerge through the ventral rami where the white rami are connected to the ganglia of the sympathetic chain (Fig. 7.19 and © of Fig. 7.20a).
3. Preganglionic fibres relay either in the /ateral ganglia (sympathetic chain) or in the collateral ganglia, e.g. the coeliac ganglion (© of Fig. 7.20a). The non-medullated post-ganglionic fibres run for some distance before reaching the organ of supply (© & @ of Fig. 7.20a). The adrenal medulla is a unique exception in the body; it is supplied by the preganglionic fibres (® of Fig. 7.20a).
4. Sympathetic nerve endings are adrenergic in nature, meaning thereby that noradrenalin is produced for neurotransmission. The only exception to this general rule are the cholinergic sympathetic nerves supplying the sweat glands and skeletal muscle vessels for vasodilatation.
5. Functionally, sympathetic nerves are vasomotor (vasoconstrictor), sudomotor (secretomotor to sweat glands), and pilomotor

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Samay
(contract the arrector pili and cause erection of hair) in the skin of limbs and body wall (@ & @ of Fig. 7.20a). In addition, sympathetic activity causes dilation of pupil, pale face, dry mouth, tachycardia, rise in blood pressure, inhibition of hollow viscera, and closure of the perineal sphincters.
The blood supply to the skeletal muscles, heart and brain is markedly increased.
Thus, sympathetic reactions tend to be 'mass reactions', widely diffused in their effect and that they are directed towards mobilization of the resources of the body for expenditure of energy in dealing with the emergencies or emotional crises (fright, fight, flight).
Fig. 7.19: Pathways of sympathetic and somatic nerves. Splanchnic afferent fibres (green). Sympathetic preganglionic efferent fibres (red). Sympathetic postganglionic efferent fibres (red dotted). Somatic efferent fibres (black). Somatic afferent fibres (green).

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1. It is also known as craniosacral outflow because it arises from the brain (mixed with III, VII, IX and X cranial nerves) and sacral 2-4 segments of the spinal cord. Thus it has a cranial and a sacral part.
a
Fig. 7.20(a): Sympathetic nervous system
2. The preganglionic fibres are very long, reaching right upto the viscera of supply. The ganglia, called terminal ganglia, are situated mostly on the viscera and, therefore, the postganglionic fibres are very short.
3. Parasympathetic nerve endings are cholinergic in nature, similar to the somatic nerves.
1.
2.
5.
Parasympathetic
|
- 4. Functionally, parasympathetic activity is seen when the subject is fully relaxed. His pupils are constricted, lenses accommodated, face flushed, mouth moist, pulse slow, blood pressure low, bladder and gut contracting, and the perineal sphincters relaxed.
In general the effects of parasympathetic activity are usually discrete and isolated, and directed towards conservation and restoration of the resources of energy in the body.
Table 7.4 shows the comparison between the two systems.
Table
7.4.
Comparison of
parasympathetic and
sympathetic nervous
systems nervous
system
All neurons
originate forming
from this

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system brain
cranial nerves)
segment and
(Ill,
VII,
1X,
X
S2-S4
of spinal
cord.
called
“craniosacral
Pre-ganglionic
So it
outflow”
fibres long
reaching are
very upto
terminal ganglia
mostly on
viscera
Post-ganglionic fibres
are short
- 3. Nerve endings are cholinergic in nature
4.
Functionally, it
is seen
when subject
is fully
relaxed.
Parasympathetic system has no
effect on
skin
Effect is
directed discrete,
towards and
restoration isolated,
conservation of
the resources
of energy
in the
body
- 6. It only supplies viscera
forming this
system originate
from
T, to
L, segment
of spinal
cord
So itis called "thoracolumbar outlow'"
Pre-ganglionic fibres
are short,
relay either
in lateral
ganglia ganglia
Post-ganglionic
Nerve endings
or fibres
collateral are
long are
adrenergic in
nature except
in sweat
gland
Functionally, sympathetic nerves are vasomotor, sudomotor and pilomotor to skin. it is seen when subject is in fear, fight and flight position. It dilates skeletal muscle blood vessels
Effect is
widely diffused
and directed
towards mobilization
and of
expenditure resources
of energy
during emergency
and emotional
crisis
It supplies visceral blood vessels, skin. Afferents from viscera and specific area of skin reach the same spinal segment to go to the cerebrum. Since pain is better appreciated from the skin, it appears to be coming from skin rather than the viscera. This is the basis of referred pain (Fig. 7.21)
is
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Fig. 7.21. (a) Referred pain area of skin where pain of viscera is felt;
(b)
Diagram of the way in which convergence in the dorsal horn cell may

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° Irritation of a motor nerve causes muscular spasm. Mild irritation of a sensory nerve causes tingling and numbness, but when severe it causes pain along the distribution of the nerve. Irritation of a mixed nerve causes combined effects.
°
Damage to a motor nerve causes muscular paralysis, and damage
to a sensory nerve causes localized anaesthesia and analgesia. Damage to a mixed nerve gives rise to both the sensory and motor losses.
Regeneration of a damaged nerve depends on the degree of injury, particularly on the continuity of the nerve. Different degrees of nerve injury are expressed by the following three terms.
(a) Neurapraxia is a minimal lesion causing transient functional block without any degeneration. Recovery is spontaneous and complete, e.g. sleeping foot.
(b) Axonotmesis is a lesion where, although continuity is preserved, true Wallerian degeneration occurs. Regeneration takes place in due course.
(c)
Neurotmesis is the complete division of a nerve. For regeneration to occur the cut ends must be sutured (Fig. 7.22).

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——
ts—izY
»
Severe pain along the distribution of a nerve is called neuralgia. Inflammation of a nerve is marked by neuralgia with sensory and motor deficits, and is called neuritis.
»
Denervation of a part produces trophic changes. The skin becomes dry (no sweating), smooth (loss of hair) and glazed; trophic ulcers may develop which do not heal easily. In patients with leprosy, repeated painless injuries to the tips of the fingers and toes makes them worn out and blunted.
A joint after denervation becomes a neuropathic (Charcot's) joint, which shows painless swelling; excessive mobility and bony destruction. The common medical diseases associated with trophic changes are leprosy, tabes dorsalis, and syringomyelia. The bed sores in paralysed patients are examples of the trophic ulcers. In general the ulcers and wounds in the denervated skin do not heal easily (Fig. 7.23).

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° Neuropathies is a group of diseases of peripheral nerve. It is of two types:
— Polyneuropathy: Several neurons are affected and usually long neurons like those supplying the feet and legs are affected first. This occurs mostly due to nutritional deficiencies (folic acid and vitamin B), metabolic disorders (diabetes mellitus), chronic diseases (renal and hepatic failure and carcinoma), infections (influenza, measles and typhoid fever) and toxic reactions (arsenic, lead, mercury and carbon tetrachloride)
ae
- — Mononeuropathy: Usually one neuron is affected and most common cause is ischaemia due to pressure. The resultant dysfunction depends on site and degree of injury.
* Bell's palsy is the compression of a facial nerve in or just outside stylomastoid foramen due to inflammation and oedema of the nerve. This causes paralysis of facial muscles and loss of facial expression on the affected side (Fig. 7.24).
Fig. 7.24. Bell's palsy on left side of face
* Acute idiopathic inflammatory polyneuropathy (Guillain-Barre syndrome) is a sudden, acute and progressive bilateral ascending paralysis which starts at the lower limb and then spreads to arms, trunks and cranial nerves. It is characterized by widespread inflammation with some demyelination of spinal and cranial nerves and the spinal ganglia.
¢ Syringomyelia is the dilation of the central canal of the spinal cord. Dilation of central canal develops pressure which causes progressive damage to sensory and motor neurons. Early effects are insensibility to heat and pain (dissociated anaesthesia) and in long term there is destruction of motor and sensory tracts leading to paralysis and loss of sensation and reflexes. This occurs most commonly in the cervical region and is associated with congenital abnormality of the distal end of the fourth ventricle.

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ay
¢ Ageing: Usually after 60-70 years or so there are changes in the brain. These are:
(a) Prominence of sulci due to cortical shrinkage.
(b) The gyri get narrow and sulci get broad.
(c) The subarachnoid space becomes wider. There is enlargement of the ventricles.
¢ Dementia: In this condition, there is slow and progressive loss of memory, intellect and personality. The consciousness of the subject is normal. Dementia usually occurs due to Alzheimer's disease.
Alzheimer’s disease:
The changes
of normal
aging are
pronounced in
the parietal
lobe, temporal
lobe, and
in the
hippocampus
(Fig.
7.25).
Fig. 7.25: Changes in the elderly brain
¢ Infections of brain: (a) Bacterial, (b) Viral, (c) Miscellaneous types.
(a) Bacterial (through blood) may cause meningitis or brain abscess. Otitis media may cause meningitis or temporal lobe abscess. Tuberculosis: TB meningitis is due to blood-borne infection.
(b) Viral infections: Most viruses enter through blood. Viruses may cause meningitis or encephalitis.
pee
Herpes simplex virus and encephalitis: This virus usually causes vesicles at angles of the mouth and alae of the nose, following cold or any other disease. In some cases it may cause encephalitis.
Herpes zoster: It presents as a vesicular rash affecting one or more dermatomes. This condition is very painful (Fig. 7.26).
Poliomyelitis: The virus has attraction for anterior (motor) horn cells, especially of the spinal cord which get damaged. The nerves arising, from these neurons get affected resulting in paresis or paralysis. There may be partial or complete recovery.
Fig. 7.26: Herpes zoster
(c) Miscellaneous types, i.e. infestations and infections.
1. Fungal infections: Primary infections of fungus of brain in healthy adults are rare. The fungus infections usually occur in AIDS (acquired immunodeficiency syndrome).

~ Malaria: Acute malaria by P. falciparum may cause cerebral malaria. It is very serious condition and may cause death unless treated well in time
— African sleeping sickness: The tsetse fly transmits T. brucei infection in man resulting in meningoencephalitis.
— Cysticercosis: The larvae of tapeworm (Taenia solium) may form a cyst in the brain. This cyst may cause epilepsy.

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a
- 3. Parkinson's disease: the extrapyramidal system which connects the higher centers and the anterior horn cells get affected in this disease. There is usually deficiency of neurotransmitter dopamine in the affected nuclei of the extrapyramidal system, including depigmentation of substantia nigra (Fig. 7.27).
The face is mask like and expressionless, the posture is bent forwards with stiff pill-rolling tremors of the hands.
Fig. 7.27: Gait in Parkinsonism
4. Encephalopathy: This condition occurs due to lack of vitamin B, especially in people who are chronic alcoholics.
5. Upper motor neuron damage: When the fibres are interrupted from their cortical origin till these synapse with anterior horn cells of the spinal cord. The tendon jerks are exaggerated and the plantar reflex is of the extensor type.
6. Lower motor neuron damage: When the anterior horn cells (motor neurons) are affected, usually by poliomyelitis virus, there is paresis or paralysis of the muscles supplied by the
ee nerves arising from the affected neurons. The affected muscles atrophy, and reflexes are absent.
- . Leprosy: There is chronic inflammation of the nerve sheaths. It is mostly associated with fibrosis and degeneration of the nerve fibres and autoamputation (Fig. 7.28).
Fig. 7.28: Leprosy
. Epilepsy: Epilepsy occurs in 1% population. There is focus of hyperexcitable neurons, which get induced by various types of stimuli, causing seizures. 25% cases of epilepsy are associated with some known disease, while 75% do not show any genetic influence.

1. Cutis (L); 2. Derma (G); 3. Integument. Compare with the terms cutaneous, dermatology and dermatomes.

Skin is the general covering of the entire external surface of the body, including the external auditory meatus and the outer surface of tympanic membrane.
It is continuous with the mucous membrane at the orifices of the body. Because of a large number of its functions, the skin is regarded as an important organ of the body (Fig. 8.1).

In an adult the surface area of the skin is 1.5—2 (average 1.7) sq. metres. In order to assess the area involved in burns, one can follow the rule of nine: head and neck 9%; each upper limb 9%; the front of the trunk 18%; the back of the trunk (including buttocks) 18%; each lower limb 18%; and perineum | % (Fig. 8.2).
The surface area of an individual can be calculated by Du Bois formula. Thus, A = W x H x 71.84, where A = surface area in sq. cm, W = weight in kg, and H = height in cm.

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The colour of the skin is determined by at least five pigments present at different levels and places of the skin. These are:
|
Fig. 8.1. Histological structure of skin
Fig. 8.2. Classification of burns
=
|
|. Melanin, brown in colour, present in the germinative zone of the epidermis.
2. Melanoid, resembles melanin, present diffusely throughout the epidermis.
3. Carotene, yellow to orange in colour, present in stratum corneum and the fat cells of dermis and superficial fascia.
4. Haemoglobin (purple).
5. Oxyhaemoglobin (red), present in the cutaneous vessels.
The amounts of first three pigments vary with the race, age, and gart of the body. In white races, the colour of the skin depends chiefly on the vascularity of the dermis and thickness (translucency) of the <eratin. The colour is red where keratin is thin (lips), and it is white where keratin is thick (palms and soles).

The skin is composed of two distinct layers, epidermis and dermis.

It is the superficial, avascular layer of stratified squamous epithelium. It is ectodermal in origin and gives rise to the appendages of the skin, namely hair, nails, sweat glands and sebaceous glands.
Structurally, the epidermis is made up ofa superficial cornified zone and a deep germinative zone. The cells of the deepest layer proliferate and pass towards the surface to replace the cornified cells lost due to wear and tear. As the cells migrate superficially, they become more and more flattened, and lose their nuclei to form the flattened dead cells of the stratum corneum. In the germinative zone, there are also "dopa' positive melanocytes (melanoblasts, dendritic cells, or clear cells) of neural crest origin, which synthesize melanin.

Dermis or corium is the deep, vascular layer of the skin, derived from mesoderm.

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a.
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It is made up of connective tissue (with variable elastic fibres) mixed with blood vessels, lymphatics and nerves. The connective tissue is arranged into a superficial papillary layer and a deep reticular layer.
The papillary layer forms conical, blunt projections (dermal papillae) which fit into reciprocal depressions on the undersurface of the epidermis. The reticular layer is composed chiefly of the white fibrous tissue arranged mostly in parallel bundles.
The direction of the bundles, constituting flexor or cleavage lines (Langer's lines), is longitudinal in the limbs and horizontal in the trunk and neck (Fig. 8.3).
Fig. 8.3. Flexure lines (Langer's lines)
In old age the elastic fibres atrophy and the skin becomes wrinkled. Overstretching of the skin may lead to rupture of the fibres, followed by scar formation. These scars appear as white streaks on the skin (e.g. lineae gravidarum).
At the flexure lines of the joints, the skin is firmly adherent to the underlying deep fascia. Dermis is the real skin, because, when dried it makes green hide, and when tanned it makes leather. Its deep surface is continuous with the superficial fascia.
Surface Irregularities of the Skin
The skin is marked by three types of surface irregularities, the tension lines, flexure lines and papillary ridges (Montagna and Lobitz, 1964).
.
1. Tension lines: Form a network of linear furrows which divide the surface into polygonal or lozenge-shaped areas. These lines to some extent correspond to variations in the pattern of fibres in the dermis.
. Flexure lines (skin creases or skin joints): Are certain permanent lines along which the skin folds during habitual movements (chiefly flexion) of the joints.
The skin along these lines is thin and firmly bound to the deep fascia.
The lines are prominent opposite the flexure of the joints, particularly on the palms, soles and digits (Fig. 8.4).
Fig. 8.4: Flexure lines
- . Papillary ridges (friction ridges): Are confined to palms and soles and their digits.
They form narrow ridges separated by fine parallel grooves, arranged in curved arrays.

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They correspond to patterns of dermal papillae. Their study constitutes a branch of science, called dermatoglyphics (Cummins and Midlo, 1961).
Three major patterns in the human fingerprints include loops, whorls and arches.
These patterns and many other minor features are determined genetically by multifactorial inheritance (Fig. 8.5).
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|
aa
Fig. 8.5. Close up of a finger tip showing characteristic ridges that form a fingerprint pattern unique for every individual

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Synonyms. (a) Onych or onycho (G); and (b) ungues (L). Compare with the terms paronychia, koilonychia and onychomycosis (Fig. 8.6).
Nails are hardened keratin plates (cornified zone) on the dorsal surface of the tips of fingers and toes, acting as a rigid support for the digital pads of terminal phalanges. Each nail has the following parts.
(a) Root is the proximal hidden part which is burried into the nail groove and is overlapped by the nail fold of the skin.
(b) Free border. is the distal part free from the skin.
(c) Body is the exposed part of the nail which is adherent to the underlying skin.
The proximal part of the body presents a white opaque crescent called Junule.
Fig. 8.6: Parts of a nail
Each lateral border of the nail body is overlapped by a fold ofa skin, termed the nail wall.
The skin (germinative zone + corium) beneath the root and body of the nail is called nail bed. The germinative zone of the nail bed beneath the root and lunule is thick and proliferative (germinal matrix), and is responsible for the growth of the nail.
The rest of the nail bed is thin (sterile matrix) over which the growing nail glides.
Under the translucent body (except lunule) of the nail, the corium is very vascular. This accounts for their pink colour.

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¢ In anaemia the nails are pale and white.
¢ In iron deficiency anaemia the nails become thin, brittle and spoonshaped (koilonychia).
° Hypertrophy of the nail bed (clubbing) occurs in chronic suppurative disease (lung abscess, bronchiectasis, osteomyelitis) and in severe type of cyanosis (Fallot's tetralogy, chronic congestive cardiac failure).
¢ Disturbances of nail growth due to acute illness or trauma give rise to transverse grooves in the nail substance, which move distally with the nail growth. Since the average rate of growth is about 0.1 mm per day or 3 mm per month, the date of the past illness can be estimated.
elt takes about 90-120 days for the whole nail (body) to grow. Therefore, in fungal diseases of the nails the course of treatment should last for not less than this period. The growth is faster in summer than in winter, in the fingers than in toes, and in the longer fingers than in the shorter ones.
° Hairs exhibit alterations in certain diseases. In malnutrition hairs become thin, dry and sparse; in hypothyroidism they become coarse and dry.
° Excessive growth of hair (hirsutism) occurs in adrenogenital syndrome. Loss of hair is known as alopecia.
° Skin is dry in 'Dhatura' poisoning, heat stroke, and diabetic coma; it is unusually moist in hypoglycaemic coma, and peripheral failure.

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They are larger than eccrine glands and produce a thicker secretion having a characteristic odour. They develop in close association with hair and their ducts typically open into the distal ends of the hair follicles.
Ceruminous glands of the external auditory meatus are modified apocrine sweat glands.
The apocrine glands also are merocrine in nature, but are regulated by a dual autonomic control. Some workers are not inclined to call them as sweat glands at all because they do not respond sufficiently to temperature changes.
In animals they produce chemical signals or pheromones, which are important in courtship and social behaviour.
On an average one litre of sweat is secreted per day; another 400 ml of water is lost through the lungs, and 100 ml through the faeces.
This makes a total of about 1500 ml, a rough estimate of the invisible loss of water per day.
However, in hot climates the secretion of sweat may amount to
3-10 litres per day, with a maximum of 1-2 litres per hour.
So long the sweat glands are intact, the skin can regenerate. If the sweat glands are lost, skin grafting becomes necessary.

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Sebaceous glands, producing an oily secretion, are widely distributed all over the dermis of the skin (Fig. 8.7), except for the palms and soles. They are especially abundant in the scalp and face, and are also very numerous around the apertures of the ear, nose, mouth, and anus.
Sebaceous glands are small and sacculated in appearance, made up of a cluster of about 2-5 piriform alveoli.
Their ducts open into the hair follicles, with the exception of lips, glans penis, inner surface of prepuce, labia minora, nipple and areola of the breast, and tarsal glands of the eyelids, where the ducts open on the surface of the skin.
Sebaceous glands are holocrine in nature, i.e. they produce their secretion by complete fatty degeneration of the central cells of the alveolus, which are then replaced by the proliferating peripheral cells.
The secretion
is under
hormonal control,
especially the
androgens.
The oily secretion of sebaceous glands is called sebum.
Fig. 8.7: Sebaceous gland between hair and arrector pilorum muscle
It lubricates skin and protects it from moisture, desiccation, and the harmful sun rays. Sebum also lubricates hair and prevents them from becoming brittle.
In addition, sebum also has some bactericidal action.
Sebum makes the skin water-proof. Water evaporates from the skin, but the fats and oils are absorbed by it.

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1. Protection. Skin protects the body from mechanical injuries, bacterial infections, heat and cold, wet and drought, acid and alkali, and the actinic rays of the sun.
. Sensory. Skin is sensory to touch, pain and temperature.
. Regulation of body temperature. Heat is lost through evaporation of sweat; and heat is conserved by the fat and hair.
4. Absorption. Oily substances are freely absorbed by the skin.
5. Secretion. Skin secretes sweat and sebum.
6. Excretion. The excess of water, salts and waste products are excreted through the sweat.
. Regulation of pH. A good amount of acid is excreted through the sweat.
. Synthesis. In the skin, vitamin D is synthesized from ergosterol by the action of ultraviolet rays of the sun.

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9. Storage. Skin stores chlorides.
10. Reparative. The cuts and wounds of the skin are quickly healed

Superficial fascia is a general coating of the body beneath the skin, made up of loose areolar tissue with varying amounts of fat.

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1. Fat is abundant in the gluteal region (buttocks), lumbar region (flanks), front of the thighs, anterior abdominal wall below the umbilicus, mammary gland (Fig. 8.8), postdeltoid region, and the cervicothoracic region.
2. In females, fat is more abundant and is more evenly distributed than in males.
3. Fat is absent from the eyelids, external ear, penis, and scrotum.
4. The subcutaneous layer of fat is called the panniculus adiposus.
In females fat is in the superficial fascia of the lower abdomen, upper thigh, whereas in males it is inside the abdominal cavity.
In general, in women fat forms a thicker and more even layer than in men.
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Fat (adipose tissue) fills the hollow spaces like axilla, orbits and ischiorectal fossa.
Fat present around the kidneys in abdomen, supports these organs.

- There are two types of fat, i.e. yellow and brown fat.
Most of the body fat is yellow, only in hibernating animals it is brown. The cells of brown fat are smaller with several small droplets, and multiple mitochondria.
Fat cells are specialised cells, and the size of fat cells increases during accumulation of fat, rather than the number of cells.
Any attempt to reduce excessive fat (obesity) must be slow and steady and not drastic, as the latter may cause harm to the body.

|. Superficial fascia is most distinct in the lower part of the anterior abdominal wall, perineum, and the limbs.
2. It is very thin on the dorsal aspect of the hands and feet, sides of the neck, face, and around the anus.
3. It is very dense in the scalp, palms, and soles.
4. Superficial fascia shows stratification (into two layers) in the lower part of anterior abdominal wall, perineum, and uppermost part of the thighs.

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(a) Subcutaneous muscles in the face, neck and scrotum
(b) Mammary gland
(c) Deeply situated sweat glands
(d) Localized groups of lymph nodes
(e) Cutaneous nerves and vessels.

1. Superficial fascia facilitates movements of the skin.
2. It serves as a soft medium for the passage of the vessels and nerves to the skin.
3. It conserves body heat because fat is a bad conductor of heat.
—

Deep fascia is a fibrous sheet which invests the body beneath the superficial fascia. It is devoid of fat, and is usually inelastic and tough (Fig. 8.9).
Fig. 8.9. Cross-section of an arm showing the arrangement of and fascia.
superficial deep

1. Deep fascia is best defined in the limbs where it forms tough and tight sleeves, and in the neck where it forms a collar.
2. It is ill-defined on the trunk and face.

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1. Extensions (prolongations) of the deep fascia form:
(a) The intermuscular septa which divide the limb into compartments (Fig. 8.10).
(b) The fibroareolar sheaths for the muscles, vessels and nerves.
2. Thickenings of the deep fascia form:
(a) Retinacula (retention bands) around certain joints like (Fig. 8.11) wrist and ankle.
i|
Fig. 8.10: Intermuscular septa forming compartments
Fig. 8.11: Flexor reticulum at the wrist
(b) The palmar and plantar aponeuroses for protection of nerves and blood vessels.
3. Interruptions in the deep fascia on the subcutaneous bones. Deep 'ascia never crosses a subcutaneous bone. Instead it blends with its periosteum and is bound down to the bone.

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1. Forms the intermuscular septa separating functionally different group of muscles into separate compartments (Fig. 8.10).
2. Covers each muscle as epimysium which sends in the septa to enclose each muscle fasciculus known as perimysium. From the perimysium septa pass to enclose each muscle fibre. These fine
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General
Anatomy septa are the endomysium. Through all these connective tissue septa, e.g. epimysium, perimysium and endomysium, arterioles, capillaries, venules, lymphatics and nerves traverse to reach each muscle fibre (Fig. 4.6).
3. Deep fascia covers each nerve as epineurium, each nerve fascicle as perineurium and individual nerve fibre as endoneurium. These connective tissue coverings support the nerve fibres and carry capillaries and lymphatics (Fig. 7.10).
4. Forms sheaths around large arteries, e.g., carotid sheath, axillary sheath. The deep fascia is dense around the artery and rather loose around the vein to give an allowance for the vein to distend (Fig. 8.12).
5. Modified to form the capsule, synovial membrane and bursae in relation to the joints.
6. Forms tendon sheaths wherever tendons cross over a joint. This mechanism prevents wear and tear of the tendon (Fig. 8.13).
7. In the region of palm and sole it is modified to form aponeuroses, e.g. palmar and plantar aponeuroses which afford protection to the underlying-structures (Fig. 8.14). It also forms septa between various muscles. These septa are specially well developed in the
Fig. 8.12: Carotid sheath: (a) Longitudinal section, (b) transverse section
calf muscles of lower limb. The contraction of calf muscles in the tight sleeve of deep fascia helps in pushing the venous blood and lymph towards the heart.
Thus the deep fascia helps in venous and lymphatic return from the lower limb (Fig. 8.15).
Fig. 8.13: Tendon sheaths or bursae in the palm
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Fig. 8.15: Transverse section through leg showing three compartments.
Posterior compartment is subdivided into three regions by two fascial septa
8. In the forearm and leg, the deep fascia is modified to form the interosseous membrane, which keeps:
(a) The two bones at optimum distance.
(b) Increases surface area for attachment of muscles (Fig. 8.16)
(c) Transmits weight from one bone to other.
Fig. 8.16: Interosseous membrane of the forearm
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1. Deep fascia keeps the underlying structures in position and preserves the characteristic surface contour of the limbs and neck.
2. It provides extra surface for muscular attachments.
. Ithelps in venous and lymphatic return. Bw
. It assists muscles in their action by the degree of tension and pressure it exerts upon their surfaces.
5. The retinacula act as pulleys and serve to prevent the loss of power. In such situations the friction is minimized by the synovial sheaths of the tendons.

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Skin is the outer garment of the body and is subjected to following maladies.
* Dermatitis or eczema: There is redness, swelling, itching and exudation in acute cases. It usually becomes chronic. Dermatitis may be allergic due to soaps and cosmetics.
* Albinism: There is no melanin pigment. It is usually an inherited condition.
* Herpes zoster virus: This virus causes vesicular lesion around the nasal and oral orifices and along a dermatome. It is also responsible for causing chicken pox (Fig. 7.26).
* Pressure sores: The skin slowly dies over the pressure sites, e.g. pressure sores in the lower back when patient lies on the back for prolonged periods due to illness (Fig. 7.23).
*
Burns: It is a condition which occurs due to too much heat or cold acids, alkalies and electricity, etc. If only epidermis is affected, the burn is called superficial. If both dermis and epidermis are affected the burn is called deep. Burn results in dehydration, shock and contractures.
«Benign pigmented naevus or mole: Melanin pigment cells are found in small numbers in the basal layer of skin. These neuroectodermal cells may proliferate at the dermoepidermal junction, in the dermis, to form naevi of different sizes and forms.
*
Colour: Skin is pale in anaemia, yellow in jaundice and blue in cyanosis.

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‘4
- ° Boil (furuncle): Boil is an infection and suppuration of the hair follicle and the sebaceous gland.
Skin incisions: These should be made parallel to the lines of cleavage. This will result in small scars (Fig. 8.3).
Sebaceous cyst is common in the scalp. It is due to obstruction of the duct of a sebaceous gland, caused either by trauma or infection (Fig. 8.17). If the duct of sebaceous gland of cheek is blocked, it leads to closed comedones or acne (Fig. 8.18). If the condition gets severe, the condition is acne vulgaris (Fig. 8.19).
- Scabies is a mite infection. It is commonly seen in genital region (Fig. 8.20) and in interdigital cleft (Fig. 8.21).
Fig. 8.18: Comedones or acne on the cheek
Fig. 8.21: Scabies in interdigital cleft
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(a
» Keloid is overgrowth of connective tissue at site of injury or burn (Fig. 8.22).
°
Fungal infection
of nail
is common
(Fig.
8.23).
It may
occur in
between the toes also.
Fig. 8.23: Fungal infection of the nail
« Vitiligo is an autoimmune disease leading to white patches on skin (Fig. 8.24).
Baldness is related to hormones. Alopecia areata (Fig. 8.25) is an autoimmune disease.
Deep fascia of the leg helps in venous return from the legs. The muscular contractions press on the deep veins and form an effective mechanism of venous return. This contraction becomes more effective within the tight sleeve of deep fascia.
Fig. 8.25: Alopecia areata
Planes: The deep fascia forms planes and the fluid or pus tracks along these fascial planes. The tubercular abscess of the cervical vertebrae passes along the prevertebral fascia into the posterior triangle of neck or into the axilla.

Retinacula keep the tendons and nerves in position. Sometimes the delicate nerve may get compressed as it traverses under the retinacula. Median nerve may get compressed deep to the flexor retinaculum, leading to the Carpal tunnel syndrome (Fig. 8.11).
*Similarly tibial nerve may get compressed under the flexor retinaculum of leg leading to Tarsal tunnel syndrome.
;
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Connective tissue is a widely distributed general type of tissue which supports, binds and protects the special (well differentiated) tissues of the body.
It has both the cellular and extracellular components.
The cellular component of connective tissue plays the role of active defence, whereas the extracellular component (fibres and ground substance) serves a number of mechanical functions of support and protection against the mechanical stresses and strains.
The ordinary type of connective tissue is distributed all over the body, but the special type of connective tissue forms certain well differentiated tissues, like the bone and cartilage.
A number of cell types of the connective tissue are also found in the blood and lymph.
The greater part of connective tissue develops from embryonic mesoderm.
The cells of the connective tissue are widely separated by the abundance of extracellular matrix.

Cells are fibroblast, macrophage, plasma cell, mast cell, fat cell, and pigment cell.
2
a

The matrix has a fibrous and a non-fibrous element. The fibrous element has three types of fibres — collagen, elastin and reticulin. The nonfibrous element is formed by the ground substance.

Different types of connective tissue are found in different parts of the body according to the local functional requirements. These types are based on predominance of the cell type, concentration and arrangement of the fibre type, and character of ground substance. The connective tissues are classified as follows.

Its types are: Areolar tissue, adipose tissue, myxomatous tissue and reticular tissue. These are described in Garg, Kaul, Bahl: 4 Textbook of Histology—A Colour Atlas and Text, 4 edn, CBS Publishers & Distributors, New Delhi.

1. Ordinary -tendon
2. Specialised -cartilage and bone which have been described in Chapter 2.

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1. As a packing material, connective tissue provides a supporting matrix for many highly organized structures.
2. It forms restraining mechanism of the body in the form of retinacula, check ligaments (Fig. 9.1) and fibrous pulley (Fig. 9.2).
3. The ensheathing layer of deep fascia preserves the characteristic contour of the limbs and aids circulation in the veins and lymphatics (Fig. 5.8).
4. It provides surface coating of the body in the form of superficial fascia which stores fat and conserves body heat.
5. It provides additional surface for the attachment of muscles in the form of deep fascia, intermuscular septa and interosseous membranes (Figs 8.15, 8.16).
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197
Fig. 9.1: Check ligaments in the orbit
Fig. 9.2: Fibrous pulley to change the direction of muscle
6. It forms fascial planes which provide convenient pathways for vessels (blood vessels and lymphatics) and nerves.
7. In places where it is loose in texture (loose connective tissue) it facilitates movements between the adjacent structures, and by forming bursal sacs it minimizes friction and pressure effects (Fig. 8.13).
8. Connective tissue helps in the repair of injuries whereby the fibroblasts lay down collagen fibres to form the scar tissue.
a

9. The macrophages of connective tissue serve a defensive function against the bacterial invasion by their phagocytic activity. They also act as scavengers in removing the cell debris and foreign material.
The plasma cells are capable of producing antibodies against specific antigens (foreign proteins).
The mast cells, by producing histamine and serotonin, are responsible for the various inflammatory, allergic and hypersensitivity reactions. Pigment cells protect the skin against ultraviolet radiation, so that the inflammatory changes typical of sunburn do not occur, and the chromosomal damage in the dividing cells of epidermis is avoided.
. Connective tissue contains mesenchymal cells of embryonic type. These are capable of transformation into each type of the connective tissue cells with their discrete functions.

Ligaments are fibrous bands which connect the adjacent bones, forming integral parts of the joints. They are tough and unyielding, but at the same time are flexible and pliant, so that the normal movements can occur without any resistance, but the abnormal movements are prevented.

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A.According to their composition
|. Most of the ligaments are made up of collagen fibres. These are inelastic and unstretchable (Fig. 9.3).
2. A few ligaments, like the ligamenta flava and ligaments of auditory ossicles, are made up of elastin fibres (predominantly). These are elastic and stretchable (Fig. 9.4).
B. According to their relation to the joint
1. Intrinsic ligaments surround the joint, and may be extracapsular or intracapsular.
2. Extrinsic ligaments are independent of the joint; and lie away from it (Fig. 9.5).
Connective
Tissue,
Ligaments and
Raphe
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199
Fig. 9.3: Collagenous, deltoid ligament
Fig. 9.4: Elastin, anterior ligament of malleus

Ligaments are usually considered as degenerated tendons of the related muscles.
1. Tibial collateral ligament is degenerated tendon of adductor magnus muscle.

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Fig. 9.5: Some of the extrinsic and intrinsic ligaments of knee joint
2. Sacrotuberous ligament is degenerated tendon of long head of biceps femoris.
3. Sacrospinous ligament is degenerated part of coccygeus muscle.
4. Long plantar ligament is part of peroneus longus.
Their tendinous nature is evident in some animal ancestors.

The blood vessels and nerves of the joint ramify on its ligaments and supply them.
Most ligaments serve as sense organs because of their rich nerve supply. They act as important reflex mechanisms and are important in monitoring the position and movements of the joint.

1. Ligaments are important agents in maintaining the stability at the joint.
2. Their sensory function makes them important reflex organs, so that their joint stabilizing role is far more efficient.
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201

A raphe is a linear fibrous band formed by interdigitation of the tendinous or aponeurotic ends of the muscles. It differs from a ligament in that it is stretchable.
Examples: linea alba, pterygomandibular raphe, mylohyoid raphe, median pharyngeal raphe (Fig. 9.6), anococcygeal raphe, etc.

° Collagen diseases include rheumatic fever, rheumatoid arthritis, disseminated lupus erythematosus, scleroderma, dermatomyositis, polyarteritis nodosa, and serum sickness. These are the diseases of connective tissue characterized by its fibrinoid necrosis.
»
Scleroderma is a slowly progressive rheumatic disease accompanied by vascular lesions, especially in the skin, lungs and kidneys. It is characterized by deposition of fibrous tissue in the skin. This leads to thickness and firmness of the affected areas. It is a autoimmune disease of connective tissue.

a
- ¢ Dupuytren's contracture: Occurs due to contraction of fibrous tissue of palmar aponeurosis. The disease results in flexion deformities of fingers, especially ring finger and little finger (Fig. 9.7).

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°Inflammations (fibrositis) and injuries (pulls and sprains) of the connective tissue are very painful because of its rich nerve supply or the associated muscle spasm. Relief (healing) of pain in these disorders is markedly delayed due to poor blood supply of the connective tissue.
° Marfan's syndrome is a hereditary disease causing mesodermal and ectodermal dysplasia. It is characterized by excessive height. arachnodactyly, high arched palate, dislocated eye lenses, and congenital heart disease.
° Tendons at the back of wrist are enveloped by synovial sheath. At times the sheath may form a swelling at back of wrist. This is called the "simple ganglion" (Fig. 9.8).
Fig. 9.8: Ganglion at the back of wrist ee
Tissue,
Raphe
|
203
Ganglion is a cystic swelling resulting from mucoid degeneration of synovial sheaths around the tendons.

» Undue stretching and tearing of the fibres of a ligament due to an injury is known as 'sprain'. It causes severe pain and effusion into the ligament and joint. The bones are normal as seen in the X-ray film.
¢ The joint stability is lost in the neuropathic joints, as occurs in tabes dorsalis, syringomyelia, leprosy, etc.

X-ray are a kind of electromagnetic waves which are used extensively in medicine for both diagnostic and therapeutic purposes.
All electromagnetic waves (X-rays, ultraviolet rays, infrared rays and radio waves) are produced by acceleration of electrons.

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X-rays were discovered accidentally on November 8, 1895, by Wilhelm Konrad Roentgen.
Roentgen was a German physicist from the University of Wurzburg. He was engaged in studying the behaviour of an electron beam when passed through a vacuum to strike a tungsten plate. To his surprise, he observed that, in addition to electrons, certain unknown rays were also produced, which could pass through the glass envelope of his apparatus and caused a glow on a distant fluorescent screen. He was able to photograph the bones of his hand by placing the hand over a photographic plate and then shining the rays on it. For his unique discovery, Roentgen was awarded the first Nobel Prize in Physics in 1901.
The discovery of X-rays provided a new dimension to the advancement of medical and other sciences.
The medical uses of X-rays are both diagnostic and therapeutic.
As a diagnostic tool, radiography has proved of great value in detection of the early stages of deep-seated diseases, when the possibility of cure is greatest.
Therapeutically, X-rays (radiotherapy) are used in the treatment of cancer (selected cases) because, the rays can destroy cancer cells much more easily without destroying the adjacent normal cells.

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X-rays form a part of the spectrum of electromagnetic radiation. They closely resemble the visible lightrays in having a similar photographic effect. But they differ from the lightrays in being invisible and in having a shorter wavelength. The wavelength of X-rays is 1/10,000 of that of the light rays, i.e. 7.5 x 10° — 1.71 x 10°? cm. It is this property of shorter wavelength which gives them the power of penetration of different materials.
When X-rays pass through the matter, the rays are absorbed to varying extents. The degree of absorption depends on the density (atomic weight) of the matter. Radiography is based on the differential absorption of the X-rays. Dense tissues, like the bone, absorb X-rays far more readily than do the soft tissues of the body. Structures which are easily penetrated by the X-rays are described as radiolucent, and the structures which are penetrated with difficulty or are not penetrated at all are described as radiopaque.
The various structures can be arranged in a scale of increasing radiopacity.
(a) Air, in the respiratory passages, stomach and intestines.
(b) Fat.
(c) Soft tissues, e.g. muscles, vessels, nerves, and viscera.
(d) Bones, due to their calcium content.
(e) Enamel of teeth, and
(f) Dense foreign bodies, e.g. metallic fillings in the teeth, and radiopaque contrast media.

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When X-rays strike a photosensitive film, the film gets photosensitized. When such a film is developed and fixed chemically, a radiography image is obtained.
X-ray film is made up of cellulose acetate, which is coated on its both sides with silver bromide (photosensitive) emulsion 0.001 inch thick. The film is blue tinted and transparent.
:
.
i
An X-ray image (picture) is called skiagram (skia = shadow), radiograph, or roentgenogram. Radiolucent structures produce black shadows and the radiopaque structures produce white shadows in the usual negative film. It is useful to remember that gas shadows are black shadows and bone shadows are the white shadows.

When X-rays strike certain metallic salts (phosphorous, zinc, cadmium, sulphide), the rays cause them to fluoresce, that is, light rays are produced. This property of X-rays is utilized in fluoroscopy.

X-rays can destroy abnormal cells (e.g. malignant cells) much more easily than the adjacent normal cells. This property of X-rays is utilized in the treatment of various cancers.
However, X-rays are potentially dangerous. On repeated exposures, they can cause burns, tumours, and even mutations. Therefore, adequate protective measures must be taken against repeated exposures to X-rays.

Radiographs ofa part taken in more than one view give a more complete information about the entire structure by eliminating some particular overlapping shadows in particular views.
The 'view' expresses the direction of flow of the X-rays. In AP (anteroposterior) view the rays pass from the anterior to the posterior surface, and the posterior surface faces the X-ray plate. In PA (posteroanterior) view the X-rays pass from the posterior to the anterior surface, and the anterior surface faces the X-ray plate. The part of the body facing the X-ray plate (i.e. near the X-ray plate) casts a sharper shadow than the part facing the X-ray tube.
The chest skiagrams are usually taken in PA view, but for visualizing the thoracic spine, AP view is preferred.
The 'view' can also be expressed by mentioning the surface facing the X-ray plate. Thus AP view can also be called as posterior view, and the PA view as the anterior view.

Similarly, when right surface of the body faces the plate, it is called the right lateral view, and when the left surface of the body faces the plate it is called the /eft lateral view.
Oblique and other special views are taken to visualize certain special structures.

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Fluoroscopy is of special advantage in observing the movements of the organs (lungs, stomach, intestines, etc.), and in changing the position of the subject during the examination.
Fluoroscopy is done in a dark room.
The fluoroscopic image is visualized directly on the fluorescent screen which is covered with a sheet of lead glass to absorb the X-rays and to protect the fluoroscopist.
The sharpness of the fluoroscopic image is inferior to that of a radiograph.
Fluoroscopic image is photographed by a camera in mass miniature radiography (MMR) by which the masses can be surveyed for the detection of diseases such as tuberculosis.

A natural X-ray image, obtained directly without using any contrast medium, is called a plain skiagram or a plain radiograph. Plain radiography is particularly useful in the study of normal and abnormal bones, lungs, paranasal air sinuses and gaseous shadows in the abdomen (Fig. 10.1).

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The various hollow viscera and body cavities cannot be visualized in plain radiographs due to their poor differential radiopacity. However, their contrast can be accentuated by filling such organs or cavities with either a radiopaque or a radiolucent substance. Radiography done after artificial accentuation of the contrast is called contrast radiography .
The radiopaque compounds used in contrast radiography are:
1. Barium sulphate suspension (emulsion) in water for gastrointestinal tract (Fig. 10.2).
Fig. 10.1: Plain radiography of abdomen
Fig. 10.2: The stomach and small intestine visualised by barium meal
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- 2. The aqueous solution of appropriate iodine compounds, for urinary and biliary passages and the vascular system.

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- 1. Computerized tomography (CT scanning): Computerized tomography is a major technological breakthrough in radiology, especially neuroradiology (Fig. 10.3).
It provides images comparable to anatomical slices (3-6 mm thick) of the brain, in which one can distinguish tissues with even slight differences in their radiodensity, viz. CSF, blood, white and grey matter, and the neoplasms.
Differentiation between vascular and avascular areas can be enhanced by simultaneous injection of a radiopaque medium in the vessels.
Thus CT scanning helps in the diagnosis of the exact location and size of the tumours, haemorrhage, infarction and malformations, including hydrocephalus, cerebral atrophy, etc.
This technique is also called as CAT (computerized axial tomography) scanning because it provides images in transverse, or axial plane.
Fig. 10.3: CT scan of the thorax
- 2. Ultrasonograph: Ultrasonic diagnostic echography is a safe procedure because instead of X-rays the high frequency sound waves are used. These sound waves are reflected by the acoustic
interface (different tissues) back to their source and are recorded in a polarised camera.
The sound waves used are above the range of human hearing, i.e. above 20,000 cycles per second, or 20 kilohertz (hertz = cycles per second). As the technique is quite safe, it is especially valuable in obstetric and gynaecological problems. Figures 10.4 and 10.5 show the ultrasound of the neck and thyroid gland
Fig. 10.4: Ultrasound of the neck and thyroid gland

The MRI uses a strong magnet and pulses of radiowaves. A pulse of radiowaves of the appropriate frequency displaces hydrogen nuclei from their new alignment. They return to their position immediately after the pulse ceases. At the same time they release the energy absorbed as a radio signal of the same frequency which is detected. The signal returned is proportional to the concentration of the protons. This is converted by computer into an analogue image presented ona screen. MRI can produce

Fig. 10.5: Magnetic resonance imaging of brain.
axial images resembling those of CT. It has the great advantage that images can also be produced in any other plane. This can also used for tissue characterization and blood flow imaging.

#/texts/6244 #/texts/6245 #/texts/6246 #/texts/6247 #/texts/6248 #/texts/6249

#/body #/groups/186 #/groups/186 #/groups/186 #/groups/186 #/groups/186

Interventional radiology involves a wide variety of procedures. These include:
*Percutaneous catheterization and embolisation in the treatment of tumours is done to reduce tumour size and vascularity prior to operation in difficult cases.
* Percutaneous transluminal dilatation and arterial stenosis for the treatment of localized stenosis in the arteries.
*Needle biopsy under imaging control for lung tumours and abdominal masses.
* Transhepatic catheterization of the bile ducts for draining in obstructive jaundice.
Needle puncture and drainage of cysts in the kidney or other organs using control by simple X-ray or ultrasound.

#/texts/6276 #/texts/6277 #/texts/6278 #/texts/6279 #/texts/6280 #/texts/6281 #/texts/6282 #/texts/6283 #/texts/6284 #/texts/6285 #/texts/6286 #/texts/6287 #/texts/6288 #/texts/6289 #/texts/6290 #/texts/6291 #/texts/6292 #/texts/6293 #/texts/6294 #/texts/6295 #/texts/6296 #/texts/6297 #/texts/6298 #/texts/6299 #/texts/6300 #/texts/6301 #/texts/6302 #/texts/6303 #/texts/6304 #/texts/6305 #/texts/6306 #/texts/6307 #/texts/6308 #/texts/6309 #/texts/6310 #/texts/6311 #/texts/6312 #/texts/6313 #/texts/6314 #/texts/6315 #/texts/6316 #/texts/6317 #/texts/6318 #/texts/6319 #/texts/6320 #/texts/6321 #/texts/6322 #/texts/6323 #/texts/6324 #/texts/6325 #/texts/6326 #/texts/6327 #/texts/6328 #/texts/6329 #/texts/6330 #/texts/6331 #/texts/6332 #/texts/6333 #/texts/6334 #/texts/6335 #/texts/6336 #/texts/6338 #/texts/6339 #/texts/6340 #/texts/6341 #/texts/6342

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(G)
(L)
(G)
(G)
(L)
(G)
(L)
(L)
(G)
(L)
(L)
(L)
Afferent
coming towards
Anus
ring
Artery
Articulation
joint
Arytenoid
like a pitcher
Ascites
bag-like (fluid collection)
Astrocyte
star-shaped
Atavism
a remote ancestor (epiphysis)
Atelectasis
incomplete expansion
Atheroma
tumour
Atlas
carry earth on head — Ist cervical vertebra
Atresia
no hole
Atrium
central open part
Atrophy
ill-nourished
Auditory
related to hearing
Auerbach
Auerbach’s plexus of autonomic nerves
(German anatomist)
Auricle
Auscultation
Autonomic
Autopsy
Avulsion
Axilla
Axis
between longitudinal and circular coats of
gastrointestinal tract
diminutive of ear
to hear with attention
self-controller
self-seeing
to tear away
armpit
carry (pillar)—2nd cervical vertebra
214
|
General
Anatomy

#/tables/11 #/texts/6558 #/texts/6559 #/texts/6560 #/texts/6561 #/texts/6562 #/texts/6563 #/texts/6564 #/texts/6565 #/texts/6566 #/texts/6567 #/texts/6568 #/texts/6569 #/texts/6570 #/texts/6572 #/texts/6573 #/texts/6574 #/texts/6575 #/texts/6576 #/texts/6577 #/texts/6578 #/texts/6579 #/texts/6580 #/texts/6581 #/texts/6582

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

of female breast. (L) Circular sinus Circle of Willis, 1 = sinus around pituitary arterial circle at base of brain
(L)
(G)
(G)
(L)
(L)
(G)
(L)
(L)
(G)
(L)
(G)
(G)
(G)
(L)
(L)
(L)
(L)
(G)
(L)
(L)
(L)
(G)
(L)
(G)

#/texts/6585 #/texts/6586 #/texts/6587 #/texts/6588 #/texts/6589 #/texts/6590 #/texts/6591 #/texts/6592 #/texts/6593 #/texts/6594 #/texts/6595 #/texts/6596 #/texts/6597 #/texts/6598 #/texts/6599 #/texts/6600 #/texts/6601 #/texts/6602 #/texts/6603 #/texts/6604 #/texts/6605 #/texts/6606 #/texts/6607 #/texts/6608 #/texts/6609 #/texts/6610 #/texts/6611 #/texts/6612 #/texts/6613 #/texts/6614 #/texts/6615 #/texts/6616 #/texts/6617 #/texts/6618 #/texts/6619 #/texts/6620 #/texts/6621 #/texts/6622 #/texts/6623 #/texts/6625 #/texts/6626 #/texts/6627 #/texts/6628 #/texts/6629 #/texts/6630 #/texts/6631 #/texts/6632 #/texts/6633 #/texts/6634 #/texts/6635 #/texts/6636 #/texts/6637 #/texts/6638 #/texts/6639 #/texts/6640 #/texts/6641 #/texts/6642 #/texts/6643 #/texts/6644 #/texts/6645 #/texts/6646 #/texts/6647 #/texts/6648 #/texts/6649 #/texts/6650 #/texts/6651

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Circulation motion in circle
Circum
Cirrhosis
Cisterna
Claustrum
Clavicle
Cledio
Climacteric
Clinic
Clinoid
Clitoris
Clivus
Cloaca
Clonus
Coarctation
Coccyx
Cochlea
Coelenterate
Coeliac
Coelome
Cohnheim (scientist)
Colic
Collagen
Colloidion
Colon
Colostrum
Colpotomy
Commissure
Complement
Concha
Concussion
Condyle
Conjugate
Conjunctiva
Conus
Coracoid
Corneum
Corona
around, e.g. circumflex artery to turn reddish yellow
reservoir (at base of brain)
barrier diminutive of key
closes the thorax, clavipectoral fascia step of a stair (menopause)
at the bed side surround pituitary fossa like four posts of bed
tender (female external genitalia)
slope of a hill -part of cranial fossae drain or sewer (dilated part of hindgut)
confused motion to curdle
to press together (coarctation of aorta)
cuckoo (coccyx resembles bill of a cuckoo) - lowest part of vertebral column
snail (internal ear)
hollow (internal)
belly (c. axis artery for stomach)
hollow
Cohnheim's areas in skeletal muscle pain in intestine
glue producing substance glue-like
large intestine first milk secreted by breasts
cutting through vagina join together
I fill up shell (in lat. wall of nose)
a shaking knob formed by knuclle of any joint
yoked together mucus membrane of eye
cone a crow-like process of scapula
most superficial layer of epithelium of skin crown, e.g. corona radiata and coronal suture
corona glandis
(coronary arteries,
coronary sulcus)
body, e.g. corpus callosum, corpus luteum

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

(L)
Corpuscle
a
little body,
corpuscle wrinkler
outer bark (grey matter)
rind
(outer layer)
rib violent
expulsion
Cowper’s gland
near hip
to contract
skull
(flesh)
a
non-protein from flesh
a
suspender (of testis)
a
little noise
a
chalk
(CaCO,)
congenital myxoedema sieve-like,
e.g.
cribiform ring,
e.g.
cricoid crest
cock's comb shin
bone or
leg cross-like
underground vault (hidden)
clear ice
elbow cube-like
growth wedge-shaped
dome-shaped
a
course of
study point of a spear (cusp of valve)
skin ducts
of
Cuvier one-eyed giant
bladder, cystic duct spread outside the nucleus
tear-drop finger inflammation
histamine discovered by Dale skin
scabs fascia
cartilage leg
(L)
(L)
(L)
(L)
(A)
(S)
(L)
(G)
(G)
(L)
(F)
(L)
(G)
(L)
(L)
(L)
(L)
(L)
(G)
(L)
(G)
(L)
(L)
(L)
(L)
(L)
(F)
(G)
(L)
(G)
(G)
(E)
Corrugator
Cortex
Cortex
Costa
Cough
Cowper
Coxa
Cramp
Cranium
Creatine
Cremaster
Crepitus
Creta
Cretin
Cribriform
Cricoid
Crista
Crista
Crus
Crural
Cruciate
Crypt
Crystal
Cubitus
Cuboid
Culture
Cuneiform
Cupola
Curriculum
Cusp
Cutis
Cuvier
(anatomist)
Cyclops
Cyst
Cytoplasm
Dacryocyte
Dactylitis
Dale

#/texts/6772 #/texts/6773 #/texts/6774 #/texts/6775 #/texts/6776 #/texts/6777 #/texts/6778 #/texts/6779

(English physician)
Dandruff galli
pacinian corpuscle,
thymic urethra
upper end
of male
nitrogenous substance
of thigh

#/texts/6781 #/texts/6782 #/texts/6783 #/texts/6784 #/texts/6785 #/texts/6786 #/texts/6787 #/texts/6788 #/texts/6789 #/texts/6790 #/texts/6791 #/texts/6792 #/texts/6793 #/texts/6794 #/texts/6795 #/texts/6796 #/texts/6797 #/texts/6798 #/texts/6799 #/texts/6800 #/texts/6801 #/texts/6802 #/texts/6803 #/texts/6804 #/texts/6805 #/texts/6806 #/texts/6807 #/texts/6808 #/texts/6809 #/texts/6810 #/texts/6811 #/texts/6812 #/texts/6813 #/texts/6814 #/texts/6815 #/texts/6816 #/texts/6817 #/texts/6818 #/texts/6819 #/texts/6820 #/texts/6821 #/texts/6822 #/texts/6823 #/texts/6824 #/texts/6825 #/texts/6826 #/texts/6827 #/texts/6828 #/texts/6829 #/texts/6830 #/texts/6831 #/texts/6832 #/texts/6833 #/texts/6834 #/texts/6835 #/texts/6836 #/texts/6837 #/texts/6838 #/texts/6839 #/texts/6840 #/texts/6841 #/texts/6842 #/texts/6843 #/texts/6844 #/texts/6845 #/texts/6846 #/texts/6847 #/texts/6848 #/texts/6849 #/texts/6850 #/texts/6851 #/texts/6852 #/texts/6853 #/texts/6854 #/texts/6855 #/texts/6856 #/texts/6857 #/texts/6858 #/texts/6859 #/texts/6860 #/texts/6861 #/texts/6862 #/texts/6863 #/texts/6864 #/texts/6865 #/texts/6866 #/texts/6867 #/texts/6868 #/texts/6869 #/texts/6870 #/texts/6871 #/texts/6872 #/texts/6873 #/texts/6874 #/texts/6875 #/texts/6876 #/texts/6877 #/texts/6878 #/texts/6879 #/texts/6880 #/texts/6881 #/texts/6882 #/texts/6883

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(S)
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(G)
(L)
(F)
(G)
(L)
(L)
(Gr)
(L)
(L)
(L)
(G)
(G)
(G)
(G)
(G)
(G)
(G)
(G)
(G)
(G)
(G)
(G)
(G)
Dartos
Darwin tubercle
Decalcify
Deci
Deciduous
Decompression
Decubitus
Decussate
Defecate
Degenerate
Deglutition
Deiters
Deltoid
Dementia
Demilune
Dendron
Denonvilliers
(French surgeon)
Dens
Dentate
Dermis
Descemet
Desquamate
Detrusor
Dexter
Diagnosis
Dialysis
Diapedesis
Diaphragm
Diaphysis
Diarrhoea
Diarthrosis
Diastole
Diathermy
Dichotomous
Didelphys
Diencephalon
Diet
leather
projection in upper part of ear
process which extracts Ca**
one tenth
falling off
decreased pressure
lying down
intersection of two lines
to evacuate the bowels
structural impairment of a tissue
action of swallowing
Deiters cells in internal ear
triangular in shape, deltoid muscle, deltoid
ligament
to be mad
half moon, demilune of Giannuzzi
tree, dendrites of neuron
fascia between rectum and prostate is
Denonvilliers’ fascia
tooth-like dens of 2nd vertebra/axis
tooth-like, dentate lig. of pia mater, dentate
gyrus of brain
skin
Descemet’s membrane of cornea
to scale off
to thrust away, detrusor muscle of urinary
bladder
right
thro knowledge
to loose from one another
leading through

#/texts/6884 #/texts/6885 #/texts/6886 #/texts/6887 #/texts/6888 #/texts/6889 #/texts/6890 #/texts/6891 #/texts/6892 #/texts/6893

#/groups/188 #/groups/188 #/groups/188 #/groups/188 #/groups/188 #/groups/188 #/groups/188 #/groups/188 #/groups/188 #/groups/188

a partition
growing through
flowing through
joint
a pause
very hot
cut in half
double uterus
between brain
a way of living

#/texts/6895 #/texts/6896 #/texts/6897 #/texts/6898 #/texts/6899 #/texts/6900 #/texts/6901 #/texts/6902 #/texts/6903 #/texts/6904 #/texts/6905 #/texts/6906 #/texts/6907 #/texts/6908 #/texts/6909 #/texts/6910 #/texts/6911 #/texts/6912 #/texts/6913 #/texts/6914 #/texts/6915 #/texts/6916 #/texts/6917 #/texts/6918 #/texts/6919 #/texts/6920 #/texts/6921 #/texts/6922 #/texts/6923 #/texts/6924 #/texts/6925 #/texts/6926 #/texts/6927 #/texts/6928 #/texts/6929 #/texts/6930 #/texts/6931 #/texts/6932 #/texts/6933 #/texts/6934 #/texts/6935 #/texts/6936 #/texts/6937 #/texts/6938 #/texts/6939 #/texts/6940 #/texts/6941 #/texts/6942 #/texts/6943 #/texts/6944 #/texts/6945 #/texts/6946 #/texts/6947 #/texts/6948 #/texts/6949 #/texts/6950 #/texts/6951 #/texts/6952 #/texts/6953 #/texts/6954 #/texts/6955 #/texts/6956 #/texts/6957 #/texts/6958 #/texts/6959 #/texts/6960 #/texts/6961 #/texts/6962 #/texts/6963 #/texts/6964 #/texts/6965 #/texts/6966 #/texts/6967 #/texts/6968 #/texts/6969 #/texts/6970 #/texts/6971 #/texts/6972 #/texts/6973 #/texts/6974 #/texts/6975 #/texts/6976 #/texts/6977 #/texts/6978 #/texts/6979 #/texts/6980 #/texts/6981 #/texts/6982 #/texts/6983 #/texts/6984 #/texts/6985 #/texts/6986 #/texts/6987 #/texts/6988 #/texts/6989 #/texts/6990 #/texts/6991 #/texts/6992 #/texts/6993 #/texts/6994 #/texts/6995 #/texts/6996 #/texts/6997 #/texts/6998 #/texts/6999 #/texts/7000 #/texts/7001 #/texts/7002 #/texts/7003 #/texts/7004

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@
(L)
(L)
(G)
(G)
(F)
(L)
(L)
(G)
(L)
(L)
(G)
(L)
(It)
(L)
(L)
(L)
(G)
(G)
(G)
(G)
(G)
(L)
(L)
(G)
(G)
(G)
(G)
(G)
(L)
(L)
(L)
(F)
Digastric
Digestion
Digit
Diphtheria
Diploe
Disease
Dislocation
Dissect
Diuresis
Diverticulum
Doctor
Dolichocephalic
Dorsum
Douche
Douglas
(Scottish anatomist)
Duct
Duodenum
Dupuytren
Duramater
Dys
(prefix)
Ectoderm
Ectopia
Ectropion
Eczema
Edinger
Effector
Efferent
Element
Elephantiasis
Embed
Embolus
Embryo
Emesis
Emissary
Empirical
Emulsion
Enamel
double belly
finger
leather-like membrane
double layers (deiploe of some of the skull
bones)
not well
pulled out of place
cut apart
increased urination
a small cul-de-sac
a teacher and a healer
long head
back
to pour
pouch of Douglas (rectouterine pouch)
to conduct
width of 12 fingers
Dupuytren’s contracture
hard mother
bad, e.g. dysentery, dyspepsia,
dysmenorrhoea
outside skin
displacement (out of place)
to turn from
anything thrown out by heat
Edinger-Westphal nuclei of III N
to effect
going away
a rudiment
elephant-like legs
holding in place
a plug
something that grows in another’s body
vomiting
escape channels (emissary veins connecting

intracranial sinuses with extracranial veins)
experienced, not scientific
milk-like mixture
coating on metal

(G), 2 = upon nipple. (G), 1 = Epoophoron. (G), 2 = upon egg-bearing. , 1 = Erb. , 2 = Erb's palsy (C 5, 6). , 1 = (German neurologist). , 2 = . (L), 1 = Erector. (L), 2 = to stand up. (G), 1 = Erotic. (G), 2 = love. (L), 1 = Eructation. (L), 2 = throwing upwards. (G), 1 = Ethmoid. (G), 2 = sieve-like. (G), 1 = Etiology. (G), 2 = giving the cause. (G), 1 = Etymology. (G), 2 = true analysis of a word. (G), 1 = Euthanasia. (G), 2 = painless death. (L), 1 = Eustachius. (L), 2 = eustachian valve. , 1 = (Italian anatomist). , 2 = . (L), 1 = Evolution. (L), 2 = to unroll. (G), 1 = Exacerbation. (G), 2 = to irritate. (L), 1 = Exogenous. (L), 2 = on the outside
|
Sa

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#/body #/body #/body #/body #/body #/body #/body #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190

;
Anatomical
Word
Meanings and
Historical
Names
225
(L)
(L)
(L)
(L)
(L)
(L)
(G)
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(L)
()
()
(L)
(G)
(G)
(G)
(G)
(G)
(G)
(L)
(L)
(F)
(L)
(L)
()
(G)
(G)
(G)
Infant
Infarct
Infection
Inflame
Infra
Inguinal
Inion
Injection
Innominate
Inoculate
Inquest
Insanity
Insemination
In
situ
Instrument
Insufflation
Insula
Internuncial
Intestine
Intoxication
Intra
Intrinsic
Intussusception
Invagination
Involution
Iodine
Iris
Ischaemia
Ischium
Isotonic
Isotope
Isthmus
Iter
Jaundice
Jejunum
Joint
Jugular
Jugam
Karyo
Keith
(English anatomist)
Keratin
Kilo
not speaking
necrotic
a bending inward
to set aflame
below
the groin—inguinal canal
below occiput
putting in
unnamed
to ingraft
inquire
unsound mind
seed
manner of lying in local position
to equip
to blow into
island
inter-messenger
internal
to smear with poison
inside
special to the thing itself
within receive
enclose in a sheath
to roll up
violet
coloured membrane of eye
lack of blood supply
bone (part of hip bone)
equal tension
equal place
narrow, isthmus of fallopian tube
a passage (iter cerebri)
yellowness
empty or fasting
to join
the throat
yoke

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#/groups/190 #/groups/190 #/groups/190 #/groups/190 #/groups/190

a nut (nucleus)
Keith’s SA node
horn-in hairy
layer of skin
one thousand

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

228
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(G)
(G)
(G)
(Gr)
(Gr)
(Gr)
(Gr)
(G)
(G)

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Mast to feed
Mastos
Mastication
Matrix
Mature
Maxilla
McBurney
(American surgeon)
Meatus
Meckel
Meatus
Median
Mediastinum
Medicine
Medulla
Medusa
(Greek goddess)
Mega
Meibom
Meiosis
Meissner
Melan
Melanin
Meninges
Meningocoele
Meniscus
Men
(prefix)
Merkel
Mesencephalon
Mesenchyme
Mesentery
Mesoderm
Mesonephros
Mesothelium
(G)
Metacarpus
(G) Metamorphosis
(G) Metanephros
(G)
Metastasis
(G)
(G)
Metencephalon
Metopic breast
to chew mould
to ripe—maturation
cheek
McBurney point for appendicectomy canal
Meckel's cave for 5th N. ganglion, Meckel's cartilage
canal central
middle space the art of healing
marrow caput medusa
big meibomian glands
lessening
Meissner's plexus in submucous coat of GIT
black black pigment
a membrane memb. + hernia
crescent (med. cat. menisci in knee joint)
month, e.g.
menopause, menorrhagia.
menstruation, menarche corpuscle sensory nerve ending
mid-brain middle infusion or juice
middle intestine middle skin
middle kidney middle nipple
from wrist changed form
after kidney removal from one place
after brain frontal,
space between
eyes—M.
suture

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Petit's triangle. Petit, 1 = . Petit, 2 = . (French surgeon) (L), 1 = Petrous. (French surgeon) (L), 2 = stony, rock, P. temporal bone. Peyer, 1 = . Peyer, 2 = . (Swiss, 1 = anatomist). (Swiss, 2 = Peyer's patch (ileum). (G), 1 = Phagocytosis Phalanx. (G), 2 = eat + cell + osis (fullness), i.e., eating cells. (G) (G), 1 = Pharynx. (G) (G), 2 = closely knit row musculo memb. sac behind the mouth. (L), 1 = Philtrum. (L), 2 = a love charm. (Gr), 1 = Phimosis. (Gr), 2 = stopping up (in relation. (Gr), 1 = . (Gr), 2 = to prepuce of penis) sound or voice. , 1 = Phonation. , 2 = 
(Gr)
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(Gr)
(Gr)
(Gr)
(Gr)
(L)
(L)
(L)
(L)
(Gr)
(Gr)
(L)
(Gr)
(Gr)
(L)
(L)
(L)
(L)
(L)
(L)
(Gr)
(Gr)
(L)
(L)
(Gr)
(Gr)
(L)
(Gr)

#/texts/8502 #/texts/8503 #/texts/8504 #/texts/8505 #/texts/8506 #/texts/8507 #/texts/8508 #/texts/8509 #/texts/8510 #/texts/8511 #/texts/8512 #/texts/8513 #/texts/8514 #/texts/8515 #/texts/8516 #/texts/8517 #/texts/8518 #/texts/8519 #/texts/8520 #/texts/8521 #/texts/8522 #/texts/8523 #/texts/8524 #/texts/8525 #/texts/8526 #/texts/8527 #/texts/8528 #/texts/8529 #/texts/8530 #/texts/8531 #/texts/8532 #/texts/8533 #/texts/8534 #/texts/8535 #/texts/8536 #/texts/8537 #/texts/8538 #/texts/8539 #/texts/8540 #/texts/8541 #/texts/8542 #/texts/8543 #/texts/8544 #/texts/8545 #/texts/8546 #/texts/8547 #/texts/8548 #/texts/8549 #/texts/8550 #/texts/8551 #/texts/8552 #/texts/8553 #/texts/8554 #/texts/8555 #/texts/8556 #/texts/8557 #/texts/8558 #/texts/8559 #/texts/8560 #/texts/8561 #/texts/8562 #/texts/8563 #/texts/8564 #/texts/8565 #/texts/8566 #/texts/8567 #/texts/8568 #/texts/8569 #/texts/8570 #/texts/8572

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Physiotherapy nature + treatment
Pia mater
Pineal
Pinna
Piriform
Pisiform
Pituitary
Placenta
Planes
Plantar
Platysma
Pleo
Plethora
Pleura
Plexus
Plica
Plumbus
Pneumo
Podagra
Podalic
Polarity
Polio
Pollex
Poly
Pons
Popliteus
Porta
Post
Poupart
(French anatomist)
Pregnant
Prepuce
Presbyopia
Proposis
Process
Procidentia
Prodo
Prodromal
Progesterone
Prognosis
Prolapse
Proliferate
Promontory soft mother
pine cone ear
pear-shaped pea-shaped
mucus secretion flat
cake flat
sole of
foot flat
more fullness
serous memb.
woven to
fold lead
gas foot
foot relating
to pole
grey matter + inflammation thumb (strong)
many bridge
ham gate
behind
Poupart's ligament (inguinal ligament)
with child
foreskin old age hypermetropia
elephant's trunk advance
parts that fall out of place anus
in advance before to bear
to know beforehand falling
create or reproduce in quick succession prominence
enfolding lung
234

#/tables/14 #/texts/8750 #/texts/8751 #/texts/8752 #/texts/8753 #/texts/8754 #/texts/8755 #/texts/8756 #/texts/8757 #/texts/8758 #/texts/8759 #/texts/8760 #/texts/8761 #/texts/8762 #/texts/8763 #/texts/8764 #/texts/8765

Pronator = Puke. (L), to bend forward = beating. (L), Pronator = Pulvinar. (L), to bend forward = cushion, pillow. (L), Pronator = Punctum. (L), to bend forward = point. (L), Pronator = Putamen. (L), to bend forward = cutting. (Gr), Pronator = Pyelo. (Gr), to bend forward = basin. CGr), Pronator = Pylorus. CGr), to bend forward = gatekeeper. (Gr), Pronator = Pyramid. (Gr), to bend forward = swelling. (Gr), Pronator = Pyrexia. (Gr), to bend forward = fever. (L), Pronator = Quadri. (L), to bend forward = four. , Pronator = Quadratus. , to bend forward = square. , Pronator = Quadriceps/quadri. , to bend forward = geminus four heads/two twins. (G), Pronator = Quartz. (G), to bend forward = rock crystal. (L), Pronator = Rabies. (L), to bend forward = rage/madness. (L), Pronator = Racemose. (L), to bend forward = cluster of grapes (glands). (Gr), Pronator = Rachitis. (Gr), to bend forward = spine. (L), Pronator = Radical. (L), to bend forward = roots. (L), Pronator = Radium. (L), to bend forward = radioactive element. (L), Pronator = Radius. (L), to bend forward = small bone of forearm. (L), Pronator = Ramus. (L), to bend forward = branch
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(L)
(Gr)
(Gr)
(Gr)
(Gr)
(Gr)
(L)
(L)
(L)

#/texts/8767 #/texts/8768 #/texts/8769 #/texts/8770 #/texts/8771 #/texts/8772 #/texts/8773 #/texts/8774 #/texts/8775 #/texts/8776 #/texts/8777 #/texts/8778 #/texts/8779 #/texts/8780 #/texts/8781 #/texts/8782 #/texts/8783 #/texts/8784 #/texts/8785 #/texts/8786 #/texts/8787 #/texts/8788 #/texts/8789 #/texts/8790 #/texts/8791 #/texts/8792 #/texts/8793 #/texts/8794 #/texts/8795 #/texts/8796 #/texts/8797 #/texts/8798 #/texts/8799 #/texts/8800 #/texts/8801 #/texts/8802 #/texts/8803 #/texts/8804 #/texts/8805 #/texts/8806 #/texts/8807 #/texts/8808 #/texts/8809 #/texts/8810 #/texts/8811 #/texts/8812 #/texts/8813 #/texts/8814 #/texts/8815 #/texts/8816 #/texts/8817 #/texts/8818 #/texts/8819 #/texts/8820 #/texts/8821 #/texts/8822 #/texts/8823 #/texts/8824 #/texts/8825 #/texts/8826 #/texts/8827 #/texts/8828 #/texts/8829 #/texts/8831 #/texts/8832 #/texts/8833 #/texts/8834 #/texts/8835 #/texts/8836 #/texts/8837 #/texts/8838 #/texts/8839 #/texts/8840 #/texts/8841 #/texts/8842 #/texts/8843 #/texts/8844 #/texts/8845 #/texts/8846 #/texts/8847 #/texts/8848 #/texts/8849 #/texts/8850 #/texts/8851 #/texts/8852 #/texts/8853 #/texts/8854 #/texts/8855

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/groups/191 #/groups/191 #/groups/191 #/groups/191 #/groups/191 #/groups/191 #/groups/191 #/groups/191 #/groups/191 #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Ranvier node of Ranvier
(French histologist)
Raphe
Rash
Rathke
Receptor
Rectum
Recurrent
Refraction
Reid
Reil
(Greek physiologist)
Reissner
(Greek anatomist)
Remarc
(German neurologist)
Resection
Resin
Restiform body
Rete
Reticulum
Retinaculum
Retort
Retro (prefix)
Retzius
(Swedish scientist)
Rheumatism
Rhinencephalon
Rhinoplasty
Rhomboid
Rhonchus
Rigor
Rima
Risus
Robertson
(Scottish ophthalmologist)
Robin
(German histologist)
suture eruption of skin
Rathke's pouch to receive
upright (misnomer)
running back broken (bend)
R. base (from lower margin of orbit through centre of ext. aud. meatus)
island of Reil-Insula
R. fibres running through the length of brainstem and spinal cord
R. fibre/non-medullated nerve fibre cut off
to flow rope-shape (inferior cerebellar peduncle)
anet, R. mirabile — a wonderful network, R. testis — tubular network
a little net to hold back
twisted back behind, retroverted, retroflexed uterus, retroph.
space space of Retzius
a liability
nose + brain
nose moulding
rhombus-like
snoring
rigidity
slit, rima glottidis
to laugh, risus sardonicus
Argyll Robertson pupil (pupil sign)
perivas/space in brain
(L)
(L)
(L)
(L)
(L)
(L)
(Gr)
(Gr)
(Gr)
(L)
(L)
(Gr)
(Gr)
(L)
(Gr)
(L)
(L)
(Gr)
(Gr)
(L)
(L)
(L)
(L)
(L)
(L)

#/texts/8856 #/texts/8857 #/texts/8858 #/texts/8859 #/texts/8860 #/texts/8861 #/texts/8862 #/texts/8863 #/texts/8864 #/texts/8865 #/texts/8866 #/texts/8867 #/texts/8868 #/texts/8869 #/texts/8870 #/texts/8871 #/texts/8872 #/texts/8873 #/texts/8874 #/texts/8875 #/texts/8876 #/texts/8877 #/texts/8878 #/texts/8879 #/texts/8880 #/texts/8881 #/texts/8882 #/texts/8883 #/texts/8884 #/texts/8885 #/texts/8886 #/texts/8887 #/texts/8888 #/texts/8889 #/texts/8890 #/texts/8891 #/texts/8892 #/texts/8893 #/texts/8894 #/texts/8895 #/texts/8896 #/texts/8897 #/texts/8898 #/texts/8899 #/texts/8900 #/texts/8901 #/texts/8902 #/texts/8903 #/texts/8904 #/texts/8905 #/texts/8906 #/texts/8907 #/texts/8908 #/texts/8909 #/texts/8910 #/texts/8911 #/texts/8912 #/texts/8913 #/texts/8914 #/texts/8915 #/texts/8916 #/texts/8917 #/texts/8918 #/texts/8919 #/texts/8920 #/texts/8921 #/texts/8922 #/texts/8923 #/texts/8924 #/texts/8925 #/texts/8926 #/texts/8927

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/groups/192 #/groups/192 #/groups/192 #/groups/192 #/groups/192 #/groups/192 #/groups/192 #/groups/192 #/groups/192 #/groups/192 #/groups/192 #/groups/192 #/groups/192

(L)
(L)
(Gr)
Rolando
(Italian anatomist)
Rosenmuller
Rostrum
Rebella
Ruffini
Rugat
Saccharin
Sacrum
Sagittal
Salpina
Santorini
Saphenous
Sarco
Sartorius
Scala
Scalenus
Scalpel
Scaphoid
Scapula
Scarpa
Italian anatomist)
Schizophrenia
Schlemm
Schwann
Sciatica
Scirrhus
Sclera
Scoliosis
Scrotum
Sebum
Segmentation
Sella turcica
Semen
Semi
Septum
Serratus
Sesamoid fissure of Rolando
fossa of Rosenmuller (Lat. pharyngeal recess)
beak of a bird red
nerve endings of skin wrinkled
sugar holy
arrow trumpet
Santorini's cartilage, Santorini's duct accessory duct of pancreas
clear, easily seen. S. vein flesh, sarcolemma
tailor stairway (scala tympani, scala vestibuli)
irregular knife
boat-shaped shoulder blades
Scarpa's fascia, (deeper membranous layer of sup. fascia) Scarpa's ganglia 8th N. ganglia
split mind
Schlemm's canal at corneo-scleral junction cell of Schwann
pain in loins
hard
hard
curvature (lateral)
skin or hide
grease
to cut
Turkish saddle
that which is sown
half
a dividing wall
like saw
seed-like (patella)

#/texts/8929 #/texts/8930 #/texts/8931 #/texts/8932 #/texts/8933 #/texts/8934 #/texts/8935 #/texts/8936 #/texts/8937 #/texts/8938 #/texts/8939 #/texts/8940 #/texts/8941 #/texts/8942 #/texts/8943 #/texts/8944 #/texts/8945 #/texts/8946

#/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193 #/groups/193

instrument of listening the ausculatory sounds
to prick
mouth
mouth bed
squinting
covering
furrow
contraction
harsh
bed
pointed
under
under clavicle
essence
sweat
furrow
over, above
bent backwards

#/texts/9071 #/texts/9072 #/texts/9073 #/texts/9074 #/texts/9075

#/groups/194 #/groups/194 #/groups/194 #/groups/194 #/groups/194

calf of leg
hand + work (operator)
support (sustentacular tali)
sewing together
Sylvius’ fissure, Lat. fissure of the brain

#/texts/9307 #/texts/9308 #/texts/9309 #/texts/9310 #/texts/9311 #/texts/9312 #/texts/9313 #/texts/9314 #/texts/9315 #/texts/9316 #/texts/9317 #/texts/9318 #/texts/9319 #/texts/9320 #/texts/9321 #/texts/9322 #/texts/9323 #/texts/9324 #/texts/9325 #/texts/9326 #/texts/9327 #/texts/9328 #/texts/9329 #/texts/9330 #/texts/9331 #/texts/9332 #/texts/9333 #/texts/9334 #/texts/9335 #/texts/9336 #/texts/9337 #/texts/9338 #/texts/9339 #/texts/9340 #/texts/9341 #/texts/9342 #/texts/9343 #/texts/9344 #/texts/9345 #/texts/9346 #/texts/9347 #/texts/9348 #/texts/9349 #/texts/9350 #/texts/9351 #/texts/9352 #/texts/9353 #/texts/9354 #/texts/9355 #/texts/9356 #/texts/9357 #/texts/9358 #/texts/9359 #/texts/9360 #/texts/9361 #/texts/9362 #/texts/9363 #/texts/9364 #/texts/9365 #/texts/9366 #/texts/9367 #/texts/9368 #/texts/9369 #/texts/9370 #/texts/9371 #/texts/9372 #/texts/9373 #/texts/9374 #/texts/9375 #/texts/9376 #/texts/9377 #/texts/9378 #/texts/9379 #/texts/9380 #/texts/9381 #/texts/9382 #/texts/9383 #/texts/9384 #/texts/9385 #/texts/9386 #/texts/9387 #/texts/9388 #/texts/9389 #/texts/9390 #/texts/9391 #/texts/9392 #/texts/9393 #/texts/9394 #/texts/9395 #/texts/9396 #/texts/9397 #/texts/9398 #/texts/9399 #/texts/9400 #/texts/9401 #/texts/9402 #/texts/9403 #/texts/9404 #/texts/9405 #/texts/9406 #/texts/9407 #/texts/9408 #/texts/9409 #/texts/9410 #/texts/9411 #/texts/9412 #/texts/9413 #/texts/9414 #/texts/9415 #/texts/9416 #/texts/9417 #/texts/9418 #/texts/9419 #/texts/9420

#/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/groups/195 #/body #/body

Gr)
(Gr)
(Gr)
(L)
(L)
(Gr)
(Gr)
()
(Gr)
(Gr)
(L)
(Gr)
(Gr)
(L)
«)
Gr)
(Gr)
(L)
(Gr)
(L)
(Gr)
(Gr)
(L)
(Gr)
(Gr)
(L)
a)
(L)
(L)
Tissue
Tone
Tooth
Topography
Torticollis
Torus
Toxin
Trabeculae
Trachea
Tract
Tragus
Transfusion
Trapezium
Trauma
Treitz.
(Austrian physician)
Trema
Tremor
Trendelenburg
(German surgeon)
Trephine
Treves
(English surgeon)
Triceps
Trichiasis
Tricuspid
Trigone
Triquetral
Trocar
Trochanter
Trochlea
Trophic
Tropism
Tube
Tumour
Turbinate
Tympanum
Typhoid
Tyson
woven
which can be stretched
an organ of mastication
a place + description
twisted
bulging place
instrument for turning
poison
a little beam
wind pipe
pathway
ear
pouring out
table
wound
lig of Treitz at duo-jej.flexure
hole
shaking
Trendelenburg’s position, Trendelenburg’s
sign and Trendelenburg’s test
a saw for cutting out circular piece of bone
esp. skull
bloodless fold of Treves
having three heads
hair (trichionis)
three cusps
three + twin-like (3 divisions)
triangle
having 3 corners
3 quarters
bony process
pulley
nourishment (trophoblast)
burning
a trumpet
swelling
spinning top
kettle drum
fever typhus like
Tyson’s glands, seb. glands on inner side of
prepuce
240
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#/texts/9535 #/texts/9536 #/texts/9537 #/texts/9538 #/texts/9539 #/texts/9540 #/texts/9541 #/texts/9542 #/texts/9543 #/texts/9544 #/texts/9545 #/texts/9546 #/texts/9547 #/texts/9548 #/texts/9549 #/texts/9550 #/texts/9551 #/texts/9552 #/texts/9553 #/texts/9554 #/texts/9555 #/texts/9556 #/texts/9557 #/texts/9558 #/texts/9559 #/texts/9560 #/texts/9561 #/texts/9562 #/texts/9563 #/texts/9564 #/texts/9565 #/texts/9566 #/texts/9567 #/texts/9568 #/texts/9569 #/texts/9570 #/texts/9571 #/texts/9572 #/texts/9573 #/texts/9574 #/texts/9575 #/texts/9576 #/texts/9577 #/texts/9578 #/texts/9579 #/texts/9580 #/texts/9581 #/texts/9582 #/texts/9583 #/texts/9584 #/texts/9585 #/texts/9586 #/texts/9587 #/texts/9588 #/texts/9589 #/texts/9590 #/texts/9591 #/texts/9592 #/texts/9593 #/texts/9594 #/texts/9595 #/texts/9596 #/texts/9597 #/texts/9598 #/texts/9599 #/texts/9600 #/texts/9601 #/texts/9602

#/body #/body #/body #/body #/body #/body #/body #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196

Word
Names
|
241
(L)
(L)
(L)
(L)
(L)
()
(L)
()
()
(L)
(L)
()
(L)
(L)
(L)
(L)
()
()
Vesalius
(Belgian anatomist)
Vesica
Vestibule
Vestigeal
Veterinary
Vidius
(Italian physician)
Villus
Virchow
(German pathologist)
Virus
Viscus/viscera
Vision
Vital
Vitamin
Vitelline
Vocal cords
Volar
Volkmann
(German physician)
Voluntary
Volvulus
Vomer
Vulva
Waldeyer
Waller
Westphal
Wharton
Whisky
Whitlow
Whooping cough
Widal
(French physician)
Willis
(English Anatomist)
Winslow
(Danish anatomist)
Wirsung

#/texts/9604 #/texts/9605 #/texts/9606 #/texts/9607 #/texts/9608 #/texts/9609 #/texts/9610 #/texts/9611 #/texts/9612 #/texts/9613 #/texts/9614 #/texts/9615 #/texts/9616 #/texts/9617 #/texts/9618 #/texts/9619 #/texts/9620 #/texts/9621 #/texts/9622 #/texts/9623 #/texts/9624 #/texts/9625 #/texts/9626 #/texts/9627 #/texts/9628 #/texts/9629 #/texts/9630 #/texts/9631 #/texts/9632 #/texts/9633 #/texts/9634 #/texts/9635 #/texts/9636

#/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196 #/groups/196

bladder
enclosed space
remnant of something formerly present
cattle doctor
vidian nerve, nerve of pterygoid canal
tuft of hair
V.
robin space
poison
vital organ/plural
act of seeing
to life
life + amine
yolk of egg
uttering of voice
palm of hand
V.
canal
willing
to roll
thin plate of bone between nostrils
to roll, to turn around
W. ring at oropharyngeal isthmus
Wallerian nerve degeneration
W. nucleus (part of oculomotor complex)
W. duct (Submand. duct), Wharton’s jelly
water of life
painful swelling in finger
to call/shout
W. reaction
circle of Willis
foramen of Winslow
pancreatic duct

#/texts/9638 #/texts/9639 #/texts/9640 #/texts/9641 #/texts/9644 #/texts/9645 #/texts/9646 #/texts/9647 #/texts/9648 #/texts/9649 #/texts/9650 #/texts/9651 #/texts/9652 #/texts/9653 #/texts/9654 #/texts/9655 #/texts/9656 #/texts/9657 #/texts/9658 #/texts/9659 #/texts/9660 #/texts/9661 #/texts/9662 #/texts/9663 #/texts/9664 #/texts/9665 #/texts/9666 #/texts/9667 #/texts/9668 #/texts/9669 #/texts/9670 #/texts/9671 #/texts/9672 #/texts/9673 #/texts/9674 #/texts/9675 #/texts/9676 #/texts/9677 #/texts/9678 #/texts/9679 #/texts/9680 #/texts/9681 #/texts/9682 #/texts/9683

#/body #/body #/body #/body #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197 #/groups/197

3
ag
(Gr)
(Gr)
Wistar
(American anatomist)
Wolff
Wright
Xanthine
Xeroderma
Xiphoid
Xylol
Yellow fever
Young
Zenker
Zinn
Zona
Zoology
Zuckerkandl
(Austrian anatomist)
Zygoma
Zymogen
pyramids (in kidneys)
Wolfian or mesonephric
W. stain
yellow
dry or parched
sword-like
wood + oil
an infectious viral fever
Young’s rule
Zenker’s solution
annulus of Zinn (origin of rectus)
zone
animal + treatise
Zuckerkandl’s gyrus, subcallosal gyrus
like a yoke
ferment producer, Z. granules in serous acini

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"

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Antagonists
97
Anterior
13,
Anti-spin 73
Antrum 25
Aplasia 27
Apocrine glands 179
Aponeurosis 21, 85
Appendicular skeleton 37
Appositional growth 46
Arches 175
Arcuate swing 73
Arrectores pilorum 178
Arterial anastomosis 113
Arterial pulsations 111
Arteries 21, 102
Arteriosclerosis 118
Arteriovenous anastomosis 113
Arteritis and phlebitis 118
Arthritis 57
Arthrology 1, 57
Articular capsule 65
Articular cartilage 65
Astrocytes 144
Atavistic epiphysis 41
Atheroma 118
Atlanto-occipital joints 69
Atrophy 27
Auscultation 2
Autonomic nervous system 156
Axial skeleton 37
Axillary sheath 186
Axis cylinder 153
15
Abdominal aorta 107
Abduction 16, 17, 60
Accessory 36
Accessory ligaments 65
Acetylcholine 143
Achondroplasia 52
Acne vulgaris 178
Acromioclavicular 66
Acute phlebothrombosis 120
Adduction 16, 60
Adduction of digits 17
Adjunct rotation 73
African sleeping sickness 167
Ageing 166
AIDS 167
Albinism 189
Alopecia 177
Alzheimer's disease 166
Amphiarthrosis 58
Anaemia 135, 177
Anaesthesia 27
Analgesia 27
Anastomosis 22, 113
Anatomical position 10
Anatomy |
Aneurysm 118
Angina pectoris 99
Angiology 1
Angular movements 16
Ankle joint 66

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

B-cells 126
B-Lymphocytes 133
Baldness 192
Ball-and-socket 64
Band 86
Basle nomina anatomica 9
Bed sores 164
Bell's palsy 165
Belly 21
Benign 27
Between femur and patella 69
Biaxial 72
Biological effect 207
Bipennate 90
Bipolar 142
Birmingham revision 10
Blood capillaries 124
Blood channels 130
Blood pressure 117
Blood-brain barrier 146
Boil 190
Bone marrow 31
Bone marrow biopsy 56
Bone tumour 56
Brachial 107
Buerger's disease 119
Burns 189
Bursa 75
Cc
Cadaveric anatomy 1
Calcaneocuboid joint 69
Cancellous spongy 37
Cancer 28
Capillaries 22, 103
Capitulum 25
Capsular 65
210
Capsular or true ligaments 65
Carcinoma 27
Cardinal swing 73
Cardiovascular system 101
Carotene 173
Carotid sheath 186
Carpal tunnel syndrome 193
Carpals 30
Carpometacarpal 66
Cartilage 48
Cartilaginous bones 36
Cartilaginous joints 58
Catecholamines 144
Caudal 13, 16
Cell body 139
Cement 39
Central nervous system 138
Cerebrospinal nervous system 139
Ceruminous glands 180
Cervical lymph nodes 128
Circulating pool of lymphocytes

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200 #/groups/200

Circulus vasculosus 76
Circumduction 17, 60
Circumduction of thumb 17
Circumpennate 90
Cirrhosis of liver 135
Clavicle 30
Cleavage lines 174
Cleidocranial dysostosis 51
Clinical anatomy 4
Close packed position 74
Closure of the mouth 19
Clubbing 177
Co-spin 73
Coma 27
Common carotid 107
Compact bone 37
Comparative anatomy 4
Complex joint 60
Composite/hybrid muscle 95
Compound joint 60
Compound joints 72
Computerized tomography 210
Condrosternal 66
Condylar joint 64
Conjunct rotation 73
Connective tissue 195
Continuous capillaries 112
Contralateral 13
Contrast radiography 208
Convalescence 28
Cornified zone 173
Coronal plane 13
Coronary arteries blockage 118
Cortex 40, 130
Costochondral joints 64
Costotransverse 66
Costovertebral 66
Cranial 13
Cricoarytenoid joint 66
Cricothyroid joint 66
CT scan 8
Cysticerocosis 167

#/texts/10197 #/texts/10198 #/texts/10199 #/texts/10200 #/texts/10201 #/texts/10202 #/texts/10203 #/texts/10204

#/groups/201 #/groups/201 #/groups/201 #/groups/201 #/groups/201 #/groups/201 #/groups/201 #/groups/201

Damage 163
Dark bands 86
Deep 13
Degeneration 26
Degrees of freedom 72
Dementia 166
Dendrite 141
Dense irregular connective tissue

#/texts/10206 #/texts/10207 #/texts/10208 #/texts/10209 #/texts/10210 #/texts/10211 #/texts/10212 #/texts/10213 #/texts/10214 #/texts/10215

#/groups/201 #/groups/201 #/groups/201 #/groups/201 #/groups/201 #/groups/201 #/groups/201 #/groups/201 #/groups/201 #/groups/201

Dentine 39
Dermatitis 189
Dermatoglyphics 175
Dermatome 149
Dermis or corium 173
Diaphysis 32
Diarthrosis 57, 58
Dislocated 77
Disseminated lupus erythematosus
201

#/texts/10217 #/texts/10218 #/texts/10219 #/texts/10220 #/texts/10221 #/texts/10222 #/texts/10223 #/texts/10224 #/texts/10225 #/texts/10226 #/texts/10227

Distributing vessels 103
Disuse atrophy 99
Dorsal 13, 15
Dorsal ramus 149
Dorsal root 149
Dorsalis pedis 107
Dorsiflexion of foot 18
Du bois formula 171
Dupuytren's contracture 202
Dynamic polarity 143
Dystrophy 27

#/texts/10229 #/texts/10230 #/texts/10231 #/texts/10232 #/texts/10233 #/texts/10234 #/texts/10235 #/texts/10236 #/texts/10237 #/texts/10238 #/texts/10239 #/texts/10240 #/texts/10241 #/texts/10242 #/texts/10243 #/texts/10244 #/texts/10245 #/texts/10246 #/texts/10247 #/texts/10248 #/texts/10249 #/texts/10250 #/texts/10251 #/texts/10252 #/texts/10253 #/texts/10254 #/texts/10255 #/texts/10256 #/texts/10258 #/texts/10259 #/texts/10260 #/texts/10261 #/texts/10262

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/groups/202 #/groups/202 #/groups/202 #/groups/202

Eccrine glands 179
Elastic cartilage 49
Elbow joint 66
Electro-myography 2
Elephantiasis 134
Ellipsoid joint 64, 69
Embolism 26, 117
Embryology 3
Encephalopathy 168
End-arteries 116
Endochondral ossification 46
Endomysium 87
Endoneurium 147
Endoscopy 2
Epicondyle 22
Epidermis 173
Epilepsy 169
Epimysium 87
Epineurium 147
Epiphyses 32
Epiphysial arteries 45
Epiphysial plate of cartilage 43
Ethmoid 30
Evagination 13
Eversion of foot 18
Exchange vessels 103
Experimental anatomy 4
Extension 60
256
Extensor 15
Extracellular matrix 196
Extrafusal muscle fibres 94
Extrinsic ligaments 198

#/texts/10265 #/texts/10266 #/texts/10267 #/texts/10268 #/texts/10269 #/texts/10270 #/texts/10271 #/texts/10272 #/texts/10273 #/texts/10274 #/texts/10275 #/texts/10276 #/texts/10277 #/texts/10278 #/texts/10279 #/texts/10280 #/texts/10281 #/texts/10282 #/texts/10283 #/texts/10284 #/texts/10285 #/texts/10286 #/texts/10287 #/texts/10288 #/texts/10289 #/texts/10290 #/texts/10291 #/texts/10292 #/texts/10293 #/texts/10294 #/texts/10295

Facial 107
Fallot's tetralogy 177
Female surface 72
Femoral 107
Femur 30
Fenestrated capillaries 112
Fibrillation 99
Fibrocartilage 49
Fibrous astrocytes 144
Fibrous bone 39
Fibrous capsule 65
Fibrous joints 57
Fibula 30
First carpometacarpal joint 69
First chondrosternal joint 64
Fistula 26
Fixators 97
Flat bones 32
Flexion 59
Flexor 15
Flexure lines 174, 175
Fluorescent effect 207
Foetal medicine 8
Fontanelles 61
Foramen 22
Foreign body giant cells 132
Fossa 22
Fracture 52
Frontal 30
Fungal infections 167
Fusiform 88

#/texts/10297 #/texts/10298 #/texts/10299 #/texts/10300 #/texts/10301 #/texts/10302 #/texts/10303 #/texts/10304 #/texts/10305 #/texts/10306 #/texts/10307 #/texts/10308 #/texts/10309 #/texts/10310 #/texts/10311 #/texts/10312 #/texts/10313 #/texts/10314 #/texts/10315 #/texts/10316 #/texts/10317 #/texts/10318

#/groups/203 #/groups/203 #/groups/203 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204 #/groups/204

Gaba
143
Ganglion 203
Gangrene 27
Gardner 76
General somatic afferent 155
General somatic efferent 155
General visceral afferent 155
General visceral efferent 155
Genetics 4
Germinative zone 173
Gift 8
Glioblasts 145
Glycine 143
Golgi typeI 142
Golgi type II 142
Gomphosis 61
Groove 22
Group A fibre 156
Group B fibre 156
Group C fibre 156
Guillain-Barre syndrome 165

#/texts/10320 #/texts/10321 #/texts/10322 #/texts/10323 #/texts/10324 #/texts/10325 #/texts/10326 #/texts/10327 #/texts/10328 #/texts/10329 #/texts/10330 #/texts/10331 #/texts/10332 #/texts/10333 #/texts/10334 #/texts/10335 #/texts/10336 #/texts/10337 #/texts/10338 #/texts/10339 #/texts/10340 #/texts/10341 #/texts/10342 #/texts/10343 #/texts/10346 #/texts/10347 #/texts/10348 #/texts/10349 #/texts/10350 #/texts/10351 #/texts/10352

Haemal nodes 131
Haemoglobin 173
Haemorrhage 26, 117
Hair 178
Hair follicle 178
Hamulus 22
Haversian fatty pads 75
Heart 102
Hemiplegia 27
Herniation of the disc 77
Herpes zoster 167
Herpes zoster virus 189
Herpes simplex 167
Heterotopic bones 36
Hinge joint 64
Hip bone 30
Hip joint 71, 80
Hirsutism 177
Histamine 143
Histology
3
Hodgkin's disease
Human genome 8
132
Humerus 30
Hyaline cartilage 49, 75
Hyoid 30
Hyperaesthesia 27
Hyperplasia 27, 99
Hypertrophy 27, 99
Hypoplasia 27

#/texts/10357 #/texts/10358 #/texts/10359 #/texts/10360 #/texts/10361 #/texts/10362 #/texts/10363 #/texts/10364 #/texts/10365 #/texts/10366 #/texts/10367 #/texts/10368 #/texts/10369 #/texts/10370 #/texts/10371 #/texts/10372 #/texts/10373 #/texts/10374 #/texts/10375 #/texts/10376 #/texts/10377 #/texts/10378 #/texts/10379 #/texts/10380 #/texts/10381 #/texts/10382 #/texts/10383 #/texts/10384 #/texts/10385 #/texts/10386 #/texts/10387 #/texts/10388 #/texts/10389 #/texts/10390 #/texts/10391

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

ncudomalleolar joint 69
ncudostapedial joint 71
ncus 30
nfarction 27
nferior 13
nferior nasal choncha 30
nflammation 26
nsertion 83
nspection 2
ntercarpal joints 66
nterchondral joint 66
ntermediate fibres 88
ntermetacarpal 66
ntermetatarsal 66
ntermuscular septa 184
nternode 154
nterosseous membrane 188
nterphalangeal joints 66
ntertarsal joints 66
nterventional radiology 212
ntervertebral joints 64
ntra-articular fibrocartilages 75
Intrafusal fibres 94
Intramembranous 46
Intrinsic ligaments 198
Invagination 13
Inversion of foot 18
Ipsilateral 13
Irregular bones 32
Irritation of
a
motor nerve
Isometric contraction 97
163
Isotonic contraction 97

#/texts/10393 #/texts/10394 #/texts/10395 #/texts/10397 #/texts/10398 #/texts/10399 #/texts/10400

Jena nomina anatomica 10
Joint cavity 65
Junctura 57
Keloid 192
Knee joint 68, 80
Koilonychia 177
Kupffer's cells 132

#/texts/10402 #/texts/10403 #/texts/10404 #/texts/10405 #/texts/10406 #/texts/10407 #/texts/10408 #/texts/10409 #/texts/10410 #/texts/10411 #/texts/10412 #/texts/10413 #/texts/10414 #/texts/10415 #/texts/10416 #/texts/10417 #/texts/10418 #/texts/10419 #/texts/10420 #/texts/10421 #/texts/10422 #/texts/10423 #/texts/10424 #/texts/10425 #/texts/10426 #/texts/10427 #/texts/10428 #/texts/10429 #/texts/10430 #/texts/10432 #/texts/10433 #/texts/10434

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Lacrimal 30
Lamellar bone 39
Language of anatomy 10
Lanugo 179
Large arteries 105
Lateral 13
Lateral border 15
Lateral flexion 16, 20
Lateral rotation 16, 20
Leprosy 164, 169
Leukopenia 135
Ligamenta flava 198
Ligaments 198
Light bands 86
Lineae gravidarum 174
Lithotomy position 12
Long bones 32
Long plantar ligament 200
Loops 175
Loose connective tissue 196
Loose packed 75
Lower motor neuron damage 168
Lymph capillaries 124
Lymph nodes 127
Lymph vessels 124
Lymphadenitis 133
Lymphangitis 133
Lymphatic channels 130
|
258
Lymphatic follicle 127
Lymphatic system 123

#/texts/10436 #/texts/10437 #/texts/10438 #/texts/10439 #/texts/10440 #/texts/10441 #/texts/10442 #/texts/10443 #/texts/10444 #/texts/10445 #/texts/10446 #/texts/10447 #/texts/10448 #/texts/10449 #/texts/10450 #/texts/10451 #/texts/10452 #/texts/10453 #/texts/10454 #/texts/10455 #/texts/10456 #/texts/10457 #/texts/10458 #/texts/10459 #/texts/10460 #/texts/10461 #/texts/10462 #/texts/10463 #/texts/10464 #/texts/10465 #/texts/10466 #/texts/10467 #/texts/10468 #/texts/10469 #/texts/10470 #/texts/10471 #/texts/10472 #/texts/10473 #/texts/10474 #/texts/10475 #/texts/10476 #/texts/10477 #/texts/10478 #/texts/10479 #/texts/10480 #/texts/10481 #/texts/10482 #/texts/10483 #/texts/10484 #/texts/10485 #/texts/10486 #/texts/10487 #/texts/10488 #/texts/10489 #/texts/10490 #/texts/10491 #/texts/10492 #/texts/10493 #/texts/10494 #/texts/10495 #/texts/10496 #/texts/10497 #/texts/10498 #/texts/10499 #/texts/10500

M band 86
Macrophages 132
Magnetic resonance imaging 211
Malaria 135, 167
Male surface 72
Malignant 27
Malleolus 22
Malleus 30
Mandible 30
Manubriosternal joint 64
Marfan's syndrome 202
Mass miniature 208
Mass miniature radiography 208
Maxilla 30
Medial 13
Medial border 15
Medial rotation 16
Median 12
Median atlanto-axial joint 66
Median or midsagittal plane 12
Medium and small arteries 105
Medulla 130
Medullary cavity 40
Melanin 173
Melanocytes 173
Melanoid 173
Membrane (dermal) bones 36
Membrano-cartilaginous bones 36
Menigocele 54
Meningo-myelocele 54
Meningocytes 132
Metacarpals 30
Metacarpophalangeal joints 69
Metaphysial arteries 45
Metaphysis 80
Metastasis 28
Metatarsals 30
Microcirculatory unit 116
Microglia 145
re
SR
ha ieee
Microglial cells 132
Midsagittal plane 12
Mole 189
Monocytes of blood 132
Mononeuropathy 165
Monoplegia 27
Motor point 95
Motor unit 95
Multi-axial 72
Multi-unit type 96
Multipennate 90
Multipolar 142
Muscle fibre 86
Muscle spindles 94
Muscular spasm 98
Myasthenia gravis 99, 135
Myelin sheath 153
Myelinated 152
Myelinated fibres 153
Myelinated or medullated fibres 152
Myocardial ischaemia 100
Myofibrils 86
Myology 1

#/texts/10502 #/texts/10503 #/texts/10504 #/texts/10505 #/texts/10506 #/texts/10507 #/texts/10508 #/texts/10509 #/texts/10510 #/texts/10511 #/texts/10512 #/texts/10513 #/texts/10514 #/texts/10515 #/texts/10516 #/texts/10517 #/texts/10518 #/texts/10519 #/texts/10520 #/texts/10521 #/texts/10522 #/texts/10523 #/texts/10524 #/texts/10525 #/texts/10526 #/texts/10527 #/texts/10528 #/texts/10529

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Nail bed 177
Nail wall 177
Nails 176
Nasal 30
Necrosis 26
Nerve plexuses for limbs 150
Nervi nervorum 152
Nervi vascularis 107
Nervorum 152
Neuralgia 164
Neurapraxia 163
Neuroglia 144
Neurolemmal 153
Neurolemmal sheath 154
Neurology 1
Neuron 139
Neuropathic (Charcot's) joint 164
Neuropathic joint 82
Bata in
:
ae
Neurotmesis 163
Nodes of Ranvier 153
Nomina anatomica 10
Non-myelinated 152
Non-myelinated fibres 154
Notch 22
Nutrient artery 43

#/texts/10531 #/texts/10532 #/texts/10533 #/texts/10534 #/texts/10535 #/texts/10536 #/texts/10537 #/texts/10538 #/texts/10539 #/texts/10540 #/texts/10541 #/texts/10542 #/texts/10543 #/texts/10544 #/texts/10545 #/texts/10546 #/texts/10547

Occipital 30
Oedema 26
Oligodendrocytes 145
Ontogeny 3
Opening the mouth 19
Opposition of thumb 17
Origin 83
Ossification 29, 46
Osteoarthritis 78
Osteology 1
Osteoma 29
Osteomalacia 29, 55
Osteomyelitis 29
Osteoporosis 55
Ovoid 73
Ovoid of motion 73
Oxyhaemoglobin 173

#/texts/10549 #/texts/10550 #/texts/10551 #/texts/10552 #/texts/10553 #/texts/10554 #/texts/10555 #/texts/10556 #/texts/10557 #/texts/10558 #/texts/10559 #/texts/10560 #/texts/10561 #/texts/10562 #/texts/10563 #/texts/10564 #/texts/10565 #/texts/10566 #/texts/10567 #/texts/10568 #/texts/10569 #/texts/10570 #/texts/10571 #/texts/10572 #/texts/10573 #/texts/10574 #/texts/10575 #/texts/10576 #/texts/10577 #/texts/10578 #/texts/10579 #/texts/10580 #/texts/10581 #/texts/10582 #/texts/10583 #/texts/10584 #/texts/10585 #/texts/10586 #/texts/10587 #/texts/10588 #/texts/10589 #/texts/10590 #/texts/10591 #/texts/10592 #/texts/10593 #/texts/10594 #/texts/10595 #/texts/10596 #/texts/10597 #/texts/10598 #/texts/10599 #/texts/10600 #/texts/10601 #/texts/10602 #/texts/10603 #/texts/10604 #/texts/10605 #/texts/10606 #/texts/10607 #/texts/10610 #/texts/10612 #/texts/10613 #/texts/10614 #/texts/10615 #/texts/10616 #/texts/10617 #/texts/10618 #/texts/10619 #/texts/10620 #/texts/10621 #/texts/10622 #/texts/10623 #/texts/10624 #/texts/10625 #/texts/10626 #/texts/10627 #/texts/10628 #/texts/10629 #/texts/10630 #/texts/10631 #/texts/10632 #/texts/10633

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/groups/205 #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Palatine 30
Palmar 15
Palpable arteries 107
Palpation 2
Panniculus adiposus 182
Papillary layer 174
Papillary ridges 175
Paraesthesia 27
Paralysis 27, 98
Paranasal air sinuses 31
Paraplegia 27
Parasympathetic nervous system 158
Parietal 30
Parkinson's disease
168
Patella 30
Penetrating power 206
Percussion 2
Perimysium 87
Perineurium 147
Peripheral autonomic nervous 139
Peripheral heart 112
Peripheral lymphoid organs 127
Peripheral nerves 147
Phalanges 30
Phlebitis 118
Photographic effect 206
Phylogeny 3
Physical anthropology 4
Physiology
1
Pigmentation of skin 171
Pivot joint 64
Plane 64
Plantar aponeuroses 185, 186
Plantarflexion of foot 18
Pneumatic bones 32
Poliomyelitis 167
Polyarthric muscles 94
Polymyositis 99
Polyneuropathy 164
Portal circulation 103
Posterior 13, 15
Posterior tibial 107
Potential arterial anastomosis 113
Precapillary sphincter 105
Pressure epiphysis 41
Pressure lamellae 38
Pressure sores 189
Prime movers 97
Pronation 16, 17
Prone position 10
Protoplasmic astrocytes 144
Protraction 16, 19
Proximal 13, 15
Pseudo-unipolar 142
Puberty 26
Pulmonary (lesser) circulation 103
Pyrexia 26
Quadriplegia 27
Radial 107
Radiographic and imaging anatomy
3
Radiographic views 207
Radiography 2
Radius 30
Ramus 22
Raphe 21, 201
Raynaud's phenomenon 119
Referred pain 161
Reflex arc 146
Regeneration 163
Regional anatomy 1
Replaced hip joints 80
Reservoir (capacitance) vessels 103
Resistance vessels 103
Reticular layer 174
Reticulo-endothelial system 131
Retinacula 184, 193
Retraction 16, 19
Rheumatic fever 77
Rheumatic fever 201

#/texts/10634 #/texts/10635 #/texts/10636 #/texts/10637 #/texts/10638 #/texts/10639 #/texts/10640 #/texts/10642 #/texts/10643 #/texts/10644 #/texts/10645 #/texts/10646 #/texts/10647 #/texts/10648 #/texts/10649 #/texts/10650 #/texts/10651 #/texts/10652 #/texts/10653 #/texts/10654 #/texts/10655 #/texts/10656 #/texts/10657 #/texts/10658 #/texts/10659 #/texts/10660 #/texts/10661 #/texts/10662 #/texts/10663 #/texts/10664 #/texts/10665 #/texts/10666 #/texts/10667 #/texts/10668 #/texts/10669 #/texts/10670 #/texts/10671 #/texts/10672 #/texts/10673 #/texts/10674 #/texts/10675 #/texts/10676 #/texts/10677 #/texts/10678 #/texts/10679 #/texts/10680 #/texts/10681 #/texts/10682 #/texts/10683 #/texts/10684 #/texts/10685 #/texts/10686 #/texts/10687 #/texts/10688 #/texts/10689 #/texts/10690 #/texts/10692 #/texts/10693 #/texts/10694 #/texts/10695 #/texts/10696 #/texts/10697 #/texts/10698 #/texts/10699 #/texts/10700 #/texts/10701 #/texts/10702 #/texts/10703 #/texts/10704 #/texts/10705 #/texts/10706 #/texts/10707 #/texts/10708 #/texts/10709 #/texts/10710 #/texts/10711 #/texts/10712 #/texts/10713 #/texts/10714 #/texts/10715 #/texts/10716 #/texts/10717 #/texts/10718 #/texts/10719 #/texts/10720 #/texts/10721 #/texts/10722 #/texts/10723 #/texts/10724 #/texts/10725 #/texts/10726

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206

Rheumatoid arthritis 78, 201
Ribs 30
Rickets 53
Right lymphatic duct 125
Rolling 74
Rostral 16
Rotation 16, 60
Sacroiliac 66
Sacrospinous ligament 200
Sacrotuberous ligament 200
Saddle joint 64
Sagittal plane 12
Sarcolemma 86
Sarcoma 27
Sarcomere 86
Scabies 190
Scapula 30
Schwann cell 154
Scleroderma 201
Scurvy 54
Sebaceous cyst 190
Sebaceous glands 180
Sebum 180
Sellar 73
Serotonin 143
Sesamoid bones 32
Short bones 32
Shoulder joint 71
Shunts 103
Simple joint 60, 72
Single-unit type 96
Sinus 25, 26
Sinusoids 112
Skeletal muscles 83
Skin incisions 190
Slide 74
Smallest arteries 105
Somatic bones 37
Somatic muscles 83
Special movements 16
Special somatic afferent 155
Special visceral afferent 155
Special visceral efferent 155
Spheno-occipital joint 64
Sphenoid 30
Spin 73
Spina bifida occulta 54
Spinal ganglion 149
Spinal nerve 149
Spinal nerves 147
Spiral 91
Splanchnology 1
Splenomegaly 135
Spondylitis 78
Stapes 30
Sternoclavicular joint 69
Sternum
30
Strap like 88
Strap like with tendinous
intersections 88
Stretch reflexes 147
Striated muscles 83
Striped muscles 83
Structure of skin 173
Styloid process 22
Subluxation 77
Superficial fascia 182
Superior
13
Superior and inferior radio-ulnar
joints 66
Superior tibiofibular 66
Supination 16, 17
Supine position 10
Surface anatomy 3
Sutures 61
Sweat glands 179
Swing 73
Sympathetic nervous system 156
Symphyses 64
Symphysis menti 64
Symphysis pubis 64
Synapse 143
Synarthrosis 58
Synarthrosis 57
Synchondrosis 63
Syndesmology 57
Syndesmosis 61
Syndrome 27

#/texts/10727 #/texts/10728 #/texts/10729 #/texts/10730 #/texts/10731 #/texts/10732 #/texts/10733 #/texts/10734 #/texts/10735 #/texts/10736 #/texts/10737 #/texts/10738 #/texts/10739 #/texts/10740 #/texts/10741 #/texts/10742 #/texts/10743 #/texts/10744 #/texts/10745 #/texts/10746 #/texts/10747 #/texts/10748 #/texts/10749 #/texts/10750 #/texts/10751 #/texts/10752 #/texts/10753 #/texts/10754 #/texts/10755 #/texts/10756 #/texts/10757 #/texts/10758 #/texts/10759 #/texts/10760 #/texts/10761 #/texts/10762 #/texts/10763 #/texts/10764 #/texts/10765 #/texts/10766 #/texts/10767 #/texts/10768 #/texts/10770 #/texts/10771 #/texts/10772 #/texts/10773 #/texts/10774

#/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/groups/206 #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/groups/207 #/groups/207 #/groups/207 #/groups/207 #/groups/207

Synergists 97
Synovial fluid 75
Synovial joints 58
Syringomyelia 165
Syringomyelia 164
Systemic (greater) circulation 103
T
Tabes dorsalis 164
Talo-calcaneonavicular joint 71
Tarsals 30
Tarsometatarsal 66
Temporal 30
Temporomandibular joint 68
Tendon 21
Tendon 85
Tension lamellae 38
Tension lines 175
Terminal hair 179
Terms related to body movements 16
Therapy 28
Thermanaesthesia 27
Thoracic duct 125
Thoroughfare channels 115
Thrombocytopenia 135
Thrombosis 26, 117
Thymus 127
Tibia 30
Tibial collateral ligament 199
Tissue culture 8
Traction epiphysis 41
Translation 72
Triangular 90
Trochanter 22
Trochlea 25
Trophic changes 164
True ligaments 65
Tumour 27
Tunica adventitia 106
Tunica intima 106
Tunica media 106
Type I (slow) fibres 88
Types of fats 183
Ulcer 26
Ulna 30
Ultrasonograph 210
Uniaxial 72
Unipennate 90

#/texts/10777 #/texts/10778 #/texts/10779 #/texts/10780 #/texts/10781 #/texts/10785 #/texts/10786 #/texts/10787 #/texts/10788 #/texts/10789 #/texts/10790 #/texts/10791 #/texts/10792 #/texts/10793 #/texts/10794 #/texts/10795 #/texts/10796 #/texts/10797 #/texts/10798 #/texts/10799 #/texts/10800 #/texts/10801 #/texts/10802 #/texts/10803

#/groups/208 #/groups/208 #/body #/body #/body #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209 #/groups/209

Unipolar 142
Upper motor neuron damage 168
Voluntary muscles 83
Vomer 30
V
Varicose veins 120
Vasa vasorum 106, 125
Veins 22, 102
Vellus 179
Venous anastomosis 113
Venous valves 111
Ventral 13, 15
Ventral ramus 149, 150
Ventral root 149
Visceral bones 37
Vitiligo 192
Whorls 175
Wolff’s law 38
Woven bone 39
Wrist joint 69
X
X-rays 205
Z
Zygomatic 30

#/texts/10807 #/texts/10808 #/texts/10809 #/texts/10813 #/texts/10814

This handbook aims to meet the requirements of medical and dental students who are newly admitted to colleges. It thoroughly introduces the reader to anatomic terminology along with various structures which constitute the human body. Since teaching of general anatomy lays down the foundation of the entire subject of medicine and surgery, this handbook has been written in a short, simple and comprehensive language. The text has been classified into small parts making it easier for the students to follow and Tulle ae
The text has been broadly divided into ten chapters. Besides the introductory chapter, the other chapters are: Skeleton, Joints, Muscles, Cardiovascular System, Lymphatic System, Nervous System, Skin and Fasciae, and Connective Tissue, Ligaments and Raphe. A new chapter Principles of Radiography has been added to introduce radiologic anatomy to the young students. Clinical anatomy has been vividly illustrated through colour diagrams.
This edition features fresh, simple, coloured diagrams which extensively illustrate each chapter. The text has been revised to supplement these for easy learning. The appendix on anatomical word meanings and historic names makes a rich source of information that is basic to the study of medicine.
|
ISBN: 978-81-239-1654-5 |

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Arun Kumar Paul BSc MBBS DA MS (Anaesth) Ex-Professor, Dept of Anaesthesiology Medical College and Hospitals Kolkata, India
JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD
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All rights reserved. No part of this publication should be reproduced, stored in a retrieval system, or transmitted in any form or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the author and the publisher.
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First Edition:
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daughter, Sushmita for the love, support and encouragement
The Manual of Anaesthesia is intended to provide a convenient, concise, readable and rapid source of information relevant to anaesthesiology. The book uses a point-wise format for easy reading and better understanding of the text.
The subject matter is discussed in some details and made interesting as well as practicable to grasp the fundamentals of clinical anaesthesia. On the whole, the text will serve as primer and the students, trainee and practitioners will find it suitable.
I wish to thank Mrs Kanyakumari Paul and Sushmita Paul for their cooperation and support in the preparation of this book.
Iam also indebted to Shri Jitendar P Vij, (Chairman and Managing Director) and Mr Tarun Duneja (Director-Publishing) of Jaypee Brothers Medical Publishers (P) Ltd, New Delhi who offered the concept of the book with enthusiasm and published the volume.
Arun Kumar Paul
Ree
RRP
BR
R
BPR
NNNN

1., 1 = Preanaesthetic Evaluation ............................................................................................................... 1. 2., 1 = Preanaesthetic Medication .............................................................................................................. 6. 3., 1 = Conduct of Anaesthesia................................................................................................................. 12. 4., 1 = Inhalational Anaesthetic Agents..................................................................................................19. 5., 1 = Intravenous Anaesthetic Agents .................................................................................................. 26. 6., 1 = Local Anaesthetic Drugs ............................................................................................................... 36. 7. Muscle Relaxants, 1 = ............................................................................................................................ 42. 8. Monitoring, 1 = during Anaesthesia ................................................................................................... 53. 9. Anaesthetic, 1 = Equipment.................................................................................................................. 56. 10. Spinal Anaesthesia, 1 = ......................................................................................................................... 92. 11. Epidural Anaesthesia, 1 = ..................................................................................................................... 98. 12., 1 = Caudal Anaesthesia ..................................................................................................................... 103. 13. Peripheral, 1 = Nerve Blocks ............................................................................................................ 106. 14., 1 = Complications of Anaesthesia ................................................................................................... 115. 15., 1 = Difficult Intubation ...................................................................................................................... 131. 16. Eye, 1 = and ENT Anaesthesia ......................................................................................................... 135. 17., 1 = Obstetric Anaesthesia ................................................................................................................. 143. 18. Paediatric Anaesthesia, 1 = ................................................................................................................ 155. 19., 1 = Anaesthesia for Day-case Surgery ........................................................................................... 165. 20. Anaesthesia, 1 = in Obese Patients ................................................................................................. 170. 21. Geriatric Anaesthesia, 1 = .................................................................................................................. 173. 22. CardiopulmonaryResuscitation, 1 = ................................................................................................ 176. 23. Anaesthesia, 1 = and Common Coexisting Diseases ................................................................... 180. , 1 = Appendix.......................................................................................................................................... 201. Bibliography, 1 = .................................................................................................................................... 207. Index, 1 = ................................................................................................................................................ 209

ONATARwWHN
. History and physical examination of the patient
. To review medical records including past anaesthetic records
. To get laboratory studies
. To determine physical status and anticipate difficulties
For planning of anaesthesia management
. To ensure obtaining consent
. To develop anaesthetist-patient rapport
. Tomake advanced preparation relating facilities, equipment and expertise, to enhance patient safety and minimise the chance of errors.

1. Patient’s medications: Aspirin, insulin, antihypertensives, MAO inhibitors, lithium, anticoagulants, etc.
Daa Any addiction: Smoking, alcohol, drugs
RON . Any allergy . Past hospitalisation, any anaesthesia/operation
. Family history
. Any associated illness: Metabolic, endocrinal, renal, hepatic, cardiac, pulmonary disease,
sexually transmitted disease, hepatitis, HIV
. Menstrual history in case of females.

. Central nervous system: Cerebrovascular insufficiency, convulsion.
. Cardiovascular system: Chest pain, myocardial infarction, hypertension, rheumatic fever, palpitations, dysrhythmias.
. Respiratory system: Dyspnoea, cough, bronchospasm, pneumonia, smoking, upper respiratory infection.

d. Liver: Alcoholism, hepatitis, jaundice
e Kidney: Polyuria, pyuria
f. Endocrine system: Diabetes mellitus, adrenal dysfunction, thyroid dysfunction.
g Miscellaneous: Allergy, bleeding tendency, myalgia, arthritis, osteoporosis, strabismus, etc.

RR POD . Digitalis: Dysrhythmias
ANAUARWNPE
R N . Anticoagulant therapy: Excessive bleeding.
. Aspirin: Bleeding tendency, platelet dysfunction
. Alcohol abuse: Resistance to anaesthetic drugs
. Antibiotics: Potentiation of muscle relaxants
. Antihypertensives: Impaired sympathetic nervous system activity, hypertensive crisis
. Beta blockers: Bradycardia, bronchospasm
Benzodiazepines: Potentiation of muscle relaxants, resistance to anaesthetic drugs
. Calcium channel blockers: Interaction with muscle relaxants, hypotension
MAO inhibitors: Increased response to sympathomimetic drugs
. Lithium: Potentiation of muscle relaxants, impaired thyroid function
. Diuretics: Hypotension, hypovolaemia, hypokalaemia

eo . Haematologic: Bruising, anaemia, bleeding.
10. Urinary system: Nocturia, dysuria, haematuria, incontinence.
11. Prosthesis, if any: Eye glasses, contact lens, hearing aid, ornaments, ring
. Vital signs: Pulse, respiration, blood pressure, body temperature
. General: Weight, weakness
. Airway examination:
Cervical spine: head mobility, Mobility of temporomandibular joint, Teeth : Central incisor prominency, Artificial teeth, if any, loose teeth, Attempt of visualise uvula, any lesion in mouth, hyromental distance (Normal more than 6.5 cm)
Nose and throat: Sore throat, sinusitis, epistaxis, deviated nasal septum, dysphagia, sunken cheeks, patency of nares opening of mouth. Appearance of neck (long or short).
. Respiratory: Breathing rate and pattern, any moist sound, wheezing, cough, sputum, emphysema, pneumonia, tuberculosis, bronchitis.
.
Cardiac:
Heart
rate,
rhythm,
murmur,
blood
pressure,
pulse,
peripheral
oedema,
dyspnoea,
orthopnoea,
veins,
heart
failure.
. Central nervous system: Level of consciousness, numbness, paralysis, skeletal muscle dysfunction, convulsion.
. Gastrointestinal system: Nausea/vomiting, constipation, diarrhoea, jaundice, hepatitis.
. Endocrine system: Diabetes, thyroid dysfunction.

12.
Miscellaneous:
Examination
of
eye;
examination
of
ear,
extremities:
Claudication,
thrombophlebitis, arthritis; skin examination: rashes, wounds, infection; psychiatric problems,
anxiety.
¢ Breath holding test: It is the time to hold his breath after full inspiration. Normal more
than 25 seconds. Less than 15 seconds indicates severely diminished cardiorespiratory reserve.
¢ Match test: Patient is asked to blow off the lighted match stick with his open mouth at a distance 15 cm. Failure indicates low maximum breathing capacity.

1. Blood examination
TC, DC, haemoglobin, haematocrit
. Blood biochemistry
Glucose, urea, nitrogen, creatinine, electrolytes, liver enzymes
. Urine analysis
. Coagulation studies
Prothrombin time, platelet count, partial thromboplastin time
. Chest X-ray
. Electrocardiogram
. Pulmonary function tests
FEV), vital capacity, peak expiratory flow rate
Arterial blood gas study, pH
[Normal Values :
Vital capacity 5 lits.
FEV; 4 lits.
PEFR 600 lits/min.
PaO? 75 to 100 mm Hg.
PaCO) 36 to 46 mm Hg.
]

Grade
1: Normal healthy patient
Grade 2: Patient with mild/moderate systemic disease
Grade 3: Patient with severe systemic disease that causes functional limitation
Grade 4: Patient with incapacitating systemic disease that causes a constant threat to life
Grade 5: Moribund patient not expected to survive 24 hours without operation
Emergency Operation E: Any patient in whom emergency operation is required. Here the suffix E is added.

1. Safety of the patient
. Coexisting systemic disorders
. Site of operation FWhN
. Elective or emergency procedure
. Age of the patient . Preference of the patient, if any . Ability of the anaesthetist concerned OANA
. Convenience of the surgeon.

. Psychological support, reassurance
. No food/drink for at least 6 hours before anaesthesia
. Urinary bladder should be emptied before taken to OT
. False teeth, artificial limbs, artificial eyes, contact lenses, shoes, ornaments, etc. should be taken
off
. No tight clothing, no lipstick, no nail vernish
. Adequate oral hygiene
. Identification label should be checked
. Consent for anaesthesia/surgery is mandatory
. Resuscitative drugs, fluids, etc.
. Night sedation
. Preanaesthetic medication. It should be given in adequate dose and at proper time.
¢ No routine preparation is needed in extreme emergencies
:
i. Rupture of major vessels
ii. Severe obstetric emergencies: ectopic rupture
iii. Acute upper airway obstruction
iv. Surgery on patients trapped and immobile.

1. Acute respiratory infection
. Coexisting systemic illness not under optimal control
. Lack of adequate resuscitation . Full stomach . Nonavailability of written consent Daa rk WN
. Failure to obstain recent investigation reports.

A. Anaesthesia machine
Anaesthesia breathing system
Gas cylinders
Soda lime
Vaporisers
Mechanical ventilator
Suction apparatus
Monitoring equipment.

Local anaesthetic drugs
iv inducing agents
Muscle relaxants
Opioids and opioid antagonist
Benzodiazepines and its antagonist
Anticholinergic drugs
Vasopressor drugs
Catecholamines.

Infusion set, fluids
Suction catheter, oral/nasal airway
Laryngoscope, endotracheal tubes
Nasogastric tube, Magill forceps.
D. Blood should be crossmatched and available in time.

ay . To relieve anxiety, fear and tension
CONATARWN
. To provide a degree of autonomic block
To produce sedation, analgesia and amnesia
. To reduce salivary secretions and secretions of respiratory tract
To reduce metabolic rate
To depress unwanted vagal reflex activities
. To potentiate anaesthetic drugs
. To protect the patient from the toxic effects of anaesthesia
. To reduce the incidence of postoperative complications.

DaoPrPwn . It should facilitate smooth anaesthesia . It should depress salivary and respiratory secretions . It should prevent nausea/vomiting . It should be safe and can be easily administered
. It should be anxiolytic, analgesic, sedative and anesic
. There should not be undue depression of cardiovascular, respiratory and central nervous system.

1. General condition of patient, age and weight, psychological status, level of anxiety, presence of pain, etc.
. Proposed operation: Nature of surgery, site of operation, posture during surgery, duration of operation
. Elective/emergency surgery
. Inpatient/outpatient

C©mMNaAM . Availability of nursing care . Tolerance of depressant drugs . Previous history of adverse reaction of drugs
. Availability of adequate surgical and anaesthetic management and care
. Presence of allergy.

ay . Sedatives : Barbiturates, benzodiazepines, phenothiazines
CONATARWN
. Narcotic analgesics: Opioid drugs
. Neuroleptic drugs: Droperidol
. Anticholinergic drugs: Atropine, hyoscine, glycopyrrolate
. Anxiolytic drugs: Diazepam, midazolam, lorazepam
Phenothiazine drugs: Promethazine, trimeprazine, prochlorperazine
. Antiemetic drugs: Metoclopramide, ondansetron
. Hy, antagonists: Ranitidine, cimetidine famotidine
. Antacids
e These drugs are being used eiher alone or in combination according to the need of the individual patients.

arwn . Sedative, hypnotic, anxiolytic . Overall depression of central nervous system . CSF pressure raised
aD . Direct myocardial depression, hypotension, reduction of cardiac output and stroke volume, peripheral vasodilation
. Alkaloid of opium, a standard potent analgesic
. Depression of respiration, decreased response of chemoreceptors to anoxia, depression of cough
reflex, increased bronchial muscle tone
. Nausea/vomiting, constriction of sphincters, diminished peristalsis, constipation
. Passes through placental barrier
. Constricted pupils
. May cause hyperglycaemia
. May cause addiction
. Antidiuresis
. Pruritus
. Dose: About 0:2 mg/kg im.

1. Synthetic opioid agonist one-tenth as potent as morphine
2. Dose: 1 to 2 mg/kg

3. Analgesic, sedative, euphoric, anxiolytic
4. Respiratory depressant, raise CSF pressure
5 . Atropine like effect: dry mouth
6 . Direct effect on smooth muscles of bronchioles, intestines, ureters and arteries causing reduction
of
tone
7. Can cause addiction
8. Does not cause constipation
9. May release histamine
10. Passes through placental barrier
11. Side-effects: Nausea/vomiting, hypotension, vertigo, tremor, limb tingling.

ONDAARWHN
. Synthetic narcotic analgesic, a benzomorphan derivative. A partial agonist of \-receptors
. It is short-acting and induces less respiratory depression
Dysphoria is common. Less addiction liability
. Sympathomimetic action
Nausea/vomiting : less ; constipation : less
. It crosses the placental barrier less
. It is less cumulative
. Dose: 20 to 60 mg by injection, 25 to 100 mg orally.

1. Benzodiazepine group of drug with main pharmacological actions:
a. Sedative, hypnotic, anxiolytic, amnesic
b. Muscle relaxation
c. Anxiolytic
d. Anticonvulsant.
. Not analgesic, does not cause nausea/vomiting
. May decrease blood pressure and increase heart rate
. Respiratory depression is less
. Potentiates nondepolarising muscle relaxants
. Dose: In premedication 10 to 20 mg orally or im; for induction of anaesthesia 0.5 mg/kg iv.

. Anticholinergic, parasympatholytic drug
. It crosses the blood-brain barrier and stimulates higher cortical centres and medulla
. In therapeutic doses atropine can cause stimulation of vagal centre and initially cause bradycardia. Later on this is overcome by antimuscarinic action on sinoatrial node causing tachycardia
. Bronchiolar dilatation, may increase rate and depth of respiration

onan . Salivary and bronchial secretions diminished . Diminishes tone and Peristalsis of gut
10. Reduces motor activity and tone in detrusor muscle of urinary bladder
11. Dose: 0:65 mg ; in children 0:02 mg/kg ; for vagolytic action 1-5 to 3 mg.
Can reduce the tone of lower oesophageal sphincter and lower oesophageal pressure
. Can raise body temperature, stimulate heat regulating centre, increase basal metabolic rate,
diminish sweating
. Dilates pupil, causes paralysis of accommodation (cycloplegia), significant effect on intraocular pressure in cases with narrow angle glaucoma

ay . Levorotatory alkaloid, anticholinergic, parasympatholytic, tertiary amine
CANA Pronounced antisalivary effect Can cause rise of body temperature . Marked mydriasis and cycloplegia
ARWHN . Marked sedation, amnesia, antanalgesic . Restlessness and delirium—common
Ag . Elderly patients
PRONE
ARON
E CNS toxicity
. Crosses the blood-brain barrier and placental barrier easily
. Effects on heart and gut : Weaker than atropine
. Dose: 0:3 to 0-6 mg ; in children 0-015 mg/kg.
Atropine/hyoscine should be used cautiously
. Thyrotoxicosis
. Febrile patients
Glaucoma
. Some cardiac patients: Mitral stenosis
. Tachycardiac patients.
Unwanted effects of anticholinergics
. Mydriasis and cycloplegia
. Rise of body temperature
. Tachycardia
. Drying of airway secretions
. Relaxation lower oesophageal sphincter.

. Synthetic quarternary ammonium compound with anticholinergic parasympatholytic activities
. Strong vagal bloking effect on heart
. Strong antisalivary effect
. Antagonises the muscarinic effects like bradycardia, bronchospasm, intestinal motility

5. Can suppress gastric fluid volume and its acidity
6. Does not cross blood-brain barrier and placental barrier
7. Dose: 0.1 to 0.3 mg; in children 0.004 mg/kg im.

Re . Usual dose: 25 mg ; in children 1 mg/kg
FPOSCMNDAUKRWONP
. Phenothiazine group of drug
. Sedative, antihistaminic
. Mild atropine like activities
. Depression of upper airway reflexes
. Potentiates the effects of hypnotics, narcotics and anaesthetic drugs
. Mild hypotensive effect
When used alone, it increases the sensitivity of pain (antanalgesic)
. Reduces the incidence of nausea and vomiting
. Marked local analgesic effect
. May produce extrapyramidal side effects.

DauF . Spasmolytic, antipruritic effect . Mild anticholinergic effects
WN . Sedative, antihistaminic, antiemetic
. Phenothiazine derivative
. Potentiates the effects of sedative and narcotics
. Dose: 1.5 mg/kg, in children Img/kg im
3 to 4 mg/kg orally.

ar . Does not produce hypotension
wh . Antiemetic effect is profound
. Weak antihistaminic and spasmolytic effect
. Dose: 12.5 mg, in children 2.5 to 5 mg orally.

. Derivative of procainamide
. Potent antiemetic effect
. Increases the rate of gastric emptying, relaxes pylorus, dilates the duodenal bulb, increases
the peristalsis of gut
. Inhibits vomiting centre, depresses cortex and reticular activating system

CaOnNaAn . Dose: 10 mg
No significant effect on gastric secretion and its acidity
. No significant effect on cardiovascular and respiratory system
. Side-effects: Spasm of muscles, abdominal cramps
. Caution: Atropine, opioids and antacids should be avoided during its use.

. It is a selective antagonist of the actions of 5-HT, serotonin at 5-HT3 receptor
. Highly effective for prevention and treatment of postoperative nausea/vomiting
. Dose: 4 mg iv. Prophylactic use 16 mg orally
. No serious side effects.

1. Parenteral
2. Oral route
a. Subcutaneous or intravascular route. It takes 45 to 60 minutes to get the optimum effect.
b. Intravenous route: Immediate effect
Advantages:
Early effect
More certain in effect
Disadvantages:
Pain, discomfort, infection, bleeding, broken needles, disliked by most patients particularly children.
In takes at least 90 minutes to get the optimum effect.
No pain, no discomfort, liked by all.
Effect is not certain, little risk of vomiting and aspiration, late and prolonged effect.
The conduct of general anaesthesia is broadly divided in various stages such as induction of anaesthesia, maintenance of anaesthesia and recovery from anaesthesia.
Before administering anaesthesia, one should check the availability of anaesthetic machine and other for use.
all equipment ready
PRR BwoN . Throat packs . Suction apparatus
RR . Lignocaine spray 4% . Vaseline
FPOSCMNDURWONE
. Anaesthetic machine and its accessories, gas cylinders, face masks
.
Set of endotracheal tubes
. Endotracheal tube connection, catheter mount
. Stilette, bougie
. Magill forceps
. Syringes, indwelling iv needles, infusion set
. Airway tubes
. Adhesive tape, bandage
. Common drugs, anaesthetic drugs, drugs for resuscitation.

in:
Here the inhalational anaesthetic agents are administered through face mask. This is mostly indicated
Children, uncooperative patients 1.
Upper or lower airway obstruction 2.
3. Bronchopleural fistula
4. Where iv induction is risky.
A. Procedure should be explained to the patient beforehand

B. Select the gas mixture
C. Initially 70% nitrous oxide in oxygen is given, then the anaesthesia is deepened by the
gradual administration of volatile anaesthetic
D. Halothane 1 to 3% is being popularly used
E. Maintenance of anaesthesia can also be done
F. Other agents used:
i. Cyclopropane
ii. Enflurane.

1. Slow induction
2. Salivation
3. Airway obstruction, bronchospasm
4. Laryngospasm, hiccups
5. Wastage of anaesthetic vapours
6. Pollution.

1. IV anaesthetic agents are used for induction
2.
It avoids most complications of inhalational induction
3. It is mostly pleasant, can cause rapid induction
4. Can be used even in emergency surgery
5. Note:
i. Drug should be selected carefully
ii. Dose should be calculated properly
iii. IV access is essential
iv. Suitable aesptic skin preparation is needed
v. IV entry must be confirmed with blood aspiration
vi. Slow injection should be given
vii. Avoid subcutaneous, intraarterial injection
viii. Monitoring the effects of drug on CVS and respiratory system is important.

i. Thiopentone sodium 4 to 5 mg/kg
ii. Methohexitone 1 to 1.5 mg/kg
iii. Etomidate 0.3 mg/kg
iv. Ketamine 2 mg/kg
v. Propofol 2.5 mg/kg
vi. Midazolam 0.2 mg/kg.
7. Complications:
i. Regurgitation, vomiting, aspiration pneumonitis
ii. Intraarterial injection of thiopentone
iii. Perivenous, subcutaneous injection
iv. Cardiovascular depression

v. Respiratory depression
vi. Histamine release
vii. Drug interaction
viii.
Porphyria in susceptible individuals by barbiturates
ix. Pain on injection
x. Muscular movements
xi. Hiccup.

1. Inhalational agents
2. IV anaesthetic agents
3. IV opioid
4. The above agents can be used alone or in combination.
e Inhalational anaesthesia with spontaneous ventilation.
1. N,O + O; + Halothane /enflurane/trichloroethylene
2. NO + O2 + Cyclopropane.

The stages and planes of anaesthesia are most precise in patients breathing ether in air. These are Guedel’s
classical signs.
Stage 1: Analgesia.
It is the period from the beginning of anaesthesia until loss of consciousness. Respiration is irregular and of small volume. Pupils are small and reflexes are mostly intact.
Stage 2:
Excitement:
The period from the loss of consciousness to the starting of rhythmic respiration. Respiration is of large volume and irregular. Eyelash reflexes are absent, but other reflexes are intact. Patient is restless.
struggling,
Stage 3: Surgical anaesthesia
The period extends from the onset of regular rhythmic respiration upto respiratory paralysis. It
includes 4 planes:
Plane 1:
Starts from onset of regular respiration and ends when eyeball comes in rest on central
position.
Plane 2:
Starts from cessation of eyeball movement and ends when intercostal muscle paralysis
starts.
Plane 3:
Progressive intercostal paralysis till its completion. Only diphragmatic respiration
continues.
Plane 4:
Respiration ceases.
Stage 4: Medullary paralysis, apnoea, intense muscular relaxation. Pupils dilated and fixed. This is due to excessive anaesthesia. Patient is endangered.

1. Low blood concentration of anaesthetics
2. Stimulus of low intensity — anaesthesia, but high stimulus causes muscle movements
3. Respiration regular, smooth, pupil fixed but not dilated. Muscle relaxation is not adequate.

1. High blood concentration of anaesthetics
2. Strong stimulus: No muscular movements
3. Respiration regular, pupils fixed, absent reflexes. Profound muscular relaxation.

1. Lacrimation, tears
. Eye movements, pupils reactive
. Rate of respiration increases, irregular
. Reflex muscle movements on stimulus
. Pulse rate increases
OCNDAAARWN
Poor muscle relaxation
. Sweating, swallowing, coughing
. Awareness.

1. Overdose of anaesthetics
2. Pupils dilated widely
3. Respiration — shallow, irregular
4. Poor muslce tone
5 . Collapsing
6 . Cold skin
7 . Operative field : Pale and dry.

Anaesthetic gases and vapours are administered through a tube inserted into the trachea through the nose or mouth or transtracheal route.

1. To ensure ventilation/oxygenation
2. . To ensure airway patency
3. . To protect against pulmonary aspiration
4. To provide separate ventilation to each lung
5 . To protect the airway from contamination
6 . To facilitate tracheobronchial toilet.

A. Head /neck surgery, major surgery, abdominal/thoracic surgery, long operation, operation in the oral cavity, difficult airway
B. Risk of aspiration, abnormal position: Prone position, needing profound muscle
Cc. Technique involving artificial ventilation, resuscitation of newborn, cardiac arrest, respiratory arrest.
relaxation

SCmMONDUPWNH
Re oO . Anaesthesia: Certain in effect
RP . Mostly safe.
. Securing the airway, provides free, unobstructed patent airway
. Patient’s respiratory effect diminished
. Ventilation can be controlled/assisted
. Aspiration of lungs prevented
. Muscle relaxants can be used
. Stomach is not inflated
. Resuscitation better performed
. Anaesthetist and apparatus kept well away from surgical field
. Tracheobronchial toilet possible

1. Trauma during laryngoscopy and intubation
tooth/lips/gum/larynx/epiglottis/pharynx/trachea
. Reflex disturbances: Breath holding, apnoea, bronchospasm, laryngospasm, cardiac arrhythmia hypertension, tachycardia.
. Obstruction: By mucus, blood, vomitus, foreign bodies, kinking /biting of the tube, bevel against
the tracheal wall.
. Position and type of the tube: Too long tube may enter in one bronchus, too short tube may
come out during change of posture. Narrow tube may cause undue resistance.
. Oesophageal intubation, gastric distension, barotrauma, pressure effect on tracheal
mucosa, disconnection, accidental extubation, etc.
. Postanaesthetic complications:
i.
ii. Laryngitis /laryngeal ulceration
iii. Tracheitis /tracheobronchitis
iv. Oedema glottis

v. Infections: Pneumonia, aspiration pneumonitis, atelectasis, lung abscess.
vi. Nonspecific granuloma of larynx
vii. Surgical emphysema
viii. Vocal cord paralysis
x. Dislocation of arytenoid cartilage a
x. Tracheal stenosis.

1. Needs adequate knowledge of anatomy and physiology. Needs experience and skill
2. Done in deeper plane of anaesthesia
3. Hazards of endotracheal intubation
4. Unconscious patient needs constant monitoring and special care
5 . Pharmacological depression of body systems
6 . Needs heavy and costly machine and equipment.

1. Where orotracheal tube comes in the surgical field as in surgery inside the mouth
2. Where laryngoscopy is difficult
3. When endotracheal tube is to be kept for prolonged period.

Unique to nasotracheal intubation
1. Injury to nasal mucosa: Epistaxis
2. Dislodgement of adenoids
3. Eustachian tube obstruction
4. Infection: Maxillary sinusitis
5 . Bacteremia
6. . Gastric distension.
Advantages of nasotracheal intubation
1. Tube is secured, no chance of spontaneous extubation
2. Comfortable in awake patient
3. No chance of tube biting
4 . Oral feeding possible
5 . Good oral hygiene can be maintained.

1. Needs small sized tube
2. Chance of infection more, bacteremia
3. Sinusitis
4. Epistaxis.

1. Keep the head upon a head rest (about 10 cm high), supine position
2. Head elevation and neck flexion to bring the pharyngeal and laryngeal axis into line. Extension of head and cervicooccipital joint helps alignment of the oral axis with the other two
. Open the mouth with your fingers
. Laryngoscopic blade is introduced along the right margin of tongue. The tongue is displaced to the left by rotating the blade
. The curved blade is advanced to the midline in the glossoepiglottic fold
. The glottis is exposed by displacing the tongue and epiglottis anteroinferiorly
. Glottis can be better viewed by applying backward, upward and rightward pressure on the thyroid cartilage. A good assistant is always helpful
. The endotracheal tube should be introduced from the right side
. The proximal margin of the cuff of the endotracheal tube should lie 3 to 4 cm beyond the vocal cords.

1. Sniffing position should be proper
2. . Muscular relaxation should be adequate
3. . Mouth should be opened widely
4. The blade should not go too far to the left
5 . Epiglottis should be identified
6 . Laryngoscopic blade should be introduced to proper depth
7. . Assistant should give the proper pressure on thyroid cartilage.

1. Patient’s head should be placed in exaggerated sniffing position
2. Lubricated endotracheal tube (0.5 mm smaller diameter tube than usual) is gently and slowly introduced along the nasal floor
3. If there is resistance, the tube is withdrawn a little and rotated to advance again
. Listen the breath sounds at the proximal end of the endotracheal tube. One can use one hand to advance the tube and the other to palpate the thyrohyoid membrane
. Position of the tube can be ascertained by the intensity of breath sounds, lateral bulging produced by the tip in the priform fosa, resistance to pass when it is in vallecula and no breath sounds, if it is in oesophagus
. Tube should be adjusted by twisting the tip from side-to-side, extending or flexing the neck to move the tip anteroposteriorly
. Tube inserted into the trachea can be judged by coughing, aphonia, and a normal capnograph tracing.

1. Causes rapid, smooth pleasant induction, amnesia and anaesthesia readily
. Adequate muscular relaxation
. Safe, potent, easily reversible. Absence of flammability, nonexplosive
. Readily acceptable by the patient
Cheap, readily available
. Easy to administer
Nontoxic, no side effects /complications
. Safe for the vital organs like brain, heart, lungs, liver and kidneys.
OCNATAON

1. Low blood-gas solubility. Insolubility causes quicker induction of and recovery from
anaesthesia
High potency and volatility . Resistance to biotransformation wWN
. Ether structure conveys cardiac stability . Halogenation reduces flammability age
. Compatible with adrenaline.

Nitrous oxide, Cyclopropane.
Their saturated vapour pressure are above ambient pressure and exist as liquid under pressure.
Other agents are volatile liquids with SVP below ambient pressure and these need special calibrated
vaporisers for their use.

1. Increased anaesthetic concentration
2. Increased fresh gas flow
3. Increased alveolar ventilation
4. Reduced cardiac output
5.
Second
gas
effect:
When
nitrous
oxide
is
administered
with
a
volatile
anaesthetic
agent,
the
rapid
uptake
of
nitrous
oxide
increases
the
effective
minute
during
Moreover
the
uptake
of
N2O
from
the
alveoli
decreases
the
volume
of
gas
remaining
in
the
alveoli
and
increases
the
of
remaining
gases
within
the
alveoli
and
thus
induction
is
enhanced.
At
the
end
of
N2O
anaesthesia,
the
reverse
effect
occurs
and
diffusion
hypoxia
may
develop.
This
can
be
prevented
by
supplemental
oxygen
therapy
6. Use of nonrebreathing anaesthetic circuit
7. Low blood gas solubility coefficient.

1. Increased fresh gas flow
2. Increased alveolar ventilation
3. Use of nonrebreathing circuit
4. Low blood gas solubility
5. Low tissue gas solubility
6. Decreased anaesthetic duration.

1. A standard measure of the potency of inhaled anaesthetics
2. Itis the partial pressure of an inhaled anaesthetic at 1 atmosphere that prevents skeletal muscle movement in response to noxious stimuli in 50% cases
3. Factors increase MAC:
(i) Young age (ii) hyperpyrexia (iii) alcoholics (iv) infants (v) hypernatraemia (vi) CNS stimulants like MAO inhibitors, tricyclic antidepressants, cocaine, amphetamine
4. Factors decrease MAC: (i) Hypothermia (ii) IV anaesthetics (iii) premedicants (iv) neonates (v) elderly (vi) pregnancy (vii) lithium (viii) CNS depressants like benzodiazepines, barbiturates, propofol, etc. (ix) Alpha 2 adrenergic agonists like clonidine.
5. MAC is not affected:
(i)
Sex
(ii)
duration
of
(iii)
of
anaesthetics
(iv)
hyper/hypokalaemia
(v)
thyroid
dysfunction,
etc.
6. MAC values for combinations of inhaled anaesthetics are additive.
7. Administration of nearly 1.3 MAC prevents skeletal muscle movement in almost all patients during surgery.
8. MAC of common inhaled anaesthetics
: (i) Nitrous oxide 110 (ii) halothane 0.75 (iii) isoflurane 1.15 (iv) enflurane 1.68 (v) desflurane 7.25 (vi) sevoflurane 2.05 (vii) ether 1.92 (viii) trichlorethylene 0.2 (ix) methoxyflurane 0.16 Cyclopropane 9.2.
9. Agent with minimum MAC will be the most potent.

1. Nonirritant sweet smelling gas supplied in blue colour coded cylinders. It is stored as a liquid
at
room
2. First anaesthetic use in 1844
3. Low blood gas solubility
4. Smooth and rapid induction of anaesthesia
5. Weak anaesthetic agent, cannot produce surgical anaesthesia when used alone
6. Excellent analgesia
7. Nitrous oxide 50% in oxygen is used for painless labour
8. Causes mild depression of cardiovascular system
9. May cause megaloblastic changes in bone Marrow in prolonged use
10. Prolonged N2O anaesthesia may cause diffusion of the gas into the body cavities
11. Atthe end of anaesthesia high percentage of expired volume may consist of N2O, so that onward diffusion of gas lowers alveolar partial pressure of oxygen (Diffusion hypoxia).

1. A mixture of 50% nitrous oxide in oxygen.
2. Stored as a gas in cylinders whose body is blue and shoulder in white / blue quarters under a of 1987 Ib inch
pressure per sq.
3. Under pressure some parts of mixture may remain in gaseous phase at temperature and pressure at which N,O is normally in liquid phase (Poynting effect)
4. It is stored horizontally at least above 10°C
5. It liquefies at-7°C
6. In cold climate there may be liquefaction and separation of the components. This may lead to uneven delivery of gases. To prevent this, the cylinder should be inverted several times to
ensure adequate mixing. Rewarming in a water bath may also be helpful
7. Uses:
a. For relief of pain in labour
b. Burn dressing
c. Management of chronic pain, cancer pain, etc.

ay . Supplied in an orange cylinder as liquid at a pressure of 75 lb per sq. inch
CaONAARWN
. Pleasant smell, nonirritant, potent. Not metabolised
. Rapid induction of anaesthesia, potent anaesthetic gas
. Sympathomimetic action. Arterial pressure is maintained and may increase
Can cause salivation, increased bronchial secretion and occassional bradycardia
. Flammable, highly explosive
Expensive
. Respiratoy depression, bronchospasm
. Post-cyclopropane hypotension can occur (Cyclopropane shock)

10. Emergence delirium can occur
11. Contraindications
i. Use of diathermy
ii. Use of adrenaline
iii. Asthmatic patients
iv. Pheochromocytoma.

1. Colourless, pungent volatile liquid
. Relatively nontoxic, safe, potent anaesthetic agent
. Produces good muscular relaxation Bwh
. Causes an increase in sympathoadrenal activity. Heart rate and blood pressure may increase a little
. Excessive salivation, cough, laryngospasm due to irritant vapour
. Bronchodilation
. Raises blood sugar level
. Raises CSF pressure
. Reduces body temperature
. Postoperative nausea/vomiting is common
. Induction is slow, unpleasant stage of excitement
. Explosion risk
. Ether convulsion can occur
. Cheap and can be used without sophisticated apparatus and compressed gas cylinders.

ay . Colourless liquid but coloured with waxoline blue dye for identification
CONATARWN
. Highly lipoid soluble, nonexplosive
. Potent agent with considerable analgesic effects
. Blood pressure is well-maintained but cardiac arrhythmias are common
. Nonirritant to tracheobronchial tree
. Prolonged induction and recovery rate
Muscular relaxation is not good
. Reacts with soda lime, cannot be used in closed circuit
. Cheap and mostly safe in adequately ventilated patient.

1. A halogenated hydrocarbon (not an ether as it lacks etheric bond)
2. Heavy, colourless volatile liquid with sweet smell. Needs thymol for stability
3. Potent and nonflammable, nonexplosive
4. Well-tolerated, provides rapid and smooth induction of anaesthesia

9. Causes bronchodilation
10. May cause hypotension
11. Salivation and other secretions are not increased
12. May cause marked uterine relaxation leading to postpartum haemorrhage
13. May trigger malignant hyperpyrexia
14. Early postoperative restlessness is common
15. Special calibrated vaporiser is needed.
Causes dose related fall in blood pressure, myocardial depression, sensitizes the heart to catecholamines
circulating causing arrhythmias
Potentiates competitive muscle relaxants
Post-operative shivering can occur
May be associated with nonspecific hepatotoxic damage. It may be due to its breakdown
products
either
causing
damage
directly
or
by
immunological
toxic
response

BR Clear colourless, volatile anaesthetic with characteristic fruity smell
CPNATRWN
10. May cause postoperative headache, emergence delirium
11. May trigger malignant hyperpyrexia.
Potent anaesthetic and good analgesic
Slow induction due to its low volatility. Slow recovery
Excellent muscular relaxation
Diminishes cardiac output and causes hypotension
May cause cardiac arrhythmia
No sensitisation of myocardium to catecholamines
Uterine muscles inhibited
Causes high output renal failure due to fluoride ion containing metabolites. Extensive metabolism (more than 30%)

R Potent volatile anaesthetic liquid
CS Depresses central nervous system, but in high concentration may cause epileptiform fits
APNAT Depresses cardiovascular and respiratory systems. Myocardial depression is dose dependent Nonirritant to respiratory tract, does not increase tracheobronchial secretions Good muscular relaxation, potentiates nondepolarising muscle relaxants Relaxes uterine muscles
AWN Clear, colourless, pleasant ethereal smell Noninflammable, nonexplosive, noncorrosive
PR
RP Only about 2% undergoes biotransformation in liver and metabolites are excreted in urine as
oO Increases intracranial pressure and cerebral blood flow
Produces rapid smooth induction of anaesthesia
organic or inorganic fluorides. But nephrotoxicity is extremely rare

12. No major liver and kidney dysfunction
13. Little risk of teratogenesis.

1. An isomer of enflurane, clear colourless volatile liquid. Noninflammable
ae Causes profound respiratory depression
WN Somewhat pungent and irritant, causes coughing
ND Potentiates muscle relaxants Uterine relaxation can occur
Induction and recovery are mostly rapid. Low blood gas coefficient
Produces hypotension, myocardial depression, reduction in cardiac output, fall in peripheral vascular resistance
Decreases cerebral metabolism, and increases cerebral blood flow. Intracranial pressure may
rise. Does not cause seizure activity
Only 0.2% is metabolised in liver, no significant toxic metabolites are formed.

AMR Respiratory depression can occur
WN Induction is rapid, recovery is prompt
Fluorinated ether, potent volatile anaesthetic liquid
Pungent, irritant to airway; breath holding and laryngospasm can occur
Dose dependent fall of blood pressure, fall in peripheral vascular resistance
Metabolism of desflurane to trifluorocretic acid and inorganic fluoride is about 10 times less
than that of isoflurane.

NQTPON
bP Fluorinated methyl ethyl ether
Potent anaesthetic
Nonirritant to airway
Respiratory and cardiovascular effects are mostly similar to those of halothane
Induction is rapid and recovery is prompt
Does not sensitise the myocardium to catecholamines
Instability in soda lime.

BR Fluorine containing ether, potent volatile anaesthetic liquid
a May cause uterine relaxation
FON Good analgesic Induction and recovery mostly smooth and rapid
May cause profound respiratory depression

6. Cardiac output and blood pressure are well-maintained
7. Passes through placental barrier readily
8. Increases intracranial pressure.

Volatile anaesthetic agents are mostly divided in two groups:
1. Halogenated hydrocarbons: Halothane, trichlorethylene, chloroform
2. Ethers: Enflurane, isoflurane, methoxyflurane, diethyl ether, etc.
Some physical properties of inhalational anaesthetic agents are shown in Table 4.1.
Table 4.1. Showing some physical properties of inhalational anaesthetic agents
Mol.
Wt.
Boiling
Vapour
Oil/Water
Blood/gas
MAC
Point
pressure
at
solubility
20°C
mm/Hg
Diethyl ether
74
35°C
460
3.2
12
1.92
Trichlorethylene
131
87°C
60
400
9
0.17
Halothane
197
50°C
243
220
2.5
0.75
165
105°C
23
400
13
0.16
Enflurane
184
56°C
180
120
1.9
1.68
184
49°C
250
174
1.4
1.15
Cyclopropane
42
—
33°C
78
34.4
0.45
9.2
Nitrous
oxide
44
—
88°C
680
3.2
0.47
105
¢
Hepatotoxic
volatile
:
Halothane,
chloroform
Nephrotoxic volatile anaesthetics : Methoxyflurane, sevoflurane
Epileptogenic:
Enflurane
¢ Inflammable and can cause explosion : Diethyl ether, cyclopropane.

1.
a . Produce no accumulation.
It should provide smooth, pleasant and rapid induction of anaesthesia
2. Effect is evident in one arm brain circulation
3. Rapid recovery with little hangover systems
4. Negligible effect on cardiovascular and respiratory effects
5 . Stable in solution, water solubility
6 . Nonirritant even if injected extravascularly
7 . No untoward reactions like nausea/vomiting, emergency delirium, hallucinations, tremor,
allergy, laryngospasm and so on

1. For induction of anaesthesia
2. For maintenance of anaesthesia
3. Often combined with other inhaled anaesthetics

1. Barbiturates Thiopentone
. Eugenols: Propanidid
. Steroids: Althesin, etanolone
. Phencyclidine: Ketamine
. Imidazole: Etomidate
. Di-isopropyl phenol: Propofol
. Benzodiazepine : Diazepam, midazolam
. Opioids : Fentanyl, alfentanil, sufentanil.
OANaATIEWN

Factors determining the anaesthetic effect:
1. Blood flow to the brain

Body pH and pKa of the drug
. Lipoid solubility PWN
. Protein binding
Redistribution to other tissues
. Metabolism . Excretion. NQO
¢ Only nonionised fraction of the drug is able to penetrate the blood-brain barrier
e A protein bound drug is unable to cross into the brain
e All IV anaestheics are to some extent protein bound
¢ The degree of ionisation depends on body pH and pKa of the drug.
Following IV injection of drug, it will reach the site of action quickly in high concentration and
produce rapid onset of action. Vital centres in medulla, vasomotor and respiratory centres are more likely to affect.
Distribution of drug depends on lipoid solubility and blood flow to body tissues. As the drugs are all highly fat-soluble and the heart and viscera belong to vessel rich groups, drug will rapidly enter the brain and produce unconsciousness.
Well-perfused organs will subsequenty be depleted of the drug by redistribution to lean tissues (muscle, bone). The blood supply of fat is so poor that it plays little role in the initial distribution
of the drug which results in early awakening.
Though this is the principal mechanism of recovery after a single dose of thiopentone, hepatic role.
metabolism also plays some
With nonbarbiturates (etomidate, propofol) redistribution plays a prominent role in recovery, but metabolism is also of greater importance. The rapidity of metabolism makes them suitable for continuous infusion and still with good recovery.

1. Barbiturates: Slowly metabolised, stored in muscles and fat for hours, produces prolonged mild sedation.
2. Propanidid, alphaxalone and etomidate: Removed from circulation mainly by
biotransformation, more rapid in case of propanidid.
3. Ketamine and diazepam: Converted to metabolites which themselves have sedative action,
may
give prolonged duration of action.
4. Diazepam: Undergoes enterohepatic recirculation—produces prolonged action.
5. Midazolam does not have these characteristics of diazepam, thus gives it a shorter duration of action.

Mechanism of action:
¢
Depresses reticular activating system
¢ Decreases rate of dissociation of GABA from its receptors
¢ Selectively decreases transmission of impulses through sympathetic ganglion
¢ No specific antidote for overdose.

AakROne
. Most commonly used iv induction agent
. Rapidly acting thiobarbiturate
Dose: 3 to 5 mg/kg (2.5% solution)
. Induction is rapid, smooth and quiet
. Metabolism in liver is slow (10-15% per hour)
. Effects:
Sedation, hypnosis, anaesthesia
Depression of respiration
Peripheral vasodilation
Direct myocardial depression
Cumulative effect
Acute tolerance
Antanalgesic.
. Complications:
Laryngospasm, bronchospasm
Intraarterial injection
Extravascular injection
Histamine release, anaphylactic reaction.

Porphyria
Fixed cardiac output disorders
Severe shock
Adrenocortical insufficiency
Nonavailability of resuscitative equipment.

1. Oxybarbiturate
2. Dose: 1 to 1.5 mg/kg (1% solution)
3. White powder, water-soluble
4. Duration of action: 2 to 3 min
5 . Recovery: Smooth, rapid
6. . Effects:
Sedative, hypnotic,
Circulatory depression
Abnormal muscle movements
Antanalgesia

i. Inducing agent
ii. Sole anaesthetic agent in short procedures, in day-care surgery.

8. Contraindications:
Epilepsy
Porphyria
Hypersensitive patients.

1, Oily liquid derived from oil of cloves
. Insoluble in water, solubilised in cremophor EL WN
. Ultrashort acting with rapid recovery within 5 to 8 min
. Dose: 6 to 7 mg/kg iv
. Initial hyperventilation, then marked hypoventilation.
. Involuntary muscle movements, coughing
. Mild rise of pulse rate, fall of blood pressure
. Detoxicated by plasma cholinesterase
ONaATIF
10. Anaphylactic reaction: Common
11. Not used now a days.
Action of suxamethonium prolonged
. Uses: Inducing agent
Sole anaesthetic agent for short surgical procedures, outpatient anaesthesia

CMON . Anaphylactic reaction: Common
DUPWNEH
a oO . Contraindications:
. A mixture of alphaxalone 9 mg/ml and alphadolone 3 mg/ml
. Steroid anaesthetic with no hormonal effect
. Dose: 0.05 to 0.1 ml/kg
. Rapid induction, recovery in 5 to 10 min
. Painless injection, no hangover effect
. Mild hypotension and respiratory depression
. Postoperative nausea/vomiting, excitement and euphoria can occur
Sole anaesthetic agent for short procedures
Porphyria
Liver diseases

1. Naturally occurring metabolite of progesterone
2. Insoluble in water 0.4% emulsion in 10% intralipid
3. 3 times more potent then propofol
— latest steroid anaesthetic

. Less pain during injection
. Dose: 0.6 mg/kg iv
Rapid induction, rapid recovery
. Dose-related reduction of blood pressure
. Mild respiratory depression
. Postoperative nausea /vomiting—less.
CONDE

1. Dissociative anaesthesia: Intense analgesia with only superficial sleep. EEG evidence of dissociation between thalamus and limbic system.
2. Dose:
i. 2 mg/kg iv—effect within 60 sec and lasts for 5 to 8 min
ii. 5 to 10 mg/kg im—effective in 3 min and lasts for 20 to 30 min
iii. 50 ug/kg/min in continuous infusion.
3. No cardiorespiratory depression, heart rate and blood pressure may increase
4. Side effects:
Hypertonus
Salivation
Nausea/vomiting
Emergence delirium
Hallucinations.
5. Indications:
Inducing agent
Sole anaesthetic agent.
6. Contraindications:
History of cerebrovascular accident
Raised intracranial pressure
Raised intraocular pressure
Recent penetrating eye injury
Psychotic patients
Thyroxine treatment
Severe cardiac disorders.

1. Carboxylated imidazole derivative
2. Dose: 0.2 to 0.3 mg/kg iv
3. Rapid iv induction within 30 sec
4. Rapid recovery within 6 to 8 min
5. Broken down by hydrolysis by esterases in liver and plasma
6. Maintains cardiovascular stability

7. No histamine release
8. Disadvantages:
Pain during injection
Involuntary muscle movements
Adrenocortical suppression.

ONDAORwWH
1. Insoluble in water, presented in a lipid emulsion
. Dose: 2 to 2.5 mg/kg iv
Rapid smooth induction within 30 sec
. Recovery within 4 to 8 min
No hangover effect
. Can be used in infusion 0.1 to 0.2 mg/kg/min
. Heart rate and blood pressure—decreased
Uses:
iv sedation
iv inducing agent
iv maintenance of anaesthesia.
9. Disadvantages:
Pain at the site of injection
Cardiorespiratory depression
Allergic reaction
May cause convulsion.
10. Contraindications:
Epilepsy
11. Safe in porphyria.

Produce sedation and hypnosis by affecting polysynaptic pathways in brain (midbrain reticular formation) and spinal cord
Causes emotional modification by depressing excitability of the limbic system
Act at specific receptors in CNS. This receptor stimulates the activity of inhibiting neurotransmitter GABA.

Anxiolysis
Amnesia
Sedation/hypnosis
Anticonvulsive effect
Muscle relaxation

No analgesic effect
Minimum depression of ventilation and CVS. CNS effects antagonised by flumazenil

Night sedation
Induction of anaesthesia.
¢ Most of the benzodiazepines are metabolised to active compounds with long elimination half lives as for desmethyl diazepam.

1. Insoluble in water, formulating in emulsifying agent
2. Usual inducing dose: 0.3 to 0.5 mg/kg
3. Onset of sleep-Slow, recovery prolonged
4. Little effect on cardiovascular system
5 . Mild muscular relaxation
6 . No analgesic effect
7. . Adverse effects:
Persistent drowsiness, occasionally muscle weakness, headache, vertigo, pain on injection, nausea/vomiting.

1. Water-soluble benzodiazepine
. Dose: 0.15 to 0.3 mg/kg iv
ONDAARWH
Rapid smooth induction within 80 sec
. Recovery is rapid within 6 to 8 min
Maintains cardiovascular stability
- No pain during iv injection
. Other effects:
Hypnosis, anxiolysis, muscle relaxation, anticonvulsant activity.
e Benzodiazepine antagonist
Flumazenil: Specific antagonist
Dose: 0.2 mg/kg every 60 sec upto 1 mg. Duration of actions is about 20 min, so resedation occur.
may

1. Exogenous substances that bind specifically to opioid receptors and produce some agonist responses

2. Analgesia mediated through a complex interaction of mu, delta and kappa receptors which are normally activated by endogenous endorphins
3. Affinity of most opioid agonists for receptors parallels analgesic potency
4. Binding of opioids to specific receptors results of all neurotransmissions concerned with analgesia within CNS
5. Fentanyl, sufentanil and alfentanil may be used for induction and/or maintenance of anaesthesia
6. Clinical uses:
Provision of analgesia in pre and postoperative period
Induction and/or maintenance of anaesthesia
Inhibition of sympathetic reflex activity
Supplementation of inhaled anaesthetics.

. Neuroleptanalgesia when used in combination with droperidol
. Analgesic potency is 100 times more than morphine
Onset of analgesia usually rapid within 1 to 2 min
. Duration of effect: About 30 min
Use: Anaesthesia for short surgical procedures
. Dose related respiratory depression
. Little effect on cardiovascular system
. Nausea/vomiting: Common
. Wooden chest effect. It can be overcome by use of muscle relaxants
. Histamine release may occur RR
. Dose: 5 to 15 g/kg (Ventilation should be controlled with muscle relaxants).
FPOSMNDAURWONE

R . Derivative of fentanyl
. Onset of action fast, within 1 arm-brain circulation time
Short duration of action Wh
. Low potential for postoperative respiratory depression A
Can be used as continuous infusion
Can produce muscular rigidity like fentanyl
No significant cumulative effect
. Prompt postoperative awakening with minimal depression of ventilation.
CENA

1. Most potent of the newer opioids
2. High lipoid solubility
3. Prompt onset of action

4. Brief duration of action due to subsequent redistribution to inactive tissue sites
5. Cumulative effect
6. May be used for analgesic component of balanced anaesthesia
7 . Excellent haemodynamic stability
8 . High doses 5 to 20 ug/kg is used in cardiac surgery.

1. A recent synthetic opioid with strong affinity to mu opioid receptors
5. It is 10 to 20 times more potent than alfentanil
6. Little change of blood pressure
7. It can cause muscle rigidity.
2. Metabolised by esterases in blood and other tissues
3. Antagonised by naloxone
4. It has rapid onset of action, a small volume of distribution and clearance and terminal half life 8 to 20 min

1, Naloxone is specific opioid antagonist reversing both desirable (analgesia) and nondesirable (respiratory depression) effects
. High doses can cause abrupt awakening with intense pain and activation of sympathetic stimulation causing hypertension, tachycardia, dysrhythmia
. Duration of effect is about 30 min. So continuous infusion may be helpful.

1. Features: Erythema, oedema, vasodilation, hypotension, bronchospasm
2. May occur following thiopentone, methohexitone, thiamylal, propanidid, althesin, etc.
3. Common in cremophor EL containing drugs
4. Common in patients with asthma, hay fever, eczema, with history of food or drug allergy, previous same anaesthetics
. Avoid use of drugs likely to cause reactions particularly in those considered to be at risk.

SNAT Inactive Nontoxic metabolites . Absence of toxicity /allergenicity to solvents . Minimum cardiovascular effects.
ANS . Onset of sleep should be within one circulation time . Predictable and short duration of action Rapid inactivation by plasma or hepatic metabolism Noncumulative

Thiopentone : Cumulative, prolonged recovery
Etomidate : Adrenocortical suppression
Methohexitone : Ketamine, propofol and midazolam seem to be more satisfactory for continuous infusion
The technique of iv infusion should be guided by individual sensitivity, type of surgery and presence of adjunctive drugs
It should be adopted when proper monitoring devices and reliable delivery systems are available
A clear understanding regarding drug kinetics and drug effect is essential.

1. It implies the avoidance of inhalational anaesthetics and its replacement with only iv anaesthetics
Methohexitone and thiopentone were used to provide total iv anaesthesia, but these agents usually prolong recovery
Propanidid is no longer used due to its adverse reactions to stabilising agent cremophor EL.
Etomidate is not used as it causes adrenocortical suppression
Ketamine has gained some popularity, but may lead to dysphoria in recovery phase. But it is satisfactory when combined with midazolam
Benzodiazepines appear to act in synergism with opioids. Midazolam can be used with
fentanyl
or
alfentanil.
Delayed
recovery
from
midazolam
can
be
antagonised
with
flumazenil
Propofol seems to have satisfactory pharmacokinetic profile in this respect and is most satisfactory to provide iv anaesthesia
The essential points to note that there should be improved methods of control of concentration of drugs and the drug administration must be titrated to achieve the desired effect for the individual patient and the degree of surgical stimulation.
The local anaesthetic drugs are usually in solution as the hydrochloride salts
These are weak bases and exist in equilibrium between lipid-soluble anionic uncharged form and water-soluble cationic form.
The local anaesthetic drug penetrates the nerve fibre following injection close to the nerve and the cationic form blocks the sodium channels to prevent sodium influx. This causes blocking of membrane depolarisation. The block is mostly dependent on concentration of the local anaesthetic. Thus when the concentration falls below a critical minimum level, the block gradually terminates. This the local anaesthetics are able to cause temporary block of nerve conduction when applied locally close to the nerve
All local anaesthetic drugs have three essential functional components, an aromatic group joined to an amine group by an ester or amide linkage. Thus the local anaesthetics are divided into two groups:
1. Amides: Lignocaine, mepivacaine, bupivacaine, etiodocaine, prilocaine, ropivacaine, etc.
2. Esters: Procaine, chloroprocaine, tetracaine.
Local anaesthetics can also be classified on their potency and duration of action.

1. Weak potency and short duration of action: Procaine, chloroprocaine
2. Intermediate potency and medium duration of action: Lignocaine, mepivacaine, prilocaine.
3. High potency and long duration of action: Tetracaine, bupivacaine, etidocaine, ropivacaine.
Criteria
for
a
good
local
:
Rapid
onset
of
action
Adequate duration of action Complete recovery FON
Safe
Devoid of toxic and allergic reactions. A
Potency depends on
1. Lipoid solubility
2. Protein binding
3. State of ionisation

e Addition of adrenaline 1 : 200000, 5ug/ml to the local anaesthetic solution prolongs the analgesic effect the of the and increases the of block
by delaying absorption drug quality
¢ Contraindications for use adrenaline containing anaesthetic solution:
1. Unstable angina pectoris
2. Cardiac dysrhythmias
3. iv regional anaesthesia
4. Uteroplacental insufficiency
5. Site that lacks collateral blood flow (digits, penis.
e
Safe maximum single dose of some local anaesthetics in adults in shown in Table 6.1.
¢ Physiochemical properties of some local anaesthetics are shown in Table 6.2

RR POD . Presence of adrenaline . Liver diseases
OND . Injection in inflamed site . Vascularity of the site
UF . Inadvertent intravascular injection
WNH . Type of local anaesthetic . Concentration of the drug
a nN . Concomitant use of anticholinesterase drugs may increase the toxicity of ester local anaesthetic.
. Total quantity of the drug
. Rate of absorption
. Physical status of the patient, nutrition, age, body weight
. Hypersensitivity of the drug

i. Central nervous system stimulation: Circumoral numbness, excitement, restlessness, tinnitus,
iv. Allergic reactions
vertigo, headache, tremor, muscle twitching, convulsion
. Followed by CNS depression: Unconsciousness, cardiorespiratory collapse
iii. Cardiovascular system
Bradycardia, extreme pallor, sweating, hypotension.
Cocaine causes tachycardia.
Urticaria, angioneurotic oedema, bronchospasm
Palpitations, tachycardia, tachypnoea, hypertension. These are mostly due to adrenaline containing anaesthesit solution.

1. Dose should be accurate.
ae Resuscitative drugs and equipment should be available
ON Adequate skill of the anaesthetist needed
Proper premedication and assessment
Avoid hyperventilation as it can elevate seizure threshold by lowering PaCOy.

Dae iv fluids Vasopressors
ON Patent airway, endotracheal intubation
Head down tilt
IPPV with 100% oxygen
. Convulsion should be treated with diazepam, curare, or thiopentone sodium.
° The minimum concentration of the local anaesthetic that will reliably produce ‘effective block’, should be used to reduce the risk of toxicity. The smallest volume to produce effective anaesthesia
should also be used. The extent of block depends on the volume of the anaesthetic solution.
Nerve fibres vary in diameter and the large nerves need higher concentration to be anaesthetised than the smaller fibres.
Type and function of different nerve fibres:
Aa motor function, proprioception
B touch, pressure sensation
Y motor to spindles, muscle tone
5 pain, touch, temperature sensation
B preganglionic autonomic axons
C postganglionic autonomic, pain sensation.
A fibres vary in diameter 2 to 20 um. Ad is thinnest. B fibres are myelinated with diameter less
than 3um. C fibres are nonmyelinated with diameter 0.5 to 1 um

_ S Not much used now a days.
BR Extracted from the leaves of coca tree
Stimulates CNS: Restlessness, excitement, euphoria, tachycardia, tachypnoea, hypertension
Sudden cardiac arrest or profound cardiovascular collapse can occur Causes vasoconstriction PWN
Mucosal surface anaesthesia in eye: Mydriasis, blanching of conjunctiva Intranasal cocaine: Vasoconstriction. AT
4 to 25% solution is used. Not more than 200 mg should be administered.
Acute intoxication can occur CON
Toxicity: Convulsion, dysrhythmia, myocardial ischaemia, coma, shock, cardiac arrest.

1. Widely used local anaesthetic agent.
5. Maximum dose: 500 to 1000 mg
6. Toxic reaction: Convulsion, cardiovascular collapse
7. Not much used now a days.
2. Rapid onset and medium duration of action
3. Action may last for 60 to 90 min when used with adrenaline
4. Dose: Infiltration 0.25 to 1%
Nerve block 1 to 2 %

1. Amide local anaesthetic, highly lipid-soluble
N . Adrenaline (in 250000) may be added to prolong the effect
9. Does not cross the placental barrier.
2. Commonly used local anaesthetic
3. Available in a wide range of concentration from 0.5 to 4%
4 . Total dose: 500 to 750 mg
5 . Dose:
For infiltration 0.5 to 1%
For nerve block 1 to 1.5%
For spinal block 4% (heavy)
For epidural block 1.5 to 2%
For corneal analgesia 4%
. Lignocaine has little effect on blood vessels
. Other uses: Antiarrhythmic drug, in management of neonatal convulsion,
parenteral analgesic

1. More potent and less toxic and more widespread than lignocaine. Wider margin of safety
2. Duration of analgesia 2.5 to 3 hours
3. Uses:
oa . Can cause methaemoglobinaemia which can be reversed by methylene blue
For infiltration 0.5%
For extremity blocks 2 to 3%
For caudal/epidural block 3 to 4%.
. Safe maximum dose 400 mg; 600 mg with adrenaline
. Eutectic mixture of local anaesthetic (EMLA) is a combination of equal amounts of crystalline 2.5% prilocaine and 2.5% lignocaine. It becomes an oil and it is then emulsified with water to form EMLA cream. It is in the ionised and diffusible form.
readily

1. Potent local anaesthetic
2. Onset of action is rapid, duration ranges from 2 to 3 hours
3. Concentration may vary 0.5 to 2%

4. Uses:
For
infiltration 0.5 to 1%
For nerve blocks 1 to 1.5%
For spinal block 4% heavy
5. Side effects:
Mild tachycardia, hypotension.
6. Can pass through placental barrier.

1. Potent local anaesthetic agent of the amide group; 4 times more potent than lignocaine.
2. Slower onset of action than that of lignocaine
3. Increased lipid solubility and protein binding compared to lignocaine leading to increased duration of action (3 to 12 hours)
Low placental transfer
. Produces more sensory than motor block
Adrenaline may be added to prolong the action and improve the block
Not more than 2 mg/kg should be used
. Concentration is usually 0.5%, but 0.25 and 0.75% solution are also available
. Used in nerve blocks, spinal/epidural block.
COND

1. Potency and duration are mostly similar, but the onset time is shorter than bupivacaine
2. Cardiovascular toxicity is less, causes vasoconstriction
3. Used in infiltration analgesia, peripheral nerve block and epidural analgesia
4. Available in solutions of 0.5 to 2
5. Maximum safe dose 150 to 200 mg.
Table 6.1: Recommended safe maximum single dose of some local anaesthetics in adults
Anaesthetic
mg/kg plain
mg/kg with adrenaline
Lignocaine
4 4 2.5 7
7 7 3.2 8.5
Mepivacaine
Bupivacaine
Table 6.2: Physiochemical properties of some local anaesthetic agents. (After Yao and Artusio:
Anaesthesiology)
pKa
Protein binding %
Procaine
8.9
5.8
7.9
64.3
7.6
77.5
8.1
95.6

Muscle relaxants or neuromuscular blocking agents act by blocking the specific nicotinic receptor sites in the neuromuscular junction or motor end plate of striated muscle. Normally acetylcholine released by nerve impulses diffuses across the junctional gap and stimulates muscle fibres by acting on specific nicotinic receptor sites on the postjunctional membrane of the motor end plate.
These receptors are related to an ion channel through which sodium and calcium ions flow and depolarise the membrane. The action of acetylcholine is short as it is rapidly hydrolysed by acetylcholinesterase. There are also prejunctional nicotinic receptors, stimulation of these receptors hasten the mobilisation of stored acetylcholine and thus increases the transmitter output during high frequencies of stimulation.
Neuromuscular blocking agents act by blocking choline receptors at both sites. These agents
may blockers.
also act as ion-channel
Muscle relaxants are traditionally distinguished into two groups:
1. Depolarising (noncompetitive) agents—Suxamethonium 2. Nondepolarising (competitive) acetylcholine antagonists—Tubocurarine, gallamine, pancuronium, atracurium, vecuronium, etc. These may be further subdivided according to their duration of action.
a. Long-acting: Tubocurarine, gallamine, metocurine, pancuronium, doxacurium, pipecuronium
b. Intermediate-acting: Atracurium, vecuronium, rocuronium
c. Short-acting: Mivacurium.
¢ Depolarising muscle relaxants act by holding the ion channels open and preventing repolarisations. These are acetylcholine agonists.

1. Muscle paralysis is always preceded by strong stimulation of the motor end plates. This causes initial fasciculations of the muscles all over the body
2. Anticholinesterase drugs may make the block profound
3. Absence of tetanic fade during partial block

4. Effects are decreased by nondepolarising relaxants
An absence of posttetanic facilitation wl
6. Following repeated administration of depolarising muscle relaxant over a prolonged period, the characteristics of depolarisation block change from a depolarising to a non-depolarising nature and it leads to development of dual block (desensitising block or phase II block).
¢ Nondepolarising neuromuscular blocking drugs compete with acetylcholine for the receptors at the motor end plate, but do not have any initial stimulating action on the motor end plate. This is often called competitive neuromuscular blocking agent.

1. Noninitial muscular fasciculation
2. Block is antagonised by anticholinesterases
3. Presence of fade of successive stimuli on both tetanic (fast) and twitch (slow) nerve stimulation
4. Following a train of tetanic stimuli, single shock leads to a temporary increase in the strength of muscle twitch (posttetanic facilitation)
5. Paralysis increased by nondepolarising agents.

1. For smooth endotracheal intubation
2. For adequate muscle relaxation in surgical field
3. For smooth artificial ventilation
4. To reduce the amount of inhalational anaesthetics
5. To overcome laryngospasm, convulsion, spasticity, rigidity, etc.

1. Prevention of acetylcholine synthesis: Hemicholinium
2. Prevention of acetylcholine release: Botulinum toxin, high magnesium concentration
3. Depletion of acetylcholine stores:
tetanus toxin, B-bungarotoxins.
4. Block of acetylcholine receptors:
a. Nondepolarising muscle relaxants : Tubocurarine, gallamine, pancuronium, etc.
b. Depolarising muscle relaxants : Suxamethonium.
5. Block of cholinesterase:
a. Anticholinesterases: Neostigmine
b. Organophosphorus poisoning.
Sequence of effects following motor nerve stimulation
Nerve action potential
L
Depolarisation of nerve terminal
L
Release of acetylcholine
L

Diffusion of acetylcholine through synaptic cleft
L
Acetylcholine activation receptors in postjunctional membrane
L
Changes in permeability leading to sodium flux into and potassium flux out of cell
Depolarisation of postjunctional membrane
L
Muscle action potential
L
Muscle fibre contraction
Factors altering the response to muscle relaxants
1. Age
Newborns are generally resistant to depolarising blocking agent and more sensitive to
nondepolarisers. Use of depolarising drug may cause dual or phase II block in infants
2. General anaesthetic agents can potentiate the action of nondepolarisers
3. Neuromuscular diseases
i. Patients suffering from myasthenia gravis are much sensitive to nondepolarisers and may show resistance to depolarisers. Rapid phase II block can also occur
ii. In some myopathies (myotonia dystrophica, myotonia congenita) generalised muscle spasm may occur following use of depolarisers
iii. Muscular dystrophy, congenital myopathies, familial periodic paralysis: Response to muscle relaxants unpredictable. It is better to avoid them
iv. Amyotrophic lateral sclerosis, poliomyelitis, syringomyelia: Exaggerated response to nondepolarisers
vi. Diseases producing hypokalaemia potentiate the action of nondepolarisers
vii. Malignant hyperthermia is a dangerous condition following use of succinylcholine.
Thyrotoxic myopathy: Decrease response to succinylcholine due to increased level of pseudocholinesterase in hyperthyroidism
4. Electrolyte imbalance
i. Hyponatraemia, hypokalaemia : Increased sensitivity to muscle relaxants
ii. Hyperkalaemia: Decreased sensitivity to nondepolarising agent
iii. Hypocalcaemia, hypomagnesaemia prolong the action of muscle relaxants.
5. Temperature:
Hypothermia potentiates the action of neuromuscular blocking agents.
Intensity of block is decreased in cases of hyperthermia.
6. Hypo/hyperventilation can also alter the response of muscle relaxants.

1. Produces profound muscle relaxation due to nondepolarising myoneural block
2. Airway reflexes depressed
3. Bronchospasm may occur due to histamine release.

SANDS
. Mild hypotension, bradycardia
. Little effect on liver and kidney
. General anaesthetics potentiate the curare effect
. Actions are antagonised by use of anticholinesterase drugs like neostigmine
. Dose: 15 to 30 mg iv for endotracheal intubation. It takes 2 to 3 min for full effect, duration
of effect is usually about 30 min

i. For smooth endotracheal intubation
ii. To produce muscle relaxation
iii. For better controlled ventilation
iv. To make bronchoscopy, oesophagoscopy and other endoscopies easier
v. As an aid, in treatment of tetanus, myositis, strychnine poisoning, spasticity, rigidity, etc.

1, Synthetic nondepolarising agent, produces profound muscular relaxation
2. Dose 2 mg/kg iv, action is rapid within 2 to 3 min, lasts for about 20 min
3. Action antagonised by anticholinesterases
4. Atropine like effect (vagal blocking) on cardiac postganglionic nerve endings causing tachycardia
5. Minimum histamine release, no ganglion blocking effect
6. Passes through placental barrier
7. General anaesthetics potentiate its action
8. No direct effect on liver and kidney.

1. Nondepolarising potent muscle relaxant
2. Muscle paralysis occurs in 3 to 4 min and lasts or about 20 min
3. Minimum histamine release
4. Mild ganglion blocking effect, mild hypotension
5 . No sympathetic stimulation, no vagolytic effects
6 . It is 40% protein bound and undergoes negligible metabolism.

DAuPWNEH
. Bisquarternary aminosteroid with no hormonal effects
. Nondepolarising potent muscle relaxantes
. Acts within 2 to 3 min and lasts for about 30 min
. Usual dose: 0.1 mg/kg
. Mild sympathomimetic and vagolytic effect, mild increase in heart rate and blood pressure
. Less histamine release, no ganglion blocking effect

7. No cumulative effect
8. It is 34% bound to albumin, 53% to globulin and only 13% unbound
9. Broken down in liver, effect prolonged in cases with liver failure.

1. Potent nondepolarising muscle relaxant
2. Dose: 0.1 mg/kg
3. Muscle paralysis within 1.5 to 2 min. and lasts for about 15 min
4. General anaesthetics potentiate its action
5. Does not cause histamine release, neither ganglion blocking nor vagal bloking effect, no
sympathomimetic
effect
6. Pulse rate and blood pressure remain unaltered
7. Metabolism is mostly dependent on liver. It should be avoided in severe liver disease
8. Cumulation may occur with repeated doses
9. May show spontaneous recovery, but antagonism of the block with anticholinesterase can be achieved.

1. Bisquarternary ammonium compound, potent nondepolarising muscle relaxant
2. Dose: 0.6 mg/kg
3. Good intubating condition within 90 sec
4. Action lasts for 20 to 30 min
5. May release histamine
6. Devoid of sympathetic stimulation, vagolytic and ganglion blocking effects
7. Itis self-destroyed by Hofmann reaction at a pH of about 7.4 and at 37°C, but ester hydrolysis in liver and plasma is the main metabolic pathway
8. Atracurium shows excellent recovery spontaneously, no need of specific antidote
9. Relaxant of choice in patients with severe liver and kidney disease.

1. Synthetic nondepolarising muscle relaxant with short duration of action
2. Dose: 0.15 to 0.25 mg/kg
3. Profound muscle relaxation within 3 min and lasts for about 15 to 20 min
4. Also used in continuous infusion in a dose of 5 to 6 ug/kg/min
5. Hydrolysed by plasma cholinesterase
6. Cardiovascular side effects minimum
7. Histamine release insignificant.

1. Long-acting nondepolarising muscle relaxant
2. Intubation dose: 0.07 to 0.085 mg/kg iv

SND
Satisfactory intubating condition within 2.5 to 3 min
Duration of action ranges from 45 to 120 min
No ganglion blocking and vagolytic effects
No histamine release
Cardiovascular side effects minimal
Effect is antagonised by neostigmine.

R Potent depolarising muscle relaxant, ultrashort acting
CANA
YN Dose: 1 mg/kg iv ; 50 to 75 mg on average Satisfactory intubating condition with 60 sec and the effect lasts fo 3 to 4 min
PR oO May cause bradycardia, hypertension and even arrhythmia
RP Disadvantages:
12. Contraindications:
Causes initial generalised muscular fasciculations
Does not pass through placental barrier
Chief use is for quick and reliable intubating condition including crash induction
Destroyed by hydrolysis by plasma pseudocholinesterase
Not antagonised by neostigmine, indeed the action is potentiated by it
Newborns are resistant to the drug. Dual block may occur in infants
i. Muscle pain: More common in ambulatory patients, mostly seen in neck, shoulder girdle, abdomen, can be lessened by pretreatment with nondepolarising muscle relaxants.
Diazepam, lignocaine or dantrolene may also help; self-taming is possible
ii. Hyperkalaemia: After injection of suxamethonium, potassium leaks into extracellular fluid. Patients suffering from tissue injury from trauma, burns, neurological injuries may exhibit servere hyperkalaemia and on occasion has resulted in cardial arrest
iii. Raised intraocular pressure, raised intracranial pressure
iv. Raised intragastric pressure
v. Bradycardia, sinus arrest
vi. Malignant hyperpyrexia may be triggered in susceptible subjects
vii. Prolonged apnoea in patients with deficiency or abnormality of the enzyme
pseudocholinesterase
viii. Anaphylactoid reactions
ix. Trismus
x. Myoglobinuria.
i. Burns
ii. Muscle trauma
iii: Upper and lower motor neurone disease
iv. Patients with arrhythmias
v. Patients with deficiency or abnormality of pseudocholinesterase
vi. Patients with penetrating eye injury, glaucoma
vii. Patients susceptible to malignant hyperthermia.

It determines the duration of action of succinylcholine. Normal level in plasma is 80 to 120 units.
A level less than 25 units is regarded as low.
e High levels are found in childhood, obesity, goitre, nephrosis, depressed states, psoriasis, alcoholism
e Low levels may be seen in patients with liver disease, pregnancy, cancer, severe anaemia, uraemia,
cardiac failure, hyperpyrexia, cytotoxic drugs, acetylcholinesterase inhibitors, drugs like phenelzine, hexaflurenium, etc.

It indicates the percentage of enzyme inhibited by 10° molar concentrate of dibucaine (cinchocaine). It does not reflect the quantity but the quality that is, its ability to hydrolyse succinylcholine.
Dibucaine inhibits the normal enzyme by about 80% and an abnormal enzyme by about 20% standardised test conditions.
under
Drugs metabolised by pseudocholinesterase
1. Suxamethonium
2. Mivacurium
3. Atracurium
4. Propanidid
5. Procaine
6. Chloroprocaine
7. Amethocaine.
Side effects of nondepolarising drugs
1. Histamine release: Tubocurarine, alcuronium, atracurium
2. Ganglion blockade: Tubocurarine
3. Vagal blockade: Gallamine, fazadinum, pancuronium
4. Sympathomimetic action: Pancuronium.

1. Widely used to antagonise nondepolarising muscle relaxants
2. Dose may range from 0.5 to 2.5 mg (0.04 to 0.07 mg/kg) depending on the dose of relaxant and time of its administration. Dose should not exceed 5
mg
3. Neostigmine may cause bradycardia, miosis, excessive salivary and bronchial secretions, bronchospasm, contraction of gut, etc. To counteract these side-effects atropine should always be given at 5 minutes before injecting neostigmine. Dose of atropine 0.015 mg/kg, adult dose 1 to 1.5 mg or glycopyrrolate (0.008 mg/kg) adult dose 0.5 to 0.6 mg may also be helpful.

1. Short-acting synthetic anticholinesterase
2. Duration of actionis about5 min. So recurarisation may occur and repeat dose may be necessary
3. Muscarinic actions are not so prominent, but atropine should always be given before injecting neostigmine
4. Usual dose: 10 to 20 mg iv

1. Adequate normal tidal exchange and ability to produce vital capacity of at least 10 mg/kg
. Able to open his eyes and to keep them open
. Able to raise head and sustain for at least 5 sec
. Nojerky respiratory movements
. Effectively cough on endotracheal tube
eONaAaRON
ay Oo . Intraocular tension normal.
Muscle power normal with electrical nerve stimulation
. Adequate grip strength
. Ability to produce negative inspiratory effort of at least—20 cm HO against obstructed airway
A TOF ratio greater than 0.7 or its equivalent

1. Unability to open the eyes, ptosis
2. Jerky respiration
3. Ineffective cough on endotracheal tube
4 . Incomplete respiratory movement
5 . Tracheal tug
6. Cyanosis.
Factors increasing the duration of neuromuscular block :
1. Overdose of muscle relaxants
. Incomplete decurarisation
. Liver dysfunction KWH
. Kidney dysfunction
PR RP oO . Electrolyte changes: Hypokalaemia, hypernatraemia, hypermagnesaemia hypocalcaemia . Drug interactions:
CONAN . Hypothermia . Alkalosis
Use of general anaesthetics
. Preexisting disease: Myasthenia gravis
. Deficiency or abnormal pseudocholinesterase
i. Antibiotics, aminoglycosides like neomycin, gentamicin, kanamycin, streptomycin
ii. Local anaesthetics
iii. Antiarrhythmic drugs: Quinidine, procainamide
iv.
Hypotensive agents: Trimethaphan, nitroprusside, nitroglycerine in high doses
v. Ketamine
vi. Diuretics: Furosemide
vii. Ca channel blockers.

1. Essential for close supervision of the degree and duration of muscle paralysis and the recovery
from
it.
2. Can be performed by
i. Continuous mechanical measurement of the actual muscle strength (force of contraction) throughout the operation
ii. Electromyography and accelography to measure the electrical activity of muscle (compound electromyogram) or the acceleration of the thumb respectively
iii. Use of peripheral nerve stimulator. It is the most practical way of monitoring muscle relaxation.

The simple instrument is used to deliver an electrical stimulus to a particular nerve and the response of the muscle innervated by the same nerve is evaluated. The stimulation pattern may be single twitch, train-of-four and tetanus. Tactile or visual evaluation is done. It can also be measured by pressure transducer.
These should be used routinely in all cases where muscle relaxants are given. The ulnar, facial, posterior tibial and the common peroneal nerves are often used. The ulnar nerve at the wrist is popularly selected. Two electrodes are placed over the nerve with negative electrode distally and the positive electrode about 2 cm proximally. Supramaximal stimulus is used to stimulate the nerve. A current of 15 to 40 mA is used. Duration of stimulation is less than 0.2 ms. Observation or measurement of muscle movement (thumb, adductor pollicis muscle) can be easily performed.

1. Twitch-tetanus-twitch: Here a train single shocks are given at a rate not more than 1 every 3s followed by a tetanic train at 50 Hz or more for 3s and then a train of single shocks are repeated.
i. Normally twitch response is followed by tetanic strong sustained contraction. No potentiation of posttetanic twitches
ii. Total block: No response is obvious
iii. Partial depolarising block: Twitch responses are weak and this is followed by a weak but sustained tetanic contraction. No posttetanic potentiation is observed
iv. Partial nondepolarising block: Here the initial twitch response is weak, the tetanic train shows contraction within fades quickly and there is potentiation of posttetanic twitches.
2. Train of four
This method uses four stimuli at 2 Hz given at 0.5s intervals. It may be repeated every 10s. TOF ratio is calculated.
In train of four stimulation a twitch and train of four are alway, combined. The first twitch of TOF is always equal to the single twitch.
The train of four ratio is calculated as the ratio of the force generated by the fourth force of contraction to the first when force transducers are used. TOF ratio is the ratio of height of the fourth to the first twitch. A ratio greater than 70% indicates adequate neuromuscular transmission following competitive blockers.

Normally it is 1 (100%)
Absence of T, response implies 75% block.
Absence of T3 response implies 80% block.
Absence of T; response implies 90% block.
Fading occurs in nondepolarising block.
No fading, but all four responses diminish simultaneously in amplitude in depolarising block.
Phase II block: Patient is on succinylcholine, but shows fading.

1. Reduction of twitch height
2. No fade of TOF or tenany
3. No posttetanic facilitation.
e Features of nondepolarising block
2. Fade of TOF and tetany
3. Posttetanic facilitation.
¢ Common uses of nerve stimulation
1. Prolonged anaesthesia. Facilitates titration of muscle relaxants. Avoids excess dose
. Patients with renal and/or liver dysfunction WN
. Hypersensitive patients, Myasthenia gravis
. Infusion of muscle relaxants . Patients with myasthenia gravis or myasthenic syndrome . New muscle relaxant investigation NDFB
. To assess the residual block. Monitors adequate reversal.

d tubocurarine 0.6 mg/kg
Pancuronium 0.1 mg/kg
Atracurium 0.5 mg/kg
Doxacurium 0.05 mg/kg
Mivacurium 0.2 mg/kg
Vecuronium 0.1 mg/kg
Rocuronium 0.6 mg/kg
Pipecuranium 0.1 mg/kg
Suxamethonium 1 mg/kg
Gallamine 2 mg/kg.

Criteria:
1. Small volume of distribution
2. Rapid clearance
3. Rapid equilibration between plasma and neuromuscular junction.

Muscle relaxants for continuous infusion
:
1. Mivacurium is the relaxant of choice
2. Atracurium
3. Vecuronium.
e Priming
Intubation time can be shortened by giving a priming dose of the muscle relaxant (10 to 20% of the intubation dose) which blocks upto 60 to 70% of the motor end plate receptors but does not give any significant muscle relaxation. Sometimes after the priming dose, a large bolus of the paralysing dose can take action within a much shorter time. Muscle relaxant produces paralysis markedly faster than if the two doses were given together.
The priming
ideal time between the dose and the intubation dose is about 4 min.
Some patients may profoundly following of priming may vary from patient-to-patient.
become weak the initial dose. Moreover, the benefits
¢ Some drugs can reduce the neuromuscular block
1. Phenytoin
2. Carbamazepine
3. Calcium.
¢ Drugs used to reverse the nondepolarising block
1. Neostigmine
2. Pyridostigmine.
¢ To measure a physiological variable and to indicate prompt recognition of any change, thus enabling appropriate therapeutic action to be taken.

. Skin : Colour, capillary refill, rash, oedema
. Nail beds : Colour, cyanosis
. Mucous membrane
. Movement, position
. Eyes, pupil, tearing
. Surgical site, blood loss
Pulse
. Respiration
. Skeletal muscle tone . Body temperature a FOOD
. Blood pressure: Continuous or intermittent
. Ventilation, breath sounds PR Wn
. Heart sounds—Amplitude, rhythm.
MONAURWNE

. Precordial/oesophageal stethoscope ay
. Noninvasive blood pressure
Pulse oximeter: Heart rate, peripheral O2 saturation
. Electrocardiogram : Heart rate, rhythm, ischaemic changes
. Capnometry : End tidal COz, rate of respiration
. Expired tidal/minute volume
Body temperature
. Arterial blood gases/pH
. Central venous pressure
CONAARWHN

10 . Peripheral nerve stimulator
11 . Disconnect alarm: Automatic alarms provided with anaesthetic machine and ventilator
12 . Urine output.
1 . Myocardial ischaemia
ONDA . Echocardiography . Electroencephalography
ARWNE
Some other monitors may be needed in special situations :
. Oxygen analyser
. Pulmonary artery catheter
Mass spectrometry
Raman spectroscopy
Spirometer
. Evoked potentials to allow assessment of the functional integrity of neural pathways during
. Estimated blood loss.
Invasive monitoring methods should be used when specially indicated.
Echocardiography helps to detect/assess
:
2. Cardiac mitral or aortic valve function
3. Intracardiac air
4. Cardiac function
5 . Ejection fraction.
1 . Check, calibrate all the monitors before use
Note :
2. Set alarm limits of all monitors
3. Monitors should not be regarded a substitute for sound clinical judgement. One should
integrate
and
interpret
the
data
available
in
the
light
of
clinical
condition.
Meticulous record keeping is essential for all patients undergoing anaesthesia. It is also important for medicolegal purposes.

1. Cylinder of oxygen on the anaesthetic machine should be checked and immediately replaced when it is exhausted
2. Pipeline system should also be checked and should automatically comes in line when it is exhausted. These are usually supplied either from a liquid oxygen tank or from a bank of O2 cylinders. There should be audible alarm and a warning lamp on display panel
3. Anaesthetic machine should be fitted with a low pressure alarming device when O2 supply fails.

1. Components of the circuit should be checked
oa . During mechanical ventilation, it is essential to monitor the airway pressure. Alarms should be fitted with adjustable low-and high pressure limits
Oo . Monitor the ventilator parameters during use.
OND . Leak check of the breathing circuit should be done
2. Oy failure, kinked and obstructed airway, blocked gas exit can occur
3. Leak, disconnection, overpressure or hyperventilation can also occur
4. High airway pressure may cause pulmonary barotrauma. There should be high pressure alarm system
. Breathing circuit should be complete, undamaged and unobstructed
. Monitor the vaporisers, valves, flowmeters, etc.
The anaesthetic machine enables to deliver gas and vapour mixtures to the patient accurately and continuously. The Boyle anaesthetic machine is designed by HEG. Boyle in 1915. Modern machine differs greatly in detail and modified time-to-time, but the basic principles remain the same. It consists
of:
. Oxygen and anaesthetic gas supply
. Pressure gauges ONS
Reducing valves
Flowmeters Vaporisers NOB
Common gas outlet
. Certain other features:
Highflow oxygen flush, pressure relief valve, oxygen supply failure alarm, suction apparatus, monitoring devices.

1. Made of molybdenum steel to withstand high pressures
2. Made of different sizes (A to J). Size E cylinders are used in the anaesthetic machine
3. Oxygen is stored as gas at about 2000 Ib/inch? and nitrous oxide is stored in a liquid phase with vapour on the top at a pressure of 760 Ib/inch?. It is 75% filled with liquid nitrous oxide. Note: Filling ratio is the weight of fluid in the cylinder divided by the weight of water needed
to fill the cylinder.
4. Cylinders are colour coded.
Oxygen: Black with white shoulders
Nitrous oxide: Blue
Carbon dioxide: Grey
Entonox: Blue with white/bluequarters shoulder
Air: Grey with white/black quarters shoulder.

5. Some markings engraved on the cylinders:
Test pressure, chemical formula, Tare weight,
dates of test performed, etc.
6. Checking and testing by manufacturers at regular intervals:
i. Flatening test
ii. Bending test
iii. Impact test
iv. Pressure test
v. Tensile test.
7. Gases and vapours must be free from water vapour as it may freeze and block the exit
port temperature particularly opening
at a decreased when
8. Cylinder valve provides pin index system as a safety feature to make it almost impossible to
connect a cylinder to a wrong yoke
9. Should be stored in a dry, well-ventilated and fire proof room. Avoid dampness, corrosives and fumes near by. No oil or greese or any other flammable materials or any source of heat should be allowed
10. Full cylinders should be kept separately and should not be mixed with empty ones
11. Avoid overpressurised full cylinders.

As pecific pin configuration for each medical gas on the yoke of the anaesthetic machine. The pin will match the holes on the valve block. It permits only the correct gas cylinder to be fitted in the yoke.
Oxygen: 2 and 5
Nitrous oxide: 3 and 5
Cyclopropane: 3 and 6
Entonox: 7
Air: 1 and 5
Carbon dioxide: 1 and 6
Pressure in the cylinders
Oxygen: About 2000 Ib/inch?
Nitrous oxide: About 750 Ib/inch?
Cyclopropane: 75lb/inch? [stored in light alloy cylinders as a liquid]
Carbon dioxide: 720 Ib/inch?.

1. Mounted on the neck of the cylinder, screwed with threaded connection
. The valve can be opened or closed by an off/on spindle for gas pathway 2
3. Noninterchangeable safety device (pin index system) prevents wrong cylinder assembly
4 . Bodok seal is placed between the valve outlet and yoke of the machine to make the gas tight joint.

Fig.
9.1:
Disposition of
holes in
flush—type
Fig. 9.2: Cylinder valve—Flush type outlet valve
- 5. The cylinder valve should be fully open during use. Check the Bodok seal before use to detect any damage.

1. Gas delivered from a central supply to different locations at a pressure of about 400 kPa
2. Oxygen, nitrous oxide, entonox, compressed air and medical vacuum can be used through the pipe line system
3. Components:
i. Central supply (cylinder bank/liquid oxygen storage bank or oxygen concentrator)
ii. Pipe work (made of copper), flexible and colour coded
ii. Outlets (identified by gas colour coding, gas name and by shape) accept matching quick connect or disconnect probes with an indexing colour specific for each gas be
iv. The cylinders of each group are connected through nonreturn valve to a common pipe.
Then in
turn is
connected to
the pipe
line through
pressure regulators
- v. The cylinders should be well-housed in a well-ventilated room (fire proof) in a suitable place in hospital.

1. Oxygen can be stored and supplied in a vacum thermally insulated evaporator at a temperature of — 150° to — 170°C and at a pressure of 5 to 10 atmospheres.
2. A pressure regulator permits gas to enter the pipelines and maintains it at about 400 kPa

3. It also provides a safety valve to release the build up high pressure, if occurs
4. A control valve is also there to meet the extra demands on the system
5. Cold oxygen gas is warmed outside the vessel in copper tubing coil. The increase in temperature increases the pressure.

1.
2. The zeolite sieve selectively retains nitrogen and other unwanted constituents of air and them to atmosphere
Extracts
oxygen
from
air
when
exposed
a
zeolite
molecular
sieve
column
at
certain
pressure
releases
. Maximum oxygen concentration can be achieved is nearly 95% by volume. Argon may be main of the other constituents.

1. Can be used either clinically at a pressure of 400 kPa or to drive power tools at a pressure of about 700 kPa
. Can be supplied from cylinders or from a compressor
. For clinical use air should be cleaned by filters and separators and then dried.

1. Measures the pressure in the cylinder mounted in the front facing panel on the anaesthetic machine
. High pressure gas acts to straighten a coiled tube (Bourdon gauge). Movement of the tube causes the needle pointer to move on a calibrated dial to indicate the pressure
Fig.
9.3:
Pressure
gauge

3. Colour coded and calibrated for a particular gas or vapour
4. Indicates the contents of gas available in the cylinder
5. Nitrous oxide cylinder does not provide pressure gauge as it is stored as a liquid and vapour
6. Pressure gauge meant for pipeline is not for cylinder and vice versa. May lead to inaccuracies
and/or damage
. Misassembly is possible.

1. Reduces the variable gas pressure from cylinders to about 400 kpa
oo . Diaphragm can rupture
2. Positioned between the cylinders and the rest of anaesthesia machine
3. Provides fine control of gas flow and protects the parts of machine against high pressure
4. Prevents the pressure from increasing above a predetermined limit
5 . Contents of the cylinder should be free from water, otherwise ice can form inside the valve
6 . Earlier models are with ‘fins’ to augment acquisition of heat from the environment
7 . Mechanism of action: The gas from the cylinder enters into the chamber of reducing valve where it distorts a diaphragm to which a toggle mechanism is connected and a balance between two opposing forces maintains a constant predetermined operating pressure (60 psi)
. Relief valve is fitted downstream of the regulator. It helps to escape the gas, if regulator fails.

. Pipeline pressure increase can be controlled by using a pressure regulator on a flow restrictor . Flow restrictor provides a constriction between the pipeline supply and the remaining part of the anaesthetic machine. The constriction causes a significant pressure drop in presence of a high gas flow rate.

1. Measures flow rate of a gas per minute
2. Each gas is individually calibrated
3. Oxygen should be the last to be added in mixture
4. Calibration done at room temperature and atmospheric pressure, accuracy with an error margin +2%
. It contains: A flow control valve, a tapered (wider at the top) transparent glass or plastic tube, a light weight rotating bobbin. Bobbin stops at either end of the tube
. Flow control (needle) valves:
i. Controls the flow through the flowmeters by manual adjustment
ii. Positioned at the base of flowmeter
iii. Control knobs are labelled and colour coded, blue for nitrous oxides, white for oxygen and grey for carbon dioxide.
Fig. 9.4: Rotameter unit
7. When the gas flows, with the help of flutes the bobbin spins clears of the walls of the tube and thus avoids the errors of friction. Bobbins should be made antistatic
8. The viscosity and density of the gases can influence the gas flow in the flowmeter. Each flowmeter is calibrated for each gas
9. Flowmeter should be set in vertical position, otherwise may give incorrect reading
10. Malfunction: Broken, cracked, bobbin stuck, wrong gas, inproper alignment, back pressure, etc. 11. Other types:
i. Heidbrink flowmeter: Metal tapered tube with inverted black float
ii. Connell flowmeter: Provides round float, reading from its centre.

To administer a controlled amount of an inhalational agent after changing a liquid to vapour to the fresh gas flow.

1. Performance not affected by changes in fresh gas flow, liquid volume, ambient temperature, and pressure, decrease in temperature due to vaporisation and pressure fluctuation due to mode of respiration
2. Low resistance to flow

3. Light weight with small liquid requirement
4. Economy, safety, minimum servicing
5. Corrosion and solvent resistant
6. Quality control by authorised institution.
Boyle anaesthetic machine provides two vaporising bottles one for diethyl ether and the other for trichlorethylene. These vaporisers are of variable bypass, flow over or bubble through type. These are neither properly calibrated nor temperature compensated.
The concentration of vapour depends on the rate of gas flow, liquid surface area, the way the gas impinges on the liquid and the temperature of liquid.

1. Contains diethyl ether which vaporises to an extent governed by its saturated vapour pressure at room temperature
. The amount of gas entering the vaporiser can be altered by moving the lever
. Gas enters the vaporiser above the anaesthetic liquid to vaporise and its concentration can be altered by depressing the plunger
. Vaporisation needs latent heat of vaporisation. Larger amount of heat is needed to vaporise ether. The concentration from the vaporiser usually falls significantly as the temperature and vapour pressure of ether fall. So the total gas flow may need to pass through the vaporiser and even bubbled through it
. Sealing washer of the Boyle's bottle should be of good order
. Dark brown-coloured bottles are used as ether can be decomposed by light
Fig. 9.5: Boyel ether vaporiser

- 7. Metal parts of U tube and hood of plunger should be made of copper as it is anticatalyst and can prevent decomposition of ether.

It is similar to ether bottle but it is smaller in size and the inlet U tube and the hood of plunger are entirely chrome plated.

1. Simple vaporiser, flow-over type, no wicks
2. Neither temperature compensated, not accurately calibrated
3. Can be used inside or outside the breathing circuit
4. Control device on the top which can be rotated to alter the vaporizer output
5. Low resistance to gas flow
6 . Maximum concentration never exceeds 3%
7 . Vapour concentration can be increased by
i. Splashing (ii) incorporating wicks (iii) employing 2 vaporisers in series.
Fig. 9.6: Goldman vaporiser

1. Part of the Boyle machine on which rotameter (containing flowmeters), vaporisers (ether and trichlorethylene) and other accessories are fitted
. ‘Trilene safety interlock’ is placed at the end of back bar (Fig. 9.7)
. Provides an angled outlet with a nonreturn valve to prevent a back pressure during positive pressure ventilation
. At extreme right there is an emergency bypass which can deliver high flow of O2 directly, bypassing the rotameter

Fig. 9.7: Terilene interlock unit
- 5. Some machines may have an emergency press button unit to suppy plentiful oxygen (oxygen flush). Risk of accidental activation includes barotrauma, dilution of anaesthetic gas, awareness.

1. Various designs are available
2. Activates on the pressure of oxygen
3. No need of battery or mains power
4 . Gives audible signal sufficient to draw attention
5 . Should have pressure linked control which interrupt the flow of other gases when they come into operation.

1. Designed to maintain a constant output of volatile anaesthetic agent
2. The case is made of copper which is a good heat sink
3. Provides a bypass channel and vaporising chamber which contains wicks to increase the surface area available for vaporisation
ONA . The effect of back pressure is compensated . The calibration of each vaporiser is agent specific
. Has a temperature-compensation device. Bimetallic strip inside to compensate for the cooling of the liquid anaesthetic. When cooling takes place due to latent heat of vaporisation, the strip made of two different metals with different coefficients of expansion bend to permit a greater fraction of the total flow to enter the vaporising chamber
The gas coming out of the vaporising chamber is fully saturated
. Vaporiser filling devices are agent specific. These are geometrically coded to fit the safety filling port of the correct vaporiser and anaesthetic bottle. The fillers are also colour coded, red for halothane, orange for enflurane and purple for isoflurane.

9. Caution:
i. The liquid anaesthetic should not enter the bypsss channel
ii. The effect of back pressure must be compensated
iii. Preservative like thymol (in halothane) can deposit on wicks and interfere the efficacy
of vaporiser. Enflurane and isoflurane do not contain preservative
iv. A pressure relief valve can prevent the damage of flowmeter/vaporiser
v. Corrosion of bimetallic strip can occur.

1. Receives all gases and vapours from the machine
2. Attached to the delivery hose
3. Leak/disconnection can occur. Adequate care is needed
4. Acheck valve can be provided just proximal to outlet to prevent retrograde gas flow from flush valve or breathing system.

1. Made of metal tube. One end connected with machine outflow and the other end to the angle piece adaptor to connect corrugated rubber hose. Rebreathing bag is attached from the under surface
2. May have a valve. By turning a lever to one side, the rebreathing bag is cut out of the circuit
3. In intermittent flow machine the bag and bag mount should be removed from the machine.
Fig. 9.8: A= Bag mount

1. Made of antistatic rubber, ellipsoidal in shape
2. Capacity may vary from 2L to 0.5L, capacity of bag must exceed patient’s tidal volume. Accommodates
fresh gas flow
3. Bag movement is important in assessing or controlling the ventilation
. Limits pressure build up in the system »
5. Must be comfortable for use.

1. Flexible, light weight breathing tube. Made of antistatic rubber. Usual length about 1 metre
2. Corrugation helps acute angulation without kinking
3. Can act as reservoir in certain systems
4 . Can be used to connect the ventilator to the breathing circuit
5 . Tube diameter should be such to present low resistance to gas flow and to promote a laminar flow
. Irregular walls of the tube may cause some turbulence and allow some dirt or infective material into it.
Fig. 9.10: Corrugated rubber tube

1. Allows the exhaled gas and excess fresh gas flow out of the breathing system
2. Oneway spring loaded valves with three ports: Inlet, patient and exhaust ports. The exhaust port can be open to atomosphere

Fig. 9.11: Expiratory valve (Heidbrink type)
. The spring adjusts the pressure required to open the valve
. During spontaneous ventilation, a positive pressure in the system during expiration can
cause the valve to open
. During positive pressure ventilation an intentional leak is produced by adjusting the valve dial
during inspiration
. Malfunction:
i. May remain open/closed.
ii. If closed, a pressure relief safety mechanism should be there which is activated at a pressure of about 60 cm HO
iii. Condensation of water vapour can damage the valve.

- 1. Made of antistatic rubber or transparent plastic designed to fit the contour of face anatomically
2. Airfilled cuff ensures the snug fit over the face
3. Proximal end connects to the angle piece
4. May have some clamps for the harness to be attached
5 6 . It should have a minimum dead space. Dead space may increase by upto 200 ml in adults . Problems:
i. Difficult to achieve airtight seal over the face particularly in edentulous patients
ii. Excessive pressure by mask can damage branches of trigeminal or facial nerve.
Fig. 9.12: Face mask
Fig. 9.13: Face mask angle piece adaptor

Formerly classified as open, semiopen, semiclosed and closed systems.
1. Open system (open drop inhalation method):
Volatile
is
to
the
patient
with
atmospheric
air.
tract
has
access
to
atmosphere
all
the
time
during
both
inspiration
and
expiration
No reservoir/rebreathing bag
Schimmelbusch mask (Fig. 9.14) is used for open drop anaesthesia. It consists of a simple wire frame over which 8 to 12 layers of gauge are stretched and kept in position. Mask provides one effective surface for volatile anaesthetics and some space for confining the anaesthetic
vapour.
2. Semiopen system: Here also the anaesthetic is administered with air and respiratory tract is open to atmosphere during inspiration and expiration. But some reservoir is made over the mask (by a folded towel over the mask), but it is mostly open to the atmosphere
3. Semiclosed system:
Patient inhales from the continuous flow of gases/vapours from anaesthetic machine and it includes one reservoir/rebreathing bag
When all the expired gases escape to the atmosphere through nonrebreathing valve, then it is nonrebreathing type of semiclosed system
If part of exhaled gases escape through expiratory valve and part passes through rebreathing bag then it is partial rebreathing type of semiclosed system.
4. Closed system:
Anaesthetic gases and vapours are not voided in atmosphere. The same gases are continuously recycled after absorption of carbon dioxide by soda lime.
Oxygen utilised in metabolism and gases/vapours utilised in the body or lost are to be supplemented
Some intentional leak is always kept in expiratory valve.
i. Economy
ii. Retention of body heat and moisture

i. Circuit should be leak proof
ii. Cumbarsome and heavy equipment
iii. CO, absorption may not be adequate
iv. Resistance to breathing and dead space may be high
Vv. Heat from soda lime may affect the body
vi. Alkaline dust from soda lime may also affect
vii. Cross infection
viii. Dilution of fresh gas mixture possible.

Composition:
Sodium hydroxide 4%
Size:
Potassium hydroxide 1%
Water 14 to 19%
Silica (to prevent powdering calcium hydroxide) 95%
4 to 8 mesh
Chemical neutralisation of CO
CO, + H,O => H»CO3
H,CO3 + 2NaOH — NarCO3 + 2H2O0 + heat
HyCO3 + Ca(OH). — CaCO; + 2H2O + heat
Soda lime is pink coloured and it turns white when it becomes ineffective. There is gain in weight, when it is exhausted. Some regeneration can occur, when it is rested for 2 to 3 hours
Trichlorethylene should never be used in a closed circuit with soda lime, otherwise toxic products
like dichloroacetylene may be formed. It can produce paralysis of cranial nerves particularly 5th and 7th cranial nerve.

|
Barium hydroxide 20%
Calcium hydroxide 80%
Water bound crystalisation
No silica is needed.
Chemical neutralisation of CO»
H2CO3 + Ba(OH)2 > BaCO3 + 2H20 + heat
H»CO3 + Ca(OH). ad CaCO; + 2H2O
e Produces less heat than soda lime
e Less caustic than soda lime.

1. Absorber of to-and-fro system is made of metal, but that of circle system is usually glass or transparent plastic. Metals conduct and dissipate heat better and more resistant to corrosive effects of alkali. Plastic is light and enables to observe colour changes
2. Shape cylindrical. Wide and short canister preferred
3.
Granular
space:
Space
occupied
by
the
absorbent.
Measured
by
the
bulk
density.
It
is
0.9
gm/
cc
for
soda
lime
and
1
gm/cc
for
baralyme
4. Air space: Usually 45 to 75% of the volume of canister. It includes intergranular (void) space and intragranular (pore) space
i. Intergranular space : Usually about 45%, depends of size of granules and type of packaging
ii. Intragranular space: Space within the pores of granules. Varies directly with the weight of absorbent but inversely with its moisture conent. Usually about 25 ml/100 gm of absorbent.
5. Exhaustion time depends on exhaled COy, type of ventilation, tidal volume, absorbent capacity, degree of channelling, fresh gas flow, position and accuracy of the canister.

1. Introduced by Ralph Waters in 1923
2. A soda lime canister is place between the face mask and rebreathing bag. Both inspiration and expiration take place through the canister. Fresh gas should be very near to face mask.
Fig. 9.15: Water's canister and its attachments Key : 1=Face mask; 2=Angle piece; 3=Expiratory valve with gas feed, inlet; 4=Water’s canister; 5=Corrugated rubber tube connector; 6=Rebreathing bag; 7=Fixing device of canister

ONA . Channelling can occur. Canister needs adequate proper filling . Disadvantages: Heat production, alkaline dust, channelling, reduced efficiency
Ts . Resistance of this circuit may be more than 2 to 3 cm HO during spontaneous ventilation
. Canister: 8x13 cm, capacity 1 lb soda lime. Air space about 400 ml, intergranular space should be nearly the tidal volume of the patient
. Mechanical dead space: About 200 ml (from wire gauge of canister to face)
. Advantages: Cheap, simple, easy to operate and sterilise, low resistance, conservation of moisture and heat.

1. 2. Soda lime is used to absorb the patient’s exhaled CO}. Fresh gas flow requirement is low Soda lime canister is positioned vertically. It provides an inlet delivering fresh gas flow from machine and 2 ports — one to deliver fresh gas flow to patients and the other to receive exhaled gases from the patient. These 2 ports incorporate unidirectional valve. An expiratory valve connected to a rebreathing bag (Fig. 9.16)
. Inspiratory and expiratory tubings are connected to the canister
. A vaporiser can be incorporated in the back bar of the anaesthetic machine (outside circle) or on the expiratory limb within the circle (VIC)
. Exhaled gases are circled back to the soda lime canister. After CO, absorption the gas joins the fresh gas flow to be delivered to patient
. The system can be used for both spontaneous or controlled ventilation
. Mechanical dead space includes the space between the face piece and the beginning of double corrugated tubing. Resistance to gases is within 2 to 3 cm HO pressure
. Canister usually large, capacity 4lb soda lime, divided in two halves and this is reversible . Advantages: Efficient
Fig. 9.16: Circle carbon dioxide absorber Key : 1=Gas inlet port; 2=Gas outlet port; 3=Non-return valves; 4=ON/OFF control lever; 5=Reservoir bag mount; 6=Soda lime canister

10. Disadvantages: Bulkiness, more resistance, more difficulty in recharging, chance of cross infection
11. Malfunction: Expiratory valve stuck closed, inspiratory valve stuck closed, circle system valve stuck open, misassembled valve components, foreign body in valve assembly
12. Adequate monitoring of inspired oxygen, end tidal CO, and inhalation anaesthetic needed.

1. Positioned on the back bar
2. Can deliver high output concentration at low flow rates
3. Have high resistance to gas flow
4. Vaporisers should be efficient enough to deliver proper concentration of vapour with both high and low fresh gas flows.

1. Minimal resistance to gas flow
2. Positioned in the expiratory limb
3. Here vapour-free fresh gas flow dilutes the inspired vapour concentration
4. During spontaneous ventilation, deep anaesthesia depresses respiration and thus anaesthetic
uptake
is
reduced
and
overdose
of
is
prevented.
This
mechanism
is
absent
in
controlled

Mapleson classified the breathing system as Mapleson A, B, C, D, E and F. Now a days only A, D, E and F and some of their modifications are in common use.

R . Simple and easy to use
CRON Can be easily sterilised
ATAwWH
. Safe and efficient
Can be used in all age groups
. Can be used in either spontaneous or controlled ventilation
. Satisfactory with low fresh gas flow
No complications like barotrauma
Compact, light weight, cheap, minimum running cost, easy to maintain
. Easy removal of exhaled gas possible.

1. Mostly popular and widely used
2. Contains a fresh gas inlet connected to a reservoir bag, then attached to a corrugated tube and then connected with face mask or endotracheal tube

Fig. 9.17: Mapleson A circuit
. Not ideal for controlled /assisted ventilation. Can be used for spontaneous ventilation. Fresh gas flow should be equal to alveolar minute volume (about 10 ml/kg/min)
. Rebreathing occurs and not suitable for IPPV unless large fresh gas flow is used
oO . Most commonly used version of Mapleson A is Magill system
. Not ideal for paediatric anaesthesia.

1. Fresh gas inlet is towards patient end in between the expiratory valve and corrugated tube. Contains a reservoir bag at one end of tubing and face mask/endotracheal tube as usual
. Less efficient during spontaneous ventilation, but more efficient in conrolled
. Not used commonly
Fig. 9.18: Mapleson B circuit

1. Mostly similar to Mapleson B system
2. Here corrugated tube is absent. Reservoir bag is attached to fresh gas inlet. Expiratory valve
is in
between face
mask/endotracheal tube
and fresh
gas inlet
- . Not much used.
Fig. 9.19: Mapleson C circuit

1. Consists of face mask/endotracheal tube, fresh gas inlet nearer face mask, corrugated tube and connected with expiratory valve and reservoir bag
2. Efficient for controlled/assisted ventilation. Can be used in spontaneous ventilation
3. Bain coaxial system is the modification of Mapleson D system.

1. Identical with Ayre’s T piece system
2. Fresh gas flow is nearer to face mask/endotracheal tube, then connected to corrugated tube
3. No reservoir bag is used. No expiratory valve is there
4. It is a T-piece with 3 ports, Fresh gas flow in one port, second port to face mask, third port is meant for a tubing
. Widely used in paediatric anaesthesia (upto 25 kg body weight) a vo
. Needs high fresh gas flow to prevent rebreathing
7. As there is no expiratory valve in this system, scavenging is a problem.
Fig. 9.21: Mapleson E circuit

1. Modified version of Mapleson E system
2. Here in T-piece system a double ended bag is attached to the end of the tubing
3. Suitable for spontaneous/controlled ventilation
4. Widely used in paediatric anaesthesia (upto 25 kg)
5. Needs high fresh gas flow (2.5 to 3 times minute ventilation) to prevent rebreathing
6. Volume of reservoir bag should approximate tidal volume.

1. Coaxial version of Mapleson D system
2. Fresh gas flow through inner tube, exhaled gas through outer tube. The reservoir bag and expiratory valve mounted at the machine end

Doe w . Internal tube should have swivel mount at patient end to avoid kinking . Tubes are transparent to detect inside, and kinking, or disconnection . Efficient in controlled ventilation. Fresh gas flow rate should be 70 to 100 ml/kg/min
. Not much efficient for spontaneous ventilation. Fresh gas flow should be 200 to 300 ml/kg to prevent rebreathing
oN . Use of ventilator is possible in this system
. Usual length 180 cm
9. Advantages: Simple, light weight and easy to handle, safe and reliable. Can be easily sterilised, disposable tubing available
10. Disadvantages: Unrecognised disconnection, kinking, etc.

Tubes through which the anaesthetic gas/vapours along with respiratory gases are conveyed to and from the trachea. It is the artificial extension of larynx and a means of securing the airway
Two ends: Patient end is bevelled, machine end is not
Made of plastic (disposable) or red rubber or latex
Criteria for an ideal endotracheal tube: Nontoxic, nonallergic, smooth, properly curved, low kinkability, easily sterilised, smooth, cheap
Orotracheal tube: Short, curved and bevel angle should not be less than 45° in relation to long axis
Fig. 9.23: Plain endotracheal tubes
Upper : Nasal tube
Lower:
Oral
tube

e Nasotracheal tube: Long curve, bevel angle should not be less than 30° to long axis
¢ Size of the tube: Magill system arbitrary number from 00 to widest 10. French scale denotes the external diameter in mm multiplied by 3. It is more or less the external circumference of the tube. Internal diameter of the tube ranges from 4 to 12 mm. Usually 8.5 mm tube is meant for females and 9.5 mm for males.

i. About twice the length from the ear to his nostrils
ii. Usually about 24 to 30 cm in adults
iii. Formula for children: Age (years)/2 + 12 cm for oral tube.
Age (years)/2 + 15 cm for nasal tube.
e Diameter of the tube in children; 4.5 mm + Age (years)/4 in mm
¢ Cuff of the endotracheal tube: To provide an airtight endotracheal seal. Consists of an inflating tube and a pilot balloon. Cuff is vulcanised on the outer side of the tube about 12 mm from its end. Usually inflated with 4 to 8 ml air.
An ideal cuff should expand symmetrically and the distance between the end of cuff and the nearest part of the cuff should be 1 to 1.25 cm. Overinflated cuff may cause ulceration and necrosis and even tracheal stenosis.

i. Low volume cuff: Needs inflation to a high pressure to produce seal. High pressure cuff causes little pressure on tracheal wall
ii. High volume/low pressure cuff: Usually floppy. May fit an irregularly shaped tracheal wall. Insertion is usually difficult and traumatic
iii. Sometimes double cuffs are used for alternate inflation and deflation at regular intervals to minimise pressure effect
¢ Culf volume should be monitored and readjusted, whenever needed
e¢ NO may diffuse into the cuft filled with air and thus may increase the pressure
e Avoid cuffed tubes in children.

1. L-shaped nonkinking endotracheal tube mostly used for head and neck surgery
2. Made of rubber/plastic, cuffed or uncuffed, wall is thick
3. Bevel is oval-shaped, faces posteriorly
Fig. 9.24: Oxford nonkinking endotracheal tube

4. Stylet is needed for its insertion
5. Distance from the bevel to the curve of the tube is fixed.

DoPPwWnr
. Made of plastic or silicone
. Thick walls contain a spiral of metal wire or tough nylon
. Spiral helps to prevent kinking
. Introducer is needed for its insertion
. Tube length is fixed; risk of bronchial intubation
. Used in anaesthesia for head/neck surgery.
SII
RE
Fig. 9.25: Latex armoured endotracheal tube

1. Preformed shape to fit the mouth and nose without kinking
2. Provides a bend located just as the tube emerges at the level of chin/forehead
3. Cuffed/noncuffed variety
4. Uncuffed tubes mainly used in paediatric cases
5 . Cuffed tube may include one Murphy eye
6 . Uncuffed tube may contain two Murphy eyes
7. . Risk of bronchial intubation.

Dae w Has a flexible stainless steel body . Reflected beams from the tube defocussed to reduce the unwanted laser strike to healthy tissues . Cuff should be filled with saline instead of air to reduce the risks of ignition
. Used for anaesthesia in laser surgery on larynx/trachea
. Designed to withstand the effects of laser beams and thus helps the risk of fire or damages of the tube
. Some designs may have two cuffs.

1. Tube is of small diameter but with an adult sized cuff
2. It can be used nasally
3. Helps better exposure and surgical access to the larynx.

1. Curved plastic tubes
. Usually inserted through 2nd, 3rd, and 4th tracheal ring . Introducer needed for insertion Wn
. Provides wings on the proximal part of the tube to fix
. May be cuffed /uncuffed
. Proximal end ends in a standard 15 mm connector. Tip is cut horizontally
. Different sizes are available
. Uses:
ANaAAaAE
i. Long-term IPPV
ii. Upper airway obstruction
iii. Maintenance of airway to protect the lungs.
. Side effects:
i. Loss of voice
ii. Loss of expulsive cough
iii. Loss of nasal humidifying effect.

i. Displacement
ii. Accidental bronchial intubation
iii. Erosion of vessels
iv. Obstruction
v. Tracheal ulceration
vi.
Tracheal stenosis.
Fig. 9.27: Metal tracheostomy tubes a= Inner tube, b=Outer tube with flange for tapes

1. Made of stainless steel/silver
2. Provides inner and outer tube
3. Uncuffed
4. Inner tube usually removed for clearing at regular intervals
5 . Used in cases needing long-term IPPV
6 . After long term use, a track is formed and them the tracheostomy tube is removed
7. . Speaking version is available.

1. Allows the anaesthetist to deflate one particular lung while maintaining standard ventilation of the other
. Includes two separate lumens each with its own cuff and pilot tube and balloon
. Contains two curves, anterior and lateral. Anterior curve is usual to fit the upper airway and
the lateral curve (right or left) to fit into the right or left bronchus
. The machine end of the tube is connected to a Y-shaped catheter mount to connect the breathing system
.
Right
sided
variety
provides
an
eye
in
the
bronchial
cuff
to
ventilate
the
right
upper
lobe.
No
eyes
in
left
sided
varity
. Carlen’s tube is commonly used and it has a carinal hook. Other varieties: Robsrtshaw, Whie
etc.
. Tubes are available in various sizes
. Position of the tube must be checked just after intubation and immediately after positioning for surgery
. Problems
i. Hook can cause trauma
ii. Relatively small lumens cause increase in resistance and difficulty in suctioning
iii. Needs technical skill and experience
iv. Risk of malpositioning.

1. Curved tubes, anatomically shaped, made of metal or rubber or plastic
2. Inserted through the mouth into oropharynx above the tongue to maintain the upper airway patency
3. Prevents the fall back of the tongue in an unconscious patient
4. Curved body contains air channel. Usually flattened anteroposteriorly and curved laterally 5. Flange at the oral end
6
7
.
Bite part is straight and hard, fits in between the teeth
.
Available in various sizes and in different varieties.
i. Philips airway: Rounded rubber tube with a metal mount, flattened on cross section i.e. (Fig. 9.28)
ii. Water’s airway: Made of metal and has two holes in the sides near the pharyngeal end and right or left nipple for attachment of oxygen catheter (Fig. 9.29)
iii. Guedel airway: Made of rubber, standard pharyngeal airway (Fig. 9.30). Ee

Fig. 9.30: Guedel airway
- 8. Complications:
i.
Trauma
ii. Risk of gag reflex stimulation and vomiting.
9. Note:
i. Air channel should be as large as possible
ii. Cleaning at regular intervals is essential
iii. Proper size should be selected
iv. Patient himself clears it away when he regains consciousness. e

- 1. Here the airway tube is inserted through the nose into nasopharynx. The distal end should
be kept just above the epiglottis and below the base of tongue
- 2. Round curved tube, bevel usually left facing, flange at the proximal end

Used as an alternative to oropharyngeal airway particularly when mouth cannot be opened Ww
. Nasotracheal suction can be done
. Better tolerated Doak
i. Injury
ii. Bleeding
iii. False passage.
7. Contraindications:
i. Coagulopathy
ii. Nasal sepsis
iii. Nasal deformity.

1. Popularly used as alternative either to face mask or endotracheal tube for anaesthesia
2. Transparent tube with an elliptical cuff resembling small face mask. It can be inflated by
a
pilot
balloon
with
a
self-sealing
valve.
The
proximal
or
machine
end
provides
a
standard
15
mm
connection.
Slits
at
the
junction
between
the
tube
and
cuff
prevent
the
epiglottis
from
obstructing
the
laryngeal
mask
3. Before application, the cuff is deflated and lubricated. It is gently introduced blindly into the mouth and the cuff should lie over the laryngeal inlet. It is then inflated
4. Different sizes are available. Wide internal diameter helps reduction of flow resistance.
5. Uses:
i. As an aid in difficult intubation
ii. Reinforced version can be used for head/neck surgery.
6. Advantages:
i. Easy to apply
ii. No laryngoscopy needed.

i. Does not protect against aspiration of gastric contents
ii. Airway obstruction can occur
iii. Less reliable and predictable than using cuffed endotracheal intubation
iv. Not a substitute for tracheal intubation.
i. Vomiting prone patients
ii. Oropharyngeal mass /abscess.

1. Device used to perform direct laryngoscopy and to aid endotracheal intubation
2. Consists of a handle and a blade. Handle provides power source/batteries. Blade is either straight or curved and is fitted in the handle. A bulb is screwed on the blade. Electrical connection occurs when the blade is opened for use
. Various designs and shapes are available
. Straight bladed Magill laryngoscope (Fig. 9.33)
i. It picks up the epiglottis. Tip of the laryngoscope is advanced over the posterior border of the epiglottis and is then lifted directly to see the laryngeal inlet
ii. Usually needed for intubating the neonates, infants and children. Larger blades can be used for adults.
. Curved blade Macintosh laryngoscope (Fig. 9.34)
i. Designed to fit in the oral/oropharyngeal cavity. Usually introduced through the right angle of the oral cavity and advanced to reach the vallecula. It is then lifted upwards to elevate the larynx and see the vocal cords
ii. Left sided Macintosh blade is also available. It is meant for left handed anaesthetists and also in patients with right sided deformity, where the use of right sided blade insertion is difficult.
. Mecoy laryngoscope
Modified Macintosh laryngoscope with a hinged tip which is operated by lever mechanism present in handle. Helpful in difficult intubation.

i. Trauma
ii. Cardiovascular reflex disturbances
iii. Malfunction.
¢ Two functional laryngoscopes should always be available during endotracheal intubation.

1. 15 mm disposable connections provided with plastic disposable endotracheal tubes 2. Variety of endotracheal connections available
i. Magill connection: Curved, one end is serrated and the other end tapered to fit catheter mount. Nasal type has a sharper curve than the oral connections. Causes less resistance to gas flows. (Figs 9.35 and 9.36)
Fig. 9.35: Magill connection—Nasal
Fig. 9.36: Magill connection—Oral

Fig. 9.37: Cobb's suction union
Fig. 9.38: Nosworthy endotracheal connection
ii. Rowbotham: Right angled. The end where the tracheal tube is attached is serrated and tapered
iii. Cobb's: Cobb's suction union is a modification of a Rowbotham connector. Designed to allow a suction catheter down the tube (Fig. 9.37)
iv. Nosworthy (Fig. 9.38)
v. Worcestor.

1. Made of metal tube and its one end provides a small rubber tube
2. Machine end is metallic to be attached to the expiratory valve. Patient end attached to endotracheal tube
OND . Length contributes dead space
TB . Internal diameter wide, gas flow resistance is low
w . Standard size
Acts as an adaptor between endotracheal tube and breathing system
Acts to stabilise the endotracheal tube
. In some designs there is a built-in condenser humidifier.
Fig. 9.39: Catheter mount

1. Used to manipulate a nasotracheal or nasogastric tube through the oropharynx and into the correct position
2. Designed to be held at right hand
. Dinner fork curvature, tip is blunt, No ‘catch’ w
4. Other uses: To introduce throat pack, to remove foreign bodies,
etc.
5. Caution : Not to injure tube cuff and soft tissues.

1. Used to deposite a fine mist of local anaesthetic (lignocaine 4%) on the mucosa of the larynx and upper trachea
2. Aids to minimise the autonomic reflexes and circulatory changes related with laryngoscopy and endotracheal intubation.
Fig.
9.40:
Magill
forceps

1. It is used when the larynx cannot be visualised
2. The bougie is inserted in the trachea through the vocal cords, then the endotracheal tube is rail roaded over it
3. Bougie and the endotracheal tube should be lubricated during use.
1. The curvature of the endotracheal tube can be altered with a malleable wire or stilet.
2. It should be lubricated before use.

1. Simple, portable, robust, economical
2. Versatility: Tidal volume upto 1500 ml with respiration rate 60/min, variable I/E ratio. Can be used in different anaesthetic systems. Can deliver any gas/vapour
3. Monitors airway pressure, inspired and exhaled minute and tidal volume, respiratory rate, inspired O2 concentration
. Provision of humidification
. Alarms: Disconnection, high/low airway pressure, power failure
. Provision of other modes of ventilation: PEEP, CPAP, IMV, etc.
Easy to clean and sterilise
Easy to use and safe
. Facilities of manual operation and emergency back-up.
CONDO

1. Whether to use sophisticated machine with ancillary apparatus built-in or relatively simple machine with separate apparatus
. Ventilators suitable for ICU are almost universally constant flow generators and they need
to
be
more
versatile
than
those
in
OT
. Most important single feature of a satisfactory ventilator for ICU is that it can cope with changing, sometimes rapidly changing pulmonary mechanics.

1. A long needle (usually 10 cm) with a stilet and a transparent hub to detect the flow of CSF
2. Different gauges : From 18 G to 25 G. 25G and smaller needles usually need introducer
3. Bevel may be cutting, traumatic or pencil like, or atraumatic with a side hole just proximal to
tip
. Atraumatic needles separate rather than cut the longitudinal fibres of dura
. Uses:
i. to inject local anaesthetics/opioids in subarachnoid space
ii. to sample CSF
iii. for intrathecal injection of drugs.
. Disadvantages:
i. Bloody tap
ii. Postspinal headache.
———<§:j—<f
Fig. 9.41: Spinal needle

1. Used to identify and cannulate the epidural space, to inject the local anaesthetics / opioids in epidural space
ONDE
wW . Stilet introducer is provided
. Usual length 10 cm marked at 1 cm intervals, has a Huber point which allows a catheter to be directed along the long axis of the epidural space. Bevel designed to be slightly oblique at 20° to the shaft
. Various gauges available. 16G or 18G commonly used
Common variety — Tuohy needle
Some designs may have wings
. Shorter needles are available for paediatric use
. Needle wall is usually thin to allow a catheter to be introduced through it.
—SSa
YH
Fig. 9.42: Tuohy needle

a . should have a special low resistance plunger to detect the epidural space
. 90 cm transparent, malleable, made of nylon or teflon
. Distal end has two or more ports with closed and rounded tip
. Distal part of the catheter is marked at 5 cm intervals with additional 1 cm markings between
5 to 15 cm
. Proximal end of the catheter is connected to a Luer-lock and a filter
. Filter (0.22 micron mesh) is bacterial, viral and foreign bodies filter. Filter should be changed every 24 hours
Syringe :
. Disadvantages
i. Catheter obstruction
ii, Kinking
iii. Injury to epidural vessels
iv. Malposition
v. Infection
vi. Transection of catheter.

AaRoON . Some designs may have a side port Can be connected with nerve stimulator . Various sizes and gauges are available . Insulated needles:
. Made of steel with Luer-lock attachment, short with blunt bevel
. Transparent hub for early detection of intravascular prick
i. Teflon coated with exposed tips
ii. Current passes through the tip only
ii. Usually have a greater diameter b
iv. Higher risk of nerve injury.

1. Designed to produce visible muscle contractions, when the needle tip reaches the peripheral
nerve
or
nerve
plexus
. Provides 2 leads, one to the skin and the other to the needle
. Usually a current 0.25 to 0.5 mA is used with a frequency of 1 to 2 H,. Stimulus is given for
a short time 1 to 2 ms
. Usually battery powered.

1. Meant for noninvasive measurement of the arterial blood oxygen saturation at the level of arterioles

2.
i . Mostly accurate, simple and rapid method
N . Excessive movement, malposition of the probe, external fluorescent light can be a source of error
Includes
a
probe
with
two
light
emiting
diodes
(LED)
and
a
photodetector.
Also
provides
a
microprocessor
to
display
oxygen
saturation,
pulse
rate
and
a
plethysmographic
waveform
of
the
pulse
. Alarm limits are set for low saturation value and for high and low pulse rate
. Sources of inaccurate readings:
i. Smoking
ii. Carbon monoxide poisoning
iii. Nail polish
iv. iv use of certain dyes: Methylene blue
v. Methaemoglobinaemia.
. Hypoperfusion and vasoconstriction can affect pulse oximetry reading
. Site of the probe placement should be checked at regular intervals.

1. Carbon dioxide concentration can be estimated directly and continuously throughout the respiratory cycle. End-tidal carbon dioxide reflects accurately arterial CO, tension
w . Sampling can be done both on side stream and mainstream
5. Loose connections and system leaks can dilute end-tidal CO
6. Nitrous oxide can distort the readings.

1. Measures the tidal volume and minute volume
2. A vane manometer. A rotating vane surrounded by slits. Attached to a pointer
. Is mounted in the expiratory limbs of the circuit. It should
. Flow is unidirectional
. Rotation of the vane drives the pointer around the dial and
. Usually over read at high tidal volumes and under read at low volumes
. Function is affected in presence of moisture
. Mostly accurate +5 to
10%.
Fig. 9.43: Wright respirometer
. Works with the principle of infrared absorption by carbon dioxide
. Uses:
i. to monitor the level of ventilation
ii. to confirm endotracheal intubation
iii. to diagnose pulmonary embolism
iv. Aids to diagnose malignant hyperpyrexia.
be as near to the endotracheal tube as possible
the gas volume is recorded

1. Designed to monitor neuromuscular transmission. The type of neuromuscular block, the depth of block and adequate reversal of block can be judged
. Two surface electrodes are positioned over the nerve and connected through the leads to the nerve stimulator. Supramaximal stimulus (current of 15 to 40 mA) is used to stimulate the nerve
. Muscle contraction can be visually observed and palpated. It can be measured with the help of a pressure transducer. Electromyography can also be done.
. Common Nerves Used:
i. Ulnar nerve
ii. Facial nerve
iii. Posterior tibial nerver
iv. Common peroneal nerve.

1. Clinically blood pressure measurement is done with the help of sphygmomanometer. Systolic and diastolic pressures are determined with Korotkoff sounds.
oO . Size of the cuff, arrhythmia, external pressure, etc. can affect the reading
. Oscillometry is the popular noninvasive method to measure arterial blood pressure during anaesthesia. The systolic pressure, diastolic pressure, mean arterial pressure and pulse rate can be measured, calculated aid displayed
. A cuff with a tube is used for inflation and deflation. It is attached to a transducer and microprocessor. It provides the display and timing mechanism. Alarm limits can also be set.
. The measurement is mostly accurate and reliable
. Control of inflation/deflation is needed.

1. Provides beat-to-beat information with much reliability
2. Contains indwelling arterial cannula, a heparinised saline column, a flushing device, a
transducer,
an
amplifier
and
an
oscilloscope
. Besides blood pressure monitoring, myocardial contractility, vascular tone, stroke volume can be measured
. The arterial pressure waveform should be displayed
. Caution:
i. Damping or resonance can occur due to air bubble, clot, etc.
ii. Transducer should be kept at proper position at the level of right atrium
iii. Arterial thrombosis can occur
iv. Risk of bleeding
v. Risk of infection.

1. Filling pressure of right ventricle is CVP
2. Measured directly using a central venous catheter inserted percutaneously via internal jugular vein/subclavian vein/ basilic vein/external jugular vein/antecubital vein
oO . Normal range of values: 0 to 6 cm HzO
. CVP can be read using either a pressure transducer or water manometer
. CVP monitoring in useful aid to blood and fluid replacement
i. Airembolism
ii. Arterial puncture
iii. Sepsis
iv. Pneumothorax me
v. Nerve damage.
. CVP catheters differ in size, length, material and number of lumens.
a
Fig. 9.44: Central venous

Uses:
i . To assess the volume status of the patient when CVP is unreadable.
ii . Sampling of pure mixed venous blood
iii . Measurement of cardiac output using thermodilution method.

:
1. Poor left ventricular function
eo . Assessment of intravascular fluid volume
10. These include 4 lumens
2. . Valvular heart disease
3. . Myocardial infarction
4. ARDS
5 . Major vascular surgery
6 . Severe shock, haemorrhage
7. . Evaluation of response of fluid therapy or administration of drugs like vasopressors,
vasodilators, inotropes
. Pulmonary artery catheters differ in sizes and are usually 110 cm in length. These are marked
at 10 cm intervals
:
i. Proximal lumen: About 25 cm from tip, should lie in the right atrium. CVP can be measured
ii. Distal lumen: Situated at the tip of the catheter, lies in a major branch of pulmonary artery. Can measure the pulmonary artery and PCW pressure

iii. Another lumen contains 2 insulated wires leading to a thermistor. Measures the temperature of blood at this site. Used for measurement of cardiac output
iv. Balloon lumen permits the introduction of about 1 ml of air into the balloon which surrounds the distal tip.
11. Catheter is inserted through a large central vein into the right atrium, right ventricle, pulmonary
artery for continuous pressure monitoring.
12. Left ventricular filling pressure can also be measured.
13. Complications :
ii. Vascular injury, perforation
iii. Arrhythmias, heart block -
iv. Knotting and kinking of the catheter
v. Valvular damage
vi. Thrombosis.

. Helps to monitor heart and breath sounds continuously when fitted with a moulded earpiece
. Change in heart sounds, air entry to lungs, development of abnormal breath crepitations or rhonchi can be detected readily
sounds,
. Simple, cheap, safe, noninvasive and free from electrical interference, and reliable.

4.
. Monitoring of body temperature is essential and should be a routine procedure during surgery
. Temperature probes can be thermistors, thermocouples and infrared thermometers
. Skin temperature can be taken from axilla and core temperature can be measured from
oesophagus, rectum, tympanic membrane, nasopharynx and urinary bladder
i. temperature in nasopharynx approximates brain temp
ii. in oesophagus approximates cardiac temp
iii. on tympanic membrane—Most reliable core temp
iv. in rectum usually reads higher core temp.

1. Inspired oxygen concentration in the anaesthetic delivery system can be measured by oxygen analyser
. It should have a low oxygen concentration limit alarm. It should be set to sound when the inspired oxygen concentration decreases below 30%.

Anaesthesia is produced by injection of local anaesthetic solution into the subarachnoid space when it is mixed with cerebrospinal fluid. Patients remain awake or sedated by intravenous drugs like midazolam, propofol or opioids. Skeletal muscle relaxation is profound without the need of muscle relaxants.

Spinal column consists 7 cervical, 12 thoracic, 5 lumbar and 5 fused sacral vertebrae. Vertebra consists of its body, an arch with 2 pedicles anteriorly and 2 laminae posteriorly. Transverse process is at the junction of lamina and pedicle. The spinous process arises from the junction of laminae in posterior midline. Lamina and spinous processes are joined by ligaments, but the pedicles form a gap through which the spinal nerves emerge.

It lies within the vertebral canal from foramen magnum to sacral hiatus. It is bounded anteriorly by vertebral bodies, posteriorly by laminae and laterally by pedicles.
Extension: From brainstem to termination at L; Lz vertebral level, then continue as cauda equina in spinal canal.
Covering: Pia mater, arachnoid mater and dura mater.
Subarachnoid space: In between pia mater and arachnoid mater, extends from cranium to the level of So. Contains nerve roots and cerebrospinal fluid (Fig. 10.1).
Epidural space: In between dura mater and connective tissues covering the vertebrae and ligamentum flavum, a potential space where local anaesthetic solution is injected for epidural block. It is bounded by dura anteriorly and ligamentum flavum posteriorly, extends from the base of skull to sacral hiatus, contains nerves, fat, lymphatic vessels and the veins of epidural plexus.

The specific gravity of cerebrospinal fluid is 1.004. Most local anaesthetic solutions are of specific
gravity 1.024 or more and addition of 5% dextrose make them hyperbaric solution. Some hypobaric
and isobaric solutions are also described.
1 . Gravity
Movement of hyperbaric solution within the subarachnoid space depends on:
2. Position of the patient
3. Curvature of the spinal cord.
1. Differential nerve block: Fibre size is an important factor that governs susceptibility of a nerve to be blocked by local anaesthetics. Sympathetic fibres are blocked to a level about two segments higher the upper segmental level of sensory block. Motor block is usually extended to within two segments below of the upper level of sensory block.
ONATRWNHE
Lumbar puncture is usually made at L3 Ly or Ly Ls space and the hyperbaric solution should
spread in a caudad and cephalad direction when the position is kept horizontal.
Density of a local anaesthetic solution is the function of its concentration and the fluid in which it is dissolved. The density of CSF may range 1.001 to 1.005g/ml. Baricity is the density of the local anaesthetic solution divided the of CSF.
by density
a. Hyperbaric: Local anaesthetic solution with densities greater than 1.008 g/ml
b. Isobaric: With densities between 0.998 and 1.007 g/ml
c. Hypobaric: With densities less than 0.997 g/ml.
The dose, baricity of the local anaesthetic solution and position of the patient during and after injection are most important to determine the distribution of local anaesthetic and level of

Factors influencing the spread of local anaesthetics:
. Position of the patient during and after injection
Curvature of spine
Speed of injection
. Barbotage
Interspace chosen
. Drug dosage
. Volume of the local anaesthetic
. Specific gravity of the drug.

2. Cardiovascular system
i. Proportional to the height of block and extent of sympathetic block
ii. Hypotension due to dialation of resistance and capacitance vessels
iii. Bradycardia
iv. Reduction of hepatic and renal blood flow.

i. No effect on respiration in low spinal block
ii. High block may affect phrenic nerve and cause total apnoea
iii. Intercostal paralysis occurs in block reaching the thoracic level

4. Alimentory system
iv. Thoracic block may decrease vital capacity, decrease in expiratory reserve volume and causes reduction in cardiac output and pulmonary artery pressure. Increased
ventilation/ perfusion imbalance.
i. Constricted gut with increased peristaltic activity
ii. Nausea/vomiting.

ar . For vaginal or operative obstetric delivery where general anaesthesia is risky . For patients with medical problems like diabetes, thyrotoxicosis, respiratory or cardiovascular disorders.
wh . To get comparatively bloodless operative field . Obstetrical procedures: Forceps delivery, removal of retained placenta etc.
. Most suitable for surgery below the umbilicus

ay . Patients with anticoagulant therapy
CONAAA
WN . Hypovolaemia . Infection at the injection site
. Patients with coagulation disorders
Active bacterial or viral infections of peripheral or central nervous system
Chronic neurological disease, disseminated sclerosis, peripheral neuropathy, poliomyelitis
Where the sterility is not guaranted
Where resuscitative drugs and equipment are not available
. Unwilling patient, patient refusal.

1. Preoperative assessment
ONAARwWN . Intravenous infusion should be started before the procedure . Correct positioning of the patient is essential. This helps rapid performance of lumbar puncture . Full sterile technique should be adopted Spinal anaesthesia kit with all drugs and equipment should be kept ready . Selection of spinal needle: 22 G and 25 G should be kept ready . Selection of drug is also important .
i. To select the patient, to examine the patient carefully, to note the cardiovascular, respiratory and haematological status
ii. Patient should be explained regarding the procedure
iii. Obtain the written consent properly
Patient should be in lateral position. An assistant should help for proper positioning the patient,
the lumbar spine is flexed to spread spinous process and enlarge the interlaminar space.
i. Midline approach is mostly adopted
ii. Skin of the chosen interspace (commonly Ly, and Ls) is infiltrated

Fig. 10.1: Spinal needle in subarachnoid space
iii. Spinal needle is advanced in midline sagittal plane directed slightly cephalad (10°) toward the interlaminar space
iv. Needle will pass through skin, subcutaneous tissue, supraspinous ligament, interspinous ligament and ligamentum flavum and dura mater. A little resistance followed by a popping sensation will be felt
. Then the stilet a removed, CSF flows freely from the needle (Fig. 10.1)
. Then a very slow injection of local anaesthetic solution should be given in the subarachnoid space
vii. After injection the needle with stilet should be taken out slowly
viii. Site should be dressed and the patient should be positioned supine.
9. Vasopressors should be available
10. Monitoring of pulse rate, blood pressure, respiration and ECG is essential.

a. Lignocaine: Onset time 1 to 3 min; duration 45 to 75 min
b. Bupivacaine: Onset time 2 to 4 min; duration 120 to 180 min 0.75% hyperbaric.

1. Saddle block:
Upper level of analgesia S;
Use: Cystoscopy, perineal, anal surgery
2. Low spinal:
—
Upper level of analgesic L; Tj.
Use: Perineal/anal surgery, lower extremities.
3. Medium spinal: Upper level of analgesia Tio — Ts
Use: Lower abdomen surgery, appendix, prostate, hip surgery, lower

4. High spinal:
5. Unilateral spinal analgesia
Upper level of analgesia Ty
Use: All abdominal surgery.

. Hypotension
. Severe itching
. Confused state
. Nausea/vomiting
. Headache, postdural puncture headache
. Urinary retention
. Backache
. Labyrinthine disturbances
. Cranial nerve palsy: VI nerve pasly
. Meningitis, meningism, paraesthesia
. Transverse myelitis
. Cauda equina syndrome from adhesive arachnoiditis
. High spinal anaesthesia may cause respiratory insufficiency
. Unexpected cardiac arrest.

1. Normally occurs 2 to 7 days after lumbar puncture
2. May persist for upto 6 weeks
. Causes:
i. Leaking of CSF out of the subarachnoid space through dural puncture
ii. Brain and its supporting structures sag
iii. traction on pain sensitive vascular structures
. Pain worsened by standing, sitting and relieved by supine position
. May be frontal, occipital or both accompanied by tinnitus, vertigo, diplopia
Common in young patients, women
. Depends on the size of hole in dura, the rate of CSF losses
. Small needles 24G or lesser should be used. Use of conical tip needle
.
Blunt or rounded point tips part the dural fibres rather than transecting the dura
. Bevel of the spinal needle should be parallel to the longitudinal dural fibres

i. Bed rest, no ambulation
ii. Large fluid intake
iii. Analgesics, oral or, i.v. caffeine
iv. Extradural infusion of saline
v. Abdominal binders
vi. Extradural blood patch.

e Advantages of spinal anaesthesia
. Simple and quick to perform
Rapid onset
Profound analgesia and muscular relaxation
. Low dose needed. Systemic toxicity—low risk
. Low risk of pulmonary aspiration
. Less surgical haemorrhage
7. Physiological response to injury—less.
DAaRWNE

1. Spinal cord is about 45 cm in length. It extends from medulla oblongata to its termination at conus medullaris at LiL; level from which pia continues as filum terminale
2. Spinal dura ends at the level of S,
3. Spinal cord terminates at the lower border of L3 in the newborn and at the lower border of L, or upper border of Lz in the adult
4. There are 31 pairs of spinal nerves. Their ventral and dorsal roots pass through the dura and epidural space independently and unite at the intervertebral foramina to form spinal nerve trunks. Then they divide into anterior and posterior primary rami.
Epidural anaesthesia follows from the injection of local anaesthetic solution into the epidural space. It blocks the spinal nerve roots as they emerge from the spinal cord and traverse the epidural space. A part of local anaesthetic may enter the CSF through dura and contribute to anaesthetic effect. The block can be performed in the sacral (caudal block), lumbar, thoracic or even cervical regions. Lumbar approach is most popular.

Choice depends on duration of operation and required intensity of motor block. Lignocaine is most widely used and it is intermediate-acting. Bupivacaine and etidocaine are long acting.
Lignocaine 1 to 2% onset 5 to 15 min, duration 60 to 90 min.
Mepivacaine 1.5% onset 5 to 15 min, duration 90 to 120 min.
Bupivacaine 0.5 to 0.75% onset 10 to 20 min, duration > 180 min.
Etidocaine 1% onset 10 to 20 min duration > 150 min.
e Addition of adrenaline 5 mg/ml to local anaesthetics prolongs their effect, decreases vascular
absorption and reduces blood concentration of the drug and lessens the risk of systemic toxicity.
¢ Management of epidural anaesthesia is mostly similar to that of spinal anaesthesia. But the identification of epidural space during the procedure is most important.

1. More or less a circular space surrounding the dural sac and its extensions
2. Extends from foramen magnum to coccyx (sacrococcygeal membrane)
3. Contents: Areolar tissue, fat,spinalnerve roots with dural sleeves, spinal arteries and venous plexus
4. Structures pierced to get the epidural space in midline plane: Skin, subcutaneous tissue,
supraspinous ligament, interspinous ligament and ligamentum flavum
. Negative pressure in epidural space oa
6. Following points are helpful to detect the needle in epidural space:
i. Sudden disappearance of resistance to the advancing needle due to piercing the ligamentum flavum.

ii. Sudden ease of injection of air from the needle attached to a syringe.
iii. Hanging-drop sign: Withdrawal of hanging drop of saline placed on the hub of the
needle
iv. Capillary tube method of Odom: Movement of the bubble of air in the capillary tube attached to the hub of the needle
v. spring loaded syringe attached to needle may be unloaded due to loss of resistance
vi. Inflated balloon attached to the hub of needle will collapse at the epidural space.

1. Provides better control of duration and extent of analgesia
2. A plastic cannula is placed through the needle exactly in the epidural space
3. The needle is then withdrawn keeping the catheter in situ. Local anaesthetic solution can then be repeatedly injected in the space through the catheter.
NQF . Speed of injection . Specific gravity of the drug
WN . Site of interspace where injection is made
8. In pregnancy, spread is greater
9. In dehydration, shock, cachexia spread is extended.
In cases of inadvertent dural puncture following measures can be adopted:
1, Spinal analgesia
2. Needle withdrawn to epidural space and local anaesthetic solution can be injected
3. Another interspace may be chosen for epidural block.

1. Volume and concentration (dose) of local anaesthetic
. Position of patient after injection
. Presence or absence of adrenaline in local anaesthetic solution
. Weight, height and age of the patient and rate of injection usually seem to influence distribution of local anaesthetic in epidural space. But spread may be increased in old age as the solution cannot readily pass through the intervertebral foramen. Taller patients may need larger dose

1. Upper and/or lower abdominal surgery, operation of lower limb, perineum, obstetric cases
2. For postoperative pain relief
3. For relief of intractable pain
4. For painless labour
5. To reduce blood loss during pelvic surgery
6 . In combination with iv drugs
7. . In combination with general anaesthesia.

2. Postspinal headache absent
3. Postoperative urinary retention not so common
4. Prolonged analgesia can be obtained
5. Haemodynamic alterations are less.

ADURONB
. Causes profound analgesia and muscular relaxation
. Can be employed in patients with respiratory, metabolic disorders
. Physiological derangement, metabolic response to injury less
. Spontaneous respiration, consciousness maintained
. Nausea/vomiting less
. Duration of anaesthesia can be prolonged by catheter technique.
omparison between subarachnoid and epidural block is shown in Table 11.1.

SCMNDUFPWNEH
PRR Nr Oo . Backache may be frequent . Haematoma, extradural abscess
. Extra technical skill is needed
. Time consuming procedure
. Onset of action is slow
. Large quantity of drug is required, so there is risk of systemic toxicity
. Inadvertent dural puncture can occur
. Total spinal block can occur
. Muscle relaxation may not be complete
. Incomplete segmental block can occur
. Catheter technique may induce injury to venous plexus
. Trauma to spinal cord and nerve roots.

1. Inadvertent dural tap may lead to:
w . Hypotension: It may be due to systemic absorption, high sympathetic block, bradycardia with
CmMND . Failure of block
US . Respiratory inadequacy
¢ Total spinal block
e Postspinal headache
. Toxicity of local anaesthetics
a sympathetic block to T;4
. Inadvertent injection of chemical irritants
. Postepidural sequelae: Paraplegia, anterior spinal artery syndrome, backache
. Nerve block may be inadequate
. Haematoma in spinal canal
. Extradural infection, abscess

11. Trauma to spinal cord and nerve roots
12. Prolonged analgesia or paraesthesia
13. Nausea/vomiting, shivering
14. Horner’s syndrome
15. Introduction of a foreign body—very rare
16. Implantation dermoid—very rare
17. In catheter technique: Knotting of catheter, tear of catheter, misplaced catheter, etc.
Table 11.1: Comparison between subarachnoid and epidural block
Subarachnoid block
Epidural block
Technique
Onset of action
Site of action
Extent of block
Intensity of block
Segmental block
Success rate
Quality of block
Headache
Backache
Easy
Small
Rapid (2-8 min)
Spinal cord and
spinal nerve roots
Extensive
Usually complete
No
100%
Satisfactory
Common
Frequent
Frequent
Relatively difficult
Large
Slow (20-30 min)
Diffusion into CSF
Paravertebral nerve block
Dependent of dose and volume
May be incomplete
Yes
Missed segments, failure
may occur
Patchy block
Less common
Less frequent
¢ Epidural space is widest in the midline posteriorly, in the lumbar region it is about 5 mm.
A midline septum and midline tenting of the dura may present in some patients.
e Ligamentum flavum is composed of elastic fibre disposed in vertical direction, thinnest in cervical region and gradually thickened as it descends caudally.
e Epidural veins :
No valves
Drain the spinal cord, vertebral canal and CSF from subarachnoid space
Connect and azygos systems.
all segments of the body from pelvis to cranium by anastomotic channels involving caval

1. Mostly due to inadvertent injection into the subarachnoid space and pooling of the large quantity of drug cephalad in upper thoracic level
» Severe hypotension, dilated pupils
Respiratory failure due to phrenic nerve paralysis and medullary depression
Convulsion

5. Management
i. Early detection is vital
ii. Endotracheal intubation, IPPV with 100% oxygen
iii. iv fluids
iv. Vasopressors.
6. Precautions
i. Response to initial injection or subsequent top up injection should be seriously considered
ii. Detection of epidural space should be accurate before giving injection
iii. Resuscitation equipment and drugs should be available and kept ready
iv. iv infusion must be established before initiating the block.

BR . Infection at local site
. Severe hypovolaemia, dehydration . Raised intracranial pressure WN
. Coagulation defects, patients with anticoagulants FP
. Patient refusal
. Presence of neurologic diseases
. History of back surgery
. History of back pain
. Systemic infection.
CANDU
It is one particular type of epidural anaesthesia where the local anaesthetic solution is injected in extradural in the sacral canal.
space
It can be achieved either by a single injection or by continuous infusion through a catheter into sacral canal.
the
Lignocaine 2% can produce analgesia for about one and half hours.

1. Cardiovascular changes—Not significant
2. Pulse rate may be slowed, BP is usually unaltered
3. Respiration not much affected
4. Gastrointestinal tone may be increased little
5. Type of block
i. Low block: Block extends upto Lz
Uses: Surgery on perineum, rectum, anal canal, urethra, vagina.
ii. Mid block: Block extends upto Tio
Use: Operation on pelvic organs, hernia, lower limb
iii. High block: Block may extend upto
Ty done.
Operations on upper abdomen can be

1. Surgery of perineum
2. For painless labour
3. For relief of intractable pain in the pelvis and lower limb.
4. Therapeutic use for sciatica, low backache, etc.

1. No postoperative headache
2. Not much physiological alterations

3. Less chance of neurological complications
4. Technically easy in children
5. Location of sacral hiatus and the technicque are easy.

RB Anomalies of sacrum and caudal canal may cause technical problems
. Much time is needed to produce the block . Subarachnoid injection may occur WN
. Intravascular injection is possible F
. Control of height of analgesia is often difficult
. High block may cause hypotension
Large volume of local anaesthetic solution may increase the risk of systemic toxicity
. Muscular relaxation may not be adequate
. Infection may occur in coccyx, sacrum and epidural space.
CAND

1. Patient refusal
2. Active diseases in CNS
3. Presence of pilonidal cyst
4. Presence of anomalies on sacrum
5. Caesarean section
6. Coagulation disorders
Fig. 12.1: Caudal block (needle advanced upto the level of Sp vertebra)
Fig. 12.2: Caudal analgesia. Catheter in caudal
7. Uncooperative patients
8. Obese patients—technical problem
9. Sterility of equipment is not guaranteed.

1. Patient is in prone position
2. Sacral hiatus is identified. It is located about 3 cm from the tip of coccyx between the sacral
cornua
. Aneedle is inserted perpendicular to skin through the sacrococcygeal ligament until it touches sacrum
. The needle is slightly withdrawn and the angle reduced and then advanced about 2 cm into the caudal canal
. Aspiration test should be done to avoid subarachnoid or intravascular injection
. Subcutaneous injection should be avoided 5 ml air may be injected and the skin should be palpated for crepitations
. The needle should never be advanced beyond the second sacral vertebra as dural sac usually ends between S; and S>
. Local anaesthetic solution 1.5% lignocaine is injected slowly
. Continuous caudal block can be achieved by introducing a catheter inside the caudal canal and injecting the drug carefully.

1. Anaesthesia
2. Postoperative analgesia
3. Diagnosis and treatment of chronic pain syndromes.

1. Less physiological response to injury
2. Patient awareness
3. Intact airway reflexes
4. Profound muscle relaxation
5. Early ambulation, oral feeding possible.

1. More difficult and time consuming
2. Failure /inadequate block
3. Risk of nerve damage.

1. Noncooperation of the patient. Unwilling patients
2. Infants and children
3. Local skin infection
4. Coagulation defect
5. Preexisting neuropathy
6. Sensitivity of local anaesthetics
7. Obscured landmarks.

1. Formed by the first four cervical nerves
2. Indications :
i. Operations of the neck
ii. Chronic pain syndromes
iii. Neuralgia
iv. Tracheostomy.
3. Technique : Supine position, head turned to opposite side. A line connecting the tip of mastoid process and the anterior tubercle of transverse process of C, vertebra indicates the plane in which transverse process lie. The transverse process of C2, C3 and Cy palpated and 3 to 5 ml of local anaesthetic solution is after test.
injected aspiration
4. Complications :
i. Phrenic nerve block, vagus nerve block
ii. Subarachnoid/epidural /intravascular injection
iii. Horner’s syndrome
iv. Recurrent laryngeal nerve block.

1. Arises from anterior rami of Cs to Cg and T). These rami unite to form three trunks, then pass over the first rib and under midpoint of clavicle to reach the apex of axilla.
2. Technique :
A. Interscalene approach : 30 to 40 ml of local anaesthetic solution is injected into the interscalene groove opposite the transverse process of C,. A line extended laterally from the cricoid intersects the interscalene groove at C,. Patient lies supine with head turned to opposite side.
Fig. 13.1: Brachial plexus block (supraclavicular approach)

Advantages
:
i. Easy
ii. No risk of pneumothorax
iii. Suitable for shoulder manipulations.
Disadvantages :
i. Absence of ulnar block
ii. Subarachnoid/epidural/intravascular injection
iv. Phrenic nerve/laryngeal nerve block.
B.
Supraclavicular approach : Supine position, head turned to opposite side. A point located 1 cm above and just lateral to midclavicular point. Needle inserted immediately posterior to subclavian artery inclined at 80° to skin and directed backwards, inwards and downwards to contact first rib. About 20 ml of local anaesthetic solution is deposited just withdrawing the needle a little.
Complications :
i. Pneumothorax
ii. Block of phrenic nerve, recurrent laryngeal nerve
iii. Block of vagus nerve
iv. Risk of subarachnoid/epidural/intravascular injection.
C. Axillary approach : Arm abducted to 90° and externally rotated. Axillary artery palpated and traced towards axilla. While palpating the artery, needle is inserted just anterior to artery into the axillary sheath. Popping sensation can be felt and the needle will pulsate with arterial pulsations. 25 to 30 ml local anaesthetic solution is deposited.
Advantages :
i. No risk of phrenic nerve/vagus nerve/recurrent laryngeal nerve/stellate ganglion block
ii. No risk of subarachnoid/epidural block
iii. Least risk of pneumothorax.
Disadvantages
:
i. Risk of intravascular injection, systemic toxicity
ii. Difficult in obese people
ii. Surgery of the shoulder and upper arm not possible b
iv. Cannot be done when arm cannot be abducted.

A. At elbow Injection of about 5 ml of local anaesthetic solution is given 1 cm medial to brachial
: artery in the flexion crease.
B. At wrist : About 5 ml of local anaesthetic solution is injected just lateral or radial side to the
flexor tendon.
palmaris longus

solution is
given in
the groove
formed by
the medial
condyle of
the humerus
and the
olecranon of
the ulna
at the
elbow.
- B. At wrist : The ulnar nerve can be blocked at the wrist by injection of local anaesthetic solution just medial to the ulnar artery.

About 3 ml of local anaesthetic solution is injected at the wrist just lateral to the radial artery. A subcutaneous cuff of anaesthesia is also produced on the lateral and dorsal aspects of the radial side of the wrist to block the branches of radial nerve that have left the main nerve in the lower part of the forearm.

Sciatic nerve arises from sacral plexus which is formed by Ly4-5, S;-3. It is about 3 cm thick as it leaves pelvis.

Patient should lie on the side opposite one to be blocked. A line is drawn from the greater trochanter of the femur to the posterior superior iliac spine. Its midpoint is located. A needle is inserted about 5 cm caudal from that point. About 25 ml of local anaesthetic solution is deposited after eliciting paraesthesia. Avoid intraneural injection by withdrawing the needle a little before injecting the drug. The block can provide adequate analgesia of the foot and lower leg.
Fig. 13.2: Sciatic nerve block (posterior approach). 1. Posterior superior iliac spine; 2. Greater trochanter; 3. Site for injection

The patient is placed supine. Midpoint of the inguinal ligament is located. A line is drawn from the anterior superior iliac spine to symphysis pubis indicates inguinal ligament. Injection of 10 to 15 ml of local anaesthetic solution is given immediately lateral to the femoral nerve. Aspiration test for blood should be done before injection. Paraesthesia may be obtained.

1. Femoral, obturator and lateral cutaneous nerve of thigh are blocked with a single injection 2. Lumbar plexus is blocked as a fascial envelope surrounds the femoral nerve. It serves as conduit for carrying the local anaesthetic solution below the inguinal ligament cephalad to the level where the lumbar plexus forms
3. Patient is in supine position, hip slightly abducted and externally rotated. Femoral artery is palpated and the needle is inserted 2 cm lateral to femoral artery in slightly cephalad direction. About 25 ml of local anaesthetic solution is injected to block three major branches of lumbar plexus
4. Indicated in surgery in the medial, anterior and lateral aspects of the lower limb.

1. It can be blocked at any point along the rib, posteriorly, laterally or anteriorly. Usual site is at the angle of rib
2. In upper thoracic region it is about 5 cm from midspinous point and in lower region it is about 10 cm
3. Technique: A needle inserted over the lower border of rib at the angle perpendicular to skin till the bone is touched. It is then redirected to reach the lower border until there is a sensation of give as it pierces external intercostal fascia. About 5 ml of local anaesthetic solution is deposited there.
Nerve

i. Some operations on chest or abdomen
ii. Intercostal drainage of empyema
iii. Rib fracture to produce analgesia, rib
resection
iv. Pain relief for chest wounds.
5. Disadvantages
:
i. Time consuming
ii. Large volume of anaesthetic solution needed, risk of systemic toxicity.
Needle inserted perpendicular to rib
Needle
redirected
to
the
subcostal
Fig. 13.3: Intercostal nerve block

1. Nerve supply form 11th and 12th thoracic and 1st and 2nd lumbar nerves via iliohypogastric, ilioinguinal and genitofemoral nerves.
2. Technique
:
i. A needle is inserted 2.5 cm medial to anterior superior iliac spine perpendicular to skin to pierce the external oblique aponeurosis with a sense of click and about 20 ml of local anaesthetic deposited to block the ilioinguinal and iliohypogastric nerves

- Fig. 13.4: Cutaneous nerve supply of groin
Fig. 13.5: Landmarks for field block for inguinal hernia, 1. Anterior superior iliac spine; 2. Pubic tubercle
- ii. A long needle in inserted subcutaneously over the pubic spine and local anaesthetic solution the umbilicus to block the from side
injected upto
nerves coming
opposite
iii. A needle is passed half inch above the midinguinal point to pierce the external oblique
aponeurosis to block the genital branch of genitofemoral nerve
iv. Further infiltration along the course of incision.

- 1. Nerve supply of penis: From S2, $3 and S, through the dorsal nerve of penis (terminal branch
of pudendal
nerve).
It runs
along the
dorsum of
penis with
the arteries
and veins.
- 2. Technique
:
i. Injection of local anaesthetic to the skin on the dorsum of penis at a point 2 cm from the base, gradually passed subcutaneously around both sides of penis
ii. Further drug is injected to make a complete ring
iii. About 2 ml of the drug may be injected at the frenulum.

1. Circumcision
2. Surgery on the glans.
¢ No adrenaline in local anaesthetic solution while using this block.

- 1. Derived from anterior divisions of Sz, S3 and S,4 nerves. It is usually blocked by infiltration just medial and posterior to ischial tuberosity.

Fig. 13.6: Innervation of female perineum and transperineal pudendal nerve block

:
i. Episiotomy
ii. Forceps delivery
iii. Repair of tears of perineum
iv. Operation on piles.

:
A.
Transperineal approach: Lithotomy position. Skin wheal at the posteromedial margin of ischial tuberosity. A needle is passed through the wheal for about 1 inch and about 5 ml of local anaesthetic solution is injected.
Needle is further introduced into the ischiorectal fossa for further injection of the solution. B. Transvaginal approach: Ischial spine is located by placing the index finger in vagina. A long needle guided lateral to the finger is directed into the region of ischial spine and about 10 ml of local anaesthetic solution is deposited there. The injection is repeated on the other side.

. Formed by the fusion of the inferior cervical ganglion and first thoracic ganglion
. Situated between the transverse process of C7 vertebra and neck of 1st rib usually on the anterolateral surface of the body of C7 vertebra.
. Indications
: To neck
release vascular tone as in thrombosis, embolism and spasm of the vessels of head, and upper extremity.
. Technique : Supine position, head extended on a pillow. Retracted sternomastoid muscle laterally and located the transverse of process of C, vertebra. This is at the level of cricoid cartilage.

The needle is inserted through the skin over the transverse process and advanced 1 to 1%
inch until it makes the contact the bone, C7. The needle is withdrawn a little and local
anaesthetic solution is deposited.
5. Signs of successful block :
Horner’s syndrome:
Ptosis
Miosis, enophthalmos
Nasal congestion
Vasodilation
Anhydrosis
Increased skin temperature.

ii. Intravascular injection usually in vertebral artery
iii. Block of cardioaccelerator fibres
iv. Hoarseness
v. Spinal/epidural block
vi. Perforation of oesophagus
vii. Brachial plexus block
viii. Phrenic nerve block.

1. Largest plexus of sympathetic nervous system.
2. Innervates the abdominal organs such as pancreas, liver and stomach, etc.
3. Technique: A long needle is introduced about 8 cm from midline at the lower edge of 12th
rib and advanced to reach the body of L; vertebra at a depth of about 10 cm. It is slightly
withdrawn and redirected to slide of the vertebral body. Needle position may be confirmed
by
radiography
or
computed
tomographic
scan.
About
30
ml
of
solution
is
deposited.
Indications :
i. To relieve visceral pain/cancer pain
ii. Management of acute pancreatitis
iii. To treat pancreatic cancer pain, alcohol or phenol is used.

1. Hypotension
2. Haematoma, visceral puncture.
3. Intraarterial injection/systemic toxicity
4. Spinal/epidural anaesthesia

1. Asimple and easy method of providing anaesthesia of the arm or leg by intravenous injection of a large volume of anaesthetic solution when the circulation of the limb is occluded by a suitable tourniquet.
2. Method : An intravenous catheter is inserted on the distal part of the limb and the limb is exsanguinated by applying an Esmarch bandage.
The tourniquet is inflated to about 50 mm Hg above the systolic pressure.
The local anaesthetic solution (25 to 50 ml for arm and 100 to 200 ml for leg) is injected
slowly. Usually 0.5% lignocaine is employed. Double tourniquet may be used to eliminate tourniquet pain.
3. Onset of anaesthesia is rapid, muscle relaxation is good. Duration of anaesthesia depends on
the time of inflation of tourniquet. Recovery is good.
4. At the end of operation tourniquet is released, sensation and muscle tone return within a few minutes.
5. Complications :
i. Systemic toxicity
ii. Methaemoglobinaemia, if prilocaine is used.
6. Contraindications
:
i. History of drug sensitivity
ii. Patients with liver disease, cardiac disease
iii. In myasthenia gravis
iv. Prolonged surgery.
Anaesthesia cannot be practised without any accident and complications. But most of them can be pevented by taking adequate measures. If the complications arise, these must be diagnosed and treated accordingly in proper time.

1. Nausea common in females, and children, after prolonged anaesthesia, vomiting prone patients
Aga . Hypoxia from any cause
PWN . Pre-and postoperative medications: Morphine, pethidine Some anaesthetic agents like ether, cyclopropane
. Site of operation: Abdominal surgery, laparoscopy, strabismus surgery, orchidopexy, etc.
. Other factors: Pregnancy, gastric dilation, toxaemia.

1. Pulmonary aspiration
2. Dehydration, fluid and electrolyte imbalance, acid base imbalance
3. Hypoxia due to airway obstruction
4 . Raises intraocular, intrathoracic, intraabdominal and intracranial tensions
5 . Increases the incidence of postoperative lung complications.

1. Adequate preparation, preoperative fasting for at least 4 to 6 hours
2. Avoid morphine, pethidine, etc.
3. Avoid hypoxia, hypotension and drugs like ether, chloroform
4. Administer antiemetic drugs like prochlorperazine, metoclopramide, phenothiazines,
etc.

1. Proper tracheobronchial suction, toilet, nursing care.
2. Tonsillar position
3. Oxygenation
4. Gastric suction
5. Ondansetron 4 to 6 mg.

1. Postoperative pain
2. Hypoxaemia
3. Drug effect:
i. Ketamine
ii. Barbiturates, hyoscine, promethazine
iii. Addiction of alcohol, opium or other drugs.
. Psychological make-up of the patient oe
. Elderly patients
6. Toxic confusional state.

1. Tachycardia
2. Tachypnoea
3. Hypotension
4. Cyanosis, pallor, sweating
5. Hypoxaemia.

1. Diagnosis of specific cause and treatment accordingly.
2. Patient’s psychology needs consideration.
3. Analgesics in presence of pain. Hypoxaemia must be ruled out before prescribing analgesics
4. Oxygenation in cases with hypoxia.

1. Personality of the patient should be considered
2. Duration of surgery: The incidence is inversely related
3. Drug effect: Halothane
4. Postspinal headache (see chapter on Spinal Anaesthesia)

1. Diagnosis of causative factors and treatment accordingly
2. Sedative/hypnotic/analgesic may be helpful.

ANAK . Epiglottitis, oedema glottis
WNEH . Overweight, short necked patient
. Oedentulous patient
. Fall back of tongue
. Swelling, oedema, tumours in supraglottic region
. Congenital subglottic stenosis, vascular ring
. Foreign bodies, mucus, saliva, blood, etc.
. Laryngotracheobronchitis
. Laryngeal spasm
. Large goitre, malignant thyroid tumour
. Collapsing trachea
. Damage of recurrent laryngeal nerve
. Big haematoma in the neck
. Mechanical obstruction: Obstruction of intubated trachea: Bite, kinking, pack
pressure, overinflation of cuff, foreign bodies, tube bevel against tracheal wall, badly placed Boyle Davis gag or palatal gag, etc.

1. Bronchial asthma
2. Bronchopneumonia
3. Aspiration pneumonitis
4. Unintentional endobronchial intubation leading to hypoxaemia, bronchospasm, pulmonary collapse, etc.

1. Noisy breathing, hurried respiration
. Rocky movement of the chest WN
. Vigorous abdominal excursion
ONaATIF
. Alae nasi moving, flaring of nostrils, tracheal tug
. Suction of sternal notch and intercostal space
. Auditory signs only in partial obstruction, no phonation in complete obstruction
. Hypoxaemia, cyanosis
. Inspiratory stridor in upper airway obstruction. Expiratory stridor in lower airway obstruction.

1. Establish and/or improve the airway
2. Ventilate the patient
3. Endotracheal intubation, IPPV

4. Oxygenation transtracheally
5. Tracheostomy in extreme cases
6. Treat the cardiovascular instability
7. Further treatment according to aetiology.

1. Following induction with thiopentone
2. . Irritation caused by blood, saliva, foreign bodies
3. . Instrumentation: Laryngoscopy, intubation
4. Certain operations: Anal stretch, breast surgery, dilatation of cervix
5 . Following extubation under light plane of anaesthesia
6 . Trauma to recurrent laryngeal nerve during thyroid surgery.

1. 100% oxygenation with respiratory assistance
2. Transtracheal oxygenation
3. Intubation following use of suxamethonium on rare occasions.

1. Common in children between 2 to 6 years
2. Mostly bacterial infection
3. Total upper airway obstruction can occur at anytime
4. Clinical features:
i.
inspiratory
stridor
ii. pharyngitis
iii. fever
iv. restlessness
v. tachycardia
vi. cyanosis
vii. swollen epiglottis
viii. Blood exam: Neutrophilia.

1. Bed rest, oxygenation
2. . Urgent endotracheal intubation
3. Tracheostomy in extreme cases
4 . iv fluids, antibiotics, corticosteroids
5 . Adequate nursing care.

1. Viral infection of upper airway, common in infants
2. Clinical features: Inspiratory stridor, croupy cough, rhinorrhoea, fever, blood exam:
Lymphocytosis, lateral chest radiography reveals narrowing of subglottic area
3. Management:
i. Oxygenation
ii. Humidification of inspired gases
iii. Aerosolised racemic adrenaline
iv. Steroids
v. Fluids
vi. Endotracheal intubation in extreme cases
vii. Adequate nursing care.

1. Common in infants and children. Oedema of 1 mm in neonate can decrease laryngeal cross section by 65%.
2. Causes:
i. Trauma
ii. Infection
ii. Sensitivity of drugs b:
iv. Incautious instrumentation
v. Mechanical injury by oversized tube, overinflation of cuff etc.
vi. Aggravated in presence of anaemia, hypoxia and hypotension.
i. Keep the patient upright in bed
ii. Cool humidified mist inhalation
iii. Adrenaline through nebulizer
iv. Corticosteroid -
v. Antibiotics
vi. Tracheostomy in extreme cases.

1. Preexisting bronchial asthma, chronic obstructive pulmonary diseases
2. Following inhalation of foreign bodies, irritant vapours
3. Following instrumentation in light anaesthesia
4. Certain drugs: Propranolol
5. During development of pulmonary oedema

1. Oxygenation
2. Salbutamol, terbutaline
3. Hydrocortisone
4. Aminophylline
5. Antibiotics
6. Increase the depth of anaesthesia. Halothane may be helpful.

1. Incoordinated diaphragmatic movement due to vagal stimulation
2. Inducing agents: Methohexitone, althesin
3. Can occur in spinal/epidural block
4. Common in upper abdominal surgery
5. Hypocapnia, unduly light anaesthesia
6. Insufficient doses of nondepolarising muscle relaxants
7. Management:
i. Manual ventilation
ii. Deepen anaesthesia
iii. More muscle relaxants
iv. Stimulation of the postnasal space with a suction catheter may help.

1. Serious problem of anaesthesia. Nitrous oxide diffusion enlarges pneumothorax, produces haemodynamic disturbances. Positive pressure ventilation intensifies the problem and may result tension pneumothorax.
2. Causes:
A. Traumatic: Chest injury, rib fracture
B. Iatrogenic: Cannulation of subclavian/internal jugular vein
Brachial plexus block, inadvertent barotrauma, cervical/thoracic injury
C. Spontaneous: Congenital bullae, emphysema, lung abscess, asthma, rapid decompression of divers.

A chest drain should be given before inducing anaesthesia in presence of known pneumothorax specially when IPPV is needed.

1. Diagnosis: Unexplained tachycardia, hypotension, bronchospasm, cyanosis, altered breathing, surgical emphysema
2. Management:
i. Discontinue NO, administer 100% oxygen
ii. Tension pneumothorax can be relieved by introducing a cannula through the second
intercostal space in midclavicular line temporarily
iii. Underwater seal intercostal drain should be given.

1. Inhalation of gastric contents
2. Common in obstetric cases, children, elderly patients, patients with full stomach, vomiting
prone
subjects
and
certain
drug
effect
3. Gastric fluid volume of 25 ml or more and/or its pH less than 2.5 are needed to produce acid aspiration syndrome
4. Other causative factors may include aspiration of food material, hypertonic solutions, irritating and infective materials
5. Pathophysiology
i. Usually nonspecific in character, large intrapulmonary shunt, increased lung water, loss of pulmonary compliance
ii. Other mechanism: Reflex airway closure, alteration of surfactant, interstitial and alveolar oedema
iii. Highly acidic material causes loss of alveolar capillary permeability, intense inflammation, oedema, noncardiogenic pulmonary oedema, haemorrhage and necrosis of airways and lung parenchyma
iv. Aspiration of large volume may lead to acute respiratory failure
v. Infection, lung abscess.

1. Respiratory insufficiency, hypoxaemia, tachypnoea
. Bronchospasm, cyanosis . Tachycardia, hypotension . Chest X-ray: Areas of density DAuPWN
. Crepitations and coarse asthmatic rhonchi
. Pulmonary oedema.

1. Head down tilt, tracheobronchial toilet
. Careful bronchoscopic suction . 100% oxygenation, endotracheal intubation, IPPV WN
. Bronchodilators
. Steroids
. Broad spectrum antibiotics
. Judicious administration of fluids
. Diuretics
. Ventilatory support
. Adequate monitoring. Proper nursing care. ay Oo
CMONDAP

1. Adequate preparation for anaesthesia
2. Identify the patients at risk and take necessary precautions

3. . Adequate preoperative fasting
4 . Nasogastric suction of gastric contents before starting anaesthesia
5 . Intragastric pressure should not be allowed to increase
6 . Pharmacological approach:
i. Sodium citrate solution
ii. Use of ranitidine, cimetidine
iii, Metoclopramide, antiemetic drugs, ondansetron.

1. Hypovolaemia
2. . Cardiovascular
3. .
4. Haematologic
5 . Neurogenic
6 . Drug effect:
Oo . Posture: Sudden change of posture, abnormal posture, rough handling
ON . Spinal/epidural anaesthesia . Hypersensitivity reactions
11. Following controlled hypotension
12. Following hypothermia
13. Drug interaction.

1. The underlying cause should be detected and treated accordingly
2 . Fluid and volume replacement
3 . Blood transfusion, iv fluid therapy
4. Vasopressors
5 . Steroids
6 . Reversal of metabolic acidosis
7 . Alpha blocking drugs: Phenoxybenzamine to improve organ perfusion.
:
Blood loss, inadequate fluid replacement
:
Myocardial infarction, arrhythmias, pulmonary embolism
Respiratory : Pneumothorax, airway obstruction, prolonged IPPV with PEEP
:
Disseminated intravascular coagulation, mismatched blood transfusion
:
Neurogenic shock, vagal over activities
i. Inducing agent: Overdose, relative overdose in very young/elderly/very ill patients. isoflurane
ii. Volatile agents: Halothane, enflurane,
iii. Muscle relaxants: Tubocurarine
iv. Narcotics/sedatives/analgesics.
. Surgical: Traction of abdominal/thoracic viscera, handling of baroreceptors, obstruction
of
venous flow by abdominal packs/retractors, strabismus surgery, prolonged surgery

1. Preoperative causes: Preexisting hypertension, anxiety, pain, preeclampsia, stoppage of
antihypertensive medication
. Operative causes: Hypoxia, hypercarbia, fluid overload, thyroid overactivity, less analgesia, of adrenal tumours
manipulation
. Drug effect: Use of vasopressors, ketamine, adrenaline, ergometrine
. Postoperative causes: hypoxia, hypercarbia, after coronary artery bypass surgery, renal transplantation, enhanced sympathetic nervous system overactivity
. Drug interaction : MAO inhibitors
. Hypertensive response to laryngoscopy, coughing, straining on endotracheal tube.

1. Prevention and treatment of the underlying cause
2. Monitoring of blood pressure is essential
3. Avoid hypoxia and hypercarbia
4. iv infusion should always be cautious
5 . Adequate ventilation
6 . Antihypertensive drug therapy.

1. Hypoxia
2. Hypercarbia, spontaneous ventilation
3. Anaesthetic agents: Halothane, cyclopropane, trichloroethylene
4. Catecholamines: Local anaesthetics with adrenaline. Adrenaline induced arrhythmia is common
with halothane
5. Hypokalaemia
6. Hyperkalaemia
7. Reflex arrhythmia during light anaesthesia
8. Drug effect: Digoxin, tricyclic antidepressants, aminophylline.

1. Correction of hypercapnia, hypoxia, or hypokalaemia
2. Reduction of inspired halothane concentration
3. Avoid adrenaline or adrenaline containing drugs
4. Monitoring of pulse, blood pressure, ECG
5 . Supraventricular tachycardia: iv verapamil or beta blockers. Cardioversion in extreme cases.
6 . Atrial flutter/fibrillation: Diagoxin, lignocaine, cardioversion
7. . Ventricular tachycardia: iv lignocaine, mexiletene, procainamide, phenytoin, beta blockers.

1. Raised intracranial pressure, head injury
2. Traction of abdominal/thoracic viscera, carotid artery
3. Drugs: Halothane, suxamethonium, digitalis, blockers
4. Hyperkalaemia
5. Jaundice
6. Aggravated by muscle diseases, burns, recent hemiplegia.

1. Hypovolaemia, dehydration 2. Hypoxaemia 3. Stress, anxiety 4. Fever 5. Anaemia 6. Congestive cardiac failure 7. Pain 8. Drugs: ether, cyclopropane, gallamine, atropine 9. Hyperthyroidism.

1. May not need treatment unless associated with congestive failure
2. Treatment of the underlying cause:
i. If there is no congestive failure, propranolol iv or oral
ii. In presence of congestive failure, digitalis is indicated
iii. Specific drug therapy to tackle haemodynamically significant cardiac tachydysrhythmias: Verapamil, lignocaine. Electrical cardioversion in extreme cases.

¢ Mild rise of body temperature is common in postoperative period due to metabolic response to
injury
¢ Causes of fever:
1. Infection
2. Respiratory infection
3. Urinary tract infection
4. Thrombophlebitis
5. Any rigor or convulsion

6. Drugs: Atropine, hyoscine
7. Associated diseases: Malaria, kalaazar, influenza, bacteraemia, septicaemia, etc.

1. Bed rest
. Antipyretics
. Cooling
. Fluids, oxygenation
. Antibiotics
. Treatment of causative factors.
DaoPrPwn

1. Following anaesthesia with halothane/cyclopropane/ether/thiopentone
2. Cold ambient temperature
3. Following blood transfusion
4. In cold stage of malarial fever.

1. Metabolic rate, and oxygen consumption increase
2. Increased carbon dioxide production
3. Ventilation increases
4. Hypoxaemia.

2. Methylphenidate, pethidine
3. General supportive measures, nursing care
4, Skin surface warming, if associated with decreased body temperature.

1. Known epileptic, convulsion-prone type of patient
2. Head injury, cerebral thrombosis /embolism
3. Hypoglycaemia, hypernatraemia
4. Brain surgery, cerebral hypoxia
5. Drug effect: Ether, enflurane, halothane, local anaesthetic toxicity
6. Eclampsia
7. . Hyperpyrexia

1, Prevention: Hypoxia, electrolyte imbalance and hypoglycaemia should be prevented
. Epileptic patients should be covered by anticonvulsant drugs before and after surgery
. Maintenance of patent clear airway . Oxygenation and IPPV, whenever needed . iv diazepam, antiepileptic drugs DaoPrPwn
. Treatment of underlying cause.

1. Slipped ligature
2. Haemorrhagic disorders, disseminated intravascular coagulation, hypofibrinogenaemia, vit-K
i . Obstetric causes: Abortion, antepartum / postpartum haemorrhage, dead foetus, amniotic fluid
ONauW . Patients with anticoagulants . Hereditary haemorrhagic disorders: Haemophilia . Patients with liver disease
. Following massive blood transfusion
embolism
. Preoperative hypotension and subsequent rise of blood pressure.

1. Adequate precautionary measures
2. Preoperative specific tests: Platelet count, bleeding time, prothrombin time, partial thromboplastin time, fibrinogen content
ND . Adequate correction of clotting factors
OB . Treatment of underlying cause
Ww . iv infusion, fresh blood transfusion
. Reexploration in case of slipped ligature
. Overdose of heparin or oral anticoagulants should be reversed by protamine or vitamin K respectively.

A. Prehepatic causes:
1. Mismatched blood transfusion, extensive haematoma
2. Bile leak

3. Severe sepsis
4. Cholestasis.
B. Hepatocellular causes:
1. Severe hypotension
2. Viral hepatitis
3. Severe hypoxaemia
4. Drugs
i. Haemolysis: Methyl dopa, phenacetin
ii. Hepatotoxic: Direct dose related: Carbon tetrachloride, chloroform, paracetamol,
methotrexate
Hypersensitivity: MAO inhibitors, halothane, isoniazid
iii. Cholestasis: Anabolic steroids, oral contraceptives, androgenic steroids
iv. Mixed reaction: Phenothiazines, tricyclic antidepressants, chlordiazepoxide, erythromycin.
C. Posthepatic causes:
1. Ligation of bile duct
2. Incidental obstructive jaundice, gallstones
3. Pancreatitis, cholangitis.

1. Proper diagnosis of type of jaundice is important
i) Liver function tests should be done
ii) Drug history, if any should be noted.
2. Withdraw the hepatotoxic drugs
3. Treat hypoxia, hypotension and infection
4. General supportive measures
5. No specific treatment for viral hepatitis
6. Conservative treatment, only specific treatment in extrahepatic obstructive jaundice is surgery.

1. Inadequate arterial oxygenation or elimination of carbon dioxide. PaOz below 60 mm Hg and
PaCO, above 49 mm Hg denotes respiratory insufficiency.

i. Inadequate ventilation, prolonged anaesthesia, after upper abdominal and thoracic surgery
ii. Overdose of analgesics and anaesthetics
iii. Hypocapnia, CO? narcosis
iv. Preexisting conditions: Kyphoscoliosis, poliomyelitis, myasthenia gravis, emphysema
etc.
v. Upper airway obstruction
vi. Lower airway obstruction: Bronchospasm
vii.
Pulmonary embolism, pulmonary oedema, pneumothorax, atelectasis
viii. Hypothermia, hyperthermia, severe shock, overhydration

ix. Use and misuse of muscle relaxants, inadequate decurarisation, incomplete hydrolysis of succinylcholine.

1. Hypoxia
2. Cyanosis
3. Hypercarbia
4. Respiratory depression
5. Pulse bounding and blood pressure elevated at first but later there is hypoxic cardiac depression and cardiac arrest.

1. Adequate preventive measures should be taken
2. Maintain patent clear airway
3. Oxygenation, endotracheal intubation and IPPV, whenever needed
4. Treatment of the underlying cause.

A. Causes:
1. Overdose of narcotics, analgesics and/or anaesthetics
2. Hypoxia, hypercarbia, acid base defect, fat/air embolism, brain trauma, severe hypotension
3. Incidental diseases: Cerebrovascular accident, myocardial infarction, myxoedema,
hypopituitary states, diabetic or hypoglycaemic coma, adrenal insufficiency, uraemia, moribundity.
4. Preexisting cerebral oedema, liver failure, renal failure, meningitis, encephalitis.
B. Diagnosis of coma is most important. Careful history, thorough examination and proper laboratory investigations may be needed.

i. A clear patent airway should be maintained
ii. 100% oxygenation and IPPV in cases of repiratory inadequacy
iii Care of the mouth and eyes
iv. General supportive measures, adequate nursing care
v. Nutritional care: Maintain fluid, electrolyte and acid base balance
vi. Treatment according to underlying cause.

4. . Hypoproteinaemia
5 . Myocardial failure
6 . Water intoxication following TURP.

Elevated CVP, tachycardia, low arterial pressure, distended neck veins, a third heart sound, pulmonary crepitations, pulmonary oedema.

1 . Judicious administration of fluids
2. . Inotropic support: Dopamine, digoxin
3. Loop diuretics
4. . General supportive measures.
Pulmonary artery and pulmonary capillary wedge measurement are often needed.

1. Mismatched blood transfusion
2. Some diseases: Diabetes, jaundice, DIC, congestive cardiac failure, urinary tract infection
3. Drugs: Overdose of analgesics, dextran, methoxyflurane, tetracycline, rifampicin, cephalosporins
. Miscellaneous: Shock states, extensive burns, severe infection, biliary tract surgery in presence of obstructive jaundice, major trauma with myoglobinuria, following open heart and aortic surgery.

1. Sudden decline in renal function
Ak Anaemia . Associated gastrointestinal haemorrhage, sepsis, respiratory failure
wN . Urine volume scanty, oliguria
. Rise in plasma creatinine, hyperkalaemia, hypocalcaemia, hyperphosphataemia, acidosis
. High output nonoliguric renal failure.

1. Prevention: Judicious fluid replacement, proper grouping and cross matching of blood, avoidance of nephrotoxic drug
Ww . Peritoneal dialysis
. Prompt recognition and effected treatment of the underlying cause
. Haemodialysis.

1. Inherited neuromuscular disorder and there is a defect in calcium binding in sarcoplasmic reticulum of skeletal muscle cells. With some triggering agents like halothane, suxamethonium, etc. calcium is released into the cytoplasm producing myofibrillar contraction, increased lactic acid and CO; production, increased O2 consumption and enormous production of heat. Membrane is lost and leaks from the cells
stability potassium causing hyperkalaemia. sometimes
. Susceptibility is inherited as an autosomal dominant and myopathic syndrome may be associated.

1. Unexplained tachycardia, cardiac dysrhythmia
. Arterial hypoxaemia
. Increased body temperature
. Increased CO, production
. Masseter muscle spasm following injection of suxamethonium; muscle rigidity
. Increased plasma creatine kinase and myoglobin concentrations
. Hyperkalaemia
. Metabolic acidosis.
ONaAAKWN

1. Past history
2. Myopathic syndrome
3. Elevation of plasma creatine kinase
4. Skeletal muscle biopsy.

1. All inhalation agents and suxamethonium should be discontinued
2. Hyperventilation with 100% oxygen
3. Dentrolene 2.5 mg/kg iv initially followed by repeat doses every 5-10 min until symptoms are
CMND . Active cooling . Adequate hydration . Maintain urinary output
OBS . Lignocaine or procainamide to treat ventricular arrhythmias
. Sodium bicarbonate to combat metabolic acidosis
. Adequate monitoring is essential.

Anticipated difficulty in endotracheal intubation by standard techniques or when intubation failed within first 2 an anaesthetist.
attempts by expert
¢ Structural or mechanical obstruction to visualise larynx for direct laryngoscopy /tracheal intubation.

1. Congenital syndromes/diseases: Pierre-Robin syndrome, Treacher Collins syndrome, cleft
palate,
ocular
hyper/hypotelorism,
orofacial
clefts,
Apert’s
syndrome,
acromegaly,
etc.
2. Anatomical: Short muscular neck, receding lower jaw, micrognathia, prominent maxillary incisors, high arched palate, restricted mobility of mandible, decreased thyromental distance, decreased distance between occiput and spinous process of C;
3. Acquired: Restriction of jaw /neck movement, instability of cervical spine, swelling of neck and face, laryngeal/tracheal stenosis, neck contracture.
4. Others: Morbid obesity, intraoral tumour, instrumental fault.
¢ Incidence of difficult tracheal intubation is much higher in obstetric
cases. It
is mostly due to:
1. Decreased mobility
2. Increased size of tongue
3. Laryngeal oedema
4. Full dentition
5. Large breasts.

Malampati et al. classification of the visibility of oropharyngeal structures with maximal mouth
opening.
Grade 1:Visualise soft palate, uvula, tonsillar pillars, fauces.
Grade 2:Visualise soft palate, possibly uvula and fauces.
Grade 3:Visualise hard palate only.

Samsoon and Young’s modification
Grade 1:Visualise soft palate, uvula, tonsillar pillars and fauces.
Grade 2:Visualise soft palate, uvula and fauces.
Grade 3:Visualise soft palate, base of uvula only.
Grade 4:Visualise hard palate only.
Cormach and Lehane classified on the anatomy visualised on direct laryngoscopy.
Grade 1:Larynx easily visualised.
Grade 2:Only the posterior area of glottis seen, but cricoid pressure helps better visualisation of
larynx.
Grade 3:Only epiglottis seen.
Grade 4:Neither epiglottis nor any other airway structures are seen.

1. Identify high risk patients
. Assess airway anatomy WN
. Detect any other related anatomic features
. X-ray of the head and neck
Thyromental distance less than 6 cm may cause difficulty
. Observe adequate mouth opening, jaw /neck movement
. A second opinion in doubtful cases
. Review available old anaesthetic records, medical card, if any.
SONAR

Preparation
1. Multiple laryngoscopic blades, endotracheal tubes, introducer, stilet, light wand
2. Suction apparatus. Good table with tilting arrangement
3. Emergency equipment for
i. Transtracheal jet ventilation
ii. Cricothyrotomy /tracheostomy
iii. Fibreoptic laryngoscope.
. A trained and undistracted assistant to apply cricoid pressure
. A person qualified to perform tracheostomy Do not use iv barbiturates and muscle relaxants . Establish iv infusion and administer iv atropine ONDE
. iv lignocaine Img/kg prior to attempt intubation.
1. Avoid general anaesthesia as far as practicable, regional anaesthesia may help
2. General anaesthesia via mask/nasal airway

CONAAHA
Ww Awake intubation
. General anaesthesia via mask and then attempt intubation
. Blind nasal intubation
. Fibreoptic laryngoscopy
. Retrograde intubation over guidewire
. Light wand/lighted stilet
. Anaesthesia through laryngeal mask airway
10. Tracheostomy: Transtracheal intubation.
¢ Success may be maximised and patient mortality /morbidity minimised through
1. Patience and experience on the part of anaesthetist
2. Thorough understanding of patient's airway anatomy and instruments used in airway management.

1. Summon help
. Ventilation with 100% oxygen under face mask Wh
. Maintain cricoid pressure. Patient positioned head down on left side
. Assess adequacy of ventilation and oxygenation
. Allow the patient to recover
. General anaesthesia may be reattempted with all precautionary measures
. Regional anaesthesia or General anaesthesia under mask may be attempted
. If ventilation is impossible
ANaATFE
a. Transtracheal jet ventilation
b. Cricothyrotomy
c. Tracheostomy.
¢ Following difficult intubation
1. The complication should be recorded
2. Patient should obtain a Medical alert card to inform the potential problem in future.

1. Trauma/bleeding/obstruction in the airway
. Laryngospasm
ANaATEWN
Hypoxaemia
. Bradycardia, cardiac arrest
. Oesophageal intubation
. Gastric distension
. Aspiration of gastric contents
. Damage to cervical spine.

1. Inability to both intubate and mask ventilate make the situation grave and critical. At that time transtracheal ventilation or oxygenation may be life saving
It consists of 14 gauze over the needle catheter, Luer lock adaptor, noncompressive oxygen
tubing, connector and a source of pressurised oxygen
Cricothyroid membrane is pierced by the needle to enter the trachea. Catheter is kept in situ. It is then attached to some device to administer oxygen under pressure.

¢ Mostly similar as in other branches of surgery, but requires expertise in ocular anatomy and physiology.

i. Normal 10 to 22 mm Hg.
ii. Factors causing increase in IOP
:
a. External pressure, contraction of orbicularis oculi
b.
Variation in vascular (choroidal) volume
Raised venous pressure
Airway obstruction, coughing, straining
IPPV
Hypercarbia, hypoxia
Sudden increase in blood pressure
c. Increase in aqueous and vitreous volume
d. Certain drugs :
Atropine in narrow angled glaucoma only
Opioids /neurolepts
Ketamine
Suxamethonium.
iii. Factors causing decrease in IOP :
Low CVP
Low arterial blood pressure
Head up tilt
Low PaCOz, hyperventilation, hyperoxaemia
Reduction of vitreous volume : Diuretics
Most anaesthetic drugs.

1. A trigeminal-vagal reflex precipitated by pressure on eyeball or traction of extraocular muscles
particularly
medial
rectus
. Long and short ciliary nerves to ciliary ganglion is the afferent pathway and motor nucleus of vagus is the efferent pathway
. May occur in strabismus surgery, during retrobulbar block or following pressure on eyeball.
Predisposing
factors
include
hypoxia,
hypercarbia,
light
. Manifestations : Bradycardia, A-V block, premature ventricular contractions, ventricular fibrillation, cardiac asystole.
. Management :
i. Continuous monitoring of vital signs to detect the condition early
ii. Removal of stimulus
iii. iv atropine in presence of bradycardia.

1. Acetazolamide : Carbonic anhydrase inhibitor, diuresis, hypokalaemic metabolic acidosis. decreases IOP
. Atropine : Anticholinergic, mydriatic, central anticholinergic syndrome
. Cyclopentolate : Potent mydriatic. Can cause CNS toxicity with disorientation, psychosis and convulsion
. Ecothiopate : Long acting anticholinesterase used to treat glaucoma. Can cause bronchospasm and prolong the effect of suxamethonium
. Adrenaline : Alpha adrenergic agonist, causes mydriasis and vasoconstriction. Can cause tachycardia, hypertension and dysrhythmias
. Timolol : A beta adrenergic blocking agent used to treat glaucoma. Can induce bradycardia, and bronchospasm
. Phenylephrine : Produces capillary decongestion and mydriasis, but can induce tachycardia, rise of BP and dysrhythmias
. Acetylcholine: Miotic used afer lens extraction. Can cause bradycardia, hypotension, bronchospasm, salivation
. Cocaine: Local anaesthetic. Can induce dysrhythmia
10. Betaxolol: Beta adrenergic blocker oculospecific for glaucoma. Additive effect with other beta blockers.

1. Control of IOP
2. Intense analgesia
3. Motionless eye
4. Avoidance of oculocardiac reflex
5 . Smooth recovery without straining, coughing, vomiting
6 . Safe.

A. Premedication
i. Little effect on IOP provided it does not alter PaCO)
ii. Opioid analgesics may cause vomiting, retching and respiratory depression
iii. Anticholinergic drugs in routine premedication have no effect on glaucomatous eye the treatment is maintained.
provided topical
B. Induction
i. Thiopentone : Moderate and transient decrease in IOP
ii. Etomidate: Immediate and prolonged decrease in IOP
iii. Ketamine increases IOP.
C. Muscle relaxants
i. Suxamethonium increases IOP, maximum in 2 min and returns to baseline by 5 min
ii. Nondepolarising muscle relaxants : No direct effect
iii. Tubocurarine : Marked decrease in IOP.
D. Volatile anaesthetics
i. Halothane and enflurane : Decrease in IOP
ii. Ether and isoflurane : Decrease in IOP
E. Nitrous oxide : No effect in the absence of air in the globe.

1. Premedication : Diazepam
2. Induction of anaesthesia
:
Fentanyl
Etomidate
Suxamethonium
Larynx and trachea should be sprayed with 4% lignocaine
. Endotracheal intubation should be gentle and smooth
IPPV
N2O + O2 + halothane/enflurane
. Reversal with atropine and neostigmine at the end of anaesthesia
. iv administration of antiemetics (droperidol or ondansetron) near the end of anaesthesia can minimise the incidence of nausea and
vomiting.
OND

1. Provides effective sensory and motor block (akinesia) for intraocular surgery
2. Affects mainly the ciliary ganglion and the long and short ciliary nerves
3. Often combined with facial nerve block to produce paralysis of orbicularis oculi
4. Technique: Patient is asked to look straight forward. A needle is passed through the reflection of conjunctiva below the lateral limbus and gently advanced parallel to the floor of the orbit in a depth of about 15 mm. Then the needle tip is redirected slightly medially and upwards to enter the muscle cone at a depth of about 30 mm. After negative aspiration test 3 to 5 ml of lignocaine 2% is deposited

- 5. Complications: Retrobulbar haemorrhage, intravascular/intraneural injection, systemic toxicity, injury to globe, central retinal occlusion, subdural/subarachnoid injection, oculocardiac reflex. Brainstem anaesthesia (due to passing of local anaesthetic along optic nerve sheath into CNS).

1. A good alternative to retrobulbar block. Injection is given around the eye
2. Technique : Patient is asked to look straight forward. A needle is inserted through the conjunctival reflection below the lateral canthus vertically. It is passed parallel to the floor of orbit and tangential to globe to place the needle point outside the muscle cone. After negative aspiration test about 10 ml lignocaine 2% is injected. A second injection of about 5 ml anaesthetic solution is given through the conjunctiva in the medial canthus medial to the caruncle.
3. Advantages
:
i. Low incidence of serious complications
ii. No facial nerve block needed.
4. Disadvantages :
i. Requires two injections
ii. Onset time longer
iii. Large volume of local anaesthetic needed, risk of toxicity.

Problems
1. Suxamethonium is better avoided as it can produce sustained contraction of extraocular muscles. Malignant hyperthermia can occur in some of these patients
. Increased risk of oculocardiac reflex. Injection of atropine sulphate iv or local infiltration of the extraocular muscle with lignocaine may be used to prevent or treat the oculocardiac reflex
. Increased incidence of postoperative nausea and vomiting. Prophylactic use of iv droperidol or ondansetron may be helpful
. Risk of inducing malignant hyperthermia is always there. Adequate screening is essential.

Special considerations
:
1. Drug induced miosis should be maintained throughout the perioperative period
2. Venous congestion should be avoided
3. Adverse drug interactions should be borne in mind.
Note :
1. Miotic eyedrops should be continued on the day of operation
2. 3. Atropine can be used in routine premedication as the drug reaches the eye in negligible amount The use of anticholinergic drugs with anticholine sterase drug is also recommended during reversal of nondepolarising block
. Suxamethonium should be used cautiously, transient rise of IOP can occur
. Prolonged hypotension should always be avoided.

1. Common eye operation in the aged. These patients may suffer from concurrent systemic diseases
. Usually done with retrobulbar/peribulbar block. Complete local anaesthesia and the akinesia
of globe are essential. Nausea/vomiting, straining and retching should be avoided
. General anaesthesia may be needed in selected cases. Smooth induction, and quiet
recovery are needed. Prophylactic use of antiemetic drugs (droperidol, ondansetron) is helpful.

1. Prevention of aspiration of gastric contents
2. Prevention of increases in IOP
3. Succinylcholine should be used cautiously in rapid sequence induction of anaesthesia
4. Proper measures should be taken to blunt the cardiovascular and IOP responses to laryngoscopy and intubation
. Nausea/vomiting, straining, coughing should be avoided.
1. IOP should be kept decreased during the operation. Use of acetazolamide or manntol is helpful. 2. Traction of extraocular muscles can activate oculocardiae reflex. Monitoring of the vital signs is essential
3. Nitrous oxide anaesthesia may cause problems as it may increase the volume of the bubble created during retinal reattachment. A sulphur hexafluoride SF, bubble is injected in the vitreal cavity. It remains in place for about 10 days in position. Some newer gases may reamin for about 28 days. Nitrous oxide is 34 times more soluble than nitrogen and 117 times more soluble in SF, and it rapidly enters the bubble and increases the volume and IOP. Thus nitrous oxide should be discontinued for at least 20 minutes before SF, injection and avoided for 4 weeks after retinal operation
4. Special care is needed, if laser is used for repair. Eye protection with a protective shield or eye glasses is essential for the patient and OT personnels.
1. Facial nerve preservation: Facial neve is adjacent to structures of ear and may be injured during operation. Some skeletal muscle response to direct nerve stimulation should remain to identify and preserve the nerve even if muscle relaxant is used. Volatile anaesthetics may be helpful as it preserves the ability to identify the nerve by using peripheral nerve stimulator.
2. Use of adrenaline: Adrenaline is often used to produce vasoconstriction and to get a dry operative field. It helps to minimise the blood loss. But it can cause hypertension and cardiac dysrhythmias. Some volatile anaesthetics like halothane can cause dysrhythmias in presence of exogenous catecholamines.

3. Nitrous oxide and middle air pressure: Middle ear is an air-filled noncompliant space. Normally pressure is vented by the eustachian tube. During N2O anaesthesia, NO can accumulate in middle air and increase the pressure. This can cause rupture of tympanic membrane and dislodgement of tympanic graft. So it is recommended that inhaled N2O should be discontinued at least 30 minutes before placement of graft.
Rapid absorption of N2O after discontinuation of the gas can produce negative pressure in middle ear manifesting as serious otitis and/or transient hearing loss.
Note:
¢ Microsurgery of middle ear is delicate and anaesthesia should provide minimum bleeding, dry operative field, smooth emergence and minimum postoperative disturbances
¢ Postoperative nausea due to labyrinthine disturbances is common
e Impaired hearing, if present may make communications difficult.
1. Topical vasoconstrictors like cocaine and adrenaline are often used to minimise bleeding. These can adversely affect the patients
w . Posterior pharyngeal pack and nasal packs need special care. It should be removed before extubation
ANAS . iv infusion and blood transfusion may be needed . Extubation should be done only when protective airway reflexes return . NO can be used satisfactorily even in nasal sinus (airfilled cavities) surgery . Endoscopic sinus surgery can be done under local anaesthesia with adequate sedation . Oversedation should be avoided.
. Volatile anaesthetics are mostly safe. Hypoxia, hypercarbia and hypotension should be avoided

1. May be due to oedema, infection, tumour of the airway
2. Problems increase when operation involves the airway
3. Good surgical access and a safe ventilatory pathway are needed
4 . Adequate evaluation, airway anatomy, airway management plan are
5 . Anaesthesia:
important
i. Proper premedication. Antisialagogue agent should be given
ii. Endotracheal intubation
iii. Patency of the airway and anaesthetic delivery system ensured
iv. Hypoxia, hypercarbia and hypotension should be avoided
v. Monitoring is essential
vi. iv infusion, blood transfusion may be needed
vii. Prevent pulmonary aspiration of gastric contents.

1. Common in paediatric age group
2. Proper evaluation and routine investigations needed
3. Adequate premedication. Trimeprazine syrup
Atropine sulphate
. Endotracheal intubation. Volatile anaesthetics and suitable muscle relaxants are helpful.
Mandibular and pharyngeal muscle relaxation are needed. Laryngeal reflexes must be suppressed
. Blood loss may be significant. iv infusion of fluids, blood transfusion may be necessary
. Adenoids are curetted and the postnasal space is packed for haemostasis. After a few minutes the pack should be removed
. After the end of operation, extubation should be done when the child is awake. Patient should be kept in tonsillar position.

1. May pose a major anaesthetic problem. Patient is hypovolaemic, anaemic, hypotensive, tachycardiac and dehydrated
a . Anaesthesia may be maintained with N2O + O; + halothane or isoflurane.
. Rehydration is most important. Blood transfusion may be needed
. Blood may be swallowed and may pose full stomach; so all precautions should be taken against aspiration
. Rapid sequence induction with iv thiopentone and suxamethonium with cricoid pressure followed by quick endotracheal intubation
. Stomach may be emptied through an orogastric tube after placement of endotracheal tube.
. At the end of operation, extubation should be done when the child is awake. Rapid smooth recovery with return of protective airway reflex is most important.

1. Laser is the light amplification by stimulated emission of radiation. It can produce an intense beam of light that can be focussed for controlled coagulation, incision or vaporisation of tissues.
. Main advantages : Minimum damage of adjoining tissue, rapid healing, immediate control of bleeding.
. Indications : Polyp/tumour of vocal cord, larynx, trachea
. Anaesthesia :
i. Patient must be kept immobile
ii. Oxygen should be given below 40% concentration
iii. N2O should be avoided. Use of air oxygen mixture may be helpful
iv. Helium can be used instead of air as its thermal conductivity can delay ignition
v. Volatile anaesthetics should be avoided
vi. Cuff of the endotracheal tube should be inflated with saline.
. Hazards :
i. Atmospheric pollution
ii. Damage of normal tissues due to misdirected beam
iii. Venous gas embolism
iv. Eye protection is needed for OT personnels
v. Fire of endotracheal tube can occur. Wrapping the tube in protective silver foil or of aluminised silicone is helpful
use
vi. Airway fire can occur. Some measures should be taken promptly :
a) Remove the ignition source
b) Stop ventilation, extubate and extinguish the flamming material with water
c) Ventilation through mask with oxygen
d) Assess the damage and remove the debris
e) Examine the oropharynx and face
f) Chest X-ray is needed to evaluate lung condition.

i. Minute ventilation is increased
ii. Tidal volume and respiratory rate increase
iii. PaO increases and PaCQO) decreases, no change in pH
iv. O2 consumption increases
v. Alveolar ventilation increases
vi Functional residual capacity, expiratory reserve volume and vital capacity decrease. No change in vital capacity.
¢ Oedema, weight gain and increased breast size may pose difficult intubation
¢ Capillary engorgement of the mucosal lining of upper airway. Avoid use of nasal airways and nasal intubation.

i. Heart rate increases
ii. Stroke volume and cardiac output increase
iii. Total peripheral resistance decreases
iv. No changes in central venous pressure
v. Blood pressure (systolic and diastolic) decreases
vi.
Intravascular volume, plasma valume, RBC volume increase
¢ Supine hypotensive syndrome : Decreased venous return due to obstruction of the inferior vena cava and/or abdominal aorta by the enlarged gravid uterus
¢ Aortocaval obstruction : Causes uteroplacental insufficiency and foetal hypoxia due to decreased uterine blood flow
¢ Left uterine displacement is helpful in moving the gravid uterus of the inferior vena cava and/or aorta.

i. Gastric emptying time slowed, gastric motility decreased
ii. Intragastric pressure increased

iii. Lower oesophageal sphincter pressure decreased
iv. Stomach position more up and horizontal
v. Increased risk of pulmonary aspiration
vi. Transaminases increase
vii. Pseudocholinesterase decreases
viii. Alkaline phosphatase increases
ix. Serum cholesterol may increase.
4. Haematologic system:
i. Haemoglobin content decreases
ii. Coagulation factors may increase. Increased coagulability
iii. Platelet count less.
5. Nervous system:
i. Anaesthetic requirement less
ii. MAC value decreases
iii. Endorphin levels increase.

i. Renal blood flow/glomerular filtration rate decrease
ii. Glycosuria, proteinuria
iii. Creatinine clearance may increase.

i. Amenorrhoea
ii. Morning sickness
ii. Breast discomfort, engorgement, pigmented nipple and areola mee
iv. Uterus just felt per abdomen at about 12 weeks
v. Sonography.
B. At about 16th week
ii. Breast changes
iii. Uterus palpable per abdomen
iv. Uterine souffle
v. Internal ballotment.
vi. X-ray : Foetal shadow.
C. At about 28th weeks
Same as above with:
i. Contractions
ii. External ballotment
iii. Palpable foetal parts
iv. Foetal movements
v. Foetal heart sound.

¢ Absolute signs of pregnancy
. Palpation of foetal parts
. Foetal movements . Foetal heart sound . Radiology ARWNHH
. Ultrasonography.
¢ Assessment of foetal maturity
. Ultrasonography NPP
. Amniocentesis, analysis of amniotic fluid for it lecithin/sphingomyelin ratio to evaluate lung maturity
. Radiological assessment w
4. Clinical assessment.

Exchange of the nutrients and waste products between mother and foetus. Depends on:
1. Physical properties of the substances
2. Extent and functional integrity of exchange membrane
3. Rate of maternal and foetal blood flow.

1. Simple diffusion

1. Maternal protein binding
2. Molecular weight of the substances
3. Lipid solubility
4. Degree of ionisation
5. Concentration at placental site
6. Foetal uptake and distribution.

Mostly by diffusion, governed by Fick’s equation
Qi
= Ka x A x [Pa (m) - Pa (f)]/b
Qa =
Quantity of drug transferred per unit time
Ka
= Diffusion constant of the drug
A
= Surface area of placenta
Py(m)
= Mean drug concentration of maternal blood
Pa
(f)
= Mean drug concentration of foetal blood
b
=
Thickness of placenta

¢ Some of these factors can be controlled but factors like surface area and thickness of placenta are not within control.

Two components
:
1. Visceral pain caused by dilatation and effacement of the cervix during uterine contractions. It is mostly in the first stage of labour
2. Somatic pain occurs during stretching of the vagina and perineum during descent of foetus. It dominates the second stage.
There may be considerable overlap of the two types.
¢ Visceral pain is referred to the lower abdomen between the umbilicus and pubis and to the lower back. Intense pain may be referred to hips and thighs. These pain sensations are mediated by small unmyelinated fibres those pass from the cervix through the pelvis and hypogastric plexuses to enter the sympathetic chain at Ls to Ls. It reaches the posterior root ganglia and spinal cord through the white rami communicates of Tj9 to Li.
¢ Somatic pain starts due to descent of foetus through the vagina. Initially it is something as a need to defaecate. But with further ascent there is uncontrollable urge to bear down. This
pain
is
conveyed
mostly
through
pudendal
nerve
to
posterior
nerve
roots
S2
to
Sq.
¢ Other contributing factors : Pressure and
on bladder, urethra and rectum (sacral segments), traction of uterine adnexae (Tio to L;) stretchand pressure on muscles and ligaments of pelvis (lower lumbar and sacralsegments).

Criteria :
GT Early interaction between mother and baby just after the delivery.
PFON Pr Satisfactory analgesia for the mother Progress of labour will not be interfered Safe for both mother and foetus
Provision of safe and comfortable delivery

A. Nonpharmacological methods
i. Parental education, encouragement and reassurance by doctors, nurses and other personnels
ii. Acupuncture, hypnosis : Mostly unpredictable.

i. Opioids to control pain in first stage
ii. Buterophenol
iii. Fentanyl
iv. Pentazocine
v. Ketamine in small iv doses 0.25 mg/kg
vi. Midazolam 0.5 to 1 mg iv
iv access to provide for hydration, transfusion or rapid administration of drugs is important.

C. Inhalational analgesia
i. Low concentrations of inhaled anaesthetics, continuous 30 to 50% nitrous oxide in
oxygen
ii. Intermittent inhalation of 0.35% methoxyflurane in air
iii. Trichlorethylene 0.5% or 0.35% in air from special vaporisers.

i. Paracervical block (T;9-L) provides perineal anaesthesia only. Foetal bradycardia can
occur
ii. Lumbar epidural analgesia block (T19-Ss) is helpful in first and second stage of labour.
Can
preserve
pelvic
muscle
tone
iii. Caudal analgesia (Tjo-Ss), continuous caudal epidural block
iv. Spinal anaesthesia Tyo-S5 ; Saddle block S;-S5
v. Pudendal nerve block (S2-Sy4).
E. Other methods
i. Extradural injection of opioids (morphine, pethidine, fentanyl)
ii. Intraspinal opioids
iii. Transcutaneous nerve stimulation : Not much accepted.

Caesarean section is performed as:
A. Anelective procedure
B. An urgent procedure
i. When progress of labour is not satisfactory
ii. Deteriorating intrauterine conditions
iii. Extreme foetal distress, prolapsed umbilical cord, maternal haemorrhage, etc.

1. Spinal or epidural analgesia
2. General anaesthesia
3. Local infiltration.

¢ Preferred for caesarean delivery as:
i. Neonatal depression is insignificant
ii. Decreased risk of pulmonary aspiration
iii. Mother remains awake and can share in the birth experience
iv. But it takes some time to perform.

i. Hypovolaemia
ii. Infection near the site of injection
iii. Septicaemia

iv. Neurologic abnormality, raised intracranial tension
v. Coagulopathy
vi. Patient refusal.

A. Spinal anaesthesia :
2. Inability to control the spread of anaesthesia
3. Postspinal puncture headache.
B. Epidural anaesthesia :
1. Time needed to place the needle/catheter
2. Potential for local anaesthetic toxicity.
¢ Spinal anaesthesia is technically easy to perform and can produce rapid and reliable block. e Epidural anaesthesia is technically difficult to perform. Continuous epidural offers better control over spread and allows unlimited duration of block
¢ Subarachnoid or epidural block upto the level of Ty is mostly satisfactory for caesarean section. Continuous epidural block is also used for analgesia in labour, the level of block can be well-
extended
by
injecting
the
local
to
perform
caesarean
section
¢ Postoperative analgesia can be provided by addition of opioid in local anaesthetic solution injected in subarachnoid/epidural space.

1. Preoperative medication
i. Benzodiazepine to allay anxiety and tension
ii. Parturients are at special risk of pulmonary aspiration. Clear antacids, metoclopramide and Hy blockers may be used.
2. Preoxygenation
3. Induction of anaesthesia: Thiopentone and suxamethonium, Crash induction. Rapid endotracheal intubation
4. Maintenance of anaesthesia : Only NO until delivery, thereafter volatile anaesthetics can be added. Opioids can also be given.

1. Short preoperative preparation
2. Patient remains unconscious.

1. Maternal pulmonary aspiration
2. Neonatal depression
3. Immediate maternal bonding not possible.

A. Chronic hypertension
B. Preeclampsia/eclampsia
C. Chronic hypertension with superimposed preeclampsia
D. Gestational hypertension.
e Hypertension : A systolic pressure above 140 mm Hg or 30 mm Hg above baseline, diastolic pressure above 90 mm Hg or 15 mm Hg above baseline. A mean pressure above 105 mm Hg or a mean increase 20 mm Hg above baseline
¢ Chronic hypertension is the state that predates pregnancy, diagnosed before 20 week of pregnancy
¢ Gestational hypertension occurs after 20th week of pregnancy with no signs of preeclampsia. It is mostly essential hypertension that is unmasked in pregnancy.

Preeclampsia : Often manifest after 20th week of pregnancy with:
Oedema.
e Preeclampsia may turn to eclampsia when generalised seizures manifest.

i. Vasospasm secondary to increased levels of renin, aldosterone, angiotensin and catecholamines
ii. Aldosterone causes sodium and water retention, generalised oedema
iii. Cerebral oedema, cerebral vasospasm, raised intracranial tension, headache, hyperreflexia,
blurred
vision,
vertigo,
blindness,
convulsion,
coma
iv. Laryngeal and upper airway oedema, airway congestion, pulmonary capillary leak, intrapulmonary shunt, deterioration of alveolar arterial oxygen gradient
v. Generalised vasoconstriction, hypertension, impaired tissue perfusion, cellular hypoxia
vi. Intravascular haemoconcentration
hypovolaemia,
vii.
Left ventricular hypertrophy, dysfunction, congestive heart failure
viii. Renal blood flow reduced, renal dysfunction.
Damaged glomeruli lead for renal loss of proteins, oliguric renal failure.
ix. Hepatocellular damage, elevated liver enzymes
x. Coagulation abnormalities
xi. HELLP syndrome : (Haemolysis, elevated liver function tests and low platelet count)

i.
Control
the
disease.
Progression
should
be
prevented
ii. Bed rest, left uterine displacement
iii. Monitoring : Blood pressure, weight gain, renal function, coagulation function. CNS irritability.
iv. Restrict oral fluid and sodium intake
v. Routine use of diuretics

vi. Monitor the foetal well being
vii.
Delivery of foetus and condition of the mother should be considered.
a. Foetal causes: Foetal distress, cessation of foetal maturity
b. Maternal causes : Worsening preeclampsia, medical control not satisfactory.

i. Magnesium sulphate
ii. Antihypertensive drugs : hydralazine, trimetaphan, nitroprusside
iii. Digitalis
iv. Mannitol
Vv. Furosemide.

i. a. Continuous lumbar epidural anaesthesia
b. Prehydration is essential
c. Coagulation studies must be done before hand.
. Spinal anaesthesia may be recommended when vaginal delivery is imminent
iii.
Caesarean section may be done under general anaesthesia, endotracheal intubation may pose
difficulty, extensive monitoring is essential. Regional anaesthesia (spinal/epidural block) is often helpful.

A. Bleeding during third trimester
i. Placenta previa
ii. Abruptio placentae
iii. Placenta accreta/increta/percreta
. During labour
i. Uterine rupture
. Postpartum haemorrhage
i. Retained placenta
ii. Uterine atony
iii. Cervical /vaginal lacerations

i. Cervical pathology : Polyp, carcinoma
ii. Preeclampsia
iii. Intrauterine death of the foetus
iv. Preexisting coagulopathy.

It is the bleeding from or into the genital tract after 28th weak of pregnancy but before the birth of the baby. Commonest cause is placenta previa or abruptio placentae.

¢ Postpartum haemorrhage : Bleeding from or into the genital tract following birth of the baby upto the end of puerperium. It is mostly due to atonic uterus or retained products of conception.
e Placenta previa: Painless vaginal bleeding, bright red blood, blood clot, blood loss not concealed. Common in advanced age and/or multiple parity. Low implantation of placenta in uterus. Diagnosis confirmed by ultrasound.
e Abruptio placentae : Separation of a normally implanted placenta after 20 weeks of gestation. Painful vaginal bleeding, portwine blood, nonclotting blood. Blood loss often concealed. DIC is common. Diagnosis confirmed by ultrasound. Acute renal failure and foetal distress can occur.
v If the maternal and foetal condition is not critical, continuous epidural anaesthesia is helpful for labour and vaginal delivery. Otherwise, in critical situation general anaesthesia is needed for emergency caesarean section. Ketamine induction and maintenance with nitrous oxide and volatile anaesthetics may be indicated. Neonates are usually acidotic and hypovolaemic. They need special care.

May be
i. Spontaneous
ii. Separation of previous uterine scar
iii. Rapid spontaneous delivery
iv. Excessive oxytocin stimulation.

i. iv fluids, blood transfusion, vasopressors
ii. Urgent laparotomy
Hysterectomy
Repair and sterilisation.
¢ Retained placenta : Needs manual exploration of uterus. Inhalation anaesthesia is mostly satisfactory for manual removal of placenta. If lumbar epidural or spinal anaesthesia is used for vaginal delivery, this may be sufficient for manual exploration.

1. Disordered uterine contraction
2. Noncontracting uterus seems boggy and large
3. Continuing painless bleeding per vagina after delivery
4. Blood loss, shock.

1. Try to increase myometrial tone
Massage the uterus WN
Oxytocin
Ergometrine Ae
Prostaglandin Fya.

1. Maternal resuscitation, iv fluid, blood transfusion
2. Vasopressors
3. Hypogastric artery ligation or even hysterectomy may have to be done under general anaesthesia.

1. Avoid teratogenic drugs
2. Avoid intrauterine foetal hypoxia and acidosis
3. Prevent premature labour.

1. Avoid major surgery during pregnancy, whenever possible
2. Emergency surgery may have to be done knowing fully the risks
3. First trimester is most critical. Avoid anaesthesia and surgery during this period
4. Operation should be done by experts with minimum handling the uterus
5. Anaesthesia and monitoring and postoperative care should be meticulous.

1. Local or regional anaesthesia is mostly satisfactory. Drug doses should be carefully judged.
Avoid
2. General anaesthesia:
i. Take preventive measures of pulmonary aspiration
ii. Cautious transportation with left uterine displacement
iii. Careful evaluation of airway
iv. Preoxygenation with 100% oxygen
v. Rapid sequence induction of anaesthesia
vi. Maintenance with NO, O2, muscle relaxant and intermittent doses of analgesics
vii. Avoid hypoxia, hypercarbia and hypotension
viii. Monitoring of vital signs of mother and foetal condition throughout the procedure
ix. Some drugs may be used to stop premature labour in postoperative period. sympathomimetics (terbutaline, ritodrine) or magnesium sulphate are commonly used
Beta
x. Prostaglandin inhibitors and calcium channel blockers can also be used.
Note:
¢ Critical period of organogenesis is 15 to 56 days of gestation. Most of the anaesthetic drugs are safe. But nitrous oxide should be used in
parturients cautiously
¢ Muscle relaxants are highly ionised and thus undergo minimum placental transfer. Most of them are safe
e Ketamine can increase the uterine tone
e Halogenated volatile anaesthetics may be used as they relax the uterus.

1. Fallopian tubes are ligated or occluded for female sterilisation during the puerperium usually 24 to 48 hours following delivery
2. Technically simple, hospital stay and rest at home help to recover simultaneously
3. Can also be done beyond 3 months following delivery or abortion, preferably following the
menstrual period in proliferative phase
4. Laparoscopic sterilisation can be done 6 weeks postpartum. It is not done early as it is technically difficult
5. Anaesthesia :
i . General anaesthesia
ii . Spinal block upto Tyo
ii b: . Epidural block
iv . Local infiltration over the abdominal wall.

1. It should be done at least 6 weeks following delivery
2. . Thorough preoperative check up and routine investigations needed
3. . Valid consent for anaesthesia and ligation.
4 . Usually done under local anaesthesia
i. Infiltration of 1% lignocaine at the site of incision
ii. Lithotomy position, about 15° Trendelenburg position
iii. Pneumoperitoneum produced by Verres needle by inflating with about 2 litres of gas
iv. Trocar and laparoscope introduced and the ring is slipped into the base of the loop of the tube under direct vision. Repeated on the other side
Vv. Laparoscope removed, abdomen deflated, wound closed.

i. Can be done in OPD or even in camps
ii. Minimum (8 to 10 hrs) hospital stay
iii. Rapid wound healing
iv. Reversal anastomosis easier, if needed
v. Least damage of the fallopian tube.
like carbon dioxide, nitrous oxide or oxygen or room air

i. Risk of cardiorespiratory embarrassment, pneumothorax, injury to intestine and vessles
iv. Needs sufficient training to adopt the technique.
. Cardiac arrest
iii. Instrument is costly

1. Dilatation of the cervix and evacuation of the products of conception.
i. Incomplete abortion, inevitable abortion
ii. Medical termination of pregnancy (6 to 8 weeks)
iii. Hydatidiform mole in the way of expulsion.
3. Anaesthesia:
i. Usually done under general anaesthesia
ii. After the operation ergometrine 0.25 mg im or methargin 0.2 mg im may be needed.
4. Dangers :
i. Excessive bleeding ; shock
ii. Pelvic infection
iii. Cervical incompetence
iv. Uterine perforation.

1. Operative procedure to extract the products of conception out of the uterine cavity before 28th
week the anterior wall of uterus.
by incising
i. Medical termination of pregnancy in mid trimester.
Where other methods are failed or otherwise contraindicated.
ii. Evacuation of molar pregnancy.
Can be done under either general anaesthesia or spinal/epidural anaesthesia.
i. Uterine bleeding
ii. Peritonitis
iii. Scar endometriosis
iv. Scar rupture in subsequent pregnancy
v. Anaesthetic complications : Aspiration pneumonitis, local anaesthetic toxicity, hypotension, etc.

Neonates differ from adults in a number of ways. The first 28 days of life is neonatal period.

1. Obligate nose breathers
2. Tongue relatively large, larynx higher and more anterior, epiglottis large, floppy, Vshaped, small mouth
. Narrowest part of larynx is the cricoid ring
. Trachea is short and soft.

1. Metabolic rate is high 7-9 ml/kg/min
2. Respiratory rate is faster, about 35/min
3. Tidal volume is 7 ml/kg, vital capacity 35 ml/kg, functional residual capacity 25 ml/kg, alveolar
ventilation 130 ml/kg/min
4. Neonatal respiration relies on diaphragmatic movement
5. Small airways contribute airway resistance
6. Work of breathing is higher
7. COz production is about 6 ml/kg/min.

1. Persistent patent ductus arteriosus and patent foramen ovale shunt blood away from the lung and into the systemic circulation. The shunt may be exacerbated with hypoxaemia, hypercarbia, acidosis and
AgNRwON . Bradycardia is common Hypotension . Systolic blood pressure is about 65 mm Hg
high airway pressure
. Cardiac output may be 2 to 3 times than that of adult
. Heart rate ranges from 120 to 130 beats/min

7. Blood volume is about 85 ml/kg
8. Haemoglobin is about 17 gm%.

1. Mostly immature, poor function
. Glomerular filtration rate and renal drug clearance reduced
. Concentrating power limited
. Hepatic drug metabolism limited
. Total body water is about 75% of the weight of newborn
. Physiological jaundice is common
. Hypoglycaemia can occur easily
. Hypocalcaemia occurs commonly.
ANaAUIEwWNY

1. Foetal hemoglobin present. Slight left ward shift of oxyhaemoglobin dissociation curve
2. Physiologic anaemia is common
3. Haematocrit ranges from 50 to 60%.
4. Platelet function reduced. Reduced plasma coagulation factors. Vit K dependent factors (II, VIL, IX and X) deficient. More at risk of bleeding.

1. Infant's surface/body weight ratio is greater than that of the adult. It predisposes more rapid
heat
loss.
Thermoregulatory
mechanism
is
mostly
chemical
2. Heat production mostly depends on metabolism of brown fat. Newborns cannot produce heat by shivering
3. Babies are prone to develop hypothermia in cold exposure.

1. Myelination of nerve fibres is incomplete in newborn
2. Cerebral cortex and blood-brain barrier not much developed
3. More sensitive to opioids and general anaesthesia.

1. Total body water content is higher (about 70%) than adults (60%)
2. A relative excess of extracellular fluids (about 38% 1 month, 26% in 1 year and 27% in adult)
3. Intracellular water is about 32% in 1 month, 36% in 1 year and 33% in adults
4. Limited ability to cope with excess water and sodium.

Depends on age and personality of the child, proposed operation, parental response and hospital/ doctor/staff response.

1. Preoperative visit is vital to allay fear, anxiety and tension and to gain confidence of the child.
2.
i. Age
ii. Body weight
iii. Consent of parents/guardian
iv. Previous anaesthesia, any medication, past history of illness
v. Present illness
vi. Operative site
vii. Physical examination : General survey, colour, nutrition, hydration, oral hygiene, teeth,
cardiovascular system, respiratory system.
viii. Vital signs : Pulse, respiration, blood pressure, body temperature.

i. Haemoglobin, total count, DC bleeding time, clotting time
ii. Urine analysis
iii. Blood urea, electrolytes
iv. Acid base status
v. Liver function tests in selected cases
vi. Chest X-ray in some cases.
e Veins should be examined with relation to iv induction and/or the establishment of iv infusion.

1. Adequate reassurance, explanations and a good rapport between the child and doctor are essential
. Food should be restricted for at least 4 hours before induction of anaesthesia. Clear fluid like— 5% dextrose solution 10 ml/kg may be given in infants under 1 year and milk 10ml/kg can be in children of 1 to 5 of
given years age
. Prolonged preanaesthetic fasting may lead to hypoglycaemia.

1. Ideal premedication should be safe and easy to administer. It should allay preoperative fear, anxiety and tension, sedate, lessen secretions, prevent vomiting and undesirable vagal reflexes.
It should not affect the vital centres
. Should be according to the need of the patient. Many children may not require any premedication
. Method of Administration
i. Oral : Well-tolerated but unpredictable effect
ii. Intramuscular : Reliable, popular, may have needle fear
iii. Intravenous : Only for anticholinergics
iv. Rectal : Not well-tolerated, unpredictable.

4. Usual Drugs
i. Atropine 0.02 mg/kg
ii. Hyoscine 15 mg/kg
iii. Pethidine 2 mg
iv. Diazepam 0.4 mg/kg
Vv. Trimeprazine 2 to 4 mg/kg orally
vi. Droperidol 0.2 to 0.4 mg/kg orally
vii. Glycopyrrolate 5 mg/kg.

1. Face mask : Rendell-Baker-Saucek Mask (Fig. 18.1)—face fitting shape, low dead space
2.
- Laryngoscope light weight easily
Laryngoscope: Magill straight bladed laryngoscope is popularly used as it aids better visualisation of larynx and to lift floppy epiglottis. Small size Macintosh laryngoscope can also be used. should be and can be handled
3. Endotracheal Tubes :
i. Uncuffed tubes must be used
ii. Various types of PVC and red rubber tubes are being used
iii. Single use sterile endotracheal tubes are also available
iv. Size of the tube
Age ae
- + 4-internal diameter in mm
. Length of the tube
+ 12-length of oral tube in cm
+ 15-length of nasal tube in cm.
vi. Tube should pass 2.5 cm in vocal cords
- vii. Size of the tube must always be such to ensure a leak is present.
Fig. 18.1: Rendell-Baker-Saucek face mask for paediatric use
. Breathing System :
A. Mapleson Systems
Jackson Rees circuit
Bain circuit
i. Recommended for infants weighing less than 10 kg
ii. Light and simple
iii. No valves and CO) absorbers
iv. Fresh gas flow should be 2.5 to 3 times the minute ventilation.
B. Circle System
i. Should be used in older children
ii. Unidirectional valves prevent rebreathing and the CO) absorber removes CO, and conserve heat
iii, Complex, valves are heavy
iv. Controlled ventilation is recommended while using paediatric circle systems.
. Special Endotracheal Tubes
i. RAE tubes: Preformed disposable PVC tube with a bend at the mouth to prevent kinking. Provides a connector at the level of chin, length should be adjusted carefully.
ii. Cole tube : Wide proximal and a narrow distal part. The shouldered part of the tube
may cause
laryngeal damage.
Narrowing may
increase turbulance
and work
of breaking.

- iii, Armoured tubes: These are latex and silicone reinforced tubes, floppy, incompressible and unkinkable. Needs introducer for intubation. These cannot be cut, thus the position should be carefully checked.

i. Small T-shaped metal tube consisting of a straight tube and 2 side arms. Fresh gas inlet is at right angle to the body. Side arms are longer and wider, one end is connected to the endotracheal tube and the other end is attached to a short tube which acts as reservoir.
ii. Reduces dead space to a minimum
iii. Low resistance (no valves)
iv. Fresh gas flow should be at least 2.5 times the minute volume.
Fig. 18.2: Ayre's T-piece with its connections

Fig. 18.3: Rees modification of Ayre's T-piece.
Key : A=Rendell-Baker-Sancek face mask; B=Metal adaptor; C=Endotracheal tube; D=T-piece;
E=Corrugated rubber tube; F=Rat-tailed reservoir bag with stop cock;
G=Fresh gas inlet tube

i. T-piece with an addition of an open ended reservoir bag
ii. Simple, light weight
iii. No valves, small dead space
iv. Used for spontaneous respiration or IPPV
v. High flow of fresh gas needed
vi. Humidification is not easy
vii. Scavenging may be difficult
viii. Recommended for children under 20 kg. body weight.

- e Main criteria needed for a paediatric ventilator
i.
Accurate control of inspired Oz, ii.
Respiratory rate
- iii. Tidal volume
iv.
Inspiratory/expiratory ratio v.
Provision of humidification
vi. Special features: IMV, PEEP, CPAP
vii. Reliable alarms
viii. Safe and reliable
ix. Light weight tubing.

1. Precordial or oesophageal stethoscope
. Blood pressure : Automatic monitors wn
. Pulse oximeter . End tidal CO ark

. Temperature . Urine output SOND
Neuromuscular block
. Monitoring on special cases
:
a. CVP
b. Transcutaneous O2 and CO? monitors
c. Pulmonary artery pressure
d. Cardiac output.

e Check the equipment and circuit
e An iv infusion is established
e All monitors are attached.

i. Intravenous induction
Thiopentone 2.5 mg/kg
Ketamine, methohexitone, etomidate, propofol can also be used, but can cause pain on
injection.
ii. Inhalational induction
Halothane
Isoflurane.
iii. Intramuscular
Ketamine can be used.
iv. Rectally : Not used now a days.
2. Mask anaesthesia
Inhalational anaesthetics can be administered via a face mask. Mask must fit the face. An
oropharyngeal airway may be helpful.
3. Endotracheal intubation
i. To control the ventilation
ii. Surgery around head and neck, thoracic, abdominal neurosurgery
iii. Prolonged anaesthetic procedures
iv. To prevent aspiration
v. Airway problems
vi. Ventilatory problems
vii. Prone/sitting posture
viii. To provide adequate muscular relaxation.
Procedure :
i. Preoxygenation
ii. Laryngoscopy

iii. Introduction of endotracheal tube. A gentle cricoid pressure may help to visualise the
larynx
iv. Check the position of the tube
v. Secure the tube firmly.

i. Malposition of the tube
ii. Disconnection
iii. Obstruction in the tube
iv. Accidental extubation
v. Sore throat
vi. Stridor, subglottic oedema
vii. Trauma
viii. Infection.
4. Maintenance of anaesthesia
i. A mixture of nitrous oxide and halothane/enflurane/isoflurane
ii. Analgesia may be provided with intermittent doses of pethidine.
iii. Muscular relaxation is provided with muscle relaxants like suxamethonium, vecuronium, atracurium, pancuronium, etc.
5. Recovery from anaesthesia
i. All potent anaesthetics should be stopped
ii. Antagonism of nondepolarising muscle relaxant needed with atropine and neostigmine
iii. Extubation done gently and carefully
iv. Check the vital signs.
6. Postoperative analgesia may be needed.
i. Morphine 0.1 to 0.2 mg/kg im
ii. Pethidine Img/kg
iii. Narcotics should be avoided in children less than 6 months.

1. Any dehydration: Preoperative fasting, abnormal loss of fluids as in vomiting, burns.
2. Maintenance of fluid requirements
3. Surgical injury : Blood loss
4. Body temperature
5. Environment : OT airconditioned or not ; high ambient temperature.

i. 5% loss of fluid : Dry tongue and mouth, pale
ii. 10% loss of fluid : Dry tongue, lethargic child, skin flaccid, fontanelle sunken, tachypnoea,
tachycardia, low blood pressure, urine output diminished
iii. 15% loss of fluid : Child not responsive, dry tongue, loose skin, fontanelle sunken, thready pulse, severe hypotension, oliguria, respiratory distress, rise of body temperature

iv. Laboratory studies:
a. Hb%
b. Haematocrit
c. Electrolytes
d. Urea, creatinine
e. Osmolality of urine and blood.
v. Fluid replacement
Physiological saline
Dextrose /saline
5% albumin
Plasma Blood. pnosD

i. First 10 kg body weight
4 ml/kg/hr
ii. Second 10 kg body weight
2 ml/kg/hr
ii. Excess of 20 kg
1
ml/kg/hr

1. Correct estimation of loss should be done by weighing of swabs and mops, drapes and measuring suction material
2. Fluid is also lost by evaporation, lack of humidification
. Rise of body temperature of 1°C can increase fluid loss by 10% w
4. Estimated fluid loss
i. Superficial surgery
0-2 ml/kg /hr
ii. Mild to moderate surgery
3-5 ml/kg/hr
iii. Extensive surgery
6 ml/kg/hr.

1. More than 10% of the estimated blood volume is lost :
Blood should be replaced millilitre for millilitre
2. If crystalloid solution is used to replace the blood loss, the replacement volume is 3 to 4 times the blood loss.

A. Before induction of anaesthesia administer 20 to 25 ml/kg in children below 3 years of age and 10 to 15 ml/kg in older children
B. Thereafter,
Basic 2 ml/kg/hour
+

For minor surgery 2 ml/kg/hr
For moderate surgery 4ml/kg/hr
For extensive surgery 6 ml/kg/hr
+
Additional replacement of blood

Atropine
0.02 mg/kg
5 ug/kg
Thiopentone
4mg/kg
1
to 2 mg/kg
0.1 mg/kg
Vecuronium
0.1 mg/kg
Atracurium
0.5 mg/kg
Ketamine
2 mg/kg; 5 to 8 mg/kg im
Etomidate
0.3 mg/kg
Fentanyl
1
to 2 ug/kg
Diazepam
0.2 to 0.4 mg/kg
Trimeprazine
2 to 4 mg/kg
5 mg/kg (maximum safe dose)
2
mg/kg (maximum safe dose)

1. Should have adequate facilities to deal with sufficient number of cases
2. Should have adequate space with OT, preoperative check up, preanaesthetic room and rooms
3. Recovery room should be large enough to accommodate all cases to remain for several hours
4. All anaesthetic drugs, equipment, monitors, emergency drugs should be available
5. Should provide adequate number of anaesthetists, nurses and other personnels in both OT. and recovery room. One senior anaesthetist should take the responsibility of the unit
postanaesthetic recovery
. Should have facility for indoor admission in case of adverse situations.

1, A significant reduction of medical cost
. Increased availability of indoor beds AgNRwON Some protection from hospital-related infections . Decreased psychological problems
. Better comfort and greater control over the patient’s personal lives
. Reduced separation induced anxiety particularly in paediatric cases.

B. Related with surgical procedure
i. ASA physical status I and II and some selected cases of grade III
ii. Patient and accompanying person must be able to follow the directions and advice
iii. A capable person must take care and help the patient at home
iv. Extremes of ages should be avoided. Premature babies are at risk
v. Morbid obesity, physical and mental handicaps are difficult subject for day-case surgery.
i. Operations of short duration

ii. Surgery involving less bleeding, minimum postoperative complications and physiological derangement can be accepted
iii. Patients with risk of airway obstruction should be avoided
iv. Infected cases should be avoided
v. Emergency cases are not suitable
vi. Operations anticipating extensive blood loss, patients requiring large fluid therapy, prolonged surgery may be risky for day-case
vii. Surgical procedures anticipating much postoperative pain may be risky.

1. Identical to those for any indoor operation
2. Preanaesthetic visit is essential. Careful history, and examination should always be done
3. Medical and anaesthetic history are also important
4. Necessary laboratory studies should always be done. Routine blood and urine exam, chest X-ray and ECG may also be done. Relevant blood biochemistry (sugar, urea, NPN, creatinine)
is needed for the aged patients
. Patients should receive written special preoperative instructions such as time of last feed, reporting time, arrangements of transportation home after discharge, the need for an accompanying adult person to care for the patient
. Written consent of operation and anaesthesia is mandatory.

1. Nothing by mouth after midnight or at least 8 hours prior to anaesthesia. Clear fluids may be allowed upto 2 hours before surgery
. Hy) receptor antagonists, soluble antacids, metoclopramide may be given in patients at risk . Premedication
i. May not be needed in all patients
ii. Children should have some premedication like diazepam/trimeprazine
iii. Atropine sulphate may be indicated in some cases
. i. Should report at scheduled time and date
ii. Laboratory reports should be verified
iii. Physical condition reassessed
iv. Patient should wear OT dresses
v. No cosmetics, ornaments, shoes, contact lens, etc. are allowed.
Postponement of surgery may be indicated :
1. No written consent
2. No responsible escort available
3. Unusual late reporting in clinic
4 . Full stomach
5 . Change of medical conditions.

Choice of anaesthesia depends on:
OANaATEWNH
. Patient choice
. Surgical requirement
. Period of surgical procedure
. Age of the patient
. Physical condition of the patient
. Duration of drug action
. Requirement of postanaesthetic care
. Judgement of anaesthetist concerned.

1. Local anaesthesia, peripheral nerve block, spinal block or epidural block are mostly helpful
2. Peribulbar or retrobulbar block can be used in ophthalmic surgery
3. Local anaesthesia and mandibular block are widely used in dental surgery
4. Advantages
:
i. Anaesthesia in surgical site
ii. Patient awareness
iii. Less physiological derangements
iv. Condition at the time of discharge is mostly satisfactory.
5. Disadvantages :
i. Needs sometime to do block
ii. Failure/incomplete block can occur
iii. Awareness
iv. Specific hazards for particular block
v. Not always predictable.

1. Essential criteria : Smooth induction, easy maintenance, early uneventful recovery
2. Halothane/enflurane/isoflurane/sevoflurane mostly popular
3.
iv
agents
such
as
propofol,
etomidate,
thiopentone,
methohexitone
can
be
used
as
inducing
agent.
Propofol
can
induce
and
maintain
anaesthesia.
Midazolam,
alfentanil
or
fentanyl
can
also
be
used
with
propofol.
Ketamine
can
be
used
as
sole
agent,
but
it
can
cause
hallucinations,
dreams
and
delirium.
Total
intravenous
is
mostly
popular
for
short
surgical
4. Endotracheal intubation can be done, whenever needed
5. Muscle relaxants can be used for intubation and muscle relaxation. Suxamethonium is mostly used for endotracheal intubation. Vecuronium, atracurium, mivacurium, etc. are also suitable for outpatient anaesthesia. Patient should be fully decurarised in case of nondepolarising muscle relaxants at the end of anaesthesia

6. Advantages
:
i. Immediate speedy effect
ii. Patient comfort
iii. Generalised effect — mostly guranteed.

i. Loss of protective airway reflexes
ii. Risk of vomiting, aspiration
iii. Cardiovascular depression.

1. Local anaesthesia and monitored anaesthesia care with sedation are combined
¢ Local anaesthesia : Local infiltration, nerve blocks
¢ Physical and emotional comfort by sedative drugs
¢ Midazolam, propofol, fentanyl, alfentanil, sufentanil, remifentanil can be used cautiously.

:
i. Maintenance of protective airway reflexes
ii. Rapid recovery
iii. Patient awareness, patient can cooperate.
3. Disadvantages
:
i. Inadequate local anaesthesia can occur
ii. Side effects of sedative drugs.
4. Note:
i. Provide patient comfort
ii. Choose appropriate drugs, minimum effective dose in small increments
iii. Reassurance and psychological support.

1. Patient should be fully awake
2. Monitor the vital signs
3. Some complications can occur such as pain, nausea, vomiting, delayed awakening, restlessness,
hypotension and so on.

1. Persistent vomiting
2. Aspiration pneumonitis
3. Prolonged surgery, surgery extended beyond expectation
4. Poorly controlled coexisting medical diseases
5. Severe blood loss
6. Needs blood transfusion
7. Myocardial ischaemia
8. Airway and respiratory problems
9. Patients refuses discharge
10. Surgeon requires overnight observation.

1. Can maintain patent airway unassisted, no respiratory depression, no threat of airway
obstruction
2. Heart rate, blood pressure near normal. No hypovolaemia
3. Orientation to person, place and time
4. No drowsiness, clear vision
5. Not much pain, no vomiting, no dehydration, absence of surgical bleeding
6. No fever, no hypothermia
7. Able to sit/little walk with minimal assistance
8. Complete recovery, stable vital signs
9. Responsible adult person present to accompany and to take care
10. Recovery criteria at the time of discharge must be recorded.
¢ Instructions at the time of discharge :
i. Regarding activity, medication, care of dressing, diet, bathing restrictions, etc.
ii.
If any complications arise, they should know what to do and where to contact and report
iii. Avoid alcohol or any depressant drug
iv. Should not drive automobile and handle complex equipment.
A person is said to be obese when he exceeds the ideal weight corrected for age and sex by more than 10%. Morbid obesity is said when the body weight is more than 20% above the ideal weight.

1. Excessive neutral fat in the fat storage depots of the body
2. Common in both sexes after middle age
3. High metabolic rate, abnormal appetite
4 . Exogenous type : Superalimentation and with less muscular activity
5 . Endogenous type : Deficiency of one or more endocrine glands: hypothyroidism, hypogonadism, increased activity of adrenal cortex, Cushing syndrome
ND . Can occur after encephalitis and following psychological disturbances
10. General lassitude, dyspnoea on exertion, osteoarthritis, various aches and pains in the body, sleep apnoea
11. Pickwickian syndrome : Obesity hypoventilation syndrome characterised by massive obesity, daytime somnolence and hypoventilation ultimately leading to pulmonary hypertension and right ventricular failure
12. Hypercholesterolaemia and hypertriglyceridaemia.
. Obesity shortens the expected span of life
. Usually prone to hypertension, myocardial disease, diabetes mellitus, adrenal, renal and hepatic dysfunction, cardiomegaly
. Decreased lung functions, restrictive ventilation defect, hypoxaemia and hypercapnia, pulmonary hypertension, emphysema, right heart failure

1. Fatty and heavy, difficult to move and lift
2. Veins may be inaccessible

13. Regional anaesthesia may be difficult to achieve. Landmarks are often ill-defined.
. Coexisting diseases : Hypertension, diabetes, atherosclerosis, cardiomegaly, deranged respiratory functions, endocrine and metabolic disorders, hepatic and renal dysfunction and so on
. Laryngoscopy and endotracheal intubation may be difficult. Difficult to get a clear patent airway, needs IPPV
. Induction of anaesthesia may be hazardous
. Surgery is technically difficult, much muscle relaxants needed
. High serum cholinesterase level in obesity, so the effects of suxamethonium quickly pass off
. Risk of pulmonary aspiration. Raised intraabdominal pressure. Hiatus hernia may be
present
. Tolerate abnormal position badly
. Tolerate blood loss badly
. Postoperative lung complications, thromboembolic complications common
. Preoperative dieting may be helpful. But drastic dieting can cause acidosis, hypokalaemia and hyperuricaemia

1. Preoperative evaluation is essential
2. History of taking drugs, any addiction, any coexisting disease
3. Thorough examination, particularly cardiovascular and respiratory system
4. Laboratory investigations : Routine blood and urine test, ECG, chest X-ray, blood biochemistry
5. Blood transfusion may be needed.

1. Preoperative night sedation
2. Relieve fear, anxiety and tension
3. Atropine is useful to get antisalivary effect
4. Narcotic analgesics should be used cautiously
5. Hp receptors antagonists : Cimetidine, ranitidine as obese patients have increased risk of aspiration.

A. Induction of anaesthesia
i. Inhalation or intravenous agents with caution
ii. Decreased mandibular and cervical spine mobility may pose difficult intubation
iii, Adequate preoxygenation is always helpful
iv. Cuffed endotracheal tube should be used
v. IPPV is essential
vi. Rapid sequence of anaesthesia induction may be used.

i. Volatile anaesthetic agents can be used
ii. Controlled ventilation
iii. CO, absorption technique is the method of choice
iv. Muscle relaxant should be used cautiously and complete decurarisation is mandatory
v. Hypoxia, hypercarbia and hypotension should be avoided
vi. Adequate monitoring of vital signs
vii. Extreme postures should be avoided. Avoid prone and head down position
viii. Fluid therapy should be accurate.
at the end of anaesthesia

i. Hypoventilation, hypoxia, hypercarbia, acidosis and hypotension can occur easily.
iv. Common complications : Pulmonary complications, deep vein thrombosis, wound infection, etc.
Monitor arterial oxygenation. Oxygen therapy
. Narcotic analgesics should be used in small doses
iii. Respiratory physiotherapy and early ambulation are helpful

Arbitrarily old age starts from 65 years of age. There may not be any correlation between biologic and chronologic age. It is accompanied with unavoidable decreases in organ function, increased atrophy, rigidity and fibrosis of body tissues and decreased adaptability and growth.

Myocardial fibrosis, hypertrophy of the heart, myocardial ischaemia, cardiac enlargement, coronary artery disease.
Heart rate and cardiac output decrease, decreased responsiveness to an increase in adrenergic drive. Baroreceptor reflex diminished. Arrhythmias, conduction defect and congestive cardiac failure are common.

Impairment of mechanical ventilatory function and efficiency of gas exchange. Tidal volume and vital capacity diminish. Decreased FEV and forced vital capacity. Maximum breathing capacity decreases.
Emphysema, chronic bronchitis, asthma, chronic obstructive pulmonary disease are common.

Progressive reduction in kidney mass, renal vascular bed and renal blood flow. Diminished tubular function and glomerular filtration rate. Renal excretion of certain drugs hampers. Less able to concentrate urine with fluid deprivation, risk of hyponatraemia. Prostatic hypertrophy is common in old age.

Overall atrophy of brain, cerebral atherosclerosis, thrombosis, hypertensive encephalopathy can occur. Decline in central nervous system activity.
Slow impaired memories, labile emotions, mental psychological and personality changes. Parkinsonism, mental confusion, dementia are common. Disturbances in sleep pattern.

Endocrine dysfunction, diabetes mellitus, hypothyroidism are common.

Metabolism is low, subnormal body temperature, poor physiological response to injury. Nerve
deafness, poor vision, cataract.
Obesity, osteoarthritis, neoplastic diseases, anaemia, hypoproteinaemia, vitamin deficiency are
common.
Hepatic blood flow decreased, functions deranged. Drug clearance is delayed.
Bones rarefied, fractures are common. Old becomes edentulous and the mandible becomes shorter.

1. Coronary artery disease
2. Hypertension
3. Strokes
4. Diabetes mellitus
5. Chronic obstructive pulmonary disease
6. Renal insufficiency / dysfunction
7. Anaemia
8. Parkinson’s disease
9. Osteoarthritis
10. Congestive cardiac failure.

1. Proper history of the illness
2. Any coexisting other ailment
3. Any addiction, any medication
4. Thorough clinical examination
5. Laboratory investigations : Routine blood and urine examination, chest X-ray, ECG, blood biochemistry (glucose, urea, NPN, creatinine, electrolytes)
6. Blood transfusion may be needed
7. Psychological status needs evaluation.

1. Night sedation
. Tranquilliers like promethazine, diazepam WN
. Minimum sedative/analgesic drugs, whenever needed
. Hy receptor antagonists . Preoperative visit is essential DAuF
. Use of anticholinergic drugs is controversial. Avoid hyoscine in elderly patients.

e Anaesthetic requirement may be decreased in old age
e Aspiration of gastric contents is common due to decreased reactivity of protective airway reflexes
¢ Regional anaesthesia may be helpful in selected cases. Dose of local anaesthetics in epidural
block decreases with increasing age and there is risk of high spread due to osteoarthritic blocking
of intervertebral foramina
¢ General anaesthesia
i. Proper mask fit is difficult in edentulous patients
ii. Risk of dislodgement of loose teeth
iii. Cuffed endotracheal tube should always be used
iv. Assisted /controlled ventilation
vi. MAC value of all volatile anaesthetics is reduced in the aged
vii. Halothane, enflurane or isoflurane can be used
viii. Adequate monitoring during anaesthesia is essential
ix. Fluid therapy should be accurate. Estimated blood loss should be replaced
x. Extreme posture should always be avoided
xi. Adequate decurarisation at the end of anaesthesia. 5.

i. Monitor the vital signs
ii. Oxygenation may be needed
iii. Fluid therapy needs attention
iv. Personal hygiene, care of the eyes, skin, bowel and bladder
v. Nutritional care
vi. Early ambulation and physiotherapy recommended.

A series of well-defined steps and protocols to revive a collapsed patient, to deliver oxygen to the heart and brain and to restore native circulation and ventilation.

1. Basic cardiac life support: Resuscitation without the aid of
equipment
2. Advanced cardiac life support: Resuscitation with the help of advanced techniques and equipment.

It should be started immediately and continued till the ACLS support arrives.
It should provide:
a. Adequate patent airway
b. Artificial breathing by expired air respiration
c. Artificial circulation by external chest compression.

Tongue may fall to the back of the pharynx: Tilt the head back and lift the chin forward: Clear the mouth and airway. Airway tube may be used.

In cases of no effective breathing, artificial ventilation should be started by expired air respiration. Patient's mouth should be covered by the mouth of the rescuer while the patient's nose is pinched closed by fingers.
The expired air should be blown to the patient to raise the chest. Then the mouth should be removed to allow the patient exhale. This mouth to mouth respiration should be given at 12-15 breaths per minute.

If there is no heart beat, no pulse in big arteries like carotid, femoral, etc. the artificial circulation
in the form of closed chest cardiac massage should be started.
Chest compressions should be given on lower third of sternum using the overlapping palm of
both hands. Sternum is depressed 4 to 5 cm, about 80 times per minute. The effectiveness of compression should be verified by peripheral pulses.
e After 15 compression, 2 mouth-to-mouth respirations are to be given
e Resuscitation sequence should be continued till ACLS service comes
¢ Incase of 2 rescuers available, the chest compression should be given 60 times per minute and the second rescuer interposes expired air ventilation after every 5th compression.

Sternal compression: 2.5 to 3.87 cm depth
rate: 80 to 100/minute
Sternal compression/ventilation ratio: 5:1
Sternal compression: Depression of sternum by using 3 fingers.

Sternal compression:
1.25 to 2.5 cm depth
rate: at least 100/min.
Sternal compression /Ventilation ratio: 5:1
Sternal compression: Chest is encircled with both hands and depression of mid sternum with
thumbs.

1. Compression of cardiac ventricles
2. Increased intrathoracic pressure produces antegrade blood flow (thoracic pump mechanism)
3. Patient-related factors:
a. Heart size
b. Anteroposterior chest distance
c. Thoracic compliance
d. Compression depth and duration
e. Manual/mechanical compression.

1. No pulse in big vessels
2. Silent precordium
3. No respiration/ gasping
4. Unconscious
5. Widely dilated pupils, unresponsive to light
6. Sudden cessation of bleeding on operation site
7. Electrocardiogram
(a) Asystole: Cardiac standstill, no contraction of myocardium

(b) Ventricular fibrillation: Uncoordinated ineffectual contraction of myocardium
(c) Electromechanical dissociation: QRS complex is present, but no palpable pulse.

Use of Guedel airway
Use of nasopharyngeal airway
Tracheal intubation with a cuffed endotracheal tube.

Use of self inflating resuscitation bag
Use of anaesthetic machine and application of manual ventilation
Use of mechanical ventilators.

iv access
Administration of fluids
Vasopressors: Dopamine
i. Cardiac asystole
Adrenaline 1 mg. iv
Cardiac pacing
Consider overdose.
possible causes: Hypoxia, hyper or hypokalaemia, acidosis, hypothermia, drug

Defibrillation, 200J.
If fails, defibrillation again 200].
If fails, defibrillator with 360].
If fails, adrenaline 1 mg iv and then defibrillation with 360].
If still fails administer lignocaine 100 mg iv
Then repeat defibrillations with 360J.
Other antiarrhythmic drugs may be used ; consider termination of efforts.

a. Adrenaline 1 mg iv
b. Specific treatment should be done when the cause is known such as hypovolaemia, cardiac tamponade, hypothermia.
Pneumothorax or pulmonary embolism, massive acute myocardial infraction.
c. Calcium chloride 10 ml of 10% solution for hyperkalaemia, hypocalcaemia, overdose of calcium antagonists.

50 ml of 8.4% sodium bicarbonate may be helpful provided the effective ventilation has established. Excess sodium bicarbonate may cause alkalosis, hyperosmolality and hypernatraemia and may lead to failure of effective resuscitation.

Postresuscitation complications
1. Cerebral oedema
2. Pulmonary oedema
3. Kidney failure.

1. i. Coma
ii. Exclude hypothermia and depressant drug overdose and metabolic or endocrine
disturbances.
2. i. Absent brainstem activity
ii. Fixed and unreactive pupils.
iii, Apnoea despite normal to raised inspired PaCO 2 above 8kPa.
3. i. Absent cortical activity
ii. Flat electroencephalogram
4. Absent cerebral circulation; cerebral angiographic study.

i. Apnoea
ii. Fixed pupils
iii. Absent corneal reflex
iv. Absent vestibuloocular reflex (Caloric test):
20 ml ice cold water poured in ear drum, no movement of the eye will be detected. Patency of the ear canal should be intact.
v. Absent motor response in the cranial nerve distribution to any stimulation.
vi. Absent
gag reflex
vii.
Oculocardiac reflex: Patient’s head is turned from side-to-side, eye movement is observed.
a. Nonfunctioning brainstem: No movement of the eyes (China doll)
b. Functioning brainstem: Eyes move (swivel-eyed doll).

A systolic blood pressure more than 160 mm Hg and/or a diastolic pressure above 90 mm Hg indicates hypertension.

A. Essential or primary hypertension : Cause not known.
B. Secondary hypertension : Aetiology is known.

i. Renal disease (pyelonephritis, glomerulonephritis, chronic nephritis, renal artery stenosis, polycystic kidney
ii. Coarctation of aorta
iii. Pheochromocytoma
iv. Intracranial hypertension
v. Primary aldosteronism, Cushing syndrome
vi. Drugs : Oral contraceptives
vii. Preeclampsia, eclampsia.

1. Ischaemic heart disease, congestive heart failure, left ventricular failure
2. Renal failure
3. Cardiovascular accident
4. Hypertensive crisis
5. Retinopathy
Degree of hypertension:
¢ Mild hypertension : Diastolic blood pressure of 90 to 104 mm Hg
Moderate hypertension : Diastolic pressure of 105 to 114 mm Hg
Severe hypertension : Diastolic pressure is equal to or more than 115 mm Hg

Malignant hypertension : Presence of papilloedema on fundoscopic examination independent of the absolute level of blood pressure.

Clinical assessment : BP measurement, physical examination on end organ damage
i. Kidneys: Blood urea nitrogen, creatinine, electrolytes
ii. Heart: Cardiomegaly, ischaemia, left ventricular failure
iii. Brain: Dizziness /syncope
iv. Peripheral vascular disease: Claudication.

i. Urine analysis
ii. Haematocrit
iii. Plasma glucose, potassium, creatinine, urea, uric acid
iv.
Vv. ECG
vi. Chest X-ray.
Fasting serum cholesterol, triglycerides, calcium

au . Drug treatment
PWN . Weight reduction . Regular isotonic exercise . Avoid stress and strain
. Moderate sodium restriction
. Avoid alcohol, smoking
i. Diuretics : Thiazides (chlorthiazide, hydrochlorthiazide), Loop diuretics (furosemide, ethacrynic acid), Potassium sparing diuretics (spironolactone triamterene),
ii. Antiadrenergic drugs : Centrally acting clonidine, methyldopa, peripherally acting reserpine, guanithidine
iii. Alpha adrenergic antagonists : Phenoxybenzamine, phentolamine
iv. Beta adrenergic antagonists : Propranolol, practolol, atenolol, metopralol
v. Directly acting vasodilators : Hydralazine, minoxidil
vi. Calcium channel blockers : Nifedipine, verapamil, diltiagem
vii. Angiotensin converting enzyme inhibitors : Captopril, ramipril, enalapril.

1. Diastolic blood pressure is more than 130 mm Hg.
2. Causes : Chronic hypertension, renovascular hypertension, pheochromocytoma, eclampsia,
head injury, spinal cord injury.
Drugs : Cocaine, LSD, tricyclic antidepressants
Sudden stoppage of antihypertensive drugs.

3. Management
iv nitroprusside
Monitor intraarterial blood pressure, urine output.

A. Note:
1. The severity of end organ dysfunction
2. Present antihypertensive medication
3. Preoperative blood pressure. Adequate control of BP
4. Antihypertensive drugs should be continued on the day of surgery, otherwise rebound increase in sympathetic outflow can occur
5. Diuretics can be discontinued as they decrease intravascular volume and the patient may suffer hypotension due to blood loss, fluid restriction and vasodilation caused by volatile anaesthetics
6. Maintain BP and pulse rate within a range of 20% of baseline, otherwise ischaemia can occur in vital organs
7. Use of vasopressors and iv infusion of fluid should be cautious
8. Associated organ dysfunction should be noted
:
a. Orthostatic hypotension
b. Ischaemic cardiac dysfunction
c. Cerebrovascular disease
d. Peripheral vascular disease
e. Renal dysfunction.

1. Exaggerated BP changes can occur during laryngoscopy and endotracheal intubation
2. Surgical anaesthesia may be done with volatile anaesthetics
3. Short duration of laryngoscopy is needed
4. Laryngeal spray of lignocaine, iv lignocaine, esmolol, nitroprusside may be helpful.
C. Maintenance of anaesthesia:
1. Volatile anaesthetics are mostly satisfactory.
2. Intraoperative monitoring is essential.
D. Postoperative management:
1. Hypertension can occur
2. Monitoring should be continued.

1. Causes
:
i. Heart valve abnormalities / malfunction
ii. Impaired myocardial contractility due to ischaemic heart disease or cardiomyopathy
iii. Systemic hypertension
iv. Pulmonary hypertension

2. Features of left ventricular failure:
i. Dyspnoea, orthopnoea, paroxysmal nocturnal dyspnoea
ii. Acute pulmonary oedema
iii. Tachycardia, tachypnoea
iv. Pleural effusion
v. Oliguria.
3. Features of right heart failure:
i. Systemic venous congestion, raised CVP
ii. Enlarged liver
iii. Peripheral oedema
iv. Ascitis.
4. Management of congestive cardiac failure:
i. Digitalis
ii. Diuretics
iii. Vasodilators
iv. Beta agonists : IV infusion of dopamine and/or dobutamine.
5. Anaesthetic considerations
:
A. General anaesthesia:
i. Preoperative treatment of infection, if any
ii. Maintain a clear patent airway. IPPV
iii. Ketamine can be used for induction of anaesthesia
iv. Opioids can be used
v. Monitoring of vital signs, invasive monitoring of blood pressure
vi. Dopamine may be needed to support cardiac output
viii. Drug interaction in digitalised patients should be borne in mind
B. Regional anaesthesia : May be well-considered in cases of peripheral operation.

1. A manifestation of rheumtic heart disease affecting the mitral valve
2. Clinical features : Atrial fibrillation, arterial embolism, pulmonary hypertension, pulmonary oedema, right heart failure.
3. Problems :
i. Usually receiving digoxin, diuretics and anticoagulants. Interaction of drugs can occur
ii. Control atrial fibrillation
iii. Treat pulmonary oedema
iv. Management of anticoagulant therapy needed.
4. Anaesthesia
i. Control heart rate. Tachycardia and bradycardia should be avoided
ii. Avoid hypoxia, hypercarbia and hypotension
iii. Drugs causing vasodilation should be avoided

iv. Avoid/treat acidosis
v. Opioid analgesics should be given carefully
vi. Avoid airway obstruction
vii. Avoid increase in central blood volume. Overinfusion should be avoided.

1. A previous myocardial infarction, within 3 months increases the risk of reinfarction. Elective surgery should better be postponed at least 6 months after infarction
2. There may be symptomless ischaemic heart disease
3. Unstable angina should always be controlled before surgery with beta blockers, nitrates or calcium antagonist
4. Various factors may precipitate further infarction. Avoid increase of myocardial work and hence oxygen requirement. Avoid decrease of coronary blood flow.
5. Anaesthetic implications :
i. Treat the congestive cardiac failure with diuretics and digoxin, if necessary
ii. Anaemia should be corrected preoperatively
iii. Premedication should be adequate. Anxiolytic drugs are often helpful
iv. Avoid tachycardia/bradycardia
v. Avoid hypotension/hypertension
vi. Avoid hypoxaemia
vii. Beta blockers is maintained
viii. Maintain normocapnia. Hypercapnia may initiate arrhythmia and hypocapnia can cause
peripheral and coronary vasoconstriction and leftward shift of oxygen dissociation curve
ix. Avoid fluid overload. Monitor fluid therapy meticulously
x. Close monitoring is vital
xi.
Choice of anaesthetic agent should be carefully judged
xii. Postoperative care
:
Adequate monitoring
Management of analgesia
Control the blood pressure precisely.

¢
Laryngoscopy and endotracheal intubation should be of short duration. Blood pressure and/
or heart rate response to endotracheal intubation should be minimised by:
i. Laryngotracheal lignocaine
ii. iv lignocaine
iii. Nitroprusside iv
iv. Esmolol iv
¢
Volatile anaesthetics with N2O are mostly satisfactory
¢ Muscle relaxants : Vecuronium, doxacurium, pipecuronium are with minimum circulatory effects. Pancuronium increases heart rate and blood pressure.

1. The disease is characterised by recurrent, reversible, generalised airways obstruction mostly due to bronchial smooth muscle spasm, mucons plugs and local oedema
2. Increased responsiveness of the airways in various stimuli
3. Chronic inflammatory changes in bronchial submucosa
4. Pathophysiology :

i. Release of chemical mediators like histamine, prostaglandins, etc.
ii. May have abnormalities of autonomic nervous system to regulate airway tone in a balanced way
iii. Chronic inflammation/infection of the airway.
Classification :
A. Extrinsic : External allergen detectable
B. Intrinsic : Occurs in adults, more chronic and continuous, requires long-term steroid therapy.

i. Inhaled/ingested allergens /irritants
ii. Airway infection
iii. Strenuous exercise.

i. Wheezing
ii. Cough
iii. Dyspnoea
iv. Tachypnoea
v. FEV; and maximum midexpiratory flow rate depends on severity of the disease.
vi. PaO» decreases
vii. PaCQOy increases.
i. Bronchodilator drugs : Theophylline, B2 agonist like albuterol by metered dose inhaler
ii. Antiinflammatory drugs : Steroids, Cromoglycate by metered dose inhaler
iii. Antibiotics
iv. Oxygen.

a. Current state should be assessed by proper history, examination and function tests
pulmonary
b. Blood gas study may be helpful in severe cases
c. Chest physiotherapy, systemic hydration and suitable antibiotics and bronchodilator drugs are much useful to improve the condition.
ii. Asthma must be controlled before elective surgery
iii. Patients with steroid therapy should have adequate steroid coverage

iv. Premedication: A sedative (diazepam) with atropine may be given. Pethidine and promethazine are mostly satisfactory. Opioids, H2 receptor antagonist are better avoided. Bronchodilator should be continued
drugs
v. a. Induction of anaesthesia should be smooth
b. Cautious laryngoscopy and endotracheal intubation
c. Volatile anaesthetics like ether, halothane are better tolerated. Ketamine is bronchodilator
d. Bronchospastic drugs should to be avoided
e. Beta blockers, tubocurarine, morphine should be avoided. Extubation should be done when the patient is still unconscious.
vi. Regional anaesthesia is a better choice and mostly satisfactory
vii. Muscle relaxants those do not cause histamine release should be used
viii. Mechanical ventilation of the lungs, a prolonged expiratory phase may be needed. Inspiratory time should be adequate. Humidification may be needed, if IPPV is prolonged.

1. Airway obstruction, secretions, kinking of tube, overinflation of the cuff
2. Inadequate depth of anaesthesia
3. Endobronchial intubation
4. Aspiration of gastric contents
5. Acute asthmatic attack.
B. Differential diagnosis :
1. Pneumothorax
3. Pulmonary embolism.

1. Patent clear airway should be guaranteed
2. Oxygenation
3. Deepen anaesthesia with halothane
4. i. Aminophylline iv
ii. Salbutamol
5. iv hydrocortisone although it has no immediate effect.

1. Limitation of airflow secondary to either airway disease (chronic bronchitis) or destruction
of lung parenchyma (emphysema) or both
2. Two groups:
i. Bronchitis group (blue bloaters) : Clinical features include hypoxia, hypercapnia and right ventricular failure
ii. Emphysematous group (pink puffers) : These patients are usually severely dyspnoeic.

3. Preoperative management:
i. Active infection, if any should be treated with broad spectrum antibiotics. Chest
physiotherapy, humidification of inspired gases
ii. Treat airway obstruction : Bronchodilator therapy
ii. Chest X-ray oe:
iv. Treat congestive cardiac failure : Digoxin, diuretic therapy. me
v. Weight reduction in cases of obesity.

i. Avoid opioids
ii. Atropine sulphate to reduce secretions
iii. Diazepam.
5. Regional anaesthesia may be advantageous as it offers freedom from respiratory ill effects and
avoids
complications
of
general
6. General anaesthesia:
i. Minimum sedation, avoid opioids. Maintain spontaneous/assisted ventilation as far as practicable
ii. Controlled oxygen therapy
iii. a. Incases of major surgery, cautious endotracheal intubation. IPPV. Muscle relaxants
and anaesthetic should be used
agents carefully
b. Optimal ventilation. Avoid hyperventilation
c. High inflation pressure increases the risk of pneumothorax.
iv.
Judicious administration of fluids. Overhydration should be avoided.
v. Postoperative care :
. Elective postoperative controlled ventilation
. Adequate oxygenation. Controlled oxygen therapy
. Analgesia without respiratory depression
. Tracheobronchial toilet
Avoid fluid overload
Chest physiotherapy
. Antibiotics moog
Adequate monitoring in postoperative period is vital.
means

A. Insulin dependent diabetes mellitus:
i.
Usually starts below the age of 16 years, abrupt onset, manifested with polyphagia,
polydipsia and polyuria
ii. They need insulin exogenously. Prone to ketoacidosis
iii. Wide variations of blood glucose concentration. =

B. Noninsulin dependent diabetes mellitus. Usually starts above the age of 35 years, gradual onset, may be asymptomatic. May not need exogenous insulin. Boold glucose concentration relatively stable. Ketoacidosis may not always occur.

1. Ketoacidosis
2. Hypoglycaemia
3. Atherosclerosis
4. Nephropathy
5. Autonomic neuropathy : Impotence, orthostatic hypotension, cardiac dysrhythmia,
gastroparesis
. Sensory neuropathy : Carpal tunnel syndrome ND
. Stiff joint syndrome.

1. Diet
2. Oral hypoglycaemic agents : Tolbutamide, acetohexamide, tolazamide, glyburide
chlorpropamide,
3. Exogenous insulin : Regular insulin, semilente isophane, protamine zinc, ultralente.

1. Preoperative assessment:
i. Previously undiagnosed or controlled by diet/oral drugs/ insulin.
ii. Any evidence of complications :
angina, myocardial infarction, hypertension, postural hypotension, anaemia, neurological impairment, pain, numbness, paraesthesia, leg ulcer, skin infection, etc.
iii. Concurrent drug therapy, if any.
iv. Investigations :
Blood count, blood glucose level, urine analysis, ECG, X-ray chest.
v. There should be no glucose in urine. Blood pressure and heart rate should be near normal.
vi. Avoid hypoglycaemia, prevent hyperglycaemia, ketoacidosis and electrolyte disturbances. Exogenous insulin and glucose should be provided.
2. Diabetes must be controlled. Switch on short acting soluble insulin usually on twice daily
basis at least for 3 days prior to surgery.
3. On the day of operation:
i. No subcutaneous insulin
ii. 10% glucose 500 ml with soluble insulin 10 units and 10 mmol of potassium chloride should be started in the morning and subsequent insulin therapy should be adjusted depending on blood sugar level. Potassium should be given according to plasma potassium level.

4. Postoperative:
Infusion of glucose and insulin should be continued until oral intake is established.
Subcutaneous insulin can be then be given.
5. Anaesthesia:
Choice of drugs for induction and maintenance of anaesthesia should be carefully judged. Controlled ventilation with NO and O} is always helpful. Muscle relaxants can be used. Avoid hypoxia, hypercarbia and hypotension. Monitoring of blood glucose at regular intervals is
mandatory.
Regional anaesthesia may have some definite advantages in diabetic patients.

1. Basic pinciples:
i. Control of blood sugar by soluble insulin
ii. Correction of dehydration
iii. Electrolyte management.
2. Investigations :
Blood glucose, electrolytes, urea
Hb%, blood gas study
ECG
Chest X-ray
Urine analysis.
i. iv infusion of glucose saline with CVP monitoring
ii. Soluble insulin 20 units iv start and then iv infusion at 10 units/hour. Low doses of insulin 6 to 8 units/hr by continuous infusion is mostly satisfactory and reliable
iii. Potassium supplementation
iv. Bicarbonate in severe acidosis in doses of 1 mmol/kg
v. Broad spectrum antibiotics
vi.
Suction through nasogastric tube is helpful to prevent aspiration.
¢ Once blood sugar starts falling, it indicates that insulin is acting successfully, then and only then surgery can be started.

a. N2O, O2 and relaxant technique with IPPV is safe
b. Crash induction with cricoid pressure
c. Frequent blood gas studies
d. Reversal of relaxants may be impaired in presence of acidosis
e. Ventilatory assistance may be needed in postoperative period.
viii.
Sliding scale insulin : When acute episode subsides, insulin can be given on a sliding scale based on sugar percentage on urine test. Soluble insulin should be given afterwards when the patient is settled and is eating normal food. The dose should be based on blood sugar.

¢ Monitoring blood glucose every hour is recommended during operation. Blood glucose
less than 100 mg% indicates additional glucose and more than 250 mg% indicates
additional exogenous insulin.

1. Total plasma thyroxine (T4) level
2. Resin triiodothyronine uptake R T3 U
3. Total plasma triiodothyronine (T3) level
4. Thyroid stimulating hormone TSH
5. Thyroid scan
6. Ultrasonography
7. Antibodies to thyroid gland components.

Hyperthyroidism :
Ts, RT3U and T3 increased
TSH normal
Primary hypothyroidism
:
Tu, RT3U, T3 decreased
TSH increased
Secondary hypothyroidism
Ts, RT3U, T3 and TSH decreased.

Manifestations: Increased BMR.
Enlarged thyroid, increased pulse rate, nervousness, anxiety, tremor, fatigue, increased appetite, loss, muscle weakness, exophthalmos, heat intolerance, sweating, atrial fibrillation.
weight

1. Assess the condition carefully
2. Antithyroid drugs : Propylthiouracil, methimazole, Beta blockers (propranolol) relieves of excessive nervous
symptoms sympathetic system activity
3. Surgery : Subtotal thyroidectomy
4. Radioactive iodine.

1. Patient must be made euthyroid before elective surgery
2. Avoid tachycardia. Avoid anticholinergic drugs
3. Prepare the patient for surgery wih beta antagonist

oO . Induction of anaesthesia should be smooth
Oo . Regional anaesthesia may be considered in selected cases
OND . Vital signs should be monitored
. Upper airway should be evaluated, chance of difficult intubation
Thiopentone, suxamethonium or nondepolarising muscle relaxants that lacks cardiovascular
effects can be used.
. Anaesthesia can be maintained with N,O, isoflurane
. Avoid hypoxia, hypercarbia and hypotension
. Following subtotal thyroidectomy
i. Airway obstruction can occur due to haematoma, tracheomalacia
ii. Damage of recurrent laryngeal nerve can occur. Causes hoarseness, aspiration, etc.
iii. Hypoparathyroidism may occur due to inadvertent removal of parathyroids
iv. Laryngospasm can occur due to hypocalcaemia.

1. Abrupt exacerbation in intensity of features of thyrotoxicosis
2. Precipitated by surgery infection, ketosis, burns, trauma, etc. particularly in poorly controlled thyrotoxicosis, following radioactive iodine therapy or excessive administration of exogenous hormone.
thyroid
. Manifestations
:
High fever, tachycardia, atrial fibrillation, heart failure, sweating, dehydration, electrolyte
imbalance, confusion, delirium, agitation, stupor, coma.
. Prevention :
i. Surgery should be done is euthyroid state
ii. Hyperthyroidism should be treated adequately
iii. Beta blocker should be given in perioperative period.

1. Restoration and maintenance of vital functions :
Fluids, electrolytes, iv infusion of glucose, oxygen.
. Control of high fever : Tepid sponging, cooling fans, cooling blanket, ice cold saline.
. Antithyroid drugs : Sodium iodide, carbimazole
. Hydrocortisone
. Correction of sympathetic overactivity : Propranolol, esmolol
. Digitalis, inotropes, diuretics, IPPV to manage heart failure
. Treat the precipitating factor, if any
. Monitoring the vital signs is essential.
ANaAAFEWN

1. Causes : Hyposecretion of the hormone due to idiopathic atrophy, medical treatment of hyperthyroidism or surgical removal of gland.

i. Bradycardia, decreased cardiac output, peripheral vasoconstriction, orthostatic
hypotension, arrhythmias, pericardial effusion
ii. Ventilatory failure, pleural effusion
iii. Intolerance to cold
iv. Lethargy, myopathy, anaemia, peripheral oedema
v. Atrophy of adrenal cortex
vi. Hyponatraemia
vii. Decreased BMR.
3. Treatment :
Oral
administration
of
thyroxine
4. Management of anaesthesia
i. Needs supplemental cortisol administration
ii. IV ketamine may be used for induction of anaesthesia
iii, Maintenance of anaesthesia:
N2O with supplementation of short acting opioid, benzodiazepine or ketamine
iv. Extubation should be done when the patient is fully awake
v. Regional anaesthesia is mostly satisfactory provided intravascular fluid volume is maintained.
vi. Note:
These patients have increased sensitivity to depressant drugs
Cardiovascular system is hypodynamic
Slow metabolism of drugs ao
. Baroreceptor reflexes are unresponsive
Associated with hypervolaemia, hyponatraemia, hypothermia, anaemia, e
hypoglycaemia, adrenal insufficiency
Impaired ventilatory response to hypoxaemia or hypercarbia. ph

1. Catecholamine secreting tumour may arise in adrenal medulla or any sympathetic ganglion.
2. Manifestations :
Palpitation, paroxysmal hypertension, diaphoresis, tachycardia, headache, fits, blurred vision, increased BMR and OQ) consumption, haemoconcentration, retinopathy, increased blood concentration of glucose, lactic acid and free fatty acid.
3. Diagnosis confirmed by excessive catecholamine level in blood. Computed tomography
may also be helpful to localise the tumour. Urinary excretion of catecholamines or of their metabolites is also increased.
i. Congestive cardiac failure
ii. Myocardial infarction
iii. Intracerebral haemorrhage.

5. Treatment:
i. Surgical removal of the tumour
ii. Medical control by alpha blockers and beta blockers.
6. Management of anaesthesia:
i. Do not stimulate the sympathetic nervous system
ii. Use invasive monitoring
iii. Alpha and beta antagonist therapy should be continued
iv. Supplemental cortisol may be needed
v. Smooth iv induction of anaesthesia, maintenance may be done with N2O and isoflurane.
vi. Endotracheal intubation should be gentle and smooth. Circulatory responses should be minimised
vii. Nondepolarising muscle relaxant devoid of circulatory effects may be used
viii. Nitroprusside, esmolol, lignocaine may be needed, whenever necessary
ix. Monitoring of arterial blood gases, electrolytes and blood glucose is essential. Pulmonary
artery catheter is often helpful
x. Monitoring should be continued in postoperative period
xi. Adequate pain relief is needed in postoperative period.

1. Bilirubin level of serum (0.3-1.1 mg%)
i. Conjugated or direct bilirubin
ii. Unconjugated or indirect bilirubin.
2. Enzyme tests
i. Alkaline phosphatase 3-13 KA units/100 ml
ii. Transaminases
SGOT (aspertate aminotransferase) 5-30 IU/litre
SGPT (alanine aminotransferase) 2-53 IU/litre
iii. Lactic dehydrogenase 100-300 IU/litre
3. Plasma proteins 6-8g% albumin 3.5-5¢% ; globulin 1.5-3 g%
. Prothrombin time 14 sec »
5. Radiology
i. Oral cholecystogram
ii. Intravenous cholecystogram
iii. Percutaneous cholecystogram.
6. Radioactive scans
7. Liver biopsy.

1. Acid: base and fluid balance fluid overload is common. Hypoalbuminaemia causes oedema, ascitis. Secondary hyperaldosteronism causes sodium retention and hypokalaemia. In hepatic failure combined respiratory and metabolic alkalosis can occur.

2. Hepatorenal syndrome: Jaundiced patients may develop renal failure.
3. Bleeding problems
i. Factors I, VI, IX and X reduced
ii. Factor V and fibrinogen reduced
iii. Thrombocytopenia.
4. Drug metabolism:
i. Slow elimination of many drugs such as anaesthetic drugs, opioids, benzodiazepines, suxamethonium, local anaesthetics, etc.
ii. Some drugs have toxic effects on liver
iii. Halothane can cause hepatitis.
5. Hepatic failure:
i. Avoid all sedative drugs, opioids, benzodiazepines
ii. Need intensive management including close metabolic, fluid and electrolyte monitoring
iii. Avoid hypoglycaemia.

A. Preoperative preparation
i. Hypovolaemia and electrolyte imbalance should be corrected
ii. Vitamin K, coagulation factors, fibrinogen, platelets, fresh blood transfusion may be needed
iii. Paracentesis in cases of huge ascitis
iv. Broad spectrum antibiotics
v. Anaemia should be treated properly
vi. Monitoring of intracranial pressure is needed
vii. Control cerebral oedema.

Most of sedative and hypnotic drugs should be given in reduced doses. Prolonged effect
is common.
C. Monitoring of vital signs, urine output, CVP, pulmonary arterial pressure is essential.
Frequent determination of glucose, electrolytes, blood gases, acid-base status, coagulation status,
haematocrit are needed.
D. Regional anaesthesia:
i. Least physiologic disturbances
ii. Severe coagulopathy may limit the choice of the block
iii: Hypotension and toxic reactions can occur.
E. General anaesthesia:
i. Endotracheal intubation ensures the tidal volume and F102, reduces the risk of aspiration
ii. Rapid sequence induction and intubation is helpful
iii Avoid hypoxia, hypocarbia, hypercarbia and hypotension
iv. N2O can cause bowel distension
v. Isoflurane/desflurane in oxygen can be used with small doses of narcotics. Controlled ventilation to a normal PaCO) is important.

F. Intravenous anaesthetics should be given in small increments. Ketamine may reduce the risk of hypotension.
G. Muscle relaxants:
Pseudocholinesterase level is decreased, the effect of suxamethonium may be prolonged. Most nondepolarising muscle relaxants can be used with due caution. Pancuronium is mostly for its cardiovascular
satisfactory stability.
. i. Fluid management should always be
judicious. adequately
ii. Urine output, cardiac output, CVP and pulmonary arterial pressure should be maintained.

1. Inability of the damaged liver to detoxicate nitrogenous substances derived from gastrointestinal breakdown
2. Precipitating factors : Azotaemia, misuse of opioid, sedative, hypnotic drugs, gastro-intestinal bleeding, constipation, high protein diet, etc.
3. Grading:
Grading of hepatic encephalopathy.

EEG
Grade 1
Confusion, euphoria
Normal
Grade 2
Mood change, altered behaviour
Generalised slowing
Grade 3
Stupor, rousable
Delta activity
Grade 4
Comatose, responds to pain
Triphasic waves
Grade 5
Deep coma
Flat
4. Other factors responsible for clinical manifestations :
2. Acid-base balance disorders
3. Depletion of neurochemical transmitters
4. Increased level of short chain fatty acids.

:
i. Avoidance and correction of predisposing factors
ii. Protein restriction
iii, Mag. sulph enema once or twice daily
iv. Antibiotics : Poorly absorbed neomycin is the antibiotic of choice v. Lactulose: Synthetic disaccharide is not hydrolysed or absorbed in intestine but metabolised to lactic acid and acetic acid in colon. It increases foecal acidity and inhibits bacterial urease.
vi. Exchange blood transfusion
vii.
Steroids
viii. Charcoal haemoperfusion
ix.
Haemodialysis
x. Liver transplant.

Modified child’s grouping of the severity of liver disease
Prognostic Scoring
Score
1
Score
2
Score
3
Gr
1
and
2
Gr
3,
4,5
Bilirubin mol/lit
<25
25-40
>
40
Albumin g/lit
35
28-35
<
28
Prothrombin time (sec prolonged)
1-4
4-6
>6
Ascitis
absent
slight
moderate
v Score less than 6 denotes good risk, 7, 8 or 9 denotes moderate risk and more than 10 is poor operative risk

1. A seizure disorder resulting from the excessive discharge of an aggregate of neurones that depolarise in a synchronous fashion either localised or spread.
. Probable causes include birth injury, hypoglycaemia, hypocalcaemia, high fever, head injury / tumour, cerebrovascular disease, drug withdrawal, etc.
. Treatment of chronic epilepsy
Anticonvulsant drugs: Valproic acid, phenytoin, carbamazepine, diazepam, etc.
ii. Avoid drugs which may have cerebral excitatory effects:
iii.
iv.
. Anaesthesia in patients with chronic epilepsy
i. Maintain anticonvulsant drugs throughout the perioperative period
a) enflurane
b) methohexitone
c) atracurium.
Local anaesthetics may cause convulsion
Phenobarbitone
and
phenytoin
induce
hepatic
enzymes
and
enhance
the
of drugs metabolised in liver
. Some drugs are mostly safe and may not cause seizure activity, are:
a. Thiopentone
b. Benzodiazepine
c. Opioids
d. Halothane /isoflurane/desflurane.

1. Jaundice is due to posthepatic biliary obstruction resulting in failure to excrete bile and leading
to hyperbilirubinaemia. Commonly occurs with gallstones in common bile duct.
2. Investigations needed : Full blood count, blood bio-chemistry, urine analysis, coagulation studies, liver function tests. X-ray of chest and abdomen, cholangiogram, ultrasound and CAT may be needed in some cases.

3. Preoperative assessment:
i. Preserve hepatic function by maintaining hepatic blood flow and adequate oxygenation. ii. Risk of renal failure is high in cases of hyperbilirubinaemia. Hypovolaemia can initiate renal failure. Preoperative fasting should be kept minimum. iv fluid (glucose saline) should be started at least 4 hours before surgery. Urine volume should be at least 1 ml/ kg/hour
iii. Vit K should be given parenterally at least 24 hours before surgery
iv. Suitable antibiotic may be needed to treat endotoxaemia
v. Bradycardia is common in jaundiced patients. Atropine should be given in premedi- cation
vi. Pethidine can be given as it constricts sphincter of Oddi less.
4. Anaesthesia:
. Maintain hepatic and renal blood flow . Normocarbia should be maintained Boe:
. Patient should be well-hydrated a
. Monitoring of vital signs, CVP, ECG and urine volume 4
. Induction and maintenance of anaesthesia: <8
Thiopentone in usual doses
Pancuronium maintains cardiovascular stability <<
Atracurium : Elimination is independent of liver and kidney function, lacks major
cardiovascular
effects
Suxamethonium can cause bradycardia Ventilation should be controlled and normocapnia maintained Adequate oxygenation SANK
At the end of anaesthesia, neuromuscular block should be well-reversed.
vii. Postoperative care: Oxygenation, narcotic analgesics in low doses.

1. Itisa chronic autoimmune disease characterised by episodes of increased muscle fatiguability
caused by decreased numbers of acetylcholine receptors at the neuromuscular junction.
2. Clinical features:
i. Muscle weakness, extraocular, bulbar and facial
ii. Ptosis and diplopia
iii. Cardiomyopathy
iv. Hypothyroidism.

i. Anticholinesterase drugs (neostigmine or pyridostigmine with vagolytic agent)
ii. Steroids
iii. Thymectomy.

4. Anaesthetic management:
A. Preoperative preparation:
i. Maintain adequate respiration
ii. Antibiotics to combat infection
iii. Atropine to diminish secretion
iv. Avoid opioids
v. Chest physiotherapy
vi. Serum potassium should be kept normal.
B. Local and regional anaesthesia—mostly satisfactory.
Doses of local anaesthetics should be kept minimum.
C. General anaesthesia:
Minimum possible inducing agent.
Relaxant is better avoided. For major surgery relaxants may be given in low doses.
IPPV following endotracheal intubation.
Maintenance of anaesthesia: Volatile agents, short or intermediate acting muscle relaxants.
At the end of surgery, adequate decnurarisation is mandatory.
D. Postoperative care :
i. Monitoring of vital signs
ii. Lungs should be ventilated for 24 to 48 hours in postoperative period.
iii. Good chest physiotherapy
iv. Steroid cover
v. Anticholinesterase drugs should be continued.

1. Usually associated with carcinoma, thyrotoxicosis, Cushing syndrome, hypokalaemia,
hypocalcaemia.
. Clinical features
:
i. Proximal limb weakness, exercise improves strength
ii. Muscle pain, reflexes absent or diminished.
. Sensitive to muscle relaxants
. Poor response to anticholinesterase drugs.

1. It is also known as consumption coagulopathy often characterised by uncontrolled activation of the coagulation system with consumption of platelets and procoagulants.
2. Causes :
Eclampsia, placental abruption, retained placenta, intrauterine foetal death, amniotic fluid embolism, malignancy, burns, malaria, infection, crush injury, haemorrhagic shock, intracranial haemorrhage, incompatible blood transfusion, malignant hyperthermia, snake bite, fatembolism, pulmonary embolism, severe shock, etc.

3. Laboratory findings :
i. Reduction of fibrinogen
ii. Elevation of fibrinogen degradation products
iii. Thrombocytopenia
iv. Red cell distortion/fragmentation
v. Prothrombin time, partial thromboplastin time and thrombin time prolonged.
vi. Serum creatine phosphokinase increased ; serum lactate level increased.
4. Clinical features :
a. Haemorrhagic manifestations in different part of the body. Hypotension, shock, profound
acidosis, hypoxia, circulatory failure, respiratory failure, shock lung syndrome.
b. Thrombotic manifestations
:
i. Poor organ perfusion, oliguria, cyanosis
ii. Circulatory failure, coma.
i. Treatment of underlying cause
a. Correct hypoxia and acidosis
b. Antibiotics to treat infection
c. Drainage in case of abscess
d. Adequate blood volume expansion in hypovolaemic shock
e. Emptying of uterus in cases of retained placenta, intrauterine foetal death.
ii. Fresh frozen plasma
a. Cryoprecipitate
b. Platelets
c. Heparin
iii. Steroids
iv. Antifibrinolytic therapy: Epsilon aminocaproic acid.

1. The red cell mass is reduced below normal. A fall in the quantity of red cells or haemoglobin in a unit volume of blood. Haemoglobin concentration 10 gm/100 ml or less is regarded as anaemia
2. Iron deficiency anaemia is most common
3. Causes
:
i. Blood loss
ii. Failure of erythropoiesis : Inadequate supplies of nutrients like iron, vitamin By, folate,
protein and certain hormones to bone marrow
iii. Bone marrow infiltration in leukaemia
iv. During pregnancy due to increased demand of iron
v. Ankylostomiasis.
4. Manifestations
: Lassitude, oedema,
tiredness, anorexia, indigestion, palpitation, dyspnoea, pallor, swelling of legs, soft systolic murmur, tachycardia, etc.

5. Haematological findings :
Moderate fall in red cell count ; greater reduction of Hb ; MCV, MCH and MCHC reduced. Red
cells are microcytic, hypochromic, anisocytosis.
6. Treatment :
i. Causative factors should be diagnosed and treated
ii. Balanced diet, proteins, iron, vitamins, folic acid
iii. Digestive enzymes
iv. Specific therapy : Iron therapy oral or parenteral
v. Blood transfusion
a. increases oxygen carrying capacity of blood
b. increases and improves coagulation and immunity
c. improves circulatory status.

i. Minimum acceptabe Hb concentration is 10 gm%
ii. Transfusion of RBC can increase oxygen carrying capacity
iii. Indication of blood transfusion preoperatively depends on various factors such as duration of anaemia, cause of anaemia, intravascular fluid volume, urgency of operation, blood loos during surgery and coexiting other diseases
iv. Avoid hypotension and decrease in cardiac output
vii. Operative blood loss should be replaced promptly
Viii. Postoperative shivering or rise of body temperature can increase tissue oxygen requirement. Preventive measures should be taken.
Avoid leftward shift of oxyhaemoglobin dissociation curve that can occur in respiratory alkalosis, PaO, less than 100 mm Hg
Anaesthetic drugs should be used cautiously. Overdose should be avoided

Acid phosphatase 1-5 KA units/100 ml
Alkali reserve 24-35 mEq/L
Alkaline phosphatase 3-13 KA units/100 ml
Bicarbonate 22-28 mEq/L
Bilirubin 0.1-1 mg/100 ml
Calcium 9-11 mg/100 ml
Chloride 95-110 mg/100 ml
Cholesterol 145-280 mg/100 ml
Cortisol 9 AM : 6-26 ug/100 ml
midnight : 6-8 1g/100 ml
Creatine phosphokinase (CPK) 0-35 IU/L
Creatinine 1-2 mg/100 mg
Glucose fasting : 70-120 mg/100 mg
after meal: up to 180 mg/100 ml
I'S! uptake 11-33% at 4h
Magnesium 1.5 to 3 mEq/L
Nonprotein nitrogen 20-40 mg/100 ml
P,CO, 35-45 mm Hg
P,O2 90-100 mm Hg
pH 7.3-7.5
Phosphate 3-4.5 mg/100 ml
Potassium 3-5 mEq/L
Protein bound iodine 4-8 ug/100 ml

Albumin 4-5.5 g/100 ml
Globulin 1.5-3 g/100 ml
Fibrinogen 0.2-0.5 g/100 ml
Total 6-8 g/100 ml
Pseudocholinesterase 40-100 units/100 ml
Sodium 135-150 mEq/L
Thyroxine 5.5-12.5 ug/100 ml
T3 resin uptake 88-110%
Thyroid stimulating hormone (TSH) 0.8-3.6mU/L

SGOT : 0-25 units/L
SGPT : 0-20 units/L
Triglyceride (fasting) 60-100 mg/100 ml
Urea 15-40 mg/100 ml
Uric acid 2-6 mg/100 ml

males 13.5-18 g/100 ml
females 11.5-16.5 g/100 ml
at birth 17-20 g/100 ml
Red blood cell count
males 4.5-6.5 million/mm?
females 3.9-5.6 million/mm?
Bleeding time 2-5 min

Coagulation time
Wright (capillary) 10-15 min
Lee and White (venous) 5-7 min
Colour index 0.85-1.15
Erythrocyte Sedimentation Rate (ESR)
2-12 mm inih
Packed cell volume (PCV)
males 40-54%
females 35-47%
Mean Corpuscular Diameter (MCD) 6.7-7.7
w
Mean Corpuscular Haemoglobin (MCH) 27-32 ng
(pg)
Mean Corpuscular Haemoglobin Concentration
(MCHC) 32-36 g%
Mean Corpuscular Volume (MCV) 80-92 113
Reticulocyte count 0.2-2%
White blood count
Total 4000-11000/mm?
Neutrophils 2500-7500/mm$ (65-70%)
Lymphocytes 1500-3500/mm* (20-25%)
Eosinophils 40-400/mm/? (0-4%)
Basophils 0-100/mm? (0-1%)
Monocytes 200-800.mm3 (5-6%)
Platelet count : 250,000-500,000/mm?
Prothrombin time : 14 sec

Amount 120-150 ml
Cells 0-10/mm3
Chlorides 700-750 mg/100 ml
Pressure 70-180 mm H2,O
Protein 10-45 mg/100 ml
Sugar 40-70 mg/100 ml

Basal Metabolic Rate (BMR) + 14 to- 14
Urine
Specific gravity 1010-1025
Amount 1500-2000 ml/24 h
Catecholamines
Adrenaline in 24 h save
Noradrenaline in 24 h save
119-338
ug
3 methoxy, 4 hydroxy mandelic acid (VMA)
in 24 h save 2.6-6.5 ug
Calcium 0.1-0.3 g/24h
Chloride 170-250 mEq/24 h
pH 4.8-7.4 (average 6)
Phosphates 30-90 mEq/24 h
Potassium 35-90 mEq/24 h
Sodium 110-240 mEq/24 h
Urea 10-35 g/24h
Urobilinogen 0-4 mg/24 h
Ammonia 0.14-1.47 g¢/24 h
Creatine 0.15-0.25 gm/24 h
Creatinine 16-21 mg/kg/24 h
Uric acid 0.2-0.7 gm/24 h

Nitrogen conent 78.08%
Oxygen conent 20.93%
Carbon dioxide content 0.04%
Argon content 0.93%
Other gases-trace

Oxygen saturation 97%
PaO? 100 mm Hg
PaCO, 40 mm Hg
Oxygen capacity 19.8 vol %

Oxygen content of expired air 16.3%
Partial pressure of oxygen in air 160 mm Hg
Solubility of oxygen in arterial blood 0.3 ml/100
ml
Oxygen capacity of haemoglobin 1.34 ml/g Hb

Total body water
:
Adult male 60%
Adult female 50%
Infant 75%
Intracellular water
:
Adult male 40%
Adult female 30%
Infant 40%
Extracellular water
:
Adult male 20% (intravascular 4% +
interstitial 16%)

Adult female 20% (intravascular 4% +
Infant 35% (intravascular 5% +
interstitial 30%)

Intake :
As liquid 1500 ml
As food 1000 ml (includes 300 ml water
of oxidation)
Total 2500 ml
Output :
Skin 500 ml
Lungs 400 ml
Faeces 100 ml
Urine 1500 ml

Body weight (kg), Infant (average) = 3.5. Body weight (kg), Adult (average) = 70. Surface area (m 2 ), Infant (average) = 0.21. Surface area (m 2 ), Adult (average) = 1.9. Tracheal diameter (mm), Infant (average) = 8. Tracheal diameter (mm), Adult (average) = 18. Respiratory frequency at rest (mm), Infant (average) = 40. Respiratory frequency at rest (mm), Adult (average) = 20. Tidal volume (ml/kg), Infant (average) = 6. Tidal volume (ml/kg), Adult (average) = 7. Dead space (ml/kg), Infant (average) = 2.2. Dead space (ml/kg), Adult (average) = 2.2. Vital capacity (ml/kg), Infant (average) = 33. Vital capacity (ml/kg), Adult (average) = 52. Minute volume (ml), Infant (average) = 650. Minute volume (ml), Adult (average) = 6000. Resting alveolar ventilation (ml/kg/min), Infant (average) = 125. Resting alveolar ventilation (ml/kg/min), Adult (average) = 60. Resting oxygen consumption (ml/kg/min), Infant (average) = 6.9. Resting oxygen consumption (ml/kg/min), Adult (average) = 3.3. Lung weight (g), Infant (average) = 50. Lung weight (g), Adult (average) = 800. Left ventricular output (ml/kg/min), Infant (average) = 350. Left ventricular output (ml/kg/min), Adult (average) = 100. Arterial oxygen tension (mm Hg), Infant (average) = 75. Arterial oxygen tension (mm Hg), Adult (average) = 95. Arterial CO 2 tension (mm Hg), Infant (average) = 35. Arterial CO 2 tension (mm Hg), Adult (average) = 40

Heart rate Respiratory effort Colour Reflexes Muscle tone, 0 = absent absent blue absent limp. Heart rate Respiratory effort Colour Reflexes Muscle tone, 1 = less than 100/min slow, irregular; body pink, extremities blue grimace some flexion of extremities. Heart rate Respiratory effort Colour Reflexes Muscle tone, 2 = more than 100/min good, crying pink cough, sneeze active movements
The baby is to be evaluated individually at 1 and 5 min of birth and scored from 0 to 2 accordingly. The total score is the sum of the scores of the individual variables.

Newborn, Pulse/min = 160-180. Newborn, Resp/min = 50-40. Newborn, BP (mm Hg) = 80/50. 1-4 yrs, Pulse/min = 100-110. 1-4 yrs, Resp/min = 35-25. 1-4 yrs, BP (mm Hg) = 85/60. 5-7 yrs, Pulse/min = 80-100. 5-7 yrs, Resp/min = 25-20. 5-7 yrs, BP (mm Hg) = 90/60. 8-9 yrs, Pulse/min = 80-100. 8-9 yrs, Resp/min = 25-20. 8-9 yrs, BP (mm Hg) = 95/65. 10 yrs, Pulse/min = 80-100. 10 yrs, Resp/min = 25-20. 10 yrs, BP (mm Hg) = 100/65

Thiopentone sodium 2.5% solution 2 to 4 mg/kg iv
Methohexitone 1% solution 1 to 1.5 mg/kg iv
Suxamethonium 1 to 2 mg/kg iv
Gallamine 2 mg/kg iv
Tubocurarine 0.6 to 0.8 mg/kg iv
Pancuronium bromide 0.12 mg/kg iv
Neostigmine 0.07 mg/kg iv
Morphine 0.125 mg/kg iv or im
Pentazocine 0.25 mg/kg iv
Fentanyl 0.001 mg/kg iv
Pethidine 1 mg/kg im
Trimeprazine 1 mg/kg i.m.
Trimeprazine syrup 4 mg/kg orally
Codeine phosphate 1 mg/kg im
Promethazine 1 mg/kg im

Newborn, Approx. tube length (cm) = 10-12. Newborn, Approx. int. diam. (mm). = 3-3.5. 1-12 months, Approx. tube length (cm) = 12-16. 1-12 months, Approx. int. diam. (mm). = 3.5-5. 1-3 years, Approx. tube length (cm) = 16-18. 1-3 years, Approx. int. diam. (mm). = 5-6. 4-6 years, Approx. tube length (cm) = 18-20. 4-6 years, Approx. int. diam. (mm). = 5.5-6.5. 7-15 years, Approx. tube length (cm) = 20-24. 7-15 years, Approx. int. diam. (mm). = 6-7.5. Adults, Approx. tube length (cm) = 24-28. Adults, Approx. int. diam. (mm). = 7.5-9.5

00, French gauze = 13. 00, Ext. diam. ( mm ) = 4.4. 00, Int. diam. ( mm ) = 3.2. OA, French gauze = 15. OA, Ext. diam. ( mm ) = 4.8. OA, Int. diam. ( mm ) = 3.6. 0, French gauze = 16. 0, Ext. diam. ( mm ) = 5.5. 0, Int. diam. ( mm ) = 4.4. 1, French gauze = 19. 1, Ext. diam. ( mm ) = 6.3. 1, Int. diam. ( mm ) = 4.8. 2, French gauze = 21. 2, Ext. diam. ( mm ) = 7.1. 2, Int. diam. ( mm ) = 5.2. 3, French gauze = 24. 3, Ext. diam. ( mm ) = 7.9. 3, Int. diam. ( mm ) = 5.6. 4, French gauze = 25. 4, Ext. diam. ( mm ) = 8.3. 4, Int. diam. ( mm ) = 6. 5, French gauze = 27. 5, Ext. diam. ( mm ) = 9.1. 5, Int. diam. ( mm ) = 6.7. 6, French gauze = 29. 6, Ext. diam. ( mm ) = 9.5. 6, Int. diam. ( mm ) = 7.1. 7, French gauze = 31. 7, Ext. diam. ( mm ) = 10.3. 7, Int. diam. ( mm ) = 7.5. 8, French gauze = 33. 8, Ext. diam. ( mm ) = 10.7. 8, Int. diam. ( mm ) = 7.9. 9, French gauze = 36. 9, Ext. diam. ( mm ) = 11.9. 9, Int. diam. ( mm ) = 8.7. 10, French gauze = 38. 10, Ext. diam. ( mm ) = 12.7. 10, Int. diam. ( mm ) = 9.5

Average distance from lips to carina : 26 cm approx
Average distance from base of nose to carina : 30 cm approx
Average distance from lips to vocal cords : 14 cm approx
Length of trachea : 12-14 cm approx
Diameter of trachea : 12 mm approx.

2-4 years: 9 cm
5-7 years: 10 cm
8-10 years: 11 cm
11-12 years: 12 cm.

Length or oral tube: Age in years/2 + 12 cm
Length of nasal tube: Age in years/2 + 15 cm
Diameter of the tube: 4.5 + age in years/4, in mm.

endotracheal tube
Mapleson A system
72
Anaesthesia for day-case surgery
165
anaesthesia general
regional criteria
for anaesthesia
analgesia discharge
monitored anaesthesia
168
outpatient surgical
patient selection
167
167
169
care unit
165
postoperative care
168
preanaesthetic preparation 166
preoperative evaluation
166
Anaesthesia in
obese patients
171
pathophysiology
170
preanaesthetic assessment 171
premedication
171
problems of anaesthesia 170
Anaesthetic equipment
56
approximate length
and diameter of endotracheal
tube in children 206
pressure monitoring
89
body temperature monitoring
91
central venous pressure 90
endotracheal tubes
and airways
75
armoured endotracheal
catheter mount
77
84
double lumen
endobronchial tubes
79
170
165
connections 83
endotracheal tubes gum
elastic bougie
intubating forceps
laryngeal
81
laryngeal spray
85
laryngoscopes 82
laser resistant endotracheal tube 77
metal tracheostomy tubes
78
microlaryngeal endotracheal tube
77
nasopharyngeal airway
oropharyngeal airway
oxford endotracheal
76
RAE
(Ring,
Elwyn)
Adair and
77
stilet
85
tracheostomy tubes 78
ventilators 85
epidural catheter
87
epidural needle 86
estimation of end-tidal carbon dioxide 88
invasive arterial blood pressure monitoring 89
Mapleson's classification 72
bain circuit 74
criteria for an ideal breathing system 72
80
79
tube
75
85
85
mask airway
Mapleson B system 73
Mapleson C system 73
Mapleson D system 74
Mapleson
E
system
74
Mapleson
F
system medical gas supply 56
absorbers
70
adjustable pressure limiting valve
66
anaesthetic breathing system
68
back bar
63
bag mount
65
baralyme
69

Boyle's bottle/glass-ether vaporiser
62
circle breathing
system common gas outlet 65
compressed air 59
corrugated rubber tube 66
cylinder valve
57
cylinders
56
face mask and angle piece
67
flow restrictor
60
flowmeters 60
Goldman halothane vaporiser
63
ideal vaporiser characteristics 61
liquid oxygen 58
oxygen concentrator 59
oxygen supply failure alarm
64
71
74

pin index system 57
piped gas supply 58
plenum vaporisers
64
pressure gauge 59
pressure regulator
60
rebreathing bag
65
trichloroethylene vaporiser bottle
63
vaporiser inside the circle breathing system 72
vaporiser outside the circle breathing
system
72
vaporisers
61
Water's to-and-fro system
70
nerve block needles 87
nerve stimulator for nerve blocks
87
oesophageal stethoscope 91
oxygen analyser 91
peripheral nerve
stimulator
89
pulmonary artery pressure monitoring
90
pulse oximetry
87
spinal needle
86
Wright respirometer 88

Cardiopulmonary resuscitation
176
advanced cardiac life support
178
anaemia
199
basic cardiac life support airway
176
breathing
176
circulation
177
bronchial asthma
185
chronic obstructive airways disease
186
congestive cardiac failure

criteria for
brain death
179
anaesthesia and
common coexisting diseases 180
diabetes mellitus 187
disseminated intravascular coagulation
198
epilepsy 196
hypertension
180
hyperthyroidism 190
hypothyroidism 191
ischaemic heart disease 184
ketoacidosis 189
liver diseases
193
anaesthetic management
194
hepatic encephalopathy
195
tests problems
relevant anaesthesia
193
mitral stenosis 183
myasthenia gravis 197
myasthenic syndrome
198
jaundice
196
pheochromocytoma
192
thyroid dysfunction 190
thyroid strom/thyrotoxic crisis

Caudal anaesthesia
advantages
103
contraindications 104
disadvantages 104
physiological changes
technique of
uses
103
Complications of
anaesthesia aspiration
pneumonitis bronchospasm
103
caudal block
105
115
121
119
cardiac arrhythmias
123
delayed recovery from anaesthesia
128
epiglottitis
118
headache
116
hiccups
120
hypervolaemia
128
122
laryngospasm
118
laryngotracheobronchitis 119
malignant hyperpyrexia nausea
and vomiting
115
pneumothorax
120
postanaesthetic postanaesthetic
convulsion
125
restlessness
116
postintubation laryngeal oedema 119
postoperative bleeding 126
postoperative bradycardia 124
193
to postoperative hypertension

postoperative hypoxia
127
postoperative jaundice
126
postoperative pyrexia 124
postoperative renal
failure
129
postoperative shivering
125
postoperative tachycardia 124
respiratory obstruction 117
Conduct of
12
endotracheal anaesthesia
disadvantages 17
induction of anaesthesia inhalational induction 12
intravenous induction
13
maintenance of
anaesthesia nasotracheal
intubation
14
17
16,
17
17
disadvantages hazards
16
indications technique
16,
17
17
16,
18
signs of

prediction
131
unexpected difficult intubation
133
E
Epidural anaesthesia 98
advantages 99
complications 100
contraindications of epidural block 102
disadvantages epidural
space indications
100
98
99
local anaesthetics
98
total spinal anaesthesia 101
Eye and
ENT
135
anaesthesia for
ear surgery
eye
140
surgery
136
cataract extraction 139
15
14
criteria needed 136
effect of anaestehtics on IOP
137
general anaesthesia
glaucoma
137
139
peribulbar block
retrobulbar
138
block
137
strabismus surgery
139
anaesthesia for nose and throat surgery
adenotonsillectomy 141
compromised airway 141
laser surgery
142
postoperative tonsil
142
eye anaesthesia
135
drug interactions
136
intraocular pressure
oculocardiac
135
reflex
136
open eye
injury special considerations 139
retinal detachment
surgery
140

Geriatric anaesthesia
173
175
common co-existing diseases in the
aged
174
175
preanaesthetic assessment 174
preoperative preparation 174
systemic changes
cardiovascular system
endocrine system
genitourinary
174
system nervous
system respiratory
173
system
173

Inhalational anaesthetic agent criteria for an ideal inhaled
anaesthetic drugs
desirable features
19
true anaesthetic
gases
19
factors increase the rate of anaesthetic
induction
19
factors increase
recovery the
rate from
20
of anaesthesia
173
173
bleeding
19
minimum alveolar concentration 20
the agents
cyclopropane desflurane
24
diethyl ether
enflurane
23
entonox fluroxene
halothane
21
24
22
isoflurane
23
methoxyflurane nitrous
oxide
21
sevoflurane
24
trichloroethylene
22
physical properties 25
Intravenous anaesthetic agents 26
33
althesin
29
barbiturates
27
benzodiazepines 31
continuous infusion of iv anaesthetics
34
diazepam
32
etanolone (pregnanolone) 29
etomidate
30
fentanyl
33
hypersensitivity anaesthetics
ketamine
30
methohexitone midazolam
32
opioids 32
propanidid propofol
31
remifentanil sufentanil
29
34
33
thiopentone
28
total intravenous

L
Local anaesthetic drugs 36
minimum blocking concentration (Cm) 41
systemic toxic reactions 37
the agents
bupivacaine
40
cocaine 38
lignocaine 39
21
22
23
reactions
34
28
of iv
mepivacaine prilocaine
39
procaine
38
ropivacaine 40

Monitoring during anaesthesia clinical monitoring 53
monitoring the anaesthetic apparatus
54
special basic monitors used in
53
42
anticholinesterase drugs edrophonium
48
neostigmine
48
characteristics of depolarising block
42
characteristics of
nondepolarising block 43
factors altering
the response
to muscle relaxants 44
monitoring of neuromuscular block
50
50
the agents
alcuronium
45
atracurium
46
curare
(d-tubercurarine)
dibucaine number 48
gallamine triethiodide
mivacurium
46
pancuronium
45
pipecuronium
46
pseudocholinesterase succinylcholine
47
vecuronium
46
types of neuromuscular block
43
uses of
muscle relaxants

Obstetric anaesthesia 143
anaesthesia for caesarean section
anaesthetic techniques
147
148
regional anaesthesia 147
43

diagnosis of
pregnancy
144
dilatation and
haemorrhage evacuation
154
in the
parturient

hypertensive disorders of pregnancy
preeclampsia
/eclampsia
149
hysterotomy laparoscopic
154
sterilisation nonobstetric
153
surgery during
pregnancy 152
physiological changes of pregnancy
cardiovascular gastrointestinal
system system
haematologic system
renal system
144
144
respiratory system
143
placental transfer /exchange factors determining the rate
and extent of transfer
145
mechanism
145
pain during
labour and
delivery 146
pain relief during and labour
146
placental transfer of drugs
145
postpartum tubal ligation 153

Paediatric anaesthesia
155
equipment
158
161
blood loss replacement 163
body fluids
156
central nervous
system drugs during paediatric
anaesthesia 164
fluid management 162
haematologic system 156
fluid requirement 163
monitoring during anaesthesia
160
practical recommendations 163
155
156
143
143
144
premedication 157
preoperative evaluation 157
preoperative preparation
157
psychological aspects 156
renal and
hepatic system
155
signs of
dehydration
162
surgical fluid/loss 163
thermoregulation
156
Peripheral nerve
block
106
brachial plexus
block
107
celiac plexus block 113
common nerve
blocks
107
femoral nerve block 109
field block for repair of inguinal hernia 110
form of regional anaesthesia
106
intercostals nerve
block
110
intravenous regional
anaesthesia (Bier's block)

median nerve
block
108
penile block
111
pudendal nerve block 111
radial nerve block 109
sciatic nerve
block
109
stellate three
ganglion in
block one
block
110
ulnar nerve
block
109
Physiological data 201
Apgar scoring
system average
pulse
204
blood
201
blood pressure 204
cerebrospinal fluid
202
doses of some drugs in children

haematological 201
haemoglobin
201
metabolism proteins 201
respiration
204
size of endotracheal tubes 205
some anatomical
measurements
205
transaminases 201
Preanaesthetic evaluation
1
ASA physical status index
202
205
156
112
204
3
associated illness
1
causes of postponement of operation 4
common medications and anaesthetic implications 2
history
1
objectives
1
physical examination preanaesthetic routine
preparation
4
preoperative diagnostic
investigations 3
routine requirement before induction of anaesthesia
selection of anaesthetic technique
4
Preanaesthetic medication 6
common drugs used in premedication
7
atropine sulphate
diazepam
8
glycopyrrolate 9
hyoscine
9
metoclopramide
10
morphine sulphate
ondansetron
11
pentazocine 8
pethidine hydrochloride prochlorperazine
10
promethazine
10
trimeprazine criteria
for ideal
6
factors for proper selection of premedicant drugs 6
preanaesthetic medication or premedication 6
routes of administration 11

Spinal anaesthesia 92
anatomical considerations spinal cord 92
spine 92
common drugs used 95
different types of block 95
indications of subarachnoid block 94
technique of subarachnoid block 94
10
8
2
7
4
7

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CONCISE
FORENSIC
MEDICINE
AND
TOXICOLOGY
Fourth
Edition
By
K.S.NARAYAN
Honorary
REDDY, m.,
F.A.F.M.,
Professor
S.V.S.
Medical
(Retired
Principal,
JAYPEE>
p.c.P.,
Ph.D.
F.I.M.S.A., of
Forensic
College,
Osmania
The
New
Delhi
|
F.A.M.S.,
F.A.F.Sc.,
F.LA.M.S.
Medicine,
Mahabubnagar.
Medical
Health
London
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College,
Hyderabad)
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Forensic
First
Edition:
Fourth
2001
Edition:
2015
ISBN
978-93-5152-823-4
Printed at
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the any
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Medicine and
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USA
Inc
Clayton
Ltd
(P)
Ltd
Inc
CONTENTS
FORENSIC
RPANAMPWNS
OC
22.
23.
24.
25.
26.
27.
28.
29.
30.
Legal
MEDICINE
Procedure
Medical
Law and
Identification
Medicolegal
Autopsy
Death and
its
Postmortem
Cause
Changes
Mechanical
Injuries
Regional
Injuries
Aspects
Thermal
Deaths
Asphyxia
Impotence and
Virginity,
Sterility

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Pregnancy
Sexual and
Offences
Abortion
Infant
Deaths
Blood
Stains
Forensic
Psychiatry
Artefacts
Forensic
Science
Laboratory
General
Considerations
Agricultural
Poisons
Corrosive
Poisons
Metallic
Poisons
Inorganic
Irritant
Organic
Poisons
Irritant
Poisons
CNS
Depressants
Poisons
Stimulants
Ethics of
Wounds
Delivery
=
40
47
52
64
86
95
103
111
113
129
133
140
154
159
167
172
184
186
188
200
206
212
221
223
230
241
243
244
248
253
255
257
263
267
FORENSIC
MEDICINE
CHAPTER

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Forensic
(legal)
medicine knowledge
to aspects
deals aid
of in
administration law,
etc.
Medical such
with of
justice, as
assault, murder,
jurisprudence doctor,
i.e.
with medical
deals legal
aspects negligence,
doctors, consent,
etc.
INQUEST
:
cause of
death and
An inquest
application of
i.e.
it deals
with sexual
with legal
medical medical
offences, poisoning,
responsibilities of
of professional
is
(S.174, suspicious
(J)
POLICE
(2)
The officer
Magistrate, an
Cr.P.C).
deaths, enquiry
medicine, such
misconduct, duties
or investigation
It is
conducted such
as suicide,
INQUEST
:
-
incharge and
proceeds is.
(3)
He
(1)
as of
into the
in cases
of unnatural
murder,
It is
of conducted
police to
conducts two
witnesses of
the place
(panchas).
wounds,
The the
report accident,
etc.
throughout station,
informs
India.
the
Executive where
body of
deceased
(panchanama),
(4)
The nature
is is
of signed
suspected, authorised
by weapon
police the
body in
report includes
and apparent
officer is
Government dead
body sent
person the
presence the
cause and
for of
description of
witnesses.
autopsy doctor.
is handed
over
(2)
MAGISTRATE’S
Magistrate
(7)
to the
INQUEST
(who death.
(5)
(6)
If foulplay
with
If no
a
requisition foul
relatives
:
are deputy
Collector, to
play the
is suspected,
for disposal.
This is
revenue conducted
officials),
Tahasildar, by
an such
etc.
It is
as
District done
in cases
the
Executive
Collector, of

= . deaths can,  = . occur under such circumstances as to suggest falsely,  = . that used. Accidental deaths and suicides can occur under,  = . circumstances homicide. In a suicide case, alterations may be made,  = circumstances homicide. In a suicide case, alterations may be made. suggest,  = suggest. violence,  = violence. boo,  = boo. booksfree.com,  = booksfree.com. which booksfree.com,  = which booksfree.com. was,  = was
CHAPTER

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Medical ethics
deals with
moral principles
members of
the medical
other, their
patients conventional
and profession
the laws
medical profession.
The of
modernised
Geneva version
(1948).
Homoeopathy to
Central regulate
the profession
council.
Its
Indian to
lay down
system of
in which
their
State.
dealings
Medical of
courtesy etiquette
observed with
guide each
deals with
between of
members
Hippocratic
Council oath
is
Act,
1973:
of functions
Homoeopathy are
almost
Medicine the
of the
the
Declaration
The and
act was
passed to
similar to
constitute
Indian
Central minimum
Council standards
Act, of
medicine.
Ashtang.
INDIAN
MEDICAL
Medical
Council each
Medical cetral
Council.
1970:
education
It includes
It was
and
Ayurveda,
COUNCIL
consists practice
Siddha,
ACT, of
University routine
and work
some is
of the
Act looked
contains
India, and
medical doctors
nominated after
enacted of
Indian
Unani
1956:
elected by
Central by
the second
Functions
In this
names
Register and
(1)
Indian from
each
Government.
Registrar.
qualifications schedule
of
Indian
State,
(2)
(3)
The
The
First granted
those granted
Medical of
all doctors
are entered.
(2)
Medical
Education undergraduate
shedule by
universities outside
Council:
who are
in
India.
(1)
enrolled
:
(1)
Medical on
Register:
any
It and
State prescribes
postgraduate medical
Medical standards
education of
for the

Punishment:, 2 = erasure of name or warning. booksfree.com. (6) Appeal:, 1 = to higher Court.. (6) Appeal:, 2 = to State and Central Government. boo
17 If a doctor performs an unauthorised operation on a patient, he may be sued in a Civil Court for damages and prosecuted in Criminal Court for assault. ContRIbutoRyneGlIGenCe: It is any unreasonable conduct or absence of ordinary care onthe part of patient, which combined with doctor's negligence contributed to injury as a direct, proximate cause and without which the injury would not have occurred. examples: (1) Failure to give the doctor correct medical history. (2) Refusal to take suggested treatment. (3) Failure to follow instructions. (4) Leaving the hospital against advice. (5) Failure to seek further medical assistance if symptoms persist. defence: (1) It is good defence if both doctor and patient are negligent at the same time. (2) The doctor has to prove it. (3) Normally, it is only a partial defence, and the damages awarded vary, depending on the negligence of each party. negligence Prevention: (1) Establish good rapport with the patient, his family, fellow physicians and paramedical staff. (2) Rationale: Carry necessary investigations to make the correct diagnosis and formulate treatment. (3) Maintain complete, accurate, mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com meboo,  = meboo

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Identification is
recognition physical
characters,
CHAPTER
of an
individual based
which
CORPUS
the are
unique
DELICTI:
elements
(body of
of any
is the
criminal determination
violence to
offence, the
individual.
essence offence,
of in
a
identity particular
are conclusive
the of
of of
crime):
e.g.
murder.
the way,
at death
crime.
by
It also
the body,
injuries.
dead
a
on certain
It
The body
and particular
foul includes
clothing
The is
play, time
by means
main part
causing and
the
a
bullet or
and photographs
identification important
of
a
dead before
a
sentence
RACE
:
Cephalic
Type index
=
of skull
Dolico-cephalic of
place, persons
a
broken of
which charged
knife-blade the
deceased body
and is
passed in
with found
showing proof
of murder
Maximum in
fatal corpus
delicti trials.
breadth
Maximum
Cephalic
(long-headed)
Mesati-cephalic
(medium-headed)
Brachy-cephalic
(short-headed)
SEX:
Sex chromatin
which lies
is used
near for
females of
length of
Index
70
to
75
75
80
is
a
to
80
to
85
small nuclear
plano-convex membrane
sex and
determination.
0
to skull
|
9, skull
Race
Aryans, aborgines
Negroes.
Europeans
Chinese.
Mongolian.
mass in
It the
cell.
(Barr
Usually is
4%
in males.
found in
and body),
buccal
20
to
80%
smear of
cells in
24
Concise
Forensic
INTERSEX:
Medicine
Characters present
in one
organs and
embryonic of
both sexes
individual including
sexual behaviour.
(1)

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Gonadal ovaries
(2)
agenesis:
have never
Gonadal but
at
Nuclear developed.
dysgenesis:
puberty
External testes
syndrome:
and ovaries
Anatomical female
(chromatin
(47
(in physical
It occurs
varying degree)
are form,
reproductive due
to some
sex is
negative.
sexual fail
defect in
The structures
to structure
chromosomes).
is female,
is
The develop.
testes or
are present,
(A)
Klinefelter’s male,
but nuclear
sex
(B)
but
Turner’s nuclear
chromosome pattern
primordial sexing
chromosome syndrome:
sexing is
pattern
Anatomical is
XO
follicles
(3)
male
(45
(ovarian is
XXY
structure negative).
Ovaries dysgenesis).
True or
two hermaphroditism
ovotestis
Trait
(1)_General size:
(2)
Capacity:
(3)
(4)
(5)
(6)
(7)
(8)
Architecture:
Forehead:
Orbits:
Cheek bones:
Zygomatic arch:
Occipital area:
(9)
Mastoid process;
(10)
Palate:
(bisexuality).
are present
with
;
An
Sex do
ovary external
Traits genitalia
diagnostic of
Male
SKULL
Larger, longer,
rounded.
1500
to
1550
Rugged.
Steeper, ml.
less rounded.
Square, lower,
relatively smaller,
rounded margins.
Heavier, arched.
More pronounced.
Protuberance marked.
Medium to
large, round,
blunt.
Large, broader,
U-shape.
laterally and
testis of
sex
Female
Smaller, lighter,
rounded.
1350
to
1400
Smooth.
Vertical, ml.

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

round, infantile.
Rounded, higher,
relatively larger,
sharper margins.
Lighter, more
compressed.
Less pronounced.
Protuberance not
marked.
Small, medium,
smooth, pointed.
Small, parabola.
full, both
sexes.
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
Bony frame
General:
Ilium:
Preauricular sulcus:
Acetabulum:
Obturator foramen:
Greater sciatic
notch:
Ischial
(9)
tuberosity:
Body of
(10)
pubis:
Subpubic
(11)
(12)
(13)
(14)
angle:
Pelvic brim
inlet:
Pelvic cavity:
Sacroiliac articulation:
Sacrum;
or work:
PELVIS
Massive, rougher;
stands higher
more and
erect.
Deep
Less funnel.
vertical;
of iliac
curve crest
reaches higher
level and
is more
prominent.
Not frequent,
narrow, shallow.
Large, directed
laterally.
Large, often
oval base
upwards.
Lower, deeper.
Inverted.
Narrow, triangular.
V-shaped, sharp
angle
70°
to
75°.
Heart-shaped.
Conical and
funnel- shaped.
Larger extends
two- and-
to half
to vertebrae.
Longer, narrower,
with more
evenly distributed
curvature;
promontory well
marked.
Body of
sacral vertebra
first larger.
three with
Less massive,
25
gracile, smoother.
Flat bowl.
More vertical;
between distance
iliac crests
is less;
iliac fossae
shollow;
curve of
crest well-marked.
More frequent,
and deep.
Small directed
anterolaterally.
Small, triangular
apex forwards.
Larger, wider,
shallower.
Everted.

#/texts/1476 #/texts/1477 #/texts/1478 #/texts/1479 #/texts/1480 #/texts/1481 #/texts/1482 #/texts/1483 #/texts/1484 #/texts/1485 #/texts/1486 #/texts/1487 #/texts/1488 #/texts/1489 #/texts/1490 #/texts/1491 #/texts/1492 #/texts/1493 #/texts/1494 #/texts/1495 #/texts/1496 #/texts/1497 #/texts/1498 #/texts/1499 #/texts/1500 #/texts/1501 #/texts/1502 #/texts/1503

Broad, square.
U-shaped, rounded,
broader angle
100°.
Circular or
elliptical;
more spacious;
diameters longer.
Broad and
small, round.
oblique, extends
2
to two-and-half
vertebrae.
Shorter, wider;
half almost
curve upper
straight, forward
in half;
lower promontory
marked.
Body of
less first
small.
90°
to broad,
with

#/tables/21 #/texts/1504 #/texts/1505 #/texts/1506 #/texts/1507 #/texts/1508 #/texts/1509 #/texts/1510 #/texts/1511 #/texts/1512 #/texts/1513 #/texts/1514 #/texts/1515 #/texts/1516 #/texts/1517 #/texts/1518 #/texts/1519 #/texts/1520 #/texts/1521 #/texts/1522 #/texts/1523 #/texts/1524 #/texts/1525 #/texts/1526 #/texts/1527 #/texts/1528 #/texts/1529 #/texts/1530 #/texts/1531 #/texts/1532 #/texts/1533 #/texts/1534 #/texts/1535 #/texts/1536 #/texts/1537 #/texts/1538 #/texts/1539 #/texts/1540 #/texts/1541 #/texts/1542 #/texts/1543 #/texts/1544 #/texts/1545 #/texts/1546 #/texts/1547 #/texts/1548 #/texts/1549 #/texts/1550 #/texts/1551

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

is delayed, but in syphilis dentition appears early or may be present at birth. (13) Stunted and notched upper central incisors are seen in syphilis. mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com.resorption of root begins = 17 to 25 years 18 to 25 years. , Permanent premolars erupt in place of deciduous molars. (5) In both deciduous and permanent teeth, dentition occurs earlier by about one year in lower jaw, except lateral incisors. (6) Third molar first erupts on the left side of lower jaw. (7) the dental and skeletal ages are almost same in the male, but in the female skeletal age is usually one year ahead of the dental age. (8) eruption occurs earlier in warmer climates and in urban areas. (9) eruption of temporary teeth is more regular than permanent. (10) eruption is not always bilaterally symmetrical. (11) Heredity, environment, endocrine reactions and nutrition affect eruption and calcification. (12) In ill-nourished children and rickets, dentition is delayed, but in syphilis dentition appears early or may be present at birth. (13) Stunted and notched upper central incisors are seen in syphilis. mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com.Calcification of root completed meboo = 17 to 25 years 18 to 25 years
28
Trait
(1)
Size:
(2)
(3)
(4)
(5)
(6)
Direction:
Crown:
Neck:
Root:
Ridge:
Temporary teeth
colour, neck
is more
crown with
root.
Permanent teeth
is root.
(2)
one or
Concise
Forensic
Difference
Medicine between
temporary
Temporary and
teeth
narrow, except
temporary molars
which are
longer permanent
than premolars
replacing them.
Anterior teeth
are vertical.
China-white colour.
More constricted.
Roots of
molar and
smaller more
A
ridge divergent.
or thick

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

at the
edge junction
crown of
with the
present.
are smaller,
constricted, the
fangs lighter,
and are
(1)
narrower,
a
ridge heavier,
less constricted,
Permanent two,
crown is
seen china-white
at stronger,
no ridge
incisors and
and upper
of incised
circular and
have two
07
broader, at
junction canines
molars
3, have
and is
lower chisel-shaped,
molars cusps
years
09
11
14
years years
years canine
cubicle.
(4)
and molars
3
to
24
teeth
24
12
junction of
ivory-white of
single molars
large crown
with root,
premolars two
and
The chewing
5.
(20
deciduous teeth
(12
deciduous permanent
24
roots.
conical, surface
(3)
The premolars
of premolars
and
4
permanent).
(molars
(8
incisors teeth
(4
permanent:
molars).
28
permanent and
canines):
and
4
diciduous
(8
incisors, teeth.
first
(canines)
molars).
and
8
20
premolars and
4
permanent teeth
Haevier, stronger,
broader, except
permanent premolars
replacing temporary
molars.
inclined forward.
Ivory-white colour.
Less constricted.
Roots of
molars are
larger and
less divergent.
No ridge.
a
little

29 after eruption of second permanent molar teeth, the body of jaw grows posteriorly, and ramus is elongated. If third molars are fully erupted, the age is above 17 . Gustafson's method: after 21 years, age is estimated by teeth from physiologic age changes in each of dental tissues. (1) Attrition. (2) periodontosis: Regression of the gums and periodontal tissues occurs in old age, gradually exposing necks and roots. (3) secondary dentin: It develops from walls within the pulp cavity and diminishes its size. (4) Cementumapposition, especially near end of root occurs continuously throughout life, and forms incremental lines. (5) root resorption starts at apex and extends upwards. (6) Transparency of root is seen after 30 years. The canals in dentin are filled by mineral and dentin becomes transparent. It is the most reliable of all criteria . error is said to be ± 4 to 7 years. Growth in Individual Bones : (1) Bones develop from a number of separate centres of ossification, which is spread over a long period of time.(2) alarge number appear in foetal life and few after birth. (3) 806 centres are present in foetus at eleventh week. (4) 450 centres are present at birth. (5) Adult skeleton has 206 bones. (6) Ossification begins centrally in an epiphysis which is amorphous, rounded and pin- head sized. (7) Most bones are ossified from several separate centres. (8) carpal and tarsal bones ossify from a single centre. (9) Hyaline cartilage is present between the diaphysis and epiphysis (epiphyseal plate or growth plate or growth cartilage) . (10) The bone increases in length at the epiphyseal plate, until its final dimensions are attained. (11) The process of union of epiphysis and diaphysis is called fusion. (12) union of epiphyses in cartilaginous bones occurs earlier by about one year in the females than in males, but the opposite is mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com meboo,  = meboo meboo

#/tables/23 #/texts/1662 #/texts/1663 #/texts/1664 #/texts/1665 #/texts/1666 #/texts/1667 #/texts/1668 #/texts/1669 #/texts/1670 #/texts/1671 #/texts/1672 #/texts/1673 #/texts/1674 #/texts/1675 #/texts/1676 #/texts/1677 #/texts/1678 #/texts/1679 #/texts/1680 #/texts/1681 #/texts/1682 #/texts/1683 #/texts/1684 #/texts/1685 #/texts/1686 #/texts/1687 #/texts/1688 #/texts/1689 #/texts/1690 #/texts/1691 #/texts/1692 #/texts/1693 #/texts/1694 #/texts/1695 #/texts/1696 #/texts/1697 #/texts/1698 #/texts/1699 #/texts/1700 #/texts/1701 #/texts/1702 #/texts/1703 #/texts/1704 #/texts/1705 #/texts/1706 #/texts/1707 #/texts/1708 #/texts/1709 #/texts/1710 #/texts/1711 #/texts/1712 #/texts/1713 #/texts/1714 #/texts/1715 #/texts/1716 #/texts/1717 #/texts/1718 #/texts/1719 #/texts/1720 #/texts/1721 #/texts/1722 #/texts/1723 #/texts/1724 #/texts/1725 #/texts/1726 #/texts/1727 #/texts/1728 #/texts/1729 #/texts/1730 #/texts/1731 #/texts/1732 #/texts/1733 #/texts/1734 #/texts/1735 #/texts/1736 #/texts/1737 #/texts/1738 #/texts/1739 #/texts/1740 #/texts/1741 #/texts/1742 #/texts/1743 #/texts/1744 #/texts/1745 #/texts/1746 #/texts/1747 #/texts/1748 #/texts/1749 #/texts/1750 #/texts/1751 #/texts/1752 #/texts/1753 #/texts/1754 #/texts/1755 #/texts/1756 #/texts/1757 #/texts/1758 #/texts/1759 #/texts/1760 #/texts/1761 #/texts/1762 #/texts/1763 #/texts/1764 #/texts/1765 #/texts/1766 #/texts/1767 #/texts/1768 #/texts/1769 #/texts/1770 #/texts/1771 #/texts/1772 #/texts/1773 #/texts/1774 #/texts/1775

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

:, 2 = . 14 th year :, 1 = Crest of ilium; head and tubercles of ribs. booksfree.com. 14 th year :, 2 = Medial epicondyle of humerus; lateral epicondyle with trochlea; patella complete. Contd... booksfree.com boo
Contd...
15"
year
16"
18"
18"
21*
year year
:
:
:
to
19"
year:
year
:
Sacrum
:
Intervertebral puberty
and
Skull and
sacrum
:
(1)
two
Acromion.
Ischial tuberosity.
—_
Inner end
of clavicle.
discs becomes
It consists
halves of
occipital of
ossify single
of bone
22
mandible bone
occipital bones.
unite fuses
at the
with fifth
(5)
Basi-occiput
(6)
year.
fuses
Closure of
Coracoid with
scapula;
triradiate cartilage
of acetabulum.
Lower end
of humerus;
olecranon to
ulna;
upper end
of radius;
metacarpals;
proximal phalanges.
Head of
femur;
lesser and
greater trochanter
acromion;
of ulna
Lower upper
end end
and fibula,
humerus;
of femur;
lower end
of femur;
of tibia
head of
lower radius.
Iliac crest;
end of
inner of
clavicle;
tuberosity.
from below
upwards between
(2)
at second
squama
(4)
with the
externally.
(7)
on outer
side.
Posterior sutures
Inner end
ischial after
21
Anterior year.
to
25
years.
fontanelle
(3)
at third
Metopic suture
closes
Condylar portion
year, and
with closes
basi-sphenoid about
between begins
side on
the union
(8)

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

1/3
lower
On the
of half
:
Middle occurs
outer sagittal;
of coronal.
40
side to
50
45
sagittal temporal.
lambdoid years
and
(9)
The and
upper
5
fusion basi-
third
18
to inner
side and
to
10
years occurs
years
:
:
year.
21
years.
proceeds earlier
:
30
Anterior
Lambdoid half
order then
1/3
than to
40
years of
suture.
reliability
:
sagittal
50
and to
60
of
60
years years:
suture
Squamous closure
:
Sagittal,
31

#/tables/29 #/texts/1836 #/texts/1837 #/texts/1838 #/texts/1839 #/texts/1840 #/texts/1841 #/texts/1842 #/texts/1843 #/texts/1844 #/texts/1845 #/texts/1846 #/texts/1847 #/texts/1848 #/texts/1849 #/texts/1850 #/texts/1851 #/texts/1852 #/texts/1853 #/texts/1854 #/texts/1855 #/texts/1856 #/texts/1857 #/texts/1858 #/texts/1859 #/texts/1860 #/texts/1861 #/texts/1862 #/texts/1863 #/texts/1864 #/texts/1865 #/texts/1866 #/texts/1867 #/texts/1868 #/texts/1869 #/texts/1870 #/texts/1871 #/texts/1872 #/texts/1873 #/texts/1874 #/texts/1875 #/texts/1876 #/texts/1877 #/texts/1878 #/texts/1879 #/texts/1880 #/texts/1881 #/texts/1882 #/texts/1883 #/texts/1884 #/texts/1885 #/texts/1886 #/texts/1887 #/texts/1888 #/texts/1889 #/texts/1890 #/texts/1891 #/texts/1892 #/texts/1893 #/texts/1894 #/texts/1895 #/texts/1896 #/texts/1897 #/texts/1898 #/texts/1899 #/texts/1900 #/texts/1901 #/texts/1902 #/texts/1903 #/texts/1904 #/texts/1905 #/texts/1906 #/texts/1907 #/texts/1908 #/texts/1909 #/texts/1910 #/texts/1911 #/texts/1912 #/texts/1913 #/texts/1914 #/texts/1915 #/texts/1916 #/texts/1917 #/texts/1918 #/texts/1919 #/texts/1920 #/texts/1921 #/texts/1922 #/texts/1923 #/texts/1924 #/texts/1925 #/texts/1926 #/texts/1927 #/texts/1928 #/texts/1929 #/texts/1930 #/texts/1931 #/texts/1932 #/texts/1933 #/texts/1934 #/texts/1935 #/texts/1936 #/texts/1937

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

(9) If hair is hair bulb will be larger, irregular and swollen and sheath mebooksfree.com mebooksfree.com booksfree.com, 1 = . nonpigmented longitudinally inner layer bodies are found males. (5) non-pigmented and and (average 2.5 any part the hair pulled meboo, 1 = . Hair non- straw- mm)., 1 = . of falls out,, 1 = of falls out,. ruptured., 1 = ruptured.. booksfree.com, 1 = booksfree.com
38
Trait
(1)
General:
(2)
(3)
(4)
(5)
(6)
(7)
Cuticle:
Cortex:
Medulla:
Pigment:
Precipitin
Medullary test:
index:
M.L.
Imp clothes,
body accident.
:
(1)
Forensic
Medicine
Difference between
Human human
hair
Fine and
thin.
Cuticular scales
short, broad,
are thin
irregularly and
annular.
Thick, well-striated,
and
4
broad to
10
times as
as
Varies medulla.
considerably, usually
narrow;
continuous, fragmented,
or entirely
absent.
Evenly distributed.
Specific for
human.
Below
0.3
Helps and
in weapon.
(B)
in sexual
injury.
Stains crime
(A)
indicate offences,
(2)
and hair
salivary
Nature blubs.
hair
Animal hair
Coarse and
thick.
very large
are and
have step-like
or wavy
projections.
Thin, rarely
than twice
more as
medulla.
Continuous and
Mostly broad
as wider.
present medulla.
Above
0.5
investigation,
In rape,
the for
it remains
sodomy, bestiality
nature stains
of weapon
(3)
burns.
(5)
metals can
Singeing of
assault, in
asphyxial can
be near
animal.

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

on and
traffic e.g.
seminal deaths,
made out
indicates
Sex may
be be
detected.
TEETH
is
:
sharp and
days there
(1)
In antemortem
feathered.
is organised
burns.
determined.
(6)
tooth
(2)
A
blood early
organisation.
clot.
partially
(4)
filled new
bone, whole
but socket
out, stains
blood stains
from injuries
(4)
in of
hair
Differentiates
Identification.
(6)
loss, clot
(3)
In
In
2
to
3
weeks with
new bone.
the location
is irregular
areas filled
bony rim
scalds
Heavy of
forms
a
and week,
soft
(5)
of root
with edges
of in
socket tissues
are
In
6
months outline
new bone.
of compressed
teeth are
seen.
remaining bone,
is healed
socket is
is visible,
(6)
and
If bone,
or fracture
entire tooth
splintering of
alveolus one
to two
filled with
and socket
in one
year was
knocked of
buccal roots
or crowns
plates, of
adjacent
Concise and
_
CHAPTER

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Objects:
To find
out
(1)
Cause of
death
Manner of
death
(4)
Establish the
and
(suicide, identity.
weapon and
viability.
Rules:
(1)
Requisition
Conducted from
police
Collect or
(5)
Conducted such
from in
day-light as
jaundice, be
made homicide,
(5)
(natural accident).
To collect
criminal.
(6)
or unnatural).
(3)
Time physical
In new
born in
to mortuary
(2)
since death.
evidence to
identify determine
only, except
liverbirth spot
necessary.
inquest, accident
as far
as
P.M.
hypostasis out
in artificial
police constable.
Assistant
(3)
Avoid register,
possible,
P.M.
(2)
delay.
case sheet,
because and
colour light.
(6)
(7)
No should
never partial.
Incision:
I-shaped symphysis
pubis
Body unauthorised
note findings.
(4)
etc.
colour changes,
of contusions
should person
(9)
be cannot
identified should
Autopsy incision
in
To by
the be
present.
must extending
midline.
demonstrate affected
pneumothorax, side
between and
the wall
bubble
Air vessels
a
pocket the
chest wall
and punctured
with out
of the
knife opening
embolism:
under through
(1)
Open of
The left
in brain
for ventricle
water gas
will if
the right
be from
complete
(8)
and chin
down is
dissected skin,
and is
the water.
water.
the bubbles.
head first,
(2)
Cut contain
frothy ventricle
to on
filled the
with water,
Air and
the apex
blood.
contains if
present examine
of will
surface heart
with
(3)
air.
(4)
The heart

Pericardial knife.
will sac
float is
filled
Autopsy 41 with water and heart punctured with a scalpel and twisted a few times. Bubbles of air will escape if air is present. Heart is opened in the direction of flow of blood. Subendocardial haemorrahges: (1) They are seen in the ventricle, on the opposing papillary muscles and adjacent carnae. (2) The haemorrhages are flame- shaped, confluent and to occur in one continuous sheet rather than patches. (3) They are seen (a) after sudden severe hypotension due to severe loss of blood or from shock, (b) after intracranial damage, such as head injury, cerebral oedema, surgical craniotomy or tumours, (c) death from ectopic pregnancy, ante-partum or post-partum haemorrhage, (d) various types of poisoning, especially arsenic. Stomach is removed by appliying double ligature at both ends. It is opened along greater curvature. Small intestine is opened along the line of mesenteric attachment, and large intestine along taenia. Liver and spleen are cut in long axis, pancreas at right to long axis, and kidney is sectioned longitudinally through border into hilum. Testis is removed by incising inguinal canal pulling loop of vas through it. Duodenum is the widest and mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com,  = left columnae tend. fixed portion of small bowel. A.M. blood clots are dark red, firm and on section show alternate,  = anterior angles convex and most meboo. layers of platelets and thrombi. Postmortem clots are moist, rubbery, homogeneous, loosely or not attached to wall, and there lines of Zahn.,

(2) Traces of marrow and periosteum may remain in, or attached to the bones for several months. (3) A fairly recent bone is slightly greasy to the touch and heavy. (4) Odour is a good indication relatively recent death. (5) After the bones have lost the and the odour of decomposition is lost, the bones still (6) Repeated freezing and thawing of the bones when meboo booksfree.com booksfree.com, 45 = . tissue, fresh., 45 = . , 45 = appear buried. of, 45 = . , 45 = within few. superficially, 45 = . maycause a bone to expand and crack months., 45 = maycause a bone to expand and crack months.. boo, 45 = boo. covering, 45 = covering
It
CHAPTER

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Thanatology deals
with death
in
Somatic death
is circulation,
respiration no
legal definition
the complete
and all
its aspects.
and irreversible
brain of
functions death.
resuscitation and
The stoppage
(tripod of
life).
question organ
cells and
transplantation.
tissues of
individually, vital
Brain death
movements, consists
no spontaneous
cardiac rhythm,
of pupils,
of death
Molecular which
occurs of
(1)
There is
important death
is of
is in
death of
|
to
2
hours after
stoppage deep
unconsciousness, breathing,
(4)
(6)
no reflexes,
flat hours,
(b)
endocrine
(3)
(2)
no stoppage
(5)
EEG, of
spontaneous bilateral
provided body
temperature dilatation
all is
not disturbances.
For transplantation,
within
6
hours, in
72
skin in
cornea
24
hours.
Kidneys, after
stoppage
MODES
of above
below and
are fixation
present
32°C.
can hours,
be bone
heart, of
lungs circulation
OF
Coma
DEATH
is
:
insensibility stem.
Syncope
(1)
Coma.
in and
(c)
(a)
for no
24
metabolic removed
from
48
hours and
liver and
dead body
blood must
vessels be
(2)
which removed
Syncope.
involves is
sudden stoppage
fatal.
ASPHYXIA
:
(1)
respiration, or
is due
eliminate
CO,), to
due soon
(3)
Asphyxia.
central of
portion action
a
condition lack
to of
of heart,
caused oxygen
in which
organs by
of brain
which may
interference respired
and be
with air
(and failure
tissues are
to deprived
of

found dead or dies within 24 hours after onset of terminal illness. (2) About 50%deaths occur due to diseases of cardiovascular system. (3) mebooksfree.com mebooksfree.com, 51 = found dead or dies within 24 hours after onset of terminal illness. (2) About 50%deaths occur due to diseases of cardiovascular system. (3) mebooksfree.com mebooksfree.com
CHAPTER

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Complete loss
of circulation
and respiration
minutes is
accepted of
death.
Suspended animation
functions are
impaired
(2)
However, cases.
(3)
The of
individual dissolved
death.
P.M
changes
:
(1)
for life
continues metabolic
cell in
help
Signs of
some for
more in
rough life
are time
or
than
4
to
5
estimation not
reduced and
resuscitation state
for oxygen
body poisoning
of and
in some
as fluids.
the is
is
(4)
found as
to minimum.
is succssful
so reduced
satisfied
In brain,
the cases
in yoga.
(6)
that through
freezing of
activity start
again.
Involuntary to
half-an-hour electrocution,
of the
(5)
It in
such the
requirement the
the body
brain can
suspension or
more may
cholera, anaesthesia,
insanity,
Skin of
be use
of oxygen
or in
severe can
drug completely
be produced
stop, voluntarily
animation found
in shock,
etc.
becomes minutes.
Eyes due
:
(1)
to
(3)
pale,
Loss drying.
If eyelids
and ashy-white
of corneal
If eyelids
are mucus
for newborn
sunstroke, few
seconds infants,
cerebral and
reflex.
are closed,
open for
forms which
becomes which
a
few two
drowning, concussion,
loses elasticity
(2)
Opacity cornea
hours yellow
brown dust
sunken settles
and after
within of
cornea will
be after
death, triangles
and then
and surface
become flaccid.
on black,
clear
a
sclera few
occurs for
2
film of
each at
called becomes
(5)
“tache wrinkled.
Pupils are
cell hours.
debris side
of noir’,
(4)

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The slightly
iris, upon
eyes dilated
soon look
after as
53 death due to relaxation of muscles of iris. Later, they are constricted due to rigor mortis of constrictor muscles. (6) Retinal vessels show fragmentation or segmentation of blood columns within minutes after death, which persists for an hour. (7) A steady rise in the potassium value occurs in vitreous humour. CoolIngofBody(Algormortis): (1) For half to one hour after death, rectal temperature falls little or not at all. (2) The curve of cooling is sigmoid in pattern. (3) Body heat is lost by conduction, convection and radiation and small fraction by evaporation of fluid from skin. (4) In serious illness, circulation begins to fail before death and hands and feet become cooler, which gradually extends towards trunk. (5) A laboratory thermometer is inserted 8 to 10 cm into rectum for two minutes, or under the liver through small midline opening, or into nose up to cribriform plate or in the ear through tympanic membrane to record temperature. (6) A marked rise in temperature occurs in case of fat or air embolism, infections, heat stroke, pontine haemorrhage, and exercise or struggle before death. (7) low temperature occurs in cases of collapse, congestive heart failure, massive haemorrhage, secondary shock. During sleep rectal temperature is half to 1ºC lower. (8) In tropical climates the heat loss is roughly 0.4ºC to 0.6ºC per hour. (9) Rectal temperature of naked body reaches that of environment in about 20 hours. (10) Time in hours mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com,  = meboo meboo. Rate of temperature fall per hour. Rate of cooling is affected by : (1) difference in temperature body and medium. (2) The build of cadaver :,

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

frozen and stiff. If the body is shifted hot atmosphere, stiffness disappears, and later normal R.M occurs. mebooksfree.com mebooksfree.com booksfree.com booksfree.com, 57 = When which voltage until occur. meboo. , 57 = temperatures, to boo
58
Concise
Forensic
Production
:
Bs echanism
:
(FY
Predisposing factors
:
time
Pou
6
Muscle as
of onset
:
involved:
stiffening:
olecular death:
heat
)
has
:
stimuli importance
testis
:
:
(3)
CADAVERIC
It is
very
rigor mortis
Rigor mortis
Freezing and
and cadaveric
spasm
Cadaveric exposure
to temperature.
Known.
One to
two death.
All the
body, hours
muscles after
of the
both voluntary
and involuntary.
Not marked;
moderate force
can overcome
Occurs.
Cold.
Indicates death.
SPASM
rare
(1)
In this,
become stiff
into stage
attitude and
OR
spasm
Cannot be
by any
death.
Not clearly
produced method
after known.
Sudden death,
excitement, fear,
exhaustion, nervous
tension, etc.
Instantaneous.
Usually restricted
to
a
single group
of yoluntary
muscles.
Marked;
very great
force is
required overcome
to it.
Does not
Warm.
Muscles respond.
Indicates mode
of death,
i.e, suicide,
homicide or
accident.
INSTANTANEOUS
muscles rigid
of primary
of that
immediately relaxation.
person death,
at the
excitement, injury
RIGOR
were contracted
during after
(2)
death,
The time
of
:
life, without
change death.
fear, severe
to nervous
system, etc.
(4)
It involves
is usually
hands, be
pain, passin
preserves
(3)
It occurs

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

exhaustion, firearm
limited wound
to
a
the in
cases severe
of head,
single but
produced
(7)
very rarely
artificially.
It passes
group whole
exact of
sudden haemorrhage,
convulsant of
poisons, muscles,
body
(6)
Stiffness without
rigor interruption
disappears.
(8)
development is
and affected.
is more
into normal
Its mechanism
molecular
(5)
than usually
It as
R.M
is obscure.
death does
not and
(9)
occur, seen
passes
At and
the cannot
in
R.M.
off the
time body
when of
its is
warm.
occur.
it.
time of

clostridia group. (4) Water required for hydrolysis is obtained mainly from body tissues. If body is in mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com meboo booksfree.com booksfree.com,
= meboo

than fatty meal. (7) Head of meal reaches hepatic flexure in 6 hours, splenic flexure in 9 to 12, 1 = . hours, and pelvic colon in 12 to 18 hours. In vitreous humour , there is gradual linear increase in, 1 = . potassium concentration up to 120 hours after death. hair does not grow after booksfree.com booksfree.com, 1 = . , 1 = boo. , 1 = meboo. not, 1 = . , 1 = day.. Beard hair grows at the rate of 0.4 mmper, 1 = . death., 1 = . leaves 2 hours, medium-sized meal 3 to 4 hours., 1 = leaves 2 hours, medium-sized meal 3 to 4 hours.. flies in 3 to 5 days., 1 = flies in 3 to 5 days.. and destroy soft tissues. (3) and adult mebooksfree.com, 1 = and destroy soft tissues. (3) and adult mebooksfree.com. stomach in 2 hours. (3) A light, 1 = stomach in 2 hours. (3) A light. starting point for their inquiries, (3), 1 = starting point for their inquiries, (3). of rigor is delayed, reddish patches region, blood is bright red, tissues mebooksfree.com, 1 = of rigor is delayed, reddish patches region, blood is bright red, tissues mebooksfree.com. meboo, 1 = meboo. onset hypostatic, 1 = onset hypostatic. Maggots, 1 = Maggots. enzymes, 1 = enzymes. 4, 1 = 4. to 5 days, 1 = to 5 days
CHAPTER

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

An injury
is any
harm, illegally
caused mind,
(I)
Due reputation
or property
Classification:
to
(S.44,
Medical:
blunt force:
(1)
(4)
Fractures wounds.
to any
person
I.P.C).
(1)
Abrasions.
and
Dislocations.
(2)
Firearm
(II)
Chop wounds.
Thermal foot.
(c)
Injuries:
Immersion
(III)
alkalis.
(IV)
substances,
(B)
wounds.
(3)
(1)
(2)
Injuries:
Contusions.
in body,
(A)
(3)
Due
Stab
Due foot.
(2)
Due
Chemical
Injuries:
Lacerations.
to sharp
force:
wounds.
to cold.
to
(1)
Incised
(C)
Firearms:
(a)
Frostbite.
heat.
(a)
Burns.
(1)
Corrosive
Electricity, etc.
(V)
Explosions
Legal
:
(1)
Simple.
(2)
:
(1)
Fabricated.
(5)
Grievous.
Suicide.
Defence.
ABRASIONS
:
(1)
involving superficial
by lateral
dragged
(2)
Homicide.
(6)
Undetermined.
Abrasion layers
rubbing action
in vehicular
Some pressure
essential.
is destruction
of epidermis
by
a
(1)
(b)
Trench
(b)
Scalds.
acids.
(b)
lightning
X-rays,
Corrosive radioactive
(3)
Accident.
(4)
of only.
skin,
(2)
blow, accident,
and
a
fall usully
They on
rough fingernails,
movement
(4)
Abrasion has
only throns

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by agent
on length
and are
caused surface,
being or
teeth-bite.
the surface
breadth.
(3)
of skin
is

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

and if blade is nearly horizontal, a flap of wound is caused. Bevelling can be produced by sharp weapon only. It is usually homicidal and may indicate relative position of victim and accused. Difference between incised and lacerated wounds mebooksfree.com mebooksfree.com meboo.Lacerated wound = usually on the sides.. (2) Level:, enters obliquely, tissues will be visible at one margin, and the other margin will be undermined, and if blade is nearly horizontal, a flap of wound is caused. Bevelling can be produced by sharp weapon only. It is usually homicidal and may indicate relative position of victim and accused. Difference between incised and lacerated wounds mebooksfree.com mebooksfree.com meboo.Incised wound = High; above the thyroid cartilage.. (2) Level:, enters obliquely, tissues will be visible at one margin, and the other margin will be undermined, and if blade is nearly horizontal, a flap of wound is caused. Bevelling can be produced by sharp weapon only. It is usually homicidal and may indicate relative position of victim and accused. Difference between incised and lacerated wounds mebooksfree.com mebooksfree.com meboo.Lacerated wound = Low; on or below the thyroid cartilage. Contd... boo
72
Concise
Contd...
(3)
(4)
(5)
(6)
(7)
(8)
(9)
Number of
Edges:
Hesitation cuts:
Tailing:
Severity:
Wounds parts
in other
of body:
(10)
Defence
(11)
(12)
(13)
wounds:
Hands:
Weapon:
Vessels:
Forensic wounds:
Medicine
Above downwards
from left
and to
right in
right-handed person.
Sometimes, horizontal
cuts are
seen not
show which
do variation
depth at
in either
Multiple, end.
may
30, superficial,
and be
20
to parallel
merged with
main wound;
single.
Usually ragged
overlapping the
rarely due
to of
multiple superficial
incisions.
Present.
Present.
Usually less

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One severe.
wound sometimes
severe
2
to
Often but
3.
present wrists,
across groin,
ankles, thighs,
or rarely
knees;
and on
neek.
Absent;
unitentional cuts
may be
the found
on fingers
has if
a
been blade
used.
Weapon may
be firmly
grasped cadaveric
due spasm.
Usually present.
As head
is thrown
carotid artery
beneath back,
is drawn
sternomastoid and
against usually
the spine
and escapes
injury.
the back
of to
Transverse or
from below
upwards.
attacked
If from
the right
side of
the vicitm,
wound from
runs left
to right;
from behind,
resemble if
it may
suicidal wound.
Multiple, other
cross each
at
a
deep not
repeated of
the level;
in depths
main wound.
Sharp and
clean-cut;
bevelling may
Absent.
Absent.
More severe;
all tissues
including vertebrae
be seen.
the the
may be
No wounds
etc.
on but
severe usually
on and
the neck.
Present.
Fragments of
clothing, hair,
etc., may
be grasped.
Usually
Jugular carotid
absent.
vein and
artery likely
to are
be cut.
cut.
wrists, injuries
head
Contd...

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= . Concealed puncture wounds: (1) They are seen in parts of the body, such as nostrils, fontanelle, fornix of eyelid, axilla, vagina, rectum, nape of neck . (2) A careful is necessary to detect them. (3) Fatal injuries may be caused leaving any external marks, e.g. thrusting a needle or pin into brain through the fontanelles, through the inner canthus of the mebooksfree.com mebooksfree.com,  = blood.. or into the medulla through the nape of the neck.,  = concealed upper search without the meboo. booksfree.com,  = eye, (1) They are caused due to immediate and booksfree.com. instinctive reaction of victim to save himself , either by raising,  = arm by grasping the weapon. (2) If weapon. to prevent the attack or,  = . is blunt, bruises and abrasions are produced on,  = boo. Defence wounds:,  = . extensor or,  = . ,  = ulnar
82
Trait
(1)
Size:
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
Edges:
Contusion, abrasion
and grease
Burning,
Bleeding:
Fat:
Tissues wound:
Fibres of
clothing:
Lead ring
ring:
(10)
Spectrography:
Abrasion collar:
stretches skin
hole due
which to
rubbing epidermis
(abrasion lead
from on
skin,
entrance
Entrance wound
Smaller than
of bullet.
Inverted.
Present.
May be
seen the
wound.
Less.
No protrusion.
May be
cherry due
to
CO
gases.
Turned in
be carried
wound.
May be
around red
of explosive
and may
into seen
the wound
by radiological
More metal
is around
found entrance
wound, if
bullet only
has passed
the soft
(1)
through tissues.
As bullet
is strikes
perforated.

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

of skin,
(2)
The gyrating
body collar).
surface of
around collar
(3)
Bullet bullet,
the sorrounds
collar, defect
dirt and
as such,
strikes body
and if
it at
strikes right
exit wounds
Exit wound
Bigger than
Everted, bullet.
puckered torn.
Absent.
Absent.
More.
May protrude.
Absent.
Turned
Absent.
The exit
contain out.
wound more
a
bone may
metal is
to it.
it skin
first indents
is struck
if nearer
and abraded
of the
bullet lubrication,
barrel debris,
(grease collar.
then around
against gun
the inverted
oil etc.
from produce
or dirt
(5)
Some it
is also
angle, at
an collar).
barrel,
a
narrow
(4)
contusion called
abrasion angle,
the wound
The is
ring abrasion
present contusion
in collar.
collar is
(6)
circular is
round and
but marginal
abraded
If bullet
uniform, abrasion
or the
diameter around
or metal
within collar:
Concise
Forensic
Difference and

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produced in inner table and bevelled opening on outer table. (5) The exit wound is larger due to deformity and tumbling of bullet after entering the skull. (6) Asymmetry of bevelling indicates angle of fire. Exit wounds: The variation in the shape and size of exit wounds are dut to: (1) Bullet tumbles in the body and fails to exit nose-end first. (2) Bullet is deformed. (3) Bullet breaks up in the tissues and exits as several pieces. (4) Fragments of bone are blown out of body with the bullet. (5) Unsupported skin at the exit tears and breaks up into pieces. SHorED or SuPPorTED ExIT wounD: If the skin at the exit wound is firmly supported by an object, e.g. a belt, wrist band of trousers, brassiere, collar and tie, etc. or if the body is leaning against a wall, back of chair, etc. the exit wound appears as circular defect, surrounded by margin of abrasion resembling wound of entrance. A bullet travelling in an irregular fashion instead of travelling mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com. nose on is called a yawning bullet. A bullet that rotates end-on-end, 1 = nose on is called a yawning bullet. A bullet that rotates end-on-end. during its motion is called tumbling bullet., 1 = meboo
Injuries
85
fired, and is known as "'metallic fouling". (6) Bullet found in the body called crime bullet or exhibit bullet is compared under a comparison microscope with the one fired from suspected weapon known as test bullet. (7) The suspected weapon is fired using the same brand and type of ammunition into a roll of wool, or a bag of rags, or sand bag, or against white blotting paper. (8) Fresh pork skins, cleanly shaven are ideal for comparison with patterns on human skin.
BOMB
EXPLOSION
WOUNDS:
When explosion
occurs,
a
large volume
of
Severe victim
gas and
energy damage
are or
produced.
traumatic is
a
few metres
of
a
localised irregular

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area areas
so that
(1)
Disruptive amputations
away or
occurs.
of skin
beneath may
be with
are smaller
(2)
Burns to
different collars,
quite normal.
which can
toss
Alveolar bras,
effects:
caused.
When explosions,
are extensive
degrees.
waist
(3)
the the
mutilation which
Tight involve
clothing bands,
socks
Air person
septa oedema
(blast not
are blast:
A
through torn
protects, and
shock shoes
wave the
producing lung).
damaged.
injuries,
Homogeneous
Intracranial ruptured
from air
air causing
the skin
is produced
blunt haemorrhage
like liver
haemorrhage, stomach
embolism.
wood, brick,
injuries.
pulmonary and
contusions and
(4)
bowel may
Flying etc.
cause intimately
mixed may
on the
cause and
asphyxia.
traumatic missiles:
bruises, muscle
of are
brain, occur.
Death
Bomb abrasions
skin.
This
(6)
heart may
pieces, and
triad of
injury gravel,
puncture occur
glass, lacerations
is diagnostic.
(5)
CO
Falling asphyxia.
masonry causes
multiple
injuries

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Scalp:
(1)
Bruising of
scalp sight.
(2)
often it
is firm
edge
Multiple contusions
difficult to
often is
better of
determine feels
wound by
blunt
SKULL:
the the
like detected
scalp by
may fuse
number the
edge weapon
resembles
Fractures:
fractures or
cracks of
blows of
a
depressed an
(1)
Fissured in
bone only.
outer table
resisting
(2)
surface striking
surface
They like
or bony
buttresses.
(2)
touch than
by together,
and inflicted.
(3)
fracture.
incised
(4)
wound.
fractures:
(1)
These linear
thickness are
involving whole
caused by
forcible
Its
Scalp are
with or
inner contact
broad ground,
from
(4)
heavy weapon
with
Depressed axe.
(2)
They are
also as
the fractures:
The
a
or agent
fall
a
on feet
having or
buttocks.
(1)
small striking
a
(3)
or broad
They are
They do
not caused
,
into surface
to outer
table is
driven
a
greater extent
called pattern
Sometimes,
“
resembles the
it only
may may
and
&.g.
by
Stone, diploe,
blows stick,
inner may
weapon involve
be
(3)
or more
pieces.
fractured.
Comminuted fractures:
(2)
They surface,
striking outer
or table
agent only.
(1)
are vehicle
be which
(5)
The caused
accidents, surface,
e.g.
axe, and
cross from
hammer, is
fractured comminuted.
(3)
), caused
it.
Rarely, bone
inner is
by
a
fall from
from thick

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by etc.
(3)
fractured
(4)
table into
a
height on
weapons with
They may
3
hard
a
large result
from table
87 kick, etc. (4) Whenthere is no displacement of fragments, it resembles a spider's web or mosaic. (5) Fissured factures may radiate from area of comminution. (4) Pond or indented fractures: (1) This is simple dent of skull, which is caused by obstetric forceps blade, a blow from a blunt object, or forcible impact against protruding object. (3) They occur in skulls which are elastic, i.e. infants. (3) Inner table is not fractured, but fissured fractures may occur in outer table. (5) Gutter fractures: These are formed when part of thickness of bone is removed to form gutter, e.g. in oblique bullet wounds. Inner table may show irregular depressed fractures. (6) Ring or foramen fracture: (1) (A) Fissured fracture encircles the skull and separates anterior third with middle and posterior third. (B) Usually, fracture runs 3 to 5 cm. outside foramen magnum at back and sides of skull and passes through middle ears and roof of nose, due to which skull is separated from spine. (2) They are rare and occur after falls from a height on to feet or buttocks. (3) Asevere blow to vertex or foreceful blow on chin in traffic accident may produce ring fracture. (7) Perforating fractures: They are caused by firearms and pointed sharp weapons like daggers or knives and axe. (8) Diastatic or sutural fracture: Separation of suture of skull occurs alone only in young persons due to a blow on head with blunt weapon, but is often associated with fracture. Mechanism of cerebral injury: (1) A fatal brain injury may be caused without any damage to scalp or skull. (2) Brain injury may be caused by: (a) Penetration by a foreign object. (b) Distortion of the mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com meboo,  = meboo meboo

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Homicide:
It is
killing of
a
human being
by another
human
Type:
()
Lawful:
(1)
Murder.
(2)
(1)
Culpable amounting
to murder.
Justifiable.
homicide,
(3)
Justifiable the
(1)
(2)
Excusable.
(a)
being.
(ID)
amounting
Rash or
Unlawful:
to negligent
homicide:
circumstances
This which
led in
the administration
(2)
the maintenance
arrest or
is murder,
homicide.
the to
the of
homicide killing
justice, of
justice, killing
in course
person who
attempts
Excusable by
which of
person.
like is
(b)
not justified
This execution
e.g.
in of
in may
of suppressing
violent to
rape homicide:
an act
done crime,
her.
This in
when good
faith.
attacked, causing
death provided
is the
This there
by operation,
(4)
Culpable an
accident homicide
homicide includes
occur death
riots sentence,
or e.g.
a
woman caused
(1)
is no
other or
misadventure, committed
homicide:
act
(1)
with of
causing the
executing who
kills
a
unintentionally killing
means in
self-defence of
(3)
death by
an insane
Culpable the
homicide intention
such bodily
of causing
injury knowledge
that such
Explanations:
defence,
(2)
following lawful
person.
is causing
death, as
is likely
act is
(1)
who is
suffering
A
from accelerates
the death
his death.
who
(2)
Where caused
such likely
person death
or
(2)
to with
cause to
cause who
a
disorder, causes
the death,
death by
doing intention
or
(3)
with
(S.299, bodily

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disease, of
a
person, death
bodily or
shall be
is caused
injury by
shall injury
bodily
LP.C.).
to another
infirmity deemed
which to
have bodily
be injury,
deemed the
to have
caused person
caused

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mebooksfree.com mebooksfree.com meboo, 101 = meboo meboo
102
Trait
(1)
Margins:
(2)
(3)
(4)
(5)
(6)
(7)
Haemorrhage:
Spurting:
Extravasation:
Coagulation:
Vital reaction:
Enzyme histochemistry:
The signs
of leucocytic
vital infiltration,
by first
intention infection.
Concise
Forensic
antemortem
Antemortem and
wound
The edges
are everted,
postmortem wounds
Postmortem swollen,
retract wound
gapes.
Abundant and
usually arterial.
Signs of
arterial spurting
of blood
body, on
clothing or
vicinity present.
Staining of
the edges
of the
wound and
extravasation in
neighbouring subcutaneous
and interstitial
tissues which
cannot removed
be by
washing.
Firmly coagulated
blood in
tissues wounds
and present.
Signs of
vital reaction
present, inflammation
i.e., and
repair.
Increased activity
of
ATP, amino-peptidase,
acid and
alkaline phosphatase.
reaction are:
pus formation,
means, union
of swelling,
wound
Edges do
not closely
gape but
approximated.
Slight or
more,
No venous.
spurting
Edges of
blood.
and cellular
stained.
No clotting
No signs
not deeply
or of
reaction.
Diminished or
no enzyme
activity.
effusion evidence
two of
of repair.
granulating lymph,
Healing surfaces
without soft
clot.
vital the
in its
and

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Hypothermia is
oral or
axillary
Trench foot
exposure and
to severe
in winter
warfare,
CHAPTER
temperature of
less immersion
cold
(5
to
8°C)
especially prolonged
immersion.
Frostbite and
foot occur
due and
dampness;
in trenches,
The extremities
occurs due
affects to
extremities, formation
and if
to seen
and in
persons are
exposure nose,
gangrene maintained
due for
to anoxia.
Neonatal prevent
a
fall
(1)
There eyelids.
is
(2)
feet cold
affected.
to ears
extremes and
occurs.
24
Body hours
causes injury
in of
cold face.
Necrosis temperature
death results
temperature swelling
than
35°C.
prolonged in
soldiers exposed
to
(-2.5°C), with
of blister
27°C
from failure
from in
a
body of
failure or
less, of
vital centres
of metabolism
exposed extremities,
Respirations to
cold particularly
slow, are
red.
(3)
shallow,
Widespread is
haemorrhagic subcutaneous
pitting tendency,
to environment.
hands, irregular;
oedema and
tissue feet
and face,
hands, of
extremities.
localised overlying
Heat hyperpyrexia
more than
delirium, oedematous
(heat hardening
and
(4)
There of
skin, and
parts.
stroke):
41°C.
(2)
(1)
Neurological stupor,
to sun
it coma
is
Rectal disturbances,
and convulsions.
called temperature,
sunstroke.
temperature such
(3)
If there
(4)
Precipitating increased
humidity, and
lack of
acclimatisation.
minor
(5)
infections,
If humidity
is as
psychosis, is
direct exposure
factors are:
high muscular
is

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1 = Antemortem burns. Trait, 2 = Postmortem burns. (1) Line of redness :, 1 = Present.. (1) Line of redness :, 2 = Absent.. (2) Blister :, 1 = Contains serous fluid with proteins and chlorides. Base is red and inflamed. mebooksfree.com. (2) Blister :, 2 = Contains air and thin clear fluid. Base is dry hard and yellow. meboo. (3) Vital reaction :, 1 = Marked cellular exudation and reactive changes in the tissue cells present.. (3) Vital reaction :, 2 = Absent.. (4) Enzymes : booksfree.com, 1 = Peripheral zone of burns shows increase in enzyme reaction. (4) Enzymes : booksfree.com, 2 = Peripheral zone does not show increase in enzyme reaction. booksfree.com
108
Concise
Forensic
Medicine
110
copper
Concise Forensic
Medicine in
the dermis.
(3)
and disappear
in
|
to
They indicate
2
days vary from 3 to 30 cm, in
if person
length, often
seen in
moist creases
are true
burns and
occur the
paths survives.
and
0.3
and folds
taken by
discharge
(II)
to
2.5
Linear cm.
of skin.
in
(III)
burns width,
and
Surface beneath
due to
accident.
metallic objects.
Death are
burns is
always
STARVATION
Acute starvation
chronic starvation
Acute occurs
from
:
(1)
CHAPTER
from complete
stoppage gradual
Feeling pain
in epigastrium.
absorption deficient
of hunger
(2)
After of
supply for
30
4
of of
food.
to to
subcutaneous pupils
dilated, thick
and
5
48
days, fat
begins lips
dry and
cracked, scanty,
voice wrinkled,
hours, general
to followed
emaciation occur.
tongue whispering,
pigmented, abdomen
progressive cardiovascular
constipation,
(3)

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coated skin
dry, and
by and
Eyes sunken,
and rough,
concave, insufficiency,
urine scanty,
turbid emaciation,
offensive
(5)
Loss of
odour
40%
dirty, thin,
limbs saliva
inelastic, thin,
subnormal with
evidence before
of body
Chronic
:
progressive weight
death.
flaccid, temperature,
of acidosis,
(4)
Intellect is
Hunger-pains, loss
gross mental
Death fatal.
mental of
weight, polyuria,
retardation, occurs
Fatal period
withdrawn
:
6
and physical
pigmentation, loss
of self-respect,
from exhaustion,
:
10
to to
Autopsy
8
:
(1)
premature extreme
clear till
death.
lethargy, hypothermia,
oedema of
circulatory
12
days if
both weeks
or more
All organs
senility.
absent in
(2)
All failure
water and
if food
and organs
subcutaneous food
alone tissues
lower limbs.
or intercurrent
are completely
is show
are tissues,
reduced withdrawn.
changes similar
in size,
omentum, to
fat is
completely mesentery,
and about
food,
112
Concise internal
organs, is
small
Forensic which
from brown
atrophy of
all distended
Medicine is
never seen
atrophy;
coats;
with lungs
in wasting
collapsed, non-specific
bile, and
diseases.
pale;
ulceration of
urinary bladder
with marked
anaemia;
empty;
(3)
G.I.
tract bowel;
blood
Heart shows
gall bladder
volume osteomalacia.
reduced
CHAPTER

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HANGING
:
(1)
It is
a
form of
asphyxial suspension
of
a
constricting person
force by
ligature being
are partially
weight suspended
or any
other above
or position.
In thyroid
to occipital
cartilage region.
Causes of
Death compressive
narrowing of
tongue against
over death
which encircles
of body.
are in
a
In partial
sitting, caused
neck, hanging,
kneeling, typical
hanging, symmetrically
lying ligature
upwards
:
(1)
Asphyxia of
lumens posterior
entrance blocked,
of on
:
Air of
larynx wall
larynx.
of
(2)
if ligature
venous occurs
is pharynx
Venous broad
congestion by
the bodies
down, runs
from prone
midline both
sides is
and blocked
trachea and
and folding
congestion and
soft.
(commonest with
ligature tissues.
(5)
of neck
due to
forcing of
:
Jugular
(3)
Combined cause).
made
Vagal vertebrae.
Delayed deaths
of thin
(6)
occur
Infections.
(3)
cord
Fracture due
Oedema encephalopathy.
veins are
asphyxia
(4)
Cerebral which
and anaemia,
sinks deeply
or dislocation
to of
:
(1)
Aspiration lungs.
(6)
Abscess
Autopsy
:
3
(4)
brain.
to
5
:
minutes.
External important,
which and
:
depends multiplicity
of of
suspension.
Ligature mark
in the
on
:
(1)
into of
cervical pneumonia.
Oedema larynx.
(7)
(2)
(5)
Hypoxic brain.
neck
Composition ligature.
(4)

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(3)
Tightness
Weight is
specific of
of body
of encircling
ligature.
and
(2)
Width suspended
and ligature.
(5)
degree
Length of
may be found dribbling from angles of mouth, which is an important sign. (7) p.M lividity is seen in lower limbs and hands. (8) Eyes are protruded and firmer, conjunctivae congested and pupils dilated. (9) If the ligature knot presses on cervical sympathetic, the eye on the same side may remain open and its pupil dilated. It indicates antemortem mebooksfree.com mebooksfree.com,  = . hanging (le facie sympathique) . internal : (1) Incision of groove may show small haemorrhages in the underlying layers of skin. (2) Tissues under mark are dry, white and glistening. (3) platysma and sternomastoid rupture and intima of carotid arteries show transverse splits with extravasation of blood in 5 to 10%. (4) Hyoid bone is fractured in 15 to 20% of cases. Fracture is common in persons above 40 years and involves greater horns at junction of inner two-thirds and outer one-third. (5) Superior horn of thyroid cartilage is fractured in about 40% of cases. (6) Lungs are congested and oedematous. (7) Trachea is usually congested. petechial haemorrhages may be found on epiglottis, in the larynx and trachea. (8) Internal organs are congested. (9) Subpleural ecchymoses may be found. Unusual positions, e.g. where parts of body touched ground, kneeling or reclining, are almost diagnostic of suicide. In postmortem hanging, marks of violence and signs of may be found on body, but dribbling of saliva is not seen and from the rope are not found on hands of victim. meboo mebooksfree.com mebooksfree.com meboo,

meboo = Fracture is uncommon.. (7) Thyroid cartilage:, Mechanical Asphyxia 117 internal : (1) Severe congestion and haemorrhage into tissues in and above the area constricted is seen. (2) There may be superficial haemorrhages under the ligature mark. (3) Neck muscles are usually lacerated. (4) Rarely, there is no external mark, but extensive bruising of deeper tissues may be found. (5) If ligature is tightly applied on the neck until death occurs, bruising will be absent. (6) Intima of carotid arteries is not damaged. (7) Hyoid bone may be fractured in older persons in 10 to 15% cases. (8) Fracture of thyroid cartilage is more common. (9) Bruising of tongue and floor of mouth occur. (10) Larynx, trachea and bronchi are congested, may show haemorrhagic infiltration, and contain forthy, blood stained mucus. mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com.Hanging = Fracture is less common.. (7) Thyroid cartilage:, Mechanical Asphyxia 117 internal : (1) Severe congestion and haemorrhage into tissues in and above the area constricted is seen. (2) There may be superficial haemorrhages under the ligature mark. (3) Neck muscles are usually lacerated. (4) Rarely, there is no external mark, but extensive bruising of deeper tissues may be found. (5) If ligature is tightly applied on the neck until death occurs, bruising will be absent. (6) Intima of carotid arteries is not damaged. (7) Hyoid bone may be fractured in older persons in 10 to 15% cases. (8) Fracture of thyroid cartilage is more common. (9) Bruising of tongue and floor of mouth occur. (10) Larynx, trachea and bronchi are congested, may show haemorrhagic infiltration, and contain forthy, blood stained mucus. mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com.Strangulation by ligature meboo = Fracture is more common. boo
118
Contd...

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

(8)
Larynx
Forensic
Medicine
Fracture trachea:
Emphysematous
(9)
bullae
:
(10)
Carotid
(11)
(12)
(13)
(14)
(15)
(16)
Face:
Signs of
Tongue arteries
:
asphyxia:
:
Saliva:
Bleeding
:
Involuntary discharage
:
(17)
PALMAR
Seminal fluid
:
rare.
Not present
surface on
of the
Damage the
lungs.
may
Usually be
pale seen.
and petechiae
are common.
External marked.
Swelling and
protrusion is
marked.
Often runs
mouth.
From the
and ears
Of out
less of
nose, mouth
not faeces
less common.
and urine
common.
At glans
is more
common.
STRANGULATION:
horizontally across
its pressure
it at
the mouth
and being
right reinforced
angles, of
the
The neck.
face the
The palm
nostrils by
Fracture found.
Very common
surface of
be on
the the
Damage is
lungs.
rare.
Congested, livid
marked with
External and
petechiae.
signs marked.
more marked.
Absent.
From the
and ears
Of faeces
more nose,
mouth common.
and urine
common.
At glans
is less
common.
of one
hand without
placing heel
of the
Diffuse bruising
is congested
In cases
using the
upper is
the other
palm with
fracture with
of due
to hanging,
which the
hyoid the
bone divergence
outward
Common of
greater displacement
methods
Ligature palm
pressing on
up of
thyroid is
forced horns
of placed
fingertips, the
top of
on the
may front

#/texts/4974 #/texts/4975 #/texts/4976 #/texts/4977 #/texts/4978 #/texts/4979 #/texts/4980 #/texts/4981 #/texts/4982 #/texts/4983 #/texts/4984 #/texts/4985 #/texts/4986 #/texts/4987 #/texts/4988 #/texts/4989 #/texts/4990 #/texts/4991 #/texts/4992 #/texts/4993 #/texts/4994 #/texts/4995 #/texts/4996 #/texts/4997 #/texts/4998 #/texts/4999 #/texts/5000 #/texts/5001 #/texts/5002 #/texts/5003 #/texts/5004 #/texts/5005 #/texts/5006 #/texts/5007 #/texts/5008 #/texts/5009 #/texts/5010 #/texts/5011 #/texts/5012 #/texts/5013 #/texts/5014 #/texts/5015 #/texts/5016 #/texts/5017 #/texts/5018 #/texts/5019 #/texts/5020 #/texts/5021 #/texts/5022 #/texts/5023 #/texts/5024 #/texts/5025

be seen.
directly back-wards,
is increased,
the of
which may
pusterior homicidal
strangulation.
(5)
Mugging.
BANSDOLA
:
(1)
front and
another rope
stick due
to which
is placed
(2)
Throttling.
One small
strangulation
(3)
strong on
are
:
(1)
Bansdola.
(4)
Garrotting.
bamboo back
of neck
victim across
is front
and squeezed
of or
stick is
both ends
to neck
and death.
placed across
are tied
with
(2)
assailant
Sometimes stands
a
with
a
foot well-
signs not
less may

(2) anteroposterior compression : (1) In cases of hanging, the hyoid bone is forced directly backwards, due to which the divergence of greater horns is increased, which may fracture with outward displacement of the posterior small fragments. (2) periosteum is torn on inner side. (3) Fracture may occur either in greater horn or at its junction with body and it may be bilateral. (4) It may also be seen in ligature strangulation, run over motor vehicle accidents and blows on front of neck. (5) The fractured fragments are displaced outwards. (3) avulsion fractures (tug or traction fractures) : They occur due to muscular overactivity without direct injury to hyoid bone. SUFFOCaTiOn : it is that form of asphyxia which is caused by deprivation of oxygen either due to lack of oxygen in environment or from obstruction of air-passages at level of nose and mouth . SmOTHEring : (1) it is caused by closing external respiratory orifices either by hand or other means, or by blocking cavities of nose and mouth by foreign substances such as cloth, paper, etc. (2) Suicidal smothering by hand is impossible. It can be caused by burying face in a mattress or by tying polythene bag over the head. (3) Most deaths are accidental due to : (a) In epileptic or intoxicated person by burying his face in a pillow, etc. (b) Falling into a large quantity of semisolid or finely divided material, like mud, ashes, grain, sand, etc. (c) Air- tight place, e.g locked boxes or trunks. (d) Inhalation of irrespirable gases, e.g. CO, CO 2 , hydrogen sulphide. (e) Application of plastic bags for experiment or autoerotic exercise. Homicidal smothering occurs due to closure of the mouth and nose by a hand or cloth, or pressing the face into a pillow. mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com meboo, 121 = meboo

foreign body which caused the occlusion of air-passages will be found in mouth, throat, larynx or trachea. (6) When food or vomited material has been inhaled, particles of food material may be observed embedded in thick mucus in trachea and bronchus. TraUmaTiC aSPHyxia : it results from respiratory arrest due to mechanical fixation of chest, which prevents normal movements of chest wall. Fatal cases are only due to accident. Causes : (1) Gross compression of chest and abdomen as in stampedes. Victim may fall on ground and crushed under feet. (2) Falls of earth or stone during tunnelling or in coal mine, etc, or building collapse. (3) Run over by a vehicle or crushed by machine. (4) Indirect compression due to thighs and knees being pressed against chest, so-called jack-knife position . autopsy : (1) intense cyanosis of deep-purple or red colour of head,neckandupperchest,abovelevelofcompressionisprominent feature. (2) Belowthis level skin is pale or slightly cyanosed. (3) Areas of pallor in areas of pressure, such as collars, buttons, folds in clothes etc. (4) Multiple injuries. (5) If patient survives, purple colour will mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com meboo, 123 = meboo

mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com,
= meboo meboo. sea water drowning, lungs are ballooned and heavy, weight up to two kg, purplish or bluish, sodden and jelly-like, tend to flatten out meboo,  = . after removal from body; large amount of liquid and froth is present, and on section crepitus is not heard. If the body remains in water for several hours, differences in appearance between fresh and sea water,  = . booksfree.com booksfree.com,  = . (dry-lung drowning) are seen, due to absorption of water,  = boo. lungs,  = . ,  = dry. drowning are not clear. (9) In many cases of drowning, relatively,  = . ,  = from
is put in a test tube and covered with five times of its volume of concentrated nitric acid and left for one day to allow digestion. This process chars, blackens and destroys organic matter but not diatoms which have silica shells. (8) The tube is centrifuged, supernatant acid poured off and replaced with distilled water and centrifuged. This process is repeated two or three times, and deposit examined under phase contrast or dark ground illumination. (9) Two litres of water is obtained from accident site and examined for diatoms. (10) Findings similar diatoms in water and body tissues is in favour of drowning. (11) Finding of diatoms is not absolutely diagnostic but is of greatest value in putrefaction. Tests : (1) gettler Test is of no value. (2) The specific gravity of plasma from left side of heart is less than that of right side in drowning. The reverse is seen in non-drowning cases. (3) Serum strontium is raised in drowning. (4) With decomposition water transudes into pleural cavities. (5) In advanced decomposition signs are completely absent. Difference in lungs between fresh water and sea water drowning mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com meboo,

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Impotence is
the inability
of
a
person intercourse.
Sterility and
in female
without is
inability being
or both
to impotent
may co-exist.
conceive to
of male
children.
or he
can be
Frigidity sexual
arousal pattern
Examination:
is perform
to
A
impotent the
in sexual
beget children,
person can
without inability
female.
Obtain especially
nervous complete
and medical
mental, examination
for sensation.
Opinion:
that
If the
person in
negative history
and should
male sexual
be carried
external is
impotent.
form, finds
nothing intercourse.
Causes of
impotence may
be present
development genitalia
In stating
to suggest
to be
sterile being
sterile, start
or maintain
of previous
history.
out.
illness,
A
complete
Private are
such parts
normal, case
that that
from the
in are
it tested
cannot opinion
should be
said be
examination person
male:
much earlier
of sex
organs precocious
development are
developed coitus
of is
incapable
(1)
Age:
given male,
he of
(a)
than puberty.
is common
as in
at an
early may
diminish.
(2)
Defects
Impotency gonadal
Power sexual
of
(2)
cause erection
Poor physical
of or
age.
adrenal
(4)
In advanced
(5)
Sperms of
development occurs
Double penis
and are
impotence.
tumours, age
not
(3)
In sex
organs ability
found and
due to
absence penis
adherent before
to perform
puberty.
acquired abnormalities:
or non-development
to scrotum
(1)
of penis.
may cause
(2)
difficulty in

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

= meboo. ArtIFICIAL InSEmInAtIOn: It is artificial introduction of semen into vagina, cervix or uterus to produce pregnancy. types: (1) Homologous (semen of woman's husband is used (A.I.H.). (2) Donor (semen of any person other than husband is used (A.I.D.). (3) Pooled (semen of husband + semen of some other person (A.I.H.D). One ml. of semen is deposited by means of a syringe in or near cervix on several successive days, about the time of ovulation. Success rate is 70 to 75% within 3 to 4 months. Indications: (1) Husband impotent. (2) Epispadiasis or hypospadiasis. (3) Husband sterile. (4) Rh incompatibility between husband and wife. (5) Husband suffering from hereditary diseases. mebooksfree.com mebooksfree.com booksfree.com booksfree.com,  = meboo
132
Concise
Forensic
Precautions:
Medicine
(1)
Consent donor
kept secret.
donated and
healthy.
(3)
its of
donor
Donor result.
(5)
Donor and
should
(4)
Donor must
Consent of
woman
Pooled semen
Legal and
not his
know should
not of
be donor
to be
relative wife.
(2)
Identity to
whom mentally
of should
of semen
is and
physically either
spouse.
resemble be
inseminated to
be used.
problems:
becomes divorce.
and husband
her
(9)
Witness
(1)
illegitimate.
(3)
(4)
of donor.
Risk of
Parties
(6)
Race of
woman.
husband must
be not
guilty
Not
a
ground in
present.
of for
adultery.
nullity
(7)
writing.
(8)
(2)
Child of
marriage incest
between children
born of
A.I.
or and
children
cunrtenl4

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VIRGINITY
:
Virgin
(virgo intacta)
is
a
female who
not experienced
virginity.
Genitals:
(1)
rounded, lie
sexual intercourse.
Ina virgin,
in minora
are narrow.
Vagina
Defloration labia
majora apposition
within
(4)
and close
labia majora.
The posterior
is walls
means are
thick, firm,
vaginal
(3)
Clitoris commissure
narrow and
tight, are
about one
hymen has
loss elastic
orifice.
(2)
is small
and fourchette
mucosa is
(6)
The mm.
thick, hymen
situated consists
types of
of folds
hymen
Infantile.
rugose, reddish,
is
a
fold at
vaginal of
membrane.
are
:
(a)
(d)
Cribriform.
(8)
The margins
be mistaken
Semilunar or
(e)
of
Vertical.
hymen for
artificial symmetrical,
(b)
(d)
covered are
of and
The labia
and vestibule
are intact.
(5)
sensitive of
mucous outlet.
and membrane
The average
Hymen is
adult intact.
crescentic.
(f)
Septate.
sometimes tears.
(7)
The
(b)
Annular.
(g)
fimbriated,
(9)
occur
Natural anteriorly,
with intercourse
one or
mucous or
by
(c)
do not
membrane.
foreign both
(c)
sides, not
covered
Causes body
are or
(a)
in midline,
with mucous
of notches
(c)
Imperforate.
which may
are extend
usually to
(10)
Tears vaginal
(a)
wall, caused
usually
(b)
situated usually
by sexual
posteriorly extend

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membrane.
rupture projecting
of substance.
Masturbation.
hymen
In
:
(1)
these
(3)
Surgical pith.
(5)
Ulceration to
irritation.
cases tearing
operation.
from diphtheria,
(7)
Sanitary to
Accident, at
vaginal wall,
e.g.
of fall
perineum
(4)
Foreign fungus,
tampons.
on occurs.
body, etc.
(6)
(2)
e.g.
sola
Scratching due

= boo. fertilisation of a second,  = fertilisation of a second. : (1),  = : (1). the only certain examples of this this means,  = the only certain examples of this this means. are SUPerFoetation mebooksfree.com,  = are SUPerFoetation mebooksfree.com
CHAPTER

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

S.
375,
I.P.C.:
A
man is
(a)
penetrates urethra
or anus
other of
person;
(b)
or inserts,
being to
the makes
penis, his
penis,
a
woman any
into said
to commit
to any
or extent,
the her
to do
so
(c)
extent, makes
any
“rape”
into her
the to
object vagina,
with manipulates
penetration into
the woman
or makes
(d)
applies the
him or
do or
urethra any
any part
vagina, her
his or
makes her
circumstances other
of the
urethra, to
do mouth
to do
so with
to so
a
part if
he:
vagina, with
him of
or the
anus person;
body anus
of or
of
a
woman or
him or
the so
with falling
(1)
Against
(2)
(3)
putting under
her vagina,
him any
will,
Without her
With the
her or
of consent.
consent, or
hurt.
(4)
With husband
any her
and another
man when
person consent,
that any
so part
mouth, or
any body,
a
not woman
or as
to cause
of any
body other
anus, or
of person;
of the
her in
whom when
her to
other of
such or
a
person, following
consent woman
under the
seven descriptions:
has she
the consent
whom is
been is
interested, man
in knows
given she
is or
fear of
death that
because believes
she herself
he is
believes to
be not
her that
lawfully he
is married.

bleed on touch . (4) After 3 to 4 days, edges are congested and swollen, which heal in one week, but do not unite. (5) Rupture can be caused by fingers or foreign bodies, as such evidence of local injury is not proof of penetration. (6) In absence mebooksfree.com mebooksfree.com booksfree.com booksfree.com, 143 = meboo
UnnaTURaL OFFenCeS : Voluntary sexual against order of nature with any man, woman, or animal unnatural sexual offence (S.377, I.p.C.) . Punishment is for up to 10 years or life. Homosexuality means persistent and physical attraction to members of the same sex. SODOMY : (1) Sodomy is the mebooksfree.com mebooksfree.com,  = intercourse is imprisonment emotional anal intercourse between two meboo

sex, or to watch mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com booksfree.com,
= usually clothed parts of body of one of sexual intercourse as a source of sexual meboo meboo meboo booksfree.com

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= . The neck is very short; tail is long and tapered. Absolute proof of semen is finding of at least one unbroken or electrophoretic LHDisoenzyme detection of sperm. absence of spermatozoa, a stain which gives fluorescence in U.V. a meboo,  = . (8),  = . spermatozoon, In,  = . light, positive precipitin test, a high level of acid phosphatase booksfree.com,  = . high,  = . and,  = . semen.,  = . phosphokinase can be considered to be due to,  = . creatine,  = . booksfree.com,  = booksfree.com
ABORTION
Legally, abortion
of foetus
(miscarriage)
from mother’s
term of
pregnancy
Natural
10
CHAPTER
means premature
womb at
any is
completed.
abortion to
one
15%
of occurs
all commonly
pregnancies.
which is
done
Criminal
Justifiable in
good faith
abortion of
foetus from
is to
save the
womb therapeutic
of indication.
Medical induced
mother
It is
time expulsion
of pregnancy,
in second
before or
third or
month therapeutic
the in
abortion life
of woman.
destruction unlawfully,
usually
Termination the
Act, pregnancy
continuation and
i.e.
expulsion when
induced before
of can
Pregnancy be
of pregnancy
severe injury
there is
risk abnormality.
to
Act terminated:
endangers her
mental of
child third
there is
no month.
(M.T.P.)
(1)
life or
physical being
born
(3)
1971:
Under
Therapeutic:
of woman,
or may
health.
(2)
Eugenic:
with
Humanitarian:
Social:
(a)
Pregnancy in
a
married serious
Pregnancy resulting
from woman,
mental health,
which
(b)
mother’s is
when health.
Rules:
25
to
(1)
A
cases of
qualified
M.T.P.
obtain licence
of woman,

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

insane social
physical
When cause
When or
mental caused
by rape.
failure of
contraceptive likely
to cause
or economic
R.M.P.
can terminate
from or
of person.
(4)
methods serious
injury environment
who has
assisted pregnancy.
chief medical
her
(4)
guardian
Consent to
her can
injure in
performance
(2)
Private officer
is required
of husband
is not
of of
hospitals have
district.
(3)
Consent in
case of
minor or
an
(5)
Abortion is

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irritants. (II) General violence : It acts directly on uterus, or meboo, 1 = . indirectly by producing congestion of pelvic organs, or haemorrhages between uterus and membranes. (1) Severe pressure on abdomen by kneading, blows, kicks, etc. and massage of uterus. (2) Violent exercise, e.g. horse riding, cycling, jumping from a height, etc. (3) Cupping : mug is turned mouth downwards over a lighted wick and placed hypogastrium, and mug is pulled, which causes partial separation booksfree.com booksfree.com, 1 = . on of, 1 = on of. A, 1 = A. boo, 1 = boo. and, 1 = and
Trait
(1)
Cause:
(2)
(3)
(4)
(5)
(6)
(7)
Marks of
Genital
Distinction violence:
organs:
Foreign bodies:
Toxic effect
of drugs:
Infection:
Foetus:
(4)
Dilatation and
given i.v.,
orally replacement.
between natural
and
Natural abortion
Abortion criminal
abortion.
Criminal diseases.
on abdomen.
Injuries are
present.
tract.
Absent.
Rare.
Wounds curettage.
absent.
abortion
unmarried women
widows.
May be
present abdomen.
Injuries, such
on as
contusions, lacerations,
perforations, etc.,
may be
seen in
uterus contents
or its
and vagina.
May be
present genital
tract.
Erosions and
inflammation of
and cervix.
The urinary
vagina
G.I.
or tract
may signs
of show
Frequent.
Rarely wounds
present.
(5)
prostaglandins, or
(7)
(1)
This is
by intravaginal
Abdominal done
cannula weeks
PGE,, application.
hysterotomy.
during is
first introduced
9
to
0.6
mm.
into cannula

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

kg/sq.
may be
and fluid
PGF.,,
(6)
Amniotic
(8)
Vacuum three
months.
the uterine
is
(2)
cavity.
sufficient.
cm.
pump is
created for
evacuation aspiration
(4)
in uterine
of contents.
and collected
Doctor’s duties
aspiration
Cervix is
:
dilated
(3)
and
Between
A
negative cavity
(5)
8
by means
The in
a
bottle in
should be
criminal maintained.
induction contents
connected abortion
(2)
Ask of
the criminal
abortion.
matter.
(3)
a
Treat professional
her to
the colleague.
(5)
12
of
0.4
of
a
vacuum are
to broken
the
:
up by
cannula.
(1)
patient
Professional to
make
If she
refuses, best
If of
she secrecy
a
statement he
should his
ability.
is serious,
about not
pursue
(4)
Must he
must consult
arrange to
in the
not in
genital
|
157
or
158
record
Medicine dying
and declaration.
death certificate
(6)
If she
should
Causes of
death
Haemorrhage.
dies, police
not be
issued.
:
(1)
(3)
to
72
hours)
:
Immediate
Air
(1)
Confined local
Jaundice embolism.
:
(1)
Vagal
(4)
Fat
Septicaemia.
should be
informed, inhibition.
embolism.
(2)
infection and
Pyaemia.
and toxaemia.
renal
(II)
(3)
General
(5)
(2)
Delayed peritonitis.
Tetanus.
(II)
Remote:
(48
(4)
(1)
failure.
embolism.
(2)

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(1)
Infanticide means
of one
year.
(2)
Only
England.
(3)
murder.
(4)
when unlawful
CHAPTER
killing of
a
child mother
In
India,
Infanticide it
is the
destroyed can
be this
is does
not charged
applicable, not
include by
killing of
foetus
STILLBIRTH:
28
with under
age this
and is
death of
craniotomy prior
offence in
considered foetus
or to
birth.
(1)
A
weeks of
pregnancy, other
signs was
alive stillborn
child and
of life,
after in
frequently uterus,
among
(4)
Incidence as
which being
but decapitation.
as during
labour,
(5)
is one
Foeticide which
did not
is breathe
completely dies
during illegitimate,
is about
such is
born after
or born.
show
(2)
birth.
immature,
5%.
(5)
putrefaction of
prolonged starts
labour, i.e.
succedaneum and
severe
DEADBIRTH:
It is
born on
surface oedema
any
The
(3)
child
Stillbirths male
children in
a
and and
moulding
A
deadborn uterus
and shows
one
(1)
occur in
primiparae.
sterile condition,
extends and
inwards.
bleeding of
head child
of the
(6)
Signs into
scalp, indicate
at
(2)
MACERATION:
usual the
for delivery.
(1)
change.
It is
a
process
(2)
This
3
If to
4
days air
surrounded enters
is born
hours putrefaction
within after
occurs stillbirth.
one which
signs.
of aseptic
when dead
with liquor
occurs.
24
hours death.
had died
autolysis, child

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remains amnii
but
(4)
caput in
and in
exclusion
Maceration after
(6)
death.
Body is
of is
not
(5)
Skin soft,
seen slippage
flaccid and
is uterus
air.
(3)
if is
flattens child
seen out.
in
12
(7)
Odour

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Blood stains
are examined
Stained article
is used.
If stains
Solvents dried
are for
cyanide,
CHAPTER
in
State
Forensic
Science at
room not
dried, blood
(2)
10%
Rust stains,
stains stains
solution temperature.
Extra putrefaction
Laboratory.
heat should
sets are
:
(1)
of in.
10%
glycerine, synthetic
resemble
(1)
dye blood
Fresh
24
solution
(3)
weak stains,
stains.
stains hours,
stains brown
are are
bright-red, within
moist blood
dries dissolves
certain which
few and
days, of
not be
potassium solution
mineral of
ammonia.
and become
and sticky,
in
|
to in
older.
2
water.
(6)
Blood
Sex hours.
(5)
can black
and stiffen
vegetable reddish-brown
after long
cloth
(4)
The fresher
Fluorescence be
effused to
presence into
determined during
of in
time.
on
(2)
Fresh drying.
the decreases
from life
can be
fibrin.
powder on
Chemical of
(8)
Blood drying.
exam:
Chemical enzyme
peroxidase.
stained material
it on
presence removed
(3)
blood, as
the of
sex in
scales flowed
after death
tests
(1)
depend
Benzidine or
tease porcelain
glacial tile.
acetic acid,
If the
blood
This is
out
Add
A
drop more
of easily
it stain
becomes chromatin.
(7)
on drying,
tends due
to on
break presence
test fibres
:
Cut from
a
drop and
then is
the of
a
drop present,
best present
in dark
preliminary
a
dilution blue
the saturated
out
a
small stained
fabric solution
of
10
volumes colour
test of
one for
part up

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in blood
piece of
and place
of benzidine
hydrogen is
produced blood,
of blood
and in
peroxide.
immediately.
it detects
in three
blood when
lakhs.
If positive
into contact with this animal serum, the antibodies in the animal serum react with proteins in human serum, and a visible precipitate is formed. (4) Asuitable antiserum should react immediately or within a minute on 1 : 1000 dilution. Technique : (1) Blood is extracted which should be clear, and diluted 1:100 with normal saline. (2) two drops of undiluted antiserum are added to 0.75 ml. of dilute stain extract in a small tapering test tube. (3) Antiserum slowly settles down to bottom, and at junction of two fluids a white ring appears in case of positive reaction, which should begin in ten minutes and should be read in half hour. (4) It is a specific protein test. (5) the origin of skin, flesh, bone, saliva, milk, and semen is determined by this test. mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com,  = . Gel diffusion and double diffusion in agar gel are also used to detect human blood.,  = . M.l. aspects of blood groups: appear in a child unless present in one or other parents. (2) If an individual is homozygous for a blood group factor, it must appear in the blood of all his children. (3) If a child is homozygous for a factor, the gene for the same must have been inherited by it from each of its parents. (4) Blood group characters are specific to individual and are unchanged throughout life. Group specific substances : (1) the antigens of ABO system are also present in body tissues in a lipoidal form. (2) In about 80%, they appear in a water-soluble form and are present in all body fluids except C.S.F. (3) Persons who possess water-soluble form are known as 'secretors'. (4) Secretors posses H antigen on their red cells. (5) meboo mebooksfree.com mebooksfree.com,

= . Mand N antigens are present in body tissues in water-soluble form. (6) Rh antigens are found in body fluids, except amniotic fluid. Blood groups can be determined from soft tissues, hair, nails, dental tissue and bone. HlA consists of protein substances on surface of wide of tissues and organs, tumours, white cells and platelets. (2) are present on placenta at term and in foetal tissues at six weeks. meboo,  = . variety they (3) they are found on lymphocytes and granulocytes. M.l. aspects: (I) disputed paternity: the blood groups used booksfree.com booksfree.com,  = . to determine paternity are: (1) Red cell antigens: ABO, MNS,,  = . Rh, boo,  = Rh, boo
CHAPTER

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Psychiatry deals
with study,
diagnosis, illness.
Forensic psychiatry
which there
intelligence is
deals and
treatment with
of justice.
application
Insanity is
derangement weakened
insanity.
APHASIA:
or
Loss of
ability writing
(motor),
(sensory or
aphasia).
DELIRIUM:
(1)
orientation
It is
a
in impaired,
content is
irrelevant patient
is or
restless, uneasy
self-control, is
disease of
mental of
psychiatry of
in mind
of mental
and or
perverted.
or personality,
emotional
The to
express to
processes, law
has not
meaning understand
spoken disturbance
by defined
use of
or written
of consciousness
critical faculty
inconsistent.
speech language
in is
blunted, and
(2)
and becomes
sleepless.
excited sometimes
hallucinations fevers
and diseases,
and are
sometimes due
cerebral become
In the
which thought
early
He then
talks stage
the completely
furiously.
present.
to tumours
impulsive loses
(4)
(5)
It
Delusions usually
overwork, or
mental drug
and violent
person is
not responsible
DELUSION:
and
(2)
by and
occurs in
high stress,
intoxication.
and for
may his
(1)
It is
false which
persists
A
normal his
even after
person can
past experience
a
disorder rational
commit criminal
belief metabolic
(6)
A
person suicide.
acts.
in something
its falsity
correct and
which has
been may
(7)
Such is
clearly delusion
by of
thought being
which forces.
is by
not
a
fact, demonstrated.
his reasoning
convinced under
power, by
control of
others.
emotional
(3)
It is
but not

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= with disease.. They occur mostly in the form of anxiety, depression, or hysteria. (3) They are reaction to stressful circumstances due to adverse childhood experiences. (4) Empathy, contact with reality and insight are present. (5) It is a minor disease. mental subnormality (amentia): It is characterised by incomplete maturation of attention, perception, cognition, and social adaptability due to arrested mental development. (1) idiots are persons so defective in mind from birth or from early age that they are unable to guard themselves from ordinary physical dangers. Mentality is that of a child of 3 years. I.Q. 0 to 20. (2) imbeciles: They are incapable of managing themselves or their affairs, and are incapable of being taught. I.Q. 20 to 50. Mentality 3 to 7 years. (3) Feeble-minded (moron): Mental age 6 to 11. I.Q. 20 to 50. Dementia is a condition in which there is degeneration of mental faculties after they have been fully developed. post-epileptic insanity : (1) Stupor following epileptic fit is replaced by automatic acts of which He is confused and terrified by mebooksfree.com mebooksfree.com booksfree.com,  = patient has no recollection. (2) visual and auditory hallucinations meboo booksfree.com
Trait
(1)
Onset:
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
Motive:
Predisposing factors:
Signs and
symptoms:
Facial:
expression:
Insomnia:
Exertion:
Habits:
Frequent exam
Certification
:
:
hours are
usually peculiarly
real
Real insanity
Gradual.
Absent, e.g.,
no of
commission
Usually
Forensic and
history of
crime.
present, history
e.g., of
insanity parents
or monetary
in of
sudden loss,
Uniform and
whether grief,
present the
patient being
is observed
Peculiar, or
not.
e.g., vacant
look or
fixed look
excitement.
Present.
Patient can

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exertion of
stand of
fatigue, hunger
and several
sleep, for
days.
Dirty and
Does filthy.
not mind.
Three examinations
recommended, at
a
due single
to delirium
Mental
Psychiatry feigned
insanity
Feigned
Sudden.
Present, e.g.,
commission of
Absent.
Present only
conscious crime.
when of
observed;
being variable
and always
exaggerated.
No peculiarity,
frequently changing,
exaggerated and
voluntary.
Cannot persist.
Cannot stand
for exertion
more days
than and
Not dirty
a
few breaks
down.
and filthy.
Resents for
detection.
on different
because examination,
caused
Health person
who days
a
person either
by fever.
Act, is
in need
1987:
Mentally of
treatment other
than mental
retardation”.
RESTRAINT
OF
INSANE
done in
case of:
(1)
others, is
(2)
persons done
in under
aroom,
:
insane person
suffering personal
as from
care long
as danger
due ill
by fear
of and
different may
to effect
person reason
of behave
of drugs
is or
defined any
(1)
“a mental
Immediate disorder
restraint:
who is
dangerous delirium,
of
This is
to himself
(3)
delirium attendants,
e.g.
exists.
or tremens.
by safely
to
It locking
up etc.
insanity
177

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Medicine = any awareness of his actions, but rarely injures himself. (2) He is not asleep but in a state of dissociated consciousness, in a hallucinatory state, unrelated to his immediate environment. (3) Such persons are usually well-adjusted in life, socially well-behaved, and not aggressive. (4) The crime is not wilfil or premeditated. (5) The mental faculties are partially active. (6) Hemaycommitsucide, fall in a well, mebooksfree.com mebooksfree.com meboo. meet with an accident or commit crime. (7) There is no recollection, Concise Forensic Medicine = meet with an accident or commit crime. (7) There is no recollection. booksfree.com booksfree.com, Concise Forensic Medicine = booksfree.com booksfree.com. the event, but in some cases the events of one fit are remembered, Concise Forensic Medicine = the event, but in some cases the events of one fit are remembered. of, Concise Forensic Medicine = of
ARTEFACTS
Artefact is
any body
after death,
medicolegally change
that
CHAPTER
caused is
or feature
introduced likely
significant to
lead antemortem
(1)
Agonal
:
Regurgitation oesophagogastromalacia.
(2)
Resuscitation
:
Injection massage
of chest
causing organs.
(3)
region.
(4)
Rigor
Handling to
misinterpretation findings.
and aspiration
marks, rib
of gastric
defibrillator fractures,
of body
mortis simulating
:
Fracture
:
in of
contents;
mark, and
lacerations of
ribs,
Partial hypertrophy.
(5)
P.M.
lividity
:
Some internal
organs pneumonia,
may breaking
external of
contusion due
poisons resemble
(6)
Burns pulmonary
change disease,
:
Heat vessels,
(7)
ruptures, heat
haematoma, pseudo-strangulation
Animal
(8)
and internal
of occipital
to handling;
colour;

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

heart hypostasis
such as
coronary of
occlusion, fat
droplets mark
due insect
bites.
Decomposition
P.M.
purge,
:
(a)
(d)
groove appearance
of mistaken
internal for
(i)
Pseudobruising, in
neck
(b)
due to
to in
tight simulate
buttoned organs,
burns, separation
simulate
AM
bullet holes.
(h)
(f)
air
P.M.
in of
sutures of
lacerations, right
skull
(k)
digestion collar.
obesity, shirt,
(c)
(e)
of heart
in congested
stomach, mistaken
a
holes child,
(g)
blebs for
(j)
air embolism,
fissures produced
or splits
from in
skin maggots
resemble
(9)
Air in
blood
(10)
Skull removing
vessels fractures
skull due
due cap.
(11)
Visceral
(12)
autopsy.
(13)
(14)
contents damage
Extravasation due
to to
pulling use
to of
Fracture hyoid
Toxicological blood
bone due
during of
dura of
Artefacts in
chisel rough
and handling
due to
of cutting
of due
to block
removal to
autopsy;
contamination sagittal
hammer brain
185
line.
while and
vessels of
of liver.
during viscera.
viscera with
stomach faulty
storage or
use of
preservatives.

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Forensic science
is the
study and
application examination
Physical and
evaluation evidence
inanimate, solid,
as they
of evidences
includes liquid
or relate
to tool,
signature, fingerprints,
of scientific
for legal
purposes.
any and
gas, the
and all
the problem
firearms, objects,
relationship in
question, bullets,
hair, of
all types,
Physical fibres,
glass, bacteria,
living between
e.g.
crime.
blood and
paint, etc.
evidence or
is and
often innocence,
(2)
Locard’s contact,
there other.
Traces criminal,
and be
left it
can
(1)
be
Exchange or
all objects
Knife, seminal
oil, even
dust, odour
the burglar
stains, poisons,
microscopic are
decisive
a
material fragments
all physical
factor aid
principle is
always
a
from scene
at the
at the
scene.
and whatever
fingerprints, evidence.
in determining
in
:
locating
When transfer
any of
may be
same time,
Wherever he
leaves hair,
seminal stains.
of crime.
guilt the
criminal.
two objects
material carried
form away
traces from
a
criminal will
serve fibres
It is
Physical
(1)
Lie to
detect from
on all
goes, as
silent clothes,
actual evidence
evidence detection
lies.
come into
each object
clothes on
the or
tools or
any of
whatever evidence
broken and
cannot these
he of
may touches,
against glass,
its presence
be
:
Polygraph
(2)
pulse,
Polygraph respiration
stimuli and
in the
form person
tells
a
wrong
:
makes electrodermal
of lie
in paints,
him, e.g.
blood is
absolute and
or proof
completely
(1)
absent.
It is
an instrument
a
continuous reaction
questions.
(3)

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answer used
record of
changes
It is
to
a
based question
and
B.P., in
response on
theory there
is to
that when
fear that
lie will
CHAPTER

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A
poison is
a
substance
(solid, liquid
introduced in
part thereof
effects the
will or
living body,
or produce
brought ill-health
both.
actions,
Toxicology toxicity,
of poisons.
or gaseous)
into or
death, is
fatal dose,
‘‘Forensic aspects
of
Acts:
(3)
contact by
its which
if with
any systemic
dealing detection,
or local
with properties,
estimation, toxicology”
harmful effects
(1)
Drugs drugs
Poisons deals
of chemicals
Act,
1919.
and
Cosmetics into
Schedules.
Magic and
on
(2)
Drugs rules,
(4)
Remedies
Act,
Substances
Act, mental
function cannabis,
human and
1945.
Drugs
1954.
1985:
by its
poppy straw,
e.g.
amphetamine, treatment
and legal
beings.
Cosmetics
These
Control
Act,
(6)
Narcotic
A
psychotropic action.
Act, rules
have
1950.
Drugs
1940.
(5)
Drugs and
drug
A
is narcotic
etc.
This tranquilisers,
Ideal homicidal
colourless, and
Psychotropic one
that alters
drug means
lists
77
psychotropic barbiturates,
poison:
(1)
odourless either
in food,
change, disease,
cocoa leaf,
substances, etc.
It should
and be
cheap, tasteless,
drink highly
toxic, there
should not
the
(3)
body.
or in
capable medicine
signs without
and symptoms
not be
any by
easily available,
of being
administered producing
should antidote
chemical
(2)
Fluorine
Arsenic and
and tests
and and
must thallium
aconite
Stupefying:
satisfy are
Datura, commonly
cannabis, any

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visible resemble
no
P.M.
changes, be
rapidly several
used chloral
natural should
destroyed of
above as
in criteria.
homicidal hydrate.
poisons.

disulfiram. (e) Musty or fishy: Zinc phosphide. other substances are cyanides, phenol, opium, ether, camphor, etc. (4) The natural orifices, e.g. mouth, nostrils, rectum, and vagina may show the mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com meboo booksfree.com booksfree.com,
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193 (9) Heart: Subendocardial haemorrhages in the left ventricle occur in most cases of acute arsenic poisoning. Failure to Detect Poison: The possible explanations of negative findings are: (1) The poison may have been eliminated by vomiting and diarrhoea, e.g. in irritant poisons. (2) The whole of the poison has disappeared from the lungs by evaporation or oxidation. (3) The poison after absorption may be detoxified, conjugated, and eliminated from the system. (4) Some vegetable alkaloidal poisons cannot be detected by chemical analysis. (5) Some drugs are rapidly metabolised, making extraction difficult. (6) Biological toxins and venoms which may be protein in nature cannot be separated from body tissues. (7) Some organic poisons especially alkaloids and glucosides may by oxidation during life or due to faulty preservation or a long interval, or from decomposition of the body may deteriorate and cannot be detected chemically. (8) Many drugs may be present in very small quantity. (9) Insufficient material for analysis. (III) Experiments on Animals: Cat and dog are affected by poisons almost in the same way as man. They may be fed with suspected food, or with the poison after it is separated from the viscera and the symptoms noted. (IV) Circumstantial Evidence: Such evidence may consist of motive, the evidence of witnesses about the recent purchase of the poison, his behaviour before and after the commission of the offence, and the recovery of poison from the possession of the accused. Drug Automatism: According to this hypothesis, the patient develops a state of toxic delirium or amnesia after ingesting one or several doses of a drug (usually depressant drugs, barbiturates and alcohol), and in the delirious or automatism state, takes additional doses of the drug without realising it. Duties of medical practitioner in case of suspected poisoning: mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com meboo booksfree.com booksfree.com,  = meboo

General Considerations 195 passing the tube. (4) Patient should be lying on his left side or prone with head hanging over the edge of the bed, and face down supported by an assistant, so that the mouth is at a lower level than pharynx. (5) The end is lubricated with olive or sweet oil, or glycerine and is passed into the stomach by depressing the tongue with the finger and slowly passing it downwards till the fifty cm. mark is reached. (6) About 250 ml. of warm water (35°C) should be passed through the funnel held high up above the patient's head. (7) When funnel is empty, compress the tube below it between the finger and thumb, and lower it below the level of the stomach; its contents will be emptied by siphon action on releasing pressure on the rubber tubing. (8) Preserve this for chemical analysis. (9) gastric lavage may be done with water; 1:5000 potassium permanganate; five percent sodium bicarbonate, four percent tannic acid; one percent sodium thiosulphate; one percent sodium or potassuim iodide; one to three percent calcium lactate; saturated lime water or starch solution. (10) Next, use about half litre of suitable solution and repeat till clear and odourless fluid comes out. (11) Asmall quantity of fluid containing the antitode is left behind in the stomach, so that it may neutralise whatever small quantity of the poison is left behind in the stomach. (12) Ryle's tube or number 10 to 12 French catheter can be used for infants and children and about 25 cm. is necessary to reach stomach. Contraindications: (1) The only absolute contraindication is corrosive poisoning (except carbolic acid) due to danger of perforation . In the following conditions stomach wash can be done by taking proper precautions. (1) Convulsant poisons, after controlling the convulsions. (2) Comatose patients. The airways should be sealed by cuffed intubation and lavage done. (3) Volatile poisons which may mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com meboo, 1 = meboo

dose of three to four mg/, 197 = . kg is given. (5) Three ml. of 10% B.A.l. and 20% benzyl benzoate in arachis oil is injected deep intramuscularly fourth hourly for the, 197 = meboo. booksfree.com booksfree.com, 197 = . , 197 = boo. , 197 = till recovery. (6). , 197 = or. first two days, and then twice daily for ten days, 197 = . It is not used when liver, 197 = . is, 197 = is. damaged., 197 = damaged.
CHAPTER

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ORGANOPHOSPHORUS
POISONS:
phosphoric acid
and form
two phosphates:
(1)
(5)
Isopestox.
(8)
HETP.
(6)
Demeton.
(Follidol, kill
(2)
They are
derived series
of compounds.
TEPP
Malathion
(9)
(tetron).
(kill
Tichlorfon.
phos;
ekato).
(metacide).
(4)
Chlorothion.
Absorption:
from
(A)
Alkyl
(3)
OMPA.
bug;
(4)
bugsoline).
(B)
Aryl
(7)
phosphates:
(2)
Paraoxon.
(5)
Dimefox.
Sulfotepp.
(1)
(3)
Methly-
Diazinon
It is
absorbed mucous
membranes
Action:
(Tik
20;
by inhalation,
and the
G.I.
tract.
(1)
Organophosphorus inhibitors
of compounds
carboxylic esterase
acetylcholinesterase.
(2)
They
Parathion parathion
diazion).
through the
are enzymes,
bind inactivating
it by
firmly phosphorylation,
synapses of
in all
the ganglions.
at skin,
powerful including
to the
esterase the
(3)
myoneural
Organic parts
at of
the body
the of
parasympathetic, nerve
due enzyme,
junctions phosphates
to which
sympathetic impulses
across the
nerve-muscle and
inhibit
AChE
acetylcholine and
synapses junction
cholinesterase inhibitors.
due to
accumulates somatic
sites at
and the
is prevented.
This unhydrolysed
sympathetic transfer
autonomic
(4)
produces acetylcholine.
and ganglia
They are
a
syndrome

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(5)
parasympathetic
Symptoms:
With begin
within five
are at
maximum
Symptoms nervous
massive ingestion
minutes, or
may in
two to
system.
or inhalation,
be delayed
eight hours.
for half
at also
called of
overactivity appear
in both
symptoms to
one may
hour and

C.n.s.: Late coma, convulsions, cerebral oedema. Cause of death: Death occurs from multiorgan failure or corrosive effects in the G.I. tract. Death from oesophageal perforation mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com meboo booksfree.com booksfree.com, 203 = meboo
of
CHAPTER

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They act
by extracting
water cellular
proteins, and
convert
SULPHURIC
ACID
Symptoms:
from the
(oil
(1)
brown or
black the
sides of
The lips
streaks the
of mouth,
tissues, into
of vitriol).
are may
chin.
(2)
throat dysphagia.
usually be
swollen found
from
There is
and haematin.
and excoriated,
angles corrosion
and oesophagus,
(3)
is brown
or
Eructation, black,
the charred
coagulate and
of the
of mouth
mucous immediate
nausea and
mucoid, wall
strongly of
chalky-white.
the stomach.
(7)
Tongue abdomen
becomes hoarse,
and to
membranes burning
vomiting occur.
acid
(5)
becomes distended
the pupils
oesophageal, and
pain and
(4)
The may
Thirst contain
is swollen,
and intense.
sodden very
usually gastric
Fatal dose:
Cause and
5
to of
tender.
dilated.
pyloric
10
ml.
Death:
(1)
glottis.
(3)
Collapse
Treatment:
vomit shreds
(6)
and
(9)
of
Teeth are
black.
The
(10)
If strictures
F.P.
(8)
voice person
and
12
to
The becomes
recovers, stenoses
24
Circulatory due
(1)
hours.
collapse.
to perforation
The and
neutralised milk,
in mixed
with oxide,
aluminium carbonates
situ acid
(2)
Spasm of
late may
develop.
or oedema
stomach.
should
(4)
Toxaemia.
be immediately
by giving
one-fourth four
tablespoonfuls hydroxide
and diluted
litre of
water of
calcium gel
or calcined

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bicarbonates, as
they cause
demulcents.
which gastric
(4)
distention
Prednisolone or
magnesia.
liberate co,
and sixty
should sometimes
mg./day or
magnesium
(2)
Alkaline not
rupture.
may be
be used,
(3)
given
Give in
divided

= water meboo. and soap and a thick paste of magnesium oxide or carbonate is applied. (2) The eyes are washed with water and irrigated with dilute sodium bicarbonate solution. (3) Later, a few drops of olive oil or castor oil is put into eyes. NiTric Acid (aqua fortis, red spirit of nitre). In concentrated from, it combines with organic matter and produces an yellow discolouration of tissues, due to the production of picric acid booksfree.com booksfree.com, 207 = . (xanthoproteic, 207 = . reaction)., 207 = . , 207 = boo
CHAPTER

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ARSENIC:
Action:
It combines
and interferes
cell metabolism.
membranes
Symptoms:
and
(1)
absorbed causes
vascular failure.
(2)
The gastroenteric
poisoning.
with the
Locally remotely
sulphydryl it
depression
The death
fulminant in
enzymes causes
irritation of
the type:
of the
nervous
Large one
to
3
hours type:
(2)
ingestion.
(3)
in
Symptoms
There the
throat system.
dose from
(1)
when shock
It usually
is sweetish
and in
difficulty oesophagus,
vomiting rapidly
and resembles
appear half
peripheral bacterial
to metallic
taste.
in swallowing;
stomach and
which stools
are stinking
may food
one
(4)
hour
G.LT.:
burning bowel
be expelled
occur.
projectile frequently
and bloody,
watery after
Constriction and
colicky
Intense pain
thirst are
the constant
and involuntarily
but later
resembling infiltration.
rice-water
(6)
Renal:
red cells
and and
severe symptoms.
and are
become colourless,
stools of
cholera.
Oliguria, casts.
(7)
increased ventricular
The dark-coloured,
odourless and
(5)
uraemia.
C.V.S.:
vascular fibrillation.
tremors, convulsions,
of hair,
Hepatic:
Urine
Fatty contains
Acute circulatory
permeability,
(8)
C.N.S.:
coma, skin
eruptions.
(3)
Narcotic formication
type:
G.I.
and tenderness
F.D.:
100
to albumen,
collapse ventricular
Headache, general
with tachycardia,
vertigo, hyperthermia,
paralysis.
symptoms
(9)
are
Skin:

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slight.
of the
200
mg.
muscles,
F.P.:
One
Delayed loss
There delirium,
to two
is giddiness,
coma, days.
and death.

219 Distribution: (1) Lead is normally present in almost all tissues. (2) It is a typical cumulative poison. (3) The bones contain large amounts and also hair and nails. (4) Lead is excreted largely in the faeces and to a small extent in urine. cOPPer: Copper as a metal is not poisonous. Copper compounds are powerful inhibitors of enzymes. (1) Copper sulphate (blue vitriol) occurs in large, blue crystals. (2) Copper subacetate (verdigris), occurs in bluish-green masses or powder. symptoms: (1) Symptoms appear in 15 to 30 minutes. (2) There is metallic taste, increased salivation, burning pain in the stomach with colicky abdominal pain, thirst, nausea, eructations and repeated vomiting. (3) The vomited matter is blue or green. (4) There is diarrhoea with much straining; motions are liquid and brown but not bloody. (5) Oilguria, haematuria, albuminuria and uraemia may occur. (6) In some cases paralysis of limbs is followed by drowsiness, insensibility, coma and death. F.D.: Copper sulphate 30 g; copper subacetate 15 g. F.P.: One to three days. Treatment: (1) stomach wash with one percent solution of potassium ferrocyanide forms an insoluble cupric ferrocyanide. (2) n-penicillamine is very effective . (3) E.D.T.A. (4) B.A.L. (5) Demulcent drinks. P.M.Appearances: (1) The skin may be yellow. (2) Greenish- blue froth at mouth and nostrils. (3) The gastric mucosa and stomach contents are greenish or bluish. (4) The gastric mucosa may be mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com meboo,  = meboo

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PHOSPHORUS:
White phosphorus
occurs as
translucent waxy
cylinders.
dark, oxidises
under and
water, and
amorphous,
Smell emits
white is
highly and
taste fumes,
toxic.
solid mass.
It is
oxidised, white
is garlicky,
ignites
Red at
or yellow,
luminous
34°C., in
and phosphorus
tasteless, non-inflammable
Action:
as such
kept is
reddish-brown, odourless,
and non-luminous,
non-toxic.
It is
protoplasmic oxidation.
(1)
Fulminating g.
is taken.
poison poisoning
is
These patients
(2)
Acute abdomen,
usually
Poisoning:
with and
which seen
when die
(A)
First intense
thirst, excreta
have are
diagnostic.
third gralic-like
Skin degree
non- affects
cellular
a
dose within
stage:
exceeding
12
hours
Burning nausea,
one due
to shock.
pain vomiting,
odour.
Luminescent contact
produces burns.
(B)
Second
(C)
stage
Third in
throat diarrhoea.
and
Breath vomit
painful is
and penetrating
symptom-free
Stage:
absorbed poison.
haematemesis,
(1)
lasting
Symptoms
(2)
for two
or of
There liver
(3)
Haemorrhages due
is nausea,
tenderness occur
to injury
systemic faeces
second and
three days.
toxicity occur
prolonged vomiting,
and enlargement,
into skin,
to blood
Renal damage
sometimes vessels
results in
anuria.
(5)
and from
diarrhoea, jaundice,
mucous membrane
inhibition oliguria,
pruritus.
and of
blood haematuria,
Convulsions, casts,
delirium viscera,
clotting.

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RICINUS
COMMUNIS:
(1)
Entire plant
containing toxalbumen
(3)
The ricin.
(2)
‘press cake’
oil is
not contains
poisonous.
swallowed and
also
Toxalbumen or
bacterial toxin
in some
The seeds
ricin are
and
(4)
The unbroken
when cooked.
Phytotoxin action
haemolysis by
a
is
a
toxic and
causes and
plant.
is
Ricin, toxalbumens
antigenic.
crotin are
rich is
poisonous seeds
is poisonous,
in purgative
oil.
whereas are
castor non-poisonous
protein, when
agglutination of
red
Phytotoxin and
cells with
is abrin
snake
Symptoms:
and scorpion
Symptoms bloody
diarrhoea, abdominal
a
toxin produced
are phytotoxins.
venoms.
include abdominal
muscles, breathing,
pain, drowsiness,
uraemia
Ricin is
and excreted
F.D.:
salivation, nausea,
thirst,
Animal vomiting,
cramps in
calves delirium,
jaundice, by
5
to
10
seeds;
P.M.
of and
convulsions, dehydration,
epithelium.
ricin six
Appearances:
the
G.I.
Ricin tract
even when
occur in
internal
CROTON
mg.
F.P.:
Two produces
to several
haemorrhagic given
organs.
TIGLIUM
crotin,
a
toxalbumen is
also present.
Applied
(jamalgota which
(2)
shallow collapse
and death.
days.
inflammation subcutaneously.
Haemorrhages or
naepala):
(1)
is not
expressed
The to
oil contains
skin, is
used as
oil produces
abortifacient with
a
powerful burning,

#/texts/7690 #/texts/7691 #/texts/7692 #/texts/7693 #/texts/7694 #/texts/7695 #/texts/7696 #/texts/7697 #/texts/7698 #/texts/7699 #/tables/181

and arrow
Seeds contain
oil.
Crotonoside, vesicating
redness poison.
resin.
(3)
and vesication.
(4)
It
(4) Symptoms may appear after about half hour. (5) The patient feels sleepy, slightly intoxicated, weakness of legs, and is unable to stand or move. (6) Weakness of muscles increase, and develops into paralysis of the lower limbs. The paralysis then spreads to the trunk, and affects the head which droops. (7) This is followed by paralysis and swelling of the tongue and the larynx, due to which there is difficulty in speech and swallowing. (8) There may be extra-ocular muscle weakness, ptosis and strabismus. (9) After about two hours, the paralysis is complete. (10) Respirations become slower and the heart rate increases. (11) Though the patient is conscious, he is not able to speak. (12) Coma sets in and finally the respirations stop with or without convulsions. krait: Symptoms resemble those of cobra bite, but there is no convulsions mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com,  = . swelling or burning pain at the site of the bite and the are milder, while the feeling of drowsiness and intoxication is more intense. meboo,  = . Russle's Viper and echis Carinate: (1) More than 50% of the victims have minimal or no poisoning, as little or no venom is injected. (2) About 25% will develop serious generalised poisoning but death occurs rarely. (3) Whenvenomisinjected, the spot develops a severe pain within eight minutes. (4) The area around the bite is red and painful. (5) The onset of swelling starts within fifteen minutes and there is often bloodstained discharge from the wound. (6) When the venom injected is less, pain and swelling restricted to below,

will have only one hole in the centre of the reddened area. The symptoms produced by a neurotoxic venom is similar to cobra bite. Treatment: (1) Atorniquet should be applied above the location of sting. (2) A local anaesthetic is injected at the site to lessen pain. mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com mebooksfree.com meboo booksfree.com booksfree.com,
= meboo meboo

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Absolute contains
alcohol
90%
alcohol.
sugar or
jaggery, contains
99.95%
alcohol;
Arrack etc.
80%
from small
within
2
or in
(1)
About
20%
3
minutes is
liquor distilled
is absorbed
(2)
Alcohol of
swallowing.
blood is
reached within
Metabolism:
in
(1)
the liver,
and from
can rectified
from spirit
palm, the
rice, stomach
be
(3)
45
to
90
About
90%
the the
lungs.
remaining
(2)
In the
The minutes
detected maximum
and in
the blood
concentration after
of ingestion.
alcohol
10%
liver, alcohol
is alcohol
dehydrogenase adeninedinucleotide
(ADH)
absorbed excreted
is and
(NAD).
a
fairly uniform
rate equivalent
of about
alcoholics mg.
are
/
100
the ml.
tissues.
of
(6)
the
(3)
of about
15
able is
by oxidised
the oxidised
kidneys and
to acetaldehyde
its coenzyme,
It disappears
10
mg.
%
to from
to
/
metabolise hour.
by nicotinamide
from
15
ml.
per the
blood alcohol
(5)
During
It is
poorly same
alcohol alcohol
body at
the blood
at hour.
per
This hour.
a
oxidation soluble
weight concentration
in body
will for
have the
in the
absorption blood.
(8)
One concentration
same phase
hour after
of alcohol
faster rate
alcohol fat
a
is the
(4)
Chronic of
is
40
to not
and higher
amount is
about as
(25%
of drink.
ten drinking,
as
50
stored in
such females
higher)
(7)
percent venous
blood.
blood
Venous lower
blood than

arterial contains
the same
Symptoms: (1) Stage of Excitement: There is increased confidence and a lack of self-control, which is constant feature. (2) The person may disclose secrets. (3) Normal good manners are forgotten. (4) When jerking movement is in the direction of the gaze and independent of the position of the head, it is known as alcohol mebooksfree.com mebooksfree.com meboo,  = . gaze nystagmus , and appears at blood levels of 40 to 100 mg% (average. 80 mg. %). (5) Mental concentration is poor and judgement is impaired. (6) These effects are usually seen between 50 to 150 mg./100 ml. of blood alcohol. (2) Stage of incoordination (150 to 250 mg/100ml.): (1) The sense perceptions and skilled movements are affected. (2) He may become carefree, cheerful, ill-tempered, irritable, excitable, quarrelsome, sleepy, and so on. (3) The breath smells of alcohol. (4) The face is flushed and the pulse is increased. (5) The temperature becomes subnormal. mebooksfree.com mebooksfree.com meboo,  = . (3) Stage of coma: (1) Speech becomes thick and slurring, coordination is markedly affected, causing the patient to become giddy, stagger and possibly to fall. (2) The person passes into a state of coma with stertorous breathing. (3) The pupils are contracted, but stimulation of the person, e.g. pinching or slapping causes them to dilate with slow return (Mc Ewan sign). asphyxia due to respiratory paralysis. F.D.: 150 to 200 ml. of absolute alcohol mebooksfree.com,  = . F.P.: 12 to 24 hours. Treatment: (1) Heamodialysis or,  = . whoexperience withdrawal symptoms if they do. chronic are those who have reached a state of irreversible changes caused due to alcohol. Treatment of chronic alcoholics: (1) Antabuse given in a daily dose of three-fourth g. for two days g. for three to five days. The dosage is gradually reduced. calcium carbimide (temposil) 50 mg. once a day. (3) 20 to 25 mg. every four to six hours. booksfree.com,

= . somatic (2) Chlorpromazine booksfree.com,  = . Alcohol addicts are,  = . useful.,  = . people,  = . followed by,  = followed by. (disulfiram) is half Citrated,  = (disulfiram) is half Citrated. or,  = or. brain,  = brain. who cannot stop drinking for,  = who cannot stop drinking for. long,,  = long,. or alcoholics,  = or alcoholics. meboo,  = meboo. peritoneal dialysis is,  = peritoneal dialysis is. mebooksfree.com,  = mebooksfree.com. consumed in one,  = consumed in one. very,  = very. hour.,  = hour.. (4) Death occurs from,  = (4) Death occurs from
232, 1 = Concise Forensic Medicine. (4) The conditioned Reflex Treatment: (1) With backdrop of bottles of various alcoholic beverages, the patient is given various types of liquor, together with drugs that will cause immediate acute nausea and vomiting. After five to eight daily treatments, symptoms are brought on simply by the sight of a bottle, and the patient begins mentally to associate his painful sickness with alcohol. (2) Hypnosis and pschotherapy are also useful. DRunkEnnESS: Drunkenness is a condition produced in a person, who has taken alcohol in a quantity sufficient to cause him to lose control of his faculties to such an extent, that he is mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com, 1 = (4) The conditioned Reflex Treatment: (1) With backdrop of bottles of various alcoholic beverages, the patient is given various types of liquor, together with drugs that will cause immediate acute nausea and vomiting. After five to eight daily treatments, symptoms are brought on simply by the sight of a bottle, and the patient begins mentally to associate his painful sickness with alcohol. (2) Hypnosis and pschotherapy are also useful. DRunkEnnESS: Drunkenness is a condition produced in a person, who has taken alcohol in a quantity sufficient to cause him to lose control of his faculties to such an extent, that he is mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com
CHAPTER

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HYDROCARBONS:
Most of
the hydrocarbons
of petroleum
distillates.
The naphtha,
mineral absorbed
spirit, from
toxic light
substances gas
the
Signs and
hydrocarbons
G.I.
tract.
Symptoms:
causes chronic
itching and
inflammation.
other hydrocarbons
are derivatives
like gasoline,
oil, and
mineral
(1)
Acute eczematoid
(2)
can
Cutaneous cause
manifestations.
(3)
kerosene, sea
oil or
chronic dermatitis,
exposure second
When abdominal
pain
The breath,
produces and
to degree
ingested diarrhoea
vomit and
coughing,
CNS
F.D.:
15
to
50
ml.
of burns,
symptoms with
urine have
choking, are
poorly contact
with with
redness, gasoline
and and
are:
systemic nausea,
risk of
aspiration the
peculiar gasping,
kerosene.
P.M.Appearances are
vomiting, pneumonitis.
smell.
(4)
(5)
Aspiration bronchospasm,
F.P.:
those hypoxia
Within of
Poisoning children.
In the
can siphoning
easily be
aspirated
Symptoms:
and one
day.
asphyxia.
is usually
of in
the
(1)
Burning apathy,
lassitude, nausea,
(2)
The respiration
dyspnoeic.
accidental, gasoline
from lungs
a
especially tank,
and pain
in vomiting
is at
(3)
Vertigo, vision
are alkalosis
common.
with
(4)
marked cause
the and
first fast
ringing among
the mobile
death.
throat thirst
and in
and mouth,
are deep,
the liquid
anorexia, early
symptoms.
and later
the ears,
laboured deafness
In severe

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poisoning hyperapnoea
and and
a
impaired primary
and loss
of respiratory
CO, is
caused.
(5)

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AMPHETAMINE:
(1)
amphetamine
(benzedrine), methylphenidate
CHAPTER
The common
preparations dexamphetamine
(ritalin), lithium
give
a
feeling of
confidence false
sense of
euphoria
(4)
The onset
completed, are:
sulphate carbonate
(dexedrine), and
and mental
which of
fatigue but
there begin
to appear
awareness caffeine.
alertness.
may is
is loss
within and
be followed
(2)
They
(3)
They by
delayed of
and severe
produce
a
tasks judgement
an activity,
insomnia, tremors,
tachycardia, hour
are and
more accuracy.
after ingestion.
lessening anorexia,
of fatigue,
easily
(5)
They liveliness,
dry arrhythmias,
pupils, confusion,
death.
(6)
mouth,
Symptoms are:
increased talkativeness,
nausea, palpitation,
Fatal
(7)
dose
Dangers of
(2)
Paranoid vomiting,
anxiety, delirium,
is about
200
sweating, headache,
convulsions, mg.
misuse are:
(1)
psychosis.
during the
withdrawal.
CAFFEINE:
(1)
myocardium to
produce
(2)
It increase
fatigue.
and fatal
Overactivity
(3)
Shock dilated
coma period
about or
aggressive and
collapse.
Large doses
tachycardia, cardiac
(4)
output
Adrenaline can
increase increases
and five
days.
behaviour.
(4)
Risk can
stimulate arrhythmias
and stroke
and of
suicide directly
and volume.
noradrenaline the
basal metabolic
oxygen amounts
of secretion
rate by
the extrasystoles.
(3)
It decreases
is about
caffeine are
present
(7)
It in
tea, increased.

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ten percent.
acts as
(5)
It
(6)
It diuretic.
cola also
(8)
Significant beverages
and chocolates.
CHAPTER

#/texts/8123 #/texts/8124 #/texts/8125 #/texts/8126 #/texts/8127 #/texts/8128 #/texts/8129 #/texts/8130 #/texts/8131 #/texts/8132 #/texts/8133 #/texts/8134 #/texts/8135 #/texts/8136 #/texts/8137 #/texts/8138 #/texts/8139 #/texts/8140 #/texts/8141 #/texts/8142 #/texts/8143 #/texts/8144 #/texts/8145 #/texts/8146 #/texts/8147 #/texts/8148 #/texts/8149 #/texts/8150 #/texts/8151 #/texts/8152 #/texts/8153 #/texts/8154 #/texts/8155 #/texts/8156 #/texts/8157 #/texts/8158 #/texts/8159 #/texts/8160 #/texts/8161 #/texts/8162 #/texts/8163 #/texts/8164 #/texts/8165 #/texts/8166 #/texts/8167 #/texts/8168 #/texts/8169 #/texts/8170 #/texts/8171 #/texts/8172 #/texts/8173 #/texts/8174 #/texts/8175 #/texts/8176 #/texts/8177 #/texts/8178 #/texts/8179 #/texts/8180 #/texts/8181 #/texts/8182 #/texts/8183 #/texts/8184 #/texts/8185 #/texts/8186 #/texts/8187 #/texts/8188 #/texts/8189 #/texts/8190 #/texts/8191 #/texts/8192 #/texts/8193 #/texts/8194 #/texts/8195 #/texts/8196 #/texts/8197 #/texts/8198 #/texts/8199 #/texts/8200 #/texts/8201 #/texts/8202 #/texts/8203 #/texts/8204 #/texts/8205 #/texts/8206 #/texts/8207 #/texts/8208 #/texts/8209 #/texts/8210 #/texts/8211 #/texts/8212 #/texts/8213 #/texts/8214 #/texts/8215 #/texts/8216 #/texts/8217 #/texts/8218 #/texts/8219 #/texts/8220 #/texts/8221 #/texts/8222 #/texts/8223 #/texts/8224 #/texts/8225 #/texts/8226 #/texts/8227 #/texts/8228 #/texts/8229 #/texts/8230 #/texts/8231 #/texts/8232 #/texts/8233 #/texts/8234 #/texts/8235 #/texts/8236 #/texts/8237 #/texts/8238 #/texts/8239 #/texts/8240 #/texts/8241 #/texts/8242 #/texts/8243 #/texts/8244 #/texts/8245 #/texts/8246 #/texts/8247 #/texts/8248 #/texts/8249 #/texts/8250 #/texts/8251 #/texts/8252 #/texts/8253 #/texts/8254 #/texts/8255 #/texts/8256

DATURA
FASTUOSA:
Datura datura
niger,
a
poisonous, deep-purple
flowered especially
of hyoscine
the seeds
alba is
plant.
a
white
All and
the
(scopolamine),
Action:
fruit.
hyoscyamine
The alkaloids
first then
the motor
centres
Symptoms:
in flowered
parts
They of
these contain
and stimulate
and traces
plant, plants
and are
0.2
to
1.4%
of the
finally
(1)
Contact sensitive
persons.
with difficulty
(3)
The cause
with leaves
(2)
A
bitter in
talking, face
becomes dilated
with agitation,
higher atropine.
centres depression
of brain,
and paralysis.
or flowers
taste, causes
dryness dysphagia,
flushed, diplopia.
and patient
patient becomes
dry and
of dermatitis
mouth and
vomiting conjunctivae
(4)
Mental cannot
confused, hot,
the and
are throat,
first congested,
changes recognise
giddy, pulse
respirations are
3°C
.
(8)
rapid increased.
Muscle rash
noticed.
pupils widely
include restlessness
relatives staggers
120
to
(7)
tone or
and or
and friends.
as if
(5)
drunk.
140
per minute,
The deep
exfoliation
(9)
Delirium of
the is
restless indicated
by excitement,
his bed,
the be
silent but
picks tips
of sight
off at
of his
and in
one the
bed fingers,
hearing to
temperature reflexes
skin
(6)
The full
may are
and
The skin
is bounding,
be increased.
may be
seen and
raised
(9)
over by
2
to
A
scarlatinal most
purposeless;
talkativeness of
in its
earlier and

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

usually he
is clothes,
threads and
two unintelligent
noisy, tries
to the
body.
stages it
is speech.
tries to
run pull
imaginary imaginary
delusions hours
and occur.
needles.
The away
from threads
from
(11)
Hallucinations
(12)
This passes
excitement into
passes deep
sleep or
coma

#/texts/8286 #/texts/8287 #/texts/8288 #/texts/8289 #/texts/8290 #/texts/8291 #/texts/8292 #/texts/8293 #/texts/8294 #/texts/8295 #/texts/8296 #/texts/8297 #/texts/8298 #/texts/8299 #/texts/8300 #/texts/8301 #/texts/8302 #/texts/8303 #/texts/8304 #/texts/8305 #/texts/8306 #/texts/8307 #/texts/8308 #/texts/8309 #/texts/8310 #/texts/8311 #/texts/8312 #/texts/8313 #/texts/8314 #/texts/8315 #/texts/8316 #/texts/8317 #/texts/8318 #/texts/8319 #/texts/8320 #/texts/8321 #/texts/8322 #/texts/8323 #/texts/8324 #/texts/8325 #/texts/8326 #/texts/8327 #/texts/8328 #/texts/8329 #/texts/8330 #/texts/8331 #/texts/8332 #/texts/8333 #/texts/8334 #/texts/8335 #/texts/8336 #/texts/8337 #/texts/8338 #/texts/8339 #/texts/8340 #/texts/8341 #/texts/8342 #/texts/8343 #/texts/8344 #/texts/8345 #/texts/8346 #/texts/8347 #/texts/8348 #/texts/8349 #/texts/8350 #/texts/8351 #/texts/8352 #/texts/8353 #/texts/8354 #/texts/8355 #/texts/8356 #/texts/8357 #/texts/8358 #/texts/8359 #/texts/8360 #/texts/8361 #/texts/8362 #/texts/8363 #/texts/8364 #/texts/8365 #/texts/8366 #/texts/8367 #/texts/8368 #/texts/8369 #/texts/8370 #/texts/8371 #/texts/8372 #/texts/8373 #/texts/8374 #/texts/8375 #/texts/8376 #/texts/8377 #/texts/8378 #/texts/8379 #/texts/8380 #/texts/8381 #/texts/8382 #/texts/8383 #/texts/8384 #/texts/8385 #/texts/8386 #/texts/8387 #/texts/8388 #/texts/8389 #/texts/8390 #/texts/8391 #/texts/8392 #/texts/8393 #/texts/8394 #/texts/8395 #/texts/8396 #/texts/8397 #/texts/8398 #/texts/8399 #/texts/8400 #/texts/8401 #/texts/8402 #/texts/8403 #/texts/8404 #/texts/8405 #/texts/8406 #/texts/8407 #/texts/8408 #/texts/8409 #/texts/8410 #/texts/8411 #/texts/8412 #/texts/8413 #/texts/8414 #/texts/8415 #/texts/8416 #/texts/8417 #/texts/8418 #/texts/8419 #/texts/8420 #/texts/8421 #/texts/8422 #/texts/8423 #/texts/8424 #/texts/8425 #/texts/8426 #/texts/8427

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

(1)
Drug addiction
is
a
intoxication, produced
and is
harmful drugs
by the
to the
individual of
addiction alcohol,
are state
of repeated
periodic or
opium the
society.
and barbiturates,
Other drugs
cocaine, are
chloral dangerous.
(2)
Drug from
the
The its
chronic of
a
drug most
derivatives, cannabis,
hydrate important
pethidine,
LSD
heroin, and
and bromides.
habituation repeated
psychological
(habit)
consumption or
emotional nicotine
are is
a
of
a
drug, dependence
habit the
terms abuse):
forming
‘
drugs.
addiction’
Improper abuse,
amphetamine.
Heroin is
the most
condition in
which on
the drug.
Drug resulting
there is
a
Caffeine dependence
and habituation’.
or excessive
even useful
in the
or includes
(3)
Drug use
of and
both abuse
therapeutic absence
illegal the
use is
of drugs
addiction.
is moderate
dependence dependence
usually leads
and is
a
drugs
The use
called to
no
(substance may
be termed
of drug
harm.
psychological biological
dose and
duration the
of drug
in medically
abuse, not
even when
(4)
Addiction dependence.
phenomenon, consists
(5)
which drug
is abruptly
a
physically
a
use of
Physical depends
irrespective withdrawn,
dependent compulsive
need being,
and it
on of
personality
a
withdrawal person.
for
a
can
Indiscriminate occur
use physical
and drug
in the
type, factors.
syndrome
(6)
Psychological in
the order
If will
occur dependence
absence of
any moral
of these
worsening of
drugs of
the physical
a
state of
well- dependence.
(7)
a

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

mouth. (3) Bagging refers to inhaling and exhaling into a bag that has been filled with a small amount of a volatile substance. (4) The effects vary meboo booksfree.com booksfree.com, 251 = . , 251 = boo. from a condition resembling alcoholic intoxication, and, 251 = . , 251 = distortion. mebooksfree.com, 251 = mebooksfree.com. : About fourteen mg. meboo, 251 = : About fourteen mg. meboo. (e) blue pills., 251 = (e) blue pills.. mebooksfree.com, 251 = mebooksfree.com. Dose, 251 = Dose. mebooksfree.com, 251 = mebooksfree.com
SPINAL
POISONS
STRYCHNOS
NUX
VOMICA
alkaloid obtained
from species
of seeds
of
(3)
The the
strychnos, nux
CHAPTER
:
(1)
Strychnine
(kuchila)
seeds of
strychnos which
vomica seeds
percent each.
to
20
are found
contained contain
nux vomica
in the
jungles in
strychnine
(4)
times
All parts
more the
and of
the tree
poisonous leaves
contain brucine
Absorption ripe
and in
India.
fruit brucine
are up
toxic.
(5)
than brucine.
but no
strychnine.
and
Excretion strychnine.
(2)
Much released
again oxidised
:
(1)
All mucous
is to
taken up
blood mainly
to four
years.
Action
:
(1)
in the
spinal
Widespread in
stream the
by or
liver.
It
(4)
depresses cord
the to
are is
an other
(2)
The poisonous.
to one-and-half
Strychnine is
10
The bark,
wood membranes
liver and
muscles be
and absorb
to be
destroyed.
It may
(3)
About be
the either
80%
found inhibitory

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

and prevents
(3)
The action
It in
the the
spinal is
cord particularly
stimulates the
Symptoms cerebral
:
vomica have
noted in
the is
cadaver post-synaptic
up potentials
effects of
glycine.
results in
‘release’
in cortex.
(1)
If no
the
G.I
mouth, tract,
sense and
great are
fear preceded
and swallowed
poisonous uncrushed,
action, are
passed of
uneasiness and
(2)
excitation.
horn the
as they
are entire
in and
difficulty by
faeces.
restlessness, in
swallowing such
cells.
(4)
seeds of
nux not
dissolved
(2)
Bitter feeling
occur.
prodromal symptoms
in taste
in of
the suffocation
(3)
The as
convulsions increased
acuity of
(6)

Hypoxia causes medullary paralysis and death. mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com meboo, tetanus = perception, increased rigidity of muscles, and muscular twitchings. (4) Convulsions are at first clonic, but eventually become tonic. (5) Risus sardonicus results. (6) The convulsions are most marked in anti- gravity muscles, so that the body typically arches in hyperextension (opisthotonus) . (7) In supine position the body is supported by the heels and head in a bow-like from. (8) Sometimes, the spasm of the abdominal muscles may bend the body forward (emprosthotonus) or to the side (pleurosthotonus) . (9) Consciousness is not lost and the mind remains clear till death. (10) The duration of convulsion varies from half to two minutes. (11) In between the convulsions, the muscles are completely relaxed and the patient looks well and the breathing is resumed. (12) After 5 to 15 minutes or on slightest impulse, another convulsion occurs. (13) Death usually occurs after 4 to 5 convulsions . (14) Hypoxia causes medullary paralysis and death. mebooksfree.com mebooksfree.com meboo mebooksfree.com mebooksfree.com meboo
CHAPTER

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NERIUM
ODORUM
:
(White are
poisonous, oleandroside
(oleandrin), digitalis
in action.
Symptoms
:
Difficulty pain,
vomiting, and
weak, salivation,
several oleander)
:
All parts
cardiac and
glycosides, nerioside
in
(nerin), swallowing
and diarrhoea.
respirations spasms,
Pulse are
lockjaw, increased,
drowsiness,
F.D.:
Fifteen
Poisoning of
th plant
primarily which
articulation, is
first resemble
abdominal slow
and muscular
coma, respiratory
to
:
20
g.
of the
later rapid
twitchings, paralysis
root.
(1)
CERBERA
Suicide
(2)
F.P.:
24
to
Abortifacient.
THEVETIA
The thevetoxin,
:
seeds tetanic
and
36
hours.
(3)
Cattle
(Yellow oleander)
contain similar
to digitalis
ruvocide and
cerberin.
Symptoms:
Burning tingling
and numbness
giddiness, loss
and irregular,
of of
muscular blood
from peripheral
F.D.:
cardiac poison.
:
All glycoside
in action;
pain in
tongue, parts
thevetin, nerifolin,
the mouth,
are and
peruvocide, dryness
vomiting, power
and pressure
Eight
F.P.:
2
to
3
CERBERA
is of
throat, diarrhoea,
fainting.
low.
Heart failure.
to ten
seeds,
15
to
20
hours.
ODALLAM
cerberin, cerberoside,
F.D.:
(pilikirbir)
odollin,
Kernal of
one g.
root;
headache,
Pulse is
rapid, block,
collaps
5
weak and
to
:

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The odollotoxin,
fruit
F.P.:
10
leaves.
active thevetin
One to
two principles
and days
or are
cerapain.
more death
death.

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CARBON
MONOXIDE
greater affinity
more
:
Action for
CHAPTER
:
(1)
CO
has
200
haemoglobin to
myoglobin.
than
(2)
It displaces
with haemoglobin
and carboxyhaemoglobin.
forms that
of oxygen,
oxygen
a
(3)
death due
to anaemic
the tissues
will
It acts
anoxia.
be electron
to
300
times and
from
40
times its
stable as
chemical
(4)
cherry-red transport
Sources
:
The from
fire and
furnace stove,
lime kilns,
by blocking
common combination
compound asphyxiant
The blood
in
15
to known
as and
under the
20%
cases.
cytochrome sources
the fumes
of
CO
from waterheater,
explosion defective
burning in
(5)
oxidase.
include
:
heating oil
lamps, mines,
exhaust fumes
of internal
Symptoms:
(2)
(1)
Damage to
coal gas,
smoke appliances,
fumes detonation
combustion
The effects
the
Impairment
CNS
of higher
cerebellar damage,
delayed by
be of
engines.
of
CO
intellectual and
several separate
pressure.
are simply
e.g., of
coke explosives,
kilns, and
those the
of monoplegia
functions, severe
suboxia.
or hemiplegia.
Parkinsonism weeks
and isolated
They fingers
and swinging,
after lesions.
are caused
toes.
due
(5)
erratic furniture,
which usually
occurs
There may
apparent
(3)
changes, occur
and recovery.
These bullae
(4)
may be
Bullae are
to skin
localised hypoxia.
is tendency
movements tend
to by
external
They of
the rerely
dying inside
the gives
an impression
when
80%
room victim

#/texts/8865 #/texts/8866 #/texts/8867 #/texts/8868 #/texts/8869 #/texts/8870 #/texts/8871 #/texts/8872 #/texts/8873 #/texts/8874 #/texts/8875 #/texts/8876 #/texts/8877 #/texts/8878 #/tables/204

distrubing of
violent of
haemoglobin involve
to wild,
clothing struggle.
is and
(6)
Death saturated
with
CO.
produces skin
It and
in inhibits
the
258 Concise Forensic Medicine mebooksfree.com mebooksfree.com, 1 = 258 Concise Forensic Medicine mebooksfree.com mebooksfree.com. CoHb% mebooksfree.com, 1 = Symptoms mebooksfree.com meboo. 0 to 10%, 1 = no appreciable symptoms.. 10 to 20%, 1 = Breathlessness on moderate exertion, mild headache.. 20 to 30%, 1 = Headache, irritability, emotional instability, disturbed judgement, defective memory and rapid fatigue.. 30 to 40 %, 1 = Severe headache, nausea, vomiting, dimness of vision, confusion, cherry-red discolouration of skin.. 40 to 50 %, 1 = increasing confusion, sometimes hallucinations, severe ataxia, rapid respirations and collapse with attempts at exertion. Symptoms resemble alcoholic intoxication. mebooksfree.com mebooksfree.com meboo. 50 to 60%, 1 = Syncope or coma with intermittent convulsions, rapid respirations, tachycardia with weak pulse.. 60 to 70%, 1 = increasing depth of coma with incontinence of urine and faeces.. 70 to 80%, 1 = Deep coma with depressed or absent reflexes, a weak thready pulse, shallow and irregular respirations. booksfree.com. above 80%, 1 = rapid death from respiratory arrest.. autopsy : (1) A cherry-red colouration of the skin, mucous booksfree.com, 1 = autopsy : (1) A cherry-red colouration of the skin, mucous booksfree.com

#/texts/8880 #/texts/8881 #/texts/8882 #/texts/8883 #/texts/8884 #/texts/8885 #/texts/8886 #/texts/8887 #/texts/8888 #/texts/8889 #/texts/8890 #/texts/8891 #/texts/8892 #/texts/8893 #/texts/8894 #/texts/8895 #/texts/8896 #/texts/8897 #/texts/8898 #/texts/8899 #/texts/8900 #/texts/8901 #/texts/8902 #/texts/8903 #/texts/8904 #/texts/8905 #/texts/8906 #/texts/8907 #/texts/8908 #/texts/8909 #/texts/8910 #/texts/8911 #/texts/8912 #/texts/8913 #/texts/8914 #/texts/8915 #/texts/8916 #/texts/8917 #/texts/8918 #/texts/8919 #/texts/8920 #/texts/8921 #/texts/8922 #/texts/8923 #/texts/8924 #/texts/8925 #/texts/8926 #/texts/8927 #/texts/8928 #/texts/8929 #/texts/8930 #/texts/8931 #/texts/8932 #/texts/8933 #/texts/8934 #/texts/8935 #/texts/8936 #/texts/8937 #/texts/8938 #/texts/8939 #/texts/8940 #/texts/8941 #/texts/8942 #/texts/8943 #/texts/8944 #/texts/8945 #/texts/8946 #/texts/8947 #/texts/8948 #/texts/8949 #/texts/8950 #/texts/8951 #/texts/8952 #/texts/8953 #/texts/8954 #/texts/8955 #/texts/8956 #/texts/8957 #/texts/8958 #/texts/8959 #/texts/8960 #/texts/8961 #/texts/8962 #/texts/8963 #/texts/8964 #/texts/8965 #/texts/8966 #/texts/8967 #/texts/8968 #/texts/8969 #/texts/8970 #/texts/8971 #/texts/8972 #/texts/8973 #/texts/8974 #/texts/8975 #/texts/8976 #/texts/8977 #/texts/8978 #/texts/8979 #/texts/8980 #/texts/8981 #/texts/8982 #/texts/8983 #/texts/8984 #/texts/8985 #/texts/8986 #/texts/8987 #/texts/8988 #/texts/8989 #/texts/8990 #/texts/8991 #/texts/8992 #/texts/8993 #/texts/8994 #/texts/8995 #/texts/8996 #/texts/8997 #/texts/8998 #/texts/8999 #/texts/9000 #/texts/9001 #/texts/9002 #/texts/9003 #/texts/9004 #/texts/9005 #/texts/9006 #/texts/9007

The term
food poisoning
which result
products.
from in
CHAPTER
its wider
sense includes
ingestion
But due
the term
to the
bacterial
Causes of
is usually
infection
:
of
(I)
foods containing
restricted of
food
Poisoning vegetable
(2)
Poisonous berries,
such
Paspalam due
origin
(natural mushroooms.
all illnesses
non-bacterial to
acute or
drink.
to bacteria
food and
poisons)
(3)
Rye, as
atropa gastroenteritis
toxins.
:
(1)
oats,
(II)
Poisons
Lathyrus barley,
belladonna.
scrobiculatum.
(9)
Groundnuts.
(5)
(7)
(10)
Argemone
Vitia
Soyabean.
(14)
added, such
fava.
Sweet sativus.
etc.
Lolium
(4)
Poisonous temulentum.
mexicana.
(11)
Cabbage.
clover.
as flavouring
preservatives,
(III)
agents extraction
Accidentally of
(6)
(8)
Cotton seeds.
(12)
Chemical in
processed
Solanine.
:
(13)
(1)
food, fat
by solvents
added, of
such as
pesticides foods.
(4)
Radionucleides.
Bacterial into
Food
Poisoning two
groups.
within body
(1)
Intentionally colouring
agents, like
hydrocarbons.
and
:
insecticides.
Bacterial
Infection of
type, pathogenic
‘toxin’
type, substances
which which
organisms, follows
have infection
(2)
(3)
Products food
poisoning follows
contained the
been ingestion
formed type,
organisms e.g.
S.
enteritidis less
commonly results
is divided
the multiplication
in of
food due
to bacterial
belong of
Gaertner,

#/texts/9008 #/texts/9009 #/texts/9010 #/texts/9011 #/texts/9012 #/texts/9013 #/texts/9014 #/texts/9015 #/texts/9016 #/texts/9017 #/texts/9018 #/texts/9019 #/texts/9020 #/texts/9021 #/texts/9022 #/texts/9023 #/texts/9024 #/texts/9025 #/texts/9026 #/texts/9027 #/texts/9028 #/texts/9029

S.
the from
the paratyphoid
ingestion the
foods.
(2)
The in
which poisonous
proliferation.
mainly to
the
In the
Salmonella typhimurium,
group,
S.
bacilli.
cholerasuis
Bacterial of
contaminated food,
and food
poisoning uncooked
food

#/tables/210 #/texts/9030 #/texts/9031 #/texts/9032 #/texts/9033 #/texts/9034 #/texts/9035 #/texts/9036 #/texts/9037 #/texts/9038 #/texts/9039 #/texts/9040 #/texts/9041 #/texts/9042 #/texts/9043 #/texts/9044 #/texts/9045 #/texts/9046 #/texts/9047 #/texts/9048 #/texts/9049 #/texts/9050 #/texts/9051 #/texts/9052 #/texts/9053 #/texts/9054 #/texts/9055 #/texts/9056 #/texts/9057 #/texts/9058 #/texts/9059 #/texts/9060 #/texts/9061 #/texts/9062 #/texts/9063 #/texts/9064 #/texts/9065 #/texts/9066 #/texts/9067 #/texts/9068 #/texts/9069 #/texts/9070 #/texts/9071 #/texts/9072 #/texts/9073 #/texts/9074 #/texts/9075 #/texts/9076 #/texts/9077 #/texts/9078 #/texts/9079 #/texts/9080 #/texts/9081 #/texts/9082 #/texts/9083 #/texts/9084 #/texts/9085 #/texts/9086 #/texts/9087 #/texts/9088 #/texts/9089 #/texts/9090 #/texts/9091 #/texts/9092 #/texts/9093 #/texts/9094 #/texts/9095 #/texts/9096 #/texts/9097 #/texts/9098 #/texts/9099 #/texts/9100 #/texts/9101 #/texts/9102 #/texts/9103 #/texts/9104 #/texts/9105 #/texts/9106 #/texts/9107 #/texts/9108 #/texts/9109 #/texts/9110 #/texts/9111 #/texts/9112 #/texts/9113 #/texts/9114 #/texts/9115 #/texts/9116 #/texts/9117 #/texts/9118 #/texts/9119 #/texts/9120 #/texts/9121 #/texts/9122 #/texts/9123 #/texts/9124 #/texts/9125 #/texts/9126 #/texts/9127 #/texts/9128 #/texts/9129 #/texts/9130 #/texts/9131 #/texts/9132 #/texts/9133 #/texts/9134 #/texts/9135 #/texts/9136 #/texts/9137 #/texts/9138 #/texts/9139

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

= . poisonous.,  = . are,  = are. booksfree.com,  = booksfree.com. a combination of both. . Fatal Period : 24 hours.,  = a combination of both. . Fatal Period : 24 hours.. mushrooms,  = mushrooms. or 2 to 3,  = or 2 to 3. neurotic : mebooksfree.com,  = neurotic : mebooksfree.com
Abortion,
Abrasions,
A
154
64
Abrasion collar,
82
Abrus precatorius,
Accident register,
Acid phosphatase,
Aconite,
224
99
53
256
Acrodynia,
216
Adipocere,
Affiliation
61
cases,
138
Age, in
identification, criminal
responsibility, foetus,
33
m.l.
importance,
Alcohol, ethyl,
231
Aldrich-Mees lines,
Algor mortis,
53
Alkalis, caustic,
Aluminium phosphide,
Amensia, in
head
Amphetamine,
210
205
injury,
243
Anoxia,
48
Anthropometry,
35
Antidotes,
196
Aphasia,
172
Arborescent markings,
Arcus senilis,
Argemone
32
mexicana,
Arrow poisons,
Arsenic,
189
212
Artefacts,
184
Artificial bruise,
88
109
265
68
33
33
214
26
INDEX
Artificial insemination,
Asphyxia,
48
Assault,
99
Atavism,
138
Automatism,
182
Autopsy, medicolegal,
Avulsion,
41
64
B
Back spatter,
B.A.L.,
197
Bagging,
251
Bansdola,
118
Barberio’s test,
Barbiturate,
152
239
Basophilic stippling,
Battered baby,
Bestiality,
164
148
Bhang,
245
Bite marks,
Blast lung,
Blood
39
85
stains,
Body

#/texts/9140 #/texts/9141 #/texts/9142 #/texts/9143 #/texts/9144 #/texts/9145 #/texts/9146 #/texts/9147 #/texts/9148 #/texts/9149 #/texts/9150 #/texts/9151 #/texts/9152 #/texts/9153 #/texts/9154 #/texts/9155 #/texts/9156 #/texts/9157 #/texts/9158 #/texts/9159 #/texts/9160 #/texts/9161 #/texts/9162 #/texts/9163 #/texts/9164 #/texts/9165 #/texts/9166 #/texts/9167 #/texts/9168 #/texts/9169 #/texts/9170 #/texts/9171 #/texts/9172 #/texts/9173 #/texts/9174 #/texts/9175 #/texts/9176 #/texts/9177 #/texts/9178 #/texts/9179 #/texts/9180 #/texts/9181 #/texts/9182 #/texts/9183 #/texts/9184 #/texts/9185 #/texts/9186 #/texts/9187 #/texts/9188 #/texts/9189 #/texts/9190 #/texts/9191 #/texts/9192 #/texts/9193 #/texts/9194 #/texts/9195 #/texts/9196 #/texts/9197 #/texts/9198 #/texts/9199 #/texts/9200 #/texts/9201 #/texts/9202 #/texts/9203 #/texts/9204 #/texts/9205 #/texts/9206 #/texts/9207 #/texts/9208 #/texts/9209 #/texts/9210 #/texts/9211 #/texts/9212 #/texts/9213 #/texts/9214 #/texts/9215 #/texts/9216 #/texts/9217 #/texts/9218 #/texts/9219 #/texts/9220 #/texts/9221 #/texts/9222 #/texts/9223 #/texts/9224 #/texts/9225 #/texts/9226 #/texts/9227 #/texts/9228 #/texts/9229 #/texts/9230 #/texts/9231 #/texts/9232 #/texts/9233 #/texts/9234 #/texts/9235 #/texts/9236 #/texts/9237 #/texts/9238 #/texts/9239 #/texts/9240 #/texts/9241 #/texts/9242 #/texts/9243 #/texts/9244 #/texts/9245 #/texts/9246 #/texts/9247 #/texts/9248 #/texts/9249 #/texts/9250 #/texts/9251 #/texts/9252 #/texts/9253 #/texts/9254 #/texts/9255 #/texts/9256 #/texts/9257 #/texts/9258 #/texts/9259 #/texts/9260 #/texts/9261 #/texts/9262 #/texts/9263 #/texts/9264 #/texts/9265 #/texts/9266 #/texts/9267 #/texts/9268 #/texts/9269 #/texts/9270 #/texts/9271 #/texts/9272 #/texts/9273 #/texts/9274 #/texts/9275 #/texts/9276 #/texts/9277 #/texts/9278

167
packer syndrome,
252
Bomb explosion
wounds,
85
Bones, examination,
Bore of
firearm,
76
Botulism,
264
Boxing injuries,
Brain death,
47
Brain mapping,
Breslau’s
187
second test,
life
162
81
94
131
212
43
Bromides,
240
Brown sugar,
Bruises,
250
66
Brush burn,
65
Buccal coitus,
Buggery,
147
Bullets,
78
Burking,
123
Burns,
Cadaveric
104
Cc spasm,
Cafe coronary,
57
122
Caffey’s syndrome,
Calotropis,
164
225
Capsicum annum,
Cannabis
224
indica,
245
Cantharides,
225
Caput succedaneum,
Carbamates,
163
201
Carbine,
77
Carbolic acid,
Carboluria,
209
210
Carbon monoxide,
Cartridges,
77
Casper dictum,
61
Catamite,
147
Cephalhaematoma,
Cephalic index,
23
Cerbera odallam,
Cerbera
255
thevetia,
Chain of
custody,
Charas,
245
Chelating agent,
Chemical
197
antidotes,
255
5
148
257
163
196
268
Concise
Chloral
Forensic hydrate,
240
Choking,
122
of firearms,
77
Chop wounds,
74
Chromolachryorrhoea,
201
Civil
Civil negligence,
14
responsibility and
insanity,
179
Cobra, poisoning
Cocaine, by,
247
Cocaine bugs,
Cognisable
247
offence,
Cold stiffening,
Coma,
57
48
Common witness,
5
Concealed puncture
wounds,
75
Concussion of
brain,
Conduct money,
Consent,
2
19
Consumer

#/texts/9279 #/texts/9280 #/texts/9281 #/texts/9282 #/texts/9283 #/texts/9284 #/texts/9285 #/texts/9286 #/texts/9287 #/texts/9288 #/texts/9289 #/texts/9290 #/texts/9291 #/texts/9292 #/texts/9293 #/texts/9294 #/texts/9295 #/texts/9296 #/texts/9297 #/texts/9298 #/texts/9299 #/texts/9300 #/texts/9301 #/texts/9302 #/texts/9303 #/texts/9304 #/texts/9305 #/texts/9306 #/texts/9307 #/texts/9308 #/texts/9309 #/texts/9310 #/texts/9311 #/texts/9312 #/texts/9313 #/texts/9314 #/texts/9315 #/texts/9316 #/texts/9317 #/texts/9318 #/texts/9319 #/texts/9320 #/texts/9321 #/texts/9322 #/texts/9323 #/texts/9324 #/texts/9325 #/texts/9326 #/texts/9327 #/texts/9328 #/texts/9329 #/texts/9330 #/texts/9331 #/texts/9332 #/texts/9333 #/texts/9334 #/texts/9335 #/texts/9336 #/texts/9337 #/texts/9338 #/texts/9339 #/texts/9340 #/texts/9341 #/texts/9342 #/texts/9343 #/texts/9344 #/texts/9345 #/texts/9346 #/texts/9347 #/texts/9348 #/texts/9349 #/texts/9350 #/texts/9351 #/texts/9352 #/texts/9353 #/texts/9354 #/texts/9355 #/texts/9356 #/texts/9357 #/texts/9358 #/texts/9359 #/texts/9360 #/texts/9361 #/texts/9362 #/texts/9363 #/texts/9364 #/texts/9365 #/texts/9366 #/texts/9367 #/texts/9368 #/texts/9369 #/texts/9370 #/texts/9371 #/texts/9372 #/texts/9373 #/texts/9374 #/texts/9375 #/texts/9376 #/texts/9377 #/texts/9378 #/texts/9379 #/texts/9380 #/texts/9381 #/texts/9382 #/texts/9383 #/texts/9384 #/texts/9385 #/texts/9386 #/texts/9387 #/texts/9388 #/texts/9389 #/texts/9390 #/texts/9391 #/texts/9392 #/texts/9393 #/texts/9394 #/texts/9395 #/texts/9396 #/texts/9397 #/texts/9398 #/texts/9399 #/texts/9400 #/texts/9401 #/texts/9402 #/texts/9403 #/texts/9404 #/texts/9405 #/texts/9406 #/texts/9407 #/texts/9408 #/texts/9409 #/texts/9410 #/texts/9411 #/texts/9412 #/texts/9413 #/texts/9414 #/texts/9415 #/texts/9416 #/texts/9417 #/texts/9418 #/texts/9419 #/texts/9420 #/texts/9421 #/texts/9422 #/texts/9423 #/texts/9424 #/texts/9425 #/texts/9426 #/texts/9427 #/texts/9428

Protection
Act,
21
Contrecoup, injury,
Contributory negligence,
17
Contusions,
66
Contusion collar,
Cooling of
Copper,
Corona,
Corpus body,
219
81
delicti,
Cot death,
166
Courts of
Covering,
Criminal,
Criminal law,
82
53
24
1
11
negligence,
15
responsibility in
insanity,
181
in ralation
to age,
Cross-examination,
33
7
Croton, poisoning
by,
Crush syndrome,
100
223
88
2
88
Medicine
Cutis anserina,
Cut-throat
124
wounds,
Cyanides,
71
208
D
Dactylography,
35
Dangerous injury,
Datura,
244
Dead born
child,
Death, brain,
47
modes of,
molecular,
47
47
somatic,
47
sudden,
50
time of,
63
Decomposition,
59
Defence wounds,
Defloration,
75
133
Delirium,
172
Delirium tremens,
Delivery,
138
Delusions,
172
Dementia,
175
Diatoms,
126
Dichotomy,
11
Dipsomania,
DMSA,
174
198
DNA
fingerprinting,
Disciplinary
170
control,
Dowry death,
Drowning,
96
123
Drug, addiction,
248
Drug habituation,
Drunkenness,
248
232
Dry drowning,
Duties,
123
of patient,
in abortion,
of
13
157
physician,
12
in suspected
poison,
193
in witness
box,
Dying declaration,
4
11
98
159
226
235
8
EDTA,
198
Effacement of
Electric burns,
Electric mark,
Electrocution, identity,
42
108
108
judicial,
Embalming,
62
Embolism, air,
fat,
101

#/texts/9429 #/texts/9430 #/texts/9431 #/texts/9432 #/texts/9433 #/texts/9434 #/texts/9435 #/texts/9436 #/texts/9437 #/texts/9438 #/texts/9439 #/texts/9440 #/texts/9441 #/texts/9442 #/texts/9443 #/texts/9444 #/texts/9445 #/texts/9446 #/texts/9447 #/texts/9448 #/texts/9449 #/texts/9450 #/texts/9451 #/texts/9452 #/texts/9453 #/texts/9454 #/texts/9455 #/texts/9456 #/texts/9457 #/texts/9458 #/texts/9459 #/texts/9460 #/texts/9461 #/texts/9462 #/texts/9463 #/texts/9464 #/texts/9465 #/texts/9466 #/texts/9467 #/texts/9468 #/texts/9469 #/texts/9470 #/texts/9471 #/texts/9472 #/texts/9473 #/texts/9474 #/texts/9475 #/texts/9476 #/texts/9477 #/texts/9478 #/texts/9479 #/texts/9480 #/texts/9481 #/texts/9482 #/texts/9483 #/texts/9484 #/texts/9485 #/texts/9486 #/texts/9487 #/texts/9488 #/texts/9489 #/texts/9490 #/texts/9491 #/texts/9492 #/texts/9493 #/texts/9494 #/texts/9495 #/texts/9496 #/texts/9497 #/texts/9498 #/texts/9499 #/texts/9500 #/texts/9501 #/texts/9502 #/texts/9503 #/texts/9504 #/texts/9505 #/texts/9506 #/texts/9507 #/texts/9508 #/texts/9509 #/texts/9510 #/texts/9511 #/texts/9512 #/texts/9513 #/texts/9514 #/texts/9515 #/texts/9516 #/texts/9517 #/texts/9518 #/texts/9519 #/texts/9520 #/texts/9521 #/texts/9522 #/texts/9523 #/texts/9524 #/texts/9525 #/texts/9526 #/texts/9527 #/texts/9528 #/texts/9529 #/texts/9530 #/texts/9531 #/texts/9532 #/texts/9533 #/texts/9534 #/texts/9535 #/texts/9536 #/texts/9537 #/texts/9538 #/texts/9539 #/texts/9540 #/texts/9541 #/texts/9542 #/texts/9543 #/texts/9544 #/texts/9545 #/texts/9546 #/texts/9547 #/texts/9548 #/texts/9549 #/texts/9550 #/texts/9551 #/texts/9552 #/texts/9553 #/texts/9554 #/texts/9555 #/texts/9556 #/texts/9557 #/texts/9558 #/texts/9559 #/texts/9560 #/texts/9561 #/texts/9562 #/texts/9563 #/texts/9564 #/texts/9565

Emetics,
195
Emphysema aquosum,
Endrin,
204
Entomology,
Eonism,
63
150
Epileptic psychosis,
Epiphyses,
175
union with
shafts,
30
Erasure of
name,
Ergot,
224
Erotomania,
173
Eunuch,
147
Euthanasia,
19
Examination-in-chief,
Exhibitionism,
151
Exhumation,
46
Exit wound
of bullet,
Expert witness,
83
6
Extradural haemorrhage,
89
Eye, m.l.
52
F
Fabricated
Fall, from
Feigned wounds,
76
height,
92
insanity,
Fellatio,
177
151
Fetichism,
150
Filigree burns,
Firearms,
76
Fish-tailing,
75
109
E
101
125
11
109
6
Flashback phenomenon,
Flash burns,
105
Florence test,
Fodere’s
152
test,
161
Foetus, development
of,
33
Food poisoning,
Forensic
263
psychiatry,
laboratory,
Frost bite,
103
Friction burn,
Frigidity,
129
Frotteurism,
152
G
Gagging,
121
Ganja,
245
Garrotting,
118
Gas rigidity,
Gastric
60
lavage,
194
Gauge of
firearm,
Gerantophilia,
76
147
Glass-blower's shakes,
Graze,
65
Grievous injury,
Gun powder,
216
98
78
Gustafson’s method,
H
Haemin crystal
test,
Haemochromogen crystal
test,
168
Haemodialysis,
198
Hair,
37
Hallucination,
Hallucinogenic
Hanging,
113
judicial,
115
Hatter’s shakes,
216
Heat haematoma,
Heat
Heat
106

#/texts/9566 #/texts/9567 #/texts/9568 #/texts/9569 #/texts/9570 #/texts/9571 #/texts/9572 #/texts/9573 #/texts/9574 #/texts/9575 #/texts/9576 #/texts/9577 #/texts/9578 #/texts/9579 #/texts/9580 #/texts/9581 #/texts/9582 #/texts/9583 #/texts/9584 #/texts/9585 #/texts/9586 #/texts/9587 #/texts/9588 #/texts/9589 #/texts/9590 #/texts/9591 #/texts/9592 #/texts/9593 #/texts/9594 #/texts/9595 #/texts/9596 #/texts/9597 #/texts/9598 #/texts/9599 #/texts/9600 #/texts/9601 #/texts/9602 #/texts/9603 #/texts/9604 #/texts/9605 #/texts/9606 #/texts/9607 #/texts/9608 #/texts/9609 #/texts/9610 #/texts/9611 #/texts/9612 #/texts/9613 #/texts/9614 #/texts/9615 #/texts/9616 #/texts/9617 #/texts/9618 #/texts/9619 #/texts/9620 #/texts/9621 #/texts/9622 #/texts/9623 #/texts/9624 #/texts/9625 #/texts/9626 #/texts/9627 #/texts/9628 #/texts/9629 #/texts/9630 #/texts/9631 #/texts/9632 #/texts/9633 #/texts/9634 #/texts/9635 #/texts/9636 #/texts/9637 #/texts/9638 #/texts/9639 #/texts/9640 #/texts/9641 #/texts/9642 #/texts/9643 #/texts/9644 #/texts/9645 #/texts/9646 #/texts/9647 #/texts/9648 #/texts/9649 #/texts/9650 #/texts/9651 #/texts/9652 #/texts/9653 #/texts/9654 #/texts/9655 #/texts/9656 #/texts/9657 #/texts/9658 #/texts/9659 #/texts/9660 #/texts/9661 #/texts/9662 #/texts/9663 #/texts/9664 #/texts/9665 #/texts/9666 #/texts/9667 #/texts/9668 #/texts/9669 #/texts/9670 #/texts/9671 #/texts/9672 #/texts/9673 #/texts/9674 #/texts/9675 #/texts/9676 #/texts/9677 #/texts/9678 #/texts/9679 #/texts/9680 #/texts/9681 #/texts/9682 #/texts/9683 #/texts/9684 #/texts/9685 #/texts/9686 #/texts/9687 #/texts/9688 #/texts/9689 #/texts/9690 #/texts/9691 #/texts/9692 #/texts/9693 #/texts/9694 #/texts/9695 #/texts/9696 #/texts/9697 #/texts/9698 #/texts/9699 #/texts/9700 #/texts/9701 #/texts/9702 #/texts/9703 #/texts/9704 #/texts/9705 #/texts/9706 #/texts/9707

hyperpyrexia,
103
stiffening,
57
173
drugs,
65
186
251
172
29
168
250
Hermaphroditism,
Heroin,
250
Hesitation cuts,
Hippus,
256
Hijrah,
147
Holograph
Homicide,
Homicidal ideal,
will,
180
culpable,
95
poison,
188
Hostile witness,
Hurt,
98
Hydrochloric
Hydrocyanic
6
acid, acid,
208
259
acid,
208
Hydrogen sulphide,
Hydrostatic
259
test,
161
Huffing,
251
Hymen,
133
Hyoid bone,
fracture
I
Identification,
23
Identity, destruction,
Idiot,
175
Illusions,
174
Immersion syndrome,
sterility,
129
Impulses,
174
Incest,
146
Injury,
64
Inscised wounds,
Inscised-like
70
wounds,
Indecent assault,
Index,
69
152
267
Indian
Medical
Council,
Informed consent,
Infanticide,
9
19
159
Infantile whiplash
syndrome,
165
Injury,
64
Inhibition of
heart,
73
of,
24
120
42
123
50
Index
Inquest,
Magistrate, police,
1
Instantaneous
Intersexuality, rigor,
24
Intracerebral haemorrhage,
J
Joule burn,
Judicial
108
execution,
Jury,
1
K
Kastle-Meyer
Kerosene,
241
Kleptomania, test,
174
Klinefelter’s syndrome,
24
Knockout drops,
240
L
Lacerated wounds,
Lathyrism,
69
205
Lead,
216
Leading question,
Legal procedure,
Legitimacy,
137
Lesbianism,
Lie detector,
148
186

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Ligature mark,
Lightning,
114
death
Live birth,
from,
160
Locard’s princple
exchange,
Loco
Love
LSD,
Lucid of
186
parentis,
21
philter,
189
251
interval,
Lust murder,
174
149
Lynching,
115
M
Maceration of
foetus,
Mc
Ewan sign,
231
|
7
269
1
57
90
261
168
109
159
270
Concise
Mc
Naughten
Forensic rules,
Magnan’s
181
symptom,
Malingering,
21
Malpraxis,
14
Mania,
176
Manual strangulation,
Masquerades, m.1.,
7
Masochism,
150
Mastrubation,
151
Meconium,
163
Medical certificates,
Medical
Medical
Medical
Medical education,
9
ethics,
9
etiquette,
9
evidence,
Medical
3
indemnity insurance,
19
Medical maloccurrence,
Medical
Medical
15
records,
21
register,
M.T.P.
Act,
154
Medico-legal reports,
M.L.
masquerades,
Melancholia,
10
4
7
176
Mental
Health
Act,
177
Mental subnormality,
Mercury,
175
215
Mercurialentis,
216
Metal fume
fever,
219
Methaemoglobinaemia,
220
Methaquolone,
Methyl alcohol,
Mugging,
240
235
119
Multidose charcoal,
Mummification,
62
Munchausen’s syndrome,
Murder,
96
Mushrooms,
Musket,
77
265
9,
247
Medicine
N
Narco-analysis,
187
Necrophagia,
149
Necrophilia,
150
Neonatal cold
injury,
Nerium odorum,
Neuroses,
255
175
Negative autopsy,
49

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Negligence, criminal,
medical,
14
Nitric acid,
207
Nitrites,
220
Novus actus
interveniens,
15
Nystagmus, alcohol
gaze,
233
oO
Obscure autopsy,
Obsession,
174
Oochronosis,
210
Opium,
237
Ophitoxaemia,
226
Oral evidence,
4
Organophosphates,
Ossific centres
of skeleton,
30
Osteometric board.
Overlaying,
Oxalic acid,
Paederasty,
122
208
P
147
Palmar strangulation,
Paltauf’s haemorrhages,
Paracetamol,
242
Paradox guns,
Paraquat,
77
203
Parathion,
200
Paternalism,
20
165
3
119
196
50
103
15
200
45
118
125
Pelvis, sex,
24
198
Peritoneal dialysis,
Perjury,
6
Pethidine,
250
Phenol,
209
Phenolphthalein
Phobia,
174
Phosphorus,
221
Physical antidotes,
Physical
196
evidence,
Physiological
186
197
Phytotoxin,
Phossy
223
jaw,
222
Pistols, wounds
Ploucquet's from,
test,
Point blank
81
161
shot,
Poisons
Act,
189
diagnosis,
190
failure to
detect, treatment,
194
Polygraph,
186
Poroscopy,
35
P.M.
caloricity, changes,
52
interval, hypostasis,
63
54
Posthumous child,
Precipitate labour,
Precipitin test,
Pregnancy,
168
134
Primary flaccidity,
Primers,
78
Privileged communications,
Products liability,
18
Professional death
sentence,
11
81
189
193
137
164
55
198

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

test,
53
168
13
Professional miscounduct,
11
Professional negligence,
14
Professional secrecy,
Pseudocyesis,
136
Pseudostrangulation,
116
Psychopath,
175
Psychoses,
175
Ptomaines,
265
Pugilistic attitude,
Punch drunk,
Punctate
105
94
basophilia,
Puncture
217
wounds,
74
Putrefaction,
59
Q
Quod,
Railway
130
R
spine,
Rape,
140
accused,
145
victim,
143
Res ipsa
loquitur,
Respondent
14
superior,
Restraint of
Revolvers,
18
insane,
177
wounds,
Ricochet, of
bullets,
Ricinus communis,
Rifling
Rights
Rigor of
firearms, of
81
83
223
76
doctors,
12
mortis,
Rule of
Rule
56
Haase, of
34
nincs,
Run
Sadism,
Scalds, amok,
105
246
Ss
149
108
Scars,
36
Schizophrenia,
Scorpion,
229
91
176
12
Scoptophilia,
Self-inflicted
151
wounds,
Semecarpus anacardium,
225
Seminal fluid,
152
Serious professional
misconduct,
Sex,
11
differences in
skeleton,
24
Sexual asphyxias,
Sexual
Sexual
Sexual
Sexual
Sewer
Sin of
128
offences,
140
harassment,
141
oralism,
151
perversions,
149
gas,
259
gomorrah,
Shock,
100
Shotguns, wounding
by,
78
Shored exit
wound,
Skeleton, sex,
24
Skeletonisation,
60
Skull, age
and sutures,
firearm wounds,
78
fractures,
86
identification from,
24
Smothering,
121
Snakes,
226

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Sodomy,
147
Solvent abuse,
251
Somnambulism,
182
Sonography,
136
Souvenir bullet,
84
Spalding’s sign,
Spectroscopic
136,
160
exam,
168
Spinal cord,
concussion of,
91
Stab wounds,
Stalking,
74
141
Starvation,
111
State
Medical
Council, of
148
83
sex
76
31
10
Stature,
34
Statutory rape,
Index
271
142
Steering wheel
injury,
93
Still birth,
159
Stomach-bowel test,
Stomach, impact
162
emptying time,
63
Strangulation,
116
Strychnine,
253
Subarachnoid haemorrhage,
90
Subdural haemorrhage,
Subendocardial haemorrhages,
Subpoena,
2
Sudden infant
death syndrome,
166
Suffocation,
121
Suicide,
96
Sulphuric acid,
Summons,
206
2
Sunstroke,
103
Superfecundation,
137
Superfoetation,
137
Superimposition,
36
Supposititious children,
138
Suicidal poisons,
ideal,
189
Suspended animation,
Swallow tails,
70
Syncope,
47
T
Tail-gating,
94
Takayama test,
Tandem
168
bullet,
Tardieu
84
spots,
Tattoo
49
marks,
37
Teeth, eruption,
Teichmann’s
27
test,
168
90
41
52

Professor (Emeritus) of Biochemistry
Purdue University
West Lafayette, Indiana

Professor (Emeritus) of Nutritional Biochemistry
University College London
London, United Kingdom

Department of Comparative Biomedical Sciences
Royal Veterinary College
University of London
NewYork

Professor
Virginia Tech
Blacksburg, Virginia

Professor
Department of Molecular Physiology & Biophysics
Vanderbilt University
Nashville, Tennessee
SanFrancisco
Athens
NewDelhi
Madrid
Toronto
Mexico
City

McMaster University
Hamilton, Ontario, Canada

Professor and Head
Christian Medical College
Bagayam, Vellore, Tamil Nadu, India

Professor (Emeritus) of Veterinary Biochemistry

University of Toronto
Toronto, Ontario, Canada

Senior Associate Scientist
Division of Haematology/Oncology
Hospital for Sick Children, Toronto, and Professor
University of Toronto, Toronto, Canada

Professor
This page intentionally left blank

SECTION
SS Tera dtic-eeca iain
Proteins
&
Enzymes
1
- 1. Biochemistry & Medicine
1
Victor W. Rodwell, PhD, & Robert K. Murray, MD, PhD
- 2 Water &pH 6
Peter J. Kennelly, PhD & Victor W. Rodwell, PhD
- 3 Amino Acids & Peptides 14
- 4 Proteins: Determination of Primary
Structure
23
- 5 Proteins: Higher Orders of Structure 33
Enzymes: Kinetics,
Mechanism, Regulation, &
Role of Transition Metals 47
- 6 Proteins: Myoglobin & Hemoglobin 47
- 7 Enzymes: Mechanism of Action 56
- 8 Enzymes: Kinetics 68
Victor W. Rodwell, PhD
- 9 Enzymes: Regulation of Activities 82
- 10 The Biochemical Roles of Transition
Metals
92
Peter J. Kennelly, PhD
Bioenergetics 105
- 11 Bioenergetics: The Role of ATP 105
Kathleen M. Botham, PhD, DSc & Peter A. Mayes, PhD, DSc
- 12. Biologic Oxidation 111
- 13. The Respiratory Chain & Oxidative
Phosphorylation 117
Metabolism of Carbohydrates
129
- 14 Overview of Metabolism & the Provision of
Metabolic Fuels 129
David A. Bender, PhD & Peter A. Mayes, PhD, DSc
- 15 Carbohydrates of Physiological
Significance 141

- 16 The Citric Acid Cycle: The Central Pathway
of
Carbohydrate,
Lipid,
&
Amino
Acid
150
David A. Bender, PhD & Peter A. Mayes, PhD, DSc vi
CONTENTS
17 Glycolysis & the Oxidation of Pyruvate 157
18 Metabolism of Glycogen 164
19 Gluconeogenesis & the Control of
20 The Pentose Phosphate Pathway & Other
7)
21 Lipids of Physiologic Significance 195
22 Oxidation of Fatty Acids: Ketogenesis 207
23 Biosynthesis of Fatty Acids &
24 Metabolism of Acylglycerols &
25 Lipid Transport & Storage 236
26 Cholesterol Synthesis, Transport, &
27
ECTION
Blood Glucose 172
Pathways of Hexose Metabolism 182
Metabolism of Lipids 195
Eicosanoids 216
Sphingolipids 229
Excretion 249
Metabolism of Proteins &
Amino Acids 263
Biosynthesis of the Nutritionally Nonessential
29
30

32
33 Metabolism of Purine & Pyrimidine
34 Nucleic Acid Structure & Function 338
35
36 RNA Synthesis, Processing, &
37 Protein Synthesis & the Genetic Code 393
38
39
of
Proteins
&
of
Amino
Acid
Nitrogen 269
of
the
Carbon
Skeletons
of
Amino Acids 280
Conversion of Amino Acids to Specialized
Products 296
Victor W. Rodwell, PhD & Robert K. Murray, MD, PhD
Structure, Function, &
Replication of Informational
Macromolecules 319
Nucleotides
319
Nucleotides 327
P. Anthony Weil, PhD
DNA
Organization,
Replication,
&
Repair 350
Modification 374
Regulation of Gene Expression 409
Molecular Genetics, Recombinant DNA, &
Genomic Technology 432

Function 459
- 41 The Diversity of the Endocrine
System 480

Absorption 519
- 44 Micronutrients: Vitamins &
Minerals 527
David A. Bender, PhD
- 45 Free Radicals & Antioxidant
Nutrients 541
- 46 Glycoproteins 546
David A. Bender, PhD & Robert K. Murray, MD, PhD
- 47 Metabolism of Xenobiotics 556
- 48 Clinical Biochemistry 560
SECTION
Special Topics (B) 573
49 Intracellular Traffic & Sorting of Proteins 573
Kathleen M. Botham, PhD, DSc & Robert K. Murray, MD, PhD

Peter J. Kennelly, PhD, Robert K. Murray, MD, PhD,
Molly Jacob, MBBS, MD, PhD & Joe Varghese, MBBS, MD
53
Red
Blood
Cells
646
Peter J. Kennelly, PhD & Robert K. Murray, MD, PhD

Special Topics (C) 669
55
Hemostasis
&Thrombosis
669
Peter L. Gross, MD, MSc, FRCP(C),
P. Anthony Weil, PhD & Margaret L. Rand, PhD
56 Cancer: An Overview 681
Molly Jacob, MD, PhD, Joe Varghese, MBBS, MD &
57. The Biochemistry of Aging 707
58 Biochemical Case Histories 719
The Answer Bank 731
Index 735
The authors and publishers are pleased to present the thirty-first edition of Harper' Illustrated Biochemistry. The first edition, entitled Harper's Biochemistry, was published in 1939 under the sole authorship of Dr Harold Harper at the University of California School of Medicine, San Francisco, California. Presently entitled Harper' Illustrated Biochemistry, the book continues, as originally intended, to provide a concise survey of aspects of biochemistry most relevant to the study of medicine. Various authors have contributed to subsequent editions of this medically oriented biochemistry text, which is now observing its 79th year.

The illustration on the cover of the thirty-first edition, the structure of Zika virus protein determined at 3.8 A resolution, was generously prepared and provided by Lei Sun. The supporting data appeared in: Sirohi D, Chen Z, Sun L, Klose T, Pierson TC, Rossmann MG, Kuhn RJ: "The 3.8 A resolution cryo-EM structure of Zika virus protein', Science 2016;352:497-470. Together with the Zika virus, first recovered in the Zika valley of Uganda, the viruses responsible for yellow fever, West Nile fever, and dengue fever are members of the Flavivridae family of positive-strand DNA viruses. The cover illustration indicates the resolving power of cryo-electron microscopy (cryo-EM). More importantly, it recognizes the medical significance of infection by the Zika virus, which in pregnant women can result in a significant risk of congenital microcephaly and associated severe mental impairment. While Zika virus typically is transmitted by the bite of an infected mosquito, emerging evidence suggests that under certain conditions the Zika virus may also be transmitted between human subjects.

As always, Harper's Illustrated Biochemistry continues to emphasize the close relationship of biochemistry to the understanding of diseases, their pathology and the practice of medicine. The contents of most chapters have been updated and provide to the reader the most current and pertinent information. Toward that end, we have replaced Chapter 10 "Bioinformatics and Computational Biology,' most of whose programs and topics (for example protein and nucleotide sequence comparisons and in silico approaches in drug design) are available on line or are now common knowledge. Its replacement, new Chapter 10 "Biochemistry of Transition Metals,' incorporates material from several chapters, notably those of blood cells and plasma, which contained extensive content on metal ion adsorption and trafficking, especially of iron and copper. Since approximately a third of all proteins are metalloproteins, new Chapter 10 explicitly addresses the importance and overall pervasiveness of transition metals. Given the overlap with the topics of protein structure and of enzyme reaction mechanisms, new Chapter 10 now follows the three chapters on enzymes as the final chapter in Section II, now renamed Enzymes: Kinetics, Mechanism, Regulation, & Role of Transition Metals.

All 58 chapters of the thirty-first edition place major emphasis on the medical relevance of biochemistry. Topics are organized under eleven major headings. Both to assist study and to facilitate retention of the contained information, Questions follow each Section. An Answer Bankk follows the Appendix.
Section I includes a brief history of biochemistry, and emphasizes the interrelationships between biochemistry and medicine. Water, the importance of homeostasis of intracellular pH are reviewed, and the various orders of protein structure are addressed. Section II begins with a chapter on hemoglobin. Four chapters next address the kinetics, mechanism of action, and metabolic regulation of enzymes, and the role of metal ions in multiple aspects of intermediary metabolism. Section III addresses bioenergetics and the role of high energy phosphates in energy capture and transfer, the oxidation-reduction reactions involved in biologic oxidation, and metabolic details of energy capture via the respiratory chain and oxidative phosphorylation. Section IV considers the metabolism of carbohydrates via glycolysis, the citric acid cycle, the pentose phosphate pathway, glycogen metabolism, gluconeogenesis, and the control of blood glucose. Section V outlines the nature of simple and complex lipids, lipid transport and storage, the biosynthesis and degradation of fatty acids and more complex lipids, and the reactions and metabolic regulation of cholesterol biosynthesis and transport in human subjects. Section VI discusses protein catabolism, urea biosynthesis, and the catabolism of amino acids and stresses the medically significant metabolic disorders associated with their incomplete catabolism. The final chapter considers the biochemistry of the porphyrins and bile pigments.
Section XI includes hemostasis and thrombosis, an overview of cancer, the biochemistry of aging, and a selection of case histories.

The authors thank Michael Weitz for his role in the planning of this edition and Peter Boyle for overseeing its preparation for publication. We also thank Surbhi Mittal and Jyoti Shaw at Cenveo Publisher Services for their efforts in managing editing, typesetting, and artwork. We gratefully acknowledge numerous suggestions and corrections received from students and colleagues from around the world, especially those of Dr. Karthikeyan Pethusamy of the All India Institute of Medical Sciences, New Delhi, India.
Victor W. Rodwell
David A. Bender
Kathleen M. Botham
Peter J. Kennelly
P. Anthony Weil

. Understand the importance of the ability of cell-free extracts of yeast to ferment sugars, an observation that enabled discovery of the intermediates of fermentation, glycolysis, and other metabolic pathways.
: Appreciate the scope of biochemistry and its central role in the life sciences,
and that
biochemistry and
medicine are
intimately related
disciplines.
earth. = Appreciate that biochemistry integrates knowledge of the chemical processes in living cells with strategies to maintain health, understand disease, identify potential therapies, and enhance our understanding of the origins of life on
=
Describe how genetic approaches have been critical for elucidating many areas of biochemistry, and how the Human Genome Project has furthered advances in numerous aspects of biology and medicine.

Biochemistry and medicine enjoy a mutually cooperative relationship. Biochemical studies have illuminated many aspects of health and disease, and the study of various aspects of health and disease has opened up new areas of biochemistry. The medical relevance of biochemistry both in normal and abnormal situations is emphasized throughout this book. Biochemistry makes significant contributions to the fields of cell biology, physiology, immunology, microbiology, pharmacology, toxicology, and epidemiology, as well as the fields of inflammation, cell injury, and cancer. These close relationships emphasize that life, as we know it, depends on biochemical reactions and processes.

Although the ability of yeast to "ferment" various sugars to ethyl alcohol has been known for millennia, only comparatively recently did this process initiate the science of biochemistry. The great French microbiologist Louis Pasteur maintained that fermentation could only occur in intact cells. However, in 1899, the brothers Biichner discovered that fermentation could occur in the absence of intact cells when they stored a yeast extract in a crock of concentrated sugar solution, added as a preservative. Overnight, the contents of the crock fermented, spilled over the laboratory bench and floor,

and dramatically demonstrated that fermentation can proceed in the absence of an intact cell. This discovery unleashed an avalanche of research that initiated the science of biochemistry. Investigations revealed the vital roles of inorganic phosphate, ADP, ATP, and NAD(H), and ultimately identified the phosphorylated sugars and the chemical reactions and enzymes that convert glucose to pyruvate (glycolysis) or to ethanol and CO, (fermentation). Research beginning in the 1930s identified the intermediates of the citric acid cycle and of urea biosynthesis, and revealed the essential roles of certain vitamin-derived cofactors or "coenzymes" such as thiamin pyrophosphate, riboflavin, and ultimately coenzyme A, coenzyme Q, and cobamide coenzyme. The 1950s revealed how complex carbohydrates are synthesized from, and broken down into simple sugars, and the pathways for biosynthesis of pentoses, and the catabolism of amino acids and fatty acids. Investigators employed animal models, perfused intact organs, tissue slices, cell homogenates and their subfractions, and subsequently purified enzymes. Advances were enhanced by the development of analytical ultracentrifugation, paper and other forms of chromatography, and the post-World War II availability of radioisotopes, principally "C, *H, and *P, as "tracers" to identify the intermediates in complex pathways such as that of cholesterol biosynthesis. X-ray crystallography was then used to solve the three-dimensional structures of numerous proteins, polynucleotides, enzymes, and viruses. Genetic advances that followed the realization that DNA was a double helix include the polymerase chain reaction, and transgenic animals or those with gene knockouts. The methods used to prepare, analyze, purify, and identify metabolites and the activities of natural and recombinant enzymes and their threedimensional structures are discussed in the following chapters.

the interrelationship of biochemistry and medicine is a wide, two-way street. Biochemical studies have illuminated many aspects of health and disease, and conversely, the study of various aspects of health and disease has opened up new areas of biochemistry (Figure 1-1). An early example of how investigation of protein structure and function revealed the single difference in amino acid sequence between normal hemoglobin and sickle cell hemoglobin. Subsequent analysis of numerous variant sickle cell and other hemoglobins has contributed significantly to our understanding of the structure and function both of hemoglobin and of other proteins. During the early 1900s the English physician Archibald Garrod studied patients with the relatively rare disorders of alkaptonuria, albinism, cystinuria, and pentosuria, and established that these conditions were genetically determined. Garrod designated these conditions as inborn errors of metabolism. His insights provided a foundation for the development of the field of human biochemical genetics. A more recent example was investigation of the genetic and molecular basis of familial hypercholesterolemia, a disease that results in early-onset atherosclerosis. In addition to clarifying different genetic mutations responsible for this disease, this provided a deeper understanding of cell receptors and mechanisms of uptake, not only of cholesterol but also of how other molecules cross cell membranes. Studies of oncogenes and tumor suppressor genes in cancer cells have directed attention to the molecular mechanisms involved in the control of normal cell growth. These examples illustrate how the study of disease can open up areas of basic biochemical research. Science provides physicians and other workers in health care and biology with a foundation that impacts practice, stimulates curiosity, and promotes the adoption of scientific approaches for continued learning.

The two major concerns for workers in the health sciences— and particularly physicians—are the understanding and maintenance of health and effective treatment of disease. Biochemistry impacts both of these fundamental concerns, and

The World Health Organization (WHO) defines health as a state of "complete physical, mental, and social well-being and not merely the absence of disease and infirmity.' From a biochemical
FIGURE 1-1 A two-way street connects biochemistry and medicine. Knowledge of the biochemical topics listed above the green line of the diagram has clarified our understanding of the diseases shown below the green line. Conversely, analyses of the diseases have cast light on many areas of biochemistry. Note that sickle cell anemia is a genetic disease, and that both atherosclerosis and diabetes mellitus have genetic components.
viewpoint, health may be considered that situation in which all of the many thousands of intraand extracellular reactions that occur in the body are proceeding at rates commensurate with the organism's survival under pressure from both internal and external challenges. The maintenance of health requires optimal dietary intake of vitamins, certain amino acids and fatty acids, various minerals, and water. Understanding nutrition depends to a great extent on knowledge of biochemistry, and the sciences of biochemistry and nutrition share a focus on these chemicals. Recent increasing emphasis on systematic attempts to maintain health and forestall disease, or preventive medicine, includes nutritional approaches to the prevention of diseases such as atherosclerosis and cancer.

Apart from infectious organisms and environmental pollutants, many diseases are manifestations of abnormalities in genes, proteins, chemical reactions, or biochemical processes, each of which can adversely affect one or more critical biochemical functions. Examples of disturbances in human biochemistry responsible for diseases or other debilitating conditions include electrolyte imbalance, defective nutrient ingestion or absorption, hormonal imbalances, toxic chemicals or biologic agents, and DNA-based genetic disorders. To address these challenges, biochemical research continues to be interwoven with studies in disciplines such as genetics, cell biology, immunology, nutrition, pathology, and pharmacology. In addition, many biochemists are vitally interested in contributing to solutions to key issues such as the ultimate survival of mankind, and educating the public to support use of the scientific method in solving environmental and other major problems that confront our civilization.

Initially unanticipated rapid progress in the late 1990s in sequencing the human genome led in the mid-2000s to the announcement that over 90% of the genome had been sequenced. This effort was headed by the International Human Genome Sequencing Consortium and by Celera Genomics. Except for a few gaps, the sequence of the entire human genome was completed in 2003, just 50 years after the description of the double-helical nature of DNA by Watson and Crick. The implications for biochemistry, medicine, and indeed for all of biology, are virtually unlimited. For example, the ability to isolate and sequence a gene and to investigate its structure and function by sequencing and "gene knockout" experiments have revealed previously unknown genes and their products, and new insights have been gained concerning human evolution and procedures for identifying diseaserelated genes.
Major advances in biochemistry and understanding human health and disease continue to be made by mutation of the genomes of model organisms such as yeast, the fruit fly Drosophila melanogaster, the roundworm Caenorhabditis elegans, and the zebra fish, all organisms that can be genetically manipulated to provide insight into the functions of individual genes. These advances can potentially provide clues to curing human diseases such as cancer and Alzheimer disease. Figure 1-2 highlights areas that have developed or accelerated as a direct result of progress made in the Human Genome Project (HGP). New "-omics" fields focus on comprehensive study of the structures and functions of the molecules with which each is concerned. The products of genes (RNA molecules and proteins) are being studied using the techniques of transcriptomics and proteomics. A spectacular example of the speed of progress in transcriptomics is the explosion of knowledge about small RNA molecules as regulators of gene activity. Other -omics fields include glycomics, lipidomics, metabolomics, nutrigenomics, and pharmacogenomics. To keep pace with the information generated, bioinformatics has received much attention. Other related fields to which the impetus from the HGP has carried over are biotechnology, bioengineering, biophysics, and bioethics.

FIGURE 1-2 The Human Genome Project (HGP) has influenced many disciplines and areas of research. Biochemistry is not listed since it predates commencement of the HGP, but disciplines such as bioinformatics, genomics, glycomics, lipidomics, metabolomics, molecular diagnostics, proteomics, and transcriptomics are nevertheless active areas of biochemical research.
Definitions of these -omics fields and other terms appear in the Glossary of this chapter. Nanotechnology is an active area, which, for example, may provide novel methods of diagnosis and treatment for cancer and other disorders. Stem cell biology is at the center of much current research. Gene therapy has yet to deliver the promise that it appears to offer, but it seems probable that ultimately will occur. Many new molecular diagnostic tests have developed in areas such as genetic, microbiologic, and immunologic testing and diagnosis. Systems biology is also burgeoning. The outcomes of research in the various areas mentioned above will impact tremendously the future of biology, medicine, and the health sciences. Synthetic biology offers the potential for creating living organisms, initially small bacteria, from genetic material in vitro that might carry out specific tasks such as cleansing petroleum spills. All of the above make the 21st century an exhilarating time to be directly involved in biology and medicine.

■, 1 = Biochemistry is the science concerned with the molecules present in living organisms, individual chemical reactions and their enzyme catalysts, and the expression and regulation of each metabolic process. Biochemistry has become the basic language of all biologic sciences.. ■, 1 = Despite the focus on human biochemistry in this text, biochemistry concerns the entire spectrum of life forms, from viruses, bacteria, and plants to complex eukaryotes such as human beings.. ■, 1 = Biochemistry, medicine, and other health care disciplines are intimately related. Health in all species depends on a harmonious balance of the biochemical reactions occurring in the body, while disease reflects abnormalities in biomolecules, biochemical reactions, or biochemical processes.. ■, 1 = Advances in biochemical knowledge have illuminated many areas of medicine, and the study of diseases has often revealed previously unsuspected aspects of biochemistry.. ■, 1 = Biochemical approaches are often fundamental in illuminating the causes of diseases and in designing appropriate therapy. Biochemical laboratory tests also represent an integral component of diagnosis and monitoring of treatment.. ■, 1 = Asound knowledge of biochemistry and of other related basic disciplines is essential for the rational practice of medicine and related health sciences.. ■, 1 = Results of the HGP and of research in related areas will have a profound influence on the future of biology, medicine, and other health sciences.. ■, 1 = Genomic research on model organisms such as yeast, the fruit fly D. melanogaster , the roundworm C. elegans , and the zebra fish provides insight into understanding human diseases.

Bioengineering: The application of engineering to biology and medicine.
Bioethics: The area of ethics that is concerned with the application of moral and ethical principles to biology and medicine.

Shown are selected examples of databases that assemble, annotate, and analyze data of biomedical importance.
ENCODE: ENCyclopedia Of DNA Elements. A collaborative effort that combines laboratory and computational approaches identify every functional element in the human genome.
to
GenBank: Protein sequence database of the National Institutes of
Health (NIH) stores all known biologic nucleotide sequences and their translations in a searchable form.
HapMap: Haplotype Map, an international effort to identify single nucleotide polymorphisms (SNPs) associated with common
human diseases and differential responses to pharmaceuticals.
ISDB: International Sequence DataBase that incorporates DNA
databases of Japan and of the European Molecular Biology
Laboratory (EMBL).
PDB: Protein DataBase. Three-dimensional structures of proteins, polynucleotides, and other macromolecules, including proteins
bound to substrates, inhibitors, or other proteins.

Vm T Ole ICM UER aCe aA you should be able to:
. Describe the properties of water that account for its surface tension, viscosity, liquid state at ambient temperature, and solvent power.
: Use structural formulas to represent several organic compounds that can serve as hydrogen bond donors or acceptors.
m= Explain the role played by entropy in the orientation, in an aqueous
environment, of the polar and nonpolar regions of macromolecules.
= Indicate the quantitative contributions of salt bridges, hydrophobic
interactions, and van der Waals forces to the stability of macromolecules.
m= Explain the relationship of pH to acidity, alkalinity, and the quantitative
determinants that
characterize weak
and strong
acids.
= Calculate the shift in pH that accompanies the addition of a given quantity of acid or base to the pH of a buffered solution.
= Describe what buffers do, how they do it, and the conditions under which a
buffer is
most effective
under physiologic
or other
conditions.
- = Illustrate how the Henderson-Hasselbalch equation can be used to calculate the net charge on a polyelectrolyte at a given pH.

the pH of extracellular fluid between 7.35 and 7.45. Suspected disturbances of acid-base balance are verified by measuring the pH of arterial blood and the CO, content of venous blood. Causes of acidosis (blood pH <7.35) include diabetic ketosis and lactic acidosis. Alkalosis (pH >7.45) may follow vomiting of acidic gastric contents.

A water molecule is an irregular, slightly skewed tetrahedron with oxygen at its center (Figure 2-1). The two hydrogens and the unshared electrons of the remaining two sp*-hybridized orbitals occupy the corners of the tetrahedron. The 105° angle between the two hydrogen atoms differs slightly from the ideal tetrahedral angle, 109.5°. Ammonia is also tetrahedral, with a 107° angle between its three hydrogens. The strongly electronegative oxygen atom in a water molecule attracts electrons away from the hydrogen nuclei, leaving them with a partial

positive charge, while its two unshared electron pairs constitute a region of local negative charge.
A molecule with electrical charge distributed asymmetrically about its structure is referred to as a dipole. Water's strong dipole is responsible for its high dielectric constant. As described quantitatively by Coulomb's law, the strength of interaction F between oppositely charged particles is inversely proportionate to the dielectric constant ¢ of the surrounding medium. The dielectric constant for a vacuum is essentially unity; for hexane it is 1.9; for ethanol, 24.3; and for water at 25°C, 78.5. Water therefore greatly decreases the force of attraction between charged and polar species relative to waterfree environments with lower dielectric constants. Its strong dipole and high dielectric constant enable water to dissolve large quantities of charged compounds such as salts.

A partially unshielded hydrogen nucleus covalently bound to an electron-withdrawing oxygen or nitrogen atom can interact with an unshared electron pair on another oxygen or nitrogen atom to form a hydrogen bond. Since water molecules contain both of these features, hydrogen bonding favors the selfassociation of water molecules into ordered arrays (Figure 2-2). Hydrogen bonding profoundly influences the physical properties of water and accounts for its relatively high viscosity, surface tension, and boiling point. On average, each molecule in liquid water associates through hydrogen bonds with 3.5 others. These bonds are both relatively weak and transient, with a half-life of a few picoseconds. Rupture of a hydrogen bond in liquid water requires only about 4.5 kcal/mol, less than 5% of the energy required to rupture a covalent O—H bond. Hydrogen bonding enables water to dissolve many organic biomolecules that contain functional groups which
FIGURE 2-2 Water molecules self-associate via hydrogen bonds. Shown are the association of two water molecules (left) and a hydrogen-bonded cluster of four water molecules (right). Notice that water can serve simultaneously both as a hydrogen donor and as a hydrogen acceptor.
FIGURE 2-3 Additional polar groups participate in hydrogen bonding. Shown are hydrogen bonds formed between alcohol and water, between two molecules of ethanol, and between the peptide carbonyl oxygen and the peptide nitrogen hydrogen of an adjacent amino acid.
can participate in hydrogen bonding. The oxygen atoms of aldehydes, ketones, and amides, for example, provide lone pairs of electrons that can serve as hydrogen acceptors. Alcohols, carboxylic acids, and amines can serve both as hydrogen acceptors and as donors of unshielded hydrogen atoms for formation of hydrogen bonds (Figure 2-3).

The covalent bond is the strongest force that holds molecules together (Table 2-1). Noncovalent forces, while of lesser magnitude, make significant contributions to the structure, stability, and functional competence of macromolecules in living cells. These forces, which can be either attractive or repulsive, involve interactions both within the biomolecule and between it and the water that forms the principal component of the surrounding environment.

O-O, Energy (kcal/mol) = 34. O-O, BondType = O-O -. O-O, Energy (kcal/mol) = 96. S-S, Energy (kcal/mol) = 51. S-S, BondType = C-H. S-S, Energy (kcal/mol) = 99. C-N, Energy (kcal/mol) = 70. C-N, BondType = C-S -. C-N, Energy (kcal/mol) = 108. S-H, Energy (kcal/mol) = 81. S-H, BondType = O-H. S-H, Energy (kcal/mol) = 110. C-C, Energy (kcal/mol) = 82. C-C, BondType = C-C -. C-C, Energy (kcal/mol) = 147. C-O, Energy (kcal/mol) = 84. C-O, BondType = C-N -. C-O, Energy (kcal/mol) = 147. N-H, Energy (kcal/mol) = 94. N-H, BondType = C-O -. N-H, Energy (kcal/mol) = 164

Most biomolecules are amphipathic; that is, they possess regions rich in charged or polar functional groups as well as regions with hydrophobic character. Proteins tend to fold with the R-groups of amino acids with hydrophobic side chains in the interior. Amino acids with charged or polar amino acid side chains (eg, arginine, glutamate, serine, see Table 3-1) generally are present on the surface in contact with water. A similar pattern prevails in a phospholipid bilayer where the charged "head groups" of phosphatidylserine or phosphatidylethanolamine contact water while their hydrophobic fatty acyl side chains cluster together, excluding water (see Figure 40-5). This pattern maximizes the opportunities for the formation of energetically favorable charge-dipole, dipole-dipole, and hydrogen bonding interactions between polar groups on the biomolecule and water. It also minimizes energetically unfavorable contacts between water and hydrophobic groups.

Hydrophobic interaction refers to the tendency of nonpolar compounds to self-associate in an aqueous environment. This selfassociation is driven neither by mutual attraction nor by what are sometimes incorrectly referred to as "hydrophobic bonds" Self-association minimizes the disruption of energetically favorable interactions between the surrounding water molecules.
While the hydrogens of nonpolar groups such as the methylene groups of hydrocarbons do not form hydrogen bonds, they do affect the structure of the water that surrounds them. Water molecules adjacent to a hydrophobic group are restricted in the number of orientations (degrees of freedom) that permit them to participate in the maximum number of energetically favorable hydrogen bonds. Maximal formation of multiple hydrogen bonds, which maximizes enthalpy, can be maintained only by increasing the order of the adjacent water molecules, with an accompanying decrease in entropy.
It follows from the second law of thermodynamics that the optimal free energy of a hydrocarbon-water mixture is a function of both maximal enthalpy (from hydrogen bonding) and highest entropy (maximum degrees of freedom). Thus, nonpolar molecules tend to form droplets that minimize exposed surface area and reduce the number of water molecules whose motional freedom becomes restricted. Similarly, in the aqueous environment of the living cell the hydrophobic portions of biopolymers tend to be buried inside the structure of the molecule, or within a lipid bilayer, minimizing contact with water.

Interactions between charged groups help shape biomolecular structure. Electrostatic interactions between oppositely charged groups within or between biomolecules are termed salt bridges. Salt bridges are comparable in strength to hydrogen bonds but act over larger distances. They therefore often facilitate the binding of charged molecules and ions to proteins and nucleic acids.
FIGURE 2-4 The strength of van der Waals interactions varies with the distance, R, between interacting species. The force of interaction between interacting species increases with decreasing distance between them until they are separated by the van der Waals contact distance (see arrow marked A). Repulsion due to interaction between the electron clouds of each atom or molecule then supervenes. While individual van der Waals interactions are extremely weak, their cumulative effect is nevertheless substantial for macromolecules such as DNA and proteins which have many atoms in close contact.

van der Waals forces arise from attractions between transient dipoles generated by the rapid movement of electrons of all neutral atoms. Significantly weaker than hydrogen bonds but potentially extremely numerous, van der Waals forces decrease as the sixth power of the distance separating atoms (Figure 2-4). Thus, they act over very short distances, typically 2 to 4 A,

The DNA double helix illustrates the contribution of multiple forces to the structure of biomolecules. While each individual DNA strand is held together by covalent bonds, the two strands of the helix are held together exclusively by noncovalent interactions such as hydrogen bonds between nucleotide bases (Watson-Crick base pairing) and van der Waals interactions between the stacked purine and pyrimidine bases. The double helix presents the charged phosphate groups and polar hydroxyl groups from the ribose sugars of the DNA backbone to water while burying the relatively hydrophobic nucleotide bases inside. The extended backbone maximizes the distance between negatively charged phosphates, minimizing unfavorable electrostatic interactions (see Figure 34-2).

Metabolic reactions often involve the attack by lone pairs of electrons residing on electron-rich molecules termed nucleophiles upon electron-poor atoms called electrophiles. Nucleophiles and electrophiles do not necessarily possess a formal negative or positive charge. Water, whose two lone pairs of
sp electrons bear a partial negative charge (see Figure 2-1), is an excellent nucleophile. Other nucleophiles of biologic importance include the oxygen atoms of phosphates, alcohols, and carboxylic acids; the sulfur of thiols; and the nitrogen atom of amines and of the imidazole ring of histidine. Common electrophiles include the carbonyl carbons in amides, esters, aldehydes, and ketones and the phosphorus atoms of phosphoesters.
Nucleophilic attack by water typically results in the cleavage of the amide, glycoside, or ester bonds that hold biopolymers together. This process is termed hydrolysis. Conversely, when monomer units are joined together to form biopolymers, such as proteins or glycogen, water is a product, for example, during the formation of a peptide bond between two amino acids.
While hydrolysis is a thermodynamically favored reaction, the amide and phosphoester bonds of polypeptides and oligonucleotides are stable in the aqueous environment of the cell. This seemingly paradoxical behavior reflects the fact that the thermodynamics that govern the equilibrium point of a reaction do not determine the rate at which it will proceed toward its equilibrium point. In the cell, protein catalysts called enzymes accelerate the rate of hydrolytic reactions when needed. Proteases catalyze the hydrolysis of proteins into their component amino acids, while nucleases catalyze the hydrolysis of the phosphoester bonds in DNA and RNA. Careful control of the activities of these enzymes is required to ensure that they act only at appropriate times.

Many of the enzymic reactions responsible for synthesis and breakdown of biomolecules involve the transfer of a chemical group G from a donor D to an acceptor A to form an acceptor group complex, A—G:

The hydrolysis and phosphorolysis of glycogen, for example, involve the transfer of glucosyl groups to water or to orthophosphate. The equilibrium constant for the hydrolysis of covalent bonds strongly favors the formation of split products. Conversely, many group transfer reactions responsible for the biosynthesis of macromolecules involve the thermodynamically unfavored formation of covalent bonds. Enzyme catalysts play a critical role in surmounting these barriers by virtue of their capacity to directly link two normally separate reactions together. By linking an energetically unfavorable group transfer reaction with a thermodynamically favorable reaction, such as the hydrolysis of ATP, a new coupled reaction can be generated whose net overall change in free energy favors biopolymer synthesis.
Given the nucleophilic character of water and its high concentration in cells, why are biopolymers such as proteins and DNA relatively stable? And how can synthesis of biopolymers occur in an aqueous environment that favors hydrolysis? Central to both questions are the properties of enzymes. In the absence of enzymic catalysis, even reactions that are highly favored thermodynamically do not necessarily take place rapidly. Precise and differential control of enzyme activity and the sequestration of enzymes in specific organelles determine the physiologic circumstances under which a given biopolymer will be synthesized or degraded. The ability of enzyme active sites to sequester substrates in an environment from which water can be excluded facilitates biopolymer synthesis.

The ability of water to ionize, while slight, is of central importance for life. Since water can act both as an acid and as a base, its ionization may be represented as an intermolecular proton transfer that forms a hydronium ion (H,O*) and a hydroxide ion (OH'):

The transferred proton is actually associated with a cluster of water molecules. Protons exist in solution not only as H,O*, but also as multimers such as H,O,* and H,O,*. The proton is nevertheless routinely represented as H*, even though it is in fact highly hydrated.
Since hydronium and hydroxide ions continuously recombine to form water molecules, an individual hydrogen or oxygen cannot be stated to be present as an ion or as part of a water molecule. At one instant it is an ion; an instant later it is part of a water molecule. Individual ions or molecules are therefore not considered. We refer instead to the probability that at any instant in time a given hydrogen will be present as an ion or as part of a water molecule. Since 1 g of water contains 3.46 x 10" molecules, the ionization of water can be described statistically. To state that the probability that a hydrogen exists as an ion is 0.01 means that at any given moment in time, a hydrogen atom has 1 chance in 100 of being an ion and 99 chances out of 100 of being part of a water molecule. The actual probability of a hydrogen atom in pure water existing as a hydrogen ion is approximately 1.8 x 10°. The probability of its being part of a water molecule thus is almost unity. Stated another way, for every hydrogen ion or hydroxide ion in pure water, there are 0.56 billion or 0.56 x 10° water molecules. Hydrogen ions and hydroxide ions nevertheless contribute significantly to the properties of water.
For dissociation of water,
where the brackets represent molar concentrations (strictly speaking, molar activities) and K is the dissociation constant. Since 1 mole (mol) of water weighs 18 g, 1 liter (L) (1000 g) of water contains 1000 + 18 = 55.56 mol. Pure water thus is 55.56 molar. Since the probability that a hydrogen in pure water will exist as a hydrogen ion is 1.8 x 10~°, the molar concentration of H* ions (or of OH~ ions) in pure water is the

product of the probability, 1.8 x 10~, times the molar concentration of water, 55.56 mol/L. The result is 1.0 x 10-7 mol/L. We can now calculate the dissociation constant K for pure water:
(H*][(OH]_
[107]
[107]
{H,O]
[55.56]
=0.018x10 4 =1.8x10 '° mol/L
The molar concentration of water, 55.56 mol/L, is too great to be significantly affected by dissociation. It is therefore considered to be essentially constant. This constant may therefore be incorporated into the dissociation constant K to provide a useful new constant K, termed the ion product for water. The relationship between K, and K is shown below:
K,,
=1.00x10-*(mol/LY
Note that the dimensions of K are moles per liter and those of K, are moles' per liter'. As its name suggests, the ion product K, is numerically equal to the product of the molar concentrations of H* and OH':

At 25°C, K, = (10°), or 10% (mol/L)'. At temperatures below 25°C, K, is somewhat less than 107", and at temperatures above 25°C it is somewhat greater than 10-'*. Within the stated limitations of temperature, K, equals 10-'* (mol/L)? for all aqueous solutions, even solutions of acids or bases. We use K, to calculate the pH of acidic and basic solutions.

The term pH was introduced in 1909 by Sérensen, who defined it as the negative log of the hydrogen ion concentration:

This definition, while not rigorous, suffices for many biochemical purposes. To calculate the pH of a solution:
1. Calculate the hydrogen ion concentration [H'*].
2. Calculate the base 10 logarithm of [H*].
3. pH is the negative of the value found in step 2.
For example, for pure water at 25°C,
pH=-log[H*]=—log10' =(-7)=7.0
This value is also known as the power (English), puissant (French), or potennz (German) of the exponent, hence the use «> of the term "p'
Low pH values correspond to high concentrations of H* and high pH values correspond to low concentrations of H*. Acids are proton donors and bases are proton acceptors. Strong acids (eg, HCI, H,SO,) completely dissociate into anions and protons even in strongly acidic solutions (low pH). Weak acids dissociate only partially in acidic solutions. Similarly, strong bases (eg, KOH, NaOH), but not weak bases like Ca(OH),, are completely dissociated even at high pH. Many biochemicals are weak acids. Exceptions include phosphorylated intermediates, whose phosphoryl group contains two dissociable protons, the first of which is strongly acidic. The following examples illustrate how to calculate the pH
of acidic and basic solutions.
Example 1: What is the pH of a solution whose hydrogen ion concentration is 3.2 x 10~* mol/L?
pH=—log[H"]
=—log(3.2x10“)
=—log(3.2)—log(10*)
=-0.5+4.0
=3.5
Example 2: What is the pH of a solution whose hydroxide ion concentration is 4.0 x 10 mol/L? We first define a quantity pOH that is equal to -log[OH™] and that may be derived from the definition of K,:

Therefore, or
pH+pOH=14
To solve the problem by this approach:
[OH ]=4.0x10"
pOH =-log[OH ]
=—log(4.0x10“*)
=—log(4.0)—log(10™)
=-0.60+4.0
=3.4
pH=14—pOH=14-3.4
=10.6
The examples above illustrate how the logarithmic pH scale facilitates recording and comparing hydrogen ion concentrations that differ by orders of magnitude from one another, 0.00032 M (pH 3.5) and 0.000000000025 M (pH 10.6). Example 3: What are the pH values of (a) 2.0 x 10° mol/L KOH and of (b) 2.0 x 10°° mol/L KOH? The OH arises from two sources, KOH and water. Since pH is determined by the total [H*]
Now
log{H*]+log[OH™ ]=log10""*
(and pOH by the total [OH-]), both sources must be considered. In the first case (a), the contribution of water to the total [OH] is negligible. The same cannot be said for the second case (b):
Molarity of KOH, Concentration (mol/L).(a) = 2.0 × 10 -2. Molarity of KOH, Concentration (mol/L).(b) = 2.0 × 10 -6. [OH - ] from KOH, Concentration (mol/L).(a) = 2.0 × 10 -2. [OH - ] from KOH, Concentration (mol/L).(b) = 2.0 × 10 -6. [OH - ] from water, Concentration (mol/L).(a) = 1.0 × 10 -7. [OH - ] from water, Concentration (mol/L).(b) = 1.0 × 10 -7. Total [OH - ], Concentration (mol/L).(a) = 2.00001 × 10 -2. Total [OH - ], Concentration (mol/L).(b) = 2.1 × 10 -6

Once a decision has been reached about the significance of the contribution
of water,
pH
may be
calculated as
above.
The above examples assume that the strong base KOH is completely dissociated in solution and that the concentration of OH" ions was thus equal to that due to the KOH plus that present initially in the water. This assumption is valid for dilute solutions of strong bases or acids, but not for weak bases or acids. Since weak electrolytes dissociate only slightly in solution, we must use the dissociation constant to calculate the concentration of [H*] (or [OH™]) produced by a given molarity of a weak acid (or base) before calculating total [H*] (or total [OH-]) and subsequently pH.

Many biochemicals possess functional groups that are weak acids or bases. Carboxyl groups, amino groups, and phosphate esters, whose second dissociation falls within the physiologic range, are present in proteins and nucleic acids, most coenzymes, and most intermediary metabolites. Knowledge of the dissociation of weak acids and bases thus is basic to understanding the influence of intracellular pH on structure and biologic activity. Charge-based separations such as electrophoresis and ion exchange chromatography are also best understood in terms of the dissociation behavior of functional groups.
We term the protonated species (HA or R—NH,") the acid and the unprotonated species (AT or R—NH,) its conjugate base. Similarly, we may refer to a base (A~ or R—NH,) and its conjugate acid (HA or R—NH,,*).
We express the relative strengths of weak acids and bases in terms of their dissociation constants. Shown below are the expressions for the dissociation constant (K,) for two representative weak acids, R-COOH and R—NH,".
Since the numeric values of K, for weak acids are negative exponential numbers, we express K, as pK,, where

Note that pK, is related to K, as pH is to [H*]. The stronger the acid, the lower is its value.
pK, Representative weak acids (left), their conjugate bases
(center), and pK, values (right) include the following:
pK, is used to express the relative strengths of both acids and bases. For any weak acid, its conjugate is a strong base. Similarly, the conjugate of a strong base is a weak acid. The relative strengths of bases are expressed in terms of the pK, of their conjugate acids. For polyprotic compounds containing more than one dissociable proton, a numerical subscript is assigned to each dissociation, numbered starting from unity in decreasing order of relative acidity. For a dissociation of the type

the pK, is the pH at which the concentration of the acid R—NH,_ equals that of the base R-NH,,.
From the above equations that relate K, to [H*] and to the concentrations of undissociated acid and its conjugate base,
when
or when then

Thus, when the associated (protonated) and dissociated (conjugate base) species are present at equal concentrations, the prevailing hydrogen ion concentration [H*] is numerically equal to the dissociation constant, K,. If the logarithms of both sides of the above equation are taken and both sides are multiplied by —1, the expressions would be as follows:

Since —log K, is defined as pK,, and —log [H*] defines pH, the equation may be rewritten as

that is, the pK, of an acid group is the pH at which the protonated and unprotonated species are present at equal
concentrations. The pK, for an acid may be determined by adding 0.5 equivalent of alkali per equivalent of acid. The resulting pH will equal the pK, of the acid.

The Henderson-Hasselbalch equation is derived below. A weak acid, HA, ionizes as follows:
The equilibrium constant for this dissociation is
Cross-multiplication gives
Divide both sides by [A>]:
Take the log of both sides:
Multiply through by -1:
Substitute pH and pK, for —log [H*] and —log K,, respectively; then
Inversion of the last term removes the minus sign and gives the Henderson-Hasselbalch equation
The Henderson-Hasselbalch equation has great predictive value in protonic equilibria. For example,
- 1. When an acid is exactly half-neutralized, [A-] = [HA]. Under these conditions,
Therefore, at half-neutralization, pH = pK..
FIGURE
2-5
Titration curve
for an
acid of
the type
heavy dot in the center of the curve indicates the pK,, 5.0.
2. When the ratio [A~]/[HA] = 100:1,
3. When the ratio [A~]/[HA] = 1:10,
If the equation is evaluated at ratios of [A~]/[HA] ranging from 10° to 10° and the calculated pH values are plotted, the resulting graph describes the titration curve for a weak acid (Figure 2-5).

Solutions of weak acids or bases and their conjugates exhibit buffering, the ability to resist a change in pH following addition of strong acid or base. Many metabolic reactions are accompanied by the release or uptake of protons. Oxidative metabolism produces CO,, the anhydride of carbonic acid, which if not buffered would produce severe acidosis. Biologic maintenance of a constant pH involves buffering by phosphate, bicarbonate, and proteins, which accept or release protons to resist a change in pH. For laboratory experiments using tissue extracts or enzymes, constant pH is maintained by the addition of buffers such as MES ([2-N-morpholino]ethanesulfonic acid, pK, 6.1), inorganic orthophosphate (pK,, 7.2), HEPES (N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pK, 6.8), or Tris (tris[hydroxymethyl]aminomethane, pK, 8.3). The value of pK, relative to the desired pH is the major determinant of which buffer is selected. Buffering can be observed by using a pH meter while titrating a weak acid or base (Figure 2-5). We can also calculate the pH shift that accompanies addition of acid or base to a buffered solution. In the example below, the buffered solution (a weak acid, pK, = 5.0, and its conjugate base) is initially at one of four pH values. We will calculate the pH shift
HA.
The
that results when 0.1 meq of KOH is added to 1 meq of each solution:

= 0.60. log ([A - ]/[HA]) final, 3 = 0.95. log ([A - ]/[HA]) final, 4 = 1.69. Final pH, 1 = 5.18. Final pH, 2 = 5.60. Final pH, 3 = 5.95. Final pH, 4 = 6.69. ΔpH, 1 = 0.18. ΔpH, 2 = 0.60. ΔpH, 3 = 0.95. ΔpH, 4 = 1.69
Notice that ApH, the change in pH per milliequivalent of OHadded, depends on the initial pH. The solution resists changes in pH most effectively at pH values close to the pK,. A solution of a weak acid and its conjugate base buffers most

Figure 2-5 also illustrates how the net charge on one molecule of the acid varies with pH. A fractional charge of —0.5 does not mean that an individual molecule bears a fractional charge but that the probability is 0.5 that a given molecule has a unit negative charge at any given moment in time. Consideration of the net charge on macromolecules as a function of pH provides the basis for separatory techniques such as ion exchange chromatography and electrophoresis (see Chapter 4).

Many acids of biologic interest possess more than one dissociating group. The presence of local negative charge hinders proton release from nearby acidic groups, raising their pK,. This is illustrated by the pK, values of the three dissociating groups of phosphoric acid and citric acid (Table 2-2). The effect of adjacent charge decreases with distance. The second pK, for succinic acid, which has two methylene groups between its carboxyl groups, is 5.6, whereas the second pK, for glutaric acid, which has one additional methylene group, is 5.4.

The pK, of a functional group is also profoundly influenced by the surrounding medium. The medium may either raise or lower the pK, relative to its value in water, depending on whether the undissociated acid or its conjugate base is the charged species. The effect of dielectric constant on pK, may be observed by adding ethanol to water. The pK, ofa carboxylic
CHAPTER2
Water
&
pH
TABLE 2-2 Relative Strengths of Monoprotic, Diprotic, and Triprotic Acids
Lactic acid, 1 = p K = 3.86. Lactic acid, 2 = . Lactic acid, 3 = . Acetic acid, 1 = p K = 4.76. Acetic acid, 2 = . Acetic acid, 3 = . Ammonium ion, 1 = p K = 9.25. Ammonium ion, 2 = . Ammonium ion, 3 = . Carbonic acid, 1 = p K 1 = 6.37;. Carbonic acid, 2 = p K 2 = 10.25. Carbonic acid, 3 = . Succinic acid, 1 = p K 1 = 4.21;. Succinic acid, 2 = p K 2 = 5.64. Succinic acid, 3 = . Glutaric acid, 1 = p K 1 = 4.34;. Glutaric acid, 2 = p K 2 = 5.41. Glutaric acid, 3 = . Phosphoric acid, 1 = p K 1 = 2.15;. Phosphoric acid, 2 = p K 2 = 6.82;. Phosphoric acid, 3 = p K 3 = 12.38. Citric acid, 1 = p K 1 = 3.08;. Citric acid, 2 = p K 2 = 4.74;. Citric acid, 3 = p K 3 = 5.40
Note: Tabulated values are the pK, values (-log of the dissociation constant).
acid increases, whereas that of an amine decreases because ethanol decreases the ability of water to solvate a charged species. The pK, values of dissociating groups in the interiors of proteins thus are profoundly affected by their local environment, including the presence or absence of water.

Water forms hydrogen-bonded clusters with itself and with other proton donors or acceptors. Hydrogen bonds account for the surface tension, viscosity, liquid state at room temperature, and solvent power of water.
™
Compounds
that
contain
O
or
N
can
serve
as
hydrogen
bond
donors
and/or
acceptors.
= Entropic forces dictate that macromolecules expose polar regions to an aqueous interface and bury nonpolar regions.
@ Salt bridges, hydrophobic interactions, and van der Waals forces participate in maintaining molecular structure.
™ pH is the negative log of [H*]. A low pH characterizes an acidic
solution, and a high pH denotes a basic solution.
™ The strength of weak acids is expressed by pK,, the negative log of the acid dissociation constant. Strong acids have low pK, values and weak acids have high pK, values.
™ Buffers resist a change in pH when protons are produced or consumed. Maximum buffering capacity occurs +1 pH unit on either side of pK,. Physiologic buffers include bicarbonate, orthophosphate, and proteins.

Reese KM: Whence came the symbol pH. Chem & Eng News
2004;82:64.
Segel IM: Biochemical Calculations. Wiley, 1968.
Skinner JL: Following the motions of water molecules in aqueous
solutions. Science 2010;328:985.
Stillinger FH: Water revisited. Science 1980;209:451.
Suresh SJ, Naik VM: Hydrogen bond thermodynamic properties of
water from dielectric constant data. J Chem Phys 2000;113:9727.
Wiggins
PM:
Role
of
water
in
some
Microbiol
Rev 1990;54:432.

After studying this chapter, you should be able to:
2 Diagram the structures and write the threeand one-letter designations for each of the amino acids present in proteins.
Provide examples of how each type of R group of the protein amino acids .
their chemical
properties.
= List additional important functions of amino acids and explain how certain amino acids in plant seeds can severely impact human health.
= Name the ionizable groups of the protein amino acids and list their
approximate pK, values as free amino acids in aqueous solution.
= Calculate the pH of an unbuffered aqueous solution of a polyfunctional amino acid and the change in pH that occurs following the addition of a given quantity of strong acid or alkali.
= Define pl and explain its relationship to the net charge on a polyfunctional electrolyte.
= Explain how pH, pK, and pl can be used to predict the mobility of a
polyelectrolyte, such
as an
amino acid,
in
a
direct-current electrical
field.
Describe the directionality, nomenclature, and primary structure of peptides.
Describe the conformational consequences of the partial double-bond
character of the peptide bond and identify the bonds in the peptide backbone that are free to rotate.

acids normally appear in the urine because amino acids are almost totally reabsorbed in the proximal tubule, conserving them for protein synthesis and other vital functions. Certain microorganisms secrete free D-amino acids, or peptides that may contain both pand L-a-amino acids. Several of these bacterial peptides are of therapeutic value, including the antibiotics bacitracin and gramicidin A, and the antitumor agent bleomycin. Certain other microbial peptides are, however, toxic. The cyanobacterial peptides microcystin and nodularin are lethal in large doses, while small quantities promote the formation of hepatic tumors. The ingestion of certain amino acids present in the seeds of legumes of the genus Lathyrus can result in lathyrism, a tragic irreversible disease in which individuals lose control of their limbs. Certain other plant seed amino acids have also been implicated in neurodegenerative disease in natives of Guam.

Although more than 300 amino acids occur in nature, proteins are synthesized almost exclusively from the set of 20 L-a-amino acids encoded by nucleotide triplets called codons (see Table 37-1). While the three-letter genetic code could potentially accommodate more than 20 amino acids, the genetic code is redundant since several amino acids are specified by multiple codons. Scientists frequently represent the sequences of peptides and proteins using oneand three-letter abbreviations for each amino acid (Table 3-1). The R groups of amino acids can be characterized as being either hydrophilic or hydrophobic (Table 3-2); properties that affect their location in a protein's mature folded conformation (see Chapter 5). Some proteins contain additional amino acids that arise by the posttranslational modification of an amino acid already present in a peptide. Examples include the conversion of peptidyl proline and peptidyl lysine to 4-hydroxyproline and 5-hydroxylysine; the conversion of peptidyl glutamate to y-carboxyglutamate; and the methylation, formylation, acetylation, prenylation, and phosphorylation of certain aminoacyl residues. These modifications significantly extend the biologic diversity of proteins by altering their solubility, stability, catalytic activity, and interaction with other proteins.

Selenocysteine (Figure 3-1) is an L-a-amino acid present in proteins from every domain of life. Humans contain approximately two dozen selenoproteins that include certain peroxidases and reductases, selenoprotein P, which circulates in the plasma, and the iodothyronine deiodinases responsible for converting the prohormone thyroxine (T,,) to the thyroid hormone 3,3'5-triiodothyronine (T,) (see Chapter 41). Peptidyl

Arginine Asparagine Aspartic acid Cysteine Glutamic acid Glutamine Glycine Histidine Lysine Serine Threonine, Hydrophobic = Alanine Isoleucine Leucine Methionine Phenylalanine Proline Tryptophan Tyrosine Valine
The distinction is based on the tendency to associate with, or to minimize contact with, an aqueous environment.
selenocysteine is not the product of a posttranslational modification, but is inserted directly into a growing polypeptide during translation. Selenocysteine thus is commonly termed the "21st amino acid.' However, unlike the other 20 protein amino acids, incorporation of selenocysteine is specified by a large and complex genetic element for the unusual tRNA called tRNA'S* which utilizes the UGA anticodon that normally signals STOP. However, the protein synthetic apparatus can identify a selenocysteine-specific UGA codon by the presence of an accompanying stem-loop structure, the selenocysteine insertion element, in the untranslated region of the mRNA (see Chapter 27).

With the sole exception of glycine, the a-carbon of every amino acid is chiral. Although some protein amino acids are dextrorotatory and some levorotatory, all share the absolute configuration of L-glyceraldehyde and thus are defined as L-aamino acids. Even though almost all protein amino acids are (R), the failure to use (R) or (S) to express absolute stereochemistry is no mere historical aberration. L-Cysteine is (S) since the atomic mass of the sulfur atom on C3 exceeds that of the amino group on C2. More significantly, in mammals the biochemical reactions of L-a-amino acids, their precursors, and their catabolites are catalyzed by enzymes that act exclusively on L-isomers, irrespective of their absolute configuration.
FIGURE 3-1 Cysteine (left) & selenocysteine (right). pK, for the selenyl proton of selenocysteine is 5.2. Since this is 3 pH units lower than that of cysteine, selenocysteine represents a better nucleophile at or below pH 7.4.
FIGURE 3-2 4-Hydroxyproline & 5-hydroxylysine.

While some prokaryotes incorporate pyrrolysine into proteins, and plants can incorporate azetidine-2-carboxylic acid, an analog of proline, a set of just 21 L-a-amino acids clearly suffices for the formation of most proteins. Posttranslational modifications can, however, generate novel R groups that impart further properties. In collagen, protein-bound proline and lysine residues are converted to 4-hydroxyproline and 5-hydroxylysine (Figure 3-2). The carboxylation of glutamyl residues of proteins of the blood coagulation cascade to y-carboxyglutamyl residues (Figure 3-3) forms a chelating group for the calcium ion essential for blood coagulation. The amino acid side chains of histones are subject to numerous modifications, including acetylation and methylation of lysine and methylation and deamination of arginine (see Chapters 35 and 37). It is also now possible in the laboratory to genetically introduce many different unnatural amino acids into proteins, generating proteins via recombinant gene expression with new or enhanced properties and providing a new way to explore protein structure-function relationships.

In February 2013, the explosion of an approximately 20,000 metric ton meteor in the skies above Chelyabinsk, Western Siberia, dramatically demonstrated its potential destructive power. However, some meteorites, the remnants of asteroids that have reached earth, contain traces of several a-amino acids. These include the protein amino acids Ala, Asp, Glu, Gly, Ile, Leu, Phe, Ser, Thr, Tyr, and Val, as well as biologically important nonprotein a-amino acids such as N-methylglycine (sarcosine) and B-alanine.
Extraterrestrial amino acids were first reported in 1969 following analysis of the famous Murchison meteorite from southeastern Australia. The presence of amino acids in other meteorites, including some pristine examples from Antarctica, has now been amply confirmed. Unlike terrestrial amino acids,
FIGURE 3-3 y-Carboxyglutamic acid.
these meteorites contain racemic mixtures of Dand L-isomers of 3to 5-carbon amino acids, as well as many additional amino acids that lack terrestrial counterparts of biotic origin. Nucleobases, activated phosphates, and molecules related to sugars have also been detected in meteorites. These findings offer potential insights into the prebiotic chemistry of Earth, and impact the search for extraterrestrial life. Some speculate that meteorites may have contributed to the origin of life on our planet, by delivering extraterrestrially generated organic molecules to our earth.

-a-Amino acids fulfill vital metabolic roles in addition to serving as the "building blocks" of proteins. For example, tyrosine is a precursor of thyroid hormone, and both tyrosine and phenylalanine are metabolized to epinephrine, norepinephrine, and dihydroxyphenylalanine (DOPA). Glutamate is both a neurotransmitter and the precursor of y-aminobutyric acid (GABA). Ornithine and citrulline are intermediates in urea biosynthesis, and homocysteine, homoserine, and glutamate-y-semialdehyde are intermediates in the metabolism of the protein amino acids.

The consumption of plants that contain certain nonprotein amino acids can adversely impact human health. The seeds and seed products of three species of the legume Lathyrus have been implicated in the genesis of neurolathyrism, a profound neurologic disorder characterized by progressive and_ irreversible spastic paralysis of the legs. Lathyrism occurs widely during famines, when Lathyrus seeds represent a major contribution to the diet. L-a-Amino acids that have been implicated in human neurologic disorders, notably neurolathyrisms, include t-homoarginine and $-N-oxalyl-L-a,b-diaminopropionic acid (B-ODAP Table 3-3). The seeds of the "sweet pea, a Lathyrus legume that is widely consumed during famines, contain the osteolathyrogen y-glutamyl-B-aminopropionitrile (BAPN), a glutamine derivative of B-aminopropionitrile (structure not shown). The seeds of certain Lathyrus species also contain a,y-diaminobutyric acid, which as an analog of ornithine inhibits the hepatic urea cycle enzyme ornithine transcarbamylase, disrupts the urea cycle, and leads to ammonia toxicity. Finally, -8-methylaminoalanine, a neurotoxic amino acid that is present in Cycad seeds, has been implicated as a risk factor for neurodegenerative diseases including amyotrophic lateral sclerosis—Parkinson dementia complex in natives of Guam who consume either fruit bats that feed on cycad flour made from seeds.
fruit, or cycad

p-Amino acids that occur naturally include free p-serine and D-aspartate in human brain tissue, D-alanine and p-glutamate in the cell walls of gram-positive bacteria, and p-amino acids in certain peptides and antibiotics produced by bacteria, fungi, reptiles, and amphibians. Bacillus subtilis excretes

Homoarginine H 2 N NH 2 NH OH O N H, Medical Relevance = Cleaved by arginase to l -lysine and urea. Implicated in human neurolathyrism.. a - N -Oxalyl diaminopropionic acid ( a -ODAP) HO NH 2 O OH O O H N, Medical Relevance = A neurotoxin. Implicated in human neurolathyrism.. a - N -Glutamylamino-propiononitrile (BAPN) H 2 N H 3 C NH 2 OH C N O O NH, Medical Relevance = An osteolathyrogen.. 2,4-Diaminobutyric acid NH 2 NH 2 OH O, Medical Relevance = Inhibits ornithine transcarbamylase, resulting in ammonia toxicity.. a -Methylaminoalanine OH O HN NH CH 3, Medical Relevance = Possible risk factor for neurodegenerative diseases.
p-methionine, D-tyrosine, D-leucine, and D-tryptophan to trigger biofilm disassembly, and Vibrio cholerae incorporates p-leucine and p-methionine into the peptide component of its peptidoglycan layer.

In aqueous solution, the charged and uncharged forms of the ionizable weak acid groups —COOH and —NH,,° exist in dynamic protonic equilibrium:
While both R-COOH and R-NH, are weak acids, R-COOH is a far stronger acid than R—-NH,_*. Thus, at physiologic pH (pH 7.4), carboxyl groups exist almost entirely as R-COOand amino groups predominantly as R—-NH,*. The imidazole group of histidine and the guanidino group of arginine
FIGURE 3-4 Resonance hybrids of the protonated R groups of histidine (top) and arginine (bottom).
exists as resonance hybrids with positive charge distributed between two nitrogens (histidine) or three nitrogens (arginine) (Figure 3-4). Figure 3-5 illustrates the effect that the pH of the aqueous environment has on the charged state of aspartic acid. Molecules that contain an equal number of positively and negatively charged groups bear no net charge. These ionized neutral species are termed zwitterions. Amino acids in blood and most tissues thus should be represented as in A, below.
Structure B cannot exist in aqueous solution because at any PH low enough to protonate the carboxyl group, the amino group would also be protonated. Similarly, at any pH sufficiently high for an uncharged amino group to predominate, a carboxyl group will be present as R—COO-. The uncharged representation B is, however, often used when diagramming reactions that do not involve protonic equilibria.

The strengths of weak acids are expressed as their pK,. For molecules with multiple dissociable protons, the pK, for each acidic group is designated by replacing the subscript "a" with a number. The net charge on an amino acid—the algebraic sum of all the positively and negatively charged groups present—
depends on the pK, values of its functional groups and the pH of the surrounding medium. In the laboratory, altering the charge on amino acids and their derivatives by varying the PH facilitates the physical separation of amino acids, peptides, and proteins (see Chapter 4).

Zwitterions are one example of an isoelectric species—the form of a molecule that has an equal number of positive and negative charges and thus is electrically neutral. The isoelectric pH, also called the pl, is the pH midway between pK, values for the ionizations on either side of the isoelectric species. For an amino acid such as alanine that has only two dissociating groups, there is no ambiguity. The first pK, (R—COOH) is 2.35 and the second pK, (R—NH,) is 9.69. The isoelectric pH (pl) of alanine thus is
For polyprotic acids, pI is also the pH midway between the pK, values on either side of the isoionic species. For example, the pl for aspartic acid is
For lysine, pI is calculated from:
Similar considerations apply to all polyprotic acids (eg, proteins), regardless of the number of dissociable groups present. In the clinical laboratory, knowledge of the pI guides selection of conditions for electrophoretic separations. Electrophoresis at pH 7.0 will separate two molecules with pI values of 6.0 and 8.0, because the molecule with a pl of 6.0 will have a net positive charge, and that with a pl of 8.0 a net negative charge. Similar considerations underlie chromatographic separations on ionic supports such as diethylaminoethyl (DEAE) cellulose (see Chapter 4).

The environment
of
a
dissociable group
affects its
pK,
(Table 3-4). A nonpolar environment, which possesses less

α-Carboxyl, p K a Range = 3.5-4.0. Non-α COOHof Asp or Glu, p K a Range = 4.0-4.8. Imidazole of His, p K a Range = 6.5-7.4. SH of Cys, p K a Range = 8.5-9.0. OHofTyr, p K a Range = 9.5-10.5. α-Amino, p K a Range = 8.0-9.0. e-Amino of Lys, p K a Range = 9.8-10.4. Guanidinium of Arg, p K a Range = ~12.0
capacity than water for stabilizing charged species, thus raises the pK, of a carboxyl group making it a weaker acid, but lowers the pK, of an amino group, making it a stronger acid. Similarly, the presence of an adjacent oppositely charged group can stabilize, or of a similarly charged group can destabilize, a developing charge. Therefore, the pK, values of the R groups of free amino acids in aqueous solution (see Table 3-1) provide only an approximate guide to their pK, values when present in proteins. The pK, of a dissociable R group will depend on its location within a protein. pK, values that diverge from aqueous solution by as much a 3 pH units are common at the active sites of enzymes. An extreme example, a buried aspartic acid of thioredoxin, has a pK, above 9—a shift of more than 6 pH units!

The charges conferred by the dissociable functional groups of amino acids ensure that they are readily solvated by—and thus soluble in-polar solvents such as water and ethanol, but insoluble in nonpolar solvents such as benzene, hexane, or ether. Amino acids do not absorb visible light and thus are colorless. However, tyrosine, phenylalanine, and tryptophan absorb high-wavelength (250-290 nm) ultraviolet light. Because it absorbs ultraviolet light about 10 times more efficiently than either phenylalanine or tyrosine, tryptophan makes the major contribution to the ability of most proteins to absorb light in the region of 280 nm (Figure 3-6).

Each functional group of an amino acid exhibits all of its
characteristic chemical reactions. For carboxylic acid groups,
these
reactions
include
the
formation
of
esters,
amides,
and
acid anhydrides; for amino groups, acylation, amidation, and
FIGURE 3-6 Ultraviolet absorption spectra of tryptophan, tyrosine, and phenylalanine.
esterification; and for —OH and —SH groups, oxidation and esterification. Since glycine, the smallest amino acid, can be accommodated in places inaccessible to other amino acids, it often occurs where peptides bend sharply. The hydrophobic R groups of alanine, valine, leucine, and isoleucine and the aromatic R groups of phenylalanine, tyrosine, and tryptophan typically occur primarily in the interior of cytosolic proteins. The charged R groups of basic and acidic amino acids stabilize specific protein conformations via ionic interactions, or salt bridges. These interactions also function in "charge relay" systems during enzymatic catalysis and electron transport in respiring mitochondria. Histidine plays unique roles in enzymatic catalysis. The pK, of its imidazole proton permits histidine to function at neutral pH as either a base or an acid catalyst without the need for any environmentally induced shift. The primary alcohol group of serine and the primary thioalcohol (—SH) group of cysteine are excellent nucleophiles, and can function as such during enzymatic catalysis. For selenocysteine, its pK, of 5.2 is 3 pH units lower than that of cysteine, 8.3. At a distinctly acidic pH, selenocysteine thus should be the better nucleophile. However, the secondary alcohol group of threonine, while a good nucleophile, is not known to serve this role in catalysis. The —OH groups of serine, tyrosine, and threonine frequently serve as the points of covalent attachment for phosphoryl groups that regulate protein function (see Chapter 9).

Amino acids are linked together by peptide bonds.
The number and order of the amino acid residues in a polypeptide constitute its primary structure. Amino acids present in peptides, called aminoacyl residues, are referred to by replacing the ate or ine suffixes of free amino acids with yl (eg, alanyl, aspartyl, tyrosyl). Peptides are then named as derivatives of the carboxy terminal aminoacyl residue. For example, Lys-Leu-Tyr-Gln is called lysyl-leucyl-tyrosyl-glutamine. The ine ending on the carboxy-terminal residue (eg, glutamine) indicates that its a-carboxyl group is not involved in a peptide bond. Three-letter abbreviations linked by straight lines represent an unambiguous primary structure. Lines are omitted when using single-letter abbreviations.
Glu—Ala—Lys—Gly—Tyr—Ala
E
A
K
G
Y
A
Prefixes like trior octadenote peptides with three or eight residues, respectively. By convention, peptides are written with the residue that bears the free a-amino group at the left. This convention was adopted long before it was discovered that peptides are synthesized in vivo starting from the amino-terminal residue.

To draw a peptide, use a zigzag to represent the main chain or backbone. Add the main chain atoms, which occur in the repeating order: a-nitrogen, a-carbon, carbonyl carbon. Now add a hydrogen atom to each a-carbon and to each peptide nitrogen, then add an oxygen to the carbonyl carbon. Finally, add the appropriate R groups (shaded) to each a-carbon atom.

In mammals, peptide hormones typically contain only the 20 codon-specified a-amino acids linked by standard peptide bonds. Other peptides may, however, contain nonprotein amino acids, derivatives of the protein amino acids, or amino acids linked by an atypical peptide bond. For example, the amino terminal glutamate of glutathione, a tripeptide that participates in the metabolism of xenobiotics (see Chapter 47) and the reduction of disulfide bonds, is linked to cysteine by a non-a peptide bond (Figure 3-7). The amino terminal glutamate of thyrotropin-releasing hormone (TRH) is cyclized to pyroglutamic acid, and the carboxyl group of the carboxyl terminal prolyl residue is amidated. The nonprotein amino acids p-phenylalanine and ornithine are present in the cyclic
FIGURE 3-7 Glutathione (y-glutamyl-cysteinyl-glycine). Note the non-a peptide bond that links Glu to Cys.
peptide antibiotics tyrocidin and gramicidin S, while the heptapeptide opioids dermorphin and deltophorin in the skin of South American tree frogs contain D-tyrosine and p-alanine.

Although peptide structures are written as if a single bond linked the a-carboxyl and a-nitrogen atoms, this bond in fact exhibits partial double-bond character:
The bond that connects a carbonyl carbon to the a-nitrogen therefore cannot rotate, as this would require breaking the partial double bond. Consequently, the O, C, N, and H atoms of a peptide bond are coplanar. The imposed semirigidity of the peptide bond has important consequences for the manner in which peptides and proteins fold to generate higher orders of structure. Encircling brown arrows indicate free rotation about the remaining bonds of the polypeptide backbone (Figure 3-8).

Folding of a peptide probably occurs coincident with its biosynthesis (see Chapter 37). The mature, physiologically active conformation reflects the collective contributions of the amino acid sequence, noncovalent interactions (eg, hydrogen bonding, hydrophobic interactions), and the minimization of steric hindrance between residues. Common repeating conformations include a-helices and B-pleated sheets (see Chapter 5).

The peptide bond is uncharged at any pH of physiologic interest. Formation of peptides from amino acids is therefore accompanied by a net loss of one positive and one negative charge per peptide bond formed. Peptides nevertheless are charged at physiologic pH, owing to their terminal carboxyl and amino groups and, where present, their acidic or basic
FIGURE 3-8 Dimensions of a fully extended polypeptide chain. The four atoms of the peptide bond are coplanar. Free rotation can occur about the bonds that connect the a-carbon with the a-nitrogen and with the a-carbonyl carbon (brown arrows). The extended polypeptide chain is thus a semirigid structure with twothirds of the atoms of the backbone held in a fixed planar relationship to one another. The distance between adjacent a-carbon atoms is 0.36 nm (3.6 A). The interatomic distances and bond angles, which are not equivalent, are also shown. (Redrawn and reproduced, with permission, from Pauling L, Corey LP, Branson HR: The structure of proteins: two hydrogen-bonded helical configurations of the polypeptide chain. Proc Natl Acad Sci USA 1951;37:205.)
R groups. As for amino acids, the net charge on a peptide depends on the pH of its environment and on the pK, values of its dissociating groups.

- ® Both p-amino acids and non-a-amino acids occur in nature,
but proteins are synthesized using only L-a-amino acids.
p-Amino acids do, however, serve metabolic roles, not only in bacteria, but also in humans.
™ 1-a-Amino acids serve vital metabolic functions in addition to protein synthesis. Examples include the biosynthesis of urea, heme, nucleic acids, and hormones such as epinephrine and DOPA.
™
The presence in meteorites of trace quantities of many of the protein amino acids lends credence to the hypothesis that asteroid strikes might have contributed to the development of life on earth.
™ Certain of the L-a-amino acids present in plants and plant seeds can have deleterious effects on human health, for example, in lathyrism.
@ The R groups of amino acids determine their unique biochemical functions. Amino acids are classified as basic, acidic, aromatic, aliphatic, or sulfur-containing based on the composition and properties of their R groups.
™ The partial double-bond character of the bond that links the carbonyl carbon and the nitrogen of a peptide render the four atoms of the peptide bond coplanar, and hence restrict the number of possible peptide conformations.
™ Peptides are named for the number of amino acid residues present, and as derivatives of the carboxyl terminal residue. The primary structure of a peptide is its amino acid sequence, starting from the amino-terminal residue, a direction in which peptides actually are synthesized in vivo.
= Allamino acids possess at least two weakly acidic functional groups, R—NH,* and R—COOH. Many also possess additional weakly acidic functional groups such as phenolic —-OH, —SH, guanidino, or imidazole moieties.
= The pK, values of all functional groups of an amino acid or of a peptide dictate its net charge at a given pH. pl, the isoelectric pH, is the pH at which an amino acid bears no net charge and thus does not move in a direct current electrical field.
=
The pK, values of free amino acids at best only approximates their pK, values when present in a protein, and can differ widely due to the influence of their surroundings in a protein.

Bender DA: Amino Acid Metabolism, 3rd ed. Wiley, 2012.
Burton AS, Stern JC, Elsila JE, et al: Understanding prebiotic chemistry through the analysis of extraterrestrial amino acids
and nucleobases in meteorites. Chem Soc Rev 2012;41:5459.
deMunck E, Mujioz-Sdez E, Miguel BG, et al: B-N-Methylamino-
- L-alanine causes neurological and pathological phenotypes
mimicking amyotrophic
lateral sclerosis
(ALS):
The first
step towards
model for
sporadic
ALS.
Environ
Toxicol Pharmacol 2013;36:243.
- Kolodkin-Gal I: p-Amino acids trigger biofilm disassembly. Science 2010;328:627.

- = Cite three examples of posttranslational modifications that commonly occur during the maturation of a newly synthesized polypeptide.
Name four chromatographic methods commonly employed for the isolation of proteins from biologic materials. :
= Describe how electrophoresis in polyacrylamide gels can be used to determine the purity, subunit composition, relative mass, and isoelectric point of a protein.
= Describe the basis on which quadrupole and time-of-flight (TOF) spectrometers determine molecular mass.
= Compare the respective strengths and weaknesses of DNA cloning and mass
spectrometry
(MS)
as tools
for determining
protein primary
significance. m= Explain what is meant by "the proteome" and cite examples of its potential
m™ Describe the advantages and limitations of gene chips as a tool for monitoring protein expression.
= Outline three strategies for resolving individual proteins and peptides from
complex biologic
samples to
facilitate their
identification by
MS.
= Comment on the contributions of genomics, computer algorithms, and
given protein. databases to the identification of the open reading frames (ORFs) that encode a

Proteins are physically and functionally complex macromolecules that perform multiple critically important roles. An internal protein network, the cytoskeleton (see Chapter 51) maintains a cell's shape and physical integrity. Actin and myosin filaments form the contractile machinery of muscle (see Chapter 51). Hemoglobin transports oxygen (see Chapter 6), while circulating antibodies defend against foreign invaders (see Chapter 52). Enzymes catalyze reactions that generate energy, synthesize and degrade biomolecules, replicate and transcribe genes, process mRNAs, etc (see Chapter 7). Receptors enable cells to sense and respond to hormones and other extracellular cues (see Chapters 41 and 42). Proteins are subject to physical and functional changes that mirror the life cycle of the organisms in which they reside. A typical protein is "born" at translation (see Chapter 37), matures through posttranslational processing events such as selective proteolysis (see Chapters 9 and 37), alternates between working and resting states through the intervention of regulatory factors (see Chapter 9), ages through oxidation, deamidation, etc (see Chapter 58), and "dies" when degraded to its component amino acids (see Chapter 29). An important goal of molecular medicine is to identify biomarkers such as proteins and/or modifications to proteins whose presence, absence, or deficiency is associated with specific physiologic states or diseases (Figure 4-1).

Highly purified protein is essential for the detailed examination of
its physical
and functional
Cells contain
thou- sands of different proteins, each in widely varying amounts.
The isolation of a specific protein in quantities sufficient for
FIGURE 4-1 Diagrammatic representation of the life cycle of a hypothetical protein. (1) The life cycle begins with the synthesis on a ribosome of a polypeptide chain, whose primary structure is dictated by an mRNA. (2) As synthesis proceeds, the polypeptide begins to fold into its native conformation (blue). (3) Folding may be accompanied by processing events such as proteolytic cleavage of an N-terminal leader sequence (Met-Asp-Phe-Gln-Val) or the formation of disulfide bonds (S—S). (4) Subsequent covalent modifications may, for example, attach a fatty acid molecule (yellow) for (5) translocation of the modified protein to a membrane. (6) Binding an allosteric effector (red) may trigger the adoption of a catalytically active conformation. (7) Over time, proteins get damaged by chemical attack, deamidation, or denaturation, and (8) may be "labeled" by the covalent attachment of several ubiquitin molecules (Ub). (9) The ubiquitinated protein is subsequently degraded to its component amino acids, which become available for the synthesis of new proteins.
analysis of its properties thus presents a formidable challenge that may require successive application of multiple purification techniques. Selective precipitation exploits differences in relative solubility of individual proteins as a function of pH (isoelectric precipitation), polarity (precipitation with ethanol or acetone), or salt concentration (salting out with ammonium sulfate). Chromatographic techniques separate one protein from another based on the difference in their size (size-exclusion chromatography), charge (ion-exchange chromatography), hydrophobicity (hydrophobic interaction chromatography), or ability to bind a specific ligand (affinity chromatography).

In column chromatography, the stationary phase matrix consists of small beads loaded into a cylindrical container of glass, plastic, or steel called a column. Liquid-permeable frits confine the beads within this space while allowing the mobilephase liquid to flow or percolate through the column. The stationary phase beads can be chemically derivatized to coat their surface with the acidic, basic, hydrophobic, or ligandlike groups required for ion exchange, hydrophobic interaction, or affinity chromatography. As the mobile-phase liquid emerges from the column, it is automatically collected as a series of small portions called fractions. Figure 4-2 depicts the basic arrangement of a simple bench-top chromatography system.
First-generation column chromatography matrices consisted of long, intertwined oligosaccharide polymers shaped into spherical beads roughly a tenth of a millimeter in diameter. Unfortunately, their relatively large size perturbed mobilephase flow and limited the available surface area for accommodating added charged or ligand-like groups. Resolution could be increased by reducing particle size. However, the resistance created by a more tightly packed matrix required the use of very high pressures that would crush beads made from soft and spongy materials such as polysaccharide or acrylamide. Eventually, methods were developed to manufacture silicon particles of the necessary size and shape, to derivatize their surface with various functional groups, and to pack them into stainless steel columns capable of withstanding pressures of several thousand psi. Because of their greater resolving power, HPLC systems have largely displaced the once familiar glass columns in the protein purification laboratory.

Size-exclusion or, as it is sometimes still referred to, gelfiltration chromatography separates proteins on the basis of their Stokes radii. Stokes radius is a measure of the effective volume occupied by a protein as it rapidly tumbles in
FIGURE 4-2 Components of a typical liquid chromatography apparatus. Shown are the key components of a programmable liquid chromatography system consisting of A: reservoirs of mobilephase liquids (yellow, light blue), B: microprocessor-controlled pumps (purple), C: mixing chamber (red), D: injection port for loading analyte (dark blue); E: glass, metal, or plastic column containing stationary phase matrix (gray), F: spectrophotometric, fluorometric, refractive index, or electrochemical detector (orange), and G: fraction collector for collecting portions, called fractions, of the eluent liquid (green) in a series of separate test tubes, vials, or wells in a microtiter plate. The microprocessor can be programmed to pump liquid from only one reservoir (isocratic elution), to switch reservoirs at some predetermined point to generate a step gradient, or to mix liquids from the two reservoirs in proportions that vary over time to generate either a multistep or a continuous gradient.
free solution. Stokes radius is thus a function of both molecular mass and shape. Like the propeller on an airplane, as it tumbles, an elongated protein occupies a larger effective volume than would a globular protein of the same mass. Size-exclusion chromatography employs porous beads (Figure 4-3) whose pores are analogous to indentations in a river bank. As objects move downstream, the motion of any object that enters an indentation is retarded until they drift back into the current. Similarly, proteins with Stokes radii too large to enter the pores (excluded proteins) remain in the flowing mobile phase, and emerge before proteins that can enter some or all of the pores (included proteins). Proteins thus emerge from a gel filtration column in descending order of their Stokes radii.

In ion-exchange chromatography, proteins interact with the stationary phase by charge-charge interactions. Proteins with a net positive charge at a given pH will tightly adhere to beads
with negatively charged functional groups such as carboxylates or sulfates (cation exchangers). Similarly, proteins with a net negative charge will adhere to beads with positively charged functional groups, typically tertiary or quaternary amines (anion exchangers). Nonadherent proteins flow through the matrix and are washed away. Bound proteins then are selectively displaced by gradually raising the ionic strength of the mobile phase, thereby weakening charge-charge interactions. Proteins elute in inverse order of the strength of their interactions with the stationary phase.

Hydrophobic interaction chromatography separates proteins based on their tendency to associate with a stationary phase matrix coated with hydrophobic groups (eg, phenyl Sepharose, octyl Sephadex). Proteins with exposed hydrophobic surfaces adhere to the matrix via hydrophobic interactions that are enhanced by employing a mobile phase of high ionic strength. After nonadherent proteins are washed away, the polarity of the mobile phase is decreased by gradually lowering its salt concentration. If the interaction between protein and stationary phase is particularly strong, ethanol or glycerol may be added to the mobile phase to decrease its polarity and further weaken hydrophobic interactions.

Affinity chromatography exploits the high selectivity displayed by most proteins for their ligands. Enzymes may be purified by affinity chromatography using immobilized substrates, products, coenzymes, or inhibitors. In theory, only proteins that interact with the immobilized ligand adhere. Bound proteins
are then eluted either by competition with free, soluble ligand or, less selectively, by disrupting protein-ligand interactions using urea, guanidine hydrochloride, mildly acidic pH, or high salt concentrations. Stationary phase matrices containing analogs of frequently encountered ligands, such as NAD* or ATP, are commercially available. Recombinantly expressed proteins are frequently purified by using vectors that add a fusion domain onto the encoded protein designed to interact with a specific affinity matrix (see Chapter 7).

The most widely used method for determining the purity of a protein is SDS-PAGE—polyacrylamide gel electrophoresis (PAGE) in the presence of the anionic detergent sodium dodecyl sulfate (SDS). Electrophoresis separates charged biomolecules based on the rates at which they migrate in an applied electrical field. For SDS-PAGE, acrylamide is polymerized and cross-linked to form a porous matrix. SDS binds to proteins at a ratio of one molecule of SDS per two peptide bonds, causing the polypeptide to unfold or denature. When used in conjunction with 2-mercaptoethanol or dithiothreitol to reduce and break disulfide bonds (Figure 4-4), SDS-PAGE separates the component polypeptides of multimeric proteins. The large number of anionic SDS molecules, each bearing a charge of -1, overwhelms the charge contributions of the amino acid functional groups endogenous to a typical polypeptide. Since this renders the charge-to-mass ratio of each SDS-polypeptide complex approximately equal, the physical
FIGURE 4-4 oxidative cleavage of adjacent polypeptide chains linked by disulfide bonds (highlighted in blue) by performic acid (left) or reductive cleavage by B-mercaptoethanol (right) forms two peptides that contain cysteic acid residues or cysteinyl residues, respectively.
FIGURE 4-5 Use of SDS-PAGE to observe successive purification of a recombinant protein. The gel was stained with Coomassie Blue. Shown are protein standards (lane S) of the indicated M, in kDa, crude cell extract (E), cytosol (C), high-speed supernatant liquid (H), and the DEAE-Sepharose fraction (D). The recombinant protein has a mass of about 45 kDa.

resistance each peptide encounters as it moves through the acrylamide matrix determines its rate of migration. Large complexes encounter greater resistance, causing polypeptides to separate based on their relative molecular mass (M ). Individual polypeptides trapped in the acrylamide gel after removal of the electrical field are visualized by staining with dyes such as Coomassie Blue (Figure 4-5).

Ionic buffers called ampholytes and an applied electric field are used to generate a pH gradient within a polyacrylamide matrix. Applied proteins migrate until they reach the region of the matrix where the pH matches their isoelectric point (pI), the pH at which a molecule's net charge is 0. IEF frequently is used in conjunction with SDS-PAGE for two-dimensional electrophoresis, which separates polypeptides based on pl in one dimension and on M, in the second (Figure 4-6). Two-dimensional electrophoresis is particularly well suited for separating the components within complex mixtures of proteins.

Mature insulin consists of the 21-residue A chain and the 30-residue B chain linked by disulfide bonds. Frederick Sanger reduced the disulfide bonds (Figure 4-4), separated the A and B chains, and cleaved each chain into smaller peptides using trypsin, chymotrypsin, and pepsin. The resulting peptides were then isolated and hydrolyzed into a mixture of smaller peptides by treatment with acid. Each peptide in the mixture
FIGURE 4-6 Two-dimensional IEF-SDS-PAGE. The gel was stained with Coomassie Blue. A crude bacterial extract was first subjected to isoelectric focusing (IEF) in a pH 3-10 gradient. The IEF gel was then placed horizontally on the top of an SDS-PAGE gel, and the proteins then further resolved by SDS-PAGE. Notice the greatly improved resolution of distinct polypeptides relative to ordinary SDS-PAGE gel (Figure 4-5).
was isolated and treated with 1-fluoro-2,4-dinitrobenzene (Sanger reagent), which reacts with the exposed a-amino groups of the amino-terminal residues. The amino acid content of each peptide was then determined and the aminoterminal amino acid was identified. The e-amino group of lysine also reacts with Sanger reagent; but since an aminoterminal lysine reacts with 2 mol of Sanger reagent, it is readily distinguished from a lysine from the interior of a peptide. Working from diand tripeptides up through progressively larger fragments, Sanger was able to reconstruct the complete sequence of insulin, an accomplishment for which he received a Nobel Prize in 1958. Sanger, who received his second Nobel prize for his development of techniques for DNA sequencing, died in 2013 at the age of 95.

Pehr Edman introduced phenyl isothiocyanate (Edman reagent) to selectively label the amino-terminal residue of a peptide. In contrast to Sanger reagent, the phenylthiohydantoin (PTH) derivative can be removed under mild conditions to generate a new amino-terminal residue (Figure 4-7). Successive rounds of derivatization with Edman reagent can therefore be used to sequence many residues of a single sample of peptide. Even with the benefit of Edman reagent, the determination of the complete sequence of a protein by chemical methods remains a timeand labor-intensive process.
Phenylisothiocyanate
(Edman reagent)
and
a
peptide
A
phenylthiohydantoic acid
A phenylthiohydantoin and a peptide shorter by one residue
FIGURE 4-7 The Edman reaction. Phenyl isothiocyanate derivatizes the amino-terminal residue of a peptide as a phenylthiohydantoic acid. Treatment with acid in a nonhydroxylic solvent releases a phenylthiohydantoin, which is subsequently identified by its chromatographic mobility, and a peptide one residue shorter. The process is then repeated.
The heterogeneous chemical properties of the amino acids meant that every step in the procedure represented a compromise between efficiency for any particular amino acid or set of amino acids and the flexibility needed to accommodate all 20. Consequently, each step in the process operates at less than 100% efficiency, which leads to the accumulation of polypeptide fragments with varying N-termini. Eventually, it becomes impossible to distinguish the correct PTH amino acid for that position in the peptide from the out-of-phase contaminants. As a result, the read length for Edman sequencing varies from 5 to 30 amino acid residues depending on the quantity and purity of the peptide.
In order to determine the complete sequence of a polypeptide several hundred residues in length, a protein must first be cleaved into smaller peptides, using either a protease or a reagent such as cyanogen bromide. Following purification by reversed-phase HPLC, these peptides are then analyzed by

Edman sequencing. In order to assemble these short peptide sequences to solve the complete sequence of the intact polypeptide, it is necessary to analyze peptides whose sequences overlap one another. This is accomplished by generating multiple sets of peptides using more than one method of cleavage. The large quantities of purified protein required to test multiple protein fragmentation and peptide purification conditions constitute the second major drawback of direct chemical protein sequencing techniques.

While the reactions that sequentially derivatize and cleave PTH amino acids from the amino-terminal end of a peptide typically are conducted in an automated sequenator, DNA sequencing is far more rapid and economical. Recombinant techniques permit researchers to manufacture a virtually infinite supply of DNA from even minute quantities of template present in the original sample (see Chapter 39). DNA sequencing methods, whose underlying chemistry was also developed by Sanger, routinely enable automated sequencers to "read" sequences several thousand deoxyribonucleotides in length. The sequence of the encoded polypeptide is then determined by simply translating the sequence of nucleotide triplets encoded by its gene. Conversely, early molecular biologists designed complementary oligonucleotide probes to identify the DNA clone containing the gene of interest by reversing this process and using a segment of chemically determined amino acid sequence as template. The advent of DNA cloning thus ushered in the widespread use of a hybrid approach in which Edman chemistry was employed to sequence a small portion of the protein, then exploiting this information to determine the remaining sequence by DNA
cloning and polydeoxyribonucleotide sequencing.

Today the number of organisms for which the complete DNA sequence of their genomes has been determined and made available to the scientific community numbers in the thousands. Thus, for most research scientists, particularly those working on commonly used "model organisms" such as Homo sapiens, mouse, rat, Escherichia coli, Drosophila melanogaster, Caenorhabditis elegans, yeast, etc, the genetically encoded sequence of the protein(s) with which they are working has already been determined and lies waiting to be accessed in a database such as GenBank. All that a scientist needs to make an unambiguous identification is the amino acid sequence for some segment, sometimes as little as five or six consecutive residues, of the protein of interest. While the requisite amino

Phosphorylation, Mass Increase (Da) = 80. Hydroxylation, Mass Increase (Da) = 16. Methylation, Mass Increase (Da) = 14. Acetylation, Mass Increase (Da) = 42. Myristylation, Mass Increase (Da) = 210. Palmitoylation, Mass Increase (Da) = 238. Glycosylation, Mass Increase (Da) = 162
acid sequence information once was obtained using the Edman technique, today mass spectrometry (MS) has emerged as the method of choice for protein identification.

The superior sensitivity, speed, and versatility of MS have replaced the Edman technique as the principal method for determining the sequences of peptides and proteins. MS is significantly more sensitive and tolerant of variations in sample quality. Moreover, since mass and charge are common properties of a wide range of biomolecules, MS can be used to analyze metabolites, carbohydrates, and lipids, and to detect posttranslational modifications such as phosphorylation or hydroxylation that add readily identified increments of mass to a protein (Table 4-1). These modifications are difficult to detect using the Edman technique and are undetectable in the DNA-derived amino acid sequence.

In a simple, single quadrupole mass spectrometer, a sample is placed under vacuum and allowed to vaporize in the presence ofa proton donor to impart a positive charge. An electrical field then propels the cations toward a curved flight tube where they encounter a magnetic field, which deflects them at a right angle to their original direction of flight (Figure 4-8). The current powering the electromagnet that generates this field is gradually increased until the path of each ion is bent sufficiently to strike a detector mounted at the end of the flight tube. For ions of identical net charge, the force required to bend their path to the same extent is proportionate to their mass. Time-of-flight (TOF) mass spectrometers employ a linear flight tube. Following vaporization of the sample in the presence of a proton donor, an electric field is briefly applied to accelerate the ions toward a detector at the end of the flight tube.
FIGURE 4-8 Basic components of a simple mass spectrometer. A mixture of molecules, represented by a red circle, green triangle, and blue diamond, is vaporized in an ionized state in the sample chamber. These molecules are then accelerated down the flight tube by an electrical potential applied to the accelerator grid (yellow). An adjustable field strength electromagnet applies a magnetic field that deflects the flight of the individual ions until they strike the detector. The greater the mass of the ion, the higher the magnetic field required to focus it onto the detector.
For molecules of identical charge, the velocity to which they are accelerated, and hence the time required to reach the detector, is inversely proportional to their mass.
Quadrupole mass spectrometers are generally used to determine the masses of molecules of 4000 Da or less, whereas TOF mass spectrometers are used to determine the large masses of complete proteins. Various combinations of multiple quadrupoles, or reflection of ions back down the linear flight tube of a TOF mass spectrometer, are used to create more sophisticated instruments.

The analysis of peptides and proteins by MS initially was hindered by difficulties in volatilizing these large organic molecules. While small organic molecules could be readily vaporized by heating in a vacuum (Figure 4-9), proteins, oligonucleotides, etc decomposed upon heating. Only when reliable techniques were devised for dispersing peptides, proteins, and other large biomolecules into the vapor phase it was possible to apply MS for their structural analysis and sequence determination. Three commonly used methods for dispersion into the vapor phase are electrospray ionization, matrix-assisted laser desorption and ionization (MALDI), and fast atom bombardment (FAB). In electrospray ionization, the molecules to be analyzed are dissolved in a volatile solvent and introduced into the sample chamber in a minute stream through a capillary (Figure 4-9). As the droplet of liquid emerges into the sample chamber, the solvent rapidly disperses, leaving the macromolecule suspended in the gaseous phase. The charged probe serves to ionize the sample. Electrospray ionization is frequently used to analyze peptides
FIGURE 4-9 Three common methods for vaporizing molecules in the sample chamber of a mass spectrometer.
and proteins as they elute from an HPLC or other chromatography column, already dissolved in a volatile solvent. In MALDI, the sample is mixed with a liquid matrix containing a light-absorbing dye and a source of protons. In the sample chamber, the mixture is excited using a laser, causing the surrounding matrix to disperse into the vapor phase so rapidly as to avoid heating embedded peptides or proteins (Figure 4-9). In fast atom bombardment, large macromolecules dispersed in glycerol or another protonic matrix are bombarded by a stream of neutral atoms, for example, xenon, that have been accelerated to a high velocity. "Soft" ionization by FAB is frequently applied to volatilize large macromolecules intact.

Peptides inside the mass spectrometer can be broken down into smaller units by collisions with neutral helium or argon atoms (collision-induced dissociation) and the masses of the individual fragments determined. Since peptide bonds are much more labile than carbon-carbon bonds, the most abundant fragments will differ from one another by increments of one or two amino acids. Since—with the exceptions of (1) leucine and isoleucine and (2) glutamine and lysine—the molecular mass of each amino acid is unique, the sequence of the peptide can be reconstructed from the masses of its fragments.

Complex peptide mixtures can be analyzed, without prior purification, by tandem MS, which employs the equivalent of two mass spectrometers linked in series. For this reason, analysis by tandem instruments is often referred to as MS-MS, or MS'. The first mass spectrometer separates individual peptides based on their differences in mass. By adjusting the field strength of the first magnet, a single peptide can be directed into the second mass spectrometer, where fragments are generated and their masses are determined. Alternatively, they can be held in an electromagnetic ion trap located between the two quadrupoles and selectively delivered to the second quadrupole instead of being lost when the first quadrupole is set to select ions of a different mass.
Tandem MS can be used to screen blood samples from newborns for the presence and concentrations of amino acids, fatty acids, and other metabolites. Abnormalities in metabolite levels can serve as diagnostic indicators for a variety of genetic disorders, such as phenylketonuria, ethylmalonic encephalopathy, and glutaric acidemia type 1.

While the sequence of the human genome is known, the picture provided by genomics is both static and incomplete. As genes are switched on and off, proteins are synthesized in particular cell types at specific times of growth or differentiation and in response to external stimuli. Muscle cells express proteins not expressed by neural cells, and the type of subunits present in the hemoglobin tetramer undergo change preand postpartum. Many proteins undergo posttranslational modifications during maturation into functionally competent forms or as a means of regulating their properties. In order to obtain a more complete and dynamic molecular description of living organisms, scientists are working to determine the
proteome, a term that refers to the identity, abundance, and state of modification of the entire suite of proteins expressed by an individual cell at a particular time. Since the proteome for each component cell of an organism is distinct and changes with time and circumstances, the ultimate, comprehensive human proteome constitutes a target of formidable size and complexity.

A key goal of proteomics is the identification of proteins whose levels of expression or modification correlate with medically significant events. In addition to their potential as diagnostic indicators, these protein biomarkers may provide important clues concerning the root causes and mechanisms of a specific physiologic condition or disease. First-generation proteomics employed SDS-PAGE or two-dimensional electrophoresis to resolve the proteins in one biologic sample from another, followed by determination of the amino acid sequence of their amino terminus by the Edman method. Identities were determined by searching available polypeptide sequences for proteins that contained a matching N-terminal sequence and were predicted to possess a similar M, and, for 2D gels, pl.
These early efforts were constrained by the limited number of polypeptide sequences available and the difficulties in isolating polypeptides from the gels in sufficient quantities for Edman analysis. Attempts to increase resolving power and sample yield by increasing the size of the gels were only marginally successful. Eventually, the development of mass spectrometric techniques provided a means for protein sequence determination whose sensitivity was compatible with electrophoretic separation approaches.
Knowledge of the genome sequence of the organism in question greatly facilitated identification by providing a comprehensive set of DNA-encoded polypeptide sequences. It also provided the nucleotide sequence data from which to construct gene arrays, sometimes called DNA chips, containing hundreds of distinct oligonucleotide probes. These chips could then be used to detect the presence of mRNAs containing complementary nucleotide sequences. While changes in the expression of the mRNA encoding a protein do not necessarily reflect comparable changes in the level of the corresponding protein, gene arrays were both less technically demanding and more sensitive than first-generation proteomic approaches, particularly with respect to low abundance proteins.

Second-generation proteomics coupled newly developed nanoscale chromatographic techniques with MS. The proteins in a biologic sample are first treated with a protease to hydrolyze them into smaller peptides that are then subject to reversed-phase, ion-exchange, or size-exclusion chromatography to apportion the vast number of peptides into smaller subsets more amenable to analysis. These subsets are analyzed by injecting the column eluent directly into a double quadrupole or TOF mass spectrometer. Multidimensional protein identification technology (MudPIT) employs successive rounds of chromatography to resolve the peptides produced from the digestion of a complex biologic sample into several simpler fractions that can be analyzed separately by MS. Today, the suspension of complex peptide mixtures within the mass spectrometer itself and subsequently exporting small subsets for final analysis using ion traps often enables even complex mixtures to be analyzed directly by MS without prior chromatographic fractionation. Efforts also continue to refine methods for analysis of mRNA and protein expression in individual cells.

The functions of a large proportion of the proteins encoded by the human genome are presently unknown. Efforts continue to develop protein arrays or chips for directly testing the potential functions of proteins on a mass scale. However, while some protein functions are relatively easy to assay, such as protease or esterase activity, others are much less tractable. Data mining via bioinformatics permits researchers to compare amino acid sequences of unknown proteins with those whose functions have been determined. This provides a means to uncover clues to their potential properties, physiologic roles, and mechanisms of action. Algorithms exploit the tendency of nature to employ variations of a structural theme to perform similar functions in several proteins [eg, the Rossmann nucleotide binding fold to bind NAD(P)H, nuclear targeting sequences, and EF hands to bind Ca'*]. These domains generally are detected in the primary structure by conservation of particular amino acids at key positions. Insights into the properties and physiologic role of a newly discovered protein thus may be inferred by comparing its primary structure with that of known proteins.

■, 1 = Long amino acid polymers or polypeptides constitute the basic structural unit of proteins, and the structure of a protein provides insight into how it fulfills its functions.. ■, 1 = Proteins undergo posttranslational alterations during their lifetime that influence their function and determine their fate.. ■, 1 = By generating a new amino terminus, Edman reagent permitted the determination of lengthy segments of amino acid sequence.. ■, 1 = Polyacrylamide gels provide a porous matrix for separating proteins on the basis of their mobility in an applied direct current electrical field.. ■, 1 = The nearly constant ratio at which the anionic detergent SDS binds proteins enables SDS-PAGE to separate polypeptides predominantly on the basis of relative size.
■, 1 = Because mass is a universal property of all biomolecules and their derivatives, MS has emerged as a versatile technique applicable to the determination of primary structure, identification of posttranslational modifications, and the detection of metabolic abnormalities.. ■, 1 = DNAcloning coupled with protein chemistry provided a hybrid approach that greatly increased the speed and efficiency for determination of primary structures of proteins.. ■, 1 = Genomics, the determination of entire polynucleotide sequences, provides researchers with a blueprint for every genetically encoded macromolecule in an organism.. ■, 1 = Proteomic analysis utilizes genomic data to identify the entire complement of proteins in a biologic sample from partial amino acid sequence data obtained by coupling protein and peptide separation methods with sequencing by MS.. ■, 1 = Amajor goal of proteomics is the identification of proteins and their posttranslational modifications whose appearance or disappearance correlates with physiologic phenomena, aging, or specific diseases.. ■, 1 = Bioinformatics refers to the development of computer algorithms designed to infer the functional properties of macromolecules through comparison of sequences of novel proteins with others whose properties are known.

- . Indicate the advantages and drawbacks of several approaches to classifying
proteins.
m= Explain and illustrate the primary, secondary, tertiary, and quaternary structure of proteins.
= Identify the major recognized types of secondary structure and explain supersecondary motifs.
= Describe the kind and relative strengths of the forces that stabilize each order of protein structure.
Describe the information summarized by a Ramachandran plot.
Summarize the basic operating principles underlying three key methods for determining protein structure: X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy.
= Indicate the present state of knowledge concerning the stepwise process by which proteins are thought to attain their native conformation.
= Identify the physiologic roles in protein maturation of chaperones, protein disulfide isomerase, and peptidylproline cis-trans isomerase.
= Describe the principal biophysical techniques used to study tertiary and quaternary structure of proteins.
= Explain how genetic and nutritional disorders of collagen maturation illustrate the close linkage between protein structure and function.
= For the prion diseases, outline the overall events in their molecular pathology and name the life forms each affects.

In nature, form follows function. In order for a newly synthesized polypeptide to mature into a biologically functional protein capable of catalyzing a metabolic reaction, powering cellular motion, or forming the macromolecular rods and cables that provide structural integrity to hair, bones, tendons, and teeth, it must fold into a specific three-dimensional arrangement, or conformation. In addition, during maturation, posttranslational modifications may add new chemical groups or remove transiently needed peptide segments. Genetic or nutritional deficiencies that impede protein maturation are deleterious to health. Examples of the former include Creutzfeldt-Jakob disease, scrapie, Alzheimer disease, and bovine spongiform encephalopathy ("mad cow disease"). Examples of the latter include scurvy (ascorbic acid) and Menkes syndrome (Cu). Conversely, next generation therapeutics for viral diseases such as hepatitis C block the maturation of virally encoded proteins by inhibiting the activity of the cyclophilins, a family of peptidyl protein cis-trans isomerases.

The terms configuration and conformation are often confused. Configuration refers to the geometric relationship between a given set of atoms, for example, those that distinguish Lfrom
Ueno Cap you should be able to:
p-amino acids. Interconversion of configurational alternatives requires breaking (and reforming) covalent bonds. Conformation refers to the spatial relationship of every atom in a molecule. Interconversion between conformers occurs with retention of configuration, generally via rotation about single bonds.

Scientists initially approached the elucidation of structurefunction relationships in proteins by separating them into classes based on properties such as solubility, shape, or the presence of nonprotein groups. For example, the proteins that can be extracted from cells using aqueous solutions of physiologic pH and ionic strength are classified as soluble. Extraction of integral membrane proteins requires dissolution of the membrane with detergents. Globular proteins are compact, roughly spherical molecules that have axial ratios (the ratio of their shortest to longest dimensions) of not over three. Most enzymes are globular proteins. By contrast, many structural proteins adopt highly extended conformations. These fibrous proteins may possess axial ratios of 10 or more.
Lipoproteins and glycoproteins contain covalently bound lipid and carbohydrate, respectively. Myoglobin, hemoglobin, cytochromes, and many other metalloproteins contain tightly associated metal ions. While more precise classification schemes have emerged based on similarity, or homology, in amino acid sequence and three-dimensional structure, many early classification terms remain in use.

Proteins perform complex physical and catalytic functions by positioning specific chemical groups in a precise threedimensional arrangement. The polypeptide scaffold containing these groups must adopt a conformation that is both functionally efficient and physically strong. At first glance, the biosynthesis of polypeptides comprised of tens of thousands of individual atoms would appear to be extremely challenging. When one considers that a typical polypeptide can potentially adopt = 10° distinct conformations, folding into the conformation appropriate to their biologic function would appear to be even more difficult. As described in Chapters 3 and 4, synthesis of the polypeptide backbones of proteins employs a small set of common building blocks or modules, the amino acids, joined by a common linkage, the peptide bond. Similarly, a stepwise modular pathway simplifies the folding and processing of newly synthesized polypeptides into mature proteins.

The modular nature of protein synthesis and folding are embodied in the concept of orders of protein structure: primary structure—the sequence of amino acids in a polypeptide chain; secondary structure—the folding of short (3-30 residue), contiguous segments of polypeptide into geometrically ordered units; tertiary structure—the assembly of secondary structural units into larger functional units such as the mature polypeptide and its component domains; and quaternary structure—the number and types of polypeptide units of oligomeric proteins and their spatial arrangement.

Free rotation is possible about only two of the three types of covalent bonds comprising the polypeptide backbone: the bond linking the a-carbon (Ca) to the carbonyl carbon (Co) and the bond linking Ca to nitrogen (see Figure 3-8). The partial double-bond character of the peptide bond that links Co to the a-nitrogen requires that the carbonyl carbon, carbonyl oxygen, and a-nitrogen remain coplanar, thus preventing rotation. The angle about the Ca—N bond is termed the phi (#) angle, and that about the Co—Ca bond the psi (y) angle. In peptides, for amino acids other than glycine, most combinations of phi and psi angles are disallowed because of steric hindrance (Figure 5-1). The conformations of proline are even more restricted as its cyclic structure prevents free rotation of the N—Ca bond.
Regions of ordered secondary structure arise when a series of aminoacyl residues adopt similar phi and psi angles. Extended segments of polypeptide (eg, loops) can possess a variety of such angles. The angles that define the two most
FIGURE 5-1 Ramachandran plot. The blue regions indicate sterically permissible combinations of phi-psi angles for nonglycine and nonproline amino acids in a polypeptide chain. The deeper the blue, the more thermodynamically favorable the phi-psi combination. Phi-psi angles corresponding to specific types of secondary structures are labeled.
common types of secondary structure, the a helix and the 6 sheet, fall within the lower and upper left-hand quadrants of a Ramachandran plot, respectively (Figure 5-1).

The polypeptide backbone of an a helix is twisted by an equal amount about each a-carbon with a phi angle of approximately -57° and a psi angle of approximately -47°. A complete turn of the helix contains an average of 3.6 aminoacy]l residues, and the distance it rises per turn (its pitch) is 0.54 nm (Figure 5-2). The R groups of each aminoacyl residue in an a helix face outward (Figure 5-3). Proteins contain only L-amino acids, for which a right-handed a helix is by far the more stable, and only right-handed a helices are present in proteins. Schematic diagrams of proteins represent a helices as coils or cylinders. The stability of helix arises primarily from hydrogen an a bonds formed between the oxygen of the peptide bond carbonyl and the hydrogen atom of the peptide bond nitrogen of the fourth residue down the polypeptide chain (Figure 5-4). The ability to form the maximum number of hydrogen bonds, supplemented by van der Waals interactions in the core of this tightly packed structure, provides the thermodynamic driving force for the formation of an a helix. Since the peptide bond nitrogen of proline lacks a hydrogen atom, it is incapable of forming a hydrogen bond with a carbonyl oxygen. Consequently, proline can only be stably accommodated within
FIGURE 5-2 orientation of the main chain atoms of a peptide about the axis of an a helix.
FIGURE 5-3 View down the axis of a polypeptide a helix. The side chains (R) are on the outside of the helix. The van der Waals radii of the atoms are larger than shown here; hence, there is almost no free space inside the helix.
the first turn of an a helix. When present elsewhere, proline disrupts the conformation of the helix, producing a bend. Because it possesses such a small R group, glycine also frequently induces bends within a helices.
FIGURE 5-4 Hydrogen bonds (dotted lines) formed between H and O atoms stabilize a polypeptide in an a-helical conformation.

Many a helices have predominantly hydrophobic R groups projecting from one side of the axis of the helix and predominantly hydrophilic R groups projecting from the other side. These amphipathic helices are well adapted to the formation of interfaces between polar and nonpolar regions such as the hydrophobic interior of a protein and its aqueous environment. Clusters of amphipathic helices can create channels, or pores, through hydrophobic cell membranes that permit specific polar molecules to pass.

The second (hence "beta') recognizable regular secondary structure in proteins is the B sheet. The amino acid residues of a B sheet, when viewed edge-on, form a zigzag or pleated pattern in which the R groups of adjacent residues project in opposite directions. Unlike the compact backbone of the a helix, the peptide backbone of the B sheet is highly extended. However, like the a helix, B sheets derive much of their stability from hydrogen bonds between the carbonyl oxygens and amide hydrogens of peptide bonds. However, in contrast to the a helix, these bonds are formed with adjacent segments of the B sheet (Figure 5-5).
FIGURE 5-5 Spacing and bond angles of the hydrogen bonds of antiparallel and parallel pleated B sheets. Arrows indicate the direction of each strand. Hydrogen bonds are indicated by dotted lines with the participating a-nitrogen atoms (hydrogen donors) and oxygen atoms (hydrogen acceptors) shown in blue and red, respectively. Backbone carbon atoms are shown in black. For clarity in presentation, R groups and hydrogen atoms are omitted. Top: Antiparallel 8 sheet. Pairs of hydrogen bonds alternate between being close together and wide apart and are oriented approximately perpendicular to the polypeptide backbone. Bottom: Parallel B sheet. The hydrogen bonds are evenly spaced but slant in alternate directions.
Interacting ® sheets can be arranged either to form a parallel B sheet, in which the adjacent segments of the polypeptide chain proceed in the same direction amino to carboxyl, or an antiparallel sheet, in which they proceed in opposite directions (Figure 5-5). Either configuration permits the maximum number of hydrogen bonds between segments, or strands, of the sheet. Most f sheets are not perfectly flat but tend to have a right-handed twist. Clusters of twisted strands of 6 sheet, sometimes referred to as B barrels, form the core of many globular proteins (Figure 5-6). Schematic diagrams represent B sheets as arrows that point in the amino to the car- boxy] terminal direction.

Roughly half of the residues in a "typical" globular protein reside in a helices or B sheets, and half in loops, turns, bends, and other extended conformational features. Turns and bends refer to short segments of amino acids that join two units of the secondary structure, such as two adjacent strands of an antiparallel B sheet. A 6 turn involves four aminoacy] residues, in which the first residue is hydrogen-bonded to the fourth, resulting in a tight 180° turn (Figure 5-7). Proline and glycine often are present in 6 turns.
Loops are regions that contain residues beyond the minimum number necessary to connect adjacent regions of secondary structure. Irregular in conformation, loops nevertheless serve key biologic roles. For many enzymes, the loops that bridge domains responsible for binding substrates often contain aminoacyl residues that participate in catalysis. Helix-loop-helix motifs provide the oligonucleotidebinding portion of many DNA-binding proteins such as repressors and transcription factors. Structural motifs such as the helix-loop-helix motif or the E-F hands of calmodulin (see Chapter 51) that are intermediate in scale between secondary and tertiary structures are often termed supersecondary structures. Since many loops and bends reside on the surface of proteins, and are thus exposed to solvent, they constitute readily accessible sites, or epitopes, for recognition and binding of antibodies.
While loops lack apparent structural regularity, many adopt a specific conformation stabilized through hydrogen bonding, salt bridges, and hydrophobic interactions with other portions of the protein. However, not all portions of proteins are necessarily ordered. Proteins may contain "disordered" regions, often at the extreme amino or carboxyl terminal, characterized by high conformational flexibility. In many instances, these disordered regions assume an ordered conformation upon binding of a ligand. This structural flexibility enables such regions to act as ligand-controlled switches that affect protein structure and function.

The term "tertiary structure" refers to the entire three-dimensional conformation of a polypeptide. It indicates, in threedimensional space, how secondary structural features—helices,
FIGURE 5-6 Examples of the tertiary structure of proteins. Left: The enzyme triose phosphate isomerase complexed with the substrate analog 2-phosphoglycerate (red). Note the elegant and symmetrical arrangement of alternating 8 sheets (gray) and a helices (green), with the B sheets forming a B-barrel core surrounded by the helices. (Adapted from Protein Data Bank ID no. 105x.) Right: Lysozyme complexed with the substrate analog penta-N-acetyl chitopentaose (red). The color of the polypeptide chain is graded along the visible spectrum from purple (N-terminal) to tan (C-terminal). Note, the concave shape of the domain forms a binding pocket for the pentasaccharide, the lack of B sheet, and the high proportion of loops and bends. (Adapted from Protein Data Bank ID no. 1sfb.)
sheets, bends, turns, and loops—assemble to form domains and how these domains relate spatially to one another. A domain is a section of the protein structure sufficient to perform a particular chemical or physical task such as binding of a substrate or other ligand. Most domains are modular in nature, that is, contiguous in both primary sequence and

three-dimensional space (Figure 5-8). Simple proteins, particularly those that interact with a single substrate or other ligand, such as lysozyme, triose phosphate isomerase (Figure 5-6), or the oxygen storage protein myoglobin (see Chapter 6), often consist of a single domain. By contrast, lactate dehydrogenase is comprised of two domains, an N-terminal NAD*-binding domain and a C-terminal binding domain for the second substrate, pyruvate (Figure 5-8). Lactate dehydrogenase is one of the family of oxidoreductases that share a common N-terminal NAD(P)*-binding domain known as the Rossmann fold. By fusing a segment of DNA coding for a Rossmann fold domain to that coding for a variety of C-terminal domains, a large family of oxidoreductases have evolved that utilize NAD(P)*/ NAD(P)H for the oxidation and reduction of a wide range of metabolites. Examples include alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, quinone oxidoreductase, 6-phosphogluconate dehydrogenase, p-glycerate dehydrogenase, and formate dehydrogenase.
FIGURE 5-7 A turn that links two segments of antiparallel B sheet. The dotted line indicates the hydrogen bond between the first and fourth amino acids of the four-residue segment
Ala-Gly-Asp-Ser.
Not all domains bind substrates. Hydrophobic domains anchor proteins to membranes or enable them to span membranes. Localization sequences target proteins to specific subcellular or extracellular locations such as the nucleus, mitochondria, secretory vesicles, etc. Regulatory domains trigger changes in protein function in response to the binding of allosteric effectors or covalent modifications (see Chapter 9). Combining the genetic material coding for individual domain modules provides

FIGURE 5-8 Polypeptides containing two domains. Left: Shown is the three-dimensional structure of a monomer unit of the tetrameric enzyme lactate dehydrogenase with the substrates NADH (red) and pyruvate (blue) bound. Not all bonds in NADH are shown. The color of the polypeptide chain is graded along the visible spectrum from blue (N-terminal) to orange (C-terminal). Note how the N-terminal portion of the polypeptide forms a contiguous domain, encompassing the upper portion of the enzyme, responsible for binding NADH. Similarly, the C-terminal portion forms a contiguous domain responsible for binding pyruvate. (Adapted from Protein Data Bank ID no. 3ldh.) Right: Shown is the three-dimensional structure of the catalytic subunit of the cAMP-dependent protein kinase (see Chapter 42) with the substrate analogs ADP (red) and peptide (purple ribbon) bound. The color of the polypeptide chain is graded along the visible spectrum from blue (N-terminal) to orange (C-terminal). Protein kinases transfer the y-phosphoryl group of ATP to protein and peptide substrates (see Chapter 9). Note how the N-terminal portion of the polypeptide forms a contiguous domain rich in B sheet that binds ADP. Similarly, the C-terminal portion forms a contiguous, a-helix-rich domain responsible for binding the peptide substrate. (Adapted from Protein Data Bank ID no. 1jbp.)

a facile route for generating proteins of great structural complexity and functional sophistication (Figure 5-9). Proteins containing multiple domains can also be assembled through the association of multiple polypeptides, or protomers. Quaternary structure defines the polypeptide composition of a protein and, for an oligomeric protein, the spatial relationships between its protomers or subunits. Monomeric proteins consist of a single polypeptide chain. Dimeric proteins contain two polypeptide chains. Homodimers contain two copies of the same polypeptide chain, while in a heterodimer the polypeptides differ. Greek letters (a, B, y, etc) are used to distinguish different subunits of a hetero-oligomeric protein, and subscripts indicate the number of each subunit type. For example, a, designates a homotetrameric protein, and a,8,y, a protein with five subunits of three different types.

The three-dimensional structure for literally thousands of proteins can be accessed through the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) and other repositories. While one can obtain the images that indicate the position of every atom, since even small proteins contain many thousands of atoms, such depictions are generally too complex to be readily interpreted. Therefore, textbooks, journals, websites, etc oftentimes utilize simplified schematic diagrams designed to highlight specific features of a protein's tertiary and quaternary structure. Ribbon diagrams (Figures 5-6 and 5-8) trace the conformation of the polypeptide backbone, with cylinders and arrows indicating regions of a helix and 6 sheet, respectively. In an even simpler representation, line segments
Forkhead transcription
factor
Residue number
FIGURE 5-9 Some multidomain proteins. The rectangles represent the polypeptide sequences of a forkhead transcription factor; 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, a bifunctional enzyme whose activities are controlled in a reciprocal fashion by allosteric effectors and covalent modification (see Chapter 19); phenylalanine hydroxylase (see Chapters 27 and 29), whose activity is stimulated by phosphorylation of its regulatory domain; and a receptor for atrial natriuretic peptide receptor, whose intracellular domain transmits signals through protein-protein interactions with heterotrimeric GTP-binding proteins (see Chapter 42). Regulatory domains are colored orange, catalytic domains in blue or purple, protein-protein interaction domains (Pr-Pr) in green, DNA-binding domains in gray, nuclear localization sequences in red, ligand-binding domains in light yellow, and transmembrane domains in black. The kinase and bisphosphatase activities of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase are catalyzed by the N(PFK-2) and C-terminal (FBP2-ase) proximate catalytic domains, respectively.
that link the a-carbons of each amino acid residue indicate the path of the polypeptide backbone. In order to emphasize specific structure-function relationships, these schematic diagrams often depict the side chains of selected amino acids.

of a peptide, while interpolypeptide disulfide bonds stabilize the quaternary structure of certain oligomeric proteins.

Higher orders of protein structure are stabilized primarily—and often exclusively—by noncovalent interactions. Principal among these are hydrophobic interactions that drive most hydrophobic amino acid side chains into the interior of the protein away from the surrounding water. Other significant contributors include hydrogen bonds and salt bridges between the carboxylates of aspartic and glutamic acid and the oppositely charged side chains of protonated lysyl, argininyl, and histidyl residues. These interactions are individually weak—1 to 5 kcal/mol relative to 80 to 120 kcal/mol for a covalent bond. However, just as a Velcro fastener harnesses the cumulative strength of a multitude of tiny plastic loops and hooks, collectively these individually weak but numerous interactions confer a high degree of stability upon the biologically functional conformation of a protein.
Some proteins contain covalent disulfide (S—S) bonds that link the sulfhydryl groups of cysteinyl residues. Formation of disulfide bonds involves oxidation of the cysteiny] sulfhydryl groups and requires oxygen. Intrapolypeptide disulfide bonds further enhance the stability of the folded conformation

Following the solution of the three-dimensional structure of myoglobin by John Kendrew in 1960, x-ray crystallography has revealed the structures of thousands of biologic macromolecules ranging from proteins to oligonucleotides and viruses. For the solution of its structure by x-ray crystallography, a protein is first precipitated under conditions that form well-ordered crystals. To establish appropriate conditions, crystallization trials use a few microliters of protein solution and a matrix of variables (temperature, pH, presence of salts or organic solutes such as polyethylene glycol) to establish optimal conditions for crystal formation. Crystals mounted in quartz capillaries are first irradiated with monochromatic x-rays of approximate wavelength 0.15 nm to confirm that they are comprised of protein, not salt. Protein crystals may then be frozen in liquid nitrogen for subsequent collection of a high-resolution data set. Early crystallographers collected the circular patterns formed by the diffracted x-rays on film and analyzed them by hand. Today, the patterns are recorded electronically using an area detector, then analyzed using a
mathematical approach termed a Fourier synthesis, which summates wave functions. The wave amplitudes are related to spot intensity, but since the waves are not in phase, the relationship between their phases must next be determined in order to extrapolate the positions of the atoms that gave rise to the diffraction pattern.
The traditional approach to solution of the "phase problem" employs isomorphous displacement. Prior to irradiation, an atom with a distinctive x-ray "signature, such as mercury or uranium, is introduced into a crystal at known positions in the primary structure of the protein. An alternative approach uses the expression of plasmid-encoded recombinant proteins in which selenium replaces the sulfur of methionine. Expression uses a bacterial host auxotrophic for methionine biosynthesis and a defined medium in which selenomethionine replaces methionine. If the unknown structure is similar to one that has already been solved, molecular replacement on an existing model provides an attractive way to phase the data without the use of heavy atoms. Finally, the results from the phasing and Fourier summations provide an electron density profile or three-dimensional map of how the atoms are connected or related to one another. The ability of some crystallized enzymes to act as catalysts suggests that their crystal structures faithfully reflect that of the enzyme in free solution.

Nuclear magnetic resonance (NMR) spectroscopy, a powerful complement to x-ray crystallography, measures the absorbance of radio frequency electromagnetic energy by certain atomic nuclei. "NMR-active" isotopes of biologically relevant elements include 'H, °C, '°N, and *!P. The frequency, or chemical shift, at which a particular nucleus absorbs energy is a function of both the functional group within which it resides and the proximity of other NMR-active nuclei. Once limited to metabolites and relatively small macromolecules, today proteins and protein complexes of > 100 kDa can be analyzed by NMR. By measuring the proximity of NMR-active nuclei to one another, twodimensional NMR spectroscopy permits a three-dimensional representation of a protein to be constructed. Since NMR spectroscopy analyzes proteins in aqueous solution, it obviates the need to form crystals (a particular advantage when dealing with difficult to crystallize membrane proteins) and renders it possible to observe the changes in conformation that accompany ligand binding or catalysis in real time. Since NMR is noninvasive and nondestructive, it also offers the possibility of one day being able to observe the structure and dynamics of proteins (and metabolites) within living cells.

The development of the microscope in the 1600s by van Leeuwenhoek triggered a revolution in biology. Scientists were now able to obtain two-dimensional images that revealed the cellular nature of living tissue and the existence of microbial
organisms. However, the resolution of microscopic analyses was limited by the relatively long wavelength of the available sources of electromagnetic radiation, generally visible light (4-7 x 10°" m). By coating materials spread in a monolayer with uranyl acetate or some other heavy metal-containing compound, electron microscopy (EM) can employ highenergy electrons with wavelengths of 1-10 x 10-'? m to generate two-dimensional projection images at a resolution of a few Angstroms.

Biochemists have long dreamed of directly viewing proteins and other biological macromolecules in the same manner that microbiologists and cell biologists can directly view living cells. However, the resolution of optical microscopes was inherently limited by the wavelength of light. In the mid-20" century, scientists developed electron microscopes which use beams of electrons as their source of electromagnetic radiation. The lower wavelength of their electron beams enabled electron microscopes to magnify objects more than a million times more than using an optical microscope—sufficient to visualize large macromolecules such as ribosomes and DNA plasmids. However, the high energy of the electron beam and the need to work under high vacuum conditions were incompatible with the survival of organic molecules.
In 2017, Jacques Dubochet, Joachim Frank, and Richard Henderson were awarded a Nobel Prize for the development of cryo electron microscopy (cryo-EM). Cryo-EM employs ultracold media such as liquid nitrogen (T around —195°C), liquid ethane, or liquid helium to stabilize biomolecules in a hydrated state and protect them from heating when bombarded by the electron beam. This technique allows large macromolecules and macromolecular complexes to be visualized. The extremely low temperatures conferred the added benefit of stabilizing these macromolecules in a particular conformational state, thus revealing different structural conformations. Tomography generates three-dimensional images of biomacromolecular complexes in a given conformational state by using computer algorithms to generate a composite using the twodimensional images of complexes captured in several different orientations on the EM sample grid. The effect of factors that trigger changes in conformational state can thus be determined and compared. The front cover of this book illustrates one example of cryo-EM, a5 A depiction of the proteins of the Zika virus.

A valuable adjunct to the empirical determination of the threedimensional structure of proteins is the use of computer technology for molecular modeling. When the three-dimensional structure is known, molecular dynamics programs can be used to simulate the conformational dynamics of a protein and the manner in which factors such as temperature, pH, ionic strength, or amino acid substitutions influence these motions. Molecular docking programs simulate the interactions that
take place when a protein encounters a substrate, inhibitor, or other ligand. Virtual screening for molecules likely to interact with key sites on a protein of biomedical interest is extensively used to facilitate the discovery of new drugs.
Molecular modeling is also employed to infer the structure of proteins for which x-ray crystallographic or NMR structures are not yet available. Secondary structure algorithms weigh the propensity of specific residues to become incorporated into a helices or 6 sheets in previously studied proteins to predict the secondary structure of other polypeptides. In homology modeling, the known three-dimensional structure of a protein is used as a template upon which to erect a model of the probable structure of a related protein. Scientists are working to devise computer programs that will reliably predict the three-dimensional conformation of a protein directly from its primary sequence, thereby permitting determination of the structures of the many unknown proteins for which templates currently are lacking.

Proteins are conformationally dynamic molecules that can fold into their functionally competent conformation in a time frame of milliseconds. Moreover, they often can refold if their conformation becomes disrupted, a process called renaturation. How are the remarkable speed and fidelity of protein folding attained? In nature, folding into the native state occurs too rapidly to be the product of a random, haphazard search of all possible structures. Denatured proteins are not just random coils. Native contacts are favored, and regions of the native structure persist even in the denatured state. Discussed below are factors that facilitate and are basic mechanistic features of protein folding-refolding.

The number of distinct combinations of phi and psi angles specifying potential conformations of even a relatively small— 15 kDa—polypeptide is unbelievably vast. Proteins are guided through this vast labyrinth of possibilities by thermodynamics. Since the biologically relevant—or native—conformation of a protein generally is the one that is most energetically favored, knowledge of the native conformation is specified in the primary sequence. However, if one were to wait for a polypeptide to find its native conformation by random exploration of all possible conformations, the process would require billions of years to complete. Clearly, in nature, protein folding takes place in a more orderly and guided fashion.

Protein folding generally occurs via a stepwise process. In the first stage, as the newly synthesized polypeptide emerges from the ribosome, short segments fold into secondary structural units that provide local regions of organized structure. Folding is now reduced to the selection of an appropriate arrangement of this relatively small number of secondary structural elements (analogous to the manner in which the classic "animal, mineral, or vegetable" query typically is used to start a round of the parlor game "20 questions"). In the second stage, the hydrophobic regions segregate into the interior of the protein away from solvent, forming a "molten globule,' a partially folded polypeptide in which the secondary structure modules rearrange until the mature conformation of the protein is attained. This process is orderly, but not rigid. Considerable flexibility exists in the ways and in the order in which elements of secondary structure can be rearranged. In general, each element of the secondary or supersecondary structure facilitates proper folding by directing the folding process toward the native conformation and away from unproductive alternatives. For oligomeric proteins, individual protomers tend to fold before they associate with other subunits.

Under appropriate laboratory conditions, many proteins will spontaneously refold after being denatured (ie, unfolded) by treatment with acid or base, chaotropic agents, or detergents. However, refolding under these conditions is slow—minutes to hours. Moreover, most proteins fail to spontaneously refold in vitro. Instead they form insoluble aggregates, disordered complexes of unfolded or partially folded polypeptides held together predominantly by hydrophobic interactions. Aggregates represent unproductive dead ends in the folding process. Cells employ auxiliary proteins to speed the process of folding and to guide it toward a productive conclusion.

Chaperone proteins participate in the folding of over half of all mammalian proteins. The hsp70 (70-kDa heat shock protein) family of chaperones binds short sequences of hydrophobic amino acids that emerge while a new polypeptide is being synthesized, shielding them from solvent. Chaperones prevent aggregation, thus providing an opportunity for the formation of appropriate secondary structural elements and their subsequent coalescence into a molten globule. The hsp60 family of chaperones, sometimes called chaperonins, differs in sequence and structure from hsp70 and its homologs. Hsp60 acts later in the folding process, often together with an hsp70 chaperone. The central cavity of the donut-shaped hsp60 chaperone provides a sheltered nonpolar environment in which a polypeptide can fold until all hydrophobic regions are buried in its interior, thus preempting any tendency toward aggregation.

Disulfide bonds between and within polypeptides stabilize tertiary and quaternary structures. The process is initiated by the enzyme protein sulfhydryl oxidase, which catalyzes the oxidation of cysteine residues to form disulfide bonds. However, disulfide bond formation is nonspecific—a given cysteine can form a disulfide bond with any accessible cysteinyl residue. By catalyzing disulfide exchange, the rupture of an S—S bond and
FIGURE 5-10 Isomerization of the N-a, prolyl peptide bond from a cis to a trans configuration relative to the backbone of the polypeptide.
its reformation with a different partner cysteine, protein disulfide isomerase facilitates the formation of disulfide bonds that stabilize a protein's native conformation. Since many eukaryotic sulfhydryl oxidases are flavin-dependent, dietary riboflavin deficiency often is accompanied by an increased incidence of improper folding of disulfide-containing proteins.

All X-Pro peptide bonds—where X represents any residue— are synthesized in the trans configuration. However, of the X-Pro bonds of mature proteins, approximately 6% are cis. The cis configuration is particularly common in f turns. Isomerization from trans to cis is catalyzed by proline-cis, transisomerases, a family of enzymes also known as cyclophilins (Figure 5-10). In addition to promoting the maturation of native proteins, cyclophilins also participate in the folding of proteins expressed by viral invaders. Consequently, cyclophilins are being pursued as targets for the development of drugs such as cyclosporine and Alisporivir for the treatment of HIV, hepatitis C, and other virally transmitted diseases.

The transmissible spongiform encephalopathies, or prion diseases, are fatal neurodegenerative diseases characterized by spongiform changes, astrocytic gliomas, and neuronal loss resulting from the deposition of insoluble protein aggregates in neural cells. They include Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy (mad cow disease) in cattle. A variant form of Creutzfeldt-Jacob disease (vCJD) that afflicts younger patients is associated with early-onset psychiatric and behavioral disorders. Prion diseases may manifest themselves as infectious, genetic, or sporadic disorders. Because no viral or bacterial gene encoding the pathologic prion protein could be identified, the source and mechanism of transmission of prion disease long remained elusive.
Today it is recognized that prion diseases are protein conformation diseases transmitted by altering the conformation, and hence the physical properties, of proteins endogenous to the host. Human prion-related protein (PrP), a glycoprotein encoded on the short arm of chromosome 20, normally is monomeric and rich in a helix. Pathologic prion proteins serve as the templates for the conformational transformation of normal PrP, known as PrPc, into PrPsc. PrPsc is rich in 6 sheet with many hydrophobic aminoacy] side chains exposed to solvent. As each new PrPsc molecule is formed, it triggers the production of yet more pathologic variants in a conformational chain reaction. Because PrPsc molecules associate strongly with one another through their exposed hydrophobic regions, the accumulating PrPsc units coalesce to form insoluble protease-resistant aggregates. Since one pathologic prion or prion-related protein can serve as template for the conformational transformation of many times its number of PrPc molecules, prion diseases can be transmitted by the protein alone without involvement of DNA or RNA.

Refolding or misfolding of another protein endogenous to human brain tissue, B-amyloid, is a prominent feature of the Alzheimer disease. While the main cause of the Alzheimer disease remains elusive, the characteristic senile plaques and neurofibrillary bundles contain aggregates of the protein B-amyloid, a 4.3-kDa polypeptide produced by proteolytic cleavage of a larger protein known as amyloid precursor protein. In Alzheimer disease patients, levels of B-amyloid become elevated, and this protein undergoes a conformational transformation from a soluble a helix-rich state to a state rich in 8 sheet and prone to selfaggregation. Apolipoprotein E has been implicated as a potential mediator of this conformational transformation.

Thalassemias are caused by genetic defects that impair the synthesis of one of the polypeptide subunits of hemoglobin (see Chapter 6). During the burst of hemoglobin synthesis that occurs during erythrocyte development, a specific chaperone called a-hemoglobin-stabilizing protein (AHSP) binds to free hemoglobin a-subunits awaiting incorporation into the hemoglobin multimer. In the absence of this chaperone, free a-hemoglobin subunits aggregate, and the resulting precipitate has cytotoxic effects on the developing erythrocyte. Investigations using genetically modified mice suggest a role for AHSP in modulating the severity of B-thalassemia in human subjects.

The maturation of proteins into their final structural state often involves the cleavage or formation (or both) of covalent bonds, a process of posttranslational modification.
Many polypeptides are initially synthesized as larger precursors called proproteins. The "extra" polypeptide segments in these proproteins often serve as leader sequences that target a polypeptide to a particular organelle or facilitate its passage through a membrane. Other segments ensure that the potentially harmful activity of a protein such as the proteases trypsin and chymotrypsin remains inhibited until these proteins reach their final destination. However, once these transient requirements are fulfilled, the now superfluous peptide regions are removed by selective proteolysis. Other covalent modifications may add new chemical functionalities to a protein. The maturation of collagen illustrates both of these processes.

Collagen is the most abundant of the fibrous proteins that constitute more than 25% of the protein mass in the human body. Other prominent fibrous proteins include keratin and myosin. These fibrous proteins represent a primary source of structural strength for cells (ie, the cytoskeleton) and tissues. Skin derives its strength and flexibility from an intertwined mesh of collagen and keratin fibers, while bones and teeth are buttressed by an underlying network of collagen fibers analogous to steel strands in reinforced concrete. Collagen also is present in connective tissues such as ligaments and tendons. The high degree of tensile strength required to fulfill these structural roles requires elongated proteins characterized by repetitive amino acid sequences and a regular secondary structure.

Tropocollagen, the repeating unit of a mature collagen fiber, consists of three collagen polypeptides, each containing about 1000 amino acids, bundled together in a unique conformation, the collagen triple helix (Figure 5-11). A mature collagen fiber forms an elongated rod with an axial ratio of about 200. Three intertwined polypeptide strands, which twist to the left, wrap around one another in a right-handed fashion to form the collagen triple helix. The opposing handedness of this superhelix and its component polypeptides makes the collagen triple helix highly resistant to unwinding—a principle also applied to the steel cables of suspension bridges. A collagen triple helix has 3.3 residues per turn and a rise per residue nearly twice that of an a helix. The R groups of each polypeptide strand of the triple helix pack so closely that, in order to fit, one of the three must be H. Thus, every third amino acid residue in collagen is a glycine residue. Staggering of the three strands provides
FIGURE 5-11 Primary, secondary, and tertiary structures of collagen.
appropriate positioning of the requisite glycines throughout the helix. Collagen is also rich in proline and hydroxyproline, yielding a repetitive Gly-X-Y pattern (Figure 5-11) in which Y generally is proline or hydroxyproline.
Collagen triple helices are stabilized by hydrogen bonds between residues in different polypeptide chains, a process helped by the hydroxyl groups of hydroxyprolyl residues. Additional stability is provided by covalent cross-links formed between modified lysyl residues both within and between polypeptide chains.

Collagen is initially synthesized as a larger precursor polypeptide, procollagen. Numerous prolyl and lysyl residues of procollagen are hydroxylated by prolyl hydroxylase and lysyl hydroxylase, enzymes that require ascorbic acid (vitamin C; see Chapters 27 and 44). Hydroxyprolyl and hydroxylysyl residues provide additional hydrogen-bonding capability that stabilizes the mature protein. In addition, glucosyl and galactosyl transferases attach glucosyl or galactosyl residues to the hydroxyl groups of specific hydroxylysyl residues.
The central portion of the precursor polypeptide then associates with other molecules to form the characteristic triple helix. This process is accompanied by the removal of the globular amino terminal and carboxyl terminal extensions of the precursor polypeptide by selective proteolysis. Certain lysyl residues are modified by lysyl oxidase, a coppercontaining protein that converts e-amino groups to aldehydes. The aldehydes can either undergo an aldol condensation to form a C=C double bond or to form a Schiff base (eneimine) with the e-amino group of an unmodified lysyl residue, which is subsequently reduced to form a C—N single bond. These covalent bonds cross-link the individual polypeptides and imbue the fiber with exceptional strength and rigidity.

The complex series of events in collagen maturation provide a model that illustrates the biologic consequences of incomplete polypeptide maturation. The best-known defect in collagen biosynthesis is scurvy, a result of a dietary deficiency of vitamin C required by prolyl and lysyl hydroxylases. The resulting deficit in the number of hydroxyproline and hydroxylysine residues undermines the conformational stability of collagen fibers, leading to bleeding gums, swelling joints, poor wound healing, and ultimately death. Menkes syndrome, characterized by kinky hair and growth retardation, reflects a dietary deficiency of the copper required by lysyl oxidase, which catalyzes a key step in the formation of the covalent cross-links that strengthen collagen fibers.
Genetic disorders of collagen biosynthesis include several forms of osteogenesis imperfecta, characterized by fragile bones. In the Ehlers-Danlos syndrome, a group of connective
tissue disorders that involve impaired integrity of supporting structures, defects in the genes that encode a collagen-1, procollagen N-peptidase, or lysyl hydroxylase result in mobile joints and skin abnormalities (see Chapter 50).

Proteins may be classified based on their solubility, shape, or function or on the presence of a prosthetic group, such as heme.
™ The gene-encoded primary structure of a polypeptide is the sequence of its amino acids. Its secondary structure results from folding of polypeptides into hydrogen-bonded motifs such as the a helix, the B-pleated sheet, 6 bends, and loops. Combinations of these motifs can form supersecondary motifs.
™
Tertiary structure concerns the relationships between secondary structural domains. Quaternary structure of proteins with two or more polypeptides (oligomeric proteins) concerns the spatial relationships between various types of polypeptides.
™ Primary structures are stabilized by covalent peptide bonds. Higher orders of structure are stabilized by weak forces— multiple hydrogen bonds, salt (electrostatic) bonds, and association of R
hydrophobic groups.
™
plot, respectively.
The phi (#) angle of a polypeptide is the angle about the C_—N bond; the psi (w) angle is that about the C —C, bond. Most combinations of phi-psi angles are disallowed due to steric hindrance. The phi-psi angles that form the a helix and the 6 sheet fall within the lower and upper left-hand quadrants of a Ramachandran
™
Protein folding is a poorly understood process. Broadly speaking, short segments of newly synthesized polypeptide fold into secondary structural units. Forces that bury hydrophobic regions from solvent then drive the partially folded polypeptide into a "molten globule" in which the modules of the secondary structure are rearranged to give the native conformation of the protein.
| Proteins that assist folding include protein disulfide isomerase, proline-cis, trans-isomerase, and the chaperones that participate in the folding of over half of mammalian proteins. Chaperones shield newly synthesized polypeptides from solvent and provide an environment for elements of secondary structure to emerge and coalesce into molten globules.
™ Biomedical researchers are currently working to develop agents that interfere with the folding of viral proteins and prions as drugs for the treatment of hepatitis C and a range of neurodegenerative disorders.

@ X-ray crystallography and NMR are key techniques used to study higher orders of protein structure.
™ While lacking the atomic-level resolution of x-ray crystallography or NMR, cryo-EM has emerged as a powerful tool for analyzing the macromolecular dynamics of biologic macromolecules in heterogeneous samples.
=
Prions—protein particles that lack nucleic acid—cause fatal transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease, scrapie, and bovine spongiform encephalopathy. Prion diseases involve an altered secondarytertiary structure of a naturally occurring protein, PrPc. When PrPc interacts with its pathologic isoform PrPsc, its conformation is transformed from a predominantly a-helical structure to the B-sheet structure characteristic of PrPsc.
™ Collagen illustrates the close linkage between protein structure and biologic function. Diseases of collagen maturation include Ehlers-Danlos syndrome and the vitamin C deficiency disease scurvy.

- D. Its ability to dissolve hydrocarbons.
1. Select the one of the following statements that is NOT CORRECT.
10. Select the one of the following statements that is NOT CORRECT.
A. Fermentation and glycolysis share many common
biochemical features.
B. Louis Pasteur first discovered that cell-free yeast
preparations could convert sugars to ethanol and carbon
dioxide.
C. Organic orthophosphate (P,) is essential for glycolysis.
D. “C is an important tool for detecting metabolic
intermediates.
E. Medicine and biochemistry provide mutual insights to one another.
. Select the one of the following statements that is NOT
CORRECT.
A. The vitamin derivative NAD is essential for conversion of
glucose to pyruvate.
B. The term “Inborn errors of metabolism” was coined by the
physician Archibald Garrod.
C. Mammalian tissue slices can incorporate inorganic
ammonia into urea.
D. Realization that DNA is a double helix permitted Watson &
Crick to describe the polymerase chain reaction (PCR).
E. Mutation of the genome of a “model organisms” can provide
insight into biochemical processes.
. Explain how the Biichner’s observation in the early part of the
20th century led to the discovery of the details of fermentation.
. Name some of the earliest discoveries that followed the
realization that a cell-free preparation of yeast cells could catalyze the process of fermentation.
. Name some of the kinds of tissue preparations that early 20th century biochemists employed to study glycolysis and urea biosynthesis, and to discover the roles of vitamin derivatives.
. Describe how the availability of radioactive isotopes facilitated
the
identification of metabolic intermediates.
. Name several of the “inborn errors of metabolism” identified by
the physician Archibald Garrod.
. Cite an example in lipid metabolism for which the linking of
biochemical and genetic approaches has contributed to the
advance of medicine and biochemistry.
. Name several of the intact “model organisms” whose genomes can
be selectively altered to provide insight into biochemical processes.
The propensity of water molecules to form hydrogen bonds

with one another is the primary factor responsible for all of the
following properties of water EXCEPT:
A. Its atypically high boiling point.
B. Its high heat of vaporization.
C. Its high surface tension.
ll.
12. Select the one of the following statements that is NOT
13. Select the one of the following statements that is NOT CORRECT.
14. Select the one of the following statements that is NOT
E. Its expansion upon freezing.
Select the one of the following statements that is NOT
CORRECT.
A. The side-chains of the amino acids cysteine and methionine
absorb light at 280 nm.
B. Glycine is often present in regions where a polypeptide
forms a sharp bend, reversing the direction of a polypeptide.
C. Polypeptides are named as derivatives of the C-terminal
aminoacyl residue.
D. The C,N, O, and H atoms of a peptide bond are coplanar.
E. A linear pentapeptide contains four peptide bonds.
CORRECT.
A. Buffers of human tissue include bicarbonate, proteins, and
orthophosphate.
B. A weak acid or a weak base exhibits its greatest buffering
capacity when the pH is equal to its pK, plus or minus one pH unit.
C. The isoelectric pH (pl) of lysine can be calculated using the formula (pK, + pK,)/2.
D. The mobility of a monofunctional weak acid in a direct
current electrical field reaches its maximum when the pH of
its surrounding environment is equal to its pK,,.
E. For simplicity, the strengths of weak bases are generally
expressed as the pK, of their conjugate acids.
A. If the pK, of a weak acid is 4.0, 50% of the molecules will
be in the dissociated state when the pH of the surrounding
environment is 4.0.
B. A weak acid with a pK, of 4.0 will be a more effective buffer than
at pH 3.8 at pH 5.7.
C. Ata pH equal to its pI, a polypeptide carries no charged groups.

D. Strong acids and bases are so named because they undergo
complete dissociation when dissolved in water.
E. The pK, of an ionizable group can be influenced by the physical and chemical properties of its surrounding
CORRECT.
A. A major objective of proteomics is to identify all of the
proteins present in a cell under different conditions as well
as their states of modification.
B. Mass spectrometry has largely replaced the Edman method
for sequencing of peptides and proteins.
C.
Sanger reagent was an improvement on Edman’s because the
former generates a new amino terminus, allowing several
consecutive cycles of sequencing to take place.
D. Since mass is a universal property of all atoms and
molecules, mass spectrometry is ideally suited to the
detection of posttranslational modifications in proteins.
E. relationship
Time-of-flight mass spectrometers take advantage of the
F = ma.
15. Why does olive oil added to water tend to form large droplets?
16. What distinguishes a strong base from a weak base?
17. Select the one of the following statements that is NOT CORRECT.
18. Select the one of the following statements that is NOT
19. Estimate pl for a polyelectrolyte that contains three carboxyl groups and three amino groups whose pK, values are 4.0, 4.6, 6.3, 7.7, 8.9, and 10.2.
20. State one drawback of the categorization of the protein amino
21. Select the one of the following statements that is NOT
A. Ion-exchange chromatography separates proteins based
upon the sign and magnitude of their charge at a given pH.
B. Two-dimensional gel electrophoresis separates proteins first
on
the
basis
of
their
pI
values
and
second
on
their
charge-to-
mass
ratio
using
SDS-PAGE.
C.
Affinity
exploits
the
selectivity
of
protein-
ligand interactions to isolate a specific protein from a
complex mixture.
D.
Many
proteins
are
expressed
with
an

domain
fused
to
their
N-
or
C-terminus.
One
common
component
of
these
fusion
domains
is
a
ligand-binding
site
designed
expressly
to
purification
by
affinity
chromatography.
E. Following purification by classical techniques, tandem mass spectrometry typically is used to analyze individual homogeneous peptides derived from a complex protein mixture.
CORRECT.
A. Protein folding is assisted by intervention of specialized
auxiliary proteins called chaperones.
B. Protein folding tends to be modular, with areas of local secondary structure forming first, then coalescing into a molten globule.
C. Protein folding is driven first and foremost by the
thermodynamics of the water molecules surrounding the
nascent
polypeptide.
D. The formation of S-S bonds in a mature protein is facilitated
by the enzyme protein disulfide isomerase.
E. Only a few unusual proteins, such as collagen, require posttranslational processing by partial proteolysis to attain their mature conformation.
acids simply as “essential” or “nonessential”?
CORRECT.
A.
Posttranslational
modifications
of
proteins
can
affect
both
their function and their metabolic fate.
B. The native conformational state generally is that which is
thermodynamically favored.
C. The complex three-dimensional structures of most proteins
are
formed
and
stabilized
by
the
cumulative
effects
of
a
large
number
of
weak
interactions.
D. Research scientists employ gene arrays for the high-throughput detection of the presence and expression level of proteins.
E, Examples of weak interactions that stabilize protein folding
include hydrogen bonds, salt bridges, and van der Waals forces.
22. Select the one of the following statements that is NOT CORRECT.
23. Select the one of the following statements that is NOT
24. What advantage does the acidic group of phosphoric acid that associated with pK, offer for buffering in human tissues?
25. The dissociation constants for a previously uncharacterized
26. A biochemical buffer is a compound which tends to resist

27. Name two amino acids whose posttranslational modification confers significant new properties to a protein.
28. Explain why diets deficient in (a) copper (Cu) or (b) ascorbic
29. Describe the role of N-terminal signal sequences in the biosynthesis of certain proteins.
A. Changes in configuration involve the rupture of covalent
bonds.
B.
Changes
in
involve
the
rotation
of
one
or
more
single
bonds.
C. The Ramachandran plot illustrates the degree to which
steric hindrance limits the permissible angles of the single
bonds in the backbone of a peptide or protein.
D. Formation of an a helix is stabilized by the hydrogen bonds
between
each
peptide
bond
carboxyl
oxygen
and
the
N-H
group
of
the
next
peptide
bond.
E. Ina sheet the R groups of adjacent residues point in
opposite directions relative to the plane of the sheet.
CORRECT.
A. The descriptor a,8,y, denotes a protein with seven subunits
of three different types.
B. Loops are extended regions that connect adjacent regions of secondary structure.
C. More than half of the residues in a typical protein reside in
either a helices or B sheets.
D. Most f sheets have a right-handed twist.
E. Prions are viruses that cause protein-folding diseases that
attack the brain.
is
racemic
amino
acid
discovered
in
a
meteor
have
been
determined
to
be
pK,
=
2.0,
pK,
=
3.5,
pK,
=
6.3,
pK,
=
8.0,
pK,
=
9.8,
and
pK,
=
10.9:
A. What carboxyl or amino functional group would you expect
to be associated with each dissociation?
B. What would be the approximate net charge on this amino
acid at pH 2?
C. What would be its approximate net charge at pH 6.3?

D. During direct current electrophoresis at pH 8.5, toward
which electrode would this amino acid be likely to move?
changes in pH even when acids or bases are added. What two properties are required of an effective physiologic buffer? In addition to phosphate, what other physiologic compounds meet these criteria?
acid lead to incomplete posttranslational processing of collagen.

. Describe the most important structural similarities and differences between myoglobin and hemoglobin.
delivery. = Explain the role of 2,3-bisphosphoglycerate (BPG) in oxygen binding and
6
thalassemias.
Sketch binding curves for the oxygenation of myoglobin and hemoglobin.
Identify the covalent linkages and other close associations between heme and globin in oxymyoglobin and oxyhemoglobin.
= Explain why the physiologic function of hemoglobin requires a sigmoidal rather than hyperbolic O,-binding curve.
m= Explain the role of the distal histidine on the ability of hemoglobin to bind carbon monoxide (CO).
Define P,, and indicate its significance in oxygen transport and delivery.
Describe the structural and conformational changes in hemoglobin that accompany its oxygenation and subsequent deoxygenation.
= Explain how the Bohr effect enhances the ability of red blood cells to transport CO, and deliver it to the lungs.
Describe the structural consequences to hemoglobin S (HbS) of lowering Po,.
Identify the metabolic defect that occurs as a consequence of a and

The efficient delivery of oxygen from the lungs to the peripheral tissues and the maintenance of tissue reserves to protect against anoxic episodes are essential to health. In mammals, these functions are performed by the homologous heme proteins hemoglobin and myoglobin, respectively. Myoglobin, a monomeric protein of red muscle, binds oxygen tightly as a reserve against oxygen deprivation. The multiple subunits of hemoglobin, a tetrameric protein of erythrocytes, interact in a cooperative fashion that enables this transporter to offload a high proportion of bound O, in peripheral tissues while simultaneously retaining the capacity to bind it efficiently in the lungs. In addition to delivering O,, proton binding to hemoglobin aids in the transport of CO,, a major product of respiration, to the lungs for disposal. Oxygen delivery is enhanced by the binding of 2,3-bisphosphoglycerate (BPG), which stabilizes the quaternary structure of deoxyhemoglobin.
Hemoglobin and myoglobin illustrate both protein structure— function relationships and the molecular basis of genetic disorders such as sickle cell disease and the thalassemias. Cyanide and carbon monoxide (CO) kill because they disrupt the physiologic function of the heme proteins cytochrome oxidase and hemoglobin, respectively.

Myoglobin and hemoglobin contain heme, an iron-containing cyclic tetrapyrrole consisting of four molecules of pyrrole linked by methyne bridges. This planar network of conjugated double bonds absorbs visible light and colors heme deep red. The substituents at the B-positions of heme are methyl (M), vinyl (V), and propionate (Pr) groups arranged in the order M, V, M, V, M, Pr, Pr, M (Figure 6-1). An atom of ferrous iron (Fe**) resides at the center of the planar tetrapyrrole. Oxidation of the Fe** of myoglobin or hemoglobin to Fe** destroys their biologic activity. Other proteins with metal-containing tetrapyrrole prosthetic groups include the cytochromes (Fe and Cu) and chlorophyll (Mg) (see Chapter 31).

Oxygen stored in red muscle myoglobin is released during O, deprivation (eg, severe exercise) for use in muscle mitochondria for aerobic synthesis of ATP (see Chapter 13). A 153-aminoacyl residue polypeptide (MW 17,000), the compactly folded myoglobin molecule measures 4.5 x 3.5 x 2.5 nm (Figure 6-2). An unusually high proportion, about 75%,
FIGURE 6-1 Heme. The pyrrole rings and methyne bridge carbons are coplanar, and the iron atom (Fe"*) resides in almost the same plane. The fifth and sixth coordination positions of Fe?* are directly perpendicular to—and directly above and below—the plane of the heme ring. Observe the nature of the methyl (blue), vinyl (green), and propionate (orange) substituent groups on the 6 carbons of the pyrrole rings, the central iron atom (red), and the location of the polar side of the heme ring (at about 7 o'clock) that faces the surface of the myoglobin molecule.
FIGURE 6-2 Three-dimensional structure of myoglobin. Shown is a ribbon diagram tracing the polypeptide backbone of myoglobin. The color of the polypeptide chain is graded along the visible spectrum from blue (N-terminal) to tan (C-terminal). The heme prosthetic group is red. The a-helical regions are designated A through H. The distal (E7) and proximal (F8) histidine residues are highlighted in blue and orange, respectively. Note how the polar propionate substituents (Pr) project out of the heme toward solvent. (Adapted from Protein Data Bank ID no. 1a6n.)

of the residues are present in eight right-handed 7 to 20 residue a helices. Starting at the amino terminal, these are termed helices A through H. Typical of globular proteins, the surface of myoglobin is rich in amino acids bearing polar and potentially charged side chains, while—with two exceptions—the interior contains residues that possess nonpolar R groups (eg, Leu, Val, Phe, and Met). The exceptions are the seventh and eighth residues in helices E and F, His E7 and His F8, which lie close to the heme iron, the site of O, binding.

The heme of myoglobin lies in a crevice between helices E and F oriented with its polar propionate groups facing the surface of the globin (Figure 6-2). The remainder resides in the nonpolar interior. The fifth coordination position of the iron is occupied by a nitrogen from the imidazole ring of the proximal histidine, His F8. The distal histidine, His E7, lies on the side of the heme ring opposite to His F8.

The iron of unoxygenated myoglobin lies 0.03 nm (0.3 A) outside the plane of the heme ring, toward His F8. Consequently, the heme "puckers" slightly. When O, occupies the sixth coordination position, the iron moves to within 0.01 nm (0.1 A) of the plane of the heme ring. Oxygenation of myoglobin thus is accompanied by motion of the iron, of His F8, and of residues linked to His F8.
FIGURE 6-3 Angles for bonding of oxygen and carbon monoxide (CO) to the heme iron of myoglobin. The distal E7 histidine hinders bonding of CO at the preferred (90°) angle to the plane of the heme ring.

When O, binds to myoglobin, the bond that links the first and second oxygen atoms lies at an angle of 121° to the plane of the heme, orienting the second oxygen away from the distal histidine (Figure 6-3, left). This permits maximum overlap between the iron and one of the lone pairs of electrons on the sp? hybridized oxygen atoms, which lie at an angle of roughly 120° to the axis of the O=O double bond (Figure 6-4, left), and protects oxygen from being displaced by CO. Minute quantities of CO arise from a variety of biologic sources, including the catabolism of red blood cells within the human body, as well as via the incomplete combustion of fossil fuels. CO binds to the iron in free heme 25,000 times more strongly than oxygen. So why is it that CO does not completely displace O, from the heme iron present in myoglobin and hemoglobin? The accepted explanation is that the apoproteins of myoglobin and hemoglobin create a hindered environment for their gaseous ligands. When CO binds to free heme, all three atoms (Fe, C, and O) lie perpendicular to the plane of the heme. This geometry maximizes the overlap between the lone pair of electrons on the sp hybridized carbon of the CO molecule and the Fe** iron (Figure 6-4, right). However, in myoglobin and
FIGURE 6-4 orientation of the lone pairs of electrons relative to the O=O and C=O bonds of oxygen and carbon monoxide. In molecular oxygen, formation of the double bond between the two oxygen atoms is facilitated by the adoption of an sp? hybridization state by the valence electron of each oxygen atom. As a consequence, the two atoms of the oxygen molecule and each lone pair of electrons are coplanar and separated by an angle of roughly 120° (left). By contrast, the two atoms of carbon monoxide are joined by a triple bond, which requires that the carbon and oxygen atoms adopt an sp hybridization state. In this state, the lone pairs of electrons and triple bonds are arranged in a linear fashion, where they are separated an of 180°
by angle (right).

hemoglobin the distal histidine sterically precludes this preferred, high-affinity orientation of CO while still permitting O, to attain its most favorable orientation. Binding at a less favored angle reduces the strength of the heme-CO bond to about 200 times that of the heme-O, bond (Figure 6-3, right). Therefore O,, which is present in great excess over CO, normally dominates. Nevertheless, about 1% of human myoglobin typically is present combined with CO.

Why is myoglobin unsuitable as an O, transport protein but well suited for O, storage? The relationship between the concentration, or partial pressure, of O, (Po,) and the quantity of O, bound is expressed as an O, saturation isotherm (Figure 6-5). The oxygen-binding curve for myoglobin is hyperbolic. Myoglobin therefore loads O, readily at the Po, of the lung capillary bed (100 mm Hg). However, since myoglobin releases only a small fraction of its bound O, at the Po, values typically encountered in active muscle (20 mm Hg) or other tissues (40 mm Hg), it represents an ineffective vehicle for delivery of O,. However, when strenuous exercise lowers the Po, of muscle tissue to about 5 mm of Hg, the dissociation of O, from myoglobin permits mitochondrial synthesis of ATP, and hence muscular activity, to continue. By contrast, hemoglobin behaves as if it were two proteins. At high Po,, 100 mm Hg and above, it displays a high affinity for oxygen that enables it to bind oxygen to nearly every available heme iron when in the lungs. This form of the protein is commonly referred to as R, for relaxed, state hemoglobin.
Gaseous pressure of oxygen (mm Hg)
FIGURE 6-5 Oxygen-binding curves of both hemoglobin and myoglobin. Arterial oxygen tension is about 100 mm Hg; mixed venous oxygen tension is about 40 mm Hg; capillary (active muscle) oxygen tension is about 20 mm Hg; and the minimum oxygen tension required for cytochrome oxidase is about 5 mm Hg. Association of chains into a tetrameric structure (hemoglobin) results in much greater oxygen delivery than would be possible with single chains. (Modified, with permission, from Scriver CR, et al (editors): The Molecular and Metabolic Bases of Inherited Disease, 7th ed. McGraw-Hill; 1995.)

At the much lower Po, values encountered in peripheral tissues, 40 mm Hg and below, hemoglobin exhibits a much lower apparent affinity for oxygen. Transition of hemoglobin to this low affinity, taut or T state enables it to release a large proportion of the oxygen previously picked up in the lungs. This dynamic interchange between the R and T state serves as the foundation for hemoglobin's sigmoidal O,-binding curve.

Hemoglobins are tetramers composed of pairs of two different polypeptide subunits (Figure 6-6). Greek letters are used to designate each subunit type. The subunit composition of the principal hemoglobins are a,8, (HbA; normal adult hemoglobin), a,y, (HbF; fetal hemoglobin), a,8°, (HbS; sickle cell hemoglobin), and a,5, (HbA,; a minor adult hemoglobin). The primary structures of the B, y, and 6 chains of human hemoglobin are highly conserved.

The properties of individual hemoglobins are consequences of their quaternary as well as secondary and tertiary structures. The quaternary structure of hemoglobin confers striking additional properties, absent from monomeric myoglobin, which adapts it to its unique biologic roles. The allosteric (Gk allos "other," steros "space") properties of hemoglobin provide, in addition, a model for understanding other allosteric proteins (see Chapter 17).
Despite differences in the kind and number of amino acids present, myoglobin and the 6 polypeptide of hemoglobin A share almost identical secondary and tertiary structures. Similarities include the location of the heme and the helical regions, and the presence of amino acids with similar properties at comparable locations. Although it possesses seven rather than eight helical regions, the a polypeptide of hemoglobin also closely resembles myoglobin.
FIGURE 6-6 Hemoglobin. Shown is the three-dimensional structure of deoxyhemoglobin with a molecule of 2,3-bisphosphoglycerate (dark blue) bound. The two a subunits are colored in the darker shades of green and blue, the two f subunits in the lighter shades of green and blue, and the heme prosthetic groups in red. (Adapted from Protein Data Bank ID no. 1b86.)

Hemoglobins can bind up to four molecules of O, per tetramer, one per heme. In addition, hemoglobin will bind a molecule of O, more readily if other O, molecules are already bound (Figure 6-5). Termed cooperative binding, this phenomenon permits hemoglobin to maximize both the quantity of O, loaded at the Po, of the lungs and the quantity of O, released at the Po, of the peripheral tissues. These cooperative interactions, an exclusive property of certain multimeric proteins, are critically important to aerobic life.

The quantity P,,, a measure of O, concentration, is the partial pressure of O, at which a given hemoglobin reaches halfsaturation. Depending on the organism, P., can vary widely, but in all instances, it exceeds the normal Po, of the peripheral tissues. For example, the values of P,, for HbA and HbF are 26 and 20 mm Hg, respectively. In the placenta, this difference enables HbF to extract oxygen from the HbA in the mother's blood. However, HbF is suboptimal postpartum since its higher affinity for O, limits the quantity of O, delivered to the tissues.
The subunit composition of hemoglobin tetramers undergoes complex changes during development. The human fetus initially synthesizes a ££, tetramer. By the end of the first trimester, § and ¢ subunits have been replaced by a and y subunits, forming HbF (a,y,), the hemoglobin of late fetal life. While synthesis of 8 subunits begins in the third trimester, the replacement of y subunits by B subunits to yield adult HbA (a,8,) does not reach completion until several weeks postpartum (Figure 6-7).
FIGURE 6-7 Developmental pattern of the quaternary structure of fetal and newborn hemoglobins. (Reproduced, with permission, from Ganong WF: Review of Medical Physiology, 20th ed. McGraw-Hill; 2001.)
FIGURE 6-8 On oxygenation of hemoglobin the iron atom moves into the plane of the heme. Histidine F8 and its associated aminoacyl residues are pulled along with the iron atom. For a representation of this motion, see http://www.rcsb.org/pdb/101/motm. do?momID=41.

The binding of the first molecule of O, to deoxyHb shifts the heme iron toward the plane of the heme ring (Figure 6-8). This motion is transmitted through the proximal (F8) histidine and the residues attached thereto to the entire tetramer, triggering the rupture of salt bridges formed by the carboxyl terminal residues of all four subunits. As a result, one pair of a/B subunits rotates 15° with respect to the other, compacting the tetramer (Figure 6-9). Profound changes in secondary,
FIGURE 6-9 During transition of the T form to the R form of hemoglobin, the a,B, pair of subunits (green) rotates through 15° relative to the pair of a,B, subunits (yellow). The axis of rotation is eccentric, and the a8, pair also shifts toward the axis somewhat. In the representation, the tan a,B, pair is shown fixed while the green a8, pair of subunits both shifts and rotates.
T
R
structure
FIGURE 6-10 Transition from the T structure to the R structure. In this model, salt bridges (red lines) linking the subunits in the T structure break progressively as oxygen is added, and even those salt bridges that have not yet ruptured are progressively weakened (wavy red lines). The transition from T to R does not take place after a fixed number of oxygen molecules have been bound but becomes more probable as each successive oxygen binds. The transition between the two structures is influenced by protons, carbon dioxide, chloride, and 2,3-bisphosphoglycerate (BPG); the higher their concentration, the more oxygen must be bound to trigger the transition. Fully oxygenated molecules in the T structure and fully deoxygenated molecules in the R structure are not shown because they are unstable. (Modified and redrawn, with permission, from Perutz MF: Hemoglobin structure and respiratory transport. Sci Am [Dec] 1978;239:92.)

tertiary, and quaternary structures accompany the O,-induced transition of hemoglobin from the low-affinity T state to the high-affinity R state. These changes significantly increase the affinity of the remaining unoxygenated hemes for O,, as subsequent binding events require the rupture of fewer salt bridges (Figure 6-10). The terms T and R also are used to refer to the low-affinity and high-affinity conformations of allosteric enzymes, respectively.

In addition to transporting O, from the lungs to peripheral tissues, red blood cells must scavenge CO,, the byproduct of respiration, from peripheral tissues for disposal in the lungs. About 15% of the CO, in venous blood is carried by hemoglobin as carbamates formed with the amino terminal nitrogens of the polypeptide chains:
T-state hemoglobin binds two protons per tetramer. Not only does proton binding help buffer against the acidification of the blood in the peripheral circulation that results from the formation of carbonic acid, it enhances the quantity of CO, absorbed by the red blood cells by favoring the conversion of CO, to carbonic acid and hence to bicarbonate. CO,, in turn, enhances the delivery of O, to respiring tissues by stabilizing the T state by inducing carbamation and lowering the pH. In lungs, this process reverses. As O, binds to deoxyhemoglobin, the resulting transition to the R state triggers release of bound protons and the breakdown of carbamates, releasing CO,. The liberated protons combine with bicarbonate to form carbonic acid. H,CO, is subsequently dehydrated, a process catalyzed by the enzyme carbonic anhydrase, to form CO,, which is exhaled (Figure 6-11). This coupling of the interconversion of CO, and H,CO, with the reciprocal binding of protons and O, by Tand R-state hemoglobin, respectively, is termed the Bohr effect.
Carbamate formation changes the charge on amino terminals from positive to negative, favoring salt bridge formation between a and f chains. The remaining CO, is carried mostly as bicarbonate, which is formed in erythrocytes by the hydration of CO, to carbonic acid (H,CO,), a process catalyzed by carbonic anhydrase. At the pH of venous blood, H,CO, dissociates into bicarbonate and a proton.

The Bohr effect is dependent on the cooperative interactions among the subunits, and their associated heme groups, of the hemoglobin tetramer. In the lungs, the partial pressure of oxygen drives the conversion of hemoglobin from the T to the R state, breaking the salt bridges involving B-chain residue His 146. The subsequent dissociation of protons from His 146 then drives the conversion of bicarbonate to carbonic acid (Figure 6-11). In respiring tissues, the high concentration of CO, drives the formation of carbonic acid and the acidification of red blood cells. Upon the release of O,, the T structure and its salt bridges reform. This conformational change increases the pK, of the B-chain His 146 residues, which bind protons. By stabilizing the formation of T-state hemoglobin,
FIGURE 6-11 The Bohr effect. Carbon dioxide generated in peripheral tissues combines with water to form carbonic acid, which dissociates into protons and bicarbonate ions. Deoxyhemoglobin acts as a buffer by binding protons and delivering them to the lungs. In the lungs, the uptake of oxygen by hemoglobin releases protons that combine with bicarbonate ion, forming carbonic acid, which when dehydrated by carbonic anhydrase becomes carbon dioxide, which then is exhaled.
the CO,-driven increase in proton concentration enhances the release of O,,.

In peripheral tissues, CO,-induced decreases in red blood cell pH promotes the synthesis of 2,3-BPG in erythrocytes. When hemoglobin is in the T state, a central cavity forms at the interface of its four subunits that can bind one molecule of BPG (Figure 6-6), which forms salt bridges with the terminal amino groups of both 6 chains via Val NA1 and with Lys EF6 and His H21 (Figure 6-12). BPG therefore stabilizes deoxygenated (T-state) hemoglobin by forming additional salt bridges that must be broken prior to conversion to the R state. Synthesis of BPG from the glycolytic intermediate 1,3-BPG is catalyzed by the bifunctional enzyme 2,3-bisphosphogylcerate synthase/2-phosphatase (BPGM). BPG is hydrolyzed to 3-phosphoglycerate by the 2-phosphatase activity of BPGM and to 2-phosphoglycerate by a second enzyme, multiple inositol polyphosphate phosphatase (MIPP). The activities of these enzymes, and hence the level of BPG in erythrocytes, are sensitive to pH. As a consequence, the binding of BPG reinforces the impact of carbonic acid-derived protons in shifting the R-T equilibrium in favor of the T state, increasing the quantity of O, released in peripheral tissues. In the fetal hemoglobin, residue H21 of the y subunit is
Ser rather than His. Since Ser cannot form a salt bridge, BPG
FIGURE 6-12 Mode of binding of 2,3-bisphosphoglycerate (BPG) to human deoxyhemoglobin. BPG interacts with three positively charged groups on each 8 chain. (Based on Arnone A: X-ray diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhemoglobin. Nature 1972;237:146. Copyright © 1972. Adapted by permission from Macmillan Publishers Ltd.)
binds more weakly to HbF than to HbA. The lower stabilization afforded to the T state by BPG accounts for HbF having a higher affinity for O, than HbA.

Physiologic changes that accompany prolonged exposure to high altitude include increases in the number of erythrocytes, the concentration of hemoglobin within them, and the synthesis of BPG. Elevated BPG lowers the affinity of HbA for O, (increases P.,), which enhances the release of O, at peripheral tissues.

Mutations in the genes that encode the a or B subunits of hemoglobin potentially can affect its biologic function. However, almost all of the over 1100 known genetic mutations affecting human hemoglobins are both rare and benign, presenting no clinical abnormalities. When a mutation does compromise biologic function, the condition is termed a hemoglobinopathy. It is estimated that more than 7% of the globe's population are carriers for hemoglobin disorders. The URL http:// globin.cse.psu.edu/ (Globin Gene Server) provides information about—and links for—normal and mutant hemoglobins. Selected examples are described below.

In methemoglobinemia, the heme iron is ferric rather than ferrous, rendering methemoglobin unable to bind or transport O,.
Normally, the Fe** of methemoglobin is returned to Fe** state through the action of the enzyme methemoglobin reductase. Methemoglobin levels can rise to pathophysiologially significant levels from a number of causes: oxidation of Fe** to Fe** as a side effect of agents such as sulfonamides, reductions in the activity of methemoglobin reductase, or inheritance of the gene for a altered form of called HbM.
mutationally hemoglobin In hemoglobin M, histidine F8 (His F8) has been replaced by tyrosine. The iron of HbM forms a tight ionic complex with the phenolate anion of tyrosine that stabilizes the Fe** form. In a-chain hemoglobin M variants, the R-T equilibrium favors the T state. Oxygen affinity is reduced, and the Bohr effect is absent. B-Chain hemoglobin M variants exhibit R-T switching, and the Bohr effect is therefore present. Mutations that favor the R state (eg, hemoglobin Chesapeake) increase O, affinity. These hemoglobins therefore fail to deliver adequate O, to peripheral tissues. The resulting tissue hypoxia leads to polycythemia, an increased concentration of erythrocytes.

In HbS, the nonpolar amino acid valine has replaced the polar surface residue Glu6é of the B subunit, generating a hydrophobic "sticky patch" on the surface of the 6 subunit of both oxyHbS and deoxyHbS. Both HbA and HbS contain a complementary sticky patch on their surfaces that is exposed only in the deoxygenated T state. Thus, at low Po,, deoxyHbS can polymerize to form long, insoluble fibers. Binding of deoxyHbA terminates fiber polymerization, since HbA lacks the second sticky patch necessary to bind another Hb molecule (Figure 6-13). These twisted helical fibers distort the erythrocyte into a characteristic sickle shape, rendering it vulnerable to lysis in the interstices of the splenic sinusoids. They also cause multiple secondary clinical effects. A low Po,, such as that at high altitudes, exacerbates the tendency to polymerize. The terms sickle cell trait and sickle cell disease refer to persons in whom either one or both beta subunit genes are mutated, respectively. Emerging treatments for sickle cell disease include inducing HDF expression to inhibit the polymerization of HbS, stem cell transplantation, and, in the future, gene therapy.

Following massive crush injury to skeletal muscle followed by renal damage, released myoglobin may appear in the urine. Myoglobin can be detected in plasma following a myocardial infarction, but assay of serum troponin, lactate dehydrogenase isozymes, or creatine kinase (see Chapter 7) provides a more sensitive index of myocardial injury.

Anemias, reductions in the number of red blood cells or of hemoglobin in the blood, can reflect impaired synthesis of hemoglobin (eg, in iron deficiency; see Chapter 53) or impaired production of erythrocytes (eg, in folic acid or vitamin B,, deficiency; see Chapter 44). Diagnosis of anemias begins with spectroscopic measurement of blood hemoglobin levels.

The genetic defects known as thalassemias result from the partial or total absence of one or more a or f chains of hemoglobin. Over 750 different mutations have been identified, but only three are common. Either the a chain (a thalassemias) or 6 chain (6 thalassemias) can be affected. A superscript indicates whether a subunit is completely absent (a° or B°) or whether its synthesis is reduced (a> or B-). Apart from marrow transplantation, treatment is symptomatic.
Certain mutant hemoglobins are common in many populations, and a patient may inherit more than one type. Hemoglobin disorders thus present a complex pattern of clinical phenotypes. The use of DNA probes for their diagnosis is considered in Chapter 39.
FIGURE 6-13 Polymerization of deoxyhemoglobin S. The dissociation of oxygen from hemoglobin S (HbS) unmasks a sticky patch (red triangle) on the surface of its B subunits (green) that can adhere to a complementary site on the B subunits of other molecules of deoxyHbS. Polymerization toa fibrous polymer is interrupted deoxyHbA, whose B subunits (lavender) lack the sticky patch required for binding additional HbS subunits.

Blood glucose that enters the erythrocytes can form a covalent adduct with the e-amino groups of lysyl residues and the N-terminal valines of hemoglobin 6 chains, a process referred to as glycation. Unlike glycosylation (see Chapter 46), glycation is not enzyme-catalyzed. The fraction of hemoglobin glycated, normally about 5%, is proportionate to blood glucose concentration. Since the half-life of an erythrocyte is typically 60 days, the level of glycated hemoglobin (HbA,_) reflects the mean blood glucose concentration over the preceding 6 to 8 weeks. Measurement of HbA,, therefore provides valuable information for management of diabetes mellitus.

= Myoglobin is monomeric; hemoglobin is a tetramer of two subunit types (a,8, in HbA). Despite having different primary structures, myoglobin and the subunits of hemoglobin have nearly identical secondary and tertiary structures.
= Heme, an essentially planar, slightly puckered, cyclic tetrapyrrole has a central Fe** linked to all four nitrogen atoms of the heme, to histidine F8, and, in oxyMb and oxyHb, also to O..
| The O,-binding curve for myoglobin is hyperbolic, but for hemoglobin it is sigmoidal, a consequence of cooperative interactions in the tetramer.
™ Cooperativity arises from the ability of hemoglobin to exist in two different conformational states, a relaxed or R state in which all four subunits exhibit a high affinity for oxygen and a taut or T state where all four subunits display a low affinity for oxygen.
@ The high levels of O, in the lungs drive the R-T equilibrium in favor of the R state, while acidification of the red blood cells generated from the catalytic hydration of CO, in the peripheral tissues favors the T state. Cooperativity thus maximizes the ability of hemoglobin both to load O, at the Po, of the lungs and to deliver O, at the Po, of the tissues.
@
Relative affinities of different hemoglobins for oxygen are expressed as P.,,, the Po, that half-saturates them with O,,. Hemoglobins saturate at the partial pressures of their respective respiratory organ, for example, the lung or placenta.
™ On oxygenation of hemoglobin, the iron and histidine F8 move toward the heme ring. The resulting conformational changes in the hemoglobin tetramer include the rupture of salt bonds and loosening of the quaternary structure that facilitates binding of additional O,.
™ 2,3-BPG in the central cavity of deoxyHb forms salt bonds with the 6 subunits that stabilize T-state hemoglobin. On oxygenation,
the central cavity contracts upon transition to the R state, BPG is extruded, and the quaternary structure loosens.

= Hemoglobin assists in CO, transport from peripheral tissues to the lungs via the formation of carbamates and the Bohr effect, a consequence of the binding of protons to T-state, but not R-state, hemoglobin. Proton binding enhances the conversion of CO, to water-soluble carbonic acid and bicarbonate. In the lungs, the release of protons from oxygenated R-state hemoglobin favors the conversion of bicarbonate and carbonic acid to co, which is exhaled.
@ In sickle cell hemoglobin (HbS), Val replaces Glu, of the 6 subunit of HbA, creating a "sticky patch" that has a complement on deoxyHb (but not on oxyHb). DeoxyHbS polymerizes at low O, concentrations, forming fibers that distort erythrocytes into sickle shapes.
™ aand f Thalassemias are anemias that result from reduced production of a and B subunits of HbA, respectively.

O B J E C T I V E S, 1 = ■ Describe the structural relationships between specific B vitamins and certain coenzymes.. After studying this chapter, you should be able to:, 1 = ■ Outline the four principal catalytic mechanisms and how they can be combined by enzymes to facilitate chemical reactions.. , 1 = ■ Explain the concept of an'induced fit' and how it facilitates catalysis.. , 1 = ■ Outline the underlying principles of enzyme-linked immunoassays.. , 1 = ■ Describe how coupling an enzyme to the activity of a dehydrogenase can simplify many enzyme assays.. , 1 = ■ Identify proteins whose plasma levels are used for diagnosis and prognosis.. , 1 = ■ Describe the application of restriction endonucleases and of restriction fragment length polymorphisms in the detection of genetic diseases.. , 1 = ■ Illustrate the utility of site-directed mutagenesis for the identification of aminoacyl residues that are involved in the recognition of substrates or allosteric effectors, or in the mechanism of catalysis.. , 1 = ■ Describe how'affinity tags'can facilitate purification of a protein expressed from its cloned gene.. , 1 = ■ Indicate the function of specific proteases in the purification of affinity-tagged enzymes.. , 1 = ■ Discuss the events that led to the discovery that RNAs can act as enzymes, and briefly describe the evolutionary concept of an'RNA world.'

Enzymes, which catalyze the chemical reactions that make life on the earth possible, participate in the breakdown of nutrients to supply energy and chemical building blocks; the assembly of those building blocks into proteins, DNA, membranes, cells, and tissues; and the harnessing of energy to power cell motility, neural function, and muscle contraction. Almost all enzymes are proteins. Notable exceptions include ribosomal RNAs and a handful of RNA molecules imbued with endonuclease or nucleotide ligase activity known collectively as ribozymes. The ability to detect and to quantify the activity of specific enzymes in blood, other tissue fluids, or cell extracts provides information that complements the physician's ability to diagnose many diseases. Many pathologic conditions are the direct consequence of changes in the quantity or in the catalytic activity of key enzymes that result from genetic defects, nutritional deficits, tissue damage, toxins, or infection by viral or bacterial pathogens (eg, Vibrio cholerae).
In addition to serving as the catalysts for all metabolic processes, the impressive catalytic activity, substrate specificity, and stereospecificity of enzymes enables them to fulfill unique roles in human health and well-being. The protease rennin, for example, is utilized in the production of cheeses, while lactase is employed to remove lactose from milk to benefit lactoseintolerant individuals. Proteases and amylases augment the capacity of detergents to remove dirt and stains, while other enzymes can participate in the stereospecific synthesis of complex drugs or antibiotics.

The enzymes that catalyze the conversion of one or more compounds (substrates) into one or more different compounds (products) generally enhance the rates of the corresponding noncatalyzed reaction by factors of 10° or more. Enzymes may undergo transient modification during catalysis, but are neither consumed nor permanently altered. In addition to being highly efficient, enzymes are also extremely selective. Unlike most catalysts used in synthetic chemistry, enzymes are specific not simply for the type of reaction catalyzed, but also for a single substrate or a small set of closely related substrates. Most enzymes are stereospecific catalysts that typically act upon only one stereoisomer of a given compound—for example, pbut not L-sugars, Lbut not p-amino acids. Since they bind substrates through at least "three points of attachment; enzymes also can produce chiral products from nonchiral substrates. Figure 7-1 illustrates why the enzyme-catalyzed reduction of the nonchiral substrate pyruvate produces exclusively L-lactate, not a racemic mixture of Dand t-lactate. The exquisite specificity of enzyme catalysts imbues living cells with the ability to simultaneously conduct and independently control a broad spectrum of biochemical processes.

Some of the names for enzymes first described in the earliest days of biochemistry persist in use to this day. Examples include pepsin, trypsin, and amylase. Early biochemists generally named newly discovered enzymes by adding the suffix -ase to a descriptor for the type of reaction catalyzed. For example, enzymes that remove the elements of hydrogen, H, or H" plus H* generally are referred to as dehydrogenases, enzymes that hydrolyze proteins as proteases, and enzymes that catalyze rearrangements in configuration as isomerases. In many cases, these general descriptors were supplemented with terms
FIGURE 7-1 Planar representation of the "three-point attachment" of a substrate to the active site of an enzyme. Although atoms 1 and 4 are identical, once atoms 2 and 3 are bound to their complementary sites on the enzyme, only atom 1 can bind. Once bound to an enzyme, apparently identical atoms thus may be distinguishable, permitting a stereospecific chemical change.
indicating the particular substrate on which the enzyme acts (xanthine oxidase), its source (pancreatic ribonuclease), its mode of regulation (hormone-sensitive lipase), or a characteristic feature of its mechanism of action (cysteine protease). Where needed, alphanumeric designators can be added to identify multiple forms, or isozymes, of an enzyme (eg, RNA polymerase III; protein kinase CB).
As more enzymes were discovered, these early naming conventions increasingly resulted in the inadvertent designation of some enzymes by multiple names or the assignment of duplicate names to enzymes exhibiting similar catalytic capabilities. To address these problems, the International Union of Biochemistry (IUB) developed a system of enzyme nomenclature in which each enzyme has a unique name and code number that identify the type of reaction catalyzed and the substrates involved. Enzymes are grouped into the following six classes:
- 1. Oxidoreductases—enzymes that catalyze oxidations and reductions.
2.
Transferases—enzymes that
catalyze transfer
of moieties
such as
glycosyl, methyl,
or phosphoryl
groups.

3. Hydrolases—enzymes that catalyze hydrolytic cleavage of C—C, C—O, C—N, and other covalent bonds.
4. Lyases—enzymes that catalyze cleavage of C—C, C—O, C—N, and other covalent bonds by atom elimination, generating double bonds.
5. Isomerases—enzymes that catalyze geometric or structural changes within a molecule.
6. Ligases—enzymes that catalyze the joining together (ligation) of two molecules in reactions coupled to the hydrolysis of ATP.
The IUB name of hexokinase is ATP:D-hexose 6-phosphotransferase E.C. 2.7.1.1. This name identifies hexokinase as a member of class 2 (transferases), subclass 7 (transfer of a phosphoryl group), sub-subclass 1 (alcohol is the phosphoryl acceptor), and "hexose-6" indicates that the alcohol phosphorylated is on carbon six of a hexose. While EC numbers have proven particularly useful to differentiate enzymes with similar functions or similar catalytic activities, IUB names tend to be lengthy and cumbersome. Consequently, hexokinase and many other enzymes commonly are referred to using their traditional, albeit sometimes ambiguous names.

Many enzymes contain small molecules or metal ions that participate directly in substrate binding or in catalysis. Termed prosthetic groups, cofactors, and coenzymes, they extend the repertoire of catalytic capabilities beyond those afforded
by the functional groups present on the aminoacyl side chains of peptides.

Prosthetic groups are tightly and stably incorporated into a protein's structure by covalent or noncovalent forces. Examples include pyridoxal phosphate, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamin pyrophosphate, lipoic acid, biotin, and transition metals such as Fe, Co, Cu, Mg, Mn, and Zn. Metal ions that participate in redox reactions generally are bound as organometallic complexes such as the prosthetic groups heme or iron-sulfur clusters (see Chapter 10). Metals also may facilitate the binding and orientation of substrates, the formation of covalent bonds with reaction intermediates (Co** in coenzyme B,,, see Chapter 44), or by acting as Lewis acids or bases to render substrates more electrophilic (electron-poor) or nucleophilic (electron-rich), and hence more reactive (see Chapter 10).

Cofactors serve functions similar to those of prosthetic groups. The major difference between the two is operational, not chemical. Cofactors bind weakly and transiently to their cognate enzymes or substrates, forming dissociable complexes. Therefore, unlike associated prosthetic groups, cofactors must be present in the surrounding environment to promote complex formation in order for catalysis to occur. The most common cofactors also are metal ions. Enzymes that require a metal ion cofactor are termed metal-activated enzymes to distinguish them from the metalloenzymes for which bound metal ions serve as prosthetic groups.

The water-soluble B vitamins supply important components of numerous coenzymes. Nicotinamide is a component of the redox coenzymes NAD and NADP (Figure 7-2); riboflavin is a component of the redox coenzymes FMN and FAD; pantothenic acid is a component of the acyl group carrier coenzyme A. As its pyrophosphate thiamin participates in the decarboxylation of a-keto acids while folic acid and cobamide coenzymes function in one-carbon metabolism. In addition, several coenzymes contain the adenine, ribose, and phosphoryl moieties of AMP or ADP (Figure 7-2).

Coenzymes serve as recyclable shuttles that transport many substrates from one point within the cell to another. The function of these shuttles is twofold. First, they stabilize species such as hydrogen atoms (FADH,) or hydride ions (NADH) that are too reactive to persist for any significant time in the presence of
FIGURE 7-2 Structure of NAD* and NADP*. For NAD*, OR = —OH. For NADP*, —OR = —OPO,".
the water, oxygen, or the organic molecules that permeate cells. Second, they increase the number of points of contact between substrate and enzyme, which increases the affinity and specificity with which small chemical groups such as acetate (coenzyme A), glucose (UDP), or hydride (NAD*) are bound by their target enzymes. Other chemical moieties transported by coenzymes include methyl groups (folates) and oligosaccharides (dolichol).

An important early 20th-century insight into enzymic catalysis sprang from the observation that the presence of substrates renders enzymes more resistant to the denaturing effects of an elevated temperature. This observation led Emil Fischer to propose that enzymes and their substrates interact to form an enzyme-substrate (ES) complex whose thermal stability is greater than that of the enzyme itself. This insight profoundly shaped our understanding of both the chemical nature and kinetic behavior of enzymic catalysis.
Fischer reasoned that the exquisitely high specificity with which enzymes discriminate their substrates when forming an ES complex was analogous to the manner in which a mechanical lock distinguishes the proper key. The analogy to enzymes is that the "lock" is formed by a cleft or pocket on the surface of the enzyme called the active site (see Figures 5-6 and 5-8). As implied by the adjective "active," the active site is much more than simply a recognition site for binding substrates; it provides the environment wherein chemical transformation takes place. Within the active site, substrates are brought into
FIGURE 7-3 Two-dimensional representation of a dipeptide substrate, glycyl-tyrosine, bound within the active site of carboxypeptidase A.
close proximity with one another in optimal alignment with the cofactors, prosthetic groups, and aminoacyl side chains that participate in catalyzing the transformation of substrates into products (Figure 7-3). Catalysis is further enhanced by the capacity of the active site to shield substrates from water and generate an environment whose polarity, hydrophobicity, acidity, or alkalinity can differ markedly from that of the surrounding cytoplasm.

Enzymes use combinations of four general mechanisms to achieve dramatic enhancements of the rates of chemical reactions.

For molecules to interact, they must come within bond-forming distance of one another. The higher their concentration, the more frequently they will encounter one another, and the greater will be the rate at which they react. When an enzyme binds substrate molecules at its active site, it creates a region of high local substrate concentration in which the substrate molecules are oriented in a position ideal for them to chemically interact. This results in rate enhancements of at least a thousandfold over the same nonenzyme-catalyzed reaction.

In addition to contributing to the ability of the active site to bind substrates, the ionizable functional groups of aminoacyl side chains and, where present, of prosthetic groups, can contribute to catalysis by acting as acids or bases. We distinguish two types of acid-base catalysis. Specific acid or base catalysis refers to reactions for which the only participating acids or bases are protons or hydroxide ions. The rate of reaction thus is sensitive to changes in the concentration of protons or hydroxide ions, but is independent of the concentrations of other acids (proton donors) or bases (proton acceptors) present in the solution or at the active site. Reactions whose rates are responsive to all the acids or bases present are said to be subject to general acid catalysis or general base catalysis.

For catalysis of lytic reactions, which involve breaking a covalent bond, enzymes typically bind their substrates in a conformation that weakens the bond targeted for cleavage through physical distortion and electronic polarization. This strained conformation mimics that of the transition state intermediate, a transient species that represents the midway point in the transformation of substrates to products. Nobel Laureate Linus Pauling was the first to suggest a role for transition state stabilization as a general mechanism by which enzymes accelerate the rates of chemical reactions. Knowledge of the transition state of an enzyme-catalyzed reaction is frequently exploited by chemists to design and synthesize more effective enzyme inhibitors, called transition state analogs, as potential pharmacophores.

The process of covalent catalysis involves the formation of a covalent bond between the enzyme and one or more substrates. The modified enzyme thus becomes a reactant. Covalent catalysis provides a new reaction pathway whose activation energy is lower—and rate of reaction therefore faster—than the pathways available in homogeneous solution. The chemically modified state of the enzyme is, however, transient. Completion of the reaction returns the enzyme to its original, unmodified state. Its role thus remains catalytic. Covalent catalysis is particularly common among enzymes that catalyze group transfer reactions. Residues on the enzyme that participate in covalent catalysis generally are cysteine or serine, and occasionally histidine. Covalent catalysis often follows a "ping-pong" mechanism—one in which the first substrate is bound and its product released prior to the binding of the second substrate (Figure 7-4).
Pyr
FIGURE 7-4 "Ping-pong" mechanism for transamination. E—CHO and E—CH,NH, represent the enzyme-pyridoxal phosphate and enzyme-pyridoxamine complexes, respectively. (Ala, alanine; Glu, glutamate; KG, a-ketoglutarate; Pyr, pyruvate.)
FIGURE 7-5 Two-dimensional representation of Koshland's induced fit model of the active site of a lyase. Binding of the substrate A—B induces conformational changes in the enzyme that align catalytic residues which participate in catalysis and strain the bond between A and B, facilitating its cleavage.

While Fischer's "lock and key model" accounted for the exquisite specificity of enzyme-substrate interactions, the implied rigidity of the enzyme's active site failed to account for the dynamic changes that accompany catalytic transformations. This drawback was addressed by Daniel Koshland's induced fit model, which states that as substrates bind to an enzyme, they induce a conformational change that is analogous to placing a hand (substrate) into a glove (enzyme) (Figure 7-5). The enzyme in turn induces reciprocal changes in its substrates, harnessing the energy of binding to facilitate the transformation of substrates into products. The induced fit model has been amply confirmed by biophysical studies of enzyme motion substrate during binding.

Enzymes of the aspartic protease family, which includes the digestive enzyme pepsin, the lysosomal cathepsins, and the protease produced by the human immunodeficiency virus (HIV), share a common mechanism that employs two conserved aspartyl residues as acid-base catalysts. In the first stage of the reaction, one aspartate functions as a general base (Asp X, Figure 7-6) that extracts a proton from a water molecule to make it more nucleophilic. The resulting nucleophile then attacks the electrophilic carbonyl carbon of the peptide bond targeted for hydrolysis, forming a tetrahedral transition state intermediate. A second aspartate (Asp Y, Figure 7-6) then facilitates the decomposition
FIGURE 7-6 Mechanism for catalysis by an aspartic protease such as HIV protease. Curved arrows indicate directions of electron movement. @ Aspartate X acts as a base to activate a water molecule by abstracting a proton. @ The activated water molecule attacks the peptide bond, forming a transient tetrahedral intermediate. @ Aspartate Y acts as an acid to facilitate breakdown of the tetrahedral intermediate and release of the split products by donating a proton to the newly formed amino group. Subsequent shuttling of the proton on Asp X to Asp Y restores the protease to its initial state.
of this tetrahedral intermediate by donating a proton to the amino group produced by rupture of the peptide bond. The two active site aspartates can act simultaneously as a general base or as a general acid because their immediate environment favors ionization of one, but not the other.

While catalysis by aspartic proteases involves the direct hydrolytic attack of water on a peptide bond, catalysis by the serine protease chymotrypsin involves formation of a covalent
acyl-enzyme intermediate. A conserved seryl residue, serine 195, is activated via interactions with histidine 57 and aspartate 102. While these three residues are far apart in primary structure, in the active site of the mature, folded protein they reside within bond-forming distance of one another. Aligned in the order Asp 102-His 57-Ser 195, this trio forms a linked charge-relay network that acts as a "proton shuttle."
Binding of substrate initiates proton shifts that in effect transfer the hydroxyl proton of Ser 195 to Asp 102 (Figure 7-7). The enhanced nucleophilicity of the seryl oxygen facilitates its attack on the carbonyl carbon of the peptide bond of the substrate, forming a covalent acyl-enzyme intermediate. The proton on Asp 102 then shuttles via His 57 to the amino group liberated when the peptide bond is cleaved. The portion of the original peptide with a free amino group then leaves the active site and is replaced by a water molecule. The charge-relay network now activates the water molecule by withdrawing a proton through His 57 to Asp 102. The resulting hydroxide ion attacks the acyl-enzyme intermediate, and a reverse proton shuttle returns a proton to Ser 195, restoring its original state. While modified during the process of catalysis, chymotrypsin emerges unchanged on completion of the reaction. The proteases trypsin and elastase employ a similar catalytic mechanism, but the numbering of the residues in their Ser-His-Asp proton shuttles differ.

Fructose-2,6-bisphosphatase, a regulatory enzyme of gluconeogenesis (see Chapter 19), catalyzes the hydrolytic release of the phosphate on carbon 2 of fructose-2,6-bisphosphate. Figure 7-8 illustrates the roles of seven active site residues. Catalysis involves a "catalytic triad" consisting of one Glu and two His residues, of which one His forms a covalent phosphohistidyl intermediate.

Members of an enzyme family such as the aspartic or serine proteases employ a similar mechanism to catalyze a common reaction type, but act on different substrates. Most enzyme families appear to have arisen through gene duplication events that created a second copy of the gene that encodes a particular enzyme. The two genes, and consequently their encoded proteins, can then evolve independently, forming divergent homologs that recognize different substrates. The result is illustrated by chymotrypsin, which cleaves peptide bonds on the carboxyl terminal side of large hydrophobic amino acids, and trypsin, which cleaves peptide bonds on the carboxy] terminal side of basic amino acids. Proteins that diverged from a common ancestor are said to be homologous to one another. The common ancestry of enzymes can be inferred from the presence of specific amino acids in the same relative position in each family member. These residues are said to be evolu- tionarily conserved.
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FIGURE 7-7 Catalysis by chymotrypsin. @ The charge-relay system removes a proton from Ser 195, making it a stronger nucleophile. @ Activated Ser 195 attacks the peptide bond, forming a transient tetrahedral intermediate. @ Release of the amino terminal peptide is facilitated by donation of a proton to the newly formed amino group by His 57 of the charge-relay system, yielding an acyl-Ser 195 intermediate. @ His 57 and Asp 102 collaborate to activate a water molecule, which attacks the acyl-Ser 195, forming a second tetrahedral intermediate. © The charge-relay system donates a proton to Ser 195, facilitating breakdown of the tetrahedral intermediate to release the carboxyl terminal peptide ©.

Higher organisms often elaborate several physically distinct of a given enzyme, each of which catalyzes the same versions reaction. Like the members of other protein families, these
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FIGURE 7-8 Catalysis by fructose-2,6-bisphosphatase. (1) Lys 356 and Arg 257, 307, and 352 stabilize the quadruple negative charge of the substrate by charge-charge interactions. Glu 327 stabilizes the positive charge on His 392. (2) The nucleophile His 392 attacks the C-2 phosphoryl group and transfers it to His 258, forming a phosphoryl-enzyme intermediate. Fructose-6-phosphate now leaves the enzyme. (3) Nucleophilic attack by a water molecule, possibly assisted by Glu 327 acting as a base, forms inorganic phosphate. (4) Inorganic orthophosphate is released from Arg 257 and Arg 307. (Reproduced, with permission, from Pilkis SJ, et al: 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolic signaling enzyme. Annu Rev Biochem 1995;64:799. © 1995 by Annual
Reviews, www.annualreviews.org.)
protein catalysts or isozymes arise through gene duplication. While the homologous proteases described act on different substrates, isozymes differ in auxiliary features such as sensitivity to particular regulatory factors (see Chapter 9) or subcellular location that adapt them to specific tissues or circumstances rather than distinct substrates. Isozymes that catalyze the identical reaction may also enhance survival by providing a "backup" copy of an essential enzyme.

The relatively small quantities of enzymes typically contained in cells hamper determination of their presence and abundance. However, the ability to rapidly transform thousands of molecules of a specific substrate into products imbues each enzyme with the ability to amplify its presence. Under appropriate conditions (see Chapter 8), the rate of the catalytic reaction being monitored is proportionate to the amount of enzyme present, which allows its concentration to be inferred.
Assays of the catalytic activity of enzymes are frequently used both in research and clinical laboratories.

The limited sensitivity of traditional enzyme assays necessitates the use ofa large group, or ensemble, of enzyme molecules in order to produce measurable quantities of product. The data obtained thus reflects the average activity of individual enzymes across multiple cycles of catalysis. Recent advances in nanotechnology and imaging have made it possible to observe catalytic events involving discrete enzyme and substrate molecules. Consequently, scientists can now measure the rate of individual catalytic events, and sometimes a specific step in catalysis, by a process called single-molecule enzymology, an example of which is illustrated in Figure 7-9.

Enzymes are frequent targets for the development of drugs and other therapeutic agents. These generally take the form of enzyme inhibitors (see Chapter 8). The discovery of new drugs is greatly facilitated when a large number of potential pharmacophores can be simultaneously assayed in a rapid, automated fashion—a process referred to as high-throughput screening (HTS). HTS employs robotics, optics, data processing, and microfluidics to simultaneously conduct and monitor thousands of parallel enzyme assays. HTS devices employ 4 to 100 pL volumes in 96, 384, or 1536 well plastic plates using
FIGURE 7-9 Direct observation of single DNA cleavage events catalyzed by a restriction endonuclease. DNA molecules immobilized to beads (blue) are placed in a flowing stream of buffer (black arrows), which causes them to assume an extended conformation. Cleavage at one of the restriction sites (orange) by an endonuclease leads to a shortening of the DNA molecule, which can be observed directly in a microscope since the nucleotide bases in DNA are fluorescent. Although the endonuclease (red) does not fluoresce, and hence is invisible, the progressive manner in which the DNA molecule is shortened (14) reveals that the endonuclease binds to the free end of the DNA molecule and moves along it from site to site.
industrial robots to dispense substrates, coenzymes, enzymes, and potential inhibitors in a multiplicity of combinations and concentrations. HTS provides a perfect complement to combinatorial chemistry, a method for generating large libraries of chemical compounds spanning all possible combinations of a given set of chemical precursors. Enzyme assays that produce a chromogenic or fluorescent product are ideal since optical detectors can readily monitor multiple samples.

The sensitivity of enzyme assays can be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunosorbent assays (ELISAs) use antibodies covalently linked to a "reporter enzyme" such as alkaline phosphatase or horseradish peroxidase whose products are readily detected, generally by the absorbance of light or by fluorescence. Serum or other biologic samples to be tested are placed in plastic, multiwell microtiter plates, where the proteins adhere to the plastic surface and are immobilized. Any exposed plastic that remains is subsequently "blocked" by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody is then determined by adding the substrate for the reporter enzyme, and measuring the rate of its conversion to product.

The physicochemical properties of the reactants in an enzymecatalyzed reaction dictate the options for the assay of enzyme activity. Spectrophotometric assays exploit the ability of a substrate or product to absorb light. The reduced coenzymes NADH and NADPH, written as NAD(P)H, absorb light at a wavelength of 340 nm, whereas their oxidized forms NAD(P)* do not (Figure 7-10). When NAD(P)* is reduced, the absorbance at 340 nm therefore increases in proportion to—and at a rate determined by—the quantity of NAD(P)H produced. Conversely, when a dehydrogenase catalyzes the oxidation of NAD(P)H, a decrease in absorbance at 340 nm will be observed. In each case, the rate of change in absorbance at 340 nm will be proportionate to the quantity of the enzyme present.
The assay of enzymes whose reactions are not accompanied by a change in absorbance or fluorescence is generally more difficult. In some instances, either the product or remaining substrate can be transformed into a more readily detected compound, although the reaction product may have to be separated from unreacted substrate prior to measurement. An alternative strategy is to devise a synthetic substrate whose product absorbs light or fluoresces. For example, hydrolysis of the phosphoester bond in p-nitrophenyl phosphate (pNPP), an artificial substrate molecule, is catalyzed at a measurable rate by numerous phosphatases, phosphodiesterases, and serine proteases. While pNPP does not absorb visible light, the anionic form of the p-nitrophenol (pK, 6.7) generated upon
FIGURE 7-10 Absorption spectra of NAD* and NADH. Densities are for a 44-mg/L solution in a cell with a 1-cm light path. NADP* and NADPH have spectra analogous to NAD* and NADH, respectively.
its hydrolysis strongly absorbs light at 419 nm, and thus can be quantified.

Another quite general approach is to employ a "coupled" assay (Figure 7-11). Typically, a dehydrogenase whose substrate is the product of the enzyme of interest is added in catalytic excess. The rate of appearance or disappearance of NAD(P)H then depends on the rate of the enzyme reaction to which the dehydrogenase has been coupled.

The analysis of enzymes in blood plasma has played a central role in the diagnosis of several disease processes. Many enzymes are functional constituents of blood. Examples include pseudocholinesterase, lipoprotein lipase, and components of the
FIGURE 7-11 Coupled enzyme assay for hexokinase activity. The production of glucose-6-phosphate by hexokinase is coupled to the oxidation of this product by glucose-6-phosphate dehydrogenase in the presence of added enzyme and NADP*. When an excess of glucose-6-phosphate dehydrogenase is present, the rate of formation of NADPH, which can be measured at 340 nm, is governed by the rate of formation of glucose-6-phosphate by hexokinase.
cascades that trigger blood clotting, clot dissolution, and opsonization of invading microbes. Several enzymes are released into plasma following cell death or injury. While these latter enzymes perform no physiologic function in plasma, they can serve as biomarkers, molecules whose appearance or levels can assist in the diagnosis and prognosis of diseases and injuries affecting specific tissues. The plasma concentration of an enzyme or other protein released consequent to injury may rise early or late, and may decline rapidly or slowly. Cytoplasmic proteins tend to appear more rapidly than those from subcellular organelles. The speed with which enzymes and other proteins are removed from plasma depends in part upon their susceptibility to proteolysis.
Quantitative analysis of the activity of released enzymes or other proteins, typically in plasma or serum but also in urine or various cells, provides information concerning diagnosis, prognosis, and response to treatment. Assays of enzyme activity typically employ standard kinetic assays of initial reaction rates. Table 7-1 lists several enzymes of value in clinical diagnosis. Note that these enzymes are not absolutely specific for the indicated disease. For example, elevated blood levels of prostatic acid phosphatase are associated typically with prostate cancer, but also may occur with certain other cancers and noncancerous conditions. Interpretation of enzyme assay data must make due allowance for the sensitivity and the diagnostic specificity of the enzyme test, together with other factors elicited through a comprehensive clinical examination that includes patient's age, sex, prior history, and possible drug use.

An enzyme useful for diagnostic enzymology should be relatively specific for the tissue or organ under study, and should appear in the plasma or other fluid at a time useful

Alanine aminotransferase (ALT), Major Diagnostic Use = Viral hepatitis. Amylase, Major Diagnostic Use = Acute pancreatitis. Ceruloplasmin, Major Diagnostic Use = Hepatolenticular degeneration (Wilson disease). Creatine kinase, Major Diagnostic Use = Muscle disorders. γ-Glutamyl transferase, Major Diagnostic Use = Various liver diseases. Lactate dehydrogenase isozyme 5, Major Diagnostic Use = Liver diseases. Lipase, Major Diagnostic Use = Acute pancreatitis. β-Glucocerebrosidase, Major Diagnostic Use = Gaucher disease. Phosphatase, alkaline (isozymes), Major Diagnostic Use = Various bone disorders, obstructive liver diseases
ALT, alanine aminotransferase.
Note: Many of the above enzymes are not specific to the disease listed.
for diagnosis (the "diagnostic window"). In the case of a myocardial infarction (MI), detection must be possible within a few hours of a preliminary diagnosis to permit initiation of appropriate therapy. The first enzymes used to diagnose MI were aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH). Diagnosis using LDH exploits the tissue-specific variations in its quaternary structure (Figure 7-12). However, it is released relatively slowly following injury. Creatine kinase (CK) has three tissuespecific isozymes: CK-MM (skeletal muscle), CK-BB (brain), and CK-MB (heart and skeletal muscle), along with a more optimal diagnostic window. As with LDH, individual CK isozymes are separable by electrophoresis. Assay of plasma CK levels presently continues in use to assess skeletal muscle disorders such as Duchene muscular dystrophy.

Troponin is a complex of three proteins present in the contractile apparatus of skeletal and cardiac muscle but not in smooth muscle (see Chapter 51). Troponin levels rise for 2 to 6 hours after an MI, and remain elevated for 4 to 10 days. Immunological measurements of plasma levels of cardiac troponins and T thus provide sensitive and specific indicators of damage to heart muscle. Since other sources of heart muscle damage also elevate serum troponin levels, cardiac troponins thus provide a general marker of cardiac injury.

Enzymes are employed in the clinical laboratory to determine the presence and the concentration of critical metabolites. For example, glucose oxidase frequently is utilized to measure plasma glucose concentration. Enzymes also are employed with increasing frequency for the treatment of injury and disease. Examples include tissue plasminogen activator (tPA) or streptokinase for treatment of acute MI, and trypsin for treatment of cystic fibrosis. Intravenous infusion of recombinantly produced glycosylases can be used to treat lysosomal storage syndromes such as Gaucher disease (B-glucosidase), Pompe disease (a-glucosidase), Fabry disease (a-galactosidase A), and Sly disease (B-glucuronidase).

Many diagnostic techniques take advantage of the specificity and efficiency of the enzymes that act on oligonucleotides such as DNA. Enzymes known as restriction endonucleases, for example, cleave double-stranded DNA at sites specified by a sequence of four, six, or more base pairs called restriction sites. Cleavage of a sample of DNA with a restriction enzyme produces a characteristic set of smaller DNA fragments (see Chapter 39). Deviations in the normal product pattern, called
restriction fragment length polymorphisms (RFLPs), occur if a mutation renders a restriction site unrecognizable to its cognate restriction endonuclease or, alternatively, generates a new recognition site. RFLPs are currently utilized to facilitate prenatal detection of a number of hereditary disorders, including sickle cell trait, B-thalassemia, infant phenylketonuria, and Huntington disease.
all animal proteins can be expressed in their appropriately folded, functionally competent form in microbial cells as these organisms cannot perform certain posttranslational processing tasks specific to higher organisms. In these instances, options include expression of recombinant genes in cultured animal cell systems or by employing the baculovirus expression vector of cultured insect cells. For more details concerning recombinant DNA techniques, see Chapter 39.

As described in Chapter 39, the polymerase chain reaction (PCR) employs a thermostable DNA polymerase and appropriate oligonucleotide primers to produce thousands of copies of a defined segment of DNA from a minute quantity of starting material. PCR enables medical, biological, and forensic scientists to detect and characterize DNA present initially at levels too low for direct detection. In addition to screening for genetic mutations, PCR can be used to detect pathogens and parasites such as Trypanosoma cruzi, the causative agent of Chagas disease, and Neisseria meningitides, the causative agent of bacterial meningitis, through the selective amplification of their DNA.

Highly purified samples of enzymes are essential for the study of their structure and function. The isolation of an individual enzyme, particularly one present in low concentration, from among the thousands of proteins present in a cell can be extremely difficult. By cloning the gene for the enzyme of interest, it generally is possible to produce large quantities of its encoded protein in Escherichia coli or yeast. However, not

Recombinant DNA technology can also be used to generate proteins specifically modified to render them readily purified by affinity chromatography. The gene of interest is linked to an additional oligonucleotide sequence that encodes a carboxyl or amino terminal extension to the protein of interest. The resulting fusion protein contains a new domain tailored to interact with an appropriately modified affinity support. One popular approach is to attach an oligonucleotide that encodes six consecutive histidine residues. The expressed "His tag" protein binds to chromatographic supports that contain an immobilized divalent metal ion such as Ni** or Cd**. This approach exploits the ability of these divalent cations to bind His residues. Once bound, contaminating proteins are washed off and the His-tagged enzyme is eluted with buffers containing high concentrations of free histidine or imidazole, which compete with the polyhistidine tails for binding to the immobilized metal ions. Alternatively, the substrate-binding domain of glutathione S-transferase (GST) can serve as a "GST tag" Figure 7-13 illustrates the purification of a GST-fusion protein using an affinity support containing bound glutathione. The addition of an N-terminal fusion domain may also help induce proper folding of the remainder of the recombinant polypeptide. Most fusion domains also possess a cleavage
FIGURE 7-13 Use of glutathione S-transferase (GST) fusion proteins to purify recombinant proteins. (GSH, glutathione.)
site for a highly specific protease such as thrombin in the region that links the two portions of the protein to permit its eventual removal.

Once the ability to express a protein from its cloned gene has been established, it is possible to employ site-directed mutagenesis to change specific aminoacyl residues by altering their codons. Used in combination with kinetic analyses and x-ray crystallography, this approach facilitates identification of the specific roles of given aminoacyl residues in substrate binding and catalysis. For example, the inference that a particular aminoacyl residue functions as a general acid can be tested by replacing it with an aminoacyl residue incapable of donating a proton.

The participation of enzyme catalysts in the posttranslational maturation of certain proteins has analogies in the RNA world. Many RNA molecules undergo processing reactions that remove segments of oligonucleotide and religate the remaining segments to form the mature polynucleotide product (see Chapter 36). Not all of these catalysts are proteins, however.
While examining the processing of ribosomal RNA (rRNA) molecules in the ciliated protozoan Tetrahymena in the early 1980s, Thomas Cech and his coworkers observed that processing of the 26S rRNA proceeded smoothly in vitro even in the total absence of protein. The source of this splicing activity was traced to a 413 bp catalytic segment that retained its catalytic activity even when replicated in Escherichia coli (see Chapter 39). Prior to that time, it had been thought that polynucleotides served solely as information storage and transmission entities, and that catalysis was restricted solely to proteins. Several other ribozymes have since been discovered. The vast majority catalyze nucleophilic displacement reactions that target the phosphodiester bonds of the RNA backbone. In small self-cleaving RNAs, such as hammerhead or hepatitis delta virus RNA, the attacking nucleophile is water and the result is hydrolysis. For the large group I intron ribozymes, the attacking nucleophile is the 3'-hydroxyl of the terminal ribose of another segment of RNA and the result is a splicing reaction.

The ribosome was the first example of a "molecular machine" to be recognized. A massive complex comprised of scores of protein subunits and several large ribosomal RNA molecules, the ribosome performs the vitally important and highly complex process of synthesizing long polypeptide chains following the instructions encoded in messenger RNA (mRNA) molecules (see Chapter 37). For many years, it was assumed that rRNAs played a passive, structural role, or perhaps assisted in the recognition of cognate mRNAs through a base pairing mechanism. It was thus somewhat surprising when it was discovered that rRNAs were both necessary and sufficient for catalyzing peptide synthesis.

The discovery of ribozymes has had a profound influence on evolutionary theory. For many years, scientists had hypothesized that the first biologic catalysts were formed when amino acids contained in the primordial soup coalesced to form simple proteins. With the realization that RNA could both carry information and catalyze chemical reactions, a new "RNA World" hypothesis emerged in which RNA constituted the first biologic macromolecule. Eventually, a more chemically stable oligonucleotide, DNA, superseded RNA for long-term information storage, while proteins, by virtue of their greater chemical functional group and conformational diversity dominated catalysis. If one assumes that some sort of RNA-protein hybrid was formed as an intermediate in the transition from ribonucleotide to polypeptide catalysts, one needs to look no further than the ribosome to find the presumed missing link. Why did not proteins take over all catalytic functions? Presumably, in the case of the ribosome the process was both too complex and too essential to permit much opportunity for possible competitors to gain a foothold. In the case of the small self-cleaving RNAs and self-splicing introns, they may
represent one of the few cases in which RNA autocatalysis is more efficient than development of a new protein catalyst.

Enzymes are efficient catalysts whose stringent specificity extends to the kind of reaction catalyzed, and typically, to a single substrate.
™
Organic and inorganic prosthetic groups, cofactors, and coenzymes play important roles in catalysis. Coenzymes, many of which are derivatives of B vitamins, serve as "shuttles" for commonly used groups such as amines, electrons, and acetyl groups.
™ During catalysis, enzymes redirect the conformational changes induced by substrate binding to effect complementary changes that facilitate transformation into product.
™
Catalytic mechanisms employed by enzymes include the introduction of strain, approximation of reactants, acid-base catalysis, and covalent catalysis. HIV protease illustrates acidbase catalysis; chymotrypsin and fructose-2,6-bisphosphatase illustrate covalent catalysis.
™
enzyme
Aminoacyl residues that participate in catalysis are highly conserved through the evolution of enzymes. Site-directed mutagenesis, used to change residues suspected of being important in catalysis or substrate binding, provides insights into mechanisms of action.
™ The catalytic activity of enzymes reveals their presence, facilitates their detection, and provides the basis for enzymelinked immunoassays. Many enzymes can be assayed spectrophotometrically by coupling them to an NAD(P)*dependent dehydrogenase.
= Combinatorial chemistry generates extensive libraries of potential
enzyme activators and inhibitors that can be tested by HTS.
@ Assay of plasma enzymes aids diagnosis and prognosis of acute pancreatitis and of various bone and liver disorders. Serum enzymes have, however, been replaced by assay of plasma troponins as diagnostic of a myocardial infarction.
@ Restriction endonucleases facilitate diagnosis of genetic diseases by revealing restriction fragment length polymorphisms.
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or glutathione.
Specific proteases
can then
remove affinity

“tags”
and generate the native enzyme.
= Notall enzymes are proteins. Several ribozymes are known that can cut and resplice the phosphodiester bonds of RNA. In the ribosome, it is the rRNA and not the polypeptide components that are primarily responsible for catalysis.

Bese RUM
you should be able to:
= Describe the scope and objectives of enzyme kinetic analysis.
Indicate whether AG, the overall change in free energy for a reaction, is dependent on reaction mechanism.
Indicate whether AG is a function of the rates of reactions.
Explain the relationship between Koy concentrations of substrates and products at equilibrium, and the ratio of the rate constants k,/k ,.
= Outline how the concentration of hydrogen ions, of enzyme, and of substrate affect the rate of an enzyme-catalyzed reaction.
= Utilize collision theory to explain how temperature affects the rate of a chemical reaction.
= Define initial rate conditions and explain the advantage obtained from measuring the velocity of an enzyme-catalyzed reaction under these conditions.
= Describe the application of linear forms of the Michaelis-Menten equation to estimate K, and V,,..
™ Give one reason why a linear form of the Hill equation is used to evaluate how substrate-binding influences the kinetic behavior of certain multimeric enzymes.
= Contrast the effects of an increasing concentration of substrate on the kinetics of simple competitive and noncompetitive inhibition.
= Describe how substrates add to, and products depart from, an enzyme that follows a ping-pong mechanism.
= Describe how substrates add to, and products depart from, an enzyme that follows a rapid-equilibrium mechanism.
m= Provide examples of the utility of enzyme kinetics in ascertaining the mode of action of drugs.

a . A complete and balanced set of enzyme activities is required for maintaining homeostasis. Enzyme kinetics, the quantita-
and the systematic study of factors that affect these rates, tive measurement of the rates of enzyme-catalyzed reactions
stitutes a central tool for the analysis, diagnosis, and treatment the
enzymic numerous
human diseases. For example, kinetic analysis can reveal the number
of imbalances
that underlie
and order of the individual steps by which enzymes transform substrates into products, and in conjunction with site-directed mutagenesis, kinetic analyses can reveal details of the catalytic mechanism of a given enzyme. In the blood, the appearance or surge in the levels of particular enzymes serves as clinical indicon-cators for pathologies such as myocardial infarctions, prostate cancer, and damage to the liver. The involvement of enzymes jn virtually all physiologic processes makes them the targets of choice for drugs that cure or ameliorate human disease.
_
Applied enzyme kinetics represents the principal tool by which scientists identify and characterize therapeutic agents that selectively inhibit the rates of specific enzyme-catalyzed processes. Enzyme kinetics thus plays a central and critical role in drug discovery, in comparative to pharmacodynamics, and in elucidating the mode of action of drugs.

A balanced chemical equation lists the initial chemical species (substrates) present and the new chemical species (products) formed for a particular chemical reaction, all in their respective proportions or stoichiometry. For example, balanced equation (1) indicates that one molecule each of substrates A and B reacts to form one molecule each of products P and Q:

(1)
The double arrows indicate reversibility, an intrinsic property of all chemical reactions. Thus, for reaction (1), if A and B can form P and Q, then P and Q can also form A and B. Designation of a particular reactant as a "substrate" or "product" is therefore somewhat arbitrary since the products for a reaction written in one direction are the substrates for the reverse reaction. The term "products" is, however, often used to designate the reactants whose formation is thermodynamically favored. Reactions for which thermodynamic factors strongly favor formation of the products to which the arrow points often are represented with a single arrow as if they were "irreversible":

(2)
Unidirectional arrows are also used to describe reactions in living cells where the products of reaction (2) are immediately consumed by a subsequent enzyme-catalyzed reaction or rapidly escape the cell, for example, CO,. The rapid removal of product P or Q therefore effectively precludes occurrence of the reverse reaction, rendering equation (2) functionally irreversible under physiologic conditions.

The Gibbs free-energy change AG (also called either free energy or Gibbs energy) describes in quantitative form both the direction in which a chemical reaction will tend to proceed and the concentrations of reactants and products that will be present at equilibrium. AG for a chemical reaction equals the sum of the free energies of formation of the reaction products AG, minus the sum of the free energies of formation of the substrates AG,. A similar but different quantity designated by AG? denotes the change in free energy that accompanies and for reaction (5)
=
transition from the standard state, one-molar concentrations of substrates and products, to equilibrium. A more useful biochemical term is AG", which defines AG? at a standard state of 10°? M protons, pH 7.0. If the free energy of formation of the products is Jower than that of the substrates, the signs of AG° and AG" will be negative, indicating that the reaction as written is favored in the direction left to right. Such reactions are referred to as spontaneous. The sign and the magnitude of the free-energy change determine how far the reaction will proceed.
Equation (3) illustrates the relationship between the equilibrium constant K,, and AG®:
K,, where R is the gas constant (1.98 cal/mol°K or 8.31 J/mol°K) and Tis the absolute temperature in degrees Kelvin. K,, is equal to the product of the concentrations of the reaction products, each raised to the power of their stoichiometry, divided by the product of the substrates, each raised to the power of their stoichiometry:
For the reaction A+B @P+Q
[P}[Q]
«q=
*
[AJ[B]
A+A2P
[P]
=
"af
(4)
(5)
6 6)

AG? may be calculated from equation (3) if the molar concentrations of substrates and products present at equilibrium are known. If AG° is a negative number, K,, will be greater than unity, and the concentration of products at equilibrium will exceed that of the substrates. If AG? is positive, K,, will be less than unity, and the formation of substrates will be favored. Note that, since AG? is a function exclusively of the initial and final states of the reacting species, it can provide information only about the direction and equilibrium state of the reaction. AG® is independent of the mechanism of the reaction, and provides no information concerning rates of reactions. Consequently—and as explained below—although a reaction may have a large negative AG° or AG", it may nevertheless take place at a negligible rate.

The concept of the transition state is fundamental to understanding the chemical and thermodynamic basis of catalysis. Equation (7) depicts a group transfer reaction in which an
entering group E displaces a leaving group L, attached initially toR:
E+R-L@E-R+L
(7)
The net result of this process is to transfer group R from L to E. Midway through the displacement, the bond between R and L has weakened but has not yet been completely severed, and the new bond between E and R is yet incompletely formed. This transient intermediate—in which neither free substrate nor product exists—is termed the transition state, E...R...L. Dotted lines represent the "partial" bonds that are undergoing formation and rupture. Figure 8-1 provides a more detailed illustration of the transition state intermediate formed during the transfer of a phosphoryl group.
Reaction (7) can be thought of as consisting of two "partial reactions,' the first corresponding to the formation (F) and the second to the subsequent decay (D) of the transition state intermediate. As for all reactions, characteristic changes in free energy, AG, and AG, are associated with each partial reaction:
E+R-L2E...R...L
AG,
(8)
E...R..L2E-R+L
AG,
E+R-LeE-R+L
AG=AG,+AG,
—

FIGURE 8-1 Formation of a transition state intermediate during a simple chemical reaction, A + B > P + Q. Shown are three stages of a chemical reaction in which a phosphoryl group is transferred from leaving group L (green) to entering group E (blue). Top: Entering group E (bracket A) approaches the other reactant, L-phosphate (bracket B). Notice how the three oxygen atoms linked by the triangular lines and the phosphorus atom of the phosphoryl group form a pyramid. Center: As E approaches L-phosphate, the new bond between E and the phosphoryl group begins to form (dotted line) as that linking L to the phosphoryl group weakens. These partially formed bonds are indicated by dotted lines. Bottom: Formation of the new product, E-phosphate (bracket P), is now complete as the leaving group L (bracket Q) exits. Notice how the geometry of the phosphoryl group differs between the transition state and the substrate or product. The phosphorus and three oxygen atoms that occupy the four corners of a pyramid in the substrate and product become coplanar, as emphasized by the triangle, in the transition state.
(9)
(10)
the overall reaction (10), AG is the numeric sum of AG, AG,,.. As for any equation of two terms, it is not possible to from their resultant AG, either the sign or the magniAG, or AG,,.
Many reactions successive transition states, each with an associated change in free energy. For these reactions, the overall AG represents the sum of all of the freeenergy changes associated with the formation and decay of all of the transition states. It therefore is not possible to infer from the overall AG the number or type of transition states through which the reaction proceeds. Stated another way, overall reaction thermodynamics tells us nothing about mechanism or kinetics.
For and deduce tude of involve several

Regardless of the sign or magnitude of AG, AG, for the overwhelming majority of chemical reactions has a positive sign, which indicates that formation of the transition state requires surmounting one or more energy barriers. For this reason, AG, for reaching a transition state is often termed the activation energy, E,. The ease—and hence the frequency—with which this barrier is overcome is inversely related to E,_,. The thermodynamic parameters that determine how fast a reaction proceeds thus are the AG, values for formation of the transition states through which the reaction proceeds. For a simple reaction, where « means "proportionate to,"
The activation energy for the reaction proceeding in the opposite direction to that drawn is equal to -AG,,.

The kinetic theory—also called the collision theory—of chemical kinetics states that for two molecules to react, they (1) must approach within bond-forming distance of one another, or "collide," and (2) must possess sufficient kinetic energy to overcome the energy barrier for reaching the transition state. It therefore follows that conditions that tend to increase the frequency or energy of collision between substrates will tend to increase the rate of the reaction in which they participate.

Raising the ambient temperature increases the kinetic energy of molecules. As illustrated in Figure 8-2, the total number of molecules whose kinetic energy exceeds the energy barrier E,,, (vertical bar) for formation of products increases from low (A) through intermediate (B) to high (C) temperatures. Increasing the kinetic energy of molecules also increases their rapidity of motion, and therefore the frequency with which they collide. This combination of more frequent and more highly energetic, and hence productive, collisions increases the reaction rate.
FIGURE 8-2 Representation of the energy barrier for chemical reactions. (See text for discussion.)

The frequency with which molecules collide is directly proportionate to their concentrations. For two different molecules A and B, the frequency with which they collide will double if the concentration of either A or B is doubled. If the concentrations of both A and B are doubled, the probability of collision will increase fourfold.
For a chemical reaction proceeding at a constant temperature that involves one molecule each of A and B,
A+BP
(12)
the fraction of the molecules possessing a given kinetic energy will be a constant. The number of collisions between molecules whose combined kinetic energy is sufficient to produce product P therefore will be directly proportionate to the number of collisions between A and B, and thus to their molar concentrations, denoted by the square brackets:
=
subscripts 1 and -1 refer to the forward and reverse reactions, respectively:
Rate,
=
k,[A]"[B]”
(20)
Rate
=k
,[P]
(21)
The sum of the molar ratios of the reactants defines the kinetic order of the reaction. Consider reaction (5). The stoichiometric coefficient for the sole reactant, A, is 2. Therefore, the rate of production of P is proportional to the square of [A] and the reaction is said to be second order with respect to reactant A. In this instance, the overall reaction is also second order. Therefore, k, is referred to as a second-order rate constant.
Reaction (12) describes a simple second-order reaction between two different reactants, A and B. The stoichiometric coefficient for each reactant is 1. Therefore, while the reaction is second order, it is said to be first order with respect to A and first order with respect to B.
In the laboratory, the kinetic order of a reaction with respect to a particular reactant, referred to as the variable reactant or substrate, can be determined by maintaining the concentration of the other reactants in large excess over the variable reactant. Under these pseudo-first-order conditions, the concentration of the "fixed" reactant remains virtually constant. Thus, the rate of reaction will depend exclusively on the concentration of the variable reactant, sometimes also called the limiting reactant. The concepts of reaction order and pseudo-first-order conditions apply not only to simple chemical reactions but also to enzyme-catalyzed reactions.

Rate
e
[A]
[B]
(13)
Similarly, for the reaction represented by
A+2B—P
(14)
which can also be written as
A+B+B4P
(15)
the corresponding rate expression is
Rate c [A] [B] [B]
(16)
or
Rate << [A] [B]}?
(17)
For the general case, when n molecules of A react with m molecules of B,
nA
+mB-P
(18)
the rate expression is
Rate e [A]" [B]”
(19)
Replacing the proportionality sign with an equals sign by introducing a rate constant, k, characteristic of the reac- tion under study gives equations (20) and (21), in which the
While all chemical reactions are to some extent reversible, at equilibrium the overall concentrations of reactants and prod- ucts remain constant. At equilibrium, the rate of conversion of substrates to products therefore equals the rate at which prod- ucts are converted to substrates:
Rate,
=
Rate
,
(22)
k,
=
[A]"[B]"
=
k_[P]
(23)
and
kth
(4)
AL
k,
[A]"B)”
The ratio of k, to k , is equal to the equilibrium constant, K,,. The following important properties of a system at equilibrium must be kept in mind.
1. The equilibrium constant is a ratio of the reaction rate con- stants (not the reaction rates).
2. At equilibrium, the reaction rates (not the rate constants) of the forward and back reactions are equal.
3. The numeric value of the equilibrium constant K,, can be calculated either from the concentrations of substrates and products at equilibrium or from the ratio k,/k_,.
4, Equilibrium is a dynamic state. Although there is no net change in the concentration of substrates or products, individual substrate and product molecules are continually being interconverted. Interconvertibility can be proved by adding to a system at equilibrium a trace of radioisotopic product, which can then be shown to result in the appearance of radiolabelled substrate.

All enzymes accelerate reaction rates by lowering AG, for the formation of transition states. However, they may differ in the way this is achieved. While the sequence of chemical steps at the active site parallels those which occur when the substrates react in the absence of a catalyst, the environment of the active site lowers AG, by stabilizing the transition state intermediates. To put it another way, the enzyme can be envisioned as binding to the transition state intermediate (Figure 8-1) more tightly than it does to either substrates or products. As discussed in Chapter 7, stabilization can involve (1) acid-base groups suitably positioned to transfer protons to or from the developing transition state intermediate, (2) suitably positioned charged groups or metal ions that stabilize developing charges, or (3) the imposition of steric strain on substrates so that their geometry approaches that of the transition state. HIV protease (see Figure 7-6) illustrates catalysis by an enzyme that lowers the activation barrier in part by stabilizing a transition state intermediate.
Catalysis by enzymes that proceeds via a unique reaction mechanism typically occurs when the transition state intermediate forms a covalent bond with the enzyme (covalent catalysis). The catalytic mechanism of the serine protease chymotrypsin (see Figure 7-7) illustrates how an enzyme utilizes covalent catalysis to provide a unique reaction pathway possessing a more favorable E....

While enzymes undergo transient modifications during the process of catalysis, they always emerge unchanged at the completion of the reaction. The presence of an enzyme therefore has no effect on AG for the overall reaction, which is a function solely of the initial and final states of the reactants. Equation (25) shows the relationship between the equilibrium constant for a reaction and the standard free-energy change for that reaction:

(25)
This principle is perhaps most readily illustrated by including the presence of the enzyme (Enz) in the calculation of the equilibrium constant for an enzyme-catalyzed reaction:
A+B+Enz=P+Q+Enz
(26)
Since the enzyme on both sides of the double arrows is present in equal quantity and identical form, the expression for the equilibrium constant,
[P][Q\[Enz]
_
[AB]
[Enz]
(27)
reduces to
one of the enzyme:
«>
identical to
that for
[PI[Q]
_
“TAIB]
absence
(28)
Enzymes therefore have no effect on K,,.

Raising the temperature increases the rate of both uncatalyzed and enzyme-catalyzed reactions by increasing the kinetic energy and the collision frequency of the reacting molecules. However, heat energy can also increase the conformational flexing of the enzyme to a point that exceeds the energy barrier for disrupting the noncovalent interactions that maintain its three-dimensional structure. The polypeptide chain then begins to unfold, or denature, with an accompanying loss of the catalytic activity. The temperature range over which an enzyme maintains a stable, catalytically competent conformation depends on—and typically moderately exceeds—the normal temperature of the cells in which it resides. Enzymes from humans generally exhibit stability at temperatures up to 45 to 55°C. By contrast, enzymes from the thermophilic microorganisms that reside in volcanic hot springs or undersea hydrothermal vents may be stable at temperatures up to or even above 100°C.
The temperature coefficient (Q_,) is the factor by which the rate of a biologic process increases for a 10°C increase in temperature. For the temperatures over which enzymes are stable, the rates of most biologic processes typically double for a 10°C rise in temperature (Q\, = 2). Changes in the rates of enzymecatalyzed reactions that accompany a rise or fall in body temperature constitute a prominent survival feature for "cold-blooded" life forms such as lizards or fish, whose body temperatures are dictated by the external environment. However, for mammals and other homeothermic organisms, changes in enzyme reaction rates with temperature assume physiologic importance only in circumstances such as fever or hypothermia.

The rate of almost all enzyme-catalyzed reactions exhibits a significant dependence on hydrogen ion concentration. Most intracellular enzymes exhibit optimal activity at pH values between 5 and 9. The relationship of activity to hydrogen ion concentration (Figure 8-3) reflects the balance between enzyme denaturation at high or low pH and effects on the the
reaction in
the
FIGURE 8-3 Effect of pH on enzyme activity. Consider, for example, a negatively charged enzyme (E>) that binds a positively charged substrate (SH*). Shown is the proportion (%) of SH* [\\\] and of E [///) as a function of pH. Only in the cross-hatched area do both the enzyme and the substrate bear an appropriate charge.
charged state of the enzyme, the substrates, or both. For enzymes whose mechanism involves acid-base catalysis, the residues involved must be in the appropriate state of protonation for the reaction to proceed. The binding and recognition of substrate molecules with dissociable groups also typically involves the formation of salt bridges with the enzyme. The most common charged groups are carboxylate groups (negative) and protonated amines (positive). Gain or loss of critical charged groups adversely affects substrate binding and thus will retard or abolish catalysis.

Most measurements of the rates of enzyme-catalyzed reactions employ relatively short time periods, conditions that are considered to approximate initial rate conditions. Under these conditions, only traces of product accumulate, rendering the rate of the reverse reaction negligible. The initial velocity (v,) of the reaction thus is essentially that of the rate of the forward reaction. Assays of enzyme activity almost always employ a large (10°-10°) molar excess of substrate over enzyme. Under these conditions, v, is proportionate to the concentration of enzyme, that is, it is pseudo-first-order with respect to enzyme. Measuring the initial velocity therefore permits one to estimate the quantity of enzyme present in a biologic sample.

In what follows, enzyme reactions are treated as if they had only a single substrate and a single product. For enzymes with multiple substrates, the principles discussed below apply with equal validity. Moreover, by employing pseudo-first-order conditions
=
FIGURE 8-4 Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction.
(see above), scientists can study the dependence of reaction rate on an individual reactant through the appropriate choice of fixed and variable substrates. In other words, under pseudo-firstorder conditions the behavior of a multisubstrate enzyme will imitate one having a single substrate. In this instance, however, the observed rate constant will be a function both of the rate constant k, for the reaction and of the concentration of the fixed substrate.
For a typical enzyme, as substrate concentration is increased, y, increases until it reaches a maximum value V_, (Figure 8-4). When further increases in substrate concentration fail to increase v, the enzyme is said to be "saturated" with the substrate. Note that the shape of the curve that relates activity to substrate concentration (Figure 8-4) is hyperbolic. At any given instant, only substrate molecules that are combined with the enzyme as an enzyme-substrate (ES) complex can be transformed into a product. Since the equilibrium constant for the formation of the enzyme-substrate complex is not infinitely large, only a fraction of the enzyme may be present as an ES complex even when the substrate is present in considerable excess (points A and B of Figure 8-5). At points A or B, increasing or decreasing [S] therefore will increase or decrease the number of ES complexes with a corresponding change in v,. At point C (Figure 8-5), however, essentially all the enzyme is present as the ES complex. Since no free enzyme remains available for forming ES, further increases in [S] cannot increase the rate of the reaction. Under these saturating conditions, v, depends solely on—and thus is limited by—the rapidity with which product dissociates from the enzyme so that it may combine with more substrate.

The Michaelis-Menten equation (29) illustrates in mathematical terms the relationship between initial reaction velocity v, and substrate concentration [S], shown graphically in Figure 8-4:
FIGURE 8-5 Representation of an enzyme in the presence of a concentration of substrate that is below K,, (A), at a concentration equal to K,, (B), and at a concentration well above K_(C). Points A, B, and C correspond to those points in Figure 8-4.
The Michaelis constant K,, is the substrate concentration at which v, is half the maximal velocity (V,, /2) attainable at a particular concentration of the enzyme. K, thus has the dimensions of substrate concentration. The dependence of initial reaction velocity on [S] and K,, may be illustrated by evaluating the Michaelis-Menten equation under three conditions.
- 1. When [S] is much less than K, (point A in Figures 8-4 and 8-5), the term K,, + [S] is essentially equal to K,. Replacing K, + [S] with K,, reduces equation (29) to
A Linear Form of the Michaelis-Menten Equation Is Used to Determine K_, & V, max The direct measurement of the numeric value of V_., and therefore the calculation of K,, often requires impractically high concentrations of substrate to achieve saturating conditions. A linear form of the Michaelis-Menten equation circumvents this difficulty and permits V_. and K,, to be extrapolated from initial velocity data obtained at less than saturating concentrations of the substrate. Start with equation (29),
where ~ means "approximately equal to." Since V_.. and K,, are both constants, their ratio is a constant. In other words, when [S] is considerably below K,, v, is proportionate to k[S]. The initial reaction velocity therefore is directly proportional to [S].
- 2. When [S] is much greater than K, (point C in Figures 8-4 and 8-5), the term K_ + [S] is essentially equal to [S]. Replacing K, + [S] with [S] reduces equation (29) to

Thus, when [S] greatly exceeds K,, the reaction velocity is maximal (V,_,.) and unaffected by further increases in the substrate concentration.
- 3. When [S] = K,, (point B in Figures 8-4 and 8-5):
Equation (32) states that when [S] equals K, , the initial velocity is half-maximal. Equation (32) also reveals that K, is—and may be determined experimentally from—the substrate concentration at which the initial is half-maximal.
velocity
invert factor
and simplify
Equation (35) is the equation for a straight line, y = ax + b, where y = 1/v, and x = 1/[S]. A plot of 1/v, as y as a function of 1/[S] as x therefore gives a straight line whose y intercept is 1/V_,, and whose slope is K,/V,. Such a plot is called a double reciprocal or Lineweaver-Burk plot (Figure 8-6). Setting the y term of equation (36) equal to zero and solving for x reveals that the x intercept is -1/K,:
can be calculated from the slope and intercept, but is per- most calculated from the x intercept.
K,, y haps readily negative
FIGURE 8-6 Double-reciprocal or Lineweaver-Burk plot of 1/v, versus 1/[S] used to evaluate K, and V,__.

The greatest virtue of the Lineweaver-Burk plot resides in the facility with which it can be used to determine the kinetic mechanism of an enzyme inhibitor (see below). However, in using a double-reciprocal plot to determine kinetic constants, it is important to avoid the introduction of bias through the clustering of data at low values of 1/[S]. This bias can be readily avoided in the laboratory as follows. Prepare a solution of substrate whose dilution into an assay will produce the maximum desired concentration of the substrate. Now prepare dilutions of the stock solution by factors of 1:2, 1:3, 1:4, 1:5, etc. Data generated using equal volumes of these dilutions will then fall on the 1/[S] axis at equally spaced intervals of 1, 2, 3, 4, 5, etc. A single-reciprocal plot such as the Eadie-Hofstee (v, vs v/[S]) or Hanes-Woolf ([S]/v, vs [S]) plot can also be used to minimize data clustering.

Several parameters may be used to compare the relative activity of different enzymes or of different preparations of the same enzyme. The activity of impure enzyme preparations typically is expressed as a specific activity (V,. divided by the protein concentration). For a homogeneous enzyme, one may calculate its turnover number (V_,, divided by the moles of enzyme present). However, if the number of active sites present is known, the catalytic activity of a homogeneous enzyme is best expressed as its catalytic constant, k_,, (V,,,, divided by the number of active sites, S,):
Since the units of concentration cancel out, the units of k_,, are reciprocal time.

By what measure should the efficiency of different enzymes, different substrates for a given enzyme, and the efficiency with which an enzyme catalyzes a reaction in the forward and reverse directions be quantified and compared? While the maximum capacity of a given enzyme to convert substrate to product is important, the benefits of a high k_,, can only be realized if K, is sufficiently low. Thus, catalytic efficiency of enzymes is best expressed in terms of the ratio of these two kinetic constants, k_,./K,,.
For certain enzymes, once substrate binds to the active site, it is converted to product and released so rapidly as to render these events effectively instantaneous. For these exceptionally efficient catalysts, the rate-limiting step in catalysis is the formation of the ES complex. Such enzymes are said to be diffusion-limited, or catalytically perfect, since the fastest possible rate of catalysis is determined by the rate at which molecules move or diffuse through the solution. Examples of enzymes for which k_,/K, approaches the diffusion limit of 108-10? M-'s" include triosephosphate isomerase, carbonic anhydrase, acetylcholinesterase, and adenosine deaminase.
In living cells, the assembly of enzymes that catalyze successive reactions into multimeric complexes can circumvent the limitations imposed by diffusion. The geometric relationships of the enzymes in these complexes are such that the substrates and products do not diffuse into the bulk solution until the last step in the sequence of catalytic steps is complete. Fatty acid synthetase extends this concept one step further by covalently attaching the growing substrate fatty acid chain to a biotin tether that rotates from active site to active site within the complex until synthesis of a palmitic acid molecule is complete (see Chapter 23).

The dissociation strate affinity of an enzyme for its substrate is the inverse of the constant K, for dissociation of the enzyme-subcomplex ES:
Stated another way, the smaller the tendency of the enzyme and its substrate to dissociate, the greater the affinity of the enzyme for its substrate. While the Michaelis constant K,, often approximates the dissociation constant K,, this should not be assumed, for it is by no means always the case. For a typical enzyme-catalyzed reaction:
The value of [S] that gives v,= V_, /2 is
Hence, 1/K, only approximates 1/K, under conditions where the association and dissociation of the ES complex are rapid
relative to catalysis. For the many enzyme-catalyzed reactions for which k_, + k, is not approximately equal to k_,, 1/K,, will underestimate 1/K,.

While most enzymes display the simple saturation kinetics depicted in Figure 8-4 and are adequately described by the Michaelis-Menten expression, some enzymes bind their substrates in a cooperative fashion analogous to the binding of oxygen by hemoglobin (see Chapter 6). Cooperative behavior is an exclusive property of multimeric enzymes that bind substrate at multiple sites.
For enzymes that display positive cooperativity in binding the substrate, the shape of the curve that relates changes in v, to changes in [S] is sigmoidal (Figure 8-7). Neither the Michaelis-Menten expression nor its derived plots can be used to evaluate cooperative kinetics. Enzymologists therefore employ a graphic representation of the Hill equation originally derived to describe the cooperative binding of O, by hemoglobin. Equation (44) represents the Hill equation arranged in a form that predicts a straight line, where k' is a complex constant:
Equation (44) states that when [S] is low relative to k', the initial reaction velocity increases as the nth power of [S]. A graph of log v,/(V,,,. — v,) versus log[S] gives a straight line (Figure 8-8). The slope of the line, n, is the Hill coefficient, an empirical parameter whose value is a function of the number, kind, and strength of the interactions of the multiple substratebinding sites on the enzyme. When n = 1, all binding sites behave independently and simple Michaelis-Menten kinetic behavior is observed. If n is greater than 1, the enzyme is said to exhibit positive cooperativity. Binding of substrate to one
FIGURE 8-7 Representation of sigmoid substrate saturation kinetics.
FIGURE 8-8 A graphical representation of a linear form of the Hill equation is used to evaluate Soy the substrate concentration that produces half-maximal velocity, and the degree of cooperativity n.

site then enhances the affinity of the remaining sites to bind additional substrate. The greater the value for n, the higher the degree of cooperativity and the more markedly sigmoidal will be the plot of v, versus [S]. A perpendicular dropped from the point where the y term log v/(V.. — v,) is zero intersects the x-axis at a substrate concentration termed S.., the substrate concentration that results in half-maximal velocity, S_,, thus is analogous to the P,, for oxygen binding to hemoglobin (see Chapter 6).

Inhibitors of the catalytic activities of enzymes provide both pharmacologic agents and research tools for the study of the mechanism of enzyme action. The strength of the interaction between an inhibitor and an enzyme depends on forces important in protein structure and ligand binding (hydrogen bonds, electrostatic interactions, hydrophobic interactions, and van der Waals forces; see Chapter 5). Inhibitors can be classified on the basis of their site of action on the enzyme, on whether they chemically modify the enzyme, or on the kinetic parameters they influence. Compounds that mimic the transition state of an enzyme-catalyzed reaction (transition state analogs) or that take advantage of the catalytic machinery of an enzyme (mechanism-based inhibitors) can be particularly potent inhibitors. Kinetically, we distinguish two classes of inhibitors based on whether raising the substrate concentration does or does not overcome the inhibition.

The effects of competitive inhibitors can be overcome by raising the concentration of substrate. Most frequently, in competitive inhibition, the inhibitor (I) binds to the substrate-binding

portion of the active site thereby blocking access by the substrate. The structures of most classic competitive inhibitors therefore tend to resemble the structure of a substrate, and thus are termed substrate analogs. Inhibition of the enzyme succinate dehydrogenase by malonate illustrates competitive inhibition by a substrate analog. Succinate dehydrogenase catalyzes the removal of one hydrogen atom from each of the two methylene carbons of succinate (Figure 8-9). Both succinate and its structural analog malonate (OQOC—CH,—COO ) can bind to the active site of succinate dehydrogenase, forming an ES or an EI complex, respectively. However, since malonate contains only one methylene carbon, it cannot undergo dehydrogenation.
The formation and dissociation of the EI complex is a dynamic process described by
for which the equilibrium constant K, is
In effect, a competitive inhibitor acts by decreasing the number of free enzyme molecules available to bind substrate, that is, to form ES, and thus eventually to form product, as described below.
A competitive inhibitor and substrate exert reciprocal effects on the concentration of the EI and ES complexes. Since the formation of ES complexes removes free enzyme available to combine with the inhibitor, increasing [S] decreases the concentration of the EI complex and raises the reaction velocity. The extent to which [S] must be increased to completely overcome the inhibition depends on the concentration of the inhibitor present, its affinity for the enzyme (K,), and the affin- ity, K,, of the enzyme for its substrate.

Double-reciprocal plots typically are used both to distinguish between competitive and noncompetitive inhibitors and to simplify evaluation of inhibition constants. v, is determined at several substrate concentrations both in the presence and in the absence of the inhibitor. For classic competitive inhibition, the lines that connect the experimental data point converge at the y-axis (Figure 8-10). Since the y intercept is equal to
=
FIGURE 8-10 Lineweaver-Burk plot of simple competitive inhibition. Note the complete relief of inhibition at high [S] (ie, low 1/[S]).
1/V,» this pattern indicates that when 1/[S] approaches 0, v, is independent of the presence of inhibitor. Note, however, that the intercept on the x-axis does vary with inhibitor concentration and that, since -1/K',, is smaller than -1/K,, K',, (the "apparent K,") becomes larger in the presence of increasing concentrations of the inhibitor. Thus, a competitive inhibitor has no effect on V, but raises K',, the apparent K, for the substrate. For a simple competitive inhibition, the intercept on the x-axis is
Once K,, has been determined in the absence of inhibitor, K, can be calculated from equation (47). K, values are used to compare different inhibitors of the same enzyme. The lower the value for K, the more effective the inhibitor. For example, the statin drugs that act as competitive inhibitors of 3-hydroxy3-methylglutaryl coenzyme A (HMG-CoA) reductase (see Chapter 26) have K, values several orders of magnitude lower than the K,, for the substrate, HMG-CoA.

In strict noncompetitive inhibition, binding of the inhibitor does not affect binding of the substrate. Formation of both EI and enzyme inhibitor substrate (EIS) complexes is therefore possible. However, while the enzyme-inhibitor complex can still bind the substrate, its efficiency at transforming substrate to product, reflected by V,., is decreased. Noncompetitive inhibitors bind enzymes at sites distinct from the substratebinding site and generally bear little or no structural resemblance to the substrate.
For simple noncompetitive inhibition, E and EI possess identical affinity for the substrate, and the EIS complex generates product at a negligible rate (Figure 8-11). More complex noncompetitive inhibition occurs when binding of the inhibitor does affect the apparent affinity of the enzyme for
FIGURE 8-11 Lineweaver-Burk plot for simple noncompetitive inhibition.
the substrate, causing the lines to intercept in either the third or fourth quadrants of a double-reciprocal plot (not shown). While certain inhibitors exhibit characteristics of a mixture of competitive and noncompetitive inhibition, the evaluation of these inhibitors exceeds the scope of this chapter.

A Dixon plot is sometimes employed as an alternative to the Lineweaver-Burk plot for determining inhibition constants. The initial velocity (v,) is measured at several concentrations of the inhibitor, but at a fixed concentration of the substrate (S). For a simple competitive or noncompetitive inhibitor, a plot of 1/v, versus inhibitor concentration [I] yields a straight line. The experiment is repeated at different fixed concentrations of the substrate. The resulting set of lines intersects to the left of the y-axis. For competitive inhibition, a perpendicular dropped to the x-axis from the point of intersection of the lines gives —K, (Figure 8-12, top). For noncompetitive inhibition, the intercept on the x-axis is —K, (Figure 8-12, bottom). Pharmaceutical publications frequently employ Dixon plots to illustrate the comparative potency of competitive inhibitors.
A less rigorous alternative to K, as a measure of inhibitory potency is the concentration of inhibitor that produces 50% inhibition, IC,,. Unlike the equilibrium dissociation constant K, the numeric value of IC,, varies as a function of the specific circumstances of substrate concentration, etc under which it is determined.

Some inhibitors bind to enzymes with such high affinity, K, < 10" M, that the concentration of inhibitor required to measure K, falls below the concentration of enzyme typically present in an assay. Under these circumstances, a significant fraction of the total inhibitor may be present as an EI complex. If so, this violates the assumption, implicit in classical steady-state kinetics, that the concentration of free inhibitor is independent of the concentration of enzyme. The kinetic analysis of these
FIGURE 8-12 Applications of Dixon plots. Top: Competitive inhibition, estimation of K, Bottom: Noncompetitive inhibition, estimation of K, tightly bound inhibitors requires specialized kinetic equations that incorporate the concentration of enzyme to estimate K, or IC,, and to distinguish competitive from noncompetitive tightly bound inhibitors.

In the above examples, the inhibitors form a dissociable, dynamic complex with the enzyme. Fully active enzyme can therefore be recovered simply by removing the inhibitor from the surrounding medium. However, a variety of other inhibitors act irreversibly by chemically modifying the enzyme. These modifications generally involve making or breaking covalent bonds with aminoacyl residues essential for substrate binding, catalysis, or maintenance of the enzyme's functional conformation. Since these covalent changes are relatively stable, an enzyme that has been "poisoned" by an irreversible inhibitor such as a heavy metal atom or an acylating reagent remains inhibited even after the removal of the remaining inhibitor from the surrounding medium.

"Mechanism-based" or "suicide" inhibitors are specialized substrate analogs that contain a chemical group that can be transformed by the catalytic machinery of the target enzyme. After binding to the active site, catalysis by the enzyme generates a highly reactive group that forms a covalent bond to and blocks the function of a catalytically essential residue. The specificity and persistence of suicide inhibitors, which are both enzyme-specific and unreactive outside the confines of the enzyme's active site, render them promising leads for the
development of enzyme-specific drugs. The kinetic analysis of suicide inhibitors lies beyond the scope of this chapter. Neither the Lineweaver-Burk nor the Dixon approach is applicable since suicide inhibitors violate a key boundary condition common to both approaches, namely that the activity of the enzyme does not decrease during the course of the assay.

While several enzymes have a single substrate, many others have two—and sometimes more—substrates and products. The fundamental principles discussed above, while illustrated for single-substrate enzymes, apply also to multisubstrate enzymes. The mathematical expressions used to evaluate multisubstrate reactions are, however, complex. While a detailed analysis of the full range of multisubstrate reactions exceeds the scope of this chapter, some common types of kinetic behavior for two-substrate, two-product reactions (termed "Bi-Bi'" reactions) are considered below.

In sequential reactions, both substrates must combine with the enzyme to form a ternary complex before catalysis can proceed (Figure 8-13, top). Sequential reactions are sometimes referred to as single-displacement reactions because the group undergoing transfer is usually passed directly, in a single step, from one substrate to the other. Sequential Bi-Bi reactions can be further distinguished on the basis of whether the
FIGURE 8-13 Representations of three classes of Bi-Bi reaction mechanisms. Horizontal lines represent the enzyme. Arrows indicate the addition of substrates and departure of products. Top: an ordered Bi-Bi reaction, characteristic of many NAD(P)H-dependent oxidoreductases. Center: a random Bi-Bi reaction, characteristic of many kinases and some dehydrogenases. Bottom: a ping-pong reaction, characteristic of aminotransferases and serine proteases.
=
two substrates add in a random or in a compulsory order. For random-order reactions, either substrate A or substrate B may combine first with the enzyme to form an EA or an EB complex (Figure 8-13, center). For compulsory-order reactions, A must first combine with E before B can combine with the EA complex. One explanation for why some enzymes follow a compulsory-order mechanism can be found in Koshland's induced fit hypothesis: the addition of A induces a conformational change in the enzyme that aligns residues that recognize and bind B.

The term "ping-pong" applies to mechanisms in which one or more products are released from the enzyme before all the substrates have been added. Ping-pong reactions involve covalent catalysis and a transient, modified form of the enzyme (see Figure 7-4). Ping-pong Bi-Bi reactions are often referred to as double displacement reactions. The group undergoing transfer is first displaced from substrate A by the enzyme to form product P and a modified form of the enzyme (F). The subsequent group transfer from F to the second substrate B, forming product Q and regenerating E, constitutes the second displacement (Figure 8-13, bottom).

Most Bi-Bi reactions conform to a somewhat more complex form of Michaelis-Menten kinetics in which V,,. refers to the reaction rate attained when both substrates are present at saturating levels. Each substrate has its own characteristic K,, value, which corresponds to the concentration that yields half-maximal velocity when the second substrate is present at saturating levels. As for single-substrate reactions, doublereciprocal plots can be used to determine V_, and K,. v, is measured as a function of the concentration of one substrate (the variable substrate) while the concentration of the other substrate (the fixed substrate) is maintained constant. If the lines obtained for several fixed-substrate concentrations are plotted on the same graph, it is possible to distinguish a pingpong mechanism, which yields parallel lines (Figure 8-14), from a sequential mechanism, which yields a pattern of intersecting lines (not shown).
Product inhibition studies are used to complement kinetic analyses and to distinguish between ordered and random Bi-Bi reactions. For example, in a random-order Bi-Bi reaction, each product will act as a competitive inhibitor in the absence of its coproducts regardless of which substrate is designated the variable substrate. However, for a sequential mechanism (Figure 8-13, top), only product Q will give the pattern indicative of competitive inhibition when A is the variable substrate, while only product P will produce this pattern with B as the variable substrate. The other combinations of product inhibitor and variable substrate will produce forms of complex noncompetitive inhibition.
1
[Si]
FIGURE 8-14 Lineweaver-Burk plot for a two-substrate ping-pong reaction. Increasing the concentration of one substrate (S,) while maintaining that of the other substrate (S,) constant alters both the x and y intercepts, but not the slope.

The goal of pharmacology is to identify agents that can:
1. Destroy or impair the growth, invasiveness, or development of invading pathogens.
2. Stimulate endogenous defense mechanisms.
3. Halt or impede aberrant molecular processes triggered by genetic, environmental, or biologic stimuli with minimal perturbation of the host's normal cellular functions.
By virtue of their diverse physiologic roles and high degree of substrate selectivity, enzymes constitute natural targets for the development of pharmacologic agents that are both potent and specific. Statin drugs, for example, lower cholesterol production by inhibiting the enzyme HMG-CoA reductase (see Chapter 26), while emtricitabine and tenofovir disoproxil fumarate block replication of the human immunodeficiency virus by inhibiting the viral reverse transcriptase (see Chapter 34). Pharmacologic treatment of hypertension often includes the administration of an inhibitor of angiotensinconverting enzyme, thus lowering the level of angiotensin II, a vasoconstrictor (see Chapter 42).

Enzyme kinetics plays a crucial role in drug discovery. Knowledge of the kinetic behavior of the enzyme of interest is necessary, first and foremost, to select appropriate assay conditions for detecting the presence of an inhibitor. The concentration of substrate, for example, must be adjusted such that sufficient product is generated to permit facile detection of the enzyme's activity without being so high that it masks the presence of an inhibitor. Second, enzyme kinetics provides the means for quantifying and comparing the potency of different inhibitors and defining their mode of action. Noncompetitive inhibitors are particularly desirable, because—by contrast to competitive inhibitors—their effects can never be completely overcome by increases in substrate concentration.

Drug development often involves more than the kinetic evaluation of the interaction of inhibitors with the target enzyme. In order to minimize its effective dosage, and hence the potential for deleterious side effects, a drug needs to be resistant to degradation by enzymes present in the patient or pathogen, a process termed drug metabolism. For example, penicillin and other B-lactam antibiotics block cell wall synthesis in bacteria by irreversibly inactivating the enzyme alanyl alanine carboxypeptidase-transpeptidase. Many bacteria, however, produce B-lactamases that hydrolyze the critical B-lactam function in penicillin and related drugs. One strategy for overcoming the resulting antibiotic resistance is to simultaneously administer a B-lactamase inhibitor with a B-lactam antibiotic.
Metabolic transformation is sometimes required to convert an inactive drug precursor, or prodrug, into its biologically active form (see Chapter 47). 2'-Deoxy-5-fluorouridylic acid, a potent inhibitor of thymidylate synthase, a common target of cancer chemotherapy, is produced from 5-fluorouracil via a series of enzymatic transformations catalyzed by a phosphoribosyl transferase and the enzymes of the deoxyribonucleoside salvage pathway (see Chapter 33). The effective design and administration of prodrugs requires knowledge of the kinetics and mechanisms of the enzymes responsible for transforming them into their biologically active forms.

™ The study of enzyme kinetics—the factors that affect the rates of enzyme-catalyzed reactions—reveals the individual steps by which enzymes transform substrates into products.
= AG, the overall change in free energy for a reaction, is independent of reaction mechanism and provides no information concerning rates of reactions.
a Ky a ratio of reaction rate constants, may be calculated from
the
concentrations
of
and
products
at
equilibrium
or
from
the
ratio
k,/k_,.
Enzymes
do
not
affect
K,,.
™ Reactions proceed via transition states, for whose formation the activation energy is referred to as AG,, Temperature, hydrogen ion concentration, enzyme concentration, substrate concentration, and inhibitors all affect the rates of enzyme- catalyzed reactions.
Measurement of the rate of an enzyme-catalyzed reaction generally employs initial rate conditions, for which the virtual absence of product effectively precludes the reverse reaction from taking place.
Linear forms of the Michaelis-Menten equation simplify
determination of
K, and
V,
,..
A linear form of the Hill equation is used to evaluate the cooperative substrate-binding kinetics exhibited by some multimeric enzymes. The slope n, the Hill coefficient, reflects the number, nature, and strength of the interactions of the substrate-binding sites. A value of n greater than 1 indicates positive cooperativity.
The effects of simple competitive inhibitors, which typically resemble substrates, are overcome by raising the concentration of the substrate. Simple noncompetitive inhibitors lower V, but do not affect K,,.
For simple competitive and noncompetitive inhibitors, the inhibitory constant K, is equal to the dissociation constant for the relevant enzyme-inhibitor complex. A simpler and less rigorous term widely used in pharmaceutical publications for evaluating the effectiveness of an inhibitor is IC,,, the concentration of inhibitor that produces 50% inhibition under the circumstances of an
particular experiment.
Substrates may add in a random order (either substrate may combine first with the enzyme) or in a compulsory order (substrate A must bind before substrate B).
=
™ In ping-pong reactions, one or more products are released from the enzyme before all the substrates have been added.

™ Applied enzyme kinetics facilitate the identification, characterization, and elucidation of the mode of action of drugs that selectively inhibit specific enzymes.
™ Enzyme kinetics plays a central role in the analysis and
optimization
of
drug
a
key
of
drug
efficacy.

A
PT
ER
- Explain the concept of whole-body homeostasis.
be close to K_. Discuss why the cellular concentrations of substrates for most enzymes tend to
List multiple mechanisms by which active control of metabolite flux is achieved.
State the advantages of synthesizing certain enzymes as proenzymes.
to its active form. Describe typical structural changes that accompany conversion of a proenzyme
activity. = Indicate two general ways in which an allosteric effector can influence catalytic
= Outline the roles of protein kinases, protein phosphatases, and of regulatory
and hormonal
and second
messengers in
regulating metabolic
= Explain how the substrate requirements of lysine acetyltransferases and sirtuins can trigger shifts in the degree of lysine acetylation of metabolic enzymes.
= Describe two ways by which regulatory networks can be constructed in cells.

The 19th-century physiologist Claude Bernard enunciated the conceptual basis for metabolic regulation. He observed that living organisms respond in ways that are both quantitatively and temporally appropriate to permit them to survive the multiple challenges posed by changes in their external and internal environments. Walter Cannon subsequently coined the term "homeostasis" to describe the ability of animals to maintain a constant internal environment despite changes in their external surroundings. At the cellular level, homeostasis is maintained by adjusting the rates of key metabolic reactions in response to internal changes. Examples include the levels of key metabolic intermediates such as 5'-AMP and NAD*, or external factors such as hormones acting through receptorcontrolled signal transduction cascades.

Perturbations of the sensor-response machinery responsible for maintaining homeostatic balance can be deleterious to human health. Cancer, diabetes, cystic fibrosis, and Alzheimer disease, for example, are all characterized by regulatory dysfunctions triggered by the interplay between pathogenic agents, genetic mutations, nutritional inputs, and lifestyle practices. For example, many oncogenic viruses contribute to the initiation of cancer by elaborating protein-tyrosine kinases that modify proteins responsible for controlling patterns of gene expression. The cholera toxin produced by Vibrio cholerae disables sensor-response pathways in intestinal epithelial cells by catalyzing the addition of ADP-ribose to the GTP-binding proteins (G-proteins) that link cell-surface receptors to adenylyl cyclase. The ADP-ribose induced activation of the cyclase leads to the unrestricted flow of water into the intestines, resulting in massive diarrhea and dehydration. Yersinia pestis, the causative agent of plague, elaborates a protein-tyrosine phosphatase that hydrolyzes phosphoryl groups on key cytoskeletal proteins, thereby disabling the phagocytic machinery of protective macrophages. Dysfunctions in the proteolytic systems responsible for the degradation of defective or abnormal proteins are believed to play a role in neurodegenerative diseases such as Alzheimer and Parkinson. In addition to their immediate function as regulators of enzyme activity, protein degradation, etc, covalent modifications such as phosphorylation, acetylation, and ubiquitination provide a protein-based code for the storage and transmission of information (see Chapter 35). Such DNA-independent hereditary information is referred to as epigenetic. Knowledge of factors that control the rates of enzyme-catalyzed
C
H
reactions thus is essential to an understanding of the molecular basis of disease and its transmission. This chapter outlines the mechanisms by which metabolic processes are controlled, and provides illustrative examples. Subsequent chapters provide additional examples.

Enzymes that operate at their maximal rate cannot increase throughput to accommodate surges in substrate availability, and will reduce their output of product only if substrate concentration decreases precipitously. The K,, values for most enzymes, therefore, tend to be close to the average intracellular concentration of their substrates, so that changes in substrate concentration generate corresponding changes in metabolite flux (Figure 9-1). Responses to changes in substrate level represent an important but passive means for coordinating metabolite flow. The mechanisms that regulate enzyme efficiency in an active manner in response to internal and external signals are discussed below.

Despite the existence of short-term oscillations in metabolite concentrations and enzyme levels, living cells exist ina dynamic steady state in which the mean concentrations of metabolic intermediates remain relatively constant over time. While all chemical reactions are to some extent reversible, in living cells the reaction products of one enzyme-catalyzed reaction serve as substrates for, and are removed by, other enzyme-catalyzed reactions (Figure 9-2). Under these circumstances many nominally reversible reactions occur unidirectionally. This succession of coupled enzyme-catalyzed reactions is accompanied by an overall change in free energy that favors unidirectional metabolite flow analogous to the flow of water through a pipe in which one end is lower than the other. Flow of water through the pipe remains unidirectional despite the presence
FIGURE 9-1 Differential response of the rate of an enzyme-catalyzed reaction, AV, to the same incremental change in substrate concentration at a substrate concentration close to K,, (AV,) or far above K,, (AV,).
FIGURE 9-2 An idealized cell in steady state. Note that
metabolite flow is unidirectional.
of bends or kinks, which simulate steps with a small or even an unfavorable change in free energy, due to the overall change in height, which corresponds to the pathway's overall change in free energy (Figure 9-3).

In eukaryotes, the anabolic and catabolic pathways that synthesize and break down common biomolecules often are physically separated from one another. Certain metabolic pathways reside only within specialized cell types or inside separate subcellular compartments. For example, fatty acid biosynthesis occurs in the cytosol, whereas fatty acid oxidation takes place within mitochondria (see Chapters 22 and 23), while many degradative enzymes are contained inside organelles called lysosomes. In addition, apparently antagonistic pathways can coexist in the absence of physical barriers provided that thermodynamics dictates that each proceeds with the formation of one or more unique intermediates. For any reaction or series of reactions, the change in free energy that takes place when metabolite flow proceeds in the "forward" direction is equal in magnitude but opposite in sign from that required to proceed in the "reverse" direction. Some enzymes within these pathways catalyze reactions, such as isomerizations, for which the difference in free energy between substrates and products
FIGURE 9-3 Hydrostatic analogy for a pathway with a ratelimiting step (A) and a step with a AG value near 0 (B).
is close to zero. These catalysts act bidirectionally, depending on the ratio of substrates to products. However, virtually all metabolic pathways possess one or more steps for which AG is significant. For example, glycolysis, the breakdown of glucose to form two molecules of pyruvate, has a favorable overall AG of -96 kJ/mol, a value much too large to simply operate in "reverse" in order to convert excess pyruvate to glucose. Consequently, gluconeogenesis proceeds via a pathway in which the three most energetically disfavored steps in glycolysis are circumvented using alternative, thermodynamically favorable reactions distinct (see 19).
catalyzed by enzymes Chapter The ability of enzymes to discriminate between the structurally similar coenzymes NAD* and NADP* also results in a form of compartmentation. The reduction potentials of both coenzymes are similar. However, most of the reactions that generate electrons destined for the electron transport chain reduce NAD*, while enzymes that catalyze the reductive steps in many biosynthetic pathways generally use NADPH as the electron donor.

While the flux of metabolites through metabolic pathways involves catalysis by numerous enzymes, active control of homeostasis is achieved by the regulation of only a select subset of these enzymes. The ideal enzyme for regulatory intervention is one whose quantity or catalytic efficiency dictates that the reaction it catalyzes is slow relative to all others in the pathway. Decreasing the catalytic efficiency or the quantity of the catalyst participating in the "bottleneck" or rate-limiting reaction will immediately reduce metabolite flux through the entire pathway. Conversely, an increase in either its quantity or catalytic efficiency will elicit an increase in flux through the pathway as a whole. As natural "governors" of metabolic flux, the enzymes that catalyze rate-limiting steps also constitute promising drug targets. For example, "statin" drugs curtail synthesis of cholesterol by inhibiting HMG-CoA reductase, catalyst of the rate-limiting reaction of cholesterogenesis.

The overall catalytic capacity of the rate-limiting step in a metabolic pathway is the product of the concentration of enzyme molecules and their intrinsic catalytic efficiency. It therefore follows that catalytic capacity can be controlled by changing the quantity of enzyme present, altering its intrinsic catalytic efficiency, or a combination thereof.

By measuring the rates of incorporation and subsequent loss of 'SN-labeled amino acids into proteins, Schoenheimer deduced that proteins exist in a state of "dynamic equilibrium" where they are continuously synthesized and degraded—a process referred to as protein turnover. Even constitutive proteins, those whose aggregate concentrations remain essentially constant over time, are subject to continual turnover. However, the concentrations of many other enzymes are subject to dynamic shifts in response to hormonal, dietary, pathologic, and other factors that may affect the overall rate constants for their synthesis (k,), degradation (k, deg)? or both.

The synthesis of certain enzymes depends on the presence of inducers, typically substrates or structurally related compounds that stimulate the transcription of the gene that encodes them (see Chapters 36 and 37), or transcription factors. Escherichia coli grown on glucose will, for example, only catabolize lactose after addition of a B-galactoside, an inducer that triggers synthesis of a B-galactosidase and a galactoside permease. Inducible enzymes of humans include tryptophan pyrrolase, threonine dehydratase, tyrosine-a-ketoglutarate aminotransferase, enzymes of the urea cycle, HMG-CoA reductase, A-aminolevulinate synthase, and cytochrome P450. Conversely, an excess of a metabolite may curtail synthesis of its cognate enzyme via repression. Both induction and repression involve cis elements, specific DNA sequences located upstream of regulated genes, and trans-acting regulatory proteins. The molecular mechanisms of induction and repression are discussed in Chapter 38. The activity of transcription factors, on the other hand, is controlled by the hormones and other extracellular signals and their corresponding cell receptors. Detailed information on the control of protein synthesis in response to hormonal stimuli can be found in Chapter 42.

In animals many proteins are degraded by the ubiquitinproteasome pathway. Degradation takes place in the 26S proteasome, a large macromolecular complex made up of more than 30 polypeptide subunits arranged in the form of a hollow cylinder. The active sites of its proteolytic subunits face the interior of the cylinder, thus preventing indiscriminate degradation of cellular proteins. Proteins are targeted to the interior of the proteasome by the covalent attachment of one or more molecules of ubiquitin, a small, approximately 8.5-kDa protein that is highly conserved among eukaryotes. "Ubiquitination" is catalyzed by a large family of enzymes called E3 ligases, which attach ubiquitin to the side-chain amino group of lysyl residues on their targets.
The ubiquitin-proteasome pathway is responsible both for the regulated degradation of selected cellular proteins, for example, cyclins (see Chapter 35), and for the removal of defective or aberrant protein species. The key to the versatility and selectivity of the ubiquitin-proteasome system resides in the variety of intracellular E3 ligases and their ability to discriminate between the different physical or conformational
states of target proteins. Thus, the ubiquitin-proteasome pathway can selectively degrade proteins whose physical integrity and functional competency have been compromised by the loss of or damage to a prosthetic group, oxidation of cysteine or histidine residues, partial unfolding, or deamidation of asparagine or glutamine residues (see Chapter 58). Recognition by proteolytic enzymes also can be regulated by covalent modifications such as phosphorylation; binding of substrates or allosteric effectors; or association with membranes, oligonucleotides, or other proteins. Dysfunctions of the ubiquitin-proteasome pathway sometimes contribute to the accumulation and subsequent aggregation of misfolded proteins characteristic of several neurodegenerative diseases.

In humans the induction of protein synthesis is a complex multistep process that typically requires hours to produce significant changes in overall enzyme level. By contrast, changes in intrinsic catalytic efficiency triggered by binding of dissociable ligands (allosteric regulation) or by covalent modification occur within fractions of seconds. Consequently, changes in protein level generally dominate when meeting long-term adaptive requirements, whereas changes in catalytic efficiency are favored for rapid and transient alterations in metabolite flux.

The governing principle of allosteric regulation is that the properties of the enzyme-effector complex differ from those of the separated enzyme and effector. In some cases, the end product of a multistep biosynthetic pathway binds to and inhibits an enzyme catalyzing one of the early steps in that pathway, a process referred to as feedback regulation. In most cases, feedback inhibitors bind to the enzyme that catalyzes the first committed step in a particular biosynthetic sequence. In the following example, the biosynthesis of D from A is catalyzed by enzymes Enz, through Enz,:
In the absence of regulatory control, if the cell no longer needs metabolite D it will continue to accumulate it until equilibrium is reached. The resulting accumulation of C will in turn cause B to accumulate. Feedback inhibition provides an alternative to simply allowing the pathway to back up like a freeway at rush hour by having D bind to Enz, and inhibit the conversion of A to B. In this example, D acts as a negative allosteric effector of Enz,. Generally, a feedback inhibitor such as D binds at an allosteric site, one spatially distinct from the catalytic site of the target enzyme. Feedback inhibitors thus typically bear little or no structural similarity to the substrates, for example, "A," of the enzymes they inhibit. For example, NAD*
FIGURE 9-4 Sites of feedback inhibition in a branched biosynthetic pathway. S, to S, are intermediates in the biosynthesis of end products A to D. Straight arrows represent enzymes catalyzing the indicated conversions. Curved red arrows represent feedback loops and indicate sites of feedback inhibition by specific end products.

and 3-phosphoglycerate, the substrates for 3-phosphoglycerate dehydrogenase, which catalyzes the first committed step in serine biosynthesis, bear no resemblance to the feedback inhibitor serine. In branched biosynthetic pathways, such as those responsible for nucleotide biosynthesis (see Chapter 33), the initial reactions supply intermediates required for the synthesis of multiple end products. Figure 9-4 shows a hypothetical branched biosynthetic pathway in which curved arrows lead from feedback inhibitors to the enzymes whose activity they inhibit. The sequences S, A, S, > B, S, > C, and S, > — D represent linear reaction sequences that are feedback inhibited by their end products. Branch point enzymes thus can be targeted to direct later stages of metabolite flow.
The kinetics of feedback inhibition may be competitive, noncompetitive, partially competitive, or mixed. Layering multiple feedback loops can provide additional fine control. For example, as shown in Figure 9-5, the presence of excess product B decreases the requirement for substrate S,. However, S, is also required for synthesis of A, C, and D. Therefore, for this pathway, excess B curtails synthesis of all four end products, regardless of the need for the other three. To circumvent this potential difficulty, each end product may only partially inhibit catalytic activity. The inhibitory effect of two or more end products in excess may be strictly additive or, alternatively, greater than their individual effect (cooperative feedback inhibition). Alternatively, in the branched pathway responsible for the synthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan in bacteria, multiple isoforms of an enzyme
FIGURE 9-5 Multiple feedback inhibition in a branched biosynthetic pathway. Superimposed on simple feedback loops (dashed red arrows) are multiple feedback loops (solid red arrows) that regulate enzymes common to biosynthesis of several end products.
evolved, each of which is sensitive to a different pathway end product. High levels of any one end product will inhibit catalysis by only a single isoform, reducing, but not eliminating, flux through the shared portion of the pathway.

Aspartate transcarbamoylase (ATCase), the catalyst for the first reaction unique to pyrimidine biosynthesis (see Figure 33-9), is a target of feedback regulation by two nucleotide triphosphates: cytidine triphosphate (CTP) and adenosine triphosphate. CTP, an end product of the pyrimidine biosynthetic pathway, inhibits ATCase, whereas the purine nucleotide ATP activates it. Moreover, high levels of ATP can overcome inhibition by CTP, enabling synthesis of pyrimidine nucleotides to proceed when purine nucleotide levels are elevated.

Jacques Monod proposed the existence of allosteric sites that are physically distinct from the catalytic site. He reasoned that the lack of structural similarity between most feedback inhibitors and the substrate(s) for the enzymes whose activities they regulate indicated that these effectors are not isosteric with a substrate but allosteric ("occupy another space"). Allosteric enzymes thus are those for which catalysis at the active site may be modulated by the presence of effectors at an allosteric site. The existence of spatially distinct active and allosteric sites has since been verified in several enzymes using many lines of evidence. For example, x-ray crystallography revealed that the ATCase of E coli consists of six catalytic subunits and six regulatory subunits, the latter of which bind the nucleotide triphosphates that modulate activity. In general, binding of an allosteric regulator influences catalysis by inducing a conformational change that encompasses the active site.

max To refer to the kinetics of allosteric inhibition as "competitive" or "noncompetitive" with substrate carries misleading mechanistic implications. We refer instead to two classes of allosterically regulated enzymes: K-series and V-series enzymes. For K-series allosteric enzymes, the substrate saturation kinetics is competitive in the sense that K, is raised without an effect on V_. For V-series allosteric enzymes, the allosteric inhibitor lowers V_,. Without affecting the K,. Alterations in K, or V often are the product of conformational changes at the catalytic site induced by binding of the allosteric effector at its site. For a K-series allosteric enzyme, this conformational change may weaken the bonds between substrate and substrate-binding residues. For a V-series allosteric enzyme, the primary effect may be to alter the orientation or charge of catalytic residues, lowering V_,.. Intermediate effects on K, and V., however, may be observed consequent to these conformational changes.

In both mammalian and bacterial cells, some pathway end products "feed back" to control their own synthesis, in many instances by feedback inhibition of an early biosynthetic enzyme. We must, however, distinguish between feedback regulation, a phenomenologic term devoid of mechanistic implications, and feedback inhibition, a mechanism for regulation of enzyme activity. For example, while dietary cholesterol decreases hepatic synthesis of cholesterol, this feedback regulation does not involve feedback inhibition. HMG-CoA reductase, the rate-limiting enzyme of cholesterogenesis, is affected, but cholesterol does not inhibit its activity. Rather, regulation in response to dietary cholesterol involves curtailment by cholesterol or a cholesterol metabolite of the expression of the gene that encodes HMG-CoA reductase (enzyme repression) (see Chapter 26). As mentioned above, ATP, a product of the purine nucleotide pathway, stimulates the synthesis of pyrimidine nucleotides by activating aspartate transcarbamoylase, a process sometimes referred to as "feed forward" regulation.

Nerve impulses and the binding of many hormones to cell surface receptors elicit changes in the rate of enzyme-catalyzed reactions within target cells by inducing the release or synthesis of specialized allosteric effectors called second messengers. The primary, or "first?" messenger is the hormone molecule or nerve impulse. Second messengers include 3', 5'-cAMP, synthesized from ATP by the enzyme adenylyl cyclase in response to the hormone epinephrine, and Ca"*, which is stored inside the endoplasmic reticulum of most cells. Membrane depolarization resulting from a nerve impulse opens a membrane channel that releases calcium ions into the cytoplasm, where they bind to and activate enzymes involved in the regulation of muscle contraction and the mobilization of stored glucose from glycogen to supply the increased energy demands of muscle contraction. Other second messengers include 3',5'cGMP, nitric oxide, and the polyphosphoinositols produced by the hydrolysis of inositol phospholipids by hormone-regulated phospholipases. Specific examples of the participation of second messengers in the regulation of cellular processes can be found in Chapters 18, 42, and 50.

In mammalian cells, a wide range of regulatory covalent modifications occur. Partial proteolysis and phosphorylation, for example, are frequently employed to regulate the catalytic
activity of enzymes. On the other hand, histones and other DNA-binding proteins in chromatin are subject to extensive modification by acetylation, methylation, ADP-ribosylation, as well as phosphorylation. The latter modifications, which modulate the manner in which the proteins within chromatin interact with each other as well as the DNA itself, constitute the basis for the "histone code. The resulting changes in chromatin structure within the region affected can render genes more accessible to the proteins responsible for their transcription, thereby enhancing gene expression or, on a larger scale, facilitating replication of the entire genome (see Chapter 38). On the other hand, changes in chromatin structure that restrict the accessibility of genes to transcription factors, DNA-dependent RNA polymerases, etc, thereby inhibiting transcription, are said to silence gene expression.

The "histone code" represents a classic example of epigenetics, the hereditary transmission of information by a means other than the sequence of nucleotides that comprise the genome. In this instance, the pattern of gene expression within a newly formed "daughter" cell will be determined, in part, by the particular set of histone covalent modifications embodied in the chromatin proteins inherited from the "parental" cell.

Acetylation, ADP-ribosylation, methylation, and phosphorylation are all examples of "reversible" covalent modifications. In this context, reversible refers to the fact that the modified protein can be restored to its original, modification-free state, not the mechanism by which restoration takes place. Thermodynamics dictates that if the enzyme-catalyzed reaction by which the modification was introduced is thermodynamically favorable, simply reversing the process will be rendered impractical by the correspondingly unfavorable free-energy change. The phosphorylation of proteins on seryl, threonyl, or tyrosyl residues, catalyzed by protein kinases, is thermodynamically favored as a consequence of utilizing the highenergy gamma phosphoryl group of ATP. Phosphate groups are removed, not by recombining the phosphate with ADP to form ATP, but by a hydrolytic reaction catalyzed by enzymes called protein phosphatases. Similarly, acetyltransferases employ a high-energy donor substrate, NAD*, while deacetylases catalyze a direct hydrolysis that generates free acetate.

Certain proteins are synthesized as inactive precursor proteins known as proproteins. Selective, or "partial? proteolysis of a proprotein by one or more successive proteolytic "clips" converts it to a form that exhibits the characteristic activity of the mature protein, for example, its catalytic activity.
The proprotein forms of enzymes are termed proenzymes or zymogens. Proteins synthesized as proproteins include the hormone insulin (proprotein = proinsulin), the digestive enzymes pepsin, trypsin, and chymotrypsin (proproteins = pepsinogen, trypsinogen, and chymotrypsinogen, respectively), several factors of the blood clotting and complement cascades (see Chapters 52 and 55), and the connective tissue protein collagen (proprotein = procollagen).
Proteolytic activation of proproteins constitutes a physiologically irreversible modification because reunification of the two portions of a protein produced by hydrolysis of a peptide bond is entropically disfavored. Once a proprotein is activated, it will continue to carry out its catalytic or other functions until it is removed by degradation or some other means. Zymogen activation thus represents a simple and economical, albeit one way, mechanism for restraining the latent activity of a protein until the appropriate circumstances are encountered. It is therefore not surprising that partial proteolysis is employed frequently to regulate proteins that work in the gastrointestinal tract or bloodstream rather than in the interior of cells.

Certain physiologic processes such as digestion, blood clot formation, and tissue remodeling take place on an intermittent, but relatively frequent, basis. Each of these processes makes extensive use of proteases, which are synthesized as catalytically inactive proenzymes to protect tissues from their degradative effects. In pancreatitis, premature activation of digestive proteases such as trypsinogen and chymotrypsinogen leads to autodigestion of healthy tissue rather than ingested proteins. Blood clot formation, clot dissolution, and tissue repair are brought "on line" only in response to pressing physiologic or pathophysiologic need. Zymogens offer a ready, rapidly activated source of clotting proteins when synthesis is insufficiently rapid to respond to a pressing pathophysiologic demand such as the loss of blood (see Chapter 55). However, in order to prevent clot formation from spreading beyond the site of injury, the processes of blood clot formation and dissolution clearly must be temporally coordinated.

Selective proteolysis involves one or more highly specific proteolytic clips that may or may not be accompanied by separation of the resulting peptides, but generally trigger conformational changes. For example, in a-chymotrypsin, the catalytically essential residues His 57 and Asp 102 reside on the B peptide while Ser 195 resides on the C peptide (Figure 9-6). The hydrolysis of key peptide bonds in chymotrypsinogen triggers conformational changes that align these three residues of the charge-relay network (see Figure 7-7), forming the catalytic site. Note also that contact and catalytic residues can
FIGURE 9-6 Two-dimensional representation of the sequence o' f proteolytic events that ultimately result in formation of the catalytic site of chymotrypsin, which includes the Asp102-His57-Ser195 catalytic triad (see Figure 7-7). Successive proteolysis forms prochymotrypsin (pro-CT), m-chymotrypsin (m-Ct), and ultimately a-chymotr' associated by covalent interchain disulfide bonds. ypsin (a-CT), an active protease whose three peptides (A, B, C) remain be located on different peptide chains but still be within bondforming distance of bound substrate.
the protein can be regenerated by hydrolytic removal of phosphoryl groups, a thermodynamically favorable reaction catalyzed by protein phosphatases.

Mammalian proteins are the targets of a wide range of covalent modification processes. Modifications such as prenylation, glycosylation, hydroxylation, and fatty acid acylation introduce unique structural features into newly synthesized proteins that persist for the lifetime of the protein. Some covalent modifications regulate protein function. The most common ones by far are phosphorylation-dephosphorylation and acetylationdeacetylation. Protein kinases phosphorylate proteins by catalyzing transfer of the terminal phosphoryl group of ATP to the hydroxyl groups of seryl, threonyl, or tyrosyl residues, forming O-phosphoseryl, O-phosphothreonyl, or O-phosphotyrosyl residues, respectively (Figure 9-7). The unmodified form of
FIGURE 9-7 Covalent modification of a regulated enzyme by phosphorylation-dephosphorylation of a seryl residue.

A typical mammalian cell possesses thousands of phosphorylated proteins and several hundred protein kinases and protein phosphatases that catalyze their interconversion. The ease of interconversion of enzymes between their phosphoand dephosphoforms accounts, in part, for the frequency with which phosphorylation-dephosphorylation is utilized as a mechanism for regulatory control. Unlike structural modifications, covalent phosphorylation persists only as long as the affected functional properties of the modified protein serve a specific need. Once the need has passed, the enzyme can be converted back to its original form, poised to respond to the next stimulatory event. A second factor underlying the widespread use of protein phosphorylation-dephosphorylation lies in the chemical properties of the phosphoryl group itself. In order to alter an enzyme's functional properties, any modification of its chemical structure must influence the protein's three-dimensional configuration. The high charge density of protein-bound phosphoryl groups, generally -2 at physiologic pH, their propensity to form strong salt bridges with arginyl and lysyl residues, and their high exceptional hydrogen-bonding capacity renders them potent agents for modifying protein structure and function. Phosphorylation generally influences an enzyme' intrinsic catalytic efficiency or other properties by inducing conformational changes. Consequently, the amino acids modified by phosphorylation can be and typically are relatively distant from the catalytic site itself.

Covalent acetylation-deacetylation has long been associated with histones and other nuclear proteins. In recent years, however, proteomic studies have revealed that thousands of other
mammalian proteins are subject to modification by covalent acetylation, including nearly every enzyme in such core metabolic pathways as glycolysis, glycogen synthesis, gluconeogenesis, the tricarboxylic acid cycle, B-oxidation of fatty acids, and the urea cycle. The potential regulatory impact of acetylationdeacetylation has been established for only a handful of these proteins. However, they include many metabolically important enzymes, such as acetyl-CoA synthetase, long-chain acyl-CoA dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, glutamate dehydrogenase, carbamoyl phosphate synthetase, and ornithine transcarbamoylase.
Lysine acetyltransferases catalyze the transfer of the acetyl group of acetyl-CoA to the e-amino groups of lysyl residues, forming N-acetyl lysine. In addition, some proteins, particularly those in the mitochondria, become acetylated by reacting with acetyl-CoA directly, that is, without the intervention of an enzyme catalyst. Acetylation not only increases the steric bulk of the lysine side chain, it transforms a basic and potentially positively charged primary amine into a neutral, nonionizable amide. Two classes of protein deacetylases have been identified: histone deacetylases and sirtuins. Histone deacetylases catalyze the removal by hydrolysis of acetyl groups, regenerating the unmodified form of the protein and acetate as products. Sirtuins, on the other hand, use NAD* as substrate, which yields O-acetyl ADP-ribose and nicotinamide as products in addition to the unmodified protein.

In many respects, the sites of protein phosphorylation, acetylation, and other covalent modifications can be considered another form of allosteric site. However, in this case, the "allosteric ligand" binds covalently to the protein. Phosphorylationdephosphorylation, acetylation-deacetylation, and feedback inhibition provide short-term, readily reversible regulation of metabolite flow in response to specific physiologic signals. All three act independently of changes in gene expression. As with feedback inhibition, protein phosphorylation-dephosphorylation generally targets an early enzyme in a protracted metabolic pathway. Feedback inhibition, however, involves a single protein that is influenced indirectly, if at all, by hormonal or neural signals. By contrast, regulation of mammalian enzymes by phosphorylation-dephosphorylation involves one or more protein kinases and protein phosphatases, and is generally under direct neural and hormonal control.
Acetylation-deacetylation, on the other hand, targets multiple proteins in a pathway. It has been hypothesized that the degree of acetylation of metabolic enzymes is modulated to a large degree by the energy status of the cell. Under this model, the high levels of acetyl-CoA (the substrate for lysine acetyltransferases and the reactant in nonenzymatic lysine acetylation) present in a well-nourished cell would promote lysine acetylation. When nutrients are lacking, acetyl-CoA levels drop and the ratio of NAD*/NADH rises, favoring protein deacetylation.

Protein phosphorylation-dephosphorylation is a highly versatile and selective process. Not all proteins are subject to phosphorylation, and of the many hydroxyl groups on a protein's surface, only one or a small subset are targeted. While the most common protein function affected is an enzyme' catalytic efficiency, phosphorylation can also alter its location within the cell, susceptibility to proteolytic degradation, or responsiveness to regulation by allosteric ligands. While phosphorylation of some increases their catalytic activity, the phosphorylated form of other enzymes may be catalytically inactive (Table 9-1).
Many proteins can be phosphorylated at multiple sites. Others are subject to regulation both by phosphorylation-dephosphorylation and by the binding of allosteric ligands, or by phosphorylation-dephosphorylation and another covalent modification. Phosphorylation-dephosphorylation at any one site can be catalyzed by multiple protein kinases or protein phosphatases. Many protein kinases and most protein phosphatases act on more than one protein and are themselves interconverted between active and inactive forms by the binding of second messengers or by covalent modification by phosphorylation-dephosphorylation.
The interplay between protein kinases and protein phosphatases, between the functional consequences of phosphorylation at different sites, between phosphorylation sites and allosteric sites, or between phosphorylation sites and other sites of covalent modification provides the basis for regulatory networks that integrate multiple inputs to evoke an appropriate coordinated cellular response. In these sophisticated regulatory networks, individual enzymes respond to different internal and environmental signals. For example, if an enzyme can be phosphorylated at a single site by more than one protein kinase, it can be converted from a catalytically efficient to an inefficient (inactive) form, or vice versa, in response to any one of several signals. If the protein kinase is activated in response to a signal different from the signal that activates the protein phosphatase,

Acetyl-CoA carboxylase, Activity State.Low = EP. Acetyl-CoA carboxylase, Activity State.High = E. Glycogen synthase, Activity State.Low = EP. Glycogen synthase, Activity State.High = E. Pyruvate dehydrogenase, Activity State.Low = EP. Pyruvate dehydrogenase, Activity State.High = E. HMG-CoA reductase, Activity State.Low = EP. HMG-CoA reductase, Activity State.High = E. Glycogen phosphorylase, Activity State.Low = E. Glycogen phosphorylase, Activity State.High = EP. Citrate lyase, Activity State.Low = E. Citrate lyase, Activity State.High = EP. Phosphorylase b kinase, Activity State.Low = E. Phosphorylase b kinase, Activity State.High = EP. HMG-CoA reductase kinase, Activity State.Low = E. HMG-CoA reductase kinase, Activity State.High = EP
Abbreviations: E, dephosphoenzyme; EP, phosphoenzyme.
FIGURE 9-8 A simplified representation of the G, to S checkpoint of the eukaryotic cell cycle. The circle shows the various stages in the eukaryotic cell cycle. The genome is replicated during S phase, while the two copies of the genome are segregated and cell division occurs during M phase. Each of these phases is separated by a G, or growth, phase characterized by an increase in cell size and the accumulation of the precursors required for the assembly of the large macromolecular complexes formed during S and M phases.
the phosphoprotein becomes a decision node, whose functional output, generally catalytic activity, reflects its phosphorylation state. This state or degree of phosphorylation is determined by the relative activities of the protein kinase and protein phosphatase, a reflection of the presence and relative strength of the environmental signals that act through each activity.

and the enzymes responsible for their interconversion act, not as isolated "on" and "off" switches, but as binary elements within integrated biomolecular information processing networks.
The ability of many protein kinases and protein phosphatases to target more than one protein provides a means for an environmental signal to coordinately regulate multiple metabolic processes. For example, the enzymes 3-hydroxy3-methylglutaryl-CoA reductase and acetyl-CoA carboxylase— the rate-controlling enzymes for cholesterol and fatty acid biosynthesis, respectively—are phosphorylated and inactivated by the AMP-activated protein kinase. When this protein kinase is activated either through phosphorylation by yet another protein kinase or in response to the binding of its allosteric activator 5'-AMP, the two major pathways responsible for the synthesis of lipids from acetyl-CoA are both inhibited.

Cells carry out a complex array of metabolic processes that must be regulated in response to a broad spectrum of internal and external factors. Hence, interconvertible enzymes
One well-studied example of such a network is the eukaryotic cell cycle that controls cell division. Upon emergence from the G, or quiescent state, the extremely complex process of cell division proceeds through a series of specific phases designated G,, S, G,, and M (Figure 9-8). Elaborate monitoring systems, called checkpoints, assess key indicators of progress to ensure that no phase of the cycle is initiated until the prior phase is complete. Figure 9-8 outlines, in simplified form, the checkpoint that controls the initiation of DNA replication, called S phase. A protein kinase called ATM is associated with the genome. ATM binds to and is activated by regions of chromatin-containing double-stranded breaks in the DNA. Upon activation, one subunit of the activated ATM dimer dissociates and initiates a series, or cascade, of protein phosphorylation-dephosphorylation events mediated by the CHK1 and CHK2 protein kinases, the Cdc25 protein phosphatase, and finally a complex between a cyclin and a cyclin-dependent protein kinase, or Cdk. Activation of the Cdk-cyclin complex blocks the G, to S transition, thus preventing the replication of damaged DNA. Failure at this checkpoint can lead to mutations in DNA that may lead to cancer or other diseases. Each step in the cascade provides a conduit for monitoring additional indicators of cell status prior to entering S phase.

- Homeostasis involves maintaining a relatively constant
intracellular and
intraorgan environment
despite wide
fluctuations in the external environment. This is achieved via appropriate
changes in
the rates
reactions in
physiologic need.
= 'The substrates for most enzymes are usually present at a concentration close to their K,,. This facilitates passive adjustments to the rates of product formation in response to changes in levels of metabolic intermediates.
= Most metabolic control mechanisms target enzymes that catalyze an early, committed, and rate-limiting reaction. Control can be exerted by varying the concentration of the target protein, its functional efficiency, or some combination of the two.
™ Secretion of inactive proenzymes or zymogens facilitates rapid mobilization of activity via partial proteolysis in response to injury or physiologic need while protecting the tissue of origin (eg, autodigestion by proteases).
™ Binding of metabolites and second messengers to sites distinct from the catalytic sites of enzymes triggers conformational changes that alter V_,, or K,.
= Phosphorylation by protein kinases of specific seryl, threonyl, or tyrosyl residues—and subsequent dephosphorylation by protein phosphatases—regulates the activity of many human in to hormonal and neural
enzymes response signals.
- = Numerous metabolic enzymes are modified by the acetylation-deacetylation of lysine residues. The degree of acetylation of these proteins is thought to be modulated by
|
the availability of acetyl-CoA, the acetyl donor substrate for lysine acetyltransferases, and NAD', a substrate for the sirtuin deacetylases.
The capacity
of protein
kinases, protein
phosphatases, lysine
acetylases, and
lysine deacetylases
to target
both multiple
proteins and multiple sites on proteins is key to the formation of
integrated regulatory
networks that
process complex
environmental information
response.

: : eee RU alle) 10
= Explain why essential transition metals are often referred to as micronutrients.
Understand the importance of multivalency to the ability of transition metals to participate in electron transport and oxidation-reduction reactions.
Understand how Lewis and Bronsted-Lowry acids differ.
Define the term complexation as it refers to metal ions.
Provide a rationale for why zinc is a common prosthetic group in enzymes that catalyze hydrolytic reactions.
= List four benefits obtained by incorporating transition metals into
organometallic
complexes
in
vivo.
= Cite examples of the ability of a given transition metal to function as an electron carrier in one protein, an oxygen carrier in another, and a redox catalyst in yet another.
m= Explain how the possession of multiple metal ions enables the metalloenzymes cytochrome oxidase and nitrogenase to catalyze the reduction of molecular oxygen and nitrogen, respectively.
= Describe two mechanisms by which excess levels of transition metals can be harmful to living organisms.
= Provide an operational definition of the term “heavy metal,’ and list three
strategies
for
treating
acute
heavy
metal
= Describe the processes by which Fe, Co, Cu, and Mo are absorbed in the human gastrointestinal tract.
= Describe the metabolic role of sulfite oxidase and the pathology of sulfite
oxidase
deficiency.
= Describe the function of zinc finger motifs and provide an example of their role in metal ion metabolism.

Maintenance of human health and vitality requires the ingestion of trace levels of numerous inorganic elements, among them the transition metals iron (Fe), manganese (Mn), zinc (Zn), cobalt (Co), copper (Cu), nickel (Ni), molybdenum (Mo), vanadium (V), and chromium (Cr). In general, transition metals are sequestered in organometallic complexes within our bodies, enabling their properties to be controlled and directed where needed, and their propensity to promote the generation of harmful reactive oxygen species is minimized. Transition metals are key components of numerous enzymes and
—
=
_
electron transport proteins as well as the oxygen transport proteins hemoglobin and hemocyanin. Zinc finger motifs provide the DNA-binding domains for many transcription factors, while Fe-S clusters are found in many of the enzymes that participate in DNA replication and repair. Nutritionally or genetically induced deficiencies of these metals are associated with a variety of pathologic conditions including pernicious anemia (Fe), Menkes disease (Cu), and sulfite oxidase deficiency (Mo). When ingested in large quantities, most heavy metals, including several of the nutritionally essential transition metals, are highly toxic and nearly all are potentially carcinogenic.

The organic elements oxygen, carbon, hydrogen, nitrogen, sulfur, and phosphorous typically account for slightly more than 97% of the mass of the human body. Calcium, the majority of which is contained in bones, teeth, and cartilage, contributes a further = 2%. The remaining 0.4 to 0.5% is accounted for by numerous inorganic elements (Table 10-1). Many of these are essential for health, albeit in minute quantities, and thus are commonly classified as micronutrients. Examples of physiologically essential micronutrients include iodine, which is required for the synthesis of triand tetraiodothyronine (see Chapter 41); selenium, which is required for the synthesis of the amino acid selenocysteine (see Chapter 27); and vitamins (see Chapter 44). The focus of the current chapter will be on the physiologic roles of the nutritionally essential transition metals iron (Fe), manganese (Mn), zinc (Zn), cobalt (Co), copper (Cu), nickel (Ni), molybdenum (Mo), vanadium (V), and chromium (Cr).

through the addition or donation of electrons, and therefore to function as an electron carrier during oxidation—-reduction (redox) reactions. That transitional metals can also act as acids further expanding their biologic roles.

In addition to serving as electron carriers, the functional capabilities of the nutritionally essential transition metals are enhanced by their ability to act as Lewis acids. Protic (Bronsted-Lowry) acids can donate a proton (H*) to an acceptor with a lone pair of electrons, for example, a primary amine or a molecule of water. Lewis acids, by contrast, are aprotic. Like Ht' ions, Lewis acids possess empty valence orbitals capable of noncovalently associating with or "accepting" a lone pair of electrons from a second, "donor" molecule. The ferrous (Fe'*) iron of myoglobin and hemoglobin act as Lewis bases when they bind oxygen or other diatomic gases such carbon monoxide (see Chapter 8). Divalent Zn** or Mn** can serve as Lewis acids during catalysis by hydrolytic enzymes, specifically by enhancing the nucleophilicity of active-site water molecules.

One common characteristic of metals is their propensity to undergo oxidation, a process in which they donate one or more electrons from their outer or valence shell to an electronegative acceptor species, for example, molecular oxygen. Oxidation of an alkali or alkaline earth metal (Figure 10-1) results in a single ionized species, for example, Na*, K*, Lit, Mg"*, or Ca**. By contrast, the oxidation of transition metals can yield multiple valence states (Table 10-2). This capability enables transition metals to undergo dynamic transitions between valence states
Most heavy metals, a loosely defined term for metallic elements with densities greater than 5 g/cm? or atomic numbers > 20, are toxic. Some well-known examples include arsenic, antimony, lead, mercury, and cadmium, whose toxicity can arise via a number of mechanisms.

The ability of a heavy metal to displace a functionally essential metal can readily lead to loss or impairment of function. Classic

Potassium, Mass = 20 ng. Potassium, Essential = -. Sodium, Mass = 100 g. Sodium, Essential = +. Sodium, Element = Cobalt. Sodium, Mass = 3 μg. Sodium, Essential = +. Sodium, Element = Yttrium. Sodium, Mass = 0.6 μg. Sodium, Essential = -. Chlorine, Mass = 95 g. Chlorine, Essential = +. Chlorine, Element = Vanadium. Chlorine, Mass = 0.1 μg. Chlorine, Essential = +. Chlorine, Element = Cerium. Chlorine, Mass = 40 μg. Chlorine, Essential = -. Magnesium, Mass = 19 g. Magnesium, Essential = +. Magnesium, Element = Silicon. Magnesium, Mass = 1.0 mg. Magnesium, Essential = Possibly. Magnesium, Element = Bromine. Magnesium, Mass = 260 μg. Magnesium, Essential = -. Iodine, Mass = 20 μg. Iodine, Essential = +. Iodine, Element = Fluorine*. Iodine, Mass = 2.6 g. Iodine, Essential = -. Iodine, Element = Lead*. Iodine, Mass = 120 μg. Iodine, Essential = -
Data for a 70-kg (150 Ib) human are from Emsley, John, The Elements, 3rd ed. Clarendon Press, Oxford, 1998. Essentiality of chromium is based on its proposed biologic role.
FIGURE 10-1 Periodic table of the elements. Transition metals occupy columns 3 to 11, also labeled 1B to 8B.
examples include the displacement of iron by gallium in the enzymes ribonucleotide reductase and Fe, Cu superoxide dismutase. Ga**, while of similar size and identical charge to Fe**, lacks the multivalence capability of iron. Replacement of Fe** by Ga** therefore renders affected enzymes catalytically inert.

impairment of function. Examples include the inhibition of 6-aminolevulinate synthase by Pb (see Chapter 31) and the inactivation of the pyruvate dehydrogenase complex by arsenic or mercury. In pyruvate dehydrogenase, the heavy metals react with the sulfhydryl on the essential prosthetic group, lipoic acid (see Chapter 18), rather than with a peptidyl cysteine.

Heavy metals readily form adducts with free sulfhydryl groups. If present in proteins, formation of these adducts undermines a protein's structural integrity, with concomitant

Cobalt, Potential Valences = Co -1 , Co 0 , Co + , Co 2+ , Co 3+ , Co 4+. Chromium, Potential Valences = Cr -4 , Cr -2 , Cr - , Cr 0 , Cr + , Cr 2+ , Cr 3+ , Cr 4+ , Cr 5+ , Cr 6+. Copper, Potential Valences = Cu 0 , Cu + , Cu 2+. Iron, Potential Valences = Fe 0 , Fe + , Fe 2+ , Fe 3+ , Fe 4+ , Fe 5+ , Fe 6+. Manganese, Potential Valences = Mn 3- , Mn 2- , Mn - , Mn 0 , Mn + , Mn 2+ , Mn 3+ , Mn 4+ , Mn 5+ , Mn 6+ , Mn 7+. Molybdenum, Potential Valences = Mo 4- , Mo 2- , Mo - , Mo 0 , Mo + , Mo 2+ , Mo 3+ , Mo 4+ , Mo 5+ , Mo 6+. Nickel, Potential Valences = Ni 2- , Ni - , Ni 0 , Ni + , Ni 2+ , Ni 4+. Vanadium, Potential Valences = V - , V 0 , V + , V 2+ , V 3+ , V 4+ , V 5+. Zinc, Potential Valences = Zn 2- , Zn 0 , Zn + , Zn 2+
Shown are the possible valence states for each of the nutritionally essential transition states. Biochemically and physiologically relevant valence states are highlighted in red.

Heavy metals can induce the formation of reactive oxygen species (ROS), which can then damage DNA, membrane lipids, and other biomolecules (see Chapter 58). Oxidative damage to DNA can cause genetic mutations that may lead to cancer or other pathophysiologic conditions. ROS-mediated peroxidation of lipid molecules (see Figure 21-23) can lead to the loss of membrane integrity. The resulting dissipation of action potentials and disruption of various cross-membrane transport processes can be particularly deleterious to neurologic and neuromuscular functions. It has also been reported that rats fed with excessive levels of heavy metals are prone to develop cancerous tumors.

While nutritionally essential, several transition metals are nonetheless harmful if present in the body in excess (Table 10-3). Consequently, higher organisms exert strict control over both the uptake and excretion of transition metal ions. Examples include the hepcidin system for regulation of iron (see Figure 52-8) to avoid its accumulation to damaging levels. These mechanisms can be circumvented to some degree when transition metals enter via inhalation or absorption through the skin or mucous membranes, and can be overwhelmed by ingestion of massive,

Aluminum Manganese Bismuth Molybdenum Calcium Potassium Cesium Rubidium Iron Sodium Lithium Strontium Magnesium, Toxicity.Low = Barium Tin Cerium Ytterbium Germanium Yttrium Gold Rhodium Scandium Terbium. Aluminum Manganese Bismuth Molybdenum Calcium Potassium Cesium Rubidium Iron Sodium Lithium Strontium Magnesium, Toxicity.Medium to High = Antimony Niobium Beryllium Palladium Cadmium Platinum Chromium Selenium Cobalt Thorium Copper Titanium Indium Tungsten Lead Uranium Mercury Vanadium Polonium Zirconium Nickel Zinc
Nutritionally essential transition metals are in red type. US Geological Circular 1133 (1995).
supraphysiologic levels. Typical symptoms of acute metal poisoning by heavy metals or by transitional metals include abdominal pain, vomiting, muscle cramps, confusion, and numbness. Treatments include administration of metal chelating agents, diuretics, or—should kidney function be compromised—hemodialysis.

Chapter 6). Free transition metal ions are vulnerable to oxidation by O, and agents inside the cell. Not only are free transition metal ions vulnerable to nonspecific oxidation, their interaction with oxidizing agents such as O,, NO, and H,O, generally results in the generation of even more reactive ROS (see Figure 58-2). Incorporation into organometallic complexes thus protects both the functionally relevant oxidation state of the transition metal and the potential for generating harmful ROS.

The levels of free transition metals in the body are, under normal circumstances, extremely low. The vast majority are found either associated directly with proteins via the oxygen, nitrogen, and sulfur atoms found on the side chains of amino acids such as aspartate, glutamate, histidine, or cysteine (Figure 10-2); or with other organic moieties such as porphyrin (see Figure 6-1), corrin (see Figure 44-10), or pterins (Figure 10-3). The sequestration of transition metals into organometallic complexes confers multiple advantages that include protection against oxidation, suppression of ROS production, enhancement of solubility, control of reactivity, and assembly of multimetal units (Figure 10-4).

Physiologic function of transition metal-containing cofactors and prosthetic groups is dependent on maintaining a multivalent ion's appropriate oxidation state. For example, the porphyrin ring and proximal and distal histidyl residues of the globin polypeptide chain that complex the Fe** atoms in hemoglobin protects them from oxidation to Fe**-containing methemoglobin, which is incapable of binding to and transporting oxygen (see

In organometallic complexes, the position and identity of the surrounding ligands can modify or tune the redox potential and Lewis acid potency of transition metal ions, thereby optimizing them for specific tasks (Table 10-4). For example, both cytochrome c and myoglobin are small, 12 to 17 kDa, monomeric proteins that contain a single heme iron. While
FIGURE 10-2 Ribbon diagram of a consensus C,H, zinc finger domain. Shown are the bound Zn" (purple) and the R groups of the conserved phenylalanyl (F), leucyl (L) cysteinyl (C), and histidinyl (H) residues with their carbon atoms in green. The polypeptide backbone is shown as a ribbon, with alpha helical portions highlighted in red. The sulfur and nitrogen atoms of the R groups of the cysteinyl and histidyl residues are shown in yellow and blue, respectively.
FIGURE 10-3 Molybdopterin. Shown are the oxidized (left) and reduced (right) forms of molybdopterin.
the iron in myoglobin is optimized by its surroundings, both to bind oxygen and to maintain a constant, Fe', valence state, the iron atom in cytochrome c is optimized to cycle between the +2 and +3 valence states so the protein can carry electrons between complexes III and IV of the electron transport chain. The superoxide dismutases (SODs) illustrate how complexation can adapt different transition metals as catalysts for a common chemical reaction, the disproportionation of H,O, into H,O and O,. Each of the four distinct, nonhomologous SODs contain different transition metals whose atomic symbols are used to designate each family: Fe-SODs, Mn-SODs, Ni-SODs, and Cu, Zn-SODs.
The presence of two Fe and two Cu atoms in cytochrome oxidase enables the complex IV of the electron transport chain to accumulate the four electrons needed to carry out the reduction of oxygen to water. Similarly, the bacterial enzyme nitrogenase employs an 8Fe-7S prosthetic group called the P-cluster and a unique Fe, Mo-cofactor to carry out the eight-electron reduction of atmospheric nitrogen to ammonia.

Iron is one of the most functionally versatile of the physiologically essential transition metals. Both in hemoglobin and myoglobin the heme-bound Fe" iron is used to bind a diatomic gas, O,, for transport and storage, respectively (see Chapter 6). Similarly, in marine invertebrates, the iron present in the diiron center of hemerythrin (Figure 10-5) can bind and transport oxygen. By contrast, the iron atoms contained in the heme groups of the band c-type cytochromes and the Fe-S clusters (see Figure 13-4) and Rieske iron centers (Figure 10-6) of other electron transport chain components transport electrons by cycling between their ferrous (+2) and ferric state (+3).

The iron atoms of metalloproteins participate in catalysis of oxidation—reduction, or redox, reactions. Stearoyl-acyl carrier protein A°-desaturase and type 1 ribonucleotide reductase employ hemerythrin-like diiron centers to catalyze the reduction of carbon-carbon double bonds and an alcohol, respectively, to methylene groups. Methane monooxygenase uses a similar diiron center for the oxidation of methane to methanol. The members of the cytochrome P450 family generate Fe = O**. This is a powerful oxidant that participates in the reduction and neutralization of a broad range of xenobiotics via the two-electron reduction of O,, a complex process during which the heme iron cycles between +2, +3, +4, and +5 oxidation states.

Purple acid phosphatases, bimetallic enzymes containing one atom of iron matched with a second metal, such as Zn, Mn, Mg, or a second Fe, catalyze the hydrolysis of phosphomonoesters. Myeloperoxidase employs heme iron to catalyze the condensation of H,O, with Cl ions to generate hypochlorous

The formation of organometallic complexes also allows multiple metal ions to be assembled together in a single functional unit with capabilities that lie beyond those obtainable with a single transition metal ion. In the plant enzyme urease, which catalyzes the hydrolysis of urea in plants, the presence of two Ni atoms within the active site enables the enzyme to simultaneously polarize electrons in the C-N bond targeted for hydrolysis and to activate the attacking water molecule (Figure 10-4).
FIGURE 10-4 Hydrolysis of urea requires the cooperative influence of two active site Ni atoms. The figure depicts the formation of the transition state intermediate for the hydrolysis of the first C-N bond in urea (red) by the enzyme urease. Note how the Ni atoms chelate a water molecule to form a nucleophilic hydroxide and weaken the C-N bond through Lewis acid interactions with lone pairs of electrons on the O and one of the N atoms of urea.

FIGURE 10-5 Diiron center of the deoxy (left) and oxy (right) forms of hemerythrin. Shown are the side chains of the histidine, glutamate, and aspartate residues responsible for binding the metal ions to the polypeptide.
FIGURE 10-6 Structure of a Rieske iron center. Rieske iron centers are a type of 2Fe-2S cluster in which histidine residues replace two of the cysteine residues that normally bind the prosthetic group to the polypeptide chain.
acid, HOCI, a potent bacteriocide used by macrophages to kill entrapped microorganisms. It recently has been shown that many enzymes involved in DNA replication and repair, including DNA helicase, DNA primase, several DNA polymerases, some glycosylases and endonucleases, and several transcription factors contain Fe-S clusters. While their elimination generally results in a loss of protein function, the role(s) performed by these Fe-S centers remains cryptic. However, since most are located in the DNA binding, rather than the catalytic domains of these proteins, it has been proposed that these Fe-S centers may function as electrochemical detectors for the identification of damaged DNA. Others speculate that these clusters serve as redox-sensitive modulators of catalytic activity or DNA binding, or simply as stabilizers of the threedimensional structure of these proteins.

Humans contain a handful of Mn-containing enzymes, the majority of which are located within the mitochondria. These include isocitrate dehydrogenase from the tricarboxylic acid (TCA) cycle, two key players in nitrogen metabolism: glutamate synthetase and arginase, and the gluconeogenic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase, isopropyl malate synthase, and the mitochondrial isozyme of superoxide dismutase. In most of these enzymes, Mn is present in the +2 oxidation state and is presumed to act as a Lewis acid. By contrast, some bacterial organisms employ Mn in several enzymes responsible for catalyzing redox reactions, where it cycles between the +2 and +3 oxidation states in, for example, Mn-superoxide dismutase (Mn-SOD), Mn-ribonucleotide reductase, and Mn-catalase.

Unlike the divalent (+2) ions of other first-row transition metals (Figure 10-1), the valence shell of Zn** possesses a full set of electrons. As a consequence, Zn** ions do not adopt alternate oxidation states under physiologic conditions, rendering it unsuitable to participate in electron transport processes or as a catalyst for redox reactions. On the other hand, redox inert Zn* ions also pose a minimal risk of generating harmful ROS species. Its unique status among the physiologically essential transition metals renders Zn** an ideal candidate as a ligand for stabilizing the protein conformation. It has been estimated that the human body contains 3000 zinc-containing metalloproteins. The vast majority of these are transcription factors and other DNAand RNA-binding proteins that contain anywhere from one to thirty copies of a Zn**-containing polynucleotide-binding domain known as a zinc finger. Zinc fingers consist of a polypeptide loop whose conformation is stabilized by the interactions between Zn** and lone pairs of electrons donated by the sulfur and nitrogen atoms contained in two conserved cysteine and two conserved histidine residues (see Figure 38-16). Zinc fingers bind polynucleotides with a high degree of site specificity that is conferred, at least in part, by variations in the sequence of amino acids that make up the remainder of the loop. Scientists are working to exploit this combination of small size and binding specificity to construct sequence-specific nucleases for use in genetic engineering and, eventually, gene therapy.
Zn** is also an essential component of several metalloenzymes, including carboxypeptidase A, carbonic anhydrase II, adenosine deaminase, alkaline phosphatase, phospholipase C, leucine aminopeptidase, the cytosolic form of superoxide dismutase, and alcohol dehydrogenase. Zn"* is also a component of the type II B-lactamases used by bacteria to neutralize penicillin and other lactam antibiotics. These metalloenzymes exploit the Lewis acid properties of Zn** to stabilize the development of negatively charged intermediates, polarize the distribution of electrons in carbonyl groups, and enhance the nucleophilicity of water (Figure 10-7).

The predominant and, thus far, only known biochemical function of dietary cobalt is as the core component of 5'-deoxyadenosylcobalamin, otherwise known as vitamin B,, (see Figure 44-10). The Co* in this cofactor resides at the center of a tetrapyrrole corrin ring where it acts as a Lewis base that binds to and facilitates the transfer of one-carbon, methyl or methylene groups. In humans, this includes the enzyme catalyzed transfer of a -CH, group from tetrahydrofolate to homocysteine, the final step in the synthesis of the amino acid methionine (see Figure 44-13), and the rearrangement of methylmalonyl-CoA to form succinyl-CoA during the catabolism of the propionate generated from the metabolism of isoleucine and lipids containing odd numbers of amino acids (see Figure 19-2). During the latter reaction, the Co** is transiently reduced to the 2+ oxidation state by abstracting an electron to generate a reactive methylene radical, R-CH,. More information of Co and vitamin B,, can be found in Chapter 44.

Copper is a functionally essential component of approximately 30 different metalloenzymes in humans, including cytochrome oxidase, dopamine -hydroxylase, tyrosinase, the cytosolic form of superoxide dismutase (Cu, Zn-SOD), and lysyl oxidase. Dopamine B-hydroxylase and tyrosinase are both
FIGURE 10-7 Role of Zn* in catalytic mechanisms of B-lactamase II. Zn is bound to the enzyme via the nitrogen atoms present in the side chains of multiple histidine (H) residues. Left: Zn?* activates a water molecule, one of whose protons is accommodated by aspartic acid (D) residue 120, which (middle) executes a nucleophilic attack on carbonyl C of the lactam ring of the antibiotic. Right: D120 then donates the bound proton to the lactam nitrogen, facilitating the cleavage of the C-N bond in the tetrahedral intermediate.
catecholamine oxidases, enzymes that oxidize the ortho-position in the phenol rings of t-dopamine (see Figure 41-10) and tyrosine, respectively. The former is the final step in the pathway by which epinephrine is synthesized in the adrenal gland, while the latter is the first and rate-limiting step in the synthesis of melanin. Both dopamine B-hydroxylase and tyrosinase are members of the type-3 family of copper proteins, which share a common dicopper center. As shown in Figure 10-8,
_
_
—
FIGURE 10-8 Reaction mechanism of the catecholamine oxidases.
the copper atoms in catecholamine oxidases chelate a molecule of molecular oxygen, activating it for attack on a phenol ring. During this process the copper atoms cycle between the +2 and +1 oxidation states. Another type-3 copper protein is hemocyanin. Unlike the catecholamine oxidases, the dicopper center of hemocyanin serves to transport oxygen in invertebrate animals such as mollusks that lack hemoglobin.

In Cu, Zn-SOD, the Cu** atom in the bimetallic center abstracts an electron from superoxide, O,, an extremely reactive and cytotoxic reactive oxygen species, forming O, and Cu'*. The Cu atom in the enzyme is then restored to its original, +2, valence state by donating an electron to a second molecule of superoxide, generating H,O,. While hydrogen peroxide also is a ROS, it is considerably less reactive than O,, a radical anion. Moreover, it can be subsequently converted to water and O, through the action of a second detoxifying enzyme, catalase (see Chapter 12).
Lysyl oxidase employs a single atom of Cu' to convert the epsilon amino groups on lysine side chains in collagen or elastin to aldehydes using molecular oxygen. The aldehyde groups on the side chain of the resulting amino acid, allysine (2-amino-6-oxo-hexanoic acid), then chemically react with the side chains of other allysine or lysine residues on adjacent polypeptides to generate the chemical crosslinks essential to the exceptional tensile strength of mature collagen and elastin fibers. Another essential feature of the enzyme is the presence of a modified amino acid, 2,4,5-trihydroxyphenylalanine quinone, in the active site. This modification is generated by the autocatalytic oxidation of the side chain of conserved tyrosine residue by lysyl oxidase itself.

Several nickel-containing enzymes are present in bacterial organisms, where they catalyze redox reactions, for example, Ni, Fe hydrogenase and methyl-coenzyme M reductase, transferase reactions, for example, acetyl-CoA synthase, and disproportionation reactions, for example, superoxide dismutase. Ni is a key component of urease, an enzyme found in bacteria, fungi, and plants (Figure 10-4). However, the molecular basis of the dietary requirement for nickel in humans and other mammals has yet to be discovered.

Molybdenum is a key component of the phylogenetically universal cofactor molybdopterin (Figure 10-3). In animals, molybdopterin serves as a catalytically essential prosthetic group for many enzymes, including xanthine oxidase, aldehyde oxidase, and sulfite oxidase. Xanthine oxidase, which also contains flavin, catalyzes the final two oxidative steps in the pathway by which uric acid is synthesized from purine nucleotides: the oxidation of hypoxanthine to xanthine and the oxidation of xanthine to uric acid (see Chapter 33). Catalysis of this two-stage process is facilitated by the ability of the bound Mo atom to cycle among the +4, +5, and +6 valence states. In addition to molybdopterin and flavin, aldehyde oxidase also contains an Fe-S cluster. Its complex suite of prosthetic groups enables the enzyme to oxidize a broad range of substrates, including many heterocyclic organic compounds. It has therefore been suggested that aldehyde oxidase participates, like the cytochrome P450 system, in the detoxification of xenobiotics (see Chapter 47).

The Feand Mo-containing metalloenzyme sulfite oxidase is located in the mitochondria, where it catalyzes the oxidation of the sulfite (SO,*) generated by the catabolism of sulfurcontaining biomolecules to sulfate, SO,"-. As for xanthine oxidase, the ability of the molybdenum ion to transition between the +6, +5, and +4 oxidation states is critical to providing a catalytic route by which the two electrons removed from the sulfite molecule can be sequentially transferred to two molecules of cytochrome c, each of which can carry only a single electron (Figure 10-9). Mutations in any one of three genes— MOCS1, MOCS2, or GPNH—whose protein products catalyze key steps in the synthesis of molybdopterin can lead to sulfite oxidase deficiency. Individuals who suffer from this autosomal inherited inborn error of metabolism are incapable of breaking down the sulfur-containing amino acids cysteine and methionine. The resulting accumulation of these amino acids and their derivatives in neonatal blood and tissues produce severe physical deformities and brain damage that leads to intractable seizures, severe mental retardation, and—in most cases—death during early childhood.

Although nutritionally essential, the role of vanadium in living organisms remains cryptic. No vanadium-containing cofactor has been identified to date. Vanadium is found throughout the body in both its +4, for example, HVO,", H,VO,, etc, and +5 oxidation states, for example, VO**, HVO*, etc. Various plasma proteins are known to bind oxides of vanadium, including albumin, immunoglobulin G, and transferrin. Although vanadate, a phosphate analog, is known to inhibit protein-tyrosine
FIGURE 10-9 Reaction mechanism of sulfite oxidase showing oxidation states of enzyme-bound iron and molybdenum atoms.
phosphatases and alkaline phosphatase in vitro, it is unclear whether these interactions are of physiologic significance.

The role of Cr in humans remains unknown. In the 1950s, a Cr** containing "glucose tolerance factor" was isolated from brewer's yeast whose laboratory effects implicated this transition metal as a cofactor in the regulation of glucose metabolism. However, decades of research have failed to uncover either a Cr-containing biomolecule or a Cr-related genetic disease in animals. Nevertheless, many persons continue to ingest Cr-containing dietary supplements, such as Cr**-picolinate, for its alleged weight-loss properties.

In general, intestinal uptake of most transition metals is relatively inefficient. Only a small portion of the transition metals ingested each day is absorbed into our bodies. In addition, some transition metals, such as Ni, can be readily absorbed through the lungs when present in contaminated air or as a component of cigarette smoke. The perceived "inefficiency" of intestinal absorption may reflect the combination of the human body's modest requirements for these elements and the need to buffer against the accumulation of excessive amounts of these potentially toxic heavy metals. While we understand the pathways by which some transition metals, such as Fe (see Chapter 52), are taken up in great detail, in other instances, little hard evidence has been uncovered.
Fe** is absorbed directly via a transmembrane protein, the divalent metal ion transport protein (DMT-1), in the proximal duodenum. DMT-1 is also postulated to constitute the primary vehicle for the uptake of Mn**, Ni**, and—to a lesser extent—Cu'*. As most of the iron in the stomach is in the ferric state, Fe**, it must be reduced to the ferrous, Fe", state in order to be absorbed. This reaction is catalyzed by a ferric reductase also present on the cell surface, duodenal cytochrome b (Dcytb). Dcytb also is responsible for reducing Cu* to Cu'* prior to transport by the high affinity Cu transport protein Ctrl. Molybdenum and vanadium are absorbed in the gut as the oxyanions vanadate, HVO,", and molybdate, HMoO,", by the same nonspecific anion transporter responsible for the absorption of their structural analogs phosphate, HPO,', and sulfate, SO,*-. Similarly, excess levels of Zn can trigger potentially lethal anemia by inhibiting absorption of a second essential nutrient transition metal, Cu. Cobalt absorbed the organometallic complex cobala- is as min, that is, vitamin B,,, via a dedicated pathway involving two secreted cobalamin-binding proteins, haptocorrin and intrinsic factor, and a cell surface receptor, cubilin. In the stomach, cobalamin released from ingested foods binds to haptocorrin,

which protects the coenzyme from the extreme pH of its surroundings. As the cobalamin-haptocorrin complex moves into the duodenum, the pH increases, inducing dissociation of the complex. The released cobalamin is then bound by a haptocorrin homolog known as intrinsic factor. The resulting cobalamin-intrinsic factor complex is then recognized and internalized by cubilin receptors present on the surface of intestinal epithelial cells.

commonly utilized to increase the nucleophilicity of water by enzymes that catalyze hydrolytic reactions.
@ The Mo atoms in xanthine oxidase and sulfite oxidase cycle between three different valence states during catalysis.
@ The buildup of the S-containing amino acids methionine and cysteine in persons suffering from a deficiency in the Fe, Mo-metalloenzyme sulfite oxidase causes serious developmental defects and death in infancy.
= In humans, the only known function of Co is as a component of 5'-deoxyadenosylcobalamin, vitamin B pa cofactor involved in the transfer of one-carbon groups. Co is absorbed into the body as its vitamin B,, complex.

Ba LA, Doering M, Burkholz T, Jacob C: Metal trafficking: from maintaining the metal homeostasis to future drug design.
Metallomics 2009;1:292.

6. Select the one of the following statements that is NOT CORRECT.
A. As used in biochemistry, the standard state concentration
for products and reactants other than protons is 1 molar.
1.
Rapid
shallow
breathing
can
lead
to
hyperventilation,
a
condition
B. AGis a function of the logarithm of K,,.
wherein carbon dioxide is exhaled from the lungs more rapidly
than
it
is
produced
by
the
tissues.
Explain
how
can lead to an increase in the pH of the blood.
. A protein engineer desires to alter the active site of chymotrypsin
so that it will cleave peptide bonds to the C-terminal side of
aspartyl
and
glutamy]
residues.
The
protein
engineer
will
be
most
likely to succeed if he replaces the hydrophobic amino acid at the bottom of the active site pocket with:
Threonine
Glutamine
Lysine
Proline
BOOS
. Select the one of the following statements that is NOT CORRECT.
A.
Many
mitochondrial
proteins
are
modified
by
the
acetylation
of
the
epsilon
amino
groups
of
lysine
residues.
B. Protein acetylation is an example of a covalent modification
that can be “reversed” under physiologic conditions.
C. Increased levels of acetyl-CoA tend to favor protein acetylation.
D. Acetylation increases the steric bulk of the amino acid side
chains that are subject to this modification.
E. The side chain of an acetylated lysyl residue is a stronger base than that of an unmodified lysyl residue.
A. Acid-base catalysis is a prominent feature of the catalytic
mechanism of the HIV protease.

B. Fischer's lock-and-key model explains the role of transition state stabilization in enzymic catalysis.
C. Hydrolysis of peptide bonds by serine proteases involves the transient formation of a modified enzyme.
D. Many enzymes employ metal ions as prosthetic groups or cofactors.
E. In general, enzymes bind transition state analogs more
tightly than substrate analogs.
CORRECT.
A. To calculate K, , the equilibrium constant for a reaction,
divide the initial rate of the forward reaction (rate 1) by the
initial velocity of the reverse reaction (rate 1).
B. The presence of an enzyme has no effect on K,,.
C. Fora reaction conducted at constant temperature, the
fraction of the potential reactant molecules possessing
sufficient kinetic energy to exceed the activation energy of
the reaction is a constant.
D. Enzymes and other catalysts lower the activation energy of
E. The algebraic sign of AG, the Gibbs free energy change for a reaction, indicates the direction in which a reaction will proceed.
C.
As
used
in
reaction
kinetics,
the
term
“spontaneity”
refers
to
whether the reaction as written is favored to proceed from
left to right.
D. AG? denotes the change in free energy that accompanies
transition from the standard state to equilibrium.
E. Upon reaching equilibrium, the rates of the forward and
reverse reaction both drop to zero.
A. Enzymes lower the activation energy for a reaction.
B. Enzymes often lower the activation energy by destabilizing transition state intermediates.
C. Active site histidyl residues frequently aid catalysis by acting
as proton donors or acceptors.
D. Covalent catalysis is employed by some enzymes to provide
an alternative reaction pathway.
E. The presence of an enzyme has no effect on AG®.
A. For most enzymes, the initial reaction velocity, v,, exhibits a
hyperbolic dependence on [S].

B. When [S] is much lower than K,, the term K,, + [S] in the
Michaelis-Menten equation closely approaches K,. Under these conditions, the rate of catalysis is a linear function of [S].
C. The molar concentrations of substrates and products are
equal
when
the
rate
of
an
enzyme-catalyzed
reaction
reaches
half
of
its
potential
maximum
value
(V,,,./2).
D.
An
enzyme
is
said
to
have
become
saturated
with
substrate
when successively raising [S] fails to produce a significant
increase in v,.
E. When making steady-state rate measurements, the
concentration of substrates should greatly exceed that of the enzyme catalyst.
CORRECT.
A. Certain monomeric enzymes exhibit sigmoidal initial rate
kinetics.
B. The Hill equation is used to perform quantitative analysis of
the
cooperative
behavior
of
enzymes
or
carrier
proteins
such
as hemoglobin or calmodulin.
C. For an enzyme that exhibits cooperative binding of
substrate, a value of n (the Hill coefficient) greater than
unity is said to exhibit positive cooperativity.
D. An enzyme that catalyzes a reaction between two or more
substrates is said to operate by a sequential mechanism if the
substrates must bind in a fixed order.
E. Prosthetic groups enable enzymes to add chemical groups beyond those present on amino acid side chains.
ll. Select the one of the following statements that is NOT CORRECT.
12. Select the one of the following statements that is NOT CORRECT.
13. Select the one of the following statements that is NOT
14. Select the one of the following statements that is NOT CORRECT.
A. IC,, is a simple operational term for expressing the potency
of an inhibitor.
B. Lineweaver-Burk and Dixon plots employ rearranged
versions
of
the
equation
to

generate
linear
representations
of
kinetic
behavior
and
C. A plot of 1/y, versus 1/[S] can be used to evaluate the type and affinity for an inhibitor.
D. Simple noncompetitive inhibitors lower the apparent K,, for a substrate.
E. Noncompetitive inhibitors typically bear little or no
structural resemblance to the substrate(s) of an enzyme-
catalyzed reaction.
A. Fora given enzyme, the intracellular concentrations of its
tend
to
be
close
to
their
K,,
values.
B. The sequestration of certain pathways within intracellular organelles facilitates the task of metabolic regulation.
C. The earliest step in a biochemical pathway where regulatory
control can be efficiently exerted is the first committed step.
D. Feedback regulation refers to the allosteric control of an
early step in a biochemical pathway by the end product(s) of that pathway.
E. Metabolic control is most effective when one of the more
rapid steps in a pathway is targeted for regulation.
A.
The
Bohr
effect
refers
to
the
release
of
protons
that
occurs
when oxygen binds to deoxyhemoglobin.
B. Shortly after birth of a human infant, synthesis of the a-chain undergoes rapid induction until it comprises 50% of the hemoglobin tetramer.
C. The B-chain of fetal hemoglobin is present throughout
gestation.
D. The term thalassemia refers to any genetic defect that results
in partial or total absence of the a- or B-chains of hemoglobin.
E. The taut conformation of hemoglobin is stabilized by several salt that form between the subunits.
bridges
CORRECT.
A. Steric hindrance by histidine E7 plays a critical role in
weakening the affinity of hemoglobin for carbon monoxide
(CO).
B. Carbonic anhydrase plays a critical role in respiration by
virtue of its capacity to break down 2,3-bisphosphoglycerate
in the lungs.
C. Hemoglobin S is distinguished by a genetic mutation that

substitutes Glu6 on the B subunit with Val, creating a sticky
patch on its surface.
D. Oxidation of the heme iron from the +2 to the +3 state
abolishes the ability of hemoglobin to bind oxygen.
E. The functional differences between hemoglobin and
myoglobin reflect, to a large degree, differences in their
quaternary structure.
A. The charge-relay network of trypsin makes the active site
serine a stronger nucleophile.
B. The Michaelis constant is the substrate concentration at
which the rate of the reaction is half-maximal.
15. Select the one of the following statements that is NOT CORRECT.
16. Which of the following is NOT a benefit obtained by
17. Which of the following is NOT a potential function of the
18. Acute heavy metal poisoning can be treated by:
19. Which of the following is the name of a common organometallic
C. During transamination reactions, both substrates are bound to the enzyme before either product is released.
D. Histidine residues act both as acids and as bases during catalysis by an aspartate protease.
E. Many coenzymes and cofactors are derived from vitamins.
A. Interconvertible enzymes fulfill key roles in integrated
regulatory networks.
B. Phosphorylation of an enzyme often alters its catalytic
efficiency.
C. “Second messengers” act as intracellular extensions or
surrogates for hormones and nerve impulses impinging on
cell surface receptors.
D. The ability of protein kinases to catalyze the reverse reaction
that
removes
the
phosphoryl
group
is
key
to
the
versatility
of
this molecular regulatory mechanism.
E. Zymogen activation by partial proteolysis is irreversible
under physiologic conditions.
incorporating physiologically essential transition metal ions into
organometallic complexes?
A. Optimization of Lewis acid potency of the bound metal.
B. Ability to construct complexes containing multiple
transition metal ions.
C. Attenuation of the production of reactive oxygen species.
D. Protection against unwanted oxidation.
E. To render the bound transition metal multivalent.

physiologically essential transition metals?
A. Binding diatomic gas molecules
B. Proton carrier
C. Stabilizing protein conformation
D. Enhancing the nucleophilicity of water
E. Electron carrier
Administration of diuretics >
Ingestion of chelating agents
All of the above
of the above
BOO
DNA-binding motif?
Zinc finger >
Molybdopterin
Fe-S center
BOOP

= State the first and second laws of thermodynamics and understand how they apply to biologic systems.
= Explain what is meant by the terms free energy, entropy, enthalpy, exergonic, and endergonic.
= Appreciate how reactions that are endergonic may be driven by coupling to those that are exergonic in biologic systems.
= Explain the role of transfer potential, adenosine triphosphate (ATP),
group and other nucleotide triphosphates in the transfer of free energy from exergonic to endergonic processes, enabling them to act as the "energy currency" of cells.

Bioenergetics, or biochemical thermodynamics, is the study of the energy changes accompanying biochemical reactions. Biologic systems are essentially isothermic and use chemical energy to power living processes. The way in which an animal obtains suitable fuel from its food to provide this energy is basic to the understanding of normal nutrition and metabolism. Death from starvation occurs when available energy reserves are depleted, and certain forms of malnutrition are associated with energy imbalance (marasmus). Thyroid hormones control the metabolic rate (rate of energy release), and disease results if they malfunction. Excess storage of surplus energy causes obesity, an increasingly common disease of Western society which predisposes to many diseases, including cardiovascular disease and diabetes mellitus type 2, and lowers life expectancy.

Gibbs change in free energy (AG) is that portion of the total energy change in a system that is available for doing work—that is, the useful energy, also known as the chemical potential.

The first law of thermodynamics states that the total energy of a system, including its surroundings, remains constant. It implies that within the total system, energy is neither lost nor gained during any change. However, energy may be transferred from one part of the system to another, or may be transformed into another form of energy. In living systems,
chemical energy may be transformed into heat or into electrical, radiant, or mechanical energy.
The second law of thermodynamics states that the total entropy of a system must increase if a process is to occur spontaneously. Entropy is the extent of disorder or randomness of the system and becomes maximum as equilibrium is approached. Under conditions of constant temperature and pressure, the relationship between the free-energy change (AG) of a reacting system and the change in entropy (AS) is expressed by the following equation, which combines the two laws of thermodynamics:

where AH is the change in enthalpy (heat) and T is the absolute temperature. In biochemical reactions, since AH is approximately equal to the total change in internal energy of the reaction or AE, the above relationship may be expressed in the following way:

If AG is negative, the reaction proceeds spontaneously with loss of free energy, that is, it is exergonic. If, in addition, AG is of great magnitude, the reaction goes virtually to completion and is essentially irreversible. On the other hand, if AG is positive, the reaction proceeds only if free energy can be gained, that is, it is endergonic. If, in addition, the magnitude of AG is great, the system is stable, with little or no tendency for a reaction to occur. If AG is zero, the system is at equilibrium and no net change takes place. When the reactants are present in concentrations of 1.0 mol/L, AG? is the standard free-energy change. For biochemical reactions, a standard state is defined as having a pH of 7.0. The standard free-energy change at this standard state is denoted by AG". The standard free-energy change can be calculated from the equilibrium constant K,|..

where R is the gas constant and T is the absolute temperature (see Chapter 8). It is important to note that the actual AG may be larger or smaller than AG" depending on the concentrations of the various reactants, including the solvent, various ions, and proteins.
In a biochemical system, an enzyme only speeds up the attainment of equilibrium; it never alters the final concentrations of the reactants at equilibrium.

The vital processes—for example, synthetic reactions, muscular contraction, nerve impulse conduction, and active transport—obtain energy by chemical linkage, or coupling, to oxidative reactions. In its simplest form, this type of coupling
FIGURE 11-1 Coupling of an exergonic to an endergonic reaction.
may be represented as shown in Figure 11-1. The conversion of metabolite A to metabolite B occurs with release of free energy and is coupled to another reaction in which free energy is required to convert metabolite C to metabolite D. The terms exergonic and endergonic, rather than the normal chemical terms "exothermic" and "endothermic, are used to indicate that a process is accompanied by loss or gain, respectively, of free energy in any form, not necessarily as heat. In practice, an endergonic process cannot exist independently, but must be a component of a coupled exergonic-endergonic system where the overall net change is exergonic. The exergonic reactions are termed catabolism (generally, the breakdown or oxidation of fuel molecules), whereas the synthetic reactions that build up substances are termed anabolism. The combined catabolic and anabolic processes constitute metabolism. If the reaction shown in Figure 11-1 is to go from left to right, then the overall process must be accompanied by loss of free energy as heat. One possible mechanism of coupling could be envisaged if a common obligatory intermediate (I) took part in both reactions, that is,

Some exergonic and endergonic reactions in biologic systems are coupled in this way. This type of system has a built-in mechanism for biologic control of the rate of oxidative processes since the common obligatory intermediate allows the rate of utilization of the product of the synthetic path (D) to determine by mass action the rate at which A is oxidized. Indeed, these relationships supply a basis for the concept of respiratory control, the process that prevents an organism from burning out of control. An extension of the coupling concept is provided by dehydrogenation reactions, which are coupled to hydrogenations by an intermediate carrier (Figure 11-2). An alternative method of coupling an exergonic to an endergonic process is to synthesize a compound of high-energy potential in the exergonic reaction and to incorporate this
FIGURE 11-2 Coupling of dehydrogenation and hydrogenation reactions by an intermediate carrier.
new compound into the endergonic reaction, thus effecting a transference of free energy from the exergonic to the endergonic pathway. The biologic advantage of this mechanism is that the compound of high potential energy, ~®, unlike I in the previous system, need not be structurally related to A, B, C, or D, allowing © to serve as a transducer of energy from a wide range of exergonic reactions to an equally wide range of endergonic reactions or processes, such as biosyntheses, muscular contraction, nervous excitation, and active transport. In the living cell, the principal high-energy intermediate or carrier compound is ATP (Figure 11-3).

In order to maintain living processes, all organisms must obtain supplies of free energy from their environment. Autotrophic organisms utilize simple exergonic processes; for example, the energy of sunlight (green plants), the reaction Fe** > Fe** (some bacteria). On the other hand, heterotrophic organisms obtain free energy by coupling their metabolism to the breakdown of complex organic molecules in their environment. In all these organisms, ATP plays a central role in the transference of free energy from the exergonic to the endergonic processes. ATP is a nucleotide consisting of the nucleoside adenosine (adenine linked to ribose) and three phosphate groups (see Chapter 32). In its reactions in the cell, it functions as the Mg** complex (Figure 11-3).
The importance of phosphates in intermediary metabolism became evident with the discovery of the role of ATP,
FIGURE 11-3 Adenosine triphosphate (ATP) is shown as the magnesium complex.
adenosine diphosphate (ADP), and inorganic phosphate (P.) in glycolysis (see Chapter 17).

The standard free energy of hydrolysis of a number of biochemically important phosphates is shown in Table 11-1. An estimate of the comparative tendency of each of the phosphate groups to transfer to a suitable acceptor may be obtained from the AG* of hydrolysis at 37°C. This is termed the group transfer potential. The value for the hydrolysis of the terminal phosphate of ATP divides the list into two groups. Lowenergy phosphates, having a low group transfer potential, exemplified by the ester phosphates found in the intermediates of glycolysis, have G" values smaller than that of ATP, while in high-energy phosphates, with a more negative G', the value is higher than that of ATP. The components of this latter group, including ATP, are usually anhydrides (eg, the 1-phosphate of 1,3-bisphosphoglycerate), enol phosphates (eg, phosphoenolpyruvate), and phosphoguanidines (eg, creatine phosphate, arginine phosphate).
The symbol ~®) indicates that the group attached to the bond, on transfer to an appropriate acceptor, results in transfer of the larger quantity of free energy. Thus, ATP has a high group transfer potential, whereas the phosphate in adenosine

′.kcal/mol = -3.3. Glycerol-3-phosphate, Δ G 0 ′.kJ/mol = -9.2. Glycerol-3-phosphate, Δ G 0 ′.kcal/mol = -2.2
Abbreviations: PP, pyrophosphate; P, inorganic orthophosphate.
Note: All values are taken from Jencks WP: Free energies of hydrolysis and decarboxylation. In: Handbook of Biochemistry and Molecular Biology, vol 1. Physical and Chemical Data. Fasman GD (editor). CRC Press, 1976:296-304, except that for PP, which is from Frey PA, Arabshahi A: Standard free-energy change for the hydrolysis of the alpha, beta-phosphoanhydride bridge in ATP. Biochemistry 1995;34:11307. Values differ between investigators, depending on the precise conditions under which the measurements were made.
FIGURE 11-4 Structure of ATP, ADP, and AMP showing the position and the number of high-energy phosphates (~®).
monophosphate (AMP) is of the low-energy type since it is a normal ester link (Figure 11-4). In energy transfer reactions, ATP may be converted to ADP and P. or, in reactions requiring a greater energy input, to AMP + PP, (Table 11-1). The intermediate position of ATP allows it to play an important role in energy transfer. The high free-energy change on hydrolysis of ATP is due to relief of charge repulsion of adjacent negatively charged oxygen atoms and to stabilization of the reaction products, especially phosphate, as resonance hybrids (Figure 11-5). Other "high-energy compounds" are thiol esters involving coenzyme A (eg, acetylCoA), acyl carrier protein, amino acid esters involved in protein synthesis, S-adenosylmethionine (active methionine), uridine diphosphate glucose (UDPGlc), and 5-phosphoribosyl1-pyrophosphate (PRPP).
FIGURE 11-5 The free-energy change on hydrolysis of ATP to ADP.

FIGURE 11-6 Role of ATP/ADP cycle in transfer of highenergy phosphate.

The high group transfer potential of ATP enables it to act as a donor of high-energy phosphate to form those compounds below it in Table 11-1. Likewise, with the necessary enzymes, ADP can accept phosphate groups to form ATP from those compounds above ATP in the table. In effect, an ATP/ADP cycle connects those processes that generate ~®) to those processes that utilize ~® (Figure 11-6), continuously consuming and regenerating ATP. This occurs at a very rapid rate since the total ATP/ADP pool is extremely small and sufficient to maintain an active tissue for only a few seconds.
conservation
There are three major sources of ~® taking part in energy or energy capture:
1. Oxidative phosphorylation is the greatest quantitative source of ~®) in aerobic organisms. ATP is generated in the mitochondrial matrix as O, is reduced to H,O by electrons passing down the respiratory chain (see Chapter 13).
2. Glycolysis. A net formation of two ~@®) results from the formation of lactate from one molecule of glucose, generated in two reactions catalyzed by phosphoglycerate kinase and pyruvate kinase, respectively (see Figure 17-2).
3. The citric acid cycle. One ~© is generated directly in the cycle at the succinate thiokinase step (see Figure 16-3).
Phosphagens act as storage forms of group transfer potential and include creatine phosphate, which occurs in vertebrate skeletal muscle, heart, spermatozoa, and brain, and arginine phosphate, which occurs in invertebrate muscle. When ATP is rapidly being utilized as a source of energy for muscular contraction, phosphagens permit its concentrations to be maintained, but when the ATP/ADP ratio is high, their concentration can increase to act as an energy store (Figure 11-7).
FIGURE 11-7 Transfer of high-energy phosphate between ATP and creatine.
When ATP acts as a phosphate donor to form compounds of lower free energy of hydrolysis (Table 11-1), the phosphate group is invariably converted to one of low energy. For example, the phosphorylation of glycerol to form glycerol-3-phosphate:

Endergonic reactions cannot proceed without an input of free energy. For example, the phosphorylation of glucose to glucose-6-phosphate, the first reaction of glycolysis (see Figure 17-2):
Glucose + P, + Glucose-6-phosphate + H,O
(AG') = +13.8 kJ/mol
(1)
is highly endergonic and cannot proceed under physiologic conditions. Thus, in order to take place, the reaction must be coupled with another—more exergonic—reaction such as the hydrolysis of the terminal phosphate of ATP.
- ATP — ADP + P, (AG® = -30.5 kJ/mol)
(2)
When (1) and (2) are coupled in a reaction catalyzed by hexokinase, phosphorylation of glucose readily proceeds in a highly exergonic reaction that under physiologic conditions is irreversible. Many "activation" reactions follow this pattern.

This enzyme is present in most cells. It catalyzes the following reaction:
ATP + AMP
~«——————->
_
2ADP
Adenylyl kinase is important for the maintenance of energy homeostasis in cells because it allows:
1, The group transfer potential in ADP to be used in the syn- thesis of ATP.
2.
The
AMP
formed
as
a
of
activating
reactions
involving
ATP
to
rephosphorylated
to
ADP.
3. AMP to increase in concentration when ATP becomes depleted so that it is able to act as a metabolic (allosteric) signal to increase the rate of catabolic reactions, which in turn lead to the generation of more ATP (see Chapter 14).

ATP can also be hydrolyzed directly to AMP, with the release of PP, (Table 11-1). This occurs, for example, in the activation of long-chain fatty acids (see Chapter 22).
ATP +CoA* SH + R * COOH
——————>
AMP + PP; + R*® CO — SCoA
This reaction is accompanied by loss of free energy as heat, which ensures that the activation reaction will go to the right, and is further aided by the hydrolytic splitting of PP,, catalyzed by inorganic pyrophosphatase, a reaction that itself has a large AG" of -19.2 kJ/mol. Note that activations via the pyrophosphate pathway result in the loss of two ~® rather than one, as occurs when ADP and P, are formed.
A combination of the above reactions makes it possible for phosphate to be recycled and the adenine nucleotides to interchange (Figure 11-8).
FIGURE 11-8 Phosphate cycles and interchange of adenine nucleotides.

By means of the nucleoside diphosphate (NDP) kinases, UTP, GTP, and CTP can be synthesized from their diphosphates, for example, UDP reacts with ATP to form UTP.
ATP +UDP
~<————»
ADP+UTP
(uridine triphosphate)
All of these triphosphates take part in phosphorylations in the cell. Similarly, specific nucleoside monophosphate (NMP) kinases catalyze the formation of NDP from the corresponding monophosphates.
Thus, Adenylyl kinase is a specialized NMP kinase.

™ Biologic systems use chemical energy to power living processes.
™ Exergonic reactions take place spontaneously with loss of free
energy
(AG
is
negative).
Endergonic
reactions
require
the
gain
of
free
energy
(AG
is
positive)
and
occur
only
when
coupled
to
exergonic reactions.
™ ATP acts as the “energy currency” of the cell, transferring free
energy derived from substances of higher energy potential to
those of lower energy potential.

Haynie D: Biological Thermodynamics. Cambridge University Press,
2008.
Nicholls DG, Ferguson S: Bioenergetics, 4th ed. Academic Press,
2013.

- . Explain the meaning of redox potential and how it can be used to predict the
direction of
flow of
electrons in
biologic systems.
Identify the four classes of enzymes (oxidoreductases) involved in oxidation and reduction reactions. :
= Describe the action of oxidases and provide examples of where they play an important role in metabolism.
m= Indicate the two main functions of dehydrogenases and explain the importance of nicotinamide adenine dinucleotide (NAD)and riboflavin-linked dehydrogenases in metabolic pathways such as glycolysis, the citric acid cycle, and the respiratory chain.
= Identify the two types of enzymes classified as hydroperoxidases; indicate the reactions they catalyze and explain why they are important.
™ Give the two steps of reactions catalyzed by oxygenases and identify the two subgroups of this class of enzymes.
synthesis. m= Appreciate the role of cytochrome P450 in drug detoxification and steroid
= Describe the reaction catalyzed by superoxide dismutase and explain how it protects tissues from oxygen toxicity.

Chemically, oxidation is defined as the removal of electrons and reduction as the gain of electrons. Thus, oxidation of a molecule (the electron donor) is always accompanied by reduction of a second molecule (the electron acceptor). This principle of oxidation-reduction applies equally to biochemical systems and is an important concept underlying understanding of the nature of biologic oxidation. Note that many biologic oxidations can take place without the participation of molecular oxygen, for example, dehydrogenations. The life of higher animals is absolutely dependent on a supply of oxygen for respiration, the process by which cells derive energy in the form of ATP from the controlled reaction of hydrogen with oxygen to form water. In addition, molecular oxygen is incorporated into a variety of substrates by enzymes designated as oxygenases; many drugs, pollutants, and chemical carcinogens (xenobiotics) are metabolized by enzymes of this class, known as the cytochrome P450 system. Administration of oxygen can be lifesaving in the treatment of patients with respiratory or circulatory failure.

In reactions involving oxidation and reduction, the free energy change is proportionate to the tendency of reactants to donate or accept electrons. Thus, in addition to expressing free energy change in terms of AG" (see Chapter 11), it is possible, in an analogous manner, to express it numerically as an oxidation-reduction or redox potential (E',). Chemically, the redox potential of a system (E,) is usually compared with the potential of the hydrogen electrode (0.0 V at pH 0.0). However, for biologic systems, the redox potential

H + /H 2, E ′ 0 Volts = -0.42. NAD + /NADH, E ′ 0 Volts = -0.32. Lipoate; ox/red, E ′ 0 Volts = -0.29. Acetoacetate/3-hydroxybutyrate, E ′ 0 Volts = -0.27. Pyruvate/lactate, E ′ 0 Volts = -0.19. Oxaloacetate/malate, E ′ 0 Volts = -0.17. Fumarate/succinate, E ′ 0 Volts = +0.03. Cytochrome b ; Fe 3+ /Fe 2+, E ′ 0 Volts = +0.08. Ubiquinone; ox/red, E ′ 0 Volts = +0.10. Cytochrome c 1 ; Fe 3+ /Fe 2+, E ′ 0 Volts = +0.22. Cytochrome a ; Fe 3+ /Fe 2+, E ′ 0 Volts = +0.29. Oxygen/water, E ′ 0 Volts = +0.82
(E',) is normally expressed at pH 7.0, at which pH the electrode potential of the hydrogen electrode is -0.42 V. The redox potentials of some redox systems of special interest in mammalian biochemistry are shown in Table 12-1. The relative positions of redox systems in the table allow prediction of the direction of flow of electrons from one redox couple to another. Enzymes involved in oxidation and reduction are called oxidoreductases and are classified into four groups: oxidases, dehydrogenases, hydroperoxidases, and oxygenases.

Oxidases catalyze the removal of hydrogen from a substrate using oxygen as a hydrogen acceptor.* They form water or hydrogen peroxide as a reaction product (Figure 12-1).

Cytochrome oxidase is a hemoprotein widely distributed in many tissues, having the typical heme prosthetic group present in myoglobin, hemoglobin, and other cytochromes (see Chapter 6). It is the terminal component of the chain of respiratory carriers found in mitochondria (see Chapter 13) and transfers electrons resulting from the oxidation of substrate molecules by dehydrogenases to their final acceptor, oxygen. The action of the enzyme is blocked by carbon monoxide, cyanide, and hydrogen sulfide, and this causes poisoning by preventing cellular respiration. The cytochrome oxidase enzyme complex comprises heme a, combined with another
"The term "oxidase" is sometimes used collectively to denote all enzymes that catalyze reactions involving molecular oxygen.
FIGURE 12-1 oxidation of a metabolite catalyzed by an oxidase (A) forming H,0 and (B) forming H,O,.
heme, heme a, in a single protein and so is also termed cytochrome aa,. It contains two molecules of heme, each having one Fe atom that oscillates between Fe** and Fe** during oxidation and reduction. Furthermore, two atoms of copper are present, one associated with each heme unit.

Flavoprotein enzymes contain flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as prosthetic groups. FMN and FAD are formed in the body from the vitamin riboflavin (see Chapter 44). FMN and FAD are usually tightly— but not covalently—bound to their respective apoenzyme proteins. Metalloflavoproteins contain one or more metals as essential cofactors. Examples of flavoprotein oxidases include L-amino acid oxidase, an enzyme found in kidney with general specificity for the oxidative deamination of the naturally occurring L-amino acids; xanthine oxidase, which contains molybdenum and plays an important role in the conversion of purine bases to uric acid (see Chapter 33), and is of particular significance in uricotelic animals (see Chapter 28); and aldehyde dehydrogenase, an FAD-linked enzyme present in mammalian livers, which contains molybdenum and nonheme iron and acts on aldehydes and N-heterocyclic substrates. The mechanisms of oxidation and reduction of these enzymes are complex. Evidence suggests a two-step reaction as shown in Figure 12-2.

There are a large number of enzymes in the dehydrogenase class. Their two main functions are as follows:
1. Transfer of hydrogen from one substrate to another in a coupled oxidation-reduction reaction (Figure 12-3). These dehydrogenases often utilize common coenzymes or hydrogen carriers, for example, nicotinamide adenine dinucleotide (NAD*). This type of reaction in which one substrate is oxidized/reduced at the expense of another is freely reversible, enabling reducing equivalents to be transferred within the cell and oxidative processes to occur in the absence of oxygen, such as during the anaerobic phase of glycolysis (see Figure 17-2).
- 2. Transfer of electrons in the respiratory chain of electron transport from substrate to oxygen (see Figure 13-3).
(FADH,)
FIGURE 12-2 Oxidoreduction of isoalloxazine ring in flavin nucleotides via a semiquinone intermediate. In oxidation reactions, the flavin (eg, FAD) accepts two electrons and two H* in two steps, forming the semiquinone intermediate followed by the reduced flavin (eg, FADH,) and the substrate is oxidized. In the reverse (reduction) reaction, the reduced flavin gives up two electrons and two H* so that it becomes oxidized (eg, to FAD) and the substrate is reduced.

These dehydrogenases use NAD* or nicotinamide adenine dinucleotide phosphate (NADP*)—or both—which are formed in the body from the vitamin niacin (see Chapter 44). The structure of NAD* is shown in Figure 12-4. NADP* has a phosphate group esterified to the 2' hydroxyl of its adenosine moiety, but otherwise is identical to NAD*. The oxidized forms of both nucleotides have a positive charge on the nitrogen atom of the nicotinamide moiety as indicated in Figure 12-4. The coenzymes are reduced by the specific substrate of the dehydrogenase and reoxidized by a suitable electron acceptor. They are able to freely and reversibly dissociate from their respective apoenzymes.
leads to the formation of ATP (see Chapter 13). NADP-linked dehydrogenases are found characteristically in biosynthetic pathways where reductive reactions are required, as in the extramitochondrial pathway of fatty acid synthesis (see Chapter 23) and steroid synthesis (see Chapter 26)—and also in the pentose phosphate pathway (see Chapter 20).
Generally, NAD-linked dehydrogenases catalyze oxidoreduction reactions of the

When a substrate is oxidized, it loses two hydrogen atoms and two electrons. One H* and both electrons are accepted by NAD* to form NADH and the other H* is released (Figure 12-4). Many such reactions occur in the oxidative pathways of metabolism, particularly in glycolysis (see Chapter 17) and the citric acid cycle (see Chapter 16). NADH is generated in these pathways via the oxidation of fuel molecules, and NAD* is regenerated by the oxidation of NADH as it transfers the electrons to O, via the respiratory chain in mitochondria, a process which
FIGURE 12-3 Oxidation of a metabolite catalyzed by coupled dehydrogenases.
FIGURE 12-4 Oxidation and reduction of nicotinamide coenzymes. Nicotinamide coenzymes consist of a nicotinamide ring linked to an adenosine via a ribose and a phosphate group, forming a dinucleotide. NAD*/NADH are shown, but NADP*/NADPH are identical except that they have a phosphate group esterified to the 2' OH of the adenosine. An oxidation reaction involves the transfer of two electrons and one H* from the substrate to the nicotinamide ring of NAD* forming NADH and the oxidized product. The remaining hydrogen of the hydrogen pair removed from the substrate remains free as a hydrogen ion. NADH is oxidized to NAD* by the reverse reaction.
113

The flavin groups such as FMN and FAD are associated with dehydrogenases as well as with oxidases as described above. FAD is the electron in reactions of the acceptor type:
FAD accepts two electrons and two H* in the reaction (Figure 12-2), forming FADH,,. Flavin groups are generally more tightly bound to their apoenzymes than are the nicotinamide coenzymes. Most of the riboflavin-linked dehydrogenases are concerned with electron transport in (or to) the respiratory chain (see Chapter 13). NADH dehydrogenase acts as a carrier of electrons between NADH and the components of higher redox potential (see Figure 13-3). Other dehydrogenases such as succinate dehydrogenase, acyl-CoA dehydrogenase, and mitochondrial glycerol-3-phosphate dehydrogenase transfer reducing equivalents directly from the substrate to the respiratory chain (see Figure 13-5). Another role of the flavin-dependent dehydrogenases is in the dehydrogenation (by dihydrolipoyl dehydrogenase) of reduced lipoate, an intermediate in the oxidative decarboxylation of pyruvate and a-ketoglutarate (see Figures 13-5 and 17-5). The electron-transferring flavoprotein (ETF) is an intermediary carrier of electrons between acyl-CoA dehydrogenase and the respiratory chain (see Figure 13-5).

The cytochromes are iron-containing hemoproteins in which the iron atom oscillates between Fe** and Fe** during oxidation and reduction. Except for cytochrome oxidase (previously described), they are classified as dehydrogenases. In the respiratory chain, they are involved as carriers of electrons from flavoproteins on the one hand to cytochrome oxidase on the other (see Figure 13-5). Several identifiable cytochromes occur in the respiratory chain, that is, cytochromes J, c,, c, and cytochrome oxidase (aa,). Cytochromes are also found in other locations, for example, the endoplasmic reticulum (cytochromes P450 and b.), and in plant cells, bacteria, and yeasts.

Two types of enzymes found both in animals and plants fall into the hydroperoxidase category: peroxidases and catalase. Hydroperoxidases play an important role in protecting the body against the harmful effects of reactive oxygen species (ROS). ROS are highly reactive oxygen-containing molecules such as peroxides, which are formed during normal metabolism, but can be damaging if they accumulate. They are believed to contribute to the causation of diseases such as cancer and atherosclerosis, as well as the aging process in general (see Chapters 21, 44, 54).

Peroxidases are found in milk and in leukocytes, platelets, and other tissues involved in eicosanoid metabolism (see Chapter 23). Their prosthetic group is protoheme. In the reaction catalyzed by peroxidase, hydrogen peroxide is reduced at the expense of several substances that act as electron acceptors, such as ascorbate (vitamin C), quinones, and cytochrome c. The reaction catalyzed by peroxidase is complex, but the overall reaction is as follows:
H,02+
AH,
———————>
2H,0+A
In erythrocytes and other tissues, the enzyme glutathione peroxidase, containing selenium as a prosthetic group, catalyzes the destruction of H,O, and lipid hydroperoxides through the conversion of reduced glutathione to its oxidized form, protecting membrane lipids and hemoglobin against oxidation by peroxides (see Chapter 21).

Catalase is a hemoprotein containing four heme groups. It can act as a peroxidase, catalyzing reactions of the type shown above, but it is also able to catalyze the breakdown of H,O, formed by the action of oxygenases to water and oxygen:
This reaction uses one molecule of H,O, as a substrate electron donor and another molecule of H,O, as an oxidant or electron acceptor. It is one of the fastest enzyme reactions known, destroying millions of potentially damaging H,O, molecules per second. Under most conditions in vivo, the peroxidase activity of catalase seems to be favored. Catalase is found in blood, bone marrow, mucous membranes, kidney, and liver. Peroxisomes are found in many tissues, including liver. They are rich in oxidases and in catalase. Thus, the enzymes that produce H,O, are grouped with the enzyme that breaks it down. However, mitochondrial and microsomal electron transport systems as well as xanthine oxidase must be considered as additional sources of H,O,.

Oxygenases are concerned with the synthesis or degradation of many different types of metabolites. They catalyze the incorporation of oxygen into a substrate molecule in two steps:
(1) oxygen is bound to the enzyme at the active site and (2) the bound oxygen is reduced or transferred to the substrate. Oxygenases may be divided into two subgroups, dioxygenases and monooxygenases.

Examples include the liver enzymes, homogentisate dioxygenase (oxidase) and 3-hydroxyanthranilate dioxygenase (oxidase), which contain iron; and L-tryptophan dioxygenase (tryptophan pyrrolase) (see Chapter 29), which utilizes heme.

The other oxygen atom is reduced to water, an additional electron donor or cosubstrate (Z) being necessary for this purpose:

Cytochromes P450 are an important superfamily of hemecontaining monooxygenases, and more than 50 such enzymes have been found in the human genome. They are located mainly in the endoplasmic reticulum in the liver and intestine, but are also found in the mitochondria in some tissues. The cytochromes participate in an electron transport chain in which both NADH and NADPH may donate reducing equivalents. Electrons are passed to cytochrome P450 in two types of reaction involving FAD or FMN. Class I systems consist of an FAD-containing reductase enzyme, an iron sulfur (Fe,S,) protein, and the P450 heme protein, while class II systems contain cytochrome P450 reductase, which passes electrons from FADH, to FMN (Figure 12-5). Class I and II systems are well characterized, but in recent years, other cytochromes P450, which do not fit into either category, have been identified. In the final step, oxygen accepts the electrons from cytochrome P450 and is reduced, with one atom being incorporated into H,O and the other into the substrate, usually resulting in its hydroxylation. This series of enzymatic reactions, known as the hydroxylase cycle, is illustrated in Figure 12-6. In the endoplasmic reticulum of the liver, cytochromes P450 are found together with another heme-containing protein, cytochrome b, (Figure 12-5) and together they have a major role in drug metabolism and detoxification. Cytochrome b, also has an important role as a fatty acid desaturase. Together, cytochromes P450 and b, are responsible for about 75% of the modification and degradation of drugs, which occurs in the body. The rate of detoxification of many medicinal drugs by cytochromes P450 determines the duration of their action. Benzpyrene, aminopyrine, aniline, morphine, and benzphetamine are hydroxylated, increasing their solubility and aiding their excretion. Many drugs such as phenobarbital have the ability to induce the synthesis of cytochromes P450.

Mitochondrial cytochrome P450 systems are found in steroidogenic tissues such as adrenal cortex, testis, ovary, and placenta and are concerned with the biosynthesis of steroid hormones from cholesterol (hydroxylation at C,, and C,, in side-chain cleavage and at the 11 and 18 positions).
Class
|
P450
FIGURE 12-5 cytochromes P450 and b, in the endoplasmic reticulum. Most cytochromes P450 are class | or class Il. In addition to cytochrome P450, class | systems contain a small FAD-containing reductase and an iron sulfur protein, and class Il contains cytochrome P450 reductase, which incorporates FAD and FMN. Cytochromes P450 catalyze many steroid hydroxylation reactions and drug detoxification steps. Cytochrome 6, acts in conjunction with the FAD-containing cytochrome b, reductase in the fatty acyl-CoA desaturase (eg, stearoyl-CoA desaturase) reaction and also works together with cytochromes P450 in drug detoxification. It is able to accept electrons from cytochrome P450 reductase via cytochrome b, reductase and donate them to cytochrome P450.
FIGURE 12-6 Cytochrome P450 hydroxylase cycle. The system shown is typical of steroid hydroxylases of the adrenal cortex. Liver microsomal cytochrome P450 hydroxylase does not require the iron-sulfur protein Fe,S,. Carbon monoxide (CO) inhibits the indicated step.
In addition, renal systems catalyzing 1aand 24-hydroxylations of 25-hydroxycholecalciferol in vitamin D metabolism—and cholesterol 7a-hydroxylase and sterol 27-hydroxylase involved in bile acid biosynthesis from cholesterol in the liver (see Chapters 26, 41)—are P450 enzymes.

In this reaction, superoxide acts as both oxidant and reductant. Thus, superoxide dismutase protects aerobic organisms against the potential deleterious effects of superoxide. The enzyme occurs in all major aerobic tissues in the mitochondria and the cytosol. Although exposure of animals to an atmosphere of 100% oxygen causes an adaptive increase in SOD, particularly in the lungs, prolonged exposure leads to lung damage and death. Antioxidants, for example, a-tocopherol (vitamin E), act as scavengers of free radicals and reduce the toxicity of oxygen (see Chapter 44).
Transfer of a single electron to O, generates the potentially damaging superoxide anion-free radical (O,), which gives rise to free-radical chain reactions (see Chapter 21), amplifying its destructive effects. The ease with which superoxide can be formed from oxygen in tissues and the occurrence of superoxide dismutase (SOD), the enzyme responsible for its removal in all aerobic organisms (although not in obligate anaerobes), indicate that the potential toxicity of oxygen is due to its conversion to superoxide.
Superoxide is formed when reduced flavins—present, for example, in xanthine oxidase—are reoxidized univalently by molecular oxygen:
Enz — Flavin — H, + O, > Enz — Flavin-— H + O,-+ H*
Superoxide can reduce oxidized cytochrome c
O, +Cytc(Fe**) > O, + Cytc(Fe"* )
or be removed by superoxide dismutase, which catalyzes the conversion of to oxygen and hydrogen peroxide.

™ In biologic systems, as in chemical systems, oxidation (loss of electrons) is always accompanied by reduction of an electron acceptor.
™ Oxidoreductases have a variety of functions in metabolism;
oxidases and
dehydrogenases play
major roles
in respiration;
hydroperoxidases protect
the radicals;
and oxygenases
body mediate
against the
damage by
free hydroxylation
of drugs
and steroids.
- ™@ Tissues are protected from oxygen toxicity caused by the superoxide free radical by the specific enzyme superoxide dismutase.

Nelson DL, Cox MM: Lehninger Principles of Biochemistry, 6th ed.
Macmillan Higher Education, 2013.
Nicholls DG, Ferguson SJ: Bioenergetics, 4th ed. Academic Press,
2013.

ee S ele B Xe gle 18
NUE Telt ehel ele aves
■, 1 = Describe the double membrane structure of mitochondria and indicate the location of various enzymes.. ■, 1 = Appreciate that energy from the oxidation of fuel substrates (fats, carbohydrates, amino acids) is almost all generated in mitochondria via a process termed electron transport in which electrons pass through a series of complexes (the respiratory chain) until they are finally reacted with oxygen to form water.. ■, 1 = Describe the four protein complexes involved in the transfer of electrons through the respiratory chain and explain the roles of flavoproteins, iron-sulfur proteins, and coenzyme Q.. ■, 1 = Explain how coenzyme Qaccepts electrons from NADHvia Complex I and from FADH 2 via Complex II.. ■, 1 = Indicate how electrons are passed from reduced coenzyme Qto cytochrome c via Complex III in the Qcycle.. ■, 1 = Explain the process by which reduced cytochrome c is oxidized and oxygen is reduced to water via Complex IV.. ■, 1 = Describe how electron transport generates a proton gradient across the inner mitochondrial membrane, leading to the buildup of a proton motive force that generates ATP by the process of oxidative phosphorylation.. ■, 1 = Describe the structure of the ATP synthase enzyme and explain how it works as a rotary motor to produce ATP from ADP and Pi.. ■, 1 = Explain that oxidation of reducing equivalents via the respiratory chain and oxidative phosphorylation are tightly coupled in most circumstances, so that one cannot proceed unless the other is functioning.. ■, 1 = Indicate examples of common poisons that block respiration or oxidative phosphorylation and identify their site of action.. ■, 1 = Explain, with examples, how uncouplers may act as poisons by dissociating oxidation via the respiratory chain from oxidative phosphorylation, but may also have a physiologic role in generating body heat.. ■, 1 = Explain the role of exchange transporters present in the inner mitochondrial membrane in allowing ions and metabolites to pass through while preserving electrochemical and osmotic equilibrium.

Aerobic organisms are able to capture a far greater proportion of the available free energy of respiratory substrates than anaerobic organisms. Most of this takes place inside mitochondria, which have been termed the "powerhouses" of the cell. Respiration is coupled to the generation of the high-energy intermediate, ATP (see Chapter 11), by oxidative phosphorylation. A number of drugs (eg, amobarbital) and poisons (eg, cyanide, carbon monoxide) inhibit oxidative phosphorylation, usually with fatal consequences. Several inherited defects of mitochondria involving components of the respiratory chain and oxidative phosphorylation have been reported. Patients present with myopathy and encephalopathy and often have lactic acidosis.

The mitochondrial matrix (the internal compartment) is enclosed by a double membrane. The outer membrane is permeable to most metabolites and the inner membrane is selectively permeable (Figure 13-1). The outer membrane is characterized by the presence of various enzymes, including acyl-CoA synthetase (see Chapter 22) and glycerol phosphate acyltransferase (see Chapter 24). Other enzymes, including adenylyl kinase (see Chapter 11) and creatine kinase (see Chapter 51) are found in the intermembrane space. The phospholipid cardiolipin is concentrated in the
FIGURE 13-1 Structure of the mitochondrial membranes. Note that the inner membrane contains many folds or cristae.
inner membrane together with the enzymes of the respiratory chain, ATP synthase, and various membrane transporters.

Most of the energy liberated during the oxidation of carbohydrate, fatty acids, and amino acids is made available within mitochondria as reducing equivalents (—H or electrons) (Figure 13-2). The enzymes of the citric acid cycle and B-oxidation (see Chapters 22 and 16), the respiratory chain complexes, and the machinery for oxidative phosphorylation are all found in mitochondria. The respiratory chain collects and transports reducing equivalents, directing them to their final reaction with oxygen to form water, and oxidative phosphorylation is the process by which the liberated free energy is trapped as high-energy phosphate.

Electrons flow through the respiratory chain through a redox span of 1.1 V from NAD*/NADH to O,/2H,0 (see Table 12-1), passing through three large protein complexes: NADH-Q oxidoreductase (Complex I), where electrons are transferred from NADH to coenzyme Q (Q) (also called ubiquinone); Q-cytochrome c oxidoreductase (Complex III), which passes the electrons on to cytochrome c; and cytochrome c oxidase (Complex IV), which completes the chain, passing the electrons to O, and causing it to be reduced to H,O (Figure 13-3). Some substrates with more positive redox potentials than NAD*/NADH (eg, succinate) pass electrons to Q via a fourth complex, succinate-Q reductase (Complex II), rather than Complex I. The four complexes are embedded in the inner mitochondrial membrane, but Q and cytochrome c are mobile. Q diffuses rapidly within the membrane, while cytochrome c is a soluble protein.

Flavoproteins (see Chapter 12) are important components of Complexes I and II. The oxidized flavin nucleotide (flavin mononucleotide [FMN] or flavin adenine dinucleotide [FAD]) can be reduced in reactions involving the transfer of two electrons (to form FMNH, or FADH,), but they can also accept one electron to form the semiquinone (see Figure 12-2). Ironsulfur proteins (nonheme iron proteins, Fe-S) are found in Complexes I, II, and III. These may contain one, two, or four Fe atoms linked to inorganic sulfur atoms and/or via cysteine-SH groups to the protein (Figure 13-4). The Fe-S take part in single
CHAPTER
13
FIGURE 13-2 Role of the respiratory chain of mitochondria in the conversion of food energy to ATP. Oxidation of the major foodstuffs leads to the generation of reducing equivalents (2H) that are collected by the respiratory chain for oxidation and coupled generation of ATP.

electron transfer reactions in which one Fe atom undergoes oxi- doreduction between Fe* and Fe**.

NADH-Q oxidoreductase or Complex I is a large L-shaped multisubunit protein that catalyzes electron transfer from NADH to Q, and during the process four H* are transferred across the membrane into the intermembrane space:
Electrons are transferred from NADH to FMN initially, then to a series of Fe-S centers, and finally to Q (Figure 13-5). In Complex II (succinate-Q reductase), FADH, is formed during the conversion of succinate to fumarate in the citric acid cycle (see Figure 16-3) and electrons are then passed via several Fe-S centers to Q (Figure 13-5). Glycerol-3-phosphate (generated in the breakdown of triacylglycerols or from glycolysis, see Figure 17-2) and acyl-CoA also pass electrons to Q via different pathways involving flavoproteins (Figure 13-5).

Electrons are passed from QH, to cytochrome c via Complex III (Q-cytochrome c oxidoreductase):
The process is believed to involve cytochromes c,, b,, and b,, and a Rieske Fe-S (an unusual Fe-S in which one of the
Fe atoms is linked to two histidine residues rather than two cysteine residues) (Figure 13-5) and is known as the Q cycle (Figure 13-6). Q may exist in three forms: the oxidized quinone, the reduced quinol, or the semiquinone (Figure 13-6). The semiquinone is formed transiently during the cycle, one turn of which results in the oxidation of 2QH, to Q, releasing 4H* into the intermembrane space, and the reduction of one Q to QH,, causing 2H* to be taken up from the matrix (Figure 13-6). Note that while Q carries two electrons, the cytochromes carry only one, thus the oxidation of one QH, is coupled to the reduction of two molecules of cytochrome c via the
Q cycle.

Reduced cytochrome c is oxidized by Complex IV (cytochrome c oxidase), with the concomitant reduction of O, to two molecules of water:
Four electrons are transferred from cytochrome c to O, via two heme groups, a and a,, and Cu (Figure 13-5). Electrons are passed initially to a Cu center (Cu,), which contains 2Cu atoms linked to two protein cysteine-SH groups (resembling an Fe-S), then in sequence to heme a, heme a,, a second Cu center, Cu,, which is linked to heme a,, and finally to O,. Eight H* are removed from the matrix, of which four are used to form two water molecules and four are pumped into the
FIGURE 13-5 Flow of electrons through the respiratory chain complexes, showing the entry points for reducing equivalents from important substrates. Q and cyt c are mobile components of the system as indicated by the dotted arrows. The flow through Complex Ill (the Q cycle) is shown in more detail in Figure 13-6. (cyt, cytochrome; ETF, electron transferring flavoprotein; Fe-S, iron-sulfur protein; Q, coenzyme Q or
QHpg: Reduced (quinol) form (QH2)
Q: Fully oxidized (quinone) form
*Q@™:
Semiquinone
(free radical)
form
FIGURE 13-6 The Q cycle. During the oxidation of QH, to Q, one electron is donated to cyt c via a Rieske Fe-S and cyt c, and the second to a Q to form the semiquinone via cyt b, and cyt 6,, with 2H* being released into the intermembrane space. A similar process then occurs with a second QH,, but in this case the second electron is donated to the semiquinone, reducing it to QH,, and 2H* are taken up from the matrix. (cyt, cytochrome; Fe-S, iron-sulfur protein; Q, coenzyme Q or ubiquinone.)

intermembrane space. Thus, for every pair of electrons passing down the chain from NADH or FADH,, 2H* are pumped across the membrane by Complex IV. The O, remains tightly bound to Complex IV until it is fully reduced, and this minimizes the release of potentially damaging intermediates such as superoxide anions or peroxide which are formed when O, accepts one or two electrons, respectively (see Chapter 12).

The flow of electrons through the respiratory chain generates ATP by the process of oxidative phosphorylation. The chemiosmotic theory, proposed by Peter Mitchell in 1961, postulates that the two processes are coupled by a proton gradient across the inner mitochondrial membrane so that the proton motive force caused by the electrochemical potential difference (negative on the matrix side) drives the mechanism of ATP synthesis. As we have seen, Complexes I, III, and IV act as proton pumps, moving H* from the mitochondrial matrix to the intermembrane space. Since the inner mitochondrial membrane is impermeable to ions in general and particularly to protons, these accumulate in the intermembrane space, creating the proton motive force predicted by the chemiosmotic theory.
The proton motive force drives a membrane-located ATP synthase that forms ATP in the presence of P, + ADP. ATP synthase is embedded in the inner membrane, together with the respiratory chain complexes (Figure 13-7). Several subunits of the protein form a ball-like shape arranged around an axis known as F,, which projects into the matrix and contains the phosphorylation mechanism (Figure 13-8). F, is attached to a membrane protein complex known as F,, which also consists of several protein subunits. F, spans the membrane and forms a proton channel. As protons flow through F, driven by the proton gradient across the membrane it rotates, driving the production of ATP in the F, complex (Figures 13-7 and 13-8). This is thought to occur by a binding change mechanism in which the conformation of the B subunits in F, is changed as the axis rotates from one that binds ATP tightly to one that releases ATP and binds ADP and P; so that the next ATP can be formed. As indicated above, for each NADH oxidized, Complexes I and III translocate four protons each and Complex IV translocates two.

ADP captures, in the form of high-energy phosphate, a significant proportion of the free energy released by catabolic processes. The resulting ATP has been called the
FIGURE 13-7 The chemiosmotic theory of oxidative phosphorylation. Complexes |, Ill, and IV act as proton pumps creating a proton gradient across the membrane, which is negative on the matrix side. The proton motive force generated drives the synthesis of ATP as the protons flow back into the matrix through the ATP synthase enzyme (Figure 13-8). Uncouplers increase the permeability of the membrane to ions, collapsing the proton gradient by allowing the H* to pass across without going through the ATP synthase, and thus uncouple electron flow through the respiratory complexes from ATP synthesis. (cyt, cytochrome; Q, coenzyme Q or ubiquinone.)
energy "currency" of the cell because it passes on this free energy to drive those processes requiring energy (see Figure 11-5).
FIGURE 13-8 Mechanism of ATP production by ATP synthase. The enzyme complex consists of an F, subcomplex which is a disk of "C" protein subunits. Attached is a y subunit in the form of a"bent axle." Protons passing through the disk of "C" units cause it and the attached y subunit to rotate. The y subunit fits inside the F, subcomplex of three a and three B subunits, which are fixed to the membrane and do not rotate. ADP and P, are taken up sequentially by the B subunits to form ATP, which is expelled as the rotating y subunit squeezes each B subunit in turn and changes its conformation. Thus, three ATP molecules are generated per revolution. For clarity, not all the subunits that have been identified are shown—eg, the "axle" also contains an € subunit.

There is a net direct capture of two high-energy phosphate groups in the glycolytic reactions (see Table 17-1). Two more high-energy phosphates per mole of glucose are captured in the citric acid cycle during the conversion of succinyl-CoA to succinate (see Chapter 16). All of these phosphorylations occur at the substrate level. For each mol of substrate oxidized via Complexes I, III, and IV in the respiratory chain (ie, via NADH), 2.5 mol of ATP are formed per 0.5 mol of O, consumed, that is, the P:O ratio = 2.5 (Figure 13-7). On the other hand, when 1 mol of substrate (eg, succinate or 3-phophoglycerate) is oxidized via Complexes II, II, and IV, only 1.5 mol of ATP are formed, that is, P:O = 1.5. These reactions are known as oxidative phosphorylation at the respiratory chain level. Taking these values into account, it can be estimated that nearly 90% of the high-energy phosphates produced from the complete oxidation of 1 mol glucose is obtained via oxidative phosphorylation coupled to the respiratory chain (see Table 17-1).

The rate of respiration of mitochondria can be controlled by the availability of ADP. This is because oxidation and phosphorylation are tightly coupled; that is, oxidation cannot proceed via the respiratory chain without concomitant phosphorylation of ADP. Table 13-1 shows the five conditions

State 1, Conditions Limiting the Rate of Respiration = Availability of ADP and substrate. State 2, Conditions Limiting the Rate of Respiration = Availability of substrate only. State 3, Conditions Limiting the Rate of Respiration = The capacity of the respiratory chain itself, when all substrates and components are present in saturating amounts. State 4, Conditions Limiting the Rate of Respiration = Availability of ADP only. State 5, Conditions Limiting the Rate of Respiration = Availability of oxygen only
controlling the rate of respiration in mitochondria. Most cells in the resting state are in state 4, and respiration is controlled by the availability of ADP. When work is performed, ATP is converted to ADP, allowing more respiration to occur, which in turn replenishes the store of ATP. Under certain conditions, the concentration of inorganic phosphate can also affect the rate of functioning of the respiratory chain. As respiration increases (as in exercise), the cell approaches state 3 or 5 when either the capacity of the respiratory chain becomes saturated or the PO, decreases below the K,, for heme a,. There is also the possibility that the ADP/ATP transporter, which facilitates entry of cytosolic ADP into and ATP out of the mitochondrion, becomes rate limiting.
whole is sufficiently exergonic to be removed from equilibrium, allowing continuous unidirectional flow and constant provision of ATP. It also contributes to maintenance of body temperature.

Much information about the respiratory chain has been obtained by the use of inhibitors, and, conversely, this has provided knowledge about the mechanism of action of several poisons (Figure 13-9). They may be classified as inhibitors of the respiratory chain, inhibitors of oxidative phosphorylation, or uncouplers of oxidative phosphorylation.
Barbiturates such as amobarbital inhibit electron transport via Complex I by blocking the transfer from Fe-S to Q. At sufficient dosage, they are fatal. Antimycin A and dimercaprol inhibit the respiratory chain at Complex III. The classic poisons HS, carbon monoxide, and cyanide inhibit Complex IV and can therefore totally arrest respiration. Malonate is a competitive inhibitor of Complex II.
Atractyloside inhibits oxidative phosphorylation by inhibiting the transporter of ADP into and ATP out of the mitochondrion (Figure 13-10). The antibiotic oligomycin completely blocks oxidation and phosphorylation by blocking the flow of protons through ATP synthase (Figure 13-9).
Thus, the manner in which biologic oxidative processes allow the free energy resulting from the oxidation of foodstuffs to become available and to be captured is stepwise, efficient, and controlled—rather than explosive, inefficient, and uncontrolled, as in many nonbiologic processes. The remaining free energy that is not captured as high-energy phosphate is liberated as heat. This need not be considered "wasted" since it ensures that the respiratory system as a
Uncouplers dissociate oxidation in the respiratory chain from phosphorylation (Figure 13-7). These compounds are toxic, causing respiration to become uncontrolled, since the rate is no longer limited by the concentration of ADP or P.. The uncoupler that has been used most frequently is 2,4dinitrophenol, but other compounds act in a similar manner. Thermogenin (or the uncoupling protein) is a physiologic uncoupler found in brown adipose tissue that functions to
FIGURE 13-9 Sites of inhibition (©) of the respiratory chain by specific drugs, chemicals, and antibiotics. (BAL, dimercaprol; TTFA, an Fe-chelating agent. Other abbreviations as in Figure 13-5.)

FIGURE 13-10 Transporter systems in the inner mitochondrial membrane. © Phosphate transporter, @ pyruvate symport, @ dicarboxylate transporter, ® tricarboxylate transporter, © a-ketoglutarate transporter, © adenine nucleotide transporter. N-Ethylmaleimide, hydroxycinnamate, and atractyloside inhibit (©) the indicated systems. Also present (but not shown) are transporter systems for glutamate/aspartate (Figure 13-13), glutamine, ornithine, neutral amino acids, and carnitine (see Figure 22-1).
generate body heat, particularly for the newborn and during hibernation in animals (see Chapter 25).

The electrochemical potential difference across the membrane, once established as a result of proton translocation, inhibits further transport of reducing equivalents through the respiratory chain unless discharged by back-translocation of protons across the membrane through the ATP synthase. This in turn depends on availability of ADP and P..
Uncouplers (eg, dinitrophenol) are amphipathic (see Chapter 21) and increase the permeability of the lipoid inner mitochondrial membrane to protons, thus reducing the electrochemical potential and short-circuiting the ATP synthase (Figure 13-7). In this way, oxidation can proceed without phosphorylation.

Exchange diffusion systems involving transporter proteins that span the membrane are present in the membrane for exchange of anions against OH" ions and cations against H* ions. Such systems are necessary for uptake and output of ionized metabolites while preserving electrical and osmotic equilibrium. The inner mitochondrial membrane is freely permeable to uncharged small molecules, such as oxygen, water, CO,, NH,, and to monocarboxylic acids, such as 3-hydroxybutyric, acetoacetic, and acetic, especially in their undissociated, more lipid soluble form. Long-chain fatty acids are transported into mitochondria via the carnitine system (see Figure 22-1), and there is also a special carrier for pyruvate involving a symport that utilizes the H* gradient from outside to inside the mitochondrion (Figure 13-10). However, dicarboxylate and tricarboxylate anions (eg, malate, citrate) and amino acids require specific transporter or carrier systems to facilitate their passage across the membrane.
The transport of diand tricarboxylate anions is closely linked to that of inorganic phosphate, which penetrates readily as the HPO, ion in exchange for OH-. The net uptake of malate by the dicarboxylate transporter requires inorganic phosphate for exchange in the opposite direction. The net uptake of citrate, isocitrate, or cis-aconitate by the tricarboxylate transporter requires malate in exchange. a-Ketoglutarate transport also requires an exchange with malate. The adenine nucleotide transporter allows the exchange of ATP and ADP, but not AMP. It is vital for ATP exit from mitochondria to the sites of extramitochondrial utilization and for the return of ADP for ATP production within the mitochondrion (Figure 13-11). Since in this translocation four negative charges are removed
FIGURE 13-11 Combination of phosphate transporter @ with the adenine nucleotide transporter @ in ATP synthesis. The H*/P, symport shown is equivalent to the P/OH" antiport shown in Figure 13-10.

FIGURE 13-12 Glycerophosphate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion.
from the matrix for every three taken in, the electrochemical gradient across the membrane (the proton motive force) favors the export of ATP. Na* can be exchanged for H*, driven by the proton gradient. It is believed that active uptake of Ca** by mitochondria occurs with a net charge transfer of 1 (Ca* uniport), possibly through a Ca**/H* antiport. Calcium release from mitochondria is facilitated by exchange with Na*.

Ionophores are lipophilic molecules that complex specific cations and facilitate their transport through biologic membranes, for example, valinomycin (K*). The classic uncouplers such as dinitrophenol are, in fact, proton ionophores.

Energy-linked transhydrogenase, a protein in the inner mitochondrial membrane, couples the passage of protons down the electrochemical gradient from outside to inside the mitochondrion with the transfer of H from intramitochondrial NADH
to NADPH for intramitochondrial enzymes such as glutamate dehydrogenase and hydroxylases involved in steroid synthesis.

NADH cannot penetrate the mitochondrial membrane, but it is produced continuously in the cytosol by 3-phosphoglyceraldehyde dehydrogenase, an enzyme in the glycolysis sequence (see Figure 17-2). However, under aerobic conditions, extramitochondrial NADH does not accumulate and is presumed to be oxidized by the respiratory chain in mitochondria. The transfer of reducing equivalents through the mitochondrial membrane requires substrate pairs, linked by suitable dehydrogenases on each side of the mitochondrial membrane. The mechanism of transfer using the glycerophosphate shuttle is shown in Figure 13-12. Since the mitochondrial enzyme is linked to the respiratory chain via a flavoprotein rather than NAD, only 1.5 mol rather than 2.5 mol of ATP are formed per atom of oxygen consumed. Although this shuttle is present in some tissues (eg, brain, white muscle), in others (eg, heart muscle) it is deficient. It is therefore believed that the malate shuttle system (Figure 13-13) is of more universal utility. The complexity of this system is due to the impermeability of the
FIGURE 13-13 Malate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion. @ a-Ketoglutarate transporter and @ glutamate/aspartate transporter (note the proton symport with glutamate).
mitochondrial membrane to oxaloacetate, which must react with glutamate to form aspartate and a-ketoglutarate by transamination before transport through the mitochondrial membrane and reconstitution to oxaloacetate in the cytosol.

Mitochondria maintain or accumulate cations such as K*, Na'*, Ca**, Mg", and P.. It is assumed that a primary proton pump drives cation exchange.

The creatine phosphate shuttle (Figure 13-14) augments the functions of creatine phosphate as an energy buffer by acting as a dynamic system for transfer of high-energy phosphate from mitochondria in active tissues such as heart and skeletal
FIGURE 13-14 The creatine phosphate shuttle of heart and skeletal muscle. The shuttle allows rapid transport of high-energy phosphate from the mitochondrial matrix into the cytosol. (CK, creatine kinase concerned with large requirements for ATP, eg, muscular contraction; CK, creatine kinase for maintaining equilibrium between creatine and creatine phosphate and ATP/ADP; CK, creatine kinase coupling glycolysis to creatine phosphate synthesis; CK_, mitochondrial creatine kinase mediating creatine phosphate production from ATP formed in oxidative phosphorylation; P, pore protein in outer mitochondrial membrane.)
muscle. An isoenzyme of creatine kinase (CK,) is found in the mitochondrial intermembrane space, catalyzing the transfer of high-energy phosphate to creatine from ATP emerging from the adenine nucleotide transporter. In turn, the creatine phosphate is transported into the cytosol via protein pores in the outer mitochondrial membrane, becoming available for generation of extramitochondrial ATP.

The condition known as fatal infantile mitochondrial myopathy and renal dysfunction involves severe diminution or absence of most oxidoreductases of the respiratory chain. MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke) is an inherited condition due to NADH-Q oxidoreductase (Complex I) or cytochrome oxidase (Complex IV) deficiency. It is caused by a mutation in mitochondrial DNA and may be involved in Alzheimer disease and diabetes mellitus. A number of drugs and poisons act by inhibition of oxidative phosphorylation (see above).

@ Virtually all energy released from the oxidation of carbohydrate, fat, and protein is made available in mitochondria as reducing equivalents (—H or e'). These are funneled into the respiratory chain, where they are passed down a redox gradient of carriers to their final reaction with oxygen to form water.
@ The redox carriers are grouped into four respiratory chain complexes in the inner mitochondrial membrane. Three of the four complexes are able to use the energy released in the redox gradient to pump protons to the outside of the membrane, creating an electrochemical potential between the matrix and the inner membrane space.
=| ATP synthase spans the membrane and acts like a rotary motor using the potential energy of the proton gradient or proton motive force to synthesize ATP from ADP and P.. In this way, oxidation is closely coupled to phosphorylation to meet the energy needs of the cell.
™ Since the inner mitochondrial membrane is impermeable to protons and other ions, special exchange transporters span the membrane to allow ions such as OH", ATP*, ADP*, and metabolites to pass through without discharging the electrochemical gradient across the membrane.
= Many well-known poisons such as cyanide arrest respiration by inhibition of the respiratory chain.

Kocherginsky N: Acidic lipids, H(+)-ATPases, and mechanism of oxidative phosphorylation. Physico-chemical ideas 30 years after P. Mitchell's Nobel Prize award. Prog Biophys Mol Biol 2009;99:20.
Mitchell P: Keilin's respiratory chain concept and its chemiosmotic consequences. Science 1979;206:1148.
Nakamoto RK, Baylis Scanlon JA, Al-Shawi MK: The rotary mechanism
of the
ATP
synthase.
Arch
Biochem
Biophys
2008;476:43.

1. Which one of the following statements about the free energy
change (AG) in a biochemical reaction is CORRECT?
A. If AG is negative, the reaction proceeds spontaneously with a
loss of free energy.
B. In an exergonic reaction, AG is positive.
C. The standard free energy change when reactants are
present in concentrations of 1.0 mol/L and the pH is 7.0 is
represented as AG®.
D. In an endergonic reaction there is a loss of free energy.
E. Ifa reaction is essentially irreversible, it has a high positive AG.
2. Ifthe AG ofa reaction is zero:
A. The reaction goes virtually to completion and is essentially
B. The reaction is endergonic.
C. The reaction is exergonic.
D. The reaction proceeds only if free energy can be gained.
E. The system is at equilibrium and no net change occurs.
3. AG? is defined as the standard free energy charge when:
A. The reactants are present in concentrations of 1.0 mol/L.
B. The reactants are present in concentrations of 1.0 mol/L at pH 7.0.
C. The reactants are present in concentrations of 1.0 mmol/L at
pH 7.0.
D. The reactants are present in concentrations of 1.0 umol/L.
E. The reactants are present in concentrations of 1.0 mol/L at
pH 7.4.
4. Which of the following statements about ATP is CORRECT?
It contains three high-energy phosphate bonds. >
It is needed in the body to drive exergonic reactions.
It is used as an energy store in the body.
It functions in the body as a complex with Mg”*.
It is synthesized by ATP synthase in the presence of uncouplers such as UCP-1 (thermogenin).
BOOf
5. Which one of the following enzymes uses molecular oxygen as a
hydrogen acceptor?
Cytochrome c oxidase >
Isocitrate dehydrogenase
Homogentisate dioxygenase
Catalase
Superoxide dismutase
BOOR

6. Which one of the following statement about cytochromes is
INCORRECT?
A. They are hemoproteins that take part in oxidation—
reduction reactions.
B. They contain iron which oscillates between and during the reactions they participate in.
Fe** Fe**
C. They act as electron carriers in the respiratory chain in mitochondria.
D. They have an important role in the hydroxylation of steroids
in the endoplasmic reticulum.
E. They are all dehydrogenase enzymes.
10. A number of compounds inhibit oxidative phosphorylation—the
ll. A number of compounds inhibit oxidative phosphorylation—the
. Which one of the following statement about cytochromes P450 is
A.
They are able to accept electrons from either NADH or NADPH.
B. They are found only in the endoplasmic reticulum.
C. They are monooxygenase enzymes.
D. They play a major role in drug detoxification in the liver.
E. Insome reactions they work in conjunction with cytochrome b5.
. As one molecule of NADH is oxidized via the respiratory chain:
A. 1.5 molecules of ATP are produced in total.
B. 1 molecule of ATP is produced as electrons pass through complex IV.
C. 1 molecule of ATP is produced as electrons pass through
complex II.
D. 1 molecule of ATP is produced as electrons pass through
complex III.
E. 0.5 of a molecule of ATP is produced as electrons pass
through complex I.
. The number of ATP molecules produced for each molecule of
FADH, oxidized via the respiratory chain is:
A. 1
B. 2.5
Cc. 15
D. 2
E. 0.5
synthesis of ATP from ADP and inorganic phosphate linked to
oxidation of substrates in mitochondria. Which of the following
describes the action of oligomycin?
A. It discharges the proton gradient across the mitochondrial
inner membrane.
B. It discharges the proton gradient across the mitochondrial
outer membrane.
C. It inhibits the electron transport chain directly by binding
to one of the electron carriers in the mitochondrial inner
membrane.

D. It inhibits the transport of ADP into, and ATP out of, the
E. It inhibits the transport of protons back into the mitochondrial
matrix through ATP synthase.
synthesis of ATP from ADP and inorganic phosphate linked to oxidation of substrates in mitochondria. Which of the following
describes the action of an uncoupler?
membrane.
matrix
through the stalk of the primary particle.
128
SECTION
III
Bioenergetics
12. A student takes some tablets she is offered at a disco, and without
13. The flow of electrons through the respiratory chain and the
14. Which of the following statements about ATP synthase is
asking
what
they
are
she
swallows
them.
A
short
time
later
she
starts
to
hyperventilate,
and
becomes
very
hot.
What
is
the
most
likely
action
of
the
tablets
she
has
taken?
A. An inhibitor of mitochondrial ATP synthesis
B. An inhibitor of mitochondrial electron transport
C.
An
inhibitor
of
the
transport
of
ADP
into
mitochondria
to
be phosphorylated
D. An inhibitor of the transport of ATP out of mitochondria
into the cytosol
E. An uncoupler of mitochondrial electron transport and
oxidative phosphorylation
production of ATP are normally tightly coupled. The processes
are uncoupled by which of the following?
Cyanide
Oligomycin
Thermogenin
Hydrogen sulphide
rOOP>
A. It is located in the inner mitochondrial membrane.

B. It requires a proton motive force to form ATP in the presence of ADP and Pi.
15. The chemiosmotic theory of Peter Mitchell proposes a
C. ATP is produced when part of the molecule rotates.
D. One ATP molecule is formed for each full revolution of the
molecule.
E. The
F,
subcomplex is fixed to the membrane and does not
rotate.
mechanism for the tight coupling of electron transport via the
respiratory chain to the process of oxidative phosphorylation.
Which of the following options is NOT predicted by the theory?
A. A proton gradient across the inner mitochondrial
membrane
generated
by
electron
transport
drives
ATP
B. The electrochemical potential difference across the inner
mitochondrial membrane caused by electron transport is
positive on the matrix side.
C. Protons are pumped across the inner mitochondrial
membrane as electrons pass down the respiratory chain.
D. An increase in the permeability of the inner mitochondrial
membrane to protons uncouples the processes of electron transport and oxidative phosphorylation.
E. ATP synthesis occurs when the electrochemical potential
difference across the membrane is discharged by
translocation of protons back across the inner mitochondrial
membrane through an ATP synthase enzyme.

m= Explain what is meant by anabolic, catabolic, and amphibolic metabolic pathways.
= Describe in outline the metabolism of carbohydrates, lipids, and amino
acids at the level of tissues and organs, and at the subcellular level, and the interconversion
fuels.
= Describe the ways in which flux of metabolites through metabolic pathways is regulated.
™ Describe how a supply of metabolic fuels is provided in the fed and fasting states; the formation of metabolic fuels reserves in the fed state and their mobilization in fasting.

Metabolism is the term used to describe the interconversion of chemical compounds in the body, the pathways taken by individual molecules, their interrelationships, and the mechanisms that regulate the flow of metabolites through the pathways. Metabolic pathways fall into three categories. (1) Anabolic pathways are involved in the synthesis of larger and more complex compounds from smaller precursors—for example, the synthesis of protein from amino acids and the synthesis of reserves of triacylglycerol and glycogen. Anabolic pathways are endothermic. (2) Catabolic pathways are involved in the breakdown of larger molecules, commonly involving oxidative reactions; they are exothermic, producing reducing equivalents, and, mainly via the respiratory chain (see Chapter 13), ATP. (3) Amphibolic pathways occur at the "crossroads" of metabolism, acting as links between anabolic and catabolic pathways, for example, the citric acid cycle (see Chapter 16). Knowledge of normal metabolism is essential for an understanding of abnormalities that underlie disease. Normal metabolism includes adaptation to periods of fasting, starvation, and exercise, as well as pregnancy and lactation. Abnormal metabolism may result from nutritional deficiency, enzyme deficiency, abnormal secretion of hormones, or the actions of drugs and toxins.
A 70-kg adult human being requires about 8 to 12 MJ (1920-2900 kcal) from metabolic fuels each day, depending on physical activity. Larger animals require less per kilogram body weight, and smaller animals more. Growing children and animals have a proportionally higher requirement to allow for the energy cost of growth. For human beings, this energy requirement is met from carbohydrates (40-60%), lipids (mainly triacylglycerol, 30-40%), and protein (10-15%), as well as alcohol. The mix of carbohydrate, lipid, and protein being oxidized varies, depending on whether the subject is in the fed or fasting state, and on the duration and intensity of physical work. There is a constant requirement for metabolic fuels throughout the day; average physical activity increases metabolic rate by about 40 to 50% over the basal or resting metabolic rate. However, most people consume their daily intake of

metabolic fuels in two or three meals, so there is a need to form reserves of carbohydrate (glycogen in liver and muscle), lipid (triacylglycerol in adipose tissue), and labile protein stores during the period following a meal, for use during the intervening time when there is no intake of food.
If the intake of metabolic fuels is consistently greater than energy expenditure, the surplus is stored, largely as triacylglycerol in adipose tissue, leading to the development of obesity and its associated health hazards. By contrast, if the intake of metabolic fuels is consistently lower than energy expenditure, there are negligible reserves of fat and carbohydrate, and amino acids arising from protein turnover are used for energy-yielding metabolism rather than replacement protein synthesis, leading to emaciation, wasting, and, eventually, death (see Chapter 43). In the fed state, after a meal, there is an ample supply of carbohydrate, and the metabolic fuel for most tissues is glucose. In the fasting state, glucose must be spared for use by the central nervous system (which is largely dependent on glucose) and the red blood cells (which are wholly reliant on glucose). Therefore, tissues that can use fuels other than glucose do so; muscle and liver oxidize fatty acids and the liver synthesizes ketone bodies from fatty acids to export to muscle and other tissues. As glycogen reserves become depleted, amino acids arising from protein turnover are used for gluconeogenesis (see Chapter 19).
The formation and utilization of reserves of triacylglycerol and glycogen, and the extent to which tissues take up and oxidize glucose, are largely controlled by the hormones insulin and glucagon. In diabetes mellitus, there is either impaired synthesis and secretion of insulin (type I diabetes, sometimes called juvenile onset, or insulin-dependent diabetes) or impaired sensitivity of tissues to insulin action (type II diabetes, sometimes called adult-onset or noninsulin-dependent diabetes), leading to severe metabolic derangement. In cattle, the demands of heavy lactation can lead to ketosis, as can the demands of twin pregnancy in sheep.

The nature of the diet sets the basic pattern of metabolism. There is a need to process the products of digestion of dietary carbohydrate, lipid, and protein. These are mainly glucose, fatty acids and glycerol, and amino acids, respectively. In ruminants (and, to a lesser extent, other herbivores), dietary cellulose is fermented by symbiotic microorganisms to shortchain fatty acids (acetic, propionic, butyric), and metabolism in these animals is adapted to use these fatty acids as major substrates. All the products of digestion are metabolized to a common product, acetyl-CoA, which is then oxidized by the citric acid cycle (see Chapter 16) (Figure 14-1).

Glucose is the major fuel of most tissues (Figure 14-2). It is metabolized to pyruvate by the pathway of glycolysis
FIGURE 14-1 Outline of the pathways for the catabolism of carbohydrate, protein, and fat. All these pathways lead to the production of acetyl-CoA, which is oxidized in the citric acid cycle, ultimately yielding ATP by the process of oxidative phosphorylation.
(see Chapter 17). Aerobic tissues metabolize pyruvate to acetyl-CoA, which can enter the citric acid cycle for complete oxidation to CO, and H,0, linked to the formation of ATP in the process of oxidative phosphorylation (see Figure 13-2). Glycolysis can also occur anaerobically (in the absence of oxygen) when the end product is lactate.
Glucose and its metabolites also take part in other processes, for example, the synthesis of the storage polymer glycogen in skeletal muscle and liver (see Chapter 18) and the pentose phosphate pathway, an alternative to part of the pathway of glycolysis (see Chapter 20). It is a source of reducing equivalents (NADPH) for fatty acid synthesis (see Chapter 23) and the source of ribose for nucleotide and nucleic acid synthesis (see Chapter 33). Triose phosphate intermediates in glycolysis give rise to the glycerol moiety of triacylglycerols. Pyruvate and intermediates of the citric acid cycle provide the carbon skeletons for the synthesis of nonessential or dispensable amino acids (see Chapter 27), and acetyl-CoA is the precursor of fatty acids (see Chapter 23) and cholesterol (see Chapter 26) and hence of all the steroid hormones synthesized in the body. Gluconeogenesis (see Chapter 19) is the process of synthesizing glucose from noncarbohydrate precursors such as lactate, amino acids, and glycerol.

The source of long-chain fatty acids is either dietary lipid or de novo synthesis from acetyl-CoA derived from carbohydrate or amino acids. Fatty acids may be oxidized to acetyl-CoA
FIGURE 14-2 Overview of carbohydrate metabolism showing the major pathways and end products. Gluconeogenesis is not shown.
(B-oxidation) or esterified with glycerol, forming triacylglycerol as the body's main fuel reserve.
Acetyl-CoA formed by -oxidation of fatty acids may undergo three fates (Figure 14-3):
1. As with acetyl-CoA arising from glycolysis, it is oxidized to CO, + H,O via the citric acid cycle.
2. It is the precursor for synthesis of cholesterol and other steroids.
3. In the liver, it is used to form the ketone bodies, acetoacetate, and 3-hydroxybutyrate (see Chapter 22), which are important fuels in prolonged fasting and starvation.

The amino acids are required for protein synthesis (Figure 14-4). Some must be supplied in the diet (the essential or indispensable amino acids), since they cannot be synthesized in the body.
“amino
FIGURE 14-3 Overview of fatty acid metabolism showing the major pathways and end products. The ketone bodies are acetoacetate, 3-hydroxybutyrate, and acetone (which is formed nonenzymically by decarboxylation of acetoacetate).
The remainder are nonessential or dispensable amino acids, which are supplied in the diet, but can also be formed from metabolic intermediates by transamination using the amino group from other amino acids (see Chapter 27). After deamination, amino nitrogen is excreted as urea, and the carbon skeletons that remain after transamination may (1) be oxidized to CO, via the citric acid cycle, (2) be used to synthesize glucose (gluconeogenesis, see Chapter 19), or (3) form ketone bodies or acetyl-CoA, which may be oxidized or used for synthesis of fatty acids (see Chapter 28).
Several amino acids are also the precursors of other compounds, for example, purines, pyrimidines, hormones such as epinephrine and thyroxine, and neurotransmitters.

In addition to studies in the whole organism, the location and integration of metabolic pathways is revealed by studies at two levels of organization. At the tissue and organ level, the nature of the substrates entering and metabolites leaving tissues and organs can be measured. At the subcellular level, each cell organelle (eg, the mitochondrion) or compartment (eg, the cytosol) has specific roles that form part of a subcellular pattern of metabolic pathways.
FIGURE 14-4 Overview of amino acid metabolism showing the major pathways and end products.

Amino acids resulting from the digestion of dietary protein and glucose resulting from the digestion of carbohydrates are absorbed via the hepatic portal vein. The liver has the role of regulating the blood concentration of these water-soluble metabolites (Figure 14-5). In the case of glucose, this is achieved by taking up glucose in excess of immediate requirements and using it to synthesize glycogen (glycogenesis, Chapter 18) or fatty acids (lipogenesis, Chapter 23). Between meals, the liver acts to maintain the blood glucose concentration by breaking
FIGURE 14-5 Transport and fate of major carbohydrate and amino acid substrates and metabolites. Note that there is little free glucose in muscle, since it is rapidly phosphorylated following uptake.
down glycogen (glycogenolysis, Chapter 18) and, together with the kidney, by converting noncarbohydrate metabolites such as lactate, glycerol, and amino acids to glucose (gluconeogenesis, Chapter 19). The maintenance of an adequate blood concentration of glucose is essential for those tissues for which it is either the major fuel (the brain) or the only fuel (erythrocytes). The liver also synthesizes the major plasma proteins (eg, albumin) and deaminates amino acids that are in excess of requirements, synthesizing urea, which is transported to the kidney and excreted (see Chapter 28).
A, D, E, and K (see Chapter 44). Unlike glucose and amino acids absorbed from the small intestine, chylomicron triacylglycerol is not taken up directly by the liver. It is first metabolized by tissues that have lipoprotein lipase, which hydrolyzes the triacylglycerol, releasing fatty acids that are incorporated into tissue lipids or oxidized as fuel. The chylomicron remnants are cleared by the liver. The other major source of longchain fatty acids is synthesis (lipogenesis) from carbohydrate, in adipose tissue and the liver (see Chapter 23).

Skeletal muscle utilizes glucose as a fuel, both aerobically, forming CO,, and anaerobically, forming lactate. It stores glycogen as a fuel for use in muscle contraction and synthesizes muscle protein from plasma amino acids. Muscle accounts for approximately 50% of body mass and consequently represents a considerable store of protein that can be drawn upon to supply amino acids for gluconeogenesis in starvation (see Chapter 19). The main dietary lipids (Figure 14-6) are triacylglycerols that are hydrolyzed to monoacylglycerols and fatty acids in the gut, then reesterified in the intestinal mucosa. Here they are packaged with protein and secreted into the lymphatic system and thence into the bloodstream as chylomicrons, the largest of the plasma lipoproteins (see Chapter 25). Chylomicrons also contain other lipid-soluble nutrients, including vitamins
Adipose tissue triacylglycerol is the main fuel reserve of the body. It is hydrolyzed (lipolysis) and glycerol and nonesterified (free) fatty acids are released into the circulation. Glycerol is a substrate for gluconeogenesis (see Chapter 19). The fatty acids are transported bound to serum albumin; they are taken up by most tissues (but not brain or erythrocytes) and either esterified to triacylglycerols for storage or oxidized as a fuel. In the liver, newly synthesized triacylglycerol and triacylglycerol from chylomicron remnants (see Figure 25-3) is secreted into the circulation in very low-density lipoprotein (VLDL). This triacylglycerol undergoes a fate similar to that of chylomicrons. Partial oxidation of fatty acids in the liver leads to ketone body production (ketogenesis, Chapter 22). Ketone bodies are exported to extrahepatic tissues, where they provide a fuel in prolonged fasting and starvation.

Compartmentation of pathways in separate subcellular compartments or organelles permits integration and regulation of metabolism. Not all pathways are of equal importance in all cells. Figure 14-7 depicts the subcellular compartmentation of metabolic pathways in a liver parenchymal cell.
The central role of the mitochondrion is immediately apparent, since it acts as the focus of carbohydrate, lipid, and amino acid metabolism. It contains the enzymes of the citric
acid cycle (see Chapter 16), B-oxidation of fatty acids and ketogenesis (see Chapter 22), as well as the respiratory chain and ATP synthase (see Chapter 13). Glycolysis (see Chapter 17), the pentose phosphate pathway (see Chapter 20), and fatty acid synthesis (see Chapter 23) all occur in the cytosol. In gluconeogenesis (see Chapter 19), substrates such as lactate and pyruvate, which are formed in the cytosol, enter the mitochondrion to yield oxaloacetate as a precursor for the synthesis of glucose in the cytosol. The membranes of the endoplasmic reticulum contain the enzyme system for triacylglycerol synthesis (see Chapter 24), and the ribosomes are responsible for protein synthesis (see Chapter 37).

Regulation of the overall flux through a pathway is important to ensure an appropriate supply of the products of that pathway. It is achieved by control of one or more key reactions in the pathway, catalyzed by regulatory enzymes. The physicochemical factors that control the rate of an enzyme-catalyzed reaction, such as substrate concentration, are of primary importance in the control of the overall rate of a metabolic pathway (see Chapter 9).

Ina reaction at equilibrium, the forward and reverse reactions occur at equal rates, and there is therefore no net flux in either direction.

equilibrium or nonequilibrium, but fall somewhere between the two extremes.

The flux-generating reaction can be identified as a nonequilibrium reaction in which the K, of the enzyme is considerably lower than the normal concentration of substrate. The first reaction in glycolysis, catalyzed by hexokinase (see Figure 17-2), is such a flux-generating step because its K, for glucose of 0.05 mmol/L is well below the normal blood glucose concentration of 3 to 5 mmol/L. Later reactions then control the rate of flux through the pathway.

In vivo, under "steady-state" conditions, there is a net flux from left to right because there is a continuous supply of substrate A and continuous removal of product D. In practice, there are normally one or more nonequilibrium reactions in a metabolic pathway, where the reactants are present in concentrations that are far from equilibrium. In attempting to reach equilibrium, large losses of free energy occur, making this type of reaction essentially irreversible. Such a pathway has both flow and direction. The enzymes catalyzing nonequilibrium reactions are usually present in low concentration and are subject to a variety of regulatory mechanisms. However, most reactions in metabolic pathways cannot be classified as
In the metabolic pathway shown in Figure 14-8,

reactions A © B and C © D are equilibrium reactions and B => C is a nonequilibrium reaction. The flux through this pathway can be regulated by the availability of substrate A. This depends on its supply from the blood, which in turn depends on either food intake or key reactions that release substrates from tissue reserves into the bloodstream, for example, glycogen phosphorylase in liver (see Figure 18-1) and hormone-sensitive lipase in adipose tissue (see Figure 25-8). It also depends on the transport of substrate A into the cell.
FIGURE 14-8 Mechanisms of control of an enzyme-catalyzed reaction. Circled numbers indicate possible sites of action of hormones: ® alteration of membrane permeability; @ conversion of an inactive to an active enzyme, usually involving phosphorylation/dephosphorylation reactions; @ alteration of the rate translation of mRNA at the ribosomal level; ® induction of new mRNA formation; and © repression of mRNA formation. © and @ are rapid mechanisms of regulation, whereas ®, ®, and © are slower.
Muscle and adipose tissue only take up glucose from the bloodstream in response to the hormone insulin. Flux is also determined by removal of the end product D and the availability of cosubstrates or cofactors represented by X and Y. Enzymes catalyzing nonequilibrium reactions are often allosteric proteins subject to the rapid actions of "feed-back" or "feed-forward" control by allosteric modifiers, in immediate response to the needs of the cell (see Chapter 9). Frequently, the end product of a biosynthetic pathway inhibits the enzyme catalyzing the first reaction in the pathway. Other control mechanisms depend on the action of hormones responding to the needs of the body as a whole; they may act rapidly by altering the activity of existing enzyme molecules, or slowly by altering the rate of enzyme synthesis (see Chapter 42).

to as ketogenic, because in prolonged fasting and starvation, much of the acetyl-CoA is used for synthesis of ketone bodies in the liver.

Erythrocytes lack mitochondria and hence are wholly reliant on (anaerobic) glycolysis and the pentose phosphate pathway at all times. The brain can metabolize ketone bodies to meet about 20% of its energy requirements; the remainder must be supplied by glucose. The metabolic changes that occur in the fasting state and starvation serve to preserve glucose and the body's limited glycogen reserves for use by the brain and red blood cells, and to provide alternative metabolic fuels for other tissues. In pregnancy, the fetus requires a significant amount of glucose, as does the synthesis of lactose in lactation (Figure 14-9).

For several hours after a meal, while the products of digestion are being absorbed, there is an abundant supply of metabolic fuels. Under these conditions, glucose is the major fuel for oxidation in most tissues; this is observed as an increase in the respiratory quotient (the ratio of carbon dioxide produced/ oxygen consumed) from about 0.8 in the fasting state to near 1

Glucose uptake into muscle and adipose tissue is controlled by insulin, which is secreted by the B-islet cells of the pancreas in response to an increased concentration of glucose in the portal blood. In the fasting state, the glucose transporter of muscle and adipose tissue (GLUT-4) is in intracellular vesicles. An early response to insulin is the migration of these vesicles to the cell surface, where they fuse with the plasma membrane, exposing active glucose transporters. These insulin-sensitive tissues only take up glucose from the bloodstream to any significant extent in the presence of the hormone. As insulin secretion falls in the fasting state, so the transporters are internalized again, reducing glucose uptake. However, in skeletal muscle, the increase in cytoplasmic calcium ion concentration in response to nerve stimulation stimulates the migration of the vesicles to the cell surface and exposure of active glucose transporters whether there is significant insulin stimulation or not.
The uptake of glucose into the liver is independent of insulin, but liver has an isoenzyme of hexokinase (glucokinase) with a high K_, so that as the concentration of glucose entering the liver increases, so does the rate of synthesis of glucose-6-phosphate. This is in excess of the liver's requirement for energy-yielding metabolism, and is used mainly for
FIGURE 14-9 Metabolic interrelationships among adipose tissue, the liver, and extrahepatic tissues. In tissues such as heart, metabolic fuels are oxidized in the following order of preference: ketone bodies > fatty acids > glucose. (LPL, lipoprotein lipase; NEFA, nonesterified fatty acids; VLDL, very low-density lipoproteins.)
synthesis of glycogen. In both liver and skeletal muscle, insulin acts to stimulate glycogen synthetase and inhibit glycogen phosphorylase. Some of the additional glucose entering the liver may also be used for lipogenesis and hence triacylglycerol synthesis. In adipose tissue, insulin stimulates glucose uptake, its conversion to fatty acids, and their esterification to triacylglycerol. It inhibits intracellular lipolysis and the release of nonesterified fatty acids.

The products of lipid digestion enter the circulation as chylomicrons, the largest of the plasma lipoproteins, which are especially rich in triacylglycerol (see Chapter 25). In adipose tissue and skeletal muscle, extracellular lipoprotein lipase is synthesized and activated in response to insulin; the resultant nonesterified fatty acids are largely taken up by the tissue and used for synthesis of triacylglycerol, while the glycerol remains in the bloodstream and is taken up by the liver

Carbohydrate, Energy Yield (kJ/g) = 16. Carbohydrate, O 2 Consumed (L/g) = 0.829. Carbohydrate, CO 2 Produced (L/g) = 0.829. Carbohydrate, RQ (CO 2 Produced/ O 2 Consumed) = 1.00. Carbohydrate, Energy (kJ)/L O 2 = ~20. Protein, Energy Yield (kJ/g) = 17. Protein, O 2 Consumed (L/g) = 0.966. Protein, CO 2 Produced (L/g) = 0.782. Protein, RQ (CO 2 Produced/ O 2 Consumed) = 0.81. Protein, Energy (kJ)/L O 2 = ~20. Fat, Energy Yield (kJ/g) = 37. Fat, O 2 Consumed (L/g) = 2.016. Fat, CO 2 Produced (L/g) = 1.427. Fat, RQ (CO 2 Produced/ O 2 Consumed) = 0.71. Fat, Energy (kJ)/L O 2 = ~20. Alcohol, Energy Yield (kJ/g) = 29. Alcohol, O 2 Consumed (L/g) = 1.429. Alcohol, CO 2 Produced (L/g) = 0.966. Alcohol, RQ (CO 2 Produced/ O 2 Consumed) = 0.66. Alcohol, Energy (kJ)/L O 2 = ~20
and used for either gluconeogenesis and glycogen synthesis or lipogenesis. Fatty acids remaining in the bloodstream are taken up by the liver and reesterified. The lipid-depleted chylomicron remnants are cleared by the liver, and the remaining triacylglycerol is exported, together with that synthesized in the liver, in very low-density lipoprotein.
and glucose is released into the bloodstream for use by the brain and erythrocytes.

Under normal conditions, the rate of tissue protein catabolism is more or less constant throughout the day; it is only in cachexia associated with advanced cancer and other diseases that there is an increased rate of protein catabolism. There is net protein catabolism in the fasting state, when the rate of protein synthesis falls, and net protein synthesis in the fed state, when the rate of synthesis increases by 20 to 25%. The increased rate of protein synthesis in response to increased availability of amino acids and metabolic fuel is again a response to insulin. Protein synthesis is an energy expensive process; it may account for up to 20% of resting energy expenditure after a meal, but only 9% in the fasting state.

There is
a
small fall
in plasma
glucose in
the fasting
state, and
then
Plasma little
change nonesterified
rise little
as fasting
more in
starvation;
is prolonged
increase as
into starvation.
fasting ketone
butyrate)
bodies increases
In the
markedly fasting
portal state,
blood secretion
is prolonged,
but then
the
(acetoacetate
(Table as
the coming
from decreases,
take up
less by
a-cells and
glucose.
and
14-2,
Figure concentration
the plasma
3-hydroxy-
14-10).
of glucose
small intestine
skeletal
The of
the muscle
increase pancreas
in the
falls, insulin
and adipose
in secretion
inhibits activates
glycogen phosphorylase
glycogen in
tissue of
glucagon synthetase,
and the
liver.
The resulting
glucose-6-phosphate is
hydrolyzed by
glucose-6-phosphatase,
Muscle glycogen cannot contribute directly to plasma glucose, since muscle lacks glucose-6-phosphatase, and the primary use of muscle glycogen is to provide a source of glucose-6-phosphate for energy-yielding metabolism in the muscle itself. However, acetyl-CoA formed by oxidation of fatty acids in muscle inhibits pyruvate dehydrogenase, leading to an accumulation of pyruvate. Most of this is transaminated to alanine, at the expense of amino acids arising from breakdown of muscle protein. The alanine, and much of the keto acids resulting from this transamination are exported from muscle, and taken up by the liver, where the alanine is transaminated to yield pyruvate. The resultant amino acids are largely exported back to muscle, to provide amino groups for formation of more alanine, while the pyruvate provides a substrate for gluconeogenesis in the liver.

In adipose tissue, the decrease in insulin and increase in glucagon results in inhibition of lipogenesis, inactivation and internalization of lipoprotein lipase, and activation of intracellular hormone-sensitive lipase (see Chapter 25). This leads to release from adipose tissue of increased amounts of glycerol (which is a substrate for gluconeogenesis in the liver) and nonesterified fatty acids, which are used by liver, heart, and skeletal muscle as their preferred metabolic fuel, so sparing glucose.
Although muscle preferentially takes up and metabolizes nonesterified fatty acids in the fasting state, it cannot meet all of its energy requirements by B-oxidation. By contrast, the liver has a greater capacity for B-oxidation than is required to meet its own energy needs, and as fasting becomes more prolonged, it forms more acetyl-CoA than can be oxidized. This acetyl-CoA is used to synthesize the ketone bodies (see Chapter 22), which are major metabolic fuels for skeletal and heart muscle and can meet up to 20% of the brain's energy needs. In prolonged

Glucose, Fed = 5.5. Glucose, 40-h Fasting = 3.6. Glucose, 7 Days Starvation = 3.5. Nonesterified fatty acids, Fed = 0.30. Nonesterified fatty acids, 40-h Fasting = 1.15. Nonesterified fatty acids, 7 Days Starvation = 1.19. Ketone bodies, Fed = Negligible. Ketone bodies, 40-h Fasting = 2.9. Ketone bodies, 7 Days Starvation = 4.5
starvation, glucose may represent less than 10% of whole body energy-yielding metabolism.
Were there no other source of glucose, liver and muscle
glycogen
would
be
exhausted
after
about
18
hours
fasting.
As
fasting
becomes
more
prolonged,
so
an
amount
of
the
amino
acids
released
as
a
result
of
protein
is
uti-
lized
in
the
liver
and
kidneys
for
gluconeogenesis
(Table
14-3).

FIGURE 14-10 Relative changes in plasma hormones and metabolic fuels during the onset of starvation.
In prolonged starvation, as adipose tissue reserves are depleted, there is a very considerable increase in the net rate of protein catabolism to provide amino acids, not only as substrates for gluconeogenesis, but also as the main metabolic fuel of all tissues. Death results when essential tissue proteins are catabolized and not replaced. In patients with cachexia as a result of release of cytokines in response to tumors and disease, there is an increase in the rate of tissue protein catabolism, as well as a considerably increased metabolic rate, so they are in a state of advanced starvation. Again, death results when essential tissue proteins are catabolized and not replaced. The high demand for glucose by the fetus, and for lactose synthesis in lactation, can lead to ketosis. This may be seen as

Substrates = Glucose, glycogen. Fast twitch muscle, Major Products Exported = Lactate, (alanine and ketoacids in fasting). Fast twitch muscle, Specialist Enzymes = -. Slow twitch muscle, Major Pathways = β-Oxidation and citric acid cycle. Slow twitch muscle, Main Substrates = Ketone bodies, chylomicron, andVLDL triacylglycerol. Slow twitch muscle, Major Products Exported = -. Slow twitch muscle, Specialist Enzymes = Lipoprotein lipase, very active electron transport chain. Kidney, Major Pathways = Gluconeogenesis. Kidney, Main Substrates = Nonesterified fatty acids, lactate, glycerol, glucose. Kidney, Major Products Exported = Glucose. Kidney, Specialist Enzymes = Glycerol kinase, phosphoenolpyruvate carboxykinase. Erythrocytes, Major Pathways = Anaerobic glycolysis, pentose phosphate pathway. Erythrocytes, Main Substrates = Glucose. Erythrocytes, Major Products Exported = Lactate. Erythrocytes, Specialist Enzymes = Hemoglobin, enzymes of pentose phosphate pathway
mild ketosis with hypoglycemia in human beings; in lactating cattle and in ewes carrying a twin pregnancy, there may be very pronounced ketoacidosis and profound hypoglycemia. In poorly controlled type 1 diabetes mellitus, patients may become hyperglycemic, both as a result of lack of insulin to stimulate uptake and utilization of glucose, and because in the absence of insulin to antagonize the actions of glucagon, there is increased gluconeogenesis from amino acids in the liver. At the same time, the lack of insulin to antagonize the actions of glucagon results in increased lipolysis in adipose tissue, and the resultant nonesterified fatty acids are substrates for ketogenesis in the liver.

Utilization of the ketone bodies in muscle (and other tissues) may be impaired because of the lack of oxaloacetate (all tissues have a requirement for some glucose metabolism to maintain an adequate amount of oxaloacetate for citric acid cycle activity). In uncontrolled diabetes, the ketosis may be severe enough to result in pronounced acidosis (ketoacidosis); acetoacetate and 3-hydroxybutyrate are relatively strong acids. Coma results from both the acidosis and also the considerably increased osmolality of extracellular fluid (mainly as a result of the hyperglycemia, and diuresis resulting from the excretion of glucose and ketone bodies in the urine).

™ The products of digestion provide the tissues with the building blocks for the biosynthesis of complex molecules and also with the fuel for metabolic processes.
@ Nearly all products of digestion of carbohydrate, fat, and protein are metabolized to a common metabolite, acetyl-CoA, before oxidation to CO, in the citric acid cycle.
™ Acetyl-CoA is also the precursor for synthesis of long-chain fatty acids and steroids (including cholesterol) and ketone bodies.
™ Glucose provides carbon skeletons for the glycerol of triacylglycerols and nonessential amino acids.
Water-soluble products of digestion are transported directly to the liver via the hepatic portal vein. The liver regulates the concentrations of glucose and amino acids available to other tissues. Lipids and lipid-soluble products of digestion enter the bloodstream from the lymphatic system, and the liver clears the remnants after extrahepatic tissues have taken up fatty acids.
Pathways are compartmentalized within the cell. Glycolysis, glycogenesis, glycogenolysis, the pentose phosphate pathway, and lipogenesis occur in the cytosol. The mitochondria contain the enzymes of the citric acid cycle and for B-oxidation of fatty acids, as well as the respiratory chain and ATP synthase. The membranes of the endoplasmic reticulum contain the enzymes for a number of other processes, including triacylglycerol synthesis and drug metabolism.
Metabolic pathways are regulated by rapid mechanisms affecting the activity of existing enzymes, that is, allosteric and covalent modification (often in response to hormone action) and slow mechanisms that affect the synthesis of enzymes.
Dietary carbohydrate and amino acids in excess of requirements can be used for fatty acid and hence triacylglycerol synthesis.
In fasting and starvation, glucose must be provided for the brain and red blood cells; in the early fasting state, this is supplied from glycogen reserves. In order to spare glucose, muscle and other tissues do not take up glucose when insulin secretion is low; they utilize fatty acids (and later ketone bodies) as their preferred fuel.
Adipose tissue releases nonesterified fatty acids in the fasting state. In prolonged fasting and starvation, these are used by the liver for synthesis of ketone bodies, which are exported to provide the major fuel for muscle.

Most amino acids, arising from the diet or from tissue protein turnover, can be used for gluconeogenesis, as can the glycerol from triacylglycerol.
Neither fatty acids, arising from the diet or from lipolysis of adipose tissue triacylglycerol, nor ketone bodies, formed from fatty acids in the fasting state, can provide substrates for gluconeogenesis.

VAT ULC NCR UIER SCO aA
2 glycomics. Explain what is meant by the glycome, glycobiology, and the science of
. Explain what is meant by the terms monosaccharide, disaccharide,
oligosaccharide, and polysaccharide.
m= Explain the different ways in which the structures of glucose and other
monosaccharides can be represented, and describe the various types of
isomerism
of
sugars
and
the
pyranose
and
furanose
ring
= Describe the formation of glycosides and the structures of the important disaccharides and polysaccharides.
Explain what is meant by the glycemic index of a carbohydrate.
Describe the roles of carbohydrates in cell membranes and lipoproteins.

Carbohydrates are widely distributed in plants and animals; they have important structural and metabolic roles. In plants, glucose is synthesized from carbon dioxide and water by photosynthesis and stored as starch or used to synthesize the cellulose of the plant cell walls. Animals can synthesize carbohydrates from amino acids, but most are derived ultimately from plants. Glucose is the most important carbohydrate; most dietary carbohydrate is absorbed into the bloodstream as glucose formed by hydrolysis of dietary starch and disaccharides, and other sugars are converted to glucose in the liver. Glucose is the major metabolic fuel of mammals (except ruminants) and a universal fuel of the fetus. It is the precursor for synthesis of all the other carbohydrates in the body, including glycogen for storage, ribose and deoxyribose in nucleic acids, galactose for synthesis of lactose in milk, in glycolipids, and in combination with protein in glycoproteins (see Chapter 46) and proteoglycans. Diseases associated with carbohydrate metabolism include diabetes mellitus, galactosemia, glycogen storage diseases, and lactose intolerance.
Glycobiology is the study of the roles of sugars in health and disease. The glycome is the entire complement of sugars of an organism, whether free or in more complex molecules. Glycomics, an analogous term to genomics and proteomics, is the comprehensive study of glycomes, including genetic, physiological, pathological, and other aspects.
A very large number of glycoside links can be formed between sugars. For example, three different hexoses may be linked to each other to form over 1000 different trisaccharides. The conformations of the sugars in oligosaccharide chains vary depending on their linkages and proximity to other molecules with which the oligosaccharides may interact. Oligosaccharide chains encode biological information that depends on their constituent sugars, sequences, and linkages.

Carbohydrates are classified as follows:
1. Monosaccharides are those sugars that cannot be hydrolyzed into simpler carbohydrates. They may be classified as trioses, tetroses, pentoses, hexoses, or heptoses, depending on the number of carbon atoms (3-7), and as aldoses or ketoses, depending on whether they have an aldehyde or ketone group. Examples are listed in Table 15-1. In addition to aldehydes and ketones, the polyhydric alcohols (sugar alcohols or polyols), in which the aldehyde or ketone group has been reduced to an alcohol group, also occur naturally in foods. They are synthesized

Trioses (C 3 H 6 O 3 ), Aldoses = Glycerose (glyceraldehyde). Trioses (C 3 H 6 O 3 ), Ketoses = Dihydroxyacetone. Tetroses (C 4 H 8 O 4 ), Aldoses = Erythrose. Tetroses (C 4 H 8 O 4 ), Ketoses = Erythrulose. Pentoses (C 5 H 10 O 5 ), Aldoses = Ribose. Pentoses (C 5 H 10 O 5 ), Ketoses = Ribulose. Hexoses (C 6 H 12 O 6 ), Aldoses = Glucose, galactose, mannose. Hexoses (C 6 H 12 O 6 ), Ketoses = Fructose. Heptoses (C 7 H 14 O 7 ), Aldoses = -. Heptoses (C 7 H 14 O 7 ), Ketoses = Sedoheptulose
by reduction of monosaccharides for use in the manufacture of foods for weight reduction and for diabetics. They are poorly absorbed, and have about half the energy yield of sugars.
2. Disaccharides are condensation products of two monosaccharide units, for example, lactose, maltose, isomaltose, sucrose, and trehalose.
3. Oligosaccharides are condensation products of 3 to 10 monosaccharides. Most are not digested by human enzymes.
4. Polysaccharides are condensation products of more than 10 monosaccharide units; examples are the starches and dextrins, which may be linear or branched polymers. Polysaccharides are sometimes classified as hexosans or pentosans, depending on the constituent monosaccharides (hexoses or pentoses, respectively). In addition to starches and dextrins (which are hexosans), foods contain a wide variety of other polysaccharides that are collectively known as nonstarch polysaccharides; they are not digested by human enzymes, and are the major component of dietary fiber. Examples are cellulose from plant cell walls (a glucose polymer; see Figure 15-13) and inulin, the storage carbohydrate in some plants (a fructose polymer; see Figure 15-13).

The straight-chain structural formula (aldohexose; Figure 15-1A) can account for some of the properties of glucose, but a cyclic structure (a hemiacetal formed by reaction between the aldehyde group and a hydroxyl group) is thermodynamically favored and accounts for other properties. The cyclic structure is normally drawn as shown in Figure 15-1B, the Haworth projection, in which the molecule is viewed from the side and above the plane of the ring; the bonds nearest to the viewer
FIGURE 15-1 -Glucose. (A) Straight-chain form. (B) a-pglucose; Haworth projection. (C) a-p-glucose; chair form.
are bold and thickened, and the hydroxyl groups are above or below the plane of the ring. The hydrogen atoms attached to each carbon are not shown in this figure. The ring is actually in the form of a chair (Figure 15-1C).

Glucose, with four asymmetric carbon atoms, can form 16 isomers. The more important types of isomerism found with glucose are as follows.
1. pand L-isomerism: The designation of a sugar isomer as the p form or its mirror image as the L form is determined by its spatial relationship to the parent compound of the carbohydrates, the three-carbon sugar glycerose (glyceraldehyde). The x and p forms of this sugar, and of glucose, are shown in Figure 15-2. The orientation of the —H and —OH groups around the carbon atom adjacent to the terminal alcohol carbon (carbon 5 in glucose) determines whether the sugar belongs to the p or L series. When the —OH group on this carbon is on the right (as seen in Figure 15-2), the sugar is the p-isomer; when it is on the left, it is the L-isomer. Most of the naturally occurring monosaccharides are D sugars, and the enzymes responsible for their metabolism are specific for this configuration.
2. The presence of asymmetric carbon atoms also confers optical activity on the compound. When a beam of plane-polarized light is passed through a solution of an optical isomer, it rotates either to the right, dextrorotatory (+), or to the left, levorotatory (-). The direction of

rotation of polarized light is independent of the stereochemistry of the sugar, so it may be designated p(-), p(+), L(-), or L(+). For example, the naturally occurring form of fructose is the p(—) isomer. Confusingly, dextrorotatory (+) was at one time called d-, and levorotatory (-) l-. This nomenclature is obsolete, but may sometimes be found; it is unrelated to pand L-isomerism. In solution, glucose is dextrorotatory, and glucose solutions are sometimes known as dextrose.
3. Pyranose and furanose ring structures: The ring structures of monosaccharides are similar to the ring structures of either pyran (a six-membered ring) or furan (a fivemembered ring) (Figures 15-3 and 15-4). For glucose in solution, more than 99% is in the pyranose form.
4. Alphaand beta-anomers: The ring structure of an aldose is a hemiacetal, since it is formed by reaction between an aldehyde and an alcohol group. Similarly, the ring structure of a ketose is a hemiketal. Crystalline glucose is a-p-glucopyranose. The cyclic structure is retained in the solution, but isomerism occurs about position 1, the carbonyl or anomeric carbon atom, to give a mixture
a-D-Glucopyranose a-D-Glucofuranose
FIGURE 15-3 Pyranose and furanose forms of glucose.
FIGURE 15-4 Pyranose and furanose forms of fructose.
of a-glucopyranose (38%) and B-glucopyranose (62%). Less than 0.3% is represented by aand B-anomers of glucofuranose.

5. Epimers: Isomers differing as a result of variations in configuration of the —OH and —H on carbon atoms 2, 3, and 4 of glucose are known as epimers. Biologically, the most important epimers of glucose are mannose (epimerized at carbon 2) and galactose (epimerized at carbon 4) (Figure 15-5).
6. Aldose-ketose isomerism: Fructose has the same molecular formula as glucose but differs in that there is a potential keto group in position 2, the anomeric carbon of fructose, whereas in glucose there is a potential aldehyde group in position 1, the anomeric carbon. Examples of aldose and ketose sugars are shown in Figures 15-6 and 15-7, respectively. Chemically, aldoses are reducing compounds, and are sometimes known as reducing sugars. This provides the basis for a simple chemical test for glucose in urine in poorly controlled diabetes mellitus, by reduction of an alkaline copper solution (see Chapter 48).

Derivatives of trioses, tetroses, and pentoses and of the sevencarbon sugar sedoheptulose are formed as metabolic intermediates in glycolysis (see Chapter 17) and the pentose phosphate pathway (see Chapter 20). Pentoses are important in nucleotides, nucleic acids, and several coenzymes (Table 15-2). Glucose, galactose, fructose, and mannose are physiologically the most important hexoses (Table 15-3). The biochemically important aldoses are shown in Figure 15-6, and ketoses in 15-7.
Figure Inaddition, carboxylicacid derivatives of glucoseareimportant, including p-glucuronate (for glucuronide formation and in glycosaminoglycans), its metabolic derivative, L-iduronate (in glycosaminoglycans, Figure 15-8) and 1-gulonate (an intermediate in the uronic acid pathway; see Figure 20-4).
a-D-Glucose a-D-Galactose
a-D-mannose

CHO
CHO,  = CHO. CHO, CHO = H HO C. CHO, CHO = OH H C. CHO, CHO = H HO C. CHO, CHO = OH H C. CHO, OH H C = H HO C. CHO, H HO C = H HO C. CHO, OH H C = H HO C. OH H C,  = OH H C. OH H C, CHO = H HO C. OH H C, CHO = H HO C. OH H C, CHO = OH H C. OH H C, CHO = OH H C. OH H C, OH H C = H HO C. OH H C, H HO C = OH H C. OH H C, OH H C = OH H C. CH OH,  = OH H C. CH OH, CHO = OH H C. CH OH, CHO = OH H C. CH OH, CHO = OH H C. CH OH, CHO = OH H C. CH OH, OH H C = OH H C. CH OH, H HO C = OH H C. CH OH, OH H C = OH H C. D-Glycerose,  = CH 2 OH. D-Glycerose, CHO = CH 2 OH. D-Glycerose, CHO = CH 2 OH. D-Glycerose, CHO = CH 2 OH. D-Glycerose, CHO = CH 2 OH. D-Glycerose, OH H C = CH 2 OH. D-Glycerose, H HO C = CH 2 OH. D-Glycerose, OH H C = CH 2 OH. ( D-glyceraldehyde),  = D-Erythrose. ( D-glyceraldehyde), CHO = D-Lyxose. ( D-glyceraldehyde), CHO = D-Xylose. ( D-glyceraldehyde), CHO = D-Arabinose. ( D-glyceraldehyde), CHO = D-Ribose. ( D-glyceraldehyde), OH H C = D-Galactose. ( D-glyceraldehyde), H HO C = D-Mannose. ( D-glyceraldehyde), OH H C = D-Glucose
FIGURE 15-6 Examples of aldoses of physiological significance.
CH,OH

CH,OH
CH,OH
c=0 |
|
|
c=o
c=o
HO—C—H
|
CHOH
HO-C~H
H—G—OH
H—G—OH
G=o
H—~G—OH
H—G—OH
H—G—OH
CH,OH
CH,OH
CH,OH
CH,OH
Dihydroxyacetone
D-Xylulose
D-Ribulose

d -Ribose, Source = Nucleic acids and metabolic intermediate. d -Ribose, Biochemical and Clinical Importance = Structural component of nucleic acids and coenzymes, including ATP, NAD(P), and flavin coenzymes. d -Ribulose, Source = Metabolic intermediate. d -Ribulose, Biochemical and Clinical Importance = Intermediate in the pentose phosphate pathway. d -Arabinose, Source = Plant gums. d -Arabinose, Biochemical and Clinical Importance = Constituent of glycoproteins. d -Xylose, Source = Plant gums, proteoglycans, glycosaminoglycans. d -Xylose, Biochemical and Clinical Importance = Constituent of glycoproteins. l -Xylulose, Source = Metabolic intermediate. l -Xylulose, Biochemical and Clinical Importance = Excreted in the urine in essential pentosuria

d -Glucose, Source = Fruit juices, hydrolysis of starch, cane or beet sugar, maltose and lactose. d -Glucose, Biochemical Importance = The main metabolic fuel for tissues; 'blood sugar'. d -Glucose, Clinical Significance = Excreted in the urine (glucosuria) in poorly controlled diabetes mellitus as a result of hyperglycemia. d -Fructose, Source = Fruit juices, honey, hydrolysis of cane or beet sugar and inulin, enzymic isomerization of glucose syrups for food manufacture. d -Fructose, Biochemical Importance = Readily metabolized either via glucose or directly. d -Fructose, Clinical Significance = Hereditary fructose intolerance leads to fructose accumulation and hypoglycemia. d -Galactose, Source = Hydrolysis of lactose. d -Galactose, Biochemical Importance = Readily metabolized to glucose; synthesized in the mammary gland for synthesis of lactose in milk. A constituent of glycolipids and glycoproteins. d -Galactose, Clinical Significance = Hereditary galactosemia as a result of failure to metabolize galactose leads to cataracts. d -Mannose, Source = Hydrolysis of plant mannan gums. d -Mannose, Biochemical Importance = Constituent of glycoproteins. d -Mannose, Clinical Significance = 
CH,OH
| ¢ =O
HO—C—H
|
H—C—OH
|
H—~G—OH
4 G—OH
CH,OH
D-Sedoheptulose

Glycosides are formed by condensation between the hydroxyl group of the anomeric carbon of a monosaccharide, and a second compound that may be another monosaccharide or, in the case of an aglycone, not a sugar. If the second group is also a hydroxyl, the O-glycosidic bond is an acetal link because it results from a reaction between a hemiacetal group (formed from an aldehyde and an —OH group) and another —OH group. If the hemiacetal portion is glucose, the resulting compound is a glucoside; if galactose, a galactoside; and so on. If the second group is an amine, an N-glycosidic bond is formed, for example, between adenine and ribose in nucleotides such as ATP (see Figure 11-4).
Glycosides are widely distributed in nature; the aglycone may be methanol, glycerol, a sterol, a phenol, or a base such as adenine. The glycosides that are important in medicine because of their action on the heart (cardiac glycosides), all contain steroids as the aglycone. These include derivatives of digitalis and strophanthus such as ouabain, an inhibitor of the Na*-K*-ATPase of cell membranes. Other glycosides include antibiotics such as streptomycin.

Deoxy sugars are those in which one hydroxyl group has been replaced by hydrogen. An example is deoxyribose (Figure 15-9) in DNA. The deoxy sugar L-fucose (see Figure 15-15) occurs in glycoproteins; 2-deoxyglucose is used experimentally as an inhibitor of glucose metabolism.

The amino sugars include p-glucosamine, a constituent of hyaluronic acid (Figure 15-10), p-galactosamine (also known as chondrosamine), a constituent of chondroitin, and
FIGURE 15-9 2-Deoxy-p-ribofuranose (B-form).
FIGURE 15-10 Glucosamine (2-amino-p-glucopyranose) (a-form). Galactosamine is 2-amino-p-galactopyranose. Both glucosamine and galactosamine occur as N-acetyl derivatives in complex carbohydrates, for example, glycoproteins.
D-mannosamine. Several antibiotics (eg, erythromycin) contain amino sugars, which are important for their antibiotic activity.

The disaccharides are sugars composed of two monosaccharide residues linked by a glycoside bond (Figure 15-11). The physiologically important disaccharides are maltose, sucrose, and lactose (Table 15-4). Hydrolysis of sucrose yields a mixture of glucose and fructose called "invert sugar" because fructose is strongly levorotatory and changes (inverts) the weaker dextrorotatory action of sucrose.

Polysaccharides include a number of physiologically important carbohydrates.
Starch is a homopolymer of glucose forming an a-glucosidic chain, called a glucosan or glucan. It is the most important dietary carbohydrate in cereals, potatoes, legumes, and other vegetables. The two main constituents are amylose (13-20%), which has a nonbranching helical structure, and amylopectin (80-87%), which consists of branched chains, consists of 24 to 30 glucose residues with al — 4 linkages in the chains and by al — 6 linkages at the branch points (Figure 15-12).
The extent to which starch in foods is hydrolyzed by amylase is determined by its structure, the degree of crystallization or hydration (the result of cooking), and whether it is enclosed in intact (and indigestible) plant cell walls. The glycemic index of a starchy food is a measure of its digestibility, based on the extent to which it raises the blood concentration of glucose compared with an equivalent amount of glucose or a reference food such as white bread or boiled rice. Glycemic index ranges from 1 (or 100%) for starches that are readily hydrolyzed in the small intestine to 0 for those that are not hydrolysed at all.
Glycogen is the storage polysaccharide in animals and is sometimes called animal starch. It is a more highly branched structure than amylopectin, with chains of 12 to 15 a-pglucopyranose residues (in al — 4 glucosidic linkage) with

branching by means of al — 6 glucosidic bonds. Muscle glycogen granules (6-particles) are spherical and contain up to 60,000 glucose residues; in liver there are similar granules and also rosettes of glycogen granules that appear to be aggregated -particles.
Inulin is a polysaccharide of fructose (a fructosan) found in tubers and roots of dahlias, artichokes, and dandelions. It is readily soluble in water and is used to determine the glomerular filtration rate, but it is not hydrolyzed by intestinal enzymes, so has no nutritional value. Dextrins are intermediates in the hydrolysis of starch. Cellulose is the chief constituent of plant cell walls. It is insoluble and consists of B-p-glucopyranose units linked by B1 — 4 bonds to form long, straight chains strengthened by cross-linking hydrogen bonds. Mammals lack any enzyme that hydrolyzes the B1 — 4 bonds, and so cannot digest cellulose. It is the major component of dietary fiber. Microorganisms in the gut of ruminants and other herbivores can hydrolyze the linkage and ferment the products to

Sucrose, Composition = O-α- d -glucopyranosyl-(1 → 2)-β- d -fructofuranoside. Sucrose, Source = Cane and beet sugar, sorghum and some fruits and vegetables. Sucrose, Clinical Significance = Rare genetic lack of sucrase leads to sucrose intolerance-diarrhea and flatulence. Lactose, Composition = O-β- d -galactopyranosyl-(1 → 4)-β- d -glucopyranose. Lactose, Source = Milk (and many pharmaceutical preparations as a filler). Lactose, Clinical Significance = Lack of lactase (alactasia) leads to lactose intolerance-diarrhea and flatulence; may be excreted in the urine in pregnancy. Maltose, Composition = O-α- d -glucopyranosyl-(1 → 4)-α- d -glucopyranose. Maltose, Source = Enzymic hydrolysis of starch (amylase); germinating cereals and malt. Maltose, Clinical Significance = . Isomaltose, Composition = O-α- d -glucopyranosyl-(1 → 6)-α- d -glucopyranose. Isomaltose, Source = Enzymic hydrolysis of starch (the branch points in amylopectin). Isomaltose, Clinical Significance = . Lactulose, Composition = O-α- d -galactopyranosyl-(1 → 4)-β- d -fructofuranose. Lactulose, Source = Heated milk (small amounts), mainly synthetic. Lactulose, Clinical Significance = Not hydrolyzed by intestinal enzymes, but fermented by intestinal bacteria; used as a mild osmotic laxative. Trehalose, Composition = O-α- d -glucopyranosyl-(1 → 1)-α- d -glucopyranoside. Trehalose, Source = Yeasts and fungi; the main sugar of insect hemolymph. Trehalose, Clinical Significance = 

FIGURE 15-12 The structure of starch and glycogen. Amylose is a linear polymer of glucose residues linked a1—>4, which coils into a helix. Amylopectin and glycogen consist of short chains of glucose residues linked a1—>4 with branch points formed by a1—6 glycoside bonds. The glycogen molecule is a sphere ~21 nm in diameter that can be seen in electron micrographs. It has a molecular mass of ~10' Da and consists of polysaccharide chains, each containing about 13 glucose residues. The chains are either branched or unbranched and are arranged in 12 concentric layers. The branched chains (each has two branches) are found in the inner layers and the unbranched chains in the outermost layer. The blue dot at the center of the glycogen molecule is glycogenin, the primer molecule for glycogen synthesis.
short-chain fatty acids as a major energy source. There is some bacterial metabolism of cellulose in the human colon. Chitin is a structural polysaccharide in the exoskeleton of crustaceans and insects, and also in mushrooms. It consists of N-acetylp-glucosamine units joined by B1 — 4 glycosidic bonds. Pectin occurs in fruits; it is a polymer of galacturonic acid linked a1 — 4, with some galactose an/or arabinose branches, and is partially methylated (Figure 15-13).
are glycosylated. The sialic acids are Nor O-acyl derivatives of neuraminic acid (Figure 15-15). Neuraminic acid is a nine-carbon sugar derived from mannosamine (an epimer of glucosamine) and pyruvate. Sialic acids are constituents of both glycoproteins and gangliosides.

Glycosaminoglycans (mucopolysaccharides) are complex carbohydrates containing amino sugars and uronicacids. They may be attached to a protein molecule to form a proteoglycan. Proteoglycans provide the ground or packing substance of connective tissue (see Chapter 50). They hold large quantities of water and occupy space, thus cushioning or lubricating other structures, because of the large number of —OH groups and negative charges on the molecule, which, by repulsion, keep the carbohydrate chains apart. Examples are hyaluronic acid, chondroitin sulfate, and heparin (Figure 15-14).
Glycoproteins (also known as mucoproteins) are proteins containing branched or unbranched oligosaccharide chains (Table 15-5), including fucose (Figure 15-15). They occur in cell membranes (see Chapters 40 and 46) and many proteins

Approximately 5% of the weight of cell membranes is the carbohydrate part of glycoproteins (see Chapter 46) and glycolipids. Their presence on the outer surface of the plasma membrane (the glycocalyx) has been shown with the use of plant lectins, proteins that bind specific glycosyl residues. For example, concanavalin A binds a-glucosyl and a-mannosyl residues. Glycophorin is a major integral membrane glycoprotein of human erythrocytes. It has 130 amino acid residues and spans the lipid membrane, with polypeptide regions outside both the external and internal (cytoplasmic) surfaces. Carbohydrate chains are attached to the amino terminal portion outside the external surface. Carbohydrates are also present in apoprotein B of plasma lipoproteins.
148
SECTIONIV

FIGURE 15-14 Structure of some complex polysaccharides and glycosaminoglycans.
Acetyl hexosamines, Mannose (Man), Galactose (Gal) = N -Acetylglucosamine (GlcNAc), N -acetylgalactosamine (GalNAc). Pentoses, Mannose (Man), Galactose (Gal) = Arabinose (Ara), Xylose (Xyl). Methyl pentose, Mannose (Man), Galactose (Gal) = l -Fucose (Fuc, see Figure 15-15). Sialic acids, Mannose (Man), Galactose (Gal) = N -Acyl derivatives of neuraminic acid; the predominant sialic acid is N -acetylneuraminic acid (NeuAc, see Figure 15-15)

The glycome is the entire complement of sugars of an organism, whether free or present in more complex molecules. Glycomics is the study of glycomes, including genetic, physiological, pathological, and other aspects.
™ Carbohydrates are major constituents of animal food and
animal tissues.
They are
the type
and number
of monosaccharide residues in their molecules.
™ Glucose is the most important carbohydrate in mammalian biochemistry because nearly all carbohydrate in food is converted to glucose for metabolism.
™ Sugars have large numbers of stereoisomers because they
contain several
asymmetric carbon
atoms.
=
The physiologically important monosaccharides include glucose, the "blood sugar,' and ribose, an important constituent of nucleotides and nucleic acids.
=
The important disaccharides include maltose (glucosyl-
glucose),
an
intermediate
in
the
digestion
of
starch;
sucrose
(glucosyl-fructose),
important
as
a
dietary
containing fructose; and lactose (galactosyl-glucose), in milk.
Starch and glycogen are storage polymers of glucose in plants
and
animals,
Starch
is
the
major
metabolic
fuel
in
the
diet.
Complex carbohydrates contain other sugar derivatives such
as
amino
sugars,
uronic
acids,
and
sialic
acids.
They
include
proteoglycans
and
glycosaminoglycans,
which
are
with
elements
of
the
tissues,
and
which
are
proteins
oligosaccharide
chains;
they
are
found
in
many
situations
including
the
cell
membrane.
Oligosaccharide chains encode biological information, depending on their constituent sugars and their sequence and linkages.

After studying this chapter,
- . Describe the reactions of the citric acid cycle and the reactions that lead to the production of reducing equivalents that are oxidized in the mitochondrial electron transport chain to yield ATP.
Explain the importance of vitamins in the citric acid cycle.
Explain how the citric acid cycle provides both a route for catabolism of amino acids and also a route for their synthesis.
= Describe the main anaplerotic pathways that permit replenishment of
citric acid cycle intermediates, and how the withdrawal of oxaloacetate for gluconeogenesis is controlled.
Describe the role of the citric acid cycle in fatty acid synthesis.
oxidized cofactors. Explain how the activity of the citric acid cycle is controlled by the availability of
= Explain how hyperammonemia can lead to loss of consciousness.

The citric acid cycle (the Krebs or tricarboxylic acid cycle) is a sequence of reactions in mitochondria that oxidizes the acetyl moiety of acetyl-CoA to CO, and reduces coenzymes that are reoxidized through the electron transport chain (see Chapter 13), linked to the formation of ATP.
considerably impaired ATP formation in the central nervous system.
The citric acid cycle is the final common pathway for the oxidation of carbohydrate, lipid, and protein because glucose, fatty acids, and most amino acids are metabolized to acetylCoA or intermediates of the cycle. It also has a central role in gluconeogenesis, lipogenesis, and interconversion of amino acids. Many of these processes occur in most tissues, but liver is the only tissue in which all occur to a significant extent. The repercussions are therefore profound when, for example, large numbers of hepatic cells are damaged as in acute hepatitis or replaced by connective tissue (as in cirrhosis). The few genetic defects of citric acid cycle enzymes that have been reported are associated with severe neurological damage as a result of very
Hyperammonemia, as occurs in advanced liver disease, leads to loss of consciousness, coma, and convulsions as a result of impaired activity of the citric acid cycle, leading to reduced formation of ATP. Ammonia both depletes citric acid cycle intermediates (by withdrawing a-ketoglutarate for the formation of glutamate and glutamine) and also inhibits the oxidative decarboxylation of a-ketoglutarate.

The cycle starts with reaction between the acetyl moiety of acetyl-CoA and the four-carbon dicarboxylic acid oxaloacetate, forming a six-carbon tricarboxylic acid, citrate. In the subsequent reactions, two molecules of CO, are released and
FIGURE 16-1 The citric acid cycle, illustrating the catalytic role of oxaloacetate.
oxaloacetate is regenerated (Figure 16-1). Only a small quantity of oxaloacetate is needed for the oxidation of a large quantity of acetyl-CoA; it can be considered as playing a catalytic role, since it is regenerated at the end of the cycle. The citric acid cycle provides the main pathway for ATP formation linked to the oxidation of metabolic fuels. During the oxidation of acetyl-CoA, coenzymes are reduced, then reoxidized in the respiratory chain, linked to the formation of ATP (oxidative phosphorylation, Figure 16-2; see also Chapter 13). This process is aerobic, requiring oxygen as the final oxidant of the reduced coenzymes. The enzymes of the citric acid cycle are located in the mitochondrial matrix, either free or attached to the inner mitochondrial membrane and the crista membrane, where the enzymes and coenzymes of the respiratory chain are also found (see Chapter 13).

The initial reaction between acetyl-CoA and oxaloacetate to form citrate is catalyzed by citrate synthase, which forms a carbon-carbon bond between the methyl carbon of acetylCoA and the carbonyl carbon of oxaloacetate (Figure 16-3). The thioester bond of the resultant citryl-CoA is hydrolyzed, releasing citrate and CoASH—an exothermic reaction.
Citrate is isomerized to isocitrate by the enzyme aconitase (aconitate hydratase); the reaction occurs in two steps: dehydration to cis-aconitate and rehydration to isocitrate. Although citrate is a symmetrical molecule, aconitase reacts with citrate asymmetrically, so that the two carbon atoms that are lost in subsequent reactions of the cycle are not those that were added from acetyl-CoA. This asymmetric behavior is the result of channeling—transfer of the product of citrate synthase directly onto the active site of aconitase, without
F,
Flavoprotein
Cyt

The citric acid cycle: the major catabolic pathway for acetyl-CoA. Acetyl-CoA, the product of carbohydrate, protein, and lipid catabolism, enters the cycle by forming citrate, and is oxidized to CO, with the reduction of coenzymes. Reoxidation of the coenzymes in the respiratory chain leads to phosphorylation of ADP to ATP. For one turn of the cycle, nine ATP are generated via oxidative phosphorylation and one ATP (or GTP) arises at substrate level from the conversion of succinyl-CoA to succinate.
entering free solution. This provides integration of citric acid cycle activity with the provision of citrate in the cytosol as a source of acetyl-CoA for fatty acid synthesis. Citrate is only available in free solution to be transported from the mitochondria to the cytosol for fatty acid synthesis when aconitase is inhibited by accumulation of its product, isocitrate.
The poison fluoroacetate is found in some of the plants, and their consumption can be fatal to grazing animals. Some fluorinated compounds used as anticancer agents and industrial chemicals (including pesticides) are metabolized to fluoroacetate. It is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits aconitase, causing citrate to accumulate.
*

FIGURE 16-3 The citric acid (Krebs) cycle. Oxidation of NADH and FADH, in the respiratory chain leads to the formation of ATP via oxidative phosphorylation. In order to follow the passage of acetyl-CoA through the cycle, the two carbon atoms of the acetyl moiety are shown labeled on the carboxyl carbon (*) and on the methyl carbon (+). Although two carbon atoms are lost as co, in one turn of the cycle, these atoms are not derived from the acetyl-CoA that has immediately entered the cycle, but from that portion of the citrate molecule that was derived from oxaloacetate. However, on completion of a single turn of the cycle, the oxaloacetate that is regenerated is now labeled, which leads to labeled CO, being evolved during the second turn of the cycle. Because succinate is a symmetrical compound, "randomization" of label occurs at this step so that all four carbon atoms of oxaloacetate appear to be labeled after one turn of the cycle. During gluconeogenesis, some of the label in oxaloacetate is incorporated into glucose and glycogen (see Figure 20-1). The sites of inhibition (©) by fluoroacetate, malonate, and arsenite are indicated.

Isocitrate undergoes dehydrogenation catalyzed by isocitrate dehydrogenase to form, initially, oxalosuccinate, which remains enzyme bound and undergoes decarboxylation to a-ketoglutarate. The decarboxylation requires Mg** or Mn** ions. There are three isoenzymes of isocitrate dehydrogenase. One, which uses nicotinamide adenine dinucleotide (NAD", is found only in mitochondria. The other two use NADP* and are found in mitochondria and the cytosol. Respiratory chain-linked oxidation of isocitrate occurs through the NAD*-dependent enzyme. a-Ketoglutarate undergoes oxidative decarboxylation in a reaction catalyzed by a multienzyme complex similar to that involved in the oxidative decarboxylation of pyruvate (see Figure 17-5). The a-ketoglutarate dehydrogenase complex requires the same cofactors as the pyruvate dehydrogenase complex—thiamin diphosphate, lipoate, NAD', flavin
adenine dinucleotide (FAD), and CoA—and results in the formation of succinyl-CoA. The equilibrium of this reaction is so much in favor of succinyl-CoA formation that it must be considered to be physiologically unidirectional. As in the case of pyruvate oxidation (see Chapter 17), arsenite inhibits the reaction, causing the substrate, a-ketoglutarate, to accumulate. High concentrations of ammonia inhibit a-ketoglutarate dehydrogenase.

Succinyl-CoA is converted to succinate by the enzyme succinate thiokinase (succinyl-CoA synthetase). This is the only example of substrate-level phosphorylation in the citric acid cycle. Tissues in which gluconeogenesis occurs (the liver and kidney) contain two isoenzymes of succinate thiokinase, one specific for GDP and the other for ADP. The GTP formed is used for the decarboxylation of oxaloacetate to phosphoenolpyruvate in gluconeogenesis, and provides a regulatory link between citric acid cycle activity and the withdrawal of oxaloacetate for gluconeogenesis. Nongluconeogenic tissues have only the isoenzyme that phosphorylates ADP.
When ketone
bodies are
being metabolized
in extrahepatic
tissues, there
is an
CoA-acetoacetate-CoA
reaction catalyzed
transferase transfer
of
CoA
from by
succinyl-
(thiophorase), succinyl-CoA
involving to
acetoacetate, forming
acetoacetyl-CoA
and succinate
(see
Chapter
22).
The onward metabolism of succinate, leading to the regeneration of oxaloacetate, is the same sequence of chemical reactions as occurs in the B-oxidation of fatty acids: dehydrogenation to form a carbon-carbon double bond, addition of water to form a hydroxyl group, and a further dehydrogenation to yield the oxo-group of oxaloacetate.

The first dehydrogenation reaction, forming fumarate, is catalyzed by succinate dehydrogenase, which is bound to the inner surface of the inner mitochondrial membrane. The enzyme contains FAD and iron-sulfur (Fe-S) protein, and directly reduces ubiquinone in the electron transport chain. Fumarase (fumarate hydratase) catalyzes the addition of water across the double bond of fumarate, yielding malate. Malate is oxidized to oxaloacetate by malate dehydrogenase, linked to the reduction of NAD*. Although the equilibrium of this reaction strongly favors malate, the net flux is to oxaloacetate because of the continual removal of oxaloacetate (to form citrate, as a substrate for gluconeogenesis, or to undergo transamination to aspartate) and also the continual reoxidation of NADH.

As a result of oxidations catalyzed by the dehydrogenases of the citric acid cycle, three molecules of NADH and one of FADH, are produced for each molecule of acetyl-CoA catabolized in one turn of the cycle. These reducing equivalents are transferred to the respiratory chain (see Figure 13-3), where reoxidation of each NADH results in formation of ~2.5 ATP, and of FADH,, ~1.5 ATP. In addition, 1 ATP (or GTP) is formed by substrate-level phosphorylation catalyzed by suc-
cinate thiokinase.

Four of the B vitamins (see Chapter 44) are essential in the citric acid cycle and hence energy-yielding metabolism: riboflavin, as FAD, is the cofactor for succinate dehydrogenase; niacin, as NAD*, is the electron acceptor for isocitrate dehydrogenase, a-ketoglutarate dehydrogenase, and malate dehydrogenase; thiamin (vitamin B,), as thiamin diphosphate, is the coenzyme for decarboxylation in the a-ketoglutarate dehydrogenase reaction; and pantothenic acid, as part of coenzyme A, is esterified to carboxylic acids to form acetyl-CoA and succinyl-CoA.

The citric acid cycle is not only a pathway for oxidation of two carbon units, but is also a major pathway for interconversion of metabolites arising from transamination and deamination of amino acids (see Chapters 28 and 29), and providing the substrates for amino acid synthesis by transamination (see Chapter 27), as well as for gluconeogenesis (see Chapter 19) and fatty acid synthesis (see Chapter 23). Because it functions in both oxidative and synthetic processes, it is amphibolic (Figure 16-4).

All the intermediates of the cycle are potentially glucogenic, since they can give rise to oxaloacetate, and hence production of glucose (in the liver and kidney, which carry out gluconeogenesis; see Chapter 19). The key enzyme that catalyzes transfer out of the cycle into gluconeogenesis is phosphoenolpyruvate carboxykinase, which catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate, with GTP acting as the phosphate donor (see Figure 19-1). The GTP required for this reaction is provided by the GDP-dependent isoenzyme of succinate thiokinase. This ensures that oxaloacetate will not be withdrawn from the cycle for gluconeogenesis if this would lead to depletion of citric acid cycle intermediates, and hence reduced generation of ATP.
Net transfer into the cycle occurs as a result of several reactions. Among the most important of such anaplerotic reactions is the formation of oxaloacetate by the carboxylation of pyruvate, catalyzed by pyruvate carboxylase (Figure 16-4). This reaction is important in maintaining an adequate concentration of oxaloacetate for the condensation reaction with acetyl-CoA. If acetyl-CoA accumulates, it acts as both an
FIGURE
16-4
involvement of
the citric
acid cycle
in transamination
and gluconeogenesis.
The bold
arrows indicate
of gluconeogenesis.
allosteric activator of pyruvate carboxylase and an inhibitor of pyruvate dehydrogenase, thereby ensuring a supply of oxaloacetate. Lactate, an important substrate for gluconeogenesis, enters the cycle via oxidation to pyruvate and then carboxylation to oxaloacetate. Glutamate and glutamine are important anaplerotic substrates because they yield a-ketoglutarate as a result of the reactions catalyzed by glutaminase and glutamate dehydrogenase. Transamination of aspartate leads directly to the formation of oxaloacetate, and a variety of compounds that are metabolized to yield propionyl CoA, which can be carboxylated and isomerized to succinyl CoA, are also important anaplerotic substrates.

Aminotransferase (transaminase) reactions form pyruvate from alanine, oxaloacetate from aspartate, and a-ketoglutarate from glutamate. Because these reactions are reversible, the cycle also serves as a source of carbon skeletons for the synthesis of these amino acids. Other amino acids contribute to gluconeogenesis because their carbon skeletons give rise to citric acid cycle intermediates. Alanine, cysteine, glycine, hydroxyproline, serine, threonine, and tryptophan yield pyruvate; arginine, histidine, glutamine, and proline yield a-ketoglutarate; isoleucine, methionine, and valine yield succinyl-CoA; tyrosine and phenylalanine yield fumarate (Figure 16-4).
The citric acid cycle itself does not provide a pathway for the complete oxidation of the carbon skeletons of amino acids that give rise to intermediates such as a-ketoglutarate, succinyl
CoA, fumarate, and oxaloacetate, because this results in an increase in the amount of oxaloacetate. For complete oxidation to occur, oxaloacetate must undergo phosphorylation and carboxylation to phosphoenolpyruvate (at the expense of GTP), then dephosphorylation to pyruvate (catalyzed by pyruvate kinase), and oxidative decarboxylation to acetyl-Co (catalyzed by pyruvate dehydrogenase).
In ruminants, whose main metabolic fuel is short-chain fatty acids formed by bacterial fermentation, the conversion of propionate, the major glucogenic product of rumen fermentation, to succinyl-CoA via the methylmalonyl-CoA pathway (see Figure 19-2) is especially important.

Acetyl-CoA, formed from pyruvate by the action of pyruvate dehydrogenase, is the major substrate for long-chain fatty acid synthesis in nonruminants (Figure 16-5). (In ruminants, acetylCoA is derived directly from acetate.) Pyruvate dehydrogenase is a mitochondrial enzyme, and fatty acid synthesis is a cytosolic pathway; the mitochondrial membrane is impermeable to acetylCoA. For acetyl-CoA to be available in the cytosol, citrate is transported from the mitochondrion to the cytosol, then cleaved in a reaction catalyzed by citrate lyase (Figure 16-5). Citrate is only available for transport out of the mitochondrion when the
main pathway
FIGURE 16-5 Participation of the citric acid cycle in provision of cytosolic acetyl-CoA for fatty acid synthesis from glucose. See also Figure 23-5.
aconitase is inhibited by its product and therefore saturated with its substrate, so that citrate cannot be channeled directly from citrate synthase onto aconitase. This ensures that citrate is used for fatty acid synthesis only when there is an adequate amount to ensure continued activity of the cycle.

The oxaloacetate released by citrate lyase cannot reenter the mitochondrion, but is reduced to malate, at the expense of NADH, and the malate undergoes oxidative decarboxylation to pyruvate, reducing NADP* to NADPH. This reaction, catalyzed by the malic enzyme, is the source of half the NADPH required for fatty acid synthesis (the remainder is provided by the pentose phosphate pathway, see Chapter 20). Pyruvate enters the mitochondrion and is carboxylated to oxaloacetate by pyruvate carboxylase, an ATP-dependent reaction in which the coenzyme is the vitamin biotin.
In most tissues, where the primary role of the citric acid cycle is in energy-yielding metabolism, respiratory control via the respiratory chain and oxidative phosphorylation regulates citric acid cycle activity (see Chapter 13). Thus, activity is immediately dependent on the supply of NAD*, which in turn, because of the tight coupling between oxidation and phosphorylation, is dependent on the availability of ADP and hence, ultimately on the utilization of ATP in chemical and physical work. In addition, individual enzymes of the cycle are regulated. The main sites for regulation are the nonequilibrium reactions catalyzed by pyruvate dehydrogenase,
to form glutamate (catalyzed by glutamate dehydrogenase) and then glutamine (catalyzed by glutamine synthetase), leading to lowered concentrations of all citric acid cycle intermediates, and hence reduced generation of ATP. The equilibrium of glutamate dehydrogenase is finely poised, and the direction of reaction depends on the ratio of NAD*: NADH and the concentration of ammonium ions. In addition, ammonia inhibits a-ketoglutarate dehydrogenase, and possibly also pyruvate dehydrogenase.

■, 1 = The citric acid cycle is the final pathway for the oxidation of carbohydrate, lipid, and protein. Their common end- metabolite, acetyl-CoA, reacts with oxaloacetate to form citrate. By a series of dehydrogenations and decarboxylations, citrate is degraded, reducing coenzymes, releasing two CO 2 , and regenerating oxaloacetate.. ■, 1 = The reduced coenzymes are oxidized by the respiratory chain linked to formation of ATP. Thus, the cycle is the major pathway for the formation of ATP and is located in the matrix of mitochondria adjacent to the enzymes of the respiratory chain and oxidative phosphorylation.. ■, 1 = The citric acid cycle is amphibolic, since in addition to oxidation it is important in the provision of carbon skeletons for gluconeogenesis, acetyl-CoA for fatty acid synthesis, and interconversion of amino acids.

- 2 Describe the pathway of glycolysis and its control, and explain how glycolysis
can operate under anaerobic conditions.
- Describe the reaction of pyruvate dehydrogenase and its regulation.
- Explain how inhibition of pyruvate metabolism leads to lactic acidosis.

Most tissues have at least some requirement for glucose; in the brain, the requirement is substantial—even in prolonged fasting the brain can meet no more than about 20% of its energy needs from ketone bodies. Glycolysis is the main pathway of glucose (and other carbohydrate) metabolism. It occurs in the cytosol of all cells, and can function either aerobically or anaerobically, depending on the availability of oxygen and the electron transport chain (and hence of the presence of mitochondria). Erythrocytes, which lack mitochondria, are completely reliant on glucose as their metabolic fuel, and metabolize it by anaerobic glycolysis.

The ability of glycolysis to provide ATP in the absence of oxygen allows skeletal muscle to perform at very high levels of work output when oxygen supply is insufficient, and it allows tissues to survive anoxic episodes. However, heart muscle, which is adapted for aerobic performance, has relatively low glycolytic activity and poor survival under conditions of ischemia. Diseases in which enzymes of glycolysis (eg, pyruvate kinase) are deficient are mainly seen as hemolytic anemias or, if the defect affects skeletal muscle (eg, phosphofructokinase), as fatigue. In fast-growing cancer cells, glycolysis proceeds at a high rate, forming large amounts of pyruvate, which is reduced to lactate and exported. This produces a relatively acidic local environment in the tumor. The lactate is used for gluconeogenesis in the liver (see Chapter 19), an energy-expensive process, which is responsible for much of the hypermetabolism seen in cancer cachexia. Lactic acidosis results from various causes, including impaired activity of pyruvate dehydrogenase, especially in thiamin (vitamin B,) deficiency.

Early in the investigations of glycolysis, it was realized that fermentation in yeast was similar to the breakdown of glycogen in muscle. When a muscle contracts under anaerobic conditions, glycogen disappears and lactate appears. When oxygen is admitted, aerobic recovery takes place and lactate is no longer produced. If muscle contraction occurs under aerobic conditions, lactate does not accumulate and pyruvate is the end product of glycolysis. Pyruvate is oxidized further to CO, and water (Figure 17-1). When oxygen is in short supply, mitochondrial reoxidation of NADH formed during glycolysis is impaired, and NADH is reoxidized by reducing pyruvate to lactate, so permitting glycolysis to continue. While glycolysis can occur under anaerobic conditions, this has a price, for it limits the amount of ATP formed per mole of glucose oxidized, so that much more glucose must be metabolized under anaerobic than aerobic conditions (Table 17-1). In yeast and some other microorganisms, pyruvate formed in anaerobic glycolysis is not reduced to lactate, but is decarboxylated and reduced to ethanol.

The overall equation for glycolysis from glucose to lactate is as follows:
Glucose + 2 ADP + 2 P, > 2 Lactate + 2 ATP + 2 H,O
All of the enzymes of glycolysis (Figure 17-2) are cytosolic. Glucose enters glycolysis by phosphorylation to
FIGURE 17-1 summary of glycolysis. ©, blocked under anaerobic conditions by absence of mitochondria key or containing respiratory enzymes, as in erythrocytes.

glucose-6-phosphate, catalyzed by hexokinase, using ATP as the phosphate donor. Under physiological conditions, the phosphorylation of glucose to glucose-6-phosphate can be regarded as irreversible. Hexokinase is inhibited allosterically by its product, glucose-6-phosphate. In tissues other than the liver (and pancreatic B-islet cells), the availability of glucose for glycolysis (or glycogen synthesis in muscle, Chapter 18, and lipogenesis in adipose tissue, Chapter 23) is controlled by transport into the cell, which in turn is regulated by insulin. Hexokinase has a high affinity (low K,) for glucose, and in the liver, it is saturated under normal conditions, and so acts at a constant rate to provide glucose-6-phosphate to meet the liver's needs. Liver cells also contain an isoenzyme of hexokinase, glucokinase, which has a K, very much higher than the normal intracellular concentration of glucose. The function of glucokinase in the liver is to remove glucose from the hepatic portal blood following a meal, so regulating the concentration of glucose available to peripheral tissues. This provides more glucose-6-phosphate than is required for glycolysis; it is used for glycogen synthesis and lipogenesis. Glucokinase is also found in pancreatic B-islet cells, where it functions to detect high concentrations of glucose in the portal blood. As more glucose is phosphorylated by glucokinase, there is increased glycolysis, leading to increased formation of ATP. This leads to closure of an ATP-potassium channel, causing membrane depolarization and opening of a voltage-gated calcium channel. The resultant influx of calcium ions leads to fusion of the insulin secretory granules with the cell membrane and the release of insulin. Glucose-6-phosphate is an important compound at the
junction of several metabolic pathways: glycolysis, gluconeogenesis (see Chapter 19), the pentose phosphate pathway

Respiratory chain oxidation of 2 NADH. , ATP per mol of Glucose = 5. , Reaction Catalyzed by = . , Method of ATP Formation = . , ATP per mol of Glucose = Net 25. , Reaction Catalyzed by = Total per mol of glucose under aerobic conditions. , Method of ATP Formation = . , ATP per mol of Glucose = 32. , Reaction Catalyzed by = Total per mol of glucose under anaerobic conditions. , Method of ATP Formation = . , ATP per mol of Glucose = 2
*This assumes that NADH formed in glycolysis is transported into mitochondria by the malate shuttle (see Figure 13-13). If the glycerophosphate shuttle is used, then only 1.5 ATP will be formed per mol of NADH. There is a considerable advantage in using glycogen rather than glucose for anaerobic glycolysis in muscle, since the product of glycogen phosphorylase is glucose-1-phosphate (see Figure 18-1), which is interconvertible with glucose-6-phosphate. This saves the ATP that would otherwise be used by hexokinase, increasing the net yield of ATP from 2 to 3 per glucose.
FIGURE 17-2 The pathway of glycolysis. (®, —PO,"; P,, HOPO,"; ©, inhibition.) Carbons 1-3 of fructose bisphosphate form dihydroxyacetone phosphate, and carbons 4-6 form glyceraldehyde-3-phosphate.

(see Chapter 20), glycogenesis, and glycogenolysis (see Chapter 18). In glycolysis, it is converted to fructose-6-phosphate by phosphohexose isomerase, which involves an aldose-ketose isomerization. This reaction is followed by another phosphorylation catalyzed by the enzyme phosphofructokinase (phosphofructokinase-1) forming fructose 1,6-bisphosphate. The phosphofructokinase reaction is irreversible under physiological conditions. Phosphofructokinase is both inducible and subject to allosteric regulation, and has a major role in regulating the rate of glycolysis. Fructose 1,6-bisphosphate is cleaved by aldolase (fructose 1,6-bisphosphate aldolase) into two triose phosphates, glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, which are interconverted by the enzyme phosphotriose isomerase.
In the next reaction, catalyzed by phosphoglycerate kinase, phosphate is transferred from 1,3-bisphosphoglycerate onto ADP, forming ATP (substrate-level phosphorylation) and 3-phosphoglycerate. Since two molecules of triose phosphate are formed per molecule of glucose metabolized, 2x ATP are formed in this reaction per molecule of glucose undergoing glycolysis. The toxicity of arsenic is the result of competition of arsenate with inorganic phosphate (P,) forming 1-arseno-3-phosphoglycerate, which undergoes spontaneous hydrolysis to 3-phosphoglycerate without forming ATP. 3-Phosphoglycerate is isomerized to 2-phosphoglycerate by phosphoglycerate mutase. It is likely that 2,3-bisphosphoglycerate (diphosphoglycerate, DPG) is an intermediate in this reaction.

Glycolysis continues with the oxidation of glyceraldehyde3-phosphate to 1,3-bisphosphoglycerate. The enzyme catalyzing this oxidation, glyceraldehyde-3-phosphate dehydrogenase, is NAD dependent. Structurally, it consists of four identical polypeptides (monomers) forming a tetramer. Four —SH groups are present on each polypeptide, derived from cysteine residues within the polypeptide chain. One of the —SH groups is found at the active site of the enzyme (Figure 17-3). The substrate initially combines with this —SH group, forming a thiohemiacetal that is oxidized to a thiol ester; the hydrogens removed in this oxidation are transferred to NAD*. The thiol ester then undergoes phosphorolysis; inorganic phosphate (P.) is added, forming 1,3-bisphosphoglycerate, and the free —SH group.
The subsequent step is catalyzed by enolase and involves a dehydration, forming phosphoenolpyruvate. Enolase is inhibited by fluoride, and when blood samples are taken for measurement of glucose, glycolysis is inhibited by taking the sample into tubes containing fluoride. Enolase is also dependent on the presence of either Mg** or Mn** ions. The phosphate of phosphoenolpyruvate is transferred to ADP in another substrate-level phosphorylation catalyzed by pyruvate kinase to form 2x ATP per molecule of glucose oxidized. The reaction of pyruvate kinase is essentially irreversible under physiological conditions, partly because of the large free-energy change involved and partly because the immediate product of the enzyme-catalyzed reaction is enolpyruvate,
FIGURE 17-3 Mechanism of oxidation of glyceraldehyde-3-phosphate. (Enz, glyceraldehyde-3-phosphate dehydrogenase.) The enzyme is inhibited by the —SH poison iodoacetate, which is thus able to inhibit glycolysis. The NADH produced on the enzyme is not so firmly bound to the enzyme as is NAD*. Consequently, NADH is easily displaced by another molecule of NAD*.

which undergoes spontaneous isomerization to pyruvate, so that the product of the reaction is not available to undergo the reverse reaction.
The availability of oxygen now determines which of the two pathways is followed. Under anaerobic conditions, the NADH cannot be reoxidized through the respiratory chain, and pyruvate is reduced to lactate catalyzed by lactate dehydrogenase. This permits the oxidization of NADH, permitting another molecule of glucose to undergo glycolysis. Under aerobic conditions, pyruvate is transported into mitochondria and undergoes oxidative decarboxylation to acetyl-CoA then oxidation to CO, in the citric acid cycle (see Chapter 16). The reducing equivalents from the NADH formed in glycolysis are taken up into mitochondria for oxidation via either the malate-aspartate shuttle or the glycerophosphate shuttle (see Chapter 13).
glycolysis and produce lactate include brain, gastrointestinal tract, renal medulla, retina, and skin. Lactate production is also increased in septic shock, and many cancers also produce lactate. The liver, kidneys, and heart normally take up lactate and oxidize it, but produce it under hypoxic conditions. When lactate production is high, as in vigorous exercise, septic shock, and cancer cachexia, much is used in the liver for gluconeogenesis (see Chapter 19), leading to an increase in metabolic rate to provide the ATP and GTP needed. The increase in oxygen consumption as a result of increased oxidation of metabolic fuels to provide the ATP and GTP needed for gluconeogenesis is seen as oxygen debt after vigorous exercise.

This is true of skeletal muscle, particularly the white fibers, where the rate of work output, and hence the need for ATP formation, may exceed the rate at which oxygen can be taken up and utilized. Glycolysis in erythrocytes always terminates in lactate, because the subsequent reactions of pyruvate oxidation are mitochondrial, and erythrocytes lack mitochondria. Other tissues that normally derive much of their energy from
Under some conditions, lactate may be formed in the cytosol, but then enter the mitochondrion to be oxidized to pyruvate for onward metabolism. This provides a pathway for the transfer of reducing equivalents from the cytotol into the mitochondrion for the electron transport chain in addition to the glycerophosphate (see Figure 13-12) and malate-aspartate (see Figure 13-13) shuttles.

Although most of the reactions of glycolysis are freely reversible, three are markedly exergonic and must therefore be considered to be physiologically irreversible. These reactions,
catalyzed by hexokinase (and glucokinase), phosphofructokinase, and pyruvate kinase, are the major sites of regulation of glycolysis. Phosphofructokinase is significantly inhibited at normal intracellular concentrations of ATP; as discussed in Chapter 19, this inhibition can be rapidly relieved by 5'AMP that is formed as ADP begins to accumulate, signaling the need for an increased rate of glycolysis. Cells that are capable of gluconeogenesis (reversing the glycolytic pathway, Chapter 19) have different enzymes that catalyze reactions to reverse these irreversible steps: glucose-6-phosphatase, fructose 1,6-bisphosphatase and, to reverse the reaction of pyruvate kinase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase. The reciprocal regulation of phosphofructokinase in glycolysis and fructose 1,6-bisphosphatase in gluconeogenesis is discussed in Chapter 19.
Fructose enters glycolysis by phosphorylation to fructose-1-phosphate, and bypasses the main regulatory steps, so resulting in formation of more pyruvate and acetyl-CoA than is required for ATP formation. In the liver and adipose tissue, this leads to increased lipogenesis, and a high intake of fructose may be a factor in the development of obesity.

In erythrocytes, the reaction catalyzed by phosphoglycerate kinase may be bypassed to some extent by the reaction of bisphosphoglycerate mutase, which catalyzes the conversion of 1,3-bisphosphoglycerate to 2,3-bisphosphoglycerate, followed by hydrolysis to 3-phosphoglycerate and P,, catalyzed by 2,3-bisphosphoglycerate phosphatase (Figure 17-4). This pathway involves no net yield of ATP from glycolysis, but provides 2,3-bisphosphoglycerate, which binds to hemoglobin, decreasing its affinity for oxygen, so making oxygen more readily available to tissues (see Chapter 6).

Pyruvate is transported into the mitochondrion by a proton symporter. It then undergoes oxidative decarboxylation to acetyl-CoA, catalyzed by a multienzyme complex that is associated with the inner mitochondrial membrane. This pyruvate dehydrogenase complex is analogous to the a-ketoglutarate dehydrogenase complex of the citric acid cycle (see Chapter 16). Pyruvate is decarboxylated by the pyruvate dehydrogenase component of the enzyme complex to a hydroxyethyl derivative of the thiazole ring of enzyme-bound thiamin diphosphate, which in turn reacts with oxidized lipoamide, the prosthetic group of dihydrolipoyl transacetylase, to form acetyl lipoamide (Figure 17-5). In thiamin (vitamin B,, see Chapter 44) deficiency, glucose metabolism is impaired, and
FIGURE 17-4 The 2,3-bisphosphoglycerate pathway in erythrocytes.
there is significant (and potentially life-threatening) lactic and pyruvic acidosis. Acetyl lipoamide reacts with coenzyme A to form acetyl-CoA and reduced lipoamide. The reaction is completed when the reduced lipoamide is reoxidized by a flavoprotein, dihydrolipoyl dehydrogenase, containing flavin adenine dinucleotide (FAD). Finally, the reduced flavoprotein is oxidized by NAD*, which in turn transfers reducing equivalents to the respiratory chain. The overall reaction is:
Pyruvate + NAD* + CoA > Acetyl-CoA + NADH + H* + CO,
The pyruvate dehydrogenase complex consists of a number of polypeptide chains of each of the three component enzymes and the intermediates do not dissociate, but are channeled from one enzyme site to the next. This increases the rate of reaction and prevents side reactions.

Pyruvate dehydrogenase is inhibited by its products, acetyl-CoA and NADH (Figure 17-6). It is also regulated by phosphorylation (catalyzed by a kinase) of three serine residues on the pyruvate dehydrogenase component of the multienzyme complex, resulting in decreased activity and by dephosphorylation
FIGURE 17-5 Oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase complex. Lipoic acid is joined by an amide link to a lysine residue of the transacetylase component of the enzyme complex. It forms a long flexible arm, allowing the lipoic acid prosthetic group to rotate sequentially between the active sites of each of the enzymes of the complex. (FAD, flavin adenine dinucleotide; NAD*, nicotinamide adenine dinucleotide.)
FIGURE 17-6 Regulation of pyruvate dehydrogenase (PDH). Arrows with wavy shafts indicate allosteric effects. (A) Regulation by endproduct inhibition. (B) Regulation by interconversion of active and inactive forms.
(catalyzed by a phosphatase) that causes an increase in activity. The kinase is activated by increases in the [ATP]/[ADP], [acetyl-CoA]/[CoA], and [NADH]/[NAD*] ratios. Thus, pyruvate dehydrogenase, and therefore glycolysis, is inhibited both when there is adequate ATP (and reduced coenzymes for ATP formation) available, and also when fatty acids are being oxidized. In fasting, when nonesterified fatty acid concentrations increase, there is a decrease in the proportion of the enzyme in the active form, leading to a sparing of carbohydrate. In adipose tissue, where glucose provides acetyl-CoA for lipogenesis, the enzyme is activated in response to insulin.

Arsenite and mercuric ions react with the —SH groups of lipoic acid and inhibit pyruvate dehydrogenase, as does a dietary deficiency of thiamin (see Chapter 44), allowing pyruvate to accumulate. Many alcoholics are thiamin deficient (both because of a poor diet and also because alcohol inhibits thiamin absorption) and may develop potentially fatal pyruvic and lactic acidosis. Patients with inherited pyruvate dehydrogenase deficiency, which can be the result of defects in one or more of the components of the enzyme complex, also present with lactic acidosis, particularly after a glucose load. Because of the dependence of the brain on glucose as a fuel, these metabolic defects commonly cause neurological disturbances.
Inherited aldolase A deficiency and pyruvate kinase deficiency in erythrocytes cause hemolytic anemia. The exercise capacity of patients with muscle phosphofructokinase deficiency is low, particularly if they are on high-carbohydrate diets.

■, 1 = Glycolysis is the cytosolic pathway in all mammalian cells for the metabolism of glucose (or glycogen) to pyruvate and lactate.. ■, 1 = It can function anaerobically by regenerating oxidized NAD + (required in the glyceraldehyde-3-phosphate dehydrogenase reaction), by reducing pyruvate to lactate.. ■, 1 = Lactate is the end product of glycolysis under anaerobic conditions (eg, in exercising muscle) and in erythrocytes, where there are no mitochondria to permit further oxidation of pyruvate.. ■, 1 = Glycolysis is regulated by three enzymes catalyzing nonequilibrium reactions: hexokinase, phosphofructokinase, and pyruvate kinase.. ■, 1 = In erythrocytes, the first site in glycolysis for generation of ATP may be bypassed, leading to the formation of 2,3-bisphosphoglycerate, which is important in decreasing the affinity of hemoglobin for O 2 .. ■, 1 = Pyruvate is oxidized to acetyl-CoA by a multienzyme complex, pyruvate dehydrogenase, which is dependent on the vitamin- derived cofactor thiamin diphosphate.. ■, 1 = Conditions that involve an impairment of pyruvate metabolism frequently lead to lactic acidosis.

- . Describe the structure of glycogen and its importance as a carbohydrate
reserve.
: processes are
Describe the synthesis and breakdown of glycogen and how the regulated in response to hormone action.
mT ULe\ INCRE Reel aA
- ™ Describe the various types of glycogen storage diseases.

Glycogen is the major storage carbohydrate in animals, corresponding to starch in plants; it is a branched polymer of a-D-glucose (see Figure 15-12). It occurs mainly in liver and muscle, with modest amounts in the brain. Although the liver content of glycogen is greater than that of muscle, because the muscle mass of the body is considerably greater than that of the liver, about three-quarters of total body glycogen is in muscle (Table 18-1).
Muscle glycogen provides a readily available source of glucose-1-phosphate for glycolysis within the muscle itself. Liver glycogen functions as a reserve to maintain the blood glucose concentration in the fasting state. The liver concentration of glycogen is about 450 mmol/L glucose equivalents after a meal, falling to about 200 mmol/L after an overnight fast; after 12 to 18 hours of fasting, liver glycogen is almost totally depleted. Although muscle glycogen does not directly yield free glucose (because muscle lacks glucose-6-phosphatase), pyruvate formed by glycolysis in muscle can undergo transamination to alanine, which is exported from muscle and used for gluconeogenesis in the liver (see Figure 19-4). Glycogen storage diseases are a group of inherited disorders characterized by deficient mobilization of glycogen or deposition of abnormal forms of glycogen, leading to liver damage and muscle weakness; some result in early death.
The highly branched structure of glycogen (see Figure 15-12) provides a large number of sites for glycogenolysis, permitting rapid release of glucose-1-phosphate for muscle activity. Endurance athletes require a slower, more sustained release of glucose-1-phosphate. The formation of branch points in glycogen is slower than the addition of glucose units to a linear chain, and some endurance athletes practice carbohydrate loading—exercise to exhaustion (when muscle glycogen in largely depleted) followed by a high-carbohydrate meal, which results in rapid glycogen synthesis, with fewer branch points than normal.

As in glycolysis, glucose is phosphorylated to glucose6-phosphate, catalyzed by hexokinase in muscle and glucokinase in liver (Figure 18-1). Glucose-6-phosphate is isomerized to glucose-1-phosphate by phosphoglucomutase. The enzyme itself is phosphorylated, and the phosphate group takes part in a reversible reaction in which glucose 1,6-bisphosphate is an intermediate. Next, glucose-1-phosphate reacts with uridine triphosphate (UTP) to form the active nucleotide uridine diphosphate glucose (UDPGlIc) and pyrophosphate (Figure 18-2), catalyzed by UDPGlc pyrophosphorylase. The reaction proceeds in the direction of UDPGlc formation because pyrophosphatase catalyzes hydrolysis of pyrophosphate to 2 x phosphate, so removing one of the reaction products. UDPGlc pyrophosphorylase has a low K,, for glucose-1-phosphate and is present in relatively large amounts, so that it is not a regulatory step in glycogen synthesis. The initial steps in glycogen synthesis involve the protein glycogenin, a 37-kDa protein that is glucosylated on a specific tyrosine residue by UDPGlc. Glycogenin catalyzes the transfer of a further seven glucose residues from UDPGlc, in 1 — 4 linkage, to form a glycogen primer that is the substrate
Liver glycogen, Storage of Carbohydrate.Percentage of Tissue Weight = 5.0. Liver glycogen, in a.Tissue Weight = 1.8 kg. Liver glycogen, 70-kg Person.Body Content (g) = 90. Muscle glycogen, Storage of Carbohydrate.Percentage of Tissue Weight = 0.7. Muscle glycogen, in a.Tissue Weight = 35 kg. Muscle glycogen, 70-kg Person.Body Content (g) = 245. Extracellular glucose, Storage of Carbohydrate.Percentage of Tissue Weight = 0.1. Extracellular glucose, in a.Tissue Weight = 10 L. Extracellular glucose, 70-kg Person.Body Content (g) = 10

for glycogen synthase. The glycogenin remains at the core of the glycogen granule (see Figure 15-12). Glycogen synthase catalyzes the formation of a glycoside bond between C-1 of the glucose of UDPGle and C-4 of a terminal glucose residue of glycogen, liberating UDP. The addition of a glucose residue to a preexisting glycogen chain, or "primer, occurs at the nonreducing, outer end of the molecule, so that the branches of the glycogen molecule become elongated as successive 14 linkages are formed (Figure 18-3).
FIGURE 18-2 Uridine diphosphate glucose (UDPGIc).

When a growing chain is at least 11 glucose residues long, branching enzyme transfers a part of the 1 — 4 chain (at least six glucose residues) to a neighboring chain to form a 1 > 6
|
FIGURE 18-1 Pathways of glycogenesis and glycogenolysis in the liver. (®, Stimulation; ©, inhibition.) Insulin decreases the level of cAMP only after it has been raised by glucagon or epinephrine; that is, it antagonizes their action. Glucagon acts on heart muscle but not in skeletal muscle. "Glucan transferase and debranching enzyme appear to be two separate activities of the same enzyme.
FIGURE 18-3 The biosynthesis of glycogen. The mechanism of branching as revealed by feeding 'C-labeled glucose and examining liver glycogen at intervals.
linkage, establishing a branch point. The branches grow by further additions of 1 — 4-glucosyl units and further branching.

Glycogen phosphorylase catalyzes the rate-limiting step in glycogenolysis—the phosphorolytic cleavage of the 1 > 4 linkages of glycogen to yield glucose-1-phosphate (Figure 18-4). There are different isoenzymes of glycogen phosphorylase in liver, muscle, and brain, encoded by different genes. Glycogen phosphorylase requires pyridoxal phosphate (see Chapter 44) as its coenzyme. Unlike the reactions of amino acid metabolism (see Chapter 28), in which the aldehyde group of the coenzyme is the reactive group, in phosphorylase it is the phosphate group that is catalytically active.
FIGURE 18-4 Steps in glycogenolysis.
The terminal glucosyl residues from the outermost chains of the glycogen molecule are removed sequentially until approximately four glucose residues remain on either side of a 1 > 6 branch (Figure 18-4). The debranching enzyme has two catalytic sites in a single polypeptide chain. One is a glucan transferase that transfers a trisaccharide unit from one branch to the other, exposing the 1 > 6 branch point. The other is a 1,6-glycosidase that catalyzes hydrolysis of the 1 + 6-glycoside bond to liberate free glucose. Further phosphorylase action can then proceed. The combined action of phosphorylase and these other enzymes leads to the complete breakdown of glycogen.
The reaction
catalyzed by
phosphoglucomutase is
revers- ible, so that glucose-6-phosphate can be formed from glucose1-phosphate. In liver, but not muscle, glucose-6-phosphatase catalyzes hydrolysis of glucose-6-phosphate, yielding glucose that is exported, leading to an increase in the blood glucose concentration. Glucose-6-phosphatase is in the lumen of the smooth endoplasmic reticulum, and genetic defects of the glucose6-phosphate transporter can cause a variant of type I glycogen storage disease (Table 18-2).

Glycogen granules can also be engulfed by lysosomes, where acid maltase catalyzes the hydrolysis of glycogen to glucose. This may be especially important in glucose homeostasis in neonates. Genetic lack of lysosomal acid maltase causes type II glycogen storage disease (Pompe disease, Table 18-2). The lysosomal catabolism of glycogen is under hormonal control.

The principal enzymes controlling glycogen metabolism— glycogen phosphorylase and glycogen synthase—are regulated in opposite directions by allosteric mechanisms and covalent modification by reversible phosphorylation and dephosphorylation of enzyme protein in response to hormone action
activity. Phosphorylation is increased in response to cyclic AMP (cAMP) (Figure 18-5) formed from ATP by adenylyl cyclase at the inner surface of cell membranes in response to hormones such as epinephrine, norepinephrine, and glucagon. cAMP is hydrolyzed by phosphodiesterase, so terminating hormone action; in liver insulin increases the activity of phosphodiesterase.

accumulation of glycogen in liver; mild hypoglycemia; generally good prognosis. VII, Name = Tarui disease. VII, Enzyme Deficiency = Muscle and erythrocyte phosphofructokinase 1. VII, Clinical Features = Poor exercise tolerance; muscle glycogen abnormally high (2.5-4%); blood lactate very low after exercise; also hemolytic anemia. VIII, Name = . VIII, Enzyme Deficiency = Liver phosphorylase kinase. VIII, Clinical Features = Hepatomegaly; accumulation of glycogen in liver; mild hypoglycemia; generally good prognosis. IX, Name = . IX, Enzyme Deficiency = Liver and muscle phosphorylase kinase. IX, Clinical Features = Hepatomegaly; accumulation of glycogen in liver and muscle; mild hypoglycemia; generally good prognosis. X, Name = . X, Enzyme Deficiency = cAMP-dependent protein kinase A. X, Clinical Features = Hepatomegaly; accumulation of glycogen in liver
Abbreviation: cAMP, cyclic adenosine monophosphate.
(see Chapter 9). Phosphorylation of glycogen phosphorylase increases its activity; phosphorylation of glycogen synthase reduces its

In the
liver, the
role of
glycogen is
to provide
free glucose
for export
the blood
glucose;
in muscle, the role of glycogen is to provide a source of glucose-
6-phosphate for
glycolysis in
the need
for
ATP
for muscle
contraction.
In both
tissues, the
enzyme is
activated by phosphorylation catalyzed by phosphorylase kinase (to yield phosphorylase a) and inactivated by dephosphorylation
catalyzed by phosphoprotein phosphatase (to yield phosphorb), in to hormonal and other ylase response signals. There is instantaneous overriding of this hormonal control. Active phosphorylase a in both tissues is allosterically inhibited by ATP and glucose-6-phosphate; in liver, but not muscle, free glucose is also an inhibitor. Muscle phosphorylase differs from the liver isoenzyme in having a binding site for 5' AMP (Figure 18-5), which acts as an allosteric activator of the (inactive) dephosphorylated b-form of the enzyme. 5' AMP acts as a potent signal of the energy state of the muscle cell; it is formed as the concentration of ADP begins to increase (indicating the need for increased substrate metabolism to permit ATP formation), as a result of the reaction of adenylate kinase: 2 x ADP <> ATP + 5' AMP.

Phosphorylase kinase is activated in response to cAMP (Figure 18-6). Increasing the concentration of cAMP activates cAMP-dependent protein kinase, which catalyzes the
(cAMP)
FIGURE 18-5 The formation and hydrolysis of cyclic AMP (3',5'-adenylic acid, cAMP).
phosphorylation by ATP of inactive phosphorylase kinase b to active phosphorylase kinase a, which in turn, phosphorylates phosphorylase b to phosphorylase a. In the liver, cAMP is formed in response to glucagon, which is secreted in response to falling blood glucose. Muscle is insensitive to glucagon; in muscle, the signal for increased cAMP formation is the action of norepinephrine, which is secreted in response to fear or fright, when there is a need for increased glycogenolysis to permit rapid muscle activity.

Glycogenolysis in muscle increases several 100-fold at the onset of contraction; the same (increased signal cytosolic Ca** ion concentration) is responsible for initiation of both
contraction and glycogenolysis. Muscle phosphorylase kinase, which activates glycogen phosphorylase, is a tetramer of four different subunits, a, B, y, and 5. The a and B subunits contain serine residues that are phosphorylated by cAMP-dependent protein kinase. The 6 subunit is identical to the Ca**-binding protein calmodulin (see Chapter 42) and binds four Ca'. The binding of Ca'* activates the catalytic site of the y subunit even while the enzyme is in the dephosphorylated b state; the phosphorylated a form is only fully activated in the presence of high concentrations of Ca'*.

In the liver, there is cAMP-independent activation of glycogenolysis in response to stimulation of a, adrenergic receptors by epinephrine and norepinephrine. This involves mobilization of Ca** into the cytosol, followed by the stimulation of a Ca**/calmodulin-sensitive phosphorylase kinase. cAMPindependent glycogenolysis is also activated by vasopressin, oxytocin, and angiotensin II acting either through calcium or the phosphatidylinositol bisphosphate pathway (see Figure 42-10).

Both phosphorylase a and phosphorylase kinase a are dephosphorylated and inactivated by protein phosphatase-1. Protein phosphatase-1 is inhibited by a protein, inhibitor-1, which is active only after it has been phosphorylated by cAMP-dependent protein kinase. Thus, cAMP controls both the activation and inactivation of phosphorylase (Figure 18-6). Insulin reinforces this effect by inhibiting the activation of phosphorylase b. It does this indirectly by increasing uptake of glucose, leading to increased formation of glucose-6-phosphate, which is an inhibitor of phosphorylase kinase.

There are different isoenzymes of glycogen synthase in liver, muscle, and brain. Like phosphorylase, glycogen synthase exists in both phosphorylated and nonphosphorylated states, and the effect of phosphorylation is the reverse of that seen in phosphorylase (Figure 18-7). Active glycogen synthase a is dephosphorylated and inactive glycogen synthase b is phosphorylated.
Six different protein kinases act on glycogen synthase, and there are at least nine different serine residues in the enzyme that can be phosphorylated. Two of the protein kinases are Ca**/ calmodulin dependent (one of these is phosphorylase kinase). Another kinase is cAMP-dependent protein kinase, which allows cAMP-mediated hormone action to inhibit glycogen synthesis synchronously with the activation of glycogenolysis.
- FIGURE 18-6 Control of glycogen phosphorylase in muscle. The sequence of reactions arranged as a cascade allows amplification of the hormonal signal at each step. (G6P, glucose-6-phosphate; n, number of glucose residues.)
FIGURE 18-7 Control of glycogen synthase in muscle. (G6P, glucose-6-phosphate; GSK, glycogen synthase kinase; n, number of glucose residues.)
Insulin also promotes glycogenesis in muscle at the same time as inhibiting glycogenolysis by raising glucose-6-phosphate concentrations, which stimulates the dephosphorylation and activation of glycogen synthase. Dephosphorylation of glycogen synthase b is carried out by protein phosphatase-1, which is under the control of cAMP-dependent protein kinase.

phosphatase-1 is inhibited by cAMP-dependent protein kinase via inhibitor-1. Thus, glycogenolysis can be terminated and glycogenesis can be stimulated, or vice versa, synchronously, because both processes are dependent on the activity of cAMPdependent protein kinase. Both phosphorylase kinase and glycogen synthase may be reversibly phosphorylated at more than one site by separate kinases and phosphatases. These secondary phosphorylations modify the sensitivity of the primary sites to phosphorylation and dephosphorylation (multisite phosphorylation). Also, they allow insulin, by way of increased glucose6-phosphate, to have effects that act reciprocally to those of cAMP (Figures 18-6 and 18-7).
At the same time, as phosphorylase is activated by a rise in concentration of cAMP (via phosphorylase kinase), glycogen synthase is converted to the inactive form; both effects are mediated via cAMP-dependent protein kinase (Figure 18-8). Thus, inhibition of glycogenolysis enhances net glycogenesis, and inhibition of glycogenesis enhances net glycogenolysis. Also, the dephosphorylation of phosphorylase a, phosphorylase kinase, and glycogen synthase b is catalyzed by a single enzyme with broad specificity—protein phosphatase-1. In turn, protein

Glycogen storage disease is a generic term to describe a group of inherited disorders characterized by deposition of an abnormal type or quantity of glycogen in tissues, or failure to mobilize glycogen. The principal diseases are summarized in Table 18-2.
™
[_Phosphodiesterase_|
FIGURE 18-8 Coordinated control of glycogenolysis and glycogenesis by cAMP-dependent protein kinase. The reactions that lead to glycogenolysis as a result of an increase in CAMP concentrations are shown with bold arrows, and those that are inhibited by activation of protein phosphatase-1 are shown with dashed arrows. The reverse occurs when cAMP concentrations decrease as a result of phosphodiesterase activity, leading to glycogenesis.

™
Cyclic AMP integrates the regulation of glycogenolysis and
™ Glycogen represents the principal storage carbohydrate in the body, mainly in the liver and muscle.
@ In the liver, its major function is to provide glucose for
extrahepatic tissues. In muscle, it serves mainly as a source
of metabolic fuel for use in muscle. Muscle lacks glucose-6-
phosphatase and cannot release free glucose from glycogen.
™ Glycogen is synthesized from glucose by the pathway of glycogenesis. It is broken down by a separate pathway, glycogenolysis.
glycogenesis by promoting the simultaneous activation of
phosphorylase and inhibition of glycogen synthase. Insulin
acts reciprocally
by inhibiting
glycogenolysis and
stimulating glycogenesis.
- Inherited deficiencies of enzymes of glycogen metabolism in both liver and muscle cause glycogen storage diseases.

Explain the importance of gluconeogenesis in glucose homeostasis.
Describe the pathway of gluconeogenesis, how irreversible of
reciprocally. enzymes glycolysis are bypassed, and how glycolysis and gluconeogenesis are regulated
= Explain how plasma glucose concentration is maintained within narrow limits in the fed and fasting states.

Gluconeogenesis is the process of synthesizing glucose from noncarbohydrate precursors. The major substrates are the glucogenic amino acids (see Chapter 29), lactate, glycerol, and propionate. Liver and kidney are the major gluconeogenic tissues; the kidney may contribute up to 40% of total glucose synthesis in the fasting state and more in starvation. The key gluconeogenic enzymes are expressed in the small intestine, but it is unclear whether or not there is significant glucose production by the intestine in the fasting state, although propionate arising from intestinal bacterial fermentation of carbohydrates is a substrate for gluconeogenesis in enterocytes. A supply of glucose is necessary especially for the nervous system and erythrocytes. After an overnight fast, glycogenolysis (see Chapter 18) and gluconeogenesis make approximately equal contributions to blood glucose; as glycogen reserves are depleted, so gluconeogenesis becomes progressively more important.
Failure of gluconeogenesis is usually fatal. Hypoglycemia causes brain dysfunction, which can lead to coma and death. Glucose is also important in maintaining adequate concentrations of intermediates of the citric acid cycle (see Chapter 16) even when fatty acids are the main source of acetyl-CoA in the tissues. In addition, gluconeogenesis clears lactate produced by muscle and erythrocytes, and glycerol produced by adipose tissue. In ruminants, propionate is a product of rumen metabolism of carbohydrates, and is a major substrate for gluconeogenesis.
Excessive gluconeogenesis occurs in critically ill patients in response to injury and infection, contributing to hyperglycemia which is associated with a poor outcome. Hyperglycemia leads to changes in osmolality of body fluids, impaired blood flow, intracellular acidosis, and increased superoxide radical production (see Chapter 45), resulting in deranged endothelial and immune system function and impaired blood coagulation. Excessive gluconeogenesis is also a contributory factor to hyperglycemia in type 2 diabetes because of impaired downregulation in response to insulin.

Three nonequilibrium reactions in glycolysis (see Chapter 17), catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase, prevent simple reversal of glycolysis for glucose synthesis (Figure 19-1). They are circumvented as follows.

Reversal of the reaction catalyzed by pyruvate kinase in glycolysis involves two endothermic reactions. Mitochondrial pyruvate carboxylase catalyzes the carboxylation of pyruvate to oxaloacetate, an ATP-requiring reaction in which the vitamin biotin is the coenzyme. Biotin binds CO, from bicarbonate as carboxybiotin prior to the addition of the CO, to pyruvate (see Figure 44-14). The resultant oxaloacetate is reduced to malate, exported from the mitochondrion into the cytosol and there oxidized back to oxaloacetate. A second
FIGURE 19-1 Major pathways and regulation of gluconeogenesis and glycolysis in the liver. Entry points of glucogenic amino acids after transamination are indicated by arrows extended from circles (see also Figure 16-4). The key gluconeogenic enzymes are shown in double-bordered boxes. The ATP required for gluconeogenesis is supplied by the oxidation of fatty acids. Propionate is important only in ruminants. Arrows with wavy shafts signify allosteric effects; dash-shafted arrows, covalent modification by reversible phosphorylation. High concentrations of alanine act as a "gluconeogenic signal" by inhibiting glycolysis at the pyruvate kinase step.
enzyme, phosphoenolpyruvate carboxykinase, catalyzes the decarboxylation and phosphorylation of oxaloacetate to phosphoenolpyruvate using GTP as the phosphate donor. In liver and kidney, the reaction of succinate thiokinase in the citric acid cycle (see Chapter 16) produces GTP (rather than ATP
as in other tissues), and this GTP is used for the reaction of phosphoenolpyruvate carboxykinase, thus providing a link between citric acid cycle activity and gluconeogenesis, to prevent excessive removal of oxaloacetate for gluconeogenesis, which would impair citric acid cycle activity.

The conversion of fructose 1,6-bisphosphate to fructose6-phosphate, for the reversal of glycolysis, is catalyzed by fructose 1,6-bisphosphatase. Its presence determines whether a tissue is capable of synthesizing glucose (or glycogen) not only from pyruvate but also from triose phosphates. It is present in liver, kidney, and skeletal muscle, but is probably absent from heart and smooth muscle.

The conversion of glucose-6-phosphate to glucose is catalyzed by glucose-6-phosphatase. It is present in liver and kidney, but absent from muscle, which, therefore, cannot export glucose into the bloodstream.

The breakdown of glycogen to glucose-1-phosphate is catalyzed by phosphorylase. Glycogen synthesis involves a different pathway via uridine diphosphate glucose and glycogen synthase (see Figure 18-1).
The relationships between gluconeogenesis and the glycolytic pathway are shown in Figure 19-1. After transamination or deamination, glucogenic amino acids yield either pyruvate or intermediates of the citric acid cycle. Therefore, the reactions described above can account for the conversion of both lactate and glucogenic amino acids to glucose or glycogen.
Propionate is a major precursor of glucose in ruminants; it enters gluconeogenesis via the citric acid cycle. After esterification with CoA, propionyl-CoA is carboxylated to p-methylmalonyl-CoA, catalyzed by propionyl-CoA carboxylase, a biotin-dependent enzyme (Figure 19-2). Methylmalonyl-CoA racemase catalyzes the conversion of p-methylmalonyl-CoA to t-methylmalonyl-CoA, which then undergoes isomerization to succinyl-CoA catalyzed by methylmalonyl-CoA mutase. In nonruminants, including human beings, propionate arises from the B-oxidation of oddchain fatty acids that occur in ruminant lipids (see Chapter 22), as well as the oxidation of isoleucine and the side chain of cholesterol, and is a (relatively minor) substrate for gluconeogenesis. Methylmalonyl-CoA mutase is a vitamin B,,-dependent enzyme, and in deficiency, methylmalonic acid is excreted in the urine (methylmalonic aciduria).
Glycerol is released from adipose tissue as a result of lipolysis of lipoprotein triacylglycerol in the fed state; it may be used for reesterification of free fatty acids to triacylglycerol, or may be a substrate for gluconeogenesis in the liver. In the fasting state, glycerol released from lipolysis of adipose tissue triacylglycerol is used as a substrate for gluconeogenesis in the liver and kidneys.

Changes in the availability of substrates are responsible for most changes in metabolism either directly or indirectly acting via changes in hormone secretion. Three mechanisms are responsible for regulating the activity of enzymes concerned in carbohydrate metabolism: (1) changes in the rate of enzyme synthesis, (2) covalent modification by reversible phosphorylation, and (3) allosteric effects.

The changes in enzyme activity in the liver that occur under various metabolic conditions are listed in Table 19-1. The enzymes involved catalyze physiologically irreversible

Glucocorticoids, glucagon, epinephrine. Pyruvate carboxylase, Repressor = Insulin. Pyruvate carboxylase, Activator = Acetyl-CoA. Pyruvate carboxylase, Inhibitor = ADP. Phosphoenolpyruvate carboxykinase, Activity in.Carbohydrate Feeding = ↓. Phosphoenolpyruvate carboxykinase, Activity in.Fasting and Diabetes = ↑. Phosphoenolpyruvate carboxykinase, Inducer = Glucocorticoids, glucagon, epinephrine. Phosphoenolpyruvate carboxykinase, Repressor = Insulin. Phosphoenolpyruvate carboxykinase, Activator = Glucagon. Phosphoenolpyruvate carboxykinase, Inhibitor = . Glucose-6-phosphatase, Activity in.Carbohydrate Feeding = ↓. Glucose-6-phosphatase, Activity in.Fasting and Diabetes = ↑. Glucose-6-phosphatase, Inducer = Glucocorticoids, glucagon, epinephrine. Glucose-6-phosphatase, Repressor = Insulin. Glucose-6-phosphatase, Activator = . Glucose-6-phosphatase, Inhibitor = 
nonequilibrium reactions. The effects are generally reinforced because the activity of the enzymes catalyzing the reactions in the opposite direction varies reciprocally (see Figure 19-1). The enzymes involved in the utilization of glucose (ie, those of glycolysis and lipogenesis) become more active when there is a superfluity of glucose, and under these conditions the enzymes of gluconeogenesis have low activity. Insulin, secreted in response to increased blood glucose, enhances the synthesis of the key enzymes in glycolysis. It also antagonizes the effect of the glucocorticoids and glucagon-stimulated cAMP, which induce synthesis of the key enzymes of gluconeogenesis.

Glucagon and epinephrine, hormones that are responsive to a decrease in blood glucose, inhibit glycolysis and stimulate gluconeogenesis in the liver by increasing the concentration of cAMP. This in turn activates cAMP-dependent protein kinase, leading to the phosphorylation and inactivation of pyruvate kinase. They also affect the concentration of fructose
2,6-bisphosphate and therefore glycolysis and gluconeogenesis, as described below.

In gluconeogenesis, pyruvate carboxylase, which catalyzes the synthesis of oxaloacetate from pyruvate, requires acetyl-CoA as an allosteric activator. The addition of acetyl-CoA results in a change in the tertiary structure of the protein, lowering the K_ for bicarbonate. This means that as acetyl-CoA is formed from pyruvate, it automatically ensures the provision of oxaloacetate, by activating pyruvate carboxylase. The activation of pyruvate carboxylase and the reciprocal inhibition of pyruvate dehydrogenase by acetyl-CoA derived from the oxidation of fatty acids explain the action of fatty acid oxidation in sparing the oxidation of pyruvate (and hence glucose) and stimulating gluconeogenesis. The reciprocal relationship between these two enzymes alters the metabolic fate of pyruvate as the tissue changes from carbohydrate oxidation (glycolysis) to gluconeogenesis during the transition from the fed to fasting state (see Figure 19-1). A major role of fatty acid
FIGURE 19-3 The inhibition of phosphofructokinase-1 by ATP and relief of inhibition by ATP. The yellow bar shows the normal range of the intracellular concentration of ATP.
oxidation in promoting gluconeogenesis is to supply the ATP that is required. Phosphofructokinase (phosphofructokinase-1) occupies a key position in regulating glycolysis and is also subject to feedback control. It is inhibited by citrate and by normal intracellular concentrations of ATP and is activated by 5' AMP. At the normal intracellular [ATP] the enzyme is about 90% inhibited; this inhibition is reversed by 5'AMP (Figure 19-3). 5' AMP acts as an indicator of the energy status of the cell. The presence of adenylyl kinase in liver and many other tissues allows rapid equilibration of the reaction fructose-6-phosphate. It inhibits fructose 1,6-bisphosphatase by increasing the K_ for fructose 1,6-bisphosphate. Its concentration is under both substrate (allosteric) and hormonal control (covalent modification) (Figure 19-4).

Thus, when ATP is used in energy-requiring processes, resulting in the formation of ADP, [AMP] increases. A relatively small decrease in [ATP] causes a severalfold increase in [AMP], so that [AMP] acts as a metabolic amplifier of a small change in [ATP], and hence a sensitive signal of the energy state of the cell. The activity of phosphofructokinase-1 is thus regulated in response to the energy status of the cell to control the quantity of carbohydrate undergoing glycolysis prior to its entry into the citric acid cycle. At the same time, AMP activates glycogen phosphorylase, so increasing glycogenolysis. A consequence of the inhibition of phosphofructokinase-1 by ATP is an accumulation of glucose-6-phosphate, which in turn inhibits further uptake of glucose in extrahepatic tissues by inhibition of hexokinase.

The most potent positive allosteric activator of phosphofructokinase-1 and inhibitor of fructose 1,6-bisphosphatase in liver is fructose 2,6-bisphosphate. It relieves inhibition of phosphofructokinase-1 by ATP and increases the affinity for
Fructose 2,6-bisphosphate is formed by phosphorylation of fructose-6-phosphate by phosphofructokinase-2. The same enzyme protein is also responsible for its breakdown, since it has fructose 2,6-bisphosphatase activity. This bifunctional enzyme is under the allosteric control of fructose6-phosphate, which stimulates the kinase and inhibits the phosphatase. Hence, when there is an abundant supply of glucose, the concentration of fructose 2,6-bisphosphate increases, stimulating glycolysis by activating phosphofructokinase-1 and inhibiting fructose 1,6-bisphosphatase. In the fasting state, glucagon stimulates the production of cAMP, activating cAMP-dependent protein kinase, which in turn inactivates phosphofructokinase-2 and activates fructose 2,6-bisphosphatase by phosphorylation. Hence, gluconeogenesis is stimulated by a decrease in the concentration of fructose 2,6-bisphosphate, which inactivates phosphofructokinase-1 and relieves the inhibition of fructose 1,6-bisphosphatase. Xylulose 5-phosphate, an intermediate of the pentose phosphate pathway (see Chapter 20) activates the protein phosphatase that dephosphorylates the bifunctional enzyme, so increasing the formation of fructose 2,6-bisphosphate and increasing the rate of glycolysis. This leads to increased flux through glycolysis and the pentose phosphate pathway and increased fatty acid synthesis (see Chapter 23).

The control points in glycolysis and glycogen metabolism involve a cycle of phosphorylation and dephosphorylation catalyzed by glucokinase and glucose-6-phosphatase;
FIGURE 19-4 Control of glycolysis and gluconeogenesis in the liver by fructose 2,6-bisphosphate and the bifunctional enzyme PFK-2/F-2,6-Pase (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase). (F-1,6-Pase, fructose 1,6-bisphosphatase; PFK-1, phosphofructokinase-1 [6-phosphofructo-1-kinase].) Arrows with wavy shafts indicate allosteric effects.

phosphofructokinase-1 and fructose 1,6-bisphosphatase; pyruvate kinase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase; and glycogen synthase and phosphorylase. It would seem obvious that these opposing enzymes are regulated in such a way that when those involved in glycolysis are active, those involved in gluconeogenesis are inactive, since otherwise there would be cycling between phosphorylated and nonphosphorylated intermediates, with net hydrolysis of ATP. While this is so, in muscle both phosphofructokinase and fructose 1,6-bisphosphatase have some activity at all times, so that there is indeed some measure of (wasteful) substrate cycling. This permits the very rapid increase in the rate of glycolysis necessary for muscle contraction. At rest the rate of phosphofructokinase activity is some 10-fold higher than that of fructose 1,6-bisphosphatase; in anticipation of muscle contraction, the activity of both enzymes increases, fructose 1,6-bisphosphatase 10 times more than phosphofructokinase, maintaining the same net rate of glycolysis. At the start of muscle contraction, the activity of phosphofructokinase increases further, and that of fructose 1,6-bisphosphatase falls, so increasing the net rate of glycolysis (and hence ATP formation) as much as a 1000-fold.

In the postabsorptive state, the concentration of blood glucose is maintained between 4.5 and 5.5 mmol/L. After the ingestion of a carbohydrate meal, it may rise to 6.5 to 7.2 mmol/L, and in starvation, it may fall to 3.3 to 3.9 mmol/L. A sudden decrease in blood glucose (eg, in response to insulin overdose) causes convulsions, because of the dependence of the brain on a supply of glucose. However, much lower concentrations can be tolerated if hypoglycemia develops slowly enough for adaptation to occur. The blood glucose level in birds is considerably higher (14 mmol/L) and in ruminants considerably lower (~ 2.2 mmol/L in sheep and 3.3 mmol/L in cattle). These lower normal levels appear to be associated with the fact that ruminants ferment virtually all dietary carbohydrate to short-chain fatty acids, and these largely replace glucose as the main metabolic fuel of the tissues in the fed state.

The digestible dietary carbohydrates yield glucose, galactose, and fructose that are transported to the liver via the hepatic portal vein. Galactose and fructose are readily converted to glucose in the liver (see Chapter 20).
Glucose is formed from two groups of compounds that undergo gluconeogenesis (see Figures 16-4 and 19-1): (1) those that involve a direct net conversion to glucose, including most amino acids and propionate and (2) those that are the products of the metabolism of glucose in tissues. Thus, lactate, formed by glycolysis in skeletal muscle and erythrocytes, is transported to the liver and kidney where it reforms glucose, which again becomes available via the circulation for oxidation in the tissues. This process is known as the Cori cycle, or the lactic acid cycle (Figure 19-5).
In the fasting state, there is a considerable output of alanine from skeletal muscle, far in excess of the amount in the muscle proteins that are being catabolized. It is formed by transamination of pyruvate produced by glycolysis of muscle glycogen, and is exported to the liver, where, after transamination back to pyruvate, it is a substrate for gluconeogenesis. This glucosealanine cycle (see Figure 19-5) provides an indirect way of utilizing muscle glycogen to maintain blood glucose in the fasting state. The ATP required for the hepatic synthesis of glucose from pyruvate is fromed by the oxidation of fatty acids.
FIGURE 19-5 The lactic acid (Cori cycle) and glucose-alanine cycles.
Glucose is also formed from liver glycogen by glycogenolysis (see Chapter 18).

The maintenance of a stable blood glucose concentration is one of the most finely regulated of all homeostatic mechanisms, involving the liver, extrahepatic tissues, and several hormones. Liver cells are freely permeable to glucose in either direction (via the GLUT 2 transporter), whereas cells of extrahepatic tissues (apart from pancreatic B-islets) are relatively impermeable, and their unidirectional glucose transporters are regulated by insulin. As a result, uptake from the bloodstream is the rate-limiting step in the utilization of glucose in extrahepatic tissues. The role of various glucose transporter proteins found in cell membranes is shown in Table 19-2.
Hexokinase has a low K,, for glucose, and in the liver it is saturated and acting at a constant rate under all normal conditions. It thus acts to ensure an adequate rate of glycolysis to meet the liver's needs. Glucokinase is an allosteric enzyme with a considerably higher apparent K,, (lower affinity) for glucose, so that its activity increases with increases in the concentration of glucose in the hepatic portal vein (Figure 19-6). In the fasting state, glucokinase is located in the nucleus. In response to an increased intracellular concentration of glucose it migrates into the cytosol, mediated by the carbohydrate response element-binding protein (CREBP). It thus permits hepatic uptake of large amounts of glucose after a carbohydrate meal, for glycogen and fatty acid synthesis, so that while the concentration of glucose in the hepatic portal vein may reach 20 mmol/L after a meal, that leaving the liver into the peripheral circulation does not normally exceed 8 to 9 mmol/L. Glucokinase is

Facilitative bidirectional transporters,  = Facilitative bidirectional transporters. Facilitative bidirectional transporters, Functions = . GLUT 1,  = Brain, kidney, colon, placenta, erythrocytes. GLUT 1, Functions = Glucose uptake. GLUT 2,  = Liver, pancreatic β cell, small intestine, kidney. GLUT 2, Functions = Rapid uptake or release of glucose. GLUT 3,  = Brain, kidney, placenta. GLUT 3, Functions = Glucose uptake. GLUT 4,  = Heart and skeletal muscle, adipose tissue. GLUT 4, Functions = Insulin-stimulated glucose uptake. GLUT 5,  = Small intestine. GLUT 5, Functions = Absorption of fructose. Sodium-dependent unidirectional transporter,  = Sodium-dependent unidirectional transporter. Sodium-dependent unidirectional transporter, Functions = . SGLT 1,  = Small intestine and kidney. SGLT 1, Functions = Active uptake of glucose against a concentration gradient
FIGURE 19-6 Variation in glucose phosphorylating activity of hexokinase and glucokinase with increasing blood glucose concentration. The K_, for glucose of hexokinase is 0.05 mmol/L and of glucokinase is 10 mmol/L.
absent from the liver of ruminants, which have little glucose entering the portal circulation from the intestines.
At normal peripheral blood glucose concentrations (4.55.5 mmol/L), the liver is a net producer of glucose. However, as the glucose level rises, the output of glucose ceases, and there is a net uptake.

acids, ketone bodies, glucagon, secretin, and the sulfonylurea drugs tolbutamide and glyburide. These drugs are used to stimulate insulin secretion in type 2 diabetes mellitus via the ATP-sensitive K* channels. Epinephrine and norepinephrine block the release of insulin. Insulin acts to lower blood glucose immediately by enhancing glucose transport into adipose tissue and muscle by recruitment of glucose transporters (GLUT 4) from the interior of the cell to the plasma membrane. Although it does not affect glucose uptake into the liver directly, insulin does enhance long-term uptake as a result of its actions on the enzymes controlling glycolysis, glycogenesis, and gluconeogenesis (see Chapter 18 and Table 19-1).
Glucagon is the hormone produced by the a cells of the pancreatic islets in response to hypoglycemia. In the liver, it stimulates glycogenolysis by activating glycogen phosphorylase. Unlike epinephrine, glucagon does not have an effect on muscle phosphorylase. Glucagon also enhances gluconeogenesis from amino acids and lactate. In all these actions, glucagon acts via generation of cAMP (Table 19-1). Both hepatic glycogenolysis and gluconeogenesis contribute to the hyperglycemic effect of glucagon, whose actions oppose those of insulin. Most of the endogenous glucagon (and insulin) is cleared from the circulation by the liver (Table 19-3).

In addition to the direct effects of hyperglycemia in enhancing the uptake of glucose into the liver, the hormone insulin plays a central role in regulating blood glucose. It is produced by the 6 cells of the islets of Langerhans in the pancreas in response to hyperglycemia. The B-islet cells are freely permeable to glucose via the GLUT 2 transporter, and the glucose is phosphorylated by glucokinase. Therefore, increasing blood glucose increases metabolic flux through glycolysis, the citric acid cycle, and the generation of ATP. The increase in [ATP] inhibits ATP-sensitive K* channels, causing depolarization of the cell membrane, which increases Ca'* influx via voltagesensitive Ca** channels, stimulating exocytosis of insulin. Thus, the concentration of insulin in the blood parallels that of the blood glucose. Other substances causing release of insulin from the pancreas include amino acids, nonesterified fatty
The anterior pituitary gland secretes hormones that tend to elevate blood glucose and therefore antagonize the action of insulin. These are growth hormone, adrenocorticotropic hormone (ACTH), and possibly other "diabetogenic" hormones. Growth hormone secretion is stimulated by hypoglycemia; it decreases glucose uptake in muscle. Some of this effect may be indirect, since it stimulates mobilization of nonesterified fatty acids from adipose tissue, which themselves inhibit glucose utilization. The glucocorticoids (11-oxysteroids) are secreted by the adrenal cortex, and are also synthesized in an unregulated manner in adipose tissue. They act to increase gluconeogenesis as a result of enhanced hepatic catabolism of amino acids, due to induction of aminotransferases (and other enzymes such as tryptophan dioxygenase) and key enzymes of gluconeogenesis. In addition, glucocorticoids inhibit the utilization of glucose in extrahepatic tissues. In all these actions, glucocorticoids act in a manner antagonistic to insulin. A number of cytokines

Increased by insulin, Liver = Fatty acid synthesis Glycogen synthesis Protein synthesis. Increased by insulin, Adipose Tissue = Glucose uptake Fatty acid synthesis. Increased by insulin, Muscle = Glucose uptake Glycogen synthesis Protein synthesis. Decreased by insulin, Liver = Ketogenesis Gluconeogenesis. Decreased by insulin, Adipose Tissue = Lipolysis. Decreased by insulin, Muscle = . Increased by glucagon, Liver = Glycogenolysis Gluconeogenesis Ketogenesis. Increased by glucagon, Adipose Tissue = Lipolysis. Increased by glucagon, Muscle = 
secreted by macrophages infiltrating adipose tissue also have insulin antagonistic actions; together with glucocorticoids secreted by adipose tissue, this explains the insulin resistance that commonly occurs in obese people.
Epinephrine is secreted by the adrenal medulla as a result of stressful stimuli (fear, excitement, hemorrhage, hypoxia, hypoglycemia, etc.) and leads to glycogenolysis in liver and muscle owing to stimulation of phosphorylase via generation of cAMP. In muscle, glycogenolysis results in increased glycolysis, whereas in liver, it results in the release of glucose into the bloodstream.

When the blood glucose concentration rises above about 10 mmol/L, the kidney also exerts a (passive) regulatory effect. Glucose is continuously filtered by the glomeruli, but is normally completely reabsorbed in the renal tubules by active transport. The capacity of the tubular system to reabsorb glucose is limited to a rate of about 2 mmol/min, and in hyperglycemia (as occurs in poorly controlled diabetes mellitus), the glomerular filtrate may contain more glucose than can be reabsorbed, resulting in glucosuria when the renal threshold for glucose is exceeded.

During pregnancy, fetal glucose consumption increases and there is a risk of maternal, and possibly fetal, hypoglycemia, particularly if there are long intervals between meals or at night. Furthermore, premature and low-birth-weight babies are more susceptible to hypoglycemia, since they have little adipose tissue to provide nonesterified fatty acids. The enzymes of gluconeogenesis may not be fully developed at this time, and gluconeogenesis is anyway dependent on a supply of nonesterified fatty acids for ATP formation. Little glycerol, which would normally be released from adipose tissue, is available for gluconeogenesis.

Glucose tolerance is the ability to regulate the blood glucose concentration after the administration of a test dose of glucose (normally 1 g/kg body weight) (Figure 19-7).
Diabetes mellitus (type 1, or insulin-dependent diabetes mellitus [IDDM]) is characterized by impaired glucose tolerance as a result of decreased secretion of insulin because of progressive destruction of pancreatic f-islet cells. Glucose tolerance is also impaired in type 2 diabetes mellitus (noninsulin-dependent diabetes [NIDDM]) as a result of reduced sensitivity of tissues to insulin action. Insulin resistance associated with obesity (and especially abdominal obesity) leading to the development of hyperlipidemia, then atherosclerosis and coronary heart disease, as well as overt diabetes, is known as the metabolic syndrome. Impaired glucose tolerance also occurs in conditions where the liver is damaged, in some infections, and in response to some drugs, as well as in conditions that lead to hyperactivity of the pituitary gland or adrenal cortex because of the hormones secreted by these glands that antagonize the action of insulin. Administration of insulin (as in the treatment of diabetes mellitus) lowers the blood glucose concentration and increases
FIGURE 19-7 Glucose tolerance test. Blood glucose curves of a normal and a diabetic person after oral administration of 1 g of glucose/kg body weight. Note the initial raised concentration in the fasting diabetic. A criterion of normality is the return to the initial value within 2 hours.
its utilization and storage in the liver and muscle as glycogen. An excess of insulin may cause hypoglycemia, resulting in convulsions and even death unless glucose is administered promptly. Increased tolerance to glucose is observed in pituitary or adrenocortical insufficiency, attributable to a decrease in the antagonism to insulin by the hormones normally secreted by these glands.

Very low carbohydrate diets, providing only 20 g per day of carbohydrate or less (compared with a desirable intake of 100120 g/day), but permitting unlimited consumption of fat and protein, have been promoted as an effective regime for weight loss, although such diets are counter to all advice on a prudent diet for health. Since there is a continual demand for glucose, there will be a considerable amount of gluconeogenesis from amino acids; the associated high ATP cost must then be met by oxidation of fatty acids.

■, 1 = Gluconeogenesis is the process of synthesizing glucose or glycogen from noncarbohydrate precursors. It is of particular importance when carbohydrate is not available from the diet. The main substrates are amino acids, lactate, glycerol, and propionate.. ■, 1 = The pathway of gluconeogenesis in the liver and kidney utilizes those reactions in glycolysis that are reversible plus four additional reactions that circumvent the irreversible nonequilibrium reactions.. ■, 1 = Since glycolysis and gluconeogenesis share the same pathway but operate in opposite directions, their activities must be regulated reciprocally.. ■, 1 = The liver regulates the blood glucose concentration after a meal because it contains the high K m glucokinase that promotes increased hepatic utilization of glucose.. ■, 1 = Insulin is secreted as a direct response to hyperglycemia; it stimulates the liver to store glucose as glycogen and increases uptake of glucose into extrahepatic tissues.. ■, 1 = Glucagon is secreted as a response to hypoglycemia and activates both glycogenolysis and gluconeogenesis in the liver, causing release of glucose into the blood.

- . Describe the pentose phosphate pathway and its roles as a source of NADPH and of ribose for nucleotide synthesis.
: vitamin C. Describe the uronic acid pathway and its importance for synthesis of glucuronic acid for conjugation reactions and (in animals for which it is not a vitamin)
Describe and explain the consequences of large intakes of fructose.
Describe the synthesis and physiological importance of galactose.
Explain the consequences of genetic defects of glucose-6-phosphate dehydrogenase (favism), the uronic acid pathway (essential pentosuria), and fructose and galactose metabolism.

The pentose phosphate pathway is an alternative route for the metabolism of glucose. It does not lead to formation of ATP but has two major functions: (1) the formation of NADPH for synthesis of fatty acids (see Chapter 23) and steroids (see Chapter 26), and maintaining reduced glutathione for antioxidant activity, and (2) the synthesis of ribose for nucleotide and nucleic acid formation (see Chapter 32). Glucose, fructose, and galactose are the main hexoses absorbed from the gastrointestinal tract, derived from dietary starch, sucrose, and lactose, respectively. Fructose and galactose can be converted to glucose, mainly in the liver.
Genetic deficiency of glucose-6-phosphate dehydrogenase, the first enzyme of the pentose phosphate pathway, causes acute hemolysis of red blood cells, resulting in hemolytic anemia. Glucuronic acid is synthesized from glucose via the uronic acid pathway, of minor quantitative importance, but of major significance for the conjugation and excretion of metabolites and foreign chemicals (xenobiotics, see Chapter 47) as glucuronides. A deficiency in the pathway leads to the condition of essential pentosuria. The lack of one enzyme of the pathway (gulonolactone oxidase) in primates and some other animals explains why ascorbic acid (vitamin C, see Chapter 44) is a dietary requirement for human beings but not most other mammals. Deficiencies in the enzymes of fructose and galactose metabolism lead to metabolic diseases such as essential fructosuria, hereditary fructose intolerance, and galactosemia.

The pentose phosphate pathway (hexose monophosphate shunt, Figure 20-1) is a more complex pathway than glycolysis (see Chapter 17). Three molecules of glucose-6-phosphate give rise to three molecules of CO, and three five-carbon sugars. These are rearranged to regenerate two molecules of glucose-6-phosphate and one molecule of the glycolytic intermediate, glyceraldehyde-3-phosphate. Since two molecules of glyceraldehyde-3-phosphate can regenerate glucose6-phosphate, the pathway can account for the complete oxidation of glucose.

Like glycolysis, the enzymes of the pentose phosphate pathway are cytosolic. Unlike glycolysis, oxidation is achieved by dehydrogenation using NADP*, not NAD*, as the
C
H
A
PTE
R
[
FIGURE 20-1 Flowchart of pentose phosphate pathway and its connections with the pathway of glycolysis. The full pathway, as indicated, consists of three interconnected cycles in which glucose-6-phosphate is both substrate and end product. The reactions above the broken line are nonreversible, whereas all reactions under that line are freely reversible apart from that catalyzed by fructose 1,6-bisphosphatase.
hydrogen acceptor. The sequence of reactions of the pathway may be divided into two phases: an irreversible oxidative phase and a reversible nonoxidative phase. In the first phase, glucose-6-phosphate undergoes dehydrogenation and decarboxylation to yield a pentose, ribulose-5phosphate. In the second phase, ribulose-5-phosphate is converted back to glucose-6-phosphate by a series of reactions involving mainly two enzymes: transketolase and transaldolase (Figure 20-1).

Dehydrogenation of glucose-6-phosphate to 6-phosphoglu- conate occurs via the formation of 6-phosphogluconolac-
tone, catalyzed by glucose-6-phosphate dehydrogenase,
an NADP-dependent enzyme (Figures 20-1 and 20-2). The hydrolysis of 6-phosphogluconolactone is accomplished by the enzyme gluconolactone hydrolase. A second oxidative step is catalyzed by 6-phosphogluconate dehydrogenase, which also requires NADP* as hydrogen acceptor. Decarboxylation forms the ketopentose ribulose-5-phosphate.
In the endoplasmic reticulum, an isoenzyme of glucose6-phosphate dehydrogenase, hexose-6-phosphate dehydrogenase, provides NADPH for hydroxylation (mixed function oxidase) reactions, and also for 11-B-hydroxysteroid dehydrogenase-1. This enzyme catalyzes the reduction of (inactive) cortisone to (active) cortisol in liver, the nervous system, and oO
FIGURE 20-2 The pentose phosphate pathway. (P, —PO,"; PRPP, 5-phosphoribosyl 1-pyrophosphate.)
adipose tissue. It is the major source of intracellular cortisol in these tissues and may be important in obesity and the metabolic syndrome.

Ribulose-5-phosphate is the substrate for two enzymes. Ribulose-5-phosphate 3-epimerase alters the configuration about carbon 3, forming the epimer xylulose 5-phosphate, also a ketopentose. Ribose-5-phosphate ketoisomerase converts ribulose-5-phosphate to the corresponding aldopentose, ribose-5-phosphate, which is used for nucleotide and nucleic acid synthesis. Transketolase transfers the two-carbon unit comprising carbons 1 and 2 of a ketose onto the aldehyde carbon of an aldose sugar. It therefore effects the conversion of a ketose sugar into an aldose with two carbons less and an aldose sugar into a ketose with two carbons more. The reaction requires Mg" and thiamin diphosphate (vitamin B,) as coenzyme. Measurement of erythrocyte transketolase and its activation by thiamin diphosphate provides an index of vitamin B, nutritional status (see Chapter 44). The two-carbon moiety is transferred as glycolaldehyde bound to thiamin diphosphate. Thus, transketolase catalyzes the transfer of the two-carbon unit from xylulose 5-phosphate to ribose-5-phosphate, producing the seven-carbon ketose sedoheptulose-7-phosphate and the aldose glyceraldehyde-3-phosphate. These two products then undergo transaldolation. Transaldolase catalyzes the transfer of a three-carbon dihydroxyacetone moiety (carbons 1-3) from the ketose sedoheptulose-7-phosphate onto the aldose glyceraldehyde-3-phosphate to form the ketose fructose-6-phosphate and the four-carbon aldose erythrose-4-phosphate. Transaldolase has no cofactor, and the reaction proceeds via the intermediate formation of a Schiff base of dihydroxyacetone to the e-amino group of a lysine residue in the enzyme. In a further reaction catalyzed by transketolase, xylulose 5-phosphate serves

as a donor of glycolaldehyde. In this case, erythrose-4-phosphate is the acceptor, and the products of the reaction are fructose-6-phosphate and glyceraldehyde-3-phosphate.
In order to oxidize glucose completely to CO, via the pentose phosphate pathway, there must be enzymes present in the tissue to convert glyceraldehyde-3-phosphate to glucose6-phosphate. This involves reversal of glycolysis and the gluconeogenic enzyme fructose 1,6-bisphosphatase. In tissues that lack this enzyme, glyceraldehyde-3-phosphate follows the normal pathway of glycolysis to pyruvate.

Although glucose-6-phosphate is common to both pathways, the pentose phosphate pathway is markedly different from glycolysis. Oxidation utilizes NADP* rather than NAD*, and
CO,, which
is not
produced in
glycolysis, is
produced.
No
ATP
is generated in the pentose phosphate pathway, whereas it is a product of glycolysis.
The two pathways are, however, connected. Xylulose 5phosphate activates the protein phosphatase that dephosphorylates the 6-phosphofructo-2-kinase/fructose 2,6-bisphophatase bifunctional enzyme (see Chapter 17). This activates the kinase and inactivates the phosphatase, leading to increased formation of fructose 2,6-bisphosphate, increased activity of phosphofructokinase-1, and hence increased glycolytic flux. Xylulose 5-phosphate also activates the protein phosphatase that initiates the nuclear translocation and DNA binding of the carbohydrate response element-binding protein, leading to increased synthesis of fatty acids (see Chapter 23) in response to a high-carbohydrate diet.

The pentose phosphate pathway is active in liver, adipose tissue, adrenal cortex, thyroid, erythrocytes, testis, and lactating mammary gland. Its activity is low in nonlactating mammary gland and skeletal muscle. Those tissues in which the pathway is active use NADPH in reductive syntheses, for example, of fatty acids, steroids, amino acids via glutamate dehydrogenase, and reduced glutathione. The synthesis of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase may also be induced by insulin in the fed state, when lipogenesis increases.

Little or no ribose circulates in the bloodstream, so tissues have to synthesize the ribose they require for nucleotide and nucleic acid synthesis using the pentose phosphate pathway (Figure 20-2). It is not necessary to have a completely functioning pentose phosphate pathway for a tissue to synthesize ribose5-phosphate. Muscle has only low activity of glucose-6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, but, like most other tissues, it is capable of synthesizing ribose-5-phosphate by reversal of the nonoxidative phase of the pentose phosphate pathway utilizing fructose-6-phosphate.

In red blood cells, the pentose phosphate pathway is the sole source of NADPH for the reduction of oxidized glutathione catalyzed by glutathione reductase, a flavoprotein containing flavin adenine dinucleotide (FAD). Reduced glutathione removes H,O, in a reaction catalyzed by glutathione peroxidase, an enzyme that contains the selenium analog of cysteine (selenocysteine)
FIGURE 20-3 Role of the pentose phosphate pathway in the glutathione peroxidase reaction of erythrocytes. (GSH, reduced glutathione; GSSG, oxidized glutathione; Se, selenium-containing enzyme.)
at the active site (Figure 20-3). The reaction is important since accumulation of H,O, may decrease the life span of the erythrocyte by causing oxidative damage to the cell membrane, leading to hemolysis. In other tissues, NADPH can also be generated by the reaction catalyzed by the malic enzyme.

In liver, the uronic acid pathway catalyzes the conversion of glucose to glucuronic acid, ascorbic acid (except in human beings and other species for which ascorbate is a vitamin, vitamin C), and pentoses (Figure 20-4). It is also an alternative oxidative pathway for glucose that, like the pentose phosphate pathway, does not lead to the formation of ATP. Glucose6-phosphate is isomerized to glucose-1-phosphate, which then reacts with uridine triphosphate (UTP) to form uridine diphosphate glucose (UDPGlc) in a reaction catalyzed by UDPGlc pyrophosphorylase, as occurs in glycogen synthesis (see Chapter 18). UDPGlc is oxidized at carbon 6 by NADdependent UDPGlc dehydrogenase in a two-step reaction to yield UDP-glucuronate.
UDP-glucuronate is the source of glucuronate for reactions involving its incorporation into proteoglycans (see Chapter 46) or for reaction with substrates such as steroid hormones, bilirubin, and a number of drugs that are excreted in urine or bile as glucuronide conjugates (see Figure 31-13 and Chapter 47).
Glucuronate is reduced to L-gulonate, the direct precursor of ascorbate in those animals capable of synthesizing this vitamin, in an NADPH-dependent reaction. In human beings and other primates, as well as guinea pigs, bats, and some birds and fishes, ascorbic acid cannot be synthesized because of the absence of t-gulonolactone oxidase. L-Gulonate is oxidized to 3-keto-L-gulonate, which is then decarboxylated to L-xylulose. L-Xylulose is converted to the p-isomer by an NADPH-dependent reduction to xylitol, followed by oxidation in an NAD-dependent reaction to p-xylulose. After conversion to p-xylulose 5-phosphate, it is metabolized via the pentose phosphate pathway.

Diets high in sucrose or in high-fructose syrups (HFS) used in manufactured foods and beverages lead to large amounts of fructose (and glucose) entering the hepatic portal vein.
Fructose undergoes more rapid glycolysis in the liver than does glucose because it bypasses the regulatory step catalyzed by phosphofructokinase (Figure 20-5). This allows fructose to flood the pathways in the liver, leading to increased fatty acid synthesis, esterification of fatty acids, and secretion of verylow-density lipoprotein (VLDL), which may raise serum triacylglycerols and ultimately raise LDL cholesterol concentrations. Fructokinase in liver, kidney, and intestine catalyzes the phosphorylation of fructose to fructose-1-phosphate. This enzyme does not act on glucose, and, unlike glucokinase, its activity is not affected by fasting or by insulin, which may explain why fructose is cleared from the blood of diabetic patients at a normal rate. Fructose-1-phosphate is cleaved to p-glyceraldehyde and dihydroxyacetone phosphate by aldolase B, an enzyme found in the liver, which also functions in glycolysis in the liver by cleaving fructose 1,6-bisphosphate. p-Glyceraldehyde enters glycolysis via phosphorylation to glyceraldehyde-3-phosphate catalyzed by triokinase. The two triose phosphates, dihydroxyacetone phosphate, and glyceraldehyde-3-phosphate, may either be degraded by glycolysis or may be substrates for aldolase and hence gluconeogenesis, which is the fate of much of the fructose metabolized in the liver.

In extrahepatic tissues, hexokinase catalyzes the phosphorylation of most hexose sugars, including fructose, but glucose inhibits the phosphorylation of fructose since it is a better substrate for hexokinase. Nevertheless, some fructose can be metabolized in adipose tissue and muscle. Fructose is found in seminal plasma and in the fetal circulation of ungulates and whales. Aldose reductase is found in the placenta of the ewe and is responsible for the secretion of sorbitol into the fetal blood. The presence of sorbitol dehydrogenase in the liver, including the fetal liver, is responsible for the conversion of sorbitol into fructose. This pathway is also responsible for the occurrence of fructose in seminal fluid.
FIGURE 20-4 Uronic acid pathway. («Indicates the fate of carbon 1 of glucose.)

Galactose is derived from intestinal hydrolysis of the disaccharide lactose, the sugar found in milk. It is readily converted in the liver to glucose. Galactokinase catalyzes the phosphorylation of galactose, using ATP as phosphate donor (Figure 20-6).
Galactose-1-phosphate reacts with UDPGlc to form uridine diphosphate galactose (UDPGal) and glucose-1-phosphate, in a reaction catalyzed by galactose-1-phosphate uridyl transferase. The conversion of UDPGal to UDPGlc is catalyzed by UDPGal 4-epimerase. The reaction involves oxidation, and then reduction, at carbon 4, with NAD* as a coenzyme. The UDPGlc is then incorporated into glycogen (see Chapter 18). The epimerase reaction is freely reversible, so glucose can be converted to galactose, and galactose is not a dietary essential. Galactose is required in the body not only for the
FIGURE 20-5 Metabolism of fructose. Aldolase A is found in all tissues, whereas aldolase B is the predominant form in liver. (*Not found in liver.)
formation of lactose in lactation but also as a constituent of glycolipids (cerebrosides), proteoglycans, and glycoproteins. In the synthesis of lactose in the mammary gland, UDPGal condenses with glucose to yield lactose, catalyzed by lactose synthase (Figure 20-6).

Amino sugars are important components of glycoproteins (see Chapter 46), of certain glycosphingolipids (eg, gangliosides; see Chapter 21), and of glycosaminoglycans (see Chapter 50). The major amino sugars are the hexosamines glucosamine, galactosamine, and mannosamine, and the nine-carbon compound sialic acid. The principal sialic acid found in human tissues is N-acetylneuraminic acid (NeuAc). A summary of the metabolic interrelationships among the amino is shown in 20-7.
sugars Figure

Genetic defects of glucose-6-phosphate dehydrogenase, with consequent impairment of the generation of NADPH, are common in populations of Mediterranean and Afro-Caribbean origin. The gene is on the X chromosome, so it is mainly males who are affected. Some 400 million people carry a mutated gene for glucose-6-phosphate dehydrogenase, making it the most common genetic defect, but most are asymptomatic. In some populations, glucose-6-phosphatase deficiency is common enough for it to be regarded as a genetic polymorphism. The distribution of mutant genes parallels that of malaria, suggesting that being heterozygous confers resistance against malaria. The defect is manifested as red cell hemolysis (hemolytic anemia) when susceptible individuals are subjected to oxidative stress
FIGURE 20-6 Pathway of conversion of (A) galactose to glucose in the liver and (B) glucose to lactose in the lactating mammary gland.
(see Chapter 45) from infection, drugs such as the antimalarial primaquine, and sulfonamides, or when they have eaten fava beans (Vicia faba—hence the name of the disease, favism). Many different mutations are known in the gene for glucose-6-phosphate dehydrogenase, leading to two main variants of favism. In the Afro-Caribbean variant, the enzyme is unstable, so that while average red-cell activities are low, it is only the older erythrocytes that are affected by oxidative stress, and the hemolytic crises tend to be self-limiting. By contrast, in the Mediterranean variant the enzyme is stable, but has low activity in all erythrocytes. Hemolytic crises in these people are more severe and can be fatal. Glutathione peroxidase is dependent on a supply of NADPH, which in erythrocytes can only be formed via the pentose phosphate pathway. It reduces organic peroxides and H,O, as part of the body's defense against lipid peroxidation. Measurement of erythrocyte glutathione reductase, and its activation by FAD is used to assess vitamin B, nutritional status (see Chapter 44).

to reduce xylulose to xylitol. Although pentosuria is benign, with no clinical consequences, xylulose is a reducing sugar and can give false-positive results when urinary glucose is measured using alkaline copper reagents (see Chapter 48). Various drugs increase the rate at which glucose enters the uronic acid pathway. For example, administration of barbital or chlorobutanol to rats results in a significant increase in the conversion of glucose to glucuronate, L-gulonate, and ascorbate. Aminopyrine and antipyrine increase the excretion of xylulose in pentosuric subjects. Pentosuria also occurs after consumption of relatively large amounts of fruits such as pears that are rich sources of pentoses (alimentary pentosuria).

In the rare benign hereditary condition essential pentosuria, considerable quantities of xylulose appear in the urine, because of a lack of xylulose reductase, the enzyme necessary

FIGURE 20-7 summary of the interrelationships in metabolism of amino sugars. (Analogous to UDPGlc.) Other purine or pyrimidine nucleotides may be similarly linked to sugars or amino sugars. Examples are thymidine diphosphate (TDP)-glucosamine and TDP-N-acetylglucosamine.
causes sequestration of inorganic phosphate in fructose1-phosphate and diminished ATP synthesis. As a result, there is less inhibition of de novo purine synthesis by ATP, and uric acid formation is increased, causing hyperuricemia, which is the cause of gout (see Chapter 33). Since fructose is absorbed from the small intestine by (passive) carrier-mediated diffusion, high oral doses may lead to osmotic diarrhea.

Diets low in fructose, sorbitol, and sucrose are beneficial for both conditions. One consequence of hereditary fructose intolerance and of a related condition as a result of fructose 1,6-bisphosphatase deficiency is fructose-induced hypoglycemia despite the presence of high glycogen reserves, because fructose-1-phosphate and 1,6-bisphosphate allosterically inhibit liver glycogen phosphorylase. The sequestration of inorganic phosphate also leads to depletion of ATP and hyperuricemia.
A lack of hepatic fructokinase causes essential fructosuria, which is a benign and asymptomatic condition. The absence of aldolase B, which cleaves fructose-1-phosphate, leads to hereditary fructose intolerance, which is characterized by profound hypoglycemia and vomiting after consumption of fructose (or sucrose, which yields fructose on digestion).

Both fructose and sorbitol are found in the lens of the eye in increased concentrations in diabetes mellitus and may be involved in the pathogenesis of diabetic cataract. The sorbitol (polyol) pathway (not found in liver) is responsible for
fructose formation from glucose (Figure 20-5) and increases in activity as the glucose concentration rises in those tissues that are not insulin-sensitive—the lens, peripheral nerves, and renal glomeruli. Glucose is reduced to sorbitol by aldose reductase, followed by oxidation of sorbitol to fructose in the presence of NAD* and sorbitol dehydrogenase (polyol dehydrogenase). Sorbitol does not diffuse through cell membranes, but accumulates, causing osmotic damage. Simultaneously, myoinositol levels fall. In experimental animals, sorbitol accumulation and myoinositol depletion, as well as diabetic cataract, can be prevented by aldose reductase inhibitors. A number of inhibitors are undergoing clinical trials for prevention of adverse effects of diabetes.

Inability to metabolize galactose occurs in the galactosemias, which may be caused by inherited defects of galactokinase, uridyl transferase, or 4-epimerase (Figure 20-6A), though deficiency of uridyl transferase is best known. Galactose is a substrate for aldose reductase, forming galactitol, which accumulates in the lens of the eye, causing cataract. The condition is more severe if it is the result of a defect in the uridyl transferase since galactose-1-phosphate accumulates and depletes the liver of inorganic phosphate. Ultimately, liver failure and mental deterioration result. In uridyl transferase deficiency, the epimerase is present in adequate amounts, so that the galactosemic individual can still form UDPGal from glucose.
This explains how it is possible for normal growth and development of affected children to occur despite the galactose-free diets used to control the symptoms of the disease.

■, 1 = The pentose phosphate pathway, present in the cytosol, can account for the complete oxidation of glucose, producing. ■, 1 = The pathway has an oxidative phase, which is irreversible and generates NADPH, and a nonoxidative phase, which is reversible and provides ribose precursors for nucleotide synthesis. The complete pathway is present mainly in those tissues having a requirement for NADPHfor reductive syntheses, for example, lipogenesis or steroidogenesis, whereas the nonoxidative phase is present in all cells requiring ribose.. ■, 1 = In erythrocytes, the pathway has a major function in preventing hemolysis by providing NADPHto maintain glutathione in the reduced state as the substrate for glutathione peroxidase.. ■, 1 = The uronic acid pathway is the source of glucuronic acid for conjugation of many endogenous and exogenous substances before excretion as glucuronides in urine and bile.. ■, 1 = Fructose bypasses the main regulatory step in glycolysis, catalyzed by phosphofructokinase, and stimulates fatty acid synthesis and hepatic triacylglycerol secretion.. ■, 1 = Galactose is synthesized from glucose in the lactating mammary gland and in other tissues where it is required for the synthesis of glycolipids, proteoglycans, and glycoproteins.

1. Which of the following is not a reducing sugar?
Erythrose
Fructose
Galactose
Glucose
Ribose
BOOR>
2. Which of the following is the composition of sucrose?
O-a-D-galactopyranosyl-(1—4)-B-D-glucopyranose
O-a-D-glucopyranosyl-(1—2)-B-D-fructofuranoside
O-a-D-glucopyranosyl-(1—4)-a-D-glucopyranose
O-a-D-glucopyranosyl-(1—1)-a-D-glucopyranoside
O-a-D-glucopyranosyl-(1—6)-a-D-glucopyranose
BOOP
>
3. Which of the following is not a pentose?
Fructose
Ribose
Ribulose
Xylose
Xylulose
ROOD
4. Ablood sample is taken from a 50-year-old woman after an
overnight
fast.
Which
one
of
the
following
will
be
at
a
higher
concentration than after she had eaten a meal?
Glucose >
Insulin
Nonesterified fatty acids
Triacylglycerol
BOOP
5. Ablood sample is taken from a 25-year-old man after he has eaten
three slices of toast and a boiled egg. Which one of the following
will be at a higher concentration than if the blood sample had been
taken after an overnight fast?
A. Alanine
B. Glucagon
C.
Glucose
D. Ketone bodies
E. Nonesterified fatty acids
6. Ablood sample is taken from a 40-year-old man has been fasting
for
a
week,
drinking
only
water.
Which
of
the
following
will
be
at
a
higher
than
after
a
normal
overnight
fast?
Glucose
Insulin

BORD
7. Which one of following statements about the fed and fasting
metabolic states is correct?
A. In the fasting state glucagon acts to increase the activity of
lipoprotein lipase in adipose tissue.
B. In the fasting state, glucagon acts to increase the synthesis of
glycogen from glucose.
C. In the fed state insulin acts to increase the breakdown of
glycogen to maintain blood glucose.
10. Which one of following statements about the fed and fasting
ll. Which one of following statements about the fed and fasting
D. In the fed state there is decreased secretion of insulin in
response to increased glucose in the portal blood.
E. Ketone bodies are synthesized in liver in the fasting state, and the amount synthesized increases as fasting extends into starvation.
. Which one of following statements about the fed and fasting
A. In the fed state muscle can take up glucose for use as a
metabolic fuel because glucose transport in muscle is
stimulated
in
response
to
glucagon.
B. In the fed state there is decreased secretion of glucagon in
C. In the fed state, glucagon acts to increase the synthesis of
D. Plasma glucose is maintained in starvation and prolonged
fasting by gluconeogenesis from ketone bodies.
E. There is an increase in metabolic rate in the fasting state.
A. In the fasting state muscle synthesizes glucose from amino acids.
B. In the fed state adipose tissue can take up glucose for
synthesis of triacylglycerol because glucose transport in
adipose tissue is stimulated in response to glucagon.
C. Ketone bodies are synthesized in muscle in the fasting state,
and the amount synthesized increases as fasting extends into starvation.
D. Ketone bodies provide an alternative fuel for red blood cells
in the fasting state.
E. is in starvation fasting by gluconeogenesis from fatty acids.
Plasma glucose maintained and prolonged
A. In the fasting state adipose tissue synthesizes glucose from

the glycerol released by the breakdown of triacylglycerol.
B. In the fasting state adipose tissue synthesizes ketone bodies.
C. In the fasting state the main fuel for red blood cells is fatty acids released from adipose tissue.
D. Ketone bodies provide the main fuel for the central nervous
system in the fasting state.
E. Plasma glucose is maintained in starvation and prolonged
fasting by gluconeogenesis in the liver from the amino acids released by the breakdown of muscle protein.
A. Fatty acids and triacylglycerol are synthesized in the liver in
the fasting state.
B. In the fasting state the main fuel for the central nervous
system is fatty acids released from adipose tissue.
C. In state main most tissues
the fasting the metabolic fuel for
comes from fatty acids released from adipose tissue.
D. In the fed state muscle cannot take up glucose for use as
a metabolic fuel because glucose transport in muscle is
in
response
to
glucagon.
fasting by gluconeogenesis in adipose tissue from the glycerol released from triacylglycerol.
12. A 25-year-old man visits his GP complaining of abdominal
13. Which one of following statements about glycolysis and
14. Which one of following statements about the step in glycolysis
15. Which one of following statements about this step in glycolysis
16. Which one of the following statements about glucose metabolism
cramps and diarrhea after drinking milk. What is the most likely
cause of his problem?
Bacterial and yeast overgrowth in the large intestine Infection with the intestinal parasite Giardia lamblia Lack of pancreatic amylase Lack of small intestinal lactase Lack of small intestinal sucrase-isomaltase moUOR>
is
correct?
A. All the reactions of glycolysis are freely reversible for
B. Fructose cannot be used for gluconeogenesis in
the liver because it cannot be phosphorylated to
fructose-6-phosphate.
C. Glycolysis can proceed in the absence of oxygen only if

pyruvate is formed from lactate in muscle.
D. Red blood cells only metabolize glucose by anaerobic
glycolysis (and the pentose phosphate pathway).
E. The reverse of glycolysis is the pathway for gluconeogenesis in skeletal muscle.
catalyzed by hexokinase and in gluconeogenesis by glucose-6-
phosphatase is correct?
A. Because hexokinase has a low K,, its activity in liver
increases as the concentration of glucose in the portal blood increases.
B. Glucose-6-phosphatase is mainly active in muscle in the fasting state.
C. Ifhexokinase and glucose-6-phosphatase are both equally
active at the same time, there is net formation of ATP from ADP and phosphate.
D. Liver contains an isoenzyme of hexokinase, glucokinase,
which is especially important in the fed state.
E. Muscle can release glucose into the circulation from its
glycogen reserves in the fasting state.
catalyzed by phosphofructokinase and in gluconeogenesis by fructose 1,6-bisphosphatase is correct?
A. Fructose 1,6-bisphosphatase is mainly active in the liver in
the fed state.
B. Fructose 1,6-bisphosphatase is mainly active in the liver in the fed state.
C. If phosphofructokinase and fructose 1,6-bisphosphatase are
both
equally
active
at
the
same
time,
there
is
a
net
formation
of
ATP
from
ADP
and
D. Phosphofructokinase is inhibited more or less completely by
physiologic concentrations of ATP.
E. Phosphofructokinase is mainly active in the liver in the
fasting state.
in maximum exertion is correct?
A. Gluconeogenesis from lactate requires less ATP than is
formed during anaerobic glycolysis.
B. In maximum exertion, pyruvate is oxidized to lactate in
muscle.
17. Which one of following statements is correct?
18. Which one of following statements about the pyruvate

19. Which one of following statements about the pentose phosphate
20. Which one of following statements about glycogen metabolism is
C. Oxygen debt is caused by the need to exhale carbon dioxide
produced in response to acidosis.
D. Oxygen debt reflects the need to replace oxygen that has
been used in muscle during vigorous exercise.
E. There is metabolic acidosis as a result of vigorous exercise.
A. Glucose-1-phosphate may be hydrolyzed to yield free
glucose in liver.
B. Glucose-6-phosphate can be formed from glucose, but not
from glycogen.
C. Glucose-6-phosphate cannot be converted to glucose-1-
phosphate in liver.
D. Glucose-6-phosphate is formed from glycogen by the action
of the enzyme glycogen phosphorylase.
E. In liver and red blood cells, glucose-6-phosphate may enter
into either glycolysis or the pentose phosphate pathway.
dehydrogenase multienzyme complex is correct?
A. In thiamin (vitamin B,) deficiency, pyruvate formed in
muscle cannot be transaminated to alanine.
B. In thiamin (vitamin B,) deficiency, pyruvate formed in muscle cannot be carboxylated to oxaloacetate.
C. The reaction of pyruvate dehydrogenase involves
decarboxylation and oxidation of pyruvate, then formation
of acetyl-CoA.
D. The reaction of pyruvate dehydrogenase is readily reversible, so that acetyl-CoA can be used for the synthesis of pyruvate, and hence glucose.
E. The reaction of pyruvate dehydrogenase leads to the
oxidation of NADH to NAD*, and hence the formation of ~2.5 x ATP per mol of pyruvate oxidized.
pathway is correct?
A. In favism red blood cells are more susceptible to oxidative
stress
because
of
a
lack
of
NADPH
for
fatty
acid
B. People who lack glucose-6-phosphate dehydrogenase cannot

synthesize
fatty
acids
because
of
a
lack
of
NADPH
in
liver
and adipose tissue.
C. The pentose phosphate pathway is especially important in tissues that are synthesizing fatty acids.
D. The pentose phosphate pathway is the only source of
NADPH for fatty acid synthesis.
E. The pentose phosphate pathway provides an alternative to
glycolysis only in the fasting state.
correct?
A. Glycogen is synthesized in the liver in the fed state, then
exported to other tissues in low-density lipoproteins.
B. Glycogen reserves in liver and muscle will meet energy
requirements for several days in prolonged fasting.
C. Liver synthesizes more glycogen when the hepatic portal
blood concentration of glucose is high because of the
activity of glucokinase in the liver.
D. Muscle synthesizes glycogen in the fed state because
glycogen phosphorylase is activated in response to insulin.
E. The plasma concentration of glycogen increases in the fed state.
194
SECTIONIV
21. Which one of following statements about gluconeogenesis is
22. Which one of following statements about carbohydrate
23. Which one of following statements about glycogen metabolism is
24. Which one of following statements about glucose metabolism is
correct?
A. Because they form acetyl-CoA, fatty acids can be a substrate
for gluconeogenesis.
B. If oxaloacetate is withdrawn from the citric acid cycle for
gluconeogenesis, then it can be replaced by the action of
pyruvate dehydrogenase.
C. The reaction of phosphoenolpyruvate carboxykinase
is important to replenish the pool of citric acid cycle intermediates.
D. The use of GTP as the phosphate donor in the
phosphoenolpyruvate carboxykinase reaction provides a
link between citric acid cycle activity and gluconeogenesis.
E. There is a greater yield of ATP in anaerobic glycolysis than the cost for synthesis of glucose from lactate.
metabolism is correct?
A. A key step in the biosynthesis of glycogen is the formation of

UDP-glucose.
B. Glycogen can be broken down to glucose-6-phosphate in
muscle, which then releases free glucose by the action of the
enzyme glucose-6-phosphatase.
Glycogen is stored mainly in the liver and brain. Insulin inhibits the biosynthesis of glycogen. moo
Phosphorylase kinase is an enzyme that phosphorylates
the enzyme glycogen phosphorylase and thereby decreases glycogen breakdown.
correct?
A. Glycogen synthase activity is increased by glucagon.
B. Glycogen phosphorylase is an enzyme that can be activated
by
phosphorylation
of
serine
residues.
C. Glycogen phosphorylase cannot be activated by calcium
ions.
D. cAMP activates glycogen synthesis.
E. Glycogen phosphorylase breaks the a1-4 glycosidic bonds by hydrolysis.
correct?
A. Glucagon increases the rate of glycolysis.
B. Glycolysis requires NADP*.
C. In glycolysis, glucose is cleaved into two three-carbon
D. Substrate-level phosphorylation takes place in the electron
transport system.
E. The main product of glycolysis in red blood cells is pyruvate.
25. Which one of following statements about metabolism of sugars is
26. In glycolysis, the conversion of 1 mol of fructose
27.
28. The disaccharide lactulose is not digested, but is fermented by
correct?
A. Fructokinase phosphorylates fructose to
B. Fructose is an aldose sugar-like glucose.
C. Fructose transport into cells is insulin dependent.
D. Galactose is phosphorylated to galactose-1-phosphate by
galactokinase.
E. Sucrose can be biosynthesized from glucose and fructose in
the liver.
1,6-bisphosphate to 2 mol of pyruvate results in the formation of:
A. 1 mol NAD* and 2 mol of ATP

B. 1 mol NADH and 1 mol of ATP
C. 2 mol NAD* and 4 mol of ATP
D. 2 mol NADH and 2 mol of ATP
E. 2 mol NADH and 4 mol of ATP
Which of the following will provide the main fuel for muscle
contraction during short-term maximum exertion?
Muscle glycogen
Muscle reserves of triacylglycerol
Plasma glucose
Plasma nonesterified fatty acids
Triacylglycerol in plasma very-low-density lipoprotein
FOOD
bacteria,
to
yield
4
mol
of
lactate
plus
four
protons.
Ammonium
(NH,_’)
is
in
with
ammonia
(NH,)
in
the
bloodstream.
Which
of
the
following
best
explains
how lactulose acts to treat hyperammonemia (elevated blood
ammonium concentration)?
A. Fermentation of lactulose increases the acidy of the
bloodstream so that there is more ammonium and less
ammonia is available to cross the gut wall.
B. Fermentation of lactulose results in acidification of the gut
contents so that ammonia diffuses from the bloodstream into
the gut and is trapped as ammonium that cannot cross back.
C. Fermentation of lactulose results in acidification of the gut
contents so that ammonia produced by intestinal bacteria is
trapped as ammonium that cannot diffuse into the bloodstream.
D. Fermentation of lactulose results in an eightfold increase
in
the
of
the
gut
contents,
so
that
there
is
more
water
for
ammonia
and
ammonium
to
dissolve
in,
so
that
less is absorbed into the bloodstream.
E. Fermentation of lactulose results in an eightfold increase
in the osmolality of the gut contents, so that there is more water for ammonia and ammonium to dissolve in, so that more will diffuse for the bloodstream into the
gut.

- = Define simple and complex lipids and identify the lipid classes in each group.
Bele Ree i you should be able to:
Indicate the structure of saturated and unsaturated fatty acids, explain how the chain length and degree of unsaturation influence their melting point, give examples, and explain the nomenclature.
Explain the difference between cis and trans carbon-carbon double bonds.
Describe how eicosanoids are formed by modification of the structure of unsaturated fatty acids; identify the various eicosanoid classes and indicate their functions.
Outline the general structure of triacylglycerols and indicate their function.
Outline the general structure of phospholipids and glycosphingolipids and indicate the functions of the different classes.
= Appreciate the importance of cholesterol as the precursor of many biologically important steroids, including steroid hormones, bile acids, and vitamins D.
Recognize the cyclic nucleus common to all steroids.
Explain why free radicals are damaging to tissues and identify the three stages in the chain reaction of lipid peroxidation that produces them continuously.
= Describe how antioxidants protect lipids from peroxidation by either inhibiting chain initiation or breaking the chain.
m= Recognize that many lipid molecules are amphipathic, having both
hydrophobic and hydrophilic groups in their structure, and explain how this influences their behavior in an aqueous environment and enables certain classes, including phospholipids, sphingolipids, and cholesterol, to form the basic structure of biologic membranes.

The lipids are a heterogeneous group of compounds, including fats, oils, steroids, waxes, and related compounds, that are related more by their physical than by their chemical properties. They have the common property of being (1) relatively insoluble in water and (2) soluble in nonpolar solvents such as ether and chloroform. They are important dietary constituents not only because of the high energy value of fats but also because essential fatty acids, fat-soluble vitamins, and other lipophilic micronutrients are contained in the fat of natural foods. Dietary supplementation with long-chain w3 fatty acids is believed to have beneficial effects in a number of chronic diseases, including cardiovascular disease, rheumatoid arthritis, and dementia. Fat is stored in adipose tissue, where it also serves as a thermal insulator in the subcutaneous tissues and around certain organs. Nonpolar lipids act as electrical insulators, allowing rapid propagation of depolarization waves along myelinated nerves. Lipids are transported in the blood combined with proteins in lipoprotein particles (see Chapters 25 and 26). Lipids have essential roles in nutrition and health and knowledge of lipid biochemistry is necessary for the understanding of many important biomedical conditions, including obesity, diabetes mellitus, and atherosclerosis.

1. Simple lipids include fats and waxes which are esters of fatty acids with various alcohols:
a. Fats: Esters of fatty acids with glycerol. Oils are fats in the liquid state.
b. Waxes: Esters of fatty acids with higher molecular weight monohydric alcohols.
2. Complex lipids are esters of fatty acids, which always con- tain an alcohol and one or more fatty acids, but which also have other groups. They can be divided into three types:
a. Phospholipids: Contain a phosphoric acid residue. They frequently have nitrogen-containing bases (eg, choline) and other substituents. In many phospholip- ids the alcohol is glycerol (glycerophospholipids), but in sphingophospholipids it is sphingosine, which con- tains an amino group.
b. Glycolipids (glycosphingolipids): Contain a fatty acid, sphingosine, and carbohydrate.
c. Other complex lipids: These include lipids such as sulfolipids and amino lipids. Lipoproteins may also be placed in this category.
3. Derived lipids are formed from the hydrolysis of both simple and complex lipids. They include fatty acids, glyc- erol, steroids, other alcohols, fatty aldehydes, ketone bod- ies (see Chapter 22), hydrocarbons, lipid-soluble vitamins and micronutrients, and hormones. Some of these (eg, free fatty acids, glycerol) also act as precursor lipids in the formation of simple and complex lipids.
Because they are uncharged, acylglycerols (glycerides), choles- terol, and cholesteryl esters are termed neutral lipids.
A saturated fatty acid (palmitic acid, C16)
AAD
A monounsaturated fatty acid (oleic acid, C18:1)
ASSNSI
NINN
coon
OO
A polyunsaturated fatty acid (linoleic acid, C18:2)
SNS

FIGURE 21-1 Fatty acids. Examples of a saturated (palmitic acid), monounsaturated (oleic acid), and a polyunsaturated (linoleic acid) fatty acid are shown.

Fatty acids occur in the body mainly as esters in natural fats and oils, but are found in the unesterified form as free fatty acids, a transport form in the plasma. Fatty acids that occur in natural fats usually contain an even number of carbon atoms. The chain may be saturated (containing no double bonds) or unsaturated (containing one or more double bonds) (Figure 21-1).

The most frequently used systematic nomenclature names the fatty acid after the hydrocarbon with the same number and arrangement of carbon atoms, with -oic being substituted for the final -e (Genevan system). Thus, saturated acids end in -anoic, for example, octanoic acid (C8), and unsaturated acids with double bonds end in -enoic, for example, octadecenoic acid (oleic acid, C18).
Carbon atoms are numbered from the carboxyl carbon (carbon no. 1). The carbon atoms adjacent to the carboxyl car- bon (nos. 2, 3, and 4) are also known as the a, B, and y carbons, respectively, and the terminal methyl carbon is known as the wor n-carbon.
Various conventions use A for indicating the number and position of the double bonds (Figure 21-2); for example, A° indicates a double bond between carbons 9 and 10 of the fatty acid; w9 indicates a double bond on the ninth carbon counting from the w-carbon. In animals, additional double bonds can be introduced only between an existing double bond at the w9, w6, or w3 position and the carboxyl carbon, leading to three series of fatty acids known as the w9, w6, and w3 families, respectively.
1817 161514 13 121110 9 1 18:1;9 or A9 18:1 CH,CH,CH,CH,CH,CH,CH,CH,CH—= CH(CH,),COOH oornt 2 3 4 5 67 8 9 10 18
FIGURE 21-2 Nomenclature for number and position of double bonds in unsaturated fatty acids. Illustrated using oleic acid as an example. n —9 is equivalent to w9.

Saturated fatty
acids may
be
(CH,—COOH)
as the
CH,—
is envisaged
first progressively
—COOH
member added
groups.
Examples higher
members in
waxes.
between as
of the
are shown
of the
series are
A
few branched-chain
based on
acetic the
series terminal
in known
fatty acid
in which
CH,—
Table and
21-1.
to acids
occur,
Other particularly
have also
been iso-
lated from
both plant
and animal
sources.

Unsaturated fatty acids (Figure 21-1, Table 21-2, for examples) may be further subdivided as follows:
1.
Monounsaturated
(monoethenoid, monoenoic)
acids, containing
one double
Polyunsaturated bond.
(polyethenoid, taining
two or
more
Eicosanoids:
polyenoic)
double bonds.
These compounds,
(20-carbon)
polyenoic prise
the derived
prostanoids,
(LXs).
Prostanoids acids,
con- from
(see eicosa
Chapter leukotrienes
include
(LTs),
23), com-
and prostaglandins
lipoxins
(PGs), pros-

Acetic, Number of C Atoms = 2. Acetic, Occurrence = Major end product of carbohydrate fermentation by rumen organisms. Butyric, Number of C Atoms = 4. Butyric, Occurrence = In certain fats in small amounts (especially butter). An end product of carbohydrate fermentation by rumen organisms a. Valeric, Number of C Atoms = 5. Valeric, Occurrence = . Caproic, Number of C Atoms = 6. Caproic, Occurrence = . Lauric, Number of C Atoms = 12. Lauric, Occurrence = Spermaceti, cinnamon, palm kernel, coconut oils, laurels, butter. Myristic, Number of C Atoms = 14. Myristic, Occurrence = Nutmeg, palm kernel, coconut oils, myrtles, butter. Palmitic, Number of C Atoms = 16. Palmitic, Occurrence = Commonin all animal and plant fats. Stearic, Number of C Atoms = 18. Stearic, Occurrence = 
*Also formed in the cecum of herbivores and to a lesser extent in the colon of humans.
Prostaglandins exist in virtually every mammalian tissue, acting
as local
hormones;
they have
important physiologic
tacyclins
(PGIs), and
thromboxanes
(TXs).
and pharmacologic
They are
synthesized in
vivo

double bonds), Family = . Hexaenoic acids (six double bonds), Common Name = . Hexaenoic acids (six double bonds), Systematic Name = . Hexaenoic acids (six double bonds), Occurrence = . 22:6;4,7,10,13,16,19, Family = ω3. 22:6;4,7,10,13,16,19, Common Name = Cervonic. 22:6;4,7,10,13,16,19, Systematic Name = all- cis -4,7,10,13,16,19- Docosahexaenoic. 22:6;4,7,10,13,16,19, Occurrence = Fish oils, algal oils, phospholipids in brain
2.
3.
198

by cyclization of the center of the carbon chain of 20-carbon (eicosanoic) polyunsaturated fatty acids (eg, arachidonic acid) to form a cyclopentane ring (Figure 21-3). A related series of compounds, the thromboxanes, have the cyclopentane ring interrupted with an oxygen atom (oxane ring) (Figure 21-4). Three different eicosanoic fatty acids give rise to three groups of eicosanoids characterized by the number of double bonds in the side chains (see Figure 23-12), for example, prostaglandin (PG),, PG,, and PG.,,. Different substituent groups attached to the rings give rise to series of prostaglandins and thromboxanes labeled A, B, etc (see Figure 23-13)—for example, the "E" type of prostaglandin (as in PGE,) has a keto group in position 9, whereas the "F" type has a hydroxyl group in this position. The leukotrienes and lipoxins (Figure 21-5) are a third group of eicosanoid derivatives formed via the lipoxygenase pathway (see Figure 23-13). They are characterized by the presence of three or four conjugated double bonds, respectively. Leukotrienes cause bronchoconstriction as well as being potent proinflammatory agents, and play a part in asthma.

The carbon chains of saturated fatty acids form a zigzag pattern when extended at low temperatures (Figure 21-1). At higher temperatures, some bonds rotate, causing chain shortening, which explains why biomembranes become thinner with increases in temperature. A type of geometric isomerism occurs in unsaturated fatty acids, depending on the orientation of atoms or groups around the axes of double bonds, which do not allow rotation. If the acyl chains are on the same side of the bond, it is cis-, as in oleic acid; if on opposite sides, it is trans-, as in elaidic acid, the trans isomer of oleic acid (Figure 21-6). Double bonds in naturally occurring unsaturated long-chain fatty acids are nearly all in the cis configuration, the molecules being "bent" 120° at the double bond. Thus, oleic acid has a V shape, whereas elaidic acid remains "straight" Increase in the number of cis double bonds in a fatty acid leads to a variety of possible spatial configurations of the molecule—for example, arachidonic acid, with four cis double bonds, is bent into a U shape (Figure 21-7). This has profound significance for molecular packing in cell membranes (see Chapter 40) and on the positions occupied by fatty
FIGURE 21-4 Thromboxane A, (TXA,).
FIGURE 21-5 Leukotriene and lipoxin structure. Examples shown are leukotriene A, (LTA,) and lipoxin A, (LXA,).
acids in more complex molecules such as phospholipids. Trans double bonds alter these spatial relationships. Trans fatty acids are present in certain foods, arising as a by-product of the saturation of fatty acids during hydrogenation, or "hardening," of natural oils in the manufacture of margarine. An additional small contribution comes from the ingestion of ruminant fat that contains trans fatty acids arising from the action of microorganisms in the rumen. Consumption of trans fatty acids is now known to be detrimental to health and is associated with increased risk of diseases including cardiovascular disease and diabetes mellitus. This has led to improved technology to produce soft margarine low in trans fatty acids or containing none at all.

The melting points ofeven-numbered carbon fatty acids increase with chain length and decrease according to unsaturation.
FIGURE 21-6 Geometric isomerism of A', 18:1 fatty acids (oleic and elaidic acids). There is no rotation around carbon-carbon double bonds. In the cis configuration, the acyl chains are on the same side of the bond, while in trans form they are on opposite sides.
FIGURE 21-7 Arachidonic acid. Four double bonds in the cis configuration bend the molecule into a U shape.
A triacylglycerol containing three saturated fatty acids of 12 or more carbons is solid at body temperature, whereas if the fatty acid residues are polyunsaturated, it is liquid to below 0°C. In practice, natural acylglycerols contain a mixture of fatty acids tailored to suit their functional roles. For example, membrane lipids, which must be fluid at all environmental temperatures, are more unsaturated than storage lipids. Lipids in tissues that are subject to cooling, for example, during hibernation or in the extremities of animals, are also more unsaturated.

Long-chain w3 fatty acids such as a-linolenic (ALA) (found in plant oils), eicosapentaenoic (EPA) (found in fish oil), and docosahexaenoic (DHA) (found in fish and algal oils) (Table 21-2) have anti-inflammatory effects, perhaps due to their promotion of the synthesis of less inflammatory prostaglandins and leukotrienes as compared to w6 fatty acids (see Figure 23-12). In view of this, their potential use as a therapy in severe chronic disease where inflammation is a contributory cause is under intensive investigation. Current evidence suggests that diets rich in w3 fatty acids are beneficial, particularly for cardiovascular disease, but also for other chronic degenerative diseases such as cancer, rheumatoid arthritis, and Alzheimer disease.

The triacylglycerols (Figure 21-8) are esters of the trihydric alcohol glycerol and fatty acids. Monoand diacylglycerols, wherein one or two fatty acids are esterified with glycerol, are
"According to the standardized terminology of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry, the monoglycerides, diglycerides, and triglycerides should be designated monoacylglycerols, diacylglycerols, and triacylglycerols, respectively. However, the older terminology is still widely used, particularly in clinical medicine.
FIGURE 21-8 (A) Triacylglycerol. (B) Projection formula showing triacyl-sn-glycerol.
also found in the tissues. These are of particular significance in the synthesis and hydrolysis of triacylglycerols (see Chapters 24 and 25).

To number the carbon atoms of glycerol unambiguously, the -sn (stereochemical numbering) system is used. It is important to realize that carbons 1 and 3 of glycerol are not identical when viewed in three dimensions (shown as a projection formula in Figure 21-8B). Enzymes readily distinguish between them and are nearly always specific for one or the other carbon; for example, glycerol is always phosphorylated on sn-3 by glycerol kinase to give glycerol-3-phosphate and not glycerol1-phosphate (see Figure 24-2).

Many phospholipids are derivatives of phosphatidic acid (Figure 21-9), in which the phosphate is esterified with one OH group of glycerol and the other two OH groups are esterified to two long-chain fatty acids (glycerophospholipids). Phosphatidic acid is important as an intermediate in the synthesis of triacylglycerols as well as phosphoglycerols (see Figure 24-2) but is not found in any great quantity in tissues. Sphingolipids, such as sphingomyelin, in which the phosphate is esterified to sphingosine, a complex amino alcohol (Figure 21-10), are also important membrane components. Both glycerophospholipids and sphingolipids have two long-chain hydrocarbon tails which are important for their function in forming the lipid bilayer in cell membranes (see Chapter 40), but in the former both are fatty acid chains while in the latter one is a fatty acid and the second is part of the sphingosine molecule (Figure 21-11).
FIGURE 21-9 Phospholipids. The O— shown shaded in phosphatidic acid is substituted by the substituents shown to form the phospholipids: (A) 3-phosphatidylcholine, (B) 3-phosphatidylethanolamine, (C) 3-phosphatidylserine, (D) 3-phosphatidylinositol, and (E) cardiolipin (diphosphatidylglycerol).

Glycerophospholipids containing choline (Figure 21-9), (phosphatidylcholines, commonly called lecithins) are the most abundant phospholipids of the cell membrane and represent a large proportion of the body's store of choline.

Choline is important in nervous transmission, as acetylcholine, and as a store of labile methyl groups. Dipalmitoyl lecithin is a very effective surface-active agent and a major constituent of the surfactant preventing adherence, due to surface tension, of the inner surfaces of the lungs. Its absence from the lungs of premature infants causes respiratory distress syndrome. Most phospholipids have a saturated acyl radical in the sn-1 position but an unsaturated radical in the sn-2 position of glycerol.
Phosphatidylethanolamine (cephalin) and phosphatidylserine (found in most tissues) are also found in cell membranes and differ from phosphatidylcholine only in that ethanolamine or serine, respectively, replaces choline (Figure 21-9). Phosphatidylserine also plays a role in apoptosis (programmed cell death).
Sphingomyelins are found in the outer leaflet of the cell membrane lipid bilayer and are particularly abundant in specialized areas of the plasma membrane known as lipid rafts (see Chapter 40). They are also found in large quantities in the myelin sheath that surrounds nerve fibers. They are believed to play a role in cell signaling and in apoptosis. Sphingomyelins contain no glycerol, and on hydrolysis they yield a fatty acid, phosphoric acid, choline, and sphingosine (Figure 21-10). The combination of sphingosine plus fatty acid is known as ceramide, a structure also found in the glycosphingolipids (see next section below).

The inositol is present in phosphatidylinositol as the stereoisomer, myoinositol (Figure 21-9). Phosphorylated phosphatidylinositols (phosphoinositides) are minor components of cell membranes, but play an important part in cell signaling and membrane trafficking. Phosphoinositides may have 1, 2, or 3 phosphate groups attached to the inositol ring. Phosphatidylinositol 4,5-bisphosphate (PiP,), for example, is cleaved into diacylglycerol and inositol trisphosphate upon stimulation by a suitable hormone agonist, and both of these act as internal signals or second messengers.
A sphingomyelin
FIGURE 21-11 Comparison of glycerophospholipid and sphingolipid structures. Both types of phospholipid have two hydrocarbon tails, in glycerophospholipids both are fatty acid chains (a phosphatidylcholine with one saturated and one unsaturated fatty acid is shown) and in sphingolipids one is a fatty acid chain and the other is part of the sphingosine moiety (a sphingomyelin is shown). The two hydrophobic tails and the polar head group are important for the function of these phospholipids in the lipid bilayer in cell membranes (see Chapter 40).

interconversion of phospholipids. This compound is also found in oxidized lipoproteins and has been implicated in some of their effects in promoting atherosclerosis.
Phosphatidic acid is a precursor of phosphatidylglycerol, which in turn gives rise to cardiolipin (Figure 21-9). This phospholipid is found only in mitochondria and is essential for the mitochondrial function. Decreased cardiolipin levels or alterations in its structure or metabolism cause mitochondrial dysfunction in aging and in pathologic conditions including heart failure, hypothyroidism, and Barth syndrome (cardioskeletal myopathy).

These are phosphoacylglycerols containing only one acyl radical, for example, lysophosphatidylcholine (lysolecithin) (Figure 21-12), which is important in the metabolism and
FIGURE 21-12 Lysophosphatidylcholine (lysolecithin).

These compounds constitute as much as 10 to 30% of the phospholipids of brain and heart. Structurally, the plasmalogens resemble phosphatidylethanolamine but possess an ether link on the sn-1 carbon instead of the ester link found in acylglycerols. Typically, the alkyl radical is an unsaturated alcohol (Figure 21-13). In some instances, choline, serine, or inositol may be substituted for ethanolamine. The function of plasmalogens remain poorly understood, but it has been suggested that they may have a protective effect against reactive oxygen species.
FIGURE 21-13 Plasmalogen.

Glycolipids are lipids with an attached carbohydrate or carbohydrate chain. They are widely distributed in every tissue of the body, particularly in nervous tissue such as brain. They occur particularly in the outer leaflet of the plasma membrane, where they contribute to cell surface carbohydrates which form the glycocalyx (see Chapter 15).
The major glycolipids found in animal tissues are glycosphingolipids. They contain ceramide and one or more sugars. Galactosylceramide (Figure 21-14) is a major glycosphingolipid of brain and other nervous tissue, found in relatively low amounts elsewhere. It contains a number of characteristic C24 fatty acids, for example, cerebronic acid.
Galactosylceramide can be converted to sulfogalactosylceramide (sulfatide) which has a sulfo group attached to the O in the three position of galactose and is present in high amounts in myelin. Glucosylceramide resembles galactosylceramide, but the head group is glucose rather than galactose. It is the predominant simple glycosphingolipid of extraneural tissues, also occurring in the brain in small amounts. Gangliosides are complex glycosphingolipids derived from glucosylceramide that contain in addition one or more molecules of a sialic acid. Neuraminic acid (NeuAc, see Chapter 15) is the principal sialic acid found in human tissues. Gangliosides are also present in nervous tissues in high concentration. They function in cell-cell recognition and communication and as receptors for hormones and bacterial toxins such as cholera toxin. The simplest ganglioside found in tissues is GM,, which contains ceramide, one molecule of glucose, one molecule of galactose, and one molecule of NeuAc. In the shorthand nomenclature used, G represents ganglioside; M is a monosialo-containing species; and the subscript 3 is a number assigned on the basis of chromatographic migration. GM1 (Figure 21-15), a more
FIGURE 21-14 Structure of galactosylceramide.

FIGURE 21-15 GM1 ganglioside, a monosialoganglioside, the receptor in human intestine for cholera toxin.
complex ganglioside derived from GM,, is of considerable biologic interest, as it is known to be the receptor in human intestine for cholera toxin. Other gangliosides can contain anywhere from one to five molecules of sialic acid, giving rise to di-, trisialogangliosides, etc.

Although cholesterol is probably best known by most people for its association with atherosclerosis and heart disease, it has many essential roles in the body. It is the precursor of a large number of equally important steroids that include the bile acids, adrenocortical hormones, sex hormones, vitamin D (see Chapters 26, 41, 44), and cardiac glycosides.
All steroids have a similar cyclic nucleus resembling phenanthrene (rings A, B, and C) to which a cyclopentane ring (D) is attached. The carbon positions on the steroid nucleus are numbered as shown in Figure 21-16. It is important to realize that in structural formulas of steroids, a simple hexagonal ring denotes a completely saturated six-carbon ring with all valences satisfied by hydrogen bonds unless shown otherwise; that is, it is not a benzene ring. All double bonds are shown as such. Methyl side chains are shown as single bonds unattached at the farther (methyl) end. These occur typically at positions 10 and 13 (constituting C atoms 19 and 18). A side chain at position 17 is usual (as in cholesterol). If the compound has one or more hydroxyl groups and no carbonyl or carboxyl groups, it is a sterol, and the name terminates in -ol.
FIGURE 21-16 The steroid nucleus.
LO
FIGURE 21-17 Conformations of stereoisomers of the steroid nucleus.

Each of the six-carbon rings of the steroid nucleus is capable of existing in the three-dimensional conformation either of a "chair" or a "boat" (Figure 21-17). In naturally occurring steroids, virtually all the rings are in the "chair" form, which is the more stable conformation. With respect to each other, the rings can be either cis or trans (Figure 21-18). The junction between the A and B rings may be cis or trans in naturally occurring steroids. The junction between B and C is trans, as is usually the C/D junction. Bonds attaching substituent groups above the plane of the rings (8 bonds) are shown with bold solid lines, whereas those bonds attaching groups below (a bonds) are indicated with broken lines. The A ring of a 5a steroid (ie, the hydrogen at position 5 is in the a configuration) is always trans to the B ring, whereas it is cis in a 56 steroid (ie, the hydrogen at position 5 is in the B configuration). The methyl groups attached to C10 and C13 are invariably in the 6 configuration.

Cholesterol (Figure 21-19) is widely distributed in all cells of the body but particularly in nervous tissue. It is a major constituent of the plasma membrane (see Chapter 40) and of plasma lipoproteins (see Chapter 26). It is often found as cholesteryl ester, where the hydroxyl group on position 3 is
FIGURE 21-18 Generalized steroid nucleus, showing (A) an all-trans configuration between adjacent rings and (B) a cis configuration between rings A and B.

esterified with a long-chain fatty acid. It occurs in animals but not in plants or bacteria.

Ergosterol occurs in plants and yeast and is important as a dietary source of vitamin D (Figure 21-20). When irradiated with ultraviolet light in the skin, ring B is opened to form vitamin D, in a process similar to the one that forms vitamin D, from 7-dehydrocholesterol in the skin (see Figure 44-3).

Polyprenoids are not steroids but are related to them because they are synthesized, like cholesterol (see Figure 26-2), from five-carbon isoprene units (Figure 21-21). They include ubiquinone (see Chapter 13), which participates in the respiratory chain in mitochondria, and the long-chain alcohol dolichol (Figure 21-22), which takes part in glycoprotein synthesis by transferring carbohydrate residues to asparagine residues of the polypeptide (see Chapter 46). Plant-derived polyprenoids include rubber, camphor, the fat-soluble vitamins A, D, E, and K, and f-carotene (provitamin A).

Peroxidation (auto-oxidation) of lipids exposed to oxygen is responsible not only for deterioration of foods (rancidity), but also for damage to tissues in vivo, where it may be a cause of cancer, inflammatory diseases, atherosclerosis, and aging. The deleterious effects are considered to be caused by free radicals,
FIGURE 21-20 Ergosterol.
CH, |
—CH=C—CH=CH—

molecules that have unpaired valence electrons, making them highly reactive. Free radicals containing oxygen (eg, ROO', RO', OH') are termed reactive oxygen species (ROS). These are produced during peroxide formation from fatty acids containing methylene-interrupted double bonds, that is, those found in the naturally occurring polyunsaturated fatty acids (Figure 21-23). Lipid peroxidation is a chain reaction in which free radicals formed in the initiation stage in turn generate more (propagation), and thus it has potentially devastating effects. The processes of initation and propagation can be depicted as follows:
1. Initiation:
ROOH + Metal" * —» ROO" + Metal""* + H*
X'+RH-R'+ XH
2. Propagation:
R'+0°?>ROO’
ROO' + RH > ROOH + R', etc
The process may be terminated at the third stage:
3. Termination:
ROO' + ROO' > ROOR + O,
ROO' + R' > ROOR
R'+R'>RR
Antioxidants are used to control and reduce lipid peroxidation, both by humans in their activities and in nature. Propyl gallate, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) are antioxidants used as food additives. Naturally occurring antioxidants include vitamin E (tocopherol) (see Chapter 44), which is lipid soluble, and urate and vitamin C, which are water soluble. B-Carotene is an antioxidant at low PO,. Antioxidants fall into two classes: (1)

preventive antioxidants, which reduce the rate of chain initiation (stage 1 above) and (2) chain-breaking antioxidants, which interfere with chain propagation (stage 2 above). Preventive antioxidants include catalase and other peroxidases such as glutathione peroxidase (see Figure 20-3) that react with ROOH; selenium, which is an essential component of glutathione peroxidase and regulates its activity, and chelators of metal ions such as ethylenediaminetetraacetate (EDTA) and diethylenetriaminepentaacetate (DTPA). In vivo, the principal chain-breaking antioxidants are superoxide dismutase, which acts in the aqueous phase to trap superoxide free radicals (O,*) urate, and vitamin E, which acts in the lipid phase to trap ROO' radicals.
Peroxidation is also catalyzed in vivo by heme compounds and by lipoxygenases (see Figure 23-13) found in platelets and leukocytes. Other products of auto-oxidation or enzymic oxidation of physiologic significance include oxysterols (formed from cholesterol) and the prostaglandin-like isoprostanes (formed from the peroxidation of polyunsaturated fatty acids such as arachidonic acid) which are used as reliable markers of oxidative stress in humans.

In general, lipids are insoluble in water since they contain a predominance of nonpolar (hydrocarbon) groups. However, fatty acids, phospholipids, sphingolipids, bile salts, and, to a lesser
ROOH
FIGURE 21-23 Lipid peroxidation. The reaction is initiated by an existing free radical (X*), by light, or by metal ions. Malondialdehyde is only formed by fatty acids with three or more double bonds and is used as a measure of lipid peroxidation together with ethane from the terminal two carbons of w3 fatty acids and pentane from the terminal five carbons of w6 fatty acids.
FIGURE 21-24 Formation of lipid membranes, micelles, emulsions, and liposomes from amphipathic lipids, for example,

extent, cholesterol contain polar groups. Therefore, a part of the molecule is hydrophobic, or water insoluble, and a part is hydrophilic, or water soluble. Such molecules are described as amphipathic (Figure 21-24). They become oriented at oilwater interfaces with the polar group in the water phase and the nonpolar group in the oil phase. A bilayer of such amphipathic lipids is the basic structure in biologic membranes (see Chapter 40). When a critical concentration of these lipids is present in an aqueous medium, they form micelles. Liposomes may be formed by sonicating an amphipathic lipid in an aqueous medium. They consist of spheres of lipid bilayers that enclose part of the aqueous medium. Aggregation of bile salts into micelles and liposomes and the formation of mixed micelles with the products of fat digestion are important in facilitating absorption of lipids from the intestine. Liposomes are of potential clinical use—particularly when combined with tissue-specific antibodies—as carriers of drugs in the circulation, targeted to specific organs, for example, in cancer therapy. In addition, they are used for gene transfer into vascular cells and as carriers for topical and transdermal delivery of drugs and cosmetics. Emulsions are much larger particles, formed usually by nonpolar lipids in an aqueous medium. These are stabilized by emulsifying agents such as amphipathic lipids
(eg, phosphatidylcholine), which form a surface layer separating the main bulk of the nonpolar material from the aqueous phase (Figure 21-24).

™ Lipids have the common property of being relatively insoluble in water (hydrophobic) but soluble in nonpolar solvents. Amphipathic lipids also contain one or more polar groups, making them suitable as constituents of membranes at lipidwater interfaces.
® Lipids of major physiologic significance include fatty acids and their esters, together with cholesterol and other steroids.
™ Long-chain fatty acids may be saturated, monounsaturated, or polyunsaturated, according to the number of double bonds present. Their fluidity decreases with chain length and increases to of unsaturation.
according degree
™ Eicosanoids are formed from 20-carbon polyunsaturated fatty acids and make up an important group of physiologically and pharmacologically active compounds known as prostaglandins, thromboxanes, leukotrienes, and lipoxins.
@ 'The esters of glycerol are quantitatively the most significant lipids, represented by triacylglycerol ("fat"), a major constituent of some lipoprotein classes and the storage form of lipid
in adipose
tissue.
are amphipathic
Glycerophospholipids lipids
constituents and
and of
have important
membranes and
sphingolipids roles—as
the outer
major layer
as surfactant
in the
lung, as
and as constituents of nervous tissue.
™ Glycolipids are also important constituents of nervous tissue such as brain and the outer leaflet of the cell membrane, where they contribute to the carbohydrates on the cell surface.
= Cholesterol, an amphipathic lipid, is an important component of membranes. It is the parent molecule from which all other steroids in the hormones such as the
body, including major
adrenocortical and sex hormones, D vitamins, and bile acids, are synthesized.
- : Peroxidation of lipids containing polyunsaturated fatty acids leads to generation of free radicals that damage tissues and cause disease.

Eljamil AS: Lipid Biochemistry: For Medical Sciences. iUniverse,
2015.
Gurr MI, Harwood JL, Frayn KN, et al: Lipids, Biochemistry,
Biotechnology and Health. Wiley-Blackwell, 2016.

VmT eS INCRUIERS Clee you should be able to:
2 Describe the processes by which fatty acids are transported in the blood,
1: Outline the B-oxidation pathway by which fatty acids are metabolized to
to obtain energy.
of
ATP.
regulated. = Indicate the three stages in the metabolism of fatty acids where ketogenesis is
activated
and
transported
into
the
matrix
of
the
for
breakdown
acetyl-CoA
and
explain
how
this
leads
to
the
of
large
quantities
=
Identify the three compounds termed “ketone bodies” and describe the reactions by which they are formed in liver mitochondria.
m Recognize that ketone bodies are important fuels for extrahepatic tissues and
indicate
the
in
which
their
synthesis
and
use
are
favored.
= Indicate that overproduction of ketone bodies leads to ketosis and, if
prolonged, ketoacidosis, and identify pathologic conditions when this occurs.
™ Give examples of diseases associated with impaired fatty acid oxidation.

Fatty acids are broken down in mitochondria by oxidation to acetyl-CoA in a process that generates large amounts of energy. When this pathway is proceeding at a high rate, three compounds, acetoacetate, D-3-hydroxybutyrate, and acetone, known collectively as the ketone bodies, are produced by the liver. Acetoacetate and D-3-hydroxybutyrate are used as fuels by extrahepatic tissues in normal metabolism, but overproduction of ketone bodies causes ketosis. Increased fatty acid oxidation and consequently ketosis is a characteristic of starvation and of diabetes mellitus. Since ketone bodies are acidic, when they are produced in excess over long periods, as in diabetes, they cause ketoacidosis, which is ultimately fatal. Because gluconeogenesis is dependent on fatty acid oxidation, any impairment in fatty acid oxidation leads to hypoglycemia. This occurs in various states of carnitine deficiency or deficiency of essential enzymes in fatty acid oxidation, for example, carnitine palmitoyltransferase, or inhibition of fatty acid oxidation by poisons, for example, hypoglycin.

Although acetyl-CoA is both an end point of fatty acid catabolism and the starting substrate for fatty acid synthesis, breakdown is not simply the reverse of the biosynthetic pathway, but an entirely separate process taking place in a different compartment of the cell. The separation of fatty acid oxidation in mitochondria from biosynthesis in the cytosol allows each process to be individually controlled and integrated with tissue requirements. Each step in fatty acid oxidation involves acyl-CoA derivatives, is catalyzed by separate enzymes, utilizes NAD* and FAD as coenzymes, and generates ATP. It is an aerobic process, requiring the presence of oxygen.

Free fatty acids (FFAs)—also called unesterified (UFA) or non- esterified (NEFA) fatty acids (see Chapter 21)—are fatty acids
that are in the unesterified state. In plasma, longer-chain FFA are combined with albumin, and in the cell they are attached to a fatty acid-binding protein, so that in fact they are never really "free." Shorter-chain fatty acids are more water soluble and exist as the unionized acid or as a fatty acid anion.

Fatty acids must first be converted to an active intermediate before they can be catabolized. This is the only step in the complete degradation of a fatty acid that requires energy from ATP. In the presence of ATP and coenzyme A, the enzyme acyl-CoA synthetase (thiokinase) catalyzes the conversion of a fatty acid (or FFA) to an "active fatty acid" or acyl-CoA, using one highenergy phosphate and forming AMP and PP, (Figure 22-1). The PP, is hydrolyzed by inorganic pyrophosphatase with the loss of a further high-energy phosphate, ensuring that the overall reaction goes to completion. Acyl-CoA synthetases are
FIGURE 22-1 Role of carnitine in the transport of longchain fatty acids through the inner mitochondrial membrane. Long-chain acyl-CoA formed by acyl-CoA synthetase enters the intermembrane space. For transport across the inner membrane, acyl groups must be transferred from CoA to carnitine by carnitine palmitoyltransferase-I. The acylcarnitine formed is then carried into the matrix by a translocase enzyme in exchange for a free carnitine and acyl-CoA is reformed by carnitine palmitoyltransferase-ll.
found in the endoplasmic reticulum, peroxisomes, and on the outer membrane of mitochondria.

Carnitine (6-hydroxy-y-trimethylammonium _ butyrate), (CH,), N*—CH,—CH(OH)—CH,—COO,, is widely distributed and is particularly abundant in muscle. Long-chain acyl-CoA (or FFA) cannot penetrate the inner membrane of mitochondria. In the presence of carnitine, however, carnitine palmitoyltransferase-I, located in the outer mitochondrial membrane, transfers the long-chain acyl group from CoA to carnitine, forming acylcarnitine and releasing CoA. Acylcarnitine is able to penetrate the inner membrane and gain access to the B-oxidation system of enzymes via the inner membrane exchange transporter carnitine-acylcarnitine translocase. The transporter binds acylcarnitine and transports it across the membrane in exchange for carnitine. The acyl group is then transferred to CoA so that acyl-CoA is reformed and carnitine is liberated. This reaction is catalyzed by carnitine palmitoyltransferase-II, which is located on the inside of the inner membrane (Figure 22-1).

In the Figure 22-2, pathway for the oxidation of fatty acids, two carbons at a time are cleaved from acyl-CoA molecules, starting at the carboxyl end. The chain is broken between
FIGURE 22-2 Overview of B-oxidation of fatty acids.
the a(2)and B(3)-carbon atoms—hence the the process is termed f-oxidation. The two-carbon units formed are acetylCoA; thus, palmitoyl(C16)-CoA forms eight acetyl-CoA molecules.

Several enzymes, known collectively as "fatty acid oxidase,' are found in the mitochondrial matrix or inner membrane adjacent to the respiratory chain. These catalyze the oxidation of acyl-CoA to acetyl-CoA via the B-oxidation pathway. The system proceeds in cyclic fashion which results in the degradation of long fatty acids to acetyl-CoA. In the process, large quantities of the reducing equivalents FADH, and NADH are generated and are used to form ATP by oxidative phosphorylation (see Chapter 13) (Figure 22-3).
The first step is the removal of two hydrogen atoms from the 2(a)and 3(B)-carbon atoms, catalyzed by acyl-CoA dehydrogenase and requiring flavin adenine dinucleotide (FAD). This resultsin the formation of A*-trans-enoyl-CoA and FADH,, Water is added to saturate the double bond and form 3-hydroxyacylCoA, catalyzed by A?-enoyl-CoA hydratase. The 3-hydroxy derivative undergoes further dehydrogenation on the 3-carbon catalyzed by L-3-hydroxyacyl-CoA dehydrogenase to form the corresponding 3-ketoacyl-CoA compound. In this case, NAD* is the coenzyme involved. Finally, 3-ketoacyl-CoA is split at the 2,3-position by thiolase (3-ketoacyl-CoA-thiolase), forming acetyl-CoA and a new acyl-CoA two carbons shorter than the original acyl-CoA molecule. The shorter acyl-CoA formed in the cleavage reaction reenters the oxidative pathway at reaction 2 (Figure 22-3). In this way, a long-chain fatty acid with an even number of carbons may be degraded completely to acetyl-CoA (C, units). For example, after seven cycles, the C16 fatty acid, palmitate, would be converted to eight acetyl-CoA molecules. Since acetyl-CoA can be oxidized to CO, and water via the citric acid cycle (which is also found within the mitochondria), the complete oxidation of fatty acids is achieved.

Fatty acids with an odd number of carbon atoms are oxidized by the pathway of B-oxidation described above producing acetyl-CoA until a three-carbon (propionyl-CoA) residue remains. This compound is converted to succinyl-CoA, a constituent of the citric acid cycle (see Figure 16-2). Hence, the propiony] residue from an odd-chain fatty acid is the only part of a fatty acid that is glucogenic.

Each cycle of B-oxidation generates one molecule of FADH, and one of NADH. The breakdown of 1 mol of the C16 fatty acid, palmitate, requires seven cycles and produces 8 mol of acetyl-CoA. Oxidation of the reducing equivalents via the respiratory chain leads to the synthesis of 28 mol of ATP (Table 22-1 and see Chapter 13) and oxidation of acetyl-CoA
2c0,
FIGURE 22-3 £-Oxidation of fatty acids. Long-chain acylCoA is cycled through reactions ® to ©, acetyl-CoA being split off, each cycle, by thiolase (reaction ©). When the acyl radical is only four carbon atoms in length, two acetyl-CoA molecules are formed in reaction ©.

Activation, Product = . Activation, Amount Product Formed (mol)/mol Palmitate = -. Activation, ATP Formed (mol)/ mol Product = . Activation, Total ATP Formed (mol)/mol Palmitate = . Activation, ATP Used (mol)/ mol Palmitate = 2. β-Oxidation, Product = FADH 2. β-Oxidation, Amount Product Formed (mol)/mol Palmitate = 7. β-Oxidation, ATP Formed (mol)/ mol Product = 1.5. β-Oxidation, Total ATP Formed (mol)/mol Palmitate = 10.5. β-Oxidation, ATP Used (mol)/ mol Palmitate = -. β-Oxidation, Product = NADH. β-Oxidation, Amount Product Formed (mol)/mol Palmitate = 7. β-Oxidation, ATP Formed (mol)/ mol Product = 2.5. β-Oxidation, Total ATP Formed (mol)/mol Palmitate = 17.5. β-Oxidation, ATP Used (mol)/ mol Palmitate = -. Citric acid cycle, Product = Acetyl-CoA. Citric acid cycle, Amount Product Formed (mol)/mol Palmitate = 8. Citric acid cycle, ATP Formed (mol)/ mol Product = 10. Citric acid cycle, Total ATP Formed (mol)/mol Palmitate = 80. Citric acid cycle, ATP Used (mol)/ mol Palmitate = -. , Product = Total ATP formed (mol)/mol palmitate. , Amount Product Formed (mol)/mol Palmitate = Total ATP formed (mol)/mol palmitate. , ATP Formed (mol)/ mol Product = . , Total ATP Formed (mol)/mol Palmitate = 108. , ATP Used (mol)/ mol Palmitate = . , Product = Total ATP used (mol)/mol palmitate. , Amount Product Formed (mol)/mol Palmitate = Total ATP used (mol)/mol palmitate. , ATP Formed (mol)/ mol Product = . , Total ATP Formed (mol)/mol Palmitate = . , ATP Used (mol)/ mol Palmitate = 2

The table shows how the oxidation of 1 mol of the C16 fatty acid, palmitate, generates 106 mol of ATP (108 formed in total—2 used in the activation step).
via the citric acid cycle produces 80 mol of ATP (Table 22-1 and see Chapter 16). The breakdown of 1 mol of palmitate, therefore, yields a gross total of 108 mol of ATP. However, two high-energy phosphates are used in the initial activation step (Figure 22-3), thus there is a net gain of 106 mol of ATP per mole of palmitate used (Table 22-1), or 106 x 30.5* = 3233 kJ. This represents 33% of the free energy of combustion of palmitic acid.
isomerized (A*cis — A?-trans-enoyl-CoA isomerase) to the corresponding A?-trans-CoA stage of B-oxidation for subsequent hydration and oxidation. Any A*-cis-acyl-CoA either remaining, as in the case of linoleic acid, or entering the pathway at this point after conversion by acyl-CoA dehydrogenase to A?-trans-A*-cis-dienoyl-CoA, is then metabolized as indicated in Figure 22-4.

A modified form of B-oxidation is found in peroxisomes and leads to the breakdown of very-long-chain fatty acids (eg, C20, C22) with the formation of acetyl-CoA and H,O,, which is broken down by catalase (see Chapter 12). This sytem is not linked directly to phosphorylation and the generation of ATP, and also does not attack shorter-chain fatty acids; the B-oxidation sequence ends at octanoyl-CoA. Octanoyl and acetyl groups are both further oxidized in mitochondria. The peroxisomal enzymes are induced by high-fat diets and in some species by hypolipidemic drugs such as clofibrate. Another role of peroxisomal B-oxidation is to shorten the side chain of cholesterol in bile acid formation (see Chapter 26). Peroxisomes also take part in the synthesis of ether glycerolipids (see Chapter 24), cholesterol, and dolichol (see Figure 26-2).

The CoA esters of unsaturated fatty acids are degraded by the enzymes normally responsible for B-oxidation until either a A*-cis-acyl-CoA compound or a A*-cis-acyl-CoA compound is formed, depending on the position of the double bonds (Figure 22-4). The former compound is
"AG for the ATP reaction, as explained in Chapter 11.

Under metabolic conditions associated with a high rate of fatty acid oxidation, the liver produces considerable quantities of acetoacetate and p-3-hydroxybutyrate (}-hydroxybutyrate). Acetoacetate continually undergoes spontaneous decarboxylation to yield acetone. These three substances are collectively known as the ketone bodies (also called acetone bodies or [incorrectly*] "ketones") (Figure 22-5). Acetoacetate and 3-hydroxybutyrate are interconverted by the mitochondrial enzyme pD-3-hydroxybutyrate dehydrogenase; the equilibrium is controlled by the mitochondrial [NAD*]/[NADH] ratio, that is, the redox state. The concentration of total ketone bodies in the blood of well-fed mammals does not normally exceed 0.2 mmol/L. However, in ruminants, 3-hydroxybutyrate is formed continuously from butyric acid (a product of ruminal fermentation) in the rumen wall. In nonruminants, the liver appears to be the only organ that adds significant quantities of ketone bodies to the blood. Extrahepatic tissues utilize acetoacetate and 3-hydroxybutyrate as respiratory substrates. Acetone is a waste product which, as it is volatile, can be excreted via the lungs. Because there is active synthesis but little utilization of ketone bodies in the liver, while they are used but not produced in extrahepatic tissues, there is a net flow of the compounds to the extrahepatic tissues (Figure 22-6).
"The term ketones should not be used as there are ketones in blood
that are not ketone bodies, for example, pyruvate and fructose.
NXP
Acetyl-CoA

FIGURE 22-4 Sequence of reactions in the oxidation of unsaturated fatty acids, for example, linoleic acid. A*-cis-fatty acids or fatty acids forming A*-cis-enoyl-CoA enter the pathway at the position shown. NADPH for the dienoyl-CoA reductase step is supplied by intramitochondrial sources such as glutamate dehydrogenase, isocitrate dehydrogenase, and NAD(P)H transhydrogenase.
FIGURE
22-5
interrelationships of
the ketone
bodies.
p-3-Hydroxybutyrate dehydrogenase is a mitochondrial enzyme.

The enzymes responsible for ketone body formation (ketogenesis) are associated mainly with the mitochondria. AcetoacetylCoA is formed when two acetyl-CoA molecules produced via fatty acid breakdown condense to form acetoacetyl-CoA by a reversal of the thiolase reaction (Figure 22-3), and may also arise directly from the terminal four carbons of a fatty acid during $-oxidation (Figure 22-7). Condensation of acetoacetyl-CoA with another molecule of acetyl-CoA by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase forms HMG-CoA.HMG-CoA lyase then causes acetyl-CoA to split off from the HMG-CoA, leaving free acetoacetate. Both enzymes must be present in mitochondria for ketogenesis to take place. In mammals, ketone bodies are formed solely in the liver and in the rumen epithelium. p-3-Hydroxybutyrate is quantitatively the predominant ketone body present in the blood and urine in ketosis.

While an active enzymatic mechanism produces acetoacetate from acetoacetyl-CoA in the liver, acetoacetate once formed can only be reactivated directly in the cytosol, where it is used in a different, much less active pathway as a precursor in cholesterol synthesis (see Chapter 26). This accounts for the net production of ketone bodies by the liver.
In extrahepatic tissues, acetoacetate is activated to acetoacetyl-CoA by succinyl-CoA-acetoacetate-CoA transferase. CoA is transferred from succinyl-CoA to form acetoacetylCoA (Figure 22-8). In a reaction requiring the addition of a CoA, two acetyl-CoA molecules are formed by the splitting of acetoacetyl-CoA by thiolase and these are oxidized in the
FIGURE 22-6 Formation, utilization, and excretion of ketone bodies. (The main pathway is indicated by the solid arrows.)
FIGURE 22-7 Pathways of ketogenesis in the liver. (FFA, free fatty acids.)

citric acid cycle. 1 mol of acetoacetate or 3-hydroxbutyrate yields 19 or 21.5 mol of ATP, respectively, by these pathways. If the blood level of ketone bodies rises to a concentration of ~12 mmol/L, the oxidative machinery becomes saturated and at this stage, a large proportion of oxygen consumption may be accounted for by their oxidation.
fatty acid taken up is esterified. CPT-I activity is low in the fed state, leading to depression of fatty acid oxidation, and high in starvation, allowing fatty acid oxidation to
In moderate ketonemia, the loss of ketone bodies via the urine is only a few percent of the total ketone body production and utilization. Since there are renal threshold-like effects (there is not a true threshold) that vary between species and individuals, measurement of the ketonemia, not the ketonuria, is the preferred method of assessing the severity of ketosis.

1. Ketosis does not occur in vivo unless there is an increase in the level of circulating FFAs arising from lipolysis of triacylglycerol in adipose tissue. FFAs are the precursors of ketone bodies in the liver. Both in fed and in fasting conditions, the liver extracts ~30% of the FFAs passing through it, so that at high concentrations the flux passing into the organ is substantial. Thus, the factors regulating mobilization of FFA from adipose tissue are important in controlling ketogenesis (Figures 22-9 and 25-8).
2. After uptake by the liver, FFAs are either oxidized to CO, or ketone bodies or esterified to triacylglycerol and phospholipid. There is regulation of entry of fatty acids into the oxidative pathway by carnitine palmitoyltransferase-I (CPT-I) (Figure 22-1), and the remainder of the
FIGURE 22-9 Regulation of ketogenesis. ® to @ show three crucial steps in the pathway of metabolism of free fatty acids (FFA) that determine the magnitude of ketogenesis. (CPT-I, carnitine palmitoyltransferase-I.)
FIGURE 22-10 Regulation of long-chain fatty acid oxidation in the liver. (FFA, free fatty acids; VLDL, very-low-density lipoprotein.) Positive (®) and negative (©) regulatory effects are represented by broken arrows and substrate flow by solid arrows.

increase. Malonyl-CoA, the initial intermediate in fatty acid biosynthesis (see Figure 23-1) is a potent inhibitor of CPT-I (Figure 22-10). In the fed state, therefore, FFAs enter the liver cell in low concentrations and are nearly all esterified to acylglycerols and transported out of the liver in very-low-density lipoproteins (VLDLs). However, as the concentration of FFA increases with the onset of starvation, acetyl-CoA carboxylase is inhibited directly by acyl-CoA, and (malonyl-CoA) decreases, releasing the inhibition of CPT-I and allowing more acyl-CoA to be B-oxidized. These events are reinforced in starvation by a decrease in the (insulin)/(glucagon) ratio. Thus, B-oxidation from FFA is controlled by the CPT-I gateway into the mitochondria, and the balance of the FFA uptake not oxidized is esterified.
are produced when acetoacetate is the end product and only 16 mol when 3-hydroxybutyrate is the end product. Thus, ketogenesis may be regarded as a mechanism that allows the liver to oxidize increasing quantities of fatty acids within the constraints of a tightly coupled system of oxidative phosphorylation.
- 3. In turn, the acetyl-CoA formed in B-oxidation is oxidized in the citric acid cycle, or it enters the pathway of ketogenesis via aetoacetyl-CoA to form ketone bodies. As the level of serum FFA is raised, proportionately more of the acetylCoA produced from their breakdown is converted to ketone bodies and less is oxidized via the citric acid cycle to CO,. The partition of acetyl-CoA between the ketogenic pathway and the pathway of oxidation to CO, is regulated so that the total free energy captured in ATP which results from the oxidation of FFA remains constant as their concentration in the serum changes. This may be appreciated when it is realized that complete oxidation of 1 mol of palmitate involves a net production of 106 mol of ATP via B-oxidation and the citric acid cycle (see above), whereas only 26 mol of ATP

A fall in the concentration of oxaloacetate, particularly within the mitochondria, can impair the ability of the citric acid cycle to metabolize acetyl-CoA and divert fatty acid oxidation toward ketogenesis. Such a fall may occur because of an increase in the (NADH)/(NAD*) ratio caused when increased B-oxidation alters the equilibrium between oxaloacetate and malate so that the concentration of oxaloacetate is decreased, and also when gluconeogenesis is elevated due to low blood glucose levels. The activation by acetyl-CoA of pyruvate carboxylase, which catalyzes the conversion of pyruvate to oxaloacetate, partially alleviates this problem, but in conditions such as starvation and untreated diabetes mellitus, ketone bodies are overproduced and cause ketosis.

Impaired Oxidation of Fatty Acids Gives Rise to Diseases Often Associated With Hypoglycemia
Carnitine deficiency can occur particularly in the newborn— and especially in preterm infants—owing to inadequate
biosynthesis or renal leakage. Losses can also occur in hemodialysis. This suggests there may be a vitamin-like dietary requirement for carnitine in some individuals. Symptoms of deficiency include hypoglycemia, which is a consequence of impaired fatty acid oxidation, and lipid accumulation with muscular weakness. Treatment is by oral supplementation with carnitine.
Inherited CPT-I deficiency affects only the liver, resulting in reduced fatty acid oxidation and ketogenesis, with hypoglycemia. CPT-II deficiency affects primarily skeletal muscle and, when severe, the liver. The sulfonylurea drugs (glyburide [glibenclamide] and tolbutamide), used in the treatment of type 2 diabetes mellitus, reduce fatty acid oxidation and, therefore, hyperglycemia by inhibiting CPT-I.
Inherited defects in the enzymes of B-oxidation and ketogenesis also lead to nonketotic hypoglycemia, coma, and fatty liver. Defects have been identified in longand short-chain 3-hydroxyacyl-CoA dehydrogenase (deficiency of the longchain enzyme may be a cause of acute fatty liver of pregnancy). 3-Ketoacyl-CoA thiolase and HMG-CoA lyase deficiency also affect the degradation of leucine, a ketogenic amino acid (see Chapter 29).

Jamaican vomiting sickness is caused by eating the unripe fruit of the akee tree, which contains the toxin hypoglycin. This inactivates mediumand short-chain acyl-CoA dehydrogenase, inhibiting B-oxidation and causing hypoglycemia. Dicarboxylic aciduria is characterized by the excretion of C,—C,, w-dicarboxylic acids and by nonketotic hypoglycemia, and is caused by a lack of mitochondrial medium-chain acyl-CoA dehydrogenase. Refsum disease is a rare neurologic disorder caused by a metabolic defect that results in the accumulation of phytanic acid, which is found in dairy products and ruminant fat and meat. Phytanic acid is thought to have pathologic effects on membrane function, protein prenylation, and gene expression. Zellweger (cerebrohepatorenal) syndrome occurs in individuals with a rare inherited absence of peroxisomes in all tissues. They accumulate C,,—C,, polyenoic acids in brain tissue and also exhibit a generalized loss of peroxisomal functions. The disease causes severe neurologic symptoms, and most patients die in the first year of life.

Higher than normal quantities of ketone bodies present in the blood or urine constitute ketonemia (hyperketonemia) or ketonuria, respectively. The overall condition is called ketosis.
The basic form of ketosis occurs in starvation and involves depletion of available carbohydrate coupled with mobilization of FFA. This general pattern of metabolism is exaggerated to produce the pathologic states found in diabetes mellitus, the type 2 form of which is increasingly common in Western countries; twin lamb disease; and ketosis in lactating cattle. Nonpathologic forms of ketosis are found under conditions of high-fat feeding and after severe exercise in the postabsorptive state. Acetoacetic and 3-hydroxybutyric acids are both moderately strong acids and are buffered when present in blood or other tissues. However, their continual excretion in quantity progressively depletes the alkali reserve, causing ketoacidosis. This may be fatal in uncontrolled diabetes mellitus.

™ Fatty acid oxidation in mitochondria leads to the generation of large quantities of ATP by a process called B-oxidation that cleaves acetyl-CoA units sequentially from fatty acyl chains. The acetyl-CoA is oxidized in the citric acid cycle, generating further ATP.
= The ketone bodies (acetoacetate, 3-hydroxybutyrate, and acetone) are formed in hepatic mitochondria when there is a high rate of fatty acid oxidation. The pathway of ketogenesis involves synthesis and breakdown of HMG-CoA by two key enzymes: HMG-CoA synthase and HMG-CoA lyase.
@ Ketone bodies are important fuels in extrahepatic tissues.
™ Ketogenesis is regulated at three crucial steps: (1) control of FFA mobilization from adipose tissue; (2) the activity of carnitine palmitoyltransferase-I in liver, which determines the proportion of the fatty acid flux that is oxidized rather than esterified; and (3) partition of acetyl-CoA between the pathway of ketogenesis and the citric acid cycle.
™ Diseases associated with impairment of fatty acid oxidation
lead to
hypoglycemia, fatty
infiltration of
organs, and
hypoketonemia.
- ® Ketosis is mild in starvation but severe in diabetes mellitus and ruminant ketosis.

Eljamil AS: Lipid Biochemistry: For Medical Sciences. iUniverse, 2015.
Gurr MI, Harwood JL, Frayn KN et al: Lipids, Biochemistry,
Biotechnology and Health. Wiley-Blackwell 2016.
- Houten SM, Wanders RJA: A general introduction to the biochemistry of mitochondrial fatty acid B-oxidation. J Inherit
Metab Dis 2010;33:469.

= Describe the reaction catalyzed by acetyl-CoA carboxylase and understand the mechanisms by which its activity is regulated to control the rate of fatty acid synthesis.
Outline the structure of the fatty acid synthase multienzyme complex, .
indicating the
enzymes in
the two
peptide chains
of the
homodimer.
= Explain how long-chain fatty acids are synthesized by the repeated condensation of two carbon units, with formation of the 16-carbon palmitate being favored in most tissues, and identify the cofactors required.
Indicate the sources of reducing equivalents (NADPH) for fatty acid synthesis.
Explain how fatty acid synthesis is regulated by nutritional status and identify other control mechanisms that operate in addition to modulation of the activity of acetyl-CoA carboxylase.
= Identify the nutritionally essential fatty acids and explain why they cannot be formed in the body.
= Explain how polyunsaturated fatty acids are synthesized by desaturase and elongation enzymes.
= Outline the cyclooxygenase and lipoxygenase pathways responsible for the formation of the various classes of eicosanoids.

are synthesized
by an
extramitochondrial which
from for
the acetyl-CoA
is in
the the
primary substrate
is acetate,
the
Critical main
fuel diseases
of complete
cytosol.
system, synthesis
of palmitate
In most
for mammals,
lipogenesis, molecule
but glucose
in they
ruminants obtain
the pathway
humans.
have inhibition
(insulin-dependent)
not been
of diabetes
lipogenesis mellitus,
it the
diet.
reported
(C,,) fatty acids, which give rise to the eicosanoids prostaglandins, thromboxanes, leukotrienes, and lipoxins. Prostaglandins mediate inflammation, pain, induce sleep, and also regulate blood coagulation and reproduction. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin and ibuprofen act by inhibiting prostaglandin synthesis. Leukotrienes have muscle contractant and chemotactic properties and are important in allergic reactions and inflammation.
occurs and
in in
type variations
1
in the
activity of
the process
affect the
nature and
extent of
obesity.
Unsaturated fatty acids in phospholipids of the cell membrane are important in maintaining membrane fluidity (see Chapter 40). A high ratio of polyunsaturated fatty acids to saturated fatty acids (P:S ratio) in the diet is considered to be beneficial in preventing coronary heart disease. Animal tissues have limited capacity for desaturating fatty acids, and require certain dietary polyunsaturated fatty acids derived from plants. These essential fatty acids are used to form eicosanoic

This system is present in many tissues, including liver, kidney, brain,
lung, mammary
gland, and
adipose tissue.
Its cofac-
tor requirements include NADPH, ATP, Mn'*, biotin, and
HCO, (as a source of CO,). Acetyl-CoA is the immediate substrate, and free palmitate is the end product.

Bicarbonate as a source of CO, is required in the initial reaction for the carboxylation of acetyl-CoA to malonyl-CoA in the presence of ATP and acetyl-CoA carboxylase. This enzyme has a major role in the regulation of fatty acid synthesis (see below). Acetyl-CoA carboxylase has a requirement for the B vitamin biotin and is a multienzyme protein containing biotin, biotin carboxylase, biotin carboxyl carrier protein, and a carboxyl transferase, as well as a regulatory allosteric site. One subunit of the complex contains all the components, and variable number of subunits form polymers in the active enzyme (see Figure 23-6). The reaction takes place in two steps: (1) carboxylation of biotin involving ATP and (2) transfer of the carboxyl group to acetyl-CoA to form malonyl-CoA (Figure 23-1).

After the formation of malonyl-CoA, fatty acids are formed by the fatty acid synthase enzyme complex. The individual enzymes required for fatty acid synthesis are linked in this multienzyme polypeptide complex that incorporates the acyl carrier protein (ACP), which has a similar function to that of CoA in the B-oxidation pathway (see Chapter 22) It contains the vitamin pantothenic acid in the form of 4'-phosphopantetheine (see Figure 44-15). In the primary structure of the protein, the enzyme domains are linked in the sequence as shown in Figure 23-2. X-ray crystallography of the three-dimensional structure, however, has shown that the complex is a homodimer, with two identical subunits, each containing six enzymes and an ACP, arranged in an X shape (Figure 23-2). The use of one multienzyme functional unit has the advantages of achieving compartmentalization of the process within the cell without the necessity for permeability barriers, and synthesis of all enzymes in the complex is coordinated since it is encoded by a single gene.

Initially, a priming molecule of acetyl-CoA combines with a cysteine —SH group (Figure 23-3, reaction 1a), while malonyl-CoA combines with the adjacent —SH on the 4'-phosphopantetheine of ACP of the other monomer (reaction 1b). These reactions are catalyzed by malonyl acetyl transacylase, to form acetyl (acyl)-malonyl enzyme. The acetyl group attacks the methylene group of the malonyl residue, catalyzed by 3-ketoacyl synthase, and liberates CO,, forming 3-ketoacyl enzyme (acetoacetyl enzyme) (reaction 2), freeing the cysteine —SH group. Decarboxylation allows the reaction to go to completion, pulling the whole sequence of reactions in the forward direction. The 3-ketoacyl group is reduced, dehydrated, and reduced again (reactions 3-5) to form the corresponding saturated acyl enzyme (product of reaction 5). A new malonyl-CoA molecule combines with the —SH of 4'-phosphopantetheine, displacing the saturated acyl residue onto the free cysteine —SH group. The sequence of reactions is repeated six more times until a saturated 16-carbon acyl
FIGURE 23-1 Biosynthesis of malonyl-CoA by acetyl carboxylase. Acetyl carboxylase is a multienzyme complex containing two enzymes, biotin carboxylase (E1) and a carboxyltransferase (E2) and the biotin carrier protein (BCP). Biotin is covalently linked to the BCP. The reaction proceeds in two steps. In step 1, catalysed by E1, biotin is carboxylated as it accepts a COO" group from HCO, and ATP is used. In step 2, catalyzed by E2, the COO: is transferred to acetyl-CoA forming malonyl-CoA.
Sequence enzyme
primary fatty
synthase
Fatty acid synthase homodimer

FIGURE 23-2 Fatty acid synthase multienzyme complex. The complex is a dimer of two identical polypeptide monomers in which six enzymes and the acyl carrier protein (ACP) are linked in the primary structure in the sequence shown. X-ray crystallography of the threedimensional structure has demonstrated that the two monomers in the complex are arranged in an X-shape.
radical (palmitoyl) has been assembled. It is liberated from the enzyme complex by the activity of the sixth enzyme in the complex, thioesterase (deacylase). The free palmitate must be activated to acyl-CoA before it can proceed via any other metabolic pathway. Its possible fates are esterification into acylglycerols, chain elongation, desaturation, or esterification into cholesteryl ester. In mammary gland, there is a separate thioesterase specific for acyl residues of C,, C,,, or C,,, which are subsequently found in milk lipids.
possess an active pentose phosphate pathway. Moreover, both metabolic pathways are found in the cytosol of the cell, so there are no membranes or permeability barriers against the transfer of NADPH. Other sources of NADPH include the reaction that converts malate to pyruvate catalyzed by the NADP malate dehydrogenase (malic enzyme) (Figure 23-4) and the extramitochondrial isocitrate dehydrogenase reaction (a substantial source in ruminants).
The equation for the overall synthesis of palmitate from acetyl-CoA and malonyl-CoA is
CH,CO—S—CoA + 7HOOCCHCO—S—CoA + 14NADPH + 14H*
— CH,(CH,),, COOH + 7CO, + 6H,O + 8CoA—SH + 14NADP*

The acetyl-CoA used as a primer forms carbon atoms 15 and 16 of palmitate. The addition of all the subsequent C, units is via malonyl-CoA. Propionyl-CoA instead of acetylCoA is used as the primer for the synthesis of long-chain fatty acids with an odd number of carbon atoms, which are found particularly in ruminant fat and milk.

NADPH is involved as a donor of reducing equivalents in the reduction of the 3-ketoacyl and the 2,3-unsaturated acyl derivatives (Figure 23-3, reactions 3 and 5). The oxidative reactions of the pentose phosphate pathway (see Chapter 20) are the chief source of the hydrogen required for the synthesis of fatty acids. Significantly, tissues specializing in active lipogenesis—that is, liver, adipose tissue, and the lactating mammary gland—also

Acetyl-CoA is formed from glucose via the oxidation of pyruvate in the matrix of the mitochondria (see Chapter 17). However, as it does not diffuse readily across the mitochondrial membranes, its transport into the cytosol, the principal site of fatty acid synthesis, requires a special mechanism involving citrate. After condensation of acetyl-CoA with oxaloacetate in the citric acid cycle within mitochondria, the citrate produced can be translocated into the extramitochondrial compartment via the tricarboxylate transporter, where in the presence of CoA and ATP, it undergoes cleavage to acetyl-CoA and oxaloacetate by ATP-citrate lyase, which increases in activity in the well-fed state. The acetyl-CoA is then available for malonyl-CoA formation and synthesis of fatty acids (Figures 23-1 and 23-3), and the oxaloacetate can form malate via NADHlinked malate dehydrogenase, followed by the generation of NADPH and pyruvate via the malic enzyme. The NADPH becomes available for lipogenesis, and the pyruvate can be used to regenerate acetyl-CoA after transport into the mitochondrion (Figure 23-4). This pathway is a means of transferring reducing equivalents from extramitochondrial NADH to NADP. Alternatively, malate itself can be transported into
KEY:
Ga).
individual monomers of fatty acid synthase
FIGURE 23-3 Biosynthesis of long-chain fatty acids. After the initial priming step in which acetyl-CoA is bound to a cysteine-SH group on the fatty acid synthase enzyme (reaction 1a) in each cycle the addition of a malonyl residue causes the acyl chain to grow by two carbon atoms. (Cys, cysteine residue; Pan, 4'-phosphopantetheine.) The blocks highlighted in blue contain initially a C, unit derived from acetyl-CoA (as illustrated) and subsequently the Cn unit formed in reaction 5.
"Shows that the carbon in the CO, initially incorporated into malonyl-CoA is then released as CO, in reaction 2.
the mitochondrion, where it is able to reform oxaloacetate.
|

enzyme in ruminants, probably because in these species ace-
Note that the citrate (tricarboxylate) transporter in the mito- chondrial membrane requires malate to exchange with citrate
(see Figure 13-10). There is little ATP-citrate lyase or malic
_
_
tate (derived from carbohydrate digestion in the rumen and activated to acetyl-CoA extramitochondrially) is the main
source of acetyl-CoA.
FIGURE 23-4 The provision of acetyl-CoA and NADPH for lipogenesis. (K, a-ketoglutarate transporter; P, pyruvate transporter;
PPP, pentose phosphate pathway; T, tricarboxylate transporter.)

This pathway (the "microsomal system") elongates saturated and unsaturated fatty acyl-CoAs (from C,, upward) by two carbons, using malonyl-CoA as the acetyl donor and NADPH as the reductant, and is catalyzed by the microsomal fatty acid elongase system of enzymes (Figure 23-5). Elongation of stearyl-CoA in brain increases rapidly during myelination in order to provide C,, and C,, fatty acids for sphingolipids.

Excess carbohydrate is stored as fat in many animals in anticipation of periods of caloric deficiency such as starvation, hibernation, etc, and to provide energy for use between meals in animals, including humans, that take their food at spaced intervals. Lipogenesis converts surplus glucose and intermediates such as pyruvate, lactate, and acetyl-CoA to fat, assisting the anabolic phase of this feeding cycle. The nutritional state of the organism is the main factor regulating the rate of lipogenesis. Thus, the rate is high in the well-fed animal whose diet contains a high proportion of carbohydrate. It is depressed by restricted caloric intake, high-fat diet, or a deficiency of insulin, as in diabetes mellitus. These latter conditions are associated with increased concentrations of plasma-free fatty acids, and an inverse relationship has been demonstrated between hepatic lipogenesis and the concentration of serum-free fatty acids. Lipogenesis is increased when sucrose is fed instead of glucose because fructose bypasses the phosphofructokinase control point in glycolysis and floods the lipogenic pathway (see Figure 20-5).

Long-chain fatty acid synthesis is controlled in the short term by allosteric and covalent modification of enzymes and in the long term by changes in gene expression governing rates of synthesis of enzymes.
FIGURE 23-5 Microsomal elongase system for fatty acid chain elongation. NADH may also be used by the reductases, but NADPH is preferred.

Acetyl-CoA carboxylase is an allosteric enzyme and is activated by citrate, which increases in concentration in the wellfed state and is an indicator of a plentiful supply of acetyl-CoA. Citrate promotes the conversion of the enzyme from an inactive dimer (two subunits of the enzyme complex) to an active polymeric form, with a molecular mass of several million. Inactivation is promoted by phosphorylation of the enzyme and by long-chain acyl-CoA molecules, an example of negative
FIGURE 23-6 Regulation of acetyl-CoA carboxylase. AcetylCoA carboxylase is activated by citrate, which promotes the conversion of the enzyme from an inactive dimer to an active polymeric form. Inactivation is promoted by phosphorylation of the enzyme and by long-chain acyl-CoA molecules such as palmitoyl-CoA. In addition, acyl-CoA inhibits the tricarboxylate transporter, which transports citrate out of mitochondria into the cytosol, thus decreasing the citrate concentration in the cytosol and favoring inactivation of the enzyme.
feedback inhibition by a product of a reaction (Figure 23-6). Thus, if acyl-CoA accumulates because it is not esterified quickly enough or because of increased lipolysis or an influx of free fatty acids into the tissue, it will automatically reduce the synthesis of new fatty acid. Acyl-CoA also inhibits the mitochondrial tricarboxylate transporter, thus preventing activation of the enzyme by egress of citrate from the mitochondria into the cytosol (Figure 23-6). Acetyl-CoA carboxylase is also regulated by hormones such as glucagon, epinephrine, and insulin via changes in its phosphorylation state (details in Figure 23-7).

Acyl-CoA causes an inhibition of pyruvate dehydrogenase by inhibiting the ATP-ADP exchange transporter of the inner mitochondrial membrane, which leads to increased intramitochondrial (ATP)/(ADP) ratios and therefore to conversion of active to inactive pyruvate dehydrogenase (see Figure 17-6), thus regulating the availability of acetyl-CoA for lipogenesis. Furthermore, oxidation of acyl-CoA due to increased levels of free fatty acids may increase the ratios of (acetyl-CoA)/(CoA) and (NADH)/(NAD*) in mitochondria, inhibiting pyruvate dehydrogenase.

Insulin stimulates lipogenesis by several other mechanisms as well as by increasing acetyl-CoA carboxylase activity. It increases the transport of glucose into the cell (eg, in adipose tissue), increasing the availability of both pyruvate for
FIGURE 23-7 Regulation of acetyl-CoA carboxylase by phosphorylation/dephosphorylation. The enzyme is inactivated by phosphorylation by AMP-activated protein kinase (AMPK), which in turn is phosphorylated and activated by AMP-activated protein kinase kinase (AMPKK). Glucagon and epinephrine increase cAMP, and thus activate this latter enzyme via cAMP-dependent protein kinase. The kinase kinase enzyme is also believed to be activated by acyl-CoA. Insulin activates acetyl-CoA carboxylase via dephosphorylation of AMPK.
fatty acid synthesis and glycerol-3-phosphate for triacylglycerol synthesis via esterification of the newly formed fatty acids (see Figure 24-2), and also converts the inactive form of pyruvate dehydrogenase to the active form in adipose tissue, but not in liver. Insulin also—by its ability to depress the level of intracellular cAMP—inhibits lipolysis in adipose tissue, reducing the concentration of plasma-free fatty acids and, therefore, long-chain acyl-CoA, which are inhibitors of lipogenesis.

These enzymes adapt to the body's physiologic needs via changes in gene expression which lead to increases in the total amount of enzyme protein present in the fed state and decreases during intake of a high-fat diet and in conditions such as starvation, and diabetes mellitus. Insulin plays an important role, promoting gene expression and induction of enzyme biosynthesis, and glucagon (via cAMP) antagonizes this effect. Feeding fats containing polyunsaturated fatty acids coordinately regulates the inhibition of expression of key enzymes of glycolysis and lipogenesis. These mechanisms for longer-term regulation of lipogenesis take several days to become fully manifested and augment the direct and immediate effect of free fatty acids and hormones such as insulin and glucagon.

Certain long-chain unsaturated fatty acids of metabolic significance in mammals are shown in Figure 23-8. Other C,,, C,,, and C,, polyenoic fatty acids may be derived from oleic, linoleic, and a-linolenic acids by chain elongation. Palmitoleic and oleic acids are not essential in the diet because the tissues can introduce a double bond at the A' position of a saturated fatty acid. Linoleic and a-linolenic acids are the only fatty acids known to be essential for the complete nutrition of many species of animals, including humans, and are termed the nutritionally essential fatty acids. In humans and most other
FIGURE 23-8 Structure of some unsaturated fatty acids. Although the carbon atoms in the molecules are conventionally numbered—that is, numbered from the carboxyl terminal—the w numbers (eg, w7 in palmitoleic acid) are calculated from the reverse end (the methyl terminal) of the molecules. The information in parentheses shows, for instance, that a-linolenic acid contains double bonds starting at the third carbon from the methyl terminal, has 18 carbons and 3 double bonds, and has these double bonds at the 9th, 12th, and 15th carbons from the carboxyl terminal. *Nutritionally essential fatty acids in humans.

mammals, arachidonic acid can be formed from linoleic acid. Double bonds can be introduced at the A', A®, A®, and A' positions (see Chapter 21) in most animals, but never beyond the A' position. In contrast, plants are able to synthesize the nutritionally essential fatty acids by introducing double bonds at the A" and A" positions.

Several tissues including the liver are considered to be responsible for the formation of nonessential monounsaturated fatty acids from saturated fatty acids. The first double bond introduced into a saturated fatty acid is nearly always in the A' position. An enzyme system—A' desaturase (Figure 23-9)—in the endoplasmic reticulum catalyzes the conversion of palmitoyl-CoA or stearoyl-CoA to palmitoleoyl-CoA or oleoyl-CoA, respectively. Oxygen and either NADH or NADPH are necessary for the reaction. The enzymes appear to be similar to a mono-oxygenase system involving cytochrome b, (see Chapter 12).

Additional double bonds introduced into existing monounsaturated fatty acids are always separated from each other by a methylene group (methylene interrupted) except in bacteria. Since animals have a A? desaturase, they are able to synthesize the w9 (oleic acid) family of unsaturated fatty acids completely bya combination of chain elongation and desaturation (Figures 23-9 and 23-10) after the formation of saturated fatty acids by the pathways described in this chapter. However, as indicated above, linoleic (w6) or a-linolenic (w3) acids are required for the synthesis of the other members of the w6 or w3 families (pathways shown in Figure 23-10) and must be supplied in the diet. Linoleic acid is converted to arachidonic acid (20:4 w6) via y-linolenic acid (18:3 6). The nutritional requirement for arachidonate may thus be dispensed with if there is adequate linoleate in the diet. Cats, however, cannot carry out this conversion owing to the absence of A' desaturase and must obtain arachidonate in their diet. The desaturation and
FIGURE 23-10 Biosynthesis of the w9, w6, and w3 families of polyunsaturated fatty acids. In animals, the w9, w6, and w3 families of polyunsaturated fatty acids are synthesized in the endoplasmic reticulum from oleic, linoleic, and B-linolenic acids, respectively, by a series of elongation and desaturation reactions. The production of 22:5 w6 (osbond acid) or 22:6 w3 (docosahexanoic acid [DHA]), however, requires one cycle of B-oxidation, which takes place inside peroxisomes after the formation of 24:5 w6 or 24:6 w3. (AA, arachidonic acid; E, elongase; DS, desaturase; EFA, essential fatty acids; EPA, eicosapentaenoic acid; GLA, y-linolenic acid; ©, inhibition.)

chain elongation system are greatly diminished in the starving state, in response to glucagon and epinephrine administration, and in the absence of insulin as in type 1 diabetes mellitus.

Rats fed a purified nonlipid diet containing vitamins A and D exhibit a reduced growth rate and reproductive deficiency which may be cured by the addition of linoleic, a-linolenic, and arachidonic acids to the diet. These fatty acids are found in high concentrations in vegetable oils (see Table 21-2) and in small amounts in animal carcasses. Essential fatty acids are required for prostaglandin, thromboxane, leukotriene, and lipoxin formation (see below), and they also have various other functions that are less well defined. They are found in the structural lipids of the cell, often in the position 2 of phospholipids, and are concerned with the structural integrity of the mitochondrial membrane.
Arachidonic acid is present in membranes and accounts for 5 to 15% of the fatty acids in phospholipids. Docosahexaenoic acid (DHA; w3, 22:6), which is synthesized to a limited extent from a-linolenic acid or obtained directly from fish oils, is present in high concentrations in retina, cerebral cortex, testis, and sperm. DHA is particularly needed for development of the brain and retina and is supplied via the placenta and milk. Patients with retinitis pigmentosa are reported to have low blood levels of DHA. In essential fatty acid deficiency, nonessential polyenoic acids of the w9 family, particularly A**!!-eicosatrienoic acid (w9 20:3) (Figure 23-10), replace the essential fatty acids in phospholipids, other complex lipids, and membranes. The triene:tetraene ratio in plasma lipids can be used to diagnose the extent of essential fatty acid deficiency.

Arachidonate and some other C,, polyunsaturated fatty acids give rise to eicosanoids, physiologically and pharmacologically active compounds known as prostaglandins (PG), thromboxanes (TX), leukotrienes (LT), and lipoxins (LX) (see Chapter 21). Physiologically, they are considered to act as local hormones functioning through G-protein-linked receptors to elicit their biochemical effects.
There are three groups of eicosanoids that are synthesized from C,, eicosanoic acids derived from the essential fatty acids linoleate and a-linolenate, or directly from dietary arachidonate and eicosapentaenoate (Figure 23-11). Arachidonate, which may be obtained from the diet, but is usually derived from the position 2 of phospholipids in the plasma membrane by the action of phospholipase A, (Figure 24-5), is the substrate for the synthesis of the PG,, TX, series (prostanoids) by the cyclooxygenase pathway, or the LT, and LX, series by the lipoxygenase pathway, with the two pathways competing for the arachidonate substrate (Figure 23-11).

Prostanoids (see Chapter 21) are synthesised by the pathway summarized in Figure 23-12. In the first reaction, catalysed by cyclooxygenase (COX) (also called prostaglandin H synthase), an enzyme that has two activities, a cyclooxygenase and peroxidase, two molecules of O, are consumed. COX is present as two isoenzymes, COX-1 and COX-2. The product, an endoperoxide (PGH), is converted to prostaglandins D and Eas well as to a thromboxane (TXA,) and prostacyclin (PGI,). Each cell type produces only one type of prostanoid.

Thromboxanes are synthesized in platelets and upon release cause vasoconstriction and platelet aggregation. Their synthesis is specifically inhibited by low-dose aspirin. Prostacyclins (PGL,) are produced by blood vessel walls and are potent inhibitors of platelet aggregation.
Thus, thromboxanes and prostacyclins are antagonistic. PG, and TX,, formed from eicosapentaenoic acid (EPA), inhibit the release of arachidonate from phospholipids and the formation of PG, and TX,. PGI, is as potent an antiaggregator of platelets as PGI,, but TXA, is a weaker aggregator than TXA,, changing the balance of activity and favoring longer clotting times. As little as 1 ng/mL of plasma prostaglandins causes contraction of smooth muscle ianimals.

The role of essential fatty acids in membrane formation is unrelated to prostaglandin formation. Prostaglandins do not relieve symptoms of essential fatty acid deficiency, and an essential fatty acid deficiency is not caused by inhibition of prostaglandin synthesis.

"Switching off" of prostaglandin activity is partly achieved by a remarkable property of cyclooxygenase—that of self-catalyzed destruction; that is, it is a "suicide enzyme.' Furthermore, the inactivation of prostaglandins by 15-hydroxyprostaglandin dehydrogenase is rapid. Blocking the action of this enzyme
FIGURE 23-11 The three groups of eicosanoids and their biosynthetic origins. (®, cyclooxygenase pathway; @), lipoxygenase pathway; LT, leukotriene; LX, lipoxin; PG, prostaglandin; PGI, prostacyclin; TX, thromboxane.) The subscript denotes the total number of double bonds in the molecule and the series to which the compound belongs.
with sulfasalazine or indomethacin can prolong the half-life of prostaglandins in the body.
formed by the combined action of more than one lipoxygenase (Figure 23-13).

The leukotrienes are a family of conjugated trienes formed from eicosanoic acids in leukocytes, mastocytoma cells, platelets, and macrophages by the lipoxygenase pathway in response to both immunologic and nonimmunologic stimuli. Three different lipoxygenases (dioxygenases) insert oxygen into the 5, 12, and 15 positions of arachidonic acid, giving rise to hydroperoxides (HPETE). Only 5-lipoxygenase forms leukotrienes (details in Figure 23-13). Lipoxins are a family of conjugated tetraenes also arising in leukocytes. They are

In adults
subsisting on
ordinary diets,
no signs
of essential
fatty acid deficiencies have been reported. However, infants receiv-
ing formula diets low in fat and patients maintained for long periods exclusively by intravenous nutrition low in essential
fatty acids show deficiency symptoms that can be prevented by
an essential
fatty acid
intake of
1
to
2%
of the
total caloric
requirement.
FIGURE 23-12 Conversion of arachidonic acid to prostaglandins and thromboxanes of series 2. (HHT, hydroxyheptadecatrienoate; PG, prostaglandin; PGI, prostacyclin; TX, thromboxane.) (*Both of these starred activities are attributed to the cyclooxgenase enzyme [prostaglandin H synthase]. Similar conversions occur in prostaglandins and thromboxanes of series 1 and 3.)

effects in the promotion of hypercholesterolemia and atherosclerosis (see Chapter 26).
Abnormal metabolism of essential fatty acids, which may be connected with dietary insufficiency, has been noted in cystic fibrosis, acrodermatitis enteropathica, hepatorenal syndrome, Sjégren-Larsson syndrome, multisystem neuronal degeneration, Crohn disease, cirrhosis and alcoholism, and Reye syndrome. Elevated levels of very-long-chain polyenoic acids have been found in the brains of patients with Zellweger syndrome (see Chapter 22). Diets with a high P:S (polyunsaturated:saturated fatty acid) ratio reduce serum cholesterol levels and are considered to be beneficial in terms of the risk of development of coronary heart disease.

Small amounts of trans-unsaturated fatty acids are found in ruminant fat (eg, butter fat has 2-7%), where they arise from the action of microorganisms in the rumen, but the main source in the human diet is from partially hydrogenated vegetable oils (eg, margarine) (see Chapter 21). Trans fatty acids compete with essential fatty acids and may exacerbate essential fatty acid deficiency. Moreover, they are structurally similar to saturated fatty acids (see Chapter 21) and have comparable

Aspirin is a nonsteroidal anti-inflammatory drug (NSAID) that inhibits COX-1 and COX-2. Other NSAIDs include indomethacin and ibuprofen, and these usually inhibit cyclooxygenases by competing with arachidonate. Since inhibition of COX-1 causes the stomach irritation often associated with taking NSAIDs, attempts have been made to develop drugs that selectively inhibit COX-2 (coxibs). Unfortunately, however, the success of this approach has been limited and some coxibs have been withdrawn or suspended from the market due to undesirable side effects and safety issues. Transcription of COX-2—but not of COX-1—is completely inhibited by anti-inflammatory corticosteroids.

Potential therapeutic uses of prostanoids include prevention of conception, induction of labor at term, termination of pregnancy, prevention or alleviation of gastric ulcers, control of inflammation and of blood pressure, and relief of asthma and nasal congestion. In addition, PGD, is a potent
FIGURE 23-13 Conversion of arachidonic acid to leukotrienes and lipoxins of series 4 via the lipoxygenase pathway. Some similar conversions occur in series 3 and 5 leukotrienes. (0), peroxidase; @, leukotriene A, epoxide hydrolase; ©, glutathione S-transferase; @), y-glutamyltranspeptidase; ©), cysteinyl-glycine dipeptidase; HETE, hydroxyeicosatetraenoate; HPETE, hydroperoxyeicosatetraenoate.)
sleep-promoting substance. Prostaglandins increase cAMP in platelets, thyroid, corpus luteum, fetal bone, adenohypophysis, and lung but reduce cAMP in renal tubule cells and adipose tissue (see Chapter 25).
Leukotrienes are vasoactive, and 5-lipoxygenase has been found in arterial walls. Evidence supports an anti-inflammatory role for lipoxins in vasoactive and immunoregulatory function, for example, as counterregulatory compounds (chalones) of the immune response.

Slow-reacting substance of anaphylaxis (SRS-A) is a mixture of leukotrienes C,, D,, and E,. This mixture of leukotrienes is a potent constrictor of the bronchial airway musculature. These leukotrienes together with leukotriene B, also cause vascular permeability and attraction and activation of leukocytes and are important regulators in many diseases involving inflammatory or immediate hypersensitivity reactions, such as asthma.

@ The synthesis of long-chain fatty acids (lipogenesis) is carried out by two enzyme systems: acetyl-CoA carboxylase and fatty acid synthase.
@ The pathway converts acetyl-CoA to palmitate and requires
NADPH, ATP, Mn", biotin, and pantothenic acid as cofactors.
™ Acetyl-CoA carboxylase converts acetyl-CoA to malonyl-CoA, and then fatty acid synthase, a multienzyme complex consisting
of two identical polypeptide chains, each containing six separate enzymatic activities and ACP, catalyzes the formation of palmitate from one acetyl-CoA and seven malonyl-CoA molecules.
- Lipogenesis is regulated at the acetyl-CoA carboxylase step
by allosteric
modifiers, phosphorylation/dephosphorylation,
- and induction and repression of enzyme synthesis. The
enzyme is
allosterically activated
by citrate
and deactivated
by long-chain
acyl-CoA.
promotes its
activity,
Dephosphorylation while
(eg, phosphorylation
by insulin)
(eg, by
glucagon epinephrine)
is inhibitory.
Biosynthesis of unsaturated long-chain fatty acids is achieved by desaturase and elongase enzymes, which introduce double bonds and lengthen existing acyl chains, respectively.
Higher animals have A', A°, A°, and A? desaturases but cannot insert new double bonds beyond the position 9 of fatty acids.
or
Thus, the essential fatty acids linoleic (w6) and a-linolenic (w3) must be obtained from the diet.
Eicosanoids are derived from C,, (eicosanoic) fatty acids
synthesized from the essential fatty acids and make up
important groups of physiologically and pharmacologically
active compounds, including the prostaglandins,
thromboxanes, leukotrienes,
and lipoxins.

2015.
Smith WL, Murphy RC: The eicosanoids: cyclooxygenase, lipoxygenase,
and epoxygenase
pathways.
In
Biochemistry of Lipids, Lipoproteins and Membranes, 6th ed. Ridgway N,
McLeod R (editors). Academic Press, 2015:260-296.

- 7
Explain that the catabolism of triacylglycerols involves hydrolysis to free fatty acids and glycerol and indicate the fate of these metabolites.
Indicate that glycerol-3-phosphate is the substrate for the formation of both triacylglycerols and phosphoglycerols and that a branch point at phosphatidate leads to the synthesis of inositol phospholipids and cardiolipin or/and triacylglycerols and other phospholipids. :
= Explain that plasmalogens and platelet-activating factor (PAF) are formed by a complex pathway starting from dihydroxyacetone phosphate.
= Illustrate the role of various phospholipases in the degradation and remodeling of phospholipids.
Explain that ceramide is the precursor from which all sphingolipids are formed.
Indicate how sphingomyelin and glycosphingolipids are produced by the reaction of ceramide with phosphatidylcholine or sugar residue(s), respectively.
m= Identify examples of disease processes caused by defects in phospholipid or sphingolipid synthesis or breakdown.

Acylglycerols constitute the majority of lipids in the body. Triacylglycerols are the major lipids in fat deposits and in food, and their roles in lipid transport and storage and in various diseases such as obesity, diabetes, and hyperlipoproteinemia will be described in subsequent chapters. The amphipathic nature of phospholipids and sphingolipids makes them ideally suitable as the main lipid component of cell membranes.
A dozen or so glycolipid storage diseases have been described (eg, Gaucher disease and Tay-Sachs disease), each due to a genetic defect in the pathway for glycolipid degradation in the lysosomes.
Phospholipids also take part in the metabolism of many other lipids. Some phospholipids have specialized functions; for example, dipalmitoyl lecithin is a major component of lung surfactant, which is lacking in respiratory distress syndrome of the newborn. Inositol phospholipids in the cell membrane act as precursors of hormone second messengers, and platelet-activating factor (PAF) is an alkylphospholipid. Glycosphingolipids, which contain sphingosine and sugar residues as well as a fatty acid are found in the outer leaflet of the plasma membrane with their oligosaccharide chains facing outward. They form part of the glycocalyx of the cell surface and are important (1) in cell adhesion and cell recognition, (2) as receptors for bacterial toxins (eg, the toxin that causes cholera), and (3) as ABO blood group substances.

Triacylglycerols must be hydrolyzed by a lipase to their constituent fatty acids and glycerol before further catabolism can proceed. Much of this hydrolysis (lipolysis) occurs in adipose tissue with release of free fatty acids into the plasma, where they are found combined with serum albumin (see Figure 25-7). This is followed by free fatty acid uptake into tissues (including liver, heart, kidney, muscle, lung, testis, and adipose tissue, but not readily by brain), where they are oxidized to obtain energy or reesterified. The utilization of glycerol depends on whether such tissues have the enzyme glycerol kinase, which is found in significant amounts in liver, kidney, intestine, brown adipose tissue, and the lactating mammary gland.

The major pathways of triacylglycerol and phosphoglycerol biosynthesis are outlined in Figure 24-1. Important substances such as triacylglycerols, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and cardiolipin, a constituent of mitochondrial membranes, are formed from glycerol-3-phosphate. Significant branch points in the pathway occur at the phosphatidate and diacylglycerol steps. Phosphoglycerols containing an ether link (—C—O—C—), the best known of which are plasmalogens and PAF, are derived from dihydroxyacetone phosphate. Glycerol-3-phosphate and dihydroxyacetone phosphate are intermediates in glycolysis, making a very important connection between carbohydrate and metabolism lipid (see Chapter 14).

Both glycerol and fatty acids must be activated by ATP before they can be incorporated into acylglycerols. Glycerol kinase catalyzes the activation of glycerol to sn-glycerol 3-phosphate. If the activity of this enzyme is absent or low, as in muscle or adipose tissue, most of the glycerol-3-phosphate is formed from dihydroxyacetone phosphate by glycerol-3-phosphate dehydrogenase (Figure 24-2).

Two molecules of acyl-CoA, formed by the activation of fatty acids by acyl-CoA synthetase (see Chapter 22), combine with glycerol-3-phosphate to form phosphatidate (1,2-diacylglycerol phosphate). This takes place in two stages, catalyzed by glycerol-3-phosphate acyltransferase and_ 1-acylglycerol3-phosphate acyltransferase. Phosphatidate is converted by
Overview of acylglycerol biosynthesis. (PAF,
FIGURE 24-1 platelet-activating factor.)
phosphatidate phosphohydrolase (also called phosphatidate phosphatase [PAP]) and diacylglycerol acyltransferase (DGAT) to 1,2-diacylglycerol and then triacylglycerol. Lipins, a family of three proteins, have PAP activity and they also act as transcription factors which regulate the expression of genes involved in lipid metabolism. DGAT catalyzes the only step specific for triacylglycerol synthesis and is thought to be rate limiting in most circumstances. In intestinal mucosa, monoacylglycerol acyltransferase converts monoacylglycerol to 1,2-diacylglycerol in the monoacylglycerol pathway. Most of the activity of these enzymes resides in the endoplasmic reticulum, but some is found in mitochondria. Although phosphatidate phosphohydrolase protein is found mainly in the cytosol, the active form of the enzyme is membrane bound.

In the biosynthesis of phosphatidylcholine and phosphatidylethanolamine (Figure 24-2), choline or ethanolamine must first be activated by phosphorylation by ATP followed by linkage to CDP. The resulting CDP-choline or CDP-ethanolamine reacts with 1,2-diacylglycerol to form either phosphatidylcholine or phosphatidylethanolamine, respectively. Phosphatidylserine is formed from phosphatidylethanolamine directly by reaction with serine (Figure 24-2). Phosphatidylserine may reform phosphatidylethanolamine by decarboxylation. An alternative pathway in liver enables phosphatidylethanolamine to give rise directly to phosphatidylcholine by progressive methylation of the ethanolamine residue. In spite of these sources of choline, it is considered to be an essential nutrient in many mammalian species, although this has not been established in humans.
The regulation of triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine biosynthesis is driven by the availability of free fatty acids. Those that escape oxidation are preferentially converted to phospholipids, and when this requirement is satisfied, they are used for triacylglycerol synthesis.
Cardiolipin (diphosphatidylglycerol; see Figure 21-9) is a phospholipid present in mitochondria. It is formed from phosphatidylglycerol, which in turn is synthesized from CDPdiacylglycerol (Figure 24-2) and glycerol-3-phosphate. Cardiolipin, found in the inner membrane of mitochondria, has a key role in mitochondrial structure and function, and is also thought to be involved in programmed cell death (apoptosis).

In glycerol ether phospholipids, one or more of the glycerol carbons is attached to a hydrocarbon chain by an ether linkage rather than an ester bond. Plasmalogens and PAF are important examples of this type of lipid. The biosynthetic pathway is located in peroxisomes. Dihydroxyacetone phosphate is the precursor of the glycerol moiety (Figure 24-3). It combines with acyl-CoA to give 1-acyldihydroxyacetone phosphate, and the ether link is formed in the next reaction, producing 1-alkyldihydroxyacetone phosphate, which is then converted to 1-alkylglycerol 3-phosphate. After further acylation in the
FIGURE 24-2 Biosynthesis of triacylglycerol and phospholipids. ©, monoacylglycerol pathway; ®, glycerol phosphate pathway. Phosphatidylethanolamine may be formed from ethanolamine by a pathway similar to that shown for the formation of phosphatidylcholine from choline.
PAF
FIGURE 24-3 Biosynthesis of ether lipids, including plasmalogens, and platelet-activating factor (PAF). In the de novo pathway for PAF synthesis, acetyl-CoA is incorporated at stage', avoiding the last two steps in the pathway shown here.
2 position, the resulting 1-alkyl-2-acylglycerol 3-phosphate (analogous to phosphatidate in Figure 24-2) is hydrolyzed to give the free glycerol derivative. Plasmalogens, which comprise much of the phospholipid in mitochondria, are formed by desaturation of the analogous 3-phosphoethanolamine derivative (Figure 24-3). PAF (1-alkyl-2-acetyl-sn-glycerol3-phosphocholine) is synthesized from the corresponding 3-phosphocholine derivative. It is formed by many blood cells and other tissues and aggregates platelets at concentrations as low as 10°"! mol/L. It also has hypotensive and ulcerogenic properties and is involved in a variety of biologic responses, including inflammation, chemotaxis, and protein phosphorylation.

Although phospholipids are actively degraded, each portion of the molecule turns over at a different rate—for example, the turnover time of the phosphate group is different from that of the 1-acyl group. This is due to the presence of enzymes that allow partial degradation followed by resynthesis (Figure 24-4). Phospholipase A, catalyzes the hydrolysis of glycerophospholipids to form a free fatty acid and lysophospholipid, which in turn may be reacylated by acyl-CoA in the presence of an acyltransferase. Alternatively, lysophospholipid (eg, lysolecithin) is attacked by lysophospholipase, forming the corresponding glyceryl phosphoryl base, which may then be split by a hydrolase liberating glycerol-3-phosphate plus base. Phospholipases A, A, B, C, and D attack the bonds indicated in Figure 24-5. Phospholipase A, is found in pancreatic fluid and snake venom as well as in many types of cells; phospholipase C is one of the major toxins secreted by bacteria; and phospholipase D is known to be involved in mammalian signal transduction. Lysolecithin (lysophosphatidylcholine) may be formed by an alternative route that involves lecithin: cholesterol acyltransferase (LCAT). This enzyme, found in plasma, catalyzes the transfer of a fatty acid residue from the 2 position

of lecithin to cholesterol to form cholesteryl ester and lysolecithin, and is considered to be responsible for much of the cholesteryl ester in plasma lipoproteins (see Chapter 25). Long-chain saturated fatty acids are found predominantly in the 1 position of phospholipids, whereas the polyunsaturated fatty acids (eg, the precursors of prostaglandins) are incorporated more frequently into the 2 position. The
FIGURE 24-5 Sites of the hydrolytic activity of phospholipases ona phospholipid substrate.
incorporation of fatty acids into lecithin occurs in three ways; by complete synthesis of the phospholipid; by transacylation between cholesteryl ester and lysolecithin; and by direct acylation of lysolecithin by acyl-CoA. Thus, a continuous exchange of the fatty acids is possible, particularly with regard to introducing essential fatty acids into phospholipid molecules.

Ceramide (see Chapter 21) is synthesized in the endoplasmic reticulum from the amino acid serine as shown in Figure 24-6. Ceramide is an important signaling molecule (second messenger) regulating pathways including programmed cell death (apoptosis), the cell cycle, and cell differentiation and senescence. Sphingomyelins (see Figure 21-10) are phospholipids and are formed when ceramide reacts with to phosphatidylcholine
FIGURE 24-7 Biosynthesis of (A) sphingomyelin, (B) galactosylceramide and its sulfo derivative. (PAPS, "active sulfate,' adenosine 3'-phosphate-5'-phosphosulfate.)
form sphingomyelin plus diacylglycerol (Figure 24-7A). This occurs mainly in the Golgi apparatus and to a lesser extent in the plasma membrane.

The simplest glycosphingolipids (cerebrosides) are galactosylceramide (GalCer) (see Figure 21-14) and glucosylceramide (GlcCer). GalCer is a major lipid of myelin, whereas GlcCer is the major glycosphingolipid of extraneural tissues and a precursor of most of the more complex glycosphingolipids. GalCer (Figure 24-7B) is formed in a reaction between ceramide and uridine diphosphate galactose (UDPGal) (formed by epimerization from UDPGlc—Figure 20-6).
Glycosphingolipids are constituents of the outer leaflet of plasma membranes and are important in cell adhesion and cell recognition. Some are antigens, for example, ABO blood group substances. Certain gangliosides function as receptors for bacterial toxins (eg, for cholera toxin, which subsequently activates adenylyl cyclase).

Lung surfactant is composed mainly of lipid with some proteins and carbohydrate and prevents the alveoli from collapsing. The phospholipid dipalmitoyl-phosphatidylcholine decreases surface tension at the air-liquid interface and thus greatly reduces the work of breathing, but other surfactant lipid and protein components are also important in surfactant function. Deficiency of lung surfactant in the lungs of many preterm newborns gives rise to infant respiratory distress syndrome (IRDS). Administration of either natural or artificial surfactant is of therapeutic benefit.

Sulfogalactosylceramide and other sulfolipids such as the sulfo(galacto)-glycerolipids and the steroid sulfates are formed after further reactions involving 3'-phosphoadenosine5'-phosphosulfate (PAPS; "active sulfate"). Gangliosides are synthesized from ceramide by the stepwise addition of activated sugars (eg, UDPGlc and UDPGal) and a sialic acid, usually N-acetylneuraminic acid (Figure 24-8). A large number of gangliosides of increasing molecular weight may be formed. Most of the enzymes transferring sugars from nucleotide sugars (glycosyl transferases) are found in the Golgi apparatus.
Certain diseases are characterized by abnormal quantities of these lipids in the tissues, often in the nervous system. They may be classified into two groups: (1) true demyelinating diseases and (2) sphingolipidoses.
In multiple sclerosis, which is a demyelinating disease, there is loss of both phospholipids (particularly ethanolamine plasmalogen) and of sphingolipids from white matter. Thus, the lipid composition of white matter resembles that of gray matter. The fluid shows raised levels.
cerebrospinal phospholipid
The sphingolipidoses (lipid storage diseases) are a group of inherited diseases that are caused by a genetic defect in the catabolism of lipids containing sphingosine. They are part of
FIGURE 24-8 Biosynthesis of gangliosides. (NeuAc, N-acetylneuraminic acid.)

Tay-Sachs disease, Enzyme Deficiency = Hexosaminidase A. Tay-Sachs disease, Lipid Accumulating = Cer-Glc-Gal(NeuAc) GalNAc G M2 Ganglioside. Tay-Sachs disease, Clinical Symptoms = Mental retardation, blindness, muscular weakness. Fabry disease, Enzyme Deficiency = α-Galactosidase. Fabry disease, Lipid Accumulating = Cer-Glc-Gal- Gal Globotriaosylceramide. Fabry disease, Clinical Symptoms = Skin rash, kidney failure (full symptoms only in males; X-linked recessive). Metachromatic leukodystrophy, Enzyme Deficiency = Arylsulfatase A. Metachromatic leukodystrophy, Lipid Accumulating = Cer-Gal- OSO 3 3-Sulfogalactosylceramide. Metachromatic leukodystrophy, Clinical Symptoms = Mental retardation and psychologic disturbances in adults; demyelination. Krabbe disease, Enzyme Deficiency = β-Galactosidase. Krabbe disease, Lipid Accumulating = Cer- Gal Galactosylceramide. Krabbe disease, Clinical Symptoms = Mental retardation; myelin almost absent. Gaucher disease, Enzyme Deficiency = β-Glucosidase. Gaucher disease, Lipid Accumulating = Cer- Glc Glucosylceramide. Gaucher disease, Clinical Symptoms = Enlarged liver and spleen, erosion of long bones, mental retardation in infants. Niemann-Pick disease, Enzyme Deficiency = Sphingomyelinase. Niemann-Pick disease, Lipid Accumulating = Cer- P-choline Sphingomyelin. Niemann-Pick disease, Clinical Symptoms = Enlarged liver and spleen, mental retardation; fatal in early life. Farber disease, Enzyme Deficiency = Ceramidase. Farber disease, Lipid Accumulating = Acyl- Sphingosine Ceramide. Farber disease, Clinical Symptoms = Hoarseness, dermatitis, skeletal deformation, mental retardation; fatal in early life

Abbreviations: Cer, ceramide; Gal, galactose; Glc, glucose; NeuAc, N-acetyIneuraminic acid; +, site of deficient enzyme reaction.
a larger group of lysosomal disorders and exhibit several constant features: (1) complex lipids containing ceramide accumulate in cells, particularly neurons, causing neurodegeneration and shortening the life span. (2) The rate of synthesis of the stored lipid is normal. (3) The enzymatic defect is in the lysosomal degradation pathway of sphingolipids. (4) The extent to which the activity of the affected enzyme is decreased is similar in all tissues. There is no effective treatment for many of the diseases, although some success has been achieved with enzyme replacement therapy and bone marrow transplantation in the treatment of Gaucher and Fabry diseases. Other promising approaches are substrate deprivation therapy to inhibit the synthesis of sphingolipids and chemical chaperone therapy. Gene therapy for lysosomal disorders is also currently under investigation. Some examples of the more important lipid storage diseases are shown in Table 24-1. Multiple sulfatase deficiency results in accumulation of sulfogalactosylceramide, steroid sulfates, and proteoglycans owing to a combined deficiency of arylsulfatases A, B, and C and steroid sulfatase.

™ Triacylglycerols are the major energy-storing lipids, whereas phosphoglycerols, sphingomyelin, and glycosphingolipids are amphipathic and have structural functions in cell membranes as well as other specialized roles.
™ Triacylglycerols and some phosphoglycerols are synthesized by progressive acylation of glycerol-3-phosphate. The pathway
bifurcates at phosphatidate, forming inositol phospholipids and cardiolipin on the one hand and triacylglycerol and choline and ethanolamine phospholipids on the other.
@ Plasmalogens and PAF are ether phospholipids formed from dihydroxyacetone phosphate.
=
Sphingolipids
are
formed
from
ceramide
(N-acylsphingosine).
is
present
in
membranes
of
organelles
involved
in
secretory
processes
(eg,
Golgi
apparatus).
The
simplest
glycosphingolipids
are
a
combination
of
ceramide
plus
a
sugar
residue
(eg,
GalCer
in
myelin).
Gangliosides
are
more
complex
more
sugar
residues
plus
sialic
acid.
They
are
present
in
the
outer
layer
of
the
plasma
membrane,
where
they
contribute
to
the
glycocalyx
and
are
important
as
antigens
and
cell
= Phospholipids and sphingolipids are involved in several disease processes, including infant respiratory distress syndrome (lack of lung surfactant), multiple sclerosis (demyelination), and sphingolipidoses (inability to break down sphingolipids in lysosomes due to inherited defects in hydrolase enzymes).

2015.
Futerman AH: Sphingolipids. In Biochemistry of Lipids, Lipoproteins and
Membranes,
6th ed.
Ridgway
N,
McLeod
R
(editors).
Academic
Press,
2015:297-327.
Ridgway ND: Phospholipid synthesis in mammalian cells. In
Biochemistry of
Lipids,
Lipoproteins and
6th ed.
Academic Press, 2015:210-236.

- 2 classes they carry. Identify the four major groups of plasma lipoproteins and the four major lipid
Illustrate the structure of a lipoprotein particle.
classes. Indicate the major types of apolipoprotein found in the different lipoprotein
m= Explain that triacylglycerol from the diet is carried to the liver in chylomicrons and from the liver to extrahepatic tissues in very-low-density lipoprotein (VLDL), and that these particles are synthesized in intestinal and liver cells, respectively, by similar processes.
the liver. = Illustrate the processes by which chylomicrons are metabolized by lipases to form chylomicron remnants, which are then removed from the circulation by
= Explain how VLDL is metabolized by lipases to intermediate-density lipoprotein (IDL) which may be cleared by the liver or converted to low-density lipoprotein (LDL), which functions to deliver cholesterol from the liver to extrahepatic tissues via the LDL (apoB100, E) receptor.
= Explain how high-density lipoprotein (HDL) is synthesized, indicate the mechanisms by which it accepts cholesterol from extrahepatic tissues and returns it to the liver in reverse cholesterol transport.
= Describe how the liver plays a central role in lipid transport and metabolism
and how
hepatic
VLDL
secretion is
regulated by
the diet
and hormones.
= Indicate the roles of LDL and HDL in promoting and retarding, respectively, the development of atherosclerosis.
Indicate the causes of alcoholic and nonalcoholic fatty liver disease (NAFLD).
Explain the processes by which fatty acids are released from triacylglycerol stored in adipose tissue.
m= Understand the role of brown adipose tissue in the generation of body heat.

Fat absorbed from the diet and lipids synthesized by the liver and adipose tissue must be transported between the various tissues and organs for utilization and storage. Since lipids are insoluble in water, the problem of how to transport them in the aqueous blood plasma is solved by associating nonpolar lipids (triacylglycerol and cholesteryl esters) with amphipathic lipids (phospholipids and cholesterol) and proteins to make water-miscible lipoproteins.
In a meal-eating omnivore such as the human, excess cal- ories are ingested in the anabolic phase of the feeding cycle,
followed by a period of negative caloric balance when the organism draws on its carbohydrate and fat stores. Lipoproteins mediate this cycle by transporting lipids from the intestines as chylomicrons—and from the liver as very-low-density lipoproteins (VLDL)—to most tissues for oxidation and to adipose tissue for storage. Lipid is mobilized from adipose tissue as free fatty acids (FFAs) bound to serum albumin. Abnormalities of lipoprotein metabolism cause various hypoor hyperlipoproteinemias. The most common of these is in diabetes mellitus, where insulin deficiency causes excessive mobilization of FFA and underutilization of chylomicrons and VLDL, leading to hypertriacylglycerolemia. Most other pathologic conditions
affecting lipid transport are due primarily to inherited defects, some of which cause hypercholesterolemia and premature atherosclerosis (see Table 26-1). Obesity—particularly abdominal obesity—is a risk factor for increased mortality, hypertension, type 2 diabetes mellitus, hyperlipidemia, hyperglycemia, and various endocrine dysfunctions.

Plasma lipids consist of triacylglycerols (16%), phospholipids (30%), cholesterol (14%), and cholesteryl esters (36%) and a much smaller fraction of unesterified long-chain fatty acids (or FFAs) (4%). This latter fraction, the FFA, is metabolically the most active of the plasma lipids.

Since fat is less dense than water, the density of a lipopro- tein
decreases as
the proportion
of lipid
to protein
increases
(Table
25-1).
Four major
groups of
lipoproteins have
been identified that are important physiologically and in clinical diagnosis. These are (1) chylomicrons, derived from intestinal absorption of triacylglycerol and other lipids; (2) VLDL, derived from the liver for the export of triacylglycerol; (3) low-density lipoproteins (LDL), representing a final stage in the catabolism of VLDL; and (4) high-density lipoproteins, (HDL), involved in cholesterol transport and also in VLDL and chylomicron metabolism. Triacylglycerol is the predominant lipid in chylomicrons and VLDL, whereas cholesterol and phospholipid are the predominant lipids in LDL and HDL, respectively (Table 25-1). Lipoproteins may also be classified according to their electrophoretic properties into a(HDL), B(LDL), and pre-B (VLDL)-lipoproteins.

The nonpolar lipid core consists of mainly triacylglycerol and cholesteryl ester and is surrounded by a single surface layer of amphipathic phospholipid and cholesterol molecules (Figure 25-1). These are oriented so that their polar groups face outward to the aqueous medium, as in the cell membrane (see Chapters 21 and 40). The protein moiety of a lipoprotein is known as an apolipoprotein or apoprotein,

Composition.Lipid (%) = . Preβ-HDL c, Main Lipid Components = . Preβ-HDL c, Apolipoproteins = A-I. Albumin/free fatty acids, Source = Adipose tissue. Albumin/free fatty acids, Diameter (nm) = . Albumin/free fatty acids, Density (g/mL) = >1.281. Albumin/free fatty acids, Composition.Protein (%) = 99. Albumin/free fatty acids, Composition.Lipid (%) = 1. Albumin/free fatty acids, Main Lipid Components = Free fatty acids. Albumin/free fatty acids, Apolipoproteins = 
*Secreted with chylomicrons but transfers to HDL.
®Associated with HDL, and HDL, subfractions.
Abbreviations: HDL, high-density lipoproteins; IDL, intermediate-density lipoproteins; LDL, low-density lipoproteins; VLDL, very-low-density lipoproteins.
"Part of a minor fraction known as very-high-density lipoproteins (VHDL).
FIGURE 25-1 Generalized structure of a plasma lipoprotein. Small amounts of cholesteryl ester and triacylglycerol are found in the surface layer and a little free cholesterol in the core.
constituting nearly 70% of some HDL and as little as 1% of chylomicrons.

One or more apolipoproteins are present in each lipoprotein. They are usually abbreviated as apo followed by the letter A, B, C, etc (Table 25-1). Some apolipoproteins are integral and cannot be removed (eg, apo B), whereas others are bound to the surface and are free to transfer to other lipoproteins, eg, apos C and E). The major apolipoproteins of HDL (a-lipoprotein) are apo As (Table 25-1). The main apolipoprotein of LDL (6-lipoprotein) is apo B (B-100), which is found also in VLDL. Chylomicrons contain a truncated form (48% of apo B-100) of apo B (B-48) that is synthesized in the intestine, while B-100 is synthesized in the liver. Apo B-100 is one of the longest single polypeptide chains known, having 4536 amino acids and a molecular mass of 550,000 Da. To produce Apo B-48, a stop signal is introduced into the mRNA transcript for apo B-100 by an RNA editing enzyme. Apos C-I, C-II, and C-III are smaller polypeptides (molecular mass 7000-9000 Da) freely transferable between several different lipoproteins. Apo E, found in VLDL, HDL, chylomicrons, and chylomicron remnants, is also freely transferable; it accounts for 5 to 10% of total VLDL apolipoproteins in normal subjects.
Apolipoproteins carry out several roles: (1) they can form part of the structure of the lipoprotein, for example, apo B; (2) they are enzyme cofactors, for example, C-II for lipoprotein lipase, A-I for lecithin:cholesterol acyltransferase (LCAT), or enzyme inhibitors, for example, apo A-II and apo C-III for lipoprotein lipase, apo C-I for cholesteryl ester transfer protein; and (3) they act as ligands for interaction with lipoprotein receptors in tissues, for example, apo B-100 and apo E for the

LDL receptor, apo E for the LDL-receptor-related protein-1 (LRP-1), which recognizes remnant lipoproteins (see below), and apo A-I for the HDL receptor. Apo A-IV is thought to have a role in chylomicron metabolism and may also act as a regulator of satiety and glucose homeostasis, making it a potential therapeutic target for the treatment of diabetes and obesity, while apo D is believed to be an important factor in human neurodegenerative disorders.

The FFAs (also termed nonesterified fatty acids [NEFAs] or unesterified fatty acids) arise in the plasma from the breakdown of triacylglycerol in adipose tissue or as a result of the action of lipoprotein lipase on the plasma triacylglycerols. They are found in combination with albumin, a very effective solubilizer. Levels are low in the fully fed condition and rise to 0.7 to 0.8 mEq/mL in the starved state. In uncontrolled diabetes mellitus, the level may rise to as much as 2 mEq/mL. FFAs are removed from the blood extremely rapidly by the tissues and oxidized (fulfilling 25-50% of energy requirements in starvation) or esterified to form triacylglycerol. In starvation, esterified lipids from the circulation or in the tissues are also oxidized, particularly in heart and skeletal muscle cells, where considerable stores of lipid are found.
The FFA uptake by tissues is related directly to the plasmaFFA concentration, which in turn is determined by the rate of lipolysis in adipose tissue. After dissociation of the fatty acid-albumin complex at the plasma membrane, fatty acids bind to a membrane fatty acid transport protein that acts as a transmembrane cotransporter with Na*. On entering the cytosol, FFAs are bound by intracellular fatty acid-binding proteins. The role of these proteins in intracellular transport is thought to be similar to that of serum albumin in extracellular transport of long-chain fatty acids.

By definition, chylomicrons are found in chyle formed only by the lymphatic system draining the intestine. They are responsible for the transport of all dietary lipids into the circulation. Small quantities of VLDL are also to be found in chyle; however, most VLDL in the plasma are of hepatic origin. They are the vehicles of transport of triacylglycerol from the liver to the extrahepatic tissues.
There are striking similarities in the mechanisms of formation of chylomicrons by intestinal cells and of VLDL by hepatic parenchymal cells (Figure 25-2), perhaps because— apart from the mammary gland—the intestine and liver are
FIGURE 25-2 The formation and secretion of (A) chylomicrons by an intestinal cell and (B) very-low-density lipoproteins by a hepatic cell. (C, chylomicrons; E, endothelium; G, Golgi apparatus; N, nucleus; RER, rough endoplasmic reticulum; SD, space of Disse, containing blood plasma; SER, smooth endoplasmic reticulum; VLDL, very-low-density lipoprotein.) Apolipoprotein B, synthesized in the RER, is incorporated into particles with triacylglycerol, cholesterol, and phospholipids in the SER. After the addition of carbohydrate residues in G, they are released from the cell by reverse pinocytosis. Chylomicrons pass into the lymphatic system. VLDL are secreted into the space of Disse and then into the hepatic sinusoids through fenestrae in the endothelial lining.

the only tissues from which particulate lipid is secreted. Newly secreted or "nascent" chylomicrons and VLDL contain only a small amount of apolipoproteins C and E, and the full complement is acquired from HDL in the circulation (Figures 25-3 and 25-4). Apo B, however, is an integral part of the lipoprotein particles. It is incorporated into the particles during their assembly inside the cells and is essential for chylomicron and VLDL formation. In abetalipoproteinemia (a rare disease), lipoproteins containing apo B are not formed and lipid droplets accumulate in the intestine and liver.
_
|
_
_
_
FIGURE 25-3 Metabolic fate of chylomicrons. (A, apolipoprotein A; B-48, apolipoprotein B-48; C, apolipoprotein C; C, cholesterol and cholesteryl ester; E, apolipoprotein E; HDL, high-density lipoprotein; HL, hepatic lipase; LRP, LDL-receptor-related protein; PL, phospholipid; TG, triacylglycerol.) Only the predominant lipids are shown.
FIGURE 25-4 Metabolic fate of very-low-density lipoproteins (VLDL) and production of low-density lipoproteins (LDL). (A, apolipoprotein A; B-100, apolipoprotein B-100; C, apolipoprotein C; C, cholesterol and cholesteryl ester; E, apolipoprotein E; HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; PL, phospholipid; TG, triacylglycerol.) Only the predominant lipids are shown. It is possible that some IDL is also metabolized via the low-density lipoprotein receptor-related protein-1 (LRP-1.)

not react readily with chylomicrons or VLDL but is involved in chylomicron remnant and HDL metabolism (see below). cofactors
The clearance of chylomicrons from the blood is rapid, the half-time of disappearance being under 1 hour in humans. Larger particles are catabolized more quickly than smaller ones. Fatty acids originating from chylomicron triacylglycerol are delivered mainly to adipose tissue, heart, and muscle (80%), while ~20% goes to the liver. However, the liver does not metabolize native chylomicrons or VLDL significantly; thus, the fatty acids in the liver must be secondary to their metabolism in extrahepatic tissues.

Lipoprotein lipase is an enzyme located on the walls of blood capillaries, anchored to the endothelium by negatively charged proteoglycan chains of heparan sulfate. It has been found in heart, adipose tissue, spleen, lung, renal medulla, aorta, diaphragm, and lactating mammary gland, although it is not active in adult liver. It is not normally found in blood; however, following injection of heparin, lipoprotein lipase is released from its heparan sulfate-binding sites into the circulation. Hepatic lipase is bound to the sinusoidal surface of liver cells and is also released by heparin. This enzyme, however, does
Both phospholipids and apo C-II are required as for lipoprotein lipase activity, while apo A-II and apo C-III act as inhibitors. Hydrolysis takes place while the lipoproteins are attached to the enzyme on the endothelium. Triacylglycerol is hydrolyzed progressively through a diacylglycerol to a monoacylglycerol and finally to FFA plus glycerol. Some of the released FFA return to the circulation, attached to albumin, but the bulk is transported into the tissue (Figures 25-3 and 25-4). Heart lipoprotein lipase has a low K,, for triacylglycerol, about one-tenth of that for the enzyme in adipose tissue. This enables the delivery of fatty acids from triacylglycerol to be redirected from adipose tissue to the heart in the starved state when the plasma triacylglycerol decreases. A similar redirection to the mammary gland occurs during lactation, allowing uptake of lipoprotein triacylglycerol fatty acid for milk fat synthesis. The VLDL receptor plays an important part in the delivery of fatty acids from VLDL triacylglycerol to adipocytes by binding VLDL and bringing it into close contact with lipoprotein lipase. In adipose tissue, insulin enhances lipoprotein lipase synthesis in adipocytes and its translocation to the luminal surface of the capillary endothelium.

Reaction with lipoprotein lipase results in the loss of 70 to 90% of the triacylglycerol of chylomicrons and in the loss of apo C (which returns to HDL) but not apo E, which is retained. The resulting chylomicron remnant is about half the diameter of the parent chylomicron and is relatively
enriched in cholesterol and cholesteryl esters because of the loss of triacylglycerol (Figure 25-3). Similar changes occur to VLDL, with the formation of VLDL remnants (also called intermediate-density lipoprotein (IDL) (Figure 25-4).

Chylomicron remnants are taken up by the liver by receptormediated endocytosis, and the cholesteryl esters and triacylglycerols are hydrolyzed and metabolized. Uptake is mediated by apo E (Figure 25-3), via two apo E-dependent receptors, the LDL (apo B-100, E) receptor and LDL receptor-related protein-1 (LRP-1). Hepatic lipase has a dual role: (1) it acts as a ligand to facilitate remnant uptake and (2) it hydrolyzes remnant triacylglycerol and phospholipid.
After VLDL has been converted to IDL, the remnant particles may be taken up by the liver directly via the LDL (apo B-100, E) receptor, or they may be further metabolized to LDL in the circulation. Only one molecule of apo B-100 is present in each of these lipoprotein particles, and this is conserved during the transformations. Thus, each LDL particle is derived from a single precursor VLDL particle (Figure 25-4). In humans, a relatively large proportion of IDL forms LDL, accounting for the increased concentrations of LDL in humans compared with many other mammals.

The liver and many extrahepatic tissues express the LDL (apo B-100, E) receptor. It is so designated because it is specific for apo B-100 but not B-48, which lacks the carboxyl terminal domain of B-100 containing the LDL receptor ligand, and it also takes up lipoproteins rich in apo E. Approximately 30% of LDL is degraded in extrahepatic tissues and 70% in the liver. A positive correlation exists between the incidence of atherosclerosis and the plasma concentration of LDL cholesterol. The LDL (apoB-100, E) receptor is defective in familial hypercholesterolemia, a genetic condition in which blood LDL cholesterol levels are increased, causing premature atherosclerosis (Table 26-1). For further discussion of the regulation of the LDL receptor, see Chapter 26.

HDL is synthesized and secreted from both liver and intestine (Figure 25-5). However, apo C and apo E are synthesized in the liver and transferred from liver HDL to intestinal HDL when the latter enters the plasma. A major function of HDL is
FIGURE 25-5 Metabolism of high-density lipoprotein (HDL) in reverse cholesterol transport. (A-I, apolipoprotein A-l; ABCA1, ATPbinding cassette transporter A1; ABCG1, ATP-binding cassette transporter G1; C, cholesterol; CE, cholesteryl ester; LCAT, lecithin:cholesterol acyltransferase; PL, phospholipid; SR-B1, scavenger receptor B1.) Pre8-HDL, HDL,, HDL,—see Table 25-1. Surplus surface constituents from the action of lipoprotein lipase on chylomicrons and VLDL are another source of preB-HDL.
of atherosclerosis, possibly because they reflect the efficiency of reverse cholesterol transport. HDL, (HDL,) is found in the blood of diet-induced hypercholesterolemic animals. It is rich in cholesterol, and its sole apolipoprotein is apo E. It appears that all plasma lipoproteins are interrelated components of one or more metabolic cycles that together are responsible for the complex process of plasma lipid transport.

The liver carries out the following major functions in lipid metabolism:
1. Facilitation of the digestion and absorption of lipids by the production of bile (see Chapter 26).
2. Active synthesis and oxidation of fatty acids (see Chapters 22 and 23) and also synthesis of triacylglycerols and phospholipids (see Chapter 24).
3. Conversion of fatty acids to ketone bodies (ketogenesis) (see Chapter 22).
- 4. Synthesis and metabolism of plasma lipoproteins.

The cellular events involved in hepatic VLDL formation and secretion are shown in Figure 25-6. VLDL assembly requires the synthesis of apo B-100 and a source of triacylglycerol. Apo B-100 is synthesized on polyribosomes and translocated to the lumen of the endoplasmic reticulum (ER) as it is formed. As the protein enters the lumen, it is lipidated with phospholipid with the aid of the microsomal triacylglycerol transfer protein (MTP), which also facilitates the transfer of triacylglycerol across the ER membrane, and apo B-containing VLDL2 (or precursor VLDL) particles are formed. The triacylglycerol is derived from lipolysis of cytosolic triacylglycerol lipid droplets followed by reesterification in a pathway requiring phospholipid derivatives and diacylglycerol acyl transferases. Triacylglycerol not used for VLDL1 formation is recycled to the cytosolic droplets. After assembly in the ER, VLDL2 are carried in coat protein II (COPII) vesicles (see Chapter 49) to the golgi, where they fuse with triacylglycerol-rich lipid droplets to produce VLDLI. Phosphatidic acid produced by the action of phospholipase D after activation by a small GTP-binding protein called ADP-ribosylation factor-1 (ARF-1) is needed for the formation of the triacylglycerol—rich particles and/ or VLDL2. Although some VLDL2 particles may be secreted without fusion, most particles which leave the cell are in the form of VLDL1. These nascent VLDL then acquire apolipoproteins C and E from HDL in the circulation to become mature VLDL.
ApoC,
E
from
HDL

FIGURE 25-6 The assembly of very-low-density lipoprotein (VLDL) in the liver. (Apo, apolipoprotein; ARF-1, ADP-ribosylation factor-1; FFA, free fatty acids; HDL, high-density lipoproteins; MTP, microsomal triacylglycerol transfer protein; PA, phosphatidic acid; PL, phospholipid; PLD, phospholipase D; TG, triacylglycerol.) The pathways indicated underlie the events depicted in Figure 25-2. Apo B-100 is synthesized on polyribosomes and is lipidated with PL by MTP as it enters the ER lumen. Any excess is degraded in proteasomes. TG derived from lipolysis of cytosolic lipid droplets followed by resynthesis is transferred into the ER lumen with the aid of MTP and interacts with apo B-100 forming VLDL2. Excess TG is recycled to the cytosolic lipid droplets. VLDL2 are translocated to the golgi in COPII vesicles where they fuse with TG-rich particles to form VLDL1. PA is produced by activation of PLD by ARF-1 and is incorporated into the TG-rich VLDL1 and/or VLDL2. Both VLDL1 and VLDL2 may be secreted into the blood. Insulin inhibits VLDL secretion by inhibiting apo B-100 synthesis and the formation of VLDL1 from VLDL2.

Triacylglycerol for VLDL formation is synthesized from FFA. The fatty acids used are derived from two possible sources: (1) de novo synthesis within the liver from acetylCoA derived mainly from carbohydrate (perhaps not so important in humans) and (2) uptake of FFA from the circulation. The first source is predominant in the well-fed condition, when fatty acid synthesis is high and the level of circulating FFAs is low. As triacylglycerol does not normally accumulate in the liver in these conditions, it must be inferred that it is transported from the liver in VLDL as rapidly as it is synthesized. FFAs from the circulation are the main source during starvation, the feeding of high-fat diets, or in diabetes mellitus, when hepatic lipogenesis is inhibited. Factors that enhance both the synthesis of triacylglycerol and the secretion of VLDL by the liver include (1) the fed state rather than the starved state; (2) the feeding of diets high in carbohydrate (particularly if they contain sucrose or fructose), leading to high rates of lipogenesis and esterification of fatty acids; (3) high levels of circulating FFA; (4) ingestion of ethanol; and (5) the presence of high concentrations of insulin and low concentrations of glucagon, which enhance fatty acid synthesis and esterification and inhibit their oxidation.
Insulin suppresses hepatic VLDL secretion both by inhibiting apo B-100 synthesis and the conversion of the smaller VLDL2 into VLDLI by fusion with bulk triacylglycerol. Some other factors which are known to inhibit or prevent VLDL
assembly in the liver include the antibiotic brefeldin A, which inhibits the action of ARF-1; the sulfonylurea hypoglycemic drug, tolbutamide, dietary w3 fatty acids (see Chapter 21), and orotic acid, an intermediate in the synthesis of pyrimidines (see Chapter 33) decrease the rate of triacylglycerol lipolysis; and a defect in the MTP gene. The regulation of VLDL formation in the liver is complex and involves interactions between hormonal and dietary factors that are not yet fully understood.

For a variety of reasons, lipid—mainly as triacylglycerol—can accumulate in the liver (Figure 25-6). Extensive accumulation causes fatty liver and is regarded as a pathologic condition. Nonalcoholic fatty liver disease (NAFLD) is the most common liver disorder worldwide. When accumulation of lipid in the liver becomes chronic, inflammatory and fibrotic changes may develop leading to nonalcoholic steatohepatitis (NASH), which can progress to liver diseases including cirrhosis, hepatocarcinoma, and liver failure.
Fatty livers fall into two main categories. The first type is associated with raised levels of plasma-free fatty acids
resulting from mobilization of fat from adipose tissue or from the hydrolysis of lipoprotein triacylglycerol by lipoprotein lipase in extrahepatic tissues. The production of VLDL does not keep pace with the increasing influx and esterification of free fatty acids, allowing triacylglycerol to accumulate, which in turn causes a fatty liver. This occurs during starvation and the feeding of high-fat diets. The ability to secrete VLDL may also be impaired (eg, in starvation). In uncontrolled diabetes mellitus, twin lamb disease, and ketosis in cattle fatty infiltration is sufficiently severe to cause visible pallor (fatty appearance) and enlargement of the liver with possible liver dysfunction.
The second type of fatty liver is usually due to a metabolic block in the production of plasma lipoproteins, thus allowing triacylglycerol to accumulate. Theoretically, the lesion may be due to (1) a block in apolipoprotein synthesis (or an increase in its degradation before it can be incorporated into VLDL), (2) a block in the synthesis of the lipoprotein from lipid and apolipoprotein, (3) a failure in provision of phospholipids that are found in lipoproteins, or (4) a failure in the secretory mechanism itself.

One type of fatty liver that has been studied extensively in rats is caused by a deficiency of choline, which has therefore been called a lipotropic factor. Orotic acid also causes fatty liver; it is believed to interfere with glycosylation of VLDL, thus inhibiting release, and may also impair the recruitment of triacylglycerol to the particles. A deficiency of vitamin E enhances the hepatic necrosis of the choline deficiency type of fatty liver. Added vitamin E or a source of selenium has a protective effect by combating lipid peroxidation. In addition to protein deficiency, essential fatty acid and vitamin deficiencies (eg, linoleic acid, pyridoxine, and pantothenic acid) can cause fatty infiltration of the liver.

Alcoholic fatty liver is the first stage in alcoholic liver disease (ALD) which is caused by alcoholism and ultimately leads to cirrhosis. The fat accumulation in the liver is caused by a combination of impaired fatty acid oxidation and increased lipogenesis, which is thought to be due to changes in the [NADH]/[NAD*] redox potential in the liver, and also to interference with the action of transcription factors regulating the expression of the enzymes involved in the pathways. Oxidation of ethanol by alcohol dehydrogenase leads to excess production of NADH, which competes with reducing equivalents from other substrates, including fatty acids, for the respiratory chain. This inhibits their oxidation and causes increased esterification of fatty acids to form triacylglycerol, resulting in the fatty liver. Oxidation of ethanol leads to the formation of acetaldehyde, which is oxidized by aldehyde dehydrogenase, producing acetate. The increased (NADH)/(NAD') ratio also causes increased (lactate)/(pyruvate), resulting in hyperlacticacidemia, which decreases excretion of uric acid, aggravating gout.
Some metabolism of ethanol takes place via a cytochrome P450-dependent microsomal ethanol oxidizing system (MEOS) involving NADPH and O,. This system increases in activity in chronic alcoholism and may account for the increased metabolic clearance of ethanol in this condition, but may also promote the development of ALD. Ethanol also inhibits the metabolism of some drugs, for example, barbiturates, by competing for cytochrome P450-dependent enzymes. populations and alcohol
In some Asian Native Americans, consumption results in increased adverse reactions to acetaldehyde owing to a genetic defect of mitochondrial aldehyde dehydrogenase.

Triacylglycerols are stored in adipose tissue in large lipid droplets and are continually undergoing lipolysis (hydrolysis) and reesterification. These two processes are entirely different pathways involving different reactants and enzymes. This allows the processes of esterification or lipolysis to be regulated separately by many nutritional, metabolic, and hormonal factors. The balance between these two processes determines the magnitude of the FFA pool in adipose tissue, which in turn determines the level of FFA circulating in the plasma. Since the latter has most profound effects on the metabolism of other tissues, particularly liver and muscle, the factors operating in adipose tissue that regulate the outflow of FFA exert an influence far beyond the tissue itself. Moreover, since the discovery in the last 20 years that adipose tissue secretes hormones such as leptin and adiponectin, known as adipokines, its role as an endocrine organ has been recognized. Leptin regulates energy homeostasis by stimulating energy use and limiting food intake. If it is lacking, food intake may be uncontrolled, causing obesity. Adiponectin modulates glucose and lipid metabolism in muscle and liver, and enhances the sensitivity of tissues to insulin.
The Provision of Glycerol-3-Phosphate Regulates Esterification: Lipolysis Is Controlled by Hormone-Sensitive Lipase
Triacylglycerol is synthesized from acyl-CoA and glycerol3-phosphate (see Figure 24-2). Since the enzyme glycerol kinase is not expressed in adipose tissue, glycerol cannot be utilized for the provision of glycerol-3-phosphate, which must be supplied from glucose via glycolysis (Figure 25-7).
Triacylglycerol undergoes hydrolysis by a hormonesensitive lipase to form FFA and glycerol. This lipase is distinct

Triacylglycerol metabolism in adipose tissue. Hormone-sensitive lipase is activated by ACTH, TSH, glucagon, epinephrine, norepinephrine, and vasopressin and inhibited by insulin, prostaglandin E,, and nicotinic acid. Details of the formation of glycerol-3-phosphate from intermediates of glycolysis are shown in Figure 24-2. (FFA, free fatty acids; PPP, pentose phosphate pathway; 1G, triacylglycerol; VLDL, very-lowdensity lipoprotein.)
from lipoprotein lipase, which catalyzes lipoprotein triacylglycerol hydrolysis before its uptake into extrahepatic tissues (see above). Since the glycerol cannot be utilized, it enters the blood and is taken up and transported to tissues such as the liver and kidney, which possess an active glycerol kinase. The FFA formed by lipolysis can be reconverted in adipose tissue to acyl-CoA by acyl-CoA synthetase and reesterified with glycerol-3-phosphate to form triacylglycerol. Thus, there is a continuous cycle of lipolysis and reesterification within the tissue (Figure 25-7). However, when the rate of reesterification is not sufficient to match the rate of lipolysis, FFAs accumulate and diffuse into the plasma, where they bind to albumin and raise the concentration of plasma-free fatty acids.

When the utilization of glucose by adipose tissue is increased, the FFA outflow decreases. However, the release of glycerol continues, demonstrating that the effect of glucose is not mediated by reducing the rate of lipolysis. The effect is due to the provision of glycerol-3-phosphate, which enhances esterification of FFA. Glucose can take several pathways in adipose tissue, including oxidation to CO, via the citric acid cycle, oxidation in the pentose phosphate pathway, conversion to long-chain fatty acids, and formation of acylglycerol via glycerol-3-phosphate (Figure 25-7). When glucose utilization is high, a larger proportion of the uptake is oxidized to CO, and converted to fatty acids. However, as total glucose utilization decreases, the greater proportion of the glucose is directed to the formation of glycerol-3-phosphate for the esterification of acyl-CoA, which helps to minimize the efflux of FFA.

The rate of release of FFA from adipose tissue is affected by many hormones that influence either the rate of esterification or the rate of lipolysis. Insulin inhibits the release of FFA from adipose tissue, which results in a fall in circulating plasma-free fatty acids. Insulin also enhances lipogenesis and the synthesis of acylglycerol and increases the oxidation of glucose to CO, via the pentose phosphate pathway. All of these effects are dependent on the presence of glucose and can be explained, to a large extent, on the basis of the ability of insulin to enhance the uptake of glucose into adipose cells via the GLUT 4 transporter. In addition, insulin increases the activity of the enzymes pyruvate dehydrogenase, acetyl-CoA carboxylase, and glycerol phosphate acyltransferase, reinforcing the effects of increased glucose uptake on the enhancement of fatty acid and acylglycerol synthesis. These three enzymes are regulated in a coordinate manner by phosphorylation-dephosphorylation mechanisms (see Chapters 17, 23, and 24).
Another principal action of insulin in adipose tissue is to inhibit the activity of hormone-sensitive lipase, reducing the release not only of FFA but also of glycerol. Adipose tissue is much more sensitive to insulin than many other tissues, and is thus a major site of insulin action in vivo.

Other hormones accelerate the release of FFA from adipose tissue and raise the plasma-free fatty acid concentration by increasing the rate of lipolysis of the triacylglycerol stores (Figure 25-8). These include epinephrine, norepinephrine, glucagon, adrenocorticotropic hormone (ACTH), and -melanocyte-stimulating hormones (MSH), thyroid-stimulating hormone (TSH), growth hormone (GH), and vasopressin. Many of these activate hormonesensitive lipase. For an optimal effect, most of these lipolytic processes require the presence of glucocorticoids and thyroid hormones. These hormones act in a facilitatory or permissive capacity with respect to other lipolytic endocrine factors. The hormones that act rapidly in promoting lipolysis, that is, catecholamines (epinephrine and norepinephrine), do so by stimulating the activity of adenylyl cyclase, the enzyme that converts ATP to cAMP. The mechanism is analogous to that responsible for hormonal stimulation of glycogenolysis (see Chapter 18). cAMP, by stimulating cAMP-dependent protein kinase, activates hormonesensitive lipase. Thus, processes which destroy or preserve cAMP influence lipolysis. cAMP is degraded to 5'-AMP by a- the enzyme cyclic 3',5'-nucleotide phosphodiesterase. This enzyme is inhibited by methylxanthines such as caffeine and theophylline. Insulin antagonizes the effect of the lipolytic hormones. Lipolysis appears to be more sensitive to changes in concentration of insulin than are glucose utilization and esterification. The antilipolytic effects of insulin, nicotinic acid, and prostaglandin E, are accounted for by inhibition of the synthesis of cAMP at the adenylyl cyclase site, acting through aG, protein. Insulin also stimulates phosphodiesterase and the lipase phosphatase that inactivates hormone-sensitive lipase. The effect of growth hormone in promoting lipolysis is dependent on synthesis of proteins involved in the formation of cAMP. Glucocorticoids promote lipolysis via synthesis of new lipase protein by a cAMP-independent pathway, which may be inhibited by insulin, and also by promoting transcription of genes involved in the cAMP signal cascade. These findings help to explain the role of the pituitary gland and

the adrenal cortex in enhancing fat mobilization. The sympathetic nervous system, through liberation of norepinephrine in adipose tissue, plays a central role in the mobilization of FFA. Thus, the increased lipolysis caused by many of the factors described above can be reduced or abolished by denervation of adipose tissue or by ganglionic blockade.
FIGURE 25-8 Control of adipose tissue lipolysis. (ACTH, adrenocorticotropin hormone; FFA, free fatty acids; TSH, thyroid-stimulating hormone.) The cascade sequence of reactions enables amplification at each step. The lipolytic stimulus is "switched off" by removal of the stimulating hormone; the action of lipase phosphatase; the inhibition of the lipase and adenylyl cyclase by high concentrations of FFA; the inhibition of adenylyl cyclase by adenosine; and the removal of cAMP by the action of phosphodiesterase. ACTH, TSH, and glucagon may not activate adenylyl cyclase in vivo since the concentration of each hormone required in vitro is much higher than is found in the circulation. Positive (®) and negative (©) regulatory effects are represented by broken lines and substrate flow by solid lines.

Perilipin, a protein involved in the formation of lipid droplets in adipocytes, inhibits lipolysis in basal conditions by preventing access of the lipase enzymes to the stored triacylglycerols. On stimulation with hormones which promote triacylglycerol degradation, however, the protein becomes phosphorylated and changes its conformation, exposing the lipid droplet surface to hormone-sensitive lipase and thus promoting lipolysis. Perilipin, therefore, enables the storage and breakdown of triacylglycerol to be coordinated according to the metabolic needs of the body.

In adipose tissue, there is no significant incorporation of glucose or pyruvate into long-chain fatty acids, ATP-citrate lyase, a key enzyme in lipogenesis, does not appear to be present, and other lipogenic enzymes—for example, glucose-6-phosphate dehydrogenase and the malic enzyme—do not undergo adaptive changes. Indeed, it has been suggested that in humans there is a "carbohydrate excess syndrome" due to a unique limitation in the ability to dispose of excess carbohydrate by lipogenesis. In birds, lipogenesis is confined to the liver, where it is particularly important in providing lipids for egg formation, stimulated by estrogens.

Brown adipose tissue (BAT) is a specialized form of adipose tissue involved in metabolism, and in thermogenesis (heat generation). Thus, it is extremely active in some species, for example, during arousal from hibernation, in animals exposed to cold (nonshivering thermogenesis), and in heat production in the newborn. Though not a prominent tissue in humans, it is present in normal individuals. BAT is characterized by a welldeveloped blood supply and a high content of mitochondria and cytochromes, but low activity of ATP synthase. Metabolic emphasis is placed on oxidation of both glucose and fatty acids. Norepinephrine liberated from sympathetic nerve endings is important in increasing lipolysis in the tissue and increasing synthesis of lipoprotein lipase to enhance utilization of triacylglycerol-rich lipoproteins from the circulation. Oxidation and phosphorylation are not coupled in mitochondria of this tissue because of the presence of a thermogenic uncoupling protein, thermogenin (also called uncoupling protein 1 [UCP1]) and the phosphorylation that does occur is at the substrate level, for example, at the succinate thiokinase step and in glycolysis. Thus, oxidation produces much heat, and little free energy is trapped in ATP. Thermogenin acts as a proton conductance pathway dissipating the electrochemical potential across the mitochondrial membrane (Figure 25-9). Recent research has

Activity of the respiratory chain results in the translocation of protons from the mitochondrial matrix into the intermembrane space (see Figure 13-7). The presence of thermogenin (UCP1) in brown adipose tissue enables the protons to flow back into the matrix without passing through the F, ATP synthase and the energy generated is dissipated as heat instead of being captured as ATP. The passage of H* via thermogenin is inhibited by purine nucleotides when brown adipose tissue is unstimulated. Under the influence of norepinephrine, the inhibition is removed by the production of free fatty acids (FFA) and acyl-CoA. Note the dual role of acyl-CoA in both facilitating the action of thermogenin and supplying reducing equivalents for the respiratory chain. @ and © signify positive or negative regulatory effects.
shown that BAT activity is inversely related to body fat content, and thus is a potential target for the treatment of obesity and related metabolic disorders.

™ Since nonpolar lipids are insoluble in water, for transport between the tissues in the aqueous blood plasma, they are combined with amphipathic lipids and proteins to make watermiscible lipoproteins.
- Four major groups of lipoproteins are recognized.
Chylomicrons transport
lipids resulting
from digestion
and absorption.
VLDLs transport
triacylglycerol from
the liver.
- LDLs deliver cholesterol to the tissues, and HDLs remove
cholesterol from the tissues and return it to the liver for excretion in the process known as reverse cholesterol transport.
- Chylomicrons and VLDL are metabolized by hydrolysis of their triacylglycerol, leaving lipoprotein remnants in the circulation. These are taken up by liver, but some of the remnants (IDL)
VLDL
form
LDL
are taken
up by
the liver
and other
tissues via
the
LDL
receptor.
Apolipoproteins constitute the protein moiety of lipoproteins. They act as enzyme activators (eg, apo C-II and apo A-I) or as ligands for cell receptors (eg, apo A-I, apo E, and apo B-100).
Triacylglycerol is the main storage lipid in adipose tissue. Upon mobilization, FFA and glycerol are released. FFAs are an important fuel source.
= Brown adipose tissue is the site of "nonshivering
thermogenesis.”
It is
found in
hibernating and
newborn animals
and is
present in
adult humans.
Thermogenesis results from the presence of UCP1, thermogenin, in the inner
mitochondrial membrane.

2015.
- Francis G: High density lipoproteins: metabolism and protective
roles against atherosclerosis. In Biochemistry of Lipids,
Lipoproteins and Membranes, 6th ed. Ridgway N, McLeod R
(editors). Academic Press, 2015:437-459.
- McLeod RS, Yao Z: Assembly and secretion of triacylglycerol-
rich lipoproteins.
In
Lipids,
6th ed.
Ridgway
Press,
2015:460-488.
N,
McLeod
R
Academic

2 development. Explain the importance of cholesterol as an essential structural component of cell membranes and as a precursor of all other steroids in the body, and indicate its pathologic role in cholesterol gallstone disease and atherosclerosis
Identify the five stages in the biosynthesis of cholesterol from acetyl-CoA.
Indicate the role of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) in controlling the rate of cholesterol synthesis and explain the mechanisms by which its activity is regulated.
= Explain that cholesterol balance in cells is tightly regulated and indicate the factors involved in maintaining the correct balance.
m=
Explain the role of plasma lipoproteins, including chylomicrons, very-lowdensity lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL), in the transport of cholesterol between tissues in the plasma.
m= Name the two main primary bile acids found in mammals, outline the pathways by which they are synthesized from cholesterol in the liver.
m= Explain the importance of bile acid synthesis not only in the digestion and absorption of fats but also as a major excretory route for cholesterol.
= Indicate how secondary bile acids are produced from primary bile acids by intestinal bacteria.
= Explain what is meant by the "enterohepatic circulation" and why it is
important.
= Identify various factors related to plasma cholesterol concentrations that affect the risk of coronary heart disease, including diet and lifestyle and the class of lipoprotein in which it is carried.
™ Give examples of inherited and noninherited conditions affecting lipoprotein metabolism that cause hypoor hyperlipoproteinemia.

Cholesterol is present in tissues and in plasma either as free cholesterol or combined with a long-chain fatty acid as cholesteryl ester, the storage form. In plasma, both forms are transported in lipoproteins (see Chapter 25). Cholesterol is an amphipathic lipid and as such is an essential structural component of membranes, where it is important for the maintenance of the correct permeability and fluidity, and of the outer layer of plasma lipoproteins. It is synthesized in many tissues from acetyl-CoA and is the precursor of all other steroids in the body, including corticosteroids, sex hormones, bile acids, and vitamin D. As a typical product of animal metabolism, cholesterol occurs in foods of animal origin such as egg yolk, meat, liver, and brain. Plasma low-density lipoprotein (LDL) is the vehicle that supplies cholesterol and cholesteryl ester to many tissues. Free cholesterol is removed from tissues by plasma high-density lipoprotein (HDL) and transported to the liver, where it is eliminated from the body either unchanged or after conversion to bile acids in the process
known as reverse cholesterol transport (see Chapter 25). Cholesterol is a major constituent of gallstones. However, its chief role in pathologic processes is as a factor in the development of atherosclerosis of vital arteries, causing cerebrovascular, coronary, and peripheral vascular disease.

A little more than half the cholesterol of the body arises by synthesis (about 700 mg/d), and the remainder is provided by the average diet. The liver and intestine account for approximately 10% each of total synthesis in humans. Virtually all tissues containing nucleated cells are capable of cholesterol synthesis, which occurs in the endoplasmic reticulum and the cytosolic compartments.

Cholesterol is a 27-carbon compound consisting of four rings anda side chain (see Figure 21-19). It is synthesized from acetylCoA by a lengthy pathway that may be divided into five steps: (1) synthesis of mevalonate from acetyl-CoA (Figure 26-1);
FIGURE 26-1 Biosynthesis of mevalonate. HMG-CoA reductase is inhibited by statins. The open and solid circles indicate the fate of each of the carbons in the acetyl moiety of acetyl-CoA.
(2) formation of isoprenoid units from mevalonate by loss of CO, (Figure 26-2); (3) condensation of six isoprenoid units form squalene (Figure 26-2); (4) cyclization of squalene gives rise to the parent steroid, lanosterol; (5) formation of cholesterol from lanosterol (Figure 26-3).
Step 1—Biosynthesis of Mevalonate: HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) is formed by the reactions used in mitochondria to synthesize ketone bodies (see Figure 22-7). However, since cholesterol synthesis is extramitochondrial, the two pathways are distinct. Initially, two molecules of acetyl-CoA condense to form acetoacetyl-CoA catalyzed by cytosolic thiolase. Acetoacetyl-CoA condenses with a further molecule of acetyl-CoA catalyzed by HMGCoA synthase to form HMG-CoA, which is reduced to mevalonate by NADPH in a reaction catalyzed by HMG-CoA reductase. This last step is the principal regulatory step in the pathway of cholesterol synthesis and is the site of action of the most effective class of cholesterol-lowering drugs, the statins, which are HMG-CoA reductase inhibitors (Figure 26-1).
Step 2—Formation of Isoprenoid Units: Mevalonate is phosphorylated sequentially using ATP by three kinases, and after decarboxylation (Figure 26-2) of the active isoprenoid unit, isopentenyl diphosphate, is formed.

Step 3—Six Isoprenoid Units Form Squalene: Isopentenyl diphosphate is isomerized by a shift of the double bond to form dimethylallyl diphosphate, and then condensed with another molecule of isopentenyl diphosphate to form the 10-carbon intermediate geranyl diphosphate (Figure 26-2). A further condensation with isopentenyl diphosphate forms farnesyl diphosphate. Two molecules of farnesyl diphosphate condense at the diphosphate end to form squalene. Initially, inorganic pyrophosphate is eliminated, forming presqualene diphosphate, which is then reduced by NADPH with elimination of a further inorganic pyrophosphate molecule.
Step 4—Formation of Lanosterol: Squalene can fold into a structure that closely resembles the steroid nucleus (Figure 26-3). Before ring closure occurs, squalene is converted to squalene 2,3-epoxide by a mixed-function oxidase in the endoplasmic reticulum, squalene epoxidase. The methyl group on C.,, is transferred to C,, and that on C, to C, , as cyclization occurs, catalyzed by oxidosqualene-lanosterol cyclase.
Step 5—Formation of Cholesterol: The formation of cholesterol from lanosterol takes place in the membranes of the endoplasmic reticulum and involves changes in the steroid nucleus and the side chain (Figure 26-3). The methyl groups on C,, and C, are removed to form 14-desmethyl lanosterol and then zymosterol. The double bond at C,—C, is subsequently moved to C,—C, in two steps, forming desmosterol. Finally, the double bond of the side chain is reduced, producing cholesterol.

The polyisoprenoids, dolichol (see Figure 21-22 and Chapter 46) and ubiquinone (see Figure 13-6) are formed from farnesyl
ADP
shunt
FIGURE 26-2 Biosynthesis of squalene, ubiquinone, dolichol, and other polyisoprene derivatives. (HMG, 3-hydroxy-3-methylglutaryl.) A farnesyl residue is present in heme of a cytochrome oxidase. The carbon marked with an asterisk becomes C,, or C,, in squalene. Squalene synthetase is a microsomal enzyme; all other enzymes indicated are soluble cytosolic proteins, and some are found in peroxisomes.
diphosphate by the further addition of up to 16 (dolichol) or 3 to 7 (ubiquinone) isopentenyl diphosphate residues (Figure 26-2). Some GTP-binding proteins in the cell membrane are prenylated with farnesyl or geranylgeranyl
(20 carbon) residues. Protein prenylation is believed to facilitate the anchoring of proteins into lipoid membranes and may also be involved in protein-protein interactions and membrane-associated protein trafficking.
252
SECTIONV
Lipids
Triparanol
FIGURE 26-3 Biosynthesis of cholesterol. The numbered positions are those of the steroid nucleus and the open and solid circles indicate the fate of each of the carbons in the acetyl moiety of acetyl-CoA. ("Refer to labeling of squalene in Figure 26-2.)

Cholesterol synthesis is tightly controlled by regulation at the HMG-CoA reductase step. The activity of the enzyme is inhibited by mevalonate, the immediate product of the reaction, and by cholesterol, the main product of the pathway. Thus, increased intake of cholesterol from the diet leads to a decrease in de novo synthesis, especially in the liver. Regulatory mechanisms include both modulation of the synthesis of enzyme protein and posttranslational modification. Cholesterol and metabolites repress transcription HMG-CoA reductase mRNA via inhibition of a sterol regulatory element-binding protein (SREBP) transcription factor. SREBPs are a family of proteins that regulate the transcription of a range of genes involved in the cellular uptake and metabolism of cholesterol and other lipids. SREBP activation is inhibited by insulininduced gene (Insig) , a protein whose expression, as it is name indicates, is induced by insulin and is present in the endoplasmic reticulum. Insig also promotes degradation of HMG-CoA reductase. A diurnal variation occurs both in cholesterol synthesis and reductase activity. Short-term changes in enzyme activity, however, are brought about by posttranslational modification (Figure 26-4). Insulin or thyroid hormone increases HMG-CoA reductase activity, whereas glucagon or glucocorticoids decrease it. Activity is reversibly modified by phosphorylation-dephosphorylation mechanisms, some
FIGURE 26-4 Possible posttranslational mechanisms in the regulation of cholesterol synthesis by HMG-CoA reductase. Insulin has a dominant role compared with glucagon. (AMPK, AMP-activated protein kinase; AMPKK, AMP-activated protein kinase kinase.) "See Figure 18-6.
of which may be cAMP-dependent and therefore immediately responsive to glucagon. AMP-activated protein kinase (AMPK) (formerly called HM-CoA reductase kinase) phosphorylates and inactivates HMG-CoA reductase. AMPK is activated via phosphorylation by AMPK kinase (AMPKK) and allosteric modification by AMP.

In tissues, cholesterol balance is regulated as follows (Figure 26-5): An increase in cell cholesterol is caused by uptake of cholesterol-containing lipoproteins by receptors, for example, the LDL receptor or scavenger receptors such as CD36, uptake of free cholesterol from cholesterol-rich lipoproteins to the cell membrane, cholesterol synthesis, and hydrolysis of cholesteryl esters by the enzyme cholesteryl ester hydrolase. A decrease is due to efflux of cholesterol from the membrane to HDL via the ABCA1, ABCGI, or SR-B1 (see Figure 25-5); esterification of cholesterol by ACAT (acyl-CoA:cholesterol acyltransferase); and utilization of cholesterol for synthesis of other steroids, such as hormones, or bile acids in the liver.

LDL (apo B-100, E) receptors occur on the cell surface in pits that are coated on the cytosolic side of the cell membrane with a protein called clathrin. The glycoprotein receptor spans the membrane, the B-100 binding region being at the exposed amino terminal end. After binding, LDL is taken up intact by endocytosis. The apoprotein and cholesteryl ester are then hydrolyzed in the lysosomes, and cholesterol is translocated into the cell. The receptors are recycled to the cell surface. This influx of cholesterol inhibits the transcription of the genes encoding HMG-CoA synthase, HMG-CoA reductase, and other enzymes involved in cholesterol synthesis, as well as the LDL receptor itself, via the SREBP pathway, and thus coordinately suppresses cholesterol synthesis and uptake. ACAT activity is also stimulated, promoting cholesterol esterification. In addition, recent research has shown that the protein proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates the recycling of the receptor to the cell surface by targeting it for degradation. By these mechanisms, LDL receptor activity on the cell surface is regulated by the cholesterol requirement for membranes, steroid hormones, or bile acid synthesis, and the free cholesterol content of the cell is kept within relatively narrow limits (Figure 26-5).

Cholesterol is transported in plasma in lipoproteins, with the greater part in the form of cholesteryl ester (Figure 26-6), and in humans the highest proportion is found in LDL.
FIGURE 26-5 Factors affecting cholesterol balance at the cellular level. Reverse cholesterol transport may be mediated via the ABCA1 transporter protein (with preB-HDL as the exogenous acceptor) or the SR-B1 or ABCG1 (with HDL, as the exogenous acceptor). (ACAT, acyl-CoA:cholesterol acyltransferase; A-I, apolipoprotein A-I; C, cholesterol; CE, cholesteryl ester; LCAT, lecithin:cholesterol acyltransferase; LDL, low-density lipoprotein; PL, phospholipid; VLDL, very-low-density lipoprotein.) LDL and HDL are not shown to scale.
Dietary cholesterol equilibrates with plasma cholesterol in days and with tissue cholesterol in weeks. Cholesteryl ester in the diet is hydrolyzed to cholesterol, which is then absorbed by the intestine together with dietary unesterified cholesterol and other lipids. With cholesterol synthesized in the intestines, it is then incorporated into chylomicrons (see Chapter 25). Of the cholesterol absorbed, 80 to 90% is esterified with long-chain fatty acids in the intestinal mucosa. Ninety-five percent of the chylomicron cholesterol is delivered to the liver in chylomicron remnants, and most of the cholesterol secreted by the liver in very-low-density lipoprotein (VLDL) is retained during the formation of intermediate-density lipoprotein (IDL) and ultimately LDL, which is taken up by the LDL receptor in liver and extrahepatic tissues (see Chapter 25).

Lecithin: cholesterol acyltransferase (LCAT) activity is associated with HDL containing apo A-I. As cholesterol in HDL becomes esterified, it creates a concentration gradient and draws in cholesterol from tissues and from other lipoproteins (Figures 26-5 and 26-6), thus enabling HDL to function in reverse cholesterol transport (see Figure 25-5).

Cholesteryl ester transfer protein, associated with HDL, is found in plasma of humans and many other species. It facilitates transfer of cholesteryl ester from HDL to VLDL, IDL, and LDL in exchange for triacylglycerol, relieving product inhibition of the LCAT activity in HDL. Thus, in humans, much of the cholesteryl ester formed by LCAT finds its way to the liver via VLDL remnants (IDL) or LDL (Figure 26-6). The triacylglycerol-enriched HDL, delivers its cholesterol to the liver in the HDL cycle (see Figure 25-5).

Cholesterol is excreted from the body via the bile either in the unesterified form or after conversion into bile acids in the liver. Coprostanol is the principal sterol in the feces; it is formed from cholesterol by the bacteria in the lower intestine.

FIGURE 26-6 Transport of cholesterol between the tissues in humans. (ACAT, acyl-CoA:cholesterol acyltransferase; A-I, apolipoprotein A-I; C, unesterified cholesterol; CE, cholesteryl ester; CETP, cholesteryl ester transfer protein; HDL, high-density lipoprotein; HL, hepatic lipase; IDL, intermediate-density lipoprotein; LCAT, lecithin:cholesterol acyltransferase; LDL, low-density lipoprotein; LPL, lipoprotein lipase; LRP, LDL receptor-related protein-1; TG, triacylglycerol; VLDL, very-low-density lipoprotein.)

The primary bile acids are synthesized in the liver from cholesterol. These are cholic acid (found in the largest amount in most mammals) and chenodeoxycholic acid (Figure 26-7). The 7a-hydroxylation of cholesterol is the first and principal regulatory step in the biosynthesis of bile acids and is catalyzed by cholesterol 7a-hydroxylase, a microsomal cytochrome P450 enzyme-designated CYP7A1 (see Chapter 12). A typical monooxygenase, it requires oxygen, NADPH, and cytochrome P450. Subsequent hydroxylation steps are also catalyzed by monooxygenases. The pathway of bile acid biosynthesis divides early into one subpathway leading to cholylCoA, characterized by an extra a-OH group on position 12, and another pathway leading to chenodeoxycholyl-CoA (Figure 26-7). A second pathway in mitochondria involving the 27-hydroxylation of cholesterol by the cytochrome P450 sterol 27-hydroxylase (CYP27A1) as the first step is responsible for a significant proportion of the primary bile acids synthesized. The primary bile acids (Figure 26-7) enter the bile as glycine or taurine conjugates. Conjugation takes place in liver peroxisomes. In humans, the ratio of the glycine to the taurine conjugates is normally 3:1. In the alkaline bile (pH 7.6-8.4), the bile acids and their conjugates are assumed to be in a salt form—hence the term "bile salts." Primary bile acids are further metabolized in the intestine by the activity of the intestinal bacteria. Thus, deconjugation and 7a-dehydroxylation occur, producing the secondary bile acids, deoxycholic acid, and lithocholic acid.

Although products of fat digestion, including cholesterol, are absorbed in the first 100 cm of small intestine, the primary and secondary bile acids are absorbed almost exclusively in the ileum, and 98 to 99% is returned to the liver via the portal circulation. This is known as the enterohepatic circulation (Figure 26-6). However, lithocholic acid, because of its insolubility, is not reabsorbed to any significant extent. Only a small fraction of the bile salts escapes absorption and is therefore eliminated in the feces. Nonetheless, this represents a major
256
FIGURE 26-7 Biosynthesis and degradation of bile acids. A second pathway in mitochondria involves hydroxylation of cholesterol by sterol 27-hydroxylase. "Catalyzed by microbial enzymes.
pathway for the elimination of cholesterol. Each day the pool of bile acids (about 3-5 g) is cycled through the intestine 6 to 10 times and an amount of bile acid equivalent to that lost in the feces is synthesized from cholesterol, so that a pool of bile acids of constant size is maintained. This is accomplished by a system of feedback controls.
CYP7AI gene is suppressed. Chenodeoxycholic acid is particularly important in activating FXR. CYP7A1 activity is also enhanced by cholesterol of endogenous and dietary origin and regulated by insulin, glucagon, glucocorticoids, and thyroid hormone.

The principal rate-limiting step in the biosynthesis of bile acids is at the CYP7A1 reaction (Figure 26-7). The activity of the enzyme is feedback regulated via the nuclear bile acid-binding receptor, farnesoid X receptor (FXR). When the size of the bile acid pool in the enterohepatic circulation increases, FXR is activated, and transcription of the

Atherosclerosis is an inflammatory disease characterized by the deposition of cholesterol and cholesteryl ester from
the plasma lipoproteins into the artery wall and is a major
cause of heart disease. Elevated plasma cholesterol levels (> 5.2 mmol/L) are one of the most important factors in promoting atherosclerosis, but it is now recognized that elevated blood triacylglycerol is also an independent risk factor. Diseases in which there is a prolonged elevation of levels of VLDL, IDL, chylomicron remnants, or LDL in the blood (eg, diabetes mellitus, lipid nephrosis, hypothyroidism, and other conditions of hyperlipidemia) are often accompanied by premature or more severe atherosclerosis. There is also an inverse relationship between HDL (HDL,) concentrations and coronary heart disease, making the LDL:HDL cholesterol ratio a good predictive parameter. This is consistent with the function of HDL in reverse cholesterol transport. Susceptibility to atherosclerosis varies widely among species, and humans are one of the few in which the disease can be induced by diets high in cholesterol.

Hereditary factors play the most important role in determining the serum cholesterol concentrations of individuals; however, dietary and environmental factors also play a part, and the most beneficial of these is the substitution in the diet of polyunsaturated and monounsaturated fatty acids for saturated fatty acids. Plant oils such as corn oil and sunflower seed oil contain a high proportion of w6 polyunsaturated fatty acids, while olive oil contains a high concentration of monounsaturated fatty acids. w3 fatty acids found in fish oils are also beneficial (see Chapter 21). On the other hand, butter fat, beef fat, and palm oil contain a high proportion of saturated fatty acids. Sucrose and fructose have a greater effect in raising blood lipids, particularly triacylglycerols, than do other carbohydrates.
One of the mechanisms by which unsaturated fatty acids lower blood cholesterol levels is by the upregulation of LDL receptors on the cell surface, causing an increase in the catabolic rate of LDL, the main atherogenic lipoprotein. In addition, w3 fatty acids have anti-inflammatory and triacylglycerol-lowering effects. Saturated fatty acids also cause the formation of smaller VLDL particles that contain relatively more cholesterol, and they are utilized by extrahepatic tissues at a slower rate than are larger particles—tendencies that may be regarded as atherogenic.

Additional factors considered to play a part in coronary heart disease include high blood pressure, smoking, male gender, obesity (particularly abdominal obesity), lack of exercise, and drinking soft as opposed to hard water. Factors associated with elevation of plasma-free fatty acids (FFAs) followed by increased output of triacylglycerol and cholesterol into the circulation in VLDL include emotional stress and coffee drinking. Premenopausal women appear to be protected against many of these deleterious factors, and this is thought to be related to the beneficial effects of estrogen. There is an association between moderate alcohol consumption and a lower incidence of coronary heart disease. This may be due to elevation of HDL concentrations resulting from increased synthesis of apo A-I and changes in activity of cholesteryl ester transfer protein. It has been claimed that red wine is particularly beneficial, perhaps because of its content of antioxidants. Regular exercise lowers plasma LDL but raises HDL. Triacylglycerol concentrations are also reduced, due most likely to increased insulin sensitivity, which enhances the expression of lipoprotein lipase.

A family of drugs known as statins have proved highly efficacious in lowering plasma cholesterol and preventing heart disease. Statins act by inhibiting HMG-CoA reductase and upregulating LDL receptor activity. Examples currently in use include atorvastatin, simvastatin, fluvastatin, and pravastatin. Ezetimibe reduces blood cholesterol levels by inhibiting the absorption of cholesterol by the intestine by blocking uptake via the Niemann-Pick C-like 1 protein. Other drugs used include fibrates such as clofibrate, gemfibrozil, and nicotinic acid, which act mainly to lower plasma triacylglycerols by decreasing the secretion of triacylglycerol and cholesterolcontaining VLDL by the liver. Since PCSK9 reduces the number of LDL receptors exposed on the cell membrane, it has the effect of raising blood cholesterol levels, thus drugs that inhibit its activity are potentially antiatherogenic and two such compounds have recently been approved for use and others are currently in clinical trials.

Inherited defects in lipoprotein metabolism lead to the primary condition of either hypoor hyperlipoproteinemia (Table 26-1). For example, familial hypercholesterolemia (FH), causes severe hypercholesterolemia and is also associated with premature atherosclerosis. The defect is most often in the gene for the LDL receptor, so that LDL is not cleared from the blood. In addition, diseases such as diabetes mellitus, hypothyroidism, kidney disease (nephrotic syndrome), and atherosclerosis are associated with secondary abnormal lipoprotein patterns that are very similar to one or another of the primary inherited conditions. Virtually all of the primary conditions are due to a defect at a stage in lipoprotein formation, transport, or degradation (see Figures 25-4, 26-5, and 26-6). Not all of the abnormalities are harmful.

andVLDL remnants of density <1.019 (β-VLDL). Causes hypercholesterolemia, xanthomas, and atherosclerosis.. Familial hypertriacylglycerolemia (type IV), Defect = Overproduction of VLDL often associated with glucose intolerance and hyperinsulinemia.. Familial hypertriacylglycerolemia (type IV), Remarks = Cholesterol levels rise with the VLDL concentration. LDL and HDL tend to be subnormal. This type of pattern is commonly associated with coronary heart disease, type 2 diabetes mellitus, obesity, alcoholism, and administration of progestational hormones.. Familial hyperalphalipoproteinemia, Defect = Increased concentrations of HDL.. Familial hyperalphalipoproteinemia, Remarks = A rare condition apparently beneficial to health and longevity.. Hepatic lipase deficiency, Defect = Deficiency of the enzyme leads to accumulation of large triacylglycerol-rich HDL andVLDL remnants.. Hepatic lipase deficiency, Remarks = Patients have xanthomas and coronary heart disease.. Familial lecithin:cholesterol acyltransferase (LCAT) deficiency, Defect = Absence of LCAT leads to block in reverse cholesterol transport. HDL remains as nascent disks incapable of taking up and esterifying cholesterol.. Familial lecithin:cholesterol acyltransferase (LCAT) deficiency, Remarks = Plasma concentrations of cholesteryl esters and lysolecithin are low. Present in an abnormal LDL fraction, lipoprotein X, found also in patients with cholestasis. VLDL is abnormal (β-VLDL).. Familial lipoprotein(a) excess, Defect = Lp(a) consists of 1 mol of LDL attached to 1 mol of apo(a). Apo(a) shows structural homologies to plasminogen.. Familial lipoprotein(a) excess, Remarks = Premature coronary heart disease due to atherosclerosis, plus thrombosis due to inhibition of fibrinolysis.
"There is an association between patients possessing the apo E4 allele and the incidence of Alzheimer disease. Apparently, apo E4 binds more avidly to B-amyloid found in neuritic
plaques.

Abbreviations: LDL, low-density lipoprotein; LPL, lipoprotein lipase; HDL, high-density lipoprotein; VLDL, very-low-density lipoprotein.

™ Cholesterol is the precursor of all other steroids in the body, for example, corticosteroids, sex hormones, bile acids, and vitamin D. It also plays an important structural role in membranes and in the outer layer of lipoproteins.
Cholesterol is synthesized in the body entirely from acetyl-
CoA.
Three molecules
of acetyl-CoA
form mevalonate
via the
important regulatory
HMG-CoA
reaction reductase.
formed, and
undergoes for
the
Next, pathway,
a
six of
these cyclization
which, after
five-carbon condense
to the
loss forms
of cholesterol.
catalyzed isoprenoid
to form
form the
three parent
methyl by
unit is
squalene.
Squalene steroid
lanosterol, groups
and other
changes,
=
Cholesterol synthesis
in the
liver cholesterol
maintained in
the diet.
between
(eg, synthesis,
the
In the
uptake factors
causing cholesteryl
receptor tissues,
factors via
is regulated
cholesterol causing
the partly
by balance
gain
LDL
is of
or loss
of cholesterol
scavenger cholesterol
ester formation,
is modulated
this balance.
cholesterol receptors)
(eg, and
steroid excretion).
by synthesis,
The activity
cellular
In reverse
from it
in the
core taken
up
IDL, or
cholesterol cholesterol
of the
by the
of the
LDL
levels to
transport, and
LCAT
particles.
liver,
LDL
via
HDL
esterifies
The cholesteryl
either directly
the
Excess cholesteryl
is or
bile achieve
takes it
up and
deposits ester
in or
after
HDL
transfer ester
excreted from
salts.
A
large transfer
the is
to
VLDL, protein.
liver in
proportion of
the bile
bile salts
as is

absorbed into the portal circulation and returned to the liver as part of the enterohepatic circulation.
Elevated levels of cholesterol present in VLDL, IDL, or LDL are associated with atherosclerosis, whereas high levels of HDL have a protective effect.
Inherited defects in lipoprotein metabolism lead to a
primary condition of hypoor hyperlipoproteinemia.
Conditions such
as diabetes
mellitus, hypothyroidism,
kidney disease, and atherosclerosis exhibit secondary abnormal lipoprotein patterns that resemble certain primary

- . Carnitine is needed for fatty acid oxidation BECAUSE:
1. Which one of the following statements concerning fatty acid
molecules is CORRECT?
A. They consist of a carboxylic acid head group attached to a
B. They are called polyunsaturated when they contain one or more carbon-carbon double bonds.
BOOP
pS Sphingomyelin
carbohydrate chain.
Their melting points increase with increasing unsaturation.
They almost always have their double bonds in the cis
configuration when they occur naturally.
They occur in the body mainly in the form of free
(nonesterified) fatty acids.
. Which one of the following is NOT a phospholipid?
Plasmalogen
Cardiolipin
Galactosylceramide
Lysolecithin
. Which one of the following statements about gangliosides is
A. They are derived from galactosylceramide.
B. They contain one or more molecules of sialic acid.
C.
D. . The ganglioside GM1 is the receptor for cholera toxin in the human intestine.
E. They function in cell-cell recognition.
BOOP
They are present in nervous tissue in high concentrations.
. Which one of the following is a chain-breaking antioxidant?
Glutathione peroxidase
Selenium
EDTA
Catalase
. After they are produced from acetyl-CoA in the liver, ketone
bodies are mainly used for which one of the following processes?
BOOP
Excretion as waste products
Energy generation in the liver
Conversion to fatty acids for storage of energy
Generation of energy in the tissues
Generation of energy in red blood cells
. The subcellular site of the breakdown of long-chain fatty acids to
acetyl-CoA via B-oxidation is:
BOOS
>
The
cytosol
The matrix of the mitochondria
The endoplasmic reticulum
The mitochondrial intermembrane space
The Golgi apparatus
9. Malonyl-CoA, the first intermediate in fatty acid synthesis, is an
10. a-Linolenic acid is considered to be nutritionally essential in

A. It is a cofactor for acyl-CoA synthetase, which activates fatty
acids for breakdown.
B. Long-chain acyl-CoA (“activated fatty acids”) need to enter
the mitochondrial matrix to be oxidized, but cannot cross
the
outer
membrane.
Transfer
of
the
acyl
group from CoA to carnitine enables translocation to occur.
C. Acylcarnitine, formed when long-chain acyl groups are
transferred from CoA to carnitine is the substrate for the
first step in the B-oxidation pathway.
D.
Long-chain acyl-CoA (“activated fatty acids”) need to enter
the mitochondrial intermembrane space to be oxidized,
but cannot cross the inner mitochondrial membrane.
Transfer of the acyl group from CoA to carnitine enables
translocation to occur.
E. It prevents the breakdown of long-chain fatty acyl-CoA in the mitochondrial intermembrane
space.
. The breakdown of one molecule of a C16 fully saturated fatty
acid (palmitic acid) by B-oxidation lead to the formation of:
8 FADH,, 8 NADH, and 8 acetyl-CoA molecules
7
FADH,,
7
NADH,
and
7
molecules
8 FADH,, 8 NADH, and 7 acetyl-CoA molecules
7
FADH,,
8 NADH, and 8 acetyl-CoA molecules
7
FADH,,
7
NADH,
and
8
molecules
BOOP
important regulator of fatty acid metabolism BECAUSE:
A. Its formation from acetyl-CoA and bicarbonate by the
enzyme acetyl-CoA carboxylase is the main rate-limiting step in fatty acid synthesis.
B. It prevents entry of fatty acyl groups into the matrix of the
because
it
is
a
potent
inhibitor
of
carnitine
palmitoyl transferase-I.
C.

It prevents entry of fatty acyl groups into the matrix of the
mitochondria because it is a potent inhibitor of carnitine
palmitoyl transferase-II.
D. It prevents entry of fatty acyl groups into the matrix of the mitochondria because it is a potent inhibitor of carnitine— acylcarnitine translocase.
E. It inhibits the synthesis of fatty acyl-CoA.
humans BECAUSE:
A. Itis an w3 fatty acid.
B. It contains three double bonds.
C. In humans, double bonds cannot be introduced into fatty
acids beyond the A9 position.
D. In humans, double bonds cannot be introduced into fatty
acids beyond the A12 position.
E. Human tissues are unable to introduce a double bond in the
A9 position of fatty acids.
261
Exam Questions
ll. Inactivation of acetyl-CoA carboxylase is favored WHEN:
12. Which one of the following eicosanoids is synthesized from linoleic acid via the cyclooxygenase pathway?
13. Which one of the following enzymes is inhibited by the
14. Which one of the following is the major product of fatty acid
15. Fatty acids are broken down by repeated removal of two
16. Hormone-sensitive lipase, the enzyme which mobilizes fatty
Cytosolic citrate levels are high. >
It is in a polymeric form.
Palmitoyl-CoA levels are low.
The tricarboxylate transporter is inhibited.
It is dephosphorylated.
BOOS
Prostaglandin E, (PGE,) Leukotriene A, (LTA,) Prostaglandin E, (PGE,) Lipoxin A, (LXA,) BOOS
Thromboxane A, (TXA,)
nonsteroidal anti-inflammatory drug (NSAID) aspirin?
Lipoxygenase >
Prostacyclin synthase
Cyclooxygenase
Thromboxane synthase
A6 desaturase
BOOR
synthase?
Oleate
Palmitoyl-CoA
Palmitate
BOOP

carbon fragments as acetyl-CoA in the B-oxidation cycle, and
synthesized by repeated condensation of acetyl-CoAs until a
long-chain saturated fatty acid with an even number of carbons
is formed. Since fatty acids need to be broken down when energy
is short supply and synthesized when it is plentiful, there are
important differences between the two processes which help
cells to regulate them efficiently. Which one of the following
statements concerning these differences is INCORRECT?
A. Fatty acid breakdown takes place inside mitochondria, while
synthesis occurs in the cytosol.
B. Fatty acid breakdown uses NAD* and produces NADH,
while synthesis uses NADPH and produces NADP.
C. Fatty acyl groups are activated for breakdown using CoA
and for synthesis using acyl carrier protein.
D. Transport across the mitochondrial membrane of fatty acyl
groups is required for fatty acid breakdown, but not for
E. Glucagon promotes fatty acid synthesis and inhibits fatty acid breakdown.
acids from triacylglycerol stores in adipose tissue is inhibited by:
Glucagon >
ACTH
Prostaglandin E
BOO
17. Which one of the following best describes the action of
18. Tay-Sachs disease is a lipid storage disease caused by a genetic
19. Which of the plasma lipoproteins is best described as
20. Which of the plasma lipoproteins is best described as follows:
21.
22. Which of the following will be elevated in the bloodstream about
phospholipase C?
A. It releases the fatty acyl chain from the sn-2 position of a
phospholipid.
B. It cleaves a phospholipid into its phosphate-containing head
group and a diacylglycerol.
C. It releases the head group of a phospholipid, leaving
phosphatidic acid.
D. It releases the fatty acyl chain from the sn-1 position of a
E. It releases the fatty acyl chains from the sn-1 and sn-2
positions of a phospholipid.

defect in which one of the following enzymes:
A. B-Galactosidase
B. Sphingomyelinase
C. Ceramidase
D. Hexosaminidase A
E. B-Glucosidase
follows: synthesized in the intestinal mucosa, contains a high concentration of triacylglycerol, and is responsible for the
transport of dietary lipids in the circulation?
Chylomicrons
High-density lipoprotein
Intermediate-density lipoprotein
Low-density lipoprotein
Very-low-density lipoprotein
FOOD
synthesized in the liver, contains a high concentration of
triacylglycerol, and is mainly cleared from the circulation by
adipose tissue and muscle?
mOOR>
Which
of
the
plasma
is
best
described
as
follows:
formed in the circulation by removal of triacylglycerol from
very-low-density lipoprotein, contains apo B-100, delivers cholesterol to tissues?
extrahepatic
rPOOR>
2 hours after eating a high-fat meal?
moOORD
262
23. Which of the following will be elevated in the bloodstream about
24. Which one of the following processes is NOT involved in the
25. Which one of the following statements concerning chylomicrons
26. Which one of the following statements concerning the biosynthesis
27. The class of drugs called statins have proved very effective
4 hours after eating a high-fat meal?
BOOP
transfer
of
from

tissues
and
its
delivery
to the liver for excretion by HDL?
A. Efflux of cholesterol from tissues to pre}-HDL via ABCA1.
B. Esterification of cholesterol to cholesteryl ester by LCAT to
form
HDL,.
C. Transfer of cholesteryl ester from HDL to VLDL, IDL, and
LDL
by
the
action
of
cholesteryl
ester
transfer
protein
(CETP).
D. Efflux of cholesterol from tissues to HDL, via SR-B1 and
ABCGI.
E. Selective uptake of cholesteryl ester from HDL, by the liver
via SR-B1.
is CORRECT?
A. Chylomicrons are made inside intestinal cells and secreted
into lymph, where they acquire apolipoproteins B and C.
B. The core of chylomicrons contains triacylglycerol and phospholipids.
C. The enzyme hormone-sensitive lipase acts on chylomicrons
to release fatty acids from triacylglycerol when they are
bound to the surface of endothelial cells in blood capillaries.
D. Chylomicron remnants differ from chylomicrons in
that they are smaller and contain a lower proportion of
triacylglycerol and a higher proportion of cholesterol.
E. Chylomicrons are taken up by the liver.
of cholesterol is CORRECT?
A. The rate-limiting step is the formation of 3-hydroxy-3-
methylglutaryl-CoA (HMG-CoA) by the enzyme HMG-CoA synthase.
B. Synthesis occurs in the cytosol of the cell.
C.
All
the
carbon
atoms
in
the
originate
from acetyl-CoA.
D. Squalene is the first cyclic intermediate in the pathway.
E. The initial substrate is mevalonate.
against hypercholesterolemia, a major cause of atherosclerosis and associated cardiovascular disease. These drugs reduce plasma cholesterol levels by:
A. Preventing absorption of cholesterol from the intestine.

B. Increasing the excretion of cholesterol from the body via conversion to bile acids.
28. Which of the following statements about bile acids (or bile salts)
29. A 35-year-old man with severe hypercholesterolemia has a family
30. The recently discovered protein, proprotein convertase subtilisin/
C. Inhibiting the conversion of 3-hydroxy-3-methylglutaryl-
CoA to mevalonate in the pathway for cholesterol
D. Increasing the rate of degradation of 3-hydroxy-3-
methylglutaryl-CoA reductase.
E. Stimulating the activity of the LDL receptor in the liver.
is
A. Primary bile acids are synthesized in the liver from
B. Bile acids are needed for the breakdown of fats by pancreatic lipase.
C. Secondary bile acids are produced by modification of
primary bile acids in the liver.
D. Bile acids facilitate the absorption of the products of lipid
digestion in the jejunum.
E. Bile acids are recirculated between the liver and the small intestine in the enterohepatic circulation.
history
of
deaths
at
a
young
age
from
heart
disease
and
stroke.
Which of the following genes is likely to be defective?
Apolipoprotein E
The LDL receptor
lipase
PCSK9
LCAT
FOOD
kexin type 9 (PCSK9), has been identified as a potential target for antiatherogenic drugs BECAUSE:
A. It decreases the number of LDL receptors exposed at the cell surface, thus LDL uptake is lowered and blood cholesterol levels rise.
B. It inhibits the binding of apo B to the LDL receptor, thus
blocking uptake of the lipoprotein and raising blood
cholesterol levels.
C. It increases the absorption of cholesterol from the
D. It prevents the breakdown of cholesterol to bile acids in the
liver.
E. It increases the synthesis and secretion of VLDL in the liver, leading to increased LDL formation in the blood.

C H A PT ER
= Explain why the absence from the diet of certain amino acids that are present in most proteins is not deleterious to human health.
= Appreciate the distinction between the terms "essentia I" \" and "nutritionally essentia amino acids, and identify the amino acids that are nutritionally nonessential.
= Name the intermediates of the citric acid cycle and of glycolysis that are precursors of aspartate, asparagine, glutamate, glutamine, glycine, and serine.
Illustrate the key role of transaminases in amino acid metabolism.
Explain the process by which the 4-hydroxyproline and 5-hydroxylysine of proteins such as collagen are formed.
= Describe the clinical presentation of scurvy, and provide a biochemical
explanation for why a severe deprivation of vitamin C (ascorbic acid) results in this nutritional disorder.
= Appreciate that, despite the toxicity of selenium, selenocysteine is an essential component of several mammalian proteins.
Define and outline the reaction catalyzed by a mixed-function oxidase.
Identify the role of tetrahydrobiopterin in tyrosine biosynthesis.
Indicate the role of modified transfer RNA (tRNA) in the cotranslational
a insertion of selenocysteine into proteins.

Amino acid deficiency states can result if nutritionally essential amino acids are absent from the diet, or are present in inadequate amounts. Examples in certain regions of West Africa include kwashiorkor, which results when a child is weaned onto a starchy diet poor in protein, and marasmus, in which both caloric intake and specific amino acids are deficient. Patients with short bowel syndrome unable to absorb sufficient quantities of calories and nutrients suffer from significant nutritional and metabolic abnormalities. Both the nutritional disorder scurvy, a dietary deficiency of vitamin C, and specific genetic disorders are associated with an impaired ability of connective tissue to form peptidyl 4-hydroxyproline and peptidyl 5-hydroxylysine. The resulting conformational instability of collagen is accompanied by bleeding gums, swelling joints, poor wound healing, and ultimately in death. Menkes syndrome, characterized by kinky hair and growth retardation, results from a dietary deficiency of copper, an essential cofactor for the enzyme lysyl oxidase that functions in formation of the covalent cross-links that strengthen collagen fibers. Genetic disorders of collagen biosynthesis include
several forms of osteogenesis imperfecta, characterized by fragile bones, and Ehlers-Danlos syndrome, a group of connective tissue disorders that result in mobile joints and skin abnormalities due to defects in the genes that encode enzymes, including procollagen-lysine 5-hydroxylase.

While often
employed with
reference to
the terms
“essential”
and mon
amino amino
“nonessential”
acids acids,
best are
misleading are
essential
8
must be
to present
termed
“nutritionally are
“nutritionally in
since ensure
all health.
in the
human essential?
nonessential”
the diet
(Table classes
of amino
ing human
diet,
The other
since
27-1).
The acids
they
20
Of com-
these and
20
thus
12
amino need
distinction was
established subjects
Subsequent purified
biochemical and
intermediates acids.
in amino
acids investigations
involved
Amino acid
regions of
not are
acids be
present between
these the
1930s in
place two
by feed-
of revealed
in the
the biosynthesis
West are
poor
Africa sources
disorders disorders
where protein.
reactions of
all are
endemic diets
rely of
tryptophan include
heavily and
kwashiorkor,
20
amino in
certain on
grains lysine.
which
These results
that nutritional
when
a
child is
weaned onto
a
starchy diet
poor in
protein, and
marasmus, in
which both caloric intake and specific amino acids are deficient.

The existence of nutritional requirements suggests that depen-
dence on an external supply of a given nutrient can be of greater

Arginine a, Nutritionally Nonessential = Alanine. Histidine, Nutritionally Nonessential = Asparagine. Isoleucine, Nutritionally Nonessential = Aspartate. Leucine, Nutritionally Nonessential = Cysteine. Lysine, Nutritionally Nonessential = Glutamate. Methionine, Nutritionally Nonessential = Glutamine. Phenylalanine, Nutritionally Nonessential = Glycine. Threonine, Nutritionally Nonessential = Hydroxyproline b. Tryptophan, Nutritionally Nonessential = Hydroxylysine b. Valine, Nutritionally Nonessential = Proline. , Nutritionally Nonessential = Serine. , Nutritionally Nonessential = Tyrosine
*Nutritionally "semiessential." Synthesized at rates inadequate to support growth of children.
'Not necessary for protein synthesis, but is formed during posttranslational processing

Enzymes Required to Synthesize.Nutritionally Nonessential = Pro a. Leu, Number of Enzymes Required to Synthesize.Nutritionally Nonessential = 3. Phe, Number of Enzymes Required to Synthesize.Nutritionally Essential = 10. Phe, Number of Enzymes Required to Synthesize.Nutritionally Nonessential = Ser. Phe, Number of Enzymes Required to Synthesize.Nutritionally Nonessential = 3. Trp, Number of Enzymes Required to Synthesize.Nutritionally Essential = 5 (8 shared). Trp, Number of Enzymes Required to Synthesize.Nutritionally Nonessential = Gly e. Trp, Number of Enzymes Required to Synthesize.Nutritionally Nonessential = 1. , Number of Enzymes Required to Synthesize.Nutritionally Essential = 59 (total). , Number of Enzymes Required to Synthesize.Nutritionally Nonessential = Cys f. , Number of Enzymes Required to Synthesize.Nutritionally Nonessential = 2. , Number of Enzymes Required to Synthesize.Nutritionally Essential = . , Number of Enzymes Required to Synthesize.Nutritionally Nonessential = Tyr g. , Number of Enzymes Required to Synthesize.Nutritionally Nonessential = 1. , Number of Enzymes Required to Synthesize.Nutritionally Essential = . , Number of Enzymes Required to Synthesize.Nutritionally Nonessential = . , Number of Enzymes Required to Synthesize.Nutritionally Nonessential = 17 (total)
*From Glu.
From Asp.
“From
Lys.
“From
Pro.
From
Ser.
'From Ser plus sulfate.
°From Phe.

survival value than the ability to biosynthesize it. Why? Ifa specific nutrient is present in the food, an organism that can synthesize it will transfer to its progeny genetic information of negative survival value. The survival value is negative rather than nil because ATP and nutrients are required to synthesize "unnecessary" DNA—even if specific encoded genes are no longer expressed. The number of enzymes required by prokaryotic cells to synthesize the nutritionally essential amino acids is large relative to the number of enzymes required to synthesize the nutritionally nonessential amino acids (Table 27-2). This suggests a survival advantage in retaining the ability to manufacture "easy" amino acids while losing the ability to make "difficult" amino acids. The metabolic pathways that form the nutritionally essential amino acids occur in plants and bacteria, but not in humans, and thus are not discussed. This chapter addresses the reactions and intermediates involved in the biosynthesis by human tissues of the 12 nutritionally nonessential amino acids and selected nutritional and metabolic disorders associated with their metabolism.

Glutamate, the precursor of the so-called “glutamate family”
of amino acids, is formed by the reductive amidation of
the citric acid cycle a-ketoglutarate, a reaction catalyzed by
FIGURE 27-1 The reaction catalyzed by glutamate dehydrogenase (EC 1.4.1.3).
mitochondrial glutamate dehydrogenase (Figure 27-1). The reaction strongly favors glutamate synthesis, which lowers the concentration of cytotoxic ammonium ion.

The amidation of glutamate to glutamine catalyzed by glutamine synthetase (Figure 27-2) involves the intermediate formation of y-glutamyl phosphate (Figure 27-3). Following the ordered binding of glutamate and ATP, glutamate attacks the y-phosphorus of ATP, forming y-glutamyl phosphate and ADP. NH," then binds, and uncharged NH, attacks y-glutamyl phosphate. Release of P, and of a proton from the y-amino group of the tetrahedral intermediate then allows release of the product, glutamine.

Transamination of pyruvate forms alanine (Figure 27-4). Similarly, transamination of oxaloacetate forms aspartate.
Glutamate Dehydrogenase, Glutamine Synthetase, & Aminotransferases Play Central Roles in Amino Acid Biosynthesis
The combined action of the enzymes glutamate dehydrogenase, glutamine synthetase, and the aminotransferases (Figures 27-1, 27-2 and 27-4) results in conversion of inorganic ammonium ion into the a-amino nitrogen of amino acids.

The conversion of aspartate to asparagine, catalyzed by asparagine synthetase (Figure 27-5), resembles the glutamine
MANY
FIGURE 27-2 The reaction catalyzed by glutamine synthetase (EC 6.3.1.2).
FIGURE 27-3 y-Glutamyl phosphate.
synthetase reaction (Figure 27-2), but glutamine, rather than ammonium ion, provides the nitrogen. Bacterial asparagine synthetases can, however, also use ammonium ion. The reaction involves the intermediate formation of aspartyl phosphate (Figure 27-6). The coupled hydrolysis of PP, to P, by pyrophosphatase, EC 3.6.1.1, ensures that the reaction is strongly favored.

Oxidation of the a-hydroxyl group of the glycolytic intermediate 3-phosphoglycerate, catalyzed by 3-phosphoglycerate dehydrogenase, converts it to 3-phosphohydroxypyruvate. Transamination and subsequent dephosphorylation then form serine (Figure 27-7).

Glycine aminotransferases can catalyze the synthesis of glycine from glyoxylate and glutamate or alanine. Unlike most aminotransferase reactions, these strongly favor glycine synthesis. Additional important mammalian routes for glycine formation are from choline (Figure 27-8) and from serine (Figure 27-9).

The initial reaction of proline biosynthesis converts the y-carboxyl group of glutamate to the mixed acid anhydride of glutamate y-phosphate (Figure 27-3). Subsequent reduction forms glutamate y-semialdehyde, which following spontaneous cyclization is reduced to L-proline (Figure 27-10).

While not nutritionally essential, cysteine is formed from methionine, which is nutritionally essential. Following conversion of methionine to homocysteine (see Figure 29-18), homocysteine and serine form cystathionine, whose hydrolysis forms cysteine and homoserine (Figure 27-11).
FIGURE 27-4 Formation of alanine by transamination of pyruvate. The amino donor may be glutamate or aspartate. The other product thus is a-ketoglutarate or oxaloacetate.
266
FIGURE 27-5 The reaction catalyzed by asparagine synthetase (EC 6.3.5.4). Note similarities to and differences from the glutamine synthetase reaction (Figure 27-2).

Phenylalanine hydroxylase converts phenylalanine to tyrosine (Figure 27-12). If the diet contains adequate quantities of the nutritionally essential amino acid phenylalanine, tyrosine is nutritionally nonessential. However, since the phenylalanine hydroxylase reaction is irreversible, dietary tyrosine cannot replace phenylalanine. Catalysis by this mixed-function oxidase incorporates one atom of O, into the para position of phenylalanine and reduces the other atom to water. Reducing power, provided as tetrahydrobiopterin, derives ultimately from NADPH.

Hydroxyproline and hydroxylysine occur principally in collagen. Since there is no tRNA for either hydroxylated amino acid, neither dietary hydroxyproline nor dietary hydroxylysine is incorporated during protein synthesis. Peptidyl hydroxyproline and hydroxylysine arise from proline and lysine, but only after these amino acids have been incorporated into peptides. Hydroxylation of peptidyl prolyl and peptidyl lysyl residues, catalyzed by prolyl hydroxylase and lysyl hydroxylase of skin, skeletal muscle, and granulating wounds requires, in addition to the substrate, molecular O,, ascorbate, Fe**, and a-ketoglutarate (Figure 27-13). For every mole of proline or lysine hydroxylated, one mole of a-ketoglutarate is decarboxylated to succinate. The hydroxylases are mixedfunction oxidases. One atom of O, is incorporated into proline or lysine, the other into succinate (Figure 27-13). A deficiency of the vitamin C required for these two hydroxylases results in scurvy, in which bleeding gums, swelling joints, and impaired wound healing result from the impaired stability of collagen (see Chapters 5 and 50).

While leucine, valine, and isoleucine are all nutritionally essential amino acids, tissue aminotransferases reversibly interconvert all
FIGURE 27-7 Serine biosynthesis. Oxidation of 3-phosphoglycerate is catalyzed by 3-phosphoglycerate dehydrogenase (EC 1.1.1.95). Transamination converts phosphohydroxypyruvate to phosphoserine. Hydrolytic removal of the phosphoryl group catalyzed by phosphoserine hydrolase (EC 3.1.3.3) then forms t-serine.
CH,
FIGURE 27-8 Formation of glycine from choline. Catalysts include choline dehydrogenase (EC 1.1.3.17), betaine aldehyde dehydrogenase (EC 1.2.1.8), betaine-homocysteine N-methyltransferase (EC 2.1.1.157), sarcosine dehydrogenase (EC 1.5.8.3), and dimethylglycine dehydrogenase (EC 1.5.8.4).
FIGURE 27-9 interconversion of serine and glycine, catalyzed by serine hydroxymethyltransferase (EC 2.1.2.1). The reaction is freely reversible. (H, folate, tetrahydrofolate.)
FIGURE 27-10 Biosynthesis of proline from glutamate. Catalysts for these reactions are glutamate-5-kinase (EC 2.7.2.11), glutamate-5-semialdehyde dehydrogenase (EC 1.2.1.41), and pyrroline-5-carboxylate reductase (EC 1.5.1.2). Ring closure of glutamate semialdehyde is spontaneous.
three amino acids and their corresponding a-keto acids. These a-keto acids thus can replace their amino acids in the diet.

While the occurrence of selenocysteine (Figure 27-14) in proteins
is uncommon,
at least
25
human selenoproteins
are known. Selenocysteine is present at the active site of several
human enzymes
that catalyze
redox reactions.
Examples include
thioredoxin reductase,
glutathione peroxidase,
and the deiodinase that converts thyroxine to triiodothyronine.
FIGURE 27-11 Conversion of homocysteine and serine to homoserine and cysteine. The sulfur of cysteine derives from methionine and the carbon skeleton from serine. The catalysts are cystathionine B-synthase (EC 4.2.1.22) and cystathionine y-lyase (EC 44.1.1).
Where present, selenocysteine participates in the catalytic mechanism of these enzymes. Significantly, the replacement of selenocysteine by cysteine can actually reduce catalytic activity. Impairments in human selenoproteins have been implicated
FIGURE 27-12 Conversion of phenylalanine to tyrosine by phenylalanine hydroxylase (EC 1.14.16.1). Two distinct enzymatic activities are involved. Activity Il catalyzes reduction of dihydrobiopterin by NADPH, and activity | the reduction of O, to H,O and of phenylalanine to tyrosine. This reaction is associated with several defects of phenylalanine metabolism discussed in Chapter 29.
AAW
FIGURE 27-13 Hydroxylation of a proline-rich peptide. Molecular oxygen is incorporated into both succinate and proline. Procollagen-proline 4-hydroxylase (EC 1.14.11.2) thus is a mixedfunction oxidase. Procollagen-lysine 5-hydroxylase (EC 1.14.11.4) catalyzes an analogous reaction.

in tumorigenesis and atherosclerosis, and are associated with selenium deficiency cardiomyopathy (Keshan disease). Biosynthesis of selenocysteine requires serine, selenate (SeO,7), ATP, a specific tRNA, and several enzymes. Serine provides the carbon skeleton of selenocysteine. Selenophosphate, formed from ATP and selenate (Figure 27-14), serves as the selenium donor. Unlike 4-hydroxyproline or 5-hydroxylysine, selenocysteine arises cotranslationally during its incorporation into peptides. The UGA anticodon of the unusual tRNA called tRNA'* normally signals STOP. The ability of the protein synthetic apparatus to identify a selenocysteine-specific UGA codon involves the selenocysteine insertion element, a stem-loop structure in the untranslated region of the mRNA. tRNA** is first charged with serine by the ligase that charges tRNA". Subsequent replacement of the serine oxygen by selenium involves selenophosphate formed by selenophosphate synthetase (Figure 27-14). Successive enzyme-catalyzed reactions convert cysteyl-tRNA'* to aminoacrylyl-tRNA'* and then to selenocysteyl-tRNA**. In the presence of a specific elongation factor that recognizes selenocysteyl-tRNA**, selenocysteine can then be incorporated into proteins.
FIGURE 27-14 Selenocysteine (top) and the reaction catalyzed by selenophosphate synthetase (EC 2.7.9.3) (bottom).

All vertebrates can form certain amino acids from amphibolic intermediates or from other dietary amino acids. The intermediates and the amino acids to which they give rise are a-ketoglutarate (Glu, Gln, Pro, Hyp), oxaloacetate (Asp, Asn), and 3-phosphoglycerate (Ser, Gly).
™ Cysteine, tyrosine, and hydroxylysine are formed from nutritionally essential amino acids. Serine provides the carbon skeleton and homocysteine the sulfur for cysteine biosynthesis.
= Inscurvy, a nutritional disease that results from a deficiency of vitamin C, impaired hydroxylation of peptidyl proline and peptidyl lysine results in a failure to provide the substrates for cross-linking of maturing collagens.
@™ Phenylalanine hydroxylase converts phenylalanine to tyrosine. Since the reaction catalyzed by this mixed function oxidase is irreversible, tyrosine cannot give rise to phenylalanine.
= Neither dietary hydroxyproline nor hydroxylysine is incorporated into proteins because no codon or tRNA dictates their insertion into peptides.
= Peptidyl hydroxyproline and hydroxylysine are formed by hydroxylation of peptidyl proline or lysine in reactions catalyzed by mixed-function oxidases that require vitamin C as cofactor.
™ Selenocysteine, an essential active site residue in several mammalian enzymes, arises by cotranslational insertion from a previously modified tRNA.

Beckett GJ, Arthur JR: Selenium and endocrine systems. J
Endocrinol 2005;184:455.
Donovan J, Copeland PR: The efficiency of selenocysteine
incorporation is regulated by translation initiation factors. J Mol Biol 2010;400:659.
Kilberg MS: Asparagine synthetase chemotherapy. Annu Rev Biochem
2006;75:629.
Ruzzo EK, Capo-Chichi JM, Ben-Zeev B, et al: Deficiency of asparagine
synthetase causes congenital microcephaly and a progressive form of encephalopathy. Neuron 2013;80:429.
Stickel F, Inderbitzin D, Candinas D: Role of nutrition in liver
transplantation for end-stage chronic liver disease. Nutr Rev
2008;66:47.
Turanov AA, Shchedrina VA, Everley RA, et al: Selenoprotein
S is involved in maintenance and transport of multiprotein
complexes. Biochem J 2014;462:555.

2 Describe protein turnover, indicate the mean rate of protein turnover in healthy individuals, and provide examples of human proteins that are degraded at rates greater than the mean rate.
1: Outline the events in protein turnover by both ATP-dependent and ATP- independent pathways, and indicate the roles in protein degradation played by the proteasome, ubiquitin, cell surface receptors, circulating asialoglycoproteins, and lysosomes.
symptoms. m= Explain why metabolic defects in different enzymes of urea biosynthesis, although distinct at the molecular level, present similar clinical signs and
= Indicate how the ultimate end products of nitrogen catabolism in mammals differ from those in birds and fish.
= Illustrate the central roles of transaminases (aminotransferases), of glutamate dehydrogenase, and of glutaminase in human nitrogen metabolism.
= Use structural formulas to represent the reactions that convert NH,, CO,, and the amide nitrogen of aspartate into urea, and identify the subcellular locations of the enzymes that catalyze urea biosynthesis.
= Indicate the roles of allosteric regulation and of acetylglutamate in the regulation of the earliest steps in urea biosynthesis.
= Describe both the classical approaches and the role of tandem mass spectrometry in screening neonates for inherited metabolic diseases.

In normal adults, nitrogen intake matches nitrogen excreted. Positive nitrogen balance, an excess of ingested over excreted nitrogen, accompanies growth and pregnancy. Negative nitrogen balance, where output exceeds intake, may follow surgery, advanced cancer, and the nutritional disorders kwashiorkor and marasmus. Genetic disorders that result from defects in the genes that encode ubiquitin, ubiquitin ligases, or deubiquitinating enzymes that participate in the degradation of certain proteins include Angelman syndrome, juvenile Parkinson disease, von Hippel-Lindau syndrome, and congenital polycythemia. This chapter describes how the nitrogen of amino acids is converted to urea, and the metabolic disorders that accompany defects in this process. Ammonia, which is highly toxic, arises in humans primarily from the a-amino nitrogen of amino acids. Tissues therefore convert ammonia to the amide nitrogen of the nontoxic amino acid glutamine. Subsequent deamination of glutamine in the liver releases ammonia, which is efficiently converted to urea, which is not toxic. However, if liver function is compromised, as in cirrhosis or hepatitis, elevated blood ammonia levels generate clinical signs and symptoms. Each enzyme of the urea cycle provides examples of metabolic defects and their physiologic consequences. In addition, the urea cycle provides a useful molecular model for the study of other human metabolic defects.

The continuous degradation and synthesis (turnover) of cellular proteins occur in all forms of life. Each day, humans turn over 1 to 2% of their total body protein, principally muscle
protein. High rates of protein degradation occur in tissues that are undergoing structural rearrangement, for example, uterine tissue during pregnancy, skeletal muscle in starvation, and tadpole tail tissue during metamorphosis. While approximately 75% of the amino acids liberated by protein degradation are reutilized, the remaining excess free amino acids are not stored for future use. Amino acids not immediately incorporated into new protein are rapidly degraded. The major portion of the carbon skeletons of the amino acids is converted to amphibolic intermediates, while in humans the amino nitrogen is converted to urea and excreted in the urine.

The relative susceptibility of a protein to degradation is expressed as its half-life (t,,,), the time required to lower its concentration to half of its initial value. Half-lives of liver proteins range from under 30 minutes to over 150 hours. Typical "housekeeping" enzymes such as those of glycolysis, have t,,, values of over 100 hours. By contrast, key regulatory enzymes may have t,,, values as low as 0.5 to 2 hours. PEST sequences, regions rich in proline (P), glutamate (E), serine (S), and threonine (T), target some proteins for rapid degradation. Intracellular proteases hydrolyze internal peptide bonds. The resulting peptides are then degraded to amino acids by endopeptidases that hydrolyze internal peptide bonds, and by aminopeptidases and carboxypeptidases that remove amino acids sequentially from the aminoand carboxyl-termini, respectively.

Degradation of blood glycoproteins (see Chapter 46) follows loss of a sialic acid moiety from the nonreducing ends of their oligosaccharide chains. Asialoglycoproteins are then internalized by liver-cell asialoglycoprotein receptors and degraded by lysosomal proteases. Extracellular, membrane-associated, and long-lived intracellular proteins are also degraded in lysosomes by ATP-independent processes.

Degradation of regulatory proteins with short half-lives and of abnormal or misfolded proteins occurs in the cytosol, and requires ATP and ubiquitin. Named based on its presence in all eukaryotic cells, ubiquitin is a small (8.5 kDa, 76 residue) polypeptide that targets many intracellular proteins for degradation. The primary structure of ubiquitin is highly conserved. Only 3 of 76 residues differ between yeast and human ubiquitin. Figure 28-1 illustrates the three-dimensional structure of ubiquitin. Ubiquitin molecules are attached by non-a-peptide bonds formed between the carboxyl terminal of ubiquitin and the e-amino groups of lysyl residues in the target protein (Figure 28-2). The residue present at its amino terminus affects whether a protein is ubiquitinated. Amino terminal Met or Ser residues retard, whereas Asp or Arg
FIGURE 28-1 Three-dimensional structure of ubiquitin. Shown are a-helices (blue), B-strands (green), and the R-groups of lysyl residues (orange). Lys48 & Lys63 are sites for attachment of additional ubiquitin molecules during polyubiquitination. Created by Rogerdodd at Wikipedia using PyMOL, PDB id 1ubi, and crediting the European Bioinformatics Institute.
accelerate ubiquitination. Attachment of a single ubiquitin molecule to transmembrane proteins alters their subcellular localization and targets them for degradation. Soluble proteins undergo polyubiquitination, the ligase-catalyzed attachment of four or more additional ubiquitin molecules (Figure 28-1). Subsequent degradation of ubiquitin-tagged proteins takes place in the proteasome, a macromolecule that also is ubiquitous in

FIGURE 28-2 Reactions involved in the attachment of ubiquitin (Ub) to proteins. Three enzymes are involved. E1 is an activating enzyme, E2 a transferase, and E3 a ligase. While depicted as single entities, there are several types of E1, and over 500 types of E2. The terminal COOH of ubiquitin first forms a thioester. The coupled hydrolysis of PP, by pyrophosphatase ensures that the reaction will proceed readily. A thioester exchange reaction now transfers activated ubiquitin to E2. E3 then catalyzes the transfer of ubiquitin to the €-amino group of a lysyl residue of the target protein. Additional rounds of ubiquitination result in subsequent polyubiquitination.
FIGURE 28-3 Representation of the structure of a proteasome. The upper ring is gated to permit only polyubiquitinated proteins to enter the proteosome, where immobilized internal proteases degrade them to peptides.
eukaryotic cells. The proteasome consists of a macromolecular, cylindrical complex of proteins, whose stacked rings form a central pore that harbors the active sites of proteolytic enzymes. For degradation, a protein thus must first enter the central pore. Entry into the core is regulated by the two outer rings that recognize polyubiquitinated proteins (Figures 28-3 and 28-4).
For the discovery of ubiquitin-mediated protein degradation, Aaron Ciechanover and Avram Hershko of Israel and Irwin Rose of the United States were awarded the 2004 Nobel Prize in Chemistry. Genetic disorders that result from defects in the genes that encode ubiquitin, ubiquitin ligases, or deubiquitinating enzymes include Angelman syndrome, autosomal recessive juvenile Parkinson disease, von Hippel-Lindau syndrome, and congenital polycythemia. For additional aspects of protein degradation and of ubiquitination, including its role in the cell cycle, see Chapters 4 and 35.

The maintenance of steady-state concentrations of circulating plasma amino acids between meals depends on the net balance between release from endogenous protein stores and
FIGURE 28-4 An end-on view of a proteasome. Created by Rogerdodd at Wikipedia and crediting the European Bioinformatics Institute.
utilization by various tissues. Muscle generates over half of the total body pool of free amino acids, and liver is the site of the urea cycle enzymes necessary for disposal of excess nitrogen. Muscle and liver thus play major roles in maintaining circulating amino acid levels.
Figure 28-5 summarizes the postabsorptive state. Free amino acids, particularly alanine and glutamine, are released from muscle into the circulation. Alanine is extracted primarily by the liver, and glutamine is extracted by the gut and the kidney, both of which convert a significant portion to alanine. Glutamine also serves as a source of ammonia for excretion by the kidney. The kidney provides a major source of serine for uptake by peripheral tissues, including liver and muscle.
FIGURE 28-5 interorgan amino acid exchange in normal of postabsorptive humans. The key role alanine in amino acid output from muscle and gut and uptake by the liver is shown.
FIGURE 28-6 The glucose-alanine cycle. Alanine is synthesized in muscle by transamination of glucose-derived pyruvate, released into the bloodstream, and taken up by the liver. In the liver, the carbon skeleton of alanine is reconverted to glucose and released into the bloodstream, where it is available for uptake by muscle and resynthesis of alanine.
Branched-chain amino acids, particularly valine, are released by muscle and taken up predominantly by the brain.

Alanine is a key gluconeogenic amino acid (Figure 28-6). The rate of hepatic gluconeogenesis from alanine is far higher than from all other amino acids. The capacity of the liver for gluconeogenesis from alanine does not reach saturation until the alanine concentration reaches 20 to 30 times its normal physiologic level. Following a protein-rich meal, the splanchnic tissues release amino acids (Figure 28-7) while the peripheral muscles extract amino acids, in both instances predominantly branched-chain amino acids. Branched-chain amino acids thus serve a special role in nitrogen metabolism. In the fasting
FIGURE 28-7 summary of amino acid exchange between organs immediately after feeding.
state, they provide the brain with an energy source, and postprandially they are extracted predominantly by muscle, having been spared by the liver.

Depending on their ecological niche and physiology, different animals excrete excess nitrogen as ammonia, uric acid, or urea. The aqueous environment of teleostean fish, which are ammonotelic (excrete ammonia), permits them to excrete water continuously to facilitate excretion of ammonia, which is highly toxic. While this approach is appropriate for an aquatic animal, birds must both conserve water and maintain low weight. Birds, which are uricotelic, address both problems by excreting nitrogen-rich uric acid (see Figure 33-11) as semisolid guano. Many land animals, including humans, are ureotelic and excrete nontoxic, highly water-soluble urea. Since urea is nontoxic to humans, high blood levels in renal disease are a consequence, not a cause, of impaired renal function.

Urea biosynthesis occurs in four stages: (1) transamination, (2) oxidative deamination of glutamate, (3) ammonia transport, and (4) reactions of the urea cycle (Figure 28-8). The expression in liver of the RNAs for all the enzymes of the urea cycle increases severalfold in starvation, probably secondary to enhanced protein degradation to provide energy.

Transamination reactions interconvert pairs of a-amino acids and a-keto acids (Figure 28-9). Transamination reactions, which are freely reversible, also function in amino acid
FIGURE 28-8 Overall flow of nitrogen in amino acid catabolism.
FIGURE 28-9 Transamination. The reaction is freely reversible with an equilibrium constant close to unity.
biosynthesis (see Figure 27-4). All of the common amino acids except lysine, threonine, proline, and hydroxyproline participate in transamination. Transamination is not restricted to a-amino groups. The 5-amino group of ornithine (but not the e-amino group of lysine) readily undergoes transamination. Alanine-pyruvate aminotransferase (alanine aminotransferase, EC 2.6.1.2) and glutamate-a-ketoglutarate aminotransferase (glutamate aminotransferase, EC 2.6.1.1) catalyze the transfer of amino groups to pyruvate (forming alanine) or to a-ketoglutarate (forming glutamate).
Each aminotransferase is specific for one pair of substrates, but nonspecific for the other pair. Since alanine is also a substrate for glutamate aminotransferase, the a-amino nitrogen from all amino acids that undergo transamination can be concentrated in glutamate. This is important because L-glutamate is the only amino acid that undergoes oxidative deamination at an appreciable rate in mammalian tissues. The formation of ammonia from a-amino groups thus occurs mainly via the a-amino nitrogen of L-glutamate.
Transamination occurs via a "ping-pong" mechanism characterized by the alternate addition of a substrate and release of a product (Figure 28-10). Following removal of its a-amino nitrogen by transamination, the remaining carbon "skeleton" of an amino acid is degraded by pathways discussed in Chapter 29.
FIGURE 28-11 Structure of a Schiff base formed between pyridoxal phosphate and an amino acid.
associated with elevated serum levels of aminotransferases (see Table 7-1).

Transfer of amino nitrogen to a-ketoglutarate forms L-glutamate. Hepatic L-glutamate dehydrogenase (GDH), which can use either NAD* or NADP', releases this nitrogen as ammonia (Figure 28-12). Conversion of a-amino nitrogen to ammonia by the concerted action of glutamate aminotransferase and GDH is often termed "transdeamination." Liver GDH activity is allosterically inhibited by ATP, GTP, and NADH, and is activated by ADP. The GDH reaction is freely reversible, and also functions in amino acid biosynthesis (see Figure 27-1).

t-Amino acid oxidase of liver and kidney convert an amino acid to an a-imino acid that decomposes to an a-keto acid with release of ammonium ion (Figure 28-13). The reduced flavin is reoxidized by molecular oxygen, forming hydrogen peroxide (H,O,), which then is split to O, and H,O by catalase, EC 1.11.1.6.
Pyridoxal phosphate (PLP), a derivative of vitamin B,, is present at the catalytic site of all aminotransferases, and plays a key role in catalysis. During transamination, PLP serves as a "carrier" of amino groups. An enzyme-bound Schiff base (Figure 28-11) is formed between the oxo group of enzymebound PLP and the a-amino group of an a-amino acid. The Schiff base can rearrange in various ways. In transamination, rearrangement forms an a-keto acid and enzyme-bound pyridoxamine phosphate. As noted earlier, certain diseases are

The ammonia produced by enteric bacteria and absorbed into portal venous blood and the ammonia produced by tissues are rapidly removed from circulation by the liver and converted to urea. Thus, normally, only traces (10-20 g/dL) are present in peripheral blood. This is essential, since ammonia is toxic
FIGURE 28-10 "Ping-pong" mechanism for transamination. E—CHO and E—CH,NH, represent enzyme-bound pyridoxal phosphate and pyridoxamine phosphate, respectively. (Ala, alanine; Glu, glutamate; KG, a-ketoglutarate; Pyr, pyruvate.)
FIGURE 28-12 The reaction catalyzed by glutamate dehydrogenase, EC 1.4.1.2. NAD(P)* means that either NAD* or NADP* can serve as the oxidoreductant. The reaction is reversible, but strongly favors glutamate formation.
to the central nervous system. Should portal blood bypass the liver, systemic blood ammonia may reach toxic levels. This occurs in severely impaired hepatic function or the development of collateral links between the portal and systemic veins in cirrhosis. Symptoms of ammonia intoxication include tremor, slurred speech, blurred vision, coma, and ultimately death. Ammonia may be toxic to the brain in part because it reacts with a-ketoglutarate to form glutamate. The resulting depletion of a-ketoglutarate then impairs function of the tricarboxylic acid (TCA) cycle in neurons.

Formation of glutamine is catalyzed by mitochondrial glutamine synthetase (Figure 28-14). Since amide bond synthesis is coupled to the hydrolysis of ATP to ADP and P,, the reaction strongly favors glutamine synthesis. During catalysis, glutamate attacks the y-phosphoryl group of ATP, forming y-glutamyl phosphate and ADP. Following deprotonation of NH,,*, NH, attacks y-glutamyl phosphate, and glutamine and P, are released. In addition to providing glutamine to serve as a carrier of nitrogen, carbon and energy between organs (Figure 28-5), glutamine synthetase plays a major role both in ammonia detoxification and in acid-base homeostasis. A rare
FIGURE 28-13 Oxidative deamination catalyzed by t-amino acid oxidase (.-a-amino acid:O, oxidoreductase, EC 1.4.3.2). The a-imino acid, shown in brackets, is not a stable intermediate.
FIGURE 28-14 Formation of glutamine, catalyzed by glutamine synthetase, EC 6.3.1.2.
deficiency in neonate glutamine synthetase results in severe brain damage, multiorgan failure, and death.

There are two human isoforms of mitochondrial glutaminase, termed liver-type and renal-type glutaminase. Products of different genes, the glutaminases differ with respect to their structure, kinetics, and regulation. Hepatic glutaminase levels rise in response to high protein intake while renal kidney-type glutaminase increases in metabolic acidosis. Hydrolytic release of the amide nitrogen of glutamine as ammonia, catalyzed by glutaminase (Figure 28-15), strongly favors glutamate formation. An analogous reaction is catalyzed by L-asparaginase (EC 3.5.1.1). The concerted action of glutamine synthetase and glutaminase thus catalyzes the interconversion of free ammonium ion and glutamine.
FIGURE 28-15 The reaction catalyzed by glutaminase, EC 3.5.1.2. The reaction proceeds essentially irreversibly in the direction of glutamate and NH,* formation. Note that the amide nitrogen, not the a-amino nitrogen, is removed.

Excretion into urine of ammonia produced by renal tubular cells facilitates cation conservation and regulation of acidbase balance. Ammonia production from intracellular renal amino acids, especially glutamine, increases in metabolic acidosis and decreases in metabolic alkalosis.

others serve as carriers of the atoms that ultimately become urea. The major metabolic role of ornithine, citrulline, and argininosuccinate in mammals is urea synthesis. Urea synthesis is a cyclic process. While ammonium ion, CO,, ATP, and aspartate are consumed, the ornithine consumed in reaction 2 is regenerated in reaction 5. Thus, there is no net loss or gain of ornithine, citrulline, argininosuccinate, or arginine. As indicated in Figure 28-16, some reactions of urea synthesis occur in the matrix of the mitochondrion, and other reactions in the cytosol.
Synthesis of 1 mol of urea requires 3 mol of ATP, 1 mol each of ammonium ion and of aspartate, and employs five enzymes (Figure 28-16). Of the six participating amino acids, N-acetylglutamate functions solely as an enzyme activator. The

Condensation of CO,, ammonia, and ATP to form carbamoyl phosphate is catalyzed by mitochondrial carbamoyl phosphate synthetase I (EC 6.3.4.16). A cytosolic form of this
FIGURE 28-16 Reactions and intermediates of urea biosynthesis. The nitrogen-containing groups that contribute to the formation of urea are shaded. Reactions (0) and ®@ occur in the matrix of liver mitochondria and reactions ® ® and © in liver cytosol. CO, (as bicarbonate), ammonium ion, ornithine, and citrulline enter the mitochondrial matrix via specific carriers (see red dots) present in the inner membrane of liver mitochondria.
enzyme, carbamoyl phosphate synthetase II, uses glutamine rather than ammonia as the nitrogen donor and functions in pyrimidine biosynthesis (see Figure 33-9). The concerted action of glutamate dehydrogenase and carbamoyl phosphate synthetase I thus shuttles amino nitrogen into carbamoyl phosphate, a compound with high group transfer potential. Carbamoyl phosphate synthetase I, the rate-limiting enzyme of the urea cycle, is active only in the presence of N-acetylglutamate, an allosteric activator that enhances the affinity of the synthetase for ATP. Synthesis of 1 mol of carbamoyl phosphate requires 2 mol of ATP. One ATP serves as the phosphoryl donor for formation of the mixed acid anhydride bond of carbamoyl phosphate. The second ATP provides the driving force for synthesis of the amide bond of carbamoyl phosphate. The other products are 2 mol of ADP and 1 mol of P, (reaction 1, Figure 28-16). The reaction proceeds stepwise. Reaction of bicarbonate with ATP forms carbonyl phosphate and ADP. Ammonia then displaces ADP, forming carbamate and orthophosphate. Phosphorylation of carbamate by the second ATP then forms carbamoyl phosphate.

L-Ornithine transcarbamoylase (EC 2.1.3.3) catalyzes transfer of the carbamoyl group of carbamoyl phosphate to ornithine, forming citrulline and orthophosphate (reaction 2, Figure 28-16). While the reaction occurs in the mitochondrial matrix, both the formation of ornithine and the subsequent metabolism of citrulline take place in the cytosol. Entry of ornithine into mitochondria and exodus of citrulline from mitochondria therefore involve mitochondrial inner membrane permeases (Figure 28-16).

Argininosuccinate synthetase (EC 6.3.4.5) links aspartate and citrulline via the amino group of aspartate (reaction 3, Figure 28-16) and provides the second nitrogen of urea. The reaction requires ATP and involves intermediate formation of citrullyl-AMP. Subsequent displacement of AMP by aspartate then forms argininosuccinate.

Cleavage of argininosuccinate is catalyzed by argininosuccinate lyase (EC 4.3.2.1). The reaction proceeds with retention of all three nitrogens in arginine and release of the aspartate skeleton as fumarate (reaction 4, Figure 28-16). Subsequent addition of water to fumarate forms L-malate, whose subsequent NAD*-dependent oxidation forms oxaloacetate. These two reactions are analogous to reactions of the citric acid cycle, but are catalyzed by cytosolic fumarase and malate dehydrogenase. Transamination of oxaloacetate by glutamate aminotransferase then reforms aspartate. The carbon skeleton of aspartate-fumarate thus acts as a carrier of the nitrogen of glutamate into a precursor of urea.

Hydrolytic cleavage of the guanidino group of arginine, catalyzed by liver arginase (EC 3.5.3.1), releases urea (reaction 5, Figure 28-16). The other product, ornithine, reenters liver mitochondria and participates in additional rounds of urea synthesis. Ornithine and lysine are potent inhibitors of arginase, and compete with arginine. Arginine also serves as the precursor of the potent muscle relaxant nitric oxide (NO) ina Ca**-dependent reaction catalyzed by NO synthetase.

The activity of carbamoyl phosphate synthetase I is determined by N-acetylglutamate, whose steady-state level is dictated by the balance between its rate of synthesis from acetyl-CoA and glutamate and its rate of hydrolysis to acetate and glutamate, reactions catalyzed by N-acetylglutamate synthetase (NAGS) and N-acetylglutamate deacylase (hydrolase), respectively.
Acetyl-CoA + L-glutamate — N-acetyl-L-glutamate + CoASH N-acetyl-L-glutamate + H,O — t-glutamate + acetate
Major changes in diet can increase the concentrations of individual urea cycle enzymes 10to 20-fold. For example, starvation elevates enzyme levels, presumably to cope with the increased production of ammonia that accompanies enhanced starvation-induced degradation of protein.

The comparatively rare, but well-characterized and medically devastating metabolic disorders associated with the enzymes of urea biosynthesis illustrate the following general principles of inherited metabolic diseases.
1. Similar or identical clinical signs and symptoms can accom- pany various genetic mutations in a gene that encodes a given enzyme or in enzymes that catalyze successive reac- tions in a metabolic pathway.
2. Rational therapy is based on an understanding of the rel- evant biochemical enzyme-catalyzed reactions in both nor- mal and impaired individuals.
3. The identification of intermediates and of ancillary prod- ucts that accumulate prior to a metabolic block provides the basis for metabolic screening tests that can implicate the reaction that is impaired.
4. Definitive diagnosis involves quantitative assay of the activity of the enzyme suspected to be defective.
5. The DNA sequence of the gene that encodes a given mutant enzyme is compared to that of the wild-type gene to identify the specific mutation(s) that cause the disease.
6. The exponential increase in DNA sequencing of human genes has identified dozens of mutations of an affected gene that are benign or are associated with symptoms of varying severity of a given metabolic disorder.

Five well-documented diseases represent defects in the biosynthesis of enzymes of the urea cycle. Molecular genetic analysis has pinpointed the loci of mutations associated with each deficiency, each of which exhibits considerable genetic and phenotypic variability (Table 28-1).
Urea cycle disorders are characterized by hyperammonemia, encephalopathy, and respiratory alkalosis. Four of the five metabolic diseases, deficiencies of carbamoyl phosphate synthetase I, ornithine carbamoyl transferase, argininosuccinate synthetase, and argininosuccinate lyase, result in the accumulation of precursors of urea, principally ammonia and glutamine. Ammonia intoxication is most severe when the metabolic block occurs at reactions 1 or 2 (Figure 28-16), for if citrulline can be synthesized, some ammonia has already been removed by being covalently linked to an organic metabolite.
sufficient protein, arginine, and energy to promote growth and development while simultaneously minimizing the metabolic perturbations.

N-Acetylglutamate is essential for the activity of carbamoyl phosphate synthetase I, EC 6.3.4.16 (reaction 1, Figure 28-16). Defects in carbamoyl phosphate synthetase I are responsible for the relatively rare (estimated frequency 1:62,000) metabolic disease termed "hyperammonemia type 1.

N-Acetylglutamate synthetase, EC 2.3.1.1 (NAGS), catalyzes the formation from acetyl-CoA and glutamate of the Nacetylglutamate essential for carbamoyl phosphate synthetase I activity.
L-Glutamate + acetyl-CoA — N-acetyl-L-glutamate + CoASH
While the clinical and biochemical features of NAGS deficiency are indistinguishable from those arising from a defect in carbamoyl phosphate synthetase I, a deficiency in NAGS may respond to administered N-acetylglutamate.

Clinical symptoms common to all urea cycle disorders include vomiting, avoidance of high-protein foods, intermittent ataxia, irritability, lethargy, and severe mental retardation. The most dramatic clinical presentation occurs in full-term infants who initially appear normal, then exhibit progressive lethargy, hypothermia, and apnea due to high plasma ammonia levels. The clinical features and treatment of all five disorders are similar. Significant improvement and minimization of brain damage can accompany a low-protein diet ingested as frequent small meals to avoid sudden increases in blood ammonia levels. The goal of dietary therapy is to provide
The hyperornithinemia, hyperammonemia, and homocitrullinuria (HHH) syndrome results from mutation of the ORNT1 gene that encodes the mitochondrial membrane ornithine permease. The failure to import cytosolic ornithine into the mitochondrial matrix renders the urea cycle inoperable, with consequent hyperammonemia, and hyperornithinemia due to the accompanying accumulation of cytosolic ornithine. In the absence of its normal acceptor (ornithine), mitochondrial carbamoyl phosphate carbamoylates lysine to homocitrulline, resulting in homocitrullinuria.

The X-chromosome-linked deficiency termed "hyperammonemia type 2" reflects a defect in ornithine transcarbamoylase (reaction 2, Figure 28-16). The mothers also exhibit hyperammonemia and an aversion to high-protein foods. Levels

Carbamoyl-phosphate synthetase 1, Enzyme Catalog Number = 6.3.4.16. Carbamoyl-phosphate synthetase 1, OMIM a Reference = 237300. Carbamoyl-phosphate synthetase 1, Figure and Reaction = 28-13 ➀. Ornithine carbamoyl transferase, Enzyme Catalog Number = 2.1.3.3. Ornithine carbamoyl transferase, OMIM a Reference = 311250. Ornithine carbamoyl transferase, Figure and Reaction = 28-13 ➁. Argininosuccinate synthetase, Enzyme Catalog Number = 6.3.4.5. Argininosuccinate synthetase, OMIM a Reference = 215700. Argininosuccinate synthetase, Figure and Reaction = 28-13 ➂. Argininosuccinate lyase, Enzyme Catalog Number = 4.3.2.1. Argininosuccinate lyase, OMIM a Reference = 608310. Argininosuccinate lyase, Figure and Reaction = 28-13 ➃. Arginase, Enzyme Catalog Number = 3.5.3.1. Arginase, OMIM a Reference = 608313. Arginase, Figure and Reaction = 28-13 ➄
Online Mendelian inheritance in man database: ncbi.nlm.nih.gov/omim/
of glutamine are elevated in blood, cerebrospinal fluid, and urine, probably as a result of enhanced glutamine synthesis in response to elevated levels of tissue ammonia.

In addition to patients who lack detectable argininosuccinate synthetase activity (reaction 3, Figure 28-16), a 25-fold elevated K_, for citrulline has been reported. In the resulting citrullinemia, plasma and cerebrospinal fluid citrulline levels are elevated, and 1 to 2 g of citrulline are excreted daily.

Argininosuccinic aciduria, accompanied by elevated levels of argininosuccinate in blood, cerebrospinal fluid, and urine, is associated with friable, tufted hair (trichorrhexis nodosa). Both earlyand late-onset types are known. The metabolic defect is in argininosuccinate lyase (reaction 4, Figure 28-16). Diagnosis by the measurement of erythrocyte argininosuccinate lyase activity can be performed on umbilical cord blood or amniotic fluid cells.

Hyperargininemia is an autosomal recessive defect in the gene for arginase (reaction 5, Figure 28-16). Unlike other urea cycle disorders, the first symptoms of hyperargininemia typically do not appear until age 2 to 4 years. Blood and cerebrospinal fluid levels of arginine are elevated. The urinary amino acid pattern, which resembles that of lysine-cystinuria (see Chapter 29), may reflect competition by arginine with lysine and cysteine for reabsorption in the renal tubule.

Metabolic diseases caused by the absence or functional impairment of metabolic enzymes can be devastating. Early dietary intervention, however, can in many instances ameliorate the otherwise inevitable dire effects. The early detection of such metabolic diseases is thus is of primary importance. Since the initiation in the United States of newborn screening programs in the 1960s, all states now conduct metabolic screening of newborn infants. The powerful and sensitive technique of tandem mass spectrometry (MS) (see Chapter 4) can in a few minutes detect over 40 analytes of significance in the detection of metabolic disorders. Most states employ tandem MS to screen newborns to detect metabolic disorders such as organic acidemias, aminoacidemias, disorders of fatty acid oxidation, and defects in the enzymes of the urea cycle. An article in Clinical Chemistry 2006 39:315 reviews the theory of tandem Ms, its application to the detection of metabolic disorders, and situations that can yield false positives, and includes a lengthy table of detectable analytes and the relevant metabolic diseases.

Despite results in animal models using an adenoviral vector to treat citrullinemia, at present gene therapy provides no effective solution for human subjects. However, direct CRISPR/ Cas9-based modification of a defective enzyme can restore functional enzyme activity of cultured human pluripotent stem cells.

Human subjects degrade 1 to 2% of their body protein daily at rates that vary widely between proteins and with physiologic state. Key regulatory enzymes often have short half-lives.
™ Proteins are degraded by both ATP-dependent and ATPindependent pathways. Ubiquitin targets many intracellular proteins for degradation. Liver cell surface receptors bind and internalize circulating asialoglycoproteins destined for lysosomal degradation.
= Polyubiquitinated proteins are degraded by proteases on the inner surface of a cylindrical macromolecule, the proteasome. Entry into the proteasome is gated by a donut-shaped protein pore that rejects entry to all but polyubiquitinated proteins.
™ Fishes excrete highly toxic NH, directly. Birds convert NH, to uric acid. Higher vertebrates convert NH, to urea.
| Transamination channels amino acid nitrogen into glutamate. GDH occupies a central position in nitrogen metabolism.
™ Glutamine synthetase converts NH, to nontoxic glutamine.
Glutaminase releases
NH, for
use in
urea synthesis.
= NH,,CO,, and the amide nitrogen of aspartate provide the atoms of urea.
™ Hepatic urea synthesis takes place in part in the mitochondrial matrix and in part in the cytosol.
™ Changes in enzyme levels and allosteric regulation of
carbamoyl phosphate
I
by
N-acetylglutamate regulate
urea biosynthesis.
™ Metabolic diseases are associated with defects in each enzyme of the urea cycle, of the membrane-associated ornithine permease, and of NAGS.
@ The metabolic disorders of urea biosynthesis illustrate six
general principles
of all
metabolic disorders.
- = Tandem mass spectrometry is the technique of choice for screening neonates for inherited metabolic diseases.

- Adam S, Almeida MF, Assoun M et al: Dietary management of
urea cycle
disorders:
European practice.
Mol
Genet
Metab
2013;110:439.
Burgard P, Kélker S, Haege G, et al. Neonatal mortality and outcome at the end of the first year of life in early onset urea cycle
disorders. J Inherit Metab Dis. 2016;39:219.
Dwane
L,
Gallagher
WM,
Ni
Chonghaile
T, et
al:
of non-traditional
ubiquitination
Chem 2017;292:3543.
The in
oncogenic emerging
pathways.
role
J
Biol
Haberle J, Pauli S, Schmidt E, et al: Mild citrullinemia in caucasians is an allelic variant of argininosuccinate synthetase deficiency (citrullinemia type 1). Mol Genet Metab 2003;80:302.
Jiang YH, Beaudet AL: Human disorders of ubiquitination and proteasomal degradation. Curr Opin Pediatr 2004;16:419.
Monné M, Miniero DV, Dabbabbo L, et al: Mitochondrial transporters for ornithine and related amino acids: a review.
Amino Acids 2015;9:1963.
A,
Young
MA,
Donato
NJ:
Emerging potential
of therapeutic
targeting of ubiquitin-specific proteases in the treatment of cancer. Cancer Res 2014;14:721.
Pal
CHAPTER
28
Pickart CM: Mechanisms underlying ubiquitination. Annu Rev
Biochem 2001;70:503.
Sylvestersen KB, Young C, Nielsen ML: Advances in characterizing ubiquitylation sites by mass spectrometry. Curr Opin Chem Biol
2013317:49.
Waisbren SE, Gropman AL: Improving long term outcomes in urea cycle disorders. J Inherit Metab Dis 2016;39:573.

C H A PT ER
2 Name the principal catabolites of the carbon skeletons of the protein amino acids and the major metabolic fates of these catabolites.
. Write an equation for an aminotransferase (transaminase) reaction and illustrate the role played by the coenzyme.
disorders. = Outline the metabolic pathways for each of the protein amino acids, and identify reactions associated with clinically significant metabolic
m= Provide examples of aminoacidurias that arise from defects in glomerular
tubular reabsorption,
and the
consequences of
impaired intestinal
absorption of
symptoms. m= Explain why metabolic defects in different enzymes of the catabolism of a specific amino acid can be associated with similar clinical signs and
= Describe the implications of a metabolic defect in A'-pyrroline5-carboxylate dehydrogenase for the catabolism of proline and of 4-hydroxyproline.
m= Explain how the a-amino nitrogen of proline and of lysine is removed by processes other than transamination.
= Draw analogies between the reactions that participate in the catabolism of fatty acids and of the branched-chain amino acids.
=
Identify the specific metabolic defects in hypervalinemia, maple syrup urine disease, intermittent branched-chain ketonuria, isovaleric acidemia, and methylmalonic aciduria.

thus are essential. The ability to detect the activities of enzymes in cultured amniotic fluid cells facilitates prenatal diagnosis by amniocentesis. In the United States, all states conduct screening tests of newborns for up to 40 metabolic diseases, including disorders associated with defects in the catabolism of amino acids. The most reliable screening tests use tandem mass spectrometry to detect, in a few drops of neonate blood, catabolites suggestive of a given metabolic defect, and thereby implicate the absence or lowered activity of one or more specific enzymes.
Mutations either of a gene or of associated regulatory regions of DNA can result either in the failure to synthesize the encoded enzyme or in synthesis of a partially or completely nonfunctional enzyme. Mutations that affect enzyme activity, those that compromise its three-dimensional structure, or that disrupt its catalytic or regulatory sites, can have severe metabolic consequences. Low catalytic efficiency of a mutant enzyme can result from impaired positioning of residues involved in catalysis, or in binding a substrate, coenzyme, or metal ion. Mutations may also impair the ability of certain enzymes to respond appropriately to the signals that modulate their activity by altering an enzyme' affinity for an allosteric regulator of activity. Since different mutations can have similar effects on any of the above factors, various mutations may give rise to the same clinical signs and symptoms. At a molecular level, these, therefore, are distinct molecular diseases. Present treatment of metabolic disorders of amino acid metabolism consists primarily of feeding diets low in the amino acid whose catabolism is impaired. Ultimately, however, genetic engineering may be able to permanently correct a given metabolic defect.

Nutritional studies in the period 1920 to 1940, reinforced and confirmed by studies using isotopically labeled amino acids conducted from 1940 to 1950, established the interconvertibility of the carbon atoms of fat, carbohydrate, and protein. These studies also revealed that all or a portion of the carbon skeleton of every amino acid is convertible either to carbohydrate, fat, or both fat and carbohydrate (Table 29-1). Figure 29-1 outlines overall aspects of these interconversions.
Removal of a-amino nitrogen by transamination, catalyzed by a transaminase (see Figure 28-9), is the first catabolic reaction of most of the protein amino acids. The exceptions are proline, hydroxyproline, threonine, and lysine, whose a-amino groups do not participate in transamination. The hydrocarbon skeletons that remain are then degraded to amphibolic intermediates as outlined in Figure 29-1.

All four carbons of asparagine and of aspartate form oxaloacetate via reactions catalyzed by asparaginase (EC 3.5.1.1) and a transaminase.
+
H,O
—
Aspartate
+
NH,*
Aspartate + Pyruvate — Alanine + Oxaloacetate

Successive reactions catalyzed by glutaminase (EC 3.5.1.2) and a transaminase form a-ketoglutarate
Glutamine + H,O — Glutamate + NH,* Glutamate + Pyruvate > Alanine + a-Ketoglutarate
While both glutamate and aspartate are substrates for the same transaminase, metabolic defects in transaminases, which fulfill central amphibolic functions, may be incompatible with life. Consequently, no known metabolic defect is associated with these two short catabolic pathways that convert asparagine and glutamine to amphibolic intermediates.

The catabolism of proline takes place in mitochondria. Since proline does not participate in transamination, its a-amino nitrogen is retained throughout a two-stage oxidation to glutamate. Oxidation to A'-pyrroline-5-carboxylate is catalyzed by proline dehydrogenase, EC 1.5.5.2. Subsequent oxidation to glutamate is catalyzed by A'-pyrroline-5-carboxylate dehydrogenase (also called glutamate-y-semialdehyde dehydrogenase, EC 1.2.1.88) (Figure 29-2). There are two metabolic disorders of proline catabolism. Inherited as autosomal recessive traits, both are consistent with a normal adult life. The metabolic block in type I hyperprolinemia is at proline dehydrogenase. There is no associated impairment of hydroxyproline catabolism. The metabolic block in type II hyperprolinemia is at A'-pyrroline-5-carboxylate dehydrogenase, which also participates in the catabolism of arginine, ornithine, and hydroxyproline (see below). Since proline and hydroxyproline catabolism are affected, both A'-pyrroline-5-carboxylate and A!-pyrroline3-hydroxy-5-carboxylate (see Figure 29-11) are excreted.

The initial reactions in arginine catabolism are conversion to ornithine followed by transamination of ornithine to glutamate-y-semialdehyde (Figure 29-3). Subsequent catabolism of glutamate-y-semialdehyde to a-ketoglutarate occurs as described for proline (Figure 29-2). Mutations in ornithine 5-aminotransferase (ornithine transaminase, EC 2.6.1.13) elevate plasma and urinary ornithine, and are associated with gyrate atrophy of the choroid and retina. Treatment involves restricting dietary arginine. In the hyperornithinemia-hyperammonemia syndrome, a defective mitochondrial ornithine-citrulline antiporter (see Figure 28-16) impairs transport of ornithine into mitochondria, where it participates in urea synthesis.

Catabolism of histidine proceeds via urocanate, 4-imidazolone5-propionate, and N-formiminoglutamate (Figlu). Formimino group transfer to tetrahydrofolate forms glutamate, then
|
A'-Pyrolline-5-carboxylate
L-Glutamate-y-semialdehyde
FIGURE 29-2 Catabolism of proline. Red bars and circled numerals indicate the locus of the inherited metabolic defects in @ type-I hyperprolinemia and @) type-II hyperprolinemia. In this and subsequent figures, blue highlights emphasize the portions of the molecules that are chemical undergoing change.
a-ketoglutarate (Figure 29-4). In folic acid deficiency, transfer of the formimino group is impaired, and Figlu is excreted. Excretion of Figlu following a dose of histidine thus can be used to detect folic acid deficiency. Benign disorders of histidine catabolism include histidinemia and urocanic aciduria associated with impaired histidase and urocanase, respectively.

The glycine cleavage complex of liver mitochondria splits glycine to CO, and NH,* and forms N°,N'°-methylene tetrahydrofolate.
FIGURE 29-3 Catabolism of arginine. Arginase-catalyzed cleavage of L-arginine forms urea and L-ornithine. This reaction (red bar) represents the site of the inherited metabolic defect in hyperargininemia. Subsequent transamination of ornithine to glutamate-y-semialdehyde is followed by its oxidation to a-ketoglutarate.
Glycine + H,folate + NAD* > CO, + NH, + 5,10-CH,-H, folate + NADH + H*
The glycine cleavage system (Figure 29-5) consists of three enzymes and an "H-protein" that has a covalently attached dihydrolipoyl moiety. Figure 29-5 also illustrates the individual reactions and intermediates in glycine cleavage. In nonketotic hyperglycinemia, a rare inborn error of glycine degradation, glycine accumulates in all body tissues including the central nervous system. The defect in primary hyperoxaluria is the failure to catabolize glyoxylate formed by the deamination of glycine. Subsequent oxidation of glyoxylate to oxalate results in urolithiasis, nephrocalcinosis, and early mortality from renal failure or hypertension. Glycinuria results from a defect in renal tubular reabsorption.

Following conversion to glycine, catalyzed by glycine hydroxymethyltransferase (EC 2.1.2.1), serine catabolism merges with that of glycine (Figure 29-6).

Transamination of a-alanine forms pyruvate. Probably on account of its central role in metabolism, there is no known metabolic defect of a-alanine catabolism.

Cystine is first reduced to cysteine by cystine reductase, EC 1.8.1.6 (Figure 29-7). Two different pathways then convert
FIGURE 29-4 Catabolism of .-histidine to a-ketoglutarate. (H, folate, tetrahydrofolate.) The red bar indicates the site of an inherited metabolic defect.
cysteine to pyruvate (Figure 29-8). There are numerous abnormalities of cysteine metabolism. Cystine, lysine, arginine, and ornithine are excreted in cystine-lysinuria (cystinuria), a defect in renal reabsorption of these amino acids. Apart from cystine calculi, cystinuria is benign. The mixed disulfide of L-cysteine and L-homocysteine (Figure 29-9) excreted by cystinuric patients is more soluble than cystine and reduces formation of cystine calculi.
Several metabolic defects result in vitamin B,-responsive or vitamin B,-unresponsive homocystinurias. These include
FIGURE
29-5
The glycine
cleavage system
of liver
mito- chondria.
and an
“H-protein”
Catalysts cleavage
complex that
has consists
covalently for
the numbered
nase
(decarboxylating), of
attached reactions
three enzymes
dihyrolipoate.
are
@
glycine
@)
transferase, an
dehydroge- ammonia-forming
and
@)
dihydrolipoamide tetrahydrofolate).
NHe"
oH.
ll fal
oO
L-Serine
FIGURE
29-6
interconversion cine
(H,
NH3*
NH,*
|
CH
~C7™
aminomethyl- dehydrogenase.
(H, folate,
Methylene
H, folate
—""_, of
serine
NHa"
CHa
Ao ll
oO
Glycine and
glycine by

folate, tetrahydrofolate.)
L-Cystine
NADH
o
ll
2CHy
|
SH
oO
L-Cysteine
FIGURE 29-7 Reduction of cystine to cysteine by cystine reductase.
+
H*
H, folate
__»_
gly-
FIGURE
29-8
Two pathways
catabolize cysteine:
the cyste-
ine sulfinate
way pathway
(bottom).
—
CH,
FIGURE 29-9 Structure of the mixed disulfide of cysteine and homocysteine.
(top)
and the
3-mercaptopyruvate path-
a deficiency in the reaction catalyzed by cystathionine B-synthase, EC 4.2.1.22:
Serine + homocysteine — cystathionine + H,O
Consequences include osteoporosis and mental retardation. Defective carrier-mediated transport of cystine results in cystinosis (cystine storage disease) with deposition of cystine crystals in tissues and early mortality from acute renal failure. Epidemiologic and other data link plasma homocysteine levels to cardiovascular risk, but the role of homocysteine as a causal cardiovascular risk factor remains controversial.

Threonine aldolase (EC 4.1.2.5) cleaves threonine to glycine and acetaldehyde. Catabolism of glycine is discussed above. Oxidation of acetaldehyde to acetate is followed by formation of acetyl-CoA (Figure 29-10).

Catabolism of 4-hydroxy-L-proline forms, successively, L-A'pyrroline-3-hydroxy-5-carboxylate, y-hydroxy-.-glutamatey-semialdehyde, erythro-y-hydroxy-.-glutamate, and a-ketoy-hydroxyglutarate. An aldol-type cleavage then forms glyoxylate plus pyruvate (Figure 29-11). A defect in 4-hydroxyproline
FIGURE 29-10 intermediates in the conversion of threonine to glycine and acetyl-CoA.
FIGURE
29-11
Intermediates in
hydroxyproline catabolism.
(a-AA, a-amino
acid;
a-KA, a-keto
acid.)
the inherited
metabolic defects
in
@® type Il hyperprolinemia.
Red bars
indicate the
sites
@)
of hyperhydroxyprolinemia
and
dehydrogenase results in hyperhydroxyprolinemia, which is benign. There is no associated impairment of proline catabolism. A defect in glutamate-y-semialdehyde dehydrogenase is accompanied by excretion of A'-pyrroline-3-hydroxy5-carboxylate.

Figure 29-12 illustrates the intermediates and enzymes that participate in the catabolism of tyrosine to amphibolic intermediates. Following transamination of tyrosine to p-hydroxyphenylpyruvate, successive reactions form homogentisate, maleylacetoacetate, fumarylacetoacetate, fumarate, acetoacetate, and ultimately acetyl-CoA and acetate.
Several metabolic disorders are associated with the tyrosine catabolic pathway. The probable metabolic defect in type I tyrosinemia (tyrosinosis) is at fumarylacetoacetate hydrolase, EC 3.7.1.12 (reaction 4, Figure 29-12). Therapy employs a diet low in tyrosine and phenylalanine. Untreated acute and chronic tyrosinosis leads to death from liver failure. Alternate metabolites of tyrosine are also excreted in type II tyrosinemia (Richner-Hanhart syndrome), a defect in tyrosine aminotransferase (reaction 1, Figure 29-12), and in neonatal tyrosinemia, due to lowered activity of p-hydroxyphenylpyruvate hydroxylase, EC 1.13.11.27 (reaction 2, Figure 29-12). Therapy employs a diet low in protein.
The metabolic defect in alkaptonuria is a defective homogentisate oxidase (EC 1.13.11.5), the enzyme that catalyzes reaction 3 of Figure 29-12. The urine darkens on exposure to air due to oxidation of excreted homogentisate. Late in the disease, there is arthritis and connective tissue pigmentation (ochronosis) due to oxidation of homogentisate to benzoquinone acetate, which polymerizes and binds to connective tissue. First described in the 16th century based on the observation that the urine darkened on exposure to air, alkaptonuria provided the basis for Sir Archibald Garrod's early 20th century classic ideas concerning heritable metabolic disorders. Based on the presence of ochronosis and on chemical evidence, the earliest known case of alkaptonuria is, however, its 1977 detection in an Egyptian mummy dating from 1500 B.c.

Phenylalanine is first converted to tyrosine (see Figure 27-12). Subsequent reactions are those of tyrosine (Figure 29-12). Hyperphenylalaninemias arise from defects in phenylalanine hydroxylase, EC 1.14.16.1 (type I, classic phenylketonuria [PKU], frequency 1 in 10,000 births), in dihydrobiopterin reductase (types II and III), or in dihydrobiopterin biosynthesis (types IV and V) (see Figure 27-12). Alternative catabolites are excreted (Figure 29-13). A diet low in phenylalanine can prevent the mental retardation of PKU.
DNA probes facilitate prenatal diagnosis of defects in phenylalanine hydroxylase or dihydrobiopterin reductase. Elevated blood phenylalanine may not be detectable until 3 to 4 days postpartum. False positives in premature infants may reflect delayed maturation of enzymes of phenylalanine catabolism. An older and less reliable screening test employs FeCl, to detect urinary phenylpyruvate. FeCl, screening for PKU of the urine of newborn infants is compulsory in many countries, but in the United States has been largely supplanted by tandem mass spectrometry.

Removal of the e-nitrogen of lysine proceeds via initial formation of saccharopine and subsequent reactions that also liberate the a-nitrogen. The ultimate product of the carbon skeleton is crotonyl-CoA. Circled numerals refer to the corresponding numbered reactions of Figure 29-14. Reactions 1 and 2 convert the Schiff base formed between a-ketoglutarate and the e-amino group of lysine to L-a-aminoadipate-5-semialdehyde. Reactions 1 and 2 both are catalyzed by a single bifunctional enzyme, aminoadipate-5-semialdehyde synthase (EC 1.5.1.8) whose N-terminal and C-terminal domains contain lysine-a-ketoglutarate reductase and saccharopine dehydrogenase activity, respectively. Reduction of L-a-aminoadipate5-semialdehyde to L-a-aminoadipate (reaction 3) is followed by transamination to a-ketoadipate (reaction 4). Conversion to the thioester glutaryl-CoA (reaction 5) is followed by the decarboxylation of glutaryl-CoA to crotonyl-CoA (reaction 6). Reduction of crotonyl-CoA by crotanoyl-CoA reductase, EC 1.3.1.86, forms butanoyl-CoA:
Crotonyl-CoA + NADPH + H*-— butanoyl-CoA + NADP*
Subsequent reactions are those of fatty acid catabolism (see Chapter 22).
Hyperlysinemia can result from a metabolic defect in either the first or second activity of the bifunctional enzyme aminoadipate-5-semialdehyde synthase, but this is accompanied by elevated levels of blood saccharopine only if the defect involves the second activity. A metabolic defect at reaction 6 results in an inherited metabolic disease that is associated with striatal and cortical degeneration, and is characterized by elevated concentrations of glutarate and its metabolites glutaconate and 3-hydroxyglutarate. The challenge in management of these metabolic defects is to restrict dietary intake of L-lysine without producing malnutrition.

Tryptophan is degraded to amphibolic intermediates via the kynurenine-anthranilate pathway (Figure 29-15). Tryptophan 2,3-dioxygenase, EC 1.13.11.11 (tryptophan pytrolase) opens the indole ring, incorporates molecular
- FIGURE 29-12 intermediates in tyrosine catabolism. Carbons are numbered to emphasize their ultimate fate. (a-KG, a-ketoglutarate; Glu, glutamate; PLP, pyridoxal phosphate.) Red bars indicate the probable sites of the inherited metabolic defects in type Il tyrosinemia; neonatal tyrosinemia; ® alkaptonuria; and @) type | tyrosinemia, or tyrosinosis. @) alkaptonuria; and @ type | tyrosinemia, or tyrosinosis.
FIGURE 29-13 Alternative pathways of phenylalanine catabolism in phenylketonuria. The reactions also occur in normal liver tissue but are of minor significance.
oxygen, and forms N-formylkynurenine. Tryptophan oxygenase, an iron porphyrin metalloprotein that is inducible in liver by adrenal corticosteroids and by tryptophan, is feedback inhibited by nicotinic acid derivatives, including NADPH. Hydrolytic removal of the formyl group of N-formylkynurenine, catalyzed by kynurenine formylase (EC 3.5.1.9), produces kynurenine. Since kynureninase (EC 3.7.1.3) requires pyridoxal phosphate, excretion of xanthurenate (Figure 29-16) in response to a tryptophan load is diagnostic of vitamin B, deficiency. Hartnup disease reflects impaired intestinal and renal transport of tryptophan and other neutral amino acids. Indole derivatives of unabsorbed tryptophan formed by intestinal bacteria are excreted. The defect limits tryptophan availability
FIGURE 29-14 Reactions and intermediates in the catabolism of lysine.
for niacin biosynthesis and accounts for the pellagra-like signs and symptoms.

Methionine reacts with ATP forming S-adenosylmethionine, "active methionine" (Figure 29-17). Subsequent reactions form propionyl-CoA (Figure 29-18), whose conversion to succinyl-CoA occurs via reactions 2, 3 & 4 of Figure 19-2.

The first three reactions of the catabolism of isoleucine, leucine, and valine (Figure 29-19) are analogous to reactions of fatty acid catabolism (see Figure 22-3). Following transamination (Figure 29-19, reaction 1), the carbon skeletons of the resulting a-keto acids undergo oxidative decarboxylation and conversion to coenzyme A thioesters. This multistep process is catalyzed by the mitochondrial branched-chain a-ketoacid dehydrogenase complex, whose components are functionally identical to those of the pyruvate dehydrogenase
FIGURE 29-15 Reactions and intermediates in the catabolism of tryptophan. (PLP, pyridoxal phosphate.)

Xanthurenate
FIGURE 29-16 Formation of xanthurenate in vitamin B, deficiency. Conversion of the tryptophan metabolite 3-hydroxykynurenine to 3-hydroxyanthranilate is impaired (see Figure 29-15). A large portion is therefore converted to xanthurenate.
complex (PDH) (see Figure 18-5). Like PDH, the branchedchain a-ketoacid dehydrogenase complex consists of five components.
El: thiamin pyrophosphate (TPP)-dependent branchedchain a-ketoacid decarboxylase
E2: dihydrolipoyl transacylase (contains lipoamide)
E3: dihydrolipoamide dehydrogenase (contains FAD)
As for pyruvate dehydrogenase, the protein kinase and protein phosphatase regulate activity of the branched-chain a-ketoacid dehydrogenase complex via phosphorylation (inactivation) and dephosphorylation (activation). Dehydrogenation of the resulting coenzyme A thioesters (reaction 3, Figure 29-19) proceeds like the dehydrogenation of lipid-derived fatty acyl-CoA thioesters (see Chapter 22). Subsequent reactions that are unique for each amino acid skeleton are given in Figures 29-20, 29-21, and 29-22.

As the name implies, the odor of urine in maple syrup urine disease (branched-chain ketonuria, or MSUD) suggests maple syrup, or burnt sugar. The biochemical defect in MSUD involves the a-ketoacid decarboxylase complex (reaction 2, Figure 29-19). Plasma and urinary levels of leucine, isoleucine, valine, and their a-keto acids and a-hydroxy acids (reduced a-keto acids) are elevated, but the urinary keto acids derive principally from leucine. Signs and symptoms of MSUD often include fatal ketoacidosis, neurological derangements, mental retardation, and a maple syrup odor of urine. The mechanism of toxicity is unknown. Early diagnosis by enzymatic analysis is essential to avoid brain damage and early mortality by replacing dietary protein by an amino acid mixture that lacks leucine, isoleucine, and valine.
The molecular genetics of MSUD are heterogeneous. MSUD can result from mutations in the genes that encode Ela, Elf, E2, and E3. Based on the locus affected, genetic subtypes of MSUD are recognized. Type IA MSUD arises from mutations in the Ela gene, type IB in the Elf gene, type II in the E2 gene, and type III in the E3 gene (Table 29-2). In intermittent branched-chain ketonuria, the a-ketoacid decarboxylase retains some activity, and symptoms occur later in life. In isovaleric acidemia, ingestion of protein-rich foods elevates isovalerate, the deacylation product of isovaleryl-CoA. The impaired enzyme in isovaleric acidemia is isovaleryl-CoA
FIGURE 29-19 The first three reactions in the catabolism of leucine, valine, and isoleucine. Note the analogy of reactions 2 and 3 to reactions of the catabolism of fatty acids (see Figure 22-3). The analogy to fatty acid catabolism continues, as shown in subsequent figures.

dehydrogenase, EC 1.3.8.4 (reaction 3, Figure 29-19). Vomiting, acidosis, and coma follow ingestion of excess protein. Accumulated isovaleryl-CoA is hydrolyzed to isovalerate and excreted.

E1α, Branched-Chain α-Ketoacid Decarboxylase Component = α-Ketoacid decarboxylase. E1α, OMIM a Reference = 608348. E1α, Maple Syrup Urine Disease = Type 1A. E1β, Branched-Chain α-Ketoacid Decarboxylase Component = α-Ketoacid decarboxylase. E1β, OMIM a Reference = 248611. E1β, Maple Syrup Urine Disease = Type 1B. E2, Branched-Chain α-Ketoacid Decarboxylase Component = Dihydrolipoyl transacylase. E2, OMIM a Reference = 608770. E2, Maple Syrup Urine Disease = Type II. E3, Branched-Chain α-Ketoacid Decarboxylase Component = Dihydrolipoamide dehydrogenase. E3, OMIM a Reference = 238331. E3, Maple Syrup Urine Disease = Type III
Online Mendelian Inheritance in Man database: ncbi.nlm.nih.gov/omim/.
FIGURE 29-22 Subsequent catabolism of the methacrylylCoA formed from t-valine (see Figure 29-19). (a-AA, a-amino acid; a-KA, a-keto acid.)
Table 29-3 summarizes the metabolic disorders associated with the catabolism of amino acids, and lists the impaired enzyme, its IUB enzyme catalog (EC) number, a cross-reference to a specific figure, and numbered reaction in this text, and a numerical link to the Online Mendelian Inheritance in Man database (OMIM).

= 29-3 ➁. Phenylalanine hydroxylase, Enzyme Catalog Number = 1.14.16.1. Phenylalanine hydroxylase, OMIM a Reference = 261600. Phenylalanine hydroxylase, Major Signs and Symptoms = Type I (classic) phenylketonuria. Phenylalanine hydroxylase, Figure and Reaction = 27-9 ➀. Proline dehydrogenase, Enzyme Catalog Number = 1.5.5.2. Proline dehydrogenase, OMIM a Reference = 606810. Proline dehydrogenase, Major Signs and Symptoms = Type I hyperprolinemia. Proline dehydrogenase, Figure and Reaction = 29-2 ➀. Δ 1 -Pyrroline-5-carboxylate dehydrogenase, Enzyme Catalog Number = 1.2.1.88. Δ 1 -Pyrroline-5-carboxylate dehydrogenase, OMIM a Reference = 606811. Δ 1 -Pyrroline-5-carboxylate dehydrogenase, Major Signs and Symptoms = Type II hyperprolinemia & hyper 4-hydroxyprolinemia. Δ 1 -Pyrroline-5-carboxylate dehydrogenase, Figure and Reaction = 29-2 ➁. Saccharopine dehydrogenase, Enzyme Catalog Number = 1.5.1.7. Saccharopine dehydrogenase, OMIM a Reference = 268700. Saccharopine dehydrogenase, Major Signs and Symptoms = Saccharopinuria. Saccharopine dehydrogenase, Figure and Reaction = 29-14 ➁. Tyrosine aminotransferase, Enzyme Catalog Number = 2.6.1.5. Tyrosine aminotransferase, OMIM a Reference = 613018. Tyrosine aminotransferase, Major Signs and Symptoms = Type II tyrosinemia. Tyrosine aminotransferase, Figure and Reaction = 29-12 ➀
*Online Mendelian Inheritance in Man database: ncbi.nlm.nih.gov/omim/.

■, 1 = Excess amino acids are catabolized to amphibolic intermediates that serve as sources of energy or for the biosynthesis of carbohydrates and lipids.. ■, 1 = Transamination is the most common initial reaction of amino acid catabolism. Subsequent reactions remove any additional nitrogen and restructure hydrocarbon skeletons for conversion to oxaloacetate, α-ketoglutarate, pyruvate, and acetyl-CoA.. ■, 1 = Metabolic diseases associated with glycine catabolism include glycinuria and primary hyperoxaluria.. ■, 1 = Two distinct pathways convert cysteine to pyruvate. Metabolic disorders of cysteine catabolism include cystine-lysinuria, cystine storage disease, and the homocystinurias.. ■, 1 = Threonine catabolism merges with that of glycine after threonine aldolase cleaves threonine to glycine and acetaldehyde.. ■, 1 = Following transamination, the carbon skeleton of tyrosine is degraded to fumarate and acetoacetate. Metabolic diseases of tyrosine catabolism include tyrosinosis, Richner-Hanhart syndrome, neonatal tyrosinemia, and alkaptonuria.. ■, 1 = Metabolic disorders of phenylalanine catabolism include PKU and several hyperphenylalaninemias.. ■, 1 = Neither nitrogen of lysine participates in transamination. The same net effect is, however, achieved by the intermediate formation of saccharopine. Metabolic diseases of lysine
catabolism include periodic and persistent forms of hyperlysinemia-ammonemia. ™ The catabolism of leucine, valine, and isoleucine presents many analogies to fatty acid catabolism. Metabolic disorders of branched-chain amino acid catabolism include hypervalinemia, maple syrup urine disease, intermittent branched-chain ketonuria, isovaleric acidemia, and methylmalonic aciduria.

Bliksrud
YT,
Brodtkorb
E,
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PA, et
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I, de novo mutation in liver tissue suppressing an inborn splicing defect. J Mol Med 2005;83:406.
Dobrowolski
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AL,
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R, et
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Biochemical characterization
of mutant phenylalanine hydroxylase enzymes and correlation with clinical presentation in hyperphenylalaninaemic patients. J
Inherit Metab Dis 2009;32:10.
Garg U, Dasouki M: Expanded newborn screening of inherited metabolic disorders by tandem mass spectrometry. Clinical and
laboratory aspects. Clin Biochem 2006;39:315.
Geng J, Liu A: Heme-dependent dioxygenases in tryptophan oxidation. Arch Biochem Biophys 2014;44:18.
Heldt
K,
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I, et
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urine disease
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screening allows
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without extraneous detoxification. Mol Genet Metab 2005;84:313.
Houten SM, Te Brinke H, Denis S, et al: Genetic basis of hyperlysinemia. Orphanet J Rare Dis 2013;8:57.
Lamp J, Keyser B, Koeller DM, et al: Glutaric aciduria type 1
metabolites impair the succinate transport from astrocytic to neuronal cells. J Biol Chem 2011;286:17-777.
Mayr JA, Feichtinger RG, Tort F, et al: Lipoic acid biosynthesis defects. J Inherit Metab Dis 2014;37:553.
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C H A PT ER
2 Cite examples how amino acids participate in a variety biosynthetic
.
coenzyme A. = Indicate the contribution of cysteine and of B-alanine to the structure of
and sarcosine. Document the role of glycine in the biosynthesis of heme, purines, creatine,
histamine. = Identify the reaction that converts an amino acid to the neurotransmitter
of of processes other than protein synthesis.
Outline how arginine participates in the biosynthesis of creatine, nitric oxide (NO), putrescine, spermine, and spermidine.
Discuss the role played by glycine in drug catabolism and excretion.
Document the role of S-adenosylmethionine in metabolism.
Recognize the structures of tryptophan metabolites serotonin, melatonin, tryptamine, and indole 3-acetate.
Describe how tyrosine gives rise to norepinephrine and epinephrine.
Illustrate the key roles of peptidyl serine, threonine, and tyrosine in metabolic regulation and signal transduction pathways.
= Diagram the roles of glycine, arginine, and S-adenosylmethionine in the biosynthesis of creatine.
Explain the role of creatine phosphate in energy homeostasis.
Illustrate the formation of y-aminobutyrate (GABA) and the rare metabolic disorders associated with defects in GABA catabolism.

Certain proteins contain amino acids that have been posttranslationally modified to permit them to perform specific functions. Examples include the carboxylation of glutamate to form y-carboxyglutamate, which functions in Ca** binding, the hydroxylation of proline for incorporation into the collagen triple helix, and the hydroxylation of lysine to 5-hydroxylysine, whose subsequent modification and crosslinking stabilize maturing collagen fibers. In addition to serving as the building blocks for protein synthesis, amino acids serve as precursors of biologic materials as diverse and important as heme, purines, pyrimidines, hormones, neurotransmitters, and biologically active peptides. Histamine plays a central role in many allergic reactions. Neurotransmitters derived from amino acids include y-aminobutyrate (GABA), 5-hydroxytryptamine (serotonin), dopamine, norepinephrine, and epinephrine. Many drugs used to treat neurologic and psychiatric conditions act by altering the metabolism of these neurotransmitters. Discussed below are the metabolism and metabolic roles of selected aand non-a-amino acids.

Alanine serves as a carrier of ammonia and of the carbons of pyruvate from skeletal muscle to liver via the Cori cycle
FIGURE 30-1 Arginine, ornithine, and proline metabolism. Reactions with solid arrows all occur in mammalian tissues. Putrescine and spermine synthesis occurs in both mammals and bacteria. Arginine phosphate of invertebrate muscle functions as a phosphagen analogous to creatine phosphate of mammalian muscle.
(see Chapters 19 & 28), and together with glycine constitutes
a major fraction of the free amino acids in plasma.

Figure 30-1 summarizes the metabolic fates of arginine. In addition to serving as a carrier of nitrogen atoms in urea biosynthesis (see Figure 28-16), the guanidino group of arginine is incorporated into creatine, and following conversion to ornithine, its carbon skeleton becomes that of the polyamines putrescine and spermine (see below).
The reaction catalyzed by nitric oxide synthase, EC 1.14.13.39 (Figure 30-2), a five-electron oxidoreductase with multiple cofactors, converts one nitrogen of the guanidine group of arginine to L-ornithine and nitric oxide, an intercellular signaling molecule that serves as a neurotransmitter, smooth muscle relaxant, and vasodilator (see Chapter 51).

Cysteine participates in the biosynthesis of coenzyme A (see Chapter 44) by reacting with pantothenate to form 4-phosphopantothenoylcysteine. Taurine, formed from cystreine, can displace the coenzyme A moiety of cholyl-CoA to form the bile acid taurocholic acid (see Chapter 26). The conversion of cysteine to taurine involves catalysis by the nonheme Fe** enzyme cysteine dioxygenase, (EC 1.13.11.20), sulfinoalanine decarboxylase (EC 4.1.1.29), and hypotaurine dehydrogenase
(EC 1.8.1.3) (Figure 30-3).
FIGURE
30-3
cysteine to
taurine.
The reac-
tions are catalyzed by cysteine dioxygenase, cysteine sulfinate decar- boxylase, and hypotaurine decarboxylase, respectively.
FIGURE 30-4 Biosynthesis of hippurate. Analogous reactions occur with many acidic drugs and catabolites.

Many relatively apolar metabolites are converted to water-soluble glycine conjugates. An example is the hippuric acid formed from the food additive benzoate (Figure 30-4). Many drugs, drug metabolites, and other compounds with carboxyl groups are conjugated with glycine. This makes them more water soluble and thereby facilitates their excretion in the urine. Glycine is incorporated into creatine, and the nitrogen and a-carbon of glycine are incorporated into the pyrrole rings and the methylene bridge carbons of heme (see Chapter 31), and the entire glycine molecule becomes atoms 4, 5, and 7 of the purines (see Figure 33-1).

Decarboxylation of histidine to histamine is catalyzed by the pyridoxal 5'-phosphate-dependent enzyme histidine decarboxylase, EC 4.1.1.22 (Figure 30-5). A biogenic amine that functions in allergic reactions and gastric secretion, histamine is present in all tissues. Its concentration in the brain hypothalamus varies in
FIGURE 30-5 The reaction catalyzed by histidine decarboxylase.
Homocarnosine
FIGURE 30-6 Derivatives of histidine. Colored boxes surround the components not derived from histidine. The SH group of ergothioneine derives from cysteine.
accordance with a circadian rhythm. Histidine compounds present in the human body include carnosine, and dietarily derived ergothioneine and anserine (Figure 30-6). Carnosine (-alanylhistidine) and homocarnosine (y-aminobutyryl-histidine) are major constituents of excitable tissues, brain, and skeletal muscle. Urinary levels of 3-methylhistidine are unusually low in patients with Wilson disease.

The major nonprotein fate of methionine is conversion to S-adenosylmethionine, the principal source of methyl groups in the body. Biosynthesis of S-adenosylmethionine from methionine and ATP is catalyzed by methionine adenosyltransferase (MAT), EC 2.5.1.6 (Figure 30-7). Human tissues contain three MAT isozymes: MAT-1 and MAT-3 of liver and MAT-2 of nonhepatic tissues. Although hypermethioninemia can result from severely decreased hepatic MAT-1 and MAT-3 activity, if there is residual MAT-1 or MAT-3 activity and MAT-2 activity is normal, a high tissue
FIGURE
30-7
Biosynthesis lyzed
by of
S-adenosylmethionine, cata-
methionine adenosyltransferase.
concentration of methionine will ensure synthesis of adequate amounts of S-adenosylmethionine. Following decarboxylation of S-adenosylmethionine by methionine decarboxylase (EC 4.1.1.57), three carbons and the a-amino group of methionine contribute to the biosynthesis of the polyamines spermine and spermidine. These polyamines function in cell proliferation and growth, are growth factors for cultured mammalian cells, and stabilize intact cells, subcellular organelles, and membranes. Pharmacologic doses of polyamines are hypothermic and hypotensive. Since they bear multiple positive charges, polyamines readily associate with DNA and RNA. Figure 30-8 summarizes the biosynthesis of polyamines from methionine and ornithine, and Figure 30-9 the catabolism of polyamines.
Spermine
FIGURE 30-9 Catabolism of polyamines.

Serine participates in the biosynthesis of sphingosine (see Chapter 24), and of purines and pyrimidines, where it provides carbons 2 and 8 of purines and the methyl group of thymine (see Chapter 33). Genetic defects in cystathionine B-synthase (EC 4.2.1.22)
Serine + Homocysteine — Cystathionine + H,O
a heme protein that catalyzes the pyridoxal 5'-phosphatedependent condensation of serine with homocysteine to form cystathionine, result in homocystinuria. Finally, serine (not cysteine) serves as the precursor of peptidyl selenocysteine (see Chapter 27).

Following hydroxylation of tryptophan to 5-hydroxytryptophan by liver tryptophan hydroxylase (EC 1.14.16.4), subsequent decarboxylation forms serotonin (5-hydroxytryptamine), a potent vasoconstrictor and stimulator of smooth muscle contraction. Catabolism of serotonin is initiated by deamination to 5-hydroxyindole-3-acetate, a reaction catalyzed by monoamine oxidase, EC 1.4.3.4 (Figure 30-10). The psychic stimulation that follows administration of iproniazid results from its ability to prolong the action of serotonin by inhibiting monoamine oxidase. In carcinoid (argentaffinoma), tumor cells overproduce serotonin. Urinary metabolites of serotonin in patients with carcinoid include N-acetylserotonin glucuronide and the glycine conjugate of 5-hydroxyindoleacetate. Serotonin and 5-methoxytryptamine are metabolized to the corresponding acids by monoamine oxidase. N-Acetylation of serotonin, followed by its O-methylation in the pineal body, forms melatonin. Circulating melatonin is taken up by all tissues, including brain, but is rapidly metabolized by hydroxylation followed by conjugation with sulfate or with glucuronic acid. Kidney tissue, liver tissue, and fecal bacteria all convert tryptophan to tryptamine, then to indole 3-acetate. The principal normal urinary catabolites of tryptophan are 5-hydroxyindoleacetate and indole 3-acetate (Figure 30-10).

Neural cells convert tyrosine to epinephrine and norepinephrine (Figure 30-11). While dopa is also an intermediate in the formation of melanin, different enzymes hydroxylate tyrosine in melanocytes. DOPA decarboxylase (EC 4.1.1.28), a pyridoxal phosphate-dependent enzyme, forms dopamine. Subsequent hydroxylation, catalyzed by dopamine B-oxidase (EC 1.14.17.1), then forms norepinephrine. In the adrenal medulla, phenylethanolamine N-methyltransferase (EC 2.1.1.28) utilizes S-adenosylmethionine to methylate the primary amine of norepinephrine, forming epinephrine (Figure 30-11). Tyrosine is also a precursor of triiodothyronine and thyroxine (see Chapter 41).

The phosphorylation and dephosphorylation of specific seryl, threonyl, or tyrosyl residues of proteins regulate the activity of certain enzymes of lipid and carbohydrate metabolism and of proteins that participate in signal transduction cascades (see Chapter 42).

Thebiosynthesisand catabolism ofsarcosine (N-methylglycine) occur in mitochondria. Formation of sarcosine from dimethyl glycine is catalyzed by the flavoprotein dimethyl glycine dehydrogenase EC 1.5.8.4, which requires reduced pteroylpentaglutamate (TPG).
Dimethylglycine + FADH, + H,TPG + H,O — Sarcosine + N-formyl-TPG
Traces of sarcosine can also arise by methylation of glycine, a reaction catalyzed by glycine N-methyltransferase, EC 2.1.1.20.
Glycine + S-Adenosylmethionine — Sarcosine
+ S-Adenosylhomocysteine
301
FIGURE 30-10 Biosynthesis and metabolism of serotonin and melatonin. ([NH,*], by transamination; MAO, monoamine oxidase; ~CH,, from S-adenosylmethionine.)
Catabolism of sarcosine to glycine, catalyzed by the flavoprotein sarcosine dehydrogenase EC 1.5.8.3, also requires reduced pteroylpentaglutamate.

Sarcosine
+
FAD
+
H,TPG
+
H,O
—
Glycine
+
FADH,
+
N-formyl-TPG
The demethylation reactions that form and degrade sarcosine represent important sources of one-carbon units. FADH, is reoxidized via the electron transport chain (see Chapter 13).
Creatinine is formed in muscle from creatine phosphate by irreversible, nonenzymatic dehydration, and loss of phosphate (Figure 30-12). Since the 24-hour urinary excretion of creatinine is proportionate to muscle mass, it provides a measure of whether a complete 24-hour urine specimen has been collected. Glycine, arginine, and methionine all participate in creatine biosynthesis. Synthesis of creatine is completed by methylation of guanidoacetate by S-adenosylmethionine (Figure 30-12).
[chen
FIGURE 30-11 Conversion of tyrosine to epinephrine and norepinephrine in neuronal and adrenal cells. (PLP, pyridoxal phosphate.)

Non-a-amino acids present in tissues in a free form include B-alanine, B-aminoisobutyrate, and GABA. B-Alanine is also present in combined form in coenzyme A, and in the B-alanyl dipeptides carnosine, anserine, and homocarnosine (see below).

B-Alanine and B-aminoisobutyrate are formed during catabolism of the pyrimidines uracil and thymine, respectively (see Figure 33-9). Traces of B-alanine also result from the

Conversion of glycine and the guanidine group of arginine to creatine and creatine phosphate. Also shown is the nonenzymic hydrolysis of creatine phosphate to creatinine.
hydrolysis of B-alanyl dipeptides by the enzyme carnosinase, EC 3.4.13.20. B-Aminoisobutyrate also arises by transamination of methylmalonate semialdehyde, a catabolite of L-valine

The initial reaction of B-alanine catabolism is transamination to malonate semialdehyde. Subsequent transfer of coenzyme A from succinyl-CoA forms malonyl-CoA semialdehyde, which is then oxidized to malonyl-CoA and decarboxylated to the amphibolic intermediate acetyl-CoA. Analogous reactions characterize the catabolism of B-aminoisobutyrate. Transamination forms methylmalonate semialdehyde, which is converted to the amphibolic intermediate succinyl-CoA by
FIGURE 30-13 Metabolism of y-aminobutyrate. (a-AA, a-amino acids; a-KA, a-keto acids; PLP, pyridoxal phosphate.)
reactions 8V and 9V of Figure 29-22. Disorders of B-alanine and B-aminoisobutyrate metabolism arise from defects in enzymes of the pyrimidine catabolic pathway. Principal among these are disorders that result from a total or partial deficiency of dihydropyrimidine dehydrogenase (see Chapter 33).

The f-alanyl dipeptides carnosine and anserine (N-methylcarnosine) (Figure 30-6) activate myosin ATPase (EC 3.6.4.1), chelate copper, and enhance copper uptake. B-Alanyl-imidazole buffers the pH of anaerobically contracting skeletal muscle. Biosynthesis of carnosine is catalyzed by carnosine synthetase (EC 6.3.2.11) in a two-stage reaction that involves initial formation of an enzyme-bound acyl-adenylate of B-alanine and subsequent transfer of the B-alanyl moiety to L-histidine.
ATP + B-Alanine — B-Alanyl-AMP + PP,
B-Alanyl-AMP + L-Histidine > Carnosine + AMP
Hydrolysis of carnosine to B-alanine and t-histidine is catalyzed by carnosinase. The heritable disorder carnosinase deficiency is characterized by carnosinuria.
Homocarnosine (Figure 30-6, present in human brain at higher levels than carnosine, is synthesized in brain tissue by carnosine synthetase. Serum carnosinase does not hydrolyze homocarnosine. Homocarnosinosis, a rare genetic disorder, is associated with progressive spastic paraplegia and mental retardation.

GABA functions in brain tissue as an inhibitory neurotransmitter by altering transmembrane potential differences. GABA is formed by decarboxylation of glutamate by L-glutamate decarboxylase, EC 4.1.1.15 (Figure 30-13). Transamination of GABA forms succinate semialdehyde, which can be reduced to y-hydroxybutyrate by t-lactate dehydrogenase, or be oxidized to succinate and thence via the citric acid cycle to CO, and H,O (Figure 30-13). A rare genetic disorder of GABA metabolism involves a defective GABA aminotransferase EC 2.6.1.19, an enzyme that participates in the catabolism of GABA subsequent to its postsynaptic release in brain tissue. Defects in succinic semialdehyde dehydrogenase, EC 1.2.1.24 (Figure 30-13) are responsible for 4-hydroxybutyric aciduria, a rare metabolic disorder of GABA catabolism characterized by the presence of 4-hydroxybutyrate in urine, plasma, and cerebrospinal fluid (CSF). No present treatment is available for the accompanying mild-to-severe neurologic symptoms.

- ® In addition to serving structural and functional roles in
proteins, a-amino acids participate in a wide variety of other biosynthetic processes.
- Arginine provides the formamidine group of creatine and the
nitrogen of
NO.
Via ornithine,
arginine provides
the skeleton
of the polyamines putrescine, spermine, and spermidine.

™ Cysteine provides the thioethanolamine portion of coenzyme A, and following its conversion to taurine, is part of the bile acid taurocholic acid.
™ Glycine participates in the biosynthesis of heme, purines, creatine, and N-methylglycine (sarcosine). Many drugs and drug metabolites are excreted as glycine conjugates. This enhances their water solubility for urinary excretion.
™ Decarboxylation of histidine forms the neurotransmitter histamine. Histidine compounds present in the human body include ergothioneine, carnosine, and anserine.
m S-Adenosylmethionine, the principal source of methyl groups in metabolism, contributes its carbon skeleton to the biosynthesis of the polyamines spermine and spermidine.
® In addition to its roles in phospholipid and sphingosine
biosynthesis, serine provides carbons 2 and 8 of purines and the methyl group of thymine.
= Key tryptophan metabolites include serotonin and melatonin. Kidney and liver tissue, and also fecal bacteria, convert tryptophan to tryptamine and thence to indole 3-acetate. The principal tryptophan catabolites in urine are indole 3-acetate and 5-hydroxyindoleacetate.
| Tyrosine forms norepinephrine and epinephrine, and following iodination the thyroid hormones triiodothyronine and thyroxine.
™ The enzyme-catalyzed interconversion of the phosphoand dephosphoforms of peptide-bound serine, threonine, and tyrosine plays key roles in metabolic regulation, including signal transduction.
™ Glycine, arginine, and S-adenosylmethionine all participate in the biosynthesis of creatine, which as creatine phosphate serves as a major energy reserve in muscle and brain tissue. Excretion in the urine of its catabolite creatinine is proportionate to muscle mass.
™ £-Alanine and B-aminoisobutyrate both are present in tissues
as free
amino acids.
B-Alanine also
occurs in
bound form
in coenzyme
A.
Catabolism of
B-alanine involves
stepwise conversion

to acetyl-CoA.
Analogous reactions
catabolize
B-aminoisobutyrate to succinyl-CoA. Disorders of B-alanine
and f-aminoisobutyrate metabolism arise from defects in enzymes of pyrimidine catabolism.
®@ Decarboxylation of glutamate forms the inhibitory neurotransmitter GABA. Two rare metabolic disorders are associated with defects in GABA catabolism.

. amphibolic condensation initiates heme biosynthesis.
Write the structural formulas of the two intermediates whose
VTC NCR UER aCe aA you should be able to:
: biosynthesis. Identify the enzyme that catalyzes the key regulated enzyme of hepatic heme
m= Explain why, although porphyrinogens and porphyrins both are tetrapyrroles, porphyrins are colored whereas porphyrinogens are colorless.
biosynthesis. = Specify the intracellular locations of the enzymes and metabolites of heme
Outline the causes and clinical presentations of various porphyrias.
catabolism. Identify the roles of heme oxygenase and of UDP-glucosyl transferase in heme
= Define jaundice, name some of its causes, and suggest how to determine its biochemical basis.
= Specify the biochemical basis of the clinical laboratory terms "direct bilirubin"
and
“indirect bilirubin.’

The biochemistry of the porphyrins and of the bile pigments are closely related topics. Heme is synthesized from porphyrins and iron, and the products of degradation of heme are the bile pigments and iron. The biochemistry of the porphyrins and of heme is basic to understanding the varied functions of hemoproteins, and the porphyrias, a group of diseases caused by abnormalities in the pathway of porphyrin biosynthesis. A much more common clinical condition is jaundice, a consequence of an elevated level of plasma bilirubin, due either to overproduction of bilirubin or to failure of its excretion. Jaundice occurs in numerous diseases ranging from hemolytic anemias to viral hepatitis and to cancer of the pancreas.
pyrrole rings. Examples include iron porphyrins such as the heme of hemoglobin and the magnesium-containing porphyrin chlorophyll, the photosynthetic pigment of plants. Heme proteins are ubiquitous in biology and serve diverse functions including, but not limited to, oxygen transport and storage (eg, hemoglobin and myoglobin) and electron transport (eg, cytochrome c and cytochrome P450). Hemes are tetrapyrroles, of which two types, heme b and heme c, predominate (Figure 31-2). In heme c the vinyl groups of heme b are replaced by covalent thioether links to an apoprotein, typically via cysteinyl residues. Unlike heme b, heme c thus does not readily dissociate from its apoprotein. Proteins that contain heme are widely distributed in nature (Table 31-1). Vertebrate heme proteins generally bind one mole of heme c per mole, although those of nonvertebrates may bind significantly more heme.

Porphyrins are cyclic compounds formed by the linkage of four pyrrole rings through methyne (~HC—) bridges (Figure 31-1). Various side chains can replace the eight numbered hydrogen atoms of the pyrrole rings.
Porphyrins can form complexes with metal ions that form coordinate bonds to the nitrogen atom of each of the four

The biosynthesis of heme involves both cytosolic and mitochondrial reactions and intermediates. Heme biosynthesis occurs in most mammalian cells except mature erythrocytes,
Porphyrin
(CaoHyaNa)
FIGURE 31-1 The porphyrin molecule. Rings are labeled |, II, III, and IV. Substituent positions are labeled 1 through 8. The four methyne bridges (=HC—) are labeled a, , y, and 6.
which lack mitochondria. Approximately 85% of heme synthesis occurs in erythroid precursor cells in the bone marrow, and the majority of the remainder in hepatocytes. Heme biosynthesis is initiated by the condensation of succinyl-CoA and glycine in a pyridoxal phosphate-dependent reaction catalyzed by mitochondrial 5-aminolevulinate synthase (ALA synthase, EC 2.3.1.37).
(1) Succinyl-CoA + glycine > 5-aminolevulinate
+
CoA-SH
+
CO,
Humans express two isozymes of ALA synthase. ALAS1 is ubiquitously expressed throughout the body, whereas ALAS2 is expressed in erythrocyte precursor cells. Formation of 6-aminolevulinate is rate-limiting for porphyrin biosynthesis in mammalian liver (Figure 31-3).
FIGURE 31-2 Structures of heme b and heme c.

Hemoglobin, Function = Transport of oxygen in blood. Myoglobin, Function = Storage of oxygen in muscle. Cytochrome c, Function = Involvement in the electron transport chain. Cytochrome P450, Function = Hydroxylation of xenobiotics. Catalase, Function = Degradation of hydrogen peroxide. Tryptophan pyrrolase, Function = Oxidation of tryptophan
*The functions of the above proteins are described in various chapters of this text.
Following the exit of 5-aminolevulinate into the cytosol, the reaction catalyzed by cytosolic ALA dehydratase (EC 4.2.1.24; porphobilinogen synthase) condenses two molecules of ALA, forming porphobilinogen:
(2) 2 8-Aminolevulinate > porphobilinogen + 2 H,O
(Figure 31-4). A zinc metalloprotein, ALA dehydratase is sensitive to inhibition by lead, as can occur in lead poisoning. The third reaction, catalyzed by cytosolic hydroxymethylbilane synthase (uroporphyrinogen I synthase, EC 2.5.1.61) involves head-to-tail condensation of four molecules of porphobilinogen to form the linear tetrapyrrole hydroxymethylbilane (Figure 31-5, top):
(3) 4 Porphobilinogen + H,O — hydroxymethylbilane + 4 NH, Subsequent cyclization of hydroxymethylbilane, catalyzed by cytosolic uroporphyrinogen III synthase, EC 4.2.1.75:
(4) Hydroxymethylbilane > uroporphyrinogen III + H,O

forms uroporphyrinogen III (Figure 31-5, bottom right). Hydroxymethylbilane can undergo spontaneous cyclization forming uroporphyrinogen I (Figure 31-5, bottom left), but under normal conditions, the uroporphyrinogen formed is almost exclusively the type III isomer. The type I isomers of porphyrinogens are, however, formed in excess in certain porphyrias. Since the pyrrole rings of these uroporphyrinogens are connected by methylene (—CH,—) rather than by
FIGURE 31-3 Synthesis of 5-aminolevulinate (ALA). This mitochondrial reaction is catalyzed by ALA synthase.
FIGURE 31-4 Formation of porphobilinogen. Cytosolic porphobilinogen synthase converts two molecules of 5-aminolevulinate to porphybilinogen.
methyne bridges (—=HC—), the double bonds do not form a conjugated system. Porphyrinogens thus are colorless. They are, however, readily auto-oxidized to colored porphyrins.
FIGURE 31-5 Synthesis of hydroxymethylbilane and its subsequent cyclization to porphobilinogen III. Cytosolic hydroxymethylbilane synthase (ALA dehydratase) forms a linear tetrapyrrole, which cytosolic uroporphyrinogen synthase cyclizes to form uroporphyrinogen Ill. Notice the asymmetry of the substituents on ring 4, so that the highlighted acetate and propionate substituents are reversed in uroporphyrinogens | and Ill. (A, acetate [—CH,COO]; P, propionate [—CH,CH,COO'].)
FIGURE 31-6 Decarboxylation of uroporphyrinogen Ill to coproporphyrinogen III. Shown is a representation of the tetrapyrrole to emphasize the conversion of four attached acetyl groups to methyl groups. (A, acetyl; M, methyl; P, propionyl.)

All four acetate moieties of uroporphyrinogen III next undergo decarboxylation to methyl (M) substituents, forming coproporphyrinogen III in a cytosolic reaction catalyzed by uroporphyrinogen decarboxylase, EC 4.1.1.37 (Figure 31-6): (5) Uroporphyrinogen III — coproporphyrinogen III + 4 CO, This decarboxylase can also convert uroporphyrinogen I, if present, to coproporphyrinogen I. The final three reactions of heme biosynthesis all occur in mitochondria. Coproporphyrinogen III enters the mitochondria and is converted, successively, to protoporphyrinogen III, and then to protoporphyrin III. These reactions are catalyzed by coproporphyrinogen oxidase (EC 1.3.3.3), which decarboxylates and oxidizes the two propionic acid side chains to form protoporphyrinogen III:
(6) Coproporphyrinogen III + O, + 2 H* > protoporphyrinogen III + 2 CO, + 2H,O
This oxidase is specific for type III coproporphyrinogen, so type I protoporphyrins generally do not occur in humans. Protoporphyrinogen III is next oxidized to protoporphyrin III in a reaction catalyzed by protoporphyrinogen oxidase, EC 1.3.3.4:
(7)
Protoporphyrinogen
III
+
3
O,
—
protoporphyrin
III
+3H,0,
The eighth and final step in heme synthesis involves the incorporation of ferrous iron into protoporphyrin III in a reaction catalyzed by ferrochelatase (heme synthase, EC 4.99.1.1), (Figure 31-7):
(8) Protoporphyrin III + Fe** + heme + 2 H*
Figure 31-8 summarizes the stages of the biosynthesis of the porphyrin derivatives from porphobilinogen. For the above reactions, numbers correspond to those in Figure 31-8 and in Table 31-2.

ALA Synthase Is the Key Regulatory Enzyme in Hepatic Biosynthesis of Heme Unlike ALAS2, which is expressed exclusively in erythrocyte precursor cells, ALAS1 is expressed throughout body tissues.
FIGURE 31-7 Biosynthesis from porphobilinogen of the indicated porphyrin derivatives.
The reaction catalyzed by ALA synthase 1 (Figure 31-3) is rate-limiting for biosynthesis of heme in liver. Typically for an enzyme that catalyzes a rate-limiting reaction, ALAS1 has a short half-life. Heme, probably acting through an aporepressor molecule, acts as a negative regulator of the synthesis of ALAS1 (Figure 31-8). Synthesis of ALAS1 thus increases greatly in the absence of heme, but diminishes in its presence. Heme also affects translation of ALAS] and its translocation from its cytosolic site of synthesis into the mitochondrion. Many drugs whose metabolism requires the hemoprotein cytochrome P450 increase cytochrome P450 biosynthesis. The resulting depletion of the intracellular heme pool induces synthesis of ALAS1, and the rate of heme synthesis rises to meet metabolic demand. By contrast, since ALAS2 is not feedback regulated by heme, its biosynthesis is not induced by these drugs.

While porphyrinogens are colorless, the various porphyrins are colored. The conjugated double bonds in the pyrrole rings and linking methylene groups of porphyrins (absent in the porphyrinogens) are responsible for their characteristic absorption and fluorescence spectra. The visible and the ultraviolet spectra of porphyrins and porphyrin derivatives are useful for their identification (Figure 31-9). The sharp absorption band near 400 nm, a distinguishing feature shared by all porphyrins, is termed the Soret band after its discoverer, the French physicist Charles Soret.
Porphyrins dissolved in strong mineral acids or in organic solvents and illuminated by ultraviolet light emit a strong red fluorescence, a property often used to detect small amounts of free porphyrins. The photodynamic properties of porphyrins have suggested their possible use in the treatment of certain types of cancer, a procedure called cancer phototherapy. Since tumors often take up more porphyrins than do normal tissues, hematoporphyrin or related compounds are administered to a patient with an appropriate tumor. The tumor is then exposed to an argon laser to excite the porphyrins, producing cytotoxic effects.

Coproporphyrins and uroporphyrins are excreted in increased amounts in the porphyrias. When present in urine or feces, they can be separated by extraction with appropriate solvents, then identified and quantified using spectrophotometric methods.
coproporphyrin. biosynthesis Chapter
FIGURE 31-8 Intermediates, enzymes, and regulation of heme synthesis. The numbers of the enzymes that catalyze the indicated reactions are those used in the accompanying text and in column 1 of Table 31-2. Enzymes 1, 6, 7, and 8 are mitochondrial, but enzymes 2 to 5 are cytosolic. Regulation of hepatic heme synthesis occurs at ALA synthase (ALAS1) by a repression—derepression mechanism mediated by heme and a hypothetical aporepressor (not shown). Mutations in the gene in the genes encoding enzymes 2 to 8 give rise to the porphyrias. encoding enzyme 1 cause X-linked sideroblastic anemia. Mutations

- 1. Similar or identical clinical signs and symptoms can arise from different mutations in genes that encode either a given enzyme or an enzyme that catalyzes a successive reaction.
Disorders of heme biosynthesis may be genetic or acquired. An example of an acquired defect is lead poisoning. Lead can inactivate ferrochelatase and ALA dehydratase by combining with essential thiol groups. Signs include elevated levels of protoporphyrin in erythrocytes and elevated urinary levels of ALA and
Genetic disorders of heme metabolism and of bilirubin metabolism (see below) share the following features with metabolic disorders of urea (see 28):
2. Rational therapy requires an understanding of the biochemistry of the enzyme-catalyzed reactions in both normal and impaired individuals.
3, Identification of the intermediates and side products that accumulate prior to a metabolic block can provide the basis for metabolic screening tests that can implicate the impaired reaction.
_4, Definitive diagnosis involves quantitative assay of the activity of the enzyme(s) suspected to be defective. To this might

Porphyria cutaneatarda (hepatic) (OMIM 176100). 5. Uroporphyrinogen decarboxylase EC 4.1.1.37, Major Signs and Symptoms = Photosensitivity. 5. Uroporphyrinogen decarboxylase EC 4.1.1.37, Results of Laboratory Tests = Urinary uroporphyrin I increased. 6. Coproporphyrinogen oxidase EC 1.3.3.3, Type, Class, and OMIMNumber = Hereditary coproporphyria (hepatic) (OMIM 121300). 6. Coproporphyrinogen oxidase EC 1.3.3.3, Major Signs and Symptoms = Photosensitivity, abdominal pain, neuropsychiatric symptoms. 6. Coproporphyrinogen oxidase EC 1.3.3.3, Results of Laboratory Tests = Urinary ALA, PBG, and coproporphyrin III and fecal coproporphyrin III increased. 7. Protoporphyrinogen oxidase EC 1.3.3.4, Type, Class, and OMIMNumber = Variegate porphyria (hepatic) (OMIM 176200). 7. Protoporphyrinogen oxidase EC 1.3.3.4, Major Signs and Symptoms = Photosensitivity, abdominal pain, neuropsychiatric symptoms. 7. Protoporphyrinogen oxidase EC 1.3.3.4, Results of Laboratory Tests = Urinary ALA, PBG, and coproporphyrin III and fecal protoporphyrin IX increased. 8. Ferrochelatase EC 4.99.1.1, Type, Class, and OMIMNumber = Protoporphyria (erythropoietic) (OMIM 177000). 8. Ferrochelatase EC 4.99.1.1, Major Signs and Symptoms = Photosensitivity. 8. Ferrochelatase EC 4.99.1.1, Results of Laboratory Tests = Fecal and red cell protoporphyrin IX increased
*Only the biochemical findings in the active stages of these diseases are listed. Certain biochemical abnormalities are detectable in the latent stages of some of the above condi-

- *The numbering of the enzymes in this Table corresponds to that used in Figure 31-8.
tions. Conditions 3, 5, and 8 are generally the most prevalent porphyrias. Condition 2 is rare.
'X-linkedsideroblastic anemia is not a porphyria but is included here because ALA synthase is involved.
"This enzyme is also called PBG deaminase or hydroxymethylbilane synthase.
°PBG = porphyrobilinogen Ill.
Abbreviations: ALA, 5-aminolevulinic acid; PBG, porphobilinogen.
be added consideration of the as yet incompletely identified factors that facilitate translocation of enzymes and intermediates between cellular compartments.
- 5. Comparison of the DNA sequence of the gene that encodes
a given mutant enzyme to that of the wild-type gene can identify the specific mutation(s) that cause the disease.

The signs and symptoms of porphyria result either from a deficiency of intermediates beyond the enzymatic block, or
FIGURE 31-9 Absorption spectrum of hematoporphyrin. The spectrum is of a dilute (0.01%) solution of hematoporphyrin in 5% HCl.
from the accumulation of metabolites prior to the block. Table 31-2 lists six major types of porphyria that reflect low or absent activity of enzymes that catalyze reactions 2 through 8 of Figure 31-8. Possibly due to potential lethality, there is no known defect of ALAS1. Individuals with low ALAS2 activity develop anemia, not porphyria (Table 31-2). Porphyria consequent to low activity of ALA dehydratase, termed ALA dehydratase-deficient porphyria, is extremely rare.

While most porphyrias are inherited in an autosomal dominant manner, congenital erythropoietic porphyria is inherited in a recessive mode. The defective enzyme in congenital erythropoietic porphyria is uroporphyrinogen III synthase (Figure 31-5, bottom). The photosensitivity and severe disfigurement exhibited by some victims of congenital erythropoietic porphyria has suggested them as prototypes of so-called werewolves.

The defective enzyme in acute intermittent porphyria is hydroxymethylbilane synthase (Figure 31-5, bottom). ALA and porphobilinogen accumulate in body tissues and fluids (Figure 31-10).

Blocks later in the pathway result in the accumulation of the porphyrinogens indicated in Figures 31-8 and 31-10. Their

oxidation products, the corresponding porphyrin derivatives, cause photosensitivity to visible light of about 400-nm wavelength. Possibly as a result of their excitation and reaction with molecular oxygen, the resulting oxygen radicals injure lysosomes and other subcellular organelles, releasing proteolytic enzymes that cause variable degrees of skin damage, including scarring.

Porphyrias may be termed erythropoietic or hepatic based on the organs most affected, typically bone marrow and the liver (Table 31-2). Different and variable levels of heme, toxic precursors, or metabolites probably account for why specific porphyrias differentially affect some cell types and organs. Alternatively, porphyrias may be classified as acute or cutaneous based on their clinical features. The diagnosis of a specific type of porphyria involves consideration of the clinical and family history, physical examination, and appropriate laboratory tests. Table 31-2 lists the major signs, symptoms, and relevant laboratory findings in the six principal types of porphyria.

Certain drugs (eg, barbiturates, griseofulvin) induce the production of cytochrome P4590. In patients with porphyria, this can precipitate an attack of porphyria by depleting heme levels. The compensating derepression of synthesis of ALAS1 then results in increased levels of potentially harmful heme precursors.

Present treatment of porphyrias is essentially symptomatic: avoiding drugs that induce production of cytochrome P450, ingestion of large amounts of carbohydrate, and administration of hematin to repress ALAS1 synthesis to diminish production of harmful heme precursors. Patients exhibiting photosensitivity benefit from sunscreens and possibly from administered B-carotene, which appears to lessen production of free radicals, decreasing photosensitivity.

Human adults normally destroy about 200 billion erythrocytes per day. A 70-kg human therefore turns over approximately 6 g of hemoglobin daily. All products are reused. The globin is degraded to its constituent amino acids, and the released iron enters the iron pool. The iron-free porphyrin portion of heme is also degraded, mainly in the reticuloendothelial cells of the liver, spleen, and bone marrow.
The catabolism of heme from all heme proteins takes place in the microsomal fraction of cells by heme oxygenase, EC 1.14.18.18. Heme oxygenase synthesis is substrate-inducible, and heme also serves both as a substrate and as a cofactor for the reaction. The iron of the heme that reaches heme oxygenase has usually been oxidized to its ferric form (hemin). Conversion of one mole of heme-Fe** to biliverdin, carbon monoxide, and Fe** consumes three moles of O,, plus seven electrons provided by NADH and NADPH -cytochrome P450 reductase:

Despite its high affinity for heme-Fe** (see Chapter 6), the carbon monoxide produced does not severely inhibit heme oxygenase. Birds and amphibians excrete the green-colored biliverdin directly. In humans, biliverdin reductase (EC 1.3.1.24) reduces the central methylene bridge of biliverdin to a methyl group, producing the yellow-pigment bilirubin (Figure 31-11):

Since 1 g of hemoglobin yields about 35 mg of bilirubin, human adults form 250 to 350 mg of bilirubin per day. This is derived principally from hemoglobin, and also from ineffective erythropoiesis and from catabolism of other heme proteins. Conversion of heme to bilirubin by reticuloendothelial cells can be observed visually as the purple color of the heme in a hematoma slowly converts to the yellow pigment of bilirubin.

Unlike bilirubin, which is only sparingly water soluble, bilirubin bound to serum albumin is readily transported to the liver. Albumin appears to have both high-affinity and low-affinity sites for bilirubin. The high-affinity site can bind approximately 25 mg of bilirubin/100 mL of plasma. More loosely bound bilirubin can readily be detached and diffused into tissues, and antibiotics and certain other drugs can compete with and displace bilirubin from albumin's high-affinity site.
FIGURE 31-11 Conversion of ferric heme to biliverdin, and then to bilirubin. (1) Conversion of ferric heme to biliverdin is catalyzed by the heme oxygenase system. (2) Subsequently, biliverdin reductase reduces bilirubin to bilirubin.

Hepatic catabolism of bilirubin takes place in three stages: uptake by the liver, conjugation with glucuronic acid, and secretion in the bile.

Bilirubin is removed from albumin and taken up at the sinusoidal surface of hepatocytes by a large capacity, saturable facilitated transport system. Even under pathologic conditions, transport does not appear to be rate-limiting for the metabolism of bilirubin. The net uptake of bilirubin depends on its removal by subsequent metabolism. Once internalized, bilirubin binds to cytosolic proteins such as glutathione S-transferase, previously known as a ligandin, to prevent bilirubin from reentering the bloodstream.

Bilirubin is nonpolar, and would persist in cells (eg, bound to lipids) if not converted to a more water-soluble form. Bilirubin is converted to a more polar molecule by conjugation with glucuronic acid (Figure 31-12). A bilirubin-specific UDP-glucuronosyltransferase (EC 2.4.1.17) of the endoplasmic reticulum catalyzes stepwise transfer to bilirubin of two glucosyl moieties from UDP-glucuronate:
Bilirubin + UDP-glucuronate — bilirubin monoglucuronide + UDP
Bilirubin monoglucuronide + UDP-glucuronate = bilirubin diglucuronide + UDP

Secretion of conjugated bilirubin into the bile occurs by an active transport mechanism, which probably is rate-limiting for the entire process of hepatic bilirubin metabolism. The protein involved is a multispecific organic anion transporter (MOAT) located in the plasma membrane of the bile canaliculi. A member of the family of ATP-binding cassette transporters, MOAT transports a number of organic anions. The hepatic transport of conjugated bilirubin into the bile is inducible by the same drugs that can induce the conjugation of bilirubin. Conjugation and excretion of bilirubin thus constitute a coordinated functional unit.
Most of the bilirubin excreted in the bile of mammals is bilirubin diglucuronide. Bilirubin UDP-glucuronosyltransferase activity can be induced by several drugs, including phenobarbital. However, when bilirubin conjugates exist abnormally in human plasma (eg, in obstructive jaundice), they are predominantly monoglucuronides. Figure 31-13 summarizes
FIGURE 31-12 Bilirubin diglucuronide. Glucuronate moieties are attached via ester bonds to the two propionate groups of bilirubin. Clinically, the diglucuronide is also termed "direct reacting" bilirubin.
FIGURE 31-13 Diagrammatic representation of the three major processes (uptake, conjugation, and secretion) involved in the transfer of bilirubin from blood to bile. Certain proteins of hepatocytes bind intracellular bilirubin and may prevent its efflux into the bloodstream. The processes affected in certain conditions that cause jaundice are also shown.
the three major processes involved in the transfer of bilirubin from blood to bile. Sites that are affected in a number of conditions causing jaundice are also indicated.

When conjugated bilirubin reaches the terminal ileum and the large intestine, the glucuronosyl moieties are removed by specific bacterial B-glucuronidases (EC 3.2.1.31). Subsequent reduction by the fecal flora forms a group of colorless tetrapyrroles called urobilinogens. Small portions of urobilinogens are reabsorbed in the terminal ileum and large intestine and subsequently are reexcreted via the enterohepatic urobilinogen cycle. Under abnormal conditions, particularly when excessive bile pigment is formed or when liver disease disrupts this intrahepatic cycle, urobilinogen may also be excreted in the urine. Most of the colorless urobilinogens formed in the colon are oxidized there to colored urobilins and excreted in the feces. Fecal darkening upon standing in air results from the oxidation of residual urobilinogens to urobilins.

Quantitation of bilirubin employs a colorimetric method based on the reddish-purple color formed when bilirubin reacts with diazotized sulfanilic acid. An assay conducted in the absence of added methanol measures "direct bilirubin," which is bilirubin glucuronide. An assay conducted in the presence of added methanol measures total bilirubin. The difference between total bilirubin and direct bilirubin is known as "indirect bilirubin, and is unconjugated bilirubin.

Hyperbilirubinemia, a blood level that exceeds 1 mg of bilirubin per dL (17 mol/L), may result from production of more bilirubin than the normal liver can excrete, or from the failure of a damaged liver to excrete normal amounts of bilirubin. In the absence of hepatic damage, obstruction of the excretory ducts of the liver prevents the excretion of bilirubin, and will also cause hyperbilirubinemia. In all these situations, when the blood concentration of bilirubin reaches 2 to 2.5 mg/dL, it diffuses into the tissues, which turn yellow, a condition termed jaundice or icterus.

Forms of hyperbilirubinemia include retention hyperbilirubinemia due to overproduction of bilirubin, and regurgitation hyperbilirubinemia due to reflux into the bloodstream because of biliary obstruction.
Because of its hydrophobicity, only unconjugated bilirubin can cross the blood-brain barrier into the central nervous system. Encephalopathy due to hyperbilirubinemia (kernicterus) thus occurs only with unconjugated bilirubin, as in retention hyperbilirubinemia. Alternatively, because of its water solubility, only conjugated bilirubin can appear in urine. Accordingly, choluric jaundice (choluria is the presence of bile pigments in the urine) occurs only in regurgitation hyperbilirubinemia, and acholuric jaundice occurs only in the presence of an excess of unconjugated bilirubin. Table 31-3 lists some causes of unconjugated and conjugated hyperbilirubinemia. A moderate hyperbilirubinemia accompanies hemolytic anemias.

Hemolytic anemias, Conjugated = Obstruction of the biliary tree. Neonatal'physiological jaundice', Conjugated = Dubin-Johnson syndrome. Crigler-Najjar syndromes types I and II, Conjugated = Rotor syndrome. Gilbert syndrome, Conjugated = Liver diseases such as the various types of hepatitis. Toxic hyperbilirubinemia, Conjugated = 
These causes are discussed briefly in the text. Common causes of obstruction of the biliary tree are a stone in the common bile duct and cancer of the head of the pancreas. Various liver diseases (eg, the various types of hepatitis) are frequent causes of predominantly conjugated hyperbilirubinemia.
Hyperbilirubinemia is usually modest (< 4 mg bilirubin per dL; < 68 umol/L) despite extensive hemolysis, due to the high capacity of a healthy liver to metabolize bilirubin.

The unconjugated hyperbilirubinemia of neonatal "physiologic jaundice" results from accelerated hemolysis and an immature hepatic system for the uptake, conjugation, and secretion of bilirubin. In this transient condition, bilirubinglucuronosyltransferase activity, and probably also synthesis of UDP-glucuronate, are reduced. When the plasma concentration of unconjugated bilirubin exceeds that which can be tightly bound by albumin (20-25 mg/dL), bilirubin can penetrate the blood-brain barrier. If left untreated, the resulting hyperbilirubinemic toxic encephalopathy, or kernicterus, can result in mental retardation. Exposure of jaundiced neonates to blue light (phototherapy) promotes hepatic excretion of unconjugated bilirubin by converting some to derivatives that are excreted in the bile, and phenobarbital, a promoter of bilirubin metabolism, may be administered.

Glucuronosyltransferases (EC 2.4.1.17), a family of enzymes with differing substrate specificities, increase the polarity of various drugs and drug metabolites, thereby facilitating their excretion. Mutations in the gene that encodes bilirubin UDPglucuronosyltransferase can result in the encoded enzyme having reduced or absent activity. Syndromes whose clinical presentation reflects the severity of the impairment include Gilbert syndrome and two types of Crigler-Najjar syndrome.

Providing that about 30% of the bilirubin UDP-glucuronosyltransferase activity is retained in Gilbert syndrome, the condition is harmless.

The severe congenital jaundice (over 20 mg bilirubin per dL serum) and accompanying brain damage of type I CriglerNajjar syndrome reflect the complete absence of hepatic UDP-glucuronosyltransferase activity. Phototherapy reduces plasma bilirubin levels somewhat, but phenobarbital has no beneficial effect. The disease is often fatal within the first 15 months of life.

In type II Crigler-Najjar syndrome, some bilirubin UDPglucuronosyltransferase activity is retained. This condition thus is more benign than the type I syndrome. Serum bilirubin tends not to exceed 20 mg/dL of serum, and patients respond to treatment with large doses of phenobarbital.

Unconjugated hyperbilirubinemia can result from toxininduced liver dysfunction caused by, for example, chloroform, arsphenamines, carbon tetrachloride, acetaminophen, hepatitis virus, cirrhosis, or Amanita mushroom poisoning. These acquired disorders involve hepatic parenchymal cell damage, which impairs bilirubin conjugation.

Conjugated hyperbilirubinemia commonly results from blockage of the hepatic or common bile ducts, most often due to a gallstone or to cancer of the head of the pancreas (Figure 31-14). Bilirubin diglucuronide that cannot be excreted regurgitates into the hepatic veins and lymphatics, conjugated bilirubin appears in the blood and urine (choluric jaundice), and the stools typically are a pale color.
The term cholestatic jaundice includes both all cases of extrahepatic obstructive jaundice and also conjugated hyperbilirubinemia due to micro-obstruction of intrahepatic biliary ductules by damaged hepatocytes, such as may occur in infectious hepatitis.
FIGURE 31-14 Major causes of jaundice. Prehepatic jaundice indicates events in the bloodstream, major causes being various hemolytic anemias. Hepatic jaundice arises from hepatitis or other liver diseases (eg, cancer). Posthepatic jaundice refers to events in the biliary tree, for which the major causes are obstruction of the common bile duct by a gallstone (biliary calculus) or by cancer of the head of the pancreas.

Condition
Serum Bilirubin
Urine Urobilinogen
Urine Bilirubin
Fecal Urobilinogen
Normal
Direct: 0.1-0.4 mg/dL
0-4 mg/24h
Absent
40-280 mg/24h
Indirect: 0.2-0.7 mg/dL
Tindirect
Increased
Absent
Increased
Hepatitis
TDirect and indirect
Decreased if micro-obstruction
Present if micro-obstruction
Decreased
is present
occurs.
Obstructive jaundice®
TDirect
Absent
Present
Trace to absent
*The most common causes of obstructive (posthepatic) jaundice are cancer of the head of the pancreas and a gallstone lodged in the common bile duct. The presence of bili- rubin in the urine is sometimes referred to as choluria—therefore, hepatitis and obstruction of the common bile duct cause choluric jaundice, whereas the jaundice of hemolytic anemia is referred to as acholuric. The laboratory results in patients with hepatitis are variable, depending on the extent of damage to parenchymal cells and the extent of micro-obstruction to bile ductules. Serum levels of alanine aminotransferase and aspartate aminotransferase are usually markedly elevated in hepatitis, whereas serum levels of alkaline phosphatase are elevated in obstructive liver disease.

This benign autosomal recessive disorder consists of conju- gated hyperbilirubinemia in childhood or during adult life. The hyperbilirubinemia is caused by mutations in the gene encoding the protein involved in the secretion of conjugated bilirubin into bile.

When levels of conjugated bilirubin remain high in plasma, a fraction can bind covalently to albumin. This fraction, termed 6-bilirubin, has a longer half-life in plasma than does con- ventional conjugated bilirubin, and remains elevated during recovery from obstructive jaundice. Some patients therefore continue to appear jaundiced even after the circulating conju- gated bilirubin level has returned to normal.

In complete obstruction of the bile duct, bilirubin has no access to the intestine for conversion to urobilinogen, so no urobilinogen is present in the urine. The presence of conju- gated bilirubin in the urine without urobilinogen suggests intrahepatic or posthepatic obstructive jaundice.
In jaundice secondary to hemolysis, the increased pro- duction of bilirubin leads to increased production of urobilin- ogen, which appears in the urine in large amounts. Bilirubin is not usually found in the urine in hemolytic jaundice, so the combination of increased urobilinogen and absence of bilirubin is suggestive of hemolytic jaundice. Increased blood destruction from any cause brings about an increase in urine urobilinogen.
Table 31-4 summarizes laboratory results obtained in patients with jaundice due to prehepatic, hepatic, or pos- thepatic causes: hemolytic anemia (prehepatic), hepatitis (hepatic), and obstruction of the common bile duct (posthe- patic); see Figure 31-14. Laboratory tests on blood (evalua- tion of the possibility of a hemolytic anemia and measurement
of prothrombin time) and on serum (eg, electrophoresis of proteins; alkaline phosphatase and alanine aminotransferase and aspartate aminotransferase activities) also help to distin- guish between prehepatic, hepatic, and posthepatic causes of jaundice.

@
The
heme
of
hemoproteins
such
as
and
the
cytochromes
is
an
iron-containing
porphyrin
of
four
pyrrole
rings
joined
by
methyne
bridges.
The eight methyl, vinyl, and propionyl substituents on the four pyrrole rings of heme are arranged in a specific sequence. The metal ion (Fe** in hemoglobin; Mg”* in chlorophyll) is linked to the four nitrogen atoms of the pyrrole rings.
Biosynthesis of the heme ring involves eight enzyme-catalyzed reactions, some of which occur in mitochondria, others in the cytosol.
Synthesis of heme commences with the condensation of succinyl-CoA and glycine to form ALA. This reaction is catalyzed by ALAS1, the regulatory enzyme of heme biosynthesis.
Synthesis of ALAS] increases in response to a low level of available heme. For example, certain drugs (eg, phenobarbital) indirectly trigger enhanced synthesis of ALAS1 by promoting synthesis of the heme protein cytochrome P450, which thereby depletes the heme pool. By contrast, ALAS2 is not regulated by heme levels, and consequently not by drugs that promote synthesis of cytochrome P450.
Genetic abnormalities of seven of the eight enzymes of heme biosynthesis result in inherited porphyrias. Erythrocytes and liver are the major sites of expression of the porphyrias. Photosensitivity and neurologic problems are common complaints. Intake of certain toxins (eg, lead) can cause acquired porphyrias. Increased amounts of porphyrins or their precursors can be detected in blood and urine, facilitating diagnosis.
of
the
heme
ring,
initiated
by
the
enzyme
heme
oxygenase,
produces
the
linear
tetrapyrrole,
biliverdin.
Subsequent
reduction
of
biliverdin
in
the
cytosol
forms
bilirubin.

® Bilirubin binds to albumin for transport from peripheral tissues to the liver, where it is taken up by hepatocytes. The iron of heme is released and reutilized.
™ The water solubility of bilirubin is increased by the addition of two moles of the highly polar glucuronosyl moiety, derived from UDP-glucuronate, per mole of bilirubin. Attachment of the glucuronosyl moieties is catalyzed by bilirubin UDPglucuronosyltransferase, one of a large family of enzymes with differing substrate specificity that increases the polarity of various drugs and their metabolites, thereby facilitating their excretion.
™ Mutations in the encoding gene result in reduced or
absent activity of bilirubin UDP-glucuronosyltransferase activity. Clinical presentations that reflect the severity of the mutation(s) include Gilbert syndrome and two types of
Crigler-Najjar syndrome, conditions whose severity depend on
the extent of remaining glucuronosyltransferase activity.
@ Following secretion of bilirubin from the bile into the gut, bacterial enzymes convert bilirubin to urobilinogen and urobilin, which are excreted in the feces and urine.
= Colorimetric measurement of bilirubin employs the color formed when bilirubin reacts with diazotized sulfanilic acid. Assays conducted in the absence of added methanol measure "direct bilirubin" (ie, bilirubin glucuronide). Assays conducted in the presence of added methanol measure total bilirubin. The difference between total bilirubin and direct bilirubin, termed "indirect bilirubin,' is unconjugated bilirubin.
™ Jaundice results from an elevated level of plasma bilirubin.
The causes
of jaundice
can be
distinguished as
prehepatic
(eg, hemolytic
anemias),
(eg, hepatic
obstruction of
(eg, hepatitis),
the of
plasma total
and urobilinogen
and enzymes,
and common
bile or
posthepatic duct).
nonconjugated bilirubin,
the
Measurements bilirubin,
of the
activity analysis

6. Select the one of the following statements that is NOT CORRECT:
1. Select the one of the following statements that is NOT
CORRECT:
A. A'-Pyrroline-5-carboxylate is an intermediate both in the
biosynthesis and in the catabolism of L-proline.
B. Human tissues can form dietarily nonessential amino acids
from amphibolic intermediates or from dietarily essential amino acids.
C. Human liver tissue can form serine from the glycolytic
intermediate 3-phosphoglycerate.
D. The reaction catalyzed by phenylalanine hydroxylase
interconverts phenylalanine and tyrosine.
E. The reducing power of tetrahydrobiopterin derives
ultimately
from
NADPH.
2. Identify the metabolite that does NOT serve as a precursor of a
dietarily essential amino acid:
3-Phosphoglycerate
Glutamate
Aspartate
Histamine
BOOP
3. Select the one of the following statements that is NOT
CORRECT:
A. Selenocysteine is present at the active sites of certain human
enzymes.
B. Selenocysteine is inserted into proteins by a
posttranslational process.
C. Transamination of dietary a-keto acids can replace the
dietary essential amino acids leucine, isoleucine, and valine.
D. Conversion of peptidyl proline to peptidyl-4-hydroxyproline
is accompanied by the incorporation of oxygen into
succinate.
E. Serine and glycine are interconverted in a single reaction in which tetrahydrofolate derivatives participate.
4. Select the CORRECT answer:
The first reaction in the degradation of most of the protein amino
acids involves the participation of:
NAD* >
Thiamine pyrophosphate (TPP)
Pyridoxal phosphate
FAD
NAD* and TPP
BOOS
5. Identify the amino acid that is the major contributor to the
transport of nitrogen destined for excretion as urea:
Alanine
Glutamine
Glycine

Lysine
Ornithine
BOOS
A. Angelman syndrome is associated with a defective ubiquitin
E3 ligase.
B.
Following a protein-rich meal, the splanchnic tissues release
predominantly branched-chain amino acids. which are
taken up by peripheral muscle tissue.
C.
The rate of hepatic gluconeogenesis from glutamine exceeds
that of any other amino acid.
D. The L-a-amino oxidase-catalyzed conversion of an a-amino
acid to its corresponding a-keto acid is accompanied by the
release of NH,*.
E. Similar or even identical signs and symptoms can be
associated with different mutations of the gene that encodes
a given enzyme.
7.
Select the one of the following statements that is NOT CORRECT:
A. PEST sequences target some proteins for rapid degradation.
B. ATP and ubiquitin typically participate in the degradation
of membrane-associated proteins and other proteins with half-lives.
long
C. Ubiquitin molecules are attached to target proteins via
non-a peptide bonds.
D. The discoverers of ubiquitin-mediated protein degradation
received a Nobel Prize.
E. Degradation of ubiquitin-tagged proteins takes place in the
proteasome, a multi-subunit macromolecule present in all
8. For metabolic disorders of the urea cycle, which statement is
NOT CORRECT:
A.
Ammonia intoxication is most severe when the metabolic
block in the urea cycle occurs prior to the reaction catalyzed
by argininosuccinate synthase.
B. Clinical include mental retardation and the
symptoms avoidance protein-rich
of foods.
C. Clinical signs can include acidosis.
D. Aspartate provides the second nitrogen of
argininosuccinate.
E. Dietary management focuses on a low-protein diet ingested
as frequent small meals.
9. Select the one of the following statements that is NOT
CORRECT:
A. One metabolic function of glutamine is to sequester
nitrogen in a nontoxic form.
B. Liver glutamate dehydrogenase is allosterically inhibited by

ATP and activated by ADP.
C. Urea is formed both from absorbed ammonia produced
by enteric bacteria and from ammonia generated by tissue
metabolic activity.
D.
The concerted action of glutamate dehydrogenase
and glutamate aminotransferase may be termed
transdeamination.
E. Fumarate generated during biosynthesis of
argininosuccinate ultimately forms oxaloacetate in reactions
in mitochondria catalyzed successively by fumarase and
malate dehydrogenase.
10. Select the one of the following statements that is NOT
11. Select the one of the following statements that is NOT
12. What distinguishes the routes by which each of the following
13. What evolutionary advantage might be gained by the fact that
14. What explanation can you offer to explain that metabolic defects
15. Which of the following is NOT a hemoprotein?
CORRECT:
A. Threonine provides the thioethanol moiety for biosynthesis
of coenzyme A.
B. Histamine arises by decarboxylation of histidine.
C. Ornithine serves as a precursor of both spermine and
spermidine.
D. Serotonin and melatonin are metabolites of tryptophan.
E. Glycine, arginine, and methionine each contribute atoms for
biosynthesis of creatine.

A. Excreted creatinine is a function of muscle mass, and can
be used to determine whether a patient has provided a
complete 24-hour urine specimen.
B. Many drugs and drug catabolites are excreted in urine as
glycine conjugates.
C. The major nonprotein metabolic fate of methionine is
conversion to S-adenosylmethionine.
D. The concentration of histamine in brain hypothalamus
exhibits a circadian rhythm.
E.
Decarboxylation of glutamine forms the inhibitory neurotransmitter GABA (y-aminobutyrate).
amino acids appears in human proteins?
5-Hydroxylysine
y-Carboxyglutamate
certain amino acids are dietarily essential for human subjects?
that result in the complete absence of the activity of glutamate dehydrogenase have not been detected?
Myoglobin
Cytochrome c
Catalase
Cytochrome P450
Albumin
BOOP
16. A 30-year-old man presented at clinic with a history of
17. Select the one of the following statements that is NOT
18.
19. Clinical laboratories typically use diazotized sulfanilic acid to
intermittent
abdominal
pain
and
episodes
of
confusion
and
psychiatric
problems.
tests
revealed
increases
of
urinary
6-aminolevulinate
and
porphobilinogen.
Mutational
analysis
revealed
a
mutation
in
the
gene
for
I
synthase
(porphobilinogen
deaminase).
The
probable
diagnosis
was:
Acute intermittent porphyria. D
X-linked sideroblastic anemia.
Congenital erythropoietic porphyria.
Porphyria cutanea tarda.
Variegate porphyria.
MOOR
CORRECT:
A. Bilirubin is a cyclic tetrapyrrole.
B. Albumin-bound bilirubin is transported to the liver.
C.
High
levels
of
bilirubin
can
cause
damage

to
the
brains
of
newborn infants.
D. Bilirubin contains methyl and vinyl groups.
B. Bilirubin does not contain iron.
A
62-year-old
female
presented
at
clinic
with
intense
jaundice,
steadily
over
the
preceding
3
months.
She
gave
a
history
of
severe
upper
abdominal
pain.
radiating
to
the
back.
and had lost considerable weight. She had noted that her stools
had
been
very
pale
for
some
time.
Lab
tests
revealed
a
very
high
level
of
direct
and
also
elevated
urinary
The
plasma
level
of
alanine
aminotransferase
(ALT)
was
only
slightly
elevated,
whereas
the
level
of
alkaline
was
markedly
elevated.
Abdominal
ultrasonography
revealed
no
evidence
of
gallstones.
Of
the
following,
which
is
the
most
likely
diagnosis?
A. Gilbert syndrome
B. Hemolytic anemia
C. Type 1 Crigler-Najjar syndrome
D. Carcinoma of the pancreas
E. Infectious hepatitis
measure serum bilirubin and its derivatives. What is the physical basis that permits the laboratory to report results to the physician in terms of these two forms of bilirubin?
20. What signals the synthesis of heme to take place?

.
Write structural formulas to represent the aminoand oxo-tautomers of a purine and of a pyrimidine and state which tautomer predominates under physiologic conditions.
: Reproduce the structural formulas for the principal nucleotides present in DNA and in RNA and the less common nucleotides 5-methylcytosine, 5-hydroxymethylcytosine, and pseudouridine (1p).
Represent D-ribose or 2-deoxy-D-ribose linked as either a syn or an anti conformer to a purine, name the bond between the sugar and the base, and indicate which conformer predominates under most physiologic conditions.
Number the C and N atoms of a pyrimidine ribonucleoside and of a purine deoxyribonucleoside, including using a primed numeral for C atoms of the sugars.
Compare the phosphoryl group transfer potential of each phosphoryl group of a nucleoside triphosphate.
Outline the physiologic roles of the cyclic phosphodiesters cAMP and cGMP.
= Appreciate that polynucleotides are directional macromolecules composed of mononucleotides linked by 3' > 5'-phosphodiester bonds.
Be familiar with the abbreviated representations of polynucleotide structures such as pTpGpT or TGCATCA, for which the 5'-end is always shown at the left and all phosphodiester bonds are 3' > 5'.
For specific synthetic analogs of purine and pyrimidine bases and their derivatives that have served as anticancer drugs, indicate in what ways these compounds inhibit metabolism.

In addition to serving as precursors of nucleic acids, purine and pyrimidine nucleotides participate in metabolic functions as diverse as energy metabolism, protein synthesis, regulation of enzyme activity, and signal transduction. When linked to vitamins or vitamin derivatives, nucleotides form a portion of many coenzymes. As the principal donors and acceptors of phosphoryl groups in metabolism, nucleoside triand diphosphates such as ATP and ADP are the principal players in the energy transductions that accompany metabolic interconversions and oxidative phosphorylation. Linked to sugars or lipids, nucleosides constitute key biosynthetic intermediates. The sugar derivatives UDP-glucose and UDP-galactose participate in sugar interconversions and in the biosynthesis of starch and glycogen. Similarly, nucleoside-lipid derivatives such as CDPacylglycerol are intermediates in lipid biosynthesis. Roles that nucleotides perform in metabolic regulation include ATPdependent phosphorylation of key metabolic enzymes, allosteric regulation of enzymes by ATP, ADP, AMP, and CTP, and control by ADP of the rate of oxidative phosphorylation. The cyclic nucleotides cAMP and cGMP serve as the second messengers in hormonally regulated events, and GTP and GDP play key roles in the cascade of events that characterize signal transduction pathways. In addition to the central roles that nucleotides play in metabolism, their medical applications include the use of synthetic purine and pyrimidine analogs that contain halogens, thiols, or additional nitrogen atoms in the chemotherapy of cancer and AIDS, and as suppressors of the immune response during organ transplantation.

Purines and pyrimidines are nitrogen-containing heterocycles, cyclic structures that contain, in addition to carbon, other (hetero) atoms such as nitrogen. Note that the smaller pyrimidine molecule has the longer name and the larger purine molecule the shorter name, and that their six-atom rings are numbered in opposite directions (Figure 32-1). Purines or
Purine
Pyrimidine
FIGURE 32-1 Purine and pyrimidine. The atoms are numbered according to the international system.
NH»
NH
FIGURE 32-2 tautomerism of the oxo and amino functional groups of purines and pyrimidines.
pyrimidines with an 'NH, group are weak bases (pK, values 3-4), although the proton present at low pH is associated, not as one might expect with the exocyclic amino group, but with a ring nitrogen, typically N1 of adenine, N7 of guanine, and N3 of cytosine. The planar character of purines and pyrimidines facilitates their close association, or "stacking," that stabilizes double-stranded DNA (see Chapter 34). The oxo and amino groups of purines and pyrimidines exhibit keto-enol and amine-imine tautomerism (Figure 32-2), although physiologic conditions strongly favor the amino and oxo forms.

Nucleosides are derivatives of purines and pyrimidines that have a sugar linked to a ring nitrogen of a purine or pyrimidine. Numerals with a prime (eg, 2' or 3') distinguish atoms of the sugar from those of the heterocycle. The sugar in ribonucleosides is D-ribose, and in deoxyribonucleosides is 2-deoxy-D-ribose. Both sugars are linked to the heterocycle by an a-N-glycosidic bond, almost always to the N-1 of a pyrimidine or to N-9 of a purine (Figure 32-3).

Mononucleotides are nucleosides with a phosphoryl group esterified to a hydroxyl group of the sugar. The 3'and 5'-nucleotides are nucleosides with a phosphoryl group on the 3'or 5'-hydroxyl group of the sugar, respectively. Since most nucleotides are 5'-, the prefix "5'-" usually is omitted when naming them. UMP and dAMP thus represent nucleotides with a phosphoryl group on C-5 of the pentose. Additional phosphoryl groups, ligated by acid anhydride bonds to the phosphoryl group of a mononucleotide, form nucleoside diphosphates and triphosphates (Figure 32-4).

Steric hindrance by the heterocycle dictates that there is no freedom of rotation about the B-N-glycosidic bond of nucleosides or nucleotides. Both therefore exist as noninterconvertible syn or anti conformers (Figure 32-5). While both syn and anti conformers occur in nature, the anti conformers predominate. Table 32-1 lists the major purines and pyrimidines and their nucleoside and nucleotide derivatives. Single-letter abbreviations are used to identify adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), whether free or
CHAPTER
32
FIGURE 32-3 Ribonucleosides, drawn as the syn conformers.
present in nucleosides or nucleotides. The prefix "d" (deoxy) indicates that the sugar is 2'-deoxy-D-ribose (eg, in dATP) (Figure 32-6).

Small quantities of additional purines and pyrimidines occur in DNA and RNAs. Examples include 5-methylcytosine of bacterial and human DNA, 5-hydroxymethylcytosine of bacterial and viral nucleic acids, and monoand the di-N-methylated adenine and guanine of mammalian messenger RNAs (Figure 32-7) that function in oligonucleotide recognition and in regulating the half-lives of RNAs. Free heterocyclic bases include hypoxanthine, xanthine, and uric acid (Figure 32-8), intermediates in the catabolism of adenine and guanine (see Chapter 33). Methylated heterocycles of plants include the xanthine derivatives caffeine of coffee, theophylline of tea, and theobromine of cocoa (Figure 32-9).
FIGURE 32-4 arp, its diphosphate, and its monophosphate.
The primary and secondary phosphoryl groups of nucleosides have pK, values of about 1.0 and 6.2, respectively. Nucleotides therefore bear significant negative charge at physiologic pH. The pK, values of the secondary phosphoryl groups are such that they can serve either as proton donors or as proton acceptors at pH values approximately two or more units above or below neutrality.

The conjugated double bonds of purine and pyrimidine derivatives absorb ultraviolet light. While their spectra are pH-dependent, at pH 7.0 all the common nucleotides absorb light at a wavelength close to 260 nm. The concentration of nucleotides and nucleic acids thus often is expressed in terms of "absorbance at 260 nm?' The mutagenic effect of ultraviolet light is due to its absorption by nucleotides in DNA that results in chemical modifications (see Chapter 35).

In addition to their roles as precursors of nucleic acids, ATP, GTP, UTP, CTP, and their derivatives each serve unique
NH,
FIGURE 32-5 The syn and anti conformers of adenosine differ with respect to orientation about the N-glycosidic bond.
321

NH 2 N N, X = H = Adenine. NH 2 N N, X = Ribose = Adenosine. NH 2 N N, X = Ribose Phosphate = Adenosine monophosphate (AMP). O, X = H = Guanine. O, X = Ribose = Guanosine. O, X = Ribose Phosphate = Guanosine monophosphate (GMP). NH 2, X = H = Cytosine. NH 2, X = Ribose = Cytidine. NH 2, X = Ribose Phosphate = Cytidine monophosphate (CMP). O, X = H = Uracil. O, X = Ribose = Uridine. O, X = Ribose Phosphate = Uridine monophosphate (UMP). dX O N H N O CH 3, X = H = Thymine. dX O N H N O CH 3, X = Ribose = Thymidine. dX O N H N O CH 3, X = Ribose Phosphate = Thymidine monophosphate (TMP)
physiologic functions discussed in other chapters. Selected examples include the role of ATP as the principal biologic transducer of free energy, and the second messenger cAMP (Figure 32-10). The mean intracellular concentration of ATP, the most abundant free nucleotide in mammalian cells, is about 1 mmol/L. Since little cAMP is required, the intracellular cAMP concentration (about 1 nmol/L) is six orders of magnitude below that of ATP. Other examples include adenosine 3'-phosphate-5'-phosphosulfate (Figure 32-11), the sulfate donor for sulfated proteoglycans (see Chapter 50) and
FIGURE 32-6 Structures of AMP, dAMP, UMP, and TMP.
UMP
TMP
NH,
5-Methylcytosine
Ho
Dimethylaminoadenine
5-Hydroxymethylcytosine
7-Methylguanine
FIGURE
32-7
Four uncommon naturally occurring pyrimi- dines and purines.

for sulfate conjugates of drugs; and the methyl group donor S-adenosylmethionine (Figure 32-12). GTP serves as an allosteric regulator and as an energy source for protein synthesis, and cGMP (Figure 32-10) serves as a second messenger in response to nitric oxide (NO) during relaxation of smooth muscle (see Chapter 51).
UDP-sugar derivatives participate in sugar epimerizations and in biosynthesis of glycogen (see Chapter 18), glucosyl disaccharides, and the oligosaccharides of glycoproteins and proteoglycans (see Chapters 46 & 50). UDP-glucuronic acid forms the urinary glucuronide conjugates of bilirubin (see Chapter 31) and of many drugs, including aspirin. CTP participates in biosynthesis of phosphoglycerides, sphingomyelin, and other substituted sphingosines (see Chapter 24). Finally, many coenzymes incorporate nucleotides as well as structures similar to purine and pyrimidine nucleotides (Table 32-2).
°
0
HN
a
N
\
|
»
H
Hypoxanthine
HN
oX,
|
Pe
H
N
\
H
Xanthine
(6-oxopurine)
(2,6-dioxopurine)
Uric acid (2,6,8-trioxypurine)
FIGURE 32-8 Structures of hypoxanthine, xanthine, and uric acid, drawn as the oxo tautomers.
CHAPTER
32
323
FIGURE 32-9 Caffeine, a trimethylxanthine. The dimethylxanthines theobromine and theophylline are similar but lack the methyl group at N-1 and at N-7, respectively.
oO
HN
ww,
O——CH, o-beo
N
\
L
oO
L,
OH
FIGURE 32-10 cAmp, 3',5'-cyclic AMP, and cGMP, 3', 5'-cyclic GMP.

FIGURE 32-11 Adenosine 3'-phosphate-5'-phosphosulfate.
FIGURE 32-12 s-Adenosylmethionine.

Active methionine, R = Methionine a. Active methionine, R /DEL = H. Active methionine, R è = H. Active methionine, n = 0. Amino acid adenylates, R = Amino acid. Amino acid adenylates, R /DEL = H. Amino acid adenylates, R è = H. Amino acid adenylates, n = 1. Active sulfate, R = SO 3 2-. Active sulfate, R /DEL = H. Active sulfate, R è = PO 3 2-. Active sulfate, n = 1. 3 ′ ,5 ′ -Cyclic AMP, R = . 3 ′ ,5 ′ -Cyclic AMP, R /DEL = H. 3 ′ ,5 ′ -Cyclic AMP, R è = PO 3 2-. 3 ′ ,5 ′ -Cyclic AMP, n = 1. NAD b, R = Nicotinamide. NAD b, R /DEL = H. NAD b, R è = H. NAD b, n = 2. NADP b, R = Nicotinamide. NADP b, R /DEL = PO 3 2-. NADP b, R è = H. NADP b, n = 2. FAD, R = Riboflavin. FAD, R /DEL = H. FAD, R è = H. FAD, n = 2. Coenzyme A, R = Pantothenate. Coenzyme A, R /DEL = H. Coenzyme A, R è = PO 3 2-. Coenzyme A, n = 2
*Replaces phosphoryl group.
®R is a vitamin B derivative.

Nucleotide triphosphates have two acid anhydride bonds and one ester bond. Unlike esters, acid anhydrides have a high-group transfer potential. AG" for the hydrolysis of each of the two terminal (6 and y) phosphoryl groups of a nucleoside triphosphate is about -7 kcal/mol (-30 kJ/mol). This high group transfer potential not only permits purine and pyrimidine nucleoside triphosphates to function as group transfer reagents, most commonly of the y-phosphoryl group, but also on occasion transfer of a nucleotide monophosphate moiety with an accompanying release of PP.. Cleavage of an acid anhydride bond typically is coupled with a highly endergonic process such as covalent bond synthesis, for example, the polymerization of nucleoside triphosphates to form a nucleic acid (see Chapter 34).

Synthetic analogs of purines, pyrimidines, nucleosides, and nucleotides modified in the heterocyclic ring or in the sugar moiety have numerous applications in clinical medicine. Their toxic effects reflect either inhibition of enzymes essential for nucleic acid synthesis or their incorporation into nucleic acids with resulting disruption of base pairing. Oncologists employ 5-fluoroor 5-iodouracil, 3-deoxyuridine, 6-thioguanine and 6-mercaptopurine, 5or 6-azauridine, 5or 6-azacytidine, and 8-azaguanine (Figure 32-13), which are incorporated into DNA prior to cell division. The purine analog allopurinol, used in treatment of hyperuricemia and gout, inhibits purine
FIGURE 32-13 Selected synthetic pyrimidine and purine analogs.

biosynthesis and xanthine oxidase activity. Cytarabine is used in chemotherapy of cancer, and azathioprine, which is catabolized to 6-mercaptopurine, is employed during organ transplantation to suppress immunologic rejection (Figure 32-14).

Synthetic, non-hydrolyzable analogs of nucleoside triphosphates (Figure 32-15) allow investigators to distinguish the effects of nucleotides due to phosphoryl transfer from effects mediated by occupancy of allosteric nucleotide-binding sites on regulated enzymes (see Chapter 9).

The 5'-phosphoryl group of a mononucleotide can esterify a second hydroxyl group, forming a phosphodiester. Most commonly, this second hydroxyl group is the 3'-OH of the pentose

B,y-Imino derivative
FIGURE 32-15 synthetic derivatives of nucleoside triphosphates incapable of undergoing hydrolytic release of the terminal phosphoryl group. (Pu/Py, a purine or pyrimidine base; R, ribose or deoxyribose.) Shown are the parent (hydrolyzable) nucleoside triphosphate (top) and the unhydrolyzable B-methylene (center) and y-imino derivatives (bottom).
of a second nucleotide. This forms a dinucleotide in which the pentose moieties are linked by a 3',5'-phosphodiester bond to form the "backbone" of RNA and DNA. The formation of a dinucleotide may be represented as the elimination of water between two mononucleotides. Biologic formation of dinucleotides does not, however, occur in this way because the reverse reaction, hydrolysis of the phosphodiester bond, is strongly favored on thermodynamic grounds. However, despite an extremely favorable AG, in the absence of catalysis by phosphodiesterases hydrolysis of the phosphodiester bonds of DNA occurs only over long periods of time. DNA therefore persists for considerable periods, and has been detected even in fossils. RNAs are far less stable than DNA since the 2'-hydroxyl group of RNA (absent from DNA) functions as a nucleophile during hydrolysis of the 3',5'-phosphodiester bond.
Posttranslational modification of preformed polynucleotides can generate additional structures such as pseudouridine, a nucleoside in which D-ribose is linked to C-5 of uracil by a carbon-to-carbon bond rather than by the usual B-Nglycosidic bond. The nucleotide pseudouridylic acid (y) arises by rearrangement of a UMP of a preformed tRNA. Similarly, methylation by S-adenosylmethionine of a UMP of preformed tRNA forms TMP (thymidine monophosphate), which contains ribose rather than deoxyribose.

Directional 3' > 5' phosphodiester bonds link the monomers of polynucleotides. Since each end of a polynucleotide thus is distinct, we refer to the "5'-end" or the "3'-end" of a polynucleotide. Since the phosphodiester bonds all are 3' > 5', the representation pGpGpApTpCpA indicates that only the 5'-hydroxyl is phosphorylated. More concisely, the representation GGATC, which shows only the base sequence, is by convention written with the 5'-base (G) at the Jeft and the 3'-base (C) at the right.

- Under physiologic conditions, the amino and oxo tautomers of
purines, pyrimidines, and their derivatives predominate.
- ™ Nucleic acids contain, in addition to A, G, C, T, and U, traces
of
5-methylcytosine,
5-hydroxymethylcytosine, pseudouridine
(y), and
N-methylated heterocycles.
= Most nucleosides contain D-ribose or 2-deoxy-D-ribose linked to N-1 of a pyrimidine or to N-9 of a purine by a B-glycosidic bond whose syn conformers predominate.
= A primed numeral indicates the hydroxyl to which the phosphoryl group of the sugars of mononucleotides (eg, 3'-GMP, 5'-dCMP) is attached. Additional phosphoryl groups linked to the first by acid anhydride bonds form nucleoside diphosphates and triphosphates.
= Nucleoside triphosphates have high group transfer potential and participate in covalent bond syntheses. The cyclic phosphodiesters cAMP and cGMP function as intracellular second messengers.
™ Mononucleotides linked by 3' > 5'-phosphodiester bonds form polynucleotides, directional macromolecules with distinct 3'and 5'-ends. When represented as pTpGpT or TGCATCA, the 5'-end is at the left, and all phosphodiester bonds are 3' > 5'.
™@ Synthetic analogs of purine and pyrimidine bases and their derivatives serve as anticancer drugs either by inhibiting an enzyme of nucleotide biosynthesis or by being incorporated into DNA or RNA.

- Adams RLP, Knowler JT, Leader DP: The Biochemistry of the Nucleic Acids, 11th ed. Chapman & Hall, 1992.
Blackburn GM, Gait MJ, Loaks D, et al: Nucleic Acids in Chemistry and Biology, 3rd ed., RSC Publishing, 2006.
Pacher P, Nivorozhkin A, Szabo C: Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol. Pharmacol Rev 2006;58:87.

. Compare and contrast the roles of dietary nucleic acids and of de novo biosynthesis in the production of purines and pyrimidines destined for polynucleotide biosynthesis.
: Explain why antifolate drugs and analogs of the amino acid glutamine inhibit purine biosynthesis.
triphosphates. = Outline the sequence of reactions that convert inosine monophosphate (IMP), first to AMP and GMP, and subsequently to their corresponding nucleoside
Describe the formation from ribonucleotides of deoxyribonucleotides (dNTPs).
Indicate the regulatory role of phosphoribosyl pyrophosphate (PRPP) in hepatic purine biosynthesis and the specific reaction of hepatic purine biosynthesis that is feedback inhibited by AMP and GMP.
biosynthesis. ™ State the relevance of coordinated control of purine and pyrimidine nucleotide
Identify the reactions discussed that are inhibited by anticancer drugs.
disease. Write the structure of the end product of purine catabolism. Comment on its solubility and indicate its role in gout, Lesch-Nyhan syndrome, and von Gierke
symptoms. = Identify reactions whose impairment leads to modified pathologic signs and
catabolism. m Indicate why there are few clinically significant disorders of pyrimidine

Despite a diet that may be rich in nucleoproteins, dietary purines and pyrimidines are not incorporated directly into tissue nucleic acids. Humans synthesize the nucleic acids and their derivatives ATP, NAD', coenzyme A, etc, from amphibolic intermediates. However, injected purine or pyrimidine analogs, including potential anticancer drugs, may nevertheless be incorporated into DNA. The biosyntheses of purine and pyrimidine ribonucleotide triphosphates (NTPs) and dNTPs are precisely regulated events. Coordinated feedback mechanisms ensure their production in appropriate quantities and at times that match varying physiologic demand (eg, cell division). Human diseases that involve abnormalities in purine metabolism include gout, Lesch-Nyhan syndrome, adenosine deaminase deficiency, and purine nucleoside phosphorylase deficiency. Diseases of pyrimidine biosynthesis are rarer, but include orotic acidurias. Unlike the low solubility of uric acid formed by catabolism of purines, the end products of pyrimidine catabolism (carbon dioxide, ammonia, f-alanine, and y-aminoisobutyrate) are highly water soluble. One genetic disorder of pyrimidine catabolism, 6-hydroxybutyric aciduria, is due to total or partial deficiency of the enzyme dihydropyrimidine dehydrogenase. This disorder of pyrimidine catabolism, also known as combined uraciluria-thyminuria, is also a disorder of B-amino acid biosynthesis, since the formation of B-alanine and of B-aminoisobutyrate is
impaired. A nongenetic form can be triggered by administration of 5-fluorouracil to patients with low levels of dihydropyrimidine dehydrogenase.

Normal human tissues can synthesize purines and pyrimidines from amphibolic intermediates in quantities and at times appropriate to meet variable physiologic demand. Ingested nucleic acids and nucleotides therefore are dietarily nonessential. Following their degradation in the intestinal tract, the resulting mononucleotides may be absorbed or converted to purine and pyrimidine bases. The purine bases are then oxidized to uric acid, which may be absorbed and excreted in the urine. While little or no dietary purine or pyrimidine is incorporated into tissue nucleic acids, injected compounds are incorporated. The incorporation of injected [*H] thymidine into newly synthesized DNA thus can be used to measure the rate of DNA synthesis.

With the exception of parasitic protozoa, all forms of life synthesize purine and pyrimidine nucleotides. Synthesis from amphibolic intermediates proceeds at controlled rates appropriate for all cellular functions. To achieve homeostasis, intracellular mechanisms sense and regulate the pool sizes of NTPs, which rise during growth or tissue regeneration when cells are rapidly dividing.
Purine and pyrimidine nucleotides are synthesized in vivo at rates consistent with physiologic need. Early investigations of nucleotide biosynthesis first employed birds, and later Escherichia coli. Isotopic precursors of uric acid fed to pigeons established the source of each atom of a purine (Figure 33-1) and initiated study of the intermediates of purine biosynthesis.
FIGURE 33-1 Sources of the nitrogen and carbon atoms of the purine ring. Atoms 4, 5, and 7 (blue highlight) derive from glycine.
Avian tissues also served as a source of cloned genes that encode enzymes of purine biosynthesis and the regulatory proteins that control the rate of purine biosynthesis. The three processes that contribute to purine nucleotide order of biosynthesis are, in decreasing importance:
1. Synthesis from amphibolic intermediates (synthesis de novo)
2. Phosphoribosylation of purines
3. Phosphorylation of purine nucleosides

The initial reaction of purine biosynthesis, transfer of two phosphoryl groups from ATP to carbon 1 of ribose 5-phosphate forming phosphoribosyl pyrophosphate (PRPP), is catalyzed by PRPP synthetase, EC 2.7.6.1. The end product of the ten subsequent enzyme-catalyzed reactions is IMP (Figure 33-2).
Following synthesis of IMP, separate branches lead to AMP and GMP (Figure 33-3). Subsequent phosphoryl transfer from ATP converts AMP and GMP to ADP and GDP, respectively. Conversion of GDP to GTP involves a second phosphoryl transfer from ATP, whereas conversion of ADP to ATP is achieved primarily by oxidative phosphorylation (see Chapter 13).

In prokaryotes, each reaction of Figure 33-2 is catalyzed by a different polypeptide. By contrast, the enzymes of eukaryotes are polypeptides that possess multiple catalytic activities whose adjacent catalytic sites facilitate channeling of intermediates between sites. Three distinct multifunctional enzymes catalyze reactions @, @, and ©; reactions @ and ®; and reactions @ and @ of Figure 33-2.

The carbons added in reactions @ and @ of Figure 33-2 are contributed by derivatives of tetrahydrofolate. Purine deficiency states, while rare in humans, generally reflect a deficiency of folic acid. Compounds that inhibit formation of tetrahydrofolates and therefore block purine synthesis have been used in cancer chemotherapy. Inhibitory compounds and the reactions they inhibit include azaserine (reaction ©, Figure 33-2), diazanorleucine (reaction @, Figure 33-2),
FIGURE 33-2 Purine biosynthesis from ribose 5-phosphate and ATP. See the text for explanations. (®, PO,* or PO,~.)

6-mercaptopurine (reactions ® and @, Figure 33-3), and mycophenolic acid (reaction @, Figure 33-3).

Conversion of purines, their ribonucleosides, and their deoxyribonucleosides to mononucleotides involves "salvage reactions" that require far less energy than de novo synthesis. The more important mechanism involves phosphoribosylation by PRPP (structure II, Figure 33-2) of a free purine (Pu) to form a purine 5'-mononucleotide (Pu-RP).

Phosphoryl transfer from PRPP catalyzed by adenosineand hypoxanthine-phosphoribosyl transferases (EC 2.4.2.7 & EC 2.4.2.8, respectively), converts adenine, hypoxanthine, and guanine to their mononucleotides (Figure 33-4). A second salvage mechanism involves phosphoryl transfer from ATP to a purine ribonucleoside (Pu-R):

Phosphorylation of the purine nucleotides, catalyzed by adenosine kinase (EC 2.7.1.20), converts adenosine and deoxyadenosine to AMP and dAMP. Similarly, deoxycytidine kinase (EC 2.7.1.24) phosphorylates deoxycytidine and 2'-deoxyguanosine, forming dCMP and dGMP, respectively.
Liver, the major site of purine nucleotide biosynthesis, provides purines and purine nucleosides for salvage and for utilization by tissues incapable of their biosynthesis. Human brain tissue has a low level of PRPP glutamyl amidotransferase, EC 2.4.2.14 (reaction @, Figure 33-2) and hence depends in part on exogenous purines. Erythrocytes and polymorphonuclear leukocytes cannot synthesize 5-phosphoribosylamine (structure III, Figure 33-2), and therefore also utilize exogenous purines to form nucleotides.

Biosynthesis of IMP is energetically expensive. In addition to ATP, glycine, glutamine, aspartate, and reduced tetrahydrofolate derivatives all are consumed. Thus, it is of survival advantage to closely regulate purine biosynthesis in response to varying physiologic need. The overall determinant of the rate of de novo purine nucleotide biosynthesis is the concentration of PRPP. This, in turn, depends on the rate of PRPP synthesis, utilization, degradation, and regulation. The rate of PRPP synthesis depends on the availability of ribose 5-phosphate and on the activity of PRPP synthetase, EC 2.7.6.1 (reaction @ Figure 33-5), an enzyme whose activity is feedback inhibited by AMP, ADP, GMP, and GDP. Elevated levels of these nucleoside phosphates thus signal a physiologically appropriate overall decrease in their biosynthesis.
NH,
FIGURE 33-4 Phosphoribosylation of adenine, hypoxanthine, and guanine to form AMP, IMP, and GMP, respectively.

In addition to regulation at the level of PRPP biosynthesis, additional mechanisms that regulate conversion of IMP to ATP and GTP are summarized in Figure 33-6. AMP feedback inhibits adenylosuccinate synthetase, EC 6.3.4.4 (reaction ®, Figure 33-3), and GMP inhibits IMP dehydrogenase, EC 1.1.1.205 (reaction @, Figure 33-3). Furthermore, conversion of IMP to adenylosuccinate en route to AMP (reaction @, Figure 33-3) requires GTP, and conversion of xanthinylate (XMP) to GMP requires ATP. This cross-regulation between the pathways of IMP metabolism thus serves to balance the biosynthesis of purine nucleoside triphosphates by decreasing the synthesis of one purine nucleotide when there is a deficiency of the other nucleotide. AMP and GMP also inhibit hypoxanthine-guanine phosphoribosyltransferase, which converts hypoxanthine and guanine to IMP and GMP (Figure 33-4), and GMP feedback inhibits PRPP glutamyl amidotransferase (reaction @, Figure 33-2).
FIGURE 33-5 Control of the rate of de novo purine nucleotide biosynthesis. Reactions @ and @ are catalyzed by PRPP synthetase and by PRPP glutamyl amidotransferase, respectively. Solid lines represent chemical flow. Broken red lines represent feedback inhibition by intermediates of the pathway.
FIGURE 33-6 Regulation of the conversion of IMP to adenosine nucleotides and guanosine nucleotides. Solid lines represent chemical flow. Broken green lines represent positive feedback loops @, and broken red lines represent negative feedback loops ©. (AMPS, adenylosuccinate; XMP, xanthosine monophosphate; their structures are given in Figure 33-3.)
FIGURE 33-7 Reduction of ribonucleoside diphosphates to 2'-deoxyribonucleoside diphosphates.

Reduction of the 2'-hydroxyl of purine and pyrimidine ribonucleotides, catalyzed by the complex that includes ribonucleotide reductase, EC 1.17.4.1 (Figure 33-7), provides the deoxyribonucleoside diphosphates (ANDPs) needed for both the synthesis and repair of DNA (see Chapter 35). The enzyme complex is functional only when cells are actively synthesizing DNA. Reduction requires reduced thioredoxin, thioredoxin reductase (EC 1.8.1.9), and NADPH. The immediate reductant, reduced thioredoxin, is produced by NADPH-dependent reduction of oxidized thioredoxin (Figure 33-7). The reduction of ribonucleoside diphosphates (NDPs) to dNDPs is subject to complex regulatory controls that achieve balanced production of dNTPs for synthesis of DNA (Figure 33-8).

Figure 33-9 illustrates the intermediates and enzymes of pyrimidine nucleotide biosynthesis. The catalyst for the initial reaction is cytosolic carbamoyl phosphate synthetase II (EC 6.3.5.5), a different enzyme from the mitochondrial carbamoyl phosphate synthetase I of urea synthesis (see Figure 28-16). Compartmentation thus provides an independent pool of carbamoyl phosphate for each process. Unlike in purine biosynthesis where PRPP serves as a scaffold for assembly of the purine ring (Figure 33-2), PRPP participates in pyrimidine biosynthesis only subsequent to assembly of the pyrimidine ring. As for the biosynthesis of pyrimidines, purine nucleoside biosynthesis is energetically costly.

Five of the first six enzyme activities of pyrimidine biosynthesis reside on multifunctional polypeptides. CAD, a single
FIGURE 33-8 Regulatory aspects of the biosynthesis of purine and pyrimidine ribonucleotides and reduction to their respective 2-deoxyribonucleotides. The broken green line represents a positive feedback loop. Broken red lines represent negative feedback loops. Abbreviations are provided for the intermediates in the biosynthesis of pyrimidine nucleotides whose structures are given in Figure 33-9. (CAA, carbamoyl aspartate; DHOA, dihydroorotate; OA, orotic acid; OMP, orotidine monophosphate; PRPP phosphoribosyl pyrophosphate.)
polypeptide named for the the first letters of its enzyme activities, catalyzes the first three reactions of Figure 33-9. A second bifunctional enzyme catalyzes reactions ® and © of Figure 33-9. The close proximity of multiple active sites on a multifunctional polypeptide facilitates efficient channeling of the intermediates of pyrimidine biosynthesis.

Adenine, guanine, and hypoxanthine released during the turnover of nucleic acids, notably messenger RNAs, are reconverted to nucleoside triphosphates via so-called salvage pathways. While mammalian cells reutilize few free pyrimidines, "salvage reactions" convert the pyrimidine ribonucleosides
FIGURE 33-9 The biosynthetic pathway for pyrimidine nucleotides.
uridine and cytidine and the pyrimidine deoxyribonucleosides thymidine and deoxycytidine to their respective nucleotides.

Guanine + PRPP — GMP + PP,
Hypoxanthine + PRPP — IMP + PP,
Phosphoryltransferases (kinases) catalyze transfer of the y-phosphoryl group of ATP to the diphosphates of the dNDPs 2'-deoxycytidine, 2'-deoxyguanosine, and 2'-deoxyadenosine, converting them to the corresponding nucleoside triphosphates.
NDP + ATP > NTP + ADP dNDP + ATP > dNTP + ADP
The reaction catalyzed by thymidylate synthase, EC 2.1.1.45 (reaction ® of Figure 33-9) is the only reaction of pyrimidine nucleotide biosynthesis that requires a tetrahydrofolate derivative. During this reaction, the methylene group of N'°,N'°-methylene-tetrahydrofolate is reduced to the methyl group that is transferred to the 5-position of the pyrimidine ring, and tetrahydrofolate is oxidized to dihydrofolate. For further pyrimidine synthesis to occur, dihydrofolate must be reduced back to tetrahydrofolate. This reduction,
catalyzed by dihydrofolate reductase (EC 1.5.1.3), is inhibited by methotrexate. Dividing cells, which must generate TMP and dihydrofolate, thus are especially sensitive to inhibitors of dihydrofolate reductase such as the anticancer drug methotrexate.

Allopurinol and the anticancer drug 5-fluorouracil (see Figure 32-13) are alternate substrates for orotate phosphoribosyltransferase, EC 2.4.2.10 (reaction ©, Figure 33-9). Both drugs are phosphoribosylated, and allopurinol is converted to a nucleotide in which the ribosyl phosphate is attached to N! of the pyrimidine ring.

CAD represents the primary focus for regulation of pyrimidine biosynthesis. Expression of the CAD gene is regulated at the level both of transcription and of translation. At the level of enzyme activity, the carbamoyl phosphate synthetase II (CPS) activity of CAD is activated by PRPP and is feedback inhibited by UTP. The effect of UTP is, however, abolished by phosphorylation of serine 1406 of CAD.

Purine and pyrimidine biosynthesis parallel one another quantitatively, that is, mole for mole, suggesting coordinated control of their biosynthesis. Several sites of cross-regulation characterize the pathways that lead to the biosynthesis of purine and pyrimidine nucleotides. PRPP synthetase (reaction @, Figure 33-2), which forms a precursor essential for both processes, is feedback inhibited by both purine and pyrimidine nucleotides, as is the conversion of both pyrimidine and purine nucleotides NDPs to NTPs (Figure 33-10).

Humans convert adenosine and guanosine to uric acid (Figure 33-11). Adenosine is first converted to inosine by adenosine deaminase, EC 3.5.4.4. In mammals other than
FIGURE 33-10 Regulation of the conversion of purine and pyrimidine NDPs to NTPs. Solid lines represent chemical flow.
Broken lines indicate targets of positive ® or negative © feedback inhibition.
higher primates, uricase (EC 1.7.3.3) converts uric acid to the water-soluble product allantoin. However, since humans lack uricase, the end product of purine catabolism in humans is uric acid.

Various genetic defects in PRPP synthetase (reaction ©, Figure 33-2) present clinically as gout. Each defect—for example, an elevated V_, increased affinity for ribose 5-phosphate, or resistance to feedback inhibition—results in overproduction and overexcretion of purine catabolites. When serum urate levels exceed the solubility limit, sodium urate crystalizes in soft tissues and joints and causes an inflammatory reaction, gouty arthritis. However, most cases of gout reflect abnormalities in renal handling of uric acid.
While purine deficiency states are rare in human subjects, there are numerous genetic disorders of purine catabolism. Hyperuricemias may be differentiated based on whether patients excrete normal or excessive quantities of total urates. Some hyperuricemias reflect specific enzyme defects. Others are secondary to diseases such as cancer or psoriasis that enhance tissue turnover.

The Lesch-Nyhan syndrome, an overproduction hyperuricemia characterized by frequent episodes of uric acid lithiasis and a bizarre syndrome of self-mutilation, reflects a defect in hypoxanthine-guanine phosphoribosyl transferase, an enzyme of purine salvage (Figure 33-4). The accompanying rise in intracellular PRPP results in purine overproduction. Mutations that decrease or abolish hypoxanthine-guanine
FIGURE 33-11 Formation of uric acid from purine nucleosides by way of the purine bases hypoxanthine, xanthine, and guanine. Purine deoxyribonucleosides are degraded by the same catabolic pathway and enzymes, all of which exist in the mucosa of the mammalian gastrointestinal tract.
phosphoribosyltransferase activity include deletions, frameshift mutations, base substitutions, and aberrant mRNA splicing.

Purine overproduction and hyperuricemia in von Gierke disease (glucose-6-phosphatase deficiency) occurs secondary to enhanced generation of the PRPP precursor ribose 5-phosphate. An associated lactic acidosis elevates the renal threshold for urate, elevating total body urates.

Hypouricemia and increased excretion of hypoxanthine and xanthine are associated with a deficiency in xanthine oxidase, EC 1.17.3.2 (Figure 33-11) due to a genetic defect or to severe liver damage. Patients with a severe enzyme deficiency may exhibit xanthinuria and xanthine lithiasis.

Adenosine deaminase deficiency (Figure 33-11) is associated with an immunodeficiency disease in which both thymus-derived lymphocytes (T cells) and bone marrowderived lymphocytes (B cells) are sparse and dysfunctional. Patients suffer from severe immunodeficiency. In the absence of enzyme replacement or bone marrow transplantation, infants often succumb to fatal infections. Defective activity of purine nucleoside phosphorylase (EC 2.4.2.1) is associated with a severe deficiency of T cells, but apparently normal B-cell function. Immune dysfunctions appear to result from accumulation of dGTP and dATP, which inhibit ribonucleotide reductase and thereby deplete cells of DNA precursors. Table 33-1 summarizes known disorders of purine metabolism.

Unlike the low solubility products of purine catabolism, catabolism of the pyrimidines forms highly water-soluble products— CO,, NH,, B-alanine, and B-aminoisobutyrate (Figure 33-12). Excretion of B-aminoisobutyrate increases in leukemia and severe x-ray radiation exposure due to increased destruction of DNA. However, many persons of Chinese or Japanese ancestry routinely excrete B-aminoisobutyrate.
Disorders of B-alanine and B-aminoisobutryrate metabolism arise from defects in enzymes of pyrimidine catabolism. These include 6-hydroxybutyric aciduria, a disorder due to total or partial deficiency of the enzyme dihydropyrimidine dehydrogenase, EC 1.3.1.2 (Figure 33-12). The genetic disease reflects an absence of the enzyme. A disorder of

toxicity to 5-fluorouracil, also a substrate for this dehydrogenase. Dihydropyrimidine dehydrogenase, Figure and Reaction = 33-12 ➁. Orotate phosphoribosyl transferase and orotidylic acid decarboxylase, Enzyme Catalog Number = 2.4.2.10 and 4.1.1.23. Orotate phosphoribosyl transferase and orotidylic acid decarboxylase, OMIMReference = 258900. Orotate phosphoribosyl transferase and orotidylic acid decarboxylase, Major Signs and Symptoms = Orotic acid aciduria type 1; megaloblastic anemia. Orotate phosphoribosyl transferase and orotidylic acid decarboxylase, Figure and Reaction = 33-9 ➄ and ➅. Orotidylic acid decarboxylase, Enzyme Catalog Number = 4.1.1.23. Orotidylic acid decarboxylase, OMIMReference = 258920. Orotidylic acid decarboxylase, Major Signs and Symptoms = Orotic acid aciduria type 2. Orotidylic acid decarboxylase, Figure and Reaction = 33-9 ➅
pyrimidine catabolism, known also as combined uraciluriathyminuria, is also a disorder of B-amino acid metabolism, since the formation of B-alanine and of 6-aminoisobutyrate is impaired. When caused by a genetic error, there are serious neurologic complications. A nongenetic form is triggered by the administration of the anticancer drug 5-fluorouracil (see Figure 32-13) to patients with low levels of dihydropyrimidine dehydrogenase.

mitochondria to utilize carbamoyl phosphate, which then becomes available for cytosolic overproduction of orotic acid. Type I orotic aciduria reflects a deficiency of both orotate phosphoribosyltransferase (EC 2.1.3.3) and orotidylate decarboxylase, EC 4.1.1.23 (reactions © and ©, Figure 33-9). The rarer Type II orotic aciduria is due to a deficiency only of orotidylate decarboxylase (reaction ©, Figure 33-9).

No human enzyme catalyzes hydrolysis or phosphorolysis of the pseudouridine (y) derived from the degradation of RNA molecules. This unusual nucleotide therefore is excreted unchanged in the urine of normal subjects. Pseudouridine was indeed first isolated from human urine (Figure 33-13).

Since the end products of pyrimidine catabolism are highly water soluble, pyrimidine overproduction results in few clinical signs or symptoms. Table 33-1 lists exceptions. In hyperuricemia associated with severe overproduction of PRPP, there is overproduction of pyrimidine nucleotides and increased excretion of B-alanine. Since N°,N'°-methylene-tetrahydrofolate is required for thymidylate synthesis, disorders of folate and vitamin B,, metabolism result in deficiencies of TMP.

The orotic aciduria that accompanies the Reye syndrome probably is a consequence of the inability of severely damaged

Increased excretion of orotic acid, uracil, and uridine accompanies a deficiency in liver mitochondrial ornithine transcarbamoylase (see reaction @, Figure 28-16). Excess carbamoyl phosphate exits to the cytosol, where it stimulates pyrimidine nucleotide biosynthesis. The resulting mild orotic aciduria is increased by high-nitrogen foods.

Allopurinol (see Figure 32-13), an alternative substrate for orotate phosphoribosyltransferase (reaction ©, Figure 33-9), competes with orotic acid. The resulting nucleotide product also inhibits orotidylate decarboxylase (reaction ©, Figure 33-9), resulting in orotic aciduria and orotidinuria. 6-Azauridine, following conversion to 6-azauridylate, also competitively inhibits orotidylate decarboxylase (reaction ©, Figure 33-9), enhancing excretion of orotic acid and orotidine. Four genes that encode urate transporters have been identified. Two of the encoded proteins are localized to the apical membrane of proximal tubular cells.
FIGURE 33-12 Catabolism of pyrimidines. Hepatic B-ureidopropionase catalyzes the formation of both B-alanine and B-aminoisobutryrate from their pyrimidine precursors.

™
Ingested nucleic acids are degraded to purines and pyrimidines.
Purines and pyrimidines are formed from amphibolic intermediates and thus are dietarily nonessential.
- ™ Several reactions of IMP biosynthesis require folate derivatives and glutamine. Consequently, antifolate drugs and glutamine analogs inhibit purine biosynthesis.
CHAPTER
33

= IMP isa precursor both of AMP and GMP. Glutamine provides the 2-amino group of GMP, and aspartate the 6-amino group of AMP.
= Phosphoryl transfer from ATP converts AMP and GMP to ADP and GDP. A second phosphoryl transfer from ATP forms GTP, but ADP is converted to ATP primarily by oxidative phosphorylation.
™ Hepatic purine nucleotide biosynthesis is stringently regulated by the pool size of PRPP and by feedback inhibition of PRPP glutamyl amidotransferase by AMP and GMP.
™ Coordinated regulation of purine and pyrimidine nucleotide biosynthesis ensures their presence in proportions appropriate for nucleic acid biosynthesis and other metabolic needs.
=
Humans catabolize purines to uric acid (pK, 5.8), present as the relatively insoluble acid at acidic pH or as its more soluble sodium urate salt at a pH near neutrality. Urate crystals are diagnostic of gout. Other disorders of purine catabolism include Lesch-Nyhan syndrome, von Gierke disease, and hypouricemias.
™ Since pyrimidine catabolites are water soluble, their overproduction does not result in clinical abnormalities. Excretion of pyrimidine precursors can, however, result from a deficiency of ornithine transcarbamoylase because excess carbamoyl phosphate is available for pyrimidine biosynthesis.

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Fu R, Jinnah HA: Genotype-phenotype correlations in Lesch-Nyhan
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Vim ToS ACM UER ese] oem el] should be able to:
C H A PT ER
. eukaryotic cells. Understand the chemical monomeric and polymeric structure of the genetic material, deoxyribonucleic acid, or DNA, which is found within the nucleus of
Explain why genomic DNA is double stranded and highly negatively charged.
duplicated. Understand the outline of how the genetic information of DNA can be faithfully
= Describe how the genetic information of DNA is transcribed, or copied, into myriad, distinct forms of ribonucleic acid (RNA).
= Appreciate that one form of information-rich RNA, the so-called messenger RNA (mRNA), can be subsequently translated into proteins, the molecules that form the structures, shapes, and ultimately functions of individual cells, tissues, and organs.

The discovery that genetic information is coded along the length of a polymeric molecule composed of only four types of monomeric units was one of the major scientific achievements of the 20th century. This polymeric molecule, deoxyribonucleic acid (DNA), is the chemical basis of heredity and is organized into genes, the fundamental units of genetic information. The basic information pathway— that is, DNA, which directs the synthesis of RNA, which in turn both directs and regulates protein synthesis—has been elucidated. Genes do not function autonomously; rather their replication and function are controlled by various gene products, often in collaboration with components of various signal transduction pathways. Knowledge of the structure and function of nucleic acids is essential in understanding genetics and many aspects of pathophysiology as well as the genetic basis of disease.

The demonstration that DNA contained the genetic information was first made in 1944 in a series of experiments by Avery, MacLeod, and McCarty. They showed that the genetic determination of the character (type) of the capsule of a specific pneumococcus bacterium could be transmitted to another of a different capsular type by introducing purified DNA from the former pneumococcus into the latter. These authors referred to the agent (later shown to be DNA) accomplishing the change as "transforming factor" Subsequently, this type of genetic manipulation has become commonplace. Conceptually similar experiments now are regularly performed utilizing a variety of eukaryotic cells, including human cells and mammalian embryos as recipients and molecularly cloned DNA as the donor of genetic information.

The chemical nature of the monomeric deoxynucleotide units of DNA—deoxyadenylate, deoxyguanylate, deoxycytidylate, and thymidylate—is described in Chapter 32. These monomeric units of DNA are held in polymeric form by 3',5'-phosphodiester bonds constituting a single strand, as depicted in Figure 34-1. The informational content of DNA (the genetic code) resides in the sequence in which these monomers—purine and pyrimidine deoxyribonucleotides— are ordered. The polymer as depicted possesses a polarity; one end has a 5'-hydroxyl or phosphate terminus while the other has a 3'-phosphate or hydroxyl terminus. The importance of this polarity will become evident. Since the genetic information
FIGURE 34-1 A segment of one strand of a DNA molecule in which the purine and pyrimidine bases guanine (G), cytosine (C), thymine (T), and adenine (A) are held together by a phosphodiester backbone between 2'-deoxyribosyl moieties attached to the nucleobases by an N-glycosidic bond. Note that the phosphodiester backbone is negatively charged and has a polarity (ie, a direction). Convention dictates that a single-stranded DNA sequence is written in the 5' to 3' direction (ie, pGpCpTpAp, where G, C, T, and A represent the four bases and p represents the interconnecting phosphates).

resides in the order of the monomeric units within the polymers, there must exist a mechanism of reproducing or replicating this specific information with a high degree of fidelity. That requirement, together with x-ray diffraction data from the DNA molecule generated by Franklin, and the observation of Chargaff that in DNA molecules the concentration of deoxyadenosine (A) nucleotides equals that of thymidine (T) nucleotides (A = T), while the concentration of deoxyguanosine (G) nucleotides equals that of deoxycytidine (C) nucleotides (G = C), led Watson, Crick, and Wilkins to propose in the early 1950s a model of a double-stranded DNA molecule. The model they proposed is depicted in Figure 34-2. The two strands of this double-stranded helix are held in register by both hydrogen bonds between the purine and pyrimidine bases of the respective linear molecules and by van der Waals and hydrophobic interactions between the stacked adjacent base pairs. The pairings between the purine and pyrimidine nucleotides on the opposite strands are very specific and are dependent on hydrogen bonding of A with T and G with C (Figure 34-2). A-T and G-C base pairs are often referred to as Watson-Crick base pairs.

form a spiral in a clockwise direction. In the double-stranded molecule, restrictions imposed by the rotation about the phosphodiester bond, the favored anti-configuration of the glycosidic bond (see Figure 32-5), and the predominant tautomers (see Figure 32-2) of the four bases (A, G, T, and C) allow A to pair only with T, and G only with C, as depicted in Figure 34-3. These base-pairing restrictions explain the earlier observation that in a double-stranded DNA molecule the content of A equals that of T and the content of G equals that of C. The two strands of the double-helical molecule, each of which possesses a polarity, are antiparallel; that is, one strand runs in the 5' to 3' direction and the other in the 3' to 5' direction. Within a particular gene in the double-stranded DNA molecules, the genetic information resides in the sequence of nucleotides on one strand, the template strand. This is the strand of DNA that is copied, or transcribed, during ribonucleic acid (RNA) synthesis. It is sometimes referred to as the noncoding strand. The opposite strand is considered the coding strand because it matches the sequence of the RNA transcript (but containing uracil in place of thymine; Figure 34-8) that encodes the protein.
This common form of DNA is said to be right handed because as one looks down the double helix, the base residues
The two strands, in which opposing bases are held together by interstrand hydrogen bonds, wind around a central axis in

FIGURE 34-2 A diagrammatic representation of the Watson and Crick model of the double-helical structure of the B form of DNA. The horizontal arrow indicates the width of the double helix (20 A), and the vertical arrow indicates the distance spanned by one complete turn of the double helix (34 A). One turn of B-DNA includes 10 base pairs (bp), so the rise is 3.4 A per bp. The central axis of the double helix is indicated by the vertical rod. The short arrows designate the polarity of the antiparallel strands. The major and minor grooves are depicted. (A, adenine; C, cytosine; G, guanine; P, phosphate; S, sugar [deoxyribose]; T, thymine.) Hydrogen bonds between A/T and G/C bases indicated by short, red, horizontal lines.
FIGURE 34-3 Classic Watson-Crick DNA base pairing between complementary deoxynucleotides involves the formation of hydrogen bonds. Two such H-bonds form between adenine and thymine, and three H-bonds form between cytidine and broken lines H-bonds.
guanine. The represent the form of a double helix. In the test tube, double-stranded DNA can exist in at least six forms (A-E and Z). These different forms of DNA differ with regard to intraand interstrand interactions and involve structural rearrangements within the monomeric units of DNA. The B form is usually found under physiologic conditions. A single turn of B form DNA about the long axis of the molecule contains 10 bp. The distance spanned by one turn of B-DNA is 3.4 nm (34 A). The width (helical diameter) of the double helix in B-DNA is 2 nm (20 A).

As depicted in Figure 34-3, three hydrogen bonds (see Figure 2-2), formed by hydrogen bonded to electronegative N or O atoms, hold the deoxyguanosine nucleotide to the deoxycytidine nucleotide, whereas the other pair, the A-T pair, is held together by two hydrogen bonds. Note that the four DNA nucleotide bases ([dG, dA] purines and [dT, dC] pyrimidines; see Figures 32-1 and Table 32-1]) are flat, planar molecules. These fundamental properties of the nucleotide bases allow them to closely stack within duplex DNA (Figure 34-2). The atoms within the aromatic, heterocyclic bases are highly polarizable, and, coupled with the fact that many of the atoms within the bases contain partial charges, allows for the stacked bases to form van der Waals and electrostatic interactions. These forces are collectively referred to as base-stacking forces or interactions. Base-stacking interactions between adjacent G-C (or C-G) base pairs are stronger than A-T (or T—A) base pairs. Thus, the G-C rich DNA sequences are more resistant to denaturation, or strand separation, termed "melting," than A-T-rich regions of DNA.

The double-stranded structure of DNA can be separated into two component strands in solution by increasing the temperature or decreasing the salt concentration. Not only do the two stacks of bases pull apart, but the bases themselves unstack while still connected in the polymer by the phosphodiester backbone. Concomitant with this denaturation of the DNA molecule is an increase in the optical absorbance of the purine and pyrimidine bases—a phenomenon referred to as hyperchromicity of denaturation. Because of the stacking of the bases and the hydrogen bonding between the stacks, the double-stranded DNA molecule exhibits properties of a rigid rod and in solution is a viscous material that loses its viscosity upon denaturation.
The strands of a given molecule of DNA separate over a temperature range. The midpoint is called the melting temperature, or T,. The T,, is influenced by the base composition of the DNA and by the salt concentration (or other solutes, see below) of the solution. DNA rich in G-C pairs, which have three hydrogen bonds, melts at a higher temperature than DNA rich in A-T pairs, which have two hydrogen bonds. A 10-fold increase of monovalent cation concentration increases the T|, by 16.6°C by neutralizing the intrinsic interchain repulsion between the highly negatively charged
phosphates of the phosophodiester backbone. Conversely, the organic solvent formamide, which is commonly used in recombinant DNA experiments, destabilizes hydrogen bonding between bases, thereby lowering the T_. Formamide addition allows the strands of DNA or DNA-RNA hybrids to be separated at much lower temperatures and minimizes the phosphodiester bond breakage and chemical damage to nucleotides that can occur upon extended incubation at higher temperatures.

Importantly, separated strands of DNA will renature or reassociate when appropriate physiologic temperature and salt conditions are achieved; this reannealing process is often referred to as hybridization. The rate of reassociation depends on the concentration of the complementary strands. Reassociation of the two complementary DNA strands of a chromosome after transcription is a physiologic example of renaturation (see below). At a given temperature and salt concentration, a particular nucleic acid strand will associate tightly only with a complementary strand. Hybrid molecules will also form under appropriate conditions. For example, DNA will form a hybrid with a complementary DNA (cDNA) or with a cognate complementary RNA (eg, mRNA; see below). When hybridization is combined with gel electrophoresis techniques that separate nucleic acids by size, coupled with radioactive or fluorescent complementary probe labeling to provide a detectable signal, the resulting analytic techniques are called Southern (DNA/DNA) and Northern (RNA-DNA) blotting, respectively. These procedures allow for very distinct, high-sensitivity identification of specific nucleic acid species from complex mixtures of DNA or RNA (see Chapter 39).

Examination of the model depicted in Figure 34-2 reveals a major groove and a minor groove winding along the molecule parallel to the phosphodiester backbones. In these grooves, proteins often interact specifically with exposed atoms of the nucleotides (via specific hydrophobic and ionic interactions), thereby recognizing and binding to specific nucleotide sequences as well as the unique shapes formed therefrom. Binding usually occurs without disrupting the base pairing of the double-helical DNA molecule. As discussed in Chapters 35, 36, and 38, regulatory proteins that control DNA replication, repair, and recombination as well as the transcription of specific genes occur through such protein-DNA interactions.

In some organisms such as bacteria, bacteriophages, many DNA-containing animal viruses, as well as organelles such as mitochondria (see Figure 35-8), the ends of the
DNA molecules are joined to create a closed circle with no covalently free ends. This of course does not destroy the polarity of the molecules, but it eliminates all free 3' and 5' hydroxyl and phosphoryl groups. Closed circles exist in relaxed or supercoiled forms. Supercoils are introduced when a closed circle is twisted around its own axis or when a linear piece of duplex DNA, whose ends are fixed, is twisted. This energy-requiring process puts the molecule under torsional stress, and the greater the number of supercoils, the greater the stress or torsion (test this by twisting a rubber band). Negative supercoils are formed when the molecule is twisted in the direction opposite from the clockwise turns of the right-handed double helix found in B-DNA. Such DNA is said to be underwound. The energy required to achieve this state is, in a sense, stored in the supercoils. The transition to another form that requires energy is thereby facilitated by the underwinding (see Figure 35-19). One such transition is strand separation, which is a prerequisite for DNA replication and transcription. Supercoiled DNA is therefore a preferred form in biologic systems. Enzymes that catalyze topologic changes of DNA are called topoisomerases. Topoisomerases can relax or insert supercoils, using ATP as an energy source. Homologs of this enzyme exist in all organisms and are important targets for cancer chemotherapy. Supercoils can also form within linear DNAs if particular segments of DNA are constrained by interacting tightly with nuclear proteins that establish two boundary sites defining a topologic domain.

The genetic information stored in the nucleotide sequence of DNA serves two purposes. It is the source of information for the synthesis of all protein molecules of the cell and organism, and it provides the information inherited by daughter cells or offspring. Both of these functions require that the DNA molecule serve as a template—in the first case for the transcription of the information into RNA and in the second case for the replication of the information into daughter DNA molecules.
When each strand of the double-stranded parental DNA molecule separates from its complement during replication, each independently serves as a template on which a new complementary strand is synthesized (Figure 34-4). The two newly formed double-stranded daughter DNA molecules, each containing one strand (but complementary rather than identical) from the parent double-stranded DNA molecule, are then sorted between the two daughter cells during mitosis (Figure 34-5). Each daughter cell contains DNA molecules with information identical to that which the parent possessed; yet, in each daughter cell, the DNA molecule of the parent cell has been only semiconserved.
FIGURE 34-4 DNA synthesis maintains the sequence and structure of the original template DNA. The double-stranded structure of DNA and the template function of each old parental strand (orange) on which a new complementary daughter strand (blue) is synthesized.

RNA is a polymer of purine and pyrimidine ribonucleotides linked together by 3',5'-phosphodiester bonds analogous to those in DNA (Figure 34-6). Although sharing many features with DNA, RNA possesses several specific differences:
1. In RNA, the sugar moiety to which the phosphates and purine and pyrimidine bases are attached is ribose rather than the 2'-deoxyribose of DNA (see Figures 19-2 and 32-3).
2. The pyrimidine components of RNA can differ from those of DNA. Although RNA contains the ribonucleotides of adenine, guanine, and cytosine, it does not possess thymine except in the rare case mentioned below. Instead of thymine, RNA contains the ribonucleotide of uracil.
FIGURE 34-5 DNA replication is semiconservative. During a round of replication, each of the two strands of DNA is used as a template for synthesis of a new, complementary strand. The semiconservative nature of DNA replication has implications for the biochemical (see Figure 35-16), cytogenetic (see Figure 35-2), and epigenetic control of gene expression (see Figures 38-8 and 38-9).
3. RNA typically exists as a single strand, whereas DNA exists as a double-stranded helical molecule. However, given the proper complementary base sequence with opposite polarity, the single strand of RNA—as demonstrated in Figures 34-7 and 34-11—is capable of folding back on itself like a hairpin and thus acquiring double-stranded characteristics: G pairing with C, and A pairing with U.
4. Since the RNA molecule is a single strand complementary to only one of the two strands of a gene, its guanine content does not necessarily equal its cytosine content, nor does its adenine content necessarily equal its uracil content.
5. RNA can be hydrolyzed by alkali to 2', 3' cyclic diesters of the mononucleotides, compounds that cannot be formed from alkali-treated DNA because of the absence of a 2'-hydroxyl group. The alkali lability of RNA is useful both diagnostically and analytically.

Information within the single strand of RNA is contained in its sequence ("primary structure") of purine and pyrimidine nucleotides within the polymer. The sequence is complementary to the template strand of the gene from which it was transcribed. Because of this complementarity, an RNA molecule can
FIGURE 34-6 A segment of a ribonucleic acid (RNA) molecule in which the purine and pyrimidine bases—guanine (G), cytosine (C), uracil (U), and adenine (A)—are held together by phosphodiester bonds between ribosyl moieties attached to the nucleobases by N-glycosidic bonds. Note that negative charge(s) on the phosphodiester backbone are not illustrated (ie, Figure 34-1) and that the polymer has a polarity as indicated by the labeled 3'and 5'-attached phosphates.
FIGURE 34-7 Diagrammatic representation of the secondary structure of a single-stranded RNA molecule in which a stem loop, or "hairpin," has been formed. Formation of this structure is dependent on the indicated intramolecular base pairing (colored horizontal lines between bases). Note that G pairs with C as in DNA, but in RNA A pairs with U via hydrogen bonds.
bind specifically via the base-pairing rules to its template DNA strand (A-T, G-C, C-G, U-A; RNA base bolded); it will not bind ("hybridize") with the other (coding) strand of its gene. The sequence of the RNA molecule (except for U replacing T) is the same as that of the coding strand of the gene (Figure 34-8).

Those cytoplasmic RNA molecules that serve as templates for protein synthesis (ie, that transfer genetic information from DNA to the protein-synthesizing machinery) are designated messenger RNAs (mRNAs). Many other very abundant cytoplasmic RNA molecules (ribosomal RNAs [rRNAs]) have structural roles wherein they contribute to the formation and function of ribosomes (the organellar machinery for protein synthesis) or serve as adapter molecules (transfer RNAs [tRNAs)]) for the translation of RNA information into specific sequences of polymerized amino acids.
Interestingly, some RNA molecules have intrinsic catalytic activity. The activity of these RNA enzymes, or ribozymes, often involves the cleavage of a nucleic acid. Two ribozymes are the peptidyl transferase that catalyzes peptide bond formation on the ribosome, and ribozymes involved in the RNA splicing. In all eukaryotic cells, there are small nuclear RNA (snRNA) species that are not directly involved in protein
RNA
FIGURE 34-8 The relationship between the sequences of an RNA transcript and its gene, in which the coding and template strands are shown with their polarities. The RNA transcript with a 5' to 3' polarity is complementary to the template strand with its 3' to 5' polarity. Note that the sequence in the RNA transcript and its polarity is the same as that in the coding strand, except that the U of the transcript replaces the T of the gene; the initiating nucleotide of RNAs contain a terminal 5-triphosphate (ie, pppA-above).
synthesis but play pivotal roles in RNA processing, particularly mRNA processing. These relatively small molecules vary in size from 90 to about 300 nucleotides (Table 34-1). The properties of the several classes of cellular RNAs are detailed below.

The genetic material for some animal and plant viruses is RNA rather than DNA. Although some RNA viruses never have their information transcribed into a DNA molecule, many animal RNA viruses—specifically, the retroviruses (eg, the human immunodeficiency, or HIV virus)—are transcribed by viral RNA-dependent DNA polymerase, the socalled reverse transcriptase, to produce a double-stranded DNA copy of their RNA genome. In many cases, the resulting double-stranded DNA transcript is integrated into the host genome and subsequently serves as a template for gene expression and from which new viral RNA genomes and viral mRNAs can be transcribed. Genomic insertion of such integrating "proviral" DNA molecules can, depending on the site involved, be mutagenic, inactivating a gene or disregulating its expression (see Figure 35-11).

As noted above, in all prokaryotic and eukaryotic organisms, four main classes of RNA molecules exist: mRNA, tRNA, rRNA, and small RNAs. Each differs from the others by abundance, size, function, and general stability.

U1, Length (nucleotides) = 165. U1, Molecules per Cell = 1 × 10 6. U1, Localization = Nucleoplasm. U2, Length (nucleotides) = 188. U2, Molecules per Cell = 5 × 10 5. U2, Localization = Nucleoplasm. U3, Length (nucleotides) = 216. U3, Molecules per Cell = 3 × 10 5. U3, Localization = Nucleolus. U4, Length (nucleotides) = 139. U4, Molecules per Cell = 1 × 10 5. U4, Localization = Nucleoplasm. U5, Length (nucleotides) = 118. U5, Molecules per Cell = 2 × 10 5. U5, Localization = Nucleoplasm. U6, Length (nucleotides) = 106. U6, Molecules per Cell = 3 × 10 5. U6, Localization = Perichromatin granules. 4.5S, Length (nucleotides) = 95. 4.5S, Molecules per Cell = 3 × 10 5. 4.5S, Localization = Nucleus and cytoplasm. 7SK, Length (nucleotides) = 280. 7SK, Molecules per Cell = 5 × 10 5. 7SK, Localization = Nucleus and cytoplasm
This class is the most heterogeneous in abundance, size, and stability; for example, in brewer's yeast, specific mRNAs are present in 100s/cell to, on average, <0.1/mRNA/cell in a genetically homogeneous population. As detailed in Chapters 36 and 38, both specific transcriptional and posttranscriptional mechanisms contribute to this large dynamic range in mRNA content. In mammalian cells, specific mRNA abundance likely varies over a 10*-fold range. All members of this RNA class function as messengers conveying the information in a gene to the protein-synthesizing machinery, where each mRNA serves as a template on which a specific sequence of amino acids is polymerized to form a specific protein molecule, the ultimate gene product (Figure 34-9).

Eukaryotic mRNAs have unique chemical characteristics. The 5' terminus of mRNA is "capped" by a 7-methylguanosine triphosphate that is linked to an adjacent 2'-O-methyl ribonucleoside at its 5'-hydroxyl through the three phosphates (Figure 34-10). The mRNA molecules frequently contain internal 6-methyladenine and other 2'-O-ribose-methylated nucleotides. The cap is involved in the recognition of mRNA by the translation machinery, and also helps stabilize the mRNA by preventing the nucleolytic attack by 5'-exoribonucleases. The protein-synthesizing machinery begins translating the mRNA into proteins beginning downstream of the 5' or capped terminus. At the other end of almost all eukaryotic mRNA
DNA
FIGURE 34-10 The cap structure attached to the 5' terminal of most eukaryotic messenger RNA molecules. A 7-methylguanosine triphosphate (black) is attached at the 5' end of the mRNA (red), which usually also contains a 2'-O-methylpurine nucleotide. These modifications (the cap and methyl group) are added after the mRNA is transcribed from DNA. Note that the yand B-phosphates of the GTP added to form the cap (black in figure) are lost upon cap addition while the y-phosphate of the initiating nucleotide (here an A-residue; red in figure) is lost during cap addition.
molecules, the 3'-hydroxyl terminus has an attached, nongenetically encoded polymer of adenylate residues 20 to 250 nucleotides in length. The poly(A) "tail" at the 3'-end of mRNAs maintains the intracellular stability of the specific mRNA by preventing the attack of 3'-exoribonucleases and also facilitates translation (see Figure 37-7). Both the mRNA "cap" and "poly(A) tail" are added posttranscriptionally by nontemplatedirected enzymes to mRNA precursor molecules (pre-mRNA). mRNA 2 to 5% of total cellular RNA.

be formed by processing from the precursor, or pre-mRNA before entering the cytoplasm. Thus, in mammalian cell nuclei, the immediate products of gene transcription (primary transcripts) are very heterogeneous and can be greater than 10to 50-fold longer than mature mRNA molecules. As discussed in Chapter 36, pre-mRNA molecules are processed to generate mRNA molecules, which then enter the cytoplasm to serve as templates for protein synthesis.
represents eukaryotic In mammalian cells, including cells of humans, the mRNA molecules present in the cytoplasm are not the RNA products immediately synthesized from the DNA template but must

tRNA molecules vary in length from 74 to 95 nucleotides, like many other RNAs, are also generated by nuclear processing of
a precursor molecule (see Chapter 36). The tRNA molecules serve as adapters for the translation of the information in the sequence of nucleotides of the mRNA into specific amino acids. There are at least 20 species of tRNA molecules in every cell, at least one (and often several) corresponding to each of the 20 amino acids required for protein synthesis. Although each specific tRNA differs from the others in its sequence of nucleotides, the tRNA molecules as a class have many features in common. The primary structure—that is, the nucleotide sequence—of all tRNA molecules allows extensive folding and intrastrand complementarity to generate a secondary structure that appears in two dimensions like a cloverleaf (Figure 34-11). All tRNA molecules contain four main double-stranded arms or stems, connected by single-stranded loops named for their respective nucleotide composition or function. The acceptor arm terminates in the nucleotides CpCpA,,,,. These three nucleotides are added posttranscriptionally by a specific nucleotidyl transferase enzyme. The tRNA-appropriate amino acid is attached, or "charged," onto the 3'-OH group of the A moiety of the acceptor arm through the action of specific aminoacyl tRANA synthetases (see Figure 37-1). The D, TyC, and extra arms help define a specific tRNA. tRNAs compose roughly 20% of total cellular RNA.

A ribosome is a cytoplasmic nucleoprotein structure that acts as the machinery for the synthesis of proteins from the mRNA templates. On the ribosomes, the mRNA and tRNA molecules interact to translate the information transcribed from the gene during mRNA synthesis into a specific protein. During periods of active protein synthesis, many ribosomes can be associated with any mRNA molecule to form an assembly called the polysome (see Figure 37-7).
The components of the mammalian ribosome, which has a molecular weight of about 4.2 x 10° and a sedimentation velocity coefficient of 80S (S = Svedberg units, a parameter sensitive to molecular size and shape) are shown in Table 34-2. The mammalian ribosome contains two major nucleoprotein subunits—a larger one with a molecular weight of 2.8 x 10° (60S) and a smaller subunit with a molecular weight of 1.4 x 10° (40S). The 60S subunit contains a 5S rRNA, a 5.8S rRNA, and a 28S rRNA; there are also more than 50 specific polypeptides. The 40S subunit is smaller and contains a single 18S rRNA and approximately 30 distinct polypeptide chains. All of the rRNA molecules except the 5S rRNA, which is independently transcribed, are processed from a single 45S precursor RNA molecule in the nucleolus (see Chapter 36). The highly methylated rRNA molecules are packaged in the nucleolus with the specific ribosomal proteins. In the cytoplasm, the ribosomes remain quite stable and capable of many translation cycles. The exact functions of the rRNA molecules in the ribosomal particle are not fully understood, but they are necessary for ribosomal assembly and also play key roles in the binding of mRNA to ribosomes and its translation. Recent studies indicate that the large rRNA component performs the peptidyl transferase activity and thus is a ribozyme. The rRNAs (28S + 18S) represent roughly 70% of total cellular RNA.

A large number of discrete, highly conserved small RNA species are found in eukaryotic cells; some are quite stable. Most of these molecules are complexed with proteins to form ribonucleoproteins and are distributed in the nucleus, the cytoplasm, or both. They range in size from 20 to 1000 nucleotides and are present in 100,000 to 1,000,000 copies per cell, collectively representing <5% of cellular RNA.

snRNAs, a subset of the small nuclear RNAs (Table 34-1), are significantly involved in rRNA and mRNA processing and gene regulation. Of the several snRNAs, U1, U2, U4, U5, and U6 are involved in intron removal and the processing of mRNA precursors into mRNA within the nucleus (see Chapter 36). The U7 snRNA is involved in production of the correct 3' ends of histone mRNA—which lacks a poly(A) tail. 7SK RNA associates with several proteins to form a ribonucleoprotein complex, termed P-TEFb, that modulates mRNA gene transcription elongation by RNA polymerase II (see Chapter 36).
Large & Small Noncoding Regulatory RNAs: Micro-RNAs (miRNAs), Silencing RNAs (siRNAs), Long Noncoding RNAs (IncRNAs), and CircularRNAs (circRNAs)
One of the most exciting and unanticipated discoveries in the last decade of eukaryotic regulatory biology has been the identification and characterization of regulatory nonprotein coding RNAs (ncRNAs). NcRNAs exist in two general size classes, large (50-1000nt) and small (20-22nt). Regulatory ncRNAs have been described in most eukaryotes (see Chapter 38).
The small ncRNAs termed miRNAs and siRNAs typically inhibit gene expression at the level of specific protein production by targeting mRNAs through one of several distinct mechanisms. miRNAs are generated by specific nucleolytic processing of the products of distinct genes/transcription units (see Figure 36-17). miRNA precursors, which are 5'-capped and 3'-polyadenylated, usually range in size from about 500 to 1000 nucleotides.
By contrast, siRNAs are generated by the specific nucleolytic processing of large dsRNAs that are either produced from other endogenous RNAs, or dsRNAs introduced into the cell by, for example, RNA viruses. Both siRNAs and miRNAs hybridize via the formation of RNA-RNA hybridization to their targeted mRNAs (see Figure 38-19). To date, hundreds of distinct miRNAs and siRNAs have been described in humans; estimates suggest that there are ~1000 human miRNA-encoding genes. Given their exquisite genetic specificity both miRNAs and

Primary Structure (1°)
5’ pGCGGAUUUAGCUCAGUUGGGAGAGCGCCAGACUGAAGAUCUGGAGGUCCUGUGUUCGAUCCACAGAAU UCGCACCAon 3
Secondary
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(2°)
Tertiary Structure (3°)
3°
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FIGURE
34-11
Structure
of
a
mature,
tRNA,
yeast
phenylalanyl-tRNA
(tRNA).
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are
primary
(1°),
secondary
(2°),
and
tertiary
(3°)
(top,
lower
left,
and
lower
right,
respectively)
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the
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nucleotide-long
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primary
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numbering
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end
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the
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contains
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a
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underlined
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shown
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U,
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and
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S
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form with the same strand polarity (ie, 5’ to 3’ and 3’ to 5’) as base-paired regions of DNA. The three bases of the anticodon loop are shown in red. In the case of amino acid-charged tRNAs, an aminoacyl moiety is esterified to the 3’-CCA,,, terminus (brown; in this case the amino acid would be phenylalanine; not shown). Blue type highlights nontraditional nucleotides introduced by posttranslational modification, abbreviated as follows: m’G = 2-methylguanosine; D = 5,6-dihydrouridine; m?,G = N2-dimethylguanosine; C,, = O2'-methylcytidine; G_, = 02’- methylguanosine; T = 5-methyluridine; Y = wybutosine; = pseudouridine; m>C = 5-methylcytidine; m’G = 7-methylguanosine; m'A = 1-methyladenosine. Essentially all tRNAs fold into similar, characteristic, tertiary structures (3°) as shown, lower right. The distinct portions of the molecule in 2° (insert) and 3° configurations are color-coded in this image for clarity. tRNA** was the first nucleic acid whose structure was determined by x-ray crystallography. Such distinct three-dimensional tRNA structures bind specifically to important functional sites on both aminoacyl tRNA synthetases and the ribosomes during protein synthesis (see Chapter 37). The tRNA"™* secondary and tertiary structure images are open source figures from Wikipedia (wikimedia.org/wikipedia/commons/b/ba/TRNA-Phe_yeast_1ehz.png).

40S subunit, Mass (MW) = 1.4 × 10 6. 40S subunit, Protein.Number = 33. 40S subunit, Protein.Mass = 7 × 10 5. 40S subunit, RNA.Size = 18S. 40S subunit, RNA.Mass = 7 × 10 5. 40S subunit, RNA.Bases = 1900. 60S subunit, Mass (MW) = 2.8 × 10 6. 60S subunit, Protein.Number = 50. 60S subunit, Protein.Mass = 1 × 10 6. 60S subunit, RNA.Size = 5S. 60S subunit, RNA.Mass = 3.5 × 10 4. 60S subunit, RNA.Bases = 120. , Mass (MW) = . , Protein.Number = . , Protein.Mass = . , RNA.Size = 5.8S. , RNA.Mass = 4.5 × 10 4. , RNA.Bases = 160. , Mass (MW) = . , Protein.Number = . , Protein.Mass = . , RNA.Size = 28S. , RNA.Mass = 1.6 × 10 6. , RNA.Bases = 4700
Note: The ribosomal subunits are defined according to their sedimentation velocity in Svedberg (S) units (40S or 60S). The number of unique proteins and their total mass (MW) and the RNA components of each subunit in size (Svedberg units), mass, and number of bases are listed.
siRNAs represent exciting new potential agents for therapeutic drug development. siRNAs are frequently used to decrease or "knockdown" specific protein levels (via siRNA homology-directed mRNA degradation) in experimental contexts in the laboratory, an extremely useful and powerful alternative to gene-knockout technology (see Chapter 39). Indeed, several siRNA-based therapeutic clinical trials are in progress to test the efficacy of these novel molecules as drugs for treating human disease.

Other exciting recent observations in the RNA realm are the identification and characterization of two classes of larger noncoding RNAs, the circular RNAs (circRNAs) and the long noncoding RNAs, or IncRNAs. Many circRNAs have recently been discovered and characterized. circRNAs appear to be produced by RNA splicing-type reactions from a wide range of precursor RNAs, both mRNA precursors and nonprotein IncRNA precursors (see below for more information on IncRNAs). Though not an abundant class of RNA molecules in most cells, circRNAs have been detected in all eukaryotes tested, particularly in metazoans. While the functions of circRNAs are still being elucidated they seem to be particularly abundant in cells of the nervous system. Similar to IncRNAs, these molecules likely play important roles in cellular biology by regulating gene expression at multiple levels. LncRNAs, which as their name implies, do not code for protein, and range in size from ~300 to 1000s of nucleotides in length. These RNAs are typically transcribed from the large regions of eukaryotic genomes that do not encode for protein (ie, the mRNA encoding genes). In fact, transcriptome analyses indicate that >90% of all eukaryotic genomic DNA is transcribed. ncRNAs make up a significant portion of this transcription. ncRNAs play many roles ranging from contributing to structural aspects of chromatin to regulation of mRNA gene transcription by RNA polymerase II. Future work will further characterize this important, newly discovered class of RNA molecules.
Interestingly, bacteria also contain small, heterogeneous regulatory RNAs termed sRNAs. Bacterial sRNAs range in size from 50 to 500 nucleotides, and like eukaryotic mi/si/ IncRNAs, also control a large array of genes. sRNAs often repress, but sometimes activate protein synthesis by binding to specific mRNA.

Enzymes capable of degrading nucleic acids have been recognized for many years. These nucleases can be classified in several ways. Those that exhibit specificity for DNA are referred to as deoxyribonucleases. Those nucleases that specifically hydrolyze RNA are ribonucleases. Some nucleases degrade both DNA and RNA. Within both of these classes are enzymes capable of cleaving internal phosphodiester bonds to produce either 3'-hydroxyl and 5'-phosphory] terminals or 5'-hydroxyl and 3'-phosphoryl terminals. These are referred to as endonucleases. Some are capable of hydrolyzing both strands of a double-stranded molecule, whereas others can only cleave single strands of nucleic acids. Some nucleases can hydrolyze only unpaired single strands, while others are capable of hydrolyzing single strands participating in the formation of a doublestranded molecule. There exist classes of endonucleases that recognize specific sequences in DNA. One class of these DNA cleaving enzymes, the restriction endonucleases, also termed restriction enzymes, do so directly by binding contiguous DNA base pairs (typically 4, 5, 6, or 8 bp) and cleaving both strands of DNA, usually DNA within the binding/recognition sequence element. The second class of enzymes, which are ribonucleoprotein complexes, utilizes a "guide RNA" of specific nucleotide sequence that targets a nuclease to cleave distinct DNA or RNA sequences. These are the CRISPR-Cas family of enzymes. Both classes of DNA-cleaving enzyme are described in greater detail in Chapter 39. These enzyems represent critically important tools in molecular genetics and medical sciences. Some nucleases are capable of hydrolyzing a nucleotide only when it is present at a terminal of a molecule; these are referred to as exonucleases. Exonucleases act in one direction (3' > 5' or 5' 3') only. In bacteria, a 3' > 5' exonuclease is an integral part of the DNA replication machinery and there serves to edit—or proofread—the most recently added deoxynucleotide for base-pairing errors.

=
DNA
consists of
four bases—A,
G,
C, and
T—that are
held linear array by phosphodiester bonds through the 3' and 5'
positions of adjacent deoxyribose moieties.
in
■, 1 = DNAis organized into two strands by the pairing of bases Ato T and Gto C on complementary strands. These strands form a double helix around a central axis.. ■, 1 = The ~3 × 10 9 bp of DNAin humans are organized into the haploid complement of 23 chromosomes. The exact sequence of these 3 billion nucleotides defines the uniqueness of each individual.. ■, 1 = DNAprovides a template for its own replication and thus maintenance of the genotype and for the transcription of the roughly 25,000 protein coding human genes as well as a large array of nonprotein coding regulatory ncRNAs.. ■, 1 = RNAexists in several different single-stranded structures, most of which are directly or indirectly involved in protein synthesis or its regulation. The linear array of nucleotides in RNAconsists of A, G, C, and U, and the sugar moiety is ribose.. ■, 1 = The major forms of RNA include mRNA, rRNA, tRNA, and snRNAs and regulatory ncRNAs. Certain RNA molecules act catalysts (ribozymes).

. Appreciate that the roughly 3 x 10° base pairs of DNA that compose the haploid genome of humans are divided uniquely between 23 linear DNA units, the chromosomes. Humans, being diploid, have 23 pairs of these linear chromosomes: 22 autosomes and two sex chromosomes.
m= Understand that human genomic DNA, if extended end-to-end, would be meters in length, yet still fits within the nucleus of the cell, an organelle that is only microns (us; 10° meters) in diameter. Such condensation in DNA length, in part, is induced following its association with the highly positively charged histone proteins resulting in the formation of a unique DNA-histone complex termed the nucleosome. Nucleosomes have DNA wrapped around the surface of an octamer of histones.
=
Explain that strings of nucleosomes form along the linear sequence of genomic DNA to form chromatin, which itself can be more tightly packaged and condensed, this ultimately leads to the formation of the chromosomes.
for life. = Appreciate that while the chromosomes are the macroscopic functional units for DNA transcription, replication, recombination, gene assortment, and cellular division, it is DNA function at the level of the individual nucleotides that composes regulatory sequences linked to specific genes that are essential
= Describe the steps, phase of the cell cycle, and the molecules responsible for the replication, repair, and recombination of DNA, and understand the negative effects that errors in any of these processes can have upon cellular and organismal integrity and health.

The genetic information in the DNA of a chromosome can be transmitted by exact replication or it can be exchanged by a number of processes, including crossing over, recombination, transposition, and gene conversion. These processes provide a
"So far as possible, the discussion in this chapter and in Chapters 36, 37, and 38 will pertain to mammalian organisms, which are, of course, among the higher eukaryotes. At times, it will be necessary to refer to observations in prokaryotic organisms such as bacteria and viruses, or lower eukaryotic model systems such as Drosophila, Caenorhabditis elegans, or, yeast, Saccharomyces cerevisae. However, in such cases the information will be of a kind that can be readily extrapolated to mammalian organisms.
means of ensuring adaptability and diversity for the organism, but when they go awry, can also result in disease. A number of enzyme systems are involved in DNA replication, alteration, and repair. Mutations are due to a change in the base sequence of DNA and may result from the faulty replication, transposition, or repair of DNA and occur with a frequency of about one in every 10° cell divisions. Abnormalities in gene products (either in RNA, protein function, or amount) can be the result of mutations that occur in transcribed protein coding, and nonprotein coding DNA, or nontranscribed regulatoryregion DNA. A mutation in a germ cell is transmitted to offspring (so-called vertical transmission of hereditary disease). A number of factors, including viruses, chemicals, ultraviolet light, and ionizing radiation, increase the rate of mutation.
Mutations often affect somatic cells and so are passed on to successive generations of cells, but only within an organism (ie, horizontally). It is becoming apparent that a number of diseases—and perhaps most cancers—are due to the combined effects of vertical transmission of mutations as well as horizontal transmission of induced mutations.

Chromatin consists of very long double-stranded DNA (dsDNA) molecules and a nearly equal mass of small basic proteins termed histones as well as a smaller amount of nonhistone proteins (most of which are acidic and larger than histones) and a small quantity of RNA. The nonhistone proteins include enzymes involved in DNA replication and repair, and the proteins involved in RNA synthesis, processing, and transport to the cytoplasm. The dsDNA helix in each chromosome has a length that is thousands of times the diameter of the cell nucleus. One purpose of the molecules that comprise chromatin, particularly the histones, is to condense the DNA; however, it is important to note that the histones also integrally participate in gene regulation (Chapters 36, 38, and 42); indeed, histones contribute importantly to all DNA-directed molecular transactions. Electron microscopic studies of chromatin have demonstrated dense spherical particles called nucleosomes, which are approximately 10 nm in diameter and connected by DNA filaments (Figure 35-1). Nucleosomes are composed of DNA wound around an octameric complex of histone molecules.

Histones are a small family of closely related basic proteins. HI histones are the ones least tightly bound to chromatin (Figures 35-1, 35-2, and 35-3) and are, therefore, easily removed with a salt solution, after which chromatin becomes more soluble. The organizational unit of this soluble chromatin is the nucleosome. Nucleosomes contain four major types of histones: H2A, H2B, H3, and H4. The sequence and structures of all four histones, H2A, H2B, H3, and H4, the so-called core histones that form the nucleosome, have been highly conserved between species, although variants of the histones exist and are used for specialized purposes. This extreme conservation implies that the function of histones is identical in all eukaryotes and that the entire molecule is involved quite specifically in carrying out this function. The carboxyl terminal two-thirds of the histone molecules are hydrophobic, while their amino terminal thirds are particularly rich in basic amino acids. These four core histones
FIGURE 35-1 Electron micrograph of chromatin showing individual nucleosomes (white, ball-shaped) attached to strands of DNA (thin, gray line); see also Figure 35-2. (Reproduced, with permission, from Shao Z: Probing nanometer structures with atomic force microscopy. News Physiol Sci 1999;14:142-149. Courtesy of Professor Zhifeng Shao, University of Virginia.)
are subject to at least six types of covalent modification or posttranslational modifications (PTMs): acetylation, methylation, phosphorylation, ADP-ribosylation, monoubiquitylation, and sumoylation. These histone modifications play important roles in chromatin structure and function, as illustrated in Table 35-1.
The histones interact with each other in very specific ways. H3 and H4 form a tetramer containing two molecules of each (H3-H4),, while H2A and H2B form dimers (H2AH2B). Under physiologic conditions, these histone oligomers associate to form the histone octamer of the composition (H3-H4),-(H2A-H2B)..

FIGURE 35-2 Model for the structure of the nucleosome. DNA is wrapped around the surface of a protein cylinder consisting of two each of histones H2A, H2B, H3, and H4 that form the histone octamer. The ~145 bp of DNA, consisting of 1.75 superhelical turns, are in contact with the histone octamer. The position of histone H1, when it is present, is indicated by the dashed outline at the bottom of the figure. Note that histone H1 interacts with DNA as it enters and exits the nucleosome.
FIGURE 35-3 Extent of DNA packaging in metaphase chromosomes (top) to noted duplex DNA (bottom). Chromosomal DNA is packaged and organized at several levels as shown (see Table 35-2). Each phase of condensation or compaction and organization (bottom to top) decreases overall DNA accessibility to an extent that the DNA sequences in metaphase chromosomes are likely almost totally transcriptionally inert. In toto, these five levels of DNA compaction result in nearly a 10*-fold linear decrease in end-to-end DNA length. Complete condensation and decondensation of the linear DNA in chromosomes occur in the space of hours during the normal replicative cell cycle (see Figure 35-20).

- 1. Acetylation of histones H3 and H4 is associated with the activation or inactivation of
gene transcription.
2. core histones is associated with chromosomal
Acetylation of assembly during DNA replication.
3. Phosphorylation of histone H1 is associated with the
condensation of chromosomes during the replication cycle.
4. ADP-ribosylation of histones is associated with DNA repair.
5. Methylation of histones is correlated with activation and
repression of
6. Monoubiquitylation is associated with gene activation, repression, and heterochromatic gene silencing.
7. Sumoylation of histones (SUMO; small ubiquitin-related modifier) is associated with transcription repression.

When the histone octamer is mixed with purified dsDNA under appropriate ionic conditions, the same x-ray diffraction pattern is formed as that observed in freshly isolated chromatin. Biochemical and electron microscopic studies confirm the existence of reconstituted nucleosomes. Furthermore, the reconstitution of nucleosomes from DNA and histones H2A, H2B, H3, and H4 is independent of the organismal or cellular origin of the various components. Neither the histone H1, nor the nonhistone proteins are necessary for the reconstitution of the nucleosome core.
In the nucleosome, the DNA is supercoiled in a lefthanded helix over the surface of the disk-shaped histone octamer (Figure 35-2). The majority of core histone proteins interact with the DNA on the inside of the supercoil without protruding, although the amino terminal tails of all the histones are thought to extend outside of this structure and are available for regulatory PTMs (Table 35-1).
The (H3-H4), tetramer itself can confer nucleosomelike properties on DNA and thus has a central role in the formation of the nucleosome. The addition of two H2A-H2B dimers stabilizes the primary particle and firmly binds two additional half-turns of DNA previously bound only loosely to the (H3-H4),. Thus, 1.75 superhelical turns of DNA are wrapped around the surface of the histone octamer, protecting 145 to 150 bp of DNA and forming the nucleosome core particle (Figure 35-2). In chromatin, core particles are separated by a roughly 30-bp region of DNA termed "linker." Most of the DNA is in a repeating series of these structures, giving chromatin a repeating "beads-on-a-string" appearance when examined by electron microscopy (Figure 35-1).
In vivo the assembly of nucleosomes is mediated by one of several nuclear chromatin assembly factors facilitated by histone chaperones, a group of proteins that exhibit high affinity for binding histones. As the nucleosome is assembled, histones are released from the histone chaperones. Nucleosomes appear to exhibit preference for certain regions on specific

DNA molecules, but the basis for this nonrandom distribution, termed phasing, is not yet completely understood. Phasing is likely related both to the relative physical flexibility of particular nucleotide sequences to accommodate the regions of kinking within the supercoil, as well as the presence of other DNA-bound factors that limit the sites of nucleosome deposition.

Electron microscopy of chromatin reveals two higher orders of structure—the 10-nm fibril and the 30-nm chromatin fiber— beyond that of the nucleosome itself. The disk-like nucleosome structure has a 10-nm diameter and a height of 5 nm. The 10-nm fibril consists of nucleosomes arranged with their edges separated by a small distance (30 bp of linker DNA) with their flat faces parallel to the fibril axis (Figure 35-3). The 10-nm fibril is probably further supercoiled with six or seven nucleosomes per turn to form the 30-nm chromatin fiber (Figure 35-3). Each turn of the supercoil is relatively flat, and the faces of the nucleosomes of successive turns would be nearly parallel to each other. H1 histones appear to stabilize the 30-nm fiber, but their position and that of the variable length linker DNA are not clear. It is probable that nucleosomes can form a variety of packed structures. In order to form a mitotic chromosome, the 30-nm fiber must be compacted in length another 100-fold (see below).
In interphase chromosomes, chromatin fibers appear to be organized into 30,000 to 100,000 bp loops or domains anchored in a scaffolding, or supporting matrix within the nucleus, the so-called nuclear matrix. Within these domains, some DNA sequences may be located nonrandomly. It has been suggested that each looped domain of chromatin corresponds to one or more separate genetic functions, containing both coding and noncoding regions of the cognate gene or genes. This nuclear architecture is likely dynamic, having important regulatory effects upon gene regulation. Recent data suggest that certain genes or gene regions are mobile within the nucleus, moving obligatorily to discrete loci within the nucleus upon activation. Further work will determine what molecular mechanisms are responsible.

Generally, every cell of an individual metazoan organism contains the same genetic information. Thus, the differences between cell types within an organism must be explained by differential expression of the common genetic information. Chromatin containing active genes (ie, transcriptionally
or potentially transcriptionally active chromatin) has been shown to differ in several ways from that of inactive regions. The nucleosome structure of active chromatin appears to be altered, sometimes quite extensively, in highly active regions. DNA in active chromatin contains large regions (about 100,000 bases long) that are relatively more sensitive to digestion by a nuclease such as DNase I. DNase I makes single-strand cuts in nearly any segment of DNA due to its low-sequence specificity. It will digest DNA that is not protected, or bound by protein, into its component deoxynucleotides. The sensitivity to DNase I of active chromatin regions reflects only a potential for transcription rather than transcription itself, and in several different cellular systems can be correlated with a relative lack of 5-methyldeoxycytidine (meC; see Figure 32-7) in the DNA, and particular histone variants and/or histone PTMs (phosphorylation, acetylation, etc; Table 35-1).
Within the large regions of active chromatin there exist shorter stretches of 100 to 300 nucleotides that exhibit an even greater (another 10-fold) sensitivity to DNase I. These hypersensitive sites probably result from a structural conformation that favors access of the nuclease to the DNA. These regions are often located immediately upstream from the active gene and are the location of interrupted nucleosomal structure caused by the binding of nonhistone regulatory transcription factor proteins (enhancer-binding transcriptional activator proteins; see Chapters 36 and 38). In many cases, it seems that if a gene is capable of being transcribed, it very often has a DNase-hypersensitive site(s) in the chromatin immediately upstream. As noted above, nonhistone regulatory proteins involved in transcription control and those involved in maintaining access to the template strand lead to the formation of hypersensitive sites. Such sites often provide the first clue about the presence and location of a transcription control element.

By contrast, transcriptionally inactive chromatin is densely packed during interphase as observed by electron microscopic studies and is referred to as heterochromatin; transcriptionally active chromatin stains less densely and is referred to as euchromatin. Generally, euchromatin is replicated earlier than heterochromatin in the mammalian cell cycle (see below). The chromatin in these regions of inactivity is often high in meC content, and histones therein contain relatively lower levels of certain "activating" covalent modifications and higher levels of "repressing" histone PTMs.
There are two types of heterochromatin: constitutive and facultative. Constitutive heterochromatin is always condensed and thus essentially inactive. It is found in the regions near the chromosomal centromere and at chromosomal ends (telomeres). Facultative heterochromatin is at times condensed, but at other times it is actively transcribed and, thus, uncondensed and appears as euchromatin. Of the two members of the X-chromosome pair in mammalian females, one X chromosome is almost completely inactive transcriptionally and is heterochromatic. However, the heterochromatic X
chromosome decondenses during gametogenesis and becomes transcriptionally active during early embryogenesis—thus, it is facultative heterochromatin.
Certain cells of insects, for example, Chironomus and Drosophila, contain giant chromosomes that have been replicated for multiple cycles without separation of daughter chromatids. These copies of DNA line up side by side in precise register and produce a banded chromosome containing regions of condensed chromatin and lighter bands of more extended chromatin. Transcriptionally active regions of these polytene chromosomes are especially decondensed into "puffs" that can be shown to contain the enzymes responsible for transcription and to be the sites of RNA synthesis (Figure 35-4). Using highly sensitive fluorescently labeled hybridization probes, specific gene sequences can be mapped, or "painted," within the nuclei of human cells, even without polytene chromosome formation, using fluorescence in situ hybridization (FISH; see Chapter 39) techniques.

FIGURE 35-4 Illustration of the tight correlation between the presence of RNA polymerase II (Table 36-2) and messenger RNA synthesis. A number of genes, labeled A, B (top), and 5C, but not genes at locus (band) BR3 (5C, BR3, bottom) are activated when midge fly Chironomus tentans larvae are subjected to heat shock (39°C for 30 minutes). (A) Distribution of RNA polymerase II in isolated chromosome IV from the salivary gland (at arrows). The enzyme was detected by immunofluorescence using a fluorescently labeled antibody directed against the polymerase. The 5C and BR3 are specific bands of chromosome IV, and the arrows indicate puffs (ie, A, B, 5C). (B) Autoradiogram of a chromosome IV that was incubated in *H-uridine to label the RNA. Note the correspondence of the immunofluorescence and presence of the radioactive RNA (black dots) (ie, A, B, 5C). Bar = 7 um. (Reproduced, with permission, from Sass H: RNA polymerase B in polytene chromosomes. Cell 1982;28:274. Copyright © 1982. Reprinted with permission from Elsevier.)

At metaphase, mammalian chromosomes possess a twofold symmetry, with the identical duplicated sister chromatids connected at a centromere, the relative position of which is characteristic for a given chromosome (Figure 35-5). The centromere is an adenine-thymine (A-T)-rich region containing repeated DNA sequences that range in size from 10? (brewers' yeast) to 10° (mammals) base pairs (bp). Metazoan centromeres are bound by nucleosomes containing the histone H3 variant protein CENP-A and other specific centromere-binding proteins. This complex, called the kinetochore, provides the anchor for the mitotic spindle. Thus, it is an essential structure for chromosomal segregation during mitosis.
The ends of each chromosome contain structures called telomeres. Telomeres consist of short TG-rich repeats. Human telomeres have a variable number of repeats of the sequence 5'-TTAGGG-3', which can extend for several kilobases. Telomerase, a multisubunit RNA _ templatecontaining complex related to viral RNA-dependent DNA polymerases (reverse transcriptases), is the enzyme responsible for telomere synthesis and thus for maintaining the length of the telomere. Since telomere shortening has been associated with both malignant transformation (Chapter 56) and aging
FIGURE 35-5 The two sister chromatids of mitotic human chromosome 12. The location of the A+T-rich centromeric region connecting sister chromatids is indicated, as are two of the four telomeres residing at the very ends of the chromatids that are attached one to the other at the centromere. (Reprinted with permission from Biophoto Associates/Photo Researchers, Inc.)

Naked double-helical DNA, Packing Ratio = ~1.0. 10-nm fibril of nucleosomes, Packing Ratio = 7-10. 30-nm chromatin fiber of superhelical nucleosomes, Packing Ratio = 40-60. Condensed metaphase chromosome loops, Packing Ratio = 8000
(Chapter 58), this enzyme has become an attractive target for cancer chemotherapy and drug development (see Chapter 56). Each sister chromatid contains one dsDNA molecule. As schematized in Figure 35-3, during interphase, the packing of the DNA molecule is less dense than it is in the condensed chromosome during metaphase. Metaphase chromosomes are nearly completely transcriptionally inactive.
The human haploid genome consists of about 3 x 10° bp and about 1.7 x 10' nucleosomes. Thus, each of the 23 chromatids in the human haploid genome would contain on the average 1.3 x 10° nucleotides in one dsDNA molecule. Therefore, the length of each DNA molecule must be compressed about 8000-fold to generate the structure of a condensed metaphase chromosome. In metaphase chromosomes, the 30-nm chromatin fibers are also folded into a series of looped domains, the proximal portions of which are anchored to the nuclear matrix, likely through interactions with proteins termed lamins that constitute integral components of the inner nuclear membrane within the nucleus (Figures 35-3 and 49-4). The packing ratios of each of the orders of DNA structure are summarized in Table 35-2. The packaging of nucleoproteins within chromatids is not random, as evidenced by the characteristic patterns observed when chromosomes are stained with specific dyes such as quinacrine or Giemsa stain (Figure 35-6).
From individual to individual within a single species, the pattern of staining (banding) of the entire chromosome complement is highly reproducible; nonetheless, it differs significantly between species, even those closely related. Thus, the packaging of the nucleoproteins in chromosomes of higher eukaryotes must in some way be dependent on species-specific characteristics of the DNA molecules.

A combination of specialized staining techniques and high-resolution microscopy has allowed cytogeneticists to quite precisely map many genes to specific regions of mouse and human chromosomes. With the recent elucidation of the human and mouse genome sequences (among others), it has become clear that many of these visual mapping methods were remarkably accurate.

The protein coding regions of DNA, the transcripts of which ultimately appear in the cytoplasm as single mRNA
FIGURE 35-6 A human karyotype (of a man with a normal 46,XY constitution), in which the metaphase chromosomes have been stained by the Giemsa method and aligned according to the Paris Convention. (Reprinted with permission from H Lawce and F Conte.)
molecules, are usually interrupted in the eukaryotic genome by large intervening sequences of nonprotein-coding DNA. Accordingly, the primary transcripts or mRNA precursors (originally termed hnRNA because this species of RNA was quite heterogeneous in size [length] and mostly restricted to the nucleus), contain noncoding intervening sequences of RNA that must be removed in a process which also joins together the appropriate coding segments to form the mature mRNA. Most coding sequences for a single mRNA are interrupted in the genome (and thus in the primary transcript) by at least one—and in some cases as many as 50—noncoding intervening sequences (introns). In most cases, the introns are much longer than the coding regions (exons). The processing of the primary transcript, which involves precise removal of introns and splicing of adjacent exons, is described in Chapter 36.
The function of the intervening sequences, or introns, is not totally clear. However, mRNA precursor molecules can be differentially spliced thereby increasing the number of distinct (yet related) proteins produced by a single gene and its corresponding primary mRNA gene transcript. Introns may also serve to separate functional domains (exons) of coding information in a form that permits genetic rearrangement by recombination to occur more rapidly than if all coding regions for a given genetic function were contiguous. Such an enhanced rate of genetic rearrangement of functional domains might allow more rapid evolution of biologic function. In some instances, other protein-coding or noncoding RNAs are localized within the intronic DNA of certain genes (see Chapter 34). The relationships among chromosomal DNA, gene clusters on the chromosome, the exon-intron structure of genes, and the final mRNA product are illustrated in Figure 35-7.

The haploid genome of each human cell consists of 3.3 x 10° bp of DNA subdivided into 23 chromosomes. The entire haploid genome contains sufficient DNA to code for nearly 1.5 million average-sized protein coding genes (i.e., ~2200 bp of proteincoding DNA). However, early studies of mutation rates and of the complexities of the genomes of higher organisms suggested that humans have significantly fewer than 100,000 proteins encoded by the ~1% of the human genome that is composed of exonic DNA. Indeed, current estimates based on sequencing of the human genome suggest there are about 25,000 proteincoding genes in humans. This implies that most genomic DNA is nonprotein coding—that is, its information is never translated into an amino acid sequence of a protein molecule. Certainly, some of the excess DNA sequences serve to regulate the expression of genes during development, differentiation, and adaptation to the environment, either by serving as binding sites for regulatory proteins or by encoding regulatory ncRNAs. Some excess clearly makes up the intervening sequences or introns that split the coding regions of genes, and another portion of the excess appears to be composed of many families of repeated sequences for which clear functions have yet to be defined, though some small RNAs transcribed from these repeats can modulate transcription, either directly by
FIGURE 35-7 The relationship between chromosomal DNA and mRNA. The human haploid DNA complement of 3 x 10° bp is unequally distributed between 23 chromosomes (see Figure 35-6). Genes are often clustered on these chromosomes. An average gene is 2 x 10* bp in length, including the regulatory region (red-hatched area), which is often located at the 5' end of the gene. The regulatory region is shown here as being adjacent to the transcription initiation site (bent arrow). Most eukaryotic genes have alternating exons and introns. In this example, there are nine exons (blue-colored areas) and eight introns (green-colored areas). The introns are removed from the primary transcript by processing reactions, and the exons are ligated together in sequence to form the mature mRNA through a process termed RNA splicing. (nt, nucleotides.)

interacting with the transcription machinery or indirectly by affecting the activity of the chromatin template. Interestingly, the ENCODE Project Consortium (see Chapters 10 and 39) has shown that most of the genomic sequence is indeed transcribed in at least some human cell types, albeit at a low level. A large fraction of such transcription appears to generate the IncRNAs (see Chapter 34). Further research will elucidate the role(s) played by such transcripts.
The DNA in a eukaryotic genome can be divided into two broad "sequence classes." These are unique-sequence DNA, or nonrepetitive DNA and repetitive-sequence DNA. In the haploid genome, unique-sequence DNA generally includes the single copy genes that code for proteins. The repetitive DNA in the haploid genome includes sequences that vary in copy number from 2 to as many as 10' copies per cell.

This estimation and genome-wide organization of repetitive sequence DNA was based on a variety of techniques, and most recently on direct genomic DNA sequencing. Similar techniques were used to determine the number of proteinencoding genes. In brewers' yeast (Saccharomyces cerevisiae, a lower eukaryote), about two-thirds of its 6200 genes are expressed, but only ~one-fifth are required for viability under laboratory growth conditions. In typical tissues in a higher eukaryote (eg, mammalian liver and kidney), between 10,000 and 15,000 genes are actively expressed. Different combinations of genes are expressed in each tissue of course, and how this is accomplished is one of the major unanswered questions in biology.

Repetitive-sequence DNA can be broadly classified as moderately repetitive or as highly repetitive. The highly repetitive sequences consist of 5 to 500 base pair lengths repeated many times in tandem. These sequences are often clustered in centromeres and telomeres of the chromosome and some are present in about 1 to 10 million copies per haploid genome. The majority of these sequences are transcriptionally inactive and some of these sequences play a structural role in the chromosome (Figure 35-5; see Chapter 39).
The moderately repetitive sequences, which are defined as being present in numbers of less than 10° copies per haploid genome, are not clustered but are interspersed with unique sequences. In many cases, these long interspersed repeats are transcribed by RNA polymerase II and contain 5-Cap structures indistinguishable from those on mRNA. Depending on their length, moderately repetitive sequences are classified as long interspersed nuclear elements (LINEs) or short interspersed nuclear elements (SINEs). Both types appear to be retroposons; that is, they arose from movement from one location to another (transposition) through an RNA intermediate by the action of reverse transcriptase that transcribes an RNA template into DNA. Mammalian genomes contain 20,000 to 50,000 copies of the 6 to 7 kbp LINEs. These

represent species-specific families of repeat elements. SINEs are shorter (70-300 bp), and there may be more than 100,000 copies per genome. Of the SINEs in the human genome, one family, the Alu family, is present in about 500,000 copies per haploid genome and accounts for ~10% of the human genome. Members of the human Alu family and their closely related analogs in other animals can be transcribed as integral components of mRNA precursors or as discrete RNA molecules, including the well-studied 4.55 RNA and 7S RNA. These particular family members are highly conserved within a species as well as between mammalian species. Components of the short-interspersed repeats, including the members of the Alu family, may be mobile elements, capable of jumping into and out of various sites within the genome (see below). These transposition events can have disastrous results, as exemplified by the insertion of Alu sequences into a gene, which, when so mutated, causes neurofibromatosis. Additionally, Alu B1 and B2 SINE RNAs have been shown to regulate mRNA production at the levels of transcription and mRNA splicing.

members can be rapidly screened for a certain microsatellite polymorphism. The association of a specific polymorphism with a gene in affected family members—and the lack of this association in unaffected members—may be the first clue about the genetic basis of a disease.
Trinucleotide sequences that increase in number (microsatellite instability) can cause disease. The unstable (CGG), repeat sequence is associated with the fragile X syndrome. Other trinucleotide repeats that undergo dynamic mutation (usually an increase) are associated with Huntington chorea (CAG), myotonic dystrophy (CTG), spinobulbar muscular atrophy (CAG), and Kennedy disease (CAG). The advent of next-generation DNA sequencing technologies (see Chapter 39) has dramatically impacted both the speed, accuracy, and precision with which scientists and clinicians can analyze human genome structure. Some newly instituted clinical tests involve targeted genomic DNA sequencing prepared either from tissues or serum samples.

One category of repeat sequences exists as both dispersed and grouped tandem arrays. The sequences consist of 2 to 6 bp repeated up to 50 times. These microsatellite sequences most commonly are found as dinucleotide repeats of AC on one strand and TG on the opposite strand, but several other forms occur, including CG, AT, and CA. The AC repeat sequences occur at 50,000 to 100,000 locations in the genome. At any locus, the number of these repeats may vary on the two chromosomes, thus providing heterozygosity of the number of copies of a particular microsatellite number in an individual. This is a heritable trait, and because of their number and the ease of detecting them using the polymerase chain reaction (PCR) (see Chapter 39), such repeats are useful in constructing genetic linkage maps. Most genes are associated with one or more microsatellite markers, so the relative position of genes on chromosomes can be assessed, as can the association of a gene with a disease. Using PCR, a large number of family
The majority of the polypeptides in mitochondria (about 54 out of 67) are encoded by nuclear genes, while the rest are coded by genes found in mitochondrial (mt) DNA. Human mitochondria contains 2 to 10 copies of a small circular ~16 kbp dsDNA molecule that makes up approximately 1% of total cellular DNA. This mtDNA codes for mt-specific ribosomal and transfer RNAs and for 13 proteins that play key roles in the respiratory chain (see Chapter 13). The linearized structural map of the human mitochondrial genes is shown in Figure 35-8. Some of the features of mtDNA are shown in Table 35-3.
An important feature of human mitochondrial mtDNA is that—because all mitochondria are contributed by the ovum during zygote formation—it is transmitted by maternal nonmendelian inheritance. Thus, in diseases resulting from mutations of mtDNA, an affected mother would in theory pass the disease to all of her children but only her daughters

FIGURE 35-8 Map of human mitochondrial genes. The maps represent the so-called light (L; upper) and heavy (H; lower) strands of the 16,569 base pair linearized mitochondrial (mt) DNA The maps show the mt genes encoding subunits of NADH-coenzyme Q oxidoreductase (ND1 through ND6), cytochrome c oxidase (COX1 through COX3), cytochrome b (cyt b), ATP synthase (ATPase 6 and 8) and the 12S and 16S mt ribosomal rRNAs. Mt transfer RNA (tRNA) encoding genes are denoted by small yellow boxes and the three-letter code indicating the cognate amino acids which they specify during mt translation. The origin of heavy-strand (O,,), and light-strand (O,) DNA replication, as well as the promoters for the initiation of heavy-strand (P,,, and P,,,), and light-strand (P,) transcription are indicated by arrows and letters (see also Table 57-3). Figure generated using Homo sapiens mitochondrion, complete genome; Sequence: NCBI Reference NC_012920.1 and annotations therein.

- + Is circular, double-stranded, and composed of heavy (H) and
light (L) chains or strands
+ Contains 16,569 bp
+ Encodes 13 protein subunits of the respiratory chain (of a total of about 67) Seven subunits of NADH dehydrogenase (complex I) Cytochrome 6 of complex III Three subunits of cytochrome oxidase (complex IV) Two subunits of ATP synthase
+ Encodes large (16S) and small (12S) mt ribosomal RNAs
+ Encodes 22 mt tRNA molecules
+ Genetic code differs slightly from the standard code
UGA (standard stop codon) is read as Trp AGA and AGG (standard codons for Arg) are read as stop codons
+ Contains very few untranslated sequences
+ High mutation rate (5-10 times that of nuclear DNA)
+ Comparisons of mtDNA sequences provide evidence about evolutionary origins of primates and other species
Source: Adapted from Harding AE: Neurological disease and mitochondrial genes. Trends Neurol Sci 1991;14:132. Copyright © 1991. Reprinted with permission from Elsevier.
would transmit the trait. However, in some cases, deletions in mtDNA occur during oogenesis and thus are not inherited from the mother. A number of diseases have now been shown to be due to mutations of mtDNA. These include a variety of myopathies, neurologic disorders, and some forms of diabetes mellitus.

An alteration in the sequence of purine and pyrimidine bases in a gene due to a change—a removal or an insertion—of one or more bases may result in an altered gene product or alteration of gene expression if nonprotein coding DNA is involved. Such insertions or deletions are termed indels. Indels often result in a mutation whose consequences are discussed in detail in Chapter 37.

Genetic information can be exchanged between similar or homologous chromosomes. The exchange, or recombination event, occurs primarily during meiosis in mammalian cells and requires alignment of homologous metaphase chromosomes, an alignment that almost always occurs with great exactness. A process of crossing over occurs as shown in Figure 35-9. This usually results in an equal and reciprocal exchange of genetic information between homologous chromosomes. If
—_—_
FIGURE 35-9 The process of crossing over between homologous metaphase chromosomes to generate recombinant chromosomes. See also Figure 35-12.
the homologous chromosomes possess different alleles of the same genes, the crossover may produce noticeable and heritable genetic linkage differences. In the rare case where the alignment of homologous chromosomes is not exact, the crossing over or recombination event may result in an unequal exchange of information. One chromosome may receive less genetic material and thus a deletion, while the other partner of the chromosome pair receives more genetic material and thus an insertion or duplication. One well-studied example of unequal crossing that occurs in humans involves the genes encoding hemoglobins. Unequal crossing over results in a human hemoglobinopathy designated Lepore and anti-Lepore

The farther apart any two genes are on an individual chromosome, the greater the likelihood of a crossover recombination event. This is the basis for genetic mapping methods. Unequal crossover affects tandem arrays of repeated DNAs whether they are related globin genes, as in Figure 35-10, or more abundant repetitive DNA. Unequal crossover through slippage in the pairing can result in expansion or contraction in the copy number of the repeat family and may contribute to the expansion and fixation of variant members throughout the repeat array.
FIGURE 35-10 The process of unequal crossover in the region of the mammalian genome that harbors the structural genes encoding hemoglobins and the generation of the unequal recombinant products hemoglobin delta-beta Lepore and betadelta anti-Lepore. The examples given show the locations of the crossover regions within amino acid coding regions of the indicated genes (ie, B and 5 globin genes). (Redrawn and reproduced, with permission, from Clegg JB, Weatherall DJ: 8° Thalassemia: time for a reappraisal? Lancet 1974;2:133. Copyright © 1974. Reprinted with permission from Elsevier.)

Some bacterial viruses (bacteriophages) are capable of recombining with the DNA ofa bacterial host in such a way that the genetic information of the bacteriophage is incorporated in a linear fashion into the genetic information of the host. This integration, which is a form of recombination, occurs by the mechanism illustrated in Figure 35-11.
FIGURE 35-11 The integration of a circular genome from a virus (with genes A, B, and C) into the DNA molecule of a host (with genes 1 and 2) and the consequent ordering of the genes.
The backbone of the circularized bacteriophage genome is broken, as is that of the DNA molecule of the host; the appropriate ends are resealed with the proper polarity. The bacteriophage DNA is figuratively straightened out ("linearized") as it is integrated into the bacterial DNA molecule—frequently a closed circle as well. The site at which the bacteriophage genome integrates or recombines with the bacterial genome is chosen by one of two mechanisms. If the bacteriophage contains a DNA sequence homologous to a sequence in the host DNA molecule, then a recombination event analogous to that occurring between homologous chromosomes can occur. However, some bacteriophages synthesize proteins that bind specific sites on bacterial chromosomes to a nonhomologous site characteristic of the bacteriophage DNA molecule. Integration occurs at the site and is said to be "site specific." Many animal viruses, particularly the oncogenic viruses—either directly or, in the case of RNA viruses such as HIV that causes AIDS, double-stranded DNA copies generated by the action of the viral RNA-dependent DNA polymerase, or reverse transcriptase—can be integrated into chromosomes of the mammalian cell. Integration of the viral DNA into the genome of the infected cells generally is not "site specific" but does display site preferences; such insertions can be mutagenic.

In eukaryotic cells, small DNA elements that clearly are not viruses are capable of transposing themselves in and out of the host genome in ways that affect the function of neighboring DNA sequences. These mobile elements, sometimes called "jumping DNA," or jumping genes, can carry flanking regions of DNA and, therefore, profoundly affect evolution. As mentioned above, the Alu family of moderately repeated DNA sequences has structural characteristics similar to the termini of retroviruses, which would account for the ability of the latter to move into and out of the mammalian genome.
Direct evidence for the transposition of other small DNA elements into the human genome has been provided by the discovery of "processed genes" for immunoglobulin molecules, a-globin molecules, and many others. These processed genes consist of DNA sequences identical or nearly identical to those of the messenger RNA for the appropriate gene product. That is, the 5'-nontranslated region, the coding region without intron representation, and the 3' poly(A) tail are all present contiguously. This particular DNA sequence arrangement must have resulted from the reverse transcription of an appropriately processed messenger RNA molecule from which the intron regions had been removed and the poly(A) tail added. The only recognized mechanism that this reverse transcript could have used to integrate into the genome would have been a transposition event. In fact, these "processed genes" have
short terminal repeats at each end, as do known transposed sequences in other organisms. In the absence of their transcription and thus genetic selection for function, many of the processed genes have been randomly altered through evolution so that they now contain nonsense codons that preclude their ability to encode a functional, intact protein even if they could be transcribed (see Chapter 37). Thus, they are referred to as
“pseudogenes.”

Besides unequal crossover and transposition, a third mechanism can effect rapid changes in the genetic material. Similar sequences on homologous or nonhomologous chromosomes may occasionally pair up and eliminate any mismatched sequences between them. This may lead to the accidental fixation of one variant or another throughout a family of repeated sequences and thereby homogenize the sequences of the members of repetitive DNA families. This process is referred to as gene conversion.

In diploid eukaryotic organisms such as humans, after cells progress through the S phase, they contain a tetraploid content of DNA. This is in the form of sister chromatids of chromosome pairs (Figure 35-6). Each of these sister chromatids contains identical genetic information since each is a product of the semiconservative replication of the original parent DNA molecule of that chromosome. Crossing over can occur between these genetically identical sister chromatids. Of course, these sister chromatid exchanges (Figure 35-12) have no genetic consequence as long as the exchange is the result of an equal crossover.

In mammalian cells, some interesting gene rearrangements occur normally during development and differentiation. For example, the V, and C, genes, which encode for the immunoglobulin G (IgG) light-chain variable (V,) and constant (C,) portions of the IgG light chain in a single IgG molecule (see Chapters 38, 52), are widely separated in the germ line DNA. In the DNA of a differentiated IgG-producing (plasma) cell, the same V, and C, genes have been moved physically closer, and linked together in the genome within a single transcription unit. However, even then, this rearrangement of DNA during differentiation does not bring the V, and C, genes into contiguity in the DNA. Instead, the DNA contains an intron of about 1200 bp at or near the junction of the V and C regions. This intron sequence is transcribed into RNA along with the V, and C, exons, and the interspersed, intronic non-IgG sequence information is removed from the RNA during its nuclear processing (see Chapters 36 and 38).

human chromosomes. The exchanges are detectable by Giemsa staining of the chromosomes of cells replicated for two cycles in the presence of bromodeoxyuridine. The arrows indicate some regions of exchange. (Reprinted with permission from S Wolff and J
Bodycote.)

The primary function of DNA replication is the provision of progeny with the genetic information possessed by the parent. Thus, the replication of DNA must be complete and carried out in such a way as to maintain genetic stability within the organism and the species. The process of DNA replication is complex and involves many cellular functions and several verification procedures to ensure fidelity in replication. About 30 proteins are involved in the replication of the Escherichia coli chromosome, and this process is more complex in eukaryotic organisms.
In all cells, replication can occur only from a singlestranded DNA (ssDNA) template. Therefore, mechanisms must exist to target the site of initiation of replication and to unwind the dsDNA in that region. The replication complex must then form. After replication is complete in an area, the parent and daughter strands must reform dsDNA. In eukaryotic cells, an additional step must occur. The dsDNA must reform the chromatin structure, including nucleosomes that existed prior to the onset of replication. Although this entire process is not completely understood in eukaryotic cells, replication has been quite precisely described in prokaryotic cells, and the general principles are the same in both. The major

steps are listed in Table 35-4, illustrated in Figure 35-13, and discussed, in sequence, below. A number of proteins, most with specific enzymatic action, are involved in this process (Table 35-5).

At the origin of replication (ori), there is an association of sequence-specific dsDNA-binding proteins with a series of direct repeat DNA sequences. In E. coli, the oriC is bound by the protein dnaA, which forms a complex consisting of 150 to 250 bp of DNA and multimers of the DNA-binding protein. This leads to the local denaturation and unwinding of an adjacent A+T-rich region of DNA. Functionally similar autonomously replicating sequences (ARS) or replicators have been identified in yeast cells. The ARS contains a somewhat degenerate 11-bp sequence called the origin replication element (ORE). The ORE binds a set of proteins, analogous to the dnaA protein of E. coli, the group of proteins is collectively called the origin recognition complex (ORC). ORC homologs have been found in all eukaryotes examined. The ORE is located adjacent to an approximately 80-bp A+T-rich sequence that is easy to unwind. This is called the DNA unwinding element
eee
=SSB
FIGURE 35-13 Steps involved in DNA replication. This figure describes DNA replication in an E. coli cell, but the general steps are similar in eukaryotes. A specific interaction of a protein (the dnaA protein) to the origin of replication (oriC) results in local unwinding of DNA at an adjacent A+T-rich region. The DNA in this area is maintained in the single-strand conformation (ssDNA) by single-strand-binding proteins (SSBs). This allows a variety of proteins, including helicase, primase, and DNA polymerase, to bind and to initiate DNA synthesis. The replication fork proceeds as DNA synthesis occurs continuously (long red arrow) on the leading strand and discontinuously (short black arrows) on the lagging strand. The nascent DNA is always synthesized in the 5' to 3' direction, as DNA polymerases can add a nucleotide only to the 3' end of a DNA strand.

DNApolymerases, Function = Deoxynucleotide polymerization. Helicases, Function = ATP-driven processive unwinding of DNA. Topoisomerases, Function = Relieve torsional strain that results from helicase-induced unwinding. DNAprimase, Function = Initiates synthesis of RNA primers. Single-strand binding proteins (SSBs), Function = Prevent premature reannealing ssDNA strands to form dsDNA. DNAligase, Function = Seals the single-strand nick between the nascent chain and Okazaki fragments on lagging strand
(DUE). The DUE is the origin of replication in yeast and is bound by the MCM protein complex.
Consensus sequences similar to ori or ARS in structure have not been precisely defined in mammalian cells, though several of the proteins that participate in ori recognition and function have been identified and appear quite similar to their yeast counterparts in both amino acid sequence and function.

The interaction of proteins with ori defines the start site of replication and provides a short region of ssDNA essential for initiation of synthesis of the nascent DNA strand. This process requires the formation of a number of protein-protein and protein-DNA interactions. A critical step is provided by a DNA helicase that allows for processive unwinding of DNA. This function is provided by a complex of dnaB helicase and the dnaC protein. Single-stranded DNA-binding proteins (SSBs) stabilize this complex.

A replication fork consists of four components that form in the following sequence: (1) the DNA helicase unwinds a short segment of the parental duplex DNA; (2) SSBs bind to ssDNA and prevent premature reannealing of ssDNA to dsDNA; (3) a primase initiates synthesis of an RNA molecule that is essential for priming DNA synthesis; and (4) the DNA polymerase initiates nascent, daughter-strand synthesis.
The DNA polymerase III enzyme (the dnaE gene product in E. coli) binds to template DNA as part of a multiprotein complex that consists of several polymerase accessory factors (B', y, 5, 5', and t). DNA polymerases only synthesize DNA in the 5' to 3' direction, and only one of the several different types of polymerases is involved at the replication fork. Because the DNA strands are antiparallel (see Chapter 34), the polymerase functions asymmetrically. On the leading (forward) strand, the DNA is synthesized continuously. On the lagging (retrograde) strand, the DNA is synthesized in short (1-5 kb; see Figure 35-16) fragments, the so-called Okazaki fragments, so named after the scientist who discovered them.
Several Okazaki fragments (up to 1000) must be sequentially synthesized for each replication fork. To ensure that this happens, the helicase acts on the lagging strand to unwind dsDNA in a5' to 3' direction. The helicase associates with the primase to afford the latter proper access to the template. This allows the RNA primer to be made and, in turn, the polymerase to begin replicating the DNA. This is an important reaction sequence since DNA polymerases cannot initiate DNA synthesis de novo. The mobile complex between helicase and primase has been called a primosome. As the synthesis of an Okazaki fragment is completed and the polymerase is released, a new primer has been synthesized. The same polymerase molecule remains associated with the replication fork and proceeds to synthesize the next Okazaki fragment.

A number of different DNA polymerase molecules engage in DNA replication. These share three important properties: (1) chain elongation, (2) processivity, and (3) proofreading. Chain elongation accounts for the rate (in nucleotides per second; nt/s) at which polymerization occurs. Processivity is an expression of the number of nucleotides added to the nascent chain before the polymerase disengages from the template. The proofreading function identifies copying errors and corrects them. In E. coli, DNA polymerase III (pol III) functions at the replication fork. Of all polymerases, it catalyzes the highest rate of chain elongation and is the most processive. It is capable of polymerizing 0.5 Mb of DNA during one cycle on the leading strand. Pol III is a large (>1 MDa), multisubunit protein complex in E. coli. DNA pol III associates with the two identical 6 subunits of the DNA sliding "clamp"; this association dramatically increases pol III-DNA complex stability, processivity (100 to >50,000 nucleotides) and rate of chain elongation (20-50 nt/s) generating the high degree of processivity the enzyme exhibits.
Polymerase I (pol I) and II (pol II) are mostly involved in proofreading and DNA repair. Eukaryotic cells have counterparts for each of these enzymes plus a large number of additional DNA polymerases primarily involved in DNA repair. A comparison is shown in Table 35-6.
In mammalian cells, the polymerase is capable of polymerizing at a rate that is somewhat slower than the rate of polymerization of deoxynucleotides by the bacterial DNA polymerase complex. This reduced rate may result from interference by nucleosomes.

The initiation of DNA synthesis (Figure 35-14) requires priming by a short length of RNA, about 10 to 200 nucleotides long. In E. coli this is catalyzed by dnaG (primase), in eukaryotes DNA Pol a synthesizes these RNA primers. The priming process involves nucleophilic attack by the 3'-hydroxyl group of the RNA primer on the phosphate of the first entering

I, Eukaryotic = . I, Function = Gap filling following DNAreplication, repair, and recombination. II, Eukaryotic = . II, Function = DNAproofreading and repair. , Eukaryotic = β. , Function = DNArepair. , Eukaryotic = γ. , Function = Mitochondrial DNAsynthesis. III, Eukaryotic = ε. III, Function = Processive, leading strand synthesis. DnaG, Eukaryotic = α. DnaG, Function = Primase. , Eukaryotic = δ. , Function = Processive, lagging strand synthesis

deoxynucleoside triphosphate (N in Figure 35-14) with the splitting off of pyrophosphate; this transition to DNA synthesis is catalyzed by the appropriate DNA polymerases (DNA pol III in E. coli; DNA pol 6 and ¢ in eukaryotes). The 3'-hydroxyl group of the recently attached deoxyribonucleoside monophosphate is then free to carry out a nucleophilic attack on the next entering deoxyribonucleoside triphosphate (N + 1 in Figure 35-14), again at its a phosphate moiety, with the splitting off of pyrophosphate. Of course, selection of the proper deoxyribonucleotide whose terminal 3'-hydroxyl group is to be attacked is dependent on proper base pairing with the other strand of the DNA molecule according to Watson and Crick base pairing rules (Figure 35-15). When an adenine deoxyribonucleoside monophosphory] moiety is in the template position, a thymidine triphosphate will enter and its a phosphate will be attacked by the 3'-hydroxyl group of the deoxyribonucleoside monophosphoryl most recently added to the polymer. By this stepwise process, the template dictates which deoxyribonucleoside triphosphate is complementary and by hydrogen bonding holds it in place while the 3'-hydroxyl group of the growing strand attacks and incorporates the new nucleotide into the polymer. These segments of DNA attached to an RNA primer component are the Okazaki fragments (Figure 35-16). In mammals, after many Okazaki fragments are generated, the replication complex begins to remove the RNA primers, to fill in the gaps left by their removal with the proper base-paired deoxynucleotide, and then to seal the fragments of newly synthesized DNA by enzymes referred to as DNA ligases.

As has already been noted, DNA molecules are double stranded and the two strands are antiparallel. The replication of DNA in prokaryotes and eukaryotes occurs on both strands simultaneously. However, an enzyme capable of polymerizing DNA in the 3' to 5' direction does not exist in any organism, so that both of the newly replicated DNA strands cannot grow in the same direction simultaneously. Nevertheless, in bacteria the same enzyme does replicate both strands at the same time (in eukaryotes pol ¢ and pol 6 catalyze leading and lagging strand synthesis; see Table 35-6. The single enzyme replicates one strand ("leading strand") in a continuous manner in the 5' to 3' direction, with the same overall forward direction. It replicates the other strand ("lagging strand") discontinuously while polymerizing the nucleotides in short spurts of 150 to 250 nucleotides, again in the 5' to 3' direction, but at the same time it faces toward the back end of the preceding RNA primer rather than toward the unreplicated portion. This process of semidiscontinuous DNA synthesis is shown diagrammatically in Figures 35-13 and 35-16.

Replication of the circular bacterial chromosome, composed of roughly 5 x 10° bp of DNA proceeds from a single ori. This process is completed in about 30 minutes, a replication rate of 3 x 10° bp/min. The entire mammalian genome replicates in approximately 9 hours, the average period required for formation of a tetraploid genome from a diploid genome in a replicating cell. If a mammalian genome (3 x 10° bp) replicated at the same rate as bacteria (ie, 3 x 10° bp/min) from but a single ori, replication would take over 150 hours! Metazoan organisms get around this problem using two strategies. First, replication is bidirectional. Second, replication proceeds from multiple origins in each chromosome (a total of as many as 100 in humans). Thus, replication occurs in both directions along all of the chromosomes, and both strands are replicated simultaneously. This replication process generates "replication bubbles" (Figure 35-17).
The multiple ori sites that serve as origins for DNA replication in eukaryotes are poorly defined except in a few animal viruses and in yeast. However, it is clear that initiation is regulated both spatially and temporally, since clusters of adjacent sites initiate replication synchronously. Replication firing, or DNA replication initiation at a replicator/ori, is influenced by a number of distinct properties of chromatin structure that are just beginning to be understood. It is clear, however, that there are more replicators and excess ORC than needed to replicate the mammalian genome within the time of a typical S phase. Therefore, mechanisms for controlling the excess ORC-bound replicators must exist. Understanding the control of the formation and firing of replication complexes is one of the major challenges in this field.

During the replication of DNA, there must be a separation of the two strands to allow each to serve as a template by hydrogen bonding its nucleotide bases to the incoming deoxynucleoside triphosphate. The separation of the DNA strands is promoted by SSBs in E. coli, and a protein termed replication protein A (RPA) in eukaryotes. These molecules stabilize the single-stranded structure as the replication fork progresses. The stabilizing proteins bind cooperatively and stoichiometrically to the single strands without interfering with the abilities of the nucleotides to serve as templates (Figure 35-13). In addition to separating the two strands of the double helix, there must be an unwinding of the molecule (once every 10 nucleotide pairs) to allow strand separation.
FIGURE 35-14 The initiation of DNA synthesis upon a primer of RNA and the subsequent attachment of the second deoxyribonucleoside triphosphate.
FIGURE 35-15 The RNA-primed synthesis of DNA demonstrating the template function of the complementary strand of parental DNA.
Ki
FIGURE 35-16 The discontinuous polymerization of deoxyribonucleotides on the lagging strand; formation of Okazaki fragments during lagging strand DNA synthesis is illustrated. Okazaki fragments are 100 to 250 nucleotides long in eukaryotes, 1000 to 2000 nucleotides in prokaryotes.
FIGURE 35-17 The generation of "replication bubbles" during the process of DNA synthesis. The bidirectional replication and the proposed positions of unwinding proteins at the replication forks are depicted.

The hexameric DNAB protein complex unwinds DNA in E. coli, whereas the hexameric MCM complex unwinds eukaryotic DNA. This unwinding happens in segments adjacent to the replication bubble. To counteract this unwinding, there are multiple "swivels" interspersed in the DNA molecules of all organisms. The swivel function is provided by specific enzymes that introduce "nicks" in one strand of the unwinding double helix, thereby allowing the unwinding process to proceed. The nicks are quickly resealed without requiring energy input, because of the formation of a high-energy covalent bond between the nicked phosphodiester backbone and the nicking-sealing enzyme. The nicking-resealing enzymes are called DNA topoisomerases. This process is depicted diagrammatically in Figure 35-18 and there compared with the ATP-dependent resealing carried out by the DNA ligases (see Table 39-2). Topoisomerases are also capable of unwinding supercoiled DNA. Supercoiled DNA is a higher-ordered structure occurring in circular DNA molecules wrapped around a core, as depicted in Figures 35-2 and 35-19. There exists in one species of animal viruses (retroviruses) a class of enzymes capable of synthesizing a single-stranded and then a dsDNA molecule from a single-stranded RNA template. This polymerase, termed RNA-dependent DNA polymerase, or "reverse transcriptase," first synthesizes a

FIGURE 35-18 Two types of DNA nick-sealing reactions. Two forms of nick sealing are represented: ATP-independent (top) and ATPdependent (bottom). Nick sealing processes proceeds in multiple steps: (i)-substrate to — (iv)-product. The enzymes involved are signified by E (top, bottom), while small molecule reactants and products are indicated as Phosphate (P); Pyrophosphate (PP), inorganic Phosphate (Pi) generated from PP by the action of ubiquitous pyrophosphatases, Ribose (R), and Adenine (A). The nick-sealing reaction at the top is catalyzed by DNA topoisomerase | and is ATP-energy independent because the energy for reformation of DNA phosphodiester bonds is stored within the covalent attachment of topoisomerase | to DNA (P-E; top; step ii). Bond reformation is accomplished by the nucleophilic attack of the 3'OH group (green arrow, step iii) to the phosphate of the P-E complex. This reaction releases free topoisomerase | (E) and intact double stranded DNA (step iv). The overall enzyme reaction is schematized at the bottom of the figure (steps | — iv). The nick-sealing reaction catalyzed by DNA ligase (bottom) repairs single strand DNA breaks in the phosphodiester backbone that are a result of DNA replication and/or DNA repair (step i; bottom). The complete DNA ligase reaction requires hydrolysis of two of the high-energy phosphodiester bonds of ATP. The overall reaction scheme of DNA ligase nick-sealing from nick, to enzyme-DNA binding, to enzyme activation that releases Pyrophosphate (PP) to release of free enzyme, AMP and intact DNA is depicted (bottom; as noted in the text, PP is rapidly converted to 2 moles of Pi by the action of ubiquitous pyrophosphatases). The activated ligase (E-P-R-A) reacts with the 5'P at the nick site to form a transient DNA-P-P-R-A complex (note: P-R-A = AMP) that liberates free DNA Ligase Enzyme (E). Nucleophilic attack of the free 3'OH group with the 5'P of the DNA-5'P-AMP complex (green arrow, step iii) reseals

the nick and liberates AMP. The overall enzyme reaction converting nicked DNA to intact DNA (E + ATP + E+ AMP + 2Pi) is schematized at the bottom of the figure (steps | — iv).
FIGURE 35-19 supercoiling of DNA. A left-handed toroidal (solenoidal) supercoil, at left, will convert to a right-handed interwound supercoil, at right, when the cylindric core is removed. Such a transition is analogous to that which occurs when nucleosomes are disrupted by the high salt extraction of histones from chromatin.
DNA-RNA hybrid molecule utilizing the RNA genome as a template. A specific virus-encoded nuclease, RNase H, degrades the hybridized template RNA strand. Subsequently, the remaining DNA strand in turn serves as a template to form a dsDNA molecule containing the genetic information originally present in the RNA genome of the animal virus. The resulting dsDNA can then integrate into the host genome.

There is evidence that nuclear organization and chromatin structure are involved in determining the regulation and initiation of DNA synthesis. As noted above, the rate of polymerization in eukaryotic cells, which have chromatin and nucleosomes, is slower than that in prokaryotic cells, which lack canonical nucleosomes. It is also clear that chromatin structure must be reformed after replication. Newly replicated DNA is rapidly assembled into nucleosomes, and the preexisting and newly assembled histone octamers are randomly distributed to each arm of the replication fork. These reactions are facilitated through the actions of histone chaperone proteins working in concert with chromatin assembly and remodeling complexes.

In eukaryotic cells, including human cells, the replication of the DNA genome occurs only at a specified time during the life span of the cell. This period is referred to as the synthetic
FIGURE 35-20 Progress through the mammalian cell cycle is continuously monitored via multiple cell-cycle checkpoints. DNA, chromosome, and chromosome segregation integrity are continuously monitored throughout the cell cycle. If DNA damage is detected in either the G, or the G, phase of the cell cycle, if the genome is incompletely replicated, or if normal chromosome segregation machinery is incomplete (ie, a defective spindle), cells will not progress through the phase of the cycle in which defects are detected. In some cases, if the damage cannot be repaired, such cells undergo programmed cell death (apoptosis). Note that cells can reversibly leave the cell cycle during G, entering a nonreplicative state termed G, (not shown, but see Figure 9-8). When appropriate signals/conditions occur, cells reenter G, and progress normally through the cell cycle as depicted.
or S phase. This is usually temporally separated from the mitotic, or M phase, by nonsynthetic periods referred to as gap 1 (G ,) and gap 2 (G,) phases, occurring before and after the S phase, respectively (Figure 35-20). Among other things, the cell prepares for DNA synthesis in G,, and for mitosis in G,. The cell regulates DNA synthesis by allowing it to occur only once per cell cycle, and only during S phase, in cells preparing to divide by a mitotic process.
All eukaryotic cells have gene products that govern the transition from one phase of the cell cycle to another. The cyclins are a family of proteins whose concentration increases and decreases at specific times, that is, "cycle" during the cell cycle—thus their name. The cyclins thus activate, at the appropriate time, different cyclin-dependent protein kinases (CDKs) that phosphorylate substrates essential for progression through the cell cycle (Figure 35-21). For example, cyclin D levels rise in late G, phase and allow progression beyond the start (yeast) or restriction point (mammals), the point beyond which cells irrevocably proceed into the S or DNA synthesis phase.

The D cyclins activate CDK4 and CDK6. These two kinases are also synthesized during G, in cells undergoing active division. The D cyclins and CDK4 and CDKé6 are nuclear proteins that assemble as a complex in late G, phase. The cyclin-CDK complex is now an active serine-threonine protein kinase. One substrate for this kinase is the retinoblastoma (Rb) protein. Rb is a cell-cycle regulator because it binds
FIGURE 35-21 Schematic illustration of the points during the mammalian cell cycle during which the indicated cyclins and cyclin-dependent kinases are activated. The thickness of the various colored lines is indicative of the extent of activity.
to and inactivates a transcription factor (E2F) necessary for the transcription of certain genes (histone genes, DNA replication proteins, etc) needed for progression from G, to S phase. The phosphorylation of Rb by CDK4 or CDK6 results in the release of E2F from Rb-mediated transcription repression—thus, gene transcription activation ensues and cell-cycle progression takes place.
Other cyclins and CDKs are involved in different aspects of cell-cycle progression (Table 35-7). Cyclin E and CDK2 form a complex in late G,. Cyclin E is rapidly degraded, and the released CDK2 then forms a complex with cyclin A. This sequence is necessary for the initiation of DNA synthesis in S phase. A complex between cyclin B and CDK1 is ratelimiting for the G,/M transition in eukaryotic cells.
Many of the cancer-causing viruses (oncoviruses) and cancer-inducing genes (oncogenes) are capable of alleviating or disrupting the restriction that normally controls the entry of mammalian cells from G, into the S phase. From the foregoing, one might have surmised that excessive production of a cyclin, loss of a specific CDK inhibitor (see below), or

D, Kinase = CDK4, CDK6. D, Function = Progression past restriction point at G 1 /S boundary. E, A, Kinase = CDK2. E, A, Function = Initiation of DNAsynthesis in early S phase. B, Kinase = CDK1. B, Function = Transition from G 2 to M
production or activation of a cyclin/CDK at an inappropriate time might result in abnormal or unrestrained cell division. Similarly, the oncoproteins (or transforming proteins) produced by several DNA viruses target the Rb transcription repressor for inactivation, inducing cell division inappropriately, while inactivation of Rb, itself a tumor suppressor gene, leads to uncontrolled cell and tumor formation.
growth During the S phase, mammalian cells contain greater quantities of DNA polymerase than during the nonsynthetic phases of the cell cycle. Furthermore, those enzymes responsible for formation of the substrates for DNA synthesis—that is, deoxyribonucleoside triphosphates—are also increased in activity, and their expression drops following the synthetic phase until the reappearance of the signal for renewed DNA synthesis. During the S phase, the nuclear DNA is completely replicated once and only once. Once chromatin has been replicated, it is marked so as to prevent its further replication until it again passes through mitosis. This process is termed replication licensing. The molecular mechanisms for this phenomenon in human cells involves dissociation and/or cyclin-CDK phosphorylation and subsequent degradation of several origin binding proteins that play critical roles in replication complex formation. Consequently, origins fire only once per cell cycle. In general, a given pair of chromosomes will replicate simultaneously and within a fixed portion of the S phase upon every replication. On a chromosome, clusters of replication units replicate coordinately. The nature of the signals that regulate DNA synthesis at these levels is unknown, but the regulation does appear to be an intrinsic property of each individual chromosome that is mediated by the several replication origins contained therein.

Repair of damaged DNA is critical for maintaining genomic integrity and thereby preventing the propagation of mutations, either horizontally (somatic cells), or vertically (germ cells). DNA is subjected to a huge array of chemical, physical, and biologic assaults on a daily basis (Table 35-8), hence efficient recognition and repair of DNA lesions is essential. Consequently, eukaryotic cells contain five major DNA repair pathways, each of which contain multiple, sometimes shared proteins; these DNA repair proteins typically have orthologues in prokaryotes. The mechanisms of DNA repair include nucleotide excision repair (NER); mismatch repair (MMR); base excision repair (BER); homologous recombination (HR); and nonhomologous end-joining (NHEJ) repair pathways (Figure 35-22). The experiment of testing the importance of many of these DNA repair proteins to human biology has been performed by nature—mutations in a large number of these genes lead to human disease (Table 35-9). Moreover, systematic genedirected "knockout" experiments (see Chapter 39) with laboratory mice and cells in culture have clearly ascribed critical gene integrity maintenance functions to these genes as well. In these

A. Depurination
B. Deamination of cytosine to uracil
C. Deamination of adenine to hypoxanthine
D. Alkylation of base
E. Base-analog incorporation

A. UV light-induced thymine-thymine (pyrimidine) dimer
B. Bifunctional alkylating agent cross-linkage

A. lonizing radiation
B. Radioactive disintegration of backbone element
C. Oxidative free-radical formation

A. Between bases in same or opposite strands
B. Between DNA and protein molecules (eg, histones)
genetic studies, it was observed that indeed targeted mutations within these genes induce defects in DNA repair while often also dramatically increasing susceptibility to cancer.

Severe combined immunodeficiency disease (SCID)
Radiation-sensitive severe combined immunodeficiency disease
(RS-SCID)

AT-like disorder (ATLD)
Nijmegen breakage syndrome (NBS)
Bloom syndrome (BS)
Werner syndrome (WS)
Rothmund-Thomson syndrome (RTS)
Breast cancer susceptibility 1 and 2 (BRCA1, BRCA2)

Xeroderma pigmentosum (XP)
Cockayne syndrome (CS)
Trichothiodystrophy (TTD)

Hereditary nonpolyposis colorectal cancer (HNPCC)
One of the most intensively studied mechanisms of DNA repair is the mechanism used to repair DNA double-strand breaks (DSBs); these will be discussed in some detail here. There are two pathways, HR and NHBEJ, that eukaryotic cells utilize to remove DSBs. The choice between the two depends on the phase of the cell cycle (Figures 35-20 and 35-21) and the exact type of DSB breaks to be repaired (Table 35-8). During the
G,/G, phases of the cell cycle, DSBs are corrected by the NHEJ pathway, whereas during S, G,, and M phases of the cell cycle HR is utilized. All steps of DNA damage repair are catalyzed by evolutionarily conserved molecules, which include DNA damage sensors, transducers, and damage repair mediators. Collectively, these cascades of proteins participate in the cellular response to DNA damage. Importantly, the ultimate cellular
FIGURE 35-22 Mammals use multiple DNA repair pathways of variable accuracy to repair the myriad forms of DNA damage genomic DNA is subjected to. Listed are the major types of DNA damaging agents, the DNA lesions so formed (schematized and listed), the DNA repair pathway responsible for repairing the different lesions, and the relative fidelity of these pathways. (Modified, with permission, from: "DNA-damage response in tissue-specific and cancer stem cells" Cell Stem Cell 8:16-29 (2011) copyright © 2011 Elsevier Inc.
outcomes of DNA damage, and cellular attempts to repair DNA damage, range from cell-cycle delay to allow for DNA repair, to cell-cycle arrest, to apoptosis or senescence (Figure 35-23; and further detail below). The molecules involved in these complex and highly integrated processes range from damage-specific histone modifications (ie, dimethylated lysine 20 histone H4; H4K20me2) and incorporation of histone isotype variants such as histone H2AX into nucleosomes at the site of DNA damage (Table 35-1), poly ADP ribose polymerase (PARP), the MRN protein complex (Mrell-Rad50-NBS1 subunits); to DNA

damage-activated kinase recognition/signaling proteins (ATM {ataxia telangiectasia, mutated] and ATM-related kinase (ATR), the multisubunit DNA-dependent protein kinase [DNA-PK and Ku70/80], and checkpoint kinases 1 and 2 [CHK1, CHK2]). These multiple kinases phosphorylate and consequently modulate the activities of dozens of proteins, such as numerous DNA repair, checkpoint control, and cell-cycle control proteins like CDC25A, B, C, Weel, p21, p16, and p19 (all cyclin-CDK regulators [see Figure 9-8; and below]; various exoand endonucleases; DNA single-strand-specific DNA-binding proteins [RPA];
FIGURE 35-23 The multistep mechanism of DNA double-strand break repair. Shown top to bottom are the proteins (protein complexes) that identify DSBs in genomic DNA (sensors), transduce, and amplify the recognized DNA damage (transducers and mediators), as well as the molecules that dictate the ultimate outcomes of the DNA damage response (effectors). Damaged DNA can be (a) repaired directly (DNA repair), or, via p53-mediated pathways and depending upon the severity of DNA damage and p53-activated genes induced, (b), cells can be arrested in the cell cycle by p21/WAF1 the potent CDK-cyclin complex inhibitor to allow time for extensively damaged DNA to be repaired, or (c), and (d) if the extent of DNA damage is too great to repair, cells can either apotose or senesce; both of these processes prevent the cell containing such damaged DNA from ever dividing and hence inducing cancer or other deleterious biologic outcomes. (Based on: "DNA-damage response in tissue-specific and cancer stem cells" Cell Stem Cell 8:16-29 (2011) copyright © 2011 Elsevier Inc.)
PCNA and specific DNA polymerases [DNA pol 6 and n]). Several of these (types of) proteins/enzymes have been discussed above in the context of DNA replication. DNA repair and its relationship to cell-cycle control are very active areas of research given their central roles in cell biology and potential for generating and preventing cancer.

Given the importance of normal DNA and chromosome function to survival, it is not surprising that eukaryotic cells have developed elaborate mechanisms to monitor the integrity of the genetic material. As detailed above, a number of complex multisubunit enzyme systems have evolved to repair damaged DNA at the nucleotide sequence level. Similarly, DNA mishaps at the chromosome level are also monitored and repaired. As shown in Figure 35-20, both DNA and chromosomal integrity are continuously monitored throughout the cell cycle. The four specific steps at which this monitoring occurs have been termed checkpoint controls. If problems are detected at any of these checkpoints, progression through the cycle is interrupted and transit through the cell cycle is halted until the damage is repaired. The molecular mechanisms underlying detection of DNA damage during the G, and G, phases of the cycle are understood better than those operative during S and M phases.

The tumor suppressor p53, a protein of apparent MW 53 kDa on SDS-PAGE, plays a key role in both G, and G, checkpoint control. Normally a very unstable protein, p53 is a DNA-binding transcription factor, one of a family of related proteins (ie, p53, p63, and p73) that is somehow stabilized in response to DNA damage, perhaps by direct p53-DNA interactions. Like the histones discussed above, p53 is subject to a panoply of regulatory PTMs, all of which likely modify its multiple biologic activities. Increased levels of p53 activate transcription of an ensemble of genes that collectively serve to delay transit through the cycle. One of these induced proteins, p21, is a potent CDK-cyclin inhibitor (CK) that is capable of efficiently inhibiting the action of all CDKs. Clearly, inhibition of CDKs will halt progression through the cell cycle (see Figures 35-20 and 35-21). If DNA damage is too extensive to repair, the affected cells undergo apoptosis (programmed cell death) in a p53-dependent fashion. In this case, p53 induces the activation of a collection of genes that induce apoptosis. Cells lacking functional p53 fail to undergo apoptosis in response to high levels of radiation or DNA-active chemotherapeutic agents. It may come as no surprise, then, that p53 is one of the most frequently mutated genes in human cancers (see Chapter 56). Indeed, recent genomic sequencing studies of a multitude of tumor DNA samples suggest that over 80% of human cancers carry p53 loss-of-function mutations. Additional research into the mechanisms of checkpoint control will prove invaluable for the development of effective anticancer therapeutic options.

DNA in eukaryotic cells is associated with a variety of proteins, resulting in a structure called chromatin.
™ Much of the DNA is associated with histone proteins to form a structure called the nucleosome. Nucleosomes are composed of an octamer of histones around which about 150 bp of DNA is wrapped.
™
Histones are
subject to
an extensive
array of
dynamic covalent
modifications that
have
Nucleosomes
™
and important
regulatory higher-order
structures consequences.
them serve
to compact
the
DNA.
= DNA in transcriptionally active regions is relatively more sensitive to nuclease attack in vitro; some regions, so-called hypersensitive sites are exceptionally sensitive and are often found to contain transcription control sites.
® Highly transcriptionally active DNA (genes) is often clustered in regions of each chromosome. Within these regions, genes may be separated by inactive DNA in nucleosomal structures. In many eukaryotic transcription units (ie, the portion of a gene that is copied by RNA polymerase) often consists of coding regions of DNA (exons) interrupted by intervening sequences of noncoding DNA (introns). This is particularly true for mRNA-encoding genes.
= After transcription, during RNA processing, introns are removed and the exons are ligated together to form the mature mRNA that appears in the cytoplasm; this process is termed RNA splicing.
™ DNA in each chromosome is exactly replicated according to the rules of base pairing during the S phase of the cell cycle.
™ Each strand of the double helix is replicated simultaneously but by somewhat different mechanisms. A complex of proteins, including DNA polymerase, replicates the leading strand continuously in the 5' to 3' direction. The lagging strand is replicated discontinuously, in short pieces of 100 to 250 nucleotides by DNA polymerase synthesizing in the 5' > 3' direction.
™
DNA replication is initiated at special sites termed origins to generate replication bubbles. Each eukaryotic chromosome contains multiple origins. The entire process takes about 9 hours in a typical human cell and only occurs during the S phase of the cell cycle.
@ A variety of mechanisms that employ different enzyme systems repair damaged cellular DNA after exposure of cells to chemical and physical mutagens.
= Normal cells containing DNA that cannot be repaired undergo programmed cell death.

Braunschweig U, Gueroussov S, Plocik AM, Graveley BR, Blencowe BJ:
Dynamic integration of splicing within gene regulatory pathways.
Cell
2013;152:1252.
Burgers
PMJ,
Kunkel
TA:
DNA
replication fork.
Annu
Rev Biochem 2017;86:417.
Chabot B, Shkreta L: Defective control of pre-messenger RNA splicing in human disease. J Cell Biol 2016;212:13.

VRIES NCR UER ee eA you should be able to:
C H A PT ER
Describe the molecules involved and the mechanism of RNA synthesis.
molecules. Explain how eukaryotic DNA-dependent RNA polymerases, in collaboration with an array of specific accessory factors, can differentially transcribe genomic DNA to produce specific messenger RNA (mRNA) precursor
= Describe the structure of eukaryotic mRNA precursors, which are highly modified internally and at both termini.
= Appreciate the fact that the majority of mammalian mRNA-encoding genes are interrupted by multiple nonprotein coding sequences termed introns, which
are interspersed
between protein
coding regions
termed exons.
= Explain that since intron RNA does not encode protein, the intronic RNA must be specifically and accurately removed in order to generate functional mRNAs from the mRNA precursor molecules in a series of precise molecular events termed RNA splicing.
= Explain the steps and molecules that catalyze mRNA splicing, a process
that converts
the end-modified
precursor molecules
into mRNAs
that are
functional for
translation.

and maintained. Errors or changes in synthesis, processing, splicing, stability, or function of mRNA transcripts are a cause of disease.

All eukaryotic cells have two major classes of RNA (Table 36-1), the protein coding RNAs, or mRNAs, and two forms of abundant nonprotein coding RNAs delineated on the basis of size: the large ribosomal RNAs (rRNAs) and long noncoding RNAs (IncRNAs) and small noncoding transfer RNAs (tRNAs), the small nuclear RNAs (snRNAs) and the micro and silencing RNAs (miRNAs and siRNAs). The mRNAs, rRNAs, and tRNAs are directly involved in protein synthesis while the other RNAs participate in either mRNA splicing (snRNAs) or modulation of gene expression by altering mRNA function (mi/siRNAs) and/or expression (IncRNAs). These RNAs differ in their diversity, stability, and abundance in cells.

Protein Coding RNAs, Types = . Protein Coding RNAs, Abundance = . Protein Coding RNAs, Stability = . Messenger (mRNA), Types = ≥10 5 Different species. Messenger (mRNA), Abundance = 2%-5% of total. Messenger (mRNA), Stability = Unstable to very stable. Nonprotein Coding RNAs (ncRNAs), Types = . Nonprotein Coding RNAs (ncRNAs), Abundance = . Nonprotein Coding RNAs (ncRNAs), Stability = . Large ncRNAs, Types = . Large ncRNAs, Abundance = . Large ncRNAs, Stability = . Ribosomal (rRNA), Types = 28S, 18S. Ribosomal (rRNA), Abundance = 80% of total. Ribosomal (rRNA), Stability = Very stable. lncRNAs, Types = ~1000s. lncRNAs, Abundance = ~1%-2%. lncRNAs, Stability = Unstable to very stable. Small ncRNAs, Types = . Small ncRNAs, Abundance = . Small ncRNAs, Stability = . Small ribosomal RNAs, Types = 5.8S, 5S. Small ribosomal RNAs, Abundance = ~2%. Small ribosomal RNAs, Stability = Very stable. Transfer RNAs, Types = ~60 Different species. Transfer RNAs, Abundance = ~15% of total. Transfer RNAs, Stability = Very stable. Small nuclear (snRNA), Types = ~30 Different species. Small nuclear (snRNA), Abundance = ≤1% of total. Small nuclear (snRNA), Stability = Very stable. Micro/Silencing (mi/SiRNAs), Types = 100s-1000. Micro/Silencing (mi/SiRNAs), Abundance = <1% of total. Micro/Silencing (mi/SiRNAs), Stability = Stable

The processes of DNA and RNA synthesis are similar in that they involve (1) the general steps of initiation, elongation, and termination with 5'-3' polarity; (2) large, multicomponent initiation and polymerization complexes; and (3) adherence to Watson-Crick base-pairing rules. However, DNA and RNA synthesis do differ in several important ways, including the following: (1) ribonucleotides are used in RNA synthesis rather than deoxyribonucleotides; (2) U replaces T as the complementary base for A in RNA; (3) a primer is not involved in RNA synthesis as RNA polymerases have the ability to initiate synthesis de novo; (4) in a given cell only portions of the genome are vigorously transcribed or copied into RNA, whereas the entire genome must be copied, once and only once during DNA replication; and (5) there is no highly active, efficient proofreading function during transcription.
The process of synthesizing RNA from a DNA template has been characterized best in prokaryotes. Although in mammalian cells, the regulation of RNA synthesis and the processing of the RNA transcripts are different from those in prokaryotes, the process of RNA synthesis per se is quite similar in these two classes of organisms. Therefore, the description of RNA synthesis in prokaryotes, where it is best understood, is applicable to eukaryotes even though the enzymes involved and the regulatory signals, though related, are different.

The sequence of ribonucleotides in an RNA molecule is complementary to the sequence of deoxyribonucleotides in one strand of the double-stranded DNA molecule (see Figure 34-8). The strand that is transcribed or copied into an RNA molecule is referred to as the template strand of the DNA. The other DNA strand, the nontemplate strand, is frequently referred to as the coding strand of that gene. It is called this because, with the exception of T for U changes, it corresponds exactly to the sequence of the mRNA primary transcript, which encodes the (protein) product of the gene. In the case of a double-stranded DNA molecule containing many genes, the template strand for each gene will not necessarily be the same strand of the DNA double helix (Figure 36-1). Thus, a given strand of a doublestranded DNA molecule will serve as the template strand for some genes and the coding strand of other genes. Note that the nucleotide sequence of an RNA transcript will be the same (except for U replacing T) as that of the coding strand. The information in the template strand is read out in the 3'-5' direction. Though not shown in Figure 36-1, there are instances of genes embedded within other genes.

DNA-dependent RNA polymerase (RNAP) is the enzyme responsible for the polymerization of ribonucleotides into a sequence complementary to the template strand of the gene
FIGURE 36-1 Genes can be transcribed off both strands of DNA. The arrowheads indicate the direction of transcription (polarity). Note that the template strand is always read in the 3'-5' direction. The opposite strand is called the coding strand because it is identical (except for T for U changes) to the mRNA transcript (the primary transcript in eukaryotic cells) that encodes the protein product of the gene.
FIGURE 36-2 RNA polymerase catalyzes the polymerization of ribonucleotides into an RNA sequence that is complementary to the template strand of the gene. The RNA transcript has the same polarity (5'-3') as the coding strand but contains U rather than T. Bacterial RNAP consists of a core complex of two B subunits (6 and 8') and two a subunits. The holoenzyme contains the o subunit bound to the a, BB' core assembly. The w subunit is not shown. The transcription "bubble" is an approximately 20-bp area of melted DNA, and the entire complex covers 30 to 75 bp of DNA depending on the conformation of RNAP.
(Figures 36-2 and 36-3). The enzyme attaches at a specific site—the promoter—on the DNA template. This is followed by initiation of RNA synthesis at the starting point, andthe process continues until a termination sequence is reached

(Figure 36-3). A transcription unit is defined as that region of DNA that includes the signals for transcription initiation, elongation, and termination. The RNA product, which is synthesized in the 5'-3' direction, is the primary transcript. Transcription frequency varies from gene to gene but can be quite high. An electron micrograph of transcription in action is presented in Figure 36-4. In prokaryotes, this can represent the product of several contiguous genes; in mammalian cells, it usually represents the product _of a single gene. The 5' termini of the primary RNA transcript and the mature cytoplasmic RNA are identical. Thus, the transcription start site (TSS) corresponds to the 5' nucleotide of the mRNA. This is designated position +1, as . . ta: is the corresponding nucleotide in the DNA. The numbers increase positively as the sequence proceeds downstream from the start site. This convention makes it easy to locate particular regions, such as intron and exon boundaries. The nucleotide in the promoter adjacent to the transcription initiation site in the upstream direction is designated -1, and these negative numbers increase as the sequence proceeds upstream, away from the TSS. This +/-numbering system provides a conventional way of defining the location of regulatory elements in a gene (Figure 36-5).
|
—_

FIGURE 36-3 The transcription cycle. The transcription cycle can be described in six steps: (1) Template binding and closed RNA polymerase-promoter complex formation: RNAP binds to DNA and then locates a promoter (P) DNA sequence element, (2) Open promoter complex formation: Once bound to the promoter, RNAP melts the two DNA strands to form an open promoter complex; this complex is also referred to as the preinitiation complex or PIC. Strand separation allows the polymerase to access the coding information in the template strand of DNA (3) Chain initiation: Using the coding information of the template, RNAP catalyzes the coupling of the first base (often a purine) to the second, template-directed ribonucleoside triphosphate to form a dinucleotide (in this example forming the dinucleotide 5' pppApN,,, 3'). (4) Promoter clearance: After RNA chain length reaches ~10 to 20 nt, the polymerase undergoes a conformational change and then is able to move away from the promoter, transcribing down the transcription unit. On many genes o-factor is released from RNAP at this phase of the transcription cycle. (5) Chain elongation: Successive residues are added to the 3'-OH terminus of the nascent RNA molecule until a transcription termination DNA sequence element (T) is encountered. (6) Chain termination and RNAP release: Upon encountering the transcription termination site, RNAP undergoes an additional conformational change that leads to release of the completed RNA chain, the DNA template, and RNAP. RNAP can rebind to DNA beginning the promoter search process and the cycle is repeated. Note that all of the steps in the transcription cycle are facilitated by additional proteins, and indeed are often subjected to regulation by positiveand/or negative-acting factors.

FIGURE 36-4 Schematic representation of an electron photomicrograph of multiple copies of amphibian rRNA-encoding genes in the process of being transcribed. The magnification is about 6000x. Note that the length of the transcripts increases as the RNA polymerase molecules progress along the individual rRNA genes from transcription start sites (filled circles) to transcription termination sites (open circles). RNA polymerase | (not visualized here) is at the base of the nascent rRNA transcripts. Thus, the proximal end of the transcribed gene has short transcripts attached to it, while much longer transcripts are attached to the distal end of the gene. The arrows indicate the direction (5'-43') of transcription.
The primary transcripts generated by RNA polymerase II— one of the three distinct nuclear DNA-dependent RNA polymerases in eukaryotes—are promptly modified by the addition of 7-methylguanosine triphosphate caps (see Figure 34-10), which persist and eventually appear on the 5' end of mature cytoplasmic mRNA. These caps are necessary for the subsequent processing of the primary transcript to mRNA, for the translation of the mRNA, and for protection of the mRNA against nucleolytic attack by 5'-exonucleases.

The basic DNA-dependent RNA polymerase of the bacterium Escherichia coli exists as an approximately 400-kDa core complex consisting of two identical a subunits, two large B and B' subunits, and an w subunit. The B subunit binds Mg** ions and composes the catalytic subunit (Figure 36-2). The core RNA polymerase, B6'a,a, often termed E, associates with a specific protein factor (the sigma [o] factor) to form holoenzyme, 6B'a,o, or Eo. The genes encoding all these proteins are essential for viability with an exception of w-encoding gene. The o subunit enables the core enzyme to recognize and bind the promoter region (Figure 36-5) to form the preinitiation complex (PIC). There are multiple, distinct o-factor encoding genes in all bacterial species. Sigma factors have a dual role in the process of promoter recognition; o association with core RNA polymerase decreases its affinity for nonpromoter DNA, while simultaneously increasing holoenzyme affinity for promoter DNA. The multiple o-factors compete for interaction with limiting core RNA polymerase (ie, E). Each of these unique o-factors act as a regulatory protein that modifies the promoter recognition specificity of the resulting unique RNA polymerase holoenzyme (ie, Eo,, Eo,,...). The appearance of different o-factors and their association with core RNA polymerase to form novel holoenzyme forms Eo,, Eo,,..., can be correlated temporally with various programs of gene expression in prokaryotic systems such as sporulation, growth in various poor nutrient sources, and the response to heat shock.

FIGURE 36-5 Prokaryotic promoters share two regions of highly conserved nucleotide sequence. These regions are located 35and 10-bp upstream of the TSS, which is indicated as +1. By convention, all nucleotides upstream of the transcription initiation site (at +1) are numbered in a negative sense and are referred to as 5'-flanking sequences, while sequences downstream of the +1 TSS are numbered in a positive sense. Also by convention, the promoter DNA regulatory sequence elements such as the —35 and the —10 TATA elements are described in the 5'3' direction and as being on the coding strand. These elements function only in double-stranded DNA. Other transcriptional regulatory elements, however, can often act in a direction independent fashion, and such cis-elements are drawn accordingly in any schematic (Figure 36-8). Note that the transcript produced from this transcription unit has the same polarity or "sense" (ie, 5'->3' orientation) as the coding strand. Termination cis-elements reside at the end of the transcription unit (see Figure 36-6 for more detail). By convention, the sequences downstream of the site at which transcription termination occurs are termed 3'-flanking sequences.

I, Sensitivity to α-Amanitin = Insensitive. I, Major Products = rRNA. II, Sensitivity to α-Amanitin = High sensitivity. II, Major Products = mRNA, lncRNA, miRNA, snRNA. III, Sensitivity to α-Amanitin = Intermediate sensitivity. III, Major Products = tRNA, 5s rRNA, some snRNAs

Some of the distinguishing properties of mammalian nuclear RNA polymerases are described in Table 36-2. Each of these DNA-dependent RNA polymerases is responsible for transcription of different sets of genes. The sizes of the RNA polymerases range from MW 500 to 600 kDa. These enzymes exhibit more complex subunit profiles than prokaryotic RNA polymerases. They all have two large subunits, which remarkably bear strong sequence and structural similarities to prokaryotic B and f' subunits, and a number of smaller subunits—as many as 14 in the case of RNA pol III. The functions of each of the subunits are not yet fully understood. A peptide toxin from the mushroom Amanita phalloides, a-amanitin, is a specific differential inhibitor of the eukaryotic nuclear DNA-dependent RNA polymerases and as such has proved to be a powerful research tool (Table 36-2). a-Amanitin blocks the translocation of RNA polymerase during phosphodiester bond formation.

The process of RNA synthesis in bacteria—depicted in Figure 36-3—is cyclical and involves multiple steps. First RNA polymerase holoenzyme (Eo) must locate and then specifically bind a promoter (P; Figure 36-3). Once the promoter is located, the Eo-promoter DNA complex undergoes a temperature-dependent conformational change and unwinds, or melts the DNA in and around the transcription start site (at +1). This complex is termed the PIC. Unwinding allows the active site of the Eo to access the template strand, which of course dictates the sequence of ribonucleotides to be polymerized into RNA. The first nucleotide (typically, though not always a purine) then associates with the nucleotide-binding site of the enzyme, and in the presence of the next appropriate nucleotide bound to the polymerase, RNAP catalyzes the formation of the first phosphodiester bond, and the nascent chain is now attached to the polymerization site on the B subunit of RNAP. This reaction is termed initiation. The analogy to the A and P sites on the ribosome should be noted; see Figure 37-9. The nascent dinucleotide retains the 5'-triphosphate of the initiating nucleotide (Figure 36-3, ATP).

RNA polymerase continues to incorporate nucleotides +3 to ~+10, at which point the polymerase undergoes another conformational change and moves away from the promoter; this reaction is termed promoter clearance. On many genes the o-factor dissocates from the Bf'a, assembly at this point. The elongation phase then commences, and here the nascent RNA molecule grows 5'-3' as consecutive NTP incorporation steps continue cyclically, antiparallel to the template. The enzyme polymerizes the ribonucleotides in the specific sequence dictated by the template strand and interpreted by Watson-Crick base-pairing rules. Pyrophosphate (PP,) is released following each cycle of polymerization. As for DNA synthesis, this PP, is rapidly degraded to two molecules of inorganic phosphate (P,) by ubiquitous pyrophosphatases, thereby providing irreversibility on the overall synthetic reaction. The decision to stay at the promoter in a poised or stalled state, or transition to elongation can be an important regulatory step in both prokaryotic and eukaryotic mRNA gene transcription.
As the elongation complex containing RNA polymerase progresses along the DNA molecule, DNA unwinding must occur in order to provide access for the appropriate base pairing to the nucleotides of the coding strand. The extent of this transcription bubble (ie, DNA unwinding) is constant throughout transcription and has been estimated to be about 20 bp per polymerase molecule (Figure 36-2). Thus, the size of the unwound DNA region is dictated by the polymerase and is independent of the DNA sequence in the complex. RNA polymerase has an intrinsic "unwindase" activity that opens the DNA helix (ie, see PIC formation above). The fact that the DNA double helix must unwind, and the strands part at least transiently for transcription implies some temporary disruption of the nucleosome structure of eukaryotic cells. Topoisomerase both precedes and follows the progressing RNA polymerase to prevent the formation of superhelical tension that would serve to increase the energy required to unwind the template DNA ahead of RNAP.

Termination of the synthesis of RNA in bacteria is signaled by sequences in the template DNA (Figure 36-3; T) and sequences within the transcript. On many genes RNAP alone efficiently terminates transcription. However, on a subset of genes a termination protein termed rho (p) factor is required to mediate transcription termination. After termination both free core RNAP (E) and product RNA dissociate from the DNA template. The resulting free core RNAP (E) is able to associate with s-factor to reform Es, and re-enter the transcription cycle. In eukaryotic cells, termination is less well understood; however, the proteins catalyzing RNA processing, termination, and polyadenylation all appear to load onto RNA polymerase II soon after initiation. More than one RNA polymerase molecule may transcribe the same template
strand of a gene simultaneously, but the process is phased and spaced in such a way that at any one moment each is transcribing a different portion of the DNA sequence (Figures 36-1 and 36-4).

Analysis of the DNA sequence of specific genes has allowed the recognition of a number of sequences important in gene transcription. From the large number of bacterial genes studied, it is possible to construct consensus models of transcription initiation and termination signals.
The question, "How does RNAP find the correct site to initiate transcription?" is not trivial when the complexity of the genome is considered. E. coli has about 4 x 10° transcription initiation sites (ie, gene promoters) within the 4.2 x 10° bp genome. The situation is even more complex in humans, where as many as 150,000 distinct transcription initiation sites are distributed throughout 3 x 10° bp of DNA. Bacterial RNAP can bind, with low affinity, to many regions of DNA, but it scans the DNA sequence—at a rate of 210° bp/s—until it recognizes certain specific regions of DNA to which it binds with higher affinity. These regions are termed promoters, and it is the base-specific association of RNAP with promoters that ensures accurate initiation of transcription. The promoter recognition-utilization process is the target for regulation in both bacteria and humans.

Bacterial promoters are approximately 40 nucleotides (40 bp or four turns of the DNA double helix) in length, a region small enough to be covered by an E. coli RNA holopolymerase molecule. In a consensus promoter, there are two short conserved sequence elements. Approximately 35-bp upstream of the transcription start site there is a consensus sequence of eight nucleotide pairs (consensus: 5'-TGTTGACA-3') to which the RNAP binds to form the so-called closed complex. More proximal to the transcription start site—about 10 nucleotides upstream—is a six-nucleotide-pair A+T-rich sequence (consensus: 5'-TATAAT-3'). These conserved sequence elements together comprise the promoter, and are shown schematically in Figure 36-5. The latter sequence has a lower melting temperature because of its lack of GC nucleotide pairs. Thus, the so-called "TATA box" is thought to ease the dissociation of the two DNA strands so that RNA polymerase bound to the promoter region can have access to the nucleotide sequence of its immediately downstream template strand. Once the process of strand separation occurs, the combination of RNA polymerase plus promoter is called the open complex. Other bacteria have slightly different consensus sequences in their promoters, but all generally have two components to the promoter; these tend to be in the same position relative to the TSS, and in all cases the sequences between the two promoter elements have no similarity but still provide critical spacing functions that facilitate recognition of -35 and -10 sequences by the cognate RNA polymerase holoenzyme. Within a bacterial cell, different sets of genes are often coordinately regulated. One important way that this is accomplished is through the fact that these coregulated genes share particular -35 and -10 promoter sequences. These unique sets of promoters are recognized by different o-factors bound to core RNA polymerase (ie, Eo,, Eo,,...).
Bacterial RNAP alone has the intrinsic ability to specifically terminate transcription on about 50% of cellular genes. On the remaining bacterial genes the accessory p termination factor is required. Proposed mechanisms for p-independent and p-dependent transcription termination events are presented in Figure 36-6. The majority of eukaryotic mRNA gene transcription events are accessory transcription factor dependent.

As discussed in detail in Chapter 38, bacterial gene transcription is controlled through the action of repressor and activator proteins. These proteins typically bind to unique and specific DNA sequences that lie adjacent to promoters. These repressors and activators affect the ability of the RNA polymerase to bind promoter DNA and/or form open complexes. The net effect is to stimulate or inhibit PIC formation and transcription initiation—consequently blocking or enhancing specific RNA synthesis.

There are two types of TSS proximal signals in DNA that control transcription in eukaryotic cells. One of these, the promoter, defines where transcription is to initiation transcription on the DNA template, while the other, DNA elements that stimulate and repress transcription contribute to the mechanisms that control how frequently transcription is to occur. For example, in the thymidine kinase (tk) gene of the herpes simplex virus (HSV), which utilizes transcription factors of its mammalian host for its early gene expression program, there is a single unique TSS, and accurate transcription initiation from this site depends on a nucleotide sequence located about 25 nucleotides upstream from the start site (ie, at -25) (Figure 36-7). This region has the sequence of TATAAAAG and bears remarkable similarity to the functionally related TATA box that is located about 10-bp upstream from the prokaryotic mRNA TSS (Figure 36-5). Mutation of the TATA box markedly reduces transcription of the HSV tk gene, and many other cellular genes that contain this consensus cis-active element (Figures 36-7 and 36-8). The TATA box is usually located 25to 30-bp upstream from the transcription start site in mammalian genes that contain it. The consensus sequence for a TATA box is TATAAA, though numerous variations have been characterized. The human TATA box is bound by the 34-kDa TATA-binding protein (TBP), a subunit in at
least two multisubunit complexes, TFIID and SAGA/P-CAEF. The non-TBP subunits of TFIID are proteins called TBPassociated factors (TAFs). Binding of the TBP-TAF TFIID complex to the TATA box sequence is thought to represent a first step in the formation of the transcription complex on the promoter.
A large number of eukaryotic mRNA-encoding genes lack a consensus TATA box. In such instances, additional DNA cis-elements, an initiator sequence (Inr) and/or the downstream promoter element (DPE), direct the RNA polymerase

II transcription machinery to the promoter and serve to direct RNA pol II to start transcription from the correct site. The Inr element spans the start site (from -3 to +5) and consists of the general consensus sequence TCA"! G/T T 1/c (A*' indicates the first nucleotide transcribed, ie, TSS). The proteins that bind to Inr in order to direct pol II binding include TFIID. Promoters that have both a TATA box and an Inr may be "stronger, or more frequently transcribed, than those that have just one of these elements. The DPE has the consensus sequence A/GGa/TCGTG and is localized about 25-bp downstream of
FIGURE 36-7 Transcription elements and binding factors in the herpes simplex virus thymidine kinase (tk) gene. DNA-dependent RNA polymerase II (not shown) binds to the region encompassing the TATA box (which is shown here bound by transcription factor TFIID) and TSS at +1 (see also Figure 36-9) to form a multicomponent PIC capable of initiating transcription at a single nucleotide (+1 TSS). The frequency of this event is increased by the presence of upstream cis-acting elements (the GC and CAAT boxes) located either near to the promoter (promoter proximal) or distant from the promoter (distal elements; see Figure 36-8). Proximal and distal DNA cis-elements are bound by trans-acting transcriptional activating factors, in this example Sp1 and CTF (also called C/EBP, NF1, NFY). These cis-elements can function independently of orientation (arrows).

the +1 TSS. Like the Inr, DPE sequences are also bound by the TAF subunits of TFIID. In a survey of hundreds of thousands of eukaryotic protein coding genes, roughly 30% contained a TATA box and Inr, 25% contained Inr and DPE, 15% contained all three elements, whereas ~30% contained just the Inr. Sequences generally, though not always, just upstream from the start site contribute importantly to how frequently transcription occurs. Not surprisingly, mutations in these regions reduce the frequency of transcription initiation 10-fold to 20-fold. Typical of these DNA elements are the GC and CAAT boxes, so named because of the DNA sequences involved. As illustrated in Figure 36-7, each of these DNA elements are bound by a specific protein, Sp1 in the case of the
GC box and CTF by the CAAT box; both bind through their distinct DNA-binding domains (DBDs). The frequency of transcription initiation is a consequence of these protein-DNA interactions and complex interactions between particular domains of the transcription factors (distinct from the DBD domains—so-called activation domains; ADs) and the rest of the transcription machinery (RNA polymerase II, the basal, or general factors, GIFs, TFIIA, B, D, E, KE, H and other coregulatory factors such as mediator, chromatin remodelers, and chromatin modifying factors). (See Figures 36-9 and 36-10.) The protein-DNA interactions at the TATA box involving RNA polymerase II and other components of the basal transcription machinery ensures the fidelity of initiation.

FIGURE 36-8 Schematic showing the transcription control regions in a hypothetical mRNA-producing eukaryotic gene transcribed by RNA polymerase II. Such a gene can be divided into its coding and regulatory regions, as defined by the transcription start site (arrow; +1). The coding region contains the DNA sequence that is transcribed into mRNA, which is ultimately translated into protein, typically after extensive mRNA processing via splicing (Figures 36-12 to 36-16). The regulatory region consists of two classes of elements. One is responsible for ensuring basal expression. The "promoter,' often composed of the TATA box and/or Inr and/or DPE elements (see Table 36-3), directs the RNA polymerase Il transcription machinery to the correct site (fidelity). However, in certain genes that lack a consensus TATA, the so-called TATA-less promoters, an initiator (Inr) and/or DPE elements may direct the polymerase to this site. Another component, the upstream elements, specifies the frequency of initiation; such elements can either be proximal (50-200 bp) or distal (1000-10° bp) to the promoter as shown. Among the best studied of the proximal elements is the CAAT box, but several other elements (bound by the transactivator proteins Sp1, NF1, AP1, etc; Table 36-3) may be used in various genes. The distal elements enhance or repress expression, several of which mediate the response to various signals, including hormones, heat shock, heavy metals, and chemicals. Tissue-specific expression also involves specific sequences of this sort. The orientation dependence of all the elements is indicated by the arrows within the boxes. For example, the proximal promoter elements (TATA box, INR, DPE) must be in the 5'-3' orientation, while the proximal upstream elements often work best in the 5'—>3' orientation, most can be reversed. The locations of some elements are not fixed with respect to the transcription start site. Indeed, some elements responsible for regulated expression can be located interspersed with the upstream elements or can be located downstream from the start site, within, or even downstream of the regulated gene itself.
TSS

FIGURE 36-9 The eukaryotic basal transcription complex. Formation of the basal transcription complex begins when TFIID binds, via its TATA binding protein (TBP) subunit and several of its 14 TBP-associated factor (TAF) subunits, to the TATA box. TFIID then directs the assembly of several other components by protein-DNA and protein-protein interactions: TFIIA, B, E, F, H, and polymerase II (pol Il). The entire complex spans DNA from about positions —30 to +30 relative to the TSS at +1 (marked by bent arrow). The atomic level, X-ray—derived structures of RNA polymerase II alone and of the TBP subunit of TFIID bound to TATA promoter DNA in the presence of either TFIIB or TFIIA have all been solved at 3-A resolution. The structures of mammalian and yeast PICs have also recently been determined at 10-A resolution by electron microscopy. Thus, the molecular structures of the transcription machinery in action are beginning to be elucidated. Much of this structural information is consistent with the models presented here.

Together, the promoter plus promoter-proximal cis-active upstream elements confer fidelity and modulate the frequency of initiation upon a gene respectively. The TATA box has a particularly rigid requirement for both position and orientation. As with bacterial promoters, single-base changes in any of these cis-elements can have dramatic effects on function by reducing the binding affinity of the cognate trans-factors (either TFIID/TBP or Spl, CTE, and similar factors). The spacing of the TATA box, Inr, and DPE is also critical. A third class of elements also increase decrease that the termination signals exist far downstream of the protein coding sequence of eukaryotic genes. For example, the transcription termination signal for mouse B-globin occurs at several positions 1000 to 2000 bases beyond the site at which the mRNA poly(A) tail will eventually be added. Less is known about the termination process or whether specific termination factors similar to the bacterial p-factor might be involved. However, it is known that formation of the mRNA 3' terminus, which is generated posttranscriptionally, is somehow coupled to events or structures formed at the time and site of initiation. Moreover, mRNA formation, and in this case mRNA 3'-end formation depends on a special structure present on the C-terminus of the largest subunit of RNA polymerase II, the carboxy-terminal domain, or CTD, and this process appears to involve at least two steps as follows. After RNA polymerase II has traversed the region of the transcription unit encoding the 3' end of the transcript, RNA endonucleases cleave the primary transcript at a position about 15 bases 3' of the consensus sequence AAUAAA that serves in eukaryotic transcripts as a cleavage and polyadenylation signal. Finally, this newly formed 3' terminal is polyadenylated in the nucleoplasm, as described below.

A complex apparatus consisting of as many as 50 unique proteins provides accurate and regulatable transcription of eukaryotic genes. The RNA polymerase enzymes (pol I, pol II, and pol III) transcribe information contained in the template strand of DNA into RNA. These polymerases must recognize a specific site in the promoter in order to initiate transcription at the proper nucleotide. In contrast to the situation in prokaryotes though, eukaryotic RNA polymerases alone are not able to discriminate between promoter sequences and other, nonpromoter regions of DNA in the test tube. All eukaryotic RNA
sequence or the rate of transcription of eukaryotic genes. These elements are called either enhancers or repressors (or silencers), depending on how they effect transcription. They have been found in a variety of locations, both upstream and downstream of the transcription start site, and even within the transcribed protein coding portions of some genes. Enhancers and silencers can exert their effects when located thousands or even many tens of thousands of bases away from transcription units located on the same chromosome. Surprisingly, enhancers and silencers can function in an orientation-independent fashion. Literally, hundreds of these elements have been described. In some cases, the sequence requirements for binding are rigidly constrained; in others, considerable sequence variation is allowed. Some sequences bind only a single protein; however, the majority of these regulatory sequences are bound by several different proteins. Together, these many transfactors binding to promoter distal and proximal cis-elements, regulate transcription in response to a vast array of biologic signals. Such transcriptional regulatory events contribute importantly to control of gene expression.

The signals for the termination of transcription by eukaryotic RNA polymerase II are only poorly understood. It appears

FIGURE 36-10 Nucleosome covalent modifications, remodeling, and eviction by chromatin-active coregulators modulate PIC formation and transcription. Shown in (A), is an inactive mRNA encoding gene (see X over TSS) with a single dimeric transcription factor (Activator-1; violet ovals) bound to its cognate enhancer binding site (Activator-1). This particular enhancer element was nucleosome-free and hence available for interaction with its cognate activator binding protein. However, this gene is still inactive (X over TSS) due to the fact that a portion of its enhancer (in this illustration the enhancer is bipartite and composed of Activator-1 and Activator-2, DNA-binding sites) and the promoter are covered by nucleosomes. Recall that nucleosomes have ~150 bp of DNA wound around the histone octamer. Hence, the single nucleosome over the promoter will occlude access of the transcription machinery (pol Il + GTFs) to the TATA, Inr and/or DPE promoter elements. (B) Enhancer DNA-bound Activator-1 interacts with any of a number of distinct ATP-dependent chromatin remodelers and chromatin-modifying coregulator complexes. These coregulators together have the ability to both move, or remodel (ie, change the octameric histone content, and/or remove nucleosomes) through the action of various ATP-dependent remodelers as well as to covalently modify nucleosomal histones using intrinsic acetylases (HAT; resulting in acetylation [Ac]) and methylases (SET; resulting in methylation [Me], among other PTMs; Table 35-1) carried by subunits of these complexes. (C) The resulting changes in nucleosome position and nucleosome occupancy (ie, nucleosomes -4, 0 and +1), composition (nucleosome —1 and nucleosome +2; replacement of nucleosomal H2A with histone H2AX[Z]) thus allows for the binding of the second Activator-2 dimer to Activator-2 DNA sequences, which ultimately leads to the binding of the transcription machinery (TFIIA, B, D, E, F, H; polymerase Il and mediator) to the promoter

(TATA-INR-DPE) and the formation of an active PIC, which leads to activated transcription
(large arrow
at
TSS).
polymerases require other proteins known as general transcription factors or GTFs in order to catalyze specific transcription. RNA polymerase II requires TFIIA, B, D (or TBP), E, FE and H to both facilitate promoter-specific binding of the enzyme and formation of the PIC. RNA polymerases I and III require their own polymerase-specific GTFs. Within the cell the transcription machinery (RNA polymerase II and GTFs) and activator, proteins interact with another set of proteins— the coactivators (also known as coregulators). Coregulators bridge between enhancer DNA-bound activator proteins and the transcription machinery, and in so doing regulate the rate of transcription.

In bacteria, a o-factor-polymerase holoenzyme complex, Eo, selectively and directly binds to promoter DNA to form the PIC. The situation is much more complex in eukaryotic genes. mRNA-encoding genes, which are transcribed by pol II, are described as an example. In the case of pol II-transcribed genes, the function of o-factors is assumed by a number of proteins. PIC formation requires pol II and the six GTFs (TFHA, TFIIB, TFIID, TFIE, TFIIF, TFIIH). These GTFs serve to promote RNA polymerase II transcription on essentially all genes. Some of these GTFs are composed of multiple subunits. The 15-subunit TFIID complex (TFIID consists of 15 subunits, TBP, and 13-14 TBP TAFs), which binds to the TATA box promoter element through its TBP and TAF subunits. TFIID is the only GTF that is independently capable of specific, high-affinity binding to promoter DNA.
TBP binds to the TATA box in the minor groove of DNA (most transcription factors bind in the major groove) and causes an approximately 100° bend or kink of the DNA helix. This bending is thought to facilitate the interaction of TAFs with other components of the transcription initiation complex, the multicomponent eukaryotic promoter, and possibly with factors bound to upstream elements. Although initially defined as a component solely required for transcription of pol II gene promoters, TBP, by virtue of its association with distinct, polymerase-specific sets of TAFs, is also an important component of pol I and pol III transcription initiation complexes even if they do not contain TATA boxes.

The binding of TFIID marks a specific promoter for transcription. Of several subsequent in vitro steps, the first is the binding of TFIIA, then TFIIB to the TFIID-promoter complex. This results in a stable multiprotein-DNA complex, which is more precisely located and more tightly bound at the transcription initiation site. This complex attracts and tethers the pol II and TFIIF complex to the promoter. Addition of TFIIE and TFIIH are the final steps in the assembly of the PIC. Each of these binding events extends the size of the complex so that finally about 60 bp (from -30 to +30 relative to the +1 TSS) are covered (Figure 36-9). The PIC is now complete and capable of basal transcription initiated from the correct nucleotide. In genes that lack a TATA box, the same factors are required. In such cases, the Inr and/or DPE serve to position the complex for accurate initiation of transcription (see Figure 36-8).

On certain eukaryotic genes, the transcription machinery (pol II, etc) cannot access promoter sequences (ie, TATA-INRDPE) because these essential promoter elements are wrapped up in nucleosomes (see Figures 35-2, 35-3, and 36-10). Only after transcription factors bind to enhancer DNA upstream of the promoter and recruit chromatin remodeling and modifying coregulatory factors, such as the Swi/Snf, SRC-1, p300/ CBP (see Chapter 42), P/CAF or other factors, are the repressing nucleosomes removed (Figure 36-10). Once the promoter is "open" following nucleosome eviction, GTFs and RNA polymerase II can bind and initiate mRNA gene transcription. Note that the binding of transactivators and coregulators can be sensitive to, and/or directly control the composition and/ or covalent modification status of the DNA and the histones within the nucleosomes in and around the promoter and enhancer, and thereby increase or decrease the ability of all the other components required for PIC formation to interact with a particular gene. This so-called epigenetic code of DNA, histone and protein modifications can contribute importantly to gene transcription control. Indeed, mutations in proteins that catalyze (code writers), remove (code erasers), or differentially bind (code readers) modified DNA and/or histones can lead to human disease.

Eukaryotic pol II consists of 12 subunits. As noted above, the two largest subunits of pol II (MW 220 and 150 kDa) are homologous to the bacterial 6' and 6 subunits. In addition to the increased number of subunits, eukaryotic pol II differs from its prokaryotic counterpart in that it has a series of heptad repeats with consensus sequence Tyr-Ser-ProThr-Ser-Pro-Ser at the carboxyl terminus of the largest pol II subunit, the so-called CTD or C-terminal Domain. This CTD has 26 repeated units in yeast and 52 units in mammals. The CTD is a substrate for several enzymes (kinases, phosphatases, prolyl isomerases, glycosylases); phosphorylation of the CTD was the first CTD PTM discovered. Among other proteins the kinase subunit of TFITH can modify the CTD. Covalently modified CTD is the binding site, or platform, for a wide array of proteins, and it has been shown to interact with many mRNA modifying and processing enzymes and nuclear transport proteins. The association of these factors with the CTD of RNA polymerase II (and other components of the basal machinery) thus serves to couple transcription initiation with mRNA capping, splicing, 3'-end formation, and transport to the cytoplasm. Pol II polymerization
is activated when phosphorylated on the Ser and Thr residues and displays reduced activity when the CTD is dephosphorylated. CTD phosphorylation/dephosphorylation is critical for promoter clearance, elongation, termination, and even appropriate mRNA processing. Pol II lacking the CTD tail is incapable of activating transcription, in vitro and cells expressing pol II lacking the CTD are inviable. These results underscore the importance of this domain to mRNA biogenesis.
Pol II can associate with other proteins termed mediator or Med proteins to form a complex sometimes referred to as the pol II holoenzyme; this complex can form on the promoter or in solution prior to PIC formation. The Med proteins (over 30 proteins; Med1-Med31) are essential for appropriate regulation of pol II transcription by serving myriad roles, both activating and repressing transcription. Thus, mediator, like TFIID is a transcriptional coregulator (Figure 36-11).

TFIID was originally considered to be a single protein, TBP. However, several pieces of evidence led to the discovery that TFIID is actually a complex consisting of TBP and the 14 TAFs. The first evidence that TFIID was more complex than just the TBP molecules came from the observation that TBP binds to a 10-bp segment of DNA, immediately over the TATA box of the gene, whereas native holo-TFIID covers a 35-bp or larger region (Figure 36-9). Second, purified recombinant E. coli-expressed TBP has a molecular mass of 20 to 40 kDa (depending on the species), whereas the native TFIID complex has a mass of about 1000 kDa. Finally, and perhaps most importantly, TBP supports basal transcription but not the augmented transcription provided by certain activators, for example, Sp1 bound to the GC box. TFIID, on the other hand, supports both basal and enhanced transcription by Sp1, Oct1, AP1, CTE, ATE etc. (Table 36-3). The TAFs are essential for this activator-enhanced transcription. There are likely several forms of TFIID in metazoans that differ slightly in their complement of TAFs. Thus, different combinations of TAFs with TBP—or one of several recently discovered TBP-like factors (TLFs)—bind to different promoters, and recent reports suggest that this may account for the tissueor cell-selective gene activation noted in various promoters and for the different strengths of certain promoters. TAFs, since they are required for the action of activators, are often called coactivators or coregulators. There are thus three classes of transcription factors involved in the regulation of pol II genes: pol II and GTFs, coregulators, and DNA-binding activator or repressor proteins (Table 36-4). How these classes of proteins interact to govern both the site and frequency of transcription is a question of central importance and active investigation. It is currently thought that coregulators both act as a bridge between the DNA-binding transactivators and pol II/GTFs, and act to modify chromatin.

FIGURE 36-11 Models for the formation of an RNA polymerase II preinitiation complex. Shown at top is a typical MRNA gene transcription unit: enhancer-promoter (TATA)-TSS (bent arrow) and ORF (open reading frame; sequences encoding the protein) within the transcribed region. PICs have been shown to form by at least two distinct mechanisms in vitro: (A) the stepwise binding of GTFs, pol Il, and mediator (Med), or (B) by the binding of a single multiprotein complex composed of pol Il, Med, and the six GTFs. DNA-binding transactivator proteins specifically bind enhancers and in part facilitate PIC formation (or PIC function) by binding directly to the TFIID-TAF subunits, or Med subunits of mediator (not shown, see Figure 36-10), or other components of the transcription machinery. The molecular mechanism(s) by which such protein-protein interactions stimulate transcription remain a subject of intense investigation.

The formation of the PIC described above is based on the sequential addition of purified components as observed through in vitro experiments. An essential feature of this model is that PIC assembly takes place on a DNA template where the transcription proteins all have ready access to DNA. Accordingly, transcription activators, which have autonomous DNA binding and activation domains (DBDs and ADs; see Chapter 38), are thought to function by stimulating PIC formation. Here the TAF or mediator complexes are viewed as bridging factors that communicate between the upstream-bound

TATA box, Consensus Sequence = TATAAA. TATA box, Factor = TBP/TFIID. Inr, Consensus Sequence = t / ct / c AN t / at / ct / c. Inr, Factor = TFIID. DPE, Consensus Sequence = a / g G a / t CGTG. DPE, Factor = TFIID. CAAT box, Consensus Sequence = CCAATC. CAAT box, Factor = C/EBP*, NF-Y*. GC box, Consensus Sequence = GGGCGG. GC box, Factor = Sp1*. E-box, Consensus Sequence = CAACTGAC. E-box, Factor = Myo D. κB motif, Consensus Sequence = gggactttcc. κB motif, Factor = NF-κB. Ig octamer, Consensus Sequence = ATGCAAAT. Ig octamer, Factor = Oct1, 2, 4, 6*. AP1, Consensus Sequence = TGA g / c TC/AA. AP1, Factor = Jun, Fos, ATF*. Serum response, Consensus Sequence = GATGCCCATA. Serum response, Factor = SRF. Heat shock, Consensus Sequence = (NGAAN) 3. Heat shock, Factor = HSF
Note: All elements listed are written 5' to 3' and only the top strand of the duplex element is shown. A complete list would include hundreds of examples. The asterisks signify that there are several members of this family. Nucleotides separated by a / indicate that either of two nucleotides can be at that position (ie, T/C in the Inr first position implies that either T or C can occupy that position; N implies any of the four DNA bases A, G, C, or T can occupy that particular position in the indicated cis-element.
activators, and the GTFs and pol II. This view assumes that there is stepwise assembly of the PIC—promoted by various interactions between activators, coactivators, and PIC components, and is illustrated in panel A of Figure 36-11. This model is supported by observations that many of these proteins can indeed bind to one another in vitro.

Recent evidence suggests that there is another possible mechanism of PIC formation and thus transcription regulation. First, large preassembled complexes of GTFs and pol II are found in cell extracts, and these complexes can associate with the promoter in a single step. Second, the rate of transcription achieved when activators are added to limiting concentrations of pol II holoenzyme can be matched by atificially increasing the concentration of pol II and GTFs

Basal components, Specific Components = RNA polymerase II, TBP, TFIIA, B, D, E, F, and H. Coregulators, Specific Components = TAFs (TBP +TAFs) =TFIID; certain genes. , Specific Components = Mediator, Meds. , Specific Components = Chromatin modifiers (pCAF, p300/CBP). , Specific Components = Chromatin remodelers (Swi/Snf). Activators, Specific Components = SP1, ATF, CTF, AP1, etc
in the absence of activators. Thus, at least in vitro, one can establish conditions where activators are not in themselves absolutely essential for PIC formation. These observations led to the "recruitment" hypothesis, which has now been tested experimentally. Simply stated, the role of activators and some coactivators may be solely to recruit a preformed holoenzyme-GTF complex to the promoter. The requirement for an activation domain is circumvented when either a component of TFIID or the pol II holoenzyme is artificially tethered, using recombinant DNA techniques, to the DBD of an activator. This anchoring, through the DBD component of the activator molecule, leads to a transcriptionally competent structure, and there is no further requirement for the activation domain of the activator. In this view, the role of activation domains is to direct preformed holoenzyme-GTF complexes to the promoter; they do not assist in PIC assembly (see panel B, Figure 36-11). In this model, the efficiency of the recruitment process directly determines the rate of transcription at a given promoter.

In prokaryotic organisms, the primary transcripts of mRNAencoding genes begin to serve as translation templates even before their transcription has been completed. This can occur because the site of transcription is not compartmentalized into the nucleus as it is in eukaryotic organisms. Thus, transcription and translation are coupled in prokaryotic cells. Consequently, prokaryotic mRNAs are subjected to little processing prior to carrying out their intended function in protein synthesis. Indeed, appropriate regulation of some genes (eg, the Trp operon) relies on this coupling of transcription and translation. Prokaryotic rRNA and tRNA molecules are transcribed in units considerably longer than the ultimate molecule. In fact, many of the tRNA transcription units encode more than one tRNA molecule. Thus, in prokaryotes, the processing of these rRNA and tRNA precursor molecules is required for the generation of the mature functional molecules.
Nearly all eukaryotic RNA primary transcripts undergo extensive processing between the time they are synthesized and the time at which they serve their ultimate function, whether it be as mRNA, miRNAs, or as a component of the translation machinery such as rRNA or tRNA. Processing occurs primarily within the nucleus. The processes of transcription, RNA processing, and even RNA transport from the nucleus are highly coordinated. Indeed, a transcriptional coactivator termed SAGA in yeasts and P/CAF in human cells is thought to link transcription activation to RNA processing by recruiting a second complex termed transcription export (TREX) to transcription elongation, splicing, and nuclear export. TREX represents a likely molecular link between transcription elongation complexes, the RNA

FIGURE 36-12 RNA polymerase II-mediated mRNA gene transcription is cotranscriptionally coupled to RNA processing and transport. Shown is RNA pol II actively transcribing an mRNA encoding gene (elongation top to bottom of figure). RNA processing factors (ie, SR/RRM-motif-containing splicing factors as well as polyadenylation and termination factors) interact with the C-terminal domain (CTD, composed of multiple copies of a heptapeptide with consensus sequence -YSPTSPS-) of pol Il, while mRNA packaging and transport factors such as THO/TREX complex (pink ovals) are recruited to the nascent mRNA primary transcript either through direct pol Il interactions as shown or through interactions with SR/splicing factors (brown circles) resident on the nascent mRNA. Note that the CTD is not drawn to scale. The evolutionarily conserved CTD of the Rpb1 subunit of pol Il is in reality 5 to 10 times the length of the polymerase due to its many prolines and consequent unstructured nature, and thus a significant docking site for RNA processing and transport proteins. In both cases, nascent mRNA chains are thought to be more rapidly and accurately processed due to the immediate recruitment of these many factors to the growing mRNA (precursor) chain. Following appropriate mRNA processing, the mature mRNA is delivered to the nuclear pores (Figures 36-17, 49-4) dotting the nuclear membrane, where, upon transport through the pores, the mRNAs can be engaged by ribosomes and translated into protein.
splicing machinery, and nuclear export (Figure 36-12). This coupling presumably dramatically increases both the fidelity and rate of processing and movement of mRNA to the cytoplasm for translation.

The RNA sequences that appear in mature RNAs are termed exons. In mRNA encoding genes these exons are often interrupted by long sequences of DNA that neither appear in mature mRNA, nor contribute to the genetic information ultimately translated into the amino acid sequence of a protein molecule (see Chapter 35). In fact, these sequences often interrupt the coding region of protein-encoding genes. These intervening sequences, or introns, exist within most but not all mRNA encoding genes of higher eukaryotes. Human mRNAencoding gene exons average ~150 nt, while introns are much more heterogeneous, ranging from 10 to 30,000 nucleotides in length. The intron RNA sequences are cleaved out of the transcript, and the exons of the transcript are appropriately spliced together in the nucleus before the resulting mRNA molecule is transported to the cytoplasm for translation (Figures 36-13 and 36-14).
FIGURE 36-13 The processing of the primary transcript to mRNA. In this hypothetical transcript, the 5' (left) end of the intron is cut (>) and a structure resembling a lariat forms between the G at the 5' end of the intron and an A near the 3' end, in the consensus sequence UACUAAC. This sequence is called the branch site, and it is the 3' most A that forms the 5'-2' bond with the G. The 3' (right) end of the intron is then cut (1). This releases the lariat, which is digested, and exon 1 is joined to exon 2 at G residues.
Consensus
FIGURE 36-14 Consensus sequences at splice junctions. The 5' (donor; left) and 3' (acceptor; right) sequences are shown. Also shown is the yeast consensus sequence (UACUAAC) for the branch site. In mammalian cells, this consensus sequence is PyNPyPyPuAPy, where Py is a pyrimidine, Pu is a purine, and N is any nucleotide. The branch site is located 20 to 40 nucleotides upstream from the 3'-splice site.

Several different splicing reaction mechanisms for intron removal have been described. The one most frequently used in eukaryotic cells is described below. Although the sequences of nucleotides in the introns of the various eukaryotic transcripts—and even those within a single transcript— are quite heterogeneous, there are reasonably conserved sequences at each of the two exons-intron (splice) junctions and at the branch site, which is located 20 to 40 nucleotides upstream from the 3'-splice site (see consensus sequences in Figure 36-14). A special multicomponent complex, the spliceosome, is involved in converting the primary transcript into mRNA. Spliceosomes consist of the primary transcript, five snRNAs (U1, U2, U4, U5, and U6), and more than 60 proteins, many of which contain conserved RRM (RNA recognition) and SR (serine-arginine) protein motifs. Collectively, the five snaRNAs and RRM/SR-containing proteins form a small nuclear ribonucleoprotein termed an snRNP complex. It is likely that this penta-snRNP spliceosome forms prior to interaction with mRNA precursors. snRNPs are thought to position the exon and intron RNA segments for the necessary splicing reactions. The splicing reaction starts with a cut at the junction of the 5' exon (donor on left) and intron (Figure 36-13). This is accomplished by a nucleophilic attack by an adenylyl residue in the branch point sequence located just upstream from the 3' end of this intron. The free 5' terminus then forms a loop or lariat structure that is linked by an unusual 5'-2' phosphodiester bond to the reactive A in the PyYNPyPyPuA Py branch site sequence (Figure 36-14). This adenylyl residue is typically located 20 to 30 nucleotides upstream from the 3' end of the intron being removed. The branch site identifies the 3'-splice site. A second cut is made at the junction of the intron with the 3' exon (donor on right). In this second transesterification reaction, the 3' hydroxy] of the upstream exon attacks the 5' phosphate at the downstream exon-intron boundary and the lariat

structure containing the intron is released and hydrolyzed. The 5' and 3' exons are ligated to form a continuous sequence.
The snRNAs and associated proteins are required for formation of the various structures and intermediates. U1 within the snRNP complex binds first by base pairing to the 5' exon-intron boundary. U2 within the snRNP complex then binds by base pairing to the branch site, and this exposes the nucleophilic A residue. U4/U5/U6 within the snRNP
complex mediates an ATP-dependent protein-mediated unwinding that results in disruption of the base-paired U4-U6 complex with the release of U4. U6 is then able to interact first with U2, then with U1. These interactions serve to approximate the 5'~splice site, the branch point with its reactive A, and the 3'-splice site. This alignment is enhanced by U5. This process also results in the formation of the loop or lariat structure. The two ends are cleaved, probably by the U2-U6 within the snRNP complex. U6 is certainly essential, since yeast cells deficient in this snRNA are not viable. It is important to note that RNA serves as the catalytic agent. This sequence of events is then repeated in genes containing multiple introns. In such cases, a definite pattern is followed for each gene, though the introns are not necessarily removed in sequence—1, then 2, then 3, etc.
Alternative Splicing Provides for Production of Different mRNAs from a Single mRNA Primary Transcript, Thereby Increasing the Genetic Potential of an

The processing of mRNA molecules is a site for regulation of gene expression. Alternative patterns of mRNA splicing result from tissue-specific adaptive and developmental control mechanisms. Interestingly, recent studies suggest that alternative splicing is controlled, at least in part, through chromatin epigenetic marks (ie, Table 35-1). This form of coupling of transcription and mRNA processing may either be kinetic and/or mediated through interactions between specific histone PTMs and alternative splicing factors that can load onto nascent mRNA gene transcripts during the process of transcription (Figure 36-12).
As mentioned above, the sequence of exon-intron splicing events generally follows a hierarchical order for a given gene. The fact that very complex RNA structures are formed during splicing—and that a number of snRNAs and proteins are involved—affords numerous possibilities for a change of this order and for the generation of different mRNAs. Similarly, the use of alternative termination-cleavage polyadenylation sites also results in mRNA variability. Some schematic examples of these processes, all of which occur in nature, are shown in Figure 36-15.
Not surprisingly, defects in mRNA splicing can cause disease. One of the first examples of the critical importance of accurate splicing was the discovery that one form of
FIGURE 36-15 Mechanisms of alternative processing of mRNA precursors. This form of mRNA processing involves the selective inclusion or exclusion of exons, the use of alternative 5'-donor or 3'-acceptor sites, and the use of different polyadenylation sites, and dramatically increases the differential protein coding potential of the genome.
6-thalassemia, a disease in which the B-globin gene of hemoglobin is severely underexpressed results from a nucleotide change at an exon-intron junction. This mutation precludes removal of the intron, altering the translational reading frame of B-globin mRNA, thereby blocking the 6-chain protein production and hence hemoglobin.

Tissue-specific regulation of gene expression can be provided by alternative splicing, as noted above, by regulatory DNA control elements in the promoter or by the use of alternative promoters. The glucokinase (GK) gene consists of 10 exons interrupted by 9 introns. The sequence of exons 2 to 10 is identical in liver and pancreatic 6 cells, the primary tissues in which GK protein is expressed. Expression of the GK gene is regulated very differently—by two different promoters—in these two tissues. The liver promoter and exon IL are located near exons 2 to 10; exon
1L is ligated directly to exon 2. By contrast, the pancreatic B-cell promoter is located about 30-kbp upstream. In this case, the 3' boundary of exon 1B is ligated to the 5' boundary of exon 2. The liver promoter and exon 1L are excluded and removed during the splicing reaction (Figure 36-16). The existence of multiple distinct promoters allows for celland tissue-specific expression patterns of a particular gene (mRNA). In the case of GK, insulin and cAMP (see Chapter 42) control GK transcription in liver, while glucose controls GK expression in B cells. Moreover, as noted above, such variation in spliced mRNAs can also change the protein coding protein potential of these mRNAs.

In mammalian cells, the three rRNA molecules (28S, 18S, 5.8S) are transcribed as part of a single large 45S precursor molecule. The precursor is subsequently processed in the nucleolus to provide these three RNA components for the ribosome subunits found in the cytoplasm. The rRNA genes are located in the nucleoli of mammalian cells. Hundreds of copies of these genes are present in every cell. This large number of genes is required to synthesize sufficient copies of each type of rRNA to form the 10' ribosomes required for round of cell duplication. Whereas a single mRNA molecule may be copied into 10° protein molecules, providing a large amplification, the rRNAs are end products. This lack of amplification requires both a large number of genes and a high transcription rate, typically synchronized with cell growth rate. Similarly, tRNAs are often synthesized as precursors, with extra sequences both 5' and 3' of the sequences comprising the mature tRNA. A small fraction of tRNAs contain introns.

As introduced in the description of tRNAs (see Figure 34-11), essentially all RNAs are covalently modified after transcription. It is clear that at least some of these modifications are regulatory.
FIGURE 36-16 Alternative promoter use in the liver and pancreatic B-cell glucokinase (GK) genes. Differential regulation of the glucokinase gene is accomplished by the use of tissue-specific promoters. The B-cell GK gene promoter and exon 1B are located about 30-kbp upstream from the liver promoter and exon 1L. Each promoter has a unique structure and is regulated differently. Exons 2 to 10 are identical in the two genes, and the GK proteins encoded by the liver and B-cell mRNAs have identical kinetic properties.

Eukaryotic mRNAs contain a 7-methylguanosine cap structure at their 5' terminus (see Figure 34-10), and most have a poly(A) tail at the 3' terminus. The cap structure is added to the 5' end of the newly transcribed mRNA precursor in the nucleus very soon after synthesis and prior to transport of the mRNA molecule to the cytoplasm. The 5' cap of the RNA transcript is required both for efficient translation initiation (see Figure 37-7) and protection of the 5' end of mRNA from attack by 5' — 3' exonucleases. The secondary methylations of mRNA molecules, those on the 2'-hydroxy and the N' of adenylyl residues, occur after the mRNA molecule has appeared in the cytoplasm.
Poly(A) tails are added to the 3' end of mRNA molecules in a posttranscriptional processing step. The mRNA is first cleaved about 20 nucleotides downstream from an AAUAA recognition sequence. Another enzyme, poly(A) polymerase, adds a poly(A) tail which is subsequently extended to as many as 200 A residues. The poly(A) tail both protects the 3' end of mRNA from 3' — 5' exonuclease attack and facilitates translation (see Figure 37-7). The presence or absence of the poly(A) tail does not determine whether a precursor molecule in the nucleus appears in the cytoplasm, because all poly(A)-tailed nuclear mRNA molecules do not contribute to cytoplasmic mRNA, nor do all cytoplasmic mRNA molecules contain poly(A) tails (histone mRNAs are most notable in this regard). Following nuclear transport, cytoplasmic enzymes in mammalian cells can both add and remove adenylyl residues from the poly(A) tails; this process has been associated with an alteration of mRNA stability and translatability.

The size of some cytoplasmic mRNA molecules, even after the poly(A) tail is removed, is still considerably greater than the size required to code for the specific protein for which it is a template, often by a factor of 2 or 3. The extra nucleotides occur in untranslated (nonprotein coding) exonic regions both 5' and 3' of the coding region; the longest untranslated sequences are usually at the 3' end. The 5' UTR and 3' UTR sequences have been implicated in RNA processing, transport, storage, degradation, and translation; each of these reactions potentially contributes additional levels of control of gene expression. Some of these posttranscriptional events involving mRNAs occur in cytoplasmic organelles termed P bodies (see Chapter 37).

The majority of miRNAs are transcribed by RNA pol II into primary transcripts termed pri-miRNAs. pri-miRNAs are 5'-capped and 3'-polyadenylated (Figure 36-17). primiRNAs are synthesized from transcription units encoding one or several distinct miRNAs; these transcription units are either located independently in the genome or within the intronic DNA of other genes. Given this organization miRNA-encoding genes must therefore minimally possess a distinct promoter, coding region, and polyadenylation/ termination signals. pri-miRNAs have extensive 2° structure, and this intramolecular structure is maintained following processing by the Drosha-DGCR8 nuclease; the portion containing the RNA hairpin is preserved, transported through the nuclear pore via the action of exportin 5, and once in the cytoplasm, further processed by the heterodimeric dicer nuclease-TRBP complex to a 21 or 22-mer. Ultimately, one of the two strands is selected for loading into the RNAinduced silencing complex (RISC), which is composed of one of four Argonaute proteins (Ago 1-4), to form a mature, functional 21-22 nt single stranded miRNA. siRNAs are produced similarly. Once in the RISC complex, miRNAs can modulate mRNA function by one of three mechanisms: (a) promoting mRNA degradation directly; (b) stimulating CCR4/NOT complex-mediated poly(A) tail degradation; or (c) inhibition of translation by targeting the 5'-methyl cap binding translation factor eIF4 (see Figures 37-7 and 37-8) or the ribosome directly. Recent data suggest that at least some regulatory miRNA-encoding genes may be linked, and hence coevolve, with their target genes.

The central dogma states that for a given gene and gene product there is a linear relationship between the coding sequence in DNA, the mRNA sequence, and the protein sequence (see Figure 35-7). Changes in the DNA sequence should be reflected in a change in the mRNA sequence and, depending on codon usage, in protein sequence. However, exceptions to this dogma have been documented. Coding information can be changed at the mRNA level by RNA editing. In such cases, the coding sequence of the mRNA differs from that in the cognate DNA. An example is the apolipoprotein B (apoB) gene and mRNA. In liver, the single apoB gene is transcribed into an mRNA that directs the synthesis of a 100-kDa protein, apoB100. In the intestine, the same gene directs the synthesis of the identical mRNA primary transcript; however, a cytidine deaminase converts a CAA codon in the mRNA to UAA ata single specific site. Rather than encoding glutamine, this codon becomes a termination signal (see Table 37-1), and hence production of a truncated 48-kDa protein (apoB48) results. ApoB100 and apoB48 have different functions in the two organs. A growing number of other examples include a glutamine to arginine change in the glutamate receptor and several changes in trypanosome mitochondrial mRNAs, generally involving the addition or deletion of uridine. The exact extent of RNA editing genomewide is unknown, but current estimates suggest that perhaps 0.01% of mRNAs are edited in this fashion. Recently, editing of miRNAs has been described suggesting that these two forms of posttranscriptional control mechanisms could cooperatively contribute to gene regulation.

FIGURE 36-17 Biogenesis of micro (mi) and silencing (si)RNAs. (Left) miRNA encoding genes are transcribed by RNA pol Il into a primary miRNA (pri-miRNA), which is 5'-capped and polyadenylated as is typical of mRNA coding primary transcripts. This pri-miRNA is subjected to processing within the nucleus by the action of the Drosha-DGCR8 nuclease, which trims sequences from both 5' and 3' ends to generate the pre-miRNA. This partially processed double-stranded RNA is transported through the nuclear pore by exportin-5. The cytoplasmic pre-miRNA is then trimmed further by the action of the heterodimeric nuclease termed Dicer (TRBP-Dicer), to form the 21-22 nt miRNA duplex. One of the two resulting 21-22 nucleotide-long RNA strands is selected, the duplex unwound, and the selected strand loaded into the RNAinduced silencing complex, or RISC complex thereby generating the mature, functional miRNA. Following target mRNA location and sequencespecific miRNA-mRNA annealing, the functional miRNA can modulate mRNA function by one of three mechanisms: translational repression, mRNA destabilization by mRNA deadenylation, or mRNA degradation. (Right) The siRNA pathway generates functional siRNAs from large double-stranded RNAs that are formed either intracellularly by RNA-RNA hybridization (interor intramolecular) or from extracellular sources such as RNA viruses. These viral dsRNAs are again processed to ~22 nt siRNA dsRNA segments via the heterodimeric Dicer nuclease, loaded into the Ago2-containing RISC complex, one strand is then selected to generate the siRNA that locates target RNA sequences via sequence-specific siRNA-RNA annealing. This ternary target RNA-siRNA-Ago2 complex induces RNA cleavage, which inactivates the target RNA.

As described in Chapters 34 & 37, the tRNA molecules serve as adapter molecules for the translation of mRNA into protein sequences. The tRNAs contain many modifications of the standard bases A, U; G, and C, including methylation, reduction, deamination, and rearranged glycosidic bonds. Further posttranscriptional modification of the tRNA molecules includes nucleotide alkylations and the attachment of
—
the characteristic CpCpA,,, terminal at the 3' end of the molecule by the enzyme nucleotidyl transferase. The 3 OH of the A ribose is the point of attachment for the specific amino acid that is to enter the polymerization reaction of protein synthesis, The methylation of mammalian tRNA precursors probably —_gccurs in the nucleus, whereas the cleavage and attachment of CpCpA,,,, are cytoplasmic functions, since the terminal nucletides turn over more rapidly than do the tRNA molecules themselves. Specific aminoacyl tRNA synthetases within the cytoplasm of mammalian cells are required for the attachment
of the
different amino
acids to
the
CpCpA,,, residues (see
Chapter 37).

In addition to the catalytic action served by the snRNAs in the formation of mRNA, several other enzymatic functions have been attributed to RNA. Ribozymes are RNA molecules with catalytic activity. These generally involve transesterification reactions, and most are concerned with RNA metabolism (splicing and endoribonuclease). Recently, a rRNA component has been implicated in hydrolyzing an aminoacyl ester and thus to play a central role in peptide bond function (peptidyl transferases; see Chapter 37). Collectively, these observations, made using RNA molecules derived from the organelles from plants, yeast, viruses, and higher eukaryotic cells, show that RNA can act as an enzyme, and have revolutionized thinking about enzyme action and the origin of life itself.

RNA is synthesized from a DNA template by the enzyme DNA-dependent RNA polymerase.
™ While bacteria contain but a single RNA polymerase (BBa,ow), there are three distinct nuclear DNA-dependent RNA polymerases in mammals: RNA polymerases I, II, and III. These enzymes catalyze the transcription of rRNA (pol I), mRNA/mi/siRNAs/IncRNAs (pol II), and tRNA and 5S rRNA (pol III) encoding genes.
™ RNA polymerases interact with unique cis-active regions of genes, termed promoters, in order to form PICs capable of initiation. In eukaryotes, the process of pol II PIC formation requires, in addition to polymerase, multiple general transcription factors (GTFs), TFIIA, B, D, E, F, and H.
@ Eukaryotic PIC formation can occur on accessible promoters either stepwise—by the sequential, ordered interactions of GTFs and RNA polymerase with DNA promoters—or in one step by the recognition of the promoter by a preformed GTFRNA polymerase holoenzyme complex.
| Transcription exhibits three phases: initiation, elongation, and termination. All are dependent on distinct DNA cis-elements and can be modulated by distinct trans-acting protein factors.
™ The presence of nucleosomes can either enhance or occlude the binding of both transfactors and the transcription machinery to their cognate DNA cis-elements, thereby with modulating transcription.
= Most eukaryotic RNAs are synthesized as precursors that contain excess sequences which are removed prior to the
generation of mature, functional RNA. These processing reactions provide additional potential steps for regulation of gene expression.
| Eukaryotic mRNA synthesis results in a pre-mRNA precursor that contains extensive amounts of excess RNA
(introns) that must be precisely removed by RNA splicing to generate functional, translatable mRNA composed of exonic coding and 5' and 3' noncoding sequences.
= All steps—from changes in DNA template, sequence, and accessibility in chromatin to RNA stability and translatability— are subject to modulation and hence are potential control sites for eukaryotic gene regulation.

VR TOle\ NCR UER eel aA you should be able to:
C H A PT ER
. translation. Understand that the genetic code is a three-letter nucleotide code, which is contained within the linear array of the exon DNA (composed of triplets of A, G, C, andT) of protein coding genes, and that this three-letter code is translated into mRNA (composed of triplets of A, G, C, and U) to specify the linear order of amino acid addition during protein synthesis via the process
of
= Appreciate that the universal genetic code is degenerate, unambiguous,
nonoverlapping, and punctuation free.
m= Explain that the genetic code is composed of 64 codons, 61 of which encode
amino acids while 3 induce the termination of protein synthesis.
= Describe how the transfer RNAs (tRNAs) serve as the ultimate informational agents that decode the genetic code of messenger RNAs (mRNAs).
m= Understand the mechanism of the energy-intensive process of protein
synthesis that
occurs on
RNA-protein complexes
termed ribosomes.
- inputs. = Appreciate that protein synthesis, like DNA replication and transcription, is precisely controlled through the action of multiple accessory factors that are responsive to multiple extraand intracellular regulatory signaling

The letters A, G, T, and C correspond to the nucleotides found in DNA. Within the protein-coding genes, these nucleotides are organized into three-letter code words called codons, and the collection of these codons, once transcribed into mRNA, makes up the genetic code. It was impossible to understand protein synthesis—or to explain mutations—before the genetic code was elucidated. The code provides a foundation for explaining the way in which protein defects may cause genetic disease and for the diagnosis and perhaps the treatment of these disorders. In addition, the pathophysiology of many viral infections is related to the ability of these infectious agents to disrupt host cell protein synthesis. Many antibacterial drugs are effective because they selectively disrupt protein synthesis in the invading bacterial cell but do not affect protein synthesis in eukaryotic cells.

The genetic information within the nucleotide sequence of DNA is transcribed in the nucleus into the specific nucleotide sequence of an mRNA molecule. The sequence of nucleotides in the RNA transcript is complementary to the nucleotide sequence of the template strand of its gene in accordance with the base-pairing rules. Several different classes of RNA combine to direct the synthesis of proteins.
In prokaryotes there is a linear correspondence between the gene, the messenger RNA (mRNA) transcribed from the gene, and the polypeptide product. The situation is more complicated in higher eukaryotic cells, in which the primary transcript is much larger than the mature mRNA. The large mRNA precursors contain coding regions (exons) that will form the mature mRNA and long intervening sequences (introns)
that separate the exons. The mRNA is processed within the nucleus, and the introns, which make up much more of this RNA than the exons, are removed. Exons are spliced together to form mature mRNA, which is transported to the cytoplasm, where it is translated into protein (see Chapter 36).
The cell must possess the machinery necessary to translate information accurately and efficiently from the nucleotide sequence of an mRNA into the sequence of amino acids of the corresponding specific protein. Clarification of our understanding of this process, which is termed translation, awaited deciphering of the genetic code. It was realized early that mRNA molecules themselves have no affinity for amino acids and, therefore, that the translation of the information in the mRNA nucleotide sequence into the amino acid sequence of a protein requires an intermediate adapter molecule. This adapter molecule must recognize a specific nucleotide sequence on the one hand as well as a specific amino acid on the other. With such an adapter molecule, the cell can direct a specific amino acid into the proper sequential position of a protein during its synthesis as dictated by the nucleotide sequence of the specific mRNA. In fact, the functional groups of the amino acids do not themselves actually come into contact with the mRNA template.

Twenty different amino acids are required for the synthesis of the cellular complement of proteins; thus, there must be at least 20 distinct codons that make up the genetic code. Since there are only four different nucleotides in mRNA, each codon must consist of more than a single purine or pyrimidine nucleotide. Codons consisting of two nucleotides each could provide for only 16 (ie, 4*) distinct codons, whereas codons of three nucleotides could provide 64 (4°) specific codons.
It is now known that each codon consists of a sequence of three nucleotides; that is, it is a triplet code (Table 37-1). The initial deciphering of the genetic code depended heavily on in vitro synthesis of nucleotide polymers, particularly triplets in repeated sequence. These synthetic triplet ribonucleotides were used as mRNAs to program protein synthesis in the test tube, which allowed investigators to deduce the genetic code.

Three of the 64 possible codons do not code for specific amino acids; these have been termed nonsense codons. These nonsense codons are utilized in the cell as translation termination

= Thr. , Second Nucleotide.A = Asn. , Second Nucleotide.G = Ser. , Third.Nucleotide = C. , Second Nucleotide.U = Ile a. , Second Nucleotide.C = Thr. , Second Nucleotide.A = Lys. , Second Nucleotide.G = Arg b. , Third.Nucleotide = A. , Second Nucleotide.U = Met. , Second Nucleotide.C = Thr. , Second Nucleotide.A = Lys. , Second Nucleotide.G = Arg b. , Third.Nucleotide = G. G, Second Nucleotide.U = Val. G, Second Nucleotide.C = Ala. G, Second Nucleotide.A = Asp. G, Second Nucleotide.G = Gly. G, Third.Nucleotide = U. , Second Nucleotide.U = Val. , Second Nucleotide.C = Ala. , Second Nucleotide.A = Asp. , Second Nucleotide.G = Gly. , Third.Nucleotide = C. , Second Nucleotide.U = Val. , Second Nucleotide.C = Ala. , Second Nucleotide.A = Glu. , Second Nucleotide.G = Gly. , Third.Nucleotide = A. , Second Nucleotide.U = Val. , Second Nucleotide.C = Ala. , Second Nucleotide.A = Glu. , Second Nucleotide.G = Gly. , Third.Nucleotide = G
"The terms first, second, and third nucleotide refer to the individual nucleotides of a triplet codon read 5'-3', left to right. A, adenine nucleotide; C, cytosine nucleotide; G, guanine nucleotide; Term, chain terminator codon; U, uridine nucleotide. AUG, which codes for Met, serves as the initiator codon in mammalian cells and also encodes for internal methionines in a protein. (Abbreviations of amino acids are explained in Chapter 3.)
'In mammalian mitochondria, AUA codes for Met and UGA for Trp, and AGA and AGG serve as chain terminators.

signals; they specify where the polymerization of amino acids into a protein molecule is to stop. The remaining 61 codons code for the 20 naturally occurring amino acids (Table 37-1). Thus, there is "degeneracy" in the genetic code—that is, multiple codons decode the same amino acid. Some amino acids are encoded by several codons; for example, six different codons, UCU, UCC, UCA, UCG, AGU, and AGC all specify serine. Other amino acids, such as methionine and tryptophan, have a single codon. In general, the third nucleotide in a codon is less important than the first two in determining the specific amino acid to be incorporated, and this accounts for most of the degeneracy of the code. However, for any specific codon, only a single amino acid is specified; with rare exceptions, the genetic code is unambiguous—that is, given a specific codon, only a single amino acid is indicated. The distinction between ambiguity and degeneracy is an important concept. The unambiguous but degenerate code can be explained in molecular terms. The recognition of specific codons in the mRNA by the tRNA adapter molecules is dependent on the tRNA anticodon region and specific base-pairing rules that dictate tRNA-mRNA codon binding. Each tRNA molecule contains a specific sequence, complementary to a codon,
which is termed its anticodon. For a given codon in the mRNA, only a single species of tRNA molecule possesses the proper anticodon. Since each tRNA molecule can be charged with only one specific amino acid, each codon therefore specifies only one amino acid. However, some tRNA molecules can utilize the anticodon to recognize more than one codon. With few exceptions, given a specific codon, only a specific amino acid will be incorporated—although, given a specific amino acid, more than one codon may be used.
As discussed below, the reading of the genetic code during the process of protein synthesis does not involve any overlap of codons. Thus, the genetic code is nonoverlapping. Furthermore, once the reading is commenced at a specific start codon, there is no punctuation between codons, and the message is read in a continuing sequence of nucleotide triplets until a translation stop codon is reached.

+ Degenerate
+
Unambiguous
+ Nonoverlapping
+ Not punctuated
+ Universal
often produced in nonhuman cells using recombinant DNA technology (see Chapter 39). The main features of the genetic code are listed in Table 37-2.
Until recently, the genetic code was thought to be universal. It has now been shown that the set of tRNA molecules in mitochondria (which contain their own separate and distinct set of translation machinery) from lower and higher eukaryotes, including humans, reads four codons differently from the tRNA molecules in the cytoplasm of even the same cells. As noted in a footnote to Table 37-1, in mammalian mitochondria the codon AUA is read as Met, and UGA codes for Trp. In addition, in mitochondria, the codons AGA and AGG are read as stop or chain terminator codons rather than as Arg. As a result of these organelle-specific changes in genetic code, mitochondria require only 22 tRNA molecules (see Figure 35-8 for the location of these genes in mrDNA) to read their genetic code, whereas the cytoplasmic translation system possesses a full complement of 31 tRNA species. These exceptions noted, the genetic code is universal. The frequency of use of each amino acid codon varies considerably between species and among different tissues within a species. The specific tRNA levels generally mirror these codon usage biases. Thus, a particular abundantly used codon is decoded by a similarly abundant-specific tRNA which recognizes that particular codon. Tables of codon usage are quite accurate now that many genomes have been sequenced and such information is vital for large-scale production of proteins for therapeutic purposes (ie, insulin, erythropoietin). Such proteins are

tRNA molecules have extraordinarily similar functions and three-dimensional structures. The adapter function of the tRNA molecules requires the charging of each specific tRNA with its specific amino acid. Since there is no affinity of nucleic acids for specific functional groups of amino acids, this recognition must be carried out by a protein molecule capable of recognizing both a specific tRNA molecule and a specific amino acid. At least 20 specific enzymes are required for these specific recognition functions and for the proper attachment of the 20 amino acids to specific tRNA molecules. This energy requiring process of recognition and attachment, tRNA amino acid charging, proceeds in two steps and is catalyzed by one enzyme for each of the 20 amino acids. These enzymes are termed aminoacyl-tRNA synthetases. They form an activated intermediate of aminoacyl-AMP-enzyme complex (Figure 37-1). The specific aminoacyl-AMP-enzyme complex then recognizes a specific tRNA to which it attaches the aminoacyl moiety at the 3'-hydroxyl adenosyl terminal. The charging reactions have an error rate of less than 10~ and so are quite accurate. The amino acid remains attached to its specific tRNA in an ester linkage until it is incorporated at a specific position during the synthesis of a polypeptide on the ribosome. The of the tRNA molecule referred to in regions Chapter 34 (and illustrated in Figure 34-11) now become
FIGURE 37-1 Formation of aminoacyl-tRNA. A two-step reaction, involving the enzyme aminoacyl-tRNA synthetase, results in the formation of aminoacyl-tRNA. The first reaction involves the formation of an AMP-amino acid—enzyme complex. This activated amino acid is next transferred to the corresponding tRNA molecule. The AMP and enzyme are released, and the latter can be reutilized. The charging reactions have an error rate (ie, esterifying the incorrect amino acid on tRNA***) of less than I1mischaging event out of 10* amino acid charging events.
Phenylalanyl-tRNA

FIGURE 37-2 Recognition of the codon by the anticodon. One of the codons for phenylalanine is UUU. tRNA charged with phenylalanine (Phe) has the complementary sequence AAA; hence, it forms a base-pair complex with the codon. The anticodon region (arm) typically consists of a sequence of seven nucleotides: variable (N), modified purine (Pu*), X, Y, Z (here, A A A), and two pyrimidines in the 3' to 5' direction.
(Py)
important. The ribothymidine pseudouridine cytidine (TyC) arm is involved in binding of the aminoacyl-tRNA to the ribosomal surface at the site of protein synthesis. The tRNA D arm is one of the sites important for the proper recognition of a given tRNA species by its proper aminoacyltRNA synthetase. The tRNA acceptor arm, located at the 3'-hydroxyl adenosy] terminal, is the site of attachment of the specific amino acid.
The anticodon region (arm) consists of seven nucleotides, and it recognizes the three-letter codon in mRNA (Figure 37-2). The sequence read from the 3' to 5' direction in that anticodon loop consists of a variable base (N)-modified purine (Pu')-XYZ (the anticodon)-pyrimidine (Py)-pyrimidine (Py)-5'. Note that this direction of reading the anticodon is 3'-5', whereas the genetic code in Table 37-1 is read 5'-3', since the codon and the anticodon loop of the mRNA and tRNA molecules, respectively, are antiparallel in their complementarity just like all other intermolecular interactions between nucleic acid strands.

The degeneracy of the genetic code resides mostly in the last nucleotide of the codon triplet, suggesting that the base pairing between this last nucleotide and the corresponding nucleotide of the anticodon is not strictly by the Watson-Crick tule. This is called wobble; the pairing of the codon and anticodon can "wobble" at this specific nucleotide-to-nucleotide pairing site. For example, the two codons for arginine, AGA and AGG, can bind to the same anticodon having an uracil at its 5' end (UCU). Similarly, three codons for glycine—GGU, GGC, and GGA—can form a base pair from one anticodon, 3' CCI 5' (ie, I, inosine, can base pair with U, C, and A). Inosine is generated by deamination of adenine (see Figure 33-2 for
structure).

Although the initial change may not occur in the template strand of the double-stranded DNA molecule for that gene, after replication, daughter DNA molecules with mutations in the template strand will segregate and appear in the population of organisms.

Single-base changes (point mutations) may be transitions or transversions. In the former, a given pyrimidine is changed to the other pyrimidine or a given purine is changed to the other purine. Transversions are changes from a purine to either of the two pyrimidines or the change of a pyrimidine into either of the two as shown in 37-3.
when translated into protein:
purines, Figure When the nucleotide sequence of a protein-coding gene containing the mutation is transcribed into an mRNA molecule, then the RNA molecule will of course possess the base change at the corresponding location. Single-base changes in the mRNA may have one of several effects
1. There may be no detectable effect because of the degeneracy of the code; such mutations are often referred to as silent mutations. This would be most likely if the changed base in the mRNA molecule were to be at the third nucleotide of a codon. Because of wobble, the translation of a codon is least sensitive to a change at the third position.
2. A missense effect will occur when a different amino acid is incorporated at the corresponding site in the protein molecule. This mistaken amino acid—or missense, depending on its location in the specific protein—might be acceptable, partially acceptable, or unacceptable to the function of that protein molecule. From a careful examination of the genetic code, one can conclude that most single-base changes would result in the replacement of one amino acid by another with rather similar functional groups. This is an effective genetic "buffering" mechanism to avoid drastic
FIGURE 37-3 Diagrammatic representation of transition and transversion mutations.

Acceptable missense, Protein molecule = Hb A, β chain Hb Hikari, β chain. Acceptable missense, Amino acid = 61 Lysine Asparagine. Acceptable missense, Codons = AAA AAU AAG or or AAC. Partially acceptable missense, Protein molecule = Hb A, β chain Hb S, β chain. Partially acceptable missense, Amino acid = 6 Glutamate Valine. Partially acceptable missense, Codons = GAA GUA GAG or or GUG. Unacceptable missense, Protein molecule = Hb A, α chain Hb M (Boston), α chain. Unacceptable missense, Amino acid = 58 Histidine Tyrosine. Unacceptable missense, Codons = CAU UAU CAC or or UAC
FIGURE 37-4 Examples of three types of missense mutations resulting in abnormal hemoglobin chains. The amino acid alterations and possible alterations in the respective codons are indicated. The hemoglobin Hikari B-chain mutation has apparently normal physiologic properties but is electrophoretically altered. Hemoglobin S has a B-chain mutation and partial function; hemoglobin S binds oxygen but precipitates when deoxygenated; this causes red blood cells to sickle, and represents the cellular and molecular basis of sickle cell disease (see Figure 6-13). Hemoglobin M Boston, an a-chain mutation, permits the oxidation of the heme ferrous iron to the ferric state and so will not bind oxygen at all.
change in the physical properties of a protein molecule. If an acceptable missense effect occurs, the resulting protein molecule may not be distinguishable from the normal one. A partially acceptable missense will result in a protein molecule with partial but abnormal function. If an unacceptable missense effect occurs, then the protein molecule will not be capable of functioning normally.
Not only is the sequence of amino acids distal to this deletion garbled, but reading of the message can also result in the appearance of a nonsense codon and thus the production of a polypeptide both garbled and prematurely terminated (example 3, Figure 37-5).

- 3. A nonsense codon may appear that would then result in the premature termination of translation and the production of only a fragment of the intended protein molecule. The probability is high that a prematurely terminated protein molecule or peptide fragment will not function in its normal role. Examples of the different types of mutations, and their effects on the coding potential of mRNA are presented in Figures 37-4 and 37-5.

The deletion of a single nucleotide from the coding strand of a gene results in an altered reading frame in the mRNA. The machinery translating the mRNA does not recognize that a base was missing, since there is no punctuation in the reading of codons. Thus, a major alteration in the sequence of polymerized amino acids, as depicted in example 1, Figure 37-5, results. Altering the reading frame results in a garbled translation of the mRNA distal to the single nucleotide deletion.
If three nucleotides or a multiple of three nucleotides are deleted from a coding region, translation of the corresponding mRNA will generate a protein that is missing the corresponding number of amino acids (example 2, Figure 37-5). Because the reading frame is a triplet, the reading phase will not be disturbed for those codons distal to the deletion. If, however, deletion of one or two nucleotides occurs just prior to or within the normal termination codon (nonsense codon), the reading of the normal termination signal is disturbed. Such a deletion might result in reading through the now "mutated" termination signal until another nonsense codon is encountered (not shown here).
Insertions of one or two or nonmultiples of three nucleotides into a gene result in an mRNA in which the reading frame is distorted upon translation, and the same effects that occur with deletions are reflected in the mRNA translation. This may result in garbled amino acid sequences distal to the insertion and the generation of a nonsense codon at, or distal to the insertion, or perhaps reading through the normal termination codon. Following a deletion in a gene, an insertion (or vice versa) can reestablish the proper reading frame (example 4, Figure 37-5). The corresponding mRNA, when translated, would contain a garbled amino acid sequence between the insertion and deletion. Beyond the reestablishment of the
3
Garbled
FIGURE 37-5 Examples of the effects of deletions and insertions in a gene on the sequence of the mRNA transcript and of the polypeptide chain translated therefrom. The arrows indicate the sites of deletions or insertions, and the numbers in the ovals indicate the number of nucleotide residues deleted or inserted. Colored type indicates the correct amino acids in the correct order.

reading frame, the amino acid sequence would be correct. One can imagine that different combinations of insertions or deletions (ie, indels), or of both would result in formation of a protein wherein a portion is abnormal, but this portion is surrounded by the normal amino acid sequences. Such phenomena have been demonstrated convincingly in a number of human diseases.

The above discussion of the altered protein products of gene mutations is based on the presence of normally functioning tRNA molecules. However, in prokaryotic and lower eukaryotic organisms, abnormally functioning tRNA molecules have been discovered that are themselves the results of mutations. Some of these abnormal tRNA molecules are capable of binding to and decoding altered codons, thereby suppressing the effects of mutations in distinct mutated mRNA-encoding structural genes. These suppressor tRNA molecules, usually formed as a result of alterations in their anticodon regions, are capable of suppressing certain missense mutations, nonsense mutations, and frameshift mutations. However, since the suppressor tRNA molecules are not capable of distinguishing between a normal codon and one resulting from a gene mutation, their presence in the cell usually results in decreased viability. For instance, the nonsense suppressor tRNA molecules can suppress the normal termination signals to allow a read-through when it is not desirable. Frameshift suppressor tRNA molecules may read a normal codon plus a component of a juxtaposed codon to provide a frameshift, also when it is not desirable. Suppressor tRNA molecules may exist in mammalian cells, since read-through of translation has on occasion been observed. In the laboratory context, such suppressor tRNAs, coupled with mutated variants of aminoacyl-tRNA synthetases, can be utilized to incorporate unnatural amino acids into defined locations within altered genes that carry
engineered nonsense mutations. The resulting labeled proteins can be used for in vivo and in vitro cross-linking and biophysical studies. This new tool adds significantly to biologists interested in studying the mechanisms of a wide range of biologic processes.

The general structural characteristics of ribosomes are discussed in Chapter 34. These particulate entities serve as the machinery on which the mRNA nucleotide sequence is translated into the sequence of amino acids of the specified protein. The translation of the mRNA commences near its 5' end with the formation of the corresponding amino terminus of the protein molecule. The message is decoded from 5' to 3', concluding with the formation of the carboxyl terminus of the protein. Again, the concept of polarity is apparent. As described in Chapter 36, the transcription of a gene into the corresponding mRNA or its precursor first forms the 5' end of the RNA molecule. In prokaryotes, this allows for the beginning of mRNA translation before the transcription of the gene is completed. In eukaryotic organisms, the process of transcription is a nuclear one, while mRNA translation occurs in the cytoplasm, precluding simultaneous transcription and translation in eukaryotic organisms and enabling the processing necessary to generate mature mRNA from the primary transcript.

Initiation of eukaryotic protein synthesis requires that an mRNA molecule be selected for translation by a ribosome (Figure 37-6). Once the mRNA binds to the ribosome, the ribosome must locate the initiation codon thereby setting the correct reading frame on the mRNA, and translation begins. This process involves tRNA, rRNA, mRNA, and at least 10 eukaryotic initiation factors (eIFs), some of which have multiple (three to eight) subunits. Also involved are GTP, ATP, and amino acids. Initiation can be divided into three steps, all of which are obligitorialy preceeded by dissociation of the 80S ribosome into its constituent 40S and 80S subunits: (1) binding of a ternary complex consisting of the initiator methionyltRNA, (met-tRNA'), GTP, and eIF-2 to the 40S ribosome to form the 43S preinitiation complex; (2) binding of mRNA to the 40S preinitiation complex to form the 48S initiation complex; and (3) combination of the 48S initiation complex with the 60S ribosomal subunit to form the 80S initiation complex.

Prior to initiation, 80S ribosomes dissociate into component 40S and 60S subunits during translation termination
(see below). Dissociation allows these components to participate in subsequent rounds of translation. Two initiation factors, eIF-3, eIF-1, and eIF-1A, bind to the newly dissociated 40S ribosomal subunit. This delays its reassociation with the 60S subunit and allows other translation initiation factors to associate with the 40S subunit.

The first step of translation initiation involves the binding of GTP by elF-2. This binary complex then binds to methtionyl tRNA', a tRNA specifically involved in binding to the initiation codon AUG. It is important to note that there are two tRNAs for methionine. One specifies methionine for the initiator codon, the other for internal methionines. Each has a unique nucleotide sequence; both are aminoacylated by the same methionyl-tRNA synthetase. The GTP-elF-2-tRNA' ternary complex binds to the 40S ribosomal subunit to form the 43S preinitiation complex. The ternary complex—40S subunit complex is stabilized by eIF-3 and eIF-1A and the subsequent binding of eIF5.
eIF-2 is one of two control points for protein synthesis initiation in eukaryotic cells. eIF-2 consists of a, B, and y subunits. eIF-2a is phosphorylated (on serine 51) by at least four different protein kinases (HCR, PKR, PERK, and GCN2) that are activated when a cell is under stress and when the energy expenditure required for protein synthesis would be deleterious. Such conditions include amino acid or glucose starvation, virus infection, intracellular presence of large quantities of misfolded proteins (endoplasmic reticulum [ER] stress), serum deprivation, hyperosmolality, and heat shock. PKR is particularly interesting in this regard. This kinase is activated by viruses and provides a host defense mechanism that decreases protein synthesis, including viral protein synthesis, thereby inhibiting viral replication. Phosphorylated eIF-2a binds tightly to and inactivates the GTP-GDP recycling protein eIF-2B,thus, preventing formation of the 43S preinitiation complex and blocking protein synthesis.

As described in Chapter 36, the 5' termini of mRNA molecules in eukaryotic cells are "capped." The ""*G-cap facilitates the binding of mRNA to the 43S preinitiation complex. A cap-binding protein complex, eIF-4F (4F), which consists of eIF-4E (4E) and the eIF-4G (4G)-eIF-4A (4A) complex, binds to the cap through the 4E protein. Then eIF-4B (4B) binds and reduces the complex secondary structure of the 5' end of the mRNA through its ATP-dependent helicase activity. The association of mRNA with the 43S preinitiation complex to form the 48S initiation complex requires ATP hydrolysis. elF-3 is a key protein because it binds with high affinity to the 4G component of 4F, and it links this complex to the 40S ribosomal subunit. Following association of the 43S preinitiation complex with the mRNA cap, and reduction ("melting") of the secondary structure near the 5' end of the mRNA through the action of the 4B helicase and ATP, the complex translocates 5' > 3' and scans the mRNA for a suitable initiation codon.
1.
Re-formation of
the
80S
initiation complex:

FIGURE 37-6 Diagrammatic representation of the initiation phase of protein synthesis on an eukaryotic mRNA. Eukaryotic mRNAs contain a 5'"™°G-cap (Cap) and 3' poly(A) terminal [(A),] as shown. Translation preinitiation complex formation proceeds in several steps: (1) Dissociation of the 80S complex to component 40S and 60S subunits, a process facilitated by binding of factors elF1, elF1A, and elF3 to the ribosomal 40S subunit (top). (2) Formation of the 43S Preinitiation Complex, a ternary complex consisting of met-tRNAi and GTP-bound to the initiation factor elF-2 (elF-2-GTP; left). This complex is then bound by the elF5 initiation factor forming the complete 43S Preinitiation Complex. (3) Activation of 'capped' mRNA and formation of the 48S Initiation Complex. mRNA is bound via its 5'-Cap by elF4F (composed of elF4E, elF4G and elF4A factors) and 3' Poly(A) tail by Poly A Binding Protein forming the 485 Initiation Complex. ATP hydrolysis-dependent 5' to 3' mRNA scanning enables location of the initiation codon AUG, which is then bound by met-tRNAi. (4) Following addition of GTP-bound el5B and dissociation of elF1, elF2-GDP, elF3 and elF5, formation of the 80S Initiation Complex occurs when a recycled 60S ribosomal subunit joins the 48S complex. This reaction positions the initiator met-tRNAi within the P-Site of the active 80S Initiation Complex formation induces dissociation of elF1A and GDP-bound elF5B (see text for details). This complex is now competent for translation initiation. (GTP, +; GDP") The various initiation factors appear in abbreviated form as circles or squares, for example, elF-3, (@), elF-4F, (4F), (45). 4-F is a complex consisting of 4E and 4A bound to 4G (see Figure 37-7). Note that the "circular" structure of mRNA illustrated in Figure

37-7 is thought to be the actual form of mRNA upon which steps 1 to 4 actually occur.
Generally, this is the 5'-most AUG, but the precise initiation codon is determined by so-called Kozak consensus sequences that surround the AUG initiation codon:
Most preferred is the presence of a purine (Pu) at positions —3 and a Gat position +4.

Biochemical and genetic experiments have revealed that the 3' poly(A) tail and the poly(A) binding protein, PAB, are both required for efficient initiation of protein synthesis. Further studies showed that the poly(A) tail stimulates recruitment of the 40S ribosomal subunit to the mRNA through a complex set of interactions. PAB (Figure 37-7) bound to the poly(A) tail, interacts with eIF-4G, and 4E subunits of cap-bound elF-4F to form a circular structure that helps direct the 40S ribosomal subunit to the 5' end of the mRNA and also likely stabilizes mRNAs from exonucleolytic degradation. This helps explain how the cap and poly(A) tail structures have a synergistic effect on protein synthesis. Indeed, differential protein—protein interactions between general and specific mRNA translational repressors and eIF-4E result in m'G cap-dependent translation control (Figure 37-8).

The binding of the 60S ribosomal subunit to the 48S initiation complex involves hydrolysis of the GTP bound to elF-2 by eIF5. This reaction results in release of the initiation factors bound to the 48S initiation complex (these factors then are recycled) and the rapid association of the 40S and 60S subunits to form the 80S ribosome. At this point, the met-tRNA' is on the P site of the ribosome, ready for the elongation cycle to commence.

The 4F complex is particularly important in controlling the rate of protein translation. As described above, 4F is a complex consisting of 4E, which binds to the m'G cap structure at the 5' end of the mRNA, and 4G, which serves as a scaffolding protein. In addition to binding 4E, 4G binds to eIF-3, which
FIGURE 37-7 Schematic illustrating the circularization of mRNA through protein-protein interactions between "™°G capbound elF4F and poly(A) tail-bound poly(A) binding protein. elF4F, composed of elF4A, 4E, and 4G subunits binds the mRNA 5'-7"°G "Cap" ('"°GpppxX-) upstream of the translation initiation codon (AUG) with high affinity. The elF4G subunit of the complex also binds poly(A) binding protein (PAB) with high affinity. Since PAB is bound tightly to the mRNA 3'-poly(A) tail (5'-(X),A(A), AAAAAAA.,,, 3'), circularization results. Shown are multiple 80S ribosomes that are in the process of translating the circularized mRNA into protein (black curlicues), forming a polysome. Upon encountering a termination codon (here UAA), translation termination occurs leading to release of the newly translated protein and dissociation of the 80S ribosome into 60S, 40S subunits. Dissociated ribosomal subunits can recycle through another round of translation (see Figure 37-6 and 37-10).

FIGURE 37-8 Activation of elF-4E by insulin and formation of the cap-binding elF-4F complex. The 4F-cap mRNA complex is depicted as in Figures 37-6 and 37-7. The 4F complex consists of elF-4E (4), elF-4A, and elF-4G. 4E is inactive when bound by one of a family of binding proteins (4E-BPs). Insulin and mitogenic growth polypeptides, or growth factors (eg, IGF-1, PDGF, interleukin-2, and angiotensin Il) activate the PI3 kinase/AKT kinase signaling pathways, which activate the mTOR kinase, and results in the phosphorylation of 4E-BP (see Figure 42-8). Phosphorylated 4E-BP dissociates from 4E, and the latter is then able to form the 4F complex and bind to the mRNA cap. These growth polypeptides also induce phosphorylation of 4G itself by the mTOR and MAP kinase pathways (see Chapter 42). Phosphorylated 4F binds much more avidly to the cap than does nonphosphorylated 4F, which stimulates 48S initiation complex formation and hence translation.
links the complex to the 40S ribosomal subunit. It also binds 4A and 4B, the ATPase-helicase complex that helps unwind the RNA (Figure 37-8).
4E is responsible for recognition of the mRNA cap structure, a rate-limiting step in translation. This process is further regulated by phosphorylation (Figure 37-8). Insulin and mitogenic growth factors result in the phosphorylation of 4E on Ser209 (or Thr210). Phosphorylated 4E binds to the cap much more avidly than does the nonphosphorylated form, thus enhancing the rate of initiation. Components of the MAP kinase, PI3K, mTOR, RAS, and S6 kinase signaling pathways (see Figure 42-8) can all, under appropriate conditions, be involved in these regulatory phosphorylation reactions.

The activity of 4E is modulated in a second way, and this also involves phosphorylation; a set of proteins bind to and inactivate 4E. These proteins include 4E-BP1 (BP1, also known as PHAS-1) and the closely related proteins 4E-BP2 and 4E-BP3. BP1 binds with high affinity to 4E. The 4E-BP1 association prevents 4E from binding to 4G (to form 4F).
Since this interaction is essential for the binding of 4F to the ribosomal 40S subunit and for correctly positioning it on the capped mRNA, BP-1 effectively inhibits translation initiation.
Insulin and other growth factors result in the phosphorylation of BP-1 at seven unique sites. Phosphorylation of BP-1 results in its dissociation from 4E, and it cannot rebind until critical sites are dephosphorylated. These effects on the activation of 4E explain in part how insulin causes a marked posttranscriptional increase of protein synthesis in liver, adipose, and muscle tissue.

Elongation is a cyclic process on the ribosome in which one amino acid at a time is added to the nascent peptide chain (Figure 37-9). The peptide sequence is determined by the order of the codons in the mRNA. Elongation involves several steps catalyzed by proteins called elongation factors (EFs). These steps are (1) binding of aminoacyl-tRNA to the A site, (2) peptide bond formation, (3) translocation of the ribosome on the mRNA, and (4) expulsion of the deacylated tRNA from the Pand E-sites.

In the complete 80S ribosome formed during the process of initiation, both the A site (aminoacyl or acceptor site) and E site (deacylated tRNA exit site) are free (Figure 37-6). The binding of the appropriate aminoacyl-tRNA in the A site requires proper codon recognition. Elongation factor 1A (EF1A) forms a ternary complex with GTP and the entering aminoacyl-tRNA (Figure 37-9). This complex then allows the correct aminoacyl-tRNA to enter the A site with the release of EF1A-GDP and phosphate. GTP hydrolysis is catalyzed by an active site on the ribosome; hydrolysis induces a conformational change in the ribosome concomitantly increasing affinity for the tRNA. As shown in Figure 37-9, EF1A-GDP then recycles to EF1A-GTP with the aid of other soluble protein factors and GTP.

The a-amino group of the new aminoacyl-tRNA in the A site carries out a nucleophilic attack on the esterified carboxyl group of the peptidyl-tRNA occupying the P site (peptidyl or polypeptide site). At initiation, this site is occupied by the initiator met-tRNA\. This reaction is catalyzed by a peptidyl transferase, a component of the 28S RNA of the 60S ribosomal subunit. This is another example of ribozyme activity and indicates an important—and previously unsuspected— direct role for RNA in protein synthesis (Table 37-3). Because the amino acid on the aminoacyl-tRNA is already "activated? no further energy source is required for this reaction. The reaction results in attachment of the growing peptide chain to the tRNA in the A site.
5’
Cap
Peptidyl-tRNA
+
GTP
+
A
+
EF2
-”
FIGURE 37-9 Diagrammatic representation of the peptide elongation process of protein synthesis. The small circles labeled n-1,n,n +1, etc, represent the amino acid residues of the newly formed protein molecule (in N-terminal to C-terminal orientation) and the corresponding codons in the mRNA. EFIA and EF2 represent elongation factors 1 and 2, respectively. The peptidyl-tRNA, aminoacyl-tRNA, and exit sites on the ribosome are represented by P site, A site, and E site, respectively.

•, 1 = Ribosomes can make peptide bonds (albeit inefficiently) even when proteins are removed or inactivated.. •, 1 = Certain parts of the rRNA sequence are highly conserved in all species.. •, 1 = These conserved regions are on the surface of the RNA molecule.. •, 1 = RNA can be catalytic in many other chemical reactions.. •, 1 = Mutations that result in antibiotic resistance at the level of protein synthesis are more often found in rRNA than in the protein com- ponents of the ribosome.. •, 1 = X-ray crystal structure of large subunit bound to tRNAs suggests detailed mechanism.

The now deacylated tRNA is attached by its anticodon to the P site at one end and by its open 3' CCA tail to the E site on the large ribosomal subunit (middle portion of Figure 37-9). At this point, elongation factor 2 (EF2) binds to and displaces the peptidyl tRNA from the A site to the P site. In turn, the deacylated tRNA is on the E site, from which it leaves the ribosome. The EF2-GTP complex is hydrolyzed to EF2GDP, effectively moving the mRNA forward by one codon and leaving the A site open for occupancy by another ternary complex of amino acid tRNA-EF1A-GTP and another cycle of elongation.
The charging of the tRNA molecule with the aminoacyl moiety requires the hydrolysis of an ATP to an AMP, equivalent to the hydrolysis of two ATPs to two ADPs and phosphates. The entry of the aminoacyl-tRNA into the A site results in the hydrolysis of one GTP to GDP. Translocation of the newly formed peptidyl-tRNA in the A site into the P site by EF2 similarly results in hydrolysis of GTP to GDP and phosphate. Thus, the energy requirements for the formation of one peptide bond include the equivalent of the hydrolysis of two ATP molecules to ADP and of two GTP molecules to GDP, or the hydrolysis of four high-energy phosphate bonds. A eukaryotic ribosome can incorporate as many as six amino acids per second; prokaryotic ribosomes incorporate as many as 18 per second. Thus, the energy requiring process of peptide synthesis occurs with great speed and accuracy until a termination codon is reached.

In comparison to initiation and elongation, termination is a relatively simple process (Figure 37-10). After multiple cycles of elongation culminating in polymerization of the specific amino acids into a protein molecule, the stop or terminating codon of mRNA (UAA, UAG, UGA) appears in the A site. Normally, there is no tRNA with an anticodon capable of recognizing such a termination signal. Releasing factor 1 (RF1) recognizes that a stop codon resides in the A site (Figure 37-10). RF1 is bound by a complex consisting of releasing factor 3
FIGURE 37-10 Diagrammatic representation of the termination process of protein synthesis. The 60S ribosomal peptidyl-tRNA, aminoacyl-tRNA, and exit sites are indicated as P site, A site, and E site, respectively. The termination (stop) codon is indicated by the three vertical bars and STOP. Releasing factor RF1 binds to the stop codon in the A site. Releasing factor RF3, which bound GTP, binds to RF1. Hydrolysis of the peptidyl-tRNA complex is shown by the entry of water (H,O); arrow. N and C indicate the aminoand carboxy-terminal amino acids of the nascent polypeptide chain, respectively, and illustrate the polarity of protein synthesis. Termination results in release of the mRNA, the newly synthesized protein (Nand C-termini; N, C), free tRNA, 40S and 60S subunits, as well as RF1, GDP-bound RF3, and inorganic P, as shown at bottom.
(RF3) with bound GTP. This complex, with the peptidyl transferase, promotes hydrolysis of the bond between the peptide and the tRNA occupying the P site. Thus, a water molecule rather than an amino acid is added. This hydrolysis releases the protein and the tRNA from the P site. Upon hydrolysis and release, the 80S ribosome dissociates into its 40S and 60S

subunits, which are then recycled (Figure 37-7). Therefore, the releasing factors are proteins that hydrolyze the peptidyltRNA bond when a stop codon occupies the A site. The mRNA is then released from the ribosome, which dissociates into its component 40S and 60S subunits, and another cycle can be repeated.

Many ribosomes can translate the same mRNA molecule simultaneously. Because of their relatively large size, the ribosome particles cannot attach to an mRNA any closer than 35 nucleotides apart. Multiple ribosomes on the same mRNA molecule form a polyribosome, or "polysome" (Figure 37-7). In an unrestricted system, the number of ribosomes attached to an mRNA (and thus the size of polyribosomes) correlates positively with the length of the mRNA molecule.
Polyribosomes actively synthesizing proteins can exist as free particles in the cellular cytoplasm or may be attached to sheets of membranous cytoplasmic structures referred to as ER. Attachment of the particulate polyribosomes to the ER is responsible for its "rough" appearance as seen by electron microscopy. The proteins synthesized by the attached polyribosomes are extruded into the cisternal space between the sheets of rough ER and are exported from there. Some of the protein products of the rough ER are packaged by the Golgi apparatus for eventual export (see Figures 49-2, 49-6). The polyribosomal particles free in the cytosol are responsible for the synthesis of proteins required for intracellular functions.

mRNAs, bound by specific packaging proteins and exported from the nucleus as ribonucleoproteins particles (mRNPs) sometimes do not immediately associate with ribosomes to be translated. Instead, specific mRNAs can associate with the protein constituents that form P bodies, small dense compartments that incorporate mRNAs as mRNPs (Figure 37-11). These cytoplasmic organelles are related to similar small mRNA-containing granules found in neurons and certain maternal cells. P bodies are sites of mRNA metabolism. Over 35 distinct proteins have been suggested to reside exclusively or extensively within P bodies. These proteins range from mRNA decapping enzymes, RNA helicases, and RNA exonucleases (5'-3' and 3'-5'), to components involved in miRNA function and mRNA quality control. However, incorporation of an mRNP is not an unequivocal mRNA "death sentence." Indeed, though the mechanisms are not yet fully understood, certain mRNAs appear to be temporarily stored in P bodies and then retrieved and utilized for protein translation. This suggests that an equilibrium exists where the cytoplasmic functions of mRNA (translation and degradation) are controlled by the dynamic interaction of mRNA with polysomes and P bodies.

Ferritin, an iron-binding protein, prevents ionized iron (Fe**) from toxic levels within cells. Elemental
The Respond reaching
FIGURE 37-11 The P body is a cytoplasmic organelle involved in mRNA metabolism. Shown is a photomicrograph of two mammalian cells in which a single distinct protein constituent of the P body has been visualized using the cognate-specific fluorescently labeled antibody. P bodies appear as small red circles of varying size throughout the cytoplasm. The cell plasma membranes are indicated by a solid white line, nuclei by a dashed line. Nuclei were counterstained using a fluorescent dye with different fluorescence excitation/emission spectra from the labeled antibody used to identify P bodies; the nuclear stain intercalates between the DNA base pairs and appears as blue/green. Modified from http://www. mcb.arizona.edu/parker/WHAT/what.htm. (Used with permission of Dr Roy Parker.)
iron stimulates ferritin synthesis by causing the release of a cytoplasmic protein that binds to a specific region in the 5' nontranslated region of ferritin mRNA. Disruption of this protein-mRNA interaction activates ferritin mRNA and results in its translation. This mechanism provides for rapid control of the synthesis of a protein that sequesters Fe**, a potentially toxic molecule (see Figures 52-7, 52-8). Similarly, environmental stress and starvation inhibit the positive roles of mTOR (Figure 37-8; Figure 42-8) on promoting activation of eIF-4F and 48S complex formation.

The protein synthesis machinery can also be modified in deleterious ways. Viruses replicate by using host cell processes, including those involved in protein synthesis. Some viral mRNAs are translated much more efficiently than those of the host cell (eg, encephalomyocarditis virus). Others, such as reovirus and vesicular stomatitis virus, replicate efficiently, and thus their very abundant mRNAs have a competitive advantage over host cell mRNAs for limited translation factors. Other viruses inhibit host cell protein synthesis by preventing the association of mRNA with the 40S ribosome.
Poliovirus and other picornaviruses gain a selective advantage by disrupting the function of the 4F complex. The mRNAs of these viruses do not have a cap structure to direct the binding of the 40S ribosomal subunit (see above). Instead, the 40S ribosomal subunit contacts an internal ribosomal entry site (IRES) in a reaction that requires 4G but not 4E. The virus gains
FIGURE 37-12 Picornaviruses disrupt the 4F complex. The 4E-4G complex (4F) directs the 40S ribosomal subunit to the typical capped mRNA (see text). However, 4G alone is sufficient for targeting the 40S subunit to the internal ribosomal entry site (IRES) of certain viral mRNAs. To gain selective advantage, some viruses (eg, poliovirus) express a protease that cleaves the 4E binding site from the amino terminal end of 4G. This truncated 4G can direct the 40S ribosomal subunit to mRNAs that have an IRES but not to those that have a cap (ie, host cell mRNAs). The widths of the arrows indicate the rate of translation initiation from the AUG codon in each example. Other viruses utilize distinct processes to effect selective initiation of translation on their cognate viral mRNAs via IRES elements.

a selective advantage by having a protease that attacks 4G and removes the amino terminal 4E binding site. Now the 4E-4G complex (4F) cannot form, so the 40S ribosomal subunit cannot be directed to host capped mRNAs, abolishing host cell protein synthesis. The 4G fragment can direct binding of the 40S ribosomal subunit to IRES-containing mRNAs, so viral mRNA translation is very efficient (Figure 37-12). These viruses also promote the dephosphorylation of BP1 (PHAS-1), thereby decreasing cap (4E)-dependent translation (Figure 37-8).

Some animal viruses, notably HIV, poliovirus, and hepatitis virus, synthesize long polycistronic proteins from one long mRNA molecule. The viral protein molecules translated from these long mRNAs are subsequently cleaved at defined sites to provide the several specific viral proteins required for viral function. In animal cells, many cellular proteins are synthesized from the mRNA template as a precursor molecule, which then must be modified to achieve the active protein. The prototype is insulin, a small protein having two polypeptide chains with interchain and intrachain disulfide bridges. The molecule is synthesized as a single chain precursor, or prohormone, which folds to allow specific disulfide bridges to form. A specific protease then clips out the segment that connects the two chains which form the functional insulin molecule (see Figure 41-12).
Many other peptides are synthesized as precursor proproteins that require modifications before attaining biologic activity. Many of the posttranslational modifications involve the removal of amino terminal amino acid residues by specific aminopeptidases (see Figure 41-14). By contrast, collagen, an abundant protein in the extracellular spaces of higher eukaryotes, is synthesized as procollagen. Three procollagen polypeptide molecules, frequently not identical in sequence, align themselves in a particular way that is dependent on the existence of specific amino terminal peptides (see Figure 5-11). Specific enzymes then carry out hydroxylations and oxidations of specific amino acid residues within the procollagen molecules to provide cross-links for greater stability. Amino terminal peptides are cleaved off the molecule to form the final product—a strong, insoluble collagen molecule. Many other posttranslational modifications of proteins occur. Covalent modification by acetylation, phosphorylation, methylation, ubiquitylation, and glycosylation is common, for example (see Chapter 5; Table 35-1).

Ribosomes in bacteria and in the mitochondria of higher eukaryotic cells differ from the mammalian ribosome described in Chapter 34. The bacterial ribosome is smaller (70S vs 80S) and has a different, somewhat simpler complement of RNA and protein molecules. This difference can be exploited for
clinical purposes because many effective antibiotics interact specifically with the proteins and RNAs of prokaryotic ribosomes and thus only inhibit bacterial protein synthesis. This results in growth arrest or death of the bacterium. The most useful members of this class of antibiotics (eg, tetracyclines, lincomycin, erythromycin, and chloramphenicol) do not interact with components of eukaryotic ribosomes and thus are not toxic to eukaryotes. Tetracycline prevents the binding of aminoacyl-tRNAs to the bacterial ribosome A site. Chloramphenicol works by binding to 23S rRNA, which is interesting in view of the newly appreciated role of rRNA in peptide bond formation through its peptidyl transferase activity. It should be mentioned that the close similarity between prokaryotic and mitochondrial ribosomes can lead to complications in the use of some antibiotics.
Other antibiotics inhibit protein synthesis on all ribosomes (puromycin) or only on those of eukaryotic cells (cycloheximide). Puromycin (Figure 37-13) is a structural analog of tyrosinyl-tRNA. Puromycin is incorporated via the A site on the ribosome into the carboxyl terminal position of a peptide but causes the premature release of the polypeptide. Puromycin, as a tyrosinyl-tRNA analog, effectively inhibits protein synthesis in both prokaryotes and eukaryotes. Cycloheximide inhibits peptidyl transferase in the 60S ribosomal
FIGURE 37-13 The comparative structures of the antibiotic puromycin (top) and the 3' terminal portion of tyrosinyl-tRNA (bottom).
subunit in eukaryotes, presumably by binding to an rRNA component.

Diphtheria toxin, an exotoxin of Corynebacterium diphtheriae infected with a specific lysogenic phage, catalyzes the ADP-ribosylation of EF-2 on the unique amino acid diphthamide (a posttranslationally modified version of histidine) in mammalian cells. This modification inactivates EF-2 and thereby specifically inhibits mammalian protein synthesis. Many animals (eg, mice) are resistant to diphtheria toxin. This resistance is due to inability of diphtheria toxin to cross the cell membrane rather than to insensitivity of mouse EF-2 to diphtheria toxin-catalyzed ADP-ribosylation by NAD.
Ricin, an extremely toxic molecule isolated from the castor bean, inactivates eukaryotic 28S ribosomal RNA by catalyzing the N-glycolytic cleavage or removal of a single adenine. Many of these compounds—puromycin and cycloheximide in particular—are not clinically useful but have been important in elucidating the role of protein synthesis in the regulation of metabolic processes, particularly enzyme induction by hormones.

The flow of genetic information generally follows the sequence DNA > RNA > protein.
= Ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger
RNA
(mRNA)
are directly
@ The information in mRNA is a continuous array of codons, each of which is three nucleotides long.
= The mRNA is read continuously from a start (AUG) to
termination (UAA, UAG, UGA) codon.
|
The open reading frame, or ORF, of the mRNA is the series of contiguous codons (AUG to STOP), each specifying a certain amino acid, that determines the precise amino acid sequence of the protein.
| Protein synthesis, like DNA and RNA synthesis, follows the 5' to 3' polarity of mRNA and can be divided into three processes: initiation, elongation, and termination.
= Mutant proteins arise when single-base substitutions result in codons that specify a different amino acid at a given position, when a stop codon results in a truncated protein, or when base additions or deletions alter the reading frame, so different codons are read.
™ A variety of compounds, including several antibiotics, inhibit protein synthesis by affecting one or more of the steps involved in protein synthesis.

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VCMT ULES NCR UIERS Cle aA
. gene product. Explain that the many steps involved in the vectorial processes of gene expression, which range from targeted modulation of gene copy number, to gene rearrangement, to transcription, to mRNA processing and transport from the nucleus, to translation, to protein subcellular compartmentalization, to posttranslational modification and degradation, are all subject to regulatory control, both positive and negative. Changes in any, or multiple of these processes, can increase or decrease the amount and/or activity of the cognate
= Appreciate that DNA-binding transcription factors, proteins that bind to specific DNA sequences that are physically linked to their target transcriptional
promoter elements,
can either
activate or
repress gene
= Recognize that DNA-binding transcription factors are often modular proteins that are composed of structurally and functionally distinct domains, which can directly or indirectly control messenger RNA (mRNA) gene transcription, either through contacts with RNA polymerase and its cofactors, or through interactions with coregulators that modulate nucleosome structure, composition, and position via histone covalent modifications and/or nucleosome displacement.
m= Understand that nucleosome-directed regulatory events typically increase or decrease the accessibility of the underlying DNA such as enhancer or promoter sequences, although nucleosome modification can also create new binding sites for other coregulators.
= Describe how the processes of gene transcription, RNA processing, and nuclear export of RNA are all coupled.

Organisms alter expression of genes in response to genetic developmental cues or programs, environmental challenges, or disease, by modulating the amount, the spatial, and/or the temporal patterns of gene expression. The mechanisms controlling gene expression have been studied in detail and often involve modulation of gene transcription. Control of transcription ultimately results from changes in the mode of interaction of specific regulatory molecules, usually proteins, with various regions of DNA in the regulated gene. Such interactions can either have a positive or negative effect on transcription. Transcription control can result in tissue-specific gene expression, and gene regulation can be influenced by a range of physiologic, biologic, environmental, and pharmacologic agents.
In addition to transcription level controls, expression gene can also be modulated by gene amplification, gene rearrangement, posttranscriptional modifications, RNA stabilization, translational control, protein modification, protein compartmentalization, and protein stabilization or degradation. Many of the mechanisms that control gene expression are used to respond to developmental cues, growth factors, hormones, environmental agents, and therapeutic drugs. Dysregulation of gene expression can lead to human disease. Thus, a molecular
understanding of these processes will lead to development of therapeutics that can alter pathophysiologic mechanisms or inhibit the function or arrest the growth of pathogenic organisms.

The genetic information present in each normal somatic cell of a metazoan organism is practically identical. The genetically reproducible, hardwired exceptions are found in those few cells that have amplified or rearranged genes in order to perform specialized cellular functions. Of course, in various disease states chromosome integrity is altered (ie, cancer; Figure 56-11) sometimes even at the whole chromosome level (eg, trisomy 21, that causes Down syndrome). Expression of the genetic information must be regulated during ontogeny and differentiation of the organism and its cellular components. Furthermore, in order for the organism to adapt to its environment and to conserve energy and nutrients, the expression of genetic information must be cued to extrinsic signals and respond only when necessary. As organisms have evolved, more sophisticated regulatory mechanisms have appeared which provide the organism and its cells with the responsiveness necessary for survival in a complex environment. Mammalian cells possess about 1000 times more genetic information than does the bacterium Escherichia coli. Much of this additional genetic information is likely involved in regulation of gene expression during the differentiation of tissues and biologic processes in the multicellular organism and in ensuring that the organism can respond to complex environmental challenges.
In simple terms, there are only two types of gene regulation: positive regulation and negative regulation (Table 38-1). When the expression of genetic information is quantitatively increased by the presence of a specific regulatory element, regulation is said to be positive; when the expression of genetic information is diminished by the presence of a specific regulatory element, regulation is said to be negative. The element or molecule mediating negative regulation is said to be a negative regulator, a silencer or repressor; the element that mediating

Regulator present, Rate of Gene Expression.Negative Regulation = Decreased. Regulator present, Rate of Gene Expression.Positive Regulation = Increased. Regulator absent, Rate of Gene Expression.Negative Regulation = Increased. Regulator absent, Rate of Gene Expression.Positive Regulation = Decreased
positive regulation is a positive regulator, an enhancer or activator. However, a double negative has the effect of acting as a positive. Thus, an effector that inhibits the function of a negative regulator will appear to bring about a positive regulation. Many regulated systems that appear to be induced are in fact derepressed at the molecular level. (See Chapter 9 for additional discussion of these terms.)

Figure 38-1 depicts the extent or amount of gene expression in three types of temporal responses to an inducing signal. A type A response is characterized by an increased extent of gene expression that is dependent on the continued presence of the inducing signal. When the inducing signal is removed, the amount of gene expression diminishes to its basal level, but
FIGURE 38-1 Diagrammatic representations of the responses of the extent of expression of a gene to specific regulatory signals as a function of time.
the amount repeatedly increases in response to the reappearance of the specific signal. This type of response is commonly observed in prokaryotes in response to sudden changes of the intracellular concentration of a nutrient. It is also observed in many higher organisms after exposure to inducers such as hormones, nutrients, or growth factors (see Chapter 42). A type B response exhibits an increased amount of gene expression that is transient even in the continued presence of the regulatory signal. After the regulatory signal has terminated and the cell has been allowed to recover, a second transient response to a subsequent regulatory signal may be observed. This phenomenon of response-desensitization recovery characterizes the action of many pharmacologic agents, but it is also a feature of many naturally occurring processes. This type of response commonly occurs during development of an organism, when only the transient appearance of a specific gene product is required although the signal persists. The type C response pattern exhibits, in response to the regulatory signal, an increased extent of gene expression that persists indefinitely even after termination of the signal. The signal acts as a trigger in this pattern. Once expression of the gene is initiated in the cell, it cannot be terminated even in the daughter cells; it is therefore an irreversible and inherited alteration. This type of response typically occurs during the development of differentiated function in a tissue or organ.

Analysis of the regulation of gene expression in prokaryotic cells helped establish the principle that information flows from the gene to a messenger RNA to a specific protein molecule. These studies were aided by the advanced genetic analyses that could be performed in prokaryotic and lower eukaryotic organisms such as baker's yeast, Saccharomyces cerevisiae, and the fruit fly, Drosophila melanogaster, among others. In recent years, the principles established in these studies, coupled with a variety of molecular biology techniques, have led to remarkable progress in the analysis of gene regulation in higher eukaryotic organisms, including mammals. In this chapter, the initial discussion will center on prokaryotic systems. The impressive genetic studies will not be described, but the physiology of gene expression will be discussed. However, nearly all of the conclusions about this physiology have been derived from genetic studies and confirmed by molecular genetic and biochemical experiments.

Before the physiology of gene expression can be explained, a few specialized genetic and regulatory terms must be defined for prokaryotic systems. In prokaryotes, the genes involved in a metabolic pathway are often present in a linear array called an operon, for example, the Jac operon. An operon can be regulated by a single promoter or regulatory region. The cistron is the smallest unit of genetic expression. A single mRNA that encodes more than one separately translated protein is referred to as a polycistronic mRNA. For example, the polycistronic lac operon mRNA is translated into three separate proteins (see below). Operons and polycistronic mRNAs are common in bacteria but not in eukaryotes.
An inducible gene is one whose expression increases in response to an inducer or activator, a specific positive regulatory signal. In general, inducible genes have relatively low basal rates of transcription. By contrast, genes with high basal rates of transcription are often subject to downregulation by repressors. The expression of some genes is constitutive, meaning that they are expressed at a reasonably constant rate and not known to be subject to regulation. These are often referred to as housekeeping genes. As a result of mutation, some inducible gene products become constitutively expressed. A mutation resulting in constitutive expression of what was formerly a regulated gene is called a constitutive mutation.

Jacob and Monod in 1961 described their operon model in a classic paper. Their hypothesis was to a large extent based on observations on the regulation of lactose metabolism by the intestinal bacterium E. coli. The molecular mechanisms responsible for the regulation of the genes involved in the metabolism of lactose are now among the best-understood in any organism. B-Galactosidase hydrolyzes the B-galactoside lactose to galactose and glucose. The gene encoding B-galactosidase (lacZ) is clustered with the genes encoding lactose permease (JacY) and thiogalactoside transacetylase (lacA). The genes encoding these three enzymes, along with the lac promoter and Jac operator (a regulatory region), and the lacI gene encoding the Lacl repressor are physically linked and constitute the Jac operon as depicted in Figure 38-2.
cf
FIGURE 38-2 The positional relationships of the protein coding and regulatory elements of the ~6kbp /ac operon. lacZ encodes f-galactosidase, lacY encodes a permease, and /acA encodes a thiogalactoside transacetylase. /acl encodes the /ac operon repressor protein. Also shown is the transcription start site for lac operon transcription (TSS). Note that the binding site for the Lacl protein (ie, lac repressor)—the /ac operator (Operator) overlaps the /ac promoter. Immediately upstream of the /ac operon promoter is the binding site (CRE) for the cAMP-binding protein, CAP, the positive regulator of lac operon transcription. See Figure 38-3 for more detail.
This genetic arrangement of the lac operon allows for coordinate expression of the three enzymes concerned with lactose metabolism. Each of the linked operon genes is transcribed into one large polycistronic mRNA molecule that contains multiple independent translation start (AUG) and stop (UAA) codons for each of the three cistrons. Thus, each protein is translated separately, and they are not processed from a single large precursor protein.

Itis now conventional to consider that a gene includes regulatory sequences as well as the region that encodes the primary transcript. Although there are many historical exceptions, a gene is generally italicized in lower case and the encoded protein, when abbreviated, is expressed in roman type with the first letter capitalized. For example, the gene lacI encodes the repressor protein LacI. When E. coli is presented with lactose or some specific lactose analogs under appropriate nonrepressing conditions (eg, high concentrations of lactose, no or very low glucose in media; see below), the expression of the activities of B-galactosidase, galactoside permease, and thiogalactoside transacetylase is increased 100-fold to 1000-fold. This is a type A response, as depicted in Figure 38-1. The kinetics of induction can be quite rapid; lac-specific mRNAs are fully induced within ~5 minutes after addition of lactose to a culture; B-galactosidase protein is maximal within 10 minutes. Under fully induced conditions, there can be up to 5000 B-galactosidase molecules per cell, an amount about 1000 times greater than the basal, uninduced level. Upon removal of the signal, that is, the inducer, the synthesis of these three enzymes declines.

When E. coli is exposed to both lactose and glucose as sources of carbon, the cells first metabolize the glucose and then temporarily stop growing until the genes of the Jac operon become induced to provide the ability to metabolize lactose as a usable energy source. Although lactose is present from the beginning of the bacterial growth phase, the cell does not induce those enzymes necessary for catabolism of lactose until glucose has been exhausted. This phenomenon was first thought to be attributable to repression of the lac operon by some catabolite of glucose; hence, it was termed catabolite repression. It is now known that catabolite repression is in fact mediated by a catabolite activator protein (CAP) in conjunction 3',5' cyclic Adenosine monophosphate (cAMP; see Figure 18-5). This protein is also referred to as the cAMP regulatory protein (CRP). The expression of many inducible enzyme systems or operons in E. coli and other prokaryotes is sensitive to catabolite repression, as discussed below.
The physiology of induction of the lac operon is well understood at the molecular level (Figure 38-3). Expression of the normal Jacl gene of the lac operon is constitutive; it is expressed at a constant rate, resulting in formation of the subunits of the lac repressor. Four identical subunits with molecular weights of 38,000 assemble into a tetrameric Lac repressor molecule. The Lacl repressor protein molecule, the product of lacI, has a very high affinity (dissociation constant, K, about 10°" mol/L) for the operator locus. The operator locus is a region of double-stranded DNA that exhibits a twofold rotational symmetry and an inverted palindrome (indicated by arrows about the dotted axis) in a region that is 21-bp long, as shown below:

.
At any one time, only two of the four subunits of the repressor appear to bind to the operator, and within the 21-base-pair region nearly every base of each base pair is involved in Lacl recognition and binding. The binding occurs mostly in the major groove without interrupting the base-paired, doublehelical nature of the operator DNA. The operator locus is between the promoter site, at which the DNA-dependent RNA polymerase attaches to commence transcription, and the transcription initiation site of the lacZ gene, the structural gene for B-galactosidase (Figure 38-2). When bound to the operator locus, the Lacl repressor molecule prevents transcription of the distal structural genes, lacZ, lacY, and lacA by interfering with the binding of RNA polymerase to the promoter; RNA polymerase and Lacl repressor cannot be effectively bound to the lac operon at the same time. Thus, the Lacl repressor molecule is a negative regulator, and in its presence (and in the absence of inducer; see below), expression from the lacZ, lacY, and lacA genes is very, very low. There are normally about 30 repressor tetramer molecules in the cell, a concentration (3 x 10° mol/L) of tetramer sufficient to effect, at any given time, >95% occupancy of the one lac operator element in a bacterium, thus ensuring low (but not zero) basal Jac operon gene transcription in the absence of inducing signals.
A
lactose analog
that is
capable of
inducing the
Jac operon

while not itself serving as a substrate for B-galactosidase is an example of a gratuitous inducer. An example is isopropylthiogalactoside (IPTG). The addition of lactose or of a gratuitous inducer such as IPTG to bacteria growing on a poorly utilized carbon source (such as succinate) results in prompt induction of the lac operon enzymes. Small amounts of the gratuitous inducer or of lactose are able to enter the cell even in the absence of permease. The LaclI repressor molecules— both those attached to the operator loci and those free in the cytosol—have a high affinity for the inducer. Binding of the inducer to repressor molecule induces a conformational change in the structure of the repressor that causes a decrease in operator DNA occupancy because its affinity for the operator is now 10' times lower (K, about 10° mol/L) than that of Lacl in the absence of IPTG. DNA-dependent RNA polymerase can now bind to the promoter (ie, Figures 36-3 and 36-8), and transcription will begin, although this process is relatively inefficient (see below). In such a manner, an inducer derepresses the lac operon and allows transcription of the genes encoding -galactosidase, galactoside permease, and thiogalactoside transacetylase. Translation of the polycistronic mRNA can occur even before transcription is completed. Derepression of the lac operon allows the cell to synthesize the enzymes necessary to catabolize lactose as an energy source. Based on the physiology just described, IPTG-induced expression of

FIGURE 38-3 The mechanism of repression, derepression, and activation of the lac operon. When no inducer is present (A), the constitutively synthesized /ac! gene products form a repressor tetramer that binds to the operator. Repressor-operator binding prevents the binding of RNA polymerase and consequently prevents transcription of the /acZ, lacY, and lacA structural genes into a polycistronic mRNA. When inducer is present, but glucose is also present in the culture medium (B), the tetrameric repressor molecules are conformationally altered by inducer, and cannot efficiently bind to the operator locus (affinity of binding reduced >1000-fold). However, RNA polymerase will not efficiently bind the promoter and initiate transcription because positive protein-protein interactions between CRE-bound CAP protein fail to occur; thus, the /ac operon is not efficiently transcribed. However, when inducer is present, and glucose is depleted from the medium (C), adenylyl cylase is activated and cAMP is produced. This cAMP binds with high affinity to its binding protein the cyclic AMP activator protein, or CAP. The CAP-cAMP complex binds to its recognition sequence (CRE, the cAMP response element) at Jac operon nucleotide coordinate -50. Direct protein-protein contacts between the CRE-bound CAP and the RNA polymerase increases promoter binding >20-fold; hence RNAP will efficiently transcribe the lac operon and the polycistronic lacZ-lacY-lacA mRNA molecule formed can be translated into the corresponding protein molecules B-galactosidase, permease, and transacetylase as shown. This protein production enables cellular catabolism of lactose as the sole carbon source for growth.
transfected plasmids
bearing the
Jac operator-promoter
ligated to
appropriate bioengineered
constructs is
commonly used

to express mammalian recombinant proteins in E. coli. In order for the RNA polymerase to form a PIC at the promoter site most efficiently, the cAMP-CAP complex must also be present in the cell. By an independent mechanism, the bacterium accumulates cAMP only when it is starved for a source of carbon. In the presence of glucose—or of glycerol in concentrations sufficient for growth—the bacteria will lack sufficient cAMP to bind to CAP because glucose inhibits adenylyl cyclase, the enzyme that converts ATP to cAMP (see Chapter 42). Thus, in the presence of glucose or glycerol, cAMP-saturated CAP is lacking, so that the DNA-dependent RNA polymerase cannot initiate transcription of the lac operon at the maximal rate. However, in the presence of the CAP-cAMP complex, which binds to CAP Response Element (CRE) DNA just upstream of the promoter site, transcription occurs at maximal levels (Figure 38-3). Studies indicate that a region of CAP directly contacts the RNA polymerase (RNAP) a subunit, and these protein-protein interactions facilitate the binding of RNAP to the promoter. Thus, the CAP-cAMP regulator is acting as a positive regulator because its presence is required for optimal gene expression. The lac operon is therefore controlled by two distinct, ligand-modulated DNAbinding trans-factors; one that acts positively (CAMP-CRP complex) to facilitate productive binding of RNA polymerase to the promoter and one that acts negatively (LacI repressor) that antagonizes RNA polymerase promoter binding. Maximal activity of the Jac operon occurs when glucose levels are low (high cAMP with CAP activation) and lactose is present (Lacl is prevented from binding to the operator).

When the Jacl gene has been mutated so that its product, Lacl, is not capable of binding to operator DNA, the organism will exhibit constitutive expression of the lac operon. In a contrary manner, an organism with a lacI gene mutation that produces a LacI protein which prevents the binding of lactose or other small molecule inducer to the repressor will remain repressed even in the presence of the inducer molecule, because such ligands cannot bind to the repressor on the operator locus in order to derepress the operon. Similarly, bacteria harboring mutations in their /ac operator locus such that the operator sequence will not bind a normal repressor molecule will constitutively express the Jac operon genes. Mechanisms of positive and negative regulation comparable to those described here for the lac system have been observed in eukaryotic cells (see below).
The Genetic Switch of Bacteriophage Lambda (A) Provides Another Paradigm for Understanding the Role of ProteinDNA Interactions in Transcriptional Regulation in Eukaryotic Cells
Like some eukaryotic viruses (eg, herpes simplex virus and HIV), certain bacterial viruses can either reside in a dormant state within the host chromosomes or can replicate within the bacterium and eventually lead to lysis and killing of the bacterial host. Some E. coli harbor such a "temperate" virus, bacteriophage lambda (\). When lambda infects an organism of that species, it injects its 45,000-bp, double-stranded, linear DNA genome into the cell (Figure 38-4). Depending on the

FIGURE 38-4 Alternate lifestyles of bacteriophage lambda. Infection of the bacterium E. coli by phage lambda begins when a virus particle attaches itself to specific receptors on the bacterial cell surface (1) and injects its DNA (dark green line) into the cell (2), where the phage genome then circularizes (3). Infection can take either of two courses depending on which of two sets of viral genes is turned on. In the lysogenic pathway, the viral DNA becomes integrated into the bacterial chromosome (red) (4, 5), where it is replicated passively as part of the bacterial DNA during E. coli cell division. This dormant, genomically integrated virus is called a prophage, and the cell that harbors it is called a lysogen. In the alternative, lytic mode of infection, the viral DNA excises from the E. coli chromosome and replicates itself (6) in order to direct the synthesis of viral proteins (7). About 100 new virus particles are formed. The proliferating viruses induce lysis of the cell (8). A prophage can be "induced" by a DNA damaging agent such as ultraviolet radiation (9). The inducing agent throws a switch (see text and Figure 38-5; the A "molecular switch.'), so that a different set of viral genes is turned on. Viral DNA loops out and is excised from the E. coli chromosome (10) and replicates; the virus then proceeds along the lytic pathway. (Reproduced, with permission, from Ptashne M, Johnson AD, Pabo CO: A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)
nutritional state of the cell, the lambda DNA will either integrate into the host genome (lysogenic pathway) and remain dormant until activated (see below), or it will commence replicating until it has made about 100 copies of complete, protein-packaged virus, at which point it causes lysis of its host (lytic pathway). The newly generated virus particles can then infect other susceptible host cells. Poor growth conditions favor lysogeny while good growth conditions promote the lytic pathway of lambda growth.

When integrated into the host genome in its dormant state, lambda will remain in that state until activated by exposure of its bacterial host to DNA-damaging agents. In response to such a noxious stimulus, the dormant bacteriophage becomes "induced" and begins to transcribe and subsequently translate those genes of its own genome that are necessary for its excision from the host chromosome, its DNA replication, and the synthesis of its protein coat and lysis enzymes. This event acts like a trigger or type C (Figure 38-1) response; that is, once dormant lambda has committed itself to induction, there is no turning back until the cell is lysed and the replicated bacteriophage released. This switch from a dormant or prophage state to a lytic infection is well understood at the genetic and molecular levels and will be described in detail here; though less well understood at the molecular level, HIV and herpes viruses can behave similarly, transitioning from dormant to active states in infected humans.
Thus, when the cI repressor gene is on, the cro gene is off, and when the cro gene is on, the cI repressor gene is off. As we shall see, these two genes regulate each other's expression and thus, ultimately, the decision between lytic and lysogenic growth of lambda. This decision between repressor gene transcription and cro gene transcription is a paradigmatic example of a molecular transcriptional switch.

The 80-bp lambda right operator, O,,, can be subdivided into three discrete, evenly spaced, 17-bp cis-active DNA elements that represent the binding sites for either of two bacteriophage lambda regulatory proteins. Importantly, the nucleotide sequences of these three tandemly arranged sites are similar but not identical (Figure 38-5B). The three related cis-elements, termed operators O,1, O,2, and O,3, can be bound by either cl or Cro proteins. However, the relative affinities of cl and Cro for each of the sites vary, and this differential binding affinity is central to the appropriate operation of the lambda phage lytic or lysogenic "molecular switch." The DNA region between the cro and repressor genes also contains two promoter sequences that direct the binding of RNA polymerase in a specified orientation, where it commences transcribing adjacent genes. One promoter directs RNA polymerase to transcribe in the rightward direction and, thus, to transcribe cro and other distal genes, while the other promoter directs the transcription of the cl repressor gene in the leftward direction (Figure 38-5B).
The lytic/lysogenic genetic switching event in lambda is centered around an 80-bp region in its double-stranded DNA genome referred toas the "right operator" (O,) (Figure 38-5A). The right operator is flanked on its left side by the gene for the lambda repressor protein, cI, and on its right side by the gene encoding another regulatory protein, the cro gene. When lambda is in its prophage state—that is, integrated into the host genome—the cI repressor gene is the only lambda gene that is expressed. When the bacteriophage is undergoing lytic growth, the cI repressor gene is not expressed, but the cro gene—as well as many other lambda genes—is expressed.

The product of the cI repressor gene, the 236-amino-acid \ cl repressor protein is a two-domain molecule with amino terminal DNA-binding domain (DBD) and carboxylterminal dimerization domain. Association of one repressor protein with another forms a dimer. cI repressor dimers bind to operator DNA much more tightly than do monomers (Figure 38-6A to 38-6C).
The product of the cro gene, the 66-amino-acid, 9-kDa Cro protein, has a single domain but also binds the operator DNA more tightly as a dimer (Figure 38-6D). The Cro protein's single domain mediates both operator binding and dimerization.
FIGURE 38-5 Genetic organization of the lambda lifestyle "molecular switch." Right operator (O,) is shown in increasing detail in this series of drawings. The operator is a region of the viral DNA some 70-bp long (A). To its left lies the gene encoding lambda repressor (c/), to its right the gene (cro) encoding the regulator protein Cro. When the operator region is enlarged (B), it is seen to include three subregions termed operators: O,1, 0,2, and O,3, each 17-bp long. These three DNA elements are recognition sites to which both A cl repressor and Cro proteins can bind. The recognition sites overlap two divergent promoters—sequences of bases to which RNA polymerase binds in order to transcribe these genes into mRNA (wavy lines) that are translated into protein. Site O,1 is enlarged (C) to show its base sequence. (Reproduced, with permission, from Ptashne M, Johnson AD, Pabo CO: A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)

FIGURE 38-6 Schematic molecular structures of lambda regulatory proteins cl and Cro. The lambda repressor protein is a 236-amino-acid polypeptide. The chain folds itself into a dumbbell shape with two substructures: an amino terminal (NH,) domain and a carboxyl-terminal (COOH) domain. The two domains are linked by a region of the chain that is less structured and susceptible to cleavage by proteases (indicated by the two arrows in A). Single repressor molecules (monomers) tend to reversibly associate to form dimers. (B) A dimer is held together mainly by contact between the carboxyl-terminal domains (hatching). cl repressor dimers bind to (and can dissociate from) the recognition sites in the operator region; they display differential affinites for the three operator sites, O,1 > O,2 > 0,3 (C). It is the DNA-binding domain (DBD) of the repressor molecule that makes contact with the DNA (hatching). Cro (D) has a single domain that promotes cro-cro dimerization, and a DBD that promotes binding of dimers to operator. It is important that cro exhibits the highest affinity for O,3, opposite the sequence binding preference of the cl protein. (Reproduced, with permission, from Ptashne M, Johnson AD, Pabo CO: A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)

In a lysogenic bacterium—that is, a bacterium containing an integrated dormant lambda prophage—the lambda repressor dimer binds preferentially to O,1 but in so doing, by a cooperative interaction, enhances the binding (by a factor of 10) of another repressor dimer to O,2 (Figure 38-7). The affinity of repressor for O,3 is the least of the three operator subregions. The binding of repressor to O,1 has two major effects. First, occupancy of O,1 by repressor blocks the binding of RNA polymerase to the rightward promoter and in that way prevents expression of cro. Second, as mentioned above, repressor dimer bound to O,1 enhances the binding of repressor dimer to O,2. The binding of repressor to O,2 has the important added effect of enhancing the binding of RNA polymerase to the leftward promoter that overlaps O,3 and thereby enhances transcription and subsequent expression of the repressor gene. This enhancement of transcription is mediated through direct protein-protein interactions between promoter-bound RNA polymerase and O,2-bound repressor, much as described above for CAP protein and RNA polymerase on the lac operon. Thus, the \ cI protein is both a negative regulator, by preventing transcription of cro, and a positive regulator, by enhancing transcription of its own gene, cI. This dual effect of repressor is responsible for the stable state of the dormant lambda bacteriophage; not only does the repressor prevent expression of the genes necessary for lysis, but it also promotes expression of itself to stabilize this state of differentiation. In the event that intracellular repressor protein concentration becomes very high, the excess repressor will bind to O,3 and by so doing diminish transcription of the repressor gene from the leftward promoter, by blocking RNAP binding to the cI promoter, until the repressor concentration drops and repressor dissociates from O,3. Similar examples of repressor proteins also having the ability to activate transcription have been observed in eukaryotes.

With such a stable, repressive, cl-mediated, lysogenic state, one might wonder how the lytic cycle could ever be entered. However, this process does occur quite efficiently. When a DNA-damaging signal, such as ultraviolet light, strikes the lysogenic host bacterium, fragments of single-stranded DNA are generated that activate a specific coprotease coded by a bacterial gene and referred to as recA (Figure 38-7). The activated recA protease hydrolyzes the portion of the repressor protein that connects the amino-terminal and carboxylterminal domains of that molecule (see Figure 38-6A). Such cleavage of the repressor domains causes the repressor dimers to dissociate, which in turn causes dissociation of the repressor molecules from O,2 and eventually from O,1. The effects of removal of repressor from O,1 and O,2 are predictable. RNA polymerase immediately has access to the rightward promoter and commences transcribing the cro gene, and the enhancement effect of the repressor at O,2 on leftward transcription is lost (Figure 38-7).
The resulting newly synthesized Cro protein also binds to the operator region as a dimer, but as noted above, its order of preference is opposite to that of repressor (Figure 38-7). That is, Cro binds most tightly to 0,3, but there is no cooperative effect of Cro at O,3 on the binding of Cro to O,2. At increasingly higher concentrations of Cro, the protein will bind to O,2 and eventually to O,1.
Occupancy of O,3 by Cro immediately turns off transcription from the leftward cI promoter and in that way prevents any further expression of the repressor gene. The molecular switch is thus completely "thrown" in the lytic direction. The cro gene is now expressed, and the repressor gene is fully turned off. This event is irreversible, and the expression of other lambda genes begins as part of the lytic cycle. When Cro repressor concentration becomes quite high, it will eventually occupy O,1 and in so doing reduce the expression of its own

FIGURE 38-7 Configuration of the lytic/lysogenic switch is shown at four stages of the lambda life cycle. The lysogenic pathway (in which the virus remains dormant as a prophage) is selected when a repressor dimer binds to O,1, thereby making it likely that O,2 will be bound immediately by another dimer due to the cooperative nature of cl-O, DNA binding. In the prophage (top), the repressor dimers bound at O,1 and O,2 prevent RNA polymerase from binding to the rightward cro promoter and so block the synthesis of cro (negative control). Simultaneously these DNA-bound cl proteins enhance the binding of polymerase to the leftward promoter (positive control), with the result that the repressor gene is transcribed into RNA (initiation at c/ gene transcription start site; TSS) and more repressor is synthesized, maintaining the lysogenic state. The prophage is induced (middle) when ultraviolet radiation activates the protease recA, which cleaves cl repressor monomers. Induction (1) The equilibrium of free monomers, free dimers, and bound dimers is thereby shifted by mass action, and dimers dissociate from the operator sites. RNA polymerase is no longer stimulated to bind to the leftward promoter, so that repressor is no longer synthesized. As induction proceeds, Induction (2) all the operator sites become vacant, thus polymerase can bind to the rightward promoter and cro is synthesized (cro TSS shown). During early lytic growth, a single cro dimer binds to O,3 (light blue shaded circles), the site for which it has the highest affinity thereby occluding the c/ promoter. Consequently, RNA polymerase cannot bind to the leftward promoter, but the rightward promoter remains accessible. Polymerase continues to bind there, transcribing cro and other early lytic genes. Lytic growth ensues (bottom). (Reproduced, with permission, from Ptashne M, Johnson AD, Pabo CO: A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)
gene, a process that is necessary in order to effect the final
stages of the lytic cycle.
The three-dimensional structures of Cro and of the
lambda repressor protein have been determined by x-ray
__

crystallography, and models for their binding and effecting the above-described molecular and genetic events have been proposed and tested. Both bind to DNA using helix-turnhelix DBD motifs (see below). Along with regulation of the
expression of the Jac operon, the \ molecular switch described here provides arguably the best understanding of the molecular events involved in gene transcription activation and repression.
Detailed analysis of the \ repressor led to the important concept that transcription regulatory proteins have several functional domains. For example, lambda repressor binds to DNA with high affinity. Repressor monomers form dimers, cooperatively interact with each other, and repressor interacts with RNA polymerase, to enhance or block promoter binding or RNAP open complex formation (see Figure 36-3). The protein-DNA interface and the three protein-protein interfaces all involve separate and distinct domains of the repressor molecule. As will be noted below (see Figure 38-19), this is a characteristic that is typical of most molecules that regulate transcription.

Most of the DNA in prokaryotic cells is organized into genes, and since the DNA is not compacted with nucleosomal histones, it always has the potential to be transcribed if appropriate positive and negative trans-factors are present in a given cell in an active form. A very different situation exists in eukaryotic cells where relatively little of the total DNA is organized into mRNA-encoding genes and their associated regulatory regions. The function of the extra DNA is being actively investigated (ie, Chapter 39; the ENCODE Projects). More importantly, as described in Chapter 35, the DNA in eukaryotic cells is extensively folded and packed into the protein-DNA complex called chromatin. Histones are an important part of this complex since they both form the structures known as nucleosomes (see Chapter 35) and also factor significantly into gene regulatory mechanisms as outlined below.

Chromatin structure provides an additional level of control of gene transcription. As discussed in Chapter 35, large regions of chromatin are transcriptionally inactive while others are either active or potentially active. With few exceptions, each cell contains the same complement of genes, hence, the development of specialized organs, tissues, and cells, and their function in the intact organism depend on the differential expression of genes.
Some of this differential expression is achieved by having different regions of chromatin available for transcription in cells from various tissues. For example, the DNA containing the B-globin gene cluster is in "active" chromatin in the reticulocyte but in "inactive" chromatin in muscle cells.
All the factors involved in the determination of active chromatin have not been elucidated. The presence of nucleosomes and of complexes of histones and DNA (see Chapter 35) certainly provides a barrier against the ready association of transcription factors with specific DNA regions. The dynamics of the formation and disruption of nucleosome structure are therefore an important part of eukaryotic gene regulation.

Histone covalent modification, also dubbed the histone code, is an important determinant of gene activity. Histones are subjected to a wide range of specific posttranslational modifications (see Table 35-1). These modifications are dynamic and reversible. Histone acetylation and deacetylation are best understood. The surprising discovery that histone acetylase and other enzymatic activities are associated with the coregulators involved in regulation of gene transcription (see Chapter 42 for specific examples) has provided a new concept of gene regulation. Acetylation is known to occur on lysine residues in the amino terminal tails of histone molecules, and has been consistently correlated with either active transcription, or alternatively, transcriptional potential. Histone acetylation reduces the positive charge of these tails and likely contributes to a decrease in the binding affinity of histone for the negatively charged DNA. Moreover, such covalent modification of the histones creates new binding, or docking sites for additional proteins such as ATP-dependent chromatin remodeling complexes that contain subunits that carry structural domains that specifically bind to histones that have been subjected to coregulator-deposited PTMs. These complexes can increase accessibility of adjacent DNA sequences by removing or otherwise altering nucleosomal histones. Together then coregulators (chromatin modifiers and chromatin remodellers), working in conjunction, can "open up" gene promoters and regulatory regions, facilitating binding of other trans-factors and RNA polymerase II and GTFs (see Figures 36-10 and 36-11). Histone deacetylation catalyzed by transcriptional corepressors would have the opposite effect. Different proteins with specific acetylase and deacetylase activities are associated with various components of the transcription apparatus. The proteins that catalyze the histone PTMs are sometimes referred to as "code writers" while the proteins that recognize, bind, and thus interpret these histone PTMs are termed "code readers" and the enzymes that remove histone PTMs are called "code erasers." (The analogy to signal transduction, with its kinases, phosphatases and phospho-amino acid binding proteins should be apparent—see Chapter 42.) Collectively then, these histone PTMs represent a very dynamic, potentially information-rich source of regulatory information. The exact rules and mechanisms defining the specificity of these various processes are under investigation. Some specific

examples are illustrated in Chapter 42. A variety of commercial enterprises are working to develop drugs that specifically alter the activity of the proteins that orchestrate the histone code.
to the
histone code
(see
Chapter
35)
and its
effects on all DNA-mediated reactions, the methylation of deoxycytidine residues, 5MeC, (in the sequence 5'-"*CpG-3') in DNA has important effects on chromatin, some of which
lead to a decrease in gene transcription. For example, in mouse liver, only the unmethylated ribosomal genes can be expressed, and there is evidence that many animal viruses are not transcribed when their DNA is methylated. Acute demethylation of 5MeC residues in specific regions of steroid hormone inducible genes has been associated with an increased rate of transcription of the gene. However, it is not yet possible to generalize that methylated DNA is transcriptionally inactive, that all inactive chromatin is methylated, or that active DNA is not methylated.
Finally, the binding of specific transcription factors to cognate DNA elements may result in disruption of nucleosomal structure. Most eukaryotic genes have multiple proteinbinding DNA elements. The serial binding of transcription factors to these elements—in a combinatorial fashion—may either directly disrupt the structure of the nucleosome, prevent its reformation, or recruit, via protein-protein interactions, multiprotein coregulator complexes that have the ability to covalently modify and/or remodel nucleosomes. These reactions result in chromatin-level structural changes that in the end increase or decrease DNA accessibility to other factors and the transcription machinery.
Eukaryotic DNA that is in an "active" region of chromatin can be transcribed. As in prokaryotic cells, a promoter dictates where the RNA polymerase will initiate transcription, but the promoter in mammalian cells (see Chapter 36) is more complex. Additional complexity is added by elements or factors that enhance or repress transcription, define tissuespecific expression, and modulate the actions of many effector molecules. Finally, recent results suggest that gene activation and repression might occur when particular genes move into or out of different subnuclear compartments or locations.

The molecules and regulatory biology described above contributes importantly to transcriptional regulation. Indeed, in recent years the role of covalent modification of DNA and histone (and nonhistone) proteins and the newly discovered ncRNAs has received tremendous attention in the field of gene regulation research, particularly through investigation into how such chemical modifications and/or molecules stably alter gene expression patterns without altering the underlying DNA gene sequence. This field of study has been termed epigenetics. As mentioned in Chapter 35, one aspect of these mechanisms, PTMs of histones has been dubbed the histone code or histone epigenetic code. The term "epigenetics" means "above genetics" and refers to the fact that these regulatory mechanisms do not change the underlying regulated DNA sequence, but rather simply the expression patterns, or function, of this DNA. Epigenetic mechanisms play key roles in the establishment, maintenance, and reversibility of transcriptional states. A key feature of epigenetic mechanisms is that the controlled transcriptional on/off states can be maintained through multiple rounds of cell division. This observation indicates that there must be robust, biochemically based mechanisms to maintain and stably propagate these epigenetic states.
Two forms of epigenetic signals, cisand transepigenetic signals, can be described; these are schematically illustrated in Figure 38-8. A simple trans-signaling event composed of positive transcriptional feedback mediated by an abundant, diffusible transactivator that efficiently partitions roughly equally between mother and daughter cell at each division is depicted in Figure 38-8A. So long as the indicated, transcription factor is expressed at a sufficient level to allow all subsequent daughter cells to inherit the trans-epigenetic signal (transcription factor), such cells will have the cellular or molecular phenotype dictated by the other target genes of this transcriptional activator. Shown in Figure 38-8 panel B is an example of how a cis-epigenetic signal (here as a specific 5MeCpG methylation mark) can be stably propagated to the two daughter cells following cell division. The hemimethylated (ie, only one of the two DNA strands is 5MeC modified) DNA mark generated during DNA replication directs the methylation of the newly replicated strand through the action of ubiquitous maintenance DNA methylases. This 5MeC methylation results in both DNA daughter strands having the complete cis-epigenetic mark.

Both cisand trans-epigenetic signals can result in stable and hereditable expression states, and therefore generally represent type C gene expression responses (ie, Figure 38-1). However, it is important to note that both states can be reversed if either the transor cis-epigenetic signals are removed by, for example, extinguishing the expression of the enforcing transcription factor (trans-signal) or by completely removing a DNA cis-epigenetic signal (via DNA demethylation). Enzymes have been described that can remove both protein PTMs and 5MeC modifications.
Stable transmission of epigenetic on/off states can be affected by multiple molecular mechanisms. Shown in Figure 38-9 are three ways by which cis-epigenetic marks can be propagated through a round of DNA replication. The first example of epigenetic mark transmission involves the propagation of DNA 5MeC marks, and occurs as described above in Figure 38-8. The second example of epigenetic state transmission illustrates how a nucleosomal histone PTM (in this example, Lysine K-27 trimethylated histone H3; H3K27me3) can be propagated. In this example immediately following DNA replication, both H3K27me3-marked and H3-unmarked nucleosomes randomly reform on both daughter DNA strands. The polycomb repressive complex 2 (PRC2), composed of EEDSUZ12-EZH2 and RbAP subunits, binds to the nucleosome containing the preexisting H3K27me3 mark via the EED subunit. Binding of PRC2 to this histone mark stimulates the methylase activity of the EZH2 subunit of PRC2, which results in the local methylation of nucleosomal H3. Histone H3 methylation thus causes the full, stable transmission of the H3K27me3 epigenetic mark to both chromatids. Finally, locus/sequence-specific targeting of nucleosomal histone
B
------
>
P
Cis epigenetic
signal

FIGURE 38-8 isand trans-epigenetic signals. (A) An example of an epigenetic signal that acts in trans. A DNA-binding transactivator protein (yellow circle) is transcribed from its cognate gene (yellow bar) located on a particular chromosome (blue). The expressed protein is freely diffusible between nuclear and cytoplasmic compartments. Note that excess transactivator reenters the nucleus following cell division, binds to its own gene, and activates transcription in both daughter cells. This cycle reestablishes the positive feedback loop in effect prior to cell division, and thereby enforces stable expression of this transcriptional activator protein in both cells. (B) A cis-epigenetic signal; a gene (pink) located on a particular chromosome (blue) carries a cis-epigenetic signal (small yellow flag) within the regulatory region upstream of the pink gene transcription unit. In this case, the epigenetic signal is associated with active gene transcription and subsequent gene product production (pink circles). During DNA replication, the newly replicated chromatid serves as a template that both elicits and templates the introduction of the same epigenetic signal, or mark, on the newly synthesized, unmarked chromatid. Consequently, both daughter cells contain the pink gene in a similarly cis-epigenetically marked state, which ensures expression in an identical fashion in both cells. See text for more detail. (Image taken from: Bonasio, R, Tu S, Reinberg D: Molecular signals of epigenetic states. Science 2010;330:612-616. Reprinted with permission from AAAS.)
epigenetic cis-signals can be attained through the action of IncRNAs as depicted in Figure 38-9, panel C. Here a specific ncRNA interacts with target DNA sequences and the resulting RNA-DNA complex is recognized by RBP, an RNA-binding protein. Then, likely through a specific adaptor protein (A), the RNA-DNA-RBP complex recruits a chromatin modifying complex (CMC) that locally modifies nucleosomal histones. Again, this mechanism leads to the transmission of a stable epigenetic mark.

Additional work will be required to establish the complete molecular details of these epigenetic processes, determine how ubiquitously these mechanisms operate, identify the full complement of molecules involved, and genes controlled. Epigenetic signals are critically important to gene regulation as evidenced by the fact that mutations and/or overexpression of many of the molecules that contribute to epigenetic control
lead to human disease.

In addition to gross changes in chromatin affecting transcriptional activity, certain DNA elements facilitate or enhance initiation at the promoter and hence are termed enhancers. Enhancer elements, which typically contain multiple binding sites for transactivator proteins, differ from the promoter in notable ways. They can exert their positive influence on transcription even when separated by tens of thousands of base pairs from a promoter; they work when oriented in either
FIGURE 38-9 Mechanisms for the transmission and propagation of epigenetic signals following a round of DNA replication. (A) Propagation of a 5MeC signal (yellow flag; see Figure 38-8B). (B) Propagation of a histone PTM epigenetic signal (H3K27me) that is mediated through the action of the PRC2, a chromatin modifying complex, or CMC. PRC2 is composed of EED, EZH2 histone methylase, RbAP, and SUZ12 subunits. Note that in this context, PRC2 is both a histone code reader (via the methylated histone-binding domain in EED) and histone code writer (via the SET domain histone methylase within EZH2). Location-specific deposition of the histone PTM cis-epigenetic signal is targeted by the recognition of the H3K27me marks in preexisting nucleosomal histones (yellow flag). (C) Another example of the transmission of a histone epigenetic signal (yellow flag) except here signal-targeting is mediated through the action of small ncRNAs that work in concert with an RNAbinding protein (RBP), an adaptor (A) protein, and a CMC. See text for more detail. (Image taken from: Bonasio R, Tu S, Reinberg D: Molecular signals of epigenetic states. Science 2010;330:612-616. Reprinted with permission from AAAS.)

direction; and they can work upstream (5') or downstream (3') from the promoter. Experimentally, enhancers can be shown to be promiscuous, in that they can stimulate transcription of any promoter in their vicinity, and may act on more than one promoter. The viral SV40 enhancer can exert an influence on, for example, the transcription of B-globin by increasing its transcription 200-fold in cells containing both the SV40 enhancer and the $-globin gene on the same plasmid (see below and Figure 38-10); in this case the SV40 enhancer B-globin gene was constructed using recombinant DNA technology—see Chapter 39. The enhancer element does not produce a product that in turn acts on the promoter, since it is active only when it exists within the same DNA molecule as (ie, in cis, or physically linked to) the promoter. Enhancerbinding proteins are responsible for this effect. The exact mechanisms by which these transcription activators work are subject to intensive investigation. Enhancer-binding transfactors, some of which are cell-type specific, while others are ubiquitously expressed, have been shown to interact with a plethora of other transcription proteins. These interactions include chromatin-modifying coactivators, mediator, as well as the individual components of the basal RNA polymerase II transcription machinery. Ultimately, transfactor-enhancer DNA-binding events result in an increase in the binding and/or activity of the basal transcription machinery on the linked promoter. Enhancer elements and associated

FIGURE 38-10 A schematic illustrating the methods used to study the organization and action of enhancers and other cis-acting regulatory elements. These model chimeric genes, all constructed by recombinant DNA techniques in vitro (see Chapter 39), consist of a reporter gene that encodes a protein that can be readily assayed, and that is not normally produced in the cells to be studied, a promoter that ensures accurate initiation of transcription, and the indicated enhancer (regulatory response) elements. In all cases, high-level transcription from the indicated chimeras depends on the presence of enhancers, which stimulate transcription =>100-fold over basal transcriptional levels (ie, transcription of the same chimeric genes containing just promoters fused to the indicated reporter genes). Examples (A) and (B) illustrate the fact that enhancers (eg, here SV40) work in either orientation and upon a heterologous promoter. Example (C) illustrates that the metallothionein (mt) regulatory element (which under the influence of cadmium or zinc induces transcription of the endogenous mt gene and hence the metal-binding mt protein) will work through the herpes simplex virus (HSV) thymidine kinase (tk) gene promoter to enhance transcription of the human growth hormone (hGH) reporter gene. In a separate experiment, this engineered genetic construct was introduced into the male pronuclei of single-cell mouse embryos and the embryos placed into the uterus of a surrogate mother to develop as transgenic animals. Offspring have been generated under these conditions, and in some the addition of zinc ions to their drinking water effects an increase in growth hormone expression in liver. In this case, these transgenic animals have responded to the high levels of growth hormone by becoming twice as large as their normal litter mates. Example (D) illustrates that a glucocorticoid response element (GRE) enhancer will work through homologous (PEPCK gene) or heterologous gene promoters (not shown; ie, HSV tk promoter, SV40 promoter, B-globin promoter, etc) to drive expression of the chloramphenicol acetyltransferase (CAT) reporter gene.

binding proteins often convey nuclease hypersensitivity to those regions where they reside (see Chapter 35). A summary of the properties of enhancers is presented in Table 38-2. One of the best-understood mammalian enhancer systems is that of the B-interferon gene. This gene is induced on viral infection of mammalian cells. One goal of the cell, once virally infected, is to attempt to mount an antiviral response— if not to save the infected cell, then to help to save the entire organism from viral infection. Interferon production is one

•, TABLE 38-2 Summary of the Properties of Enhancers = Work when located long distances from the promoter. •, TABLE 38-2 Summary of the Properties of Enhancers = Work when upstream or downstream from the promoter. •, TABLE 38-2 Summary of the Properties of Enhancers = Work when oriented in either direction. •, TABLE 38-2 Summary of the Properties of Enhancers = Can work with homologous or heterologous promoters. •, TABLE 38-2 Summary of the Properties of Enhancers = Work by binding one or more proteins. •, TABLE 38-2 Summary of the Properties of Enhancers = Work by recruiting chromatin-modifying coregulatory complexes. •, TABLE 38-2 Summary of the Properties of Enhancers = Work by facilitating binding or function of the basal transcription complex at the cis -linked promoter

mechanism by which this is accomplished. This family of proteins is secreted by virally infected cells. Secreted interferon interacts with neighboring cells to cause an inhibition of viral replication by a variety of mechanisms, thereby limiting the extent of viral infection. The enhancer element controlling induction of the B-interferon gene, which is located between nucleotides —110 and —45 relative to the transcription start site (+1), is well characterized. This enhancer consists of four distinct clustered cis-elements, each of which is bound by unique trans-factors. One cis-element is bound by the transacting factor NF-«B (see Figures 42-10 and 42-13) one by a member of the interferon regulatory factor (IRF) family of transactivator factors, and a third by the heterodimeric leucine zipper factor ATF-2/c-Jun (see below). The fourth factor is the ubiquitous, abundant architectural transcription factor known as HMG I(Y). Upon binding to its A + T-rich binding sites, HMG I[(Y) induces a significant bend in the DNA. There are four such HMG I(Y) binding sites interspersed throughout the enhancer. These sites play a critical role in forming a particular 3D structure, along with the aforementioned three transfactors, by inducing a series of critically spaced DNA bends. Consequently, HMG I(Y) induces the cooperative formation of a unique, stereospecific, 3D structure within which all four factors are active when viral infection signals are sensed by the cell. The structure formed by the cooperative assembly of these four factors is termed the B-interferon enhanceosome (Figure 38-11), so named because of its obvious structural similarity to the nucleosome, which is also a unique threedimensional protein-DNA structure that wraps DNA about a core assembly of proteins (see Figures 35-1 and 35-2). The enhanceosome, once formed, induces a large increase in B-interferon gene transcription upon virus infection. It is not simply the protein occupancy of the linearly apposed ciselement sites that induces B-interferon gene transcription— rather, it is the formation of the enhanceosome proper that provides appropriate surfaces for the efficient recruitment of coactivators that results in the enhanced formation of the PIC on the cis-linked promoter and thus transcription activation.

cis-Acting DNA elements that decrease the expression of specific genes are termed silencers. Silencers have also been identified in a number of eukaryotic genes. However, because fewer of these elements have been intensively studied, it is not possible to formulate accurate generalizations about their

FIGURE 38-11 Formation and putative structure of the enhanceosome formed on the human B-interferon gene enhancer. Diagrammatically represented at the top is the distribution of the multiple cis-elements (HMG, PRDIV, PRDI-III, PRDII, NRDI) composing the B-interferon gene enhancer. The intact enhancer mediates transcriptional induction of the B-interferon gene (/FNB7) over 100-fold upon virus infection of human cells. The cis-elements of this modular enhancer represent the binding sites for the transfactors HMG I(Y), cJun-ATF-2, IRF3-IRF7, and NF-xB, respectively. The factors interact with these DNA elements in an obligatory, ordered, and highly cooperative fashion as indicated by the arrow. Initial binding of four HMG I(Y) proteins induces sharp DNA bends in the enhancer, causing the entire 70to 80-bp region to assume a high level of curvature. This curvature is integral to the subsequent highly cooperative binding of the other trans-factors since bending enables the DNA-bound factors to make critical direct protein-protein interactions that both contribute to the formation and stability of the enhanceosome and generate a unique 3D surface that serves to recruit chromatin-modifying coregulators that carry enzymatic activities (eg, Swi/Snf: ATPase, chromatin remodeler and P/CAF: histone acetyltransferase) as well as the general transcription machinery (RNA polymerase II and GTFs). Although four of the five cis-elements (PRDIV, PRDI-III, PRDII, NRDI) independently can modestly stimulate (~10-fold) transcription of a reporter gene in transfected cells (see Figures 38-10 and 38-12), all five cis-elements, in appropriate order, are required to form an enhancer that can appropriately stimulate transcription of /FNB7 (ie, >100-fold) in response to viral infection of a human cell. This distinction indicates the strict requirement for appropriate enhanceosome architecture for efficient trans-activation. Similar enhanceosomes, involving distinct cisand trans-factors and coregulators, are proposed to form on many other mammalian genes.

mechanism of action. That said, it is clear that as for gene activation, chromatin level covalent modifications of histones, and other proteins, by silencer-recruited repressors and corecruited multisubunit corepressors likely play central roles in these regulatory events.

Most genes are now recognized to harbor enhancer elements in various locations relative to their coding regions. In addition to being able to enhance gene transcription, some of these enhancer elements clearly possess the ability to do so in a tissue-specific manner. By fusing known or suspected tissuespecific enhancers or silencers to reporter genes (see below) and introducing these chimeric enhancer-reporter constructs via microinjection into single-cell embryos, one can create a transgenic animal (see Chapter 39), and rigorously test whether a given test enhancer or silencer truly modulates expression in a cellor tissue-specific fashion. This transgenic animal approach has proved useful in studying tissue-specific gene expression.

By ligating regions of DNA suspected of harboring regulatory sequences to various reporter genes (the reporter or chimeric gene approach) (Figures 38-10, 38-12, and 38-13), one can determine which regions in the vicinity of structural genes have an influence on their expression. Pieces of DNA thought to harbor regulatory elements, often identified by bioinformatic sequence alignments, are ligated to a suitable reporter gene and introduced into a host cell (Figure 38-12). Expression of the reporter gene will be increased if the DNA contains a particular enhancer. For example, addition of different hormones to separate cultures will increase expression of the reporter gene if the DNA contains a particular hormone response DNA element (HRE) (Figure 38-13; see also Chapter 42). The location of the element can be pinpointed by using progressively shorter pieces of DNA, deletions, or point mutations (Figure 38-13).
This strategy, typically performed using transfected cells in culture (ie, cells induced to take up exogenous DNAs), has led to the identification of hundreds of enhancers, silencers/ repressors such as tissue-specific elements, and hormone, heavy metal, and drug-response elements. The activity of a gene at any moment reflects the interaction of these numerous cis-acting DNA elements with their respective trans-acting factors. Overall, transcriptional output is determined by the balance of positive and negative signaling to the transcription machinery. The challenge now is to figure out exactly how this regulation occurs at the molecular level so that we might ultimately have the ability to modulate gene transcription therapeutically.

Prokaryotic genes are often regulated in an on-off manner in response to simple environmental cues. Some eukaryotic genes are regulated in the simple on-off manner, but the process in most genes, especially in mammals, is much more complicated. Signals representing a number of
complex environmental stimuli may converge on a single gene. The response of the gene to these signals can have several physiologic characteristics. First, the response may extend over a considerable range. This is accomplished by having additive and synergistic positive responses counterbalanced by negative or repressing effects. In some cases, either the positive or the negative response can be dominant. Also required is a mechanism whereby an effector, such as a hormone, can activate some genes in a cell while repressing others and leaving still others unaffected. When all of these processes are coupled with tissue-specific element factors, considerable flexibility is afforded. These physiologic variables obviously require an arrangement much more complicated than an on-off switch. The collection and organization of DNA elements in a promoter specifies—via associated factors—how a given gene will respond, and how long a particular response is maintained. Some simple examples are illustrated in Figure 38-14.

The large number of genes in eukaryotic cells and the complex arrays of transcription regulatory factors present an organizational problem. Why are some genes available for transcription in a given cell whereas others are not? If enhancers can regulate several genes from tens of kilobase distances and are not positionand orientation-dependent, how are they prevented from triggering transcription of all cis-linked genes in the vicinity? Part of the solution to these problems is arrived at by having the chromatin arranged in functional units that restrict patterns of gene expression. This may be achieved by having the chromatin
form a structure with the nuclear matrix or other physical entity or compartment within the nucleus. Alternatively, some regions are controlled by complex DNA elements called locus control regions (LCRs). An LCR—with associated bound proteins— controls the expression of a cluster of genes. The best-defined LCR regulates expression of the globin gene family over a large region of DNA. Another mechanism is provided by insulators. These DNA elements, also in association with one or more proteins, prevent an enhancer from acting on a promoter on the other side of an insulator in another transcription domain. Insulators thus serve as transcriptional boundary elements. In the globin gene cluster, and many other genes, enhancer and promoter sequences are brought into physical contact via specific DNA looping events. The rules controlling such chromosome looping are currently under intense study.

The specificity involved in the control of transcription requires that regulatory proteins bind with high affinity and specificity to the correct region of DNA. Three unique motifs—the helixturn-helix, the zinc finger, and the leucine zipper—account

Helix-turn-helix, Organism = E. coli Phage Mammals. Helix-turn-helix, Regulatory Protein = lac repressor, CAP λcI, cro, and 434 repressors Homeobox proteins Pit-1, Oct1, Oct2. Zinc finger, Organism = E. coli Yeast Drosophila Xenopus Mammals. Zinc finger, Regulatory Protein = Gene 32 protein Gal4 Serendipity, hunchback TFIIIA Steroid receptor family, Sp1. Leucine zipper, Organism = Yeast Mammals. Leucine zipper, Regulatory Protein = GCN4 C/EBP, fos, Jun, Fra-1, CRE binding protein (CREB), c- myc , n- myc , I- myc
for many of these specific protein-DNA interactions. Examples of proteins containing these motifs are given in Table 38-3. Comparison of the binding activities of the proteins that contain these motifs leads to several important generalizations.
1. Binding must be of high affinity to the specific site and of low affinity to other DNA.
2. Small regions of the protein make direct contact with DNA; the rest of the protein, in addition to providing the trans-activation domains, may be involved in the dimerization of monomers of the binding protein, may provide a contact surface for the formation of heterodimers, may provide one or more ligand-binding sites, or may provide surfaces for interaction with coactivators, corepressors, or the transcription machinery.
3. The protein-DNA interactions made by these proteins are maintained by hydrogen bonds, ionic interactions, and van der Waals forces.
4, The motifs found in these proteins are class-specific; their presence in a protein of unknown function suggests that the protein may bind to DNA.
5. Proteins with the helix-turn-helix or leucine zipper motifs form dimers, and their respective DNA-binding sites are symmetric palindromes. In proteins with the zinc finger motif, the binding site is repeated two to nine times. These features allow for cooperative interactions between binding sites and enhance the degree and affinity of binding.

The first motif described was the helix-turn-helix. Analysis of the 3D structure of the lambda cro transcription regulator has revealed that each monomer consists of three antiparallel 6 sheets and three a helices (Figure 38-15). The dimer forms
FIGURE 38-15 A schematic representation of the 3D structure of Cro protein and its binding to DNA by its helix-turn-helix motif (left). The Cro monomer consists of three antiparallel B sheets (B,-B,) and three a helices (a,-a,). The helix-turn-helix (HTH) motif is formed because the a, and a, helices are held at about 90° to each other by a turn of four amino acids. The a, helix of Cro is the DNA recognition surface (shaded). Two monomers associate through interactions between the two antiparallel 8, sheets to form a dimer that has a twofold axis of symmetry (right). A Cro dimer binds to DNA through its a, helices, each of which contacts about 5 bp on the same face of the major groove (see Figures 34-2 and 38-6). The distance between comparable points on the two DNA a helices is 34 A, the distance required for one complete turn of the double helix. (Reprinted with permission from B Mathews.)
by association of the antiparallel 8, sheets. The a, helices form the DNA recognition surface, and the rest of the molecule appears to be involved in stabilizing these structures. The average diameter of an a helix is 1.2 nm, which is the approximate width of the major groove in the B form of DNA. The DNA recognition domain of each Cro monomer interacts with 5 bp and the dimer-binding sites span 3.4 nm, allowing fit into successive half turns of the major groove on the same surface of DNA (Figure 38-15). X-ray analyses of the A cI repressor, CAP (the cAMP receptor protein of E. coli), tryptophan repressor, and phage 434 repressor, all also display this dimeric helix-turn-helix structure that is present in eukaryotic DNA-binding proteins as well (Table 38-3).

the DNA helix, with successive fingers alternatively positioned in one turn in the major groove. As is the case with the recognition domain in the helix-turn-helix protein, each TFIIIA zinc finger contacts about 5 bp of DNA. The importance of this motif in the action of steroid hormones is underscored by an "experiment of nature" A single amino acid mutation in either of the two zinc fingers of the 1,25(OH),-D, receptor

The zinc finger was the second DNA-binding motif whose atomic structure was elucidated. It was known that the protein TFIIIA, a positive regulator of 5S RNA gene transcription, required zinc for activity. Structural and biophysical analyses revealed that each TFIIIA molecule contains nine zinc ions ina repeating coordination complex formed by closely spaced cysteine-cysteine residues followed 12 to 13 amino acids later by a histidine-histidine pair (Figure 38-16). In some instances— notably the steroid-thyroid nuclear hormone receptor family—the His-His doublet is replaced by a second Cys-Cys pair. The zinc finger motifs of the protein lie on one face of
FIGURE 38-16 Zinc fingers are a series of repeated domains (two to nine) in which each is centered on a tetrahedral coordination with zinc. In the case of the DNA-binding transcription factor TFIIIA, the coordination is provided by a pair of cysteine residues (C) separated by 12 to 13 amino acids from a pair of histidine (H) residues. In other zinc finger proteins, the second pair also consists of C residues. Zinc fingers bind in the major groove, with adjacent fingers making contact with 5 bp along the same face of the helix.

Analysis of a 30-amino-acid sequence in the carboxyl-terminal region of the enhancer-binding protein C/EBP revealed a novel structure, the leucine zipper motif. As illustrated in Figure 38-17, this region of the protein forms an a helix in which there is a periodic repeat of leucine residues at every seventh position. This occurs for eight helical turns and four leucine repeats. Similar structures have been found in a number of other proteins associated with the regulation of transcription in all eukaryotes tested. This structure allows two identical or nonidentical monomers (eg, Jun-Jun or Fos-Jun) to "zip together" in a coiled coil and form a tight dimeric complex (Figure 38-17). This proteinprotein interaction serves to enhance the association of the separate DBDs with their target DNA sites (Figure 38-17).

DNA binding could result in a general conformational change that allows the bound protein to activate transcription, alternatively these two functions could be served by separate and
FIGURE 38-17 The leucine zipper motif. (A) Shown is a helical wheel analysis of a carboxyl-terminal portion of the DNA-binding protein C/EBP (see Table 36-3). The amino acid sequence is displayed end-to-end down the axis of a schematic a helix (see Figures 5-2 to 5-4). The helical wheel consists of seven spokes that correspond to the seven amino acids that comprise every two turns of the a helix. Note that leucine residues (L) occur at every seventh position (in this schematic C/EBP amino acid residues 1, 8, 15, 22; see arrow). Other proteins with "leucine zippers" have a similar helical wheel pattern. (B) A schematic model of the DNA-binding domain of C/EBP. Two identical C/EBP polypeptide chains are held in dimer formation by the leucine zipper domain of each polypeptide (denoted by the white rectangles and attached orangeshaded ovals). This association is required to hold the DNA-binding domains of each polypeptide (the green-shaded rectangles) in the proper conformation and register for DNA binding. (Reprinted with permission from S McKnight.)
protein results in resistance to the action of this hormone and the clinical syndrome of rickets.
independent domains. Domain swap experiments suggest that the latter is typically the case.

The GALI product is involved in galactose metabolism gene in yeast. Transcription of this gene is positively regulated by the Gal4 protein, which binds to an upstream activator sequence (UAS), or enhancer, through an amino terminal DBD. To systematically test the contributions of the Gal4 AD and DBD to GALI gene transcription activation, a series of domain swap experiements were performed (Figure 38-18). The amino terminal 73-amino-acid DBD of Gal4 was removed and replaced with the DBD of LexA, an E. coli DNA-binding protein. This domain swap resulted in a molecule that did not bind to the GALI UAS and of course, did not activate the GAL1 gene (Figure 38-18). If, however, the lexA operator—the DNA sequence normally bound by the LexA DBD—was inserted into the promoter region of the GAL gene to replace the normal GAL1 enhancer, the hybrid protein bound to this promoter (at the substituted lexA operator) and it activated transcription of GAL1. This general experiment has been repeated many times with different heterologous DBDs. The results demonstrate that the carboxyl-terminal region of Gal4 contains a transcriptional activation domain, or AD. These data also demonstrate that the DBD and AD can function independently. The hierarchy involved in assembling gene transcription-activating complexes includes proteins that bind DNA and transactivate; others that form proteinprotein complexes which bridge DNA-binding proteins to transactivating proteins; and others that form protein-protein complexes with components of coregulators or the basal
FIGURE 38-19 Proteins that regulate transcription have several domains. This hypothetical transcription factor has a DBD that is distinct from a ligand-binding domain (LBD) and several activation domains (ADs) (1-4). Other proteins may lack the DBD or LBD and all may have variable numbers of domains that contact other proteins, including coregulators and those of the basal transcription complex (see also Chapters 41 and 42).

transcription apparatus. A given protein may thus have several modular surfaces or domains that serve different functions (Figure 38-19). (Not shown here, but DNA-binding repressor proteins are organized similarly with separable DBDs and silencing domains, SDs. As described in Chapter 36, the primary purpose of these molecules is to facilitate the assembly and/or activity of the basal transcription apparatus on the cis-linked promoter.

In addition to transcription, eukaryotic cells employ a variety of mechanisms to regulate gene expression (Table 38-4). Many more steps, especially in RNA processing, are involved in the expression of eukaryotic genes than of prokaryotic genes, and these steps provide additional sites for regulatory influences that cannot exist in prokaryotes. These RNA processing steps in eukaryotes, described in detail in Chapter 36, include capping of the 5' ends of the primary transcripts,
TABLE 38-4 Gene Expression Is Regulated by Transcription and in Numerous Other Ways in Eukaryotic Cells
+ Gene amplification
+ Gene rearrangement
+ RNA
+ Alternate mRNA splicing
+ Transport of mRNA from nucleus to cytoplasm
+ Regulation of mRNA stability
+ Compartmentalization
+ ncRNA silencing and activation
addition of a polyadenylate tail to the 3' ends of transcripts, and excision of intron regions to generate spliced exons in the mature mRNA molecule. To date, analyses of eukaryotic gene expression provide evidence that regulation occurs at the level of transcription, nuclear RNA processing, nuclear transport, mRNA stability, and translation. In addition, gene amplification and rearrangement influence gene expression. Owing the advent of recombinant DNA technology and to high throughput DNA and RNA sequencing and other genetic tools (see Chapter 39), much progress has been made in recent years in our understanding of eukaryotic gene expression. However, because most eukaryotic organisms contain so much more genetic information than do prokaryotes and because manipulation of their genes is more difficult, molecular aspects of eukaryotic gene regulation are less well understood than the examples discussed earlier in this chapter. This section briefly describes a few different types of eukaryotic gene regulation.

As noted in Chapter 35, the recently discovered class of ubiquitous large and small eukaryotic non-protein-coding ncRNAs contribute importantly to the control of gene expression. The mechanism of action of the small miRNA and siRNAs are best understood. These ~22 nucleotide RNAs regulate the function/expression of specific mRNAs by either inhibiting translation or inducing mRNA degradation via different mechanisms; in a very few cases miRNAs have been shown to stimulate mRNA function. At least a portion of the miRNA-driven modulation of mRNA activity is thought to occur in the P body (see Figure 37-11). miRNA action can result in dramatic changes in protein production and hence gene expression. These small ncRNAs have been implicated in numerous human diseases such as heart disease, cancer, muscle wasting, viral infection, and diabetes.
miRNAs and siRNAs, like the DNA-binding transcription factors described in detail above, are transactive, and once synthesized and appropriately processed, interact with specific proteins and bind target mRNAs (see Figure 36-17). Binding of miRNAs to mRNA targets is directed by normal base-pairing rules. In general, if miRNA-mRNA base pairing has one or more mismatches, translation of the cognate "target" mRNA is inhibited, whereas if miRNA-mRNA base pairing is perfect over all 22 nucleotides, the corresponding mRNA is degraded. Given the tremendous and evergrowing importance of miRNAs, many scientists and biotechnology companies are actively studying miRNA biogenesis, transport, and function in hopes of curing human disease. Time will tell the magnitude and universality of ncRNA-mediated gene regulation.

During early development of metazoans, there is an abrupt increase in the need for specific molecules such as ribosomal
RNA and messenger RNA molecules for proteins that make up specific cell or tissue types. One way to increase the rate at which such molecules can be formed is to increase the number of genes available for transcription of these specific molecules. Among the repetitive DNA sequences within the genome are hundreds of copies of ribosomal RNA genes. These genes preexist repetitively in the DNA of the gametes and thus are transmitted in high copy numbers from generation to generation. In some specific organisms such as the fruit fly (Drosophila), there occurs during oogenesis an amplification of a few preexisting genes such as those for the chorion (eggshell) proteins. Subsequently, these amplified genes, presumably generated by a process of repeated initiations during DNA synthesis, provide multiple sites for gene transcription (Figures 36-4 and 38-20). The dark side of specific gene amplification is the fact that in human cancer cells drug resistance can develop upon extended therapeutic treatment due to the amplification and increased expression of genes that encode proteins that either degrade, or pump drugs from target cells.
As noted in Chapter 36, the coding sequences responsible for the generation of specific protein molecules are frequently not contiguous in the mammalian genome. In the case of antibody encoding genes, this is particularly true. As described in detail in Chapter 52, immunoglobulins are composed of two polypeptides, the so-called heavy (about 50 kDa) and light (about 25 kDa) chains. The mRNAs encoding these two protein subunits are encoded by gene sequences that are subjected to extensive DNA sequence-coding changes. These DNA coding changes are integral to generating the requisite recognition diversity central to appropriate immune function.

IgG heavyand light-chain mRNAs are encoded by several different segments that are tandemly repeated in the germline. Thus, for example, the IgG light chain consists of variable (V,), joining (J,), and constant (C,) domains or segments. For particular subsets of IgG light chains, there are roughly 300 tandemly repeated V, gene coding segments, 5 tandemly arranged J, coding sequences, and roughly 10 C, gene coding segments. All of these multiple, distinct coding sequences are located in the same region of the same chromosome, and each type of coding segment (V,, J,, and C,) is tandemly repeated
FIGURE 38-20 Schematic representation of the amplification of chorion protein genes s36 and s38. (Reproduced, with permission, from Chisholm R: Gene amplification during development. Trends Biochem Sci 1982;7:161. Copyright © 1982. Reprinted, with permission, from Elsevier.)
in head-to-tail fashion within the segment repeat region. By having multiple V,, J,, and C, segments to choose from, an immune cell has a greater repertoire of sequences to work with to develop both immunologic flexibility and specificity. However, a given functional IgG light-chain transcription unit—like all other "normal" mammalian transcription units—contains only the coding sequences for a single protein. Thus, before a particular IgG light chain can be expressed, single V,, J,, and C, coding sequences must be recombined to generate a single, contiguous transcription unit excluding the multiple nonutilized segments (ie, the other approximately 300 unused V, segments, the other 4 unused J, segments, and the other 9 unused C, segments). This deletion of unused genetic information is accomplished by selective DNA recombination that removes the unwanted coding DNA while retaining the required coding sequences: one V,, one J,, and one C, sequence. (V, sequences are subjected to additional point mutagenesis to generate even more variability—hence the name.) The newly recombined sequences thus form a single transcription unit that is competent for RNA polymerase II-mediated transcription into a single monocistronic mRNA. Although the IgG genes represent one of the best-studied instances of directed DNA rearrangement modulating gene expression, other cases of gene regulatory DNA rearrangement have been described.

In addition to affecting the efficiency of promoter utilization, eukaryotic cells employ alternative RNA processing to control gene expression. This can result when alternative promoters, intron-exon splice sites, or polyadenylation sites are used. Occasionally, heterogeneity within a cell results, but more commonly the same primary transcript is processed differently in different tissues. A few examples of each of these types of regulation are presented below.
result in mRNAs that are either 2700 bases long (u,) or 2400 bases long (u.). This results in a different carboxylterminal region of the encoded proteins such that the y,, protein remains attached to the membrane of the B lymphocyte and the », immunoglobulin is secreted. Alternative splicing and processing results in the formation of seven unique a-tropomyosin mRNAs in seven different tissues. It is not yet fully understood how these processing-splicing decisions are made or exactly how these steps can be regulated.

Although most mRNAs in mammalian cells are very stable (half-lives measured in hours), some turn over very rapidly (half-lives of 10-30 minutes). In certain instances, mRNA stability is subject to regulation. This has important implications since there is usually a direct relationship between mRNA amount and the translation of that mRNA into its cognate protein. Changes in the stability of a specific mRNA can therefore have major effects on biologic processes.
Messenger RNAs exist in the cytoplasm as ribonucleoprotein particles (RNPs). Some of these proteins protect the mRNA from digestion by nucleases, while others may under certain conditions promote nuclease attack. It is thought that mRNAs are stabilized or destabilized by the interaction of proteins with these various structures or sequences. Certain effectors, such as hormones, may regulate mRNA stability by increasing or decreasing the amount of these mRNA-binding proteins.
The use of alternative transcription start sites results in a different 5' exon on mRNAs encoding mouse amylase and myosin light chain, rat glucokinase, and Drosophila alcohol dehydrogenase and actin. Alternative polyadenylation sites in the « immunoglobulin heavy-chain primary transcript
It is known that the ends of mRNA molecules are involved in mRNA stability (Figure 38-21). The 5'-cap structure in eukaryotic mRNA prevents attack by 5' exonucleases, and the poly(A) tail prohibits the action of 3' exonucleases. In mRNA molecules with those structures, it is presumed that a single endonucleolytic cut allows exonucleases to attack and digest the entire molecule. Other structures (sequences) in the 5'-untranslated region (5' UTR), the coding region, and the 3' UTR are thought to promote or prevent this initial endonucleolytic action (Figure 38-21). Much of this mRNA metabolism likely occurs in cytoplasmic P bodies.

FIGURE 38-21 Structure of a typical eukaryotic mRNA showing elements that are involved in regulating mRNA stability. The typical eukaryotic mRNA has a 5'-noncoding sequence (NCS), or untranslated exonic region (5' UTR), a coding region, and a 3'-exonic untranslated NCS region (3' UTR). Essentially all mRNAs are capped at the 5' end, and most have a 100 to 200 nt polyadenylate sequence at their 3' end. The 5' cap and 3' poly(A) tail protect the mRNA against exonuclease attack and are bound by specific proteins that interact to facilitate translation (see Figure 37-7). Stem-loop structures in the 5' and 3' NCS, and the AU-rich region in the 3' NCS are thought to represent the binding sites for specific proteins that modulate mRNA stability.
Thus, it is clear that a number of mechanisms are used to regulate mRNA stability and hence function—just as several mechanisms are used to regulate the synthesis of mRNA. Coordinate regulation of these two processes confers on the cell remarkable adaptability.

■, 1 = The genetic constitutions of metazoan somatic cells are nearly all identical.. ■, 1 = Phenotype (tissue or cell specificity) is dictated by differences in gene expression of the cellular complement of genes.. ■, 1 = Alterations in gene expression allow a cell to adapt to environmental changes, developmental cues, and physiologic signals.. ■, 1 = Gene expression can be controlled at multiple levels by changes in transcription, mRNAprocessing, localization, and stability or translation. Gene amplification and rearrangements also influence gene expression.. ■, 1 = Transcription controls operate at the level of protein-DNA and protein-protein interactions. These interactions display protein domain modularity and high specificity.. ■, 1 = Several different classes of DBD have been identified in transcription factors.. ■, 1 = Chromatin and DNAmodifications contribute importantly in eukaryotic transcription control by modulating DNA accessibility and specifying recruitment of specific coactivators and corepressors to target genes.. ■, 1 = Several epigenetic mechanisms for gene control have been described and the molecular mechanisms through which these processes operate are being elucidated at the molecular level.. ■, 1 = ncRNAs modulate gene expression. The short miRNAs and siRNAs modulate mRNAtranslation and stability.

Ambrosi C, Manzo M, Baubec T: Dynamics and context-dependent roles of DNA methylation. J Mol Biol 2017;429(10):1459-1475. Browning DF, Busby SJ: Local and global regulation of transcription inititiation in bacteria. Nat Rev Microbiol 2016;14:638-650.

VATU INCREAS Ce aA
2 Understand the basic procedures and methods involved in recombinant DNA technology and genetic engineering.
. Appreciate the rationale behind the methods used to synthesize, analyze, and sequence DNA and RNA.
= Describe how to identify and quantify individual proteins, both soluble
and insoluble
(ie, membrane
bound or
compartmentalized intracellularly)
- DNA or RNA. proteins, as well as proteins bound to specific sequences of genomic

The development of recombinant DNA techniques, highdensity DNA microarrays, high-throughput screening, low-cost genome-scale DNA and RNA sequencing, and other molecular genetic methodologies has revolutionized biology and is having an increasing impact on clinical medicine. Although much has been learned about human genetic disease from pedigree analysis and study of affected proteins, in many cases where the specific genetic defect is unknown, these approaches cannot be used. The new technologies circumvent these limitations by going directly to cellular DNA and RNA molecules for information. Manipulation of a DNA sequence and the construction of chimeric molecules—so-called genetic engineering—provide a means of studying how a specific segment of DNA controls cellular function. New biochemical and molecular genetic tools allow investigators to query and manipulate genomic sequences as well as to examine the entire complement of cellular RNA, protein, and protein PTM status at the molecular level, even in single cells.
(2) Human proteins can be produced in abundance for therapy (eg, insulin, growth hormone, tissue plasminogen activator). (3) Proteins for preparation of vaccines (eg, hepatitis B) and for diagnostic testing (eg, Ebola and AIDS tests) can be readily obtained. (4) This technology is used both to diagnose existing diseases as well as to predict the risk of developing a given disease and individual response to pharmacologic therapeutics—so called personalized medicine. (5) Special techniques have led to remarkable advances in forensic medicine, which have allowed for the molecular diagnostic analysis of DNA from single cells. (6) Finally, in extremely well understood diseases, potentially curative gene therapy for diseases caused by a single-gene deficiency such as sickle cell disease, the thalassemias, adenosine deaminase deficiency, and others may be devised.
Understanding molecular genetics technology is important for several reasons: (1) it offers a rational approach to understanding the molecular basis of disease. For example, familial hypercholesterolemia, sickle cell disease, the thalassemias, cystic fibrosis, muscular dystrophy as well as more complex multifactorial diseases like vascular and heart disease, Alzheimer disease, cancer, obesity, and diabetes.
"See glossary of terms at the end of this chapter.

Isolation and manipulation of DNA, including end-to-end joining of sequences from very different sources to make chimeric molecules (eg, molecules containing both human and bacterial DNA sequences in a sequence-independent fashion), is the essence of recombinant DNA research. This involves several unique techniques and reagents.

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Restriction enzymes are named after the bacterium from which they are isolated. For example, EcoRI is from Escherichia coli, and BamHI is from Bacillus amyloliquefaciens (Table 39-1). The first three letters in the restriction enzyme name consist of the first letter of the genus (E) and the first two letters of the species (co) in the case of the restriction enzyme EcoRI derived from E. coli strain R. These designations may be followed by a strain designation (R) and a roman numeral (J) to indicate the order of discovery (eg, EcoRI and EcoRI). Each enzyme recognizes and cleaves a specific double-stranded DNA sequence that is typically 4to 8-bp long. These DNA cuts result in blunt ends (eg, Hpal) or overlapping (sticky or cohesive) ends (eg, BamHI) (Figure 39-1), depending on the mechanism used by the enzyme. Sticky ends are particularly useful in constructing hybrid or chimeric DNA molecules (see below). If the four nucleotides are distributed randomly in a given DNA molecule, one can calculate how frequently a given enzyme will cut a length of DNA. For each position in the DNA molecule, there are four possibilities (A, C, G, and T); therefore, a restriction enzyme that recognizes a 4-bp sequence cuts DNA, on average, once every 256 bp (4°), whereas another enzyme that recognizes a 6-bp sequence cuts once every 4096 bp (4°). A given piece of DNA has a characteristic linear array of sites for the various enzymes dictated by the linear sequence of its bases; hence, a restriction map can be constructed. When DNA is digested with

BamHI, Sequence Recognized Cleavage Sites Shown = ↓ G GATCC CCTAC C. BamHI, Bacterial Source = Bacillus amyloliquefaciens H. BgIII, Sequence Recognized Cleavage Sites Shown = ↓ A GATCT TCTAG A ↑. BgIII, Bacterial Source = Bacillus globigii. EcoRI, Sequence Recognized Cleavage Sites Shown = ↓ G AATTC CTTAA C. EcoRI, Bacterial Source = Escherichia coli RY13. EcoRII, Sequence Recognized Cleavage Sites Shown = ↓ CCTGG GGACC ↑. EcoRII, Bacterial Source = Escherichia coli R245. HindIII, Sequence Recognized Cleavage Sites Shown = ↓ A AGCTT TTCGA A ↑. HindIII, Bacterial Source = Haemophilus influenzae R d. HhaI, Sequence Recognized Cleavage Sites Shown = ↓ GCG C C GCG ↑. HhaI, Bacterial Source = Haemophilus haemolyticus. HpaI, Sequence Recognized Cleavage Sites Shown = ↓ GTT AAC CAA TTC ↑. HpaI, Bacterial Source = Haemophilus Parainfluenza. MstII, Sequence Recognized Cleavage Sites Shown = ↓ CC TnAGG GGAnT CC ↑. MstII, Bacterial Source = Microcoleus strain. PstI, Sequence Recognized Cleavage Sites Shown = ↓ CTGCA G G ACGTC ↑. PstI, Bacterial Source = Providencia stuartii 164. TaqI, Sequence Recognized Cleavage Sites Shown = ↓ T CGA AGC T. TaqI, Bacterial Source = Thermus aquaticus YTI

Abbreviations: A, adenine; C, cytosine; G, guanine, T, thymine. Arrows show the site of cleavage; depending on the site, the ends of the resulting cleaved doublestranded DNA are termed sticky ends (BamHI) or blunt ends (Hpal). The length of the recognition sequence can be 4 bp (Taq/), 5 bp (EcoRI), 6 bp (EcoRI), or 7 bp (Mstll) or longer. By convention, these are written in the 5' to 3' direction for the upper strand of each recognition sequence, and the lower strand is shown with the opposite (ie, 3'-5') polarity. Note that most recognition sequences are palindromes (ie, the sequence reads the same in opposite directions on the two strands). A residue designated n means that any nucleotide is permitted.
B.
FIGURE 39-1 Results of restriction endonuclease digestion. Digestion with a restriction endonuclease can result in the formation of DNA fragments with sticky, or cohesive, ends (A), or blunt ends (B); phosphodiester backbone, black lines; interstrand hydrogen bonds between purine and pyrimidine bases, blue. Generating fragments whose ends have particular structures (ie, blunt, cohesive) is an important consideration in devising cloning strategies.
a particular enzyme, the ends of all the fragments have the same DNA sequence. The fragments produced can be isolated by electrophoresis on agarose or polyacrylamide gels (see the discussion of blot transfer, below); this is an essential step in DNA cloning as well as various DNA analyses, and a major use of these enzymes. A number of other enzymes that act on DNA and RNA are an important part of recombinant DNA technology. Many of these are referred to in this and subsequent chapters (Table 39-2).
Restriction Enzymes, Endonucleases, Recombinases, & DNA Ligase Are Used to Engineer and Prepare Chimeric DNA Molecules
Sticky, or complementary cohesive-end ligation of DNA fragments is technically easy, but some special techniques are often required to overcome problems inherent in this approach. Sticky ends of a vector may reconnect with themselves, with

′ ends). Terminal transferase, Reaction = Adds nucleotides to the 3 ′ ends of DNA. Terminal transferase, Primary Use = Homopolymer tailing. Recombinases (CRE, INT, FLP), Reaction = Catalyze site-specific recombination between DNA containing homologous target sequences. Recombinases (CRE, INT, FLP), Primary Use = Generation of specific chimeric DNAmolecules, work both in vitro and in vivo. CRISPER-Cas9/C2c2, Reaction = RNA-targeted DNA-, or RNA-directed nuclease. CRISPER-Cas9/C2c2, Primary Use = Genome editing and with variations modulation of gene expression at DNAand RNA levels
no net gain of DNA. Sticky ends of fragments also anneal so that heterogeneous tandem inserts can form. Also, sticky-end sites may not be available or in a convenient position. To alleviate these problems, an enzyme that generates blunt ends can be used. Blunt ends can be ligated directly; however, ligation is not directional. To circumvent this problem new DNA ends of specific sequence can be added to by direct blunt-end ligation using the bacteriophage T4 enzyme DNA ligase. Alternatively, convenient RE recognition sites can be added to a DNA fragmaent through the use of polymerase chain reaction (PCR) amplification (see below).

As an adjunct to the use of restriction endonucleases to combine and engineer DNA fragments, scientists have begun utilizing recombinases such as bacterial lox P sites, which are recognized by the CRE recombinase, bacteriophage } att sites recognized by the A phage encoded INT protein or yeast FRT sites recognized by the yeast Flp recombinase. These recombinase systems all catalyze specific incorporation of two DNA fragments that carry the appropriate recognition sequences and carry out homologous recombination (see Figure 35-9) between the relevant recognition sites. A novel DNA editing/gene regulatory system termed CRISPR-Cas9 (clustered regularly interspersed short palindromic repeats— associated gene 9) first discovered in 2012, has revolutionized genomic DNA studies. The CRISPR system, found in many bacteria, represents a form of acquired, or adaptive immunity (see Chapters 52, 54) to prevent reinfection of a bacterium by specific bacteriophages. CRISPR complements the system of restriction endonucleases and methylases described above. CRISPR uses RNA-based targeting to bring the Cas9 nuclease to foreign (or any complementary) DNA. Within bacteria this CRISPR-RNA-Cas9 complex then degrades and inactivates the targeted DNA. The CRISPR system has been adapted for use in eukaryotic cells, including human cells, where it has been shown to be an RNA-directed site-specific nuclease just as it is in bacteria. Variations on the use of CRISPR allow for gene deletion, gene editing, gene visualization, and even modulation of gene transcription. Thus, CRISPR has added an exciting new, highly efficient, and very specific technology to the toolbox of methods for the manipulation of DNA and genetic analysis of mammalian cells. The basic aspects of CRISPR-Cas9 function are outlined in Figure 39-2.

The similarities of the CRISPR-Cas RNA-directed targeting and gene inactivation method and mi/siRNA-mediated repression of expression in higher eukaryotes are notable. Both methodologies are being actively pursued for experimental and therapeutic purposes. Interestingly, a variant of the CRISPR-Cas system, C2c2, has been shown to site-specifically cleave RNA. This exciting discovery paves the way for potential specific alteration of mRNA/ncRNA levels in human cells absent the ethical and technical challenges inherent in genome editing with the CRISPR-Cas9 system.

A clone is a large population of identical molecules, cells, or organisms that arise from a common ancestor. Molecular cloning allows for the production of a large number of identical DNA molecules, which can then be characterized or
FIGURE 39-2 Overview of the mechanism of CRISPR-Cas9. Shown is the two-domain CRISPR-Cas9 nuclease protein bound to target genomic DNA (red, blue) and specific guide RNA (green), which through base complementarity (20 nts) locates its genomic target, which is adjacent to a short protospacer adjacent motif, or PAM. The guide RNA binding, and nuclease domains are labeled. Once specifically localized, the two distinct Cas9 nuclease active centers cleave both strands of the targeted genomic DNA (cleavage; arrows) immediately downstream of the PAM, which results in DNA double-strand break. Subsequent DNA repair by cellular acitivities (see Chapter 35) can introduce mutations thereby inactivating the targeted gene. Variations on the use of CRISPR-Cas9 are numerous and allow for the sculpting of the structure and expression of genomic DNA.
(recombinant
DNA
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FIGURE 39-3 Use of restriction endonucleases to make new recombinant or chimeric DNA molecules. When inserted back into a bacterial cell (by the process called DNA-mediated transformation), typically only a single plasmid is taken up by a single cell, and the plasmid DNA replicates clonally, not only itself, but also the physically linked new DNA insert. Since recombining the sticky ends, as indicated, typically regenerates the same DNA sequence recognized by the original restriction enzyme, the cloned DNA insert can be cleanly cut back out of the recombinant plasmid circle with this endonuclease. Alternatively, the insert sequences can be specifically amplified from the purified chimeric plasmid DNA by PCR (Figure 39-7). If a mixture of all of the DNA pieces created by treatment of total human DNA with a single restriction nuclease is used as the source of human DNA, a million or so different types of recombinant DNA molecules can be obtained, each pure in its own bacterial clone. (Modified and reproduced, with permission, from Cohen SN: The manipulation of genes. Sci Am [July] 1975;233:25. Copyright © The Estate of Bunji Tagawa.)

used for other purposes. This technique is based on the fact that chimeric or hybrid DNA molecules can be constructed in cloning vectors—typically bacterial plasmids, phages, or cosmids (hybrid plasmids that also contain specific phage sequences)—which then continue to replicate clonally in a single host cell under their own control systems. In this way, the chimeric DNA is amplified. The general procedure is illustrated in Figure 39-3.
specific for the restriction site into which the original piece of DNA was inserted.
Bacterial plasmids are small, circular, duplex DNA molecules whose natural function is to confer antibiotic resistance to the host cell. Plasmids have several properties that make them extremely useful as cloning vectors. They exist as single or multiple copies within the bacterium and replicate independently from the bacterial DNA as episomes (ie, a genome above or outside the bacterial genome) while using primarily the host replication machinery. The complete DNA sequence of thousands of plasmids is known; hence, the precise location of restriction enzyme cleavage sites for inserting the foreign DNA is available. Plasmids are smaller than the host chromosome and are therefore easily biochemically separated from the latter, and the desired plasmid-inserted DNA can be readily removed by cutting the plasmid with the enzyme
Phages (bacterial viruses) often have linear DNA genomes into which foreign DNA can be inserted at unique restriction enzyme sites. The resulting chimeric DNA is collected after the phage proceeds through its lytic cycle and produces mature, infective phage particles. A major advantage of phage vectors is that while plasmids accept DNA pieces up to about 10-kb long, phages can readily accept DNA fragments up to ~20-kb long. The ultimate insert size is imposed by the amount of DNA that can be packed into the phage head during virus propagation.

Larger fragments of DNA can be cloned in cosmids, DNA cloning vectors that combine the best features of plasmids and phages. Cosmids are plasmids that contain the DNA sequences, so-called cos sites, required for packaging lambda DNA into the phage particle. These vectors grow in the plasmid form in bacteria, but since much of the unnecessary lambda DNA has been removed, more chimeric DNA can be packaged into the particle head. Cosmids can carry inserts of chimeric DNA that are 35to 50-kb long. Even larger pieces of DNA can be incorporated into bacterial artificial chromosome

Plasmid pUC19, DNAInsert Size (kb) = 0.01-10. Lambda charon 4A, DNAInsert Size (kb) = 10-20. Cosmids, DNAInsert Size (kb) = 35-50. BAC, P1, DNAInsert Size (kb) = 50-250. YAC, DNAInsert Size (kb) = 500-3000
(BAC), yeast artificial chromosome (YAC), or E. coli bacteriophage P1-derived artificial chromosome (PAC) vectors. These vectors will accept and propagate DNA inserts of several hundred kilobases or more, and have largely replaced the plasmid, phage, and cosmid vectors for some cloning and eukaryotic gene mapping/expression applications. A comparison of these vectors is shown in Table 39-3.
is commonly used as the insertion site for a piece of foreign DNA. In addition to having sticky ends (Table 39-1 and Figure 39-1), the DNA inserted at this site disrupts the ORF of the B-lactamse-encoding bla gene. B-lactamase, a secreted enzyme degrades and inactivates ampicillin. A bacterium carrying such a plasmid will be Amp-sensitive (Amp'). Thus, cells carrying the parental plasmid, which provides resistance to both antibiotics, can be readily distinguished, and separated from cells carrying the chimeric plasmid, which is resistant only to tetracycline (Figure 39-4). YACs contain selection, replication, and segregation functions that work in both bacteria and yeast cells and therefore can be propagated in either organism.
Because insertion of DNA into a functional region of the vector will interfere with the action of this region, care must be taken not to interrupt an essential function of the vector. This concept can be exploited, however, to provide a powerful double positive/negative selection technique. For example, a common early plasmid vector pBR322 has genes conferring resistance to both tetracycline (Tet) and ampicillin (Amp), that is, Tet'-and Amp' resistant growth, respectively. A single PstI restriction enzyme site within the Amp resistance gene

In addition to the vectors described in Table 39-3 that are designed primarily for propagation in bacterial cells, vectors for mammalian cell propagation and insert gene (cDNA)/ protein expression have also been developed. These vectors are all based on various eukaryotic viruses that are composed of RNA or DNA genomes. Notable examples of such viral vectors are those utilizing adenoviral (Ad), or adenovirusassociated viral (AAV) (DNA-based) and retroviral (RNA based) genomes. Though somewhat limited in the size of DNA sequences that can be inserted, such mammalian viral cloning vectors make up for this shortcoming because they will efficiently infect a wide range of different cell types. For this reason, various mammalian viral vectors, some with both positive and negative selection genes (aka selectable "markers" as noted above for pBR322) are being investigated for
FIGURE 39-4 A method of screening recombinants for inserted DNA fragments. Using the plasmid pBR322, a piece of DNA is inserted into the unique Pst! site. This insertion disrupts the codon reading frame for the gene coding for a protein that provides ampicillin resistance to the host bacterium. Hence, cells carrying the chimeric plasmid will no longer grow/survive when cultured in liquid, or plated on a substrate medium that contains this antibiotic. The differential sensitivity to tetracycline and ampicillin can therefore be used to distinguish clones of plasmid that contain an insert. A similar scheme relying on production of an in-frame fusion of a newly inserted DNA producing a peptide fragment capable of complementing an inactive, N-terminally truncated form of the enzyme $-galactosidase, a component of the Jac operon (see Figure 38-2) allows for blue-white colony formation on agar plates containing a dye hydrolyzable by B-galactoside. 8-galactosidase-positive colonies are blue; such colonies contain plasmids in which a DNA was successfully inserted.
use in gene therapy and are commonly used for laboratory experiments.

The combination of restriction enzymes and various cloning vectors allows the entire genome of an organism to be individually packed, in small segments, into a vector. A collection of these different recombinant clones is called a library. A genomic library is prepared from the total DNA of a cell line or tissue that has been fragmented using either restriction endonucleases, or shearing and adaptor ligation to the resulting fragments. A cDNA library comprises complementary DNA copies of the population of mRNAs in a tissue. Genomic DNA libraries are often prepared by performing partial digestion of total DNA with a restriction enzyme that cuts DNA frequently (eg, a fourbase cutter such as TaqI). The idea is to generate rather large fragments so that most genes will be left intact. The BAC, YAC, and P1 vectors are preferred since they can accept very large fragments of DNA and thus offer a better chance of isolating an intact eukaryotic mRNA-encoding gene on a single DNA fragment.
A vector in which the protein coded by the gene introduced by recombinant DNA technology is actually synthesized is known as an expression vector. Such vectors are now commonly used to detect specific cDNA molecules in libraries and to produce proteins by genetic engineering techniques. These vectors are specially constructed to contain very active inducible promoters, proper in-phase translation initiation codons, transcription and translation termination signals, and appropriate protein processing signals, if needed. Some expression vectors even contain genes that code for protease inhibitors, so that the final yield of product is enhanced. Interestingly, as the cost of synthetic DNA synthesis has dropped, many investigators now often simply synthesize an entire cDNA (gene) of interest (in 100150 nt segments) incorporating the codon preferences of the host used for expression in order to maximize protein production. New efficiencies in synthetic DNA synthesis now allow for the de novo synthesis of complete genes and even genomes. These advances usher in new and exciting possibilities in synthetic biology while concomitantly introducing potential ethical conundrums.

A variety of molecules can be used to "probe" libraries in search of a specific gene or cDNA molecule or to define and quantitate DNA or RNA separated by electrophoresis through various gels. Probes are generally pieces of DNA or RNA labeled with a *P-containing nucleotide—or fluorescently labeled nucleotide(s) (more commonly now). Importantly, neither modification (*P or fluorescent-label)
affects the hybridization properties of the resulting labeled nucleic acid probes. The probe must recognize a complementary sequence to be effective. A cDNA synthesized from a specific mRNA (or a synthetic oligonucleotide) can be used to screen either a cDNA library for a longer cDNA or a genomic library for a complementary sequence in the coding region of a gene. cDNA/oligonucleotide/cRNA probes are used to detect DNA fragments on Southern blot transfers and to detect and quantitate RNA on Northern blot transfers (see below).

Visualization of a specific DNA or RNA fragment (or protein, see below) among the many thousands of "contaminating" nontarget molecules in a complex sample requires the convergence of a number of techniques, collectively termed blot transfer. Figure 39-5 illustrates the Southern (DNA), Northern (RNA), and Western (protein) blot transfer procedures. The first technique is named for the person who devised the technique, Edward Southern; the other names began as laboratory jargon, but are now accepted terms. These procedures are useful in determining how many copies of a gene are in a given tissue or whether there are any alterations in a gene (deletions, insertions, or rearrangements) because the requisite, initial gel electrophoresis step separates the molecules on the basis of size. Occasionally, if a specific base is changed and a restriction site is altered, these procedures can detect a point mutation (ie, Figure 39-9 below). The Northern and Western blot transfer techniques are used to size and quantify specific RNA and protein molecules, respectively. A fourth technique, the Southwestern or overlay blot, which examines protein-nucleic acid interactions or protein-protein interactions, respectively are variants of the Southern/Northern/ Western blotting methods (not shown). In these last two techniques, proteins are separated by electrophoresis, blotted to a membrane, renatured, and analyzed for an interaction with a particular DNA or RNA sequence or protein by incubation with a specific labeled nucleic acid probe (Southwestern) or protein probe (overlay assay) using either a labeled protein, or alternatively protein-protein interactions are detected using a specific antibody.
All of the nucleic acid-based hybridization procedures discussed in this section depend on the specific base-pairing properties of complementary nucleic acid strands (see Chapter 34). Perfect matches hybridize readily and withstand high temperatures and/or low ionic strength buffer in the hybridization and washing reactions. Less than perfect matches do not tolerate such stringent conditions (ie, elevated temperatures and low-salt concentrations); thus, hybridization either never occurs or is disrupted during the washing step. Hybridization conditions capable of detecting just a single base-pair (bp) mismatch between probe and target have been devised.
ona’

FIGURE 39-5 The blot transfer procedure. In a Southern, or DNA blot transfer, DNA isolated from a cell line or tissue is digested with one or more restriction enzymes. This mixture is pipetted into a well in an agarose or polyacrylamide gel and exposed to a direct electrical current. DNA, being negatively charged, migrates toward the anode; the smaller fragments move the most rapidly. After a suitable time, the DNA within the gel is denatured by exposure to mild alkali and transferred, via capillary action (or electrotransfer—not shown), to nitrocellulose or nylon paper, resulting in an exact replica of the pattern on the gel, using the blotting technique devised by Southern blot. The DNA is bound to the paper by exposure to heat or UV, and the paper is then exposed to the labeled DNA probe, which hybridizes to complementary strands on the filter. After thorough washing, the paper is exposed to x-ray film or an imaging screen, which is developed to reveal several specific bands corresponding to the DNA fragment(s) that were recognized (hybridized to) the sequences in the DNA probe. The RNA, or Northern, blot is conceptually similar. RNA is subjected to electrophoresis before blot transfer. This requires some different steps from those of DNA transfer, primarily to ensure that the RNA remains intact, and is generally somewhat more difficult. In the protein, or Western, blot, proteins are electrophoresed and transferred to special paper that avidly binds proteins and then probed with a specific antibody or other probe molecule. (Asterisks signify labeled probes, either radioactive or fluorescent.) In the case of Southwestern blotting (see the text; not shown), a protein blot similar to that shown above under "Western" is exposed to labeled nucleic acid, and protein-nucleic acid complexes formed are detected by autoradiography or imaging.

The segments of specific DNA molecules obtained by recombinant DNA technology can be analyzed to determine their nucleotide sequence. DNA sequencing depends on having a population of identical DNA molecules. This requirement can be satisfied by cloning the fragment of interest, either using the techniques described above, or by using PCR methods (see below). The manual enzymatic Sanger method employs specific dideoxynucleotides that terminate DNA strand synthesis at specific nucleotides as the strand is synthesized on purified single-stranded template DNA. The reactions are adjusted so that a population of DNA fragments representing termination at every nucleotide is obtained. By having a radioactive label incorporated at the termination site, one can separate the fragments according to size using polyacrylamide gel electrophoresis. An autoradiograph is made, and each of the fragments produces an image (band) on an x-ray film or imaging plate. These are read in order to give the DNA sequence (Figure 39-6). Techniques that do not require the use of radioisotopes are employed in automated DNA sequencing. Most commonly employed is an automated procedure in which four different fluorescent labels, one representing each nucleotide, are used. Each emits a specific signal upon excitation by a laser beam of a particular wavelength that is measured by sensitive detectors, and these signals can be recorded by a computer. The newest DNA sequencing machines use fluorescently labeled nucleotides but detect incorporation using microscopic optics. These machines have reduced the cost of DNA sequencing by orders of magnitude. These reductions in cost have ushered in the era of personalized genome sequencing. Indeed, using this new technology, the genome sequence of the codiscoverer of the double helix, James Watson, was completely determined.

Automated chemical synthesis of moderately long oligonucleotides (~100 nucleotides) of precise sequence is now a routine laboratory procedure. Each synthetic cycle takes but minutes such that very large DNA molecules can be made by synthesizing relatively short segments that are then be ligated to one another. As mentioned above for DNA sequencing, the process has been miniaturized and can be significantly parallelized to allow the synthesis of hundreds to thousands of defined sequence oligonucleotides simultaneously. Oligonucleotides are now indispensable for DNA sequencing, library screening, protein-DNA binding assays, the PCR (see below), site-directed mutagenesis, complete synthetic gene synthesis as well as complete (bacterial) genome synthesis, and numerous other applications.

PCR is a method of amplifying a target sequence of DNA. The development of PCR has revolutionized the ways in which both DNA and RNA can be studied. PCR provides a sensitive, selective, and extremely rapid means of amplifying any desired sequence of DNA. Specificity is based on the use of two oligonucleotide primers that hybridize to complementary sequences on opposite strands of DNA and flank the target sequence (Figure 39-7). The DNA sample is first heat

-A-
G-T-C-T-T-G-G-A-G-C-T-3'
ne
FIGURE 39-6 Sequencing of DNA by the chain termination method devised by Sanger. The ladder-like arrays represent, from bottom to top, all of the successively longer fragments of the original DNA strand. Knowing which specific dideoxynucleotide reaction was conducted to produce each mixture of fragments, one can determine the sequence of nucleotides from the unlabeled end toward the labeled end (*) by reading up the gel. The base-pairing rules of Watson and Crick (A-T, G-C) dictate the sequence of the other (complementary) strand. (Asterisks signify site of radiolabeling.) Schematically shown (left, middle) are the terminated synthesis products of a hypothetical fragment of DNA, sequence listed (middle, top). An autoradiogram (right) of an actual set of DNA sequencing reactions that utilized the four **P-labeled dideoxynucleotides indicated at the top of the scanned autoradiogram (ie, dideoxy(dd)G, ddA, ddT, ddC). Electrophoresis was from top to bottom. The deduced DNA sequence is listed on the right side of the gel. Note the log-linear relationship between distance of migration (ie, top to bottom of gel) and DNA fragment length. Current state-of-the-art DNA sequencers no longer utilize gel electrophoresis for fractionation of labeled synthesis products. Moreover, in the majority of NGS sequencing platforms, synthesis is followed by monitoring incorporation of the four fluorescently labeled dXTPs.

denatured (>90°C) to separate the two strands of the template DNA containing the target sequence; the primers, added in excess, are allowed to anneal to the DNA (typically at 50-75°C) in order to generate the required template-primer complex. Subsequently, each strand is copied by a DNA polymerase, starting at the primer sites in the presence of all four dXTPs. The two DNA strands each serve as a template for the synthesis of new DNA from the two primers. Repeated cycles of heat denaturation, annealing of the primers to their complementary sequences, and extension of the annealed primers with DNA polymerase result in the exponential amplification of DNA segments of defined length. Product DNA doubles with each PCR cycle. DNA synthesis is catalyzed by a heat-stable DNA polymerase purified from one of a number of different thermophilic bacteria, organisms that grow at 70 to 80°C. Thermostable DNA polymerases withstand short incubations at over 90°, temperatures required to completely denature DNA. These thermostable DNA polymerases have made automation of PCR possible.

DNA sequences as short as 50 to 100 bp and as long as 10 kb can readily be amplified by PCR. Twenty cycles provide an amplification of 10° (ie, 2") and 30 cycles, 10° (2*°). Each cycle takes <5 to 10 minutes so that even large DNA molecules can be amplified fairly rapidly. Because of subtle differences in DNA sequence with each new PCR target, the exact conditions for amplification must be empirically optimized. The workhorse PCR technique is central to many DNA/RNA sequencing technologies. The PCR method allows the DNA in a single cell, hair follicle, or spermatozoon to be amplified and analyzed. Thus, the applications of PCR to forensic medicine are obvious. PCR is also used (1) to detect and quantify infectious agents, especially latent viruses; (2) to make prenatal genetic diagnoses; (3) to detect allelic polymorphisms ranging from single base pair changes to large and small indels and gene amplification; (4) to establish precise tissue types for transplants; and (5) to study evolution, using DNA from archeological samples (6) for quantitative RNA analyses after RNA copying and mRNA quantitation by the so-called RTPCR method (cDNA copies of mRNA generated by a retroviral reverse transcriptase) or (7) to score in vivo protein-DNA occupancy using chromatin immunoprecipitation assays (see below). New uses of PCR are developed every year.
FIGURE 39-7 The polymerase chain reaction technique is used to amplify specific gene sequences. Double-stranded DNA is heated to separate it into individual strands. These bind two distinct primers that are directed at specific sequences on opposite strands and that define the segment to be amplified. DNA polymerase extends the primers in each direction and synthesizes two strands complementary to the original two. This cycle is repeated 30 or more times, giving an amplified product of defined length and sequence. Note that the 4 dXTPs and the two primers are present in excess to minimize the possibility that these components are limiting for polymerization/amplification. It is important to note though that as cycle number increases incorporation rates can drop, and mutation/ error rates can increase.

The isolation of a specific (ca 1000 bp) mRNA-encoding gene from an entire human genome requires a technique that will discriminate one part in a million. The identification of a regulatory region that may be only 10 bp in length requires a sensitivity of one part in 3 x 10%; a disease such as sickle cell anemia is caused by a single base change, or one part in 3 x 10°. DNA technology is powerful enough to accomplish all these things.

Gene localization can define a map of the human genome. This is already yielding useful information in the definition of human disease. Somatic cell hybridization and in situ hybridization are two techniques historically used to accomplish this. In in situ hybridization, the simpler and more direct procedure, a radioactive probe is added to a metaphase spread of chromosomes on a glass slide. The exact area of hybridization is localized by layering photographic emulsion over the slide and, after exposure, lining up the grains with some histologic identification of the chromosome. Fluorescence in situ hybridization (FISH), which utilizes fluorescent rather than radioactively labeled probes, is a very sensitive technique that is also used for this purpose. This often places the gene at a location on a given band or region on the chromosome. Some of the human genes localized using these techniques are listed in Table 39-4. This table represents only a sampling of mapped genes since tens of thousands of genes have been mapped as a result of the sequencing of human genomes. Once the defect is localized to a region of DNA that has the characteristic structure of a gene, a synthetic cDNA copy of the gene can be constructed, which contains only mRNA-encoding exons, and expressed in an appropriate vector and its function can be assessed—or the putative polypeptide, deduced from the open reading frame in the coding region, can be synthesized. Antibodies directed against this protein or peptide fragments derived therefrom can be used to assess whether this protein is expressed in normal persons and whether it is absent, or altered in those with the genetic syndrome.

A practical goal of recombinant DNA research is the production of materials for biomedical applications. This technology has two distinct merits: (1) it can supply large amounts of material that could not be obtained by conventional purification methods (eg, interferon, tissue plasminogen activating factor, etc); and (2) it can provide human proteins (eg, insulin and growth hormone). The advantages in both cases are obvious. Although the primary aim is to supply products—generally

Insulin, Chromosome = 11p15. Insulin, Disease = Diabetes. Prolactin, Chromosome = 6p23-q12. Prolactin, Disease = Sheehan syndrome. Growth hormone, Chromosome = 17q21-qter. Growth hormone, Disease = Growth hormone deficiency. α-Globin, Chromosome = 16p12-pter. α-Globin, Disease = α-Thalassemia. β-Globin, Chromosome = 11p12. β-Globin, Disease = β-Thalassemia, sickle cell. Adenosine deaminase, Chromosome = 20q13-qter. Adenosine deaminase, Disease = Adenosine deaminase deficiency. Phenylalanine hydroxylase, Chromosome = 12q24. Phenylalanine hydroxylase, Disease = Phenylketonuria. Hypoxanthine-guanine phosphoribosyltransferase, Chromosome = Xq26-q27. Hypoxanthine-guanine phosphoribosyltransferase, Disease = Lesch-Nyhan syndrome. DNAsegment G8, Chromosome = 4p. DNAsegment G8, Disease = Huntington chorea
"This table indicates the chromosomal location of several genes and the diseases associated with deficient or abnormal production of the gene products. The chromosome involved is indicated by the first number or letter. The other numbers and letters refer to precise localizations, as defined in McKusick VA: Mendelian Inheritance in Man: Catalogs of Autosomal Dominant, Autosomal Recessive, and X-Linked Phenotypes. Baltimore: Johns Hopkins University Press; 1983.
proteins—for treatment (insulin) and diagnosis (AIDS testing) of human and other animal diseases and for disease prevention (hepatitis B vaccine), there are other potential commercial applications, especially in agriculture. An example of the latter is the attempt to engineer plants that are more resistant to drought or temperature extremes, more efficient at fixing nitrogen, or that produce seeds containing the complete complement of essential amino acids (rice, wheat, corn, etc).

There is a normal variation of DNA sequence just as it is true of more obvious aspects of human structure. Variations of DNA sequence, polymorphisms, occur approximately once in every 500 to 1000 nucleotides. A recent comparison of the nucleotide sequence of the genome of James Watson, the codiscoverer of DNA structure, identified about 3,300,000 single-nucleotide polymorphisms (SNPs) relative to the "standard" initially sequenced human reference genome. Interestingly, >80% of the SNPs found in Watson's DNA had already been identified in other individuals. There are also genomic deletions and insertions of DNA (ie, copy number variations; CNVs) as well as single-base substitutions. In healthy people, these alterations obviously occur in noncoding regions of DNA or at sites that cause no change in function of the encoded protein. This heritable polymorphism of
DNA structure can be associated with certain diseases within a large kindred and can be used to search for the specific gene involved, as is illustrated below. It can also be used in a variety of applications in forensic medicine.

Classic genetics taught that genetic diseases were due to point mutations that resulted in an impaired protein. This may still be true, but if on reading previous chapters one predicted that genetic disease could result from derangement of any of the steps leading from replication to transcription to RNA processing/transport and protein synthesis, PTMs, and/or subcellular localization and physical state (ie, aggregation and polymerization), one would have made a proper assessment. This point is again nicely illustrated by examination of the B-globin gene. This gene is located in a cluster on chromosome 11 (Figure 39-8), and an expanded version of the gene is illustrated in Figure 39-9. Defective production of 6-globin results in a variety of diseases and is due to many different lesions in and around the B-globin gene (Table 39-5).

The classic example is sickle cell disease, which is caused by mutation of a single base out of the 3 x 10° in the genome, a T-to-A DNA substitution, which in turn results in an A-to-U change in the mRNA corresponding to the sixth codon of the 6-globin gene. The altered codon specifies a different amino acid (valine rather than glutamic acid), and this causes a structural abnormality of the B-globin molecule leading to hemoglobin aggregation and red blood cell "sickling" Other point mutations in and around the B-globin gene result in decreased or, in some instances, no production of B-globin; B-thalassemia is the result of these mutations. (The thalassemias are characterized by defects in the synthesis of hemoglobin subunits, and so B-thalassemia results when there is insufficient production of B-globin.) Figure 39-9 illustrates that point mutations affecting each of the many processes involved in generating a normal mRNA (and therefore a normal protein) have been implicated as a cause of B-thalassemia.

Studies of bacteria, viruses, yeasts, fruit flies, and now humans show that pieces of DNA can move, or transpose, from one place to another within a genome via the process of DNA transposition. The deletion of a critical piece of DNA, the rearrangement of DNA within a gene, or the insertion or amplification of a piece of DNA within a coding or regulatory region can all cause changes in gene expression resulting in disease. Again, a molecular analysis of thalassemias produces numerous examples of these processes—particularly deletions—as causes of disease (Figure 39-8). The globin gene clusters seem particularly prone to this lesion. Deletions in the a-globin cluster, located on chromosome 16, cause a-thalassemia. There is a strong ethnic association for many of

FIGURE 39-8 Schematic representation of the B-globin gene cluster and of the lesions in some genetic disorders. The B-globin gene is located on chromosome 11 in close association with the two y-globin genes and the 6-globin gene. The B-gene family is arranged in the order 5'-€-Gy-Ay-B-5-B-3'. The € locus is expressed in early embryonic life (as a,,). The y-genes are expressed in fetal life, making fetal hemoglobin (HbF, a.y,). Adult hemoglobin consists of HbA (a,8,) or HbA 2 (a,6,). The 8 is a pseudogene that has sequence homology with B but contains mutations that prevent its expression. A locus control region (LCR), a powerful enhancer located upstream (5') of these six genes, controls the rate of transcription of the entire B-globin gene cluster. Deletions (solid darker bars, lower) within the B locus cause B-thalassemia (deficiency or absence [B°] of B-globin). Meiotic recombination between 6 and B causes hemoglobin Lepore, and results in DNA deletion and 6-B coding sequence fusions reducing the levels of HbB (see Figures 6-7 and 35-10). An inversion (AydB)° in this region (largest bar) disrupts gene function and also results in thalassemia (type Ill). Each type of thalassemia tends to be found in a certain group of people, for example, the (Ay5B)° deletion inversion occurs in persons from India. Many more deletions in this region have been mapped, and each causes some type of thalassemia.

these deletions, so that northern Europeans, Filipinos, blacks, and Mediterranean peoples have different lesions all resulting in the absence of hemoglobin A and a-thalassemia. A similar analysis could be made for a number of other diseases. Point mutations are usually defined by sequencing the gene in question, though occasionally, if the mutation destroys or creates a restriction enzyme site, the technique of restriction fragment analysis can be used to pinpoint the lesion. Deletions or insertions of DNA larger than 50 bp can often be detected by the Southern blotting procedure while PCR-based assays can detect much smaller changes in DNA structure.

Sickle cell disease again provides an excellent example of how recombinant DNA technology can be applied to the study of human disease. The substitution of T for A in the template strand of DNA in the B-globin gene changes the sequence in the region that corresponds to the sixth codon from
Other MstII sites 5' and 3' from this site (Figure 39-10) are not affected and so will be cut. Therefore, incubation of DNA from normal (AA), heterozygous (AS), and homozygous (gS) individuals results in three different patterns on Southern blot transfer (Figure 39-10). This illustrates how a DNA pedigree can be established using the principles discussed in this chapter. Pedigree analysis has been applied to a number
FIGURE 39-9 Mutations in the a-globin gene causing B-thalassemia. The B-globin gene is shown in the 5' to 3' orientation. The crosshatched areas indicate the 5' and 3' untranslated regions (UTRs). Reading from the 5' to 3' direction, the shaded areas are exons 1 to 3 (E,, E,, E,) and the white areas between exons are introns 1 (I,) and 2 (I,). Mutations that affect transcription control (+) are located in the 5' flanking-region DNA. Examples of nonsense mutations (A), mutations in RNA processing (©), and RNA cleavage mutations (O) have been identified and are indicated. In some regions, many distinct mutations have been found. These are indicated by the size and location of the brackets.

Point mutations, Function Affected = Protein folding. Point mutations, Disease = Sickle cell disease. Point mutations, Function Affected = Transcriptional control. Point mutations, Disease = β-Thalassemia. Point mutations, Function Affected = Frameshift and nonsense mutations. Point mutations, Disease = β-Thalassemia. Point mutations, Function Affected = RNA processing. Point mutations, Disease = β-Thalassemia. Deletion, Function Affected = mRNAproduction. Deletion, Disease = β 0 -Thalassemia. Deletion, Function Affected = . Deletion, Disease = Hemoglobin Lepore. Rearrangement, Function Affected = mRNAproduction. Rearrangement, Disease = β-Thalassemia type III
of genetic diseases and is most useful in those caused by deletions and insertions or the rarer instances in which a restriction endonuclease cleavage site is affected, as in the example cited here. Such analyses are now facilitated by the PCR reaction, which can amplify and hence provide sufficient DNA for analysis from just a few nucleated cells.

If the genetic lesion is understood and a specific probe is available, prenatal diagnosis is possible. DNA from cells collected from a small volume of amniotic fluid (or by chorionic villus biopsy) can be analyzed by Southern blot transfer, and much smaller volumes if PCR-based assays are used. A fetus with the restriction pattern AA in Figure 39-10 neither has sickle
FIGURE
39-10
restriction enzyme
1.15- and
Pedigree analysis
of sickle
cell disease.
The top
part of
the figure
(A)
shows the
first part
of the
B-globin sites
0.2-kb
B-globin long
gene;
a
in the
normal in
hence,
Southern blot.
normal
a
(A)
and individuals.
single
(B)
restriction
Pedigree sickle
cell
The
T-to-A
fragment analysis
shows
(S)
B-globin change
genes.
Digestion in
1.35
three kb
in length
possibilities with
with sickle
is generated
for
(AS):
AA, normal
(open circle);
AS, heterozygous
(half-solid four
circles, prenatal diagnosis of sickle cell disease (dash-sided square).
the restriction
cell upon
offspring
O,, half-solid
O,, square);
SS, disease
enzyme abolishes
cleavage with
one
Mstll.
This
O, and
O,, given
homozygous
Mstil results
of gene
in and
the
Mstll
DNA
the size
Parental
(solid
(P,, square).
three fragments
Mstll difference
sites is
around easily
P,)
the detected
heterozygous
This on
geneotypes approach
can allow
for
cell disease, nor is it a carrier. A fetus with the SS pattern will develop the disease. Probes are now available for this type of analysis of many genetic diseases.

The differences in DNA sequence cited above can result in variations of restriction sites and thus in the length of restriction fragments. Similarly, SNPs can be detected by the sensitive PCR method. An inherited difference in the pattern of restriction enzyme digestion (eg, a DNA variation occurring in more than 1% of the general population) is known as a restriction fragment length polymorphism (RFLP). Extensive RFLP and SNP maps of the human genome have been constructed. This is proving useful in the Human Genome Analysis Project and is an important component of the effort to understand various single-gene and multigenic diseases. RFLPs result from single-base changes (eg, sickle cell disease) or from deletions or insertions (CNVs) of DNA into a restriction fragment (eg, the thalassemias) and have proved to be useful diagnostic tools. They have been found at known gene loci and in sequences that have no known function; thus, RFLPs or SNPs may disrupt the function of the gene or may have no apparent biologic consequences. As mentioned above, 80% of the SNPs in the genome of a single known individual had already been mapped independently through the efforts of the SNP-mapping component of the International HapMap Project and now supplemented by genomic sequencing.
procedure is repeated sequentially until the desired sequence is obtained. Collections of mapped, overlapping BACor PACcloned human genomic DNAs are commercially available. The X chromosome-linked disorders are particularly amenable to the approach of chromosome walking since only a single allele is expressed. Hence, 20% of the defined RFLPs are on the X chromosome and a complete linkage map (and genomic sequence) of this chromosome have been determined. The gene for the X-linked disorder, Duchenne-type muscular dystrophy, was found using RFLPs. Similarly, the defect in Huntington disease was localized to the terminal region of the short arm of chromosome 4, and the defect that causes polycystic kidney disease is linked to the a-globin locus on chromosome 16. Genomic sequencing depends on this "overlap" between sequenced DNA fragments to assemble complete genomic DNA sequences.

Short (2-6 bp), inherited, tandem repeat units of DNA occur about 50,000 to 100,000 times in the human genome (see Chapter 35). Because they occur more frequently, and in view of the routine application of sensitive PCR methods, they are replacing RFLPs as the marker loci for various genome searches.

RFLPs and SNPs are inherited, and they segregate in a mendelian fashion. A major use of SNPs/RFLPs is in the definition of inherited diseases in which the functional deficit is unknown. SNPs/RFLPs can be used to establish linkage groups, which in turn, by the processes of pedigree analyses and chromosome walking, will eventually define the disease locus. In chromosome walking (Figure 39-11), a fragment representing one end of a long piece of DNA is used to isolate another that overlaps but extends the first. The direction of extension is determined by restriction mapping, and the
Variable numbers of tandemly repeated (VNTR) units are one common type of "insertion" that results in an RFLP. The VNTRs can be inherited, in which case they are useful in establishing genetic association with a disease in a family or kindred; or they can be unique to an individual and thus serve as a molecular fingerprint of that person.

As noted above, recent advances in DNA-sequencing technology, the so-called next-generation (NGS), or high-throughput sequencing (HTS) platforms, have dramatically reduced the per base cost of DNA sequencing. The initial sequence of the
FIGURE 39-11 The technique of chromosome walking. Gene X is to be isolated from a large piece of DNA. The exact location of this gene is not known, but a probe (* ) directed against a fragment of DNA (shown at the 5' end in this representation) is available, as is a library of clones containing a series of overlapping DNA insert fragments. For the sake of simplicity, only five of these are shown. The initial probe will hybridize only with clones containing fragment 1, which can then be isolated and used as a probe to detect fragment 2. This procedure is repeated until fragment 4 hybridizes with fragment 5, which contains the entire sequence of gene X. A conceptually similar method of DNA sequence overlap is used to assemble the contiguous sequence reads generated by direct NGS/high-throughput sequencing of genomic
DNA fragments.
human genome costs roughly $350,000,000 (US). The cost of sequencing the same 3 x 10° bp diploid human genome using the new NGS platforms is estimated to be <0.03% of the original. Very recently, the technology has been developed to allow for human genome sequencing for $1000 (US). This dramatic reduction in cost has stimulated various international initiatives to sequence the entire genomes of thousands of individuals of various racial and ethnic backgrounds in order to determine the true extent of DNA/genome polymorphisms present within the human population. The resulting cornucopia of genetic information, and the ever-decreasing cost of genomic DNA sequencing is dramatically increasing our ability to diagnose and, ultimately treat human disease. Obviously, when personal genome sequencing does become commonplace, dramatic changes in the practice of medicine will result because therapies will ultimately be custom tailored to the exact genetic makeup of each individual.

Diseases caused by deficiency of a single gene product (Table 39-4) are all theoretically amenable to replacement therapy. The strategy is to clone a normal copy of the relevant gene (eg, the gene that codes for adenosine deaminase) into a vector that will readily be taken up and incorporated into the genome of a host cell. Bone marrow precursor cells are being investigated for this purpose because they presumably will resettle in the marrow and replicate there. The introduced gene would begin to direct the expression of its protein product, and this would correct the deficiency in the host cell.
As an alternative to "replacing" defective genes to cure human disease, many scientists are investigating the feasibility of identifying and characterizing pluripotent stem cells that have the ability to differentiate into any cell type in the body. Recent results in this field have shown that adult human somatic cells can readily be converted into apparent induced pluripotent stem cells (iPSCs) by transfection with cDNAs encoding a handful of DNA-binding transcription factors. These and other new developments in the fields of gene therapy and stem cell biology promise exciting new potential therapies for curing human disease. Finally, generating iPSCs from diseased patient cells also offer the opportunity to create authentic human cell-based models for laboratory studies of the molecular basis of human disease.

and in discovering genes involved in development, a process that heretofore has been difficult to study in mammals. The transgenic approach has been used to correct a genetic deficiency in mice. Fertilized ova obtained from mice with genetic hypogonadism were injected with DNA containing the coding sequence for the gonadotropin-releasing hormone (GnRH) precursor protein. This gene was expressed and regulated normally in the hypothalamus of a certain number of the resultant mice, and these animals were in all respects normal. Their offspring also showed no evidence of GnRH deficiency. This is, therefore, evidence of somatic cell expression of the transgene and of its maintenance in germ cells.

Various technical advances have allowed for the precise, targeted modification of mammalian genes. The exact methods used for genetically engineering mammalian genomes have evolved from tedious, low-efficiency methods based on positive and negative drug selections and homologous recombination (Knockout/Knockin) to the recently described CRISPR-Cas9 system described above. The goal of all of these methods is ultimately to generate a family of genetic variants of a gene of interest that are (a) a null, or complete loss-of-function allele; (b) recessive, loss-of-function alleles; and (c) ideally, dominant gain-of-function alleles. These genetic alterations are generated in pluripotent stem cells, which ultimately allow for introduction and propagation in whole model organisms (flys, fish, worms, rodents, etc). Having all three of these genetic variants allows one to precisely dissect the mechanism of action of any gene. However, complicating genetic analyses of many genes is the fact that their function(s) are essential for viability. To get around this problem, cell-type or tissue-specific genetic variants must be generated. This hurdle has been overcome through the use of celland tissue-specific enhancers that can drive the conditional (ie, experimentally controlled) expression of the targeting recombinases (ie, CRE-lox) and/or or nuclease (CRISPR-Cas9) that generate the altered genes, either or lossor gain-of-function alleles. Alternatively, selected loss-of-function alleles can be generated through equivalent siRNA expression to knock down production of a specific gene product. Collectively, these methods allow for sophisticated genetic and biochemical tests of gene function and allow scientists to probe the structure and function of mammalian genes in physiologic contexts. Tremendous molecular mechanistic insights have been, and will continue to be, obtained into the molecular etiology of human disease through these and other biochemical approaches. Exciting times lie ahead!

The "-omic" revolution of the last decade has culminated in the determination of the complete nucleotide sequence
of tens of thousands of genomes, including those of budding and fission yeasts, numerous bacteria, the fruit fly, the worm Caenorhabditis elegans, plants, the mouse, rat, chicken, monkey, and, most notably, humans. Additional genomes are being sequenced at an accelerating pace. The availability of all of this DNA sequence information, coupled with engineering advances, has led to the development of several revolutionary methodologies, most of which are based on high-density microarray technology and NGS sequencing platforms. In the case of microarrays, it is now possible to deposit thousands of specific, known, defined DNA sequences on a glass microscope-style slide, or other inert support, in the space of a few square centimeters. By coupling such DNA microarrays with highly sensitive detection of hybridized fluorescently labeled nucleic acid probes derived from mRNA, investigators can rapidly and accurately generate profiles of gene expression (eg, specific cellular mRNA content) from cell and tissue samples as small as 1 g or less. Thus, entire transcriptome information (the entire collection of cellular RNAs) for such cell or tissue sources can readily be obtained in only a few days. In the case of NGS sequencing, mRNAs are converted to cDNAs using reverse transcription, and these cDNAs are amplified and directly sequenced; this method is termed RNA-Seq. These methods allow for the quantitative description of the entire transcriptome. Recent reports in the literature have used RNA-Seq to describe the transcriptome of single cells, and when coupled with high-sensitivity ribosome profiling (see below) and mass spectrometry-based proteomics (see below), confidently define gene expression profiles at the mRNA and protein levels.

Recent methodologic advances (PRO-Segq, Precision RunOn sequencing, and NET-seq, native elongating transcript sequencing) allow for sequencing of RNA within elongating RNA polymerase-DNA-RNA ternary complexes, thereby allowing nucleotide-level descriptions, genome-wide, of active transcription in living cells. A parallel method termed ribosome profiling allows investigators to use high-throughput DNA sequencing to determine both the identity and number of cellular mRNAs in the process of being actively translated—thereby defining the cellular proteome. Transcriptome information allows one to quantitatively predict the collection of proteins that might be expressed in a particular cell, tissue, or organ in normal and disease states based on the mRNAs present in those cells, while ribosome profiling allows for the quantitative measurement of the actual cellular proteome.

Complementing the very high-throughput, genome-wide expression profiling methods described above is the development of methods to map the location, or occupancy of specific proteins bound to discrete DNA sequences within living cells. This method, illustrated in Figure 39-12, is termed chromatin immunoprecipitation (ChIP). Proteins are cross-linked in situ in cells or tissues, the cellular chromatin is isolated, sheared, and specific cross-linked protein-DNA complexes purified using antibodies that recognize a particular protein, or protein isoform. DNA bound to this protein is recovered and amplified using PCR and analyzed using gel electrophoresis imaging, microarray hybridization (ChIP-chip), or direct sequencing. There are two versions of the DNA sequencing assay readout. In the first, the immunopurified DNA is directly subjected to NGS/high-throughput DNA sequencing (ChIP-Seq); in the second version, the immunopurified, cross-linked proteinDNA complex is treated with exonucleases to remove crosslinked DNA sequences that are not in intimate contact with the protein of interest; this is termed ChIP-Exo. Collectively ChIP-chip and ChIP-Seq methods allow investigators to identify the locations of a single protein genome-wide throughout all the chromosomes. ChIP-Exo has the added advantage of allowing investigators to map in vivo protein occupancy at single nucleotide-level resolution. Finally, methods for highsensitivity, high-throughput mass spectrometry of metabolites (metabolomics), various small molecules (lipids, lipidomics; carbohydrates, glycomics, etc), and complex protein samples (proteomics) have been developed. Newer mass spectrometry methods allow scientists to identify hundreds to thousands of proteins in complex samples extracted from very small numbers of cells (<1 g). Such analyses can now be used to quantify the relative amounts of proteins in two samples, as well as the level of certain PTMs, such as phosphorylation, acetylation etc; and with the use of specific antibodies, define specific proteinprotein interactions. This critical information tells investigators which of the many mRNAs detected in transcriptome mapping studies are being translated into protein, generally the ultimate dictator of phenotype.

New genetic means for identifying protein-protein interactions and protein function have also been devised. Systematic genome-wide gene expression knockdown using siRNAs, synthetic lethal genetic interaction screens, or most recently CRISPR-Cas9 knockdown have been used to assess the contribution of individual genes to a variety of processes in model systems (yeast, worms, and flies) and mammalian cells (human and mouse). Specific network mappings of protein-protein interactions, on a genome-wide basis, have been identified using high-throughput variants of the two-hybrid interaction test (Figure 39-13). This simple yet powerful method can be performed in bacteria, yeast, or metazoan cells, and allows for the detection of specific protein-protein interactions in living cells. Reconstruction experiments indicate that protein-protein interactions with binding affinities of >K, ~10~° mol/L can readily be detected with this method. Together, these technologies provide powerful tools with which to dissect the intricacies of human biology.

Microarray techniques, high-throughput genomic DNA sequencing, RNA-Seq, ribosome profiling, mass spectrometry, ChIP-Seq/ChIP-Exo, genome-wide two-hybrid screens,

FIGURE 39-12 Outline of the chromatin immunoprecipitation (ChIP) technique. This method allows for the precise localization of a particular protein (or modified protein if an appropriate antibody is available; eg, n = anti-phosphorylated or acetylated histones, transcription factors, etc) on a particular sequence element in living cells. Depending on the method used to analyze the immunopurified DNA, quantitative or semiquantitative information, at near nucleotide level resolution, can be obtained. Protein-DNA occupancy can be scored genome-wide in two ways. First, by ChIP-chip, a method that uses a hybridization readout. In ChIP-chip total genomic DNA is labeled with one particular fluorophore and the immunopurified DNA is labeled with a spectrally distinct fluorophore. These differentially labeled DNAs are mixed and hybridized to microarray "chips" (microscope slides) that contain specific DNA fragments, or more commonly now, synthetic oligonucleotide 50 to 70 nucleotides long. These gene-specific oligonucleotides are deposited and covalently attached at predetermined, known X,Y position/coordinates on the slide. The labeled DNAs are hybridized, the slides washed and hybridization to each gene-specific oligonucleotide probe is scored using differential laser scanning and sensitive photodetection at micron resolution. The hybridization signal intensities are quantified, and the ratio of IP DNA/genomic DNA signals is used to score occupancy levels. The second method, termed ChIP-Seq, directly sequences immunopurified DNAs using NGS sequencing methods. Two variants of ChIP-Seq are shown: "standard" ChIP-Seq and ChIP-Exo. These two approaches differ in their ability to resolve and map the locations of the bound protein on genomic DNA. Standard ChIP-Seq resolution is 50 to 100 nt resolution, while ChIP-Exo has near single nt level resolution. Both approaches rely on efficient bioinformatic algorithms to deal with the very large datasets that are generated. ChIP-chip and ChIP-Seq techniques provide a (semi-) quantitative measure of in vivo protein

occupancy. Though not schematized here, similar methods termed RIP (RNA immunoprecipitation) or CLIP (cross-linking protein-RNA and immunoprecipitation), which differ primarily in the method of protein-RNA cross-linking, can score the in vivo binding of specific proteins to specific RNA species (typically mRNAs, though any RNA species can be analyzed by these techniques).
genetic knockdown, and synthetic lethal screens coupled with mass spectrometric protein and metabolite identification experiments have led to the generation of enormous amounts of data. Appropriate data management, analysis, and interpretation of the deluge of information forthcoming from such studies have relied on the application of statistical methods and novel algorithms for analyzing or "mining" and visualizing such huge datasets. This has led to the development
(1)

FIGURE 39-13 Overview of two hybrid system for identifying and characterizing protein-protein interactions. Shown are the basic components and operation of the two hybrid systems, originally devised by Fields and Song (Nature 340:245-246 [1989]) to function in the baker's yeast system. (1) A reporter gene, either a selectable marker (ie, a gene conferring prototrophic growth on selective media, or producing an enzyme for which a colony colorimetric assay exists, such as B-galactosidase) that is expressed only when a transcription factor binds upstream to a cis-linked enhancer (dark red bar). (2) A"bait" fusion protein (DBD-X) produced from a chimeric gene expressing a modular DNA-binding domain (DBD; often derived from the yeast Gal4 protein or the bacterial Lex A protein, both high-affinity, high-specificity DNA-binding proteins) fused in-frame to a protein of interest, here X. In two hybrid experiments, one is testing whether any protein can interact with protein X. Prey protein X may be fused in its entirety or often alternatively just a portion of protein X is expressed in-frame with the DBD. (3) A "prey" protein (Y-AD), which represents a fusion of a specific protein fused in-frame to a transcriptional activation domain (AD; often derived from either the herpes simplex virus VP16 activator protein or the yeast GAL4 protein). This system serves as a useful test of protein-protein interactions between proteins X and Y because in the absence of a functional transactivator binding to the indicated enhancer, no transcription of the reporter gene occurs (ie, see Figure 38-16). Thus, one observes transcription only if protein X-protein Y interaction occurs, thereby bringing a functional AD to the cis-linked transcription unit, in this case activating transcription of the reporter gene. In this scenario, protein DBD-X alone fails to activate reporter transcription because the X-domain fused to the DBD does not contain an AD. Similarly, protein Y-AD alone fails to activate reporter gene transcription because it lacks a DBD to target the Y-AD protein to the enhancer-promoter-reporter gene.

Only when both proteins are expressed in a single cell and bind the enhancer and, via DBD-X-Y-AD protein-protein interactions, regenerate a functional transactivator binary "protein,' does reporter gene transcription result in activation and mRNA synthesis (green line from AD to reporter gene).
of the
field of
bioinformatics
(see also
Chapter
11).
These new
technologies, tal
data, coupled
with have
further systems
biology, analyze,
led to
the flood
the
a
of development
discipline and
integrate information
whose this
experimen- of
the field
of goal
is to
flood in
order to
gain pathologic
quantitatively of
novel
Future biologically
insights important
into biology
work at
the and
its intersection
of bioinformatics, engineering, biophysics, genetics, transcript, protein, protein PTM profiling, and systems biology will revolutionize our understanding of physiology and medicine and ultimately human health.

@ In DNA cloning, a particular segment of DNA is either directly synthesized, or is removed from its normal environment using PCR or one of many DNA-directed endonucleases. Such DNA is then ligated into a vector in which the DNA segment can be individually amplified and produced in abundance.
™ Manipulation of the DNA to change its sequence, so-called genetic engineering, is a key element in cloning (eg, the construction of chimeric molecules) and can also be used to study the function of a certain fragment of DNA and to analyze how genes are regulated.
™@ A variety of very sensitive techniques can now be applied to the isolation and characterization of genes and to the quantitation of gene products in both static (ie, equilibrium) and dynamic (kinetic) modes. These methods allow for the identification of the genes responsible for diseases, and for the study of how faulty genes/gene regulation causes disease.
= Mammalian genomes can now be precisely engineered to knockin (add/replace a gene), knockout (delete or inactivate), and/or actively and conditionally manipulate specific genes using miRNAs and SiRNAs, and novel genome editing enzymes (recombinases) and enzyme-RNA systems (CRISPR-Cas).

ability polymerase
degrade
RT-PCR: Amethod used to quantitate mRNAlevels that relies on a first step of cDNA copying of mRNAs catalyzed by reverse transcriptase prior to PCR amplification and quantitation., 1 = . Signal: The end product observed when a specific sequence of DNA or RNA is detected by autoradiography or some other method. Hybridization with a complementary radioactive polynucleotide (eg, by Southern or Northern blotting) is commonly used to generate the signal. SINES: Short interspersed repeat sequences. siRNAs: Silencing RNAs, 21 to 25 nt in length generated by selective, 1 = . nucleolytic degradation of double-stranded RNAs of cellular or viral origin. siRNAs anneal to various specific sites within target in RNAs leading to mRNAdegradation, hence gene 'knockdown.', 1 = . SNP: Single-nucleotide polymorphism. Refers to the fact that single-nucleotide genetic variation in genome sequence exists at discrete loci throughout the chromosomes., 1 = . Measurement of allelic SNP differences is useful for gene mapping studies., 1 = . Southern blot: Amethod for transferring DNAfrom an agarose gel to nitrocellulose filter, on which the DNAcan be detected by a suitable probe (eg, complementary DNAor RNA)., 1 = . blot: Amethod for detecting protein-DNA, 1 = . Southwestern interactions by applying a labeled DNAprobe to a, 1 = . membrane that contains a renatured protein., 1 = . , 1 = transfer

C. Cytosine and ribose
D. Thymine and deoxyribose
1. Which of the following statements about B,y-methylene and
£,y-imino derivatives of purine and pyrimidine triphosphates is
CORRECT?
A. They are potential anticancer drugs.
B. They are precursors of B vitamins.
C. They readily undergo hydrolytic removal of the terminal
D. They can be used to implicate involvement of nucleotide
triphosphates by effects other than phosphoryl transfer.
E. They serve as polynucleotide precursors.
2. Which of the following statements about nucleotide structures is
NOT CORRECT?
A. Nucleotides are polyfunctional acids.
B. Caffeine and theobromine differ structurally solely with
respect to the number of methyl groups attached to their
ring nitrogens.
C. A purine is a heterocyclic aromatic molecule composed of a
pyrimidine ring fused to an imidazole ring.
D. NAD*, FMN, S-adenosylmethionine, and coenzyme A all
are derivatives of ribonucleotides.
E. 3’,5’-Cyclic AMP and 3;5’-cyclic GMP (cAMP and cGMP) serve as second messengers in human physiology.
3. Which of the following statements about purine nucleotide
metabolism is NOT CORRECT?
A. An early step in purine biosynthesis is the formation of
PRPP (phosphoribosyl 1-pyrophosphate).
B. Inosine monophosphate (IMP) is a precursor of both AMP and GMP.
E. A phosphate group and adenine
. The backbone of a DNA molecule consists of which of the
following?
Alternating sugars and nitrogenous bases R>
Nitrogenous bases alone
Phosphate groups alone Alternating phosphate and sugar groups BOO
Five carbon sugars alone
. Which of the following is the interconnecting bond that connects
the nucleotides of RNA and DNA ?
A. N-glycosidic bonds

B. 3’-5’ phosphodiester linkages
C.
Phosphomonoesters
D. -2’
phosphodiester linkages
E. Peptide nucleic acid bonds
. Which component of the DNA duplex causes the molecule to
have a net negative charge at physiologic pH?
A. Deoxyribose
B. Ribose
C. Phosphate groups
D. Chlorine ion
E, Adenine
. Which molecular feature listed causes duplex DNA to exhibit a
near-constant width along its long axis?
A. A purine nitrogenous base always pairs with another purine
nitrogenous base.
B. A pyrimidine nitrogenous base always pairs with another
pyrimidine nitrogenous base.
C. Orotic acid is an intermediate in pyrimidine nucleotide
C. A pyrimidine nitrogenous base always pairs with a purine
D. Humans catabolize uridine and pseudouridine by analogous
D. Repulsion between phosphate groups keeps the strands a
E. Ribonucleotide reductase converts nucleoside diphosphates
to the
corresponding deoxyribonucleoside diphosphates.
4. Which of the following statements is NOT CORRECT?
A. Metabolic disorders are only infrequently associated with
defects in the catabolism of purines.
B. Immune dysfunctions are associated both with a defective
adenosine deaminase and with a defective purine nucleoside phosphorylase.
C. The Lesch-Nyhan syndrome reflects a defect in
hypoxanthine-guanine phosphoribosyl transferase.
D. Xanthine lithiasis can be due to a severe defect in xanthine
oxidase.
E. Hyperuricemia can result from conditions such as cancer characterized by enhanced tissue turnover.
5. Which of the following components are found in DNA? Choose
the most complete answer.
A. A phosphate group, adenine, and ribose
B. A phosphate group, guanine, and deoxyribose
10. The model for DNA replication first proposed by Watson and

uniform distance apart.
E. Attraction between phosphate groups keeps the strands a
Crick posited that every newly replicated double-stranded
daughter duplex DNA molecule.
A. Was composed of the two strands from the parent DNA
molecule
B. Contained solely the two newly synthesized strands of DNA
C.
Contained
two
strands
that
are
random
mixtures
of
new
and
old DNA within each strand
D. Was composed of one strand derived from the original
parental DNA duplex and one strand that was newly
E. Was composed of nucleotide sequences completely distinct from either parental DNA strand
454
11. Name the mechanism through which RNAs are synthesized from
12. Which of the forces or interactions listed below play the
13. Name the enzyme that synthesizes RNA from a double-stranded
14. Define the most notable characteristic difference with regard to
15. Which entry below correctly describes the approximate
16. What is the approximate number of base pairs associated with a
17. All but one of the following histones are found located within the
DNA.
Replicational duplication >
Translation
Translesion repair
Transesterification
Transcription
BOOS
predominant role in driving RNA secondary and tertiary
structure formation?
Hydrophilic repulsion
Formation of complementary base pair regions
Hydrophobic interaction
van der Waals interactions
Salt bridge formation
BOOP
DNA template.
RNA-dependent RNA polymerase >
DNA-dependent RNA convertase
RNA-dependent replicase
DNA-dependent RNA polymerase
Reverse transcriptase
BOOS
gene expression between eukaryotes and prokaryotes.
Ribosomal RNA nucleotide lengths
Mitochondria
Lysosomes and peroxisomes
Sequestration of the genomic material in the nucleus
BOOP
number of bp of DNA.
»
which
is
separated
into
chromosomes in a typical diploid human cell in a nonreplicating
state?
64 billion, 23
6.4 trillion, 46
23 billion, 64
64 billion, 46
6.4 billion, 46

BOOS
single nucleosome?
146
292
73
1460
900
BOOP
superhelix formed between DNA and the histone octamer; which
of the following is this histone?
Histone H2B Histone H3 Histone H1 Histone H3 Hisone H4 BOOR
18. Chromatin can be broadly defined as active and repressed.
19. Which of the following hypothesizes that the physical and
20. What is the name of the unusual repeated stretch of DNA
21. Given that DNA polymerases are unable to synthesize DNA
22. Which of the following terms is used for the discontinuous DNA
23. What molecule or force supplies the energy that drives the relief
24. What is the name of the phase of the cell cycle between the
Which of the following is termed as a subclass of chromatin that
is
inactivated
at
certain
times
within
an
organism's
life and/or in particular sets of differentiated cells?
A. Constitutive euchromatin
B. Facultative heterochromatin
C. Euchromatin
D. Constitutive heterochromatin
functional status of a certain region of genomic chromatin is
dependent
on
the
patterns
of
specific
histone
posttranslational
modifications (PTMs), and/or DNA methylation status?
Morse code
PTM hypothesis
Nuclear body hypothesis
Epigenetic code
Genetic code
BOOP
localized at the tips of all eukaryotic chromosomes?
Kinetochore >
Telomere
Centriole
Chromomere
Micromere
BOOS
without a primer, what molecule serves as the primer for these
enzymes during DNA replication?
Five carbon sugars
Deoxyribose alone
A short RNA molecule
Proteins with free hydroxyl groups
Phosphomonoester
BOOS
replication that occurs during replication is catalyzed via the
production of small DNA segments?
Okazaki fragments
Toshihiro pieces
Onishi oligonucleotides
Crick strands
Watson fragments
BOOS
of mechanical strain by DNA gyrase?
. Pyrimidine to purine conversion

Hydrolysis of GTP APS
. Hydrolysis of ATP
. Glycolysis mo
A proton gradient molecule or force
conclusion of cell division and the beginning of DNA synthesis?
G 1
BOOP S
G
25. At what stage of the cell cycle are key protein kinases, like cyclin-
26. What disease is often associated with a breakdown ofa cell’s
27. What is the molecular mechanism that is responsible for the
28. Which of the following is the site to which RNA polymerase
29. The large eukaryotic rRNA genes, such as 18S and 28S RNA-
30. Eukaryotic RNA polymerases all have a requirement for a large
31. The DNA segment from which the primary transcript is copied
dependent kinase, activated?
Right before mitosis
At the beginning of S phase
Near the end of G, phase
At the end of the G, phase
BOOP
ability to regulate/control its own division?
Kidney disease
Cancer Diabetes moUOR>
Emphysema
quick decrease in the Cdk activity that leads to exit from the
M phase and the entry into G,?
Drop in mitotic cyclin concentration >
Decreased G, cyclin concentration
Rise in G, cyclin concentration
Rise in mitotic cyclin concentration
BOOR
binds on the DNA template prior to the initiation of
transcription?
Intron/exon junction >
Open reading frame DNA
Terminator
Initiator methionine codon
Promoter
BOOR
encoding genes, are transcribed by which of the following RNA polymerases?
RNA polymerase III >
RNA-dependent RNA polymerase 5
RNA polymerase I
RNA polymerase II
Mitochondrial RNA polymerase
BOOR
variety of accessory proteins to enable them to bind promoters
and form physiologically relevant transcription complexes. What
are these proteins termed as?
A. Basal or general transcription factors
B. Activators
C. Accessory factors
D. Elongation factors
E. Facilitator polypeptides
or transcribed is called which of the following?

Coding strand >
Initiator methionine domain
Translation unit
Transcriptome
Initial codon
BOOP
32. What class of DNA sequences are the eukaryotic genes that
33. How the modifications to the nucleotides of the pre-tRNAs, pre-
34. RNA polymerase II promoters are located on which side of the
35. With regard to eukaryotic mRNAs, which one of the following is
36. Which of the following is the bond connecting the initiation
37. What sequence feature of mature mRNAs listed below is thought to protect mRNAs from degradation?
38. What could the consequences of inaccurate mRNA splicing be
encode rRNAs?
A. Single-copy DNA
B. Highly repetitive DNA
C. Moderately repetitive DNA
D. Mixed sequence DNA
rRNAs, and pre-mRNAs occur?
A. Postprandially
B. Postmitotically
C. Pretranscriptionally
D. Posttranscriptionally
E. Prematurely
transcription unit?
Internal
3’ downstream
Nearest to the C-terminus
Nearest to the N-terminus
5’ upstream
mOOR>
not a normal property of mRNAs?
A. Eukaryotic mRNAs have special modifications at their 5’
(cap) and 3’ (poly(A) tail) termini.
B. Are attached to ribosomes when they are translated.
C. They are found in the cytoplasm within peroxisomes.
D. Most have a significant noncoding segment that does not
direct assembly of amino acids.
E. Contain continuous nucleotide sequences that encode a
particular polypeptide.
nucleotide of the mRNA with the 5™:-G Cap structure?
3’-5’ phosphodiester bridge
5’-5’ triphosphate bridge
3’-3’ triphosphate bridge
3’-5’ triphosphate bridge
5’-3’ triphosphate bridge
BOOP
Special posttranslational modifications >
3’ Poly(C), tail
5™°-G

Cap
Introns
Lariat structures
mOOR
for the RNA?
A. A single base error at a splice junction will cause a large
deletion.
B. A single base error at a splice junction will cause a large
insertion.
C. A single base error at a splice junction will cause a large
inversion.
D. CandE.
E. A single base error at a splice junction will change the
reading frame and result in mRNA mistranslation.
39. What is the macromolecular complex that associates with introns
40. What reaction does reverse transcriptase catalyze?
Al. RNAi or dsRNA-mediated RNA interference mediates which of
42. While the genetic code has 64 codons, there are only 20 naturally
43. The genetic code contains how many termination codons?
44,
45, What is on the 3’ end of all functional, mature tRNAs?
during mRNA splicing?
Splicer
Dicer
Nuclear body
Spliceosome
Slicer
BOOP
A. Translation of RNA to DNA
Transcription of DNA to RNA Conversion of ribonucleotides into deoxyribonucleotides Transcription of RNA to DNA moas
Conversion of a ribonucleotide to deoxynucleotides in the
DNA double helix
the following?
RNA ligation RNA silencing RNA restoration BOOP
RNA
inversion
RNA quelling
occurring
amino
acids.
some
amino
acids
are
encoded
by
more
than
one
codon.
This
feature
of
the
genetic
code is an illustration of the genetic code being which of the
A. Degenerative
B. Duplicative
C. Nonoverlapping
D. Overlapping
E. Redundant
A. 3
B. 21
Cc. 61
D. 64
E. 20
Ifa
tRNA
has
the
sequence
5’-CAU-3’,
what
codon
would
it
recognize (ignore wobble base pairing).

3’-UAC-5’ >
3’-AUG-5’
5’-ATG-3’
5’-AUC-3’
5’-AUG-3’
BOO
A. The cloverleaf loop
B. The anticodon
C. The sequence CCA
D. The codon
46. Most aminoacyl-tRNA synthetases possess an activity
that is shared with DNA polymerases. This activity is a
function.
Proofreading >
Hydrogenase
Proteolytic
Helicase
Endonucleolytic
MOOR
. Which of the following is the CORRECT order for the three
distinct phases of protein synthesis?
A.
Initiation, termination, elongation
B. Termination, initiation, elongation
C.
Initiation, elongation, termination
D. Elongation, initiation, termination
E. Elongation, termination, initiation
. Which amino acid is the initiating amino acid for essentially all

Cysteine >
Threonine
Tryptophan
Glutamic acid
BOOR
. The initiator tRNA is placed within the active 80S complex at
which of the three-canonical ribosomal “sites” during protein
synthesis?
E
site
I site
P site
A site
Releasing factor binding site
BOOR
>
. Name the enzyme that forms the peptide bond during protein
synthesis and define its chemical composition.
Pepsynthase, protein
Peptidyl transferase, RNA
Peptidase, glycolipid
Peptidyl transferase, protein
GTPase, glycopeptide
BOOP
. What is the term used for mutations in the middle of an open
reading frame that create a stop codon?
Frameshift mutation >
Missense mutation
No-nonsense mutation
Point mutation
BOO
. What is the directionality of polypeptide synthesis?
.
C-terminal to N-terminal direction
N-terminal to 3’ direction
N-terminal to C-terminal direction
3’ to 5’ direction
MOORS
5' to 3' direction
53. Which of the following cis-acting elements typically resides
54. What is the term applied to a segment of a bacterial chromosome
55. What is the term applied to the complete collection of proteins
56. How does nucleosome formation on genomic DNA affect the
57. Which types of molecules interact with eukaryotic mRNA
58. Most eukaryotic transcription factors contain at least two
59. Transcription factors bound at enhancers stimulate the initiation
adjacent to or overlaps with many prokaryotic promoters?
Regulatory gene
Structural gene(s)
Repressor
Operator
BOOP
where genes for the enzymes of a particular metabolic pathway
are clustered and subject to coordinate control?
Operon
Operator
Promoter
Terminal controller
Origin
BOOP
present in a particular cell type?
Genome >
Peptide collection
Translatome
Proteome
BOOR
initiation and/or elongation phases of transcription?

A. Nucleosomes inhibit access of enzymes involved in all
phases of transcription.
B. Nucleosomes recruit histone and DNA-modifying enzymes,
and enzymes of
the actions of these recruited affect the access
transcription proteins to DNA.
C. Nucleosomes induce DNA degradation where the DNA
contacts the histones.
D. Nucleosomes have no significant effect on transcription.
gene core promoter sites to facilitate the association of RNA
polymerase II?
Termination factors >
Sequence-specific transcription factors (transactivators)
Elongation factors
GTPases
General, or basal transcription factors (ie, the GTFs)
BOO
domains, each of which mediate different aspects of transcription factor function. Which of the are these domains?
following
RNA-binding domain and repression domain >
Activation domain and repression domain
DNA-binding domain and activation domain
DNA-binding domain and ligand-binding domain
RNA-binding domain and the activation domain
BOO
of transcription at the cis-linked core promoter through the
action of intermediaries are termed as which of the following?
Coactivators >
Cotranscription proteins
Corepressors
Receptors
Coordinators
BOOP
60. What reactions among transcription proteins greatly expand the
61. The gene region containing the TATA box and extending to
62.
63. Which of the following histone amino acids are typically
64.
diversity of regulatory factors that can be generated from a small
number of polypeptides?
Recombination >
Homodimerization
Heterozygosity
Heterodimerization
Trimerization
MOOR
the transcription start site (TSS) is often termed which of the
Polymerase home
Initiator
Start selector
Core promoter
Operator
mopOR>
Which
of
the
following
possible
mechanisms
for
how
enhancers
can stimulate transcription from great distances are currently
thought to be CORRECT?
A. Enhancers can reversibly excise the intervening DNA
between enhancers and promoters.
B. RNA polymerase II binds avidly to enhancer sequences.
C. Enhancers unwind DNA.
D. Enhancers can search through DNA and bind directly to the

associated core promoter.
E. Enhancers and core promoters are brought into close
proximity through DNA loop formation mediated by DNA- binding proteins.
acetylated?
Lysine
Arginine
Histidine
Leucine
BOOP
Place the following steps in order; what are the steps that occur
sequentially during a transcription activation event following
the binding of a transcriptional activator to its cognate activator-
binding site on genomic DNA.
1.
The chromatin remodeling complex binds to the core histones
at the target region.
2. The combined
actions
of
the
various
molecular
com-
plexes increase promoter accessibility to the transcriptional machinery.
3.
The activator recruits a coactivator to a region of chromatin
targeted for transcription.
4.
Transcriptional machinery assembles at the site where tran-
scription will be initiated.
5. The coactivator acetylates the core histones of nearby nucleosomes.
A. 1-2-3-4-5
B. 3-1-5-2-4
C.
3-5-1-2-4
D. 5-3-1-2-4
E. 3-5-1-4-2
65. What strategy in transcription factor research allows for the
66. Which sequences extend between the 5’-methylguanosine cap
simultaneous
of
all
of
the
genomic
sites
bound
by a given transcription factor under a given set of physiologic
conditions?
A. Systematic deletion mapping
B. DNAse I sensitivity
C. Chromatin immunoprecipitation-sequencing (ChIP-seq)
D. FISH
E. Fluorescence lifetime imaging microscopy
present on eukaryotic mRNAs to the AUG initiation codon?
Stop codon
Last exon
Last intron
3’
UTR
5’UTR
mopOR>
67. Which of the following features of eukaryotic mRNA contribute
importantly to message half-life?
5’-UTR sequences
The promoter
The operator
3’ UTR and poly(A) tail
The first intron
BOOP

. Know that biologic membranes are mainly composed of a lipid bilayer and associated proteins and glycoproteins. The major lipids are phospholipids, cholesterol, and glycosphingolipids.
: Appreciate that membranes are asymmetric, dynamic structures containing a mixture of integral and peripheral proteins.
Describe the widely accepted fluid mosaic model of membrane structure.
Understand the concepts of passive diffusion, facilitated diffusion, active transport, endocytosis, and exocytosis.
= Recognize that transporters, ion channels, the Na* — gap junctions are important participants in membrane function. K*-ATPase, receptors, and
= Be aware that a variety of disorders result from abnormalities of membrane structure and function, including familial hypercholesterolemia, cystic fibrosis, hereditary spherocytosis, among others.

Membranes also form specialized compartments within
Membranes are dynamic, highly fluid structures consisting of a lipid bilayer and associated proteins. Plasma membranes form closed compartments around the cytoplasm to define cell boundaries. The plasma membrane has selective permeabilities and acts as a barrier, thereby maintaining differences in composition between the inside and outside of the cell. Selective membrane molecular permeability is generated through the action of specific transporters and ion channels. The plasma membrane also exchanges material with the extracellular environment by exocytosis and endocytosis, and there are special areas of membrane structure—gap junctions—through which adjacent cells may communicate by exchanging material. In addition, the plasma membrane plays key roles in cell-cell interactions and in transmembrane the cell. Such intracellular membranes help shape many of the morphologically distinguishable structures (organelles), for example, mitochondria, endoplasmic reticulum (ER), Golgi, secretory granules, lysosomes, and the nucleus. Membranes localize enzymes, function as integral elements in excitationresponse coupling, and provide sites of energy transduction, such as in photosynthesis in plants (chloroplasts) and oxidative phosphorylation (mitochondria).
Changes in membrane components can affect water balance and ion flux, and therefore many processes within the cell. Specific deficiencies or alterations of certain membrane components (eg, caused by mutations in genes encoding membrane proteins) lead to a variety of diseases (see Table 40-7). In short, normal cellular function critically depends on normal membranes.
signaling.

Life originated in an aqueous environment; enzyme reactions, cellular and subcellular processes have therefore evolved to work in this milieu, encapsulated within a cell.

Water makes up about 60% of the lean body mass of the human body and is distributed in two large compartments.

This compartment constitutes two-thirds of total body water and provides a specialized environment for the cell to (1) make, store, and utilize energy; (2) to repair itself; (3) to replicate; and (4) to perform cell-specific functions.

This compartment contains about one-third of total body water and is distributed between the plasma and interstitial compartments. The extracellular fluid is a delivery system. It brings to the cells nutrients (eg, glucose, fatty acids, and amino acids), oxygen, various ions and trace minerals, and a variety of regulatory molecules (hormones) that coordinate the functions of widely separated cells. Extracellular fluid removes CO,, waste products, and toxic or detoxified materials from the immediate cellular environment.

As illustrated in Table 40-1, the internal environment is rich
in K* and Mg”, and phosphate is its major inorganic anion.

Na +, Extracellular Fluid = 140 mmol/L. Na +, Intracellular Fluid = 10 mmol/L. K +, Extracellular Fluid = 4 mmol/L. K +, Intracellular Fluid = 140 mmol/L. Ca 2+ (free), Extracellular Fluid = 2.5 mmol/L. Ca 2+ (free), Intracellular Fluid = 0.1 μmol/L. Mg 2+, Extracellular Fluid = 1.5 mmol/L. Mg 2+, Intracellular Fluid = 30 mmol/L. Cl -, Extracellular Fluid = 100 mmol/L. Cl -, Intracellular Fluid = 4 mmol/L. HCO 3 -, Extracellular Fluid = 27 mmol/L. HCO 3 -, Intracellular Fluid = 10 mmol/L. PO 4 3-, Extracellular Fluid = 2 mmol/L. PO 4 3-, Intracellular Fluid = 60 mmol/L. Glucose, Extracellular Fluid = 5.5 mmol/L. Glucose, Intracellular Fluid = 0-1 mmol/L. Protein, Extracellular Fluid = 2 g/dL. Protein, Intracellular Fluid = 16 g/dL
The cytosol of cells contains a high concentration of protein that acts as a major intracellular buffer. Extracellular fluid is characterized by high Na* and Ca** content, and Cl is the major anion. These ionic differences are maintained due to various membranes found in cells. These membranes have unique lipid and protein compositions. A fraction of the protein constituents of membrane proteins are specialized to generate and maintain the differential ionic compositions of the extraand intracellular compartments.

We shall mainly discuss the membranes present in eukaryotic cells, although many of the principles described also apply to the membranes of prokaryotes. The various cellular membranes have different lipid and protein compositions. The ratio of protein to lipid in different membranes is presented in Figure 40-1, and is responsible for the many divergent functions of cellular organelles. Membranes are sheet-like enclosed structures consisting of an asymmetric lipid bilayer with distinct inner and outer surfaces or leaflets. These structures and
FIGURE
40-1
Membrane protein
content is
highly vari-
able.
The amount
equals or
exceeds the
quantity of
lipid in nearly all membranes. The outstanding exception is myelin, an
electrical insulator found on many nerve fibers.
surfaces are protein-studded, sheet-like, noncovalent assemblies that form spontaneously in aqueous environments due to the amphipathic nature of lipids and the proteins contained within the membrane.

Of the two major phospholipid classes present in membranes, phosphoglycerides are the more common and consist of a glycerol-phosphate backbone to which are attached two fatty acids in ester linkages and an alcohol (Figure 40-2). The fatty acid constituents are usually even-numbered carbon molecules, most commonly containing 16 or 18 carbons. They are unbranched and can be saturated or unsaturated with one or more double bonds. The simplest phosphoglyceride is phosphatidic acid, a 1,2-diacylglycerol 3-phosphate, a key intermediate in the formation of other phosphoglycerides (see Chapter 24). In most phosphoglycerides present in membranes, the 3-phosphate is esterified to an alcohol such as choline, ethanolamine, glycerol, inositol, or serine (see Chapter 21). Phosphatidylcholine is generally the major phosphoglyceride by mass in the membranes of human cells.
The second major class of phospholipids comprises sphingomyelin (see Figure 21-11), a phospholipid that contains a sphingosine rather than a glycerol backbone. A fatty acid is attached by an amide linkage to the amino group of sphingosine, forming ceramide. When the primary hydroxyl group of sphingosine is esterified to phosphorylcholine, sphingomyelin is formed. As the name suggests, sphingomyelin is prominent in myelin sheaths.

The glycosphingolipids (GSLs) are sugar-containing lipids built on a backbone of ceramide. GSLs include galactosyland glucosyl-ceramides (cerebrosides) and the gangliosides (see structures in Chapter 21), and are mainly located in the
FIGURE 40-2 A phosphoglyceride showing the fatty acids (R, and R,), glycerol, and a phosphorylated alcohol component. Saturated fatty acids are usually attached to carbon 1 of glycerol, and unsaturated fatty acids to carbon 2. In phosphatidic acid, R, is hydrogen.
plasma membranes of cells, displaying their sugar components to the exterior of the cell.

The most common sterol in the membranes of animal cells is cholesterol (see Chapter 21). The majority of cholesterol resides within plasma membranes, but smaller amounts are found within mitochondrial, Golgi complex, and nuclear membranes. Cholesterol intercalates among the phospholipids of the membrane, with its hydrophilic hydroxyl group at the aqueous interface and the remainder of the molecule buried within the lipid bilayer leaflet. From a nutritional viewpoint, it is important to know that cholesterol is not present in plants.
Lipids can
be separated
from one
another and
quantified by
techniques such
as column,
thin-layer, and
gas-liquid matography
and their
structures established
chro- by
mass trometry and other techniques (see Chapter 4).

All major lipids in membranes contain both hydrophobic and hydrophilic regions and are therefore termed amphipathic. If the hydrophobic region were separated from the rest of the molecule, it would be insoluble in water but soluble in organic solvents. Conversely, if the hydrophilic region were separated from the rest of the molecule, it would be insoluble in organic solvents but soluble in water. The amphipathic nature of a phospholipid is represented in Figure 40-3 and also
FIGURE 40-3 Diagrammatic representation of a phospholipid or other membrane lipid. The polar head group is hydrophilic, and the hydrocarbon tails are hydrophobic or lipophilic. The fatty acids in the tails are saturated (S) or unsaturated (U); the former is usually attached to carbon 1 of glycerol and the latter to carbon 2 (see Figure 40-2). Note the kink in the tail of the unsaturated fatty acid (U), which is important in conferring increased membrane fluidity. The S-U phospholipid on the left contains the C,, saturated lipid palmitic acid, and the monounsaturated C18 lipid cis-oleic acid; both are esterified to glycerol (see Figure 40-2). The S-S phospholipid schematized on the right contains the C,, saturated lipid palmitic acid and the saturated C,, lipid, stearic acid.
spec-
Figure 21-24. Thus, the polar head groups of the phospholipids and the hydroxyl group of cholesterol interface with the aqueous environment; a similar situation applies to the sugar moieties of the GSLs (see below).
Saturated fatty acids form relatively straight tails, whereas unsaturated fatty acids, which generally exist in the cis form in membranes, form "kinked" tails (Figure 40-3; see also Figures 21-1, 21-6). As the number of double bonds within the lipid side chains increase, the number of kinks in the tails increases. As a consequence, the membrane lipids become less tightly packed and the membrane more fluid. The problem caused by the presence of trans fatty acids in membrane lipids is described in Chapter 21.

Detergents are amphipathic molecules that are important in biochemistry as well as in the household. The molecular structure of a detergent is not unlike that of a phospholipid. Certain detergents are widely used to solubilize and purify membrane proteins. The hydrophobic end of the detergent binds to hydrophobic regions of the proteins, displacing most of their bound lipids. The polar end of the detergent is free, bringing the proteins into solution as detergent-protein complexes, usually also containing some residual lipids.

The amphipathic character of phospholipids suggests that the two regions of the molecule have incompatible solubilities. However, in a solvent such as water, phospholipids spontaneously organize themselves into micelles (Figure 40-4 and Figure 21-24), an assembly that thermodynamically satisfies the solubility requirements of the two chemically distinct regions of these molecules. Within the micelle the hydrophobic regions of the amphipathic phospholipids are shielded from water, while the hydrophilic polar groups are immersed in the aqueous environment. Micelles are usually relatively
FIGURE 40-4 Diagrammatic cross-section of a micelle. The polar head groups are bathed in water, whereas the hydrophobic hydrocarbon tails are surrounded by other hydrocarbons and thereby protected from water. Micelles are relatively small (compared with structures.
lipid bilayers) spherical
FIGURE 40-5 Diagram of a section of a bilayer membrane formed from phospholipids. The unsaturated fatty acid tails are kinked and lead to more spacing between the polar head groups, and hence to more room for movement. This in turn results in increased membrane fluidity.
small in size (eg, ~200 nm) and consequently are limited in their potential to form membranes. Detergents commonly form micelles.

Phospholipids and similar amphipathic molecules can form another structure, the bimolecular lipid bilayer, which also satisfies the thermodynamic requirements of amphipathic molecules in an aqueous environment. Bilayers are the key structures in biologic membranes. Bilayers exist as sheets wherein the hydrophobic regions of the phospholipids are sequestered from the aqueous environment, while the hydrophilic, charged portions are exposed to water (Figure 40-5 and Figure 21-24). The ends or edges of the bilayer sheet can be eliminated by folding the sheet back on itself to form an enclosed vesicle with no edges. The closed bilayer provides one of the most essential properties of membranes. The lipid bilayer is impermeable to most water-soluble molecules since such charged molecules would be insoluble in the hydrophobic core of the bilayer. The self-assembly of lipid bilayers is driven by the hydrophobic effect, which describes the tendency of nonpolar molecules to self-associate in an aqueous environment, while in the process excluding H,O. When lipid molecules come together in a bilayer, the entropy of the surrounding solvent molecules increases due to the release of immobilized water.
Two questions arise from consideration of the information described above. First, how many biologically important molecules are lipid-soluble and can therefore readily enter the cell? Gases such as oxygen, CO,, and nitrogen—small molecules with little interaction with solvents—readily diffuse through the hydrophobic regions of the membrane. The permeability coefficients of several ions and a number of other molecules in a lipid bilayer are shown in Figure 40-6. The electrolytes Na', K*, and Cl cross the bilayer much more slowly than water. In general, the permeability coefficients of small molecules in a lipid bilayer correlate with their solubilities in nonpolar solvents. For instance, steroids more readily traverse the lipid bilayer compared with electrolytes. The high permeability coefficient of water itself is surprising, but is partly explained by its small size and relative lack of charge. Many drugs are hydrophobic and can readily cross membranes and enter cells. The second question concerns non-lipid-soluble mole- cules.
How are
the transmembrane
concentration gradients
for

FIGURE 40-6 Permeability coefficients of water, some ions, and other small molecules in lipid bilayer membranes. The permeability coefficient is a measure of the ability of a molecule to diffuse across a permeability barrier. Molecules that move rapidly through a given membrane are said to have a high permeability coefficient.
these molecules maintained? The answer is that membranes contain proteins, many of which span the lipid bilayer. These proteins either form channels for the movement of ions and small molecules or serve as transporters for molecules that otherwise could not readily traverse the lipid bilayer (membrane). The nature, properties, and structures of membrane channels and transporters are described below.

Membrane phospholipids act as a solvent for membrane proteins, creating an environment in which the latter can function. As described in Chapter 5, the a-helical structure of proteins minimizes the hydrophilic character of the peptide bonds themselves. Thus, proteins can be amphipathic and form an integral part of the membrane by having hydrophilic regions protruding at the inside and outside faces of the membrane but connected by a hydrophobic region traversing the hydrophobic core of the bilayer. In fact, those portions of membrane proteins that traverse membranes do contain substantial numbers of hydrophobic amino acids and almost invariably have a high a-helical content. For most membranes, a stretch of ~20 amino acids in an a-helical configuration will span the lipid bilayer (see Figure 5-2).
It is possible to calculate whether a particular sequence of amino acids present in a protein is consistent with a transmembrane location. This can be done by consulting a table that lists the hydrophobicities of each of the 20 common amino acids and the free energy values for their transfer from the interior of a membrane to water. Hydrophobic amino acids have positive values; polar amino acids have negative values. The total free energy values for transferring successive sequences of 20 amino acids in the protein are plotted, yielding a so-called hydropathy plot. Values of over 20 kcal mol"! are consistent with—but do not prove—the interpretation that the hydrophobic sequence is a transmembrane segment. Another aspect of the interaction of lipids and proteins is that some proteins are anchored to one leaflet of the bilayer by covalent linkages to certain lipids; this process is termed protein lipidation. Lipidation can occur at protein termini
(Nor C-) or internally. Common protein lipidation events are C-terminal protein isoprenylation, cholesterylation, and glycophosphatidylinositol (GPI; see Figure 46-1); N-terminal protein myristoylation and internal cysteine S-prenylation and S-acylation. Such lipidation only occurs on a specific subset of proteins and typically plays key roles in their biology.

The number of different proteins in a membrane varies from less than a dozen very abundant proteins in the sarcoplasmic reticulum of muscle cells to hundreds in plasma membranes. Proteins are the major functional molecules of membranes and consist of enzymes, pumps and transporters, channels, structural components, antigens (eg, for histocompatibility), and receptors for various molecules. Because every type of membrane possesses a different complement of proteins, there is no such thing as a typical membrane structure. The enzymes associated with several different membranes are shown in Table 40-2.

Membranes and their components are dynamic structures. Membrane lipids and proteins undergo turnover, just as they do in other compartments of the cell. Different lipids have different turnover rates, and the turnover rates of individual species of membrane proteins may vary widely. In some instances, the membrane itself can turn over even more rapidly than any of its constituents. This is discussed in more detail in the section on endocytosis.
Another indicator of the dynamic nature of membranes is that a variety of studies have shown that lipids and certain proteins exhibit lateral diffusion in the plane of their membranes. Many nonmobile proteins do not exhibit lateral diffusion because they are anchored to the underlying actin cytoskeleton. By contrast, the transverse movement of lipids

Plasma, Enzyme = 5 ′ -Nucleotidase. Endoplasmic reticulum, Enzyme = Glucose-6-phosphatase. Golgi apparatus, Enzyme = . Cis, Enzyme = GlcNAc transferase I. Medial, Enzyme = Golgi mannosidase II. Trans, Enzyme = Galactosyl transferase. Trans Golgi network, Enzyme = Sialyltransferase. Inner mitochondrial membrane, Enzyme = ATP synthase
*Membranes contain many proteins, some of which have enzymatic activity. Some of these enzymes are located only in certain membranes and can therefore be used as markers to follow the purification of these membranes.
across the membrane (flip-flop) is extremely slow (see below), and does not appear to occur at an appreciable rate in the case of membrane proteins.

Proteins have unique orientations in membranes, making the outside surfaces different from the inside surfaces. An inside-outside asymmetry is also provided by the external location of the carbohydrates attached to membrane proteins. In addition, specific proteins are located exclusively on the outsides or insides of membranes.
There are also regional heterogeneities in membranes. Some, such as occur at the villous borders of mucosal cells, are almost macroscopically visible. Others, such as those at gap junctions, tight junctions, and synapses, occupy much smaller regions of the membrane and generate correspondingly smaller local asymmetries.
There is also inside-outside asymmetry of the phospholipids. The choline-containing phospholipids (phosphatidylcholine and sphingomyelin) are located mainly in the outer leaflet; the aminophospholipids (phosphatidylserine and phosphatidylethanolamine) are preferentially located in the inner leaflet. Obviously, if this lipid asymmetry is to exist at all, there must be limited transverse mobility, or 'flipflop' the membrane phospholipids. In fact, phospholipids in synthetic bilayers exhibit an extraordinarily slow rate of flipflop; the half-life of the asymmetry in these synthetic bilayers is on the order of several weeks.

microsomal membrane vesicles. Translocases (flippases) exist that transfer certain phospholipids (eg, phosphatidylcholine) from the inner to the outer leaflet. Specific proteins that preferentially bind individual phospholipids also appear to be present in the two leaflets; thus, lipid binding also contributes to the asymmetric distribution of specific lipid molecules. In addition, phospholipid exchange proteins recognize certain phospholipids and transfer them from one membrane (eg, the ER) to others (eg, mitochondrial and peroxisomal). A related issue is how lipids enter membranes. This has not been studied as intensively as the topic of how proteins enter membranes (see Chapter 49) and knowledge is still relatively meager. Many membrane lipids are synthesized in the ER. At least three pathways have been recognized: (1) transport from the ER in vesicles, which then transfer the contained lipids to the recipient membrane; (2) entry via direct contact of one membrane (eg, the ER) with another, facilitated by specific proteins; and (3) transport via the phospholipid exchange proteins (also known as lipid transfer proteins) mentioned above, which only exchanges lipids, but does not cause net transfer.
There is further asymmetry with regard to glycosphingolipids and glycoproteins; the sugar moieties of these molecules all protrude outward from the plasma membrane and are absent from its inner face.

The mechanisms involved in the lipid asymmetry are not well understood. The enzymes involved in the synthesis of phospholipids are located on the cytoplasmic side of
It is useful to classify membrane proteins into two types: integral and peripheral (Figure 40-7). Most membrane proteins fall into the integral class, meaning that they interact
FIGURE 40-7 The fluid mosaic model of membrane structure. The membrane consists of a bimolecular lipid layer with proteins inserted in it or bound to either surface. Integral membrane proteins are firmly embedded in the lipid layers. Some of these proteins completely span the bilayer and are called transmembrane proteins, while others are embedded in either the outer or inner leaflet of the lipid bilayer. Loosely bound to the outer or inner surface of the membrane are the peripheral proteins. Many of the proteins and all the glycolipids have externally exposed oligosaccharide carbohydrate chains. (Reproduced, with permission, from Junqueira LC, Carneiro J: Basic Histology: Text &
Atlas, 10th ed. McGraw-Hill, 2003.)
extensively with the phospholipids and require the use of detergents for their solubilization. Also, they generally span the bilayer as a bundle of a-helical transmembrane segments. Integral proteins are usually globular and are themselves amphipathic. They consist of two hydrophilic ends separated by an intervening hydrophobic region that traverses the hydrophobic core of the bilayer. As the structures of integral membrane proteins were being elucidated, it became apparent that certain ones (eg, transporter molecules, ion channels, various receptors, and G proteins) span the bilayer many times, whereas other simple membrane proteins (eg, glycophorin A) span the membrane only once (see Figures 42-4 and 52-5). Integral proteins are asymmetrically distributed across the membrane bilayer. This asymmetric orientation is conferred at the time of their insertion in the lipid bilayer during biosynthesis in the ER. The molecular mechanisms involved in insertion of proteins into membranes and the topic of membrane assembly are discussed in Chapter 49.

Peripheral proteins do not interact directly with the hydrophobic cores of the phospholipids in the bilayer and thus do not require use of detergents for their release. They are bound to the hydrophilic regions of specific integral proteins and head groups of phospholipids and can be released from them by treatment with salt solutions of high ionic strength. For example, ankyrin, a peripheral protein, is bound to the inner aspect of the integral protein "band 3" of the erythrocyte membrane. Spectrin, a cytoskeletal structure within the erythrocyte, is in turn bound to ankyrin and thereby plays an important role in maintenance of the biconcave shape of the erythrocyte.

Artificial membrane systems can be prepared by appropriate techniques. These systems generally consist of mixtures of one or more phospholipids of natural or synthetic origin that have been treated by using mild sonication to induce the formation of spherical vesicles in which the lipids form a bilayer. Such vesicles, surrounded by a lipid bilayer with an aqueous interior, are termed liposomes (see Figure 21-24).
The advantages
and uses
of artificial
membrane sys-
tems for
the biochemical
study of
membrane function
are as
follows:
1. The lipid content of the membranes can be varied, allowing systematic examination of the effects of varying lipid composition on certain functions.
2. Purified membrane proteins or enzymes can be incorporated into these vesicles in order to assess what factors (eg, specific lipids or ancillary proteins) the proteins require to reconstitute their function.
3. The environment of these systems can be rigidly controlled and systematically varied (eg, ion concentrations and ligands).
4. When liposomes are formed, they can be made to entrap certain compounds within the vesicle such as drugs and isolated genes. There is interest in using liposomes to distribute drugs to certain tissues, and if components (eg, antibodies to certain cell surface molecules) could be incorporated into liposomes so that they would be targeted to specific tissues or tumors, the therapeutic impact would be considerable. DNA entrapped inside liposomes appears to be less sensitive to attack by nucleases; this approach may prove useful in attempts at gene therapy.

The fluid mosaic model of membrane structure proposed in 1972 by Singer and Nicolson (Figure 40-7) is now widely accepted. The model is often likened to integral membrane protein "icebergs" floating in a sea of (predominantly) fluid phospholipid molecules. Early evidence for the model was the finding that well characterized, fluorescently labeled integral membrane proteins could be seen microscopically to rapidly and randomly redistribute within the plasma membrane of a hybrid cell formed by the artificial fusion of two different (mouse and human) parent cells (one labeled the other not). It has subsequently been demonstrated that phospholipids undergo even more rapid lateral diffusion with subsequent redistribution within the plane of the membrane. Measurements indicate that within the plane of the membrane, one molecule of phospholipid can move several micrometers per second.
The phase changes—and thus the fluidity of membranes— are largely dependent on the lipid composition of the membrane. In a lipid bilayer, the hydrophobic chains of the fatty acids can be highly aligned or ordered to provide a rather stiff structure. As the temperature increases, the hydrophobic side chains undergo a transition from the ordered state (more gel-like or crystalline phase) to a disordered one, taking on a more liquid-like or fluid arrangement. The temperature at which membrane structure undergoes the transition from ordered to disordered (ie, melts) is called the "transition temperature" (T_). Longer and more saturated fatty acid chains interact more strongly with each other via their extended hydrocarbon chains and thus cause higher values of T.—that is, higher temperatures are required to increase the fluidity of the bilayer. On the other hand, unsaturated bonds that exist in the cis configuration tend to increase the fluidity of a bilayer by decreasing the compactness of the side chain packing without diminishing hydrophobicity (Figures 40-3 and 40-5). The phospholipids of cellular membranes generally contain at least one unsaturated fatty acid with at least one cis double bond.
Cholesterol acts as a buffer to modify the fluidity of membranes. At temperatures below the T., it interferes with the interaction of the hydrocarbon tails of fatty acids and thus

increases fluidity. At temperatures above the T., it limits disorder because it is more rigid than the hydrocarbon tails of the fatty acids and cannot move in the membrane to the same extent, thus limiting, or "buffering" membrane fluidity. The fluidity of a membrane significantly affects its functions. As membrane fluidity increases, so does its permeability to water and other small hydrophilic molecules. The lateral mobility of integral proteins increases as the fluidity of the membrane increases. If the active site of an integral protein involved in a given function is exclusively in its hydrophilic regions, changing lipid fluidity will probably have little effect on the activity of the protein; however, if the protein is involved in a transport function in which transport components span the membrane, lipid-phase effects may significantly alter the transport rate. The insulin receptor (see Figure 42-8) is an excellent example of altered function with changes in fluidity. As the concentration of unsaturated fatty acids in the membrane is increased (by growing cultured cells in a medium rich in such molecules), fluidity increases. Increased fluidity alters the receptor such that it binds insulin more effectively. At normal body temperature (37°C), the lipid bilayer is in a fluid state. Underscoring the importance of membrane fluidity, it has been shown that bacteria can modify the composition of their membrane lipids to adapt to changes in temperature.

Plasma membranes contain certain specialized structures whose biochemical natures have been investigated in some detail. Lipid rafts are specialized areas of the exoplasmic (outer) leaflet of the lipid bilayer enriched in cholesterol, sphingolipids, and certain proteins (Figure 40-8). They have been
actin-binding signaling
proteins
FIGURE 40-8 Schematic diagram of a lipid raft. Shown in schematic form are multiple lipid rafts (red membrane shading) that represent localized microdomains rich in the indicated lipids and signaling proteins (blue, green, yellow). Lipid rafts are stabilized through interactions (direct and indirect) with the actin cytoskeleton (red bihelical chains; see Figure 51-3). (Figure modified from: The lipid raft hypothesis revisited—new insights on raft composition and function from super-resolution fluorescence microscopy. Bioessays 2012;34:739-747. Wiley Periodical, Inc. Copyright © 2012.)
FIGURE 40-9 Schematic diagram of a caveola. A caveola is an invagination in the plasma membrane. The protein caveolin appears to play an important role in the formation of caveolae and occurs as a dimer. Each caveolin monomer is anchored to the inner leaflet of the plasma membrane by three palmitoyl molecules (not shown).
hypothesized to be involved in signal transduction and other processes. It is thought that clustering certain components of signaling systems closely together may increase the efficiency of their function.
Caveolae may derive from lipid rafts. Many, if not all, contain the protein caveolin-1, which may be involved in their formation from rafts. Caveolae are observable by electron microscopy as flask, or tube-shaped indentations of the cell membrane into the cytosol (Figure 40-9). Proteins detected in caveolae include various components of the signal transduction system (eg, the insulin receptor and some G proteins; see Chapter 42), the folate receptor, and endothelial nitric oxide synthase (eNOS). Caveolae and lipid rafts are active areas of research, and ideas concerning them and their roles in various biologic processes are rapidly evolving.

Tight junctions are other structures found in surface membranes. They are often located below the apical surfaces of epithelial cells and prevent the diffusion of macromolecules between cells. They are composed of various proteins, including occludin, various claudins, and junctional adhesion molecules. Yet other specialized structures found in surface membranes include desmosomes, adherens junctions, and microvilli; their chemical natures and functions are not discussed here. The nature of gap junctions is described below.

If the plasma membrane is relatively impermeable, how do most molecules enter a cell? How is selectivity of this movement established? Answers to such questions are important in understanding how cells adjust to a constantly changing extracellular environment. Metazoan organisms also must have means of communicating between adjacent and distant cells, so that complex biologic processes can be coordinated. These
signals must arrive at and be transmitted by the membrane, or they must be generated as a consequence of some interaction with the membrane. Some of the major mechanisms used to accomplish these different objectives are listed in Table 40-3.

Molecules can passively traverse the bilayer down electrochemical gradients by simple diffusion or by facilitated diffusion. This spontaneous movement toward equilibrium contrasts with active transport, which requires energy because it constitutes movement against an electrochemical gradient. Figure 40-10 provides a schematic representation of these mechanisms.

Cross-membrane movement of small molecules
Diffusion (passive and facilitated)
Active transport
Cross-membrane movement of large molecules
Endocytosis
Exocytosis

Cell surface receptors
1. Signal transduction (eg, glucagon — cAMP)
2. Signal internalization (coupled with endocytosis, eg, the LDL
receptor)
- Movement to intracellular receptors (steroid hormones; a form of diffusion)
Simple diffusion is the passive flow of a solute from a higher to a lower concentration due to random thermal movement. By contrast, facilitated diffusion is passive transport of a solute from a higher to a lower concentration mediated by a specific protein transporter. Active transport is vectorial movement of a solute across a membrane against a concentration gradient, and thus requires energy (frequently derived from the hydrolysis of ATP); a specific transporter (pump) is involved. As mentioned earlier in this chapter, some solutes such as gases can enter the cell by diffusing down an electrochemical gradient across the membrane and do not require metabolic energy. Simple diffusion of a solute across the membrane

Passive (simple) diffusion is the flow of solute from a higher to a lower concentration due to random thermal movement
Facilitated diffusion is passive transport of a solute from a higher concentration to a lower concentration, mediated by a specific protein transporter
Active transport is transport of a solute across a membrane in the direction of increasing concentration, and thus requires energy (frequently derived from the hydrolysis of ATP); a specific transporter (pump) is involved
Extracellular microvesicle and exosome secretion and uptake
The other terms used in this table are explained later in this chapter or elsewhere in this text.
oe
Electrochemical gradient
eo
FIGURE 40-10 Many small, uncharged molecules pass freely through the lipid bilayer by simple diffusion. Larger uncharged molecules, and some small uncharged molecules, are transferred by specific carrier proteins (transporters) or through channels or pores. Passive transport is always down an electrochemical gradient (shown schematically, right), toward equilibrium. Active transport is against an electrochemical gradient and requires an input of energy, whereas passive transport does not. (Redrawn and reproduced, with permission, from Alberts B, et al: Molecular Biology of the Cell. Garland, 1983.)
is limited by three factors: (1) the thermal agitation of that specific molecule; (2) the concentration gradient across the membrane; and (3) the solubility of that solute (the permeability coefficient, Figure 40-6) in the hydrophobic core of the membrane bilayer. Solubility is inversely proportional to the number of hydrogen bonds that must be broken in order for a solute in the external aqueous phase to become incorporated in the hydrophobic bilayer. Electrolytes, poorly soluble in lipid, do not form hydrogen bonds with water, but they do acquire a shell of water from hydration by electrostatic interaction. The size of the shell is directly proportional to the charge density of the electrolyte. Electrolytes with a large charge density have a larger shell of hydration and thus a slower diffusion rate. Na*, for example, has a higher charge density than K*. Hydrated Na' is therefore larger than hydrated K*; hence, the latter tends to move more easily through the membrane.

The following affect net diffusion of a substance: (1) concentration gradient across the membrane—solutes move from high to low concentration; (2) electrical potential across the membrane: solutes move toward the solution that has the opposite charge. The inside of the cell usually has a net negative charge; (3) permeability coefficient of the substance for the membrane; (4) hydrostatic pressure gradient across the membrane: increased pressure will increase the rate and force of the collision between the molecules and the membrane; and (5) temperature, since increased temperature will increase particle motion and thus increase the frequency of collisions between external particles and the membrane.
Facilitated diffusion involves either certain transporters or ion channels (Figure 40-11). Active transport is mediated by other transporters most of which are ATP-driven. A multitude of transporters and channels exist in biologic membranes that route the entry of ions into and out of cells. Table 40-4 summarizes some important differences between transporters and ion channels.

Transport systems can be described in a functional sense according to the number of molecules moved and the direction of movement (Figure 40-12) or according to whether
FIGURE 40-11 A schematic diagram of the two types of membrane transport of small molecules.

Bind solute and undergo conformational changes, transferring the solute across the membrane, Ion Channels = Form pores in membranes. Involved in passive (facilitated diffusion) and active transport, Ion Channels = Involved only in passive transport. Transport is significantly slower than via ion channels, Ion Channels = Transport is significantly faster than via transporters
Note: Transporters are also known as carriers or permeases. Active transporters are often called pumps.
movement is toward or away from equilibrium. The following classification depends primarily on the former. A uniport system moves one type of molecule bidirectionally. In cotransport systems, the transfer of one solute depends on the stoichiometric simultaneous or sequential transfer of another solute. A symport moves two solutes in the same direction. Examples are the proton-sugar transporter in bacteria and the Na*-sugar transporters (for glucose and certain other sugars) and Na*-amino acid transporters in mammalian cells. Antiport systems move two molecules in opposite directions (eg, Na* in and Ca** out).
Hydrophilic molecules that cannot pass freely through the lipid bilayer membrane do so passively by facilitated diffusion or by active transport. Passive transport is driven by the transmembrane gradient of substrate. Active transport always occurs against an electrical or chemical gradient, and so it requires energy, usually in the form of ATP. Both types of transport involve specific carrier proteins (transporters) and both show specificity for ions, sugars, and amino acids. Passive and active transports resemble a substrate-enzyme interaction. Points of resemblance of both to enzyme action are as follows: (1) There is a specific binding site for the
Cotransport
FIGURE 40-12 schematic representation of types of transport systems. Transporters can be classified with regard to the direction of movement and whether one or more unique molecules are moved. A uniport can also allow movement in the opposite direction, depending on the concentrations inside and outside a cell of the molecule transported. (Redrawn and reproduced, with permission, from Alberts B, et al: Molecular Biology of the Cell. Garland, 1983.)
Solute concentration

FIGURE 40-13 A comparison of the kinetics of carriermediated (facilitated) diffusion with passive diffusion. The rate of movement in the latter is directly proportionate to solute concentration, whereas the process is saturable when carriers are involved. The concentration at half-maximal velocity is equal to the binding constant (kK, of the carrier for the solute. (V___, maximal rate.) max!
solute. (2) The carrier is saturable, so it has a maximum rate of transport (V,_,; Figure 40-13). (3) There is a binding constant (K,,) for the solute, and so the whole system has a K,, (Figure 40-13). (4) Structurally similar competitive inhibitors block transport. Transporters are thus like enzymes, but generally do not modify their substrates.
Cotransporters use the gradient of one substrate created by active transport to drive the movement of the other substrate. The Na* gradient produced by the Na*-K*-ATPase is used to drive the transport of a number of important metabolites. The ATPase is a very important example of primary transport, while the Na*-dependent systems are examples of secondary transport that rely on the gradient produced by another system. Thus, inhibition of the Na*-K*-ATPase in cells also blocks the Na*-dependent uptake of substances like glucose.

Some specific solutes diffuse down electrochemical gradients across membranes more rapidly than might be expected from their size, charge, or partition coefficient. This is because specific transporters are involved. This facilitated diffusion exhibits properties distinct from those of simple diffusion. The rate of facilitated diffusion, a uniport system, can be saturated; that is, the number of sites involved in diffusion of the specific solutes appears finite. Many facilitated diffusion systems are stereospecific but, like simple diffusion, are driven by the transmembrane electrochemical gradient.
A "ping-pong" mechanism (Figure 40-14) helps explain facilitated diffusion. In this model, the carrier protein exists in two principal conformations. In the "ping" state, it is exposed to high concentrations of solute, and molecules of the solute bind to specific sites on the carrier protein. Binding induces a conformational change that exposes the carrier to a lower concentration of solute ("pong" state). This process is completely reversible, and net flux across the membrane depends on the concentration gradient. The rate at which solutes enter a cell by facilitated diffusion is determined by (1) the concentration gradient across the membrane; (2) the amount of carrier available (this is a key control step); (3) the affinity of the solute-carrier interaction; (4) the rapidity of the conformational change for both the loaded and the unloaded carrier. Hormones can regulate facilitated diffusion by changing the number of transporters available. Insulin via a complex signaling pathway increases glucose transport in fat and
FIGURE 40-14 The"ping-pong" model of facilitated diffusion. A protein carrier (blue structure) in the lipid bilayer associates with a solute in high concentration on one side of the membrane. A conformational change ensues ("ping" to "pong"), and the solute is discharged on the side favoring the new equilibrium (solute concentration gradient shown schematically, right). The empty carrier then reverts to the original conformation ("pong' to "ping") to complete the cycle.

muscle by recruiting glucose transporters (GLUT) from an intracellular reservoir. Insulin also enhances amino acid transport in liver and other tissues. One of the coordinated actions of glucocorticoid hormones is to enhance transport of amino acids into liver, where the amino acids then serve as a substrate for gluconeogenesis. Growth hormone increases amino acid transport in all cells, and estrogens do this in the uterus. There are at least five different carrier systems for amino acids in animal cells. Each is specific for a group of closely related amino acids, and most operate as Na*-symport systems (Figure 40-12).

Natural membranes contain transmembrane channels, porelike structures composed of proteins that constitute selective ion channels. Cation-conductive channels have an average diameter of about 5 to 8 nm. The permeability of a channel depends on the size, the extent of hydration, and the extent of charge density on the ion. Specific channels for Na*, K*, Ca'*, and Cl have been identified. The functional a subunit of a Na* channel is schematically illustrated in Figure 40-15. The a subunit is composed of four domains (I-IV) each of which is formed by six contiguous transmembrane a-helices; each of these domains is connected by variable-length intraand extracellular loops. The aminoand carboxy termini of the a subunit are located in the cytoplasm. The actual pore in the channel through which Na' ions pass is formed by interactions between the four domains, generating a tertiary structure by interactions between the four sets of 5,6 a-helices of domains I to IV. Na* channels are often voltage sensitive or gated; the voltage sensor of the channel is formed through the interaction domain I to IV the four a-helices-4 formed when domains I to IV interact. This ~5 to 8 nm pore constitutes the center of the tertiary channel structure.
Ion channels are very selective, in most cases permitting the passage of only one type of ion (Na*, Ca™, etc). The selectivity filter of K* channels is made up of a ring of carbonyl groups donated by the subunits. The carbonyls displace bound water from the ion, and thus restrict its size to appropriate precise dimensions for passage through the channel. Many variations on the structural theme described above for the Na* channel have been described. However, all ion channels are basically made up of transmembrane subunits that come together to form a central pore through which ions pass selectively.
The membranes of nerve cells contain well-studied ion channels that are responsible for the generation of action potentials. The activity of some of these channels is controlled neurotransmitters; hence, channel can be

Ion are open are Gates can be controlled by opening or closing. In ligandgated channels, a specific molecule binds to a receptor and opens the channel. Voltage-gated channels open (or close) in response to changes in membrane potential. Mechanically gated channels respond to mechanical stimuli (pressure and touch). Some properties of ion channels are listed in Tables 40-4 and 40-5.
by activity regulated. channels transiently and thus "gated."
FIGURE 40-15 Diagrammatic representation of the structures of an ion channel (a Na* channel of rat brain). The Roman numerals indicate the four domains (I-IV) of the Na* channel a subunit. The a-helical transmembrane domains of each domain are numbered 1 to 6. The four blue-shaded subunits in the different domains represent the voltage-sensing portion of the a subunit. The actual pore through which the ions (Na*) pass is not shown, but is formed by apposition of the 5 and 6 transmembrane a-helices of domains | to IV (colored yellow). The specific areas of the subunits involved in the opening and closing of the channel are also not indicated. (After WK Catterall. Modified and reproduced, with permission, from Hall ZW: An Introduction to Molecular Neurobiology. Sinauer, 1992.)

*Some
•, 1 = They are composed of transmembrane protein subunits.. •, 1 = Most are highly selective for one ion; a few are nonselective.. •, 1 = They allow impermeable ions to cross membranes at rates approaching diffusion limits.. •, 1 = They can permit ion fluxes of 10 6 -10 7 /s.. •, 1 = Their activities are regulated.. •, 1 = The main types are voltage-gated, ligand-gated, and mechanically gated.. •, 1 = They are usually highly conserved across species.. •, 1 = Most cells have a variety of Na + , K + , Ca 2+ , and Cl - channels.. •, 1 = Mutations in genes encoding them can cause specific diseases. a. •, 1 = Their activities are affected by certain drugs.
diseases caused
by mutations
of ion
channels are
briefly discussed
in
Chapter 49.

There are at least four features of ion channels that must be elucidated: (1) their overall structures; (2) how they conduct ions so rapidly; (3) their selectivity; and (4) their gating properties. As described below, considerable progress in tackling these difficult problems has been made.
The K* channel (KvAP) is an integral membrane protein composed of four identical subunits, each with two transmembrane segments, creating an inverted "V"-like structure (Figure 40-16). The part of the channels that confers ion selectivity (the selectivity filter) measures 12-A long (a relatively short length of the membrane, so K* does not have far to travel in the membrane) and is situated at the wide end of the inverted "V' The large, water-filled cavity and helical dipoles
FIGURE 40-16 Schematic diagram of the structure of a K* channel (KvAP) from Streptomyces lividans. A single K* is shown in a large aqueous cavity inside the membrane interior. Two helical regions of the channel protein are oriented with their carboxylate ends pointing to where the K* is located. The channel is lined by carboxyl oxygen. (Modified, with permission, from Doyle DA, et al: The structure of the potassium channel: molecular basis of K* conduction and selectivity. Science 1998;280:69. Reprinted with permission from AAAS.)
FIGURE 40-17 Schematic diagram of the voltage-gated K* channel of Aeropyrum pernix. The voltage sensors behave like charged paddles that move through the interior of the membrane. Four voltage sensors (only two are shown here) are linked mechanically to the gate of the channel. Each sensor has four positive charges contributed by arginine residues. (Modified, with permission, from Sigworth FJ: Nature 2003;423:21. Copyright © 2003. Macmillan Publishers Ltd.)

shown in Figure 40-16 help overcome the relatively large electrostatic energy barrier for a cation to cross the membrane. The selectivity filter is lined with carbonyl oxygen atoms (contributed by a TVGYG sequence), providing a number of sites with which K* can interact. K* ions, which dehydrate as they enter the narrow selectivity filter, fit with proper coordination into the filter, but Na* is too small to interact with the carbonyl oxygen atoms in correct alignment and is rejected. Two K* ions, when close to each other in the filter, repel one another. This repulsion overcomes interactions between K* and the surrounding protein molecule and allows very rapid conduction of K* with high selectivity.
Other studies on a voltage-gated ion channel (HvAP) in Aeropyrum pernix have revealed many features of its voltagesensing and voltage-gating mechanisms. This channel is made up of four subunits, each with six transmembrane segments. One of the six segments (S4 and part of $3) is the voltage sensor. It behaves like a charged paddle (Figure 40-17), in that it can move through the interior of the membrane transferring four positive charges (due to four Arg residues in each subunit) from one membrane surface to the other in response to changes in voltage. There are four voltage sensors in each channel, linked to the gate. The gate part of the channel is constructed from S6 helices (one from each of the subunits). Movements of this part of the channel in response to changing voltage effectively close the channel or reopen it, in the latter case allowing a current of ions to cross the membrane.

Certain microbes synthesize small cyclic organic molecules, ionophores, such as valinomycin that function as shuttles for
the movement of ions (K* in the case of valinomycin) across membranes. Ionophores contain hydrophilic centers that are surrounded by peripheral hydrophobic regions. Specific ions within the hydrophilic center of the molecule, which then diffuses through the membrane efficiently delivering the ion in question to the cytosol. Other ionophores (the polypeptide antibiotic gramicidin) fold up to form hollow channels which ions can traverse the membrane.
bind through Microbial toxins such as diphtheria toxin and activated serum complement components can produce large pores in cellular membranes and thereby provide macromolecules with direct access to the internal milieu. The toxin a-hemolysin (produced by certain species of Streptococcus) consists of seven subunits that come together to form a $-barrel that allows metabolites like ATP to leak out of cells, resulting in cell lysis.

In certain cells (eg, red cells and cells of the collecting ductules of the kidney), the movement of water by simple diffusion is augmented by movement through water channels. These channels are composed of tetrameric transmembrane proteins named aquaporins. At least 10 distinct aquaporins (AP-1 to AP-10) have been identified. Crystallographic and other studies have revealed how these channels permit passage of water but exclude passage of ions and protons. In essence, the pores are too narrow to permit passage of ions. Protons are excluded by the fact that the oxygen atom of water binds to two asparagine residues lining the channel, making the water unavailable to participate in an H* relay, and thus preventing entry of protons. Mutations in the gene encoding AP-2 have been shown to be the cause of one type of nephrogenic diabetes insipidus, a condition in which there is an inability to concentrate urine.

The process of active transport differs from diffusion in that molecules are transported against concentration gradients; hence, energy is required. This energy can come from the hydrolysis of ATP, from electron movement, or from light. The maintenance of electrochemical gradients in biologic systems is so important that it consumes approximately 30% of the total energy expenditure in a cell.
As shown in Table 40-6, four major classes of ATP-driven active transporters (P, F, V, and ABC transporters) have been recognized. The nomenclature is explained in the legend to the table. The first example of the P class, the Na*-K*-ATPase, is discussed below. The Ca** ATPase of muscle is discussed in Chapter 51. The second class is referred to as F-type. The most important example of this class is the mt ATP synthase, described in Chapter 13. V-type active transporters pump protons into lysosomes and other structures. ABC transporters include the CFTR protein, a chloride channel involved in the causation of cystic fibrosis (described later in this chapter and
P-type, MajorTypesofATP-DrivenActiveTransporters = Ca 2+ ATPase (SR); Na + -K + -ATPase (PM). F-type, MajorTypesofATP-DrivenActiveTransporters = mt ATP synthase of oxidative phosphorylation. V-type, MajorTypesofATP-DrivenActiveTransporters = The ATPase that pumps protons into lysosomes and synaptic vesicles. ABC transporter, MajorTypesofATP-DrivenActiveTransporters = CFTR protein (PM); MDR-1 protein (PM)

Abbreviations: CFTR, cystic fibrosis transmembrane regulator protein, a Cltransporter, and the protein implicated in the causation of cystic fibrosis (see later in this chapter and also Chapter 57); MDR-1 protein (multidrug-resistance-1 protein), a protein that pumps many chemotherapeutic agents out of cancer cells and is thus an important contributor to the resistance of certain cancer cells to treatment; mt, mitochondrial; PM, plasma membrane; SR, sarcoplasmic reticulum of muscle.. P (in P-type) signifies phosphorylation (these proteins autophosphorylate). F (in F-type) signifies energy coupling factors. V (in V-type) signifies vacuolar. ABC signifies ATP-binding cassette transporter (all have two nucleotide-binding domains and two transmembrane segments).
in Chapter 58). Another important member of this class is the multidrug-resistance-1 protein (MDR-1 protein). This transporter will pump a variety of drugs, including many anticancer agents, out of cells. It is a very important cause of cancer cells exhibiting resistance to chemotherapy, although many other mechanisms are also implicated (see Chapter 56).

As shown in Table 40-1, cells maintain a low intracellular Na* concentration and a high intracellular K* concentration, along with a net negative electrical potential inside. The pump that maintains these ionic gradients is an ATPase that is activated by Na* and K* (Na*-K*-ATPase). The Na*-K*-ATPase pumps three Na* out and two K* into cells (Figure 40-18). This pump
FIGURE 40-18 Stoichiometry of the Na*-K*-ATPase pump. This pump moves three Na* ions from inside the cell to the outside and brings two K* ions from the outside to the inside for every molecule of ATP hydrolyzed to ADP by the membrane-associated ATPase. Ouabain and other cardiac glycosides inhibit this pump by acting on the extracellular surface of the membrane. (Reprinted with permis- sion from R Post.)

is an integral membrane protein that contains a transmembrane domain allowing the passage of ions, and cytosolic domains that couple ATP hydrolysis to transport. There are catalytic centers for both ATP and Na* on the cytoplasmic (inner) side of the plasma membrane (PM), while there are K*-binding sites located on the extracellular side of the membrane. Phosphorylation by ATP induces a conformational change in the protein leading to the transfer of three Na* ions from the inner to the outer side of the plasma membrane. Two molecules of K* bind to sites on the protein on the external surface of the cell membrane, resulting in dephosphorylation of the protein and transfer of the K* ions across the membrane to the interior. Thus, three Na* ions are transported out for every two K* ions entering. This differential ion transport creates a charge imbalance between the inside and the outside of the cell, making the cell interior more negative (an electrogenic effect). Two clinically important cardiac drugs ouabain and digitalis, inhibit the Na*-K*-ATPase by binding to the extracellular domain. This enzyme can consume significant amounts of cellular ATP energy. The Na*-K*-ATPase can be coupled to various other transporters, such as those involved in transport of glucose (see below).

The membrane enclosing neuronal cells maintains an asymmetry of inside-outside voltage (electrical potential) and is also electrically excitable due to the presence of voltage-gated channels. When appropriately stimulated by a chemical signal mediated by a specific synaptic membrane receptor (see discussion of the transmission of biochemical signals, below), channels in the membrane are opened to allow the rapid influx of Na* or Ca?* (with or without the efflux of K*), so that the voltage difference rapidly collapses, and that segment of the membrane is depolarized. However, as a result of the action of the ion pumps in the membrane, the gradient is quickly restored.
When large areas of the membrane are depolarized in this manner, the electrochemical disturbance propagates in wavelike form down the membrane, generating a nerve impulse. Myelin sheets, formed by Schwann cells, wrap around nerve fibers and provide an electrical insulator that surrounds most of the nerve and greatly speeds up the propagation of the wave (signal) by allowing ions to flow in and out of the membrane only where the membrane is free of the insulation (at the nodes of Ranvier). The myelin membrane has a very high lipid content that accounts for its excellent insulating property. Relatively few proteins are found in the myelin membrane; those present appear to hold together multiple membrane bilayers to form the hydrophobic insulating structure that is impermeable to ions and water. Certain diseases, for example, multiple sclerosis and the Guillain-Barré syndrome, are characterized by demyelination and impaired nerve conduction.

A discussion of the transport of glucose summarizes many of the points discussed above. Glucose must enter cells as the first step in energy utilization. A number of different glucose transporters (GLUTs) are involved, varying in different tissues (see Table 19-2). In adipocytes and skeletal muscle, glucose enters by a specific transport system (GLUT4) that is enhanced by insulin. Changes in transport are primarily due to alterations of V_., (presumably from more or fewer transporters), but changes in K, may also be involved.
Glucose transport in the small intestine involves some different aspects of the principles of transport discussed above. Glucose and Na* bind to different sites on a Na*-glucose symporter located at the apical surface. Na* moves into the cell down its electrochemical gradient and "drags" glucose with it (Figure 40-19). Therefore, the greater the Na* gradient, the more glucose enters; and if Na* in extracellular fluid is low, glucose transport stops. To maintain a steep Na* gradient, this Na*-glucose symporter is dependent on gradients generated by the Na*-K*-ATPase, which maintains a low intracellular Na* concentration. Similar mechanisms are used to transport other sugars as well as amino acids across the apical lumen in polarized cells such as are found in the intestine and kidney. The transcellular movement of glucose in this case involves one additional component, a uniport (Figure 40-19) that allows the glucose accumulated within the cell to move

FIGURE 40-19 The transcellular movement of glucose in an intestinal cell. Glucose follows Na* across the luminal epithelial membrane. The Na* gradient that drives this symport is established by Na*-K* exchange, which occurs at the basolateral membrane facing the extracellular fluid compartment via the action of the Na*-K*-ATPase. Glucose at high concentration within the cell moves "downhill" into the extracellular fluid by facilitated diffusion (a uniport mechanism), via GLUT2 (a glucose transporter, see Table 19-2). The sodium-glucose symport actually carries 2 Na* for each glucose.
across the basolateral membrane and involves a glucose uniporter (GLUT2).
The treatment of severe cases of diarrhea (such as is found in cholera) makes use of the above information. In cholera (see Chapter 57), massive amounts of fluid can be passed as watery stools in a very short time, resulting in severe dehydration and possibly death. Oral rehydration therapy, consisting primarily of NaCl and glucose, has been developed by the World Health Organization (WHO). The transport of glucose and Na' across the intestinal epithelium forces (via osmosis) movement of water from the lumen of the gut into intestinal cells, resulting in rehydration. Glucose alone or NaCl alone would not be effective.

The process by which cells take up large molecules is called endocytosis. Some of these molecules, when hydrolyzed inside the cell, yield nutrients (eg, polysaccharides, proteins, and polynucleotides). Endocytosis also provides a mechanism for regulating the content of certain membrane components, hormone receptors being a case in point. Endocytosis can be used to learn more about how cells function. DNA from one cell type can be used to transfect a different cell and alter the latter's function or phenotype. A specific gene is often employed in these experiments, and this provides a unique way to study and analyze the regulation of that gene. DNA transfection depends on endocytosis, which is responsible for the entry of DNA into the cell. Such experiments commonly use calcium phosphate since Ca** stimulates endocytosis and precipitates DNA, which makes the DNA a better object for endocytosis (see Chapter 39). Cells also release macromolecules by exocytosis. Endocytosis and exocytosis both involve vesicle formation with or from the plasma membrane.

Almost all eukaryotic cells are continuously recycling parts of their plaama membranes. Endocytotic vesicles are generated when segments of the plasma membrane invaginate, enclosing a small volume of extracellular fluid and its contents. The vesicle then pinches off as the fusion of plasma membranes seals the neck of the vesicle at the original site of invagination (Figure 40-20). The bilayer lipid membrane, or vesicle so generated, then fuses with other membrane structures and thus achieves the transport of its contents to other cellular compartments or even back to the cell exterior. Most endocytotic vesicles fuse with primary lysosomes to form secondary lysosomes, which contain hydrolytic enzymes and
FIGURE 40-20 Two types of pinocytosis. An endocytotic vesicle (V) forms as a result of invagination of a portion of the plasma membrane. Fluid-phase pinocytosis (A) is random and nondirected. Absorptive (receptor-mediated endocytosis) (B) is selective and occurs in coated pits (CP) lined with the protein clathrin (the fuzzy material). Targeting is provided by receptors (brown symbols) specific for a variety of molecules. This results in the formation of an internalized clathrin-coated vesicle (CV).
are therefore specialized organelles for intracellular disposal. The macromolecular contents are digested to yield amino acids, simple sugars, or nucleotides, which are transported out of the vesicles to be (re)used by the cell. Endocytosis requires (1) energy, usually from the hydrolysis of ATP; (2) Ca**; and (3) contractile elements in the cell (likely the microfilament system) (see Chapter 50).

There are two general types of endocytosis. Phagocytosis occurs only in specialized cells such as macrophages and granulocytes. Phagocytosis involves the ingestion of large particles such as viruses, bacteria, cells, or cellular debris. Macrophages are extremely active in this regard and may ingest 25% of their volume per hour. In so doing, a macrophage may internalize 3% of its plasma membrane each minute or the entire membrane every 30 minutes.
Pinocytosis ("cell drinking") is a property of all cells and leads to the cellular uptake of fluid and fluid contents. There are two types. Fluid-phase pinocytosis is a nonselective process in which the uptake of a solute by formation of small vesicles is simply proportionate to its concentration in the surrounding extracellular fluid. The formation of these vesicles is an extremely active process. Fibroblasts, for example, internalize their plasma membrane at about one-third
the rate of macrophages. This process occurs more rapidly than membranes are made. The surface area and volume of a cell do not change much, so membranes must be replaced by exocytosis or by being recycled as fast as they are removed by endocytosis.

The other type of pinocytosis, absorptive pinocytosis or receptor-mediated endocytosis, is primarily responsible for the uptake of specific macromolecules for which there are binding sites on the plasma membrane. These high-affinity receptors permit the selective concentration of ligands from the medium, minimize the uptake of fluid or soluble unbound macromolecules, and markedly increase the rate at which specific molecules enter the cell. The vesicles formed during absorptive pinocytosis are derived from invaginations (pits) that are coated on the cytoplasmic side with a filamentous material and are appropriately named coated pits. In many systems, the protein clathrin is the filamentous material. It has a three-limbed structure (called a triskelion), with each limb being made up of one light and one heavy chain of clathrin. The polymerization of clathrin into a vesicle is directed by assembly particles, composed of four adapter proteins. These interact with certain amino acid sequences in the receptors that become cargo, ensuring selectivity of uptake. The lipid phosphatidylinositol 4,5-bisphosphate (PIP,) (see Chapter 21) also plays an important role in vesicle assembly. In addition, the protein dynamin, which both binds and hydrolyzes GTP, is necessary for the pinching off of clathrin-coated vesicles from the cell surface. Coated pits may constitute as much as 2% of the surface of some cells. Other aspects of vesicles are discussed in Chapter 49.
As an example, the low-density lipoprotein (LDL) molecule and its receptor (see Chapter 25) are internalized by means of coated pits containing the LDL receptor. Endocytotic vesicles containing the LDL-bound LDL receptor complex fuse to lysosomes in the cell. The receptor is released and recycled back to the cell surface membrane, but the apoprotein of LDL is degraded and the cholesteryl esters metabolized. Synthesis of the LDL receptor is regulated by secondary or tertiary consequences of pinocytosis, for example, by metabolic products—such as cholesterol— released during the degradation of LDL. Disorders of the LDL receptor and its internalization are medically important and are discussed in Chapters 25 and 26.

membrane receptor molecules that play a role analogous to that of the LDL receptor. A galactosyl receptor on the surface of hepatocytes is instrumental in the absorptive pinocytosis of asialoglycoproteins from the circulation (see Chapter 46). Acid hydrolases taken up by absorptive pinocytosis in fibroblasts are recognized by their mannose 6-phosphate moieties. Interestingly, the mannose 6-phosphate moiety also seems to play an important role in the intracellular targeting of the acid hydrolases to the lysosomes of the cells in which they are synthesized (see Chapter 46).
There is a problematic side to receptor-mediated endocytosis in that viruses which cause such diseases as hepatitis (affecting liver cells), poliomyelitis (affecting motor neurons), and AIDS (affecting T cells) initiate their infectious cycles by entering cells via this mechanism. Iron toxicity also begins with excessive uptake due to endocytosis.

Most cells release macromolecules to the exterior by exocytosis. This process is also involved in membrane remodeling, when the components synthesized in the ER and Golgi are carried in vesicles that fuse with the plasma membrane. The signal for this "classical exocytosis" (see below) is often a hormone which, when it binds to a cell surface receptor, induces a local and transient change in Ca®* concentration. Ca** triggers exocytosis. Figure 40-21 provides a comparison of the mechanisms of exocytosis and endocytosis.
Molecules released by this mode of exocytosis have at least three fates: (1) they are membrane proteins and remain associated with the cell surface; (2) they can become part of the extracellular matrix, for example, collagen and glycosaminoglycans; (3) they can enter extracellular fluid and signal other cells. Insulin, parathyroid hormone, and the catecholamines are all packaged in granules and processed within cells, to be released on appropriate stimulation.

Absorptive pinocytosis of extracellular glycoproteins requires that the glycoproteins carry specific carbohydrate recognition signals. These recognition signals are bound by
Specific biochemical signals such as neurotransmitters, hormones, and immunoglobulins bind to integral transmembrane receptor proteins via their exposed extracellular domains, thereby transmitting information through the
FIGURE 40-21 A comparison of the mechanisms of endocytosis and exocytosis. Exocytosis involves the contact of two inside-surface (cytoplasmic side) monolayers, whereas endocytosis results from the contact of two outer-surface monolayers.
membranes to the cytoplasm. This process, called transmembrane signaling or signal transduction, involves the generation of a number of second messenger signaling molecules, including cyclic nucleotides, calcium, phosphoinositides, and diacylglycerol (see Chapter 42). Many of the steps involve phosphorylation of receptors and downstream proteins.

Gap junctions are structures that permit direct transfer of small molecules (up to ~1200 Da) from one cell to its neighbor. Gap junctions are composed of a family of proteins called connexins that form a bihexagonal structure consisting of 12 such proteins. Six connexins form a connexin hemichannel and join to a similar structure in a neighboring cell to make a complete, membrane-spanning connexon channel (Figure 40-22). One gap junction contains several connexons. Different connexins are found in different tissues. Mutations in genes encoding connexins have been found to be associated with a number of conditions, including cardiovascular abnormalities, one type of deafness, and the X-linked form of Charcot-Marie-Tooth disease (a demyelinating neurologic disorder).

In the last decade, a class of small, heterogeneous, secreted vesicles, broadly termed extracellular vesicles, have been identified and characterized. These extracellular vesicles have been implicated as a new and important mediator of cell-cell communication that likely contribute importantly to both normal and pathologic physiology. These vesicles, enclosed by a lipid bilayer, are somewhat heterogeneous in size (30-2000 nm diameter), and are generated by at least two distinct mechanisms (Figure 40-23): microvesicles are generated by budding from the plasma membrane ofa source cell, while exosomes are generated from the multivesicular body (MVB), a component of the endocytic membrane trafficking system described above (ie, see Figure 40-12). Exosomes are secreted from the source cell upon fusion of the MVB with the plasma membrane. In both cases, the released extracellular vesicles (exosomes and microvesicles) ultimately fuse to their target cell to deliver a distinct "payload." Unfortunately, given the recent discovery of extracellular vesicles, the exact names
Schematic of
connexin-mediated
cell-to-cell transport
FIGURE 40-22 Schematic diagram of a gap junction. Shown schematically are (A) the relationships between cells containing connexin; (B) open and closed complete connexin channels; and (C) the flow of molecules (blue, red arrows) between a group of three cells. One connexon is made from two hemiconnexons. Each hemiconnexon is made from six connexin molecules. Small solutes are able to diffuse through the central channel when open, thereby providing a direct mechanism of cell-cell communication. Note that connexins connect cells that are within 2 to 4 nm of each other. Image source: http://upload.wikimedia.org/wikipedia/commons/b/b7/Gap_cell_junction-en.svg.

FIGURE 40-23 Cell-cell communication via extracellular vesicles. Shown are the proposed mechanisms for the formation and production of exosomes and microvesicles via exocytosis (exosome) and membrane budding (microvesicle) from a source cell. Vesicles produced in the multivesicular body (MVB) can be exocytosed following fusion with the plasma membrane as shown, or budded into the extracellular space. All of these processes involve the collection of proteins, lipids, and signaling molecules previously implicated in exocytosis and budding (not shown). Once released from the source cell, the resulting exosomes and/or microvesicles locate their target cell, and following the types of vesicle-target cell interactions shown, release their contents (see black arrows within the target cell). Different vesicles have been shown to contain RNA (mRNA, miRNA, IncRNA; see Chapter 36) and DNA, specific bioactive proteins and lipids; antigens; and biologically active small molecules. Importantly, extracellular vesicles have been shown to have both positive and negative biologic effects on target cells in both normal and pathologic states.
and terms used to describe these vesicles, their cargos, and relevant source and target cells vary in the biomedical literature. Moreover, the terms "microvesicle" and "exosome" are often lumped together as simply "exosomes."

Vesicle content varies from source cell to source cell and has even been reported to be different from the same source cell grown under different conditions. Vesicle payloads can include a variety of cytoplasmic and nuclear proteins, membrane-bound proteins ranging from channels to receptors, major histocompatibility complex (MHC) molecules, lipid raft-interacting proteins, DNA, mRNA, large and small ncRNAs, as well as small protein and bioactive small molecules (Figure 40-23). Given the rich and broad diversity of vesicle/exosome contents, it is not surprising that these structures have been implicated in a very broad range of biology and diseases. Moreover, given their membrane protein content and the fact that extracellular vesicles appear to target specific recipient cells, the potential value of exosomes as therapeutic
delivery systems is receiving significant interest and attention in the pharmaceutical and biotechnology industries. Future work will determine whether this new and exciting area of biomedical research on extracellular vesicles lives up to this promise.

In view of the fact that membranes are located in so many organelles and are involved in so many processes, it is not surprising that mutations affecting their protein constituents should result in many diseases or disorders. While some mutations directly affect the function of membrane proteins, the majority cause protein misfolding that impair membrane trafficking at any of a number of steps from their site of synthesis in the ER to the plasma membrane or other intracellular sites/organelles (see Chapter 49). Examples of diseases or disorders due to abnormalities in membrane proteins are listed in Table 40-7. These mainly reflect mutations in proteins of the plasma membrane, with one affecting lysosomal function (I-cell disease).
Proteins in plasma membranes can be classified as receptors, transporters, ion channels, enzymes, and structural components. Members of all of these classes are often glycosylated, so that mutations affecting this process may alter their function (see Chapter 46). Mutations in receptors can cause defects in transmembrane signaling, a common occurrence in cancer (see Chapter 56). Many genetic diseases or disorders have been ascribed to mutations affecting various proteins involved in the transport of amino acids, sugars, lipids, urate, anions, cations, water, and vitamins across the plasma membrane.
Mutations in genes encoding proteins in other membranebound compartments can also have harmful consequences. For example, mutations in genes encoding mitochondrial membrane proteins involved in oxidative phosphorylation can cause neurologic and other problems (eg, Leber hereditary optic neuropathy [LHON], a condition in which some success with gene therapy has been reported).

Membrane proteins can also be affected by conditions other than mutations. Formation of autoantibodies to the acetylcholine receptor in skeletal muscle causes myasthenia gravis. Ischemia can quickly affect the integrity of various ion channels in membranes. Overexpression of P-glycoprotein (MDR-1), a plasma membrane-localized drug pump, results in multidrug resistance (MDR) in cancer cells. Abnormalities of membrane constituents other than proteins can also be harmful. With regard to lipids, excess of cholesterol (eg, in familial hypercholesterolemia), of lysophospholipid (eg, after bites by certain snakes, whose venom contains phospholipases), or of glycosphingolipids (eg, in a sphingolipidosis), can all affect membrane structure and hence function.
TABLE 40-7 Some Diseases or Pathologic States Resulting From or Attributed to Abnormalities of Membranes?

Achondroplasia (OMIM 100800), Abnormality = Mutations in the gene encoding the fibroblast growth factor receptor 3. Familial hypercholesterolemia (OMIM 143890), Abnormality = Mutations in the gene encoding the LDL receptor. Cystic fibrosis (OMIM 219700), Abnormality = Mutations in the gene encoding the CFTR protein, a Cl - transporter. Congenital long QT syndrome (OMIM 192500), Abnormality = Mutations in genes encoding ion channels in the heart. Wilson disease (OMIM 277900), Abnormality = Mutations in the gene encoding a copper- dependent ATPase. I-cell disease (OMIM 252500), Abnormality = Mutations in the gene encoding GlcNAc phosphotransferase, resulting in absence of the Man 6-P signal for lysosomal localization of certain hydrolases. Hereditary spherocytosis (OMIM 182900), Abnormality = Mutations in the genes encoding spectrin or other structural proteins in the red cell membrane. Metastasis of cancer cells, Abnormality = Abnormalities in the oligosaccharide chains of membrane glycoproteins and glycolipids are thought to be of importance. Paroxysmal nocturnal hemoglobinuria (OMIM 311770), Abnormality = Mutation resulting in deficient attachment of the GPI anchor (see Chapter 46) to certain proteins of the red cell membrane
Abbreviations: CFTR, cystic fibrosis transmembrane regulator protein; GPI, glycosylphosphatidylinositol; LDL, low-density lipoprotein. "The disorders listed are discussed further in other chapters. The table lists examples of mutations affecting two receptors, one transporter, several ion channels (ie, congenital long QT syndrome), two enzymes, and one structural protein. Examples of altered or defective glycosylation of glycoproteins are also presented. Most of the conditions listed involve the plasma membrane.

Cystic fibrosis (CF) is a recessive genetic disorder prevalent among whites in North America and certain parts of northern Europe. CF is characterized by chronic bacterial infections of the airways and sinuses, fat maldigestion due to pancreatic exocrine insufficiency, infertility in males due to abnormal development of the vas deferens, and elevated levels of chloride in sweat (>60 mmol/L). It now known that mutations in a gene encoding a protein named cystic fibrosis transmembrane regulator protein (CFTR) is responsible for CF. CFTR is a cyclic AMP-regulated Cl transporter. The major clinical features of CF and further information about the gene responsible for CF and about CFTR are presented in Case 5, Chapter 57.

■, 1 = Membranes are complex dynamic structures composed of lipids, proteins, and carbohydrate-containing molecules.. ■, 1 = The basic structure of all membranes is the lipid bilayer. This bilayer is formed by two sheets of phospholipids in which the hydrophilic polar head groups are directed away from each other and are exposed to the aqueous environment on the outer and inner surfaces of the membrane. The hydrophobic nonpolar tails of these molecules are oriented toward each other, in the direction of the center of the membrane.. ■, 1 = Membranes are very dynamic structures. Lipids and certain proteins show rapid lateral diffusion. Flip-flop is very slow for lipids and almost nonexistent for proteins.. ■, 1 = The fluid mosaic model forms a useful basis for thinking about membrane structure and function.. ■, 1 = Membrane proteins are classified as integral if they are firmly embedded in the bilayer and as peripheral if they are attached to the outer or inner membrane surface.. ■, 1 = The 20 or so membranes in a mammalian cell have different compositions and functions and they define essential compartments, or specialized environments, within the cell that have specific functions.. ■, 1 = Certain hydrophobic molecules freely diffuse across membranes, but the movement of others is restricted because of their size and/or charge.. ■, 1 = Various passive and active (usually ATP-dependent) mechanisms are employed to maintain gradients of many different molecules across different membranes.. ■, 1 = Certain solutes, for example, glucose, enter cells by facilitated diffusion along a downhill gradient from high to low concentration using specific carrier proteins (transporters).. ■, 1 = The major ATP-driven pumps are classified as P (phosphorylated), F (energy factors), V(vacuolar), and ABC transporters.. ■, 1 = Ligand- or voltage-gated ion channels are often employed to move charged molecules (Na + , K + , Ca 2+ , etc) across membranes down their electrochemical gradients.. ■, 1 = Large molecules can enter or leave cells through mechanisms such as endocytosis or exocytosis. These processes often require binding of the molecule to a receptor, which affords specificity to the process.
- ™ Extracellular vesicles, termed exosomes, also allow direct
movement of
macromolecules from
cell to
cell via

small vesicles.
Exosome payloads
can
(receptors, channels,
signaling include
specific proteins),
lipids,
DNA, proteins
RNAs, and
small bioactive molecules.
- = Mutations that affect the structure of membrane proteins may cause diseases.

C
H
A
PT
ER
Explain the basic principles of endocrine hormone action.
Understand the broad diversity and mechanisms of action of endocrine
hormones.
- = Appreciate the complex steps involved in the production, transport, and
storage of hormones.

The survival of multicellular organisms depends on their ability to adapt to a constantly changing environment. Intercellular communication mechanisms are necessary requirements for this adaptation. The nervous system and the endocrine system provide this intercellular, organism-wide communication. The nervous system was originally viewed as providing a fixed communication system, whereas the endocrine system supplied hormones, which are mobile messages. In fact, there is a remarkable convergence of these regulatory systems. For example, neural regulation of the endocrine system is important in the production and secretion of some hormones; many neurotransmitters resemble hormones in their synthesis, transport, and mechanism of action; and many hormones are synthesized in the nervous system. The word "hormone" is derived from a Greek term that means to arouse to activity. As classically defined, a hormone is a substance that is synthesized in one organ and transported by the circulatory system to act on another tissue. However, this original description is too restrictive because hormones can act on adjacent cells (paracrine action) and on the cell in which they were synthesized (autocrine action) without entering the systemic circulation. A diverse array of hormones—each with distinctive mechanisms of action and properties of biosynthesis, storage,
secretion, transport, and metabolism—has evolved to provide homeostatic responses. This biochemical diversity is the topic of this chapter.

There are over 200 types of differentiated cells in humans. Only a few produce hormones, but virtually all of the 75 trillion cells in a human are targets of one or more of the 50* known hormones. The concept of the target cell is a useful way of looking at hormone action. It was thought that hormones affected a single cell type—or only a few kinds of cells—and that a hormone elicited a unique biochemical or physiologic action. We now know that a given hormone can affect several different cell types; that more than one hormone can affect a given cell type; and that hormones can exert many different effects in one cell or in different cells. With the discovery of specific cell surface and intracellular hormone receptors, the definition of a target has been expanded to include any cell in which the hormone (ligand) binds to its receptor, whether or not a biochemical or physiologic response has yet been determined.
Several factors determine the response of a target cell to a hormone. These can be thought of in two general ways: (1) as factors that affect the concentration of the hormone at the target cell (Table 41-1) and (2) as factors that affect the actual response of the target cell to the hormone (Table 41-2).

One of the major challenges faced in making the hormonebased communication system work is illustrated in Figure 41-1. Hormones are present at very low concentrations in the extracellular fluid, generally in the femtoto nanomolar range (1075-10 mol/L). This concentration is much lower than that of the many structurally similar molecules (sterols, amino acids, peptides, and proteins) and other molecules that circulate at concentrations in the microto millimolar (10~°-10~* mol/L) range. Target cells, therefore, must distinguish not only between

The rate of synthesis and secretion of the hormones.
The proximity of the target cell to the hormone source (dilution
effect).
The affinity (dissociation constant; K,) of the hormone with specific plasma transport proteins (if any).
The conversion of inactive or suboptimally active forms of the
hormone into the fully active form.
The rate of clearance of hormone from plasma, by other tissues, or by degradation, metabolism, or excretion.

The number, relative activity, and state of occupancy of the specific receptors on the plasma membrane or in the cytoplasm or nucleus.
The metabolism (activation or inactivation) of the hormone in the cell.
target
The presence of other factors within the cell that are necessary for the hormone response.
Upor downregulation of the receptor consequent to the interaction with its ligand.
Postreceptor desensitization of the cell, including downregulation of the receptor.
different hormones present in small amounts but also between a given hormone and the 10°to 10°-fold excess of other similar molecules. This high degree of discrimination is provided by cellassociated recognition molecules called receptors. Hormones initiate their biologic effects by binding to hormone-specific receptors, and since any effective control system also must provide a means of stopping a response, hormone-induced actions generally, but not always, terminate when the effector dissociates from the receptor (see Figure 38-1; type A response). A target cell is defined by its ability to selectively bind a given hormone to its cognate receptor. Several biochemical features of this interaction are important in order for hormone-receptor interactions to be physiologically relevant: (1) binding should be specific, that is, displaceable by agonist or antagonist; (2) binding should be saturable; and (3) binding should occur within the concentration range of the expected biologic response.

All receptors have at least two functional domains. A recognition domain binds the hormone ligand and a second region
FIGURE 41-1 Specificity and selectivity of hormone receptors. Many different molecules circulate in the extracellular luid (ECF), but only a few are recognized by hormone receptors. Receptors must select these molecules from among high concentrations of the other molecules. This simplified drawing shows that a cell may have no hormone receptors (Cell type 1), have one receptor (Cell types 2+5+6), have receptors for several hormones (Cell type 3), or have a receptor but no hormone in the vicinity (Cell type 4).
generates a signal that couples hormone recognition to some intracellular function. This coupling, or signal transduction, occurs in two general ways. Polypeptide and protein hormones and the catecholamines bind to receptors located in the plasma membrane and thereby generate a signal that regulates various intracellular functions, often by changing the activity of an enzyme. By contrast, the lipophilic steroid, retinoid, and thyroid hormones interact with intracellular receptors, and it is this ligand-receptor complex that directly provides the signal, generally to specific genes whose rate of transcription is thereby affected.
The domains responsible for hormone recognition and signal generation have been identified in the protein polypeptide and catecholamine hormone receptors. Like many other DNA-binding transcription factors, the steroid, thyroid, and retinoid hormone receptors have several functional domains: one site binds the hormone; another binds to specific DNA regions; a third is involved in the interaction with various coregulator proteins that result in the activation (or repression) of gene transcription; and a fourth region may specify binding to one or more other proteins that influence the intracellular trafficking of the receptor (see Figure 38-19).
The dual functions of binding and coupling ultimately define a receptor, and it is the coupling of hormone binding to signal transduction, the so-called receptor-effector coupling—that provides the first step in amplification of the hormonal response. This dual purpose also distinguishes the target cell receptor from the plasma carrier proteins that bind hormone but do not generate a signal (see Table 41-6).

Several classes of peptide hormone receptors have been defined. For example, the insulin receptor is a heterotetramer composed of two copies of two different protein subunits (a,B,) linked by multiple disulfide bonds in which the extracellular a subunit binds insulin and the membrane-spanning B subunit transduces the signal through the tyrosine protein kinase domain located in the cytoplasmic portion of this polypeptide. The receptors for insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) are generally similar in structure to the insulin receptor. The growth hormone (GH) and prolactin (PRL) receptors also span the plasma membrane of target cells but do not contain intrinsic protein kinase activity. Ligand binding to these receptors, however, results in the association and activation of a completely different protein kinase signaling pathway, the Jak-Stat pathway. Polypeptide hormone and catecholamine receptors, which transduce signals by altering the rate of production of cAMP through G-proteins, which are guanosine nucleotide-binding proteins that are characterized by the presence of seven membranespanning domains. Protein kinase activation and the generation of cyclic AMP (cAMP, 3'5'-adenylic acid; see Figure 18-5) is a downstream action of this class of receptor (see Chapter 42 for further details).
A comparison of several different steroid receptors with thyroid hormone receptors revealed a remarkable conservation of the amino acid sequence in certain regions, particularly in the DNA-binding domains. This observation led to the realization that receptors of the steroid and thyroid type are members of a large superfamily of nuclear receptors. Many related members of this family currently have no known ligand and thus are called orphan receptors. The nuclear receptor superfamily plays a critical role in the regulation of gene transcription by hormones, as described in Chapter 42.

Hormones can be classified according to chemical composition, solubility properties, location of receptors, and the nature of the signal used to mediate hormonal action within the cell. A classification based on the last two properties is illustrated in Table 41-3, and general features of each group are illustrated in Table 41-4.
The hormones in group I are lipophilic. After secretion, these hormones associate with plasma transport or carrier proteins, a process that circumvents the problem of solubility while prolonging the plasma half-life of the hormone. The relative percentages of bound and free hormone are determined by the amount, binding affinity, and binding capacity of the transport protein. The free hormone, which is the biologically active form, readily traverses the lipophilic plasma membrane of all cells and encounters receptors in either the cytosol or nucleus of target cells. The ligand-receptor complex is the intracellular messenger in this group.
The second major group consists of water-soluble hormones that bind to specific receptors spanning the plasma membrane of the target cell. Hormones that bind to these surface receptors of cells communicate with intracellular metabolic processes through intermediary molecules called second messengers (the hormone itself is the first messenger), which are generated as a consequence of the ligand-receptor interaction. The second messenger concept arose from an observation that epinephrine binds to the plasma membrane of certain cells and increases intracellular cAMP. This was followed by a series of experiments in which cAMP was found to mediate the effects of many hormones. Hormones that employ this mechanism are shown in group II.A of Table 41-3. Atrial natriuretic factor (ANF) uses cGMP as its second messenger (group II.B). Several hormones, many of which were previously thought to affect cAMP, appear to use ionic calcium (Ca"*) or metabolites of complex phosphoinositides (or both) as the intracellular second messenger signal. These are shown in group ILC of the table. The intracellular messenger for group II.D is a protein kinase-phosphatase cascade; several have been identified, and a given hormone may use more than one kinase cascade. A few hormones fit into more than one category, and assignments change as new information is discovered.

Hormones are synthesized in discrete organs designed solely for this specific purpose, such as the thyroid (triiodothyronine),

Types, Group I = Steroids, iodothyronines, calcitriol, retinoids. Types, Group II = Polypeptides, proteins, glycoproteins, catecholamines. Solubility, Group I = Lipophilic. Solubility, Group II = Hydrophilic. Transport proteins, Group I = Yes. Transport proteins, Group II = No. Plasma half-life, Group I = Long (hours to days). Plasma half-life, Group II = Short (minutes). Receptor, Group I = Intracellular. Receptor, Group II = Plasma membrane. Mediator, Group I = Receptor- hormone complex. Mediator, Group II = cAMP, cGMP, Ca 2+ , metabolites of complex phosphinositols, kinase cascades
adrenal (glucocorticoids and mineralocorticoids), and the pituitary (TSH, FSH, LH, GH, PRL, ACTH). Some organs are designed to perform two distinct but closely related functions. For example, the ovaries produce mature oocytes and the reproductive hormones estradiol and progesterone. The testes produce mature spermatozoa and testosterone. Hormones are also produced in specialized cells within other organs such as the small intestine (glucagon-like peptide), thyroid (calcitonin), and kidney (angiotensin II). Finally, the synthesis of some hormones requires the parenchymal cells of more than one organ—for example, the skin, liver, and kidney are required for the production of 1,25(OH),-D, (calcitriol). Examples of this diversity in the approach to hormone synthesis, each of which has evolved to fulfill a specific purpose, are discussed below.

Hormones are synthesized from a wide variety of chemical building blocks. A large series is derived from cholesterol. These include the glucocorticoids, mineralocorticoids, androgens, estrogens, progestins, and 1,25(OH),-D, (Figure 41-2). In some cases, a steroid hormone is the precursor molecule for another hormone. For example, progesterone is a hormone in its own right but is also a precursor in the formation of glucocorticoids, mineralocorticoids, testosterone, and estrogens. Testosterone is an obligatory intermediate in the biosynthesis of estradiol and in the formation of dihydrotestosterone (DHT). In these examples, described in detail below, the final product is determined by the cell type and the associated set of enzymes in which the precursor exists.
The amino acid tyrosine is the starting point in the synthesis of both the catecholamines and thyroid hormones tetraiodothyronine (thyroxine; T,) and triiodothyronine (T,) (Figure 41-2). T, and T, are unique in that they require the addition of iodine (as I-) for bioactivity. Since dietary iodine is very scarce in many parts of the world, an intricate mechanism for accumulating and retaining I" has evolved.
common © subunits unique B subunits
FIGURE 41-2 Chemical diversity of hormones. (A) cholesterol derivatives; (B) tyrosine derivatives; (C) peptides of various sizes; note: pyroglutamic acid (pyro) is a cyclized variant of glutamic acid in which side chain carboxyl and free amino groups cyclize to form a lactam. (D) glycoproteins (TSH, FSH, and LH) with common a subunits and unique B subunits.

Many hormones are polypeptides or glycoproteins. These range in size from the small thyrotropin-releasing hormone (TRH), a tripeptide, to single-chain polypeptides like adrenocorticotropic hormone (ACTH; 39 amino acids), parathyroid hormone (PTH; 84 amino acids), and growth hormone (GH; 191 amino acids) (Figure 41-2). Insulin is an A-B chain heterodimer of 21 and 30 amino acids, respectively. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and chorionic gonadotropin (CG) are glycoprotein hormones of af heterodimeric structure. The a chain is identical in all of these hormones, and distinct 8 chains impart hormone uniqueness. These hormones have a molecular mass in the range of 25 to 30 kDa depending on the degree of glycosylation and the length of the 6 chain.

Some hormones are synthesized in final form and secreted immediately. Included in this class are hormones derived from cholesterol. Some, such as the catecholamines, are synthesized in final form and stored in the producing cells, while others, like insulin, are synthesized as precursor molecules in the producing cell, and then are processed and secreted upon a physiologic cue (plasma glucose concentrations). Finally, still others are converted to active forms from precursor molecules in the peripheral tissues (T, and DHT). All of these examples are discussed in more detail below.

The adrenal steroid hormones are synthesized from cholesterol, which is mostly derived from the plasma, but a small portion is synthesized in situ from acetyl-CoA via mevalonate and squalene. Much of the cholesterol in the adrenal is esterified and stored in cytoplasmic lipid droplets. Upon stimulation of the adrenal by ACTH, an esterase is activated, and the free cholesterol formed is transported into the mitochondrion, where a cytochrome P450 side chain cleavage enzyme (P450scc) converts cholesterol to pregnenolone. Cleavage of the side chain involves sequential hydroxylations, first at C,, and then at C,,, followed by side chain cleavage (removal of the six-carbon fragment isocaproaldehyde) to give the 21-carbon steroid (Figure 41-3, top). An ACTH-dependent steroidogenic acute regulatory (StAR) protein is essential for the transport of cholesterol to P450scc in the inner mitochondrial membrane.
18-hydroxylase and 19-hydroxysteroid dehydrogenises, which are required for aldosterone synthesis, are found only in the zona glomerulosa cells (the outer region of the adrenal cortex), so that the biosynthesis of this mineralocorticoid is confined to this region. A schematic representation of the pathways involved in the synthesis of the three major classes of adrenal steroids is presented in Figure 41-4. The enzymes are shown in the rectangular boxes, and the modifications at each step are shaded.

All mammalian steroid hormones are formed from cholesterol via pregnenolone through a series of reactions that occur in either the mitochondria or endoplasmic reticulum of the producing cell. Hydroxylases that require molecular oxygen and NADPH are essential, and dehydrogenases, an isomerase, and a lyase reaction are also necessary for certain steps. There is cellular specificity in adrenal steroidogenesis. For instance,
Synthesis of aldosterone follows the mineralocorticoid pathway and occurs in the zona glomerulosa. Pregnenolone is converted to progesterone by the action of two smooth endoplasmic reticulum enzymes, 36-hydroxysteroid dehydrogenase (3B-OHSD) and A**-isomerase. Progesterone is hydroxylated at the C,, position to form 11-deoxycorticosterone (DOC), which is an active (Na*-retaining) mineralocorticoid. The next hydroxylation, at C,,, produces corticosterone, which has glucocorticoid activity and is a weak mineralocorticoid (it has <5% of the potency of aldosterone). In some species (eg, rodents), it is the most potent glucocorticoid. C,, hydroxylation is necessary for both mineralocorticoid and glucocorticoid activity, but most steroids with a C,, hydroxyl group have more glucocorticoid and less mineralocorticoid action. In the zona glomerulosa, which does not have the smooth endoplasmic reticulum enzyme 17a-hydroxylase, a mitochondrial 18-hydroxylase is present. The 18-hydroxylase (aldosterone
Pregnenolone
+
isocaproaldehyde
Basic steroid
hormone
Testosterone
17a-D-Estradiol structures
Estrane group
(C18)
Androstane group
(C19)
Pregnane group
(C21)
FIGURE 41-3 Cholesterol side chain cleavage and basic steroid hormone structures. The basic sterol rings are identified by the letters A to D. The carbon atoms are numbered 1 to 21, starting with the A ring (see Figure 26-3).

FIGURE 41-4 Pathways involved in the synthesis of the three major classes of adrenal steroids (mineralocorticoids, glucocorticoids, and androgens). Enzymes are shown in the rectangular boxes, and the modifications at each step are shaded. Note that the 17a-hydroxylase and 17,20-lyase activities are both part of one enzyme, designated P450c17. (Slightly modified and reproduced, with permission, from Harding BW: In: Endocrinology, vol 2. DeGroot LJ (editor). Grune & Stratton, 1979. Copyright © 1979 Elsevier Inc. Reprinted with permission from Elsevier.)
synthase) acts on corticosterone to form 18-hydroxycorticosterone, which is changed to aldosterone by conversion of the 18-alcohol to an aldehyde. This unique distribution of enzymes and the special regulation of the zona glomerulosa by K* and angiotensin II have led some investigators to suggest that, in addition to the adrenal being two glands, the adrenal cortex is actually two separate organs.

Cortisol synthesis requires three hydroxylases located in the fasciculata and reticularis zones of the adrenal cortex that act sequentially on the C,,,C,,,andC,, positions. The first two reactions are rapid, while C,, hydroxylation is relatively slow. If the C,, position is hydroxylated first, the action of 17a-hydroxylase is impeded, and the mineralocorticoid pathway is followed (forming corticosterone or aldosterone, depending on the cell type). 17a-Hydroxylase is a smooth endoplasmic reticulum enzyme that acts on either progesterone or, more commonly, pregnenolone. 17a-Hydroxyprogesterone is hydroxylated at C,, to form 11-deoxycortisol, which is then hydroxylated at C, , to form cortisol, the most potent natural glucocorticoid hormone in humans. 21-Hydroxylase is a smooth endoplasmic
reticulum enzyme, whereas 11$-hydroxylase is a mitochondrial enzyme. Steroidogenesis thus involves the repeated shuttling of substrates into and out of the mitochondria.

The major androgen or androgen precursor produced by the adrenal cortex is dehydroepiandrosterone (DHEA). Most 17-hydroxypregnenolone follows the glucocorticoid pathway, but a small fraction is subjected to oxidative fission and removal of the two-carbon side chain through the action of 17,20-lyase. The lyase activity is actually part of the same enzyme (P450c17) that catalyzes 17a-hydroxylation. This is therefore a dual-function protein. The lyase activity is important in both the adrenals and the gonads and acts exclusively on 17a-hydroxy-containing molecules. Adrenal androgen production increases markedly if glucocorticoid biosynthesis is impeded by the lack of one of the hydroxylases (adrenogenital syndrome). DHEA is really a prohormone since the actions of 3B-OHSD and A*'-isomerase convert the weak androgen DHEA into the more potent androstenedione. Small amounts of androstenedione are also formed in the adrenal by the action of the lyase on 17a-hydroxyprogesterone. Reduction of androstenedione at the C,, position results in the formation of testosterone, the most potent adrenal androgen. Small amounts of testosterone are produced in the adrenal by this mechanism, but most of this conversion occurs in the testes.

Testicular androgens are synthesized in the interstitial tissue by the Leydig cells. The immediate precursor of the gonadal steroids, as for the adrenal steroids, is cholesterol. The ratelimiting step, as in the adrenal, is delivery of cholesterol to the inner membrane of the mitochondria by the transport protein StAR. Once in the proper location, cholesterol is acted upon by the side chain cleavage enzyme P450scc. The conversion of cholesterol to pregnenolone is identical in adrenal, ovary, and testis. In the latter two tissues, however, the reaction is promoted by LH rather than ACTH.
The conversion of pregnenolone to testosterone requires the action of five enzyme activities contained in three proteins: (1) 3B-hydroxysteroid dehydrogenase (3B-OHSD) and A®4-isomerase; (2) 17a-hydroxylase and 17,20-lyase; and (3) 17B-hydroxysteroid dehydrogenase (17B-OHSD). This sequence, referred to as the progesterone (or A*) pathway, is shown on the right side of Figure 41-5. Pregnenolone can also be converted to testosterone by the dehydroepiandrosterone (or A®) pathway, which is illustrated on the left side of Figure 41-5. The A5 route appears to be most used in human testes.
The five enzyme activities are localized in the microsomal fraction in rat testes, and there is a close functional association between the activities of 38-OHSD and A*-isomerase and between those of a 17a-hydroxylase and 17,20-lyase. These enzyme pairs, both contained in a single protein, are shown in the reaction in 41-5.
general sequence Figure

Testosterone is metabolized by two pathways. One involves oxidation at the 17-position, and the other involves reduction of the A ring double bond and the 3-ketone. Metabolism by the first pathway occurs in many tissues, including liver, and produces 17-ketosteroids that are generally inactive or less active than the parent compound. Metabolism by the second pathway, which is less efficient, occurs primarily in target tissues and produces the potent metabolite DHT.
The most significant metabolic product of testosterone is DHT, since in many tissues, including prostate, external genitalia, and some areas of the skin, this is the active form of the hormone. The plasma content of DHT in the adult male is about one-tenth that of testosterone, and ~400 ug of DHT is produced daily as compared with about 5 mg of testosterone. About 50 to 100 tg of DHT are secreted by the testes. The rest is produced peripherally from testosterone in a reaction catalyzed by the NADPH-dependent 5a-reductase (Figure 41-6). Testosterone can thus be considered a prohormone since it is converted into a much more potent compound (DHT) and since most of this conversion occurs outside the testes. Some estradiol is formed from the peripheral aromatization of testosterone, particularly in males.

The estrogens are a family of hormones synthesized in a variety of tissues. 17B-Estradiol is the primary estrogen of ovarian origin. In some species, estrone, synthesized in numerous tissues, is more abundant. In pregnancy, relatively more estriol is produced, and this comes from the placenta. The general pathway and the subcellular localization of the enzymes involved in the early steps of estradiol synthesis are the same as those involved in androgen biosynthesis. Features unique to the ovary are illustrated in Figure 41-7.
Estrogens are formed by the aromatization of androgens in a complex process that involves three hydroxylation steps, each of which requires O, and NADPH. The aromatase enzyme complex is thought to include a P450 monooxygenase. Estradiol is formed if the substrate of this enzyme complex is testosterone, whereas estrone results from the aromatization of androstenedione.
The cellular source of the various ovarian steroids has been difficult to unravel, but a transfer of substrates between two cell types is involved. Theca cells are the source of androstenedione and testosterone. These are converted by the aromatase enzyme in granulosa cells to estrone and estradiol, respectively. Progesterone, a precursor for all steroid hormones, is produced and secreted by the corpus luteum as an end-product hormone because these cells do not contain the enzymes necessary to convert progesterone to other steroid hormones (Figure 41-8).
Significant amounts of estrogens are produced by the peripheral aromatization of androgens. In human males,
FIGURE 41-5 Pathways of testosterone biosynthesis. The pathway on the left side of the figure is called the A® or dehydroepiandrosterone pathway; the pathway on the right side is called the A* or progesterone pathway. The asterisk indicates that the 17a-hydroxylase and 17,20-lyase activities reside in a single protein, P450c17.

the peripheral aromatization of testosterone to estradiol (E,) accounts for 80% of the production of the latter. In females, adrenal androgens are important substrates since as much as 50% of the E, produced during pregnancy comes from the aromatization of androgens. Finally, conversion of androstenedione to estrone is the major source of estrogens in postmenopausal women. Aromatase activity is present in adipose cells and also in liver, skin, and other tissues. Increased activity of this enzyme may contribute to the "estrogenization" that characterizes such diseases as cirrhosis of the liver, hyperthyroidism, aging, and obesity. Aromatase inhibitors show promise as therapeutic agents
fe)
FIGURE 41-6 Dihydrotestosterone is formed from testosterone through action of the enzyme 5a-reductase.
in breast cancer and possibly in other female reproductive tract malignancies.
_
processes in a manner similar to that employed by the steroid hormones.

1,25(OH),-D, is produced by a complex series of enzymatic reactions that involve the plasma transport of precursor molecules to a number of different tissues (Figure 41-9). One of these precursors is vitamin D—really not a vitamin, but this common name persists. The active molecule, 1,25(OH),D,, is transported to other organs where it activates biologic
Small amounts of the precursor for 1,25(OH),-D, synthesis _are present in food (fish liver oil, and egg yolk), but most of the precursor for 1,25(OH),-D, synthesis is produced in the malpighian layer of the epidermis from 7-dehydrocholesterol in an ultraviolet light-mediated, nonenzymatic photolysis reaction. The extent of this conversion is related directly to the intensity of the exposure and inversely to the extent of pigmentation in
_
_
_
_
FIGURE 41-7 Biosynthesis of estrogens. (Slightly modified and reproduced, with permission, from Ganong WF: Review of Medical Physiology, 21st ed. McGraw-Hill, 2005.)

the skin. There is an age-related loss of 7-dehydrocholesterol in the epidermis that may be related to the negative calcium balance associated with old age.

A specific transport protein called the vitamin D-binding protein binds vitamin D, and its metabolites and moves vitamin D, from the skin or intestine to the liver, where it undergoes 25-hydroxylation, the first obligatory reaction in the production of 1,25(OH),-D,. 25-Hydroxylation occurs in the endoplasmic reticulum in a reaction that requires magnesium, NADPH, molecular oxygen, and an uncharacterized cytoplasmic factor. Two enzymes are involved: an NADPH-dependent cytochrome P450 reductase and a cytochrome P450. This reaction is not regulated, and it also occurs with low efficiency in kidney and intestine. The 25(OH),-D, enters the circulation, where it is the major form of vitamin D found in plasma, and is transported to the kidney by the vitamin D-binding protein.

25(OH),-D, is a weak agonist and must be modified by hydroxylation at position C, for full biologic activity. This is accomplished in mitochondria of the renal proximal convoluted tubule by a three-component monooxygenase reaction
FIGURE 41-9 Formation and hydroxylation of vitamin D,. 25-Hydroxylation takes place in the liver, and the other hydroxylations occur in the kidneys. 25,26(OH),-D, and 1,25,26(OH),-D, are probably formed as well. The structures of 7-dehydrocholesterol, vitamin D,, and 1,25(OH),-D, are also shown. (Modified and reproduced, with permission, from Ganong WF: Review of Medical Physiology, 21st ed. McGraw-Hill, 2005.)
that requires NADPH, Mg", molecular oxygen, and at least three enzymes: (1) a flavoprotein, renal ferredoxin reductase; (2) an iron-sulfur protein, renal ferredoxin; and (3) cytochrome P450. This system produces 1,25(OH),-D,, which is the most potent naturally occurring metabolite of vitamin D.

Three amines—dopamine, norepinephrine, and epinephrine— are synthesized from tyrosine in the chromaffin cells of the adrenal medulla. The major product of the adrenal medulla is epinephrine. This compound constitutes about 80% of the catecholamines in the medulla, and it is not made in extramedullary tissue. In contrast, most of the norepinephrine present in organs innervated by sympathetic nerves is made in situ (about 80% of the total), and most of the rest is made in other nerve endings and reaches the target sites via the circulation. Epinephrine and norepinephrine may be produced and stored in different cells in the adrenal medulla and other chromaffin tissues. The conversion of tyrosine to epinephrine requires four sequential steps: (1) ring hydroxylation; (2) decarboxylation; (3) side chain hydroxylation to form norepinephrine; and (4) N-methylation to form epinephrine. The biosynthetic pathway and the enzymes involved are illustrated in Figure 41-10.

Tyrosine is the immediate precursor of catecholamines, and tyrosine hydroxylase is the rate-limiting enzyme in catecholamine biosynthesis. Tyrosine hydroxylase is found in both soluble and particle-bound forms only in tissues that synthesize catecholamines; it functions as an oxidoreductase, with tetrahydropteridine as a cofactor, to convert L-tyrosine to t-dihydroxyphenylalanine (t-dopa). As the rate-limiting enzyme, tyrosine hydroxylase is regulated in a variety of ways. The most important mechanism involves feedback inhibition by the catecholamines, which compete with the enzyme for the pteridine cofactor. Catecholamines cannot cross the blood-brain barrier; hence, in the brain they must be synthesized locally. In certain central nervous system diseases (eg, Parkinson disease), there is a local deficiency of dopamine synthesis. L-Dopa, the precursor of dopamine, readily crosses the blood-brain barrier and so is an important agent in the treatment of Parkinson disease.

This soluble enzyme requires pyridoxal phosphate for the conversion of L-dopa to 3,4-dihydroxyphenylethylamine

phenylethanolamine-N-methyltransferase.)
(dopamine). Compounds that resemble L-dopa, such as a-methyldopa, are competitive inhibitors of this reaction. a-Methyldopa is effective in treating some kinds of hypertension.

DBH is a monooxygenase and uses ascorbate as an electron donor, copper at the active site, and fumarate as modulator. DBH is in the particulate fraction of the medullary cells, probably in the secretion granule; thus, the conversion of dopamine to norepinephrine occurs in this organelle.

PNMT catalyzes the N-methylation of norepinephrine to form epinephrine in the epinephrine-forming cells of the adrenal medulla. Since PNMT is soluble, it is assumed that norepinephrine-to-epinephrine conversion occurs in the cytoplasm. The synthesis of PNMT is induced by glucocorticoid hormones that reach the medulla via the intra-adrenal portal system. This special system provides for a 100-fold steroid concentration gradient over systemic arterial blood, and this high intra-adrenal concentration appears to be necessary for the induction of PNMT.

The formation of triiodothyronine (T,) and tetraiodothyronine (thyroxine; T,) (see Figure 41-2) illustrates many of the principles of diversity discussed in this chapter. These hormones require a rare element (iodine) for bioactivity; they are synthesized as part of a very large precursor molecule (thyroglobulin); they are stored in an intracellular reservoir (colloid); and there is peripheral conversion of T, to T,, which is a much more active hormone.
The thyroid hormones T, and T, are unique in that iodine (as iodide) is an essential component of both. In most parts of the world, iodine is a scarce component of soil, and for that reason there is little in food. A complex mechanism has evolved to acquire and retain this crucial element and to convert it into a form suitable for incorporation into organic compounds. At the same time, the thyroid must synthesize thyronine from tyrosine, and this synthesis takes place in thyroglobulin (Figure 41-11).

Thyroglobulin is the precursor of T, and T,. It is a large iodinated, glycosylated protein with a molecular mass of 660 kDa. Carbohydrate accounts for 8 to 10% of the weight of thyroglobulin and iodide for about 0.2 to 1%, depending on the iodine content in the diet. Thyroglobulin is composed of two large subunits. It contains 115 tyrosine residues, each of which is a potential site of iodination. About 70% of the iodide in thyroglobulin exists in the inactive precursors, monoiodotyrosine (MIT) and diiodotyrosine (DIT), while 30% is in the iodothyronyl residues, T, and T,. When iodine supplies are sufficient, the T,:T, ratio is about 7:1. In iodine deficiency, this ratio decreases, as does the DIT:MIT ratio. Thyroglobulin, a large molecule of about 5000 amino acids, provides the conformation required for tyrosyl coupling and iodide organification necessary in the formation of the diaminoacid thyroid hormones. It is synthesized in the basal portion of the cell and moves to the lumen, where it is a storage form of T, and T, in the colloid; several weeks' supply of these hormones exist in the normal thyroid. Within minutes after stimulation of the thyroid by TSH, colloid reenters the cell and there is a marked increase of phagolysosome activity. Various acid proteases and peptidases hydrolyze the thyroglobulin into its constituent amino acids, including T, and T,, which are discharged into the extracellular space (Figure 41-11). Thyroglobulin is thus a very large prohormone.

The thyroid is able to concentrate I against a strong electrochemical gradient. This is an energy-dependent process and is linked to the Na*-K*-ATPase-dependent thyroidal Itransporter. The ratio of iodide in thyroid to iodide in serum (T:S ratio) is a reflection of the activity of this transporter. This activity is primarily controlled by TSH and ranges from 500:1 in animals chronically stimulated with TSH to 5:1 or less in hypophysectomized animals (no TSH). The T:S ratio in humans on a normal iodine diet is about 25:1.
The thyroid is the only tissue that can oxidize I to a higher valence state, an obligatory step in Iorganification and thyroid hormone biosynthesis. This step involves a heme-containing peroxidase and occurs at the luminal surface of the follicular cell. Thyroperoxidase, a tetrameric protein with a molecular mass of 60 kDa, requires hydrogen peroxide (H,O,) as an oxidizing agent. The H,O, is produced by an NADPH-dependent enzyme resembling cytochrome c reductase. A number of compounds inhibit I oxidation and therefore its subsequent incorporation into MIT and DIT. The most important of these are the thiourea drugs. They are used as antithyroid drugs because of their ability to inhibit thyroid hormone biosynthesis at this step. Once iodination occurs, the iodine does not readily leave the thyroid. Free tyrosine can be iodinated, but it is not incorporated into proteins since no tRNA recognizes iodinated tyrosine.
The coupling of two DIT molecules to form T,—or of an MIT and DIT to form T,—occurs within the thyroglobulin molecule. A separate coupling enzyme has not been found, and since this is an oxidative process, it is assumed that the same thyroperoxidase catalyzes this reaction by stimulating free radical formation of iodotyrosine. This hypothesis is supported by the observation that the same drugs which inhibit IT oxidation also inhibit coupling. The formed thyroid hormones remain as integral parts of thyroglobulin until the latter is degraded, as described above.

A deiodinase removes I" from the inactive mono and diiodothyronine molecules in the thyroid. This mechanism provides a substantial amount of the Iused in T, and T, biosynthesis. A peripheral deiodinase in target tissues such as pituitary, kidney, and liver selectively removes Ifrom the 5' position of T, to make T, (Figure 41-2), which is a much more active molecule. In this sense, T, can be thought of as a prohormone, though it does have some intrinsic activity.

Formation of the critical disulfide bridges in insulin requires that this hormone be first synthesized as part of a larger

FIGURE 41-11 Model of iodide metabolism in the thyroid follicle. A follicular cell is shown facing the follicular lumen (top) and the extracellular space (bottom). lodide enters the thyroid primarily through a transporter (bottom left). Thyroid hormone synthesis occurs in the follicular space through a series of reactions, many of which are peroxidase-mediated. Thyroid hormones, stored in the colloid in the follicular space, are released from thyroglobulin by hydrolysis inside the thyroid cell. (DIT, diiodotyrosine; MIT, monoiodotyrosine; Tgb, thyroglobulin; T,, triiodothyronine; T,, tetraiodothyronine; T, and T, structures are shown in Figure 41-2B.) Asterisks indicate steps or processes where inherited enzyme deficiencies cause congenital goiter and often result in hypothyroidism.
precursor molecule, proinsulin. This is conceptually similar to the example of the thyroid hormones, which can only be formed in the context of a much larger molecule. Several other hormones are synthesized as parts of large precursor molecules, not because of some special structural requirement but rather as a mechanism for controlling the available amount of the active hormone. PTH and angiotensin II are examples of this type of regulation. Another interesting example is the pro-opiomelanocortin (POMC) protein, which can be processed into many different hormones in a tissue-specific manner. These examples are discussed in detail below.

Insulin has an AB heterodimeric structure with one intrachain (A6-A11) and two interchain disulfide bridges (A7-B7 and A20-B19) (Figure 41-12). The A and B chains could be synthesized in the laboratory, but attempts at a biochemical synthesis of the mature insulin molecule yielded very poor results. The reason for this became apparent when it was discovered that insulin is synthesized as a preprohormone (molecular weight ~11,500), which is the prototype for peptides that are
FIGURE 41-12 Structure of human proinsulin. Insulin and C-peptide molecules are connected at two sites by peptide bonds. An initial cleavage by a trypsin-like enzyme (red open arrows) followed by several cleavages by a carboxypeptidase-like enzyme (green solid arrows) results in the production of the heterodimeric (AB) insulin molecule (colored), which is held together by two intrapeptide cysteine disulfide bonds; insulin C-peptide (white).
processed from larger precursor molecules. The hydrophobic 23-amino-acid pre-, or leader, sequence directs the molecule into the cisternae of the endoplasmic reticulum and then is removed. This results in the 9000-MW proinsulin molecule, which provides the conformation necessary for the proper and efficient formation of the disulfide bridges. As shown in Figure 41-12, the sequence of proinsulin, starting from the amino terminus, is B chain—connecting (C) peptide— A chain. The proinsulin molecule undergoes a series of sitespecific peptide cleavages that result in the formation of equimolar amounts of mature insulin and C-peptide. These enzymatic cleavages are summarized in Figure 41-12.

The immediate precursor of parathyroid (PTH) is proPTH, which differs from the native 84-amino-acid hormone by having a highly basic hexapeptide amino terminal extension. The primary gene product and the immediate precursor for proPTH is the 115-amino-acid preproPTH. This differs from proPTH by having an additional 25-amino-acid NH,-terminal extension that is common with the other leader or signal sequences characteristic of secreted proteins; it is predominantly hydrophobic in nature. The complete structure of preproPTH and the sequences of proPTH and PTH are illustrated in Figure 41-13. PTH, ,, has full biologic activity, and the region 25 to 34 is primarily responsible for receptor binding. The biosynthesis of PTH and its subsequent secretion are regulated by the plasma ionized calcium (Ca'*) concentration through a complex process. An acute decrease of Ca'* results in a marked increase of PTH mRNA, and this is followed by an increased rate of PTH synthesis and secretion. However, about 80 to 90% of the proPTH synthesized cannot be accounted for as intact PTH in cells or in the incubation medium of experimental systems. This finding led to the conclusion that most of the proPTH synthesized is quickly degraded. It was later discovered that this rate of degradation decreases when Ca** concentrations are low, and it increases when Ca** concentrations are high. A Ca** receptor on the surface of the parathyroid cell mediates these effects. Very specific fragments of PTH are generated during its proteolytic digestion (Figure 41-13). A number of proteolytic enzymes, including cathepsins B and D, have been identified in parathyroid tissue. Cathepsin B cleaves PTH into two fragments: PTH, ,, and PTH,, ,,. PTH,, ,, is not further degraded; however, PTH, ,, is rapidly and progressively cleaved into diand tripeptides. Most of the proteolysis of PTH occurs within the gland, but a number of studies confirm that PTH, once secreted, is proteolytically degraded in other tissues, especially the liver, by similar mechanisms.

FIGURE 41-13 Structure of bovine preproparathyroid hormone. The green arrows indicate sites of cleavage by processing enzymes in the parathyroid gland and in the liver after secretion of the hormone (numbered 1 to 5). The biologically active region of the molecule (colored) is flanked by sequence not required for activity on target receptors. (Slightly modified and reproduced, with permission, from Habener JF: Recent advances in parathyroid hormone research. Clin Biochem 1981;14:223. Copyright © 1981. Reprinted with permission from Elsevier.)

The renin-angiotensin system is involved in the regulation of blood pressure and electrolyte metabolism (through production of aldosterone). The primary hormone involved in these processes is angiotensin II, an octapeptide made from angiotensinogen (Figure 41-14). Angiotensinogen, a large a,-globulin made in liver, is the substrate for renin, an enzyme produced in the juxtaglomerular cells of the renal afferent arteriole. The position of these cells makes them particularly sensitive to blood pressure changes, and many of the physiologic regulators of renin release act through renal baroreceptors. The juxtaglomerular cells are also sensitive to changes of Na* and Cl concentration in the renal tubular fluid; therefore, any combination of factors that decreases fluid volume (dehydration, decreased blood pressure, fluid, or blood loss) or decreases NaCl concentration stimulates renin release. Renal sympathetic nerves that terminate in the juxtaglomerular cells mediate the central nervous system and postural effects on renin release independently of the baroreceptor and salt effects, a mechanism that involves the B-adrenergic receptor. Renin acts on the substrate angiotensinogen to produce the decapeptide angiotensin I.
Angiotensin-converting enzyme (ACE), a glycoprotein found in lung, endothelial cells, and plasma, removes two carboxyl terminal amino acids from the decapeptide angiotensin I to form angiotensin II in a step that is not thought to be rate-limiting. Various nonapeptide analogs of angiotensin I and other compounds act as competitive inhibitors of converting enzyme and are used to treat renin-dependent hypertension. These are referred to as ACE inhibitors. Angiotensin II increases blood pressure by causing vasoconstriction of the arteriole and is a very potent vasoactive substance. It inhibits renin release from the juxtaglomerular cells and is a potent stimulator of aldosterone production. This results in Na* retention, volume expansion, and increased blood pressure.

In some species, angiotensin II is converted to the heptapeptide angiotensin III (Figure 41-14), an equally potent stimulator of aldosterone production. In humans, the plasma level of angiotensin II is four times greater than that of angiotensin III, so most effects are exerted by the octapeptide. Angiotensins II and III are rapidly inactivated by angiotensinases.
Angiotensin II binds to specific adrenal cortex glomerulosa cell receptors. The hormone-receptor interaction does not activate adenylyl cyclase, and cAMP does not appear to mediate the
FIGURE 41-14 Formation, metabolism, and selected physiologic activities of angiotensins. The three most biologically active forms of angiotensin (Ang), Ang 1-7, Ang 1-8 (Ang Il), and Ang 2-8 (Ang Ill) are shown. The numbers represent the amino acids present in each Ang are numbered relative to the sequence of Ang 1-10 (Ang |). All Ang forms are derived by proteolysis catalyzed by a number of different proteases. Initial processing of the 400* amino acid long angiotensinogen precursor is catalyzed by renin, while several of the other proteolytic events are catalyzed by angiotensin-converting enzyme 1 (ACE1), or ACE2. The receptors bound by the different Ang forms, as well as physiologic outcomes of are shown receptor binding (bottom).
action of this hormone. The actions of angiotensin II, which are to stimulate the conversion of cholesterol to pregnenolone and of corticosterone to 18-hydroxycorticosterone and aldosterone, may involve changes in the concentration of intracellular calcium and of phospholipid metabolites by mechanisms similar to those described in Chapter 42.
POMC is synthesized as a precursor molecule of 285 amino acids and is processed differently in various regions of the pituitary.

The POMC family consists of peptides that act as hormones (ACTH, LPH, MSH) and others that may serve as neurotransmitters or neuromodulators (endorphins) (Figure 41-15).
The POMC gene is expressed in the anterior and intermediate lobes of the pituitary. The most conserved sequences between species are within the amino terminal fragment, the ACTH region, and the B-endorphin region. POMC or related products are found in several other vertebrate tissues, including the brain, placenta, gastrointestinal tract, reproductive tract, lung, and lymphocytes.
The POMC protein is processed differently in the anterior lobe than in the intermediate lobe. The intermediate lobe of the pituitary is rudimentary in adult humans, but it is active

FIGURE 41-15 Products of pro-opiomelanocortin (POMC) cleavage. (CLIP, corticotropin-like intermediate lobe peptide; LPH, lipotropin; MSH, melanocyte-stimulating hormone.)

in human fetuses and in pregnant women during late gestation and is also active in many animal species. Processing of the POMC protein in the peripheral tissues (gut, placenta, and male reproductive tract) resembles that in the intermediate lobe. There are three basic peptide groups: (1) ACTH, which can give rise to a-MSH and corticotropin-like intermediate lobe peptide (CLIP); (2) B-lipotropin (B-LPH), which can yield y-LPH, B-MSH, and B-endorphin (and thus aand y-endorphins); and (3) a large amino-terminal peptide, which generates y-MSH (not shown). The diversity of these products is due to the many dibasic amino acid clusters that are potential cleavage sites for trypsin-like enzymes. Each of the peptides mentioned is preceded by Lys-Arg, Arg-Lys, Arg-Arg, or Lys-Lys residues. After the prehormone segment is cleaved, the next cleavage, in both anterior and intermediate lobes, is between ACTH and £-LPH, resulting in an amino-terminal peptide with ACTH and a B-LPH segment (Figure 41-15). ACTH, ,, is subsequently cleaved from the amino-terminal peptide, and in the anterior lobe essentially no further cleavages occur. In the intermediate lobe, ACTH, ,, is cleaved into a-MSH (residues 1-13) and CLIP (18-39); B-LPH (42-134) is converted to y-LPH (42-101) and B-endorphin (104-134). B-MSH (84-101) is derived from y-LPH, while y-MSH (50-74) is derived from a POMC N-terminal fragment (1-74). There are extensive additional tissue-specific modifications of these peptides that affect activity. These modifications include phosphorylation, acetylation, glycosylation, and amidation. Mutations of the a-MSH receptor are linked to a common, early-onset form of obesity. This observation has redirected attention to the POMC peptide hormones.

As mentioned
above, the
steroid hormones
and
D, are
synthesized secreted
in as
they are
their made,
final active
and form.
thus there
1,25(OH),-
They is
no are
also intracellular
reservoir of these hormones. The catecholamines, also synthesized in active form, are stored in granules in the chromaffin cells in the adrenal medulla. In response to appropriate neural stimulation, these granules are released from the cell through exocytosis, and the catecholamines are released into the circulation. A several-hour reserve supply of catecholamines exists in the chromaffin cells.
PTH also exists in storage vesicles. As much as 80 to 90% of the proPTH synthesized is degraded before it enters this final storage compartment, especially when Ca** levels are high in the parathyroid cell (see above). PTH is secreted when Ca** is low in the parathyroid cells, which contain a severalhour supply of the hormone.
The human pancreas secretes about 40 to 50 units of insulin daily; this represents about 15 to 20% of the hormone stored in the 6 cells. Insulin and the C-peptide (Figure 41-12) are normally secreted in equimolar amounts. Stimuli such as glucose, which provokes insulin secretion, therefore trigger the processing of proinsulin to insulin as an essential part of the secretory response.
A several-week supply of T, and T, exists in the thyroglobulin that is stored in colloid in the lumen of the thyroid follicles. These hormones can be released upon stimulation by TSH. This is the most exaggerated example of a prohormone, as a molecule containing ~5000 amino acids must be first synthesized, then degraded, to supply a few molecules of the active hormones T, and T,.
The diversity in storage and secretion of hormones is illustrated in Table 41-5.

Steroids and 1,25(OH) 2 -D 3, Supply Stored in Cell = None. Catecholamines and PTH, Supply Stored in Cell = Hours. Insulin, Supply Stored in Cell = Days. T 3 andT 4, Supply Stored in Cell = Weeks

The class I hormones are hydrophobic in chemical nature and thus are not very soluble in plasma. These hormones, principally the steroids and thyroid hormones, have specialized plasma transport proteins that serve several purposes. First, these proteins circumvent the solubility problem and thereby deliver the hormone to the target cell. They also provide a circulating reservoir of the hormone that can be substantial, as in the case of the thyroid hormones. Hormones, when bound to the transport proteins, cannot be metabolized, thereby prolonging their plasma half-life (t,,). The binding affinity of a given hormone to its transporter determines the bound versus free ratio of the hormone. This is important because only the free form of a hormone is biologically active. In general, the concentration of free hormone in plasma is very low, in the range of 10~' to 10° mol/L. It is important to distinguish between plasma transport proteins and hormone receptors. Both bind hormones but with very different characteristics (Table 41-6).
The hydrophilic hormones—generally class II and of peptide structure—are freely soluble in plasma and do not require transport proteins. Hormones such as insulin, growth hormone, ACTH, and TSH circulate in the free, active form and have very short plasma half-lives. A notable exception is IGF-I, which is transported bound to members of a family of binding proteins.

Many of the principles discussed above are illustrated in a discussion of thyroid-binding proteins. One-half to two-thirds of T, and T, in the body is in an extrathyroidal reservoir. Most of this circulates in bound form, that is, bound to a specific binding protein, thyroxine-binding globulin (TBG). TBG, a glycoprotein with a molecular mass of 50 kDa, binds T, and T, and has the capacity to bind 20 ug/dL of plasma. Under normal circumstances, TBG binds—noncovalently—nearly all of the T, and T, in plasma, and it binds T, with greater affinity

Concentration, Receptors = Very low (thousands/cell). Concentration, Transport Proteins = Very high (billions/μL). Binding affinity ( K d ), Receptors = High (pmol/L to nmol/L range). Binding affinity ( K d ), Transport Proteins = Low (μmol/L range). Binding specificity, Receptors = Very high. Binding specificity, Transport Proteins = Low. Saturability, Receptors = Yes. Saturability, Transport Proteins = No. Reversibility, Receptors = Yes. Reversibility, Transport Proteins = Yes. Signal transduction, Receptors = Yes. Signal transduction, Transport Proteins = No

T 4 8, Percentage of Total = 0.03. T 4 8, ng/dL = ~2.24. T 4 8, Molarity = 3.0 × 10 -11. T 4 8, t 1/2 in Blood (days) = 6.5. T 3 0.15, Percentage of Total = 0.3. T 3 0.15, ng/dL = ~0.4. T 3 0.15, Molarity = 0.6 × 10 -11. T 3 0.15, t 1/2 in Blood (days) = 1.5
than T, (Table 41-7). The plasma half-life of T, is correspondingly four to five times that of T,. The small, unbound (free) fraction is responsible for the biologic activity. Thus, in spite of the great difference in total amount, the free fraction of T, approximates that of T,, and given that T, is intrinsically more active than T,, most biologic activity is attributed to T,. TBG does not bind any other hormones.

Hydrocortisone (cortisol) also circulates in plasma in proteinbound and free forms. The main plasma binding protein is an a-globulin called transcortin, or corticosteroid-binding globulin (CBG). CBG is produced in the liver, and its synthesis, like that of TBG, is increased by estrogens. CBG binds most of the hormone when plasma cortisol levels are within the normal range; much smaller amounts of cortisol are bound to albumin. The affinity of binding helps determine the biologic half-lives of various glucocorticoids. Cortisol binds tightly to CBG and has a t, of 1.5 to 2 hours, while corticosterone, which binds less tightly, has a t,, of <1 hour (Table 41-8). The unbound (free) cortisol constitutes ~8% of the total and represents the biologically active fraction. Binding to CBG is not restricted to glucocorticoids. Deoxycorticosterone and progesterone interact with CBG with sufficient affinity to compete for cortisol binding. Aldosterone, the most potent natural mineralocorticoid, does not have a specific plasma

Dihydrotestosterone, SHBG a = 1. Dihydrotestosterone, CBG a = >100. Testosterone, SHBG a = 2. Testosterone, CBG a = >100. Estradiol, SHBG a = 5. Estradiol, CBG a = >10. Estrone, SHBG a = >10. Estrone, CBG a = >100. Progesterone, SHBG a = >100. Progesterone, CBG a = ~2. Cortisol, SHBG a = >100. Cortisol, CBG a = ~3. Corticosterone, SHBG a = >100. Corticosterone, CBG a = ~5
*Affinity expressed as K, (nmol/L).
Abbreviations: CBG, corticosteroid-binding globulin; SHBG, sex hormone-binding globulin.
transport protein. Gonadal steroids bind very weakly (Table 41-8).
to CBG

Most mammals, humans included, have a plasma $-globulin that binds testosterone with specificity, relatively high affinity, and limited capacity (Table 41-8). This protein, usually called sex hormone-binding globulin (SHBG) or testosteroneestrogen-binding globulin (TEBG), is produced in the liver. Its production is increased by estrogens (women have twice the serum concentration of SHBG as men), certain types of liver disease, and hyperthyroidism; it is decreased by androgens, advancing age, and hypothyroidism. Many of these conditions also affect the production of CBG and TBG. Since SHBG and albumin bind 97 to 99% of circulating testosterone, only a small fraction of the hormone in circulation is in the free (biologically active) form. The primary function of SHBG may be to restrict the free concentration of testosterone in the serum. Testosterone binds to SHBG with higher affinity than does estradiol (Table 41-8). Therefore, a change in the level of SHBG causes a greater change in the free testosterone level than in the free estradiol level.
Estrogens are bound to SHBG and progestins to CBG. SHBG binds estradiol about five times less avidly than it binds testosterone or DHT, while progesterone and cortisol have little affinity for this protein (Table 41-8). In contrast, progesterone and cortisol bind with nearly equal affinity to CBG, which in turn has little affinity for estradiol and even less for testosterone, DHT, or estrone.
These binding proteins also provide a circulating reservoir of hormone, and because of the relatively large binding capacity, they probably buffer against sudden changes in the plasma level. Because the metabolic clearance rates of these steroids are inversely related to the affinity of their binding to SHBG, estrone is cleared more rapidly than estradiol, which in turn is cleared more rapidly than testosterone or DHT.

™ The presence of a specific receptor defines the target cells for a given hormone.
™ Receptors are proteins that bind specific hormones and generate an intracellular signal (receptor-effector coupling).
= Some hormones have intracellular receptors; others bind to receptors on the plasma membrane.
™ Hormones are synthesized from a number of precursor molecules, including cholesterol, tyrosine per se, and all the constituent amino acids of peptides and proteins.
= A number of modification processes alter the activity of
hormones.
For
example,
many
hormones
are
from
larger precursor molecules.
= The complement of enzymes in a particular cell type allows for the production of a specific class of steroid hormone.
= Most of the lipid-soluble hormones are bound to rather specific plasma transport proteins.

. Explain the roles of stimulus, hormone release, signal generation, and effector response in hormone-regulated physiologic processes.
: Describe the role of receptors and guanosine nucleotide-binding G-proteins in hormone signal transduction, particularly with regard to the generation of second messengers.
= Appreciate the complex patterns of signal transduction pathway cross-talk in mediating complicated physiologic outputs.
m= Understand the key roles that protein-ligand, protein-protein, protein posttranslational modification, and protein-DNA interactions play in mediating hormone-directed physiologic processes.
physiology. = Detail why hormone-modulated receptors, second messengers, and associated signaling molecules represent a rich source of potential drug target development given their key roles in the regulation of

The homeostatic adaptations an organism makes to a constantly changing environment are in large part accomplished through alterations of the activity and amount of proteins. Hormones provide a major means of facilitating these changes. A hormone-receptor interaction results in generation of an amplified intracellular signal that can either regulate the activity of a select set of genes that alters the amounts of certain proteins in the target cell or affect the activity of specific proteins, including enzymes, transporters, or channel proteins. Signals can influence the location of proteins in the cell, and often affects general processes such as protein synthesis, cell growth, and replication, via their effects on gene expression. Other signaling molecules—including cytokines, interleukins, growth factors, and metabolites—use some of the same general mechanisms and signal transduction pathways. Excessive, deficient, or inappropriate production and release of hormones and the other regulatory signaling molecules are major causes of disease. Many pharmacotherapeutic agents are aimed at correcting or otherwise influencing the pathways discussed in this chapter.

The general steps involved in producing a coordinated response to a particular stimulus are illustrated in Figure 42-1. The stimulus can be a challenge or a threat to the organism, to an organ, or to the integrity of a single cell within that organism. Recognition of the stimulus is the first step in the adaptive response. At the organismic level, this generally involves the nervous system and the special senses (sight, hearing, pain, smell, and touch). At the organ, tissue, or cellular level, recognition involves physicochemical factors such as pH, O, tension, temperature, nutrient supply, noxious metabolites, and osmolarity. Appropriate recognition results in the release of one or more hormones that will govern generation of the necessary adaptive response. For purposes of this discussion, the hormones are categorized as described in Table 41-4, that is, based on the location of their specific cellular receptors and the type of signals generated. Group I hormones interact with an intracellular receptor and group II hormones with receptor
a
Coordinated response to stimulus
FIGURE 42-1 Hormonal involvement in responses to a stimulus. Physiologic needs, or challenges to the integrity of the organism, elicit a response that includes the release of one or more hormones. These hormones generate signals at or within target cells and these signals regulate a variety of biologic processes that provide for a coordinated response to the stimulus or challenge. See Figure 42-8 for a specific example.
recognition sites located on the extracellular surface of the plasma membrane of target cells. The cytokines, interleukins, and growth factors should also be considered in this latter category. These molecules, of critical importance in homeostatic adaptation, are hormones in the sense that they are produced in specific cells, have the equivalent of autocrine, paracrine, and endocrine actions, bind to cell surface receptors, and activate many of the same signal transduction pathways employed by the more traditional group II hormones.

The lipophilic group I hormones diffuse through the plasma membrane of all cells but only encounter their specific, highaffinity intracellular receptors in target cells. These receptors can be located in the cytoplasm or in the nucleus of target cells. The hormone-receptor complex first undergoes an activation reaction. As shown in Figure 42-2, receptor activation occurs by at least two mechanisms. For example, glucocorticoids diffuse across the plasma membrane and encounter their cognate receptor in the cytoplasm of target cells. Ligand-receptor binding results in a conformational change in the receptor leading to the dissociation of heat shock protein 90 (hsp90). This step is necessary for subsequent nuclear localization of the glucocorticoid receptor (GR). This receptor also contains a nuclear localization sequence that is now free to assist in the translocation from cytoplasm to nucleus. The activated receptor moves into the nucleus (Figure 42-2) and binds with high affinity to a specific DNA sequence called the hormone response element (HRE). In the case of GR, this is a glucocorticoid response

FIGURE 42-2 Regulation of gene expression by two different group | hormones, thyroid hormone and glucocorticoids. The hydrophobic steroid hormones readily gain access to the cytoplasmic compartment of target cells by diffusion through the plasma membrane. Glucocorticoid hormones (solid triangles) encounter their cognate receptor (GR) in the cytoplasm where GR exists in a complex with a chaperone protein, heat shock protein 90 (hsp). Ligand binding causes dissociation of hsp90 and a conformational change of the receptor. The receptor-ligand complex then traverses the nuclear membrane and binds to DNA with specificity and high affinity at a glucocorticoid response element (GRE). This event affects the architecture of a number of transcription coregulators (green triangles), and enhanced transcription ensues. By contrast, thyroid hormones and retinoic acid (black circle) directly enter the nucleus, where their cognate heterodimeric (TR-RXR; see Figure 42-12) receptors are already bound to the appropriate response elements with an associated transcription corepressor complex (red circles). Binding of hormones occurs, which again induces conformational changes in the receptor leading to dissociation of the corepressor complex from the receptor, thereby allowing an activator complex, consisting of the TR-TRE and coactivator, to assemble. The gene is then actively transcribed.

element, or GRE. Consensus sequences for HREs are shown in Table 42-1. The DNA-bound, liganded receptor serves as a high-affinity binding target for one or more coactivator proteins, and accelerated gene transcription typically ensues when this occurs. By contrast, certain hormones such as the thyroid hormones and retinoids diffuse from the extracellular fluid across the plasma membrane and go directly into the nucleus. In this case, the cognate receptor is already bound to the HRE (the thyroid hormone response element [TRE], in this example). However, this DNA-bound receptor fails to activate transcription because it exists in complex with a corepressor. Indeed, this receptor-corepressor complex serves as a tonic repressor of gene transcription. Association of ligand with these receptors results in dissociation of the corepressor(s). The liganded receptor is now capable of binding one or more coactivators with high affinity, resulting in the recruitment of RNA polymerase II and the GTFs and activation of gene transcription as noted above for the GR-GRE complex. The relationship of hormone receptors to other nuclear receptors and to coregulators is discussed in more detail below.
By selectively affecting gene transcription and the consequent production of appropriate target mRNAs, the amounts of specific proteins are changed and metabolic processes are influenced. The influence of each of these hormones is

Glucocorticoids, HRE = GRE. Glucocorticoids, DNASequence = GGTACA nnn TGTTCT. Progestins, HRE = PRE. Progestins, DNASequence = . Mineralocorticoids, HRE = MRE. Mineralocorticoids, DNASequence = . Androgens, HRE = ARE. Androgens, DNASequence = . Estrogens, HRE = ERE. Estrogens, DNASequence = AGGTCA -TGACCT. Thyroid hormone, HRE = TRE. Thyroid hormone, DNASequence = AGGTCA n (1-5) AGGTCA. Retinoic acid, HRE = RARE. Retinoic acid, DNASequence = . Vitamin D, HRE = VDRE. Vitamin D, DNASequence = . cAMP, HRE = CRE. cAMP, DNASequence = TGACGTCA
*Letters indicate nucleotide; N means any one of the four can be used in that position. The arrows pointing in opposite directions illustrate the slightly imperfect inverted palindromes present in many HREs; in some cases, these are called "half binding sites,' or half-sites, because each binds one monomer of the receptor. The GRE, PRE, MRE, and ARE consist of the same DNA sequence. Specificity may be conferred by the intracellular concentration of the ligand or hormone receptor, by flanking DNA sequences not included in the consensus, or by other accessory elements. A second group of HREs includes those for thyroid hormones, estrogens, retinoic acid, and vitamin D. These HREs are similar except for the orientation and spacing between the half palindromes. Spacing determines the hormone specificity. VDRE (N = 3), TRE (N = 4), and RARE (N = 5) bind to direct repeats rather than to inverted repeats. Another member of the steroid receptor superfamily, the retinoid X receptor (RXR), forms heterodimers with VDR, TR, and RARE, and these constitute the functional forms of these DNA-binding transacting factors. cAMP affects gene transcription through the CRE.

FIGURE 42-3 The "information pathway." Information flows from the gene to the primary transcript to mRNA to protein. Hormones can affect any of the steps involved and can affect the rates of processing, degradation, or modification of the various products.
quite specific; generally, a given hormone directly affects <1% of the genes, mRNA, or proteins in a target cell; sometimes only a few are affected. The nuclear actions of steroid, thyroid, and retinoid hormones are quite well defined. Most evidence suggests that these hormones exert their dominant effect on modulating gene transcription, but they— and many of the hormones in the other classes discussed below—can act at any step of the "information pathway,' as illustrated in Figure 42-3, to control specific gene expression and, ultimately, a biologic response. Direct actions of steroids in the cytoplasm and on various organelles and membranes have also been described. Recently, microRNAs and IncRNAs have been implicated in mediating some of the diverse actions of hormones.

Many hormones are water soluble, have no transport proteins (and therefore have a short plasma half-life), and initiate a response by binding to a receptor located in the plasma membrane (see Tables 41-3 and 41-4). The mechanism of action of this group of hormones can best be discussed in terms of the intracellular signals they generate. These signals include cAMP (cyclic AMP; 3',5'-adenylic acid; see Figure 18-5), a nucleotide derived from ATP through the action of adenylyl cyclase; cGMP, a nucleotide formed from GTP by guanylyl cyclase; Ca**; and phosphatidylinositides; such small molecules are termed second messengers as their synthesis is triggered by the presence of the primary

FIGURE 42-4 components of the hormone receptor-G-protein effector system. Receptors that couple to effectors through G-proteins, the G-protein-coupled receptors (GPCRs), typically have seven a-helical membrane-spanning domains (here shown as long cylinders). In the absence of hormone (left), the heterotrimeric G-protein complex (a,B,y) is in an inactive guanosine diphosphate (GDP)-bound form and is probably not associated with the receptor. This complex is anchored to the plasma membrane through prenylated groups on the By subunits (wavy lines) and perhaps by myristoylated groups on a subunits (not shown). On binding of hormone (H) to the receptor, there are conformational changes within the receptor (as indicated by the tilted membrane-spanning domains) and association of the G-protein complex with the rearranged receptor—this the activates the G-protein complex. This activation results from the exchange of GDP with guanosine triphosphate (GTP) on the a subunit, after which a and By dissociate. The a subunit binds to and activates the effector (E). E can be adenylyl cyclase, Ca?*, Na*, or Cl channels (a.), or it could be a K* channel (a.), phospholipase CB (a_), or CGMP phosphodiesterase (a,); see Table 42-3. e ¥ q v The By subunit can also have direct actions on E. (Modified and reproduced, with permission, from Granner DK. In: Principles and Practice of Endocrinology and Metabolism, 2nd ed. Becker KL (editor). Lippincott, 1995.)
hormone (molecule) binding its receptor. Many of these second messengers affect gene transcription, as described in the previous paragraph; but they also influence a variety of other biologic processes, as shown in Figure 42-3, but see also Figures 42-6 and 42-8.

different genes (Table 42-3). Two parallel systems, a stimulatory (s) one and an inhibitory (i) one, converge upon a catalytic molecule (C). Each consists of a receptor, R, or R, and a regulatory G-protein complex termed G, and G,. Both G, and G, are heterotrimeric G-proteins composed of a, B, and y subunits.

Many of the group II hormones bind to receptors that couple to effectors through a guanine-binding protein (G-proteins) intermediary. These receptors typically have seven a-helical hydrophobic plasma membrane-spanning domains, here illustrated by the seven interconnected cylinders extending through the lipid bilayer in Figure 42-4. Receptors of this class, which signal through G-proteins, are known as G-protein-coupled receptors (GPCRs). To date, hundreds of GPCR genes have been identified, and represent the largest family of cell surface receptors in humans. Not surprisingly, a wide variety of responses are mediated by the GPCRs.

Cyclic AMP was the first intracellular second messenger signal identified in mammalian cells. Several components comprise a system for the generation, degradation, and action of cAMP (Table 42-2).

Different peptide hormones either stimulate (s) inhibit can or (i) the production of cAMP from adenylyl cyclase through the action of the G-proteins. G-proteins are encoded by at least 10

ACTH, HormonesThat Inhibit Adenylyl Cyclase (H I ) = Acetylcholine. ADH, HormonesThat Inhibit Adenylyl Cyclase (H I ) = α 2 -Adrenergics. β-Adrenergics, HormonesThat Inhibit Adenylyl Cyclase (H I ) = Angiotensin II. Calcitonin, HormonesThat Inhibit Adenylyl Cyclase (H I ) = Somatostatin. CRH, HormonesThat Inhibit Adenylyl Cyclase (H I ) = . FSH, HormonesThat Inhibit Adenylyl Cyclase (H I ) = . Glucagon, HormonesThat Inhibit Adenylyl Cyclase (H I ) = . hCG, HormonesThat Inhibit Adenylyl Cyclase (H I ) = . LH, HormonesThat Inhibit Adenylyl Cyclase (H I ) = . LPH, HormonesThat Inhibit Adenylyl Cyclase (H I ) = . MSH, HormonesThat Inhibit Adenylyl Cyclase (H I ) = . PTH, HormonesThat Inhibit Adenylyl Cyclase (H I ) = . TSH, HormonesThat Inhibit Adenylyl Cyclase (H I ) = 
Abbreviations: ACTH, adrenocorticotropic hormone; ADH, antidiuretic hormone; CRH, corticotropin-releasing hormone; FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; LH, luteinizing hormone; LPH, lipotropin; MSH, melanocyte-stimulating hormone; PTH, parathyroid hormone; TSH, thyroidstimulating hormone.

G s, Stimulus = . G s, Effector = . G s, Effect = . α s, Stimulus = Glucagon, β-adrenergics. α s, Effector = ↑ Adenylyl cyclase ↑ Cardiac Ca 2+ , Cl - , and Na + channels. α s, Effect = Glyconeogenesis, lipolysis, glycogenolysis Olfaction. α olf, Stimulus = Odorant. α olf, Effector = ↑ Adenylyl cyclase. α olf, Effect = . G i, Stimulus = . G i, Effector = . G i, Effect = . α i-1,2,3, Stimulus = Acetylcholine, α 2 -adrenergics. α i-1,2,3, Effector = ↓ Adenylyl cyclase ↑ Potassium channels. α i-1,2,3, Effect = Slowed heart rate. α o, Stimulus = Opioids, endorphins. α o, Effector = ↑ Potassium channels. α o, Effect = Neuronal electrical activity. α t, Stimulus = Light. α t, Effector = ↑ cGMP phosphodiesterase. α t, Effect = Vision. G q, Stimulus = . G q, Effector = . G q, Effect = . α q, Stimulus = M 1 cholinergics α 1 -Adrenergics. α q, Effector = ↑ Phospholipase C-β1. α q, Effect = ↓ Muscle contraction. α 11, Stimulus = α 1 -Adrenergics. α 11, Effector = ↑ Phospholipase C-β2. α 11, Effect = ↓ Blood pressure. G 12, Stimulus = . G 12, Effector = . G 12, Effect = . α 12, Stimulus = Thrombin. α 12, Effector = Rho. α 12, Effect = Cell shape changes

"The four major classes or families of mammalian G-proteins (G, G, G, and G,,) are based on protein sequence conservation. Representative members of each are shown, along with known stimuli, effectors, and well-defined biologic effects. Nine isoforms of adenylyl cyclase have been identified (isoforms I-IX). All isoforms are stimulated by a; a, isoforms inhibit types V and VI, and a, inhibits types | and V. At least 16 different a subunits have been identified. Source: Modified and reproduced, with permission, from Granner DK: In: Principles and Practice of Endocrinology and Metabolism, 2nd ed. Becker KL (editor). Lippincott, 1995.
Since the a subunit in G, differs from that in G, the proteins, which are distinct gene products, are designated a, and a,. The a subunits bind guanine nucleotides. The B and y subunits are likely always associated (By) and appear to function predominantly as a heterodimer. The binding of a hormone to R, or R, results in a receptor-mediated activation of G-protein, which entails the exchange of GDP by GTP on a and the concomitant dissociation of By from a.
C group of enzymes. The By complexes have been associated with K* channel stimulation and phospholipase C activation. G proteins are involved in many important biologic processes in addition to hormone action. Notable examples include olfaction (a,,,.) and vision (a,). Some examples are listed in Table 42-3. GPCRs are implicated in a number of diseases and are major targets for pharmaceutical agents.

The a, protein has intrinsic GTPase activity. The active form, a-GTP, is inactivated on hydrolysis of the GTP to GDP; the trimeric G, complex (aBy) is then reformed and is ready for another cycle of activation. Cholera and pertussis toxins catalyze the ADP ribosylation of a, and a, , (Table 42-3), respectively. In the case of a, this modification disrupts the intrinsic GTPase activity; thus, a, cannot reassociate with By and is therefore irreversibly activated. ADP ribosylation of a,_, prevents the dissociation of a, , from By, and free a,_, thus cannot be formed. a, activity in such cells is therefore unopposed. There is a large family of G-proteins, and these are part of the superfamily of GTPases. The G-protein family is classified according to sequence homology into four subfamilies, as illustrated in Table 42-3. There are 21 a, 5 8, and 8 y subunit genes. Various combinations of these subunits provide a large number of possible aBy complexes.
The a subunits and the By complex have actions independent of those on adenylyl cyclase (see Figure 42-4 and Table 42-3). Some forms of a, stimulate K* channels and inhibit Ca** channels, and some a, molecules have the opposite effects. Members of the G, family activate the phospholipase

As discussed in Chapter 38, in prokaryotic cells, cAMP binds to a specific protein called cAMP activator protein (CAP) that binds directly to DNA and influences gene expression. By contrast, in eukaryotic cells, cAMP binds to a protein kinase called protein kinase A (PKA), a heterotetrameric molecule consisting of two regulatory subunits (R) that inhibit the activity of the two catalytic subunits (C) when bound as a tetrameric complex. cAMP binding to the R,C, tetramer results in the following reaction:
The R,C, complex has no enzymatic activity, but the binding of cAMP to the R subunit induces dissociation of the R-C complex, thereby activating the latter (Figure 42-5). The active C subunit catalyzes the transfer of the y phosphate of ATP to a serine or threonine residue in a variety of proteins. The consensus PKA phosphorylation sites are -ArgArg/ Lys-X-Ser/Thrand -Arg-Lys-X-X-Ser-, where X can be any amino acid.
FIGURE 42-5 Hormonal regulation of cellular processes through cAMP-dependent protein kinase (PKA). PKA exists in an inactive form as an R,C, heterotetramer consisting of two regulatory (R) and two catalytic (C) subunits. The cAMP generated by the action of adenylyl cyclase (activated as shown in Figure 42-4) binds to the regulatory subunit of PKA. This results in dissociation of the regulatory and catalytic subunits and activation of the latter. The active catalytic subunits phosphorylate a number of target proteins on serine and threonine residues. Phosphatases remove phosphate from these residues and thus terminate the physiologic response. A phosphodiesterase can also terminate the response by converting cAMP to 5'-AMP.

Historically protein kinase activities were described as being "cAMP-dependent" or "cAMP-independent." This classification has changed, as protein phosphorylation is now recognized as being a major and ubiquitous regulatory mechanism. Several hundred protein kinases have now been described. These kinases are related in sequence and structure within the catalytic domain, but each is a unique molecule with considerable variability with respect to subunit composition, molecular weight, autophosphorylation, K_ for ATP, and substrate specificity. Both kinase and protein phosphatase activities can be targeted by interaction with specific kinase-binding proteins. In the case of PKA, such targeting proteins are termed A kinase anchoring proteins, or AKAPs. AKAPs serve as scaffolds, which localize PKA near to substrates thereby focusing PKA activity toward physiologic substrates and facilitating spatiotemporal biologic regulation while also allowing for common, shared proteins to elicit specific physiologic responses. Multiple AKAPs have been described and importantly they can bind PKA and other kinases as well as phosphatases, phosphodiesterases (which hydrolyze cAMP), and protein kinase substrates. The multifunctionality of AKAPs facilitates signaling localization, rate (production and destruction of signals), specificity, and dynamics.

The effects of cAMP in eukaryotic cells are all thought to be mediated by protein phosphorylation-dephosphorylation, principally on serine and threonine residues. The control of any of the effects of cAMP, including such diverse processes as steroidogenesis, secretion, ion transport, carbohydrate and fat metabolism, enzyme induction, gene regulation, synaptic transmission, and cell growth and replication, could be conferred by a specific protein kinase, by a specific phosphatase, or by specific substrates for phosphorylation. The array of specific substrates contributes critically to defining a target tissue, and are involved in defining the extent of a particular response within a given cell. For example, the effects of cAMP on gene transcription are mediated by the cyclic AMP response element binding protein (CREB). When CREB binds to a cAMP responsive DNA enhancer element (CRE) (see Table 42-1) in its nonphosphorylated state, it is a weak activator of transcription. However, when phosphorylated by PKA at key amino acids, CREB binds the coactivator CREB-binding protein CBP/p300 (see below) and as a result is a much more potent transcriptional activator. CBP and the related p300 contain histone acetyltransferase activities (HATs), and hence serve as chromatin-active transcriptional coregulators (see Chapters 36, 38). Interestingly, CBP/p300 can also acetylate certain transcription factors thereby stimulating their ability to bind DNA and modulate transcription.

Actions caused by hormones that increase cAMP concentration can be terminated in a number of ways, including the hydrolysis of cAMP to 5'-AMP by phosphodiesterases (see Figure 42-5). The presence of these hydrolytic enzymes ensures a rapid turnover of the signal (CAMP) and hence a rapid termination of the biologic process once the hormonal stimulus is removed. There are at least 11 known members of the phosphodiesterase family of enzymes. These are subject to regulation by their substrates, cAMP and cGMP; by hormones; and by intracellular messengers such as calcium, probably acting through calmodulin. Inhibitors of phosphodiesterase, most notably methylated xanthine derivatives such as caffeine, increase intracellular cAMP and mimic or prolong the actions of hormones through this signal.

Given the importance of protein phosphorylation, it is not surprising that regulation of the protein dephosphorylation reaction is another important control mechanism (see Figure 42-5). The phosphoprotein phosphatases are themselves subject to regulation by phosphorylation-dephosphorylation reactions and by a variety of other mechanisms, such as protein-protein interactions. In fact, the substrate specificity of the phosphoserine-phosphothreonine phosphatases may be dictated by distinct regulatory subunits whose binding is regulated hormonally. One of the best-studied roles of regulation by the dephosphorylation of proteins is that of glycogen metabolism in muscle (see Figures 18-6 to 18-8). Two major types of phosphoserinephosphothreonine phosphatases have been described. Type I preferentially dephosphorylates the § subunit of phosphorylase kinase, whereas type II dephosphorylates the a subunit. Type I phosphatase is implicated in the regulation of glycogen synthase,
phosphorylase, and phosphorylase kinase. This phosphatase is itself regulated by phosphorylation of certain of its subunits, and these reactions are reversed by the action of one of the type II phosphatases. In addition, two heat-stable protein inhibitors regulate type I phosphatase activity. Inhibitor-1 is phosphorylated and activated by cAMP-dependent protein kinases, and inhibitor-2, which may be a subunit of the inactive phosphatase, is also phosphorylated, possibly by glycogen synthase kinase-3. Phosphatases that target phosphotyrosine are also important in signal transduction (see Figure 42-8).

Cyclic GMP is made from GTP by the enzyme guanylyl cyclase, which exists in soluble and membrane-bound forms. Each of these enzyme forms has unique physiologic properties. The atriopeptins, a family of peptides produced in cardiac atrial tissues, cause natriuresis, diuresis, vasodilation, and inhibition of aldosterone secretion. These peptides (eg, atrial natriuretic factor) bind to and activate the membrane-bound form of guanylyl cyclase. This results in an increase of cGMP by as much as 50-fold in some cases, and this is thought to mediate the effects mentioned above. Other evidence links cGMP to vasodilation. A series of compounds, including nitroprusside, nitroglycerin, nitric oxide, sodium nitrite, and sodium azide, all cause smooth muscle relaxation and are potent vasodilators. These agents increase cGMP by activating the soluble form of guanylyl cyclase, and inhibitors of cGMP phosphodiesterase (eg, the drug sildenafil [Viagra]) enhance and prolong these responses. The increased cGMP activates cGMP-dependent protein kinase (PKG), which in turn phosphorylates a number of smooth muscle proteins. Presumably, this is involved in relaxation of smooth muscle and vasodilation.

Ionized calcium, Ca"*, is an important regulator of a variety of cellular processes, including muscle contraction, stimulussecretion coupling, the blood clotting cascade, enzyme activity, and membrane excitability. Ca'* is also an intracellular messenger of hormone action.

is a Ca**/proton ATPase-dependent pump that extrudes Ca'* in exchange for H*. This has a high affinity for Ca** but a low capacity and is probably responsible for fine-tuning cytosolic Ca**. Furthermore, Ca'**-ATPases pump Ca** from the cytosol to the lumen of the endoplasmic reticulum. There are three ways of changing cytosolic Ca" levels: (1) Certain hormones (class II.C, Table 41-3) by binding to receptors that are themselves Ca** channels, enhance membrane permeability to Ca'*, and thereby increase Ca** influx. (2) Hormones also indirectly promote Ca* influx by modulating the membrane potential at the plasma membrane. Membrane depolarization opens voltage-gated Ca** channels and allows for Ca** influx. (3) Ca** can be mobilized from the endoplasmic reticulum, and possibly from mitochondrial pools. An important observation linking Ca** to hormone action involved the definition of the intracellular targets of Ca** action. The discovery of a Ca®*-dependent regulator of phosphodiesterase activity provided the basis for a broad understanding of how Ca®* and cAMP interact within cells.

The calcium-dependent regulatory protein is calmodulin, a 17-kDa protein that is homologous to the muscle protein troponin C in structure and function. Calmodulin has four Ca**-binding sites, and full occupancy of these sites leads to a marked conformational change, which allows calmodulin to activate enzymes and ion channels. The interaction of Ca** with calmodulin (with the resultant change of activity of the latter) is conceptually similar to the binding of cAMP to PKA and the subsequent activation of this molecule. Calmodulin can be one of numerous subunits of complex proteins and is particularly involved in regulating various kinases and enzymes of cyclic nucleotide generation and degradation. A partial list of the enzymes regulated directly or indirectly by Ca**, probcalmodulin, is in Table 42-4.
ably through presented In addition to its effects on enzymes and ion transport, Ca**/calmodulin regulates the activity of many structural elements in cells. These include the actin-myosin complex of smooth muscle, which is under B-adrenergic control, and various microfilament-mediated processes in noncontractile cells, including cell motility, cell conformation changes, mitosis, granule release, and endocytosis.

+ Adenylyl cyclase
Ca?*-dependent protein kinases
Ca?*-Mg?*-ATPase
Ca?*-phospholipid—dependent protein kinase
Cyclic nucleotide phosphodiesterase
Some cytoskeletal proteins
Some ion channels (eg, I-type calcium channels)
Nitric oxide synthase
Phosphorylase kinase
Phosphoprotein phosphatase 2B
+ Some receptors (eg, NMDA-type glutamate receptor)

A role for Ca** in hormone action is suggested by the observations that the effect of many hormones is (1) blunted by Ca?*-free media or when intracellular calcium is depleted; (2) can be mimicked by agents that increase cytosolic Ca', such as the Ca®* ionophore A23187; and (3) influences cellular calcium flux. Again, the regulation of glycogen metabolism in liver (by vasopressin and B-adrenergic catecholamines; see Figures 18-6 and 18-7).
A number of critical metabolic enzymes are regulated by Ca', phosphorylation, or both. These include glycogen synthase, pyruvate kinase, pyruvate carboxylase, glycerol3-phosphate dehydrogenase, and pyruvate dehydrogenase among others (see Figure 19-1).

Some signal must provide communication between the hormone receptor on the plasma membrane and the intracellular
Ca** reservoirs. This is accomplished by products of phosphatidylinositol metabolism. Cell surface receptors such as those for acetylcholine, antidiuretic hormone, and a,-type catecholamines are, when occupied by their respective ligands, potent activators of phospholipase C. Receptor binding and activation of phospholipase C are coupled by the G, isoforms (Table 42-3 and Figure 42-6). Phospholipase C catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol trisphosphate (IP,) and 1,2-diacylglycerol (Figure 42-7). Diacylglycerol (DAG) is itself capable of activating protein kinase C (PKC), the activity of which also depends on Ca** (see Chapter 21 and Figures, 24-1, 24-2, and 55-1). IP,, by interacting with a specific intracellular receptor, is an effective releaser of Ca** from intracellular storage sites in the endoplasmic reticulum. Thus, the hydrolysis of phosphatidylinositol 4,5-bisphosphate leads to activation of PKC and promotes an increase of cytoplasmic Ca**. As shown in Figure 42-4, the activation of G-proteins can also have a direct action on Ca** channels. The resulting elevations of cytosolic Ca** activate Ca**-calmodulin-dependent kinases and many other Ca'*-calmodulin-dependent enzymes.

FIGURE 42-6 Certain hormone-receptor interactions result in the activation of phospholipase C (PLC). PLC activation appears to involve a specific G-protein, which also may activate a calcium channel. Phospholipase C generates inositol trisphosphate (IP,) from PIP, (phosphoinositol 4,5-bisphosphate; see Figure 42-7), which liberates stored intracellular Ca'*, and diacylglycerol (DAG), a potent activator of protein kinase C (PKC). In this scheme, the activated PKC phosphorylates specific substrates, which then alter physiologic processes. Likewise, the Ca?*-calmodulin complex can activate specific kinases, two of which are shown here. These actions result in phosphorylation of substrates, and this leads to altered physiologic responses. This figure also shows that Ca** can enter cells through voltageor ligand-gated Ca** channels. The intracellular Ca?* is also regulated through storage and release by the mitochondria and endoplasmic reticulum. (Reprinted with permission from JH Exton.)
FIGURE 42-7 Phospholipase C cleaves PIP, into diacylglycerol and inositol trisphosphate. R, generally is stearate, and R, is usually arachidonate. IP, can be dephosphorylated (to the inactive 1-1,4-P.) or phosphorylated (to the potentially active I-1,3,4,5-P,).

Steroidogenic agents—including ACTH and cAMP in the adrenal cortex; angiotensin II, K*, serotonin, ACTH, and cAMP in the zona glomerulosa of the adrenal; LH in the ovary; and LH and cAMP in the Leydig cells of the testes—have been associated with increased amounts of phosphatidic acid, phosphatidylinositol, and polyphosphoinositides (see Chapter 21) in the respective target tissues. Several other examples could be cited. The roles that Ca** and polyphosphoinositide breakdown products might play in hormone action are presented in Figure 42-6. In this scheme, the activated protein kinase C can phosphorylate specific substrates, which then alter physiologic processes. Likewise, the Ca?*-calmodulin complex can activate specific kinases. These then modify substrates and alter thereby physiologic responses.

Single protein kinases such as PKA, PKC, and Ca**-calmodulin (CaM) kinases, which result in the phosphorylation of serine and threonine residues in target proteins, play a very important role in hormone action. The discovery that the epidermal growth factor (EGF) receptor contains an intrinsic tyrosine kinase activity that is activated by the binding of the ligand EGF was an important breakthrough. The insulin and insulinlike growth factor 1 (IGF-1) receptors also contain intrinsic ligand-activated tyrosine kinase activity. Several receptors— generally those involved in binding ligands involved in growth control, differentiation, and the inflammatory response— either have intrinsic tyrosine kinase activity or are tightly associated with proteins that are tyrosine kinases. Another distinguishing feature of this class of hormone action is that these kinases preferentially phosphorylate tyrosine residues, and tyrosine phosphorylation is infrequent (<0.03% of total amino acid phosphorylation) in mammalian cells. A third distinguishing feature is that the ligand-receptor interaction that results in a tyrosine phosphorylation event initiates a cascade that
may involve
several protein
kinases, phosphatases,
other regulatory proteins.

The insulin, EGF, and IGF-1 receptors have intrinsic protein tyrosine kinase activities located in their cytoplasmic domains. These activities are stimulated when their ligands bind to the cognate receptor. The receptors are then autophosphorylated on tyrosine residues, and this phosphorylation initiates a complex series of events (summarized in simplified fashion in Figure 42-8). The phosphorylated insulin receptor next phosphorylates insulin receptor substrates (there are at least four of these molecules, called IRS 1-4) on tyrosine residues. Phosphorylated IRS binds to the Src homology 2 (SH2) domains of a variety of proteins that are directly involved in mediating different effects of insulin. One of these proteins, PI-3 kinase, links insulin receptor activation to insulin action through activation of a number of molecules, including the kinase phosphoinositide-dependent kinase 1 (PDK1). This enzyme propagates the signal through several other kinases, including PKB (also known as AKT), SKG, and aPKC (see legend to Figure 42-8 for definitions and expanded abbreviations). An alternative pathway downstream from PDK1 involves p70S6K and perhaps other as yet unidentified kinases. A second major pathway involves mTOR. This enzyme is directly regulated by amino acid levels and insulin and is essential for p70S6K activity. The mTOR-signaling system provides a distinction between the PKB and p70S6K branches downstream from PKD1. These pathways are involved in protein translocation, enzyme activation, and the regulation, by insulin, of genes involved in metabolism (Figure 42-8). Another SH2 domaincontaining protein is GRB2, which binds to IRS-1 and links tyrosine phosphorylation to several proteins, the result of which is activation ofa cascade of threonine and serine kinases. A pathway showing how this insulin-receptor interaction activates the mitogen-activated protein kinase (MAPK) pathway and the anabolic effects of insulin is illustrated in Figure 42-8. The exact roles of many of these docking proteins, kinases, and phosphatases are actively being studied.

Tyrosine kinase activation can also initiate a phosphorylation and dephosphorylation cascade that involves the action of several other protein kinases and the counterbalancing actions of phosphatases. Two mechanisms are employed to initiate this cascade. Some hormones, such as growth hormone, prolactin, erythropoietin, and the cytokines, initiate their action by activating a tyrosine kinase, but this activity is not an integral part of the hormone receptor. The hormone-receptor interaction promotes binding and activation of cytoplasmic protein tyrosine kinases, such as JAK], or JAK2 or TYK.
These kinases phosphorylate one or more cytoplasmic proteins, which then associate with other docking proteins and

FIGURE 42-8 insulin signaling pathways. The insulin signaling pathways provide an excellent example of the "recognition > hormone release — signal generation — effects" paradigm outlined in Figure 42-1. Insulin is released into the bloodstream from pancreatic B cells in response to hyperglycemia. Binding of insulin to a target cell-specific plasma membrane heterotetrameric insulin receptor (IR) results in a cascade of intracellular events. First, the intrinsic tyrosine kinase activity of the insulin receptor is activated, and marks the initial event. Receptor activation results in increased tyrosine phosphorylation (conversion of specific Y residues — Y-P) within the receptor. One or more of the insulin receptor substrate (IRS) molecules (IRS 1-4) then bind to the tyrosine-phosphorylated receptor and themselves are specifically tyrosine phosphorylated. IRS proteins interact with the activated IR via N-terminal PH (pleckstrin homology) and phosphotyrosine binding (PTB) domains. IR-docked IRS proteins are tyrosine phosphorylated and the resulting P-Y residues form the docking sites for several additional signaling proteins (ie, PI-3 kinase, GRB2, and mTOR). GRB2 and PI-3K bind to IRS P-Y residues via their SH (Src homology) domains, binding to IRS-Y-P residues leads to activation of the activity of many intracellular signaling molecules such as GTPases, protein kinases, and lipid kinases, all of which play key roles in certain metabolic actions of insulin. The two best-described pathways are shown. In detail, phosphorylation of an IRS molecule (probably IRS-2) results in docking and activation of the lipid kinase, PI-3 kinase; PI-3K generates novel inositol lipids that act as "second messenger" molecules. These, in turn, activate PDK1 and then a variety of downstream signaling molecules, including protein kinase B (PKB/AKT), SGK, and aPKC. An alternative pathway involves the activation of p70S6K and perhaps other as yet unidentified kinases. Next, phosphorylation of IRS (probably IRS-1) results in docking of GRB2/mSOS and activation of the small GTPase,

p21Ras, which initiates a protein kinase cascade that activates Raf-1, MEK, and the p42/p44 MAP kinase isoforms. These protein kinases are important in the regulation of proliferation and differentiation of many cell types. The mTOR pathway provides an alternative way of activating p70S6K and is involved in nutrient signaling as well as insulin action. Each of these cascades may influence different biologic processes, as shown (protein translocation, protein/ enzyme activity, gene transcription, cell growth). All of the phosphorylation events are reversible through the action of specific phosphatases. As an example, the lipid phosphatase PTEN dephosphorylates the product of the PI-3 kinase reaction, thereby antagonizing the pathway and terminating the signal. Representative effects of major actions of insulin are shown in each of the boxes (bottom). The asterisk after phosphodiesterase indicates that insulin indirectly affects the activity of many enzymes by activating phosphodiesterases and reducing intracellular CAMP levels. (aPKC, atypical protein kinase C; GRB2, growth factor receptor binding protein 2; IGFBP, insulin-like growth factor binding protein; IRS 1-4, insulin receptor substrate isoforms 1-4; MAP kinase, mitogen-activated protein kinase; MEK, MAP kinase and ERK kinase; mSOS, mammalian son of sevenless; mTOR, mammalian target of rapamycin; p70S6K, p70 ribosomal protein S6 kinase; PDK1, phosphoinositide-dependent kinase; PI-3 kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; PTEN, phosphatase and tensin homolog deleted on chromosome 10; SGK, serum and glucocorticoid-regulated kinase.

through binding to SH2 domains. One such interaction results in the activation of a family of cytosolic proteins called STATs, or signal transducers and activators of transcription. The phosphorylated STAT protein dimerizes and translocates into the nucleus, binds to a specific DNA element such as the interferon response element (IRE), and activates transcription. This is illustrated in Figure 42-9. Other SH2 docking events may result in the activation of PI-3 kinase, the MAP kinase pathway (through SHC or GRB2), or G-protein-mediated activation of phospholipase C (PLCy) with the attendant production of diacylglycerol and activation of protein kinase C. It is apparent that there is a potential for "cross-talk" when different hormones activate these various signal transduction pathways.
FIGURE 42-9 initiation of signal transduction by receptors linked to Jak kinases. The receptors (R) that bind prolactin, growth hormone, interferons, and cytokines lack endogenous tyrosine kinase. Upon ligand binding, these receptors dimerize and an associated, though inactive protein kinase (JAK1, JAK2, or TYK) is phosphorylated. Phospho-JAK is now activated, and proceeds to phosphorylate the receptor on tyrosine residues. The STAT proteins associate with the phosphorylated receptor and then are themselves phosphorylated by JAK-P. The phosphorylated STAT protein, STAT ®) dimerizes, translocates to the nucleus, binds to specific DNA elements, and regulates transcription. The phosphotyrosine residues of the receptor also bind to several SH2 domain-containing proteins (X-SH2), which result in activation of the MAP kinase pathway (through SHC or GRB2), PLCy, or PI-3 kinase.

The DNA-binding transcription factor NF-«B is a heterodimeric complex typically composed of two subunits termed p50 and p65 (Figure 42-10). Normally NF-« is sequestered in the cytoplasm in a transcriptionally inactive form by members of the IxB (inhibitor of NF-«) family of proteins. Extracellular stimuli such as proinflammatory cytokines, reactive oxygen species, and mitogens lead to activation of the IKK (IkB kinase) complex, which is a heterohexameric structure consisting of a, 8, and y subunits. IKK phosphorylates IkB

FIGURE 42-10 Regulation of the NF-kB pathway. NF-x8 consists of two subunits, p50 and p65, which when present in the nucleus regulates transcription of the multitude of genes important for the inflammatory response. NF-KB is restricted from entering the nucleus by IkB, an inhibitor of NF-KB. IkB binds to—and masks—the nuclear localization signal of NF-KB. This cytoplasmic protein is phosphorylated by an IKK complex which is activated by cytokines, reactive oxygen species, and mitogens. Phosphorylated IkB can be ubiquitylated and degraded, thus releasing its hold on NF-kB, and allowing for nuclear translocation. Glucocorticoids, potent anti-inflammatory agents, are thought to affect at least three steps in this process (1, 2, 3), as described in the text.
on two serine residues. This phosphorylation targets IxB for polyubiquitylation and subsequent degradation by the proteasome. Following IxB degradation, free NF-«B translocates to the nucleus, where it binds to a number of gene enhancers and activates transcription, particularly of genes involved in the inflammatory response. Transcriptional regulation by NF-KB is mediated by a variety of coactivators such as CREB-binding protein (CBP), as described below (Figure 42-13).
Glucocorticoid hormones are therapeutically useful agents for the treatment of a variety of inflammatory and immune diseases. Their anti-inflammatory and immunomodulatory actions are explained in part by the inhibition of NF-«B and its subsequent actions. Evidence for three mechanisms for the inhibition of NF-«B by glucocorticoids has been described: (1) glucocorticoids increase IkB mRNA, which leads to an increase of IxB protein and more efficient sequestration of NF-«B in the cytoplasm. (2) The glucocorticoid receptor competes with NF-«B for binding to coactivators. (3) The glucocorticoid receptor directly binds to the p65 subunit of NF-«B and inhibits its activation (Figure 42-10).

The signals generated as described above have to be translated into an action that allows the cell to effectively adapt to a challenge (Figure 42-1). Much of this adaptation is accomplished through alterations in the rates of transcription of specific genes. Many different observations have led to the current view of how hormones affect transcription. Some of these are as follows: (1) actively transcribed genes are in regions of "open" chromatin (experimentally defined as relative susceptibility to the enzyme DNase I, and containing certain histone PTMs or "marks"), which allows for the access of transcription factors to DNA. (2) Genes have regulatory regions, and transcription factors bind to these to modulate the frequency of transcription initiation. (3) The hormone-receptor complex can be one of these transcription factors. The DNA sequence to which receptors bind is called a HRE (see Table 42-1 for examples). (4) Alternatively, other hormone-generated signals can modify the location, amount, or activity of transcription factors and thereby influence binding to the regulatory or response element. (5) Members of a large superfamily of nuclear receptors act with—or in a manner analogous to—the hormone receptors described above. (6) These nuclear receptors interact with another large group of coregulatory molecules to effect changes in the transcription of specific genes.

HREs resemble enhancer elements in that they are not strictly dependent on position and location or orientation. They generally are found within a few hundred nucleotides upstream (5') of the transcription initiation site, but they may be located within the coding region of the gene, in introns. HREs were defined by the strategy illustrated in Figure 38-11. The consensus sequences illustrated in Table 42-1 were arrived at through analysis of many genes regulated by a given hormone using simple, heterologous reporter systems (see Figure 38-10). Although these simple HREs bind the hormone-receptor complex more avidly than surrounding DNA—or DNA from an unrelated source—and confer hormone responsiveness to a reporter gene, it soon became apparent that the regulatory circuitry of natural genes must be much more complicated. Glucocorticoids, progestins, mineralocorticoids, and androgens have vastly different physiologic actions. How could the specificity required for these effects be achieved through regulation of gene expression by the same HRE (Table 42-1)? Questions like this have led to experiments which have allowed for elaboration of a more complex model of transcription regulation by the steroid hormone receptor family of proteins. For example, in the vast majority of cellular genes, the HRE is found associated with other specific regulatory DNA elements (and associated binding proteins); these associations are mandatory for optimal function. The extensive sequence similarity noted between steroid hormone receptors, particularly in their DNA-binding domains (DBD), led to discovery of the nuclear receptor superfamily of proteins. These—and a large number of coregulator proteins—allow for a wide variety of DNAprotein and protein-protein interactions and the specificity necessary for highly regulated physiologic control. A schematic of such an assembly is illustrated in Figure 42-11.

The nuclear receptor superfamily consists of a diverse set of transcription factors that were discovered because of a sequence similarity in their DBDs. This family, now with >50 members, includes the nuclear hormone receptors discussed above, a number of other receptors whose ligands were discovered after the receptors were identified, and many putative or orphan receptors for which a ligand has yet to be discovered. These nuclear receptors have several common structural features (Figure 42-12). All have a centrally located DBD that allows the receptor to bind with high affinity to its cognate HRE. The DBD contains two zinc finger binding motifs (see Figure 38-14) that direct binding either as homodimers, as heterodimers (usually with a retinoid X receptor [RXR] partner), or as monomers. The target response element consists of one or two DNA half-site consensus sequences arranged as an inverted or direct repeat. The spacing between the latter helps determine binding specificity. Thus, in general, a direct repeat with three, four, or five nucleotide spacer regions specifies the binding of the vitamin D, thyroid, and retinoic acid receptors, respectively, to the same consensus response element (Table 42-1). A multifunctional ligand-binding domain (LBD) is located in the carboxyl terminal half of the receptor. The LBD binds hormones or metabolites with selectivity and thus specifies a particular biologic response. The LBD also contains domains that mediate the binding of heat shock

FIGURE 42-11 The hormone response transcriptional activation unit. The hormone response transcription unit is an assembly of DNA elements and complementary, cognate DNAbound proteins that interact, through protein-protein interactions, with a number of coactivator or corepressor molecules. An essential component is the hormone response element that is bound by the ligand (4)-bound receptor (R). Also important are the accessory factor elements (AFEs) with bound transcription factors. More than two dozen of these accessory factors (AFs), which are often members of the nuclear receptor superfamily, have been linked to hormone effects on transcription. The AFs can interact with each other, with the liganded nuclear receptors, or with coregulators. These components communicate with the basal transcription machinery, forming the polymerase II PIC (ie, RNAP II and GTFs; Figure 36-10) through a coregulator complex that can consist of one or more members of the p160, corepressor, mediator-related, or CBP/p300 families (see Table 42-6). Recall (see Chapters 36, 38) that many of the transcription coregulators carry intrinsic enzymatic activities that covalently modify the DNA, transcription proteins, and the histones present in the nucleosomes (not shown here) in and around the enhancer (HRE, AFE) and promoter. Collectively the hormone, hormone receptor, chromatin, DNA and transcription machinery integrate and process hormone signals to regulate transcription in a physiologic fashion.

proteins, dimerization, nuclear localization, and transactivation. The latter function is facilitated by the carboxyl-terminal transcription activation function, or activation domain/AD (AF-2 domain), which forms a surface required for the interaction with coactivators. A highly variable hinge region separates the DBD from the LBD. This region provides flexibility to the receptor, so it can assume different DNA-binding conformations. Finally, there is a highly variable amino-terminal region that contains another AD referred to as AF-1. The AF-1 AD likely provides for distinct physiologic functions through the binding of different coregulator proteins. This region of the receptor, through the use of different promoters, alternative splice sites, and multiple translation initiation sites, provides for receptor isoforms that share DBD and LBD identity but exert different physiologic responses because of the association of various coregulators with this variable amino terminal AF-1 AD.
It is possible to sort this large number of receptors into groups in a variety of ways. Here, they are discussed according to the way they bind to their respective DNA elements (Figure 42-12). Classic hormone receptors for glucocorticoids (GR), mineralocorticoids (MR), estrogens (ER), androgens (AR), and progestins (PR) bind as homodimers to inverted repeat sequences. Other hormone receptors such as thyroid (TR), retinoic acid (RAR), and vitamin D (VDR) and receptors that bind various metabolite ligands such as PPAR a, B, and y, FXR, LXR, PXR, and CAR bind as heterodimers, with RXR as a partner, to direct repeat sequences (see Figure 42-12 and Table 42-5). Another group of orphan receptors that as yet have no known ligand bind as homodimers or monomers to direct repeat sequences.
As illustrated in Table 42-5, the discovery of the nuclear receptor superfamily has led to an important understanding
Receptors:
Steroid
class
Binding:
Homodimers
Ligand:
Steroids
DNA
element:
Inverted
repeat

FIGURE 42-12 The nuclear receptor superfamily. Members of this family are divided into six structural domains (A-F). Domain A/B is also called AF-1, or the modulator region, because it contains an AD and is involved in activating transcription. The C domain consists of the DNA-binding domain (DBD). The D region contains the hinge, which provides flexibility between the DBD and the ligand-binding domain (LBD, region E). The C-terminal part of region E contains AF-2, another AD that also contributes importantly to activation of transcription. The F region is less well defined. The functions of these domains are discussed in more detail in the text. Receptors with known ligands, such as the steroid hormones, bind as homodimers on inverted repeat half-sites. Other receptors form heterodimers with the partner RXR on direct repeat elements. There can be nucleotide spacers of one to five bases between these direct repeats (DR1-5; see Table 42-1 for more details). Another class of receptors for which ligands have not been definitively determined (orphan receptors) bind as homodimers to direct repeats and occasionally as monomers to a single half-site.

Peroxisome,  = PPAR α. Peroxisome, Partner = RXR (DR1). Peroxisome, Ligand = Fatty acids. Peroxisome, Process Affected = Peroxisome proliferation. Proliferator-activated,  = PPAR β. Proliferator-activated, Partner = . Proliferator-activated, Ligand = Fatty acids Fatty acids. Proliferator-activated, Process Affected = . ,  = PPAR γ. , Partner = . , Ligand = . , Process Affected = Lipid and carbohydrate metabolism. ,  = . , Partner = . , Ligand = Eicosanoids, thiazolidinediones. , Process Affected = . Farnesoid X,  = FXR. Farnesoid X, Partner = RXR (DR4). Farnesoid X, Ligand = Farnesol, bile acids. Farnesoid X, Process Affected = Bile acid metabolism. Liver X,  = LXR. Liver X, Partner = RXR (DR4). Liver X, Ligand = Oxysterols. Liver X, Process Affected = Cholesterol metabolism. Xenobiotic X,  = CAR. Xenobiotic X, Partner = RXR (DR5). Xenobiotic X, Ligand = Androstanes phenobarbital xenobiotics. Xenobiotic X, Process Affected = Protection against certain drugs, toxic metabolites, and xenobiotics. ,  = PXR. , Partner = RXR (DR3). , Ligand = Pregnanes. , Process Affected = . ,  = . , Partner = . , Ligand = Xenobiotics. , Process Affected = 

*Many members of the nuclear receptor superfamily were discovered by "homology" cloning, and the corresponding ligands were subsequently identified. These ligands are not hormones in the classic sense, but they do have a similar function in that they activate specific members of the nuclear receptor superfamily. The receptors described here form heterodimers with RXR and have variable nucleotide sequences separating the direct repeat binding elements (DR1-5). These receptors regulate a variety of genes encoding cytochrome p450s (CYP), cytosolic binding proteins, and ATP-binding cassette (ABC) transporters to influence metabolism and protect cells against drugs and noxious agents.
of how a variety of metabolites and xenobiotics regulate gene expression and thus the metabolism, detoxification, and elimination of normal body products and exogenous agents such as pharmaceuticals. Not surprisingly, this area is a fertile field for investigation of new therapeutic interventions.

Chromatin remodeling (histone modifications, DNA methylation, nucleosome _ repositioning/remodeling/displacement) transcription factor modification by various enzyme activities, and the communication between the nuclear receptors and the basal transcription apparatus are accomplished by protein-protein interactions with one or more of a class of coregulator molecules. The number of these coregulator molecules now exceeds 100, not counting species variations and splice variants. The first of these to be described was the CREB-binding protein, CBP. CBP, through an aminoterminal domain, binds to phosphorylated serine 137 of CREB and mediates transactivation in response to cAMP. It thus is described as a coactivator. CBP and its close relative, p300, interact directly or indirectly with a number of DNA-binding transcription factors, including activator protein-1 (AP-1), STATs, nuclear receptors, and CREB (Figure 42-13). CBP/ p300 also binds to the p160 family of coactivators described below and to a number of other proteins, including the p90"* protein kinase and RNA helicase A. It is important to note, as mentioned above, that CBP/p300 also has intrinsic histone
FIGURE 42-13 Several signal transduction pathways converge on CBP/p300. Many ligands that associate with membrane or nuclear receptors eventually converge on CBP/p300. Several different signal transduction pathways are illustrated. (EGF, epidermal growth factor; GH, growth hormone; Prl, prolactin; TNF, tumor necrosis factor; other abbreviations are expanded in the text.)
acetyltransferase (HAT) activity. Some of the many actions of CBP/p300, which appear to depend on intrinsic enzyme activities and its ability to serve as a scaffold for the binding of other proteins, are illustrated in Figure 42-11. Other coregula- tors serve similar functions.

Several other families of coactivator molecules have been described. Members of the p160 family of coactivators, all of about 160 kDa, include (1) SRC-1 and NCoA-1; (2) GRIP 1, TIF2, and NCoA-2; and (3) p/CIP, ACTR, AIB1, RAC3, and TRAM-1 (Table 42-6). The different names for members within a subfamily often represent species variations or minor splice variants. There is about 35% amino acid iden- tity between members of the different subfamilies. The p160 coactivators share several properties. They (1) bind nuclear receptors in an agonist- and AF-2 AD-dependent manner, (2) have a conserved amino terminal basic helix-loop-helix

I. 300-kDa family of coactivators, 1 = . A. CBP, 1 = CREB-binding protein. B. p300, 1 = Protein of 300 kDa. II. 160-kDa family of coactivators, 1 = . A. SRC-1,2,3, 1 = Steroid receptor coactivator 1, 2, and 3. NCoA-1, 1 = Nuclear receptor coactivator 1. B. TIF2, 1 = Transcriptional intermediary factor 2. GRIP1, 1 = Glucocorticoid receptor- interacting protein. NCoA-2, 1 = Nuclear receptor coactivator 2. C. p/CIP, 1 = p300/CBP cointegrator- associated protein 1. ACTR, 1 = Activator of the thyroid and retinoic acid receptors. AIB, 1 = Amplified in breast cancer. RAC3, 1 = Receptor-associated coactivator 3. TRAM-1, 1 = TR activator molecule 1. III. Corepressors, 1 = . A. NCoR, 1 = Nuclear receptor corepressor. B. SMRT, 1 = Silencing mediator for RXR andTR. IV. Mediator subunits, 1 = . A. TRAPs, 1 = Thyroid hormone receptor- associated proteins. B. DRIPs, 1 = Vitamin Dreceptor- interacting proteins. C. ARC, 1 = Activator-recruited cofactor

(bHLH) motif (see Chapter 38), (3) have a weak carboxylterminal transactivation domain and a stronger aminoterminal transactivation domain in a region that is required for CBP-p160 interaction, (4) contain at least three of the LXXLL motifs required for protein-protein interaction with other coactivators, and (5) often have HAT activity. The role of HAT is particularly interesting, as mutations of the HAT domain disable many of these transcription factors. Current thinking holds that these HAT activities acetylate histones, which facilitate the remodeling of chromatin into a transcription-efficient environment. Histone acetylation/deacetylation thus plays a critical role in gene expression. Finally, it is important to note that other protein substrates for HATmediated acetylation, such as DNA-binding transcription activators and other coregulators have been reported. Such nonhistone PTM events likely also factor importantly into the overall regulatory response.
A small number of proteins, including NCoR and SMRT, comprise the corepressor family. They function, at least in part, as described in Figure 42-2. Another family includes the TRAPs, DRIPs, and ARC (Table 42-6). These proteins represent subunits of the mediator (see Chapter 36) and range in size from 80 to 240 kDa and are thought to link the nuclear receptor-coactivator complex to RNA polymerase II and the other components of the basal transcription apparatus.
The exact role of these coactivators is presently under intensive investigation. Many of these proteins have intrinsic enzymatic activities. This is particularly interesting in view of the fact that acetylation, phosphorylation, methylation, sumoylation, and ubiquitination—as well as proteolysis and cellular translocation—have been proposed to alter the activity of some of these coregulators and their targets.
It appears that certain combinations of coregulators— and thus different combinations of activators and inhibitors—are responsible for specific ligand-induced actions through various receptors. Furthermore, these interactions on a given promoter are dynamic. In some cases, complexes consisting of over 45 transcription factors have been observed on a single gene.

Hormones, cytokines, interleukins, and growth factors use a variety of signaling mechanisms to facilitate cellular adaptive responses.
@ The ligand-receptor complex serves as the initial signal for
members
of
the
nuclear
receptor
family.
™ Class II peptide/protein and catecholamine hormones,
which bind to cell surface receptors, generate a variety of
intracellular signals. These include cAMP, cGMP, Ca**,
phosphatidylinositides, and protein kinase cascades.
= Many hormone responses are accomplished through alterations
in the rate of transcription of specific genes.
@ The nuclear receptor superfamily of proteins plays a central role in the regulation of gene transcription.
hormones,
= DNA-binding nuclear receptors, which may have metabolites, or drugs as ligands, bind to specific HREs as homodimers or as heterodimers with RXR.
= Another large family of coregulator proteins remodel
chromatin, modify
other transcription
factors, and
bridge nuclear receptors to the basal transcription apparatus.

- D. Receptors that specifically recognize and bind that
particular messenger molecule
1. Regarding membrane lipids, select the one FALSE answer.
A. The major phospholipid by mass in human membranes is
generally phosphatidylcholine.
B. Glycolipids are located on the inner and outer leaflets of the plasma membrane.
C. Phosphatidic acid is a precursor of phosphatidylserine, but
not of sphingomyelin.
D. Phosphatidylcholine and phosphatidylethanolamine
are located primarily on the outer leaflet of the plasma
membrane.
E. The flip-flop of phospholipids in membranes is very slow.
2. Regarding membrane proteins, select the one FALSE answer.
A. Because of steric considerations, a-helices cannot exist in
membranes.
B. A hydropathy plot helps one to estimate whether a segment
of a protein is predominantly hydrophobic or hydrophilic.
C. Certain proteins are anchored to the outer leaflet of plasma
membranes via glycophosphatidylinositol (GPI) structures.
D. Adenylyl cyclase is a marker enzyme for the plasma
membrane.
E. Myelin has a very high content of lipid compared with protein.
3. Regarding membrane transport, select the one FALSE statement.
A. Potassium has a lower charge density than sodium and
tends to move more quickly through membranes than does sodium.
B.
The
flow
of
ions
through
ion
channels
is
an
example
of
passive transport.
C. Facilitated diffusion requires a protein transporter.
D. Inhibition of the Na*-K*-ATPase will inhibit sodium-
dependent uptake of glucose in intestinal cells.
E. Insulin, by recruiting glucose transporters to the plasma membrane, increases uptake of glucose in fat cells but not in muscle.
4. Regarding the Na*-K*-ATPase, select the one FALSE statement.
A. Its action maintains the high intracellular concentration of
sodium compared with potassium.
B. It can use as much as 30% of the total ATP expenditure of a
cell.

C. It is inhibited by digitalis, a drug that is useful in certain
cardiac conditions.
D. It is located in the plasma membrane of cells.
E. Phosphorylation is involved in its mechanism of action,
leading to its classification as a P-type ATP-driven active
transporter.
5. What molecules enable cells to respond to a specific extracellular
signaling molecule?
A. Specific receptor carbohydrates localized to the inner
plasma membrane surface
B. Plasma lipid bilayer
C.
Ion
channels
E. Intact nuclear membranes
. Indicate the term generally applied to the extracellular messenger
molecules that bind to transmembrane receptor proteins.
A. Competitive inhibitor
B. Ligand
C. Scatchard curve
D. Substrate
E. Key
. In autocrine signaling:
A. Messenger molecules reach their target cells via passage
through bloodstream.
B. Messenger molecules travel only short distances through the
extracellular space to cells that are in close proximity to the
cell that is generating the message.
C. The cell producing the messenger expresses receptors on its
surface that can respond to that messenger.
D. The messenger molecules are usually rapidly degraded and
hence can only work over short distances.
. Regardless of how a signal is initiated, the ligand-binding event is
propagated via second messengers or protein recruitment. What
is the ultimate, or final biochemical outcome of these binding events?
A. A protein in the middle of an intracellular signaling pathway
is activated.
B. A protein at the bottom of an intracellular signaling pathway
C. A protein at the top of an extracellular signaling pathway is
activated.
D. A protein at the top of an intracellular signaling pathway is
deactivated.
E. A protein at the top of an intracellular signaling pathway is
. What features of the nuclear receptor superfamily suggest that
these proteins have evolved from a common ancestor?
A. They all bind the same ligand with high affinity.
B. They all function within the nucleus.
C. They are all subject to regulatory phosphorylation.
D. They all contain regions of high amino acid sequence
similarity/identity.

E. They all bind DNA.
10. What effect does degradation of receptor-ligand complexes
after internalization have upon the ability of a cell to respond if immediately reexposed to the same hormone?
A. The cellular response is attenuated due to a decrease in
cellular receptor number.
B. Cellular response is enhanced due to reduced receptor-
ligand competition.
C. The cellular response is unchanged to subsequent stimuli.
D. Cell hormone response is now bimodal; enhanced for a
short time and thereafter inactivated.
ll. Typically, what is the first reaction after most receptor protein-
12. Where is the kinase catalytic domain of the receptor protein-
13. The subunits of the heterotrimeric G-proteins are called the
14, Of the receptors listed below, which can directly conduct a flow of
15. Which of the following is Nor a natural ligand that binds to
16. Place the events of signaling listed below in the corrECT order.
tyrosine kinases (RTKs) bind their ligand?
A. Receptor trimerization
B. Receptor degradation
C. Receptor denaturation
D. Receptor dissociation
E. Receptor dimerization
tyrosine kinases found?
A. On the extracellular surface of the receptor, immediately
adjacent to the ligand-binding domain.
B. On the cytoplasmic domain of the receptor.
C. On an independent protein that rapidly binds the receptor
upon ligand binding.
D. Within the transmembrane spanning portion of the
receptor.
__»__,
and__
subunits.
a, B, and x
a, 6, and §
a,
y,
and
6
a, 6, and y
y, 5, and n
BOOP
ions across the plasma membrane when bound to their cognate
ligand?
A. Receptor tyrosine kinases (RTKs)
B. G-protein-coupled receptors (GPCRs)
C. G-protein gamma a subunit.
D. Steroid hormone receptors
E. Ligand-gated channels
G-protein-coupled receptors?
. Hormones >

Steroid hormones
. Chemoattractants
. Opium derivatives
Neurotransmitters
MOOR
1. G-protein binds to activated receptor forming a receptor-G-
protein complex.
2.
Release of GDP by the G-protein.
3. Change in conformation of the cytoplasmic loops of the
receptor.
4. Binding of GTP by the G-protein.
5. Increase in the affinity of the receptor for a G-protein on the
cytoplasmic
surface
of
the
membrane.
6. Binding of a hormone or neurotransmitter to a G-protein-
coupled receptor.
7.
Conformational shift in the a subunit of the G-protein.
A. 6-3-5-1-2-4-7
B. 6-5-4-1-7-2-3
17. Which heterotrimeric G-proteins couple receptors to adenylyl
18.
19. Which of the following hormones termed the “flight-or-fight”
20. Which hormone is secreted by a cells in the pancreas in response
21.
22. What happens to protein kinase A (PKA) following the binding
. 6 . 6 6 moo -3- -7- -3- non -5-1-2-4 -4-7-2-1
-1-7-2-4
cyclase via the activation of GTP-bound G, subunits?
A.
G,
family
B. G,
family
C. G, D.
family
G,,,,, family
E. G, family
What must happen in order to prevent overstimulation by a
hormone?
A. Hormones must be degraded.
B. G-proteins must be recycled and then degraded.
C.
Receptors must be blocked from continuing to activate
G-proteins.
D. Receptors must dimerize.
hormone is secreted by the adrenal medulla?
A. Epinephrine
B. Oxytocin
C.
Insulin
D. Glucagon
E. Somatostatin

to low blood glucose levels?
A. Insulin
C.
Estradiol
D. Epinephrine
In liver cells, the expression of genes encoding gluconeogenic
enzymes such as phosphoenolpyruvate carboxykinase is induced
in response to which of the following molecules?
A. cGMP
B. Insulin
Cc.
ATP
D. cAMP
E. Cholesterol
of cAMP?
A. The regulatory subunits of PKA dissociate, thereby
activating the catalytic subunits.
B. PKA catalytic subunits then bind to two regulatory subunits,
thereby activating the catalytic subunits.
C.
The inhibitory regulatory subunits dissociate from the
catalytic subunits, completely inactivating the enzyme.
D. The stimulatory regulatory subunits dissociate from the
catalytic subunits, inhibiting the enzyme.
E. Phosphodiesterase binds to the catalytic subunits, which
results in enzyme inactivation.

- = Describe the digestion and absorption of carbohydrates, lipids, vitamins, and minerals.
proteins,
= Explain how requirements can be measured and estimated, and how
energy measuring the respiratory quotient permits estimation of the mix of metabolic fuels being oxidized.
= Describe the of undernutrition: marasmus, cachexia, and
consequences kwashiorkor.
= Explain how protein requirements are determined and why more of some proteins than others are required to maintain nitrogen balance.

In addition to water, the diet must provide metabolic fuels (mainly carbohydrates and lipids), protein (for growth and turnover of tissue proteins, as well as a source of metabolic fuel), fiber (for bulk in the intestinal lumen), minerals (containing elements with specific metabolic functions), and vitamins and essential fatty acids (organic compounds needed in smaller amounts for other metabolic and physiological functions). The polysaccharides, triacylglycerols, and proteins that make up the bulk of the diet must be hydrolyzed to their constituent monosaccharides, fatty acids, and amino acids, respectively, before absorption and utilization. Minerals and vitamins must be released from the complex matrix of food before they can be absorbed and utilized.
there are more overweight and obese people than undernourished people. Deficiencies of vitamin A, iron, and iodine pose major health concerns in many countries, and deficiencies of other vitamins and minerals are a major cause of ill health. In developed countries nutrient deficiency is rare, although there are vulnerable sections of the population at risk. Intakes of minerals and vitamins that are adequate to prevent deficiency may be inadequate to promote optimum health and longevity.
Globally, undernutrition is widespread, leading to impaired growth, defective immune system, and reduced work capacity. By contrast, in developed countries, and increasingly in developing countries, there is excessive food consumption, leading to obesity, and the development of diabetes, cardiovascular disease, and some cancers. Worldwide,
Excessive secretion of gastric acid, associated with Helicobacter pylori infection, can result in the development of gastric and duodenal ulcers; small changes in the composition of bile can result in crystallization of cholesterol as gallstones; failure of exocrine pancreatic secretion (as in cystic fibrosis) leads to undernutrition and steatorrhea. Lactose intolerance is the result of lactase deficiency, leading to diarrhea and intestinal discomfort when lactose is consumed. Absorption of intact peptides that stimulate antibody responses causes allergic reactions; celiac disease is an allergic reaction to wheat gluten.

The digestion of carbohydrates is by hydrolysis to liberate oligosaccharides, then free monoand disaccharides. The increase in blood glucose after a test dose of a carbohydrate compared with that after an equivalent amount of glucose (as glucose or from a reference starchy food) is known as the glycemic index. Glucose and galactose have an index of 1 (or 100%), as do lactose, maltose, isomaltose, and trehalose, which give rise to these monosaccharides on hydrolysis. Fructose and the sugar alcohols are absorbed less rapidly and have a lower glycemic index, as does sucrose. The glycemic index of starch varies between near 1 (or 100%) and near 0 as a result of variable rates of hydrolysis, and that of nonstarch polysaccharides (see Figure 15-13) is 0. Foods that have a low glycemic index are considered to be more beneficial since they cause less fluctuation in insulin secretion. Resistant starch and nonstarch polysaccharides provide substrates for bacterial fermentation in the large intestine, and the resultant butyrate and other short-chain fatty acids provide a significant source of fuel for intestinal enterocytes. There is evidence that butyrate also has antiproliferative activity, and so provides protection against colorectal cancer.

The hydrolysis of starch is catalyzed by salivary and pancreatic amylases, which catalyze random hydrolysis of a(1 > 4) glycoside bonds, yielding dextrins, then a mixture of glucose, maltose, and maltotriose and small branched dextrins (from the branchpoints in amylopectin, Figure 15-12).

The disaccharidases, maltase, sucrase-isomaltase (a bifunctional enzyme catalyzing hydrolysis of sucrose and isomaltose), lactase, and trehalase are located on the brush border of the intestinal mucosal cells, where the resultant monosaccharides and those arising from the diet are absorbed. Congenital deficiency of lactase occurs rarely in infants, leading to lactose intolerance and failure to thrive when fed on breast milk or normal infant formula. Congenital deficiency of sucrase-isomaltase occurs among the Inuit, leading to sucrose intolerance, with persistent diarrhea and failure to thrive when the diet contains sucrose.
In most mammals, and most human beings, lactase activity begins to fall after weaning and is almost completely lost by late adolescence, leading to lactose intolerance. Lactose remains in the intestinal lumen, where it is a substrate for bacterial fermentation to lactate, resulting in abdominal discomfort and diarrhea after consumption of relatively large amounts. In two population groups, people of north European origin and nomadic tribes of sub-Saharan Africa and Arabia, lactase persists after weaning and into adult life. Marine mammals secrete a high-fat milk that contains no carbohydrate, and their pups lack lactase.

Glucose and galactose are absorbed by a sodium-dependent process. They are carried by the same transport protein (SGLT 1) and compete with each other for intestinal absorption (Figure 43-1). Other monosaccharides are absorbed by carrier-mediated diffusion. Because they are not actively transported, fructose and sugar alcohols are only absorbed down their concentration gradient, and after a moderately high intake, some may remain in the intestinal lumen, acting as a substrate for bacterial fermentation. Large intakes of fructose and sugar alcohols can lead to osmotic diarrhea.

The major lipids in the diet are triacylglycerols and, to a lesser extent, phospholipids. These are hydrophobic molecules and have to be hydrolyzed and emulsified to very small droplets
FIGURE 43-1 Transport of glucose, fructose, and galactose across the intestinal epithelium. The SGLT 1 transporter is coupled to the Na*-K* pump, allowing glucose and galactose to be transported against their concentration gradients. The GLUT 5 Na*-independent facilitative transporter allows fructose, as well as glucose and galactose, to be transported down their concentration gradients. Exit from the cell for all sugars is via the GLUT 2 facilitative transporter.
(micelles, 4-6 nm in diameter) before they can be absorbed. The fat-soluble vitamins, A, D, E, and K, and a variety of other lipids (including cholesterol and carotenes) are absorbed dissolved in the lipid micelles. Absorption of carotenes and fatsoluble vitamins is impaired on a very low-fat diet.

Hydrolysis of triacylglycerols is initiated by lingual and gastric lipases, which attack the sn-3 ester bond forming 1,2-diacylglycerols and free fatty acids, which act as emulsifying agents. Pancreatic lipase is secreted into the small intestine and requires a further pancreatic protein, colipase, for activity. It is specific for the primary ester links—that is, positions 1 and 3 in triacylglycerols—resulting in 2-monoacylglycerols and free fatty acids as the major end products of luminal triacylglycerol digestion. Inhibitors of pancreatic lipase are used to inhibit triacylglycerol hydrolysis in the treatment of severe obesity. Pancreatic esterase in the intestinal lumen hydrolyzes monoacylglycerols, but they are poor substrates, and only ~25% of ingested triacylglycerol is completely hydrolyzed to glycerol and fatty acids before absorption (Figure 43-2). Bile salts, formed in the liver and secreted in the bile, permit emulsification of the products of lipid digestion into micelles together with dietary phospholipids and cholesterol secreted in the bile (about 2 g/d) as well as dietary cholesterol (about 0.5 g/d). Micelles are less than 1 um in diameter, and soluble, so they allow the products of digestion, including the fat-soluble vitamins, to be transported through the aqueous environment of the intestinal lumen to come into close contact with the brush border of the mucosal cells, allowing uptake into the epithelium. The bile salts remain in the intestinal lumen, where most are absorbed from the ileum into the enterohepatic circulation (see Chapter 26).

Within the intestinal epithelium, 1-monoacyglycerols are hydrolyzed to fatty acids and glycerol and 2-monoacylglycerols are reacylated to triacylglycerols via the monoacylglycerol pathway. Glycerol released in the intestinal lumen is absorbed into the hepatic portal vein; glycerol released within the epithelium is reutilized for triacylglycerol synthesis via the normal phosphatidic acid pathway (see Chapter 24). Long-chain fatty acids are esterified to triacylglycerol in the mucosal cells and together with the other products of lipid digestion, secreted as chylomicrons into the lymphatics, entering the bloodstream via the thoracic duct (see Chapter 25). Shortand mediumchain fatty acids are mainly absorbed into the hepatic portal vein as free fatty acids.
Cholesterol is absorbed dissolved in lipid micelles and is mainly esterified in the intestinal mucosa before being incorporated into chylomicrons. Plant sterols and stanols (in which the B ring is saturated) compete with cholesterol for esterification, but are poor substrates, so that there is an increased amount of unesterified cholesterol in the mucosal cells. Unesterified cholesterol and other sterols are actively transported out of the mucosal cells into the intestinal lumen. This means that plant sterols and stanols effectively inhibit the absorption of not only dietary cholesterol, but also the larger amount that is secreted in the bile, so lowering the whole body cholesterol content, and hence the plasma cholesterol concentration.

Native proteins are resistant to digestion because few peptide bonds are accessible to the proteolytic enzymes without prior denaturation by heat in cooking and by the action of gastric acid.

There are two main classes of proteolytic digestive enzymes (proteases), with different specificities for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases hydrolyze peptide bonds between specific amino acids throughout the molecule. They are the first enzymes to act, yielding a larger number of smaller fragments. Pepsin in the gastric juice catalyzes hydrolysis of peptide bonds adjacent to amino acids with bulky side-chains (aromatic and branched-chain amino acids and methionine). Trypsin, chymotrypsin, and elastase are secreted into the small intestine by the pancreas. Trypsin catalyzes hydrolysis of lysine and arginine esters, chymotrypsin esters of aromatic amino acids, and elastase esters of small neutral aliphatic amino acids. Exopeptidases catalyze the hydrolysis of peptide bonds, one at a time, from the ends of peptides. Carboxypeptidases, secreted in the pancreatic juice, release amino acids from the free carboxyl terminal; aminopeptidases, secreted by the intestinal mucosal cells, release amino acids from the amino terminal. Dipeptidases and tripeptidases in the brush border of intestinal mucosal cells catalyze the hydrolysis of diand tripeptides, which are not substrates for aminoand carboxypeptidases.
The proteases are secreted as inactive zymogens; the active site of the enzyme is masked by a small region of the peptide chain that is removed by hydrolysis of a specific peptide bond. Pepsinogen is activated to pepsin by gastric acid and by activated pepsin. In the small intestine, trypsinogen, the precursor of trypsin, is activated by enteropeptidase, which is secreted by the duodenal epithelial cells; trypsin can then activate chymotrypsinogen to chymotrypsin, proelastase to elastase, procarboxypeptidase to carboxypeptidase, and proaminopeptidase to aminopeptidase.

The end product of the action of endopeptidases and exopeptidases is a mixture of free amino acids, diand tripeptides, and oligopeptides, all of which are absorbed. Free amino acids are absorbed across the intestinal mucosa by sodium-dependent active transport. There are several different amino acid transporters, with specificity for the nature of the amino acid side chain (large or small, neutral, acidic, or basic). The amino acids carried by any one transporter compete with each other for absorption and tissue uptake. Dipeptides and tripeptides enter the brush border of the intestinal mucosal cells, where they are hydrolyzed
FIGURE 43-2 Digestion and absorption of triacylglycerols. The values given for percentage uptake may vary widely but indicate the relative importance of the three routes shown.
to free amino acids, which are then transported into the hepatic portal vein. Relatively large peptides may be absorbed intact, either by uptake into mucosal epithelial cells (transcellular) or by passing between epithelial cells (paracellular). Many such peptides are large enough to stimulate antibody formation—this is the basis of allergic reactions to foods.

Vitamins and minerals are released from food during digestion, although this is not complete, and the availability of vitamins and minerals depends on the type of food and, especially for minerals, the presence of chelating compounds. The fatsoluble vitamins are absorbed in the lipid micelles that are the result of fat digestion; water-soluble vitamins and most mineral salts are absorbed from the small intestine either by active transport or by carrier-mediated diffusion followed by binding to intracellular proteins to achieve concentrative uptake. Vitamin B,, absorption requires a specific transport protein, intrinsic factor (see Chapter 44); calcium absorption is dependent on vitamin D; zinc absorption requires a zinc-binding ligand secreted by the exocrine pancreas, and the absorption of iron is limited (see below).

In addition to its role in regulating calcium homeostasis, vitamin D is required for the intestinal absorption of calcium. Synthesis of the intracellular calcium-binding protein, calbindin, required for calcium absorption, is induced by vitamin D. Vitamin D also acts to recruit calcium transporters to the cell surface, so increasing calcium absorption rapidly—a process that is independent of new protein synthesis.
Phytic acid (inositol hexaphosphate) in cereals binds calcium in the intestinal lumen, preventing its absorption. Other minerals, including zinc, are also chelated by phytate. This is mainly a problem among people who consume large amounts of unleavened whole-wheat products; yeast contains an enzyme, phytase, that dephosphorylates phytate, so rendering it inactive. High concentrations of fatty acids in the intestinal lumen, as a result of impaired fat absorption, can also reduce calcium absorption by forming insoluble calcium salts; a high intake of oxalate can sometimes cause deficiency since calcium oxalate is insoluble.

Although iron deficiency is a common problem in both developed and developing countries, about 10% of the population are genetically at risk of iron overload (hemochromatosis), and in order to reduce the risk of adverse effects of nonenzymic generation of free radicals by iron salts, absorption is strictly regulated. Inorganic iron is transported into the mucosal cell by a proton-linked divalent metal ion transporter, and accumulated intracellularly by binding to ferritin. Iron leaves the mucosal cell via a transport protein ferroportin, but only if there is free transferrin in plasma to bind to. Once transferrin is saturated with iron, any that has accumulated in the mucosal cells is lost when the cells are shed. Expression of the ferroportin gene (and possibly also that for the divalent metal ion transporter) is downregulated by hepcidin, a peptide secreted by the liver when body iron reserves are adequate. In response to hypoxia, anemia, or hemorrhage, the synthesis of hepcidin is reduced, leading to increased synthesis of ferroportin and increased iron absorption (Figure 43-3). As a result of this
mucosal barrier, only ~10% of dietary iron is absorbed, and only 1 to 5% from many plant foods. Inorganic iron is absorbed in the Fe** (reduced) state, and hence, the presence of reducing agents enhances absorption. The most effective compound is vitamin C, and while intakes of 40 to 80 mg of vitamin C per day are more than adequate to meet requirements, an intake of 25 to 50 mg per meal enhances iron absorption, especially when iron salts are used to treat iron deficiency anemia. Alcohol and fructose also enhance iron absorption. Heme iron from meat is absorbed separately and is considerably more available than inorganic iron. However, the absorption of both inorganic and heme iron is impaired by calcium—a glass of milk with a meal significantly reduces iron availability.

After the provision of water, the body's first requirement is for metabolic fuels—fats, carbohydrates, and amino acids from proteins. Food intake in excess of energy expenditure leads to obesity, while intake less than expenditure leads to emaciation and wasting, marasmus, and kwashiorkor. Both obesity and severe undernutrition are associated with increased mortality. The body mass index = weight (in kg)/height? (in m) is commonly used as a way of expressing relative obesity; a desirable range is between 20 and 25.

Energy expenditure can be determined directly by measuring heat output from the body, but is normally estimated indirectly from the consumption of oxygen. There is an energy expenditure of ~20 kJ/L of oxygen consumed, regardless of whether the fuel being metabolized is carbohydrate, fat, or protein (see Table 14-1).
Measurement of the ratio of the volume of carbon dioxide produced: volume of oxygen consumed (respiratory quotient [RQ]) is an indication of the mixture of metabolic fuels being oxidized (see Table 14-1).
A more recent technique permits estimation of total energy expenditure over a period of 1 to 3 weeks, using dual isotopically labeled water, *H,'*O. *H is lost from the body only in water, while '*O is lost in both water and carbon dioxide; the difference in the rate of loss of the two labels permits estimation of total carbon dioxide production, and hence oxygen consumption and energy expenditure (Figure 43-4).
Basal metabolic rate (BMR) is the energy expenditure by the body when at rest, but not asleep, under controlled conditions of thermal neutrality, measured about 12 hours after the last meal, and depends on weight, age, and gender. Total energy expenditure depends on the BMR, the energy required for physical activity, and the energy cost of synthesizing reserves in the fed state. It is therefore possible to estimate an individual's energy requirement from body weight,
FIGURE 43-4 Dual isotopically labeled water for estimation of energy expenditure.
age, gender, and level of physical activity. Body weight affects BMR because there is a greater amount of active tissue in a larger body. The decrease in BMR with increasing age, even when body weight remains constant, is the result of muscle tissue replacement by adipose tissue, which is metabolically less active. Similarly, women have a significantly lower BMR than do men of the same body weight and age because women's bodies contain proportionally more adipose tissue.

The most useful way of expressing the energy cost of physical activities is as a multiple of BMR. This is known as the physical activity ratio (PAR) or metabolic equivalent of the task (MET). Sedentary activities use only about 1.1 to 1.2 x BMR. By contrast, vigorous exertion, such as climbing stairs, cross-country walking uphill, etc, may use 6 to 8 x BMR. The overall physical activity level (PAL) is the sum of the PAR of different activities, multiplied by the time taken for that activity, divided by 24 hours.

There is a considerable increase in metabolic rate after a meal (diet-induced thermogenesis). A small part of this is the energy cost of secreting digestive enzymes and of active transport of the products of digestion; the major part is the energy cost of synthesizing reserves of glycogen, triacylglycerol, and protein.

Marasmus can occur in both adults and children and occurs in vulnerable groups of all populations. Kwashiorkor affects only children and has been reported only in developing countries.
The distinguishing feature of kwashiorkor is that there is fluid retention, leading to edema, and fatty infiltration of the liver. Marasmus is a state of extreme emaciation; it is the outcome of prolonged negative energy balance. Not only have the body's fat reserves been exhausted, but there is wastage of muscle as well, and as the condition progresses, there is loss of protein from the heart, liver, and kidneys. The amino acids released by the catabolism of tissue proteins are used as a source of metabolic fuel and as substrates for gluconeogenesis to maintain a supply of glucose for the brain and red blood cells (see Chapter 20). As a result of the reduced synthesis of proteins, there is impaired immune response and increased risk of infection. Impairment of cell proliferation in the intestinal mucosa occurs, resulting in reduction in the surface area of the intestinal mucosa, and reduction in the absorption of such nutrients as are available.

Patients with advanced cancer, HIV infection and AIDS, and a number of other chronic diseases are frequently undernourished, a condition called cachexia. Physically, they show all the signs of marasmus, but there is considerably more loss of body protein than occurs in starvation. Unlike marasmus, where protein synthesis is reduced, but catabolism is unaffected, in cachexia the secretion of cytokines in response to infection and cancer increases the catabolism of tissue protein by the ATP-dependent ubiquitin-proteasome pathway, so increasing energy expenditure. Patients are hypermetabolic, that is, they have a considerably increased BMR. In addition to activation of the ubiquitin-proteasome pathway of protein catabolism, three other factors are involved. Many tumors metabolize glucose anaerobically to release lactate. This is then used for gluconeogenesis in the liver, which is energy consuming with a net cost of six ATP for each mol of glucose cycled (see Figure 19-4). There is increased stimulation of mitochondrial uncoupling proteins by cytokines leading to thermogenesis and increased oxidation of metabolic fuels. Futile cycling of lipids occurs because hormone-sensitive lipase is activated by a proteoglycan secreted by tumors, resulting in liberation of fatty acids from adipose tissue and ATP-expensive reesterification to triacylglycerols in the liver, which are exported in very-low-density lipoprotein (VLDL).

In addition to the wasting of muscle tissue, loss of intestinal mucosa and impaired immune responses seen in marasmus, children with kwashiorkor show a number of characteristic features. The defining feature is edema, associated with a decreased concentration of plasma proteins. In addition, there is enlargement of the liver as a result of accumulation of fat. It was formerly believed that the cause of kwashiorkor was a lack of protein, with a more or less adequate energy intake;
however, analysis of the diets of affected children shows that this is not so. Protein deficiency leads to stunting of growth, and children with kwashiorkor are less stunted than those with marasmus. Furthermore, the edema begins to improve early in treatment, when the child is still receiving a low-protein diet. Very commonly, an infection precipitates kwashiorkor. Superimposed on general food deficiency, there is probably a deficiency of antioxidant nutrients such as zinc, copper, carotene, and vitamins C and E. The respiratory burst in response to infection leads to the production of oxygen and halogen free radicals as part of the cytotoxic action of stimulated macrophages. This added oxidant stress triggers the development of kwashiorkor.

The continual catabolism of tissue proteins creates the requirement for dietary protein, even in an adult who is not growing; although some of the amino acids released can be
reutilized, much is used for gluconeogenesis in the fasting state. Nitrogen balance studies show that the average daily requirement is 0.66 g of protein per kg body weight (giving a reference intake of 0.825 g of protein/kg body weight, allowing for individual variation); ~55 g/d, or 8 to 9% of energy intake. Average intakes of protein in developed countries are of the order of 80 to 100 g/d, that is, 14 to 15% of energy intake. Because growing children are increasing the protein in the body, they have a proportionally greater requirement than adults and should be in positive nitrogen balance. Even so, the need is relatively small compared with the requirement for protein turnover. In some countries, protein intake is inadequate to meet these requirements, resulting in stunting of growth. There is little or no evidence that athletes and body builders require large amounts of protein; simply consuming more of a normal diet providing about 14% of energy from protein will provide more than enough protein for increased muscle protein synthesis—the main requirement is for an increased energy intake to permit increased protein synthesis.

One of the metabolic reactions to a major trauma, such as a burn, a broken limb, or surgery, is an increase in the net catabolism of tissue proteins, both in response to cytokines and glucocorticoid hormones, and as a result of excessive utilization of threonine and cysteine in the synthesis of acute-phase proteins. As much as 6 to 7% of the total body protein may be lost over 10 days. Prolonged bed rest results in considerable loss of protein because of atrophy of muscles. Protein catabolism may be increased in response to cytokines, and without the stimulus of exercise it is not completely replaced. Lost protein is replaced during convalescence, when there is positive nitrogen balance. Again, as in the case of athletes, a normal diet is adequate to permit this replacement protein synthesis.

nitrogen balance since there will not be enough of that amino acid for protein synthesis.
Two amino acids, cysteine and tyrosine, can be synthesized in the body, but only from essential amino acid precursors— cysteine from methionine and tyrosine from phenylalanine. The dietary intakes of cysteine and tyrosine thus affect the requirements for methionine and phenylalanine. The remaining 11 amino acids in proteins are considered to be nonessential or dispensable since they can be synthesized as long as there is enough total protein in the diet. If one of these amino acids is omitted from the diet, nitrogen balance can still be maintained. However, only three amino acids, alanine, aspartate, and glutamate, can be considered to be truly dispensable; they are synthesized by transamination of common metabolic intermediates (pyruvate, oxaloacetate, and ketoglutarate, respectively). The remaining amino acids are considered as nonessential, but under some circumstances the requirement may outstrip the capacity for their synthesis.

■, 1 = Digestion involves hydrolyzing food molecules into smaller molecules for absorption through the gastrointestinal epithelium. Polysaccharides are absorbed as monosaccharides, triacylglycerols as 2-monoacylglycerols, fatty acids and glycerol, and proteins as amino acids and small peptides.. ■, 1 = Digestive disorders arise as a result of (1) enzyme deficiency, for example, lactase and sucrase; (2) malabsorption, for example, of glucose and galactose as a result of defects in the Na + -glucose cotransporter (SGLT 1); (3) absorption of unhydrolyzed polypeptides leading to immune responses, for example, as in celiac disease; and (4) precipitation of cholesterol from bile as gallstones.. ■, 1 = In addition to water, the diet must provide metabolic fuels (carbohydrate and fat) for body growth and activity, protein for synthesis of tissue proteins, fiber for bulk in the intestinal contents, minerals for specific metabolic functions (see Chapter 44), polyunsaturated fatty acids of the n -3 and n -6 families, and vitamins-organic compounds needed in small amounts for other essential functions (see Chapter 44).. ■, 1 = Undernutrition occurs in two extreme forms: marasmus, in adults and children, and kwashiorkor in children. Chronic illness can also lead to undernutrition (cachexia) as a result of hypermetabolism.. ■, 1 = Overnutrition leads to excess energy intake and is associated with chronic noncommunicable diseases such as obesity, type 2 diabetes, atherosclerosis, cancer, and hypertension.. ■, 1 = Twenty different amino acids are required for protein synthesis, of which nine are essential in the human diet. The quantity of protein required can be determined by studies of nitrogen balance and is affected by protein quality-the amounts of essential amino acids present in dietary proteins compared with the amounts required for tissue protein synthesis.

- . Describe how reference intakes for vitamins and minerals are determined
and explain why reference intakes published by different national and international
authorities differ.
- . Define a vitamin and describe the metabolism, principal functions, deficiency
diseases associated
with inadequate
intake, and
the toxicity
of excessive
intakes of
the vitamins.
- m= Explain why mineral salts are required in the diet.

Vitamins are a group of organic nutrients, required in small quantities for a variety of biochemical functions that, generally, cannot be synthesized by the body and must therefore be supplied in the diet.
The lipid-soluble vitamins are hydrophobic compounds that can be absorbed efficiently only when there is normal fat absorption. Like other lipids, they are transported in the blood in lipoproteins or attached to specific binding proteins. They have diverse functions—for example, vitamin A, vision and cell differentiation; vitamin D, calcium and phosphate metabolism, and cell differentiation; vitamin E, antioxidant; and vitamin K, blood clotting. As well as dietary inadequacy, conditions affecting the digestion and absorption of the lipid-soluble vitamins, such as a very-low-fat diet, steatorrhea, and disorders of the biliary system, can all lead to deficiency syndromes, including night blindness and xerophthalmia (vitamin A); rickets in young children and osteomalacia in adults (vitamin D); neurologic disorders and hemolytic anemia of the newborn (vitamin E); and hemorrhagic disease of the newborn (vitamin K). Toxicity can result from excessive intake of vitamins A and D. Vitamin A and the carotenes (many of which are precursors of vitamin A), and vitamin E are antioxidants (see Chapter 45) and have possible roles in prevention of atherosclerosis and cancer, although in excess they may also act as damaging pro-oxidants.
The water-soluble
vitamins are
vitamins
B
and
C, folic
acid, biotin,
enzyme and
pantothenic cofactors.
units.
Deficiency acid;
Folic acid
of
a
single acts
as vitamin
they function
a
carrier of
mainly as
of one-carbon
the
B
complex is

rare since poor diets are most often associated with multiple deficiency states. Nevertheless, specific syndromes are characteristic of deficiencies of individual vitamins, for example, beriberi (thiamin); cheilosis, glossitis, seborrhea (riboflavin); pellagra (niacin); megaloblastic anemia, methylmalonic aciduria, and pernicious anemia (vitamin B,,); megaloblastic anemia (folic acid); and scurvy (vitamin C). Inorganic mineral elements that have a function in the body must be provided in the diet. When the intake is insufficient, deficiency signs may arise, for example, anemia (iron), and cretinism and goiter (iodine). Excessive intakes may be toxic.

For any nutrient, there is a range of intakes between that which is clearly inadequate, leading to clinical deficiency disease, and that which is so much in excess of the body's metabolic capacity that there may be signs of toxicity. Between these two extremes is a level of intake that is adequate for normal health and the maintenance of metabolic integrity. Requirements are determined in depletion/repletion studies, in which people are deprived of the nutrient until there is a metabolic change, then repleted with the nutrient until the abnormality is normalized. Individuals do not all have the same requirement for nutrients, even when calculated on the basis of body size or energy expenditure. There is a range of individual requirements of up to 25% around the mean. Therefore, in order to
assess the
level adequacy
of fers
intake of
diets, high
enough from
deficiency that
individual normal
it to
or is
at is
necessary ensure
risk requirements
fashion
a
range of
+2
to that
no of
toxicity.
are distributed
around the
observed
x
includes the
requirements ence
or set
one
If it
a
reference either
suf- is
assumed in
a
statistically mean
requirement, deviation
of recommended
requirement
(SD)
95%
the mean
of intakes
plus
2
x
the population.
are therefore
SD, and
so meet
Refer- set
at the
average or
exceed the
require- ments
of
97.5%
of the
Reference and recommended intakes of vitamins and minerals published by different national and international authorities differ because of different interpretations of the available data, and the availability of new experimental data in more recent publications.

A vitamin is defined as an organic compound that is required in the diet in small amounts for the maintenance of normal metabolic integrity. Deficiency causes a specific disease, which is cured or prevented only by restoring the vitamin to the diet (Table 44-1). However, vitamin D, which is formed in the skin from 7-dehydrocholesterol on exposure to sunlight, and niacin, which can be formed from the essential amino acid tryptophan, do not strictly comply with this definition.

Retinoids comprise retinol, retinaldehyde, and retinoic acid (preformed vitamin A, found only in foods of animal origin); carotenoids, found in plants, are a variety of carotenes and related compounds; many are precursors of vitamin A, as they can be cleaved to yield retinaldehyde, then retinol and retinoic acid (Figure 44-1). The a-, B-, and y-carotenes and cryptoxanthin are quantitatively the most important provitamin A carotenoids. B-Carotene and other provitamin A carotenoids are cleaved in the intestinal mucosa by carotene dioxygenase, yielding retinaldehyde, which is reduced to retinol, esterified and secreted in chylomicrons together with esters formed from dietary retinol. The intestinal activity of carotene dioxygenase is low, so that a relatively large proportion of ingested B-carotene may appear in the circulation unchanged. There are two isoenzymes of carotene dioxygenase. One catalyzes cleavage of the central bond of B-carotene; the other catalyzes asymmetric cleavage leading to the formation of 8'-, 10'-, and 12'-apo-carotenals, which are oxidized to retinoic acid, but cannot be used as sources of retinol or retinaldehyde.
Although it would appear that one molecule of 6-carotene should yield two of retinol, this is not so in practice; 6 ug of B-carotene is equivalent to 1 ug of preformed retinol. The total amount of vitamin A in foods is therefore expressed as micrograms of retinol equivalents = 1g preformed vitamin A + 1/6 x ug B-carotene + 1/12 x ug other provitamin A carotenoids. Before pure vitamin A was available for chemical analysis, the vitamin A content of foods was determined by biological assay and the results expressed as international units (IU). 11U =0.3 ug retinol; 1 pg retinol = 3.33 IU. Although obsolete, IU is sometimes still used in food labeling. The term retinol

FIGURE 44-1 8-Carotene and the major vitamin A vitamers. Asterisk shows the site of symmetrical cleavage of B-carotene by carotene dioxygenase, to yield retinaldehyde.
activity equivalent takes account of the incomplete absorption and metabolism of carotenoids; 1 RAE = 1 pg all-transretinol, 12 ug b-carotene, 24 tg a-carotene or B-cryptoxanthin. On this basis, 1 IU of vitamin A activity is equal to 3.6 ug B-carotene or 7.2 ug of other provitamin A carotenoids.

In the retina, retinaldehyde functions as the prosthetic group of the light-sensitive opsin proteins, forming rhodopsin (in rods) and iodopsin (in cones). Any one cone cell contains only one type of opsin and is sensitive to only one color. In the pigment epithelium of the retina, all-trans-retinol is isomerized to 11-cis-retinol and oxidized to 11-cis-retinaldehyde. This reacts with a lysine residue in opsin, forming the holoprotein rhodopsin. As shown in Figure 44-2, the absorption of light by rhodopsin causes isomerization of the retinaldehyde from 11-cis to all-trans, and a conformational change in opsin. This results in the release of retinaldehyde from the protein, and the initiation of a nerve impulse. The formation of the initial excited form of rhodopsin, bathorhodopsin, occurs within picoseconds of illumination. There is then a series of conformational changes leading to the formation of metarhodopsin II, which initiates a guanine nucleotide amplification cascade and then a nerve impulse. The final step is hydrolysis to release all-trans-retinaldehyde and opsin. The key to initiation of the visual cycle is the availability of 11-cis-retinaldehyde, and hence vitamin A. In deficiency, both the time taken to adapt to darkness and the ability to see in poor light are impaired.

A major role of vitamin A is in the control of cell differentiation and turnover. All-trans-retinoic acid and 9-cis-retinoic acid (Figure 44-1) regulate growth, development, and tissue differentiation; they have different actions in different tissues. Like the thyroid and steroid hormones and vitamin D, retinoic acid binds to nuclear receptors that bind to response elements of DNA and regulate the transcription of specific genes. There are two families of nuclear retinoid receptors: the retinoic acid receptors (RAR) bind all-trans-retinoic acid or 9-cis-retinoic acid, and the retinoid X receptors (RXR) bind 9-cis-retinoic acid. RXRs also form dimers with vitamin D, thyroid, and other nuclear acting hormone receptors. Deficiency of vitamin A impairs vitamin D and thyroid hormone function because of lack of 9-cis-retinoic acid to form active receptor dimers. Unoccupied RXRs form dimers with occupied vitamin D and thyroid hormone receptors, but not only do these not activate gene expression, they may repress it, so that vitamin A deficiency has a more severe effect on vitamin D and thyroid hormone function than simply failure to activate gene expression. Excessive vitamin A also impairs vitamin D and thyroid hormone function, because of formation of RXR homodimers, meaning that there are not enough
L_
FIGURE 44-2 The role of retinaldehyde in the visual cycle.
RXR available to form heterodimers with the vitamin D and thyroid hormone receptors.

Vitamin A deficiency is the most important preventable cause of blindness. The earliest sign of deficiency is a loss of sensitivity to green light, followed by impairment to adapt to dim light, then night blindness, an inability to see in the dark. More prolonged deficiency leads to xerophthalmia: keratinization of the cornea, and blindness. Vitamin A also has an important role in differentiation of immune system cells, and even mild deficiency leads to increased susceptibility to infectious diseases. The synthesis of retinol-binding protein, which is required to transport the vitamin in the bloodstream, is reduced in response to infection (it is a negative acute phase protein), decreasing the circulating concentration of the vitamin, and further impairing immune responses.

There is only a limited capacity to metabolize vitamin A, and excessive intakes lead to accumulation beyond the capacity of intracellular binding proteins; unbound vitamin A causes membrane lysis and tissue damage. Symptoms of toxicity affect the central nervous system (headache, nausea, ataxia, and anorexia, all associated with increased cerebrospinal fluid pressure); the liver (hepatomegaly with histological changes and hyperlipidemia); calcium homeostasis (thickening of the long bones, hypercalcemia, and calcification of soft tissues); and the skin (excessive dryness, desquamation, and alopecia).

Vitamin D is not strictly a vitamin since it can be synthesized in the skin, and under most conditions this is the major source of the vitamin. Only when sunlight exposure is inadequate is a dietary source required. Its main function is in the regulation of calcium absorption and homeostasis; most of its actions are mediated by way of nuclear receptors that regulate gene expression. It also has a role in regulating cell proliferation and differentiation. There is evidence that intakes considerably higher than are required to maintain calcium homeostasis reduce the risk of insulin resistance, obesity, and the metabolic syndrome, as well as various cancers. Deficiency, leading to rickets in children and osteomalacia in adults, continues to be a problem in northern latitudes, where sunlight exposure is inadequate.

7-Dehydrocholesterol (an intermediate in the synthesis of cholesterol that accumulates in the skin) undergoes a nonenzymic reaction on exposure to ultraviolet light, yielding previtamin D (Figure 44-3). This undergoes a further reaction over a period of hours to form cholecalciferol, which is absorbed into the bloodstream. In temperate climates, the plasma concentration of vitamin D is highest at the end of summer and lowest at the end of winter. Beyond latitudes about 40° north or south, there is very little ultraviolet radiation of the appropriate wavelength in winter.

Cholecalciferol, either synthesized in the skin or from food, undergoes two hydroxylations to yield the active metabolite, 1,25-dihydroxyvitamin D or calcitriol (Figure 44-4). Ergocalciferol from fortified foods undergoes similar hydroxylation to
FIGURE 44-3 The synthesis of vitamin D in the skin.
yield ercalcitriol. In the liver, cholecalciferol is hydroxylated to form the 25-hydroxy derivative, calcidiol. This is released into the circulation bound to a vitamin D-binding globulin, which is the main storage form of the vitamin. In the kidney, calcidiol undergoes either 1-hydroxylation to yield the active metabolite 1,25-dihydroxyvitamin D (calcitriol), or 24-hydroxylation to yield a probably inactive metabolite, 24,25-dihydroxyvitamin D (24-hydroxycalcidiol). Some tissues, other than those that are involved in calcium homeostasis, take up calcidiol from the circulation and synthesize calcitriol that acts within that cell.
in insulin secretion, synthesis and secretion of parathyroid and thyroid hormones, inhibition of production of interleukin by activated T-lymphocytes and of immunoglobulin by activated B-lymphocytes, differentiation of monocyte precursor cells, and modulation of cell proliferation. In most of these actions, it acts like a steroid hormone, binding to nuclear receptors and enhancing gene expression, although it also has a rapid effect to mobilize calcium transporters in the intestinal mucosa.

The main function of vitamin D is in the control of calcium homeostasis, and in turn, vitamin D metabolism is regulated by factors that respond to plasma concentrations of calcium and phosphate. Calcitriol acts to reduce its own synthesis by inducing the 24-hydroxylase and repressing the 1-hydroxylase in the kidney. The principal function of vitamin D is to maintain the plasma calcium concentration. Calcitriol achieves this in three ways: it increases intestinal absorption of calcium; it reduces excretion of calcium (by stimulating reabsorption in the distal renal tubules); and it mobilizes bone mineral. In addition, calcitriol is involved
There is growing evidence that higher vitamin D status is protective against various cancers, including prostate and colorectal cancer, and also against prediabetes and the metabolic syndrome. Desirable levels of intake may be considerably higher than current reference intakes, and certainly could not be met from unfortified foods. While increased sunlight exposure would meet the need, it carries the risk of developing skin cancer.

In the vitamin D deficiency disease rickets, the bones of children are undermineralized as a result of poor absorption of
FIGURE 44-4 Metabolism of vitamin D.
calcium. Similar problems occur as a result of deficiency during the adolescent growth spurt. Osteomalacia in adults results from the demineralization of bone, especially in women who have little exposure to sunlight, especially after several pregnancies. Although vitamin D is essential for prevention and treatment of osteomalacia in the elderly, there is less evidence that it is beneficial in treating osteoporosis.

Some infants are sensitive to intakes of vitamin D as low as 50 yg/d, resulting in an elevated plasma concentration of calcium. This can lead to contraction of blood vessels, high blood pressure, and calcinosis—the calcification of soft tissues. In at least a few cases, hypercalcemia in response to low intakes of vitamin D is due to genetic defects of calcidiol 24-hydroxylase, the enzyme that leads to inactivation of the vitamin. Although excess dietary vitamin D is toxic, excessive exposure to sunlight does not lead to vitamin D poisoning, because there is a limited capacity to form the precursor, 7-dehydrocholesterol, and prolonged exposure of previtamin D to sunlight leads to formation of inactive compounds.

No unequivocal unique function for vitamin E has been defined. It acts as a lipid-soluble antioxidant in cell membranes, where many of its functions can be provided by synthetic antioxidants, and is important in maintaining the fluidity of cell membranes. It also has a (relatively poorly defined) role in cell signaling. Vitamin E is the generic descriptor for two families of compounds, the tocopherols and the tocotrienols (Figure 44-5). The different vitamers have different biologic potency; the most active is D-a-tocopherol, and it is usual to express vitamin E intake in terms of milligrams p-a-tocopherol equivalents. Synthetic DL-a-tocopherol does not have the same biologic potency as the naturally occurring compound.

The main function of vitamin E is as a chain-breaking, free radical-trapping antioxidant in cell membranes and plasma lipoproteins by reacting with the lipid peroxide radicals formed by peroxidation of polyunsaturated fatty acids (see Chapter 45). The tocopheroxyl radical is relatively unreactive, and ultimately forms nonradical compounds. Commonly, the tocopheroxyl radical is reduced back to tocopherol by reaction with vitamin C from plasma. The resultant, stable, monodehydroascorbate radical then undergoes enzymic or nonenzymic reaction to yield ascorbate and dehydroascorbate, neither of which is a radical.
FIGURE 44-5 Vitamin E vitamers. In a-tocopherol and tocotrienol Ry R,, and R, are all -CH, groups. In the B-vitamers R, is H, in the y-vitamers R, is H, and in the 6-vitamers R, and R, are both H.

In experimental animals, vitamin E deficiency results in resorption of fetuses and testicular atrophy. Dietary deficiency of vitamin E in human beings is unknown, although patients with severe fat malabsorption, cystic fibrosis, and some forms of chronic liver disease suffer deficiency because they are unable to absorb or transport the vitamin, leading to nerve and muscle membrane damage. Premature infants are born with inadequate reserves of the vitamin. The erythrocyte membranes are abnormally fragile as a result of lipid peroxidation, leading to hemolytic anemia.

Vitamin K was discovered as a result of investigations into the cause of a bleeding disorder, hemorrhagic (sweet clover) disease of cattle and of chickens fed on a fat-free diet. The missing factor in the diet of the chickens was vitamin K, while the cattle feed contained dicumarol, an antagonist of the vitamin. Antagonists of vitamin K are used to reduce blood coagulation in patients at risk of thrombosis; the most widely used is warfarin.
Three compounds
have the
biological activity
of vitamin
K
(Figure 44-6): phylloquinone, the normal dietary source, found in green vegetables; menaquinones, synthesized by intestinal bacteria, with differing lengths of side chain; and menadione and menadiol diacetate, synthetic compounds that can be metabolized to phylloquinone. Menaquinones are absorbed to some extent, but it is not clear to what extent they are biologically active as it is possible to induce signs of vitamin K deficiency simply by feeding a phylloquinonedeficient diet, without inhibiting intestinal bacterial action.
Vitamin K Is the Coenzyme for Carboxylation of Glutamate in Postsynthetic Modification of Calcium-Binding Proteins
Vitamin K is the cofactor for the carboxylation of glutamate residues in the postsynthetic modification of proteins to form the unusual amino acid y-carboxyglutamate (Gla) (Figure 44-7).
FIGURE 44-6 The vitamin K vitamers. Menadiol (or menadione) and menadiol diacetate are synthetic compounds that are converted to menaquinone in the liver.
Initially, vitamin K hydroquinone is oxidized to the epoxide, which activates a glutamate residue in the protein substrate to a carbanion, which reacts nonenzymically with carbon dioxide to form y-carboxyglutamate. Vitamin K epoxide is reduced to the quinone by a warfarin-sensitive reductase, and the quinone
FIGURE 44-7 The role of vitamin K in the synthesis of y-carboxyglutamate.

is reduced to the active hydroquinone by either the same warfarin-sensitive reductase or a warfarin-insensitive quinone reductase. In the presence of warfarin, vitamin K epoxide cannot be reduced, but accumulates and is excreted. If enough vitamin K (as the quinone) is provided in the diet, it can be reduced to the active hydroquinone by the warfarin-insensitive enzyme, and carboxylation can continue, with stoichiometric utilization of vitamin K and excretion of the epoxide. A high dose of vitamin K is the antidote to an overdose of warfarin.
Prothrombin and several other proteins of the bloodclotting system (factors VII, IX, and X, and proteins C and S, Chapter 52) each contain 4 to 6 y-carboxyglutamate residues. y-Carboxyglutamate chelates calcium ions, and so permits the binding of the blood-clotting proteins to membranes. In vitamin K deficiency, or in the presence of warfarin, an abnormal precursor of prothrombin (preprothrombin) containing little or no y-carboxyglutamate, and incapable of chelating calcium, is released into the circulation.

A number of other proteins undergo the same vitamin K— dependent carboxylation of glutamate to y-carboxyglutamate, including osteocalcin and the matrix Gla protein in bone, nephrocalcin in kidney and the product of the growth arrest specific gene Gas6, which is involved in both the regulation of differentiation and development in the nervous system, and control of apoptosis in other tissues. All of these y-carboxyglutamate-containing proteins bind calcium, which causes a conformational change so that they interact with membrane phospholipids. The release into the circulation of osteocalcin provides an index of vitamin D status.

Thiamin has a central role in energy-yielding metabolism, and especially the metabolism of carbohydrates (Figure 44-8). Thiamin diphosphate is the coenzyme for three multienzyme complexes that catalyze oxidative decarboxylation reactions: pyruvate dehydrogenase in carbohydrate metabolism (see Chapter 17); a-ketoglutarate dehydrogenase in the citric acid cycle (see Chapter 16); and the branched-chain keto acid
dehydrogenase involved in the metabolism of leucine, isoleucine, and valine (see Chapter 29). In each case, the thiamin diphosphate provides a reactive carbon on the thiazole moiety that forms a carbanion, which then adds to the carbonyl group, for example, pyruvate. The addition compound is then decarboxylated, eliminating CO,. Thiamin diphosphate is also the coenzyme for transketolase, in the pentose phosphate pathway (see Chapter 20).
Thiamin triphosphate has a role in nerve conduction; it phosphorylates, and so activates, a chloride channel in the nerve membrane.

Thiamin deficiency can result in three distinct syndromes: a chronic peripheral neuritis, beriberi, which may or may not be associated with heart failure and edema; acute pernicious (fulminating) beriberi (Shoshin beriberi), in which heart failure and metabolic abnormalities predominate, without peripheral neuritis; and Wernicke encephalopathy with Korsakoff psychosis, which is associated especially with alcohol and narcotic abuse. The role of thiamin diphosphate in pyruvate dehydrogenase means that in deficiency there is impaired conversion of pyruvate to acetyl-CoA. In subjects on a relatively high carbohydrate diet, this results in increased plasma concentrations of lactate and pyruvate, which may cause life-threatening lactic acidosis.

The activation of apotransketolase (the enzyme protein) in erythrocyte lysate by thiamin diphosphate added in vitro has become the accepted index of thiamin nutritional status.

Riboflavin provides the reactive moieties of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) (see Figure 12-2). FMN is formed by ATP-dependent phosphorylation of riboflavin; FAD is synthesized by further reaction with ATP in which the AMP moiety is transferred onto FMN. The main dietary sources of riboflavin are milk and dairy products. In addition, because of its intense yellow color, riboflavin is widely used as a food additive.

These include the mitochondrial respiratory chain, key enzymes in fatty acid and amino acid oxidation, and the citric acid cycle. Reoxidation of the reduced flavin in oxygenases and mixed-function oxidases proceeds by way of formation of the flavin radical and flavin hydroperoxide, with the intermediate generation of superoxide and perhydroxyl radicals and hydrogen peroxide. Because of this, flavin oxidases make a significant contribution to the total oxidant stress in the body (see Chapter 45).

Although riboflavin is centrally involved in lipid and carbohydrate metabolism, and deficiency occurs in many countries, it is not fatal, because there is very efficient conservation of tissue riboflavin. Riboflavin released by the catabolism of enzymes is rapidly incorporated into newly synthesized enzymes. Deficiency is characterized by cheilosis, desquamation and inflammation of the tongue, and seborrheic dermatitis. Riboflavin nutritional status is assessed by measurement of the activation of erythrocyte glutathione reductase by FAD added in vitro.

Niacin was discovered as a nutrient during studies of pellagra. It is not strictly a vitamin since it can be synthesized in the body from the essential amino acid tryptophan. Two compounds, nicotinic acid and nicotinamide, have the biological activity of niacin; its metabolic function is as the nicotinamide ring of the coenzymes NAD and NADP in oxidation/reduction reactions (see Figures 7-2 and 12-4). Some 60 mg of tryptophan is equivalent to 1 mg of dietary niacin. The niacin content of foods is expressed as:
mg niacin equivalents = mg preformed niacin
+ 1/60 x mg tryptophan
Since most of the niacin in cereals is biologically unavailable, this is discounted.

In addition to its coenzyme role, NAD is the source of ADPribose for the ADP-ribosylation of proteins and polyADPribosylation of nucleoproteins involved in the DNA repair mechanism. Cyclic ADP-ribose and nicotinic acid adenine dinucleotide, formed from NAD, act to increase intracellular calcium in response to neurotransmitters and hormones.

Pellagra is characterized by a photosensitive dermatitis. As the condition progresses, there is depressive psychosis and possibly diarrhea. Untreated pellagra is fatal. Although the nutritional etiology of pellagra is well established, and either tryptophan or niacin prevents or cures the disease, additional
factors, including deficiency of riboflavin or vitamin B,, both of which are required for synthesis of nicotinamide from tryptophan, may be important. In most outbreaks of pellagra, twice as many women as men are affected, probably the result of inhibition of tryptophan metabolism by estrogen metabolites.

A number of genetic diseases that result in defects of tryptophan metabolism are associated with the development of pellagra, despite an apparently adequate intake of both tryptophan and niacin. Hartnup disease is a rare genetic condition in which there is a defect of the membrane transport mechanism for tryptophan, resulting in large losses as a result of intestinal malabsorption and failure of renal reabsorption. In carcinoid syndrome, there is metastasis of a primary liver tumor of enterochromaffin cells, which synthesize 5-hydroxytryptamine. Overproduction of 5-hydroxytryptamine may account for as much as 60% of the body's tryptophan metabolism, causing pellagra because of the diversion away from NAD synthesis.

Nicotinic acid has been used to treat hyperlipidemia when of the order of 1 to 6 g/d are required, causing dilation of blood vessels and flushing, along with skin irritation. Intakes of both nicotinic acid and nicotinamide in excess of 500 mg/d cause liver damage.

Six compounds have vitamin B, activity (Figure 44-9): pyridoxine, pyridoxal, pyridoxamine, and their 5'-phosphates. The active coenzyme is pyridoxal 5'-phosphate. Some 80% of the body's total vitamin B, is pyridoxal phosphate in muscle, mostly associated with glycogen phosphorylase. This is not available in deficiency, but is released in starvation, when glycogen reserves become depleted, and is then available, especially to liver and kidney, to meet increased requirement for gluconeogenesis from amino acids.

Pyridoxal phosphate is a coenzyme for many enzymes involved in amino acid metabolism, especially transamina- tion and decarboxylation. It is also the cofactor of glycogen phosphorylase, where the phosphate group is catalytically important. In addition, it is important in steroid hormone
FIGURE 44-9 interconversion of the vitamin B, vitamers.
action. Pyridoxal phosphate removes the hormone-receptor complex from DNA binding, terminating the action of the hormones. In vitamin B, deficiency, there is increased sensitivity to the actions of low concentrations of estrogens, androgens, cortisol, and vitamin D.

Although clinical deficiency disease is rare, there is evidence that a significant proportion of the population has marginal vitamin B, status. Moderate deficiency results in abnormalities of tryptophan and methionine metabolism. Increased sensitivity to steroid hormone action may be important in the development of hormone-dependent cancer of the breast, uterus, and prostate, and vitamin B, status may affect the prognosis.

The most widely used method of assessing vitamin B, status is by the activation of erythrocyte transaminases by pyridoxal phosphate added in vitro, expressed as the activation coefficient. Measurement of plasma concentrations of the vitamin is also used.

The development of sensory neuropathy has been reported in patients taking 2 to 7 g of pyridoxine per day for a variety of reasons. There was some residual damage after withdrawal of these high doses; other reports suggest that intakes in excess of 100 to 200 mg/d are associated with neurological damage.

The term "vitamin B,," is used as a generic descriptor for the cobalamins—those corrinoids (cobalt-containing compounds possessing the corrin ring) having the biological activity of the vitamin (Figure 44-10). Some corrinoids that are growth factors for microorganisms not only have no vitamin B,, activity, but may also be antimetabolites of the vitamin. Although it is synthesized exclusively by microorganisms, for practical purposes vitamin B,, is found only in foods of animal origin, there being no plant sources of this vitamin. This means that strict vegetarians (vegans) are at risk of developing B,, deficiency. The small amounts of the vitamin formed by bacteria on the surface of fruits may be adequate to meet requirements, but preparations of vitamin B,, made by bacterial fermentation are available.

Vitamin B,, is absorbed bound to intrinsic factor, a small glycoprotein secreted by the parietal cells of the gastric mucosa. Gastric acid and pepsin release the vitamin from protein binding in food and make it available to bind to cobalophilin, a binding protein secreted in the saliva. In the duodenum, cobalophilin is hydrolyzed, releasing the vitamin for binding to intrinsic factor. Pancreatic insufficiency can therefore be a factor in the development of vitamin B,, deficiency, resulting in the excretion of cobalophilin-bound vitamin B,,. Intrinsic
FIGURE 44-10 Vitamin B,,. Four coordination sites on the central cobalt atom are chelated by the nitrogen atoms of the corrin ring, and one by the nitrogen of the dimethylbenzimidazole nucleotide. The sixth coordination site may be occupied by CN" (cyanocobalamin), OH" (hydroxocobalamin), H,O (aquocobalamin, —CH, (methyl cobalamin), or 5'-deoxyadenosine (adenosylcobalamin).
factor binds only the active vitamin B,, vitamers and not other corrinoids. Vitamin B,, is absorbed from the distal third of the ileum via receptors that bind the intrinsic factor—vitamin B,, complex, but not free intrinsic factor or free vitamin. There is considerable enterohepatic circulation of vitamin B,,, with excretion in the bile, then reabsorption after binding to intrinsic factor in the ileum.

Methylmalonyl-CoA mutase, and methionine synthase (Figure 44-11) are vitamin B,,-dependent enzymes. Methylmalonyl-CoA is formed as an intermediate in the catabolism of valine and by the carboxylation of propionyl-CoA arising in the catabolism of isoleucine, cholesterol, and rare fatty acids with an odd number of carbon atoms, or directly from propionate, a major product of microbial fermentation ruminants. It undergoes a vitamin B,,-dependent rearrangement to succinyl-CoA, catalyzed by methylmalonyl-CoA mutase (see Figure 19-2). The activity of this enzyme is greatly reduced in vitamin B,, deficiency, leading to an accumulation of methylmalonyl-CoA and urinary excretion of methylmalonic acid, which provides a means of assessing vitamin B,, nutritional status.

Pernicious anemia arises when vitamin B,, deficiency impairs the metabolism of folic acid, leading to functional folate deficiency that disturbs erythropoiesis, causing immature precursors of erythrocytes to be released into the circulation (megaloblastic anemia). The most common cause of pernicious anemia is failure of the absorption of vitamin B,, rather than dietary deficiency. This can be the result of failure of intrinsic factor secretion caused by autoimmune disease affecting parietal cells or from production of anti-intrinsic factor antibodies. There is irreversible degeneration of the spinal cord in pernicious anemia, as a result of failure of methylation of one arginine residue in myelin basic protein. This is the
FIGURE 44-11 Homocysteine and the folate trap. Vitamin B,, deficiency leads to impairment of methionine synthase, resulting in accumulation of homocysteine and trapping folate as methyltetrahydrofolate.
substituted folates.
result of methionine deficiency in the central nervous system, rather than secondary folate deficiency.

The active form of folic acid (pteroyl glutamate) is tetrahydrofolate (Figure 44-12). The folates in foods may have up to seven additional glutamate residues linked by y-peptide bonds. In addition, all of the one-carbon substituted folates in Figure 44-12 may also be present in foods. The extent to which the different forms of folate can be absorbed varies, and folate intakes are calculated as dietary folate equivalents—the sum of ug food folates + 1.7 x pg of folic acid (used in food enrichment).

Tetrahydrofolate can carry one-carbon fragments attached to N-5 (formyl, formimino, or methyl groups), N-10 (formyl) or bridging N-5-N-10 (methylene or methenyl groups). 5-Formyl-tetrahydrofolate is more stable than folate and is therefore used pharmaceutically (known as folinic acid), and the synthetic (racemic) compound (leucovorin). The major point of entry for one-carbon fragments into substituted folates is methylenetetrahydrofolate (Figure 44-13), which is formed by the reaction of glycine, serine, and choline with tetrahydrofolate. Serine is the most important source of substituted folates for biosynthetic reactions, and the activity of serine hydroxymethyltransferase is regulated by the state of folate substitution and the availability of folate. The reaction is reversible, and in liver it can form serine from glycine as a substrate for gluconeogenesis. Methylene-, methenyl-, and 10-formyltetrahydrofolates are interconvertible. When onecarbon folates are not required, the oxidation of formyltetrahydrofolate to yield carbon dioxide provides a means of maintaining a pool of free folate.

The methylation of deoxyuridine monophosphate (UMP) to thymidine monophosphate (TMP), catalyzed by thymidylate synthase, is essential for the synthesis of DNA. The one-carbon fragment of methylene-tetrahydrofolate is reduced to a methyl group with release of dihydrofolate, which is then reduced back to tetrahydrofolate by dihydrofolate reductase. Thymidylate synthase and dihydrofolate reductase are especially active in tissues with a high rate of cell division. Methotrexate, an analog of 10-methyltetrahydrofolate, inhibits dihydrofolate reductase and is used as in cancer chemotherapy. The dihydrofolate reductases of some bacteria and parasites differ from the human enzyme; inhibitors of these enzymes can be used as antibacterial drugs (eg, trimethoprim) and antimalarial drugs (eg, pyrimethamine).
FIGURE 44-13 Sources and utilization of one-carbon substituted folates.

When acting as a methyl donor, S-adenosyl methionine forms homocysteine, which may be remethylated by methyltetrahydrofolate catalyzed by methionine synthase, a vitamin B,,-dependent enzyme (Figure 44-11). As the reduction of methylenetetrahydrofolate to methyltetrahydrofolate is irreversible and the major source of tetrahydrofolate for tissues is methyltetrahydrofolate, the role of methionine synthase is vital, and provides a link between the functions of folate and vitamin B,,. Impairment of methionine synthase in vitamin B,, deficiency results in the accumulation of methyltetrahydrofolate that cannot be used—the "folate trap" There is therefore functional deficiency of folate, secondary to the deficiency of vitamin B,,.

Deficiency of folic acid itself or deficiency of vitamin B,,, which leads to functional folic acid deficiency, affects cells that are dividing rapidly because they have a large requirement for thymidine for DNA synthesis. Clinically, this affects the bone marrow, leading to megaloblastic anemia.

Supplements of 400 g/d of folic acid begun before conception result in a significant reduction in the incidence of spina bifida and other neural tube defects. Because of this, there is mandatory enrichment of flour with folic acid in many countries. Elevated blood homocysteine is a significant risk factor for atherosclerosis, thrombosis, and hypertension. The condition is the result of an impaired ability to form methyltetrahydrofolate by methylenetetrahydrofolate reductase, causing functional folate deficiency, resulting in failure to remethylate homocysteine to methionine. People with an abnormal variant of methylenetetrahydrofolate reductase that occurs in 5 to 10% of the population do not develop hyperhomocysteinemia if they have a relatively high intake of folate. A number of placebo-controlled trials of supplements of folate (commonly together with vitamins B, and B,,) have shown the expected lowering of plasma homocysteine, but apart from reduced incidence of stroke there has been no effect on death from cardiovascular disease.
There is also evidence that low folate status results in impaired methylation of CpG islands in DNA, which is a factor in the development of colorectal and other cancers. A number of studies suggest that folic acid supplementation or food enrichment may reduce the risk of developing some cancers. However, there is also some evidence that folate supplements increase the rate of transformation of preneoplastic colorectal polyps into cancers, so that people with such polyps may be at increased risk of developing colorectal cancer if they have a high folate intake.

Folic acid supplements will rectify the megaloblastic anemia of vitamin B,, deficiency but not the irreversible nerve damage. A high intake of folic acid can thus mask vitamin B,, deficiency. This is especially a problem for elderly people, since atrophic gastritis that develops with increasing age leads to failure of gastric acid secretion, and hence failure to release vitamin B,, from dietary proteins. Because of this, although many countries have adopted mandatory enrichment of flour with folic acid to prevent neural tube defects, others have not. There is also antagonism between folic acid and some anticonvulsants used in the treatment of epilepsy, and, as noted above, there is some evidence that folate supplements may increase the risk of developing colorectal cancer among people with preneoplastic colorectal polyps.

The structures of biotin, biocytin, and carboxybiotin (the active metabolic intermediate) are shown in Figure 44-14. Biotin is widely distributed in many foods as biocytin (e-amino-biotinyl lysine), which is released on proteolysis. It is synthesized by intestinal flora in excess of requirements. Deficiency is unknown, except among people maintained for many months on total parenteral nutrition, and a very small number who eat abnormally large amounts of uncooked egg white, which contains avidin, a protein that binds biotin and renders it unavailable for absorption.
FIGURE 44-14 Biotin, biocytin, and carboxybiocytin.

Biotin functions to transfer carbon dioxide in a small number of reactions: acetyl-CoA carboxylase (see Figure 23-1), pyruvate carboxylase (see Figure 19-1), propionyl-CoA carboxylase (see Figure 19-2), and methylcrotonyl-CoA carboxylase. A holocarboxylase synthetase catalyzes the transfer of biotin onto a lysine residue of the apoenzyme to form the biocytin residue of the holoenzyme. The reactive intermediate is 1-N-carboxybiocytin, formed from bicarbonate in an ATPdependent reaction. The carboxyl group is then transferred to the substrate for carboxylation.
Biotin also has a role in regulation of the cell cycle, acting to biotinylate key nuclear proteins.

Pantothenic acid has a central role in acyl group metabolism when acting as the pantetheine functional moiety of coenzyme A (CoA) or acyl carrier protein (ACP) (Figure 44-15). The pantetheine moiety is formed after combination of pantothenate with cysteine, which provides the—SH prosthetic group of CoA and ACP. CoA takes part in reactions of the citric acid cycle (see Chapter 16), fatty acid oxidation (see Chapter 22), acetylations, and cholesterol synthesis (see Chapter 26). ACP participates in fatty acid synthesis (see Chapter 23). The vitamin is widely distributed in all foodstuffs, and deficiency has not been unequivocally reported in humans except in specific depletion studies.

Vitamin C (Figure 44-16) is a vitamin for human beings and other primates, the guinea pig, bats, passeriform birds, and
FIGURE 44-15 Pantothenic acid and coenzyme A. Asterisk shows site of acylation by fatty acids.

most fishes and invertebrates; other animals synthesize it as an intermediate in the uronic acid pathway of glucose metabolism (see Figure 20-4). In those species for which it is a vitamin, gulonolactone oxidase is absent. Both ascorbic acid and dehydroascorbic acid have vitamin activity.

Ascorbic acid has specific roles in the copper-containing hydroxylases and the a-ketoglutarate-linked iron-containing hydroxylases. It also increases the activity of a number of other enzymes in vitro, although this is a nonspecific reducing action. In addition, it has a number of nonenzymic effects as a result of its action as a reducing agent and oxygen radical quencher (see Chapter 45).
Dopamine 6-hydroxylase is a copper-containing enzyme involved in the synthesis of the catecholamines (norepinephrine and epinephrine), from tyrosine in the adrenal medulla and central nervous system. During hydroxylation the Cu* is oxidized to Cu'*; reduction back to Cu* specifically requires ascorbate, which is oxidized to monodehydroascorbate.
A number of peptide hormones have a carboxy-terminal amide that is derived from a terminal glycine residue. This glycine is hydroxylated on the a-carbon by a copper-containing enzyme, peptidylglycine hydroxylase, which, again, requires ascorbate for reduction of Cu'.
A number of iron-containing, ascorbate-requiring hydroxylases share a common reaction mechanism, in which hydroxylation of the substrate is linked to oxidative decarboxylation of a-ketoglutarate. Many of these enzymes are involved in the modification of precursor proteins. Proline and lysine hydroxylases are required for the postsynthetic modification of procollagen to collagen, and proline hydroxylase is also required in formation of osteocalcin and the Clq component of complement. Aspartate B-hydroxylase is required for the postsynthetic modification of the precursor of protein C, the vitamin K-dependent protease that hydrolyzes activated factor V in the blood-clotting cascade (see Chapter 52). Trimethyllysine and y-butyrobetaine hydroxylases are required for the synthesis of carnitine. In these enzymes ascorbate is required to reduce the iron prosthetic group after accidental oxidation during reaction; it is neither consumed stoichiometrically with the substrates nor does it have a role.

Signs of vitamin C deficiency include skin changes, fragility of blood capillaries, gum decay, tooth loss, and bone fracture, which can be attributed to impaired collagen synthesis, and psychological changes that can be attributed to impaired synthesis of catecholamines.

At intakes above about 100 mg/d, the body's capacity to metabolize vitamin C is saturated, and any further intake is excreted in the urine. However, in addition to its other roles, vitamin C enhances the absorption of inorganic iron, and this depends on the presence of the vitamin in the gut. Therefore, increased intakes may be beneficial, and it is frequently prescribed together with iron supplements to treat iron deficiency anemia. There is very little good evidence that high doses of vitamin C prevent the common cold, although they may reduce the duration and severity of symptoms.

Many of the essential minerals (Table 44-2) are widely distributed in foods, and most people eating a mixed diet are likely to receive adequate intakes. The amounts required vary from grams per day for sodium and calcium, through milligrams per day (eg, iron and zinc), to micrograms per day for the trace elements. In general, mineral deficiencies occur when foods come from one region where the soil may be deficient in some minerals (eg, iodine and selenium, deficiencies of both of which occur in many areas of the world). When

Structural function, Mineral = Calcium, magnesium, phosphate. Involved in membrane function, Mineral = Sodium, potassium. Function as prosthetic groups in enzymes, Mineral = Cobalt, copper, iron, molybdenum, selenium, zinc. Regulatory role or role in hormone action, Mineral = Calcium, chromium, iodine, magnesium, manganese, sodium, potassium. Known to be essential, but function unknown, Mineral = Silicon, vanadium, nickel, tin. Have effects in the body, but essentiality is not established, Mineral = Fluoride, lithium. May occur in foods and known to be toxic in excess, Mineral = Aluminum, arsenic, antimony, boron, bromine, cadmium, cesium, germanium, lead, mercury, silver, strontium
foods come from a variety of regions, mineral deficiency is less likely to occur. Iron deficiency is an important problem worldwide, because if iron losses from the body are relatively high (eg, from heavy menstrual blood loss or intestinal parasites), it is difficult to achieve an adequate intake to replace losses. However, 10% of the population (and more in some areas) are genetically at risk of iron overload, leading to formation of free radicals as a result of nonenzymic reactions of iron ions in free solution when the capacity of iron-binding proteins has been exceeded. Foods grown on soil containing high levels of selenium cause toxicity, and excessive intakes of sodium cause hypertension in susceptible people.

Describe the damage caused to DNA, lipids, and proteins by free radicals, and the diseases associated with radical damage.
Describe the main sources of oxygen radicals in the body.
damage. Describe the mechanisms and dietary factors that protect against radical
= Explain how antioxidants can also act as pro-oxidants, and why intervention trials of antioxidant nutrients have generally yielded disappointing results.

Free radicals are formed in the body under normal conditions. They cause damage to nucleic acids, proteins, and lipids in cell membranes and plasma lipoproteins. This can cause cancer, atherosclerosis and coronary artery disease, and autoimmune diseases. Epidemiological and laboratory studies have identified a number of protective antioxidant nutrients: selenium, vitamins C and E, B-carotene, and other carotenoids, and a variety of polyphenolic compounds derived from plant foods. Many people take supplements of one or more antioxidant nutrients. However, intervention trials show little benefit of antioxidant supplements except among people who were initially deficient, and many trials of b-carotene and vitamin E have shown increased mortality among those taking the supplements.

Free radicals are highly reactive molecular species with an unpaired electron; they persist for only a very short time (of the order of 10° to 10°" seconds) before they collide with another molecule and either abstract or donate an electron in order to achieve stability. In so doing, they generate a new radical from the molecule with which they collided. The main way in which a free radical can be quenched, so terminating this chain reaction, is if two radicals react together, when the unpaired electrons can become paired in one or other of the parent molecules. This is a rare occurrence, because of the very short half-life of an individual radical and the very low concentrations of radicals in tissues.
The most damaging radicals in biological systems are oxygen radicals (sometimes called reactive oxygen species) — especially superoxide, *O,, hydroxyl, *OH, and perhydroxyl, *O,H. Tissue damage caused by oxygen radicals is often called oxidative damage, and factors that protect against oxygen radical damage are known as antioxidants.

Interaction of radicals with bases in DNA can lead to chemical changes that, if not repaired (see Chapter 35), may be inherited in daughter cells. Radical damage to unsaturated fatty acids in cell membranes and plasma lipoproteins leads to the formation of lipid peroxides, then highly reactive dialdehydes that can chemically modify proteins and nucleic acid bases. Proteins are also subject to direct chemical modification by interaction with radicals. Oxidative damage to tyrosine residues in proteins can lead to the formation of dihydroxyphenylalanine that can undergo nonenzymic reactions leading to further formation of oxygen radicals (Figure 45-1).
The total body radical burden can be estimated by measuring the products of lipid peroxidation. Lipid peroxides can be measured by the ferrous oxidation in xylenol orange (FOX) assay. Under acidic conditions, they oxidize Fe** to Fe**, which forms a chromophore with xylenol orange. The dialdehydes formed from lipid peroxides can be measured by reaction with thiobarbituric acid, when they form a red fluorescent adduct—the results of this assay are generally reported as total thiobarbituric acid reactive substances, TBARS. Peroxidation of n-6 polyunsaturated fatty acids leads to the formation of
.

pentane, and of n-3 polyunsaturated fatty acids to ethane, both of which can be measured in exhaled air.

Radical damage to DNA in germline cells in ovaries and testes can lead to heritable mutations; in somatic cells, the result may be initiation of cancer. The dialdehydes formed as a result of radical-induced lipid peroxidation in cell membranes can also modify bases in DNA.
Chemical modification of amino acids in proteins, either by direct radical action or as a result of reaction with the products of radical-induced lipid peroxidation, leads to proteins that are recognized as nonself by the immune system. The resultant antibodies will also cross-react with normal tissue proteins, so initiating autoimmune disease.
Chemical modification of the proteins or lipids in plasma low-density lipoprotein (LDL) leads to abnormal LDL that is not recognized by the liver LDL receptors, and so is not cleared by the liver. The modified LDL is taken up by macrophage scavenger receptors. Lipid-engorged macrophages infiltrate under blood vessel endothelium (especially when there is already some damage to the endothelium), and are killed by the high content of unesterified cholesterol they have accumulated. This occurs in the development of atherosclerotic plaques, which, in extreme cases, can more or less completely occlude a blood vessel.

Ionizing radiation (x-rays and UV) can lyse water, leading to the formation of hydroxyl radicals. Transition metal ions, including Cu*, Co**, Ni'*, and Fe** can react nonenzymically with oxygen or hydrogen peroxide, again leading to the

formation of hydroxyl radicals. Nitric oxide (an important compound in cell signaling, originally described as the endothelium-derived relaxation factor) is itself a radical, and, more importantly, can react with superoxide to yield peroxynitrite, which decays to form hydroxyl radicals (Figure 45-2).
The respiratory burst of activated macrophages (see Chapter 53) is increased utilization of glucose via the pentose phosphate pathway (see Chapter 20) to reduce NADP* to NADPH, and increased utilization of oxygen to oxidize NADPH to produce oxygen (and halogen) radicals as cytotoxic agents to kill phagocytosed microorganisms. The respiratory burst oxidase (NADPH oxidase) is a flavoprotein that reduces oxygen to superoxide:

Plasma markers of radical damage to lipids increase considerably in response to even a mild infection. The oxidation of reduced flavin coenzymes in the mitochondrial (see Chapter 13) and microsomal electron transport chains proceeds through a series of steps in which the flavin semiquinone radical is stabilized by the protein to which it is bound, and forms oxygen radicals as transient intermediates. Although the final products are not radicals, because of the unpredictable nature of radicals there is considerable "leakage" of radicals, and some 3 to 5% of the daily consumption of 30 mol of oxygen by an adult human being is converted to singlet oxygen, hydrogen peroxide, and superoxide, perhydroxyl, and hydroxyl radicals, rather than undergoing complete reduction to water. This results in daily production of about 1.5 mol of reactive oxygen species.

The metal ions that undergo nonenzymic reaction to form oxygen radicals are not normally free in solution, but are bound to either the proteins for which they provide the prosthetic group, or to specific transport and storage proteins, so that they are unreactive. Iron is bound to transferrin, ferritin, and hemosiderin, copper to ceruloplasmin, and other metal ions are bound to metallothionein. This binding to transport
proteins that are too large to be filtered in the kidneys also prevents loss of metal ions in the urine. Superoxide is produced both accidentally and also as the reactive oxygen species required for a number of enzymecatalyzed reactions. A family of superoxide dismutases catalyze the reaction between superoxide and protons to yield oxygen and hydrogen peroxide:

The hydrogen peroxide is then removed by catalase and various peroxidases: 2H,0, > 2H,O + O,. Most enzymes that produce and require superoxide are contained in the peroxisomes, together with superoxide dismutase, catalase, and peroxidases.
The peroxides that are formed by radical damage to lipids in membranes and plasma lipoproteins are reduced to hydroxy fatty acids by glutathione peroxidase, a selenium-dependent enzyme (hence the importance of adequate selenium intake to maximize antioxidant activity), and the oxidized glutathione is reduced by NADPH-dependent glutathione reductase (see Figure 20-3). Lipid peroxides are also reduced to fatty acids by reaction with vitamin E, forming the tocopheroxyl radical, which is relatively stable, since the unpaired electron can be located in any one of three positions in the molecule (Figure 45-3). The tocopheroxyl radical persists long enough to undergo reduction back to tocopherol by reaction with vitamin C at the surface of the cell or lipoprotein. The resultant monodehydroascorbate radical then undergoes enzymic reduction back to ascorbate or a nonenzymic reaction of 2 mol of monodehydroascorbate to yield 1 mol each of ascorbate and dehydroascorbate.
Ascorbate, uric acid, and a variety of polyphenols derived from plant foods act as water-soluble radicaltrapping antioxidants, forming relatively stable radicals that persist long enough to undergo reaction to nonradical products. Ubiquinone and carotenes similarly act as lipidsoluble radical-trapping antioxidants in membranes and plasma lipoproteins.

Although ascorbate is an antioxidant, reacting with superoxide and hydroxyl to yield monodehydroascorbate and hydrogen peroxide or water, it can also be a source of superoxide radicals by reaction with oxygen, and hydroxyl radicals by reaction with Cu** ions (Table 45-1). However, these pro-oxidant actions require relatively high concentrations of ascorbate, which are unlikely to be reached in tissues, since once the plasma concentration of ascorbate reaches about 30 mmol/L, the renal threshold is reached, and at intakes above about 100 to 120 mg/d the vitamin is excreted in the urine quantitatively with intake.
A
considerable body
of epidemiological
evidence suggests
that carotene is protective against lung and other cancers.
However, two major intervention trials in the 1990s showed an
FIGURE 45-3 The roles of vitamins E and C in reducing lipid peroxides, and stabilization of the tocopheroxyl radical by delocalization of the unpaired electron.
increase in death from lung (and other) cancer among people who were given supplements of B-carotene. The problem is that although $-carotene is indeed a radical-trapping antioxidant under conditions of low partial pressure of oxygen, as in most tissues, at high partial pressures of oxygen (as in the lungs) and especially in high concentrations, B-carotene is an autocatalytic pro-oxidant, and hence can initiate radical damage to lipids and proteins.

Ascorbate + *O,- > H,O, + monodehydroascorbate; catalase and peroxidases catalyze the reaction: > +
2H,O, 2H,O O, H,O
Ascorbate + *OH — + monodehydroascorbate

Ascorbate + O, > *O, + monodehydroascorbate
Ascorbate + Cu?* — Cu* + monodehydroascorbate
Cur +H,0, — Cu?* + OH" +"OH
Epidemiological evidence also suggests that vitamin E is protective against atherosclerosis and cardiovascular disease. However, meta-analysis of intervention trials with vitamin E shows increased mortality among those taking (high dose) supplements. These trials have all used a-tocopherol, and it is possible that the other vitamers of vitamin E that are present in foods, but not the supplements, may be important. In vitro, plasma lipoproteins form less cholesterol ester hydroperoxide when incubated with sources of low concentrations of perhydroxyl radicals when vitamin E has been removed than when it is present. The problem seems to be that vitamin E acts as an antioxidant by forming a stable radical that persists long enough to undergo metabolism to nonradical products. This means that the radical also persists long enough to penetrate deeper in to the lipoprotein, causing further radical damage, rather than interacting with a water-soluble antioxidant at the surface of the lipoprotein.
Nitric oxide and other radicals are important in cell signaling, and especially in signaling for programmed cell death (apoptosis) of cells that have suffered DNA and other damage. It is likely that high concentrations of antioxidants, so far from protecting against tissue damage, may quench the signaling radicals, and so permit the continued survival of damaged cells, so increasing, rather than decreasing, the risk of cancer development.

- @ Free radicals are highly reactive molecular species with an unpaired electron. They can react with, and modify, proteins, nucleic acids, and fatty acids in cell membranes and plasma lipoproteins.
■, 1 = Radical damage to lipids and proteins in plasma lipoproteins is a factor in the development of atherosclerosis and coronary artery disease; radical damage to nucleic acids may induce heritable mutations and cancer; radical damage to proteins may lead to the development of autoimmune diseases.. ■, 1 = Oxygen radicals arise as a result of exposure to ionizing radiation, nonenzymic reactions of transition metal ions, the respiratory burst of activated macrophages, and the normal oxidation of reduced flavin coenzymes.. ■, 1 = Protection against radical damage is afforded by enzymes that remove superoxide ions and hydrogen peroxide, enzymic reduction of lipid peroxides linked to oxidation of glutathione, nonenzymic reaction of lipid peroxides with vitamin E, and reaction of radicals with compounds such as vitamins C and E, carotene, ubiquinone, uric acid, and dietary polyphenols that form relatively stable radicals that persist long enough to undergo reaction to nonradical products.. ■, 1 = Except in people who were initially deficient, intervention trials of vitamin E and β-carotene have generally shown increased mortality among those taking the supplements. β-Carotene is only an antioxidant at low concentrations of oxygen; at higher concentrations of oxygen it is an autocatalytic pro- oxidant. Vitamin E forms a stable radical that is capable of either undergoing reaction with water-soluble antioxidants or penetrating further into lipoproteins and tissues, so increasing radical damage.. ■, 1 = Radicals are important in cell signaling, and especially in signaling for apoptosis of cells that have suffered DNAdamage. It is likely that high concentrations of antioxidants, so far from protecting against tissue damage, may quench the signaling radicals, and so permit the continued survival of damaged cells, so increasing, rather than decreasing, the risk of cancer development.

Explain the importance of glycoproteins in health and disease.
Describe the principal sugars found in glycoproteins.
Describe the major classes of glycoproteins (N-linked, O-linked, and GPI-linked).
glycoproteins. Describe the major features of the pathways of biosynthesis and degradation of
m= Explain how many microorganisms, such as influenza virus, attach to cell surfaces via sugar chains.

The glycoproteins are proteins that contain oligosaccharide chains (glycans) covalently bound to amino acids; glycosylation (the enzymic attachment of sugars) is the most frequent posttranslational modification of proteins. Many proteins also undergo reversible glycosylation with a single sugar (N-acetylglucosamine) bound to a serine or threonine residue that is also a site for reversible phosphorylation. This is an important mechanism of metabolic regulation. Nonenzymic attachment of sugars to proteins can also occur, and is referred to as glycation. This process can have serious pathologic consequences (eg, in poorly controlled diabetes mellitus).
some of which play key roles in viral attachment to host cells. The glycoproteins have a wide range of functions (Table 46-1); their carbohydrate content ranges from 1 to over 85% by weight. The glycan structures of glycoproteins change in response to signals involved in cell differentiation, normal physiology, and neoplastic transformation. This is the result of different expression patterns of glycosyltransferases. Table 46-2 lists some of the major functions of the glycan chains of glycoproteins.
Glycoproteins are one class of glycoconjugate or complex carbohydrate—molecules containing one or more carbohydrate chains covalently linked to protein (to form glycoproteins or proteoglycans, see Chapter 50) or lipid (to form glycolipids, see Chapter 21). Almost all plasma proteins, and many peptide hormones, are glycoproteins, as are a number of blood group substances (others are glycosphingolipids). Many cell membrane proteins (see Chapter 40) contain substantial amounts of carbohydrate, and many are anchored to the lipid bilayer by a glycan chain. Evidence is accumulating that alterations in the structures of glycoproteins and other glycoconjugates on the surface of cancer cells are important in metastasis.

Glycoproteins occur in most organisms, from bacteria to human beings. Many viruses also contain glycoproteins,

The biological information in the sequence and linkages of sugars in glycans differs from that in DNA, RNA, and proteins in one important respect; it is secondary rather than primary information. The pattern of glycosylation of a given protein depends on the pattern of expression of the various glycosyltransferases in the cell that are involved in glycoprotein synthesis, the affinity of the different glycosyltransferases for their carbohydrate substrates, and the relative availability of the different carbohydrate substrates. Because of this there is microheterogeneity of glycoproteins, something that complicates their analysis. Not all of the glycan chains of a given glycoprotein are complete; some are truncated.
The information from the sugars is expressed via interactions between the glycans and proteins such as lectins (see below) or other molecules. These interactions lead to changes in cellular activity.

Structural molecule, Glycoproteins = Collagens. Lubricant and protective agent, Glycoproteins = Mucins. Transport molecule, Glycoproteins = Transferrin, ceruloplasmin. Immunological molecule, Glycoproteins = Immunoglobulins, histocompatibility antigens. Hormone, Glycoproteins = Chorionic gonadotropin, thyroid- stimulating hormone (TSH). Enzyme, Glycoproteins = Various, eg, alkaline phosphatase. Cell attachment-recognition site, Glycoproteins = Various proteins involved in cell- cell (eg, sperm-oocyte), virus-cell, bacterium-cell, and hormone-cell interactions. Antifreeze, Glycoproteins = Plasma proteins of cold-water fish. Interact with specific carbohydrates, Glycoproteins = Lectins, selectins (cell adhesion lectins), antibodies. Receptor, Glycoproteins = Cell surface proteins involved in hormone and drug action. Regulate folding of proteins that are exported from the cell, Glycoproteins = Calnexin, calreticulin. Regulation of differentiation and development, Glycoproteins = Notch and its analogs, key proteins in development. Hemostasis (and thrombosis), Glycoproteins = Specific glycoproteins on the surface membranes of platelets

Only eight monosaccharides are commonly found in glycoproteins (Table 46-3 and Chapter 15). N-acetylneuraminic acid (NeuAc) is usually found at the termini of oligosaccharide chains, attached to subterminal galactose (Gal) or

+ Change physicochemical properties of the protein such as solubil- ity, viscosity, charge, conformation, denaturation.
+ Provide binding sites for various molecules, as well as bacteria, viruses, and some parasites.
+ Provide cell surface recognition signals.
+ Protect against proteolysis.
+
Ensure
correct
folding
of
proteins
that
are
exported
from
the
cell
and
target
incorrectly
folded
proteins
for
transport
from
the
endo-
plasmic
reticulum
back
to
the
cytosol
for
catabolism.
+ Protect peptide hormones and other plasma proteins against clearance by the liver.
+ Permit anchoring of extracellular proteins in the cell membrane, and of intracellular proteins inside subcellular organelles such as endoplasmic reticulum and Golgi.
+ Direct intracellular migration, sorting and secretion of proteins.
+ Affect embryonic development and cell and tissue differentiation.
+ affect sites of metastases selected cancer cells.
May by
N-acetylgalactosamine (GalNAc) residues. The other sugars are usually found in more internal positions. Sulfate is often found in glycoproteins, usually attached to galactose, N-acetylglucosamine, or N-acetylgalactosamine. As in most biosynthetic reactions, it is not the free or phosphorylated sugar that is the substrate for glycoprotein synthesis, but the corresponding sugar nucleotide (see Figure 18-2); some contain UDP and others guanosine diphosphate (GDP) or cytidine monophosphate (CMP).

Lectins are carbohydrate-binding proteins that agglutinate cells or precipitate glycoconjugates; a number of lectins are themselves glycoproteins. Immunoglobulins that react with sugars are not considered to be lectins. Lectins contain at least two sugar-binding sites; proteins with only a single sugar-binding site will not agglutinate cells or precipitate glycoconjugates.
Lectins were
first discovered
in plants
and microor-
ganisms, but many lectins of animal origin are now known, including the mammalian asialoglycoprotein receptor. Many peptide hormones, and most plasma proteins are glycoproteins. Treatment of the protein with neuraminidase removes the terminal N-acetylneuraminic acid moiety, exposing the subterminal galactose residue. This asialoglycoprotein is cleared from the circulation very much faster than the intact glycoprotein. Liver cells contain an asialoglycoprotein receptor that recognizes the galactose moiety of many desialylated plasma proteins, leading to their endocytosis and catabolism.
Plant lectins
were formerly
called phytohemagglutinins,
because of
their ing
with the
cell undercooked
ability to
agglutinate surface
red glycoproteins.
legumes can
blood cells
by react-
Undenatured lead
to stripping
of the
lectins in
intestinal mucosa
by agglutinating
the mucosal
cells.
Lectins are used to purify glycoproteins, as tools for probing the glycoprotein profiles of cell surfaces, and as reagents for generating mutant cells deficient in certain enzymes involved in the biosynthesis of oligosaccharide chains.

Glycoproteins can be divided into three main groups, based on the nature of the linkage between the polypeptide and oligosaccharide chains (Figure 46-1); there are other minor classes of glycoprotein:
1.
Those
an
O-glycosidic
linkage
(O-linked),
involving
the
hydroxyl
side
chain
of
serine
or
threo-
nine
(and
sometimes
also
tyrosine)
and
a
sugar
such
as
N-acetylgalactosamine
(GalNAc-Ser[Thr]).
2. Those containing an N-glycosidic linkage (N-linked), involving the amide nitrogen of asparagine and N-acetyl- glucosamine (GlcNAc-Asn).

Comments = Present in both N - and O -linked glycoproteins.. N -Acetylglucosamine, Type = Aminohexose. N -Acetylglucosamine, Abbreviation = GlcNAc. N -Acetylglucosamine, Sugar Nucleotide = UDP-GlcNAc. N -Acetylglucosamine, Comments = The sugar attached to the polypeptide chain via Asn in N -linked glycoproteins; also found at other sites in the oligosaccharides of these proteins. Many nuclear proteins have GlcNAc attached to the OHof Ser or Thr as a single sugar.. Xylose, Type = Pentose. Xylose, Abbreviation = Xyl. Xylose, Sugar Nucleotide = UDP-Xyl. Xylose, Comments = Xyl is attached to the OHof Ser in many proteoglycans. Xyl in turn is attached to two Gal residues, forming a link trisaccharide. Xyl is also found in t-PA and certain clotting factors.
"The structures of these sugars are illustrated in Chapter 15.
FIGURE 46-1 The three main types of glycoprotein: (A) an O-linkage (N-acetylgalactosamine to serine), (B) an N-linkage (N-acetylglucosamine to asparagine), and (C) a glycosylphosphatidylinositol (GPI) linkage. The GPI structure shown is that linking acetylcholinesterase to the plasma membrane of the human red blood cell. The site of action of Pl-phospholipase C (PI-PLC), which releases the enzyme from membrane binding is indicated. This particular GPI contains an extra fatty acid attached to inositol and also an extra phosphorylethanolamine moiety attached to the central of the mannose residue. Variations found among different GPI structures include the identity of the carboxyl-terminal amino acid, the molecules attached to the mannose residues, and the precise nature of the lipid moiety.

- 3. Those linked to the carboxyl-terminal amino acid of a protein via a phosphorylethanolamine moiety joined to an oligosaccharide (glycan), which in turn is linked via glucosamine to phosphatidylinositol (PI). These are glycosylphosphatidylinositol-anchored (GPI-anchored) glycoproteins. Among other functions, they are involved in directing glycoproteins to the apical or basolateral areas of the plasma membrane of polarized epithelial cells (see Chapter 40 and below).
The number of oligosaccharide chains attached to one protein can vary from 1 to 30 or more, with the sugar chains ranging from one or two residues in length to much larger structures. The glycan chain may be linear or branched. Many proteins contain more than one type of sugar chain; for instance, glycophorin, an important red cell membrane glycoprotein (see Chapter 53), contains both Oand N-linked oligosaccharides.

At least four subclasses of O-glycosidic linkages are found in human glycoproteins:
1. The N-acetylgalactosamine-Ser(Thr) linkage shown in Figure 46-1 is the predominant linkage. Usually a galactose or an N-acetylneuraminic acid residue is attached to the N-acetylgalactosamine, but many variations in the sugar compositions and lengths of such oligosaccharide chains are found. This type of linkage is found in mucins (see below).
2. Proteoglycans contain a galactose-galactose-xylose trisaccharide (the so-called link trisaccharide) attached to serine.
3. Collagens (see Chapter 50) contain a galactose-hydroxylysine linkage.
4, Many nuclear and cytosolic proteins contain side chains consisting of a single N-acetylglucosamine attached to a serine or threonine residue.

Mucins are glycoproteins that are highly resistant to proteolysis because the density of oligosaccharide chains makes it difficult for proteases to access the polypeptide chain. They help to lubricate and form a protective physical barrier on epithelial surfaces.
Mucins have two distinctive characteristics: a high con- tent of O-linked oligosaccharides (the carbohydrate content is generally more than 50%); and the presence of variable numbers of tandem repeats (VNTRs) of peptide sequence
in the center of the polypeptide chain, to which the O-glycan chains are attached in clusters. These tandem repeat sequences are rich in serine, threonine, and proline; indeed, up to 60% of the dietary requirement for threonine can be accounted for by the synthesis of mucins. Although O-glycans predominate, mucins often also contain a number of N-glycan chains.
Both secretory and membrane-bound mucins occur. Mucus secreted by the gastrointestinal, respiratory, and reproductive tracts is a solution containing about 5% mucins. Secretory mucins generally have an oligomeric structure, with monomers linked by disulfide bonds, and hence a very high molecular mass. Mucus has a high viscosity and often forms a gel because of its content of mucins. The high content of O-glycans confers an extended structure. This is partly explained by steric interactions between the N-acetylgalactosamine moieties and adjacent amino acids, resulting in a chain-stiffening effect, so that the conformation of mucins often become rigid rods. Intermolecular noncovalent interactions between sugars on neighboring glycan chains contribute to gel formation. The high content of N-acetylneuraminic acid and sulfate residues found in many mucins gives them a negative charge.
Membrane-bound mucins participate in cell-cell interactions, and may also mask cell surface antigens. Many cancer cells form large amounts of mucins that mask surface antigens and protect the cancer cells from immune surveillance. Mucins also carry cancer-specific peptide and carbohydrate epitopes. Some of these have been used to stimulate an immune response against cancer cells.

Because most glycoproteins are membrane-bound or secreted, their mRNA is usually translated on membrane-bound polyribosomes (see Chapter 37). The glycan chains are built up by the sequential donation of sugars from sugar nucleotides, catalyzed by glycoprotein glycosyltransferases. Because many glycosylation reactions occur within the lumen of the Golgi apparatus, there are carrier systems (permeases and transporters) to transport sugar nucleotides (UDP-galactose, GDP-mannose, and CMP-N-acetylneuraminic acid) across the Golgi membrane. They are antiporter systems; the influx of one molecule of sugar nucleotide is balanced by the efflux of one molecule of the corresponding nucleotide (UMP, GMP, or CMP).
There are 41 different types of glycoprotein glycosyltransferases. Families of glycosyltransferases are named for the sugar nucleotide donor, and subfamilies on the basis of the linkage formed between the sugar and the acceptor substrate; transfer may occur with retention or inversion of the conformation at C-1 of the sugar. Binding of the sugar nucleotide to the enzyme causes a conformational change in the enzyme that permits binding of the acceptor substrate. Glycosyltransferases show a high degree of specificity for the acceptor substrate,
typically acting only on the product of the preceding reaction. The different stages in glycan formation, and hence the different glycosyltransferases, are located in different regions of the Golgi, so that there is spatial separation of the steps in the process. Not all of the glycan chains of a given glycoprotein are complete; some are truncated, leading to microheterogeneity. No consensus sequence is known to determine which serine and threonine residues are glycosylated, but the first sugar moiety incorporated is usually N-acetylgalactosamine.

constituting the disaccharide N-acetyllactosamine. Repeating N-acetyllactosamine units—[Gal$1-3/4GlcNAcB1-3]n (poly-N-acetyllactosaminoglycans)—are often found on N-linked glycan chains. I/i blood group substances belong to this class. A bewildering number of chains of the complex type exist, and that indicated in Figure 46-2 is only one of many. Other complex chains may terminate in galactose or fucose.
High-mannose oligosaccharides typically have two to six additional mannose residues linked to the pentasaccharide core. Hybrid molecules contain features of both of the other classes.
N-Linked glycoproteins are the major class of glycoproteins, including both membrane-bound and circulating glycoproteins. They are distinguished by the presence of the asparagine—N-acetylglucosamine linkage (Figure 46-1). There are three major classes of N-linked oligosaccharides: complex, high-mannose, and hybrid. All three classes have the same branched pentasaccharide, Man,GIcNAc,, bound to asparagine, but differ in their outer branches (Figure 46-2). Complex oligosaccharides contain two, three, four, or five outer branches. The oligosaccharide branches are often referred to as antennae, so that bi-, tri-, tetra-, and pentaantennary structures may all be found. They generally contain terminal N-acetylneuraminic acid residues and underlying galactose and N-acetylglucosamine residues, the latter often

The synthesis of all N-linked glycoproteins begins with the synthesis of a branched oligosaccharide attached to dolichol pyrophosphate (Figure 46-3) on the cytosolic side of the endoplasmic reticulum membrane, which is then translocated to the lumen of the endoplasmic reticulum, where it undergoes further glycosylation, before the oligosaccharide chain is transferred by an oligosaccharyltransferase onto an asparagine residue of the acceptor apoglycoprotein as it enters the endoplasmic reticulum during synthesis on membrane-bound polyribosomes. This is thus a cotranslational modification. In many of the N-linked glycoproteins there is a consensus
FIGURE 46-3 The structure of dolichol. The group within the brackets is an isoprene unit (n = 17-20 isoprenoid units).
sequence of Asn-X-Ser/Thr (where X = any amino acid other than proline) to determine the site of glycosylation; in others there is no clear consensus sequence for glycosylation.
processing, forming complex chains and units on the other arm.
on one arm mannose
As shown in Figure 46-4, the first step is a reaction between UDP-N-acetylglucosamine and dolichol phosphate, forming N-acetylglucosamine dolichol pyrophosphate. A second N-acetylglucosamine is added from UDP-N-acetylglucosamine, followed by the addition of five molecules of mannose from GDP-mannose. The dolichol pyrophosphate oligosaccharide is then translocated into the lumen of the endoplasmic reticulum, and further mannose and glucose molecules are added, to form the final dolichol pyrophosphate oligosaccharide, using dolichol phosphate mannose and dolichol phosphate glucose as the donors. The dolichol pyrophosphate oligosaccharide is then transferred onto the acceptor asparagine residue of the nascent protein chain.

Calnexin is a chaperone protein in the endoplasmic reticulum membrane; binding to calnexin prevents a glycoprotein from aggregating. It is a lectin, recognizing specific carbohydrate sequences in the glycan chain of the glycoprotein. Incorrectly folded glycoproteins undergo partial deglycosylation, and are targeted to undergo transport from the endoplasmic reticulum back to the cytosol for catabolism.
To form high-mannose chains, the glucose and some of the peripheral mannose residues are removed by glycosidases. To form an oligosaccharide chain of the complex type, the glucose residues and four of the mannose residues are removed by glycosidases in the endoplasmic reticulum and Golgi, then N-acetylglucosamine, galactose, and N-acetylneuraminic acid are added in reactions catalyzed by glycosyltransferases in the Golgi apparatus. Hybrid chains are formed by partial
Calnexin binds to glycoproteins that possess a monoglycosylated core from which the terminal glucose residue has been removed, leaving only the innermost glucose attached. Calnexin and the bound glycoprotein form a complex with ERp57, a homolog of protein disulfide isomerase, which catalyzes disulfide bond interchange, facilitating correct folding. The bound glycoprotein is released from its complex with calnexin-ERp57 when the sole remaining glucose is hydrolyzed by a glucosidase and is then available for secretion if it is correctly folded. If not, a glucosyltransferase recognizes
FIGURE 46-4 Pathway of biosynthesis of dolichol pyrophosphate oligosaccharide. Note that the first five internal mannose residues are donated by GDP-mannose, whereas the more external mannose residues and the glucose residues are donated by dolichol-P-mannose and dolichol-P-glucose. (Dol, dolichol; GDP, guanosine diphosphate; P, phosphate; UDP, uridine diphosphate; UMP, uridine monophosphate.)

this and reglucosylates the glycoprotein, which rebinds to the calnexin-Erp57 complex. If it is now correctly folded, the glycoprotein is again deglucosylated and secreted. If it is not capable of correct folding, it is translocated out of the endoplasmic reticulum into the cytosol for catabolism. The glucosyltransferase senses the folding of the glycoprotein and only reglucosylates misfolded proteins. The soluble endoplasmic reticulum protein calreticulin performs a similar function to that of calnexin.

The glycosylation of glycoproteins involves a large number of enzymes; about 1% of the human genome codes for genes that are involved with protein glycosylation. There are at least 10 distinct N-acetylglucosamine transferases, and multiple isoenzymes of the other glycosyltransferases. Controlling factors in the first stage of N-linked glycoprotein biosynthesis (assembly and transfer of the dolichol pyrophosphate oligosaccharide) include not only the availability of the sugar nucleotides, but also the presence of suitable acceptor sites in proteins, the tissue concentration of dolichol phosphate, and the activity of the oligosaccharide: protein transferase.
Various cancer cells are found to synthesize different oligosaccharide chains from those made in normal cells (eg, greater branching). This is due to cancer cells expressing different patterns of glycosyltransferases from those in normal cells, as a result of specific gene activation or repression. The differences in oligosaccharide chains could affect adhesive interactions between cancer cells and the normal parent tissue cells, contributing to metastasis.

Membrane-bound proteins that are anchored to the lipid bilayer by a glycophosphatidylinositol (GPI) tail (Figure 46-1) constitute the third major class of glycoproteins. GPI linkage is the commonest way in which various proteins are anchored to cell membranes.
The proteins are anchored to the outer leaflet of the plasma membrane or the inner (luminal) leaflet of the membrane in secretory vesicles by the fatty acids of phosphatidylinositol. The phosphatidylinositol is linked via N-acetylglucosamine to a glycan chain containing a variety of sugars, including mannose and glucosamine. In turn, the oligosaccharide chain is linked via phosphorylethanolamine in an amide linkage to the carboxyl-terminal amino acid of the protein. Additional constituents are found in many GPI structures; for example, that shown in Figure 46-1 contains an extra phosphorylethanolamine attached to the middle of the three mannose moieties of the glycan and an extra fatty acid attached to glucosamine.
There are three functions of this GPI linkage:
1, The GPI anchor allows greatly enhanced mobility of a protein in the plasma membrane compared with that of a protein that contains transmembrane sequences. The GPI anchor is attached only to the outer leaflet of the lipid bilayer, so that it is freer to diffuse than a protein anchored through both layers of the membrane. Increased mobility may be important in facilitating rapid responses to stimuli.
2. Some GPI anchors may connect with signal transduction pathways, so that proteins that do not have a transmembrane domain may nevertheless be receptors for hormones and other cell surface signals.
3. GPI structures can target proteins to apical or basolateral domains of the plasma membrane of polarized epithelial cells.

The GPl anchor is preformed in the endoplasmic reticulum, and is then attached to the protein after ribosomal synthesis is complete. The primary translation products of GPI-anchored proteins have not only an amino-terminal signal sequence that directs them into the endoplasmic reticulum during synthesis, but also a carboxy-terminal hydrophobic domain that acts as the signal for attachment of the GPI anchor. The first stage in synthesis of the GPI anchor is insertion of the fatty acids of phosphatidylinositol into the luminal face of the endoplasmic reticulum membrane, followed by glycosylation, starting with esterification of N-acetyl-glucosamine to the phosphate group of phosphatidylinositol. A terminal phosphoethanolamine moiety is added to the completed glycan chain. The hydrophobic carboxy-terminal domain of the protein is displaced by the amino group of ethanolamine in the transamidation reaction that forms the amide linkage between the GPI anchor and an aspartate residue in the protein.

Many proteins, including nuclear pore proteins, proteins of the cytoskeleton, transcription factors, and proteins associated with chromatin, as well as nuclear oncogene proteins and tumor suppressor proteins, undergo rapidly reversible O-glycosylation with a single sugar moiety, N-acetylglucosamine. The serine and threonine sites of glycosylation are the same as those of phosphorylation of these proteins, and glycosylation and phosphorylation occur reciprocally in response to cellular signaling.
The O-linked N-acetylglucosamine transferase that catalyzes this glycosylation uses UDP-N-acetylglucosamine as the sugar donor, and has phosphatase activity, so can directly replace a serine or threonine phosphate with N-acetylglucosamine. There is no absolute consensus sequence for the reaction, but about half the sites that are subject to reciprocal glycosylation and phosphorylation are Pro-Val-Ser. O-linked N-acetylglucosamine transferase is activated by phosphorylation in response
to insulin action, and the N-acetylglucosamine is removed by N-acetylglucosaminidase, leaving the site available for phosphorylation.
Both the activity and peptide specificity of O-linked N-acetylglucosamine transferase depend on the concentration of UDP-N-acetylglucosamine. Depending on cell type, up to 2 to 5% of glucose metabolism is by way of the hexosamine pathway leading to N-acetylglucosamine formation, giving the O-linked N-acetylglucosamine transferase a role in nutrient sensing in the cell. Excessive O-glycosylation with N-acetylglucosamine (and hence reduced phosphorylation) of target proteins is implicated in insulin resistance and glucose toxicity in diabetes mellitus, as well as neurodegenerative diseases.

Glycation is the nonenzymic attachment of sugars (mainly glucose) to amino groups of proteins (and also to other molecules including DNA and lipids), unlike glycosylation which is enzyme-catalyzed. Initially, glucose forms a Schiff base to the amino terminal of the protein, which then undergoes the Amadori rearrangement to yield ketoamines (Figure 46-5), and further reactions to yield advanced glycation end-products (AGEs). The overall series of reactions is known as the Maillard reaction, which is involved in the browning of certain foodstuffs during storage or heating, and provides much of the flavor of some foods.
Advanced glycation end-products underlie tissue damage in poorly controlled diabetes mellitus. When the blood glucose concentration is consistently elevated, there is increased glycation of proteins. Glycation of collagen and other proteins in the extracellular matrix alters their properties (eg, increasing the cross-linking of collagen). Cross-linking can lead to
FIGURE 46-5 Formation of advanced glycation endproducts from glucose.
accumulation of various plasma proteins in the walls of blood vessels; in particular, accumulation of low-density lipoprotein (LDL) can contribute to atherogenesis. AGEs appear to be involved in both microvascular and macrovascular damage in diabetes mellitus. Endothelial cells and macrophages have AGE receptors on their surfaces; uptake of glycated proteins by these receptors can activate the transcription factor NF-kB (see Chapter 52), generating a variety of cytokines and proinflammatory molecules.
Nonenzymic glycation of hemoglobin A present in red blood cells leads to the formation of HbA... It occurs normally to a small extent, and is increased in diabetic patients with poor glycemic control, whose blood glucose concentration is consistently elevated. As discussed in Chapter 6, measurement of HbA, has become a very important part of the management of patients with diabetes mellitus.

As listed in Table 46-1, glycoproteins have many different functions, including transport molecules, immunological molecules, and hormones. They are also important in fertilization and inflammation; a number of diseases are due to defects in the synthesis and catabolism of glycoproteins.

To reach the plasma membrane of an oocyte, a sperm has to traverse the zona pellucida (ZP), a thick, transparent, envelope that surrounds the oocyte. The glycoprotein ZP3 is an O-linked glycoprotein that functions as a sperm receptor. A protein on the sperm surface interacts with the oligosaccharide chains of ZP3. By transmembrane signaling, this interaction induces the acrosomal reaction, in which enzymes such as proteases and hyaluronidase and other contents of the acrosome of the sperm are released. Liberation of these enzymes permits the sperm to pass through the zona pellucida and reach the plasma membrane of the oocyte. Another glycoprotein, PH-30, is important in binding of the sperm plasma membrane to that of the oocyte, and the subsequent fusion of the two membranes, enabling the sperm to enter and fertilize the oocyte.

Leukocytes play important roles in many inflammatory and immunological processes; the first steps are interactions between circulating leukocytes and endothelial cells prior to passage of the leukocytes out of the circulation. Leukocytes and endothelial cells contain cell surface lectins, called selectins, which participate in intercellular adhesion. Selectins are single-chain Ca**-binding transmembrane proteins; the
amino terminals contain the lectin domain, which is involved in binding to specific carbohydrate ligands.
Interactions between selectins on the neutrophil cell surface and glycoproteins on the endothelial cell trap the neutrophils temporarily, so that they roll over the endothelial surface. During this the neutrophils are activated, undergo a change in shape, and now adhere firmly to the endothelium. This adhesion is the result of interactions between integrins (see Chapter 53) on the neutrophils and immunoglobulin-related proteins on the endothelial cells. After adhesion, the neutrophils insert pseudopodia into the junctions between endothelial cells, squeeze through these junctions, cross the basement membrane, and are then free to migrate in the extravascular space.
Selectins bind sialylated and fucosylated oligosaccharides. Sulfated lipids (see Chapter 21) may also be ligands. Synthesis of compounds such as monoclonal antibodies that block selectin-ligand interactions may be therapeutically useful to inhibit inflammatory responses. Cancer cells often have selectin ligands on their surfaces, which may have a role in the invasion and metastasis of cancer cells.

Leukocyte adhesion deficiency II is a rare condition due to mutations affecting the activity of a Golgi-located GDP-fucose transporter. The absence of fucosylated ligands for selectins leads to a marked decrease in neutrophil rolling. Patients suffer life-threatening recurrent bacterial infections, and also psychomotor and mental retardation. The condition may respond to oral fucose.
Paroxysmal nocturnal hemoglobinuria is an acquired mild anemia characterized by the presence of hemoglobin in urine due to hemolysis of red cells, particularly during sleep, reflecting a slight drop in plasma pH during sleep, which increases susceptibility to lysis by the complement system (see Chapter 52). The condition is due to the acquisition in hematopoietic cells of somatic mutations in the gene coding for the enzyme that links glucosamine to phosphatidylinositol in the GPI structure. This leads to a deficiency of proteins that are anchored to the red cell membrane by GPI linkage. Two proteins, decay accelerating factor and CD59 normally interact with components of the complement system to prevent the hemolysis. When they are deficient, the complement system acts on the red cell membrane to cause hemolysis.
Some of the congenital muscular dystrophies are the result of defects in the synthesis of glycans in the protein a-dystroglycan. This protein protrudes from the surface membrane of muscle cells and interacts with laminin-2 (merosin) in the basal lamina. If the glycans of a-dystroglycan are not correctly formed (as a result of mutations in genes encoding some glycosyltransferases), this results in defective interaction of a-DG with laminin.

Rheumatoid arthritis is associated with an alteration in the glycosylation of circulating immunoglobulin G (IgG) molecules (see Chapter 52), such that they lack galactose in their Fc regions and terminate in N-acetylglucosamine. Mannosebinding protein, a lectin synthesized by liver cells and secreted into the circulation, binds mannose, N-acetylglucosamine, and some other sugars. It can thus bind agalactosyl IgG molecules, which subsequently activate the complement system, contributing to chronic inflammation in the synovial membranes of joints.
Mannose-binding protein can also bind sugars when they are present on the surfaces of bacteria, fungi, and viruses, preparing these pathogens for opsonization or for destruction by the complement system. This is an example of innate immunity, not involving immunoglobulins or T lymphocytes. Deficiency of this protein in young infants as a result of mutation renders them susceptible to recurrent infections.

Mannose 6-phosphate serves to target enzymes into the lysosome. I-cell disease is a rare condition characterized by severe progressive psychomotor retardation and a variety of physical signs, with death often occurring in the first decade of life. Cells from patients with I-cell disease lack almost all of the normal lysosomal enzymes; the lysosomes thus accumulate many different types of undegraded molecules, forming inclusion bodies. The patients' plasma contains very high activities of lysosomal enzymes, suggesting that the enzymes are synthesized but fail to reach their proper intracellular destination and are instead secreted. Lysosomal enzymes from normal individuals carry the mannose 6-phosphate recognition marker, cells from patients with I-cell disease lack the Golgi-located N-acetylglucosamine phosphotransferase. Two lectins act as mannose 6-phosphate receptor proteins; both function in the intracellular sorting of lysosomal enzymes into clathrincoated vesicles in the Golgi. These vesicles then leave the Golgi and fuse with a prelysosomal compartment.

Turnover of glycoproteins involves catabolism of the oligosaccharide chains catalyzed by a number of lysosomal hydrolases, including a-neuraminidase, B-galactosidase, B-hexosaminidase, aand §-mannosidases, a-N-acetylgalactosaminidase, a-fucosidase, endo-B-N-acetylglucosaminidase, and aspartylglucosaminidase. Genetic defects of these enzymes result in abnormal degradation of glycoproteins. The accumulation in tissues of partially degraded glycoproteins leads to various diseases. Among the best recognized of these are mannosidosis, fucosidosis, sialidosis, aspartylglucosaminuria, and Schindler
disease, due respectively to deficiencies of a-mannosidase, a-fucosidase, a-neuraminidase, aspartylglucosaminidase, and a-N-acetylgalactosaminidase.

A feature of glycans that explains many of their biological actions is that they bind specifically to proteins and other glycans. One reflection of this is their ability to bind some viruses, bacteria, and parasites.
Influenza virus A binds to cell surface glycoprotein receptor molecules containing N-acetylneuraminic acid via a hemagglutinin protein. The virus also has a neuraminidase that plays a key role in allowing elution of newly synthesized progeny from infected cells. If this process is inhibited, spread of the viruses is markedly diminished. Inhibitors of this enzyme (eg, zanamivir, oseltamivir) are now available for use in treating patients with influenza. Influenza viruses are classified according to the type of hemagglutinin (H) and neuraminidase (N) that they possess. There are at least 16 types of hemagglutinin and 9 types of neuraminidase. Thus, avian influenza virus is classified as HSN1.
Human immunodeficiency virus type 1 (HIV-1), the cause of AIDS, attaches to cells via one of its surface glycoproteins (gp 120) and uses another surface glycoprotein (gp 41) to fuse with the host cell membrane. Antibodies to gp 120 develop during infection by HIV-1, and there has been interest in using the protein as a vaccine. One major problem with this approach is that the structure of gp 120 can change relatively rapidly due to mutations, allowing the virus to escape from the neutralizing activity of antibodies directed against it.
Helicobacter pylori is the major cause of peptic ulcers. It binds to at least two different glycans present on the surfaces of epithelial cells in the stomach allowing it to establish a stable attachment site to the stomach lining. Similarly, many bacteria that cause diarrhea attach to surface cells of the intestinal mucosa via glycans present in glycoproteins or glycolipids. The attachment of the malarial parasite Plasmodium falciparum to human cells is mediated by a GPI present on the surface of the parasite.

. Describe the two phases of xenobiotic metabolism, the first involving mainly
hydroxylation reactions catalyzed by cytochromes P450 and the second conjugation reactions.
Describe the metabolic importance of glutathione.
effects. Describe how xenobiotics can have toxic, immunological, and carcinogenic

We are exposed to a wide variety of compunds that are foreign to the body (xenobiotics, from the Greek xenos = foreign); naturally occurring compounds in plant foods, and synthetic compounds in medicines, food additives, and environmental pollutants. Knowledge of the metabolism of xenobiotics is essential for an understanding of pharmacology, therapeutics, and toxicology. Many of the xenobiotics in plant foods have potentially beneficial effects (eg, acting as antioxidants, Chapter 45).
process of detoxification, sometimes the metabolites of compounds that are themselves inert or harmless are biologically active. This may be desirable, as in the activation of a prodrug to the active compound, or it may be undesirable, as in the formation of a carcinogen or mutagen from an inert precursor.
Understanding the mechanisms involved in xenobiotic metabolism will permit the development of transgenic microorganisms and plants containing genes that encode enzymes that can be used to render potentially hazardous pollutants harmless. Similarly, transgenic organisms may be used for biosynthesis of drugs and other chemicals.

The main xenobiotics of medical relevance are drugs, chemical carcinogens, naturally occurring compounds in plant foods, and a wide variety of compounds that have found their way into our environment, such as polychlorinated biphenyls (PCBs), and insecticides and other pesticides. Most of these compounds are metabolized, mainly in the liver. While the metabolism of xenobiotics is generally considered to be a
The metabolism of xenobiotics occurs in two phases. In phase 1, the major reaction involved is hydroxylation, catalyzed by enzymes that are monooxygenases or cytochromes P450. In addition to hydroxylation, these enzymes catalyze a wide range of other reactions, including deamination, dehalogenation, desulfuration, epoxidation, peroxygenation, and reduction. Reactions involving hydrolysis (eg, catalyzed by esterases) and other non-P450-catalyzed reactions also occur in phase 1.
Phase 1 metabolism renders compounds more reactive, introducing groups that can be conjugated with glucuronic acid, sulfate, acetate, glutathione, or amino acids in phase 2 metabolism. This produces polar compounds that are water-soluble and can therefore be excreted in urine or bile.
In some cases, phase 1 metabolic reactions convert xenobiotics from inactive to biologically active compounds. In these instances, the original xenobiotics are referred to as prodrugs or procarcinogens. Sometimes, additional phase 1 reactions (eg, further hydroxylation reactions) convert these active compounds into less active or inactive forms prior to conjugation. In other cases, it is the conjugation reactions that convert the active products of phase 1 reactions to inactive compounds, which are excreted.

The main reaction involved in phase 1 metabolism is hydroxylation, catalyzed by a family of enzymes known as monooxygenases or cytochromes P450. There are at least 57 cytochrome P450 genes in the human genome. Cytochrome P450 is a heme enzyme. It is so named because it was originally discovered when it was noted that preparations of microsomes (fragments of the endoplasmic reticulum) that had been chemically reduced and then exposed to carbon monoxide had an absorption peak at 450 nm.
At least half of the common drugs that we ingest are metabolized by isoforms of cytochrome P450. They also act on steroid hormones, carcinogens, and pollutants. In addition, cytochromes P450 are important in the metabolism of a number of physiological compounds—for example, the synthesis of steroid hormones (see Chapter 26) and the conversion of vitamin D to its active metabolite, calcitriol (see Chapter 44).
The overall reaction catalyzed by cytochrome P450 is:

The reaction mechanism is shown in Figure 12-6. NADPH-cytochrome P450 reductase catalyzes the transfer of electrons from NADPH to cytochrome P450. Reduced cytochrome P450 catalyzes the reductive activation of molecular oxygen, one atom of which becomes the hydroxyl group in the substrate and the other is reduced to water. Cytochrome b., another hemoprotein found in the membranes of the smooth endoplasmic reticulum (see Chapter 12), may be involved as an electron donor in some cases.

There is a systematic nomenclature for the cytochromes P450 and their genes, based on their amino acid sequence homology. The root CYP denotes a cytochrome P450. This is followed by number designating the family; cytochromes P450 are included in the same family if they exhibit 40% or more amino acid sequence identity. This is followed by a capital letter indicating the subfamily; P450s are in the same subfamily if they exhibit more than 55% sequence identity. The individual P450s are then assigned numbers in their subfamily. Thus, CYP1A1 denotes a cytochrome P450 that is a member of family 1 and subfamily A and is the first individual member of that subfamily. The nomenclature for the genes encoding cytochromes P450 is the same, except that italics are used; thus, the gene encoding CYP1A1 is CYPIAI. The major cytochromes P450 involved in drug metabo- lism are CYP1 (with 3 subfamilies), CYP2 (13 subfamilies),
and CYP3 (1 subfamily). The various cytochromes P450 have overlapping substrate specificities, so that a very broad range of xenobiotics can be metabolized by one or other of the enzymes.
Cytochromes P450 are present in greatest amount in liver cells and enterocytes. In liver and most other tissues, they are present mainly in the membranes of the smooth endoplasmic reticulum, which constitute part of the microsomal fraction when tissue is subjected to subcellular fractionation. In hepatic microsomes, cytochromes P450 can comprise as much as 20% of the total protein. In the adrenal gland, where they are involved in cholesterol and steroid hormone biosynthesis, they are found in mitochondria as well as in the endoplasmic reticulum (see Chapters 26 and 41).

Most isoforms of cytochrome P450 are inducible. For instance, the administration of phenobarbital or other drugs causes hypertrophy of the smooth endoplasmic reticulum and a threeto fourfold increase in the amount of cytochrome P450 within a few days. In most cases this involves increased transcription to mRNA. However, in some cases, induction involves stabilization of mRNA or the enzyme protein itself, or increased translation of existing mRNA.
Induction of cytochrome P450 underlies drug interactions, when the effects of one drug are altered by administration of another. For example, the anticoagulant warfarin is metabolized by CYP2C9, which is induced by phenobarbital. Induction of CYP2C9 will increase the metabolism of warfarin, so reducing its efficacy, so that the dose must be increased. CYP2E1 catabolizes the metabolism of some widely used solvents and compounds found in tobacco smoke, many of which are procarcinogens; it is induced by ethanol, so increasing the risk of carcinogenicity.
Naturally occurring compounds in foods may also affect cytochrome P450. Grapefruit contains a variety of furanocoumarins, which inhibit cytochrome P450 and so affect the metabolism of many drugs. Some drugs are activated by cytochrome P450, so that grapefruit will reduce their activity; others are inactivated by cytochrome P450, so that grapefruit increases their activity. Drugs that are affected include statins, omeprazole, antihistamines, and benzodiazepine antidepressants.
Polymorphism of
P450
may explain
much of the variation in drug responses by different patients; variants with low catalytic activity will lead to slower metabolism of the substrate, and hence prolonged drug action. CYP2A6 is involved in the metabolism of nicotine to conitine. Three CYP2A6 alleles have been identified: a wild type and two inactive alleles. Individuals with the alleles, who have impaired metabolism of nicotine, are apparently protected against becoming tobacco-dependent smokers. These individuals
smoke less, presumably because their blood and brain concentrations of nicotine remain elevated longer than those of individuals with the wild-type allele. It has been speculated that inhibiting CYP2A6 may provide a novel way to help smoking cessation.

In phase 1 reactions, xenobiotics are converted to more polar, hydroxylated derivatives. In phase 2 reactions, these derivatives are conjugated with molecules such as glucuronic acid, sulfate, or glutathione. This renders them even more watersoluble, and they are eventually excreted in the urine or bile.

The glucuronidation of bilirubin is discussed in Chapter 31; xenobiotics are glucuronidated in the same way, using UDPglucuronic acid (see Figure 20-4), catalyzed by a variety of glucuronosyltransferases, present in both the endoplasmic reticulum and cytosol. Molecules such as 2-acetylaminofluorene (a carcinogen), aniline, benzoic acid, meprobamate (a tranquilizer), phenol, and many steroid hormones are excreted as glucuronides. The glucuronide may be attached to oxygen, nitrogen, or sulfur groups of the substrates.

The sulfate donor in these and other biologic and sulfation reactions (eg, sulfation of steroids, glycosaminoglycans, glycolipids, and glycoproteins) is "active sulfate'—adenosine 3'-phosphate-5'-phosphosulfate (PAPS, see Chapter 24).

The tripeptide glutathione (y-glutamylcysteinylglycine) is important in the phase II metabolism of electrophilic compounds, forming glutathione S-conjugates that are excreted in urine and bile. The reaction catalyzed by glutathione S-transferases is:

where R is an electrophilic compound.
There are four classes of cytosolic glutathione S-transferase and two classes of microsomal membrane-bound enzyme, as well as a structurally distinct kappa class that is found in mitochondria and peroxisomes. Glutathione S-transferases are homoor heterodimers of at least seven different types of subunit, and different subunits are induced by different xenobiotics.
Because glutathione S-transferases also bind a number of ligands that are not substrates, including bilirubin, steroid hormones, and some carcinogens and their metabolites, they are sometimes known as ligandin. Glutathione S-transferase binds bilirubin at a site distinct from the catalytic site, transporting it from the bloodstream to the liver, then to the endoplasmic reticulum for conjugation with glucuronic acid, and excretion in the bile (see Chapter 31). Binding of carcinogens sequesters them, so preventing their actions on DNA.
The liver has a very high activity of glutathione S-transferase; in vitro the entire pool of glutathione can be depleted within minutes on exposure to a xenobiotic substrate. The activity of glutathione S-transferase is upregulated in many tumors, leading to resistance to chemotherapy.
Glutathione conjugates may be transported out of the liver, where they are substrates for extracellular y-glutamyltranspeptidase and dipeptidases. The resultant cysteine S-conjugates are taken up by other tissues (especially the kidney) and N-acetylated to yield mercapturic acids (Nacetylcysteine S-conjugates), which are excreted in the urine. Some hepatic glutathione S-conjugates enter the bile canniculi, where they are broken down to cysteine S-conjugates that are then taken up into the liver for N-acetylation, and re-excreted in the bile.
In addition to its role in phase 2 metabolism, glutathione has a number of other roles in metabolism:

1. It provides the reductant for the reduction of hydrogen peroxide to water in the reaction catalyzed by glutathione peroxidase (see Figure 20-3).
2. Itisanimportant intracellular reductant and antioxidant, helping to maintain essential SH groups of enzymes in their reduced state.
3. A metabolic cycle involving GSH as a carrier has been implicated in the transport of some amino acids across membranes in the kidney. The first reaction of the cycle is catalyzed by y-glutamyltransferase (GGT):
Amino acid + GSH > y-Glutamyl amino acid
+ Cysteinylglycine
This reaction transfers amino acids across the plasma membrane, the amino acid being subsequently hydrolyzed from its complex with glutamate and the GSH being resynthesized from cysteinylglycine. GGT is present in the plasma membrane of renal tubular cells and bile ductule cells, and in the endoplasmic reticulum of hepatocytes. It is released into the blood from hepatic cells in various hepatobiliary diseases, providing an early indication of liver damage.

The two most important reactions other than conjugation are acetylation and methylation.

where X represents a xenobiotic or its metabolite. As for other acetylation reactions, acetyl-CoA is the acetyl donor. These reactions are catalyzed by acetyltransferases present in the cytosol of various tissues, particularly liver. The drug isoniazid, used in the treatment of tuberculosis, is subject to acetylation. There is polymorphism of acetyltransferases, resulting in individuals who are classified as slow or fast acetylators. Slow acetylators are more subject to the toxic effects of isoniazid because the drug persists for longer. Some xenobiotics are subject to methylation by methyltransferases, employing S-adenosylmethionine (see Figure 29-17) as the methyl donor.

There are very few xenobiotics, including drugs, that do not have some toxic effects if the dose is large enough. The toxic effects of xenobiotics cover a wide spectrum, but can be considered under three general headings:
1. Covalent binding of xenobiotic metabolites to macromolecules including DNA, RNA, and protein can lead to cell injury (cytotoxicity), which can be severe enough to result in cell death. In response to damage to DNA, the DNA repair mechanism of the cell is activated. Part of this involves the transfer of multiple ADP-ribose units onto DNA-binding proteins, catalyzed by poly(ADP-ribose polymerase). The source of ADP-ribose is NAD, and in response to severe DNA damage there is considerable depletion of NAD. In turn, this leads to severely impaired ATP formation, and cell death.
2. The reactive metabolite of a xenobiotic may bind to a protein, acting as a hapten, and altering its antigenicity. On its own it will not stimulate antibody production, but does so when bound to a protein. The resultant antibodies react not only with the modified protein but also with the unmodified protein, so potentially initiating autoimmune disease.
3. Reactions of some activated xenobiotics with DNA are important in chemical carcinogenesis. Some chemicals (eg, benzo[a]pyrene) require activation by cytochrome P450 in the endoplasmic reticulum to become carcinogenic (they are therefore called indirect carcinogens). The activities of the xenobiotic-metabolizing enzymes present in the endoplasmic reticulum thus help to determine whether such compounds become carcinogenic or are "detoxified""

The enzyme epoxide hydrolase in the membranes of the endoplasmic reticulum can have protection against some carcinogens. The products of the action of cytochrome P450 on some procarcinogen substrates are epoxides. Epoxides are highly reactive and mutagenic or carcinogenic. As shown in Figure 47-1, the hydrolase catalyzes hydrolysis of epoxides to much less reactive dihydrodiols.

■, 1 = Xenobiotics are chemical compounds foreign to the body, including drugs, food additives, and environmental pollutants, as well as naturally occurring compounds in plant foods.. ■, 1 = Xenobiotics are metabolized in two phases. The major reaction of phase 1 is hydroxylation catalyzed by a variety of monooxygenases, known as the cytochromes P450. In phase 2, the hydroxylated species are conjugated with a variety of hydrophilic compounds such as glucuronic acid, sulfate, or glutathione. The combined operation of these two phases converts lipophilic compounds into water-soluble compounds that can be excreted in urine or bile.. ■, 1 = Cytochromes P450 catalyze reactions that introduce one atom of oxygen derived from molecular oxygen into the substrate, yielding a hydroxylated product, and the other into water. NADPHand NADPHcytochrome P450 reductase are involved in the reaction mechanism.. ■, 1 = Cytochromes P450 are hemoproteins and generally have a wide substrate specificity, acting on many exogenous and endogenous substrates. At least 57 cytochrome P450 genes are found in human tissue.. ■, 1 = Cytochromes P450 are generally located in the endoplasmic reticulum of cells, especially in the liver.. ■, 1 = Many cytochromes P450 are inducible. This has important implications for interactions between drugs.. ■, 1 = Phase 2 conjugation reactions are catalyzed by enzymes such as glucuronyltransferases, sulfotransferases, and glutathione S -transferases, using UDP-glucuronic acid, PAPS (active sulfate), and glutathione, respectively, as donors.. ■, 1 = Glutathione not only plays an important role in phase 2 reactions but also is an intracellular reducing agent.. ■, 1 = Xenobiotics can produce a variety of biological effects, including toxicity, immunological reactions, and cancer.

Explain the importance of laboratory tests in clinical and veterinary medicine.
Explain what is meant by the reference range for the results of a test.
Explain the difference between the precision and accuracy of an assay method, and explain the sensitivity and specificity of an assay method.
m= Explain what is meant by the sensitivity, specificity, and predictive value of a laboratory test.
= List techniques that are commonly used in a diagnostic lab carrying out biochemical tests and explain the principle of each method.
m= Explain why high plasma concentrations of enzymes are considered to be indicators of tissue damage.
=
Describe in outline the different requirements for measuring an enzyme ina plasma sample and using an enzyme to measure an analyte.

Various laboratory tests are an essential part of medicine and veterinary practice. Biochemical tests can be used for screening for disease, for confirmation (or otherwise) of a diagnosis made on clinical examination, for monitoring progression of a disease and the outcome of treatment. Blood and urine samples are most commonly used; occasionally feces, saliva, or cerebrospinal fluid (CSF) may be used, and on rare occasions, tissue biopsy samples. Most of our knowledge and understanding of the underlying causes of metabolic diseases and of the effects of disease on metabolism has come from analysis of metabolites in blood and urine, and from measurement of enzymes in blood. In turn, that knowledge has permitted advances in the treatment of disease and the development of more effective drugs.
Advances in technology mean that many tests that were formerly carried out only in specialist laboratories can now be performed at the bedside, in the doctor's office, or veterinary practice, sometimes even at home by patients themselves, with automated machines or "dipsticks" that are simple to use. Other tests are still conducted in hospital laboratories or by private clinical chemistry laboratories, with samples sent in by the referring physician. Some tests that are less commonly requested and may be technically more demanding are performed only in specialist centers. These often involve specialist techniques to study rare (and sometimes newly discovered) metabolic diseases. In addition, testing of samples from athletes and race horses for performance-enhancing drugs and other banned substances is normally carried out in only a limited number of specially licensed laboratories.

A great many different conditions can lead to abnormalities of the results of laboratory tests. Tissue injury that results in damage to cell membranes and an increase in the permeability of the plasma membrane leads to leakage of intracellular material into the bloodstream (eg, leakage of creatine kinase MB into the bloodstream following a myocardial infarction). In other cases, the synthesis of proteins and hormones is increased or decreased (eg, C-reactive protein in inflammatory states, or hormones in endocrine disorders). Kidney and liver failure lead to the accumulation of a number of compounds (eg, creatinine and bilirubin, respectively) in the blood, due to an inability of the organ concerned to excrete or metabolize the compound concerned.

For any compound that is measured (an analyte), there is a range of values around the average or mean that can be considered to be normal. This is the result of biological variations between individuals. In addition, day-to-day or week-to-week variations can occur in the results for the same individual. Therefore, the first step in establishing any new laboratory test for screening for, or diagnosis of, disease, or monitoring treatment, is to determine the range of results in a population of healthy people. For some tests, this will also mean determining the normal ranges of analytes in people of different ages. The normal range of some analytes will differ between men and women, and there may be differences between different ethnic groups to be considered as well.
If the results obtained for a target healthy population group (depending on age, gender, and perhaps ethnicity) are statistically normally distributed (ie, the results show a symmetrical gaussian distribution around the mean), then the acceptable or normal range is taken to be + 2x standard deviation around the mean. This range includes 95% of the target population, and is known as the reference range. Values outside the reference range are considered to be abnormal, meriting further investigation. If the results from the healthy population are not statistically normally distributed, but skewed, then a further step of statistical manipulation is required before the 95% reference range can be established.
For some tests, the results from different laboratories will differ, usually because they use different methods of measurement. Each laboratory establishes its own set of reference ranges for the analyses it performs. Some laboratories report the results as the value; others report results as the number of standard deviations away from the mean—the socalled Z-score. This allows the physician to see how far from the mean the result is—in other words, how abnormal it is. Sometimes the results will be reported as 5 or 10 (or more) times above the upper limit of normal.

The use of the 95% range as the reference range has an unfortunate consequence. By chance, 5% of the "normal" results will be outside the reference range. This first became apparent in the 1970s, when multichannel analysers were developed that were capable of determining 20 or more analytes in each sample. Almost every sample gave one result that was outside the reference range, but if the same person gave a sample a few days later, that apparently abnormal result was now within the reference range, although by chance the result for another analyte might now be outside the reference range.

Diagnostic laboratories are subject to inspection and regulatory procedures to assess the validity of their results and ensure quality control of their reports. Such measures will ensure that the value of the concentration, activity, or amount of a substance in a specimen reported represents the best value obtainable with the method, reagents, and instruments used.
In establishing a new test, or a new method, four questions have to be answered:
1. How precise is the method? This is a measure of the reproducibility of the method. If the same sample is analyzed many times over, how much variation will be seen in the results obtained? Figure 48-1 illustrates this. In this example, one set of results is much more precise than the other (there is a difference between the two in the spread of results around the mean), even though they have the same mean result. Precision is not absolute, but subject to variations inherent in the complexity of the method used, the stability of reagents, the sophistication of the equipment used for the assay, and the skill of the technicians involved.
2. How accurate is the result? This is a measure of how close the result is to the true value. Figure 48-2 shows the results of assays by two different methods or by the same method but in two different laboratories. Both have similar precision, but their mean values are very different. It is not possible to say from this information which laboratory is correct (and this is part of the reason why laboratories establish their own reference ranges). There are a number of national or regional quality control schemes in which all participating laboratories are sent the same (pooled) blood or urine sample. Each laboratory measures the various analytes in the pooled sample. The results obtained by all laboratories are plotted as a distribution curve. The mean of these values is calculated and considered to be the "true value." Such a quality control scheme allows each participating laboratory to determine how close its results are to the "true value"
Number of results
Concentration of analyte

FIGURE 48-1 Precision of an analytical method. The graph shows the results of an analyte measured multiple times in the same sample, either by two different analytical methods or by the same method in two different laboratories. In both cases, the mean result is the same. However, one method or laboratory, shown in blue, has a low scatter of results, and hence a low standard deviation, and high precision, while the other, shown in red, has a high scatter of results, a high standard deviation, and low precision.
analyte
FIGURE 48-2 Accuracy of an analytical method. Two different analytical methods, performed on multiple samples, or the same method performed in two different laboratories, with the same scatter of results, and hence the same standard deviation and the same precision. However, the mean values of analytes obtained for the two methods or laboratories are very different; it is not possible to tell which result is closer to the true value.
3. Howsensitive is the method? In other words, how little of the analyte can be determined reliably? What is the lower limit of reliable detection? This is obviously important when results below the reference range are clinically significant, or when samples are being analyzed for narcotics or performance-enhancing substances that are banned in competitive sport.
4. How specific is the method? This question deals with the issue of confidence that the assay is actually measuring the analyte of interest. For example, the now obsolete method of measuring glucose in blood or urine used an alkaline copper (Cu'*) solution, which was reduced to Cu* by glucose. However, other reducing compounds in urine or blood, such as xylose or vitamin C, also reduce glucose giving a falsely high value. Modern methods of measuring glucose depend on the enzyme glucose oxidase, which only reacts with glucose, and so is highly specific. One of the products of the action of glucose oxidase on glucose is hydrogen peroxide; the second step in the assay is to reduce the hydrogen peroxide produced to water and oxygen, using peroxidase. A colorless compound that turns blue when it is oxidized by the oxygen produced is also present in the assay medium. High concentrations of vitamin C, as would be seen when the patient is taking vitamin supplements, reduce the dye back to its colorless form, so giving a false-negative result (Figure 48-3).

The above four criteria must be established for each analytical method. In addition, the clinical value of the test has to be
FIGURE 48-3 Specificity of an analytical method. Measurement of blood glucose by two methods. Chemical reduction of Cu?* in alkaline solution will detect not only glucose, but any other reducing sugar and other substances such as vitamin C. Enzymic oxidation of glucose using glucose oxidase is a specific reaction; no other compound will be oxidized and contribute to the value obtained.
established by taking into consideration its sensitivity, specificity, and positive and negative predictive values (Table 48-1). Here, unfortunately, the same two terms, sensitivity and specificity, are used, but with very different meanings from those used in establishing the analytical method.
The sensitivity of a test is the percentage of positive test results in patients with the disease ("true positive"). For example, the test for phenylketonuria is highly sensitive; a positive result is obtained in all who have the disease (100% sensitivity). By contrast, the carcinoembryonic antigen (CEA) test for carcinoma of the colon has lower sensitivity; only 72% of those with carcinoma of the colon test positive when the disease is extensive, and only 20% with early disease.
The specificity of a test is the percentage of negative test results among people who do not have the disease. The test for phenylketonuria is highly specific; 99.9% of normal individuals give a negative result; only 0.1% gives a false-positive result. By contrast, the CEA test has a variable specificity; about 3% of nonsmoking individuals give a false-positive result (97% specificity), whereas 20% of smokers give a falsepositive result (80% specificity).
The sensitivity and specificity of a test are inversely related to each other. If the cutoff point is set too high, then very few healthy people will give a false-positive result, but many people with the disease may give a false-negative result. The sensitivity will thus be low, but the specificity will be high. Conversely, if the cutoff point is too low, then almost all people with the disease will be detected (the test will have a high sensitivity), but more disease-free people may give a false-positive result (the test will have a low specificity).

The predictive value of a positive test (positive predictive value) is the percentage of positive results that are true positives. Similarly, the predictive value of a negative test (negative predictive value) is the percentage of negative results that are true

negatives. This is related to the prevalence of the disease. For example, in a group of patients in a urology ward, the prevalence of renal disease is higher than in the general population. In this group, the serum concentration of creatinine will have a higher predictive value than in the general population. Formulae for calculating sensitivity, specificity, and predictive values of a diagnostic test are shown in Table 48-1.

The usual samples for analysis are blood and urine. Blood is collected into tubes with or without an anticoagulant, depending on whether plasma or serum is required. Less commonly, samples of saliva, CSF, or feces may be used.
require either serum or plasma. For a serum sample, the blood is allowed to clot, then the red cells and fibrin clot are removed by centrifugation. For a plasma sample, the blood is collected into a tube containing an anticoagulant, and the red cells are removed by centrifugation. The difference between serum and plasma is that plasma contains prothrombin and the other clotting factors, including fibrinogen, while serum does not. Different anticoagulants (citrate, EDTA or oxalate, all of which chelate calcium and so inhibit coagulation) are used for collection of plasma samples, depending on the assay to be performed. Heparin, which acts by activating antithrombin III, is also used. For measurement of blood glucose, potassium fluoride is added, as an inhibitor of glycolysis by red blood cells.
There is a difference between measurement of an analyte in a blood sample and in urine. The concentration of an analyte in blood reflects levels at the time the sample was taken, whereas a urine sample represents the cumulative excretion of the analyte over a period of time. A further difference is that it is usual to report results of blood tests as amount of analyte (or enzyme activity) per milliliter or liter of blood (or plasma or serum). Reporting the concentration of the analyte in urine in the same way is not useful, since urine volume depends very largely on fluid intake. In some cases, the patient is asked to provide a complete 24-hour urine sample; this is a tedious procedure, and it is difficult to know whether there really has been a complete 24-hour collection. Alternatively, the concentration of the analyte is reported per mol of creatinine. Creatinine excretion is reasonably constant from day to day for any one individual, but varies between individuals because it depends mainly on muscle mass; creatinine is formed nonenzymically from creatine and creatine phosphate, most of which is in skeletal muscle.

Apart from measurement of blood gases, for which arterial samples are required, blood samples are usually of venous blood. Blood glucose is often measured in capillary blood from a finger prick. Some analyses use whole blood; others

Most routine clinical chemistry reactions involve linking a chemical or enzymic reaction to the development of a colored product that is measured by absorption spectrophotometry. Different compounds absorb light at different wavelengths; the energy of the absorbed light excites electrons to an unstable orbital. The absorbance of light at a specific wavelength in the visible or ultraviolet range is directly proportional to the concentration of the colored end-product, and hence to the concentration of the analyte in the sample. Although at one time such analyses were performed manually, nowadays most assays are automated, and a single instrument can carry out multiple assays on a single sample.
In absorption spectrophotometry, the excited electrons return to their basal state in a series of small quantum jumps, emitting the energy absorbed as heat. For some compounds the electrons return to a lower energy state in a single quantum jump, emitting light of a higher wavelength (lower energy) than the exciting light. This is fluorescence, and the technique is known as fluorescence spectrophotometry or
spectrophotofluorimetry. The sample is illuminated with light of a specific wavelength, and the light emitted is measured, at right angles to the direction of the illuminating wavelength. Again, the intensity of the fluorescence is proportional to the concentration of the fluorophore, and hence the concentration of the analyte. Fluorimetry permits both greater specificity and sensitivity of the assay. The specificity is greater than for absorption spectrophotometry because both the exciting wavelength and the emitted wavelength are specific for the fluorophore, while for absorption spectrophotometry there is only one wavelength to be set, that of the light that is absorbed. Fluorimetry is more sensitive because it is easier to detect the emission of a small amount of light than the absorption.
Increasingly, especially in research and specialist centers, multiple analytes are measured in the same sample using high-pressure liquid chromatography to separate analytes, followed by colorimetric, fluorimetric, or electrochemical detection, or linked to mass spectrometry to identify compounds. Such methods form the basis of metabolomics, the study of a whole array of metabolites in a single sample, and metabonomics, the study of changes in analytes in response to a drug or experimental treatment of some kind.

Historically, electrolytes such as sodium and potassium were measured by flame photometry, measuring the light emitted when the ion was introduced into a clear flame. Sodium gives a yellow flame and potassium a purple one. Nowadays these and other ions are measured using ionspecific electrodes. In some cases, metal ions are measured by atomic absorption spectrometry. Here the sample is introduced into a flame, and illuminated at a specific wavelength. The light energy absorbed excites electrons to an unstable orbital, and the absorption of light is directly proportional to the concentration of the element in the sample, as is the case for absorption spectrophotometry.

Enzymes are important in clinical chemistry in three different ways: to measure analytes in a sample; to measure the activity of enzymes themselves in a sample; and as a test of vitamin nutritional status.
Using an enzyme to measure the concentration of an analyte confers a high degree of specificity on the assay, since in general an enzyme will act on only a single substrate, or a small range of closely related substrates, while a simple chemical reaction may well respond to a variety of (possibly unrelated) analytes. For example, as shown in Figure 48-3, a variety of reducing compounds will react with an alkaline copper reagent to give a false-positive result for glucose, whereas the enzymic assay using glucose oxidase will only give a positive result for glucose, and not other reducing compounds.
When an enzyme is used to detect an analyte, the limiting factor in the assay must be the analyte itself; the enzyme and other reagents must be present in excess. More importantly, the concentration of the analyte in the sample must be adjusted to be below the K,, of the enzyme, so that there is a large change in the rate of reaction with a small change in the concentration of the analyte (region A in Figure 48-4).
When cells are damaged or die, their contents leak out into the bloodstream. Measurement of enzymes in plasma can therefore be used to detect tissue damage; information is obtained from the pattern of enzymes (and tissue-specific isoenzymes) released. The extent of the increase in enzyme activity in plasma above the normal range often indicates the severity of tissue damage. When an assay is to determine the activity of an enzyme in plasma, the limiting factor must be the enzyme itself. The concentration of substrate added must be considerably in excess of the K,, of the enzyme, so that the enzyme is acting at or near V_, and even a relatively large change in the concentration of substrate does not have a significant effect on the rate of reaction (region B in Figure 48-4). In practice, this means that the concentration of substrate added is about 20-fold higher than the K,, of the enzyme.

If an enzyme has a vitamin-derived coenzyme that is essential for activity, then measurement of the activity of the enzyme in red blood cells with and without added coenzyme can be used as an index of vitamin nutritional status. This provides an indication of functional nutritional status, while measurement of the vitamin and its metabolites may reflect recent intake rather than physiological adequacy. The underlying assumption is that red blood cells have to compete with other tissues in the body for what may be a limited supply of the coenzyme. Therefore, the extent to which the red cell enzyme
FIGURE 48-4 Use of enzymes to measure analytes and measurement of enzyme activity in biologic samples. At concentrations of substrate (analyte) at or below the K_, of the enzyme (region A in the graph), there is a very sharp increase in the rate of reaction with a small change in the concentration of analyte, so the enzyme-linked assay has greatest sensitivity over this range of concentration. At concentrations of substrate considerably above the K,, of the enzyme, as the enzyme is approaching V._,, (region B in the graph), it is the amount of enzyme in the sample that is limiting for the rate of formation of product, so that this is the appropriate range of substrate concentration to use for measurement of enzyme activity in a biological sample.

is saturated with its coenzyme will reflect the availability of the coenzyme over a period of time corresponding to the halflife of red cells. The assay consists of incubating two samples of the red cell lysate: one that has been preincubated with, and one without, addition of the coenzyme. Then substrate is added to both, and the activity of the enzyme is measured. In the sample preincubated without addition of the coenzyme, only that enzyme that had coenzyme bound (the holoenzyme) will be active. In the sample that was preincubated with the coenzyme, any apoenzyme (inactive enzyme protein without bound coenzyme) will have been activated to the holoenzyme. There is, therefore, always either no change in enzyme activity on addition of coenzyme, indicating complete saturation of the enzyme with coenzyme, or an increase in activity, reflecting the activation of the apoenzyme by added coenzyme. The results are reported as an enzyme activation coefficient (the ratio of activity in the sample preincubated with coenzyme: that without). Reference ranges for the activation coefficient are established in the same way as for any other test. Such enzyme activation assays are available for thiamin (vitamin B,, using red cell transketolase), riboflavin (vitamin B,, using red cell glutathione reductase), and vitamin B, (using one or the other of the red cell transaminases).

If there is a protein that will bind the analyte, and the bound and free analyte (ligand) can be separated and measured, then it is possible to devise an assay for the analyte. Perhaps the simplest such ligand-binding assay is that for the hormone cortisol, which is transported in the bloodstream bound to a specific cortisol-binding globulin. It is easy to prepare a plasma sample containing the binding globulin that has been stripped of its ligand (cortisol) by incubation with aluminum oxide or charcoal. This is done using a relatively large pooled plasma sample, and provides binding globulin for a large number of assays. The hormone is extracted from each sample to be analyzed, using an organic solvent, evaporated to dryness, then dissolved in ethanol and a suitable buffer, with the addition of a tracer amount of high specific activity radioactive hormone. Each sample is then incubated with the binding globulin at 37°C, then cooled to 4°C. Charcoal is added to adsorb the unbound ligand, and rapidly removed by centrifugation. The radioactivity in the supernatant is measured. This gives the amount of bound ligand, and is expressed as a percentage of the total radioactivity added to each sample. A standard curve is constructed using known amounts of hormone, so that the concentration of hormone in the samples can be determined. A wide variety of other hormones and other analytes can be measured in the same way, by raising either monoclonal antibodies or polyclonal antisera against the analyte, for example, by injecting the analyte covalently bound to a protein into an animal. The antiserum against a hormone raised in a single rabbit can be used for many thousands of assays. Each batch of antiserum must, of course, be tested for its specificity for the hormone (ensuring that it does not also bind related hormones, especially a problem with steroid hormones), and for its sensitivity. When the binding protein is an antibody or antiserum, the assay is usually called a radioimmunoassay. In a variant of the competitive binding assay, the antibody is bound covalently to the surface of beads. It is then easy to separate the bound and free ligand simply by washing the beads with ice-cold buffer, leaving the bound ligand attached to the beads for measurement of bound radioactivity. Alternatively, the antibody may

be bound covalently to the surface of the test tube, or to each well in a multiwell plate. After incubation, a sample of the incubation medium is taken for measurement of the radioactivity that is not bound.
Increasingly, in order to minimize exposure to radioactive materials, fluorescently labeled ligand or antibody is used. A further development is the sandwich assay, in which two different antibodies against the ligand are used, each of which binds to a different region (epitope) of the analyte. The first antibody is covalently bound to the surface of each well of a multiple well plate, and the sample is added and incubated. After removal of the incubation medium and washing each well, the second antibody is added, sandwiching the analyte between the two antibodies. The second antibody is labeled with a radioactive isotope or a fluorophore, thus permitting measurement of the bound second antibody, and hence bound ligand. In some cases, the second antibody is labeled with an enzyme, and measurement of bound second antibody, and hence bound ligand, is by measurement of the activity of the enzyme that is now bound to the wall of each cell of the plate, after washing to remove unbound second antibody and adding an excess of the enzyme substrate. This is the enzyme- linked immunosorbent assay (ELISA).

For a number of assays, the enzymes or antibodies and reagents can be combined on a plastic strip. For measurement of blood glucose, a finger-prick blood sample is placed on the test strip that contains glucose and the reagents shown in Figure 48-3. The intensity of the blue color formed, and hence the concentration of glucose, is measured using a handheld device called the glucometer. This provides a simple and reliable method to estimate glucose at the bedside in a hospital ward, a doctor's clinic or even at home. For urine testing, several different assays can be included as separate pellets on a plastic stick called a dipstick—for example, to detect or semiquantitatively estimate levels of glucose, ketone bodies, protein, and several other analytes at the same time. Similar dipsticks are available to detect human chorionic gonadotropin (hCG) in urine, as a home pregnancy test.

Many of the inborn errors of metabolism can lead to very severe mental retardation if treatment is not initiated early enough. For
conditions such as phenylketonuria and maple syrup urine disease, dietary restriction of the amino acids that are not metabolized normally (phenylalanine in phenylketonuria; the branched-chain amino acids leucine, isoleucine, and valine in maple syrup urine disease) is essential for management of the condition. Therefore, it is usual in most developed countries to screen neonates for such conditions. The concentration of the offending amino acid(s) is measured in a blood sample that is normally taken a week after birth, when the enzymes that are affected in the disease should have reached full expression. Most commonly, a capillary blood sample is taken by heel prick, and is blotted onto absorbent paper to be sent to the for

The first such screening test for an inborn error of metabolism was the Guthrie bacterial inhibition test. A disk from the paper containing the blood sample is laid onto an agar plate that has been seeded with a phenylalanine-requiring strain of Bacillus subtilis, together with a competitive inhibitor of phenylalanine uptake into the bacteria (6-thienylalanine) at such a concentration that it will compete with phenylalanine at levels normally found in blood, so that the bacteria will not grow. If the concentration of phenylalanine is more than that usually found in blood, it will be taken up by the bacteria despite the inhibitor, and the bacteria will form visible colonies on the agar. In most centers, the bacterial inhibition test has been superseded by chromatographic techniques that permit the detection of a variety of abnormal metabolites, and hence the detection of a variety of different inborn errors of metabolism.

Tests that provide information on the functioning of particular organs are often grouped together as organ function tests. Such grouped tests include tests of kidney, liver, and thyroid function.

A complete urinalysis includes assessment of the physical and chemical characteristics of urine. Physical characteristics to be assessed include urine volume (this requires a timed urine sample, usually 24 hours), odor, color, appearance (clear or turbid), specific gravity, and pH. Protein, glucose, blood, ketone bodies, bile salts, and bile pigments are abnormal constituents of urine that appear in different disease conditions. Urea and creatinine are excreted in urine; their serum concentrations can be used as markers of renal function because the serum concentration increases as renal function deteriorates. Creatinine is a better marker of renal function than urea because its blood concentration is not significantly affected by nonrenal factors, thus making it a specific indicator of renal function; a number of factors affect blood urea concentration.
Normally, less than 150 mg of protein, and less than 30 mg of albumin, is excreted in urine per 24 hours. This is below the limit of detection by routine tests. Presence of protein in excess of this is referred to as proteinuria and is a sign of renal disease. The most common cause of proteinuria is loss of integrity of the glomerular basement membrane (glomerular proteinuria), as seen in nephrotic syndrome and diabetic nephropathy. The major protein found in glomerular proteinuria is albumin. Microalbuminuria is defined as the presence of 30 to 300 mg of albumin in a 24-hour urine sample. It is an marker of renal in diabetes mellitus.
early damage Even though serum creatinine is a marker of renal function, a significant increase in the blood concentration is seen only when there has been about a 50% decline in the glomerular filtration rate (GFR). Measurement of serum creatinine is therefore a test with poor sensitivity. Measurement of creatinine clearance gives an estimate of the GFR, and so can be used to detect the early stages of renal failure. Clearance is the volume of plasma from which a compound is completely cleared by the kidney in unit time. It is calculated by the formula:

where U is concentration of the measured analyte in a timed sample of urine (usually 24 hours); P is plasma concentration of the analyte; and V is volume of urine produced per minute (calculated by dividing the volume of urine collected over 24 hours by [24 x 60]).
A compound that is useful for measurement of renal clearance has a fairly constant blood concentration, is excreted only in urine, is freely filtered at the glomerulus, and is neither reabsorbed nor secreted by the renal tubules. Although creatinine clearance is commonly measured, it overestimates GFR because it is secreted by the renal tubules to a small extent. Inulin clearance satisfies all the criteria essential for a compound to be used in clearance tests. However, unlike creatinine, inulin is an exogenous compound that has to be infused intravenously at a constant rate.

Liver function tests (LFTs) are a group of tests that help in diagnosis, assessing prognosis and monitoring therapy of liver disease. Each test assesses a specific aspect of liver function. An increase in serum bilirubin occurs due to many causes, and results in jaundice. In obstruction of the bile duct (obstructive jaundice), it is mainly conjugated bilirubin that increases. In hepatocellular disease, both conjugated and unconjugated bilirubin are elevated, reflecting the inability of the liver to take up, conjugate, and excrete bilirubin into the bile (see Chapter 31). Total serum protein and albumin levels are low in chronic liver diseases, such as cirrhosis. Prothrombin time (see Chapter 55) may be prolonged in acute disorders of the liver because of impaired synthesis of coagulation factors. The activities of alanine (ALT) and aspartate (AST)
serum aminotransferases (see Chapter 28) are significantly elevated several days before onset of jaundice in acute viral hepatitis. ALT is considered to be more specific for liver disease than AST, because AST is elevated in cases of cardiac or skeletal muscle injury while ALT is not. Serum alkaline phosphatase activity is elevated in obstructive jaundice. A high activity of serum alkaline phosphatase is also seen in bone disease.

The thyroid gland secretes the thyroid hormones—thyroxine or tetraiodothyronine (T,) and triiodothyronine (T,). Diseases associated with increased or decreased synthesis of thyroid hormones (hyperthyroidism and hypothyroidism, respectively) occur commonly. A clinical diagnosis of a thyroid disorder is confirmed by measurement of serum thyroid-stimulating hormone (thyrotropin, TSH) and free thyroxine and triiodothyronine. The concentration of total serum thyroxine can be affected by changes in the concentration of thyroid-binding globulin in the absence of thyroid disease. Total thyroxine is seldom measured nowadays, because assays to measure free thyroxine are available.

A clinical diagnosis of adrenal hyperfunction (Cushing syndrome) or hypofunction (Addison disease) is confirmed by adrenal function tests. Secretion of cortisol from the adrenal gland shows diurnal variation; serum cortisol is highest during the early morning hours and lowest around midnight. Loss of this diurnal variation is one of the earliest signs of adrenal hyperfunction. Measurement of serum cortisol in blood samples drawn at midnight and 8 am is, therefore, useful as a test. A diagnosis of adrenal hyperfunction is confirmed by demonstration of failure of suppression of the early morning concentration of cortisol following the administration of 1 mg dexamethasone (a potent synthetic glucocorticoid) at midnight; this is the dexamethasone suppression test.

As discussed in Chapter 25, plasma total cholesterol, and especially the ratio of LDL:HDL cholesterol provides an index of the risk of developing atherosclerosis. The plasma lipoproteins were originally separated by centrifugation, hence their classification by density. Later methods involved separation by electrophoresis. Nowadays, total plasma cholesterol is measured, then lipoproteins containing apoprotein B (see Table 25-1) are precipitated using a divalent cation, allowing measurement of the cholesterol associated with high-density lipoprotein (HDL).
An electrocardiogram may not always show typical changes following a myocardial infarction. In such a situation, elevation in serum levels of cardiac troponin or creatine kinase MB isoenzyme provides confirmation of the occurrence of a myocardial infarction, as both of these proteins are specific to cardiac muscle.

=
Laboratory tests can provide useful information for diagnosis and treatment of disease as well as providing information about normal metabolism and the pathology of disease.
™ The reference range of an analyte is the range + 2 x standard deviations around the mean value for the population group under consideration. Values outside this reference range are suggestive of an abnormality that merits further investigation.
@ The precision of an analytical method is a measure of its
reproducibility;
the accuracy
of
a
method is
a
measure of
how close
the result
is to
the true
value.

@ The sensitivity of an analytical method is a measure of how
little of the analyte can be detected. The specificity is the extent to which other compounds present in the sample may give a false-positive result.
= The sensitivity of a test refers to the percentage of patients with the disease who will give a positive result. The specificity of a test is the percentage of patients without the disease who will give a negative result.
™ Samples for analysis are usually blood and urine, although saliva, feces, and CSF may also be used. Blood samples may be collected in tubes containing an anticoagulant (for plasma samples) or without (for serum samples).
= Many laboratory tests rely on production of a colored or fluorescent product that can be measured by absorption spectrophotometry or fluorimetry.
™ Many compounds can be measured by high-pressure liquid chromatography, sometimes in conjunction with mass spectrometry. The measurement of a large number of analytes in a sample is the basis of metabolomics, and of metabonomics, which is the effect of a disease, drug, or other treatment on metabolism.
assay
™ Enzymes may be used to provide sensitive and specific methods for analytes. In this case, there must be an excess of enzyme in the sample, so that the limiting factor is the concentration of the analyte in the sample.
™
Many enzymes are released into the bloodstream from dying cells in disease, and their measurement can give useful diagnostic and prognostic information. In order to determine the activity of an enzyme in a sample, there must be an excess of substrate, so that the limiting factor is the amount of enzyme present.
™
Many analytes (and especially hormones) are measured by competitive binding assays, using either a naturally occurring binding protein or an antiserum or monoclonal antibody to bind the ligand. Trace amounts of high specific activity radioactive ligand, or fluorescently labelled ligand or binding protein are used.

Lab Tests Online: www.labtestsonline.org (A comprehensive web site provided by the American Association of Clinical Chemists that provides accurate information on many laboratory tests). MedlinePlus: http://www.nlm.nih.gov/medlineplus/encyclopedia. html (The A.D.A.M. Medical Encyclopedia includes over 4000 articles about diseases, lab tests and other matters).

1. Which of the following will be elevated in the bloodstream about
lto 2 hours after eating a high-fat meal?
moO Ketone bodies Nonesterified fatty acids
R> Chylomicrons
. Wh ich of the following will be elevated in the bloodstream about
4to 5 hours after eating a high-fat meal?
BOOS
- Whi ich of the following is the best definition of glycemic index?
> The increase in the blood concentration of glucagon after consuming the food compared with that after an amount of white bread.
. Wh ich of the following will have the highest glycemic index?
BOOP
. Wh ich one of the following statements concerning chylomicrons
isC CORRECT?
B. The core of chylomicrons contains triacylglycerol and
High density lipoprotein
Very low-density lipoprotein
equivalent
The increase in the blood concentration of glucose after consuming the food.
. The increase in the blood concentration of glucose after
consuming the food compared with that after an equivalent amount of white bread.
The increase in the blood concentration of insulin after
consuming the food.
The increase in the blood concentration of insulin after consuming the food compared with that after an equivalent amount of white bread.
ich of the following will have the lowest glycemic index?
A baked apple
A baked potato
An uncooked apple
An uncooked potato
Apple juice
An
uncooked
apple
. The enzyme hormone-sensitive lipase acts on chylomicrons
Chylomicron remnants differ from chylomicrons in
triacylglycerol.
.
Plant
sterols
and

stanols
inhibit
the
of
from the gastrointestinal tract. Which of the following best
describes how they act?
A. They are incorporated into chylomicrons in place of
B. They compete with cholesterol for esterification in the
intestinal lumen, so that less cholesterol is esterified.
. They compete with cholesterol for esterification in the
mucosal
cell,
and
unesterified
is
actively
transported out of the cell into the intestinal lumen.
They compete with cholesterol for esterification in
the mucosal cell, and unesterified cholesterol is not
incorporated into chylomicrons.
They displace cholesterol from lipid micelles, so that it is not
available for absorption.
. Which one of following statements about energy metabolism is
CORRECT?
A. Adipose tissue does not contribute to basal metabolic rate
B. Physical activity level (PAL) is the sum of physical activity
(BMR).
ratios for different activities throughout the day, multiplied by the time spent in each activity, expressed as a multiple of
BMR.
. Physical activity ratio (PAR) is the energy cost of physical
activity throughout the day.
Resting metabolic rate (RMR) is the energy expenditure of
the body when asleep.
The energy cost of physical activity can be determined by
measuring respiratory quotient (RQ) production during the
activity.
. A patient with metastatic colorectal cancer has lost 6 kg of body
weight
over
the
last
month.
Which
of
the
following
is
the
best
explanation for her weight loss?
A. Because of the tumour she is oedematous.
B. Chemotherapy has caused nausea and loss of appetite.
Cc. Her basal metabolic rate has fallen as a result of protein
catabolism caused by tumour necrosis factor and other
cytokines.
Her basal metabolic rate (BMR) has increased as a result of
anaerobic glycolysis in the tumour and the energy cost of
gluconeogenesis from the resultant lactate in her liver.
The tumour has a very high energy requirement for cell proliferation.

10. A 5-year-old child arriving at a refugee center in East Africa is
stunted in growth (only 89% of expected height for age) but not
oedematous. Would you consider him to be:
A. Suffering from kwashiorkor
B. Suffering from marasmic kwashiorkor
C. Suffering from marasmus
D. Suffering from undernutrition
E. Underfed but not considered to be clinically malnourished
11. A 5-year-old child arriving at a refugee center in East Africa is
stunted
in
growth
(only
55%
of
expected
height
for
age)
but
not
Suffering from kwashiorkor
Suffering from marasmic kwashiorkor
Suffering from marasmus
Suffering from undernutrition
Underfed but not considered to be clinically malnourished
mopOR>
12. Which of the following is the definition of nitrogen balance?
A. Protein intake as a percentage of total energy intake
B. The difference between protein intake and excretion of nitrogenous compounds
C. The ratio of excretion of nitrogenous compounds/protein
intake
D. The ratio of protein intake/excretion of nitrogenous
compounds
E. The sum of protein intake and excretion of nitrogenous
compounds
13. Which one of following statements about nitrogen balance is
CORRECT?
A. Ifthe intake of protein is greater than requirements, there
will always be positive nitrogen balance.
B. In nitrogen equilibrium, the excretion of nitrogenous
is
greater
than
the
dietary
intake
of
nitrogenous
C. In positive nitrogen balance, the excretion of nitrogenous
metabolites is less than the dietary intake of nitrogenous
D. Nitrogen balance is the ratio of intake of nitrogenous
compounds/output of nitrogenous metabolites from the body.
E. Positive nitrogen balance means that there is a net loss of
protein from the body.
14. Inaseries of experiments to determine amino acid requirements,
healthy young adult volunteers were fed mixtures of amino acids

as their sole protein source. Which of the following mixtures
would lead to negative nitrogen balance (assuming that all other amino acids are provided in adequate amounts)?
One lacking alanine, glycine, and tyrosine >
One lacking arginine, glycine, and cysteine
One lacking asparagine, glutamine, and cysteine
One lacking lysine, glycine, and tyrosine
One lacking proline, alanine, and glutamate
BOOP
15. Which of the following vitamins provides the cofactor for
reduction reactions in fatty acid synthesis?
16. Deficiency of which one of these vitamins is a major cause of blindness worldwide?
17.
18.
19. Which one of the following criteria of vitamin adequacy can be
20. Which of the following is the best definition of the reference
21. What percentage of the population will have met their
Vitamin A Vitamin B,, Vitamin D Vitamin K BOOP
Vitamin
B,
Deficiency of which one of these vitamins may lead to
megaloblastic anaemia?
Vitamin B, Vitamin B,, Vitamin E Vitamin K moUOR>
Vitamin
D
Which one of the following criteria of vitamin adequacy can
be defined as “There are no signs of deficiency under normal
conditions, but any trauma or stress reveals the precarious state of the body reserves and may precipitate clinical signs”?
A. Abnormal response to a metabolic load
B. Clinical deficiency disease
C. Covert deficiency
D. Incomplete saturation of body reserves
E. Subclinical deficiency
defined as metabolic abnormalities under normal conditions?
Abnormal response to a metabolic load >
Clinical deficiency disease
Covert deficiency
Incomplete saturation of body reserves
Subclinical deficiency
BOR
nutrient intake (RNI) or recommended daily amount (RDA), of a
vitamin
or
mineral?
A. One standard deviation above the average requirement of
the population group under consideration
B. One standard deviation below the average requirement of
C. The average requirement of the population group under consideration
D.
Two standard deviations above the average requirement of
E. Two standard deviations below the average requirement of the under consideration
population group

requirement for a vitamin or mineral if their intake is equal to the
RNI or RDA?
2.5%
5% e>
50% 95% HOO
97.5%
Folate
Niacin
Thiamin
Vitamin B,
BOOP
22. What percentage of the population will have met their
23. What percentage of the population will have met their
24.
25. For a person whose intake of a vitamin or mineral is equal to the
26. For a person whose intake of a vitamin or mineral is equal to the
27. Which one of the following is NOT a source of oxygen radicals?
28. Which one of the following provides protection against oxygen
lower reference nutrient intake (LRNI)?
2.5% >
5%
50%
95%
97.5%
BOOR
average requirement?
2.5%
5% mopOR>
50%
95%
97.5%
For a person whose intake of a vitamin or mineral is equal to the
average
requirement,
what
is
the
probability
that
this
level
of
intake is adequate to meet his/her individual requirement?
2.5% >
5% BOOR
50%
95%
97.5%
LRNI,
what
is
the
that
this
level
of
intake
is
adequate
to meet his/her individual requirement?
2.5%
5% BOOR
50%
95%
97.5%
>
RNI, what is the probability that this level of intake is adequate to
meet his/her individual requirement?
2.5% >
5% BOO
50%
95%
97.5%
Action of superoxide dismutase >
Activation of macrophages
Nonenzymic reactions of transition metal ions
Reaction of b-carotene with oxygen
Ultraviolet radiation
BOO
radical damage to tissues?
Action of superoxide dismutase

Reaction of B-carotene with oxygen
BOOP
29. Which one of the following is NOT the result of oxygen radical
30. Which of the following types of oxygen radical damage may lead
31. Which of the following types of oxygen radical damage may
32. Which of the following types of oxygen radical damage may lead
33. Which of the following types of oxygen radical damage may lead
34.
35. Which of the following best describes the glycome?
action?
A. Activation of macrophages
B. Modification of bases in DNA
C. Oxidation of amino acids in apoproteins of LDL
D. Peroxidation of unsaturated fatty acids in membranes
E. Strand breaks in DNA
to the development of autoimmune thyroid disease?
Chemical modification of DNA bases in somatic cells
Chemical modification of DNA in germ-line cells
Oxidation of amino acids in cell membrane proteins
Oxidation of amino acids in mitochondrial proteins
Oxidation of unsaturated fatty acids in plasma lipoproteins
moUOR>
lead to the development of atherosclerosis and coronary heart
disease?
Chemical modification of DNA bases in somatic cells >
Oxidation
of
amino
acids
in
cell
membrane
proteins
BOOR
to the development of cancer?
A. Chemical modification of DNA bases in somatic cells
B. Chemical modification of DNA in germ-line cells
C. Oxidation of amino acids in cell membrane proteins
D. Oxidation of amino acids in mitochondrial proteins
E. Oxidation of unsaturated fatty acids in plasma lipoproteins
to the development of hereditary mutations?
BOOR
Which
one
of
the
following
best
explains
the
action
of vitamin E?
A. It forms a stable radical that can be reduced back to active

vitamin E by reaction with vitamin C.
B. It isa radical, so that when it reacts with another radical, a
nonradical product is formed.
C. It is converted to a stable radical by reaction with vitamin C.
D.
It
is
lipid
soluble
and
can
react
with
free
radicals
in
the
blood plasma resulting from nitric oxide (NO) formation by
vascular endothelium.
E. Oxidized vitamin E can be reduced back to active vitamin E
by reaction with glutathione and glutathione peroxidase.
A. The DNA coding for glycosyltransferases
B.
The full complement of all carbohydrates in the body
C.
The full complement of free sugars in cells and tissues
D.
The full complement of glycoproteins and glycolipids in the
body
E. The full complement of glycosyltransferases in the body
36. Which of the following methods CANNOT be used to determine
37. Which of the following is NOT a function of glycoproteins?
38. Which of the following is NOT a constituent of glycoproteins?
39, Which of the following is used as a sugar donor in the synthesis
40. Which of the following is NOT used as a sugar donor in
41. Which of the following best describes the attachment of the
42. Which of the following is NOT a glycoprotein?
the structures of glycoproteins?
Carbohydrate microarrays
Degradation using endo- and exoglycosidases
Genome analysis
Sepharose-lectin chromatography
BOOP
Anchoring proteins at the cell surface Protecting plasma proteins against clearance by the liver Providing a transport system for folate into cells DORE
Providing a transport system for uptake of low-density
lipoprotein into the liver
E. Providing cell surface recognition signals
Fucose >
Galactose
Glucose
Mannose
Sucrose
BOOR
of the common pentasaccharide of N-linked glycoproteins?
CMP-N-acetylneuraminic acid
Dolichol pyrophosphate N-acetylglucosamine

Dolichol pyrophosphate-mannose
GDP-fucose
UDP-N-acetylglucosamine
BOOP
the synthesis of N-linked glycoproteins in the endoplasmic
reticulum?
Dolichol pyrophosphate fructose
Dolichol pyrophosphate galactose
Dolichol pyrophosphate mannose
Dolichol pyrophosphate N-acetylneuraminic acid
BOOP
common pentapeptide to the apoprotein in synthesis of an
N-linked glycoprotein?
A. Direct glycation of the amino terminal amino acid of the
peptide
B. Displacement of the amino terminal region of the peptide in
a transamidation reaction
C. Displacement of the amino terminal region of the peptide in
a transamination reaction
D. Displacement of the carboxy terminal region of the peptide
in a transamidation reaction
E. Displacement of the carboxy terminal region of the peptide
in a transamination reaction
Collagen >
Immunoglobulin G
Serum albumin
Thyroid-stimulating hormone
Transferrin
BOOP
43. Which one of the following statements is INCORRECT?
44, Which of the following is NOT an activity of cytochrome P450?
45. Which of the following best describes the reaction ofa
46. Which of the following is the preferred lipid component of the
47. Which of the following best describes the drug interactions
A. Calnexin ensures the correct folding of glycoproteins in the
endoplasmic reticulum.
B. Dolichol-pyrophosphate oligosaccharide donates all of the
sugars found in N-linked glycoproteins.
C. Mucins contain predominantly O-linked glycans.
D. N-Acetylneuraminic acid is commonly found at the termini
of N-linked sugar chains of glycoproteins.
E. O-linked sugar chains of glycoproteins are built up by the
stepwise addition of sugars from sugar nucleotides.
Activation of vitamin D

Hydroxylation of steroid hormone precursors
Hydroxylation of xenobiotics
Hydroxylation of retinoic acid
Methylation of xenobiotics
mopOR>

RH + O, + NADP* > R-OH + H,O + NADPH RH + O, + NAD* > R-OH + H,O + NADH RH + O, + NADPH > R-OH + H,O + NADP* RH + O, + NADPH > R-OH + H,O, + NADP* BOOP
RH + O, + NADH > R-OH + H,O + NAD*
cytochrome P450 system?
Dolichol phosphate >
Phosphatidylethanolamine
Phosphatidylinositol
Phosphatidylserine
BOR
between phenobarbital and warfarin?
A.
Phenobarbital induces CYP2C9, and this results in
decreased catabolism of warfarin.
B. Phenobarbital induces CYP2C9, and this results in
increased catabolism of warfarin.
C. Phenobarbital represses CYP2C9, and this results in
D. Warfarin induces CYP2C9, and this results in decreased
catabolism of phenobarbital.
E. Warfarin induces CYP2C9, and this results in increased
48. Which of the following best describes the effects of
49. Which of the following is NOT a function of glutathione?
50. Which of the following best describes the reference range for a
Sl. Which of the following statements about laboratory tests is
52. Which of the following is CORRECT when an enzyme is used to
polymorphisms of CYP2A6?
A. People with the active allele are less likely to become
tobacco-dependent smokers because this cytochrome
inactivates nicotine to cotinine.
B. People with the inactive (null) allele are less likely to become
C. People with the inactive (null) allele are less likely to become
activates nicotine to cotinine.

D. People with the inactive (null) allele are more likely
to become tobacco-dependent smokers because this
cytochrome inactivates nicotine to cotinine.
E. People with the inactive (null) allele are more likely to become tobacco-dependent smokers because this cytochrome activates nicotine to cotinine.
A. Coenzyme for the reduction of hydrogen peroxide
B. Conjugation of bilirubin
C.
Conjugation of some products of phase I metabolism of
xenobiotics
D. Transport of amino acids across cell membranes
E. Transport of bilirubin in the bloodstream
laboratory test?
A range +1x standard deviation around the mean value
A range +1.5x standard deviation around the mean value
A range +2x standard deviation around the mean value
A range +2.5x standard deviation around the mean value
A range +3x standard deviation around the mean value
BOOP
A. The predictive value of a test is the extent to which it will
correctly predict whether or not a person has the disease.
he sensitivity and specificity of a test are inversely related. B. T
e sensitivity of a test is a measure of how many people TI Cc.
ith the disease will give a positive result. wi
D. The specificity of a test is a measure of how many people
wi ith the disease will give a positive result.
TI e specificity of a test is a measure of how many people
wi ithout the disease will give a negative result.
measure an analyte in a blood sample?
A. The concentration of substrate must be about 20-times the
K,, of the enzyme.
B. The concentration of substrate must be equal to the K,, of the
enzyme.
C. The concentration of substrate must be equal to or lower
than
the
K,,
of
the
enzyme.
D. The concentration of the substrate in the assay is not
E. The concentration of substrate must be about 1/20th of the
K, of the enzyme.
53. Which of the following is CORRECT when an enzyme is being measured in a blood sample?
54. Which of the following best explains the use of enzyme activation

55. Which of the following would be used to prepare serum from a
56. Which of the following would be used to take a blood sample for
57. Which of the following best explains the difference between
A. The concentration of substrate must be about 20 times the
enzyme.
than the K,, of the enzyme.
assays to assess vitamin nutritional status?
A. Adding the vitamin-derived cofactor to the incubation
converts previously inactive apoenzyme into active
holoenzyme.
B. Adding the vitamin-derived cofactor to the incubation
converts previously inactive holoenzyme into active
apoenzyme.
C.
Adding
the
vitamin-derived
cofactor
to
the
incubation
converts previously active holoenzyme into inactive
D. Adding the vitamin-derived cofactor to the incubation
converts previously active apoenzyme into inactive
E. Adding the vitamin-derived cofactor to the incubation leads
to a reduction in enzyme activity.
blood sample?
A plain tube
A tube containing citrate
A tube containing EDTA
A tube containing oxalate
An evacuated tube to exclude oxygen
BOOP
blood gas analysis?
A plain tube >
BOOR
creatinine clearance and inulin clearance as tests of renal
function?
A.
Creatinine
clearance
is
higher
than
inulin
clearance
because
creatinine is actively secreted in the distal renal tubules.
B. Creatinine clearance is higher than inulin clearance because
inulin is actively secreted in the proximal renal tubules.
C. Creatinine clearance is higher than inulin clearance because
inulin is actively secreted in the distal renal tubules.
D. Creatinine clearance is lower than inulin clearance because

E. Creatinine clearance is lower than inulin clearance because inulin is not completely filtered at the glomerulus.

Kathleen M. Botham, PhD, DSc &
C
H
A
PT
ER
Robert K. Murray, MD, PhD
= Indicate that many proteins are targeted by signal sequences to their correct destinations and that the Golgi apparatus plays an important role in sorting proteins.
m™ Recognize that specialized signals are involved in sorting proteins to
mitochondria, the
nucleus, and
to peroxisomes.
= Explain that N-terminal signal peptides play a key role in directing newly synthesized proteins into the lumen of the endoplasmic reticulum (ER).
m= Explain how chaperones prevent faulty folding of other proteins, how misfolded proteins are disposed of, and how the ER acts as a quality control compartment.
Explain the role of ubiquitin as a key molecule in protein degradation.
Recognize the important role of transport vesicles in intracellular transport.
Indicate that many diseases result from mutations in genes encoding proteins involved in intracellular transport.

Proteins are synthesized on polyribosomes, but perform varied functions at different subcellular locations, including the cytosol, specific organelles, or membranes. Yet others are destined for export. Thus, there is considerable intracellular traffic of proteins. As recognized by Blobel in 1970, to enable proteins to attain their proper locations, their structure contains a signal or coding sequence that targets them appropriately. This led to the identification of numerous specific signals (Table 49-1), and also the recognition that certain diseases result from mutations that adversely affect these signals. This chapter will discuss the sorting and intracellular traffic of proteins and briefly consider some of the disorders that result when abnormalities occur.

The protein biosynthetic pathways in cells can be considered to be one large sorting system. Many proteins carry signals (usually but not always specific sequences of amino acids) that direct them to their specific subcellular destinations; these signals are a fundamental component of the sorting system. Usually, the signal sequences are recognized and interact with complementary areas of other proteins that serve as receptors which recognize the signals.

N-terminal signal peptide, Organelle Targeted = ER. Carboxyl-terminal KDEL sequence (Lys-Asp-Glu-Leu) in ER-resident proteins in COPI vesicles, Organelle Targeted = Lumen of ER. Diacidic sequences (eg, Asp-X-Glu) in membrane proteins in COPII vesicles, Organelle Targeted = Golgi membranes. Amino terminal sequence (20-50 residues), Organelle Targeted = Mitochondrial matrix. NLS (eg, Pro 2 -Lys 3 -Arg-Lys-Val), Organelle Targeted = Nucleus. PTS (eg, Ser-Lys-Leu), Organelle Targeted = Peroxisome. Mannose 6-phosphate, Organelle Targeted = Lysosome
Abbreviations: ER, endoplasmic reticulum; NLS, nuclear localization signal; PTS, peroxisomal-matrix targeting sequence.
A major sorting decision is made early in protein biosynthesis, when specific proteins are synthesized either on cytosolic (free) or membrane-bound polyribosomes (see Chapter 37). The signal hypothesis was proposed by Blobel and Sabatini in 1971 partly to explain the distinction between free and membrane-bound polyribosomes. They proposed that proteins synthesized on membrane-bound polyribosomes contain an N-terminal signal peptide, which causes them to become attached to the membranes of the ER, and facilitates protein transfer into the ER lumen. On the other hand, proteins synthesized on free polyribosomes lack the signal peptide and retain free movement in the cytosol. An important aspect of the signal hypothesis is that all ribosomes have the same structure, and that the distinction between membrane-bound and free ribosomes depends solely on the former carrying proteins that have signal peptides. Because many membrane proteins are synthesized on membrane-bound polyribosomes, the signal hypothesis plays an important role in concepts of membrane assembly. ER regions containing attached polyribosomes are called the rough ER (RER), and the distinction between the two types of ribosomes results in two branches of the protein-sorting pathway, called the cytosolic branch and the RER branch (Figure 49-1).

Proteins synthesized by cytosolic polyribosomes are directed to mitochondria, nuclei, and peroxisomes by specific signals, or remain in the cytosol if they lack a signal. Any protein that contains a targeting sequence that is subsequently removed is designated as a preprotein. In some cases, a second peptide is also removed, and in that event the original protein is known as a preproprotein (eg, preproalbumin; see Chapter 52).
Proteins synthesized and sorted in the RER branch (Figure 49-1) include many destined for various membranes (eg, of the ER, Golgi apparatus [GA], plasma membrane [PM]), and also lysosomal enzymes. In addition, proteins for export from the cell via exocytosis (secretion) are synthesized via this route. These various proteins may thus reside in
the membranes or lumen of the ER, or follow the major transport route of intracellular proteins to the GA. In the secretory or exocytotic pathway, proteins are transported from the ER > GA > PM and then released into the external environment. Secretion may be constitutive, meaning that transport occurs continuously, or regulated, where transport is switched on and off as required. Proteins destined for the GA, the PM, certain other sites, or for constitutive secretion are carried in transport vesicles (Figure 49-2) (see also below). Other proteins which are subject to regulated secretion are carried in secretory vesicles (Figure 49-2). These are particularly prominent in the pancreas and certain other glands. Passage of enzymes to the lysosomes using the mannose 6-phosphate signal is described in Chapter 46.

The GA plays two major roles in protein synthesis. First, it is involved in the processing of the oligosaccharide chains of membrane and other N-linked glycoproteins and also contains enzymes involved in O-glycosylation (see Chapter 46). Second, it is involved in the sorting of various proteins prior to their delivery to their appropriate intracellular destinations. The GA consists of cis(facing the ER), medial and transcisternae (membrane stacks), and the trans-Golgi network (TGN) (Figure 49-2). All parts of the GA participate in the first role, whereas the TGN is particularly involved in the second and is very rich in vesicles.

Molecular chaperones are proteins which stabilize unfolded or partially folded intermediates, allowing them time to fold properly, preventing inappropriate interactions, thus combating the formation of nonfunctional structures. Most chaperones exhibit ATPase activity and bind ADP and ATP. This activity is important for their effect on protein folding.
FIGURE 49-2 The rough ER branch of protein sorting. Newly synthesized proteins are inserted into the ER membrane or lumen from membrane-bound polyribosomes (small black circles studding the cytosolic face of the ER). Proteins that are transported out of the ER are carried in COPII vesicles to the cis-Golgi (anterograde transport). Proteins move through the Golgi as the cisternae (membrane sac-like structures) mature. In the trans-Golgi network (TGN), the exit side of the Golgi, proteins are segregated and sorted. For regulated secretion, proteins accumulate in secretory vesicles, while proteins destined for insertion in the plasma membrane for constitutive secretion are carried to the cell surface in transport vesicles. Clathrin-coated vesicles are involved in endocytosis, carrying cargo to late endosomes and to lysosomes. Mannose 6-phosphate (not shown; see Chapter 46) acts as a signal for transporting enzymes to lysosomes. COPI vesicles transport protein from GA the to the ER (retrograde transport) and may be involved in some intra-Golgi transport. Cargo normally passes through the ER-Golgi intermediate complex (ERGIC) compartment to the GA. (Courtesy of E Degen.)
The ADP-chaperone complex often has a high affinity for the unfolded protein, which, when bound, stimulates the replacement of ADP with ATP. The ATP-chaperone complex, in turn, releases segments of the protein that have folded properly, and the cycle involving ADP and ATP binding is repeated until the protein is released. Chaperones are required for the correct targeting of proteins to their subcellular locations. A number of important properties of these proteins are listed in Table 49-2.

studied in detail. It is polymeric, has two ring-like structures, each composed of seven identical subunits, and again ATP is involved in its action. The heat shock protein Hsp60 is the equivalent of GroEL in eukaryotes.
Chaperonins are the second major class of chaperones. They form complex barrel-like structures in which an unfolded protein is sequestered away from other proteins, giving it time and suitable conditions in which to fold properly. The structure of the bacterial chaperonin GroEL has been

Proteins synthesized via the cytosolic sorting branch either contain an uptake signal, enabling them to taken up into the correct subcellular organelle, or, if they are destined for the

cytosol, they have no targeting signal. Specific uptake signals direct proteins to the mitochondria, nucleus, and peroxisomes (Table 49-1). Since protein synthesis is complete before transport occurs, these processes are termed posttranslational translocation.

Mitochondria contain many proteins. Thirteen polypeptides (mostly membrane components of the electron transport chain) are encoded by the mitochondrial (mt) genome and synthesized in that organelle using its own proteinsynthesizing system. However, the vast majority (at least several hundreds) are encoded by nuclear genes, and synthesized outside the mitochondria on cytosolic polyribosomes, and must be imported. Most progress has been made in the study of proteins present in the mitochondrial matrix, such as the ATP synthase subunits (see Chapter 13). Only the pathway of import of matrix proteins will be discussed in any detail here.
Matrix proteins must pass from cytosolic polyribosomes through the outer and inner mitochondrial membranes to reach their destination. Passage through the two membranes is called translocation. They have an amino terminal leader sequence (presequence), about 20 to 50 amino acids in length (Table 49-1), which is amphipathic and contains many hydrophobic and positively charged amino acids (eg, Lys or Arg). The presequence is equivalent to a signal peptide mediating attachment of polyribosomes to membranes of the ER (see below), but in this instance targeting proteins to the matrix. Some general features of the passage of a protein from the cytosol to the mitochondrial matrix are shown in Figure 49-3. Translocation occurs posttranslationally, after the matrix preproteins are released from the cytosolic polyribosomes. Interactions with a number of cytosolic proteins that act as chaperones (see below) and as targeting factors occur prior to translocation.
Two distinct
translocation complexes
are situated
in the outer and inner mitochondrial membranes, referred to (respectively) as translocase of the outer membrane (TOM)

and translocase of the inner membrane (TIM). Each complex has been analyzed and found to be composed of a number of proteins, some of which act as receptors (eg, Tom20/22) for the incoming proteins and others as components (eg, Tom40) of the transmembrane pores through which these proteins must pass. Proteins must be in the unfolded state to pass through the complexes, and this is made possible by ATPdependent binding to several chaperone proteins including Hsp70 (Figure 49-3). In mitochondria, chaperones are involved in translocation, sorting, folding, assembly, and degradation of imported proteins. A proton-motive force across the inner membrane is required for import; it is made up of the electric potential across the membrane (inside negative) and the pH gradient (see Chapter 13). The positively charged leader sequence may be helped through the membrane by the negative charge in the matrix. In addition, close apposition at contact sites between the outer and inner membranes is necessary for translocation to occur.
The presequence is split off in the matrix by a matrix protease. Contact with other chaperones present in the matrix is essential to complete the overall process of import. Interaction with mt-Hsp70 (mt = mitochondrial; Hsp = heat shock protein; 70 = ~70 kDa) ensures proper import into the matrix and prevents misfolding or aggregation, while interaction with the mt-Hsp60-Hsp10 system ensures proper folding. The interactions of imported proteins with the above chaperones require hydrolysis of ATP to drive them.
The above describes the major pathway of proteins destined for the mitochondrial matrix. However, certain proteins insert into the outer mitochondrial membrane facilitated by the TOM complex. Others remain in the intermembrane space, and some insert into the inner membrane. Yet others proceed into the matrix and then return to the inner membrane or intermembrane space. A number of proteins contain two signaling sequences—one to enter the mitochondrial matrix and the other to mediate subsequent relocation (eg, into the inner membrane). Certain mitochondrial proteins do not contain presequences (eg, cytochrome c, which locates in the intermembrane space), and others contain internal presequences. Overall, proteins employ a variety of mechanisms and routes to attain their final destinations in mitochondria.

General features that apply to the import of proteins into organelles, including mitochondria and some of the other organelles to be discussed below, are summarized in Table 49-3.

It has been estimated that more than a million macromolecules per minute are transported between the nucleus and the cytoplasm in an active eukaryotic cell. These macromolecules include histones, ribosomal proteins and ribosomal subunits,
FIGURE 49-3 Entry of a protein into the mitochondrial matrix. After synthesis on cytosolic polyribosomes, an unfolded protein containing a matrix-targeting sequence interacts with the cytosolic chaperone Hsp70 and then with mitochondria (mt) via the receptor translocon of the outer membrane (Tom) 20/22. Transfer to the import channel Tom 40 followed by translocation across the outer membrane is the next step. Transport across the inner mt membrane occurs via a complex comprising Tim (translocon of the inner membrane) 23 and Tim 17 proteins. On the inside of the inner mt membrane, the protein interacts with the matrix chaperone Hsp 70, which in turn interacts with membrane protein Tim 44. The hydrolysis of ATP by mt Hsp70 probably helps drive the translocation, as does the electronegative interior of the matrix. The targeting sequence is subsequently cleaved by the matrix protease, and the imported protein assumes its final shape, sometimes with the prior aid of interaction with an mt chaperonin. At the site of translocation, the outer and inner mt membranes are in close contact. OMMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane. (Modified, with permission, from Lodish H, et al: Molecular Cell Biology, 6th ed. W.H. Freeman & Co, 2008.)

Source: Data from McNew JA, Goodman JM: The targeting and assembly of peroxisomal proteins: some old rules do not apply. Trends Biochem Sci 1998;21:54. Reprinted, with permission, from Elsevier.
transcription factors,
and mRNA
molecules.
The transport
is bidirectional
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at the NPC. Another family of proteins called Ran plays a critical regulatory role in the interaction of the complex with the NPC and in its translocation through the NPC. Ran proteins are small monomeric nuclear GTPases and, like other GTPases, exist in either GTP-bound or GDP-bound states. They are themselves regulated by guanine nucleotide exchange factors (GEFs), which are located in the nucleus, and Ran GTPase-accelerating proteins (GAPs), which are predominantly cytoplasmic. The GTP-bound state of Ran is favored in the nucleus and the GDP-bound state in the cytoplasm. The conformations and activities of Ran molecules vary depending on whether GTP or GDP is bound to them (the GTP-bound state is active; see discussion of G-proteins in Chapter 42). The asymmetry between nucleus and cytoplasm—with respect to which of these two nucleotides is bound to Ran molecules—is thought to be crucial in understanding the roles of Ran in transferring complexes unidirectionally across the NPC. When cargo molecules are released inside the nucleus, the importins recirculate to the cytoplasm to be used again. Figure 49-4 summarizes some of the principal features in the above process.
Proteins similar to importins, referred to as exportins, are involved in the export of many macromolecules (various proteins, tRNA molecules, ribosomal subunits, and certain mRNA molecules) from the nucleus. Cargo molecules for export carry nuclear export signals (NESs). Ran proteins are involved in this process also, and it is now established that the processes of import and export share a number of common features. The family of importins and exportins are referred to as

Another system is involved in the translocation of the majority of mRNA molecules. These are exported from the nucleus to the cytoplasm as ribonucleoprotein (RNP) complexes attached to a protein termed mRNP exporter that carries RNP molecules through the NPC. Ran is not involved. This system appears to use the hydrolysis of ATP by an RNA helicase (Dbp5) to drive translocation.
FIGURE 49-4 The entry of a protein into the nucleoplasm. A cargo molecule (C) in the cytoplasm interacts via its nuclear localization signal (NLS) to form a complex with an importin (I). (This may be either importin a or both importin a and importin B.) This complex binds to Ran (R)-GDP and traverses the nuclear pore complex (NPC) into the nucleoplasm. In the nucleoplasm, Ran-GDP is converted to Ran-GTP by guanine nuclear exchange factor (GEF), causing a conformational change in Ran which releases the cargo molecule. The I-Ran-GTP complex then leaves the nucleoplasm via the NPC to return to the cytoplasm. Here | is released by the action of GTPase-accelerating protein (GAP), which converts GTP to GDP, enabling it to bind to another C. The Ran-GTP is the active form of the complex, with the Ran-GDP form is inactive. Directionality is believed to be conferred on the overall process by the dissociation of Ran-GTP in the cytoplasm. (Modified, with permission, from Lodish H, et al: Molecular Cell 6th ed. W.H. Freeman &
Biology, Co, 2008.)
Other small monomeric GTPases (eg, ARE, Rab, Ras, and Rho) are important in various cellular processes such as vesicle formation and transport (ARF and Rab; see below), certain growth and differentiation processes (Ras), and formation of the actin cytoskeleton (Rho). A process involving GTP and GDP is also crucial in the transport of proteins across the membrane of the ER (see below).

The peroxisome is an important organelle involved in aspects of the metabolism of many molecules, including fatty acids and other lipids (eg, plasmalogens, cholesterol, bile acids), purines, amino acids, and hydrogen peroxide. The peroxisome is bounded by a single membrane and contains more than 50 enzymes; catalase and urate oxidase are marker enzymes for this organelle. Its proteins are synthesized on cytosolic polyribosomes and fold prior to import. The pathways of import of a number of its proteins and enzymes have been studied, some being matrix components (Figure 49-5) and others membrane components. At least two peroxisomal-matrix targeting sequences (PTSs) have been discovered. One, PTSI1, is a tripeptide (ie, Ser-Lys-Leu [SKL], but variations of this sequence have been detected) located at the carboxyl terminal of a number of matrix proteins, including catalase. Another, PTS2, is a nine amino acid sequence at the N-terminus and has been found in at least four matrix proteins (eg, thiolase). Neither of these two sequences is cleaved after entry into the matrix.
Proteins containing PTS1 sequences form complexes with a cytosolic receptor protein (Pex5) and proteins containing PTS2 sequences complex with another receptor protein (Pex7). The resulting complexes then interact with a membrane receptor complex, Pex2/10/12, which translocates them into the matrix. Proteins involved in further transport of proteins into the matrix are also present. Pex5 is recycled to the cytosol. Most peroxisomal membrane proteins have been found to contain neither of the above two targeting sequences, but seem to contain others. The import system can handle intact oligomers (eg, tetrameric catalase). Import of matrix proteins requires ATP, whereas import of membrane proteins does not.

Interest in import of proteins into peroxisomes has been stimulated by studies on Zellweger syndrome. This condition is apparent at birth and is characterized by profound neurologic impairment, victims often dying within a year. The number of peroxisomes can vary from being almost normal to being virtually absent in some patients. Biochemical findings include an accumulation of very-long-chain fatty acids, abnormalities of the synthesis of bile acids, and a marked reduction of plasmalogens. The condition is usually caused by mutations in genes encoding certain proteins—the PEX family of genes, also called peroxins—involved in various steps of peroxisome
FIGURE 49-5 Entry of a protein into the peroxisomal matrix. The protein for import into the matrix is synthesized on cytosolic polyribosomes, assumes its folded shape prior to import, and contains a C-terminal peroxisomal-targeting sequence (PTS). This binds to the cytosolic receptor protein Pex5, and the complex then interacts with a receptor on the peroxisomal membrane, Pex14. In turn, the protein-Pex 14 complex is passed to the Pex 2/10/12 complex on the peroxisomal membrane and is translocated. Pex 5 is returned to the cytosol. The protein retains its PTS in the matrix. (Modified, with permission, from Lodish H, et al: Molecular Cell Biology, 6th ed. W.H. Freeman & Co, 2008.)

Zellweger syndrome, OMIMNumber a = 214100. Neonatal adrenoleukodystrophy, OMIMNumber a = 202370. Infantile Refsum disease, OMIMNumber a = 266510. Hyperpipecolic acidemia, OMIMNumber a = 239400. Rhizomelic chondrodysplasia punctata, OMIMNumber a = 215100. Adrenoleukodystrophy, OMIMNumber a = 300100. Pseudoneonatal adrenoleukodystrophy, OMIMNumber a = 264470. Pseudo-Zellweger syndrome, OMIMNumber a = 261515. Hyperoxaluria type 1, OMIMNumber a = 259900. Acatalasemia, OMIMNumber a = 115500. Glutaryl-CoA oxidase deficiency, OMIMNumber a = 231690
2OMIM, Online Mendelian Inheritance in Man. Each number specifies a reference in which information regarding each of the above conditions can be found. Source: Reproduced, with permission, from Seashore MR, Wappner RS: Genetics in Primary Care & Clinical Medicine. Appleton & Lange, 1996.
biogenesis (such as the import of proteins described above), or in genes encoding certain peroxisomal enzymes themselves. Two closely related conditions are neonatal adrenoleukodystrophy and infantile Refsum disease. Zellweger syndrome and these two conditions represent a spectrum of overlapping features, with Zellweger syndrome being the most severe (many proteins affected) and infantile Refsum disease the least severe (only one or a few proteins affected). Table 49-4 lists these and related conditions.

As indicated above, the RER branch is the second of the two branches involved in the synthesis and sorting of proteins. In this branch, proteins have N-terminal signal peptides and are synthesized on membrane-bound polyribosomes. They are usually translocated into the lumen of the rough ER prior to further sorting (Figure 49-2). Certain membrane proteins, however, are transferred directly into the membrane of the ER without reaching its lumen.
Some characteristics of N-terminal signal peptides are summarized in Table 49-5.
There is much evidence to support the signal hypothesis, confirming that N-terminal signal peptides are involved in the process of protein translocation across the ER membrane. For example, mutant proteins containing altered signal peptides in which hydrophobic amino acids are replaced by hydrophilic ones are not inserted into the lumen of the ER. On the other hand, nonmembrane proteins (eg, a-globin) to which signal peptides have been attached by genetic engineering can be inserted into the lumen of the ER, or even secreted.

+ Usually, but not always, located at the amino terminal
+ Contain approximately 12-35 amino acids
+ Methionine is usually the amino-terminal amino acid
+ Contain a central cluster (~6-12) of hydrophobic amino acids
+
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Most nascent proteins are transferred across the ER membrane into the lumen by the cotranslational pathway, so called because the process occurs during ongoing protein synthesis. The process of elongation of the remaining portion of the protein being synthesized probably facilitates passage of the nascent protein across the lipid bilayer. It is important that proteins be kept in an unfolded state prior to entering the conducting channel—otherwise, they may not be able to gain access to the channel. The pathway involves a number of specialized proteins, including the signal recognition particle (SRP), the SRP receptor (SRP-R), and the translocon. The translocon consists of three membrane proteins (the Sec61 complex) that form a protein-conducting channel in the ER membrane through which the newly synthesized protein may pass. The channel opens only when a signal peptide is present. Closure of the channel when proteins are not being translocated prevents ions such as calcium and other molecules leaking through it, causing cell dysfunction. The process proceeds in five steps summarized below and in Figure 49-6.
Step 1: The signal sequence emerges from the ribosome and binds to the SRP. The SRP contains six proteins associated with an RNA molecule, and each of these plays a role (eg, binding of another molecule) in its function. This step temporarily stops further elongation of the polypeptide chain (elongation arrest) after some 70 amino acids have been polymerized.
Step 2: The SRP-ribosome-nascent protein complex travels to the ER membrane, where it binds to the SRP-R, an ER membrane protein composed of two subunits. The a subunit (SPR-Ra) binds the SRP complex and the membrane-spanning $ subunit (SPR-R®) anchors SPR-Ra in the ER membrane. The SRP guides the complex to the SRP-R, preventing premature expulsion of the growing polypeptide into the cytosol.
Step 3: The SRP is released, translation resumes, the ribosome binds to the translocon (Sec 61 complex), and the signal peptide inserts into the channel in the translocon. SRP and both subunits of the SRP-R bind GTP, this enables their interaction, resulting in the hydrolysis of GTP. SRP then dissociates small
+

from SRP-R and is released, and the ribosome binds to the translocon, allowing the signal peptide to enter it.
Step 4: The signal peptide induces opening of the channel in the translocon, by causing the plug (shown at the bottom on the translocon in Figure 49-6) to move. The growing polypeptide is then fully translocated across the membrane, driven by its ongoing synthesis.
Step 5: Cleavage of the signal peptide by signal peptidase occurs, and the fully translocated polypeptide/protein is released into the lumen of the ER. The signal peptide is degraded by proteases. Ribosomes are released from the ER membrane and dissociate into their two types of subunits.
Secretory proteins and soluble proteins destined for organelles distal to the ER completely traverse the membrane bilayer and are discharged into the lumen. Many secretory proteins are N-glycosylated. N-Glycan chains, if present, are added by the enzyme oligosaccharide:protein transferase (see Chapter 46) as these proteins traverse the inner part of the ER membrane—a process called cotranslational glycosylation. Subsequently, these glycoproteins are found in the lumen of the Golgi apparatus, where further changes in glycan chains occur (see Chapter 46) prior to intracellular distribution or secretion.
In contrast, proteins destined to be embedded in membranes of the ER or in other membranes along the secretory pathway only partially translocate across the ER membrane (steps 1-4, above). They are able to insert into the ER membrane by lateral transfer through the wall of the translocon (see below).

Posttranslational translocation of proteins to the ER does occur in eukaryotes, although it is less common than the cotranslational route. The process (Figure 49-7) involves the Sec61 translocon complex, the Sec62/Sec63 complex which is also membrane bound, and chaperone proteins of the Hsp70 family. Some of these prevent the protein folding in the cytosol, but one of them, binding immunoglobulin protein (BiP), is inside the ER lumen. The protein to be translocated initially binds to the translocon, and cytosolic chaperones are released. The leading end of the peptide then binds to BiP in the lumen. ATP bound to BiP interacts with Sec62/63, ATP is hydrolyzed to ADP providing energy to move the protein forwards, while the bound BiP-ADP prevents its moving backward into the cytosol. It can then be pulled through by sequential binding of BiP molecules and ATP hydrolysis. When the entire protein has entered to lumen, ADP is exchanged for ATP, allowing BiP to be released. In addition to its function in protein sorting to the ER lumen, BiP promotes proper folding by preventing aggregation and will temporarily bind abnormally folded immunoglobulin heavy chains and many other proteins, preventing them from leaving the ER.
FIGURE 49-6 Cotranslational targeting of secretory proteins to the ER. Step 1: As the signal sequence emerges from the ribosome, it is recognized and bound by the signal recognition particle (SRP) and translation is arrested. Step 2: The SRP escorts the complex to the ER membrane where it binds to the SRP receptor (SR). Step 3: The SRP is released, the ribosome binds to the translocon, translation resumes, and the signal sequence is inserted into the membrane channel. Step 4: The signal sequence opens the translocon andthe growing polypeptide chain is translocated across the membrane. Step 5: Cleavage of the signal sequence by signal peptidase releases the polypeptide into the lumen of the ER. (Reproduced, with permission, from Cooper GM, Hausman RE: The Cell: A Molecular Approach. Sinauer Associates, Inc, 2009.)

FIGURE 49-7 Posttranslational translocation of proteins into the ER. 1. Proteins synthesized in the cytosol are prevented from folding by chaperone proteins such as members of the Hsp70 family. The N-terminal signal sequence inserts into the Sec61 translocon complex and the cytosolic chaperones are released. BiP interacts with the protein and the Sec62/63 complex and its bound ATP is hydrolyzed to ADP. 2. The protein is prevented from moving back into the cytosol by the bound BiP and successive binding of BiP and ATP hydrolysis pulls the protein into the lumen. 3. When the whole protein is inside, ADP is exchanged for ATP and BiP is released.

The routes that proteins follow to be inserted into the membranes of the ER include cotranslational insertion; posttranslational insertion; retention in the GA followed by retrieval to the ER; and retrograde transport from the GA.

There is evidence that the ER membrane is involved in retrograde transport of various molecules from the ER lumen to the cytosol. These molecules include unfolded or misfolded glycoproteins, glycopeptides, and oligosaccharides. At least some of these molecules are degraded in proteasomes (see below). The involvement of the translocon in retrotranslocation is not clear; one or more other channels may be involved. Whatever the case, there is two-way traffic across the ER membrane.
Figure 49-8 shows a variety of ways in which proteins are distributed in membranes. In particular, the amino termini of certain proteins (eg, the low-density lipoprotein [LDL] receptor) can be seen to be on the extracytoplasmic face, whereas for other proteins (eg, the asialoglycoprotein receptor) the carboxyl termini are on this face. These dispositions are explained by the initial biosynthetic events at the ER membrane. Proteins like the LDL receptor enter the ER membrane in a manner analogous to a secretory protein (Figure 49-6); they partly traverse the ER membrane, the signal peptide is cleaved, and their amino terminal protrudes into the lumen (see also Figure 49-14). However, this type of protein contains a highly hydrophobic segment which acts as a haltor stop-transfer signal and causes its retention in the membrane (Figure 49-9). This sequence has its N-terminal end in the ER lumen and the C-terminal in the cytosol; the stop-transfer signal forms the single transmembrane segment of the protein and is
Transmembrane protein
type:

FIGURE 49-8 variations in the way in which proteins are inserted into membranes. This schematic representation illustrates a number of possible orientations. The orientations form initially in the ER membrane, but are retained when vesicles bud off and fuse with the plasma membrane, so that the terminal initially facing the ER lumen always faces the outside of the cell. Type | transmembrane proteins (eg, the LDL receptor and influenza hemagglutinin) cross the membrane once and have their amino termini in the ER lumen/cell exterior. Type Il transmembrane proteins (eg, the asialoglycoprotein and transferrin receptors) also cross the membrane once, but have their C-termini in the ER lumen/ cell exterior. Type Ill transmembrane proteins (eg, cytochrome P450, an ER membrane protein) have a disposition similar to type | proteins, but do not contain a cleavable signal peptide. Type IV transmembrane proteins (eg, G-protein-coupled receptors and glucose transporters) cross the membrane a number of times (7 times for the former and 12 times for the latter); they are also called polytopic membrane proteins. (C, car- boxyl terminal; N, amino terminal.)
FIGURE 49-9 insertion of a membrane protein with a cleavable signal sequence and a single stop-transfer sequence. The signal sequence is cleaved as the polypeptide chain crosses the membrane, so the amino terminus of the polypeptide chain is exposed in the ER lumen. However, translocation of the polypeptide chain across the membrane is halted when the translocon recognizes a transmembrane stoptransfer sequence. This allows the protein to exit the channel via a lateral gate and become anchored in the ER membrane. Continued translation results in a membrane-spanning protein with its carboxy terminus on the cytosolic side. (Reproduced with permission from Cooper GM, Hausman RE: The Cell: A Molecular Approach. Sinauer Associates, Inc, 2009.)
its membrane-anchoring domain. The protein is believed to exit the translocon into the membrane by a lateral gate which opens and closes continuously allowing hydrophobic sequences to enter the lipid bilayer.

The small patch of ER membrane in which the newly synthesized LDL receptor is located subsequently buds off as a component of a transport vesicle which eventually fuses with the plasma membrane so that the C-terminal faces the cytosol and the N-terminal faces the outside of the cell (Figure 49-14). In contrast, the asialoglycoprotein receptor lacks a cleavable N-terminal signal peptide, but possesses an internal insertion sequence, which inserts into the membrane but is not cleaved. This acts as an anchor, and its C-terminus is extruded through the membrane into the ER lumen. Cytochrome P450 is anchored in a similar way, but its N-terminal, rather than C-terminal, is extruded into the lumen. The more complex disposition of a transmembrane transporter (eg, for glucose) which may cross the membrane up to 12 times, can be explained by the fact that alternating transmembrane a-helices act as uncleaved insertion sequences and as halttransfer signals, respectively. Each pair of helical segments is inserted as a hairpin. Sequences that determine the structure of a protein in a membrane are called topogenic sequences. The LDL receptor, asialoglycoprotein receptor, and glucose transporter are examples of types I, II, and IV transmembrane proteins and are found in the plasma membrane, while cytochrome P450 is an example of a type III protein which remains in the ER membrane (Figure 49-8).

Proteins may enter the ER membrane posttranslationally through the lateral gate in the translocon in a similar way to cotranslationally sorted molecules. An example is cytochrome b,, which appears to enter the ER membrane subsequent to translation, assisted by several chaperones.

A number of proteins possess the amino acid sequence KDEL (Lys-Asp-Glu-Leu) at their carboxyl terminal (Table 49-1). KDEL-containing proteins first travel to the GA in vesicles coated with coat protein II (COPII) (see below). This process is known as anterograde vesicular transport. In the GA, they interact with a specific KDEL receptor protein, which retains them transiently. They then return to the ER in vesicles coated with COPI (retrograde vesicular transport), where they dissociate from the receptor, and are thus retrieved. HDEL sequences (H = histidine) serve a similar purpose. The above processes result in net localization of certain soluble proteins to the ER lumen.
Certain other non-KDEL-containing proteins also pass to the Golgi and then return, by retrograde vesicular transport, to the ER to be inserted therein. These include vesicle components that must be recycled, as well as certain ER membrane proteins. These proteins often possess a C-terminal signal located in the cytosol rich in basic residues.
Thus, proteins reach the ER membrane by a variety of routes, and similar pathways are likely to be used for other membranes (eg, the mitochondrial membranes and the plasma membrane). Precise targeting sequences have been identified in some instances (eg, KDEL sequences).
The topic of membrane biogenesis is discussed further later in this chapter.

After entering the ER, newly synthesized proteins fold with the assistance of chaperones and folding enzymes, and their folding status is monitored by chaperones and also enzymes (Table 49-6).
The chaperone calnexin is a calcium-binding protein located in the ER membrane. This protein binds a wide variety of proteins, including major histocompatibility complex (MHC) antigens and a variety of plasma proteins. As described in Chapter 46, calnexin binds the monoglucosylated species of glycoproteins that occur during processing of glycoproteins, retaining them in the ER until the glycoprotein has folded properly. Calreticulin, which is also a calcium-binding protein, has properties similar to those of calnexin, but it is not membrane-bound. In addition to chaperones, two enzymes in the ER lumen are concerned with proper folding of proteins. Protein disulfide isomerase (PDI) promotes rapid formation and reshuffling of disulfide bonds until the correct set is achieved. Peptidyl prolyl isomerase (PPI) accelerates folding of prolinecontaining proteins by catalyzing the cis—trans isomerization of X-Pro bonds, where X is any amino acid residue.
Misfolded or incompletely folded proteins interact with chaperones, which retain them in the ER and prevent them from being exported to their final destinations. If such interactions continue for a prolonged period of time, harmful buildup of misfolded proteins is avoided by ER-associated degradation (ERAD). In a number of genetic diseases, such as cystic fibrosis, misfolded proteins are retained in the ER, and in
TABLE 49-6 Some Chaperones and Enzymes Involved in Folding That Are Located in the Rough Endoplasmic Reticulum
BiP (immunoglobulin heavy-chain binding protein)
GRP94 (glucose-regulated protein)
Calnexin
Calreticulin
PDI (protein disulfide isomerase)
PPI (peptidyl prolyl cis-trans isomerase)

some cases, the retained proteins still exhibit some functional activity. As discussed later in this Chapter, drugs that will interact with such proteins and promote their correct folding and export out of the ER are currently under investigation.

Maintenance of homeostasis in the ER is important for normal cell function. Perturbation of the unique environment within the lumen of the ER (eg, by changes in ER Ca", alterations of redox status, exposure to various toxins or some viruses), can lead to reduced protein folding capacity and the accumulation of misfolded proteins. The accumulation of misfolded proteins in the ER is referred to as ER stress. The unfolded protein response (UPR) is a mechanism within cells which senses the levels of misfolded proteins and activates intracellular signaling mechanisms to restore ER homeostasis. The UPR is initiated by ER stress sensors, which are transmembrane proteins embedded in the ER membrane. Their activation causes three principal effects: (1) transient inhibition of translation so that the synthesis of new proteins is reduced, (2) induction of transcription to increase the expression of ER chaperones, and (3) increased synthesis of proteins involved in the degradation of misfolded ER proteins (discussed below). Thus, the UPR increases the ER folding capacity and prevents a buildup of unproductive and potentially toxic protein products, as well as promoting other responses to restore cellular homeostasis. However, if impairment of folding persists, cell death pathways (apoptosis) are activated. A more complete understanding of the UPR is likely to provide new approaches to treating diseases in which ER stress and defective protein folding occur (Table 49-7).
Proteins that misfold in the ER are degraded by the ERAD pathway (Figure 49-10). This process selectively transports both luminal and membrane proteins back across the ER (retrograde translocation or dislocation) into the cytosol where they are degraded in proteasomes (see Chapter 28). Chaperones present in the lumen of the ER (eg, BiP) help to target misfolded proteins to proteasomes.

The assembly of the retrotranslocation translocon comprising a number of proteins is initiated by recognition of misfolded proteins. Various types of translocons are thought to occur, but a typical one may include Sec61, Derlin, Hrd1, and SellL. As retrotranslocation occurs, misfolded proteins are polyubiquinated (see Chaper 28) by ubiquitin ligases on the cytosolic side, and are then pulled from the membrane by P97, an AAA-ATPase (ATPase associated with various cellular activities) and delivered to the proteasome for degradation with the help of ubiquitin receptor chaperones.

There are two major pathways of protein degradation in eukaryotes. One involves lysosomal proteases and does not

α 1 -Antitrypsin deficiency with liver disease, Affected Protein = α 1 -Antitrypsin. Chediak-Higashi syndrome, Affected Protein = Lysosomal trafficking regulator. Combined deficiency of factors V andVIII, Affected Protein = ERGIC53, a mannose-binding lectin. Cystic fibrosis, Affected Protein = CFTR. Diabetes mellitus (some cases), Affected Protein = Insulin receptor (α-subunit). Familial hypercholesterolemia, autosomal dominant, Affected Protein = LDL receptor. Gaucher disease, Affected Protein = β-Glucosidase. Hemophilia A and B, Affected Protein = Factors VIII and IX. Hereditary hemochromatosis, Affected Protein = HFE. Hermansky-Pudlak syndrome, Affected Protein = AP-3 adaptor complex β3A subunit. I-cell disease, Affected Protein = N -acetylglucosamine 1-phosphotransferase. Lowe oculocerebrorenal syndrome, Affected Protein = PIP 2 5-phosphatase. Tay-Sachs disease, Affected Protein = β-Hexosaminidase. von Willebrand disease, Affected Protein = von Willebrand factor
LDL,
Abbreviations:
low-density lipoprotein;
PIP,, phosphatidylinositol
4,5-bisphosphate.
*See
Schroder
M,
Rev
Kaufman
Biochem
2005;74:739
RJ:
The mammalian
and
Olkonnen unfolded
V,
Ikonen protein
E:
Genetic response.
Annu defects
of intracellular
membrane transport.
N
Eng
J
Med
2000;343:10095.

require ATP, but the major pathway involves ubiquitin and is ATP-dependent. The ubiquitin pathway is particularly associated with disposal of misfolded proteins and regulatory enzymes that have short half-lives. Ubiquitin is known to be involved in diverse important physiologic processes including cell cycle regulation (degradation of cyclins), DNA repair, inflammation and the immune response (see Chapter 52), muscle wasting, viral infections, and many others. Ubiquitin is a small (76 amino acids), highly conserved protein that tags various proteins for degradation in proteasomes. The mechanism of attachment of ubiquitin to a target protein (eg, a misfolded form of cystic fibrosis transmembrane conductance regulator [CFTR], the protein involved in the causation of cystic fibrosis; see Chapter 40) is shown in Figure 28-2 and the process is described in detail in Chapter 28.

Polyubiquitinated target proteins enter proteasomes located in the cytosol. Proteasomes are protein complexes with a relatively large cylindrical structure and are composed of
FIGURE 49-10 Simplified scheme of the events in ERAD. An ER target protein which is misfolded either in the lumen or in the membrane undergoes retrograde transport into the cytosol via a translocon which is made up of a number of proteins, typically including Sec61, Derlin, Hrd1, and Sel1L. Chaperone proteins such as BiP target misfolded proteins for retrotranslocation. As the protein enters the cytosol, it is ubiquinated by ubiquitin ligases (UL), pulled out of the membrane by P97, an AAA-ATPase, and delivered to the proteasome with the aid of ubiquitin receptor chaperones. Inside the proteasome it is degraded to small peptides that may have several fates after exit.
four rings with a hollow core containing the protease active sites, and one or two caps or regulatory particles that recognize the polyubiquinated substrates and initiate degradation (Figures 28-3 and 28-4). Target proteins are unfolded by ATPases present in the proteasome caps. Proteasomes can hydrolyze a very wide variety of peptide bonds. Target proteins pass into the core to be degraded to small peptides, which then exit the proteasome to be further degraded by cytosolic peptidases. Both normally and abnormally folded proteins are substrates for the proteasome. Liberated ubiquitin molecules are recycled. The proteasome plays an important role in presenting small peptides produced by degradation of various viruses and other molecules to MHC class I molecules, a key step in antigen presentation to T lymphocytes.

Proteins that are synthesized on membrane-bound polyribosomes and are destined for the GA or PM reach these sites inside transport vesicles. As indicated in Table 49-8, there are a number of different types of vesicles. There may be other types of vesicles still to be discovered.

COPI, Function = Involved in intra-GA transport and retrograde transport from the GA to the ER. COPII, Function = Involved in export from the ER to either ERGIC or the GA. Clathrin, Function = Involved in transport in post-GA locations including the PM, TGN, and endosomes. Secretory vesicles, Function = Involved in regulated secretion from organs such as the pancreas (eg, secretion of insulin). Vesicles from the TGN to the PM, Function = They carry proteins to the PMand are also involved in constitutive secretion
Abbreviations: ER, endoplasmic reticulum; ERGIC, ER-GA intermediate compartment; GA, Golgi apparatus; PM, plasma membrane; TGN, trans-Golgi network (the network of membranes located on the side of the Golgi apparatus distal to the ER). Note: Each vesicle has its own set of coat proteins. Clathrin is associated with various adaptor proteins forming different types of clathrin vesicles which have different intracellular targets.
Each vesicle has its own set of coat proteins. Clathrin is used in vesicles destined for exocytosis (see discussions of the LDL receptor in Chapters 25 and 26) and in some of those carrying cargo to lysosomes. This protein consists of three interlocking spirals, which interact to form a lattice around the vesicle. COPI and COPII, the vesicles involved in retrograde transport (from the GA to the ER) and anterograde transport (from the ER to the GA), respectively, however, are clathrin-free. Transport and secretory vesicles carrying cargo from the GA to the PM are also clathrin-free. Here we focus mainly on COPII, COPI, and clathrin-coated vesicles. Each type has a different complement of proteins in its coat. For the sake of clarity, the non-clathrin-coated vesicles are referred to in this text as transport vesicles. The principles concerning assembly of these different types are generally similar, although some details of assembly for COPI and clathrincoated vesicles differ from those for COPII (see below).

Vesicles lie at the heart of intracellular transport of many proteins. Genetic approaches and cell-free systems have been used to elucidate the mechanisms of vesicle formation and transport.The overall process is complex, and involves a variety of cytosolic and membrane proteins, GTP, ATP, and accessory factors. Budding, tethering, docking, and membrane fusion are key steps in the life cycles of vesicles, with the GTP-binding proteins, Sarl, ARF, and Rab acting as molecular switches. Sarl is the protein involved in step 1 of formation of COPII vesicles, whereas ARF is involved in the formation of COPI and clathrin-coated vesicles. The functions of the various proteins involved in vesicle processing and the abbreviations used are shown in Table 49-9.
There are common general steps in transport vesicle formation, vesicle targeting, and fusion with a target membrane,

•, 1 = ARF: ADP-ribosylation factor, a GTPase involved in formation of COPI and also clathrin-coated vesicles.. •, 1 = Coat proteins: A family of proteins found in coated vesicles. Different transport vesicles have different complements of coat proteins.. •, 1 = NSF: N -ethylmaleimide-sensitive factor, an ATPase.. •, 1 = Sar1: A GTPase that plays a key role in assembly of COPII vesicles.. •, 1 = Sec12p: A guanine nucleotide exchange factor (GEF) that intercon- verts Sar1·GDP and Sar1·GTP.. •, 1 = α-SNAP: Soluble NSF attachment protein. Along with NSF, this protein is involved in dissociation of SNARE complexes.. •, 1 = SNARE: SNAP receptor. SNAREs are key molecules in the fusion of vesicles with acceptor membranes.. •, 1 = t-SNARE: Target SNARE.. •, 1 = v-SNARE: Vesicle SNARE.. •, 1 = Rab proteins: A family of Ras-related proteins (monomeric GTPases) first observed in rat brain. They are active when GTP is bound. Different Rab molecules dock different vesicles to acceptor membranes.. •, 1 = Rab effector proteins: A family of proteins that interact with Rab molecules; some act to tether vesicles to acceptor membranes.

irrespective of the membrane, the vesicle forms from or its intracellular destination. The nature of the coat proteins, GTPases and targeting factors differ depending on where the vesicle forms from and its eventual destination. Anterograde transport from the ER to the Golgi involving COPII vesicles is the best studied example. The process can be considered to occur in eight steps (Figure 49-11). The basic concept is that each transport vesicle is loaded with specific cargo and also one or more v-SNARE proteins that direct targeting. Each target membrane bears one or more complementary t-SNARE proteins with which v-SNAREs interact, mediating SNARE protein-dependent vesicle-membrane fusion. In addition, Rab proteins also help direct the vesicles to specific membranes and their tethering at a target membrane.
Step 1: Budding is initiated when the GTPase Sar] is activated by binding of GTP in exchange for GDP via the action of the guanine nucleotide exchange factor (GEF) Secl2p (Table 49-9), switching it from a soluble to a membrane bound form via conformational change which exposes a hydrophobic tail. This enables it to become embedded in the ER membrane to form a focal point for vesicle assembly.
Step 2: Various coat proteins bind to Sar1-GTP. In turn, membrane cargo proteins bind to the coat proteins either directly or via intermediary proteins that attach to coat proteins, and they then become enclosed in their appropriate vesicles. Soluble cargo proteins bind to receptor regions inside the vesicles. A number of signal sequences on cargo molecules have been identified (Table 49-1). For example, KDEL sequences direct certain proteins in retrograde flow from the GA to the ER in COPI vesicles. Diacidic sequences (eg, Asp-X-Glu, X = any amino acid) and short hydrophobic sequences on membrane proteins destined for the GA membrane are involved in interactions with coat proteins of COPII vesicles. However, not all cargo molecules have a sorting signal.
(cis
Golgi)

FIGURE 49-11 Model of the steps in a round of anterograde transport involving COPII vesicles. Step 1: Sar1 is activated when GDP exchanged for GTP and it becomes embedded in the ER membrane to form a focal point for bud formation. Step 2: Coat proteins bind to Sar1-GTP and cargo proteins become enclosed inside the vesicles. Step 3: The bud pinches off, formatting a complete coated vesicle. Vesicles move through cells along microtubules or actin filaments. Step 4: The vesicle is uncoated when bound GTP is hydrolyzed to GDP by Sar1. Step 5: Rab molecules are attached to vesicles after switching of Rab.GDP to Rab.GTP, a specific GEF (see Table 49-9). Rab effector proteins on target membranes bind to Rab-GTP, tethering the vesicles to the target membrane. Step 6: v-SNAREs pair with cognate t-SNAREs in the target membrane to form a four-helix bundle which docks the vesicles and initiates fusion. Step 7: When the vand t-SNARES are closely aligned, the vesicle fuses with the membrane and the contents are released. GTP is then hydrolyzed to GDP, and the Rab-GDP molecules are released into the cytosol. An ATPase (NSF) and a-SNAP (see Table 49-9) dissociate the four-helix bundle between the vand t-SNARES so that they can be reused. Step 8: Rab and SNARE proteins are recycled for further rounds of vesicle fusion. (Adapted, with permission, from Rothman JE: Mechanisms of intracellular protein transport. Nature 1994;372:55.)

Some highly abundant secretory proteins travel to various cellular destinations in transport vesicles by bulk flow; that is, they enter into transport vesicles at the same concentration that they occur in the organelle. However, it appears that most proteins are actively sorted (concentrated) into transport vesicles and bulk flow is used by only a select group of cargo proteins. Additional coat proteins are assembled to complete bud formation. Coat proteins promote budding, contribute to the curvature of buds, and also help sort proteins.
Step 3: The bud pinches off, completing formation of the coated vesicle. The curvature of the ER membrane and proteinprotein and protein-lipid interactions in the bud facilitate pinching off from ER exit sites. Vesicles move through cells along microtubules or along actin filaments.
Step 5: Vesicle targeting is achieved by attachment of Rab molecules to vesicles. Rabs are a family of Ras-like proteins required in several steps of intracellular protein transport and also in regulated secretion and endocytosis. They are small monomeric GTPases that attach to the cytosolic faces of budding vesicles in the GTP-bound state and are also present on acceptor membranes. Rab-GDP molecules in the cytosol are switched to Rab-GTP molecules by a specific GEF (Table 49-9). Rab effector proteins on acceptor membranes bind to Rab-GTP, but not Rab. GDP molecules, thus tethering the vesicles to the membranes.
Step 4: Coat disassembly, or uncoating (involving dissociation of Sarl and the shell of coat proteins) follows hydrolysis of bound GTP to GDP by Sar1, promoted by a specific coat protein. Sarl thus plays key roles in both assembly and dissociation of the coat proteins. Uncoating is necessary for fusion to occur.

Step 6: v-SNAREs pair with cognate t-SNAREs in the target membrane to dock the vesicles and initiate fusion. Generally, one v-SNARE in the vesicle pairs with three t-SNAREs on the acceptor membrane to form a tight four-helix bundle. In synaptic vesicles one v-SNARE is called synaptobrevin. Botulinum B toxin, one of the most lethal toxins known and the most serious cause of food poisoning contains a protease that binds synaptobrevin, thus inhibiting release of acetylcholine at the neuromuscular junction and often proving fatal.
Step 7: Fusion of the vesicle with the acceptor membrane occurs once the vand t-SNARES are closely aligned. After vesicle fusion and release of contents, GTP is hydrolyzed to GDP, and the Rab-GDP molecules are released into the cytosol. When a SNARE on one membrane interacts with a SNARE on another membrane, linking the two, this is referred to as a trans-SNARE complex or a SNARE pin. Interactions of SNARES on the same membrane form a cis-SNARE complex. In order to dissociate the four-helix bundle between the vand t-SNARES so that they can be reused, two additional proteins are required. These are an ATPase (NSF) and a-SNAP (Table 49-9). NSF hydrolyzes ATP and the energy released dissociates the four-helix bundle making the SNARE proteins available for another round of membrane fusion.
Step 8: Certain components, such as the Rab and SNARE proteins, are recycled for subsequent rounds of vesicle fusion. During the above cycle, SNARES, tethering proteins, Rab, and other proteins all collaborate to deliver a vesicle and its contents to the appropriate site.

Proteins found in the apical or basolateral areas of the plasma membranes of polarized epithelial cells may be transported to these sites in various ways; in transport vesicles budding from the trans-Golgi network, with different Rab proteins directing some vesicles to apical regions and others to basolateral regions; via intial direction to the basolateral membrane, followed by endocytosis and transport across the cell by transcytosis to the apical region; or via a process involving the glycosylphosphatidylinositol (GPI) anchor described in Chapter 46. This structure is also often present in lipid rafts (see Chapter 40).
toward the trans-Golgi. COPI vesicles return Golgi enzymes (eg, glycosyltransferases) back from distal (trans-) cisternae of the GA to more proximal (cis) cisternae.

The fungal metabolite brefeldin A inhibits the formation of COPI vesicles by preventing GTP from binding to ARF. In its presence, the Golgi apparatus appears to collapse into the ER. Brefeldin A has proven to be a useful tool for examining some aspects of Golgi structure and function.

Some proteins are subjected to further processing by proteolysis while inside either transport or secretory vesicles. For example, albumin is synthesized by hepatocytes as preproalbumin (see Chapter 52). Its signal peptide is removed, converting it to proalbumin. In turn, proalbumin, while inside secretory vesicles, is converted to albumin by action of furin (Figure 49-12). This enzyme cleaves a hexapeptide from proalbumin immediately C-terminal to a dibasic amino acid site (ArgArg). The resulting mature albumin is secreted into the plasma. Hormones such as insulin (see Chapter 41) are subjected to similar proteolytic cleavages while inside secretory vesicles.

Once proteins in the secretory pathway reach the cisGolgi from the ER, they may be retained in vesicles for travel through the GA to the trans-Golgi, or they may cross by a process called cisternal maturation, in which the cisternae (the flattened membrane disks of the GA that bud off from the ER) move and transform into one another, or perhaps in some cases diffusion via intracisternal connections. In this model, vesicular elements from the ER fuse with one another to help form the cis-Golgi, which in turn can move forward
There are a number of different types of cell membranes, ranging from the plasma membrane which separates the cell contents from the external environment to the internal membranes of subcellular organelles, such a mitochondria and the ER. Although the general lipid bilayer structure is similar in all membranes, they differ in their specific protein and lipid content and each type has its own specific features (see Chapter 40). No satisfactory scheme describing the assembly of any one of these membranes is currently available. Vesicular transport and the way in which various proteins are initially inserted into the membrane of the ER have been discussed above. Some general points about membrane assembly are addressed below.
FIGURE 49-12 Processing of preproalbumin albumin. The signal peptide is removed from preproalbumin as it moves into the ER. Furin cleaves proalbumin at the C-terminal end of a basic dipeptide (ArgArg) while the protein is inside the secretory vesicle. The mature albumin is secreted into the plasma.

Vesicles formed from membranes of the ER and Golgi apparatus, either naturally or pinched off by homogenization, exhibit transverse asymmetries of both lipid and protein. These asymmetries are maintained during fusion of transport vesicles with the plasma membrane. The inside of the vesicles after fusion becomes the outside of the plasma membrane, and the cytoplasmic side of the vesicles remains the cytoplasmic side of the membrane (Figure 49-13). The enzymes responsible for the synthesis of phospholipids, the major class of lipids in
FIGURE 49-13 Fusion of a vesicle with the plasma membrane preserves the orientation of any integral proteins embedded in the vesicle bilayer. Initially, the amino terminal of the protein faces the lumen, or inner cavity, of such a vesicle. After fusion, the amino terminal is on the exterior surface of the plasma membrane. The lumen of a vesicle and the outside of the cell are topologically equivalent. (Redrawn and modified, with permission, from Lodish HF, Rothman JE: The assembly of cell membranes. Sci Am
[Jan] 1979;240:43.)
membranes (see Chapter 40) reside in the cytoplasmic surface of the cisternae (the sac-like structures) of the ER. Phospholipids are synthesized at that site, and it is thought that they self-assemble into thermodynamically stable bimolecular layers, thereby expanding the membrane and perhaps promoting the detachment of so-called lipid vesicles from it. It has been proposed that these vesicles travel to other sites, donating their lipids to other membranes. Phospholipid exchange proteins are cytosolic proteins that take up phospholipids from one membrane and release them to another are believed to play a role in regulating the specific lipid composition of various membranes.

It should be noted that the lipid compositions of the ER, Golgi, and plasma membrane differ, the latter two membranes containing higher amounts of cholesterol, sphingomyelin, and glycosphingolipids, and less phosphoglycerides than does the ER. Sphingolipids pack more densely in membranes than do phosphoglycerides. These differences affect the structures and functions of membranes. For example, the thickness of the bilayer of the GA and PM is greater than that of the ER, which affects which particular transmembrane proteins are found in these organelles. Also, lipid rafts (see Chapter 40) are believed to be formed in the GA.

It has been shown that the half-lives of the lipids of the ER membranes are generally shorter than those of its proteins, so that the turnover rates of lipids and proteins are independent. Indeed, different lipids have been found to have different half-lives. Furthermore, the half-lives of the proteins of these membranes vary widely, some exhibiting short (hours) and others long (days) half-lives. Thus, individual lipids and proteins of the ER membranes appear to be inserted into it relatively independently and this is believed to be the case for many other membranes.
The biogenesis of membranes is thus a complex process about which much remains to be learned. One indication of the complexity involved is to consider the number of posttranslational modifications that membrane proteins may be subjected prior to attaining their mature state. These may include disulfide formation, proteolysis, assembly into multimers, glycosylation, addition of a glycophosphatidylinositol (GPI) anchor, sulfation on tyrosine or carbohydrate moieties, phosphorylation, acylation, and prenylation. Nevertheless, significant progress has been made; Table 49-10 summarizes some of the major features of membrane assembly that have emerged to date.
Various Disorders Result From Mutations in Genes Encoding Proteins Involved in Intracellular Transport Some disorders reflecting abnormal peroxisomal function and abnormalities of protein synthesis in the ER and of the

+ Lipids and proteins are inserted independently into membranes.
+ Individual membrane lipids and proteins turn over independently and at different rates.
+ Topogenic sequences (eg, signal [amino terminal or internal] and stop-transfer) are important in determining the insertion and disposition of proteins in membranes.
+ Membrane proteins inside transport vesicles bud off the
endoplasmic reticulum on their way to the Golgi; final sorting of many membrane proteins occurs in the trans-Golgi network.
+ Specific sorting sequences guide proteins to particular organelles such as lysosomes, peroxisomes, and mitochondria.
synthesis of lysosomal proteins have been listed earlier in this chapter (see Tables 49-4 and 49-7, respectively). Many other mutations affecting folding of proteins and their intracellular transport to various organelles have been reported, including neurodegenerative disorders such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. The elucidation of the causes of these various conformational disorders has contributed significantly to our understanding of molecular pathology. The term "diseases of proteostasis deficiency" has also been applied to diseases due to misfolding of proteins. Proteostasis is a composite word derived from protein homeostasis. Normal proteostasis is due to a balance of many factors, such as synthesis, folding, trafficking, aggregation, and normal degradation. If any one of these is disturbed (eg, by mutation, aging, cell stress, or injury), a variety of disorders can occur, depending on the particular proteins involved.
Potential therapies for the various diseases caused by protein dysfunction due to misfolding are aimed at correcting the conformational errors. One promising approach is to employ chaperones such as Hsp70 to promote proper folding. In addition, the antibiotic geldanamycin has been shown to activate heat shock proteins. Small drug molecules that act as chemical chaperones have also been shown to prevent misfolding and restore protein function. These approaches, however, have so far been tested only in animal experiments and in vitro systems and their effectiveness in humans remains to be established.

- ™ Many proteins are targeted to their destinations by signal
sequences.
A
major sorting
decision is
made when
proteins partitioned
between polyribosomes
cytosolic by
virtue
(or free)
of the
are and
membrane-bound absence
or presence
of an
N-terminal signal
peptide.
™ Proteins synthesized on cytosolic polyribosomes are targeted by specific signal sequences to mitochondria, nuclei, peroxisomes, and the ER. Proteins which lack a signal remain in the cytosol.
™ Proteins synthesized on membrane-bound polyribosomes
initially enter
the
ER
membrane or
lumen, and
many are
ultimately destined
for other
membranes including
the
PM and that of the GA, for lysosomes and for secretion via exocytosis.
Many glycosylation reactions occur in compartments of the
Golgi, and
proteins are
further sorted
in the
trans-Golgi network.
- Molecular chaperones stabilize unfolded or partially folded
proteins.
Chaperones are
the correct
targeting of
their subcellular
locations.
- In posttranslational translocation, proteins are transported to
their target
organelles after
their synthesis
is complete.
Proteins destined
for follow
this mitochondria,
route, as
well as
the nucleus,
a
and minority
of peroxisomes
proteins targeted
the
ER.
- Most proteins enter the ER lumen by the cotranslational
pathway, where
translocation occurs
during ongoing
- Proteins embedded in the ER membrane may inserted
cotranslationally, posttranslationally,
or after
transport
GA
(anterograde transport),
transient retention,
to the
and return
to the
ER
(retrograde transport).

- Harmful buildup of misfolded proteins triggers the unfolded protein response and they are degraded via the ERAD pathway. Proteins are tagged for degradation by the addition of a number of ubiquitin molecules and then enter the cytosol where they are broken down in proteasomes.
Different types
of transport
vesicles are
coated with
different proteins.
Clathrin-coated and
lysosomes,
COPI
vesicles while
coat and
COPII
are proteins
vesicles, destined
I
for exocytosis
and which
II
are responsible
with retrograde
and anterograde
transport, respectively.
Transport vesicle processing is complex and requires many protein factors. Budding from the donor membrane is followed by movement through the cytosol, tethering, docking, and fusion with the target membrane.
Certain proteins (eg, precursors of albumin and insulin) are subjected to proteolysis while inside transport vesicles, producing the mature proteins.
Small GTPases (eg, Ran, Rab) and GEFs play key roles in many aspects of intracellular trafficking.
Vesicles formed from membranes of the ER and Golgi apparatus are asymmetrical in both lipid and protein content. The asymmetry is maintained during assembly and during fusion of transport vesicles with the plasma membrane, so that the inside of the vesicles after fusion becomes the outside of the plasma membrane, and the cytoplasmic side of the vesicles remains facing the cytosol.
Lipids and proteins are inserted independently and turn over at different rates. Exact details of the assembly process remain to be established.
Many disorders have been shown to be due to mutations in genes or to other factors that affect the folding of various proteins. These conditions have been referred to as conformational diseases, or alternatively as diseases of proteostatic deficiency. Promising therapeutic approaches include the use of chaperones such as Hsp70 and small molecules that can prevent misfolding and restore protein function.
to

Alberts B, Johnson A, Lewis J, et al: Molecular Biology of the
Cell, 6th ed. Garland Science, 2014. (An excellent textbook of cell biology, with comprehensive coverage of trafficking and
sorting.)
Cooper GM, Hausman RE: The Cell: A Molecular Approach, 7th ed.
Palgrave, 2015. (An excellent textbook of cell biology, with comprehensive coverage of trafficking and sorting.)
Hebert DN, Molinari M: In and out of the ER: protein folding, quality control, degradation and related human diseases. Physiol
Rev 2007;87:1377.

RTOS CM UER eee you should be able to:
. Indicate the importance of the extracellular matrix (ECM) and its components in health and disease.
. Describe the structural and functional properties of collagen and elastin, the
major proteins
of the
ECM.
= Indicate the major features of fibrillin, fibronectin, and laminin, other important proteins of the ECM.
= Describe the properties and general features of the synthesis and degradation of glycosaminoglycans and proteoglycans, and their contributions to the ECM.
= Give a brief account of the major biochemical features of bone and cartilage.

Most mammalian cells are located in tissues where they are surrounded by a complex extracellular matrix (ECM) often referred to as "connective tissue,' which protects the organs and also provides elasticity where required (eg, in blood vessels, lungs, and skin). The ECM contains three major classes of biomolecules: structural proteins, for example, collagen, elastin, and fibrillin; certain specialized proteins such as fibronectin and laminin, which form a mesh of fibers that are embedded in the third class, proteoglycans. The ECM is involved in many processes, both normal and pathologic, for example, it plays important roles in development, in inflammatory states, and in the spread of cancer cells. Certain components of the ECM play a part in both rheumatoid arthritis and osteoarthritis. Several diseases (eg, osteogenesis imperfecta and a number of types of the Ehlers-Danlos syndrome) are due to genetic disturbances of the synthesis of collagen, a major ECM component. Specific components of proteoglycans (the glycosaminoglycans; GAGs) are affected in the group of genetic disorders known as the mucopolysaccharidoses. Changes occur in the ECM during the aging process. This chapter describes the basic biochemistry of the three major classes of biomolecules found in the ECM and illustrates their biomedical significance. Major biochemical features of two specialized forms of ECM—bone and cartilage—and of a number of diseases involving them are also briefly considered.

Collagen, the major component of most connective tissues, constitutes approximately 25% of the protein of mammals. It provides an extracellular framework for all metazoan animals and exists in virtually every animal tissue. At least 28 types of collagen made up of over 30 distinct polypeptide chains (each encoded by a separate gene) have been identified in human tissues (Table 50-1). Although several of these are present only in small proportions, they may play important roles in determining the physical properties of specific tissues. In addition, a number of proteins (eg, the Clq component of the complement system, pulmonary surfactant proteins SPA and SPD) that are not classified as collagens have collagen-like domains in their structures; these proteins are sometimes referred to as "noncollagen collagens."

All collagen types have a triple helical structure made up of three polypeptide chain subunits (a chains). In some collagens, the entire molecule is a triple helix, whereas in others only a fraction of the structure may be in this form. Mature collagen type I belongs to the former type; each polypeptide

I, Distribution = Noncartilaginous connective tissues, including bone, tendon, skin. II, Distribution = Cartilage, vitreous humor. III, Distribution = Extensible connective tissues, including skin, lung, vascular system. IV, Distribution = Basement membranes. V, Distribution = Minor component in tissues containing collagen I. VI, Distribution = Muscle and most connective tissues. VII, Distribution = Dermal-epidermal junction. VIII, Distribution = Endothelium and other tissues. IX, Distribution = Tissues containing collagen II. X, Distribution = Hypertrophic cartilage. XI, Distribution = Tissues containing collagen II. XII, Distribution = Tissues containing collagen I. XIII, Distribution = Many tissues, including neuromuscular junctions and skin. XIV, Distribution = Tissues containing collagen I. XV, Distribution = Associated with collagens close to basement membranes in many tissues including in eye, muscle, microvessels. XVI, Distribution = Many tissues. XVII, Distribution = Epithelia, skin hemidesmosomes. XVIII, Distribution = Associated with collagens close to basement membranes, close structural homologue of XV. XIX, Distribution = Rare, basement membranes, rhabdomyosarcoma cells. XX, Distribution = Many tissues, particularly corneal epithelium. XXI, Distribution = Many tissues. XXII, Distribution = Tissue junctions, including cartilage-synovial fluid, hair follicle-dermis. XXIII, Distribution = Limited in tissues, mainly transmembrane and shed forms. XXIV, Distribution = Developing cornea and bone. XXV, Distribution = Brain. XXVI, Distribution = Testis, ovary. XXVII, Distribution = Embryonic cartilage and other developing tissues, cartilage in adults. XXVIII, Distribution = Basement membrane around Schwann cells
subunit is twisted into a left-handed polyproline helix of three residues per turn forming an a chain. Three of these are then wound into a right-handed tripleor superhelix, forming a rod-like molecule 1.4 nm in diameter and about 300-nm long (Figure 50-1). Glycine residues occur at every third position of the triple helical portion of the a chain. This is necessary because glycine is the only amino acid small enough to be

accommodated in the limited space available in the central core of the triple helix. This repeating structure, represented as (Gly-X-Y)n, is an absolute requirement for the formation of the triple helix. While X and Y can be any other amino acids, the X positions are often proline and the Y positions are often hydroxyproline. Proline and hydroxyproline confer rigidity on the collagen molecule. Hydroxyproline is formed by the posttranslational hydroxylation of peptide-bound proline residues catalyzed by the enzyme prolyl hydroxylase, whose cofactors are ascorbic acid (vitamin C) and a-ketoglutarate. Lysines in the Y position may also be posttranslationally modified to hydroxylysine through the action of lysyl hydroxylase, an enzyme with similar cofactors. Some of these hydroxylysines may be further modified by the addition of galactose or galactosyl-glucose through an O-glycosidic linkage (see Chapter 46), a glycosylation site that is unique to collagen. Some collagen types form long rod-like fibers in tissues. These are assembled by lateral association of these triple helical units into fibrils (10-300 nm in diameter) in a "quarterstaggered" alignment such that each is displaced longitudinally from its neighbor by slightly less than one-quarter of its length (Figure 50-1). Fibrils, in turn, associate into thicker fibers (1-20 um in diameter). Because the quarter staggered alignment results in regularly spaced gaps between the triple helical molecules in the array, fibers have a banded appearance in

connective tissues. In some tissues, for example tendons, fibers associate into even larger bundles, which may have a diameter of up to 500 um. Collagen fibers are further stabilized by the formation of covalent cross-links, both within and between the triple helical units. These cross-links form through the action of lysyl oxidase, a copper-dependent enzyme that oxidatively deaminates the e-amino groups of certain lysine and hydroxylysine residues, yielding reactive aldehydes. Such aldehydes can form aldol condensation products with other lysineor hydroxylysine-derived aldehydes or form Schiff bases with the e-amino groups of unoxidized lysines or hydroxylysines. These reactions, after further chemical rearrangements, result in the stable covalent cross-links that are important for the tensile strength of the fibers. Histidine may also be involved in certain cross-links.
The main fibril-forming collagens in skin and bone and in cartilage, respectively, are types I and II, although other collagens also adopt this structure. In addition, however, there are many nonfibril-forming collagens and their structures and functions are described briefly in the section below.

Several collagen types do not form fibrils in tissues (Figure 50-2). They are characterized by interruptions of the triple helix with stretches of protein lacking Gly-X-Y repeat sequences. Thus, areas of globular structure are interspersed in the triple helical structure. Network-like collagens such as type IV form networks in basement membranes; fibril-associated collagens with interrupted triple helices (FACITs), as their name indicates, have interruptions in the triple helical domains; beaded filaments consist of long chains of collagen molecules which have a regular beaded appearance; collagen VII forms the main part of anchoring fibrils in epithelial tissues; transmembrane collagens have short intracellular N-terminal domains and extracellular domains with long interrupted triple helices; multiplexins are collagens with multiple triple helix domains and interruptions.
FIGURE 50-2 Classification of collagens according to the structures they form. FACIT, fibril-associated collagen with interrupted triple helices; multiplexin, multiple triple helix domains and interruptions.

Newly synthesized collagen undergoes extensive posttranslational modification before becoming part of a mature extracellular collagen fiber (Table 50-2). Like most secreted proteins, collagen is synthesized on ribosomes in a precursor form, preprocollagen, which contains a leader or signal sequence that directs the polypeptide chain into the lumen of the endoplasmic reticulum (ER) (see Chapter 49). As it enters the ER, this leader sequence is removed enzymatically. Hydroxylation of proline and lysine residues and glycosylation of hydroxylysines in the procollagen molecule also take place at this site. The procollagen molecule contains polypeptide extensions (extension peptides) of 20 to 35 kDa at both its aminoand carboxyl-terminal ends, which are not present in mature collagen. Both extension peptides contain cysteine residues. While the amino terminal propeptide forms only intrachain disulfide bonds, the carboxyl-terminal propeptides form both intrachain and interchain disulfide bonds. Formation of these disulfide bonds assists in the registration of the three collagen molecules to form the triple helix, winding from the carboxyl-terminal end. After formation of the triple helix, no further hydroxylation of proline or lysine or glycosylation of hydroxylysines can take place. Self-assembly is a cardinal principle in the biosynthesis of collagen.
Following secretion from the cell by way of the Golgi apparatus, extracellular enzymes called procollagen aminoproteinase and procollagen carboxyproteinase remove the extension peptides at the aminoand carboxyl-terminal ends, respectively, forming the monomeric units of collagen, termed tropocollagen. Cleavage of the propeptides may occur within crypts or folds in the cell membrane. Once the propeptides are removed, the tropocollagen molecules, containing approximately 1000 amino acids per a chain, spontaneously assemble into collagen fibers. These are further stabilized by the formation of interand intrachain cross-links through the action of lysyl oxidase, as described previously.

The same cells that secrete collagen also secrete fibronectin, a large glycoprotein present on cell surfaces, in the extracellular matrix, and in blood (see below). Fibronectin binds collagen fibers during aggregation and alters the kinetics of fiber formation in the pericellular matrix. Associated with fibronectin and procollagen in this matrix are the proteoglycans heparan sulfate and chondroitin sulfate (see below). In fact, type IX collagen, a minor collagen type from cartilage, contains an attached glycosaminoglycan chain. Such interactions may serve to regulate the formation of collagen fibers and to determine their orientation in tissues. Once formed, collagen is relatively metabolically stable. However, its breakdown is increased during starvation and various inflammatory states. Excessive production of collagen occurs in a number of conditions, for example, hepatic cirrhosis.

More than 30 genes encode the collagens, and they are designated according to the procollagen type and their constituent a chains, called proa chains. Collagens may be homotrimeric, containing three identical proa chains, or heterotrimeric, where the proa chains are different. For example, type I collagen is heterotrimeric, containing two proal(I) and one proa2(I) chains (the arabic number refers to the proa chain, and the roman numeral in parentheses indicates the collagen type), while type II collagen is homotrimeric, having three proal(II) chains. Collagen genes have the prefix COL followed by the type in arabic numerals, then an A and the number of the proa chain they encode. Thus, COLIAI and COL1A2 are the genes for the proal and 2 chains of type I collagen, COL2A1 is the gene for the proal chain of type II collagen, and so on.
The pathway of collagen biosynthesis is complex, involving at least eight enzyme-catalyzed posttranslational steps. Thus, it is not surprising that a number of diseases (Table 50-3) are due to mutations in collagen genes or in genes encoding some of the enzymes involved in these posttranslational modifications. Diseases affecting bone (eg, osteogenesis imperfecta) and cartilage (eg, the chondrodysplasias) will be discussed later in this chapter.
Ehlers-Danlos syndrome (formerly called Cutis hyperelastica), comprises a group of inherited disorders whose principal clinical features are hyperextensibility of the skin, abnormal tissue fragility, and increased joint mobility. The clinical picture is variable, reflecting underlying extensive genetic heterogeneity. A number of forms of the disease caused by genetic defects in proteins involved in the synthesis and assembly of collagens type I, III, and V are known, and since 1997 the Villefranche classification of six subtypes based on their phenotype and molecular defects has been used (Table 50-4). The hypermobility, vascular and classical
TABLE 50-3 Diseases Caused by Mutations in Collagen Genes or by Deficiencies in the Activities of Enzymes Involved in the Posttranslational Biosynthesis of

COL1A1, COL1A2, Disease a = Osteogenesis imperfecta type 1 b Osteoporosis Ehlers-Danlos syndrome, subtype arthrochalasia. COL2A1, Disease a = Severe chondrodysplasia Osteoarthritis. COL3A1, Disease a = Ehlers-Danlos syndrome, subtype vascular. COL4A3-COL4A6, Disease a = Alport syndrome (autosomal and X-linked). COL7A1, Disease a = Epidermolysis bullosa, dystrophic. COL10A1, Disease a = Schmid metaphyseal chondrodysplasia. COL5A1, COL5A2, COL1A1, Disease a = Ehlers-Danlos syndrome, subtype classical. COL3A1 , tenascin XB ( TNXB ), Disease a = Ehlers-Danlos syndrome, subtype hypermobility. Lysyl hydroxylase, Disease a = Ehlers-Danlos syndrome, subtype kyphoscoliosis. ADAMmetallopeptidase with thrombospondin type 1 motif ( ADAMTS2 ) (also called procollagen N -proteinase), Disease a = Ehlers-Danlos syndrome, subtype dermatosparaxis. Lysyl oxidase, Disease a = Menkes disease c
*Genetic linkage
to collagen
genes has
been shown
to
a
few other
conditions not
listed here.
»Eight different
types of
osteogenesis imperfecta
are recognized,
caused by
the
COLIA7
and
COL1A2
genes.
"Secondary to a deficiency of copper (see Chapter 52).

subtypes are more common, while the other three, kyphoscoliosis, arthrochalasis, and dermatosparaxis are extremely rare. The vascular subtype is the most serious because of its tendency for spontaneous rupture of arteries or the bowel, reflecting abnormalities in type III collagen. Patients with kyphoscoliosis exhibit progressive curvature of the spine (scoliosis) and a tendency to ocular rupture due to a deficiency of lysyl hydroxylase. A deficiency of procollagen N-proteinase (ADAM metallopeptidase with thrombospondin type 1 motif [ADAMTS2]), causing formation of abnormal thin, irregular collagen fibrils, results in dermatosparaxis, manifested by marked fragile and sagging skin.
The Alport syndrome (hereditary nephritis) is the name given to a number of genetic disorders (both X-linked and autosomal) affecting type IV collagen, a network-like collagen which forms part of the structure of the basement membranes of the renal glomeruli, inner ear, and eye (see discussion of laminin, below). Mutations in several genes encoding type IV collagen fibers have been demonstrated.
but most
cases are

Hypermobility, Defect in = Type III collagen, tenascin X b. Hypermobility, Incidence = 1:10,000-15,000. Hypermobility, Clinical Signs = Joint hypermobility, skin abnormalities, osteoarthritis, severe pain. Classical, Defect in = Types I andV collagen. Classical, Incidence = 1:20,000-30,000. Classical, Clinical Signs = Similar to the hypermobility subtype, but with more severe skin abnormalities and less severe joint changes. Vascular, Defect in = Type III collagen. Vascular, Incidence = 1:100,000. Vascular, Clinical Signs = Fragile blood vessels and organs, small stature, thin and translucent skin, easy bruising. Kyphoscoliosis, Defect in = Lysyl hydroxylase. Kyphoscoliosis, Incidence = <60 cases. Kyphoscoliosis, Clinical Signs = Curvature of the spine (scoliosis), severe muscle weakness, fragile eyes, hyperextensible and bruisable skin. Arthrochalasis, Defect in = Type I collagen. Arthrochalasis, Incidence = <40 cases. Arthrochalasis, Clinical Signs = Very loose joints and dislocation of both hips. Dermatosparaxis, Defect in = ADAMmetallopeptidase with thrombospondin type 1 motif (ADAMTS2) c. Dermatosparaxis, Incidence = <10 cases. Dermatosparaxis, Clinical Signs = Very fragile and sagging skin
*Beighton P, De Paepe A, Steinmann B, et al: Ehlers-Danlos syndromes: revised nosology, Villefranche, Ehlers-Danlos National Foundation (USA) and Ehlers-Danlos Support
Group (UK). Am J Med Genet 1998;64:31-37.
- °A glycoprotein expressed in connective tissues such as skin, joints, and muscles.
"Also called procollagen N-proteinase.

Elastin is
a
connective tissue
protein that
for properties
of extensibility
and elastic
recoil in
tissues.
Although not
as widespread
as collagen,
elastin is
present in
large amounts in tissues that require these physical properties, for example, lung, large arterial blood vessels, and some elastic ligaments. Smaller quantities of elastin are also found in skin, ear cartilage, and several other tissues. In contrast to collagen, only one genetic type of elastin is known, although variants arise by alternative splicing (see Chapter 36) of the hnRNA for elastin. Elastin is synthesized as a soluble monomer of ~70 kDa called tropoelastin. Some of the prolines of tropoelastin are hydroxylated to hydroxyproline by prolyl hydroxylase, though hydroxylysine and glycosylated hydroxylysine are not present. Unlike collagen, tropoelastin is not synthesized in a proform with extension peptides. Furthermore, elastin does not contain repeat Gly-X-Y sequences, triple helical structure, or carbohydrate moieties.
After secretion
from the
cell, certain
lysyl residues
of tropoelastin
are oxidatively
oxidase, the
same gen.
However, desmosines,
deaminated enzyme
the to
involved major
aldehydes in
cross-links which
result these
lysine-derived from
this by
lysyl process
in formed
in colla-
elastin the
aldehydes to
form
a
tetrafunctional cross-linked
condensation with
are the
of three
an unmodified
cross-link in
its mature,
insoluble, extremely
The various
ture unique
extracellular stable,
random permit
during of
lysine to
elastin.
form, and
has
a
very coil
Once elastin
is highly
low-turnover conformations
the performance
present to
stretch and
of its
Table
50-5
summarizes physiologic

the main
rate.
in its
struc- subsequently
recoil functions.
differences between

Deletions in the elastin gene (located at 7q11.23) have been found in approximately 90% of subjects with the WilliamsBeuren syndrome, a developmental disorder affecting connective tissue and the central nervous system. The mutations, affect the synthesis of elastin, and probably play a causative role in the supravalvular aortic stenosis often found in this condition. Fragmentation or, alternatively, a decrease of elastin is found in conditions such as pulmonary emphysema, cutis laxa, and aging of the skin.

1. Many different genetic types, Elastin = One genetic type. 2. Triple helix, Elastin = No triple helix; random coil conformations permitting stretching. 3. (Gly-X-Y) n repeating structure, Elastin = No(Gly-X-Y) n repeating structure. 4. Presence of hydroxylysine, Elastin = No hydroxylysine. 5. Carbohydrate-containing, Elastin = No carbohydrate. 6. Intramolecular aldol cross-links, Elastin = Intramolecular desmosine cross-links. 7. Presence of extension peptides during biosynthesis, Elastin = No extension peptides present during biosynthesis

Microfibrils are fine fiber-like strands 10 to 12 nm in diameter which provide a scaffold for the deposition of elastin in the ECM. Fibrillins are large glycoproteins (about 350 kDa) that are major structural component of these fibers. They are secreted (subsequent to a proteolytic cleavage) into the ECM by fibroblasts and become incorporated into the insoluble microfibrils. Fibrillin-1 is the main fibrillin present, but fibrillins-2 and 3 have also been identified, and fibrillin-2 is thought to be important in deposition of microfibrils early in development. Other proteins including microfibrilassociated glycoproteins (MAGPs), fibulins, and members of the ADAMTS family are also associated with microfibrils. Fibrillin microfibrils are found in elastic fibers and also in elastin-free bundles in the eye, kidney, and tendons.

Marfan syndrome is a relatively prevalent inherited disease affecting connective tissue; it is inherited as an autosomal dominant trait. It affects the eyes (eg, causing dislocation of the lens, known as ectopia lentis), the skeletal system (most patients are tall and exhibit long digits [arachnodactyly] and hyperextensibility of the joints), and the cardiovascular system (eg, causing weakness of the aortic media, leading to dilation of the ascending aorta). Abraham Lincoln may have had this condition. Most cases are caused by mutations in the gene (on chromosome 15) for fibrillin-1. This results in abnormal fibrillin and/or lower amounts being deposited in the ECM. Since the cytokine transforming growth factor 6 (TGF-f) normally binds to fibrillin-1, decreased binding in Marfan syndrome causes disturbances in TGF-B signaling. This could potentially lead to the development of therapies for the condition using drugs that antagonize TGF-6 (eg, the angiotensin II receptor antagonist, Losartan).
FIGURE 50-3 Probable sequence of events in the causation of the major signs exhibited by patients with Marfan syndrome.
Mutations in the fibrillin-1 gene have also been identified as the cause of acromicric dysplasia and geleophysic dysplasia, which are characterized by short stature, skin thickening, and stiff joints. Congenital contractural arachnodactyly is associated with a mutation in the gene for fibrillin-2. The probable sequence of events leading to Marfan syndrome is summarized in Figure 50-3.

Fibronectin is a major glycoprotein of the extracellular matrix, also found in a soluble form in plasma. It consists of two identical subunits, each of about 230 kDa, joined by two disulfide bridges near their carboxyl terminals. The gene encoding fibronectin is very large, containing some 50 exons; the RNA produced by its transcription is subject to considerable alternative splicing, and as many as 20 different mRNAs have been detected in various tissues. Fibronectin contains three types of repeating motifs (I, II, and III), which are organized into functional domains (at least seven); functions of these domains include binding fibronectin (enabling molecules of the protein to interact) heparin (see below), fibrin, collagen, and cell surfaces (Figure 50-4). Fibronectin binds to cells via a transmembrane receptor protein which belongs to the integrin class of proteins (see Chapter 55). Fibronectin contains an Arg-Gly-Asp (RGD) sequence that binds to the integrin receptor. This sequence is shared by a number of other proteins present in the ECM that bind to integrins present in cell plasma membranes, and its presence in synthetic peptides enables them to inhibit the binding of fibronectin to cells. Figure 50-5 illustrates the interaction of collagen, fibronectin, and laminin, all major proteins of the ECM, with a typical cell (eg, fibroblast) present in the matrix.
The fibronectin receptor interacts indirectly with actin microfilaments (see Chapter 51) present in the cytosol via a number of proteins, collectively known as attachment proteins; these include talin, vinculin, a-actinin, and paxillin (Figure 50-6). Such large protein complexes form focal

FIGURE 50-4 Structure of the fibronectin monomer. Fibronectin is a dimer joined by disulfide bridges (not shown) near the carboxyl terminals of the monomers. Each monomer consists mainly of repeating motifs of type |, Il, or Ill and has a number of protein-binding domains. Four bind fibronectin and there are also domains for collagen, heparin, fibrin, and cell binding. The approximate location of the RGD sequence of fibronectin, which interacts with a variety of fibronectin integrin receptors on cell surfaces, is indicated by the arrow.
adhesions which not only anchor cells in the ECM, but also relay signals from the exterior which influence cell behavior. Thus, the interaction of fibronectin with its receptor provides one route whereby the outside of the cell can communicate with the inside. Fibronectin is also involved in cell migration, as it provides a binding site for cells and thus helps them to steer their way through the ECM. The amount of fibronectin around many transformed cells is sharply reduced, partly explaining their faulty interaction with the ECM.

Basal laminas are specialized areas of the ECM that surround epithelial and some other cells (eg, muscle cells). Laminin (a glycoprotein of about 850 kDa and 70-nm length) consists of three distinct elongated polypeptide chains (a, 8, and y chains, each of which have genetic variants) linked together to form a complex, elongated shape (see Figure 51-11, which shows laminin-2, also called merosin). In basal laminas, laminin forms networks which are attached to type IV collagen by entactin (also called nidogen), a glycoprotein containing an RGD sequence, and the heparan sulfate proteoglycan, perlecan. The collagen interacts with laminin (rather than directly with the cell surface), which in turn interacts with integrins or other proteins, such as dystroglycans (see Chapter 51) thus anchoring the lamina to the cells (Figure 50-7).
make up the glomerular membrane. This relatively thick basal lamina has an important role in glomerular filtration. The glomerular membrane allows small molecules, such as inulin (5.2 kDa), to pass through as easily as water. On the other hand, large molecules, including most plasma proteins, are not able to pass through. The reason for this is two-fold: (1) The pores in the glomerular membrane are about 8-nm across, thus molcules larger than this are unable to pass through. (2) Although some plasma proteins, such as albumin, are smaller than this pore size, they are prevented from passing through easily by the negative charges of heparan sulfate and of certain sialic acid-containing glycoproteins present in the lamina which repel most plasma proteins, which are negatively charged at the pH of blood. The normal structure of the glomerulus may be severely damaged in certain types of glomerulonephritis (eg, caused by antibodies
In the renal glomerulus, the basal lamina consists of two separate sheets of cells (one endothelial and one epithelial), each disposed on opposite sides of the lamina; these three layers

FIGURE 50-5 Schematic representation of a cell interactions with major proteins of the ECM. a and b indicate aand B-polypeptide chains of integrins.
FIGURE 50-6 Schematic representation of fibronectin interacting with actin in the cytosol via an integrin fibronectin receptor. The fibronectin dimer on the outside of the plasma membrane binds to the membrane-spanning integrin receptor via the RGD sequences. On the cytosolic side, integrin interacts with attachment proteins including talin, vinculin, a-actinin, and paxillin (shown as a complex) which interact with actin microfilaments, thus indirectly linking fibronectin in the ECM with actin in the cell cytosol.
FIGURE 50-7 Structure of the basal lamina. Laminin is attached to type IV collagen via entactin and perlecan (forming the basal lamina) and to the epithelial cell layer via integrins and dystroglycans.
directed against various components of the glomerular membrane). This affects the pores and the amounts and dispositions of the negatively charged macromolecules referred to above, and relatively massive amounts of albumin (and of certain other plasma proteins) can pass through into the urine, resulting in severe albuminuria.

Proteoglycans, proteins that contain covalently linked glycosaminoglycans (GAGs) (see also Chapters 15 and 46), are a major component of the ECM. At least 30 have been characterized, for example, syndecan, betaglycan, serglycin, perlecan, aggrecan, versican, decorin, biglycan, and fibromodulin. A proteoglycan consists of a core protein bound covalently to GAGs, and these units form large complexes with other components of the extracellular matrix, such as hyaluronic acid or collagen. Figures 50-8 and 50-9 show the general structure of such complexes. They are very large with an overall structure resembling that of a bottlebrush. The example shown in Figure 50-9 contains a long strand of hyaluronic acid (one type of GAG) (see Chapter 15) to which link proteins are attached noncovalently. In turn, the link proteins interact noncovalently with core protein molecules from which chains of other GAGs (eg, keratan sulfate and chondroitin sulfate) project. Proteoglycans vary in tissue distribution, nature of the core protein, attached GAGs, and their function. The amount of carbohydrate in a proteoglycan is usually much greater than that found in a glycoprotein, and may comprise up to 95% of its weight.
There are at least seven GAGs: hyaluronic acid (hyaluronan), chondroitin sulfate, keratan sulfates I and II, heparin, heparan sulfate, and dermatan sulfate. GAGs are unbranched polysaccharides made up of repeating disaccharides, one component of which is always an amino sugar (hence, the name GAG), either p-glucosamine or D-galactosamine. The other component of the repeating disaccharide (except in the case of keratan sulfate)

a can aggregate. The proteoglycan subunits and filamentous backbone are particularly well extended in this image. (Reproduced, with permission, from Rosenberg L, Hellman W, Kleinschmidt AK: Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage. J Biol Chem 1975;250:1877.)
is a uronic acid, either p-glucuronic acid (GIcUA) or its 5'-epimer, L-iduronic acid (IdUA). With the exception of hyaluronic acid, all the GAGs contain sulfate groups, either as O-esters or as N-sulfate (in heparin and heparan sulfate). Hyaluronic acid is also exceptional because it appears to exist as a polysaccharide in the ECM, with no covalent attachment to protein, as the definition of a proteoglycan given above specifies. Both GAGs and proteoglycans have proved difficult to work with, partly because of their complexity. However, since they are major components of the ECM and have a number of important biologic roles as well as being involved in a number of disease processes, interest in them has increased greatly in recent years.

The linkage between GAGs and their core proteins is generally one of the following three types:
1. An O-glycosidic bond between xylose (Xyl) and Ser, a bond that is unique to proteoglycans. This linkage is formed by transfer of a Xyl residue to Ser from UDP-xylose. Two residues of Gal are then added to the Xyl residue, forming a
FIGURE 50-9 Schematic representation of a proteoglycan complex. In this example, proteoglycans are attached via noncovalent bonds to link proteins which, in turn, bond noncovalently to a long strand of the glycoaminoglycan (GAG), hyaluronic acid.
link trisaccharide, Gal-Gal-Xyl-Ser. Further chain growth of the GAG occurs on the terminal Gal.
growing GAG chain away from the membrane site where catalysis occurs.
2. An O-glycosidic bond between GalNAc (N-acetylgalactosamine) and Ser (Thr) (see Figure 46-1A), present in keratan sulfate II. This bond is formed by donation to Ser (or Thr) ofa GalNAc residue, employing UDP-GalNAc as its donor.
3. An N-glycosylamine bond between GlcNAc (N-acetylglucosamine) and the amide nitrogen of Asn, as found in N-linked glycoproteins (see Figure 46-1B). Its synthesis is believed to involve dolichol-PP oligosaccharide.
The synthesis of the core proteins occurs in the ER, and formation of at least some of the above linkages also occurs there. Most of the later steps in the biosynthesis of GAG chains and their subsequent modifications occur in the Golgi apparatus.

Appropriate nucleotide sugars and highly specific Golgilocated glycosyltransferases are employed to synthesize the oligosaccharide chains of GAGs. The "one enzyme, one linkage" relationship appears to hold here, as in the case of certain types of linkages found in glycoproteins. The enzyme systems involved in chain elongation are capable of highfidelity reproduction of complex GAGs (see also Chapter 46).

This appears to result from (1) sulfation, particularly at certain positions of the sugars, and (2) the progression of the

After formation of the GAG chain, numerous chemical modifications occur, such as the introduction of sulfate groups onto GalNAc and other moieties and the epimerization of GIcUA to IdUA residues. The enzymes catalyzing sulfation are designated sulfotransferases and use 3'-phosphoadenosine5'-phosphosulfate [PAPS; active sulfate] (see Chapter 32) as the sulfate donor. These Golgi-located enzymes are highly specific, and distinct enzymes catalyze sulfation at different positions (eg, carbons 2, 3, 4, and 6) on the acceptor sugars. An epimerase catalyzes conversions of glucurony] to iduronyl residues.

Proteoglycans are found in every tissue of the body, mainly in the ECM or "ground substance." There they are associated with each other and also with the other major structural components of the matrix, collagen, and elastin, in specific ways. Some proteoglycans bind to collagen and others to elastin. These interactions are important in determining the structural organization of the matrix. Some proteoglycans (eg, decorin) can also bind growth factors such as TGF-f,
modulating their effects on cells. In addition, some of them interact with certain adhesive proteins such as fibronectin and laminin (see above), also found in the matrix. The GAGs present in the proteoglycans are polyanions and hence bind polycations and cations such as Na* and K*. This latter ability attracts water by osmotic pressure into the extracellular matrix and contributes to its turgor. GAGs also gel at relatively low concentrations. Because of the long, extended nature of the polysaccharide chains of GAGs and their ability to gel, the proteoglycans can act as sieves, restricting the passage of large macromolecules into the ECM but allowing relatively free diffusion of small molecules. Again, because of their extended structures and the huge macromolecular aggregates they often form, they occupy a large volume of the matrix relative to proteins.

protein, their tissue and subcellular distribution, and their biologic functions.
The structure (Figure 50-10), distribution, and functions of each of the GAGs will now be briefly discussed. The major features of the seven GAGs are summarized in Table 50-6.

Hyaluronic acid consists of an unbranched chain of repeating disaccharide units containing GIcUA and GlcNAc. It is present in bacteria and is found in the ECM of nearly all animal tissues, but is especially high in concentration in highly hydrated types such as skin and umbilical cord, and in bone, cartilage, joints (synovial fluid) and in vitreous humor in the eye, as well as in embryonic tissues. It is thought to play an important role in permitting cell migration during morphogenesis and wound repair. Its ability to attract water into the ECM triggers loosening of the matrix, aiding this process. The high concentrations of hyaluronic acid together with chondroitin sulfates present in cartilage contribute to its compressibility (see below).
The seven GAGs named above differ from each other in a number of the following properties: amino sugar composition, uronic acid composition, linkages between these components, chain length of the disaccharides, the presence or absence of sulfate groups and their positions of attachment to the constituent sugars, the nature of the core proteins to which they are attached, the nature of the linkage to core

Proteoglycans linked to chondroitin sulfate by the Xyl-Ser O-glycosidic bond are prominent components of cartilage (see below). The repeating disaccharide is similar to that found in hyaluronic acid, containing GIcUA but with GalNAc replacing GlcNAc. The GalNAc is substituted with sulfate at either its 4' or its 6' position, with approximately one sulfate
. 5 Hyaluronic acid:
1,4
1,3,
1,4
S
p
S
Chondroitin sulfates:
2*
4- or 6-Sulfate
Keratan
sulfates
e
and
Il:
BS
Glonac
“85
Gals
Gicnac
“8p
Gal.
6-Sulfate
6-Sulfate
Heparin
and
a4.
a4
I
at,4
B14
heparan
sulfate:
2-Sulfate
SO;
or
Ac
1,4
1,3,
1,4
Dermatan sulfate:
p
-
S
|
2-Sulfate
4-Sulfate
1,4
——>
GIcUA
—>
GIcNAc
——»>
GIcUA
—»
GIcNAc
——>
81,3
BS
aicua
28s
Gainac
“4
eicua
28>
Gai
Bs
Gai
2s
xy:
2
ser
|
not
GlcNAc
+>
Asn (keratan sulfate |)
woe
se
sche
6-Sulfate
~~.
GalNAc
——»
Thr (Ser) (keratan sulfate II)
|
Gal-NeuAc
ot.
1,3
B13
B14
—_
va
—_
Get
——>
GIcUA
—>
GIcNAc
—»>
GIcUA
—»>
Gal
—>

Gal
—>
Xyl
+,
Ser
B14
61,3
61,3
B14
SY
1sUA
a>
Gainac
“>
aicua
**>
Gainac
“>
aicua
“*>
Gal
*>
Gai
“*s
xyi
+>
ser
FIGURE 50-10 Structures of glycosaminoglycans and their attachments to core proteins. (Ac, acetyl; Asn, L-asparagine; Gal, p-galactose; GalN, p-galactosamine; GIcN, p-glucosamine; GICUA, p-glucuronic acid; IdUA, L-iduronic acid; Man, o-mannose; NeuAc, N-acetylneuraminic acid; Ser, t-serine; Thr, t-threonine; Xyl, t-xylose.) The summary structures are qualitative representations only and do not reflect, for example, the uronic acid composition of hybrid glycosaminoglycans such as heparin and dermatan sulfate, which contain both t-iduronic and p-glucuronic acid. Hyaluronic acid has no covalent attachemnt to protein. Chondroitin sulfates, heparin, heparan sulfate, and dermatan sulfate attach to a Ser on the core protein via the Gal-Gal-Xyl link trisaccharide. Keratan sulfate | links to a core protein Asn via GIcNAc and Keratan sulfate Il to a Ser (or Thr) via GalNAc.

Hyaluronic acid, Sugars = GlcNAc, GLcUA. Hyaluronic acid, Sulfate a = -. Hyaluronic acid, Protein Linkage = None. Hyaluronic acid, Location = Skin, synovial fluid, bone, cartilage, vitreous humor, embryonic tissues. Chondroitin sulfate, Sugars = GalNAc, GlcUA. Chondroitin sulfate, Sulfate a = GalNAc. Chondroitin sulfate, Protein Linkage = Xyl-Ser; associated with HA via link proteins. Chondroitin sulfate, Location = Cartilage, bone, CNS. Keratan sulfate I and II, Sugars = GlcNAc, Gal. Keratan sulfate I and II, Sulfate a = GlcNAc. Keratan sulfate I and II, Protein Linkage = GlcNAc-Asn (KS I) GalNAc-Thr (KS II). Keratan sulfate I and II, Location = Cornea, cartilage, loose connective tissue. Heparin, Sugars = Gln, IdUA. Heparin, Sulfate a = GlcN GlcN IdUA. Heparin, Protein Linkage = Ser. Heparin, Location = Mast cells, liver, lung, skin. Heparan sulfate, Sugars = GlcN, GlcUA. Heparan sulfate, Sulfate a = GlcN. Heparan sulfate, Protein Linkage = Xyl-Ser. Heparan sulfate, Location = Skin, kidney basement membrane. Dermatan sulfate, Sugars = GalNAc, IdUA, (GlcUA). Dermatan sulfate, Sulfate a = GalNAc IdUA. Dermatan sulfate, Protein Linkage = Xyl-Ser. Dermatan sulfate, Location = Skin, wide distribution
"The sulfate is attached to various positions of the sugars indicated (see Figure 50-10). Note that all GAGs except the keratan sulfates contain a uronic acid which may be glucuronic or iduronic acid.

being present per disaccharide unit. Chondroitin sulfates have an important role in maintaining the structure of the ECM. They are located at sites of calcification in endochondral bone and are a major component of cartilage. They are found in high amounts in the ECM of the central nervous system and, in addition to their structural function, are thought to act as signaling molecules in the prevention of the repair of nerve endings after injury.

As shown in Figure 50-10, the keratan sulfates consist of repeating Gal-GlcNAc disaccharide units containing sulfate attached to the 6' position of GlcNAc or occasionally of Gal. Keratan sulfate I was originally isolated from the cornea, while keratan sulfate II came from cartilage. The two GAGs differ in the structural links to the core proteins, and as I or II, the classification is based on the different linkage to the core protein (Figure 50-10). In the eye, they lie between collagen fibrils and play a critical role in corneal transparency. Changes in proteoglycan composition found in corneal scars disappear when the cornea heals.

The repeating disaccharide heparin contains glucosamine (GlcN) and either of the two uronic acids (GICUA or IGUA)
(Figure 50-11). Most of the amino groups of the GIcN residues are N-sulfated, but a few are acetylated (GlcNAc). The GIcN also carries a sulfate attached to carbon 6. The vast majority of the uronic acid residues are IdUA. Initially, all of the uronic acids are GIcUA, but a 5'-epimerase converts approximately 90% of the GlcUA residues to IdUA after the polysaccharide chain is formed. The protein molecule of the heparin proteoglycan is unique, consisting exclusively of serine and glycine residues. Approximately, two-thirds of the serine residues contain GAG chains, usually of 5 to 15 kDa but occasionally much larger. Heparin is found in the granules of mast cells and also in liver, lung, and skin. It is an important anticoagulant. It is released into the blood from capillary walls by the action of lipoprotein lipase and it binds with factors IX and XI, but its most important interaction is with plasma antithrombin (discussed in Chapter 55).
—
_
_
_
|

This molecule is present in a proteoglycan found on many extracellular cell surfaces. It contains GlcN with fewer N-sulfates than heparin, and, unlike heparin, its predominant uronic acid is GlcUA. Heparan sulfates are associated with the plasma membrane of cells, with their core proteins actually spanning that membrane. In this, they may act as receptors
__
FIGURE 50-11 Structure of heparin. Structural features typical of heparin are shown. Each repeating disaccharide contains glucosamine (GlcN) and either p-glucuronic (GIcUA) or L-iduronic acid (IdUA). A few GIcN residues are acetylated (GIcNAc). The sequence of variously substituted repeating disaccharide units has been arbitrarily selected. Non-O-sulfated or 3-O-sulfated glucosamine residues may also occur. (Modified and redrawn from Lindahl U, et al: Structure and biosynthesis of heparin-like polysaccharides. Fed Proc 1977;36:19.)
and may also participate in the mediation of the cell growth and cell-cell communication. The attachment of cells to their substratum in culture is mediated at least in part by heparan sulfate. This proteoglycan is also found in the basement membrane of the kidney along with type IV collagen and laminin (see above), where it plays a major role in determining the charge selectiveness of glomerular filtration.

This substance is widely distributed in animal tissues. Its structure is similar to that of chondroitin sulfate, except that in place of a GIcUA in B-1,3 linkage to GalNAc it contains an IdUA in an a-1,3 linkage to GalNAc. Formation of the IdUA occurs, as in heparin and heparan sulfate, by 5'-epimerization of GIcUA. Because this is regulated by the degree of sulfation and because sulfation is incomplete, dermatan sulfate contains both IdUA-GalNAc and GlcUA-GalNAc disaccharides. Dermatan sulfate has a widespread distribution in tissues, and is the main GAG in skin. Evidence suggests it may play a part in blood coagulation, wound repair, and resistance to infection.
Proteoglycans are also found in intracellular locations such as the nucleus where they are thought to have a regulatory role in functions such as cell proliferation and transport of molecules between the nucleus and the cytosol. The various functions of GAGs are summarized in Table 50-7.

Both exoand endoglycosidases degrade GAGs. Like most other biomolecules, GAGs are subject to turnover, being both synthesized and degraded. In adult tissues, GAGs generally

+ Actas structural components of the ECM
+ Have specific interactions with collagen, elastin, fibronectin, laminin, and other proteins such as growth factors
+ As polyanions, bind polycations and cations
+ Contribute to the characteristic turgor of various tissues
+ Actas sieves in the ECM
+ Facilitate cell migration (HA)
+ Have role in compressibility of cartilage in weight bearing (HA, CS)
+ Play role in corneal transparency (KS | and DS)
+ Have structural role in sclera (DS)
+ Actas anticoagulant (heparin)
+ Are components of plasma membranes, where they may act as receptors and participate in cell adhesion and cell-cell interactions (eg, HS)
+ Determine charge selectiveness of renal glomerulus (HS)
+ Are components of synaptic and other vesicles (eg, HS)
+ Have a role in nuclear functions such as cell proliferation and transport of molecules between the nucleus and the cytosol
Abbreviations: CS, chondroitin sulfate; DS, dermatan sulfate; ECM, extracellular matrix; HA, hyaluronic acid; HS, heparan sulfate; KS |, keratan sulfate |.
exhibit relatively slow turnover, their half-lives being days to weeks.
Understanding of the degradative pathways for GAGs, as in the case of glycoproteins (see Chapter 46) and glycosphingolipids (see Chapter 24), has been greatly aided by elucidation of the specific enzyme deficiencies that occur in certain inborn errors of metabolism. When GAGs are involved, these inborn errors are called mucopolysaccharidoses (MPSs) (Table 50-8).

Degradation of GAGs is carried out by a battery of lysosomal hydrolases. These include endoglycosidases, exoglycosidases, and sulfatases, generally acting in sequence. The MPSs (Table 50-8) share a common mechanism of causation involving a mutation in a gene encoding a lysosomal hydroxylase responsible for the degradation of one or more GAGs. This leads to a defect in the enzyme and the accumulation of the substrate GAGs in various tissues, including the liver, spleen, bone, skin, and the central nervous system. The diseases are usually inherited in an autosomal recessive manner, with Hurler and Hunter syndromes being perhaps the most widely studied. is common. In general, these conditions are chronic and progressive and affect multiple organs. Many patients exhibit organomegaly (eg, hepatoand splenomegaly); severe abnormalities in the development of cartilage and bone; abnormal facial appearance; and mental retardation. In addition, defects in hearing, vision, and the cardiovascular system may be present. Diagnostic tests include analysis of GAGs in urine or tissue biopsy samples; assay of suspected defective enzymes in white blood cells, fibroblasts or serum; and test for specific genes. Prenatal diagnosis is now sometimes possible using amniotic fluid cells or chorionic villus biopsy samples. In some cases, a family history of a mucopolysaccharidosis is obtained.

The term "mucolipidosis" was introduced to denote diseases that combined features common to both mucopolysaccharidoses and sphingolipidoses (see Chapter 24). In sialidosis (mucolipidosis I, ML-I), various oligosaccharides derived from glycoproteins and certain gangliosides accumulate in tissues. I-cell disease (ML-II) and pseudo-Hurler polydystrophy (MLIII) are described in Chapter 46. The term "mucolipidosis" is retained because it is still in relatively widespread clinical usage, but it is not appropriate for these two latter diseases since the mechanism of their causation involves mislocation of certain lysosomal enzymes. Genetic defects of the catabolism of the oligosaccharide chains of glycoproteins (eg, mannosidosis, fucosidosis) are also described in Chapter 46. Most of these defects are characterized by increased excretion of various fragments of glycoproteins in the urine, which accumulate because of the metabolic block, as in the case of the mucolipidoses.
Hyaluronidase is one important enzyme involved in the catabolism of both hyaluronic acid and chondroitin sulfate. It is a widely distributed endoglycosidase that cleaves hexosaminidic linkages. From hyaluronic acid, the enzyme will generate a tetrasaccharide with the structure

a = MPS IVA. Morquio syndrome A, Enzyme Defective = Galactosamine 6-sulfatase. Morquio syndrome A, GAG(s) Affected = Keratan sulfate, chondroitin 6-sulfate. Morquio syndrome A, Symptoms = Skeletal dysplasia, short stature. Morquio syndrome B, Abbreviation a = MPS IVB. Morquio syndrome B, Enzyme Defective = β-Galactosidase. Morquio syndrome B, GAG(s) Affected = Keratan sulfate. Morquio syndrome B, Symptoms = As MPS IVA. Maroteaux-Lamy syndrome, Abbreviation a = MPSVI. Maroteaux-Lamy syndrome, Enzyme Defective = N -Acetylgalactosamine 4-sulfatase c. Maroteaux-Lamy syndrome, GAG(s) Affected = Dermatan sulfate. Maroteaux-Lamy syndrome, Symptoms = Curvature of the spine, short stature, skeletal dysplasia, cardiac defects. Sly syndrome, Abbreviation a = MPSVII. Sly syndrome, Enzyme Defective = β-Glucuronidase. Sly syndrome, GAG(s) Affected = Dermatan sulfate, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate. Sly syndrome, Symptoms = Skeletal dysplasia, short stature, hepatomegaly, cloudy cornea. Natowicz syndrome, Abbreviation a = MPS IX. Natowicz syndrome, Enzyme Defective = Hyaluronidase. Natowicz syndrome, GAG(s) Affected = Hyaluronic acid. Natowicz syndrome, Symptoms = Joint pain, short stature
"The terms MPS V and MPS VIII are no longer used.
°Also called sulfaminidase.
"Also called arylsulfatase B.

(GIcUAB-1,3-GlcNAc-B-1,4),, which can be degraded further by a B-glucuronidase and B-N-acetylhexosaminidase. A genetic defect in hyaluronidase causes MPS IX, a lysosomal storage disorder in which hyaluronic acid accumulates in the joints.

Hyaluronic acid may be important in permitting tumor cells to migrate through the ECM. Tumor cells can induce fibroblasts to synthesize greatly increased amounts of this GAG, thereby facilitating their own spread. Some tumor cells have less heparan sulfate at their surfaces, and this may play a role in the lack of adhesiveness that these cells display.
The intima of the arterial wall contains hyaluronic acid and chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans. Of these proteoglycans, dermatan sulfate binds plasma low-density lipoproteins. In addition, dermatan sulfate appears to be the major GAG synthesized by arterial smooth muscle cells. Because these cells proliferate in atherosclerotic lesions in arteries, dermatan sulfate may play an important role in development of the atherosclerotic plaque.
In various types of arthritis, proteoglycans may act as autoantigens, thus contributing to the pathologic features of these conditions. The amount of chondroitin sulfate in cartilage diminishes with age, whereas the amounts of keratan sulfate and hyaluronic acid increase. These changes may contribute to the development of osteoarthritis, as may increased activity of the enzyme aggrecanase, which acts to degrade aggrecan. Changes in the amounts of certain GAGs in the skin help to account for its characteristic alterations with aging.
In the past few years, it has become clear that in addition to their structural role in the ECM, proteoglycans function as signaling molecules which influence cell behavior, and they are now believed to play a part in diverse diseases such as fibrosis, cardiovascular disease, and cancer.

Bone contains both organic and inorganic material. The organic matter is mainly protein. The principal proteins of bone are listed in Table 50-9; type I collagen is the major protein, comprising 90 to 95% of the organic material. Type V collagen is also present in small amounts, as are a number of noncollagen proteins, some of which are relatively specific to bone. These are now believed to play an active part of the mineralization process. The inorganic or mineral component is mainly crystalline hydroxyapatite—Ca,,(PO,),(OH),—along
Collagens, Comments = . Collagen type I Collagen type V, Comments = Approximately 90% of total bone protein. Composed of two α1(I) and one α2(I) chains. Minor component.. Noncollagen proteins, Comments = . Plasma proteins Proteoglycans b CS-PG I, Comments = Mixture of various plasma proteins. Contains two GAG chains; found in other. (biglycan), Comments = tissues.. CS-PG II (decorin), Comments = Contains one GAG chain; found in other tissues.. CS-PG III, Comments = Bone-specific.. Bone SPARC c protein (osteonectin), Comments = Not bone-specific.. Osteocalcin (bone Gla protein), Comments = Contains γ-carboxyglutamate (Gla) residues that bind to hydroxyapatite. Bone-specific.. Osteopontin, Comments = Not bone-specific. Glycosylated and phosphorylated.. Bone sialoprotein, Comments = Bone-specific. Heavily glycosylated, and sulfated on tyrosine.. Bone morphogenetic proteins (BMPs), Comments = A family (at least 20) of secreted proteins with a variety of actions on bone; many induce ectopic bone growth.. Osteoprotegerin, Comments = Inhibits osteoclastogenesis
*Noncollagen proteins
are involved
in the
the mineralization
process.
A
number of
other proteins
are also
present in
bone, including
a
tyrosine-rich acidic
matrix protein
(TRAMP), some
growth factors
(eg,
TGF-B), and
®CS-PG, chondroitin

sulfate-proteoglycan;
these are
enzymes similar
"SPARC, secreted protein acidic and rich in cysteine.
with sodium,
magnesium, carbonate,
mately
99%
of
Chapter
44).
resilience the
body’s calcium
is
Hydroxyapatite required
Bone cycles
of confers
for its
to the
collagen dermatan
and fluoride;
contained on
physiologic is
a
dynamic remodeling,
ization)
followed structure
synthesis sulfate
PGs approxi-
in bone
bone the
roles.
that consisting
by deposition
alization).
This undergoes
of resorption
of remodeling
physical
(eg,
(see strength
and continuing
(demineral- new
bone tissue
permits increases
in weight
bone to
bearing)
(miner- adapt
to both
signals.
The major cell types involved in bone resorption and deposition are osteoclasts and osteoblasts, respectively (Figure 50-12). Osteocytes are found in mature bone and are also involved in the maintenance of the bone matrix. They are descended from osteoblasts and are very long-lived, with an average half-life of 25 years.

Osteoclasts are multinucleated cells derived from pluripotent hematopoietic stem cells. Osteoclasts possess an apical membrane domain, exhibiting a ruffled border that plays a key role in bone resorption (Figure 50-13). A special proton-translocating ATPase expels protons across the ruffled border into the resorption area, which is the microenvironment of low pH shown in the figure. This lowers the local pH to 4.0 or less, thus increasing the solubility of hydroxyapatite and helping its breakdown into Ca', H,PO, and H,CO,, and water, thus allowing demineralization to occur. Lysosomal acid proteases such as cathepsins are also released to digest the now accessible matrix proteins. Osteoblasts— mononuclear cells derived from pluripotent mesenchymal precursors—synthesize most of the proteins found in bone (Table 50-9) as well as various growth factors and cytokines. They are responsible for the deposition of the new bone matrix (osteoid) and its subsequent mineralization.
(eg, lysyl
oxidase).
(DS-PGs)
of cartilage.
Osteoblasts control mineralization by regulating the passage of calcium and phosphate ions across their surface membranes. Alkaline phosphatase, an enzyme in the cell membrane, generates phosphate ions from organic phosphates. The mechanisms involved in mineralization are not fully understood, but a number of factors, including tissue nonspecific alkaline phosphatase (TNAP), an isoenzyme of alkaline phosphatase, matrix vesicles, which contain calcium and phosphate and bud off from the osteoblast membrane and type I collagen have been implicated. Mineralization first becomes evident in the gaps between successive collagen molecules. Acidic phosphoproteins, such as bone sialoprotein and osteopontin, are thought to act as sites of nucleation. These proteins contain RGD sequences for cell attachment and motifs (eg, poly-Asp and poly-Glu stretches) that bind calcium and may provide an initial scaffold for mineralization. Some macromolecules, such as certain proteoglycans and glycoproteins, can also act as inhibitors of nucleation.

Bone consists of two types of tissue, trabecular (also called cancellous or spongy) bone is found at the end of long bones close to the joints and is less dense than compact (or cortical) bone, which, as well as being denser, is harder and stronger. It forms the cortex (or outer shell) of most bones and accounts for about 80% of the weight of the human skeleton. It is estimated that approximately 4% of compact bone and 20% of trabecular bone is renewed annually in the typical healthy adult. Many factors are involved in the regulation of bone metabolism. Some of which stimulate osteoblast activity (eg, parathyroid hormone and 1,25-dihydroxycholecalciferol [see Chapter 44]) to promote mineralization, while others inhibit it (eg, corticosteroids). Parathyroid hormone and
FIGURE 50-12 Schematic illustration of the major cells present in the membranous bone. Osteoblasts (lighter color) are synthesizing type | collagen, which forms a matrix that traps cells. As this occurs, osteoblasts gradually differentiate to become osteocytes. (Reproduced, with permission, from Junqueira LC, Carneiro J: Basic Histology: Text & Atlas, 10th ed. McGraw-Hill, 2003.)
FIGURE 50-13 Schematic illustration of the role of the osteoclast in bone resorption. Lysosomal enzymes and hydrogen ions are released into the confined microenvironment created by the attachment between the bone matrix and the peripheral clear zone of the osteoclast. The acidification of this confined space facilitates the dissolution of calcium phosphate from bone and is the optimal pH for the activity of lysosomal hydrolases. The bone matrix is thus removed, and the products of bone resorption are taken up into the cytoplasm of the osteoclast, probably digested further, and transferred into capillaries. The chemical equation shown refers to the action of carbonic anhydrase II, described in the text. (Reproduced, with permission, from Junqueira LC, Carneiro J: Basic Histology: Text & Atlas, 10th ed. McGraw-Hill, 2003.)

1,25-dihydroxycholecalciferol also stimulate bone resorbtion by increasing osteoclast activity, whereas calcitonin and estrogens have the opposite effect.

A number of metabolic and genetic disorders affect bone, and some of the more important examples are listed in Table 50-10.
Osteogenesis imperfecta (brittle bones) is characterized by abnormal fragility of bones. The sclera of the eye is often abnormally thin and translucent and may appear blue owing to a deficiency of connective tissue. Eight types (I-VI) of this condition have been recognized. Types I to IV are caused by mutations in the COLIA1 or COL1A2 genes or both. Type I is mild, but type II is severe and infants born with the condition usually do not survive, and types III and IV are progressive and/or deforming. Over 100 mutations in these two genes have been documented and include partial gene deletions and duplications. Other mutations affect RNA splicing, and the most frequent type results in the replacement of glycine by another bulkier amino acid, affecting formation of the triple helix. In general, these mutations result in decreased expression of collagen or in structurally abnormal pro chains that assemble into abnormal fibrils, weakening the overall structure of bone. When one abnormal chain is present, it may interact with two normal chains, but folding may be prevented, resulting in enzymatic degradation of all of the chains. This is called "procollagen suicide" and is an example of a dominant negative mutation, a result often seen when a protein consists of multiple different subunits. Types V to VIII are less common and are caused by mutations in the genes for proteins involved in bone mineralization other than collagen.
Osteopetrosis (marble bone disease), characterized by increased bone density, is a rare condition characterized by inability to resorb bone. It is due to mutations in the gene (located on chromosome 8q22) encoding carbonic anhydrase II (CA ID), one of four isozymes of carbonic anhydrase present in human tissues. Deficiency of CA II in osteoclasts prevents normal bone resorption, and osteopetrosis results.

Osteoporosis is a generalized progressive reduction in bone tissue mass per unit volume, caused by an imbalance between bone resorption and deposition, and leads to skeletal weakness. The primary type 1 condition commonly occurs in women after the menopause. This is thought to be mainly due to lack of estrogen, which promotes bone resorption and decreases bone mineralization. Primary type 2 or senile osteoporosis occurs in both sexes post 75 years, although is more prevalent in women (ratio 2:1 female:male). The ratio of mineral to organic elements is unchanged in the remaining normal bone. Fractures of various bones, such as the head of the femur, occur very easily and represent a huge burden to both the affected patients and to society in general.

There are three types of cartilage. The major type is hyaline (articular) cartilage and its principal proteins are listed in Table 50-11. Type II collagen is the major protein component (Figure 50-14), and a number of other minor types of collagen are also present. In addition to these components, the second type, elastic cartilage, contains elastin, and the third, fibroelastic cartilage, contains type I collagen. Cartilage contains a number of proteoglycans, which play an important

Dwarfism, Causes = Often deficiency of growth hormone, but many other causes. Dwarfism, Condition = Osteoporosis. Dwarfism, Causes = Age-related, estrogen deficiency following menopause, mutations in genes affecting bone metabolism, a including the vitamin Dreceptor ( VDR ), estrogen receptor-α ( ER- α), and COL1A1. Rickets, Causes = Deficiency of vitamin Din childhood. Rickets, Condition = Osteoarthritis. Rickets, Causes = Age-related cartilage degeneration, mutations in various genes a including VDR , ER- α, and COL2A1. Osteomalacia, Causes = Deficiency of vitamin Din adults. Osteomalacia, Condition = Chondrodysplasias. Osteomalacia, Causes = Mutations in COL2A1. Hyperparathyroidism, Causes = Excess parathyroid hormone causing bone resorption. Hyperparathyroidism, Condition = Pfeiffer, Jackson-Weiss, and Crouzon syndromes b. Hyperparathyroidism, Causes = Mutations in the gene for fibroblast growth factor receptor (FGFR) 1 and/or 2. Osteogenesis imperfecta, Causes = Mutations in COL1A1 and COL1A2 affecting the synthesis and structure of collagen. Osteogenesis imperfecta, Condition = Achondroplasia and thanatophoric dysplasia c. Osteogenesis imperfecta, Causes = Mutation in the gene for FGFR3
*Only
a
small number
of cases.
'In the Pfeiffer, Jackson-Weiss, and Crouzon syndromes, there is premature fusion of some bones in the skull (craniosynostosis). 'Thanatophoric dysplasia in the most common neonatal lethal skeletal dysplasia.

Collagen proteins Collagen type II, Comments = 90-98% of total hyaline cartilage collagen. Composed of three α1(II) chains.. Collagens V, VI, IX, X, XI, Comments = Type IX cross-links to type II collagen. Type XI may help control diameter of type II fibrils.. Noncollagen proteins Cartilage oligomeric matrix protein (COMP), Comments = An important structural component of cartilage. Regulates cell movement and attachment.. Aggrecan DS-PG I (biglycan) a DS-PG II (decorin), Comments = The major proteoglycan of cartilage. Similar to CS-PG I of bone. Similar to CS-PG II of bone.. Chondronectin, Comments = Promotes chondrocyte attachment to type II collagen
"The core proteins of the proteoglycans DS-PG | and DS-PG II are homologous to those of CS-PG | and CS-PG II found in bone. A possible explanation is that osteoblasts lack the epimerase required to convert glucuronic acid to iduronic acid, the latter is found in dermatan sulfate.

role in its compressibility. Aggrecan (about 2 x 10° kDa) is the major proteoglycan. As shown in Figure 50-15, it has a very complex structure, containing several GAGs (hyaluronic acid, chondroitin sulfate, and keratan sulfate) and both link and core proteins. The core protein contains three domains: A, B, and C. Hyaluronic acid binds noncovalently to domain A of the core protein as well as to the link protein, which stabilizes the hyaluronate-core protein interactions. The keratan sulfate chains are located in domain B, whereas the chondroitin sulfate chains are located in domain C; both of these types of GAGs are bound covalently to the core protein. The core protein also contains both Oand N-linked oligosaccharide chains. The other proteoglycans found in cartilage have simpler structures than aggrecan. Chondronectin is involved in the attachment of type II collagen to chondrocytes (the cells in cartilage). Cartilage is an avascular tissue and obtains most of its nutrients from synovial fluid. It exhibits slow but continuous turnover. Various proteases (eg, collagenases and stromelysin) synthesized by chondrocytes can degrade collagen and the other proteins found in cartilage. Interleukin-1 (IL-1) and tumor necrosis factor a (TNF-a) appear to stimulate the production of such proteases, whereas TGF-f and insulin-like growth factor 1 (IGF-I) generally exert an anabolic influence on the cartilage.

Chondrodysplasias are a mixed group of hereditary disorders affecting cartilage. They are manifested by short-limbed dwarfism and numerous skeletal deformities. A few are due to a variety of mutations in the COL2A1 gene, leading to abnormal forms of type II collagen. One example is the Stickler syndrome, manifested by degeneration of the joint cartilage and of the vitreous body of the eye.
FIGURE 50-14 schematic representation of the molecular organization in the cartilage matrix. Link proteins noncovalently bind the core protein (red) of proteoglycans to the linear hyaluronic acid molecules (gray). The chondroitin sulfate side chains of the proteoglycan electrostatically bind to the collagen fibrils, forming a cross-linked matrix. The oval outlines the area enlarged in the lower part of the figure. (Reproduced, with permission, from Junqueira LC, Carneiro J: Basic Histology: Text & Atlas, 10th ed. McGraw-Hill, 2003.)
FIGURE 50-15 Schematic diagram of aggrecan. A strand of hyaluronic acid is shown on the left. The core protein (about 210 kDa) has three major domains. Domain A, at its amino-terminal end, interacts with approximately five repeating disaccharides in hyaluronate. The link protein interacts with both hyaluronate and domain A, stabilizing their interactions. Approximately 30 keratan sulfate chains are attached, via GalNAc-Ser linkages, to domain B. Domain C contains about 100 chondroitin sulfate chains attached via Gal-Gal-Xyl-Ser linkages and about 40 O-linked oligosaccharide chains. One or more N-linked glycan chains are also found near the carboxyl terminal of the core protein. (Moran LA, et al: Biochemistry, 2nd ed., © 1994, pp. 9-43. Adapted by permission of Pearson Education, Inc., Upper Saddle River, NJ.)

The best known of the chondrodysplasias is achondroplasia, the most common cause of short-limbed dwarfism. Affected individuals have short limbs, normal trunk size, macrocephaly, and a variety of other skeletal abnormalities. The condition is often inherited as an autosomal dominant trait, but many cases are due to new mutations. Achondroplasia is not a collagen disorder but is due to mutations in the gene encoding fibroblast growth factor receptor 3 (FGFR3). Fibroblast growth factors are a family of more than 20 proteins that affect the growth and differentiation of cells of mesenchymal and neuroectodermal origin. Their receptors are transmembrane proteins and form a subgroup of four in the family of receptor tyrosine kinases. FGFR3 is one member of this subgroup and mediates the actions of FGF3 on cartilage. In almost all cases of achondroplasia that have been investigated, the mutations were found to involve nucleotide 1138 and resulted in substitution of arginine for glycine (residue number 380) in the transmembrane domain of the protein, rendering it inactive. No such mutation was found in unaffected individuals.

Other mutations in the same gene can result in hypochondroplasia, thanatophoric dysplasia (types I and II) (other forms of short-limbed dwarfism), and the SADDAN phenotype (severe achondroplasia with developmental delay and acanthosis nigricans [the latter is a brown to black hyperpigmentation of the skin]).
As indicated in Table 50-10, other skeletal dysplasias (including certain craniosynostosis syndromes) are also due to mutations in genes encoding FGF receptors. Another type of skeletal dysplasia, diastrophic dysplasia has been found to be due to mutation in a sulfate transporter.

■, 1 = The major components of the ECMare the structural proteins collagen, elastin, and fibrillin-1, a number of specialized proteins (eg, fibronectin and laminin), and various proteoglycans.. ■, 1 = Collagen is the most abundant protein in the animal kingdom; approximately 28 types have been isolated. All collagens contain greater or lesser stretches of triple helix and the repeating structure (Gly-X-Y) n .. ■, 1 = The biosynthesis of collagen is complex, featuring many posttranslational events, including hydroxylation of proline and lysine.. ■, 1 = Diseases associated with impaired synthesis of collagen include scurvy, osteogenesis imperfecta, Ehlers-Danlos syndrome (six subtypes), and Menkes disease.. ■, 1 = Elastin confers extensibility and elastic recoil on tissues. Elastin lacks hydroxylysine, Gly-X-Y sequences, triple helical structure, and sugars, but contains desmosine and isodesmosine cross-links not found in collagen.. ■, 1 = Fibrillin-1 is located in microfibrils. Mutations in the gene encoding fibrillin-1 cause Marfan syndrome. The cytokine TGF-β appears to contribute to the cardiovascular pathology.. ■, 1 = The GAGs are made up of repeating disaccharides containing a uronic acid (glucuronic or iduronic) or hexose (galactose) and a hexosamine (galactosamine or glucosamine). Sulfate is also frequently present.. ■, 1 = The major GAGs are hyaluronic acid, chondroitin 4- and 6-sulfates, keratan sulfates I and II, heparin, heparan sulfate, and dermatan sulfate.. ■, 1 = The GAGs are synthesized by the sequential actions of specific enzymes (glycosyltransferases, epimerases, sulfotransferases,
SECTION
X_
Special
Topics
(B)

- . Describe the differences between actin-based and myosin-based regulation
of muscle contraction.
: Outline the composition and organization of the thick and thin filaments in striated muscle tissues.
= Describe the pivotal role of Ca?* in the initiation of both contraction and relaxation in muscle.
= List the various channels, pumps, and exchangers involved in regulating intracellular Ca* levels in various types of muscle.
List the major energy sources for regenerating ATP in muscle tissue.
Identify the preferred energy sources for fast and slow twitch fibers.
Understand the molecular bases of malignant hyperthermia, Duchenne and Becker muscular dystrophies, and inherited cardiomyopathies.
Explain how nitric oxide (NO) induces relaxation of vascular smooth muscle.
filaments. Know the general structures and functions of the major components of the cytoskeleton, namely microfilaments, microtubules, and intermediate
= Explain the role of mutations in the gene encoding lamin A and lamin in Hutchinson-Gilford progeria syndrome (progeria).

The shape, integrity, and internal organization of all mammalian cells is maintained by an internal network of polymeric protein fibers and associated molecular motors called the cytoskeleton. This structural and mechanical network mediates motional processes such as cytokinesis, endocytosis, exocytosis, secretion, phagocytosis, and diapedesis. Several pathogenic microorganisms, among them Yersinia, Salmonella enterica, Listeria monocytogenes, and Shigella attack or co-opt the cytoskeleton of the infected host as an integral part of their mechanisms of virulence.
Highly specialized muscle cells elaborate extensive inter- nal networks consisting of physically juxtaposed actin and
myosin polymers, or fibrils, that form the core of their contractile apparatus. This mechanically powerful contractile machinery is controlled by signal transduction pathways in which the second messenger Ca** plays a pivotal role. Muscle tissue is subject to a variety of pathologic conditions, many of them hereditary in nature, including Duchenne-type muscular dystrophy; malignant hyperthermia, a serious complication for some patients undergoing certain types of anesthesia, and cardiomyopathies. Heart failure is a very common medical condition, with a variety of causes. Its rational therapy requires understanding of the biochemistry of heart muscle. For example, many widely used vasodilators—such as nitroglycerin, used in the treatment of angina pectoris—act by increasing the formation of NO.

Muscle is a highly specialized tissue configured to convert the chemical energy of ATP potential into mechanical energy on a mass, macroscopic scale. Three types of muscle are found in vertebrates. Skeletal and cardiac muscle display a striated appearance, a consequence of the parallel alignment of the fibrils of their contractile apparatus. Smooth muscle is devoid of striations, a consequence of the (more) random orientation of its contractile fibrils. Mechanically, these differences in orientation mean that cardiac and skeletal muscle contract and exert force in only one dimension, like a coil spring, while contracting smooth muscle contracts exerts force and shortens in all directions, like the polymer skin of an inflated balloon. Skeletal muscle also is under conscious or voluntary nervous control, while both cardiac and smooth muscle work in an unconscious, involuntary manner.

along its length are bundles of parallel myofibrils, consisting of interdigitated, overlapping thick and thin filaments, embedded in intracellular fluid termed sarcoplasm. When a myofibril is examined by electron microscopy, alternating dark and light bands can be observed (Figure 51-1).
These bands are thus referred to as A and I bands, respectively. In the A band, the thin filaments are arranged around the thick (myosin) filament as a secondary hexagonal array. Each thin filament lies symmetrically between three thick filaments (Figure 51-2, center, mid cross section), and each thick filament is surrounded symmetrically by six thin filaments. The central region of the A band, a seemingly less dense region known as the H band, consists entirely of thick filaments. The I band corresponds to a zone containing only thin filaments, and is bisected by the dense and narrow Z line, which consists of a complex network of polypeptides anchoring the thin filaments together (Figure 51-2).
The sarcomere is defined as the region between two Z lines (Figures 51-1 and 51-2) and is repeated along the axis of a fibril at distances of 1500 to 2300 nm depending on the state of contraction. Most muscle fiber cells are aligned so that their sarcomeres are in parallel register (Figure 51-1).

Striated muscle is composed of multinucleated muscle fiber cells, which may extend the entire length of the muscle, surrounded by an electrically excitable plasma membrane, the sarcolemma. Within each individual muscle fiber cell oriented longitudinally
The sliding filament model was largely based on careful morphologic observations on resting, extended, and contracting muscle. When muscle contracts, the H zones and the I bands shorten (see legend to Figure 51-2). Given that the thin and
FIGURE 51-1 The structure of voluntary muscle. The sarcomere is the region between the Z lines. (Drawing by Sylvia Colard Keene. Reproduced, with permission, from Bloom W, Fawcett DW: A Textbook of Histology, 10th ed. Saunders, 1975.)
FIGURE 51-2 Arrangement of filaments in striated muscle. (A) Extended. The positions of the I, A, and H bands in the extended state are shown. The thin filaments partly overlap the ends of the thick filaments, and the thin filaments are shown anchored in the Z lines (often called Z disks). In the lower part of Figure 51-2A, "arrowheads,' pointing in opposite directions, are shown emanating from the myosin (thick) filaments. Four actin (thin) filaments are shown attached to two Z lines via a-actinin. The central region of the three myosin filaments, free of arrowheads, is called the M band (not labeled). Cross sections through the M bands, through an area where myosin and actin filaments overlap and through an area in which solely actin filaments are present, are shown. (B) Contracted. The actin filaments are seen to have slipped along the sides of the myosin fibers toward each other. The lengths of the thick filaments (indicated by the A bands) and the thin filaments (distance between Z lines and the adjacent edges of the H bands) have not changed. However, the lengths of the sarcomeres have been reduced (from 2300 to 1500 nm), and the lengths of the H and | bands are also reduced because of the overlap between the thick and thin filaments. These morphologic observations provided part of the basis for the sliding filament model of muscle contraction.

thick filaments remain intact, it was concluded that the interdigitated filaments slide past one another during contraction. As the tension developed during muscle contraction is proportionate to the degree to which the filaments overlapped, it was apparent that contraction involved a dynamic interaction between the filaments by which they were actively pulled past one another.
The F-actin fiber, also known as the thin filament, is 6to 7-nm thick and has a structure that repeats every 35.5 nm. Humans contain genes encoding 12 classes of myosins. The tissue distribution and relative amounts of these myosin isoforms can vary in different anatomic, physiologic, and pathologic situations.

Actin and myosin contribute 20 and 55% of the protein mass in muscle, respectively. The monomeric form of actin, G-actin (43 kDa; G, globular) polymerizes in the presence of Mg** to form an insoluble double helical filament called F-actin (Figure 51-3).
Myosin-I, which exists in cells as a monomer, links cytoskeletal microfilaments to the plasma membrane. The predominant form of myosin in contractile tissues is myosin II, hereafter referred to simply as myosin, an asymmetric hexamer with a molecular mass of =460 kDa. The hexamer consists of one pair of ~200 kDa heavy (H) chains, and two pairs of light (L) chains, referred to as essential and regulatory, each with a molecular mass of ~20 kDa. The two heavy chains are intertwined, forming an extended helical tail, each capped by a globular head domain to which the light chains associate (Figure 51-4). Myosin possesses low,
FIGURE 51-3 Schematic representation of the thin filament, showing the spatial configuration of its three major protein components: actin, troponin, and tropomyosin. The upper panel shows individual molecules of G-actin. The middle panel shows actin monomers assembled into F-actin. Individual molecules of tropomyosin (two strands wound around one another) and of troponin (made up of its three subunits) are also shown. The lower panel shows the assembled thin filament, consisting of F-actin, tropomyosin, and the three subunits of troponin (TpC, Tpl, and TpT).
but detectable levels of ATPase activity in vitro: the catalysis of the hydrolysis of ATP by water to form ADP and P.. Low levels of ATPase activity are a common feature of enzymes that employ ATP as a substrate. When skeletal muscle myosin is complexed to actin to form actomyosin complex, its ATPase activity greatly increases.

The tendency of myosin to polymerize under physiologic conditions frustrated attempts to map its domain organization. Therefore, researchers turned to limited proteolysis, a technique wherein proteins are treated with small quantities of proteolytic enzymes for short periods. The objective of this method is to capture individual protein domains that have been liberated by the hydrolysis of the oftentimes highly susceptible peptide bonds present in the flexible "hinge" regions that link individual functional domains.
Limited proteolysis with trypsin yielded two myosin fragments called light and heavy meromyosin (HMM). Light meromyosin (LMM) consists of the aggregated a-helical fibers from the tail of myosin (Figure 51-4). It does not hydrolyze ATP or bind to F-actin. By contrast, HMM is a soluble = 340 kDa protein that possesses both fibrous and globular regions (Figure 51-4). HMM exhibits ATPase activity and binds to F-actin. Limited proteolysis of HMM with papain cleaves it into two subfragments, S-1 and S-2, that encompass the globular and fibrous regions, respectively. Only the globular S-1 fragment, ~ 115 kDa, exhibits ATPase activity and binds both actin and myosin light chains (Figure 51-4).

In striated muscle, there are two other proteins that are minor in terms of their mass but important in terms of their function. Tropomyosin is a fibrous molecule that consists of two
FIGURE 51-4 Diagram of a myosin molecule showing the two intertwined a-helices (fibrous portion), the globular region or head (G), the light chains (L), and the effects of proteolytic cleavage by trypsin and papain. The globular head region, which contains an actinbinding site and an L chain-binding site, attaches to the remainder of the myosin molecule.
chains, a and £, that attach to F-actin in the groove between its filaments (Figure 51-3). Tropomyosin is present in all muscular and muscle-like structures. The troponin complex is unique to striated muscle and consists of three polypeptides. Troponin T (TpT) binds to tropomyosin as well as to the other two troponin components. Troponin I (Tp!) inhibits the F-actin-myosin interaction and also binds to the other components of troponin. Troponin C (TpC) is a calcium-binding polypeptide that is structurally and functionally analogous to calmodulin, an important calcium-binding protein widely distributed in nature. Up to four calcium ions can bind per molecule of troponin C or calmodulin, both of which have a molecular mass of ~ 17 kDa.
the energy unleashed, when multiplied by the 1-2 x 10'* myosin molecules in a human bicep, both great force and rapid motion can be generated. A schematic diagram of the cycle of events involved in each power stroke is presented in Figure 51-5.

Muscle contraction occurs when thick filaments translocate along adjacent thin filaments by a process analogous to climbing a rope hand over hand. The hands in this instance are the S-1 domains of the myosin head, which ascend by via a repeated cycle of attachment, an ATP-powered conformational change or power stroke, and detachment. Although individually minute in terms of both distance covered and
FIGURE 51-5 The hydrolysis of ATP drives the cyclic association and dissociation of actin and myosin in five reactions described in the text. (Reproduced with permission from McGraw-Hill Higher Education.)
At rest, the S-1 head of myosin contains bound ADP and P,, remnants of the last prior power stroke. Upon stimulation by the second messenger Ca** (see below), actin becomes accessible to the S-1 head of myosin which finds it and binds it, thereby forming a cross-bridge linking the thick and thin filaments that are spaced at intervals of 14 nm along the thick filaments. As depicted in Figure 51-2, the clusters of crossbridges (abbreviated as arrowheads with the remainder of the thin filaments not shown) at ends of a thick filament have opposite polarities. The two polar regions of the thick filament are separated by a 150-nm segment (the M band, not labeled in the figure) which does not participate in cross-bridge formation.
Once the actin:myosin:ADP:P, cross-bridge complex is formed, the bound P, is released. This is followed by release of the bound ADP, triggering a large conformational change in the head of myosin in relation to its tail (Figure 51-6), the power stroke, that pulls the actin about 10 nm toward the center of the sarcomere. This nucleotide-free actin-myosin complex represents its low-energy state.

FIGURE 51-6 Representation of the active cross-bridges between thick and thin filaments. This diagram was adapted by AF Huxley from HE Huxley: the mechanism of muscular contraction. Science 1969;164:1356. The latter proposed that the force involved in muscular contraction originates in a tendency for the myosin head (S-1) to rotate relative to the thin filament and is transmitted to the thick filament by the S-2 portion of the myosin molecule acting as an inextensible link. Flexible points at each end of S-2 permit S-1 to rotate and allow for variations in the separation between filaments. This figure is based on HE Huxley's proposal, and also incorporates elastic (the coils in the S-2 portion) and stepwise-shortening elements (depicted here as four sites of interaction between the S-1 portion and the thin filament). (See Huxley AF, Simmons RM: Proposed mechanism of force generation in striated muscle. Nature [Lond] 1971;233:533.) The strengths of binding of the attached sites are higher in position 2 than in position 1 and higher in position 3 than position 2. The myosin head can be detached from position 3 with the utilization of a molecule of ATP; this is the predominant process during shortening. The myosin head is seen to vary in its position from about 90° to about 45°, as indicated in the text. (S-1, myosin head; S-2, portion of the myosin molecule; LMM, light meromyosin) (see legend to Figure 49-4). (Reproduced from Huxley AF: Muscular contraction. J Physiol 1974;243:1. By kind permission of the author and the Journal of Physiology.)

Next, a molecule of ATP binds to the S-1 head, which drastically lowers the affinity of the myosin head for actin. Actin is thus released, or detached. Finally, in the relaxation phase of muscle contraction, the S-1 head of myosin hydrolyzes ATP to ADP and P,, but these products remain bound. The hydrolysis of ATP to ADP-P,-myosin raises myosin into a so-called high-energy conformation. The myosin:ADP:P, complex now stands ready to engage in another cycle and move another 10 nm along the thin filaments, provided Ca** levels remain elevated.
Calculations have indicated that the thermodynamic efficiency of muscle contraction is about 50%, which compares quite favorably to the 20% or less of the internal combustion engine. It should also be noted that, while hydrolysis of ATP ultimately powers the cycle, the release of ADP provides the immediate driver for the conformational change in S-1 that translates chemical energy into the mechanical power stroke. Rigor mortis, the stiffening of the body that occurs postmortem, occurs because the resulting fall in intracellular ATP levels prevents the dissociation of the myosin S-1 head from actin.

The contraction of muscles from all sources occurs by the general mechanism described above. However, the manner in which contraction is regulated differs among the various types of muscle tissue. Striated muscle relies on actin-based regulation, while regulation in smooth muscle is myosin-based. However, regardless of whether regulation is actinor myosinbased, the second messenger Ca"* plays a central role in the initiation and control of contraction.

The contractile apparatus in skeletal muscle is regulated by the troponin system, which is bound to tropomyosin and F-actin in the thin filament (Figure 51-3). Hence, regulation of skeletal muscle is said to be actin-based. In resting muscle, TpI prevents binding of the myosin head to its F-actin attachment site either by altering the conformation of F-actin via the tropomyosin molecules or by simply rolling tropomyosin into a position that directly blocks the sites on F-actin to which the myosin heads attach. Binding of Ca** to TpC relieves inhibition by TpI, permitting the power stroke cycle to proceed. When Ca" levels fall, the TpC:Ca** complex dissociates, allowing TpI to reestablish its inhibitory block on the binding of the myosin S-1 head to F-actin. The thick and thin filaments detach from one another, allowing the muscle to relax.

Smooth muscles have molecular structures similar to those in striated muscle, but the sarcomeres are not aligned so as
to generate the striated appearance. Smooth muscles contain a-actinin and tropomyosin molecules, as do skeletal muscles, but they lack the troponin system. The light chains of smooth muscle myosin molecules also differ from those of striated muscle myosin. In smooth muscle, it is the light chains that prevent the binding of the myosin head to F-actin in the resting state. This inhibitory block is released by the phosphorylation of the regulatory, or type 2, light chains in the myosin S-1 head by myosin light-chain kinase.
The activity of myosin light-chain kinase is controlled by the binding of the small, ~ 17 kDa, Ca**-binding protein, calmodulin (Figure 51-7).
Calmodulin is a ubiquitous protein that contains four EF-hand motifs, each of which can bind one molecule of calcium. Binding of calcium to all four of these sites triggers a conformational change that enables the Ca**,:calmodulin complex to bind to and activate various intracellular target enzymes, including myosin light-chain kinase and Ca**ATPase (see below). Ca** binding to calmodulin exhibits strong positive cooperativity, optimizing it to serve as a Ca'*-sensitive molecular trigger. When Ca'* levels fall, the balance of activity between myosin light-chain kinases and phosphatases shifts in favor of the latter, leading to the dephosphorylation of regulatory light chains, the detachment of myosin S-1 domains from actin, and muscle relaxation.
FIGURE 51-7 Regulation of smooth muscle contraction by Ca**. The pL-myosin is the phosphorylated light chain of myosin and L-myosin is the dephosphorylated light chain. (Adapted, with permission, from Adelstein RS, Eisenberg R: Regulation and kinetics of actin-myosin ATP interaction. Annu Rev Biochem 1980;49:921. Copyright © 1980 by Annual Reviews, www.annualreviews.org.)

Rho kinase provides a Ca®'-independent pathway for initiating contraction. Rho kinase not only phosphorylates myosin's regulatory light chains, it also phosphorylates the phosphatase that dephosphorylates them. Since phosphorylation of the phosphatase inhibits its activity, this serves to further shift the balance between the kinases and phosphatases that act on myosin in favor of the former. Table 51-1 summarizes and compares the regulation of actin-myosin interactions
(activation of myosin ATPase) in striated and smooth muscles. Phosphorylation of Rho kinase by the cAMP-dependent protein kinase may play a role in the dampening effect of this second messenger on smooth muscle contraction.
Another protein that appears to play a Ca**-dependent role in the regulation of smooth muscle contraction is caldesmon (87 kDa). This protein is ubiquitous in smooth muscle and is

Proteins of muscle filaments, Striated Muscle = Actin Myosin Tropomyosin Troponin (Tpl, TpT, TpC). Proteins of muscle filaments, Smooth Muscle (and Nonmuscle Cells) = Actin Myosin a Tropomyosin. Spontaneous interaction of F-actin and myosin alone (spontaneous activation of myosin ATPase by F-actin), Striated Muscle = Yes. Spontaneous interaction of F-actin and myosin alone (spontaneous activation of myosin ATPase by F-actin), Smooth Muscle (and Nonmuscle Cells) = No. Inhibitor of F-actin-myosin interaction (inhibitor of F-actin-dependent activation of ATPase), Striated Muscle = Troponin system (Tpl). Inhibitor of F-actin-myosin interaction (inhibitor of F-actin-dependent activation of ATPase), Smooth Muscle (and Nonmuscle Cells) = Unphosphorylated myosin light chain. Contraction activated by, Striated Muscle = Ca 2+. Contraction activated by, Smooth Muscle (and Nonmuscle Cells) = Ca 2+. Direct effect of Ca 2+, Striated Muscle = 4Ca 2+ bind to TpC. Direct effect of Ca 2+, Smooth Muscle (and Nonmuscle Cells) = 4Ca 2+ bind to calmodulin. Effect of protein-bound Ca 2+, Striated Muscle = TpC · 4Ca 2+ antagonizes Tpl inhibition of F-actin-myosin interaction (allows F-actin activation of ATPase). Effect of protein-bound Ca 2+, Smooth Muscle (and Nonmuscle Cells) = Calmodulin · 4Ca 2+ activates myosin light-chain kinase that phosphorylates myosin p-light chain. The phosphorylated p-light chain no longer inhibits F-actin-myosin interaction (allows F-actin activation of ATPase)

also found in nonmuscle tissue. At low concentrations of Ca", it binds to tropomyosin and actin. This prevents the interaction of actin with myosin, keeping muscle in a relaxed state. At higher concentrations of Ca**:calmodulin binds caldesmon, releasing it from actin. The latter is then free to bind to myosin, and contraction can occur. Caldesmon is also subject to phosphorylation-dephosphorylation; when phosphorylated, it cannot bind actin, again freeing the latter to interact with myosin.

When striated muscle is in the relaxed state, the Ca** needed to initiate contraction is kept stored, ready for release into the sarcoplasm, within a network of fibrous membrane sacks—the sarcoplasmic reticulum or SR. The SR is linked to the sarcomeres by the transverse channels of the T-tubule system. In resting muscle, the concentration of Ca** in the sarcoplasm typically ranges between 10°* and 10°' mol/L. This low resting concentration is maintained by the basal activity of the Ca** ATPase (Figure 51-8), a Ca**-activated calcium pump that uses the energy of hydrolysis of ATP to move calcium ions from regions of low to high concentration. Once inside the SR, Ca** is bound to a specific Ca**-binding protein-designated calsequestrin.
Sarcomere
FIGURE 51-8 Diagram of the relationships among the sarcolemma (plasma membrane), a T tubule, and two cisternae of the SR of skeletal muscle (not to scale). The T tubule extends inward from the sarcolemma. A wave of depolarization, initiated by a nerve impulse, is transmitted from the sarcolemma down the T tubule. It is then conveyed to the Ca** release channel (RYR), perhaps by interaction between it and the dihydropyridine receptor (slow Ca** voltage channel), which are shown in close proximity. Release of Ca** from the Ca** release channel into the cytosol initiates contraction. Subsequently, Ca'* is pumped back into the cisternae of the SR by the Ca?* ATPase (Ca'* pump) and stored there, in part bound to calsequestrin.

When the sarcolemma is excited by a nerve impulse, the excitable membranes of the T-tubule system become depolarized, opening the voltage-gated Ca** release channels in the nearby SR. Ca" rapidly floods into the sarcoplasm, raising the concentration nearly 100-fold, to 10° mol/L, where it binds to troponin C and calmodulin to initiate contraction. The voltage-gated Ca** release channel, a homotetramer of ~ 565 kDa subunits, is the target of the plant alkaloid ryanodine, which binds to it and modulates its activity. Hence, the voltage-gated Ca** channel is also referred to as the ryanodine receptor, or RYR. There are two isoforms of this receptor. RYR1 is present in skeletal muscle. RYR2 is present heart muscle and brain. The channel lies very close to another voltage-gated Ca** channel of the transverse tubule system, the dihydropyridine receptor (Figure 51-8).
The release of Ca** into the sarcoplasm also activates the Ca**-dependent ATPase, whose activity is stimulated by the binding of Ca'*,:calmodulin. This immediately sets into motion the export of Ca** from the sarcoplasm back into the SR, enabling the muscle to rapidly relax and ready for another contraction. If the concentration of ATP in the sarcoplasm falls dramatically (eg, by excessive usage during the cycle of contraction-relaxation or by diminished formation, such as might occur in ischemia), the Ca**-ATPase will cease pumping and calcium ion levels will remain high.

Some genetically predisposed patients experience a severe reaction, designated malignant hyperthermia (MH), on exposure to certain anesthetics (eg, halothane) and depolarizing skeletal muscle relaxants (eg, succinylcholine). The reaction consists primarily of rigidity of skeletal muscles, hypermetabolism, and high fever. A high cytosolic concentration of Ca" in skeletal muscle is a major factor in its causation. Unless malignant hyperthermia is recognized and treated immediately, patients may die acutely of ventricular fibrillation or succumb to other serious complications. Appropriate treatment is to stop the anesthetic and administer the drug dantrolene, a skeletal muscle relaxant that acts to inhibit release of Ca** from the SR into the cytosol, intravenously. MH also occurs in swine homozygous for MH (porcine stress syndrome). If the reaction occurs prior to slaughter, it affects the quality of the pork adversely, resulting in an inferior product.
The causes of MH in humans and livestock appear to be complex, and may involve mutations in the genes encoding the Ca** release channel, calsequestrin-1, or the dihydropyridine receptor or in the RYRI gene. Mutations in the RYR1 gene are also associated with central core disease. This is a rare myopathy presenting in infancy with hypotonia and proximal muscle weakness. Electron microscopy reveals an absence of mitochondria in the center of many type I muscle fibers. Damage to mitochondria induced by high intracellular levels of Ca** secondary
to abnormal functioning of RYRI appears to be responsible for the morphologic findings.

Cardiac muscle, like skeletal muscle, is striated and is regulated by the same actin-myosin-tropomyosin-troponin system. Unlike skeletal muscle, cardiac muscle exhibits intrinsic rhythmicity, and individual myocytes communicate with each other because of its syncytial nature. The T-tubular system (see below) is more developed in cardiac muscle, whereas the SR is less extensive and consequently the intracellular supply of Ca** for contraction is lower. Cardiac muscle thus relies on extracellular Ca®** for contraction; if isolated cardiac muscle is deprived of Ca'*, it ceases to beat within approximately 1 minute, whereas skeletal muscle can continue to contract without an extracellular source of Ca** for a longer period. Cyclic AMP plays a more prominent role in cardiac than in skeletal muscle. It modulates intracellular levels of Ca** through the activation of the protein kinases that phosphorylate various transport proteins in the sarcolemma and SR. They also target the troponin-tropomyosin regulatory complex, affecting its responsiveness to intracellular Ca'*. There is a rough correlation between the phosphorylation of Tpl and the increased contraction of cardiac muscle induced by catecholamines. This may account for the inotropic effects (increased contractility) of B-adrenergic compounds on the heart. Some differences among skeletal, cardiac, and smooth muscle are summarized in Table 51-2.

Extracellular Ca** enters cardiomyocytes via highly selective channels. The major portal of entry is the L-type (longduration current, large conductance) or slow Ca** channel, which is voltage-gated, opening during depolarization and closing when the action potential declines. These channels are equivalent to the dihydropyridine receptors of skeletal muscle (Figure 51-8). Slow Ca** channels are regulated by cAMPdependent protein kinases (stimulatory) and cGMP-dependent protein kinases (inhibitory), and can be inhibited by so-called calcium channel blockers (eg, verapamil). Fast (or T, transient) Ca** channels are also present in the plasmalemma, though in much lower numbers; they probably contribute to the early phase of increase of myoplasmic Ca'*.
When the concentration of Ca" in the myoplasm increases, it triggers the opening of the Ca" release channel of the SR. In fact, Ca**-induced Ca" release from the SR stores accounts for approximately 90% of the Ca'* that enters a stimulated cardiomyocyte. However, the 10% that enters from the cytoplasm is vitally important, as it serves as a trigger for mobilization of Ca** from the SR.
The Na*-Ca** exchanger serves as the principal route of egress of Ca** from cardiomyocytes. Na* and Ca** are exchanged at a ratio of 3:1, with movement of sodium ions into the cell from the plasma providing the energy needed to move calcium into the plasma against a concentration gradient. This exchange contributes to relaxation, but may run in the reverse direction during excitation. Consequently, anything that causes intracellular Na* to rise will secondarily cause intracellular Ca** to rise as well, causing more forceful contraction (positive inotropic effect). Digitalis promotes

1. Striated, Cardiac Muscle = 1. Striated. 1. Striated, Smooth Muscle = 1. Nonstriated. 2. No syncytium, Cardiac Muscle = 2. Syncytial. 2. No syncytium, Smooth Muscle = 2. Syncytial. 3. Small T tubules, Cardiac Muscle = 3. Large T tubules. 3. Small T tubules, Smooth Muscle = 3. Generally rudimentary T tubules. 4. Sarcoplasmic reticulum well developed and Ca 2+ pump acts rapidly. 5. Plasmalemma contains few hormone receptors. 6. Nerve impulse initiates contraction. 7. Extracellular fluid Ca 2+ not important for contraction. 8. Troponin system present. 9. Caldesmon not involved. 10. Very rapid cycling of the cross-bridges., Cardiac Muscle = 4. Sarcoplasmic reticulum present and Ca 2+ pump acts relatively rapidly. 5. Plasmalemma contains a variety of receptors (eg, α- and β-adrenergic). 6. Has intrinsic rhythmicity. 7. Extracellular fluid Ca 2+ important for contraction. 8. Troponin system present. 9. Caldesmon not involved. 10. Relatively rapid cycling of the cross-bridges.. 4. Sarcoplasmic reticulum well developed and Ca 2+ pump acts rapidly. 5. Plasmalemma contains few hormone receptors. 6. Nerve impulse initiates contraction. 7. Extracellular fluid Ca 2+ not important for contraction. 8. Troponin system present. 9. Caldesmon not involved. 10. Very rapid cycling of the cross-bridges., Smooth Muscle = 4. Sarcoplasmic reticulum often rudimentary and Ca 2+ pump acts slowly. 5. Plasmalemma contains a variety of receptors (eg, α- and β-adrenergic). 6. Contraction initiated by nerve impulses, hormones, etc. 7. Extracellular fluid Ca 2+ important for contraction. 8. Lacks troponin system; uses regulatory head of myosin. 9. Caldesmon is important regulatory protein. 10. Slow cycling of the cross-bridges permits slow, prolonged contraction and less utilization of ATP.

FIGURE 51-9 Scheme of how the drug digitalis (used in the treatment of certain cases of heart failure) increases cardiac contraction. Digitalis inhibits the Na*-K* ATPase (see Chapter 40). This results in less Na* being pumped out of the cardiac myocyte and leads to an increase of the intracellular concentration of Na*. In turn, this stimulates the Na*-Ca** exchanger so that more Na* is exchanged outward, and more Ca' enters the myocyte. The resulting increased intracellular concentration of Ca"* increases the force of muscular contraction.
the inflow of Ca** via the Ca**-Na* exchanger by inhibiting the sarcolemmal Na*-K*-ATPase, reducing the rate of Na* exit by this route, thereby increasing intracellular Na*. The resulting increase in intracellular Ca** enhances the force of cardiac contraction to the benefit of a patient experiencing heart failure

In contrast to skeletal muscle, the sarcolemmal Ca** ATPase is believed to be a minor contributor to Ca** egress as compared with the Ca'*-Na* exchanger. Cardiac muscle is rich in ion channels (see Chapter 40), and they are also important in skeletal muscle. Mutations in genes encoding ion channels have been shown to be responsible for a number of relatively rare conditions affecting muscle. These and other diseases due to mutations of ion channels have been termed channelopathies; some are listed in Table 51-3.

The amount of ATP in skeletal muscle is only sufficient to provide energy for contraction for a few seconds. Consequently, muscle cells have developed multiple mechanisms to regenerate the ATP needed to sustain the contraction-relaxation cycle: (1) by glycolysis, using blood glucose or muscle glycogen, (2) by oxidative phosphorylation, (3) from creatine phosphate, and (4) from two molecules of ADP in a reaction catalyzed by adenylyl kinase (Figure 51-10).

The sarcoplasm of skeletal muscle contains large stores of glycogen, located in granules close to the I bands. The release of glucose from glycogen is dependent on a specific muscle
TABLE 51-3 Some Disorders (Channelopathies) due to Mutations in Genes Encoding Polypeptide Constituents of lon Channels
Central core disease (OMIM 117000), Ion Channel and Major Organs Involved = Ca 2+ release channel (RYR1), skeletal muscle. Hyperkalemic periodic paralysis (OMIM 170500), Ion Channel and Major Organs Involved = Sodium channel, skeletal muscle. Hypokalemic periodic paralysis (OMIM 170400), Ion Channel and Major Organs Involved = Slow Ca 2+ voltage channel (DHPR), skeletal muscle. Malignant hyperthermia (OMIM 145600), Ion Channel and Major Organs Involved = Ca 2+ release channel (RYR1), skeletal muscle. Myotonia congenita (OMIM 160800), Ion Channel and Major Organs Involved = Chloride channel, skeletal muscle
*Other channelopathies include the long QT syndrome (OMIM 192500); pseudoaldosteronism (Liddle syndrome, OMIM 177200); persistent hyperinsulinemic hypoglycemia of infancy (OMIM 601820); hereditary X-linked recessive type Il nephrolithiasis of infancy (Dent syndrome, OMIM 300009); and generalized myotonia, recessive (Becker disease, OMIM 255700). The term "myotonia" signifies any condition in which muscles do not relax after contraction. Source: Data in part from Ackerman NJ, Clapham DE: lon channels—basic science and clinical disease. N Engl J Med 1997;336:1575.
glycogen phosphorylase (see Chapter 18), which can be activated by Ca'*, epinephrine, and AMP. Ca" also activates phosphorylase b kinase, allowing glucose mobilization to begin with the initiation of muscle contraction.

Synthesis of ATP via oxidative phosphorylation requires a supply of oxygen. Muscles that have a high demand for oxygen as a result of sustained contraction (eg, to maintain posture) store it attached to the heme moiety of myoglobin. Muscles containing myoglobin are red in color, whereas muscles with little or no myoglobin are white. Glucose, derived from the blood glucose or from endogenous glycogen, and fatty acids, derived from the triacylglycerols of adipose tissue, are the principal substrates used for aerobic metabolism in muscle.

Creatine phosphate provides a readily available source highenergy phosphate that can be used to regenerate ATP from ADP. Creatine phosphate is synthesized by creatine kinase (CK), a muscle-specific enzyme with clinical utility in the detection of acute or chronic diseases of muscle, using ATP as the phosphodonor (Figure 51-10). The equilibrium constant for this reaction is near 1. Hence, when ATP levels are high, formation of creatine phosphate is favored. However, when ATP levels drop, the equilibrium shifts in favor of synthesizing ATP at the expense of stored creatine phosphate.
FIGURE 51-10 The multiple sources of ATP in muscle.

ATP synthase (see Figure 13-8) is the primary vehicle for regenerating ATP in living cells. However, it can only synthesize ATP from ADP. In order to regenerate ATP from AMP, the latter must first be phosphorylated to form ADP, a process catalyzed by the enzyme adenylyl kinase using ATP as phosphodonor. This enzyme thus can generate two molecules of ADP from one molecule each of AMP and ATP. When other means for regenerating ATP become exhausted and nucleotide triphosphate level plummet, the equilibrium shifts in favor of synthesizing ATP at the expense of ADP. It is important to note that the by-product of this reaction is AMP. Hence, this is only a temporary expedient as the cell can only cannibalize their total adenine nucleotide pool for so long.

Different types of fibers have been detected in skeletal muscle. One classification subdivides them into type I (slow twitch), type IIA (fast twitch-oxidative), and type IIB (fast twitchglycolytic). For the sake of simplicity, we shall consider only two types: type I (slow twitch, oxidative) and type II (fast twitch, glycolytic). The type I fibers are red because they contain myoglobin and mitochondria; their metabolism is aerobic, and they maintain relatively sustained contractions. The white type II fibers lack myoglobin and contain few mitochondria: they derive their energy from anaerobic glycolysis and perform contractions of relatively short duration.
The proportion of these two types of fibers varies among the muscles of the body, depending on function and training.
For example, the number of type I fibers in certain leg muscles increases in athletes training for marathons, whereas the number of type II fibers increases in sprinters. Type I fibers rely heavily on aerobic metabolism to regenerate ATP, while type II fibers rely on oxygen-independent sources such as creatine phosphate. For this reason, many endurance athletes will engage in carbohydrate loading, the ingestion of meals that predominantly consist of foods with a high starch content, in an effort to increase the size of their glycogen stores, while some sprinters will attempt to increase the pool of creatine phosphate in their muscles by ingesting creatine as a dietary supplement.

Inherited Cardiomyopathies Can Arise from Disorders of Cardiac Energy Metabolism or Abnormal Myocardial Proteins
A cardiomyopathy is any structural or functional abnormality of the ventricular myocardium. These abnormalities can arise from a number of causes, many of them hereditary. As shown in Table 51-4, the causes of inherited cardiomyopathies fall into two broad classes: (1) disorders of cardiac energy metabolism, mainly reflecting mutations in genes encoding enzymes or proteins involved in fatty acid oxidation (a major source of energy for the myocardium) and oxidative phosphorylation; (2) mutations in genes encoding proteins involved in or affecting myocardial contraction, such as myosin, tropomyosin, the troponins, and cardiac myosin-binding protein C.

Cause
Proteins or Process Affected
Inborn errors of fatty acid oxidation
Carnitine entry into cells and
Certain enzymes of fatty acid
oxidation
Disorders of mitochondrial
Proteins encoded by
mitochondrial genes
Proteins encoded by nuclear
genes
Abnormalities of myocardial contractile and structural proteins
B-Myosin heavy chains, troponin, tropomyosin, dystrophin
*Mutations (eg, point mutations, or in some cases deletions) in the genes (nuclear or mitochondrial) encoding various proteins, enzymes, or tRNA molecules are the fundamental causes of the inherited cardiomyopathies. Some conditions are mild, whereas others are severe and may be part of a syndrome affecting other tissues. Source: Based on Kelly DP, Strauss AW: Inherited cardiomyopathies. N Engl J Med 1994;330:913.

Familial hypertrophic cardiomyopathy is one of the most commonly encountered hereditary cardiac diseases. Patients exhibit hypertrophy—often massive—of one or both ventricles, starting early in life. Most cases are transmitted in an autosomal dominant manner; the rest are sporadic. The root cause of this condition is any one of several missense mutations in the gene encoding the B-myosin heavy chain that leads to the replacement of a highly conserved amino acid with some other residue. The substitutions cluster in the head and head-rod regions of the myosin heavy chain. One hypothesis is that the mutant polypeptides ("poison polypeptides") cause formation of abnormal myofibrils, eventually resulting in compensatory hypertrophy.
Patients with familial hypertrophic cardiomyopathy can show great variation in clinical picture. This in part reflects genetic heterogeneity as it appears that mutations in a number of other genes (eg, those encoding cardiac actin, tropomyosin, cardiac troponins I and T, essential and regulatory myosin light chains, cardiac myosin-binding protein C, titin, and mitochondrial tRNA-glycine and tRNA-isoleucine) may also cause familial hypertrophic cardiomyopathy. Patients harboring mutations that are predicted to alter the charge character of the affected amino acid side chain exhibit a significantly shorter life expectancy than patients in whom the mutation produced no alteration in charge.
Mutations in the genes encoding dystrophin, muscle LIM protein (so called because it was found to contain a cysteinerich domain originally detected in three proteins: Lin-II, Isl-1, and Mec-3), the cyclic AMP response-element binding protein (CREB), desmin, and lamin have each been implicated in the causation of dilated cardiomyopathy. The first two proteins help organize the contractile apparatus of cardiac muscle cells, while CREB regulates the expression of several genes within these cells.

Other protein components of the contractile apparatus include titin, the world's largest known protein whose role is to anchor the ends of myofibrils, nebulin, a-actinin, desmin, and dystrophin. Among these proteins, dystrophin is of particular biomedical interest. Mutations in the gene that encodes it play a causative role in Duchenne muscular dystrophy and Becker muscular dystrophy and have been implicated in dilated cardiomyopathy (see below). Dystrophin bridges the actin cytoskeleton to the extracellular matrix at the interior face of the plasma membrane. Formation of this link is necessary for assembly of the synaptic junction. Duchenne muscular dystrophy appears to result from the inability of mutationally altered forms of dystrophin to support formation of functionally competent synaptic junctions. Similarly, mutations in genes encoding the glycosyltransferases that modify a-dystroglycan or those encoding polypeptide components of the sarcoglycan complex (Figure 51-11) are responsible for certain other congenital forms of muscular dystrophy such as limb-girdle.

Acetylcholine triggers the relaxation of the smooth muscle of blood vessels via a receptor-mediated signal transduction pathway. Binding of acetylcholine to its cell surface receptors on the endothelial cells surrounding vascular smooth muscle cells activates associated phospholipases on the interior surface of the plasma membrane. These enzymes hydrolyze and release the polyphosphorylated head groups, particularly 3,4,5-triphosphoinositol, from phosphatidylinositol, a quantitatively minor but functionally important phospholipid component of the plasma membrane. These polyphosphoinositol second messengers initiate the release of Ca** into the cytoplasm of these vascular epithelial cells, which in turn triggers the release of endothelium-derived relaxing factor (EDRE), which diffuses into the adjacent smooth muscle.
The identification of EDRF as NO, nitrous oxide, proved very difficult, because this diatomic gas rapidly reacts with oxygen and superoxide, resulting in a very short half-life (~ 3-4 seconds) in tissues.
NO is formed by NO synthase, a Ca'*-activated enzyme found in the cytosol. NO synthase catalyzes the five-electron oxidation of an guanidino nitrogen in the side chain of arginine, yielding citrulline and NO (Figure 51-12), a complex reaction that utilizes NADPH and four redox-active prosthetic groups: FAD, FMN, heme, and tetrahydrobiopterin. Upon diffusing into the surrounding vascular smooth muscle cells, NO binds to the heme moiety of a soluble guanylyl cyclase, activating the enzyme and elevating the intracellular levels of
Intracellular

FIGURE 51-11 Organization of dystrophin and other proteins in is part of a large oligomeric complex associated with several other protein relation to the plasma membrane of muscle cells. Dystrophin complexes. The dystroglycan complex consists of a-dystroglycan, which associates with the basal lamina protein merosin (also named laminin-2, see Chapter 50), and a-dystroglycan, which binds a-dystroglycan and dystrophin. Syntrophin binds to the carboxyl terminal of dystrophin. The sarcoglycan complex consists of four transmembrane proteins: a-, B-, y-, and 5-sarcoglycan. The function of the sarcoglycan complex and the nature of the interactions within the complex and between it and the other complexes are not clear. The sarcoglycan complex is formed only in striated muscle, and its subunits preferentially associate with each other, suggesting that the complex may function as a single unit. Mutations in the gene encoding dystrophin cause Duchenne and Becker muscular dystrophies. Mutations in the genes encoding the various sarcoglycans have been shown to be responsible for limb-girdle dystrophies (eg, OMIM 604286) and mutations in genes encoding other muscle proteins ca' use other types of muscular dystrophy. Mutations in genes encoding certain glycosyltransferases involved in the synthesis of the glycan chains of a-dystroglycan are responsible for certain congenital muscular dystrophies. (Reproduced, with permission, from Duggan DJ et al: Mutations in the sarcoglycan genes in patients with myopathy. N Engl J Med 1997;336:618. Copyright © 1997 Massachusetts Medical Society. All rights reserved.)
the second messenger 335'-cyclic GMP (cGMP). This in turn stimulates the activities of certain cGMP-dependent protein kinases, which probably phosphorylate specific muscle proteins, causing relaxation.

NO can also be formed from nitrite, derived from the metabolism of vasodilators such as glyceryl trinitrate, also known as nitroglycerin, which is commonly administered to treat angina. Another important cardiovascular effect of NO is the inhibition of platelet aggregation, a consequence of the increased synthesis of CGMP. NO has also been implicated in other physiologic processes. For example, it can reacts with superoxide to generate peroxynitrite (ONOO'), which can react with tyrosine side chains, forming nitrotyrosine, or generate the highly reactive OH radical when it decomposes.

In humans, skeletal muscle proteins are the major nonfat source of stored energy. This explains the large losses of muscle mass, particularly in adults, that accompany prolonged caloric undernutrition.
All cells perform mechanical work, albeit of lower physical magnitude than muscle cells. These include self-propulsion, cytokinesis, endocytosis, exocytosis, and phagocytosis. Like muscle cells, the core of this machinery is comprised of filamentous protein polymers called the cytoskeleton. The cytoskeleton's more quantitatively prominent filamentous structures include actin filaments (also known as microfilaments), microtubules, and intermediate filaments.

G-actin is present in most if not all cells of the body. Under physiologic conditions, G-actin monomers spontaneously polymerize to form double helical F-actin filaments, 7 to 9.5 nm in diameter, similar to those found in muscle. The actin microfilaments of the cytoskeleton generally exist as bundles within a tangled-appearing meshwork. These prominent bundles, which just underlie the plasma membranes of many cells, are referred to as stress fibers. Stress fibers
FIGURE 51-12 Diagram showing formation in an endothelial cell of nitric oxide (NO) from arginine in a reaction catalyzed by NO synthase. Interaction of an agonist (eg, acetylcholine) with a receptor (R) leads to intracellular release of Ca?* induced by inositol trisphosphate generated by the phosphoinositide pathway, resulting in activation of NO synthase. The NO subsequently diffuses into adjacent smooth muscle, where it leads to activation of guanylyl cyclase, formation of cGMP, stimulation of cGMP protein kinases, and subsequent relaxation. The vasodilator nitroglycerin is shown entering the smooth muscle cell, where its metabolism also leads to formation of NO.
disappear as cell motility increases or upon malignant transformation of cells.
Two types of actin can be found in the cytoskeletal microfilaments, called B-actin and y-actin. Although not organized as in muscle, actin filaments in nonmuscle cells can interact
with myosin to cause cellular movements.

Microtubules, an integral component of the cellular cytoskeleton, consist of cytoplasmic tubes, 25 nm in diameter that are often of extreme length (Figure 51-13). Each cylindrical tube is comprised of 13 longitudinally arranged protofilaments comprised of a-tubulin and B-tubulin, closely related proteins of ~50 kDa molecular mass. Assembly starts with the formation of tubulin dimers that assemble into protofilaments that associate with one another in parallel to form sheets and eventually cylinders. GTP is required for assembly. A microtubule-organizing center, located around a pair of centrioles, nucleates the growth of new microtubules. A third species of tubulin, y-tubulin, appears to play an important role in this process.
Microtubules are necessary for the formation and function of the mitotic spindle responsible for chromosome segregation during cell division. They are also involved in the intracellular movement of endocytic and exocytic vesicles and form the major structural components of cilia and flagella. Microtubules are a major component of axons and dendrites, in which they maintain structure and participate in the axoplasmic flow of material along these neuronal processes. Microtubules also contain a variety of microtubule-associated proteins [MAPs], one of which is tau, which also play important roles in microtubule assembly and stabilization.
Microtubules are in a state of dynamic instability, constantly assembling and disassembling. They exhibit polarity (plus and minus ends); a feature important to both in their growth from centrioles and their ability to direct intracellular movement. For instance, in axonal transport, the protein kinesin, with a myosin-like ATPase activity, uses hydrolysis of ATP to move vesicles down the axon toward the positive end of the microtubular formation. Flow of materials in the opposite direction, toward the negative end, is powered by
(Subunits as seen in negative stain preparation)

FIGURE 51-13 Schematic representation of microtubules. At left and center are shown drawings of microtubules as seen in the electron microscope following fixation with tannic acid in glutaraldehyde. The unstained tubulin subunits are delineated by the dense tannic acid. Cross sections of tubules reveal a ring of 13 subunits of dimers arranged in a spiral. Changes in microtubule length are due to the addition or loss of individual tubulin subunits. Characteristic arrangements of microtubules (not shown here) are found in centrioles, basal bodies, cilia, and flagellae. (Reproduced, with permission, from Junqueirai LC, Carneiro J, Kelley RO: Basic Histology, 7th ed. Appleton & Lange, 1992.)
cytosolic dynein, another protein with ATPase activity. Similarly, axonemal dyneins power ciliary and flagellar movement while dynamin, which uses GTP instead of ATP, is involved in endocytosis. Kinesins, dyneins, dynamin, and myosins are referred to as molecular motors.
An absence of dynein in cilia and flagella results in immotile cilia and flagella, leading to male sterility, situs inversus, and chronic respiratory infection, a condition known as Kartagener syndrome (OMIM 244400). Mutations in genes affecting the synthesis of dynein have been detected in individuals with this syndrome. Certain drugs bind to microtubules and thus interfere with their assembly or disassembly. These include colchicine (used for treatment of acute gouty arthritis), vinblastine (a vinca alkaloid used for treating certain types of cancer), paclitaxel (Taxol) (effective against ovarian cancer), and griseofulvin (an antifungal agent).

An intracellular fibrous system exists comprised of filaments with an axial periodicity of 21 nm anda diameter of 8 to 10 nm that is intermediate between that of microfilaments (6 nm) and microtubules (23 nm). At least four classes of intermediate filaments are found, as indicated in Table 51-5. Each is made up of elongated, fibrous molecules with a central rod domain, an amino-terminal head, and a carboxyl-terminal tail. These subunits assemble in a helical a manner to form repeating tetrameric units to form rope-like fibrils. Intermediate filaments are important structural components of cells that serve as are relatively stable components of the

Lamins, Molecular Mass (kDa) = . Lamins, Distributions = . A, B, and C, Molecular Mass (kDa) = 65-75. A, B, and C, Distributions = Nuclear lamina. Keratins, Molecular Mass (kDa) = . Keratins, Distributions = . Type I (acidic), Molecular Mass (kDa) = 40-60. Type I (acidic), Distributions = Epithelial cells, hair, nails. Type II (basic), Molecular Mass (kDa) = 50-70. Type II (basic), Distributions = As for type I (acidic). Vimentin-like, Molecular Mass (kDa) = . Vimentin-like, Distributions = . Vimentin, Molecular Mass (kDa) = 54. Vimentin, Distributions = Various mesenchymal cells. Desmin, Molecular Mass (kDa) = 53. Desmin, Distributions = Muscle. Glial fibrillary acid protein, Molecular Mass (kDa) = 50. Glial fibrillary acid protein, Distributions = Glial cells. Peripherin, Molecular Mass (kDa) = 66. Peripherin, Distributions = Neurons. Neurofilaments, Molecular Mass (kDa) = . Neurofilaments, Distributions = . Low (L), medium (M), and high (H) a, Molecular Mass (kDa) = 60-130. Low (L), medium (M), and high (H) a, Distributions = Neurons
*Refers to
their molecular
masses.
Note: Intermediate filaments have an approximate diameter of 10 nm and have various functions. For example, keratins are distributed widely in epithelial cells and adhere via adapter proteins to desmosomes and hemidesmosomes. Lamins provide support for the nuclear membrane.
cytoskeleton, not undergoing rapid assembly and disassembly and not disappearing during mitosis, as do actin and many microtubular filaments. An important exception to this are the lamins, which, subsequent to phosphorylation, disassemble at mitosis and reappear when it terminates. Lamins form a meshwork positioned in apposition to the inner nuclear membrane.

Mutations in the gene encoding lamin A and lamin C cause Hutchinson-Gilford progeria syndrome (progeria) [OMIM 176670]. A farnesylated form (see Figure 26-2 for the structure of farnesyl) of prelamin A accumulates in this condition, because the site at which the farnesylated portion of lamin A is normally cleaved by proteases has been altered by mutation. Lamin A is an important component of the structural scaffolding that maintains the integrity of the nucleus of a cell. It appears that the accumulation of farnesylated prelamin A destabilizes nuclei, altering their shape, somehow predisposing victims to manifest signs of premature aging. Experiments in mice have indicated that administration of a farnesyltransferase inhibitor may ameliorate the development of misshapen nuclei. Children affected by this condition often die in their teens of atherosclerosis.
Keratins form a large family consisting of about 30 members. As indicated in Table 51-5, two major types of keratins are found that assemble into heterodimers made up of one member of each class. Vimentins are widely distributed in mesodermal cells. Desmin, glial fibrillary acidic protein, and peripherin are related to them. All members of the vimentinlike family can copolymerize with each other.
Intermediate filaments
in nerve
cells, called
neurofila- ments, are classified as low, medium, and high on the basis of their molecular masses. The distribution of intermediate filaments in normal and abnormal (eg, cancer) cells can be studied by the use of immunofluorescent techniques, using antibodies of appropriate specificities. These antibodies to specific intermediate filaments can also be of use to pathologists in helping to decide the origin of certain dedifferentiated malignant tumors. These tumors may still retain the type of intermediate filaments found in their cell of origin.

A number of skin diseases, mainly characterized by blistering, have been found to be due to mutations in genes encoding various keratins. Two of these disorders are epidermolysis bullosa simplex (OMIM 131800) and epidermolytic palmoplantar keratoderma (OMIM 144200). The blistering found in these disorders probably reflects a diminished capacity of various layers of the skin to resist mechanical stresses due to abnormalities in the keratin structure.

=
The
myofibrils
of
skeletal
muscle
consist
of
thick,
myosin-based
and
thin,
actin-based
filaments.
™ During contraction, these interdigitating filaments slide past
one
another
with
cross-bridges
between
myosin
and
actin
generating
and
sustaining
tension.

™ Muscle contraction is driven by the cyclic attachment, conformational shift, and detachment of massive numbers of myosin head domains to adjacent actin fibrils.
| The hydrolysis of ATP is used to drive movement of the filaments. ATP binds to myosin heads and is hydrolyzed to ADP and P, by the ATPase activity of the actomyosin complex.
™ In striated muscle, the contractile apparatus is held in check by the troponin complex (troponins T, I, and C) until inhibition is relieved by the binding of Ca** to troponin C.
= In smooth muscle, the contractile apparatus is held in check by the regulatory light chains in myosin. This block is relieved when the regulatory light chains are phosphorylated by a Ca** :calmodulin-activated protein kinase, myosin light-chain kinase.
the
@ In skeletal muscle, the SR regulates distribution of Ca** to sarcomeres, whereas inflow of Ca?* via Ca?* channels in the sarcolemma is of major importance in cardiac and smooth muscle.
™ Ca* not only initiates contraction, it also activates the calcium efflux systems that will bring contraction to a close.
= Many cases of malignant hyperthermia in humans are due to mutations in the gene encoding the Ca** release channel.
™ Some cases of familial hypertrophic cardiomyopathy are due to missense mutations in the gene coding for the B-myosin heavy chain. Mutations in genes encoding a number of other proteins have also been detected.
@ NOisa regulator of vascular smooth muscle; blockage of its formation from arginine causes an acute elevation of blood pressure, indicating that regulation of blood pressure is one its many functions.
of
™ Duchenne-type muscular dystrophy is due to mutations in the gene, located on the X chromosome, encoding the protein dystrophin.
= Two major types of muscle fibers are found in humans: white (anaerobic) and red (aerobic).
® Nonmuscle cells contain an internal network of fibers called the cytoskeleton that provides the mechanical apparatus
needed to maintaining and changing cell shape, cell motility, phagocytosis, etc.

@ The cytoskeleton is comprised of a variety of filaments that include actin-based microfilaments, a-tubulin and f-tubulin containing microtubules, and lamin-, keratin-, and vimentin- intermediate filaments.
™ Mutations in the gene encoding lamin A cause progeria, a
condition
by
symptoms
resembling
premature
aging.
™ Mutations in genes for certain keratins cause a number of skin diseases.
™ The cytoskeleton provides a scaffold for a variety of molecular motors, such as kinesin and dynein, that participate in vesicle transport, axonal flow, flagellar motion, and morphologic changes in cell shape.

Peter J. Kennelly, PhD, Robert K. Murray, MD, PhD, Molly Jacob, MBBS, MD, PhD & Joe Varghese, MBBS, MD

- List the major functions of blood.
VR TOle\ CM UER Sele you should be able to:
Describe the principal functions of serum albumin.
iron precipitates. Explain how haptoglobin protects the kidney against formation of damaging
Describe the roles of ferritin, transferrin, and ceruloplasmin in iron homeostasis.
Describe the mechanism by which transferrin, transferrin receptors, and HFE protein regulate the synthesis of hepcidin.
certain disorders. = Explain how iron homeostasis can be perturbed by dietary deficiencies or
= Describe the general structures and functions of the five classes of
immunoglobulins.
m= Explain how up toa million different immunoglobulins can be generated utilizing fewer than 150 human genes.
Describe the activation and mode of action of the complement system.
Compare and contrast the adaptive and innate immune systems.
Define the term lectin.
Outline the key differences between polyclonal and monoclonal antibodies.
Describe the salient features of autoimmune and immunodeficiency disorders.

The proteins that circulate in blood plasma play important roles in human physiology. Albumins facilitate the transit of fatty acids, steroid hormones, and other ligands between tissues, while transferrin aids the uptake and distribution of iron. Circulating fibrinogen serves as a readily mobilized building block of the fibrin mesh that provides the foundation of the clots used to seal injured vessels. Formation of these clots is triggered by a cascade of latent proteases, or zymogens, called blood coagulation factors. Plasma also contains several proteins that function as inhibitors of proteolytic enzymes. Antithrombin helps confine the formation of clots to the vicinity of a wound, while a,-antiproteinase and a,-macroglobulin shield healthy tissues from the proteases that destroy invading pathogens and remove dead or defective cells. Circulating immunoglobulins called antibodies form the front line of the body's immune system.
Perturbances in the production of plasma proteins can have serious health consequences. Deficiencies in key components of the blood clotting cascade can result in excessive bruising and bleeding (hemophilia). Persons lacking plasma ceruloplasmin, the body's primary carrier of copper, are subject to hepatolenticular degeneration (Wilson disease), while emphysema is associated with a genetic deficiency in the production of circulating a,-antiproteinase. More than one in every 30 residents of North America suffer from an autoimmune disorder, such as type 1 diabetes, asthma, and rheumatoid arthritis, resulting from the production of aberrant immunoglobulins (Table 52-1). Insufficiencies in the production of protective antibodies, such as occur in many persons infected by the human immunodeficiency virus (HIV) or patients administered immunosuppressant drugs, render them immunocompromised, extremely susceptible to infection by microbial and viral pathogens. While the root causes of plasma protein-related diseases such as
C H A PT ER

Graves disease/ hyperthyroidism, Mean Prevalence Rate (per 100,000) = 1152. Graves disease/ hyperthyroidism, Percentage Female = 88. Rheumatoid arthritis, Mean Prevalence Rate (per 100,000) = 860. Rheumatoid arthritis, Percentage Female = 75. Thyroiditis/ hypothyroidism, Mean Prevalence Rate (per 100,000) = 792. Thyroiditis/ hypothyroidism, Percentage Female = 95. Vitiligo, Mean Prevalence Rate (per 100,000) = 400. Vitiligo, Percentage Female = 52. Type 1 diabetes, Mean Prevalence Rate (per 100,000) = 192. Type 1 diabetes, Percentage Female = 48. Pernicious anemia, Mean Prevalence Rate (per 100,000) = 151. Pernicious anemia, Percentage Female = 67. Multiple sclerosis, Mean Prevalence Rate (per 100,000) = 58. Multiple sclerosis, Percentage Female = 64. Primary glomerulonephritis, Mean Prevalence Rate (per 100,000) = 40. Primary glomerulonephritis, Percentage Female = 32. Systemic lupus erythematosus, Mean Prevalence Rate (per 100,000) = 24. Systemic lupus erythematosus, Percentage Female = 88. IgA glomerulonephritis, Mean Prevalence Rate (per 100,000) = 23. IgA glomerulonephritis, Percentage Female = 67. Sjogren syndrome, Mean Prevalence Rate (per 100,000) = 14. Sjogren syndrome, Percentage Female = 94. Myasthenia gravis, Mean Prevalence Rate (per 100,000) = 5. Myasthenia gravis, Percentage Female = 73. Addison's disease, Mean Prevalence Rate (per 100,000) = 5. Addison's disease, Percentage Female = 93. Schleroderma, Mean Prevalence Rate (per 100,000) = 4. Schleroderma, Percentage Female = 92

Source: Data from Jacobson DL, Gange SJ, Rose NR, Graham NMH: Epidemiology and estimated population burden of selected autoimmune diseases in the United States. J Clin Immunol Immunopathol 1997;84:223.
hemophilia are relatively straightforward, others—in particular many autoimmune disorders—arise due to the complex and cryptic interplay of genetic, dietary, nutritional, environmental, and medical factors.

As the primary avenue by which tissues are connected to each other and the surrounding environment, the blood that circulates throughout our body performs a variety of functions. These include delivering nutrients and oxygen, removing waste products, conveying hormones, and defending against infectious microorganisms (Table 52-2). These myriad functions are carried out by a diverse set of components that include cellular entities such as red blood cells, platelets, and leukocytes (see Chapters 53 and 54), and the water, electrolytes, metabolites, nutrients, proteins, and hormones that comprise the plasma.

Plasma contains a complex mixture of proteins. Early scientists classified these proteins into three groups, fibrinogen, albumin, and globulins, on the basis of their relative solubility in the presence of added organic solvents such as ethanol or

salting out agents such as as ammonium sulfate. Subsequently, clinical scientists employed electrophoresis within a cellulose acetate matrix to analyze the protein composition of plasma. Using this technique, salt-soluble serum protein fraction separated into five major components designated albumin and the a,-, a,-, B-, and y-globulins, respectively (Figure 52-1). Plasma proteins tend to be rich in disulfide bonds and frequently contain bound carbohydrate (glycoproteins) or lipid (lipoproteins). The relative dimensions and molecular masses of several plasma proteins are shown in Figure 52-2.

The aggregate concentration of the proteins present in human plasma typically falls in the range of 7 to 7.5 g/dL. The resulting osmotic pressure (oncotic pressure) is approximately 25 mm Hg. Since the hydrostatic pressure in the arterioles is approximately 37 mm Hg, with an interstitial (tissue) pressure of 1 mm Hg opposing it, a net outward force of about 11 mm Hg drives fluid from the plasma into the interstitial spaces. By contrast, the hydrostatic pressure in venules is about 17 mm Hg; thus, a net force of about 9 mm Hg drives water from tissues back into the circulation. The above pressures are often referred to as the Starling forces. If the concentration of plasma proteins is markedly diminished (eg, due to severe protein malnutrition), fluid will cease flowing back into the intravascular compartment and begin to accumulate in the extravascular tissue spaces, resulting in a condition known as edema.

Roughly 70 to 80% of all plasma proteins are synthesized in the liver. These include albumin, fibrinogen, transferrin, and most components of the complement and blood
FIGURE 52-1 Technique of cellulose acetate zone electrophoresis. (A) A small amount of serum or other fluid is applied to a cellulose acetate strip. (B) Electrophoresis in electrolyte buffer is performed. (C) Staining enables separated bands of protein to be visualized. (D) Densitometer scanning reveals the relative mobilities of albumin, a,-globulin, 8,-globulin, B-globulin, and y-globulin. (Reproduced, with permission, from Parslow TG et al (editors): Medical Immunology, 10th ed. McGraw-Hill, 2001.)
coagulation cascades. Two prominent exceptions are von Willebrand factor, which is synthesized in the vascular endothelium, and the y-globulins, which are synthesized in the lymphocytes. Most plasma proteins are covalently modified by the addition of either Nor O-linked oligosaccharide chains, or both (see Chapter 46). Albumin is the major exception. These oligosaccharide chains fulfill a variety of functions
FIGURE 52-2 Relative dimensions and approximate molecular masses of protein molecules in the blood.

(see Table 46-2). Loss of terminal sialic acid residues accelerates clearance of plasma glycoproteins from the circulation. As is the case for other proteins destined for secretion from a cell, the genes for plasma proteins code for an aminoterminal signal sequence that targets them to the endoplasmic reticulum. As this leader sequence emerges from the ribosome, it binds to a transmembrane protein complex in the endoplasmic reticulum called the signal recognition particle. The emerging polypeptide chain is pulled through the signal recognition particle into the lumen of the endoplasmic reticulum, during which process the leader sequence is cleaved off by an associated signal peptidase (see Chapter 49). The newly synthesized proteins then traverse the major secretory route in the cell (rough endoplasmic membrane — smooth endoplasmic membrane — Golgi apparatus — secretory vesicles) prior to entering the plasma, during which process they are subject to various posttranslational modifications (proteolysis, glycosylation, phosphorylation, etc). Transit times from the site of synthesis in the hepatocyte from to the plasma vary from 30 minutes to several hours for individual proteins.

A polymorphism is a mendelian or monogenic trait that exists in the population in at least two phenotypes, neither of which is rare (ie, it occurs with frequency of at least 1-2%). Most polymorphisms are innocuous. The ABO blood group substances (see Chapter 53) are perhaps the best known example of a human polymorphism. Other human plasma proteins that exhibit polymorphism include a,-antitrypsin, haptoglobin, transferrin, ceruloplasmin, and immunoglobulins.

The half-life of a plasma protein is the time required for 50% of the molecules present at any given moment to be degraded or otherwise cleared from the blood. For example, the half-lives of albumin and haptoglobin in healthy adults are approximately 20 and 5 days, respectively. Under normal circumstances, as older protein molecules are cleared they are replaced by newly synthesized ones, a process called turnover. During normal turnover, the total concentration of these proteins will remain constant as the countervailing processes of synthesis and clearance reach a steady state.
In certain diseases, the half-life of a protein may be markedly altered. For instance, in some gastrointestinal diseases such as regional ileitis (Crohn disease), considerable amounts of plasma proteins, including albumin, may be lost into the bowel through the inflamed intestinal mucosa. The half-life of albumin in these subjects may be reduced to as little as 1 day, a condition referred to as a protein-losing gastroenteropathy.

The liver synthesizes approximately 12 g of albumin per day, representing about 25% of total hepatic protein synthesis and half its secreted protein. About 40% of the body's albumin circulates in the plasma, where it accounts for roughly three-fifths of total plasma protein by weight (3.4-4.7 g/dL). The remainder resides in the extracellular space. Because of its relatively low molecular mass (about 69 kDa) and high concentration, albumin is thought to contribute 75 to 80% of the osmotic pressure of human plasma. Like most other secreted proteins, albumin is initially synthesized as a preproprotein. Its signal peptide is removed as it passes into the cisternae of the rough endoplasmic reticulum. A second hexapeptide is cleaved from the new N-terminus farther along the secretory pathway.
Mature human albumin consists of a single polypeptide chain, 585 amino acids in length, that is organized into three functional domains. Its ellipsoidal conformation is stabilized by a total of 17 intrachain disulfide bonds. A major role of albumin is to bind to and transport numerous ligands. These include free fatty acids (FFA), calcium, certain steroid hormones, bilirubin, copper, and tryptophan. A variety of drugs, including sulfonamides, penicillin G, dicumarol, and aspirin, also bind to albumin; a finding with important pharmacologic implications. Preparations of human albumin have been widely used in the treatment of burns and of hemorrhagic shock.
Some humans suffer from genetic mutations that impair their ability to synthesize albumin. Individuals whose plasma is completely devoid of albumin are said to exhibit analbuminemia. Surprisingly, persons suffering from analbuminemia display only moderate edema. Depressed synthesis of albumin also occurs in a variety of diseases, particularly those of the liver. The plasma of patients with liver disease often shows a decrease in the ratio of albumin to globulins (decreased albumin-globulin ratio). The synthesis of albumin decreases relatively early in conditions of protein malnutrition, such as kwashiorkor.

Table 52-3 summarizes the functions of many of the plasma proteins. C-reactive protein (CRP, so named because it reacts with the C polysaccharide of pneumococci), a,-antiproteinase, haptoglobin, a,-acid glycoprotein, and fibrinogen are classified as acute-phase proteins. Acute-phase proteins are believed to

Antiproteases, Plasma Proteins = Antichymotrypsin α 1 -Antitrypsin (α 1 -antiproteinase) α 2 -Macroglobulin Antithrombin. Blood clotting, Plasma Proteins = Various coagulation factors, fibrinogen. Enzymes, Plasma Proteins = Function in blood, for example, coagulation factors, cholinesterase Leakage from cells or tissues, eg, aminotransferases. Hormones, Plasma Proteins = Erythropoietin a. Immune defense, Plasma Proteins = Immunoglobulins, complement proteins, and β 2 -macroglobulin. Involvement in inflammatory responses, Plasma Proteins = Acute phase response proteins (eg, C-reactive protein, α 1 -acid glycoprotein [orosomucoid]). Oncofetal, Plasma Proteins = α 1 -Fetoprotein (AFP). Transport or binding proteins, Plasma Proteins = Albumin (various ligands, including bilirubin, free fatty acids, ions [Ca 2+ ], metals [eg, Cu 2+ , Zn 2+ ], metheme, steroids, other hormones, and a variety of drugs) Corticosteroid-binding globulin (transcortin) (binds cortisol) Haptoglobin (binds extracorpuscular hemoglobin) Lipoproteins (chylomicrons, VLDL, LDL, HDL) Hemopexin (binds heme) Retinol-binding protein (binds retinol) Sex-hormone-binding globulin (binds testosterone, estradiol) Thyroid-binding globulin (binds T 4 , T 3 ) Transferrin (transport iron) Transthyretin (formerly prealbumin; binds T 4 and forms a complex, with retinol-binding protein)
*Various other protein hormones circulate in the blood but are not usually designated as plasma proteins. Similarly, ferritin is also found in plasma in small amounts, but it too is not usually characterized as a plasma protein.

play a role in the body's response to inflammation. C-reactive protein stimulates the complement pathway (see below), while a,-antitrypsin neutralizes certain proteases released during acute inflammation.
The levels of acute-phase proteins may increase by 50% to as much as 1000-fold (in the case of CRP) during chronic inflammatory states and in patients with cancer. Interleukin 1 (IL-1), a polypeptide released from mononuclear phagocytic cells, is the principal—but not the sole—stimulator of acutephase reactant synthesis by hepatocytes. Additional molecules such as IL-6 also participate. Because its concentration can rise so dramatically, CRP is used as a biomarker of tissue injury, infection, and inflammation.
The small proteins such as interferons, ILs, and tumor necrosis factors that facilitate cell-cell communication between the components of the immune system are called cytokines. Cytokines can be both autocrine and paracrine in nature. One of the primary targets of IL-1 and IL-6 is nuclear factor kappa-B (NF«B), a transcription factor that regulates the expression of the genes encoding many cytokines, chemokines, growth factors, and cell adhesion molecules. NF«B, a heterodimer comprised of a 50and a 65-kDa polypeptide, normally resides in the cytosol as an inactive complex with a second protein, NF«B inhibitor-a, also known as IxBa. When stimulation by inflammation, injury, or radiation, IkBa becomes phosphorylated, which targets it for ubiquitination and degradation. Once freed from its inhibitory partner, active NF«B translocates to the nucleus where it stimulates transcription of its target genes.

Erythrocytes normally have a lifespan of approximately 120 days. Senescent or damaged erythrocytes are phagocytosed by macrophages of the reticuloendothelial system (RES) present in the spleen and liver. Around 200 billion erythrocytes are catabolized every day. Within the macrophage, heme derived from hemoglobin is converted by the enzym heme oxygenase to biliverdin (see Figure 31-13), releasing carbon monoxide and iron as byproducts. Iron released from heme is exported from phagocytic vesicles in the macrophage by NRAMP 1 (natural resistanceassociated macrophage protein 1), a transporter homologous to DMT1. Iron is subsequently secreted into the circulation by the transmembrane protein ferroportin (Figure 52-3). Thus, ferroportin plays a central role in both iron absorption by the intestine and iron secretion from macrophages.
In the blood, Fe** is oxidized to Fe** in a reaction catalyzed by the ferrioxidase ceruloplasmin (see below), a coppercontaining plasma enzyme synthesized by liver. Once oxidized, Fe** is then bound to transferrin in blood. The iron released from macrophages in this way (about 25 mg/d) is recycled, thereby reducing the need for intestinal iron absorption, which averages only 1 to 2 mg/d.
FIGURE 52-3 Recycling of iron in macrophages. Senescent erythrocytes are phagocytosed by macrophages. Hemoglobin is degraded and iron is released from heme by the action of the enzyme heme oxygenase. Ferrous iron is then transported out of the macrophage via ferroportin (Fp). In the plasma, it is oxidized to the ferric form by ceruloplasmin before binding to transferrin (Tf). Iron circulates in blood tightly bound to Tf.

During the course of red blood cell turnover, approximately 10% of an erythrocyte's hemoglobin escapes into the circulation. This free, extracorpuscular hemoglobin is sufficiently small at ~65 kDa to pass through the glomerulus of the kidney into the tubules, where it tends to form damaging precipitates. Haptoglobin (Hp) is a plasma glycoprotein that binds extracorpuscular hemoglobin (Hb), forming a tight noncovalent complex (Hb-Hp). Human haptoglobin exists in three polymorphic forms, known as Hp 1-1, Hp 2-1, and Hp 2-2 that reflect the patterns of inheritance of two genes, designated Hp' and Hp*. Homozygotes synthesize Hp 1-1 or Hp 1-2, respectively, while Hp 2-1 is synthesized by heterozygotes. Normally, the level of haptoglobin in a deciliter of human plasma is sufficient to bind 40 to 180 mg of hemoglobin. Since the resulting Hb-Hp complex is too large (2 155 kDa) to pass through the glomerulus, the kidney is protected from the formation of harmful precipitates while the loss of the iron associated with extracorpuscular hemoglobin is reduced.
Certain other plasma proteins bind heme, but not hemoglobin. They include a B,-globulin hemopexin, which binds free heme, and albumin, which binds metheme (ferric heme) to form methemalbumin. Methemalbumin subsequently transfers this metheme to hemopexin.

In situations where hemoglobin is constantly being released from red blood cells, such as occurs in hemolytic anemias, the level of haptoglobin can fall dramatically. This decrease
reflects the marked difference in the half-lives of free haptoglobin, approximately 5 days, and the Hb-Hp complex, approximately 90 minutes. The level of haptoglobin-related protein, a homologue of haptoglobin also present in plasma, is elevated in some patients with cancers, although the significance of this is not understood.

Iron is a key constituent of many human proteins, including hemoglobin, myoglobin, the cytochrome P450 group of enzymes, numerous components of the electron transport chain, and ribonucleotide reductase, which catalyzes the conversion of ribonucleotides into deoxyribonucleotides. Body iron, which is distributed as shown in Table 52-4, is highly conserved. A healthy adult loses only about 1 to 1.5 mg (< 0.05%) of their 3 to 4 g of body iron each day. However, an adult premenopausal female can experience iron deficiency due to blood loss during menstruation.
Enterocytes can absorb dietary iron in its free, ferrous Fe**, form, or as heme. Absorption of nonheme iron by enterocytes of the proximal duodenum is a highly regulated process (Figure 52-4). The transfer of iron across the apical membrane of the enterocytes is mediated via the divalent metal transporter 1 (DMT1 or SLC11A2), a relatively nonspecific transporter that also conveys Mn**, Co**,

Transferrin, 1 = 3-4 mg. Hemoglobin in red blood cells, 1 = 2500 mg. In myoglobin and various enzymes, 1 = 300 mg. In stores (ferritin), 1 = 1000 mg. Absorption, 1 = 1 mg/d. Losses, 1 = 1 mg/d
'In an adult female of similar weight, the amount in stores would generally be less (100-400 mg) and the losses would be greater (1.5-2 mg/d).
Zn**, Cu**, and Pb**. Since DMT1 is specific for divalent metal ions, free ferric iron (Fe**) must be converted to its ferrous form (Fe**) by ingested reducing agents such as vitamin C or enzymatically by a brush border membranebound ferrireductase, duodenal cytochrome b (Dcytb). Absorbed heme-bound iron is released by the enzymatic action of heme (see Chapter 31).
Once enterocytes, iron can bound to the iron-storage protein ferritin or transferred across the basolateral membrane by the iron exporter protein ferroportin, also known as iron-regulated protein 1 (IREG1 or SLC40A1). In the plasma, iron is transported in the Fe** form bound to the transport protein, transferrin. Hephaestin, oxygenase inside the either be stored

FIGURE 52-4 Nonheme iron transport in enterocytes. Ferric iron is reduced to the ferrous form by a luminal ferrireductase, duodenal cytochrome b (Dcytb). Ferrous iron is transported into the enterocyte via divalent metal transporter1 (DMT1). Within the enterocyte, iron is either stored as ferritin, or transported out of the cell by ferroportin (Fp). Ferrous iron is oxidized to its ferric form by hephaestin. The ferric iron is then bound by transferrin for transport by the blood to various sites in the body. (Based on Andrews NC: Forging a field: the golden age of iron biology. Blood 2008;112;219.)
a copper-containing ferroxidase homologous to ceruloplasmin, oxidizes Fe** to Fe** prior to export. Any excess ferritinbound iron remaining in the enterocytes is disposed of when they are sloughed off into the gut lumen.

The human body can typically store up to 1 g of iron, the vast majority of which is bound to ferritin. Ferritin (MW 440 kDa) forms a hollow ball composed of two-dozen ~19-21 kDa polypeptide subunits that can encapsulate as many as 3000 to 4500 ferric atoms. The subunits may be of the H (heavy) or the L (light) type. The H-subunit possesses ferroxidase activity, which is required for iron-loading of ferritin. The L-subunit is proposed to play a role in ferritin nucleation and stabilization. Normally, a small amount of ferritin is present in human plasma (50-200 g/dL) proportionate to the total stores of iron in the body. Plasma ferritin levels thus are used as an indicator of body iron stores. However, it is not known whether ferritin in plasma is derived from damaged cells or secretion by healthy ones. In addition, hemosiderin, a partly degraded form of ferritin may appear in tissues under conditions of iron overload (hemosiderosis).

The toxicity of free iron is largely a consequence of its ability to induce the formation of damaging reactive oxygen species (Figure 52-5). Biologic organisms minimize iron's potential toxicity by employing specialized storage and transport proteins, and maintaining it in its less reactive, Fe** state, during transport. Humans transport iron through the circulation tightly bound to transferrin (Tf), a glycoprotein synthesized by the liver. This 6,-globulin has a molecular mass of approximately 76 kDa and contains two high-affinity binding sites for Fe**. Glycosylation of transferrin is impaired in congenital disorders of glycosylation (see Chapter 46) or in chronic alcoholism. Hence, carbohydrate-deficient transferrin (CDT) is sometimes used as a biomarker of chronic alcoholism.
The concentration of Tf in plasma is approximately 300 mg/dL, sufficient to carry a total of approximately 300 ug of iron per deciliter of plasma. This figure represents the total iron-binding capacity (TIBC) of plasma. Typically, about 30% of the iron-binding sites in transferrin are occupied. Occupancy can decrease to less than 16% during severe iron
FIGURE 52-5 The Fenton reaction. Free iron is extremely toxic as it can catalyze the formation of hydroxyl radical (OH*) from hydrogen peroxide (see also Chapter 53). The hydroxyl radical is a transient but highly reactive species and that oxidize cellular macromolecules resulting in tissue damage.
deficiency and may increase to more than 45% in iron overload conditions.

For the delivery of transported iron, the recipient cell must bind circulating transferrin via a cell surface receptor, the transferrin receptor 1 (TfR1). The receptor-transferrin complex is then internalized by receptor-mediated endocytosis (see Chapter 25) and bound iron released as late endosomes become acidified. The free iron leaves the endosome via DMT1 and enters the cytoplasm. apoTf (Tf without bound iron) is then recycled. First, the transferrin receptor returns to the plasma membrane with apoTf still bound. Upon reaching the cell suface, apoTf dissociates from the receptor and reenters the plasma, where it picks up more iron for delivery to cells. This is called the transferrin cycle (Figure 52-6).
While transferrin receptor 1 is found on the surface of most cells, the homologous transferrin receptor 2 (TfR2) is encountered primarily on the surface of hepatocytes and the crypt cells of the small intestine. The affinity of TfR2 for Tf-Fe is much lower than that of TfR1, optimizing the former as a sensor, rather than an importer, for iron.

Macrophages play a key role in the turnover of red blood cells. Following phagocytosis and digestion via lysosomal hydrolases, the iron is expelled largely in the ferrous, Fe**, state. In order to be recovered via the transferrin cycle, this iron must be oxidized to the ferric, Fe**, state by the ferroxidase ceruloplasmin, a 160-kDa a,-globulin synthesized by the liver. With six, catalytically essential, copper atoms, ceruloplasmin is the major copper-containing protein in plasma.

Ceruloplasmin deficiency can arise from genetic causes as well as a lack of copper, an essential micronutrient, in the diet. When adequate quantities of catalytically functional ceruloplasmin are lacking, the body's ability to recycle Fe** becomes impaired, leading to iron accumulation in tissues. While persons suffering from hypoceruloplasmenia, a genetically heritable condition in which ceruloplasmin levels are roughly 50% of normal, generally display no clinical abnormalities, genetic mutations that abolish the ferroxidase activity of ceruloplasmin, aceruloplasminemia, can have severe physiologic consequences. If left untreated, the progressive accumulation of iron in pancreatic islet cells and basal ganglia eventually leads to the development of insulin-dependent diabetes and neurologic degeneration that may manifest as dementia, dysarthria, and dystonia.
FIGURE 52-6 The transferrin cycle. Holotransferrin (Tf-Fe) binds to transferrin receptor 1 (TfR1) present in clathrin-coated pits on the cell surface. The TfR1-Tf-Fe complex is endocytosed and the endocytic vesicles fuse to form early endosomes. The early endosomes mature in to late endosomes, which have a low internal pH. These acidic conditions cause the release of iron from transferrin. The resulting apotransferrin (apoTf) remains bound to TfR1. Ferric iron is converted to its ferrous form by the ferrireductase, Steap 3, and is then transported to the cytosol via DMT1. The TfR1-apoTf complex then is recycled back to the cell surface. At the cell surface, apoTf is released from TFR1. TfR1 then binds to new Tf-Fe. This completes the transferrin cycle.

In Wilson disease, a mutation in the gene for a copperbinding P-type ATPase (ATP7B protein) blocks the excretion of excess copper in the bile. As a consequence, copper accumulates in the liver, brain, kidney, and red blood cells. Paradoxically, rising levels of copper within the liver apparently interferes with the incorporation of this metal into newly synthesized ceruloplasmin polypeptides (apoceruloplasm) leading to a fall in plasma ceruloplasmin levels. If left untreated, patients suffering from this form of copper toxicosis may develop a hemolytic anemia or chronic liver disease (cirrhosis and hepatitis). Accumulation of copper in the basal ganglia and other centers can lead to neurologic symptoms. Wilson disease can be treated by limiting the dietary intake of copper while depleting any excess copper by the regular administration of penicillamine, which chelates copper and is subsequently excreted in the urine.

The synthesis of TfR1 and ferritin are reciprocally linked to intracellular iron levels. When iron is low, the rate of TfR1 synthesis increases and that of ferritin declines. The opposite occurs when iron is abundant and tissue needs have been sated. Control is exerted through the binding of iron regulatory proteins (IRPs) to hairpin loops structures called iron response elements (IREs) located in the 5' and 3' untranslated regions (UTRs) of the mRNAs coding for ferritin and TfR1, respectively (Figure 52-7). IRPs bind to the IREs only when intracellular iron levels are low. Binding at the 3' UTR of the mRNA for TfR1 stabilizes it, thereby increasing TfR1
FIGURE 52-7 Schematic representation of the reciprocal relationship between synthesis of ferritin and the transferrin receptor (TfR1). The mRNA for ferritin is represented on the left, and that for TfR1 on the right of the diagram. At high concentrations of iron, the iron bound to the IRP prevents that protein from binding the IREs on either type of mRNA. The mRNA for ferritin is able to be translated under these circumstances, and ferritin is synthesized. On the other hand, when the IRP is not able to bind to the IRE on the mRNA for TfR1, that mRNA is degraded. In contrast, at low concentrations of iron the IRP is able to bind to the IREs on both types of mRNA. In the case of the ferritin mRNA, this prevents it from being translated. Hence ferritin is not synthesized. In the case of the mRNA for TfR1, binding of the IRP prevents that mRNA from being degraded, enabling it to be translated and TfR1 to be synthesized. IRE, iron response element; IRP, iron regulatory protein.
synthesis, while binding of an IRP to the IRE located at the 5' UTR of ferritin mRNA blocks translation. Similarly, when iron levels are high, the IRBs dissociate. Under these circumstances translation of ferritin mRNA is facilitated and TfR1 mRNA is rapidly degraded.

The 25-amino acid peptide hepcidin plays a central role in iron homeostasis. Synthesized in the liver as an 84-amino acid precursor (prohepcidin), hepcidin binds to the cellular iron exporter, ferroportin, triggering its internalization and degradation. The consequent decrease in ferroportin produces a "mucosal block" by lowering iron absorption in the intestine and depresses recycling of the iron liberated by red blood cell turnover (Figure 52-8). Together, these result in a reduction in circulating iron levels (hypoferremia) as well as reduced placental iron transfer during pregnancy. When plasma iron levels are high, hepatic synthesis of hepcidin increases, reducing both iron absorption and recycling.

Liver cells monitor iron levels using a multicomponent "ironsensing complex" comprised of two transmembrane receptors whose cores consist of homodimers of TfR1 and T£R2, respectively, linked by a third transmembrane protein, HFE protein (Figure 52-9). HFE protein is a major histocompatibility (MHC) class 1-like molecule that binds 6,-microglobulin (a component of class I MHC molecules, not shown in Figure 52-9) and, normally, TfR1. TfR1 also binds the ironbound form of transferrin (Tf-Fe). The bindng sites for Tf-Fe and HFE overlap. Consequently, when iron is abundant and Tf-Fe levels are high, HFE is displaced from TfR1. The displaced HFE protein then binds to TfR2, forming a complex that can be further stabilized by binding of Tf-Fe. Binding of HFE to TfR2 triggers an intracellular signaling cascade that activates expression of HAMP, the gene encoding hepcidin. The gene encoding HFE protein is commonly mutated in hereditary hemochromatosis.

While bone morphogenic proteins (BMPs) act by mechanisms that are distinct from HFE protein, considerable cross-talk exists between these pathways. BMP binds to a cell surface receptor (BMPR) whose binding affinity is augmented by binding to a coreceptor, hemojuvelin (HJV). The activation of the BMPR-HJV complex triggers the phosphorylation of intracellular signaling proteins called SMADs, which stimulates the transcription of the gene that codes for hepcidin (Figure 52-9).

Synthesis of hepcidin is induced by cytokines such as interleukin 6 (IL-6) that are released as part of an inflammatory response. Binding of IL-6 to its cell surface receptor stimulates gene expression by activating the JAK-STAT (Janus kinase—signal transducer and activator of transcription) pathway (Figure 52-9). Inflamation-associated cytokines are thought to trigger the increase in hepcidin levels that accompanies anemia of inflammation (AI). AI manifests as a microcytic, hypochromic anemia that is refractory to iron supplementation.
Hepcidin expression decreases during hypoxia or B-thalassemia. The former is mediated by erythropoietin, whose synthesis is controlled by hypoxia-inducible transcription factors 1 and 2 (HIF-1 and HIF-2). In B-thalassemia, the expression of hepcidin is inhibited by growth differentiation factor 15 (GDF15) and twisted gastrulation 1 (TWSG1), which are secreted by erythroblasts.

Blood
FIGURE 52-8 Role of hepcidin in systemic iron regulation. Hepcidin binds to and triggers the internalization and degradation of ferroportin expressed on the surface of enterocytes and macrophages. This decreases iron absorption from the intestine and inhibits iron release from macrophages, leading to hypoferremia. (Based on Andrews NC: Forging a field: the golden age of iron biology. Blood 2008;112(2):219.)

levels of these and other clinically utilized biomarkers typically observed as patients progress through each stage of iron deficiency anemia.
Iron deficiency is extremely common in many parts of the world, especially in developing countries. Major causes of iron insufficiency include dietary deficiency, malabsorption, gastrointestinal bleeding, and episodic blood loss such as from menstruation. Persistent iron deficiency will lead to the progressive depletion of bodily iron stores. If the level of transferrin saturation falls to 20% or below, hemoglobin synthesis will be impaired, resulting in iron-deficient erythropoiesis. Hemoglobin levels in blood will gradually fall, resulting in iron-deficiency anemia. Patients typically present a hypochromic, microcytic blood picture that is accompanied by fatigue, pallor, and reduced exercise capacity.
The erythrocytes of persons suffering from iron deficient anemia display increased levels of surface transferrin receptor-1 and deficits in the ferrochelatase-catalyzed incorporation of iron into protoporphyrin IX. The resulting rise in the levels of soluble transferrin receptor protein (sTfR) released into the plasma by partial proteolysis of cell surface transferrin receptors and the accumulation of red-cell protoporphyrin serve as diagnostic biomarkers for iron deficiency anemia. Estimation of the serum sTfR level is especially useful for distinguishing anemia due to chronic inflammation, which does not affect the level of erythrocyte transferrin receptors, from iron deficiency anemia. Table 52-5 summarizes the

The presence of stainable iron in tissues, hemosiderosis, is characteristic of individuals suffering from hemochromatosis or iron overload. Hereditary hyperabsorption of iron by the intestines can be caused by mutations in the HFE gene or, less frequently, the genes encoding hepcidin, TfR2, HJV, or ferroportin (Table 52-6). Secondary iron overload is usually associated with ineffective erythropoiesis, as seen in the thalassemia syndromes. Repeated blood transfusions can also result in progressive iron overload, iron accumulation in tissues, and the generation of toxic reactive oxygen species.

Proteases are essential participants in tissue remodeling, blood clotting, elimination of old or diseased cells, destruction of invading pathogens, and other physiologic functions. Left unchecked, however, proteolytic enzymes that are secreted or escape into the blood can damage healthy tissue. Protection from indiscriminate proteolysis involves a battery of serum

FIGURE 52-9 Regulation of hepcidin gene expression. Tf-Fe (holotransferrin) competes with HFE for binding to TFR1. High levels of Tf-Fe displace HFE from its binding site on TfR1. Displaced HFE binds to TfR2 along with Tf-Fe to signal via the ERK/MAPK pathway to induce hepcidin expression. BMP binds to its receptor BMPR and HJV (coreceptor) to activate R-SMAD. R-SMAD dimerizes with SMAD4, then translocates to the nucleus where it binds to the BMP-RE, resulting in transcriptional activation of hepcidin as shown. IL-6, which is a biomarker of inflammation, binds to its cell surface receptor and activates the JAK-STAT pathway. STAT3 translocates to the nucleus where it binds to its response element (STAT-RE) on the hepcidin gene to induce it. BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptor; BMP-RE, BMP response element; ERK-MAPK, extracellular signal-regulated kinase/mitogen-activated protein kinase; HAMP, gene encoding hepcidin antimicrobial peptide (hepcidin); HJV, hemojuvelin; IL-6, interleukin 6; IL-6R, interleukin 6 receptor; JAK, Janus-associated kinase; SMAD, Sma and MAD (mothers against decapentaplegic)-related protein; STAT, signal transduction and activator of transcription; STAT3-RE, STAT 3 response element; TfR1, transferrin receptor 1; TfR2, transferrin receptor 2. (Redrawn from Hentz MW, Muckenthaler MU, Gali B, et al: Two to tango: regulation of mammalian iron metabolism. Cell 2010;142:24.)

RBC morphology, Negative Iron Balance = Normal. RBC morphology, Iron-Deficient Erythropoiesis = Normal. RBC morphology, Iron-Deficiency Anemia = Microcytic Hypochromic
Modified, with permission, from Figure 98-2, page 630, Harrison's Principles of Internal Medicine, 17th ed. Fauci AS, et al (editors). McGraw-Hill, 2008.

+ HFE-related hemochromatosis (type 1)
+ Non-HFE-related hemochromatosis
+ 4, Juvenile hemochromatosis (type 2)
+ Hepcidin mutation (type 2A)
+ Hemojuvelin mutation (type 2B)
+ 4, Transferrin receptor 2 mutation (type 3)
+ H, Ferroportin mutation (type 4)

- + Anemia Characterized by ineffective erythropoiesis
(eg, thalassemia major)
+ Repeated blood transfusions
+ Parenteral iron therapy
+ Dietary iron overload (Bantu siderosis)

+ Alcoholic liver disease
+ Nonalcoholic steatohepatitis
+ Hepatitis C infection
proteins that inhibit, and thereby limit the scope of, protease action.

a,-Antiproteinase, a 394-residue glycoprotein that makes up >90% of the a,-albumin fraction, is the principal serine protease inhibitor (serpin) in human plasma. Formerly called a,-antitrypsin, a,-antiproteinase inhibits trypsin, elastase, and other serine proteases by forming an inactive covalent complex with them. a,-Antiproteinase is synthesized by hepatocytes and macrophages. At least 75 polymorphic forms of this serpin, or Pi, exist. The major genotype is MM, whose phenotypic product is PiM. A deficiency in a,-antiproteinase plays a role in some cases (~ 5%) of emphysema, particularly in subjects with the ZZ genotype (who synthesize PiZ) and in PiSZ heterozygotes, both of whom secrete lower levels of serpins than PiMM individuals.

In the lungs, components of the smoke produced by burning tobacco products and industrial activities can oxidize a key methionine residue, Met,.,, located in the proteasebinding domain of a,-antiproteinase. Oxidation renders a,-antiproteinase unable to covalently bind and neutralize serine proteases. The subsequent damage produced by unchecked proteolytic activity in the lungs can contribute to the development of emphysema. Smoking can be particularly devastating for patients who already have low levels of a,-antiproteinase (eg, PiZZ phenotype). Intravenous administration of serpins (augmentation therapy) has been used as an adjunct in the treatment of patients with emphysema that exhibit a,-antiproteinase deficiency.
Individuals deficient in a,-antiproteinase are also at greater risk of lung damage from pneumonia or other conditions that induce the accumulation of polymorphonuclear white blood cells in the lung. Deficiency of a,-antiproteinase is also implicated in a,-antitrypsin deficiency liver disease, a form of cirrhosis that afflicts persons possessing the ZZ phenotype. In these individuals, substitution of Glu,,, by lysine promotes the formation of polymeric aggregates of a,-antiproteinase in the cisternae of the endoplasmic reticulum in hepatic cells.

a,-Macroglobulin, a member of the thioester plasma protein family, comprises 8 to 10% of the total plasma protein in humans. This homotetrameric glycoprotein is the most abundant member of a group of homologous plasma proteins that include complement proteins C3 and C4. a,-Macroglobulin is synthesized by monocytes, hepatocytes, and astrocytes. It mediates the inhibition and clearance of a broad spectrum of truant proteases by a "Venus flytrap" mechanism. The key components of the trap include a 35-residue "bait domain" located near the center of its polypeptide sequence and an internal cyclic thioester linking a cysteine and a glutamine residue (Figure 52-10). Cleavage of the bait domain produces a massive conformational change, triggering the envelopment of the attacking protease. The reactive thioester then reacts with the protease to covalently link the two proteins. This conformational change also exposes a sequence in a,-macroglobulin that is recognized by cell surface receptors that subsequently bind and remove the complex from the plasma.
In addition to serving as the plasma's predominant broadspectrum, or panproteinase, inhibitor, a,-macroglobulin also binds to and transports approximately 10% of the zinc in plasma (the remainder being transported by albumin) as well as cytokines such as platelet-derived growth factor and transforming growth factor B. As a cytokine transporter, a,-macroglobulin appears to be involved in targeting these effectors toward particular tissues or cells. Once taken up by cells, the cytokines dissociate, freeing them to modulate their growth and function.
FIGURE 52-10 An internal cyclic thiol ester bond, as in a,-macroglobulin. and neighboring present AA, AA, are amino acids to cysteine and glutamine.

Type
Protein Implicated
Primary
Principally light chains of immunoglobulins
Secondary
Serum amyloid A (SAA)
Familial
Transthyretin; also rarely apolipoprotein A-1,
cystain
C,
fibrinogen,
gelsolin,
lysozyme
Alzheimer’s
Amyloid 8 peptide (see Chapter 57, case no. 2)
disease
Dialysis-related
B,-microglobulin
Note: Proteins other than these listed have also been implicated in amyloidosis.

Amyloidosis refers to an impairment of tissue function that results from the accumulation of insoluble aggregates of proteins in the interstitial spaces between cells. The term is a misnomer, as it was originally thought that the fibrils were starch-like in nature. The fibrils generally are made up of proteolytic fragments of plasma proteins whose conformation is rich in B-pleated sheet. They generally also contain a P component derived from a plasma protein closely related to
C-reactive protein
called serum
amyloid
P
component.
Structural abnormalities or overproduction of more than 20 different proteins have been implicated in various types of amyloidosis. Primary amyloidosis (Table 52-7) typically is caused by a monoclonal plasma cell disorder that leads to the accumulation of protein fragments derived from immunoglobulin light chains (see below). Secondary amyloidosis results from an accumulation of fragments of serum amyloid A (SAA) consequent to chronic infections or cancer. In these instances, elevated levels of inflammatory cytokines stimulate the liver to synthesize SAA, which leads to a concomitant rise in its proteolytic degradation products. Familial amyloidosis results from accumulation of mutated forms of certain plasma proteins such as transthyretin (Table 52-3). Over 80 mutationally altered forms of this protein have been identified. Patients undergoing regular, long-term dialysis are at risk from B,-microglobulin, a plasma protein that is retained by membranes.
dialysis

The three major components of the body's immune system are B lymphocytes (B cells), T lymphocytes (T cells), and the innate immune system. B lymphocytes are mainly derived from bone marrow cells, while T lymphocytes originate from the thymus. B cells are responsible for the synthesis of circulating, humoral antibodies, also known as immunoglobulins. The T cells are involved in a variety of important cell-mediated immunologic processes such as graft rejection, hypersensitivity reactions, and defense against malignant cells and many viruses. B and T cells respond in an adaptive manner, developing a targeted response for each invader encountered. The innate immune system defends against infection in a nonspecific manner. It contains a variety of cells such as phagocytes, neutrophils, natural killer cells, and others that will be discussed in Chapter 54.

Immunoglobulins are oligomeric glycoproteins whose individual subunits traditionally have been classified as heavy (H) or light (L) based on their migration during SDSpolyacrylamide gel electrophoresis. Human immunoglobulins can be grouped into five classes abbreviated as IgA, IgD, IgE, IgG, and IgM (Table 52-8). Their respective biologic functions are summarized in Table 52-9. The most abundant of the five, IgG, consists of two identical light chains (23 kDa) and two identical heavy chains (53-75 kDa) linked together by a network of disulfide bonds.
The type of H chain determines the class of immunoglobulin and thus its effector function (see below): a (IgA), 6 (IgD), ¢ (IgE), y (IgG), and (IgM). The y chains of IgG are organized into four conserved domains: an amino-terminal variable region (V,,) and three constant regions (C,,1, C,,2, C,,3). The five types of H chains are distinguished by differences in their C,, regions. The pt and ¢ chains each have four C,, domains rather than the usual three. The Y-shaped configuration of the immunoglobulin core unit is illustrated by the IgG heterotetramer (L,H,) shown in Figure 52-11. Some immunoglobulins such as IgG exist only as the basic tetramer. Others such as IgA and IgM can form higher oligomers comprised of two, three (IgA), or five (IgM) copies of the core tetrameric unit (Figure 52-12).
The IgG light chain can be divided into a C-terminal constant region (C,) and amino-terminal variable region (V,). There are two general types of light chains, kappa (i) and lambda (}), which can be identified by their distinct C, regions. In human immunoglobulins, « chains are more common than ) chains. A given immunoglobulin molecule always contains either two x or two A light chains—never a mixture ofakandah.

IgG molecules are divalent. The tip of each Y contains an antigen-binding site made up of V,, and V, domains that form two antiparallel sheets of amino acids. The site on the antigen to which an antibody binds is termed an antigenic determinant, or epitope. Because the region between the C,,1 and C,,2 domains can be readily cleaved by pepsin or papain (Figure 52-11), it is referred to as the hinge region. The hinge region confers flexibility to the Fab arms, which facilitates binding to epitopes that may be far apart or on two separate antigens. By linking antigen particles together, large antibodyantigen clusters form that are readily recognized for disposal by phagocytic leukocytes. Cluster formation is often demonstrated in the laboratory by the formation of erythrocyte rosettes.

Transplacental passage, IgE = -. Mediation of allergic responses, IgG = -. Mediation of allergic responses, IgA = -. Mediation of allergic responses, IgM = -. Mediation of allergic responses, IgD = -. Mediation of allergic responses, IgE = +. Found in secretions, IgG = -. Found in secretions, IgA = +. Found in secretions, IgM = -. Found in secretions, IgD = -. Found in secretions, IgE = -. Opsonization, IgG = +. Opsonization, IgA = -. Opsonization, IgM = - b. Opsonization, IgD = -. Opsonization, IgE = -. Antigen receptor on B cell, IgG = -. Antigen receptor on B cell, IgA = -. Antigen receptor on B cell, IgM = +. Antigen receptor on B cell, IgD = ?. Antigen receptor on B cell, IgE = -. Polymeric form contains J chain, IgG = -. Polymeric form contains J chain, IgA = +. Polymeric form contains J chain, IgM = +. Polymeric form contains J chain, IgD = -. Polymeric form contains J chain, IgE = -
"The 11S form is found in secretions (eg, saliva, milk, and tears) and fluids of the respiratory, intestinal, and genital tracts.
- ®IgM opsonizes indirectly by activating complement. This produces C3b, which is an opsonin.
Source: Reproduced, with permission, from Levinson W, Jawetz E: Medical Microbiology and Immunology, 7th ed. McGraw-Hill, 2002.

The variable regions of the immunoglobulin light and heavy chains form the antigen-binding sites that confer antibodies with their amazing specificity. Within the variable regions of the L and H chains are a handful of hypervariable regions, short (5-10 residue) islands interspersed within the relatively invariable framework or complementarity-determining regions (CDRs) (Figure 52-13). An antigen-binding site is formed when the hypervariable regions of an H and L chain align together to form a projecting loop from the antibody surface. As their name implies, no two variable regions in the immunoglobulins from different individuals share the same amino acid sequence.
The capacity to generate various combinations of H and L chain CDRs provides a mechanism for producing combinatorial diversity, a collection of antibodies possessing different specificities. The essence of antigen-antibody interactions is mutual complementarity between the surfaces of CDRs and epitopes that involve multiple noncovalent interactions such as hydrogen bonding, salt bridges, hydrophobic interactions, and van der Waals forces (see Chapter 2).

The constant regions of the immunoglobulin molecules, particularly C,,2 and C,,3 (and C,,4 of IgM and IgE) on the Fc fragment, are responsible for the class-specific effector functions

IgG, Major Functions = Main antibody in the secondary response. Opsonizes bacteria, making them easier to phagocytose. Fixes complement, which enhances bacterial killing. Neutralizes bacterial toxins and viruses. Crosses the placenta.. IgA, Major Functions = Secretory IgA prevents attachment of bacteria and viruses to mucous membranes. Does not fix complement.. IgM, Major Functions = Produced in the primary response to an antigen. Fixes complement. Does not cross the placenta. Antigen receptor on the surface of B cells.. IgD, Major Functions = Found on the surfaces of B cells where it acts as a receptor for antigen.. IgE, Major Functions = Mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to antigen (allergen). Defends against worm infections by causing release of enzymes from eosinophils. Does not fix complement. Main host defense against helminthic infections.
FIGURE 52-11 Structure of IgG. The molecule consists of two light (L) chains and two heavy (H) chains. Each light chain consists of a variable (V) and a constant (C) region. Each heavy chain consists of a variable region (V,,) and a constant region that is divided into three domains (C1, C2, and C,3). The C2 domain contains the complement-binding site and the C,3 domain contains a site that attaches to receptors on neutrophils and macrophages. The antigen-binding site is formed by the hypervariable regions of both the light and heavy chains, which are located in the variable regions of these chains (see Figure 52-13). The light and heavy chains are linked by disulfide bonds, and the heavy chains are also linked to each other by disulfide bonds. (Reproduced, with permission, from Parslow TG, et al (editors): Medical Immunology, 10th ed. McGraw-Hill, 2001.)
of the different immunoglobulin molecules (Table 52-9, bottom part), such as complement fixation or transplacental passage.

The human genome contains fewer than 150 immunoglobulin genes. Nevertheless, each person is capable of synthesizing perhaps 1 million different antibodies, each specific for a unique antigen. Clearly, immunoglobulin expression does not follow the "one gene, one protein" paradigm. Instead, immunoglobulin diversity is generated by combinatorial mechanisms based on mixing and rearranging a finite pool of genetic information in multiple ways (see Chapters 35 and 38). Antibody diversity arises, in part, from the distribution of the coding sequence for each immunoglobulin chain among multiple genes. Each light chain is the product of three or more separate structural genes that code for the variable region (V,), joining region (J) (bearing no relationship to the J chain of IgA or IgM), and constant region (C,). Similarly, each heavy chain is the product of at least four different genes that code for a variable region (V,,), a diversity region (D), a joining region (J), and a constant region (C,,). Each gene is present in the human genome in several versions that can be assembled in a multiplicity of combinations. Diversity is further augmented through the action of the activation-induced cytidine deaminase (AID). By catalyzing the conversion of cytidine to uracil, AID massively increases the frequency of mutation of immunoglobulin V genes. AIDgenerated mutations are somatic in nature, unique to the differentiated cell where they occurred rather than to a germline cell. Consequently, activation of AID can generate unique subpopulations of B cells that harbor distinct mutations of their V genes, causing each to synthesize immunoglobulins of differing antigen specificity. In some pathologic states, the mutagenic action of AID can lead to the generation of autoantibodies that target the body's endogenous components, a phenomenon termed autoimmunity. A third mechanism for synthesizing antibodies that target novel antigens is junctional diversity. This refers to the addition or deletion of random numbers of nucleotides that takes place when certain gene segments are joined together. As is the case with AID, the mutations generated by junctional diversity are somatic in nature.

FIGURE 52-12 Schematic representation of serum IgA, secretory IgA, and IgM. Both IgA and IgM have a J chain, but only secretory IgA has a secretory component. Polypeptide chains are represented by thick lines; disulfide bonds linking different polypeptide chains are represented by thin lines. (Reproduced, with permission, from Parslow TG, et al (editors): Medical Immunology, 10th ed. McGraw-Hill, 2001.)

Immunoglobulin molecules of different classes that possess identical hypervariable and variable domains, and antigen specificity are said to share a common idiotype.
In most humoral immune responses, antibodies of different classes are generated that target the same epitope. Each class appears in a specific chronologic order following exposure to an immunogen (immunizing antigen). For instance, antibodies of the IgM class normally precede the appearance of the IgG class. The transition from the synthesis of one class to another is designated class or isotype switching. Switching involves the combining of a given immunoglobulin light chain with different heavy chains. Whereas a newly synthesized light chain will initially be mated with a chain to generate a specific IgM molecule, over time the same antigen-specific light chain will be mated with a y chain to generate an IgG whose V,, region and, consequently, antigen specificity will be identical to that of the u chain of the preceding IgM molecule. Combining this light chain with an a heavy chain, in turn, forms an IgA molecule with identical antigen specificity.

Antibodies have emerged as a major tool in biomedical research, diagnosis, and treatment. Originally, the production of antibodies against a selected antigen required that the antigen be injected into a host animal, such as a rabbit or goat, and serumcontaining plasma immunoglobulins that included (hopefully) antibodies against the antigen of interest obtained. When an antigen is injected into an animal, a mixture of B cells is induced to synthesize antibodies directed against epitopes on the antigen. Antibodies produced are thus heterogeneous or polyclonal in nature. Moreover, unless subjected to costly affinity purification, the serum will contain all antibodies produced by the host animal in addition to those against the antigen of interest.
FIGURE 52-13 Schematic model of an IgG molecule showing approximate positions of the hypervariable regions in heavy and light chains. The antigen-binding site is formed by these hypervariable regions. The hypervariable regions are also called complementarity-determining regions (CDRs). (Modified and reproduced, with permission, from Parslow TG, et al (editors): Medical Immunology, 10th ed. McGraw-Hill, 2001.)
Homogenous monoclonal antibodies that target, not just a single antigen, but a single epitope on its surface can be generated by isolating B cells from the spleen of a mouse (or another suitable animal) that has been previously injected with antigen. The cultured B cells are fused with mouse myeloma cells to generate an immortalized hybridoma cell line that secretes a single, monoclonal antibody. These antibodies are then screened to identify hybridoma lines that secrete a monoclonal antibody specific for the antigen or even the epitope of choice.
For therapeutic applications, monoclonal antibodies produced by murine cell lines can be humanized. This is accomplished by attaching the CDRs (the sites that bind antigens) onto appropriate sites in a human immunoglobulin molecule. This produces an antibody that is very similar to a human antibody, markedly reducing its immunogenicity and thus diminishing the chances of an anaphylactic reaction.

Immunoglobulins form the core of the body's adaptive immune system, a name that reflects its ability to generate antibodies against novel infectious agents. The innate immune system derive its names from the fact that the number, function, and specificity of its components are fixed and remain constant throughout life. The plasma-borne arm of the innate immune system is called the complement system, which can be activated by antibody-antigen complexes and thus acts consequent to and in support of or as a complement to the adaptive immune system.
The complement system displays features reminiscent of the blood's coagulation cascade. Both consist of sets of circulating zymogens (proproteins) that remain catalytically dormant until activated by proteolytic cleavage. These proteins, called complement factors, are synthesized by a variety of cell types, including hepatocytes, macrophages, monocytes, and intestinal endothelial cells. As is the case for clotting factors, most complement factors are proproteases (see Chapter 9) that, upon activation, target other components of the system, thereby generating a series or cascade of proteolytic activation events that amplify the production of the system's protective end products.

The classical pathway for activating the complex system is triggered when an antibody-antigen complex binds to and stimulates the protease activity of factor C1. Cl then cleaves complement factor C2 to form two smaller proteins, C2a and C2b, and cleaves complement factor C4 to form C4a and C4b (Figure 52-14). Two of the proteolytic fragments, C2a and C4b then associate together to form a new protease, the C3 convertase, which cleaves complement factor C3 into C3a and C3b. C3a now binds with the C2a:C4b heterodimer to form a heterotrimeric complex, the C5 convertase, that cleaves complement factor C5 into C5a and C5b. The C5b protein then combines with complement factors C6, C7, C8, and C9 to form the membrane attack complex (MAC). MAC kills bacterial invaders by binding to and opening a pore in their plasma membrane. Following lysis, the bacterial remains are destroyed by phagocytic macrophages. Meanwhile, the C3a and C5a proteins serve as chemoattractants that recruit leukocytes to the site of infection and stimulate an inflammatory response.
Targeting of the MAC to invading bacteria is facilitated by the presence of thioester bonds in C3 and C4. Like the thioester bond in the plasma protease inhibitor a,-macroglobulin, this highly reactive bond becomes exposed as a result of the conformational change that accompanies activation. In the case of C3 and C4, the thioester reacts with the hydroxyl groups of the bacteria's surface polysaccharides, covalently anchoring the C5 convertase complex of which they are a part to the targeted pathogen. This enables the remaining components of the MAC to be formed in close proximity to the bacterial membrane, facilitating assembly.

Activation can also be triggered via the lectin pathway, wherein the complexes formed when a complement factor known as mannose-binding lectin (MBL), also called mannan-binding protein (MBP), binds bacterial polysaccharides to generate a complex that recruits and activates C4 (Figure 52-14). The term lectin refers to any protein that binds polysaccharides. Most lectins are highly selective. MBL is specific for the mannose-containing carbohydrate moieties (mannans) of glycoproteins and lipopolysaccharides present on the surface of gram-positive bacteria, some viruses, and several fungi. Upon binding to the polysaccharide-MBL complex, C4 undergoes autoproteolysis, releasing C4a and C4b.
FIGURE 52-14 The complement cascade. Activation of the complement system can occur via three different mechanisms, referred to as the classical, lectin, and alternative pathways. Shown are the major components involved in each pathway, the products formed by proteolytic cleavage of the inactive proproteins, and the major complexes formed. Colons are used to indicate association in a complex.
In addition, C4 cleaves C2 into C2a and C2b. The remainder of cascade mirrors that for the classical pathway. MBL circulates as large, ~ 400 to 700 kDa, multivalent complexes consisting of four or more copies of a homotrimeric core unit made up of three copies of a ~ 30-kDa polypeptide. The core of the homotrimer is formed when the amino-terminal collagen-like domains in these polypeptides intertwine to generate an extended tail. The globular head of each polypeptide contains a carbohydrate-binding domain. Once formed, four or more homotrimers associate together, covalently linked by disulfide bonds between their amino-terminal tail regions, to form a "stalk" from which each of the C-terminal carbohydratebinding heads projects in a branched arrangement resembling that of an immunoglobulin (Figure 52-15).

The complement system also can be activated by the alternative pathway, in which C3 is activated by direct chemical hydrolysis, a process sometimes referred to as "ticking over" In the alternative pathway, C3b binds complement factor B, forming a C3b:B complex that is then cleaved by complex factor D. The resulting C3b:Bb complex possesses C5 convertase activity.

Dysfunctions of the innate and adaptive immune systems can have serious physiologic consequences. Deficits in the production of immunoglobulins or complement factors can leave the affected, immunocompromised individual susceptible to the
FIGURE 52-15 Schematic representation of mannosebinding lectin (MBL). Shown is a schematic diagram of a MBL comprised of four sets of MBL homotrimers. The carbohydrate-binding domains are colored. The intertwined collagen-like binding domains for each trimer are shown in blue. The stalk region, where the aminoterminal portions of the homotrimers of collagen-like domains associate together, is colored orange and yellow, with yellow marking the region where the S—S cross-links that stabilize the tetramer of homotrimers are located.
occurrence and spread of bacterial, fungal, or viral infections. Many factors can contribute to a depression in the effectiveness of the immune system. These include genetic abnormalities (eg agammaglobulinemia, in which production of IgG is markedly affected), toxins, viral infections, malnutrition, neoplastic transformation, or treatment with immunosuppressant drugs.
Overproduction and precocious activation of the immune and complement systems can also be deleterious. Failure to differentiate host cells from a foreign invader can trigger an autoimmune response in which the body's immune system attacks its own tissues and organs. The resulting damage may be cumulative, such as occurs in rheumatoid arthritis and multiple sclerosis, or acute, such as the complete destruction of pancreatic islet cells that occurs in type 1 diabetes. In North America, autoimmune disorders affect three in every hundred persons.
Table 52-1 lists several of the more commonly encountered autoimmune disorders.

Most plasma are synthesized in the liver. The majority are glycosylated.
= Albumin accounts for roughly 60%, by mass, of the protein content of plasma. As such, it is the principal determinant of intravascular osmotic pressure. Albumin also binds to and transports fatty acids, bilirubin, metal ions, and certain drugs.
@ Haptoglobin binds extracorpuscular hemoglobin to prevent its escape into the kidney, preventing the formation of damaging precipitates in the tubules.
@ Ferritin binds to and stores ferric iron inside cells.
| Transferrin transports iron to the sites where it is required.
= Ceruloplasmin, the major copper-containing protein in plasma, is a ferroxidase that plays a key role in recycling the iron released when senescent red blood cells are destroyed.
= Hepcidin regulates iron homeostasis by blocking
internalization of
the cellular
iron export
protein, ferrocidin.
™ Hepcidin expression is stimulated when binding of transferriniron complexes to type 1 transferrin receptors displaces HFE protein, which then binds to and activates type 2 transferrin receptors.
= Hereditary hemochromatosis is a genetic disease involving excessive absorption of iron.
= Autoimmune disorders result when the immune system attacks our body's own tissues.
= a,-Antitrypsin is the major serine protease inhibitor of plasma. Genetically produced deficiencies of this protein can lead to emphysema and liver disease.
™ a,-Macroglobulin is a major plasma protein that neutralizes many proteases and targets select cytokines to specific organs.
=™ Humans can synthesize immunoglobulins that specifically
target
millions
of
different
antigens.
™ ‘The core structure of the immunoglobulins is a tetramer consisting of two light and two heavy chains arranged in a “Y configuration. >
= Synthesis of diverse antibodies from a limited set of genes is made possible by combining, rearranging, and somatic mutation of immunoglobulin genes.
= ‘The ability to synthesize antibodies against novel antigens represents the defining feature of the adaptive immune system.
™ Hybridoma cells can provide monoclonal antibodies for
and
clinical
use.

= The complement system is generally activated by complexes formed between infecting microbes and protective antibodies or between mannose-rich polysaccharides on the pathogen’s surface and mannose-binding protein.
= The complement system is activated by a series of proteolytic cleavage events that transform dormant zymogens into active proteases.

Burtis CA, Ashwood EA, Bruns DE (editors): Tietz Textbook of
Clinical Chemistry and Molecular Diagnostics, 4th ed. Elsevier Saunders, 2006.
- Carroll MV, Sim RB: Complement in health and disease. Adv Drug Disc Rev 2011;63:965.
Craig WY, Ledue TB, Ritchie RF: Plasma Proteins: Clinical Utility and
Interpretation.
Foundation for
Blood
Research,
2008.
Hentz MW, Muckenthaler MU, Gali B, et al: Two to tango:
regulation of mammalian iron metabolism. Cell 2010;142:24.
Noris M, Remuzzi G: Overview of complement activation and regulation. Sem Nephrol 2013;33:479.
- Schaller H, Gerber S, Kaempfer U, et al: Human Blood Plasma
Proteins: Structure and Function. Wiley, 2008.

- Understand the concept of stem cells and their importance.
Explain why red blood cells are reliant on glucose for energy.
Describe the roles of erythropoietin, thrombopoietin, and other cytokines in the production of red blood cells and platelets.
Describe the enzyme systems that protect heme iron from oxidation and reduce methemoglobin.
Identify the major components of the erythrocyte cytoskeleton.
Summarize the causes of the major disorders affecting red blood cells.
Describe the major function of erythrocyte band 3 protein.
Explain the biochemical bases of the ABO blood group substances.
platelets. List the major components contained in the dense granules and a-granules in
= Describe the molecular bases of immune thrombocytopenic purpura and von Willebrand disease.

The evolution of a diverse array of freely circulating blood cells was critical to the development of animal life. The packaging of hemoglobin and carbonic anhydrase inside specialized cells called erythrocytes greatly amplified the capacity of circulating blood to carry oxygen to and carbon dioxide away from peripheral tissues. Anemia, a deficiency in the level of circulating hemoglobin (< 120-130 g/L), compromises health by reducing the ability of the blood to supply tissues with adequate levels of oxygen. Anemia can arise from a variety of causes that include genetic abnormalities (eg, sickle cell trait, pernicious anemia), excessive bleeding, insufficiencies of dietary iron or vitamin B,,, or the lysis of red blood cells by invading pathogens (eg, malaria). Platelets help staunch the outflow of blood from damaged tissues. Deficits in platelet number or function increase a patient's vulnerability to hemorrhage by reducing the speed of formation and structural integrity of protective clots. As is the case for anemia, a low platelet count, known as thrombocytopenia, can be triggered by a range of factors that include bacterial infection, some medications including sulfa-containing antibiotics, or autoimmune reactions such as idiopathic thrombocytopenic purpura. Other pathophysiologic syndromes, such as von Willebrand disease and Glanzmann thrombasthenia, are caused by genetic mutations that impair platelet adherence or aggregation rather than their abundance.

Both red blood cells and platelets turn over at a relatively high rate. Hence, replacements are constantly being produced from precursor stem cells. Stem cells possess a unique capacity both to produce unaltered daughter cells (self-renewal) and to generate a diverse range of specialized cell types (potency). Thus, stem cells are considered to exist in an undifferentiated state. Stem cells may be totipotent (capable of producing all the cells in an organism), pluripotent (able to differentiate into cells of any of the three germ layers), multipotent (able to produce only cells of a closely related family), or unipotent (able to produce only one type of cell). Stem cells are also classified as embryonic or adult, the latter of which are more limited in their capacity to differentiate.
- =

FIGURE 53-1 Hematopoiesis. Shown is a simplified and heavily abbreviated scheme indicating the paths by which hematopoietic stem cells differentiate to produce many of the more quantitatively prominent red and white blood cells. Only selected developmental intermediates are shown. The names for each cell type are indicated in bold type. Cell nuclei are shown in purple. Each arrow summarizes a multistage transition. The hormones and cytokines that stimulate each transition are listed next to the arrows. EPO, erythropoietin; FLT3 ligand, FMS-like tyrosine kinase 3 ligand; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; M-CSF, macrophage colony-stimulating factor; SCF, stem cell factor; TGF-B,, transforming growth factor B,; TNF-a, tumor necrosis factor a; TPO, thrombopoietin.
Differentiation of hematopoietic stem cells is regulated by a set of secreted glycoproteins called cytokines. Stem cell factor and several colony-stimulating factors collaborate with interleukins 1, 3, and 6 to stimulate the proliferation of hematopoietic stem cells in the bone marrow and induce their differentiation into one of several myeloid cell types (Figure 53-1). Erythropoietin or thrombopoietin then directs myeloid progenitor cells to eventually differentiate into erythrocytes or platelets, respectively.

The structure and composition of red blood cells reflect their highly specialized function: to deliver the maximum quantity of oxygen to tissues and aid in the removal of carbon dioxide, a waste product of cellular respiration, and urea. The interior of a red blood cell contains a massive amount of hemoglobin, roughly one-third by weight (30-34 g/dL for an adult). This extraordinary hemoglobin concentration is achieved, in part, by dispensing with the intracellular organelles normally found in eukaryotic cells (eg, nucleus, lysosome, Golgi apparatus, mitochondria). As a consequence, mature enucleated red blood cells are unable to reproduce.
Red blood cells possess an extensive cytoskeletal network responsible for maintaining their biconcave configuration (Figure 53-2). Their unusual shape enhances the exchange of
FIGURE 53-2 Red blood cells are shaped like biconcave disks. Shown are drawings of (A) a red blood cell, (B) a section through a red blood cell illustrating its biconcave shape, and (C) a red blood cell folded for passage through a narrow capillary.
oxygen and carbon dioxide between erythrocytes and tissues in two ways. First, their disc-like configuration possesses a much higher ratio of surface area to volume than more spherical shapes. Second, it enables red blood cells to fold over and squeeze through capillaries whose diameter is smaller than that of the erythrocyte itself. Both factors reduce the distance gas molecules must diffuse to and from the rapidly moving (up to 2 mm/s) erythrocytes.

Mature red blood cells lack the mitochondria that contain ATP synthase and the enzymes of the tricarboxylic acid cycle (TCA), electron transport chain, and $-oxidation pathway. They are therefore incapable of utilizing fatty acids or ketone bodies as metabolic fuel. Consequently, red blood cells are completely reliant of glycolysis to generate ATP. Glucose enters red blood cells by facilitated diffusion (see Chapter 40), a process mediated by glucose transporter 1 (GLUT1), also known as glucose permease (Table 53-1).
The glycolytic pathway in red blood cells also possesses a unique branch, or shunt, whose purpose is to isomerize 1,3-bisphosphoglycerate (1,3-BPG) to 2,3-bisphosphoglycerate (2,3-BPG). 2,3-BPG binds to and stabilizes hemoglobin in the T-state (see Chapter 6). Conversion of 1,3-BPG to 2,3-BPG is catalyzed by 2,3-BPG mutase, a bifunctional enzyme that also catalyzes the hydrolysis of 2,3-BPG to the glycolytic intermediate 3-phosphoglycerate. A second enzyme, multiple inositol polyphosphate phosphatase, also catalyzes the hydrolysis of 2,3-BPG to the glycolytic intermediate 2-bisphosphoglycerate. The activities of these enzymes are sensitive to pH, which ensures that 2,3-BPG levels rise and fall at the appropriate times during the oxygen transport cycle.
Various aspects of the metabolism of the red cell, several of
which are
discussed in
other chapters,
are summarized
in

+
The
RBC
is highly
dependent upon
glucose as
its energy
source, which
its membrane
contains high-affinity
glucose transporters.
+
Glycolysis, producing
lactate, is
the mode
of production
of
ATP.
+ Because RBCs lack mitochondria there is no production of ATP by oxidative phosphorylation.
+ The RBC has a variety of transporters that maintain ionic and water balance.
+ Production of 2,3-bisphosphoglycerate by reactions closely
associated with glycolysis is important in regulating the ability of Hb to transport oxygen.
+ The pentose phosphate pathway of the RBC metabolizes about
5%-10%
of the
total flux
of glucose)
and produces
NADPH.
due to
a
deficiency of
of glucose-
6-phosphate dehydrogenase
is common.
- + Reduced glutathione (GSH) is important in the metabolism of the
RBC, in
part to
counteract the
action of
potentially toxic
peroxides.
The
RBC
can synthesize
GSH
and the
NADPH
required to
return oxidized
(G-S-S-G)
to the
reduced state
GSH.
- + The iron of Hb must be maintained in the ferrous state. Ferric iron
is reduced
to the
ferrous state
by the
action of
an
NADH- dependent
methemoglobin reductase
system involving
cytochrome b,
reductase and
6,.

+ While biosynthesis of glycogen, fatty acids, protein, and nucleic acids does not occur in the RBC, some lipids (eg, cholesterol) in the red cell membrane can exchange with corresponding plasma lipids.
+ The RBC contains certain enzymes of nucleotide metabolism (eg, adenosine deaminase, pyrimidine nucleotidase, and adenylyl kinase). Deficiencies of these enzymes are involved in some cases of hemolytic anemia.
+ When RBCs reach the end of their lifespan, the globin is degraded to amino acids (which are reutilized in the body), the iron is released from heme and reutilized, and the tetrapyrrole component of heme is converted to bilirubin, which is mainly excreted into the bowel via the bile.

Like oxygen, the solubility of carbon dioxide in aqueous solution is low, much too low to accommodate more than a few percent of the CO, produced by metabolically active tissues. However, the solubility of the hydrated form of CO,, carbonic acid (H,CO,) and its protonic dissociation product, bicarbonate (HCO, ) is relatively high. The presence in erythrocytes of high levels of the enzyme carbonic anhydrase (see Figure 6-11) enables them both to absorb waste CO, by catalyzing its rapid conversion to carbonic acid, and to reverse this process in order to facilitate its expulsion in the lungs. While red blood cells carry some CO, in the form of hemoglobin-bound carbamates (see Chapter 6), roughly 80% is carried as dissolved carbonic acid and bicarbonate.

The 120-day lifespan of a normal red blood cell requires that nearly 1% of the 20 to 30 trillion erythrocytes in a typical for
individual must be replaced daily. This equates to a rate of production of ~ 2 million new red blood cells per second. When initially formed, differentiated red blood cells retain a portion of the ribosomes, endoplasmic reticulum, mitochondria, etc that were present in their nucleated precursors. Consequently, during the ~ 24 hours required to complete maturation to erythrocytes, these nascent red blood cells, called reticulocytes, retain the capacity to synthesize polypeptides (eg, globin) under the direction of vestigial mRNA molecules. In rare cases, genetic mutations that lead to an impairment of ribosome function, called ribomyopathies, can result in red blood cell hypoplasia. Diamond-Blackfan anemia is caused by mutations in the gene encoding for the ribosomal processing protein RPS19. 5q-syndrome, which presents a similar clinical picture, is caused by mutations that lead to an insufof ribosomal RPS14.
ficiency protein

The initial stages of erythropoiesis, the production of red blood cells, are modulated by stem cell factor, colonystimulating factors, and interleukins 1, 3, and 6. Commitment of myeloid progenitor cells to differentiation into erythrocytes is largely dependent on erythropoietin (EPO), a glycoprotein of 166 amino acids (molecular mass ~ 34 kDa). EPO, which is synthesized mainly by the kidney, is released into the bloodstream in response to hypoxia. Upon reaching the bone marrow, EPO stimulates red blood cell progenitors via a transmembrane receptor. Binding of EPO to its receptor stimulates the dimerization of the receptor and activation of associated molecules of the Jak2 protein-tyrosine kinase.
Erythropoietin is administered therapeutically to treat anemias arising from chronic kidney failure, disorders of hematopoietic stem cells (myelodysplasia), or from the collateral effects of chemical and radiologic treatments for cancer. Today, recombinant DNA technology has made it possible to produce substantial amounts of erythropoietin from cultured human cells. As described in Chapter 49, scientists are working to increase the dose effectiveness of recombinant EPO by manipulating the composition of its polysaccharide chains.

The ferrous, Fe*, iron atoms in hemoglobin are susceptible to oxidation by reactive oxygen species (ROS). Hemoglobin in which one or more heme irons has been oxidized to the ferric (Fe**) state is called methemoglobin. Ferric hemes do not bind oxygen, which not only reduces the number of O,-binding sites, but can interfere with the cooperative interactions between the subunits of the hemoglobin tetramer (see Chapter 6). The ability to rescue methemoglobin by reducing ferric iron is thus of great physiologic importance. In red blood cells, methemoglobin reduction is catalyzed by the NADH-cytochrome b, methemoglobin reductase system. The first component of the system, a flavoprotein named cytochrome b, reductase (also known as methemoglobin reductase), transfers electrons from NADH to the second component, cytochrome b,, using electrons supplied by NADH:

Reduced cytochrome b, then transfers the electrons to methemoglobin, reducing Fe** back to the Fe'* state, thereby restoring hemoglobin to a fully functional state:

The ultimate source of the electrons used to reduce methemoglobin is glycolysis, where NAD* is reduced to NADH by the action of glyceraldehyde-3-phosphate dehydrogenase. The efficiency of this system is such that only trace quantities of methemoglobin are normally present in erythrocytes.

Methemoglobinemia, the abnormal accumulation of methemoglobin, can arise from genetic abnormalities (inherited methemoglobinemia) or from the ingestion of certain drugs and chemicals (acquired methemoglobinemia) such as sulfonamides or aniline (Table 53-3). Affected patients often exhibit bluish discoloration of the skin and mucous membranes (cyanosis). The inherited form most commonly arises from a deficiency in the quantity or activity of cytochrome b, reductase, although mutations that affect the properties of cytochrome b, itself have also been encountered. In rare instances, methemoglobinemia can result from mutations in hemoglobin itself, such as those affecting the proximal and distal histidine residues (see Figure 6-3), that render it more susceptible to oxidation. Collectively referred to as hemoglobin M (HbM), these include HbM,,,,,. in which His87 in the a subunit is replaced by Tyr; HbM,,,.p,,2 in which His92 of the B subunit is replaced by Tyr; HbM.,,.,. in which His58 in the a subunits of hemoglobin is replaced by Tyr; and HbM,,,..,,,» in which His63 in the B subunit is replaced by Tyr. One exception to this pattern is HBM yi, aukeoi Which Val67 of the B subunit is replaced by Glu. All known carriers of HbM are heterozygotes.

The radical anion superoxide, O,, is generated in red blood cells by the autoxidation of hemoglobin to methemoglobin. This potent ROS can react with and damage proteins, lipids, nucleotides, and other biomolecules (see Chapter 58). Approximately 3% of the hemoglobin of human blood undergoes

GPI-anchored proteins
*OMIM members apply only to disorders with a genetic basis.
auto-oxidation each day. In addition, oxidation of the iron storage protein ferritin by superoxide can result in the release of free Fe** and the subsequent iron-catalyzed generation of OH' (see Figure 58-2). Superoxide may thus produce the tissue damage that occurs in persons suffering from abnormally high levels of iron in the body, known as iron overload. Iron overload is characteristic of individuals suffering from hereditary hemochromatosis, a genetic condition that causes the body to absorb excessive quantities of dietary iron. Another endogenous source of superoxide is the enzyme NADPH-hemoprotein reductase (a cytochrome P450 reductase, see Chapter 12), another enzyme that can catalyze the reduction of the Fe** in methemoglobin to Fe**.

The limited suite of metabolic pathways present in red blood cells renders them completely reliant on the pentose phosphate pathway (see Chapter 20) or, to be more specific, the X-linked enzyme glucose-6-phosphate dehydrogenase for the reduction of NADP* to NADPH. Glucose-6-phosphate dehydrogenase deficiency is the most common of all enzymopathies (diseases caused by abnormalities of enzymes). More than 400 million people are estimated to carry one of the over 140 genetic variants of glucose-6-phosphate dehydrogenase. This deficiency is most common among natives of tropical Africa (and their African-American descendants), the Mediterranean, and certain parts of Asia. Individuals harboring this deficiency are vulnerable to attacks of hemolytic anemia, a consequence of their inability to generate sufficient NADPH to maintain glutathione, a key intracellular antioxidant, in the reduced state (Figure 53-3).
Mutations in the gene for GGPDH
y
Decreased activity of GGPDH
y
Decreased levels of NADPH
y
Decreased regeneration of GSH from GSSG by glutathione reductase (which uses NADPH)
y
Oxidation, due to decreased levels of GSH and increased levels of intracellular oxidants (eg, O2"), of SH groups of Hb (forming Heinz bodies), and of membrane proteins, altering membrane structure and increasing susceptibility to ingestion by macrophages (peroxidative damage to lipids in the membrane also possible)
————E——)

FIGURE 53-3 summary of probable events causing hemolytic anemia due to deficiency of the activity of glucose6-phosphate dehydrogenase (OMIM 305900).
A deficiency in glucose-6-phosphate dehydrogenase renders red blood cells hypersensitive to oxidative stress and ROS-induced the formation of Heinz bodies, insoluble aggregates consisting of hemoglobin molecules whose —SH groups have become oxidized, and which stain purple with cresyl violet. Like sickle cell trait, the persistence of these genetic variants has been attributed to their potential to confer enhanced resistance to malaria.

Hemolytic anemia can be triggered by factors other than a deficiency in glucose-6-phosphate dehydrogenase (Figure 53-4). Extrinsic causes (beyond the erythrocyte membrane) include hypersplenism, in which the enlargement of the spleen causes red blood cells to become sequestered within this organ. Erythrocytes also can lyse if attacked by incompatible antibodies present in intravenously administered plasma or blood (eg, transfusion reaction). Immunologic incompatibilities may arise when an Rh' fetus is carried by an Rh" mother (Rh disease) or as a consequence of an autoimmune disorder (eg, warm or cold antibody hemolytic anemias). Some infectious and toxic agents act by directly undermining the structural integrity of the erythrocyte membrane. For example, many insect and reptile venoms of various species contain phospholipases or proteases that catalyze the hydrolytic breakdown of membrane components. Similarly, some infectious bacteria, including certain strains of Escherichia coli and clostridia, secrete lytic factors called hemolysins, which can be composed of proteins, lipids, or some combination thereof. Parasitic infections (eg, the plasmodia causing malaria) are also a major cause of hemolytic anemias in certain geographic areas.
The root cause of many hemolytic anemias is intracellular, also referred to as intrinsic. Gluocse-6-phosphate dehydrogenase deficiency falls into this category. Defects in the composition or structure of hemoglobin, called hemoglobinopathies,
FIGURE 53-4 Schematic diagram of some causes of hemolytic anemias. Extrinsic causes include hypersplenism, various antibodies, certain bacterial hemolysins, and some snake venoms. Causes intrinsic to the red cells include mutations that affect the structures of membrane proteins (eg, in hereditary spherocytosis and hereditary elliptocytosis), paroxysmal nocturnal hemoglobinuria (PNH, see Chapter 47), enzymopathies, abnormal hemoglobins, and certain parasites (eg, plasmodia causing malaria).

constitute the second major intrinsic cause of hemolysis. Most hemoglobinopathies, such as sickle cell anemia and the various thalassemias (see Chapter 6), are genetic in nature. In rare cases, hemolytic anemia can arise from an insufficiency in the enzyme pyruvate kinase. The resulting impairment of glycolysis reduces the production of the ATP required to power the export of excess water and Na*. The resulting osmotic pressure (see below) can compromise and potentially overwhelm the integrity of the erythrocyte membrane.
Mutations that affect the cytoskeletal proteins responsible for maintaining their biconcave shape and resistance to osmotic pressure are classified as membrane-specific causes of hemolytic anemia (see below). The most important of these are hereditary spherocytosis and hereditary elliptocytosis, which arise from abnormalities in the amount or structure of the cytoskeletal protein spectrin. Defects may also occur in the synthesis of the glycophosphatidylinositol groups that anchor certain proteins, such as acetylcholinesterase and decay-accelerating factor, to the surface of the erythrocyte membrane, as is the case in paroxysmal nocturnal hemoglobinuria (see Chapter 46).

Early analyses by SDS-PAGE of the polypeptides present in red blood cells revealed 10 major proteins (Figure 53-5). These proteins were initially assigned numeric designators based on their migration on SDS-PAGE. Thus, the polypeptide with the highest molecular mass, which migrates slowest, was designated band 1 protein, also known as spectrin (Table 53-4). As illustrated by Figure 53-6, certain of these proteins are glycosylated. Several proteins span the membrane bilayer (integral membrane proteins), while others associate with its surface, generally via protein-protein interactions (peripheral membrane proteins).
FIGURE 53-5 Major membrane proteins of the human red blood cell. Proteins separated by SDS-PAGE were detected by staining with Coomassie blue dye. (Reproduced, with permission, from Beck WS, Tepper RI: Hemolytic anemias III: membrane disorders. In: Hematology, 5th ed. Beck WS (editor). The MIT Press, 1991.)

(I) or Peripheral (P) = P I. 8, Approximated Molecular Mass (kDa) = 23 31, 23, and 28
*The band number refers to the relative rates of migration on SDS-PAGE (see Figure 53-5). A number of other components (eg, 4.2 and 4.9) are not listed. Source: Adapted from Lux DE, Tse WT: Hereditary spherocytosis and hereditary elliptocytosis. In: The Metabolic basis of inherited Disease, 8th ed. Scriver CR, Beaudet AL, Valle D, et al (editors). McGraw-Hill, 2001. Chapter 183.

Band 3 protein is a multipass transmembrane glycoprotein whose polypeptide chain crosses the bilayer 14 times. Band 3
FIGURE 53-6 interactions of cytoskeletal proteins with each other and with certain integral proteins of the membrane of the red blood cell. (Reproduced, with permission, from Beck WS, Tepper RI: Hemolytic anemias III: membrane disorders. In: Hematology, 5th ed. Beck WS (editor). The MIT Press, 1991.)
protein is oriented with its carboxyl terminal end projecting from the external surface of the erythrocyte membrane and its amino-terminal end from the cytosolic face. The principal function of this dimeric anion exchange protein is to provide a channel through the membrane via which chloride and bicarbonate anions can be exchanged. At the tissues, bicarbonate generated from the hydration of CO, is exchanged for chloride. At the lungs, where carbon dioxide is exhaled, this process is reversed. The amino-terminal end also serves as an anchoring point for several other red blood cell proteins, including band 4.1 and 4.2 proteins, ankyrin, hemoglobin, and several glycolytic enzymes.
Glycophorins A, B, and C are single-pass transmembrane proteins (the polypeptide chain crosses the membrane only once). The single 23-amino acid transmembrane segment is a-helical in configuration. The predominant form, glycophorin A, is comprised of a 131-amino acid polypeptide covalently modified by 16 oligosaccharide chains, 15 of them O-linked, that account for roughly 60% of its mass and nearly 90% of the sialic acid residues exposed on the surface of the red cell membrane. The carboxyl-terminal end extends into the cytosol and binds to band 4.1 protein, which in turn binds to spectrin. Polymorphism of glycophorin A serves as the basis of the MN blood group system (see below). Some viral and bacterial pathogens, such as influenza virus and Plasmodium falciparum, target erythrocytes by recognizing and binding to glycophorin A. Intriguingly, individuals whose red cells lack glycophorin A exhibit no adverse effects.

In order to maximize the efficiency of gas exchange, red blood cells must possess the structural strength to maintain their biconcave shape, yet remain sufficiently flexible to squeeze through peripheral capillaries and the sinusoids of the spleen. The structural foundation that molds and maintains the membrane's inherent fluid and deformable lipid bilayer into the erythrocyte's characteristic biconcave shape is provided by a strong but flexible network of cytoskeletal proteins (Figure 53-6).
Spectrin is the most abundant protein of the erythrocyte cytoskeleton. It is composed of two polypeptides more than 2100 residues in length: spectrin 1 (a chain) and spectrin 2 (6 chain). The a and 6 chains of each spectrin dimer intertwine in an antiparallel orientation to form a highly extended structural unit ~ 100 nm in length. Normally, two spectrin dimers self-associate head-to-head to form an approximately 200-nm long heterotetramer that is linked to the inner surface of the plasma membrane (and is bridged to other spectrin tetramers) via ankyrin, actin, and band 4.1 protein. The result is an internal mesh, the cytoskeleton, that is strong enough to maintain cell shape and resist swelling due to osmotic pressure, yet flexible enough to allow the erythrocyte to fold when needed.
Ankyrin is a pyramid-shaped protein that binds spectrin. In turn, ankyrin binds tightly to band 3, securing attachment of spectrin to the membrane. Ankyrin is sensitive to proteolysis, accounting for the appearance of bands 2.2, 2.3, and 2.6, all of which are derived from band 2.1.

Actin (band 5) exists in red blood cells as short, doublehelical filaments of F-actin. The tail end of spectrin dimers binds to actin. Actin also binds to protein 4.1. Protein 4.1 is a globular protein that binds tightly to a site near the actinbinding domain in the tail of spectrin to form a protein 4.1-spectrin-actin ternary complex. Protein 4.1 also binds to the integral membrane proteins glycophorin A and glycophorin C, as well as certain phospholipids, thereby anchoring the ternary complex to the membrane.
Certain other less quantitatively prominent proteins, such as band 4.9, adducin, and tropomyosin, also participate in cytoskeletal assembly.

Hereditary spherocytosis, a genetic disease transmitted as an autosomal dominant, is characterized by the presence of spherocytes (spherical red blood cells) in the peripheral blood, by hemolytic anemia, and by splenomegaly. Their abnormal shape, in turn, renders spherocytes less deformable and more prone to destruction in the spleen, thus greatly shortening their life in the circulation. This condition, which affects about 1 in 5000 persons of Northern European ancestry, is caused by a deficiency in the amount or abnormalities in the structure of spectrin or, less frequently, ankyrin or band 3, 4.1, or 4.2 proteins. Loss of these proteins or reductions in their capacity to associate with other cytoskeletal components weakens the links that anchor the erythrocyte membrane to the cytoskeleton, allowing the erythrocyte to swell into a spherical shape. The anemia associated with hereditary spherocytosis is generally relieved by surgical removal of the patient's spleen (splenectomy).
Hereditary elliptocytosis can readily distinguished from hereditary spherocytosis by virtue of the fact that the affected red blood cells assume an elliptic shape. This condition results from genetic abnormalities that affect spectrin or, less frequently, band 4.1 protein or glycophorin C.

Approximately 30 human blood group systems have been recognized, the best known of which are the ABO, Rh (Rhesus), and MN systems. The term "blood group" refers to a defined set of red blood cell antigens, or blood group substances, controlled by a genetic locus having a variable number of alleles (eg, A, B, and O in the ABO system). The term "blood type" refers to the antigenic phenotype, usually recognized by the use of appropriate antibodies.

The ABO system was discovered by Landsteiner in 1900 while investigating the basis of compatible and incompatible transfusions in humans. The membranes of the erythrocytes of most individuals contain one blood group substance, either type A, B, AB, or O. Individuals of type A have anti-B antibodies in their plasma that will agglutinate the erythrocytes in type B or type AB blood. Individuals of type B have anti-A antibodies that will agglutinate type A or type AB erythrocytes. Type AB blood has neither anti-A nor anti-B antibodies, and has been designated the universal recipient. Type O blood has neither A nor B antigens, and has been designated the universal donor. The above description has been simplified considerably, as further subgroups exist such as A, and A,. The genes responsible for production of the ABO substances are present on the long arm of chromosome 9. There are three alleles, two of which are codominant (A and B) and the third (O) recessive; these ultimately determine which of the four phenotypic products is synthesized: the A, B, AB, and O substances.

The ABO antigens are complex oligosaccharides present in most cells of the body and in certain secretions (Figure 53-7). These oligosaccharides are bound to membrane proteins or lipids, and are collectively referred to as ABO substances. For red blood cells, the membrane oligosaccharides that determine the antigenic natures of the ABO substances appear to be mostly present in glycosphingolipids, whereas in secretions the same oligosaccharides are present in glycoproteins. Their presence in secretions is determined by a gene designated Se (for secretor), which codes for a specific fucosyl (Fuc) transferase in secretory organs, such as the exocrine glands, but which is not active in red blood cells. Individuals of SeSe or Sese genotypes secrete either or both A and B antigens whereas individuals of the sese genotype do not. However, their red blood cells can express the A and B antigens.

Transferase, the B Gene a Gal Transferase, & the O Gene an Inactive Product
H substance, the blood group substance found in persons of type O, is the precursor of both the A and B substances (Figure 53-7). It is formed by the action of a fucosyltransferase, coded for by the H locus, that catalyzes the addition of an al + 2 linked fucose onto the terminal Gal residue of its precursor:
GDP — Fuc + Gal — B — R > Fuca1, 2—Gal-fB —-R+GDP Precursor H substance
A substance contains an additional GalNAc, while B substance contains an additional Gal, linked as indicated.
FIGURE 53-7 Diagrammatic representation of the structures of the H, A, and B blood group substances. R represents a long complex oligosaccharide chain, joined either to ceramide where the substances are glycosphingolipids, or to the polypeptide backbone of a protein via a serine or threonine residue where the substances are glycoproteins. Note that the blood group substances are biantennary; that is, they have two arms, formed at a branch point (not indicated) between the GlcNAc—R, and only one arm of the branch is shown. Thus, the H, A, and B substances each contains two of their respective short oligosaccharide chains shown above. The AB substance contains one type A chain and one type B chain.
These differences reflect the specificity of the glycosyltransferase that catalyzes the addition of the terminal monosaccharide. The A gene encodes a UDP-GalNAc-specific GalNAc transferase that adds the terminal GalNAc to H substance. The B gene encodes a UDP-Gal-specific Gal transferase that adds the Gal residue to H substance. Individuals of type AB possess both enzymes, and thus synthesize two oligosaccharide chains (Figure 53-7), one terminated by GalNAc and the other by Gal.

Anti-A antibodies are directed to the additional GalNAc residue present in the A substance, and anti-B antibodies are directed toward the additional Gal residue found in the B substance. Type O individuals harbor a frameshift mutation in the gene encoding the terminal glycosyltransferase that results in the production of an inactive protein. Thus, H substance is their ABO blood group substance.
This honeycomb of channels increases the surface area of the platelets, which are spheroidal at rest, thereby facilitating the secretion of various endocrine and coagulation factors (see Chapter 55). These factors are stored inside the platelets within densely packed secretory vesicles, called dense granules, which contain Ca**, ADP, and serotonin, and a-granules, which contain fibrinogen, fibronectin, platelet-derived growth factor, von Willebrand factor, or other coagulation factors that are released on receipt of an appropriate stimulus. Under normal circumstances, these small (2-m diameter), enucleated cells circulate at a density of 2 to 4 x 10° platelets per milliliter of blood. While platelets derive the majority of their energy from metabolizing glucose, their mitochondria enable them to generate ATP via the B-oxidation of fatty acids. The mechanisms by which platelets become activated to participate in formation of a clot are discussed in Chapter 55.
An h allele arises when a mutation in the portion of H locus that codes for the fucosyltransferase yields an inactive enzyme. While individuals of the heterozygous Hh genotype still can synthesize adequate levels of H substance, individuals of the homozygous hh genotype cannot. Since H substance is the precursor for A and B substances, all individuals carrying the hh genotype will have red blood cells of type O, referred to as the Bombay phenotype (Oh), regardless of whether or not they express an A, B, or both A and B terminal glycosyltransferase(s).

When megakaryocytes, the progenitors of red blood cells, are exposed to thrombopoietin they may fragment and form platelets (Figure 53-1). Like red blood cells, platelets lack a nucleus, but unlike erythrocytes they possess mitochondria, lysozymes, and a tubular network that forms an open canalicular system.

Abnormalities in platelet number or function can have serious physiologic consequences. For example, in acute coronary syndrome, the formation of enlarged, hyperreactive platelets results in an increased risk of blood clot formation in the circulation, known as thrombosis. The presence of larger than normal platelets also correlates with an increased frequency of myocardial infarction.
Immune thrombocytopenic purpura is an autoimmune disorder marked by depressed platelet counts (thrombocytopenia) caused by the generation of antibodies against the patient's own platelets. When platelets are decorated with antibodies on their surface, they are subject to clearance by splenic macrophages. In some instances, platelet autoantibodies will bind to differentiating megakaryocytes, depressing platelet production. Thrombocytopenia also can occur when persons who are homozygous for a mutant variant of glycoprotein IIb/II]a in which the leucine 33 is replaced by
proline receive blood from a donor that is homoor heterozygous for the wild-type form of this major platelet antigen. Exposure to the donor's platelets triggers the production of alloantibodies that attack not only the donated platelets, but the patient's endogenous platelets as well. In neonatal alloimmune thrombocytopenia, which affects roughly 1 in 200 term pregnancies, antibodies from the mother's circulation cross the placental barrier and attack platelets in the fetus' circulatory system. Thrombocytopenia also can be induced by drugs such as tamoxifen, ibuprofen, vancomycin, and many sulfonamides.
The symptoms of hemolytic-uremic syndrome, a disease of infants characterized by progressive kidney failure, include both thrombocytopenia and hemolytic anemia. By contrast, the abnormal bleeding associated with von Willebrand disease is caused by a genetic defect that compromises the ability of platelets to adhere to the endothelium, rather than a deficit in platelet number. Other bleeding disorders resulting from defects in platelet adherence include Bernard-Soulier syndrome (genetically inherited deficiency in glycoprotein 1b), and Glanzmann thrombasthenia (genetically inherited deficiency in the glycoprotein IIb/II]a complex).

The bases of the thalassemias and of many disorders of coagulation (see Chapter 55) have been greatly clarified by investigations utilizing gene cloning and DNA sequencing, while the study of oncogenes and chromosomal translocations has advanced our understanding of the leukemias. As discussed above, recombinant DNA technology has made available therapeutically useful quantities of erythropoietin and other growth factors.
The first pathophysiologic condition to be treated by gene therapy was a deficiency of adenosine deaminase. Lymphocytes are particularly sensitive to deficits in this enzyme. In 1990, Dr. William French Anderson introduced a new copy of the gene, carried on a retroviral vector, into a 4-year-old girl suffering from severe combined immunodeficiency (bubble boy disease). Although the patient is still required to take medications, the replacement gene has remained stable into adulthood.

™ Major causes of anemia include blood loss, deficiencies of iron, folate, and vitamin B,,, and various factors that cause hemolysis.
™ The shape of the red blood cell contributes to the efficiency of gas exchange and to its ability to undergo deformation when passing through capillaries.
@ The production of red cells and platelets is regulated by erythropoietin, thrombopoietin, and other cytokines.
@ Mature red blood cells, which lack internal organelles, are
glycolysis to
generate
ATP.
= 2,3-Bisphosphoglycerate mutase catalyzes the isomerization of the glycolytic intermediate 1,3-bisphosphoglycerate to form the 2,3-bisphosphoglycerate, which stabilizes T-state hemoglobin.
= Methemoglobin is unable to transport oxygen.
= Cytochrome b, reductase reduces the Fe** of methemoglobin to Fe*, restoring function.
@ The red cell contains a battery of cytosolic enzymes— superoxide dismutase, catalase, and glutathione peroxidase— that catalyze the neutralization of reactive oxygen species.
™
Deficiencies in the quantity or the activity of glucose-6phosphate dehydrogenase, which produces NADPH, constitute a major cause of hemolytic anemia.
™ Cytoskeletal proteins such as spectrin, ankyrin, and actin interacting with integral membrane proteins underlie the flexible biconcave shape of red blood cells.
™ Deficiencies or defects of spectrin can lead to hereditary
spherocytosis and
hereditary elliptocytosis,
both causes
of hemolytic
anemia.
= Band 4.1 protein facilitates the exchange of bicarbonate and chloride ions across erythrocyte membranes.
™ The ABO blood group substances of the red cell membrane are complex glycosphingolipids. The immunodominant sugar of A substance is N-acetylgalactosamine, whereas that of B substance is galactose. O substance contains neither of these sugar residues.
™ Platelets are small, enucleated fragments of larger precursor cells called megakaryocytes.
= When activated, platelets release effector molecules and

fibrinogen stored
in secretory
granules.
- ® von Willebrand disease, a bleeding disorder, is caused by a genetic mutation that impairs the ability of platelets to adhere.

- Alkrimi J, George L: Medical Diagnosis by Analysis of Blood Cell
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Israels SJ (editor): Mechanisms in Hematology, 4th ed. Core Health Sciences Inc, 2011.
Martin JE, Kristensen SD, Mathur A, et al: The causal role of megakaryocyte platelet hyperactivity in acute coronary
syndromes. Nat Rev Cardiol 2012;9:658.
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. Describe how white blood cells work in concert to combat infection and to
trigger an inflammatory response.
: phagocytosis. List the basic steps in elimination of infectious microorganisms by
Describe the role of chemotaxis in leukocyte function.
List the key components found within the granules of phagocytes and basophils and describe their primary functions.
List the reactive oxygen species produced during the respiratory burst.
oxidase system. Explain the basis for the physiologic effects caused by defects in the NADPH
Explain the molecular basis of type 1 leukocyte adhesion deficiency.
Describe how neutrophils and eosinophils entrap parasites using neutrophil extracellular traps (NETs).
Describe the role of the helper T cells in the production of new antibodies.
Define the term cytokine and describe the key characteristics of interleukins, interferons, prostaglandins, and leukotrienes.

White blood cells, or leukocytes, serve as key sentries and potent defenders against invading pathogens. Neutrophils, the most abundant type of white blood cell, ingest and destroy invading bacteria and fungi by a process known as phagocytosis, while eosinophils phagocytize larger parasites. Circulating monocytes migrate from the bloodstream to diseased tissues, where they differentiate into phagocytic macrophages. Granulocytes such as basophils and mast cells release stored effectors that attract additional leukocytes to the site of infection and trigger an inflammatory response. B lymphocytes generate and release protective antibodies with the assistance of T lymphocytes. Other lymphocytes, such as cytotoxic T cells and natural killer cells, target virally infected and malignantly transformed host cells.
Malignant neoplasms of blood-forming tissues, called leukemias, can lead to the uncontrolled production of one or more of the major classes of white blood cells. The hyperactivation of granulocytes during an allergic response can, in extreme cases, lead to anaphylaxis and death. Leukopenia, a depression in the production of white blood cells, can result from physical injury or infection of the bone marrow, chemotherapy, ionizing radiation, infection by the Epstein-Barr virus (mononucleosis), an autoimmune response (lupus), or the displacement of bone marrow cells by fibrous tissues (myelofibrosis). The resulting deficit in the levels of circulating leukocytes can leave the affected individual vulnerable to infection (immunocompromised).

The white blood cells, or leukocytes, are key participants in the acute inflammatory response, a multicomponent process that defends the body against infectious organisms and ameliorates the impact of tissue infection or morbidity. The principal steps in an inflammatory response include (1) an increase in vascular permeability, (2) entry of activated leukocytes into the tissues, (3) activation of platelets, and (4) spontaneous subsidence (resolution) if the invading microorganisms have been dealt with successfully. Basophils secrete hematologic
\Z
Histidine
Histamine

effectors such as histamines (Figure 54-1) that facilitate the accumulation of fluid within infected or damaged tissues as well as chemokines that attract additional neutrophils. The activated neutrophils encapsulate invading bacteria within membrane vesicles (phagocytosis) and destroy them using a combination of hydrolytic enzymes, reactive oxygen species (ROS), and antimicrobial peptides. Circulating monocytes are the precursors of phagocytic macrophages, which phagocytize infected and damaged host cells. Lymphocytes produce protective antibodies that target foreign invaders and tag them for elimination.
Leukocytes, unlike red blood cells and platelets, possess a full complement of internal organelles. However, the nuclei of many leukocytes exhibit marked deviations from the compact, spherical organelle typical of most eukaryotic cells. In monocytes, for example, the nuclei are unusually large and noticeably irregular in shape while in neutrophils, eosinophils, and other polymorphonuclear leukocytes they segment into multiple lobes.

Most white blood cells turn over rapidly and thus must be continually replaced. The lifetime of a circulating myeloid leukocyte, for example, ranges from a few hours to a few days, while most lymphocytes persist for only a few weeks in the blood. A notable exception to this pattern is memory lymphocytes, which may live for several years. The production of monocytes and granulocytes proceeds via the formation of a common myeloid progenitor, while differentiation of hematopoietic stem cells into lymphocytes proceeds via the formation of a common lymphoid progenitor (see Figure 53-1). The proliferation of hematopoietic stem cells and the determination of their ultimate fate is controlled by the concerted influences of multiple effector molecules. Stem cell growth factor, granulocyte-macrophage colony-stimulating factor, and interleukins 5 and 6, for example, stimulate the production of granulocytes (neutrophils, eosinophils, basophils) and monocytes, a process that proceeds via the formation of myeloid progenitor cells. Tumor necrosis factor a, transforming growth factor B,, and interleukins 2 and 7 promote the formation of lymphoid progenitor cells and their eventual maturation into B and T lymphocytes.

Leukocytes can be found throughout the body, migrating from the blood to sites of injury or infection in response to chemical signals, a process referred to as chemotaxis. Migration out of the circulation takes place via diapedesis, an amoeboid mechanism involving the cytoskeleton-mediated contortion of the cell (Figure 54-2). A thin pseudopod is extended between the cells of the capillary epithelium. Once anchored on the other side, cytoskeletal proteins squeeze the contents of the cell through the projection, which fills the distal end of the pseudopod to form a new, translocated cell body, leaving behind the deflated remains. Once within the tissues, locomotion proceeds via a similar, stepwise amoeboid mechanism.

Leukocytes are attracted to tissues by chemotactic factors such as chemokines, complement fragment C5a, small peptides
Diapedesis. Shown, from left to right, are the major steps in diapedesis, the process by which neutrophils and other traverse the capillary wall, whose cells are shown in red, in response to chemotactic signals. Cell nuclei are shown in purple and
FIGURE 54-2 leukocytes granules in green.
derived from bacteria (eg, N-formyl-methionyl-leucylphenylalanine), and several leukotrienes. These factors bind to one of several cell surface receptors that share similar transmembrane domains comprised of seven membrane-spanning a-helices. Because these receptors all are closely coupled with one or more heterotrimeric guanosine nucleotide-binding proteins (G-proteins), they are often referred to as G-proteincoupled receptors. Upon ligand binding, a signal transduction cascade is initiated in which G-proteins activate phospholipase C, which hydrolyses phosphatidylinositol 4,5-bisphosphate to produce diacylglycerols and the water-soluble second messenger inositol 1,4,5-triphosphate (IP,). The appearance of IP, triggers the release of Ca", leading to a transient increase in the level of cytoplasmic Ca'*. In neutrophils, the appearance of cytoplasmic Ca" activates the components of the actin-myosin cytoskeleton responsible for effecting cell migration and granule secretion. Diacylglycerol, together with Ca', stimulates protein kinase C and induces its translocation from the cytosol to the plasma membrane, where it catalyzes the phosphorylation of various proteins, including some involved in triggering the respiratory burst (see below).

Chemokines are small, generally 6 to 10 kDa, proteins secreted by activated white blood cells in order to attract additional neutrophils to a site of infection or injury. Chemokines can be divided into four subclasses based on the number and spacing of the cysteine residues that form the disulfide bonds that stabilize the protein's conformation (Figure 54-3). Type C chemokines are characterized by an intrachain disulfide bond formed by a pair of conserved cysteine residues. In addition to the conserved disulfide bond present in type C, the other three recognized chemokine groups possess a second disulfide bond. In type CC chemokines, one of the additional cysteine residues lies adjacent to the first of the first pair of universally conserved residues. In types CXC and CX,C, these cysteines are separated by one and three intervening amino acid residues, respectively. CX,C chemokines, the largest of the four types of cytokines, have a longer C-terminus that includes sites of covalent modification by glycosylation.

The adhesion of leukocytes to vascular endothelial cells is mediated by transmembrane glycoproteins of the integrin and selectin families (see the discussion of selectins in Chapter 46). Integrins consist of noncovalently associated a and a B® subunits, each of which contains an extracellular, transmembrane, and intracellular segment. The extracellular segments bind to various extracellular matrix proteins that possess Arg-Gly-Asp sequences, while the intracellular domains bind to cytoskeletal components such as actin and vinculin. Their ability to link the exterior of a cell with its interior enables integrins to link leukocyte responses (eg, movement and phagocytosis) to changes in the surrounding environment. Some integrins of specific interest with regard to neutrophils are listed in Table 54-1.
In type 1 leukocyte adhesion deficiency, the lack of the 8, subunit (also designated CD18) of LFA-1 and of two related integrins found in neutrophils and macrophages, Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18), impairs the ability of the affected leukocytes to adhere to vascular
FIGURE 54-3 Chemokines. This figure depicts the key structural features of type C, CC, CXC, and CX,C chemokines. The polypeptide chains are depicted in blue with their amino and carboxy termini marked by H,N and COOH, respectively. Key cysteine residues are denoted as Cys, conserved disulfide bonds at S-S, and spacer amino acids for types CXC and CX,C using X. Bound carbohydrate is depicted in green.

VLA-1 (CD49a), Cell = WBCs, others. VLA-1 (CD49a), Subunit = α1β1. VLA-1 (CD49a), Ligand = Collagen, laminin. VLA-1 (CD49a), Function = Cell-ECM adhension. VLA-5 (CD49e), Cell = WBCs, others. VLA-5 (CD49e), Subunit = α5β1. VLA-5 (CD49e), Ligand = Fibronectin. VLA-5 (CD49e), Function = Cell-ECM adhesion. VLA-6 (CD49f), Cell = WBCs, others. VLA-6 (CD49f), Subunit = α6β1. VLA-6 (CD49f), Ligand = Laminin. VLA-6 (CD49f), Function = Cell-ECM adhesion. LFA-1 (CD11a), Cell = WBCs. LFA-1 (CD11a), Subunit = αLβ2. LFA-1 (CD11a), Ligand = ICAM-1. LFA-1 (CD11a), Function = Adhesion of WBCs. Glycoprotein IIb/IIIa, Cell = Platelets. Glycoprotein IIb/IIIa, Subunit = αIIbβ3. Glycoprotein IIb/IIIa, Ligand = ICAM-2. Glycoprotein IIb/IIIa, Function = . Fibrinogen, fibronectin, von Willebrand factor, Cell = Platelet adhesion and aggregation. Fibrinogen, fibronectin, von Willebrand factor, Subunit = . Fibrinogen, fibronectin, von Willebrand factor, Ligand = . Fibrinogen, fibronectin, von Willebrand factor, Function = 

*CD, cluster of differentiation; ECM, extracellular matrix; ICAM, intercellular adhesion molecule; LFA-1, lymphocyte function-associated antigen 1; VLA, very late antigen. Note: A deficiency of LFA-1 and related integrins is found in type | leukocyte adhesion deficiency (OMIM 116920). A deficiency of platelet glycoprotein IIb/IIlb complex is found in Glazmann thrombasthenia (OMIM 273800), a condition characterized by a history of bleeding, a normal platelet count, and abnormal clot retraction. These findings illustrate how fundamental knowledge of cell surface adhesion proteins is shedding light on the causation of a number of diseases.
endothelial cells, impeding diapedesis. Since fewer white blood cells enter their infected tissues, affected individuals tend to suffer from recurrent bacterial and fungal infections.

via endocytosis (see Figure 40-21), then route them to the cell surface in association with a membrane protein called the major histocompatibility complex (MHC). The MHC serves as a scaffold for presenting potential antigens to surrounding lymphocytes in a form likely to stimulate the production of new antibodies.

White blood cells typically destroy invading microorganisms via phagocytosis (Figure 54-4). Phagocytic leukocytes recognize and bind target cells using receptors that recognize bacterial lipopolysaccharides or peptidoglycans. In most cases, however, infective pathogens are recognized indirectly, by the presence of antibodies or complement factors that have previously adhered to their surface (see Chapter 52). The process of tagging an invader with protective proteins to facilitate recognition by phagocytic leukocytes is called opsonization.
Receptor binding triggers dramatic alterations in the shape of the phagocyte, which proceeds to envelop the target cell until it is encased within an internalized membrane vesicle called a phagosome (phagolysosome). The encapsulated invader is then destroyed using a combination of hydrolytic enzymes (eg, lysozyme, proteases), antimicrobial peptides (defensins), and reactive oxygen species. These enzymes and toxins are stored in cytoplasmic vesicles known as granules, which then fuse with the phagosome (Table 54-2). Since these granules can be observed under a microscope, the cells that harbor them are referred to as granulocytes. Eventually, after digestion of the microbial invader and absorption of their component sugars, amino acids, etc, the phagosome migrates to the plasma membrane of the white blood cell, where it fuses and expels the remaining debris.
The components of this debris, which include fragments of proteins, oligosaccharides, lipopolysaccharides, peptidoglycans, and polynucleotides, provide an important source of antigens for stimulating the production of new antibodies. Helper T cells and other leukocytes absorb these materials

FIGURE 54-4 Phagocytosis. This figure depicts the destruction of an opsonized microorganism, shaded in ORANGE, by a neutrophil via phagocytosis. The multilobed nucleus of the neutrophil is shown in purple, secretory granules in green. The presence of an antibody or complement tag is indicated by a yellow triangle, with the corresponding cell surface receptor as a bright orange square. Cellular debris from the microorganism is represented as orange line segments. (A) The neutrophil binds an antigen molecule on the opsonized microbe via a receptor. (B) The neutrophil envelops the microbe. (C) Secretory granules fuse with the newly internalized phagosome, delivering their contents. (D) Granule-derived enzymes and cytotoxins destroy the microorganism. (E) The phagosome then fuses with the cell membrane, expelling any remaining debris.

Myeloperoxidase (MPO), Reaction Catalyzed or Function = H 2 O 2 + X - (halide) + H + → HOX + H 2 O where X - = Cl - , HOX = hypochlorous acid. Myeloperoxidase (MPO), Comment = Responsible for the green color of pus Genetic deficiency can cause recurrent infections. NADPH oxidase, Reaction Catalyzed or Function = 2O 2 + NADPH → 2O 2 • + NADP + H +. NADPH oxidase, Comment = Key component of the respiratory burst Deficient in chronic granulomatous disease. Lysozyme, Reaction Catalyzed or Function = Hydrolyzes link between N-acetylmuramic acid and N -acetyle- d -glucosamine found in certain bacterial cell walls. Lysozyme, Comment = Abundant in macrophages. Hydrolyzes bacterial peptidoglycans. Defensins, Reaction Catalyzed or Function = Basic antibiotic peptides of 20-33 amino acids. Defensins, Comment = Apparently kill bacteria by causing membrane damage. Lactoferrin, Reaction Catalyzed or Function = Iron-binding protein. Lactoferrin, Comment = May inhibit growth of certain bacteria by binding iron and may be involved in regulation of proliferation of myleoid cells. Elastase Collagenase Gelatinase Cathepsin G, Reaction Catalyzed or Function = Proteases. Elastase Collagenase Gelatinase Cathepsin G, Comment = Abundant in phagocytes; Breakdown protein components of infectious organisms; Generate fragments for antigen presentation

The three principal classes of phagocytic leukocytes are neutrophils, eosinophils, and macrophages. Neutrophils, which comprise roughly 60% of the white blood cells present in the circulation, phagocytize bacteria and small eukaryotic microorganisms such as fungi. The less numerous eosinophils, which make up 2 to 3% of the leukocytes in the blood, ingest larger eukaryotic microorganisms such as paramecia. Macrophages are derived from monocytes, which comprise about 5% of the leukocytes in the blood. Monocytes migrate from the bloodstream into tissues throughout the body where, upon receipt of a stimulus, they differentiate to form macrophages. While macrophages can ingest invading microbes, the signature function of these large phagocytes is to destroy human host cells that have been compromised by infection, malignant transformation, or programmed cell death, also known as apoptosis. These functionally compromised cells are recognized by the appearance of aberrant proteins and oligosaccharides on their surface. Precocious activation of macrophages is associated with the etiology of many degenerative diseases such as osteoporosis, atherosclerosis, arthritis, and cystic fibrosis. They also can facilitate the metastasis of cancer cells.

Phagocytes employ
ROS
such as
O,”,
H,O,,
OH’, and
HOCI
(hypochlorous acid)
and enzymatic
tion as
a
arsenal major
used of
the various
ROS
after internalization
electrons component
to destroy
takes of
derived from
oxygen consumption
Producing of
the ingested
place shortly
an chemical
cells.
(15-60
encapsulated
NADPH.
cell, using
The accompanying
has the
large pentose
phosphate by
the phagocyte’s
been termed
Produc- seconds)
O, and
surge the
respiratory of
pathway
NADPH
(see heavy
reliance in
burst.
required via
Chapter the
20)
on is
facilitated aerobic
generate ATP, a consequence of the low number of mitochondria contained within them. The first step in the formation of microbicidal ROS during the respiratory burst is the synthesis of superoxide, which is catalyzed by the NADPH oxidase system. Catalysis proceeds via a two-step mechanism, the reduction of molecular oxygen to form superoxide (Table 54-2):

followed by the spontaneous dismutation of hydrogen peroxide from two molecules of superoxide:

The NADPH oxidase system is comprised of cytochrome b,,. a plasma membrane-associated heterodimer, and two cytoplasmic polypeptides of 47 and 67 kDa. Upon activation, the cytoplasmic peptides are recruited to the plasma membrane where they associate with cytochrome b.., to form the active complex. Flux through the pentose phosphate cycle, the cell's primary source of NADPH, also increases markedly during phagocytosis. The cell is protected from any superoxide that may escape from the phagosomes by superoxide dismutase, which catalyzes the transformation of two superoxide radical anions into H,O, and O,. The hydrogen peroxide can be used as a substrate for myeloperoxidase (see below) or disposed of by the action of glutathione peroxidase or catalase.

The formation of hypohalous acids during the respiratory burst is catalyzed by the enzyme myeloperoxidase.
HO, +X" +H*
—————___——>
_
HOX + H,0
(xX =CI, Br, or SCN'; HOX = hypohalous acid)
Present in large amounts in neutrophil granules, this enzyme uses H,O, and to oxidize Cl and other halides to produce HOC] and other hypohalous acids. HOCI, the active ingredient of household liquid bleach, is a powerful oxidant that is highly microbicidal. When used to sterilize wounds, it reacts with primary or secondary amines present to produce various nitrogen-chlorine derivatives. These chloramines are less powerful oxidants than HOCI, allowing them to act as microbicidal agents without causing damage to the surrounding tissue.

Functionally deleterious mutations in the genes encoding any of the four polypeptides of the NADPH oxidase system can cause chronic granulomatous disease. The resulting impairment of the production of ROS undermines the ability of phagocytic leukocytes to kill ingested pathogens. Although relatively uncommon, persons suffering from this condition experience recurrent infections. They also form granulomas (chronic inflammatory lesions) in the skin, lungs, and lymph nodes as a means to wall off invading pathogens. In some cases, relief can be provided by the administration of gamma interferon, which may increase transcription of the 91-kDa component of cytochrome b,,...

In addition to ingesting small microorganisms such as bacteria by phagocytosis, neutrophils and eosinophils can assist in the elimination of larger invaders by trapping them within webs called neutrophil extracellular traps or NETs (Figure 54-5). The strands for these NETs are composed of polynucleotide strands generated by the dispersal, or decondensation, of a neutrophil's chromosomal DNA. This process involves rupture of the nuclear membrane and the disruption of the favorable charge-charge interactions that normally stabilize chromatin. Dissolution of histonepolynucleotide complexes is promoted by the enzyme peptidylarginine deiminase, which catalyzes the deimination of the strongly basic side chains of arginine residues to form neutral citrulline residues (Figure 54-6). Some chromatin proteins do remain associated with the DNA, forming cross-links between the polynucleotide strands. Granule membranes also rupture at this time, releasing their contents into the cytoplasm where they can bind to the decondensing polynucleotide strands, decorating the DNA with granule-derived proteases, antimicrobial peptides, and other factors. Eventually, the neutrophils lyse, unleashing their NETs upon invading parasites in order to immobilize them and hinder their spread.
FIGURE 54-5 Trapping parasites using NETs. The figure depicts the basic stages in the formation and deployment of a DNA-based web by a neutrophil or eosinophil to trap a parasitic microorganism. (A) Resting neutrophil. The multilobed nucleus is shown in hatched purple, intracellular granules in green, and granule enzymes and cytotoxins as orange circles and yellow triangles. (B) Upon stimulation, the membranes encasing the nucleus and granules rupture, releasing enzymes, cytotoxins, and strands of DNA (purple) from decondensing chromosomes. (C) The DNA strands form a mesh that fills the interior of the cell to which some granule-derived proteins adhere. (D) The neutrophil lyses, releasing its DNA-protein web, which entraps the parasite (orange) against surface of the epithelium (hatched).

Macrophages and other phagocytes produce numerous proteases (Table 54-2), several of which can hydrolyze elastin, various types of collagen, and other proteins present in the extracellular matrix. Although small amounts of elastase and
FIGURE 54-6 Citrullination. The enzyme peptidyl arginine deiminase displaces one of the imino groups (red) on the side chain of arginine by an oxygen atom (blue) derived from water. The net result is to replace a positive charge provided by the protonated arginine side chain by an amide, which is neutral.
other proteinases leak out into normal tissues, their activities are normally kept in check by a number of antiproteinases present in plasma and the extracellular fluid (see Chapter 52). One of these, a,-macroglobulin, forms a noncovalent complex with certain proteinases, thereby inhibiting their activity. A genetic defect that permits elastase to act upon pulmonary tissue unchecked by inhibitors of proteolysis such as a,-antiproteinase inhibitor (a,-antitrypsin) contributes significantly to the causation of emphysema.
Elevated levels of chlorinated oxidants formed during inflammation can tilt the balance between proteinases and antiproteinases in favor of the former. For instance, some of the proteinases listed in Table 54-2 are activated by HOCI, while several of the countervailing antiproteinases are inactivated by this same hypohalous acid. In addition, these inhibitory proteins can themselves be degraded by proteases. For example, tissue inhibitor of metalloproteinases and a,-antichymotrypsin can be hydrolyzed by elastase while a, -antiproteinase inhibitor can be hydrolyzed by collagenase and gelatinase. While an appropriate balance between proteinases and antiproteinases is generally maintained, in certain instances, such as when inadequate drainage leads to the accumulation of large numbers of neutrophils, considerable tissue damage can result.

The development of the immune and inflammatory responses by injured or infected tissues requires the coordinated action of leukocytes and other cells. Much of this coordination is accomplished by secreting a diverse set of small (< 25 kDa) proteins, termed cytokines, that includes interleukins, interferons, and chemokines.
The more than three dozen known interleukins derive their name from the cells in which they are synthesized and from which they are secreted. They are generally designated by the class abbreviation IL followed by an identifying number, eg, IL1, IL3, IL22. The interferons (IFN), on the other hand, derive their name from their ability to inhibit, or interfere, with the replication of infecting viruses. Approximately 10 distinct families of interferons have been identified in animals to date. Chemokines attract and activate migrating leukocytes to a site of injury or infection. Most cytokines are glycosylated. In general, they stimulate both the leukocytes from which they are secreted (autocrine signaling) as well as other types of leukocytes (paracrine signaling). Historically, cytokines have been distinguished from hormones by their close association with immunity and inflammation.
Leukocytes also secrete lipid mediators, called eicosanoids, produced by the oxidation of arachidonic acid (see Chapter 15). These lipid mediators fall into two broad classes, leukotrienes and prostaglandins. Leukotrienes are characterized by the presence of a set of three conjugated carbon-carbon double bonds. Several incorporate the amino acid cysteine into their structure. Prostaglandins, which were first isolated from the prostate gland, contain 20 carbon atoms and are distinguished by their common five-membered ring.
Histamine (Figure 54-1), which is synthesized by decarboxylating the amino acid histidine, is secreted in large amounts by activated basophils and mast cells. Histamine works with other hematologic factors, such as heparin and eicosanoids, to maintain blood flow to the site of injury or infection and stimulate the accumulation of fluid (edema). The resulting inflammation enhances an immune response as the accumulated fluid facilitates leukocyte migration.

Lymphocytes make up approximately 30% of the leukocytes present in the blood. By virtue of their capacity to produce novel protective antibodies against newly encountered antigens (see Chapter 53), lymphocytes form the cornerstone of the body's adaptive immune system. The classification of lymphocytes into B and T types originally was based on the identity of the tissues in which each form completed their maturation. In avian species, the B lymphocytes (B cells) are processed in the bursa of Fabricius. The B cells in humans, which lack this organ, mature in the bone marrow. Maturation of T lymphocytes (T cells) takes place in the thymus. B lymphocytes secrete the soluble antibodies present in bodily humors, for example, plasma and interstitial fluids, and are therefore said to confer humoral immunity.
Lymphocytes that have yet to be stimulated to produce immunoglobulins are said to be naive. Synthesis of a new antibody can be triggered by several mechanisms. Lymphocytes can bind directly to foreign invaders via one of the many receptors present on their surface configured to bind bacterial glycoproteins, lipopolysaccharide, or peptidoglycan. Alternatively, the lymphocyte can be activated by encountering an antigen that has been presented on the surface of another white blood cell in association with the major histocompatibility complex. Macrophages, neutrophils, and phagocytic lymphocytes called plasma cells all display or present fragments of macromolecules they have destroyed by phagocytosis. In addition, helper T cells present antigens on their surface, including debris ejected by phagocytes, that has been ingested by endocytosis. Helper T cells also serve as "cellular switchboards,' coordinating the immune response by receiving, processing, and sending signals from and to other components of the immune system.

Cytotoxic T cells recognize proteins that appear on the surface of host cells as a consequence of viral infection or oncogenic transformation. Once bound, they induce the lysis of the target cell using perforins, proteins that form channels in the plasma membrane, and proteases called granzymes, which mimic the action of the cathepsin proteases that normally trigger programmed cell death (apoptosis). Natural killer cells resemble cytotoxic T cells, but contain granules holding additional toxic chemicals to aid in their attack.

The elimination of infectious microorganisms involves the cumulative actions of multiple types of leukocytes, including lymphocytes, phagocytes, and basophils.
= White blood cells communicate using secreted effector molecules such as chemokines, prostaglandins, leukotrienes, interleukins, and interferons.
= Leukocytes migrate from the blood to the tissues in response
to specific chemical attractants, a process termed chemotaxis.
=
The amoeboid migration of leukocytes from blood to the tissues relies on cytoskeletally mediated cell flexibility and deformation.
= Basophils secrete histamine and heparin, which facilitate the migration of leukocytes by inducing fluid accumulation at a
site of infection or injury.
™ Integrins mediate the adhesion of white blood cells to the vascular endothelium, the first step in migration toward infected tissues.
@ Phagocytes internalize invading microorganisms inside membrane vesicles called phagosomes.
= Destruction of phagocytized microorganisms is accomplished using a combination of reactive oxygen species (the respiratory burst), hydrolytic enzymes, and cytotoxic peptides.
= Mutations in proteins of the NADPH oxidase system cause
chronic granulomatous disease.
- = Neutrophils and eosinophils defend against large parasites by immobilizing them within webs comprised of strands of chromosomal DNA.
™ Decondensation of chromosomal DNA is facilitated by citrullination of arginine side chains on histones.
= Lymphocytes produce protective immunoglobulins (antibodies) that confer humoral immunity.
™ Phagocytes and helper T cells stimulate the production of new antibodies by presenting fragments of pathogen-derived macromolecules on their surface.
™ Cytotoxic T cells and natural killer cells recognize and destroy
host
cells
that
display
cell
surface
proteins
and
glycolipids
characteristic of viral infection or malignant transformation.

1. Briefly describe the mode of action of nitroglycerin, acommon
agent for treating angina.
2. Patients being treated for heart failure oftentimes exhibit
decreased expression and defective regulation of SERCA2a, the
principal Ca**-ATPase of the sarcoplasmic reticulum. Explain
how defects in this protein might contribute to deterioration in cardiac function.
3. Select the one of the following that is NOT CORRECT:
A. The troponin system regulates contraction of smooth
B. Muscle contraction takes place via a sliding filament
muscle.
mechanism.
. Myosin light-chain kinase phosphorylates the regulatory
light chains in the myosin head domain.
F-actin is formed via the polymerization of G-actin.
Ca’* both activates muscle contraction and stimulates its
own removal by activating the Ca’ ATPase.
4. A patient anesthetized using a halothane compound exhibits
a marked rise in body temperature, a behavior indicative of
malignant hyperthermia (HT). Select the one of the following statements that is NOT CORRECT:
A. MH can arise from mutations that alter the amino acid
B. MH can arise from mutations that alter the amino acid
Cc. The muscle rigidity that occurs during MH is triggered
sequence of the Na*-K*-ATPase.
sequence of the ryanodine-sensitive Ca** release channel.
by the presence of high concentrations of Ca®* in the cytoplasm.
MH can arise from mutations that alter the amino acid
sequence of the voltage-gated, slow K-type Ca®* channel.
MH can be treated by intravenous administration of dantrolene, which inhibits release of Ca** from the sarcoplasmic reticulum into the cytosol.
5. Select the one of the following statements that is NOT
CORRECT:
A. Fast-twitch fibers rely heavily on creatine phosphate to
B. Slow-twitch fibers appear red because they contain
regenerate ATP.
Fast-twitch fibers contain relatively few mitochondria.
Marathoners try to increase the quantity of glycogen in their
muscles
by
eating
carbohydrate-rich
meals
before
an
event

(carbo loading).
Skeletal muscle serves as the major reserve of protein in the
body.
6. Select the one of the following that is NOT a feature of the
contractile cycle in striated muscle:
A. Binding of Ca** to troponin C uncovers the myosin-binding
B. The power stroke is initiated by the release of P, from the
sites on actin.
actin-myosin-ADP-P, complex.
10. Describe the role of haptoglobin in the protection of the kidneys from the potentially damaging effects of extracorpuscular hemoglobin.
ll. Briefly describe how activation of cytidine deaminase helps
12. Select the one of the following statements that is NOT CORRECT:
C. Release of ADP from the actin-myosin-ADP complex is
accompanied by a large change in the conformation of myosin’s head domain (relative to its tail domain).
D. The binding of ATP by myosin increases its affinity for
actin.
E.
Rigor mortis results for the inability of actin to release from
the actin-myosin complex when cells are deficient in ATP.
. Select the one of the following that does NOT serve as a major
energy reserve for replenishing ATP in muscle tissue:
Glycogen
Creatine phosphate
ADP (in conjunction with adenylyl kinase)
BOOP
CORRECT:
A. The drugs colchicine and vinblastine inhibit microtubule
assembly.
B. Mutations affecting keratin can lead to blistering.
C. Mutations in the gene encoding lamin A and lamin C cause
progeria (accelerated aging).
D. a- and f-tubulin are the major components of stress fibers.
E.
Molecular
motors
such
as
dynein,
kinesis,
and
dynamin
power ciliary movement, vesicle transport, and endocytosis.
CORRECT:
A. The major function of the Ca’* channels in cardiomyocytes

is
to
admit
extracellular
calcium
ions
into
the
cell
in
order
to
trigger Ca®*-induced Ca** release from the SR.
B. Digitalis increases the forcefulness of cardiac contractions by raising the level of intracellular Na*.
C. Certain types of muscular dystrophies are causes by
mutations in enzymes called glycosyltransferases.
D. Dantrolene relaxes skeletal muscle by inhibiting the release of Ca®* from the SR.
E. In the SR, Ca** is bound to a specific Ca**-binding protein called calmodulin.
generate immunoglobulins with unique antigen-binding sites.
A. Interleukin 1 stimulates the production of acute-phase
proteins.
B. Iron must be reduced to the ferrous (Fe?*) state in order to
be recovered via the transferrin cycle.
C. Many complement proteins are zymogens.
D. The type 2 transferrin receptor (TfR2) functions primarily
as an iron sensor.
E. Mannose-binding lectin binds carbohydrate groups present on the surface of invading bacteria.
CORRECT:
14. Select the one of the following statements that is NOT CORRECT:
15. You encounter a 50-year-old woman in the clinic who is pale
16. Select the one of the following that is NOT a potential cause of
17. Select the one of the following statements that is NOT CORRECT:
18. Explain the linkage how a deficiency in glucose-6-phosphate
19. Select the one of the following statements that is NOT CORRECT:
A. Albumin is synthesized as a proprotein.
B. Albumin is stabilized by multiple intrachain disulfide
Cc. Albumin is a glycoprotein.
D. Albumin facilitates the movement of fatty acids through the
E. Albumin is the major determinant of plasma osmotic
bonds.
pressure.
A. Wilson disease can be treated using copper chelators such as
B. Wilson disease is characterized by copper toxicosis
penicillamine.
(abnormally high levels of copper).

. Wilson disease is caused by mutations in the gene encoding
ceruloplasmin.
Albumin facilitates the movement of sulfonamide drugs
through the circulation.
Albumin can be lost from the body if the intestinal mucosa
becomes inflamed.
and
tired.
You
suspect
that
she
is
suffering
from
iron
anemia and prescribe a series of laboratory tests. Select the one
of
the
following
potential
test
outcomes
that
would
NOT
be
consistent with your provisional diagnosis:
BOOP
Lower than normal levels of red cell protoporphyrin
Increased saturation of transferrin
Increased expression of TfR
Increased levels of plasma hepcidin
Decreased levels of hemoglobin
amyloidosis:
A. Accumulation of B,-macroglobulin
B. Deposition of fragments derived from immunoglobulin
Cc. Accumulation of degradation products of serum amyloid A
D. Presence of mutationally altered forms of transthyretin
E. Amylase deficiency
light chains
A. All immunoglobulins contain at least two heavy-chain
B. Immunoglobulin polypeptide chains are linked together by disulfide bonds.
Cc . Immunoglobulins are multivalent.
D. Immunoglobulins are glycosylated.
E. . Immunoglobulins are primary components of the body’s
polypeptides and two light-chain polypeptides.
innate immune system.
within
erythrocytes
can
lead
to
hemolytic
anemia.
A. The high surface area of biconcave red blood cells facilitates
B. Hereditary elliptocytosis can be caused by defects in or a
gas exchange.
deficiency of spectrin.
C.
The diameter of red blood cells exceeds that of many
peripheral capillaries.
D. Protein 4.1 helps link the erythrocyte cytoskeleton to
proteins in the cell’s plasma membrane.
E. In order to pass through narrow capillaries, red blood cells
must be squeezed into a compact, spherical shape.

20. Select the one of the following statements that is NOT
CORRECT:
A. Red blood cells contain high levels of superoxide dismutase.
B. Aand B substances are formed by the addition of fucose
and N-acetylglucosamine, respectively, to H substance.
C. Platelets generate ATP exclusively via glycolysis.
D. Mature red blood cells are devoid of internal organelles.
E. Erythrocyte membranes contain high levels of the Band 3 anion exchange protein.
CORRECT:
A. Erythropoietin stimulates the formation of red blood cells
from hematopoietic stem cells.
B. Multipotent stem cells are able to differentiate into cells of a closely related type.
C. Carbonic anhydrase increases the capacity of red blood cells
to transport CO,.
D. GLUT 1 mediates the active transport of glucose into
E. Hypoxia stimulates the production of erythropoietin by the kidneys.
22. A patient recently exposed to aniline displays bluish
discoloration of their skin and mucous membranes. Select a
plausible provisional diagnosis from the list below:
Methemoglobinemia
Hereditary hemochromatosis
5q-syndrome
Immune thrombocytopenic purpura
Glanzmann thrombasthenia
BOOP
CORRECT:
A. The accumulation of fluid at a site of infection (edema)
facilitates
leukocyte migration.
B. Type 1 leukocyte adhesion deficiency is caused a lack of the 6, subunit an integrin designated LFA-1.
C.
The components of the complement cascade circulate
though the plasma as inactive zymogens.
D. Leukocytes are recruited to a site of infection by chemotaxis
toward
the
sources
of
epinephrine.
E. Neutrophils can trap large pathogens in NETS constructed,
in part, from strands of chromosomal DNA.
24.

A. Interleukins are key mediators of leukocyte production.
B. Lymphocytes produce protective antibodies.
C. Monocytes can be found in tissues throughout the body.
D. The hematologic factor histamine is synthesized by the
deamination of the amino acid histidine.
E. The term polymorphonuclear refers to leukocytes
possessing a segmented nucleus.
666
SECTION X_
Special
Topics
(B)
25. Select the one of the following statements that is NOT
CORRECT:
A. Phagocytes destroy ingested bacteria using reactive oxygen
B. Chronic granulomatous disease is caused by a deficiency in myeloperoxidase activity.
species and hydrolytic enzymes.
NADPH serves as the primary source of electrons for
generating ROS during the oxidative burst.
Neutrophils aid in the elimination of some parasites by
enmeshing them in NETs formed from their chromosomal
DNA.
Chemokines are stabilized by the formation of intrachain
disulfide
bonds.
26. Select the one of the following statements that is NOT

A. Activated leukocytes secrete lipid mediators called
B. Neutrophils facilitate production of protective antibodies
interferons.
by presenting fragments of phagocytized microbes on their surface in association with the major histocompatibility
complex (MHC).
Cytotoxic T cells use perforins to lyse infected cells.
Soluble antibodies are released into the plasma primarily by
B lymphocytes.
Emphysema can arise from the action of elastase and other
granule-derived proteases on pulmonary tissue.

A. The great majority of mitochondrial proteins are encoded
B. Ran proteins, like ARF and Ras proteins, are monomeric
by the nuclear genome.
GTPases.
. One cause of Refsum disease is mutations in genes encoding
peroxisomal proteins.
Peroxisomal proteins are synthesized on cytosolic
polyribosomes.
Import of proteins into mitochondria involves proteins
known as importins.
28. Select the one FALSE statement:
A. N-terminal signal peptides directing nascent proteins to the
ER membrane contain a hydrophobic sequence.
Posttranslational translocation of proteins to the ER does
not occur in mammalian species.
The SRP contains one RNA species.
N-glycosylation is catalyzed by oligosaccharide: protein
transferase.
Type I membrane proteins have their N-termini facing the
lumen of the ER.

A. Chaperones often exhibit ATPase activity.
B.
C. Ubiquitin is a small protein involved in protein degradation by lysosomes.
Protein
disulfide
isomerase
and
peptidyl
prolyl
isomerase
are enzymes involved in helping proteins fold properly.
Mitochondria contain chaperones.
Retrotranslocation across the ER membrane is involved in
helping dispose of misfolded proteins.
30. Select the one FALSE statement:
31. Which one of the following types of protein does NOT act as a
32. Select the one FALSE statement:

A. Rab is a small GTPase involved in vesicle targeting.
B. COPII vesicles are involved in anterograde transport of cargo from the ER to the ERGIC or Golgi apparatus.
C. Brefeldin A prevents GTP binding to ARF, and thus inhibits
D. Botulinum toxin B acts by cleaving synaptobrevin,
E. Furin converts preproalbumin to proalbumin.
formation of COPI vesicles.
inhibiting release of acetylcholine at the neuromuscular
junction.
GTPase?
BOOP
ADP ribosylation factor (ARF)
Rab proteins
N-ethylmaleimide-sensitive factor (NSF)
Sarl
Ran proteins
> Collagen has a triple helical structure, forming a right-hand superhelix.
Boas Proline and hydroxyproline confer rigidity on collagen. Collagen contains one or more O-glycosidic linkages. Collagen lacks cross-links.
Deficiency of vitamin C impairs the action of prolyl and
lysyl hydroxylases.
A. Elastin contains hydroxyproline, but not hydroxylysine.
B. Elastin contains cross-links formed by desmosines.
C. No genetic diseases due to abnormalities of elastin have as
D. Unlike collagen, there is only one gene encoding elastin.
E. Elastin does not contain any sugar molecules.
yet been identified.
A. Marfan syndrome is due to mutations in the gene encoding
B. All subtypes of Ehlers-Danlos syndrome are due to
fibrillin-1, a major constituent of microfibrils.
mutations affecting the genes encoding the various types of collagen.
Laminin is found in renal glomeruli along with entactin,
type IV collagen, and heparin or heparan sulfate.
Mutations affecting type IV collagen can cause serious renal
disease.
Mutations in the collagen 1A1 gene can cause osteogenesis
A. Most but not all GAGs contain an amino sugar and a uronic
acid.
All GAGs are sulfated.

GAGs are built up by the actions of glycosyltransferases
using sugars donated by nucleotide sugars.
Glucuronic acid can be converted to iduronic acid by an
epimerase.
The proteoglycan aggrecan contains hyaluronic acid,
keratan sulfate, and chondroitin sulfate.
- 36. A male infant is failing to thrive and, on examination, is noted
to have
hepatomegaly and
splenomegaly, among
other findings.
Urinalysis reveals
the presence
of both
dermatan sulfate
and heparan sulfate. You suspect the patient has Hurler syndrome.
From the following list, select the enzyme that you would wish to have assayed to support your diagnosis:
BOOP
6-Glucuronidase
a-L-Iduronidase
a-N-Acetylglucosaminidase
Neuraminidase
37. You see a child in clinic who is well below average height.
You
note
that
the
child
has
short
limbs,
normal
trunk
size,
macrocephaly,
and
a
variety
of
other
skeletal
abnormalities.
You suspect that the child has achondroplasia. Select from the
following list the test that would best confirm your diagnosis:
A. Measurement of growth hormone
B. Assays for enzymes involved in the metabolism of GAGs
Cc. Tests for urinary mucopolysaccharides
D.
E. Gene tests for abnormalities of growth hormone
. Gene tests for abnormalities of the fibroblast growth factor
receptor 3 (FGFR3)

- . disease. Understand the significance of hemostasis and thrombosis in health and
Outline the steps leading to platelet activation.
Identify antiplatelet drugs and their mode of inhibition.
Outline the pathways of coagulation that result in the formation of fibrin.
Identify the vitamin K-dependent coagulation factors.
Provide examples of genetic disorders that lead to bleeding.
Describe the process of fibrinolysis.

Fundamental aspects of platelet biology are described in this chapter as well as basic aspects of the proteins of the bloo coagulation system and of fibrinolysis. Hemorrhagic and thrombotic states can cause serious medical emergencies, and thromboses in the coronary and cerebral arteries are major causes of death in many parts of the world. Rational management of these conditions requires a clear understanding of the bases of platelet activation, blood coagulation, and fibrinolysis.
In hemostasis, there is initial vasoconstriction of the injured vessel, causing diminished blood flow distal to the injury. Then, hemostasis and thrombosis share three phases:

Hemostasis is the cessation of bleeding from a cut or severed vessel, whereas thrombosis occurs when the endothelium lining blood vessels is damaged or removed (eg, upon rupture of an atherosclerotic plaque). These processes involve blood vessels, platelet aggregation, and plasma proteins that cause formation or dissolution of platelet aggregates and fibrin.
1. Formation of a loose and temporary platelet aggregate at the site of injury. Platelets bind to collagen at the site of vessel wall injury, form thromboxane A, (TxA,), and release ADP, which activate other platelets flowing by the vicinity of the injury. (The mechanism of platelet activation is described below.) Thrombin, formed during coagulation at the same site, causes further platelet activation. Upon activation, platelets change shape and, in the presence of fibrinogen and/or von Willebrand factor, aggregate to form the hemostatic plug (in hemostasis) or thrombus (in thrombosis).
2. Formation ofa fibrin mesh that binds to the platelet aggregate forming a more stable hemostatic plug or thrombus.
3. Partial or complete dissolution of the hemostatic plug or thrombus by plasmin.

Three types of thrombi or clots are distinguished. All three contain fibrin in various proportions.
1. The white thrombus is composed of platelets and fibrin and is relatively poor in erythrocytes. It forms at the site of an injury or abnormal vessel wall, particularly in areas where blood flow is rapid (arteries).
2. The red thrombus consists primarily of red cells and fibrin. It morphologically resembles the clot formed in a test tube and may form in vivo in areas of retarded blood flow or stasis (eg, veins) with or without vascular injury, or it may form at a site of injury or in an abnormal vessel in conjunction with an initiating platelet plug.
3. A third type is fibrin deposits in very small blood vessels or capillaries.
We shall first describe some of the aspects of the involvement of platelets and blood vessel walls in the overall process. Then, we shall describe the coagulation pathway leading to the formation of fibrin. This separation of platelets and clotting factors is artificial since both play intimate and often mutually interdependent roles in hemostasis and thrombosis; this strategy facilitates description of the overall processes involved.

Platelets normally circulate in an unstimulated disk-shaped form. During hemostasis or thrombosis, platelets become activated and help form hemostatic plugs or thrombi (Figure 55-1). Three major steps are involved: (1) adhesion to exposed collagen in blood vessels; (2) release (exocytosis) of the contents of their storage granules; and (3) aggregation.
Platelets adhere to collagen via specific receptors on the platelet surface, including the glycoprotein complexes GPla-IIa (a2B1 integrin; see Chapter 54) and GPIb-IX-V, and GPVI. The binding of GPIb-IX-V to collagen is mediated via von Willebrand factor; this interaction is especially important in platelet adherence to the subendothelium under conditions of high shear stress that
occur in
small vessels
and partially
stenosed arteries.
Platelets that are bound to collagen change shape and spread out on the subendothelium. These adherent platelets release the contents of their storage granules (the dense granules and the alpha granules); some of the molecules released amplify the responses to vessel wall injury. Granule release is also stimulated by thrombin.

Thrombin, formed from the coagulation cascade (described below), is the most potent activator of platelets and initiates activation by interacting with its receptors protease-activated receptor-1 (PAR-1), PAR-4, and GPIb-IX-V on the platelet plasma membrane (Figure 55-1A). The further events leading to platelet activation upon binding to PAR-1 and PAR-4 are examples of outside-in transmembrane signaling, in which a chemical messenger outside the cell generates effector molecules inside the cell (see Chapter 42). In this instance, thrombin acts as the external chemical messenger (stimulus or agonist). The interaction of thrombin with its G-protein— coupled receptors PAR-1 and PAR-4 stimulates the activity of an intracellular phospholipase CB (PLC6). This enzyme hydrolyzes the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP,, a polyphosphoinositide) to form the two internal effector molecules (1,2-diacylglycerol [DAG] and 1,4,5-inositol trisphosphate [IP,]; see Figures 42-6 and 42-7). Hydrolysis of PIP, is also involved in the action of many hormones and drugs. DAG stimulates protein kinase C, which phosphorylates the protein pleckstrin (47 kDa). This results in aggregation and release of the contents of the storage granules. ADP released from dense granules can also activate platelets via its specific G-protein-coupled receptors (Figure 55-1A), resulting in aggregation of additional platelets. IP, causes release of Ca** into the cytosol mainly from the dense tubular system (or residual smooth endoplasmic reticulum from the megakaryocyte), which then interacts with calmodulin and myosin light-chain kinase, leading to phosphorylation of the light chains of myosin. These chains then interact with actin, causing changes of platelet shape.

Collagen-induced activation of a platelet cytosolic phospholipase A, (cPLA,) by increased levels of intracellular Ca?* results in liberation of arachidonic acid from platelet membrane phospholipids, leading to the formation of TxA, (see Chapters 21 and 23). TxA, in turn, by binding to its specific G-protein-coupled receptor, can further activate PLCB, promoting platelet aggregation (Figure 55-1A).
All of the aggregating agents, including thrombin, collagen, ADP, and others such as platelet-activating factor, via an inside-out signaling pathway, modify the platelet surface glycoprotein complex GPIIb-IIIa (alIbB3 integrin; see Chapters 54) so that the receptor has a higher affinity for fibrinogen or von Willebrand factor (Figure 55-1B). Molecules of divalent fibrinogen or multivalent von Willebrand factor then link adjacent activated platelets to each other, forming a platelet aggregate. von Willebrand factor-mediated platelet aggregation occurs under conditions of high shear stress. Some agents, including epinephrine, serotonin, and vasopressin, exert synergistic effects with other aggregating agents.
Activated platelets, besides forming a platelet aggregate, accelerate the activation of the coagulation factor X and prothrombin by exposing the anionic phospholipid phosphatidylserine on their membrane surface (see below for more detail). Polyphosphate, released from the dense granules, accelerates factor V activation and also accelerates factor XI activation by thrombin.

The endothelial cells in the walls of blood vessels make important contributions to the overall regulation of
FIGURE 55-1 (A) Diagrammatic representation of platelet activation by collagen, thrombin, thromboxane A, and ADP, and inhibition by prostacyclin. The external environment (Out), the plasma membrane, and the inside of a platelet (In) are depicted from top to bottom. Platelet responses include, depending on the agonist, change of platelet shape, release of the contents of the storage granules, and aggregation. (AC, adenylyl cyclase; cAMP, cyclic AMP; COX-1, cyclooxgenase-1; cPLA,, cytosolic phospholipase A,; DAG, 1,2-diacylglycerol; GP, glycoprotein; IP, prostacyclin receptor; IP,, inositol 1,4,5-trisphosphate; P2Y,, P2Y,,, purinoceptors; PAR, protease activated receptor; PIP,, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PL, phospholipid; PLCB, phospholipase CB; PLCy, phospholipase Cy; TP, thromboxane A, receptor; TxA,, thromboxane A,; VWF, von Willebrand factor.) The G-proteins that are involved are not shown. (B) Diagrammatic representation of platelet aggregation mediated by fibrinogen binding to activated GPIlb-Illa molecules on adjacent platelets. Signaling events initiated by all aggregating agents transform GPllb-llla from its resting state to an activated form that can bind divalent fibrinogen or, at the high shear that occurs in small vessels, multivalent von Willebrand factor.

hemostasis and thrombosis. As described in Chapter 23, these cells synthesize the prostanoid prostacyclin (PGI,), a potent inhibitor of platelet aggregation. Prostacyclin acts by stimulating the activity of adenylyl cyclase in the surface membranes of platelets via its G protein-coupled receptor (Figure 55-1A). The resulting increase of intraplatelet cAMP opposes the increase in the level of intracellular Ca** produced by IP, and thus inhibits platelet activation. This is in contrast with the effect of the prostanoid TxA, that is formed by activated platelets, which is that of promoting aggregation. Endothelial cells play other roles in the regulation of thrombosis. For instance, these cells possess an ADPase, which hydrolyzes ADP, and thus opposes its aggregating effect on platelets. In addition, these cells express proteoglycans such as heparan sulfate, an anticoagulant, and they also release plasminogen activators, which may help dissolve thrombi. Nitric oxide (endothelium-derived relaxing factor), another potent platelet inhibitor, is discussed in Chapter 51.

Antiplatelet drugs inhibit platelet responses. The most commonly used antiplatelet drug is aspirin (acetylsalicylic acid), which irreversibly acetylates and thus inhibits the platelet cyclooxygenase (COX-1) involved in formation of TxA, (see Chapter 21); TxA, is a potent aggregator of platelets and also a vasoconstrictor. Platelets are very sensitive to aspirin; as little as 30 mg of aspirin/day (one regular aspirin tablet contains 325 mg) effectively eliminates the synthesis of TxA, by platelets. Aspirin also inhibits production of PGI,, which opposes platelet aggregation and is a vasodilator, by endothelial cells, but unlike platelets, these cells regenerate cyclooxygenase within a few hours. Thus, the overall balance between TxA, and PGI, can be shifted in favor of the latter, opposing platelet aggregation. Indications for treatment with aspirin include management of acute coronary syndromes (angina, myocardial infarction), acute stroke syndromes (transient ischemic attacks, acute stroke), severe carotid artery stenosis, and primary prevention of these and other atherothrombotic diseases.
Other antiplatelet drugs include specific inhibitors of the P2Y,, receptor for ADP (eg, clopidogrel, prasugrel, and ticagrelor) and antagonists of ligand binding to GPIIb-IIIa (eg, abciximab, eptifibatide, and tirofiban) that interfere with fibrinogen and von Willebrand factor binding and thus platelet aggregation.

Two pathways lead to fibrin clot formation: the extrinsic and the intrinsic pathways. These pathways are not independent, as previously thought. However, this artificial distinction is retained in the following text to facilitate their description. Initiation of fibrin clot formation in response to tissue injury is carried out by the extrinsic pathway. The intrinsic pathway can be activated by negatively charged surfaces in vitro, for example, glass. Both pathways lead to the proteolytic conversion of prothrombin to thrombin. Thrombin catalyzes cleavage of fibrinogen to initiate fibrin clot formation. The extrinsic and intrinsic pathways are complex and involve many different proteins (Figures 55-2 and 55-3; Table 55-1). The coagulation factors are another example of multidomain proteins sharing conserved domains (see Figure 5-9). In general, as shown in Table 55-2, these proteins can be classified into five types: (1) zymogens of serine-dependent proteases that are activated during the process of coagulation; (2) cofactors; (3) fibrinogen; (4) a zymogen of a transglutaminase that covalently cross-links fibrin and stabilizes the fibrin clot; and (5) regulatory and other proteins.

The extrinsic pathway involves tissue factor, factors VII and X, and Ca" and results in the production of factor Xa (by convention, activated clotting factors are referred to by use of the suffix a). The extrinsic pathway is initiated at the site of tissue
FIGURE 55-2 The pathways of blood coagulation, with the extrinsic pathway indicated at the top left and the intrinsic pathway at the top right. The pathways converge in the formation of active factor X (ie. factor Xa) and culminate in the formation of cross-linked fibrin (lower right). Complexes of tissue factor and factor Vila activate not only factor X (extrinsic Xase [tenase]) but also factor IX in the intrinsic pathway (dotted arrow). In addition, thrombin feedback activates at the sites indicated (dashed arrows) and also activates factor VII to factor Vila (not shown). The three predominant complexes, extrinsic Xase, intrinsic Xase, and prothrombinase, are indicated within the large arrows; these reactions require anionic procoagulant phospholipid membrane and calcium. Activated proteases are in solid-outlined boxes; active cofactors are in dash-outlined boxes; and inactive factors are not in boxes. (HK, high-molecular-weight kininogen; PK, prekallikrein.)

FIGURE 55-3 The structural domains of selected proteins involved in coagulation and fibrinolysis. Shared domains are a result of gene duplication and exon shuffling that contributed to the molecular evolution of the coagulation system. The domains are as identified at the bottom of the figure and include signal peptide, propeptide, Gla (y-carboxyglutamate) domain, epidermal growth factor (EGF) domain, apple domain, kringle domain, fibronectin (types | and Il) domain, the zymogen activation region, aromatic amino acid stack, and the catalytic domain. Interdomain disulfide bonds are indicated, but numerous intradomain disulfide bonds are not. Sites of proteolytic cleavage in synthesis or activation are indicated by arrows (dashed and solid, respectively). (FVII, factor VII; FIX, factor IX; FX, factor X, FXI; factor XI; FXIl, factor XIl; t-PA, tissue plasminogen activator.) (Adapted, with permission, from Furie B, Furie BC: The molecular basis of blood coagulation. Cell 1988;53:505.)
injury with the exposure of tissue factor (TF; Figure 55-2), located in the subendothelium and on activated monocytes. TF interacts with and activates factor VII (53 kDa, azymogencontaining vitamin K-dependent y-carboxyglutamate [Gla] residues; see Chapter 44), synthesized in the liver. It should be noted that in the Gla-containing zymogens (factors II, VII, IX, and X), the Gla residues in the amino-terminal regions of the molecules serve as high-affinity binding sites for Ca**. TF acts as a cofactor for factor VIIa, enhancing its enzymatic activity to activate factor X (56 kDa). The reaction by which factor X is activated requires the assembly of the extrinsic tenase complex (Ca"*-TF-factor VIIa) formed on a cell membrane surface exposing the procoagulant anionic aminophospholipid phosphatidylserine. Factor VIIa cleaves

I II III, Common Name(s) = Fibrinogen Prothrombin Tissue factor. I II III, Common Name(s) = These factors are usually referred to by their common names   . IV, Common Name(s) = Ca 2+. IV, Common Name(s) = Ca 2+ is usually not referred to as a coagulation factor. V, Common Name(s) = Proaccelerin, labile factor, accelerator (Ac-) globulin. V, Common Name(s) = Proaccelerin, labile factor, accelerator (Ac-) globulin. VII a, Common Name(s) = Proconvertin, serum prothrombin conversion accelerator (SPCA), cothromboplastin. VII a, Common Name(s) = Proconvertin, serum prothrombin conversion accelerator (SPCA), cothromboplastin. VIII, Common Name(s) = Antihemophilic factor A, antihemophilic globulin (AHG). VIII, Common Name(s) = Antihemophilic factor A, antihemophilic globulin (AHG). IX, Common Name(s) = Antihemophilic factor B, Christmas factor, plasma thromboplastin component (PTC). IX, Common Name(s) = Antihemophilic factor B, Christmas factor, plasma thromboplastin component (PTC). X, Common Name(s) = Stuart-Prower factor. X, Common Name(s) = Stuart-Prower factor. XI, Common Name(s) = Plasma thromboplastin antecedent (PTA). XI, Common Name(s) = Plasma thromboplastin antecedent (PTA). XII, Common Name(s) = Hageman factor. XII, Common Name(s) = Hageman factor. XIII, Common Name(s) = Fibrin stabilizing factor (FSF), fibrinoligase. XIII, Common Name(s) = Fibrin stabilizing factor (FSF), fibrinoligase
“There is
no
VI.
Note: The numbers indicate the order in which the factors have been discovered and bear no relationship to the order in which they act.

factor an Arg-Ile bond in factor X to produce the two-chain serine protease, factor Xa. TF and factor VIIa also activate factor IX in the intrinsic pathway. Indeed, the formation of complexes between membrane-bound TF and factor VIIa is now considered to be the key process involved in initiation of blood coagulation in vivo.
Tissue factor pathway inhibitor (TFPI) is a major physiologic inhibitor of coagulation. TFPI is a protein that circulates in the blood where it directly inhibits factor Xa by binding to the enzyme near its active site. This factor Xa-TFPI complex then inhibits the factor VIla-TF complex.

The formation of factor Xa is the major site where the intrinsic and extrinsic pathways converge (Figure 55-2). The intrinsic pathway (Figure 55-2) involves factors XII, XI, IX, VIII, and X, as well as prekallikrein, high-molecular-weight (HMW) kininogen, Ca"*, and cell surface exposed phosphatidylserine. This pathway results in the production of factor Xa by the intrinsic tenase complex (see below for composition), in which factor [Xa serves as the serine protease and factor VIIa as the cofactor. As noted above, the activation of factor X provides an important link between the intrinsic and extrinsic pathways.
The intrinsic pathway can be initiated by "contact" in which prekallikrein, HMW kininogen, factors XII and XI are exposed to a negatively charged activating surface. In vivo, polymers of phosphates, such as extracellular DNA, RNA, and polyphosphate (macromolecules available only following

proteins contain Gla (γ-carboxyglutamate) residues. Thrombomodulin, 1 = Protein on the surface of endothelial cells; binds thrombin, which then activates protein C
cell damage) may serve as this negatively charged activating surface. Kaolin, a highly negatively charged hydrated aluminum silicate, can be used for in vitro tests as an initiator of the intrinsic pathway. When the components of the contact phase assemble on the activating surface, factor XII is activated to factor XIIa upon proteolysis by kallikrein. This factor XIla, generated by kallikrein, cleaves prekallikrein to generate more kallikrein, setting up a positive feedback activation loop. Factor XIla, once formed, activates factor XI to XIa and also releases bradykinin (a peptide with potent vasodilator action) from HMW kininogen.
thrombin to form factor VIIIa, which is in turn inactivated upon further cleavage by thrombin-mediated activated protein C (see below).
In the
Ca**, factor
XIa activates
factor
IX
(55
kDa, factor
a
IXa.
factor
Gla-containing zymogen),
This, in
X
to turn,
produce assembly
of factor
to also
cleaves
Xa.
components, composed
of
This called
Ca*-factor procoagulant
serine, often
is normally
the serine
an
Arg-Ile latter
reaction the
intrinsic
VIIIa-factor membrane
protease, bond
in requires
tenase
Xa, the
complex, which
surfaces activated
expressing platelets.
on the
(Note inner
leaflet forms
on phosphatidyl-
that this
side of
the phospholipid
plasma membrane
bilayer of
resting, nonactivated
platelets.)
Factor VIII (330 kDa), a circulating glycoprotein, is not
a
protease precursor
but
a
cofactor precursor
that, when
acti- vated,
serves as
a
receptor on

the platelet
surface for
factors
IXa and X. Factor VIII is activated by minute quantities of
The role of the initial steps of the intrinsic pathway in initiating coagulation has been called into question because patients with a hereditary deficiency of factor XII, prekallikrein or HMW kininogen do not exhibit bleeding problems. Similarly, patients with a deficiency of factor XI may not have bleeding problems. In thrombosis, deficiencies in the intrinsic pathway are protective. The intrinsic pathway largely serves to amplify factor Xa and ultimately thrombin formation, through feedback mechanisms (see below). The intrinsic pathway may also be important in fibrinolysis (see below) since kallikrein, factors XIIa and XIa can cleave plasminogen and kallikrein can activate single-chain urokinase.

Factor Xa, produced by either the extrinsic or the intrinsic pathway, activates prothrombin (factor II) to thrombin (factor Ila) (Figure 55-2; Table 55-1).
The activation of prothrombin, like that of factor X, occurs on a membrane surface and requires the assembly of a prothrombinase complex, consisting of Ca', and
factors Va and Xa. The assembly of the prothrombinase complex, like that of the tenase complex, takes place on the phosphatidylserine-exposing membrane surface, often activated platelets. Factor V (330 kDa) is synthesized in the liver, spleen, and kidney and is found in platelets as well as in plasma. Factor Va functions as a cofactor in a manner similar to that of factor VIIla in the tenase complex. Factor V is activated to factor Va by traces of thrombin, and binds specifically to a procoagulant membrane (often that of platelets) (Figure 55-4) and forms a complex with factor Xa and prothrombin. It is subsequently inactivated by activated protein C (see below), thereby providing a means of limiting the activation of prothrombin to thrombin. Prothrombin (72 kDa; Figure 55-4) is a single-chain glycoprotein synthesized in the liver. The aminoterminal region of prothrombin (Figure 55-3) contains 10 Gla residues, and the serine-dependent active protease site is in the catalytic domain close to the carboxyl-terminal region of the molecule. Upon binding to the complex of factors Va and Xa on the procoagulant membrane (Figure 55-4), prothrombin is cleaved by factor Xa at two sites to generate the active, two-chain thrombin molecule, which is then released from the membrane surface.

Thrombin, produced by the prothrombinase complex, in addition to having a potent stimulatory effect on platelets (see above), converts fibrinogen to fibrin (Figure 55-2). Fibrinogen (factor I, 340 kDa; see Figure 55-2 and Figure 55-5; Tables 55-1 and 55-2) is an abundant (3 mg/mL) soluble plasma glycoprotein that consists of a dimer of three polypeptide chains, (Aa, Bf, y),, that is covalently linked by 29
UREA
FIGURE 55-4 Schematic representation of the prothrombinase complex bound to the procoagulant plasma membrane. The prothrombinase complex contains factors Va, Xa, and prothrombin. A central theme in blood coagulation is the assembly of protein complexes, that is, the tenase complexes and the prothrombinase complex, in a Ca?*-dependent fashion, on membrane surfaces on which phosphatidylserine is exposed. The catalytic efficiency of zymogen activation is increased by many orders of magnitude by the membrane-bound complexes. y-carboxyglutamate residues (indicated by Y) on vitamin K-dependent proteins bind calcium and contribute to the exposure of membrane-binding sites on these proteins (black ovals, Xa and prothrombinase).
disulfide bonds. The BB and y chains contain asparaginelinked complex oligosaccharides (see Chapter 46). All three chains are synthesized in the liver; the three genes encoding these proteins are on the same chromosome where their expression is coordinately regulated in humans. The aminoterminal regions of the six chains are held in close proximity by a number of disulfide bonds (a subset is shown in Figure 55-5), while the carboxyl-terminal regions are spread apart. Thus, the fibrinogen molecule has a trinodular, elongated structure

FIGURE 55-5 Diagrammatic representation of fibrinogen. (A) Fibrinogen is a dimeric molecule, with each half composed of three polypeptide chains: Aa, BB, and y. Disulfide bonds join together the chains and the two halves of the molecule. (B) Fibrinogen forms a trinodular structure with a central E domain linked via coiled coil regions to two lateral D domains each of which contains a flexible Aa chain aC domain. The thrombin-cleaved regulatory peptides fibrinopeptide A (FPA) and fibrinopeptide B (FPB) reside within the E nodule as shown.
with a central E domain that is linked to lateral D domains via coiled coil regions (Figure 55-5 and Figure 55-6A). The N-terminal A and B portions of the Aa and Bf chains are termed fibrinopeptide A (FPA) and fibrinopeptide B (FPB), respectively; these domains are highly negatively charged as _a result of an abundance of aspartate and glutamate residues (see below). The negative charges contribute to the solubility of fibrinogen in plasma and importantly also serve to prevent
_
aggregation by causing electrostatic repulsion between fibrinogen molecules.

Thrombin (34 kDa), the serine protease formed by the prothrombinase complex, hydrolyzes the four Arg-Gly bonds between the N-terminal fibrinopeptides and the a and 6 portions of the Aa and B6 chains of fibrinogen (Figure 55-6A, B). The release of FPA and FPB by thrombin generates fibrin monomer, which has the subunit structure (a, 8, y),. Since FPA and FPB contain only 16 and 14 residues, respectively, the fibrin molecule retains 98% of the residues present in fibrinogen. The removal of the fibrinopeptides exposes binding sites within the E domain of fibrin monomers that specifically interact with complementary domains within the D domains of other fibrin monomers. In this way, fibrin monomers spontaneously polymerize in a half-staggered pattern to form long strands (protofibrils) (Figure 55-6A). Although insoluble, this initial fibrin clot is unstable, held together only by the noncovalent association of fibrin monomers.
In addition to converting fibrinogen to fibrin, thrombin also activates factor XIII to factor XIIIa. Factor XIIIa is a highly specific transglutaminase that covalently cross-links y chains and, more slowly, a chains of fibrin molecules by forming peptide bonds between the amide groups of glutamine and the e-amino groups of lysine residues (Figure 55-6C). Such cross-linking yields a more stable fibrin clot with increased resistance to proteolysis. This fibrin mesh serves to stabilize the hemostatic plug or thrombus.

Once active thrombin is formed in the course of hemostasis or thrombosis, its concentration must be carefully controlled to prevent further fibrin formation or platelet activation. This is achieved in two ways. Thrombin circulates as its inactive precursor, prothrombin, which is activated as a result of a cascade of enzymatic reactions, each converting an inactive zymogen to an active enzyme and leading finally to the conversion of prothrombin to thrombin (Figure 55-2). At each point in the cascade, feedback mechanisms produce a delicate balance of activation and inhibition. The concentration of factor XII in plasma is approximately 30 g/mL, while that of fibrinogen is 3 mg/mL, with intermediate clotting factors increasing in concentration as one proceeds down the cascade; these facts illustrate that the clotting cascade provides amplification. The second means of controlling thrombin activity is the inactivation of any formed thrombin by circulating inhibitors, the most important of which is antithrombin (see below).

Four naturally occurring thrombin inhibitors exist in normal plasma. The most important is antithrombin, which contributes approximately 75% of the antithrombin activity.

Antithrombin can also inhibit the activities of factors [Xa, Xa, Xla, XIIa, and VIla complexed with tissue factor. a,Macroglobulin contributes most of the remainder of the antithrombin activity, with heparin cofactor II and a, -antitrypsin acting as minor inhibitors under physiologic conditions. The endogenous activity of antithrombin is greatly potentiated by the presence of sulfated glycosaminoglycans (heparans) (see Chapter 50). Heparans bind to a specific cationic site of antithrombin, which induces a conformational change that promotes binding of antithrombin to thrombin and factor Xa, as well as to its other substrates. This mechanism is the basis for the use of heparin, a derivatized heparan, in clinical medicine to inhibit coagulation. The anticoagulant effects of heparin can be antagonized by strongly cationic polypeptides such as protamine, which bind strongly to heparin, thus inhibiting the bindng of heparin to antithrombin. Low-molecular-weight heparins (LMWHs), derived from enzymatic or chemical cleavage of unfractionated heparin, have more clinical use. They can be administered subcutaneously at home, have greater bioavailability than unfractionated heparin, and do not need frequent laboratory monitoring. Individuals with inherited deficiencies of antithrombin are prone to develop venous thrombosis, providing evidence that antithrombin has a physiologic function and that the coagulation system in humans is normally in a dynamic state. Thrombin is involved in an additional regulatory mechanism that operates in coagulation. It combines with thrombo- modulin, a glycoprotein present on the surfaces of endothelial cells. The complex activates protein C on the endothelial protein C receptor. In combination with protein S, activated protein C (APC) degrades factors Va and VIIIa, thereby limiting their actions in coagulation. A genetic deficiency of either protein C or protein S can cause venous thrombosis. Furthermore, patients with factor V Leiden (which has a glutamine residue in place of an arginine at position 506) have

an increased risk of venous thrombotic disease because factor V Leiden is resistant to inactivation by APC; this condition is also termed APC resistance.

The coumarin drugs (eg, warfarin), which are used as anticoagulants, inhibit the vitamin K-dependent carboxylation of Glu to Gla residues (see Chapter 44) in the amino-terminal regions of factors II, VII, IX, and X and also proteins C and S. These proteins, all of which are synthesized in the liver, are dependent on the Ca**-binding properties of the Gla residues for their normal function in the coagulation pathways. Coumarins inhibit the reduction of the quinone derivatives of vitamin K to the active hydroquinone forms (see Chapter 44). Thus, the administration of vitamin K will bypass the coumarin-induced inhibition and allow the posttranslational modification of carboxylation to occur. Reversal of coumarin inhibition by vitamin K requires 12 to 24 hours, whereas
reversal of the anticoagulant effects of heparin by protamine is almost instantaneous. Coumarin reversal is achieved faster by infusing normal coagulation factors.
Heparin and warfarin are used in the treatment of thrombotic and thromboembolic conditions, such as deep vein thrombosis and pulmonary embolism, and the prevention of stroke in patients with the heart rhythm abnormality atrial fibrillation. Heparin is administered first, because of its prompt onset of action, whereas warfarin takes several days to reach full effect. Their effect is not well predictable by dosage, and thus because of the risk of producing hemorrhage, these drugs are closely monitored by use of appropriate tests of coagulation (see below).
New oral inhibitors of thrombin (eg, dabigatran) or of factor Xa (eg, rivaroxaban, apixaban, and others) are also used in the prevention and treatment of thrombotic conditions. These drugs are advantageous because their effect is predictable based on the dose, and some do not require routine monitoring by laboratory tests. Specific reversal agents, such as antibodies or decoy molecules, are in various stages of development. Agents that target the "intrinsic" factors are being evaluated as antithrombotics.

Inherited deficiencies of the clotting system that result in bleeding are found in humans. The most common is deficiency of factor VIII, causing hemophilia A, an X chromosomelinked disease. Hemophilia B, also X chromosome-linked, is due to a deficiency of factor IX and has recently been identified as the form of hemophilia that played a major role in the history of the royal families of Europe. The clinical features of hemophilia A and B are almost identical, but these two diseases can be readily distinguished on the basis of specific assays for the two factors.
The gene for human factor VIII measures 186 kb in length, and contains 26 exons that encode a protein of 2351 amino acids. A variety of mutations in the factor VIII and IX genes have been detected leading to diminished activities of the factor VIII and IX proteins; these include partial gene deletions and point and missense mutations. Prenatal diagnosis by DNA analysis after chorionic villus sampling is now possible (see Chapter 39).
Treatment for patients with hemophilia A consisted initially, in the 1960s, of administration of cryoprecipitate (enriched in factor VIII) prepared from individual donors; in the 1970s, lyophilized factor VIII or factor IX concentrates, prepared from very large plasma pools, became available for treatment of hemophilia A and B patients, respectively. In the 1990s, factors VIII and IX prepared by recombinant DNA technology (see Chapter 39) were introduced. Such preparations are free of contaminating viruses (eg, hepatitis A, B, C, or HIV-1) found in human plasma, but are expensive; their use will increase as the cost of production decreases. Longer-acting recombinant factors with extended half-lives in the circulation are now in use, and hemophilia is a target of gene therapy approaches.
The most common hereditary bleeding disorder is von Willebrand disease, with a prevalence of up to 1% of the population. It results from a deficiency or defect in von Willebrand factor, a large multimeric glycoprotein that is secreted by endothelial cells and platelets into the plasma, where it stabilizes factor VIII. von Willebrand factor also promotes platelet adhesion at sites of vessel wall injury and platelet aggregation under conditions of high shear (see above).

As stated above, the coagulation system is normally in a state of dynamic equilibrium in which fibrin clots are constantly being laid down and dissolved. This latter process is termed fibrinolysis. Plasmin, the serine protease mainly responsible for degrading fibrin and fibrinogen, circulates in the form of its inactive zymogen, plasminogen (90 kDa), and any small amounts of plasmin that are formed in the fluid phase under physiologic conditions are rapidly inactivated by the fastacting plasmin inhibitor, a,-antiplasmin. Plasminogen binds to fibrin and thus becomes incorporated in clots as they are produced; since plasmin that is formed when bound to fibrin is protected from a.,-antiplasmin, it remains active. Activators of plasminogen of various types are found in most body tissues, and all cleave the same Arg-Val bond in plasminogen to produce the disulfide bridge-linked two-chain serine protease, plasmin (Figure 55-7). The specificity of plasmin for
fibrin is another mechanism to regulate fibrinolysis. Via one ofits kringle domains, plasmin(ogen) specifically binds lysine residues on fibrin and so is increasingly incorporated into the fibrin mesh as it cleaves it. (Kringle domains [Figure 55-3] are common protein motifs of about 100 amino acid residues in length; they have a characteristic covalent structure defined by a pattern of three disulfide bonds.) Thus, the carboxypeptidase activated thrombin activatable fibrinolysis inhibitor (TAFIa) (Figure 55-7), which removes terminal lysines from fibrin, is able to inhibit fibrinolysis. Thrombin activates TAFI to TAFla, thereby inhibiting fibrinolysis during clot formation.

Tissue plasminogen activator (t-PA) (Figures 55-3 and 55-7) is a serine protease that is released into the circulation from vascular endothelium under conditions of injury or stress and is catalytically inactive unless bound to fibrin. Upon binding to fibrin, t-PA cleaves plasminogen within the clot to generate plasmin, which in turn digests the fibrin to form soluble degradation products and thus dissolves the clot. Neither plasmin nor the plasminogen activator can remain bound to these degradation products, and so they are released into the fluid phase where they are inactivated by their natural inhibitors. Prourokinase is the precursor of a second activator of plasminogen, urokinase. Originally isolated from urine, it is now known that urokinase is synthesized by various cell types including monocytes and macrophages, fibroblasts, and epithelial cells. The main action of urokinase appears to be the degradation of extracellular matrix. Figure 55-7 indicates the sites of action of five proteins that influence the formation and action of plasmin.

t-PA, marketed as alteplase, is produced by recombinant DNA methods. It is used therapeutically as a fibrinolytic agent, as is streptokinase, an enzyme secreted by a number of streptococcal bacterial strains. However, the latter is less selective than t-PA, activating plasminogen in the fluid phase (where it can degrade circulating fibrinogen) as well as plasminogen that is bound to a fibrin clot. The amount of plasmin produced by therapeutic doses of streptokinase may exceed the capacity of the circulating a,-antiplasmin, causing fibrinogen as well as fibrin to be degraded and resulting in the bleeding often encountered during fibrinolytic therapy. Because of its relative selectivity for degrading fibrin, recombinant t-PA has been widely used to restore the patency of coronary arteries following thrombosis. If administered early enough, before irreversible damage of heart muscle occurs (about 6 hours after onset of thrombosis), t-PA can significantly reduce the mortality rate from myocardial damage following coronary thrombosis. Streptokinase has also been widely used in the treatment of coronary thrombosis, but has the disadvantage of being antigenic. t-PA has also been used in the treatment of ischemic stroke, peripheral arterial occlusion, deep vein thrombosis, and pulmonary embolism.
There are a number of disorders, including cancer and sepsis, in which the concentrations of plasminogen activators increase. In addition, the antiplasmin activities contributed by a,-antitrypsin and a,-antiplasmin may be impaired in diseases such as cirrhosis. Since certain bacterial proteins, such as streptokinase, are capable of activating plasminogen, they may be responsible for the diffuse hemorrhage sometimes observed in patients with disseminated bacterial infections.

A number of laboratory tests are available to measure the phases of hemostasis described above. The tests include platelet count, bleeding time/closure time, platelet aggregation, activated partial thromboplastin time (aPTT or PTT), prothrombin time (PT), thrombin time (TT), concentration of fibrinogen, fibrin clot stability, and measurement of fibrin degradation products. The platelet count quantitates the number of platelets. The skin bleeding time is an overall test of platelet and vessel wall function, while the closure time measured using the platelet function Analyzer PFA-100/200 is an in vitro test of platelet-related hemostasis. Platelet aggregation measures responses to specific aggregating agents. aPTT is a measure of the intrinsic pathway and PT of the extrinsic pathway, with aPTT being used to monitor heparin therapy and PT to measure the effectiveness of warfarin. The reader is referred to a textbook of hematology for a discussion of these tests.

■, 1 = Hemostasis and thrombosis are complex processes involving platelets, coagulation factors, and blood vessels.. ■, 1 = Thrombin and other agents cause platelet aggregation, which involves a variety of biochemical and morphologic events. Stimulation of phospholipase C and the polyphosphoinositide pathway is a key event in platelet activation, but other processes are also involved.. ■, 1 = Aspirin is an important antiplatelet drug that acts by inhibiting production of thromboxane A 2 .. ■, 1 = Many coagulation factors are zymogens of serine proteases, becoming activated, then inactivated during the overall process.. ■, 1 = Both extrinsic and intrinsic pathways of coagulation exist, the former initiated in vivo by tissue factor. The pathways converge at factor Xa, ultimately resulting in thrombin-catalyzed conversion of fibrinogen to fibrin, which is strengthened by covalent cross-linking, catalyzed by factor XIIIa.. ■, 1 = Genetic disorders that lead to bleeding occur; the principal disorders involve factor VIII (hemophilia A), factor IX (hemophilia B), and von Willebrand factor (von Willebrand disease).. ■, 1 = Antithrombin is an important natural inhibitor of coagulation; genetic deficiency of this protein can result in thrombosis.
680
SECTION
XI
Special
Topics
(C)
- ™ Factors II, VII, IX, and X and proteins C and S require
vitamin K-dependent y-carboxylation of certain glutamate residues to function in coagulation. This carboxylation process
can be inhibited by the anticoagulant warfarin.
- | Fibrin is dissolved by plasmin. Plasmin exists as an inactive
precursor, plasminogen, which can be activated by tissue plasminogen activator (t-PA). t-PA is widely used clinically to
treat early thrombosis in the coronary arteries.

Hoffman R, Benz EJ Jr, Silberstein LR, et al (editors): Hematology:
Basic Principles and Practice, 7th ed. Elsevier Saunders, 2017.
Israels SJ (editor): Mechanisms in Hematology, 4th ed. Core Health
Sciences Inc, 2011. (This text has many excellent illustrations of basic mechanisms in hematology.)
- Kasper DL, Fauci AS, Longo DL, et al: Harrison's Principles of
Internal Medicine, 19th ed. McGraw-Hill, 2016.
- Marder VJ, Aird WC, Bennett JS, et al (editors): Hemostasis and
Thrombosis: Basic Principles and Clinical Practice, 6th ed.
Lippincott Williams & Wilkins, 2013.
Michelson AD (editor): Platelets, 3rd ed. Elsevier, 2013.

Molly Jacob, PhD, Varghese, P. Anthony Weil, PhD
. Present an overview of carcinogenesis and important biochemical and genetic features of cancer cells.
= Describe important properties of oncogenes and tumor suppressor genes and their role in
carcinogenesis.
from normal cells. = Describe the important characteristics of cancer cells that distinguish them
= Briefly describe the concepts of genomic instability, aneuploidy, and
angiogenesis in
tumor formation
and growth.
Discuss the relevance of tumor markers.
Outline how understanding of the biology of cancer has led to the development of various new therapies.

Cancers constitute the second most common cause of death, after cardiovascular disease, in many countries. Approximately 9 million people around the world die from cancer each year, and this figure is projected to increase. Humans of all ages develop cancer, and a wide variety of organs are affected. Worldwide, the main types of cancer accounting for mortality are those involving the lung, stomach, colon, rectum, liver, and breast. Other types of cancers that lead to death include cervical, esophageal, and prostate cancers. Skin cancers are also very common, but apart from melanomas, are generally not as aggressive as those mentioned above. The incidence of many cancers increases with age. Hence, as people live longer, many more will develop the disease. Hereditary factors play a role in some types of tumors. Apart from great individual suffering caused by the disease, the economic burden to society is immense.
Cancer cells are characterized by certain key properties: they (1) proliferate rapidly; (2) display diminished growth control; (3) display increased genomic mutations at the level of the nucleotide, small and large insertions and deletions (indels), and gross chromosomal rearrangements, duplications and loss; (4) display loss of contact inhibition in culture in vitro; (5) invade local tissues and spread, or metastasize, to other parts of the body; (6) are self-sufficient in growth signals and are insensitive to antigrowth signals; (7) stimulate local angiogenesis; and (8) are often able to evade apoptosis. These properties are characteristic of cells of malignant tumors. It is metastasis that is generally responsible for the deaths of patients who have cancer. By contrast, cells of benign tumors also show diminished control of growth, but do not invade local tissue or spread to other parts of the body. These points are summarized in Figure 56-1 and amplified on in Figure 56-2.

A neoplasm refers to any abnormal new growth of tissue. It may be benign or malignant in nature. The term "cancer" is usually associated with malignant tumors. Tumors can arise in any organ in the body and result in different clinical features, depending on the location of the growth.
Central issues in oncology (the study of cancer) include elucidation of biochemical and genetic mechanisms that underlie the uncontrolled growth of cancer cells and their ability to invade and metastasize, and development of successful treatments that will destroy cancer cells, while causing minimal damage to normal cells. Considerable progress has been made in understanding the basic nature of cancer cells, and it is now generally accepted that though mutations in key genes contribute significantly to malignancies, particularly at the initiation phase of oncogenesis, other factors are
FIGURE 56-1 Six major biologic features of cancer cells. Other key properties of cancer cells are shown in Figure 56-2. (After Hanahan D, Weinberg RA. The Hallmarks of Cancer: The next generation. Cell 2011;144:646-674.)
between them contribute to the pathogenesis of malignancies. However, many aspects of the behavior of cancer cells, in particular their ability to metastasize, have yet to be fully explained. In addition, despite improvements in treatment of certain types of cancers, therapies are still often unsuccessful. This chapter aims to introduce the reader to key concepts of cancer biology. A glossary at the end of this chapter defines many of the terms used herein.

Non-lethal genetic damage is thought to be the initiating event in carcinogenesis. There are several broad classes of genes, which when mutated cause gainor loss-of-function or misregulation, and can thus result in the development of a tumor. These are proto-oncogenes, tumor suppressor genes, DNA synthesis and repair genes, genes regulating apoptosis, or genes causing evasion of immune surveillance.
also implicated in the development of malignant phenotypes. Organismal immunologic status and tissue microenvironment are two such factors. Recent work has shown that the microenvironment of host and tumor cells, and the interactions
Cancer is of clonal origin, with a single abnormal cell (often with many genetic alterations) multiplying to become a mass of cells forming a tumor. As mentioned earlier, the tissue microenvironment significantly affects tumor formation. The exact nature of these influences may vary with cell types involved, cell-cell interactions, and the presence of factors
and glycolipids

FIGURE 56-2 Some biochemical and genetic changes that occur in human cancer cells. Many changes, in addition to those indicated in Figure 56-1, can be observed in cancer cells; only some of these are shown here. The roles of mutations in activating oncogenes and inactivating tumor suppressor genes are discussed in the text. Abnormalities of cell cycle progression and of chromosome and chromatin structure, including aneuploidy, are common. Alterations of expression of specific mRNAs and regulatory ncRNAs have been reported, and the relationship of stem cells to cancer cells is a very active area of research. Telomerase activity is often detectable in cancer cells. Tumors sometimes synthesize certain fetal antigens, which may be measurable in the blood. Changes in plasma membrane constituents (eg, alteration of the sugar chains of various glycoproteins—some of which are cell adhesion molecules—and glycolipids) have been detected in many studies, and may be of importance in relation to decreased cell adhesion and metastasis. Various molecules are released from cancer cells, in either soluble or membrane-bound vesicular forms (exosomes), and can be detected in the blood or extracellular fluid; these include metabolites, lipids, carbohydrates, proteins, and nucleic acids. Angiogenic factors and various proteinases are also released by some tumors. Many changes in metabolism have been observed; for example, cancer cells often exhibit a high rate of aerobic glycolysis. (CAM, cell adhesion molecule; ECM, extracellular matrix.)
such as paracrine signals, local hypoxia, and proinflammatory responses. Carcinogenesis is, thus, a multistep process that ultimately transforms normal cells into malignant cells. Tumors often take from a few to tens of years to develop to macroscopic levels.

Genetic damage causing cancer can be due to acquired, or inherited mutations. Aquired muations occur either by errors in DNA replication or DNA repair, and are termed replication mutations (R), or by exposure to environmental carcinogens, and are termed environmental mutations (E) (radiation, chemicals and viruses—see below). The third major class of oncogenic mutation are termed hereditary mutations (H); inherited mutations are derived from one or both parents. Such hereditary abnormalities result in a number of familial conditions that predispose to hereditary cancer. These mutations are found in specific genes (eg, tumor suppressor genes; DNA repair genes, cell cycle control genes, etc) present in the germ cells, and are discussed later in this chapter. Mutations of the R, E, and H variety collectively cause the majority of human cancers, though the exact percentage of a particular type of cancer caused by these three types of mutations varies, and indeed is being actively debated in the field of medical oncology.
Spontaneous mutations, some of which may predispose to cancer, occur at a frequency of approximately 10~' to 10% per cell per generation. This rate is greater in tissues undergoing a high rate of proliferation, a dynamic that can increase the generation of cancer cells from affected parent cells. Oxidative stress (see Chapter 45), generated as a result of enhanced production of reactive oxygen species (ROS), may also be a factor in increasing mutation rates.

In general, there are three classes of environmental carcinogens that can induce tumor formation: radiation, chemicals, and certain oncogenic viruses (Figure 56-3). The first two
FIGURE 56-3 Radiation, chemical carcinogens, and certain viruses can cause cancer by damaging chromosomal DNA.

+ Formation of pyrimidine dimers
+ Formation of apurinic or apyrimidinic sites by elimination of
corresponding bases
- + Formation of singleor double-strand breaks or cross-linking of DNA strands
cause mutations in DNA, while viruses generally novel into normal cells.
We shall describe how radiation, chemicals, cause cancer.
carcinogens act by introducing genes only briefly and oncogenic viruses

Ultraviolet rays (UV), x-rays, and y-rays are mutagenic and carcinogenic. Extensive studies have shown that these agents damage DNA in a number of ways (Table 56-1; see also Table 35-8, and Figure 35-22). Mutations in DNA, if not corrected, are thought to underlie the carcinogenic effect of radiation, although the exact pathways are still under investigation. Additionally, x-rays and y-rays can induce formation of reactive oxygen species (ROS), which as noted above are also mutagenic and probably contribute to the carcinogenic effects of radiation.
Exposure to UV radiation is common due to exposure to sunlight, which is its main source. Ample evidence shows that UV radiation is linked to cancers of the skin. The risk of developing a skin cancer due to ultraviolet radiation increases with increased frequency and intensity of exposure, and decreases with increasing melanin content of skin.
As detailed in Chapter 35, DNA damage produced by environmental agents is usually removed by DNA repair mechanisms. Not surprisingly then, given the mutagenic nature of DNA damage, individuals who have an inherited inability to repair DNA have increased risk of developing a malignancy (see Table 35-9).

A wide variety of chemical compounds are carcinogenic (Table 56-2).

Polycyclic aromatic hydrocarbons, Compound = Benzo[a]pyrene, dimethylbenzanthracene. Aromatic amines, Compound = 2-Acetylaminofluorene, N -methyl-4- aminoazobenzene (MAB). Nitrosamines, Compound = Dimethylnitrosamine, diethylnitrosamine. Various drugs, Compound = Alkylating agents (eg, cyclophosphamide), diethylstilbestrol. Naturally occurring compounds, Compound = Aflatoxin B 1
Note: As listed above, some drugs used as chemotherapeutic agents (eg, cyclophosphamide) can be carcinogenic.
Most chemical carcinogens are thought to covalently modify DNA thereby forming a wide range of nucleotide adducts (see Table 35-8). Depending on the extent of damage to DNA and its repair by DNA repair systems (see Figure 35-22), a variety of mutations in DNA can result from exposure of an animal or human to chemical carcinogens, some of which contribute to the development of cancer.
Some chemicals interact directly with DNA (eg, mechlorethamine and 6-propiolactone), but others, termed procarcinogens, require conversion by enzyme action to become ultimate carcinogens. Most ultimate carcinogens are electrophiles (molecules deficient in electrons) and readily attack nucleophilic (electron-rich) groups in DNA. Conversion of chemicals to ultimate carcinogens is principally due to the actions of various species of cytochrome P450 located in the endoplasmic reticulum (ER) (see Chapter 49). This fact is used in the Ames assay (see below), in which an aliquot of postmitochondrial supernatant (containing ER) is added to the assay system as a source of cytochrome P450 enzymes.
Chemical carcinogenesis involves two stages— initiation and promotion. Initiation is the stage where exposure to a chemical cause genomic DNA damage, some of which go unrepaired, and is a necessary initial event for a cell to become cancerous. Promotion is the stage at which an initiated cell begins to grow and proliferate abnormally. The cumulative effect of these two events is a neoplasm.

Chemical carcinogens can be identified by testing for their ability to induce mutations. A simple way to do this is by using the Ames assay (Figure 56-4). This relatively simple test, which detects mutations in the bacterium Salmonella typhimurium caused by chemicals, has proven very valuable for screening purposes. A refinement of the Ames test is to add an aliquot of mammalian ER to the assay, to make it possible to identify procarcinogens. Very few, if any, compounds that have tested negative in the Ames test have been shown to cause tumors in animals. However, animal testing is required to show unambiguously that a chemical is carcinogenic.
It should be noted that compounds that alter epigenetic factors (such as DNA methylation and/or histone posttranslational modifications; see Chapters 35, 38) that might predispose to cancer, would not test positive in the Ames test, as they are not mutagenic.

FIGURE 56-4 The Ames assay to screen for mutagens. The chemical tested will increase the frequency of reversion of His" to His* cells if it is a mutagen and, therefore, a potential carcinogen. A control plate (not shown) contains the solvent in which the suspected mutagen is dissolved. (Reproduced, with permission, from Nester EW, et al: Microbiology: A Human Perspective, 5th ed. McGraw-Hill, 2007.)
viral DNA (called the provirus) into the host DNA results in various effects such as deregulation of the cell cycle, inhibition of apoptosis, and abnormalities of cell-signaling pathways (see Chapters 35, 42). All these events are discussed later in this chapter. DNA viruses often act by downregulation of the expression, and/or function of tumor suppressor genes P53 and RB (see below) and their protein products. RNA viruses often carry oncogenes in their genomes; how oncogenes act to cause malignancy is discussed below.

Epstein-Barr virus, Genome = DNA. Epstein-Barr virus, Cancer = Burkitt lymphoma, nasopharyngeal cancer, B-cell lymphoma. Hepatitis B, Genome = DNA. Hepatitis B, Cancer = Hepatocellular carcinoma. Hepatitis C, Genome = RNA. Hepatitis C, Cancer = Hepatocellular carcinoma. Human herpesvirus 8 (HHV-8), Genome = DNA. Human herpesvirus 8 (HHV-8), Cancer = Kaposi sarcoma. Humanpapilloma viruses (types 16 and 18), Genome = DNA. Humanpapilloma viruses (types 16 and 18), Cancer = Cancer of the cervix. HumanT-cell leukemia virus type 1, Genome = RNA. HumanT-cell leukemia virus type 1, Cancer = Adult T-cell leukemia

Over the past several decades, major advances have been made in understanding how cancer cells develop and grow. Two key findings were the discoveries of oncogenes and tumor suppressor genes. These discoveries pointed to specific molecular mechanisms through which cell growth and division could be dysregulated, resulting in abnormal growth.

An oncogene
can be
defined as
an altered
gene, the
product which
acts in
cell division.
mal cellular
a
dominant manner
to accelerate
Oncogenes are
generated proto-oncogenes,
lating proteins.
cell growth
by
“activation”
which
Such several
activation different
Table in
56-4
can be
(Table lists
the
RAS
the an
example oncogene.
GTPase activity
of encode
of or
of nor-
growth-stimu- effected
56-4).
of
a
point
RAS
encodes this
mutation
a
small
G-protein through
any of
occurring
GTPase.
results
Loss of
in chronic
stimu- lation
of the
adenylyl cyclase
and the
MAP
kinase

Mutation, Explanation = A classic example is a point mutation of the RAS oncogene. This results in the gene product, a small G-protein (RAS), in which the intrinsic GTPase activity of the protein is lost. Consequently, cells are subjected to constitutively active signaling via stimulation of adenylyl cyclase and the mitogen-activated protein (MAP) kinase pathway.. Promoter insertion, Explanation = Insertion of a viral promoter sequence near an oncogene activates it.. Enhancer insertion, Explanation = Insertion of a viral enhancer sequence near an oncogene activates it.. Chromosomal translocation, Explanation = Apiece of one chromosome is split off and is joined to another, resulting in activation of an oncogene at the site where insertion occurs. Classic examples of such translocations include those seen in Burkitt lymphoma (see Figure 56-5) and in the Philadelphia chromosome in chronic myelocytic leukemia (see the Glossary).. Gene amplification a, Explanation = Abnormal multiplication of a gene occurs, resulting in many copies. This can occur with oncogenes and genes involved in drug resistance.
*Regions of gene amplification may be recognized as homogeneously stained regions (HSRs) on chromosomes, or as double minute chromosomes (which are extrachromosomal in location).
pathway, leading to cell proliferation (recall that G-proteins are active when complexed with GTP and inactive when the bound GTP is hydrolyzed to GDP by GTPase; see Chapter 42). Another way an oncogene can be activated is via insertion of an enhancer and/or a strong promoter upstream of a proteincoding gene, resulting in increased transcription (and hence protein expression) of the cognate gene. Figure 56-5A illustrates how integration of a retroviral provirus (ie, the reverse transcriptase-generated dsDNA copy of the RNA genome of a tumor virus such as Rous sarcoma virus, RSV) can act as an enhancer/promoter to activate MYC, a neighboring host gene. Overproduction of the oncogenic transcription factor MYC activates the transcription of cell cycle-regulatory genes and, hence, stimulates unregulated cell proliferation.

Chromosomal translocations are found quite frequently in cancer cells; literally hundreds of different examples have been documented. The translocation found in cases of Burkitt lymphoma is illustrated in Figure 56-5B. The overall effect of this translocation is also to activate MYC gene expression, resulting in cell proliferation.
Yet another mechanism of oncogene activation is via gene amplification (see Chapter 38), a process that occurs quite commonly in various cancers. In this case, multiple copies of an oncogene are formed, which results in increased production of a growth-promoting protein.
Activated oncogenes
promote cancer
through
a
variety of
mechanisms in
as depicted
Figure
56-6.
genes affect
a
growth or
cell in
Figure
The protein
signaling factor,
56-2
products pathways,
a
receptor as
a
downstream act
and summarized
of where
for
a
growth signaling
to alter
transcription to
deregulate cell-cell
activated onco-
they may
factor, molecule.
of genes
the cell
cycle.
interactions, tively,
these act
as
a
Other crucial
Still or
G-protein, oncoproteins
for oncogenesis
other the
mechanisms of
cancer cells
replicative shown
or oncoproteins
process explain
in potential,
pathways, of
many
Figure affect
Collec- of
the
56-1, their
their ability
sion of
Certain tumor
contain oncogenes.
the retrovirus
genes.
major such
constitutively to
invade viruses
as their
activated and
spread,
(eg, features
limitless signaling
and retroviruses,
It was
the
RSV)
Further were
derived study
that study
from oncogenes,
first showed
normal which
their eva-
papovaviruses)
of such
tumor revealed
the that
many cellular
the tumor
viruses
(eg, presence
retroviral genes,
viruses of
onco- oncogenes

the so-called
had picked
proto- up
during their
passage through
host cells.

A
tumor suppressor
gene produces
a
normally suppresses
altered by
mutation, diminished.
or cell
division.
the inhibitory
This effect
loss-of-function leads
to increased
by
AG
Knudson cell
growth or
in1971, based
on
When such
a
of its
gene is
product of
a
tumor division.
is lost
suppressor
As studies
of or
gene first
suggested the
inheritance of
t(8;14)(q24;q32)

FIGURE 56-5 (A) Schematic representation of how promoter insertion may activate a proto-oncogene. (1) Normal chicken chromosome showing a nearly inactive MYC gene. (2) An avian leukemia virus has integrated in the chromosome in its proviral form (a DNA copy of its RNA genome) adjacent to the MYC gene with the RSV right-hand long terminal repeat (LTR) sequence element, which contains both a strong enhancer and promoter (see Chapter 36) now resides just upstream of the MYC gene and therefore activates MYC, resulting in vigorous transcription of the MYC mRNA. For simplicity, only one strand of DNA is depicted and other details have been omitted. (B) Schematic representation of the reciprocal translocation involved in Burkitt lymphoma. The chromosomes involved are 8 and 14. Letters p, q refer to the short and long arms of the chromosomes, while the white oval labeled CEN, is the centeromere. Only a portion of each chromosome is shown. A segment from the end of the q arm of chromosome 8 breaks off and translocates to chromosome 14. The reverse process moves a small segment from the q arm of chromosome 14 to chromosome 8; these translocations involve regions q24 (chromosome 8) and q32 (chromosome 14). The MYC gene is contained in the small piece of chromosome 8 that was transferred to chromosome 14. Thus, MYC is placed adjacent to the genes transcribing the heavy chains of immunoglobulin molecules, and MYC gene transcription is activated by the potent IgG H gene enhancer. Many other translocations have been identified, with perhaps the best known being that involved in formation of the Philadelphia chromosome (see the Glossary). (Revised and redrawn from Dalla-Favera et al. Human c-myc onc gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Nat Acad Sci USA 1982;79:7824-7827, for more detail.)
retinoblastomas, both copies of a tumor suppressor gene must be affected for it to lose its inhibitory effects on growth (ie,a mutated loss-of-function allele, rb', is recessive to a wild-type RB copy of the gene).
_

A useful distinction has been made between gatekeeper and caretaker functions of tumor suppressor genes. Gatekeeper genes (products) control cell proliferation, and include mainly genes that act to regulate the cell cycle and apoptosis.
By contrast, caretaker gene products are concerned with preserving the integrity of the genome, and include genes whose protein products are involved in recognizing and correcting DNA damage and in maintaining chromosomal integrity during cell division.
Many oncogenes
and tumor
suppressor genes
have now
been identified.
Only differences
between
a
few are
mentioned oncogenes
here.
and tumor
are listed
in
Table
56-5.
Table
56-6
lists some
properties of

Mutation in one of the two alleles is sufficient to produce oncogenic effects., Tumor Suppressor Genes = Mutations in both alleles are required to produce oncogenic effects.. Oncogenic effect is due to gain-of-function of a protein that stimulates cell growth and proliferation, Tumor Suppressor Genes = Oncogenic effect is due to loss-of-function of a protein that inhibits cell growth and proliferation.. Mutations in oncogenes arise in somatic cells, and hence are not inherited., Tumor Suppressor Genes = Mutations in tumor suppressor genes may be present in somatic or germ cells (and hence, may be inherited). Usually do not show tissue preference with respect to the type of cancers in which they are found, Tumor Suppressor Genes = Often demonstrate strong tissue preference with respect to the type of cancers in which they are found (eg, mutations in the RB gene result in retinoblastoma)
Source: Data from Levine AJ: The p53 tumor suppressor gene. N Engl J Med
1992;326:1350.

MYC, Properties = An oncogene (OMIM 190080) that encodes a transcription factor that can alter transcription of many key cellular regulatory genes. MYC is involved in stimulation of cell growth, cell cycle progression, and DNAreplication. It is mutated in a variety of tumors.. P53, Properties = A tumor suppressor gene (OMIM 191170) that is activated in response to various stimuli that produce DNAdamage. Activation of p53 induces cell cycle arrest, apoptosis, senescence, and DNArepair. It is also involved in some aspects of regulation of cellular metabolism. It has, therefore, been named'the guardian of the genome.'p53 is mutated in about 50% of human tumors.. RAS, Properties = A family of oncogenes encoding small G-proteins, specifically GTPases that were initially identified as the transforming genes present in certain murine sarcoma viruses. Important members of the family are K-RAS (Kirsten), H-RAS (Harvey) (OMIM 190020), and N-RAS (neuroblastoma). Persistent activation of these genes due to mutations contributes to the development of a variety of cancers.. RB, Properties = A tumor suppressor gene (OMIM 180200) encoding the Rb protein. Rb, a transcriptional repressor, regulates the cell cycle by binding to the activating transcription factor E2F. Rb represses the transcription of various genes involved in the transition of the G 1 phase of the cell cycle to the S phase. Mutation of the RB gene results in retinoblastoma, but it is also involved in the genesis of certain other tumors (see Chapter 35).
two of the most intensively studied oncogenes (MYC and RAS), and two of the most highly studied tumor suppressor genes (P53 and RB).
or
Chromosomal microsattelite
instability
———>

FIGURE 56-7 Multistep genetic changes associated with the development of colorectal cancers. Mutations in the APC gene initiate the formation of adenomas. One sequence of mutations in an oncogene and in various tumor suppressor genes that can result in further progression to large adenomas and cancer is indicated. Patients with familial adenomatous polyposis (OMIM 175100) inherit mutations in the APC gene and develop numerous dysplastic aberrant crypt foci (ACF), some of which progress as they acquire the other mutations indicated in the figure. The tumors from patients with hereditary nonpolyposis colon cancer (OMIM 120435) go through a similar, though not identical, series of mutations; mutations in the DNA mismatch repair system (see Chapter 35) speed up this process. K-RAS, BRAF, and PI3KCA are oncogenes; the other genes listed are tumor suppressor genes. The sequence of events shown here is not invariable in the development of all colorectal cancers. A variety of other genetic alterations have been described in a small fraction of advanced colorectal cancers. These may be responsible for the heterogeneity of biologic and clinical properties observed among different cases. Instability of chromosomes and microsatellites (see Chapter 35) occurs in many tumors, and likely involves mutations in a considerable number of genes. (Reproduced, with permission, from Bunz F, Kinzler KW, Vogelstein B: Colorectal tumors, Fig. 48-2, The Online Metabolic & Molecular Bases of Inherited Disease, www.ommbid.com)
stages of the sequence of events shown. The functions of the various genes identified are listed in Table 56-7. The overall sequence of changes can vary from that shown and other genes may also be involved. Similar studies have been performed on a number of other human tumors, revealing different patterns of activation of oncogenes, and mutations of tumor suppressor genes. Further mutations in these and other genes are involved in tumor progression, a phenomenon whereby clones of tumor cells get selected for fast growth rate and ability to metastasize. Thus, a relatively large tumor may contain a variety of cells with different genotypes, making successful treatment more difficult.

Several conclusions can be drawn from these results and those from other similar studies. First, cancer is truly a genetic disease, but in a somewhat different sense from the normal meaning of the phrase, insofar as many of the gene alterations are due to somatic mutations. Second, carcinogenesis is a multistep process. It is estimated that in most cases a minimum of five to six genes must be mutated for cancer to occur. Third, additional subsequent mutations are thought to confer selective advantages on clones of cells, some of which acquire the ability to metastasize (see below). Finally, many of the genes implicated in colorectal carcinogenesis and other types of cancers are involved in cell signaling events, showing again the central role that alterations in signaling play in the development of cancer.

APC (OMIM 611731), Action of Encoded Protein = Antagonizes WNT b signaling; if mutated, WNTsignaling is enhanced, stimulating cell growth.. β -CATENIN (OMIM 116806), Action of Encoded Protein = Encodes β-catenin, a protein present in adherens junctions, which are important for the integrity of epithelial tissues. It is an integral part of the WNTsignaling pathway.. K-RAS (OMIM 601599), Action of Encoded Protein = Involved in tyrosine kinase signaling, especially in the mitogen-activated protein (MAP) kinase pathway.. BRAF (OMIM 164757), Action of Encoded Protein = A serine/threonine kinase that acts in concert with Ras to activate the MAPkinase pathway.. SMAD4 (OMIM 600993), Action of Encoded Protein = Involved in intracellular signaling by transforming growth factor β (TGF-β c ).. TGF- β RII, Action of Encoded Protein = Acts as a receptor for TGF-β.. PI3KCA (OMIM 171834), Action of Encoded Protein = Acts as a catalytic subunit of phosphatidylinositol 3-kinase (PI3K).. PTEN (OMIM 601728), Action of Encoded Protein = A protein tyrosine phosphatase which acts as an important inhibitor of signaling via the PI3K-Akt pathway.. P53 (OMIM 191170), Action of Encoded Protein = The product, p53, is induced in response to DNAdamage and is also a transcription factor for many genes involved in cell division (see Chapter 35, Table 56-10).. BAX (OMIM 600040), Action of Encoded Protein = Acts to induce cell death (activator of apoptosis).. PRL3 (OMIM 606449), Action of Encoded Protein = A protein tyrosine phosphatase involved in cell cycle regulation.

Abbreviations: APC, adenomatous polyposis coli gene; BAX, encodes BCL2-associated X protein (BCL2 is a repressor of apoptosis); BRAF, the human homolog of an avian protooncogene; K-RAS, Kirsten-Ras-associated gene; PI3KCA, encodes the catalytic subunit of phosphatidylinositol 3-kinase; PRL3, encodes a protein tyrosine phosphatase with homology to PRL1, another protein tyrosine phosphatase found in regenerating liver; PTEN, encodes a protein tyrosine phosphatase and tensin homolog; P53, encodes p53, a polypeptide of molecular mass ~53 kDa; SMAD4, the homolog of a gene found in Drosophila.
°*K-RAS, BRAF, and PI3KCA are oncogenes; the other genes listed are either tumor suppressor genes or genes the products of which are associated with the actions of the prod- ucts of tumor suppressor genes.
'The WNT family of secreted glycoproteins is involved in a variety of developmental processes.
'TGF-B is a polypeptide (a growth factor) that regulates proliferation and differentiation in many cell types.
Note: The various genes listed are either oncogenes, tumor suppressor genes, or genes the products of which are closely associated with the products of these two types of genes. The cumulative effects of mutations in the genes listed are to drive colonic epithelial cells to proliferate and eventually become cancerous. They achieve this mainly via effects on various signaling pathways that affect cellular proliferation. Other genes and proteins not listed here are also involved. This table and Figure 56-7 vividly illustrate the importance of a detailed knowledge of cell signaling for understanding the genesis of cancer.

A large variety of polypeptide growth factors that affect human tissues and cells have been identified. Some are listed in Table 56-8. Here, we focus mostly on their relationship with cancer.
Growth factors can act in an endocrine, paracrine, or autocrine manner (see Chapter 41) and affect a wide variety of cells to produce a mitogenic response (see Chapter 42). As described earlier (see Chapter 53), growth factors play important roles in the differentiation of hematopoietic cells.
Growth inhibitory
factors also
exist.
trans- forming growth factor 6 (TGF-) exerts inhibitory effects on the growth of certain cells. Thus, chronic exposure to either increased amounts of a growth factor, or to decreased amounts of a growth inhibitory factor, can alter the balance of cellular growth.

Growth factors produce their effects by interacting with specific receptors on cell surfaces, initiating various signaling events (see Chapter 42). Genes encoding receptors for growth factors have been identified and characterized. These receptors

Epidermal growth factor (EGF), Function = Stimulates growth of many epidermal and epithelial cells. Erythropoietin (EPO), Function = Regulates development of early erythropoietic cells. Fibroblast growth factors (FGFs), Function = Promote proliferation of many different cells. Interleukins, Function = Interleukins exert a variety of effects on cells of the immune system. Nerve growth factor (NGF), Function = Trophic effect on certain neurons. Platelet-derived growth factor (PDGF), Function = Stimulates growth of mesenchymal and glial cells. Transforming growth factor α (TGF-α), Function = Similar to EGF. Transforming growth factor β (TGF-β), Function = Exerts both stimulatory and inhibitory effects on certain cells
Note: Many other growth factors have also been identified. Growth factors may be produced by a variety of cells, or may have mainly one source. Many different interleukins have now been isolated; along with the interferons and some other proteins/polypeptides, they are referred to as cytokines.
generally have short membrane-spanning segments and external and cytoplasmic domains (see Chapters 40, 42), and a number (eg, those for epidermal growth factor [EGF], insulin and platelet-derived growth factor [PDGF]) have tyrosine kinase activity. The kinase activity, located in the cytoplasmic domains of these receptors, causes autophosphorylation of the receptor protein and also phosphorylates certain other proteins.

Consideration of platelet-derivd growth factor, PDGF, illustrates how one particular growth factor brings about its effects. Interaction of PDGF with its receptor stimulates the activity of phospholipase C (PLC). PLC splits phosphatidylinositol bisphosphate (PIP,) (found in biologic cell membranes) into inositol trisphosphate (IP,) and diacylglycerol (DAG) (see Figure 42-6). Elevations in IP, levels result in increased levels of intracellular Ca**, while DAG activates protein kinase C (PKC). Hydrolysis of DAG may release arachidonic acid, which can stimulate production of prostaglandins and leukotrienes, each of which has various biologic effects. Exposure of target cells to PDGF can result in rapid (minutes to 1-2 h) activation of the expression of the genes encoding certain cellular proto-oncogenes (eg, MYC and FOS) that participate in stimulation of mitosis via effects on the cell cycle (see below). The bottom line is that growth factors interact with specific receptors to stimulate specific signaling pathways that serve to increase or decrease the activities of various genes that affect cell division.

Micro-RNAs (miRNA), discovered in 1993, are nonproteincoding RNA molecules that are ~22 nucleotides long (see Chapters 34, 36). miRNAs are expressed in different tissue and cell types and are known to be involved in regulation of gene expression by decreasing mRNA translation or mRNA stability (see Figures 36-17).
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MicroRNAs have also been shown to influence extrinsic factors that modulate tumors. These include interactions between the immune system and cancer cells, interactions of stromal cells, effect on oncoviruses, etc. It is likely that the balance of protein expression levels resulting from the exact miRNAs and their concentrations determines whether the net effect of a specific miRNA is oncogenic or tumor suppressive. The use of miRNAs in clinical applications is gaining ground. miRNAs are being investigated as biomarkers for cancer diagnosis, prognosis, and classification. These small RNAs also hold promise in therapy, where oligonucleotides are used to enhance expression of tumor suppressor miRNAs in cells or antisense oligonucleotides are employed to counter the action of oncogenic miRNAs. Several such miRNA-targeted therapeutics have been developed. These include a mimic of miR-34, which has now reached clinical trials. Interestingly, an anti-miR against miR-122 is also in clinical trials for the treatment of hepatitis.
Another important clinic-based application of miRNAs lies in their ability to potentiate therapeutic responses during anticancer chemotherapy and radiotherapy. Thus, the use of miRNAs as specific targets for treatment and/or for therapeutic supplementation in the management of cancer appears highly promising. However, as with all gene-based therapies effective delivery of stable oligonucleotides into appropriate target cells is extremely challenging. Nevertheless, tremendous advances in scientific and pharmaceutical developments are anticipated in this area in the years ahead, bringing to clinical practice a group of new and novel therapeutic agents for the treatment of cancers.

Extracellular vesicles (EVs; aka exosomes and microvesicles) are a group of small vesicles with a lipid bilayer that are released by a wide range of cells, both normal and diseased. These vesicles vary from one another in their size and the way in which they are formed (see Figure 40-23). Collectively, noncoding RNAs (ncRNA) (comprising miRNAs, long noncoding RNAs {IncRNAs], and circular RNAs [circRNAs]; see Chapters 34, 36) can be loaded into EVs, which are then secreted. Such EVassociated ncRNAs have been shown to exert paracrine effects on surrounding cells (see Figure 40-23) thereby facilitating intercellular communication. EVs have been shown to transfer information to target cells by mechanisms such as receptorligand binding, direct fusion of the EV with the plasma membrane of recipient cells, or by endocytosis. Transfer of ncRNAs in this way has been implicated in the pathogenesis of several diseases; cancer is one such condition, where laboratory studies have shown that EVs can modulate expression of crucial genes that are involved in tumorigenesis. The effects produced by ncRNA their cells on target include control of cell proliferation, induction of angiogenesis, alterations in the tumor microenvironment and tumor immunity, and even induction of drug resistance. Such EVs
have also been shown to act on distant organs, thereby affecting metastasis.
EV-associated ncRNAs are highly stable. They are found in blood, urine, and saliva. Hence, they have the potential to be used as noninvasive biomarkers to aid in diagnosis and prognosis of cancers. In addition, due to their small size, and lipid bilayer constitution, EVs can readily cross biologic membranes. Consequently, EVs may be used as effective delivery systems to transfer specific molecules (miRNA or anti-miRNA) that can be used in the treatment of disease. Further in vivo work with model systems will be required to explore this exciting new area of oncology.

There is growing evidence that epigenetic mechanisms (see Chapter 36) are involved in the causation of cancer. Such mechanisms produce nonmutational changes that affect regulation of gene expression. Methylation of specific cytosine bases in genes is implicated in turning off activities of certain genes. Changes in the normal pattern of methylation/demethylation of cytosine residues in specific genes have been detected in cancer cells. Histone posttranslational modifications (ie, acetylation, ADP-ribosylation, methylation, phosphorylation, sumoylation, and ubiquitylation; see Chapters 35, 38), for example, changes in acetylation of histones H3 and H4, which affect gene transcription, have been found in cancer cells. Mutations affecting the structure and/or activity of nucleosome remodeling complexes (ie, SWI/SNF complex) involved in chromatin remodeling can also affect gene transcription. Indeed, several of the components of the SWI/SNF complexes may act as tumor suppressor genes.
A matter of particular interest regarding epigenetic changes in gene expression is that many of these protein and DNA modifications are potentially reversible. In this regard, 5-azadeoxycytidine and decitabine are inhibitors of DNA methyltransferases (DNMTs), while valproic acid and vorinostat act to inhibit histone deacetylases (HDACs). Both of these agents have been used to treat certain types of leukemias and lymphomas and are thought to work by derepressing the transcription of certain critical growth-regulatory genes, such as tumor suppressors. The increasing use of various -omic technologies to study epigenetic changes in many types of cancers will likely add considerably to knowledge in this area, while hopefully driving the rationale for new chemotherapeutic development.

It has been known for many years that certain cancers have a hereditary basis. Depending on the specific cancer type, it has been estimated that 5 to 15% of cancers have a hereditary etiology. The discovery of oncogenes and tumor suppressor genes has allowed investigations of the basis of this phenomenon.

Knowledge of the cell cycle is necessary for understanding the molecular mechanisms involved in the development of many types cancer. It is also of importance because many anticancer drugs act only against cells that are dividing, or in a certain phase of the cycle.
Basic aspects of the cell cycle were described in Chapter 35. As shown in Figure 35-20, the cycle has four phases: G,, S, G,, and M. If cells are not in one of these phases, they are said to be in the G, phase and are termed quiescent. Cells can be recruited into the cycle from G, by various influences (eg, certain growth factors). Cancer cells usually have a shorter generation time than normal cells, and there are fewer of them in the quiescent G, phase.
The roles of various cyclins, cyclin-dependent kinases (CDKs), and a number of other important molecules that affect the cell cycle (eg, the protein products of the RB and P53 genes) were also described in Chapter 35. The points in the cycle, at which some of these molecules act, are indicated in Figures 35-20, 35-21 and Table 35-7. Because a major property of cancer cells is uncontrolled growth, many aspects of their cell cycle have been studied in

Retinoblastoma (MIM 180200), Gene = RB1. Retinoblastoma (MIM 180200), Major Function = See Table 56-6. Retinoblastoma (MIM 180200), Major Clinical Feature = Hereditary or sporadic retinoblastoma a
*In hereditary retinoblastoma, one allele is mutated in the germ line, requiring only one subsequent mutation for a tumor to form. In sporadic retinoblastoma, neither allele is mutated at birth, so that subsequent mutations in both alleles are required for a tumor to develop. Many other hereditary cancer conditions have also been identified.
Many hereditary types of cancer have now been recognized; a few of these are listed in Table 56-9. In a number of cases, where a hereditary syndrome is suspected, appropriate genetic screening of families has allowed early interventions to be made. For example, some young women who have inherited either a mutated BRCA1 or BRCA2 gene have opted for prophylactic mastectomies to prevent cancer of the breast occurring in later life.
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As referred to above and also later in this chapter, cancer cell genomes carry many mutations. One possible explanation for their genomic instability is that they have a mutator phenotype. The idea of mutator phenotypes was originally postulated by Loeb and colleagues, who argued that mutator phenotypes were caused by cancer cells having acquired mutations in genes involved in DNA replication and DNA
thus
repair, thus allowing mutations to accumulate. The concept was later expanded to include mutations that affect chromosomal segregation, DNA damage surveillance, and processes such as apoptosis. Increasing evidence suggests that collectively these various mechanisms, termed errors in genome replicative functions, contribute to a very large fraction of cancers.
The term genomic instability is frequently used to refer to two abnormalities shown by many cancer cells, microsatellite and chromosomal instability (CIN). Microsatellite instability was described briefly in Chapter 35. It involves expansion or contraction of microsatellite DNA sequences, usually due to abnormalities of mismatch repair or replication slippage. CIN occurs more often than microsatellite instability, and the two are often mutually exclusive. CIN refers to gain or loss of chromosomes caused by abnormalities of chromosomal segregation during mitosis.
Another area of interest regarding CIN is copy number variation (CNV) (see Glossary; Chapter 39). Associations of various CNVs with many cancers have been identified, and their precise roles in cancer are under investigation.
An important
aspect of
CIN
is aneuploidy,
a
very com-
mon feature of solid tumors. Aneuploidy exists when the chromosomal number of a cell is not a multiple of the haploid number. The degree of aneuploidy often correlates with a poor prognosis. This has suggested that abnormalities of chromosomal segregation may contribute to tumor progression by increasing genetic diversity. Some scientists believe that aneuploidy is a fundamental aspect of cancer.

Much research is aimed at determining the basis of CIN and aneuploidy. As shown in Figure 56-8, a number of different processes are involved in normal chromosomal segregation. Each process is complex, and involves various organelles and many individual proteins. A textbook of cell biology should be consulted for details of the process of chromosomal segregation and cell division. Studies are in progress to compare these processes in normal and tumor cells, and to determine which of the differences detected may be contributors to CIN and aneuploidy. One hope of this line of research is that it might be possible to develop drugs that diminish or even prevent CIN and aneuploidy.
FIGURE 56-8 Factors involved in chromosomal segregation, which are relevant to understanding chromosomal instability (CIN) and aneuploidy. CIN syndromes include Bloom syndrome (OMIM 210900) and others. (Based on Thompson SL, et al: Mechanisms of chromosomal instability. Curr Biol 2010;20(6):R285.)

There has been considerable interest in the involvement of telomeres in a number of diseases and also in aging (see Chapters 35, 57). With respect to cancer, when tumor cells divide rapidly their telomeres often shorten. Such hortened telomeres have been implicated as a risk factor for many, but not all, solid tumors (eg, breast cancer). Short telomeres appear to be of predictive value with regard to the progression of chronic inflammatory diseases (such as ulcerative colitis and Barrett esophagus) to cancer. Abnormalities of telomere structure and function can contribute to CIN (see above). The activity of telomerase, the main enzyme involved in synthesizing, and thus maintaining the length of telomeres, is very low in normal somatic cells, but is frequently elevated in cancer cells, providing a mechanism for overcoming telomere shortening. Hence, inhibiting telomerase activity represents an attractive target of cancer chemotherapy as most cancer cells exhibit elevated activity of this enzyme, unlike normal somatic cells that display very low activity. However, any such inhibitor would also adversely affect normal stem cells (a ubiquitous class of essential cells found in most tissue types and that require telomerase activity for their regenerative function). This is a major limitation to such an approach. However, Imetelstat (GRN163L) is one such agent that has reached clinical trials. Time will tell if telomerase is an effective target for chemotherapy.

Apoptosis is a genetically directed program, which when activated, causes cell death. The main proteins involved in apoptosis are proteolytic enzymes termed caspases that normally exist in inactive forms, the procaspases. The name caspase is derived from the fact that these enzymes utilize an active site cysteine to cleave polypeptide chains immediately after aspartic residues. About 15 human caspases are known, although not all participate in apoptosis. When those involved in apoptosis are activated (mainly caspases 2, 3, 6, 7, 8, 9, and 10), they participate in a cascade of events (compare with the coagulation cascade, Chapter 55) that ultimately kills cells by digesting various proteins and other molecules. The upstream caspases (eg, 2, 8, and 10) at the beginning of the cascade are often called initiators, and those downstream at the end of the pathway (caspases 3, 6, and 7) are called effectors or executioners. A caspase-activated DNase (CAD) fragments DNA, thereby producing a characteristic DNA laddering pattern that is readily detected by native gel electrophoresis. Microscopic features of apoptosis include condensation of chromatin, changes of nuclear shape, and membrane blebbing. The resulting dead
cells are rapidly disposed of by phagocytic activity, avoiding an inflammatory reaction.
Apoptosis differs from necrosis, a pathologic form of cell death that is not genetically programmed. Necrosis occurs on exposure to external agents, such as certain chemicals and extreme heat (eg, burns). Various hydrolytic enzymes (proteases, phospholipases, nucleases, etc) are involved in necrosis. Release of cell contents from dying cells can cause local inflammation, unlike apoptosis.
The overall process of apoptosis is complex and tightly regulated. The apoptotic regulatory pathway involves proteins that act as receptors and adapters, procaspases and caspases, and proand anti-apoptotic factors. There are two major pathways, extrinsic (death receptor) and intrinsic, with mitochondria being important participants in the latter pathway.
Figure 56-9 shows a simplified diagram of some of the key events in apoptosis.

Major features of the death receptor pathway are shown on the left-hand side of the figure. External signals initiating apoptosis include tumor necrosis factor a (TNF-a) and Fas ligand. A number of death receptors have been identified. These receptors are transmembrane proteins, some of which interact with adapter proteins (such as FADD [Fas-associated protein with death domain]). These complexes in turn interact with procaspase-8, resulting in its conversion to caspase-8 (an initiator). Caspase-3 (an effector) is activated via a series of further reactions. It digests important structural proteins such as lamin (associated with nuclear condensation), various cytoskeletal proteins, and enzymes involved in DNA repair, causing cell death.
Membrane blebs

FIGURE 56-9 Simplified scheme of apoptosis. The major molecular events in the extrinsic pathway. Death signals include TNF-a and FAS (present on the surface of lymphocytes and some other cells). The signals (ligands) interact with specific death receptors (there are a number of them) Left. The activated receptor then interacts with an adapter protein (FADD is one of a number of them), and then forms a complex with procaspase 8. (The complex is indicated by the ... between the receptor and procaspase-8 in the figure). Through a series of further steps, active caspase-3 is formed, which is a major effector (executioner) of cell damage. Regulation of the extrinsic pathway can occur due to the inhibitory effect of FLIP on the conversion of procaspase-8 to caspase-8, and also the inhibitory effect of IAP on procaspase-3. The major cellular events in the intrinsic (mt) pathway. Various cell stresses affect the permeability of the mt outer membrane, resulting in efflux of cytochrome cinto the cytoplasm. This forms a multiprotein complex with APAF-1 and procaspase-9, called an apoptosome. Through these interactions, procaspase-9 is converted to caspase-9. This, in turn, can act on procaspase-3 to convert it to its active form. Regulation of the intrinsic pathway can occur at the level of BAX, which facilitates increasing mitochondrial permeability permitting efflux of cytochrome c, and is thus proapoptotic. BCL-2 opposes this effect of BAX and is thus anti-apoptotic. IAP also inhibits procaspase-9, and this effect of IAP can be overcome by SMAC. (APAF-1, apoptotic protease activating factor-1; BAX, BCL-2-associated X protein; BCL-2, B-cell CLL/lymphoma 2 (CLL represents chronic lymphatic leukemia); FADD, FAS-associated via death domain; FAS, FAS antigen; FLICE, FADD-like ICE; FLIP, FLICE inhibitory protein; IAP, inhibitor of apoptosis proteins; ICE,

interleukin-1-B convertase; SMAC, second mitochondria-derived activator of caspase.) — signifies opposes the action
Regulation of this pathway occurs at several levels. FLIP inhibits the conversion of procaspase-8 to its active form. Inhibitors of apoptosis (IAPs) inhibit the conversion of procaspase-3 to its active form. These effects can be overcome by the protein SMAC (second mitochondria-derived activator of caspase), which is released from mitochondria.
The mitochondrial pathway can be initiated by exposure to reactive oxygen species, DNA damage and other stimuli. Initiation results in the formation of pores in the outer mitochondrial membrane, through which cytochrome C escapes into the cytoplasm. In the cytoplasm, cytochrome C interacts with APAF-1, procaspase-9, and ATP to form a multiprotein complex known as the apoptosome. As a result of this interaction, procaspase-9 is converted to its active form and, in turn, acts on procaspase-3 to produce caspase-3.
Activation of the p53 gene upregulates transcription of BAX. The BAX protein is pro-apoptotic; it causes loss of the mitochondrial membrane potential, thus helping initiate the mitochondrial apoptotic pathway. On the other hand, BCL-2 inhibits this loss of membrane potential, and is thus anti-apoptotic. IAPs inhibit conversion of procaspase-9 to caspase-9; SMAC can overcome this. Note that the death pathway uses caspase-8 as an initiator, whereas the mitochondrial pathway uses caspase-9. These two pathways can interact. In addition, there are also other pathways of apoptosis, which are not discussed here.

Cancer cells have developed mechanisms to evade apoptosis, and thus continue to grow and divide. In general, these mechanisms involve mutations that cause loss-of-function of proteins that are pro-apoptotic, or from overexpression of anti-apoptotic genes. One such example concerns lossof-function of the P53 gene, perhaps the most commonly mutated gene in cancers. Resultant loss of upregulation of transcription of pro-apoptotic BAX (see above) shifts the balance in favor of anti-apoptotic proteins. Overexpression of many anti-apoptotic genes is a frequent finding in cancers. The consequent evasion of apoptosis favors the continuing growth of cancer cells. Attempts are being made to develop drugs that will specifically turn on apoptosis in cancer cells, terminating their lifespans.
As indicated above, apoptosis is a complex, highly regulated pathway with numerous participants, many of which are not mentioned here in this abbreviated account. Apoptosis is also involved in various developmental and physiologic processes. It may seem paradoxical, but regulated cell death is as important in maintaining health, as is formation of new cells. In addition to cancer, apoptosis is implicated in other diseases, including certain autoimmune and chronic neurologic disorders, such as Alzheimer disease and Parkinson disease, where excessive cell death (rather than excessive growth) is a feature. Table 56-10 summarizes some of the principal features of apoptosis.

- + It involves a genetically programmed series of events and differs from necrosis, which is a result of direct cellular damage.
+
The series
events involved
is
a
cascade, similar
to the
process of
+ It is characterized by cell shrinking, membrane blebbing, absence of inflammation, and a distinct electrophoresis pattern (laddering) of degraded DNA.
+ Many caspases (proteinases) are involved; some are initiators and others act as effectors (executioners).
+ Apoptosis can occur either by extrinsic (death receptor-mediated) or intrinsic (mitochondrial) pathways.
+ FAS and other receptors are involved in the extrinsic pathway of apoptosis.
+ Cellular stress and other factors activate the intrinsic (mitochon-
drial)
pathway of
apoptosis;
release of
c
into the
cytoplasm is
event in
this pathway.
+ Apoptosis is regulated by a balance between inhibitors (antiapoptotic factors) and activators (pro-apoptotic factors) of the process.
+ Acquired mutations found in cancer cells enable them to evade apoptosis, thus promoting cell proliferation.

Unlike apoptosis, necrosis of tissue results in release of intracellular contents into the surrounding microenvironment. These include molecules that serve as mediators of the proinflammatory response resulting in the infiltration of tissue by immune inflammatory cells. It has been shown that such cells can have active tumor-promoting effects. Immune inflammatory cells have been reported to promote angiogenesis, cell proliferation, and invasiveness. Necrosis, which appears to counter the proliferative tendency of cancerous cells, may paradoxically benefit tumorigenesis. Thus, developing tumors appear to gain by tolerance of some degree of cell necrosis, because this results in recruitment of inflammatory cells that ultimately supply the tumor cells with growth-promoting factors via angiogenesis.

The tumor microenvironment (TME) consists not only of cancer cells, but also a variety of noncancer cells and components of the extracellular matrix. Complex intercellular and stromal interactions occurring in the TME play important roles in cancer cell proliferation, survival, spread (metastasis), and response to treatment. The major noncancer cells present in the TME include those of the immune system (T and B lymphocytes, natural killer cells, and macrophages) and mesenchymal cells (stem cells, myofibroblasts, endothelial cells, and adipocytes).
Although a variety of immune cells infiltrate tumors, their antitumor effector functions are dampened in response to tumor-derived signals. Furthermore, T lymphocytes and macrophages are reprogrammed (eg, by sustained nuclear factor kappa B [NF-«B] activation; see Figure 42-10) to promote tumor growth and survival. This allows cancer cells to escape immune surveillance and also co-opts the immune system to accelerate cancer progression. A greater understanding of the role of immune cells in the TME in driving carcinogenesis has opened up a new and important avenue of cancer treatment called immunotherapy (discussed in more detail later in the chapter).
Mesenchymal cells, such as the myofibroblasts and mesenchymal stem cells, are known to facilitate formation of cancer stem cell niches, thus aiding cancer stem cell survival and proliferation. Other mesenchymal cells, such as endothelial cells, respond to paracrine signals in the TME (eg, vascular endothelial growth factor [VEGF]) by promoting tumor angiogenesis and metastasis. Adipocytes in the TME secrete a number of growth factors that support tumor growth. In addition, the extracellular matrix of the TME also contributes to tumor progression by facilitating the formation of cancer stem cell niches, aiding tumor invasion and metastasis. Figure 56-10 shows the typical constituents of the TME.
constitute a typical tumor. Understanding the complex interactions between these cells and their microenvironment is an important aspect of ongoing research in cancer.

In order to survive, and ultimately grow and proliferate to form a tumor mass tumor cells must develop the ability to procure all necessary nutrients from typically hypoxic and nutrient-poor environments. Given these immutable facts, it is not surprising that mulitiple transcriptomic (RNA-seq) studies have shown that genes that code for proteins involved in nutrient capture, uptake and metabolism are often mutated, and/ or over-/underexpressed in different types of tumors. These observations have reinvigorated research into metabolism in general, and in cancer cells in particular.
Overall, it is important to note that tumors are not simply a collection of cancer cells; a variety of different types of cells, consisting of both transformed and nontransformed cells,
Glucose and the amino acid glutamine are two of the most abundant metabolites in plasma. Together, they account for much of the carbon and nitrogen metabolism in human cells. In 1924, the biochemist Otto Warburg and his colleagues made the discovery that cancer cells take up large amounts of glucose and metabolize it via glycolysis to lactic acid, even in the presence of oxygen. This observation was termed the Warburg effect. Based on these data, Warburg proposed two hypotheses: first, that the increased ratio of glycolysis to aerobic respiration was likely to be due to defects in the mitochondrial

FIGURE 56-10 The tumor microenvironment contributes critically to tumor cell growth. Each cell type depicted affects the tumor by secreting factors that stimulate the formation of cancer stem cells (CSCs) and helping maintain the residence of already formed CSCs in the stem cell state. Summarized here are some of the major cell types and their secreted factors, listed by cell type, that are known to have an impact on CSCs and cancer cells. (CXCL7, CXC-chemokine ligand 7; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IL-6, interleukin 6; MMP, matrix metalloproteinase; MSC, mesenchymal stem cell; OncoM, oncostatin M; PDGF, platelet-derived growth factor; PGE,, prostaglandin E,; SDF1, stromal cell-derived factor 1; TGF-B, transforming growth factor B.) (Adapted from Pattabiraman DR, Weinberg RA. Tackling the cancer stem cells—what challenges do they pose? Nat Rev Drug Discov 2014 Jul;13(7):497-512.)
respiratory chain; and second, that enhanced rates of glycolysis enabled cancer cells to preferentially proliferate in the reduced oxygen tension often seen in tumors. Furthermore, Warburg argued that the switch from aerobic to anaerobic glucose metabolism was a/the driver of tumorigenesis.

It is now thought that the reprogramming of mitochondrial respiration typically observed in tumor cells is a direct effect of at least two kinds of influences, rather than to overt defects in mitochondria. The first of these is the self-sustaining signaling by growth factors that characterizes cancer cells, which causes increased cellular proliferation (Figures 56-1 and 56-2). The second of these are the genetic changes in specific metabolic enzyme-encoding genes, metabolite transporters, and other related genes. These genetic alterations include preferential expression of certain mRNA splice variants, amplification of particular enzyme-encoding genes, as well as altered catalytic efficiencies and specificities and metabolic products. Collectively, the resulting changes elicit important metabolic rewiring as well as epigenetic changes in the activity of the transcription machinery (ie, DNA and protein methylation, acetylation, and other posttranslational modifications) that lead to more efficient cellular anabolism and alteration of the tumor microenvironment, which in toto allow tumors to proliferate.

An example of tumor cell-specific metabolic rewiring can be illustrated by the example of pyruvate kinase. There are two isozymes, PKM1 and PKM2, which are encoded by the glycolytic pyruvate kinase muscle gene, PKM. PKM1 and PKM2 are generated by alternative splicing (see Chapter 38). Unlike PKM1, which is expressed constitutively in normal cells, PKM2 is often more highly expressed in cancer cells. Likely more important though is the fact that PKM2 exists in either a dimeric form that exhibits very low catalytic activity, or a tetrametic form, with high catalytic activity. PKM2 in cancer cells is most often in the low activity, dimeric form, which results in the accumulation of glycolytic intermediates. These intermediates allow cancer cells to synthesize macromolecules that support rapid proliferation (as proposed in the original Warburg hypothesis). Such metabolic enzyme reprogramming ultimately leads to less shuttling of glucose-derived chemical energy into the production of ATP (Figure 56-11), with a concomitant shunting of such energy into building up the cellular biomass of proteins, lipids, nucleic acids, etc. These macromolecules are essential for cell proliferation (in this case, proliferation of cancer cells). Collectively, these observations help explain why a high rate of glycolysis confers a selective advantage on tumor cells. Based on such observations, a current promising approach is to analyze blood and urine samples by mass spectrometry to look for alterations in metabolite profiles that may help detect cancer at an early stage.
Solid tumors typically have localized areas of poor blood supply, and as noted above, preferentially utilize glycolytic metabolism and thus secrete lactic acid into the tumor microenvironment, which leads to local acidosis. It has been postulated that local TME acidosis allows tumor cells to invade more easily. The low oxygen tension in areas of tumors with poor blood supply stimulates the expression of hypoxia-inducible factor-1 (HIF-1). This transcription factor, which is activated by low-oxygen tension, upregulates (among other actions) transcription of at least eight genes that control synthesis of enzymes involved in glycolysis.

The pH and oxygen tension in tumors are important factors that affect responses to anticancer drugs and other treatments. For example, the anticancer efficacy of radiation treatment of cancers is significantly lower in hypoxic conditions. Chemicals have been developed to inhibit glycolysis in tumor cells, and perhaps selectively kill them (Table 56-11). These include 3-bromopyruvate (an inhibitor of hexokinase-2) and 2-deoxy-p-glucose (an inhibitor of hexokinase-1). Another compound, dichloroacetate (DCA), inhibits the activity of pyruvate dehydrogenase kinase, and thus stimulates the activity of pyruvate dehydrogenase (see Chapter 17), diverting substrate from glycolysis into the citric acid cycle. However, so far, none of these have been found to be clinically useful.

Stem cells were discussed briefly in Chapters 39 and 53. Much work is currently ongoing to investigate the role of stem cells in cancer. Cancer stem cells are believed to harbor mutations that, either by themselves or in combination with further mutations, make these cells cancerous. Stem cells can be detected by the use of specific surface markers, or other techniques. It appears that surrounding tissues (eg, components of the extracellular matrix) can significantly influence the behavior of these cells (see Figure 56-10). An important concept driving some of the research in this area is the belief that one of the reasons that cancer chemotherapy is often not successful is that a pool of cancer stem cells exists that is not susceptible to conventional chemotherapy. Reasons for this include the facts that many stem cells are relatively dormant, have active DNA repair systems (see Figure 35-23), express drug transporters that can expel anticancer drugs, and are often resistant to apoptosis.
Evidence is accumulating that cancer stem cells do indeed play key roles in many types of neoplasia. If so, development of therapies with high specificity for killing these stem cells will be of considerable value.

Tumor cells need an adequate blood supply to provide nutrients for their survival. Both tumor cells and cells in tissues surrounding tumors have been found to secrete angiogenic factors that stimulate the growth of new blood vessels. There has been much interest in tumor angiogenesis, because inhibiting this process represents a potential means of killing tumor cells.
FIGURE 56-11 Pyruvate kinase isozymes and glycolysis in normal and in cancer cells. In normal cells, the major source of ATP is oxidative phosphorylation. Some ATP is obtained from glycolysis. The major pyruvate kinase (PK) isozyme in normal cells is PKM1. In cancer cells, aerobic glycolysis is prominent, lactic acid is produced via the action of lactate dehydrogenase (LDH) and production of ATP from oxidative phosphorylation is diminished (not shown in the figure). In cancer cells, PKM2 is the major PK isozyme. For complex reasons not as yet fully understood, this change of isozyme profile in cancer cells is associated with decreased net production of ATP from glycolysis, but increased use of metabolites to build up biomass.
The growth of blood vessels that supply tumor cells can be stimulated by hypoxia and other factors. As mentioned above, hypoxia induces HIF-1, which in turn increases levels of vascular endothelial growth factor (VEGF), a family of proteins that serve as major stimulators of angiogenesis. VEGF proteins interact with specific tyrosine kinase receptors on endothelial and lymphatic cells. These interactions activate signaling pathways that cause upregulation of the NF-«B pathway (see Chapter 42), resulting in proliferation of endothelial cells and formation of new blood vessels. Blood vessels supplying tumors are not normal; their structure is often disorganized, with lower than normal levels of integrity. Consequently, they are often leakier than normal blood vessels. Molecules other than VEGFs, such as angiopoietin, B-fibroblast growth factor

(B-FGF), TGF-6, and placental growth factor, also stimulate angiogenesis. Certain other molecules also inhibit blood vessel growth (eg, angiogenin and endostatin). Monoclonal antibodies (mAbs) to one form of VEGF have been developed (eg, bevacizumab or Avastatin) and have been used in the treatment of certain types of cancer (eg, colon and breast). These mAbs bind to VEGF and block it from acting, presumably by blocking VEGF from interacting with the VEGF receptor. These therapeutic mAbs were found to increase patient survival, but most patients eventually relapsed. As with many antineoplastic therapies, it is now believed that these mAbs are best used in combination with other anticancer therapies. Monoclonal antibodies to other growth factors that stimulate angiogenesis are also being
TABLE 56-11 Some Compounds That Inhibit Glycolysis and Have Been Found to Display Variable Anticancer Activity
3-Bromopyruvate, Enzyme Inhibited = Hexokinase II. 2-Deoxy- d -glucose, Enzyme Inhibited = Hexokinase I. Dichloroacetate, Enzyme Inhibited = Pyruvate dehydrogenase kinase (PDH). Iodoacetate, Enzyme Inhibited = Glyceraldehyde-phosphate dehydrogenase
Note: The rationale for development of these agents is that glycolysis is usually much more active in tumor cells, so that inhibiting it may damage them more than normal cells. Inhibition of PDH kinase results in stimulation of PDH, diverting pyruvate away from glycolysis.
developed and are in clinical trials, as are small molecule inhibitors of angiogenesis. Inhibitors of angiogenesis are useful in other conditions, such as "wet," or age-related, macular degeneration and diabetic retinopathy, in which proliferation of blood vessels is a feature.

It has been estimated that about 85% of the mortality associated with cancer results from metastasis. Spread of cancer is usually via lymphatics or blood vessels. Metastasis is a complex process, the molecular mechanisms of which are only now beginning to be understood.
Figure 56-12 presents a simplified scheme of metastasis. The earliest event is detachment of tumor cells from the primary tumor. The cells can then gain access to the circulation (or lymphatics), a process termed intravasation. Once in the circulation, they tend to arrest in the nearest small capillary bed. In that site, they extravasate and migrate through the neighboring extracellular matrix (ECM), before finding a site to settle. Thereafter, if they survive host defense mechanisms, they grow at variable rates. To ensure growth, metastatic cells need an adequate blood supply, as discussed above.
Many studies have shown that cancer cells have an abnormal complement of proteins on their surfaces. These changes may permit decreased cell adhesion and allow individual cancer cells to detach from the parent tumor. Molecules on cell surfaces involved in cell adhesion are called cell adhesion molecules, or CAMs (Table 56-12). Decreases in the amounts of the CAM E-cadherin, a molecule of major importance in the adhesion of many normal cells, may help to account for the decreased adhesiveness of many cancer cells. Many studies have shown changes in the oligosaccharide chains of cell surface glycoproteins (see Figure 40-7), which occur due to altered activities of various glycosyltransferases (see Chapter 46). One such important change is an increase in the activity of GlcNAc transferase V. This enzyme catalyzes transfer of GlcNAc to a growing oligosaccharide chain, forming a B1-6 linkage and allowing further growth of the chain. It has been proposed that such elongated chains participate in an altered glycan lattice at the cell surface. This may cause structural reorganization of receptors and other molecules, perhaps predisposing to the spread of cancer cells.

Another important property of many cancer cells is that they can release various proteinases into the ECM. Of the four major classes of proteinases (serine, cysteine, aspartate, and metalloproteinases), in cancer, particular interest has focused on the matrix metalloproteinases (MMPs). MMPs constitute a large family of metal-dependent (usually zinc) enzymes. A number of studies have shown increased activity of MMPs, such as MMP-2 and MMP-9 (also known as gelatinases), in tumors. These enzymes are capable of degrading proteins, such as collagen, in the basement membrane and in the ECM, thereby facilitating the spread of tumor cells. Inhibitors of these enzymes have been developed, but so far these have not exhibited much clinical success.
Another factor that allows increased movement of cancer cells is epithelial-to-mesenchymal transition, or EMT. This is a change of cell morphology and function from epithelial to mesenchymal type, perhaps induced by growth factors. Mesenchymal types have more actin filaments, permitting increased movement, an essential property of cells that metastasize.
The ECM also plays an important role in metastasis. There is evidence of communication by signaling mechanisms between cancer cells and those of the ECM. The types of cells in the ECM can also affect metastasis. As mentioned above, proteinases that degrade proteins in the ECM can facilitate spread of cancer cells. In addition, the ECM contains various growth factors that can influence tumor behavior.
On their travels, tumor cells are exposed to various cells of the immune system (such as T cells, NK cells, and macrophages; see Chapter 54), and must be able to survive exposure to them. Some of these immune surveillance cells secrete various chemokines, small proteins that can attract various cells such as leukocytes, sometimes causing an inflammatory response to tumor cells.
It has been estimated that significantly fewer that 1 in 10,000 cancer cells may have the genetic capacity to successfully colonize. Certain tumor cells show a predilection to metastasize to specific organs (eg, prostate cells to bone). It is likely that specific cell surface molecules are involved in this tropism.

Various studies have shown that certain genes enhance metastasis, whereas others act as metastasis suppressor genes. Determining exactly how these genes work is the subject of intense investigation. Table 56-13 summarizes some important points regarding metastasis.

Tumor immunology is a broad area of study; due to the extensive breadth of this field, it can only be dealt with here briefly. It is likely that the normal decline in immune responsiveness that accompanies aging plays a role in the increased incidence
Proteinases
Extracellular matrix
Signaling molecules
Angiogenic
=
factors

the factors believed to be involved. (From Tannock IF, et al: The Basic Science of Oncology, 4th ed. McGraw-Hill, 2005.)
of cancer
in older
people.
A
long-standing hope
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cancer cells.
to treat
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There ing
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is the use of antibodies against certain T-lymphocyte surface
proteins. For example, antibodies developed against cytotoxic T-lymphocyte antigen 4 (anti-CTLA-4) or programmed death 1 (anti-PD1) have been shown to "remove the brakes" on these cells, thus setting them free to attack cancer cells. Other strategies using modified T cells have proven to be effective as well. The major advantage of immunotherapy is that it has a broad spectrum of action and can therefore be used against a wide variety of cancers. In addition, resistance is less likely to occur to this form of treatment. It is hoped that immunotherapy

+ Cadherins
+ ICAMS, Intercellular adhesion molecules
+ Integrins
+ Selectins
Note: CAMs may be homophilic or heterophilic. Homophilic CAMs interact with identical molecules on neighboring cells, whereas heterophilic CAMs interact with different molecules. Cadherins are homophilic, selectins and integrins are hetero- philic, and Ig CAMs may be either. Integrins are discussed briefly in Chapter 54, and selectins in Chapter 46.
will be the fourth major weapon against cancer, after surgery, radiotherapy, and chemotherapy, making it Science magazine's "Breakthrough of the Year 2013."
Chronic inflammation involves aspects of immune function. There is evidence that it can predispose to cancer (eg, the incidence of colorectal cancer is much higher than normal in individuals who have had long-standing ulcerative colitis). Some inflammatory cells produce relatively large amounts of reactive oxygen species (ROS), which can cause damage to DNA, and perhaps contribute to oncogenesis. It has also been reported that low doses of aspirin may lower the risk of development of colorectal cancer, perhaps via its anti-inflammatory action.

The association between inflammation and cancer is now well established. Inflammation is known to be a critical component of tumorigenesis. That said, the exact mechanisms linking inflammation and cancer are poorly understood. Examples of possible molecules that are involved in induction of an inflammatory process include nuclear factor kappa B (NF-«B) and signal transducer and activator of transcription 3 (STAT3). NF-KB is a transcription factor that induces expression of proteins that are involved in proinflammatory, proliferative, and

•, 1 = An epithelial-to-mesenchymal cell transition is often found in cancers, allowing detachment and spread of potentially metastatic cells.. •, 1 = Metastasis is relatively inefficient (only about 1:10,000 tumor cells may have the genetic potential to colonize).. •, 1 = Metastatic cells must evade various cells of the immune system to survive.. •, 1 = Changes in cell surface molecules (eg, CAMs and others) are involved in the process.. •, 1 = Increased proteinase activity (eg, of MMP-2 and MMP-9) facilitates invasion.. •, 1 = The existence of metastasis-enhancer and suppressor genes has been shown.. •, 1 = Some cancer cells metastasize preferentially to specific organs.. •, 1 = Metastasis-gene signatures may be detected by transcriptome/ exome analysis; such transcriptome information can be of prognostic value, potentially allowing for personalized therapeutic treatment.

reparative processes. Activation of NF-kB has been shown to occur in tumors in response to inflammatory stimuli or oncogenic mutations (see Chapter 42). Signaling via STATS is activated by interleukin 6 (IL-6), a proinflammatory cytokine that activates Janus kinase (JAK)-STAT signaling and its downstream effects (see Chapter 42). Such events are thought to be responsible for driving many hallmark features of cancer. In addition, the "inflammasome, a multiprotein complex that acts as a sensor of cellular damage, is another potential candidate that mediates inflammation. Activation of inflammasomes leads to secretion of proinflammatory cytokines, such as IL-16 and IL-18, both of which have been implicated in tumorigenesis. There is a large body of evidence to implicate other inflammatory mediators in the development of tumors. Obesity is associated with low-grade inflammation. Visceral adipose tissue is considered an important source of proinflammatory cytokines. It is now known that the microenvironment surrounding tumor cells influences tumorigenesis (see above). Inflammatory cells in the microenvironment of the tumor are considered to play a crucial role in the process. Obesity has been shown to mediate and exacerbate dysfunctional changes in the microenvironment; this has been shown to occur both in normal tissue and tumors. Such changes include alterations in factors that may be endocrine, metabolic, or inflammatory in nature. By contrast, caloric restriction has been shown to inhibit tumorigenesis in experimental models. Many cellular pathways, such as those involving growth factor signaling, inflammation, cellular homeostasis, and the tumor microenvironment are affected by such caloric restriction. These observations suggest that such targets may be considered for prevention of cancer in humans.

Biochemical tests are often helpful in the management of patients with cancer (eg, some patients with advanced cancers may have elevated levels of plasma calcium, which can cause serious problems, if not attended to). Many cancers are associated with the abnormal production of enzymes, proteins, and hormones that can be measured in plasma or serum. These molecules are known as tumor biomarkers. Some of them are listed in Table 56-14.
However, significant elevations of some of the biomarkers listed in Table 56-14 also occur in a variety of noncancerous conditions. For example, elevations of the level of prostate-specific antigen (PSA), a glycoprotein synthesized by prostate cells, occur not only in patients with cancer of the prostate, but also in patients with prostatitis and benign prostatic hyperplasia (BPH). Similarly, elevations of carcinoembryonic antigen (CEA) are found not only in patients with various types of cancer, but also in heavy smokers and people with ulcerative colitis and cirrhosis. The fact that elevations of tumor biomarkers are usually not specific for cancer has

α-Fetoprotein (AFP), Associated Cancer = Hepatocellular carcinoma, germ cell tumor. Calcitonin (CT), Associated Cancer = Thyroid (medullary carcinoma). Carcinoembryonic antigen (CEA), Associated Cancer = Colon, lung, breast, pancreas, ovary. Human chorionic gonadotropin (hCG), Associated Cancer = Trophoblastic disease, germ cell tumor. Monoclonal immunoglobulin, Associated Cancer = Myeloma. Prostate-specific antigen (PSA), Associated Cancer = Prostate. CA-125, Associated Cancer = Ovary. CA 19-9, Associated Cancer = Pancreas
Note: Most of these tumor biomarkers are also elevated in the blood of patients with noncancerous diseases. For example, CEA is elevated in a variety of noncancerous gastrointestinal disorders, and PSA is elevated in prostatitis and benign prostatic hyperplasia. This is why interpretation of elevated results of tumor markers must be made with caution and their main uses are to follow effectiveness of treatments and to detect recurrences. There are a number of other tumor biomarkers that are widely used.
meant that measurements of most of them are not used primarily for diagnosis of cancer. Their main uses have been in following the effectiveness of treatments and in detecting early recurrence. As with other laboratory tests (see Chapter 48), the entire clinical picture must be considered when interpreting the results of measurements of tumor biomarkers.
It is hoped that ongoing -omic analyses of body fluids and accessible cancer cells (in blood, serum, and biopsy samples) will provide new tumor biomarkers of increased sensitivity and specificity, and those capable of alerting one to the presence of cancers at an early stage of their development. Transcriptome and whole genome sequencing analyses (see Chapter 39 and below) of cancer cells have revealed a plethora of potentially very useful biomarkers of oncogenesis. These methods are also useful in more accurately subclassifying tumors (so-called "personalized medicine'; see Chapter 39) in order to provide more accurate diagnoses and guide more efficacious, targeted modes of therapy. Such molecular diagnostic methods are becoming the standard of care for a select subset of cancers.

Since the completion of the Human Genome Project, the technology of large-scale DNA sequencing and bioinformatic analyses and interpretation of sequence data has advanced considerably. Large-scale DNA sequencing has become both faster and cheaper with the widespread availability of nextgeneration sequencing (NGS) technologies (see Chapter 39). These advances have allowed for analyses of DNA sequences of a large number of different types of tumors. By sequencing the whole genomes and exomes of tumors, a comprehensive catalog of specific types and numbers of gene mutations found in different cancers has been determined. Such information is revolutionizing diagnostic testing and the development of custom-tailored therapy. In addition, such studies will help identify specific mutations in genes that either cause or accelerate cancers. The former type of mutations are known as driver mutations, whereas the latter types are termed as passenger mutations. Genome-modulating technologies, such as gene silencing and genome editing (based on CRISPRbased methods), are becoming important tools in such studies. Tumors often exhibit extreme heterogeneity, such that they consist of subpopulations of cells that are genetically and phenotypically distinct from one another (see Figure 56-10). It is now possible to isolate and sequence single cells obtained from tumors (single-cell sequencing), in order to understand the genetic landscape of a particular tumor. Such understanding is important to develop multimodal treatment strategies that efficiently target all the subpopulations in a given tumor. Overall, it is expected that information obtained from these new technologies will dramatically impact the next stages of cancer genomics and help in development of methods that allow for early diagnosis of cancer, identification of critical genetic changes that drive cancer progression and, ultimately, personalized therapy for cancer in individual patients. Such an individualized approach to cancer diagnosis and treatment is termed
precision oncology.

One of the great hopes of cancer research is that insights into fundamental biochemical and genetic mechanisms involved in carcinogenesis will lead to new and better therapies. This has already occurred to a certain extent and it is hoped that ongoing developments will accelerate this process.
Classical chemotherapeutic drugs include alkylating agents, platinum complexes, antimetabolites, mitotic spindle poisons, among other classes of chemical compounds. These agents will not be discussed here. Within the classes of drugs developed more recently are inhibitors of signal transduction (including tyrosine kinase inhibitors), monoclonal antibodies directed to various target molecules, inhibitors of hormone receptors, drugs that affect differentiation, antiangiogenesis agents, and biologic response modifiers. Examples of each of these are listed in Table 56-15.
The finding of widespread defects in signaling mechanisms in cancer cells, and in particular the detection of mutations in tyrosine kinases, has led to the development of inhibitors of these enzymes. The most dramatic success has probably been the introduction of imatinib (marketed as Gleevec) for the treatment of chronic myelocytic leukemia (CML). Imatinib is an orally administered drug that inhibits the tyrosine

Inhibitors of signal transduction, Example = Imatinib, an inhibitor of tyrosine kinase. Inhibitors of signal transduction, Used to Treat = Chronic myelocytic leukemia. Monoclonal antibodies, Example = Trastuzumab, a Mab to the HER2/Neu receptor. Monoclonal antibodies, Used to Treat = Late-stage breast cancer. Antiangiogenesis agents, Example = Bevacizumab, a Mab to VEGF A. Antiangiogenesis agents, Used to Treat = Colon and breast cancers. Antihormonal agents, Example = Tamoxifen, antagonist of the estrogen receptor. Antihormonal agents, Used to Treat = Breast cancer. Affect differentiation, Example = All-trans retinoic acid (ATRA), targets the retinoic acid receptor on promyelocytic leukemia cells, causing them to differentiate. Affect differentiation, Used to Treat = Promyelocytic leukemia. Affect epigenetic changes, Example = 5-Azadeoxycytidine inhibits DNAmethyltransferases; SAHA inhibits histone deacetylases. Affect epigenetic changes, Used to Treat = Certain leukemias, cutaneous T-cell lymphoma
Abbreviations: Chronic myelocytic leukemia; Mab, monoclonal antibody; SAHA, suberoylanilide hydroxamic acid (Vorinostat); VEGF A, vascular endothelial growth factor A. Note: In some cases, the above agents may have been replaced by other more effective agents. Some of the agents listed above are also used to treat conditions other than those listed.

Increased drug efflux from the cell, Example = Overexpression of transport proteins, such as multidrug resistance proteins (MDRs) (eg, P-glycoprotein or MDR1), causes efflux of cancer chemotherapeutic drugs, such as taxanes, topoisomerase inhibitors, and antimetabolites.. Decreased drug activation, Example = Decreased conversion of prodrugs (eg, 5-fluorouracil) to their active forms due to downregulation of enzymes that catalyze their activation.. Drug inactivation, Example = Platinum drugs (cisplatin and carboplatin) are inactivated by conjugation with glutathione.. Increased drug target expression, Example = Increased expression of thymidylate synthase, the target of antimetabolites such as 5-fluorouracil.. Dysfunctional apoptosis, Example = Overexpression of anti-apoptotic proteins such as the BCL2 family of proteins, and decreased expression of pro-apoptotic proteins such as BAX and BAK.. Activation of prosurvival signaling, Example = Activation of epidermal growth factor receptor (EGFR)-mediated signaling in response to various chemotherapeutic drugs.. Modification of tumor microenvironment, Example = Increased expression of integrins (proteins that attach cells to the extracellular matrix), which inhibits apoptosis and alters drug targets.
FIGURE 56-13 Examples of targets for anticancer drugs and some emerging therapies, both of which have developed from relatively recent research. Not shown in the figure are antiangiogenic agents, applications of nanotechnology, therapies directed against cancer stem cells, and immunologic approaches. Most of the targets and therapies indicated are discussed briefly in the text.

cellular, molecular, and cancer biology to develop safer and more effective agents. Intensive research over the past several decades has resulted in an improved understanding of genetic alterations that underlie the development of specific types of cancer. This knowledge has led to a shift from the use of broad-spectrum cytotoxic drugs to therapies that are specifically designed to target individual tumors. Currently, a major area of research is to identify specific driver mutations, mutations that play critical roles in development of tumors (see discussion of colorectal cancer above). Molecular profiling of cancer in individual patients allows oncologists to choose both the most appropriate drug or treatment modality that targets the molecular abnormality in each tumor, and to monitor the efficacy of such treatment(s) over time. Such personalized anticancer therapy has been shown to significantly improve clinical response to drugs and increase survival in various types of cancer. An understanding of individuals' genetic differences in metabolism of anticancer drugs may also help personalize anticancer treatments. Figure 56-13 summarizes some of the targets for drug therapy and some emerging therapies that have developed from studies of fundamental aspects of cancer.

The tremendous individual suffering caused by cancer, and the heavy economic burden it imposes on society makes it important to adopt measures to prevent the development of cancer. Modifiable risk factors have been linked to a wide variety of cancers. It is likely that a significant number of all cancers in developed countries could be prevented if the measures summarized in Table 56-17 were introduced on a population-wide basis.
Cancer 2004;90:299.
The use of tobacco, in its various forms, continues to be a major cause of cancer affecting the lungs, mouth, larynx, esophagus, and stomach. A sustained public education campaign regarding the adverse effects of tobacco use has resulted in significant decreases in the incidence of cancers associated with tobacco use. Vaccines against the human papillomavirus (HPV) (known to be associated with cancer of the uterine cervix) and hepatitis B virus (HBV) (associated with hepatocellular carcinoma) have been found to be effective in decreasing the incidence of cancers caused by these viruses.
Chemoprevention, which refers to the use of drugs to prevent the development of cancer, has been found to be effective in certain types of cancer. For example, the use of estrogen receptor modulators (like tamoxifen) has been shown to decrease the incidence of breast cancer in high-risk women by about 50%. Similarly, the use of finasteride (a drug that inhibits the enzyme, 5a-reductase, that converts testosterone to dihydrotestosterone), has been associated with reduced incidence of prostate cancer. It has also been shown that long-term use of aspirin, which is commonly prescribed as an antiplatelet agent, is associated with decreased incidence of colon cancer. In some instances, identification of genetic risk factors for cancer is opening up possibilities for newer strategies in cancer prevention. For example, women who have mutations in the breast-cancer associated genes, BRCAI and BRCA2, can undergo prophylactic mastectomy (surgery for removal of the breast) in order to mitigate any future risk of developing cancer. Overall, the rapid progression of research in cancer biology is not only opening up newer ways to treat cancer but also to decrease and/or prevent the occurrence of the disease in the first place.

- ™ Cancer is caused by mutations in the genes that control cell
growth and
duplication, cell
death
(apoptosis), and
cell-cell interactions
(eg, cell
cancer adhesion).
are defects
of angiogenesis,
Other in
cell signaling
aneuploidy, important
pathways, aspects
of stimulation
and changes
in the
cellular metabolism
and cell
microenviornment.
- = A majority of cancers are likely due to replicative errors (as broadly defined) that affect somatic cells. However, a number
of cancers have been identified that are caused by herediatary or environmental factors.
tumor
™ Major classes of genes involved in cancer are oncogenes, suppressor genes, and genes encoding proteins important for DNA synthesis, DNA repair, and chromosomal metabolism.
= Mutations affecting genes directing the synthesis and
expression of
microRNAs are
implicated in
oncogenesis.
™ Epigenetic changes that alter gene expression are increasingly being recognized in cancer (and in other diseases); one reason for interest in epigenetics is that epigenetic "marks" are potentially reversible by drugs.
™ Mechanisms of metastasis are being explored intensively; the discovery of metastasis enhancer and suppressor genes, among other findings, may lead to new therapies.
= Apoptosis, programmed cell death, plays important roles in oncogenesis. Cancer cells acquire mutations that permit them to evade apoptosis, thus prolonging and enabling their continued replication.
™ Cancer cells display various alterations of metabolism and of nutrient capture and utilization.
@ The development of cancer is a multistep process involving genetic, epigenetic, and microenvironmental changes that confer selective advantages on clones of cells, some of which eventually acquire the ability to metastasize successfully. Because of the diversity of mutations, it is possible that no two tumors have identical genomes.
™ Metastasis (the spread of cancer to distant locations) is
changes in
cell adhesion
molecules and
modification of
the extracellular
matrix which

allows cancer cells to detach and migrate to distant locations.
™@ Itis likely that the extracellular vescicles (exosomes) released by cancer cells play important roles in cancer progression and metastasis.
=| Tumor biomarkers may help in both the early diagnosis of
cancer and
treatment as
well as
detecting recurrences.
- ™ Diagnostic whole genome-, exome-, and circulating tumor-
derived
DNA
sequencing is
now able
to reveal
the important
driver and passenger mutations present in many types of cancers and is becoming a powerful complement to patient treatment.
™ Advances in understanding the molecular biology of cancer cells have led to the introduction of a number of new therapies, with others in the pipeline.
™ Several strategies have proven useful in preventing cancer. These include modification of risk factors (like decrease in tobacco use in its various forms), vaccination against tumor-causing viruses (such as HPV and HBV), use of drugs (antiestrogens in breast cancer), and risk-modifying surgery (mastectemoy in women with mutations in BRCAI and BRCA2).

Alexandrov LB, Nik-Zainal S, Wedge DC, et al: Signatures of mutational processes in human cancer. Nature 2013;500:415-421.
Aravanis AM, Lee M, Klausner RD: Next-generation sequencing of circulating tuor DNA for early cancer detection. Cell
2017;168:571-574.

American Cancer Society. http://www.cancer.org
National Cancer Institute, U.S. National Institute of Health.
http://www.cancer.gov
The Cancer Genome Atlas. http://cancergenome.nih.gov
National Cancer Institute Genomic Data Commons.
http://gdc.cancer.gov
Nature. TCGA (2011, 2012, 2013, 2014). TCGA pan-cancer analysis.
http://www.nature.com/tcga/

Describe the essential features of wear and tear theories of aging.
List four or more common environmental constituents known to damage biologic macromolecules such as proteins and DNA.
Explain why nucleotide bases are especially vulnerable to damage.
Describe the most physiologically important difference between mitochondrial and nuclear genomes.
Describe the oxidative theory of aging.
List the primary sources of reactive oxygen species (ROS) in humans.
by ROS. Describe three mechanisms by which cells prevent or repair damage inflicted
Describe the basic tenets of metabolic theories of aging.
Explain the mechanism of the telomere "countdown clock.'
Outline our current understanding of the genetic contribution to aging.
Explain the benefits of model organisms to biomedical research.

Consider the various stages in the lifespan of Homo sapiens. Infancy and childhood are characterized by continual growth in height and body mass. Basic motor and intellectual skills develop: walking, language, etc. Infancy and childhood also represent a period of vulnerability wherein a youngster is dependent on adults for water, food, shelter, protection, and instruction. Adolescence witnesses a final burst of growth in the body's skeletal framework. More importantly, a series of dramatic developmental changes occur—an accumulation of muscle mass, maturation of the gonads and brain, and the emergence of secondary sex characteristics—that transform a child into an independent and reproductively capable adult. Adulthood, the longest stage, is a period devoid of dramatic physical growth or developmental change. With the notable exception of pregnancy in females, it is not unusual for adults to maintain the same body weight, overall appearance, and general level of activity for two decades or more.
Barring fatal illness or injury, the onset of the final stage of life, old age, is signaled by a resurgence of physical and physiologic change. Muscle and bone mass progressively decrease. Hair begins to thin and lose its pigmentation. Skin loses its suppleness and accumulates blemishes. Attention span and recall decline. Eventually, inevitably, life itself comes to an end as essential bodily functions decline.
Understanding the underlying causes and instigating triggers of aging and the changes that accompany it is of great biomedical importance. Hutchison-Gilford, Werner, and Down syndrome are three human genetic diseases whose pathologies include an acceleration of many of the physiologic events associated with aging. Slowing or preventing some of the degenerative processes that cause or accompany aging can render the later stages of life more vital, productive, and fulfilling. Coopting the factors responsible for triggering cell death may enable physicians to selectively destroy harmful tumors, polyps, and cysts.

From Paleolithic to Medieval times the average life expectancy of a newborn baby oscillated a the range of 25 to 35 years. Beginning with the Renaissance, however, this number gradually increased such that, by the beginning of the 20th century, the average life expectancy of persons born in developing countries reached the mid-40s. Today, 100 years later,

1900-1902, Average Life Expectancy (Years).From Birth = 49.24. 1900-1902, Average Life Expectancy (Years).If Survived to Age 5 = 59.98. 1909-1911, Average Life Expectancy (Years).From Birth = 51.49. 1909-1911, Average Life Expectancy (Years).If Survived to Age 5 = 61.21. 1919-1921, Average Life Expectancy (Years).From Birth = 56.40. 1919-1921, Average Life Expectancy (Years).If Survived to Age 5 = 62.99. 1929-1931, Average Life Expectancy (Years).From Birth = 59.20. 1929-1931, Average Life Expectancy (Years).If Survived to Age 5 = 64.29. 1939-1941, Average Life Expectancy (Years).From Birth = 63.62. 1939-1941, Average Life Expectancy (Years).If Survived to Age 5 = 67.49. 1949-1951, Average Life Expectancy (Years).From Birth = 68.07. 1949-1951, Average Life Expectancy (Years).If Survived to Age 5 = 70.54. 1959-1961, Average Life Expectancy (Years).From Birth = 69.89. 1959-1961, Average Life Expectancy (Years).If Survived to Age 5 = 72.04. 1969-1971, Average Life Expectancy (Years).From Birth = 70.75. 1969-1971, Average Life Expectancy (Years).If Survived to Age 5 = 72.43. 1979-1981, Average Life Expectancy (Years).From Birth = 73.88. 1979-1981, Average Life Expectancy (Years).If Survived to Age 5 = 75.00. 1989-1991, Average Life Expectancy (Years).From Birth = 75.37. 1989-1991, Average Life Expectancy (Years).If Survived to Age 5 = 76.22. 1999-2001, Average Life Expectancy (Years).From Birth = 76.83. 1999-2001, Average Life Expectancy (Years).If Survived to Age 5 = 77.47
Source: Adapted from Table 12 of the National Vital Statistics Reports (2008) 57, vol. 1.

the current world average is 67 years, and that for developed nations is approaching 80. These dramatic increases have led to speculation about how long this trend might be expected to continue. Can future generations expect to live past the century mark? Is it possible that human beings possess the potential, with proper care and maintenance, to live indefinitely?
Unfortunately, such extrapolations are unlikely to be realized because they are based on a misunderstanding of the term life expectancy. Life expectancy is calculated by averaging over all births. Hence, it is dramatically influenced by infant mortality rates. While the life expectancy of a Roman child was 25 years, if one calculated the expected lifespan only for those persons who survived infancy, which we will refer to as longevity, the average nearly doubled to 48. When one factors out the dramatic decline in infant mortality rates that has taken place over the past century and a half, the predicted longevity of a 5-year-old child in the United States has increased from 70.5 in 1950 to 77.5 years in 2000 (Table 57-1). Is there some sort of upper limit to the lifespan of a properly nourished, protected, well-maintained human being?

Are aging and death nondeterminant or stochastic processes wherein living creatures inevitably reach a tipping point after a lifetime's accumulation of damage from disease, injury, and simple wear and tear? Alternatively, are aging and death genetically programmed processes analogous to puberty that have evolved through a process of natural selection? In all likelihood, aging and death are multifactorial processes to which both stochastic and programmed factors contribute.

Many theories regarding aging and mortality hypothesize that the human body eventually succumbs to the accumulation of damage over time as a result of injury and long-term exposure to environmental factors that degrade organic biomolecules. These theories note that while repair and turnover mechanisms exist to restore or replace many classes of damaged molecules, they are less than perfect. Hence, some damage inevitably leaks through—damage that will accumulate over time, particularly among those cell populations that undergo little, if any, turnover (Table 57-2). Ironically, many of these agents are essential for terrestrial life: water, oxygen, and sunlight.

Water is a relatively weak nucleophile. However, because of its ubiquity and high concentration (> 55 M, see Chapter 2), even this weak nucleophile will occasionally react with susceptible targets inside the cell. In proteins, while hydrolysis of a peptide bond can sever a polypeptide chain, the most vulnerable amide bonds are frequently found on the exposed side chains of the amino acids asparagine and glutamine. Hydrolysis transforms their neutral amides into potentially negatively charged and acidic carboxylates, producing aspartate and glutamate, respectively (Figure 57-1A and B). As most of the proteins within a living organism are subject to regular turnover, in many cases the chemically modified protein will eventually be degraded and replaced by a newly synthesized version.
The amino groups projecting from the heterocyclic aromatic rings of the nucleotide bases cytosine, adenine, and guanine are each susceptible to hydrolytic attack. In each case, the amino group is replaced by a carbonyl to form uracil, hypoxanthine, and xanthine, respectively (Figure 57-1C). The bond between the nucleotide base and the deoxyribose

Intestinal epithelium, Turnover = 34 h a. Epidermis, Turnover = 39 d b. Leukocyte, Turnover = <1 y c. Adipocytes, Turnover = 9.8 y c. Intercostal skeletal muscle, Turnover = 15.2 y c. Cardiomyocytes, Turnover = ≥100 y c
Source: Data from:
*Potten CS, Kellett M, Rew DA, et al: Proliferation in human gastrointestinal epithe- lium
using bromodeoxyuridine
in vivo.
Gut
1992;33:524.
Weinstein
GD,
McCullough
JL,
Ross
P:
Cell proliferation
in normal
epidermis.
J
Invest
Dermatol
1984;82:623.
"Spalding KL, Arner E, Westermark PO, et al: Dynamics of fat cell turnover in humans. Nature 2008;453:783.
FIGURE 57-1 Examples of hydrolytic damage to biologic macromolecules. Shown are a few of the ways in which water can react with and chemically alter proteins and DNA: (A) Net substitution of aspartic acid via hydrolytic deamidation of the neutral side chain of asparagine. (B) Net substitution of glutamic acid via hydrolytic deamidation of the neutral side chain of glutamine. (C) Net mutation of cytosine to uracil by water. (D) Formation of an abasic site in DNA via hydrolytic cleavage of a ribose-base bond.
moiety in DNA is also vulnerable to hydrolysis. In this instance the base is completely eliminated, leaving a gap in the sequence (Figure 57-1D). The hydrolysis elimination or alternation of nucleotide bases in DNA is of potentially much greater biomedical significance than those affecting proteins, as the former—if left unrepaired (see Chapter 35)— will produce a genetic mutation.

Other biologically relevant bonds that are susceptible to hydrolysis include the ester linkages that connect fatty acids to their cognate glycerolipids, the glucosidic bonds that link the monosaccharide units of carbohydrates, and the phosphodiester bonds that hold polynucleotides together and link the head groups of phospholipids to their diacylglycerol partners. In most instances, with the notable exception of polynucleotide chain breaks, the products of these reactions appear to be biologically innocuous.
side chains in collagen (see Chapter 5), the detoxification of xenobiotics by cytochrome P450 (see Chapter 47), the degradation of purine nucleotides to uric acid (see Chapter 33), the reoxidation of the prosthetic groups in the flavin-containing enzymes that catalyze oxidative decarboxylation (eg, the pyruvate dehydrogenase complex, see Chapter 17) and other redox reactions (eg, amino acid oxidases, see Chapter 28), and the generation of the chemiosmotic gradient in mitochondria by the electron transport chain (see Chapter 13). Redox enzymes frequently employ prosthetic groups such as flavin nucleotides, iron-sulfur centers, or heme-bound metal ions (see Chapters 12 and 13) to assist in generating and stabilizing the free radical and oxyanion intermediates formed during these processes.

Numerous biologic processes require enzyme-catalyzed oxidation of organic molecules by molecular oxygen (O,). These processes include the hydroxylation of proline and lysine
Occasionally, these highly reactive intermediates escape to form of ROS such as superoxide and hydrogen peroxide inside the cell (Figure 57-2A). The most common of these sources is the electron transport chain, whose high levels of electron flux and structural complexity render it vulnerable to "leakage" of ROS. In addition, many mammalian cells synthesize and release nitric oxide (NO'), a free radical containing second messenger that promotes vasodilation and muscle relaxation in the cardiovascular system (see Chapter 55).
FIGURE 57-2 Reactive oxygen species (ROS) are toxic byproducts of life in an aerobic environment. (A) Many types of ROS are encountered in living cells. (B) Generation of hydroxyl radical via the Fenton reaction. (C) Generation of hydroxyl radical by the HaberWeiss reaction.

The extremely high reactivity of ROS makes them extremely dangerous. ROS can react with and chemically alter virtually any organic compound, including proteins, nucleic acids, and lipids. ROS also display a strong tendency to form adducts— products formed by the combination of precursors—with biologic compounds that contain multiple double bonds such as nucleotide bases and polyunsaturated fatty acids (Figure 57-3). Adducts formed with nucleotide bases can be especially dangerous because of their potential, if uncorrected, to cause mutation generating errors during DNA replication.
The ease with which exposure to warm air can turn household butter rancid is a testament to the reactivity of unsaturated fats, those containing one or more carboncarbon double bonds (see Chapter 23), with ROS. The resulting peroxidation of lipids can lead to the formation of cross-linked lipid-lipid and lipid-protein adducts that may compromise membrane fluidity and integrity. In mitochondria, a loss of membrane integrity can reduce the efficiency with which the electron transport chain generates ATP and increases leakage of deleterious ROS. Accumulated damage to mitochondrial membranes may eventually lead to the efflux of cytochrome c, an inducer of programmed cell death or apoptosis (see below).

The destructiveness inherent in the high reactivity of many of ROS, particularly free radicals, is exacerbated by their capacity to participate in chain reactions in which the product of the reaction includes not just a damaged biomolecule, but another free radical species capable of producing more damage and yet another radical by-product. This chain of events will continue until a free radical intermediate is able to pair its radical electron, perhaps by encountering another free radical or a redox protectant such as reduced glutathione. Alternatively, the ROS may be eliminated by one of the cell's suite of dedicated antioxidant enzymes (see Chapters 12 and 53).
The reactivity, and hence destructiveness, of individual ROS varies. Hydrogen peroxide, for example, is less reactive than superoxide, which in turn is less reactive than hydroxyl radical (OH"). Two pathways exist by which the highly toxic hydroxyl radical can be generated from less destructive ROS. If ferrous (+2) iron is present, the Fenton reaction can transform hydrogen peroxide into hydroxyl radicals (Figure 57-2B). The ferric (+3) iron, in turn, can be reduced back to the ferrous (+2) state by other hydrogen peroxide molecules, permitting the iron to act as a catalyst for the production of additional hydroxyl radicals. Hydroxyl radical can also be generated when superoxide and hydrogen peroxide disproportionate via the Haber-Weiss reaction (Figure 57-2C).

In 1956, Denham Harmon proposed the so-called free radical theory of aging. It had been reported that the toxicity of hyperbaric oxygen treatment and radiation could be explained by a factor common to both, the generation of ROS. This report dovetailed nicely with Harmon's own observation that lifespan was inversely related to metabolic rate and, by extrapolation, respiration. He therefore postulated that the cumulative damage was caused by the continual and inescapable production of ROS.
In more recent years, the proponents of the free radical theory of aging have focused attention on the mitochondria. Not only do mitochondria harbor the dominant source of ROS in the cell, the electron transport chain, but oxidative damage to the components of this pathway could lead to increased ROS yields. Damage to the mitochondria also might adversely affect rate efficiency with which they generate ATP, perhaps to the point where it undermines cell vitality and function.
A second contributor to this mitochondrial redox damage cycle is the mitochondrion's indigenous genome. The mitochondrial genome is a much reduced, vestigial remnant of the genome of the ancient bacterium that was the precursor of the current organelle. It is presumed primitive eukaryotes became dependent on surrounding bacteria to provide certain materials, eventually cementing the relationship by endosymbiosis, the internalization of the smaller bacterium.
FIGURE 57-3 ROS react directly and indirectly with a wide range of biologic molecules. (A) Peroxidation of unsaturated lipids generates reactive products such as malondialdehyde and 4-hydroxynonenal. (B) Guanine can be directly oxidized by ROS to produce 8-oxoguanine or form an adduct, M,dG, with the ROS product malondialdehyde. (C) Common reactions of proteins with ROS, including oxidation of amino acid side chains and cleavage of peptide bonds. Oxygen atoms derived from ROS are marked in red. Carbon atoms derived from malondialdehyde in M,dG are colored blue. The complete chemical name for M,dG is 3-(2-Deoxy-D-erythro-pentofuranosyl)pyrimido(1,2-a)purin-10(3H)-one.

Over time most, but not all, of the genes contained in the bacterium's genome were either eliminated or were transferred to the nuclear DNA of the eukaryotic host. Today, the genome of the human mitochondrion encodes two ribosomal RNAs (one for each subunit), 22 tRNAs, several of the polypeptide components of complexes I, III, and IV of the electron transport chain, and parts of F,, F, ATPase (Table 57-3). The mitochondrial genome lacks the surveillance and repair enzymes that help maintain the integrity of nuclear DNA. Hence, deleterious mutations, introduced into the mitochondrially encoded portions of the electron transport chain and their consequent functional defects, become a permanent feature of a mitochondrion's genome to which additional mutations may accrue over time. Under this model, the initial ROS insult serves as the trigger for a self-perpetuating cycle of ROS leakage, DNA damage, and increased ROS leakage. While the mitochondrial hypothesis is no longer viewed as providing a unifying explanation for all of the changes that are associated with human aging and its comorbidities, this organelle remains a likely contributor. Powerful circumstantial evidence for this is provided by the central role played by this organelle in the sensor-response pathways that trigger apoptosis.

Apoptosis imbues higher organisms with the ability to selectively eliminate cells that are rendered superfluous by developmental changes, such as those that continually take place during embryogenesis, or which have been damaged beyond repair. During developmental tissue remodeling, the apoptotic cell death program is triggered by receptor-mediated signals. In the case of damaged cells, ROS, viral dsRNA, DNA damage, or heat shock may act as triggers. These signals induce the opening of the permeability transition pore complex embedded in the mitochondrial outer membrane, which allows cytochrome c, a small (~ 12.5 kDa), soluble electron carrier protein, to escape into the cytoplasm. Here, cytochrome c provides the core around which apoptosome, a multiprotein complex, coalesces. Assembly of the apoptosome initiates a cascade of proteolytic activation events targeting the proenzyme forms the caspases, a family of cysteine proteases. The terminal caspases, numbers 3 and 7, break down structural proteins in the cytoplasm and chromatin proteins in the nucleus; events that lead to the death and, eventually, phagocytosis of the morbid cell. Many researchers are working to find ways to exploit the presence of this intrinsic, receptor-mediated cell death pathway as a means for selectively eliminating cancer and other malicious cells.

The term ultraviolet (UV) radiation refers to those wavelengths of light that lie immediately below the blue end of the visible spectrum. While the human eye cannot detect them, they are strongly absorbed by organic compounds possessing aromatic rings or multiple, conjugated double bonds. These include the nucleotide bases of DNA and RNA; the side chains of phenylalanine, tyrosine, and tryptophan; polyunsaturated fatty acids; heme groups; and numerous cofactors and coenzymes including flavins, cyanocobalamin, etc. Absorption of this short wavelength, high-energy light can cause the rupture of covalent bonds in proteins, DNA, and RNA; the formation of thymine dimers in DNA (Figure 57-4); crosslinking of proteins; and the generation of free radicals. While UV radiation does not penetrate beyond the first few layers of epidermal cells, the high efficiency with which it is absorbed can lead to the rapid accumulation of damage by the skin. Because the nucleotide bases of DNA and RNA are particularly effective at absorbing UV radiation, it is highly mutagenic. Thus, prolonged exposure to intense sunlight can lead to the accumulation of multiple DNA lesions that can overwhelm a cell's intrinsic repair capacity, leading to the development of myelomas—some of which aggressively proliferate if left untreated.

When amine groups in proteins and nucleotides are exposed to a reducing sugar such as glucose, they may form an adduct by a process called glycation. The initial step in this process is the formation of a Schiff base between the aldehyde or ketone group of the sugar and the amine. Over time, the glycated macromolecule undergoes a series of rearrangements to form Amadori products, which contain a conjugated carboncarbon double bond that can react with the amino group on a neighboring protein (Figure 57-5). The net result is the formation of a covalent cross-link between two proteins or other biologic macromolecules. These same macromolecules can, in turn, be glycated further, extending the network of cross-links to include other macromolecules. These cross-linked aggregates are sometimes called advanced glycation end products or AGEs.
CHAPTER
57.
FIGURE 57-4 Formation of a thymine dimer following excitation by UV light. When consecutive thymine bases are stacked one above the other in a DNA double helix, absorption of UV light can lead to the formation of a cyclobutane ring (red, not to scale) covalently linking the two bases together to form a thymine dimer.
The physiologic impact of protein glycation can be especially pronounced when long-lived proteins such as collagen or B-crystallins are affected. Their persistence enhances the opportunity for multiple glycation and subsequent crosslinking events to occur. In vascular endothelial cells, the accumulation of cross-links in the collagen network can lead to the progressive loss of elasticity and thickening of the basement membrane in blood vessels, both of which potentiate plaque formation. The end result is an increasing workload for heart. In the eye, the accumulation of aggregated proteins compromises the opacity of the lens, eventually manifesting itself as cataracts. Diabetics, whose ability to control blood glucose levels is impaired, are particularly susceptible to the formation of AGEs. In fact, the glycation of hemoglobin and serum albumin are used as biomarkers for the diagnosis of diabetes.
For example, fruit flies that have been genetically altered to express elevated levels of superoxide dismutase exhibit significantly extended lifespans.

A corollary to the wear and tear theory of aging is that longevity reflects the effectiveness and robustness of an individual's molecular prevention, repair, and replacement mechanisms.
In the cytoplasm, the cysteine-containing tripeptide glutathione acts as a chemical redox protectant that reacts directly with ROS to generate less reactive compounds such as water. Oxidized glutathione, which consists of two tripeptides linked by an S-S bond, is then enzymatically reduced to maintain the pool of protectant (see Chapter 53). Glutathione can also react directly with cysteine sulfenic acids and disulfides on proteins to restore them to their reduced state, and form adducts with toxic xenobiotics (see Chapter 47). Ascorbic acid and vitamin E also possess antioxidant properties, which accounts for the popularity of diets that emphasize foods rich in these compounds, or supplementation with them, to combat ROS and, hopefully, slow aging.

In addition to the prophylactic measures mentioned above, living organisms possess a limited capacity to replace or repair damaged macromolecules. The largest suite of repair enzymes is devoted to maintaining the integrity of the nuclear (but not the mitochondrial) genome. This is to be
FIGURE 57-5 Protein glycation can lead to the formation of protein-protein cross-links. Shown are the sequence of reactions that generate the Amadori product on the surface of the protein marked in green, and the subsequent formation of a protein-protein cross-link via an amino group on the surface of a second, red, protein.
expected given DNAs pivotal role in inheritance, its vulnerability to chemical assault and UV radiation, and the fact that—by contrast to almost every other macromolecule— each human cell contains only one or two copies of each chromosome.
Maintaining the integrity of the genome begins at replication, where careful proofreading is performed to ensure that the new genome formed during somatic cell division faithfully replicates the template that directed its synthesis. The term somatic refers to the differentiated cells that comprise the body of an organism. In addition, most living organisms possess an impressive cadre of enzymes whose role is to inspect and correct aberrations that either escaped replicative proofreading or were subsequently generated through the action of water (double-strand breaks, depurination, and cytosine deamidation), UV radiation (thymine dimers and strand breaks), or exposure to chemical modifiers (adduct formation). This multilayered system is composed of mismatch repair enzymes, nucleotide excision repair enzymes, and base excision repair enzymes as well as the Ku system for repairing double-strand breaks in the phosphodiester backbone (see
Chapter 35). As a last resort, cells harboring damaging mutations are subject to removal by apoptosis.
Despite the many precautions taken to identify and correct errors, some mutations inevitably slip through. Indeed, a low, but finite, frequency of mutation is necessary in order to generate the genetic variability that furthers evolution. The somatic mutation theory of aging proposes that these mutations also serves as a driver of the aging process. Simply put, the accumulation of mutated somatic cells must inevitably lead to compromised biologic function that manifests itself, at least in part, as the physical changes we associate with aging.

In contrast to DNA, a cell's capacity to repair damage to other biomolecules is relatively limited. For the most part, cells appear to rely on routine turnover, wherein the global population of a given biomolecule is degraded and replaced by new synthesis on a continuing, or constitutive, basis (see Chapter 9) to remove damaged lipids, carbohydrates, and proteins. However, some proteins, particularly the fibrous proteins found in tendons, ligaments, bones, matrix, etc, undergo little, if any, turnover. These long-lived proteins tend to accumulate damage over the years, which contributes to the loss of elasticity in vascular tissues and joints, loss of lens opacity, etc. The most prominent mechanisms for repairing damaged proteins target the oxidized side chain sulfur atoms of cysteine and methionine, and the isoaspartyl groups formed when a peptide bond shifts from an a to a side chain carboxyl group.
The side chain sulfhydryl group of cysteine frequently plays important catalytic, regulatory, and structural roles (eg, cysteine disulfides, Fe-S centers) in proteins. However, both the sulfhydryl group of cysteine and the sulfur ether of methionine are extremely vulnerable to oxidation (Figure 57-3C). Cysteine disulfides, cysteine sulfenic acids, and methionine sulfoxide can be reduced by disulfide reductases and methionine sulfoxide reductases, all of which use NADPH as electron donor, or direct reaction with reduced glutathione. Unfortunately, the reduction potentials of glutathione and NADPH are only sufficient to reduce the lowest oxidation states of these sulfur atoms: cysteine disulfides or sulfenic acids and methionine sulfoxide. Cysteine sulfinic acid, cysteine sulfonic acid, and methionine sulfone are refractory to reduction by available biochemical means.

Aspartic acid possesses the precise geometry needed to bring its side chain carboxyl group into close proximity with the peptide bond involving its a-carboxyl group. The amide nitrogen can react with it to generate a cyclic diamide. This intermediate can reopen to form either the original peptide bond or an isoaspartyl residue in which the side chain carboxyl now forms part of the protein's peptide backbone (Figure 57-6). The isoaspartyl methyltransferase-catalyzed
isoaspartyl
FIGURE 57-6 Formation of an isoaspartyl linkage in a polypeptide backbone and its repair via the intervention of isoaspartyl methyltransferase. Shown is the sequence of chemical and enzyme-catalyzed reactions that lead to formation of an isoaspartyl linkage and restoration of a normal peptide linkage. The carbons corresponding to the a and side chain carboxylic acid groups in aspartic acid are colored blue and green, respectively. Red arrows denote the routes of nucleophilic attack during the cyclization and hydrolysis reactions. The methyl group added by isoaspartyl methyltransferase is colored red.
methylation of the a-carboxyl introduces a leaving group that potentiates the reformation of the cyclic diamide, which can then reopen to form the normal peptide bond linkage (Figure 57-6).

Modifications to a protein's composition or conformation that cause it to adhere to other protein molecules can lead to the formation of toxic aggregates, called amyloid. Such aggregates are a hallmark of several neurodegenerative diseases, including Parkinson, Alzheimer, Huntington disease, spinocerebellar ataxias, and the transmissible spongiform encephalopathies. The toxic effects of these insoluble aggregates are exacerbated by their persistence, as they are generally refractory to degradation by the proteases normally responsible for protein turnover.

While molecular wear and tear undoubtedly contribute to aging, several observations suggest a prominent role for programmed, deterministic mechanisms. Female menopause, for example, provides an unambiguous example of an ageassociated physiologic change that is genetically programmed and hormonally controlled. The paragraphs below describe several current theories regarding deterministic, programmed mechanisms for controlling aging and death.
One of the many variants of the famous quote attributed to the ancient Chinese philosopher Lao Tzu summarizes the salient features of metabolic theories of aging. Its origins can be traced to the observation that the larger members of the animal kingdom tend to live longer than the smaller ones (Table 57-4). Reasoning that the causal basis for this correlation derived from in something connected with size, rather than size itself, scientists focused their attention on the organ most frequently associated with life and vitality—the heart. In general, the resting heart rate of small animals such as hummingbirds, 250 beats per minute, tends to be higher than those of large animals such as whales, 10 to 30 beats per minute. Estimates of the total number of times each vertebrate animal's heart beat over the course of a lifetime exhibited an amazing convergence on 1 x 10°.
Is the vertebrate heart physically or genetically limited to 1 billion beats? A more nuanced variation of this heartbeat hypothesis was put forward by Raymond Pearl in the 1920s. Pearl's metabolic or rate of living hypothesis posited that an individual's lifespan was reciprocally linked to their basal metabolic rate. It was calculated that every vertebrate animal expends a similar amount of total metabolic energy per unit body mass, 7 x 10° J/g, over their lifetime. While intuitively appealing, identification of a mechanistic link between lifespan and energy expenditure or metabolic rate has proven elusive. Adherents of the mitochondrial

2390. Hippopotamus, Mean Expectation of Life at Maturity (years) = 16.40. African elephant, Approximate Mass (kg) = 4000. African elephant, Mean Expectation of Life at Maturity (years) = 19.10
Source: Adapted from Millar JS, Zammuto: Life histories of mammals: an analysis of life tables. Ecology 1983;64:631.
theory of aging suggest that what is being "counted" is not heartbeats or energy, but the ROS that are the by-product of respiration. Over time, the continued generation of energy and related consumption of O, leads to the accumulation of ROS-induced damage to DNA, proteins, and lipids until, eventually, a universally conserved tipping point is reached. Cells experiencing caloric deficits adjust (reprogram) their metabolic pathways to utilize available resources in a more efficient manner that concomitantly decreases the yield of collateral ROS.

A second school of thought holds that the putative countdown clock that controls aging and lifespan does not monitor heartbeats, energy, or ROS. Rather, it uses telomeres to track the number of times each somatic cell divides.
Unlike the closed circular DNA of bacterial genomes, the genomic DNA of eukaryotes is linear. If left unprotected, the exposed ends of these linear polynucleotides would be available to participate in potentially deleterious genetic recombination events. Telomeres cap the ends of eukaryotic chromosomes with a few hundred GT-rich hexanucleotide repeats. These caps also provide a source dispensable DNA to accommodate the wastage that occurs when linear chromosomes are replicated.
This wastage is a consequence of the fact that all DNA polymerases work unidirectionally, 3' to 5' (see Chapter 35). When replicating linear double-stranded DNA via discontinuous 3' to 5' synthesis, the 5' end of each new strand will generally commence 2100 bp short of the extreme 5' end of the template strand. Consequently, each time a cell replicates its genome in order to divide, its chromosomes become shorter (Figure 57-7). The telomeres provide an innocuous source of DNA whose gradual loss is of little immediate consequence to the cell. However, once the supply of telomere DNA is exhausted, roughly 100 cell divisions for humans, mitosis will cease, causing the somatic cell to enter a state of replicative senescence. Hence, as our bodies age, they progressively lose the capacity to replace lost or damaged cells.
Organisms are able to generate progeny that contain full-length telomeres through the intervention of the enzyme telomerase. Telomerase is a ribonucleoprotein that is expressed in stem cells and most cancer cells, but not in somatic cells. Using an RNA template, telomerase adds GT-rich hexanucleotide repeat sequences to the ends of linear DNA molecules that restore their telomeres to full length. In the laboratory, the operation of the proposed telomere clock has been demonstrated by genetically engineered somatic cells to express telomerase. As predicted by the telomere clock hypothesis, the engineered cells continued to divide in culture long after wild-type control cells entered replicative senescence.

Many advances in biomedical science are the product of research that uses a variety of so-called model organisms as their test subjects. The fruit fly, Drosophila melanogaster, has yielded a rich harvest of information concerning the
genes that guide cellular differentiation and organ development. Baker's yeast and the African clawed frog, Xenopus laevis, have served as the workhorses for dissecting the signal transduction circuitry that orchestrates the cell division cycle. A variety of cultured mammalian cell lines serve as surrogates for adipocytes, kidney cells, tumors, dendrites, etc. While at first glance it would appear that many of these model systems share little in common with humans, each possesses unique attributes that render them convenient vehicles for addressing particular problems or exploring specific cellular or molecular processes.
Caenorhabditis elegans is a worm that has served as an important subject for the study of developmental biology. C. elegans is transparent and grows rapidly, enabling the developmental program that generates all 959 cells in an adult to be traced back to the fertilized egg. In the early 1990s, Cynthia Kenyon and colleagues observed that worms carrying mutations of the gene encoding an insulin receptor-like molecule, daf-2, lived 70% longer than their wild-type counterparts. Equally important, the mutant worms behaved in a manner resembling that of young wild-type C. elegans for much of this period. This is an important distinction, as a bona fide "aging gene,' must do more than simply prolong existence. It must delay one or more of the physiologic changes associated with aging.
Investigation of further aging genes indicate they code for either one of a small set of transcription factors that include PHA-4 or DAF-16, which presumably control expression of aging critical genes, or signaling proteins such as DAF-2 that probably activate PHA-4, DAF-16, etc, in response to specific environmental signals. Much remains to be learned about the extent to which aging is controlled by genetically programmed events, and how these gene and their products interact with nutritional and other factors that influence vitality and longevity.

The idea that animals would have evolved mechanisms designed specifically to limit their lifespan would appear, at first glance, to be counterintuitive. If the driving force behind evolution is the selection for traits that enhance fitness and survival, should not this translate into an ever-increasing life expectancy? However, while extending lifespan may represent a desirable trait from the point of view of the individual, it does not necessarily follow that this applies to a population or species as a whole. A genetically programmed limit on lifespan could benefit the group by eliminating the drain on available resources imposed by members no longer actively involved in the production, development, and training of offspring. Indeed, the current three-generation lifespan can be rationalized as providing time (a) for newborns to develop into reproductively active young adults, (b) for these young adults to produce and nurture their offspring, and (c) for older adults to serve as a source of guidance and assistance to young adults facing the challenges of childbirth and childrearing.

™
Aging
and
longevity
are
via
the
complex
interplay
between
random
and
deterministic
factors
that
include
genetic
programming,
stresses,
lifestyle,
cellular
countdown clocks, and molecular repair processes.
™ Wear and tear theories hypothesize that the aging results from
the accumulation of damage over time.
=
Water, oxygen, and light are essential for life, but possess an
intrinsic capacity to damage biologic macromolecules.
™ ROS are continually generated as a by-product of metabolism, particularly the electron transport chain.
™ The deleterious effects of ROS are often amplified by free radical chain reactions.
| 'The reactivity of their unsaturated ring systems and ability to absorb UV light render the nucleotide bases of DNA particularly vulnerable to UV or chemical damage.
= Mutations resulting from errors caused by missing or damaged nucleotide bases can be particularly harmful, and may result in oncogenic transformation.
@ Their critical roles in energy production and apoptosis, along with their indigenous genome, render mitochondria a central player in many theories of aging and death.
™ Long repeating sequences called telomeres cap the ends of the linear chromosomes in eukaryotes.
™ Because telomeres progressively shorten with each division of a somatic cell, they are hypothesized to serve as a countdown clock.
= Model organisms provide useful vehicles for investigating biologic processes.
= Mutation of the daf-2 gene in C. elegans extends the lifespan of worms by 70%.
™ Evolutionary selection of a limited lifespan may optimize the vitality of the population rather than that of its individual members.

m= Use your knowledge to explain the underlying biochemical defects in diseases.

In this final chapter, nine case histories are presented as openended problems for you to solve, based on what you have learnt from studying this book. No solutions are provided, and there is no discussion of the cases; all that you need to know in order to explain the problems is available elsewhere in this book.
In many cases, the patient's clinical chemistry results are presented together with reference ranges. These may differ from problem to problem, because, as discussed in Chapter 48, reference ranges from different laboratories may differ.

and determined insulin in the fractions eluted from the columns both by radioimmunoassay (graph A in Figure 58-2) and by stimulation of glucose oxidation (graph B). Three molecular mass markers were used; they eluted as follows: M_ 9000 in fraction 10, M, 6000 in fraction 23, and M, 4500 in fraction 27. The investigators also measured insulin in the fractions eluted from the chromatography column after treatment of each fraction with trypsin. The results are shown in graph C. After seeing the results of these studies, they subjected the same-pooled serum sample to brief treatment with trypsin, and performed gel exclusion chromatography on the product. Again, they measured insulin by radioimmunoassay (graph D) and biologic assay (graph E).
The patient is a 5-year-old boy, who was born in 1967, at term, after an uneventful pregnancy. He was a sickly infant, and did not grow well. On a number of occasions his mother noted that he appeared drowsy, or even comatose, and said that there was a "chemical, alcohol-like" smell on his breath, and in his urine. The GP suspected diabetes mellitus, and sent him to the Middlesex Hospital in London for a glucose tolerance test. The

Blood samples were also taken for measurement of insulin at zero time and 1 hour after the glucose load. At this time, a new method of measuring insulin was being developed, radioimmunoassay (see Chapter 48), and therefore both this and the conventional biological assay were used. The biological method of measuring insulin is by its ability to stimulate the uptake and metabolism of glucose in rat muscle in vitro; this can be performed relatively simply by measuring the radioactivity in "CO, after incubating duplicate samples of the muscle with ['*C]glucose, with and without the sample containing insulin. The results are shown in Table 58-1.
As a part of their studies of the new radioimmunoassay for insulin, the team at the Middlesex Hospital performed gel exclusion chromatography of a pooled sample of normal serum,
Since these studies in the 1960s, the gene for human insulin has been cloned. Although insulin consists of two peptide chains, 21and 30-amino acids long, respectively, these are
FIGURE 58-1 Plasma glucose in the patient and a control subject after a test dose of glucose.

Biological assay, Baseline (Fasting) Blood Sample.Patient (Case 1) = 0.8. Biological assay, Baseline (Fasting) Blood Sample.Control Subjects = 6 ± 2. Biological assay, 1 Hour After Glucose Load.Patient (Case 1) = 5. Biological assay, 1 Hour After Glucose Load.Control Subjects = 40 ± 11. Radioimmunoassay, Baseline (Fasting) Blood Sample.Patient (Case 1) = 10. Radioimmunoassay, Baseline (Fasting) Blood Sample.Control Subjects = 6 ± 2. Radioimmunoassay, 1 Hour After Glucose Load.Patient (Case 1) = 50. Radioimmunoassay, 1 Hour After Glucose Load.Control Subjects = 40 ± 11
coded for by a single gene, which has a total of 330 base pairs between the initiator and stop codons. As you would expect for a secreted protein, there is a signal sequence coding for 24 amino acids at the 5' end of the gene.

What does this information suggest about the processes that occur in the synthesis of insulin?
What is likely to be the underlying biochemical basis of the patient's problem?
The patient is a 50-year-old man, 174-cm tall, and weighing 105 kg. He is an engineer, and works on secondment in one of the strict Islamic states in the Gulf, where alcohol is prohibited. At the beginning of August, he returned home for his annual leave. According to his family, he behaved as he usually did when on leave, consuming a great deal of alcohol and refusing meals. He was known to be drinking 2 L of whiskey, two or three bottles of wine, and a dozen or more cans of beer each day; his only solid food consisted of sweets and biscuits.
On September Ist, he was admitted to the emergency department, semiconscious, and with a rapid respiration rate (40/minute). His blood pressure was 90/60 and his pulse rate was 136/minute. His temperature was normal (37.1°C). Emergency blood gas analysis revealed severe acidosis: pH 7.02 and base excess —23; Po, 91 mm Hg and Pco, 10 mm Hg. He was transferred to intensive care and given intravenous bicarbonate.
His pulse rate remained high, and his blood pressure low, so emergency cardiac catheterization was performed; this revealed a cardiac output of 23 L/min (normal 4-6). A chest x-ray shows significant cardiac enlargement.
Table 58-2 shows the clinical chemistry results from a plasma sample taken shortly after he was admitted.
What is the likely biochemical basis of the patient's problem, which led to his emergency hospitalization? What additional test(s) might you request to confirm your assumption? What would emergency treatment you suggest?

Glucose, Patient in Case 2 = 10.6. Glucose, Reference Range = 3.5-5. Sodium, Patient in Case 2 = 142. Sodium, Reference Range = 131-151. Potassium, Patient in Case 2 = 3.9. Potassium, Reference Range = 3.4-5.2. Chloride, Patient in Case 2 = 91. Chloride, Reference Range = 100-110. Bicarbonate, Patient in Case 2 = 5. Bicarbonate, Reference Range = 21-29. Lactate, Patient in Case 2 = 18.9. Lactate, Reference Range = 0.9-2.7. Pyruvate, Patient in Case 2 = 2.5. Pyruvate, Reference Range = 0.1-0.2

Initial, Patient in Case 3.GSH (mmol/L) = 1.61. Initial, Patient in Case 3.GSSG (μmol/L) = 400. Initial, Control Subjects.GSH (mmol/L) = 2.01 ± 0.29. Initial, Control Subjects.GSSG (μmol/L) = 4.2 ± 0.61. + Acetylphenylhydrazine, Patient in Case 3.GSH (mmol/L) = 0.28. + Acetylphenylhydrazine, Patient in Case 3.GSSG (μmol/L) = 1540. + Acetylphenylhydrazine, Control Subjects.GSH (mmol/L) = 1.82 ± 0.24. + Acetylphenylhydrazine, Control Subjects.GSSG (μmol/L) = 190 ± 28

The patient is an African American recruit to the army. He was given the antimalarial drug primaquine, and suffered a delayed reaction with kidney pain, dark urine, and low red blood cell counts that led to anemia and weakness. Centrifugation of a blood sample showed a low hematocrit, and the plasma was red colored.
Since none of the red cell lysates showed any significant activity with NADH, it is unlikely that there is any transhydrogenase activity in erythrocytes, to reduce NADP* to NADPH at the expense of NADH. This raises the problem of the source of NADPH in erythrocytes.
Similar acute hemolytic attacks have been observed, predominantly in men of Afro-Caribbean origin, in response to primaquine and a variety of other drugs, including dapsone, the antipyretic acetylphenylhydrazine, the antibacterial bactrim/septrin, sulfonamides, and sulfones, whose only common feature is that they all undergo cyclic nonenzymic reactions in the presence of oxygen to produce hydrogen peroxide and a variety of oxygen radicals that can cause oxidative damage to membrane lipids, leading to hemolysis. Moderately severe infection can also precipitate a hemolytic crisis in susceptible people.
One way of screening for sensitivity to primaquine is based on the observation that the glutathione concentration of erythrocytes from sensitive subjects is somewhat lower than that of control subjects, and falls considerably on incubation with acetylphenylhydrazine.
Glutathione (GSH) is a tripeptide, y-glutamyl-cysteinylglycine, which readily undergoes oxidation to form a disulphide-linked hexapeptide, oxidized glutathione, generally abbreviated to GSSG. Table 58-3 shows the concentrations of GSH and GSSG in red cells from the patient and 10 control subjects, before and after incubation with acetylphenylhydrazine.

The reported K, of glutathione reductase for GSSG is 65 mol/L and for NADPH is 8.5 mol/L. Erythrocyte lysates were incubated with a saturating concentration of GSSG (1 mmol/L) and either NADPH or NADH (100 pmol/L). Each incubation contained the hemolysate from 0.5 mL packed cells (Table 58-4).

NADPH, Patient in Case 3 = 0.64. NADPH, Control Subjects = 0.63 ± 0.06. NADH, Patient in Case 3 = 0.01. NADH, Control Subjects = 0.01 ± 0.001
The dye methylene blue will oxidize NADPH; the reduced dye then undergoes nonenzymic oxidation in air, so the addition of a relatively small amount of methylene blue will effectively deplete NADPH, and would be expected to stimulate any pathway that reduces NADP* to NADPH.
Erythrocytes from control subjects were incubated with 10 mmol/L ["C]glucose with or without the addition of methylene blue; all six possible positional isomers of ['*C]glucose were used, and the radioactivity in (lactate + pyruvate) was determined after thin layer chromatography of the incubation mixture. Each incubation contained 1 mL of erythrocytes in a total incubation volume of 2 mL (Table 58-5).
In further
studies, only
of
CO, from
["C-1]glucose was measured, with the addition of:
¢ Sodium ascorbate (which undergoes a nonenzymic reac-
tion in
air to
produce
H,O,)
e
Acetylphenylhydrazine
(which hemolysis
in sensitive
is subjects,
known to
precipitate and
depletes glutathione)
- ¢ Methylene blue (which oxidizes NADPH)
The incubations were repeated with N-ethylmaleimide, which undergoes a nonenzymic reaction with the —SH group of reduced glutathione, and thus depletes the cell of total glutathione. The results are shown in Table 58-6.
Further studies
showed that
the patient's
red blood

cells contained only about 20% of the normal activity of glucose6-phosphate dehydrogenase (see Chapter 20). In order to investigate why his enzyme activity was so low, a sample of his red blood cells was incubated at 45°C for 60 minutes, then cooled to 30°C and the activity of glucose-6-phosphate dehydrogenase was determined. After the preincubation at 45°C, his red cells showed only 60% of their initial activity. By contrast, red cells from control subjects retained 90% of their initial activity after preincubation at 45°C for 60 minutes.

The patient is a 10-year-old Maltese boy. On his birthday his aunt gave him a pie made from fava beans (a local delicacy), and that evening he suffered kidney pain, and reduced

Pyruvate = 14060 ± 120. [ 14 C-5]glucose, + Methylene Blue.CO 2 = nd. [ 14 C-6]glucose, Control.Lactate + Pyruvate = 14090 ± 110. [ 14 C-6]glucose, Control.CO 2 = nd. [ 14 C-6]glucose, + Methylene Blue.Lactate + Pyruvate = 14100 ± 120. [ 14 C-6]glucose, + Methylene Blue.CO 2 = nd
Abbreviation: no, not detectable, ie, below the limits of detection. Figures show dpm, mean + sd for 5 replicate incubations.
passed dark urine. A blood film showed a low red blood cell count and the plasma was red colored. This problem is not uncommon in Malta, and indeed several of his classmates (all boys) have died when an acute crisis has been precipitated by eating fava beans, or after a moderate fever associated with an infection.
Further studies showed that his erythrocyte glucose6-phosphate dehydrogenase was only 10% of normal and had avery high K, for NADP*. Unlike the patient in Case 3, his red blood cell enzyme was as stable to incubation at 45°C as that from control subjects.
What conclusions these observations? can you draw from

The patient is a 28-week-old baby girl. She was admitted to the emergency department in a coma, having suffered a convulsion after feeding. She had a mild infection and slight fever at the time. Since birth she had been a sickly child, and had frequently vomited and become drowsy after feeding. She was bottle-fed and at one time, cows' milk allergy was suspected, although the problems persisted when she was fed on soya milk.

None, Control = 1410 ± 70. None, + N -Ethylmaleimide = 670 ± 30. Ascorbate, Control = 8665 ± 300. Ascorbate, + N -Ethylmaleimide = 2133 ± 200. Acetylphenylhydrazine, Control = 7740 ± 320. Acetylphenylhydrazine, + N -Ethylmaleimide = 4955 ± 325. Methylene blue, Control = 12230 ± 500. Methylene blue, + N -Ethylmaleimide = 11265 ± 450
Figures show dpm, mean + sd for 5 replicate incubations.
On admission, she was mildly hypoglycemic, ketotic, and her plasma pH was 7.29. Analysis of a blood sample showed normal levels of insulin, but considerable hyperammonemia (plasma ammonium ion concentration 500 umol/L; reference range 40-80 umol/L). She responded well to intravenous glucose infusion and rectal infusion of lactulose, regaining consciousness. She had poor muscle tone.
A liver biopsy sample was taken, and the activities of the enzymes of urea synthesis (see Chapter 28) were determined, and compared with activities in postmortem liver samples from six infants of the same age. The results are shown in Table 58-7. She remained well on a high-carbohydrate, lowprotein diet for several days, although the poor muscle tone and muscle weakness persisted. A second liver biopsy sample was taken after 4 days and the activity of the enzymes determined again.
TABLE 58-7 Activity of Enzymes of the Urea Synthesis Cycle in Liver Biopsy Samples From the Patient in Case 5 on Admission and After 4 Days on a High Carbohydrate, Low-Protein Diet, Compared With Activities in Postmortem Samples From 6 Infants of the Same Age

Carbamoyl phosphate synthetase, μmol Product Formed/min/mg Protein.Patient.On Admission = 0.337. Carbamoyl phosphate synthetase, μmol Product Formed/min/mg Protein.Patient.After 4 Days = 1.45. Carbamoyl phosphate synthetase, μmol Product Formed/min/mg Protein..Control Subjects = 1.30 ± 0.40. Ornithine carbamoyltransferase, μmol Product Formed/min/mg Protein.Patient.On Admission = 29.0. Ornithine carbamoyltransferase, μmol Product Formed/min/mg Protein.Patient.After 4 Days = 28.6. Ornithine carbamoyltransferase, μmol Product Formed/min/mg Protein..Control Subjects = 18.1 ± 4.9. Argininosuccinate synthetase, μmol Product Formed/min/mg Protein.Patient.On Admission = 0.852. Argininosuccinate synthetase, μmol Product Formed/min/mg Protein.Patient.After 4 Days = 0.75. Argininosuccinate synthetase, μmol Product Formed/min/mg Protein..Control Subjects = 0.49 ± 0.09. Argininosuccinase, μmol Product Formed/min/mg Protein.Patient.On Admission = 1.19. Argininosuccinase, μmol Product Formed/min/mg Protein.Patient.After 4 Days = 0.95. Argininosuccinase, μmol Product Formed/min/mg Protein..Control Subjects = 0.64 ± 0.15. Arginase, μmol Product Formed/min/mg Protein.Patient.On Admission = 183. Arginase, μmol Product Formed/min/mg Protein.Patient.After 4 Days = 175. Arginase, μmol Product Formed/min/mg Protein..Control Subjects = 152 ± 56

Her very low-protein diet was continued, but in order to ensure an adequate supply of essential amino acids for growth, she was fed a mixture of the keto acids of threonine, methionine, leucine, isoleucine, and valine. After each feed she again became abnormally drowsy and markedly ketotic, with significant acidosis. Her plasma ammonium ion concentration was within the normal range, and a glucose tolerance test was normal, with a normal increase in insulin secretion after glucose load.
High-pressure liquid chromatography of her plasma revealed an abnormally high concentration of propionic acid (24 «mol/L; reference range 0.7-3 umol /L). Urine analysis showed considerable excretion of methylcitrate (1.1 umol/mg creatinine), which is not normally detectable. She was also excreting a significant amount of short-chain acyl carnitine (mainly propionyl carnitine)—28.6 ,mol/ 24 hours, compared with a reference range of 5.7 + 3.5 umol/ 24 hours.
The metabolism of a test dose of ['*C] propionate given by intravenous infusion was determined in the patient, her parents, and a group of control subjects; skin fibroblasts were cultured and the activity of propionyl-CoA carboxylase was determined by incubation with propionate and NaH"CO,, followed by acidification and measurement of the radioactivity in products. The results are shown in Table 58-8.
The results of measuring carnitine in the first liver biopsy sample and in a muscle biopsy sample gave the results shown in Table 58-9.
What conclusions can you draw from these results?
Can you explain the biochemical basis of the patient's condition?

The patient is a 9-month-old girl, the second child of unrelated parents. She was born at term after an uneventful pregnancy, weighing 3.4 kg and was breast-fed, with gradual introduction of solids from 3 months of age onward. Her mother reported that, while she liked cheese, meat, and fish, she frequently became irritable and grizzly after meals, and became lethargic, drowsy, and "floppy" after eating relatively large amounts of protein-rich foods. Her urine had a curious odor, described by her mother as being "cat-like," on such occasions.
At 9 months of age she was admitted to the emergency department in a coma, and suffering convulsions. She had been unwell for the last 3 days, with a slight fever, and for the last 12 hours had been refusing all food and drink. At this time, she weighed 8.8 kg, and her body length was 70.5 cm. Emergency blood tests revealed moderate acidosis (pH 7.25) and severe hypoglycemia (glucose < 1 mmol/L); a dipstick test for plasma ketone bodies was negative. A blood sample was taken for full clinical chemistry tests, and she was given intravenous glucose. Within a short time, she recovered consciousness. The results of the blood tests are shown in Table 58-10.
She remained in hospital for several weeks, while further tests were performed. She was generally well through this time, but became drowsy and severely hypoglycemic, and hyperventilated, if she was deprived of food for more than about 8 to 9 hours. Her muscle tone was poor, and she was very weak, with considerably less strength (eg, in pushing her arms or legs against the pediatrician's hand) than would be expected for a girl of her age.
On one occasion her blood glucose was monitored at 30-minute intervals over 3 hours from waking, without being fed. It fell from 3.4 mmol/L on waking to 1.3 mmol/L 3 hours later. She was deprived of breakfast again the next day, and again blood glucose was measured at 30-minute intervals for 3 hours during which she received an intravenous infusion of B-hydroxybutyrate (50 umol/min/kg body weight). During the infusion of B-hydroxybutyrate, her plasma glucose remained between 3.3 and 3.5 mmol/L.

At no time were ketone bodies detected in her urine, and there was no evidence of any abnormal excretion of amino acids. However, a number of abnormal organic acids were detected in her urine, including relatively large amounts of

Total carnitine, Liver.Patient in Case 5 = 0.23. Total carnitine, Liver.Control Subjects = 0.83 ± 0.26. Total carnitine, Muscle.Patient in Case 5 = 1.56. Total carnitine, Muscle.Control Subjects = 2.29 ± 0.75. Free carnitine, Liver.Patient in Case 5 = 0.05. Free carnitine, Liver.Control Subjects = 0.41 ± 0.17. Free carnitine, Muscle.Patient in Case 5 = 0.29. Free carnitine, Muscle.Control Subjects = 1.62 ± 0.67. Short-chain acylcarnitine, Liver.Patient in Case 5 = 0.16. Short-chain acylcarnitine, Liver.Control Subjects = 0.37 ± 0.20. Short-chain acylcarnitine, Muscle.Patient in Case 5 = 1.16. Short-chain acylcarnitine, Muscle.Control Subjects = 0.58 ± 0.32. Long-chain acylcarnitine, Liver.Patient in Case 5 = 0.01. Long-chain acylcarnitine, Liver.Control Subjects = 0.05 ± 0.02. Long-chain acylcarnitine, Muscle.Patient in Case 5 = 0.11. Long-chain acylcarnitine, Muscle.Control Subjects = 0.09 ± 0.03

Glucose, mmol/L, Patient in Case 6 = 0.22. Glucose, mmol/L, Reference Range = 4-5. pH, Patient in Case 6 = 7-25. pH, Reference Range = 7-35-7-45. Bicarbonate, mmol/L, Patient in Case 6 = 11. Bicarbonate, mmol/L, Reference Range = 21-29. Ammonium, μmol/L, Patient in Case 6 = 120. Ammonium, μmol/L, Reference Range = <50. Ketone bodies, mmol/L, Patient in Case 6 = undetectable. Ketone bodies, mmol/L, Reference Range = 2.5-3.5. Nonesterified fatty acids, mmol/L, Patient in Case 6 = 2. Nonesterified fatty acids, mmol/L, Reference Range = 1.0-1.2. Insulin, mU/L, Patient in Case 6 = 5. Insulin, mU/L, Reference Range = 5-35. Glucagon, ng/mL, Patient in Case 6 = 140. Glucagon, ng/mL, Reference Range = 130-160
3-hydroxy-3-methylglutaric and 3-hydroxy-3-methylglutaconic acids. The excretion of these two acids increased considerably under two conditions:
- 1. When she was fed a relatively high-protein meal (she became grizzly, lethargic, and irritable). A blood sample taken after such a meal showed significant hyperammonemia (130 mol/L), but normal glucose (5.5 mmol/L).
2.
When she
was fasted
for more
than the
normal overnight
fast, with
or without
the infusion
of
B-hydroxybutyrate.

One obvious metabolic precursor of 3-hydroxy3-methylglutaric acid is 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is normally cleaved to yield acetoacetate and acetyl-CoA by the enzyme hydroxymethyglutarylCoA lyase (see Chapter 22). Therefore, the activity of this enzyme was determined in leukocytes from blood samples from the patient and her parents. The results are shown in Table 58-11.
Analysis of her urine also revealed considerable excretion

What is the likely biochemical basis of the patient's problem? To what extent can you account for her various metabolic problems from the information you are given?
What dietary manipulation(s) would be likely to maintain her in good health, and prevent further emergency hospital admissions?

Patient in Case 6, 1 = 1.7. Mother, 1 = 10.2. Father, 1 = 11.4. Control subjects, 1 = 19.7 ± 2.0

Total carnitine, Patient in Case 6 = 680. Total carnitine, Reference Range = 125 ± 75. Free carnitine, Patient in Case 6 = 31. Free carnitine, Reference Range = 51 ± 40. Acyl carnitine, Patient in Case 6 = 649. Acyl carnitine, Reference Range = 74 ± 40

The patient is a 9-month-old boy, the second child of unrelated parents; his brother is 5 years old, fit, and healthy. He was born at full term after an uneventful pregnancy, weighing 3.4 kg (the 50th centile), and developed normally until he was 6 months old, after when he showed some retardation of development. He also developed a fine scaly skin rash about this time, and his hair, which had been normal, became thin and sparse.
At 9 months of age, he was admitted to the emergency department in a coma; the results of clinical chemistry tests on a plasma sample are shown in Table 58-13.
The acidosis was treated by intravenous infusion of bicarbonate, and he recovered consciousness. Over the next few days he continued to show signs of acidosis (rapid respiration), and even after a meal ketone bodies were present in his urine. His plasma lactate, pyruvate, and ketone bodies remained high; plasma glucose was in the low normal range, and his plasma insulin was normal both in the fasting state and after an oral glucose load.
Urine analysis revealed the presence of significant amounts of a number of organic acids that are not normally excreted in the urine, including:
e Lactate, pyruvate, and alanine
e Propionate, hydroxypropionate, and propionyl glycine
¢ Methylcitrate
e Tiglate and tiglylglycine
e
3-Methyl crotonate,
3-methylcrotonylglycine, and
3-hydroxyisovalerate

Glucose, mmol/L, Patient in Case 7 = 3.3. Glucose, mmol/L, Reference Range = 3.5-5.5. pH, Patient in Case 7 = 6.9. pH, Reference Range = 7-35-7-45. Bicarbonate, mmol/L, Patient in Case 7 = 2.0. Bicarbonate, mmol/L, Reference Range = 21-25. Ketone bodies, mmol/L, Patient in Case 7 = 21. Ketone bodies, mmol/L, Reference Range = 1-2.5. Lactate, mmol/L, Patient in Case 7 = 7-3. Lactate, mmol/L, Reference Range = 0.5-2.2. Pyruvate, mmol/L, Patient in Case 7 = 0.31. Pyruvate, mmol/L, Reference Range = <0.15

His skin rash and hair loss were reminiscent of the signs of biotin deficiency (see Chapter 44), as caused by excessive consumption of uncooked egg white. However, his mother said that he did not eat raw or undercooked eggs at all, although he was fond of hard-boiled eggs and yeast extract (which are rich sources of biotin). His plasma biotin was 0.2 nmol/L (normal > 0.8 nmol/L), and he excreted a significant amount of biotin in the form of biocytin (see Figure 44-14) and small biocytincontaining peptides, which are not normally detectable in urine. He was treated with 5 mg of biotin per day. After 3 days the abnormal organic acids were no longer detectable in his urine, and his plasma lactate, pyruvate, and ketone bodies had returned to normal, although his excretion of biocytin and biocytin-containing peptides increased. At this stage he was discharged from hospital, with a supply of biotin tablets. After 3 weeks his skin rash began to clear, and his hair loss ceased. Three months later, at a regular out-patient visit, it was decided to cease the biotin supplements. Within a week, the abnormal organic acids were again detected in his urine, and he was treated with varying doses of biotin until the organic aciduria ceased. This was achieved at an intake of 150 pg/d (compared with the reference intake of 10-20 yg/d for an infant under 2 years old). He has continued to take 150 ug of biotin daily, and has remained in good health for the last 4 years.
Can you account for the biochemical basis of the patient's problem?

The patient is a 4-year-old girl, the only child of nonconsanguineous parents, born at term after an uneventful pregnancy. At 14 months of age she was admitted to hospital with a 1-day history of persistent vomiting, rapid shallow respiration, and dehydration. On admission, her respiration rate was 60/minute and her pulse 178/minute. The first column in Table 58-14 shows the results of clinical chemistry tests at that time. She responded rapidly to intravenous bicarbonate and a single intramuscular injection of insulin.
The results of a glucose tolerance test 3 days after admission were normal, and her plasma insulin response to an oral glucose load was within the normal range. She was discharged from hospital 7 days after admission, apparently fit and well. The second column in Table 58-14 shows the results of clinical chemistry tests taken shortly before her discharge.
She was readmitted to hospital at 16, 25, 31, and 48 months of age, suffering from restlessness, unsteady gait, rapid shallow respiration, persistent vomiting, and dehydration. Each time the crisis was preceded by a common childhood illness and decreased appetite, and she responded well to intravenous fluids and bicarbonate. A number of milder episodes were treated at home by oral fluid and bicarbonate.
During her admission at age 25 months, a skin biopsy was taken, fibroblasts were cultured, and the mitochondrial enzyme activities shown in Table 58-15 were determined.
TABLE 58-14 Clinical Chemistry Results for Plasma and Urine Samples From the Patient in Case 8 on Admission and Again 1 Week Later

Lactate, mg/g creatinine, 1 Week Later = 1.48. Lactate, mg/g creatinine, Reference Range = <0.1. Ketone bodies, using Ketostix, Acute Admission = Very high. Ketone bodies, using Ketostix, 1 Week Later = Negative. Ketone bodies, using Ketostix, Reference Range = Negative

The patient is a 5-year-old boy who is diabetic. There is a family history of diabetes, which strongly suggests a dominant pattern of inheritance. He secretes a significant amount of insulin, although less than normal subjects,
TABLE 58-15 Activities of Mitochondrial Enzymes From Cultured Skin Fibroblasts (nmol product formed/min/mg protein)
Citrate synthase, Patient in Case 8 = 32.8. Citrate synthase, Control Subjects = 76.3 ± 15.1. Cytochrome c reduction by NADH, Patient in Case 8 = 11.6. Cytochrome c reduction by NADH, Control Subjects = 16.7 ± 4.6. Cytochrome c reduction by succinate, Patient in Case 8 = 9.43. Cytochrome c reduction by succinate, Control Subjects = 12.3 ± 3.2. Cytochrome oxidase, Patient in Case 8 = 37-7. Cytochrome oxidase, Control Subjects = 50.3 ± 11.6. NADHdehydrogenase, Patient in Case 8 = 633. NADHdehydrogenase, Control Subjects = 910 ± 169. Pyruvate carboxylase, Patient in Case 8 = 0.03. Pyruvate carboxylase, Control Subjects = 1.62 ± 0.39. Pyruvate dehydrogenase, Patient in Case 8 = 1.86. Pyruvate dehydrogenase, Control Subjects = 1.72 ± 0.35. Succinate oxidase, Patient in Case 8 = 190. Succinate oxidase, Control Subjects = 210 ± 30

3.3 mmol/L glucose, Control = 3.5. 3.3 mmol/L glucose, + Mannoheptulose = 3.5. 16.6 mmol/L glucose, Control = 12.5. 16.6 mmol/L glucose, + Mannoheptulose = 3.5
Source: From data reported by Coore HG, Randle PJ: Biochemical J 1964;93:66-77.
suggesting that
is not
type
1
diabetes.
Unlike type
2
diabetes, and
the this
condition referred
develops to
as in
early childhood,
maturity-onset diabetes
of young
(MODY).
The results of studies of the secretion of insulin by rab- bit
pancreas incubated
in vitro
with two
concentrations of
glucose, with
and without
mannoheptulose, the
addition which
is an
glucose to
of the
7-carbon sugar
Two enzymes
phate the
are phosphorylation
shown catalyze
from glucose
e
Hexokinase
(see
Chapter is
cose of
~0.15
17):
in mmol/L.
Glucokinase
¢
is expressed
only in
liver and
the
6
cells in
Table
58-16.
glucose-6-phos- has
a
K,, for
glu- of
the pancreas; it has a K,, for glucose of ~20 mmol/L.
The normal range of plasma glucose is between 3.5 and 5 mmol/L, rising in peripheral blood to 8 to 10 mmol/L after a moderately high intake of glucose. After a meal, the concentration of glucose in the portal blood, coming from the small intestine to the liver, may be considerably higher than this.
What effect will changes in the plasma concentration of glucose have on the rate of formation of glucose6-phosphate catalyzed by hexokinase?
What effect
will changes
in the
plasma concentration

of glucose have on the rate of formation of glucose-
6-phosphate catalyzed by glucokinase?
What is the importance of glucokinase in the liver?
Froguel and coworkers (1993) reported studies of the glucokinase gene in a number of families affected by MODY, and also in unaffected families. They published a list of 16 variants of the glucokinase gene, shown in Table 58-17. All their patients with MODY had an abnormality of the gene.

4, Nucleotide Change = GAC ⇒ AAC. 4, Amino Acid Change = ?. 4, Effect = None. 10, Nucleotide Change = GCC ⇒ GCT. 10, Amino Acid Change = ?. 10, Effect = None. 70, Nucleotide Change = GAA ⇒ AAA. 70, Amino Acid Change = ?. 70, Effect = MODY. 98, Nucleotide Change = CAG ⇒ TAG. 98, Amino Acid Change = ?. 98, Effect = MODY. 116, Nucleotide Change = ACC ⇒ ACT. 116, Amino Acid Change = ?. 116, Effect = None. 175, Nucleotide Change = GGA ⇒ AGA. 175, Amino Acid Change = ?. 175, Effect = MODY. 182, Nucleotide Change = GTG ⇒ ATG. 182, Amino Acid Change = ?. 182, Effect = MODY. 186, Nucleotide Change = CGA ⇒ TGA. 186, Amino Acid Change = ?. 186, Effect = MODY. 203, Nucleotide Change = GTG ⇒ GCG. 203, Amino Acid Change = ?. 203, Effect = MODY. 228, Nucleotide Change = ACG ⇒ ATG. 228, Amino Acid Change = ?. 228, Effect = MODY. 261, Nucleotide Change = GGG ⇒ AGG. 261, Amino Acid Change = ?. 261, Effect = MODY. 279, Nucleotide Change = GAG ⇒ TAG. 279, Amino Acid Change = ?. 279, Effect = MODY. 300, Nucleotide Change = GAG ⇒ AAG. 300, Amino Acid Change = ?. 300, Effect = MODY. 300, Nucleotide Change = GAG ⇒ CAG. 300, Amino Acid Change = ?. 300, Effect = MODY. 309, Nucleotide Change = CTC ⇒ CCC. 309, Amino Acid Change = ?. 309, Effect = MODY. 414, Nucleotide Change = AAG ⇒ GAG. 414, Amino Acid Change = ?. 414, Effect = MODY
Source: From data reported by Froguel P, et al: N Engl J Med 1993;328:697-702.

Why do the mutations affecting codons 4, 10, and 116 have no effect on the people involved?
What conclusions can you draw from this information?
The same authors also studied the secretion of insulin in response to glucose infusion in patients with MODY and control subjects. They were given an intravenous infusion of glucose; the rate of infusion was varied so as to maintain a constant plasma concentration of glucose of 10 mmol/L. Their plasma concentrations of glucose and insulin were measured before and after 60 minutes of glucose infusion; the results are shown in Table 58-18.
What are the amino acid changes associated with each mutation in the gene?
What conclusions can you draw from this information about the probable role of glucokinase in the 6 cells of the pancreas? Can you deduce the way in which the f cells of the pancreas sense an increase in plasma glucose and signal the secretion of insulin?

Fasting, Plasma glucose (mmol/L).Patients With MODY = 7-0 ± 0.4. Fasting, Plasma glucose (mmol/L).Control Subjects = 5.1 ± 0.3. Fasting, Insulin (mU/L).Patients With MODY = 5 ± 2. Fasting, Insulin (mU/L).Control Subjects = 6 ± 2. 60-min glucose infusion, Plasma glucose (mmol/L).Patients With MODY = Maintained at 10 mmol/L by varying rate of infusion. 60-min glucose infusion, Plasma glucose (mmol/L).Control Subjects = Maintained at 10 mmol/L by varying rate of infusion. 60-min glucose infusion, Insulin (mU/L).Patients With MODY = 12 ± 7. 60-min glucose infusion, Insulin (mU/L).Control Subjects = 40 ± 11
all tissues;
it
- 5, A 15-year-old adolescent girl presented at clinic with bruises on

1. Which one of the following statements regarding the blood
coagulation pathways is NOT CORRECT?
A. The components of the extrinsic Xase (tenase) complex are
factor VIIa, tissue factor, Ca**, and factor X.
B. The components of the intrinsic Xase (tenase) complex are
factors [Xa and VIIIa, Ca”*, and factor X.
C. The components of the prothrombinase complex are factors Xa and Va, Ca”*, and factor II (prothrombin).
D. The extrinsic and intrinsic Xase complexes and
prothrombinase complex require anionic procoagulant
on
low-density
lipoprotein
(LDL)
for
their
assembly.
E. Fibrin formed by cleavage of fibrinogen by thrombin is
covalently cross-linked by the action of factor XIIIa, which itself is formed by the action of thrombin on factor XIII.
2.
On
which
one
of
the
following
factors
does
a
patient
taking warfarin for his thrombotic disorder have decreased Gla
(y-carboxyglutamate) residues?
A. Tissue factor
B. Factor XI
C. Factor V
D. Factor II (prothrombin)
E. Fibrinogen
3. A 65-year-old man suffers a myocardial infarction and is given
tissue plasminogen activator within 6 hours of onset of the
thrombosis to achieve which one of the following?
Prevent activation of the extrinsic pathway of coagulation >
Inhibit thrombin
Enhance degradation of factors VIIIa and Va
Enhance fibrinolysis
Inhibit platelet aggregation
moon
4. Which one of the following statements regarding platelet
activation in hemostasis and thrombosis is NOT CORRECT?
A. Platelets adhere directly to subendothelial collagen via
GPla-Ila and GPVI, while binding of GPIb-IX-V is mediated via von Willebrand factor.

B. The aggregating agent thromboxane is formed from
A, arachidonic acid liberated from platelet membrane phospholipids by the action of phospholipase A,.
C. The aggregating agent ADP is released from the dense granules of activated platelets.
D. The aggregating agent thrombin activates intracellular phospholipase CB, which forms the internal effector molecules 1,2-diacylglycerol and 1,4,5-inositol trisphosphate from the membrane phospholipid
phosphatidylinositol 4,5-bisphosphate.
E. The ADP receptors, the thromboxane A, receptor, the thrombin PAR-1 and PAR-4 receptors, and the fibrinogen GPIIb-IIIa receptor are all examples of G-protein-coupled receptors.
her
lower
Of
the
which
is
least
likely
to
explain the bleeding signs exhibited by this individual?
Hemophilia A >
von Willebrand disease
A low platelet count
Aspirin ingestion
A platelet disorder with absence of storage granules
mOoOp
6.
Regarding chemical carcinogenesis, select the one FALSE
statement:
A. Approximately 80% of human cancers may be due to
environmental factors.
B. In general, chemical carcinogens interact noncovalently
with DNA.
C. Some chemicals are converted to carcinogens by enzymes, usually cytochrome P450 species.
D. Most ultimate carcinogens are electrophiles and attack
nucleophilic groups in DNA.
E. The Ames assay is a useful test for screening chemicals for
mutagenicity; however, animal testing is required to show that chemical
a is carcinogenic.
7. Regarding viral carcinogenesis, select the one FALSE statement:
A. Approximately 15% of human cancers may be caused by
viruses.
B. Only RNA viruses are known to be carcinogens.
C. RNA viruses causing or associated with tumors include
hepatitis C virus.
D. Retroviruses possess reverse transcriptase, which copies
RNA to DNA.
E. Tumor viruses act by deregulating the cell cycle, inhibiting

apoptosis, and interfering with normal cell signaling
8. Regarding oncogenes and tumor suppressor genes, select the one
FALSE statement:
A. Both copies of a tumor suppressor gene must be mutated
for its product to lose its activity.
B. Mutation of an oncogene occurs in somatic cells and is not
inherited.
C. The product of an oncogene shows a gain of function that
signals cell division.
D. RB and P53 are tumor suppressor genes; MYC and RAS are oncogenes.
E. Mutation of one tumor suppressor gene or one oncogene is
thought to be sufficient to cause cancer.
9. Regarding growth factors, select the one FALSE statement:
10. Regarding the cell cycle, select the one FALSE statement:
ll.
A. They include a large number of polypeptides, most of
B. Growth factors can act in an endocrine, paracrine, or autocrine manner.
which
stimulate
cell
growth.
Certain growth factors, such as TGF-f, can act in a growth
inhibitory
manner.
Some receptors for growth factors have tyrosine kinase activity; mutations of these receptors occur in cancer cells.
PDGEF stimulates phospholipase A,, which hydrolyzes
PIP, to form DAG and IP,, both of which are second messengers.
A.
B. Cancer cells usually have a shorter generation time than
Cells
transiting
the
cell
cycle
can
reside
within
any
of
the
five
phases
of
the
cell
cycle
(ie,
G,
G,
S,G,
and
M).
normal
cells
and
there
are
fewer
of
them
in
G,
phase.
A variety of mutations in cyclins and CDKs have been reported in cancer cells.
RB is a cell cycle regulator, where it binds to transcription
factor
E2K,
thus
allowing
progression
of
the
cell
from
G,
to
S
phase.
When damage to DNA occurs, p53 increases in amount
and activates transcription of genes that delay transit
through
the

cycle.
Regarding
and
genomic
instability,
select
the
one
FALSE
A.
Cancer
cells
may
have
a
mutator
phenotype,
which
means that they have mutations in genes that affect DNA
replication and repair, chromosomal segregation, DNA
damage
surveillance,
and
Chromosomal instability refers to gain or loss of
chromosomes caused by abnormalities of chromosomal
segregation
during
mitosis.
Microsatellite instability involves expansion or contraction
of microsatellites due to abnormalities of nucleotide
excision
repair.
Aneuploidy (when the chromosomal number of a cell is not
a
multiple
of
the
haploid
number)
is
a
common
feature
of
tumor
cells.
Abnormalities of chromosome cohesion and of
kinetochore-microtubule attachment may contribute to
chromosomal instability and aneuploidy.

A. The activity of telomerase is frequently elevated in cancer cells.
B. A number of cancers have a strong hereditary
predisposition
or
susceptibility;
these
include
Li-Fraumeni
syndrome
and
retinoblastoma.
The products of BRCAI and BRCA2 (responsible for
hereditary
breast
cancer
types
I
and
II)
appear
to
be
involved
in
DNA
repair.
Tumor cells usually exhibit a high rate of anaerobic
glycolysis;
this
may
be
at
least
partly
explained
by
the
presence
in
many
tumor
cells
of
the
PK-2
isozyme,
which
is
with
lesser
of
ATP
and
possibly
increased use of metabolites to build up biomass.
Dichloroacetate, a compound found to display some

16. The number of enzymes dedicated to repairing hydrolytic,
A. Whole-genome and exome sequencing is revealing
important
new
about
the
numbers
and
types
of
mutations
in
cancer
cells.
Abnormalities of epigenetic mechanisms, such as
demethylation of cytosine residues, abnormal modification
of
histones,
and
aberrant
chromatin
remodeling
are
being
increasingly
detected
in
cancer
cells.
Persistence of cancer stem cells (which are often relatively dormant and have active DNA repair systems) may help to explain some of the shortcomings of chemotherapy.
Angiogenin is an inhibitor of angiogenesis.
Chronic inflammation, possibly via increased production of reactive oxygen species, predisposes to development of certain types of cancer.
A.
B. Cell stress and other factors activate the mitochondrial
Apoptosis
can
be
initiated
by
the
interaction
of
certain
ligands
with
specific
receptors
on
cell
surface.
pathway
of
release
of
P450
into
the
cytoplasm is an important event in this pathway.
A distinct pattern of fragments of DNA is found in
apoptotic cells; it is caused by caspase-activated DNase.
Caspase 3 digests cell proteins such as lamin, certain
cytoskeletal
proteins,
and
various
enzymes,
leading
to
cell
death.
Cancer cells have acquired various mutations that allow
them to evade apoptosis, prolonging their existence.
A.
B.
D. Cancer cells secrete metalloproteinases that degrade
E. All tumor cells have the genetic capacity to colonize.
Proteins
involved
in
cell
adhesion
include
cadherins,
integrins,
and
selectins.
Decreased
amounts
of
E-cadherin
on
the
surfaces
of
cancer
cells
may
help
account
for
the
decreased
adhesiveness
shown
by
tumor
cells.

Increased activity of GlcNAc transferase V in cancer cells may lead to an altered glycan lattice at the cell surface, perhaps predisposing to their spread.
proteins in the ECM and facilitate their spread.
oxidative,
and
photochemical
damage
to
polynucleotides
such
as
DNA
is
much
greater
than
the
number
devoted
to
repairing
damaged
proteins.
Identify
the
statement
from
the
list
below
that
is
INCONSISTENT
with
this
observation:
A.
B. Proteins contain sulfur, an element that is susceptible to
Polynucleotides
absorb
ultraviolet
light
more
efficiently
than
do
proteins.
oxidation.
In general, proteins turn over more frequently than does
DNA.
Mutations in a structural gene have the potential to alter the
proteins
they
encode
as
well
as
the
DNA
itself.
If left uncorrected, genome mutations will be passed on to succeeding generations.
anticancer activity, inhibits pyruvate carboxylase, and thus diverts pyruvate away from glycolysis.
729
17. Which of the following is NOT a feature of the mitochondrial
18. Which of the following is NOT a component of the cell’s suite of
19. Select the one of the following statements that describes an aspect
hypothesis of aging?
A. Reactive oxygen species are generated as a by-product by
the electron transport chain.
B. Mitochondria lack the capacity to repair damaged DNA.
C. Many of the complexes in the electron transport chain
are constructed from a mixture nuclearly encoded and
mitochondrially encoded subunits.
D. Damaged mitochondria form protease-resistant aggregates.
E. Damaged mitochondria can trigger apoptosis—
programmed cell death.
damage repair and prevention agents?
Superoxide dismutase >
Glutathione
Isoaspartyl methyltransferase
Catalase
Caspase 7
moO
of the metabolic theory of aging:
A. Elevated levels of plasma glucose promote the formation of

cross-linked protein aggregates.
B. Damage from ROS is multiplied by the tendency of oxygen
radicals multiply chain
to via reactions.
Calorically restricted diets promote lower and more efficient metabolic activity.
Blood flow to the heart muscle becomes restricted over
time due to the cholesterol-induced formation of arterial
plaques.
Vigorous physical activity correlates with the loss of STEM
cells.
CORRECT:
A. Telomeres prevent genetic recombination by capping the
B. Aging genes can be distinguished by their impact on an
ends of linear DNA molecules.
organism's lifespan.
The short lifespan of Caenorhabditis elegans renders them
an attractive model organism for studying aging.
Telomere shortening is a consequence of the discontinuous
nature of the process by which the “lagging strand” is
synthesized during chromosome replication.
Telomerase activity is high in both STEM cells and in many cancer cells.

1.
2.
3. That fermentation required intact cells was disproved by the discovery that a cell-free yeast extract could convert sugar to ethanol and carbon dioxide. This discovery led to the identifi- cation of the intermediates, enzymes, and cofactors of fermen- tation and glycolysis.
10. D. Hydrocarbons are water insoluble.
ll.
12. D. When present in solution at a pH equal to their pKa only half of the molecules of a monofunctional weak acid (eg, ammonium ion or acetic acid) are in the charged state. Max- imal mobility will occur either at a pH 3 or more pH units below the pKa for ammonium ion, or at a pH 3 or more pH units above the pKa for acetic acid.
13.
14.
15.
16.
17.
18.
B.
D.
Fermentation
ceased
over
time,
but
resumed
when
inorganic
orthophosphate
was
added.
This
led
to
the
isolation
of
phos-
phorylated
Other
experiments
using
heated
yeast
extract
led
to
the
discovery
of
ATP,
ADP,
and
NAD.
Preparations used to identify metabolites and enzymes included perfused liver, liver slices, and tissue homogenates fractionated by centrifugation.
. RadioactiveC, *H, and **P facilitated the isolation of inter- mediates of carbohydrate, lipid, nucleotide, and amino acid metabolism and enabled precursor product relationships between intermediates to be tracked.
. Garrod proposal that alkaptonuria, albinism, cystinuria, and pentosuria resulted from “inborn errors of metabolism” led to the field of biochemical genetics.
Regulation of cholesterol biosynthesis illustrates the link between biochemistry and genetics. Cell surface receptors internalize plasma cholesterol, which then regulates cho- lesterol biosynthesis. Defective receptors result in extreme hypercholesterolemia.
. Key model organisms include yeast, slime mold, fruit fly, and a small round worm, each with a short generation time and read- ily mutated.
A.
Of
the

protein
amino
acids,
only
phenylalanine,
tyrosine,
and
absorb
light
at
280
nm.
C.
At
its
pI
an
amino
acid
has
an
equal
number
of
positive
and
negative
charges,
but
has
no
net
overall
charge.
C.
The
Edman
technique
involves
successive
derivatization
and
removal
of
N-terminal
residues.
Self-association
in
an
aqueous
environment
as
a
large
droplet
minimizes
the
surface
area
in
contact
with
water,
and
hence
the
number
of
water
molecules
whose
degrees
of
rotational
free-
dom
are
restricted.
Strong
bases
and
acids
dissociate
essentially
in
water,
NaOH
as
Na*
and
OH.
By
contrast,
a
weak
acid
such
as
pyruvic
acid
dissociates
only
partially
in
solution.
E.
Tandem
mass
can
separate
complex
mixtures
of
peptides.
E.
Many
proteins
undergo
processing,
for
example,
insulin,
which
is
as
a
single
which
subsequent
proteolysis
converts
to
two
chains
linked
by
disulfide
bonds.
19.
20.
21. D. Gene arrays, also termed DNA chips or DNA arrays, contain multiple DNA probes with differing sequences bound at known locations on a solid support. Hybridization of complementary DNA or RNA probes at particular locations provides informa- tion about their nucleic acid composition.
22.
23.

24. Unlike pK, (6.82) of phosphoric acid, the other two dissociating groups of phosphoric acid cannot serve as effective buffers at physiologic pH because they are either completely dissociated or predominantly protonated at pH 7.
25.
26.
27.
28.
29. Signal sequences target proteins to specific subcellular loca- tions in the cell, or for secretion.
pl
is
the
pH
at
which
a
molecule
bears
no
net
charge.
In
this
example,
the
pI
is
a
pH
midway
between
the
third
and
fourth
pK,
values:
pI
=
(6.3
+
7.7)/2
=
7.0.
As
pH
is
adjusted
from
acidic
to basic, net charge will change successively as follows: +3, +2, +1, 0, -1, -2, -3.
All
of
the
protein
amino
acids
are
essential
since
all
are
required
for
protein
but
“nutritionally
essential”
amino
acids
(10
for
humans)
are
those
which
an
organism
cannot
synthe-
size.
Many
vitamins
are
“dietarily
although
vitamin
C
is
dietarily
essential
only
for
humans,
catfish,
and
certain
other
D.
A
hydrogen
bond
involves
the
residue
in
fourth
place
along
the
helix.
E.
Prions
contain
no
nucleic
acid,
just
protein.
Prion
diseases
therefore
are
transmitted
by
protein
without
involvement
of
DNA
or
RNA.
A:
Carboxyl
groups
(pK,
through
pK,)
and
amino
groups
(pK,
through
pK,)
B:

Minus
one
C:
Plus
0.5
D:
Toward
the
cathode
To
act
as
an
effective
buffer,
a
compound
should
have
a
pK,
no
less
than
0.5
pH
units
removed
from
the
desired
pH,
and
be
present
in
sufficient
quantity.
Carboxylation
of
a
glutamyl
residue
forms
y-carboxyglutamate,
a
potent
chelator
of
Ca**
required
for
blood
clotting
and
clot
dissolution.
4-Hydroxyproline
and
5-hydroxylysine
are
present
in
several
proteins.
(a)
Copper
is
an
essential
group
for
the
amine
oxidase
that
converts
lysine
to
the
hydroxylysine
that
participates
in
formation
of
covalent
crosslinks
that
strengthen
collagen.
(b)
Ascorbic
acid
is
essential
for
proline
to
con-
vert
proline
to
the
hydroxyproline,
which
provides
interchain
hydrogen
bonds
that
stabilize
the
collagen
triple
helix.

1.
Carbonic
anhydrase
catalyzes
the
hydration
of
carbon
dioxide
to
form
carbonic
acid.
A
portion
of
this
weak
acid,
in
turn,
dis-
sociates
to
produce
bicarbonate
and
a
proton.
As
the
concen-
tration
of
carbon
dioxide
falls,
carbonic
acid
is
broken
down
form
carbon
dioxide
and
water.
To
compensate
for
the
loss
of
Ane B. A.
YWd E.
carbonic acid, bicarbonate and protons recombine to restore equilibrium, leading to a net drop in [H+] and a rise in pH.
D.
7. B.
12. B.
17.
B.
8. C.
13.
B.
18. D.
9. A.
14.
C.
19. A
10. D.
15.
D.
E.
ll. E
16.
A

1 . A.A reaction with a negative AG is exergonic, it proceeds spon- taneously and free energy is released.
10. E. Oligomycin blocks oxidation and ATP synthesis as it pre- vents the flow of electrons back into the mitochondrial matrix through ATP synthase.
ll.
12. E. In the presence of an uncoupler, the energy released as elec-
13.
14. D. Three ATP molecules are generated for each revolution of the ATP synthase molecule.
15. B. The electrochemical potential difference across the inner mitochondrial membrane caused by electron transport must be negative on the matrix side so that protons are forced to reenter via the ATP synthase to discharge the gradient.
E. In an exergonic reaction AG is negative and in an ender- gonic reaction it is positive. When AG is zero, the reaction is at equilibrium.
. B. When the reactants are present in concentrations of 1.0 mol/L, AG° is the standard free-energy change. For bio- chemical reaction, the pH (7.0) is also defined and this is AG’.
D.
ATP
contains
two
phosphate
bonds
and
is
needed
to
drive
It
is
not
stored
in
the
body
and
in
the
presence
of
uncouplers
its
synthesis
is
blocked.
.
A.
Reduced
c
is
oxidized
by
c
oxidase
(complex
IV
of
the
respiratory
chain),
with
the
reduction
of
molecular
oxygen
to
two
molecules
of
water.
E. Cytochrome oxidase is not a dehydrogenase, although all other cytochromes are classed as such.
B.
Although
Cytochromes
p450
are
located
mainly
in
the
endo-
plasmic
reticulum,
they
are
found
in
in
some
tissues.

D. Oxidation of one molecule of NADH via the respiratory chain generates 2.5 molecules of ATP in total. One is formed via complex I, 1 via complex II and 0.5 via complex IV.
. C. 1.5 molecules of ATP are formed in total as FADH, is oxi- dized, 1 via complex II and 0.5 via complex IV.
A.
Uncouplers
allow
electrons
to
reenter
the
matrix
without
passing
through
ATP
synthase.
trons
flow
into
the
matrix
is
not
captured
as
ATP
and
is
dissipated
as
heat.
C.
is
a
uncoupler
found
in
brown
adipose
tissue.
Its
function
is
to
generate
body
heat.

NAP
YN
B.
8.
B.
15.
D.
22.
A.
B.
9.
B.
16.
E.
23.
B.
A.
10.
E.
17.
E.
24.
C.
Dz.
11.
C.
18.
C.
25.
D.
Cc.
12.
D.
19.
C.
26.
E.
Cc.
13.
D.
20.
C.
27.
A.
E.
14.
D.
21.
D.
28.
B.
nw
20.
21.
22.
23.
24.
1 . D.
2.
3. A. Gangliosides are derived from glucosylceramide.
4
Ye
wWwDd
»

.
D.
.
C.A,
B,
D,
and
E
are
classed
as
preventive
as
they
act by reducing the rate of chain initiation.
Dz.
B.
D.
Long
chain
fatty
acids
are
activated
by
coupling
to
CoA,
but
fatty
acyl
CoA
cannot
cross
the
inner
mem-
brane.
After
transfer
of
the
acyl
group
from
CoA
to
carnitine
by
carnitine
palmitoyl
transferase
(CPT)-I,
acylcarnitine
is
carried
across
by
carnitine-acylcarnitine
translocase
in
exchange
for
a
carnitine.
Inside
the
matrix,
CPT-II
transfers
the
acyl
group
back
to
CoA
and
carnitine
is
taken
back
into
the
intermem-
brane
space
by
the
enzyme.
E.
The
breakdown
of
palmitic
acid
(C16)
requires
7
cycles
of
each
producing
1
FADH,
and
1
NADH
mol-
ecule
and
results
in
the
formation
of
eight
2C
acetyl
CoA
B. When the action of carnitine palmitoyl transferase-I is inhibited by malonyl CoA, fatty acyl groups are unable to enter the matrix of the mitochondria where their breakdown by B-oxidation takes place.
C.
Humans
(and
most
mammals)
do
not
possess
enzymes
able
to
introduce
a
double
bond
into
fatty
acids
beyond
A9.
D.
Inhibition
of
the
acid
causes
levels

of
citrate
in
the
cytosol
to
decrease
and
favors
inactivation
of
the
enzyme.
A.
Cc.
E.
E.
Glucagon
is
released
when
blood
glucose
levels
are
low.
In
this
situation,
fatty
acids
are
broken
down
for
energy
and
fatty
acid
synthesis
is
inhibited.
.
E.
Glucagon,
ACTH,
epinephrine
and
vasopressin
promote
of
the
enzyme.
B.
Dz.
A.
are
triacylglycerol-rich
syn-
thesized
in
the
mucosa
using
fat
from
the
diet
and
secreted
into
lymph.
E.
VLDL
is
and
secreted
by
the
liver,
and
adipose
tissue
and
muscle
take
up
the
fatty
acids
released
by
the
action
of
lipase.
D.
Very
low
density
secreted
by
the
liver
is
con-
verted
to
density
and
then
to
low
den-
sity
(LDL)
by
the
action
of
lipases
and
the
transfer
of
and
proteins
from
high
density
lipoprotein.
LDL
delivers
to
tissues
and
is
also
cleared
by
the
liver.
A.
are
in
the
intestine
and
secreted
into
lymph
after
a
fat
meal.
E.
and
their
remnants
are
cleared
from
the
circu-
lation

rapidly
after
a
meal,
and
the
secretion
of
very
low
density
by
the
liver
then
Ketone
bodies
and
non-
esterified
fatty
acids
are
elevated
in
the
fasting
state.
C.
When
ester
is
transferred
from
HDL
to
other
by
the
action
of
CETP
it
is
delivered
to
the liver in VLDL, IDL, or LDL.
25.
26.
27.
28.
29.
30.
D.
are
metabolized
by
lipase
when
bound
to
the
surface
of
endothelial
cells.
This
process
releases
fatty
acids
from
which
are
then
taken
up
by
the
tissues.
The
resulting
smaller,
cholesterol-enriched
chylomi-
cron
remnant
particles
are
released
into
the
and
cleared
by
the
liver.
C.
is
in
the
endoplasmic
reticulum
from
acetyl
CoA.
The
rate-limiting
step
is
the
formation
of
mevalonate
from
3-hydroxy
3-methylglutaryl-CoA
by
HMG
CoA
reductase
and
lanosterol
is
the
first
cyclic
intermediate.
Cc.
C.
Secondary
bile
acids
are
produced
by
the
of
primary
bile
acids
in
the
B.
If
the
LDL
receptor
is
defective,
LDL
is
not
cleared
from
the
blood,
causing
severe
hypercholesterolemia.
A.

PCSK9
regulates
the
re-cycling
of
LDL
receptors
to
the
cell
surface
after
endocytosis
has
taken
place.
of
PCSK9
activity,
therefore,
increases
the
number
of
LDL
receptor
mol-
ecules
on
the
cell
surface,
leading
to
an
increased
rate
of
clear-
ance
and
lower
blood
levels.

1.
10.
ll. E. Decarboxylation of glutamate, not glutamine forms GABA.
12.
13.
D.
catalyzes
a
functionally
irrevers-
ible
reaction,
and
thus
cannot
convert
tyrosine
to
phenylalanine.
E. Histamine is a catabolite, not a precursor, of histidine.
B.
The
insertion
of
selenocysteine
into
a
peptide
occurs
during,
not
to
C.
Pyridoxal-dependent
transamination
is
the
first
reaction
in
of
all
the
common
amino
acids
except
threonine,
lysine,
proline,
and
hydroxyproline.
B.
Glutamine.
C.
The
carbon
skeleton
of
alanine
the
most
to
hepatic
.
B.
ATP
and
ubiquitin
participate
in
the
of
nonmembrane-associated
proteins
and
proteins
with
short
half-lives.
C. Due to the failure to incorporate NH," into urea, clinical signs of metabolic disorders of the urea cycle include alkalosis, not acidosis.
.
E.
Cytosolic
fumarase
and
cytosolic
malate
convert
fumarase
to
oxaloacetate
following
a
cytosolic
reaction
of
the
urea
cycle.
The
fumarase
and
malate
dehy-
drogenase
function
in
the
TCA
cycle,
not
urea
A.
Serine,
not
provides
the
thioethanol
moiety
of
coenzyme
A.

and
y-carboxyglutamate
represent
exam-
ples
of
of
peptidyl
lysyl
and
peptidyl
glutamyl
residues,
By
contrast,
sele-
nocysteine
is
incorporated
into
proteins
cotranslationally,
in
the
same
way
as
the
other
20
common
protein
amino
acids.
The
process
is
complex,
and
involves
the
unusual
tRNA
termed
tRNA**.
Biosynthesis
of
the
amino
acids
that
are
dietarily
essential
for
humans
requires
multiple
Since
human
diets
typi-
cally
contain
adequate
amounts
of
these
amino
acids,
loss
of
the
genes
that
can
encode
these
“unnecessary”
enzymes
and
the
lack
of
need
to
expend
the
energy
required
to
copy
them
pro-
vide
an
evolutionary
advantage.
14.
15.
16.
17.
18.
19.
20.
Since
glutamate
plays
multiple
central
roles
in
its
complete
absence
would
unquestionably
be
fatal.
E.
Albumin
is
not
a
hemoprotein.
In
cases
of
hemolytic
anemia,
albumin
can
bind
some
metheme,
but
unlike
the
other
proteins
listed,
albumin
is
not
a
A.
Acute
porphyria
is
due
to
mutations
in
the
gene
for
uroporphyrin
I
synthase.
A.
Bilirubin
is
a
linear

tetrapyrrole.
D.
The
severe
jaundice,
upper
abdominal
pain,
and
weight
loss
plus
the
lab
results
indicating
an
type
of
jaundice
are
consistent
with
cancer
of
the
pancreas.
The
assay
takes
advantage
of
the
different
water
of
unconjugated
and
Two
assays
are
con-
ducted,
one
in
the
absence
and
a
second
in
the
presence
of
an
organic
solvent,
typically
methanol.
The
highly
polar
gluc-
uronic
acid
groups
of
bilirubin
convey
water
solu-
bility
that
insures
that
it
will
react
with
the
colorimetric
reagent
even
in
the
absence
of
any
added
organic
solvent.
Data
from
an
assay
conducted
in
the
absence
of
added
methanol,
termed
“direct
bilirubin,
is
bilirubin
glucuronide.
A
second
assay
conducted
in
the
presence
of
added
methanol
measures
total
ie,
both
and
The
difference
between
total
bilirubin
and
direct
reported
as
“indirect
bilirubin,’
is
The
biosynthesis
of
heme
from
succinyl-CoA
and
glycine
occurs
only
when
the
availability
of
free
iron
signals
the
poten-
tial
for
synthesis
of
heme.

targets
the
first
enzyme
of
the
pathway,
5-aminolevulinate
synthase
(ALA
synthase)
rather
than
a
reaction.
This
conserves
energy
by
avoiding
wasting
a
coenzyme
A
thioester.

1.
D.
B,y-Methylene
and
§,y-imino
purine
triphos-
phates
do
not
readily
release
the
terminal
phosphate
by
hydro-
lysis
or
by
group
transfer.
Dz.
E.
Pseudouridine
is
excreted
unchanged
in
human
urine.
Its
presence there is not indicative of pathology.
A.
Metabolic
disorders
are
infrequently
with
defects
in
which
forms
water-soluble
products.
B.
21.
C.
37.
C.
53.
D.
Dz.
22.
A.
38.
E.
54,
A.
B.
23.
C.
39.
D.
55.
E.
Cc.
24,
A.
40.
D.
56.
A.
Cc.
25.
E.
41.
B.
57.
E.
D.
26.
B.
42.
A.
58.
C.
E.
27.
A.
43.
A.
59.
A.
B.
28.
E.
44.
E.
60.
D.
Dz.
29.
C.
45.
C.
61.
D.
Dz.
30.
A.
46,
A.
62.
E.
E.
31.
A.
47.
C.
63.
A.
A.
32.
C.
48.
D.
64,
C.
Cc.
33.
D.
49.
C.
65.
C.
B.
34.
E.
50.
B.
66.
E.
Dz.
35.
C.
51.
E.
67.
D.
B.
36.

1. B. Glycolipids are located on the outer leaflet.
2.
3. E. Insulin also increases glucose uptake in muscle.
.
A.
Alpha-helices
are
major
of
membrane
proteins.
4. A. Its action maintains the high intracellular concentration of
potassium compared with sodium.
5.
D.
10.
A.
15.
B.
20.
B.
6.
B.
ll. E
16.
C.
21.
D.
7.
C.
12.
B.
17.
A.
22.
A
8.
B.
13. D.
18.
C.
9. D.
14. E.
19.
A.

1A
16.
A.
31.
E.
46.
B.
2.
EB.
17.
B.
32.
A.
47.
B.
3.
C.
18.
C.
33.
B.
48.
B.
4.
D.
19.
E.
34,
A.
49.
B.
5.
E
20.
D.
35.
B.
50.
C.
6. D.
21.
E.
36.
C.
51.
D.
7. GC.
22.
A.
37.
D.
52.
C.
8. B.
23.
C.
38.
E.
53.
A.
9.
D.
24.
C.
39.
E.
54.
A.
10.
E.
25.
A.
40.
A.
55.
A.
11.
C.
26.
E.
41.
D.
56.
E.
12.
B.
27.
A.
42.
C.
57.
A.
13.
C.
28.
A.
43.
B.
14.
D.
29.
A.
44.
E.
15.
B.
30. C.
45.
C.

1 . Within the body, hydrolysis of nitroglycerin releases nitrate ions that can be reduced by mitochondrial aldehyde dehydro- genase to generate nitric oxide (NO), a potent vasodilator.
10.
11. The production of new antibodies with unique antigen-binding
12. B.
13. Cc.
. The contractile cycle of cardiac muscle is controlled by oscilla- tions in the level of cytosolic Ca**. If the reuptake of Ca** by the sarcoplasmic reticulum is slowed sufficiently by a deficiency SERCA2a activity, cardiac myocytes will be unable to clear this second messenger from their cytoplasm prior to the onset of the next cycle of excitation. The persistence of high basal levels of cytosolic Ca?* will lead to both a reduction in the amplitude of the contractile cycle and the progressive uncoupling of the excitation-contraction cycle.
A.
5. B.
7. E 8. D.
9. E.
A.
6.
B.
Haptoglobin
binds
extracorpuscular
forming
a
complex
that
is
too
large
to
pass
through
the
glomerulus
into
kidney tubules.
properties
is
reliant
on
the
recombination
and
mutation
of
the
DNA
encoding
the
hypervariable
regions
of
the
immunoglob-
ulin
heavy
and
light
chains.
Cytidine
deaminase
introduces
genetic
mutations
by
catalyzing
the
hydrolysis
of
cytosine
bases
present
in
DNA
to
uracil.
14. C.
16.
E.
15. B.
17.
E.
19.
20.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
.
deficient
in
glucose-6-phosphate
are
rendered
extremely
vulnerable
to
destruction
by
reactive
oxygen
spe-

cies
resulting
from
a
lack
of
reduced
glutathione,
an
important
agent
for
protecting
against
oxidative
stress.
This
is
a
conse-
quence of their reliance on this enzyme to generate a plentiful
supply
of
the
NADPH
used
by
reductase.
E.
21. D.
23.
D.
25. B.
Cc.
22. A.
24. D.
26. A.
E.
Importins
are
involved
in
the
import
of
proteins
into
the
nucleus.
B.
Some
mammalian
proteins
are
known
to
be
translocated
posttranslationally.
C. Ubiquitin tags proteins for degradation by proteasomes.
E.
Furin
converts
proalbumin
to
albumin.
C. NSF is an ATPase.
D.
Cross-links
are
an
important
feature
of
collagen
C.
Deletions
in
the
elastin
gene
have
been
as
respon-
sible for many cases of Williams-Beuren syndrome.
B.
syndrome
subtypes
kyphoscoliosis
and
dermatosparaxis are caused by defects in noncollagen genes.
B.
Hyaluronic
acid
(hyaluronan)
is
not
sulfated.
C. Hurler syndrome is caused by a deficiency of a-L-iduronidase.
D.
Achondroplasia
is
caused
by
mutations
in
the
FGFR3
gene.

1. Dz.
2.
ll.
12.
13.
14.
15.
16.
17.
18.
19.
20.
D.
Of
the
listed
proteins,
only
factor
II
is
a
vitamin
K-dependent
factor.
Dz.
E.
GPIlIb-IIla
(integrin
oIIbB3)
is
not
a
G
protein-coupled
receptor.
A.
A,
being
and
Xchromosome-linked
disease,
is
a
very unlikely to occur in a female.
B. Most chemical carcinogens interact covalently with DNA.
B. Certain DNA viruses are also known to be carcinogenic.
E.
Mutations
in
5
to
6
of
these
two
types
of
cancer
promoting
or
genes
are
thought
to
be
neces-
sary
for
. E. PDGF stimulates phospholipase C, not phospholipase A.
.
D.
Binding
of
RB
to
E2F
blocks
of
the
cell
from
G,
to
S
phase.
C.
Microsatellite
is
caused
by
of
mismatch
repair.
E.
Dichloroacetate
inhibits
pyruvate
kinase.
D.
Angiogenin
is
an
inhibitor
of
angiogenesis.
B. Cytochrome C is released from mitochondria.
E.
Only
about
1
in
10,000
cancer
may
have
the
capacity
to
colonize.
RODD
Note: Page numbers followed by f indicate figures; and page numbers followed by t indicate tables.
A
in citric acid cycle, 150-151, 151f, 152f,
A band, 612, 612f, 613f

A blood group substances, 653-654, 654f
A
cyclins,
369,
369f,
369t
A gene, 653-654
A kinase anchoring proteins (AKAPs), 505
A (acceptor) site, 402, 403f
AAV (adenovirus-associated viral)
genomes, 437
ABCAI (ATP-binding cassette transporter
Al), 241f, 242
ABCGI (ATP-binding cassette transporter
G1),
241f,
242
abciximab, 672
abetalipoproteinemia, 239, 258
ABO system, 653-654, 654f

biomedical importance of, 519
of carbohydrates, 520, 520f of lipids, 520-521, 522f
of proteins, 521, 523
of vitamin
B,,,
101,
536
of vitamins and minerals, 523-524, 523f absorption spectrophotometry, laboratory
tests using, 563-564
absorptive pinocytosis, 474f, 475
ACAT (acyl-CoA:cholesterol acyltransferase), 253
acceptor arm of tRNA, 346-347, 346f,
396,
396f acceptor (A) site, of 80S ribosome, 402, 403f
accuracy, of laboratory test, 561, 562f
ACE (angiotensin-converting enzyme),

aceruloplasminemia, 633
acetic acid, 13t, 197t acetoacetate, 207, 210, 211f
in ketogenesis, 211, 212f in mevalonate synthesis, 250, 250f

acetyl (acyl)-malonyl enzyme, in lipogenesis, 217, 218f, 219f
acetylation enzyme regulation by, 86-89
of histones, 351, 353t mass spectrometry detection of, 28t
of xenobiotics, 558-559
acetylcholine, synaptobrevin and, 587
cholesterol synthesis from, 250,
250f, 252f
154-155, 155f in glycolysis and gluconeogenesis
regulation, 175
in lipogenesis, 217-219, 219f, 220f, 221
pathways,
130-131,
130f,
131f,
136
phenylalanine formation of, 286, 288f pyruvate oxidation to, 161-163, 162f threonine conversion to, 285, 285f tyrosine conversion to, 286, 287f in VLDL formation, 243, 243f
in xenobiotic metabolism, 559
acetyl-CoA carboxylase in lipogenesis, 217, 217f, 219f,
221-222, 221f regulation
of,
89t,
90
N-acetylgalactosamine (GalNAc), 547,

N-acetylglucosamine in glycoproteins, 548
rapidly reversible glycosylation with,
552-553
N-acetylglutamate in urea cycle disorders, 277
in urea synthesis, 275f, 276
N-acetylglutamate deacylase, in urea cycle,
276
N-acetylglutamate synthetase (NAGS)
defects in, 277
in urea cycle, 276
N-acetylneuraminic acid (NeuAc),
547, 548t
N-acetylneuraminic acid, in gangliosides,
234, 234f acetylsalicylic acid. See aspirin
acetyltransferases, 89, 559
acholuric jaundice, 313
achondroplasia, 478t, 609
acid anhydride bonds, 320
acid anhydrides, group transfer potential

acid hydrolases, 475
acid maltase, glycogen hydrolysis by, 166
acid-base balance, ammonia role in, 275
acid-base catalysis,
enzymatic,

acidic phosphoproteins, 605
acidity, 6
acidosis ammonia role in, 275
in cancer cells, 696

conjugate, 11
as proton donors, 10
as protonated species, 11
strength of, 13, 13t strong,
10-11
transition metals as, 93
weak. See weak acids

dicarboxylic, 215
orotic, 336, 336t aconitase, in citric acid cycle, 151, 152f
ACP. See acyl carrier protein acromicric dysplasia, 597
acrosomal reaction, 553
actin, 611
F-Actin, 613, 614f
G-Actin, 613, 614f, 623-624
in muscle contraction, 613-615,

in red blood cells, 652t, 653
striated muscle regulation by, 616
actin filaments, 587, 623-624
actin (thin) filaments, 612-613, 613f, 614f activated partial thromboplastin time

activated protein C (APC), 677
activation coefficient, 565
activation domains (AD), 381, 427,
428f,
512
activation energy (E,_,), 69-70, 70f, 72
activation reaction, 501
activation-induced cytidine deaminase,
641
activators in gene expression, 410-411
transcription, 384-385
active chromatin, 353-354, 354f, 418
active site, 58-59, 59f activation energy lowering by, 72
allosteric site compared with, 86
active transport, 467, 467f, 467¢
ATP in, 472-473, 472t of bilirubin, 312
transporters involved in, 468-469, 468f,

actomyosin, 614
acute coronary syndrome, 654
acute fatty liver of pregnancy, 215
acute inflammatory response, 656
acute intermittent porphyria, 310,
3108, 311f
acute-phase proteins, 526, 630, 630f acyl carrier protein (ACP), 217,

pantothenic acid in, 539, 539f acyl enzyme, 217, 219f
S-acylation, 463
acylcarnitine, 208, 208f

formation of, 208, 208f pyruvate dehydrogenase regulation by,
221
in triacylglycerol synthesis, 230
acyl-CoA dehydrogenase, 114
in fatty acid oxidation, 209, 209f medium-chain, deficiency of, 215
acyl-CoA synthetase (thiokinase)
in fatty acid activation, 208, 208f mitochondrial compartmentalization
of,
118
in triacylglycerol synthesis, 230,
244,
245f acyl-CoA:cholesterol acyltransferase

acyl-enzyme intermediate, 61, 61f acylglycerol metabolism
acylation of triose phosphates,

biomedical importance of, 229
ceramide in,
233-234,
233f,
234f clinical aspects of, 234-235, 235t
hydrolysis, 229
phosphatidate in, 230-232,

phospholipases in, 232-233, 233f
1-acylglycerol-3-phosphate acyltransferase, 230, 231f
acylglycerols, 196
synthesis of, 230-233, 230f, 231f
AD (activation domains), 381, 427,

Ad (adenoviral) genomes, 437
adapter proteins, 475
adaptive immune system, 639, 643, 662
adducts, 710

base pairing in DNA, 339, 340f base pairing in RNA, 342, 343f
salvage pathways of, 332-333
adenine nucleotide transporter, of mitochondria, 124, 124f
adenocarcinoma, colorectal, 687-688,

in S-adenosylmethionine, 323f derivatives of, 322t
syn and anti conformers of, 321f in uric acid formation, 334, 335f
adenosine deaminase
deficiency of, 327, 335, 335f, 336t,
432, 655
gene location of, 442t adenosine diphosphate (ADP)
creatine phosphate shuttle for, 126, 126f energy capture and transfer by,
107-108, 107¢, 108f, 121-122
phosphate cycles of, 109, 109f in
platelet aggregation,
670,
671f respiratory control by, 122-123, 123t
108f,
321f adenosine monophosphate (AMP),

cyclic. See cyclic AMP
energy capture and transfer by,
107-108, 107¢, 108f
IMP conversion to, 328, 330f feedback regulation of, 331, 331f
phosphate cycles of, 109, 109f
PRPP
glutamyl amidotransferase
regulated by, 330, 331f structure of, 108f, 321f, 322f
adenosine triphosphate (ATP)
in active transport, 472-473, 472f as allosteric effector, 86
biomedical importance of, 105
as cellular energy currency, 108-110,

control of supply of, 122-123, 123t in coupling of endergonic and exergonic
reactions, 109
erythrocyte generation of, 648, 648¢
fatty acid oxidation production of,
209-210, 209f, 210t free energy of hydrolysis of, 107-108,
107t, 108f in glycolysis and gluconeogenesis
regulation, 176, 176f glycolysis production of, 157, 158f, 159,
159f,
161,
161f in muscle contraction, 614-616, 615f,
620-621, 621f oxidative phosphorylation in synthesis
of, 118, 121-122, 122f phosphate cycles of, 109, 109f
protein degradation dependent on,
270-271, 270f, 271f in purine synthesis, 328
structure of, 107f, 108f, 321f as transducer of free energy, 322
323,
323f,
324t biosynthesis of, 288, 290f, 291f,

adenoviral (Ad) genomes, 437
adenovirus-associated viral (AAV)
adenylyl cyclase, 167, 168f, 169f cAMP intracellular signal and,
503-504, 503t in lipolysis, 246, 246f
adenylyl kinase (myokinase), 109, 176
mitochondrial compartmentalization

in muscle contraction, 621
adherens junctions, 466
adhesion, glycoprotein role in, 553-554
adipocytes, 247
turnover of, 708t adipokines, 244
adiponectin, 244
adipose tissue, 196, 229

in fed state, 130, 136-138, 137f, 138t glucose uptake by, 136-137, 137f
glucose utilization by, 245
hormones and lipolysis in, 246, 246f insulin and, 245
lipase, 244-245, 245f lipogenesis and, 247
perilipin in, 247
triacylglycerol storage in, 244
ADP. See adenosine diphosphate
ADPase, in hemostasis and thrombosis,

ADP-chaperone complex, 575
ADP-ribosylation enzyme regulation by, 86-87
of histones, 351, 353t
NAD' as source for, 534
ADP-ribosylation factor-1 (ARF-1),

adrenal function tests, 567
adrenal gland, cytochromes P450 in, 557
adrenal steroidogenesis androgen synthesis, 486f, 487
glucocorticoid synthesis, 486-487, 486f mineralocorticoid synthesis,

overview of, 485, 485f adrenergic receptors, glycogenolysis
activation by, 168, 169f adrenocortical hormone, cholesterol as
precursor for, 202
adrenogenital syndrome, 487
adrenoleukodystrophy, neonatal,
580, 580t advanced glycation end-products (AGEs),
553, 553f, 712-713
aerobic conditions, 157, 158t, 160, 620
aerobic processes, 151
affinity chromatography protein and peptide purification with,

recombinant fusion protein purification with, 65-66, 66f
agammaglobulinemia,
645
AGEs (advanced glycation end-products),
553,
553f,
712-713
aggrecan, 608, 608t, 609f aggregated proteins, toxic effects of, 715
aggregates, protein, 41
aggregating agents, in platelet aggregation,

biomedical importance of, 707
cancer and, 698
extracellular matrix and, 592
metabolic theories of, 715-716, 716t mortality and, 708
as preprogrammed process, 715-717
proteoglycans and, 604
somatic mutation theory of, 714
telomerase and, 355
wear and tear theories of. See wear and tear theories of aging

model organisms of, 716-717
transcription factors, 717
aglycone, 145
AHSP (a-hemoglobin-stabilizing protein),
42

glycan role in, 555
undernutrition in, 525
AKAPs (A kinase anchoring proteins), 505
ALA. See 5-aminolevulinate; a-linolenic

carbon skeleton catabolism of, 283
circulating plasma levels of, 271-272,

metabolic pathways of, 138
specialized products of, 296-297
265,
265f transamination reactions forming,
272-273, 273f
B-alanine, biosynthesis
of,
302-303
alanine aminotransferase (ALT)
diagnostic use of, 64, 64t, 566
transamination reaction of, 273, 273f
-alanyl dipeptides, biosynthesis of, 303 Dm lbumin, 599, 627, 628, 630, 631 2
bilirubin binding
to,
311,
315
free fatty acids in combination with,
208,
236,
237t,
238
synthesis of, 588, 588f

cohol dehydrogenase in fatty liver, 244
coholic liver disease (ALD), 244
coholism case study, 720
cirrhosis and, 244
transferrin glycosylation in, 633

carbohydrates as aldehyde or ketone derivatives of, 141-142, 142t
energy yields from, 138t hydrogen bonding of, 7, 7f
iron absorption and, 523-524
sulfation of, 558
ALD (alcoholic liver disease), 244
aldehyde dehydrogenase, 112
in fatty liver, 244
ppp
2 dehyde oxidase, molybdenum in, 100

deficiency of, 163
in glycolysis, 159, 159f
2 dolase B, 186, 188f deficiency of, 190
Pp dose reductase, 191
dose-ketose isomerism, 143, 144f dosterone synthase, 485-486
PPP doses, 141-142, 142t, 143, 144f
Se imentary pentosuria, 189
lisporivir,
42
p kaline phosphatase, 64t, 605
pp kalosis, ammonia effects on, 275
kaptonuria, 286, 287f ep kylphospholipid, 229
lergic reactions, to foods, 519, 523
lopurinol, 324, 324f, 334, 336
losteric properties, of hemoglobin,
Pepe losteric effectors, 85-86, 85f, 136, 175

of enzymes, 85-86, 85f, 135-136, 135f in glycolysis and gluconeogenesis
175
a losteric site, 85-86
a [pha-anomers, 143
a
helix in myoglobin, 48, 48f
as secondary structure unit, 35-36, 35f,
37f
Alport syndrome, 595-596, 595t
ALT. See alanine aminotransferase alteplase, 679
alternative pathway, 644
alternative polyadenylation, 430
alternative transcription start site, 430
altitude, physiologic adaptations to, 53
Alu family, 358, 360
Alzheimer disease w3 fatty acids and, 199
pathologic protein conformations

respiratory chain disorders in, 126
Amadori products, 712
ambiguity and genetic code, 394
Ames assay, 684, 684f amino acid oxidases, 112, 273-274, 274f
amino acids hydrolysis of peptide bonds,
708-709, 709f missense mutations and,

nonsense codons and, 397, 398f transporter/carrier systems for, 468
tRNA association with, 395-396,

D -amino acids, free, 18
D -a-amino acids, biomedical importance

absorption of, 521, 523
biomedical importance of, 14
biosynthetic conversions of alanine, 296-297
arginine, 297, 297f, 301, 302f biomedical importance of, 296
cysteine, 297, 297f glycine, 298, 298f, 300-301, 302f
histidine, 298, 298f methionine, 298-299, 299f, 300f,
301,
302f phosphoserine, phosphothreonine,
and phosphotyrosine, 300
serine,
300
tryptophan, 300, 301f tyrosine, 300, 302f

ammonia from, 269, 272-275, 274f arginine and ornithine, 282, 283f
asparagine and aspartate, 281-282
biomedical importance of, 269,

to carbohydrate and lipid biosynthesis intermediates,
281, 281f, 281t carbon skeleton fates in, 281,

cystine and cysteine,
283-285, 284f disorders of, 276-278, 277t
end products of, 272, 275, 275f glutamate dehydrogenase in,
273,
274f glutaminase and asparaginase in,

glutamine and glutamate, 282
glutamine synthetase in, 274, 274f glycine, 282-283, 284f
histidine, 282, 283f hydroxyproline, 285-286, 285f
initiation of, 281
isoleucine, leucine, and valine, 288,
290, 292f, 293, 293f, 293t lysine,
286,
288f metabolic diseases of, 280-281,

methionine, 288, 290f, 291f phenylalanine, 286, 288f
proline, 282, 282f rate of, 269-270
serine, 283, 284f threonine,
285,
285f transamination reactions in,
272-273, 273f, 281
tryptophan, 286-288, 289f, 290f tyrosine, 286, 287f
urea synthesis in, 272-273, 272f, 273f,

chemical reactions
of,
20
271,
272f conservation of catalytic, 61
L-a-amino acids (Cont.):
deamination of, 133
deficiency of, 263-264
determination of sequences of
Edman reaction for, 27-28, 27f genomics and, 28
mass spectrometry for, 28-30, 28¢,

molecular biology techniques for, 28
proteomics and, 30-31
purification techniques for, 23-26,
25f,
26f,
27f
Sanger's work in, 26-27
essential, 14, 131, 264, 264t, 526
extraterrestrial, 17-18
functional groups of, 18-20, 19f

genetic code specification of, 15,
15t-16t, 17t, 394, 394t glucogenic, 136, 272, 272f
glucose derived from, 177
for health maintenance, 3
hydrophobic and hydrophilic, 17¢
ketogenic, 136
as metabolic fuel, 130, 136
metabolic pathways of, 130-131,

in fasting state, 138, 138f in
fed state,
137f,
138,
138¢
at tissue and organ levels, 132-133,

metabolic roles of, 18
net charge of, 18-19, 19f nonessential, 131, 264, 264, 526
nutritional requirements for, 525-526
peptide bonds between, 20
pl of, 19
pKa of, 19-20, 20t pK, values of, 15t-16t
post-translational modifications of, 15,
17, 17f properties of, 15-18, 15t-16t, 17f,

protease and peptidase generation of,
270-271, 270f, 271f protein and peptide primary structures

selenocysteine as 21st, 15, 17, 17f solubility of, 20, 20f
stereochemistry of, 17

alanine and aspartate, 265, 265f asparagine, 265, 266f
biomedical importance of, 263-264
citric acid cycle role in,

cysteine, 265, 267f glutamate, 264-265, 265f
glutamine, 265, 265f glycine, 265, 266f
hydroxyproline and hydroxylysine,
266, 268f lengthy pathways of, 264, 264t
proline, 265, 267f selenocysteine, 267-268, 268f
serine, 265, 266f tyrosine, 266, 267f
valine, leucine, and isoleucine,
266-267
toxic plant, 18, 18f ultraviolet light absorption by, 20, 20f
unusual,
21,
21f
B-amino acids, biosynthetic conversions of alanine and aminoisobutyrate, 302-303
alanyl dipeptides,
303
GABA, 303, 303f amino sugars, 145, 145f
synthesis of, 188, 190f aminoacyl residues, 21
aminoacy] site, aminoacyl-tRNA binding

aminoacyl-tRNA in protein synthesis, 395,
395f, 402, 403f aminoacyl-tRNA synthetases, 395, 395f
aminoadipate-5-semialdehyde synthase,
286,
288f y-aminobutyrate (GABA), biosynthesis of,
303, 303f
B-aminoisobutyrate, biosynthesis of,

5-aminolevulinate (ALA)
heavy metal inactivation of, 94
synthesis of, 306, 306f, 309f
5-aminolevulinate (ALA) dehydratase,
306, 307f, 309f, 3108
5-aminolevulinate (ALA) synthase,
306-308, 306f, 309f, 310t aminopeptidases, 521
aminophospholipids, membrane asymmetry and, 464
aminopyrine, uronic acid pathway effects

aminotransferases, 154, 265, 265f diagnostic use of, 64, 64t, 566
transamination reactions of, 272-273,

vitamin B, in, 273, 273f, 535
ammonia from amino acid degradation, 269,
272-275, 274f citric acid cycle and, 150, 156
intoxication with,
273-274
nitrogen excretion as, 272
in urea cycle disorders, 276-278, 277t ammonium
ion, pK,
of,
13t ammonotelic
animals,
272
amobarbital, 118, 123
AMP. See adenosine monophosphate
AMP-activated protein kinase, 90
AMP-activated protein kinase (AMPK),

amphibolic pathways, 129
citric acid cycle as, 153, 154f amphipathic helices, 36
amphipathic lipids in lipoproteins, 237-238, 238f
in membranes, 204-205, 205f,
461-462, 461f amphipathic molecules, 8
ampicillin (Amp) resistance genes, 437
AMPK (AMP-activated protein kinase),
253, 253f
AMPK kinase (AMPKK), 253, 253f amplification
in cancer, 685
in fibrin clot formation, 677

diagnostic use of, 64t starch hydrolysis by, 520
amyloid,
715
B-amyloid, in Alzheimer disease, 42
amyloidosis, 639, 639t amylopectin, 145, 147f
amylose, 145, 147f anabolic pathways, 129
anabolism, 106
anaerobic conditions, glycolysis in,
157, 158f, 158t, 160, 696, 697f analbuminemia, 630
analytes, 561
anaphylaxis, slow-reacting substance of,
227
anaplerotic substrates, 153-154

steroidogenesis ovarian, 487-489, 489f, 490f
testicular, 487, 488f synthesis of, 486f, 487
androstenedione, 487, 488f anemias,
54
causes of, 646, 650t definition, 646
Diamond-Blackfan, 649
hemolytic. See hemolytic anemias of inflammation, 635
iron deficiency, 636, 637t, 650t megaloblastic, 538, 650t
pernicious, 536-537
aneuploidy, in cancer, 691-692, 692f
ANF (atrial natriuretic factor), 482, 483t angiogenesis, in cancer, 696-698
angiotensin II, 495-496, 496f angiotensin-converting enzyme (ACE),
495
angiotensin-converting enzyme

angiotensinogen, 495, 496f anion exchange protein, 652
ankyrin, 652t, 653
anomeric carbon atom, 143
anserine, 298, 298f, 303
antennae, of glycoproteins, 550
anterior pituitary gland, hormones of, 179
anterograde transport
(COPII),
586,
587f anterograde vesicular transport, 583
anti conformers, 320-321, 321f antibiotics
amino acids in, 14
amino sugars in, 145
bacterial protein synthesis and,
406-407, 407f folate metabolism inhibitors as, 537
glycosides,
145
resistance to, 80, 98, 99f antibodies, 627. See also immunoglobulins
monoclonal, 642-643
anticancer agents. See chemotherapy anticoagulant drugs, mechanism of,

anticodon region of tRNA, 394-396, 396f antifolate
drugs, purine
nucleotide synthesis affected by, 328, 330
antigenicity, xenobiotic alteration of, 559
anti-Lepore, 359, 360f antimalarial drugs, folate metabolism
inhibitors as, 537
antimycin A, 123
antioxidants, 116
biomedical importance of, 541
classes of, 204
pro-oxidant paradox of, 544-545, 544t protective mechanisms of,

vitamin E as, 532
antiparallel loops, mRNA and tRNA, 396
antiparallel strands, DNA, 339, 340f antiparallel 6 sheet, 36, 36f
antiplatelet drugs, 672
antiport systems, 468, 468f antiporter systems, for sugar nucleotides in
glycoprotein synthesis, 549-550
a,-antiproteinase, 638
antiproteinase inhibitor, 662
antiproteinases, 662
antipyrine, uronic acid pathway effects of,

antithrombin, 627, 630t, 677
a,-antitrypsin, 662, 677
APC (activated protein C), 677
apo A-I, 237t, 238
alcohol consumption and, 257
deficiencies of, 258t in HDL metabolism, 241f, 242
apo A-II, 237t, 238, 240
apo A-IV, 237#, 238
apo B-48, 237t, 238
apo B-100, 237t, 238
in LDL metabolism, 240f, 241
regulation of, 253
apo B-100 receptor in LDL metabolism, 241
apo
C-I,
237t,
238
apo C-II, 237f, 238, 240
apo C-III, 237t, 238, 240
apo D, 237t, 238
apo E, 2371, 238, 241
apo E receptor in chylomicron remnant uptake,
240f,
241
in LDL metabolism, 241
apolipoprotein E, in Alzheimer disease, 42 apolipoproteins/apoproteins, distribution of, 237t, 238
apomyoglobin, 49, 49f apoptosis, 710
cancer evasion of, 681-682, 692-694,

cardiolipin in, 230
ceramide in, 233
DNA damage and, 371-372, 371f phosphatidylcholines in, 200
apoptosome, 694, 712
apoptotic cell death program, 712
aprotic acids, 93
aPTT. See activated partial thromboplastin time
aquaporins, 472
aqueous solutions. See water arabinose, 144f, 144t
arachidonic acid/arachidonate,
197t, 199f
eicosanoid formation and, 224, 225f,
226f,
227f for essential fatty acid deficiency, 224
nutritionally essential, 222-223, 222f
ARF-1 (ADP-ribosylation factor-1),
242, 243

defects in, 278
arginine, 16t carbon skeleton catabolism of,
282,
283f resonance hybrids of, 18-19, 19f
specialized products of, 297, 297f,
301, 302f in urea synthesis, 275f, 276
arginine phosphate, in energy conservation and capture, 108
argininosuccinate, in urea synthesis,
275-276, 275f argininosuccinate lyase
defects in, 277t, 278
argininosuccinate synthetase defects in, 277t, 278
Argonaute proteins, 391
aromatase enzyme
complex,
487-489, 489f
ARS (autonomously replicating sequences), 362
arsenic in glycolysis pathway, 159
toxicity of, 93-94
arsenite, 153
arthritis. See also osteoarthritis gouty, 334, 336t
proteoglycans in, 604
rheumatoid. See rheumatoid arthritis artificial membranes, 465
arylamines, sulfation of, 558
ascorbate, 186
atorvastatin, 257
ATP. See adenosine triphosphate
ATP synthase mitochondrial compartmentalization
of, 118
rotary motor function of, 121, 122f
ATP/ADP
cycle,
108,
108f
ATPase, 472-473, 472f chaperones exhibiting activity of, 574
in osteoclasts, 605
ATP-binding cassette transporter Al
(ABCAI), 241f, 242
ATP-binding cassette transporter G1
(ABCG1), 241f, 242
ATP-chaperone complex, 575
ATP-citrate lyase, in lipogenesis, 218, 219f
ATP-driven active
transporters,
472,
473t
ATR (ATM-related kinase), 371, 371f atractyloside, 123, 124f
atrial natriuretic factor (ANF), 482, 483t autoantibodies, 641
autocrine signaling, 662

autoimmune disease free radical damage causing, 542
xenobiotic role in, 559
autoimmune disorder, 627, 628t autoimmune response, 645
autoimmunity, 641
autonomously replicating sequences

autotrophic organisms, 107
avian influenza virus, glycans in binding

axial ratios, 34
axonemal dyneins, 625
5or 6-azacytidine, 324
5-azadeoxycytidine, 690
8-azaguanine, 324, 324f azaserine, 328, 329f
azathioprine, 325, 325f
5or 6-azauridine, 324, 324f, 336
B
B blood group substances, 653-654, 654f
B cyclins, 369, 369f, 369t
B form of DNA, 340, 340f
B gene, 653-654
B lymphocytes, 639, 662
BAC (bacterial artificial chromosome)
vector, 436-437, 437t bacitracin, 14

antibiotic and protein synthesis in,
406-407, 407f bilirubin reduction by intestinal, 313
glycans in binding of, 555
transcription cycle in, 377, 377f transcription in, 379-380, 380f
bacterial artificial chromosome (BAC)
vector, 436-437, 437t bacterial DNA-dependent RNA
polymerase, 377, 377f bacterial lox P sites, 435
bacterial plasmids, 436, 436f bacterial promoters, in transcription,

bacteriophage lambda as paradigm for protein-DNA
interactions, 414-418, 414f, 415f,
416f, 417f at sites of, 435
balanced chemical equations, 69
BamHI, 433, 433t, 434f
BAPN (y-glutamyl-
B-aminopropionitrile), 18, 18t barbital, uronic acid pathway effects of,
189
barbiturates, respiratory chain inhibition by, 118, 123
barrel-like structures, 575
Barth syndrome, 201
basal laminas, 598
basal metabolic rate (BMR), 524
base excision repair (BER), 369, 370f, 370t base pairing in DNA, 339, 340f, 341
base pairs (bp), 355
base substitution, mutations occurring by,
396-397, 396f, 397f, 398f

as proton acceptors, 10
strong, 10-11
as unprotonated species, 11
weak. See weak bases base-stacking forces, 340
basophils,
656-657,
662
BAT (brown adipose tissue), 123-124,
247,
247f
BAX, 693f, 694
BCL-2, in apoptosis, 693f, 694
Becker muscular dystrophy, 622
bends, in proteins, 36, 37f benzoate,
298,
298f
BER (base excision repair), 369,

beriberi, 534
beta-anomers, 143
B-oxidation of fatty acids, 208-210, 208f,
209f, 210t ketogenesis regulation and, 213-214,
213f, 214f modified, 210, 211f
6 barrels, 36, 37f
6 sheets, as secondary structure unit, 36,
36f, 37f
6 turns, 36, 37f
B-oxidation, 131
BglII, 433t
BHA (butylated hydroxyanisole), 204
BHT (butylated hydroxytoluene), 204
Bi-Bi reactions, 79, 79f, 80f bicarbonate, carbon dioxide conversion to,
52, 53f bicarbonate in extracellular and
intracellular fluid, 460t bifunctional enzymes, 176
bilayers, lipid, 462-463, 462f membrane proteins and, 463
thickness of, 589
bile, bilirubin secretion into,
312-313, 313f bile acids (salts), 202, 249, 255-256, 255f
bile duct
obstruction, hyperbilirubinemia
caused by, 313f, 313t,
314-315,
315t bile pigments, biomedical importance of,
305
bile salts, 521
biliary tree obstruction, hyperbilirubinemia caused by,
313f, 313t, 314-315, 315t bilirubin
conjugation of, 312, 312f, 313f elevated levels of, 305, 313-314, 313t
excretion of, 312-313, 313f from heme catabolism, 311-313,
312f,
313f intestinal bacteria reduction of, 313
metabolic disorders of, 314-315,
314f,
315¢
serum measurement of, 313, 315,

structure of, 311, 312f transport of, 311-312
§-bilirubin, 315
bilirubin diglucuronide, 312, 312f bilirubin UDP-glucuronosyltransferase,

biliverdin, 311, 312f biliverdin reductase, 311, 312f
bimolecular lipid bilayer, 462
binding change mechanism, of ATP
synthase, 121, 122f binding constant, Michaelis constant as
approximation of, 75-76
binding domains, 37, 38f binding immunoglobulin protein (BiP),
581, 582f biochemical case histories
alcoholism, 720
biotin, 724-725
carnitine, 723-724
diabetes mellitus, 719-720, 725-726
glucose-6-phosphate dehydrogenase,

glutathione, 721
biochemistry, 1-4, 2f, 3f clinical. See laboratory tests
bioenergetics
ATP as cellular energy currency,
108-110, 108f, 109f, 121-122
coupling of endergonic and exergonic reactions,
106-107,
106f,
107f,
109
energy capture and transfer by high- energy phosphates, 107-108, 107t,
108f, 121-122
free energy in biologic systems, 105-106
bioengineering, 3
bioethics, 3
bioinformatics, 3, 31
biomarkers of cardiovascular risk, 567
enzymes used as, 64, 65f tumor, 700-701, 701t
biophysics, 3
biopolymers, enzymatic synthesis of, 9
biotechnology, 3
biotin case study of, 724-725
deficiency of, 528f, 538
528t,
539
in malonyl-CoA synthesis, 217, 217f structure of, 538, 538f
BiP (binding immunoglobulin protein),
581, 582f
1,3-bisphosphoglycerate, in glycolysis,
159, 159f, 160f
2,3-bisphosphoglycerate (2,3-BPG), 648
hemoglobin binding of, 50f, 53, 53f bisphosphoglycerate
mutase,
161,
161f
2,3-bisphosphoglycerate phosphatase,
161,
161f
2,3-bisphosphoglycerate synthase/
2-phosphatase (BPGM), 53
bleeding disorders, 678
bleeding time, 679
bleomycin, 14
blindness, vitamin A deficiency causing,

bilirubin in, 313-315, 313t, 315¢
metabolism integration by, 132-133,

blood, functions of, 628, 628t. See also plasma proteins
blood cells derivation from hematopoietic stem
cells,
646-647,
647f red. See red blood cells
white. See white blood cells

formation of, 87. See also fibrin clot formation
vitamin K role in, 532-533, 533f blood coagulation. See coagulation, blood
biomedical importance of, 172
clinical aspects of, 180-181, 180f free fatty acids and, 245, 245f
glucose oxidase in measurement of, 64
glucose tolerance measurement of,
180-181, 180f glycated hemoglobin in measurement
of, 55, 553
glycogen in maintenance of, 164
red blood cells and, 648
regulation of glucokinase role in, 178-179, 179f
hormonal mechanisms, 178-180,
178t, 179t metabolic mechanisms, 178, 178t
narrow limits of, 177
renal threshold for, 180
sources of, 177-178, 178f blood group, 653-654, 654f
blood samples, 563
blood transfusion, ABO system importance in, 653
blood type, 654
blood vessels, nitric oxide affecting,
622-623, 624f blot transfer techniques, 438, 439f
blotting techniques, 438, 439f blunt end ligation, 434-435
blunt ends, 433, 434f
BMPs (bone morphogenetic proteins),
635
BMR (basal metabolic rate), 524
Bohr effect, 52, 53f bond energies, of biologically significant
atoms,
7f bonds, biomolecule stabilization by, 7, 7t
bone components of, 604-607, 605t, 606f
metabolic and genetic disorders of,
607, 607t vitamin K role in, 533

heme synthesis in, 306
transplantation of, 235
bone morphogenetic proteins (BMPs), 635
botulinum B toxin, 587
boundary elements, 425
bovine preproparathyroid hormone, 495f bp (base pairs), 355
2,3-BPG. See 2,3-bisphosphoglycerate
BPGM (2,3-bisphosphoglycerate synthase/2-phosphatase), 53
bradykinin, 674

in fasting state, 130, 138-139, 138t,
139f,
139t in fed state, 130, 136-138, 137f, 138t
glucose requirement of, 136
branch point, of glycogen, 166
branched-chain amino acids, carbon skeleton catabolism of, 288, 290,
292f, 293, 293f, 293t branched-chain ketonuria, 290, 293, 293t
branching enzyme, in glycogenesis,

brefeldin A, 243, 588
broad B-disease, 258t
3-bromopyruvate, 696, 698f brown adipose tissue (BAT), 123-124,

brush border enzymes, 520
budding of vesicles, 586-587, 587f buffering, 12
buffers
Henderson-Hasselbalch equation describing, 12, 12f
weak acids acting as, 12-13, 12f
cAMP-dependent protein kinase glycogen regulation by, 167-168, 169f,

cancer.
See also
chemotherapy angiogenesis in, 696-698
apoptosis in, 681-682, 692-694,

biomarkers of,
700-701,
701t biomedical
importance of,
681
carcinogenesis in, 682-683, 688
causes of. See carcinogens cell cycle abnormalities in, 691
cell features in, 681, 682f colorectal, 370t, 687-688, 688f, 688t
copy number
variation in,
692,
692f epigenetic mechanisms involved in, 690
extracellular vesicles
and,
690
folate metabolism inhibition in

folic acid supplements for, 538
free radical damage causing, 542
genetic analyses of, 701
genetic damage in, 683
genomic instability and aneuploidy in,

glycoproteins in, 552
growth factor role in, 689, 689¢
hereditary, 683, 688, 690-691, 691t hyperbilirubinemia caused by, 313f,

hypermetabolism in, 157
immunologic aspects of, 698-700
incidence of, 681
metabolism in, 695-696, 697f, 698t metastatic process in, 698, 699f, 700t
micro-RNA in, 689-690
molecular profiling of, 703
neoplasms and, 681-682, 682f new therapies for, 701-703, 702t, 703f
w3 fatty acids and, 199
oncogene role in, 685-688, 685t, 686f,

prevention of, 703, 703t pro-oxidant paradox and, 544-545
selectins in, 554
signaling pathways in, 689, 689¢
stem cells in, 696
telomerase activity in, 692
tumor microenvironment in, 682,

vitamin
B, and,
535
xenobiotic role in, 556-557, 559, 559f

cyclins and, 369
membrane abnormalities and, 478t cancer phototherapy, 308
CAP (catabolite activator protein), 412
cap of mRNA, 345, 345f caproic acid, 197t
carbamates, hemoglobin transport of,

carbamoyl phosphate excess, 336
in urea synthesis, 275-276, 275f carbamoyl phosphate synthetase I
defects in, 277, 277t in urea synthesis, 275-276, 275f
carbamoyl phosphate synthetase II, in pyrimidine synthesis, 332,
333f,
334
"carbohydrate excess syndrome,' 247
carbohydrate loading, 164
carbohydrate-deficient transferrin, 633
carbohydrates as aldehyde or ketone derivatives of
polyhydric alcohols, 141-142, 142t biological information encoded in, 141
biomedical importance of, 141
blood glucose derived from,

in cell membranes, 147
cell surface, glycolipids and, 202
141-142,
142t diets low in, 181
digestion and absorption of, 520, 520f disaccharides. See disaccharides
energy yields from, 138t glycoproteins. See glycoproteins
in lipoproteins, 147
metabolic pathways of, 130, 130f, 131f in fasting state, 138-139, 138f, 139t
in fed state, 136-138, 137f, 138t at tissue and organ levels, 132-133,

vitamin B, role in, 533-534
monosaccharides. See monosaccharides polysaccharides. See polysaccharides
carbon dioxide
(CO,)
carbonic anhydrase and, 648
citric acid cycle production of,

heme binding of, 49, 49f respiratory chain inhibition by, 112,
118,
123
carbonic acid carbon dioxide conversion to,
52,
53f pK,
of,
13¢
carbonic anhydrase (CA), 52, 53f, 648
carbonic anhydrase II (CA II), 607
y-carboxyglutamate, synthesis of,

y-carboxyglutamic acid, 17, 17f carboxyl transferase, 217, 217f
carboxylases, biotin coenzyme of, 539
carboxypeptidase A, active site of, 59f carboxypeptidases, 521
carboxy-terminal domain (CTD),

carcinoembryonic antigen (CEA), 700
carcinogenesis, 682-683, 688
carcinogens chemical, 556-557, 559, 559f,
683-684, 6831, 684f radiation, 683, 683t
viruses, 684-685, 684t, 686f, 703
carcinoid syndrome, 535
cardiac glycosides, 145, 202
cardiac muscle,
612
calcium channels in, 619-620, 620f, 620t contraction regulation in, 619, 619t cardiac troponins, 64
cardiolipin, 200f, 201

synthesis of, 230, 230f, 231f cardiomyocytes, turnover rate of, 708t
cardiomyopathies, 621-622, 622t cardiovascular disease
biomarkers of, 567
lipids and, 198
caretaker tumor suppressor genes,

deficiency of, 207, 214-215
in fatty acid transport, 208, 208f liver and muscle, 723
urinary excretion of, 724t carnitine palmitoyltransferase, 207
carnitine palmitoyltransferase-I (CPT-I)
deficiency of, 215
in fatty acid transport, 208, 208f in ketogenesis regulation, 213, 213f
carnitine palmitoyltransferase-II (CPT-II)
in fatty acid transport, 208, 208f carnitine-acylcarnitine
translocase,

carnosinase, deficiency of, 303
carnosine, biosynthesis of, 298, 298f, 303
carnosinuria, 303
B-carotene,
529,
529f as antioxidant, 204
as pro-oxidant, 544
carrier proteins, 468-469, 469f carrier systems, for sugar nucleotides in
cartilage, 601-602, 607-608, 608f,

caspase-activated DNase (CAD), 692
caspases, 692-694, 693f catabolic pathways, 129
catabolism, 106
respiratory chain capture of energy from, 121-122
catabolite activator protein (CAP), 412
catalase, 114, 273, 544, 649
as antioxidant, 204

acid-base, 59-60, 60f active site role in, 58-59, 59f
biomedical importance of, 56
conformational changes in, 60, 60f covalent, 59-61, 59f, 61f, 62f
enzyme detection by, 62-63, 62f, 63f free-energy changes of, 69-70, 70f
by isozymes, 61-62
kinetics of. See enzyme kinetics prosthetic
group, cofactor,
and coenzyme roles in, 57-58, 58f
by proximity, 59
residues conserved in, 61
site-directed mutagenesis in study of,

enzymes as, 9, 57, 57f oxaloacetate as, 150-151, 151f
ribozymes as, 66-67

catalytic constant (k.,,), 75
catalytic efficiency (k.,,/K,,), 75
catalytic site. See active site cataract,
diabetic,
190-191
catecholamine oxidases, copper in,

storage of, 497, 497t synthesis of, 491-492, 491f
caveolae, 466, 466f caveolin-1, 466, 466f
CBG (corticosteroid-binding globulin),
498-499, 498t
CBP (CREB-binding protein),
513-514, 514t
CBP/p300 and signal transduction pathways, 513-514, 513f, 514t

CDK-cyclin inhibitor (CKI), 372
CDKs. See cyclin-dependent kinases cDNA library, 438
CEA (carcinoembryonic antigen), 700
celiac disease, 519
cell, macromolecule transport in,

cell adhesion, glycosphingolipids in, 234
cell adhesion molecules (CAMs),

cancer abnormalities in, 691
delay or arrest of, 371, 371f phases of, 368, 368f
regulation of, 585
S phase of, DNA synthesis during,
368-369, 368f, 369f, 369t cell death. See apoptosis
cell division, checkpoints in, 90, 90f cell growth, 603
cell membranes carbohydrates in, 147
vitamin E in, 532
cell migration, 598, 601
cell recognition, glycosphingolipids in, 234

phosphatidylcholines in, 200
phosphatidylinositols in, 200
cell-cell communication with gap junctions, 476, 476f
heparan sulfate and, 603
cell-cell interactions, 459
cell-mediated immunity, 639
cellulose, 146-147, 148f cellulose acetate zone electrophoresis, 628,

central core disease, 618, 620t central nervous system (CNS), glucose
requirement of, 136
centromere, 355, 355f cephalin (phosphatidylethanolamine),

membrane asymmetry and, 464
synthesis of, 230, 230f, 231f ceramide, 200, 202f
accumulation of, 235
in membranes, 461
in sphingolipid synthesis, 233-234, 234f synthesis
of,
233,
233f cerebrohepatorenal (Zellweger) syndrome,

ceruloplasmin, 631, 631f, 633-634
diagnostic use of, 64t cervonic acid, 197t
CF (cystic fibrosis), 64, 478, 478t, 519, 585t cGMP. See cyclic GMP
chain elongation, 376f, 378. See also elongation
chain initiation, 376f, 378. See also initiation
chain termination, 376f, 378-379.

chain termination method, 439, 440f chain-breaking antioxidants, 204
chalones, 227
channeling, in citric acid cycle, 151
channelopathies, 620, 620t channels, amphipathic helices in, 36
chaperones,
41
ATP-dependent protein binding to, 576
histone, 353
misfolded proteins and, 584-585,

properties of, 576t in protein folding, 574-575
in protein sorting, 575t, 576
in quality control, 584, 584t in translocation, 576, 577f
chaperonins, 41, 575
charged groups, noncovalent interactions of,
8
charged paddle, 471, 471f charge-relay network, 61, 61f
charging, in protein synthesis, 395, 395f checkpoint
controls,
372
checkpoint kinases 1 and 2 (CHK1 and

checkpoints, in cell division, 90, 90f
Chédiak-Higashi syndrome, 585t chemical carcinogenesis, 559, 559f
chemical carcinogens, 556-557, 559, 559f,
683-684, 6831, 684f chemical chaperone therapy, 235
chemical equations balancing of, 69
kinetic order of, 71
chemical mechanisms and reactive oxygen

chemical potential, 105
chemical reactions catalysis of. See catalysis
coupling of, 106-107, 106f, 107f, 109
equilibrium of, 69, 71-72
free-energy changes of. See free energy mechanisms of, 69
multisubstrate, 79, 79f, 80f rate-limiting, 84
rates of. See rate of reaction reversibility of, 69
transition states of, 69-70, 70f chemiosmotic theory, 121, 122f, 124
chemokines, 658, 658f, 662
chemotaxis, 657-658

folate metabolism inhibitors, 537
new therapies, 701-703, 702t, 703f resistance to, 702-703, 702t
synthetic nucleotide analogs in,

chenodeoxycholic acid, 255-256, 256f chenodeoxycholyl-CoA, 255, 256f
children, kwashiorkor in, 525
chimeric gene approach, 422f, 423, 424f chimeric molecules

restriction enzymes for
DNA chain cleavage with, 433-434,
433t, 434f, 434t preparation with, 434-435
ChIP (chromatin immunoprecipitation),

ChIP-chip, 447
ChIP-Exo, 447
ChIP-Seq, 447
chitin, 147, 148f
CHK1 and CHK2 (checkpoint kinases and 2), 371, 371f
chloramines, 661
chloride (CI)
in extracellular and intracellular fluid,

permeability coefficient of, 463f chlorinated oxidants, production of, 662
chlorobutanol, uronic acid pathway effects of, 189
1
chlorophyll, 305
cholecalciferol. See vitamin D, cholera
glucose transport in treatment of, 474
toxin, 202, 202f, 234, 504
cholestatic jaundice, 314

absorption of, 521
in bile acid synthesis, 254-256, 256f biomedical importance of, 249
clinical aspects of, 256-257, 258t dietary, 250, 257
excretion of, 254-256, 256f fructose effects on, 186, 188f, 189-190
function of, 202
in high-density lipoproteins in,

HMG-CoA reductase regulation and,
86
hormone synthesis from, 202, 249, 483,

in lipoprotein, 236-237, 238f in membranes, 461, 589
fluid mosaic model and, 465-466
metabolic pathways of, 130-131, 131f

atherosclerosis and coronary heart disease and, 202, 250, 256-257
dietary changes
and,
257
drug therapy and, 257
lifestyle changes and, 257

diurnal variation in, 252
HMG-CoA reductase regulation of,
252-253, 253f tissues,
203,
203f factors influencing balance of,
253, 254f

with lipoproteins, 253-254, 255f reverse, 241f, 242, 250, 254,
254f,
257
cholesterol 7a-hydroxylase, 255, 256f cholesteryl ester hydrolase, 253
cholesteryl ester transfer protein, 238, 254,

cholesteryl esters, 203, 253-254, 255f in HDL metabolism, 241f, 242
in lipoprotein core, 236, 237, 238f cholesterylation, 463
cholic acid,
255,
256f choline,
200,
200f,
201f deficiency of, fatty liver and, 244
choluric jaundice, 313-314
cholyl-CoA, in bile acid synthesis,

chondrodysplasias, 608-609
chondroitin sulfate, 147, 148f, 595,
599, 601-602, 601f, 602t, 604,
604t, 608f in
chondronectin, 608, 608¢
chorion protein genes, 429, 429f choroid,
gyrate atrophy
of,
282
chromatids, 355, 355f, 355t, 356f,
361, 361f

active regions of, 353-354, 354f, 418
components of, 351, 351f covalent modifications of, 87
gene expression and template for,
418-419
higher order structure and compaction

inactive regions of, 354, 418
physiologic roles of, 101
reconstitution in DNA replication, 368
chromatin fibers, 352f, 353
chromatin fibrils,
352f,
353
chromatin immunoprecipitation (ChIP),
447,
448f chromatin modifying complex (CMC),

chromatography affinity, 25-26
column, 24, 25f high-pressure liquid, 24, 25f
laboratory tests using, 564
hydrophobic interaction, 25
ion-exchange, 25
protein and peptide purification with,

human requirement for, 93, 93f multivalent states of, 93, 94f, 94t
toxicity of, 95t chromosomal instability (CIN),

coding regions of, 355-356, 357f, gene conversion, 361
gene localization to, 441, 442t integration of, 359-360, 360f
integrity of, monitoring, 372
interphase, 353
metaphase, 352f, 355, 355t, 356f organization of, 355, 355f, 355t, 356f
polytene,
354
recombination of, 359, 359f, 360f scaffolding in, 352f, 353
sister chromatid exchange, 361, 361f transposition, 360, 442
chronic granulomatous disease, 661
chronic myelocytic leukemia (CML), 701
chyle, 238
chylomicron remnants, 237t, 238
formation of, 240-241
liver uptake of, 239f, 241
clofibrate, 257
cloning, 435-438, 436f, 437f, 437t cloning vectors, 436, 436f, 437t
clopidogrel, 672
closed complex, 376f, 379
closure time, 679
clotting. See fibrin clot formation
CMC (chromatin modifying complex),

CML. See chronic myelocytic leukemia
CML (chronic myelocytic leukemia), 701
CMP (cytidine monophosphate), 322t
CNS (central nervous system), glucose requirement of, 136
CNV. See copy number variation
CO,,. See carbon dioxide coactivators, transcription, 383-385, 385t
coagulation, blood
disorders of, 655
prostaglandins in, 216
coagulation factors, 672, 673f, 673t, 674t deficiencies of, 678
vitamin K-dependent carboxylation of,

coagulation system. See also hemostasis anticoagulant drug effects on, 677-678
biomedical importance
of,
669
endothelial cell actions in, 670, 672
fibrin clot formation in. See fibrin clot

inherited deficiencies of, 678
laboratory tests measuring, 679
platelet aggregation in, 670, 671f thrombi types in, 670
thrombin inhibition in, 677-678
thrombin regulation in, 677
coat proteins, 583, 586-587, 586t, 587f coated pits, 475
cobalamin. See vitamin B,, cobalophilin, 536

human requirement for, 93, 93t multivalent states of, 93, 94f, 94t
physiologic roles of, 98
toxicity of, 95t cobamide, 58
code erasers, 418
code readers, 418
code writers, 418
coding regions, 355-356, 357f coding strand
in DNA replication, 339, 344f in RNA synthesis, 375, 380f

amino acid sequence and, 394, 394t anticodon recognition of,
395-396, 396f nonsense, 394, 397-398, 398f
stop, 403-404, 404f tables of usage of, 395

cysteine conversion to, 297
pantothenic acid in, 539, 539f coenzyme Q (Q, ubiquinone), 203
250-251,
251f in respiratory chain, 118, 119f, 120,

coenzymes,
57-58,
58f laboratory tests for, 564-565
nicotinamide oxidation and reduction,

nucleotide derivatives, 323, 324t cofactors, 57-58, 58f
cognate receptor, 502
colchicine, 625
collagen,
406
abundance of, 592
in bone, 604-605, 605t in cartilage, 607-608, 608f, 608¢
chondrodysplasias, 595, 608-609
classification of, 594, 594f cross-linking of, 553
defects in, 263-264
diseases caused by abnormalities in,

elastin compared with, 597t fibril formation by, 593-594
fibronectin interaction with, 597, 598f as fibrous protein, 43
O-glycosidic linkages in, 549
nutritional and genetic disorders of,

osteogenesis imperfecta and, 607, 607t platelet adhesion to, 670, 671f
posttranslational modification of,
594-595,
594t posttranslational processing of,

structure of, 592-594, 593f, 594f synthesis of, 43
triple helix structure of, 43, 43f types of, 593t
vitamin C effects in, 539
collision theory, 70
colony-stimulating factor, 647
colorectal cancer development of, 687-688,

mismatch repair in, 370t column chromatography, protein and
peptide purification with, 24, 25f combinatorial chemistry, 63
common lymphoid progenitor, 657
common myeloid progenitor, 657
compartmentation metabolic regulation with, 83-84
of mitochondria, 118, 118f competitive inhibitors, 76-78, 77f, 78f
competitive ligand-binding assays, laboratory tests using, 565
complement, 539
complement system (cascade),
643-644, 644f complementarity
of DNA, 341, 341f, 438
of RNA, 342-343, 344f, 346
complementarity-determining regions,
640
complementary DNA (cDNA) library, 438
complex carbohydrates, glycoproteins as,
546
Complex
I.
See NADH-Q oxidoreductase
Complex
II.
See succinate-Q reductase
Complex III. See Q-cytochrome c oxidoreductase
Complex IV. See cytochrome c oxidase complex lipids, 196
complex oligosaccharides, in glycoproteins, 550-551, 550f
complexation, of transition metals, 95-97,

compulsory-order reactions, 79, 79f concanavalin A, 147
conformational disorders, 590

of enzymes, 60, 60f hemoglobin changes in, 51-52, 51f, 52f
of proteins biomedical importance of, 33
configuration versus, 33-34
native, 41
pathologic diseases of, 42
peptide bond restrictions on,

congenital contractural arachnodactyly,
597
congenital disorders of glycosylation, 633
congenital erythropoietic porphyria,

congenital forms of muscular dystrophy,
622
congenital long QT syndrome, 478
congenital muscular dystrophies, glycoprotein synthesis defects in,

conjugate acid, 11
conjugate base, 11
conjugated bilirubin, 312, 312f, 313f conjugated double bonds, in porphyrins,
308, 310f conjugated hyperbilirubinemia, 313f, 313t,
314-315
conjugation reactions, of xenobiotics, 558
connective tissue, 592. See also extracellular matrix
connexin, 476, 476f connexon channel, 476, 476f
consensus sequences, 383-384, 388f

constitutive gene expression, 411, 414
constitutive heterochromatin,
354
constitutive mutation, 411
constitutive proteins, 84
constitutive secretion, 574
contact sites, 576
contraction, muscle. See muscle contraction
convalescence, protein replacement during, 526
Coomassie Blue dye, 26, 26f cooperative binding
of hemoglobins, 51
Hill equation for, 76, 76f coordinate expression, 412
COPI vesicles, 583, 586, 586f, 588
COPII vesicles, 583, 586, 586t, 587f

absorption of, 101
in Complex IV, 120, 120f in cytochrome oxidase, 97
deficiency of, 43, 263
human requirement
for,
93,
93t multivalent
states of,
93,
94f,
94t physiologic roles of, 98-100, 99f
toxicity of, 95t copper toxicosis, 634
coproporphyrinogen III, in heme synthesis, 307, 307f, 308f, 309f
coproporphyrinogen oxidase, 307, 308f,
309f,
310¢
coprostanol (coprosterol), 254
copy number variation (CNV), 442
in cancer, 692, 692f coregulators, transcription, 383-385, 385t,

corepressor family, 514, 514t
Cori cycle, glucose derived from, 177, 178f cornea, 602
coronary (ischemic) heart disease.
See also atherosclerosis cholesterol and, 257
corrinoids, 536
corticosteroid-binding globulin (CBG),

cortisone reduction to, 184-185
laboratory tests for, 565, 567
plasma transport of, 498-499, 498¢
synthesis of, 484f, 485f, 486
cortisone, cortisol synthesis from, 184-185
cos sites, 436
cosmids, 436-437, 437t cotranslational glycosylation, 581
cotranslational insertion, 582-583,

cotranslational pathway, 580-581, 581f cotransport systems, 468, 468f
cotransporters, 469
Coulomb' law, 7
coumarin drugs, 677-678
coupled enzyme assays, 63, 63f coupling
of endergonic and exergonic reactions,
106-107, 106f, 107f, 109
of oxidation and phosphorylation in respiratory chain, 122-123, 123
covalent bonds biomolecule stabilization by, 7, 7t
in collagen, 43
membrane lipid-protein interaction and, 463

activation energy lowering by, 72
enzymatic, 59-61, 59f, 61f, 62f covalent cross-links, 593
covalent modifications. See also phosphorylation
carcinogens causing, 684
enzyme regulation by, 85-90, 88f, 89t in glycolysis and gluconeogenesis
of histones, 351, 353t mass spectrometry detection of,
in protein posttranslational processing,
42-43
of pyruvate dehydrogenase, 161-163,
162f reversibility of, 87
COX-1. See cyclooxygenase
COX-2, 224
coxibs, 226
cPLA, (cytosolic phospholipase A,), 670,
671f
CPT-I.
See carnitine palmitoyltransferase-I
CPT-II.
See carnitine
palmitoyltransferase-II
C-reactive protein (CRP), 630, 630t creatine, biosynthesis of, 301, 302f
creatine kinase, 620
in creatine phosphate shuttle, 126, 126f diagnostic use of, 64, 64t
mitochondrial compartmentalization of,
118
creatine phosphate in energy conservation and capture,
108, 109f in muscle contraction, 620, 620f
creatine phosphate shuttle, 126, 126f creatinine
biosynthesis of, 301, 302f laboratory tests for, 566
creatinine clearance,
455
CREB (cyclic AMP response element binding protein), 505
CREB-binding protein (CBP),
Creutzfeldt-Jakob disease (CJD), 42
Crigler-Najjar syndrome, 314
CRISPR-Cas, 348
CRISPR-Cas9, 434t, 435, 435f, 446-447
cro gene, 415, 415f
Cro protein, 415-416, 416f
Crohn disease, 226
cross-bridge complex, 616, 616f crossing-over,
in chromosomal
recombination, 359, 359f, 360f crotonyl-CoA, lysine formation of,

CRP (C-reactive protein), 630, 630t cryo-electron microscopy, protein
structures solved by, 40
crystallography, protein structures solved by, 39-40
CTD (carboxy-terminal domain),

C-terminal binding domain, 37
CTP (cytidine triphosphate), 86, 323
cubilin, 101
cutaneous porphyrias, 310, 311
cyanide, respiratory chain inhibition by,

cyclic 3',5'-nucleotide phosphodiesterase, in lipolysis, 246
cyclic AMP (cAMP), 322, 323f, 324t adenylyl cyclase and, 503-504, 503t
formation and hydrolysis of,
167, 168f glycogen regulation by, 166-170, 168f,
169f, 170f, 171f in hemostasis and thrombosis, 672
in muscle contraction, 619
in operon model, 414
phosphodiesterases and, 505
phosphoprotein phosphatases and,
505-506
phosphoproteins and, 505
protein kinases and, 504-505, 505f as second messenger, 482, 483t, 502
cyclic AMP regulatory protein (catabolite gene activator protein), 412
cyclic AMP response element binding protein activator protein (CREB),
505
cyclic AMP-dependent protein kinase, in lipolysis,
246,
246f cyclic GMP (cGMP), 323f
as second messenger, 323, 483, 483t,
502, 506
synthesis of, 506
cyclin-dependent kinases (CDKs)
in cancer, 691
in cell cycle, 368, 369f, 369t inhibition of, 372
cyclins, 368-369, 369f, 369t in cancer, 691
cycloheximide, 407
cyclooxygenase (COX-1), 224-225
inhibition of, 672
cyclooxygenase pathway, 224-225,
225f, 226f cyclophilins. See proline-cis,
trans-isomerases cyclosporine, 42
CYP2A6, 557
CYP2C9, 557
CYP27A\
(sterol 27-hydroxylase),
255-256, 256f cystathionine B-synthase, 285, 300
cysteine,
16t carbon skeleton catabolism of,

specialized products of, 297, 297f synthesis of, 265, 267f
cysteine sulfinate pathway, 283, 284f cystic fibrosis (CF), 64, 478, 478t,
519, 585t
cystic fibrosis transmembrane regulator protein, 478
degradation of, 585
cystine, carbon skeleton catabolism of,
283-285, 284f cystine reductase, 283, 284f
cystinosis, 285
cystinuria, 283
cytarabine, 325, 325f cytidine, 321f, 322t, 333
cytidine monophosphate (CMP), 322t cytidine triphosphate (CTP), 86, 323
cytochrome aa, 112
cytochrome b, reductase, 649
cytochrome bs 115, 557, 583, 649
cytochrome b.,.,,
660
cytochrome b,,, in respiratory chain,
120, 121f cytochrome b,, in respiratory chain,
120,
121f cytochrome c
in apoptosis, 694
iron in,
95-96
in respiratory chain, 118, 119f, 120,

cytochrome c oxidase (Complex IV), 118,
119f, 120-121, 120f deficiency of, 126
cytochrome c,, in respiratory chain,
120,
121f cytochrome oxidase, 112
transition metals in, 97
cytochrome P450 side chain cleavage enzyme (P450scc), 485, 485f
cytochrome P450 system, 111
carcinogen activation by, 684
in drug metabolism, 115-116, 115f,

inducible nature of, 557-558
iron in, 97
membrane insertion, 583
microsomal ethanol oxidizing system,
244
as monooxygenases, 115-116, 115f, 116f nomenclature of, 557
polymorphism of, 557
sterol 27-hydroxylase, 255
xenobiotic hydroxylation by, 556-558
cytochromes, as dehydrogenases, 114
cytokines, 631, 638, 647, 662
advanced glycation end-product activation of, 553
blood glucose regulation by, 179-180
cachexia resulting from, 139, 525
in cancer development, 700
cytoplasmic protein tyrosine kinases,

base pairing in DNA, 339, 340f base
pairing in
RNA,
342,
343f deoxyribonucleosides of, in pyrimidine
synthesis, 332-333
salvage pathways of, 333
cytoskeleton biomedical importance of, 611
cellular functions of, 623-625, 624f, 625t

fatty acid synthesis in, 207
glycolysis in, 134, 134f lipogenesis in, 216-220, 219f,
221, 221f pentose phosphate pathway in, 182-183
pyrimidine synthesis in, 332
cytosolic branch, of protein sorting, 574f in
cell nucleus,
575f,
576-579,
577t,
578f in mitochondria, 576, 577f, 577t
in peroxisomes, 579, 579f signals in, 574-575
cytosolic dynein, 625
cytosolic phospholipase A, (cPLA,),

cytotoxic T cells, 656, 662
cytotoxicity, of xenobiotics, 559, 559f
D
D arm of tRNA, 346f, 347, 396, 396f

cancer and, 369
in cell cycle, 368-369, 369f, 369t
DAG. See diacylglycerol; 1,2-diacylglycerol damage repair mediators, 370, 371f
dAMP, 322f dantrolene, 618
DBDs.
See
DNA-binding domains
DBH (dopamine f-hydroxylase),
491,
491f
DCA (dichloroacetate), 696, 698t
Dcytb (duodenal cytochrome b),
101, 632
deacetylases, 89
deacetylation, enzyme regulation by,

deacylase (thioesterase), 218, 218f, 219f deamination, 131
of amino acids, 133
citric acid cycle role in, 153-154, 154f in nitrogen metabolism, 273-274, 274f
death receptor pathway, of apoptosis,
692-694, 693f debranching enzyme, in glycogenolysis,
166, 166f decarboxylation, oxidative. See oxidative
decarboxylation decay accelerating factor, 554
decitabine, 690
decondensation, 661
defensins, 660t degeneracy, of genetic code, 394, 394t
dehydratase, 219f
7-dehydrocholesterol, 530, 531f dehydroepiandrosterone pathway, 487, 488f
dehydrogenases coupled enzyme assays using, 63, 63f
cytochromes as, 114
detection of NAD(P)*-dependent,
63, 63f flavin groups in, 114
nicotinamide coenzymes of, 113, 113f oxidation-reduction reactions of,
112-114, 113f dehydrogenation, hydrogenation coupling

DNA structure analysis and, 340-341
protein refolding
after,
41
temperature increases causing, 72
dense granules, 654
deoxy sugars, 145, 145f deoxyadenylate, 338-339
deoxycholic acid, synthesis of, 255
deoxycytidine residues, methylation of,

deoxycytidylate, 338-339
2-deoxy-D-glucose, 696, 698t
2-deoxy-D-ribose, 320
deoxyguanylate, 338-339
deoxynucleotides, 338-340, 339f deoxyribonucleases (DNase), 348
active chromatin and, 353
deoxyribonucleic acid. See DNA
deoxyribonucleoside diphosphates
(dNDPs), 332, 332f deoxyribonucleosides, 320, 332-333
deoxyribose, 141, 145, 145f
3-deoxyuridine,
324
dephosphorylation, enzyme regulation by,

depolarization, in nerve impulse transmission, 473
depolarization, nonpolar lipids and, 196
derepressed systems, 410
dermatan sulfate, 599, 601f, 602t, 603
A°-desaturase, 97, 223-224, 223f desmosines, 596
desmosomes, 466
desmosterol, in cholesterol synthesis,

detergents, 462
detoxification, cytochrome P450 roles in,
115-116, 115f, 116f, 556-558
developmental biology, 717
dexamethasone suppression test, 567
dextrins, 146
dextrose, 143
DGAT (diacylglycerol acyltransferase),

DHA (docosahexaenoic acid), 199, 224
DHT. See dihydrotestosterone diabetes insipidus, nephrogenic, 472
585t advanced glycation end-products in,
553, 553f case study, 719-720, 725-726
excessive gluconeogenesis in, 172
fatty liver and, 244
free fatty acid levels in, 238
glucose tolerance in, 180-181, 180f glycated hemoglobin in management of,
55, 553
diabetes mellitus (Cont.):
insulin impairments in, 130, 140
ketosis/ketoacidosis in, 215
lipid transport and storage disorders,
236-237
lipids and, 196, 198
lipogenesis and, 216, 220
diabetic cataract, 190-191
diacylglycerol (DAG), 200, 658
formation of, 230f, 234f protein kinase C and, 507, 507f
1,2-diacylglycerol (DAG), in platelet aggregation, 670, 671f
diacylglycerol acyltransferase (DGAT),
230, 231f diagnostic enzymology, 63-65, 64t, 65f,

diagnostic testing. See laboratory tests a,y-diaminobutyric acid, 18, 18¢
Diamond-Blackfan anemia, 649
diapedesis, 657, 657f diarrhea, 474, 555
diazanorleucine, 328, 329f dicarboxylic aciduria, 215
dicer nuclease-TRBP complex, 391
dichloroacetate (DCA), 696, 698t dicopper center, 99-100, 99f
dicumarol, 532
dielectric constant,
7

177-178, 178f cholesterol levels affected by, 250,
254,
257
hepatic VLDL secretion and,

high fructose, 186, 188f, 189-190
high-fat, fatty liver and, 244
low carbohydrate, 181
multiple deficiency states in, 527
requirements energy, 129-130, 524-525, 524f
protein and amino acid, 525-526
transition metals, 93, 93t vitamins
and minerals,
527-528
diethylenetriaminepentaacetate (DTPA),

facilitated. See facilitated diffusion net, 468
simple, 467-468, 467f, 4671, 468f diffusion-limited enzymes, 75

of carbohydrates,
520,
520f of lipids, 520-521, 522f
metabolism of major products of,
130-131, 130f, 131f, 132f protease activation in, 87
of vitamins and minerals, 523-524, 523f digitalis, 473
dihydrobiopterin reductase, 286, 288f dihydrofolate, methotrexate and, 333-334
dihydrofolate reductase, 537
dihydrolipoyl dehydrogenase, 114,
161, 162f dihydrolipoyl transacetylase, 161, 162f
dihydroorotic acid, 333f dihydropyridine receptor, 618, 618f
dihydropyrimidine dehydrogenase deficiency, 327, 335-336,
3361, 337f dihydrotestosterone (DHT), 483
plasma transport of, 498f, 499
synthesis of, 487, 489f dihydroxyacetone phosphate, in glycolysis,
159,
159f,
230,
231f,
232f
1,25-dihydroxyvitamin D, 530-531, 531f diiodotyrosine (DIT), 492, 493f
diiron center, of hemerythrin, 97, 97f dilated cardiomyopathy, 622
dimercaprol, 123
dimeric proteins, 38

Cro protein, 415, 416f histone, 351, 351f, 353
lambda repressor (cI) protein, 415, 416f dimethylallyl diphosphate, in cholesterol
synthesis, 250, 251f dimethylaminoadenine, 323f
2,4-dinitrophenol, 123
dinucleotide, 325
dioxygenase, 115
dipalmitoyl lecithin, 200, 229
dipeptidases, 521
diphosphomevalonate decarboxylase, 251f diphosphomevalonate kinase, 251f
diphtheria toxin, 407, 472
dipoles, 6-7, 7f dipsticks, 565
direct bilirubin, 313
disaccharidases, 520
disaccharides, 142
digestion of, 520, 520f of physiological importance, 145,
146f,
146¢

autoimmune, 627, 628t biochemical
processes underlying,
biochemistry interrelationship
2,
2f conformational, 585t
of DNA damage repair, 369, 370t genetic.
See genetic
diseases membranes and, 459, 478t
molecular analysis of, 442-445,

protein sorting and, 573
3
with, red blood cells and, 646, 650t
of skin, 3
diseases of proteostasis deficiency, 590
dislocation, 584
dispensable amino acids, 131
dissociation, 9-12
dissociation constant (K,)
definition of, 9
Michaelis constant as approximation of,

of water, 10
of weak acids and bases, 11
disulfide bonds in chemokines, 658, 658f
formation of, 41-42
in protein tertiary and quaternary structures, 39
DIT (diiodotyrosine), 492, 493f diurnal variation, in cholesterol synthesis,

divalent metal ion transport protein
(DMT-1), 101
divalent metal transporter, 632, 632f
Dixon plot, 78, 78f
DMT-1 (divalent metal ion transport protein), 101

matching of for renaturation, 341
biomedical importance of, 338
carcinogen reactions with, 684
in chromatin, 352f chromosomal, 352f, 355-356, 355f,

cloning of, 435-438, 436f, 437f, 437t coding regions of, 355-356, 357f
complementarity of, 341, 341f, 438
damage to
double-strand break, 370, 370f, 371f types of, 369, 370t
double-helix structure of, 8,
339-340, 340f enhancer elements, 420-422, 422f,

epigenetic code of, 384
free radical damage to, 541-542, 542f genetic information contained in,

in genetic information flow, 393-394
immunoglobulin in rearrangements of,

integrity monitoring of, 372
mitochondrial, 358-359, 358f, 359t mutations in, 350-351, 359-361.
See also mutations, genetic noncovalent interactions in, 8
in nucleosomes, 351, 351f, 352f, 353
partial digestion of, 438
as polynucleotides, 325
protein identification using, 28
recombinant. See recombinant DNA
relationship to mRNA, 357f relaxed form of, 341
renaturation of, base pair matching and,

study of, 369-372
types of, 369-370, 370f, 370t repetitive-sequence, 357
replication and synthesis of, 341, 342f biomedical importance of, 350-351
control of,
361-362,
362f,
362t
DNA polymerase complex in, 362f,

epigenetic signals during, 419, 420f initiation
of,
363-364,
365f,
366f origin of, 362-363, 362f
polarity of, 364, 366f reconstitution of chromatin structure

repair during, 369-370, 370t replication bubble formation and,
364, 366f, 367-368, 367f replication fork formation and, 362f,

ribonucleoside diphosphate reduction and, 332
RNA primer in, 362f, 362t,
363-364, 365f, 366f
RNA synthesis compared with, 375
in S phase of cell cycle, 368-369,

semiconservative nature of, 341, 342f semidiscontinuous, 362f, 364, 366f
steps in, 362f, 362t unwinding of, 363
restriction enzyme cleavage of,
433-434, 433t, 434f, 434t in RNA synthesis. See RNA, synthesis of
sequencing of, 439, 440f direct, 445-446
silencer elements, 422-423

components of, 338-339, 339f denaturation in analysis of, 340-341
double-helical, 339-340, 340f forms of, 341
grooves in, 340f, 341
supercoiled, 341, 367, 368f topoisomerases of, 341
transcription of, 341. See also RNA,

transposition of, 360, 442
unique-sequence (nonrepetitive), 357
unwinding of, 378
xenobiotic reactions with, 559, 559f
DNA blot transfer procedure, 438, 439f
DNA chips, 31
DNA damage sensors, 370, 371f
DNA
elements combinations of, 424, 425f
gene expression enhancement and repression by, 420-423, 422f,
4221, 423f locus control regions and insulators,
424-425
reporter genes and, 423, 424f tissue-specific expression of, 423
DNA helicase, 362f, 363, 363t
DNA lesions, 712
DNA ligase, 363t, 364
in chimeric DNA molecule preparation,
434-435
in recombinant DNA technology, 434t
DNA
methylases, sequence-specific,
433
DNA methyltransferases (DNMTs), inhibitors of, 690

prokaryotic and eukaryotic, 364t in recombinant DNA technology, 434t,

in replication, 362f, 363, 363t
DNA primase, 362f, 363t
DNA regulatory elements, 422, 423f
DNA repair mechanism, 559
DNA topoisomerases, 367, 367f
DNA transfection, endocytosis in, 474
DNA unwinding element (DUE), 362-363
DNA-binding domains (DBDs), 381
of cl repressor protein, 415
of regulatory transcription factor proteins, 425t
helix-turn-helix motif, 425-426, 426f leucine zipper motif, 427, 427f
zinc finger
motif,
426,
426f transactivation domain separation

DNA-dependent RNA polymerases initiation by, 375-377, 376f, 377f
in operon model, 412, 413f, 414
DNA-PK (DNA-dependent protein kinase), 370, 371f
DNA-protein interactions bacteriophage lambda as paradigm for,
414-418, 414f, 415f, 416f, 417f mapping of, 446-447
DNase. See deoxyribonucleases
DNase
I
in recombinant DNA technology, 434t dNDPs (deoxyribonucleoside
diphosphates), 332, 332f
DNMTs (DNA methyltransferases), inhibitors of, 690
docking, in protein sorting, 578, 586-587
docosahexaenoic acid (DHA), 199, 224

in cholesterol synthesis, 250-251, 251f dolichol pyrophosphate, in glycoprotein
synthesis, 550-551, 551f

albumin, 630
chromatin, 352f, 353, 355
DNA-binding. See DNA-binding domains
of hormone receptors, 481-482
protein, 37, 38f, 39f

acromicric, 597
colorectal, 687-688, 688f, 688t
geleophysic, 597
thanatophoric, 609
a-dystroglycan, 622, 623f
dystrophin, 622, 623f
E
E.
coli, lactose
metabolism and
operon hypothesis in, 411-412, 411f,
413f, 414
E. coli bacteriophage P1-based (PAC)
vector, 437, 437t
E cyclins, 369, 369f, 369t
E (exit) site, of 80S ribosome, 402, 403f
E',, See redox potential
E3 ligases, 84-85
E, ,. See activation energy
E-cadherin, in cancer, 698, 700t
ECE (extracellular fluid), 460, 460t
ECM. See extracellular matrix
EcoRI, 433, 433t, 436f

edema kwashiorkor causing, 525
plasma protein concentration and, 628
vitamin B, deficiency in, 534
Edman reaction, protein and peptide sequencing with, 27-28, 27f
Edman reagent, 27, 27f
EDTA (ethylenediaminetetraacetate), 204
EF1A (elongation factor 1A), 402, 403f
EF2 (elongation factor 2), 403, 403f
EFA. See essential fatty acids effectors,
of apoptosis,
692-694,
693f
EFs (elongation factors), 402-403,
403f,
403¢
EGE See epidermal growth factor
Ehlers-Danlos syndrome, 43-44, 264, 592,
595, 595t, 596t eicosanoids, 16, 197, 198, 224, 225f, 662
eicosapentaenoic acid
(EPA),
199,

eIF-1A, in protein synthesis, 399, 400f elF-2, in protein synthesis, 399, 400f
elF-3, in protein synthesis, 399, 400f elF-4F complex, in protein synthesis, 399,

elFs, in protein synthesis, 399, 400f elaidic acid, 197¢, 198, 198f
elastase, 521
covalent catalysis of, 61
elastic cartilage, 607-608, 608¢
elastin, 596, 597t, 607
electric potential, 576
electrical insulators myelin
sheets as,
473
nonpolar lipids as, 196
electrogenic effect, 473
electrolytes lipid bilayer and, 462
measurement of, 564
electron microscopy
(EM), protein
electron movement, in active transport,
472
electron transport, in respiratory chain,
118, 119f, 120-121, 120f, 122f electron-transferring flavoprotein (ETF),

electrophiles, 8
carcinogens as, 684
in enzymatic catalysis, 58
electrophoresis. See also gel electrophoresis
for plasma protein analysis, 628
electrospray ionization, 29-30, 30f electrostatic interactions, 8, 340
ELISAs. See enzyme-linked immunosorbent assays
elliptocytosis, hereditary, 651, 653
elongase, 220, 221f in polyunsaturated fatty acid synthesis,
223-224, 223f

in protein synthesis, 402-403, 403f, 403t in proteoglycan synthesis, 600
in RNA synthesis, 376f, 378
elongation arrest, 580, 581f elongation chain, in fatty acid synthesis,

elongation factor 1A (EF1A), 402, 403f elongation factor 2 (EF2), 403, 403f
elongation factors (EFs), 402-403,

EM (electron microscopy), protein structures solved by, 40
emaciation, 130
emphysema, 638
EMT (epithelial-to-mesenchymal transition), 698, 699f
emulsions, amphipathic lipids forming,
204-205, 205f encephalopathy
lactic acidosis with, 118
endergonic reactions, exergonic reaction coupling to, 106-107, 106f,

endocrine system. See also hormone receptors; hormones
biomedical importance of, 480
neural regulation of, 480
endocytosis, 459, 467t
LDL in, 253
of macromolecules, 474-475, 474f,

endoglycosidases, 603
endonucleases, 348, 433. See also restriction enzymes
434-435
endopeptidases, 521
endoplasmic reticulum (ER), 134
brefeldin A and, 588
calnexin in, 551-552
cytochromes P450 in, 557
exocytosis in, 475
fatty acid chain elongation in, 220, 221f misfolded proteins degradation in,
584-585, 585f, 585t protein attachment to, 583
protein sorting signals in, 573, 574t quality control of, 584
rough. See rough endoplasmic reticulum
signal hypothesis of polyribosome binding to, 574, 574f, 580-582, 5808, 581f, 582f
endoplasmic reticulum-associated degradation (ERAD), 584-585,
585f, 585t endosymbiosis, 710
endothelial cells in hemostasis and thrombosis,

selectins of, 553-554
endothelium-derived relaxing factor, 622
energy. See also bioenergetics; free energy for active transport, 472, 472t
human requirement for, 129-130,
524-525, 524f transduction in membranes, 459
energy balance, 105, 524-525, 524f energy capture, 108
energy conservation, 108
energy expenditure, 524, 524f energy of activation. See activation energy
energy-linked transhydrogenase, mitochondrial, 125
enhancer response element, 422f enhancer-binding proteins, 421
enhancers, in gene expression, 410
B-interferon gene, 422, 423f properties
of,
420-422,
422t study of, 420-421, 422f
tissue-specific expression of, 423
enolase, in glycolysis, 159, 159f, enoyl reductase, 218f, 219f
A?-enoyl-CoA hydratase, 209, 209f, 210
entactin, 598
enterohepatic circulation, 255-256,

enterohepatic urobilinogen cycle, 313
enthalpy, 106
entropy, 106
environmental mutations, 683
enzymatic assays clinical diagnosis using, 63-65, 64t, 65f,

coupled, 63, 63f in drug development, 80
high-throughput screening, 62-63
immunoassays, 63
initial velocity measurements with,
73,
73f single-molecule, 62, 62f
spectrophotometric, 63, 63f
enzyme inhibitors, transition state

activation energy, 69-70, 70f, 72
allosteric effects, 86
biomedical importance of, 68-69
catalytic constant and catalytic efficiency, 75
catalytic lowering of activation energy barriers, 72
in drug development, 80
equilibrium constant, 69, 71-72
free-energy changes, 69-70, 70f
Hill equation, 76, 76f inhibition, 76-79, 77f, 78f
initial velocity, 73-74, 73f kinetic order, 71
Lineweaver-Burk plots, 74-75, 75f, 77,

multisubstrate reactions, 79, 79f, 80f reaction rates. See rate of reaction
transition states, 69-70, 70f enzyme-linked immunosorbent assays

active site of, 58-59, 59f activity of, 75, 564-565, 564f
bifunctional, 176
as catalysts, 9, 57, 57f classification of, 57
clinical diagnosis using, 63-65, 64t, 65f,

coenzymes of, 57-58, 58f cofactors of, 57-58, 58f
conformational changes of, 60, 60f conservation of catalytic residues in, 61

regulation of, 84-85
detection of, 62-63, 62f, 63f digestive, 521
in DNA repair, 350
as drug targets, 80
group transfer reactions of, 9
half-lives of, 270
homologous, 61
inhibitors of. See inhibitors isozymes, 61-62
kinetics of. See enzyme kinetics mechanisms
of action
of,
56
acid-base catalysis, 59-60, 60f covalent catalysis, 59-61, 59f,

proximity, 59
66
strain,
59
membranes in localization of, 459
of neutrophils, 659, 660t
nucleases, 348
prosthetic groups of, 57-58, 58f
rate-limiting, 84
reactive oxygen species and, 713
recombinant DNA in studies of,

active or passive, 83, 83f allosteric, 85-86, 85f, 135-136, 135f
of,
82-83
compartmentation role
in,
83-84
control networks of, 90, 90f covalent modifications in, 85-90,

hormones in, 86, 135-136, 135f, multiple mechanisms of, 85
proenzymes in, 87-88, 88f quantities, 84-85
rate-limiting enzymes as targets of,
84
second messengers in, 86
regulatory, 135
replacement therapy for, 235,
RNA, 343
specificity of, 57, 57f synthesis of, regulation of, 84
enzyme-substrate (ES) complex, stability

enzymopathy, 650
eosinophils, 656, 659, 661, 661f
EPA (eicosapentaenoic acid), 199,
222f,
224
epidermal growth factor (EGF)
in insulin signal generation, 508, 509f receptor for, 482, 702
epidermis, 708t epidermolysis bullosa, 595t, 596, 625
epidermolytic palmoplantar keratoderma,

epigenetic code of DNA, 384
epigenetic mechanisms, control of gene transcription and, 419-420,

epigenetic signals, 419, 420f, 421f epigenetics,
82
in cancer development, 690
histone code as, 87
epimerases, 600
epimers, 143, 144f epinephrine. See also catecholamines
blood glucose regulation by, 180
glycogen regulation by, 167-168, 169f in glycolysis and gluconeogenesis
in lipogenesis regulation, 221, 222f synthesis of, 484f, 491f, 492
tyrosine conversion to, 300, 302f episomes, 436
epithelial-to-mesenchymal transition
(EMT), 698, 699f epitopes, 36
exocytosis, 459, 467t, 474-475, 475f,

exocytotic (secretory) pathway, 574
exoglycosidases, 603
exons, 356, 357f description of, 386
processing of, 387f, 393-394
splicing of, 386-388, 387f, 388f exonucleases, 348, 433
in recombinant DNA technology, 434t exopeptidases, 521
exosomes, 476-478, 477f cancer and, 690
exportins, 578
expression vector, 438
extra arm, of tRNA, 346f, 347
extracellular environment, membranes in

extracellular fluid (ECF), 460, 460t extracellular matrix (ECM). See also bone
aging process and, 592
biomedical importance of, 592
collagen. See collagen elastin, 596, 597t
fibrillins, 597, 597f fibronectin, 595, 597-598, 598f
glycosaminoglycans. See glycosaminoglycans
laminin, 598-599, 599f proteoglycans. See proteoglycans
extracellular vesicles (EVs), 476-478,

extramitochondrial system, fatty acid synthesis in, 216
extravasation, of cancer cells, 698, 699f extrinsic pathway, 672-673, 672f, 673f,

extrinsic tenase complex, 673
ezetimibe, for hypercholesterolemia, 257
F
FAB (fast atom bombardment), 29-30, 30f
Fabry disease, 235t facilitated diffusion, 467-468, 467f, 467t
for glucose.
See glucose
transporters hormone regulation of, 469-470
ping-pong mechanism
of,
469,
469f transporters involved in, 468-469, 468f,

facilitated transport, of bilirubin, 312
FACITs (fibril-associated collagens with interrupted triple helices), 594
factor I. See fibrinogen factor II. See prothrombin
factor III. See tissue factor factor IX, 673t, 674, 674t
coumarin drugs and, 677-678
deficiency of, 678
factor V, 673t, 674t, 675, 675f.
factor V Leiden, 677
factor VII, 673, 673t, 674t, 677-678
factor VIII, 673t, 674, 674t deficiency of, 678

extrinsic pathway activation of,
672-673, 672f, 673f intrinsic pathway activation of,
672-674, 672f, 673f prothrombin activation by,

actor XI, 673t, 674, 674t factor XII, 673t, 674, 674¢
factor XIII, 673t, 674t, 677
facultative heterochromatin, 354
FAD. See flavin adenine dinucleotide
FADH,, fatty acid oxidation generation of,

'alse-positive result, 562
amilial amyloidosis, 639
familial hypercholesterolemia (FH), 241,
257, 258t, 478, 478t, 585t familial hypertrophic cardiomyopathy, 622
Farber disease, 235t arnesoid X receptor (FXR), 256, 513t
farnesyl diphosphate, in cholesterol/
polyisoprenoid synthesis,
250-251, 251f fast atom bombardment (FAB),

'ast twitch fibers, 621
asting state, metabolic fuels in, 130,
138-139, 138¢, 139f, 139t fatal infantile mitochondrial myopathy
and renal dysfunction, 126
fatigue, glycolysis defects causing, 157
fats, 138t, 196. See also lipids fatty acid chains, elongation of, 220, 221f
atty acid elongase system, 220, 221f,

fatty acid oxidase, 209, 209f fatty acid synthase complex, 217-218,

fatty acid synthesis biomedical importance of, 216
citric acid cycle role in, 153-155, 155f in cytosol, 216-220, 217f, 218f
extramitochondrial, 216
fructose effects on, 186, 188f, 189-190
fatty acid synthetase, 75
fatty acid transport protein, membrane,

activation of, 208, 208f anti-inflammatory, 199
eicosanoids formed from, 224, 225f essential. See essential fatty acids
free. See free fatty acids for
health maintenance,
long-chain w3, 196, 199
in membranes,
461,
461f as metabolic fuel, 130, 136
130f, 131f
3
in fasting state, 138, 138¢
in fed state, 137-138, 137f, 138t at tissue and organ levels,
132-133, 133f monounsaturated.
See monounsaturated fatty acids
nomenclature of, 196, 196f oxidation of. See also ketogenesis
acetyl-CoA release and, 208-210,

biomedical importance of, 207
clinical aspects of, 214-215
hypoglycemia caused by impairment of, 214-215
impairment of, 214-215
physical/physiologic properties of,
198-199
polyunsaturated. See polyunsaturated fatty acids
saturated, 196, 196f, 197, 197t. See also saturated fatty acids
trans, 198, 226
transport of, carnitine in, 208, 208f triacylglycerols as storage form of,
199, 199f unsaturated.
See unsaturated fatty acids

alcoholism and, 244
nonalcoholic fatty liver disease, 243
nonalcoholic steatohepatitis, 243
of pregnancy, 215
triacylglycerol metabolism imbalance

avism, 189
ed state, metabolic fuels in, 130, 136-138,
137f, 138t feedback inhibition, of enzymes, 85-86,

feedback regulation, 86, 89, 136
of thrombin, 677
Fenton reaction, 633, 633f ermentation, by cell-free extract of yeast,

in heme, 53-54, 95-96
from heme catabolism, 311
ferritin, 405, 523, 523f, 632-633, 632t,

errochelatase, 307, 308f, 309f, 310t erroportin, 632, 635
ferrous iron, in heme, 48-49, 48f, 49f,

fertilization, glycoprotein role in, 553
Fe-S clusters, 97-98, 98f
Fe-S (iron-sulfur) proteins, in respiratory chain complexes, 118-120, 119f
etal hemoglobin (HbF), 50-51,

a-fetoprotein, 630
FFAs. See free fatty acids
FGER3 (fibroblast growth factor receptor 3), 609
FH (familial hypercholesterolemia), 241,
257, 258t, 478, 478t, 585t fibril-associated collagens with interrupted
triple helices (FACITs), 594
fibrillins, 597, 597f fibrils, 593-594, 594f, 607
fibrin, 670
fibrin clot formation amplification in, 677
extrinsic pathway, 672-673, 672f, 673f,
673t, 674t fibrinogen conversion to fibrin,
675-677, 675f, 676f inherited deficiencies of, 678
intrinsic pathway, 672-674, 672f, 673f,
673t, 674t plasmin dissolution of,
678-679, 678f prothrombin conversion to thrombin,

fibrin deposits, 670
fibrin monomer, 677
fibrinogen (factor I), 627, 628, 629f, 672,

conversion to fibrin, 675-677, 675f, 676f in platelet aggregation, 670, 671f
fibrinolysis, 678-679, 678f intrinsic pathway in, 674
fibrinopeptide A (FPA), 676-677, 676f fibrinopeptide B (FPB), 676-677, 676f
fibroblast growth factor receptor 3

fibroblasts, pinocytosis by, 475
fibroelastic cartilage, 607-608, 608¢
fibronectin, 595, 597-598, 598f fibrous proteins, 34
collagen as, 43
finasteride, 703
first law of thermodynamics, 105-106
first-order rate constant, 71
FISH (fluorescence in situ hybridization),
44)
fish-eye disease,
258t flame photometry, laboratory tests using,
564
flavin adenine dinucleotide (FAD), 324t in citric acid cycle, 152f, 153
in cytochromes P450, 115, 115f in dehydrogenases, 114
in oxidases, 112, 113f in respiratory chain complexes, 118
vitamin B, in synthesis of, 534
flavin mononucleotide (FMN)
vitamin
B, in
534

cytochromes P450, 115, 115f dehydrogenases, 114
oxidases, 112, 113f in respiratory chain complexes, 118
FLIP, in apoptosis, 693f, 694
flip-flop, phospholipid, membrane asymmetry and, 464
flippases, 464
fluid mosaic model, 465-466, 466f fluidity, membrane, 465-466
fluid-phase pinocytosis, 474-475, 474f fluorescence, of porphyrins, 308, 310f
fluorescence in situ hybridization (FISH),
441
fluorescence spectrophotometry, laboratory tests using, 563-564
fluoride, enolase inhibition by,
159, 159f
1-fluoro-2,4-dinitrobenzene, 27
fluoroacetate, 151, 152f
5-fluorouracil, 80, 324, 324f, 334, 336t fluvastatin, 257
flux, metabolite, 83, 83f, 89,
135-136, 135f flux-generating reactions, of metabolic
pathways, 135
FMN. See flavin mononucleotide folate trap, 536f, 538
folding protein, 41-42, 42f
of proteins, calnexin role in, 551-552

coenzymes derived from, 58
deficiency of, 282, 526t, 538
functions of, 528t, 537, 537f inhibition of, 537
structure of, 537, 537f supplements of, 538
toxicity of, 538
forensic medicine, 445
four-helix bundle, 587
Fourier synthesis, 40
FPA (fibrinopeptide A), 676-677, 676f
FPB (fibrinopeptide B), 676-677, 676f frameshift mutations, 397-398, 398f
ATP as cellular currency of, 108-110,

of ATP hydrolysis, 107-108, 107¢, 108f chemical reaction changes in, 69-70,
70f, 105-106
enzymatic effects on, 72
high-energy phosphate capture and transfer of, 107-108, 107t, 108f,
121-122
metabolic regulation and, 83-84, 83f obtainment of, 107
redox potential and, 111-112, 112t as useful energy in biologic systems,
105-106
free fatty acids (FFAs), 196, 207-208
adipose tissue
and,
244
glucose metabolism and, 245
insulin and, 236, 245
ketogenesis regulation and, 213-214,
213f,
214f
free fatty acids (FFAs) (Cont.):
lipid mobilization and, 236
lipogenesis affected by, 220, 221f in lipoproteins, 237, 237t
metabolism of, 238
in VLDL formation, 243, 243f free polyribosomes, protein synthesis on,

free radical theory of aging, 710, 712
free radicals antioxidant paradox and, 544-545, 544t
of,
541
damage caused
by,
541-542,
542f lipid peroxidation producing,
203-204, 204f mechanisms protecting against,
543-544, 544f self-perpetuating chain reactions of, 541
sources of, 542-543, 543f theory of aging, 710, 712
fructokinase, 186, 188f, 189
deficiency of, 190

absorption of, 520, 520f defective metabolism of, 190
in diabetic cataract, 190-191
in glycolysis pathway, 161
ingestion of large quantities of, 186,
188f, 189-190
physiological importance of, 144t structure of, 143, 143f
fructose 1,6-bisphosphatase deficiency of, 190
in gluconeogenesis, 173f, 174
in pentose phosphate pathway, 183f,

fructose 1,6-bisphosphate in gluconeogenesis, 173f, 174
in glycolysis, 159, 159f fructose 2,6-bisphosphatase, 176, 177f
fructose 2,6-bisphosphate, in glycolysis and gluconeogenesis regulation,
176,
177f fructose-2,6-bisphosphatase, covalent
catalysis of, 61, 62f fructose-6-phosphate
in glycolysis, 159, 159f in pentose phosphate pathway, 183f,

fucose, 147, 148f, 548t fucosyl (Fuc) transferase, 653
fumarase (fumarate hydratase), in citric acid cycle, 152f, 153
fumarate in citric acid cycle, 152f, 153
fumarylacetoacetate hydrolase, 286, 287f functional groups
of amino acids, 18-20, 19f, 20f, 20¢
weak acids acting as, 11-12
furanose ring structures, 143, 143f furin, 588, 588f
fusion of vesicles, 588
fusion proteins, 65-66, 66f futile cycles
in glycolysis and gluconeogenesis regulation, 176-177
of lipids, 525
FXR (farnesoid X receptor), 256, 513
G
AG. See Gibbs free-energy change
AG". See standard free-energy change
G1 and G2 phase, 368, 368f, 369f
G6PD. See glucose-6-phosphate dehydrogenase
GA. See Golgi apparatus
GABA (y-aminobutyrate), biosynthesis of,
303,
303f
GAGs. See glycosaminoglycans
Gal transferase, 654
GALI enhancer, 427, 428f galactokinase, 187, 189f
galactosamine, 145, 145f synthesis of, 188, 190f
galactose, 141, 143, 144f absorption of, 520, 520f
in glycoproteins,
547,
548t impaired metabolism of, 191
metabolism of, 187-188, 189f physiological importance of, 144t
galactose-1-phosphate uridyl transferase,

galactosemias, 191
galactoside, 145
galactosyl receptor, 475
galactosylceramide, 202, 202f, 234, 234f,

gallium, toxicity of, 94
gallstones, 250, 519
hyperbilirubinemia caused by, 313f,
313t,
314
GalNAc (N-acetylgalactosamine), 547,
548t, 549
GalNAc transferase, 654
y-rays, carcinogenic effect of, 683, 683t gangliosides,
202
amino sugars in, 145, 145f in membranes, 461
synthesis of, 234, 234f gangliosides.,
147
gap 1 and 2 phase, 368, 368f, 369f gap junctions, 459, 476, 476f
GAPs (GTPase-accelerating proteins), 578
gastroenteropathy, protein-losing, 630
gated ion channels, 471, 471f gatekeeper tumor suppressor genes,

Gaucher disease, 235t, 585t
GDF15 (growth differentiation factor 15),
635
GDH. See glutamate dehydrogenase
GDP, 587
gefitinib, 702
GEFs (guanine nucleotide exchange factors), 578, 578f
gel electrophoresis polyacrylamide gel, 26, 26f

geldanamycin, 590
geleophysic dysplasia, 597
gel-filtration chromatography. See size- exclusion chromatography
gemfibrozil, 257
gene amplification, in cancer, 685
gene arrays, 31
gene conversion, 361
gene expression constitutive, 411, 414
miRNA and siRNA inhibition of, 348
in pyrimidine nucleotide synthesis regulation, 334
retinoic acid role in, 529-530
silencing of, 87
gene expression regulation alternative RNA processing and, 430
amplification, 429, 429f biomedical importance of, 409-410
DNA-binding domains of regulatory transcription
factor proteins,
425t helix-turn-helix motif, 425-426, 426f
leucine zipper motif, 427, 427f zinc finger motif, 426, 426f
domain separation in, 427-428, 428f

bacteriophage lambda as paradigm for protein-DNA interactions,
414-418, 414f, 415f, 416f, 417f chromatin
template in,
418-419
DNA enhancer elements and,
420-423, 422f, 422t, 423f epigenetic mechanisms, 419-420,
420f, 421f locus control regions and insulators
in, 424-425
prokaryotes compared with, 428-430,

reporter genes, 423, 424f response diversity in, 424, 425f
special features of, 418
models of,
411
mRNA stability and, 430, 430f ncRNA alteration of mRNA function in,
428-429
operon model of, 411-412, 411f,

positive and negative, 410, 410t prokaryotic
eukaryotes compared with,
428-430, 4281, 429f, 430f on-off manner of, 424
uniqueness of, 411
RNA alternative processing for, 430
targeted, 446-447
temporal responses to, 410-411, 410f

artificial membranes and, 465
biomedical importance of, 432
for lipid storage disorders, 235
mammalian viral vectors for, 437-438
for metabolic disorders, 278
general acid-base catalysis, 59
general transcription factors (GTFs),

activators and coregulators and,
384-385
in PIC formation, 385-386
in RNA polymerase II formation,

alteration of, 359-361, 359f, 360f biomedical importance of, 338
cancer, 682, 685-688, 685t, 686f, 687f,

chromosome localization of, 441, 442t of cytochromes P450, 557
disease causing variations in, 442,

housekeeping, 411
immunoglobulin, DNA rearrangement and, 360-361
inducible, 411
knockout/knockin, 446
mapping of, 355, 441, 442t processed, 360
targeted disruption of, 446-447
genetic code, 338. See also DNA
L-a-amino acids specified by, 15,

biomedical importance of, 393
features of, 394-395, 395t triplet codes in, 394, 394t

of bone, 607, 607t gene therapy for, 446
of membrane protein mutations,

of muscles, 621-623, 622t, 623f, 624f recombinant
DNA
technology in
diagnosis of, 442-445, 443f,
444f,
444t genetic disorders
amino acid metabolism defects, 276-278,

cancer, 683, 688, 690-691, 691t clotting system deficiencies, 678
collagen defects in, 43-44
enzymes for diagnosis of, 64-65
fructose metabolism deficiencies, 190
glycogen storage diseases, 164, 166,

glycoprotein lysosomal hydrolase deficiencies, 554-555
hemoglobin mutations, 53-54, 54f hemolytic anemia, 157, 163, 182,
188-189
porphyrias, 305, 308, 310-311, 310¢, 311f pyruvate dehydrogenase deficiency, 163
respiratory chain, 126
urea cycle defects, 276-278, 277t genetic engineering, 432. See also
recombinant DNA technology genetic information, flow of, 393-394
genetic mutations. See mutations, genetic
Genevan system, for fatty acid nomenclature, 196

function of, 356-357
microsatellite repeat sequences in, 358
nonrepetitive sequences in, 357
repetitive sequences in, 357-358
targeted gene regulation in, 446-447
genomic instability,
in cancer,

genomic library, 438
genomic technology. See recombinant
DNA technology genomics, protein identification using, 28
geometric isomerism, of unsaturated fatty acids, 198, 198f
geranyl diphosphate, in cholesterol synthesis, 250, 251f
GGT. See y-glutamyltransferase
GH. See growth hormone
Gibbs free-energy change (AG),

of ATP hydrolysis, 107-108, 107t, 108f in biologic systems, 105-106
enzyme effects on, 72
equilibrium constant and, 69, 106
metabolic regulation and, 83-84, 83f redox potential and, 111-112, 112t
Gilbert syndrome, 314
GK (glucokinase) gene, 388-389, 389f
GIcN (glucosamine), 602
glibenclamide, 215
a-globin gene
defective production of, 442-443, 443f location of, 442t
B-globin gene,
421-422,
422f defective production of, 442, 443f
location of, 442t schematic representation of, cluster,
442, 443f structural alterations of, 442, 444t
globular proteins, 34
globulins, 628
glomerular filtration, 598
glomerular membrane, 598
glomerulonephritis, 598-599
glucagon,
130,
138
blood glucose regulation by, 179, 179t in cholesterol synthesis,
252-253, 253f glycogen regulation by, 167
in glycolysis and gluconeogenesis regulation, 175
in lipogenesis regulation, 221-222, 222f glucagon/insulin ratio, in ketogenesis
regulation, 214

metabolic pathways of, 130, 130f, 131f in
fasting state,
138-139,
138f,
139

as most important monosaccharide, 142
in pentose phosphate pathway.
See pentose phosphate pathway permeability coefficient of, 463f
structure of, 142, 142f synthesis of. See gluconeogenesis
transport of, 473-474, 473f glucose6-phosphate, in uronic acid
pathway, 186-187, 187f glucose oxidase, plasma glucose
concentration measurement

glucose tolerance, 180-181, 180f glucose transporters (GLUT), 178, 178t
in blood glucose regulation, 245, 245f in
erythrocytes,
648,
648¢
insulin and, 470, 473, 473f glucose uniporter (GLUT2), 473-474, 473f

gluconeogenesis and, 173f, 174
glycogen release of, 164, 166, 166f in glycogenesis, 164, 165f

deficiency of, 335
in glycogenolysis, 166

in glycogenesis, 164, 165f in glycolysis, 158, 159f
in pentose phosphate pathway,
182-185,
183f,
184f glucose-6-phosphate dehydrogenase

183-184, 183f, 184f glucose-alanine cycle, 177, 178f
glucoside, 145
glucosuria, 180
glucosylceramide, 202, 234, 234f, 461
glucuronate, 143, 145f bilirubin conjugation with,
312, 312f uronic acid pathway production of,
186-187, 187f glucuronic acid, uronic acid pathway
formation of, 182
-glucuronidases, 313
glucuronidation, of xenobiotics, 558
glucuronide conjugates, glucuronate production for, 186-187, 187f
glucuronides, 182
GLUT. See glucose transporters
GLUT2 (glucose uniporter), 473-474, 473f
GLUT4 transporter, 245

carbon skeleton catabolism of, 282
carboxylation of, 532-533, 533f in citric acid cycle, 153, 154f
synthesis of, 264-265, 265f transamination reactions forming,
272-273, 273f glutamate aminotransferase,
transamination reaction of,
273, 273f glutamate dehydrogenase (GDH)
in amino acid synthesis, 265, 265f in nitrogen metabolism, 273, 274f
glutamate-y-semialdehyde dehydrogenase,

glutamic acid,
16t glutaminase, 274, 274f
in amino acid catabolism, 282
glutamine, 16¢
ammonia fixation as, 274, 274f carbon skeleton catabolism of, 282
271
f,
272f in citric acid cycle, 153, 154f
deamination of, 274, 274f synthesis of, 265, 265f
glutamine analogs, purine nucleotide synthesis
affected by,
328,
330
glutamine synthetase, 265, 265f ammonia fixation by, 274, 274f
y-glutamyltransferase (GGT), 558
diagnostic use of, 64t y-glutamyl-B-aminopropionitrile (BAPN),

glutaric acid, pK, of, 13t glutathione, 21, 21f, 558, 713, 721, 721t
204,
544
hemolysis protection from, 185-186,

selenium in, 114, 185-186, 186f glutathione reductase, 185, 186f, 189,

glutathione S-transferase (GST)
fusion protein purification with,

in xenobiotic metabolism, 558
glyburide, 215
N-glycan chains, 581
glycans, 546, 547t, 550-551, 550f in virus, bacteria, and parasite binding,
555
glycated hemoglobin (HbA, .), 55, 553

in diabetes mellitus, 55, 553, 553f glycemic index, 145, 520
glyceraldehyde-3-phosphate in glycolysis, 159, 159f, 160f
182-185, 183f, 184f glyceraldehyde-3-phosphate
dehydrogenase, in glycolysis,
159,
159f,
160f lycerol, 196, 199 a
in gluconeogenesis, 174
permeability coefficient of, 463f glycerol ether phospholipids, synthesis of,
230, 232, 232f ycerol kinase, 229, 230, 231f, 244 98
lycerol moiety, 130 9a lycerol phosphate acyltransferase, ga
mitochondrial compartmentalization of, 118
ycerol phosphate pathway, 231f 99, go
ycerol-3-phosphate acylglycerol biosynthesis and, 230, 231f
triacylglycerol esterification and,
244-245, 245f lycerol-3-phosphate acyltransferase, a
230, 231f lycerol-3-phosphate dehydrogenase, ga
230, 231f lycerophosphate shuttle, 125, 125f 90
ycerophospholipids, 196, 199, 201f 90, lycerose, 142, 143f 99
lycine, 15¢ go carbon skeleton catabolism of,
282-283, 284f heme biosynthesis from, 305-307, 306f,
307f, 308f, 309f, 310t specialized products of, 298, 298f,
300-301, 302f synthesis of, 265, 266f
threonine conversion to, 285, 285f glycine cleavage complex, 282-283, 284f
glycine residues, 593
glycinuria, 283
glycobiology, 141
glycocalyx, 147, 202, 229
glycochenodeoxycholic acid, 256f glycocholic acid, 256f
glycoconjugate carbohydrates, glycoproteins as, 546

biomedical importance of, 164
breakdown of, 157, 158f, 158t clinical aspects of, 170
glycogenesis reaction pathway for, 132,
164-166, 165f, 166f glycogenolysis reaction pathway for,
165f, 166, 166f, 167t liver synthesis of, 136-137
metabolic pathways of, 130,
132-133,
132f in muscle contraction, 620, 621f
regulation of, 166-170, 168f, 169f, 170f,
171f storage function of, 145-146, 147f, 164,

glycogen phosphorylase, 620
in glycogenolysis, 166, 166f regulation of, 89t, 166-168, 169f,
170, 171f
lycogen primer, 164-165 ga lycogen storage diseases, 164, 166, ga

lycogen synthase go gluconeogenesis and, 173f, 174
in glycogenesis, 165, 165f, 166f regulation of, 89t, 166-168, 170,

lycogen synthase a, 168 99
lycogen synthase b, 168 ga lycogenesis, 132 go.
reaction pathway of, 164-166, 165f, 166f regulation of, 166-170, 168f, 169f,
glycogenin, in glycogenesis, 164-165, 165f

177-178, 178f induction and repression of enzymes
catalyzing,
174-175,
175t reaction pathway of, 165f, 166,
166f, 167t regulation of, 166-170, 168f,

glycolipid storage diseases, 229
glycolipids, 196, 202, 202f galactose production for, 187-188, 189f

in anaerobic conditions, 157, 158f, 158t biomedical importance of, 157
in cancer cells, 695-696, 697f, 698t clinical aspects of, 163
dihydroxyacetone phosphate in, 230,

in energy conservation and capture, 108
in erythrocytes,
161,
161f,
648,
648t flux-generating reaction in, 135
Gibbs free-energy change of, 84
gluconeogenesis regulation and,
174-177, 175t, 176f, 177f induction and repression of enzymes
catalyzing, 174-175, 175t pentose phosphate pathway connections
with, 183f, 185
pyruvate oxidation after,
161-163, 162f reactions of, 157-160, 159f, 160f
regulation of, 160-161
at subcellular level, 134, 134f thermodynamic barriers to reversal of,

glycome, 141
glycomics, 3, 141, 447
glycophorins, 147, 549, 652, 652t glycophosphatidylinositol, 463
glycoprotein complex GPIIb-IIIa, in platelet aggregation, 670, 671f
glycoprotein glycosyltransferases,
549-550

amino sugars
in,
145,
145f,
188
biologic information encoded in, 546
biomedical importance of, 546
classes of, 547, 548f, 549
degradation of, 270
in diabetes mellitus, 553, 553f disorders involving, 554-555
extracellular, absorptive pinocytosis of,

functions of, 546, 547t, 553-554
galactose production for,
187-188, 189f
N-glycosidic linkages in, 547, 548f,
550-551, 550f, 551f
O-glycosidic linkages in, 547, 548f,
549-550
as hormone precursors, 484, 484f membrane asymmetry and, 464
microfibril-associated, 597
monosaccharides commonly found in,

oligosaccharide chains in, 546, 547t,
550-551, 550f, 555
on plasma membrane, 552
protein folding role of, 551-552
purification of, 547
rapidly reversible, 552-553
regulation of, 552
synthesis of, 549-551, 551f in virus, bacteria, and parasite binding,

in,
145,
145f,
188
components of, 599, 599f, 600f functions of, 603
mucopolysaccharidoses and,
603-604, 604t properties of, 602t
structures of, 601-603, 601f synthesis of, 599-600
1,6-glycosidase, in glycogenolysis,

glycosides, 145
N-glycosides nucleosides as, 320, 321f
syn and anti conformers of, 320-321,

N-glycosidic linkages, in glycoproteins,
547, 548f, 550-551, 550f, 551f
O-glycosidic linkages, 593, 599-600
in glycoproteins, 547, 548f, 549-550
glycosphingolipids (GSLs), 196, 202, 202f,

amino sugars in, 188
membrane asymmetry and, 464, 590
synthesis of, 234, 234f
N-glycosylamine bond,
600
glycosylation, 546. See also glycoproteins congenital
disorders of,
633
cotranslational, 581
in diabetes mellitus, 553
of hydroxylysines, 594
mass spectrometry detection of, 28t rapidly reversible, 552-553
glycosylphosphatidylinositol (GPI), 588
GTEs. See general transcription factors
GTP, 323, 587, 588
GTPase-accelerating proteins (GAPs), 578
GTPases, 578-579, 587
GTP-binding proteins, 251

ROS oxidation of, 711f salvage pathways of, 332-333
guanine nucleotide exchange factors

guanine-binding protein.
See
G-proteins guanosine,
321f,
322t salvage pathways of, 332-333
in uric acid formation, 334, 335f guanosine diphosphate. See GDP
guanosine monophosphate. See GMP
guanosine triphosphate. See GTP
Guillain-Barré syndrome, 473
t-gulonolactone oxidase, 186
Guthrie bacterial inhibition test, 566

H blood group substances, 653-654, 654f
H1
histones,
351,
351f,
352f,
353,
353t
H2A histones, 351, 351f, 353
H2AX histone, 371
H2B histones, 351, 351f, 353
H3 histones, 351, 351f, 353, 353t
H4 histones, 351, 351f, 353, 353t hairpin,
342,
343f half-life
(¢,,,)
of plasma proteins, 630
of proteins, 270
halt-transfer signal, 582, 583f haptocorrin, 101
haptoglobin, 631-632, 631f haptoglobin-related protein, 632
Hartnup disease, 288, 289f, 535,
HAT (histone acetyltransferase activity),

Haworth projection, 142, 142f
Hb Hikari, 397f
HbA, (glycated hemoglobin), 55, 553
HDF
(fetal hemoglobin),
50-51,
51f,
53
HbM (hemoglobin M), 53-54, 397f
HbS
(hemoglobin
S),
54,
54f,
397f
HBV (hepatitis B virus), 703
HDACs (histone deacetylases), 89, 690
HDL. See high-density lipoproteins
HDL cycle, 241f, 242
HDL receptor, 238, 241f, 242
health, 2-4, 2f, 3f

in fed state, 130, 136-138, 137f, 138t heart failure, 611
cardiolipin in, 201
heartbeat hypothesis, 715-716
proton binding of, 52-53, 53f quaternary structure of, 50-53, 50f, 51f,

relaxed and taut states of, 49-50
salt bridges in, 52-53
secondary and tertiary structures of, 50
subunits of, 50, 50f tetrameric structure of, 50, 50f
turnover of, 311
hemoglobin (Hb) Hikari, 397
hemoglobin M (HbM), 53-54, 397f hemoglobin S (HbS), 54, 54f, 397f
hemoglobinopathies, 53-54, 54f, 651
hemoglobinopathy, 359, 360f a-hemoglobin-stabilizing protein (AHSP),

hemojuvelin (HJV), 635
a-hemolysin, 472
hemolysins, 651
hemolysis pentose phosphate pathway and
glutathione peroxidase protection against, 185-186, 186f
pentose phosphate pathway impairment leading to, 188-189
hemolytic anemias, 157, 163, 182
causes of,
651,
651f hyperbilirubinemia with, 313, 315, 315t
pentose phosphate pathway impairment in, 188-189
hemolytic-uremic syndrome, 655
hemopexin, 631, 631f hemophilia, 627
hemophilia A, 678
hemophilia B, 678
hemoproteins biomedical importance of, 305
examples of, 305, 306t hemosiderin, 633
hemosiderosis, 633, 636

biomedical importance of, 669
coagulation. See coagulation system fibrinolysis. See fibrinolysis
phases of, 669
hemostatic plugs, formation of, 670, 671f
Henderson-Hasselbalch equation, 12, 12f heparan sulfate, 595, 599, 601f,

in hemostasis and thrombosis, 672
heparin, 147, 148f, 597, 599, 602t, 677-678
lipoprotein and hepatic lipases and, 240
structure of, 601f, 602, 602f heparin cofactor II, 677
hepatic lipase in chylomicron remnant uptake,
240-241, 241f deficiency of, 258t
metabolism by, 240
hepatic porphyrias, 310#, 311
hepatic portal vein, 177
hepatic purine biosynthesis
AMP and GMP formation regulation in, 331, 331f
PRPP glutamyl amidotransferase regulation in, 330

hyperbilirubinemia caused by, 315, 315t hepatitis B virus (HBV), 703
hepatocarcinoma, 243

pinocytosis by, 475
hepatolenticular degeneration (Wilson disease), 298, 478t, 627, 634
hepcidin, 523-524, 523f, 635, 636f, 637f hepcidin system, 94
hephaestin, 632-633
heptoses, 141, 142t hereditary bleeding disorders, 678
hereditary cancers, 683, 688,

hereditary elliptocytosis, 651, 653
hereditary fructose intolerance, 190
hereditary hemochromatosis, 636,

hereditary mutations, 683
hereditary nonpolyposis colon cancer,
370t hereditary spherocytosis, 478t, 6501,

Hermansky-Pudlak syndrome, 585t herpes simplex virus, 380f
heterochromatin, 354
heterodimers, 38
heterogeneous regulatory RNAs (sRNAs),

heterotrophic organisms, 107
hexapeptide, in albumin synthesis, 630
hexokinase, 57, 726
blood glucose concentration and,

coupled enzyme assay for, 63f as flux-generating step in glycolysis, 135
in fructose metabolism, 186, 188f in glycogenesis, 164
in glycolysis, 158, 159f regulation of, 161
reversal of reaction catalyzed by,

hexosamines, 145, 145f synthesis of, 188, 190f
hexose monophosphate shunt. See pentose phosphate pathway
hexose-6-phosphate dehydrogenase,

hexoses,
141,
142t of physiological importance, 143, 144t
HFS (high-fructose syrups), 186, 188f,
189-190
HGP (Human Genome Project), 3-4, 3f
Hhal, 433t
HHH
(hyperornithinemia, hyperammonemia, and
homocitrullinuria) syndrome, 277
HIF-1 (hypoxia-inducible factor-1),
696-697
high altitude, physiologic adaptations to,
53
high-density lipoproteins (HDL),
237, 237t apolipoproteins of, 238
atherosclerosis and,
242,
257
cholesterol removal by, 249
familial deficiency of, 258t
LDL ratio to, 257
metabolism of, 241-242, 241f receptor for, 238, 241f, 242
synthesis of, 241, 241f high-density microarray technology, 447
high-energy phosphates creatine phosphate shuttle for,

cycles of, 109, 109f energy capture and transfer by,
107-108, 107, 108f, 121-122
oxidative phosphorylation in synthesis of, 118, 121-122, 122f
high-fructose syrups (HFS), 186, 188f,

high-mannose oligosaccharides, in glycoproteins, 550-551, 550f
high-pressure liquid chromatography

high-throughput sequencing (HTS),
62-63, 445-446
Hill coefficient (1), 76, 76f
Hill equation, 76, 76f
Hindlll, 433t hippuric acid, glycine conversion to,

histamine, 298, 298f, 657f, 662
histidase, 282
histidine, 16t, 657f carbon skeleton catabolism of,
282, 283f fusion protein purification with, 65
in heme, 48, 48f, 54
resonance hybrids of, 18-19, 19f specialized products of, 298, 298f
histidinemia, 282
histone acetyltransferase activity (HAT),

histone chaperones, 353
histone code, 87, 418-419
histone covalent modification,

histone deacetylases (HDACs), 89, 690
histone dimer,
351,
351f,
353
histone epigenetic code, 419
histone octamer, 351, 351f, 352f, 353
histone tetramer, 351, 351f, 353
histones, 351, 351f, 353, 353t covalent modifications of, 87
modifications of, 384
HIV. See human immunodeficiency virus
HIV protease, acid-base catalysis of,

HJV (hemojuvelin), 635
HMG-CoA. See
3-hydroxy-3-methylglutaryl-CoA
homeostasis, 82
in
ER,
584
hormone signal transduction and,
500-501,
501f intracellular iron, 634-635, 635f,

homeostatic adaptations, 500
L-homoarginine, 18, 18¢
homocarnosine, biosynthesis of, 298,

homocarnosinosis, 303
homocysteine, 283, 284f homocystinuria, 283-285, 300
homodimers, 38
homogentisate dioxygenase, 115
homogentisate oxidase, 286, 287f homologous recombination (HR), 369,

homologs, protein, 61
homology, protein classification based on,
34
homology modeling, protein structures solved by, 41
homopolymer tailing, 434t hormone receptor-G-protein effector
system, 503f hormone receptors
classification of, 482, 483t as proteins, 482
recognition and coupling domains of,
481-482
specificity and selectivity of, 481, 481f hormone response elements (HRE)
DNA
sequences of,
502t gene expression and, 423, 424f
in signal generation, 501, 502f types of, 511
hormone response transcription unit,

active form and, 484
adrenal steroidogenesis,
485-487, androgen synthesis, 486f, 487
angiotensin II, 495-496, 496f calcitriol, 489-491
catecholamines, 491-492, 491f cellular arrangements of, 483
from cholesterol, 202, 249, 483, 484f,

dihydrotestosterone, 483, 487, 489f glucocorticoids, 486-487, 486f
insulin, 493-494, 494f iodide metabolism and, 492
mineralocorticoids, 485-486, 486f
485f
hormone synthesis (Cont.):
ovarian steroidogenesis, 487-489,

parathyroid, 494, 495f peptide precursors for, 492-493
POMC family, 496-497, 497f testicular steroidogenesis, 487, 488f
tetraiodothyronine, 492, 493f triiodothyronine,
492,
493f hormone-dependent cancer, vitamin B,

active form of, 484
adenylyl cyclase and, 503t of adipose tissue, 244
amino acid precursors of, 18
biomedical importance of, 500
in blood glucose regulation, 178-180,
178t, 179t
cell surface receptor binding of,
482,
483t chemical diversity of, 483-484, 484f
definition, 480
enzyme regulation by, 86, 135-136, 135f facilitated diffusion regulation by,

features of, 483¢
242-243,
243f intracellular receptor binding of,

laboratory tests for, 565
lipogenesis regulation by, 247
lipolysis regulation by, 245-247, 246f lipophilic, 482, 483t
peptide, 546
plasma transport proteins of,

precursor molecule for, 483-484, 484f receptor interaction of, 500-501, 501f
as second messengers. See second

signal transduction by, 500-501, 501f, stimulus response of, 500-501, 501f
storage and secretion of, 497, 497t transcription modulation by,
511-514,
512f,
513f,
513t, vitamin D as, 530-531, 531f,
water-soluble, 482, 483t housekeeping genes, 411

HPLC. See high-pressure liquid chromatography
HPV (human papillomavirus), 703
HR (homologous recombination), 369,

HRE. See hormone response elements hsp (heat-shock proteins), as chaperones,
41,576
Hsp60, 575
Hsp70, 576
514t
HTS (high-throughput sequencing),
human genes, localization of, 441, 442t
Human Genome Project (HGP),
3-4,
3f human immunodeficiency virus (HIV)
undernutrition caused by, 525
human papillomavirus (HPV), 703
human B-interferon gene enhancer,
422, 423f humoral immunity, 662
Hunter syndromes, 603, 604t
Hurler syndromes, 603, 604t
Hutchinson-Gilford progeria syndrome,

hyaline cartilage, 607, 608t hyaluronic acid, 147, 148f, 599, 601, 601f

hyaluronidase, 603-604
hybrid oligosaccharides, in glycoproteins,
550-551, 550f hybridization, 341
hybridomas,
643
hydrogen bonds in collagen, 43
definition of, 7
in DNA, 339-340, 340f hydrophobic interactions and, 8
water molecule formation of, 7, 7f in a helices, 35, 35f
in B sheets, 36, 36f hydrogen ions
reaction rate response to, 72-73, 73f in water, 9-13, 12f, 13t
hydrogen peroxide, 710
amino acid oxidase generation of, 273
glutathione peroxidase removal of, 114,
185-186, 186f reduction of, 114
hydrogen sulfide, respiratory chain inhibition
by,
112,
123
hydrogenation, dehydrogenation coupling

of ATP, 107-108, 107#, 108f of bound GTP to GDP, 587
as group transfer reaction, 9
of triacylglycerols, 229
of water, 9
hydronium ions, 9-10
hydropathy plot, 463
hydroperoxidases, 112, 114
hydroperoxides, 225, 227f hydrophilic portion of lipid molecule,

hydrophobic domains, 37
hydrophobic effect, in lipid bilayer self- assembly, 462
hydrophobic interaction chromatography, protein and peptide purification
with,
25
hydrophobic interactions, 8, 339
hydrophobic portion of lipid molecule,
hydrostatic pressure,
628
hydroxide ions, 9-10

in cholesterol synthesis, 250, 251f in ketogenesis, 211, 212f
in mevalonate synthesis, 250, 251f

in ketogenesis, 211, 212f
reductase, regulation of, 90
(HMG-CoA) reductase cholesterol synthesis controlled by, 250,

regulation of, 86, 89t
(HMG-CoA) reductase kinase, regulation of, 89t
(HMG-CoA) synthase in cholesterol synthesis, 250, 251f
L-3-hydroxyacyl-CoA dehydrogenase,
209, 209f
3-hydroxyanthranilate dioxygenase,
115
hydroxyapatite, 604-605
D-3-hydroxybutyrate, 207, 210, 211f
D-3-hydroxybutyrate dehydrogenase,
210, 211f
4-hydroxybutyric aciduria, 303
6-hydroxybutyric aciduria, 327,

7a-hydroxylase, sterol, 255, 256f
17a-hydroxylase, 486, 488f
18-hydroxylase, 485-486
27-hydroxylase, sterol, 255, 256f hydroxylase cycle, 115, 116f
hydroxylases,
115
in cortisol synthesis, 486-487
vitamin C as coenzyme for, 539
hydroxylation mass spectrometry detection of, 28t
of proline, 594
of xenobiotics,
556-558
hydroxylysine, synthesis of, 266, 268f
5-hydroxylysine, 17, 17f hydroxylysines, 594
hydroxymethylbilane, in heme synthesis,
306, 307f, 308f, 309f hydroxymethylbilane synthase, 306, 307f,

deficiency in, 310, 310t
5-hydroxymethylcytosine, 321, 323f hydroxyproline,
593,
596
carbon skeleton catabolism of,
285-286, 285f synthesis of, 266, 268f
4-hydroxyproline, 17, 17f
4-hydroxyproline dehydrogenase,

19-hydroxysteroid, 485
6-hydroxy-y-trimethylammonium butyrate.
hyperalphalipoproteinemia, familial, 258t hyperammonemia, 150, 156
urea cycle disorders causing,
276-278, 277t hyperbilirubinemia, 305, 313-314, 313t
metabolic disorders underlying,

hyperbilirubinemic toxic encephalopathy,
313-314
hypercholesterolemia, 226, 237, 241, 257,

fructose role in, 186, 188f, 189-190
hyperchromicity of denaturation, 340
140,
172
hyperglycinemia, nonketotic, 283
hyperhomocysteinemia, folic acid supplements for, 538
hyperhydroxyprolinemia, 285f, 286
hyperkalemic periodic paralysis, 620t hyperketonemia, 215
hyperlacticacidemia, 244
hyperlipidemia, 257
hyperlipoproteinemias, 236, 257, 258t hyperlysinemia, 286
hypermetabolism, disease causing, 157, 525
hypermethioninemia, 298-299
hyperornithinemia, hyperammonemia, and homocitrullinuria (HHH)
syndrome, 277
hyperornithinemia-hyperammonemia syndrome,
282
hyperoxaluria, 283
hyperphenylalaninemias, 286, 288f hyperprolinemia, 282, 282f, 285f, 286
hypersensitive sites, chromatin, 354
hypersplenism, 651
hypertension, folic acid supplements for,

hyperthermia, malignant, 618-619, 620t hypertriacylglycerolemia, 236, 258t
hypertrophic cardiomyopathy, familial,

hyperuricemia, 334
fructose role in, 189-190
hypoceruloplasmenia, 633
hypoglycemia, 172, 181
fatty acid oxidation and, 207, 214-215
fructose-induced, 190
during pregnancy and in neonates, 180
hypoglycin, 207, 215
hypokalemic periodic paralysis, 620t hypolipidemic drugs, 257
hypolipoproteinemia, 236, 257, 258t hypothyroidism, cardiolipin in, 201
hypouricemia, 335
hypoxanthine,
321,
323f salvage pathways of, 332-333
hypoxanthine-guanine phosphoribosyl transferase, 334-335, 336t
gene location of, 442t hypoxia, in cancer cells, 696-697
hypoxia-inducible factor-1 (HIF-1),
696-697
hypoxic conditions, glycolysis in, 157,

I
I bands, 612, 612f, 613f
IAPs (inhibitors of apoptosis), 693f, 694
ibuprofen, 216, 226
IC,
78
T-cell disease, 478, 478t, 554, 585t
ICF (intracellular fluid), 460, 460t icterus, 313-314, 313t
idiotypes,
642
IDL (intermediate-density lipoproteins),

iduronate, 143, 145f
IEF (isoelectric focusing), protein and peptide purification with, 26, 27f
IgA, 6408, 642f
IgD, 640t
IgE, 640t
IGF-1. See insulin-like growth factor
IgG, 6408, 641f
IgM, 640t, 642f
IL-1 (interleukin-1), 631
IL-6 (interleukin-6), 635
imatinib, 701-702, 702t immune response
cancer escape from, 695, 695f glycoproteins in, 553-554

adaptive, 639, 643, 662
dysfunctions of, 644-645
innate, 639, 643
immune thrombocytopenic purpura,

immunoassays enzyme-linked, 63, 565
laboratory tests using, 565
immunocompromised state,

immunogenicity, 643
immunoglobulin genes amplification and, 429
DNA rearrangement and, 360-361
1
immunoglobulin light chains, 361,

immunoglobulins, 627, 630t binding specificity of, 640, 643f
class switching and, 642
class-specific effector functions of,

composition of, 639, 6401, 641f, 642f diversity of, 641
immunotherapy, for cancer, 698-700
IMP. See inosine monophosphate importins, 577-578, 578f
in situ hybridization, 441
inactive chromatin, 354, 418
inborn errors of metabolism of amino acid metabolism, 276-278,

early studies on, 2
gene or protein modification for, 278
general features of, 276-277
mucopolysaccharidoses,
603-604, 604t neonate screening for, 565-566
tandem mass spectrometry in screening for, 278, 280
urea cycle defects, 276-278, 277t inclusion cell (I-cell) disease, 478, 478t,

indels, 359, 398
indirect bilirubin, 313
indirect carcinogens, 559
indispensable amino acids. See essential amino
acids indole, permeability coefficient of,
463f indomethacin, cyclooxygenases affected

induced fit model, 60, 60f induced pluripotent stem cells (iPSCs),
446
inducers,
84
gratuitous, 412
in regulation of gene expression, 411
inducible gene, 411
induction, of enzyme synthesis, 84
infant respiratory distress syndrome

infantile Refsum disease, 580, 580t infectious
disease enzymes for diagnosis of, 64-65
glycoprotein role in, 555
kwashiorkor after, 525
protein loss in, 526
inflammasome, 700
inflammation advanced glycation end-products in,
553
cancer development and, 700
glycoprotein role in, 553-554
plasma proteins in, 630-631, 630t prostaglandins in, 216
inflammatory response, 511, 656
influenza virus A, glycans in binding of,

information pathway, 502, 502f inhibitor-1, 168, 170
inhibitors of angiogenesis, 697-698
competitive, 76-78, 77f, 78f of DNA methyltransferases, 690
drugs acting as, 80
feedback, 85-86, 85f, 89
of folic acid metabolism, 537
of histone deacetylases, 690
irreversible,
78
kinetic analysis of, 76-79, 77f, 78f mechanism-based, 76, 78-79
noncompetitive, 77-78, 77f, 78f of platelet activation, 671f, 672
of pyruvate dehydrogenase,
161-163, 162f of respiratory chain, 112, 118,

of thrombin, 677-678
tightly bound, 78
of tyrosine kinases, 701-702, 702t inhibitors of apoptosis (IAPs), 693f, 694
initial velocity
(v,),
73-76,
73f,
75f,
76f,

in DNA synthesis, 363-364, 365f, 366f in protein synthesis, 399-402, 400f
in RNA synthesis, 375-378, 376f, 377f
43S initiation complex, in protein synthesis, 399, 400f
48S initiation complex, in protein synthesis, 399, 400f, 401
80S initiation complex, in protein synthesis, 399, 400f, 401
initiation complexes, in protein synthesis,

initiator methionyl-tRNA, 399, 400f initiator sequence (Inr), 380
initiators, of apoptosis, 692-694, 693f innate immune system, 639, 643
innate immunity, glycoproteins in, 554
inner membrane, of mitochondria, 118,

inner mitochondrial membrane, 576
inorganic elements, human requirement

inorganic pyrophosphatase, 109, 109f inorganic pyrophosphate (PP,), 109, 109f
inosine monophosphate
(IMP)
conversion to AMP and GMP, 328, 330f,

synthesis of, 328-330, 329f, 330f inositol
229
inositol trisphosphate, 200
1,4,5-inositol trisphosphate
in chemotaxis, 658
(IP,)
in platelet aggregation, 670, 671f
Inr (initiator sequence), 380
insertions of
397-398,
398f inside-out transmembrane signaling, in
platelet aggregation, 670, 671f inside-outside asymmetry, membrane, 464
Insig (insulin-induced gene), 252
insulators in gene expression, 424-425

biological assay to measure, 719, 720t blood
glucose regulation
by,
179,
179t in
cholesterol synthesis,
252-253,
253f in diabetes mellitus, 130, 140
free fatty acids affected by, 236, 245
gene location of, 442t glucose regulation by, 136-138, 158
in glucose transport, 469-470
glycogen regulation by, 168, 170
kinase cascade signal transmission by,

in lipogenesis regulation, 221-222, 222f in lipolysis regulation, 222, 245-246,
245f, 246f protein synthesis initiation by,
402,
402f radioimmunoassay to measure,

receptor for, 482
secretion by rabbit pancreas, 726t sequencing of, 26-27
storage of, 497, 497t synthesis of, 493-494, 494f
VLDL secretion and, 243
insulin receptor substrates (IRS),

insulin resistance, glycoprotein role in, 553
insulin/glucagon ratio, in ketogenesis regulation, 214
insulin-induced gene (Insig), 252
insulin-like growth factor 1 (IGF-1), 482
in insulin signal generation, 508, 509f integral membrane proteins, 34
integral proteins, 464-465, 464f integration, chromosomal, 359-360, 360f
integrins, 554, 658-659, 659t intercellular communication, 480. See also
endocrine system intercostal skeletal muscle cells, turnover

B-interferon gene, 422, 423f interferons, 662
interleukin-1 (IL-1), 631
interleukin-6 (IL-6), 635
interleukins, 662, 700
intermediate filaments, 625, 625t intermediate-density lipoproteins (IDL),
237t, 241, 254, 257
intermembrane mitochondrial space, proteins in, 576
of water, 9-10
ionizing radiation, DNA damage caused by, 370f, 370t
ionophores, 471-472
mitochondrial, 125
ion-specific electrodes, 564
IP,. See 1,4,5-inositol trisphosphate iproniazid, 300
iPSCs (induced pluripotent stem cells), 446
IPTG
(isopropylthiogalactoside),
412
IRDS (infant respiratory distress syndrome), 234
IREGI (iron-regulated protein 1), 632
IRES (internal ribosomal entry site),
405-406, 406f iron
absorption of, 101, 523-524, 523f conservation of, 632-634,

in cytochrome oxidase, 97
in cytochromes P450, 115, 115f deficiency of, 636, 637t
heavy metal displacement of, 94
in heme, 48-49, 48f, 49f, 53-54, 95-97
human requirement for, 93, 93t intracellular homeostasis, 634-635,

macrophage recycling of, 631, 631f multivalent states of, 93, 94f, 94t
overload, 633, 636, 638t oxidation of heme, 649-651, 650f,

physiologic roles of, 97-98, 97f, 98f regulation of, 94
in sulfite oxidase, 100, 100f toxicity of, 95t
transferrin cycle, 633, 634f iron deficiency anemia, 636, 637t, 650t
iron porphyrins, 305. See also heme iron response elements, 634-635
iron toxicity, 475
iron-regulated protein 1 (IREG1), 632
iron-sulfur (Fe-S) proteins, in respiratory chain complexes, 118-120, 119f
irreversible inhibitors, 78
IRS (insulin receptor substrates), 508, 509f ischemia, 157, 478
isoaspartyl linkage in polypeptide backbone, 715f
isoaspartyl methyltransferase, 715f isocitrate, in citric acid cycle,
151-152, 152f isocitrate dehydrogenase
in citric acid cycle, 152, 152f in NADPH production, 218, 219f, 220f
regulation of, 156
isoelectric focusing (IEF), protein and peptide
purification with,
26,
27f isoelectric pH (pl), of amino acids, 19
isoleucine, 15¢
carbon skeleton catabolism of, 288, 290,
292f, 293, 293f, 293t synthesis of, 266-267
isomaltose, 146f, 146t isomerases, 57
isomerism geometric, of unsaturated fatty acids,
198, 198f of monosaccharides, 142-143, 143f, 144f
isomerization, of folding proteins,
41-42, 42f isomorphous displacement, 40
isoniazid, 559
isopentenyl diphosphate, in cholesterol synthesis, 250, 251f
isopentenyl diphosphate isomerase, 251f isoprene
units, polyprenoids
synthesized from, 203, 204f
isoprenoid units, 250, 251f, 252f isopropylthiogalactoside (IPTG), 412
isoprostanes, 204
isosteric effectors, 86
isothermic systems, 105
isotypes, 642

isovaleric acidemia, 290, 293
isovaleryl-CoA dehydrogenase, deficiency of, 290, 292f, 293
isozymes, 61-62
J
Jak/STAT pathway, 508-509, 510f
Jamaican vomiting sickness, 215
jaundice hyperbilirubinemia causing, 305,
313-314, 313t laboratory tests for, 566
314-315, 314f, 315t obstructive, 242
"jumping DNA,' 360
junctional diversity, 641
K
k, See rate constant
K'. See potassium
K* channel (KvAP), 471, 471f
Kartagener syndrome, 625
karyopherins, 578
karyotype, 356f k_,. See catalytic constant
k_,/K,,. See catalytic efficiency
K,,. See dissociation constant kDEL-containing proteins, 574f, 583
Ky See equilibrium constant keratan sulfate I, 599, 601f, 602, 602t
keratan sulfate II, 599, 601f, 602, 602t keratins, 625, 625t
kernicterus, 313-314
a-ketoacid decarboxylase complex, deficiency of, 290, 292f, 293, 293t
ketoacidosis, 140, 207, 215
3-ketoacyl enzyme, 217, 219f
3-ketoacyl reductase, 219f
3-ketoacyl synthase, 217, 219f
3-ketoacyl-CoA thiolase deficiency, 215
ketoamines, in diabetes mellitus, 553, 553f ketogenesis, 133
high rates of fatty acid oxidation and,
210-213, 211f, 212f 213f
HMG-CoA in, 211, 212f regulation of, 213-214, 213f, 214f
ketogenic amino acids, 136
a-ketoglutarate arginine and ornithine formation of,

in citric acid cycle, 152-153, 152f glutamine and glutamate formation of,
282
histidine formation of, 282, 283f in transamination reactions,
272-273, 273f a-ketoglutarate dehydrogenase complex
in citric acid cycle, 152-153, 152f regulation of, 156
ketone bodies, 207, 210, 211f brain use of, 136
free fatty acids as precursors of,
213, 213f as fuel for extrahepatic tissues, 211,
213, 213f metabolic pathways of, 131, 131f,
133, 136
in fasting state, 138-139, 138t ketonemia, 213, 215
ketonuria, 215, 290, 293, 293t ketoses, 141-142, 142t, 143, 144f
ketosis, 139-140, 207, 211, 213, 215, 244
kidney basement membrane of, 603
blood glucose threshold of, 180
calcitriol synthesis in, 490-491, 490f vitamin D metabolism in,

kidney function tests, 566
kinesin, 624
kinetic order, 71
kinetic theory, 70
kinetics, enzyme. See enzyme kinetics kinetochore, 355
K,,. See Michaelis constant
Korsakoff psychosis, 534
Kozak consensus sequences, 401
Krabbe disease, 235t
Krebs cycle. See citric acid cycle
Ku70/80, 370, 371f
KvAP (voltage-gated K* channel),

kwashiorkor, 263-264, 524-525
kynureninase,
288,
289f kynurenine formylase, 288, 289f
kynurenine-anthranilate pathway,
286-288, 289f, 290f
L

biomedical importance of, 560
for cancer, 700-701, 701t causes of abnormal results of, 560
clinical value of, 562-563, 563t
laboratory tests (Cont.):
enzymes used in, 63-65, 64t, 65f,

for hemostasis, 679
of organ function, 566-567
reference range of, 561
samples for, 563
techniques used in, 563-566, 564f, validity of, 561-563, 561f, 562f, 563¢
lac operon, 411-412, 411f, 413f, 414
lac repressor, 411-412, 413f lacA gene, 411-412, 411f, 413f
lacI gene, 411-412, 411f, 413f, 414
B-lactamase, 80, 98, 99f

in citric acid cycle, 154, 154f glucose derived from, 177, 178f
glycolysis production of, 157, 158f,
158t, 160
lactate dehydrogenase diagnostic use of, 64, 64t, 65f
pyruvate reduction by, 160
structure of, 37, 38f lactation, glucose requirement of, 136
lactic acid, pK, of, 13t lactic acid cycle, glucose derived from,

metabolism of, operon hypothesis and,
411-412,
411f,
413f,
414
physiological importance of, 145,

lactose intolerance, 519-520
lactose synthase, 188, 189f lactulose, 147¢
lacY gene, 411-412, 411f, 413f lacZ gene, 411-412, 411f, 413f
lagging (retrograde) strand, in DNA
replication, 362f, 363, 366f lambda repressor (cI) gene, 415, 415f
lambda repressor (cl) protein,

laminin, 598-599, 599f lamins, 355, 625, 625t
lanosterol, in cholesterol synthesis, 250,

lathyrism, 14, 18, 18¢
lauric acid, 197t
LBD (ligand-binding domain),
511-512, 512f
LCAT.
See lecithin:cholesterol acyltransferase
LCRs (locus control regions), 424-425
LDL. See low-density lipoproteins
LDL receptor, 238
in chylomicron remnant uptake,
240f, 241
in cotranslational insertion, 582-583,

LDL-receptor-related protein-1 (LRP-1),
238, 239f, 241
lead, toxicity of, 93-94, 306, 309
leading (forward) strand, in DNA
replication, 362f, 363
lecithin:cholesterol acyltransferase
(LCAT), 254
apolipoprotein role in, 238
familial deficiency of, 258t in HDL metabolism, 241f, 242
in phosphoglycerol degradation and remodeling, 232-233
lecithins, 200, 200f lectin pathway, 643, 644f
lectins, 147
glycoprotein purification with, 547
mannose 6-phosphate receptor proteins,
554
Lepore, 359, 360f leptin, 244
Lesch-Nyhan syndrome, 327,
334-335, 336t leucine, 15t

synthesis of, 266-267
leucine zipper motif, 427, 427f leucovorin, 537
leukemias, 655, 656, 701
leukocyte adhesion deficiency II, 554
leukocytes communication through effectors, 662
motility of, 657-659, 657f, 658f, 659t multiple types of, 656-657, 657f
turnover of, 708t leukodystrophy, metachromatic, 235¢
leukopenia, 656
leukotriene A,, 198f leukotriene B,, 227
leukotrienes (LTs), 197, 198, 198f,
216, 662
clinical significance of, 224, 227
lipoxygenase pathway in formation of,

Lewis acids, transition metals as, 93
library, 438
life expectancy, 708, 708t

body mass versus, 715-716, 716t evolution and, 717
versus longevity, 707-708
lifestyle changes, cholesterol levels affected

ligand-binding assays, laboratory tests using, 565
ligand-binding domain (LBD),
511-512, 512f ligand-gated channels, 470
ligandin, 312, 558
ligand-receptor complex, 501-502, 501f,
501t, 502f ligases, 57
DNA, 363t, 364
ubiquitin, 584, 585f light, energy source in active transport,
472
light chains, immunoglobulin, 361,
639, 642
LINEs (long interspersed nuclear elements), 357-358
Lineweaver-Burk plot, 74-75, 75f for Bi-Bi reactions, 79, 80f
inhibitor evaluation with, 77, 77f, 78f
O-linked N-acetylglucosamine transferase,

linoleic acid/linoleate, 196f, 197t in essential fatty acid deficiency, 224
nutritionally essential, 222, 222f oxidation of, 211f
synthesis of, 222-223, 223f a-linolenic acid (ALA), 197¢, 199
for essential fatty acid deficiency, 224
nutritionally essential, 222, 222f synthesis of, 223-224, 223f
y-linolenic acid,
197t,
223

diagnostic use of, 64t insulin and, 245
triacylglycerol hydrolysis of, 521
in triacylglycerol metabolism, 229,

lipid bilayer, 462-463, 462
lipid core, of lipoprotein, 237-238, 238f lipid droplets, 244, 247
lipid rafts, 200, 466, 466f, 588, 589
lipid storage disorders (lipidoses),

lipidomics, 3, 447
lipidoses (lipid storage disorders),
234-235, 235t lipids. See also fatty acids; glycolipids;
phospholipids; steroids;
triacylglycerols amphipathic, 204-205, 205f
biomedical importance of, 196
classification of, 196
complex, 196
derived, 196
digestion and absorption of,
520-521, 522f disorders associated with abnormalities

free radical damage to, 541-542, 542f futile cycling of, 525
in membranes. See also membrane

amphipathic, 461-462, 461f artificial, 465
asymmetry and, 464, 589, 589f protein association with, 463
protein ratio to, 460-461, 460f

in fed state, 137-138, 137f, 138t at tissue and organ levels, 133, 133f
metabolism of, in liver, 242-243, 243f neutral, 196
peroxidation of, 203-204, 204f,
710, 711f precursor, 196
simple, 196
solubility of, 196
transport and storage of adipose tissue and, 244-245, 245f
biomedical importance of, 236-237
brown adipose
tissue and,
247,
247f clinical aspects of, 243-244
fatty acid deficiency and, 225
lipogenesis and, 247
lipolysis and, 246, 246f as lipoproteins, 237-238, 237t, 238f
liver in,
242-243,
243f perilipin and, 247
lipid-soluble molecules, 462
lipid-soluble vitamins, 527, 528t lipins, 230, 231f
lipoamide, 161, 162f lipogenesis,
132,
133
for,
218-219, adipose tissue and, 247
ethanol and, 244
fatty acid synthase complex in, 217-218,

main pathway for, 216-217
malonyl-CoA production in, 217, 217f
NADPH for, 218, 220f regulation of
enzymes in, 217-218, 221, 221f, 222f nutritional state in, 220
shortand long-term mechanisms in,
220-222, 221f, 222f

hormone regulation of, 245-247, 246f insulin inhibition of, 222
lipase in, 244-245, 245f of triacylglycerol, 229
lipophilic hormones, 482, 483t lipoprotein lipase, 133
familial deficiency of, 258t hydrolysis by, 240, 240f
lipoprotein(a) excess, familial, 258t lipoproteins, 34, 133, 133f, 628, 630t
apolipoproteins in, 237t, 238
carbohydrates in, 147
in cholesterol transport, 253-254, 255f classification
of,
237,
237t deficiency of, fatty liver and, 244
disorders of, 257, 258t formation of, 236
remnant, 237t, 240f, 241
219f,
220f structure of, 237-238, 238f
a-lipoproteins. See also high-density lipoproteins
£-lipoproteins. See low-density lipoproteins
liposomes amphipathic lipids forming,
204-205, 205f artificial membranes and, 465
lipotropic factor, 244
lipoxin A,, 198f lipoxins (LXs), 197, 198, 198f, 216,
224, 225
clinical significance of, 227

lipoxygenase, 225, 227f reactive species produced by, 204
5-lipoxygenase, 225, 227, 227f lipoxygenase pathway, 198, 224, 225,

liquid chromatography laboratory tests using, 564

lithocholic acid, synthesis of, 255, 256f liver
amino acid levels and, 271-272,
271f,
272f bilirubin metabolism of, 311-313,

blood glucose regulation by, 178, 178t calcitriol synthesis in, 490, 490f
carbohydrate metabolism enzyme induction and repression in,
174-175, 175t cirrhosis of, 243, 244
citric acid cycle in, 150
failure of, 243
139f,
139¢
fatty. See fatty liver in fed state, 130, 136-138, 137f, 138t
fructose
2,6-bisphosphate in,
176,
177f fructose effects in, 186, 188f, 189-190
glucose uptake
by,
136-137,
137f glycogen in, 164, 165¢
glycogen phosphorylase regulation in,
167
glycogenesis in, 164-166, 165f, 166f glycogenolysis in, 166, 168
heme synthesis in, 306-308
metabolic role of, 132-133, 132f metabolism in
chylomicrons and VLDL, 240
fatty acid oxidation and ketogenesis,
210-213,
211f,
212f, 213f of lipids, 242
plasma protein synthesis in, 628-629
554-555
lysosomal proteases, in protein degradation, 584-585
lysosomal proteins, 590

in endocytosis, 474
faulty targeting of enzymes into, 554
glycogen hydrolysis by, 166
protein sorting signals in, 573, 574t

structure of, 37f lysyl hydroxylases, 43, 266, 593, 596, 596t
lysyl oxidase, 43, 594, 596
copper in, 100
lytic pathway, 414f, 415
lytic/lysogenic genetic switching event,
415, 415f

M phase, 368, 368f, 369f mAbs (monoclonal antibodies),
642-643, 697-698, 702, 702t
MAC
(membrane attack
complex),
643
a,-macroglobulin,
638,
638f,
662,
677
macromolecules cell nucleus transport of, 575f,
576-579, 577t, 578f cellular transport of, 474-475,

mitochondria import of, 576, 577f, 577t macrophages, 656, 657, 660, 662
iron recycling by, 631, 631f magnesium
(Mg”*)

porphyrins containing, 305
Maillard reaction, 553, 553f major groove, in DNA, 340f, 341
helix-turn-helix motif and,

operon model and, 412
major histocompatibility complex (MHC),
584, 659
major sorting decision, 574
malaria, glycan role in, 555

in citric acid cycle, 152f, 153
in lipogenesis, 218, 220f malate dehydrogenase, in citric acid cycle,

malate shuttle, 125-126, 125f
MALDI (matrix-assisted laser desorption and ionization), 29-30, 30f
malic enzyme, in NADPH production,

malignancy. See cancer malignant hyperthermia (MH),
618-619, 620
malonate, 77, 123
malonyl acetyl transacylase, 217, 217f,

CPT-I regulation by, 213-214, 214f in fatty acid synthesis, 217, 217f
maltose, physiological importance of, 145,

mammalian coregulator proteins, 514t mammalian DNA-dependent RNA
polymerases, 378, 378t mammalian viral cloning vectors, 437-438
manganese human requirement for, 93, 93t
multivalent states of, 93, 94f, 94t physiologic roles of, 98
toxicity of, 95t mannan-binding protein (MBP), 643
mannosamine, 145, 188, 190f mannose, 143, 144f, 144t, 548¢
mannose 6-phosphate, 554
mannose-6-phosphate, 475, 574t mannose-binding lectin (MBL),

mannose-binding protein, 554
mannosidosis, 554-555
manual enzymatic Sanger method,

MAPK (mitogen-activated protein kinase)
pathway, 508, 509f maple syrup urine disease (MSUD), 290,

marasmus, 105, 263-264, 524-525
Marfan syndrome, 597, 597f
Maroteaux-Lamy syndrome, 604
mass spectrometry (MS)
metabolic disease detection with, 278
protein analysis using, 28-30, 28¢,

proteomics with, 447
spectrometer configurations used for,
28-30, 29f, 30f tandem, 30, 278, 280
volatilization methods for, 29-30, 30f mast cells, 602, 656, 662
MAT (methionine adenosyltransferase),

matrix, mitochondrial, 118, 118f, 151
matrix metalloproteinases (MMPs), 698
matrix protease, 576
matrix proteins, 576, 579
matrix-assisted laser desorption and ionization
(MALDI),
29-30, maximal velocity (V,,,.), 73-76, 73f,

allosteric effects on, 86
in Bi-Bi reactions, 79, 80f inhibitor effects on, 77-78, 77f, 78f
MBL (mannose-binding lectin),

MBP (mannan-binding protein), 643
MDR
(multidrug resistance),
478
MDR-1 protein (multidrug-resistance-1
protein), 472
mechanically gated channels, 470
mechanism-based inhibitors, 76, 78-79
mediator (Med) proteins, 384, 385f medicine
biochemical research impacts on, 2-3
30f biochemistry importance to, 1
biochemistry interrelationship with,
2,
2f
Human Genome Project impact on,

personalized, 432
medium-chain acyl-CoA dehydrogenase, deficiency of, 215
megaloblastic anemias, 538, 650t
MELAS (mitochondrial encephalopathy, lactic
acidosis, and
stroke),
126
melatonin, tryptophan conversion to,

melting temperature. See transition temperature
membrane attack complex (MAC), 643
membrane fatty
acid transport
protein,

membrane fusion, 586
membrane lipids bilayers of, 462-463, 462f
cholesterol in, 461
glycosphingolipids in, 461
phospholipids in, 199-201, 200f,
461, 461f sterols in, 461
turnover of, 589
membrane proteins differences in, 463, 463t
integral, 34, 464-465, 464f lipid bilayer and, 463
mutations of, diseases caused by,

peripheral, 464-465, 464f structure of, dynamic, 463-464
membrane-bound polyribosomes, 580

assembly of, 574, 588-590, 589f, 590t asymmetry
of,
460,
464,
589,
589f biomedical importance of, 459
depolarization of, in nerve impulse

fluidity affecting, 465-466
gap junctions, 459, 476, 476f
Golgi apparatus in synthesis of, 574
intracellular, 460
mitochondrial, 118, 118f 119f,
576, 577f selective permeability of, 124-126,

phase changes of, 465-466
of red blood cells, 651-653, 651f, 652f,

selectivity of, 467t. See also transporters and transport systems
facilitated diffusion, 469-470, 469f ion channels, 470-471, 470f,

mechanisms of, 467-468
passive diffusion, 467-468, 467f,
467t, 468f, 468t transporters, 468-469, 468f, 469f
signal transmission across, 459, 467t,

asymmetry and, 464
fluid mosaic model of, 465-466, 466f lipids
in,
204-205,
205f,
461-463, 461f, 462f protein:lipid ratio in, 460-461, 460f
menadione,
528t,
532-533,
533f menaquinones,
528t,
532-533,
533f
Menkes syndrome, 43, 263, 596
MEOS (microsomal ethanol oxidizing

6-mercaptopurine,
324f,
325,
329f,
330
3-mercaptopyruvate pathway, 283, 284f mercury, toxicity of, 93-94
meromyosin, 614
merosin, 598, 623f messenger RNA (mRNA), 343, 374, 375¢
alternative splicing and, 388, 388f amplification of, 429, 429f
chemical characteristics of, 344-345,
345f codon assignments in, 393-395, 394t
editing of, 391
344,
344f gene expression modulation and,
428-429
in genetic information flow,

modification of, 389-390
nontranslating, 405, 405f nucleotide sequence of, 394, 394t
mutations caused by changes in,
397-398, 398f polycistronic, 411
precursors, 345-346, 356
processing of, 386-388, 387f in protein synthesis
elongation, 402-403, 402f initiation, 399, 400f, 401-402, 401f
termination, 403-404
relationship to chromosomal DNA,

transcription starting point and, 376
translocation of, 578
MET (metabolic equivalent), 524
metabolic acidosis, ammonia role in, 275
metabolic alkalosis, ammonia effects on,

metabolic disorders of amino acid metabolism, 276-278,
277t, 280-281, 293, 294t of bilirubin metabolism, 314-315, 314f,

neonate screening for, 565-566
tandem mass spectrometry in screening for,
278,
280
urea cycle defects, 276-278, 277t metabolic equivalent (MET), 524
metabolic fuels in fasting state, 130, 138-139, 138¢,

in fed state, 130, 136-138, 137f, 138t human
intake of,
129-130
interconvertible nature of, 136
processing of, 130-131, 130f,
131f,
132f metabolic rate, 105, 524
metabolic regulation active or passive, 83, 83f
allosteric, 85-86, 85f, 135-136, 135f biomedical
82-83
in,
83-84

of enzyme quantity, 84-85
flux-generating reactions in, 135
hormones in, 86, 135-136, 135f multiple mechanisms of, 85
nonequilibrium reactions as control points in, 135
proenzymes in, 87-88, 88f rate-limiting enzymes as targets of, 84
metabolic syndrome, 180
metabolic theories of aging,

metabolism, 106
amino acid roles in, 18
biomedical importance of,
129-130
in cancer cells, 695-696, 697f, 698t citric acid cycle as pivotal pathway in,
153-156, 154f, 155f clinical aspects of, 139-140
CNS and erythrocyte glucose requirements and, 136
drug. See drug metabolism energy requirements for, 129-130

in fed state, 130, 136-138, 137f, 138t free energy changes of. See bioenergetics
fructose effects on, 186,

group transfer reactions in, 9
inborn errors of, 2
interconvertible fuels in, 136
normal, 129
nucleophiles and electrophiles in, 8
pathways for processing products of digestion, 130-131, 130f,
131f,
132f regulation of. See metabolic regulation
regulation of metabolite flux through,
135-136, 135f subcellular level of, 132, 134, 134f
tissue and organ level of, 132-133,

metabolites, flux of, 83, 83f, 89,
135-136, 135f metabolomics, 3, 447, 564
metabonomics, 564
metachromatic leukodystrophy, 235t metal ions. See also transition metals
in enzyme prosthetic groups, 58
heavy metal displacement of, 93-94
metal-activated enzymes, 58
metalloenzymes, 58
metalloproteins, 34
physiologic roles of, 97-101, 97f, 98f,

metaphase chromosomes, 355, 355t, 356f metastasis
mechanisms of,
698,
699f,
700t membrane abnormalities and, 478t
tumor microenvironment role in,

methane monooxygenase, iron in, 97
methemoglobin, 53-54, 649
methemoglobin reductase, 54
methemoglobinemia, 649, 650t methionine,
16f in S-adenosylmethionine, 323f, 324t
carbon skeleton catabolism of, 288,
290f, 291f specialized products of, 298-299, 299f,
300f,
301,
302f methionine adenosyltransferase (MAT),
298, 299f methionine synthase, vitamin B,,
dependency of, 536, 536f, 538
methionyl-tRNA (met-tRNA), 399, 400f methotrexate, 333-334, 537
L-B-methylaminoalanine, 18, 18¢
methylation of deoxycytidine residues, 418-419
enzyme regulation
by,
86-87
6-methylcrotonyl-CoA, catabolism of,

321,
323f methylene bridges, in porphyrinogens,
306-307
N-methylglycine, 300-301
7-methylguanine, 323f
7-methylguanosine cap structure, mRNA,

3-methylhistidine, 298
methylmalonic aciduria, 174, 174f methylmalonyl-CoA mutase, 174,
174f, 536
mineralocorticoids, synthesis of,
485-486, 486f minerals
biomedical importance of, 527
523-524, 523f functions of, 540, 540f
nutritional requirements for, 527-528
minor groove, in DNA, 340f, 341
MIPP (multiple inositol polyphosphate phosphatase), 53
miRNAs. See micro-RNAs misfolded proteins, endoplasmic
reticulum degradation of,
584-586, 585f, 585t mismatch repair (MMR), 369, 370f, 370t
missense mutations, 396-397, 397f,

MIT (monoiodotyrosine), 492, 493f mitochondria
apoptosis in, 712
B-oxidation in, 208-210, 208f,

citric acid cycle in, 134, 134f, 151
enzyme activity of, 725t exchange transporters of, 124-126, 124f,

fatty acid oxidation in, 207-208, 208f genes encoded by genome of, 712t
genetic disorders involving, 126
membranes and compartments of,

protein sorting signals in, 573, 574t protein synthesis and import by, 576,

redox damage of, 710, 712
respiratory chain complexes in,
118, 119f mitochondrial branched-chain a-ketoacid
dehydrogenase complex, 288,
290,
292
mitochondrial DNA, 358-359, 358f, 359t mitochondrial encephalopathy, lactic
acidosis, and stroke (MELAS), 126
mitochondrial genome,
576
mitochondrial glycerol-3-phosphate dehydrogenase, 114
mitochondrial matrix, 576, 577f mitochondrial pathway, of apoptosis,
692-694, 693f mitogen-activated protein kinase (MAPK)
pathway, 508, 509f mitotic phase, 368, 368f, 369f
mitotic spindle, 624
mixed-function oxidases, 115
MMPs (matrix metalloproteinases),

MMR (mismatch repair), 369, 370f, 370t
MOAT (multispecific organic anion transporter), 312
modeling, of protein structures, 40-41
molecular biology,
protein and
peptide sequencing
in,
28
molecular chaperones. See chaperones molecular diagnostic tests, 4
molecular docking programs, protein structures solved by, 40-41
molecular dynamics, protein structures solved by, 40
molecular genetics. See recombinant DNA
technology molecular modeling, of protein structures,

molecular pathology, 590
molecular repair mechanisms, wear and tear theory of aging
aggregated proteins, 715
enzymatic and chemical mechanisms,
713
proofreading and repair mechanisms,

protein damage, 714-715, 715f molecular replacement, 40
molecular switches,
586
molybdenum absorption of, 101
human requirement for, 93, 93f multivalent
states of,
93,
94f,
94t physiologic roles of, 100, 100f
toxicity of, 95t molybdopterin, 95, 96f, 100
monoacylglycerol acyltransferase,
230,
231f monoacylglycerol pathway, 230,

2-monoacylglycerols, 231f monoamine oxidase, 300, 301 if
monoclonal antibodies (mAbs),
642-643, 697-698, 702, 702t monocytes, 656, 657
monoiodotyrosine (MIT), 492, 493f monomeric proteins, 38
mononucleotides, 320
"salvage" reactions and, 330, 331f monooxygenases
cytochromes P450 as, 115-116,

monosaccharides, 141-142, 142t absorption of, 520, 520f
amino sugars, 145, 145f deoxy sugars, 145, 145f
glucose as most important, 142
547,
548t glycosides formed from, 145
isomerism of, 142-143, 143f, 144f of physiological importance, 143,

monoubiquitylation, of histones,
351, 353t monounsaturated fatty acids, 197, 197t
dietary, cholesterol levels affected by,
257
synthesis of, 223, 223f
Morquio syndrome, 604t mortality and aging, 708
mRNA. See messenger RNA
mRNA-encoding gene, 441
mRNP 578
exporter, mRNPs (ribonucleoprotein particles), 405,

MS. See mass spectrometry
MS. See tandem MS
MS-MS. See tandem MS

in sickle cell disease, 443, 444f
MSUD maple syrup urine disease), 290,

mucolipidosis, 603
mucopolysaccharidoses, 592,
603-604, 604t mucoproteins. See glycoproteins
mucus,
549
multidimensional protein identification technology (MudPIT), 31
multidrug resistance (MDR), 478
multidrug-resistance-1 protein (MDR-1
multiple inositol polyphosphate phosphatase (MIPP), 53
multiple sclerosis, 234, 473
multiple sulfatase deficiency, 235
multiplexins, 594
multipotent stem cells, 646
multisite phosphorylation, 170
multispecific organic anion transporter

actin and myosin in, 613-615,
614f, 615f amino acid levels and, 271-272,
27
1f,
272f cardiac, 619-620, 619t, 620f
139f,
139¢
in fed state, 130, 136-138, 137f, 138t genetic disorders of, 621-623, 622t,

glucose uptake by, 136-137, 137f glycogen in, 164, 165t
167-168, 169f glycogen synthase regulation in, 170f
glycogenesis in, 164-166, 165f, 166f lactate
production by,
160
ribose synthesis in, 185
structure of, 612-613, 612f, 613f types of, 612

ATP in, 614-616, 615f, 620-621, 621f calcium role in, 611, 616-619, 617f,

energy conversion in, 615-616,
615f,
616f glycogen phosphorylase activation with,
168
nitric oxide in, 622-623, 624f relaxation phase of, 616
thin and thick filaments in,
612-613, 613f muscular dystrophy, 445, 622
glycoprotein synthesis defects in, 554
mutations, genetic, 350-351
Alu family and, 358, 360
base substitution, 396-397, 396f,

cancer-causing, 683, 701, 703
constitutive, 411
frameshift, 398-399, 398f free radical damage causing, 542
gene conversion and, 361
indels and, 359
integration and, 359-360, 360f of membrane proteins, diseases caused

missense, 396-397, 397f, 398f, 622
nonsense, 397-398, 398f point, 396-397, 396f, 397f, 398f,

premature termination, 397, 398f recombination
and,
359,
359f,
360f silent, 396
sister chromatid exchanges and,

suppressor, 398-399
transition,
396,
396f transposition and, 360
transversion, 396, 396f xenobiotic role in, 559, 559f
Zellweger syndrome and, 579-580
mutator phenotype, 691-692, 692f
MYC gene, in cancer, 685, 686f, 687t mycophenolic acid, 329f, 330

galactosylceramide in, 234
sulfatide in, 202
myelin sheath, 200
myelin sheets, 473
myelinated nerves, depolarization waves along, 196
myelodysplasia, 649
myeloid progenitor cells, 657
myeloperoxidase, 660-661, 660t iron in, 97-98
myocardial infarction (MI)
serum enzyme analysis after, 64, 65f

a helices in, 48, 48f biomedical implications of, 54
biomedical importance of, 47-48
heme in, 48-49, 48f, 49f hindered environment of, 49, 49f
iron in, 95-96
oxygen dissociation curve of, 49-50, 49f secondary and tertiary structures of, 50
myoglobinuria, 54
myokinase. See adenylyl kinase myopathy, lactic acidosis with, 118
myosin, 611
614f, 615f smooth muscle contraction regulation
by, 616-618, 617f, 617t structure and function of, 613-614,

myosin (thick) filaments, 612-613, 613f myotonia congenita, 620t
myristic acid, 197t myristoylation, 463
mass spectrometry detection of, 28t
N
n. See Hill coefficient
Nat. See sodium
NAD*. See nicotinamide adenine dinucleotide

NADH dehydrogenase, 114
NADH-Q oxidoreductase (Complex I),

deficiency of, 126
NAD-linked dehydrogenases, 113
NADP*. See nicotinamide adenine dinucleotide phosphate
NADP malate dehydrogenase, 218, 219f,

NADPH, for lipogenesis, 218, 219f, 220f
NADPH oxidase, 660, 6601, 661
NADPH-cytochrome P450 reductase,
557
NADP-linked dehydrogenases, 113
NAELD (nonalcoholic fatty liver disease),
243
NAGS. See N-acetylglutamate synthetase
Na*-K*-ATPase,
472-474,
472f,
473f nanotechnology, 4, 62, 62f
NASH (nonalcoholic steatohepatitis),
243
native conformation, protein, 41
Natowicz syndrome, 604t natural
killer cells,
656,
662
ncRNAs. See noncoding RNAs
NDP (nucleoside diphosphate) kinases,

necrosis, 693-694
negative allosteric effectors, 85
negative nitrogen balance, 525
negative predictive value, 562-563, 563t negative regulators, of gene expression,
410,
4108,
412,
414,
416
negative supercoils, DNA, 341
neonatal adrenoleukodystrophy,
580, 580t neonatal alloimmune thrombocytopenia,
655

jaundice in, 314
metabolic disease screening in, 278
tyrosinemia in, 286, 287f neoplasms, 681-682, 682f
nephrogenic diabetes insipidus, 472
NER (nucleotide excision-repair), 369,

nerve impulses, 473
NESs (nuclear export signals), 578
net diffusion, 468
NET-Seq, 447
NeuAc (N-acetylneuraminic acid),

neuraminic acid, 147, 202
neuraminidase, influenza, 555
neurodegenerative diseases, prion, 42
neurofilaments, 625, 625t neurolathyrism, 18, 18f
neurologic impairment, profound, 580
neurons, membranes of impulses transmitted along, 473
ion channels in, 470f neuropathy, vitamin B, causing, 535,
neurotransmitters, amino acids as, 18
neutral lipids, 196
neutrophil extracellular traps, 661, 661f neutrophils,
656,
657
enzymes and proteins of, 659, 660t integrins in, 658-659, 659t
myeloperoxidase in, 661
in parasite entrapment, 661, 661f newborns,
hemoglobins of,
51,
51f next-generation sequencing (NGS),
445-447, 701
NF«B (nuclear factor kappa-B),
553, 631
NEF-«B pathway, 510-511, 510f
NGS (next-generation sequencing),
NHEJ (nonhomologous end-joining), 369,

citric acid cycle need for, 153
deficiency of, 528, 534-535
functions of, 528t, 534
nicotinamide coenzymes formed from,
113
toxicity of, 535
nick translation, 434t nickel
physiologic roles of, 100
toxicity of, 95t in urease, 97, 97f
nicks and nick-sealing, in DNA
replication, 367, 367f nicotinamide, 58, 58f, 534
nicotinamide adenine dinucleotide

in citric acid cycle, 152f, 153, 155-156
as coenzyme, 324t cytochrome P450 use of, 115, 115f
dehydrogenase use of, 113, 113f in fatty liver, 244
metabolic compartmentation using, 84
niacin in synthesis of, 534
spectrophotometric assays using,

substrate shuttles in oxidation of,
125-126, 125f nicotinamide adenine dinucleotide
phosphate (NADP*)

cytochrome P450 use of, 115, 115
dehydrogenase use of, 113, 113f intramitochondrial source of, 125
pentose phosphate pathway formation of, 182-185, 183f, 184f

nicotinic acid, 534
as hypolipidemic drug, 257
Niemann-Pick C-like 1 protein, 257
Niemann-Pick disease, 235t nitric oxide (NO), 611, 622-623, 624f
arginine conversion to, 297, 297f in hemostasis and thrombosis, 672
nitric oxide synthases, 297, 297f, 622

nitrogen, metabolism of amino acid oxidases in, 273-274, 274f
ammonia from, 269, 272-275, 274f disorders of, 276-278, 277t
excreted forms of, 272, 275, 275f glutamate dehydrogenase in, 273, 274f
glutaminase and asparaginase in,

glutamine synthetase in, 274, 274f transamination
reactions in,
272-273,

nitrogen balance, 269, 525-526
nitrogenase, 97
nitroglycerin, 611
p-nitrophenyl phosphate (pNPP), 63
NLS (nuclear localization signal), 574t,
577-578, 578f
NMP (nucleoside monophosphate)
kinases, 110
NMR (nuclear magnetic resonance)
spectroscopy, protein structures solved by, 40
NO. See nitric oxide nodes of Ranvier, 473
272f, nodularin, 14
nonalcoholic fatty liver disease (NAFLD),

nonalcoholic steatohepatitis (NASH), 243
non-clathrin-coated vesicles. See transport vesicles
noncoding RNAs (ncRNAs), 347-348.
See also long noncoding RNAs;
micro-RNAs;
ribosomal
RNA;
silencing RNAs; small nuclear
RNA; transfer RNA
in cancer, 689-690
gene expression modulation by,

noncoding strand, 339, 344f noncompetitive inhibitors, 77-78, 77f, 78f
noncovalent assemblies, in membranes,

biomolecule stabilization by, 7-8, 8f peptide conformations and, 21
in protein tertiary and quaternary

nonequilibrium reactions in glycolysis regulation, 160-161
regulation of metabolic pathways with,
135
nonessential amino acids, 131, 264, 264t,

nonesterified fatty acids. See free fatty acids
nonheme iron proteins. See iron-sulfur

nonketotic hyperglycinemia, 283
non-lipid-soluble molecules, 462-463
nonoverlapping of genetic code, 395
nonpolar groups, noncovalent interactions of,
8
nonpolar lipid core, of lipoprotein,
237-238, 238f nonrepetitive (unique-sequence) DNA,

nonsense codons, 394, 397-398, 398f nonsense mutations, 397-398, 398f
nonsteroidal anti-inflammatory drugs cyclooxygenase
226
prostaglandins synthesis and,

nontemplate strand DNA, 375
non-a-peptide bonds, between ubiquitin and proteins, 270, 270f
norepinephrine. See also catecholamines glycogen regulation by, 167-168
synthesis of, 484f, 491, 491f in thermogenesis, 247, 247f
tyrosine conversion to, 300, 302f
Northern (RNA-DNA) blotting, 341
Northern RNA blot procedure, 438, 439f
NPCs (nuclear pore complexes), 577
pNPP (p-nitrophenyl phosphate), 63
N-terminal binding domain, 37
nuclear export signals (NESs), 578
nuclear factor kappa-B (NFKB), 553, 631
nuclear genes, proteins encoded by, 576
nuclear localization signal (NLS), 574¢,
577-578, 578f nuclear magnetic resonance (NMR)
nuclear pore complexes (NPCs), 577
nuclear receptor coregulators, 513-514,
513f, 514t nuclear receptor superfamily, 511-513,
512f,
513¢
nuclear receptors, 482
nucleases, 9
active chromatin and, 354
nucleic acid digestion by, 348

nuclease digestion of, 348
nucleolytic processing, of RNA,
390-391,
390f

definition of, 8
water as, 8-10
nucleophilic attack, 364, 365f nucleoproteins, packing of, 355, 355t, 356f
nucleoside diphosphate (NDP) kinases,

nucleoside monophosphate (NMP)
nucleoside triphosphates chemistry of, 320, 321f
group transfer potential of, 324
nonhydrolyzable analogs of, 325, 325f nucleosides, 321-323, 322t
biomedical importance of, 320
chemistry of, 320, 321f triphosphates, 324
nucleosomes, 351, 351f, 352f, 353
transcription machinery and, 384
nucleotide adducts, carcinogens forming,

nucleotide excision-repair (NER), 369,
370f,
370t nucleotides, 322t
biosynthesis of, purines, 328, 328f chemistry of, 320, 321f
dietarily nonessential, 328
frameshift mutations of, 397-398, 398f metabolism of, biomedical importance
of, 327-328
396-398, 396f, 397f, 398f physiologic functions of, 321-323, 323f,

as polyfunctional acids, 321
polynucleotides,
325
substitution mutations of, 396-397,

synthetic analogs of, in chemotherapy,
324-325, 324f, 325f triplet codes of, 394, 394t
ultraviolet light absorption by, 321

macromolecule transport in, 575f,
576-579, 577t, 578f protein sorting signals in, 573, 574t
nutrigenomics, 3

antioxidants and. See antioxidants biochemical research impacts on, 2-3
lipogenesis regulated by, 220
transition metals, 93, 93t vitamins and minerals, 527-528
nutritional disorders amino acid deficiency, 14, 131, 264,

collagen defects in, 43
nutritionally essential fatty acids, 222, 222f abnormal metabolism of, 226
deficiency of, 224, 225
oO
O gene, 653-654
obesity, 105, 130, 237, 519, 524
lipids and, 196
lipogenesis and, 216
obstructive jaundice, 242
octamers, histone, 351, 351f, 352f, 353
oculocerebrorenal syndrome, 585t
B-ODAP (B-N-oxalyl-L-a,
6-diaminopropionic acid),

oils, 196
Okazaki fragments, 363-364, 363t, 366f oleic acid, 196, 196f, 197¢, 198f

oligomers, import of by peroxisomes, 579
oligomycin, 123
oligonucleotide, synthesis of, 439
oligosaccharide processing, 574
oligosaccharide:protein transferase, 581
oligosaccharides, 142, 546, 547t,

long-chain, 196, 199
synthesis of, 223-224, 223f
w6 fatty acids, 196, 199
w9 fatty acids, 196
OMP (orotidine monophosphate), 333f oncogenes, 685-688, 685t, 686f, 687f, 687t,

oncology, 681, 701
oncoproteins, Rb protein and, 369
oncotic (osmotic) pressure, 628, 630
oncoviruses, cyclins
and,
369
open complex, 376f, 379
operator locus, 411f, 412
operon, 411-412, 411f, 413f, 414
operon model, 411-412, 411f, 413f, 414
opsonization, 659
optical activity, 142
optical isomers, of monosaccharides,

oral rehydration therapy, 474
ORC (origin replication complex), 362
ORE (origin replication element), 362
organ function tests, 566-567
organometallic complexes, transition metals in, 95-97, 95f, 96f,

metabolic pathways at level of, organs,
132-133, 132f, 133f origin of replication (ori), 362-363, 362f
origin replication
complex
(ORC),
362
origin replication element (ORE), 362
ornithine arginine conversion to, 297, 297f

in urea synthesis, 275-276, 275f ornithine permease, defects in, 277
ornithine transaminase, 282, 283f ornithine transcarbamoylase
defects in, 277-278
deficiency of, 336
ornithine-citrulline antiporter, defect in,

orotic acid, 243, 244, 333f orotic aciduria, 336, 336t
orotidine monophosphate (OMP), 333f orotidinuria, 336
orotidylate decarboxylase, 336
orotidylic acid decarboxylase, 336t osmotic (oncotic) pressure, 628, 630
osteoarthritis, 592, 604
osteoblasts, 605, 606f osteocalcin, 539
osteoclasts, 605, 606f osteocytes, 605
osteogenesis imperfecta, 43, 264,

osteomalacia, 532
osteopetrosis, 607
osteopontin, 605
osteoporosis, 532, 607, 607t ouabain,
145,
473
outer membrane, of mitochondria,
118, 118f outer mitochondrial membrane, 576
outside-in transmembrane signaling, in platelet aggregation, 670, 671f
489f, 490f overlay blot procedure, 438, 439f
overnutrition, 524-525, 524f. See also obesity
oxaloacetate, 134
asparagine and aspartate formation of,
281-282
in citric acid cycle, 150-154, 151f, 152f,
154f in gluconeogenesis, 172-173, 173f.
- in lipogenesis, 218-219, 220f
B-N-oxalyl-L-a, B-diaminopropionic acid

biomedical importance of, 111
definition of, 111
dehydrogenase reactions,

endergonic reaction coupling to,
106-107, 106f, 107f of
fatty acids.
See also
ketogenesis acetyl-CoA release and, 208-210,

ketogenesis regulation and,
213-214, 213f, 214f in mitochondria, 207-208, 208f
modified, 210
of heme iron, 649-651, 650f, 650t, 651f hydroperoxidase reactions, 114
of iodide, 492
oxidase reactions, 112, 112f, 113f oxygenase reactions, 111-112,
114-116, 115f, L16f in pentose phosphate pathway,
183-185, 183f, 184f phosphorylation coupling to,
122-123, 123t pyruvate. See pyruvate oxidation
redox potential
and,
111-112,
112t of reducing equivalents by respiratory
chain, 118-121, 119f, 120f, 121f substrate shuttles mediating,
125-126, 125f superoxide dismutase reactions, 116
of transition metals, 93, 94f, 94t
B-. See B-oxidation oxidative damage. See reactive oxygen

oxidative decarboxylation of amino acid oxidases,
273-274, 274f in citric acid cycle, 152, 152f
of pyruvate, 161-163, 162f oxidative phosphorylation, 620
biomedical importance of, 118
clinical aspects of, 126
control of, 122-123, 123t in energy conservation and capture,
high-energy phosphate synthesis by,
118, 121-122, 122f inhibition of, 118
in mitochondrial compartments,
118,
118f uncoupling of, 123-124
oxidoreductases,
57,
112
structure of, 37, 38f oxidosqualene:lanosterol cyclase,
250, 252f oxygen
Complex IV reduction of, 120-121
heme binding of, 48-49, 48f, 49f hemoglobin affinities for, 51, 51f
hemoglobin binding
of,
51-52,
51f,
52f toxicity of, 116. See also reactive oxygen
transport and storage of, 47
biomedical implications of, 54-55
heme and ferrous iron roles in,

hemoglobin and myoglobin suitability for, 49-50, 49f
hemoglobin conformational changes during, 50-53, 50f, 51f, 52f, 53f
hemoglobin mutations impacting,
53-54,
54f high altitude changes in, 53
oxygen debt, 160
oxygen dissociation curve of hemoglobin, 49-50, 49f
of myoglobin, 49-50, 49f oxygen radicals. See reactive oxygen

oxygen tension, in cancer cells, 696
oxygenases, 111-112, 114-116, 115f, 116f oxysterols, 204
P
P bodies, 405, 405f, 429
P component, in amyloidosis, 639
P (peptidyl) site, of 80S ribosome,

P53 gene, 6871, 691, 694
p53 protein, 372
p70S6K, 508, 509f p97, 584
p160 family of coactivators, 514, 514t
P450scc (cytochrome P450 side chain cleavage enzyme), 485, 485f
PAB (poly(A) binding protein), 401, 401f,

PAC (E. coli bacteriophage P1-based)
vector, 437, 437t paclitaxel, 625
paddle, charged, 471, 471f
PAE See platelet-activating factor
108
PAGE (polyacrylamide gel electrophoresis), 26, 26f
pain, prostaglandins in, 216
PAL (physical activity level), 524
palmitate, 217-218, 219f, 220f palmitic acid, 196f, 197t
palmitoleic acid, 197t, 222, 222f palmitoleoyl-CoA, 223
palmitoylation, mass spectrometry

palmitoyl-CoA, 223
pancreatic cancer, hyperbilirubinemia caused by, 313f, 313t, 314
pancreatic insufficiency,
vitamin deficiency in, 536
pancreatic lipase, 521
pancreatitis, protease activation in, 87
panproteinase, 638
pantothenic acid, 217
coenzymes derived from, 58, 539
deficiency of, 528t functions of, 528t, 539, 539f
PAP (phosphatidate phosphatase),

PAR (physical activity ratio), 524
PAR-1. See protease-activated receptor-1
PAR-4. See protease-activated receptor-4
paracrine signaling,
662
parallel 6 sheet, 36, 36f paramecia, 660
parasites, glycans in binding of, 555
parathyroid hormone (PTH)
storage of, 497, 497t synthesis of, 494, 495f
paroxysmal nocturnal hemoglobinuria,
478t, 554, 6508, 651
PARP (poly ADP ribose polymerase),
371,
371f partial proteolysis. See selective proteolysis
passenger mutations,
701
passive diffusion, 467-468, 467f, 467t,

pBR322, 437, 437f
P-cluster, 97
PCR. See polymerase chain reaction
PCSK9 (proprotein convertase subtilisin/
kexin type 9), 253
PDGF (platelet-derived growth factor), in cancer, 689, 689t
PDH. See pyruvate dehydrogenase
PDI (protein disulfide isomerase), 584
PDK] (phosphoinositide-dependent kinase 1), 508, 509f
pectin,
147,
148f pedigree analysis for sickle cell disease,

pellagra, 534-535
penicillamine, for Wilson disease, 634
penicillin, 80
B,,
pentose phosphate pathway, 130, 131f biomedical importance of, 182
cellular location of, 182-183
glycolysis connections
with, hemolysis protection from,
185-186, 186f impairment of, 188-189
irreversible oxidative phase of,
183-185, 183f, 184f
NADPH produced by, for lipogenesis,
218, 219f, 220f reducing equivalents generated by, 185
reversible nonoxidative phase of, 183,

pentoses, 141, 142t, 143, 144t pentosuria,
182,
189
pepsin, 521
pepsinogen, 521
peptic ulcers, 555
peptidases, proteins degradation by,

between amino acids, 20
formation of, 9, 402
hydrolysis of, 708-709, 709f partial double-bond character of,
21, 22f secondary structure restrictions from,
34-35,
34f between ubiquitin and proteins,

peptide hormone receptors, 482
peptide hormones, glycoproteins as, 546
peptides absorption of, 521, 523
L-a-amino acids in, 15, 15t-16t biomedical importance of, 14
conformations of, noncovalent forces affecting, 21
as hormone precursors, 484, 484f as
polyelectrolytes,
21-22
as precursors in hormone synthesis,

gel electrophoresis, 26, 26f, 27f isoelectric focusing, 26, 27f
sequencing of. See protein sequencing structure drawings for, 21
structure of, primary. See primary structure
unusual amino acids in, 21, 21f volatilization of, 29-30, 30f
peptidyl prolyl isomerase (PPI), 584
peptidyl (P) site, of 80S ribosome,

peptidyl transferase, 402, 403¢
peptidylarginine deiminase, 661, 661f peptidylglycine hydroxylase, vitamin C as
coenzyme for, 539
peptidyl-tRNA, 402-403, 403f, 403¢
perilipin, 247
183f,
185
periodic table, 94f peripheral proteins, 464-465, 464f
permeability coefficients, of substances in lipid bilayer, 462, 463f
pernicious anemia, 536-537
peroxidases, 114, 224, 544
peroxidation, of lipids, 203-204, 204f,
710, 711f

lipid, 541-542, 542
mechanisms protecting against,
544,
544f reduction of, 114
peroxins, 579-580
peroxisomal-matrix
(PTSs),
574t, targeting
579,
579f peroxisomes, 114
biogenesis of, 579-580
disorders due to abnormalities of, 580t,
589-590
in fatty acid oxidation, 210
in polyunsaturated fatty acid synthesis,
223f protein sorting signals in, 573, 574t
proteins imported into, 579, 579f in transcription regulation, 513t
in Zellweger syndrome, 215,

peroxynitrite, 623
personalized medicine, 432
pertussis toxin, 504
PGE, (prostaglandin E,), 197, 198f
PGI, (prostacyclin ), 670, 671f, 672
PGI, (prostaglandin I,), 224
PGIs.
See prostacyclins
PGs. See prostaglandins pH
amino acid properties and, 19, 19f biomedical importance of, 6
buffering and, 12-13, 12f in cancer cells, 696
definition and calculation of, 10-11
isoelectric,
19
pK, relationship to, 11-12
reaction rate response to, 72-73, 73f of water, 10
PH gradient, 576
phages, in recombinant DNA technology,

phagosomes, 659
pharmacogenomics, 3
phasing, of nucleosome, 353
phenobarbital, 557
phenols, sulfation of, 558
phenyl isothiocyanate, 27, 27f phenylalanine, 16t
286,
288f ultraviolet light absorption by,
20, 20f sequences
phenylalanine hydroxylase, 266, 267f,

gene location of, 442t phenylethanolamine-N-methyltransferase
(PNMT), 491f, 492
phenylketonuria (PKU), 286, 288f phi angle, 34, 34f
phosphagens, in energy conservation and capture, 108
phosphatase cascade, 482, 483t phosphatases, in recombinant DNA
technology, 434t phosphate transporter, of mitochondria,

phosphates creatine phosphate shuttle for, 126, 126f
energy capture
and transfer
by,
460t oxidative phosphorylation in synthesis
of, 118, 121-122, 122f in RNA synthesis, 378
phosphatidate, 230-232, 230f, 231f, 232f phosphatidate phosphatase (PAP),
230, 231f phosphatidate phosphohydrolase,

phosphatidic acid, 199, 200f, 461, 461f phosphatidylcholines, 200, 200f
metabolism of, 233f synthesis of, 230, 230f, 231f
phosphatidylethanolamine (cephalin),
200, 200f membrane asymmetry and, 464
synthesis of, 230, 230f, 231f phosphatidylglycerol, 200f, 201
phosphatidylinositides calcium-dependent hormone action
and, 507-508, 507f, 508f as second messenger, 200, 200f, 482,

phosphatidylinositol 4,5-bisphosphate
(PiP,), 200
in absorptive pinocytosis, 475
phospholipase C cleavage of,
507, 508f in platelet aggregation, 670, 671f
phosphatidylinositols in membranes, 200, 200f
synthesis of, 230, 230f, 231f phosphatidylserine, 200, 200f, 230,

3’-phosphoadenosine-5’-phosphosulfate
(PAPS), 322-323, 323f, 324t,
558, 600
phosphodiesterases, 167, 168f, 169f,
325, 505
phosphoenolpyruvate in gluconeogenesis, 172-173, 173f
phosphoenolpyruvate carboxykinase, 153
in gluconeogenesis, 173, 173f phosphofructokinase
in glycolysis, 159, 159f regulation of, 161, 176, 176f

phosphofructokinase-2, 176, 177f phosphoglucomutase, in glycogenesis,
164, 165f
6-phosphogluconate dehydrogenase, 183f,
184,
184f
6-phosphogluconolactone, 183-184, 183f,
184f
2-phosphoglycerate, in glycolysis,
159, 159f
3-phosphoglycerate, in glycolysis,
159, 159f
3-phosphoglycerate dehydrogenase, allosteric regulation of, 85
phosphoglycerate kinase in erythrocytes, 161
in glycolysis, 159, 159f phosphoglycerate mutase, in glycolysis,
159, 159f phosphoglycerides, in membranes, 461,

lysophospholipids in metabolism of,
201,
201f synthesis of, 230-232, 230f
phosphohexose isomerase, in glycolysis,
159, 159f phosphoinositide-dependent kinase 1
(PDK1),
508,
509f phosphoinositides, 200, 482, 483t
phospholipase A,, 232, 233f phospholipase A,, 232, 232f, 233f
phospholipase B, 232, 233f phospholipase C (PLC), 658
activation and hormone-receptor interactions of, 507, 507f
in phosphoglycerol degradation and remodeling, 232, 233f
PiP, cleavage
by,
507,
508f phospholipase CB (PLC), in platelet
aggregation, 670, 671f phospholipase D, 232, 233f
phospholipases, in phosphoglycerol degradation and remodeling,
232-233, 233f phospholipid bilayer, noncovalent
interactions in, 8
phospholipid exchange proteins, 464
phospholipids, 196
clinical aspects of, 234-235
520-521, 522f glycerol ether, synthesis of, 230,
232, 232f in lipoprotein lipase activity, 240
in lipoproteins, 236, 237
in membranes, 199-201, 200f,
461, 461f, 463, 589
asymmetry of, 464, 589, 589f in multiple sclerosis, 234
as second messenger precursors, 229
synthesis of, 230, 231f phosphomevalonate kinase, 251f
phosphoprotein phosphatases, 505-506
phosphoproteins, 505
phosphoribosyl pyrophosphate (PRPP)
in
Lesch-Nyhan syndrome,
334-335
in purine synthesis, 328, 329f, 330
in pyrimidine synthesis, 332,

phosphoric acid, pK, of, 13¢
phosphorolysis, as group transfer reaction, phosphorylase
kinase a,
168,
169f,
171f phosphorylase kinase b, 89t, 168,

phosphorylation in energy conservation and capture, 108
enzyme regulation by, 86-90, 88f, 89t in glycogen regulation, 166-168, 169f,
170, 171f in glycolysis and gluconeogenesis
multisite, 170
oxidation coupling to, 122-123, 123t oxidative. See oxidative phosphorylation
161-163, 162f respiratory chain level, 122
in RNA polymerase II activation,
384, 385f substrate level, 122
phosphoserine, 300
phosphothreonine, 300
phosphotriose isomerase, in glycolysis,

phosphotyrosine, 300
photolysis, 489-490
photosensitivity, porphyrias causing, 311
phylloquinone, 528t, 532-533, 533f physical activity
energy requirements for, 524
oxygen debt after, 160
physical activity level (PAL), 524
physical activity ratio (PAR), 524
physiologic jaundice, 314
phytanic acid, Refsum disease caused by accumulation of, 215
hytase, 523
hytohemagglutinins, 547
hytic acid, 523 TUU
pl. See isoelectric pH
PIC.
See preinitiation complex ping-pong mechanism, in facilitated
diffusion, 469, 469f ping-pong reactions, 79, 79f, 80f
in covalent
catalysis,
59,
59f transamination, 59, 59f, 273, 273f,
pinocytosis, 474-475, 474f
PiP,. See phosphatidylinositol
4,5-bisphosphate pituitary gland, hormones of, 179
PK. See pyruvate kinase
PK, of amino acids, 19-20, 20t
of L-a-amino acids, 15t-16t environmental influence on, 19-20, 20t
medium properties
affecting,
13
molecular structure effects on, 13, 13t of weak acids, 11-13, 12f, 13t, 19
PKA (protein kinase A), 504-505, 505f
PKC. See protein kinase C
PKU (phenylketonuria), 286, 288f plants, toxic amino acids of, 18, 18¢
plasma, 628
analysis of enzymes in, 63-64, 64t, 65f plasma glucose concentration. See blood

plasma membrane, 459. See also membranes
asymmetry of, 589, 589f cholesterol in, 461
glycoproteins anchored to, 552
glycosphingolipids in,
461
mutations in, diseases caused by,

deposition of, 639, 639t electrophoresis for analysis of, 628
fluid distribution
and,
628
functions of, 630
glycoproteins as, 546
half-life of, 630
haptoglobin, 631-632, 631f immunoglobulins, 639-643, 6401, 641f,

in inflammation, 630-631, 630t polymorphism of, 629
synthesis of, 133, 628-629
plasma samples, 563
plasma transport proteins, 498-499, 498t plasmalogens, 201, 201f
biosynthesis of, 230f, 232, 232f plasmids, 436, 436f, 437f
plasmin, 678-679, 678f plasminogen, 678-679, 678f
plasminogen activators, in hemostasis and thrombosis,
672
Plasmodium falciparum, glycans in binding of, 555
platelet aggregation, 670, 671f laboratory tests measuring, 679
platelet count, 679
platelet-activating factor (PAF), 229
synthesis of, 230f, 232, 232f platelet-derived growth factor (PDGF), in
cancer, 689, 689t
platelets, 646, 654-655, 659t inhibitors of activation of, 671f, 672
PLC. See phospholipase C
PLC6 (phospholipase Cf), in platelet aggregation, 670, 671f
pleckstrin, in platelet aggregation,
670, 671f
PLP (pyridoxal phosphate), 273, 273f, 535
pluripotent stem
cells, induced,
446,
646
PNMT (phenylethanolamine-N- methyltransferase), 491f, 492
pOH, 10
point mutations, 396-397, 396f, 397f, 398f,
442, 443f poisons, respiratory chain inhibitors, 112,
118,
123-124,
123f,
124f

hydrogen bonding by, 7, 7f noncovalent interactions of, 8
polar head groups, 461-462
polarity of DNA, 339, 340f
of DNA synthesis, 364, 366f of microtubules, 624
of protein synthesis, 399
poly ADP ribose polymerase (PARP),
371, 371f ly(A) binding protein (PAB), 401, 401f, ° P

poly(A) polymerase, 389
poly(A) tail of mRNA, 345, 389, 401, 401f polyacrylamide gel electrophoresis

polyadenylation, alternative, 430
polyamines, biosynthesis and catabolism of, 299, 299f, 300f
polycistronic mRNA, 411
polycomb repressive complex 2 (PRC2),
419
polycythemia, hemoglobin mutations leading to, 54
polyelectrolytes, peptides as, 21-22
polyhydric alcohols, carbohydrates as aldehyde or ketone derivatives of,
141-142, 142t polyisoprenoids, in cholesterol synthesis,
250-251, 251f polymerase chain reaction (PCR),
439-440, 441f medical applications of, 65
in microsatellite repeat sequence detection,
358

DNA. See DNA polymerases
RNA. See RNA polymerase II; RNA
polymerases polymerization, of hemoglobin S,

polymorphisms, 442
microsatellite, 358, 445-446
plasma protein, 629
polymorphonuclear leukocytes, 657
polynucleotide kinase, 434t polynucleotides
as directional macromolecules, 325
as
DNA
and
RNA,
325
functions of, 321
modification of, 321, 323f posttranslational modification of, 325
polyol pathway,
190-191
polyols, 141-142
polyprenoids, 203, 204f polyps, colorectal, 687-688, 688f, 688t
polyribosomes, 347, 401f, 405
membrane-bound, 580
protein synthesis on, 574, 574f, 575f,
576, 579, 583
signal hypothesis of binding of,
574, 574f polysaccharides, 142
storage and structural functions of,
145-147, 147f, 148f, 148¢
polysomes. See polyribosomes polytene chromosomes, 354, 354f
polyubiquitination, 270
polyunsaturated fatty acids, 196f,
197,
197t dietary, cholesterol levels affected by,
257
eicosanoids formed from, 224, 225f essential, 222, 222f
POMC peptide family, 496-497, 497f
Pompe disease, 166, 167t porcine stress syndrome, 618
porphobilinogen, in heme synthesis, 306,

porphyrias, 305, 310, 311f classification of, 310f, 311
drug-induced, 311
spectrophotometric assessment of, 308
porphyrinogens accumulation of, 310-311
colorless nature of, 308
in heme synthesis, 306-307, 307f,
porphyrins biomedical importance of, 305
biosynthesis of, 305-307, 306f, 307f,

regulation of, 307-308
catabolism of, 311-313, 312f, 313f color and fluorescence of, 308, 310f
structure of, 305, 306f positive cooperativity, 76
positive nitrogen balance, 525
positive predictive value, of laboratory tests, 562-563, 563t
positive regulators, of gene expression,
410, 4108, 414, 416
posttranslational modifications of amino acids, 15, 17, 17f
biomedical importance of, 33
of collagen, 594-595, 594t of histones, 351, 353t
mass spectrometry detection of, 28, 28t of
membrane proteins,
589
polynucleotides, 325
in protein maturation, 42-44, 43f posttranslational processing, 406
posttranslational translocation, 576,

potassium (K*)
460, 460t permeability coefficient of, 463f
potassium (K*) ion channels, 470-471,
47if power stroke, 615-616, 616f
PP.. See inorganic pyrophosphate;
pyrophosphate
PPI (peptidyl prolyl isomerase), 584
prasugrel, 672
pravastatin, 257
PRC2 (polycomb repressive complex 2),
419
precision, of laboratory test, 561, 561f precision oncology, 701
predictive value, of laboratory tests,

fatty liver of, 215
hypoglycemia during, 180
pregnenolone, 485f, 487, 488f, 490f preinitiation complex (PIC), 377, 382f
assembly of, 385-386, 385f nucleosomes and, 384
in protein synthesis, 399, 400f
43S preinitiation complex, in protein synthesis, 399, 400f
premature termination, 397, 398f prenatal diagnosis, 444-445, 445f
S-prenylation, 463
cis-prenyltransferase, 251f trans-prenyltransferase, 251f
preproalbumin, 588, 588f preprocollagen, 594
preprohormone, 493
preproprotein, 630
preproPTH, 494, 495f preproteins, 574
presequence, 576
preventive antioxidants, 204
preventive medicine, biochemical research

primaquine, 721
primary hyperoxaluria, 283
primary lysosomes, 474
primary structure amino acids and, 20-21
of collagen,
43,
43f determination of
mass spectrometry for, 28-30, 28t,
29f,
30f
primary structure, determination of (Cont.):

as order of protein structure, 34
primary structure, of RNA, 342-343, 346f primary transcripts, 356, 376,

primary transport, 469
primases, DNA, 362f, 363t pri-miRNAs,
390-391,
390f primosome, 363
prion diseases, pathologic protein conformations in, 42

proalbumin, 588, 588f probing techniques, 438, 439f
procarcinogens, 556-557, 559, 559f, 684
procaspases, 692-694, 693f processed genes, 360
prochymotrypsin, activation of, 87-88, 88f procollagen, 43, 406, 539, 594
procollagen aminoproteinase, 594
procollagen carboxyproteinase, 594
procollagen suicide, 607

in balanced chemical equations, 69
enzyme, 57
proenzymes, enzyme regulation using,

progesterone, 484f, 485f plasma transport of, 498-499, 498t
synthesis of, 487, 488f, 490f programmed cell death. See apoptosis
prohormones, 406
proinflammatory cytokines, 700

as model for study, 411
on-off manner of, 424
unique features of, 411

carbon skeleton catabolism of, 282, 282f in collagen, 593-594, 596
synthesis of, 265, 267f proline dehydrogenase, deficiency of, 282,

proline hydroxylase, vitamin C as coenzyme
for,
539
proline-cis, trans-isomerases, 42, 42f prolyl hydroxylase, 43, 266, 593, 596
promoter clearance, 378
promoter recognition specificity, 377
promoter site, in operon model,

promoters, in transcription, 376
alternative, 388-389, 389f bacterial, 379, 380f
eukaryotic, 379-381, 380f, 381f,

pro-opiomelanocortin (POMC) peptide family, 496-497, 497f
pro-oxidants, antioxidants as,
544-545, 544t prophage state, 415
propionate glucose derived from, 177
metabolism of, 173f, 174, 174f, 723t

in citric acid cycle, 153, 154f in fatty acid oxidation, 209
methionine conversion to, 288,

propionyl-CoA
174,
174f proprotein
convertase subtilisin/kexin
type 9 (PCSK9), 253
proproteins, 43, 87, 406
proPTH, 494, 495f propyl gallate, as antioxidant/food

PRO-Seq,
447
prostacyclin (PGI,), 670, 671f, 672
prostacyclins (PGIs), 197
clinical significance of, 226-227
synthesis of, 224, 226f prostaglandin E, (PGE,), 197, 198f
prostaglandin I, (PGI,), 224
prostaglandin H synthase, 224
prostaglandins (PGs), 197-198, 198f, 216,
224, 662
cyclooxygenase pathway in synthesis of,
224-225, 225f, 226f prostanoids, 197
clinical significance of, 224, 226-227

prostate-specific antigen (PSA), 700
prostatic acid phosphatase, diagnostic use

prosthetic groups of enzymes, 57-58, 58f
flavins, 112, 113f, 114-115, 115f heme. See heme
protoheme, 114
protamine, 677
protease-activated receptor-1 (PAR-1), in platelet aggregation, 670, 671f
protease-activated receptor-4 (PAR-4), in platelet aggregation, 670, 671f

as digestive enzymes, 521
using,
87-88, a2-macroglobulin and, 638, 638f
matrix, 576
phagocyte-derived, 661-662
88f in protein degradation, 584-585
proteins degradation by, 270-271, 270f,

synaptobrevin and, 587
26S proteasome, 84
proteasomes protein degradation by, 84-85,
270-271, 270f, 271f protein degradation in, 582,
584-585, 585f structure of, 270-271, 271f
protein blot procedure, 438, 439f protein C, 677
protein coding RNAs. See messenger RNA
protein damage, repairing of,
714-715, 715f
Protein Data Bank, 38
protein degradation, ubiquitin in,

protein disulfide isomerase, 41-42
protein disulfide isomerase (PDI), 584
protein folding chaperones and, 574-575
misfolded, 584-585, 585f, 585t protein genes s36 and s38, chorion, 429f
protein kinase A (PKA), 504-505, 505f, protein kinase C (PKC), 507, 507f

cAMP and, 504-505, 505f in hormonal regulation of lipolysis,
246, 246f hormone signaling and, 508-511,
509f, 510f in initiation of protein synthesis,
399
phosphorylation by, 87-90, 88f, 89t as second messengers, 482, 483
protein lipidation, 463
protein misfolding endoplasmic reticulum accumulation
of, 584, 585t ubiquitination of, 585-586, 585f
protein phosphatase-1, 168, 169f,
170, 171f protein phosphatases, dephosphorylation
by, 87-90, 88f, 89t protein prenylation, 251

protein sequencing

purification techniques for, 23-26, 25f,

protein sorting branches of, 575, 575f
cell nucleus and, 575f, 576-579,
577t, 578f
cotranslational insertion and,
582-583, 582f, 583f disorders of, 589-590
Golgi apparatus in, 574, 575f, 584
importins and exportins in, 578, 578f
KDEL amino acid sequence and,
57At, 583
membrane assembly and, 588-590,
589f,
590t misfolded proteins, 584-585,

mitochondria and, 576, 577f peroxisomes and, 579, 579f
peroxisomes/peroxisome disorders and,

quality control in, 584, 584t retrograde transport and, 582-584
rough ER branch of, 574, 574f, 575f signal hypothesis of polyribosome
binding, 574, 574f, 580-582, 580t,
581f,
582f signal sequences and, 573-575, 574t,
582f,
629
transport vesicles and, 585-588,
586t,
587f

antibiotic inhibition of, 406-407, 407f biomedical importance of, 393
elongation in,
402-403,
403f,
403t environmental threats and, 405
genetic information flow of, 393-394
in
Golgi apparatus,
574,
575f initiation of, 399-402, 400f, 401f
by mitochondria,
574t,
576
modular principles in, 34
mRNA and, 344, 344f, 394, 394t mutations and, 396-398, 396f,

polysomes in, 405, 574, 574f posttranslational processing and, 406
rate of, 269-270, 401-402, 402f with recombinant DNA technology,

regulation of, 84
reticulocytes and, 649
RNA and, 343-344
termination of, 403-404, 404f translocation and, 403
tRNA and, 395-396, 395f, 396f virus replication and, 405-406, 406f
protein turnover, 84, 269-270
in membranes, 589
proteinases, of cancer cells, 698
protein-conducting channel, 580
protein-DNA interactions bacteriophage lambda as paradigm for,
protein-losing gastroenteropathy, 630
protein-protein cross-links and protein glycation, 714f
protein-protein interactions, identification of, 447, 449f
protein-RNA complexes, in initiation,

acute phase, 526, 630, 630t aggregates of, 41
L-a-amino acids in, 15, 15t-16t, 17
asymmetry of, in membrane assembly,

biomedical importance of, 23
classification of, 34
configuration of, 33-34
conformations of, 33-35, 34f, 41-42
constitutive, 84
degradation of amino acid levels and, 271-272,

ATPand ubiquitin-dependent,
270-271, 270f, 271f
ATP-independent, 270
biomedical importance of, 269
disorders of, 276-278, 277t end products of, 272, 275, 275f
protease and peptidase role in,
270-271, 270f, 271f rate of, 269-270
denaturation of, 41
diets deficient in, 263-264
digestion and absorption of, 521, 523
domains of, 37, 38f, 39f dual-function, 487
energy yields from, 138t in extracellular and intracellular fluid,
460, 460t folding of, 41-42, 42f
calnexin role in, 551-552
free radical damage to, 541-542, 542f function of, bioinformatics and, 31
fusion, 65-66, 66f in genetic information flow,
393-394
glycoproteins. See glycoproteins half-lives of, 270
hormone receptors as, 482
identification of genomics role in, 28
mass spectrometry role in, 28-30,

by cell nucleus, 576-579, 578f by mitochondria, 576, 577t
by peroxisomes, 579, 579f intracellular traffic of, 573
ion channels as transmembrane,
470-471, 470f, 471f, 471t life
cycle of,
23,
24f lysosomal, 590
maturation of folding, 41-42, 42f
posttranslational processing, 42-44,
43f in membranes. See also membrane
proteins in artificial, 465
lipid ratio to, 460-461, 460f as
metabolic fuel,
130,
136
metabolic pathways of, 130f, 131,

in fed state, 137f, 138, 138¢t at tissue and organ levels, 132-133,

misfolded, 584-586, 585f, 585t modifications of, 384
of neutrophils, 659, 659t noncovalent interactions in, 8
nutritional requirements for,

phospholipid exchange, 464
plasma. See plasma proteins plasma transport, 498-499, 498¢
posttranslational processing of, 406
prenylation of, 251
profiling of, 446-447
purification of chromatographic techniques, 23-26,

ROS
reactions with,
711f sequences or molecules that direct,
573, 574t sequencing of. See protein sequencing
structure of
biophysical techniques for solving of,
39-41
conformation versus configuration,

folding into, 41-42, 42f gross characteristics, 34
modular nature of, 34
orders of, 34
pathologic perturbations of, 42
posttranslational processing of,
42-44, 43f primary. See primary structure
quaternary.
See quaternary
structure secondary. See secondary structure
tertiary.
See tertiary
structure volatilization of, 29-30, 30f

proteoglycans, 147
accumulation of,
235
in cartilage, 607-608, 608f, 608t components of, 599, 599f, 600f
disease and aging with, 604
functions of, 600-601, 603t galactose production for, 187-188, 189f
glucuronate production for,
186-187, 187f
proteolysis, 588
enzyme regulation by, 86-88, 88f
proteome, 30-31
proteomics, 3, 447
bioinformatics and, 31
challenges in, 31
goal of, 30-31
prothrombin, 672, 673f, 673t, 674t coumarin
drugs and,
677-678
factor Xa activation of, 674-675, 675f prothrombinase complex, 674-675, 675f
protic acids, 93
proton acceptors, bases as, 10
proton donors, acids as, 10
proton gradient, of respiratory chain,
121, 122f proton motive force, of respiratory chain,
121, 122f proton pump, respiratory chain action as,
118-121, 119f, 120f, 121f proton shuttle, 61, 61f
proton-motive force, 576
protons hemoglobin binding of, 52-53, 53f
in water, 9-10
proto-oncogenes, 685, 686f protoporphyrin III, in heme synthesis,
307, 308f, 309f protoporphyrinogen III, in heme
307,
308f,
309f protoporphyrinogen oxidase, 307, 308f,

proximity, enzymatic catalysis by, 59
PRPP. See phosphoribosyl pyrophosphate
PRPP glutamyl amidotransferase, in purine synthesis, 330-331, 331f
PRPP synthetase defect in, gout caused by, 334, 336t
in purine synthesis, 330, 331f,

in pyrimidine synthesis, 334, 334f
PSA (prostate-specific antigen), 700
pseudo-first-order conditions, 71, 73
pseudogenes, 360
pseudouridine, 336, 337f psi angle, 34, 34f
PstI, 433t
PstI site, insertion of DNA at, 437, 437f pterins, 95, 96f
PTH. See parathyroid hormone
PTSs (peroxisomal-matrix targeting sequences), 574t, 579, 579f
PTT. See activated partial thromboplastin

"puffs," polytene chromosome, 354, 354f pumps, 467, 467f, 473
punctuation of genetic code, 395
purification of glycoproteins, 547
of proteins and peptides chromatographic techniques, 23-26,

purine nucleoside phosphorylase deficiency, 327, 335, 336t

chemistry of, 320, 320f derivatives of, 320-321, 322t
dietary nonessential, 328
in DNA, 338-339, 339f gout and, 334
metabolism of biomedical importance of, 327-328
disorders of, 334-335, 336t

serine conversion to, 300
substitution mutation of, 396-397, 396f,
397f, 398f synthesis of, 328, 328f, 329f, 330, 330f
pyrimidine synthesis coordinated with, 334, 334f
regulation of, 332, 332f, 334, 334f synthetic
analogs of,
324f ultraviolet light absorption by, 321

purple acid phosphatases, iron in, 97
pyranose ring structures, 143, 143f pyridoxal. See vitamin B,
pyridoxal phosphate (PLP), 273, 273f, 535
pyridoxamine. See vitamin B, pyridoxine.
See vitamin
B, pyrimethamine, 537

in DNA, 338-339, 339f metabolism of, 335f
biomedical importance of, 327-328
diseases caused by catabolite overproduction and, 336, 336t
water-soluble metabolites of,
335-336, 337f precursors of, deficiency of, 336

synthesis of, 328, 332, 333f analogs in, 334
catalysts in, 332, 333f purine synthesis coordinated with,
334, 334f regulation of, 332, 332f, 334, 334f
synthetic analogs of, 324f ultraviolet light absorption by, 321
pyrophosphatase in fatty acid activation, 208
in glycogenesis, 164, 165f in RNA synthesis, 378
pyrophosphate (PP,)
in fatty acid activation, 208, 208f in glycogenesis, 164, 165f
in RNA synthesis, 378
pyrrole rings, of porphyrins, 305, 306f
A'-pyrroline-5-carboxylate dehydrogenase, 282, 282f
pyruvate, 136, 138
in citric acid cycle, 153, 154f in gluconeogenesis, 172-173, 173f
glycolysis production of, 157, 158f, 158¢,
159-160, 159f inhibition of metabolism of, 163
in transamination reactions,
272-273, 273f pyruvate carboxylase, 153-154, 154f
in gluconeogenesis, 172, 173f regulation of, 175
pyruvate dehydrogenase (PDH)
in pyruvate oxidation, 161, 162f regulation of, 89f, 155-156,
161-163, 162f acyl-CoA in, 221
pyruvate dehydrogenase complex,

pyruvate kinase deficiency of, 163
in glycolysis, 159-160, 159f regulation of, 161, 175

pyruvate kinase (PK)
in cancer cells, 696, 697f deficiency of, 6508, 651
pyruvate oxidation,
161-163,
162f induction and repression of enzymes
catalyzing, 174-175, 175t
Q
Q. See coenzyme Q
Q cycle, 120, 121f
Q,,See temperature coefficient
Q-cytochrome c oxidoreductase (Complex
IID), 118, 119f, 120, 120f, 121f
QT
interval, congenitally
long,
478t quadrupole mass spectrometers, 28, 29f
quality control, for laboratory tests, 561
quaternary structure, 36-37, 38f, 39f of
50-53,
50f,
51f,
52f, as order of protein structure, 34
schematic diagrams of, 38-39
stabilizing factors in, 39
R
a-R groups, of amino acids, 20
R state. See relaxed state
Rab effector proteins, 586t, 587
Rab molecules, 587
Rab proteins, 586-588, 586t radiation
carcinogenic effect of, 683, 683t
DNA damage caused by, 370f, 370t radioimmunoassays, laboratory tests
using, 565
Ramachandran plot, 34f
53f
Ran proteins, 578
rancidity, peroxidation causing, 203
random-order reactions, 79, 79f
RAS oncogene, 685, 685, 687t rate
constant
(k),
71-72
rate of reaction activation energy and, 69-70, 70f, 72
Gibbs free-energy change and, 69
hydrogen ion concentration effects on,
72-73,
73f initial velocity, 73-76, 73f, 75f, 76f,
79,
80f maximal velocity. See maximal velocity
Michaelis constant. See Michaelis constant
substrate concentration effects on, 71,

Rb protein. See retinoblastoma protein reactants. See substrates
reactive oxygen species (ROS), 649, 709
antioxidant paradox
and,
544-545,
544t in cancer development, 683, 700
chain reactions and, 710
damage caused by, 541-542, 542f enzymatic and chemical mechanisms
intercept damaging, 713
heavy metal formation of, 94
hydroperoxidase reactions
with, in
lipid peroxidation,
204, mechanisms
204f against,

reaction with biological molecules, 711f respiratory burst generation of, 660
self-perpetuating chain
541
sources of, 542-543, 543f superoxide dismutase reactions with,
116
as toxic byproducts of life, 710f receptor-corepressor complex, 502
receptor-effector coupling, 482
receptor-mediated endocytosis, 474f, 475
recombinant DNA technology

protein production, 441-442
restriction fragment length polymorphism, 445
single nucleotide polymorphisms,

targeted gene regulation, 446-447
blotting and probing techniques for,
438, 439f chimeric molecules in, 432
114
cloning in, 435-438, 436f, 437f, 437t
DNA
sequencing,
439,
440f enzyme studies using, 65-66, 66f,
genomic library for, 438
hematology and,
655
library probe for, 438
oligonucleotide synthesis, 439
polymerase chain reaction method,
439-440, 441f restriction enzymes in
for chimeric DNA molecule preparation, 434-435
DNA
with,
433-434, 434f selection of, 433-434, 433t, 434t
recombinant fusion proteins, 65-66, 66f recombinant t-PA, 679
recombinases, 434t, 435
recombination, chromosomal, 359,

recruitment hypothesis, 386
red blood cells
ABO system, 653-654, 654f biomedical importance of, 646
CO, transport by, 648
derivation from hematopoietic stem cells, 646-647, 647f
diseases affecting, 646, 650t functions of, 647-648, 647f, 648t
glycolysis of, 648, 648t lifespan of, 648-649
membrane of, 651-653, 651f, 652f, 652t replacement of, 648-649
red thrombus, 670
redox potential (E',)
free energy changes and, 111-112, 112¢
of transition metals in organometallic complexes, 95-97

free energy changes of, 111-112, 112t iron participation in, 97
redox state, 210
reducing equivalents citric acid cycle production of,
151-153, 152f pentose phosphate pathway generation

respiratory chain oxidation of,
118-121, 119f, 120f, 121f
5a-reductase, 487, 489f

112-114, 113f hydroperoxidase reactions, 114
of oxygen by Complex IV, 120-121
and,
111-112,
112t reference range, of laboratory tests, 561
215,
580,
580t regional heterogeneities, 464
regulated secretion, 574
regulation. See metabolic regulation regulatory domains, 37
regulatory enzymes, of metabolic

regulatory transcription factor proteins,
DNA-binding domains of, 425t helix-turn-helix motif, 425-426, 426f
leucine zipper
motif,
427,
427f zinc finger motif, 426, 426f
relaxation phase of muscle contraction,
616
relaxed form of DNA, 341
relaxed (R) state, of hemoglobin, 50-52,

releasing factor 1 (RF1), 403, 404f releasing factor 3 (RF3), 403-404, 404f
remnant removal disease, 258t renal glomerulus, 598
renaturation, DNA, base pair matching

rennin, 56
repair mechanisms and proofreading for
DNA, 713-714
repetitive-sequence DNA, 357-358
replication. See DNA, replication and synthesis of
replication bubbles, 364, 366f,

replication fork, 362f, 363, 366f replication
683
replication protein A (RPA), 364
replicative senescence, 716
reporter gene approach, 422f, 423, 424f repression, of enzyme synthesis, 84
repressor gene, lambda (cI), 415, 415f repressor protein, lambda (cI),

repressors in
410-412
reproduction, prostaglandins in, 216
RER. See rough endoplasmic reticulum
REs. See restriction enzymes resonance structures, of amino acids,

respiration, 111
respiratory burst, 525, 660
free radicals from, 542

catabolic energy captured by, 121-122
citric acid cycle production of substrates for, 150-151, 151f
coenzyme Q in, 118, 119f, 120,
120f,
121f complexes of, 118-121, 119f,
120f, 121f control of, 122-124, 123t
cytochrome c in, 118, 119f, 120,

dehydrogenase role in, 112
electron transport in, 118, 119f,
120-121, 120f, 122f flavoproteins and iron-sulfur proteins
in,
118-120,
119f
respiratory chain (Cont.):
inhibition of, 112, 118, 123-124,
123f, 124f in mitochondrial compartments,
118, 118f mitochondrial exchange transporters
and, 124-126, 124f, 125f, 126f oxidation of reducing equivalents by,
118-121,
119f,
120f,
121f oxygen reduction in, 120-121
proton gradient of, 121, 122f proton pump action of, 118-121, 119f

respiratory chain level phosphorylation,
122
respiratory control, 106, 155
respiratory distress syndrome, surfactant deficiency
and,
200,
229,
234
respiratory quotient (RQ), 524
restriction endonucleases clinical diagnosis using, 64
direct observation of, 62f restriction enzymes (REs), 348
434-435

selection of, 433, 433t restriction fragment length
polymorphisms (RFLPs), 65, 445
restriction map, 434-444
restriction sites, 64
reticulocytes, 649
retina, gyrate atrophy of, 282
retinaldehyde, 529, 529f retinitis pigmentosa, essential fatty acid
deficiency and, 224
retinoblastoma protein (Rb protein), oncoproteins and, 369
retinoic acid, 529-530, 529f retinol. See vitamin A
retinol activity equivalent, 529
retrograde transport (COPI),
582-584, 586
retrograde vesicular transport, 583
retroposons, 357
retrotranslocation, 584, 585f retroviral genomes, 437
retroviruses, reverse transcriptases in, 344,

reverse cholesterol transport, 241f, 242,
250, 254, 254f, 257
reverse transcriptase/reverse transcription,

Reye syndrome, 226
orotic aciduria in, 336
RFI (releasing factor 1), 403, 404f
RF3 (releasing factor 3), 403-404, 404f
RELP (restriction fragment length polymorphisms), 65, 445
rheumatoid arthritis, 592
w3
and,
199
rho (p) factor, 378
rho kinase, 617
rhodopsin, 529, 530f ribbon diagrams, 38
riboflavin. See vitamin B, riboflavin-linked dehydrogenases, 114
ribomyopathies,
649
ribonucleases, 348
ribonucleic acid. See RNA
ribonucleoprotein particles (mRNPs), 405,
405f, 430
ribonucleoside diphosphates, 332, 332f ribonucleosides, 320, 321f
ribonucleotide reductase gallium in, 94
iron in, 97
ribonucleotide reductase complex,
332, 332f ribose, 141, 144f, 144t
metabolic pathways of, 130
in nucleosides, 320, 321f pentose phosphate pathway formation

D-ribose, 320, 321f ribose 5-phosphate, in purine synthesis,
328, 329f, 330, 331f ribose-5-phosphate ketoisomerase, in
pentose phosphate pathway, 183f,
184f,
185
ribosomal dissociation, in protein synthesis, 399
ribosomal RNA (rRNA), 343, 374, 375t amplification of, 429, 429f
function of, 347
as peptidyl transferase, 402, 403t precursors for, 389
in protein synthesis elongation, 402-403, 402f
initiation, 399, 400f, 401
structure of, 347, 347t ribosomes, 134, 347, 347t
bacterial, 406-407
profiling of, 447
in protein synthesis, 399
as ribozyme, 66
structure of, 574
ribothymidine pseudouridine cytidine
(TyC) arm of tRNA, 346f, 347,
396, 396f ribozymes, 66-67, 343, 391
ribulose, 144f, 144¢
ribulose-5-phosphate, in pentose phosphate pathway, 183-185, 183f,
184,
184f ribulose-5-phosphate 3-epimerase, in
184f,
185
Richner-Hanhart syndrome,
286,
287f ricin, 407
rickets, 531-532
Rieske iron centers, 97, 98f, 120, 121f right operator, 415, 415f
right-handed tripleor superhelix,
593, 593f rigor mortis, 616
RISC (RNAinduced silencing complex),
391

biomedical importance of, 338, 374
catalysis by, 66-67
as catalysts,
391
in chromatin, 351
circular, 348
classes/species of, 344-348, 374, 375t complementarity of, 342-343,

long noncoding, 348, 374, 375t messenger. See messenger RNA
micro. See micro-RNAs modification of
of mRNA, at 5' and 3' ends, 389-390
mRNA sequence alteration, 391
pri-miRNA, 389-390, 390f tRNA processing and, 391
396-398, 396f, 397f, 398f, 411
noncoding. See noncoding RNAs as polynucleotides, 325

alternative, gene expression and, 430
alternative promoter utilization in,
388-389, 389f alternative splicing, 388, 388f
in gene expression regulation,
428, 428t of rRNAs and tRNAs, 389
sequences to remove, 386,
387f, 388f after transcription, 386
in protein synthesis, 343-344
ribosomal. See ribosomal RNA
silencing. See silencing RNAs small,
347-348
small, heterogeneous regulatory, 348
small nuclear. See small nuclear RNA
splicing of, 386-388, 387f alternative, 388, 388f
structure of, 342-343, 343f, 344f,

synthesis of, 339. See also transcription cyclical process of, 378-379
DNA synthesis compared with, 375
DNA template strand for, 375, 375f
RNA polymerase in, 375-377,
376f, 377f transfer. See transfer RNA
in viruses, 344
RNA blot procedure, 438, 439f
RNA editing, 391
384-385, 385t formation of, 383-384
phosphorylation activation of,

RNA processing and, 386, 387f termination signals for, 383
in transcription, 380-381, 380f,

RNA polymerases (RNAP)
bacterial, 377, 377f initiation by, 375-377, 376f, 377f
mammalian, 378, 378¢
RNA primer, in DNA synthesis, 362f, 362t,

RNA recognition motifs (RRM), 387
RNA world, 66-67
RNA-dependent DNA polymerase, 360
RNA-induced silencing complex (RISC),

RNAP. See RNA polymerases
RNA-RNA hybridization, 348
RNAse H, 434t
RNA-Seq, 447
ROS. See reactive oxygen species
Rossmann fold, 37
rough endoplasmic reticulum (RER)
binding to, 575f, 580-582, 580t,

in protein sorting, 574, 574f, 585f protein
405
routes of protein insertion into,

cotranslational pathway, 580-581, 581f posttranslational translocation,
581-582, 582f
N-terminal signal peptides in, 580, 580t
virus
(RSV),
685
RPA (replication protein A), 364
RQ (respiratory quotient), 524
RRM (RNA recognition motifs), 387
rRNA. See ribosomal RNA
RSV (Rous sarcoma virus), 685
ryanodine, 618
Ss
S phase of cell cycle, DNA synthesis during, 368-369, 368f,
369f, 369t
S1 nuclease, in recombinant DNA
technology, 434t

SADDAN phenotype, 609
salt bridges, 8
in hemoglobins, 52-53
"salvage" reactions in purine synthesis, 330, 331f
in pyrimidine synthesis, 332-333
samples, for laboratory analysis, 563
sandwich assay, laboratory tests using, 565
Sanfilippo syndrome, 604t
Sanger, Frederick, sequencing work of,

Sanger reagent, 27
sarcoglycan, 622, 623f sarcolemma, 612
sarcomere, 612, 612f sarcoplasm, 612
sarcoplasmic reticulum, 618, 618f sarcosine, 300-301
saturated acyl enzyme, 217, 219f saturated fatty acids, 196, 196f, 197, 197t
in membranes, 461-462, 461f saturation kinetics, 76
scaffolding, 352f, 353
scavenger receptor B1, 241f, 242
screening, for new drugs, 80
scurvy, 43, 263, 266, 540, 596
SDs (silencing domains), 428, 428f
SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel
electrophoresis), protein and peptide purification with, 26, 26f

Sec61 complex, 581
Sec61/Sec63 complex, 581
second law of thermodynamics, 106
second messengers, 482, 483t, 502. See also

enzyme regulation by, 86
phosphatidylinositide as, 200, 200f phospholipids
as,
229
secondary lysosomes, 474
secondary structure a helices, 35-36, 35f, 37f
6 sheets, 36, 36f, 37f of collagen, 43, 43f
folding into, 41
loops and bends, 36, 37f of myoglobin and hemoglobin, 50
peptide bond
restrictions on,
34-35, secondary structure, of RNA, 342,

secondary transport, 469
second-order rate constant, 71
secretory (exocytotic) pathway, 574
secretory proteins, 581
secretory vesicles, 574, 575f selectins, 553-554
selective permeability of inner mitochondrial membrane,

membrane, 459, 470, 470f selective proteolysis, enzyme regulation

selectivity filter, 470, 471
selenium,
204,
244
in glutathione peroxidase, 114,
185-186, 186f human requirement for, 93, 93t
selenocysteine, 15, 17, 17f synthesis of, 267-268, 268f
34f
signal generation, in group II hormones
(Cont.):
cGMP, 506
G-protein-coupled receptors,
503, 503f intracellular, 502-503
phosphatidylinositide, 507-508,

protein kinases, 508-511, 509f, 510f signal hypothesis of polyribosome

signal peptidase, 581, 581f, 629
signal peptide, 574
of albumin, 630
in protein sorting, 574f, 576, 577f,
580-582, 581f, 582f signal recognition particle (SRP),
580-581, 581f, 629
signal recognition particle (SRP) receptor,
580-581, 581f signal sequences, 573-575, 574t,

signal transducers and activators of transcription (STATs),
509, 510f

CBP/p300 and, 513-514, 513f of
hormones,
500-501,
501f across membranes, 459, 467t, 475-476
silencers, of gene expression, 410
DNA elements, 422-423
silencing, of gene expression, 87
silencing domains (SDs), 428, 428f silencing RNAs (siRNAs), 348, 374, 375t

synthesis of, 390-391, 390f silent mutations, 396, 396f
simple competitive inhibition, 77, 77f simple diffusion, 467-468, 467f,

simvastatin, 257
SINEs (short interspersed nuclear elements), 357-358
single nucleotide polymorphisms (SNPs),
442, 445
single quadrupole mass spectrometers,
28,
29f single-cell sequencing, in cancer research,
701
single-displacement reactions, 79, 79f single-molecule enzymology, 62, 62f
single-strand binding proteins (SSBs),
362f, 363-364, 363¢
single-stranded DNA (ssDNA), 361
siRNA-miRNA complexes, 348
siRNAs. See silencing RNAs sirtuins, 89
sister chromatids, 355, 355f, 361, 361f site-directed mutagenesis, enzyme studies

Sjégren-Larsson syndrome, 226
skeletal muscle, 612, 619t actin-based regulation in, 616
glucose uptake by, 136-137, 137f glycogen, supplies of, 620
lactate production by, 160
malignant hyperthermia of,
618-619, 620t metabolic role of, 133
as protein reserve, 623
sarcoplasmic reticulum in, 618, 618f twitch fibers of, 621

calcitriol synthesis in, 489-490, 490f diseases of, 625
essential fatty acid deficiency and, 225
vitamin D synthesis in, 530, 531f skin bleeding time, 679
sleep, prostaglandins in, 216
slow twitch fibers, 621
slow-reacting substance of anaphylaxis,

Sly syndrome, 604t
SMAC, in apoptosis, 693f, 694
small, heterogeneous regulatory RNAs
(sRNAs), 348
small nuclear RNA (snRNA), 343-344,
344t, 347, 374, 375t in alternative splicing, 388
in RNA processing, 386-388
small RNA, 347-348
smooth muscle, 612, 619t actin-myosin interactions in,
617, 617t regulation of contraction by myosin,
616-618, 617f, 617t
SNAP (soluble NSF attachment factor)
proteins, 586t, 587f, 588
SNARE pin, 588
SNARE proteins, 586-588, 586t, 587f
SNPs (single nucleotide polymorphisms),

snRNA. See small nuclear RNA
sodium (Na*)
460,
460t permeability coefficient of, 463f
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),
protein and peptide purification with, 26, 26f
sodium-potassium pump (Na*-K*-
ATPase), 472-473, 472f, 473f
SODs. See superoxide dismutases solubility
of amino acids, 20, 20f lipids,
196
of soluble NSF attachment factor (SNAP)
soluble proteins, 34, 581
solvent, water as ideal, 6-7, 7f somatic mutation theory of aging, 714
sonication, mild, 465
sorbitol, in diabetic cataract, 190-191
sorbitol dehydrogenase, 186, 188f sorbitol pathway, 190-191
Soret band, 308, 310f
Southern (DNA/DNA) blotting, 341
Southern DNA blot transfer procedure,

specialized compartments, 459
specific acid-base catalysis, 59
specific activity, 75

of enzymes, 57, 57f of hormone receptors, 481, 481f
of laboratory test, 562, 562f, 563t spectrin, 651, 652, 652f, 652t, 653
spectrophotofluorimetry, 563-564
spectrophotometry enzymatic assays using, 63, 63f
laboratory tests using, 563-564
of porphyrins, 308, 310f spectroscopy, protein structures solved

spermidine, 299, 299f, 300f spermine, 299, 299f, 300f
spherocytosis, hereditary, 478t, 650t,

sphingolipidoses, 234-235, 235t sphingolipids, 199, 201f, 229
clinical aspects of, 234-235, 235t formation of, 233-234, 234f
in multiple sclerosis, 234
sphingomyelins, 199, 200f membrane asymmetry and,

synthesis of, 233-234, 234f sphingophospholipids, 196
sphingosine, 199, 200f serine conversion to, 300
spina bifida, folic acid supplements for,

spontaneous assembly, 594
spontaneous chemical reactions, 69
spontaneous mutations, 683
squalene, in cholesterol synthesis, 250,

squalene epoxidase, in cholesterol synthesis, 250, 252f
squalene synthetase, 251f
SR (serine-arginine motifs), 387
SR-B1 (scavenger receptor B1),
2Alf, 242
Src homology 2 (SH2), 508, 509f
SREBP (sterol regulatory element-binding protein), 252
sRNAs (small, heterogeneous regulatory
RNAs), 348
SRP (signal recognition particle),
SRP receptor (SRP-R), 580-581, 581f
SSBs (single-strand binding proteins),
362f, 363-364, 363t ssDNA (single-stranded DNA), 361
staggered ends, 433, 434f standard free-energy change (AG")
of
ATP
hydrolysis,
107-108,

in biologic systems, 106
StAR (steroidogenic acute regulatory)
protein, 485, 485f starch, 145, 147f
hydrolysis of, 520
Starling forces, 628
starvation, 105
ketosis in, 215
metabolic pathways in, 138-139, 138t,
139f,
139¢
triacylglycerol redirection and, 240
statin drugs, 250, 250f, 257
statins, 84
STATs (signal transducers and activators of transcription), 509, 510f
stearic acid, 197t stearoyl-CoA, 223, 223f
stem cell biology, 4, 446
stem cell factor, 647
stem cells in cancer, 696
hematopoietic, 646-647, 647f induced pluripotent, 446
step-wise assembly, 386
stereochemical (-sn) numbering system,

stereochemistry, of L-a-amino acids, 17
stereospecificity, of enzymes, 57, 57f steroid sulfates, 234, 235
testicular, 487, 488f steroidogenic acute regulatory (StAR)
protein, 485, 485f steroids
cytochrome P450 in metabolism of,

lipid bilayer and, 462
metabolic pathways of, 131, 131f nucleus of, 202, 202f
as precursor molecule, 483, 484f receptors for, 482
roles of, 202
stereoisomers of, 203, 203f storage of, 497, 497t
vitamin B, role in actions of, 535
sterol 27-hydroxylase (CYP27A1),
255-256, 256f sterol regulatory element-binding protein
(SREBP), 252
sterols, 202, 461
Stickler syndrome, 608
sticky end ligation, 434-435
sticky ends, 433, 434f sticky patch, in hemoglobin S, 54, 54f
stochastic processes, mortality and aging

stoichiometry, 69
Stokes radii, 24-25
stop codon, 403-404, 404f stop-transfer signal, 582, 583f
storage granules, of platelets, 670
strain, enzymatic catalysis by, 59
streptokinase, 64, 679
streptomycin, 145
striated muscle, 613f, 616, 617t, 619
strong acids, dissociation of, 10-11
strong bases, dissociation of, 10-11
structural proteins, 592. See also collagen;
elastin; fibrillin substrate analogs, 77, 77f
substrate cycles, in glycolysis and gluconeogenesis

substrate deprivation therapy, 235
substrate level phosphorylation, 122
substrate shuttles, mitochondrial,
125-126,
125f

anaplerotic, 153-154
cellular concentrations of, 83
conformational changes induced by,

multiple,
79,
79f,
80f pairs of, 125, 125f
prosthetic group, cofactor, and coenzyme interactions with,

reaction rate response to concentrations of,
71,
73-74,
73f,
74f regulation of metabolic pathways with,
135
for respiratory chain substrates,
150-151, 151f succinate dehydrogenase, 114
inhibition of, 77, 77f succinate thiokinase (succinyl-CoA
synthetase), 152f, 153, 247, 247f succinate-Q reductase (Complex II), 118,
119f, 120, 120f succinic acid, pk, of, 13t
succinic semialdehyde dehydrogenase, defects in, 303, 303f
heme biosynthesis from, 305-307, 306f,
307f, 308f, 309f, 310t succinyl-CoA synthetase (succinate
thiokinase), 152f, 153, 247, 247f succinyl-CoA-acetoacetate-CoA
transferase (thiophorase),
211, 213f in citric acid cycle, 152f, 153
sucrose, physiological importance of, 145,

sugar nucleotides, in glycoprotein synthesis, 549-550
sugars, in amphipathic lipids, 462
"suicide enzyme,' cyclooxygenase as,
224-225
suicide inhibitors, 78-79
sulfate, in glycoproteins, 547
sulfatide, 202
sulfation, of xenobiotics, 558
sulfite oxidase deficiency of, 100
transition metals in, 100, 100f sulfo(galacto)-glycerolipids, 234
sulfogalactosylceramide, 202
synthesis of, 234
sulfonylurea drugs, 215
sulfotransferases, 600
sumoylation, of histones, 351, 353t supercoils, DNA, 341, 367, 368f
superoxide, 649-650
superoxide anion-free radical, 116
superoxide dismutases (SODs), 204,
649-650
gallium toxicity to, 94
oxygen toxicity protection by,
116, 544
transition metals in, 96-97
supersecondary structures, 36
suppressor mutations, 398-399
suppressor tRNA, 398
supravalvular aortic stenosis, 596
surfactant, lung, 229
deficiency of, 200, 234
SV40 enhancer, 421, 422f
Svedberg units, 347
symport systems, 468, 468f syn conformers, 320, 321f
synaptic vesicles, 587
synaptobrevin, 587
5q-syndrome, 649
synthetic biology, 4
systems biology, 4, 447-449
T
T lymphocytes, 639, 656, 662
T
state.
See taut
state t,.See half-life
T,. See triiodothyronine
T, (thyroxine). See tetraiodothyronine
TAFla (thrombin-activatable fibrinolysis inhibitor), 678f, 679
TAFs (TBP-associated factors), 380, 381f,
383-385

tandem MS (MS-MS, MS?)
metabolic disease detection with,
278, 280
protein analysis using, 30
Tangier disease, 258t
TaqI, 433t target cell, 476, 481, 481t
TATA box, in transcription control in bacteria, 379-380, 380f
in eukaryotes, 379-381, 380f, 381f
TBP binding with, 383-384
TATA-binding protein (TBP), 380
TATA box binding of, 383-384
taurine, cysteine conversion to, 297, 297f taurochenodeoxycholic acid, 256f
taurocholic acid, 256f taut (T) state, of hemoglobin, 50-52,

Tay-Sachs disease, 235t, 585t
TBG (thyroxine-binding globulin), 498
TBP. See TATA-binding protein
TBP-associated factors (TAFs), 380, 381f,

telomerase, 355, 692, 716
telomeres, 355, 355f, 716, 717f temperature
in fluid mosaic model of membrane structure, 465
reaction rate response to, 70, 7If, 72
transition, 340, 465
temperature coefficient (Q,,), 72
template binding, in transcription, 376f template strand DNA
in DNA replication, 339, 343, 344f in RNA synthesis, 375, 375f, 380f
peptide,
574,
574f,
580-582, 580t, 581f, 582f terminal transferase, 434t

of protein synthesis, 403-404, 404f in proteoglycan synthesis, 600
in transcription cycle, 376-379,

termination protein, 378
termination signals, of translation, 394, 394t tertiary structure, 36-37, 38f, 39f
of collagen, 43, 43f of myoglobin and hemoglobin,
testicular steroidogenesis, 487, 488f

metabolism of, 487, 489f plasma transport of, 498f, 499
synthesis of, 483, 484f, 485f, 487, 488f
50
testosterone-estrogen-binding globulin
(TEBG), 499
tethering, 586-587
tetracycline (Tet) resistance genes, 437
tetrahedral geometry, of water molecules,
6,
7f tetrahedral transition state intermediates,

tetrahydrofolate, 537, 537f tetraiodothyronine (thyroxine; T,)
plasma transport of, 498, 498¢
storage of, 497, 497t synthesis of, 483, 484f, 492, 493f
tetramers, histone, 351, 351f, 353
tetrapyrroles, in heme, 48, 48f, 305, 306f tetroses, 141, 142t
of physiological importance, 143
TF
(tissue factor),
673,
673t,
674t
TFIIA, 381, 381, 382f, 383-384
TFIIB, 381, 381f, 382f, 383-384
TFIID, 381, 381f, 382f components of, 384-385
383-384
TFIIE, 381, 381f, 382f, 383-384
TFIIH,
381,
381f,
382f
CTD modification by, 384

TFPI (tissue factor pathway inhibitor), 673
TGN (trans-Golgi network), 574, 575f, 588
thalassemias, 54
a-thalassemias, 650t
B-thalassemias, 442, 443f, 650t pathologic protein conformations in, 42
thanatophoric dysplasia, 609
theobromine, 321, 323f theophylline, 246, 321, 323f
thermodynamics, of biologic systems,
105-106
thermogenesis, 247, 247f

thermogenin, 123-124, 247, 247f thiamin. See vitamin B,
thiamin diphosphate, 161, 162f,
533-534
thick (myosin) filaments, 612-613, 613f thin (actin) filaments, 612-613,

thioesterase (deacylase), 218, 218f, 219f
6-thioguanine, 324, 324f thiokinase. See acyl-CoA synthetase;
succinate thiokinase thiolase, 209, 209f, 211
in mevalonate synthesis, 250, 250f thiophorase (succinyl-CoA-acetoacetate-
CoA transferase), 211, 213f in citric acid cycle, 152f, 153
thioredoxin, 332, 332f thioredoxin reductase, 332, 332f
threonine, 15t carbon skeleton catabolism of, 285, 285f

in fibrin clot formation, 672
fibrinogen conversion to fibrin by,
675-677, 675f, 676f inhibition of, 677-678
in platelet aggregation, 670, 671f prothrombin conversion to,

regulation of, 677
thrombin-activatable fibrinolysis inhibitor
(TAFla), 678f, 679
thrombocytopenia, 646, 654-655
thrombomodulin, 677
thrombopoietin, 647
thrombosis, 654
alteplase and streptokinase treatment

anticoagulant drug effects on, 677-678
fibrin clot formation in. See fibrin clot formation
laboratory tests for, 679
thrombin regulation
in,
677
thromboxane A, (TxA,), 198f, 224

in platelet aggregation, 670, 671f thromboxanes (TXs), 197, 198, 198f, 216
cyclooxygenase pathway in formation

formation of, 670, 671f types of, 670
thymidine, 322t salvage pathways of, 333
thymidine monophosphate (TMP),

thymidylate, 338-339
thymine, 322t, 337f base pairing in DNA, 339, 340f
thymine dimer formation and UV light,

thyroglobulin, 492
thyroid function tests, 567
thyroid hormones in cholesterol synthesis, 252-253, 253f
in lipolysis, 246, 246f plasma transport of, 498, 498¢
receptors for, 482
regulation of gene expression by,
501, 501f storage of, 497, 497t
synthesis of, 492, 493f thyroxine (T,). See tetraiodothyronine
thyroxine-binding globulin (TBG), 498
ticagrelor, 672
tight junctions, 466
tiglyl-CoA, catabolism of, 292f, 293f
TIM (translocase of the inner membrane),
576
time-of-flight (TOF) mass spectrometers,

timnodonic acid, 197t tirofiban, 672
tissue factor (TF), 673, 673t, 674t tissue factor pathway inhibitor (TFPI), 673
tissue injury fibrin clot formation in, 672-673, 672f,

myoglobinuria after, 54
serum enzyme analysis after, 64, 65f tissue nonspecific alkaline phosphatase
(TNAP), 605
tissue plasminogen activator (tPA), 64,

tissues differentiation of, retinoic acid role in,
529-530
139f, 139t in fed state, 130, 136-138, 137f, 138t
glucose uptake by, 136-137, 137f hypoxic,
160
metabolic pathways at level of,
132-133,
132f,
133f titration curve, for weak acids, 12, 12f
T.,. See transition temperature
TME (tumor microenvironment), 682,
694-695, 695f
TMP (thymidine monophosphate),
322f, 322¢
TNAP (tissue nonspecific alkaline phosphatase), 605
tobacco, 703
tocopherols. See vitamin E
tocotrienols. See vitamin E
TOF (time-of-flight) mass spectrometers,
28-29
tolbutamide, 215, 243
TOM (translocase of the outer membrane), 576
topogenic sequences, 583
topoisomerases, DNA, 341, 363t, 367, 367f total bilirubin, 313
total energy expenditure, 524
total iron-binding capacity, 633
totipotent stem cells, 646
toxic hyperbilirubinemia, 314

of ammonia, 273-274
bilirubin, 313-314
of folic acid, 538
heavy metal, 93-94
lead, 93-94, 306, 309
micronutrient, 527
niacin, 535
oxygen. See reactive oxygen species of plant amino acids, 18, 18t
of transition metals, 94-95, 95t vitamin
vitamin
A,
B,,
530
535
vitamin
D,
532
of xenobiotics, 559, 559f toxins
botulinum B, 587
cholera, 202, 202f, 234, 504
diphtheria, 407, 472
microbial, 472
pertussis, 504
tPA (tissue plasminogen activator), 64,

TwC arm. See ribothymidine pseudouridine cytidine arm
trans fatty acids, 198, 226, 462
transactivator proteins, 420, 421f transaldolase,
in pentose
phosphate pathway, 183-185, 183f, 184f

in amino acid catabolism, 281-282
vitamin B, assessment with, 535
transamination in amino acid metabolism, 131, 132f,
153-154, 154f ping-pong mechanism for, 59, 59f,

transcortin,
498-499,
498f transcription, 341
activators and coregulators in control of, 384-385, 385t
bacterial promoters in, 379, 380f eukaryotic promoters in, 379-381, 380f,
381f, 382f, 383
eukaryotic transcription complex,
383-386
hormone modulation of, 511-514, 512f,
513f, 513t, 514t initiation of, 375-377, 376f, 377f
promoters in, 376
alternative, 388-389, 389f protein-DNA interactions in regulation
of, 414-418, 414f, 415f, 416f, 417f reverse in retroviruses, 344, 360,
367-368
in RNA synthesis, 339
termination of, 383
transcription complex, eukaryotic.
See eukaryotic transcription complex
transcription control elements, 385t transcription factors, 385t, 717
for enzyme synthesis, 84
transcription start site (TSS), 376, 377f alternative, 430
transcription unit, 376
transcriptome information, 447
transcriptomics, 3
transcytosis, 588
transdeamination, 273, 274f transducers, 370, 371f
transfected cells in culture, 423, 424f transfer RNA (tRNA), 343, 374, 375t
amino acid association with, 395-396,

anticodon region of, 394-396, 396f.
345-346
processing of modification and, 391
precursors for, 389
initiation, 399, 400f, 401-402
structure of, 346-347, 346f suppressor, 398
transferases, 57
transferrin, 523, 523f, 627, 632-633, 632t,

transferrin cycle, 633, 634f transferrin receptor, 633, 634-635, 635f.
transgenic animal approach, 423
transgenic animals, 446
trans-Golgi network (TGN), 574,

transhydrogenase, mitochondrial, 125
transition metals
absorption and transport of, 101
of,
92
heavy metal toxicity and, 93-94
for,
93,
93t
Lewis acid properties of, 93
multivalent states of, 93, 94f, 94t, 95
in organometallic complexes, 95-97,

oxidation of, 93, 94f, 94t physiologic roles of, 97-101, 97f, 98f,

toxicity of, 94-95, 95t transition mutations, 396, 396f
transition state of chemical reactions, 69-70, 70f
enzymatic stabilization of, 72
transition state analogs, 59, 76
transition state intermediates, 59, 70, 70f acyl-enzyme, 61, 61f
tetrahedral, 60, 60f transition state stabilization, in enzyme
catalysis, 59
transition temperature (T.,), 340, 465
transketolase in pentose phosphate pathway,

vitamin B, assessment with, 534
translation, 394. See also protein synthesis translation termination signals, 394, 394t
translocase of the inner membrane (TIM),

in cancer, 685, 686f into cell nucleus, 577-578, 578f
of mRNA, 578
of proteins, 576
translocation, in protein synthesis, 403
translocation complexes, 576
translocon, 580
transmembrane collagens, 594
transmembrane proteins amino acid sequence of, 463
ion channels
as,
469-471,
470f,

transmembrane signaling, 459,
467t, 475-476
in cancer, 689, 689t in platelet aggregation, 670, 671f

coat proteins of, 586
definition of, 585
model of, 586-588, 586, 587f proteins in, 574, 575f
in trans-Golgi network, 588
transporters and transport systems
ATP in, 472-473, 472t transporters involved in, 468-469,

ATP-binding cassette, 241f, 242
ATP-driven active, 472, 473t facilitated diffusion, 467-468, 467f,

hormone regulation of, 469-470
of,
469,
469f transporters involved in, 468-469,

glucose. See glucose transporters ion channels and, 468t
membrane fatty acid, 238
of membranes, 459, 463, 469-470
of mitochondria, 124-126, 124f,

467t, 468f plasma transport proteins, 498-499,
498t reverse cholesterol, 241f, 242, 250, 254,
254f,
257
simple diffusion, 467-468, 467f,

tricarboxylate, 221
transposition, 360, 442
retroposons and, 357
transthyretin, 639
transverse asymmetry, 589
transverse movement, of lipids across membrane, 463-464
transversion mutations, 396, 396f trauma, protein loss in, 526
trehalose,
146f,
146¢
triacylglycerol as metabolic fuel, 130, 136
metabolic pathways of, 131, 131f,

in fed state, 137-138, 137f, 138t triacylglycerol synthesis, 134
T-tubular system, 619
a-tubulin, 624, 625f
B-tubulin, 624, 625f y-tubulin, 624
tumor biomarkers, 700-701, 701t tumor microenvironment (TME), 682,

tumor progression, 688
tumor suppressor genes, 685-688, 685¢,
686f, 687f, 6871, 688f, 688¢
p53, 372
tumor viruses, 684-685, 684t,

tumors benign, 681
malignant. See cancer turnover, protein, 84, 269-270
turnover number, 75
turns, in proteins, 36, 37f twin lamb disease, 215, 244
twisted gastrulation 1 (TWSG1), 635
two-dimensional electrophoresis, protein purity assessment with, 26, 27f
two-hybrid interaction test, 447, 449f
TWSGI (twisted gastrulation 1), 635
TxA,. See thromboxane A,
TXs. See thromboxanes type 1 leukocyte adhesion deficiency,

type 2 diabetes, 172
type A response, in gene expression,
410-411, 410f type B response, in gene expression,
410f, 411
type C response, in gene expression,

type I collagen, 604-605, 605t type I hyperprolinemia, 282, 282f
type I tyrosinemia, 286, 287f type II collagen, 607, 608f
type II hyperprolinemia, 282, 282f,

type II tyrosinemia, 286, 287f type IV collagen, 595-596
type IX collagen, 595
type V collagen, 604
type VII collagen, 596
tyrosinase, copper in, 98-100
tyrosine, 15t-16t carbon skeleton catabolism of,

hormone synthesis from, 483, 484f,
491-492, 491f, 493f specialized products of, 300, 302f
synthesis of, 266, 267f ultraviolet light absorption by,

tyrosine aminotransferase, 286, 287f tyrosine hydroxylase, 491, 491f
tyrosine kinases, inhibitors of, 701
tyrosinosis, 286, 287f tyrosinyl-tRNA, 407, 407f
upstream activator sequence (UAS), 427

base pairing in RNA, 342, 343f deoxyribonucleosides of, in pyrimidine
in RNA synthesis, 375
uraciluria-thyminuria, 327-328, 336
urate, as antioxidant, 204
urea laboratory tests for, 566
metabolic pathways of, 131, 133
nitrogen excretion as, 272, 275, 275f permeability coefficient of, 463f
synthesis of, 272-273, 272f, 273f,
275-276, 275f active enzymes, 722t
defects of, 276-278, 277t regulation of, 276
urease, transition metals in, 97, 97f ureotelic animals, 272

fructose effects on, 189-190
purine catabolism to, 334, 335f uricase,
334
uricemia, 336t uricotelic animals, 272
uridine, 321f, 322t, 333
uridine diphosphate galactose (UDPGal),
187-188, 189f uridine diphosphate glucose (UDPGlc)
in glycogenesis, 164-165, 165f in uronic acid pathway,
186-187, 187f uridine monophosphate (UMP),
322f, 322t uridyl transferase, deficiency of, 191
urinalysis, 566

bilirubin in, 313, 315, 315¢
glucose in, 180
myoglobin in, 54
urobilinogen in, 315, 315t xylulose in, 189
urine samples, 563
urobilinogen, 313, 315, 315t urobilins, 313
urocanic aciduria, 282
urokinase, 678f, 679
uronic acid, 599
uronic acid pathway deficiency of, 182
disruption of, 189
reactions of, 186-187, 187f uronic acids, in glycosaminoglycans,
147
uroporphyrinogen decarboxylase, 307,
307f, 308f, 309f, 310t uroporphyrinogen I, in heme synthesis,
306, 307f, 308f, 309f uroporphyrinogen I synthase, 306, 307f,
308f,
309f deficiency in, 310, 310t
uroporphyrinogen III, in heme synthesis,
306-307, 307f,
308f, 309f uroporphyrinogen III synthase, 306, 307f,

UV. See ultraviolet light
Vv vaccines, anticancer, 703
valence states, of transition metals, 93, 94f,

valine, 15t carbon skeleton catabolism of, 288, 290,
valinomycin, 125, 471-472
valproic acid, 690
van der Waals forces, 8, 8f, 339-340
vanadium absorption of, 101
physiologic roles of, 100-101
variable numbers of tandem repeats
(VNTRs), 445, 549
variant form of Creutzfeldt-Jakob disease
(vCJD), 42
vascular endothelial growth factor

vasodilators, 611, 622-623, 624f vCJD (variant form of Creutzfeldt-Jakob
disease), 42
vector cloning, 436-437, 437t
VEGEF (vascular endothelial growth factor), 697
velocity, enzyme. See initial velocity;
maximal velocity very-low-density lipoproteins (VLDLs),
133,
138,
237t atherosclerosis and, 257
hepatic secretion of, 242-243, 243f in ketogenesis regulation, 214
in lipid
transport,
236-237
metabolism of, 240-241, 240f remnants, 241, 254
in triacylglycerol transport, 238-239,

in cell-cell communication, 476-478,
477
coat proteins and, 583, 586-587, 586t,

extracellular, 476-478, 477f processing within, 588
secretory, 574, 575f synaptic, 587

targeting of, 587, 587f transport. See transport vesicles
types and functions, 586t v,.
See initial
velocity
Villefranche classification, 595, 596t vimentins,
625,
625t vinblastine, 625
viral RNA-dependent DNA polymerase,

chromosomal integration with,
359-360, 360f cyclophilins in, 42
host cell protein synthesis by,
405-406,
406f receptor-mediated endocytosis and, 475
RNA
in,
344
tumor, 684-685, 684f, 686f, 703
vision, vitamin A function in, 529, 530f vitamin
A
(retinol)
528,
530
functions of, 528t, 529-530, 530f structure of, 529, 529f
toxicity of, 530

prosthetic groups, cofactors, and coenzymes derived from, 58, 58f

deficiency of, 161, 163, 528t, 534
functions of, 528t, 533-534
pentose phosphate pathway need for,

coenzymes derived
from,
58
42,
528t,
534
flavin groups formed from, 112, 114
measurement of,
189
vitamin B, (pyridoxine, pyridoxal,

in aminotransferases, 273, 273f, 535
deficiency of, 288, 290f, 528t, 535
functions of, 528t, 535
structure of, 535, 535f toxicity of, 535
vitamin
B,,
(cobalamin)
absorption of, 101, 536
cobalt in, 98
deficiency of, 528t, 536-538, 536f functions of, 528t, 536, 536f
structure of, 536, 536f vitamin C (ascorbic acid)
as antioxidant, 204, 544, 544f in bile acid synthesis, 256f
in collagen synthesis, 43
deficiency of, 43, 263, 266, 528t,
540,
596
functions of, 528t, 539
higher intakes of, 540
human requirement for, 182, 186
as pro-oxidant, 544, 544¢
structure of, 539, 539f

as calcitriol precursor, 489
calcium absorption and, 523
in calcium
homeostasis,
531
cholesterol as precursor for, 249
528t,
531-532
ergosterol as precursor for,

528t hormone nature of, 530-531, 531f
synthesis and metabolism of,

toxicity of, 532
vitamin D, (cholecalciferol), 530, 531f as antioxidant, 203
formation and hydroxylation of,
489-491, 490f vitamin D-binding protein, 490
vitamin E (tocopherols, tocotrienols)
as antioxidant, 204, 532, 544, 544f deficiency of, 528, 532
functions of, 528t, 532
as pro-oxidant, 545
structure of, 532, 532f

deficiency of, 528t, 533
functions of, 528t, 532-533, 533f structure
of,
532-533,
533f vitamin K-dependent carboxylation, of
coagulation factors, 677-678

of,
527
523-524
laboratory tests for, 564-565
lipid(fat-) soluble, 196
lipid-soluble or water-soluble classification of, 527, 528
VLDL receptor, 238, 240
VLDLs. See very-low-density lipoproteins
Vijaxt
Se
VNTRs (variable numbers of tandem repeats ), 445, 549
voltage-gated channels, 470, 471,
471f, 618
voltage-gated K* channel (HvAP),
471, 471f von Gierke disease, 166, 167t, 335
von Willebrand disease, 585t,
655, 678
von Willebrand factor, 678
in platelet aggregation, 670, 671f vorinostat, 690
v-SNARE proteins, 586-588, 587f

Warburg effect, 695-696, 697f, 698t warfarin,
532-533
drug interactions of, 557
mechanism of, 677-678

as biologic solvent, 6-7, 7f biomedical
6
biomolecule interactions with,
7-8,
7t,
8f body compartmentalization of,

dipole formation by, 6-7, 7f dissociation of, 9-10
hydrogen bonding of, 7, 7f hydrogen
ions in,
9-13,
12f, hydrolysis
9
as nucleophile, 8-10
permeability coefficient of, 463f pH
of,
10
respiratory chain production of,

tetrahedral geometry of, 6, 7f water channels, 472
water-miscible lipoproteins, 236
water-soluble hormones, 482, 483t water-soluble molecules, 462
water-soluble vitamins, 527, 528t
Watson-Crick base pairing,

waxes,
196
weak acids
amino acids as, 18-20, 19f, 20t buffering actions of, 12-13, 12f
dissociation of, 10-12
as functional groups, 11-12
Henderson-Hasselbalch equation describing behavior of, 12, 12f
medium properties affecting, 13
molecular structure effects on,
13,
13t pk,
of,
11-13,
12f,
13t,
19

buffering actions of, 12-13, 12f dissociation of, 10-12
wear and tear theories of aging free
radicals,
710,
712
hydrolytic reactions,
708-709, 709f mitochondria and, 710, 712
molecular repair mechanisms and,
713-715
protein glycation, 712-713, 714f reactive oxygen species, 709-710,
710f, 711f ultraviolet radiation, 712, 713f
13t
weight loss, low carbohydrate diets for,
181
Wernicke encephalopathy, 534
Western protein blot procedure,
438, 439f white blood cells
biomedical importance of, 656
integrins in, 658-659, 659
multiple types of, 656-657, 657f phagocytosis, 474, 656, 657, 659-661,
659f, 660t production regulation of, 657
white thrombus, 670
Williams-Beuren syndrome, 596
Wilson disease, 298, 478t, 627, 634
wobble, 396
xX
xanthine, 321, 323f xanthine oxidase, 112
deficiency of, hypouricemia and,
335
molybdenum in, 100
xanthurenate, 288, 290f
X-chromosome pair, 354
xenobiotics biomedical importance of, 556
metabolism of acetylation and methylation, 558-559
conjugation, 558
hydroxylation by cytochrome P450,
556-558
toxic, immunologic, and carcinogenic effects of, 559, 559f
types of, 556
xerophthalmia, 530
X-linked disorders, 445
x-ray crystallography, 39-40
x-rays, carcinogenic effect of, 683, 683t xylose, 144f, 144t, 548¢
xylulose, 144f, 144t, 189
xylulose 5phosphate, 185
Y
YAC (yeast artificial chromosome) vector,
437, 437t yeast, fermentation by cell-free extract of,
1-2

yeast artificial chromosome (YAC) vector,
437, 437t yeast Flp recombinase, 435
yeast FRT sites, 435
Z
Z line, 612, 612f, 613f
Zellweger (cerebrohepatorenal) syndrome,
215, 579-580, 580t zinc
human requirement for, 93, 93t multivalent states of, 93,
94f, 94t physiologic roles of, 98, 99f
toxicity of, 95t zinc finger, 98
structure of, 95f zinc finger motif, 426, 426f
zona pellucida, 553
zwitterions, 19
zymogens, 87, 627
protease secretion as, 521

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* Thoroughly revised & updated
+ Fully revised color plates & color atlas
+ Points of identification added
* New flowcharts and tables included
* Revised added information and applied histology
* MCQs added
-Interactive Online Student Resource
Revised & Edited by
Foreword
Nachiket Shankar
SU ee me cl RUE EUE Um ee cme aC ALi eR meme tcl eR Weel meer lla eee. s)

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+ Revised added information and applied histology
+ MCQs added
+ Interactive Online Student Resource
Revised & Edited by Pushpalatha K Deepa Bhat Pushpa NB
Foreword Nachiket Shankar
|
|
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Professor Inderbir Singh, a legendary anatomist, is renowned for being a pillar in the education of generations of medical graduates across the globe. He was one of the greatest teachers of his times. He was a passionate writer who poured his soul into his work. His eagle's eye for details and meticulous way of writing made his books immensely popular amongst students. He managed to become enmeshed in millions of hearts in his lifetime. He was conferred the title of Professor Emeritus by Maharishi Dayanand University, Rohtak.
On 12th May, 2014, he was awarded posthumously with Emeritus Teacher Award by National Board of Examination for making invaluable contribution in teaching of Anatomy. This award is given to honour legends who have made tremendous contribution in the field of medical graduate. He was a visionary for his times, and the legacies he left behind are his various textbooks on Gross Anatomy, Histology, Neuroanatomy and Embryology. Although his mortal frame is not present amongst us, his genius will live on forever.

MBBS MD (PhD)
Professor and Head Department of Anatomy Jagadguru Sri Shivarathreeshwara (JSS) Medical College
Mysuru, Karnataka, India

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MBBS MD
Associate Professor Department of Anatomy Jagadguru Sri Shivarathreeshwara (JSS) Medical College Mysuru, Karnataka, India
Assistant Professor Department of Anatomy Jagadguru Sri Shivarathreeshwara (JSS) Medical College Mysuru, Karnataka, India
Foreword
JAYPEE BROTHERS MEDICAL PUBLISHERS The Health Sciences Publisher New Delhi | London

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New Delhi 110 002, India
Phone: +91-11-43574357
Fax: +91-11-43574314
Email: jaypee@jaypeebrothers.com

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J.P. Medical Ltd
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Phone: +44 20 3170 8910
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Email: info@jpmedpub.com
Website:
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© 2020, Jaypee Brothers Medical Publishers
The views and opinions expressed in this book are solely those of the original contributor(s)/author(s) and do not necessarily represent those of editor(s) of the book.
All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission in writing of the publishers.
All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book.
Medical knowledge and practice change constantly. This book is designed to provide accurate, authoritative information about the subject matter in question. However, readers are advised to check the most current information available on procedures included and check information from the manufacturer of each product to be administered, to verify the recommended dose, formula, method and duration of administration, adverse effects and contraindications. It is the responsibility of the practitioner to take all appropriate safety precautions. Neither the publisher nor the author(s)/editor(s) assume any liability for any injury and/or damage to persons or property arising from or related to use of material in this book.
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Every effort has been made where necessary to contact holders of copyright to obtain
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Inquiries for bulk sales may be solicited at: jayoee@jaypeebrothers.com
Inderbir Singh’s Textbook of Human Histology
First Edition : 1987

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Second Edition : 1992
Third Edition : 1992
Fourth Edition : 2002
Reprint : 2005
Fifth Edition : 2006
Reprint : 2008
Reprint : 2009
Sixth Edition : 2011
Seventh Edition : 2014
Eighth Edition : 2016
Ninth Edition : May 2019
Revised Reprint : Sep 2019
ISBN: 978-93-89034-97-4
Printed at
The Almighty, family, colleagues and friends within that has led to the to write a book
for their support and for kindling the fire fulfil passion
It gives me great pleasure to write the foreword for the latest edition of this well-known textbook of histology. For a number of years, this textbook was the preferred learning resource for health professional students. More recently a number of other textbooks in histology have become available. It is therefore appropriate that this edition has emerged, incorporating the changes that are required for the current generation of students, yet retaining the strengths of the older editions. Each chapter begins with the learning objectives and identification points of the related slides. The text has been presented in a concise, bulleted format that makes for easy readability. Schematic diagrams are presented alongside pictures from actual slides, making it simple for students to identify and draw the key features. The applied histology section provides a relevant clinical context for learning. Each chapter ends with multiple choice questions, which provide an opportunity for students to evaluate their learning. This edition will be ideally suited for the first batch of MBBS students undergoing the recently rolled out competency-based curriculum.
Professor
Department of Anatomy and Medical Education
St John's Medical College
Bengaluru, Karnataka, India

(Deemed to be University) Accredited 'A*' Grade by NAAC
Inderbir Singh's Textbook of Human Histology is widely used by students and faculty of health profession. Ninth edition of this book has been improvised as per current student requirements. The chapters have been revised with simplified representation. The contents are updated incorporating the recent advances that the students must be aware off.
Pushpalatha K
Well-defined learning objectives assist focussed student learning, help to choose appropriate teaching learning methods and assessment strategies. The content has been stratified to must know, good to know and nice to know categories. This intends to progressively take student from _ basic understanding to in-depth knowledge as per their needs. The content is modified and revised according to the new competency-based curriculum. Optic nerve and umbilical cord is added as per Medical Council of India (MCI) requirements.
The point-wise representation will make the reading easier and

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Deepa Bhat
The histology slides that students' observe during practical classes are similar with newly-added color plates of the textbook. Core highlight of this edition is addition of easily reproducible hand written diagrams of histology slides that student witnesses in the practical class. This will help in practical record writing and during examination.
The clinical features are retained, modified and added keeping in mind their applicability in pathology. Thus the learning carries forward in next phase of MBBS which forms the core vision of competency-based medical education that is being rolled out this academic year.
Identification points are added in the beginning of every chapter that helps the students to identify the slide, for last minute revision and during spotters.
The Multiple Choice Questions (MCQs) added will help the students in preparing for examinations. This book offers a one-stop solution for all histology-related needs.
Pushpa NB
Effort was made keeping in mind student friendliness yet maintaining the quality. Utmost care has been taken to avoid any errors or omissions. The suggestions, from learned and expertise would always be welcomed wholeheartedly to improve the efficacy of the manuscript. Scope for amendment always remains, and is invited from teachers and students.

We would like to thank all the people for the help and support they provided in the preparation of this book. Thanks to Principal and the staff of JSS Medical College, Mysuru, Karnataka, India, for this opportunity.
Special thanks to Molecular Biology Laboratory, Department of Biochemistry, JSS Medical College, Mysuru, for permitting to capture the photomicrographs that is the salient highlight of this manuscript. Thank those who have directly and indirectly assisted in completion of this task.
Finally our thanks to Shri Jitendar P Vij (Group Chairman), Mr Ankit Vij (Managing Director), Mr MS Mani (Group President), Dr Madhu Choudhary (Publishing Head—Education), Ms Pooja Bhandari (Production Head) and Ms Sunita Katla (Executive Assistant to Group Chairman and Publishing Manager) of M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, for this opportunity.

Akhilandeswari B
Professor
Bangalore Medical College and Research Institute

RG Kar Medical College
Kolkata, West Bengal, India

Ludhiana, Punjab, India

Lady Hardinge Medical College
New Delhi, India

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Indira Gandhi Medial College
Shimla, Himachal Pradesh, India
Arun Sharma
Head, Department of Anatomy
MLB Medical College
Jhansi, Uttar Pradesh, India
Ashwini C Appaji
MS Ramaiah Medical College
Avinash Thakur
ESIC Medical College and Hospital
Faridabad, Haryana, India

Jorhat Medical College
Jorhat, Assam, India

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MP Shah Medical College
Jamnagar, Gujarat, India
Doris George Yohannan
Government Medical College
Thiruvananthapuram, Kerala, India

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Chennai, Tamil Nadu, India
K Aparna Vedapriya
Osmania Medical College
Hyderabad, Telangana, India
Kanchan Kapoor
Professor
Chandigarh, Punjab, India

VCS Government Medical College
Srinagar, Uttarakhand, India

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Armed Forces Medical College
Pune, Maharashtra, India
Namita Malhotra
Mewat, Nalhar, Haryana, India
Navita Aggarwal
Adesh Institute of Medical Sciences and Research
Bathinda, Punjab, India

Hassan, Institute of Medical Sciences
Hassan, Karnataka, India

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Professor
SRM Institute of Science and Technology
Preeti Dnyandeo Sonje
Dr DY Patil Medical College

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Seth GS Medical College and KEM Hospital
Mumbai, Maharashtra, India
Purushottam Rao Manvikar
Rakesh Kumar Verma
King George’s Medical College
Lucknow, Uttar Pradesh, India

Saraswati Institute of Medical Sciences
Hapur, Uttar Pradesh, India

Professor
GCS Medical College
Ahmedabad, Gujarat, India

Pali, Rajasthan, India

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Doon Medical College
Dehradun, Uttarakhand, India
Sneh Agarwal
Director, Professor and Head
Sumedh G Sonavane
TN Medical College and BYL Nair Charitable Hospital
Vanita Gupta
Rama Medical College
and Glands

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History
Parts of a light microscope
Principle of microscopy
Practical tips in using a bright-field microscope
Types of microscopes
Tissue processing
Chapter

Structure
2:
Components of a cell
Staining of a cell
Applied histology

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3:
Epithelia
Definition
General features of epithelial tissue
Functions
Classification of epithelia
Simple epithelium
Basement membrane
Projections from the cell surface

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Classification of glands
Classification of exocrine glands
Structural organization
Development of glands
Structure of acini

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4:
General features
Classification of connective tissue
Components of connective tissue
Fibers of connective tissue
Intercellular ground substance of connective tissue
Different forms of connective tissue
Functions of connective tissue
Connective tissue with special properties
Chapter
Cartilage
5:
General features of cartilage
Perichondrium
Components of cartilage
Types of cartilage

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6:
Composition of bone tissue
Bone membranes
Classification and structure of bone
Formation of bone—ossification
How bones grow?

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Muscular Tissue
7:
Types of muscular tissue
Smooth muscle
Myoepithelial cells

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8:
Classification
Other classification
Lymph
Lymphatic vessels
Lymphocytes
Lymph nodes
Spleen
Thymus
Mucosa-associated lymphoid tissue
Tonsils
Further reading
Chapter
Nervous Tissue
9:
Structure of a neuron neurons
nerves
Neuroglia Types of Peripheral
Ganglia

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Chapter
Cardiovascular System
10:
Basic structure of a blood vessel
Structure of arteries
Arterioles
Capillaries
Veins
Venules
Nutrition and innervation of blood vessels
Heart
Chapter
Skin and its Appendages
11:
Functions
Types of skin
Structure of skin
Blood supply of the skin
Nerve supply of the skin
Appendages of the skin
Chapter
Salivary Glands
12:
Salivary glands

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Placenta and Umbilical Cord
13:
Placenta
Formation of placenta
Microscopic structure
Placental circulation and barrier
Umbilical Cord
Chapter
Respiratory System
14:
Common features of air passages
Nasal cavities
Pharynx
Larynx
Trachea and principal bronchi
Lungs
Chapter 15:
Digestive System: Oral Cavity and Related
Structures
Oral cavity
BE In eeth
Tongue
Chapter 16:
Digestive System: General Plan of
Gastrointestinal Tract, Stomach and Intestines
General structure of GIT
Esophagus
Stomach
Large intestine
Chapter
Hepatobiliary System and Pancreas
Liver
Extrahepatic biliary apparatus
Pancreas
Differences between serous salivary gland and pancreas
Chapter
Urinary System
18:
Functions
Kidneys
Ureters
Urinary bladder
Urethra
Chapter 19:
Central Nervous System: Spinal Cord, Cerebellar
Cortex and Cerebral Cortex
Spinal cord
Cerebral cortex
Cerebellar cortex
Chapter
Male Reproductive System
20:
Testis
Spermatogenesis
Epididymis
Ductus deferens
Seminal vesicle
Prostate
Penis
Chapter
Female Reproductive System
Ovary
Uterine tubes
Uterus
Cervix
Vagina
Female external genitalia
Mammary gland
Chapter
Endocrine System
22:
Hormones
Distribution of endocrine cells
Hypophysis cerebri
Thyroid gland
Suprarenal glands/adrenal glands
Pineal gland
Some other organs having endocrine functions

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Senses: Eye
23:
Structure of eyeball
Outer fibrous coat
Lens
Accessory visual organs
Chapter
Special Senses: Ear
24:
External ear
Middle ear
Internal ear
Some elementary facts about the mechanism of hearing Applied histology
Index

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*Components of a light microscope
*Principles of a conventional bright-field microscope
* Practical tips in using a bright-field microscope
* Types of microscopes
* Tissue processing
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Microscope is an instrument used to see the structures that are very small to be visualized by naked eyes. Microscopy is the science which deals with the study of small objects using microscope.
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Use of hand lens to observe small object dates back to 13th century, but it was Robert Hooke who first used the microscope which could provide magnification up to 42 times. Improved version with magnification of up to 300 times was developed by Antony van Leeuwenhoek (Father of Microscopy). Bonnannus added source of light, condenser, rack, and pinion for better focusing.

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In the histology laboratory, students use light microscope. It is also called as compound microscope or bright-field microscope (Fig. 1.1). It is mandatory to have the proper knowledge of parts of the light microscope for optimal focusing and proper handling of the microscope. Following are the essential parts of light microscope (Fig. 1.2):
* Stand
* Eyepiece
* Revolving nose piece with objective lenses
* Focus knob
* Stage
* Light source—mirror
* Diaphragm and condenser
* Mechanical stage.
Stand or base: It supports the microscope. It bears arm or limb. At the upper end of the arm, their lies body tube.
Body tube: It bears eyepiece at one end and objective lenses at the other end.
Eye piece: Eye piece or ocular lens is used to bring the image into the focus of eye. It enlarges the image produced by particular objective lens.
Objective lenses: These lenses are usually fitted into the revolving nose piece/objective turret, which allow the user to switch between objective lenses. They are powerful magnifying lenses which produces best possible image of an object. Compound microscopes nowadays have more than one objective lenses with magnification ranging from 5X to 100X and numerical aperture varying from 0.14 to 0.7, respectively. Objective lenses with very low power objective (3X, 4X, or 5X magnification) are used to scan the field, low power objective produces 10X magnification (10 times), high power objective produces 40X, and oil immersion gives 100X magnification. Objective lenses will have color coding for easy identification.
Fig. 1.1: Microscope.
Fig. 1.2: Compound microscope.
Stage: The stage is a platform below the objective which supports the slide being viewed. Center of the stage has aperture through which light passes to illuminate the specimen. The stage usually has arms to hold slides. Recent microscopes will have a mechanical stage that allows tiny movements of the slide via control knobs that reposition the slide as desired. With a mechanical stage, slides move on two horizontal axes for positioning the slide, thereby facilitating to study the specimen in detail.

Focus knobs: It includes coarse adjustment and fine adjustment knobs. Coarse adjustment knob moves the stage in vertical direction to bring the specimen into focus. Slight movement of the coarse adjustment brings about large vertical movement of the stage. Hence, coarse adjustment should only be used with low power objective. Fine adjustment knob present inside the coarse adjustment knob is used to bring the specimen into sharp focus.
Light source: Natural or artificial sources of light can be used. Daylight is directed via a mirror. Most microscopes have their own adjustable and controllable light source, most often it is a halogen bulb. Other options being illumination using laser and LEDs.
Condenser: The condenser is a lens designed to focus light from the illumination source onto the slide. Most of the times, condenser also include other features, such as a diaphragm/or filters, to manage the quality and intensity of the illumination. Thus, condenser controls the quality of image formed by the objective. With the condenser, it is always advisable to use only plane mirror to focus the light. Condenser can be moved up and down using rack and pinion. Condenser should be at optimal height for proper lighting, hence for better resolution.
Mirror: Microscopes without integral light sources are provided with mirror. Mirror will have two surfaces—plane and concave. Concave surface converts beam of light into cone of light. Hence, it is ideal to use concave mirror with low power objective. While focussing with natural light it is ideal to use plane mirror.
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Objective lens of the microscope forms enlarged image of the object under observation, this image is called as primary image (I1) and the magnification caused is called primary or initial magnification which is engraved on the objective. The primary image acts as object for the eyepiece and a secondary image (12) is formed within the barrel by first lens (field lens). The eye lens, which is above the field lens, forms a smaller image (of millimeter diameter) of the secondary image called Ramsden's disk (13). The lens of observer's eye converts this image into actual image (14) that is formed on the retina of the observer.
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1. Microscope should be placed on the level surface.
2. Switch on the power supply for light source. If there is no inbuilt light so then natural light has to be focused, so that light strikes the center of m and directed toward the condenser.
3. Adjust the condenser at optimal position. Remember only plane mirror h
be used with condenser. Open the aperture of the iris diaphragm fully.
4. Focusing starts at lower magnification in order to center the specimen b user on the stage. Place the slide on the center of the stage just belov objective, ensure that the coverslip faces upward.
5. Move the stage using coarse adjustment until the slide comes close t objective and focus the section sharp. Care should be taken not to strik objective on the slide. So, it is always better to keep the objective as ne possible to the slide and then moving it away until the specimen is focuse
6. Finally use fine adjustment for optimal focusing and better view.
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Microscopes are mainly classified based on the source of light they use. Following are some varieties of microscopes other than bright-field microscope:
1. Dark-field microscope: In this microscope, only the objects are illumil by strong oblique peripheral light, whereas the central beam of lig prevented from entering the objective lens by central patch stop fitted condenser. It is used to study extremely tiny particles (colloid suspen and transparent objects like crystals, protozoa, etc.
2. Phase-contrast microscope: It mainly works on the difference ir refractive indices of different media and object. With difference ir refractive indices, there occurs phase difference and image will be fo. due to interference between direct light (not passed through object) diffracted light (passed through the object). The phase differences converted into differences of amplitude, hence objects which causes : difference in the phase leads to more difference in amplitude, thus resu brighter image without staining.
3. Interference microscope: In this microscope, beam of light from s source is split and passed through the object and outside. Later, they recombined by beam recombiner at image plane. After recombiné difference in retardation caused by interference is used to measure refractive index, thereby measuring the thickness of the object t observation.
4. Polarization microscope: It is a conventional microscope having rot stage with 2 polarizing elements and balsam. Polarizer is interfered beloy condenser and analyzer is placed above the objective lens. Polar

, 1 = microscope is mainly used to distinguish between amorphous and crystalline biological specimens like bones, lipid droplets, etc.. 5., 1 = Fluorescence microscope: In this microscope, ultraviolet light is incident on object and made to emit the visible light. Tissues treated with fluorescent dye are examined with ultraviolet light, by emitting light of longer wavelength. They are seen as bright objects against dark background.. 6., 1 = Ultraviolet microscope: In this microscope, ultraviolet light is used as source. Amount of light absorbed is recorded photographically and the molecules in the specimen are detected. Mainly, nucleic acids, purine, and pyrimidine are detected by this kind of microscope.. 7., 1 = Electron microscope: Is a refracting microscope where a stream of high- velocity electrons is used instead of light. Since it provides resolution around 3-5 Å finer details of structure can studied. As electrons are used, the image is focused by the magnetic coils on fluorescent screen. There are two types of electron microscopes- transmission electron microscope and scanning electron microscope.
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1., 1 = Always carry the microscope in both hands. Hold the arm in one hand and support the base of the microscope with other hand.. 2., 1 = After tissue is procured, it has to be properly fixed in ideal fixative. Fixation is done in order to enable the tissue to withstand further steps of tissue processing. An ideal fixative acts rapidly, causes immediate death of the tissue and preserves life-like appearance. It also prevents autolysis, putrefaction neither shrinks nor swells the tissue, cheap, nontoxic, and provides wide range of staining options. Commonly used are alcohol, acetone, formalin, Zenker's fluid, etc.. 3., 1 = Once the tissue is properly 'fixed', it is later dehydrated using increasing strengths of alcohol ending with absolute alcohol.. 4., 1 = After tissue is dehydrated, excess of alcohol is removed by a process called 'dealcoholization' or 'clearing'. Clearing agents raises the refractive index of the tissue and makes it transparent. Commonly used are xylene, cedar wood oil, chloroform, etc.. 5., 1 = Cleared tissue is impregnated both externally and internally by embedding media (paraffin), hence blocks are prepared. Embedded tissue is sectioned with microtome into very thin slices without distortion and attached to glass

6., 1 = Mounted paraffin sections are deparaffinized before staining. In routine histology laboratory, thin sections are stained using hematoxylin and eosin stains. Hematoxylin is a basic dye, hence it combines with acid components of the cell like DNA, RNA, and renders them purple/blue color. Hematoxylin usually stains cell nuclei, myelin, elastic fibers, fibrin, neuroglia, and muscle striations. Whereas eosin is acidic dye, hence it combines with basic components of cytoplasm like proteins (amino acids) and stains it pink.. 7., 1 = After hematoxylin and eosin (H & E) staining, slide is cleared, mounted using DPX, and covered with coverslip for future use.. 8., 1 = Special stains are used when particular components have to be studied. For example, collagen is stained by Van Gieson method, elastic fibers by Verhoeff's method, and so on.
Note: Bipolar staining—in this, basal portion of the cell stained blue because of extensive rough endoplasmic reticulum and apical part stained pink because of eosinophilic zymogen granules. Bipolar staining is exhibited by pancreatic acini, serous acini of salivary gland.

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* Introduction and types of cell
*Components of a cell—plasma membrane, cell organelles, nucleus, and cytoskeleton
*Cell junctions
* Staining of a cell
* Applied histology
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Cells are the structural and functional units of all the tissues. There are two types of cells—(1) Cells which lack membrane-bound nucleus and membrane-bound organelles are called prokaryotic cells (e.g. bacteria) and (2) Cells with membrane-bound nucleus and membrane-bound organelles are called eukaryotic cells. In this chapter, we will discuss about the eukaryotic cells.
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* A cell is bounded by a cell membrane/plasma membrane within which is enclosed by a complex material called protoplasm.
* The protoplasm consists of a central, denser part called the nucleus, and an outer less dense part called the cytoplasm. The nucleus is separated from
the cytoplasm by a nuclear membrane.
* The cytoplasm has a fluid base (matrix) which is referred to as the cytosol or hyaloplasm. The cytosol contains a number of organelles which have distinctive structure and functions. Many of them are in the form of membranes that enclose spaces. These spaces are collectively referred to as the vacuoplasm.

* The membrane which separates the interior of the cell from surrounding structure is called plasma membrane. It has high permeability property and regulates the cellular metabolism.
* Structure of plasma membrane: The structure of cell membrane is called as fluid mosaic model. In electron microscopy (EM), the average cell membrane is seen to be about 7.5 nm thick. It consists of two densely stained layers separated by a lighter zone, thus creating a trilaminar appearance (Fig. 2.2).
* Cell membranes are mainly made up of lipids, proteins, and carbohydrates.

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* Tt is now known that the trilaminar structure of membranes is produced by the arrangement of lipid molecules (predominantly phospholipids) that constitute the basic framework of the membrane (Fig. 2.2).
* Each phospholipid molecule consists of an enlarged head in which the phosphate portion is located, and of two thin tails (Fig. 2.3). The head end is also called the polar end while the tail end is the nonpolar end. The head end is soluble in water and is said to be hydrophilic. The tail end is insoluble and is said to be hydrophobic.
* When such molecules are suspended in an aqueous medium, they arrange themselves so that the hydrophilic ends are in contact with the medium but not the hydrophobic ends thus, forming a bilayer.
* The dark staining parts of the membrane are formed by the heads of the molecules, while the light staining intermediate zone is occupied by the tails, thus giving the membrane its trilaminar appearance.
* Because of the manner of its formation, the membrane is to be regarded as a fluid structure that can readily reform when its continuity is disturbed. For the same reasons, proteins present within the membrane can move freely within the membrane.
Fig. 2.1: Schematic representation of eukaryotic cell and its components.
Fig. 2.2: Trilaminar structure of a cell membrane as revealed by high magnifications of electron microscopy (EM).
Fig. 2.3: Diagram showing the structure of a phospholipid molecule (phosphatidylcholine) seen in a cell membrane.

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* The proteins are present in the form of irregularly rounded masses. Most of them are embedded within the thickness of the membrane and partly project on either outer or inner surface. Some proteins occupy the entire thickness of the membrane and may project out of both its surfaces and are called as transmembrane proteins (Fig. 2.4).
* Significance of proteins of the cell membrane:
"They form an essential part of the structure of the membrane
"Play a vital role in transport across the membrane and act as pumps. Ic get attached to the protein on one surface and move with the protein to 1 other surface
™Some proteins form passive channels through which substances can diffi through the membrane
™ Some proteins act as receptors for specific hormones or neurotransmitters
™ Some proteins act as enzymes.
Fig. 2.4: Some varieties of membrane proteins.

* In addition to the phospholipids and proteins, carbohydrates are present at the surface of the membrane. They are attached either to the proteins forming glycoproteins or to the lipids forming glycolipids (Fig. 2.5).
* The carbohydrate layer is especially well developed on the external surface of the plasma membrane forming the cell boundary. This layer is referred to as the cell coat or glycocalyx.

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The glycocalyx is made up of the carbohydrate portions or glycoproteins and glycolipids present in the cell membrane. Some functions attributed to the glycocalyx are as follows:
* Special adhesion molecules present in the layer enable the cell to adhere to specific types of cells, or to specific extracellular molecules.
*The layer contains antigens. These' include major histocompatibility complex (MHC). In erythrocytes, the glycocalyx
contains blood group antigens.
- *Most molecules in the glycocalyx are negatively charged causing adjoining cells to repel one another. This force of repulsion maintains the 20 nm interval between cells. However, some molecules that are positively charged adhere to negativelycharged molecules of adjoining cells, holding the cells together at these sites.
Fig. 2.5: Glycolipid and glycoprotein molecules attached to the outer aspect of cell membrane.

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. It maintains the shape of the cell.
ii. It controls the passage of all substances into or out of the cell.
iii. The cell membrane forms a sensory surface. This function is most devel in nerve and muscle cells.
iv. The surface of the cell membrane bears receptors for hormones and enzy
Stimulation of these receptors (e.g. by the specific hormone) can pro profound effects on the activity of the cell. Receptors also play an impc role in absorption of specific molecules into the cell.
. Membrane proteins help to maintain the structural integrity of the ce giving attachment to cytoskeletal filaments. They also help to prc
adhesion between cells and extracellular materials.
- vi. Cell membranes of some cells may show a high degree of specializatic some cells. For example, the membranes of rod and cone cells of retina proteins that are sensitive to light.

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* Transport across the plasma membrane can be either active transport which requires energy or passive transport which does not require any energy.
* Passive transport can be either by simple diffusion (e.g. water, oxygen, glycerol, carbon dioxide, etc.) or facilitated diffusion (e.g. ions, urea, glucose, etc.).
* Some molecules can enter cells by passing through passive channels in the cell membrane and large molecules enter the cell by the process of endocytosis (Fig. 2.6A).
* Endocytosis is the process of actively transporting molecules into the cell by engulfing it with its membrane. In endocytosis, the molecule invaginates a part of the cell membrane and gets surrounded by it and then separates from the rest of the cell membrane to form an endocytic vesicle. This vesicle can move through the cytosol to other parts of the cell. Some cells use the process of endocytosis to engulf foreign matter (e.g. bacteria) and it is then referred to as phagocytosis (cell eating).
* Pinocytosis (cell drinking) is a process similar to endocytosis where the vesicles formed are used for absorption of fluids or other small molecules into the cell.
* Exocytosis is a form of active transport in which a cell transports molecules out of the cell. Molecules produced within the cytoplasm (e.g. secretions) are enclosed in membranes to form vesicles (exocytic vesicles) that approach the cell membrane and fuse with its internal surface. The vesicle then ruptures releasing the molecule to the exterior (Fig. 2.6B).
Fig. 2.6A: Three stages in the absorption of extracellular molecules by endocytosis.
Fig. 2.6B: Three stages in exocytosis. The fusogenic proteins facilitate adhesion of the vesicle to the cell membrane.

Cell junctions are intercellular connections between the plasma membranes of adjacent cells of animal tissues. Intercellular connections between adjacent cells resulting in varying degrees of fusion and specialized functions of animal tissues.They are formed by multiprotein complexes. These junctions can be recognized by electron microscope.

There are three major types of cell junctions:
1. Anchoring junctions or adhesive junctions: Bind cells together.
2. Occluding junctions/tight junctions.
3. Communicating junctions/gap junctions: Direct transport of some substé

* These are contacts that do not show any specialized features on EM examination. At such sites, adjoining cell membranes are held together as follows.
* Some glycoprotein molecules, present in the cell membrane, are called cell adhesion molecules (CAMs). These molecules occupy the entire thickness of the cell membrane (i.e. they are transmembrane proteins). At its cytosolic end, each CAM is in contact with an intermediate protein/link protein.
* Fibrous elements of the cytoskeleton are attached to this intermediate protein (and thus, indirectly, to CAMs). The other end of the CAM juts into the 20 nm intercellular space, and comes in contact with a similar molecule from the opposite cell membrane. In this way, a path is established through which forces can be transmitted from the cytoskeleton of one cell to another (Fig. 2.7).
* Cell adhesion molecules and intermediate proteins are of various types. Contacts between cells can be classified on the basis of the type of CAMs proteins present. The adhesion of some CAMs is dependent on the presence of calcium ions, while some others are not dependent on them (Table 2.1). Intermediate proteins are also of various types (catenins, vinculin, and a-actinin).
Fig. 2.7: Scheme to show the basic structure of an unspecialized contact between two cells.

* Anchoring junctions are cell junctions that are attached to one another and with the components of the extracellular matrix. They are commonly found in tissues that are prone to constant mechanical stress, e.g. skin and heart.
* Anchoring junctions are of the following types:
" Adhesive spots/desmosomes/macula adherens
" Adhesive belts/zonula adherens
" Adhesive strips/fascia adherens.

* These are the most common type of junctions between adjoining cells. Desmosomes are present where strong anchorage between cells is needed, e.g. between cells of the epidermis.
* At the site of a desmosome, the plasma membrane is thickened due to presence of a dense layer of proteins on its inner surface. The thickened areas of the two sides are separated by a gap of 25 nm. The region of the gap is rich in glycoproteins. The thickened areas of the two membranes are held together by fibrils that appear to pass from one membrane to the other across the gap (Fig. 2.9).
Calcium dependent, Subtypes = Cadherins (of various types). Calcium dependent, Present in = Most cells including epithelia. Calcium dependent, Subtypes = Selectins. Calcium dependent, Present in = Migrating cells, e.g. leukocytes. Calcium dependent, Subtypes = Integrins. Calcium dependent, Present in = Between cells and intercellular substances. About 20 types of integrins, each attaching to a special extracellular molecule. Calcium independent, Subtypes = Neural cell adhesion molecule (NCAM). Calcium independent, Present in = Nerve cells. Calcium independent, Subtypes = Intercellular adhesion molecule (ICAM). Calcium independent, Present in = Leukocytes
Fig. 2.8: Electron microscopy (EM) appearance of a desmosome.
Fig. 2.9: Schematic diagram to show the detailed structure of a desmosome (in the epidermis). (CAMs: cell adhesion molecules).

It is usually seen near the apices of epithelial cells. This is similar to a desmosome in being marked by thickenings of the two plasma membranes, to the cytoplasmic aspects of which fibrils are attached. However, the gap between the thickenings of the plasma membranes of the two cells is not traversed by filaments (Fig. 2.10).

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* These are similar to adhesive belts. They differ from the latter in that the areas of attachment are in the form of short strips and do not go all round the cell. These are seen in relation to smooth muscle, intercalated discs of cardiac muscle, and in junctions between glial cells and nerves. Modified anchoring junctions attach cells to extracellular material. Such junctions are seen as hemidesmosomes, or as focal spots.
* Hemidesmosomes are similar to desmosomes, but the thickening of cell membrane is seen only on one side. Hemidesmosomes are common where basal epidermal cells lie against connective tissue.
* Focal spots are also called focal adhesion plaques, or focal contacts. They represent areas of local adhesion of a cell to extracellular matrix. Such junctions are of a transient nature (e.g. between a leukocyte and a vessel
Fig. 2.10: Scheme to show a junctional complex.
wall). Such contacts may send signals to the cell and initiate cytoskeletal formation.

* Like the zonula adherens, the zonula occludens are seen most typically near the apices of epithelial cells. At such a junction, the two plasma membranes are in actual contact (Figs. 2.11A and B).
* These junctions act as barriers that prevent the movement of molecules into the intercellular spaces. For example, intestinal contents are prevented by them from permeating into the intercellular spaces between the lining cells (Fig. 2.12).

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* These junctions separate areas of cell membrane that are specialized for absorption or secretion from the rest of the cell membrane.
*In cells involved in active transport against a concentration gradient, occluding junctions prevent back diffusion of transported substances.
Apart from epithelial cells, zonula occludens are also present between endothelial cells.
In some situations, occlusion of the gaps between the adjoining cells may be incomplete and the junction may allow slow diffusion of molecules across it.
- These are referred to as leaky tight junctions.

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- * At these junctions, the plasma membranes are not in actual contact, but lie very close to each other, the gap being reduced from the normal 20 nm to 3 nm. In transmission electron micrographs, this gap is seen to contain beadlike structures (Fig. 2.11B).
Figs. 2.11A and B: (A) Zonula occludens as seen by electron microscopy (EM); (B) Gap as seen EM.
junction by
Fig. 2.12: Schematic diagram to show the detailed structure of part of an occluding junction. cell adhesion
(CAMs: molecules).
* A minute canaliculus passing through each "bead" connects the cytoplasm of the two cells thus, allowing the free passage of some substances (sodium, potassium, calcium, and metabolites) from one cell to the other (also see here). Gap junctions are, therefore, also called macula communicans. They are widely distributed in the body.
* Changes in pH or in calcium ion concentration can close the channels of gap junctions. By allowing passing of ions, they lower transcellular electrical resistance. Gap junctions form electrical synapses between some neurons.
* The wall of each channel is made up of six protein elements (called nexins, or connexons). The "inner" ends of these elements are attached to the cytosolic side of the cell membrane while the "outer" ends project into the gap between the two cell membranes (Fig. 2.13). Here, they come in contact with (and align perfectly with) similar nexins projecting into the space from the cell membrane of the opposite cell, to complete the channel.

- * The cytoplasm of a typical cell contains various structures that are referred to as organelles. They include the endoplasmic reticulum (ER), ribosomes, mitochondria, the Golgi complex, and various types of vesicles (Fig. 2.14).
Fig. 2.13: Diagram to show the constitution of one channel of a communicating junction.
- * The cytosol also contains a cytoskeleton made up of microtubules, microfilaments, and intermediate filaments. Centrioles are closely connected with microtubules.

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* Tt is a network of membranous tubules within the cytoplasm of a cell and it is continuous with the nuclear membrane. It is involved in protein and lipid synthesis.
* The membranes form the boundaries of channels that may be arranged in the form of flattened sacs (or cisternae) or of tubules. Ribosomes and enzymes are present on the "outer" surfaces of the membranes of the reticulum.
* Because of the presence of the ER, the cytoplasm is divided into two components, one within the channels and one outside them (Fig. 2.15). The cytoplasm within the channels is called the vacuoplasm, and that outside the channels is the hyaloplasm or cytosol.
* There are two types of ER within each cell: (1) Smooth endoplasmic reticulum (SER) and (2) Rough endoplasmic reticulum (RER).
* In most places, the membranes forming the ER are studded with minute particles of ribonucleic acid (RNA) called ribosomes. The presence of these ribosomes gives the membrane a rough appearance and they are called RER.
* Rough endoplasmic reticulum represents the site at which proteins are synthesized. The attached ribosomes play an important role in this process. The lumen of RER is continuous with the perinuclear space. It is also continuous with the lumen of SER.
* Smooth endoplasmic reticulum is involved in lipid metabolism and acts as the calcium store for the cell. It is involved in the synthesis of lipids, especially that of membrane phospholipids. It is often located near the periphery of the cell. SER is responsible for further processing of proteins synthesized in RER.
* Products synthesized by the ER are stored in the channels within the reticulum.
Fig. 2.14: Some features of a cell that can be seen with a light microscope.
Fig. 2.15: Schematic diagram to show the various organelles to be found in a typical cell. (Various structures shown are not drawn to scale).

* We have seen earlier that ribosomes are present in relation to RER. They may also lie free in the cytoplasm.
* They may be present singly in which case they are called monosomes or in
groups which are referred to as polyribosomes or polysomes. Each ribosome consists of proteins and RNA and is about 15 nm in diameter. Ribosomes play an essential role in protein synthesis.

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* The mitochondrion is a double membrane-bound organelle and can be seen with the light microscope in specially stained preparations. They are so called because they appear either as granules or as rods (mitos = granule; chondrium = rod).
* The number of mitochondria varies from cell to cell being greatest in cells with high metabolic activity (e.g. in secretory cells). Mitochondria vary in size, most of them being 0.5—2 yim in length. Mitochondria are large in cells with a high oxidative metabolism. 3
The mitochondrion is bounded by a smooth outer membrane within which there is an inner membrane, the two being separated by an intermembranous space. The inner membrane is highly folded on itself forming incomplete partitions called cristae. The space bounded by the inner membrane is filled by a granular material called the matrix (Fig. 2.16). *
* The matrix contains numerous enzymes. It also contains some RNA and deoxyribonucleic acid (DNA): These are believed to carry information that enables mitochondria to duplicate themselves during cell division. All mitochondria are derived from those in the fertilized ovum, and are entirely of maternal origin. ate
The most prominent roles of mitochondria are to produce the energy of the cell (power house of the cell). Primary function of mitochondria is to generate large quantities of energy in the form of adenosine triphosphate (ATP). They contain many enzymes including some that play an important part in Krebs cycle (TCA cycle). In addition to producing energy, mitochondria store calcium for cell signaling activities, generate heat, and mediate cell growth and death. A &
* Adenosine triphosphate and guanosine triphosphate (GTP) are formed in mitochondria and from there they pass to other parts of the cell and provide energy for various cellular functions. These facts can be correlated with the observation that within the cell mitochondria tend to concentrate in regions where energy requirements are greatest.
Fig. 2.16: Structure of a mitochondrion.

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* Golgi apparatus, also called Golgi complex or Golgi body, is a membranebound organelle of cells that is made up of a series of flattened, stacked pouches called cisternae. It is involved in secretion and intracellular transport.
*In light microscopic preparations, suitably treated with silver salts, the Golgi complex can be seen as a small structure of irregular shape, usually present near the nucleus (see Fig. 2.14).
* When examined with the EM, the complex is seen to be made up of membranes similar to those of SER. The membranes form the walls of a number of flattened sacs that are stacked over one another. Toward their margins, the sacs are continuous with small rounded vesicles (Fig. 2.17). The cisternae of the Golgi complex form an independent system. Their lumen is not in communication with that of ER. Material from ER reaches the Golgi complex through vesicles.
* The Golgi apparatus is made up of approximately four to eight cisternae, although in some single-celled organisms, it may consist of as many as 60 cisternae. The cisternae are held together by matrix proteins, and the whole of the Golgi apparatus is supported by cytoplasmic microtubules. The
apparatus has three regions known generally as: (1) "Cis" (cisternae nearest the endoplasmic reticulum), (2) "Medial" (central layers of cisternae), and (3) "Trans" (cisternae farthest from the ER).
Fig. 2.17: Structure of the Golgi complex.

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* Material synthesized in RER travels through the ER lumen into SER. Vesicles budding off from SER transport this material to the cis face of the Golgi complex. Some proteins are phosphorylated here. From the cis face, all these materials pass into the medial Golgi. Here, sugar residues are added to proteins to form protein-carbohydrate complexes.
* Finally, all material passes to the trans face, which performs the following functions (Fig. 2.18):
a. Proteolysis of some proteins converts them from inactive to active for
b. Like the medial Golgi, the trans face is also concerned in addin residues to proteins
c. In the trans face, various substances are sorted out and packed in app vesicles. The latter may be secretory vesicles, lysosomes, or vesicle for transport of membrane to the cell surface.
Fig. 2.18: Scheme to illustrate the role of the Golgi complex in formation of secretory vacuoles.

Materials such as lipids, or carbohydrates, may also be stored within the cytoplasm in the form of membrane-bound vesicles.

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* These vesicles contain enzymes that can destroy unwanted material present within a cell. Such material may have been taken into the cell from outside (e.g. bacteria), or may represent organelles that are no longer of use to the cell. The enzymes present in lysosomes include proteases, lipases, carbohydrases, and acid phosphatase (as many as 40 different lysosomal enzymes have been identified).
* The stages in the formation of a lysosome are as follows:
" Acid hydrolase enzymes synthesized in ER reach the Golgi complex whi
they are packed into vesicles (Fig. 2.19). The enzymes in these vesicles | inactive because of the lack of an acid medium (these are called prime lysosomes or Golgi hydrolase vesicles) (Flowchart 2.1).
Fig. 2.19: Scheme to show how lysosomes, phagolysosomes, and multivesicular bodies are
formed.
"These vesicles fuse with other vesicles derived from cell membre (endosomes). These endosomes possess the membrane proteins necessi for producing an acid medium. The product formed by fusion of the t vesicles is an endolysosome or secondary lysosome (Flowchart 2.1).
"H+ ions are pumped into the vesicle to create an acid environment. T activates the enzymes and a mature lysosome is formed.
*Lysosomes help in "digesting" the material within phagosomes. A lysosome, containing appropriate enzymes, fuses with the phagosome so that the enzymes of the former can act on the material within the phagosome. These bodies consisting of fused phagosomes and lysosomes are referred to as phagolysosomes (Fig. 2.19).
* In a similar manner, lysosomes may also fuse with pinocytotic vesicles. The structures formed by such fusion often appear to have numerous small vesicles within them and are, therefore, called multivesicular bodies.
* Lysosomes are present in all cells except mature erythrocytes. They are a prominent feature in neutrophil leukocytes.

* These are similar to lysosomes in that they are membrane-bound vesicles containing enzymes.
* The enzymes in most of them react with other substances to form hydrogen peroxide which is used to detoxify various substances by oxidizing them. The enzymes are involved in oxidation of very long chain fatty acids. Hydrogen peroxide resulting from the reactions is toxic to the cell. Other peroxisomes contain the enzyme catalase which destroys hydrogen peroxide, thus preventing the latter from accumulating in the cell. Peroxisomes are most prominent in cells of the liver and in cells of renal tubules.

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* The cytoskeleton is a network of filaments and tubules that extends throughout a cell, through the cytoplasm, except for the nucleus.
* The cytoskeleton supports the cell, gives it shape, organizes and tethers the organelles, and has role in molecule transport, cell division, and cell signaling.
* The elements that constitute the cytoskeleton consist of the following:
™ Microfilaments
™ Microtubules
"Intermediate filaments.

- * Microfilaments are made up of actin filaments so they are also called actin filaments. Their structure is two strands of actin wound in a spiral. They are about 7 nm thick, making them the thinnest filaments in the

* They facilitate in cytokinesis, cell motility, and allow single-celled organisms like amebas to move. They are also involved in cytoplasmic streaming, which is the flowing of cytosol throughout the cell. Cytoplasmic streaming transports nutrients and cell organelles.
* Microfilaments are also part of muscle cells and allow these cells to contract, along with myosin.

* Microtubules are the largest cytoskeletal filaments in cells, with a diameter of 25 nm.
* The basic constituent of microtubules is the protein tubulin. Chains of tubulin form protofilaments. The wall of a microtubule is made up of 13 protofilaments that run longitudinally (Fig. 2.20). The tubulin protofilaments are stabilized by microtubule-associated proteins (MAPs).
* Microtubules are formed in centrioles which constitute a microtubule organizing center.
Fig. 2.20: Scheme to show how a microtubule is constituted.

- * They have role in cell movement, cell division, and transporting materials

* As part of the cytoskeleton, they provide stability to the cell. They prevent tubules of ER from collapsing.
* They facilitate transport within the cell. As part of the cytoskeleton, microtubules help to move organelles inside a cell's cytoplasm.
* In dividing cells, microtubules form the mitotic spindle.
* Cilia and flagella are made up of microtubules.

* These are so called as their diameter (10 nm) is intermediate between that of microfilaments (5 nm) and of microtubules (25 nm). The proteins constituting these filaments vary in different types of cells.
* They include cytokeratin (in epithelial cells), neurofilament protein (in neurons), desmin (in muscle), glial fibrillary acidic protein (in astrocytes), lamin (in the nuclear lamina of cells), and vimentin (in many types of cells).

* Intermediate filaments link cells together. The filaments also facilitate cell attachment to extracellular elements at hemidesmosomes.
* In the epithelium of the skin, the filaments undergo modification to form keratin. They also form the main constituent of hair and of nails.
* The neurofilaments of neurons are intermediate filaments. Neurofibrils help to maintain the cylindrical shape of axons.
* The nuclear lamina consists of intermediate filaments.

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* A centriole is a small structure made up of microtubules which exists as part of the centrosome, which helps to organize microtubules in the body.
* With the light microscope, the two centrioles are seen as dots embedded in a region of dense cytoplasm which is called the centrosome.
* With the EM, the centrioles are seen to be short cylinders that lie at right angles to each other. When we examine a transverse section across a centriole by EM, it is seen to consist essentially of a series of microtubules arranged in a circle. There are nine groups of tubules, each group
consisting of three tubules (Fig. 2.21).
* Centrioles play an important role in the formation of various cellular structures that are made up of microtubules. These include the mitotic spindles of dividing cells, cilia, flagella, and some projections of specialized cells (e.g. the axial filaments of spermatozoa). It is of interest to note that cilia, flagella, and the tails of spermatozoa all have the 9 + 2 configuration of microtubules that are seen in a centriole.

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* The nucleus constitutes the central and denser parts of the cell. It is usually rounded or ellipsoid. Occasionally, it may be elongated, indented, or lobed. It is usually 4-10 pm in diameter.
*Tt is a highly specialized organelle that serves as the information processing and administrative center of the cell. It has two major functions: (1) It stores the cell's hereditary material, or DNA, and (2) It coordinates the cell's activities, which include growth, intermediary metabolism, protein synthesis, and cell division.
* In usual classroom slides stained with hematoxylin and eosin, the nucleus stains dark purple or blue while the cytoplasm is usually stained pink. In some cells, the nuclei are relatively large and light staining.
Fig. 2.21: Transverse section across a centriole (near its base). Note nine groups of tubules, each group having three microtubules.
- * Nuclei appear to be made up of a delicate network of fibers: The material making up the fibers of the network is called chromatin (because of its
affinity for dyes). At some places in the nucleus, the chromatin is seen in the form of irregular dark masses that are called heterochromatin. At other places, the network is loose and stains lightly: The chromatin of such areas is referred to as euchromatin.

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* Nuclei which are large and in which relatively large areas of euchromatin can be seen are referred to as open-face nuclei. Nuclei that are made up mainly of heterochromatin are referred to as closed-face nuclei (Figs. 2.22A and B).
*In addition to the masses of heterochromatin, the nucleus shows one or more rounded, dark staining bodies called nucleoli. The nucleolus is a membraneless organelle within the nucleus that manufactures ribosomes, the cell's protein-producing structures.
* Through the microscope, the nucleolus looks like a large dark spot within the nucleus. A nucleus may contain up to four nucleoli.
* After a cell divides, a nucleolus is formed when chromosomes are brought together into nucleolar organizing regions and during cell division, the nucleolus disappears.
* The nucleus also contains various small granules, fibers, and vesicles. The spaces between the various constituents of the nucleus described earlier are filled by a base called the nucleoplasm.
* With the EM, the nucleus is seen to be surrounded by a double-layered nuclear membrane or nuclear envelope. The outer nuclear membrane is continuous with ER. The space between the inner and outer membranes is the perinuclear space. This is continuous with the lumen of RER.
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* The inner layer of the nuclear membrane provides attachment to the ends of chromosomes. Deep to the inner membrane, there is a layer containing proteins and a network of filaments: This layer is called the nuclear lamina. Specific proteins present in the inner nuclear membrane give attachment to filamentous proteins of the nuclear lamina.
* At several points, the inner and outer layers of the nuclear membrane fuse leaving gaps called nuclear pores. Each pore is surrounded by dense protein arranged in the form of eight complexes. These proteins and the pore together form the pore complex.
* Nuclear pores represent sites at which substances can pass from the nucleus to the cytoplasm and vice versa (see Fig. 2.15). The nuclear pore is about 80 nm across. It is partly covered by a diaphragm that allows passage only to particles less than 9 nm in diameter. A typical nucleus has 3,000—4,000 pores.
* It is believed that pore complexes actively transport some proteins into the nucleus, and ribosomes out of the nucleus.
* Building blocks for building DNA and RNA are allowed into the nucleus as well as molecules that provide the energy for constructing genetic material.

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* Chromatin is made up of a substance called deoxyribonucleic acid (usually abbreviated to DNA), and of proteins. It is in the form of a long chain of nucleotides.
* Most of the proteins in chromatin are histones. Some nonhistone proteins are also present. Filaments of DNA form coils around histone complexes.
* The structure formed by a histone complex and the DNA fiber coiled around it is called a nucleosome. Nucleosomes are attached to one another forming long chains (Fig. 2.23). These chains are coiled on themselves (in a helical manner) to form filaments having 30 nm in diameter. These filaments constitute chromatin.
* The DNA fibril makes two turns around a complex formed by histones to form a nucleosome. Nucleosomes give the chromatin fiber the appearance
of a beaded string. The portion of the DNA fiber between the nucleosomes is called linkerDNA.
* Filaments of chromatin are again coiled on themselves (supercoiling), and this coiling is repeated several times. Each coiling produces a thicker filament. In this way, a filament of DNA that is originally 50 mm long can be reduced to a chromosome by only 5 pm in length.
Fig. 2.23: Scheme to show the structure of a chromatin fiber. (DNA: deoxyribonucleic acid)

* Five types of histones are recognized. These are H1, H2A, H2B, H3, and H4 (Fig. 2.24). Two molecules each of H2A, H2B, H3, and H4 join to form a granular mass, the nucleosome core. The DNA filament is wound twice around this core, the whole complex forming a nucleosome. The length of the DNA filament in one nucleosome contains 146 nucleotide pairs. One nucleosome is connected to the next by a short length of linker DNA. Linker DNA is made up of about 50 nucleotide pairs.
* Heterochromatin represents areas where chromatin fibers are tightly coiled on themselves forming "solid" masses. In contrast, euchromatin represents areas where coiling is not so marked. During cell division, the entire chromatin within the nucleus becomes very tightly coiled and takes on the appearance of a number of short, thick, and rod-like structures called chromosomes. Chromosomes are made up of DNA and proteins. Proteins stabilize the structure of chromosomes.
Fig. 2.24: Diagram showing detailed composition of a histone complex forming the nucleosome core.

* Nuclei contain one or more nucleoli and are spherical and about 1—3 pm in diameter. They stain intensely both with hematoxylin and eosin, the latter giving them a slight reddish tinge. Nucleoli are larger and more distinct in cells that are metabolically active.
* Using histochemical procedures that distinguish between DNA and RNA, it is seen that the nucleoli have a high RNA content. With the EM, nucleoli are seen to have a central filamentous zone (pars filamentosa) and an outer granular zone (pars granulosa) both of which are embedded in an amorphous material (pars amorpha) (Fig. 2.25).
* Parts of the chromosomes located within nucleoli constitute the pars chromosoma of nucleoli.
* Nucleoli are sites where ribosomal RNA is synthesized.

Many cells show projections from the cell surface. The various types of projections are described here.

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- * These can be seen, with the light microscope, as minute hair-like
projections from the free surfaces of some epithelial cells (Fig. 2.26). In the living animal, cilia can be seen to be motile.
* The free part of each cilium is called the shaft. The region of attachment of the shaft to the cell surface is called the base. The free end of the shaft tapers to a tip.
* Each cilium is 0.25 pm in diameter. It consists of: (a) an outer covering that is formed by an extension of the cell membrane; and (b) an inner core (axoneme) that is formed by microtubules arranged in a definite manner (Figs. 2.27A and B).
Fig. 2.26: Pseudostratified columnar epithelium showing cilia.
nN
Figs. 2.27A and B: Drawing of cilia as seen by scanning electron microscopy.
* It has a striking similarity to the structure of a centriole. There is a central pair of tubules that is surrounded by nine pairs of tubules. The outer tubules are connected to the inner pair by radial structures like the spokes of a wheel. Other projections pass outward from the outer tubules (Fig. 2.28).
+ As the tubules of the shaft are traced toward the tip of the cilium, it is seen that one tubule of each outer pair ends short of the tip so that near the tip each outer pair is represented by one tubule only. Just near the tip, only the central pair of tubules is seen (Fig. 2.29).
* At the base of the cilium, one additional tubule is added to each outer pair so that here the nine outer groups of tubules have three tubules each, exactly as in the centriole.
* Microtubules in cilia are bound with proteins (dynein and nexin). Nexin holds the microtubules together. Dynein molecules are responsible for bending of tubules, and thereby for movements of cilia.
Fig. 2.28: Transverse section across a cilium.

* The cilia lining an epithelial surface move in coordination with one another, the total effect being that like a wave. As a result, fluid, mucous, or small solid objects lying on the epithelium can be caused to move in a specific direction. Movements of cilia lining the respiratory epithelium help to move secretions in the trachea and bronchi toward the pharynx. Ciliary action helps in the movement of ova through the uterine tube, and of spermatozoa through the male genital tract.
* In some situations,they perform a sensory function. Cilia present on the cells in the olfactory mucosa of the nose are called olfactory cilia: They are receptors for smell. Similar structures called kinociliaare present in some parts of the internal ear.
Fig. 2.29: Longitudinal section through a cilium.

* These are somewhat larger processes having the same basic structure as cilia. In the human body, the best example of a flagellum is the tail of the spermatozoon.
* In a flagellum, movement starts at its base. The segment nearest the base bends in one direction. This is followed by bending of succeeding segments in opposite directions, so that a wave-like motion passes down the flagellum. When a spermatozoon is suspended in a fluid medium, this wave of movement propels the spermatozoon forward.
Figs. 2.30A and B: Microvilli as seen in longitudinal section. The regular arrangement of microvilli is characteristic of the striated border of intestinal absorptive cells.

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* Microvilli are finger-like projections from the cell surface that can be seen by EM (Figs. 2.30A and B).
* Each microvillus consists of an outer covering of plasma membrane and a cytoplasmic core in which there are numerous microfilaments (actin filaments). The filaments are continuous with actin filaments of the cell cortex.
Numerous enzymes and glycoproteins concerned with absorption have been located in microvilli.
* With the light microscope, the free borders of epithelial cells lining the small intestine appear to be thickened: The thickening has striations perpendicular to the surface. This striated border of light microscopy (Figs. 2.31A and B) has been shown by EM to be made up of long microvilli arranged parallel to one another.
* In some cells, the microvilli are not arranged so regularly. With the light microscope, the microvilli of such cells give the appearance of a brush border (Fig. 2.32).
Figs. 2.31A and B: Light microscopic appearance of striated border formed by microvilli.
Fig. 2.32: Light microscopic appearance of a brush border in epithelium lining renal tubules.
* Microvilli greatly increase the surface area of the cell and are, therefore, seen most typically at sites of active absorption, e.g. the intestine, and the proximal and distal convoluted tubules of the kidneys.
* Modified microvilli called stereocilia are seen on receptor cells in the internal ear, and on the epithelium of the epididymis.
* In some cells, the cell membrane over the basal or lateral aspects of the cell shows deep folds (basolateral folds). Like microvilli, basolateral folds are an adaptation to increase cell surface area.
* Basal folds are seen in renal tubular cells, and in cells lining the ducts of some glands. Lateral folds are seen in absorptive cells lining the gut (Fig. 2.32).

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Nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining.
* Structures that are stained purple (basophilic): DNA (heterochromatin and the nucleolus) in the nucleus, and RNA in ribosomes and in the RER are both acidic, and so hematoxylin binds to them and stains them purple. Some extracellular materials (i.e. carbohydrates in cartilage) are also basophilic.
* Structures that are stained pink (eosinophilic or acidophilic): Most proteins in the cytoplasm are basic, and so eosin binds to these proteins and stains them pink. This includes cytoplasm, cell membrane,
cytoplasmic filaments in muscle cells, intracellular membranes, and extracellular fibers.
Mucous cells like goblet cell produce mucus, which does not stain very darkly, so the mucous cells look almost clear. Mucous cells stain very poorly because they contain little RER and their mucin granules tend to react poorly with most stains.
Serous cells produce a watery secretion that contains a lot of proteins and stain fairly well due to the presence of RER and secretory granules in the cytoplasm.
Bf

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* Mitochondrial DNA can be abnormal. This interferes with mitochondrial and cell functions, resulting in disorders referred to as mitochondrial cytopathy syndromes. The features (which differ in intensity from patient to patient) include muscle weakness, degenerative lesions in the brain, and high levels of lactic acid. The condition can be diagnosed by EM examination of muscle biopsies. The mitochondria show characteristic paracrystalline inclusions.
Genetic defects can lead to absence of specific acid hydrolases that are normally present in lysosomes. As a result, some molecules cannot be degraded, and accumulate in lysosomes. Examples of such disorders are lysosomal glycogen storage disease in which there is abnormal accumulation of glycogen, and Tay-Sachs disease in which lipids accumulate in lysosomes and lead to neuronal degeneration. *, Os
Defects in enzymes of peroxisomes can result in metabolic disorders associated with storage of abnormal lipids in some cells (brain and adrenal). *
Cilia can be abnormal in persons with genetic defects that interfere with synthesis of ciliary proteins. This leads to the immotile cilia syndrome. As secretions are not removed from respiratory passages, the patient has repeated and severe chest infections. Women affected by the syndrome may be sterile as movement of ova along the uterine tube is affected. Ciliary proteins are present in the tails of spermatozoa, and an affected male may be sterile because of interference with the motility of spermatozoa. *
1. A structure that is continuous with rough endoplasmic reticulum (RER) is the:
a. Golgi apparatus
b. Nucleolus
c. Outer nuclear membrane
d. Euchromatin
2. Which of the following is not a membranous organelle?
a. Centrioles
b. Mitochondria
c. Lysosomes
d. Endoplasmic reticulum
3. “Cell drinking” is also called as:
a. Endocytosis
b. Exocytosis
c. Pinocytosis
d. Phagocytosis
4. Protein synthesis occurs at:
a. Ribosomes
b. Microfilaments
c. Centrioles
d. Nucleoli
5. Junction that segregates two cell compartments from mixing is:

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a. Gap junction
b. Desmosomes
c. Tight junction
d. Cell junction
6. A type of junctions that has protein extending from one cell to another:
. Gap junction
. Desmosomes . Tight junction 2009
. Cell junction

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* Definition, general features and functions of epithelial tissue
* Classification of the epithelial tissue with example
* Basement membrane
* Cell surface projections

* Definition of glands
* Classification with example
* Structure of serous and mucous Salivary glands

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One or more layers of cells that cover the outer surface (of the body) or line the luminal surface of tubular structures and cavities of the body are called epithelia (singular = epithelium).
I.
GENERAL FEATURES OF EPITHELIAL TISSUE
* Very little intercellular space (20 nm)
* Usually avascular and nourishment is by diffusion from the adjacent supporting tissue
* Cells rest on a basement membrane
* Cells show polarity
* Cells show surface modifications
* Good regenerative capacity
* Metaplasia (changes from one type to another) can take place
* Derived from all three germ layers.
Bi

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* Protection from abrasion and injury
* Absorption of the material from lumen
* Secretion of mucus, hormones, enzymes, etc.
* Exchange/transcellular transport across the epithelial layers.
i

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Epithelial tissue is classified depending on the cell arrangements and morphology (Flowchart 3.1).
An epithelium may consist of only one layer of cells when it is called a unilayered or simple epithelium. Alternatively, it may be multilayered (stratified) or it can be pseudostratified.
* Unilayered (simple) epithelia: Single layer of cells resting on a basement membrane. It may be further classified according to the shape of the cells constituting them.
* Multilayered (stratified) epithelia: Epithelia which consist of multiple layers with the basal layer resting on the basement membrane. The epithelium is named according to the shape of cells of the most superficial layer.
* Pseudostratified epithelia: Single-layered cells but giving a false appearance of stratification.
i

Based on the shape of the cells constituting them, simple epithelia can be

* Tt is composed of single layer of flattened cells, their height being very little as compared to their width. Such an epithelium is called a squamous epithelium. The cytoplasm of cells forms only a thin layer. The nuclei produce bulging of the cell surface (Plate 3.1).
* In surface view, the cells have polygonal outlines that interlock with those of adjoining cells (Plate 3.1D). In a section, the cells appear flattened and their height being much less as compared to their width (Fig. 3.1).
* Functions: It helps in rapid transport of substances, diffusion of gases, and filtration of fluids.
* With the electron microscope (EM) the junctions between cells are marked by occluding junctions. The junctions are, thus, tightly sealed, and any substance passing through the epithelium has to pass through the cells, and not between them.

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* Alveoli of the lungs.
* Free surface of the serous pericardium, the pleura, and the peritoneum; here it is called mesothelium.
* Inside the heart, where it is called endocardium and inside the blood vessels and lymphatics, where it is called endothelium.
* Squamous epithelium is also found lining some parts of the renal tubules, and in some parts of the internal ear.
Fig. 3.1: Simple squamous epithelium (schematic representation).

* When the height and width of the cells of the epithelium are more or less equal (i.e. they look like squares in section), it is described as a cuboidal epithelium.
* The nuclei are usually rounded and centrally placed (Plate 3.2).
*In sectional view, cells appear cuboidal in shape. When viewed from surface, cells are hexagonal in shape (Fig. 3.2).
* Function: It is mainly concerned with secretory, absorptive, and excretory functions.

* Tt is seen in the follicles of the thyroid gland, in the ducts of many glands, and on the surface of the ovary, the choroid plexuses, the inner surface of the lens, and the pigment cell layer of the retina.
* A cuboidal epithelium with a prominent brush border is seen in the proximal convoluted tubules (PCTs) of the kidneys.

* When the height of the cells of the epithelium is distinctly greater than their width, it is described as a columnar epithelium.
* Nuclei are elongated and located in the lower half of the cells. Nuclei of all cells are placed at the same level (Fig. 3.3 and Plate 3.3).
* In vertical section, the cells of this epithelium are rectangular. On surface

Simple squamous epithelium: (A) As seen in drawing; (B) An alveolus of the lung showing a lining of simple squamous epithelium (photomicrograph) (see arrows); (C) A capillary lined by endothelium (photomicrograph) (see arrow); (D) Surface view as seen in buccal smear (photomicrograph).

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"The cells of this epithelium are flattened in sections. They appear so thin that bulgings are produced on the surface by their nuclei
"In surface view the cells have polygonal outlines that interlock with those of adjoining cells.
Columnar epithelium can be further classified according to the nature of the free surfaces of the cells as follows:
1. Simple columnar epithelium: In some situations, the cell surface he particular specialization. A simple columnar epithelium (nonciliated) the mucous membrane of the stomach and of the large intestine. They even contain goblet cells.
Goblet cells are glandular simple columnar epithelial cells which perform the major function of secreting mucus. The name goblet is derived due to the special shape of these cells (Fig. 3.4). It has a narrow base and expanded apical portion. Since the mucus gets washed away during routine hematoxylin and eosin (H & E) staining, the upper expanded portions appear empty. The nucleus is round and pushed to the bottom due to accumulated mucinogen.
Plate 3.2: Simple cuboidal epithelium
Simple cuboidal epithelium: (A) As seen in a section (drawing); (B) Thyroid follicle lined by simple cuboidal epithelium (photomicrograph) (see arrows); (C) Duct of salivary gland (photomicrograph) (see arrow).
"This epithelium is made up of cells that look like squares (in which the length and breadth are equal). Nuclei are rounded
"A typical cuboidal epithelium lines follicles of the thyroid gland, ducts of salivary gland, and tubules of kidney.
Fig. 3.2: Simple cuboidal epithelium. Note that the cells appear cuboidal in section and hexagonal in surface view (schematic representation).
Fig. 3.3: Simple columnar epithelium. Note the basally placed oval nuclei. The cells appear hexagonal in surface view (schematic representation).
Simple columnar epithelium: (A) As seen in a section (drawing); (B) Lining the mucosa of the stomach (photomicrograph) (see arrows)

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*The height of the cells in this epithelium is much greater than their width. The nuclei are oval being elongated in the same direction as the cells. They lie near the bases of the cells. Because of this we see a zone of clear cytoplasm above the nuclei
'A simple columnar epithelium (non-ciliated) lines the mucous membrane of the stomach and of the large intestine.
Fig. 3.4: Goblet cells.
2. Ciliated columnar epithelium: In some situations, the cell surface bears e.g. lining epithelium of fallopian tube.
3. At some places, the surface is covered with microvilli. Although microvilli are visible only with the EM, they are seen as a striated bc with the light microscope (when the microvilli are arranged regularly) o1 brush border (when the microvilli are irregularly placed) (Figs. 3.5A anc

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* Mainly columnar cells have a secretory and absorption function.
* In the intestines, many of them secrete mucous that accumulates in the apical part of the cell making it very light staining. These cells acquire a characteristic shape and are called goblet cells.
Figs. 3.5A and B: (A) Columnar epithelium showing cilia; (B) Columnar epithelium showing a striated border made up of microvilli (schematic representation).
* Columnar epithelial cells can be found in the linings of the ears, nose, and on the taste buds of the tongue. These cells are designed to respond to stimuli.
* Ciliated columnar epithelium can be found in the lining of some organs, such as the fallopian tubes and in the lungs. Cilia are specialized bundles of protein which can move together to help propel material in one direction.
* Columnar epithelial cells are rather thick, which allows them to withstand mild amounts of damage.
* Microvilli increase the surface area for absorption.

* Simple columnar epithelium is present over the mucous membrane of the stomach and the large intestine.
* Columnar epithelium with a striated border is seen most typically in the small intestine, and with a brush border in the gallbladder (Plate 3.4).
* Ciliated columnar epithelium lines most of the respiratory tract, the uterus, and the uterine tubes. It is also seen in the efferent ductules of the testis, parts of the middle ear and auditory tube, and the ependyma lining the central canal of the spinal cord and the ventricles of the brain.
* In the respiratory tract, the cilia move mucous accumulating in the bronchi (and containing trapped dust particles) toward the larynx and pharynx. When excessive, this mucous is brought out as sputum during coughing. In the uterine tubes, the movements of the cilia help in the passage of ova toward the uterus.

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* It is not a true stratified epithelium but appears to be stratified. All the cells are in contact with basal lamina, but only few cells reach the surface. Tall cells have narrow base and broad apical part, but short cells have broad base and narrow apical part, and nuclei are located at the broad end of the cells, which gives appearance as many layers (Fig. 3.6 and Plate 3.5). However, the nuclei appear to be arranged in two or more layers giving the impression that the epithelium is more than one cell thick.
* The epithelium may bear cilia (ciliated epithelium) and may contain goblet cells. The cilia are capable of movement. At some sites, this epithelium may display stereocilia.
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Columnar epithelium showing striated border: (A) As seen in a section (drawing); (B)
Columnar epithelium with a striated border in the small intestine (see arrow).

"In some regions the free surfaces of the cells of columnar epithelium show a thickening with vertical striations in it, this is called a striated border
'This is seen typically in the small intestine.
Fig. 3.6: Pseudostratified columnar epithelium as seen in a section.

The tall columnar cells are secretory in nature, while the short, basal cells are stem cells which constantly replace the tall cells. The cilia help in clearance of the mucous. The stereocilia help in absorption.

* Nonciliated pseudostratified columnar epithelium is found in some parts of the auditory tube, the ductus deferens, and the male urethra (membranous and penile parts).
* Ciliated pseudostratified columnar epithelium is seen in the trachea and in large bronchi.
* Pseudostratified columnar epithelium with stereocilia (long microvilli) is seen in epididymis.

It is stratified more than one layer of cells. It is classified according to the cell shape of the superficial layer as squamous, cuboidal, and columnar.

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- * This type of epithelium is thick and made up of several layers of cells
(Plate 3.6 and Fig. 3.7).
* The cells of the deepest (or basal) layer rest on the basement membrane, they are usually columnar in shape. Lying over the columnar cells, there are polyhedral or cuboidal cells. As we pass toward the surface of the epithelium, these cells become progressively more flat, so that the most superficial cells consist of flattened squamous cells.
* Stratified squamous epithelium is found over those surfaces of the body that are subject to friction. As a result of friction the most superficial layers are constantly being removed and are replaced by the proliferation of cells from the basal layer. This layer, therefore, shows frequent mitoses.
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Pseudostratified ciliated columnar epithelium: (A) As seen in a section (drawing); (B) Pseudostratified ciliated columnar epithelium in trachea (photomicrograph) (see arrow).

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At is not a true stratified epithelium but appears to be stratified. Normally, in columnar epithelium the nuclei lie in a row, toward the basal part of the cells. Sometimes, however, the
nuclei appear to be arranged in two or more layers giving the impression that the epithelium is more than one cell thick in some situations, pseudostratified columnar epithelium bears hair-like projections called cilia
*Pseudostratified ciliated columnar epithelium is seen in trachea and in large bronchi.
The surface layer is made up of keratin which appears as fibers. No cellular outline or nuclei can be seen
'It is seen typically in epidermis of the skin.
Fig. 3.7: Stratified squamous epithelium. There is a basal layer of columnar cells that rests on the basement membrane. Overlying the columnar cells of this layer, there are a few layers of polygonal cells or rounded cells. Still more superficially, the cells undergo progressive flattening, becoming squamous (schematic representation).
Stratified squamous epithelium can be divided into two types—(1) Nonkeratinized and (2) Keratinized.
- 1. Nonkeratinized stratified squamous epithelium: Stratified epithelium, w the surface of the squamous epithelium remains moist, the most super: cells are living, and nuclei can be seen in them. This kind of epitheliv described as nonkeratinized stratified squamous epithelium (Fig. 3.8A).
Examples: It is usually wet and is found in lining the mouth, oropharynx, esophagus, true vocal folds, and vagina.
- 2. Keratinized stratified squamous epithelium: It is similar to nonkeratii epithelium, except that epithelial surface is dry, and the most super layers of epithelium are composed of dead cells whose nuclei and cytop have been replaced with keratin. This kind of epithelium consti
keratinized stratified squamous epithelium (Fig. 3.8B and Plate 3.6).
Example: Epithelium covers the skin of whole of the body and forms the epidermis.

* Tt is protective in nature
* Keratin prevents dehydration of underlying tissue.
Figs. 3.8A and B: Stratified squamous epithelium. (A) Nonkeratinized; (B) Keratinized (schematic representation).

It consists of only two layers of cuboidal cells. It is found in ducts of sweat glands.

It is composed of two layers, polyhedral to cuboidal deeper layer and superficial layer of columnar cells. It is found in conjunctiva of eye, certain large excretory ducts, like pancreas and salivary glands, and regions of male urethra.

Like all stratified epithelia, it is protective in function, and it also helps in conducting the secretion of the glands.

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This is a multilayered epithelium with four to six cells thick and which can contract and expand as needed.
* Tt is called transitional because it is transitional between stratified squamous and stratified columnar epithelia. 4,
* It differs from stratified squamous epithelium in that the cells at the surface are not squamous. The deepest cells are columnar or cuboidal. The middle layers are made up of polyhedral or pear-shaped cells. The cells of the surface layer are large and often shaped like an umbrella (Plate 3.7).
* The superficial cell layer, which lines the lumen, is the only fully differentiated layer of the epithelium. 4,
*Tt provides an impenetrable barrier between the lumen and _ the bloodstream. It is exclusively located in urinary system and named as urothelium.
* In the urinary bladder, it is seen that cells of transitional epithelium can be stretched considerably without losing their integrity. When stretched, it appears to be thinner, and the cells become flattened (Figs. 3.9A and B).

Transitional epithelium is found in the renal pelvis and calyces, the ureter, the urinary bladder, and part of the urethra. Because of this distribution, it is also called urothelium.

At the surface of the epithelium, the plasma membranes are unusual.
Embedded in the lipid layer of the membranes, there are special glycoproteins. It is believed that these glycoproteins make the membrane impervious and resistant to the toxic effects of substances present in urine and thus, afford protection to adjacent tissues. The transitional epithelium cells stretch readily.

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It is a thin, delicate membrane of protein fibers and mucopolysaccharides separating an epithelium from underlying connective tissue.
* A distinct basement membrane cannot be seen in H&E preparations, but it can be well demonstrated using the periodic acid-Schiff (PAS) method. The latter stains the glycoproteins present in the membrane.
* Under the EM a basement membrane is seen to have a basal lamina (nearest to the epithelial cells) and a reticular lamina or fibroreticular lamina (consisting of reticular tissue and merging into surrounding connective tissue). The basal lamina is divisible into the lamina densa containing fibrils and the lamina lucida which appears to be transparent. The lamina lucida lies against the cell membranes of epithelial cells.
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Points of identification
*
In this type of epithelium, several layers of round nuclei are seen
¥ The superficial cells are not flattened but are umbrella shaped
¥ Their nuclei appear rounded and may show mitotic figures
® This epithelium lines many parts of the urinary tract.
* Basal membrane also present where other cell types come in contact with connective tissues, like muscle, adipose and nervous tissue.
* Main component is type IV collagen fibers, glycoproteins laminin and entactin, and proteoglycans.
Figs. 3.9A and B: Transitional epithelium: (A) Stretched; (B) Relaxed.

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* It provides structural support to the cells. It provides adhesion on one side of epithelial cells.
* Tt acts as a barrier that limits or regulates the exchange of molecules. The barrier function varies with location (because of variations in pore size). Large proteins are prevented from passing out of blood vessels, but (in the lung) diffusion of gases is allowed.
* It influences cell polarity, regulates cell proliferation and differentiation by binding with growth factors, influences cell metabolism, and serves as pathways for cell migration.
* The membranes may influence the regeneration of peripheral nerves after injury and may play a role in reestablishment of the neuromuscular junctions.
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The free or epical surface epithelial cells show projections from the cell surface. The various types of projections are as follows:
* Cilia
* Microvilli
* Stereocilia.
Figs. 3.10A and B: Cilium. (A) Ultrastructure; (B) Internal structure (schematic representation).

Cilia are slender, long, motile, microscopic, hair-like structures, or organelles that extend from the epical surface of some epithelial cells. In the living animal, cilia can be seen to be motile.

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* The detailed structure of cilia can only be made out by electron microscopy.
* The free part of each cilium is called the shaft. The region of attachment of the shaft to the cell surface is called the base (also called the basal body, basal granule, or kinetosome). The free end of the shaft tapers to a tip. Each cilium is 10 pm in length and 0.25 pm in diameter.
It consists of: (1) an outer covering that is formed by an extension of the cell membrane; and (2) an inner core (axoneme) that is formed by microtubules arranged in a definite manner (Figs. 3.10A and B). *
It has a striking similarity to the structure of a centriole. There is a central pair of tubules that is surrounded by nine pairs of tubules. The outer tubules are connected to the inner pair by radial structures (which are like the spokes of a wheel). Other projections pass outward from the outer tubules (Fig. 3.11). *
* As the tubules of the shaft are traced toward the tip of the cilium, it is seen that one tubule of each outer pair ends short of the tip so that near the tip each outer pair is represented by one tubule only. Just near the tip, only the central pair of tubules is present.
* At the base of the cilium, one additional tubule is added to each outer pair so that here the nine outer groups of tubules have three tubules each, exactly as in the centriole. Microtubules in cilia are bound with proteins (dynein and nexin). Nexin holds the microtubules together. Dynein molecules are responsible for bending of the tubules, and thereby for the movements of cilia (Fig. 3.11). ate

The cilia lining an epithelial surface move in coordination with one another, the total effect being that like a wave. As a result fluid, mucous, or small solid objects lying on the epithelium can be caused to move in a specific

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* Movements of cilia lining the respiratory epithelium help to move secretions in the trachea and bronchi toward the pharynx.
* Ciliary action helps in the movement of ova through the uterine tube, and of spermatozoa through the male genital tract.
* In some situations, there are cilia-like structures that perform a sensory function. They may be nonmotile, but can be bent by external influences. Such "cilia" present on the cells in the olfactory mucosa of the nose are called olfactory cilia, they are receptors for smell. Similar structures called kinocilia are present in some parts of the internal ear.
Note: Flagella: These are somewhat larger processes having the same basic structure as cilia. In the human body, the best example of a flagellum is the tail of the spermatozoon. The movements of flagella are different from those of cilia. In a flagellum, movement starts at its base. The segment nearest to the base bends in one direction. This is followed by the bending of succeeding segments in opposite directions, so that a wave-like motion passes down the flagellum.
Fig. 3.11: Helical structure of microtubule (schematic representation).

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* Microvilli are small finger-like cytoplasmic projections from the free surface of the cell that can be seen by EM measuring 1—2 mm in length and about 75-90 ym in diameter. Microvilli are covered by a polysaccharide surface coat called as glycocalyx.
* Each microvillus consists of an outer covering of plasma membrane and a
cytoplasmic core in which there are numerous microfilaments (actin filaments). The filaments are continuous with actin filaments of the cell cortex.
Numerous enzymes and glycoproteins, concerned with absorption, have been located in microvilli.
In some places, like epithelial cells lining the small intestine, the microvilli appear to be thickened, the thickening has striations perpendicular to the surface and arranged regularly giving a striated border appearance.
In some places, like PCTs of kidney, the microvilli are not arranged so regularly. With the light microscope, the microvilli of such cells give the appearance of a brush border.

Microvilli are nonmotile processes which greatly increase the surface area of the cell and are, therefore, seen most typically at the sites of active absorption, e.g. the intestine, and the PCTs of the kidney (Table 3.1).
Table 3.1: Differences between cilia and microvilli., 1 = Table 3.1: Differences between cilia and microvilli.. Cilia, 1 = Microvilli. 10 μm in length and 0.25 μm in diameter, 1 = 1-2 mm in length and about 75- 90 μm in diameter. Motile, 1 = Nonmotile. Contains 9+2 pattern of microtubules, 1 = Contains numerous microfilaments (actin filaments). Concerned with the movement of ova through uterine tube and movement of secretions in trachea and bronchi toward pharynx, 1 = Concerned with absorptive functions. Seen over lining epithelium of respiratory tract and uterine tube., 1 = Seen over intestinal epithelium and proximal convoluted tubule of kidney

*Renewal of epithelial cells:Epithelial cells have a high turnover rate which is related to their location and function, and the time frame for renewal is constant for a particular cell type. For example, cells of epidermis are constantly being renewed, and it takes about 28 days for complete process from basal layer to keratinization. Small intestine cells are replaced every 4—6 days.

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*With the EM, the cells of transitional epithelium are seen to b firmly united to one another by numerous desmosomes. Becaus of these connections, the cells retain their relative position whe the epithelium is stretched or relaxed.
*The cells in the basal layer of transitional epithelium sho} occasional mitoses, but these are much less frequent than thos in stratified squamous epithelium, as there is normally littl erosion of the surface. Many cells of the superficial (lumina layers of the epithelium may contain two nuclei. In some cells, th nucleus is single, but contains multiples of the normal number ¢ chromosomes (i.e. it may be polyploid).
*According to some workers, all cells of transitional epitheliur reach the basal lamina through thin processes. Even though th cells are stratified, they retain a contact with the basemer membrane. Hence, this is a transition from unilayered multilayered epithelium. t
*The shape of epithelial cells is related to the amount of contained cytoplasm and organelles. These in turn are related to metabolic activity. Squamous cells are least active. Columnar cells contain abundant mitochondria and endoplasmic reticulum and are highly active.
*Laterally, epithelial cells are in contact with other epithelial cells. The contact between adjoining cells is generally an intimate one because of the presence of desmosomes, zonulae adherens, and zonulae occludens. The intimate contact ensures that materials passing through the epithelium have to pass through the cells, rather than between them.
* Some epithelial cells contain pigment. Such cells are present in the skin, the retina, and the iris.
*Epithelia are generally devoid of blood vessels. Their cells obtain nutrition by diffusion from blood vessels in underlying tissues. In contrast, delicate nerve fibers frequently penetrate into the intervals between epithelial cells.
*Epithelia have considerable capacity for repair after damage. They grow rapidly after injury, to repair the defect.
*It should be remembered that same epithelial cells could have very different functions at different locations. For example, cuboidal cells lining follicles of the thyroid gland have very little in common with cuboidal cells covering the surface of the ovary.

* Epithelial cells in which transport of ions is an important function (e.g. renal tubules) are marked by the presence of basolateral folds, and the presence of large numbers of mitochondria, which provide adenosine triphosphate (ATP) for ion transport. Tight junctions between the cells prevent the passive diffusion of the ions.
* Epithelial cells contain some proteins not present in nonepithelial cells. These include cytokeratin (present in intermediate filaments). Such proteins can be _ localized using immunohistochemical techniques.

Stereocilia are very long, nonmotile, and thick microvilli measuring about 510 um in length. They are seen on receptor cells in the internal ear, and on the epithelium of the epididymis. They increase the cells surface area for absorption in epididymis and probably function in signal generation in the hair cells of internal ear.
Note: Basal folds: In some cells, the cell membrane over the basal or lateral aspect of the cell shows deep folds (basolateral folds). Like microvilli, basolateral folds are an adaptation to increase cell surface area. Basal folds are seen in renal tubular cells, and in cells lining the ducts of some glands. Lateral folds are seen in absorptive cells lining the gut.

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* Each epithelium has its own unique characteristics, location, and morphology which are related to its function. Sometimes, the cell population of an epithelium may undergo metaplasia, transforming it into another epithelial type.
™ Squamous metaplasia: Pseudostratified ciliated columnar epithelium of 1 bronchi in heavy smokers may transform into stratified squamc epithelium.
@
"A tumor (or neoplasm) can arise from any tissue if there is uncontrol] growth of cells. Such a tumor may be benign, when it remains localized, may be malignant. A malignant growth invades surrounding tissues.
"A malignant tumor arising from an epithelium is called a carcinoma. I arises from a squamous epithelium, it is a squamous cell carcinoma; anc it arises from glandular epithelium, it is called an adenoma.
= Quite commonly, cells in tumors resemble those of the tissue from whi they are derived, and this is useful in pathological diagnosis. However, metastases of fast growing tumors, the cells may not show : characteristics of the tissue of origin (undifferentiated tumor), and it may difficult to find out the location of the primary growth. In such cas diagnosis can be aided by the localization of proteins that are present o1 in epithelia. As mentioned earlier, this can be done by usi immunohistochemical techniques.

"Immotile cilia syndrome (ICS) is an autosomal recessive disease w extensive genetic heterogeneity characterized by abnormal ciliary moti and impaired mucociliary clearance. Ultrastructural and functional defe of cilia result in the lack of effective ciliary motility, causing abnorn mucociliary clearance. As secretions are not removed from respiratc passages, the patient has repeated and severe chest infections. Wor affected by the syndrome may be sterile as the movement of ova along | uterine tube is affected. Ciliary proteins are present in the tails spermatozoa, and an affected male may be sterile because of interferer with the motility of spermatozoa.
" Kartagener's syndrome: It is aone type of ICS. It is an autosomal recess' genetic ciliary disorder comprising the triad of situs inversus, chro sinusitis, and bronchiectasis. The basic problem lies in the defect movement of cilia, leading to recurrent chest infections, ear/nose/thr: symptoms, and infertility.
"Ciliary action is also necessary for normal development of the tissues embryonic life. Migration of cells during embryogenesis is dependent ciliary action, and if the cilia are not motile, various congeni abnormalities can result.

Simple, 1 = Squamous. Simple, 2 = Lung alveoli, blood vessels (endothelium), lining of pericardium, pleura, and peritoneum (mesothelium). Simple, 3 = Active transport/exchange. , 1 = Cuboidal. , 2 = Thyroid follicle, covering of ovary. , 3 = Covering, secretion. , 1 = Columnar. , 2 = Gastrointestinal tract (GIT), gallbladder. , 3 = Protection, lubrication, absorption, and secretion. Pseudostratified, 1 = Pseudostratified. Pseudostratified, 2 = Trachea, bronchi, nasal cavity. Pseudostratified, 3 = Protection, secretion. Stratified, 1 = Stratifiedsquamous keratinized. Stratified, 2 = Skin. Stratified, 3 = Protection. , 1 = Stratified squamous nonkeratinized. , 2 = Oral cavity, esophagus. , 3 = Protection, secretion. , 1 = Stratified cuboidal. , 2 = Ducts of sweat glands. , 3 = Absorption, secretion. , 1 = Stratified columnar. , 2 = Conjunctiva, some large excretory ducts, and part of male urethra. , 3 = Protection, secretion, and absorption. Transitional, 1 = Transitional. Transitional, 2 = Urinary tract from renal calyces to urethra. Transitional, 3 = Protection, distensible
Bf

A gland is a group of cell body that synthesizes substances, like hormones, sweat, saliva, mucus, or acids, for release into the bloodstream or into cavities inside the body or its outer surface.
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* Glands originate from specialized secretory epithelial cells that leave the surface from where they developed and penetrate into the underlying connective tissue and form a basal lamina around them.
* Glandular epithelium is formed by the cells specialized to produce secretion. The secretory molecules are stored in the cells in small membrane-bound vesicle called secretory granules. Glandular cells may synthesize, store, and secrete proteins, lipids, or complexes of carbohydrate and proteins.
* Glands are under hormonal or nervous control through chemical messengers.
* They may be exocrine or endocrine.
i

"Unicellular: Unicellular glands are interspersed among otl (nonsecretory) epithelial cells. They can be found in the epithelium lini the intestines, e.g. goblet cells.
- " Multicellular: Most glands are multicellular. Such glands develop diverticula from epithelial surfaces. The "distal" parts of the divertic develop into secretory elements, while the "proximal" parts form du through which secretions reach the epithelial surface, e.g. lacrimal gla parotid gland, etc.

"Exocrine glands: Glands that pour their secretions onto an epithel surface, directly or through ducts, are called exocrine glands (or externa secreting glands), e.g. salivary glands.
" Endocrine glands: Some glands lose all contact with the epithelial surfé from which they develop, and they pour their secretions into blood. St glands are called endocrine glands, internally secreting glands, or ductl glands. They secrete the products through the basal lamina onto the blc stream, e.g. thyroid, pituitary, and adrenal.
" Paracrine glands:Glands that secrete a hormone which has effect only the vicinity of the gland secreting it/hormones that diffuses to sh distances to other cells. e.g. enteroendocrine cells of gastrointestinal tr. (GIT).
i

It is dispersed individually among the epithelia of the GIT and respiratory tract. They protect the linings. They derive their name from their shape like a goblet. They have thin basal and expanded apical part. Cytoplasm is filled with membrane-bound secretory droplets, which displaces the cytoplasm to the periphery and pushes the nucleus toward base.

Multicellular exocrine glands can be further classified as follows:

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"Merocrine: It is a type of exocrine gland. The secretion is released exocytosis without losing a part of the secretory cell, e.g. salivary glan some sweat glands, and pancreas.
"Apocrine: Small apical part of the cell is released along with secret« product, e.g. sweat glands in the axillae, areola and nipples of the bre<
ear canal, eyelids, wings of the nostril, perianal region, and some parts the external genitalia.
" Holocrine: As the secretory cell matures, it dies and becomes the secret« product, e.g. sebaceous gland, tarsal gland.
" Cytocrine: In testis, the cells are released as secretion.

™ Serous gland: The secretions of serous glands are protein in nature. Ce secrete an enzyme-rich watery fluid. Cells of serous acini are triangular shape with a rounded nucleus. Their nuclei are centrally placed,e pancreas.
"Mucous gland: In mucous. glands, the secretion conta mucopolysaccharides. Cells secrete mucus which is thick, viscous, é jelly-like protective lubricant known as mucin. Cell of mucous acini are t with flat nuclei at their bases. The lumen of these acini is larger than | serous acini, e.g. goblet cells, minor salivary glands of tongue and palate.

Figs. 3.12A to C: Types of glands based on the manner in which their secretions are poured out of the cells. (A) Merocrine; (B) Apocrine; (C) Holocrine (Schematic representation).
- ™ Mixed gland: Gland contains acini that secrete both mucus and serc secretions, e.g. submandibular and sublingual glands.

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" Simple: All the secretory cells of an exocrine gland discharge into one dt e.g. gastric glands, sweat glands, etc.
"Compound: There are a number of groups of secretory cells, and e< group discharging into its own duct. These ducts unite to form larger du that ultimately drain onto an epithelial surface, e.g. parotid gland, pancre etc.
* Based on the shape of the secretory unit: Both in simple and in compound glands, the secretory cells may be arranged in various ways:
" Tubular glands: Glands with secretory unit tubular in shape. The tube 1 be straight, coiled, or branched, e.g. gastric glands.
"Acinar glands: Glands with secretory unit round or oval in shape, € salivary glands.
" Alveolar glands: Glands with secretory unit flask-shaped. However, it 1 be noted that the terms acini and alveoli are often used as if they wi synonymous. Glands in which the secretory elements are greatly distenc are called saccular glands.
Note: Combinations of the earlier may be present in a single gland. From what has been said earlier, it will be seen that an exocrine gland may be:
* Unicellular
* Simple tubular
* Simple alveolar (or acinar)
* Compound tubular
* Compound alveolar
* Compound tubuloalveolar (or racemose).
Some further subdivisions of these are shown in Figures 3.13A to I.
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All exocrine glands have basically the same structural organization consisting of three components—(1) parenchyma, (2) stroma, and (3) duct system.
* Parenchyma: The secretory cells of a gland constitute its parenchyma.
* Stroma: The connective tissue in which the parenchyma lies is called the stroma. The glandular tissue is often divisible into lobules separated by connective tissue septa. Aggregations of lobules may form distinct lobes. The connective tissue covering the entire gland forms a capsule for it. Blood vessels and nerves pass along the connective tissue septa to reach the secretory elements. Their activity is under nervous or hormonal control (Fig. 3.14).
* Duct system: The ducts convey the secretory product of the gland. When a gland is divided into lobes, the ducts draining it may be intralobular (lying within a lobule), interlobular (lying in the intervals between lobules), or interlobar (lying between adjacent lobes), in increasing order of size (Fig. 3.14).

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* They are the sources of many of the body's chemical messengers, known as hormones that act at a distance from their origin.
* They are very varied in their size, location, and appearance.
* Many are solid organs, but few are made up of widely distributed single cells.
* Several endocrine glands consist of more than one type of secretory cell and may release more than one hormone product.
* They can be (a) individual cells (unicellular glands), e.g. enteroendocrine cells; (b) endocrine tissue in mixed glands (both endocrine and exocrine) as seen in pancreas and male and female reproductive organs; and (c) separate endocrine organs, e.g. pituitary gland, thyroid glands, parathyroid glands, and adrenal glands (Fig. 3.15).
Figs. 3.13A to I: Scheme to show various ways in which the secretory elements of a gland may be organized. (A) Unicellular gland; (B to G) Multicellular glands with a single duct are simple glands; and (H and 1) Multicellular glands with branching duct system are compound glands.
Fig. 3.14: Structural organization of an exocrine gland (schematic representation).

a. Cord and clump type: Endocrine glands are usually arranged in cor clumps that are intimately related to a rich network of blood capillari sinusoids
b. Follicular type:In some cases (for example, the thyroid gland), the ce form rounded follicles.
* Endocrine cells and their blood vessels are supported by delicate connective tissue, and are usually surrounded by a capsule.

The diffuse neuroendocrine system produces paracrine and endocrine hormones. They are widely spread throughout the digestive tract and in the respiratory system, they are interspersed among other secretory cells. Cells are capable of taking up precursors of amines and decarboxylating amino acids, and they are also called amine precursor uptake and decarboxylation (APUD) cells. The cells are stained with silver salts so they are also called argentaffin and argyophil cells.

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Bi
* Glands, both exocrine and endocrine, develop as diverticula of the epithelium (Fig. 3.15).
* The exocrine develops as a solid bud from the epithelium into the underlying connective tissue. Soon it elongates, undergoes canalization, and displays a secretory and conducting portion. The conducting part forms the duct and is continuous with the epithelium. Hence, an endocrine gland discharges its secretions through a duct.
* Endocrine gland also develops in a similar manner to exocrine gland, but with further development, it breaks the continuity with overlying epithelium. It appears as a clump of cells. Soon these groups of cells get surrounded by blood vessels into which they pour their secretions.
Fig. 3.15: Development of glands (schematic representation).
Note: In this chapter, we have considered the general features of glands. Further details of the structure of exocrine and endocrine glands will be considered while studying individual glands.

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Epithelia in secretory portions of glands show specializations of structure depending upon the nature of secretion as follows (Table 3.2):
* Cells that are protein secreting (e.g. hormone-producing cells) have a welldeveloped rough endoplasmic reticulum (ER), and a supranuclear Golgi complex. Secretory granules often fill the apical portions of the cells. The staining characters of the granules differ in cells producing different
secretions (the cells being described as acidophil, basophil, etc.).
* Mucin-secreting cells have a well-developed rough ER (where the protein component of mucin is synthesized) and a very well-developed Golgi complex (where proteins are glycosylated).
* Steroid-producing cells are characterized by the presence of extensive smooth ER and prominent mitochondria.

* Serous cells are usually arranged in the form of rounded acini. As a result, each cell is roughly pyramidal having a broad base and a narrow apex with a single, round, and basally located nucleus (Figs. 3.16A to C).
* Some microvilli and pinocytotic vesicles are seen at the apex of the cell. The lumen of the acinus often extends for some distance between adjacent cells: these extensions are called intercellular secretory canaliculi. Deep to these canaliculi, the cell membranes of adjoining cells are united by tight junctions. Deep to these junctions, the lateral cell margins show folds that interdigitate with those of adjoining cells. The apical cytoplasm contains secretory granules that are small, homogeneous, and electron dense. The cytoplasm also contains a prominent Golgi complex and abundant rough ER, both features indicating considerable synthetic activity. Mitochondria, lysosomes, and microfilaments are also present.

- * Mucous cells are usually arranged in the form of tubular secretory elements (Figs. 3.16A to C). The cells lining mucous cells tend to be columnar rather than pyramidal with an elongated nucleus at the basal part of the cell.
Table 3.2: Comparison between serous and mucous acini., 1 = Table 3.2: Comparison between serous and mucous acini.. Serous acini, 1 = Mucous acini. Triangular cells with rounded nucleus at the base. Cell boundaries are indistinct, 1 = Tall cells with flat nucleus at the base. Cell boundaries are distinct. Contain zymogen granules, 1 = Contain mucoid material. Darkly stained with H & E (because of the presence of zymogen granules, the color varies from pink to dark purple), 1 = Lightly stained and appear empty with hematoxylin and eosin (H & E). Thin watery secretion, 1 = Thick mucoid secretion
Figs. 3.16A to C: Types of acini. (A) Serous; (B) Mucous; (C) Mucous acini with serous demilune (schematic representation).
- * Crescents present in relation to them are located at the ends of the tubules. Their secretory granules are large and ill defined. Rough ER and Golgi complex are similar to those in serous cells, but microvilli, foldings of plasma membrane, and intercellular canaliculi are not usually seen.

From the point of view of ultrastructure, many cells of salivary glands are intermediate between serous and mucous cells. They are referred to as seromucous cells. Most of the cells identified as serous with light microscopy in the parotid and submandibular glands are really seromucous. The secretions of all types of salivary secretory cells contain protein— carbohydrate complexes. Their concentration is lowest in the cases of serous cells, very high in mucous cells, and with widely differing concentrations in seromucous cells.
8

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* Neoplasms can arise from the epithelium lining a gland. A benign growth arising in a gland is an adenoma; and a malignant growth is an adenocarcinoma.
* Sialorrhea (ptyalism): Increased flow of saliva is termed sialorrhea or ptyalism.
* Xerostomia: Decreased salivary flow is termed xerostomia.
* Sialadenitis: Inflammation of salivary glands is called as sialadenitis.
* Tumors of salivary glands:
1. Stratified squamous epithelium tissues are present in:
a. Thyroid gland and trachea
b. Lungs and kidneys
a., 1 = Pleomorphic adenoma (mixed salivary tumor) : It is the most common tumor of major and minor salivary glands. It is characterized by pleomorphic or mixed appearance in which there are epithelial elements present in a matrix of mucoid, myxoid, and chondroid tissue;. b., 1 = Mucoepidermoid carcinoma : It is the most common malignant salivary gland tumor. The tumor is composed of combination of four types of cells: (1) mucin-producing, (2) squamous, (3) intermediate, and (4) clear cells. Well-differentiated tumors have predominance of mucinous cells, while poorly differentiated have more solid and infiltrative pattern;and. c., 1 = Apudomas are tumors arising from polypeptide-secreting cells of the diffuse neuroendocrine system.

Nature of secretion, 2 = Secretion is protein in nature and watery. Nature of secretion, 3 = Pancreas. Nature of secretion, 1 = Mucous. Nature of secretion, 2 = Cells secrete mucus rich in mucopolysaccharides, which is thick, viscous, and jelly-like protective lubricant known as mucin. Nature of secretion, 3 = Goblet cells, minor salivary glands of tongue and palate. Nature of secretion, 1 = Mixed. Nature of secretion, 2 = Both mucus and serous acini present. Nature of secretion, 3 = Submandibular and sublingual glands. Exocrine gland may be: Unicellular, simple tubular, simple alveolar (or acinar), compound tubular, compound alveolar, and compound tubuloalveolar (or racemose), 1 = Exocrine gland may be: Unicellular, simple tubular, simple alveolar (or acinar), compound tubular, compound alveolar, and compound tubuloalveolar (or racemose). Exocrine gland may be: Unicellular, simple tubular, simple alveolar (or acinar), compound tubular, compound alveolar, and compound tubuloalveolar (or racemose), 2 = Exocrine gland may be: Unicellular, simple tubular, simple alveolar (or acinar), compound tubular, compound alveolar, and compound tubuloalveolar (or racemose). Exocrine gland may be: Unicellular, simple tubular, simple alveolar (or acinar), compound tubular, compound alveolar, and compound tubuloalveolar (or racemose), 3 = Exocrine gland may be: Unicellular, simple tubular, simple alveolar (or acinar), compound tubular, compound alveolar, and compound tubuloalveolar (or racemose)
c. Urinary bladder and kidneys
d. Mouth and esophagus

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Stomach
Urinary bladder Uterus 2900p
Lungs
3. What do you call the simple squamous epithelium that lines the blood vessels?
Epithelioid tissue
Mesothelium Endothelium Transitional 2905p
4. What cell type makes up the mucosa of the gallbladder?
Simple cuboidal Simple columnar Stratified squamous Transitional 2909

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Microvilli
Cilia Stereocilia 29009
Flagella
6. Reproductive cells (germinal epithelium) are made up of which of the following epit
tissue?
a. Cuboidal
b. Columnar
c. Squamous
c. Sensory
7. Urethra, vagina, and esophagus have a common inner lining of:
a Squamous epithelium
b. Ciliated epithelium
c. Columnar epithelium
d. Stratified squamous epithelium
8. Pavement epithelium is another name for:
a. Simple cuboidal epithelium
b. Simple ciliated epithelium
10. Outer layer of skin is made up of keratinized epithelium, this is because:
11. Epithelial tissues arise from:

13. The cells which is contractile and present in acini or ducts are:
14. Salivary gland is unique in that, its secretion are controlled by:

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c. Simple squamous epithelium
d. Stratified epithelium
. All of the following are the characteristic of epithelial tissues; except:
They are highly vascular They rest on basement membrane They have very little intercellular spaces Cells have surface modifications 2900p
a. Itis exposed thus, subjected to wear and tear
b. It covers the whole body
c. It is thick
d. It prevents the entry of pathogens
a. Ectoderm
b. Endoderm
c. Mesoderm
d. All of the above
Mucus
Serous Holocrine 29009
Apocrine
Serous
Mucus
Goblet
Myoepithelial 2050
a. Hormones
b. Nerves
c. Chemicals
Serous
Mucous Mixed 2909

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a. They are specialized for the synthesis, storage, and secretion of proteins
b. They contain secretory granules in the apical cytoplasm and the secretion of granu
occurs by exocytosis
c. Serous cells are pyramidal in shape

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-
™*
e
'
!
f
#
‘
a. Serous acini
b. Mucous acini
c. Serous demilunes
d. Intercalated duct

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* General features
* Classification of connective tissue (CT)
*Components of CT
* Different forms of connective tissue
*Functions of CT
* Connective tissue with special properties

Connective tissue (CT) components seen-fibers, cells, and ground substance, Adipose tissue = Signet ring-shaped cells seen-fat deposit pushes the cytoplasm to periphery with only bulge seen due to nucleus. Areolar pockets are seen due to loose arrangement of components, Adipose tissue = Cells arranged together in groups pressing against each other with thin rim of cytoplasm are seen. Collagen fibers seen-arranged in bundles and are unbranched Elastic fibers-branched, single, and wavy Fibrocytes or blasts are seen dispersed among fibers Adipocytes are seen in groups., Adipose tissue = Blood vessels are seen.

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* The term connective tissue (CT) is applied to a tissue that fills the interstices between more specialized elements.
* They are predominantly composed of intercellular substance (matrix) secreted by its cells.
* Present in almost every part of the body, it is conspicuous in some regions and scanty in others.
* This kind of CT is referred to as general CT.
* They are mesodermal in origin.
* They also have trophic and morphogenetic roles in growth and differentiation of surrounding tissues, mediate exchange of nutrients, metabolites, and waste products.
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Connective tissue can assume various forms depending upon the nature of the ground substance, and of the type of fibers and cells present. There are various kinds of classification of CTs. Based on the structure and function, it can be classified into:
* Embryonic/fetal CT:
™"Mucoid CT
™ Mesenchyme
* Connective tissue proper:
"Loose CT
= Dense CT—tegular and irregular
* Specialized CT:
" Cartilage
= Bone
" Adipose tissue
"Blood
™ Hemolymphatic tissue.

Connective tissue = Cells + Intercellular matrix (Fibers + Ground substance)

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Cells are intrinsic components of CT. They can be broadly classified into two groups:
- 1. Resident cells/intrinsic component cells: They are responsible for produ and maintenance of extracellular matrix.
Fig. 4.1: Stretch preparation of omentum showing loose areolar tissue (schematic representation).
Flowchart 4.1: Basic components of connective tissue.
" Fibroblast
™ Adult stem cells
"Fat cells
"Fixed macrophages
™ Mast cells
"Pigment cells.
2. Wandering/fluctuating/migrating cells extrinsic: They bring out
t reaction to injuries.
™ Free macrophages
™ Leukocytes
™ Plasma cells
™ Monocytes.

* These are the most numerous cells of CT.
* They are called fibroblasts because they are concerned with the production of fibers. Fibroblasts are present in close relationship to collagen fibers.
* Where associated with reticular fibers, they are usually called reticular

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* In tissue sections the cells appear spindle-shaped, and the nucleus appears to be flattened. When seen from the surface, the cells show branching processes (Figs. 4.2A and B).
* The nucleus is large, euchromatic, and has prominent nucleoli.
* They are called fibrocytes when the cytoplasm is scanty, organelles are few, and the nucleus is heterochromatic.
*In contrast to fibrocytes, active fibroblasts have abundant cytoplasm (characteristic of cells actively engaged in protein synthesis). The endoplasmic reticulum, the Golgi complex, and mitochondria become much more conspicuous.
* Myofibroblasts share the features of fibroblasts and smooth muscle. They are elongated spindle-shaped cells with the bundles of actin filaments.

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- * Fibroblasts become very active when there is a need to lay down collagen fibers; for example, during wound repair, where they form granulation tissue with budding capillaries.
Figs. 4.2A and B: Structure of a fibroblast. (A) Profile view; (B) Structure view (schematic representation).
Fig. 4.3: Mesenchymal cells (schematic representation).
* They also produce ground substance.
* Fibroblasts are targets of various growth factors which influence cell growth and differentiation.
*In glands and lymphoid tissue, fibroblasts with reticular fibers form fibrocellular network called reticular tissue.
* Myofibroblasts help in wound contraction (Fig. 4.3).

* Embryonic CT is called mesenchyme.
* Tt is made up of stellate small cells with slender branching processes that join to form a fine network.

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* They are most abundant in CT of the skin, and in the choroid and iris of the eyeball.
* Pigment cells are easily distinguished as they contain brown pigment (melanin) in their cytoplasm and are called melanocytes.
* Melanocytes have rounded cell bodies and long processes. They contain melanin granules in a membrane-bound form called—melanosomes.
The cells which cannot synthesize melanin but engulf them are called chromatophore (when they engulf melanin—melanophore).
* In contrast to melanocytes, chromatophores or melanophores are starshaped (stellate) with long branching processes. They are probably modified fibroblasts.
* When melanin pigment is brownish-black, it is called—eumelanin and pheomelanin when it is reddish-yellow.

* Variations in the number of pigment cells, and in the amount of pigment in them account for the differences in the skin color of different races, and in different individuals (Fig. 4.4).
* Pigment cells prevent light from reaching other cells. The importance of this function in relation to the eyeball is obvious.
* Pigment cells in the skin protect deeper tissues from the effects of light (especially ultraviolet light). The darker skin of races living in tropical climates is an obvious adaptation for this purpose.
* Albinism is a condition where there is a complete absence of melanin pigment.

* Fat-storing cells are called adipocytes/lipocytes.
* Aggregations of fat cells constitute adipose tissue.

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Fig. 4.5: Fat cell (schematic representation).
* Each fat cell contains a large droplet of fat, which almost fills it (Fig. 4.5). As a result, the cell becomes rounded (when several fat cells are closely packed they become polygonal because of mutual pressure).
* The cytoplasm of the cell forms a thin layer just deep to the plasma membrane.
* The nucleus is pushed against the plasma membrane and is flattened.
* In routine slide preparation, fat is dissolved by organic solvent, thus giving adipocytes signet ring appearance.
* Adipocytes are incapable of division.
* For more details, see the "Adipose Tissue" section.

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* Macrophage cells of CT are also called histiocytes or clasmatocytes (Fig. 4.6).
* Macrophage cells of CT belong to mononuclear phagocyte system (MPS).
* They are derived from monocytes which migrate from blood.
* They have the ability to phagocytose (eat up) unwanted material (both organic and inorganic). Macrophages can be demonstrated by injecting the tissue with India ink (or trypan blue, or lithium carmine) particles of it are
taken up into the cytoplasm of macrophages, thus making them easy to recognize.
* Macrophages are usually described as "fixed" when they are attached to fibers. Fixed macrophages resemble fibroblasts in appearance.
* They are called motile/nomadic macrophages when they are free, not attached to fibers. Free macrophages are rounded and have regular form.
* However, all macrophages are capable of becoming mobile when suitably stimulated.
* The nuclei of macrophages are smaller, heterochromatic and stain more intensely than those of fibroblasts. They are often kidney-shaped (Fig. 4.6).
* Cytoplasm is mildly basophilic. With the electron microscope (EM), the cytoplasm is seen to contain numerous lysosomes that help in "digesting" material phagocytosed by the macrophage.
* Sometimes, macrophages may fuse together to form multinucleated giant cells.
* Apart from direct phagocytic activity, macrophages play an important role in immunological mechanisms.

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* These are large round or oval cells (mastocytes or histaminocytes) (Fig. 4.7).
* The nucleus is small and centrally placed.
* Trregular microvilli (filopodia) are present on the cell surface.
* The distinguishing feature of these cells is the presence of numerous granules in the cytoplasm.
* The granules can be demonstrated with the periodic acid-Schiff (PAS) stain.
* In hematoxylin and eosin (H&E) stain,mast cells are seen with pale blue cytoplasm and round to oval nuclei. They also stain with dyes, such as toluidine blue or Alcian blue; with them the nuclei stain blue, but the granules stain purple to red (when components of a cell or tissue stain in a color different from that of the dye used, the staining is said to be
Fig. 4.6: Macrophage cell (histiocyte) (schematic representation).
Fig. 4.7: Mast cell (schematic representation).

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* On the basis of the staining reactions, the granules are known to contain acid mucopolysaccharides.
* With the EM, cytoplasm shows membrane-bound vesicles containing "granules."
* The granules are osmophillic substances containing heparin, histamine tryptase, and superoxide dismutase. The most important of these is histamine.
* Release of histamine is associated with the production of allergic reactions when a tissue is exposed to an antigen to which it is sensitive (because of previous exposure).
* They are most frequently seen around blood vessels and nerves.
* Mast cells are probably related in their origin to basophils of blood.
* Brain and spinal cord are devoid of mast cells, thus they are protected from harmful effects of edema and allergic reactions.

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* Lymphocytes represent one variety of leukocytes (white blood cells) present in blood.
* Large aggregations of lymphocytes are present in lymphoid tissues. They reach CT from these sources, and are especially numerous when the tissue undergoes inflammation.
* Lymphocytes are small with rounded highly heterochromatic nuclei and thin rim of cytoplasm.
* They play an important role in defense of the body against invasion by bacteria and other organisms.
* They have the ability to recognize substances that are foreign to the host body and to destroy these invaders by producing antibodies against them.

* The lymphocytes are derived from stem cells present in bone marrow.
* Based on the presence of specific cluster of differentiation (CD) proteins, three major groups of cells are described. They are: (1) T-lymphocytes, (2) B-lymphocytes and (3) Natural killer cells (for more details, refer Lymphatic Tissue chapter).

* Apart from lymphocytes, two other types of leukocytes may be seen in CT.
* Monocytes are closely related in function to the macrophages.
* Eosinophils (so called because of the presence of eosinophilic granules in the cytoplasm) are found in the CT of many organs (Fig. 4.9). They increase in number in allergic disorders.

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- * They are round, relatively large cell with basophilic cytoplasm (Fig. 4.10).
Fig. 4.9: Schematic representation of eosinophil.
Fig. 4.10: Plasma cell (schematic representation).
* Basophilic cytoplasm is due to the fact that it has many rough endoplasmic reticulum in it, except for a small region near the nucleus where a welldeveloped Golgi complex is located.
* Nucleus is spherical and eccentric.
* The chromatin in its nucleus forms four or five clumps near the periphery (of the nucleus), thus giving the nucleus a resemblance to a cartwheel appearance.
* Both these features are indicative of the fact that plasma cells are engaged in considerable synthetic activity.
* They produce antibodies that may be discharged locally, may enter the circulation, or may be stored within the cell itself in the form of inclusions called Russell's bodies.
* Very few plasma cells can be seen in normal CT. Their number increases in the presence of certain types of inflammation.
* It is believed that plasma cells represent B-lymphocytes that have matured and have lost their power of further division.

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- *When macrophages encounter foreign bodies, they may fuse to form large cell with many nuclei to engulf the foreign body. Such
cells are called Langerhans/epitheloid/foreign body giant cells. Under MPS, macrophages are given different names in different tissue as follows:
*Macrophage/histiocyte—CT
*Kupffer cell—liver
*Osteoclast—bone
*Langerhans cell—skin
*Hofbauer cell—placenta
*Microglia—central nervous system
* Alveolar macrophage—lungs.
*Mast cells and basophils arise from hemopoietic stem cell in bone marrow. At first, mast cells circulate in blood as agranular cells of monocyte appearance, after migrating into tissue, immature mast cells differentiate and produce their characteristic granules.
Two types of mast cells are described in human beings:
1. MCryc type: They have granule-associated tryptase and chymase. These cel
cytoplasmic granules with lattice-like structure. For example, mast cells of skin, in mucosa, breast, and axillary lymphnodes.
2. MC; type: They have granule-associated tryptase. These cells have cyto
granules with scroll-like structure. For example, mast cells of lung, intestinal muco
- *T-lymphocytes: These cells arise from bone marrow, then migrates to thymus for maturation, and later moves to their destination (peripheral lymphoid tissue).
li

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The most conspicuous components of CT are the fibers within it. These are of three main types (Table 4.1): (1) Collagen, (2) Reticular and (3) Elastic fibers.
Collagen fibers
Elastic fibers
White in color
Yellow in color
Arranged in bundles
Arranged individually
Bundles branch
Individual fibers branch
Have straight ends, 1 = Have curled ends. Provides strength to the structure, 1 = Provides elasticity to the structure. Produced throughout the life, 1 = Produced only during fetal period

* Collagen fibers are most numerous. They can be classified into various types. Reticular fibers were once described as a distinct variety of fibers, but they are now regarded as one variety of collagen fiber.
* With the light microscope, collagen fibers are seen in bundles (Figs. 4.11 and 4.12A). The bundles may be straight or wavy depending upon how much they are stretched. The bundles are made up of collections of individual collagen fibers which are 1-12 pm in diameter.
* The bundles often branch or anastomose with adjacent bundles, but the individual fibers do not branch.
* With the EM, each collagen fiber is seen to be made of fibrils that are 20200 nm in diameter. Each fibril consists of a number of microfibrils (3.5 nm in diameter). At high magnifications of the EM, each fibril shows characteristics of cross-striations (or periods) after every 67 nm interval (in unfixed tissue).

* Unstained collagen fibers in bundles appear white in color with the unaided eye.
* In sections stained with H&E, collagen fibers are stained light pink.
* With special methods, they assume different colors depending upon the dye used. Two commonly used methods are Masson's trichrome with which the fibers stain blue and the Van Gieson method with which they stain red. After silver impregnation, the fibers are stained brown.

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* Collagen fibers are mainly made up of a protein called collagen.
* Protein collagen in turn is made up of molecules of tropocollagen.
* Microfibrils of collagen are the chains of tropocollagen molecules. Each molecule of tropocollagen is 300 nm in length. Within a fiber, the molecules of tropocollagen are arranged in a regular overlapping pattern which is responsible for producing the cross-striated appearance of the fibers.
* Each molecule of tropocollagen is made up of three polypeptide chains. The chains are arranged in the form of a triple helix. The polypeptide chains are referred to as procollagen.
* Each procollagen chain consists of a long chain of amino acids that are
Fig. 4.11: Components of loose connective tissue (schematic representation).
Figs. 4.12A to C: Fibers of connective tissue. (A) Collagen fibers; (B) Elastic fibers; (C) Reticular fibers (schematic representation).
arranged in groups of three (triplets). Each triplet contains the amino acid glycine. The other two amino acids in each triplet are variable. Most commonly, these are hydroxyproline and hydroxylysine.

Collagen fibers can resist considerable tensile forces (i.e. stretching) without significant increase in their length. At the same time, they are pliable and can bend easily.

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These fibers are a variety of collagen fibers and are composed of collagen type III. They show periodicity (striations) of 67 nm. They differ from typical (type I) collagen fibers as follows:
* They are much finer and have uneven thickness.
* They form a network (or reticulum) by branching, and by anastomosing with each other. They do not run in bundles (Fig. 4.12C).
* They can be stained specifically by silver impregnation, which renders them black. They can, thus, be easily distinguished from type I collagen fibers which are stained brown. Because of their affinity for silver salts, reticular fibers are sometimes called argentophil fibers (Fig. 4.13).
* Reticular fibers contain more carbohydrates than type I fibers (which is probably the reason, why they are argentophil).

* Reticular fibers provide a supporting network in lymphoid organs, such as the spleen, lymph nodes, and bone marrow; most glands, including liver (Fig. 4.13) and kidneys.
* Reticular fibers form an essential component of all basement membranes.
* They are also found in relation to smooth muscle and nerve fibers. Reticular fibers are synthesized by fibroblasts and reticular cells (special variety of fibroblasts).

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* Elastic fibers are much fewer than those of collagen. They run singly (not in bundles), branch and anastomose with other fibers (Fig. 4.12B).
* Elastic fibers are thinner than those of collagen (0.1—0.2 pm) (see Fig. 4.1). In some situations, elastic fibers are thick (e.g. in the ligamenta flava). In other situations (as in walls of large arteries), they form fenestrated membranes.
* With the EM, each elastic fiber is seen to have a central amorphous core and an outer layer of fibrils (Fig. 4.14).
Fig. 4.13: Reticular fibers (black) forming a network in the liver. The white spaces represent sinusoids (schematic representation).
Fig. 4.14: Electron microscopy appearance of an elastic fiber as seen in transverse section (schematic representations).
- * The outer fibrils are made up of a glycoprotein called fibrillin. Periodic striations are not present in elastic fibers.

Elastic fibers do not stain with the usual stains for collagen. They can be demonstrated by staining with orcein, with aldehyde fuchsin, and by Verhoeff's method.

* Elastic fibers are composed mainly of a protein called elastin that forms their central amorphous core.
* Elastin is made up of smaller units called tropoelastin.
* Elastin contains a high quantity of the amino acids valine and alanine. Another amino acid called desmosine is found exclusively in elastic tissue. The outer fibrils of elastic fibers are composed of the glycoprotein fibrillin.

Elastic fibers of CT are produced by fibroblasts. In some situations, elastic tissue can be formed by smooth muscle cells.

* Elastic fibers can be stretched (like a rubber band) and return to their original length when tension is released.
* They are highly refractile and are, therefore, seen as shining lines in unstained preparations. Relaxed elastic fibers do not show birefringence, but when stretched the fibers become highly birefringent.
* Unlike collagen, elastic fibers are not affected by weak acids or alkalies, or by boiling. However, they are digested by the enzyme elastase.
Bf

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* Tt is transparent homogenous viscous solution called ground substance or matrix.
* Tt fills the space between cells and fibers.
* Ground substance is rich in protein—carbohydrate complexes or proteoglycans.
* Proteoglycans are complexes formed by protein and _ long-chained
polysaccharides called glycosaminoglycans (GAGs).
* Glycosaminoglycans are highly negatively charged and tend to take basic stain because of sulfate and carboxyl groups in them. The negative-charged molecules attract water and forms hydrated gel.
* In addition to proteoglycans, the ground substance also contains structural glycoproteins. Its main function is to facilitate adhesion between various elements of CT.
* Intercellular ground substance is synthesized by fibroblasts. Osteoblasts, chondroblasts, and even smooth muscle cells can also produce ground substance.
* Because of different sugar moieties and bondages, various GAGs are recognized. Different GAGs and the tissues in which each type is present are given in Table 4.2.

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With the exception of hyaluronic acid, all other GAGs listed in the Table 4.2 have the following features.
* They are linked with protein (to form proteoglycans).
*They carry sulfate groups (SO-) and carboxyl groups (COO.) which give them a strong negative charge.
*The proteoglycans formed by them are in the form of long chains that do not fold. Because of this, they occupy a large space (or domain) and hold a large amount of water.
* They also hold Na+ ions. Retained water and proteoglycans form a gel that gives a certain degree of stiffness to CT and helps it to resist compressive forces.
*Because of the arrangement of molecules within it, ground substance acts like a sieve. The size of the pores of the sieve can be altered (by change in the orientation of molecules, and by change in the charges on them).
*In this way, ground substance forms a selective barrier. This barrier function is very important in basement membranes. In the kidney, this barrier prevents large protein molecules from passing (from blood) into urine. However, exchange of gases is permitted in the lungs.

* They are huge molecules with thousands of sugar moieties.
* Unlike other GAGs,they are not directly linked to proteins, by indirect linkages, it forms proteoglycan aggregates. They are devoid of sulfate groups.

* These protein molecules are responsible for anchoring the extracellular matrix to cell surface and thus stabilizes it.
* They also regulate the functions of extracellular matrix.
* Examples—fibronectin, osteopontin, laminin, and tenascin.
Bi

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* Tt is so called because of the loosely arranged collagen fibers in it enclose small spaces (areolae—L. "little open space") which are filled with interstitial fluid.
* Tt has abundant ground substance, which makes it soft.
* Tt has numerous fibroblasts and macrophages along with other types of CT cells scattered between loosely arranged collagen and elastic fibers.
* Its main function is to bind different tissues together.
* Areolar tissue gets distorted easily; hence, it allows the tissue to move freely.
* Examples—endomysium, subperiosteal tissue, lamina propria of gastrointestinal tract (GIT), hypodermis, stroma of glands, and mesentery.

* Mainly contains collagen fibers (or fiber bundles) arranged in orderly fashion.
* Nuclei of some cells (mainly fibroblasts) are seen between the bundles of collagen. Nuclei are elongated in shape (elliptical).
* Ground substance is less in amount.
* Examples—Tendon of muscle, ligaments of joint, deep fascia, intermuscular septa, aponeuroses, the central tendon of the diaphragm, the fibrous pericardium, and dura mater (Plate 4.1).

Typical connective tissue, Chondroitin sulfate = +. Typical connective tissue, Dermatansulfate = ~. Typical connective tissue, Heparansulfate = -. Typical connective tissue, Heparin = =. Typical connective tissue, Keratan sulfate = =. Typical connective tissue, Hyaluronic acid = +. Cartilage, Chondroitin sulfate = =. Cartilage, Dermatansulfate = -. Cartilage, Heparansulfate = =. Cartilage, Heparin = =. Cartilage, Keratan sulfate = +. Cartilage, Hyaluronic acid = +. Bone, Chondroitin sulfate = +. Bone, Dermatansulfate = -. Bone, Heparansulfate = =. Bone, Heparin = =. Bone, Keratan sulfate = bs. Bone, Hyaluronic acid = . Skin, Chondroitin sulfate = +. Skin, Dermatansulfate = 7. Skin, Heparansulfate = . Skin, Heparin = +. Skin, Keratan sulfate = =. Skin, Hyaluronic acid = *. Basementmembrane, Chondroitin sulfate = —-. Basementmembrane, Dermatansulfate = -. Basementmembrane, Heparansulfate = +. Basementmembrane, Heparin = ~. Basementmembrane, Keratan sulfate = =. Basementmembrane, Hyaluronic acid = Pe. Others, Chondroitin sulfate = -. Others, Dermatansulfate = Blood vessels and heart. Others, Heparansulfate = Lung arteries. Others, Heparin = Mast cells, lungs, and liver. Others, Keratan sulfate = Cornea, intervertebral discs. Others, Hyaluronic acid = Synovial fluid

* In this CT, collagen bundles do not show such a regular arrangement, but interlace in various directions forming dense irregular tissue.
* Few cells (fibroblast) and less ground substance.
* Examples—Dermis, CT sheaths of muscles and nerves, capsules of glands, the sclera, the periosteum, and the adventitia of blood vessels (Plate 4.2 and Figure 4.15).

Adipose tissue is an aggregation of fat cells also called adipocytes (Fig. 4.16).

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* Tt is found subcutaneously (superficial fascia) throughout the body except over the eyelid, auricle, penis, and scrotum. This subcutaneous layer of fat is called the panniculus adiposus. In women, it forms a thicker and more even layer: this is responsible for the soft contours of the female body.
* Subcutaneous fat is not present in animals that have a thick coat of fur.
* Adipose tissue fills several hollow spaces in the body. These include the orbits, the axillae, and the ischiorectal fossae.
* In the adult, much of the space in marrow cavities of long bones is filled by fat in the form of yellow bone marrow.
* Much fat is also present in synovial folds of many joints filling spaces that would otherwise have been empty during certain phases of movement.
* Fat is present around many abdominal organs, specially the kidneys (perinephric fat).
* Considerable amounts of fat may be stored in the greater omentum, and in other peritoneal folds.

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* Adipocytes have single large fat globule, which occupies whole of the cytoplasm.
* Tn routine sections, the cells appear empty as the fat in them gets dissolved during preparation (treatment with fat solvents, such as xylene or benzene) of the section giving it a honeycomb appearance.
* The cytoplasm of the cell appears as a thin rim around the fat globule.
* The nucleus is flat and lies to one side (eccentric) giving it signet ring appearance.
* Fat cells may be scattered singly in some situations, but they are usually aggregated into groups that form lobules of adipose tissue.
* The cells are supported by reticular fibers, and the lobules are held together by areolar tissue.
* Adipose tissue is richly supplied with blood and is rich in enzyme systems.
* Fat cells are derived from specific cells (lipoblasts) arising from undifferentiated mesenchymal cells.
* Special stains, such as Sudan III, Sudan IV, stain them (fat droplets) dark (Plate 4.3).

* Tt acts as a storehouse of nutrition, fat being deposited when available in excess, and being removed when deficient in the diet.
* In many situations, fat performs a mechanical function. The fat around the kidneys keeps them in position. If there is a sudden depletion of this fat, the kidneys may become mobile (floating kidney). The fat around the eyeball performs an important supporting function and allows the eyeball to move smoothly. In the palms and soles and over the buttocks, fat has a cushioning effect protecting underlying tissues from pressure. This fat is

(Sudan IV) (schematic representation).
Plate 4.1: Dense regular connective tissue
Dense regular connective tissue (Longitudinal section through a tendon): (A) As seen in drawing; (B) Photomicrograph.

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”
Presence of collagen fibers (or fiber bundles) arranged in orderly fashion parallel to each other
* Nuclei of some cells (mainly fibroblasts) are seen between the bundles of collagen. They are elongated (elliptical)
Ground substance is less in amount.
re)
‘=:

Dense irregular connective tissue (dermis of skin): (A) Drawing; (B) Photomicrograph.

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are
Irregularly arranged bundles of collagen fibers that stain pink. In stretch preparation they seen in wavy bundles. Other fibers present (elastic, reticular) can be seen only with special stains
Few cells (fibroblast) and less ground substance.
|
eae
‘==

Adipose tissue: (A) As seen in drawing; (B) Photomicrograph (the arrows point to nuclei).

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"Presence of fat cells. In routine sections the cells appear empty as the fat in them gets dissolved during preparation of the section giving it a honeycomb appearance
'The cytoplasm of each cell is seen as a pink rim
'The nucleus is flat and lies to one side (eccentric).
* The subcutaneous fat has been regarded as insulation against heat loss and would certainly perform this function if the layer of adipose tissue is thick. The whale (a warm blooded mammal) can survive in very cold water because it has a very thick layer of subcutaneous fat.
* They also help in heat generation.

Adipose tissue is of following two types:
1. Yellow (white) or unilocular adipose tissue (adult type)
2. Brown or multilocular adipose tissue (embryonic type).
The yellow adipose tissue is the adult type which has been described in detail above.

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* Tt is brown in color.
* Cells are smaller than in typical adipose tissue.
* The fat in the cytoplasm occurs in the form of several small droplets. Hence, brown fat is also called multilocular adipose tissue (while the typical variety is described in Figs. 4.17A and B).
* The cytoplasm and nucleus of the cell are not pushed to the periphery.
* The cytoplasm contains numerous mitochondria (which are few in typical fat cells).
* Brown adipose tissue is abundant in the newborn, but most of it is lost during childhood.
* Brown fat is also abundant in hibernating animals in which it serves mainly as a heat generator when the animal comes out of hibernation.
li

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* Tt holds together structures, such as skin, muscles, blood vessels, etc. It binds together various layers of hollow viscera. In the form of areolar tissue and reticular tissue, it forms a framework that supports the cellular elements of various organs, such as the spleen, lymph nodes, and glands, and provides capsules for them.
* The looseness of areolar tissue facilitates movement between structures connected by it. The looseness of superficial fascia enables the movement of skin over deep fascia. In hollow organs, this allows for mobility and stretching. ate
*In the form of deep fascia, CT provides a tight covering for deeper structures (especially in the limbs and neck) and helps to maintain the shape of these regions and also venous return of lower limb.
* In the form of ligaments, it holds bone ends together at joints.
*In the form of deep fascia, intermuscular septa, and aponeuroses, CT provides attachment for the origins and insertions of many muscles.
* In the form of tendons, it transmits the pull of muscles to their insertion.
* Thickened areas of deep fascia form retinacula that hold tendons in place at the wrist and ankle.
* Both areolar tissue and fascial membranes provide planes along which blood vessels, lymphatics, and nerves travel. The superficial fascia provides passage to vessels and nerves going to the skin and supports them.
* In the form of dura mater, it provides support to the brain and spinal cord.
*In the form of adipose tissue, it provides a store of nutrition. In cold weather, the fat provides insulation and helps to generate heat.
* Because of the presence of cells of the immune system (macrophages and plasma cells), CT helps the body to fight against invading foreign substances (including bacteria) by destroying them, or by producing antibodies against them.
* Because of the presence of fibroblasts, CT helps in laying down collagen fibers necessary for wound repair.
* By virtue of the presence of undifferentiated mesenchymal cells, CT can help in regeneration of tissues (e.g. cartilage and bone) by providing cells from which specialized cells can be formed.

Figs. 4.17A and B: (A) Unilocular adipose tissue (white adipose tissue cell); (B) Multilocular adipose tissue (brown adipose tissue cell) (schematic representation).
i

* It is specialized dense CT formed by elastic fibers.
* In contrast to fibrous tissue, which appears white, elastic tissue is yellow in color.
* Some ligaments like ligamentum nuchae, ligamentum flava and vocal ligament are examples.

* It is loose CT made up of reticular fibers.
* In certain structures,such as lymph nodes, glands, etc., these fibers form supporting networks for the cells (see Fig. 4.13).
* In other tissues (e.g. bone marrow, spleen, and lymph nodes), the reticular network is closely associated with reticular cells. Most of these cells are fibroblasts, but some may be macrophages.

* The most conspicuous component of mucoid tissue is a jelly-like ground substance rich in hyaluronic acid.
* Scattered through this ground substance, there are star-shaped fibroblasts, some delicate collagen fibers, and some rounded cells (Fig. 4.18).
* This kind of tissue is found in the umbilical cord, vitreous humor of the eyeball.
Fig. 4.18: Mucoid tissue (schematic representation).

The mechanism of the production of collagen fibers by fibroblasts has been extensively studied. Collagen is also synthesized by chondroblasts of cartilage, osteoblasts of bone and smooth muscles of blood vessels. Amino acids necessary for the synthesis of fibers are taken into the cell. Under the influence of ribosomes, located on rough endoplasmic reticulum, the amino acids are bonded together to form polypeptide chains (a-chains) (Elowchart_ 4.2). A procollagen molecule is formed by joining together of three such chains. Molecules of procollagen are transported to the exterior of the cell where they are acted upon by enzymes (released by the fibroblast) to form tropocollagen. Collagen fibers are formed by the aggregation of tropocollagen molecules. Vitamin C and oxygen are necessary for collagen formation, and wound repair may be interfered with if either of these is deficient. In this connection, it may be noted that fibroblasts are themselves highly resistant to damaging influences and are not easily destroyed.
There are observations that indicate that the orientation of collagen fibers depends on the stresses imposed on the tissue. If fibroblasts growing in tissue culture are subjected to tension in a particular direction, the cells exposed to the tension multiply faster than others; they orientate themselves along the line of stress and lay down fibers in that direction. It follows that in the embryo collagen fibers would tend to be laid down wherever they are required to resist a tensile force. In this way, tendons, ligaments, etc., will tend to develop wherever they are required. (This cannot, of course, be a complete explanation. Genetic factors must play a prominent role in development of these structures).
ef

A single collagen molecule is made up of three polypeptide chains (triple helical structure) called as a chains. In each chain glycine is a compulsory amino acid along with any other two amino acids. The other two amino acids will be proline and hydroxyproline/ hydroxylysine usually. Different arrangement of amino acids results in formation of more than 40 types of a chain, which vary in size. Based on various combinations of a chains 25 or more types of collagen have been identified. The types and their location is briefed in Flowchart 4.2 and Table 4.3.
Bf

* Osteogenesis imperfecta: Disease of bone, due to the mutation of genes coding for collagen fibers. When collagen is not properly formed, bones are weak and break easily, one of the characteristic feature of the disease.
* Ehlers-Danlos syndrome: Here skin becomes abnormally extensible, and joints may be lax (because of improperly formed ligaments).
* Marfan's syndrome: This is due to defect in the genes coding for fibrillin, which is needed in the formation of extracellular matrix and elastic fibers. It affects almost all structures of the body.
I, Location = Skin, tendon, sclera, bone, fascia. II, Location = Vitreous humor of eye, intervertebral disc, cartilage. III, Location = Skin, blood vessels, lung. IV, Location = Basement membrane of epithelium. V, Location = Mostly found with type I collagen, forms stroma. VI, Location = Muscle, cartilage (matrix). VII, Location = Skin-mainly at the dermo-epidermal junction, uterus. VIII, Location = Endothelium of blood vessels. IX, Location = Vitreous humor, cartilage. X, Location = Growing cartilages. XI, Location = Vitreous humor, cartilage. XII, Location = Skin, placenta, tendon and bone. XIII, Location = Skin, neuromuscular junction. XIV, Location = Skin, tendon and bone. XV, Location = Surrounding the basement membrane, skeletal and cardiac muscles. XVI, Location = Wide distribution. XVII, Location = Epithelial tissue-mainly at junctions. XVIII, Location = In structures surrounding the basement membrane. XIX, Location = Basement membrane. XX, Location = Cornea. XXI, Location = Various structures containing type I collagen. XXII, Location = At the junction of two tissues. XXIII, Location = Heart, retina and metastatic cancer cells. XXIV, Location = Bone, cornea. XXV, Location = Brain. XXVI, Location = Gonads. XXVII, Location = Schwann cell, basement membrane

1. Wharton’s jelly is an example for: a. Loose areolar tissue b. Dense connective tissue c. Mucoid connective tissue d. Adipose tissue responsible a. Clasmatocytes b. Plasma cells c. Myofibroblast d. Histiocytes collagen a. IV b. Il c. | d. V 4. Reticular fibers are seen in: Liver Lung Heart . Spleen aoop Answers PWNEP an2oo
2. Cell for wound contraction/scar retraction is:
3. Type of fibers present in the basement membrane is:

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To study the:
* Features of cartilage
*Perichondrium
*Components of cartilage
* Different types of cartilage with examples

Ground glass appearance of matrix, White fibrous cartilage = Bundles of collagen fibers with little ground substance. Ground glass appearance of matrix, Elastic cartilage = Branching and anastomosing elastic fibers in all direction. Chondrocytes arranged in group (cell nest) of 2 or more, White fibrous cartilage = Chondrocytes arranged in rows (of single cell thickness) between the fibers. Chondrocytes arranged in group (cell nest) of 2 or more, Elastic cartilage = Closely packed chondrocytes surrounded by amorphous ground substance. Perichondrium surrounding the cartilage., White fibrous cartilage = Absence of perichondrium.. Perichondrium surrounding the cartilage., Elastic cartilage = Presence of perichondrium.

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* Cartilage is an avascular connective tissue that forms the "skeletal" basis of some parts of the body, e.g. the auricle of the ear, or the lower part of the nose.
* Cartilage develops from mesenchyme.
* They are not rigid but firm enough to maintain the form of the structure.
* Cartilage contains cells and fibers embedded in homogeneous ground substance.
* Cells are relatively less and are called as chondrocytes.
* Three main types of cartilage can be recognized depending on the amount and variety of fibers in the
matrix. These are: (1) hyaline cartilage, (2) fibrocartilage, and (3) elastic cartilage.
* Growth of cartilage: When cartilage grows by multiplication of cells throughout its substance, such a growth is called interstitial growth. Further, if a cartilage grows by addition of new cartilage over the surface of existing cartilage that is called oppositional growth.
* Cartilage has very limited ability for regeneration (after destruction by injury or disease). Defects in cartilage are usually filled in by fibrous tissue.

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* Perichondrium develops from mesenchymal cells that surround the developing cartilage.
* Tt has two layers: (1) inner chondrogenic layer and (2) outer fibrous layer.
* The undifferentiated mesenchymal cells in the chondrogenic layer, can proliferate and differentiate to form cartilage cells on the existing cartilage (oppositional growth).
* The cells in the outer layer differentiate to form fibroblast/ fibrocytes which in turn produces fibers (collagen) thus, converting it to fibrous layer.
i

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Cartilage = Chondrocytes + Extracellular matrix (ground substance + fibers)
Like ordinary connective tissue, cartilage is made up of:
* Cells—chondrocytes
*, od Intercellular/extracellular matrix
+. od Fibers—collagen/elastic/reticular fibers.

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' + Mature cells of cartilage are called as chondrocytes.
*, ~ They vary in size and shape with degree of maturation. Mature cells are large and rounded.
+. od They lie in spaces (or lacunae) present in the matrix.
* The nucleus is heterochromatic and organelles are less prominent. The cytoplasm of chondrocytes may also contain glycogen and lipids.
*, od Young metabolically active flat cells are called as chondroblasts. The nucleus is euchromatic. Mitochondria, endoplasmic reticulum (ER), and Golgi complex are prominent.

* Ground substance forms major component and makes up to 95% of volume of the cartilage.
* The ground substance is made up of complex molecules containing proteins and carbohydrates (proteoglycans). Along with the water content, these molecules form a firm gel that gives cartilage its characteristic consistency.
* The carbohydrates are chemically glycosaminoglycans (GAGs).
* Multiadhesive glycoproteins bind chondrocytes and the matrix molecules.

4, * The collagen fibers present in cartilage are (as a rule) chemically distinct from those in most other tissues.
* They are described as type II collagen.
* However, type I collagen is present in fibrocartilage and perichondrium. Elastic cartilage contains elastic fibers.
li

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* Hyaline cartilage is so called because it is translucent (hyalos = glass).
* Its matrix appears to be homogeneous, but using special techniques, many collagen fibers can be demonstrated.
* Hyaline cartilage has been compared to a tyre. The ground substance (corresponding to the rubber of the tyre) resists compressive forces, while the collagen fibers mainly type-II (corresponding to the treads of the tyre) resist tensional forces.
* Hyaline cartilage is surrounded by perichondrium but although articular cartilage is a hyaline cartilage, it is devoid of perichondrium.
* In hematoxylin and eosin-stained preparations, the matrix is stained blue, i.e. it is basophilic.
* The GAGs of ground substance includes chondroitin sulfate, keratin sulfate, and hyaluronic acid. In hyaline cartilage, the most important proteoglycan monomer is aggrecan (Plate 5.1).
* Toward the center of mass of hyaline cartilage, the chondrocytes are large and are usually present in groups (of two or more)—cell nest (isogenous cell groups). The groups are formed by division of a single parent cell.
* Matrix around the cells is darkly stained called as capsular matrix and around the isogenous cell group as territorial/lacunar matrix.
* Toward the periphery of the cartilage, the cells are small and elongated in a direction parallel to the surface. Just under the perichondrium, the cells become indistinguishable from fibroblasts. The pale staining matrix separating cell nests is the interstitial matrix.
* In old people, hyaline cartilage can undergo calcification and degeneration. The costal cartilages or the large cartilages of the larynx are commonly affected.
* Examples: Costal cartilage, articular cartilage, thyroid cartilage, cricoid cartilage, arytenoid cartilage, tracheal rings, and large and smaller bronchi.

* By forming structural framework that provides support to various structures
* Forms cartilage model for developing fetal skeleton
* Forms articular cartilage.

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* Elastic cartilage (or yellow fibrocartilage) is similar in many ways to hyaline cartilage.
* The main difference between hyaline cartilage and elastic cartilage is that instead of collagen fibers, the matrix contains numerous branching and anastomosing elastic fibers that form a network (Plate 5.2).
* The fibers are difficult to see in hematoxylin and eosin-stained sections, but they can be clearly visualized if special methods for staining elastic fibers are used.
* The surface of elastic cartilage is covered by perichondrium.
* Contrast to hyaline cartilage, elastic cartilage does not undergo calcification.
Plate 5.1: Hyaline cartilage
Hyaline cartilage: (A) As seen in drawing; (B) Photomicrograph.
Courtesy: Balakrishna Shetty, Sweekritha H Poonja. HISTOLOGY Practical Manual, 4th edition. New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd; 2019. p. 17-18.

"Hyaline cartilage is characterized by isogenous groups of chondrocytes called as cell nest "Chondrocytes are surrounded by a homogeneous basophilic matrix which separates the cells widely
"Chondrocytes increase in size from periphery to center
Near the surface of the cartilage the cells are flattened and merge with the cells of the overlying connective tissue. This connective tissue forms the perichondrium
"Perichondrium displays an outer fibrous and inner cellular layer.

Auricle (or pinna) and of the lateral part of the external acoustic meatus, medial part of the auditory tube, epiglottis, and two small laryngeal cartilages (corniculate and cuneiform). The apical part of the arytenoid cartilage contains elastic fibers but the major portion of it is hyaline.
Plate 5.2: Elastic cartilage

* Elastic cartilage is characterized by presence of chondrocytes within lacuna surrounded by bundles of elastic fibers
*Perichondrium is present showing an outer fibrous and inner cellular layer.

* Fibrous cartilage (also called white fibrocartilage) looks very much like dense fibrous tissue (Plate 5.3).
* Since fibrocartilage is a transitional tissue between dense connective tissue and hyaline cartilage, lack of perichondrium helps in its proper blending with surrounding structure.
* The chondrocytes are similar to that of hyaline cartilage but arranged in single rows or in isogenous groups.
Ca ae Fibrocartilage

* The matrix is pervaded by numerous collagen bundles both type I and type II, in between, there are some fibroblasts. The fibers merge with those of surrounding connective tissue. Their proportion varies with age.
* There is no perichondrium over the cartilage.
* The fibrocartilage undergoes calcification during repair process.

Intervertebral discs, symphysis pubis, menisci of the knee joint, and articular disc of sternoclavicular and temporomandibular joint.

* Offers resistance to both compression and shearing forces
* Acts as shock absorbers.

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Bi
* Osteoarthritis: Osteoarthritis is an inflammatory disorder of joint due to degeneration of articular cartilage.
* Achondroplasia: One of the causes of dwarfism in early life is due to reduced proliferation of chondrocytes in the epiphyseal plates of long bones.
* Cartilage-forming (chondroblastic) tumors: The tumors which are composed of frank cartilage or derived from cartilage-forming cells are included in this group. This group comprises benign lesions like osteocartilaginous exostoses (osteochondromas), enchondroma, chondroblastoma and chondromyxoid fibroma, and a _ malignant counterpart, chondrosarcoma.
" Osteocartilaginous exostoses or osteochondromas: These are the m common of benign cartilage-forming lesions. Exostoses arise from grov plate of long bones as exophytic lesions, most commonly lower femur a upper tibia and upper humerus.
"Enchondroma: Enchondroma is the term used for the benign cartila; forming tumor that develops centrally within the interior of the affec bone, while chondroma refers to the peripheral development of lesion.
[
"Chondroblastoma: Chondroblastoma is a relatively rare benign tun arising from the epiphysis of long bones adjacent to the epiphyseal cartilé plate. Most commonly affected bones are upper tibia and lower femur < upper humerus.
" Chondrosarcoma: Chondrosarcoma is a malignant tumor of chondroblas
~

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*Being avascular tissue, cartilage receives nutrition through blood vessels found in cartilage canals. Each canal contains a small artery surrounded by numerous venules and capillaries. Cartilage cells receive their nutrition by diffusion from vessels in the perichondrium or in cartilage canals. Cartilage canals may also play a role in the ossification of cartilage by carrying bone forming cells into it.
*The greater part of the skeleton is cartilaginous in early fetal life. The ends of most long bones are cartilaginous at the time of birth, and are gradually replaced by bone. The replacement is completed only after full growth of the individual (i.e. by about 18 years of age). Replacement of cartilage by bone is called ossification. Ossification of cartilage has to be carefully distinguished from calcification, in which the matrix hardens because of the deposition of calcium saltsin it but true bone is not formed.
*Capsular matrix contains the highest concentration of sulfated proteoglycans, hyaluronan, and many multiadhesive glycoproteins (e.g. fibronectin, laminin, and decorin). Main type of fiber istype VI collagen fibrils whereas territorial matrix—type II collagen fibers.
*The first formed chondrocyte lives within the primary lacuna, when the same cell divides it forms daughter cells. The lacunae around each daughter cell are termed as secondary lacunae. Therefore, the secondary lacunae of cell nest lie within the primary lacunae.
*Growth of cartilage: The process of formation of cartilage is known as chondrogenesis. Main gene regulating is SOX9.The
process starts with aggregation of chondrogenic mesenchymal cells. The place where hyaline cartilage is going to form there appears chondrogenic nodule, cells in them gets differentiated to form chondroblasts. Chondroblasts secrets the matrix and also lay down the essential fibers. Once they are completely surrounded by the matrix and locked in the lacunae, chondroblasts are referred as chondrocytes. The peripheral cells of the nodule form the perichondrium. Two types of growth are observed:
1. Interstitial growth: Here the chondroblasts within the matrix divides and contril increase in size of the cartilage.

- 2. Oppositional growth: Here the cells in the chondrogenic layer of the perich¢ differentiate into chondroblasts and lay down matrix and fibers on the existing cart
Appearance, Hyaline cartilage = Ground glass. Appearance, Elastic cartilage = -. Appearance, Fibrocartilage = White. Main cells, Hyaline cartilage = Chondroblasts, chondrocytes. Main cells, Elastic cartilage = | Chondroblasts, chondrocytes. Main cells, Fibrocartilage = Chondrocytes, fibroblasts. Fibers, Hyaline cartilage = Type ll collagen. Fibers, Elastic cartilage = Type Il collagen and elastic fibers. Fibers, Fibrocartilage = Type | and type Il collagen. Perichondrium, Hyaline cartilage = Present. Perichondrium, Elastic cartilage = Present. Perichondrium, Fibrocartilage = Absent. Calcification, Hyaline cartilage = Occurs. Calcification, Elastic cartilage = No. Calcification, Fibrocartilage = Only during repair. Function, Hyaline cartilage = Protects against compression, provides cushion effect. Function, Elastic cartilage = Provides flexibility. Function, Fibrocartilage = Protects against deformation. Examples, Hyaline cartilage = Tracheal rings, costal cartilages, thyroid, cricoid, and base of arytenoid cartilage. Examples, Elastic cartilage = Auditory tube, epiglottis, cuneiform and corniculate cartilage, and ear pinna. Examples, Fibrocartilage = Intervertebral disks, symphysis pubis, menisci of the knee joint, and articular disc of sternoclavicular and temporomandibular joint

Elastic cartilage contains type II collagen
Elastic cartilage provides flexibility
Perichondrium is absent in fibrocartilage Hyaline cartilage does not undergo calcification 2900p

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- Matrix around the chondrocytes Matrix surrounding the cell nests Matrix near the perichondrium Matrix between the cell nests 2050p
3. Hyaline cartilage is present in:
2050p Epiglottis Auricle Tracheal rings
Intervertebral disc
4. Gene regulating chondrogenesis:
2909 HLA-DRB1 CAPN10 BRCA1
SOX9
5. Core protein in hyaline cartilage is:
29509 Laminin Lumican
Aggrecan
Fibromodulin
6. Cartilage seen in intervertebral disc is:
2900p Hyaline cartilage Elastic cartilage White fibrocartilage of the above
7. Fibers seen in hyaline cartilage are:
2905p Reticular fibers Type II collagen fibers Type | collagen fibers

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* General features of bone and its function
*Components of bone
* Classification and structure of bone
* Formation of bone—ossification
* Growth of bone

Haversian system made up of concentric lamellae, lacunae with osteocytes, Haversian (central) canal containing blood vessels and nerves are seen, Bone (Longitudinal section) = Volkmann's canal interconnecting Haversian systems is seen. Three types of lamellae are seen-(1) circumferential, (2) concentric, and (3) interstitial Canaliculi radiate from lacunae, Bone (Longitudinal section) = Lacunae with osteocytes.

Bone is a hard and rigid form of connective tissue in which the extracellular matrix is impregnated with inorganic salts, mainly calcium phosphate and

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* Sclerous connective tissue specialized to provide support because of infiltration with inorganic salts.
* Tt is characterized by its rigidity, hardness, and power of regeneration and repair.
* Bone is a dynamic, living tissue, which constantly undergoes remodeling during an individual's life to help it adapt to changing physical, biochemical, and hormonal influences.
* Functions: (a) Bears weight; (b) Provides attachment for muscles and tendons thus, helps in movement; (c) Reservoir of minerals—calcium, phosphorus, and other ions; (c) Protects vital organs; and (d) Bone marrow within is hematopoietic tissue.
i

Like cartilage, bone is a modified connective tissue. It consists of bone cells (Figs. 6.1A to C) that are widely separated from one another by a considerable amount of intercellular substance. The latter consists of a homogeneous ground substance or matrix in which collagen fibers and mineral salts (mainly calcium and phosphorus) are deposited.
There are three types of cells in the bone: (1) osteocytes (mature bone cells), (2) osteoblast (bone forming cells), and (3) osteoclasts or bone removing cells (Figs. 6.1 and 6.2). Other cells present include osteoprogenitor cells from which osteoblasts and osteocytes are derived; cells lining the surfaces of bone; cells belonging to periosteum; and cells of blood vessels and nerves which invade bone from outside.
Figs. 6.1A to C: Bone cells. (A) Osteocyte; (B) Osteoblast; (C) Osteoclast (schematic

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= Stem cells of mesenchymal origin
=Can proliferate and convert themselves into osteoblasts whenever there need for bone formation
™Resemble fibroblasts in appearance
"In
the fetus, such cells are numerous at sites where bone formation is take place. In the adult, osteoprogenitor cells are present over bone surfat (on both the periosteal and endosteal aspects).

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= Bone forming cells derived from osteoprogenitor cells.
"Found lining growing surfaces of bone, sometimes giving an epitheliu like appearance. However, on closer examination, it is seen that the ce are of varied shapes (oval, triangular, cuboidal, etc.) and that there i numerous gaps between adjacent cells.
"The nucleus of an osteoblast is ovoid and euchromatic. The cytoplasm basophilic because of the presence of abundant rough endoplasn reticulum. This, and the presence of a well-developed Golgi compl: signifies that the cell is engaged in considerable synthetic activi Numerous slender cytoplasmic processes radiate from each cell and coi into contact with similar processes of neighboring cells.
Fig. 6.2: Photomicrograph of bone cells showing osteoblast rimming the surface of the bone; multinucleated osteoclast and osteocyte. [Ob: osteoblast (short arrow); Oc: osteoclast (long arrow); Os: osteocyte (arrowhead)]
"Osteoblasts are responsible for laying down the organic matrix of bc including the collagen fibers. They are also responsible for the calcificati of the matrix. Alkaline phosphatase present in the cell membranes osteoblasts plays an important role in this function. Osteoblasts are belie to shed off matrix vesicles that possibly serve as points around wh: formation of hydroxyapatite crystals takes place. Osteoblasts may indirec influence the resorption of bone by inhibiting or stimulating the activity osteoclasts.

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"The cells of mature bone.
"Lie in the lacunae of bone and represent osteoblasts that have beco: "imprisoned" in the matrix during bone formation. Delicate cytoplasr processes arising from osteocytes establish contacts with other osteocy and with bone lining cells present on the surface of bone.
"In contrast to osteoblasts, osteocytes have eosinophilic or lightly basophi cytoplasm. This is to be correlated with the fact that these cells he negligible secretory activity and presence of only a small amount
endoplasmic reticulum in the cytoplasm.
"Osteocytes are present in greatest numbers in young bone, the numl gradually decreasing with age.
"Functions: (1) It maintains the integrity of the lacunae and canaliculi, a thus, keeps open the channels for the diffusion of nutrition through bo: (2) They play a role in the removal or deposition of matrix and of calci when required.

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" These are bone removing cells.
"Found in relation to surfaces where bone removal is taking place (bc removal is essential for maintaining the proper shape of growing bone). such locations, the cells occupy pits called resorption bays or lacunae Howship.
"Very large cells (20-100 pm or even more in diameter). They he numerous nuclei: up to 20 or more. The cytoplasm shows numerc mitochondria and lysosomes containing acid phosphatase. At the sites bone resorption, the surface of an osteoclast shows many folds that | described as a ruffled membrane.
"Removal of bone by osteoclasts involves demineralization and removal matrix. Bone removal can be stimulated by factors secreted by osteobla: by macrophages, or by lymphocytes. It is also stimulated by the parathyri hormone.
™Recent studies have shown that osteoclasts are derived from monocytes blood. It is not certain whether osteoclasts are formed by the fusion several monocytes, or by repeated division of the nucleus, without divisi of cytoplasm.

These cells form a continuous epithelium-like layer on bony surfaces where active bone deposition or removal is not taking place. The cells are flattened. They are present on the periosteal surface as well as the endosteal surface. They also line spaces and canals within bone. It is possible that these cells can change to osteoblasts when bone formation is called for (in other words, many of them are osteoprogenitor cells).

It is made up of fibers and ground substance (matrix). Matrix in turn is made up of organic and inorganic components.

It forms 80-90% of organic component of bone. The collagen fibers are similar to those in connective tissue (type I collagen) (they are sometimes referred to as osteoid collagen). The fibers are usually arranged in layers, the fibers within a layer running parallel to one another. Collagen fibers of bone are synthesized by osteoblasts.

The ground substance (or matrix) of bone consists of an organic matrix in which mineral salts are deposited.

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* This consists of a ground substance in which collagen fibers are embedded.
* The ground substance consists of glycosaminoglycans, proteoglycans, and water.
* Two special glycoproteins osteonectin and osteocalcin are present in large quantity. They bind readily to calcium ions and, therefore, play a role in the mineralization of bone. Vitamin D stimulates the synthesis of these proteins.
* Various other substances including chondroitin sulfates, phospholipids, and phosphoproteins are also present.
* The matrix of bone shows greater density than elsewhere immediately around the lacunae, forming capsules around them, similar to those around chondrocytes in cartilage. The term osteoid is applied to the mixture of organic ground substance and collagen fibers (before it is mineralized).

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* The ions present are predominantly calcium and phosphorus (or phosphate). Magnesium, carbonate, hydroxyl, chloride, fluoride, citrate, sodium, and potassium are also present in significant amounts.
* Most of the calcium, phosphate, and hydroxyl ions are in the form of
needle-shaped crystals that are given the name _ hydroxyapatite [Ca,,(PO,),(OH,)]. Hydroxyapatite crystals lie parallel to collagen fibers and contribute to the lamellar appearance of bone. Some amorphous calcium phosphate is also present.
* About 65% of the dry weight of bone is accounted for by inorganic salts, and 35% by organic ground substance and collagen fibers (note that these percentages are for dry weight of bone. In living bone, about 20% of its weight is made up by water). About 85% of the total salts present in bone are in the form of calcium phosphate, and about 10% in the form of calcium carbonate. 97% of total calcium in the body is located in bone.
* The calcium salts present in bone are not "fixed". There is considerable interchange between calcium stored in bone and that in circulation. When calcium level in blood rises, calcium is deposited in bone; and when the level of calcium in blood falls, calcium is withdrawn from bone to bring blood levels back to normal. These exchanges take place under the influence of hormones (parathormone produced by the parathyroid glands, and calcitonin produced by the thyroid gland).
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The external and internal surface of the bone is covered by the layers of connective tissue known as periosteum and endosteum, respectively.

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* The external surface of any bone is, as a rule, covered by a membrane called periosteum (Fig. 6.3).
* The only parts of the bone surface devoid of periosteum are those that are covered with articular cartilage.
Fig. 6.3: Some features of bone structure as seen in a longitudinal section through one end of a long bone (schematic representation).
* The periosteum consists of two layers, outer and inner. The outer layer is a fibrous membrane. The inner layer is cellular. In young bones, the inner layer contains numerous osteoblasts and is called the osteogenetic layer (this layer is sometimes described as being distinct from periosteum). In the periosteum covering, the bones of an adult osteoblasts are not conspicuous, but osteoprogenitor cells present here can form osteoblasts when need arises, e.g. in the event of a fracture.
* Periosteum is richly supplied with blood. Many vessels from the periosteum enter the bone and help to supply it.

* Thin layer of vascular connective tissue lining the medullary spaces of tubular bone and marrow spaces of cancellous bone.
* Also extend as lining into canaliculi of compact bone.
* Have osteogenic potential.

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* Tt provides a medium through which muscles, tendons, and ligaments are attached to bone. In situations where very firm attachment of a tendon to bone is necessary, the fibers of the tendon continue into the outer layers of bone as the perforating fibers of Sharpey (Fig. 6.4). The parts of the fibers that lie within the bone are ossified; they have been compared to "nails" that keep the lamellae in place.
* Because of the blood vessels passing from periosteum into bone, the periosteum performs a nutritive function. This also forms reason for preserving them during bone surgeries.
* The osteoprogenitor cells in its deeper layer can form bone when required. This role is very important during development. It is also important in later life for the repair of bone after fracture.
* The fibrous layer of periosteum is sometimes described as a limiting membrane that prevents bone tissue from "spilling out" into neighboring
Fig. 6.4: Longitudinal section of compact bone.
tissues. This is based on the observation that if periosteum is torn, osteogenic cells may extend into surrounding tissue forming bony projections (exostoses). Such projections are frequently seen on the bones of old persons. The concept of the periosteum as a limiting membrane helps to explain how ridges and tubercles are formed on the surface of a bone. At sites where a tendon pulls upon periosteum, the latter tends to be lifted off from bone. The "gap" is filled by proliferation of bone leading to the formation of a tubercle (Such views are, however, hypothetical).
- * It has rich nerve supply hence sensitive to pain.
Decalcification: Mineral salts can be removed from bone by treating it with weak acids. Chelating agents (e.g. ethylenediaminetetraacetic acid or EDTA) are also used. The process, called decalcification, is necessary for preparing sections of bone. When mineral salts are removed by decalcification, the tissue becomes soft and pliable. This shows that the rigidity of bone is mainly due to the presence of mineral salts in it.

Calcination: Conversely, the organic substances present in bone can be destroyed by heat (as in burning). The process is called calcination. The form of the bone remains intact, but the bone becomes very brittle and breaks easily. It follows that the organic matter contributes substantially to the strength of bone. Resistance to tensile forces is mainly due to the presence of collagen fibers.
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* Based on the gross appearance of bone: Compact bone and spongy bone.
* Based on maturity: Mature/lamellar bone and immature/ woven bone.
* Based on ossification: Cartilaginous, membranous, and membranocartilaginous.

The wall of the tube is made up of a hard dense material that appears, on naked eye examination, and shows a uniform smooth texture with no obvious spaces in it. This kind of bone is called compact bone (Plates 6.1 and 6.2).
The section of bone consists of the compact following:
- * Lamellae (lamella—singular): Thin plate of bone consisting of collagen fibers and mineral salts that are deposited in a gelatinous ground substance.
Even the smallest piece of bone is made up of several lamellae placed over one another (Fig. 6.5). They are of three types:

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1. Circumferential lamellae: Arranged parallel to the surface. Outer the periosteum and inner around marrow cavity.
2. Concentric lamellae: Arranged in the form of concentric rings that : a narrow Haversian canal.
3. Interstitial lamellae: Located between osteons at the angular interve are the remnants of earlier formed osteons.
* Lacunae: Between adjoining lamellae, we see small flattened spaces or lacunae. Each lacuna contains one osteocyte. Spreading out from each lacuna, there are fine canals or canaliculi that communicate with those from other lacunae (Fig. 6.6). The canaliculi are occupied by delicate cytoplasmic processes of osteocytes. They network with each other and thus facilitate the diffusion of metabolic products between osteocytes.
* The unit of compact bone is Haversian system or osteon. Most of the lamellae are arranged in the form of concentric rings that surround a narrow Haversian canal present at the center of each ring. The Haversian canal is occupied by blood vessels, nerve fibers, and some cells. One Haversian canal and the lamellae around it constitute a Haversian system or osteon (Figs. 6.7 and 6.8). Compact bone consists of several such osteons.
* The longitudinal sections of compact bone (Fig. 6.8) has Haversian canals (and, therefore, the osteons) that run predominantly along the length of the bone. The canals branch and anastomose with each other. They also communicate with the marrow cavity, and with the external surface of the bone through channels that are called the canals of Volkmann. Blood vessels and nerves pass through all these channels so that compact bone is permeated by a network of blood vessels that provide nutrition to it.
* Cement line: The place where the periphery of one osteon comes in contact with another osteon (or with interstitial lamellae) is marked by the presence of a cement line. Along this line there are no collagen fibers, the line consisting mainly of inorganic matrix.
Fig. 6.5: How lamellae constitute bone (schematic representation).
Fig. 6.6: Relationship of osteocytes to bone lamellae (schematic representation).

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- * The shaft is tubular and encloses a large marrow cavity. The end of long bone and diploé of flat bones the marrow cavity does not extend. Instead made up of trabeculated network of tiny rods or plates of bone and containing numerous spaces, the whole appearance resembles to that of a sponge. This kind of bone is called spongy or cancellous bone (cancel = cavity). They do not have Haversian system.
Fig. 6.8: Transverse section and longitudinal section through compact bone to show Haversian canals and the Volkmann's canal (schematic representation).
* The spongy bone at the bone ends is covered by a thin layer of compact bone, thus providing the bone ends with smooth surfaces (see Fig. 6.3). Small bits of spongy bone are also present over the wall of the marrow cavity.
* Cancellous bone is made up of a meshwork of bony plates or rods called
trabeculae. Each trabeculus is made up of a number of lamellae (described above) between which there are lacunae containing osteocytes. Canaliculi, containing the processes of osteocytes, radiate from the lacunae (Fig. 6.9). The trabeculae enclose wide spaces that are filled in by bone marrow (Fig. 6.9 and Plate 6.1). They receive nutrition from blood vessels in the bone marrow.
- * The trabeculae are covered externally by vascular endosteum containing osteoblasts, osteoclasts, and osteoprogenitor cells.
The marrow cavity and the spaces of spongy bone (present at the bone ends) are filled by a highly vascular tissue called bone marrow.
* Red marrow: At the bone ends, the marrow is red in color. Apart from blood vessels, this contains numerous masses of blood forming cells (hematopoietic tissue).
* Yellow marrow: In the shaft of the bone of an adult, the marrow is yellow. This is made up predominantly of fat cells. Some islands of hematopoietic tissue may be seen here also.
Fig. 6.9: Structure of cancellous bone (schematic representation).
Compact Bone (Transverse section): (A) As seen in drawing; (B) Ground section; (C)
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system made up of concentric lamellae, lacunae with osteocytes, Haversian (central) canal containing blood vessels and nerves are seen types of lamellae are seen—(1) circumferential, (2) concentric, and (3) interstitial
radiate from lacunae.
"Haversian 'Three *Canaliculi
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ae
Compact bone (Longitudinal section): (A) Hand drawing; (B) Photomicrograph.

In bones of a fetus or of a young child, the entire bone marrow is red. The marrow in the shaft is gradually replaced by yellow marrow with increasing age.

There is an essential similarity in the structure of cancellous and compact bone. Both are made up of lamellae. The difference lies in the relative volume occupied by bony lamellae and by the spaces. In compact bone the spaces are small, and the solid bone is abundant with Haversian system, whereas in cancellous bone the spaces are large, and actual bone tissue is sparse (lamella do not form Haversian system) (Plate 6.2).
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*During bone formation, the first formed osteons do not have a clear lamellar structure, but consist of woven bone. Such osteons are described as primary osteons (or atypical Haversian systems). Subsequently, the primary osteons are replaced by secondary osteons (or typical Haversian systems) having the structure already described.
*The osteons run in a predominantly longitudinal direction (i.e. along the long axis of the shaft). However, this does not imply that osteons lie parallel to each other. They may follow a spiral course, may branch, or may join other osteons. In transverse sections, osteons may appear circular, oval, or ellipsoid.
*The number of lamellae in each osteon is highly variable. The average number is six.
*In an osteon collagen fibers in one lamellus usually run either longitudinally or circumferentially (relative to the long axis of the osteon). Typically, the direction of fibers in adjoining lamellae is alternately longitudinal and circumferential. It is because of this difference in the direction of fibers that the lamellar arrangement is obvious in sections. It has been claimed that in fact fibers in all lamellae follow a spiral course, the "longitudinal" fibers belonging
to a spiral of a long "pitch"; and the "circumferential" fibers to the spirals of a short "pitch".
*The various lacunae within an osteon are connected with one another through canaliculi that also communicate with the Haversian canal. The peripheral canaliculi of the osteon do not (as a rule) communicate with those of neighboring osteons: They form loops and turn back into their own osteon. A few canaliculi that pass through the cement line provide communications leading to interstitial lamellae.
*The lacunae and canaliculi are only partially filled in by osteocytes and their processes. The remaining space is filled by a fluid that surrounds the osteocytes. This space is in communication with the Haversian canal and provides a pathway along which substances can pass from blood vessels in the Haversian canal to osteocytes.

*When a transverse section of compact bone is examined with polarized light, each osteon shows two bright bands that cross each other. This phenomenon is called birefringence. It is an indication of the very regular arrangement of collagen fibers (and the crystals related to them) within the lamellus.
*The lamellar appearance (see Fig. 6.5) of bone depends mainly on the arrangement of collagen fibers. The fibers of one lamellus run parallel to each other, but those of adjoining lamellae run at varying angles to each other. The ground substance of a lamellus is continuous with that of adjoining lamellae.
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* Lamellar bone: Bone matrix contains thin sheets called as lamellae arranged parallel. It is also called secondary bone.
*, od Woven bone: The newly formed bone does not have a lamellar structure. The collagen fibers are present in bundles that appear to run randomly in different directions, interlacing with each other. Because of the interlacing of fiber bundles, this kind of bone is called woven bone. It is also known as primary bone. All newly formed bone is woven bone. It is later replaced by lamellar bone.
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* All bones are of mesodermal origin. The process of bone formation is called ossification.
* Types: (a) Endochondral ossification: The formation of most of the bones is preceded by the formation of a cartilaginous model, which is subsequently replaced by bone. This kind of ossification is called endochondral ossification; and bones formed in this way are called cartilage bones, (b) In some situations (e.g. the vault of the skull), formation of bone is not preceded by the formation of a cartilaginous model. Instead bone is laid down directly in a fibrous membrane. This process is called intramembranous ossification; and bones formed in this way are called membrane bones. The bones of the vault of the skull, the mandible, and the clavicle are membrane bones.

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The various stages in intramembranous ossification are as follows:
* At the site where a membrane bone is to be formed the mesenchymal cells become densely packed (i.e. a mesenchymal condensation is formed). The region becomes highly vascular. Some of the mesenchymal cells lay down the bundles of collagen fibers in the mesenchymal condensation. In this way a membrane is formed.
* Some mesenchymal cells (possibly those that had earlier laid down the collagen fibers) enlarge and acquire a basophilic cytoplasm and may now be called osteoblasts (Fig. 6.10A). They come to lie along the bundles of collagen fibers. These cells secrete a gelatinous matrix in which the fibers get embedded. The fibers also swell up. Hence the fibers can no longer be seen distinctly. This mass of swollen fibers and matrix is called osteoid (Fig. 6.10B).
* Under the influence of osteoblasts, calcium salts are deposited in osteoid. As soon as this happens, the layer of osteoid can be said to have become one lamellus of bone (Fig. 6.10C).
* Over this lamellus, another layer of osteoid is laid down by osteoblasts. The osteoblasts move away from the lamellus to line the new layer of osteoid. However, some of them get caught between the lamellus and the osteoid (Fig. 6.10D). The osteoid is now ossified to form another lamellus.
The cells trapped between the two lamellae become osteocytes (Fig. 6.10D).
* In this way a number of lamellae are laid down one over another, and these lamellae together form a trabeculus of bone (Fig. 6.10E).
* The first formed bone may not be in the form of regularly arranged lamellae. The elements are irregularly arranged and form woven bone.
Figs. 6.10A to E: Scheme to show how bony lamellae are laid down over one another intramembranous ossification.

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The essential steps in the formation of bone by endochondral ossification are as follows:
* At the site where the bone is to be formed, the mesenchymal cells become closely packed to form a mesenchymal condensation (Fig. 6.11A).
* Some mesenchymal cells become chondroblasts and lay down hyaline cartilage (Fig. 6.11B). Mesenchymal cells on the surface of the cartilage form a membrane called the perichondrium. This membrane is vascular and contains osteoprogenitor cells.
* The cells of the cartilage are at first small and irregularly arranged. However, in the area where bone formation is to begin, the cells enlarge considerably (Fig. 6.11C).
* The intercellular substance between the enlarged cartilage cells becomes calcified, under the influence of alkaline phosphatase, which is secreted by the cartilage cells. The nutrition to the cells is thus cut off, and they die, leaving behind empty spaces called primary areolae (Fig. 6.11C).
* Some blood vessels of the perichondrium (which may be called periosteum as soon as bone is formed) now invade the cartilaginous matrix. They are accompanied by osteoprogenitor cells. This mass of vessels and cells is called the periosteal bud. It eats away much of the calcified matrix forming the walls of the primary areolae, and thus creates large cavities called secondary areolae (Fig. 6.11D).
* The walls of secondary areolae are formed by thin layers of calcified matrix that have not dissolved. The osteoprogenitor cells become osteoblasts and arrange themselves along the surfaces of these bars, or plates, of calcified matrix (Fig. 6.11EF).
These osteoblasts now lay down a layer of ossein fibrils embedded in a gelatinous ground substance (i.e. osteoid), exactly as in intramembranous ossification (Fig. 6.11F). This osteoid is calcified, and a lamellus of bone is formed (Fig. 6.11G).

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* Osteoblasts now lay down another layer of osteoid over the first lamellus. This is also calcified. Thus two lamellae of bone are formed. Some osteoblasts that get caught between the two lamellae become osteocytes. As more lamellae are laid down, bony trabeculae are formed (Figs. 6.11H and I).
*Tt may be noted that the process of bone formation in endochondral
ossification is exactly the same as in intramembranous ossification. The calcified matrix of cartilage only acts as a support for the developing trabeculae and is not itself converted into bone.
* At this stage the ossifying cartilage shows a central region where bone has been formed. As we move away from this area, we see:
= A region where cartilaginous matrix has been calcified and surrounds de and dying cartilage cells.
= A zone of hypertrophied cartilage cells in an uncalcified matrix.
™ Normal cartilage in which there is considerable mitotic activity.
In this way the formation of new cartilage keeps pace with the loss due to replacement by bone. The total effect is that the ossifying cartilage progressively increases in size.

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All newly formed bone is cancellous. It is converted into compact bone as follows.
* Each space between the trabeculae of cancellous bone comes to be lined by a layer of osteoblasts. The osteoblasts lay down lamellae of bone as already described. The first lamellus is formed over the inner wall of the original space and is, therefore, shaped like a ring. Subsequently, concentric lamellae are laid down inside this ring thus forming an osteon. The original space becomes smaller and smaller and persists as a Haversian canal.
* The first formed Haversian systems are called atypical Haversian systems or primary osteons. These osteons do not have a typical lamellar structure, and their chemical composition may also be atypical.
Figs. 6.11A to I: Endochondral ossification. Formation of cartilaginous model (schematic representation). (A and B) Formation of cartilaginous model; (C) Early stage, the cartilage cells are separated by matrix; (D) The matrix has calcified. Cartilage cells are dead leaving empty spaces called primary areolae; (E) Some primary areolae have fused to form larger spaces called secondary areolae; (F to |) Four stages in formation of bony lamellae.
- * Primary osteons are soon invaded by blood vessels and by osteoclasts that bore a new series of spaces through them. These new spaces are again filled in by bony lamellae, under the influence of osteoblasts, to form secondary osteons (or typical Haversian systems). The process of
formation and destruction of osteons takes place repeatedly as the bone enlarges in size and continues even after birth. In this way the internal structure of the bone can be repeatedly remodeled to suit the stresses imposed on the bone.

* Interposed in between osteons of the newest series, there will be remnants of previous generations of osteons. The interstitial lamellae of compact bone represent such remnants.
* When a newly created cavity begins to be filled in by lamellae of a new osteon, the first formed layer is atypical in that it has a very high density of mineral deposit. This layer can subsequently be identified as a cement line that separates the osteon from previously formed elements. As the cement line represents the line at which the process of bone erosion stops and at which the process of bone formation begins, it is also called a reversal line. The cement lines are never present around primary osteons but are always present around subsequent generations of osteons.
Bf

A hard tissue like bone can grow only by the deposition of new bone over existing bone, i.e. by apposition.

* In the bones of the vault of the skull (e.g. the parietal bone), ossification begins in one or more small areas called centers of ossification and forms following the usual process.
* At first, it is in the form of narrow trabeculae or spicules. These spicules increase in length by the deposition of bone at their ends. As the spicules lengthen, they radiate from the center of ossification to the periphery. Gradually the entire mesenchymal condensation is invaded by this spreading process of ossification, and the bone assumes its normal shape. However, even at birth, the radiating arrangement of trabeculae is obvious.
* The mesenchymal cells lying over the developing bone differentiate to form the periosteum.
* The embryonic parietal bone, formed as described above, has to undergo considerable growth. After ossification has extended into the entire membrane representing the embryonic parietal bone, this bone is separated from neighboring bones by intervening fibrous tissue (in the region of the

+. od Growth in size of the bone can occur by the deposition of bone on the edges adjoining sutures (Figs. 6.12 and 6.13). Growth in thickness and size of the bone also occurs when the overlying periosteum forms bone (by the process of intramembranous ossification described above) over the outer surface of the bone.
* Simultaneously, there is the removal of bone from the inner surface. In this way, as the bone grows in size, there is simultaneous increase in the size of the cranial cavity.

*. od In the region where a long bone is to be formed the mesenchyme first lays down a cartilaginous model of the bone (Figs. 6.14A to C). This cartilage is covered by perichondrium. Endochondral ossification starts in the central part of the cartilaginous model (i.e. at the center of the future shaft). This area is called the primary center of ossification (Fig. 6.14D). Gradually, bone formation extends from the primary center toward the ends of shaft. This is accompanied by progressive enlargement of the cartilaginous model.
, a Soon after the appearance of the primary center, and the onset of endochondral ossification in it, the perichondrium (which may now be called periosteum) becomes active. The osteoprogenitor cells in its deeper layer lay down bone on the surface of the cartilaginous model by intramembranous ossification. This periosteal bone completely surrounds the cartilaginous shaft and is, therefore, called the periosteal collar (Figs. 6.14D and E). The periosteal collar is first formed only around the region of the primary center, but rapidly extends toward the ends of the cartilaginous model (Figs. 6.14F and G). It acts as a splint and gives strength to the cartilaginous model at the site where it is weakened by the formation of secondary areolae. We shall see that most of the shaft of the bone is derived from this periosteal collar and is, therefore, membranous in origin.
A ate * At about the time of birth the developing bone consists of a part called the diaphysis (or shaft), that is bony, and has been formed by extension of the primary center of ossification, and ends that are cartilaginous (Fig. 6.14F). At varying times after birth, secondary centers of endochondral
ossification appear in the cartilages forming the ends of the bone (Fig. 6.14G). These centers enlarge until the ends become bony (Fig. 6.14H). More than one secondary center of ossification may appear at either end. The portion of bone formed from one secondary center is called an epiphysis.

- * For a considerable time after birth the bone of the diaphysis and the bone of any epiphysis are separated by a plate of cartilage called the epiphyseal cartilage, or epiphyseal plate. This is formed by cartilage into which ossification has not extended either from the diaphysis or from the epiphysis. We shall see that this plate plays a vital role in growth of the bone.
Figs. 6.12A to D: Scheme to show how skull bones grow.
Fig. 6.13: Growth of skull bones at sutures.

* A growing bone increases both in length and in girth. The periosteum lays down a layer of bone around the shaft of the cartilaginous model. This periosteal collar gradually extends to the whole length of the diaphysis. As more layers of bone are laid down over it, the periosteal bone becomes thicker and thicker. However, it is neither necessary nor desirable for it to become too thick. Hence, osteoclasts come to line the internal surface of the shaft and remove bone from this aspect.
* As bone is laid down outside the shaft, it is removed from the inside. The shaft thus grows in diameter, and at the same time, its wall does not become too thick. The osteoclasts also remove trabeculae lying in the center of the bone that were formed by endochondral ossification. In this way a marrow cavity is formed.
* As the shaft increases in diameter, there is a corresponding increase in the size of the marrow cavity. This cavity also extends toward the ends of the diaphysis but does not reach the epiphyseal plate. Gradually, most of the bone formed from the primary center (i.e. of endochondral origin) is removed, except near the bone ends, so that the wall of the shaft is ultimately made up entirely of periosteal bone formed by the process of intramembranous ossification.
* To understand how a bone grows in length, we will now take a closer look at the epiphyseal plate. Depending on the arrangement of cells, three zones

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= Zone of resting cartilage: Here the cells are small and irregularly arrange
"Zone of proliferating cartilage: This is also called the zone of cartilc growth. In this zone the cells are larger and undergo repeated mitosis. they multiply, they come to be arranged in parallel columns, separated bars of intercellular matrix.
Figs. 6.14A to H: Formation of a typical long bone (schematic representation). (A to C) Establishment of cartilaginous model; (D and E) Formation of primary center of ossification and periosteal collar; (F and G) Formation of secondary centers of ossification; (H) Formation of bony epiphyses and epiphyseal plates.
Fig. 6.15: Structure of an epiphyseal plate (schematic representation).
"Zone of calcification: This is also called the zone of cartilc transformation. In this zone the cells become still larger, and the mat becomes calcified. Next to the zone of calcification, there is a zone whi cartilage cells are dead, and the calcified matrix is being replaced by bo: Growth in length of the bone takes place by continuous transformation the epiphyseal cartilage to bone in this zone (i.e. on the diaphyseal surfe of the epiphyseal cartilage). At the same time the thickness of © epiphyseal cartilage is maintained by the active multiplication of cells in: zone of proliferation.
* When the bone has attained its full length, cells in the cartilage stop proliferating. The process of calcification, however, continues to extend
into it until the whole of the epiphyseal plate is converted into bone. The bone substance of the diaphysis and that of the epiphysis then become continuous. This is called fusion of the epiphysis.

The portion of the diaphysis adjoining the epiphyseal plate is called the metaphysis. It is a region of active bone formation and, for this reason, it is highly vascular. The metaphysis does not have a marrow cavity. Numerous muscles and ligaments are usually attached to the bone in this region. Even after bone growth has ceased, the calcium turnover function of bone is most active in the metaphysis, which acts as a storehouse of calcium. The metaphysis is frequently the site of infection (osteomyelitis) because blood vessels show hairpin bends, and blood flow is sluggish.

Wolff's law: The structure of each part of a bone is adapted to these stresses bone being thickest along the lines of maximum stress, and absent in areas where there is no stress. This applies not only to the gross structure of a bone but also to its microscopic structure. The fact that the trabeculae of cancellous bone are arranged along the lines of stress has been recognized since long: this fact is known as Wolff's law. In some situations (e.g. at the upper end of the femur) the trabeculae appear to be arranged predominantly in two planes at right angles to each other. It has been suggested that trabeculae in one plane resist compressive forces, and those in the other direction resist tensile forces. In this context, it is interesting to note that a clear pattern of trabeculae is seen in the femur of a child only after it begins to walk.

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Bi
* Osteopetrosis: It is a disorder affecting osteoclasts in which they cannot resorb bone, which creates an imbalance between bone formation and bone resorption. These persons with display increased bone density.
* Osteoporosis
* Rickets/Acromegaly
* A benign tumor arising from osteoblasts is called an osteoma. A malignant tumor arising from the same cells is called an osteosarcoma. Osteosarcomas are most commonly seen in bones adjoining the knee joint. They can spread to distant sites in the body through the blood stream.
* Paget's disease of bone or osteitis deformans: Abnormal persistence of woven bone is a feature of Paget's disease. It was first described by Sir
James Paget in 1877. Paget's disease of bone is an osteolytic and osteosclerotic bone disease of uncertain etiology involving one (monostotic) or more bones (polyostotic). The condition affects predominantly males over the age of 50 years. The bones are weak, and there may be deformities.
1. Which of the following provide a pathway for the diffusion of nutrients within compact bo
Canailiculi
Lamellae Periosteum Lacunae 2900p
2. Which of the following cells is the precursor to bone forming cells?
a. Osteogenic
b. Osteoblast
c. Chondroblast
d. Osteoclast
3. When there is too little calcium in blood, which cells begin resorption of bone to re calcium to the blood?
a. Osteocytes
c. Osteocyte
d. Osteoblast
4. Which of these features indicates that a bone is not mature?
. Articular cartilage
Epiphyseal plate Medullary cavity . Spongy bone 2009
5. What is compact bone?
a. Dense
b. Spongy
c. Cancellous
d. Woven
6. What sits in a lacuna?
a. Osteoclast
b. Osteogenic

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2900p Osteon
Osteoclast
Osteogenic
8. What is the hollow area underneath an osteoclast called?
2905p Lacuna Howship’s lacuna Osteon

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*Types of muscular tissue
* Skeletal muscle
* Cardiac muscle
*» Smooth muscle
* Myoepithelial cells

Long cylindrical fibers, Smooth muscle = Spindle-shaped cells with central flat nucleus. Long cylindrical fibers, Cardiac muscle = Branched muscle fibers. Alternate dark and light band causing striations, Smooth muscle = Absence of striations.. Alternate dark and light band causing striations, Cardiac muscle = Central round single nucleus. Peripherally located multiple nuclei., Smooth muscle = . Peripherally located multiple nuclei., Cardiac muscle = Presence of striations.
Bf

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Muscle tissue is one of the basic tissues that are specialized to shorten in length by contraction causing movement. It is in this way that virtually all movements within the body, or of the body in relation to the environment, are ultimately produced.
* Muscle tissue is made up basically of cells that are called myocytes. Myocytes are elongated in one direction and are, therefore, often referred to as muscle fibers.
* Myocytes are mesodermal in origin; exception: arrector pilorum, muscles of iris and myoepithelial cells which are ectodermal in origin.
* Each muscle fiber contains contractile proteins actin and myosin.
* The various cell organelles of muscle fibers have been given special terms, plasma membrane-sarcolemma, cytoplasm-sarcoplasm, smooth endoplasmic reticulum (ER)-sarcoplasmic reticulum, and mitochondriasarcosome.
* Each muscle fiber is closely invested by connective tissue that is continuous with that around other muscle fibers. Because of this fact, the force generated by different muscle fibers gets added together. In some cases, a movement may be the result of simultaneous contraction of thousands of muscle fibers. The connective tissue framework of muscle also provides pathways along which blood vessels and nerves reach muscle fibers.
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"Present mainly in the limbs and in relation to the body wall.
"Because of its close relationship to the bony skeleton, it is called skele muscle.
"When examined under a microscope, fibers of skeletal muscle shi prominent transverse striations. Skeletal muscleis, therefore, also cal striated muscle.
"Skeletal muscle can normally be made to contract under our will perform movements we desire). It is, therefore, also called volunt muscle. Skeletal muscle is supplied by somatic motor nerves.

" Present mainly in relation to viscera.
"It is seen most typically in the walls of hollow viscera.
"As fibers of this variety do not show transverse striations, it is cal smooth muscle, or nonstriated muscle.
"As a rule, contraction of smooth muscle is not under our control; a smooth muscle is, therefore, also called involuntary muscle. It is suppl by autonomic nerves.

™ Present exclusively in the heart.
"It resembles smooth muscle in being involuntary; but it resembles striai muscle in that the fibers of cardiac muscle also show transverse striations
™ Cardiac muscle has an inherent rhythmic contractility the rate of which ¢ be modified by autonomic nerves that supply it.
Note: Some "skeletal" muscle has no relationship to the skeleton being present in situations such as the wall of the esophagus, or of the anal canal. The term striated muscle is usually treated as being synonymous with skeletal muscle, but we have seen that cardiac muscle also has striations. In many instances, the contraction of skeletal muscle may not be strictly voluntary (e.g. in sneezing or coughing; respiratory movements; maintenance of posture). Conversely, contraction of smooth muscle may be produced by voluntary effort as in passing urine.

* Skeletal muscle is made up essentially of long, cylindrical "fibers". The length of the fibers is highly variable, the longest being as much as 30 cm in length. The diameter of the fibers also varies considerably (10-60 pm; usually 50— 60 pm). Each "fiber" is really a syncytium (Fig. 7.1A) with hundreds of nuclei along its length (The "fiber" is formed, during development, by fusion of numerous myoblasts).
* The nuclei are elongated and lie along the periphery of the fiber, just under the cell membrane (which is called the sarcolemma). The cytoplasm (or sarcoplasm) is filled with numerous longitudinal fibrils that are called myofibrils (Fig. 7.1B). This pushes the nuclei to periphery underneath

* The most striking feature of skeletal muscle fibers is the presence of transverse striations in them (Plate 7.1). After staining with hematoxylin, the striations are seen as alternate dark and light bands that stretch across the muscle fiber. The dark bands are called A-bands (anisotropic), while the light bands are called I-bands (isotropic) (Fig. 7.3). (As an aid to memory, note that "A" and "I" correspond to the second letters in the words dark and light).
In addition to myofibrils, the sarcoplasm of a muscle fiber contains the usual cell organelles that tend to aggregate near the nuclei. Mitochondria are numerous. Substantial amounts of glycogen are also present. Glycogen provides energy for contraction of muscle. + a
Skeletal muscle fibers are multinucleated because they are syncytium. In the embryo they are uninucleated entities in myoblasts. Later during development myoblasts fuse together and gives rise to multinucleated muscle fiber. Larger the length of fiber more will be the number of nuclei. This in turn helps to sufficient translation of mRNA to proteins and enzymes. + a
Organization: Each muscle fiber is closely invested by connective tissue. This connective tissue supports muscle fibers and unites them to each other. Each muscle fiber is surrounded by delicate connective tissue that is called the endomysium. Individual fasciculi are surrounded by astronger sheath of connective tissue called the perimysium. The entire muscle is surrounded by connective tissue called the epimysium (Fig. 7.2 and Plate 7.2). At the junction of a muscle with a tendon, the fibers of the endomysium, the perimysium, and the epimysium become continuous with the fibers of the tendon. 4 '

Within a muscle, the muscle fibers are arranged in the form of bundles or fasciculi. The numbers of fasciculi in a muscle, and the number of fibers in each fasciculus, are both highly variable. In small muscles concerned with fine movements (like those of the eyeball, or those of the vocal folds), the fasciculi are delicate and few in number. In large muscles (in which strength of contraction is the main consideration), fasciculi are coarse and numerous.
- * Ultrastructure of striated muscle[under electron microscope (EM)]: Each muscle fiber is covered by a plasma membrane that is called the sarcolemma. The sarcolemma is covered on the outside by a basement membrane (also called the external lamina) that establishes an intimate connection between the muscle fiber and the fibers (collagen, reticular) of the endomysium. The cytoplasm (sarcoplasm) is permeated with myofibrils that push the elongated nuclei to a peripheral position. Between the myofibrils, there is an elaborate system of membrane-lined tubes called the sarcoplasmic reticulum. Elongated mitochondria (sarcosomes) and clusters of glycogen are also scattered among the myofibrils. Perinuclear Golgi bodies, ribosomes, lysosomes, and lipid vacuoles are also present.
Transverse section through skeletal muscle: (A) As seen in drawing; (B) Photomicrograph.

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The transverse section of a skeletal muscle fiber is characterized by:
* Fibers seen as irregularly round structures with peripheral nuclei
* Muscle fibers grouped into numerous fasciculi
* Dots within the fibers are myofibrils which are seen at higher magnification.
- The connective tissue of the muscle consists of:
Epimysium: Connective tissue sheath of muscle (not seen in photomicrograph)
* Perimysium: Connective tissue covering of each fascicle
* Endomysium: Loose connective tissue surrounding each muscle fiber.
* Structure of myofibrils (Fig. 7.3): When examined by EM, each myofibril is seen to be made of fine myofilaments. Each myofibril has alternating I and A bands seen under light microscopy. In good preparations (especially if the fibers are stretched), some further details can be made out. Running across the middle of each I-band there is a thin dark line called the Z-band. The middle of the A-band is traversed by a lighter band, called the H-band (or H-zone). Running through the center of the Hband, a thin dark line can be made out. This is the M-band. These bands appear to run transversely across the whole muscle fiber because corresponding bands in adjoining myofibrils lie exactly in alignment with one another. The part of a myofibril situated between two consecutive Zbands is called a sarcomere. The cross striations seen in a myofibril under electron microscopy is due to the presence of orderly arrangement of myofilaments (contractile protein filaments) within it.
Fig. 7.2: Connective tissue present in relation to transverse section of skeletal muscle (schematic representation).
Fig. 7.3: Terminology of transverse bands in a myofibril. Note that the A-band is confined to one sarcomere, but the I-band is made up of parts of two sarcomeres that meet at the Z-band (schematic representation).

* Structure of myofilaments: There are two types of myofilaments in skeletal muscle, myosin and actin, made up of molecules of corresponding proteins (each myosin filament is about 12 nm in diameter, while an actin filament is about 8 nm in diameter. They are, therefore, referred to as thick and thin filaments, respectively). The arrangement of myosin and actin filaments within a sarcomere is shown in Figure 7.4. It will be seen that myosin filaments are confined to the A-band, the width of the band being equal to the length of the myosin filaments. The actin filaments are attached at one end to the Z-band. From here, they pass through the I-band and extend into the "outer" parts of the A-band, where they interdigitate with the myosin filaments. Note that the I-band is made up of actin filaments alone. The H-band represents the part of the A-band into which actin filaments do not extend. The Z-band is really a complicated network at which the actin filaments of adjoining sarcomeres meet. The M-band is produced by fine interconnections between adjacent myosin filaments.

We have seen that the light and dark bands of myofibrils (or of muscle fibers) are designated I-bands and A-bands, respectively. The letters "I" and "A" stand for the terms isotropic and anisotropic, respectively. These terms refer to the way in which any material (e.g. a crystal) behaves with regard to the transmission of light through it. Some materials refract light equally in all directions: they are said to be isotropic. Other materials that do not refract light equally in different planes are anisotropic. These qualities depend on the arrangement of the elements making up the material. In the case of muscle fibers, the precise reason for alternate bands being isotropic and anisotropic is not understood. The phenomenon is most probably due to peculiarities in arrangement of molecules within them. Although striations can be made out in unstained material using ordinary light, they are much better seen through a microscope using polarized light.

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We have seen that the part of a myofibril between the two Z-bands is called a sarcomere. In other words, a Z band is a plate lying between two sarcomeres. The letter "Z" is from the German word zwischenschiebe (zwischen = between; schiebe = disc). The M-band is a plate lying in the middle of the sarcomere. The letter "M" is from the German word mittleschiebe (mittle = middle). The H-band (or zone) is named after Hensen who first described it.
Each myosin filament is made up of a large number of myosin molecules. Each molecule is made up of two units, each unit having a head and a tail (Fig. 7.5). The tails are coiled over each other. A myosin filament is a "bundle" of the tails of such molecules (Fig. 7.6). The heads project outward from the bundle as projections of the myosin filament. The projecting heads are arranged in a regular helical manner.
Because of the manner in which it is formed, each myosin fibril can be said to have a head end and a tail end. The tail end is attached to the M-line.
Each actin filament is really composed of two subfilaments that are twisted round each other (Fig. 7.7). Each subfilament is a chain of globular (rounded) molecules. These globular molecules are G-actin, and the chain formed by them is designated as F-actin. Each actin filament has a head end (that extends into the A-band) and a tail end that is anchored to the Z-line (through a protein called a-actinin). The filament also contains two other proteins called tropomyosin and troponin. Tropomyosin is in the form of a long fiber that winds around actin and stabilizes it.
Fig. 7.4: Arrangement of myofilaments in sarcomere. Note that the width of the I-band becomes less, and that the H-zone disappears when the myofibril contracts (schematic representation).

Fig. 7.5: Structure of a myosin molecule. Each molecule has two components (shown in red and blue) each consisting of a head and a tail. The tails are coiled over each other. The parts shown in red or blue are heavy myosin. Light myosin is shaded yellow (schematic representation).
Fig. 7.6: Myosin filament made up of several molecules of myosin (schematic representation).
Fig. 7.7: Actin filament (F-actin) made up of globular molecules of G-actin (schematic representation).
Troponin is a complex made up of several fractions. These complexes are arranged regularly over the actin fiber and represent sites at which myosin binds to actin.

During contraction, there is no shortening of individual thick and thin myofilaments; but there is an increase in the degree of overlap between the filaments. In an uncontracted myofibril, overlap between actin and myosin filaments is minimal. During contraction under the influence of energy released by adenosine triphosphate (ATP) and calcium released from sarcoplasmic reticulum, the fibril shortens by sliding in of actin filaments more and more into the intervals between the myosin filaments. As a result, the width of the I-band decreases, but that of the A-band is unchanged. The H-bands are obliterated in a contracted fibril.

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Several proteins other than actin and myosin are present in muscle. Some of them are as follows.
* Actinin is present in the region of Z discs. It binds the tail ends of actin filaments to this disc.
* Myomesin is present in the region of the M disc. It binds the tail ends of myosin filaments to this disc.
* Titin links the head ends of myosin filaments to the Z disc. This is a long and elastic protein that can lengthen and shorten as required. It keeps the myosin filament in proper alignment.
* Desmin is present in intermediate filaments of the cytoskeleton. It links myofibrils to each other, and also to the cell membrane.

In the intervals between myofibrils, the sarcoplasm contains an elaborate system of tubules called the sarcoplasmic reticulum (Fig. 7.8). The larger elements of this reticulum run in planes at right angles to the long axes of the myofibrils, and form rings around each myofibril. At the level of every junction between an A and I band, the myofibril is encircled by a set of three closely connected tubules that constitute a muscle triad.

For purposes of description, each such triad can be said to be composed of an upper, a middle, and a lower tubule (Fig. 7.8). The upper and lower tubules of the triad are connected to the tubules of adjoining triads through a network of smaller tubules. There is one such network opposite each A-band, and another opposite each I-band. These networks, along with the upper and lower tubules of the triad, constitute the sarcoplasmic reticulum. This reticulum is a closed system of tubes.
The middle tube of the triad is an entity independent of the sarcoplasmic reticulum. It is called a centrotubule and belongs to what is called the Tsystem of membranes. The centrotubules are really formed by invagination of the sarcolemma into the sarcoplasm. Their lumina are, therefore, in communication with the exterior of the muscle fiber.
Contraction of muscle is dependent on release of calcium ions into myofibrils. In a relaxed muscle, these ions are strongly bound to the membranes of the sarcoplasmic reticulum. When a nerve stimulus reaches a motor endplate, the sarcolemma is depolarized. The wave of depolarization is transmitted to the interior of the muscle fiber through the centrotubules. As a result of this wave, calcium ions are released from the sarcoplasmic reticulum into the myofibrils causing their contraction.

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* From morphological, histochemical, and functional point of view, skeletal muscle fibers are of two types (Table 7.1):
1. Red muscle fiber (because they are red in color).
2. White muscle fiber.
* The color of red fibers is due to the presence (in the sarcoplasm) of a pigment called myoglobin. This pigment is similar (but not identical with) hemoglobin. It is present also in white fibers, but in much lesser quantity.
* As compared to white fibers, the contraction of red fibers is relatively slow. Hence, red fibers are also called slow twitch fibers, or type I fibers; while white fibers are also called fast twitch fibers or type II fibers.
* Red fibers are narrower than white fibers. Relative to the volume of the myofibrils, the sarcoplasm is more abundant. Probably because of this fact, the myofibrils, and striations, are less well-defined; and the nuclei are not always at the periphery, but may extend deeper into the fiber. Mitochondria are more numerous in red fibers, but the sarcoplasmic reticulum is less extensive. The sarcoplasm contains more glycogen. The capillary bed around red fibers is richer than around white fibers. Differences have also been described in enzyme systems and the respiratory mechanisms in the two types of fibers. Fibers intermediate between red and white fibers have also been described.
Fig. 7.8: Relationship of the sarcoplasmic reticulum and the T-tubes to a myofibril (schematic representation).

Table 7.1: Characteristics of red and white muscle fibers., 1 = Table 7.1: Characteristics of red and white muscle fibers.. Red muscle fiber, 1 = White muscle fiber. Rich in myoglobin and cytochrome hence red in color, 1 = Less myoglobin and cytochrome hence white in color. Narrow in diameter with less defined striations and nuclei not always placed at the periphery, 1 = Broader in diameter, with well-defined striations and nuclei placed at the periphery. Volume of sarcoplasm is more than myofibrils. Sarcoplasm contains more glycogen, 1 = Volume of myofibrils is more. Sarcoplasm contains less glycogen. Numerous mitochondria but sarcoplasmic reticulum is less extensive, 1 = Few mitochondria with extensive sarcoplasmic reticulum. Slow and continuous contraction (not easily fatigued), 1 = Rapid contraction (easily fatigued). Rich blood supply, 1 = Poor blood supply. For example, predominate in postural muscles which have to remain contracted over long periods, e.g. erector spinae, 1 = For example, predominate in muscles responsible for sharp active movements, e.g. extraocular muscles and flight muscles in birds
- * In some animals, complete muscles may consist exclusively of red or white
fibers, but in most mammals, including man, muscles contain an admixture of both types. Although red fibers contract slowly, their contraction is more sustained, and they fatigue less easily. They predominate in the socalled postural muscles (which have to remain contracted over long periods), while white fibers predominate in muscles responsible for sharp active movements.
* Type II (white) fibers may be divided into type IIA and type IIB, the two types differing in their enzyme content, and in the chemical nature of their myosin molecules.
*In addition to color and speed of contraction, there are several other differences between red and white fibers. In comparison to white fibers, red fibers differ as follows.

* Skeletal muscle is richly supplied with blood vessels. The main artery to the muscle enters it at the neurovascular hilus. Several other arteries may enter the muscle at its ends or at other places along its length.
* The arteries form a plexus in the epimysium and in the perimysium, and end in a network of capillaries that surrounds each muscle fiber. This network is richer in red muscle than in white muscle.
* Veins leaving the muscle accompany the arteries. A lymphatic plexus extends into the epimysium and the perimysium, but not into the endomysium.

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* The nerve supplying a muscle enters it (along with the main blood vessels) at an area called the neurovascular hilus. This hilus is usually situated nearer the origin of the muscle than the insertion.
* After entering the muscle, the nerve breaks up into many branches that run through the connective tissue of the perimysium and endomysium to reach each muscle fiber.
* The nerve fibers supplying skeletal muscle are axons arising from large neurons in the anterior (or ventral) gray columns of the spinal cord (or of corresponding nuclei in the brainstem). These alpha-efferents have a large diameter and are myelinated. Because of repeated branching of its axon, one anterior gray column neuron may supply many muscle fibers all of
which contract when this neuron "fires".
* One anterior gray column neuron and the muscle fibers supplied by it constitute one motor unit (Fig. 7.9). The number of muscle fibers in one motor unit is variable. The units are smaller where precise control of muscular action is required (as in ocular muscles), and much larger in limb muscles where force of contraction is more important. The strength with which a muscle contracts at a particular moment depends on the number of motor units that are activated.
* The junction between a muscle fiber and the nerve terminal that supplies it is highly specialized and is called a motor endplate (discussed in detail later).
* Apart from the alpha efferents described earlier, every muscle receives smaller myelinated gamma-efferents that arise from gamma neurons in the ventral gray column of the spinal cord.
* These fibers supply special muscle fibers that are present within sensory receptors called muscle spindles. These special muscle fibers are called intrafusal fibers. Nerves to muscles also carry autonomic fibers that supply smooth muscle present in the walls of blood vessels.
Fig. 7.9: Motor unit consisting of a number of muscle fibers innervated by a single motor neuron (schematic representation).
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The structure of cardiac muscle has many similarities to that of skeletal muscle; but there are important differences as well.

* Like skeletal muscle, cardiac muscle is made up of elongated "fibers" within which there are numerous myofibrils (Plate 7.3). The myofibrils (and, therefore, the fibers) show transverse striations similar to those of skeletal muscle. A, I, Z, and H bands can be made out in the striations.
* The connective tissue framework, and the capillary network around cardiac muscle fibers are similar to those in skeletal muscle.
* With the EM, it is seen that myofibrils of cardiac muscle have the same structure as those of skeletal muscle and are made up of actin and myosin filaments. A sarcoplasmic reticulum, T-system of centrotubules, numerous mitochondria, and other organelles are present.

- * The fibers of cardiac muscle do not run in strict parallel formation, but branch and anastomose with other fibers to form a network.
Plate 7.3: Cardiac muscle

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*The fibers are made up of "cells" each of which has a centrally placed nucleus and transverse striations
*A clear space called perinuclear halo is seen around the nucleus
* Adjacent cells are separated from one another by transverse lines called intercalated discs
* Fibers show branching
* Blood vessels are also seen.
*, ~ Each fiber of cardiac muscle is not a multinucleated syncytium as in skeletal muscle, but is a chain of cardiac muscle cells (or cardiac myocytes) each having its own nucleus. Each myocyte is about 80 pm long and about 15 um broad.
oa The nucleus of each myocyte is located centrally (and not peripherally as in skeletal muscle).
, ate '= The sarcoplasm of cardiac myocytes is abundant and contains numerous large mitochondria. The myofibrils are relatively few. At places, the myofibrils merge with each other. As a result of these factors, the myofibrils and striations of cardiac muscle are not as distinct as those of skeletal muscle. In this respect, cardiac muscle is closer to the red variety of skeletal muscle than to the white variety. Other similarities with red muscle are the presence of significant amounts of glycogen and of myoglobin, and the rich density of the capillary network around the fibers.
* With the EM, it is seen that the sarcoplasmic reticulum is much less prominent than in skeletal muscle. The centrotubules of the T-system lie opposite the Z-bands (and not at the junctions of A and I-bands as in skeletal muscle). The tubules are much wider than in skeletal muscle. Typical triads are not present. They are often replaced by dyads having one T-tube and one tube of the sarcoplasmic reticulum.

, ate '= With the light microscope, the junctions between adjoining cardiac myocytes are seen as dark staining transverse lines running across the muscle fiber. These lines are called intercalated discs. Sometimes, these discs do not run straight across the fibers, but are broken into a number of "steps" (Plate 7.3). The discs always lies opposite the I-bands. They are made of 3 types of cell junctions-Desmosomes/Macula densa (binds the filaments and joins the cells together there by prevents their seperation during contraction), Fascia adherens (anchoring sites for actin and connects neigbhouring sarcomeres) and gap junctions (helps in spread of action potential).
Cardiac muscle is involuntary and is innervated by autonomic fibers (in contrast to skeletal muscle that is innervated by cerebrospinal nerves). Nerve endings terminate near the cardiac myocytes, but motor endplates are not seen.
*, od Isolated cardiac myocytes contract spontaneously in a rhythmic manner. In
the intact heart, the rhythm of contraction is determined by a pacemaker located in the sinoatrial node. From here, the impulse spreads to the entire heart through a conducting system made up of a special kind of cardiac muscle.

With the EM, it is seen that the intercalated discs are formed by cell membranes of adjacent myocytes, and by a layer of particularly dense cytoplasm present next to the cell membrane. The ends of actin filaments are embedded in this dense cytoplasm (Eig. 7.10). The cell membranes of adjoining myocytes are connected by numerous desmosomes, gap junctions, and tight junctions. Desmosomes link intermediate filaments present in the cytoskeleton of adjacent cells. Actin filaments of the cells end in relation to tight junctions. Gap junctions allow electrical continuity between adjacent myocytes, and thus, convert the cardiac muscle into a physiological syncytium.
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* Smooth muscle (also called nonstriated, involuntary, or plain muscle) is made up of long spindle-shaped cells (myocytes) having a broad central part and tapering ends.
* The nucleus, which is oval or elongated, lies in the central part of the cell. The length of smooth muscle cells (often called fibers) is highly variable (15-500 pm).
* With the light microscope, the sarcoplasm appears to have indistinct longitudinal striations, but there are no transverse striations.
* Smooth muscle is not striated because myofilaments are not arranged regularly along the length of spindle shaped cell.
* Smooth muscle cells are usually aggregated to form bundles, or fasciculi, that are further aggregated to form layers of variable thickness. In such a layer, the cells are so arranged that the thick central part of one cell is opposite the thin tapering ends of adjoining cells (Fig. 7.11). Aggregations of smooth muscle cells into fasciculi and layers are facilitated by the fact that each myocyte is surrounded by a network of delicate fibers (collagen, reticular, and elastic) that holds the myocytes together (Plate 7.4). The fibers between individual myocytes become continuous with the more abundant connective tissue that separates fasciculi or layers of smooth muscle.
Fig. 7.10: Electron microscopic structure of part of an intercalated disc (schematic representation).

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* Each smooth muscle cell is bounded by a plasma membrane. Outside the plasma membrane, there is an external lamina to which the plasma membrane is adherent. Connective tissue fibers are attached to the lamina (through special proteins).
* Adjacent smooth muscle cells communicate through gap junctions.
* The longitudinal striations (seen with the light microscope) are due to the presence of delicate myofilaments. These myofilaments are composed mainly of the proteins actin and myosin, but these do not have the highly ordered arrangement seen in striated muscle. The actin filaments are also different from those in skeletal muscle. Troponin is not present.
* Apart from myofibrils, the sarcoplasm also contains mitochondria, a Golgi complex, some granular endoplasmic reticulum, free ribosomes, and intermediate filaments.
* A sarcoplasmic reticulum, similar to that in skeletal muscle, is present, but is not as developed.
* Numerous invaginations (caveolae) resembling endocytic vesicles are seen near the surface of each myocyte, but no endocytosis occurs here.

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The mechanism of contraction of smooth muscle is different from that of skeletal muscle as follows:
* The myosin is chemically different from that in skeletal muscle. It binds to actin only if its light chain is phosphorylated. This phosphorylation of myosin is necessary for contraction of smooth muscle.
* As compared to skeletal muscle, smooth muscle needs very little ATP for contraction.
* The mechanisms regulating the flow of calcium ions into smooth muscle are different from those for skeletal muscle. Caveolae present on the surface of smooth muscle cells play a role in this process.
* Actin and myosin form bundles that are attached at both ends to the points on the cell membrane called anchoring points (or focal densities). When the muscle contracts, these points are drawn closer to each other. This converts an elongated smooth muscle cell in one that is oval.
Fig. 7.11: Smooth muscle cells (schematic representation).

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* Smooth muscle is seen most typically in the walls of hollow viscera including the stomach, the intestines, the urinary bladder, and the uterus.
* Tt is present in the walls of several structures that are in the form of narrow tubes, e.g. arteries, veins, bronchi, ureters, deferent ducts, uterine tubes, and the ducts of several glands.
* The muscles that constrict and dilate the pupil are made up of smooth muscle.
* Some smooth muscle is present in the orbit (orbitalis); in the upper eyelid (Muller's muscle); in the prostate; and in the skin of the scrotum (Dartos
muscle). In the skin, delicate bundles of smooth muscle are present in relation to hair follicles. These bundles are called the arrector pili muscles.

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Smooth muscle fibers may be arranged in a variety of ways depending on functional requirements.
* In some organs (e.g. the gut), smooth muscle is arranged in the form of two distinct layers: (1) an inner circular and (2) an outer longitudinal. Within each layer, the fasciculi lie parallel to each other. Such an arrangement allows peristaltic movements to take place for propulsion of contents along the tube.
*In some organs (e.g. the ureter), the arrangement of layers may be reversed, the longitudinal layer being internal to the circular one. In yet other situations, there may be three layers: inner and outer longitudinal with a circular layer in between (e.g. the urinary bladder and vas deferens).
*In some regions (e.g. urinary bladder, uterus), the smooth muscle is arranged in layers, but the layers are not distinctly demarcated from each other. Even within layers, the fasciculi tend to run in various directions and may form a network.
Plate 7.4: Smooth muscle
Smooth muscle: (A) As seen in drawing; (B) Photomicrograph.

In the drawing, muscle is seen cut longitudinally as well as transversely.
Loose connective tissue is seen above and below the layers of muscle.

*The smooth muscle fibers are spindle-shaped cells with tapering ends
- > : The nucleus is elongated and centrally placed
>

*The spindle-shaped cells are cut at different places along the length resulting in various shapes and sizes of the cells
*The nucleus is seen in those cells which are cut through the center. Others do not show nuclei.
* In some tubes (e.g. the bile duct), a thick layer of circular muscle may surround a segment of the tube forming a sphincter. Contraction of the sphincter occludes the tube.
* In the skin, and in some other places, smooth muscle occurs in the form of narrow bands.

Smooth muscle is innervated by autonomic nerves, both sympathetic and parasympathetic. The two have opposite effects. For example, in the iris, parasympathetic stimulation causes constriction of the pupil, and sympathetic stimulation causes dilatation. It may be noted that sympathetic or parasympathetic nerves may cause contraction of muscle at some sites, and relaxation at other sites.

Blood vessels and lymphatics are present in smooth muscle, but the density of blood vessels is much less than in skeletal muscle (in keeping with much less activity) (Table 7.2).

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* Apart from muscle, myoepithelial cells show the presence of contractile proteins (actin and myosin).
* Myoepitheliocytes (or myoepithelial cells) are present in close relation to secretory elements of some glands. They help to squeeze secretions out of secreting elements. Myoepithelial cells may be stellate, forming baskets around acini, or may be fusiform.
* Myoepitheliocytes are seen in salivary glands, the mammary glands, and sweat glands. These cells are of ectodermal origin.
* With the EM, they are seen to contain actin and myosin filaments. They
can be localized histochemically, because they contain the protein desmin that is specific to muscle.
- * Myoepithelial cells are innervated by autonomic nerves.
Bf

* Muscle dystrophy: Each muscle fiber contains a cytoskeleton. The fibers of the cytoskeleton are linked to actin fibers. The cytoskeleton is also linked to the external lamina through glycoproteins present in the cell membrane. Forces generated within the fiber are, thus, transmitted to the external lamina. The external lamina is in turn attached to connective tissue fibers around the muscle fiber. A number of proteins are responsible for these linkages. Genetic defects in these proteins can result in abnormalities in muscle (muscle dystrophy). One such protein is dystrophin, and its absence is associated with a disease called Duchenne muscular dystrophy.
* Myasthenia gravis (MG) is a neuromuscular disorder of autoimmune origin in which the acetylcholine receptors in the motor endplates of the muscles are damaged (Figs. 7.12A and B). The term myasthenia means "muscular weakness" and gravis implies "serious"; thus, both together denote the clinical characteristics of the disease. MG may be found at any age but adult women are affected more often than adult men in the ratio of 3:2. The condition presents clinically with muscular weakness and fatigability, initially in the ocular musculature but later spreads to involve the trunk and limbs.

Muscle fiber, Skeletal muscle = Long, cylindrical, and unbranched. Muscle fiber, Cardiac muscle = Short, narrow, and branched. Muscle fiber, Smooth muscle = Fusiform/spindle-shaped and unbranched. Control, Skeletal muscle = Voluntary. Control, Cardiac muscle = Involuntary. Control, Smooth muscle = Involuntary. Location, Skeletal muscle = Muscle of skeleton, tongue, esophagus, and diaphragm. Location, Cardiac muscle = Heart, pulmonary veins, and superior and inferior vena cava. Location, Smooth muscle = Vessels, organs, and viscera. Striations, Skeletal muscle = Present (well-defined). Striations, Cardiac muscle = Present (poorly defined). Striations, Smooth muscle = Absent. Nuclei, Skeletal muscle = Multiple, flat, and located at periphery. Nuclei, Cardiac muscle = _Single, oval, and present in center. Nuclei, Smooth muscle = Single, oval, and present in center. Sarcoplasmic reticulum, Skeletal muscle = Present (form triads). Sarcoplasmic reticulum, Cardiac muscle = Present (form dyad). Sarcoplasmic reticulum, Smooth muscle = Absent. Intercalated disc, Skeletal muscle = Absent. Intercalated disc, Cardiac muscle = Present. Intercalated disc, Smooth muscle = Absent. Regeneration after injury, Skeletal muscle = Seen (limited). Regeneration after injury, Cardiac muscle = Not seen. Regeneration after injury, Smooth muscle = Seen
Figs. 7.12A and B: Neuromuscular junction in normal transmission. (A) In myasthenia gravis (MG); and (B) The junction in MG shows reduced number of acetylcholine receptors (AChRs), flattened and simplified postsynaptic folds, and a widened synaptic space but a normal nerve terminal (schematic representation).

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* Skeletal muscle tumors: Rhabdomyoma and rhabdomyosarcoma are the benign and malignant tumors, respectively of striated muscle.
* Muscle hypertrophy: All varieties of muscle can hypertrophy when exposed to greater stress. Hypertrophy takes place by enlargement of existing fibers, and not by formation of new fibers. Skeletal muscle hypertrophies with exercise. Cardiac muscle hypertrophies, if the load on a chamber of the heart, is increased for any reason. An example is the hypertrophy of muscle in the wall of the left ventricle in hypertension. Hypertrophy of smooth muscle is seen most typically in the uterus where myocytes may increase from a length of about 15-20 um at the beginning of pregnancy to as much as 500 ptm toward the end of pregnancy.
* Smooth muscle and cardiac muscle have very little capacity for regeneration. Any defects produced by injury or disease are usually repaired by formation of fibrous tissue.
* Skeletal muscle fibers can undergo some degree of regeneration. They cannot divide to form new fibers. However, satellite cells present in relation to them (just deep to the external lamina) can give rise to new muscle fibers. Satellite cells are regarded as persisting myoblasts. When large segments of a muscle are destroyed, the gap is filled in by fibrous tissue.
* Excessive activity of smooth muscle is responsible for many symptoms. Constriction of bronchi leads to asthma. Spasm of smooth muscle can give rise to severe pain (colic) that may originate in the intestines (intestinal colic), ureter (renal colic), or bile duct (biliary colic). These symptoms can

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1. All the following are skeletal muscle type, except:
. Biceps brachii
. Diaphragm . Esophagus 2009
. Arrector pili
2. The part of a myofibril situated between two consecutive Z-bands is called:
a. Sarcosome
b. Sarcomere
c. Sarcolemma
d. Sarcoplasm
3. Origin of myocytes:
Ectoderm
Endoderm Mesoderm Neural crest 2905p
4. Contraction of muscle is dependent on release of:
a.
Ca+
a.
K+
c.
Mg*
d.
Cl~

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* Overview of lymphatic system
* Classification
*Lymph, lymph vessels and lymphocytes
*Lymphoid organs—structure of lymph node, spleen, thymus and palatine tonsil

Q Capsule with subcapsular sinus
QO Well demarcated cortex and medulla
Q Cortex with aggregation of lymphatic follicles
Q Medulla containing cords of lymphocytes with medullary sinuses.

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Q Presence of white pulpand red pulp
Q White pulp with eccentric arteriole
Q Cords of Billroth
Q Numerous trabeculae with arteriole.
Presence of tonsillar crypts
Q Lined by stratified squamous nonkeratinized epithelium
Q Subepithelial aggregation of lymphatic follicles
= Mucous glands (if present).

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Q Capsule with trabeculae
0 Presence of incomplete lobules
Q Lobules with cortex and medulla
Q Hassall's corpuscles.
Mf

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* The lymphoid system includes lymphatic vessels and lymphoid tissue.
* When circulating blood reaches the capillaries, part of its fluid content
passes into the surrounding tissues as tissue fluid. Most of this fluid reenters the capillaries at their venous ends. Some of it is, however, returned to the circulation through a separate system of lymphatic vessels (usually called lymphatics).
* The fluid passing through the lymphatic vessels is called lymph.
* The smallest lymphatic (or lymph) vessels are lymphatic capillaries that join together to form larger lymphatic vessels.
* The largest lymphatic vessel in the body is the thoracic duct. It drains lymph from the greater part of the body. The thoracic duct ends by joining the left subclavian vein at its junction with the internal jugular vein. On the right side, there is the right lymphatic duct that has a similar termination.
* The lymphatic system plays an important role in providing immunity.
i

Lymphoid tissue constitutes group of cells and organ like lymphocytes, thymus, bone marrow, spleen, etc. which are involved in protection of body surface and internal environment by combating noxious substances.
* Primary lymphoid organs: Here production and maturation of Tand Blymphocytes takes place. They are thymus and bone marrow.
* Secondary lymphoid organs: Here the lymphocytes come in contact with the antigens, and then particular group of lymphocytes proliferate. They are lymphnode, spleen, mucosa-associated lymphoid tissue (MALT) such as Peyer's patch, vermiform appendix, and tonsil.
i

Lymphoid tissue may be broadly classified as:
* Diffuse lymphoid tissue
* Dense lymphoid tissue.

Diffuse lymphoid tissue consists of diffusely arranged lymphocytes and plasma cells in the mucosa of large intestine, trachea, bronchi, and urinary tract.

It consists of an aggregation of lymphocytes arranged in the form of nodules. These nodules are found either as discrete encapsulated organs or in close association to the lining epithelium of the gut. Dense lymphoid tissue can therefore be divided as:
* Discrete lymphoid organs: These include thymus, lymph nodes, spleen, and tonsils.
* Mucosa-associated lymphoid tissue: Small numbers of lymphocytes may be present almost anywhere in the body, but significant aggregations are seen in relation to the mucosa of the respiratory, alimentary, and urogenital tracts. These aggregations are referred to as MALT. In the respiratory system the aggregations are relatively small and are present in the walls of the trachea and large bronchi called bronchus-associated lymphoid tissue (BALT). MALT in the alimentary system also called gut-associated lymphoid tissue (GALT) and includes Peyer's patches of ileum, adenoids (located in the roof of pharynx), lingual tonsils in posterior one-third of tongue, palatine tonsils, and lymphoid nodules in vermiform appendix.

* Lymph is a transudate from blood and contains the same proteins as in plasma, but in smaller amounts and different proportions.
* Suspended in lymph, there are cells that are chiefly lymphocytes.
* Most of these lymphocytes are added to lymph as it passes through lymph nodes, but some are derived from tissues drained by the nodes.
* Large molecules of fat (chylomicrons) that are absorbed from the intestines enter lymph vessels. After a fatty meal, these fat globules may be so numerous that lymph becomes milky (and is then called chyle) (Fig. 8.1).

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* Lymph capillaries (or lymphatic capillaries) begin blindly in tissues where they form a network.
* The structure of lymph capillaries is basically similar to that of blood capillaries but has greater permeability.
* There is an inner lining of endothelium. The basal lamina is absent or poorly developed.
* Pericytes or connective tissue are not present around the capillary.
* Much larger molecules can pass through the walls of lymph capillaries. These include colloidal material, fat droplets, and particulate matter, such as bacteria.
* It is believed that these substances pass into lymph capillaries through gaps between endothelial cells lining the capillary, or by pinocytosis.
* Lymph capillaries are present in most tissues of the body. They are absent in avascular tissues (e.g. the cornea, hair, nails), in the splenic pulp, and in the bone marrow.
Fig. 8.1: Circulation of T-lymphocytes (schematic representation).

* The structure of the thoracic duct and of other larger lymph vessels is similar to that of veins.
* A tunica intima, media, and adventitia can be distinguished. Elastic fibers are prominent and can be seen in all the three layers.
* The media, and also the adventitia, contain some smooth muscle. In most vessels the smooth muscle is arranged circularly, but in the thoracic duct, the muscle is predominantly longitudinal.
* Numerous valves, similar to those in veins, are present in small as well as large lymphatic vessels. They are more numerous than in veins. The valves often give lymph vessels a beaded appearance.

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* Lymphocytes are an essential part of the immune system of the body that is responsible for defense against invasion by bacteria and other organisms.
* In the embryo, lymphocytes are derived from mesenchymal cells present in the wall of the yolk sac, in the liver, and in the spleen. These stem cells later migrate to bone marrow. Lymphocytes formed from these stem cells (in bone marrow) enter the blood.
* Lymphocytes which mature in the thymus are called T-lymphocytes ("T" from thymus). These T-lymphocytes, that have been "processed" in the thymus, re-enter the circulation to reach lymphoid tissue in lymph nodes, spleen, tonsils, and intestines. There are four main types of T lymphocytes —CD4+ helper cells, CD8+ cytotoxic cells, memory T cells and natural killer T cells.
* Lymphocytes of a second group arising from stem cells in bone marrow enter the bloodstream and directly go to lymphoid tissues (other than the thymus). Such lymphocytes are called B-lymphocytes ("B" from bursa of Fabricus, a diverticulum of the cloaca in birds: in birds, B-lymphocytes are formed here). B-lymphocytes are seen in lymphatic nodules. The germinal centers are formed by actively dividing B-lymphocytes. While the dark rims of lymphatic nodules are formed by dense aggregations of resting B-lymphocytes. Like T-lymphocytes, B-lymphocytes also circulate between lymphoid tissues and the bloodstream (Fig. 8.2).
Fig. 8.2: Circulation of B-lymphocytes (schematic representation).

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* Lymphocytes help to destroy microbes by producing substances called antibodies. These are protein molecules that have the ability to recognize a "foreign" protein (i.e. a protein not normally present in the individual). The foreign protein is usually referred to as an antigen.
* Antigen can be neutralized only by a specific antibody. This function of antibody production is done by B-lymphocytes. When stimulated by the presence of antigen, the cells enlarge and get converted to plasma cells. The plasma cells produce antibodies. Antibodies are also called immunoglobulins (Igs). Igs are of five main types, viz. IgG, IgM, IgA, IgE, and IgD. B-lymphocytes defend the body through blood-borne antibodies thus provides humoral immunity.
T-lymphocytes have surface receptors that recognize specific antigens (there being many varieties of T-lymphocytes; each type recognizing a specific antigen). When exposed to a suitable stimulus, the T-lymphocytes multiply and form large cells that can destroy abnormal cells by direct contact, or by producing cytotoxic substances called cytokines or *, ate '=
lymphokines. T-lymphocytes are responsible for cell-mediated immune responses (cellular immunity).
i

* Lymph nodes are small masses of lymphatic tissue along the course of lymphatic vessels.
* Lymph from any part of the body (with some exceptions) passes through one or more lymph nodes before entering the bloodstream. Lymphocytes are added to lymph in these nodes.
Lymph nodes act as filters removing bacteria and other particulate matter from lymph. Each group of lymph nodes has a specific area of drainage. *, te
* Each lymph node consists of a connective tissue framework, and of numerous lymphocytes and other cells, that fill the interstices of the network. The entire node is bean shaped, the concavity constituting a hilum through which blood vessels enter and leave the node. Several lymph vessels enter the node on its convex aspect. Usually, a single lymph vessel leaves the node through its hilum (Fig. 8.3). he

- * When a section through a lymph node is examined (at low magnification), it is seen that the node has an outer zone that contains densely packed lymphocytes, and therefore stains darkly: this part is the cortex. The cortex does not extend into the hilum.
Plate 8.1: Lymph node
Lymph node: (A) As seen in drawing; (B) Photomicrograph.

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'A thin capsule surrounds the lymph node and sends in trabeculae
'Just beneath the capsule a clear space is seen. This is the subcapsular sinus
'A lymph node has an outer cortex and an inner medulla
'The cortex is packed with lymphocytes. A number of rounded lymphatic follicles (or nodules) are present. Each nodule has a pale staining germinal center surrounded by a zone of densely packed lymphocytes
'Within the medulla the lymphocytes are arranged in the form of anastomosing cords. Several blood vessels can be seen in the medulla.

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Fig. 8.3: Some features of the structure of a lymph node (schematic representation).
* Surrounded by the cortex, there is a lighter staining zone in which lymphocytes are fewer, called medulla (Plate 8.1).
* A lymph node is surrounded by a capsule. The capsule consists mainly of collagen fibers. Some elastic fibers and some smooth muscle may be present.
* Just beneath the capsule is the subcapsular sinus (Fig. 8.3).
* A number of septa (or trabeculae) extend into the node from the capsule and divide the node into lobules.
* The hilum is occupied by a mass of dense fibrous tissue.
* A delicate network of reticular fibers occupies the remaining spaces forming a fibrous/reticular framework within the node. Associated with the network, there are reticular cells, macrophages, dendritic cells, and follicular dendritic cells.

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* Within the cortex, there are several rounded areas that are called lymphatic follicles or lymphatic nodules within the reticular framework.
* Lymphatic nodules which are aggregations of small lymphocytes are called primary nodules, and those with germinal center are called secondary nodules.
* Germinal center is the central lightly stained region of the lymphatic nodules. They mainly contain immature proliferating lymphocytes (lymphoblasts). Germinal center indicates the response of the nodules to the antigen.
* Small lymphocytes encircle this germinal center and form a ring called as mantle zone/corona.
*Tt is mainly in the superficial part of the cortex where the lymphatic nodules are seen so it is otherwise called as nodular cortex/superficial cortex.
* Region between the nodular cortex and the medulla is called deep/paracortex. It is mainly populated by the major portion of Tlymphocytes of lymph node; hence, it is also called as thymus-dependent cortex.

* Within the medulla, the lymphocytes are arranged in the form of branching and anastomosing cords called medullary cords. The cords are separated by medullary sinuses.
* Contained within the reticular framework, the medullary cords contain mainly B-lymphocytes, macrophages, dendritic cells, and plasma cells.

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* The entire lymph node is pervaded by a network of reticular fibers. Most of the spaces of this network are packed with lymphocytes. At some places, however, these spaces contain relatively few cells and form channels through which lymph circulates.
* These channels known as sinuses are lined by endothelium, but their walls allow free movement of lymphocytes and macrophages into and out of the channels.
* Afferent lymphatics reaching the convex outer surface of the node enter an extensive subcapsular sinus (Fig. 8.3). From this sinus a number of radial cortical sinuses run through the cortex toward the medulla.
* Reaching the medulla the sinuses join to form larger medullary sinuses. In tum the medullary sinuses join to form (usually) one, or more than one, efferent lymph vessel through which lymph leaves the node.
* Lymph passing through the system of sinuses comes into intimate contact with macrophages present in the node. Bacteria and other particulate matter are removed from lymph by these cells. Lymphocytes freely enter or leave the node through these channels.

* Arteries enter the lymph node at the hilum. They pass through the medulla to reach the cortex where they end in arterioles and capillaries. These arterioles and capillaries are arranged as loops that drain into venules.
* Postcapillary venules in lymph nodes are unusual in that they are lined by cuboidal endothelium (they are, therefore, called high endothelial venules).
* This "high" endothelium readily allows the passage of lymphocytes between the bloodstream and the surrounding tissue.
* These endothelial cells bear receptors that are recognized by circulating lymphocytes. Contact with these receptors facilitates passage of lymphocytes through the vessel wall.

* Lymph nodes are the centers of lymphocyte production. Both Blymphocytes and T-lymphocytes are produced here by multiplication of pre-existing lymphocytes. These lymphocytes pass into lymph and thus reach the bloodstream.
* Bacteria and other particulate matter are removed from lymph through phagocytosis by macrophages. Antigens thus carried into these cells are "presented" to lymphocytes stimulating their proliferation. In this way, lymph nodes play an important role in the immune response to antigens.
* Plasma cells (representing fully mature B-lymphocytes) produce antibodies against invading antigens, while T-lymphocytes attack cells that are "foreign" to the host body.
i

* The spleen is the largest lymphoid organ of the body.
*Tt is a blood-forming organ in fetal life and blood-destroying organ in postnatal life [graveyard of red blood cells (RBCs)].
* Since it is in the bloodstream, it filters the blood from bloodborne antigens and microorganisms.

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* Except at the hilum, the surface of the spleen is covered by a layer of peritoneum (referred to as the serous coat). Deep to the serous layer, the organ is covered completely by a capsule (Plate 8.2).
* Trabeculae arising from the capsule extend into the substance of the spleen and form network.
* The capsule and trabeculae are made up of fibrous tissue in which elastic fibers are abundant.
* The spaces between the trabeculae are pervaded by a network of reticular fibers, embedded in an amorphous matrix. ate
* Fibroblasts (reticular cells) and macrophages are also present in relation to the reticulum. The interstices of the reticulum are pervaded by lymphocytes, blood vessels and blood cells, and by macrophages.

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Knowledge of vasculature is essential to understand the histology of spleen.
* On reaching the hilum of the spleen, the splenic artery divides into about five branches that enter the organ independently.
* Each branch divides and subdivides as it travels through the trabecular network.
* Arterioles arising from this network leave the trabeculae to pass into the intertrabecular spaces. For some distance, each arteriole is surrounded by a dense sheath of lymphocytes. These lymphocytes constitute the white pulp of the spleen.
* The arteriole then divides into a number of straight vessels that are called penicilli.
* Penicillar arterioles either open into the red pulp (open circulation) or they open into the splenic sinusoids (closed circulation) (Fig. 8.4).
* Veins from these sinusoids and the red pulp end in the trabecular veins.
* The sinusoids of the spleen are lined by modified endothelium. The cells are elongated and are banana-shaped called as stave cells. With the electron microscopy a system of ultramicroscopic fibrils is seen to be present in their cytoplasm. The fibrils may help to alter the shape of the endothelial cells thus opening or closing gaps between adjoining cells. he
* The spleen acts as a filter for worn-out RBCs. Normal erythrocytes can change shape and pass easily through narrow passages in penicilli and ellipsoids. However, cells that are aged are unable to change shape and are trapped in the spleen where they are destroyed by macrophages.

The interior of the spleen shows round white areas (white pulp) surrounded by red matrix (red pulp).

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* The white pulp is made up of aggregations of lymphocytes that surround a small artery or arterioles.
* In hematoxylin and eosin (H&E) staining, white pulp appears basophilic because of dense heterochromatin of the lymphocytes.
eee Spleen
'arteriole occupies an eccentric position in the nodule.
Note: Observe that this organ is full of lymphocytes. In the drawing also see the cords of densely packed lymphocytes around arteriole.
Fig. 8.4: The splenic circulation (schematic representation).
* Within the white pulp, the branch of splenic artery is called central artery (though eccentric in position). The lymphocyte aggregation around the central artery constitutes the periarterial lymphatic sheath (PALS).
* Localized expansions of PALS are called as nodules. The splenic nodules which have quite large germinal enters in reaction to antigen exposures are called as malpighian bodies. More than one arteriole may be present in relation to one germinal center.
* The functional significance of the white pulp is similar to that of cortical tissue of lymph nodes. Lymphatic nodules of the white pulp are the aggregations of B-lymphocytes. The germinal centers are the areas where B-lymphocytes are dividing. Lymphocytes of the PALS are chiefly Tlymphocytes.

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* Red pulp is a modified lymphoid tissue infiltrated with all the cells of circulating blood.
*Tt is like a sponge permeated by spaces (splenic sinusoids) lined by
reticular cells.
* The intervals between the spaces are filled by B-lymphocytes, Tlymphocytes, macrophages, and blood cells.
* Endothelium lining the sinusoids has processes, which makes contact with the adjacent cells.
* These cells appear to be arranged as cords (splenic cords of Billroth). The cords form a network.
* The zone of red pulp immediately surrounding white pulp is the marginal zone. This zone has a rich network of sinusoids. Numerous antigenpresenting cells (APCs) are found close to the sinusoids.
* This region seems to be specialized for bringing antigens confined to circulating blood (e.g. some bacteria) into contact with lymphocytes in the spleen so that an appropriate immune response can be started against the antigens (Such contact does not take place in lymph nodes. Antigens reach lymph nodes from tissues, through lymph). Surgical removal of the spleen (splenectomy) reduces the ability of the body to deal with bloodborne infections.
* The central artery like in white pulp divides and redivides to form straight arterioles then capillaries. Some capillaries are surrounded by macrophages and are called as sheathed capillaries.
* Blood from these capillaries directly drain into reticular meshwork of splenic cords and then into splenic sinuses. This type of circulation is called as open circulation, which effectively exposes the blood to macrophages before they reach the splenic sinuses.
* In other species, blood from the sheathed capillaries directly opens into splenic sinuses, which is called as closed circulation.
* Blood from the spaces of red pulp is collected by wide sinusoids that drain into trabecular veins.

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* Like other lymphoid tissues, the spleen is a center where both Blymphocytes and T-lymphocytes multiply and play an important role in immune responses. As stated above, the spleen is the only site where an immune response can be started against antigens present in circulating blood (but not present in tissues).
* The spleen contains the largest aggregations of macrophages of the mononuclear phagocyte system. In the spleen the main function of these cells is the destruction of red blood corpuscles that have completed their useful life. This is facilitated by the intimate contact of blood with the macrophages because of the presence of an open circulation. Macrophages also destroy worn-out leukocytes, and bacteria.
* In fetal life the spleen is a center for the production of all blood cells. In later life, only lymphocytes are produced here.
* The spleen is often regarded as a store of blood that can be thrown into the circulation when required. This function is much less important in man than in some other species.
§

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* The thymus is a lymphoepithelial bilobed organ which is usually not seen in dissection hall cadavers (because of atrophy in old people, and because of rapid autolysis after death).
* The organ is also not accessible for clinical examination (as it lies deep to the manubrium sterni).
* At birth the thymus weighs 10-15 g. The weight increases to 30-40 g at puberty. Subsequently, much of the organ is replaced by fat. However, the thymus is believed to produce T-lymphocytes throughout life.
* The thymus has rich blood supply. It does not receive any lymph vessels but gives off efferent vessels.
* All other lymphoid organs originate exclusively from mesenchyme but thymus has dual embryonic origin.

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* The thymus consists of right and left lobes that are joined together by fibrous tissue.
* Each lobe has a connective tissue capsule. Connective tissue septa passing inward from the capsule incompletely subdivide the lobe into a large number of lobules (Plate 8.3).
* The capsule and trabeculae contain blood vessels, efferent lymphatic vessels and nerves.
Each lobule is about 2 mm in diameter. It has an outer cortex and an inner medulla. The medulla of adjoining lobules is continuous.
* Thymic cortex is darkly stained because of the densely packed developing T-lymphocytes called thymocytes.
* Both the cortex and medulla contain cells of two distinct lineages as described below.

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* Embryologically these cells are derived from endoderm lining the third pharyngeal pouch. The cells form sheets that cover the internal surface of the capsule, the surfaces of the septa, and the surfaces of blood vessels.
* The epitheliocytes lying deeper in the lobule develop processes that join similar processes and form a reticulum. It may be noted that this reticulum is cellular, unlike the reticulum formed by reticular fibers in lymph nodes and spleen. Epithelial cells of the thymus are not phagocytic.
* These cells share features of reticular by providing structural reticulum and epithelial cells by having intercellular junctions and intermediate fibers.
* On the basis of structural differences, six types of epitheliocytes are recognized.
* Type 1 epitheliocytes line the inner aspect of the capsule, the septa, and blood vessels and form blood-thymus barrier that prevents antigens (present in blood and connective tissue) from reaching lymphocytes present in the thymus. Hence, blood—thymus barrier is formed by:
= Tight junction between endothelial cells
™ Basal lamina of endothelial cells
" Perivascular connective tissue
™ Basal lamina of epitheliocytes
"Type 1 epitheliocytes.
* Type 2 epitheliocytes are star shaped with prominent nucleus. Type 2 and type 3 cells are present in the outer and inner parts of the cortex, respectively. They compartmentalize the cortex for developing lymphocytes. Cortical epitheliocytes are also described as thymic nurse cells. They destroy lymphocytes that react against self-antigens.
Type 4 cells lie in the deepest parts of the cortex, and also in the medulla. They form barrier between cortex and medulla.

* Type 5 cells have darkly stained nucleus and provide structural framework in the medulla.
* Type 6 cells form the characteristic feature of the thymic medulla— Hassall's corpuscles. These corpuscles are pink stained small rounded structures with concentrically arranged epitheliocytes intermingled with keratohyalin and lipid droplets. The central core is formed by degenerating epitheliocytes and macrophages. The functional significance of the corpuscles of Hassall is not understood. Recent studies have shown that they produce substances which help in lymphocyte differentiation and maturation.

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'The thymus is made up of lymphoid tissue arranged in the form of distinct lobules. The presence of this lobulation enables easy distinction of the thymus from all other lymphoid organs
'The lobules are partially separated from each other by connective tissue septae
'In each lobule an outer darkly stained cortex (in which lymphocytes are densely packed);
and an inner lightly stained medulla (in which the cells are diffuse) are present
"Whereas the cortex is confined to one lobule, the medulla is continuous from one lobule to another
'The medulla contains pink staining rounded masses called the corpuscles of Hassall.

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*In the cortex of each lobule of the thymus, the reticulum formed by epithelial cells is densely packed with lymphocytes. Stem cells formed in bone marrow travel to the thymus.
* In the thymus, they lie in the superficial part of the cortex and divide repeatedly to form small lymphocytes. Lymphatic nodules are not present in the normal thymus.
* The medulla of each lobule also contains lymphocytes, but these are less densely packed than in the cortex. As a result, the epithelial reticulum is more obvious in the medulla than in the cortex.
* As thymocytes divide, they pass deeper into the cortex, and into the medulla.
* Ultimately, they leave the thymus by passing into blood vessels and lymphatics.

* Apart from epithelial cells and lymphocytes, the thymus contains a fair number of macrophages (belonging to the mononuclear phagocyte system).
* They are placed subjacent to the capsule, at the corticomedullary junction, and in the medulla.
* The subcapsular macrophages are highly phagocytic. Deeper lying macrophages are dendritic cells. Their significance is considered below.

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* Stem cells (from bone marrow) that reach the superficial part of the cortex divide repeatedly to form smaller lymphocytes. Total 90% of lymphocytes formed in the thymus are destroyed within 3-4 days. The remaining lymphocytes, which react only against proteins foreign to the body, are thrown into the circulation as circulating, immunologically competent Tlymphocytes. They lodge themselves in secondary lymph organs, such as lymph nodes, spleen, etc., where they multiply to form further Tlymphocytes of their own type when exposed to the appropriate antigen. Hence thymus is regarded as a primary lymphoid organ (along with bone marrow), where schooling of lymphocytes takes place to recognize self and foreign antigen.
* Within the thymus, lymphocytes are not allowed to come into contact with foreign antigens, because of the presence of the blood—thymic barrier. It has also been said that because of this, thymocytes do not develop into large lymphocytes or into plasma cells and do not form lymphatic nodules.
* The proliferation of T-lymphocytes and their conversion into cells capable of reacting to antigens probably takes place under the influence of hormones produced by epithelial cells of the thymus. Hormones produced by the thymus may also influence lymphopoiesis in peripheral lymphoid organs. This influence appears to be especially important in early life, as lymphoid tissues do not develop normally if the thymus is removed. Thymectomy has much less influence after puberty as the lymphoid tissues have fully developed by then.
* A number of hormones produced by the thymus have now been identified as follows. They are produced by epitheliocytes.
" Thymulin enhances the function of various types of T-cells, especially tl of suppressor cells.
" Thymopoietin stimulates the production of cytotoxic T-cells. The combir action of thymulin and thymopoietin allows precise balance of the activ of cytotoxic and suppressor cells.
"Thymosin alpha-1 stimulates lymphocyte production, and also i production of antibodies.
" Thymosin beta-4 is produced by mononuclear phagocytes.
"Thymic humoral factor controls the multiplication of helper ¢ suppressor T-cells.

Apart from their actions on lymphocytes, hormones (or other substances) produced in the thymus probably influence the adenohypophysis and the ovaries. In turn the activity of the thymus is influenced by hormones produced by the adenohypophysis, by the adrenal cortex, and by sex hormones.
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* Aggregations of lymphocytes seen in relation to the mucosa of the respiratory, alimentary, and urogenital tracts are referred to as MALT.
* The total volume of MALT is more or less equal to that of the lymphoid tissue present in lymph nodes and spleen.
* Mucosa-associated aggregations of lymphoid tissue have some features in common, which are as follows:
"These aggregations are in the form of one or more lymphatic follic (nodules) having a structure similar to nodules of lymph nodes. Germi: centers may be present.
" Diffuse lymphoid tissue (termed the parafollicular zone) is present in | intervals between the nodules. The significance of the nodules and of ° diffuse aggregations of lymphocytes is the same as already described in | case of lymph nodes.
"The nodules consist predominantly of B-lymphocytes, while the diffi areas contain T-lymphocytes.
"These masses of lymphoid tissue are present in very close relationship the lining epithelium of the mucosa in the region concerned and lie in | substantia propria. Larger aggregations extend into the submuco Individual lymphocytes may infiltrate the epithelium and may pass throu it into the lumen.
"The aggregations are not surrounded by a capsule, nor do they he connective tissue septa. A supporting network of reticular fibers is presen
"As a rule, these masses of lymphoid tissue do not receive afferent lym vessels and have no lymph sinuses. They do not, therefore, serve as filt of lymph. However, they are the centers of lymphocyte producti: Lymphocytes produced here pass into lymph nodes of the region throu efferent lymphatic vessels. Some lymphocytes pass through the overlyi epithelium into the lumen.

In the respiratory system the aggregations are relatively small and are present in the walls of the trachea and large bronchi. The term BALT is applied to these aggregations.

This is also called GALT. In the alimentary system, examples of aggregations of lymphoid tissue are tonsils, Peyer's patches, and lymphoid nodules in vermiform appendix.
Bi

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* Near the junction of the oral cavity with the pharynx, there are a number of collections of lymphoid tissues that are referred to as tonsils.
* The largest of these are the right and left palatine tonsils, present on either side of the oropharyngeal isthmus. (In common usage the word—tonsils— refers to the palatine tonsils). Another midline collection of lymphoid tissue, the pharyngeal tonsil, is present on the posterior wall of the pharynx.
* Smaller collections are present on the dorsum of the posterior part of the tongue (lingual tonsils), and around the pharyngeal openings of the auditory tubes (tubal tonsils).
* They together form Waldeyer's ring.
Fig. 8.5: Coronal section of palatine tonsil (schematic representation).

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* Each palatine tonsil consists of diffuse lymphoid tissue in which lymphatic nodules are present.
* The lymphoid tissue is covered by stratified squamous nonkeratinized epithelium, continuous with that of the mouth and pharynx (Fig. 8.5 and Plate 8.4).
* This epithelium extends into the substance of the tonsil in the form of several tonsillar crypts.
* The lumen of a crypt usually contains some lymphocytes that have traveled into it through the epithelium.
* Desquamated epithelial cells and bacteria are also frequently present in the lumen of the crypt.
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* Distributed widely through the body, there are a series of cells that share the property of being able to phagocytose unwanted matter, including bacteria and dead cells.
* These cells also play an important role in defense mechanisms, and in carrying out this function, they act in close collaboration with lymphocytes.
* With the discovery of a close relationship between these cells and mononuclear leukocytes of blood, the term mononuclear phagocyte system (or monocyte phagocyte system) has come into common usage.

The various cells that are usually included in the mononuclear phagocyte system are as follows.
* Monocytes of blood and their precursors in bone marrow (monoblasts, promonocytes).
* Macrophage cells (histiocytes) of connective tissue.
Plate 8.4: Palatine tonsil

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"Palatine tonsil is an aggregation of lymphoid tissue that is readily recognized by the fact that it is covered by a stratified squamous epithelium
"At places the epithelium dips into the tonsil in the form of deep crypts
"Deep to the epithelium there is diffuse lymphoid tissue in which typical lymphatic nodules can be seen.
* Littoral cells (von Kupffer cells) interspersed among cells lining the sinusoids of the liver; and cells in the walls of sinusoids in the spleen and lymph nodes.
Microglial cells of the central nervous system.
* Macrophages in pleura, peritoneum, alveoli of lungs, spleen, and in synovial joints.
* Free macrophages present in pleural, peritoneal, and synovial fluids.
* Dendritic cells of the epidermis and similar highly branched cells in lymph nodes, spleen, and thymus. These are now grouped as APCs (see below).

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All cells of the mononuclear phagocyte system have some features in common.
* They are large (15-25 wm) in diameter. The nucleus is euchromatic. Granular and agranular endoplasmic reticulum, Golgi complex, and mitochondria are present, as are endocytic vesicles and lysosomes. The cells have irregular surfaces that bear filopodia (irregular microvilli). Most of the cells are more or less oval in shape, but the dendritic cells are highly branched.
* Macrophages often form aggregations. In relation to the peritoneum and pleura, such aggregations are seen as milky spots; and in the spleen, they form ellipsoids around small arteries. When they come in contact with large particles, macrophages may fuse to form multinuclear giant cells (foreign body giant cells).
*In the presence of organisms, such as tubercle bacilli, the cells may transform to epithelioid cells. (These are involved in T-cell-mediated immune responses).
From a functional point of view, mononuclear phagocytes are divided into two main types.
1. With the exception of dendritic cells, all the cell types are classified as hi phagocytic cells.
2. The dendritic mononuclear phagocytes (now called dendritic APCs' capable of phagocytosis, but their main role is to initiate immune reactio lymphocytes present in lymph nodes, spleen, and thymus (in the mé discussed above). It has been postulated that all dendritic cells are prim located in the skin. From here they pick up antigens and migrate to lymf tissues where they stimulate lymphocytes against these antigens. The
therefore referred to as APCs. Most APCs are derived from monocytes some are derived from other sources.

As already stated, the cells have the ability to phagocytose particulate matter, dead cells, and organisms. In the lungs, alveolar macrophages engulf inhaled particles and are seen as dust cells. In the spleen and liver, macrophages destroy aged and damaged erythrocytes.

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* All mononuclear phagocytes bear antigens on their surface [class II major histocompatibility complex (MHC) antigens]. Antigens phagocytosed by macrophages are partially digested by lysosomes. Some remnants of these pass to the cell surface where they form complexes with the MHC antigens. This complex has the ability to stimulate T-lymphocytes.
* Certain T-lymphocytes produce macrophage-activating factors (including interleukin-2) that influence the activity of macrophages. Macrophages when thus stimulated synthesize and secrete cytokines that stimulate the proliferation and maturation of further lymphocytes.
* When foreign substances (including organisms) enter the body, antibodies are produced against them (by lymphocytes). These antibodies adhere to the organisms. Macrophages bear receptors (on their surface) that are able to recognize these antibodies. In this way, macrophages are able to selectively destroy such matter by phagocytosis, or by the release of lysosomal enzymes.
From the above, it will be seen that lymphocytes and macrophages constitute an integrated immune system for defense of the body.
* When suitably stimulated mononuclear phagocytes secrete a tumornecrosing factor which is able to kill some neoplastic cells.
* Macrophages influence the growth and differentiation of tissues by producing several growth factors and differentiation factors.
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APPLIED HISTOLOGY

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* Lymphangitis is inflammation of the lymphatics. Lymphangitis may be acute or chronic.
*, ote '= Acute lymphangitis occurs in the course of many bacterial infections. The most common organisms are b-hemolytic streptococci and staphylococci. When this happens in vessels of the skin, the vessels are seen as red lines that are painful.
* te ' Chronic lymphangitis occurs due to persistent and recurrent acute lymphangitis or from chronic infections, such as tuberculosis, syphilis, and actinomycosis.
* Lymphedema is the swelling of soft tissues due to localized increase in the quantity of lymph. It may be primary (idiopathic) or secondary (obstructive). Secondary lymphedema is more common and may be due to lymphatic invasion by malignant tumor, Postirradiation fibrosis, Parasitic infestations, e.g. in filariasis of lymphatics producing elephantiasis. Rupture of dilated large lymphatics may result in the escape of milky chyle into the peritoneum (chyloperitoneum), into the pleural cavity (chylothorax), into pericardial cavity (chylopericardium), and into the urinary tract (chyluria). 3
Infection in any part of the body can lead to enlargement and inflammation of the lymph nodes draining the area. The inflammation of lymph nodes is called lymphadenitis. A ate *
Carcinoma (cancer) usually spreads from its primary site either by growth of malignant cells along lymph vessels, or by "loose" cancer cells passing through lymph to nodes into which the area drains. This leads to enlargement of the lymph nodes of the region. Examination of lymph nodes gives valuable information about the stage of the cancer. In surgical excision of cancer, lymph nodes draining the region are usually removed. A ate *

Splenomegaly: Enlargement of spleen is termed splenomegaly. It occurs in a wide variety of disorders which increase the cellularity and vascularity of the organ. Many of the causes are exaggerated forms of normal splenic function.

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* Infections: Malaria, leishmaniasis.
* Disorders of immunoregulation: Rheumatoid arthritis, systemic lupus erythematosus.
* Altered splenic blood flow: Cirrhosis of liver, portal vein obstruction, splenic vein obstruction.
* Lymphohematogenous malignancies: Hodgkin's disease, non-Hodgkin's lymphomas.
* Diseases with abnormal erythrocytes: Thalassemias, spherocytosis, sickle cell disease.
* Storage diseases: Gaucher's disease, Niemann-Pick's disease.
* Miscellaneous: Amyloidosis, primary and metastatic splenic tumors.

* Enlargement of the thymus is often associated with a disease called myasthenia gravis. In this condition, there is great weakness of skeletal muscle.
* Myasthenia gravis is due to disturbance of the immune system. There are some proteins to which acetyl choline released at motor end plates gets attached. In myasthenia gravis, antibodies are produced against these proteins rendering them ineffective. Myasthenia gravis is, thus, an example of a condition in which the immune system begins to react against one of the body's own proteins. Such conditions are referred to as autoimmune diseases.
* In many such cases the thymus is enlarged, and removal of the thymus may result in considerable improvement in some cases.

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Tonsillitis is the infection of palatine tonsils. The palatine tonsils are often infected. It is a common cause of sore throat. Frequent infections can lead to considerable enlargement of the tonsils, especially in children. Such enlarged tonsils may become a focus of infection, and their surgical removal (tonsillectomy) may then become necessary.
1. Blood—thymus barrier is formed by:
a. Type 3 epitheliocytes
b. Type 2 epitheliocytes
c. Type 1 epitheliocytes
d. Type 4 epitheliocytes
2. Modified endothelial cells lining the splenic sinusoids are:
a. Pericytes
b. Stave cells
c. Thymocytes
d. Reticulocytes

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a. Thymus
b. Spleen
c. Tonsil
d. Lymph node
4. Following are true about white pulp, except:
a. Contains eccentric arteriole
b. Also known as Malpighian corpuscles
c. Formed by aggregation of lymphocytes
d. They are found in thymus

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. Type 3 epitheliocytes
. Type 6 epitheliocytes . Type 1 epitheliocytes a2009
. Type 4 epitheliocytes
6. Schooling of lymphocytes occurs at:
a. Thymus
b. Lymph node
c. Spleen
d. Tonsil
7. Cords of Billroth are present in:
. Thymus Lymph node . Spleen . Tonsil 2009

Activation of lymphocytes Antibody production Differentiation of plasma cells Degeneration of lymphocytes 2050p

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*Components of nervous system with classification
* Structure of a neuron and its types
* Neuroglia—types, structure, and function
*Myelination
* Structure of peripheral nerve/optic nerve
* Ganglia—autonomic and spinal

Q Each nerve fiber is covered by endoneurium Q Each bundle of nerve fibers is covered by perineurium Q Each nerve is covered by epineurium Q Cut section of axon with myelin sheath and Schwann cell nucleus., Optic nerve = Q Covered by meninges—dura mater, arachnoid mater, and pia mater Q Central retinal artery and vein are seen Q Pialsepta dividesthenerve into bundles.. Q Each nerve fiber is covered by endoneurium Q Each bundle of nerve fibers is covered by perineurium Q Each nerve is covered by epineurium Q Cut section of axon with myelin sheath and Schwann cell nucleus., Sensory/dorsal nerve root ganglia = QO Unipolar neurons with centrally placed nucleus Q Neurons are presented in a group between the bundles of nerve fibers Prominent satellite cells are seen.. Q Each nerve fiber is covered by endoneurium Q Each bundle of nerve fibers is covered by perineurium Q Each nerve is covered by epineurium Q Cut section of axon with myelin sheath and Schwann cell nucleus., Autonomic ganglia = QQ Multipolar neurons with eccentrically placed nucleus Neurons are scattered between the nerve fibers.
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The nervous system is a specialized type of tissue that has property of ability to conduct impulses rapidly from one part of the body to another. Nerve cells can convert information obtained from the environment into codes that can be transmitted along their axons. By such coding, the same neuron can transmit different kinds of information.
The total number of neurons in the human brain is estimated at more than 1 trillion. Nervous tissue is composed of neurons and neuroglia.
* Neurons are the specialized cells that constitute the structural and functional unit of nervous system.
* Neuroglia: Supporting connective tissue cells present in nervous system.
It has been taught that lymph vessels are not present, but the view has recently been challenged.
The nervous system may be divided into the central nervous system (CNS), made up of the brain and spinal cord, the peripheral nervous system (PNS), consisting of the peripheral nerves and the ganglia associated with them, and the autonomic nervous system, consisting of the sympathetic and the parasympathetic nervous systems (Flowchart 9.1).
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Neurons are responsible for the receptive, integrative, and motor functions of the nervous system. Neurons vary considerably in size, shape, and other features. However, most of them have some major features in common, and these are described below.

* Cell body/soma/perikaryon
* Processes/neurites—single axon and multiple dendrites

A neuron consists of a cell body that gives off a variable number of processes (Fig. 9.1).
Flowchart 9.1: Anatomical classification of nervous system.
Fig. 9.1: Some parts of a neuron (schematic representation).
Cytoplasm: Like a typical cell, it consists of a mass of cytoplasm surrounded by a cell membrane. The cytoplasm contains a large central nucleus with a prominent nucleolus, highly developed rough endoplasmic reticulum, numerous mitochondria, lysosomes, and a Golgi complex (Fig. 9.2).

* The cytoplasm shows the presence of a granular material that stains intensely with basic dyes; this material is the Nissl substance (also called Nissl bodies or granules) (Fig. 9.2).
* When examined by electron microscope (EM), these bodies are seen to be composed of rough surfaced endoplasmic reticulum (Fig. 9.3). The presence of abundant granular endoplasmic reticulum is an indication of the high level of protein synthesis in neurons. The proteins are needed for maintenance and repair, and for the production of neurotransmitters and enzymes.
Fig. 9.2: Neuron stained to show Niss! substance. Note that the NissI substance extends into the dendrites but not into the axon.

- * Another distinctive feature of neurons is the presence of a network of

- * These neurofibrils are seen when prepared by silver impregnation.
Fig. 9.3: Some features of the structure of a neuron as seen by electron microscope (schematic representation).
Fig. 9.4: Neuron stained to show neurofibrils. Note that the fibrils extend into both axons and dendrites.
* When seen under EM, it shows microfilaments, microtubules and neurofilaments (The centrioles present in neurons may be concerned with the production and maintenance of microtubules).

- * The axon hillock and the initial segment of the axon are of special functional significance. This is the region where action potentials are generated (spike generation), resulting in conduction along the axon. The initial segment is unmyelinated. It often receives axo-axonal synapses that are inhibitory. The plasma membrane here is rich in voltage-sensitive channels.
Some neurons contain pigment granules, e.g. neuromelanin in neurons of the substantia nigra. Aging neurons contain a pigment lipofuscin made up of residual bodies derived from lysosomes.

The processes arising from the cell body of a neuron are called neurites. These are of two kinds, number of short branching processes called dendrites and one longer process called an axon (Table 9.1).

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The axon may extend for a considerable distance away from the cell body.
* The longest axons may be as much as 1 meter long.
* Each axon has a uniform diameter and is devoid of Nissl substance. The Nissl-free zone extends for a short distance into the cell body: this part of the cell body is called the axon hillock. The part of the axon just beyond the axon hillock is called the initial segment.
* The cytoplasm within the axon is called axoplasm, and its cell membrane is called axolemma.
* The axoplasm contains all the cell organelles of neurons cell body except ribosomes. Hence, proteins synthesized in the cell body are continuously transported toward the axon terminals by a process called axoplasmic transport.
* Tn an axon the impulse travels away from the cell body.
* Axons constitute what are commonly called nerve fibers. The bundles of nerve fibers found in CNS are called as nerve tracts, while the bundles of

An axon (or its branches) can terminate in two ways. Within the CNS, it always terminates by coming in intimate relationship with another neuron, the junction between the two neurons being called a synapse. Outside the CNS, the axon may end in relation to an effector organ (e.g. muscle or gland), or may end by synapsing with neurons in a peripheral ganglion.
Axon is a single, long, and thin process of a nerve cell, which terminates away from the nerve cell body, Dendrites = Dendrites are multiple, short, thick, and tapering processes of the nerve cell which terminate near the nerve cell body. Axon rarely branches at the right angle collaterals) but ends by dividing into many fine processes called axon terminals, Dendrites = (axonDendrites are highly branched. Their branching pattern forms a dendritic tree. It has uniform diameter and smooth surface, Dendrites = The thickness of dendrite reduces as it divides repeatedly Its surface is not smooth, but it bears many small spine-like projections for making synaptic contacts with the axons of other nerve cells. It is free of Nissl granules, Dendrites = Nissl granules are present in dendrites. The nerve impulses travel away from the cell body, Dendrites = The nerve impulses travel toward the cell body

* They are short processes of neuron and terminate near the cell body
* They are irregular in thickness, and Nissl granules extend into them
* They bear numerous small spines that are of variable shape
* In a dendrite, the nerve impulse travels toward the cell body (Table 9.1).

The cytoplasm of neurons is in constant motion. Movements of various materials occur through axons. This axoplasmic flow takes place both away from and toward the cell body. The flow away from the cell body is greater. Some materials travel slowly (0.1-2 mm a day) constituting a slow transport. In contrast, other materials (mainly in the form of vesicles) travel 100-400 mm a day constituting a rapid transport. The proteins present in dendrites and axons are not identical. This fact is used for the immunocytochemical identification of dendrites in tissue sections. A protein microtubuleassociated protein-2 (MAP-2) is present exclusively in dendrites and helps in their identification.
B

In addition to neurons, the nervous system contains several types of supporting cells called neuroglia (Flowchart 9.2).

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* Astrocytes, oligodendrocytes, and microglia found in the parenchyma of the brain and spinal cord and ependymal cells lining the ventricular system.
* Schwann cells (lemmocytes) or peripheral glia forming myelin sheaths around axons of peripheral nerves. It is important to note that both neurilemma and myelin sheaths are the components of Schwann cells.
* Capsular cells or satellite cells or capsular gliocytes that surround neurons in peripheral ganglia.
* All neuroglial cells are much smaller in size than neurons. However, they are far more numerous. It is interesting to note that the number of glial cells in the brain and spinal cord is 10-50 times as much as that of neurons. Neurons and neuroglia are separated by a very narrow extracellular space.
* In ordinary histological preparations, only the nuclei of neuroglial cells are seen. Their processes can be demonstrated by special techniques.

* These are small star-shaped cells that give off a number of processes (Fig. 9.5). The processes are often flattened into leaf-like laminae that may partly surround neurons and separate them from other neurons. The processes frequently end in expansions in relation to blood vessels or in relation to the surface of the brain. Small swellings called gliosomes are present on the processes of astrocytes. These swellings are rich in mitochondria.
* The processes of astrocytes are united to those of other astrocytes through gap junctions. Astrocytes communicate with one another through calcium channels. Such communication plays a role in the regulation of synaptic activity and in the metabolism of neurotransmitters and neuromodulators.
* Astrocytes are of two types: (1) Fibrous and (2) Protoplasmic.
a. Fibrous astrocytes are seen mainly in white matter. Their processes

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Fig. 9.5: Astrocytes and microglial cells (schematic representation).
b. Protoplasmic astrocytes are seen mainly in gray matter. Their proce
thicker than those of fibrous astrocytes and are symmetrical.
c. Intermediate forms between fibrous and protoplasmic astrocytes |
present. Protoplasmic extensions of astrocytes surround the nodes of I

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* Biochemical support of endothelial cells
* They help in the formation of blood-brain barrier
* Provision of nutrients to the nervous tissue
* Maintenance of extracellular ion balance
* Help in repair process by forming cellular scar tissue (gliosis)
* They serve as insulators and prevent neuronal impulses from spreading in unwanted directions
Fig. 9.6: Oligodendrocyte giving off a process that forms a segment of the myelin sheath of an axon (schematic representation).
- * They are believed to help in maintaining a suitable metabolic environment for the neurons. They can absorb neurotransmitters from synapses, thus, terminating their action.

* These cells are rounded or pear-shaped bodies with relatively few processes (oligo—scanty) (Fig. 9.6).
* These cells provide myelin sheaths to nerve fibers that lie within the brain
and spinal cord. Their relationship to nerve fibers is basically similar to that of Schwann cells to peripheral nerve fibers.

Oligodendrocytes provide myelin sheaths to nerve fibers within the CNS for fast conduction of nerve impulses.

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* Ependymal cells are low columnar epithelial cells lining the ventricles of the brain and central canal of the spinal cord.
* The specialized ependymal cells in choroid plexuses (choroidal epithelial cells) secrete cerebrospinal fluid (CSF).
* In some regions, these cells are ciliated that helps in the movement of CSF.
* Modifications of the ependymal cells in the ventricle help in the formation of choroid plexus and CSF.
* The ependymal cells lining the floor of the fourth ventricle have long basal processes and are termed "tanycytes"'.

* Tt secretes CSF and helps in maintaining the chemical composition of the CSF.
* Ependymal cells are concerned in the exchange of material between the brain and the CSF at the brain— CSF barrier. The blood in the capillaries of the choroid plexus is filtered through choroid epithelial cells at the blood— CSF barrier to secrete CSF.

* These are the smallest neuroglial cells (Fig. 9.5). The cell body is flattened with short processes.
* They are the members of the mononuclear phagocyte system and mesodermal in origin.
* They are more numerous in gray matter than in white matter.

* They act as phagocytes in clearing debris and damaged structures and become active after damage to nervous tissue by trauma or disease.
* They also protect nervous system from viruses, microorganisms, and tumor formation.

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* Schwann cells are located in PNS and envelop axons.
* They are flattened cells with flattened nucleus.
* They form either myelinated or unmyelinated coverings over the axons.
* They form myelin sheath in peripheral nervous system.
* Axon may be myelinated or unmyelinated. Axons having a myelin sheath are called myelinated axons.
* They are derived from neural crest.

* Satellite glial cells are the principal glial cells found in the PNS, specifically in sensory, sympathetic, and parasympathetic ganglia.
* They help to regulate and stabilize the environment around ganglion cell bodies.
* They supply nutrients to the surrounding neurons and also act as protective, cushioning cells (Fig. 9.2).

* It is an insulating sheath surrounding the axons outside the axolemma.
* In PNS, myelin sheath is formed by Schwann cells and in CNS by an oligodendrocyte.
* Many Schwann cells surround the axons to form a myelin sheath in PNS, and single oligodendrocyte provides myelin sheath for many axons in CNS.

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- * An axon lying near a Schwann cell invaginates into the cytoplasm of the Schwann cell. In this process, the axon comes to be suspended by a fold
cell membrane of the Schwann cell: this fold is called the mesaxon (Figs. 9.7A and B). In some situations, the mesaxon becomes greatly elongated and comes to be spirally wound around the axon, which is thus surrounded by several layers of cell membrane.
Figs. 9.7A and B: Myelin sheath: (A) Transverse section; (B) Longitudinal section (schematic representation).
* Lipids are deposited between the adjacent layers of the membrane. These layers of the mesaxon, along with the lipids, form the myelin sheath.
* Outside the myelin sheath, a thin layer of Schwann cell cytoplasm persists to form an additional sheath that is called the neurilemma (neurilemmal sheath or Schwann cell sheath).
* The presence of a myelin sheath increases the velocity of conduction. It also reduces the energy expended in the process of conduction.
* An axon is related to a large number of Schwann cells over its length (Fig. 9.8). Each Schwann cell provides the myelin sheath for a short segment of the axon. At the junction of any two such segments, there is a short gap in the myelin sheath. These gaps are called the nodes of Ranvier and it is the junction between the myelin sheath of two different Schwann cells.
* The nodes of Ranvier have great physiological importance. When an impulse travels down a nerve fiber, it jumps from one node to the next. This is called saltatory conduction (in unmyelinated neurons, the impulse travels along the axolemma. Such conduction is much slower than saltatory conduction and consumes more energy).
* The segment of myelin sheath between two nodes of Ranvier is called internode, and a single Schwann cell will myelinate an internode.
* Myelin contains protein, lipids, and water. The main lipids present include cholesterol, phospholipids, and glycosphingolipids. Other lipids are present in smaller amounts.

Fig. 9.8: Stages in the formation of the myelin sheath by a Schwann cell (schematic representation).

* The presence of a myelin sheath increases the velocity of conduction.
* Tt reduces the energy expended in the process of conduction.
* It is responsible for the color of the white matter of the brain and spinal cord and provides insulation to the neurons.

There are the axons that are devoid of myelin sheaths. These unmyelinated axons invaginate into the cytoplasm of Schwann cells, but the mesaxon does not spiral around them (Figs. 9.9 and 9.10), and several such axons may invaginate into the cytoplasm of a single Schwann cell (Table 9.2).
Fig. 9.9: One Schwann cell forms a short segment of the myelin sheath (schematic representation).
Fig. 9.10: Relationship of unmyelinated axons to Schwann cells (schematic representation).
Table 9.2: Differences between myelinated and unmyelinated nerve fibers., 1 = Table 9.2: Differences between myelinated and unmyelinated nerve fibers.. Myelinated nerves, 1 = Unmyelinated nerves. Axons are of large diameter, 1 = Axons are of small diameter. Axons surrounded by concentric layers of Schwann cell plasma membrane, 1 = Axons surrounded by cytoplasm of Schwann cells. Single axon is surrounded by many Schwann cells, 1 = Single or group of axons are invaginated by single Schwann cell. Nerve impulse jumps from one node to other node called as saltatory conduction, 1 = Nerve impulse travels uniformly along the axolemma. Conduction is fast and consumes less energy, 1 = Conduction is slow and consumes more energy

* Unipolar/Pseudounipolar: These neurons have single process. After a very short course, this process divides into two. One of the divisions represents the axon; the other is functionally a dendrite, but its structure is indistinguishable from that of an axon, e.g. neurons in dorsal root ganglion (Fig. 9.11).
* Bipolar neurons: These neurons have only one axon and one dendrite, e.g. neurons in vestibular and spiral ganglia.
* Multipolar neurons: It is the most common type of neurons; the neuron gives off several processes, i.e. these neurons have one axon and many dendrites, e.g. motor neurons (Fig. 9.11).

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- * Sensory neuron: They carry impulses from receptor organ to the CNS.
Fig. 9.11: Unipolar, bipolar, and multipolar neurons (schematic representation).
Table 9.3: Morphological classification of neurons., 1 = Table 9.3: Morphological classification of neurons.. Morphology, 1 = Location and example. According to polarity :, 1 = According to polarity :. Unipolar/ pseudounipolar, 1 = Sensory neurons of dorasl root ganglia and cranial nerve ganglia. Bipolar, 1 = Bipolar cells of retina, and sensory ganglia of vestibulocochlear nerve. Multipolar, 1 = Motor neuron and interneuron. According to size of nerve fiber :, 1 = According to size of nerve fiber :. Golgi type I, 1 = Purkinje cells of cerebellum, anterior horn cells of spinal cord, and pyramidal cells of cerebral cortex. Golgi type II, 1 = Cerebral and cerebellar cortex
* Motor neuron: They transmit impulses from the CNS to the muscles and glands.
III. On the basis of length of axons:
* Golgi type I: These neurons have long axons and connect remote regions,
e.g. pyramidal cells of motor cortex in cerebrum.
* Golgi type II: These neurons have short axons which end near the cell body, e.g. stellate cells, cells of Martinotti of cerebral cortex and granule cells of cerebellar cortex.
i

Peripheral nerves are the bundles of nerve fibers (axons) outside the CNS. These may be myelinated or unmyelinated.
* Nerve fibers carry impulses from the spinal cord or brain to peripheral structures, such as muscle or gland, are called efferent or motor fibers. Efferent fibers are axons of neurons located in the gray matter of the spinal cord or of the brainstem.
* Nerve fibers carry impulses from peripheral organs to the brain or spinal cord are called afferent fibers. Afferent nerve fibers are processes of neurons that are located in ganglia.

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* Nerve fibers (myelinated or unmyelinated) are arranged in the form of bundles surrounded by connective tissue coverings.
* Each nerve fiber with its Schwann cell and basal lamina is surrounded by a layer of connective tissue called the endoneurium (Fig. 9.12 and Plate 9.1). The endoneurium contains type II collagen fibers, fibroblasts, Schwann cells, endothelial cells, and macrophages.
* Many nerve fibers together form bundles or fasciculi. Endoneurium holds adjoining nerve fibers together and facilitates their aggregation to form fasciculi. Each fasciculus is surrounded by a thicker layer of connective tissue called the perineurium. The perineurium is made up of layers of flattened cells separated by the layers of collagen fibers. Flattened cells are joined at their edges by tight junctions, this controls diffusion of substances in and out of axons (nerve-blood barrier).
* Many fasciculi are held together by a fairly dense layer of connective tissue that surrounds the entire nerve and is called the epineurium. It is made up of fibroblast, collagen fibers, and adipose tissue which give cushioning effect to the nerve.
* Nuclei seen in the section of axon is of Schwann cells and fibrocytes. The
Schwann cells myelinate and surrounds individual axons, or encloses unmyelinated axons.

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* Tt belongs to CNS. It is second cranial nerve—a special sensory nerve.
*Tt is an outgrowth of brain—hence covered by meninges: dura mater, arachnoid mater, and pia mater which are separated from each other by subdural and subarachnoid spaces.
* Tt is made up of bundles of myelinated axons of ganglion cells of retina.
* In the center of the intraorbital part of the optic nerve lie the central retinal vessels enclosed in their envelope.
* The myelin sheath of optic nerve is formed by oligodendrocytes as it belongs to CNS.

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*
ia
Each nerve fiber is covered by endoneurium
*
Each bundle of nerve fibers is covered by perineurium
Each nerve is covered by epineurium
*
Cut section of axon with myelin sheath and Schwann cell nucleus.
e
Covered by meninges—dura mater, arachnoid mater, and pia mater
* Central retinal artery and vein are seen
* Pial septa divides the nerve into bundles.
* The pia mater, attached directly to the optic nerve sends septa which divides the nerve into bundles/fascicles and guide the blood vessels to the nerve fibers.
* At the eyeball, the dura fuses with the sclera while the arachnoid and pia mater merge with the choroid. Connective tissue septa, which arise from the pia mater, separate the fiber bundles in the optic nerve.
* Since it is an extension of CNS, any changes in intracranial pressure is reflected in optic disc and nerve.

Synapses are the sites of junction between the neurons. Synapses can be broadly classified into:
*Chemical synapses: Synapses involving the release of neurotransmitters are referred to as chemical synapses.
* Electrical synapses: At some sites, one cell may excite another without the release of a transmitter. At such sites, adjacent cells have the direct channels of communication through which ions can pass from one cell to another altering their electrical status. Such synapses are called electrical synapses.

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Synapses may be of various types depending upon the parts of the neurons that come in contact.
* Axodendritic synapse: \t is the most common type of synapse. In this type, an axon terminal establishes contact with the dendrite of a receiving neuron to form a synapse (Fig. 9.13A).
* Axosomatic synapse: The axon terminal synapses with the cell body (Fig. 9.13B).
* Axoaxonal synapse: The axon terminal synapses with the axon of the receiving neuron. An axoaxonal synapse may be located either on the initial segment (of the receiving axon) or just proximal to an axon terminal (Fig. 9.13C).
* Dendroaxonic synapse: |n some parts of the brain (for example, the thalamus), we see some synapses in which the presynaptic element is a dendrite, instead of an axon, which synapses with the axon of the receiving neuron.
* Dendrodendritic synapse: Synapse between two dendrites.
* Somatosomatic synapse: The soma of a neuron may synapse with the soma of another neuron.
*Somatodendritic synapse: Synapse between a soma and a dendrite.

Aggregations of cell bodies of neurons, placed outside the CNS, are known as ganglia. Ganglia are of two main types: sensory and autonomic.

* Sensory ganglia are the collection of cell bodies of sensory neurons.
* Sensory ganglia are associated with the dorsal nerve roots of spinal nerves called dorsal nerve root ganglia and with 5th, 7th, 8th, 9th, and 10th cranial nerves.
Figs. 9.13A to C: Various types of chemical synapses. (A) Axodendritic synapse; (B) Axosomatic synapse; and (C) Axo-axonal synapse (schematic representation).
Ca Sensory ganglia

® Unipolar neurons with centrally placed nucleus
® Neurons are presented in a group between the group of nerve fibers
* Prominent satellite cells are seen.
* Neurons in these ganglia are of the unipolar type (except in the case of ganglia associated with the vestibulocochlear nerve in which they are

- * The peripheral process of each neuron forms an afferent (or sensory) fiber of a peripheral nerve. The central process enters the spinal cord or brainstem.

* The ganglion is covered the outside by a connective tissue capsule, and the entire ganglion is pervaded by fine connective tissue.
* The unipolar neurons with centrally placed nucleus are large and arranged in groups chiefly at the periphery of the ganglion (Plate 9.2). The groups of cells are separated by the groups of myelinated nerve fibers.
* The cell body of each neuron is surrounded by a layer of flattened capsular cells or satellite cells. Outside the satellite cells, there is a layer of delicate connective tissue (The satellite cells are continuous with the Schwann cells covering the processes arising from the neuron. The connective tissue covering each neuron is continuous with the endoneurium).

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* They are concermed with the nerve supply of smooth muscle or of glands.
* The pathway for this supply consists of two neurons: (1) preganglionic and (2) postganglionic.
* The cell bodies of preganglionic neurons are always located within the spinal cord or brainstem. Their axons leave the spinal cord or brainstem and terminate by synapsing with postganglionic neurons, the cell bodies of which are located in autonomic ganglia.
* Autonomic ganglia are, therefore, aggregations of the cell bodies of postganglionic neurons.
* Autonomic ganglia are subdivisible into two major types: (1) sympathetic and (2) parasympathetic.
* The neurons of autonomic ganglia are multipolar neurons, and they are smaller than those in sensory ganglia (Plate 9.3).

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- * The ganglion is covered outside by a thin capsule and permeated by
connective tissue (intramural ganglia have no capsule).
* The multipolar neurons with eccentrically placed nucleus are not arranged in definite groups as in sensory ganglia but are scattered throughout the ganglion. The nerve fibers are nonmyelinated and thinner. They are, therefore, much less conspicuous than in sensory ganglia.
* Satellite cells are present around the neurons of autonomic ganglia, but they are not so well defined.
* The Nissl substance of the neurons is much better defined in autonomic ganglia than in sensory ganglia.
* In sympathetic ganglia the neuronal cytoplasm synthesizes catecholamines; and in parasympathetic ganglia, it synthesizes acetylcholine (Table 9.4).
Table 9.4: Difference between sensory and autonomic ganglia., 1 = Table 9.4: Difference between sensory and autonomic ganglia.. Sensory ganglia, 1 = Autonomic ganglia. Cell bodies are large, 1 = Cell bodies are smaller. Unipolar type of neuron except in vestibulocochlearMultipolar nerve (bipolar), 1 = type of neuron. Neurons with centrally placed nucleus, 1 = Neurons with eccentrically placed nucleus. Cell bodies of the neurons are arranged in groups, 1 = Cell bodies of the neuron does not form definite groups. Myelinated nerve fibers are arranged in bundles, 1 = Unmyelinated nerve fibers are scattered. Prominent satellite cells, 1 = Satellite cells are less prominent. Well-defined connective tissue capsule, 1 = Poorly defined or absent connective tissue capsule
Bf

* The regeneration of neurons do not occur in CNS as myelination is by oligodendrocytes. Hence in multiple sclerosis, myelin formed from oligodendrocytes undergoes degeneration, while myelin from Schwann cells is spared.
* The epineurium contains fat that cushions nerve fibers. Loss of this fat in bedridden patients can lead to pressure on nerve fibers and paralysis.
* Blood vessels to a nerve travel through the connective tissue that surrounds it. Severe reduction in blood supply can lead to ischemic neuritis and pain.

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- * Precursors of neural cells can give rise to medulloblastomas. Once mature
neurons are formed, they lose the power of mitosis and do not give origin to tumors.
* Certain tumors called germinomas appear near the midline, mostly near the third ventricle. They arise from germ cells that also give rise to teratomas.
* Most tumors of the brain arise from neuroglial cells. Astrocytomas are most common. Oligodendromas are also frequent.
* Tumors can also arise from ependyma and from Schwann cells (Figs. 9.14A and B).
Plate 9.4: Autonomic ganglia

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*
Multipolar neurons with eccentrically placed nucleus
*
Neurons are scattered between the nerve fibers.
Figs. 9.14A and B: (A) Structure of a typical chemical synapse as seen under electron microscope; (B) Mechanism of synaptic transmission (Schematic representation).

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. Spinal ganglia have unipolar neurons
. Autonomic ganglia has centrally placed nucleus
. Autonomic ganglia have multipolar neurons . Spinal ganglia have continuous sheath of satellite cells a2009
2. All of the following statements regarding myelination are true, except:
Schwann cells are responsible for myelination in PNS Oligodendrocytes are responsible for myelination in CNS Initial segments are myelinated 2959
Myelination helps in saltatory conduction of impulses
3. The gap between two adjacent myelin segments along an axon is called the:
. Axolemma
. Axonal cleft . Choroid plexus Node of Ranvier oop
4. Schwann cell membrane wrapped many times around an individual axon is called:
. Axolemma
. Myelin sheath . Perikaryon . Node of Ranvier a2009
5. A group of nerve cell bodies located outside the CNS is called a:
a. Ganglion
b. Nucleus c. Node d. Myelin 6. White matter appears white because of the: a. Absence of blood vessels b. Absence of glial cells c. Presence of many collagen fibers d. Presence of many myelinated axons 7. A single Schwann cell forms myelin around one and only one axon, while a single oligodendroglial cell forms myelin around several separate axons., 1 = . 8. Endoneurium, perineurium, and epineurium are the connective tissues which ensheath axons, axon bundles, and peripheral nerves., 1 = . Which of the following cell types lines the central canal of the spinal cord and the system of the brain?, 1 = . 10. ventricular a. Astroglia, 1 = . 1., 1 = d c. 2. 3., 1 = d b. 4., 1 = a. 5. 6., 1 = d. , 1 = a. 7., 1 = . , 1 = a. , 1 = c. 8., 1 = . , 1 = d. 10., 1 = . 9., 1 = 

*Components of cardiovascular system
* Structure of blood vessels

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The tunica intima, media and adventitia can be made out, and are sharply demarcated, Medium-sized artery = The tunica intima, media, and adventitia can be made out and can be sharply demarcated. The tunica intima, media and adventitia can be made out, and are sharply demarcated, Large-sized vein = The vein has a thinner wall and a larger lumen than the artery. Media made up mainly of elastic fibers in the form of fenestrated concentric membranes, Medium-sized artery = Intima is well developed and internal elastic lamina is prominent. Media made up mainly of elastic fibers in the form of fenestrated concentric membranes, Large-sized vein = The tunica intima, media, and adventitia can be made out, but are not distinctly demarcated. The internal elastic lamina is not distinct., Medium-sized artery = Media predominantly is made up of smooth muscle fibers arranged concentrically.. The internal elastic lamina is not distinct., Large-sized vein = The media is thin and contains a much larger quantity of collagen fibers than arteries. The amount of elastic tissue is less. Adventitia is relatively thin with greater proportion of elastic fibers., Medium-sized artery = . Adventitia is relatively thin with greater proportion of elastic fibers., Large-sized vein = The adventitia is relatively thick and contains bundles of longitudinal muscle fibers.
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* The cardiovascular system consists of the heart and blood vessels.
* The blood vessels that take blood from the heart to various tissues are called arteries. The smallest arteries are called arterioles. Arterioles open into a network of capillaries that pervade the tissues. Exchanges of various substances between the blood and the tissues take place through the walls of capillaries. At some places, capillaries are replaced by slightly different vessels called sinusoids.
* Blood from capillaries (or from sinusoids) is collected by small venules that join to form veins.
* The veins return blood to the heart.
* Blood vessels deliver nutrients, oxygen, and hormones to the cells of the body and remove metabolic base products and carbon dioxide from them.

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Wall of arteries/veins is made of three layers. From within outward, they are as follows:
1. Tunica intima: It is the innermost layer, made of three components:
i. Endothelium—simple squamous epithelium lining the intima
ii. Basal lamina—basement membrane of the lining cells
iii. Subendothelial connective tissue—loose connective tissue bene endothelium.
Tunica intima is limited by a fenestrated layer of elastic tissue called internal elastic lamina.
2. Tunica media: This layer consists of concentrically arranged smooth m cells, elastic fibers, and reticular fibers. Their concentration depends o1 nature of the vessel, i.e. artery/vein. It is separated from tunica adventit fenestrated layer of elastic tissue external elastic lamina.
3. Tunica adventitia: It is the outer most layer of a vessel wall. It consis longitudinally arranged collagen and reticular fibers, which gradually mi: with the connective tissue of the surrounding structures. Thus it m anchors the blood vessel to the neighboring structures. In large vessels. layer contains network of blood vessels providing nutrition to them c
vasa vasorum and network of autonomic nerves controlling their h diameter, nervi vasorum.

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* They are monolayer of flattened polygonal cells, resting on basement membrane.
* Tt lines the inner surface of the heart and blood vessels.
* Many endothelial cells show invaginations of the cell membrane (on both internal and external surfaces). Sometimes the inner and _ outer invaginations meet to form channels passing right across the cell (seen typically in small arterioles). These features are seen in situations where vessels are highly permeable.
* Adjoining endothelial cells are linked by tight junctions, and also by gap junctions.
* Externally, they are supported by a basal lamina.

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Apart from providing a smooth internal lining to heart and blood vessels, endothelial cells perform a number of other functions as follows:
* Maintenance of selective permeability barrier between the blood and intestinal fluid. Hence, they allow simple diffusion of O,, CO,; active transport of glucose, amino acids, electrolytes, and receptor-mediated endocytosis of low-density lipoprotein (LDL), cholesterol.
* Luminal surface is coated with glycocalyx; it also secretes anticoagulants, and antithrombogenic agents, such as heparin, tissue plasminogen activator (TPA), etc. form nonthrombogenic barrier.
* Modulates blood flow and vascular resistance by secreting vasoconstrictors [endothelin, angiotensin-converting enzyme (ACE)] and vasodilators [nitric oxide (NO), prostacyclins].
* Secretes and stores Von Willebrand factor (VWF) as rod-like inclusions like Weibel—Palade bodies. vWF plays an important role in platelet adhesion during vessel wall injury.
* Regulates immune response.
* Synthesizes growth factors.
* Maintains extracellular matrix (ECM).
* Antibodies to VWF serve as marker for tumors arising from endothelium.

* It is thin but variable.
* It is also termed as lamina propria. In smallest vessels, they are replaced by pericytes.
*Tt contains fibrocollagenous ECM, few fibroblasts, and occasionally smooth muscle cells.
* Von Willebrand factor concentrates in this layer and participates in clotting whenever endothelium is injured.

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* They are spindle-shaped cells with elongated nuclei.
* Contraction of smooth muscle will cause reduction of vessel's lumen and hence raises the pressure on the proximal side.
* They synthesize and secrete elastin, collagen, and other components of ECM.
* Alters rigidity of vessel wall without causing constriction and thus affects distensibility of wall and propagates pulse.
* Mesenchymal cells are multifunctional in arteries, migrates and proliferates to replace damaged endothelium.
* They undergo fatty degeneration and form atheromatous plaques.
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* They are large conducting vessels.
"Tunica intima is limited by a layer of elastic tissue internal elastic lamii which is less prominent due to underlying tunica media.
* Outside the tunica intima lies the thickest middle layer tunica media.
* The tunica media consists predominantly of elastic tissue and few smooth muscle fibers. The elastic fibers form fenestrated concentric lamellae alternating with smooth muscle cells. The number of lamellae of elastic fibers is directly proportional with the diameter of the artery and age of the individual.
* Some collagen fibers are also present in this layer, but fibroblasts are absent.
* In hypertension, the size and number of elastic lamellae increases due to increased blood pressure (BP).
* Tunica media is limited by a membrane formed by elastic fibers—external elastic lamina.
* The outermost layer is called the tunica adventitia. This coat consists of connective tissue in which collagen fibers are prominent. This layer prevents undue stretching or distension of the artery (Plate 10.1).
* Examples—aorta, carotid artery, subclavian artery, axillary artery, and iliac artery.
* The innermost layer is called the tunica intima (tunica = coat). It consists of:
™ An endothelial lining.
"A thin layer of glycoprotein which lines the external aspect of | endothelium and is called the basal lamina.
" A delicate layer of subendothelial connective tissue— consisting contract smooth muscle cells which is responsible for forming ECM, collagen < elastic fibers of this layer.
Plate 10.1: Elastic artery
Elastic artery: (A) As seen in drawing; (B) Photomicrograph.

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Elastic artery is characterized by presence of:
"Tunica intima consisting of endothelium, subendothelial connective tissue, and internal elastic lamina
"The first layer of elastic fibers is called the internal elastic lamina. The internal elastic lamina is not distinct from the elastic fibers of media
"Well developed subendothelial layer in tunica intima
Thick tunica media with many elastic fibers and some smooth muscle fibers
"Tunica adventitia containing collagen fibers with several elastic fibers
"Vasa vasorum in the tunica adventitia (Not seen in this slide).

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* As the arterial tree divides and redivides, the size of the artery decreases. After the elastic artery, the next member in the hierarchy is muscular artery.
* The muscular arteries have more smooth muscle fibers in the media than elastic fibers.
* The transition from elastic to muscular arteries is not abrupt. There is a gradual reduction in elastic fibers and increase in smooth muscle content in the media from elastic artery to muscular artery.
* Muscular arteries can alter the size of the lumen because of the contraction or relaxation of smooth muscle in its wall; therefore, they regulate the amount of blood flowing into the regions supplied by them; hence, they are also called as distributing arteries.
* Tunica intima: The structure of tunica intima is like elastic artery with endothelium, lining the lumen (Table 10.1). Endothelial cells resting on basement membrane with underlying connective tissue. The internal elastic lamina in the muscular arteries is more prominent due to underlying smooth muscle fibers in the muscular media.
* Tunica media: It is made up mainly of smooth muscles and some elastic fibers in between. Longitudinally arranged muscle is present in the media of arteries that undergo repeated stretching or bending. Examples of such arteries are the coronary, carotid, axillary, and palmar arteries.
* Tunica adventitia: It mainly consists of collagen fibers, fibroblasts, and elastic fibers. In addition, they will have vasa vasorum and nervosa. The adventitia of muscular artery is relatively thicker than that of elastic artery (Plate 10.2).

Adventitia, Table 10.1: Differences between elastic artery and muscular artery..Elastic artery = It is relatively thin with greater proportion of elastic fibers. Adventitia, Table 10.1: Differences between elastic artery and muscular artery..Muscular artery = It consists of thin layer of fibroelastic tissue. Media, Table 10.1: Differences between elastic artery and muscular artery..Elastic artery = It is made up mainly of elastic tissue in the form of fenestrated concentric membranes. There may be as many as 50 layers of elastic membranes. Media, Table 10.1: Differences between elastic artery and muscular artery..Muscular artery = It is made up mainly of smooth muscles arranged in circular manner. Intima, Table 10.1: Differences between elastic artery and muscular artery..Elastic artery = It is made up of endothelium, subendothelial connective tissue, and internal elastic lamina the subendothelial. Intima, Table 10.1: Differences between elastic artery and muscular artery..Muscular artery = Intima is well developed, especially internal elastic
connective tissue contains more elastic fibers. The
lamina which stands out prominently
internal elastic lamina is not distinct.
§

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* When traced distally, muscular arteries progressively decrease in caliber till they have a diameter of about 100 pm.
* They become continuous with arterioles (Fig. 10.1). The larger or muscular arterioles are 100—50um in diameter.
* Arterioles less than 50 jm in diameter are called terminal arterioles.
* All the three layers, i.e. (1) tunica adventitia, (2) tunica media, and (3) tunica intima are thin as compared to arteries.
* Tn arterioles, the adventitia is made up of a thin network of collagen fibers.
* Arterioles are the main regulators of peripheral vascular resistance.
* Contraction and relaxation of the smooth muscles present in the walls of the arterioles can alter the peripheral vascular resistance (or BP) and the blood flow.
Muscular arterioles can be distinguished from true arteries:
* By their small diameter.
* They do not have an internal elastic lamina. They have a few layers of smooth muscle in their media.
Terminal arterioles can be distinguished from muscular arterioles as follows:
* They have a diameter less than 50 pm, the smallest terminal arterioles having a diameter as small as 12 pm.
* They have only a thin layer of muscle in their walls.
* They give off lateral branches (called meta-arterioles) to the capillary bed.
Fig. 10.1: Photomicrograph showing an arteriole and a venule.
ee PAE oe et (medium size) artery
Muscular (medium size) artery: (A) As seen in drawing; (B) Photomicrograph.

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'In muscular arteries, the tunica intima is made up of endothelium and internal elastic lamina, which is thrown into wavy folds due to contraction of smooth muscle in the media 'Tunica media is composed mainly of smooth muscle fibers arranged circularly 'Tunica adventitia contains collagen fibers and few elastic fibers.
The initial segment of each lateral branch is surrounded by a few smooth muscle cells. These muscle cells constitute the precapillary sphincter. This sphincter regulates the flow of blood to the capillaries.
* Terminal arterioles are continued into a capillary plexus that pervades the tissue supplied.
* Capillaries are the smallest blood vessels.
* The average diameter of a capillary is 8 pm. ate
* Exchanges (of oxygen, carbon dioxide, fluids, and various molecules) between blood and tissue take place through the walls of the capillary plexus (and through postcapillary venules).
* The arrangement of the capillary plexus and its density varies from tissue to tissue, the density being greatest in tissues having high metabolic activity.

* The wall of a capillary is lined by endothelial cells resting on the basal lamina.
* Overlying the basal lamina there is a delicate network of reticular fibers and cells.
* In some capillaries, specialized branching cells may be present called as pericytes.
* Pericyte or adventitial cells contain contractile filaments in the cytoplasm. They mainly provide support to the capillary wall and control the lumen size.

There are three types of capillaries:
1. Continuous
2. Discontinuous
3. Fenestrated.

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- *In this type, the ends of endothelial cells fuse and form continuous capillary wall.
|B
Figs. 10.2A and B: Structure of continuous capillary. (A) Circular section; (B) Longitudinal section (schematic representation).
* In continuous capillaries, exchange of material between blood and tissue take place through the cytoplasm of endothelial cells
* Continuous capillaries are seen in the skin, connective tissue, muscle, lungs, and brain.

* In this type, the capillary wall has fenestrations.
* The "fenestrations" are closed by a thin diaphragm (which may represent greatly thinned out cytoplasm of an endothelial cell, or only the basal lamina).
* Some fenestrations represent areas where endothelial cell cytoplasm has pores passing through the entire thickness of the cell.
* Fenestrated capillaries are seen in renal glomeruli, intestinal villi, endocrine glands, and pancreas.

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* They are organ specific, also called as sinusoidal capillaries (Figs. 10.4A and B).
* They are found in spleen, liver, and bone marrow.
* They show gaps between the neighboring endothelial cells.
* They are more irregular and of larger diameter.
* They are associated with specialized cells of the tissue.
B

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The basic structure of veins is similar to that of arteries. The tunica intima, media, and adventitia can be distinguished, especially in large veins. The structure of veins differs from that of arteries in the following aspects:
Figs. 10.3A and B: Structure of fenestrated capillary. (A) Circular section; (B) Longitudinal section (schematic representation).
Figs. 10.4A and B: Structure of sinusoid. (A) Circular section; (B) Longitudinal section (schematic representation).
* The wall of a vein is thinner than that of an artery having the same-sized lumen.
* The tunica media contains more collagen fibers than that of arteries. The amount of elastic fibers or of smooth muscle is much less.
* Because of the above mentioned differences, the wall of a vein is easily compressed. After death, veins are usually collapsed. In contrast, arteries retain their patency.
*In arteries the tunica media is usually thicker than the adventitia. In contrast the adventitia of veins is thicker than the media (especially in large veins). In some large veins (e.g. the inferior vena cava), the adventitia contains a considerable amount of elastic and muscle fibers that run in a predominantly longitudinal direction. These fibers facilitate elongation and shortening of the vena cava with respiration. This is also facilitated by collagen fibers in the adventitia which form a meshwork that spirals around the vessel.
* A clear distinction between the tunica intima, media, and adventitia cannot be made out in small veins as all these layers consist predominantly of fibrous tissue.
Muscle is conspicuous by its complete absence in venous spaces of erectile tissue, in veins of cancellous bone, dural venous sinuses, retinal veins, and placental veins (Plate 10.3).

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* Most veins contain valves that allow the flow of blood toward the heart, but prevent its regurgitation in the opposite direction.
* Typically, each valve is made up of two semilunar cusps. Each cusp is a fold of endothelium within which there is some connective tissue that is rich in elastic fibers.
* Valves are absent in very small veins, venae cavae, veins within the cranial cavity, and vertebral canal.
* Flow of blood through veins is assisted by the contractions of muscle in their walls. It is also assisted by the contraction of surrounding muscles, especially when the latter are enclosed in deep fascia.
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* The smallest veins, into which capillaries drain, are called venules (Fig. 10.1).
* They are 20-30 pm in diameter. Their walls consist of endothelium, basal lamina, and a thin adventitia consisting of longitudinally running collagen fibers.
* Flattened or branching cells called pericytes may be present outside the basal laminae of small venules (called postcapillary venules), while some muscle may be present in larger vessels (muscular venules).
* The walls of venules (especially those of postcapillary venules) have considerable permeability, and exchanges between blood and surrounding tissues can take place through them.
* In particular, venules are the sites at which lymphocytes and other cells may pass out of (or into) the bloodstream.
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- * The walls of small blood vessels receive adequate nutrition by diffusion from blood in their lumina.
The walls of largeand medium-sized vessels are supplied by small arteries called vasa vasorum (literally "vessels of vessels"; singular = vas vasis).
* Vasa vasorum supply the adventitia and the outer part of the media (since diffusion becomes difficult from lumen if wall thickness > 1mm). These layers of the vessel wall also contain many lymphatic vessels.
* Blood vessels have a fairly rich supply by autonomic nerves (sympathetic).
* The nerves (Nervi vasorum) are unmyelinated. Most of the nerves are vasomotor and supply smooth muscle. Their stimulation causes vasoconstriction in some arteries, and vasodilatation in others. Some myelinated sensory nerves are also present in the adventitia.
* Coronary arteries are the largest vaso vasorum in the body.
See Large-sized vein
Large-sized vein: (A) As seen in drawing; (B) Photomicrograph (low magnification).

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*The vein has a thinner wall and a larger lumen than the artery
*The tunica intima, media, and adventitia can be made out, but they are not sharply demarcated
"The media is thin and contains a much larger quantity of collagen fibers than arteries. The amount of elastic tissue or of muscle is much less
The adventitia is relatively thick and contains considerable amount of elastic and muscle fibers.
Note: The luminal surface appears as a dark line, with an occasional nucleus along it.
Arteriovenous Anastomoses
* In many parts of the body, small arteries and veins are connected by direct channels that constitute arteriovenous anastomoses. These channels may be straight or coiled.
* Their walls have a thick muscular coat that is richly supplied with sympathetic nerves.
* When the anastomoses are patent, blood is short circuited from the artery to the vein so that very little blood passes through the capillary bed.
* However, when the muscle in the wall of the anastomosing channel contracts, its lumen is occluded so that all blood now passes through the capillaries.
* Arteriovenous anastomoses are found in the skin of the nose, lips, fingertips, and external ear, mucous membrane of the alimentary canal, nose, and the erectile tissue of penis and clitoris.
* They are also seen in the tongue, thyroid, and sympathetic ganglia.
* In some regions, we see arteriovenous anastomoses of a special kind. The vessels taking part in these anastomoses are in the form of a rounded bunch covered by connective tissue. This structure is called a glomus (Fig. 10.5). Each glomus consists of an afferent artery, one or more coiled (S-shaped) connecting vessels, and an efferent vein.

Arteriovenous anastomoses in the skin help in regulating body temperature, by increasing blood flow through capillaries in warm weather and decreasing it in cold weather to prevent heat loss.
Fig. 10.5: An arteriovenous anastomosis (glomus) (Schematic representation).
B

The heart is a muscular organ that pumps blood throughout the blood vessels to various parts of the body by repeated rhythmic contractions.

There are three layers in the wall of the heart:
1. The innermost layer is called the endocardium. It corresponds to the ti intima of blood vessels. It consists of a layer of endothelium that rests thin layer of delicate connective tissue. Outside this, there is a th subendocardial layer of connective tissue.
2. The main thickness of the wall of the heart is formed by a thick lay cardiac muscle called myocardium.
3. The external surface of the myocardium is covered by the epicardiun visceral layer of serous pericardium). It consists of a layer of conne tissue that is covered, on the free surface, by a layer of flattened mesotl cells.

They are few and inefficient in the newborn. In old age, again, arteriovenous anastomoses of the skin decrease considerably in number. It is because of this reason, temperature regulation is not efficient in the newborn as well as in old persons.

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Arteriovenous anastomoses control blood flow through relatively large segments of the capillary bed. Much smaller segments can be individually controlled as follows:
* Capillaries arise as side branches of terminal arterioles. The initial segment of each such branch is surrounded by a few smooth muscle cells that constitute a precapillary sphincter (Fig. 10.6). Blood flow, through any part of the capillary bed, can be controlled by the precapillary sphincter.
In many situations, arterioles and venules are connected (apart from capillaries) by some channels that resemble capillaries but have a larger caliber. These channels run a relatively direct course between the arteriole and venule. Isolated smooth muscle fibers may be present on their walls. These are called thoroughfare channels (Fig. 10.6). + a
At times when most of the precapillary sphincters in the region are contracted (restricting flow through capillaries), blood is short circuited from arteriole to venule through the thoroughfare channels. A thoroughfare channel and the capillaries associated with it are sometimes referred to as a microcirculatory unit. *, ate '=
Bf
Fig. 10.6: Precapillary sphincters and thoroughfare channels (schematic representation).

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1. Atherosclerotic lesions develop primarily in the tunica intima of large e! arteries following endothelial injury, which leads to endothelial dysfun and the formation of atheromatous plaques, which is the main risk factc myocardial infarction, stroke, etc.
2. Chronic hypertension (BP >140/90 mm Hg) causes pressure overloa the cardiac muscle, leading to left ventricular hypertrophy. In small mus arteries and arterioles, lumen size is reduced because of both contractior hyperplasia of smooth muscles in the tunica media, thereby increasin; vascular resistance.
3. Varicose veins are permanently dilated and tortuous superficial veins o lower extremities, especially the long saphenous vein and its tributi About 10-12% of the general population develops varicose veins of | legs, with the peak incidence in fourth and fifth decades of life. Adult fer are affected more commonly than the males, especially during pregn This is attributed to venous stasis in the lower legs because of compre: on the iliac veins by pregnant uterus.
4. Angioma benign tumors derived from the cells of vessel
(blood/lymphatic vessel). Hemangioma benign tumor of small blood ve:
Types—capillary and cavernous hemangioma.
- 5. A glomus tumor is a rare neoplasm arising from the glomus body
mainly found under the nail, on the fingertip or in the foot.
1. Following are the layers of blood vessels, except:
. Tunica adventitia
. Tunica media . Tunica albuginea . Tunica intima a2009
2. Pericytes are seen in:
Elastic artery Muscular artery Large-sized vein 2959
Postcapillary venules
3. Vessels responsible for peripheral resistance are:
. Elastic artery
. Arterioles . Large-sized vein Venules oop
4. Deep vein thrombosis mainly involves:
Elastic artery Medium-sized vein Large-sized vein 2905p

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*Functions of skin
* Types of skin with location
* Layers of skin
* Glands related to skin
* Appendages of skin
* Blood and nerve supply of skin

Skin is made up of epidermis and dermis, Thick (non-hairy ) skin = Skin is made up of epidermis and dermis. Epidermis has five layers: stratum basale, granulosum (single layer), spinosum (thin), lucidum (thin or absent) and corneum (thin), Thick (non-hairy ) skin = Epidermis has five layers: stratum basale, granulosum (4 to 5 layers), spinosum (prominent), lucidum (thick) and corneum (very thick). Epidermis is stratified squamous keratinized epithelium Hair follicles, arrector pili, sebaceous and sweat glands are present in the dermis., Thick (non-hairy ) skin = Dermis is thinner and does not contain hairs, sebaceous glands, or apocrine sweat glands.
Bf
INTRODUCTION
The skin forms the external covering of the body. It is the largest organ constituting 15-20% of total body mass.
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*Tt provides covering for the underlying soft tissues: Protection against injury, bacterial invasion, and desiccation.
* Absorption of ultraviolet rays from sun and the synthesis of vitamin D: Melanin—a dark-colored light-sensitive pigment that is found in the skin.
* Thermoregulation.
* Excretion through sweat glands.
* Largest sensory organ: Pressure/touch, heat/cold, and pain through somatic sensory receptors.
* Immunity, ie. the role of the skin within the immune system by Langerhans cells of epidermis, phagocytic cells, and epidermal dendritic cells.
* Palm and sole have ridges and sulci and the pattern assumed by these ridges and sulci are known as dermatoglyphics. They are unique for each individual, and it is used for personal identification.
Mi

There are two types of skin (Table 11.1):
1. Thin or hairy skin: In this type of skin, epidermis is very thin. It con hair and is found in all other parts of body except palms and soles ( 11.1).
2. Thick or glabrous skin: In this type of skin, it has no hair and epidern very thick with a thick layer of stratum corneum. It is found in paln hands and soles of feet (Plate 11.2).
Table 11.1: Difference between thick and thin skin., 1 = Table 11.1: Difference between thick and thin skin.. Thin skin, 1 = Thick skin. Location : Entire body except palm and sole, 1 = Location : Where there is a lot of abrasion-fingertips, palm, and the sole. Epidermis is thin with only four layers (stratum lucidum layer is absent), 1 = Epidermis is thick with all five layers. Dermis is thicker, which makes thin skin easier to, 1 = Dermis is thinner and does not
suture, if it gets damaged. Contains hair follicle, sebaceous, and apocrine sweat glands, 1 = contain hairs, sebaceous glands, or apocrine sweat glands. Sweat glands are more, 1 = Sweat glands are less. Arrector pili muscle is present, 1 = Arrector pili muscle is absent

Thin skin or hairy skin: (A) As seen in drawing; (B) Photomicrograph (low magnification).

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Presence of thin epidermis made up of keratinized stratified squamous epithelium (stratum corneum is thin)
*Hair follicles, sebaceous glands and sweat glands are present in the dermis
"It is found in all others parts of body except palms and soles.
Thick skin: (A) As seen in drawing; (B) Photomicrograph (low magnification).
* Presence of thick epidermis made up of keratinized stratified squamous epithelium (stratum corneum is very thick)
* Hair follicles and sebaceous glands are absent in dermis
> . . Sweat glands are present in the dermis
"It is found in palms of hands and soles of feet.
Fig. 11.1: Thin skin (schematic representation).
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The skin consists of two layers.
* A superficial layer, the epidermis, is made up of stratified squamous epithelium.
* A deeper layer, the dermis, is made up of connective tissue (Fig. 11.1).
* The dermis rests on subcutaneous tissue (subcutis). This is sometimes described as a third layer of skin.
* Tn sections through the skin, the line of junction of the two layers is not straight but is markedly wavy because of the presence of numerous fingerlike projections of dermis upward into the epidermis. These projections are called dermal papillae. The downward projections of the epidermis (in the intervals between the dermal papillae) are sometimes called epidermal papillae (Fig. 11.2).
Fig. 11.2: Dermal and epidermal papillae (schematic representation).

The epidermis is derived from ectoderm and composed of stratified squamous keratinized epithelium (Figs. 11.3 and 11.4).

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It is made up of five layers from within outward:
1. Stratum basale
2. Stratum spinosum
3. Stratum granulosum
4. Stratum lucidum
5. Stratum corneum.
Fig. 11.3: Epidermal ridges (schematic representation).
Fig. 11.4: Section through skin showing the layers of epidermis (schematic representation).

* It is the deepest or basal layer of epidermis.
* It is made up of a single layer of cuboidal to columnar cells with baso cytoplasm and large nucleus that rests on a basal lamina.
* Tt contains stem cells that undergo mitosis and are responsible fc renewal of epidermal cells. Human epidermis is renewed about ever 30 days depending on the region and age. The basal layer is the called the germinal layer.

* Above the basal layer, there are several layers of polygonal keratinc that constitute the stratum spinosum (or malpighian layer).
*The cells of this layer are attached to one another by nur desmosomes. During routine preparation of tissue for sectioning the retract from each other except at the desmosomes. As a result the appear to have a number of "spines." Hence, this layer is called the st spinosum, and the keratinocytes of this layer are also called prickle (Fig. 11.5).
* Some mitoses may be seen in the deeper cells of the stratum spin hence is included with the basal cell layer as the germinative zone | epidermis.

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* Above the stratum spinosum, there are one to five layers of flattenec that are characterized by the presence of deeply staining granules ir cytoplasm called as keratohyalin granules.
* The granules in them consist of a protein called keratohyalin (precur keratin).
* The nuclei of cells in this layer are condensed and dark staining (pykn
"Stratum lucidum: Superficial to the stratum granulosum, there is | stratum lucidum (lucid = clear). This layer is so called because it appe homogeneous, the cell boundaries being extremely indistinct. Traces flattened nuclei are seen in some cells.
Fig. 11.5: Cells of the stratum spinosum showing typical spines (schematic representation).

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* It is the most superficial layer of the epidermis.
* It is acellular.
*It is made up of flattened scale-like elements (squames)/dead containing keratin filaments embedded in protein. The squames are together by a glue-like material which contains lipids and carbohyc The presence of lipid makes this layer highly resistant to permeati: water.
"The thickness of the stratum corneum is greatest where the skin is expos to maximal friction, e.g. on the palm and sole.
Note: The stratum basale and stratum granulosum are called as germinative zone, andstratum corneum, the stratum lucidum, and the stratum granulosum are collectively referred to as the zone of keratinization, or as the cornified zone. The stratum granulosum and the stratum lucidum are well formed only in thick nonhairy skin (e.g. on the palms). They are usually absent in thin hairy skin.

The epidermis consists of two types of cells: (1) keratinocytes and (2)
nonkeratinocytes (melanocytes, dendritic cell of Langerhans, and cells of Merkel).

* Keratinocytes are the predominant cell type of epidermis.
* They produce keratin.
* They originate from stem cells present in basal layer.
* After entering the stratum spinosum, some keratinocytes may undergo further mitoses. Such cells are referred to as intermediate stem cells. Thereafter, keratinocytes do not undergo further cell division.

* Melanocytes are derived from melanoblasts that arise from the neural crest.
* They produce melanin pigment that imparts coloration to skin.
* They may be present among the cells of the germinative zone, or at the junction of the epidermis and the dermis. Each melanocyte gives off many processes; each of which is applied to a cell of the germinative zone.
* Melanin granules formed in the melanocyte are transferred to surrounding nonmelanin-producing cells through these processes. Because of the presence of processes, melanocytes are also called dendritic cells (Fig. 11.6).

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* Langerhans cells are antigen-presenting cells located among the cells of the stratum spinosum.
* Represent 24% of cell population.
* Called dendritic cells because of the long processes. These cells are also found in oral mucosa, vagina, and thymus. These cells belong to the mononuclear phagocyte system.
* The dendritic cells of Langerhans originate in bone marrow and are a part of the mononuclear phagocytic system.
* They play an important role in protecting the skin against viral and other infections.
* Tt is believed that when the cells are stimulated, they take up antigens in
the skin and transport them to lymphoid tissues.
* Under the electron microscopy (EM), dendritic cells are seen to contain characteristic elongated vacuoles that have been given the name Langerhans bodies, or Birbeck bodies.
* They play a role in controlling the rate of cell division in the epidermis. They increase in number in chronic skin disorders, particularly those resulting from allergy.

- * The basal layer of the epidermis contains specialized sensory cells called the cells of Merkel which may serve as mechanoreceptor.
Fig. 11.6: Melanocyte showing dendritic processes (schematic representation).
* They are derived from neural crest cells.
* Sensory nerve endings are present in relation to these cells.

The dermis is made up of irregular collagenous dense connective tissue. It is derived from mesoderm and divided into two layers.
1. Papillary layer: The papillary layer forms the superficial layers of dermi: includes the dense connective tissue of the dermal papillae. These papilla best developed in the thick skin of the palms and soles. Each papilla con a capillary loop. Some papillae contain tactile corpuscles.
2. Reticular layer: The reticular layer of the dermis is the deep layer of de and consists mainly of thick bundles of type I collagen fibers. It also con considerable numbers of elastic fibers. Intervals between the fiber bundle usually occupied by adipose tissue. The dermis rests on the superficial f through which it is attached to deeper structures. It contains epiderr derived structures, such as sweat glands, hair follicles, and sebaceous gla
li

* Blood vessels to the skin are derived from a number of arterial plexuses. The deepest plexus is present over the deep fascia and one more plexus just below the dermis (rete cutaneum or reticular plexus), and a third plexus just below the level of the dermal papillae (rete subpapillare, or papillary plexus). Capillary loops arising from this plexus pass into each dermal papilla.
* Blood vessels do not penetrate into the epidermis. The epidermis derives nutrition entirely by diffusion from capillaries in the dermal papillae. Veins from the dermal papillae drain into a venous plexus lying on deep fascia.
* A special feature of the blood supply of the skin is the presence of numerous arteriovenous anastomoses that regulate blood flow through the capillary bed and thus help in maintaining body temperature.

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* The skin is richly supplied with sensory nerves, and dense networks of nerve fibers are seen in the superficial parts of the dermis.
* Sensory nerves end in relation to various types of specialized terminals,such as free nerve endings, Meissner's corpuscles, Pacinian corpuscles, and Ruffini's corpuscles.
* In contrast to blood vessels, some nerve fibers do penetrate into the deeper parts of the epidermis.
* Skin also receives autonomic nerves that supply smooth muscle in the walls of blood vessels, the arrectores pilorum muscles, and myoepithelial
cells present in relation to sweat glands. They also provide a secretomotor supply to sweat glands. In some regions (nipple, scrotum), nerve fibers innervate smooth muscle present in the dermis.

The appendages of the skin are the hair, nails, sebaceous glands, and sweat glands. The mammary glands may be regarded as highly specialized appendages of the skin.

* A hair may be regarded as a modified part of the stratum corneum of the skin.
* Hair is present on the skin covering almost the whole body. The sites where they are not present include the palms, the soles, the ventral surface and sides of the digits, and some parts of the male and female external genitalia.

* Each hair consists of a part (of variable length) that is seen on the surface of the body, and a part anchored in the thickness of the skin. The visible part is called the shaft, and the embedded part is called the root.
* The root has an expanded lower end called the bulb. The bulb is invaginated from below by part of the dermis that constitutes the hair papilla. The root of each hair is surrounded by a tubular sheath called the hair follicle (Fig. 11.7). The follicle is made up of several layers of cells that are derived from the layers of the skin.
* Hair roots are always attached to skin obliquely. As a result, the emerging hair is also oblique and easily lies flat on the skin surface.

It consists of three layers:
1. Cuticle: The surface of the hair is covered by a thin membrane calle cuticle that is formed by flattened cornified cells.
2. Cortex: It lies deep to the cuticle. The cortex is acellular and is made 1

- 3. Medulla: An outer cortex and an inner medulla can be made out in large but there is no medulla in thin hair. In thick hair the medulla consis cornified cells of irregular shape.
Fig. 11.7: Scheme to show some details of a hair follicle (schematic representation).
The cornified elements making up the hair contain melanin that is responsible for their color. Both in the medulla and in the cortex of a hair, minute air bubbles are present: they influence its color. The amount of air present in a hair increases with age and, along with the loss of pigment, is responsible for graying of hair.

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The hair follicle may be regarded as a part of the epidermis that has been invaginated into the dermis around the hair root. Its innermost layer that immediately surrounds the hair root is, therefore, continuous with the surface of the skin; while the outermost layer of the follicle is continuous with the dermis.
The wall of the follicle consists of three main layers. From within outward, it is as follows (Fig. 11.8):
1. The inner root sheath present only in the lower part of the follicle
2. The outer root sheath that is continuous with the stratum spinosum
3. A connective tissue sheath derived from the dermis.
Note: The inner and outer root sheaths are derived from epidermis.

* The innermost layer is called the cuticle. It lies against the cuticle of the hair and consists of flattened cornified cells.
* Next, there are one to three layers of flattened nucleated cells that constitute Huxley's layer, or the stratum epithelial granulosum. Cells of this layer contain large eosinophilic granules (trichohyaline granules).
* The outer layer (of the inner root sheath) is made up of a single layer of cubical cells with flattened nuclei. This is called Henle's layer, or the stratum epithelial pallidum.

* The outer root sheath is continuous with the stratum spinosum of the skin. At the lower end of the follicle, the cells of this layer become continuous with the hair bulb.
* The cells of the hair bulb also correspond to those of the stratum spinosum and constitute the germinative matrix. They give rise to cells of the inner root sheath.
* The outer root sheath is separated from the connective tissue sheath by a basal lamina that appears structureless and is, therefore, called the glassy membrane.
Fig. 11.8: Various layers to be seen in a hair follicle (schematic representation).

* The connective tissue sheath is made up of tissue continuous with that of the dermis.
* The tissue is highly vascular, and contains numerous nerve fibers that form a basket-like network round the lower end of the follicle.

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* These are bands of smooth muscle attached at one end to the dermis, just below the dermal papillae, and at the other end, to the connective tissue sheath of a hair follicle.
* The arrector pili muscles pass obliquely from the lower part of the hair follicle toward the junction of the epidermis and dermis.
* Tt lies on that side of the hair follicle that forms an obtuse angle with the skin surface. A sebaceous gland lies in the angle between the hair follicle and the arrector pili.
* Contraction of the muscle pulls the hair follicle vertical (from its original
oblique position) relative to the skin surface. Simultaneously the skin surface overlying the attachment of the muscle becomes depressed, while surrounding areas become raised and the skin takes on the appearance of "goose flesh". These reactions are seen during exposure to cold, or during emotional excitement, when the "hair stand on end".
* Upon the contraction of the arrector pili muscle the sebaceous gland is pressed upon, and its secretions are squeezed out into the hair follicle.
* The arrector pili muscles receive a sympathetic innervation.

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* Sebaceous glands are holocrine type of glands and present in dermis in close association with hair follicles.
* Each gland consists of a number of alveoli that are connected to a broad duct that opens into a hair follicle (Plates 11.1 and 11.3). Each alveolus is pear shaped. It consists of a solid mass of polyhedral cells and has hardly any lumen (Fig. 11.9).
* The outermost cells are small and rest on a basement membrane. The inner cells are larger, more rounded, and filled with lipid.
* The secretion of sebaceous glands is called sebum. Its oily nature helps to keep the skin and hair soft. It helps to prevent the dryness of the skin and also makes it resistant to moisture.
* At some places, sebaceous glands occur independently of hair follicles. Such glands open directly on the skin surface. They are found around the lips, and in relation to some parts of the male and female external genitalia.
* The tarsal (Meibomian) glands of the eyelid are modified sebaceous glands.
* Montgomery's tubercles present in the skin around the nipple (areola) are also sebaceous glands.
* Secretion by sebaceous glands is not under nervous control.

Sweat glands produce sweat or perspiration. They are present in the skin over most of the body. They are of two types:
- 1. Typical or merocrine sweat glands

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* Typical sweat glands are of the merocrine variety. They are most numerous in the palm and sole, the forehead and scalp, and the axillae.
* The sweat glands are simple, coiled tubular glands whose duct opens at the skin surface.
* The lower end of the tube is highly coiled on itself and forms the body or fundus or secretory part of the gland. Secretory part is present in the reticular layer of the dermis (Fig. 11.10).
Fig. 11.9: Sebaceous gland (schematic representation).
Plate 11.3: Hair follicle and sebaceous gland

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*
In figures small areas of skin at higher magnification are shown
* The parts of a sebaceous gland and hair follicle containing a hair root can be seen
*
Each sebaceous gland consists of a number of alveoli that open into a hair follicle
* Each alveolus is pear shaped. It consists mainly of a solid mass of polyhedral cells.
*In the secretory part is lined by cuboidal to low columnar cells. Sometimes, the epithelium may appear to be pseudostratified.
* The part of the tube connecting the secretory element to the skin surface is the duct. It runs upward through the dermis to reach the epidermis. Within the epidermis the duct follows a spiral course to reach the skin surface. The orifice is funnel shaped. On the palms, soles, and digits the openings of sweat glands lie in rows on epidermal ridges.
* In larger sweat glands flattened contractile, myoepithelial cells (Fig. 11.11) are present between the epithelial cells and their basal lamina.
* In the duct is lined by stratified cuboidal epithelium.
* Sweat glands (including the myoepithelial cells) are innervated by cholinergic nerves.
* Two types of cells are seen in the secretory part. Dark cells are pyramidal shape and contain secretory granules and clear cells without any granules. Dark cells secretion is mucoid, whereas clear cells secretion is watery.
Fig. 11.10: Parts of a typical sweat gland (schematic representation).

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* Atypical sweat glands are of the apocrine variety (apical parts of the secretory cells are shed off as part of their secretion).
* They are present in some parts of the body, such as the axilla, the areola and nipple, the perianal region, the glans penis, and some parts of the female external genitalia.
* Apocrine sweat glands are much larger compared to merocrine they are fully developed only after puberty.
* Unlike merocrine, the secretory part of these glands branch.
* Their ducts open not on the skin surface but into hair follicles.
* Secretory part is lined by epithelium of varying height: it may be squamous, cuboidal, or columnar.
* The secretions of apocrine sweat glands are viscous and contain proteins. They are odorless, but after bacterial decomposition, they give off body odors that vary from person to person.
* Innervated by an adrenergic nerve endings.
* Wax-producing ceruminous glands of the external acoustic meatus and ciliary glands of the eyelids are modified apocrine sweat glands.

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* Nails are present on fingers and toes.
* They provide a rigid support for the finger tips. This support increases the sensitivity of the finger tips and increases their efficiency in carrying out delicate movements.
* The nail represents a modified part of the zone of keratinization of the epidermis. It is usually regarded as a much thickened continuation of the stratum lucidum, but it is more like the stratum corneum in structure. The nail substance consists of several layers of dead, cornified, "cells" filled with keratin.
* Nail consists of body and root. The main part of a nail is called its body. The proximal part of the nail is implanted into a groove on the skin and is called the root (or radix).
* The tissue on which the nail rests is called the nail bed. The nail bed is highly vascular, and that is why, the nails look pink in color (Figs. 11.12 and 11.13).
Fig. 11.11: Sweat gland (schematic representation in high power view).
[

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*Basal cells are mitotically active, and it shows mitotic figures. Mitosis occurs during night, and histological specimens are procured during day so they are not seen in the slides.
*The color of skin is influenced by the amount of melanin present. It is also influenced by some other pigments present in the epidermis, and by pigments hemoglobin and oxyhemoglobin present in blood circulating through the skin. The epidermis is sufficiently translucent for the color of blood to show through, especially in light-skinned individuals. That is why the skin becomes pale in anemia, blue when oxygenation of blood is insufficient, and pink while blushing.
*The time elapsing between the formation of a keratinocyte in the basal layer of the epidermis and its shedding off from the surface of the epidermis is highly variable. It is influenced by many
factors including skin thickness, and the degree of friction on the surface. On the average, it is 40-50 days. In some situations, it is seen that flakes of keratin in the stratum corneum are arranged in regular columns (one stacked above the other). It is believed that localized areas in the basal layer of the epidermis contain groups of keratinocytes, all derived from a single stem cell. It is also believed that all the cells in the epidermis overlying this region are derived from the same stem cell. Such groups of cells, all derived from a single stem cell, and stacked in layers passing from the basal layer to the surface of the epidermis, constitute epidermal proliferation units. One dendritic cell (see below) is present in close association with each such unit.
- * Growth of nails: Nails undergo constant growth by proliferation of cells in the germinal matrix. Growth is faster in hot weather than in cold. Finger nails grow faster than toe nails. Nail growth can be disturbed by serious illness or by injury over the nail root, resulting in transverse grooves or white patches in the nails. These grooves or patches slowly grow toward the free edge of the nail. If a nail is lost by injury, a new one grows out of the germinal matrix if the latter is intact.
Bf

- * Psoriasis: There is an increase in the number of proliferating cells and decrease in the cycle time of the cells in the stratum basale and spinosum leading to increase in the epidermal thickness and rapid renewal of epidermis.
Fig. 11.12: Parts of a nail as seen in a longitudinal section (Schematic representation).
Fig. 11.13: Lunule of a nail (schematic representation).

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"It
is a very common chronic inflammatory disease involving the sebacec glands and hair follicles, found predominantly in adolescents in both sexe
"The flow of the sebum is continuous and a disturbance in the norn secretion and flow leading to obstruction and results in acne.
* Albinism: Hereditary inability of the melanocytes to synthesize melanin. Skin is not protected from solar radiation therefore increased incidence of skin cancers.
* Vitiligo: It is a disease that results from the immune system killing melanocytes. This results in the depigmentation of skin.
* Onychia: It is the inflammation of nail folds and shedding of nail resulting due to the introduction of microscopic pathogens through small wounds.
"Paronychia: It is caused due to bacterial or fungal infection produci change in the shape of nail plate.
*Koilonychia: It is caused due to iron deficiency or vitamin B,, deficier
and is
abnormal thinness
and concavity
(spoonshape)
the nails.
* The fiber bundles in the reticular layer of the dermis mostly lie parallel to one another. In the limbs the predominant direction of the bundles is along the long axis of the limb; while on the trunk and neck, the direction is transverse. The lines along which the bundles run are often called cleavage lines. The cleavage lines are of importance to the surgeon as incisions in the direction of these lines gape much less than those at right angles to them.
* The dermis contains considerable amounts of elastic fibers. Atrophy of elastic fibers occurs with age and is responsible for the loss of elasticity and wrinkling of the skin.
* Linea gravidarum: If skin is rapidly stretched, fiber bundles in the dermis may rupture scar tissue which can be seen in the form of prominent white lines (anterior abdominal wall in pregnancy).

"Malignant melanoma: Tumors arising from melanocytes.
™ Basal cell carcinoma:Tumors arising fromthe basal cells of stratum basa
"Squamous cell carcinoma: Tumors from the squamous cells of strat spinosum. Squamous cell carcinoma may arise on any part of the skin a mucous membranes lined by squamous epithelium but are more likely occur on sun-exposed parts in older people.

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. Lucidum
- . Corneum . Spinosum . Granulosum 2009
2. Which of the following statements about the sebaceous glands is true?
They control the body temperature They are abundant on the face only They are a source of new hairs 2050p
They are always associated with hair follicles
3. The dermis is made up of two layers:
Adipose tissue and basal layer
Papillary layer and horny layer Papillary and reticular layers 2909
Inner layer and granular layer

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a. Pathogenic bacteria
b. Electric heat
c. Steam heat
d. Ultraviolet rays
5. The main function of sebum is to prevent the skin from:
Losing moisture
Becoming dirty Losing color 2900p
Becoming blemished
6. Name the sublayers of the epidermis from the inside outward:
Basal, corneum, spinosum, lucidum, and granulosum Basal, spinosum, granulosum, lucidum, and corneum Basal, spinosum, granulosum, corneum, and lucidum Basal, granulosum, lucidum, corneum, and spinosum 2905p

* Classification of salivary glands with example
*Composition of salivary gland
* Structure of serous, mixed, and mucous Salivary glands

Only serous acini are present, which contain basophilic zymogen granules and are darkly stained, Submandibular gland = Mixed salivary gland, predominantly serous with a few mucous acini. Only serous acini are present, which contain basophilic zymogen granules and are darkly stained, Sublingual gland = Predominantly mucous acini are present with few serous acini. Intercalated and striated (intralobular) ducts are seen, Submandibular gland = Serous demilunes are present. Intercalated and striated (intralobular) ducts are seen, Sublingual gland = Intercalated and interlobular duct can be seen. Interlobular duct can be seen, Submandibular gland = Intercalated and striated (intralobular) ducts are seen. Interlobular duct can be seen, Sublingual gland = It also contains adipocytes.. It also contains adipocytes., Submandibular gland = Interlobular duct can be seen Striated ducts are more prominent than those in parotid gland.. It also contains adipocytes., Sublingual gland = 
SALIVARY GLANDS
There are two main groups of salivary glands: (1) major and (2) minor.
1. Major salivary glands: Three paired glands: (1) parotid, (2) submandi and (3) sublingual. They are branched tubuloalveolar glands.
2. Minor salivary glands are numerous and are widely distributed ir mucosa of oral cavity. Some of the minor salivary glands are the Von Eb gland in tongue, buccal glands in cheeks and labial glands in lips.

Basically, a salivary gland consists of stroma, parenchyma, and a duct system which carries the secretions into the oral cavity.

The connective tissue capsule provides septa and divides the glands into lobes and lobules. The blood vessels and nerves traverse through the connective tissue septa. Large ducts of the glands are also present in it.

Parenchyma has two components: (1) the secretory part and (2) conducting part.

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* Salivary glands are compound tubuloalveolar glands (racemose glands). Their secretory elements (also referred to as end pieces or as the portio terminalis) may be rounded (acini), pear-shaped (alveoli), tubular, or a mixture of these (tubuloacinar, tubuloalveolar).
* Salivon—Basic secretary unit which includes acini, intercalated duct, and exceretory duct.
* The acini are made up of either serous or mucous cells. A salivary gland may have only one type of acini, or there may be a mixture of both serous and mucous acini, these are called mixed acini.
* A secretory unit, or gland, with only one type of cell (serous or mucous) is said to be homocrine. If it contains more than one variety of cells, it is said to be heterocrine.
"Serous acini: Serous cells are usually arranged in the form of rounc acini. As a result, each cell is roughly pyramidal having a broadbase an
narrow apex with a single, round, and basally located nucleus. The nucle is pushed to basal region due to accumulated secretory granules of api region. The apical region is eosinophilic due to accumulation of zymog granules; cytoplasm is basophilic due to cell organelles. Hence exhi bipolar staining.

"Mucous cells: Mucous cells are usually arranged in the form of tubu secretory. The cells lining mucous cells tend to be columnar rather tt pyramidal with a flattened nucleus at the basal part of the cell. 1 cytoplasm is filled completely with a light-staining, secretory prod called mucus. This pushes the nucleus to the base of the cytoplas Lumens of these acini are larger than serous.
"Seromucous cells: Some salivary glands contain both serous and mucc acini in same secretory component. They are referred to as seromucc cells. In the submandibular glands, mucous acini are often capped by crescent or moon-shaped cap of serous cells over them called a serc demilunes of Giannuzzi. The serous cells of a demilune drain into | lumen of the acinus through fine canaliculi passing through the interv between mucous cells.
™ Myoepithelial cells (basket cells): Myoepithelial cells are contractile, th contraction helping to squeeze out secretion from secretory or conducti portion of the gland. They are present in relation to acini, intercalat intralobular and extralobular ducts. They are located between the ba lamina and acinar or ductal cells. They contain actin and myosin filament

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The secretory elements lead into a series of ducts through which their secretions are poured into the oral cavity. In sections through salivary glands, we see a large number of closely packed acini with ducts scattered between them. These elements are supported by the connective tissue that also divides the glands into lobules and forms capsules around them. Blood vessels, lymphatics, and nerves run in the connective tissue that may at places contain some adipose tissue.
* Duct system: The ducts are highly branched and range from very small intercalated ducts to very large terminal ducts. Secretions produced in acini pass along a system of ducts, different parts of which have differing structure.
* The smallest ducts are called intercalated ducts to which secretory acini are attached. These are lined by cuboidal or flattened cells.
* Many intercalated ducts join to form striated ducts, and they are lined by cuboidal to low columnar cells. They are so called because the basal parts of the cells show vertical striations.
* Striated ducts join with each other and form intralobular ducts of increasing caliber. Ducts arising from lobules unite to form interlobular ducts, which in turn forms intralobar and interlobar ducts. The interlobular are lined by simple columnar epithelium. Terminal part toward oral cavity gets lined by nonkeratinized stratified squamous epithelium.

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Secretion by salivary glands is under hormonal as well as neural control.
* A local hormone plasmakinin formed by secretory cells influences vasodilation.
* Salivary glands are innervated by autonomic nerves, both parasympathetic (cholinergic) and sympathetic (adrenergic). Parasympathetic nerves travel to secretory elements along ducts, while sympathetic nerves travel along arteries. Synaptic contacts between nerve terminals and effector cells form neuroeffector junctions.
Two types of junction, epilemmal and hypolemmal., are present.
1. At epilemmal junctions, the nerve terminal is separated from the secreto effector cell by the basal lamina.
2. At hypolemmal junctions, the nerve terminal pierces the basal lamina comes into direct contact with the effector cell.
Nerve impulses reaching one effector cell spread to others through intercellular contacts. Classically, salivary secretion has been attributed to parasympathetic stimulation. While this is true, it is believed that sympathetic nerves can also excite secretion either directly, or by vasodilation. Autonomic nerves not only stimulate secretion, but also appear to determine its viscosity and other characteristics. Autonomic nerve terminals are also seen on myoepithelial cells and on cells lining the ducts of salivary glands. The latter probably influence the reabsorption of sodium by cells lining the ducts.
Salivary glands are sensitive to pain, and must therefore have a sensory innervation as well.

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* Parotid gland, the large serous salivary gland, located in the parotid region. Its excretory duct is called Stensen's duct, opens at vestibule of mouth into a small elevation on the mucosal surface of the cheek opposite the second upper molar tooth.
* Tt is compound tubuloacinar or racemose gland.
* The parotid gland is covered by a capsule from which arise numerous interlobular connective tissue septa that subdivide the gland into lobes and lobules. Blood vessels and interlobular excretory ducts are located in the connective tissue septa.
* Each lobule is made up of a cluster of secretory cells that form the serous acini. The cells of acini are pyramid shaped with spherical nuclei located at the base of the slightly basophilic cytoplasm. At high power, small secretory granules occupy the cell apices of the serous acini.
* The acini are surrounded by thin, contractile myoepithelial cells located between the basement membrane and acinar cells.
* The lobules may contain numerous adipose cells.
* The secretions of the acini flow to narrow channels called the intercalated that are lined by a simple squamous or low cuboidal epithelium.
* The secretion drains into larger striated ducts having larger lumens lined by simple columnar cells with basal striations. Striated ducts are large and conspicuous in parotid gland. The striations are formed by deep infolding of the basal cell membrane. Longitudinally oriented, elongated mitochondria are enclosed in between the infoldings. They empty their product into the intralobular excretory ducts which in turn drain into interlobular ducts located in the connective tissue septa.
* The lumen of ducts become progressively wider, and the epithelium get taller as they increase in size. The epithelium changes from columnar to pseudostratified or even stratified columnar in large lobar ducts close to their termination.

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It is a mixed gland, containing both serous and mucous acini, with serous acini predominating.
* The submandibular salivary gland is also a compound tubuloacinar gland.
* The serous acini have smaller, darker-stained pyramidal cells, spherical basal nuclei, and apical secretory granules. The mucous acini are larger with wide lumen exhibiting more variation in size and shape. The mucous cells are columnar with pale or almost colorless cytoplasm. The nuclei are flattened and pressed against the base of the cell membrane.
* The serous demilunes are dispersed among the acini. The mucous acini have a cap of serous cells that are thought to secrete into the highly convoluted intercellular space between the mucous cells.
* The thin, contractile myoepithelial cells are seen around serous, mucous acini, and intercalated ducts.
* The duct system of the submandibular gland is similar to that of the parotid gland.

Fig. 12.1: Some features of serous cell in a salivary gland (schematic representation).
Fig. 12.2: Some features of mucous cells in salivary glands (schematic representation).

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The parotid gland is a serous salivary gland. The characteristic features are:
* Only serous acini are present which contain basophilic zymogen granules and are darkly stained
*Intercalated and striated (intralobular) ducts are seen
* Interlobular duct can be seen
* It also contains adipocytes.
red
‘=:

Submandibular gland (Low magnification): (A) As seen in drawing; (B) Photomicrograph.

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*The
submandibular gland is a mixed salivary gland, predominantly serous with a few mucous acini
"Serous cells are frequently located at the periphery of mucous acini in the form of a crescent and called as demilunes
" Striated ducts are more than those in
prominent parotid gland.

Submandibular gland (High magnification): (A) As seen in drawing; (B) Photomicrograph.

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"In the high power view the serous and mucous acini can be identified by their staining reaction, and shape and position of nucleus
- "The serous acini are darkly stained and have rounded nucleus placed near the center of the cell
"The mucous acini are lightly stained with flat nucleus placed toward the basement membrane
"The mucous acini are often associated with darkly stained crescentic patch of serous cells called serous demilune.
*The sublingual gland is predominantly a mucous gland but few serous acini may also be seen
- * Serous demilunes may be present.
Fig. 12.3: Electron microscopic structure of a cell from striated duct (schematic representation).

*TIt is also a compound, mixed tubuloacinar gland. Mucous acini are predominantly seen with few serous acini. The light-stained mucous acini are conspicuous in the section.
* In comparison with other salivary glands, the duct system of the sublingual gland is somewhat different. The intercalated ducts are short or absent, and not easily visualized in the section. In contrast, the nonstriated intralobular excretory ducts are more prevalent in the sublingual glands. The interlobular connective tissue septa are abundant in the sublingual compared to other two.
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Tumors of salivary glands are common in the major salivary glands. Majority of them are benign and originate in the parotid gland (pleomorphic adenoma).
1. About 60% of the saliva is secreted in oral cavity by
:
Parotid
Submandibular Sublingual Lingual 2050p

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b. They contain secretory granules in the apical cytoplasm, and the secretion of granu
- c. Serous cells are pyramidal in shape

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* Structure and functions of placenta
*Components and formation of placenta
* Placental circulation and barrier
* Microscopic structure
* Umbilical cord

Section of tertiary villi of various sizes and shapes are seen, Umbilical cord = Amnion lining the section of cord is made of simple squamous, cuboidal epithelium. Intervillous space containing maternal blood, Umbilical cord = Two umbilical arteries and one umbilical vein are seen. Anchoring villi, floating villi are seen, Umbilical cord = Wharton's jelly -mucoid connective tissue is seen.. Section of tertiary villi has core of mesoderm surrounded by cytotrophoblast and syncytiotrophoblast., Umbilical cord = . PLACENTA, Umbilical cord = PLACENTA
Bf

Placenta is a fetomaternal organ. It connects growing fetus to the wall of gravid uterus. It is a disk-shaped organ and weighs 500 g. It has two surfaces —(1) An irregular maternal surface that is divided into 15-20 small lobules called maternal cotyledons and (2) A smooth fetal surface covered with amnion. The umbilical cord is attached at or near the center of this surface (Fig. 13.1).

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a. Nutrition: It maintains nutrition of the fetus through blood vessels that t
in the umbilical cord. It has several functions that facilitate growth o fetus. It acts as a temporary organ that allows transport of oxygen nutrients from the mother to the developing fetus.
b. Excretion: It eliminates carbon dioxide and waste products like urea fetus into the maternal circulation.
c. Immunological barrier: Maternal immunoglobulin protects fetus ag some infections.
d. Barrier function: It acts as a selective barrier allowing transfer of substé that facilitate growth and development of embryo. It prevents entr harmful substances including certain drugs, viruses, and toxins.
e. Hormone production: Placenta synthesizes several hormones. progesterone secreted helps to maintain the pregnancy after 4th month estrogens secreted promotes uterine growth, development of mammary g
i

It is a disk-shaped organ having structural components of fetal and maternal origins.
The maternal component is contributed by decidua and fetal component by chorion.

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- * The uterine endometrium is called decidua after the attachment (implantation) of fertilized embryo (blastocyst) to it (Fig. 13.2).
Fig. 13.1: Maternal and fetal surfaces of placenta showing maternal cotyledons and attachment of umbilical cord.
Fig. 13.2: Implantation and growing embryo in the wall of uterus.
Parts of decidua: The endometrium (decidua) that contributes for placenta
is called decidua and its parts are:
" Decidua basalis: Part contributing for placenta
" Decidua capsularis: Part surrounding embryo
" Decidua parietalis: Part lining the cavity of uterus.

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* The fertilized ovum undergoes series of cleavage divisions increasing the number of cells (blastomeres) that get reorganized into peripheral trophoblast and inner embryoblast. The embryoblast gives rise to the embryo.
* The trophoblast along with adjacent extraembryonic mesoderm contributes to the formation of chorion all around the developing embryo. Later blood vessels develop in the extraembryonic mesoderm forming chorionic vessels.
* That part of chorion coming in contact with decidua basalis is called chorion frondosum whereas the part in contact with decidua capsularis is called chorion laeve (Fig. 13.2).
* Parts of chorion:
" Chorion frondosum: Contributes for fetal component of placenta
" Chorion laeve: Covers the decidua capsularis.
* The trophoblast presents outer layer of syncytiotropho-blast and an inner layer of cytotrophoblast. The syncytiotrophoblast contains number of cell layers without cellular outlines. The cytotrophoblast consists of single layer of cuboidal cells.

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* The maternal part of placenta is contributed by decidua basalis of endometrium that sends septa dividing it into 15-20 maternal cotyledons.
* The fetal part is contributed by chorion frondosum that is seen as a plate called chorionic plate. From the chorionic plate, 40-60 extensions fetal cotyledons arise and extend toward the maternal part called decidua basalis.
* Each extension is called stem villus/truncus chorii that shows ramifications into number of branches (ramus chorii, ramuli chorii) like the
branches of a tree. Their terminal ramifications are called chorionic villi (Fig. 13.3).
* Villi attached to decidua basalis are called anchoring villi. Villi in the intervillous spaces bathed by maternal blood are called floating villi. The structural components of chorionic villous differs at different stages of embryonic development are of three types. The primary villi have innermost cytotrophoblast surrounded by Syncytiotrophoblast, while secondary villi have in addition a central core extraembryonic mesoderm; tertiary villi have three layers—an outer trophoblast (cyto and syncytio), middle layer of extraembryonic mesoderm with central core of chorionic vessels.
* The villi are separated by irregular spaces called intervillous spaces.
Bi

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* Microscopic structure of placenta at term present cut sections of tertiary villi of different sizes and shapes with maternal blood cells in between villi (Plate 13.1).
"If a section contains chorionic plate and amniotic membrane, extraembryonic mesoderm with blood vessels can be identified.
* Each villous has a central core of mesoderm with fetal capillaries lined with simple squamous epithelium and containing nucleated fetal red blood cell (RBC). In addition, macrophages (Hofbauer cells) and fibroblasts are present in the mesodermal core. It is covered with thin layer of intensely stained syncytiotrophoblast and discontinuously arranged cytotrophoblast (Fig. 13.4). Because of the disappearance of cytotrophoblast and thinning of syncytiotrophoblast, the fetal capillaries move toward the periphery thereby reducing the thickness of placental barrier.
Fig. 13.3: Figure showing maternal and fetal components of placenta and cotyledons. Ramifications of fetal cotyledons are: Anchoring villi, ttuncus chorii, ramus chorii, ramuli chorii, floating villi in intervillous space.
* In early placenta at low magnification, a large numbers of villi projecting into the lacunae filled with maternal blood are seen; some villi show evidence of branching. Higher magnification shows a core of primitive mesenchyme invested by an inner layer of cytotrophoblast cells and an outer syncytiotrophoblast layer (Fig. 13.4).
* The term placenta at low magnification shows a huge numbers of villi cut in various planes and varying in diameter from large main stem villi to very small terminal branch villi. The villous pattern get highly developed and extensive branched as the placenta enlarges. Term placenta also shows the syncytial knots formed by syncytiotrophoblast nuclei that are aggregated together in clusters leaving zones of thin cytoplasm devoid of nuclei between.
* The proximity of blood in fetal capillaries to maternal blood reduces. There is a thin layer of syncytiotrophoblast only and the capillaries tend to be located in the periphery of the villi.
ll

The circulation of blood in the placenta is unique in that maternal blood in the intervillous space and fetal blood in fetal capillaries of chorionic villi will be traversing side by side without mixing and in opposite directions. Yet the exchange takes place between the two circulations by the process of diffusion across the structural components forming the villi known as blood placental barrier.

The tissues intervening between maternal and fetal blood constitutes the barrier. The exchange of gases, nutrients, and waste products occurs across this barrier. The thickness of the barrier reduces with progression of pregnancy to facilitate the increased efficiency of transport across the membrane to growing fetus.

Structures constituting the barrier from maternal to fetal blood are as follows:
Plate 13.1: Placenta (Low magnification)
Placenta (Low magnification): (A) As seen in drawing; (B) Photomicrograph.

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*
Tertiary villi of various size and shapes seen
* Inter villous space containing maternal blood
*Truncus chorii, ramus chorii, ramuli chorii and floating villi in intervillous space seen
* Section of tertiary villi has core of mesoderm surrounded by cytoand syncytiotrophoblast.
* Endothelium of fetal blood vessels and its basement membrane
* Mesoderm
* Cytotrophoblast and its basement membrane
* Syncytiotrophoblast.

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Barrier gets thinner and improves the efficiency of exchange.
* Cytotrophoblast disappears
* Syncytiotrophoblast becomes thinner
* Connective tissue core gets edematous
* Fetal blood vessels move to periphery.
Fig. 13.4: Photomicrograph of sections of floating tertiary villi at high magnification.
Bi

* Abortions: Initially corpus luteum of pregnancy secretes hormone progesterone and continues pregnancy. Once placenta is formed it takes over the function to maintain pregnancy. Any derangement during switch over might cause termination of pregnancy, hence abortion.
* Placental insufficiency: Placenta is essential for growth of the fetus and any abnormality hamper growth and development. Early pregnancy changes can lead to reduction in growth of the fetus, termed 'intrauterine
growth restriction' (IugR) several medical conditions can lead to placental insufficiency and IugR: Maternal malnutrition, diabetes, twin or triplet pregnancies, pre-eclampsia or eclampsia, infections and abnormal implantation of the placenta in the uterus.

™ Placenta accreta: The placenta attaches too deeply into the uterine wall | does not penetrate the myometrium.
"Placenta increta: Placental villi penetrate deep into the muscular layer the myometrium.
" Placenta percreta: Penetrates through the entire uterine wall and attaches another organ such as the bladder, rectum, intestines, or large blood vesse

Umbilical cord is a tubular structure that connects developing fetus with placenta. This helps to conduit oxygen and nutrients from mother and takes away the fetal waste products.
* At birth, one end of the cord is attached to the anterior abdominal wall of the fetus and other end at fetal surface of placenta. The folding of embryo leads to formation of connecting stalk. It gets surrounded by a sleeve of amnion thus, forming umbilical cord.
* At full term, it is around 50 cm in length. But the length may vary. It is twisted to forming knots which may be due to movements of the fetus.

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* The cross-section of umbilical cord shows a covering of single-layered amnion which is simple cuboidal epithelium.
*Tt is filled with a gelatinous or mucous connective tissue, called as Wharton's jelly. This stroma supports the blood vessels and is derived from primary mesoderm. The ground substance is made up of primarily hyaluronic acid and chondroitin sulfate with a low abundance of collagen or reticular fibers.
* The mucoid connective tissue consists of a wide-meshed network of long, stretched fibrocytes made up of many interconnecting cell processes. They
are stellate and spindle-like mesenchymal cells (indistinguishable from resting fibroblasts).
* The gelatinous tissue is embedded with a single umbilical vein and the two umbilical arteries. The umbilical arteries have thick muscular walls with double-layered muscular walls without an elastic layer. They carry deoxygenated blood from the fetus to the placenta. umbilical veins deliver oxygenated blood from the placenta to the fetus. It has a thick layer of circular smooth muscle unlike adult veins that characteristically show thick longitudinal layer. In early pregnancy, umbilical veins are two in number but later right umbilical vein disappears.
* The remnant of allantois may be seen in the center.
1. The following feature is found in a primary placental villus:
a. Cytotrophoblast cells filling the central core of a villus
b. Fetal capillaries
c. Mesodermal core
d. Outer covering of syncytiotrophoblast
2. The portion of the decidua where the placenta is to be formed:
a. Decidua basalis
Fig. 13.5: Photomicrograph of umbilical cord.
b. Decidua capsularis
c. Decidua parietalis
3. Human placenta is:
a. Epitheliochorial
b. Hemoendothelial
c. Hemochorial
d. Syndesmochorial

Maternal blood
Fetal blood Amniotic fluid 200

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* Structure of nasal cavity, pharynx
* Structure of larynx (epiglottis)
* Structure of bronchial tree
*Lung—composition, microscopic structures, alveoli

Lined by pseudostratified ciliated columnar epithelium, Trachea = Pseudostratified ciliated columnar epithelium with goblet cells. Lined by pseudostratified ciliated columnar epithelium, Lung = Presence of alveolar ducts. Lamina propria with serous and mucous glands, Trachea = Lamina propria. Lamina propria with serous and mucous glands, Lung = Alveolar sac lined by simple squamous epithelium. Central core of elastic tissue., Trachea = Submucosa with serous and mucous glands. Central core of elastic tissue., Lung = Presence of intrapulmonary bronchus with varying amount of islands cartilage and smooth muscles. , Trachea = Presence of hyaline cartilage and trachealis. , Lung = Respiratory bronchioles with simple cuboidal epithelial lining lacking cilia and goblet cells.

The respiratory system consists of:
* Conducting part that includes the nasal cavities, the pharynx, the trachea, the bronchi, and their intrapulmonary continuations, responsible for providing passage of air and conditioning the inspired air.
* Respiratory part that includes the lungs is involved in the exchange of oxygen and carbon dioxide between blood and inspired air.
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* Their walls have a skeletal framework made up of bone, cartilage, and connective tissue, which keeps the passages always patent.
* Smooth muscle present in the walls of the trachea and bronchi enables some alterations in the size of the lumen.
* The interior of the passages is lined over most of its extent by pseudostratified, ciliated, and columnar epithelium.
* The epithelium is kept moist by the secretions of numerous serous glands.
* Numerous goblet cells and mucous glands cover the epithelium with a protective mucoid secretion that serves to trap dust particles present in inhaled air.
* This mucous (along with the dust particles in it) is constantly moved toward the pharynx by the action of cilia.
* When excessive mucous accumulates, it is brought out by coughing, or is swallowed.
* Deep to the mucosa, there are numerous blood vessels that serve to warm the inspired air.

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Bi
* The nasal cavity is the beginning of the respiratory system.
* These are paired chambers separated by septum.
* Tt extends from the nostrils in front to the posterior nasal apertures behind.
* Each nasal cavity is a hollow organ composed of bone, cartilage, and connective tissue covered by mucous membrane.
Histologically, the wall of each half of the nasal cavity is divisible into three distinct regions.
1. Vestibule
2. Olfactory mucosa
3. Respiratory mucosa.

* Tt is the anterior dilated part of the nasal cavity.
* The vestibule is lined by skin continuous with that on the exterior of the nose.
* Hair and sebaceous glands are present.

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* Apart from their respiratory function, the nasal cavities serve as end organs for smell.
* Receptors for smell are located in the olfactory mucosa which is confined to arelatively small area on the superior nasal concha, and on the adjoining part of the nasal septum.
* Olfactory mucosa is yellow in color, in contrast to the pink color of the respiratory mucosa.
* Tt consists of a lining epithelium and a lamina propria.
* The olfactory epithelium is pseudostratified epithelium made of olfactory cells, sustentacular cells, and basal cells.

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The rest of the wall of each half of the nasal cavity is covered by respiratory mucosa lined by pseudostratified ciliated columnar epithelium. In the epithelium, the following cells are present (Fig. 14.1):
* Ciliated cells are the columnar cells with cilia on their free surfaces and are the most abundant cell type.
* Goblet cells (flask-shaped cells) scattered in the epithelium produce mucous.
* Nonciliated columnar cells with microvilli on the free surface probably secrete a serous fluid that keeps the mucosa moist.
* Basal cells lying near the basal lamina help to replace those lost cells.
Fig. 14.1: Structure of respiratory part of nasal mucosa (schematic representation).

The lamina propria of nasal mucosa contains lymphocytes, plasma cells, macrophages, a few neutrophils, and eosinophils. Eosinophils increase greatly in number in persons suffering from allergic rhinitis.
B

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* The pharynx consists of nasal, oral, and laryngeal parts.
* The nasal part is purely respiratory in function, but the oral and laryngeal parts are more intimately concerned with the alimentary system.
* The wall of the pharynx is fibromuscular.
*In the nasopharynx, the epithelial lining is ciliated columnar, or pseudostratified ciliated columnar. The inferior surface of the soft palate and over the oropharynx and laryngopharynx, the epithelium is stratified
squamous.
* Subepithelial aggregations of lymphoid tissue are present on the posterior wall of the nasopharynx, and around the orifices of the auditory tubes, forming the nasopharyngeal and tubal tonsils.
* Numerous mucous glands are present in the submucosa, including that of the soft palate.
B

* Larynx is a specialized organ responsible for the production of voice. It houses the vocal cords. The wall of the larynx has a complex structure made up of a number of cartilages, membranes, and muscles.
* The epithelium lining the mucous membrane of the larynx is predominantly pseudostratified ciliated columnar, but in some parts that come in contact with swallowed food, the epithelium is lined by stratified squamous (anterior surface and upper part of the posterior surface of epiglottis, and the upper parts of the aryepiglottic folds, vocal folds).
* Numerous goblet cells and subepithelial mucous glands provide a mucous covering to the epithelium.

The larynx has a cartilaginous framework which is made of nine cartilages (three paired and three unpaired) that are connected to each other by membranes and ligaments (Fig. 14.2). The cartilages are either hyaline or elastic in nature. These are as follows:

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" Epiglottis (unpaired)
™ Cuneiform (paired)
™ Corniculate (paired).
With advancing age, calcification may occur in hyaline cartilage, but not
- in elastic cartilage.

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* The epiglottis has a central core of elastic cartilage.
* Overlying the cartilage, there is mucous membrane.
* The greater part of the mucous membrane is lined by stratified squamous epithelium (nonkeratinizing).
* The mucous membrane over the lower part of the posterior surface of the epiglottis is lined by pseudostratified ciliated columnar epithelium. Some taste buds are present in the epithelium of the epiglottis.
* Numerous glands, predominantly mucous, are present in the mucosa deep to the epithelium. Some of them lie in depressions present on the epiglottic cartilage (Plate 14.1).
Fig. 14.2: Anterior view of the larynx (schematic representation).
i

* Trachea extends from the lower border of cricoid cartilage (C6) to its level of bifurcation (T4) into right and left bronchi.
* The trachea is a fibroelastic cartilaginous tube. The trachea consists of four layers (Plate 14.2).

* Mucosa of the tracheal lumen is lined by pseudostratified ciliated columnar epithelium. It also contains goblet cells, brush cells, small granule cell, and basal cells that lie next to the basement membrane. Numerous lymphocytes are seen in deeper parts of the epithelium.
* Cells: (a) Ciliated cells—the most numerous extending through the full thickness of the epithelium. They create mucociliary current to remove the inhaled dust particles, (b) Brush cells—they are columnar cells that bear blunt microvilli, (c) Small granule cells (Kulchitsky cells)— they are like enteroendocrine cells of gastrointestinal tract (GIT), secrete catecholamine, serotonin, etc., (d) Mucous cells—they are similar in appearance to intestinal goblet interspersed among the ciliated cells and also extend through the full thickness of the epithelium, and (e) Basal cells—reserve cells. ate
* Basement membrane is thick.
* Lamina propria contains lymphocytes, eosinophils, plasma cells, and fibroblasts with diffuse and nodular lymphatic aggregation[bronchusassociated lymphoid tissue (BALT)].

* The subepithelial connective tissue contains numerous elastic fibers.
* Submucosal glands help to keep the epithelium moist and provide a covering of mucous in which dust particles get caught. he
* Numerous aggregations of lymphoid tissue are present in the subepithelial connective tissue. Eosinophil leukocytes are also present.

- * The skeletal basis of the trachea is made up of 16—20 tracheal cartilages.
Plate 14.1: Epiglottis

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*
Lined by pseudostratified ciliated columnar epithelium
*
Lamina propria with serous and mucous glands
*
Central core of elastic tissue.
- ® Pseudostratified ciliated columnar epithelium with goblet cells
®
Lamina propria
® Submucosa with serous and mucous glands
® Presence of hyaline cartilage and trachealis muscle.
* Each of these is a C-shaped mass of hyaline cartilage.
* The open end of the "C" is directed posteriorly. The gaps between the cartilage ends, present on the posterior aspect, are filled in by smooth muscle and fibrous tissue.
The intervals between the cartilages are filled by fibrous tissue that becomes continuous with the perichondrium covering the cartilages.
- * The connective tissue in the wall of the trachea contains many elastic fibers.

It is made of fibroelastic connective tissue containing blood vessels and nerves.

The trachea divides at the level of T4 into right and left principal bronchi (primary or main bronchi). They have a structure similar to that of the trachea.
B

The lungs are the principal respiratory organs that are situated on either side of mediastinum in the thoracic cavity. They are covered by visceral pleura. Structure of the lung includes the structure of intrapulmonary bronchial tree and alveoli.

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* On entering the lung, the principal bronchus divides into secondary, or lobar bronchi (one for each lobe).
* Each lobar bronchus divides into tertiary, or segmental bronchi (one for each segment of the lobe).
* The segmental bronchi divide into smaller and smaller bronchi, which ultimately end in bronchioles.
* The lung substance is divided into numerous lobules each of which receives a lobular bronchiole.
* The lobular bronchiole gives off a number of terminal bronchioles (Fig. 14.3). As indicated by their name, the terminal bronchioles represent the most distal parts of the conducting passage.
* Each terminal bronchiole ends by dividing into respiratory bronchioles. These are so called because they are partly respiratory in function as some
air sacs (see below) arise from them.
* Each respiratory bronchiole ends by dividing into a few alveolar ducts. Each alveolar duct ends in a passage, the atrium, which leads into a number of rounded alveolar sacs.
* Each alveolar sac is studded with a number of air sacs or alveoli. The alveoli are blind sacs having very thin walls through which oxygen passes from air into blood, and carbon dioxide passes from blood into air.
The structure of the larger intrapulmonary bronchi is similar to that of the trachea. As these bronchi divide into smaller ones, the following changes in structure are observed:
* The cartilages in the walls of the bronchi become irregular in shape and are progressively smaller.
* Cartilage is absent in the walls of bronchioles. This is the criterion that distinguishes a bronchiole from a bronchus.
* The amount of muscle in the bronchial wall increases as the bronchi become smaller. The presence of muscle in the walls of bronchi is of considerable clinical significance. Spasm of this muscle constricts the bronchi and can cause difficulty in breathing.
Fig. 14.3: Some terms used to describe the terminal ramifications of the bronchial tree
(schematic representation).

* Subepithelial lymphoid tissue increases in quantity as bronchi become smaller. Glands become fewer and are absent in the walls of bronchioles.
* The trachea and larger bronchi are lined by pseudostratified ciliated columnar epithelium. As the bronchi become smaller, the epithelium first becomes simple ciliated columnar, then nonciliated columnar, and finally cuboidal (in respiratory bronchioles). The cells contain lysosomes and numerous mitochondria.
Apart from typical ciliated columnar cells, various other types of cells are to be seen in the epithelium lining the air passages. They are goblet cells, nonciliated serous cells, basal cells, cells of Clara (nonciliated cells of terminal bronchiole) whose secretion reduces surface tension, and cells similar to diffuse endocrine cells of the gut (Figs. 14.4A to G).
The differences between bronchus and bronchioles are given in Table 14.1.

There are about 200 million alveoli in a normal lung. The total area of the alveolar surface of each lung is extensive. It has been estimated to be about 75 m*. The total capillary surface area available for gaseous exchanges is about 125 m2.

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* Each alveolus has a very thin wall. The wall is lined by simple squamous epithelium resting on the basement membrane (Plate 14.3).
* Deep to the basement membrane, there is a layer of delicate connective tissue through which pulmonary capillaries run. These capillaries have the usual endothelial lining that rests on a basement membrane.
* The barrier between air and blood is made up of the epithelial cells and their basement membrane, by endothelial cells and their basement membrane, and by intervening connective tissue. At many places, the two basement membranes fuse greatly reducing the thickness of the barrier.
* The endothelial cells lining the alveolar capillaries are remarkable for their extreme thinness.
* With the electron microscopy (EM), they are seen to have numerous projections extending into the capillary lumen. These projections greatly increase the surface of the cell membrane that is exposed to blood and is, therefore, available for the exchange of gases.
* At many places, the basement membrane of the endothelium fuses with that of the alveolar epithelium greatly reducing the thickness of the barrier between blood and air in alveoli.
Diameter, Characteristics Bronchus = Larger diameter (more than 1 mm). Diameter, Bronchiole = Smaller diameter (less than 1 mm). Lining epithelium, Characteristics Bronchus = Pseudostratified ciliated columnar epithelium with goblet cells. Lining epithelium, Bronchiole = Large-sized bronchioles : Simple columnar cells with few cilia and few goblet cells Small-sized bronchioles : Simple columnar or simple cuboidal cells with no cilia or no goblet cells. Smooth muscle layer, Characteristics Bronchus = Present between mucosa and cartilage layer. Smooth muscle layer, Bronchiole = Smooth muscles and elastic fibers form a well-defined layer beneath mucosa. Cartilage, Characteristics Bronchus = Present in irregular patches. Cartilage, Bronchiole = Absent. Glands in submucosa, Characteristics Bronchus = Both serous and mucous acini present between cartilage and muscle layer. Glands in submucosa, Bronchiole = Absent

Figs. 14.4A to G: Various types of cells to be seen lining the respiratory passages: (A) Typical ciliated columnar; (B) Basal; (C) Goblet; (D) Serous; (E) Brush; (F) Clara; (G) Argyrophil (schematic representation).
Courtesy (B): Ivan Damjanov. Atlas of Histopathology, 1st edition. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2012. p. 37.

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* Presence of alveolar ducts
* Alveolar sac lined by simple squamous epithelium
* Presence of intrapulmonary bronchus with varying amount of islands cartilage and smooth muscles
”
Respiratory bronchioles with simple cuboidal epithelial lining lacking cilia and goblet cells.

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Electron microscopy studies have shown that the cells forming the lining epithelium of alveoli (pneumocytes) are of various types (Fig. 14.5).
* The most numerous (lines 90% of the alveolar surface) cells are simple squamous cells called type I alveolar epithelial cells. Except in the region of the nucleus, these cells are reduced to a very thin layer (0.05—-0.2 jm).
* Scattered in the epithelial lining, there are rounded secretory cells bearing microvilli on their free surfaces. These are designated as type II alveolar epithelial cells (Figs. 14.5 and 14.6). Their cytoplasm contains secretory granules (multilamellar bodies). These cells secrete pulmonary surfactant that reduces surface tension and prevents collapse of the alveolus during expiration. Surfactant contains phospholipids, proteins, and glycosaminoglycans produced in type II cells (a similar fluid is believed to be produced by the cells of Clara present in bronchial passages). Type II cells may multiply to replace damaged type I cells.
Fig. 14.5: Some cells to be seen in relation to an alveolus (schematic representation).
- * Type ITI alveolar cells, or brush cells, of doubtful function, have also been described.
Different types of cells present in the respiratory system are summarized in Flowchart 14.1.

* The connective tissue framework of alveolus contains collagen fibers and numerous elastic fibers continuous with those of bronchioles.
* Fibroblasts, histiocytes, mast cells, lymphocytes, and plasma cells may be present. Pericytes are present in relation to capillaries.
* Alveolar macrophages which swallow dust particles are called as dust cells. In congestive heart failure (in which pulmonary capillaries become overloaded with blood), these macrophages phagocytose erythrocytes that escape from capillaries. The cells, therefore, acquire a brick red color and are then called heart failure cells. Macrophages also remove excessive surfactant and secrete several enzymes.

The pleura is lined by flat mesothelial cells that are supported by loose connective tissue rich in elastic fibers, blood vessels, nerves, and lymphatics. There is considerable adipose tissue under parietal pleura.
Fig. 14.6: Type II pneumocytes (schematic representation).
Flowchart 14.1: Different types of cells of respiratory system.

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*The olfactory epithelium is pseudostratified. It is much thicker than the epithelium lining the respiratory mucosa (about 100 pm). Within the epithelium, there is a superficial zone of clear cytoplasm below which there are several rows of nuclei (Fig. 14.7).
*Using special methods, three types of cells can be recognized in the epithelium (Fig. 14.8):
1. Olfactory cells are modified neurons. Each cell has a central part containing a 1 nucleus. Two processes, distal and proximal, arise from this central part. Thi process (representing the dendrite) passes toward the surface of the olfactory epit It ends in a thickening (called the rod or knob) from which a number of ng olfactory cilia arise and project into a layer of fluid covering the epithelium. The p process of each olfactory cell represents the axon. It passes into the su connective tissue where it forms one fiber of the olfactory nerve.
2. The sustentacular cells support the olfactory cells. Their nuclei are oval and lie 1 free surface of the epithelium. The free surface of each cell bears numerous ni (embedded in overlying mucous). The cytoplasm contains yellow pigment (lipofus¢ gives olfactory mucosa its yellow color. In addition to their supporting f sustentacular cells may be phagocytic, and the pigment in them may represent re of phagocytosed olfactory cells.
3. The basal cells lie deep in the epithelium and do not reach the luminal surfac: divide to form new olfactory cells to replace the dead cells. Some basal cells supporting function too.

- *In vertebrates, olfactory cells are unique in being the only neurons that have cell bodies located in an epithelium.
* Olfactory cells are believed to have a short life. Dead olfactory cells are replaced by new cells produced by the division of basal cells. This is the only example of regeneration of neurons in mammals.

The lamina propria, lying deep to the olfactory epithelium, consists of connective tissue within which blood capillaries, lymphatic capillaries, and olfactory nerve bundles are present. It also contains serous glands (of Bowman), the secretions of which constantly "wash" the surface of the olfactory epithelium. This fluid may help in transferring smell carrying substances from air to receptors on olfactory cells.
Fig. 14.7: Olfactory mucosa seen in section stained by routine methods (schematic representation).
Fig. 14.8: Cells to be seen in olfactory epithelium (Schematic representation).

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Bi
+, oa Acute laryngitis: This may occur as a part of the upper or lower respiratory tract infection. Atmospheric pollutants, such as cigarette smoke, exhaust fumes, industrial and domestic smoke, etc. predispose the larynx to acute bacterial and viral infections. Streptococci and Haemophilus influenzae cause acute epiglottitis which may be lifethreatening.
od . Chronic laryngitis: Chronic laryngitis may occur from the repeated attacks of acute inflammation, excessive smoking, chronic alcoholism, or vocal abuse. The surface is granular due to swollen mucous glands. There may be extensive squamous metaplasia due to heavy smoking, chronic bronchitis, and atmospheric pollution.
4 ' Acute respiratory distress syndrome (ARDS) is a severe, at times lifethreatening, form of progressive respiratory insufficiency which involves pulmonary tissues diffusely, i.e. involvement of the alveolar epithelium, alveolar lumina, and interstitial tissue. ARDS exists in two forms: (1) neonatal type and (2) adult type. Both have the common morphological feature of the formation of the hyaline membrane in the alveoli, and hence, it is also termed as hyaline membrane disease (HMD).
, ate '= Bacterial pneumonia: Bacterial infection of the lung parenchyma is the most common cause of pneumonia or consolidation of one or both the lungs. Two types of acute bacterial pneumonias are distinguished—(1) lobar pneumonia and (2) broncho (lobular) pneumonia, each with distinct etiologic agent and morphologic changes.
oo Chronic bronchitis is a common condition defined clinically as persistent cough with expectoration on most days for at least 3 months of the year for 2 or more consecutive years. The cough is caused by the oversecretion of mucous. In spite of its name, chronic inflammation of the bronchi is not a prominent feature. The condition is more common in middle-aged males than females.
Asthma is a disease of airways that is characterized by increased responsiveness of the tracheobronchial tree to a variety of stimuli, resulting in widespread spasmodic narrowing of the air passages which may be relieved spontaneously or by therapy. Asthma is an episodic disease manifested clinically by paroxysms of dyspnea, cough, and wheezing.

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However, a severe and unremitting form of the disease termed status asthmaticus may prove fatal.
- *Immotile cilia syndrome that includes Kartagener's syndrome (bronchiectasis, situs inversus, and sinusitis) is characterized by ultrastructural changes in the microtubules causing immotility of cilia of the respiratory tract epithelium, sperms, and other cells. Males in this syndrome are often infertile.
1. Lining epithelium of alveoli is:
. Simple columnar . Simple squamous . Simple cuboidal Qoop
Pseudostratified columnar
2. Surfactant is secreted by:
a. Goblet cells
b. Type | pneumocytes
c. Type II pneumocytes
d. Clara cells

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a. Phagocytosis
b. Secretion of surfactant
c. Exchange of gases
d. Absorption
4. Skeletal framework of epiglottis is formed by:
. Elastic cartilage
. Hyaline cartilage . White fibrocartilage cartilage . Reticular tissue Qa2009

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* Oral cavity
* Structure of lip
* Tooth:
°General structure
* Structure of enamel
*Structure and types of dentin
* Structure of cementum and pulp
* Structure of tongue:
*Mucous membrane
* Structure and types of papillae
* Structure of taste bud

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Presence of mucocutaneous junction, Tooth = Presence of enamel and dentin. Presence of mucocutaneous junction, Tongue = Mucous membrane showing numerous papillae
Subepithelial connective tissue containing serous and mucous glands.
Pulp cavity lined by odontoblasts
Presence of taste buds
‘Presence of
“Serous glands of von Ebner
granular layer of
Tomes
“Apical
foramen
at
the
“Central core formed by skeletal muscles.
apex of the root.
Bf

The abdominal part of the alimentary canal (consisting of the stomach and intestines) is often referred to as the gastrointestinal tract. Closely related to the alimentary canal, there are several accessory organs that form part of the alimentary system. These include the structures of the oral cavity (lips, teeth, tongue, and salivary glands), liver, and pancreas.
The oral cavity is divided into a vestibule and the oral cavity proper. The vestibule is the space between the lips, cheeks, and teeth. The oral cavity proper lies behind the teeth and is bounded by the hard and soft palates above, the tongue and the floor of the mouth below, and the oropharynx posteriorly.

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* The wall of the oral cavity is made up partly of bone (jaws and hard palate), and partly of muscle and connective tissue (lips, cheeks, soft palate, and floor of mouth).
* These structures are lined by mucous membrane which is lined by stratified squamous epithelium that rests on connective tissue, similar to that of the dermis. The epithelium differs from that on the skin in that it is not keratinized (exception—gums).
* Deep to the epithelium, there lies lamina propria containing vessels and nerves.
* Papillae of connective tissue (similar to dermal papillae) extend into the epithelium. The size of these papillae varies considerably from region to region.
* Over the alveolar processes (where the mucosa forms the gums), and over the hard palate, the mucous membrane is closely adherent to underlying
periosteum. Elsewhere, it is connected to underlying structures by dense reticular layer. In the cheeks, this connective tissue contains many elastic fibers and much fat (especially in children).

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* Lips are fleshy folds which on the "external" surface are lined by skin, and on "internal" surface are lined by mucous membrane.
* The substance of each lip (upper or lower) is predominantly muscular (skeletal muscle) (Fig. 15.1).
* The upper and lower lips close along the red margin which represents the mucocutaneous junction. There is a transitional zone between the skin and mucous membrane referred as the vermilion, because of its pink color in fair-skinned individuals. This part meets the skin along a distinct edge.
* The "external" surface of the lip is lined by true skin in which hair follicles and sebaceous glands can be seen.
* The mucous membrane is lined by stratified squamous nonkeratinized epithelium (Plate 15.1). This epithelium is much thicker than that lining the skin (especially in infants). The epithelium has a well-marked fingerlike projections rete ridge system that extends into underlying connective
Fig. 15.1: Some relationships of the lips (schematic representation).
tissue and anchors the epithelium to underlying connective tissue. A similar arrangement is seen in the mucosa over the palate. This arrangement helps to withstand friction.
- * Beneath the epithelium, the mucosa has a layer of connective tissue (corresponding to the dermis), and a deeper layer of loose connective tissue. The latter contains numerous mucous glands, sebaceous glands (not associated with hair follicles). Their secretions prevent dryness and cracking of the exposed part of the mucosa.
LF
Fre
‘==

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Longitudinal section through the lip as seen in drawing:
*The substance of the lip is formed by a mass of muscle
* Each lip has an "external" surface covered by skin and an "internal" surface lined by mucous membrane
* There is a transitional zone between the skin and mucous membrane known as vermilion border.
Fig. 15.2: Vertical section through a tooth (schematic representation).

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A tooth consists of an "upper" part, the crown, which is seen in the mouth, and of one or more roots which are embedded in sockets in the jaw bone (mandible or maxilla). It is composed of three calcified tissues, namely: enamel, dentin, and the pulp (Figs. 15.2 and 15.3).
* The greater part of the tooth is formed by a bone-like material called dentin.
* Within the dentin, there is a pulp canal (or pulp cavity) that contains a mass of cells, blood vessels, and nerves that constitute the pulp. The blood vessels and nerves enter the pulp canal through the apical foramen (Fig. 15.3) which is located at the apex of the root.
* In the region of the crown, the dentin is covered by a much harder white material called the enamel.
* Over the root, the dentin is covered by a thin layer of cementum. The cementum is united to the wall of the bony socket in the jaw by a layer of fibrous tissue called the periodontal ligament.

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* Enamel is the hardest material in the body. It is made up almost entirely (96%) of inorganic salts.
* These salts are mainly in the form of complex crystals of hydroxyapatite (as in bone). The crystals contain calcium phosphate and calcium carbonate. Some salts are also present in amorphous form. The crystals of hydroxyapatite are arranged in the form of rod-shaped prisms, which run from the deep surface of the enamel (in contact with dentin) to its superficial (or free) surface.
* Prisms are separated by interprismatic material. Structure of prism and interprismatic material is same but they differ in their appearance due to difference in the orientation of hydroxyapatite crystals in them. The most superficial part of the enamel is devoid of prisms.
* During development, enamel is laid down in the form of layers. When seen in section, these layers can be distinguished as they are separated by lines running more or less parallel to the surface of the enamel. These lines are called the incremental lines or the lines of Retzius (Fig. 15.4).
* In some teeth, in which enamel formation takes place partly before birth and partly after birth (e.g. in milk teeth), one of the incremental lines is particularly marked. It represents the junction of enamel formed before birth with that formed after birth, and is called the neonatal line.
* At places, the enamel is penetrated by extraneous material. Projections entering the enamel from the dentin-enamel junction are called enamel tufts, and projections entering it from the free surface are called enamel lamellae (because of their shape). Dentinal tubules (see below) may extend into the enamel forming enamel spindles.
Fig. 15.3: Dried bone as seen in drawing.
Fig. 15.4: Part of a tooth to show some features of the structure of enamel and dentin (schematic representation).

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* Dentin is a hard material having several similarities to bone. It is made up basically of calcified ground substance (glycosaminoglycans) in which there are numerous collagen fibers (type 1).
* The calcium salts are mainly in the form of hydroxyapatite. Amorphous salts are also present.
* The inorganic salts account for 70% of the weight of dentin.
* Like bone, dentin is laid down in layers that are parallel to the pulp cavity.
The layers may be separated by less mineralized tissue that forms the incremental lines of Von Ebner.
* Dentin is permeated by numerous fine canaliculi that pass radially from the pulp cavity toward the enamel (or toward cement). These are the dentinal tubules. The tubules may branch especially near the enamel-dentin junction. Dentinal tubules extend into the enamel as enamel spindles.
* The ground substance of dentin is more dense (than elsewhere) immediately around the dentinal tubules, and forms the peritubular dentin or the dentinal sheath (of Neumann).
* Each dentinal tubule contains a protoplasmic process arising from cells called odontoblasts that line the pulp cavity. These protoplasmic processes are called the fibers of Tomes.
* Near the surface of the root of the tooth (i.e. just deep to the cementum), the dentin contains minute spaces that give a granular appearance. This is the granular layer of Tomes.

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1. Predentin: Unmineralized dentin at the junction of dentin and the cavity.
2. Mantle dentin: Dentin near the enamel-dentin junction is less minera (than elsewhere).
3. Circumpulpal dentin: Main part of dentin, i.e. between predentin and m dentin (Fig. 15.4).
4. Primary dentin: Dentin formed before eruption of the tooth.
5. Secondary dentin: Dentin formed after eruption of the tooth.

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* Cementum is the portion of tooth which covers the dentin at the root of tooth and is the site where periodontal ligament is attached. In old people, cementum may be lost, exposing the dentin.
* Cementum is similar to bone in morphology and composition and may be regarded as a layer of true bone that covers the root of the tooth.
* Toward the apex of the tooth, the cementum contains lacunae and canaliculi as in bone. The lacunae are occupied by cells similar to osteocytes (cementocytes). Some parts of cementum are acellular.
*Cementum is covered by a fibrous membrane called the periodontal membrane (or ligament). This membrane may be regarded as the periosteum of the cementum.
* Collagen fibers from this membrane extend into the cementum, and also into the alveolar bone (forming the socket in which the root lies) as fibers of Sharpey.
* The periodontal membrane fixes the tooth in its socket. It contains numerous nerve endings that provide sensory information.

* Dental pulp lies inner to dentin and occupies the pulp cavity and root canal.
* The dental pulp is made up of very loose connective tissue resembling embryonic mesenchyme (mucoid tissue).
* The ground substance is gelatinous and abundant. In it, there are many spindle-shaped and star-shaped cells.
* Delicate collagen fibers, numerous blood vessels, lymphatics, and nerve fibers are present. The nerve fibers are partly sensory and partly sympathetic.

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* The tongue is a conical muscular organ lies in the floor of the oral cavity.
*Tt has a dorsal surface that is free, and a ventral surface that is free anteriorly, but is attached to the floor of the oral cavity posteriorly.
* The dorsal and ventral surfaces become continuous at the lateral margins, and at the tip (or apex) of the tongue.
* Near its posterior end, the dorsum of the tongue is marked by a V-shaped groove called the sulcus terminalis (Fig. 15.5).
* The apex of the "V" points backward and is marked by a depression called the foramen cecum.
* The limbs of the sulcus terminalis run forward and laterally. The sulcus terminalis divides the tongue into a larger (two-thirds) anterior, or oral part, and a smaller (one-third) posterior, or pharyngeal part.
* The substance of the tongue is made up chiefly of skeletal muscle supported by connective tissue (Plate 15.2). The muscle is arranged in
bundles that run in vertical, transverse, and longitudinal directions. This arrangement of muscle permits intricate movements of the tongue associated with the chewing and swallowing of food, and those necessary for speech. The substance of the tongue is divided into right and left halves by a connective tissue septum.
Fig. 15.5: Dorsal surface of the tongue (schematic representation).
- * The surface of the tongue is covered by mucous membrane.

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* Mucous membrane of tongue is lined by stratified squamous epithelium. The epithelium is supported by a layer of connective tissue.
* On the ventral surface, the mucous membrane resembles that lining the rest of the oral cavity, and the epithelium is not keratinized.
* The mucous membrane covering the dorsum of the tongue is different over the anterior and posterior parts.
* Anterior part of the dorsal surface shows numerous projections called papillae.
* The mucous membrane of the posterior (pharyngeal) part of the dorsum of the tongue is devoid of papillae but bears numerous rounded elevations (lingual tonsil). Mucous membrane is separated from the underlying
muscles by submucous coat which contains serous and mucous glands, aggregation of lymphatic follicles called lingual tonsil. Each lymphatic follicle is aggregation of lymphocytes with central recess, to which the ductules of mucous glands open.

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Each papilla consists of a lining epithelium and a core of connective tissue. The epithelium over the papillae is partially keratinized (parakeratinized). The papillae are of four types (Figs. 15.6A to D):
* Filiform papillae: They are the most numerous papillae present on the anterior two-thirds of the tongue. They lie in rows parallel to sulcus terminalis and transversely oriented toward the apex. They are small and conical in shape. The epithelium at the tips of these papillae is keratinized, thus makes the surface rough and helps to grasp the food particles. They lack taste buds.
* Fungiform papillae: These are larger mushroom-shaped reddish papillae, with rounded summits and narrower bases present at the apex of the tongue and along its lateral margins. Fungiform papillae bear taste buds (described below). In contrast to the filiform papillae, the epithelium on fungiform papillae is not keratinized.
* Circumvallate papillae: These are the largest papillae of the tongue about 8-12 in number. They are arranged in a row just anterior and parallel to the sulcus terminalis. When viewed from the surface, each papilla is seen to have a circular top demarcated from the rest of the mucosa by a groove (Figs. 15.6A to D and Plate 15.3). Taste buds are present on the inner and outer walls of the groove trench and the floor receives the opening of ducts on serous glands. The secretion of serous glands makes the food soluble and also flushes it and thereby helps in the stimulation of taste buds in the groove. ate
Tongue (Anterior part): (A) As seen in drawing; (B) Photomicrograph.

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*The tongue is covered on both surfaces by stratified squamous epithelium (nonkeratinized)
*The ventral surface of the tongue is smooth, but on the dorsum the surface shows numerous projections or papillae
'Each papilla has a core of connective tissue covered by epithelium. Some papillae are pointed (filiform), while others are broad at the top (fungiform). A third type of papilla is circumvallate, the top of this papilla is broad and lies at the same level as the surrounding mucosa
The main mass of the tongue is formed by skeletal muscle seen below the lamina propria.
Muscle fibers run in various directions so that some are cut longitudinally and some transversely
'Numerous serous and mucous glands are present amongst the muscle fibers.
Figs. 15.6A to D: Papillae: (A) Filiform; (B) Fungiform; (C) Circumvallate; (D) Foliate (schematic representation).
- * Foliate papillae: These are three to five vertical folds of mucous membrane occasionally present in man, along the posterior part of lateral margin of the tongue. They contain taste buds. The folate papillae are more prominent in rabbits.

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* Numerous mucous and serous glands (of Von Ebner) are present in the connective tissue deep to the epithelium of the tongue.
* Mucous glands are most numerous in the pharyngeal part, in relation to the masses of lymphoid tissue. They open into recesses of mucosa that dip into the masses of lymphoid tissue.
* The serous glands are present mainly in relation to circumvallate papillae, and open into the furrows surrounding the papillae.
* The largest glands in the tongue are present on the ventral aspect of the apex. They contain both mucous and serous acini and are referred to as the anterior lingual glands of Blandin and Nuhn.
* Numerous serous and mucous glands are present among the muscle fibers.

Taste buds are present in relation to circumvallate papillae, fungiform papillae, and foliate papillae. Taste buds are also present on the soft palate, the epiglottis, the palatoglossal arches, and the posterior wall of the

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*In H&E staining, they are pale staining oval structures which extend through the entire thickness of the epithelium.
ate * Each bud has a small cavity that opens to the surface through a gustatory pore. The cavity is filled by a material rich in polysaccharides.
*, ate '= They are made up of modified epithelial cells (Fig. 15.7). The cells are elongated and are vertically orientated; those toward the periphery being curved like crescents (Fig. 15.8).
*, ate '= Each cell has a central broader part containing the nucleus and tapering ends.
he *The cells are of two basic types—receptor cells/gustatory cells/neuroepithelial cells and supporting cells/ sustentacular cells.
* Gustatory cells are chemoreceptors, present in the central portion of the taste bud. They are spindle-shaped with large spherical nucleus. They form basal synapse with special afferent nerves of the tongue.
* Supporting cells are barrel-shaped cells, usually present toward the periphery, and form an envelope for the taste bud.
* The average life of cells is about 10 days.
Fig. 15.7: Taste buds. 1—elongated cells; 2—pore; and 3— stratified squamous epithelium (schematic representation).
Vallate papilla: (A) As seen in drawing; (B) Photomicrograph.

*Circumvallate papillae are characterized by their dome-shaped structure lined by stratified squamous epithelium
"* Numerous oval-shaped lightly stained taste buds can be seen on the lateral wall of the papillae
*The underlying connective tissue contains serous glands of von Ebner

Fig. 15.8: Arrangement of cells in a taste bud (schematic representation).
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* Fordyce's granules: Fordyce's granules are symmetric, small, light yellow macular spots on the lips and buccal mucosa, and represent collections of sebaceous glands.
* Pyogenic granuloma: This is an elevated, bright red swelling of variable size occurring on the lips, tongue, buccal mucosa, and gingiva. It is a vasoproliferative inflammatory lesion. Pregnancy tumor is a variant of pyogenic granuloma.
* Cheilitis: Inflammation of lips.

* Dental caries: The most common disease of dental tissues, causing destruction of the calcified tissues of the teeth. Caries occurs chiefly in the areas of pits and fissures, mainly of the molars and premolars, where food retention occurs, and in the cervical part of the tooth.
* Pulpitis: It refers to the inflammation of the pulp. It may be acute, which is accompanied by severe pain and requires immediate intervention, or it may be chronic, where the pulp is exposed widely. It may protrude through the cavity forming polyp of the pulp.

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* Fissured tongue: It is a genetically-determined condition characterized by numerous small furrows or grooves on the dorsum of the tongue.
* Hairy tongue: In this condition, the filiform papillae are hypertrophied and elongated. These "hairs" (papillae) are stained black, brown, or yellowishwhite by food, tobacco, oxidizing agents, or by oral flora.
* Glossitis refers to inflammation of tongue.
* Bald tongue/atrophic glossitis: Tongue without papillae, usually seen in nutritional deficiencies.
* Geographic tongue: It appears as patches on the tongue without papillae.
li

It can be divided into three parts—masticatory mucosa, lining mucosa, and specialized mucosa.
* Masticatory mucosa: It lines the gum and hard palate which has keratinized (lacks stratum lucidum) and _ parakeratinized epithelium (superficial cells retain nuclei).
* Specialized mucosa: That presents on the dorsal surface of tongue which contains taste buds.
* Lining mucosa: The rest of the oral cavity has lining mucosa. It has fewer anchoring papillae so that it can adjust to the movement of underlying structures.

* Each tooth may be regarded as a highly modified form of the stratified squamous epithelium covering the developing jaw (alveolar process).
* A thickening of epithelium grows downward into the underlying connective tissue and enlarges to form an enamel organ (Figs. 15.9A to

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"Bud stage (Fig. 15.9A): The enamel organ resembles a small bud, whict surrounded by the condensation of ectomesenchymal cells. In this stage, 1 enamel organ is made up of peripherally located low columnar cells é centrally located polygonal cells.
"Cap stage (Fig. 15.9B): The enamel organ then proliferates to form a ¢ over the central condensation of ectomesenchymal cells which is called : dental papilla. The dental papilla and the dental sac become well-defin: Three layers are differentiated from the enamel organ are as follows:
1. Inner dental/inner enamel epithelium
2. Stellate reticulum
3. Outer dental/outer enamel epithelium.
" Early bell stage (Fig. 15.9C): The enamel organ resembles bell shape a result of deepening of the undersurface of the epithelial cap. A cell la forms in between the inner dental epithelium and stellate reticulum cal the stratum intermedium.
Figs. 15.9A to D: Stages of tooth development: (A) Bud stage; (B) Cap stage; (C) Early bell stage; (D) Advance bell stage (schematic representation).
The inner dental epithelium differentiates into tall columnar cells called the ameloblasts. The peripheral cells of the dental papilla differentiate into odontoblasts.
Under form the organizing influence of inner dental epithelium, ameloblasts enamel and odontoblasts produce dentin.
"Advanced bell stage (Fig. 15.8D): In this stage, apposition of dental hi tissues occurs. Odontoblasts are dentin forming cells; ameloblasts . enamel forming cells. A layer of predentin is secreted by the odontobla: Both ameloblasts and odontoblasts behave in a way very similar to that osteoblasts and lay down layer upon layer of enamel, or of dentin. T deposition of enamel and dentin continues until the crown formation complete.

The formation of layers of enamel and dentin results in separation of ameloblasts and odontoblasts. The original line at which enamel and dentin formation begins is known as the enamel-dentin junction. At last, ameloblasts come to line the external aspect of the enamel. The odontoblasts persist as a lining for the pulp cavity.
When examined by EM, both ameloblasts and odontoblasts show the features typical of actively secreting cells. They have prominent Golgi complexes and abundant rough ER. The apical part of each cell is prolonged into a process. In the case of odontoblasts, the process runs into the proximal part of a dentinal tubule. In ameloblasts, the projection is called Tomes process. This process contains numerous microtubules and many secretory vesicles. Other smaller processes are present near the base of Tomes process. The organic matrix of enamel is released mainly by Tomes process, which also appears to be responsible for forming prisms of enamel.

Root formation is carried out by Hertwig's epithelial root sheath which is formed by the cervical portion of the enamel organ. It molds the shape of the roots and initiates radicular dentin formation.
The root sheath encloses the dental pulp except at apical portion. The rim of root sheath, the epithelial diaphragm surrounds the primary apical foramen. The root apex remains wide open until about 2 to 3 years after the eruption of the tooth, when the root development is completed.

a. Inner border of lip
b. Outer border of lip
c. Mucocutaneous junction
d. Angle of mouth

Predentin
Mantle dentin Circumpulpal dentin Primary dentin 2905p

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a. Dentin
b. Enamel
c. Cementum
d. Pulp tissue
7. Structure which covers the dentin at the root is:
a. Crown

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* General structure of GIT
*Layers of GIT with detailed structure
* Structure of esophagus
* Layers, cells, and functions of stomach
* Structure of small intestine: Duodenum, jejunum, ileum
* Differences between duodenum, jejunum, and ileum
* Structure of colon and vermiform appendix
* Structure of rectum and anal canal

villi, lined by simple columnar epithelium Q with numerous goblet cells Q Lymphatic follicles dispersed in the lamina propria Q Q Presence of Taenia coli, appendices epiploicae. Q, Stomach (Fundus) Stomach = Wall made up of four layers Mucosa—absence of villi, lined by simple columnar epithelium with numerous goblet cells Submucosa packed with numerous lymphatic follicles, extending into lamina propria Absence of Taenia coli, appendices epiploicae.. Q Wall made up of four layers Q Q Mucosa—absence of villi, lined by simple columnar epithelium Q with numerous goblet cells Q Lymphatic follicles dispersed in the lamina propria Q Q Presence of Taenia coli, appendices epiploicae. Q, (Pylorus) = Wall made up of four layers Mucosa—absence of villi, lined by simple columnar epithelium with numerous goblet cells Submucosa packed with numerous lymphatic follicles, extending into lamina propria Absence of Taenia coli, appendices epiploicae.
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* The gastrointestinal tract (GIT) or alimentary canal is a long muscular tube that begins at the oral cavity and ends in the anus.
* Different parts of the tract are specialized to perform different functions, and hence structural modifications are seen in various parts of the GIT.
* The esophagus and anal canal are merely transport passages.
* The part of the alimentary canal from the stomach to the rectum is the proper digestive tract, responsible for digestion and absorption of food.
* Reabsorption of secreted fluids is an important function of the large intestine.

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* The structure of the alimentary canal, from the esophagus up to the anal canal, shows several features that are common to all these parts.
* From the upper end of the esophagus up to the lower end of the anal canal, the alimentary canal has the form of a fibromuscular tube.
* The wall of the tube is made up of the following layers (from inner to outer side) (Fig. 16.1):
= The innermost layer is the mucous membrane that is made up of:
A lining epithelium
*A layer of connective tissue, the lamina propria, that support epithelium
* A thin layer of smooth muscle called the muscularis mucosae.
"The mucous membrane rests on a layer of loose areolar tissue called 1 submucosa.
= Thick layer of muscle muscularis externa that surrounds the submucosa
"Covering the muscularis externa, there is a serous layer or an adventit layer.
Primarily, it is the mucosa in which changes are seen in the alimenti tract; the other layers remain almost the same.

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"
The lining epithelium is columnar all over the gut; except in the esophag and in the lower part of the anal canal, where it is stratified squamous.
"This stratified squamous epithelium has a protective function in the structures.
™ The cells of the columnar epithelium are either absorptive or secretory.
"The epithelium of the gut presents an extensive absorptive surface.
Fig. 16.1: Layers of the gut (schematic representation).
factors contributing to the increase in the surface are as follows:

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* The presence of numerous folds involving the entire thickness mucous membrane. These folds can be seen by naked eye. The subm
extends into the folds (Plica circularis of small intestine).
* At numerous places the epithelium dips into the lamina propria fo: crypts.
* In the small intestine, the mucosa bears numerous finger-like proc that project into the lumen. These processes are called villi. Each vilh a surface lining of epithelium and a core formed by an extension | connective tissue of the lamina propria. The luminal surfaces c epithelial cells bear numerous microvilli.
"The epithelium of the gut also performs a very important secretc function. The secretory cells are arranged in the form of numerous glar as follows:
* Some glands are unicellular, the secretory cells being scattered amo! cells of the lining epithelium.
* In some organs, the epithelium dips into the lamina propria forming s tubular glands (These are the crypts referred to above).
* In other structures (e.g. in the esophagus, duodenum) there are comp tubuloalveolar glands lying in the submucosa. They open into the |] of the gut through ducts traversing the mucosa.
* Finally, there are glands—salivary glands, pancreas, and liver that distinct organs lying outside the gut wall. They pour their secretion the lumen of the gut through large ducts (in this respect, these glan: similar to the salivary glands). The liver and pancreas, and most « salivary glands, are derivatives of the epithelial lining of the Embryologically, this epithelium is derived from endoderm.

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=
The lamina propria is made up of collagen and reticular fibers embedded a glycosaminoglycan matrix. Some fibroblasts, blood capillaries, lym vessels, and nerves are seen in this layer.
"In the small intestine, the lamina propria forms the core of each villus. surrounds and supports glandular elements and the overlying epithelium.
"Mucosal glands also extend into lamina propria of the gut and are read seen in certain organs like esophagus and anal canal. They mainly lubric the mucosa and protect it from chemical injury.
"Prominent aggregations of lymphoid tissue (as well as scattel lymphocytes) are present in the lamina propria. Some of this lymph
tissue extends into the submucosa and is called as gut-associated lymph« tissue (GALT).

" This is a thin layer of smooth muscle that separates the connective tissue the lamina propria from the submucosa.
"It consists of an inner layer in which the muscle fibers are arrang circularly (around the lumen) and an outer layer in which the muscle fib run longitudinally.
= The muscularis mucosa extends into mucosal folds, but not into villi.
™Contractions of the muscularis mucosae are important for the local mixi of intestinal contents and also facilitate absorption and secretion.

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* This layer made up of dense irregular connective tissue connects the mucosa to the muscularis externa.
* Numerous blood vessels, lymphatics, and nerve fibers traverse the submucosa.
* Smaller branches of larger blood vessels enter the mucous membrane, muscularis externa, and serosa.
* Submucosal plexus of unmyelinated nerve fibers and ganglion cells constitute Meissner's plexus in this layer.
*In addition, at certain regions, submucosa contains mucous glands (esophagus and proximal part of duodenum).

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* Over the greater part of the gut, the muscularis externa consists of smooth muscle. The exceptions are the upper part of the esophagus and anal canal where this layer contains striated muscle fibers.
* The muscle layer consists (typically) of an inner layer of circular layer and an outer longitudinal layer of smooth muscle fibers.
* Both layers actually consist of spirally arranged fasciculi, the turns of the spiral being compact in the circular layer, and elongated in the longitudinal layer.
* The arrangement of muscle fibers shows some variation from region to region. In the stomach, an additional oblique layer is present. In the colon,
the longitudinal fibers are gathered to form prominent bundles called the taenia coli.
* Localized thickenings of circular muscle fibers form sphincters that can occlude the lumen of the gut. For example, the pyloric sphincter is present around the pyloric end of the stomach, and the internal anal sphincter surrounds the anal canal. A functional sphincter is seen at the junction of the esophagus with the stomach. A valvular arrangement at the ileocaecal junction (ileocaecal valve) prevents regurgitation of cecal contents into the ileum.
* Thin layer of connective tissue is present between the two muscle layers. It contains plexus of nerves called Myenteric plexus/Auerbach's plexus. It also contains blood vessels and lymphatics.
* Contraction of muscles in this layer helps in mixing and propulsion of luminal contents by slow rhythmic movement called Peristalsis.

* Outermost layer of the gut, covering the muscle coat.
* It is a serous membrane lined by simple squamous epithelium called mesothelium and minimal amount of connective tissue. This layer is nothing but visceral peritoneum that covers most parts of the gastrointestinal tract and extends over the abdominal wall to form parietal peritoneum.
* Large blood vessels, lymphatics, and nerve trunk traverse through serosa.
* In some places where a peritoneal covering is absent (e.g. thoracic part of the esophagus, duodenum, etc.) the muscle coat is covered by an adventitia made up of connective tissue only.

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The gut is richly innervated by autonomic nervous system (enteric nervous system). The nerve fibers form two distinct plexus:
1. The submucosal plexus of Meissner, that lies in the submucosa (nec junction with the circular muscle layer) has only parasympathetic f which are secretomotor.
2. The myenteric plexus of Auerbach, that lies between the circular longitudinal coats of muscularis externa has both sympathetic
parasympathetic fibers but only cell bodies of parasympathetic neurons.
These plexus are both under intrinsic and extrinsic control. Nerve fibers in these plexuses are both afferent and efferent.
i

* The esophagus is a long muscular tube beginning at the end of cricoid cartilage and opens into the cardiac end of stomach.
* Tt conducts chewed food (bolus) and liquids to stomach.
* Its length is about 25 cm.

The wall of esophagus (Plate 16.1) has the usual four layers viz., mucosa, submucosa, muscularis externa, and an external adventitia. The esophagus does not have a serous covering except over a short length near its lower end.

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* The mucous membrane of the esophagus shows several longitudinal folds that disappear when the tube is distended.
* The mucosa is lined by stratified squamous nonkeratinized epithelium. Occasionally melanocytes and endocrine cells are encountered.
* Finger-like processes (or papillae) of the connective tissue of the lamina propria project into the epithelial layer (just like dermal papillae). This helps to prevent separation of epithelium from underlying connective tissue.
* At the upper and lower ends of the esophagus, some tubuloalveolar mucous glands are present in the lamina propria which are called as esophageal cardiac glands.
* The muscularis mucosae are made of longitudinally oriented smooth muscle fibers which are thick in the upper end of the esophagus and help in swallowing.

- * The only special feature of the submucosa is the presence of compound tubuloalveolar mucous glands called esophageal glands proper, more
concentrated in the upper half of the esophagus. They help to lubricate the luminal wall.
- * Small aggregations of lymphoid tissue may be present in the submucosa, especially near the lower end. Some plasma cells and macrophages are also present.

* Consists of inner circular and outer longitudinal layer of smooth muscle fibers. But in the upper third of the esophagus, it is made of only skeletal muscle fibers.
* In the middle third, it is a combination of both smooth muscle and skeletal muscle fibers.
* In the lower third, it is made up of only smooth muscle fibers like the rest of the GIT.
* Myenteric plexus is present between outer and inner muscular layer which produces peristalsis by stimulating muscularis externa.

* The muscle layer of the esophagus is surrounded by dense fibrous tissue that forms an adventitial coat for the esophagus.
* The lowest part of the esophagus is intra-abdominal and has a covering of peritoneum.

The circular muscle fibers present at the lower end of the esophagus could possibly act as a sphincter guarding the cardioesophageal junction. However, the circular muscle is not thicker here than elsewhere in the esophagus, and its role as a sphincter is not generally accepted. However, a physiological sphincter does appear to exist. The anatomical factors that could account for this sphincteric action are not agreed upon.

* Stomach is a hollow muscular organ that receives food bolus from esophagus.
* The food passes through the esophagus and enters the stomach where it is converted into a thick paste known as chyme.
* Anatomically, stomach is divided into four regions: (1) cardia, (2) fundus,

- * Histologically fundus and body of stomach have a similar structure.
Fig. 16.2: Anatomical regions of stomach (schematic representation).

* Four layers of gastrointestinal tract (GIT): Mucosa, submucosa, muscularis externa, adventitia seen
* Lining epithelium is stratified squamous nonkeratinized epithelium
* Submucosa is studded with mucus-secreting esophageal glands.

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* Tt acts as temporary reservoir of food.
* Secretes copious amount of mucous, so that prevents autodigestion of the tissues.
* Helps in digestion of proteins.
* Acidic environment of stomach provides protection against microbes.
* Intrinsic factor secreted in the stomach aids in absorption of vitamin B,,.

The wall of the stomach has the four basic layers, (Plates 16.2 and 16.3 and Table 16.1) from within outward they are: (1) mucous membrane, (2) submucosa, (3) muscularis externa, and (4) serosa.

* As seen with the naked eye, (Fig. 16.3) the mucous membrane is thick, soft, and velvety, presents numerous vertical folds (or rugae) that disappear when the stomach is distended.
* Mucous membrane consists of lining epithelium, supported by lamina propria and muscularis mucosa.

* The surface and gastric pits are lined by simple columnar epithelium resting on basement membrane.
* They are mucus-secreting cells with apical large mucin granules which are usually removed during processing of tissues so that the cells look empty (or vacuolated).
* Mucus secreted by cells of the lining epithelium protects the gastric mucosa against acid and enzymes produced by the mucosa itself.
* At numerous places, the lining epithelium dips into the lamina propria and forms numerous depressions called gastric pits (Fig. 16.3). Deep to the gastric pits, the mucous membrane is packed with numerous gastric glands. Depending on their presence in different regions of stomach, these glands are of three types: (1) Gastric (fundic), (2) cardiac, and (3) pyloric.

The main gastric glands are present over most of the stomach except in the pyloric region and in a small area near the cardiac end. Though they are

Fig. 16.3: Basic structure of the mucous membrane of the stomach (schematic representation).
Table 16.1: Salient features of each region of stomach., 1 = Table 16.1: Salient features of each region of stomach.. Table 16.1: Salient features of each region of stomach., 2 = Table 16.1: Salient features of each region of stomach.. Cardia, 1 = Fundus and body. Cardia, 2 = Pylorus. Shallow gastric pits, 1 = Shallow gastric pits occupying superficial one- fourth or less of the mucosa. Shallow gastric pits, 2 = Deep gastric pits occupying two-thirds of the depth of the mucosa. Presence of cardiac glands (mucous secreting glands) in the mucosa, 1 = Presence of gastric glands in the mucosa. Presence of cardiac glands (mucous secreting glands) in the mucosa, 2 = Presence of pyloric glands in the mucosa. Cardiac glands are either simple tubular, or compound tubuloalveolar, 1 = Gastric glands are simple or branched tubular glands. They secrete enzymes and hydrochloric acid. Cardiac glands are either simple tubular, or compound tubuloalveolar, 2 = Pyloric glands (mucous glands) are simple or branched tubular glands that are coiled. Change of epithelium from stratified squamous of the esophagus to simple columnar epithelium in stomach, 1 = Epithelium is simple columnar.. Change of epithelium from stratified squamous of the esophagus to simple columnar epithelium in stomach, 2 = Circular muscle layer is thick and is called as pyloric sphincter
ral ee

- *® Presence of four layers—mucosa, submucosa, muscularis externa, and serosa
*®
- Shallow gastric pits occupying superficial one-fourth or less of the mucosa
>

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Stomach (Pylorus): (A) As seen in drawing; (B) Photomicrograph (low power); (C)
Photomicrograph (high power).
Presence of four layers—mucosa, submucosa, muscularis externa, and serosa
*Deep gastric pits occupying two-thirds of the depth of the mucosa
Presence of pyloric glands (mucous glands) in the mucosa that are simple/branched tubular glands which are coiled.
* Simple or branched tubular glands that lie at right angles to the mucosal surface.
* Open into gastric pits, each pit receiving the openings of several glands. Here the gastric pits occupy the superficial one-fourth or less of the mucosa, the remaining thickness being closely packed with gastric glands.
The following types of cells are present in the epithelium lining the glands.

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"They are most numerous cells particularly in the basal parts of the glands
"The cells are cuboidal or low columnar. Their cytoplasm is basophilic.
"With special methods, the chief cells are seen to contain promin: secretory granules in the apical parts of their cytoplasm. The granu contain pepsinogen that is a precursor of pepsin.
= With the EM, the cytoplasm is seen to contain abundant rough endoplasn reticulum and a prominent Golgi complex. The luminal surfaces of the ce bear small irregular microvilli.
"Chief cells secrete the digestive enzymes of the stomach including peps Pepsin is produced by action of gastric acid on pepsinogen. Pepsin bre< down proteins into small peptides. It is mainly through the action of pep. that solid food is liquefied.

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" They are large, ovoid or polyhedral, with a large central nucleus (Fig. 16.
"They are present singly, sandwiched between peptic cells and basem: membrane. They are more numerous in the upper half of the gland than its lower half.
" They are called oxyntic cells because they stain strongly with eosin.
"They are called parietal cells as they lie against the basement membra and often bulge outward (into the lamina propria) creating a beac appearance.
"The EM shows that each parietal cell has a narrow apical part that react the lumen of the gland. The apical cell membrane shows seve invaginations into the cytoplasm, producing tortuous intracellular canalic that communicate with the glandular lumen. The walls of the canalic bear microvilli that project into the canaliculi. The cytoplasm (in | intervals between the canaliculi) is packed with mitochondria. 1 mitochondria are responsible for the granular appearance and eosinophi of the cytoplasm (seen with the light microscope). Secretory granules . not present.
"Oxyntic cells secrete hydrochloric acid. They also produce an intrin factor of Castle (a glycoprotein) that combines with vitamin B,, (present
Fig. 16.4: Electron microscope structure of an oxyntic cell (schematic representation).
ingested food and constituting an extrinsic factor) to form a comp) necessary for normal formation of erythrocytes.

"They are large cells with a clear cytoplasm found near the upper end "neck") of the glands, also called mucous neck cells.
"The nucleus is flattened and is pushed to the base of the cell accumulated mucous.
"The supranuclear part of the cell contains prominent granules. The chemi structure of the mucus secreted by these cells is different from that secre by mucous cells lining the surface of the gastric mucosa.

"Near the basal parts of the gastric glands there are endocrine cells t contain membrane bound neurosecretory granules.
"As the granules stain with silver salts, these have been called argentaf, cells in the past.
"These cells are flattened. They do not reach the lumen, but lie between i chief cells and the basement membrane.
" These cells probably secrete the hormone gastrin. Some of the cells can shown to contain serotonin (5HT).

"Some undifferentiated cells (stem cells) that multiply to replace other ce are also present.
"They increase in number when the gastric epithelium is damaged ( example, when there is a gastric ulcer), and play an important role healing.

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* These are confined to a small area (cardia) near the opening of the esophagus.
* In this region, the mucosa is relatively thin.
* Gastric pits are shallow (as in the body of the stomach).
* The cardiac glands are either simple tubular, or compound tubule-alveolar.
* They are mainly mucus-secreting. An occasional oxyntic or peptic cell may be present.

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* In the pyloric region of the stomach, the gastric pits are deep and occupy two-thirds of the depth of the mucosa.
* The pyloric glands that open into these pits are short and occupy the deeper one-third of the mucosa.
* They are simple or branched tubular glands that are coiled.
* The glands are lined by mucus-secreting cells. Occasional oxyntic and argentaffin cells may be present.
* In addition to other substances, pyloric glands secrete the hormone gastrin.

* The lamina propria is relatively scanty.
* Made of reticular fibers and other connective tissue cells.
* Occasional aggregations of lymphoid tissue are present in it.

* The muscularis mucosae of the stomach is thin.
* Apart from the usual circular (inner) and longitudinal
(outer) layers, an additional circular layer may be present outside the longitudinal layer.
- * The smooth muscle helps in the expulsion of the secretion of gastric glands.

Consists of loose areolar tissue with blood vessels, lymphatics, and nerve plexus.

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- * The muscularis externa of the stomach is well-developed.
Made of three layers: (1) inner oblique, (2) middle circular, and (3) longitudinal layer.
* The appearance of the layers in sections is, however, highly variable depending upon the part of the stomach sectioned.
* Longitudinal layer covers the entire organ and is more thick along the curvatures. At the pyloric end, it forms two strata: (1) superficial and (2) deep. Superficial layer is continuous with the corresponding layer of duodenum, whereas deep layer forms dilator component of pyloric sphincter. This layer helps in expulsion of gastric contents into duodenum.
* The circular fibers are greatly thickened at the pylorus where they form the pyloric sphincter (sphincteric component). This layer retains the gastric content in situ till completion of digestion. Unlike the longitudinal layer, it is not continuous with that of duodenum.
* Both circular and longitudinal layer are continuous with corresponding muscle of esophagus but does not form any anatomical sphincter.
* Oblique layer covers the fundus and the body. Has inverted "U" shaped loop of smooth muscle fibers covering the adjoining surfaces. Along the lesser curvature it forms free margin, contraction of which makes the cardioesophageal angle more acute, thereby prevents regurgitation of gastric contents into esophagus.
* Between the layers of muscles there exists, myenteric/Aurebach's plexus of nerves.

Gastric serosa is derived from peritoneum and is continuous with the parietal peritoneum of abdominal cavity via greater omentum and visceral peritoneum of liver via lesser omentum. But the lesser curvature, greater curvature, and bare area of stomach are devoid of serosa.

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The small intestine is a tube about 5 m long. It is divided into three parts.
In the craniocaudal sequence, these are:
* Duodenum (about 25 cm long)
* Jejunum (about 2 m long)
* Tleum (about 3 m long).
Small intestine is the principal site for absorption of products of digestion.
It also secretes some hormones through enteroendocrine cells. Complete digestion of ingested food occurs in small intestine.

* The wall of the small intestine is made up of the four layers: (1) mucosa, (2) submucosa, (3) muscularis externa, and (4) serosa (Fig. 16.5).
* The serosal and muscular layers correspond exactly to the general structure of alimentary canal.
* The submucosa is also typical except in the duodenum, where it contains the glands of Brunner.
* The mucous membrane exhibits several special features that are discussed below.

Lumen-wider, wall-thicker, Table 16.2: Distinguishing features of duodenum, jejunum, and ileum (Plates 16.4 to 16.6)..Jejunum = Lumen-wider, wall- thicker. Lumen-wider, wall-thicker, Table 16.2: Distinguishing features of duodenum, jejunum, and ileum (Plates 16.4 to 16.6)..Ileum = Lumen-narrow, wall- thinner. Large, thick circular folds in the lumen, Table 16.2: Distinguishing features of duodenum, jejunum, and ileum (Plates 16.4 to 16.6)..Jejunum = Large, thick circular folds in the lumen. Large, thick circular folds in the lumen, Table 16.2: Distinguishing features of duodenum, jejunum, and ileum (Plates 16.4 to 16.6)..Ileum = Small, few circular folds in the lumen. Numerous broad villi, Table 16.2: Distinguishing features of duodenum, jejunum, and ileum (Plates 16.4 to 16.6)..Jejunum = Numerous tall villi. Numerous broad villi, Table 16.2: Distinguishing features of duodenum, jejunum, and ileum (Plates 16.4 to 16.6)..Ileum = Only few, short and slender villi. Mucus-secreting Duodenal glands or glands of Brunner, Table 16.2: Distinguishing features of duodenum, jejunum, and ileum (Plates 16.4 to 16.6)..Jejunum = Very few Peyer patches extending into submucosa-only in the distal part. Mucus-secreting Duodenal glands or glands of Brunner, Table 16.2: Distinguishing features of duodenum, jejunum, and ileum (Plates 16.4 to 16.6)..Ileum = Numerous Peyer's patches extending into mucosa
Fig. 16.5: Basic structure of the small intestine (schematic representation).

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Mucous membrane is made up of lining epithelium resting on basement membrane, lamina propria, and muscularis mucosa. The surface area of the mucous membrane of the small intestine is extensive (to allow adequate absorption of food). This is achieved by the following features.
* The considerable length of the intestine.
** The presence of numerous circular folds (plicae circularis) in the mucosa.
* The presence of numerous finger-like processes, or villi, that project from the surface of the mucosa into the lumen.
** The presence of numerous depressions or crypts that invade the lamina propria.
* The presence of microvilli on the luminal surfaces of the cells lining the

"They are permanent circular folds, called the Valves of Kerckring. I folds are large and readily seen with the naked eye. Sometimes they may semilunar or spiral.
"Each fold is made up of all layers of the mucosa (lining epithelium, lam: propria, and muscularis mucosae). The submucosa also extends into | folds (Fig. 16.6).
"They are absent in the first 1 or 2 inches of the duodenum. They « prominent in the rest of the duodenum, and in the whole of the jejunu The folds gradually become fewer and less marked in the ileum. T terminal parts of the ileum have no such folds.
Fig. 16.6: Longitudinal section through a part of the small intestine seen at a very low magnification to show a circular mucosal fold (schematic representation). Note the submucosa extending into the mucosal fold.

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Wall made up of four layers
"*Mucosa—presence of numerous broad villi, lined by simple columnar epithelium with microvilli and goblet cells
”
Presence of submucous Brunner's gland.
"Wall made up of four layers
*Mucosa—presence of numerous tall slender villi, ined by simple columnar epithelium with microvilli and goblet cells
* Absence of submucous Brunner's gland.
*Mucosa—presence of short, small fewer villi, lined by simple columnar epithelium with
”
microvilli and numerous goblet cells
Absence of submucous Brunner's gland.
"Apart from adding considerably to the surface area of the mucc membrane, the circular folds tend to slow down the passage of conte through the small intestine thus facilitating absorption.

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"The villi are, typically, finger-like projections consisting of a core reticular tissue covered by a surface epithelium (Fig. 16.6).
"The connective tissue core contains numerous blood capillaries formin; plexus.
"The endothelium lining the capillaries is fenestrated thus allowing raj absorption of nutrients into the blood.
"Each villus contains a central lymphatic vessel called a lacteal. Lympha capillary is blind toward the tip and ends in a plexus of lymphatic vess present in the lamina propria. They are concerned with fat absorpti: Occasionally, the lacteal may be double.
"Some muscle fibers derived from the muscularis mucosae extend into 1 villus core and surround the lacteal.
"Lamina propria contains reticular fibers and other connective tissue ce. Plasma cells of lamina propria provide surface immunity by secreting Ig/
3. Crypts: The crypts (of Lieberkiihn) are tubular invaginations of epithelium into the lamina propria. They are simple tubular intestinal gl which open between the bases of adjacent villi.

* The epithelium lining the luminal surface (villi, and areas intervening between them) is mainly columnar, specialized for absorption. The tall columnar cells are called enterocytes. Scattered amongst the columnar cells there are mucus-secreting goblet cells.
*The cells lining the crypts (intestinal glands) are predominantly undifferentiated. These cells multiply to give rise to absorptive columnar
cells and to goblet cells.
* Near the bases of the crypts there are Paneth cells that secrete enzymes. Endocrine cells (bearing membrane bound granules filled with various neuroactive peptides) are also present.

"Each cell has an oval nucleus located in its lower part. When seen with : light microscope the luminal surface of the cell appears to be thickened < to have striations in it, perpendicular to the surface (Fig. 16.7).
"This striated border is made up of microvilli arranged in a very regu manner as seen through electron microscope. The presence of microv greatly increases the absorptive surface of the cell.
"Each microvillus has a wall of plasma membrane containing fine filamer These filaments extend into the apical part of the cell where they beco! continuous with a plexus of similar filaments and form terminal web. '1 surface of each microvillus is covered by a layer of fine fibrils and mucc (glycocalyx).
"The plasma membrane on the lateral sides of absorptive cells shows fo that interdigitate with those of adjoining cells. Adjacent cells are united typical junctional complexes and by scattered desmosomes. Intercellu canals may be present between adjacent cells. The cytoplasm of absorpt cells contains the usual organelles, including lysosomes and smooth E These cells are responsible for absorption of amino acids, carbohydrat and lipids present in digested food.

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"Each goblet cell has an expanded upper part that is distended with mu granules (Figs. 16.8 and 16.9).
= The nucleus is flattened and is situated near the base of the cell.
Fig. 16.7: Cells of small intestine (schematic representation).
Fig. 16.8: Columnar epithelium lining the small intestine. Note the striated border and some goblet cells (schematic representation).
Fig. 16.9: High power view of an intestinal crypt showing Paneth cells with red staining supranuclear granules (schematic representation).
Courtesy: Dr V Subhadra Devi, SVIMS-SPMC (W), Tirupati.
"Goblet cells are mucus-secreting cells. In consonance with their secretc function, these cells have a prominent Golgi complex and abundant rou endoplasmic reticulum toward the basal region.
™ The luminal surface of the cell bears some irregular microvilli.
"In routine hematoxylin and eosin (H&E) staining, the apical part of got cells appears pale, as the mucin granules are washed out duri preparations. It stains brightly with the PAS technique.
™ Mucous cells increase in number as we pass down the small intestine, bei few in the duodenum and most numerous in the terminal ileum.

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"These are columnar cells present in the walls of intestinal crypts. They « similar to absorptive cells, but their microvilli and terminal webs are not well developed. The cytoplasm contains secretory granules.
"Undifferentiated cells proliferate actively by mitosis. The newly form cells migrate upward from the crypt to reach the walls of villi.
"Here they differentiate either into typical absorptive cells, or into got cells. These cells migrate toward the tips of the villi where they are st off. In this way, the epithelial lining is being constantly replaced, each c
having a life of only a few days.
- "The term "intermediate cells" has been applied to differentiating stem ce that show features intermediate between those of stem cells and fu differentiated cells.

" These cells are found only in the deeper parts of intestinal crypts.
"They contain prominent eosinophilic secretory granules (Figs. 16.8 ¢ 16.9).
= With the EM, Paneth cells are seen to contain considerable Golgi apparat rough endoplasmic reticulum, and other organelles.
"They are known to produce lysozyme that destroys bacteria. They may a produce other enzymes.

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"Cells containing membrane-bound vesicles filled with neuroact substances are present in the epithelial lining of the small intestine.
"They are most numerous near the lower ends of crypts. As the granules them stain with silver salts, these cells have, in the past, been term argentaffin cells (Fig. 16.10).
Fig. 16.10: High power view of intestinal crypt showing argentaffin cell with black staining that are infranuclear granules mostly (schematic representation).
- "Some of them also give a positive chromaffin reaction, so also cal enterochromaffin cells. With the introduction of immunohistochemi techniques, it has now been demonstrated that these cells are of varic functional types, and contain many amines having an endocrine function.

"They are epithelial cells lying on the Peyer's patch or large lymphe aggregation.
"They possess microfolds instead of microvilli, through which they eng the microbes gaining access to the lumen.
"In the form of vesicles, these microbes are transported to the basolate surface to the vicinity of T lymphocytes in the intercellular space, her stimulating the GALT. Hence they are also called as antigen-transporti cells.

Made of meshwork of reticular fibers; is thickly populated with lymphatic aggregations, lymphocytes, plasma cells and other components of immune system apart from connective tissue associated fibroblasts, eosinophils, leukocytes, macrophages, and mast cells.
* Solitary and aggregated lymphatic follicles (Peyer's patches) are present in the lamina propria of the small intestine constituting GALT. The solitary follicles become more numerous, and the aggregated follicles larger, in proceeding caudally along the small intestine. They are most prominent in the terminal ileum (Plate 16.6). Their lymphoid tissue may occasionally extend into the submucosa. Villi are few or missing in the mucosa overlying aggregated follicles.
* Peyer's patches (see later; Plate 16.6) can be seen by naked eye, and about 200 of them can be counted in the human gut. The mucosa overlying them is devoid of villi or may have rudimentary villi. Peyer's patches always lie along the antimesenteric border of the intestine and measure 2 cm to 10 cm.
* Lymphocytes are also present between the epithelial cells.

Consists of inner circular and outer longitudinal layers of smooth muscles.
* Few fibers extend to the mucosa and help in the movement of villi and expulsion of secretions from the intestinal glands.

* Consists of loose areolar tissue containing blood vessels, lymphatics, and nerve plexus (Meissner's plexus).
* Submucosal glands of Brunner are the characteristic feature of duodenum. They are branched tubular glands, secreting mucus which is rich in bicarbonate ions.

* Made of inner circular and outer longitudinal layer.
* Contains myenteric plexus between these two layers.
* Inner circular mainly concerned with segmentation movement (local mixing of chime). Peristalsis involves coordinated contraction of both layers for propulsion of intestinal contents.

* Is derived from peritoneum surrounding the tube except at the attachment of mesentery.
* Tt is incomplete in duodenum.

Bi
*Tt consists of the cecum, appendix, colon (ascending, transverse, descending, and sigmoid), rectum, and anal canal.
* The main functions of the large intestine are absorption of water and conversion of the liquid, undigested material into solid feces.
* It harbors some nonpathogenic bacteria that produce vitamin B,, necessary for hemopoiesis and vitamin K required for coagulation of blood.

The structure of the colon conforms to the general description of the structure of the gut by having four layers from inside to outside—mucosa, submucosa, muscularis externa, and serosa (Table 16.3 and Plate 16.7).

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* The mucous membrane of the colon shows numerous crescent-shaped folds but Villi are absent.
* The mucosa shows numerous closely arranged tubular glands or crypts similar to those in the small intestine.
* The mucosal surface and the glands are lined by an epithelium made up predominantly of columnar cells. Their main function is to absorb excess water and electrolytes from intestinal contents.
* Numerous goblet cells are present, their number increasing in proceeding caudally. The mucous secreted by them serves as a lubricant that facilitates the passage of semisolid contents through the colon.
* Some endocrine cells and stem cells are seen but Paneth cells are absent.
* The epithelium overlying solitary lymphatic follicles (present in the lamina
Fig. 16.11: Basic features of the structure of the mucous membrane of the large intestine (schematic representation).
propria) contains M-cells similar to those described in the small intestine. Scattered cells bearing tufts of long microvilli are also seen. They are probably sensory cells.
- * Lamina propria is made up of thick coat of collagen fibers present between the basal lamina of epithelium and venous plexus. It helps in regulation of water and electrolyte absorption.

The submucosa often contains fat cells. Some cells that contain PAS-positive granules, termed muciphages, are also present. These are most numerous in the rectum.

* The longitudinal layer of muscle is unusual. Most of the fibers in it are collected to form three thick bands, the Taenia coli (Fig. 16.12).
* A thin layer of longitudinal fibers is present in the intervals between the taenia. The taeniae are shorter in length than other layers of the wall of the colon. This results in the production of sacculations (Houstra coli) on the wall of the colon.
Table 16.3: Differences between small intestine and large intestine., 1 = Table 16.3: Differences between small intestine and large intestine.. Small intestine, 1 = Large intestine. Narrow lumen, 1 = Wider lumen. Villi present in the mucosa, 1 = Villi absent in the mucosa. Crypts of Lieberkühn contains columnar, goblet, and Paneth cells, 1 = Long and numerous crypts containing predominantly goblet cells. Paneth cells absent. Numerous Peyer patches in the ileum along antimesenteric border, 1 = Lymphatic follicles dispersed along different parts. Taenia coli, sacculations, and appendices epiploicae are absent, 1 = Taenia coli, sacculations, and appendices epiploicae are present
Fig. 16.12: Segment of the colon (schematic representation).

The serous layer is missing over the posterior aspect of the ascending and descending colon. In many situations, the peritoneum forms small pouch-like processes that are filled with fat. These yellow masses are called the appendices epiploicae.

Appendix is a finger-like extension from the cecum. It is the narrowest part of the gut. From inside to outside, it is made up of—mucosa, submucosa, muscularis externa, and serosa.
Plate 16.7: Colon

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* Wall made up of four layers
* Mucosa—absence of villi, lined by simple columnar epithelium with numerous goblet cells
* Submucosa packed with numerous lymphatic follicles, extending into lamina propria
* Absence of taenia coli, appendices epiploicae.
* Mucous membrane: Is devoid of villi, poorly formed crypts. Epithelium is lined by simple columnar cells, numerous goblet cells, and occasional enterochromaffin cells. Epithelium rests on basement membrane, supported by lamina propria and muscularis mucosae.
* Submucosa: Contains loose areolar tissue and abundant lymphatic follicles. The lymphoid tissue is not present at birth. It gradually increases and is best seen in children about 10 years old. Subsequently, there is progressive reduction in quantity of lymphoid tissue. Hence, it is also called abdominal tonsil.
* Muscularis externa: Consists of inner circular and outer longitudinal layers of smooth muscle. At places the muscle coat is deficient and is called as hiatus muscularis. Taenia coli are absent.
* Serosa: Derived from peritoneum covers the entire organ except at the attachment of mesoappendix.

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The structure of the rectum is similar to that of the colon except for the following:
* Mucous membrane has temporary (longitudinal) and permanent (horizontal) folds.
* Permanent folds/Houston's valves are semilunar in shape, made of mucosa, submucosa, and thickened circular muscle coat.
* A continuous coat of longitudinal muscle is present. There are no taenia coli.
* Peritoneum covers the front and sides of the upper one-third of the rectum; and only the front of the middle third. The rest of the rectum is devoid of a serous covering.
* There are no appendices epiploicae.

The anal canal is about 4 cm long. The upper 3 cm are lined by mucous membrane and the lower 1 cm by skin (Fig. 16.13).

* The mucous membrane of the upper 15 mm of the canal is lined by columnar epithelium. The mucous membrane of this part shows 6 to 12 longitudinal folds that are called the anal columns. The lower ends of the anal columns are united to each other by short transverse folds called the anal valves. The anal valves together form a transverse line that runs all around the anal canal, this is the pectinate/dentate line. Above each anal valve there is a depression called the anal sinus. Mucus-secreting anal glands open into each sinus.
* The mucous membrane of the next 15 mm of the rectum is lined by nonkeratinized stratified squamous epithelium. This region does not have anal columns. The mucosa has a bluish appearance because of the presence of a dense venous plexus between it and the muscle coat. This region is called the pecten or transitional zone. The lower limit of the pecten forms the white line of Hilton.
* The lowest 8 to 10 mm of the anal canal are lined by true skin in which hair follicles, sebaceous glands, and sweat glands (circumanal) are present.
Fig. 16.13: Some features in the interior of the anal canal (schematic representation).

* Prominent venous plexuses are present in the submucosa of the anal canal.
* The internal hemorrhoidal plexus lies above the level of the pectinate line, while the external hemorrhoidal plexus lies near the lower end of the canal.
* Anal glands also extend into the submucosa.

* The anal canal is surrounded by circular and longitudinal layers of muscle continuous with those of the rectum. The circular muscle is thickened to form the internal anal sphincter.
* Outside the layer of smooth muscle, there is the external anal sphincter that is made up of striated muscle.
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* Mucous glands near the gastric end of esophagus help to protect the regurgitated gastric content which causes heartburn. If the condition persists for longer time it can cause gastroesophageal reflux disease (GERD).
* One of the common causes of GERD is abnormal relaxation of lower esophageal sphincter.
* Barrett's esophagus: This is a condition in which, following reflux esophagitis, stratified squamous epithelium of the lower esophagus is replaced by columnar epithelium (columnar metaplasia). The condition is seen more commonly in later age and is caused by factors producing gastroesophageal reflux disease.
* Achalasia (cardiospasm): Achalasia of the esophagus is a neuromuscular dysfunction due to which the cardiac sphincter fails to relax during swallowing and results in progressive dysphagia and dilatation of the esophagus (megaesophagus).

* Gastritis: The term "gastritis" is commonly employed for any clinical condition with upper abdominal discomfort like indigestion or dyspepsia in which the specific clinical signs and radiological abnormalities are absent. The condition is of great importance due to its relationship with peptic ulcer and gastric cancer.
* Certain clinical conditions (like autoimmune disorders) can cause achlorhydria, where there is absence of HCl due to damaged gastric mucosa. Achlorhydria can in turn lead to vitamin B,, deficiency due to lack of intrinsic factor.
* Gastric ulcer: Gastric ulcer may occur due to damage to the gastric mucosa barrier. It is most common along the lesser curvature and pyloric antrum. Food-pain pattern, vomiting, significant weight loss, and deep tenderness in the midline in epigastrium are the main presentations.
* Zollinger—Ellison syndrome: Caused by excessive secretion of gastrin by enter enteroendocrine cells, which in turn continuously stimulate parietal cells to secret HCl thus causing gastric and duodenal ulcers.

* Crohn's disease or regional enteritis is an idiopathic chronic ulcerative inflammatory bowel disease, characterized by transmural, non-caseating granulomatous inflammation, affecting most commonly the segment of terminal ileum and/or colon, though any part of the gastrointestinal tract may be involved.
* Celiac sprue is the most important cause of primary malabsorption occurring in temperate climates. The condition is characterized by significant loss of villi in the small intestine and thence diminished absorptive surface area. The condition occurs in two forms:
1. Childhood form, seen in infants and children and is commonly referred to as coeliac disease.
2. Adult form, seen in adolescents and early adult life and used to be called idiopathic steatorrhea.

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* The lining epithelium of the stomach, and of the small and large intestines, contains scattered cells that have an endocrine function. These were recognized by early workers because of the presence, in them, of infranuclear granules that blackened with silver salts. They were, therefore, termed argentaffin cells.
*The granules also show a positive chromaffin reaction and have, therefore, also been called enterochromaffin cells.
*More recently, by the use of immunohistochemical methods, several other biologically active substances (amines or polypeptides) have been located in these cells.
*Many of these substances are also found in the nervous system where they function as neurotransmitters.
*They also act as hormones. This action is either local on neighboring cells (paracrine effect); or on cells at distant sites (through the bloodstream).
*Very similar cells are also to be seen in the pancreas. All these cells are now grouped together under the term gastroenteropancreatic endocrine system.
*Some features of this system are similar to those of amineproducing cells in other organs. All these are included under the term APUD cell system.

* Ulcerative colitis is an idiopathic form of acute and chronic ulceroinflammatory colitis affecting chiefly the mucosa and submucosa of the rectum and descending colon, though sometimes it may involve the entire length of the large bowel.
Acute inflammation of the appendix, acute appendicitis, is the most common acute abdominal condition confronting the surgeon. The condition is seen more commonly in older children and young adults, and is uncommon at the extremes of age. The disease is seen more frequently in the West and in affluent societies which may be due to variation in diet —a diet with low bulk or cellulose and high protein intake more often , a

- * Hemorrhoids or piles are the varicosities of the hemorrhoidal veins. They are called "internal piles" if dilatation is of superior hemorrhoidal plexus covered over by mucous membrane, and "external piles" if they involve inferior hemorrhoidal plexus covered over by the skin. They are common lesions in elderly and pregnant women. They commonly result from increased venous pressure.
Some of the cell types recognized, and their secretory products are given in the Table 16.4.

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. Simple squamous epithelium
. Stratified squamous nonkeratinized epithelium
. Transitional epithelium Pseudostratified epithelium 200M
3. Mucous glands of esophagus are found in:
Lining epithelium Submucosa Muscularis externa Adventitia 2050p
4. Nerve plexus of muscularis externa is called as:
» Meissner’s plexus
b. Aurebach’s plexus
c. Barrett's plexus
d. Brachial plexus
5. Brunner’s gland are found in the mucosa of:
a. Stomach
b. Colon
c. Duodenum
d. Esophagus
6. Following cells are found in small intestine, except:
Stem cells
Goblet cells Neck cells Paneth cells 2900p
7. Cells which give beaded cells to the mucosa of the stomach are:
Stem cells
Chief cells Neck cells Parietal cells 2905p
8. Peyer's patches are characteristic feature of:
Stomach »
b. Colon
c. Ileum

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* Structure and function of liver
* Bile
* Structure of gallbladder
* Structure of pancreas
* Differences between serous salivary gland and pancreas

Central vein surrounded by hexagonal plate of hepatocytes, Gallbladder = Wall made up of three layers- mucosa, fibromuscular layer, and serosa. Central vein surrounded by hexagonal plate of hepatocytes, Pancreas = Presence of serous acini, lined by pyramidal cells with basal rounded nucleus. Presence of portal triad consisting of arteriole branch of hepatic artery, bile ductule, and venule branch of portal vein, Gallbladder = Mucosa showing honeycombed appearance, lined by simple tall columnar epithelium with striated border.. Presence of portal triad consisting of arteriole branch of hepatic artery, bile ductule, and venule branch of portal vein, Pancreas = Presence of islets of Langerhans. Hepatic sinusoids between the hepatocytes., Gallbladder = . Hepatic sinusoids between the hepatocytes., Pancreas = Presence of lightly stained squamous- centroacinar cells.
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The hepatobiliary system comprises of liver, gallbladder, and extrahepatic ducts.
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* Liver is the largest gland of the body situated mainly in the right hypochondrium, below the right dome of diaphragm in the abdomen.
* The liver may be regarded as a modified exocrine gland.
* The functions are:
™ Metabolism of carbohydrates, proteins, and fats.
"Synthesis of plasma proteins, fibrinogen, and prothrombin, and : regulation of blood glucose and lipids.
"Storage of glucose (as glycogen), lipids, vitamin A, metabolism of vitan D, and processing of vitamin K.
"Storage and metabolism of iron, degradation of various substan (including drugs and alcohol).
™ Removal of bile pigments and their excretion through bile.
"Modification of thyroxine, growth hormone; degradation of insulin a glucagon.
™ During fetal life, the liver is the center for hemopoiesis.
"The macrophage cells (of Kupffer) lining the sinusoids of the liver hav: role similar to that of other cells of the mononuclear phagocyte syste They are of particular importance as they are the first cells of this syst that come in contact with materials absorbed through the gut. They a remove damaged erythrocytes from blood.
* Blood supply of liver: Liver has dual blood supply from portal vein (venous blood) and hepatic artery. Around 70% of nutrition is provided by portal vein which carries blood from gut, pancreas, and spleen containing absorbed substances, destroyed blood cells, and secretions of pancreas and intestinal cells. Remaining 30% of nutrition is conveyed by hepatic artery. Both the vessels enter the liver at porta hepatis, where the bile duct and lymphatics leave the organ. Hence, blood and bile travel in opposite direction within the parenchyme. Within the organ, the branches of hepatic artery and portal vein supply the liver sinusoids. Sinusoids drain into
hepatic venule (central vein). Central veins of adjacent lobules unite to form interlobular veins. Finally, blood from the liver is drained into inferior vena cava via hepatic veins.

- * Macroscopic structure of liver: The liver is covered by a thin layer of connective tissue capsule called Glisson's capsule. This connective tissue extends into the liver substance through the portal canals (portal triads) and surrounds them. The liver substance is divisible into large lobes, each of which consists of numerous lobules (Plate 17.1).

The liver is an accessory gland of gastrointestinal system made up of stroma and parenchyma.
* Stroma: It is formed by extensions from Glisson's capsule. Blood vessels, nerves, lymphatic vessels, and bile ducts travel within the connective tissue stroma. It branches extensively and divides the parenchyma into basic structural units called classical liver lobule.
* Parenchyma: Consists of organized plates of hepatocytes, which in the adult are normally one cell thick and are separated by sinusoidal capillaries.
* Hepatic sinusoids: They are present between the hepatic laminae and also between the cords of hepatocytes. They are fenestrated sinusoids lined by endothelial cells and Kupffer cells. Adjacent sinusoids communicate thereby maintains optimal intersinusoidal pressure by acting as vascular pressure. Blood from the sinusoids open into a central vein that occupies the center of the lobule. Hence, hepatic sinusoids work in centripetal direction. Finally, central vein drains into hepatic veins (which leave the liver to end in the inferior vena cava).

The parenchyma of liver is divided into units based on three ways:

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™ Hepatic lobules are hexagonal in shape with 1 mm width (Fig. 17.1).
"Lobules are surrounded by scanty connective tissue (in humans). Cent axis of the lobule is occupied by central vein (tributary of hepatic vein).
"From the central vein, cords of hepatocytes radiate outward as plates/she
of cells called hepatic laminae. Each lamina is one cell thick, cells witl the laminae branch and anastomose. About 20 hepatocytes are present each lamina.
Fig. 17.1: Classic liver lobule (schematic representation).
"Hepatocytes are separated by spaces, known as hepatic lacunae which i occupied by hepatic sinusoids. Hepatocytes are bathed in the sinusoi: blood.
" At the periphery, the adjacent hepatic laminae join and form limiting pl which overlies on portal canals.
"Limiting plates have perforations through which the terminal branches venules open into sinusoid. Branches of hepatic artery either join | branches of venules or directly open into the sinusoid.
"In some animals, e.g. pig, the classic lobule can be easily recogniz because of well-differentiated, relatively thick layers of connective tiss forming structural units. But in humans due to scanty connective tiss around the portal canals, hepatocytes are continuous with adjacent lamin Hence, hepatic lobules may not be considered as structural/functional ur but an independent venous unit drained by particular central vein.
"Portal canal: These are angular intervals at the periphery of each lob filled by connective tissue. Each "canal" contains: A radicle of portal ve
hepatic arteriole, and interlobular bile duct. These three structu collectively form a portal triad (Fig. 17.2).
- "Blood from the branch of the portal vein, and from the branch of | hepatic artery, enters the sinusoids at the periphery of the lobule and pas: toward its center. Blood vessels and hepatic ducts present in portal can are surrounded by a narrow interval called the space of Mall. Lymphat of liver begin in this space as blind radicles.
Plate 17.1: Liver (Panoramic view)

"Each such area contains a branch of the portal vein, a branch of the hepatic artery, and an interlobular bile duct
'These three constitute a portal triad.
Fig. 17.2: Portal triad (schematic representation).

"They are triangular territory of liver tissue with portal triad at center a central vein at each corer of the triangle (Fig. 17.3).
"It is called the nutritional lobule of the liver, obtained by joining | central vein of three adjacent hepatic lobules.

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"It is a diamond-shaped smaller unit of liver tissue supplied by one hepe arteriole (Fig. 17.4) running along the line of junction of two hepa lobules. Two central veins lie at the ends of the acinus. They are functio unit of liver.
" The long axis of the acinus is formed by joining two central veins and sh by joining adjacent portal triads.
" According to the gradient of blood supply, acinus can be divided into thi zones—inner zone/zone 1 (around the hepatic arteriole, hence w oxygenated), intermediate zone/zone 2 (moderately oxygenated), and ou zone/zone 3 (least oxygenated). Hence, cells in zone 1 are the first to : not only oxygen, nutrients but also toxins from the sinusoidal blood. Hen show morphologic changes after bile duct occlusion early. They are last perish if circulation is impaired and the first to regenerate. Zone 3, on |
other hand, is first to show ischemic necrosis on reduced perfusion and a fat accumulation. They are the last to be affected by toxic substances a bile stasis.
Fig. 17.3: Scheme to show the concept of portal lobules (pink). Hepatic lobules are shaded green. Note that the portal lobule is made up of parts of three hepatic lobules.
Fig. 17.4: Scheme to show the concept of portal acini (pink).

- * Canaliculi are merely spaces present between plasma membranes of adjacent liver cells into which bile secreted by liver cells is poured.
At the periphery of a lobule, the canaliculi become continuous with delicate intralobular ductules, which in turn become continuous with larger interlobular ductules of portal triads. Hence, bile canaliculi functions in centrifugal direction.
- * The interlobular ductules are lined by cuboidal epithelium. Some smooth muscle is present in the walls of larger ducts.

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" Hepatocyte is a large cell with a round open faced nucleus, with promin: nucleoli (Plate 17.2).
"The cytoplasm of liver cells contains numerous mitochondria, abund. rough and smooth endoplasmic reticulum, a well-developed Golgi compl: lysosomes, and vacuoles containing various enzymes. Numerous f ribosomes are present. These features are to be correlated with the hi metabolic activity of liver cells.
"Stored glycogen, lipids, and iron (as crystals of ferritin and hemosider are usually present.
"Glycogen is often present in relation to smooth endoplasmic reticulu Many hepatocytes show two nuclei, or a single polyploid nucleus.
"Liver cells are arranged in the form of anastomosing plates, one cell thi separated by sinusoids (Fig. 17.5). In this way, each liver cell has a sinus on two sides.
"The surface of a hepatocyte can show three kinds of specialization (Fi 17.6 and 17.7):
* Sinusoidal surface: The cell surfaces adjoining sinusoids bear mic that project into the space of Disse. The cell surface here also shows coated pits that are concerned with exocytosis. Both these features h active transfer of materials from sinusoids to hepatocytes, and vice ' About 70% of the surface of hepatocytes is of this type.
* Canalicular surface: Areas of cell membrane bear longitt depressions that are opposed to similar depressions on neight hepatocytes and form the wall of a bile canaliculus. Irregular mic) project into the canaliculus. On either side of the canaliculus, th membranes of adjoining cells are united by junctional complexe
forms blood-bile barrier, which prevents escaping of bile into interce space. About 15% of the hepatocyte surface is canalicular.
* Intercellular surface: These are areas of cell surface where ad hepatocytes are united to each other just as in typical cells. Communi: junctions allow exchanges between the cells. About 15% of the hepai surface is intercellular.

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"Space of Disse: The surface of the liver cell is separated from | endothelial lining of the sinusoid by a narrow perisinusoidal space Disse) (Fig. 17.8). Microvilli of the liver cells extend into this space and intimately related to circulating blood. Some fat cells and Ito cells are a seen in this space. They are intralobular, in contrast to space of Mi which are interlobular. The portal triad is bordered by the outerm hepatocytes of the lobule (Fig. 17.2). A small space between the connecti tissue stroma and hepatocytes at the edges of triad is called the peripor space or space of Mall. The lymph is thought to be originated from t space.
Fig. 17.5: Relationship of bile capillaries to liver cells (schematic representation).
Fig. 17.6: Three functional specializations of cell surface of a hepatocyte (schematic representation).
Plate 17.2: Liver (High magnification)
Liver (High magnification): (A) As seen in drawing; (B) Photomicrograph.

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On high magnification:
'The lobule is made up of polygonal liver cells arranged in the form of radiating cords
'The central round nucleus of hepatocyte is surrounded by abundant pink cytoplasm
'The cords are separated from each other by spaces called sinusoids
'The sinusoids are lined by endothelial cells and Kupffer cells (macrophage cells)
'Each portal tract contains a hepatic arteriole, portal venule, and one or two interlobular bile ducts. Normally, these structures are surrounded by fibroconnective tissue and a few

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"They are star-/stellate-shaped cells, found lining the walls of hepa sinusoids.
" Derived from bone marrow, they belong to mononuclear phagocyte syst (MPS), hence clears the cellular debris and microbes from the blood.
Fig. 17.7: Scheme to show the presence of several branches of the portal vein around a hepatic lobule. The manner in that they open into sinusoids is shown. The intervals between the sinusoids are occupied by liver cells (schematic representation).
Fig. 17.8: Space of Disse and bile canaliculus (schematic representation).
- "Recent studies have confirmed their role in final breakdown of senile ¢ damaged RBCs.

" They are also stellate-shaped, present in the perisinusoidal space of Disse
= They are the primary storehouse of vitamin A.
"It helps in remodeling of extracellular matrix during recovery from li: damage.
"Under pathological conditions, they differentiate into myofibroblasts a synthesize collagen.

* The exocrine secretion of the liver cells is called bile. Bile is poured out from liver cells into very delicate bile canaliculi that are present in intimate relationship to the cells.
* From the canaliculi, bile drains into progressively larger ducts that end in the bile duct. This duct conveys bile into the duodenum where bile plays a role in digestion of fat.
i

The extrahepatic biliary apparatus consists of the gallbladder and the extrahepatic bile ducts.

* Gallbladder is a blind muscular sac situated on the visceral surface of liver in the fossa on inferior surface.
* The gallbladder stores and concentrates bile.
* This bile is discharged into the duodenum through cystic duct, which joins common hepatic duct to form common bile duct when required.

The wall of the gallbladder is made up of: (a) Inner mucous membrane, (b) Middle fibromuscular coat, and (c) Outer serous layer that cover part of the organ (Plate 17.3).

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* Mucous membrane is thrown into numerous folds, which gives the surface honeycomb appearance.
* The mucous membrane of the gallbladder is lined by simple tall columnar epithelium with microvilli showing striated border.
* Do not contain goblet cells in contrast to small intestine.
* Mucosa contains lamina propria, which lacks the mucosal glands and lymphatic vessels.
* Muscularis mucosa and submucosa are absent.
* Sometimes, deep mucosal diverticula can extend into the muscularis externa and they are called as Rokitansky—Aschoff sinuses. If present, they may contribute as risk factor for stone formation and infection.
Plate 17.3: Gallbladder
Gallbladder: (A) As seen in drawing; (B) Photomicrograph.

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"The mucous membrane is lined by tall columnar cells with striated border
"The mucosa is highly folded and some of the folds might look-like villi
"Crypts may be found in lamina propria
*Submucosa is absent
The muscle coat is poorly developed with numerous connective tissue fibers interspersed among muscle. This is called as fibromuscular coat
"A serous covering lined by flattened mesothelium is seen.
Note: Gallbladder can be differentiated from small intestine by absence of villi, goblet cells, submucosa, and proper muscularis externa.

* Muscularis externa is made up of mainly connective tissue (collagen and elastic fibers) with some amount of smooth muscle.
* Smooth muscle fibers are arranged in random direction.

* The serosal layer has a lining of mesothelium (peritoneum) resting on connective tissue.
* This layer also contains large blood vessels, lymphatic network, and nerve plexus along with elastic fibers and adipose tissue.
* The fundus and lower surface of body of gallbladder is covered by serosa, whereas the upper surface is attached to the fossa for gallbladder by means of connective tissue (adventitia).

These are the right, left, and common hepatic ducts; the cystic duct; and the bile duct. All of them have a common structure. They have a mucosa surrounded by a wall made up of connective tissue, in which some smooth muscle may be present. The mucosa is lined by a tall columnar epithelium with a striated border.

* At its lower end, the bile duct is joined by the main pancreatic duct, the two usually forming a common hepatopancreatic duct (or ampulla) that opens into the duodenum at the summit of the major duodenal papilla.
* The mucosa of the hepatopancreatic duct is highly folded. These folds are believed to constitute a valvular mechanism that prevents duodenal contents from entering the bile and pancreatic ducts.
* Sphincter of Oddi:
™ Well-developed smooth muscle is present in the region of the lower end

"From a functional point of view, this sphincter consists of three separ parts. The sphincter choledochus surrounds the lower end of the bile dt It is always present, and its contraction is responsible for filling of | gallbladder. A less developed sphincter pancreaticus surrounds terminal part of the main pancreatic duct (Fig. 17.9). A third sphinc surrounds the hepatopancreatic duct (or ampulla) and often forms a ri round the lower ends of both the bile and pancreatic ducts. This is 1 sphincter ampullae.
"The sphincter ampullae and the sphincter pancreaticus are often missing.
Fig. 17.9: Section through the major duodenal papilla to show the components of the sphincter of Oddi (schematic representation).
B

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* Pancreas is a retroperitoneal organ, extends from the concavity of the duodenum to the hilum of the spleen.
* Tt is a gland that is partly exocrine, and partly endocrine, the main bulk of the gland being constituted by its exocrine part (Plate 17.4).
* The exocrine pancreas secretes enzymes that play a very important role in
the digestion of carbohydrates, proteins, and fats. After digestion and absorption through the gut, these products are carried to the liver through the portal vein.
* The endocrine pancreas produces two very important hormones, insulin, and glucagon. These two hormones are also carried through the portal vein to the liver where they have a profound influence on the metabolism of carbohydrates, proteins, and fats.
* The functions of the exocrine and endocrine parts of the pancreas are, thus, linked. The linkage between the two parts is also seen in their common embryonic derivation from the endodermal lining of the gut.

The exocrine pancreas is in the form of compound tubuloalveolar serous gland. Its general structure is very similar to that of parotid gland.
Plate 17.4: Pancreas
Pancreas: (A) As seen in drawing; (B) Photomicrograph.

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Made up of serous acini
*The cells forming the acini of the pancreas are highly basophilic (bluish staining). The lumen of the acinus is very small
Some acini may show pale staining (centroacinar cell) in the center
*Among the acini, some ducts are seen
*The ducts have a distinct lumen, lined by cuboidal epithelium
"At some places, the acini are separated by areas where we see aggregations of cells quite different from those of the acini
*These aggregations form the pancreatic islets: pale staining cells arranged as groups, surrounded by blood vessels
4n the photomicrograph, an interlobular duct lined by cuboidal epithelium is surrounded by lobules of acinar cells which have small basal nuclei and granular eosinophilic or slightly basophilic cytoplasm. Islets of Langerhans appear as groups of small cells with lightly stained cytoplasm.
* Capsule: A delicate capsule surrounds the pancreas. Septa extend from the capsule into the gland and divide it into lobules. Each lobule consists of ramified ducts ending in numerous alveoli/acini.
* Pancreatic acini: The secretory elements of the exocrine pancreas are long and tubular (but they are usually described as acini as they appear rounded or oval in sections). Their lumen is small (Fig. 17.10) and they have following cells: Secretory cells:
" The cells lining the alveoli are pyramidal in shape.
" They have spherical nuclei located toward basal region.
"With hematoxylin and eosin staining, the cytoplasm is highly basophi (blue) particularly in the basal part as the basal region is packed with rou endoplasmic reticulum and shows striations. Even the spherical nucleus located toward the basement membrane.
"With suitable fixation and staining, numerous secretory (or zymogi granules can be demonstrated in the apical part of cytoplasm. The granules are eosinophilic. They decrease considerably after the cell |} poured out its secretion.

" The junction between the alveoli and intercalated ducts is lined by cuboi: cells called centroacinar cells.
"They are called so because they appear to be located near the center of : acinus.
" Actually these cells belong to the intercalated ducts that are invagina into the secretory elements.
Some cell bodies of autonomic neurons and undifferentiated cells are also present in relation to the secretory elements.

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™Secretions produced in the acini are poured into intercalated ducts (a called intralobular ducts).
"These ducts are lined by flattened pale-stained epithelium and . invaginated deeply into the secretory elements (Fig. 17.10). As a result this invagination, the intercalated ducts are not conspicuous in sections.
"From the intercalated ducts, the secretions pass into larger, interlobu ducts. These are lined by low columnar cells, endocrine and occasioi goblet cells.
"They finally pass into the duodenum through the main pancreatic duct 2 the accessory pancreatic duct.
"The cells lining the pancreatic ducts control the bicarbonate and wa content of pancreatic secretion. These actions are under hormonal ¢ neural control.
"The walls of the larger ducts are formed mainly of fibrous tissue. They i lined by a columnar epithelium.
"The terminal part of the main pancreatic duct is surrounded by a sphinct A similar sphincter may also be present around the terminal part of | accessory pancreatic duct.
Fig. 17.10: Pancreatic serous acinus.

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* The endocrine pancreas is in the form of numerous rounded collections of cells that are embedded within the exocrine part.
* These collections of polyhedral cells are called the pancreatic islets, or the islets of Langerhans. The human pancreas has about one million islets. They are most numerous in the tail of the pancreas.
* Each islet is separated from the surrounding alveoli by a thin layer of reticular tissue.
* The islets are very richly supplied with blood through a dense capillary plexus. The intervals between the capillaries are occupied by cells arranged in groups or as cords.
* In ordinary preparations stained with H and E, all the cells appear similar, but with the use of special procedures, three main types of cells can be distinguished.

* In islets of the human pancreas, the alpha cells (or A-cells) tend to be arranged toward the periphery (or cortex) of the islets.
* They form about 20% of the islet cells. They contain smaller granules that stain brightly with acid fuchsin. They do not stain with aldehyde fuchsin.
* When seen with electron microscopy, the granules of alpha cells (A2) appear to be round or ovoid with high electron density.
* The alpha cells secrete the hormone glucagon.

* The beta cells (or B-cells) tend to lie near the center (or medulla) of the islet.
* About 70% of the cells of islet are of this type. The beta cells contain granules (larger than alpha cells) that can be stained with aldehyde fuchsin.
* When seen with electron microscopy, the granules of beta cells are fewer, larger, and of less electron density than those of alpha cells.
* The beta cells secrete the hormone insulin.

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* Delta cells (or D-cells), like alpha cells, are also peripherally placed.
* The delta cells (also called type III cells) stain black with silver salts (i.e. they are argyrophile).
* They resemble alpha cells in having granules that stain with acid fuchsin, and are, therefore, sometimes called A1 cells in distinction to the glucagon producing cells that are designated A2. The two can be distinguished by the fact that A2 cells are not argyrophile.
* When seen with electron microscopy, the granules of delta cells (A1) appear to be round or ovoid with low electron density.
* The delta cells probably produce the hormones gastrin and somatostatin. Somatostatin inhibits the secretion of glucagon by alpha cells, and (to a lesser extent) that of insulin by beta cells. Pancreatic islets are richly innervated by autonomic nerves. Noradrenaline and acetylcholine released at nerve endings influence secretion by islet cells.

Table 17.1: Differences between serous salivary gland and pancreas., 1 = Table 17.1: Differences between serous salivary gland and pancreas.. Serous salivary gland, 1 = Pancreas. Contains only exocrine part, 1 = Contains both exocrine and endocrine parts. Islets of Langerhans are absent, 1 = Islets of Langerhans are present. Striated ducts are present, 1 = Striated ducts are present. Centroacinar cells are absent, 1 = Centroacinar cells are present within the acini. Myoepithelial cells are seen between the basement membrane and acinar cells, 1 = Myoepithelial cells are absent

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- *The secretory cells produce two types of secretion: One of these is watery and rich in bicarbonate. Bicarbonate is probably added to pancreatic secretion by cells lining the ducts. It helps to neutralize the acid contents entering the duodenum from the stomach. Production of this secretion is stimulated mainly by the
hormone secretin liberated by the duodenal mucosa. The other secretion is thicker and contains numerous enzymes (including trypsinogen, chymotrypsinogen, amylase, lipases, etc.). The production of this secretion is stimulated mainly by the hormone cholecystokinin (pancreozymin) liberated by endocrine cells in the duodenal mucosa.
* Secretion by cells of the exocrine pancreas and the composition of the secretion are influenced by several other amines produced either in the gastrointestinal mucosa or in pancreatic islets (these include gastrin, vasoactive intestinal polypeptide, and pancreatic polypeptide). Secretion is also influenced by autonomic nerves, mainly parasympathetic.
*The enzymes are synthesized in the rough endoplasmic reticulum. From here, they pass to the Golgi complex where they are surrounded by membranes, and are released into the cytoplasm as secretory granules.
*The granules move to the luminal surface of the cell where the secretions are poured out by exocytosis.
*Within the cell, the enzymes are in an inactive form. They become active only after mixing with duodenal contents. Activation is influenced by enzymes present in the epithelium lining the duodenum.
* Apart from the three main types of cells described earlier, some other types are also present in the islets of Langerhans. These are the PP cells containing pancreatic polypeptide (and located mainly in the head and neck of the pancreas), and D1 cells (or type IV cells) probably containing vasoactive _ intestinal polypeptide (or a similar amine).
* A few cells secreting serotonin, motilin, and substance P are also present. Hence, in the cellular mass of islets, two zones are noted. Homocellular medulla in the center containing mainly beta cells and heterocellular cortex containing mixture of alpha, delta, and PP cells.

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* Inflammation in the liver is called hepatitis. It is frequently caused by viruses (viral hepatitis), and by a protozoan parasite Entamoeba histolytica (amebic hepatitis). An abscess may form in the liver as a sequel of amebic hepatitis.
* Cirrhosis of the liver is a disease in which many hepatocytes are destroyed, the areas being filled by fibrous tissue. This gradually leads to collapse of the normal architecture of the liver.
* Fish liver oils (e.g. cod liver oil) are important nutritional sources of vitamin A.
* Hepatocytes have lifespan of about 5 months. Also, they are capable of considerable regeneration when liver substance is damaged due to hepatotoxic processes, disease, or surgery.
* One effect of cirrhosis of the liver is to disrupt the flow of blood (reduction of vascular sponge) through the liver sinusoids. As a result of increased resistance to blood flow, there is increased blood pressure in the portal circulation (portal hypertension). In portal hypertension, anastomoses between the portal and systemic veins dilate to form varices (e.g. at the lower end of the esophagus). Rupture of these varices can result in fatal bleeding.
* When a large number of hepatocytes are destroyed, this leads to liver failure. Hepatic failure may be acute or chronic. Accumulation of waste products in blood (due to lack of detoxification by the liver) ultimately leads to unconsciousness (hepatic coma) and death.

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* Inflammation of the gallbladder is called cholecystitis.
* Stones may form in the gallbladder (gallstones; cholelithiasis). A gallstone passing through the bile duct can cause severe pain. This pain is biliary colic. In such cases, surgical removal of the gallbladder may be necessary (cholecystectomy).
* Blockage of the bile duct (by inflammation, by a gallstone, or by carcinoma) leads to accumulation of bile in the biliary duct system, and
within the bile capillaries.
* As pressure in the passages increases, bile passes into blood leading to jaundice. The sclera, the skin, and the nails appear to be yellow in color, and bile salts and pigments are excreted in urine. Jaundice occurring as a result of such obstruction is called obstructive jaundice. Jaundice is seen in the absence of obstruction in cases of hepatitis.

* Acute pancreatitis: Acute pancreatitis is an acute inflammation of the pancreas presenting clinically with "acute abdomen". The condition occurs in adults between the age of 40 years and 70 years and is more common in females than in males. The onset of acute pancreatitis is sudden, occurring after a bout of alcohol or a heavy meal. Characteristically, there is elevation of serum amylase level within the first 24 hours and elevated serum lipase level after 3-4 days.
* Chronic pancreatitis: Chronic pancreatitis or chronic relapsing pancreatitis is the progressive destruction of the pancreas due to repeated mild and subclinical attacks of acute pancreatitis. Most patients present with recurrent attacks of severe abdominal pain at intervals of months to years. Weight loss and jaundice are often associated.
* Diabetes mellitus is due to insulin resistance or decreased secretion of insulin by beta cells.

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Hepatocytes
Cholangiocytes
Ito cells 2900p
- 2. Macrophages in the liver are:
Hepatocytes Ito cells Cholangiocytes ao fp

Canal of Cloquet Canal of Nuck Canal of Hering Canal of Schlemm 2050p

» Hepatic lobule
b. Portal triad
c. Liver acinus
d. Portal lobule

a. Simple squamous
b. Simple columnar
c. Simple cuboidal
d. Pseudostratified ciliated columnar

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* Microscopic structure of kidney
* Structure of nephron
* Structure of juxtaglomerular apparatus
* Structure of ureters
* Structure of urinary bladder

Presence of cortex and medulla, Ureters = Star-shaped lumen is seen. Presence of cortex and medulla, Urinary bladder = Wall is made up of mucous, muscular, and adventitia layers. Medullary rays are seen in the cortex, Ureters = Wall is made up of mucous, muscular, and serous layers. Medullary rays are seen in the cortex, Urinary bladder = Mucosa is thrown into folds. Number of glomerulus is seen, Ureters = It is lined by transitional epithelium. Number of glomerulus is seen, Urinary bladder = It is lined by transitional epithelium. Cut section of proximal convoluted tubule (PCT) lined by columnar epithelium is seen, Ureters = It has inner longitudinal and outer circular layer of smooth muscles.. Cut section of proximal convoluted tubule (PCT) lined by columnar epithelium is seen, Urinary bladder = It has inner and outer longitudinal and middle circular layer of smooth muscle.. Cut section of distal convoluted, Ureters = . Cut section of distal convoluted, Urinary bladder = 

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The urinary organs are:
* A pair of kidneys
* A pair of ureters
* The urinary bladder
* The urethra.
§

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* The urinary organs are responsible for the production, storage, and passing of urine.
* Toxic byproducts of metabolism are removed from blood through urine by kidney. These include urea and creatinine that are end products of protein metabolism.
* Many drugs, or their breakdown products, are also excreted in urine.
* Conserves salts, glucose, proteins, and water.
* Kidneys help to regulate blood pressure, hemodynamics, and the acid-base balance of the body.
* Urine production, and the control of its composition, is exclusively the function of the kidneys. The urinary bladder is responsible for storage of urine until it is voided. The ureter and urethra are simple passages for transport of urine.
Note: In diseased conditions, urine can contain glucose as in diabetes mellitus, or proteins in kidney disease, the excretion of which is normally prevented.

* Kidneys are bean-shaped organs situated retroperitoneally on the posterior abdominal wall.
* The kidney is invested by thin adhering capsule consisting of dense irregular collagen fibers.

On coronal section, the kidney consists of an outer cortex and an inner medulla. Cortex appears dark brown and granular and medulla contains 8-12 pyramidal-shaped pale striated region called renal pyramids.

* Medulla is made up of 8-10 triangular areas of renal tissue called the renal pyramids which are striated, pale, and conical structure (Fig. 18.1). Each pyramid has a base directed toward the cortex; and an apex or papilla that is directed toward the renal pelvis, and fits into a minor calyx.
* Pyramids show striations that pass radially toward the apex. Striations are caused by series of U-shaped loops of Henle, collecting tubules, and ducts of Bellini and arteriolae recti and venae recti.

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The renal cortex consists of the following:
* Cortex is granular in appearance and consists of renal columns and cortical arches.
* Tissue lying between the bases of the pyramids and the surface of the kidney is called the cortical arches or cortical lobules.
* Cortical arches show light and dark striations. The light lines are called medullary rays. Medullary ray is a conical mass, the apex is directed toward the surface of the kidney and the base is continuous with the striations of the pyramid. Each ray is made up of collecting tubules and ducts of Bellini (Plate 18.1).
* Tissue lying between adjacent pyramids is called the renal columns.
* One pyramid is surrounded by a "shell" of cortex. The pyramid capped with cortex is known as the lobe of kidney.
* The area of the cortical arch bounded on each side by interlobular blood vessels with central medullary ray is known as the lobule of the kidney.
The kidney has a convex lateral margin; and a concavity on the medial side that is called the hilum. The hilum leads into a space called the renal sinus. The renal sinus is occupied by the upper expanded part of the ureter called the renal pelvis.
Fig. 18.1: Some features seen in a coronal section through the kidney (schematic representation).
Within the renal sinus, the pelvis divides into two or three major calyces and each major calyx divides into a three to four minor calyces (Fig. 18.1). The dilated outer end of each minor calyx presents a cup-shaped depression to receive renal papillae. The renal papilla is the location where the renal pyramids in the medulla empty urine into the minor calyx.

* The kidney is composed of 1-4 million closely packed uriniferous tubules.
* Each uriniferous tubule consists of a secreting part which develops from metanephric blastema called the nephron and collecting part which develops from ureteric bud called a collecting tubule and ducts of Bellini.
* Urinary tubules are held together by scanty connective tissue. Blood vessels, lymphatics, and nerves lie in this connective tissue.

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* These are the structural and functional unit of kidney and there are about 1-4 million nephrons in each kidney. Length of nephron varies from 50 mm to 55 mm (Fig. 18.2).
* The nephron consists of a renal corpuscle (or Malpighian corpuscle) for filtration and a long renal tubule for selective reabsorption. Renal tubule is made up of three parts:
Fig. 18.2: Parts of a nephron. A collecting duct is also shown (schematic representation).
nce eee Kidney (Low magnification)
Kidney (Low magnification): (A) As seen in drawing; (B) Photomicrograph of cortex.
Courtesy (B): Balakrishna Shetty, Sweekritha H Poonja. HISTOLOGY Practical Manual, 4th edition. New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd; 2019. p. 108.

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*The kidney is covered by a capsule
'Deep to the capsule there is the cortex
Deep to the cortex there is the medulla of the kidney in the cortex we see circular structures called renal corpuscles surrounding which there are tubules cut in various shapes
*The dark pink stained tubules are parts of the proximal convoluted tubules (PCT): their lumen is small and indistinct. It is lined by cuboidal epithelium with brush border
'Lighter staining tubules, each with a distinct lumen, are the distal convoluted tubules (DCT). They are lined by simple cuboidal epithelium
*PCT are more in number than DCT
in the medulla we see very light staining, elongated, parallel running tubules. These are collecting
ducts and
loop of
Henle.
Some of
them extend
into the
cortex forming
a
medullary ray. The collecting ducts are lined by simple cuboidal epithelium and loop of Henle (thin
segments)
are lined
by simple
squamous epithelium
Cut sections of blood vessels are seen both in the cortex and medulla.
Note: When we look at a section of the kidney we see that most of the area is filled with a very large number of tubules. These are of various shapes and have different types of epithelial lining. This fact by itself suggests that the tissue is the kidney.
1. The proximal convoluted tubule
2. Loop of Henle
3. The distal convoluted tubule.
* Renal corpuscles and the greater parts of the proximal and distal convoluted tubules are located in the cortex of the kidney. The loops of Henle and the collecting ducts lie in the medullary rays and in the substance of the pyramids.
* Two types of nephrons are found in the kidney. The cortical/superficial nephrons whose corpuscles located in the outer cortex and juxtamedullary nephrons whose corpuscles located in the inner cortex near medulla or corticomedullary junction and its tubular parts extend into the medulla.

* The renal corpuscle is also called a Malpighian body consists of a rounded tuft of blood capillaries called the glomerulus and a cup-like doublelayered covering for the glomerulus called the glomerular capsule/ Bowman's capsule (Fig. 18.3).
* The glomerular capsule represents the cup-shaped blind beginning of the renal tubule. Between the two layers of the capsule, there is a urinary space that is continuous with the lumen of the renal tubule.

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The glomerulus is a lobulated tuft of anastomosing fenestrated capillaries formed by afferent and efferent arterioles, which project into the Bowman's capsule (Plate 18.2). Blood enters the tuft through an afferent arteriole and leaves it through an efferent arteriole.
Fig. 18.3: Basic structure of a renal corpuscle (schematic representation).
* The afferent and efferent glomerular arterioles are approximated to each other to form the vascular pole of the renal corpuscle.
* Each lobule consists of a capillary loop which is suspended in the capsular space by a mesentery derived from visceral layer.
* Mesentery contains mesangium that is made up of mesangial cells surrounded by a noncellular mesangial matrix.
* Mesangial cells contain filaments similar to myosin and they bear angiotensin II receptors. It is believed that stimulation by angiotensin causes the fibrils to contract. This suggests that mesangial cells play a role in controlling blood flow through the glomerulus. Mesangial cells also play a role in phagocytosis, support to the glomerulus, and synthesize extracellular matrix.
* The mesangium becomes prominent in a disease called glomerulonephritis.

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It is the dilated blind end of the renal tubule which is invaginated by the glomerular plexus.
* The capsule is a double-layered cup consists of outer parietal and inner visceral layers. The two layers of which are separated by the urinary space.
* The parietal layer is lined by continuous flattened epithelium resting on basement membrane. The visceral layer is lined by discontinuous polyhedral cells known as podocytes.
* The urinary space becomes continuous with the lumen of the renal tubule at the urinary pole of the renal corpuscle and this pole lies opposite the vascular pole.
* The podocytes are so called because they possess foot-like processes. The podocytes are separated from the basement membrane by filtrate-filled spaces.
* The podocytes have numerous long tentacle like major/ primary process that are parallel to the basement membrane. Each major has numerous secondary processes called pedicels (or end feet) that rest on the basal lamina. The cell body of the podocyte comes in contact with the basal lamina only through the pedicels (Fig. 18.4).
* The pedicles of adjacent podocytes interdigitate and form spaces called filtration slits. These slits are covered by a thin membrane called slit diaphragm through which the filtration takes place.
EC EW eee meen ty (High magnification)
Renal cortex (High magnification): (A) As seen in drawing; (B) Photomicrograph.

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"In the high power view of renal cortex large renal corpuscles can be identified
"The renal corpuscle consists of a tuft of capillaries that form a rounded glomerulus, and an outer wall, the glomerular capsule (Bowman's capsule)
- "A urinary space between the glomerulus and the capsule is seen
convoluted tubules are dark staining. They are lined by cuboidal cells with a
"Proximal prominent brush border. Their lumen is indistinct
"Distal convoluted tubules are lighter staining. The cuboidal cells lining them do not have a brush border. Their lumen is distinct.
Fig. 18.4: Relationship of podocytes to a glomerular capillary. Note that the entire surface of the capillary is covered by processes of podocytes, the bare areas being shown only for sake of clarity (schematic representation).

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* As compared to typical membranes, the glomerular basement membrane is very thick (about 300 nm). The membrane acts as filtration barrier that separates urinary space from blood in the capillaries.
* Tt is made up of three layers. There is a central electron dense layer called lamina densa, and inner and outer electron lucent layers called lamina rara interna and externa.
* The lamina densa contains a network of collagen (type IV) fibrils, and thus acts as a physical barrier. The electron lucent layers contain the glycosaminoglycan heparan sulfate and bear the negative charges. The glomerular basement membrane is, therefore, both a physical barrier and an electrical barrier to the passage of large molecules.
* Glomerular basement membrane acts as an ultrafilter and allows filtration of water and simple solutes of blood plasma into capillary space. The glomerular filtrate consists of blood plasma without proteins, lipid droplets, and blood cells. Macromolecules greater than 4 nm cannot pass through the filtration slits (Fig. 18.5).
* As shown in Figure 18.6, the glomerular basement membrane and
podocyte does not go all round a glomerular capillary and the gap is filled in by mesangium.
- * Glomerular filtration barrier separates blood in the capillaries from urinary space. It is formed by fenestrated endothelium of capillaries, glomerular basement membrane, and pedicles of podocytes.
Fig. 18.5: Filtration slits (schematic representation).
Fig. 18.6: Relationship of a glomerular capillary to podocytes, basement membrane, and mesangium (schematic representation).
The renal tubule consists of the PCT, the loop of Henle consisting of a descending limb, a loop, and an ascending limb and the DCT, which ends by joining a collecting tubule.

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* Urinary space continuous as PCT at the urinary pole. The PCTs are 40-60 yum in diameter with small lumen.
* The initial part of PCT is convoluted and present in the cortex and the terminal part is straight and descends into the medulla to become continuous with the descending limb of the loop of Henle (Table 18.1).
* The junction of the PCT with the glomerular capsule is narrow and is referred as the neck. The neck is lined by simple squamous epithelium which continuous with that of the glomerular capsule.
* Proximal convoluted tubule is lined by cuboidal or low columnar cells resting on the basement membrane. The cells have acidophilic cytoplasm and apex of the cells have microvilli (brush border). The nuclei are central and euchromatic.
* Electron microscopic structure: The basal part of the cell shows a vertical striation. These striations are produced by infoldings of the basal plasma membrane and by numerous mitochondria that lie longitudinally in the cytoplasm intervening between the folds (Fig. 18.7). Only close to the apex, the cells are connected by tight junctions and rest of the surfaces they are separated by intercellular clefts (Plate 18.3).
* The presence of microvilli, and of the basal infoldings greatly increases the surface area available for transport.
Proximal convoluted tubule, 1 = Distal convoluted tubule. Begins from the Bowman's capsule, 1 = Begins from ascending limb of loop of Henle. Lengthy so more number of PCT sections are seen in cortex of kidney, 1 = Shorter so less number of DCT are seen in cortex of kidney section. Continuous with loop of Henle, 1 = Continuous with collecting duct. Lined by cuboidal epithelium with microvilli, 1 = Lined by cuboidal epithelium without microvilli. Cytoplasm is more acidophilic, 1 = Cytoplasm is less acidophilic/ basophilic. More convoluted, 1 = Less convoluted. Main function is reabsorption, 1 = Main function is secretion
Fig. 18.7: Some features of the ultrastructure of a cell lining a proximal convoluted tubule (schematic representation).

* About 80% of the filtrate is reabsorbed by PCT.
* Active reabsorption of water, glucose, amino acids, sodium, calcium chloride, phosphate, and bicarbonate from the filtrate back to the blood.

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* The loop of Henle is U-shaped and consists of the descending thin limb, the loop, and ascending thin limb. The descending limb enters the renal pyramid as a continuation of the proximal tubule. The loop of the juxtamedullary nephron reaches close to the apical zone of the pyramid and of the cortical nephrons reaches up to the basal zone of the pyramid. The ascending limb reenters the cortex and reaches close to the vascular pole.
* The descending limb and the loop constitutes thin segment of the loop with a diameter of 15 pm. It is lined by a squamous epithelium resting on basement membrane.
* The ascending limb constitutes the thick segment of the loop of Henle with diameter of 30 jm in diameter. It is lined by cuboidal epithelium.
* The loops are surrounded by arteriolae rectae and venae rectae and the capillary between them. The loop of Henle is also called the ansa nephroni.
* With the electron microscopy (EM), the flat cells lining the thin segment of the loop of Henle show very few organelles indicating that the cells play only a passive role in ionic movements across them. In some areas, the lining epithelium may show short microvilli, and some basal and lateral infoldings.

*. Od About 5% of the fluid is absorbed by loop of Henle.
* Ascending limb actively pumps out chloride and sodium ion passively into the tissue space.
* Descending limb diffuses water out and some chloride and sodium ion enters the loop.
J od The loops of Henle maintain a graded osmolarity of the interstitial fluid of the pyramid. From apex to base of the pyramid, the interstitial fluid becomes successively hypertonic, isotonic, and hypotonic.

- +. od Distal convoluted tubule begins from the vascular pole of the nephron as a continuation of the ascending limb of loop of Henle and terminates in the collecting tubule.
Plate 18.3: Renal medulla (High magnification)

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"A high power view of a part of the renal medulla shows a number of collecting ducts cut longitudinally or transversely
'They are lined by a cuboidal epithelium, the cells of which stain lightly. Cell boundaries are usually distinct. The lumen of the tubule is also distinct
"Sections of the thin segment of the loop of Henle are seen. They are lined by flattened cells, the walls being very similar in appearance to those of blood capillaries
"Sections through the thick segments of loops of Henle are seen. They are lined by cuboidal epithelium.
In the photomicrograph, collecting ducts and thick segments and thin segments of the loops of Henle can be identified.
* The distal convoluted tubule has a straight part continuous with the ascending limb of the loop of Henle, and a convoluted part lying in the cortex. At the junction between the two parts, the distal tubule lies very close to the vascular pole of the renal corpuscle of the nephron to which it belongs.
* The terminal part of the DCT is called the junctional tubule or connecting tubule, which joins the collecting duct.
* The distal convoluted tubules are 20-50 pm in diameter and lined by cuboidal epithelium without brush border.
* At the junction of the straight and convoluted parts of the DCTs, the cells show specializations that are described here in connection with the juxtaglomerular apparatus (Fig. 18.8).

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* Active absorption of about 15% of water from the filtrate.
* Absorbs sodium ions in exchange of excretion of potassium ions. This is regulated by aldosterone.
* Distal tubule receives hypotonic fluid and it helps in acidification of urine and isotonic fluid enters collecting tubule.
Note: Students may be confused by somewhat different terminology used in some books. The straight part of the PCT is sometimes described as part of the loop of Henle, and is termed the descending thick segment, in distinction to the ascending thick segment. Some workers regard the thin segment alone to be the loop of Henle. They include the descending and ascending thick segments with the proximal and DCTs, respectively.
Fig. 18.8: Some features of ultrastructure of a cell lining a distal convoluted tubule (schematic representation).

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* The collecting tubules pass through the medullary rays, receive many junctional tubules and enter the pyramids as ducts of Bellini.
* Three are cortical, medullary, and papillary (ducts of Bellini) collecting tubules. Cortical collecting tubules are located in the medullary rays; medullary tubules are formed by the union of many cortical tubules.
* The smallest collecting tubules are 40-50 pm in diameter, and the largest as much as 200 pm.
* Both collecting tubule and ducts of Bellini are lined by a simple cuboidal epithelium.
* The walls of cortical and medullary collecting tubules are lined by two types of cells called principal/clear cells with very few organelles, a few microvilli, and some basal infoldings and some dark cells or intercalated cells with microvilli.
* The cells of the collecting ducts do not have microvilli or lateral infoldings of plasma membrane.
Ducts of Bellini are surrounded by hypertonic tissue fluid of renal pyramids. Collecting tubules and ducts play a role in final concentration of urine.
- * Collecting tubules and ducts convey and modify the ultrafiltrate from the nephron to the minor calyces.

The only real barrier across which filtration occurs is the basal lamina or the glomerular basement membrane that is thickened at the filtration slits by the glomerular slit diaphragm. The efficacy of the barrier is greatly enhanced by the presence of a high negative charge in the basement membrane and in podocyte processes. Loss of this charge, in some diseases, leads to excessive leakage of protein through the barrier.

Juxtaglomerular apparatus is a specialized organ situated near the glomerulus of each nephron (juxta = near). The juxtaglomerular apparatus is formed by three different structures:
a. Macula densa of the DCT.
b. Juxtaglomerular cells of the afferent arteriole.
c. Lacis cells/extraglomerular mesangial cells/cells of polkissen.

* These are the modified cells of DCT near afferent arteriole.
* The wall of the DCTs comes in close contact with the afferent arterioles where the basement membranes of the tubules disappear. Here the lining cells are densely packed together, and are columnar rather than cuboidal as in the rest of the tubule. These cells form the macula densa.
* The cells of the macula densa lie in close contact with the juxtaglomerular cells.
* The cells of the macula densa monitor the ionic constitution of the fluid passing across tubule and influence the release of renin by the juxtaglomerular cells.

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* Tt is a modified smooth muscle cells located in the tunica media of afferent
glomerular arterioles and are situated very close to the macula densa.
* A part of the DCT at the junction of its straight and convoluted parts lies close to the vascular pole of the renal corpuscle, between the afferent and efferent arterioles. In this region, the muscle cells in the wall of the afferent arteriole are modified and are large and rounded (epithelioid) with spherical nuclei. These are called juxtaglomerular cells.
* They are innervated by unmyelinated adrenergic nerve fibers.
* Juxtamedullary cells are regarded, by some, as highly modified myoepithelial cells as they contain contractile filaments in the cytoplasm.

* It secretes an enzyme renin. The juxtaglomerular cells act as baroreceptors reacting to a fall in blood pressure by release of renin. Secretion of renin is also stimulated by low sodium blood levels and by sympathetic stimulation.
* Renin acts on a substance called angiotensinogen present in blood and converts it into angiotensin I. Angiotensin-converting enzyme (ACE) (present mainly in the lungs) converts angiotensin I into angiotensin II.
* Angiotensin II reduces the diameter of blood vessels and increases blood pressure. It also stimulates the secretion of aldosterone by the adrenal cortex, thus influencing the reabsorption of sodium ions by the DCTs and that of water through the collecting ducts. In this way, it helps to regulate plasma volume and blood pressure.
Note: In addition to renin, the kidney produces the hormone erythropoietin (which stimulates erythrocyte production). Some workers have claimed that erythropoietin is produced by juxtaglomerular cells, but the site of production of the hormone is uncertain.

These are polyhedral cluster of cells lie between the macula densa, afferent and efferent arterioles, and vascular pole of nephron. These cells are so called as they bear processes that form a lace-like network. The function of lacis cells is unknown.

* At the hilum of the kidney, each renal artery divides into a number of lobar arteries one for each pyramid. Each lobar artery divides into two or more interlobar arteries that enter the tissue of the renal columns and run toward the surface of the kidney. At the base of the pyramids, the interlobar arteries divide dichotomously into arcuate arteries which arch over the base of the pyramids (Fig. 18.9).
* The arcuate arteries run at right angles to the parent interlobar arteries. They lie parallel to the renal surface at the junction of the pyramid and the cortex. They give off a series of interlobular arteries that run through the cortex at right angles to the renal surface to form subcapsular plexus. Each interlobular artery gives off a series of arterioles that enter glomeruli as afferent arterioles. Blood from these arterioles circulates through glomerular capillaries that join to form efferent arterioles that emerge from glomeruli.
*Efferent arterioles arising from the cortical glomeruli divide into capillaries that surround the proximal and DCTs. These capillaries drain into interlobular veins and through them into arcuate veins and interlobar
Fig. 18.9: Arrangement of arteries within the kidney (schematic representation).

+. od Efferent arterioles arising from juxtamedullary glomeruli divide into 12— 25 straight vessels that descend into the medulla. These are the descending vasa recta (Fig. 18.10). Side branches arising from the vasa recta join a capillary plexus that surrounds the descending and ascending limbs of the loop of Henle and also the collecting tubules. The capillary plexus consists predominantly of vessels running longitudinally along the tubules. It is drained by ascending vasa recta that run upward parallel to the descending vasa recta to reach the cortex and they drain into interlobular or arcuate veins.
, ate '= There are two sets of arterioles and capillaries intervene between the renal artery and vein. The first capillary system, present in glomeruli, is concerned exclusively with the removal of waste products from blood. It does not supply oxygen to renal tissues. An exchange of gases between blood and renal tissue is entirely through the second capillary system present around tubules.
*, ote '= It has also been held that interlobular arteries divide the renal cortex into small lobules. Each lobule is defined as the region of cortex lying between two adjacent interlobular arteries. A medullary ray, containing a collecting duct, runs vertically through the middle of the lobule (midway between the two arteries) (Fig. 18.11). Glomeruli lie in a zone adjacent to the arteries while other parts of the nephron lie nearer the center of the lobule.
Fig. 18.10: Behavior of efferent arterioles of glomeruli in the superficial and deeper parts of the renal cortex (schematic representation).

The space between the tubules and blood vessels form the renal interstitium. It occupies a small volume in cortex but increases in medulla. It contains scanty amount of connective tissue with fibroblasts, interstitial dendritic cells, collagen fibers, and ground substances rich in proteoglycan. In medulla, the secreting interstitial cells are present and they help in the synthesis of prostaglandins and prostacyclins.
B®

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* Ureters are muscular tubes that conduct urine from renal pelvis to the urinary bladder. Each ureter is about 3-4 mm in diameter and approximately 25-30 cm in length.
* The wall of the ureter has three layers:
1. An inner mucous membrane
2. A middle layer of smooth muscle
3. An outer fibrous coat: Adventitia.
Fig. 18.11: The concept of cortical lobules (schematic representation).

* The mucosa shows a number of longitudinal folds that give the lumen a star-shaped appearance in transverse section. The folds disappear when the ureter is distended.
* The mucous membrane has a lining of transitional epithelium that is four to five cells thick and an underlying dense, irregular connective tissue the lamina propria. The epithelium is separated from lamina propria by a basal lamina.

* The muscle coat is composed of two inseparable smooth muscle layers—
(1) An inner longitudinal layer and (2) An outer circular layer. A third layer of longitudinal fibers is present outside the circular coat in the lower one-third of the ureter.
* Traced above the circular muscle surrounds the renal papillae and exerts milking action to squeeze urine from ducts to minor calyces. Traced below outer two layers are continuous with the detrusor muscle of bladder and inner longitudinal layer forms muscle of Bell.
* Muscle cells of the ureter intercommunicate with each other and form a syncytium. The waves of peristalsis pass downward through muscle and not depend on the nerve supply.

* Adventitia is the outer fibrous coat consisting of loose connective tissue. It contains numerous blood vessels, nerves, lymphatics, and some fat cells.
* At proximal and distal parts of the ureter, it blends with the capsule of the kidney and connective tissue of the urinary bladder wall, respectively (Plate 18.4).

Reflux of urine from the urinary bladder into the ureters is prevented by the oblique path followed by the terminal part of the ureter, through the bladder wall. When the musculature of the bladder contracts this part of the ureter is compressed. This mechanism constitutes a physiological sphincter.

* Urinary bladder is a muscular bag, where urine is stored temporarily and is discharged periodically via urethra during micturition.
* The wall of the urinary bladder from inside outward consists of four layers —(1) Mucosa (2) Submucosa (3) Muscular layer (4) Outer serosa.

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- * The mucous membrane is lined by transitional epithelium/urothelium which is urine proof, devoid of muscularis mucosae. Surface epithelium is polyhedral with abundant cytoplasm and prominent nuclei. Some nuclei have polyploid number of chromosomes. The cytoplasm of surface cells
spread like umbrella to allow distension of the bladder without tension of individual cells, hence called umbrella cells.
* In the empty bladder, the mucous membrane is thrown into numerous folds that disappear when the bladder is distended.
* The lamina propria has superficial dense irregular and deeper loose connective tissue. It contains some mucous glands near the internal urethral orifice.
* When the bladder is distended, the lining epithelium becomes thinner due to the ability of the epithelial cells to change shape and shift over one another (Plate 18.5).
Note: Umbrella cells haave intercellular junctional complexes and surface their is covered by double lipid layer with uroplakins (integral membrane protein) together called Urothelial plaques. These plaques are discontinuous and termed as asymmetric unit membrane. This arrangement helps to protect underlying cells from cytotoxic and hypertonic effects of urine

It consists of loose areolar tissue all over the bladder except at the trigone where mucous membrane is adherent to the muscular layer.

* The muscle layer is thick and composed of three interlaced layers of smooth muscles. They are arranged as internal and external layers of longitudinal fibers and in between them there is a thicker layer of circular (or oblique) fibers.
* The muscle is called detrusor muscle and contraction of this muscle is responsible for emptying of the bladder.
* At the internal urethral orifice/neck of bladder, the circular fibers are thickened to form the sphincter vesicae/internal sphincter muscle in men.
* The external longitudinal layer continues to the muscle layer of prostate in men and to the external urethral meatus in women.

- * The serous layer is composed of dense irregular connective tissue. The
superior surface of the bladder is covered by mesothelium of peritoneum, forming serous layer.

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"The ureter can be recognized because it is tubular and its mucous membrane is lined by transitional epithelium
"The epithelium rests on a layer of connective tissue (lamina propria)
"The mucosa shows folds that give the lumen a star-shaped appearance
>
The muscle coat has an inner layer of longitudinal fibers and an outer layer of circular fibers. This arrangement is the reverse of that in the gut
"The muscle coat is surrounded by connective tissue—adventitia in which blood vessels and fat cells are present.
red

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The urinary bladder is easily recognized because the mucous membrane is lined by
transitional epithelium
'The epithelium rests on lamina propria
"The muscle layer is thick. It has inner and outer longitudinal layers between which there is layer of circular or oblique fibers. The distinct muscle layers may not be distinguishable
a
"The outer surface is lined in parts by peritoneum (serosa) (not seen in the photomicrograph).
- * The inferior part of the bladder is covered with adventitia which is made of fibroelastic connective tissue carrying blood vessels, nerves, and lymphatics.
Bi

* Urethra is a tube that carries urine from bladder to the exterior. In males, semen also passes through the urethra. Although the male urethra is much longer than the female urethra, the structure of the two is the same.
* The wall of the urethra is composed from inside outward— mucous membrane, submucosa, and muscular layer.
*In male, the prostatic urethra is surrounded by prostatic tissue, and the penile urethra by erectile tissue of the corpus spongiosum.

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* The mucous membrane consists of a lining epithelium that rests on connective tissue. The epithelium varies in different parts of the urethra.
*In female, it is lined by stratified squamous nonkeratinized epithelium along the length except near the bladder it is by transitional epithelium.
* In male, prostatic urethra is lined by transitional epithelium, membranous urethra by stratified columnar epithelium, spongy/penile urethra by stratified columnar interspersed with patches of pseudostratified columnar and stratified squamous nonkeratinized epithelia.
* The mucosa shows invaginations or recesses into which mucous glands open. Littre's glands are the mucous glands found along the entire length of the urethra and most abundant at the spongy part of the male urethra. Its
secretion lubricates the epithelial lining of the urethra.

* The muscle coat consists of an inner longitudinal layer and an outer circular layer of smooth muscle. This coat is better defined in the female urethra. In the male urethra, it is well-defined only in the membranous and prostatic parts, the penile part being surrounded by occasional fibers only.
*In addition to this smooth muscle, the membranous part of the male urethra, and the corresponding part of the female urethra are surrounded by striated muscle that forms the external urethral sphincter.
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* Defects in the glomerular basement membrane are responsible for the nephrotic syndrome in which large amounts of protein are lost through urine. The regular arrangement of podocyte processes is also disorganized in this condition.
* The presence of albumin in the urine (albuminuria) is the result of increased permeability of the glomerular endothelium. Causes of albuminuria are hypertension, vascular injury, and bacterial infections.
* Glomerulonephritis or Bright's disease is the term used for diseases that primarily involve the renal glomeruli.
* Nephrotic syndrome is a constellation of features in different diseases having varying pathogenesis; it is characterized by findings of massive proteinuria, hypoalbuminemia, edema, hyperlipidemia, lipiduria, and hypercoagulability.
* Hydronephrosis is the term used for dilatation of renal pelvis and calyces due to partial or intermittent obstruction to the outflow of urine. Hydronephrosis develops if one or both the pelviureteric sphincters are incompetent, as otherwise there will be dilatation and hypertrophy of the urinary bladder but no hydronephrosis. Hydroureter nearly always accompanies hydronephrosis. Hydronephrosis may be unilateral or

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* Ureterocele is cystic dilatation of the terminal part of the ureter which lies within the bladder wall. The cystic dilatation lies beneath the bladder mucosa and can be visualized by cystoscopy.
* Cystitis is the inflammation of the urinary bladder. Cystitis is caused by a variety of bacterial and fungal infections. Cystitis, like urinary tract infection (UTI), is more common in females because of the shortness of urethra which is liable to fecal contamination and due to mechanical trauma during sexual intercourse.
1. What makes the renal corpuscle?
Bowman’s capsule and glomerulus
Bowman's capsule and tubules Glomerulus and tubules 2905p
Bowman’s capsule, glomerulus, and tubules
2. Renal corpuscles are found in:
Cortex
Medulla Both cortex and medulla a2905f
Pyramid
3. Podocytes are the cells that lines the:
. Glomerulus
. Bowman's capsule . Tubules . Ducts 2009
4. What is the function of the brush border on the proximal convoluted tubule?
. Decrease the surface area
. Secretion
. Motility a2009
. Absorption
5. Renin is secreted by which cell?
. Macula densa
. Juxtaglomerular . Interstitial Lacis 29009
6. Ureter
7. Proximal convoluted tubule is lined by which epithelium?

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a. Squamous
b. Stratified squamous
c. Transitional
d Pseudostratified
b. Cuboidal
c. Columnar
d. Transitional
. Which hormone regulates the reabsorption of sodium and potassium ions at convoluted tubule?
a. Aldosterone
b. Angiotensin |
c. Angiotensin II
d. ACE
. The ultrafiltrate leaving the thin ascending loop of Henle is to plasma.
Hypotonic
lsotonic Hypertonic 29509
Kidney Ureter Bladder Urethra 2900p
a. JG cells are modified smooth cells of efferent arteriole
b. Macula densa are modified cells of distal convoluted tubule near afferent arteriole
c. Lacis cells are extraglomerular mesangial cells
d. JG apparatus secretes angiotensin
Visceral layer of Bowman’s capsule—podocytes Parietal layer of Bowman’s capsule—simple squamous epithelium Distal tubule cells—brush border 2909
Thin segment of Henle’s loop—simple squamous epithelium
13 . A small protein passes through the glomerular basement membrane into the glome

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filtrate. Cells of which of the following tissues would remove the protein from the filtrate?
Visceral epithelium of the renal corpuscle
Distal convoluted tubule Proximal convoluted tubule 2900p
Parietal layer of the renal corpuscle
a. Renal tubules
b. Minor calyces
c. Major calyces
d. Renal pelvis
It consists of cortical tissue
Interlobular arteries pass through them It is located adjacent to the renal pyramids 2909
It is part of the medulla of the kidney
a. Glomerulus
b. Calyx
c. Renal pyramid
d. Renal sinus
Proximal convoluted tubule
Loop of Henle
Distal convoluted tubule
Papillary duct 2050
Lobule
Lobe Renal columns Medullary ray 2900p
DarWNP
ovraT799

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* Structure of spinal cord
*Layers of cerebrum
*Cells of cerebrum
*Layers of cerebellum
*Neurons of cerebellum

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Presence of outer white and inner gray matter, Cerebrum = Presence of outer gray and inner white matter. Presence of outer white and inner gray matter, Cerebellum = Presence of outer gray and inner white matter. Gray matter consists of anterior horn and posterior horn, Cerebrum = Cerebral cortex is made up of six laminae from superficial to deep: Plexiform, outer granula, outer pyramidal, inner granular, inner pyramidal, and pleomorphic layer. Gray matter consists of anterior horn and posterior horn, Cerebellum = Cerebellar cortex is made up of three layers from superficial to deep: molecular, Purkinje cell, and granular layer
Sentral canalis present in the gray commissure White matter consists of anterior, lateral, and posterior
Pyramidal, stellate, granule, cells of Martinotti, and horizontal cells of Cajal are present
Purkinje, granule, Golgi, stellate, and basket cells are present
__
Pyramidal cells are present in almost all the layers.
Purkinje single
cells are flask
shaped and present as a
stratum.
funiculi.
Bf

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* The central nervous system (CNS) consists of brain and spinal cord floating in cerebrospinal fluid. They are protected by the skull and vertebrae and is surrounded by meninges. The classification of nervous system is depicted in Flowchart 19.1.
* Section through CNS shows gray and white matter.
* Gray matter consists of the aggregation of cell bodies of neurons, dendrites, unmyelinated/initial part of axons, and neuroglial cells. Fibers within the gray matter are unmyelinated, and this causes these regions to appear gray.
* White matter consists predominantly of myelinated fibers along with some unmyelinated fibers and neuroglial cells. It is the reflection of light by myelin that gives this region its whitish appearance.
* Blood vessels are present in both gray and white matter.
*In the cerebrum and cerebellum, gray matter present on the surface forming cerebral and cerebellar cortex. Deep to the cortex, there is white matter, but within the latter, several isolated masses of gray matter are present. Such aggregations of neuronal cell bodies embedded in white matter in the CNS are called nuclei.
* In the spinal cord and brainstem, the white matter is peripheral and the gray matter is central. It assumes a shape of H in the spinal cord.
* The axons arising from in one mass of gray matter terminate by synapsing with neurons in other masses of gray matter. Such axons connecting two or more masses of gray matter form bundles, and the aggregations of such fibers are called tracts. Larger collections of fibers are also referred to as funiculi, fasciculi, or lemnisci. Large bundles of fibers connecting the cerebral or cerebellar hemispheres to the brainstem are called peduncles.
i

* The spinal cord is an elongated and cylindrical part of CNS and located within the upper two-thirds of the vertebral canal. It is the continuation of medulla oblongata and ends as tapering/conical structure called the conus medullaris. The conus is continuous and, below, with a fibrous cord called the filum terminale (modification of pia mater).
* Tt is surrounded by meninges: Outer dura, middle arachnoid, and inner pia mater. On cross-section, the spinal cord presents outer white and inner gray matter (Fig. 19.1 and Plate 19.1).

Fig. 19.1: Main features to be seen in a transverse section through the spinal cord (schematic representation).

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* Tt consists of cell bodies of nerve cells, neuroglia, and blood vessels and arranged in H-shaped column (Plate 19.1).
* The gray matter presents a pair of larger anterior/ventral gray column called anterior/ventral horn and a narrow elongated posterior/dorsal gray column called posterior/ dorsal horn, an intermediate region intervenes between anterior and posterior horns, and a gray commissure connects the symmetrical halves of gray matter across the midline.
* Anterior gray column is broad and short and presents head and base. Posterior gray column is longer and narrower than the anterior column and consists of base, neck, head and apex ventrodorsally. Base is continuous with the intermediate region of gray matter.
Plate 19.1: Spinal cord
Spinal cord: (A) As seen in drawing; (B) Gray matter (photomicrograph).

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"The spinal cord has a characteristic oval shape.
"It is made up of white matter (containing mainly of myelinated fibers), and gray matter (containing neurons and unmyelinated fibers).
"The gray matter lies toward the center and is surrounded all round by white matter.
"The gray matter consists of a centrally placed mass and projections (horns) that pass
* Intermediate region presents between the base of anterior and posterior horns. In thoracic and upper lumbar region of the spinal cord, it forms a small lateral projection from the base of anterior horn called as the lateral gray column.
* The gray commissure is traversed by the central canal, which contains cerebrospinal fluid and it is lined by ependyma. Central canal is continuous with fourth ventricle through the canal of medulla. Within the conus medullaris, the central canal is enlarged to form terminal ventricle. Central canal divides the gray commissure into ventral and dorsal parts.
* The cell bodies of neurons differ in size and in the prominence of Nissl substance in different regions of spinal gray matter. They are most prominent in the anterior gray column.

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* Tt consists of nerve fibers, neuroglia, and blood vessels. It occupies the periphery of H-shaped gray matter.
* Tt is divided into right and left halves by a deep anterior median fissure and posterior median septum. Each half of the white matter of the spinal cord is arranged into anterior, lateral, and posterior funiculi.
* Anterior funiculus extends on each side from anterior median fissure to the most lateral fibers of the ventral nerve roots, and it is medial and ventral to the anterior gray column. Anterior white commissure connects the anterior funiculi of both sides, and it is present in front of ventral gray commissure.
* Lateral funiculus intervenes between the emerging of the ventral roots of spinal nerves and posterolateral sulcus where dorsal nerve roots emerge and present lateral to and between the anterior and posterior gray columns. The anterior and lateral funiculi are collectively referred to as the anterolateral funiculus.
* Posterior funiculus extends on each side from the posterolateral sulcus to the posteromedian septum and presents medial to the dorsal gray matter.
* The white matter of the right and left halves of the spinal cord is continuous across the middle line through the ventral white commissure
which lies anterior to the gray commissure. The white matter contains tracts (ascending or descending) that connect gray matter at different levels of the spinal cord. Some tracts ascend into (or descend from) the brainstem, the cerebellum, or the cerebral cortex. Details of such tracts are given in books of neuroanatomy.
- * The cross-section of spinal cord varies depending on the region of spinal cord. The features detailed in Table 19.1.
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* Cerebral hemispheres are separated from each other by longitudinal fissure.
* A thin layer of gray matter present on the surface of the cerebrum forming the cerebral cortex. Cerebral hemisphere comprises a mantle of gray matter at the surface, white matter beneath the cortex, and some collections of subcortical gray matter masses in the white matter known as basal ganglia.
* Gray matter of the cerebral cortex contains the cell bodies of neurons along with their processes, neuroglia, and blood vessels. The neurons are of various sizes and shapes. They establish extremely intricate connections with each other and with axons reaching the cortex from other masses of gray matter.
* The cortex is subdivided into allocortex/old cortex which form about 10% of the cortex and isocortex/ neocortex which comprises 90% of the cortex.
* For accommodation in the rigid cranial cavity, the cerebral cortex is folded into numerous gyri separated by sulci.
Gray matter, Cervical = Q More Q Ventral horn is bulbous and broad OQ Lateral horn is absent. Gray matter, Thorax = O Less Q Ventral and dorsal horns are slender 2 Lateral horn is present. Gray matter, Lumbar = Q More Q Ventral and dorsal horns are bulbous and broad 2 Lateral horn is present in only upper two segments. Gray matter, Sacral = Q More Q Ventral and dorsal horns are bulbous and broad 2 Lateral horn is absent. White matter (volume of white matter is progressively increasing from below upward), Cervical = Abundant. White matter (volume of white matter is progressively increasing from below upward), Thorax = Abundant but lessthan cervical. White matter (volume of white matter is progressively increasing from below upward), Lumbar = Less. White matter (volume of white matter is progressively increasing from below upward), Sacral = Least. Position of centralcanal, Cervical = _—- Ventral. Position of centralcanal, Thorax = Ventral. Position of centralcanal, Lumbar = Central. Position of centralcanal, Sacral = Dorsal. Cross-section, Cervical = Oval. Cross-section, Thorax = Round and small. Cross-section, Lumbar = Oval but smaller than cervical. Cross-section, Sacral = Round and very small

* The neocortical gray matter thickness ranges from 1.5 mm to 4 mm, thick over gyri and thin over sulci.
* The cortical gray matter presents horizontal lamination and consists of six superimposed layers.

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Cortical neurons vary in size, in the shape of their cell bodies, and in the lengths, branching patterns, and orientation of their processes (Fig. 19.2). Cortical neurons are of four basic types:
1. Pyramidal
2. Stellate/granule cells
3. Cells of Martinotti
4. Horizontal cells of Cajal.

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* Pyramidal cells are about 5.5 billion in number, and it constitutes about two-thirds of all cortical neurons. Their cell bodies are pyramidal in shape, with the apex generally directed toward the surface of the cortex.
* A large apical dendrite arises from the apex and extends toward superficial layer of the cortex and divides horizontally. Numerous basal dendrites arise from basal angles and extend horizontally. All the dendrites are provided with numerous dendritic spines, which establish synaptic contacts with the projection fibers from other cortical neurons.
Fig. 19.2: Some of the cell types to be seen in the cerebral cortex (schematic representation). (B: basket cells; F: fusiform cells; H: horizontal cell of Cajal; M: cells of Martinotti; N: neuroglia form cell; P: pyramidal cell; S: stellate cell)
* The axon arises from the base of the pyramid and gives rise to efferent projection, association, or commissural fibers. The larger cell axons possess longer axons and form projection fibers to the basal ganglia, brainstem, or spinal cord. Small-sized cells provide association or commissural fibers. The collateral branches of the axons projects back the cortex and makes synapse with the stellate cells or other interneurons.
* Once committed to perform specific function, pyramidal cells do not change the modus operandi.

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* The stellate neurons are relatively small and multipolar and belong to type II neurons. They form about one-third of the total neuronal population of the cortex.
* Their axons are short and end within the cortex. Their processes extend chiefly in a vertical direction within the cortex, but in some cases, they
may be oriented horizontally.
* Dendrites make synapses with afferent projection fibers and with the collaterals of pyramidal axons. The axons of stellate cells form ascending and descending branches and establish synapses with the dendritic spines of pyramidal neurons or other interneurons.
* Fusiform cells are modified stellate neurons.

They are multipolar small neurons present in almost all layers of the cortex but more in sixth layer. They have smaller dendrites and elongated axons which run vertically outward to ramify in the superficial layer.

They are located in first layer. Their neurites extend tangentially and integrate numerous columnar units of cortex.

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* The cells of the cortex are arranged in horizontal lamination and also in vertical columns. Vertical columns are the functional units of the cortex, since within the columns pyramidal cells, stellate cells, and other interneurons communicate with one another by short synaptic loops resulting in large output.
* Axon collaterals of pyramidal cells excite some interneurons of the column and inturn it discharge repeated excitatory impulses to the aforesaid pyramidal cells of that column. Simultaneously, the axon collaterals of pyramidal cells inhibit some other interneurons of the column and latter inhibit the pyramidal cells of the surrounding columns. The neuronal sharpening is achieved by a group of excited neurons in an inhibitory surround.
The horizontal
lamination
(Fig.
19.3
and
Plate
19.2)
of the
neocortex is
arranged from
outside inward,
which is
as
"Lamina I: Plexiform or molecular layer
" Lamina IT: External/outer granular layer
™ Lamina III: Outer pyramidal cell layer
"Lamina IV: Internal/inner granular layer
follows:
Lamina V: Ganglionic layer/inner pyramidal layer

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"Lamina VI: Polymorphic/pleomorphic layer.
* The plexiform layer is made up predominantly of horizontal nerve fibers with the occasional horizontal cells of Cajal. Fibers are derived from apical dendrites of pyramidal cells, axons of stellate and Martinotti cells, and neuritis of horizontal cells of Cajal.
* Outer granular layer is packed with numerous stellate cells and small number of pyramidal cells.
* Outer pyramidal layer predominantly contains pyramidal cells and few stellate cells. Small-sized pyramidal cells lie in the superficial part and mediumsized cells in the deeper part of the layer.
* Inner granular layer densely packed with stellate cells with medium-sized pyramidal cells dispersed in irregular manner. Inner part of this layer is traversed by tangential fibers of the outer band of Baillarger more marked in visual cortex of occipital lobe, hence it is also called striate cortex.
+ a Ganglionic/inner pyramidal layer contains medium-and large-sized pyramidal cells. Large neurons called as giant pyramidal cells of Betz lie in the deeper zone of the layer. In neocortex, small pyramidal cells are located superficially; medium-sized cells in more deep, and large cells lie further deeply. Tangential fibers of inner band of Baillarger traverse at the outer part of this layer.
Polymorphic layer is the deepest layer and contains multipolar neurons, which are probably modified pyramidal cells. Cells of Martinotti are more in this layer with axons run vertically to ramify in the molecular layer. *, te
*In addition to the cell bodies of neurons, the cortex contains abundant nerve fibers. Many of these are vertically oriented. In addition to the vertical fibers, the cortex contains transversely running fibers called the external band of Baillarger and internal band of Baillarger. ate

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* Most of the afferent fibers from the specific nuclei of thalamus make synapses in the laminae I-IV. Afferent projection fibers from other nuclei of thalamus and ascending reticular system terminate in all the layers of cortex. Laminae IJ and IV are concerned with sensory, laminae HI and V with somatomotor or visceral motor activities. Laminae I and VI are for the association of sensory and motor behavior. 3
Efferent projection fibers from pyramidal cells of fifth layer give rise to corticostriate, corticobulbar, corticopontine, and corticospinal fibers. Modified pyramidal cells of sixth layer give rise to corticothalamic fibers. Pyramidal cells of third layer give rise to association and commissural fibers. 4, &
Fig. 19.3: Laminae of cerebral cortex (schematic representation).
®
*
Cerebral cortex: (A) As seen in drawing; (B) Photomicrograph.

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| slide of cerebral cortex shows outer gray and inner white matter
Multipolar neurons of various shapes are arranged in six layers in the gray matter
*
Axons of these neurons are present in the white matter
Neuroglia and blood vessels are present in gray and white matter.

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*The structure of the cerebral cortex shows considerable variation from region to region, both in terms of thickness and in the prominence of the various laminae described above. Finer variations form the basis of the subdivisions into Brodmann's areas. These are as follows:
*In the agranular cortex, the external and internal granule laminae are inconspicuous. This type of cortex is seen mo; typically in the precentral gyrus (area 4) and is, therefore believed to be typical of "motor" areas. It is also seen in som other areas.
*In the granular cortex, the granular layers are highly develope while the pyramidal and ganglionic layers are poorly developed 4 absent. In the visual area, the external band of Baillarger prominent and forms a white line that can be seen with the nake eye when the region is freshly cut across. This stria of Genna gives the name striate cortex to the visual cortex.
*Between the two extremes represented by the agranular an granular varieties of cortex, three intermediate types describe are frontal cortex, parietal cortex, and polar cortex. The fronti type is nearest to the agranular cortex, the pyramidal cells bein prominent, while the polar type is nearest to the granular cortex.
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* The cerebellum lies in the posterior cranial fossa. The cerebellum has a superficial layer of gray matter, the cerebellar cortex, a medullary core of white matter, and four pairs of cerebellar nuclei embedded in the white matter.
* The cortex is thrown into numerous transverse folds called folia separated by fissures.
* The central core of each cerebellar hemisphere is formed by white matter which is continuous with the peduncles. The white matter of the two sides is connected by a thin lamina of fibers that are closely related to the fourth ventricle. The upper part of this lamina forms the superior medullary velum, and its inferior part forms the inferior medullary velum. Both of these take part in forming the roof of the fourth ventricle.
* The cerebellar nuclei are as follows (Fig. 19.4):
"The dentate nucleus lies in the center of each cerebellar hemisphere. It made up of a thin lamina of gray matter that is folded upon it so tha resembles a crumpled purse.
" The emboliform nucleus lies on the medial side of the dentate nucleus.
" The globose nucleus lies medial to the emboliform nucleus.
" The fastigial nucleus lies close to the middle line in the anterior part of | superior vermis.

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* The cerebellar cortex is entirely uniform in structure, and their neurons with processes are arranged in geometrical configuration.
* The cortex consists of three layers as follows (Plate 19.3 and Fig. 19.5):
1. Outer molecular layer
2. Middle Purkinje cell layer
3. Inner granular layer, which rests on white matter.
* The neurons of the cerebellar cortex are of five main types. All of them are inhibitory except granule cells.
. Purkinje cells, forming the layer named after them
. Basket cells, present in the molecular layer
. Granule cells, forming the granular layer . Stellate cells, present in molecular layer bBWNrR
. Golgi cells, present in the granular layer. Oa
1. Molecular layer: Very few nuclei of neurons seen. Many cell processes present. Appears pale
2. Purkinje cell layer: Single layer of big flask-shaped pink neurons
3. Granular cell layer: Appears very dark blue because of presence of abundant nuclei of neurons
Fig. 19.4: Cerebellar nuclei (schematic representation).

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The section of cerebellum shows leaf-like folia
The cortex is covered by pia mater which appears as a thin layer of collagen fibers. Blood vessels may be seen just beneath the pia mater
Outer gray matter is arranged in three layers from without inwards:
Inner white matter shows axons which appear as pink fibers

Fig. 19.5: Arrangement of neurons in the cerebellar cortex (schematic representation).

* The molecular layer is the superficial layer of the cortex and situated just below the pia mater.
* This layer predominantly consists of unmyelinated nerve fibers which are derived from the parallel fibers axons of the granule, stellate,and basket cells, sensory climbing fibers, dendrites of Purkinje, and Golgi cells. This layer stains light as it consists of few cells and more of nerve fibers.
* Two types of cells are found in this layer: (1) stellate cells (situated in the superficial part of the molecular layer) and (2) basket cells (situated in the deeper layer).

The Purkinje cell layer contains flask-shaped cell bodies of Purkinje cells which are arranged as a single stratum.
- * A dendrite arises from the "neck" of the "flask" and passes "upward" into the molecular layer, and axons make synaptic connections with the deep cerebellar nuclei.

*Tt is the innermost layer and packed with cell bodies and dendrites of granule cells and a few Golgi cells with their processes and brush cells.
* Tt also contains sensory mossy fibers with their synaptic glomeruli. The granular layer stains deeply with hematoxylin as it is densely packed with granular cell.

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* Cell bodies of Purkinje cells are large and flask shaped and present in the middle layer as a single stratum.
* The dendrites arise from the neck of the flask and pass upward into the molecular layer at right angles to the long axis of the cerebellar folium. Here it divides and subdivides to form an elaborate dendritic tree. First three orders of the dendritic tree are smooth, and subsequent branches are provided with spines for synaptic contacts.
* The axon arises from the bottom of the cell and passes downward through the granular layer to enter into the white matter and makes synaptic contact with deep cerebellar nuclei. The proximal part of unmyelinated axon makes synaptic contact with collaterals of basket cells. Recurrent collateral from Purkinje cells makes synapses with the stellate, basket, Golgi, and other Purkinje cells.
* Purkinje cells receive input from parallel fibers of granule cells, axons of stellate cells, axons of basket cells, climbing fibers, and recurrent axon collaterals of other Purkinje cells.
* Purkinje cells give output to deep cerebellar nuclei, lateral vestibular nucleus, stellate, basket, Golgi, and to other Purkinje cells through axon collaterals.
* They act as sole output neurons from the cerebellar cortex and exert
inhibitory influence to the deep cerebellar and lateral vestibular nuclei.

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* Granule cells are very small with rounded cell body and four to five dendrites. It is present in the granular layer. It is the excitatory neuron.
* The spaces not occupied by granular cells in the granular layer are called cerebellar islands/glomeruli (Fig. 19.6).
* The dendrites radiate in the granular layer and synapse in glomeruli as claw-like expansion around the rosette of mossy fibers.
* The axons of granule cell enter into molecular layer, where it bifurcates in T-shaped manner and runs in opposite direction along the long axis of the folium and covers a territorial plane of about 500 Purkinje cells.
* The granule cells receive inputs from afferent mossy fibers through glomeruli, collaterals of climbing fibers, axons of Golgi cells, and recurrent axons of Purkinje cells.
* The parallel fibers of granule cells excite the inhibitory response of Purkinje, stellate, basket, and Golgi cells.

* These cells are present in the molecular layer. Their cell bodies and processes lie at right angles to the long axis of folium.
* Their dendrites synapse with parallel fibers of granule cells and collateral climbing fibers, while their axons synapse with dendritic spine of Purkinje cells near their origin.

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* These cells lie in the deeper part of the molecular layer, and their arrangements and connections are almost similar to stellate cells.
* Their dendrites ramify in the molecular layer and are intersected by parallel fibers with which they synapse. They also receive recurrent collaterals from Purkinje cells, climbing fibers, and mossy fibers.
* The axons of these cells pass at right angles to the axis of the folium and cover the territory of 10-12 Purkinje cells. The axon collaterals branch and form networks or baskets around the cell bodies of Purkinje cells and synapse at the preaxon of the Purkinje cells.
* Both stellate and basket cells are inhibitory neurons, and they suppress the inhibitory response of Purkinje cells if excited by parallel fibers.
Fig. 19.6: Structure of cerebellar glomeruli. The outer capsule is not shown (schematic representation).

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* These are the largest neurons of cerebellar cortex. The cell bodies are located in the outer part of granular layer beneath the Purkinje cells.
* Their dendrites enter the molecular layer and granular layer. In molecular layer, they synapse with the parallel fibers, climbing fibers, and recurrent axon collateral of Purkinje cells, and in granular layer they synapse with mossy fibers.
* They receive inputs from parallel fibers and mossy fibers which excite the Golgi cells and recurrent axon collateral of Purkinje cells which inhibit the Golgi cells.
* They give output to dendrites of granule cells.
* Golgi cells are inhibitory neurons, and they diminish the excitability of granule cells.
Afferent fibers entering the Cerebellar Cortex
The afferent fibers to the cerebellar cortex are of two types, and they reach the cerebellum through peduncles: (1) Mossy fibers and (2) Climbing fibers, and both the fibers are excitatory.

* All fibers entering the cerebellum other than olivocerebellar end as mossy fibers which synapse with many granule and few Golgi cells.
* Mossy fibers originate from the vestibular nuclei (vestibulocerebellar), pontine nuclei (pontocerebellar), and spinal cord (spinocerebellar) and terminate in the granular layer of the cortex within glomeruli.
* Each mossy fiber divides into 30-40 terminal swellings called the rosette, which forms the central component of glomeruli. A single rosette is surrounded by and makes synapses with the dendrites of 15 granule cells.
* A single mossy fiber activates about 450 granule cells, and their parallel fibers excite about 450-500 Purkinje cells (Fig. 19.6). Afferent inputs through mossy fibers pass through granule cells to reach the Purkinje cells.

* These fibers are derived mainly from the inferior olivary complex (Fig. 19.5). They pass through the granular and Purkinje cell layer to reach the molecular layer. Each climbing fiber exerts specific influence on one Purkinje cell.
* Each fiber provides collateral branches to synapse with the deep cerebellar nuclei and makes monosynaptic connections with the single Purkinje cells. In molecular layer the collaterals of climbing fibers synapse with all other types of neurons.

* Efferent fibers of the cerebellar cortex are from Purkinje cells into deep cerebellar or vestibular nuclei. Axons of the Purkinje cells are inhibitory to cerebellar nuclei.
* The fibers from dentate, emboliform, and globose nuclei leave cerebellum through the superior cerebellar peduncle. The fibers from the fastigial nucleus leave the cerebellum through inferior cerebellar peduncle.

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Glomerulus is covered by Bergmann glia cells. It is composed of:
* A rosette of mossy fiber in the center forming presynaptic element
* Claw-like dendritic expansions of granule cells enveloping the rosette forming postsynaptic element
* Axon terminal of Golgi cells which come in contact with dendrites of granule cells as presynaptic element.
B

* Parkinson's disease: Slowly progressive neurologic disorder caused by the loss of dopamine-secreting cells in the substantia nigra and basal ganglia of the brain.
* Cerebellar ataxia: Purkinje cells in the cerebellar cortex are damaged leading to the development of poor coordination of voluntary movement.
* Motor neuron disease: A group of neurodegenerative disorders that selectively affect motor neurons, the cells which control voluntary muscles of the body.

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*The climbing fibers when stimulated directly facilitate the inhibitory function of Purkinje cells to the deep cerebellar nuclei, but at the same time stellate and basket cells are stimulated by the climbing fibers exert their inhibitory function on the same Purkinje cells. This type of alteration of response of Purkinje cells is called feed forward inhibition.
*When mossy fibers and granule cells are stimulated and the excitatory waves are conveyed by the parallel fibers to the Purkinje, stellate, basket, and Golgi cells results in feed forward inhibition. Inhibitory Golgi cells on facilitation suppresses the excitation of the granule cells, this type of alteration of excitatory influence of granule cells is called feed backward inhibition.
*When the sensory input to the cerebellum is excitatory, the output from the cerebellar cortex is inhibitory.
*The row of excited Purkinje cell is surrounded by the zones of inhibition at the periphery. This helps in neuronal sharpening.
1. All of the following are the components of gray matter; except:
Cell bodies
Myelinated nerve fiber
Neuroglia Dendrites 2959
2.
Lateral horn is present in segment of spinal cord.
a. Cervical
b. Thorax
c. Lumbar
d. Sacral
3. Which of the following is not a layer of cerebral cortex?
External granular External pyramidal Plexiform Purkinje 2905p
4. Outer band of Baillarger is present in which layer of cerebral cortex?
a. Outer pyramidal
b. Outer granular
c. Inner pyramidal
d. Inner granular

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a. Purkinje
b. Stellate
c. Granule
d. Golgi
6. Cerebellar glomerulus is composed of all, except:
Mossy fibers Climbing fibers Granule cells Golgi cells 2959

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* Structure of testis, seminiferous tubules, and Sertoli cells
* Spermatogenesis
* Structure of epididymis, vas deferens, and seminal vesicle
* Structure of prostate

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cellsand supporting cells of Sertoli Q Between the tubules, fibrous connective tissue is present Q Interstitial cells of Leydig are present between the tubules., Prostate = Q Fibromuscular glandular tissue Q Glandular tissue is made up of numerous follicles of compound tubuloalveolar glands Q Follicles are lined by pseudostratified columnar epithelium.
The male reproductive system consists of (Fig. 20.1):
* A pair of testis
* Genital ducts: The epididymis, the ductus deferens, and ejaculatory ducts
* Accessory sex glands: A pair of seminal vesicle, prostate, and a pair of bulbourethral glands
* Male urethra
* Penis.
The testes are responsible for the formation of male gametes called spermatozoa and synthesis and storage of testosterone. The glands secrete the noncellular portion of semen. Ducts deliver the semen to the female genital tract.

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* Each testis is about 4 cm long and is located in the scrotum and produces spermatozoa and testosterone. The adult testes are placed outside the body in scrotum for normal spermatogenesis as the temperature is 2—3°C less than the body temperature.
* Testis is surrounded by a capsule from outside inward: visceral layer of tunica vaginalis, tunica albuginea, and tunica vasculosa.
Fig. 20.1: Male reproductive system (schematic representation).
* Tunica albuginea is a thick fibrous membrane which covers the entire testis
and projects into the interior from posterior border as an incomplete partition known as mediastinum testis. The visceral layer of tunica vaginalis covers the tunica albuginea, except in the region of the mediastinum testis.
* From the anterior surface of the mediastinum testis, numerous septa radiate and divide the substance of the testis into a number of lobules. About 200300 lobules are present in each testis. Each lobule is roughly conical, the apex of the cone being directed toward the mediastinum testis.
Individual lobule of the testis is lined by an aereolar and vascular membrane called tunica vasculosa. , a
* Each lobule contains two to three highly convoluted seminiferous tubules. Near the apex of the lobule, the seminiferous tubules lose their convolutions and join one another to form about 20-30 larger, straight tubules (or tubuli recti) (Fig. 20.2). These straight tubules enter the fibrous tissue of the mediastinum testis and unite to form a network called the rete testis. .
* At the upper end the rete testis gives off 12-20 efferent ductules and enters into the head of the epididymis. This duct is highly coiled on itself and forms the body and tail of the epididymis. The head of the epididymis is made up of highly convoluted continuations of the efferent ductules. At the lower end of the head of the epididymis, these tubules join to form a single tube called the duct of the epididymis. The duct continuous as ductus deferens forms the tail of epididymis (Plate 20.1).

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* Seminiferous tubules are highly convoluted structures present in each lobule of the testes. Each testis has about 200 lobules with one to three seminiferous tubules per lobule, and the total number of tubules is between 400 and 600.
* When stretched out, each tubule is 70-80 cm in length and has a diameter of about 150 pm. The combined length of all seminiferous tubules in one testis is between 300 mt and 900 mt.
* Within a lobule, the spaces between seminiferous tubules are filled by very loose connective tissue, containing blood vessels and lymphatics.
* Wall of the each seminiferous tubule is covered by fibrous connective tissue and lined by a generation of spermatogenic cells and supporting cells of Sertoli. The connective tissue and epithelium are separated by a basement membrane.
Fig. 20.2: Basic structure of the testis (schematic representation).
Details of cells lining a seminiferous tubule seen at a high magnification. Note that the cell boundaries are indistinct, and nuclei are prominent.
* The outermost row of nuclei belongs to sustentacular cells (Sertoli) and to spermatogonia; some of which are undergoing mitosis (note very dense nucleus of irregular shape).
* Passing inward toward the center of the tubule, we have large darkly staining nuclei of spermatocytes, and many smaller nuclei of spermatids.
* Toward the center of the tubule a number of developing spermatozoa are seen. The sperms are often found in clusters embedded in the cytoplasm of Sertoli cells.
* In the adult testis, sustentacular cells are less prominent than germ cells.
rr)

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They are more prominent than germ cells before puberty and in old age.
1. Spermatogenic cells which produce spermatozoa
2. Sustentacular (Sertoli) cells which have a supportive function.
‘==
Testis (Low magnification): (A) As seen in drawing; (B) Photomicrograph.

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The testis has an outer fibrous layer, the tunica albuginea deep to which:
"A number of seminiferous tubules cut in various directions are seen
*The tubules are separated by connective tissue, containing blood vessels and groups of interstitial cells of Leydig
Each seminiferous tubule is lined by several layers of cells
*Cells are of two types:
Note: The appearance of the cellular lining of the seminiferous tubules is characteristic and a student who has studied sections through them carefully (even at low magnification) is not likely to mistake the seminiferous tubules for anything else. The points to note are: (a) the many layers of cells; (b) the great variety in size and shape of the cells and of their nuclei; (c) the lack of a well-defined margin of the lumen; and (d) inconspicuous cell boundaries.

* Spermatogenic cells represent the various stages of maturation of spermatozoa. They are arranged in developmentally higher order from the basal lamina to the lumen, namely spermatogonia, spermatocytes, spermatids, and spermatozoa (Plate 20.2). However, all types of cells are not seen in any one part of the seminiferous tubule at a given time.
* In a given segment of the tubule, there is a gradual change in the type of cells encountered. Over a period of time, waves of maturation of germ cells pass along the length of a seminiferous tubule, and this is referred to as spermatogenic cycle.

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* These are tall, elongated columnar/pyramidal-shaped cells. It has basally located, oval nucleus with centrally placed prominent nucleolus. The base of each sustentacular cell rests on the basement membrane, spermatogonia being interposed among the sustentacular cells.
* The apex of the sustentacular cell reaches the lumen of the seminiferous tubule. Numerous spermatids, at various stages of differentiation, are embedded in the apical part of the cytoplasm (Fig. 20.3 and Plate 20.2).
* Near the basement membrane, spermatocytes and spermatogonia indent the sustentacular cell cytoplasm.
* The lateral surface of the adjoining sustentacular cells is connected by tight junctions that divide the wall of the seminiferous tubule into deep basal and superficial adluminal compartments. The basal occluded junctions form the blood-testis barrier that isolates the adluminal compartment from connective tissue. This protects the developing spermatozoa from immune system. Otherwise the newly formed germ cells would be considered foreign cells (as they have different chromosome number and surface membrane receptor), and antispermic antibody will produce and lead to the suppression of spermatogenesis.
* The basal compartment contains spermatogonia cells, and the adluminal compartment contains primary and secondary spermatocytes and spermatids. The spermatids are drawn within the upper part of cells for spermiogenesis until mature spermatozoa is released into the lumen.
* Sustentacular cells contain abundant mitochondria, endoplasmic reticulum, and other organelles. It also contains an abundant microfilaments and microtubules for providing structural support to the developing spermatozoa.
* In the adult testis, sustentacular cells are less prominent than germ cells but more prominent than germ cells before puberty and in old age.

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* They provide physical and nutritional support to the developing germ cells.
* The phagocytosis of the residual cytoplasm that remains after conversion of spermatids to spermatozoa.
* They establish blood—testis barrier and protect the developing spermatozoa from immune system.
* They secrete fructose-rich fluid that helps to transport the spermatozoa along the seminiferous tubules. This fluid is rich in testosterone that may stimulate activity of cells lining the epididymis.
* Sertoli cells secrete a Miillerian inhibitory substance or anti-Miillerian hormone that suppresses the development of the Miillerian ducts in male fetuses. They are also believed to produce a substance that inhibits spermatogenesis before puberty.
* Follicle-stimulating hormone (FSH) from anterior pituitary activates Sertoli cells to secrete androgen-binding protein (ABP), and luteinizing hormone stimulates interstitial cells to secrete testosterone. ABP binds to
testosterone and increases its concentration within seminiferous tubules. It also secretes an inhibin hormone which inhibits the secretion of FSH. Testosterone, FSH, and ABP along with low scrotal temperature is necessary for proper spermatogenesis (Flowchart 20.1).
Fig. 20.3: A sustentacular cell and some related germ cells (schematic representation).
Plate 20.2: Testis (High magnification)

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'The outermost row of nuclei belongs to sustentacular cells (Sertoli) and to spermatogonia, some of which are undergoing mitosis (note very dense nucleus of irregular shape)
'Passing inwards toward the center of the tubule we have large darkly staining nuclei of spermatocytes, and many smaller nuclei of spermatids
'Toward the center of the tubule a number of developing spermatozoa are seen. The sperms are often found in clusters embedded in the cytoplasm of Sertoli cells
'In the adult testis, sustentacular cells are less prominent than germ cells. They are more prominent than germ cells before puberty and in old age
'In the drawing, groups of interstitial cells can be seen in the connective tissue between the seminiferous tubules (not seen in photomicrograh).
Note: In the practical class you may not be able to recognize these cells. Observe that the presence of many cells located at different levels gives the appearance of a stratified epithelium which are actually the spermatogonia at different stages of maturation.

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* The interstitial cells of Leydig are the large, round, or polyhedral cells lying in the connective tissue that intervenes between the coils of seminiferous tubules within the lobule (Plate 20.1A). They have single eccentrically placed nucleus and the cytoplasm stains lightly and often has a foamy appearance.
* They are typically steroid-secreting cells which have mitochondria with tubular cristae, numerous smooth endoplasmic reticulum, and welldeveloped Golgi complex. Rod-shaped Reinke's crystalloids are also present in the cytoplasm. Yellow-brown pigment (lipofuscin) is seen in some cells.
* The Leydig cells are abundant if fetal testis disappear after birth and reappear at puberty and persist throughout the reproductive life. The growth of the cells in fetal life is regulated by placental gonadotropin hormone and in puberty by interstitial stimulating hormone.
* Interstitial cells secrete male sex hormone testosterone. Secretion is stimulated by the interstitial cell stimulating hormone from hypophysis cerebri. Interstitial cells help in the descent of testis in fetal life, and in puberty, they stimulate the growth of secondary sex characters, initiate heterosexual drive, and play a role in growth of epithelial cells of prostate and seminal vesicles.
* Some interstitial cells may be present in the mediastinum testis, in the epididymis, or even in the spermatic cord. Apart from interstitial cells, the interstitial tissue contains collagen fibers, fibroblasts, macrophages, mast cells, blood vessels, and lymphatics.

(FSH: follicle-stimulating hormone; ICSH: interstitial cell-stimulating hormone; LH: luteinizing hormone)

* The rete testis consists of anastomosing tubules that are lined by flattened or cuboidal cells. They bear microvilli. The epithelium is surrounded by connective tissue of the mediastinum testis.
* The efferent ductules are lined by ciliated columnar epithelium, and some nonciliated cells bearing microvilli are also present. The tubules are surrounded by circularly placed smooth muscle. Movement of spermatozoa through the tubules is facilitated by ciliary action, and by peristaltic contraction of smooth muscle.

The process of the formation of spermatozoa from type A spermatogonia is called spermatogenesis (Flowchart 20.2). This process occurs in waves along the length of seminiferous tubules, and it takes about 64 days to complete. It consists of several stages as described in Figure 20.4.

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* The stem cells from which all stages in the development of spermatozoa are derived are called spermatogonia which are derived from primitive male sex cells. Spermatogonia lie near the basal lamina and contain diploid chromosomes (46, XY). Spermatogonia undergo several mitotic divisions and give rise to more spermatogonia, and to primary spermatocytes.
* Spermatogonia consists of dark type-A, paletype-A, and type-B types. Each germ cell divides into two dark type-A spermatocytes, which acts as stem cells. Each dark A divides into one dark A cell as a reserve and one pale A cell. Pale A undergo mitosis into two pale A cells, one as a reserve and another one divides into type B cell. Each type B cell undergoes mitosis, and four generations of type B spermatogonia are formed. B cells undergo mitotic division to form primary spermatocytes with diploid chromosomes.
Flowchart 20.2: Spermatogenesis.
* Primary spermatocytes are the largest cells with large spherical nuclei occupying the middle region of the seminiferous epithelium. Primary spermatocytes undergo first meiotic division (reduction division) to form two secondary spermatocytes with haploid chromosomes (23 X/23 Y) and 2n deoxyribonucleic acid (DNA). Primary spermatocytes enter into prolonged prophase of first meiotic division.
* Secondary spermatocytes are smaller than primary spermatocytes and undergo second meiotic division to form two spermatids with haploid chromosomes with 1n DNA and some residual bodies between them. There is no further reduction in chromosome number. They are scarcely seen in sections as they undergo second meiotic division as soon as they are formed.
* Each spermatid is a rounded cell with a spherical nucleus and present near the inner zone of seminiferous tubule epithelium. Older spermatids are plunge into the cytoplasm of Sertoli cells until the mature spermatozoa is released into the lumen of seminiferous tubules. The spermatid undergoes changes in shape and in the orientation of its organelles to form a spermatozoon. This process is called spermiogenesis.

Fig. 20.4: Some stages in spermatogenesis as seen in the walls of seminiferous tubules
Flowchart 20.3: Stages of spermatogenesis.

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* The process by which a spermatid becomes a spermatozoon is called spermiogenesis (or spermateleosis). The spermatid is a more or less circular cell containing a nucleus, Golgi complex, centriole, and mitochondria. All these components take part in forming the spermatozoon.
* The nucleus undergoes condensation and changes shape to form the head. The Golgi complex is transformed into the acrosomic cap that comes to lie over one side of the nucleus. The acrosome marks the future anterior pole of the spermatozoon.
* The centriole divides into two parts and migrates away from acrosome to the pole of the cell. The axial filament grows out of the distal centriole. The region occupied by the two centrioles later becomes the neck of the spermatozoon. The proximal centriole probably forms the basal body.
* The part of the axial filament between the head and the annulus becomes surrounded by mitochondria and together with them forms the middle piece.
Most of the cytoplasm of the spermatid is shed and is phagocytozed by Sertoli cells. The cell membrane persists as a covering for the spermatozoon.

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* The spermatozoon has a head, neck, and tail.
The tail is made of three pieces, i.e. middle piece, principal piece, and endpiece.
* The head is covered by a cap called the acrosomic cap, anterior nuclear cap, or galea capitis (Fig. 20.5). It is flattened from before backward so that it is oval when seen from the front but appears to be pointed or in section. It is occupied by nucleus with extremely condensed chromatin. This condensation makes it highly resistant to various physical stresses.
* The neck of the spermatozoon is narrow. It contains a funnel-shaped basal body and a spherical centriole.
* The basal body also called the connecting piece helps to establish an intimate union between the head and the remainder of the spermatozoon. It is composed of cylindrical arrangement of the nine columns of the connecting piece that encircles the centriole. Each of the columns is continuous distally with one coarse fiber of the axial filament.
* An axial filament begins just behind the centriole. It passes through the middle piece and extends into the tail. At the point where the middle piece joins the tail, this axial filament passes through a ring-like annulus. The axial filament that lies in the middle piece is surrounded by a spiral sheath made up of mitochondria.
Fig. 20.5: Structure of spermatozoon as seen by electron microscope (schematic
representation).

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* The axial filament is composed of several fibrils arranged as illustrated in Figure 20.6. There is a pair of central fibrils, surrounded by nine pairs or doublets arranged in a circle around the central pair. In addition to these doublets, there are nine coarser petal-shaped fibrils present outside each doublet. These coarse fibrils are present in the middle piece and most of the tail, but do not extend into the terminal part of the tail. These fibrils are surrounded by fibrous sheath, and the fibrous sheath is surrounded by spirally arranged mitochondria. The entire sperm is enclosed in a plasma membrane.
* The part of tail connected to neck is middle piece. The middle piece contains the mitochondrial sheath and provides energy for sperm maturation. Principal piece contains the 9 + 2 pattern of microtubules in the central core surrounded by nine coarse fibers enclosed in a fibrous sheath (Fig. 20.6). End piece contains 9 + 2 axonema enclosed by plasma membrane.
Maturation and capacitation of spermatozoa
Maturation
* As fully formed spermatozoa pass through the male genital passages, they undergo a process of maturation. Spermatozoa acquire some motility only after passing through the epididymis. The secretions of the epididymis, seminal vesicles, and the prostate have a stimulating effect on sperm motility, but spermatozoa become fully motile only after ejaculation.
* When introduced into the vagina, spermatozoa reach the uterine tubes much sooner than their own motility would allow, suggesting that contractions of uterine and tubal musculature exert a sucking effect.
Fig. 20.6: Transverse section across the tail of a spermatozoon to show the arrangement of fibrils (schematic representation).

* Spermatozoa acquire the ability to fertilize the ovum only after they have been in the female genital tract for sometime. This final step in their maturation is called capacitation. During capacitation, some proteins and glycoproteins are removed from the plasma membrane overlying the acrosome.
* When the sperm reaches near the ovum, changes take place in membranes over the acrosome and enable release of lysosomal enzymes present within the acrosome. This is called the acrosome reaction. The substances released include hyaluronidase that helps in separating corona radiata cells
present over the ovum. Trypsin-like substances and a substance called acrosin help in digesting the zona pellucida and penetration of the sperm through it. Changes in the properties of the zona pellucida constitute the zona reaction.
B

* The epididymis is a highly convoluted tubule and a comma-shaped structure present on the posterolateral aspect of testis.
* Tt consists of head, body, and tail. The head is formed by the union of the 10-20 efferent ductules, becomes highly coiled and continuous body. These are lined by ciliated columnar epithelium (Plate 20.3). At the lower end of the head of the epididymis, these tubules join to form a single tube called the duct of the epididymis.
EWA Epididymis
Epididymis: (A) As seen in drawing; (B) Photomicrograph.

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'The body of the epididymis is a long convoluted duct
'A section shows number of tubules lined by pseudostratified columnar epithelium in which there are tall columnar cells and shorter basal cells that do not reach the lumen. The
columnar cells bear stereocilia
'Smooth muscles are present in the wall of the duct
'Clumps of spermatozoa are present in the lumen of the duct.
* The body and tail of the epididymis are made up of the duct of the epididymis that is greatly coiled on itself. The terminal part of the tail which stores spermatozoa loses its convolutions and continuous with ductus deferens.
* The duct is lined by pseudostratified columnar epithelium which is composed of two types of cells called tall columnar principal cells and shorter basal cell.
* The basal cells are short, and they do not reach the lumen. Basal cells are pyramidal or polyhedral with rounded nuclei with large accumulation of heterochromatin, and it stains dark. The basal cells function as stem cells to replace the principal and basal cells.
* The tall principal cells have irregular, oval nuclei with one or two nucleoli. The nuclei stains light compared to basal cells and are located at the base of the cell. The cytoplasm has abundant rough endoplasmic reticulum, lysosomes, and a prominent Golgi complex. The luminal surfaces of each columnar cell bear long, branched, irregular microvilli, and nonmotile projections stereocilia, absorbs 90% of fluid and creates fluid current that moves the immobile sperm from the seminiferous tubles.
* These cells are supported on a basal lamina by circularly arranged smooth muscle fibers and loose connective tissue rich in blood capillaries. This muscle layer increases in thickness gradually from head to tail and may be organized into the inner circular and outer longitudinal layers in the tail region. This muscle layer helps to move the sperm along the duct.

* Phagocytosis of defective spermatozoa and residual bodies that are eliminated during spermatogenesis
* Absorption of excess fluid
* Secretion of substances (sialic acid, glycerylphosphorylcholine) that play a role in maturation of spermatozoa.
li

The ductus deferens (deferent duct or vas deferens) is a thick cord-like muscular tube, about 45 cm in length extending from the lower end of epididymis to the prostatic urethra. The wall of the ductus deferens consists of three layers from inside outward:
1. Mucous membrane
2. Muscular layer
3. Connective tissue

The mucous membrane shows a number of longitudinal folds so that the lumen appears to be stellate in section, and it is lined by simple columnar throughout and pseudostratified at the distal end of the vas deferens. Epithelium is similar to epididymis but principal cells are shorter. The cells are ciliated in the extra abdominal part of the duct. The epithelium is supported by a lamina propria in which there are many elastic fibers.

The muscle coat is very thick and consists of three layers of smooth muscle. It is arranged in the form of an inner circular layer and an outer longitudinal layer. An inner and outer longitudinal layer and middle circular layer are present.

The fibroelastic connective tissue forms the adventitial layer containing blood vessels and nerves.
The terminal dilated part of the ductus deferens is called the ampulla, which joints the duct of seminal vesicles to form ejaculatory duct. It has the same structure as that of the seminal vesicle (Plate 20.4).
i

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* The seminal vesicle (Plate 20.5) is a sac-like mass, located adjacent to the posterior wall of the prostate gland, and secretes a viscous fluid that constitutes about 70% of the ejaculate.
* The wall of the seminal vesicle consists of three layers from inside outwardwhich are as follows:

* The mucous membrane is highly convoluted, forming labyrinth-like culde-sac with lumen. The lumen is lined by pseudostratified columnar epithelium consists of short basal cells and low columnar cells. Goblet cells are also present in the epithelium.
* Columnar cells have numerous short microvilli, and cytoplasm contains rough endoplasmic reticulum, Golgi apparatus, numerous mitochondria, and abundant secretory granules. The height of the cell varies directly with the blood testosterone level.

The seminal vesicles consist of a thin intermediate layer of smooth muscles. The muscle layer contains outer longitudinal and inner circular fibers.

The outer covering of loose connective tissue forms the adventitial layer containing blood vessels and nerves.

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* The seminal vesicles produce a thick secretion that forms about 70% of the semen. The secretion contains fructose which provides nutrition to spermatozoa.
* Tt also contains amino acids, proteins, prostaglandins, fibrinogen, ascorbic acid, and citric acid. This secretion is expelled during ejaculation by the contraction of the smooth muscle of the vesicle wall.
* Prostaglandins stimulate activity in the female genital tract. Fibrinogen allows semen to coagulate after ejaculation.
Plate 20.4: Ductus deferens
*
>
Ductus deferens: (A) As seen in drawing; (B) Photomicrograph.
Jaypee Brothers Medical Publishers (P) Ltd; 2019. p. 120.

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This tubular structure displays:
A small irregular lumen
Mucous membrane lined by pseudostratified columnar epithelium with underlying lamina propria
*The muscle coat is very thick. Three layers, inner longitudinal, middle circular, and outer longitudinal are seen
* Outer most layer is adventitia composed of collagen fibers and containing blood vessels.
rr

Seminal vesicle (Low magnification): (A) As seen in drawing; (B) Photomicrograph.

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* The seminal vesicle is made up of a convoluted tubule
* The tube has an outer covering of connective tissue, a thin layer of smooth muscle and an inner mucosa
*The mucosal lining is thrown into numerous folds that branch and anastomose to form a network
* The lining epithelium is usually simple columnar or pseudostratified.
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* The prostate is the largest accessory sex gland. It is a dense conical organ extending from the neck of the urinary bladder and surrounds the beginning of urethra.
* Tt is made up of 30-50 compound tubuloalveolar glands that are embedded in the fibromuscular stroma. The glandular part of the prostate is poorly developed at birth. It undergoes considerable proliferation at puberty and degenerates in old age.
* The prostate is covered by inner true and outer false capsules. True capsule is formed by condensation of the fibrous stroma of the gland, and false capsule is derived from visceral layer of pelvic fascia. The space between the two capsules is occupied by prostatic venous plexus.
* The prostate consists of one-fourth of fibrous, one-fourth of muscular, and half of glandular tissue.
* In sections, the glandular tissue is seen in the form of numerous follicles that are lined by simple to pseudostratified columnar epithelium (Plate 20.6). The epithelium is thrown into numerous folds along with some underlying connective tissue. The foldings are formed after puberty, and in old age, infoldings of follicles disappear. The follicles drain into 12-20 excretory ducts that open into the prostatic urethra. The ducts are lined by a double layered epithelium. The superficial luminal layer is columnar, and the deeper layer is cuboidal.
* Gland consists of four zones: The transition zone surrounds the proximal part of urethra and comprises of 5% of the glandular tissue. The central zone comprises of 20% and surrounds ejaculatory duct behind the transition zone. The peripheral zone comprises of 70%, and it is cupshaped which surrounds the central and transition zones as well as prostatic
urethra except in front. The fibromuscular stroma with no glandular tissue is present in front of prostatic urethra between the peripheral zones.
* The glands of prostate are arranged in three concentric layers around the urethra and from within outward (Figs. 20.7 and 20.8):
1. Inner mucous glands: They are simple tubular surrounds the pre urethra open into the prostatic sinuses above the colliculus.

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2. Intermediate submucous glands with less follicle and smaller du into the sinuses at the level of colliculus.
3. Outer main glands are long branched glands with numerous follic longer ducts open in the prostatic sinuses below the colliculus. Di with the urethra.
* Small rounded masses of uniform or lamellated structure are found within the lumen of the follicles called amyloid bodies or corpora amylacea. These are more abundant in older individuals. They are rich in condensed glycoprotein and keratin sulfate without any clinical significance. They are often calcified.
* The fibromuscular tissue forms a conspicuous feature of sections of the prostate. It contains collagen fibers and smooth muscle. Within the gland the fibromuscular tissue forms septa that separate the glandular elements. These septa are continuous with a fibrous capsule that surrounds the prostate. The capsule contains numerous veins and parasympathetic ganglion cells.
* The prostate gland is traversed by the prostatic urethra and the ejaculatory ducts.

* The prostate produces a secretion that forms a considerable part of semen. The secretion is serous white fluid and rich in enzymes (acid phosphatase, amylase, and protease) and in citric acid.
* The prostate also produces prostaglandins that have numerous actions.

- * The penis is the erectile copulatory organ in males and consists of two parts, root and a body/corpus. The root is fixed to the perineum, and the

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* The penis is covered all round by thin skin that is attached loosely to underlying tissue (Fig. 20.9).
* The substance of the penis is made up of three columns of erectile tissue, two dorsal and one ventral, and each enclosed by its own dense fibrous connective tissue capsule, the tunica albuginea. The dorsal columns are the right and left corpora cavernosa, while the ventral column is the corpus spongiosum.
* The corpus spongiosum ends distally in an enlarged, bulbous portion, the glans penis.
* Erectile tissue of the penis contains numerous variably shaped, endothelially lined spaces, and they are separated from each other by connective tissue trabeculae and smooth muscle cells. The vascular spaces of the corpora cavernosa are larger centrally and smaller peripherally, whereas spaces of the spongiosum are similar in size throughout. The trabeculae of corpus spongiosum contain more elastic fibers and fewer muscle fibers than corpora cavernosa.
Plate 20.6: Prostate (Low magnification)
Prostate (low magnification): (A) As seen in drawing (Low magnification); (B) Photomicrograph (low magnification).

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'The prostate consists of glandular tissue embedded in prominent fibromuscular stroma "The glandular tissue is in the form of follicles with serrated edges. They are lined by columnar epithelium. The lumen may contain amyloid bodies
'The follicles are separated by broad bands of fibromuscular tissue.
* The spaces are in communication with arteries and veins but normally they are empty. They are filled with blood under pressure during erection of the penis.
* Each corpus cavernosum is surrounded by a dense sheath containing collagen fibers, elastic fibers, and some smooth muscle. In the midline the sheaths of the right and left corpora cavernosa fuse to form a median septum. The corpora cavernosa lie side by side and are separated only by a median fibrous septum.
Fig. 20.7: Arrangement of glandular tissue in prostate (schematic representation).
* The corpus spongiosum is placed in the midline ventral to the corpora cavernosa. The corpus spongiosum is also surrounded by a sheath, but this sheath is much thinner than that around the corpora cavernosa. The corpus spongiosum is traversed by the penile urethra throughout its length.
*The tip of urethra at glans penis is lined by stratified squamous nonkeratinized epithelium. Many small mucous glands of Littre are scattered along the length of urethra, which secrete mucus and have lubricating functions.
* Many sensory nerve endings are present in the penis, particularly on the glans.
Fig. 20.8: Lobes of prostate gland.
Fig. 20.9: Transverse section of penis (schematic representation).

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Bl
* Testicular tumors can arise from germ cells (germ cell tumors), stroma (sex cord stromal tumors), or both (combined germ cell-sex cord-stromal tumors). Examples of germ cell tumors are seminoma, embryonal carcinoma, teratoma, and choriocarcinoma; sex cord stromal tumors are Leydig cell tumor, Sertoli cell tumor, and granulosa cell tumor.
Gonadoblastoma is an example of combined germ cell-sex cord-stromal tumors.
* Germ cell tumors comprise approximately 95% of all testicular tumors and are more frequent before the age of 45 years. Testicular germ cell tumors are almost always malignant.
* Fructose content in seminal fluid: in the cases of male infertility, on semen analysis, the absence of fructose suggests congenital absence of seminal vesicle or portion of the ductal system or both.
* Benign prostatic hypertrophy: It mainly affects the transition zone, and the enlargement of the prostate is a very common condition in men and considered by some as normal aging process. Enlargement of the prostate can compress the urethra leading to difficulty in micturition.
* Carcinoma of prostate: It mostly arises from the peripheral zone, and many a times, carcinoma of the prostate is small and detected as microscopic foci in a prostate removed for benign enlargement of prostate or found incidentally at autopsy.
* Central zone is rarely involved in any disease.
1. Which of the following structure is incorrectly matched with their lining epithelium?
a. Rete testis—simple cuboidal epithelium
b. Ductuli efferentes—pseudostratified epithelium
c. Ductus epididymidis—pseudostratified columnar with stereocilia
d. Ductus deferens—simple columnar ciliated epithelium

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Sertoli
Leydig Spermatogonia Spermatocytes 2900p
3. All of the following are the functions of Sertoli cells except:
a. Physical and nutritional support to the developing germ cells
b. Phagocytosis of the residual cytoplasm
c. Establish blood—testis barrier
d. They secrete fructose less fluid
4. Which of the following is true?
There is one corpus cavernosa and one corpus spongiosum There is one corpus cavernosa and two corpora spongiosum There are two corpora cavernosa and one corpus spongiosum 2050p
There are two corpora cavernosa and two corpora spongiosum
. What is the most commonly seen type of epithelium in the prostate?
a. Transitional
b. Pseudostratified squamous
c. Simple squamous
d. Simple cuboidal
. What are the spherical structures seen in some prostatic alveoli called?
Corpora arenacea Hassall's corpuscles Prostatic concretions 29509
Pacinian corpuscles
. What type of gland composes the prostate?
Simple tubular gland Simple alveolar gland Compound tubular gland 2900p
Compound tubuloalveolar gland

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Loose irregular connective tissue
Smooth muscle Fibromuscular Adipose tissue 2905p
. How many seminiferous tubules are found in each testis of an average man?
a. 4-6
b. 40-60
c. 400-600
d. 4,000-6,000

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Sertoli
Androgen-binding protein Testosterone All of the above 20509
11. What is the surface modification seen on the cells of the epididymis?
a. Flagella and microvilli
b. Stereocilia and cilia
c. Cilia and microvilli
L

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* Structure of ovary
* Steps in oogenesis
* Formation of ovarian follicles and ovulation
* Structure of uterine tube
* Structure of uterus
* Structure of cervix, vagina and external genitalia
* Structure of mammary gland

epithelium Q Presence of cortex and medulla Q The medulla consists of loose connective tissue, smooth muscles, blood vessels, lymphatics, and nerves Q Cortex contains ovarian follicle at different stages of development: Primary, primordial, secondary, and mature Graafian follicles Q Presence of corpus luteum and atretic follicle in the cortex., Mammary gland.Resting phase = Q Presence of tubuloalveolar glands separated by connective and fatty tissues Q Contain more connective tissue and less glandular tissue Q Presence of few poorly developed alveoli Q Alveoliis lined by cuboidal epithelium and wider lumen Q Presence of narrow empty ducts.. Q Covered by single layer of cuboidal cells as germinal epithelium Q Presence of cortex and medulla Q The medulla consists of loose connective tissue, smooth muscles, blood vessels, lymphatics, and nerves Q Cortex contains ovarian follicle at different stages of development: Primary, primordial, secondary, and mature Graafian follicles Q Presence of corpus luteum and atretic follicle in the cortex., Mammary gland.Lactating phase = Q Presence of compound tubuloalveolar glands separated by connective and fatty tissues Q Contain more glandular and less connective tissue Q Presence of numerous well- developed alveoli Q Alveoli is lined by columnar epithelium and narrow lumen © Presence of wider ducts and filled with secretions.
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The female reproductive system includes (Fig. 21.1):
* A pair of ovaries
* A pair of uterine tubes
* Uterus
* Vagina
* External genitalia
* Mammary glands.
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* The ovaries are the female gonads, responsible for the formation of ova.
* Each ovary is an oval structure about 3 cm long in diameter and situated on each side of the uterus and attached to the posterior layer of the broad ligament below and behind the uterine tubes.
* Before puberty, its surface is smooth; thereafter due to repeated ovulations and cicatrization, the surface becomes irregular.
* They also produce hormones estrogen and progesterone that are responsible for the development of the female secondary sex characters, and produce marked cyclical changes in the uterine endometrium.
Fig. 21.1: Parts of female reproductive system (schematic representation).

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* The ovary is covered by a single layer of cubical cells known as germinal epithelium. This epithelium is continuous with the mesothelium lining the peritoneum, and represents a modification of the latter.
* The germinal epithelium rests on a connective tissue layer called the tunica albuginea which is thinner and less dense than that of the testis. Tunica albuginea is a dense, irregular collagenous connective tissue capsule and collagen fibers run parallel to ovarian surface.
* The substance of the gland consists of inner medulla and outer cortex (Fig. 21.2 and Plate 21.1):
Cortex: Cortex composed of connective tissue stroma made up of reticular fibers and numerous fusiform cells that resemble mesenchymal cells called stromal/ interstitial cells. Stroma also contains ovarian follicles at various stages of development.
" At birth: Cortex contains a numerous primordial follicles. Each primorc follicle consists of a centrally placed sex cell known as primary oocyte @
surrounded by a single layer of follicular cells derived from the some tissue.
"In childbearing period: During this period, the primordial follicles . divided into primary, secondary, and tertiary follicular stages. Duri reproductive life, the cortex contains the ovarian follicles and corpora lu at different stages of development, atretic follicles, and corpora albicantiz
"After menopause: The cortex becomes fibrotic and contains interstit cells and corpora albicantia.
Medulla: It consists of loose connective tissue, smooth muscles, blood vessels, lymphatics, and nerves. The hilum of the ovary is the site for entry of blood vessels and lymphatics and it is continuous with the medulla. The hilum also contains some remnants of the mesonephric ducts, and hilus cells that are similar to interstitial cells of the testis and secrete androgens.
Fig. 21.2: Histological structure of ovary showing follicles at various stages of development (schematic representation).
Just deep to the tunica albuginea, many primordial follicles each of which contains a developing ovum surrounded by flattened follicular cells are present
Large follicles have a follicular cavity surrounded by several layers of follicular cells

The cells surrounding the ovum constitute the cumulus oophorus
'The follicle is surrounded by a condensation of connective tissue which forms a capsule for it 'The capsule consists of an inner cellular part (the theca interna), and an outer fibrous part (the theca externa) collectively called as theca folliculi. The follicle is surrounded by a stroma made up of reticular fibers and fusiform cells.
Note: The term germinal epithelium is a misnomer. The epithelium does not produce germ cells. The cells of this epithelium bear microvilli, and contain numerous mitochondria. They become larger in pregnancy.

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The process of formation of ovum from the stem cells is called oogenesis (Flowchart 21.1).
* Oogonia are large round stem cells from which ova are derived and they are present in the cortex of the ovary. Oogonia develop in the yolk sac endoderm from primordial stem cells and undergo mitotic division and during 6th week of gestation migrate to the cortex of developing ovary.
* Oogonia undergo mitotic division till 5th month of gestation and about 5 to 7 million oogonia are formed. Only about 1 million of oogonia is surrounded by follicular cells and survive till the birth and remaining oogonia undergo atresia, degenerate, and die.
* All oogonia to be used throughout the life of a woman are produced before birth and do not multiply thereafter.
Flowchart 21.1: Stages of oogenesis.
* An oogonium enlarges to form a primary oocyte. The primary oocyte contains the diploid number of chromosomes, i.e. 46. It is spherical with acentric nucleus containing single nucleolus.
* Primary oocyte is arrested in the prophase stage of 1st meiosis. It completes the 1st meiotic division just before ovulation to form two daughter cells each of which has 23 chromosomes. The primary oocytes remain in prophase and do not complete their first meiotic division until they begin to mature and are ready to ovulate.
* With each menstrual cycle, about 5 to 30 primary oocytes begin to mature and complete the first meiotic division just before ovulation.
* The cytoplasm of the primary oocyte is not equally divided. Most of it goes to one daughter cell that is large and is called the secondary oocyte. The second daughter cell has hardly any cytoplasm, and forms the first polar body.
The secondary oocyte immediately enters the second meiotic division. Ovulation takes place while the oocyte is in metaphase. The secondary oocyte remains arrested in metaphase till fertilization occurs.

- * The second meiotic division is completed only if fertilization occurs. The secondary oocyte undergoes the second meiotic division and the daughter cells being again unequal in size. The larger daughter is the mature ovum and the smaller daughter cell which has hardly any cytoplasm is the second polar body. Thus, one primary oocyte ultimately gives rise to only one ovum.

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* Ovarian follicles consist of a developing ova surrounded by follicular cells. The development and maturation of an ovarian follicle passes through four stages (primordial, primary, secondary, and Graafian) and the process is called folliculogenesis (Figs. 21.2 and 21.3).
* Primordial follicle (Fig. 21.3A) is the most primitive follicles. It is composed of primary oocyte surrounded by a single layer of flattened cells called follicular cells/ granulosa cells. Primordial follicles are the smallest and simplest in structure located at the periphery of the cortex. Numerous primordial follicles are present in the ovary at birth. They undergo further development only at puberty.
* A primordial follicle develops into primary follicle. The flattened follicular cells become cuboidal. Oocyte surrounded by a single layer of cuboidal follicular cells is called as unilaminar primary follicle. The follicular cells proliferate and stratify to form several layers of cells and now the primary follicle with many layers of follicular cells is called as multilaminar primary follicle (Fig. 21.3B).
* A homogeneous membrane, the zona pellucida, appears between the follicular cells and the oocyte. Cytoplasmic extensions from follicular cells invade the zona pellucida, come in contact with the oocyte plasmalemma and form gap junctions through which they communicate with oocyte.
* Stromal cells organize around multilaminar primary follicle forming an inner cellular layer called theca interna and outer fibrous layer called theca externa. Granulosa cells are separated from the theca interna by thick basal lamina. Theca interna cells express receptors for luteinizing hormone (LH) to produce androstenedione which gets converted to

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* Secondary follicle: Multilaminar primary follicle develops and increases in size and the follicular cells proliferate to form several layers of cells that constitute the membrana granulosa. The oocyte enlarges and reaches its maximum size (125 pm) (Figs. 21.3A and B).
* Intercellular spaces develop within the masses of granulosa cells and filled with the fluid called liquor folliculi and the follicle is called as secondary follicle. Under the influence of FSH, number of layers of granulosa cells increase as well as the fluid-filled cavity. As more fluid is produced, individual cavity coalesces to form single cavity called antrum.
Figs. 21.3A and B: (A) Primordial follicle; (B) Primary follicle (schematic representation).
- * Graafian follicle: The granulosa cells are rearranged and now the primary oocyte lies eccentrically and surrounded by small group of granulosa cells
that projects into the cavity called as cumulus oophorus. The granulosa cells that attach the oocyte to the wall of the follicle constitute the discus proligerus (Fig. 21.4). The innermost layer of cumulus oophorus that lies directly adjacent to the zona pellucida is called corona radiata.

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* By 14th day of menstrual cycle, blood estrogen level increases causes surge of LH. The ovarian follicle is at first very small compared to the thickness of the ovarian cortex. As the follicle enlarges, it becomes so big that it not only reaches the surface of the ovary, but forms a bulging in this situation. As a result, the stroma and the theca on this side of the follicle are stretched and become very thin (Fig. 21.5).
* An avascular area (stigma) appears over the most convex point of the follicle. At the same time, the cells of the cumulus oophorus become loosened by accumulation of fluid between them. The follicle ultimately ruptures and the ovum is shed from the ovary.
* The shedding of the ovum is called ovulation. The "ovum" that is shed from the ovary is not fully mature. It is really a secondary oocyte surrounded by zona pellucida and corona radiata (Fig. 21.6).
Fig. 21.4: Mature ovarian follicle (schematic representation).
Fig. 21.5: Relationship of a growing ovarian follicle to the ovary (schematic representation).

* The ovum is carried into the fallopian tube partly by the follicular fluid discharged from the follicle and partly by the activity of ciliated cells lining the tube.
* The ovum slowly travels through the tube toward the uterus, taking three to four days to do so.
"Tf fertilization takes place, then the ovum begins to develop into an
embryo. It travels to the uterus and gets implanted in its wall. If the ovum is not fertilized, it dies in 12 to 24 hours and it passes through the uterus into the vagina and is discharged.

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* After ovulation, the follicle ruptures and its wall collapses and becomes folded. Sudden reduction in pressure caused by rupture of the follicle results in bleeding into the follicle. The follicle filled with blood is called the corpus hemorrhagicum. At this stage, the follicular cells are small and rounded.
* The cells now enlarge rapidly and as they increase in size, their walls press against those of neighboring cells so that the cells acquire a polyhedral shape (Figs. 21.7 and 21.8). Their cytoplasm becomes filled with a yellow pigment called lutein. The presence of this yellow pigment gives the structure a yellow color, and that is why it is called the corpus luteum (= yellow body) which functions as endocrine gland.
Fig. 21.6: Structure of ovum at the time of ovulation (schematic representation).
* The granulosa cells are modified into large pale staining cells called granulosa-lutein cells, which constitutes about 80% of cell population and present in the center of the follicle. They produce progesterone and convert androgens produced by theca cells to estrogen.
* The theca interna cells modified into dark staining cells called theca lutein cells, which constitutes about 20% of cell population and present near the periphery. They produce progesterone, some estrogen, and androgens.
Fig. 21.7: Corpus luteum. Note the large hexagonal cells filled with yellow granules (schematic representation).
Fig. 21.8: Corpus luteum (high magnification) (schematic representation).
* The subsequent fate of the corpus luteum depends on whether the ovum is fertilized or not. If the ovum is not fertilized, the corpus luteum persists for about 14 days. During this period, it secretes progesterone. It remains relatively small and is called the corpus luteum of menstruation.

* If the ovum is fertilized and pregnancy results, the corpus luteum persists for three to four months. It is larger than the corpus luteum of menstruation, and is called the corpus luteum of pregnancy. The progesterone secreted by it is essential for the maintenance of pregnancy in the first few months. After the fourth month, the corpus luteum is no longer needed, as the placenta begins to secrete progesterone.
* The corpus luteum is invaded by fibroblasts, becomes fibrotic and ceases to function and the mass of fibrous tissue is called as the corpus albicans (= white body) (Fig. 21.9). The remnants of the corpus albicans persist as a scar on the surface of the ovary.
Fig. 21.9: Comparison of fate of ovarian follicles that shed an ovum and of those that do (schematic representation).
not

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* The series of changes that begin with the formation of an ovarian follicle and end with the degeneration of the corpus luteum constitute what is called an ovarian cycle.
* Tn each ovarian cycle, one follicle reaches maturity, sheds an ovum, and becomes a corpus luteum. At the same time, several other follicles also begin to develop but do not reach maturity. These follicles do not persist into the next ovarian cycle but undergo degeneration and eventually phagocytosed by macrophages. Follicles that undergo degeneration are known as atretic follicles.
* The ovum and granulosa cells of each follicle disappear. The cells of the theca interna, however, proliferate to form the interstitial glands, also called the corpora atretica. These glands are believed to secrete estrogens. After a period of activity, each gland becomes a mass of scar tissue indistinguishable from the corpus albicans formed from the corpus luteum.
* The cortex of an ovary of woman in the reproductive period can show ovarian follicles at various stages of maturation (corpora lutea, corpora albicantia, and corpora atretica).
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* Uterine tubes are paired muscular tubes which convey the ova from ovary to uterine cavity and they are called fallopian tubes/oviducts.
* Each uterine tube has a medial or uterine end attached to the uterus, and a lateral end that opens into the peritoneal cavity near the ovary.
* From medial to lateral side, the tube has:
" A uterine/intramural part that passes through the thick uterine wall
"A relatively narrow, thick-walled part called the isthmus
" A thin-walled dilated part called the ampulla
"Funnel-shaped infundibulum. It is prolonged into a number of finger-l processes or fimbriae.

The wall of the uterine tube consists of mucous, muscular, and serous layers

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* Mucous membrane shows three to six longitudinal folds, which give rise to a number of secondary and tertiary folds that almost fill the lumen of the tube (Plate 21.2). These folds are most conspicuous in the ampulla. It is lined by columnar epithelium that rests on a basement membrane. The epithelium consists of ciliated, secretory, and intercalary cells.
" Ciliated cells help to move the ova.
"Secretory cells are also called as peg cells, they contain secretory granu and are not ciliated and their surface shows microvilli. Secretion from | cells provides nutrition to the fertilized ovum and the spermatozoa.
"Intercalary cells are sandwiched between the basement membrane and | epithelium. They act as precursors of the ciliated and secretory cells.
* Muscular layer consists of outer longitudinal and inner circular layers of smooth muscles. The intramural part may have innermost longitudinal layer. The circular muscle is abundant at the isthmus where the lumen is narrow and suggesting of existence of anatomical sphincter.
* Serosa layer is derived from the peritoneum and covers the entire tube except the lower border and the intramural part.

* The uterine tube conveys ova, shed by the ovary, to the uterus. Ova enter the tube at its fimbriated end. Spermatozoa enter the uterine tube through the vagina and uterus.
* Fertilization normally takes place in the ampulla. When fertilization occurs, the fertilized ovum travels toward the uterus through the tube.
* Secretions present in the tubes provide nutrition, oxygen, and other requirements for ova and spermatozoa passing through the tube.

* Uterus is an inverted pear-/pyriform-shaped muscular organ located in the pelvic cavity between urinary bladder below urinary bladder in front and sigmoid colon, rectum behind.
* On each side, it receives the opening of the uterine tube at the superolateral angle/cornu and communicates below with vagina through external os.
* The uterus consists of three parts from above downward: Fundus, body, and cervix. The fundus is the upper dome-shaped part which is above the attachment of the fallopian tube. The body extends from the fundus to the isthmus; the isthmus is a narrow constricted part separating the body of the uterus from the cervix. Below the isthmus, the uterus becomes cylindrical in shape; this part is known as cervix.
* The uterus has a very thick wall consists of endometrium, myometrium, and perimetrium from within outward.

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* The mucous membrane of the uterus is called the endometrium.
*The endometrium consists of a lining epithelium and lamina propria/stroma. Numerous uterine glands are present in the stroma.
"Lining epithelium is columnar. Before menarche, the cells are ciliat during reproductive life, it is lined by simple columnar epithelium becai cilia cannot grow due to repeated destruction of superficial part endometrium.
"Lamina propria/stroma contains embryonic stromal cells, numerc blood vessels, lymphatics, nerves, and tubular uterine glands.
Plate 21.2: Fallopian tube

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"The uterine tube is characterized by the presence of numerous branching mucosal folds that almost fill the lumen of the tube
"The mucosa is lined by ciliated columnar epithelium
"The uterine tube has a muscular wall with an inner circular and outer longitudinal muscle layer.
"The glands are lined by simple columnar epithelium and extend up to | inner surface of myometrium.
* Functionally, endometrium consists of outer basal layer and inner functional layer.
"Functional layer undergoes changes during the menstrual cycle and it c off in each month during menstruation. Basal layer remains practica unaltered and helps in regeneration of the epithelial lining af menstruation.
" The basal layer is supplied by straight basal arteries from myometrium a functional layer is supplied by spiral arteries which allow diminished blc flow and undergo vasoconstriction before menstruation.

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* The endometrium undergoes marked cyclical changes that constitute the menstrual cycle. The cycle is calculated from the beginning of one menstrual bleeding to the beginning of the next bleeding and an average of 28 days duration. Ovulation takes place at the middle of the cycle. Preovulatory cycle is regulated by estrogen of ovarian follicle and postovulatory cycle is regulated by progesterone of corpus luteum.
* The endometrial changes are divided into menstrual, postmenstrual/regenerative, proliferative, and secretory (Table 21.1).
= The menstrual phase is manifested by uterine bleeding which persists fo to 4 days. There is exfoliation of functional layer of endometrium due sudden withdrawal of progesterone after the regression of corpus luteum.
"In the postmenstrual phase, the endometrium is thin. It progressiv increases in thickness being thickest at the end of the secretory phase.
"The regenerative phase takes 1 to 3 days to complete. The epithelial ce from the uterine glands proliferate by mitosis and cover the denuc surface of mucous membrane. Estrogen helps in regeneration.
"During proliferative/follicular/estrogenic phase, the functional la gradually increases in thickness from 1 mm to 3 mm before the ovulati: The proliferation is stimulated by estrogen. There is increase in | population of stroma cells, elongation of uterine glands, and spiral arter of the endometrium. The uterine glands are straight in the proliferat phase. Proliferative phase may extend 1-2 days after ovulation till 1 formation of corpus luteum (Plate 21.3).

"In secretory/luteal phase, the functional layer is increased in thickness 2 3 times more than the proliferative phase. Enlargements of the stroma ce due to appearance of cytoplasmic glycogen, accumulation of tissue fl around the stroma cells, and by distension of uterine glands with secreti showing raggedy appearance of the epithelial cells lining the glandu lumen. As the endometrium increases in thickness, the glands elongé increase in diameter, and become twisted on themselves and they havi saw-toothed appearance in sections. Glycogen-filled stroma cells . known as decidual cells which provide nutrition to the embedded ovu Secretory phase is under the control of estrogen and progestero Implantation of fertilized ovum takes place in secretory phase (Plate 21.4
"Premenstrual phase is 1 to 2 days prior to the onset of next menstn cycle. In this phase, there is a reduction in thickness of the functional la due to regression of corpus luteum. The spiral arteries shorten and becoi more coiled. Blood appear in the uterine lumen with shedding of flakes
Table 21.1: Differences between proliferative and secretory phase., 1 = Table 21.1: Differences between proliferative and secretory phase.. Table 21.1: Differences between proliferative and secretory phase., 2 = Table 21.1: Differences between proliferative and secretory phase.. , 1 = Proliferative phase. , 2 = Secretory phase. Duration, 1 = 6th to 13th day in a 28-day cycle 15th to 28th day in a 28-day cycle. Duration, 2 = 6th to 13th day in a 28-day cycle 15th to 28th day in a 28-day cycle. Thickness of endometrium It is about 2-3 mm thick, 1 = Thickness of endometrium It is about 2-3 mm thick. Thickness of endometrium It is about 2-3 mm thick, 2 = It is about 5 mm thick

stroma, Simple columnar = Highly cellular and nonedematous. stroma, Simple columnar = Highly vascular and edematous. Uterine glands, Simple columnar = Uterine glands are elongated, straight, and tubular without secretion. Uterine glands, Simple columnar = Uterine glands are dilated, tortuous, and giving a saw-toothed appearance with watery secretion. spiral artery, Simple columnar = Less coiled, do not reach the superficial part of endometrium. spiral artery, Simple columnar = More coiled, reach superficial part of endometrium. Hormones, Simple columnar = Estrogen is secreted. Hormones, Simple columnar = Progesterone is secreted
endometrium (Fig. 21.10).
* At the time of menstruation, the greater parts of the uterine glands are lost along with the entire lining epithelium leaving behind only their most basal parts.
* The lining epithelium is reformed after the cessation of menstruation by proliferation of epithelial cells in the basal parts of the glands.

- * The muscle layer of the uterus is called the myometrium and it consists of bundles of smooth muscle. Along with, there is connective tissue, blood vessels, nerves, and lymphatics.
Plate 21.3: Uterus (Proliferative phase)
Uterus (Proliferative phase): (A) As seen in drawing; (B) Photomicrograph.

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'The wall of the uterus consists of a mucous membrane (called the endometrium) and a very thick layer of muscle (the myometrium). The thickness of the muscle layer helps to identify the uterus easily
'The endometrium has a lining of columnar epithelium that rests on a stroma of connective tissue
Numerous tubular uterine glands dip into the stroma
'The appearance of the endometrium varies considerably depending upon the phase of the menstrual cycle. The endometrium is thin and progressively increases in thickness. The uterine glands are straight and tubular in this phase.
Plate 21.4: Uterus (Secretory phase)

*The thickness of the endometrium is much increased
*The uterine glands elongate, become dilated, and tortuous as a result of which they have saw-toothed margins in sections

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Fig. 21.10: Uterine glands at various stages of menstrual cycle. The thickness of endometrium is also indicated (schematic representation).
* The muscle fibers run in various directions and distinct layers are difficult to define. The myometrium consists of three ill-defined layers as outer longitudinal, middle circular, and inner reticular. In the middle layer, there is a mixture of bundles running in various directions.
"The longitudinal muscles are continuous with the corresponding muscles the uterine tubes. Longitudinal muscle helps in the anchorage of uterus ¢ expels the uterine contents to the exterior.
™ Middle layer contains vascular bed, the stratum vasculare. Circular mus of this layer forms a sphincter to retain the uterine contents (product conception).
"Endometrial vessels pass through the interstices of inner reticu arrangement of smooth muscle layer. On contraction, the muscle stops ° bleeding in menstruation or during placental separation. Inner layer acts living ligature of the uterus.
"Growth of myometrium is stimulated by estrogen. The muscle ce increase in number (hyperplasia) and in size (hypertrophy) duri
pregnancy. Contractions of the myometrium are responsible for expulsi of the fetus at the time of childbirth.

* The perimetrium/serous coat is derived from the peritoneum and covers the entire organ except two lateral borders, anterior surface of the supravaginal part of the cervix and the vaginal part of the cervix.
* Posterior surface of the uterus is adherent to the underlying muscle but in the anterior surface, it is adherent in the upper part but separated from the muscle by loose areolar tissue in the lower part.

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Hormones influencing ovulation and menstruation
The changes taking place in the uterine endometrium during the menstrual cycle occur under the influence of:
*estrogens produced by the thecal gland (theca interna) and by the interstitial gland cells, and possibly by granulosa cells.
* Progesterone produced by the corpus luteum.
The development of the ovarian follicle and of the corpus luteum is in turn dependent on hormones produced by the anterior lobe of the hypophysis cerebri. These are:
*The follicle-stimulating hormone (FSH), which stimulates the formation of follicles and the secretion of estrogens by them, and the luteinizing hormone (LH), which helps to convert the ovarian follicle into the corpus luteum, and stimulates the secretion of progesterone.
*Secretion of FSH and LH is controlled by a gonadotropinreleasing hormone (GnRH) produced by the hypothalamus. Production of LH is also stimulated by feedback of estrogens secreted by follicular cells of the ovary. A sudden increase (surge) in the level of LH takes place near the middle of the menstrual cycle, and stimulates ovulation that takes place about 36 hours after the surge.
Apart from hormones, nervous and emotional influences may affect the ovarian and menstrual cycles. An emotional disturbance may delay or even prevent menstruation.

The cervix is the lower, narrow cylindrical part of the uterus. The cervical canal is narrow and communicates with the uterine cavity at its upper end and with vagina at its lower end. The upper and lower openings are referred to as internal and external os, respectively. The lower portion of cervix, which projects into vagina, is called as portio vaginalis.

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* The structure of the cervix of the uterus is somewhat different from that of the body. Here, the mucous membrane (or endocervix) has a number of obliquely placed palmate folds. It contains deep branching glands that secrete mucous.
* The mucosa also shows small cysts that probably represent glandular elements that are distended with secretion. These cysts are called the Ovula Nabothi.
* The mucous membrane of the upper two-thirds of the cervical canal is lined by ciliated columnar epithelium, but over its lower one-third the epithelium is nonciliated columnar. Near the external os, the canal is lined by stratified squamous epithelium.
* The part of the cervix that projects into vagina has an external surface that is covered by stratified squamous epithelium.
* The cervix has few smooth muscle fibers and mainly (85%) consists of dense connective tissue.
* The mucosa does not undergo remarkable changes during the menstrual cycle and does not desquamate during menstruation.
* During pregnancy, the cervical mucous glands proliferate and secrete a more viscous and abundant mucus.
* The lumen of the cervix is normally a narrow canal. It has tremendous capacity for dilation and, at the time of childbirth, it becomes large enough for the fetal head to pass through.

* The vagina is the female organ of copulation and forms the lower part of the birth canal.
* The vagina is a fibromuscular elastic tube that extends from lower part of the cervix to the external genitalia. It is about 8 cm long. It is capable of considerable elongation and distension, this being helped by the rich network of elastic fibers in its wall.

From within outward, the wall of the vagina consists of mucous, muscular, and serous layers (Plate 21.5).

* The mucous membrane shows numerous longitudinal folds, and is firmly fixed to the underlying muscle layer. It consists of lamina propria and epithelium.
* It is lined by stratified squamous nonkeratinized epithelium and devoid of mucous glands. The vaginal surface being kept moist by secretions of glands in the cervix of the uterus. The vaginal fluid is derived from the transudate present in the lamina propria and secretions from cervix.
* The epithelial cells are rich in glycogen. Naturally occurring vaginal bacterial flora metabolize the glycogen forming lactic acid and protects the vagina from invasion of pathogenic bacteria. Bacterial infection of the vagina is common during childhood and after menopause as the acidity is low.
* The lamina propria is composed of loose connective tissue and contains plexus of blood vessels, lymphatics, and nerves. The tissue is rich in elastic fibers.

* The muscle coat is made up of an outer longitudinal and a much thinner inner circular layers of smooth muscles layers.
* Many elastic fibers are present among the muscle fibers.
* The lower end of the vagina is surrounded by striated muscle fibers (bulbospongiosus muscle) that form the sphincter at its external opening.

The muscle wall is surrounded by an adventitia made up of fibrous tissue containing many elastic fibers. It is derived from peritoneum and covers only the posterior fornix of vagina.
Mf

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The external genitalia consist of labia majora, labia minora, vestibule, and clitoris.
* Labia majora are two folds of skin with adipose tissue and a thin layer of smooth muscle. It is covered by coarse hair on their external surface and is devoid of hair on the inner surface. Numerous sebaceous and sweat glands are present. The homolog of this is scrotum in the male.
* Labia minora are present medial to and slightly deep to the labia majora. They are two smaller folds of skin without hair follicles and adipose tissue. The mucous membrane is made up of a core of connective tissue that is covered by stratified squamous epithelium. Modified sebaceous glands are present. It is the homolog of urethral surface of penis in male.
* The cleft between the two labia minora is the vestibule. It receives the secretions of the glands of Bartholin, which are paired mucous glands. Orifices of vagina and urethra are located here.
* Clitoris is regarded as a miniature penis, with the important difference that the urethra does not pass through it. Two erectile bodies called corpora cavernosa and a glans are present. The surface of the clitoris is covered by mucous membrane and not skin that is lined by stratified squamous epithelium. The mucosa is richly supplied with nerves.
Vagina:
(A)
As seen
in drawing;
(B)
Photomicrograph.

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'The vagina is a fibromuscular structure consisting of an inner mucosa, a middle muscular layer, and an outer adventitia
"The mucosa consists of stratified squamous nonkeratinized epithelium and loose fibroelastic connective tissue lamina propria with many blood vessels and no glands
'The mucosa of vagina is rich in glycogen and hence, the cells are pale stained which distinguishes it from esophagus
"Muscular layer consists of smooth muscle fibers.
MAMMARY GLAND
Bf

* The mammary glands are present in both sexes but they remain rudimentary in the male and are well-developed in females after puberty.
* Mammary glands secrete milk which contains protein, lipids, lactose, lymphocytes and monocytes, antibodies, minerals, and fat-soluble vitamins to provide nourishment for the newborn.
* Mammary gland is a modified sweat gland. Each gland is a soft rounded elevation present over the pectoral region. The skin over the center of the elevation shows a darkly pigmented circular area called the areola and overlying the central part of the areola there is a projection called the nipple.
* Each mammary gland is covered by skin and deep to which it is made up of glandular, fibrous, and interlobar fatty tissue. The glandular masses are separated by connective tissue and of adipose tissue (Plate 21.6). The fascia covering the gland is connected to overlying skin by fibrous processes called the suspensory ligaments of Cooper.

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The gland is made up of stroma and parenchyma. Parenchyma is mainly constituted by lactiferous glands with their ducts.
* The glandular tissue are of compound tubule alveolar type and arranged as 15 to 20 pyramidal lobes, each being drained by a separate lactiferous duct. The lobes are arranged in radiating manner and converge toward the areola. Near the areola, each duct dilates to form the lactiferous sinus and act as a reservoir of milk and finally the ducts open on to the nipple. Each duct drains a smaller segmental ducts and lobules. Each segmental duct divides into number of terminal ducts and from the latter numerous secretory glands pouch out to form alveoli.
* The area of parenchyma drained by one terminal duct is known as the lobule. The lobules are separated by fibro-fatty dense connective tissue and the alveoli are embedded in this intralobular connective tissue.
* The cells lining the alveoli vary in appearance in accordance with functional activity. In the "resting" phase, they are cuboidal and in actively producing alveoli, the cells become columnar. Myoepithelial cells are present between the cells and the basement membrane. Once the secretions
are poured into lumen, reduction in the size lining cells to cuboid is observed but never to initial size. The cells are filled with secretory vacuoles.

The structure of the glandular elements of the mammary gland varies considerably at different periods of life as follows:
* Before the onset of puberty, the glandular tissue consists entirely of ducts.
* Between puberty and the first pregnancy, the duct system proliferates. At the end of each duct, solid masses of polyhedral cells are formed, but proper alveoli are few or absent. The bulk of the breast consists of connective tissue and fat that widely separate the glandular elements.
* Resting/nonsecreting mammary glands have same structure as the lactating gland except that they are smaller and they contain more connective tissue and less glandular tissue. The alveoli are lined by cuboidal epithelium and have a large lumen (Plate 21.6).

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"Mammary glands are activated by elevated level of estrogen ¢ progesterone during pregnancy to become lactating glands.
" During pregnancy, there is a proliferation and epithelial growth of termi ducts and lobules with increase in the number of alveoli per lobule. T terminal portions of the ducts branch and grow and the alveoli develop ¢ mature. Each lobe is filled with compound tubuloalveolar gland.
"The development of breast tissue during pregnancy takes place under ° influence of hormones produced by the hypophysis cerebri. Cells lini glandular tissue bear receptors for these hormones.
"As the pregnancy progress, there is a hypertrophy of the glandu parenchyma and engorgement with colostrum, a protein-rich fluid. Ti milk secretion starts after a few days of delivery with the suckling of 1 newborn.
"The alveoli are lined by columnar epithelium and surrounded myoepithelial cells. The secretory cells possess abundant rou endoplasmic reticulum, mitochondria, several Golgi apparatus, lij
fr

droplets, and numerous vesicles. Different alveoli display varying degri of preparation for synthesis of milk substance.
- " Alveolar cells secrete lipid and proteins. Lipids are stored as droplets in1 cytoplasm and released from the cells by apocrine mode of exocyto: Proteins are secreted from the cells by merocrine mode of exocytosis.
“=:

'Mammary gland consists of lobules of glandular tissue separated by considerable quantity of connective tissue and fat 'Nonlactating mammary glands contain more connective tissue and less glandular tissue 'The glandular elements or alveoli are distinctly tubular. They are lined by cuboidal epithelium and have a large lumen so that they look like ducts. Some of them may be in form of solid cords of cells
'Extensive branching of duct system seen.
" After lactation stops, the alveoli shrink, remaining milk is absorbed, and | glandular tissue returns to the resting condition. It undergoes atrophy af menopause.

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- * Each lobe drains into a lactiferous duct that opens at the summit of the nipple (Fig. 21.11). Beneath the nipple, the lactiferous duct dilates into lactiferous sinus, which functions as a reservoir of milk.
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Plate 21.7: Mammary gland (Lactating)
“=:
Mammary gland (Lactating): (A) As seen in drawing; (B) Photomicrograph.

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'In lactating mammary gland, the glandular elements proliferate so that they become relatively more prominent than the connective tissue
'The interlobular connective tissue septum is very thin
'The lobules are formed by compactly arranged alveoli
'The alveoli are lined by simple cuboidal secretory epithelium and associated myoepithelial cells. Their lumen contains eosinophilic secretory material which appears vacuolated due to the of fat presence droplets.
- * The smaller ducts are lined by columnar epithelium but the larger ducts are lined by two or more layers of columnar epithelium and near their openings on the nipple, the lining becomes stratified squamous keratinized epithelium. Between the epithelium and the basement membrane of the
ducts, the myoepithelial cells are present.
- * Difference between structure of resting and lactating mammary gland is provided in Table 21.2.
Fig. 21.11: Human female breast (schematic representation).

Stroma, Table 21.2: Differences between resting and lactating mammary gland..Resting mammary gland = Contains more connective tissue and less glandular tissue. Stroma, Table 21.2: Differences between resting and lactating mammary gland..Lactating mammary gland = Contains more glandular tissue and less connective tissue. Secretory units, Table 21.2: Differences between resting and lactating mammary gland..Resting mammary gland = Secretory units are few and poorly developed. Secretory units, Table 21.2: Differences between resting and lactating mammary gland..Lactating mammary gland = Secretory units are numerous and are more developed. Alveoli, Table 21.2: Differences between resting and lactating mammary gland..Resting mammary gland = Lined by cuboidal epithelium and wider lumen. Alveoli, Table 21.2: Differences between resting and lactating mammary gland..Lactating mammary gland = Lined by columnar epithelium and narrow lumen. Excretory ducts, Table 21.2: Differences between resting and lactating mammary gland..Resting mammary gland = Narrow and empty. Excretory ducts, Table 21.2: Differences between resting and lactating mammary gland..Lactating mammary gland = Wider and filled with milk

* The nipple is the conical projection below the center of the breast and is pierced by 15—20 lactiferous ducts.
* The nipple is covered by keratinized stratified squamous epithelium. It consists of dense connective tissue and smooth muscles arranged circularly and longitudinally. A circular muscle erects the nipple for suckling and longitudinal muscle retracts the nipple.
* Nipple has rich nerve supply and provided with sensory receptors for

* The pigmented skin around the nipple is called areola. The pigmentation is irreversibly darkened after pregnancy.
* The skin of the areola lacks hair follicles. Outer margin of the areola contains a number of modified sebaceous glands, which are enlarged during pregnancy and lactation and are known as tubercles of Montgomery. Oily secretion of these glands provides lubrication during lactation.
* Areola also contains sweat and accessory mammary glands. Skin of areola is devoid of hair and subcutaneous fat.
[

*In the resting mammary gland, glandular epithelium is surrounded by an avascular zone containing fibroblasts. It has been claimed that this zone constitutes an epitheliostromal junction that controls passage of materials to glandular cells.
*In the male, the mammary gland is rudimentary and consists of ducts that may be represented by solid cords of cells. The ducts do not extend beyond the areola.
* During menstrual cycle, at the time of ovulation, there is increase in the estrogen level which leads to proliferation of the cells of the ducts.
*Premenstrual phase breast engorgement is due to greater hydration of connective tissue.

* Ovarian tumors: 70% of ovarian tumors arise from the epithelial surface.
* Polycystic ovarian disease: Ovaries are filled with numerous fluid-filled follicular cysts. This is associated with anovulation, irregular, or scanty menstruation. Also called as Stein—Leventhal syndrome.
* Ovulation pain: During ovulation, about 45% of women experience midcycle pain ("mittelschmerz") which is sharp, lower abdominal that lasts from a few minutes to as long as 24 hours.
* Pregnancy test: Human chorionic gonadotropin (hCG) produced by corpus luteum of pregnancy can be detected in urine as early as 10 to 14 days of pregnancy. It forms the basis of urine pregnancy test kits.

Ectopic tubal pregnancy: The implantation of a fertilized ovum in the uterine tube is called as ectopic pregnancy. The most frequent site of tubal pregnancy is the ampullary portion and the least common is interstitial pregnancy.

* Dysfunctional uterine bleeding: Dysfunctional uterine bleeding (DUB) is defined as excessive bleeding occurring during or between menstrual periods without a causative uterine lesion such as tumor, polyp, infection, hyperplasia, trauma, blood dyscrasia, or pregnancy.
* Endometriosis: Endometriosis refers to the presence of endometrial glands and stroma in abnormal locations outside the uterus. Most common site being ovary followed by pouch of Douglas.
* Adenomyosis: Adenomyosis is defined as abnormal distribution of histologically benign endometrial tissue within the myometrium along with myometrial hypertrophy, due to either a metaplasia or estrogenic stimulation due to endocrine dysfunction of the ovary.

Vaginitis: The most common causes of vaginitis are Candida (moniliasis) and Trichomonas (trichomoniasis). These infections are particularly common in pregnant and diabetic women and may involve both vulva and vagina. However, the adult vaginal mucosa is relatively resistant to gonococcal infection because of its structure.

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- * Fibroadenoma: Fibroadenoma or adenofibroma is a benign tumor of fibrous and epithelial elements. It is the most common benign tumor of the female breast. Though it can occur at any age during reproductive life, most patients are between 15 and 30 years of age. Clinically, fibroadenoma
generally appears as a solitary, discrete, and freely mobile nodule within the breast.
- * Carcinoma of the breast: Cancer of the breast is among the most common of human cancers throughout the world. The incidence of breast cancer is highest in the perimenopausal age group and is uncommon before the age of 25 years. Clinically, the breast cancer usually presents as a solitary, painless, and palpable lump which is detected quite often by selfexamination.
1. What are developing gametes called?
a. Oogenesis
b. Ovary
c. Oocyte
d. Ova
2. Which stage of the follicle is arrested in prophase?
a. Primordial follicle
b. Primary follicle
c. Secondary follicle
d. Mature follicle
3. What is the cavity within a secondary follicle?
a. Graafian follicle
b. Theca folliculi
c. Zona pellucida
d. Antrum
4. Which of these correctly describe the sequence of the follicular phase?
a. Graafian follicle stage, secondary (antral follicle) stage, growing follicle stage, an follicle stage
b. Graafian follicle stage, growing follicle stage, primary follicle stage, and secondé follicle) stage
c. Growing follicle stage, primary follicle stage, secondary (antral follicle) stage, anc follicle stage
d. Primary follicle stage, growing follicle stage, secondary (antral follicle) stage, and follicle stage
5. Which of these secrete androgens in corpus luteum?
a. Connective septa
b. Granulosa-lutein cells

c., 1 = Theca lutein cells. 6., 1 = d. Corpus albicans Which of these secrete progesterone in corpus luteum?. , 1 = c. Corpus albicans What is the name given to the type of cells surrounding oocyte which are a. Granulosa b. Leydig c. Sertoli d.. a., 1 = Spermatogonia Oogonia. b., 1 = Granulosa-lutein cells Theca lutein cells. d., 1 = . 7., 1 = cuboidal?. , 1 = Theca. 8., 1 = What is the layer which is thick glycoprotein and acid proteoglycan between oocyte granulosa cells?. , 1 = Antrum Mature follicle. a. b., 1 = . , 1 = Theca. c. d., 1 = Zona pellucida. 9., 1 = of the following cells have a haploid chromosome number?. Which, 1 = . b. c. d., 1 = Primary oocyte Secondary. , 1 = spermatocyte. 10., 1 = duct-the. , 1 = True. , 1 = False. 11., 1 = bacterial growth?. , 1 = What types of glands are found in the areola that alter pH and discourages Sweat. , 1 = Montgomery Eccrine. a. b. c. d., 1 = Ebner's. 12., 1 = . , 1 = epithelium.. , 1 = Uterine tube is lined by --------------. , 1 = Simple columnar. a. b., 1 = Pseudostratified columnar. , 1 = Ciliated columnar Stratified columnar. c., 1 = . d., 1 = . , 1 = ducts drain into duct.. , 1 = interlobular. , 1 = ducts. , 1 = which. , 1 = join to. , 1 = form a. , 1 = Intralobular single excretory lactiferous. a. b., 1 = 

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* Chemical structure of hormones
* Distribution of endocrine cells
* Hypophysis cerebri—parts, cells, secretion, and function
* Blood supply of pituitary gland
* Thyroid gland—cells, synthesis of hormone, and function
*Parathyroid gland—cells, secretion, and function
*Suprarenal gland (cortex and medulla)—layers, cells, secretion, and function
* Pineal gland
* Paraganglia, aortic bodies, and carotid body

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Pars anterior, pars intermedia, and pars posterior, Thyroid gland = Colloid-filled follicles are seen. Pars anterior, pars intermedia, and pars posterior, Adrenal gland = Outer cortex and inner medulla. Presence of chromophils and chromophobes in the anterior lobe, Thyroid gland = Follicles are lined by cuboidal/ columnar epithelium. Presence of chromophils and chromophobes in the anterior lobe, Adrenal gland = Cortex is divided into zona glomerulosa, zona fasciculata, and zona reticularis. Presence of unmyelinated, Thyroid gland = Parafollicular cells between. Presence of unmyelinated, Adrenal gland = Chromaffin cells are seen
nerve fibers with pituicytes and
follicular cells and basement
in medulla.
Herring bodies in the posterior
membrane/ in between follicles
lobe
are seen.
¥ollicles in pars intermedia.

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* Endocrine glands are ductless glands whose secretions are carried to specific destinations through vascular system.
* The secretions of endocrine cells are called hormones (Gr—hormaein = to excite). They interact with specific hormone receptors to alter biologic activity of the target cells.
* Along with the autonomic nervous system, the endocrine organs coordinate and control the metabolic activities and the internal environment of the body.
* Feedback mechanisms (positive or negative) regulate hormonal function.
* Endocrine tissues are highly vascular. The secretory pole of an endocrine cell is toward the wall of a capillary (or sinusoid).
* Endocrine gland includes pituitary, suprarenal, thyroid, and parathyroid glands, etc.
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* Hormones are chemical messengers that are produced by endocrine glands and delivered by bloodstream to target cells or organs.
* A hormone acts on cells that bear specific receptors for it. Some hormones act only on one organ or on one type of cell, while other hormones may have widespread effects.
* On the basis of their chemical structure, hormones belong to four main types:
1. Amino acid derivatives, for example, adrenalin, noradrenalin, and thy
2. Small peptides, for example, encephalin, vasopressin, and thyroid-1 hormone.
3. Proteins, for example, insulin, parathormone, and_ thyroid-sti hormone.
4. Steroids, for examples, progesterone, estrogens, testosterone, and cor

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Bi
Endocrine cells are distributed in three different ways:
1. Some organs are entirely endocrine in function. They are referred | endocrine glands (or ductless glands). Example are the hypop. cerebri/pituitary, the pineal gland, the thyroid gland, the parathyroid gl. and the suprarenal/adrenal glands.
2. Groups of endocrine cells may be present in organs that have | functions. They include the islets of the pancreas, the interstitial cells o testes, and the follicles and corpora lutea of the ovaries. Hormones are produced by some cells in the kidneys, the thymus, and the placenta. $ authors describe the liver as being partly an endocrine gland.
3. Isolated endocrine cells may be distributed in the epithelial lining c organ like gastrointestinal tract (GIT) and respiratory tract. Cells in 1 locations in the body produce amines that have endocrine functions. Mai these amines also act as neurotransmitters or as neuromodulators. T widely distributed cells are grouped together as the neuroendocrine sy or the amine precursor uptake and decarboxylation (APUD) cell system.
Bi

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* The hypophysis cerebri is also called the pituitary gland and is approximately the size of a pea.
* Tt is suspended from the floor of the third ventricle (of the brain) by a narrow funnel-shaped stalk called the infundibulum, and lies in a depression on the upper surface of the sphenoid bone, called sella turcica.
* Tt produces several hormones some of which profoundly influence the activities of other endocrine tissues and is sometimes referred as "master endocrine gland". Its own activity is influenced by the hypothalamus, and by the pineal body.
* The hypophysis cerebri is divided into adenohypophysis and neurohypophysis (Fig. 22.1).
™ Adenohypophysis/anterior lobe: The pars anterior also called the pe distalis and the pars intermedia, are both made up of cells having a dir secretory function. They are collectively referred to as | adenohypophysis. An extension of the pars anterior surrounds the cent
nervous core of the infundibulum. Because of its tubular shape, t extension is called the pars tuberalis. The pars tuberalis is part of adenohypophysis.
Fig. 22.1: Subdivisions of the hypophysis cerebri (schematic diagram).
- = Neurohypophysis/posterior lobe: The pars posterior contains numerc nerve fibers. It is directly continuous with the central core of | infundibular stalk which is made up of nervous tissue. The pars poster and infundibular stalks are together referred to as the neurohypophy: The area in the floor of the third ventricle (tuber cinereum) immediat adjoining the attachment to it of the infundibulum is called the medi eminence. Some authorities include the median eminence in | neurohypophysis.

* Anterior lobe is covered by fibrous capsule and consists of cords of cells separated by fenestrated sinusoids.
* The parenchymal cells of the pars anterior consist of chromophils (cells have affinity for dyes) and chromophobes (cells have no affinity for dyes). (a) Chromophil cells that have brightly staining granules in their cytoplasm and (b) Chromophobe cells in which granules are not prominent.

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* Chromophil cells have affinity for histological dyes and are further classified as acidophil when their granules stain with acid dyes (like eosin or orange G), or basophil when the granules stain with basic dyes (like hematoxylin). Basophil granules are also periodic acid-Schiff (PAS) stain positive. The acidophil cells are often called alpha cells, and the basophils are called beta cells (Plate 22.2).
* Electron microscope (EM) examination shows that both acidophil and basophil cells contain abundant dense cored vesicles in the cytoplasm.
* Acidophils: Acidophils are most abundant cells in pars anterior. Acidophils can be divided into subtypes on the basis of the size and shape of the granules in them. Types of acidophil cells:
" Somatotrophs produce the somatotropic hormone/ somatotropin (STH), growth hormone (GH). This hormone controls body growth, especia before puberty.
"Mammotrophs/lactotrophs produce the mammotropic hormone [a called mammotropin, prolactin (PRL), lactogenic hormone, or LT which stimulates the growth and activity of the female mammary gle during pregnancy and lactation.
* Basophil cells: Basophils stain blue with basic dyes and it is usually located at the periphery of pars distalis.

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"Corticotrophs (or _ corticotropes) produce' the corticotro; hormone/adrenocorticotropin or ACTH. This hormone stimulates | secretion of some hormones of the adrenal cortex. The staining charact of these cells are intermediate between those of acidophils and basoph Other corticotropic hormones that have been identified are B-lipotropin | LPH), a-melanocyte-stimulating hormone (a-MSH), and B-endorphin.
"Thyrotrophs (or thyrotropes) produce the thyrotropic hormoa (thyrotropin or TSH) which stimulates the activity of the thyroid gland.
"Gonadotrophs (gonadotropes, or delta basophils) produce two types hormones, each type having a different action in the male and female.
* In the female, the first of these hormones stimulates the growth of ov follicles. It is, therefore, called the follicle-stimulating hormone (FS also stimulates the secretion of estrogens by the ovaries. In the mal same hormone stimulates spermatogenesis.
* In the female, the second hormone stimulates the maturation of the c luteum, and secretion of progesterone by it. It is called the luteii hormone (LH). In the male, the same hormone stimulates the product androgens by the interstitial cells of the testes, and is called the inter cell-stimulating hormone (ICSH).

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* These cells do not stain darkly as they contain very few granules in their cytoplasm. Immunocytochemistry shows that they represent cells similar to the various types of chromophils mentioned earlier (including mammotrophs, somatotrophs, thyrotrophs, gonadotrophs, or corticotrophs).
* They are probably degranulated chromophils.
i. Somatotrophs constitute about 50%, mammotrophs about 25 corticotrophs 15-20%, and gonadotrophs about 10% of the cell populat of the pars anterior.
|
f
=
Plate 22.1: Hypophysis cerebri

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The hypophysis cerebri consists of three main parts:
Pars anterior is cellular
*®
*
Pars intermedia is variable in structure
*
Pars posterior consists of fibers, and is lightly stained.
1. The pink staining cells are alpha cells or acidophils rr

2. The cells with bluish cytoplasm are beta cells or basophils
3. Cells in which the cytoplasm is not conspicuous, and the nuclei are closely packed, are chromophobe cells
Na

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It consists of groups or cords of cells.
*
The cells are of three types:
* Numerous sinusoids are present between the groups of cells.
- ii. Somatotrophs are located mainly in the lateral parts of the anterior lo Thyrotrophs are concentrated in the anterior, median part; | corticotrophs in the posterior, median part. Gonadotrophs j mammotrophs are scattered throughout the anterior lobe.

The pars tuberalis surrounds the infundibulum of neurohypophysis and consists mainly of undifferentiated cells. Some acidophil and basophil cells are also present.

* This is poorly developed in the human hypophysis. In ordinary preparations, the most conspicuous feature is the presence of colloid-filled vesicles/follicles (Fig. 22.2). The vesicles are remnants of the pouch of Rathke.
* Beta cells, other secretory cells, and chromophobe cells are present. Some cells of the pars intermedia produce the melanocyte-stimulating hormone (MSH) which causes increased pigmentation of the skin. Other cells produce ACTH. Endorphins are present in the cytoplasm of secretory cells.
Note: The secretion of hormones from the adenohypophysis is under control of the hypothalamus as described later.

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* The pars posterior consists of numerous unmyelinated nerve fibers which are the axons of secretory neurons located in supraoptic and paraventricular nuclei of the hypothalamus (Fig. 22.2).
* Situated between these axons, there are supporting cells of a special type called pituicytes. These cells have long dendritic processes many of which lie parallel to the nerve fibers.
* The pars posterior of the hypophysis is associated with the release of vasopressin and oxytocin. The axons descending into the pars posterior from the hypothalamus end in terminals closely related to capillaries.
* The neurosecretions/hormones secreted by supraoptic and paraventricular nuclei travel through these neurons and stored along the axons as well as at dilated nerve terminals. Collection of secretory granules at nerve terminal is called Herring bodies (Fig. 22.3).
* Hormones released at pars nervosa are vasopressin from supraoptic and oxytocin from paraventricular nuclei. Here, they are released into the capillaries of the region and enter the general circulation.
" Vasopressin (also called the antidiuretic hormone or ADH): This hormc controls reabsorption of water by kidney tubules.
" Oxytocin: It controls the contraction of smooth muscle of the uterus <¢ also of the mammary gland.

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* The hypophysis cerebri is supplied by superior and inferior branches arising from the internal carotid arteries. Some branches also arise from the anterior and posterior cerebral arteries.
* The inferior hypophyseal arteries are distributed mainly to the pars posterior.
* Branches from the superior set of arteries supply the median eminence and infundibulum.
* Superior hypophyseal arteries divide into a plexus of fenestrated capillaries. These plexus join to form portal vessels. These portal vessels descend through the infundibular stalk and end in the pars anterior as secondary capillary plexus called sinusoids. The sinusoids are drained by veins that end in neighboring venous sinuses.
* The earlier arrangement is unusual in that two sets of capillaries intervene between the arteries and veins. One of these is in the median eminence and the upper part of the infundibulum. The second set of capillaries is represented by the sinusoids of the pars anterior. This arrangement is referred to as the hypothalamohypophyseal portal system (Fig. 22.4).
Fig. 22.3: The relationship between hypothalamus and the pars posterior of the hypophysis cerebri (schematic representation).

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* The secretion of hormones by the adenohypophysis takes place under higher control of neurons in the hypothalamus, notably those in the median eminence and in the infundibular nucleus. The axons of these neurons end in relation to capillaries in the median eminence and in the upper part of the infundibulum.
* Different neurons produce specific releasing factors (or releasing
hormones) for each hormone of the adenohypophysis. These factors are released into the capillaries.
* Portal vessels arising from the capillaries carry these factors to the pars anterior of the hypophysis. Here, they stimulate the release of appropriate hormones.
* Some factors inhibit the release of hormones. The synthesis and discharge of releasing factors by the neurons are under nervous control.
* As these neurons serve as intermediaries between nerve impulse and hormone secretion, they have been referred to as neuroendocrine transducers. Some cells called tanycytes, present in ependyma, may transport releasing factors from neurons into the cerebrospinal fluid (CSF), or from CSF to blood capillaries. They may, thus, play a role in control of the adenohypophysis.
* The entire neurohypophysis (from the median eminence to the pars posterior) is permeated by a continuous network of capillaries in which blood may flow in either direction. The capillaries provide a route through which hormones released in the pars posterior can travel back to the hypothalamus, and into CSF. Some veins draining the pars posterior pass into the adenohypophysis. Secretions by the adenohypophysis may, thus, be controlled not only from the median eminence, but by the entire neurohypophysis. Blood flow in veins connecting the pars anterior and pars posterior may be _ reversible providing a feedback from adenohypophysis to the neurohypophysis.
Fig. 22.4: Hypothalamo-hypophyseal portal system (schematic representation).
Bi

* Thyroid is a bilobed gland. Each lobe is situated on either side of trachea, below larynx, and in lower neck.
* The two lobes are connected to each other by isthmus in front of trachea.
* Peculiarities: (a) stores hormone in the form of colloid, (b) superficially located, and (c) depends on environment for secretion (iodine).

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* The thyroid gland is covered by a dense irregular connective tissue capsule derived from deep cervical fascia. Parathyroid glands are embedded within the capsule on posterior aspect of the thyroid gland. Septa extending into the gland from the capsule divide it into lobules.
* On microscopic examination, each lobule is seen to be made up of an aggregation of follicles.
* Each follicle is lined by follicular cells that rest on a basement membrane.
* The follicle is filled with a homogeneous material called colloid which appears pink in hematoxylin and eosin-stained sections (Plate 22.3).
* The spaces between the follicles are filled by a stroma made up of delicate connective tissue with numerous capillaries and lymphatics. The capillaries lie in close contact with the walls of follicles.
* Apart from follicular cells, the thyroid gland contains C-cells (or parafollicular cells) which intervene between the follicular cells and the basement membrane and also between the follicles in the connective tissue stroma (Plate 22.3).

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* The follicular cells vary in shape from squamous, cuboidal, and to columnar depending on the level of their activity (Fig. 22.5).
* Highly active follicle is lined by columnar cells with scanty colloid, moderate active follicle is lined by cuboidal cells with moderate colloid, and inactive follicle is lined by squamous cells with abundant colloid. Different follicles may show different levels of activity (Fig. 22.5).
* Follicular cells have a round to ovoid nucleus with two nucleoli and basophilic cytoplasm.
* With the EM, a follicular cell shows the presence of apical microvilli, abundant granular endoplasmic reticulum, and a prominent supranuclear Golgi complex. Lysosomes, microtubules, and microfilaments are also present. The apical part of the cell contains many secretory vacuoles (Fig. 22.6).
* The activity of follicular cells is influenced by the thyroid-stimulating hormone (TSH or thyrotropin) produced by the hypophysis cerebri.
* The follicular cells secrete triiodothyronine (T3) with three atoms of iodine and tetraiodothyronine (T4) or thyroxine with four atoms of iodine in each molecule. These hormones influence the rate of metabolism.

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* They are also called clear cells, or light cells. C-cells share features of the APUD cell system and derived from neural crest cells.
* The cells are polyhedral with oval eccentric nuclei.
* Typically, they lie between the follicular cells and their basement membrane. They may, however, lie between adjoining follicular cells, but
they do not reach the lumen and may also lie in the connective tissue between the follicles in groups.
* With the EM, the cells show well-developed granular endoplasmic reticulum, Golgi complexes, numerous mitochondria, and membranebound secretory granules.
* C-cells secrete the hormone calcitonin.
* This hormone has an action opposite to that of the parathyroid hormone on calcium metabolism.
Fig. 22.5: Variations in appearance of thyroid follicles at different levels of activity (schematic representation).
Crear Thyroid gland
'The thyroid gland is made up of follicles lined by cuboidal epithelium
- 'In photomicrograph, in low magnification, it can be seen that follicles vary in shape and size
‘Each follicle
is filled
with
a
homogenous pink
colloid proteinaceous
material composed
primarily of thyroglobulin that has been produced by the follicular epithelial cells
- 'Parafollicular cells are present in relation to the follicles and also as groups in the connective tissue
'In the intervals between the follicles, there is some connective tissue and blood vessels between follicles.
* It mainly lowers the blood calcium level when serum calcium level is high by suppressing release of calcium ions from bone. This is achieved by suppressing bone resorption by osteoclasts.
* Secretion of calcitonin is stimulated by increase in blood calcium level.

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* The synthesis of thyroid hormones is regulated by iodine levels and TSH binding to TSH receptors of follicular cells.
* The synthesis and accumulation of thyroid hormone takes place in four stages: Synthesis of thyroglobulin, uptake of iodine from the blood, activation of iodine, and iodination of the tyrosine residues of thyroglobulin (Flowchart 22.1).
"Synthesis of thyroglobulin: Thyroglobulin (a glycoprotein) is synthesiz by granular endoplasmic reticulum and is packed into secretory vacuoles the Golgi complex. The vacuoles travel to the luminal surface where tt release thyroglobulin into the follicular cavity by exocytosis.
"The uptake of circulating iodine by follicular cells by a membre transport protein.
"Activation of iodine: Iodine is oxidized by thyroid peroxidase ¢ transported into the follicle. Here, the thyroglobulin combines with iod' to form colloid. Colloid is iodinated thyroglobulin.
Flowchart 22.1: Some steps in the formation of hormones by the thyroid gland.
"Todination: Within the colloid, the iodination of the tyrosine residues thyroglobulin resulting in the formation of monoiodothyronine ¢ diiodothyronine. The coupling of these molecules produces T3 and hormones.
* Release of thyroid hormones: When stimulated by TSH, follicular cells take up the colloid into the cell by endocytosis. Within the cell, the iodinated thyroglobulin is acted upon by enzymes present in lysosomesreleasing hormones T3 and T4 which pass basally through the cell and are released into blood capillaries.
* Hormone produced in the thyroid gland is mainly T4. In the liver, the kidneys, and some other tissues, T4 is converted to T3 by removal of one iodine molecule. T3 and T4 circulating in blood are bound to a protein called thyroxine-binding globulin, TBG and the bound form of hormone is not active.
*T4 is more abundant than T3, constitutes almost 90% of circulating hormones but T3 acts more rapidly and is more potent.
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PARATHYROID GLANDS

The parathyroid glands are located behind the thyroid gland, one at each end of upper and lower pole and within the capsule of thyroid gland.

* Gland is covered by connective tissue capsule from which septa extend into the gland substance and merge with reticular fibers which supports the secretory cells.
* With increasing age, secretory cells are replaced with adipocytes and almost it constitutes more than 50% of the gland in older age (Plate 22.4).
* The endocrine cells are arranged in cords. Numerous sinusoids lie in close relationship to the cells.
* There are two types of cells: Chief cells and oxyphil cells.

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* The chief cells are more numerous than the oxyphil cells.
* With the light microscope, the chief cells are seen to be small polygonal cells with vesicular nucleus and light staining and _ slightly eosinophil/acidophilic cytoplasm.
* Sometimes, the cell accumulates glycogen and lipids and looks "clear".
* Three types of chief cells: light, dark, and clear have been described.
Cree Parathyroid gland
Parathyroid gland: (A) As seen in drawing; (B) Photomicrograph.
Courtesy: Damjanov I. Atlas of Histopathology, 1st edition. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2012. p. 290.

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*These glands are made up of masses of cells with numerous capillaries in between
*Most of the cells (of which only nuclei are seen) are the chief cells which appear as small basophilic cells
"Oxyphil cells appear as large as eosinophilic (pink) cells
- *Oxyphil cells are few in number
*
Adipose cells are also seen.
* With the EM, active chief cells are seen to have abundant granular endoplasmic reticulum and well-developed Golgi complexes. Small secretory granules are seen, especially in parts of the cytoplasm near adjacent blood sinusoids.
* The chief cells produce the parathyroid hormone or parathormone.
* Parathormone tends to increase the serum calcium level by: Increasing the bone resorption through stimulation of osteoclastic activity, calcium resorption from renal tubules, and calcium absorption from the gut.

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* The oxyphil cells are much larger than the chief cells and contain granules that stain strongly with acid dyes.
* Their nuclei are smaller and stain more intensely than those of chief cells.
* They are less numerous than the chief cells.
* The oxyphil cells are absent in the young and appear a little before the age of puberty.
* With the EM, it is seen that the granules of oxyphil cells are really mitochondria, large numbers of which are present in the cytoplasm.
* True secretory granules are not present. The functions of oxyphil cells are unknown.
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* Suprarenal glands are paired organs and lie near the superior poles of the kidney in the abdomen. In many animals, they do not occupy a "supra" renal position, but lie near the kidneys therefore, called as adrenal glands.
* The gland is made up of two functionally distinct parts—a yellow superficial part called the cortex, and a reddish-brown deep part called the medulla. The volume of the cortex is about ten times that of the medulla.
* Cortex and medulla have distinct origins, functions, and morphological characteristics that are united during development.

Gland is covered by a connective tissue capsule from which septa extend into the gland substance. Stroma has network of reticular fibers that supports the secretory cells.

* The suprarenal cortex is made up of cells arranged in cords. Sinusoids intervene between the cords.
* On the basis of the arrangement of the cells, the cortex can be divided into three layers: (a) Outermost zona glomerulosa, (b) Middle zona fasciculata, and (c) Inner zona reticularis (Plate 22.5).
* The cells of adrenal cortex have the typical structure of steroid-secreting cells. They do not store the secretory products but they secrete when needed.

* Tt is the outermost layer and constitutes the outer one-fifth of the cortex.
* Here, the cells are arranged in closely packed inverted U-shaped cords or rounded like acinus groups and surrounded by capillaries.
* With the light microscope, the cells of the zona glomerulosa are seen to be small polyhedral or columnar with basophilic cytoplasm and deeply staining nuclei.
* Produce the mineralocorticoid hormones' aldosterone and deoxycorticosterone. These hormones influence the electrolyte and water balance of the body. The secretion of aldosterone is influenced by renin secreted by juxtaglomerular cells of the kidney. The secretion of hormones by the zona glomerulosa appears to be largely independent of the hypophysis cerebri.

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* Tt is the middle layer and forms the middle three-fifths of the cortex.
* The cells are arranged in straight columns with two cells thick at right angles to the surface. Sinusoids intervene between the columns.
* With the light microscope, the cells of the zona fasciculata are seen to be large, polyhedral with basophilic cytoplasm, and vesicular nuclei.
* The cells of the zona fasciculata are very rich in lipids. With routine staining methods, the lipids are dissolved giving the cells an "empty" or
vacuolated appearance. Because of their vacuolization, the cells are also called spongiocytes. These cells also contain considerable amounts of vitamin C.
- * Produce the glucocorticoids cortisone and cortisol (dihydrocortisone). These hormones have widespread effects, including those on carbohydrate metabolism and protein metabolism. They appear to decrease antibody response and have an anti-inflammatory effect. The zona fasciculata also produces small amounts of dehydroepiandrosterone (DHA) which is an androgen.

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* The inner one-fifth layer of the cortex is called the zona reticularis.
* Tt is called as reticularis because it is made up of cords that branch and anastomose with each other to form a kind of reticulum.
* The cells in this layer are smaller and more acidophilic.
* With the light microscope, the cells of the zona reticularis are seen to be similar to those of the zona fasciculata but the lipid content is less. Their cytoplasm is often eosinophilic. The cells often contain brown lipofuscin pigment granules.
* With the EM, the cells in all layers of the cortex are characterized by the presence of abundant smooth endoplasmic reticulum. The Golgi complex is best developed in cells of the zona fasciculata. Mitochondria are elongated in the glomerulosa, spherical in the fasciculata, and unusual with tubular cisternae (instead of the usual plates) in the reticularis (Fig. 22.7).
Plate 22.5: Suprarenal gland
Suprarenal gland: (A) As seen in drawing; (B) Photomicrograph.

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"The suprarenal gland is made up of a large number of cells arranged in layers. It consists of an outer cortex and an inner medulla
"The cortex is divisible into three zones
"The zona glomerulosa is most superficial. Here, the cells are arranged in the form of inverted U-shaped structures or acinus-like groups
"In the zona fasciculata, the cells are arranged in straight columns (typically two-cell thick). Sinusoids intervene between the columns
"The zona reticularis is made up of cords of cells that branch and form a network
"The medulla is made up of groups of cells separated by wide sinusoids. Some sympathetic neurons are also present.
- * Produce some glucocorticoids, and sex hormones, both estrogens and androgens.
Fig. 22.7: Some features of ultrastructure of a cell from the adrenal cortex (schematic representation).

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* Both functionally and embryologically, the medulla of the suprarenal gland is distinct from the cortex.
* Functions as a modified sympathetic ganglion with postganglionic sympathetic cells without axons and dendrites.
* Tt has chromaffin cells and sympathetic ganglion cells which are scattered throughout.
* They are derived from neural crest cells. Cells are nothing but modified postganglionic neurons that have a secretory function.
* Chromaffin cells are columnar or polyhedral and have a _ basophilic cytoplasm arranged in cords or clumps. The cell groups or columns are separated by wide sinusoids.
* With the EM, the cells of the adrenal medulla are seen to contain abundant granular endoplasmic reticulum and a prominent Golgi complex (Fig. 22.8). The cells also contain membrane-bound secretory vesicles. In some
cells, these vesicles are small and electron dense while in others, they are large and not so dense. The former contains noradrenalin and the latter adrenalin.
* In contrast to the suprarenal cortex, the medulla is not essential for life as its functions can be performed by other chromaffin tissues.
* Chromaffin cells secrete noradrenalin (norepinephrine) and adrenalin (epinephrine) into the blood. This secretion takes place mainly at times of stress (fear and anger) and results in widespread effects similar to those of stimulation of the sympathetic nervous system (e.g. increase in heart rate and blood pressure).
* A secretion of suprarenal medulla is controlled by splanchnic nerves.
Fig. 22.8: Some features of ultrastructure of a cell from the adrenal medulla (schematic representation).

* The pineal gland (or pineal body) is a small piriform structure present in relation to the posterior wall of the third ventricle of the brain. It is also called the epiphysis cerebri.
* The secretions of the gland are influenced by the light and dark periods of the day and it is most active during night.

* The gland is covered by pia mater forming a capsule from which septa extends into the gland and divides it into incomplete lobules.
* Sections of the pineal gland stained with hematoxylin and eosin shows a mass of cells among which there are blood capillaries and nerve fibers.
* A distinctive feature of the pineal in sections is the presence of irregular masses made up mainly of calcium salts. These masses constitute the corpora arenacea or brain sand (Fig. 22.9).
* The gland composed of pinealocytes and interstitial cells.

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* Pinealocytes cell have a polyhedral body containing a spherical, oval, or irregular nucleus.
* The cell body gives off long processes with expanded terminal buds that end in relation to the walls of capillaries, or in relation to the ependyma of the third ventricle.
* The cell bodies of pinealocytes contain both granular and agranular endoplasmic reticulum, a well-developed Golgi complex, and many mitochondria. An organelle of unusual structure made up of groups of microfibrils and perforated lamellae may be present (canaliculate lamellar bodies).
* Hormone produced by pinealocytes: The pinealocytes produce a number of hormones which are chemically indolamines or polypeptides. The best known hormone of the pineal gland is the amino acid melatonin (so called because it causes changes in skin color in amphibia). Large concentrations of melatonin are present in the pineal gland. It is released at night and inhibits the release of growth hormone and gonadotropin hormones. It also induces the feeling of sleepiness and can be used as a supplement to combat sleep disorders.
Fig. 22.9: Pineal body as seen with a light microscope (schematic representation).

* The pinealocytes are separated from one another by neuroglial cells that resemble astrocytes in structure.
* They lie in proximity to blood vessel and pinealocytes.
* Cells are dark staining elongated nucleus with well-developed rough endoplasmic reticulum.

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- * Because of the light-mediated response, the pineal gland may act as a kind of biological clock which may produce circadian
rhythms (variations following a 24-hour cycle) in various parameters.
*The suprachiasmatic nucleus of the hypothalamus plays an important role in the cyclic activity of the pineal gland.
*This nucleus receives fibers from the retina. In turn, it projects to the tegmental reticular nuclei located in the brainstem. Reticulospinal fibers arising in these nuclei influence the sympathetic preganglionic neurons located in the first thoracic segment of the spinal cord. Axons of these neurons reach the superior cervical ganglion from where the nervus conarii arises and supplies the pineal gland.
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* Agegregations of cells similar to those of the adrenal medulla are to be found at various sites. They are collectively referred to as paraganglia because most of them are present in close relation to autonomic ganglia.
* The cells of paraganglia give a positive chromaffin reaction, receive a preganglionic sympathetic innervation, and have secretory granules containing catecholamines in their cytoplasm.
* Like the cells of the adrenal medulla, paraganglia are believed to develop from cells of the neural crest.
* Paraganglia are richly vascularized. They are regarded as endocrine glands that serve as alternative sites for the production of catecholamines in the fetus, and in early postnatal life, when the adrenal medulla is not yet fully differentiated.
* Most of the paraganglia retrogress with age, but some persist into adult life.
Note: Some workers include the para-aortic bodies and carotid bodies among paraganglia.

* These are two elongated bodies that lie, one on each side of the aorta, near
the origin of the inferior mesenteric artery.
* The two masses may be united to each other by a band passing across the aorta.
* These bodies have a structure similar to that of the adrenal medulla. The cells secrete noradrenalin. The aortic bodies retrogress with age.

* These are small oval structures, present one on each side of the neck, at the bifurcation of the common carotid artery (i.e. near the carotid sinus).
* The carotid bodies contain a network of capillaries in the intervals between which there are several types of cells.

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* The most conspicuous cells of the carotid body are called glomus cells (or type I cells) (Fig. 22.10). These are large cells that have several similarities to neurons as follows:
" They give off dendritic processes.
"Their cytoplasm contains membrane-bound granules which contain number of neuropeptides. In the human carotid body, the most promin: peptide present is encephalin. Others present include dopamine, seroton catecholamines, vasoactive intestinal peptide (VIP), and substance P.
"The cells are in synaptic contact with afferent nerve terminals of | glossopharyngeal nerve. Chemoreceptor impulses pass through these fib to the brain. Some glomus cells also show synaptic connections with | endings of preganglionic sympathetic fibers, and with other glomus cells.
"The organization of endoplasmic reticulum in them shows similarities that of Nissl substance.
" They are surrounded by sheath cells that resemble neuroglial elements.
* Because of these similarities to neurons, and because of the possibility that the cells release dopamine (and possibly other substances), they are sometimes described as neuroendocrine cells (and are included in the APUD cell category).
* The exact significance of the glomus cells, and of their nervous connections, is not understood at present. They could possibly be sensory receptors sensitive to oxygen and carbon dioxide tension. Dopamine
released by them may influence the sensitivity of chemoreceptor nerve endings. They may also serve as interneurons.
* Apart from the glomus cells, other cells present in the carotid bodies are as follows:
™ Sheath cells (or type II cells) that surround the glomus cells.
" A few sympathetic and parasympathetic postganglionic neurons.
"Endothelial cells of blood vessels, and muscle cells in the walls arterioles.
= Some connective tissue cells.
Fig. 22.10: Structure of the carotid body (schematic representation).

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The carotid body is richly innervated as follows:
* Afferent nerve terminals from the glossopharyngeal nerve form synapses with glomus cells.
* Preganglionic sympathetic and parasympathetic fibers end on the corresponding ganglion cells. Some preganglionic sympathetic fibers end
by synapsing with glomus cells.
* Postganglionic fibers arising from the sympathetic and parasympathetic nerve cells within the carotid body innervate muscle in the walls of arterioles.

* The main function of the carotid bodies is that they act as chemoreceptors that monitor the oxygen and carbon dioxide levels in blood.
* They reflexly control the rate and depth of respiration through respiratory centers located in the brainstem.
* Tn addition to this function, the carotid bodies are also believed to have an endocrine function.
Note: The precise mechanism by which the carotid bodies respond to changes in oxygen and carbon dioxide tension is not understood. It is not certain as to which cells, or nerve terminals are responsible for this function.

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*Apart from the discrete endocrine organs considered in this chapter, there are groups of endocrine cells scattered in various parts of the body. These cells share some common characteristics with each other, and also with the cells of some discrete endocrine organs.
* All these cells take up precursor substances from the circulation and process them (by decarboxylation) to form amines or peptides. They are, therefore, included in what is called the APUD cell system. These peptides or amines serve as hormones.
*Many of them also function as neurotransmitters. Hence, the APUD cell system is also called the diffuse neuroendocrine system. The cells of this system contain spherical or oval membrane-bound granules with a dense core. There is an electron-lucent halo around the dense core.
* The diffuse neuroendocrine system is regarded as representing a
link between the autonomic nervous system on the one hand, and the organs classically recognized as endocrine on the other, as it shares some features of both.

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* The effects of the amines or peptides produced by the cells of the system are sometimes "local" (like those of neurotransmitters) and sometimes widespread (like those of better known hormones).
The cell types included in the APUD cell system are as follows:
*Various cells of the adenohypophysis.
*Neurons in the hypothalamus that synthesize the hormones of the neurohypophysis (oxytocin, vasopressin), and the cells that synthesize releasing factors controlling the secretion of hormones by the adenohypophysis.
*The chief cells of the parathyroid glands producing parathyroid hormone.
*The C-cells (parafollicular cells) of the thyroid, producing calcitonin.
* Cells of the adrenal medulla (along with some outlying chromaffin tissues) that secrete adrenalin and noradrenalin. These include the SIF cells of sympathetic ganglia.
*Cells of the gastroenteropancreatic endocrine system which includes cells of pancreatic islets producing insulin, glucagon, and some other amines. It also includes endocrine cells scattered in the epithelium of the stomach and intestines producing one or more of the following: 5-hydroxytryptamine, glucagon, dopamine, somatostatin, substance P, motilin, gastrin, cholecystokinin, secretin, vasoactive intestinal polypeptide (VIP), and some other peptides.
*Glomus cells of the carotid bodies producing dopamine and noradrenalin.
*Melanocytes of the skin producing promelanin.
*Some cells in the pineal gland, the placenta, and modified myocytes of the heart called myoendocrine cells.
*Renin-producing cells of the kidneys.

* Gigantism: When growth hormone (GH) excess occurs prior to epiphyseal closure, gigantism is produced. The main clinical feature in gigantism is the excessive and proportionate growth of the child. There is enlargement as well as thickening of the bones resulting in considerable increase in height and enlarged thoracic cage.
Acromegaly: Acromegaly results when there is overproduction of GH in adults following cessation of bone growth and is more common than gigantism. The term "acromegaly" means increased growth of extremities (acro = extremity). There is enlargement of hands and feet, coarseness of facial features with increase in soft tissues, prominent supraorbital ridges, and a more prominent lower jaw which when clenched results in protrusion of the lower teeth in front of upper teeth (prognathism). , a
Diabetes insipidus: Deficient secretion of ADH due to inflammatory and neoplastic lesions of the hypothalamohypophyseal axis, destruction of neurohypophysis due to surgery, radiation, head injury, etc., causes diabetes insipidus. The main features of diabetes insipidus are excretion of a very large volume of dilute urine of low specific gravity (below 1.010), polyuria, and polydipsia. *
* The vessels descending through the infundibular stalk are easily damaged in severe head injuries. This leads to loss of function in the anterior lobe of the hypophysis cerebri. ate

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* Hyperthyroidism: Hyperthyroidism, also called thyrotoxicosis, is a hypermetabolic clinical and biochemical state caused by excess production of thyroid hormones. The condition is more frequent in females.
* Hypothyroidism: Hypothyroidism is a hypometabolic clinical state resulting from inadequate production of thyroid hormones for prolonged periods, or rarely, from resistance of the peripheral tissues to the effects of thyroid hormones. Depending upon the age at onset of disorder, it is divided into two forms:
1. Cretinism or congenital hypothyroidism is the development o:
hypothyroidism during infancy and childhood.
2. Myxedema is the adulthood hypothyroidism.
* Graves' disease: Graves' disease, also known as Basedow's disease, primary hyperplasia, exophthalmic goiter, and diffuse toxic goiter, is characterized by a triad of features:
" Hyperthyroidism (thyrotoxicosis)
" Diffuse thyroid enlargement
™ Ophthalmopathy.
The disease is more frequent between the age of 30 years and 40 years and has five-fold increased prevalence among females.

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* Hyperparathyroidism: Hyperfunction of the parathyroid glands occurs due to excessive production of parathyroid hormone. It is classified into three types—primary, secondary, and tertiary.
1. Primary hyperparathyroidism occurs from oversecretion of pal hormone due to disease of the parathyroid glands.
2. Secondary hyperparathyroidism is caused by diseases in other pat body.
3. Tertiary hyperparathyroidism develops from secondary hyperpla removal of the cause of secondary hyperplasia.
* Hypoparathyroidism: Deficiency or absence of parathyroid hormone secretion causes hypoparathyroidism.

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* Addison's disease: Condition with decreased secretion of the adrenocortical hormones due to destruction of suprarenal cortex.
* Cushing's disease: A small tumors in the basophils of the anterior pituitary lead to increase in ACTH output leading to enlargement of suprarenal cortex resulting in overproduction of cortisol.
1. Which of the following is not a characteristic of the endocrine system?
Products secreted into blood
Glands with ducts Secretes hormones aoo0p
Nonlocalized response
2. Calcitonin is a hormone of which of following?
. Adrenal cortex
. Thyroid gland . Pituitary gland 2009
. Thymus gland
3. Calcium level in the blood is regulated by the:
. Thyroid
. Parathyroid
. Posterior pituitary
. Adrenal medulla
aandb
9200
4. Which gland is called the “master gland”?
. Adrenal cortex . Thyroid . Pituitary 2009
5. Which cell type secretes ACTH?
a. Lactotropic
b. Thyrotropic
c. Corticotropic
d. Gonadotropic
6. Which cell type is involved in the secretion of thyroglobulin?
a. Principal cells
b. Oxyphil cells
c. Parafollicular cells
d. Follicular cells
7. Which part of the adrenal gland secretes glucocorticoids?
a. Chromaffin cells
b. Zona reticularis
c. Zona glomerulosa

8. Which part of pituitary gland contains Herring bodies?
Pars nervosa
Pars intermedia Pars anterior Pars distalis 2050p

The cornea is made up of five layers: Corneal epithelium, anterior limiting membrane, or Bowman's membrane, corneal stroma, posterior limiting lamina, and endothelium, Retina = Made up of ten layers: Pigment epithelium, layer of rods and cones, external limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglionic cell layer, nerve fiber layer, and internal limiting membrane. The corneal epithelium is nonkeratinized stratified squamous epithelium, Retina = Outer pigment epithelium is made up of simple cuboidal epithelium. The substantia propria is made up of collagen fibers embedded in a ground substance, Retina = The outer nuclear layer is thicker with densely packed nuclei than in the inner nuclear layer
'Endothelium is lined by a single layer Ganglion cell layer is made up of a single row of cells of flattened or cuboidal cells. of varying size.
Bf

* The eyes are peripheral organs for vision and are located in the bony orbit.
* Each eyeball is like a camera. It has a lens that produces images of objects that we look at. The images fall on a light sensitive membrane called the retina. Cells in the retina convert light images into nerve impulses that pass through the optic nerve, and other parts of the visual pathway to reach visual areas in the cerebral cortex. It is in the cortex that vision is actually perceived.
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The wall of an eyeball consists of three layers:
1. Outer fibrous coat: That includes sclera and cornea
2. Middle vascular coat: That includes choroid, ciliary body, and iris
3. Inner nervous coat: Called retina.
** The space between the iris and the cornea is called the anterior chamber, while the space between the iris and the front of the lens is called the posterior chamber. These chambers are filled with a fluid called the aqueous humor. The part of the eyeball behind the lens is filled by a jellylike substance called the vitreous body.
** The main parts of eyeball (as seen in section) are shown in Figure 23.1.
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* The outer wall of the eyeball is formed (in its posterior five-sixths) by a thick white opaque membrane called the sclera. The sclera consists of white fibrous tissue (collagen). Some elastic fibers and connective tissue cells (mainly fibroblasts) are also present. Some of the cells are pigmented.
* Externally, the sclera is covered in its anterior part by the ocular conjunctiva, and posteriorly by a fascial sheath (or episclera). The deep surface of the sclera is separated from the choroid by the perichoroidal space. Delicate connective tissue present in this space constitutes the suprachoroid lamina (or lamina fusca).
* Anteriorly, the sclera becomes continuous with the cornea at the corneoscleral junction (also called sclerocorneal junction or limbus). A circular channel called the sinus venosus sclerae (or canal of Schlemm) is located in the sclera just behind the corneoscleral junction (Fig. 23.2). A triangular mass of scleral tissue projects toward the cornea just medial to this sinus. This projection is called the scleral spur.
* The optic nerve is attached to the back of the eyeball, a short distance medial to the posterior pole. Here, the sclera is perforated like a sieve, and the area is, therefore, called the lamina cribrosa. Bundles of optic nerve fibers pass through the perforations of the lamina cribrosa.
Fig. 23.1: Section across the eyeball to show its main parts (schematic representation).

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* The sclera (along with the cornea) collectively forms the fibrous tunic of
the eyeball and provides protection to delicate structures within the eye.
* Tt resists intraocular pressure and maintains the shape of the eyeball.
* Its smooth external surface allows eye movements to take place with ease.
* The sclera also provides attachment to muscles that move the eyeball.
Fig. 23.2: Some features of the eyeball to be seen at the junction of the cornea with the sclera (schematic representation).

In the anterior one-sixth of the eyeball, the sclera is replaced by a transparent disc called the cornea. The cornea is convex forward. It is colorless and avascular but has a very rich nerve supply.

The cornea is made up of five layers:
- 1. Anterior corneal epithelium:
"The outermost layer is of nonkeratinized stratified squamous epithelit

"The cells in the deepest layer of the epithelium are columnar; in the mid layers, they are polygonal; and in the superficial layers, they are flatten The cells are arranged with great regularity.
= With the electron microscope (EM), the cells on the superficial surface the epithelium show projections either in the form of microvilli or folds plasma membrane. These folds are believed to play an important role retaining a film of fluid over the surface of the cornea.
= The corneal epithelium regenerates rapidly after damage.

"The corneal epithelium rests on the anterior limiting lamina (also cal Bowman's membrane).
= With the light microscope, this lamina appears to be structureless, but w the EM, it is dense homogeneous, made up of fine collagen fibi embedded in matrix.
"It gives great stability and strength to the cornea. It also acts as barrier spread of infections.
"Once damaged, it cannot regenerate, leading to scar formation a permanent impairment of vision.

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"Most of the thickness of the cornea is formed by the substantia propria corneal stroma).
"The substantia propria is made up of type 1 collagen fibers embedded i) ground substance containing sulfated glycosaminoglycans. They . arranged with great regularity and form lamellae (around 200-250 layers'
"The fibers within one lamellus are parallel to one another, but the fibers adjoining lamellae run in different directions forming obtuse angles w each other.
"The transparency of the cornea is due to the regular arrangement of fibe and because of the fact that the fibers and the ground substance have © same refractive index.
"Fibroblasts are present in the substantia propria. They appear to be flatter
in vertical sections through the cornea, but are seen to be star-shaped surface view. They are also called keratocytes or corneal corpuscles.

"Deep to the substantia propria, there is a thin homogeneous acellular la called the posterior limiting lamina (or Descemet's membrane). It is a t basement membrane.
"They can regenerate after injury.
"At the margin of the cornea, the posterior limiting membrane becon continuous with fibers that form a network in the angle between the corr and the iris (iridocorneal angle).
"The spaces between the fibers of the network are called the spaces of | iridocorneal angle. Some of the fibers of the network pass onto the iris the pectinate ligament (Fig. 23.2).

"The posterior surface of the cornea is lined by a single layer of flatter cells that constitute the endothelium of the anterior chamber. This layer in contact with the aqueous humor of the anterior chamber.
"The endothelial cells are adapted for transport of ions. They possi numerous mitochondria.
"They are united to neighboring cells by desmosomes and by occludi junctions. The cells pump out excessive fluid from cornea, and thus, ens! its transparency (Plate 23.1).

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* Avascularity
* The regular arrangement of fibers in substantia propria
* The collagen fibers and the ground substance have the same refractive index
* Uniform and regular arrangement of anterior epithelium
* Relative state of corneal dehydration.

* Lacrimal fluid forming thin capillary layer on the surface of cornea
* Aqueous humor in anterior chamber
* Blood vessels at sclerocorneal junction.

Deep to the sclera, there is a vascular coat (uvea) that consists of:
* Choroid
* Ciliary body
* Tris.

'The cornea is made up of five layers: Corneal epithelium, anterior limiting membrane, or Bowman's membrane, corneal stroma, posterior limiting lamina, and endothelium 'The corneal epithelium is nonkeratinized stratified squamous epithelium 'The substantia propria is made up of collagen fibers embedded in a ground substance "Endothelium is lined by a single layer of flattened or cuboidal cells.

The choroid consists of:
* Choroid proper
* Suprachoroid lamina that separates the choroid proper from the sclera
* Basal lamina (membrane of Bruch), which intervenes between the choroid proper and the retina (Fig. 23.3).

* The choroid proper consists of a network of blood vessels supported by connective tissue in which many pigmented cells are present, giving the choroid a dark color. This color darkens the interior of the eyeball. The pigment also prevents reflection of light within the eyeball. Both these factors help in formation of sharp images on the retina.
* The choroid proper is made up of an outer vascular lamina containing small arteries and veins, and lymphatics, and an inner capillary lamina (or choroidocapillaris). The connective tissue supporting the vessels of the vascular lamina is the choroidal stroma. Apart from collagen fibers, it contains melanocytes, lymphocytes, and mast cells. The capillary lamina is not pigmented. Nutrients diffusing out of the capillaries pass through the basal lamina to provide nutrition to the outer layers of the retina.

The suprachoroid lamina is also called the lamina fusca. It is nonvascular. It is made up of delicate connective tissue containing collagen, elastic fibers, and branching cells containing pigment. A plexus of nerve fibers is present. Some neurons may be seen in the plexus.
Fig. 23.3: Various layers of the eyeball (schematic representation).

With the light microscope, the basal lamina (or membrane of Bruch) appears to be a homogeneous layer. However, with the EM, the membrane is seen to have a middle layer of elastic fibers, on either side of which there is a layer of delicate collagen fibers. The basal lamina is said to provide a smooth surface on which pigment cells and receptors of the retina can be arranged in precise orientation.

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* The ciliary body represents an anterior continuation of the choroid. It is a ring-like structure continuous with the periphery of the iris.
* Tt is connected to the lens by the suspensory ligament.
* The ciliary body is made up of vascular tissue, connective tissue, and muscle.
* The muscle component constitutes the ciliaris muscle. The ciliaris muscle is responsible for producing alterations in the convexity of the lens (through the suspensory ligament) enabling the eye to see objects at
varying distances from it. In other words, the ciliaris is responsible for accommodation.
* The inner surface of the ciliary body is lined by a double-layered epithelium. The outer cell layer is pigmented, whereas the inner cell layer (facing the posterior chamber) is nonpigmented. The cells of the inner layer secrete aqueous humor.
* The anterior part of the inner surface of the ciliary body has short processes toward the lens, known as ciliary processes.

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* The iris is the most anterior part of the vascular coat of the eyeball.
*Tt forms a diaphragm placed immediately in front of the lens. At its periphery, it is continuous with the ciliary body. In its center, there is an aperture the pupil. The pupil regulates the amount of light passing into the eye.
* The iris is composed of a stroma of connective tissue containing numerous pigment cells, and in which are embedded blood vessels and smooth muscle.
* Some smooth muscle fibers are arranged circularly around the pupil and constrict it. They form the sphincter pupillae. Other fibers run radially and form the dilator pupillae.
* The posterior surface of the iris is lined by a double layer of epithelium continuous with that over the ciliary body. This epithelium represents a forward continuation of the retina. The cells of this epithelium are deeply pigmented.

This is the inner coat of eyeball and lines its posterior three-fourths of surface. The retina contains photoreceptors (rods and cones) which are essential for vision.

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To understand the structure of the retina, brief reference to its development is necessary (Figs. 23.4A and B).
- * The retina develops as an outgrowth from the brain (diencephalon). The
proximal part of the diverticulum remains narrow and is called the optic stalk. It later becomes the optic nerve. The distal part of the diverticulum forms a rounded hollow structure called the optic vesicle.
* This vesicle is invaginated by the developing lens (and other surrounding tissues) so that it gets converted into a two-layered optic cup.
* At first, each layer of the cup is made up of a single layer of cells. The outer layer persists as a single-layered epithelium that becomes pigmented. It forms the pigment cell layer of the retina. Over the greater part of the optic cup, the cells of the inner layer multiply to form several layers of cells that become the nervous layer of the retina.
* In the anterior part, both layers of the optic cup remain single layered. These two layers line: (1) the inner surface of the ciliary body forming the ciliary part of the retina, and (2) the posterior surface of the iris forming the iridial part of the retina.
* Retina has a specialized area where vision is most acute, called as fovea centralis or macula (Fig. 23.5). This area contains only cones which are essentially bare (the overlying layers are pushed to the side).
* The retina also has a "blind spot", the optic disc, where the optic nerve leaves the eye and there are no photoreceptor cells.
* Opposite the posterior pole of the eyeball, the retina shows a central region about 6 mm in diameter. This region is responsible for sharp vision. In the center of this region, an area about 2 mm in diameter has a yellow color
Figs. 23.4a and B: Some features of the developing eye: (A) Early stage; (B) Later stage (schematic representation).
and is called the macula lutea (Fig. 23.5). In the center of the macula lutea, there is a small depression that is called the fovea centralis. The floor of the fovea centralis is often called the foveola. This is the area of clearest vision.

Fig. 23.5: Some features of the retina as seen through an ophthalmoscope (schematic representation). Note the arteries emerging through the optic disc.
- * The optic nerve is attached to the eyeball a short distance medial to the posterior pole. The nerve fibers arising from the retina converge to this region, where they pass through the lamina cribrosa. When viewed from the inside of the eyeball, this area of the retina is seen as a circular area called the optic disc.

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When we examine sections through the retina (stained by hematoxylin and eosin, Fig. 23.2), a number of layers can be distinguished. The significance of the layers becomes apparent, however, only if we study the retina using special methods. A highly schematic presentation of the layers of the retina, and of the cells present in them is shown in Figure 23.6. The retina can be said to have an external surface that is in contact with the choroid, and an internal surface that is in contact with the vitreous.
From outside to inside, retina consists of following ten layers:
- 1. Pigment cell layer: It is the outermost layer of retina which is separated choroid by Bruch's membrane. This consists of a single layer of low cub cells containing melanin pigment. Processes from pigment cells extend the next layer.
Fig. 23.6: Layers of the retina and the main structures therein (schematic representation).
This layer performs the following functions:
"It absorbs and prevents back reflection of light that has passed through 1 neural layers of the retina.
"The pigment cells phagocytose the shed membranous discs of the ou segment of rods and cones.
" These cells also produce melanin.
"They may play a role in regular spacing of rods and cones and may prov. mechanical support to them.
"They have a phagocytic role. They "eat up" the ends of rods and cor which are constantly.
Pigment cells appear to be rectangular in vertical section, their width being greater than their height (Fig. 23.7). In surface view, they are hexagonal. The nucleus is basal in position. The pigment in the cytoplasm is melanin. With the EM, it can be seen that the surface of the cell shows large microvilli that contain pigment. These microvilli project into the intervals between the processes of rods and cones. Each pigment cell is related to about a dozen rods and cones. The plasma membrane at the base of the cell shows numerous infoldings.

2. Layer of rods and cones: The rods are processes of rod cells, and cone processes of cone cells. The peripheral process is rod-shaped in the ca rod cells, and cone-shaped in the case of cone cells.
3. Outer limiting membrane or lamina: Between second and third layer, is presence of a pink linear marking called as outer limiting membrai lamina. This results because of zonula adherens of the glial cells (M cells) with the cell bodies of photoreceptor cells. The Miiller cells supporting cells of retina. They have long slender body that is rac oriented in retina.
4. External nuclear layer: The external nuclear layer contains the cell bi and nuclei of rod cells and of cone cells.
Fig. 23.7: Some features of a pigment cell of the retina (schematic representation).

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"These cells are photoreceptors that convert the stimulus of light into ne) impulses.
"Each rod cell or cone cell can be regarded as a modified neuron. It consi
of a cell body, a peripheral (or external) process, and a central (or intern process. The peripheral processes lie in the layer of rods and cor described above. The nuclei of these cells are arranged in several layers the form of external nuclear layer. This layer is darkly stained. The cent process of each rod cell or cone cell is an axon. It extends into the exten plexiform layer where it synapses with dendrites of bipolar neurons.
"There are about 7 million cones in each retina. The rods are far m numerous. They number more than 100 million. The cones respond best bright light (photopic vision). They are responsible for sharp vision and the discrimination of color. Rods can respond to poor light (scotopic visic and especially to movement across the field of vision.
"Each rod is about 50 pm in length and about 2 pm thick. Cones are abi 40 um in length and 3—5 pm thick (Fig. 23.8).
" The pigment in the rods is rhodopsin, and that in the cones is iodopsin.

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The ultrastructure of rod cells and of cone cells is similar and is, therefore, considered together.
Fig. 23.8: The main parts of rods and cones (schematic representation).
Each rod or cone cell consists of a cell body containing the nucleus, and external and internal processes, an inner fiber and spherule (Fig. 23.9). T parts of cone cells are almost same except the terminal part which is cal pedicle instead of spherule.
"The cell body (lying in the external nuclear layer) gives off two "fiber inner and outer. The outer fiber passes outward up to the external limiti membrane and becomes continuous with the rod process, or the cc process.
"The process itself can be divided into an inner segment, and an ou segment. The outer segment is the real photoreceptor element. It contain large number of membranous discs stacked on one another. It is belie that the discs are produced by the cilium (see below) and gradually mc toward the tip of the outer segment. Here, old discs are phagocytosed pigment cells.
"The outer segments of rods and cones contain photosensitive pigments t! are concerned with the conversion of light into nerve impulses. T pigments are believed to be bound to the membranes of the sacs of the ou segments.
™Cones are believed to be of three types, red sensitive, green sensitive, a blue sensitive. Iodopsin has, therefore, to exist in three forms, one for e< of these colors. However, the three types of cones cannot be distinguist from one another on the basis of their ultrastructure.
Fig. 23.9: Structure of a rod cell as seen by electron microscope (schematic representation).

5. External plexiform layer: The external plexiform layer (or outer syn zone) consists only of nerve fibers that form a plexus. The axons of rod: cones synapse here with dendrites of bipolar neurons and horizontal « This layer stains lightly.
6. Internal nuclear layer: The internal nuclear layer contains the cell bi and nuclei of three types of neurons:
i. Bipolar cells: They give off dendrites that enter the external plexifc layer to synapse with the axons of rod and cone cells, and axons that er the internal plexiform layer where they synapse with dendrites of gang] cells. The bipolar cells are oriented perpendicular to the layers of retina.

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*The inner segment of the rod or cone process is wider than the outer segment. It contains a large number of mitochondria that are concentrated in a region that is called the ellipsoid.
* At the junction of the inner and outer segments of the rod or cone process, there is an indentation of the plasma membrane on one side, so that the connection becomes very narrow. This narrow part contains a fibrillar cilium in which the microfibrils are orientated as in cilia elsewhere. This cilium is believed to give rise to the flattened discs of the outer segment.
*The part of the rod cell between the cell body and the external limiting membrane is the outer fiber. The length of the outer fiber varies from rod to rod, being greatest in those rods that have cell bodies placed "lower down" in the external nuclear layer. The outer fiber is absent in cones, the inner segment of the cone process being separated from the cone cell body only by a slight constriction.
* The cell bodies of rod cells and of cone cells show no particular peculiarities of ultrastructure.
*The inner fibers of rod and cone cells resemble axons. At its termination, each rod axon expands into a spherical structure called the rod spherule, while cone axons end in expanded terminals called cone pedicles (Figs. 23.10 and 23.11). The rod spherules and cone pedicles form complex synaptic junctions
with the dendrites of bipolar neurons, and with processes of horizontal cells. Each rod spherule synapses with processes of two bipolar neurons, and with processes of horizontal neurons. *Each cone pedicle has numerous synapses with processes from one or more bipolar cells, and with processes of horizontal cells. In many situations, the cone pedicle bears several invaginations that are areas of synaptic contacts. Each such area receives one process from a bipolar dendrite, and two processes, one each from two horizontal neurons. Such groups are referred to as triads. Each cone pedicle has 24 such triads. Apart from triads, the cone pedicle bears numerous other synaptic contacts in areas intervening between the triads. These areas synapse with dendrites of diffuse bipolar cells. Some pedicles also establish synaptic contacts with other cone pedicles.

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ii. Horizontal cells: They give off processes that run parallel to the reti surface. These processes enter the outer plexiform layer and synapse w rods, cones, and dendrites of bipolar cells. The horizontal cells oriented parallel to the layers of retina. Horizontal neurons are of two types, rod horizontals and cc horizontals, depending on whether they synapse predominantly with ri or cones. Each horizontal cell gives off one long process, and a numbel short processes (7 in case of rod horizontal cells, and 10 in case of c horizontal cells). The short processes are specific for the type of c those of rod horizontals synapse with a number of rod spherules, ; those of cone horizontals synapse with cone pedicles. The long proces synapse with both rods and cones (which are situated some distance av from the cell body of the horizontal neuron). The long and short proces of horizontal cells cannot be distinguished as dendrites or axons, and e process probably conducts in both directions.
Fig. 23.10: Rod spherule synapsing with terminals of rod bipolar cells and horizontal cells (schematic representation).
Fig. 23.11: Cone pedicle showing a number of synaptic areas, each area receiving three terminals
the inner plexiform layer where they synapse with axons of bipolar ce and with dendrite of ganglion cells.
The term amacrine is applied to neurons that have no true axon. L the processes of horizontal cells, those of amacrine neurons also cond impulses in both directions. Each cell gives off one or two th processes that divide further into a number of branches. The amacr cells are believed to play a very important role in the interaction betw adjacent areas of the retina resulting in production of sharp images. T] are also involved in the analysis of motion in the field of vision.

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Miiller cells: Apart from bipolar, horizontal, and amacrine neurons, the internal nuclear layer also contains the nuclei of retinal gliocytes or cells of Miller (Fig. 23.6). These cells give off numerous protoplasmic processes that extend through almost the whole thickness of the retina. Externally, they extend to the junction of the layer of rods and cones with the external nuclear layer. Here, the processes of adjoining gliocytes meet to form a thin external limiting membrane. Internally, the gliocytes extend to the internal surface of the retina where they form an internal limiting membrane. The retinal gliocytes are neuroglial in nature. They support the neurons of the retina and may ensheath them. They probably have a nutritive function as well.
7. Internal plexiform layer: The internal plexiform layer (or inner syn zone) consists of synapsing nerve fibers. The axons of bipolar cells syr with dendrites of ganglion cells, and both these processes synapse processes of amacrine cells. The internal plexiform layer also contains : horizontally placed internal plexiform cells, and also a few ganglion cell
8. Layer of ganglion cells: The layer of ganglion cells contains the cell br of ganglion cells. The dendrites of these cells enter the internal plexi layer to synapse with processes of bipolar cells and of amacrine cells. ganglion cell gives off an axon that forms a fiber of the optic nerve.
9. Layer of optic nerve fibers: The layer of optic nerve fibers is made 1 axons of ganglion cells. The fibers converge on the optic disc where pass through foramina of the lamina cribrosa to enter the optic nerve.
10. Internal limiting membrane: The processes of adjoining Miiller cells e> to the internal surface of the retina to form an internal limiting membi This membrane separates the retina from the vitreous.

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* The inner and outer nuclear layers can be made out even at low magnification.
* The outer nuclear layer is thicker, and the nuclei in it are more densely packed than in the inner nuclear layer. This (outer nuclear) layer contains the nuclei of rods and cones. The cone nuclei are oval and lie in a single row adjoining the layer of rods and cones. The remaining nuclei are those of rods.
* The nuclei in the inner nuclear layer belong to bipolar cells, horizontal cells, amacrine cells, and gliocytes.
* The layer of ganglion cells is (at most places) made up of a single row of cells of varying size. The cell outlines are indistinct, but the nuclei can be made out. They are of various sizes. On the whole, they are larger and stain more lightly than nuclei in the inner and outer nuclear layers.
* The layer of pigment cells resembles a low cuboidal epithelium. All the nuclei in this layer are of similar size, and lie in a row.
* The remaining layers (layers of rods and cones, inner and outer plexiform layers, and the layer of optic nerve fibers) are seen as light staining areas in which no detail can be made out. The layer of rods and cones may show vertical striations.

The blood vessels that ramify in the retina do not supply the rods and cones. These are supplied by diffusion from choroidal vessels. The endothelial cells of capillaries in the retina are united by tight junctions to prevent diffusion of substances into the rods and cones. This is referred to as the blood-retinal barrier.

The lens of the eye is a transparent biconvex avascular structure. It is suspended between the iris and the vitreous by the zonules, which connect the lens with the ciliary body. It is surrounded by an elastic capsule which is a semipermeable membrane. The posterior surface of the lens is more curved than the anterior surface.

1. Lens capsule
2. Lens epithelium
3. Lens substance (Fig. 23.12).

'Made up of ten layers: Pigment epithelium, layer of rods and cones, external limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglionic cell layer, nerve fiber layer, and internal limiting membrane 'Outer pigment epithelium is made up of simple cuboidal epithelium 'The outer nuclear layer is thicker with densely packed nuclei than in the inner nuclear layer 'Ganglion cell layer is made up of a single row of cells of varying size.
Fig. 23.12: Section through part of the lens near its margin (schematic representation).

* Tt is a transparent, homogeneous, and highly elastic collagenous basement membrane. It is made up mainly of type IV collagen and glycoproteins.
* The capsule is thicker in front than behind.
* Tt is secreted by the lens epithelium.

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* Deep to the capsule, the lens is covered on its anterior surface by a lens epithelium.
* The cells of the epithelium are cuboidal. However, toward the periphery of
the lens, the cells become progressively longer. Ultimately, they are converted into long fibers that form the substance of the lens.
* The cells of epithelium are metabolically active, contain Nat-K+-ATPase, and generate adenosine triphosphate (ATP) to meet the energy demand of the lens.
* The cells show high mitotic activity and form new cells which migrate toward the equator. The lens epithelial cells continue to divide and develop into the lens fibers.

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* The lens fibers develop from the lens epithelial cells that continue to divide and get elongated and transformed into lens fibers.
* They are mainly composed of soluble proteins called crystallins.
* The fibers formed earlier lie in the deeper plane (nucleus of the lens), the newer ones occupy a more superficial plane.
Fig. 23.13: Arrangement of fibers within the lens. Note the Y-shaped lines on the front and back of the lens (schematic representation).
- * When the lens is examined from the front, or from behind, three faint lines
are seen radiating from the center to the periphery. In the fetus, these lines form a "Y" that is upright on the front of the lens, and inverted at the back (Fig. 23.13). The lines become more complex in the adult. These lines are called sutural lines. They are made up of amorphous material. The ends of lens fibers are attached at these lines. Each lens fiber starts on one surface at such a line, and follows a curved course to reach the opposite surface where it ends by joining another such line.
i

The accessory visual organs include the extraocular muscles and related fascia, the eyebrows, the eyelids, the conjunctiva, and the lacrimal gland.

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Eyelids are two movable skin folds that protect the eye from injury and keep the cornea clean and moist.
The basic structure of an eyelid is shown in Figure 23.14.
* Anteriorly, there is a layer of true skin with which a few small hair and sweat glands are associated. The skin is thin.
* Deep to the skin, there is a layer of delicate connective tissue that normally does not contain fat.
* Considerable thickness of the lid is formed by fasciculi of the palpebral part of the orbicularis oculi muscle (skeletal muscle).
* The "skeleton" of each eyelid is formed by a mass of fibrous tissue called the tarsus, or tarsal plate.
* On the deep surface of the tarsal plate, there are a series of vertical grooves in which tarsal glands (or meibomian glands) are lodged. Occasionally, these glands may be embedded within the tarsal plate. Each gland has a duct that opens at the free margin of the lid. The tarsal glands are modified sebaceous glands. They produce an oily secretion, a thin film of which spreads over the lacrimal fluid (in the conjunctival sac) and delays its evaporation.
* Modified sweat glands, called ciliary glands (or glands of Moll), are present in the lid near its free edge. Sebaceous glands present in relation to eyelashes constitute the glands of Zeis. They open into hair follicles. Accessory lacrimal glands are often present just above the tarsal plate (glands of Wolfring).
* The inner surface of the eyelid is lined by the palpebral conjunctiva.
Fig. 23.14: Eyelid (schematic representation).

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- * The conjunctiva is a thin transparent membrane that covers the inner
surface of each eyelid (palpebral conjunctiva) and the anterior part of the sclera (ocular conjunctiva).
* At the free margin of the eyelid, the palpebral conjunctiva becomes continuous with skin, and at the margin of the comea, the ocular conjunctiva becomes continuous with the anterior epithelium of the cornea.
* When the eyelids are closed, the conjunctiva forms a closed conjunctival sac. The line along which palpebral conjunctiva is reflected onto the eyeball is called the conjunctival fornix: superior, or inferior.
* Conjunctiva consists of an epithelial lining that rests on connective tissue. Over the eyelids, this connective tissue is highly vascular and contains much lymphoid tissue. It is much less vascular over the sclera.
* The epithelium lining the palpebral conjunctiva is typically two layered. There is a superficial layer of columnar cells, and a deeper layer of flattened cells. At the fornix, and over the sclera, the epithelium is three layered; there being an additional layer of polygonal cells between the two layers mentioned above. The three-layered epithelium changes to stratified squamous at the sclerocomeal junction.
Fig. 23.15: Lacrimal gland (schematic representation).

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- * The lacrimal gland is a compound tubuloalveolar gland and consists of a number of lobes that drain through about 20 ducts. It is a tear-secreting gland.
The structure of the lacrimal gland is similar to that of a serous salivary gland (Fig. 23.15). But it can be distinguished from those of serous salivary glands because of the following features:
"The acini are larger, and have wider lumina.
" All cells appear to be of the same type. They are low columnar in shape a stain pink with hematoxylin and eosin.
" The profiles of the acini are often irregular or elongated.
"The walls of adjacent acini within a lobule may be pressed together, th being very little connective tissue between them. However, the acini different lobules are widely separated by connective tissue. Myoepithel cells are present as in salivary glands.
* Small ducts of the lacrimal gland are lined by cuboidal or columnar epithelium. Larger ducts have a two-layered columnar epithelium or a pseudostratified columnar epithelium.
* Electron microscope studies on the human lacrimal gland reveal that the secretory cells may be of several types, including both mucous and serous cells.
* The ducts of the lacrimal gland open into the lateral part of the superior conjunctival fornix. Lacrimal fluid keeps the conjunctiva moist.
* Accessory lacrimal glands are present near the superior conjunctival fornix (glands of Krause).

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The density of rods and cones in different parts of the retina is shown schematically in Figure 23.16. Note the following points:
- *The density of cones is greatest in the fovea (about 1.5 million/mmz2). Their density decreases sharply in proceeding to
the margin of the central area, but thereafter the density is uniform up to the ora serrata (about 5,000/mmg).
*The density of rods is greatest at the margin of the central area (about 1.5 million/mmg2). It decreases sharply on proceeding
toward the margin of the central area. There are no rods in the foveola. The density of rods also decreases in passing toward the

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Bipolar cells of the retina are of various types. The terminology used for them is confusing as it is based on multiple criteria. The main points to note are as follows:
*The primary division is into bipolars that synapse with rods (rod bipolars), and those that synapse with cones (cone bipolars).
*As there are three types of cones, responding to the colors red, green, and blue, we can distinguish three corresponding types of cone bipolars (red cone bipolar, green cone bipolar, and blue cone bipolar).
*When a photoreceptor (rod or cone) is exposed to light, it releases neurotransmitter at its synapse with the bipolar cell. Some bipolars respond to neurotransmitter by depolarization (and secretion of neurotransmitter at their synapses with ganglion cells). These are called ON bipolars as they are "switched on" by light. Other bipolars respond to release of neurotransmitter by hyperpolarization. In other words, they are "switched off" by light and are called OFF bipolars.
*On the basis of structural characteristics, and the synapses established by them, cone bipolars are divided into three types: midget, blue cone, and diffuse.
*A midget bipolar establishes synapses with a single cone (whic may be red or green sensitive). Some midget bipolars synaps with indented areas on cone pedicles forming triads (Fig. 23.11 These are ON bipolars. Other midget bipolars establish "fla synapses with the cone pedicle (and are also referred to as flé bipolars). These are OFF bipolars.
*A blue cone bipolar connects to one blue cone, and establishe triads. It may be of the ON or OFF variety.
*Diffuse cone bipolars establish synapses with several con pedicles. They are not color specific.
Axons of rod bipolar neurons synapse with up to four ganglion cells, but those of one midget bipolar neuron synapse with only one (midget) ganglion cell, and with amacrine neurons.

*The dendrites of ganglion cells synapse with axons of bipolar
cells, and also with processes of amacrine cells. The axons arising from ganglion cells constitute the fibers of the optic nerve. *Ganglion cells are of two main types. Those that synapse with only one bipolar neuron are monosynaptic, while those that synapse with many bipolar neurons are _ polysynaptic. Monosynaptic ganglion cells are also called midget ganglion cells. Each of them synapses with one midget bipolar neuron. We have seen that midget bipolars in turn receive impulses from a single cone. This arrangement is usual in the central region of the retina, and allows high resolution of vision to be attained.
*Polysynaptic ganglion cells are of various types. Some of them synapse only with rod bipolars (rod ganglion cells). Others have very wide dendritic ramifications that may synapse with several 100 bipolar neurons (diffuse ganglion cells). This arrangement allows for summation of stimuli received through very large numbers of photoreceptors facilitating vision in poor light. On physiological grounds, ganglion cells are also classified as "ON" or "OFF" cells.

Horizontal neurons establish numerous connections between photoreceptors (Fig. 23.17). Some of them are excitatory, while others are inhibitory. In this way, these neurons play a role in integrating the activity of photoreceptors located in adjacent parts of the retina. As they participate in synapses between photoreceptors and bipolar neurons, horizontal neurons may regulate synaptic transmission between these cells.

Different types of amacrine neurons are recognized depending upon the pattern of branching. We have seen that the processes of amacrine neurons enter the internal plexiform layer where they may synapse with axons of several bipolar cells, and with the dendrites of several ganglion cells (Fig. 23.18). They also synapse with other amacrine cells. At many places, an amacrine process synapsing with a ganglion cell is accompanied by a bipolar cell axon. The two are referred to as a dyad.
Internal plexiform cells (present in the internal plexiform layer) represent a third variety of horizontally oriented neurons in the retina.

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*Apart from integration of impulses from rods and cones, horizontal, amacrine, and internal plexiform cells act as "gates" that can modulate passage of inputs from rods and cones to ganglion cells. In this connection, it is to be noted that processes of amacrine neurons are interposed between processes of bipolar cells and ganglion cells, while processes of horizontal
cells are interposed between photoreceptors and bipolar cells.
*While there are well over a 100 million photoreceptors in each retina, there are only about 1 million ganglion cells, each giving origin to one fiber of the optic nerve (the bipolar cells are intermediate in number between photoreceptors and ganglion cells). In passing from the photoreceptors to the ganglion cells, there has, therefore, to be considerable convergence of impulses (Fig. 23.19). Each ganglion cell would be influenced by impulses originating in several photoreceptors. On _ functional considerations, it would be expected that such convergence would be most marked near the periphery of the retina, and that it would involve the rods much more than the cones. It has been estimated that in the peripheral parts of the retina, one ganglion cell may be connected to as many as 300 rods or to 10 cones. Convergence leads to summation of impulses arising in many photoreceptors and allows vision even in very dim light. It would also be expected that convergence would be minimal in the macula, and absent in the foveola to allow maximal resolution.
*The second highly important fact about intraretinal connections is the presence of numerous arrangements for interaction of adjacent regions of the retina as follows:
*Firstly, cone pedicles establish numerous contacts with othe cone pedicles and with adjacent rod spherules.
*Except in the fovea, most photoreceptors are connected to mon than one bipolar cell. In turn, each bipolar cell is usual connected to more than one ganglion cell.
*The vertically arranged elements of the retina (photoreceptor; bipolar cells, and ganglion cells) are intimately interconnected t adjacent elements through horizontal neurons and amacrin neurons.

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*When no light falls on the retina, photoreceptors are depolarized. Exposure to light causes hyperpolarization.
*When a photoreceptor is depolarized, it releases inhibitor at its junction with a bipolar neuron. This prevents the bipolar neuron
from firing. Release of inhibitor is controlled by voltage-gated calcium channels.
- *Hyperpolarization of photoreceptor, caused by exposure to light, leads to closure of Ca++ gates and release of inhibitor is
stopped. This causes the bipolar neuron to fire. As explained earlier, this description applies to ON bipolars.
- *Rhodopsin, present in photoreceptors, is a complex of a protein opsin and cis-retinal that is sensitive to light. When exposed to light, cis-retinal is transformed to trans-retinal. This leads to decrease in concentration of cyclic GMP that in turn leads to closure of sodium channels. Closure of sodium channels results in hyperpolarization of photoreceptor (Elowchart 23.1).
Fig. 23.16: Scheme to show the relative number of rods and cones in different parts of the retina. The figures represent number of receptors per mm 2 scale. . The diagram is not drawn to
Fig. 23.17: Connections of a cone horizontal neuron (schematic representation).
Fig. 23.18: Connections of an amacrine neuron (schematic representation).
Fig. 23.19: How impulses arising in several photoreceptors concentrate on one ganglion cell

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Corneal ulcer: It is an inflammatory or infective condition of the cornea involving disruption of epithelial layer and corneal stroma.
Keratitis: It is an inflammation of cornea, may be or may not be associated with infection. Noninfectious keratitis may be due to minor injuries or contact lens, long wear, or foreign body.
Corneal opacity: It occurs when the cornea becomes scarred. This stops light from passing through the cornea to the retina and may cause the cornea to appear white or clouded over.
Corneal abrasion: The loss of the surface epithelial layer of the eye's cornea as a result of trauma to the surface of the eye.
Corneal neovascularization: Excessive ingrowth of blood vessels from the limbal vascular plexus into the cornea, caused by deprivation of oxygen from the air.
Keratoconus: A degenerative disease, the cornea thins and changes shape to be more like a cone.
Wilson's disease: Copper pigment deposited in the Descemet's membrane.

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* Retinal detachment: It is the separation of the neurosensory retina from the retinal pigment epithelium. It may occur spontaneously in older individuals past 50 years of age or may be secondary to trauma in the region of head and neck. There are three pathogenetic mechanisms of retinal detachment:
1. Pathologic processes in the vitreous or anterior segment
Collection of serous fluid in the subretinal space N
3. Accumulation of vitreous under the retina through a hole or a tec retina.
, ate '= Retinitis pigmentosa: It is a group of systemic and ocular diseases of unknown etiology, characterized by degeneration of the retinal pigment epithelium. The earliest clinical finding is night blindness due to loss of rods and may progress to total blindness.
Retinoblastoma: This is the most common malignant ocular tumor in children. It may be present at birth or recognized in early childhood before the age of 4 years. About 60% cases of retinoblastoma are sporadic and the remaining 40% are familial. Familial tumors are often multiple and multifocal and transmitted as an autosomal dominant trait by retinoblastoma gene (Rb gene) located on chromosome 13. Such individuals have a higher incidence of bilateral tumors and have increased risk of developing second primary tumor, particularly osteogenic sarcoma. Clinically, the child presents with leukokoria, i.e. white pupillary reflex. , ate '=

Cataract: It is the opacification of the normally crystalline lens which leads to gradual painless blurring of vision. The various causes of cataract are: Senility, congenital (e.g. Down syndrome, rubella, galactosemia), traumatic (e.g. penetrating injury, electrical injury), metabolic (e.g. diabetes, hypoparathyroidism), drug-associated (e.g. long-term corticosteroid therapy), smoking, and heavy alcohol consumption.

* Stye or "external hordeolum" is an acute suppurative inflammation of the sebaceous glands of Zeis, the apocrine glands of Moll, and the eyelash follicles.
* Chalazion is a very common lesion and is the chronic inflammatory process involving the meibomian glands. It occurs as a result of obstruction to the drainage of secretions. The inflammatory process begins with destruction of meibomian glands and duct and subsequently involves tarsal plate.

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a. Choroid b. Ciliary body
c. Iris d. Cornea
- 2. What type of neurons is present in the retina?
a. Unipolar b. Pseudounipolar
c. Bipolar d. Multipolar

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Epithelium
- Substantia propria
Endothelium Descemet's membrane ao"
- 4. Which of the following is the receptor for color?
a. Rods b. Cones
c. Bipolar cells
d. Ganglion cells
5. Nutrition of cornea are all, except:
a. Lacrimal fluid on the surface of cornea
b. Aqueous humor in anterior chamber
c. Blood vessels at sclerocorneal junction
d. Central artery of retina
6. All of the following are the functions of pigment layer, except:
2009 Convert light to electrical energy Provide mechanical support to them
It absorbs and prevents back reflection of light
They have a phagocytic role
7. Cell that form the internal limiting membrane is:
2050 Bipolar Amacrine
Ganglion
Muller

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*Parts of ear
*Components and structure of external ear
*Components of middle ear
* Bony labyrinth and its parts
*Membranous labyrinth and its parts
* Specialized end organs in membranous labyrinth
*Mechanism of hearing

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The pinna has a core of elastic cartilage covered on both sides by true skin in which hair follicles and sweat glands are seen., Cochlea = The cochlea is embedded in the petrous temporal bone. , Cochlea = It is in the form of a spiral canal and is, therefore, cut up six times. , Cochlea = The cone-shaped mass of bone surrounded by these turns of the cochlea is called the modiolus which contains a canal through which fibers of the cochlear nerve pass
A mass of neurons belonging to the spiral ganglion lies to the inner side of each turn of the cochlea
Fhe parts to be identified in each turn of the cochlea are the scala vestibuli, scala media, the scala tympani, the vestibular membrane, the basilar membrane, the membrana tectoria, and the organ of Corti, and the spiral lamina
®uter wall of the cochlear turn is the spiral ligament and it is lined by avascularized epithelium (stria vascularis).
Bf

* Ear is the peripheral sense organ concerned with hearing and equilibrium.
* Anatomically speaking, the ear is made up of three main parts called the external ear, the middle ear, and the internal ear.
* The external and middle ears are concerned exclusively with hearing. The internal ear has a cochlear part concerned with hearing, and a vestibular part which provides information to the brain regarding the position and movements of the head.
The main parts of the ear are shown in Figure 24.1.

* The external ear consists of auricle or pinna and external acoustic meatus (external auditory canal).
* The part of the ear that is seen on the surface of the body (i.e. the part that the lay person calls the ear) is anatomically speaking, the auricle or pinna. Leading inward from the auricle, there is a tube called the external acoustic meatus.
* The inner end of the external acoustic meatus is closed by a thin membranous diaphragm called the tympanic membrane. This membrane separates the external acoustic meatus from the middle ear.

* The auricle consists of a thin plate of elastic cartilage covered on both sides by true skin (Plate 24.1).
* The skin is closely adherent to the cartilage on its lateral surface while it is comparatively loose on medial surface. Epithelium is squamous

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The pinna has a core of elastic cartilage covered on both sides by true skin in which hair follicles and sweat glands are seen.
* External auditory canal (EAC) measures about 24 mm and extends from the concha to the tympanic membrane.
* EAC is usually divided into two parts: (1) cartilaginous and (2) bony. Its outer one-third (8 mm) is cartilaginous and its inner two-thirds (16 mm) is bony.
* Cartilaginous EAC: It is a continuation of the cartilage that forms the framework of the pinna. The skin of the cartilaginous canal is thick and contains hair follicles, ceruminous and pilosebaceous glands that secrete wax. The ceruminous glands secrete the wax of the ear. They are modified sweat glands lined by a columnar, cuboidal, or squamous epithelium.
* Bony EAC: It is mainly formed by the tympanic portion of temporal bone but roof is formed by the squamous part of the temporal bone. Skin of the bony EAC is thin and continuous over the tympanic membrane. Skin is devoid of subcutaneous layer, hair follicles, and ceruminous glands.
* Isthmus: Approximately 6 mm lateral to tympanic membrane, bony EAC has a narrowing called the isthmus.

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* Its dimensions are: 9-10 mm height and 8—9 mm width. It is 0.1 mm thick.
* Tympanic membrane (TM) is a partition wall between the EAC and the middle ear. It is positioned obliquely. It forms angle of 55° with deep EAC.
* Structure: Tympanic membrane consists of the following three layers:
" Outer epithelial layer: It is continuous with the EAC skin.
" Middle fibrous layer: The middle layer is made up of fibrous tissue, whi is lined on the outside by skin (continuous with that of the external acous meatus), and on the inside by mucous membrane of the tympanic cavity.
"The fibrous layer contains collagen fibers and some elastic fibers. T fibers are arranged in two layers. In the outer layer, they are placed radial while in the inner layer, they run circularly.
"Inner mucosal layer: The mucous membrane is lined by an epithelir which may be cuboidal or squamous. It is said that the mucosa over ' upper part of the tympanic membrane may have patches of ciliat columnar epithelium, but this is not borne out by EM studies.
* Otoscopy: Normal tympanic membrane is shiny and pearly gray in color.

* The middle ear is a small space placed deep within the petrous part of the temporal bone. It is also called the tympanum.
* Medially, the middle ear is closely related to parts of the internal ear. It is lined with mucous membrane.
* The cavity of the middle ear is continuous with that of the nasopharynx through a passage called the auditory tube. Within the cavity of the middle ear, there are three small bones or ossicles: the malleus, the incus, and the stapes. They form a chain that is attached on one side to the tympanic membrane, and at the other end to a part of the internal ear.

* The walls of the tympanic cavity are formed by bone which is lined by mucous membrane.
* The mucous membrane also covers the ossicles.
* The lining epithelium varies from region to region.
Typically, it is cuboidal or squamous. At places, it may be ciliated columnar. The ossicles of the middle ear consist of compact bone, but do not have marrow cavities.

* It is a channel connecting the tympanic cavity with the nasopharynx. The length of Eustachian tube (ET) is 36 mm. Its lateral third is bony and medial two-thirds (i.e. 24 mm) is fibrocartilaginous.
* The bone or cartilage is covered by mucous membrane which is lined by ciliated columnar epithelium. Near the pharyngeal end of the tube, the epithelium becomes pseudostratified columnar.
* Goblet cells and tubuloalveolar mucous glands are also present. A substantial collection of lymphoid tissue, present at the pharyngeal end, forms the tubal tonsil.
Bf

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The internal ear is in the form of a complex system of cavities lying within the petrous temporal bone. It has sense organs for both hearing and balance.
* Tt has a central part called the vestibule. Continuous with the front of the vestibule, there is a spiral-shaped cavity called the cochlea. Posteriorly, the vestibule is continuous with three semicircular canals.
* Because of the complex shape of these intercommunicating cavities, the internal ear is also called the labyrinth. It consists of a bony labyrinth contained within the petrous part of temporal bone (Fig. 24.2). The space bounded by bone is bony labyrinth. Its wall is made up of bone that is denser than the surrounding bone. Its inner surface is lined by periosteum. Lying within the bony labyrinth, there is a system of ducts which constitute the membranous labyrinth.
* The space within the membranous labyrinth is filled by a fluid called the endolymph. The space between the membranous labyrinth and the bony labyrinth is filled by another fluid called the perilymph.
Fig. 24.2: Basic structure of internal ear as seen in a section through a semicircular canal (schematic representation).

The bony labyrinth consists of three parts:
2. Semicircular canals
3. The bony cochlea.

Vestibule is the central part (Fig. 24.3). It is continuous anteriorly with the cochlea, and posteriorly with three semicircular canals.

* There are three semicircular canals (SCCs): Lateral (horizontal), posterior, and superior (anterior).
* Each canal occupies two-thirds of a circle and has a diameter of 0.8 mm. They lie in planes at right angles to one another.
* Each canal has two ends: Ampullated and nonampullated. All the three ampullated ends and nonampullated end of lateral SCC open independently and directly into the vestibule. The nonampullated ends of posterior and superior canals join and form a crus commune (4 mm length), which then opens into the medial part of vestibule. So, the three SCCs open into the vestibule by five openings.

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- * The cochlea has a striking resemblance to a snail shell. It is basically a tube that is coiled on itself for two and three-fourth turns. The "turns" rest on a solid core of bone called the modiolus.
Fig. 24.3: Bony labyrinth as seen from the lateral side (schematic representation).
Note: Because of the spiral nature of the cochlea, the mutual relationships of the structures within it differ in different parts of the cochlea. A structure that is "inferior? in the upper part of the canal becomes "superior" in the lower part. For descriptive convenience, the structures lying next to the modiolus are described as "inner" and those away from it as "outer". These terms as used here are not equivalents of "medial" and "lateral" as normally used. The words "superior" and "inferior" indicate relationships as they exist in the lowest (or basal) turn of the cochlea. In sections, through the middle of the cochlea, the cochlear canal is cut up six times as shown in Plate 24.2.
* The cochlear canal is partially divided into two parts by a bony lamina that projects outward from the modiolus. This bony projection is called the spiral lamina.
* Passing from the tip of the spiral lamina to the opposite wall of the canal, there is the basilar membrane.
* The spiral lamina and the basilar membrane together divide the cochlear canal into three parts—scala vestibuli, scala tympani, and scala media (membranous cochlea). The lower most channel is the scala tympani. When traced proximally, the scala tympani opens in the medial wall of the middle ear through an aperture fenestra cochleae (round window), which is closed by the secondary tympanic membrane.
* The part of the cochlear canal above the basilar membrane is further divided into two parts by an obliquely placed vestibular membrane (of
Reissner). The part above the vestibular membrane is the scala vestibuli. When traced proximally, it becomes continuous with the vestibule (Fig. 24.4).

- * At the apex of the cochlea, the scala vestibuli becomes continuous with the scala tympani called helicotrema. Both scala vestibuli and scala tympani are filled with perilymph.
Cochlea: (A) As seen in drawing (low magnification); (B) As seen in drawing (magnified view).

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'The cochlea is embedded in the petrous temporal bone
It is in the form of a spiral canal and is, therefore, cut up six times
The cone-shaped mass of bone surrounded by these turns of the cochlea is called the modiolus which contains a canal through which fibers of the cochlear nerve pass
'A mass of neurons belonging to the spiral ganglion lies to the inner side of each turn of the cochlea
The parts to be identified in each turn of the cochlea are the scala vestibuli, scala media, the scala tympani, the vestibular membrane, the basilar membrane, the membrana tectoria, and the organ of Corti, and the spiral lamina
'Outer wall of the cochlear turn is the spiral ligament and it is lined by avascularized epithelium (stria vascularis).
Fig. 24.4: Structure of cochlear canal to show scala vestibuli and tympani (schematic representation).
* The triangular space between the basilar and vestibular membranes is called the duct of the cochlea. This duct represents the membranous labyrinth of the cochlea and contains endolymph.
* The vestibular membrane consists of a basal lamina lined on either side by squamous cells. Some of the cells show an ultrastructure indicative of a fluid transport function. The cells of the membrane form a barrier to the flow of ions between endolymph and perilymph so that these two fluids have different concentrations of electrolytes.
* The basilar membrane is divisible into two parts. The part supporting the
organ of Corti is the zona arcuata. The part lateral to the zona arcuata is the zona pectinata. The zona arcuata is made up of a single layer of delicate filaments of collagen. The zona pectinata is made up of three layers of fibers.
- * Aqueduct of cochlea: The scala tympani is connected with the subarachnoid space through the aqueduct of cochlea. It is thought to regulate perilymph and pressure in bony labyrinth.

Perilymph fills the space between bony and membranous labyrinth while endolymph fills the entire membranous labyrinth.

* It resembles extracellular fluid and is rich in sodium ions. The aqueduct of cochlea provides communication between scala tympani and subarachnoid space. Perilymph percolates through the arachnoid type connective tissue present in the aqueduct of cochlea.
* Source: There are two theories:
1. Filtrate of blood serum from the capillaries of spiral ligament.
2. CSF reaching labyrinth via aqueduct of cochlea.

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* It resembles intracellular fluid and is rich in potassium ions. Protein and glucose contents are less than in perilymph.
* Source: They are believed to be following:
* Stria vascularis
™ Dark cells of utricle and ampullated ends of semicircular ducts.
* Absorption: There are following two opinions regarding the absorption of endolymph:
1. Endolymphatic sac: The longitudinal flow theory believes that from duct, endolymph reaches saccule, utricle, and endolymphatic duct an absorbed by endolymphatic sac.
2. Stria vascularis: The radial flow theory believes that endolymph is

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* Utricle and saccule within vestibule
* Cochlear duct in the cochlea
* Three semicircular ducts within each semicircular canal
* Endolymphatic duct and sac.
The wall of the membranous labyrinth is trilaminar. The outer layer is fibrous and is covered with perilymphatic cells. The middle layer is vascular. The inner layer is epithelial, the lining cells being squamous or cuboidal. Some of the cells (called dark cells) have an ultrastructure indicative of active ionic transport. They probably control the ionic composition of endolymph.

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* The utricle, which is oblong and irregular, has anteriorly upward slope at an approximate angle of 30°.
* Tt lies in the posterior part of bony vestibule and receives the five openings of the three semicircular ducts. The utricle (4.33 mm2) is bigger than saccule (2.4 mm2) and lies superior to saccule.
* The utricle is connected with the saccule through utriculosaccular duct.
Fig. 24.5: Parts of the membranous labyrinth (schematic representation).
- *Tts sensory epithelium, which is called macula, is concerned with linear acceleration and deceleration.

* The saccule lies anterior to the utricle opposite the stapes footplate in the bony vestibule.
*Tts sensory epithelium, macula, responds to linear acceleration and deceleration. The saccule is connected to the cochlea through the thin reunion duct.

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The cochlear duct is a triangular canal lying between the basilar membrane and the vestibular membrane (Figs. 24.4 to 24.6).
* The endosteum on the outer wall of the cochlear canal is thickened. This thickened endosteum forms the outer wall of the duct of the cochlea. The basilar and vestibular membranes are attached to this endosteum.
* The thickened endosteum shows a projection in the region of attachment of the basilar membrane: This projection is called the spiral ligament.
* A little above the spiral ligament the thickened endosteum shows a much
Fig. 24.6: Structure of the organ of Corti (schematic representation).
larger rounded projection into the cochlear duct: This is the spiral prominence. The spiral prominence forms the upper border of a concavity called the outer spiral sulcus.

* Between the spiral prominence and the attachment of the vestibular membrane, the thickened endosteum is covered by a specialized epithelium that is called the stria vascularis. The region is so called because there are capillaries within the thickness of the epithelium (this is the only such epithelium in the whole body). The epithelium of the stria vascularis is made up of three layers of cells: Marginal, intermediate, and basal.
* The cells of the marginal layer are called dark cells. They are in contact with the endolymph filling the duct of the cochlea. These cells have a structure and function similar to that of the dark cells already described in the planum semilunatum. These dark cells may be responsible for the formation of endolymph. The basal parts of the dark cells give off processes that come into intimate contact with the intraepithelial capillaries. The capillaries are also in contact with processes arising from cells in the intermediate and basal layers of the stria vascularis.
* Spiral lamina is a bony projection into the cochlear canal. Near the attachment of the spiral lamina to the modiolus, there is a spiral cavity in which the spiral ganglion is lodged. This ganglion is made up of bipolar cells. Central processes arising from these cells form the fibers of the cochlear nerve. Peripheral processes of the ganglion cells pass through canals in the spiral lamina to reach the spiral organ of Corti.
* The periosteum on the upper surface of the spiral lamina is greatly thickened to form a mass called the limbus lamina spiralis (or spiral limbus). The limbus is roughly triangular in shape. It has a flat "lower" surface attached to the spiral lamina, a convex "upper" surface to which the vestibular membrane is attached, and a deeply concave "outer" surface. The concavity is called the internal spiral sulcus. This sulcus is bounded above by a sharp vestibular lip and below by a tympanic lip which is fused to the spiral lamina.

The internal ear is a highly specialized end organ that performs the dual functions of hearing and of providing information about the position and movements of the head. The impulses in question are converted into nerve impulses by a number of structures that act as transducers.
* These are spiral organ (of Corti) for hearing and maculae (singular = macula) present in the utricle and saccule for changes in position of the head (Fig. 24.7).
* Information about angular movements of the head is provided by end organs called the ampullary crests (or cristae ampullae). One such crest is present in each semicircular duct. One end of each semicircular duct is dilated to form an ampulla, and the end organ lies within this dilatation. These end organs are described here.

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- * They lie in otolith organs (utricle and saccule). Macula of the utricle is situated in its floor in a horizontal plane in the dilated superior portion of the utricle. Macula of saccule is situated in its medial wall in a vertical plane. The macula utriculi (approximately 33,000 hair cells) are larger than saccular macula (approximately 18,000 hair cells).
Fig. 24.7: End organs in the membranous labyrinth (schematic representation).
- * The striola, which is a narrow curved line in center, divides the macula into two areas. They appreciate position of head in response to gravity and linear acceleration. A macula consists mainly of two parts: A sensory neuroepithelium and an otolith membrane.
1.
Sensory neuroepithelium:
It is
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2
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concentration along
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the utricular
in the
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and whereas
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in saccule,
shape of
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the correction.
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receptors range
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2. Otolithic membrane: The otolithic membrane consists of a gelatinous subgelatinous space, and the crystals of calcium carbonate called (otoconia or statoconia) (Fig. 24.8). The otoconia, which are mull small cylindrical and hexagonally-shaped bodies with pointed ends, of an organic protein matrix together with crystallized calcium ce The otoconia (3-19 rm long) lie on the top of the gelatinous mass. * of hair cells project into the gelatinous layer. The linear, gravitatic head tilt movements result into the displacement of otolithic me which stimulate the hair cells lying in different planes.
* The maculae give information about the position of the head and are organs of static balance. In contrast, the ampullary crests are organs of kinetic balance.
* The macula of the saccule may be concerned with the reception of low frequencies of sound. Impulses arising from the ampullary crests and the maculae influence the position of the eyes. They also have an influence on body posture (through the vestibular nuclei).
Fig. 24.8: Macula of otolith organs utricle and saccule (schematic representation).

* This sensory organ of the hearing is situated on the basilar membrane. It is spread like a ribbon along the entire length of basilar membrane (Figs. 24.4 and 24.6).
* The spiral organ of Corti is so called because (like other structures in the cochlea) it extends in a spiral manner through the turns of the cochlea. It is made up of epithelial cells that are arranged in a complicated manner.
* The cells are divisible into the true receptor cells or hair cells, and supporting elements which are given different names depending on their

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* The cells of the spiral organ are covered from above by a gelatinous mass called the membrana tectoria. It consists of delicate fibers embedded in a gelatinous matrix. This material is probably secreted by cells lining the vestibular lip of the limbus lamina spiralis.
* The tunnel of Corti, which is situated between the inner and outer rod cells and contains a fluid called cortilymph (Fig. 24.9). The base of the tunnel lies over the basilar membrane.
* To the internal side of the inner rod cells, there is a single row of inner hair cells. The inner hair cell is supported by tall cells lining the tympanic lip of the internal spiral sulcus.
* On the outer side of each external rod cell, there are three or four outer hair cells. The outer hair cells do not lie directly on the basilar membrane, but are supported by the phalangeal cells (of Deiters) which rest on the basilar membrane.
* To the outer side of the outer hair cells and the phalangeal cells, there are tall supporting cells (cells of Hensen). Still more externally, the outer spiral sulcus is lined by cubical cells (cells of Claudius).
* A narrow space the cuniculum externum intervenes between the outermost hair cells and the cells of Hensen. A third space, the cuniculum medium (or space of Nuel) lies between the outer rod cell and the outer hair cells. The spaces are filled with perilymph (or cortilymph).

* Each rod cell (or pillar cell) has a broad base (or footplate, or crus) that rests on the basilar membrane, an elongated middle part (rod or scapus), and an expanded upper end called the head or caput.
* The bases of the rod cells are greatly expanded and contain their nuclei. The bases of the inner and outer rod cells meet each other forming the base of the tunnel of Corti.
* The heads of these cells also meet at the apex of the tunnel. Here, a convex prominence on the head of the outer rod cell fits into a concavity on the head of the inner rod cell. The uppermost parts of the heads are expanded into horizontal plates called the phalangeal processes. These processes join similar processes of neighboring cells to form a continuous membrane

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* These important receptor cells of hearing transduce sound energy into electrical energy.
* The hair cells are so called because their free "upper" or apical ends bear a number of "hairs". The hairs are really stereocilia.
* Each cell is columnar or piriform. The hair cells are distinctly shorter than the rod cells.
* Their apices are at the level of the reticular lamina. Their lower ends (or bases) do not reach the basilar membrane. They rest on phalangeal cells. The plasma membrane at the base of each hair cell forms numerous synaptic contacts with the terminations of the peripheral processes of neurons in the spiral ganglion. Some efferent terminals are also present. The apical surface of each hair cell is thickened to form a cuticular plate the edges of which are attached to neighboring cells.
* There are two types of hair cells—inner and outer ('Table 24.1).
1. Inner hair cells: Inner hair cells (IHCs) form a single row and a supplied by afferent cochlear fibers. These are flask-shaped c relatively short (Fig. 24.10). They are very important in the transm: auditory impulses. Their nerve fibers are mainly afferent.
. Outer hair cells: Outer hair cells (OHCs) are arranged in three or ft
Fig. 24.9: The cells in the organ of Corti (schematic representation).
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a

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“W”
(instead of
a
“V”).
Cells numbers, Inner hair cells = 3,500. Cells numbers, Outer hair cells = 12,000. Rows, Inner hair cells = One. Rows, Outer hair cells = Three or four. Shape, Inner hair cells = Flask. Shape, Outer hair cells = Cylindrical. Nerve supply, Inner hair cells = Mainly afferent fibers. Nerve supply, Outer hair cells = Mainly efferent fibers. Development, Inner hair cells = Early. Development, Outer hair cells = Late. Function, Inner hair cells = Transmit auditory stimuli. Function, Outer hair cells = Modulate function of inner hair cells. Ototoxicity, Inner hair cells = More resistant. Ototoxicity, Outer hair cells = More sensitive and easily damaged. High intensity noise, Inner hair cells = More resistanty. High intensity noise, Outer hair cells = More sensitive and easily damaged. Generation of otoacoustic emissions, Inner hair cells = No. Generation of otoacoustic emissions, Outer hair cells = Yes

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* These are the cells that support the outer hair cells.
* They lie lateral to the outer rod cells. Their bases rest on the basilar membrane. Their apical parts have a complicated configuration. The greater part of the apex forms a cup-like depression into which the base of an outer hair cell fits.
* Arisin g from one side (of the apical part) of the cell, there is a thin rod-like
phalangeal process. This process passes "upward", in the interval between hair cells, to reach the level of the apices of hair cells. Here, the phalangeal process expands to form a transverse plate called the phalanx.
Fig. 24.10: Structure of the inner hair cell (schematic representation).
Fig. 24.11: Structure of the outer hair cell (schematic representation).
- * The edges of the phalanges of adjoining phalangeal cells unite with each other to form a membrane called the reticular lamina (the reticular lamina also receives contributions from the heads of hair cells). The apices of hair cells protrude through apertures in this lamina. The cell edges forming the reticular lamina contain bundles of microtubules embedded in dense cytoplasm. Adjacent cell margins are united by desmosomes, occluding junctions, and gap junctions. The reticular lamina forms a_ barrier impermeable to ions except through the cell membranes. It also forms a rigid support between the apical parts of hair cells thus, ensuring that the hair cells rub against the membrana tectoria when the basilar membrane vibrates.

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- * One ampullary crest is present in the ampullated end of each of the three semicircular ducts. Each crest is an elongated ridge projecting into the ampulla, and reaching almost up to the opposite wall of the ampulla. The long axis of the crest lies at right angles to that of the semicircular duct.
The crest is lined by a columnar epithelium in which two kinds of cells are present. These are hair cells which are specialized mechanoreceptors, and supporting (or sustentacular) cells.
* Hair cells: The hair cells occupy only the upper half of the epithelium. The luminal surface of each hair cell bears "hairs". When examined by EM, the "hairs" are seen to be of two types as follows:
1. There is one large kinocilium which is probably nonmotile.
2. There are a number of stereocilia (large microvilli).
* These "hair" extend into a gelatinous (protein polysaccharide) material which covers the crest and is called the cupula. The hair processes of the hair cells are arranged in a definite pattern, the orientation being specific for each semicircular duct. This orientation is of functional importance.
* Each hair cell is innervated by terminals of afferent fibers of the vestibular nerve. Efferent fibers that can alter the threshold of the receptors are also present.
* Hair cells can be divided into two types depending on their shape and on the pattern of nerve endings around them. Type I hair cells (inner hair cell) are flask shaped. They have a rounded base and a short neck. The nucleus lies in the expanded basal part (outer hair cell). The basal part is surrounded by a goblet-shaped nerve terminal (or calix). Type I hair cells are columnar. Both types of hair cells receive nerve terminals which are
Fig. 24.12: Structure of an ampullary crest (schematic representation).
afferent (nongranular) as well as efferent (granular).

*, ~ Both in the ampullae of semicircular ducts, and in the maculae of the utricle and saccule, each hair cell is polarized with regard to the position of the kinocilium relative to the stereocilia. Each hair cell (in an ampulla) can be said to have a side that is toward the utricle, and a side that faces in the opposite direction. In the lateral semicircular duct, the kinocilia lie on the side of the cells which are toward the utricle, while in the anterior and posterior semicircular ducts, the kinocilia lie on the opposite side. When stereocilia are bent toward the kinocilium, the cell is hyperpolarized. It is depolarized when bending is away from the kinocilium. Depolarization depends on the opening up of Ca++ channels.
The supporting (or sustentacular) cells are elongated and may be shaped-like hourglasses (narrow in the middle and wide at each end). They support the hair cells and provide them with nutrition. They may also modify the composition of endolymph.
, ate '= Functioning of ampullary crests: The ampullary crests are stimulated by movements of the head (especially by acceleration). When the head moves, a current is produced in the endolymph of the semicircular ducts (by inertia). This movement causes deflection of the cupula to one side distorting the hair cells. It appears likely that distortion of the crest in one direction causes stimulation of nerve impulses, while distortion in the opposite direction produces inhibition. In any given movement, the cristae of some semicircular ducts are stimulated while those of others are inhibited. Perception of the exact direction of movement of the head depends on the precise pattern formed by responses from the various cristae.

+. od Sound waves traveling through air pass into the external acoustic meatus and produce vibrations in the tympanic membrane. These vibrations are transmitted through the chain of ossicles to perilymph in the vestibule.
+. od In this process, the force of vibration undergoes considerable amplification because: (a) the chain of ossicles acts as a lever, and (b) the area of the tympanic membrane is much greater than that of the footplate of the stapes
(increasing the force per unit area).

* Movement of the stapes (toward the vestibule) sets up a pressure wave in the perilymph. This wave passes from the vestibule into the scala vestibuli, and travels through it to the apex of the cochlea. At this point (called the helicotrema), the scala vestibuli is continuous with the scala tympani. The pressure wave passes into the scala tympani and again traverses the whole length of the cochlea to end by causing an outward bulging of the secondary tympanic membrane.
* In this way, vibrations are set up in the perilymph and through it in the basilar membrane. Movements of the basilar membrane produce forces that result in friction between the "hairs" of hair cells against the membrana tectoria. This friction leads to bending of the "hairs". This bending generates nerve impulses that travel through the cochlear nerve to the brain.
* The presence of efferent terminals on the hair cells probably controls the afferent impulses reaching the brain. It can also lead to sharpening of impulses emanating from particular segments of the spiral organ by suppressing impulses from adjoining areas.
* It has to be remembered that the transverse length of the basilar membrane is not equal in different parts of the cochlear canal. The membrane is shortest in the basal tur of the cochlea, and longest in the apical tum (quite contrary to what one might expect). Different segments of the membrane vibrate most strongly in response to different frequencies of sound thus, providing a mechanism for differentiation of sound frequencies. Low frequency sounds are detected by hair cells in the organ of Corti lying near the apex of the cochlea, while high frequency sounds are detected by hair cells placed near the base of the cochlea. The intensity of sound depends on the amplitude of vibration.

With the EM, the "hairs" of hair cells are seen to be similar to microvilli. Each hair has a covering of plasma membrane within which there is a core of microfilaments. Each hair is cylindrical over most of its length, but it is much narrowed at its base. The hair can, therefore, bend easily at this site. The hair on each hair cell is arranged in a definite manner. When viewed from "above", they are seen to be arranged in the form of the letter "V" or "U". Each limb of the "V" has three rows of hairs. The hairs in the three rows are of unequal height being tallest in the "outer" row, intermediate in the middle row, and shortest in the "inner" row. The "V" formed by the hairs of various hair cells are all in alignment, the apex of the "V" pointing toward the "outer" wall of the cochlear canal. At the point corresponding to the apex of the "V", there is a centriole lying just under the apical cell membrane, but a true kinocilium is not present (unlike hair cells of ampullary crests).
The earlier description applies to both inner and outer hair cells.
The direction of the "V" is of functional importance. Like hair cells of the maculae and cristae, those of the cochlea are polarized. Bending of stereocilia toward the apex of the "Vv" causes depolarization, while the reverse causes hyperpolarization. lonic gradients associated with depolarization and hyperpolarization are maintained because apices of hair cells and surrounding cells are tightly sealed by occluding junctions.

On each side of each ampullary crest, the epithelium of the semicircular duct shows an area of thickened epithelium that is called the planum semilunatum. The importance of this area is that (among other cells) it contains certain dark cells that have an ultrastructure similar to cells (elsewhere in the body) that are specialized for ionic transport. The cells bear microvilli and have deep infoldings of their basal plasma membrane. The areas between the folds are occupied by elongated mitochondria (compare with structure of cells of the distal convoluted tubules of the kidney). The dark cells are believed to control the ionic content of the endolymph. Similar cells are also present elsewhere in the membranous labyrinth. The planum semilunatum may secrete endolymph.
Bi

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* Grafts in rhinoplasty: The conchal cartilage is frequently used to correct depressed nasal bridge. The composite grafts of the skin and cartilage can be used for repair of defects of ala of nose.
* Grafts in tympanoplasty: Tragal and conchal cartilage and perichondrium and fat from lobule are often used during tympanoplasty operations.
* Fissures of Santorini: Transverse slits in the floor of cartilaginous EAC called "fissures of Santorini" provide passages for infections and neoplasms to and from the surrounding soft tissue (especially parotid gland). The parotid and mastoid infections can manifest in the EAC.
* Hair follicles are present only in the outer cartilaginous canal and, therefore, furuncles (staphylococcal infection of hair follicles) are seen only in the cartilaginous EAC.
* Foreign body impacted medial to bony isthmus of EAC is difficult to remove.
* Foramen of MHuschke: In children and occasionally in adults, anteroinferior bony EAC may have a deficiency that is called foramen of
Huschke. Foramen of Huschke permits spread of infections to and from EAC and parotid.
Note: The skin of EAC has a unique self-cleansing mechanism. This migratory process continues from the medial to lateral side. The sloughed epithelium is extruded out as a component of cerumen.
1. Outer epithelial layer of tympanic membrane is lined by this epithelium.
b. Pseudostratified
c. Stratified squamous keratinized d. Stratified squamous nonkeratinized
2. All are parts of membranous labyrinth, except:
a. Vestibule
b. Saccule
c. Semicircular duct
d. Cochlear duct
3. Cochlea is a tube that is coiled on itself for:
a. One and three-fourth
b. Two and three-fourth
c. Three and three-fourth
d. Four and three-fourth
4. Organ of Corti is located on this membrane
a. Vestibular
b. Basilar

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The study of histology is very important for the understanding of the normal functioning of the human body. It also forms the essential basis for the study of the changes in various tissues and organs in disease (This is the science of pathology). From these points of view the study of histology is best done taking one organ system at a time. That is the approach most teachers prefer to take in practical classes of histology. It is also the basis on which the chapters of this book have been organized.
However, in practical examinations, the emphasis is on the ability of the student to recognize a tissue or organ that is being viewed through a microscope. Here it becomes necessary to know how to distinguish between similar looking tissues or organs belonging to different systems. This atlas has been organized to serve this objective. Tissues and organs that have a similar appearance are considered in one lot. For example, if a slide presents something that looks like a tube, whether it be an artery or the ureter or the ductus deferens, these are considered together. This makes the grouping unusual, but this is exactly what the student needs at the time of an examination.
At the same time it is true that an organ can be composed of several tissues (or layers), and the ability to recognize them can go a long way in arriving at a correct diagnosis of the organ being seen. We will, therefore, first try to study and identify the various tissues that make up different organs. We will then have a good basis for identifying any organ that we are required to recognize.
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BASIC TISSUES THAT CAN BE RECOGNIZED IN

The outer surface of the body, and the luminal surfaces of cavities (big or small) lying within the body are lined by one or more layers of cells that completely cover them. Such layers of cells are called epithelia. Epithelial tissue forms the lining of the general body surfaces, passages and cavities within the body. Basement membrane connects the epithelium to the underline subepithelial tissues.
Classification of epithelial tissue is based on shape of the cells, number of cell layers and special modifications seen on the cells. Epithelia may be simple, when they consist of only one layer of cells, or stratified when there are several layers of cells. Epithelial cells may be flat (or squamous), cuboidal, or columnar.
Several types of epithelia can be recognized. Learning to identify an epithelium can be of considerable help in finding out what organ you are seeing.

EPITHELIA AND GLANDS
Simple Squamous Epithelium
Fig. A1.1: An alveolus of the lung showing a lining of simple squamous epithelium (see arrows).
Fig. A1.2: A capillary lined by endothelium (see arrow).

* The cells of this epithelium are flattened in sections. They appear so thin that bulgings are produced on the surface by their nuclei
* In surface view the cells have polygonal outlines that interlock with those of adjoining cells.

Fig. A1.4: Thyroid follicle lined by simple cuboidal epithelium (see arrows).

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* This epithelium is made up of cells that look like squares (in which the length and breadth are equal). Nuclei are rounded
* A typical cuboidal epithelium lines follicles of the thyroid gland, ducts of salivary gland, and tubules of kidney.
Simple Columnar Epithelium
Fig. A1.5: Lining the mucosa of the stomach (see arrows).
* The height of the cells in this epithelium is much greater than their width. The nuclei are oval being elongated in the same direction as the cells. They lie near the bases of the cells. Because of this we see a zone of clear cytoplasm above the nuclei
» A simple columnar epithelium (non-ciliated) lines the mucous membrane of the stomach and of the large intestine.
Columnar Epithelium Showing Striated Border
Fig. A1.6: Columnar epithelium with a striated border in the small intestine (see arrow).
* In some regions the free surfaces of the cells of columnar epithelium show a thickening with vertical striations in it, this is called a striated border
* This is seen typically in the small intestine.
Pseudostratified Ciliated Columnar Epithelium
Fig. A1.7: Pseudostratified ciliated columnar epithelium in trachea (see arrow).
* It is not a true stratified epithelium but appears to be stratified. Normally, in columnar epithelium the nuclei lie in a row, toward the basal part of the cells. Sometimes, however, the nuclei appear to be arranged in two or more layers giving the impression that the epithelium is more than one cell thick
* In some situations, pseudostratified columnar epithelium bears hair-like projections called cilia
* Pseudostratified ciliated columnar epithelium is seen in trachea and in large bronchi.
Stratified Squamous Epithelium
Fig. A1.8: Nonkeratinized stratified squamous epithelium: As seen in esophagus (see arrow).

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® Although this is called stratified squamous epithelium, only the most superficial cells are squamous (flattened)
*The cells in the deepest (or basal) layer are columnar. In the middle layers, they are polyhedral, while the more superficial layers show increasing degrees of flattening
*The nuclei are oval in the basal layer, rounded in the middle layer, and transversely elongated in the superficial layers
* The surface layer shows squamous cells with flattened nuclei
* This kind of epithelium is seen lining some internal organs, like the esophagus or the vagina
*Here the deeper layers are covered by additional layers that represent stages in the conversion of cells into nonliving fibers. This process is called keratinization (or cornification).
Fig. A1.9: Keratinized stratified squamous epithelium.
* The surface layer is made up of keratin which appears as fibers. No cellular outline or nuclei can be seen
- * It is seen typically in epidermis of the skin.
Transitional Epithelium
Fig. A1.10: Transitional epithelium seen at high magnification in ureter (See arrows).
* In this type of epithelium, several layers of round nuclei are seen
* The superficial cells are not flattened but are umbrella shaped
* Their nuclei appear rounded and may show mitotic figures
* This epithelium lines many parts of the urinary tract.
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GENERAL CONNECTIVE TISSUE
Dense Regular Connective Tissue
* Presence of collagen fibers (or fiber bundles) arranged in orderly fashion parallel to each other
Dense Irregular Connective Tissue (Dermis of Skin)
Fig. A2.2: Dense irregular connective tissue as seen in dermis of skin.
* Irregularly arranged bundles of collagen fibers that stain pink. In stretch preparation they are seen in wavy bundles. Other fibers present (elastic, reticular) can be seen only with special stains
Adipose Tissue
Fig. A2.3: Adipose tissue.

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* Presence of fat cells. In routine sections the cells appear empty as the fat in them gets dissolved during preparation of the section giving it a honeycomb appearance
* The cytoplasm of each cell is seen as a pink rim
* The nucleus is flat and lies to one side (eccentric).
CARTILAGE
Hyaline cartilage

Courtesy: Balakrishna Shetty, Sweekritha H Poonja. HISTOLOGY Practical Manual, 4th edition. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2019. p. 18.

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* Hyaline cartilage is characterized by isogenous groups of chondrocytes called as cell nest
* Chondrocytes are surrounded by a homogeneous basophilic matrix which separates the cells widely
* Chondrocytes increase in size from periphery to center
*Near the surface of the cartilage the cells are flattened and merge with the cells of the overlying connective tissue. This connective tissue forms the perichondrium
* Perichondrium displays an outer fibrous and inner cellular layer.

* Perichondrium is present showing an outer fibrous and inner cellular layer.

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* Presence of prominent collagen fibers arranged in bundles with rows of chondrocytes intervening between the bundles
* Perichondrium is absent
* This kind of cartilage can be confused with the appearance of a tendon. However, the chondrocytes in fibrocartilage are rounded but in a tendon, fibrocytes are flattended and elongated.
BONE
Compact Bone (Transverse Section)
Fig. A4.2: Haversian system.
* Haversian system made up of concentric lamellae, lacunae with osteocytes, Haversian (central) canal containing blood vessels and nerves are seen
* Three types of lamellae are seen—(1) circumferential, (2) concentric, and (3) interstitial
* Canaliculi radiate from lacunae.
Compact Bone (Longitudinal Section)
Fig. A4.3: Compact bone: Transverse section.
- * Volkmann's canal interconnecting Haversian systems is seen
*
Lacunae with osteocytes.
Hl
MUSCULAR TISSUE
Longitudinal Section Through Skeletal Muscle
Fig. A5.1: Longitudinal section through skeletal muscle (low power).
* In a longitudinal section through skeletal muscle, the fibers are easily distinguished as they show characteristic transverse striations
* The fibers are long and parallel without branching
* Many flat nuclei are placed at the periphery
* The muscle fibers are separated by some connective tissue.
Transverse Section Through Skeletal Muscle
Fig. A5.2: Transverse section through skeletal muscle.

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* epimysium: Connective tissue sheath of muscle (not seen in photomicrograph)
* perimysium: Connective tissue covering of each fascicle
* endomysium: Loose connective tissue surrounding each muscle fiber.
Cardiac Muscle
Fig. A5.3: Cardiac muscle.

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* A clear space called perinuclear halo is seen around the nucleus
® Fibers show branching

* Loose connective tissue is seen above and below the layers of muscle.

* The smooth muscle fibers are spindle-shaped cells with tapering ends
* The nucleus is elongated and centrally placed
* No striations are seen.

* The spindle-shaped cells are cut at different places along the length resulting in various shapes and sizes of the cells
* The nucleus is seen in those cells which are cut through the center. Others do not show nuclei.
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* A thin capsule surrounds the lymph node and sends in trabeculae
* Just beneath the capsule a clear space is seen. This is the subcapsular sinus
* A lymph node has an outer cortex and an inner medulla
* The cortex is packed with lymphocytes. A number of rounded lymphatic follicles (or nodules) are present. Each nodule has a pale staining germinal center surrounded by a zone of densely packed lymphocytes
* Within the medulla the lymphocytes are arranged in the form of anastomosing cords. Several blood vessels can be seen in the medulla.
Note: All lymphoid tissue are easily recognized due to presence of aggregation of dark staining nuclei. The nuclei belong to lymphocytes.

*The spleen is characterized by a thick capsule with trabeculae extending from it into the organ (not shown in photomicrograph)
*The substance of the organ is divisible into the red pulp in which there are diffusely distributed lymphocytes and numerous sinusoids; and the white pulp in which dense aggregations of lymphocytes are present. The latter are in the form of cords surrounding arterioles
* When cut transversely the cords resemble the lymphatic nodules of lymph nodes, and like them they have germinal centers surrounded by rings of densely packed lymphocytes. However, the nodules of the spleen are easily distinguished from those of lymph nodes because of the presence of an arteriole in each nodule
* This arteriole occupies an eccentric position in the nodule.

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*The thymus is made up of lymphoid tissue arranged in the form of distinct lobules. The presence of this lobulation enables easy distinction of the thymus from all other lymphoid organs
* The lobules are partially separated from each other by connective tissue septae
* In each lobule an outer darkly stained cortex (in which lymphocytes are densely packed); and an inner lightly stained medulla (in which the cells are diffuse) are present
* Whereas the cortex is confined to one lobule, the medulla is continuous from one lobule to another
* The medulla contains pink staining rounded masses called the corpuscles of Hassall.

* Palatine tonsil is an aggregation of lymphoid tissue that is readily recognized by the fact that it is covered by a stratified squamous epithelium
* At places the epithelium dips into the tonsil in the form of deep crypts
* Deep to the epithelium there is diffuse lymphoid tissue in which typical lymphatic nodules can be seen.
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* Each nerve fiber is covered by endoneurium
* Each bundle of nerve fibers is covered by perineurium
* Each nerve is covered by epineurium
* Cut section of axon with myelin sheath and Schwann cell nucleus.

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* Covered by meninges—dura mater, arachnoid mater, and pia mater
Sensory Ganglia
Fig. A7.3: Sensory ganglia.
* Unipolar neurons with centrally placed nucleus
* Neurons are presented in a group between the group of nerve fibers
Autonomic Ganglia
Fig. A7.4: Autonomic ganglia.
* Multipolar neurons with eccentrically placed nucleus
* Neurons are scattered between the nerve fibers.
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CARDIOVASCULAR SYSTEM
Elastic Artery
Fig. A8.1: Elastic artery.
* Tunica intima consisting of endothelium, subendothelial connective tissue, and internal elastic lamina
* The first layer of elastic fibers is called the internal elastic lamina. The internal elastic lamina is not distinct from the elastic fibers of media
* Well developed subendothelial layer in tunica intima
* Thick tunica media with many elastic fibers and some smooth muscle fibers
* Tunica adventitia containing collagen fibers with several elastic fibers
* Vasa vasorum in the tunica adventitia (Not seen in this slide).

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* In muscular arteries, the tunica intima is made up of endothelium and internal elastic lamina, which is thrown into wavy folds due to contraction of smooth muscle in the media
* Tunica media is composed mainly of smooth muscle fibers arranged circularly
* Tunica adventitia contains collagen fibers and few elastic fibers.
Large-Sized Vein
Fig. A8.3: Large-sized vein.
* The vein has a thinner wall and a larger lumen than the artery
*The tunica intitma, media, and adventitia can be made out, but they are not sharply demarcated
* The media is thin and contains a much larger quantity of collagen fibers than arteries. The amount of elastic tissue or of muscle is much less
*The adventitia is relatively thick and contains considerable amount of elastic and muscle fibers.
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Thin Skin or Hairy skin
Fig. A9.1: Thin skin or hairy skin (low magnification).

* Presence of thin epidermis made up of keratinized stratified squamous epithelium (stratum corneum is thin)
* Hair follicles, sebaceous glands and sweat glands are present in the dermis
* It is found in all others parts of body except palms and soles.

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* Sweat glands are present in the dermis
* It is found in palms of hands and soles of feet.
Hair Follicle and sebaceous gland
Fig. A9.4: Hair follicle and sebaceous gland (High magnification).
* In figures small areas of skin at higher magnification are shown
* Each sebaceous gland consists of a number of alveoli that open into a hair follicle

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* Intercalated and striated (intralobular) ducts are seen
Submandibular Gland (Low Magnification)
Fig. A10.2: Submandibular gland (Low magnification).
*The submandibular gland is a mixed salivary gland, predominantly serous with a few mucous acini
*Serous cells are frequently located at the periphery of mucous acini in the form of a crescent and called as demilunes
* Striated ducts are more prominent than those in parotid gland.
Submandibular Gland (High Magnification)
Fig. A10.3: Submandibular gland (High magnification).
* In the high power view the serous and mucous acini can be identified by their staining reaction, and shape and position of nucleus
* The serous acini are darkly stained and have rounded nucleus placed near the center of the cell
*The mucous acini are lightly stained with flat nucleus placed toward the basement membrane
* The mucous acini are often associated with darkly stained crescentic patch of serous cells called serous demilune.
Sublingual Salivary Gland
Fig. A10.4: Sublingual salivary gland.
* The sublingual gland is predominantly a mucous gland but few serous acini may also be seen
* Serous demilunes may be present.
PLACENTA
Placenta (Low Magnification)
Fig. A11.1: Placenta (Low magnification).
* Tertiary villi of various size and shapes seen
* Truncus chorii, ramus chorii, ramuli chorii and floating villi in intervillous space seen
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* Lined by pseudostratified ciliated columnar epithelium
* Lamina propria with serous and mucous glands
* Central core of elastic tissue.
Trachea
Fig. A12.2: Trachea.
* Pseudostratified ciliated columnar epithelium with goblet cells
* Lamina propria
* Submucosa with serous and mucous glands
* Presence of hyaline cartilage and trachealis muscle.
Courtesy: lvan Damjanov. Atlas of Histopathology, 1st edition. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2012. p. 37.
* Respiratory bronchioles with simple cuboidal epithelial lining lacking cilia and goblet cells.
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DIGESTIVE SYSTEM: ORAL CAVITY AND RELATED

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* The tongue is covered on both surfaces by stratified squamous epithelium (nonkeratinized)
* Each papilla has a core of connective tissue covered by epithelium. Some papillae are pointed (filiform), while others are broad at the top (fungiform). A third type of papilla is circumvallate, the top of this papilla is broad and lies at the same level as the surrounding mucosa
* The main mass of the tongue is formed by skeletal muscle seen below the lamina propria. Muscle fibers run in various directions so that some are cut longitudinally and some transversely
*» Numerous serous and mucous glands are present amongst the muscle fibers.
Vallate Papilla
Fig. A13.2: Vallate papilla.
* Circumvallate papillae are characterized by their dome-shaped structure lined by stratified squamous epithelium
* Numerous oval-shaped lightly stained taste buds can be seen on the lateral wall of the papillae
* The underlying connective tissue contains serous glands of von Ebner
® Skeletal muscle can be seen extending into the papillae.
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DIGESTIVE SYSTEM: GENERAL PLAN OF
GASTROINTESTINAL TRACT, STOMACH AND INTESTINES
ESOPHAGUS
Fig. A14.1: Esophagus.
*Four layers of gastrointestinal tract (GIT): Mucosa, submucosa, muscularis externa, adventitia seen

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* Presence of four layers—mucosa, submucosa, muscularis externa, and serosa
* Shallow gastric pits occupying superficial one-fourth or less of the mucosa
* Presence of gastric glands in the mucosa
* Gastric glands with numerous oxyntic cells which give beaded appearance.
* Deep gastric pits occupying two-thirds of the depth of the mucosa
* Presence of pyloric glands (mucous glands) in the mucosa that are simple/branched tubular glands which are coiled.

* Mucosa—presence of numerous broad villi, lined by simple columnar epithelium with microvilli and goblet cells
* Presence of submucous Brunner's gland.

* Mucosa—presence of numerous tall slender villi, lined by simple columnar epithelium with microvilli and goblet cells

* Mucosa—presence of short, small fewer villi, lined by microvilli and numerous goblet cells simple columnar epithelium with

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* Lymphatic follicles dispersed in the lamina propria
* Presence of taenia coli, appendices epiploicae.
Vermiform Appendix
Fig. A14.10: Vermiform appendix.
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* The panoramic view of liver shows many hexagonal areas called hepatic lobules. The lobules are partially separated by connective tissue
* Each lobule has a small round space in the center. This is the central vein
* A number of broad irregular cords of cells seem to pass from this vein to the periphery of the lobule. These cords are made up of polygonal liver cells—hepatocytes
* Along the periphery of the lobules, there are angular intervals filled by connective tissue
* Each such area contains a branch of the portal vein, a branch of the hepatic artery, and an interlobular bile duct
* These three constitute a portal triad.
Liver (High Magnification)
Fig. A15.2: Liver (High magnification).
* The lobule is made up of polygonal liver cells arranged in the form of radiating cords
* The central round nucleus of hepatocyte is surrounded by abundant pink cytoplasm
* The cords are separated from each other by spaces called sinusoids
* The sinusoids are lined by endothelial cells and Kupffer cells (macrophage cells)
* Each portal tract contains a hepatic arteriole, portal venule, and one or two interlobular bile ducts. Normally, these structures are surrounded by fibroconnective tissue and a few lymphocytes.

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* The mucous membrane is lined by tall columnar cells with striated border
* The mucosa is highly folded and some of the folds might look-like villi
* Crypts may be found in lamina propria
* Submucosa is absent
* The muscle coat is poorly developed with numerous connective tissue fibers interspersed among muscle. This is called as fibromuscular coat
» A serous covering lined by flattened mesothelium is seen.

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* Made up of serous acini
* The cells forming the acini of the pancreas are highly basophilic (bluish staining). The lumen of the acinus is very small
* Some acini may show pale staining (centroacinar cell) in the center
® Among the acini, some ducts are seen
* The ducts have a distinct lumen, lined by cuboidal epithelium
* At some places, the acini are separated by areas where we see aggregations of cells quite different from those of the acini
* In the photomicrograph, an interlobular duct lined by cuboidal epithelium is surrounded by lobules of acinar cells which have small basal nuclei and granular eosinophilic or slightly basophilic cytoplasm. Islets of Langerhans appear as groups of small cells with lightly stained cytoplasm.
Bf

Courtesy: Balakrishna Shetty, Sweekritha H Poonja. HISTOLOGY Practical Manual, 4th edition. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2019. p. 108.

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* The kidney is covered by a capsule, deep to which is the cortex
* Deep to the cortex there is the medulla
* In the cortex we see circular structures called renal corpuscles surrounding which there are tubules cut in various shapes
* Lighter staining tubules, each with a distinct lumen, are the distal convoluted tubules (DCT). They are lined by simple cuboidal epithelium
* PCT are more in number than DCT
* In the medulla we see very light staining, elongated, parallel running tubules. These are collecting ducts and loop of Henle. Some of them extend into the cortex forming a medullary ray. The collecting ducts are lined by simple cuboidal epithelium and loop of Henle (thin segments) are lined by simple squamous epithelium
* Cut sections of blood vessels are seen both in the cortex and medulla.
Renal cortex (High Magnification)
Fig. A16.2: Renal cortex (High magnification).
* In the high power view of renal cortex large renal corpuscles can be identified
* The renal corpuscle consists of a tuft of capillaries that form a rounded glomerulus, and an outer wall, the glomerular capsule (Bowman's capsule)
* A urinary space between the glomerulus and the capsule is seen
* Proximal convoluted tubules are dark staining. They are lined by cuboidal cells with a prominent brush border. Their lumen is indistinct
* Distal convoluted tubules are lighter staining. The cuboidal cells lining them do not have a brush border. Their lumen is distinct.
Renal Medulla (High Magnification)
Fig. A16.3: Renal medulla (High magnification).

* A high power view of a part of the renal medulla shows a number of collecting ducts cut longitudinally or transversely
* They are lined by a cuboidal epithelium, the cells of which stain lightly. Cell boundaries are usually distinct. The lumen of the tubule is also distinct
* Sections of the thin segment of the loop of Henle are seen. They are lined by flattened cells, the walls being very similar in appearance to those of blood capillaries
® Sections through the thick segments of loops of Henle are seen. They are lined by cuboidal epithelium.

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* The ureter can be recognized because it is tubular and its mucous membrane is lined by transitional epithelium
* The epithelium rests on a layer of connective tissue (lamina propria)
* The mucosa shows folds that give the lumen a star-shaped appearance
* The muscle coat has an inner layer of longitudinal fibers and an outer layer of circular fibers. This arrangement is the reverse of that in the gut
* The muscle coat is surrounded by connective tissue—adventitia in which blood vessels and fat cells are present.

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*The urinary bladder is easily recognized because the mucous membrane is lined by transitional epithelium
* The epithelium rests on lamina propria
* The muscle layer is thick. It has inner and outer longitudinal layers between which there is a layer of circular or oblique fibers. The distinct muscle layers may not be distinguishable
*The outer surface is lined in parts by peritoneum (serosa) (not seen in the photomicrograph).
i CENTRAL NERVOUS SYSTEM: SPINAL CORD, CEREBELLAR CORTEX AND CEREBRAL CORTEX
Spinal Cord
Fig. A17.1: Spinal cord: Gray matter.
* The spinal cord has a characteristic oval shape
*|It is made up of white matter (containing mainly of myelinated fibers), and gray matter (containing neurons and unmyelinated fibers)
* The gray matter lies toward the center and is surrounded all round by white matter
*The gray matter consists of a centrally placed mass and projections (horns) that pass forwards and backwards.

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Outer granular layer
Pyramidal cell layer
Inner granular layer
Ganglionic layer
{
{
Polymorphous layer
Fig. A17.2: Cerebral cortex.

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* A slide of cerebral cortex shows outer gray and inner white matter
* Multipolar neurons of various shapes are arranged in six layers in the gray matter
* Axons of these neurons are present in the white matter
* Neuroglia and blood vessels are present in gray and white matter.
Cerebellar Cortex
Fig. A17.3: Cerebellar cortex.
* The section of cerebellum shows leaf-like folia
* The cortex is covered by pia mater which appears as a thin layer of collagen fibers. Blood vessels may be seen just beneath the pia mater
* Outer gray matter is arranged in three layers from without inwards:
1. Molecular layer: Very few nuclei of neurons seen. Many cell processes present. Appi
3. Granular cell layer: Appears very dark blue because of presence of abundant neurons
* Inner white matter shows axons which appear as pink fibers
* Nuclei of neuroglia are present both in gray and white matter.
Mf

Testis (Low Magnification)
Fig. A18.1: Testis (Low magnification).

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* A number of seminiferous tubules cut in various directions are seen
* Each seminiferous tubule is lined by several layers of cells
* Cells are of two types:
Testis (High Magnificatione)

Courtesy: lvan Damjanov. Atlas of Histopathology, 1st edition. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2012. p. 203.

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* The outermost row of nuclei belongs to sustentacular cells (Sertoli) and to spermatogonia, some of which are undergoing mitosis (note very dense nucleus of irregular shape)
* Passing inwards toward the center of the tubule we have large darkly staining nuclei of spermatocytes, and many smaller nuclei of spermatids
* Toward the center of the tubule a number of developing spermatozoa are seen. The sperms are often found in clusters embedded in the cytoplasm of Sertoli cells
* In the adult testis, sustentacular cells are less prominent than germ cells. They are more prominent than germ cells before puberty and in old age.
* The body of the epididymis is a long convoluted duct
* A section shows number of tubules lined by pseudostratified columnar epithelium in which there are tall columnar cells and shorter basal cells that do not reach the lumen. The columnar cells bear stereocilia
* Smooth muscles are present in the wall of the duct
* Clumps of spermatozoa are present in the lumen of the duct.

Courtesy: Balakrishna Shetty, Sweekritha H Poonja. HISTOLOGY Practical Manual, 4th edition. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2019. p. 120.

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® A small irregular lumen
*Mucous membrane lined by pseudostratified columnar epithelium with underlying lamina propria
Seminal Vesicle (Low Magnification)
Prostate (Low Magnification)
Fig. A18.6: Prostate (Low Magnification).
* The prostate consists of glandular tissue embedded in prominent fibromuscular stroma
*The glandular tissue is in the form of follicles with serrated edges. They are lined by columnar epithelium. The lumen may contain amyloid bodies
* The follicles are separated by broad bands of fibromuscular tissue.
Bi

Courtesy: Balakrishna Shetty, Sweekritha H Poonja. HISTOLOGY Practical Manual, 4th edition. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2019. p. 126.

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* The surface is covered by a cuboidal epithelium. Deep to the epithelium, there is a layer of connective tissue that constitutes the tunica albuginea
*The substance of the ovary has an outer cortex in which follicles of various sizes are present, and an inner medulla consisting of connective tissue containing numerous blood vessels
* Just deep to the tunica albuginea, many primordial follicles each of which contains a developing ovum surrounded by flattened follicular cells are present
* Large follicles have a follicular cavity surrounded by several layers of follicular cells
* The cells surrounding the ovum constitute the cumulus oophorus
* The follicle is surrounded by a condensation of connective tissue which forms a capsule for it
* The capsule consists of an inner cellular part (the theca interna), and an outer fibrous part (the theca externa) collectively called as theca folliculi. The follicle is surrounded by a stroma made up of reticular fibers and fusiform cells.

*The uterine tube is characterized by the presence of numerous branching mucosal folds that almost fill the lumen of the tube
* The mucosa is lined by ciliated columnar epithelium
* The uterine tube has a muscular wall with an inner circular and outer longitudinal muscle layer.

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* The wall of the uterus consists of a mucous membrane (called the endometrium) and a very thick layer of muscle (the myometrium). The thickness of the muscle layer helps to identify the uterus easily
* The endometrium has a lining of columnar epithelium that rests on a stroma of connective tissue
*» Numerous tubular uterine glands dip into the stroma
* The appearance of the endometrium varies considerably depending upon the phase of the menstrual cycle. The endometrium is thin and progressively increases in thickness. The uterine glands are straight and tubular in this phase.
Uterus (Secretory Phase)
Fig. A19.5: Uterus (Secretory phase).

* The thickness of the endometrium is much increased
* The uterine glands elongate, become dilated, and tortuous as a result of which they have sawtoothed margins in sections
* Blood vessels extend in the upper portion of endometrium.

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* The vagina is a fibromuscular structure consisting of an inner mucosa, a middle muscular layer, and an outer adventitia
* The mucosa consists of stratified squamous nonkeratinized epithelium and loose fibroelastic connective tissue lamina propria with many blood vessels and no glands
*The mucosa of vagina is rich in glycogen and hence, the cells are pale stained which distinguishes it from esophagus
» Muscular layer consists of smooth muscle fibers.
Mammary gland (Resting)
* Mammary gland consists of lobules of glandular tissue separated by considerable quantity of connective tissue and fat
* Nonlactating mammary glands contain more connective tissue and less glandular tissue
*The glandular elements or alveoli are distinctly tubular. They are lined by cuboidal epithelium and have a large lumen so that they look like ducts. Some of them may be in form of solid cords of cells
* Extensive branching of duct system seen.
Mammary gland (lactating)
Lactiferous duct
Lobule
Interlobular connective tissue
Fig. A19.8: "Mammary gland (Lactating)
* In lactating mammary gland, the glandular elements proliferate so that they become relatively more prominent than the connective tissue
* The interlobular connective tissue septum is very thin
* The lobules are formed by compactly arranged alveoli
* The alveoli are lined by simple cuboidal secretory epithelium and associated myoepithelial cells. Their lumen contains eosinophilic secretory material which appears vacuolated due to the presence of fat droplets.
Bf
ENDOCRINE SYSTEM
Hypophysis Cerebri
Fig. A20.1: Hypophysis cerebri.
* Pars anterior is cellular
* Pars intermedia is variable in structure
* Pars posterior consists of fibers, and is lightly stained.

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* It consists of groups or cords of cells.
* The cells are of three types:
. The pink staining cells are alpha cells or acidophils 1
3 . Cells in which the cytoplasm is not conspicuous, and the nuclei are closely pa chromophobe cells
Thyroid Gland
Fig. A20.3: Thyroid gland (Low magnification).
* The thyroid gland is made up of follicles lined by cuboidal epithelium
* In photomicrograph, in low magnification, it can be seen that follicles vary in shape and size
* Each follicle is filled with a homogenous pink colloid proteinaceous material composed primarily of thyroglobulin that has been produced by the follicular epithelial cells
* Parafollicular cells are present in relation to the follicles and also as groups in the connective tissue
* In the intervals between the follicles, there is some connective tissue and blood vessels between follicles.
Parathyroid Gland
Fig. A20.4: Parathyroid gland.
* These glands are made up of masses of cells with numerous capillaries in between
* Most of the cells (of which only nuclei are seen) are the chief cells which appear as small basophilic cells
* Oxyphil cells appear as large as eosinophilic (pink) cells
* Oxyphil cells are few in number
* Adipose cells are also seen.

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* The suprarenal gland is made up of a large number of cells arranged in layers. It consists of an outer cortex and an inner medulla
* The cortex is divisible into three zones
*The zona glomerulosa is most superficial. Here, the cells are arranged in the form of inverted U-shaped structures or acinus-like groups
* In the zona fasciculata, the cells are arranged in straight columns (typically two-cell thick). Sinusoids intervene between the columns
* The zona reticularis is made up of cords of cells that branch and form a network
* The medulla is made up of groups of cells separated by wide sinusoids. Some sympathetic neurons are also present.
i

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* The cornea is made up of five layers: corneal epithelium, anterior limiting membrane, or Bowman's membrane, corneal stroma, posterior limiting lamina, and endothelium
* The corneal epithelium is nonkeratinized stratified squamous epithelium
* The substantia propria is made up of collagen fibers embedded in a ground substance
* Endothelium is lined by a single layer of flattened or cuboidal cells.
Fig. A21.2: Retina.
*Made up of ten layers: pigment epithelium, layer of rods and cones, external limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglionic cell layer, nerve fiber layer, and internal limiting membrane
* Outer pigment epithelium is made up of simple cuboidal epithelium
* The outer nuclear layer is thicker with densely packed nuclei than in the inner nuclear layer
* Ganglion cell layer is made up of a single row of cells of varying size.
i
SPECIAL SENSES: EAR

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* The cochlea is embedded in the petrous temporal bone
* It is in the form of a spiral canal and is, therefore, cut up six times
*The cone-shaped mass of bone surrounded by these turns of the cochlea is called the modiolus which contains a canal through which fibers of the cochlear nerve pass
* A mass of neurons belonging to the spiral ganglion lies to the inner side of each turn of the cochlea
* The parts to be identified in each turn of the cochlea are the scala vestibuli, scala media, the scala tympani, the vestibular membrane, the basilar membrane, the membrana tectoria, and the organ of Corti, and the spiral lamina
* Outer wall of the cochlear turn is the spiral ligament and it is lined by avascularized epithelium (stria vascularis).

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Abortions 164
Acetylcholine receptors 97f
Achalasia 209
Achlorhydria 209
Achondroplasia 65
Acid hydrolase enzymes 14
Acidophil 291
cells, types of 291
Acinar glands 38
Acini
structure of 41
types of 41f
Acinus 214
Acne vulgaris 150
Acoustic meatus, external 325, 326
Acromegaly 305
Acrosome reaction 260
Actin 87, 88
filament 89f
Actinin 89
Active transport 8
Acute respiratory distress syndrome 176
Addison's disease 306
Adenocarcinoma 42
Adenohypophysis 291
Adenoma 42
Adenomyosis 288
Adenosine triphosphate 13, 89
Adhesion spots 9
Adhesive belts 100
Adhesive junctions 9
Adhesive strips 10
Adipocytes 46
Adipose tissue 44, 46, 53, 56
cell 57f section 47
Adrenocorticotropin 291
Advanced bell stage 186
Adventitia 171, 190, 191, 219, 235, 282
Afferent fibers 121
entering cerebellar cortex 250
Agranular cortex 247
Air passages, common features of 166
Albinism 150
Albuminuria 238
causes of 238
Aldehyde fuchsin 51
Aldosterone 300
Alimentary canal 177
Allocortex 243
Alpha cells 221, 291
Alpha efferents 91
Alveolar ducts 171
Alveolar epithelial cells 174
Alveolar glands 38
Alveolar sacs 171
Alveolar wall, structure of 172
Alveoli 171, 172, 284, 287
Alveolus 174f
Amacrine cells 317
Amacrine neurons 322
connections of 323f
Ameloblasts 186
Amine precursor uptake and decarboxylation 40
Amino acid derivatives 289
Ampulla 262, 276, 332
Ampullary crest 332, 335
functioning of 335
structure of 335f
Amyloid bodies 265
Amyloidosis 112
Anal canal 190, 208
interior of 208f
Anal columns 208
Anal sinus 208

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Anal sphincter, internal 190, 208
Anal valves 208
Anchoring junctions 9
Anchoring villi 162, 162f
Androgen binding protein 255
Angioma 138
Angiotensin 233
converting enzyme 130
Angiotensinogen 233
Ansa nephroni 230
Antibodies 100
Antigen 100
presenting cells 105
Anti-Miillerian hormone 255
Antrum 273
Apical foramen, primary 187 Apocrine 37, 38, 284 Appendices epiploicae 205 Appendicitis, acute 209 APUD cell system 305 Areola 284, 287 Areolae primary 76 secondary 76 Areolar tissue 52 loose 45f, 52 Argentaffin cells 40, 196, 203f, 209 Argentophil fibers 51 Argyophil cells 40 Arrector pili muscles 94, 147 Arterioles 129, 132, 132f afferent 233 efferent 234 Arteriovenous anastomoses 137, 137f Artery 129, 132 arcuate 233 elastic 130-132, 132t
interlobar 233
large-sized 130
medium-sized 132, 133
structure of 130
within kidney, arrangement of 233f
Asthma 176
Astrocytes 16, 116, 117f
fibrous 116
Atherosclerotic lesions 13
Atretic follicles 275
Atrium 171
Auditory canal, external 325
Auditory tube 327
Auerbach’s plexus 190
Auricle 325
Autoimmune diseases 112
Autonomic ganglia 126, 126f, 127
Autonomic nervous system 113
Axoaxonal synapse 124, 124f
Axodendritic synapse 124, 124f
Axoneme 18, 34
Axons 115, 116, 116t
hillock 115
on basis of length of 120
unmyelinated 119
Axoplasmic flow 116
Axosomatic synapse 124, 124f
Bacteria 103
Balt 109
Barrett's esophagus 209
Basal body 34
Basal cells 167, 168, 175, 261
carcinoma 151
Basal folds 36
Basal granule 34
Basal lamina 32, 129, 130, 311

#/texts/17516 #/texts/17517 #/texts/17518 #/texts/17519 #/texts/17520 #/texts/17521 #/texts/17522 #/texts/17523 #/texts/17524 #/texts/17525 #/texts/17526 #/texts/17527 #/texts/17528 #/texts/17529 #/texts/17530 #/texts/17531 #/texts/17532 #/texts/17533 #/texts/17534 #/texts/17535 #/texts/17536 #/texts/17537 #/texts/17538 #/texts/17539 #/texts/17540 #/texts/17541 #/texts/17542 #/texts/17543 #/texts/17544 #/texts/17545 #/texts/17546 #/texts/17547 #/texts/17548 #/texts/17549 #/texts/17550 #/texts/17551 #/texts/17552 #/texts/17553

#/body #/body #/body #/body #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/groups/1115 #/body #/body

Basement membrane 31f, 32, 52, 229f functions of 33
Basilar membrane 328
Basket cells 153, 244f, 247, 250
Basolateral folds 20 Basophil cells 291 types of 291 Bell stage, early 185, 186f Beta cells 222, 291 Bile 217 canaliculus 217f capillaries 215f Biliary apparatus, extrahepatic 217 Biliary duct system 215 Bipolar cells 315 Bipolar neurons 120, 120f, 321 firing of 322, 323fc Birbeck bodies 144 Blastomeres 161 Blood 44 monocyte of 109 placental barrier 162 pressure 130 retinal barrier 317 testis barrier 255 thymic barrier 108 thymus barrier 106 vessels 91, 96 basic structure of 129 innervation of 135 B-lymphocytes 48, 100 circulation of 100f Body 150 tube 1 Bone 44, 52, 67 cells 67f, 68, 68f mature 67
classification of 71
deformans 81
formation of 75
forming cells 67
gross appearance of 71
lamellae 71f
lining cells 69
marrow 72, 98, 108, 109
matrix 69
membranes 69
of vault of skull, growth of 78
osteoblast rimming surface of 68f
structure 7Of, 71
tissue, composition of 67
woven 75
Bony cochlea 328
Bony labyrinth 327, 328, 328f
Bony lamellae 76f
Bowman’s capsule 227
Bowman’s membrane 309
Brain sand 302
Breast
carcinoma of 288
human female 287f
Bright’s disease 238
Brodmann’s areas 247
Bronchial tree 171f
Bronchioles 171
Bronchitis, chronic 176
Brown adipose tissue 56
cell 57f
Brush border 20, 7, 35
Brush cells 168, 174
Bud stage 185, 186f

#/texts/17554 #/texts/17555 #/texts/17556 #/texts/17557 #/texts/17558 #/texts/17559 #/texts/17560 #/texts/17561 #/texts/17562 #/texts/17563 #/texts/17564 #/texts/17565 #/texts/17566 #/texts/17567 #/texts/17568 #/texts/17569 #/texts/17570 #/texts/17571 #/texts/17572 #/texts/17573 #/texts/17574 #/texts/17575 #/texts/17576 #/texts/17577 #/texts/17578 #/texts/17579 #/texts/17580 #/texts/17581 #/texts/17582 #/texts/17583 #/texts/17584 #/texts/17585 #/texts/17586 #/texts/17587 #/texts/17588 #/texts/17589 #/texts/17590

#/groups/1116 #/groups/1116 #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/groups/1117

Calcification 65 zone of 81 Calcination 71 Calcitonin 296 Calcium dependent 9 independent 9 Calix 335 Canal of Schlemm 308 Canal of Volkmann 72 Canalicular surface 215 Canaliculate Canaliculi 71 Cancellous bone 72, 74, 76 structure of 72f Cap stage 185, 186f Capillary 129, 133 lamina 311 structure of 134 types of 134 Capsule 39, 102, 103, 221 Cardiospasm 209 Cardiovascular system 129 Carotid body 304 cells of 304 functions of 305 nerve supply of 305 structure of 304f
lamellar bodies 302
Cartilaginous
Cartwheel
Castle,
Cataract
C-cells
Celiac
```
bones 75 cells 61 components of 60 epiphyseal 78 fibers of 61 forming tumors 65 general features of 60 growth of 60, 65 zone of 80 hyaline 60-62, 66, 168 transformation, zone of 81 types of 59, 61 zone of proliferating 80 resting 80 model, establishment of 80f appearance 49 intrinsic factor of 196 324 296 sprue 209 adhesion molecules 9, 9f, 10f, 11f types of 9t arrangement of 23, 185f basal parts of 153 body 113, 115, 316 toward 116 capsular 116
```
components of 5
drinking 8
eating 8
elongated 183f flask-shaped 167
from adrenal cortex, ultrastructure of 302f
medulla, ultrastructure of 302f from striated duct, electron microscopic structure of 159f
highly phagocytic 111
horizontal 316, 316f intercalated 232
intermediate 203
junctions 9
classification of 9
lining distal convoluted tubule, ultrastructure of 232f lining proximal convoluted tubule, ultrastructure of 230f
membrane 5, 7f, 8f carbohydrates of 7
functions of 8
lipids in 5
outer aspect of 7f protein in 7
significance of proteins of 7
trilaminar structure of 6f morphology 23
nest 61
number of 37
organelles 11
power house of 13
staining of 21
structure of 5, 111
surface, projections from 18, 33
system 290
types of 144, 172f, 175
undifferentiated 203

#/texts/17591 #/texts/17592 #/texts/17593 #/texts/17594 #/texts/17595 #/texts/17596 #/texts/17597 #/texts/17598

#/groups/1117 #/groups/1117 #/groups/1117 #/body #/body #/body #/body #/body

Cells of Cajal 245
horizontal 244, 244f Cells of Clara 172 Cells of Claudius 333 Cells of Hensen 333 Cells of Martinotti 244, 244f Cells of Merkel 144 Cells of Miiller 317 Cells of Polkissen 233 Cells of Sertoli 255 Cementocytes 180 Cementum 180 Central nervous system 110, 113, 240 Centrioles 16 Centroacinar cells 221 Centrosome 16 Centrotubule 89 Cerebellar ataxia 251 cortex 240, 247, 248, 249f cortex, structure of 247 glomeruli 250 glomeruli, structure of 250f islands 250 nuclei 247, 247f Cerebellum, cells of 249 Cerebral cortex 240, 243, 244f, 246 lamina of 244, 245f neurons in 244 Cerebrospinal fluid 118 Cervix 281
Chalazion 324
Cheilitis 185
Chemical nature 49, 51
Chemical synapses 124
classification of 124f structure of typical 128f types of 124f Chief cells 196, 298 Cholecystectomy 223 Cholelithiasis 223 Cholesterol 130 Chondroblastoma 65 Chondroblasts 61, 76 Chondrocytes 60, 61 Chondrogenic layer 60 inner 60 Chondroitin sulfate 52 Chondrosarcoma 65 Chorion 161 frondosum 161 laeve 161 Chorionic plate 161 Chorionic villi 162 Choroid 307, 311 Choroidal epithelial cells 118 Choroidal stroma 311 Choroidocapillaris 311 Chromatin 17 fiber, structure of 18f nature of 17 Chromatophores 46 Chromophil cells 291 Chromophobe cells 291
Chromosomes 18

#/texts/17599 #/texts/17600 #/texts/17601 #/texts/17602 #/texts/17603 #/texts/17604 #/texts/17605 #/texts/17606 #/texts/17607 #/texts/17608 #/texts/17609 #/texts/17610 #/texts/17611 #/texts/17612 #/texts/17613 #/texts/17614 #/texts/17615 #/texts/17616 #/texts/17617 #/texts/17618 #/texts/17619 #/texts/17620 #/texts/17621 #/texts/17622 #/texts/17623

#/body #/body #/body #/body #/body #/body #/body #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118

Chyle 99 Chylomicrons 99 Chylopericardium 111 Chyloperitoneum 111 Chylothorax 111 Chyluria 111 Chyme 191 Cilia 18, 19f, 27f, 33, 34, 35, 35t abnormalities of 36 functions of 19 Ciliaris muscle 311 Ciliary body 307, 311 Ciliary glands 149, 320 Ciliary processes 312 Ciliated cells 167, 168, 276 Ciliated columnar epithelium 27, 28 Cilium 19f, 34f, 316 Circular layer, inner layer of 190 Circumvallate papillae 181 Cisternae, central layers of 14 Clasmatocytes 47 Clear cells 148, 232 Clitoris 282 Closed-face nuclei 17 Cochlea 327-329 aqueduct of 330 duct of 330 Cochlear canal, structure of 330f Collagen 49 fibers 49, 49t, 50f, 60, 69 types of 59t Collecting tubule OS 032
Colloid 296
Colon 188, 204, 206, 209
segment of 205f
Colostrum 284
Columnar cells absorptive 202 basal layer of 31f Columnar epithelial cells 28 Columnar epithelium 24, 25, 27f, 28 lining small intestine 203f stratified 31 Communicating junctions 9, 11, 12f Compact bone 71, 72f, 74, 76 longitudinal section of 70f, 74 transverse section 73 Compound tubuloalveolar glands 189 mucous glands 191 serous gland 219 Concentric lamellae 71 Condenser 2 Cone horizontal neuron, connections of 323f pedicles 316, 316 Conjunctiva 320 Conjunctival fornix 320 Conjunctival sac 320 Connecting tubule 232 basic components of 45fc classification of 44 of
components 44
loose 50f
diseases of 59
fibers of 49, 50f
framework, structure 103
functions of 56
histiocytes of 109
intercellular ground substance of 51
loose 44, 52
macrophage cells of 109
sheath 147
subendocardial layer of 137
typical 52
Connexons 11
Conus medullaris 241
Cord and clump type 40
Cornea 307, 308, 310, 323
junction of 308f
nutrition of 309

#/texts/17624 #/texts/17625 #/texts/17626 #/texts/17627 #/texts/17628 #/texts/17629 #/texts/17630 #/texts/17631 #/texts/17632 #/texts/17633 #/texts/17634 #/texts/17635 #/texts/17636 #/texts/17637 #/texts/17638 #/texts/17639 #/texts/17640 #/texts/17641 #/texts/17642 #/texts/17643 #/texts/17644 #/texts/17645 #/texts/17646 #/texts/17647 #/texts/17648 #/texts/17649 #/texts/17650 #/texts/17651 #/texts/17652 #/texts/17653 #/texts/17654 #/texts/17655 #/texts/17656 #/texts/17657 #/texts/17658 #/texts/17659 #/texts/17660 #/texts/17661 #/texts/17662 #/texts/17663 #/texts/17664 #/texts/17665 #/texts/17666 #/texts/17667 #/texts/17668 #/texts/17669 #/texts/17670

#/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/groups/1118 #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

transparency of 309
Corneal abrasion 324
Corneal epithelium, anterior 309
Corneal neovascularization 324
Comeal opacity 323
Corneal stroma 309
Corneal ulcer 323
Corneoscleral junction 308
Corona radiata 273
Corpora amylacea 265
Corpora arenacea 302
Corpora atretica 275
Corpora cavernosa 265, 282
Corpus albicans 275
Corpus hemorrhagicum 274
Corpus luteum 274, 274f
Corpus spongiosum 265
Cortex 102, 224, 225, 270
Corti, spiral organ of 332, 333
Cortical arches 225
Cortical glomeruli 234
Cortical lobules 225
concept of 234f
Cortical structure, variations in 247
Corticotropes 291
Cortisol 300
Cortisone 300
Cotyledons, components of 162f
Cretinism 306
Cristae 13
ampullae 332
Crohn's disease 209
Crown 179
Crypts 202
Crystals, needle-shaped 69
Cuboidal epithelium 24
simple 24, 26, 26f stratified 31
Cumulus oophorus 273
Cupula 335
Cushing's disease 306
Cystitis 238
Cytocrine 37
Cytokeratin 16
Cytokines 100
Cytoplasm 5, 114
Cytoskeleton 15
Cytosol 5, 12

#/texts/17671 #/texts/17672 #/texts/17673 #/texts/17674 #/texts/17675 #/texts/17676 #/texts/17677 #/texts/17678 #/texts/17679 #/texts/17680 #/texts/17681 #/texts/17682 #/texts/17683 #/texts/17684 #/texts/17685 #/texts/17686 #/texts/17687 #/texts/17688 #/texts/17689 #/texts/17690 #/texts/17691 #/texts/17692 #/texts/17693 #/texts/17694 #/texts/17695 #/texts/17696 #/texts/17697 #/texts/17698 #/texts/17699 #/texts/17700 #/texts/17701 #/texts/17702

Dark cells 148, 232, 330, 331 Dartos muscle 94 Decalcification 70 Decidua 160 basalis 161 Deep fascia 53 Defense mechanisms, participation in 111 Dehydroepiandrosterone 300 Delta basophils 291 Delta cells 222 Dendrites 115, 116, 116t Dendritic cells 144 Dendritic processes 144f Dendroaxonic synapse 124 Dendrodendritic synapse 124 Dense connective tissue 52 irregular 44, 53, 55 regular 44, 52, 54 Dense lymphoid tissue 98, 99 Dental caries 185 Dentate nucleus 247 Dentin 180 types of 180 Dentinal sheath 180 Deoxycorticosterone 300 Deoxyribonucleic acid 13, 17, 18, 18f Dermal papillae 142, 142f Dermatan sulfate 52 Dermis 142, 145
papillary layer 145
reticular layer 145
Descemet's membrane 309
Desmin 89, 96
Desmosome 9
electron microscopy appearance of 10f structure of 10f
Detrusor muscle 235
Diabetes insipidus 306
Diaphysis 78
Digestive system 177, 188
Dihydrocortisone 300
Dilator pupillae 312
Distal convoluted tubule 230
Domain 52
Duchenne muscular dystrophy 97
Duct of Bellini 225, 232
Ducts 148
branching of 38
intercalated 153, 221
interlobular 153, 221
intralobar 153
intralobular 221
system 39, 153, 221, 285
Ductules, efferent 257
Ductus deferens 253, 262, 263
Duodenal papilla 219f
Duodenum 188, 189, 197, 198, 198t, 199
part of 190
Dust cells 111, 174
Dyads 93
Dysfunctional uterine bleeding 287

#/texts/17704 #/texts/17705 #/texts/17706 #/texts/17707 #/texts/17708 #/texts/17709 #/texts/17710 #/texts/17711 #/texts/17712 #/texts/17713 #/texts/17714 #/texts/17715 #/texts/17716 #/texts/17717 #/texts/17718 #/texts/17719 #/texts/17720 #/texts/17721 #/texts/17722 #/texts/17723 #/texts/17724 #/texts/17725 #/texts/17726 #/texts/17727 #/texts/17728 #/texts/17729 #/texts/17730 #/texts/17731 #/texts/17732 #/texts/17733 #/texts/17734 #/texts/17735 #/texts/17736 #/texts/17737 #/texts/17738 #/texts/17739

#/groups/1119 #/groups/1119 #/groups/1119 #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

external 325
main parts of 326f middle 325, 327 Ehlers-Danlos syndrome 59 Elastase 51 Elastic lamina external 129, 131 internal 129, 130 Elastin 51 Electrical synapses 124 Electron microscopy 5, lif high magnifications of 6f scanning 19f Ellipsoid 316 Embryonic connective tissue 44 Enamel 179 dentin, structure of 180f lamellae 180 organ 185 spindles 180 structure of 180f tufts 180 Enchondroma 65 Endocardium 24, 137 Endocervix 281 Endochondral ossification 75, 76, 77f secondary centers of 78 Endocrine cells 196, 203, 209 distribution of 290 groups of 290 of
in gut, types 210¢t
isolated 290
functions 303
gland 37, 39, 40, 290
pancreas 221
system 289
Endocytic vesicle 8
Endocytosis 8, 8f
Endolymph 330
Endolymphatic sac 330
Endolysosome 15
Endometriosis 287
Endometrium 276
thickness of 278, 281f
Endomysium 84, 86
Endoneurium 121
Endoplasmic reticulum 12, 41
Endorphins 294
Endosomes 15
Endosteum 70
functions of 70
Endothelial cells 106
basal lamina of 106
Endothelial venules, high 103
Endothelin 130
Endothelium 24, 129, 130, 309
functions of 130
Entamoeba histolytica 222
Enterochromaffin cells 204, 209
Enterocytes 202
Eosinophil 48, 48f
Ependymal cells 118
Epicardium 137
Epidermal papillae 142, 142f

```
Epidermal proliferation units 150 Epidermal ridges 142f Epidermis 10f, 142 cells of 144 layer of 142, 143f Epididymis 253, 260, 261 duct of 261 Epiglottis 168 Epilemmal junctions 153 Epimysium 85, 86 Epineurium 121 Epiphyseal plate 78, 80 structure of 81f Epiphysis 78 cerebri 302 fusion of 81 Episclera 308 Epithelia 23, 35 classification of 23, 24fc functions 23 stratified 23 unilayered 23 Epithelial cells 35 renewal of 35 shape of 35 Epithelial diaphragm 187 Epithelial layer, outer 327 Epithelial lining 202 Epithelial tissue, general features of 23 Epitheliocytes 106 basal lamina of 106 Epithelioid cells 111 Epithelioreticular cells 106
```
```
Epithelium 23, 208 lining 172, 276, 278 intestines 42 renal tubules 21f simple 23 stratified 29 Epitheloid 49 Erythrocytes, abnormal 1 Esophagus 188-192, 08, 23 part of 190 Estrogens 281 Euchromatic nucleus 17f Euchromatin 17 Eukaryotic cell 6f Eustachian tube 327 Excretion 160 Excretory ducts 287 Exocrine 40 gland 37, 39 classification of 37 structural organization of 40f pancreas 219 Exocytic vesicles 8 Exocytosis 8 stages in 8f Exostoses 70 External piles 209 Extracellular molecules, absorption of 8f Extraglomerular mesangial cells 233 Extrahepatic ducts 211, 219 Eye piece 1 Eyeball 308f of
```
layer 311f

Fabricus, bursa of 100 F-actin 88 Fallopian tube 276, 277, 287 Fascia adherens 10 Fasciculi 121, 240 Fast twitch fibers 89 Fastigial nucleus 247 Fat cells 45, 46, 47f, 53f Fenestra cochleae 328 Fenestrated capillary, structure of 134f Fetal connective tissue 44 cotyledons, ramifications of 162f Fibers 60, 69 efferent 121, 251 elastic 49, 49t, 50f, 51, 51f, 60 intrafusal 91 of Tomes 180 within lens, arrangement of 319f Fibrillin 51 Fibrils, arrangement of 260f Fibroadenoma 288 Fibroblast 45 structure of 46f Fibrocartilage 60, 63, 64, 66 Fibromuscular coat 219 lamina 32
stroma 265

#/texts/17747 #/texts/17748 #/texts/17749 #/texts/17750 #/texts/17751 #/texts/17752 #/texts/17753 #/texts/17754 #/texts/17755 #/texts/17756 #/texts/17757 #/texts/17758 #/texts/17759 #/texts/17760 #/texts/17761 #/texts/17762 #/texts/17763 #/texts/17764 #/texts/17765 #/texts/17766 #/texts/17767 #/texts/17768 #/texts/17769 #/texts/17770 #/texts/17771 #/texts/17772 #/texts/17773 #/texts/17774 #/texts/17775 #/texts/17776 #/texts/17777 #/texts/17778 #/texts/17779 #/texts/17780 #/texts/17781 #/texts/17782 #/texts/17783 #/texts/17784 #/texts/17785 #/texts/17786 #/texts/17787 #/texts/17788

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stimulating hormone 257f, 281, 291
Fibrous coat, outer 307 Fibrous layer 60 middle 327 outer 60 Filiform papillae 181 Filtration slits 227, 229f Filum terminale 241 Fimbriae 276 Finger-like processes 191 First meiotic division 258 First polar body 272 Fissure 247 anterior median 243 Fissures of Santorini 337 Flagella 20, 34 Floating villi 162, 162f Fluids inner ear 330 mosaic model 5 Focal spots 10 Focus knobs 2 Folia 247 Foliate papillae 183 Follicle 295 primary 272, 273f secondary 273 Follicular cells 272, 296 Follicular type 40 Foramen cecum 181 Foramen of Huschke 337
globular molecules of 89f
211, 217, 218, 223
microscopic structure 217
Fordyce's granules 185 Free macrophages 45 Fungiform 183f papillae 181 Funiculi 240 Funiculus anterior 243 anterolateral 243 posterior 243 Fusiform 96 cells 244f Fusogenic proteins 8f G-actin, Galea capitis 259 Gallbladder Gallstones 223 Gamma-efferents 91 Ganglia 124 Ganglion cell 322, 323f diffuse 322 layer of 317 Gap junctions 9, 11 Gastric glands 38, 42, 193 pits 193
ulcer 209
Gastrin 196
Gastritis 209
Gastroenteropancreatic endocrine system 209
Gastrointestinal tract 177
enteroendocrine cells of 37
general plan of 188, 189
Gaucher’s disease 112
General connective tissue 44
Genital ducts 252
Genitalia, female external 282
Germ cell 255, 255f
tumors 268
Germinal center 102
Germinal layer 143
Germinative matrix 146
Germinative zone 143
Giant cells 47
foreign body 49, 111
Gigantism 305
Glabrous skin 139
Gland 23, 37, 189
adrenal 300
anterior lingual 183
cardiac 197
ceruminous 149
characteristic features 37
classification of 37, 38fc
development of 40, 40f
ductless 37, 290
externally secreting 37
intermediate submucous 265
internally secreting 37
interstitial 275
intestinal 202
lacrimal 321, 321f

#/texts/17789 #/texts/17790 #/texts/17791 #/texts/17792 #/texts/17793 #/texts/17794 #/texts/17795 #/texts/17796 #/texts/17797 #/texts/17798 #/texts/17799 #/texts/17800 #/texts/17801 #/texts/17802 #/texts/17803 #/texts/17804 #/texts/17805 #/texts/17806 #/texts/17807 #/texts/17808 #/texts/17809 #/texts/17810 #/texts/17811 #/texts/17812 #/texts/17813 #/texts/17814 #/texts/17815 #/texts/17816 #/texts/17817 #/texts/17818 #/texts/17819 #/texts/17820 #/texts/17821 #/texts/17822 #/texts/17823 #/texts/17824 #/texts/17825 #/texts/17826 #/texts/17827 #/texts/17828 #/texts/17829 #/texts/17830 #/texts/17831 #/texts/17832 #/texts/17833 #/texts/17834 #/texts/17835 #/texts/17836 #/texts/17837 #/texts/17838 #/texts/17839 #/texts/17840 #/texts/17841 #/texts/17842 #/texts/17843 #/texts/17844 #/texts/17845 #/texts/17846 #/texts/17847 #/texts/17848 #/texts/17849 #/texts/17850 #/texts/17851 #/texts/17852 #/texts/17853 #/texts/17854 #/texts/17855 #/texts/17856

#/groups/1120 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/groups/1121 #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

lactating 284
mixed 38
multicellular 37, 39f
outer 265
secretory elements of 39f
types of 38f
Glands of Brunner 198
Glands of Krause 321
Glands of Moll 320
Glands of Wolfring 320
Glands of Zeis 320
Glandular elements, structure of 284
Glandular tissue 284
arrangement of 267f
Glans penis 265
Glassy membrane 147
Glial fibrillary acidic protein 16
Gliocytes, capsular 116
Gliosis 117
Gliosomes 116
Glisson’s capsule 212
Globose nucleus 247
Glomerular basement membrane 229
Glomerular capsule 227
Glomerular
barrier
229
Glomeruli, efferent arterioles of 234f
Glomerulonephritis 238
Glomerulus 227, 251
Glomus 137, 137f, 138
cells 304
tumor 138
Glossitis, atrophic 185
Glucagon 219
Glucose, storage of 211
Glycocalyx 7, 35, 202
Glycolipid and glycoprotein molecules 7f
Glycoprotein 196
structural 52
Glycosaminoglycans 52, 61
types of 52t
Golgi apparatus 13, 14, 284
Golgi cells 247, 250
Golgi complex 13, 14, 215, 262
role of 14f structure of 14f
Golgi hydrolase vesicles 15
Gonadotrophs 291
Gonadotropin-releasing hormone 281
Graafian follicle 273
Granular cortex 247
Granular layer 249
inner 245
outer 245
Granular layer of Tomes 180
Granule 48
cells 244, 247, 250
small 168
Granulosa cells 272, 274
Graves' disease 306
Gravid uterus, wall of 160
Gravis 96
Gray matter 240, 241
Ground substance 51, 61, 69
Growth, interstitial 60, 65
Guanosine triphosphate 13
Gustatory cells 183

#/texts/17863 #/texts/17864 #/texts/17865 #/texts/17866 #/texts/17867 #/texts/17868 #/texts/17869 #/texts/17870 #/texts/17871 #/texts/17872 #/texts/17873 #/texts/17874 #/texts/17875 #/texts/17876 #/texts/17877 #/texts/17878 #/texts/17879 #/texts/17880 #/texts/17881 #/texts/17882 #/texts/17883 #/texts/17884 #/texts/17885 #/texts/17886 #/texts/17887 #/texts/17888 #/texts/17889 #/texts/17890 #/texts/17891 #/texts/17892 #/texts/17893 #/texts/17894 #/texts/17895 #/texts/17896 #/texts/17897 #/texts/17898 #/texts/17899 #/texts/17900 #/texts/17901 #/texts/17902 #/texts/17903 #/texts/17904 #/texts/17905 #/texts/17906 #/texts/17907 #/texts/17908 #/texts/17909 #/texts/17910 #/texts/17911 #/texts/17912 #/texts/17913 #/texts/17914 #/texts/17915 #/texts/17916

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

cell 333, 335, a3 inner 333, outer 333, i follicle 145, 146, op ae, "ire. 337 structure of 146 papilla 145 parts of 145 shaft, structure of 145 Hairy skin 139, 140 Hand lens, use of 1 Hassall's corpuscles 106 Haversian canal 71, 72f Haversian system 71, 72, 72f, 74 atrophic 74, 77 typical 74, 77 Hearing, mechanism of 336 Heart 137 failure cells 174 Helicotrema 336 Hemangioma 138 Hematopoietic tissue 72 Hemidesmosomes 10 Hemolymphatic tissue 44 Hemorrhoids 209 Henle's layer 146 Heparan sulfate 52
H
Heparin 52
Hepatic coma 223
Hepatic lobules 212, 214f
Hepatic sinusoids 212
Hepatobiliary system 211
Hepatocyte 215
cells surface of 215f
Hepatopancreatic duct 219
Herring bodies 294
Hertwig's epithelial root sheath 186
Heterochromatic nucleus 17f
Heterochromatin 17
Heterocrine 152
Hilum 225
Hilus cells 272
Histaminocytes 47
Histiocyte 47, 47f
Histone 17
complex forming nucleosome core, composition of 18f
Hodgkin's disease 112
Hofbauer cell 49
Holocrine 37, 38f
Homocrine 152
Hordeolum, external 324
Hormones 39, 278, 289
antidiuretic 294
control of secretion of 295
corticotropic 291
formation of 298fc inhibin 257
luteinizing 257, 281, 291
production 160
Horn, anterior 241
Houstra coli 205
Huxley's layer 146
Hyaline membrane disease 176
Hyaloplasm 5, 12
Hyaluronic acid 52
Hydronephrosis 238
Hydroxylysine 50
Hydroxyproline 50
Hyperparathyroidism 306
primary 306
secondary 306
Hyperplasia 281
Hypertension 138, 238
Hyperthyroidism 306
Hypertrophy 281
benign prostatic 268
Hypolemmal junctions 153
Hypoparathyroidism 306
Hypophysis cerebri 290, 292, 294f blood supply of 294
pars posterior of 295f subdivisions of 290f

#/texts/17917 #/texts/17918 #/texts/17919 #/texts/17920 #/texts/17921 #/texts/17922 #/texts/17923 #/texts/17924 #/texts/17925 #/texts/17926 #/texts/17927 #/texts/17928 #/texts/17929 #/texts/17930 #/texts/17931 #/texts/17932 #/texts/17933 #/texts/17934 #/texts/17935 #/texts/17936 #/texts/17937 #/texts/17938 #/texts/17939 #/texts/17940 #/texts/17941 #/texts/17942 #/texts/17943 #/texts/17944 #/texts/17945 #/texts/17946 #/texts/17947 #/texts/17948 #/texts/17949 #/texts/17950 #/texts/17951 #/texts/17952 #/texts/17953 #/texts/17954 #/texts/17955 #/texts/17956 #/texts/17957 #/texts/17958 #/texts/17959 #/texts/17960 #/texts/17961 #/texts/17962 #/texts/17963 #/texts/17964 #/texts/17965 #/texts/17966 #/texts/17967 #/texts/17968 #/texts/17969 #/texts/17970 #/texts/17971 #/texts/17972 #/texts/17973 #/texts/17974 #/texts/17975 #/texts/17976 #/texts/17977 #/texts/17978 #/texts/17979 #/texts/17980 #/texts/17981 #/texts/17982

#/body #/body #/body #/body #/body #/body #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/groups/1122 #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Hypothalamo-hypophyseal portal system 295, 295f
Hypothalamus 295f
Hypothyroidism 306
Immotile cilia syndrome 21, 36, 17
Immune responses, role in 111
system 100
Immunity
cellular 100
humoral 100
Immunoglobulins 100
Immunoregulation, disorders of 112
Infections 112
Infundibulum 276, 290
Inner band of Baillarger 245
Insulin 219
Intercalated disc 93
electron microscopic structure of part of 93f
Intercellular secretory canaliculi 41
Intercellular substance 69
Interlobular arteries 233
Intermediate filaments 15, 16
functions of 16
Internal ear 325, 327
basic structure of 328f
Internode 119
Interstitial cell of Leydig 257
Interstitial cells 303
stimulating hormone 257f, 291
Intervillous space 162, 162f
Intestinal absorptive cells 20f
Intestine 188, 204, 205, 205t
Intramembranous ossification 75, 76f, 78
Iodine, activation of 298
Ions, inorganic 69
Iris 307, 312
Islets of Langerhans 221
Isogenous cell groups 61
Isthmus 276, 327
Joint, ligaments of 53
Junctional complex 10f
Junctional tubule 232
Juxtaglomerular apparatus 232
Juxtaglomerular cells 233
functions of 233
Juxtamedullary glomeruli 234 234
Juxtamedullary nephrons 227
Kartagener's syndrome 36
Keratan sulfate 52
Keratin 31
precursor of 143
Keratinization, zone of 144
Keratinized stratified squamous epithelium 31
Keratinocytes 144
Keratitis 323
Keratoconus 324
Keratohyalin 143
Kidney 224, 225f, 226
floating 56
macroscopic structure 224
proximal convoluted tubules of 24
Kinetic balance 333
Kinetosome 34
Kinocilia 20, 34
Koilonychia 151
Krebs cycle 13
Kulchitsky cells 168

#/texts/17984 #/texts/17985 #/texts/17986 #/texts/17987 #/texts/17988 #/texts/17989

```
Labyrinth 327 Lacis cells 233 Lactiferous duct 284, 285 sinus 284, 285 Lactogenic hormone 291 Lacunae 61, 71 Lamellae 71 constitute bone 71f interstitial 71 Lamellar bone 75 Lamina 314 cribrosa 308 densa 32, 229 fusca 308, 311 lucida 32 rara interna 229 stroma 276 Langerhans bodies 144 cell 49 dendritic cells of 144 Laryngitis acute 176 chronic 176
```
Larynx 167 anterior view of 168f cartilage of 167 Lateral funiculus 243 Lateral gray column 243 Leishmaniasis 112 Lemmocytes 116 Lemnisci 240 Lens 307, 317, 324 capsule 319 epithelium 319 fibers 319 objective 1 Leukocytes 45, 48 Light microscope, parts of 1 Light source 2 Limbus lamina spiralis 332 Limiting membrane, outer 314 Linea gravidarum 151 Lines of Retzius 179 Lingual tonsil 109, 181 Lip 177, 178f, 185 longitudinal section through 178 Lipoblasts 53 Lipofuscin 257 Lipoprotein, low-density 130 Liquor folliculi 273 Littoral cells 110 Littre’s glands 238 Liver 211, 213, 216, 222 blood supply of 211 cells of 215, 215f
cirrhosis of 112
lobule 212 classical 212, 212f macroscopic structure of 212 Lobar arteries 233 Lobar bronchi 171 Lobe 225 posterior 291 Lobular bronchiole 171 Lobule 284 classical 212 Long bone, growth of 79 Loop of Henle 230 Lung 171, 173 Lunule of nail 150f Lutein 274 Lymph 98, 99 capillaries 99 vessels, larger 99 Lymph node 100-103, 111 blood supply of 102 structure of 102f Lymphadenitis 112 Lymphangitis 111 acute 111 chronic 111 Lymphatic capillaries 99 Lymphatic duct, right 98 Lymphatic follicles 102 Lymphatic nodules 102 Lymphatic vessels 98, 99 Lymphedema 111 Lymph node 98 Lymphoblasts 102
malignancies 112
glycogen storage disease 21

#/texts/17990 #/texts/17991 #/texts/17992 #/texts/17993 #/texts/17994 #/texts/17995 #/texts/17996 #/texts/17997 #/texts/17998 #/texts/17999 #/texts/18000 #/texts/18001 #/texts/18002 #/texts/18003 #/texts/18004 #/texts/18005 #/texts/18006 #/texts/18007 #/texts/18008 #/texts/18009 #/texts/18010 #/texts/18011 #/texts/18012 #/texts/18013 #/texts/18014 #/texts/18015 #/texts/18016 #/texts/18017 #/texts/18018 #/texts/18019 #/texts/18020 #/texts/18021 #/texts/18022 #/texts/18023 #/texts/18024 #/texts/18025 #/texts/18026 #/texts/18027 #/texts/18028 #/texts/18029 #/texts/18030 #/texts/18031 #/texts/18032 #/texts/18033 #/texts/18034 #/texts/18035 #/texts/18036 #/texts/18037 #/texts/18038 #/texts/18039

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Lymphocytes 48, 48f, 100 functions of 100 Lymphohematogenous Lymphoid organ discrete 99 primary 98, 108 secondary 98 Lymphoid system 98 Lymphoid tissue 98 bronchial-associated 99 diffuse 98, 99 gut-associated 99 Lymphokines 100 Lysosomal Lysosome 14, 15f, 215 primary 15 secondary 15 types of 15fc
M cells 204
Macrophage 57, 108, 110
cells 47, 47f, 211
fixed 45
Macula 332
adherens 9
communicans 11
densa 233
Malaria 112
Male genital system, control of 257/fc
Male reproductive system 252, 253f
Malpighian bodies 105
Malpighian corpuscle 225
Malpighian layer 143
Malt 99, 108
Mammary gland 284-286, 288
active 284
lactating 287t
Mammotropin 291
Marfan's syndrome 59
Marrow cavity 72, 79
Masson's trichrome 49
Mast cells 45, 47, 47f
Masticatory mucosa 185
Mastocytes 47
Maternal cotyledons 160, 161f
Matrix 13, 51
capsular 61, 65
extracellular 60, 130
intercellular 60
unterstitial 61
vesicles 68
Maturation 260
Mediastinum testis 253
Medulla 102, 145, 224, 270
Medullary rays 225
Meibomian glands 320
Meiotic division, second 258
Meissner's plexus 190, 204
Melanocyte 144, 144f stimulating hormone 294
Melanoma, malignant 151
Melanophores 46
Melatonin 303
Membrana granulosa 273
Membrana tectoria 333
Membrane bones 75
internal limiting 317
of Bruch 311
outer 13

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#/body #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124 #/groups/1124

proteins 7f ruffled 69 T-system of 89 Membranous labyrinth 327, 330, 332f parts of 331f Menstrual cycle 278 endometrial changes in 278 stages of 281f Merocrine 37, 38f, 285 Mesangium 227, 229f Mesaxon 118 Mesenchymal cells 46f, 165 undifferentiated 46 Mesenchymal condensation 75 Mesenchyme 44 Mesothelium 24, 190 Metaphysis 81 Metastatic splenic tumors 112 Microcirculatory unit 138 Microfilaments 15 Microglia 49, 118 Microglial cells 110, 117f Microscope 1, 1f compound 2f dark-field 3 electron 3, 24, 115f, 260f fluorescence 3
interference 3
types of 3
Microscopy, principle of 2
Microtubules 15, 16f
Microvilli 20, 20f, 27, 33, 35, 35t, 189
Midget ganglion cells 322
Milky spots 111
Mitochondria 13
Mitochondrial cytopathy syndromes 21
Mitochondrion, structure of 13f
Mittleschiebe 88
Monoblasts 109
Monocyte 45, 48
phagocyte system 109
Mononuclear phagocyte system 47, 109, 217
cells of 109
functions of 111
Monosomes 13
Montgomery, tubercles of 287
Mossy fibers 250
Motor endplate 91
Motor fibers 121
Motor neuron 120
disease 251
single 91f
Motor unit 91
Muciphages 205
Mucocutaneous junction 177
Mucoepidermoid carcinoma 42
Mucoid connective tissue 44, 165
Mucoid tissue 57, 58f
Mucosa 168, 189, 191, 208
lining 185

#/texts/18075 #/texts/18076 #/texts/18077 #/texts/18078 #/texts/18079 #/texts/18080 #/texts/18081 #/texts/18082 #/texts/18083 #/texts/18084 #/texts/18085 #/texts/18086 #/texts/18087 #/texts/18088 #/texts/18089 #/texts/18090 #/texts/18091 #/texts/18092 #/texts/18093 #/texts/18094 #/texts/18095 #/texts/18096 #/texts/18097 #/texts/18098 #/texts/18099 #/texts/18100 #/texts/18101 #/texts/18102 #/texts/18103 #/texts/18104 #/texts/18105 #/texts/18106 #/texts/18107 #/texts/18108 #/texts/18109 #/texts/18110 #/texts/18111 #/texts/18112 #/texts/18113 #/texts/18114 #/texts/18115 #/texts/18116 #/texts/18117 #/texts/18118 #/texts/18119 #/texts/18120 #/texts/18121 #/texts/18122 #/texts/18123 #/texts/18124 #/texts/18125 #/texts/18126 #/texts/18127 #/texts/18128 #/texts/18129 #/texts/18130 #/texts/18131 #/texts/18132 #/texts/18133 #/texts/18134 #/texts/18135

#/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/groups/1125 #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/groups/1126 #/groups/1126 #/groups/1126 #/groups/1126 #/groups/1126 #/groups/1126 #/groups/1126 #/groups/1126 #/groups/1126 #/groups/1126 #/groups/1126 #/groups/1126

Mucosa-associated lymphoid tissue 98, 99, 108
in alimentary system 109
in respiratory system 109
Mucosal layer, inner 327
Mucous 21, 153
acini 41, 41f, 41t
gland 37
inner 265
layer, lamina propria of 235
membrane 181, 189, 193, 198, 204, 208, 235, 238, 262, 276, 282
neck cells 196
secreting Goblet cells 193, 202
structure of 205f
Miiller’s cells 317
Miiller’s muscle 94
Miillerian inhibitory substance 255
Multiadhesive glycoproteins 52, 61
Multilamellar bodies 174
Multilaminar primary follicle 273
Multilayered epithelia 23
Multilocular
adipose
tissue
56,
57f
Multipolar neurons 120, 120f
Multivesicular bodies 15, 15f
Muscle 262
bundle 84f
coat 235, 238, 282
dystrophy 96
fiber 83, 84f
layer contains striated 190
number of 91f
white 90, 90t hypertrophy 97
involuntary 84
Muscular artery 132, 132t, 133
Muscularis mucosae 189, 190, 197, 204
Myelin sheath 118, 118f, 119
of axon, segment of 117f plain 93 spindles 91 tendon of 53 tissue 83 triad 89 Muscular arterioles 132 Muscular coat 235 Muscular tissue 83 types of 83 Muscular venules 135 Myasthenia 96 gravis 96, 97f, 112 formation of 118, 119f functions of 119 short segment of 119f Myelinated axons 118
Myelinated nerves 120
Myenteric plexus 190
of Auerbach 190
Myocardium 137
Myocytes 83
cardiac 93
Myoepithelial cells 96, 148, 153
Myoepitheliocytes 96
Myofibril 84
contracts 88f
structure of 87
Myofilaments 87
in sarcomere, arrangement of 88f
Myoglobin 89
Myomesin 89
Myometrium 278
Myosin 87, 88f
filament 88, 88f
molecule, structure of 88f
Myxedema 306

#/texts/18137 #/texts/18138 #/texts/18139 #/texts/18140 #/texts/18141 #/texts/18142 #/texts/18143 #/texts/18144 #/texts/18145 #/texts/18146 #/texts/18147 #/texts/18148 #/texts/18149 #/texts/18150 #/texts/18151 #/texts/18152 #/texts/18153 #/texts/18154 #/texts/18155 #/texts/18156 #/texts/18157 #/texts/18158 #/texts/18159 #/texts/18160 #/texts/18161 #/texts/18162 #/texts/18163 #/texts/18164 #/texts/18165 #/texts/18166 #/texts/18167 #/texts/18168 #/texts/18169 #/texts/18170 #/texts/18171 #/texts/18172 #/texts/18173 #/texts/18174 #/texts/18175 #/texts/18176 #/texts/18177 #/texts/18178 #/texts/18179 #/texts/18180 #/texts/18181 #/texts/18182 #/texts/18183 #/texts/18184 #/texts/18185 #/texts/18186 #/texts/18187 #/texts/18188 #/texts/18189 #/texts/18190 #/texts/18191 #/texts/18192 #/texts/18193 #/texts/18194 #/texts/18195 #/texts/18196 #/texts/18197 #/texts/18198 #/texts/18199 #/texts/18200 #/texts/18201 #/texts/18202 #/texts/18203 #/texts/18204 #/texts/18205 #/texts/18206 #/texts/18207 #/texts/18208 #/texts/18209 #/texts/18210 #/texts/18211 #/texts/18212 #/texts/18213 #/texts/18214

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bed 150
growth of 150
parts of 150f
Nasal cavities 166
Natural killer cells 48
Neocortex, structure of 244
Neonatal line 180
Nephron 225
parts of 225f
Nephrotic syndrome 238
Nerve
fibers 115
myelinated 120t
unmyelinated 120t
plexuses 190
tracts 115
unmyelinated 120
Nervi vasorum 130
Nervous system 113
anatomical classification of 114fc
classification of 241fc
Nervous tissue 113
tumors of 126
Nervus conarii 303
Neurilemma 119
Neurilemmal sheath 119
Neurites 115
ischemic 126
Neuroeffector junctions 153
Neuroendocrine system 290
diffuse 40, 305
Neuroendocrine transducers 295
Neuroepithelial cells 183
Neurofibrils 114, 115f
Neurofilament protein 16
Neuroglia 113, 116
form cell 244f
found, types of 117/fc
Neuroglial cells 116, 118
Neurohypophysis 291, 294
Neuron 113, 114f
arrangement of 249f
horizontal 322
morphological classification of 120t
structure of 113, 115f
types of 120
Neurovascular hilus 91
Nexins 11
Niemann-Pick’s disease 112
Nipple 284, 287
Nissl bodies 114
Nissl granules 116
Nissl substance 114, 114f
Nodes of Ranvier 119
Nodules primary 102
secondary 102
Nonciliated columnar cells 167
Non-Hodgkin's lymphomas 112
Nonpolar end 5
Nonstriated muscle 83
Normal transmission 97f
Nuclear envelope 17
Nuclear lamina 17
Nuclear layer external 314
internal 315
Nuclear membrane 17
Nuclear pores 17
Nucleoli 17, 18
pars chromosoma of 18
Nucleolus, electron microscopy structure of 18f
Nucleoplasm 17
Nucleosome 17
core 18
Nucleus 5, 16, 240
emboliform 247
Nutrition 135, 160
Nutritional lobule 214
Ocular conjunctiva 320

#/texts/18215 #/texts/18216 #/texts/18217 #/texts/18218 #/texts/18219 #/texts/18220 #/texts/18221 #/texts/18222 #/texts/18223 #/texts/18224 #/texts/18225 #/texts/18226 #/texts/18227 #/texts/18228 #/texts/18229 #/texts/18230 #/texts/18231 #/texts/18232 #/texts/18233 #/texts/18234 #/texts/18235 #/texts/18236 #/texts/18237 #/texts/18238 #/texts/18239 #/texts/18240 #/texts/18241 #/texts/18242 #/texts/18243 #/texts/18244 #/texts/18245 #/texts/18246 #/texts/18247 #/texts/18248 #/texts/18249 #/texts/18250 #/texts/18251 #/texts/18252 #/texts/18253 #/texts/18254 #/texts/18255 #/texts/18256 #/texts/18257 #/texts/18258 #/texts/18259 #/texts/18260 #/texts/18261 #/texts/18262 #/texts/18263 #/texts/18264 #/texts/18265 #/texts/18266

#/body #/body #/body #/body #/body #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/groups/1129 #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Oddi, sphincter of 219, 219f
Odontoblasts 180, 186
Olfactory cells 175
Olfactory cilia 20, 34
Olfactory epithelium 167, 175, 175f Olfactory mucosa 167, 175f Oligodendrocyte 117, 117f, 118 Onychia 150 Oocyte primary 272 secondary 272 Oogenesis 272 stages of 272fc Oogonia 272 Open-face nuclei 17 Oppositional growth 60, 65 Optic cup 312 Optic nerve 121, 123 fibers, layer of 317 Optic stalk 312 Optic vesicle 312 Oral cavity 177 proper 177 Oral mucosa 185 Organ of Corti cell in 333f structure of 331f Organelles 5 Organic ground substance, mixture of 69 Organic matrix 69 Organization 84 Organs 290 Ossification 65, 75
centers of 78
primary center of 78, 80f
Osteitis deformans 81
Osteoarthritis 65
Osteoblast 67, 67f, 68, 68f, 75
Osteocalcin 69
Osteocartilaginous exostoses 65
Osteochondromas 65
Osteoclast 49, 67f, 68, 68f
multinucleated 68f
Osteogenesis imperfecta 59
Osteogenetic layer 70
Osteoid 69, 75
collagen 69
Osteoma 81
Osteonectin 69
Osteons
primary 74, 77
secondary 74, 77
Osteopetrosis 81
Osteoprogenitor cells 68
Osteosarcoma 81
Otolithic membrane 332
Otoscopy 327
Ovarian cycle 275
Ovarian follicle 270, 272, 274f
fate of 275, 275f
formation of 272
mature 273f
Ovarian tumors 287
Ovary 269, 271, 274f, 287
histological structure of 270f
Oviducts 276
Ovula nabothi 281
Ovulation 273
pain 287
time of 274f
Ovum fate of 274
mature 272
structure of 274f
Oxyntic cell 196
electron microscope structure of 196f
Oxyphil cells 300
Oxytocin 294
Paget's disease 81
Palatine tonsil 109, 110
coronal section of 109f

#/texts/18267 #/texts/18268 #/texts/18269 #/texts/18270 #/texts/18271 #/texts/18272 #/texts/18273 #/texts/18274 #/texts/18275 #/texts/18276 #/texts/18277 #/texts/18278 #/texts/18279 #/texts/18280 #/texts/18281 #/texts/18282 #/texts/18283 #/texts/18284 #/texts/18285 #/texts/18286 #/texts/18287 #/texts/18288 #/texts/18289 #/texts/18290 #/texts/18291 #/texts/18292 #/texts/18293 #/texts/18294 #/texts/18295 #/texts/18296 #/texts/18297 #/texts/18298 #/texts/18299 #/texts/18300 #/texts/18301 #/texts/18302 #/texts/18303 #/texts/18304 #/texts/18305 #/texts/18306 #/texts/18307 #/texts/18308 #/texts/18309 #/texts/18310 #/texts/18311 #/texts/18312 #/texts/18313 #/texts/18314 #/texts/18315 #/texts/18316 #/texts/18317 #/texts/18318 #/texts/18319 #/texts/18320 #/texts/18321 #/texts/18322 #/texts/18323 #/texts/18324 #/texts/18325 #/texts/18326 #/texts/18327 #/texts/18328 #/texts/18329 #/texts/18330 #/texts/18331 #/texts/18332 #/texts/18333 #/texts/18334 #/texts/18335 #/texts/18336 #/texts/18337 #/texts/18338 #/texts/18339 #/texts/18340 #/texts/18341

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130 #/groups/1130

Palms 144
Palpebral conjunctiva 320
types of 210t
Pancreatic acini 221
accessory 221
main 221
Pancreatic islets 221
Pancreatic serous acinus 221f
Pancreatitis acute 223
chronic 223
Paneth cells 202, 203, 203f
Panniculus adiposus 53
U
Papillae 181, 183f
Papillary plexus 145
Para-aortic bodies 303
Paracrine glands 37
Parafollicular cells 296
Parafollicular zone 108
Paraganglia 303
Parasympathetic nervous systems 113
Parathormone 299
Parathyroid gland 298, 299, 306
hormone 299
Parenchyma 39, 103, 1 Cn 2, 212
Parietal cells 196
Parkinson's disease 251
Paronychia 150
Parotid gland 155
pancreas 38
Parotid salivary gland 154
Pars amorpha 18
Pars anterior 291, 293
Pars distalis 291
Pars filamentosa 18
Pars granulosa 18
Pars intermedia 294, 294f
Pars nervosa 294
Pars tuberalis 291, 293
Passive transport 8
Pecten 208
Peduncles 240
Peg cells 276
Penicilli 103
Penis 265
transverse section of 267f
Peptic cells 196
Peptides, small 289
Perforating fibers of Sharpey 70
Periarterial lymphatic sheath 105
Perichondrium 60
Perichoroidal space 308
Pericytes 134, 135
Perilymph 330
Perilymphatic cells 330
Perimetrium 281
Perimysium 84, 86
Perinephric fat 53
Perineurium 121
Perinuclear space 17
Periodic acid-Schiff
method 32
stain 48
Periodontal ligament 179
Periodontal membrane 180
Periosteal bud 76
Periosteal collar 78, 80f
Periosteum 69
Peripheral nerves 115, 120, 122
basic anatomy of 121f
microscopic structure of 121
Peripheral nervous system 113, 117fc
Peristalsis 190
Peritoneum 219

#/texts/18342 #/texts/18343 #/texts/18344 #/texts/18345 #/texts/18346 #/texts/18347 #/texts/18348 #/texts/18349 #/texts/18350 #/texts/18351 #/texts/18352 #/texts/18353 #/texts/18354 #/texts/18355 #/texts/18356

#/groups/1130 #/groups/1130 #/groups/1130 #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

Peritubular dentin 180
Perivascular connective tissue 106
Peroxisomes 15
Peyer's patch 98, 204
Phagocytosis 8
Phagolysosomes 15, 15f
Phalangeal cells 333, 334
Pharyngeal tonsil 109
Phospholipid molecule, structure of 7f
Pharynx 167 Phosphatidylcholine 7f Physiological syncytium 93 Pia mater, modification of 241 Pigment cell 45, 46, 46f layer 312, 313 Pillar cell 333 Pineal body 303f Pineal gland 302 cyclic activity of 303 Pinealocytes 302 Pinna 325, 326 Pinocytosis 8 Pituicytes 294 Pituitary gland 290, 305 Placenta 160, 163 accreta 164 components of 160, 162f early 162 fetal surfaces of 161f formation of 161 functions 160 increta 164 maternal surfaces of 161f percreta 164 synthesizes 160
term 162
Placental abnormalities, structural 164
circulation and barrier 162
Placental Placental insufficiency 164 Planum semilunatum 336 Plaques, atheromatous 138 membrane 5 structure of 5 Plasmakinin 153 Plasmatocytes 48 Pleomorphic adenoma 42, 159 Pleura 174 Plexiform cells, internal 317, 322 Plexiform layer 245 external 315 internal 317 Plica circularis 189, 198 Pneumocytes 174, 174f Pneumonia, bacterial 176 Podocytes 227, 229f Polar body, second 272 Polar end 5 Polycystic ovarian disease 287 Polygonal cells 31f Polyribosomes 13 Polysomes 13 Pore complex 17 Portal acini 214 concept of 214f Portal canal 212 Portal hypertension 223 Portal lobules 214f Portal triad 212, 214f
Portal vein obstruction 112

#/texts/18357 #/texts/18358 #/texts/18359 #/texts/18360 #/texts/18361 #/texts/18362 #/texts/18363 #/texts/18364 #/texts/18365 #/texts/18366 #/texts/18367 #/texts/18368 #/texts/18369 #/texts/18370 #/texts/18371 #/texts/18372 #/texts/18373 #/texts/18374 #/texts/18375 #/texts/18376 #/texts/18377 #/texts/18378 #/texts/18379 #/texts/18380 #/texts/18381 #/texts/18382 #/texts/18383 #/texts/18384 #/texts/18385 #/texts/18386 #/texts/18387 #/texts/18388 #/texts/18389 #/texts/18390 #/texts/18391 #/texts/18392 #/texts/18393 #/texts/18394 #/texts/18395 #/texts/18396 #/texts/18397 #/texts/18398 #/texts/18399 #/texts/18400 #/texts/18401 #/texts/18402 #/texts/18403 #/texts/18404 #/texts/18405 #/texts/18406 #/texts/18407

#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131

Portio vaginalis 281 Postcapillary venules 135 Postmenstrual phase 278 Precapillary sphincter 133, 137, 138 Pregnancy corpus luteum of 275 ectopic tubal 287 test 287 tumor 185 Prickle cells 143 Primordial follicle 272, 273f Principal bronchi 171 Principal cells 232, 261, 262, 298 Principal respiratory organs 171 Prisms 179 Progesterone 281 Prognathism 305 Prolactin 291 Proliferative phase 278t Promonocytes 109 Prostaglandins 265 Prostate 265, 266, 267f carcinoma of 268 gland, lobes of 267f Protein 289 intermediate 9 link 9 microtubule-associated 15, 116 tubulin 15 Proteoglycans 52 Protofilaments 15
Protoplasm 5
Protoplasmic astrocytes 117
Proximal convoluted tubule 229, 230
Pseudostratified ciliated columnar epithelium 29
Pseudostratified columnar epithelium 19f, 29f
Pseudostratified epithelium 28
Psoriasis 150
Ptyalism 42
Pulmonary surfactant 174
Pulp 179, 180
canal 179
cavity 179
white 103
Pulpitis 185
Pupil 312
Purkinje cell 247, 249
layer 249
Pyloric glands 197
Pyloric sphincter 190
Pyogenic granuloma 185
Pyramidal cell 244, 244f
Pyramidal layer, outer 245
Ramus chorii 162f
Rapid transport 116
Receptor cells 183
Receptors 8
Rectum 208, 209
Red marrow 72
Red muscle fiber 90, 90t
Red pulp 103, 105
Renal blood vessels 233
Renal columns 225
consists of 225
Renal corpuscle 225, 227
basic structure of 227f
Renal cortex 228
deeper parts of 234f
superficial of 234f
Renal interstitium 234
Renal medulla 231
Renal pelvis 225
Renal pyramids 224
Renal sinus 225
Renal tubule 35, 225, 229
Renin 233
Reproductive system, female 269, 270f
Resident cells 44
Resorption bays 68
Respiratory bronchioles 171
Respiratory mucosa 167

#/texts/18408 #/texts/18409 #/texts/18410 #/texts/18411 #/texts/18412 #/texts/18413 #/texts/18414 #/texts/18415 #/texts/18416 #/texts/18417 #/texts/18418 #/texts/18419 #/texts/18420 #/texts/18421 #/texts/18422 #/texts/18423

#/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/groups/1131 #/body #/body #/body

Respiratory part of nasal mucosa, structure of 167f
Respiratory system 166
types of cells of 174 fc
Rete cutaneum 145
Rete ridge system 178
Rete subpapillare 145
Rete testis 253
structure of 257
Reticular cells 45, 57
Reticular fibers 49, 50, 50f, 51f, 60 |O
Reticular lamina 32, 333, 335, 33 I
Reticular plexus 145
Reticular tissue 57
basic structure of 313 ciliary part of 312 different parts of 323f iridial part of 312 layer of 313f nervous layer of 312 pigment cell of 314f Retinal detachment 324 Retinitis pigmentosa 324 Retinoblastoma 324 Rheumatoid arthritis 112 Rhinoplasty 336 Rhodopsin 314 Ribonucleic acid 12 Ribosomes 12, 13 Rod and cone 314 cells, ultrastructure of 314 density of 321 layer of 314 main parts of 315f Rod bipolar cells, terminals of 316f Rod cell 333 structure of 315f Rod ganglion cells 322 Rod spherule 316 synapsing 316f Rokitansky-Aschoff sinuses 219 Root 150, 179 formation 187 Root sheath inner 146
outer 146
Russell's bodies 49

#/texts/18426 #/texts/18427 #/texts/18428 #/texts/18429 #/texts/18430 #/texts/18431 #/texts/18432 #/texts/18433 #/texts/18434 #/texts/18435 #/texts/18436 #/texts/18437 #/texts/18438 #/texts/18439 #/texts/18440 #/texts/18441 #/texts/18442 #/texts/18443 #/texts/18444 #/texts/18445 #/texts/18446 #/texts/18447 #/texts/18448 #/texts/18449 #/texts/18450 #/texts/18451 #/texts/18452

#/groups/1132 #/groups/1132 #/groups/1132 #/groups/1132 #/groups/1132 #/groups/1133 #/groups/1133 #/groups/1133 #/groups/1133 #/groups/1133 #/groups/1133 #/groups/1133 #/groups/1133 #/groups/1133 #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body

cells of 154
innervation of 153 major 152, 159 minor 152 serous cell in 154f tumors of 42, 159 Salivary tumor, mixed 42 Saltatory conduction 119 Sarcolemma 84, 87 Sarcoplasm 84 Sarcoplasmic reticulum 89, 90f Satellite cells 116, 118 Scala tympani 328 Scala vestibuli 328, 330f sheath 119 Sclera 307 junction of 308f Scleral spur 308 Sclerous connective tissue 67 Sebaceous gland 42, 147, 147f, 148 Sebum 147 Secretion mode of 37
nature of 37
site of 37
Secretory cells 221, 276
Secretory component 153
Secretory function 189
Secretory part 152
Secretory phase 278t
Secretory unit 287
shape of 38
Secretory vacuoles, formation of 14f
Section across eyeball 308f Segmental bronchi 171 Sella turcica 290 Semicircular canals 327, 328, 328f Seminal vesicle 262, 264 Seminiferous tubules 253 general structure of 253 walls of 258f Sensory 126t ganglia 124-126 neuroepithelium 332 neuron 120 Seromucous cells 42, 153 Serous 41t acini 41, 153 cell 21, 41, 152, 153 coat 103 demilunes 153 gland 37 layer 189, 235 pericardium, visceral layer of 137 salivary gland 222, 222t Sertoli cells, cytoplasm of 258 Serum amylase 223
Sex glands, accessory 252
Sialadenitis 42
Sialorrhea 42
Sickle cell disease 112
Signet ring appearance 53
Sinus venosus sclerae 308
Sinusoid, structure of 135f
Sinusoidal surface 215
Sinusoids 129
contraction of 89, 94
fibers, types of 89
innervation of 91
lymphatic of 91
proteins in 89 structure of 84f transverse section of 86, 87f tumors 96 Skin 52 appendages of 145 blood supply of 145 dermis of 53, 55 functions 139 nerve supply of 145 thick 139, 139t, 141 thin 139, 139t, 140, 142f types of 139 Skull bones, growth of 79f Slit diaphragm 227 Slow transport 116 basic structure of 198f cells of 202, 202f structure 198

#/texts/18453 #/texts/18454 #/texts/18455 #/texts/18456 #/texts/18457 #/texts/18458 #/texts/18459 #/texts/18460 #/texts/18461 #/texts/18462 #/texts/18463

93-96, 96t Smooth muscle 83,
```
cells 94f, 130 aggregations of 94 layer 168, 172 Somatodendritic synapse 124 Somatosomatic synapse 124 Space of Disse 217 Special senses ear 325 eye 307 Spermatocytes primary 258 secondary 258 Spermatogenesis 257, 258f, 258fc stages of 257, 259fc Spermatogenic cells 253, 255 Spermatogonia 257 Spermatozoa 252 capacitation of 260 maturation of 260 Spermatozoon 260f structure of 260f mature 259 Spermiogenesis 258, 259 Spherocytosis 112 Sphincter 96, 190 ampullae 219 choledochus 219 muscle, internal 235 pancreaticus 219 pupillae 312 vesicae 235
```
Spinal cord 240, 241f, 242
cross-section of 243t
Spine 143
typical 143f Spiral artery 278 Spiral ganglion 331 Spiral lamina 328 Spiral ligament 331 Spiral limbus 332 Spiral prominence 331 Spiral sheath 260 Spiral sulcus internal 332 outer 331 Spleen 98, 103, 104, 112 Splenectomy 105 Splenic circulation 105f Splenic cords 105 Splenic sinusoids 105 Splenic tumors, primary 112 Splenic vein obstruction 112 Splenomegaly 112 causes of 112 Spongiocytes 300 Spongy bone 71, 72 Squamous cell carcinoma 151 Squamous epithelium 24 simple 24, 24f, 25
Squamous metaplasia 36
Squamous nonkeratinized epithelium, stratified 178, 191
Stave cells 103
Stellate cell 244f, 247
inner 250

#/texts/18465 #/texts/18466 #/texts/18467 #/texts/18468 #/texts/18469 #/texts/18470 #/texts/18471 #/texts/18472 #/texts/18473 #/texts/18474 #/texts/18475 #/texts/18476 #/texts/18477 #/texts/18478 #/texts/18479 #/texts/18480 #/texts/18481 #/texts/18482 #/texts/18483 #/texts/18484 #/texts/18485 #/texts/18486 #/texts/18487 #/texts/18488 #/texts/18489 #/texts/18490 #/texts/18491 #/texts/18492 #/texts/18493 #/texts/18494 #/texts/18495 #/texts/18496 #/texts/18497 #/texts/18498 #/texts/18499 #/texts/18500 #/texts/18501 #/texts/18502 #/texts/18503 #/texts/18504 #/texts/18505 #/texts/18506 #/texts/18507 #/texts/18508 #/texts/18509

#/groups/1134 #/groups/1134 #/groups/1134 #/groups/1134 #/groups/1134 #/groups/1134 #/groups/1134 #/groups/1134 #/groups/1134 #/groups/1134 #/groups/1134 #/groups/1134 #/groups/1134 #/groups/1134 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135 #/groups/1135

Stellate neurons 244
Stem cells 46, 108, 197
adult 45
intermediate 144
Stem villus 162
Stereocilia 21, 33, 36
Steroids 289
Stigma 273
Stomach 188, 191, 209
anatomical regions of 191f
basic structure of mucous membrane of 193f
functions 193 fundus 188, 194 microscopic features 193 pylorus 188, 195 region of 193t Storage diseases 112 Storage vesicles 14 Stratum basale 143 Stratum corneum 144 Stratum epithelial granulosum 146 pallidum 146 Stratum germinativum 143 Stratum granulosum 143 Stratum intermedium 186 Stratum lucidum 143, 144 Stratum spinosum 143 cells of 143f Stria vascularis 330, 331
Striate cortex 247
Striated border 20, 27, 20 INO
appearance 35
Striated ducts 153
Striated muscle 83
ultrastructure of 85
Stye 324
Subcapsular sinus 102
Subcutis 142
Subendothelial connective tissue 129, 130
Sublingual salivary gland 158, 159
Submandibular gland 154, 156, 157
Submucosa 168, 189-191, 197, 204, 205, 208, 238
glands in 172
Submucosal plexus of Meissner 190
Submucous layer 235
Sulcus terminalis 181
Supercoiling 17
Supporting cells 183, 335
of Sertoli 253
Suprachoroid lamina 308, 311
Supranuclear granules 203f
Suprarenal cortex 300
Suprarenal gland 300, 301, 306
Suprarenal medulla 302
Suspensory ligaments 284
Sweat gland 38, 42, 147, 149f
atrophic 149
parts of typical 149f
typical 147
Synapse 116, 124
Synaptic transmission, mechanism of 128f
Synaptic zone, inner 317

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#/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/body #/groups/1136 #/groups/1136 #/groups/1136 #/groups/1136 #/groups/1136 #/groups/1136 #/groups/1137 #/groups/1137 #/groups/1137 #/groups/1137 #/groups/1137 #/groups/1137

Taenia coli 190, 205
Tanycytes 118, 295
Tarsal gland 42, 320
Tarsal plate 320
Tarsus 320
Taste bud 183, 183f, 185f
Tay-Sachs disease 21
Teeth 177, 179
Tendon, transverse section through 53f
Terminal arterioles 132
Terminal bronchioles 171
Terminal buds 302
Terminal web 202
Tertiary hyperparathyroidism 306
Testicular tumors 268
Testis 252, 254, 256
basic structure of 253f general structure of 252
Testosterone 252
Thalassemias 112
Theca externa 273
Theca interna 273
Thoracic duct 98
Thoroughfare channels 137
Thymic humoral factor 108
Thymic nurse cells 106
Thymocytes 106, 108
Thymopoietin 108
Thymosin
alpha-1 108
beta-4 108
Thymulin 108 Thymus 98, 106, 107, 112 functions of 108 lymphocytes of 108 Thyroglobulin, synthesis of 298 Thyroid follicles, appearance of 296f gland 295, 297, 298fc, 306 follicular cell of 296f hormone, synthesis of 298 Thyrotropes 291 Thyrotropic hormone 291 Thyrotropin 291 Tight junctions, functions of 11 Tissue elastic 57 plasminogen activator 130 processing 3 T-lymphocytes 48, 49, 100 circulation of 99f Tomes process 186 Tongue 177, 181, 182f, 185 bald 185 dorsal surface of 181f fissured 185 geographic 185 glossitis 185 hairy 185
and palate, minor salivary glands of 42
mucous glands of 183
serous glands of 183
Tonsil 98
Tonsillar crypts 109
Tonsillectomy 112
Tonsillitis 112
Tonsils 109, 112
Tooth 185

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development, stages in 185, 186f part of 180f Trabeculae 103 Trachea 168, 170 Transitional zone 208 Transmembrane proteins 7 Triads 316 Trichohyaline granules 146 Trophoblast 161 Tropocollagen 49 Tropoelastin 51 Tropomyosin 88 Troponin 88, 94 Truncus chorii 162, 162f Trypsin-like substances 260 Tubal tonsils 109 Tubular glands 38 Tubules 16f Tubuli recti 253 Tubuloalveolar glands, branched 152 Tumor 36, 151 chondroblastic 65 Tunica adventitia 129, 131, 132 Tunica albuginea 252, 270 Tunica intima 129, 132 Tunica media 129, 130, 132
Tunica vaginalis 252
Tunica vasculosa 252, 253
Tympani 330f
Tympanic cavity 327
Tympanic lip 332
Tympanic membrane 327
secondary 328
Tympanum 327
Typical long bone development of 78
formation of 80f
Ulcerative colitis 209
Ultraviolet microscope 3
Umbilical arteries 165
Umbilical cord 160, 161f, 164, 165f microscopic section of 164
Umbrella cells 235
Unicellular gland 37, 39f
Unicellular goblet cells 37
Unilaminar primary follicle 272
Unilocular adipose tissue 57f
Unipolar neurons 120, 120f
Ureter 234, 236
Ureterocele 238
Urethra 238
female 238
Urethral sphincter, external 238
Urinary bladder 94, 235, 237
Urinary pole 227
Urinary space 227
Urinary system 224
functions 224, 238
Urothelium 32
Uterine glands 278, 281f
Uterine part 276
Uterine tubes 276
Uterus 276, 279, 280, 287
living ligature of 281
wall of 161f
Utricle 330
Uvea 309
Vacuoplasm 5, 12
Vagina 282, 283, 288
Vaginitis 288
Vallate papilla 184f van Gieson method 49
Varicose veins 138
Vas deferens 94
Vasa recta ascending 234
descending 234

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Vasa vasorum 135
Vascular coat 309
Vascular lamina 311
Vascular pole 227
Vasopressin 294
Vasorum 130
Vein 129, 134
arcuate 234
interlobar 234
interlobular 234
large-sized 136
valves of 135
Ventral horn 241
Ventral white commissure 243
Venule 132f, 129, 135
Verhoeff’s method 51
Vermiform appendix 98, 188, 205, 207
Vermilion 178
Vestibular lip 332
Vestibule 167, 282, 327
Villi 189, 198, 202
Vimentin 16
Visual organs, accessory 319
Vitiligo 150
Vocal folds 167
Volkmann’s canal 72f
Voluntary muscle 83
von Kupffer cells 110
von Willebrand factor 130
Waldeyer's ring 109
Weibel-Palade bodies 130
Wharton's jelly 164
White fibrocartilage 63
White line of Hilton 208
White matter 240, 243
Wilson's disease 324
Wolff's law 81

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Yellow marrow 72
Zollinger-Ellison syndrome 209
Zona arcuata 330
Zona fasciculata 300
Zona glomerulosa 300
Zona pectinata 330
Zona pellucida 273
Zona reaction 260
Zona reticularis 300
Zonula adherens 10
Zonula occludens 11, 11f
Zwischenschiebe
88
Zymogen cells 196, 203

PL Dhingra ° Shruti Dhingra
Assisted by Deeksha Dhingra

Emeritus Consultant
Indraprastha Apollo Hospital, New Delhi
Formerly Director, Professor & Head
Department of Otolaryngology and Head & Neck Surgery
Maulana Azad Medical College and
Associated LNJP & GB Pant Hospitals, New Delhi

Member, International Medical Sciences Academy
Fellow, Laryngology and Voice Disorders
Assistant Professor, Department of Otolaryngology and Head & Neck Surgery,
BPS Govt. Medical College for Women, Haryana

Deeksha Dhingra, MD, PGDHA, MPH (University of Sydney)
Diseases of Ear, Nose and Throat & Head and Neck Surgery, 6/e
PL Dhingra, Shruti Dhingra and Deeksha Dhingra

Reed Elsevier India Private Limited.
© 2014 Elsevier, a division of Reed Elsevier India Private Limited.
First Edition 1992
Second Edition 1998
Third Edition 2004
Fourth Edition 2007
Fifth Edition 2010
Reprinted 2011 (Twice), 2012, 2013
Sixth Edition 2014
All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher.

Medical knowledge is constantly changing. As new information becomes available, changes in treatment, procedures, equipment and the use of drugs become necessary. The authors, editors, contributors and the publisher have, as far as it is possible, taken care to ensure that the information given in this text is accurate and up-to-date. However, readers are strongly advised to confirm that the information, especially with regard to drug dose/usage, complies with current legislation and standards of practice. Please consult full prescribing information before issuing prescriptions for any product mentioned in the publication.
Published by Elsevier, a division of Reed Elsevier India Private Limited.
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Printed and bound at Replica Press (P) Ltd., Kundli, Haryana.
Dedicated to all students: and who my past, present future are the inspiring force behind this work.
I reproduce below the invocation from our great ancient scripture—the Kathopanishad which shows the relationship between the teacher and the taught.
- S we at aadi we at ward we ay ecard! tafe At aia aq m fafemad:) S wmf: ent: wf:
"O God, the almighty, bless us both (the teacher and the student) together, develop us both together, give us strength
together. Let the knowledge acquired by us be bright and illuminant, and second to none. Let both of us live
together with love, affection and harmony. O God, let there be physical, mental and spiritual peace."

(17 September 1917 to 5 September 2013)
Obeisance to My Mentor, Friend and Guide
A household name in Mumbai, Dr LH Hiranandani contributed a great deal to the speciality of ENT and annexed Head & Neck Surgery to it. He was truly known as Father of Otolaryngology in India. He was awarded Padma Bhushan in 1972, Millennium Award in 2001, and SAARC, ENT Award in 2001 in recognition of his academic and research contributions and social causes. He was a great surgeon and teacher par excellence. To his credit, amongst other innovative surgical techniques, is the "Tongue Flap Operation" for closure of pharynx. His book on Histopathological Study of Middle Ear Cleft and Its Clinical Applications has received wide acclaim.
PL Dhingra
With this sixth edition, the book completes 22 years of service to its readers. The speciality of ENT, often called otolaryngology or otorhinolaryngology has diversified into several subspecialities of otology, otoneurology, rhinology, laryngology, bronchoesophagology, paediatric otolaryngology, skull base surgery, and now emerging subspeciality of neuro-rhinology where the brain tumours related to skull base are being treated by endoscopic nasal approaches. The growth in several branches owe their emergence to rapid strides being made in technology such as imaging techniques from simple X-ray to CT, MRI, MR angiography, PET-CT and simultaneous PET-MRI. The development of endoscopes from 4 mm to nearly 1 mm with various viewing angles, lasers, computers and miniature cameras which can be fixed on the tip of the flexible endoscopes have further revolutionized surgery. This further has given birth to minimally invasive surgery, navigation surgery and robotic surgery. With the growth of the speciality both in breadth and depth, it throws several challenges to authors on how much to introduce the subject yet to be concise but comprehensive, and not to lose sight of the basic fundamentals and clinical applications to students entering medical profession in a readable form.
In the present edition, all the chapters have been revised, updated and augmented. Some have been completely rewritten and expanded. New chapters have been added

on thyroid disorders, thyroid surgery and proptosis. Several new diagrams, algorithms, tables, flowcharts and clinical photographs have been added to make the subject easily understandable. The chapter on "Nuggets for Rapid Review" provides useful tips which help solve several MCQs often set in the university or board examinations.
Mnemonics set here and there are useful as aide memoire to recall and reproduce the subject for exam going students. The book is clinically oriented with practical approach to the patient as before and provides broad insight into the subject for undergraduates. It is hoped that this edition of the book will also prove useful to students of DLO, MS/MD and DNB (Diplomate National Board) as a foundation course before they take recourse to comprehensive volumes of the subject. It will also be useful to general practitioners, students of nursing, audiology and speech therapy, and to those studying alternative systems of medicine such as Ayurveda, Sidha, Unani and Tibbia, Homeopathy and Physiotherapy. The authors will feel gratified if the above objectives are fulfilled.
There is always scope for improvementin any work and the authors will welcome any suggestions and comments from learned teachers and students at pldhingra@gmail.com or indiacontact@elsevier.com.
PL Dhingra
Shruti Dhingra
In bringing out the sixth edition of this book, we owe a great deal of indebtedness to several eminent professors, teachers, faculty members, contributors and students for their support, encouragement, inspiration and suggestions. It may be difficult to name them all but we will be remiss if some of them are not mentioned. We extend our heartfelt thanks to:

e Dr Arun Agarwal, Director—Professor, Department of ENT and Head & Neck Surgery and ex-Dean, Maulana Azad Medical College, New Delhi; Dr Anoop Raj, Dr Achal Gulati, Dr JC Passey, Dr Ishwar Singh, Dr PK Rathore, Dr Ravi Meher and Dr Vikas Malhotra who form a galaxy of distinguished teachers.
¢ The entire faculty of University College of Medical Sciences, Delhi—Dr PP Singh, Dr Laxmi Vaid, Dr HC Taneja and Dr Neelima Gupta.
¢ Dr JK Sahni, Dr TS Anand and Dr Sunil Kumar, Lady Harding Medical College, Delhi.
e Dr SC Sharma, Professor and Head and Dr Alok Thakar, Department of ENT, AIIMS, New Delhi.
¢ Dr Arjan Das, Professor and Head, Department of ENT, Medical College, Chandigarh.
¢ Dr (Prof.) Naresh Panda, Dr (Prof.) AK Gupta, Dr Satyawati Mohindra and Dr Jayamanti Bakshi, Postgraduate Institute of Medical Sciences, Chandigarh.
e Dr Anirban Biswas, Neuro-otologist, Kolkata for his reviews of the book in Indian Journal of Otolaryngology.
¢ Dr AK Singhal, Dean, Professor and Head, FH Medical College, Tundla, Agra.
¢ Dr (Prof.) Amrik Singh, Guru Ram Das Medical College, Amritsar.
e Dr Karan Singh, Professor and Head, Medical College, Amritsar.
¢ Dr RC Yadav, Professor and Head, Medical College, Kota, Rajasthan.
e Dr Saurabh Varshney, Professor and Head, AIIMS, Rishikesh, Uttarakhand.
¢ Dr (Prof.) Rohit Saxena, Department of ENT, Sharda University, Greater Noida, Uttar Pradesh.
¢ Dr (Prof.) Hemant Chopra and Dr Munish Munjal, Dayanand Medical College, Ludhiana.
¢ Dr Dalbir Singh, Professor, Department of ENT, Govt. Medical College and Rajendra Hospital, Patiala, Punjab.

PL Dhingra
Dedication, v
Obeisance, vi
Preface, vii
Acknowledgements,
ix

1 Anatomy of Ear, 2
2 Peripheral Receptors and Physiology of Auditory and Vestibular Systems, 13
co Eustachian Tube and Its Disorders, 57
oN
O Assessment of Vestibular Functions, 41
U Hearing Loss, 29
FPF Assessment of Hearing, 21
W Audiology and Acoustics, 19
11 Cholesteatoma and Chronic Otitis Media, 67
12 Complications of Suppurative Otitis Media, 75
13 Otosclerosis (Syn. Otospongiosis),
14 Facial Nerve and Its Disorders, 90
15 Méniére’s Disease, 100
16 Tumours of External Ear, 106
17 Tumours of Middle Ear and Mastoid,
18 Acoustic Neuroma, 112
19 The Deaf Child, 115
20 Rehabilitation of the Hearing Impaired, 121
21 Otalgia (Earache), 128
22 Tinnitus, 130
Disorders of Vestibular System, 45
Diseases of External Ear, 48
Disorders of Middle Ear, 62
86
109

23 Anatomy of Nose, 134
24 Physiology of Nose, 140
25 Diseases of External Nose and Nasal Vestibule, 143
26 Nasal Septum and Its Diseases, 147
27 Acute and Chronic Rhinitis, 152
28 Granulomatous Diseases of Nose, 156
29 Miscellaneous Disorders of Nasal Cavity, 161
30 Allergic Rhinitis, 166
31 Vasomotor and Other Forms of Nonallergic Rhinitis, 170
32 Nasal Polypi, 172
33 Epistaxis, 176
34 Trauma tothe Face, 181
35 Anatomy and Physiology of Paranasal Sinuses, 187
36 Acute Sinusitis, 191
37 Chronic Sinusitis, 195
38 Complications of Sinusitis, 198
39 Benign and Malignant Neoplasms of Nasal Cavity, 202
40 Neoplasms of Paranasal
Sinuses, 205
41 Proptosis, 211
SECTION III DISEASES OF ORAL CAVITY AND SALIVARY GLANDS
42 Anatomy of Oral Cavity, 216
43
Common Disorders of Oral Cavity, 217
xii
CONTENTS
44 Tumours of Oral Cavity,
223
45 Non-neoplastic Disorders of Salivary
Glands,
231
46 Neoplasms of Salivary Glands, 234
SECTION IV DISEASES OF PHARYNX
47 Anatomy and Physiology of Pharynx, 238
48 Adenoids and Other Inflammations
of
Nasopharynx,
243
49 Tumours of Nasopharynx, 246
50 Acute and Chronic Pharyngitis, 254
51 Acute and Chronic Tonsillitis, 257
52 Head and Neck Space Infections, 263
53 Tumours of Oropharynx, 269
54 Tumours of the Hypopharynx and Pharyngeal
Pouch, 273
55 Snoring and Sleep Apnoea, 276
SECTION
V
DISEASES
OF
LARYNX
AND TRACHEA
56 Anatomy and Physiology of Larynx, 282
57 laryngotracheal Trauma, 287

58 Acute and Chronic Inflammations of Larynx, 289
59 Congenital Lesions of Larynx and Stridor, 295
60 Laryngeal Paralysis, 298
61 Benign Tumours of Larynx, 303
62 Cancer Larynx, 307
63 Voice and Speech Disorders, 313
64 Tracheostomy and Other Procedures for Airway Management, 316
65 Foreign Bodies of Air Passages, 321
SECTION VI THYROID GLAND AND ITS DISORDERS
66 Thyroid Gland and Its Disorders, 326

67 Anatomy and Physiology of Oesophagus, 340
68 Disorders of Oesophagus, 342
69 Dysphagia, 347
70 Foreign Bodies of Food Passage, 349
SECTION VIII RECENT ADVANCES
71 Laser Surgery, Radiofrequency Surgery and Hyperbaric Oxygen Therapy, 354
72 Cryosurgery, 360
73 Radiotherapy in Head and Neck Cancer, 362
74
Chemotherapy
for
Head
and
Neck
Cancer,
367
75 HIV Infection/AIDS and ENT Manifestations, 369
SECTION IX CLINICAL METHODS IN ENT
AND NECK MASSES
76 Clinical Methods in ENT, 374
77
Neck
Masses,
390

78 Myringotomy, 398
79 Cortical Mastoidectomy, 400
80 Radical Mastoidectomy, 403
81 Modified Radical Mastoidectomy, 405
82 Myringoplasty, 406
83 Proof Puncture (Syn. Antral Lavage), 408
84 Intranasal Inferior Meatal Antrostomy, 410
85 Caldwell-Luc (Anterior Antrostomy)
Operation,
411
86 Submucous Resection of Nasal Septum (SMR Operation), 413
87 Septoplasty, 415
88 Diagnostic Nasal Endoscopy, 417
89 Endoscopic Sinus Surgery, 419
90 Direct Laryngoscopy,
91 Bronchoscopy,
92 Oesophagoscopy,
93 Tonsillectomy,
94 Adenoidectomy,
95 Radiology in ENT,
422
424
426
428
431
433

APPENDIX |
Some Memorable Nuggets for Rapid
Review,
446
APPENDIX II Instruments,
451
Index,
465

The ear is divided into:
1. External ear
2. Middle ear
3. Internal ear or the labyrinth

The external ear consists of the (i) auricle or pinna, (ii) external acoustic canal and (iii) tympanic membrane (Figure 1.1A).

The entire pinna except its lobule and the outer part of external acoustic canal are made up of a framework of a single piece of yellow elastic cartilage covered with skin. The latter is closely adherent to the perichondrium on its lateral surface while it is slightly loose on the medial cranial surface. The various elevations and depressions seen on the lateral surface of pinna are shown in Figure 1.1B. There is no cartilage between the tragus and crus of the helix, and this area is called incisura terminalis (Figure 1.1C). An incision made in this area will not cut through the cartilage and is used for endaural approach in surgery of the external auditory canal or the mastoid. Pinna is also the source of several graft materials for the surgeon. Cartilage from the tragus, perichondrium from the tragus or concha and fat from the lobule are frequently used for reconstructive surgery of the middle ear. The conchal cartilage has also been used to correct the depressed nasal bridge while the composite grafts of the skin and cartilage from the pinna are sometimes used for repair of defects of nasal ala.

It extends from the bottom of the concha to the tympanic membrane and measures about 24 mm along its posterior wall. It is not a straight tube; its outer part is directed upwards, backwards and medially while its inner part is directed downwards, forwards and medially. Therefore, to see the tympanic membrane, the pinna has to be pulled upwards, backwards and laterally so as to bring the two parts in alignment.
The canal is divided into two parts: (i) cartilaginous and
(ii) bony.

It forms outer one-third (8 mm) of the canal. Cartilage is a continuation of the cartilage which forms the framework

of the pinna. It has two deficiencies—the "fissures of Santorini" in this part of the cartilage and through them the parotid or superficial mastoid infections can appear in the canal or vice versa. The skin covering the cartilaginous canal is thick and contains ceruminous and pilosebaceous glands which secrete wax. Hair is only confined to the outer canal and therefore furuncles (staphylococcal infection of hair follicles) are seen only in the outer onethird of the canal.

It forms inner two-thirds (16 mm). Skin lining the bony canal is thin and continuous over the tympanic membrane. It is devoid of hair and ceruminous glands. About 6 mm lateral to tympanic membrane, the bony meatus presents a narrowing called isthmus. Foreign bodies, lodged medial to the isthmus, get impacted, and are difficult to remove. Anteroinferior part of the deep meatus, beyond the isthmus, presents a recess called anterior recess, which acts as a cesspool for discharge and debris in cases of external and middle ear infections (Figure 1.2). Anteroinferior part of the bony canal may present a deficiency (foramen of Huschke) in children up to the age of four or sometimes in adults, permitting infections to and from the parotid.

It forms the partition between the external acoustic canal and the middle ear. It is obliquely set and as a result, its posterosuperior part is more lateral than its anteroinferior part. It is 9-10 mm tall, 8-9 mm wide and 0.1 mm thick. Tympanic membrane can be divided into two parts:

It forms most of tympanic membrane. Its periphery is thickened to form a fibrocartilaginous ring called annulus tympanicus, which fits in the tympanic sulcus. The central part of pars tensa is tented inwards at the level of the tip of malleus and is called wmbo. A bright cone of light can be seen radiating from the tip of malleus to the periphery in the anteroinferior quadrant (Figures 1.3 and 1.4).

This is situated above the lateral process of malleus between the notch of Rivinus and the anterior and posterior malleal folds (earlier called malleolar folds). It is not so taut and may appear slightly pinkish. Various landmarks seen on the lateral surface of tympanic membrane are shown in Figure 1.4.
Figure 1.2 Anterior recess of the meatus. It is important to clean discharge and debris from this area.

Tympanic membrane consists of three layers:
- ¢ Outer epithelial layer, which is continuous with the skin
lining the meatus.
- ¢ Inner mucosal layer, which is continuous with the mucosa
of the middle ear.
¢
Middle fibrous
layer, which
encloses the
handle of
mal- leus and has three types of fibres—the radial, circular and
parabolic (Figure 1.5).
Figure
1.3
Coronal section
through tympanic
membrane and
exter- nal
ear canal
showing structures
of pars
tensa and
pars flaccida
of tympanic membrane. Scutum forms a part of lateral attic wall.
Fibrous layer in the pars flaccida is thin and not organized
into various fibres as in pars tensa.

Superiorly: Middle cranial fossa
Posteriorly: Mastoid air cells and the facial nerve
Inferiorly: Parotid gland
Anteriorly: Temporomandibular joint
Posterosuperior part of deeper canal near the tympanic membrane is related to the mastoid antrum. "Sagging" of
this area may be noticed in acute mastoiditis.

- 1. Greater auricular nerve (Cys) supplies most of the
medial surface of pinna and only posterior part of the lateral
surface
(Figure
1.6).
- 2. Lesser occipital (Cy) supplies upper part of medial surface.
Figure
1.4
Landmarks of anormal tympanic membrane of right side.
Figure
1.5
Radial, circular and parabolic fibres of pars tensa of tym- panic membrane.
3. Auriculotemporal (V3) supplies tragus, crus of helix and the adjacent part of the helix.
4. Auricular branch of vagus (CN X), also called Arnold's
nerve, supplies
the concha
and corresponding
eminence on
the medial
surface.
- 5. Facial nerve, which is distributed with fibres of auricular branch of vagus, supplies the concha and retroauricular groove.

1. Anterior wall and roof: auriculotemporal (Vs).
2. Posterior wall and floor: auricular branch of vagus (CN X).
3. Posterior wall of the auditory canal also receives sen-
sory fibres
of
CN
VII
through auricular
branch of
vagus
(see Hitzelberger's sign on p. 112).
- In herpes zoster oticus, lesions are seen in the distribution of facial nerve, i.e. concha, posterior part of tympanic membrane and postauricular region.

1. Anterior half of lateral surface: auriculotemporal (V3).
2. Posterior half of lateral surface: auricular branch of vagus (CN X).
3. Medial surface: tympanic branch of CN IX (Jacobson's nerve).

The middle ear together with the eustachian tube, aditus, antrum and mastoid air cells is called middle ear cleft (Figure 1.7). It is lined by mucous membrane and filled with air. The middle ear extends much beyond the limits of tympanic membrane which forms its lateral boundary and is sometimes divided into: (i) mesotympanum (lying opposite the pars tensa), (ii) epitympanum or the attic (lying above the pars tensa but medial to Shrapnell's membrane and the bony lateral attic wall) and (iii) hypotympanum (lying below the level of pars tensa) (Figure 1.8). The portion of middle ear around the tympanic orifice of the eustachian tube is sometimes called protympanum. Middle ear can be likened to a six-sided box with a roof, a floor, medial, lateral, anterior and posterior walls (Figure 1.9).
Figure
1.6
Nerve supply of pinna. (A) Lateral surface of pinna. (B) Medial or cranial surface of pinna.
Figure
1.7
Middle ear
cleft.
Figure
1.8
Divisions of middle ear into epi-, mesoand hypo- tympanum.
The roof is formed by a thin plate of bone called tegmen tympani. It also extends posteriorly to form the roof of the aditus and antrum. It separates tympanic cavity from the middle cranial fossa.
The floor is also a thin plate of bone, which separates tympanic cavity from the jugular bulb. Sometimes, it is congenitally deficient and the jugular bulb may then project into the middle ear; separated from the cavity only by the mucosa.
The anterior wall has a thin plate of bone, which separates the cavity from internal carotid artery. It also has two openings; the lower one for the eustachian tube and the upper one for the canal of tensor tympani muscle.

The posterior wall lies close to the mastoid air cells. It presents a bony projection called pyramid through the summit of which appears the tendon of the stapedius muscle to get attachment to the neck of stapes. Aditus, an opening through which attic communicates with the antrum, lies above the pyramid. Facial nerve runs in the posterior wall just behind the pyramid. Facial recess or the posterior sinus is a depression in the posterior wall lateral to the pyramid. It is bounded medially by the vertical part of VIIth nerve, laterally by the
1., Figure 1.9 = Canal for tensor tympani. 1., Walls of middle ear and the structures related to them. = Canal for tensor tympani. 1., Walls of middle ear and the structures related to them. = 7. Facial nerve. 2., Figure 1.9 = Opening of eustachian tube. 2., Walls of middle ear and the structures related to them. = Opening of eustachian tube. 2., Walls of middle ear and the structures related to them. = 8. Pyramid. 3., Figure 1.9 = Oval window. 3., Walls of middle ear and the structures related to them. = Oval window. 3., Walls of middle ear and the structures related to them. = 9. Aditus. 4., Figure 1.9 = Round window. 4., Walls of middle ear and the structures related to them. = Round window. 4., Walls of middle ear and the structures related to them. = 10. Chorda tympani. 5., Figure 1.9 = Processus cochleariformis. 5., Walls of middle ear and the structures related to them. = Processus cochleariformis. 5., Walls of middle ear and the structures related to them. = 11. Carotid artery. 6., Figure 1.9 = Horizontal canal. 6., Walls of middle ear and the structures related to them. = Horizontal canal. 6., Walls of middle ear and the structures related to them. = 12. Jugular bulb
chorda tympani and above, by the fossa incudis (Figure 1.10). Surgically, facial recess is important, as direct access can be made through this into the middle ear without disturbing posterior canal wall (intact canal wall technique, see p. 73).

The medial wall (Figure 1.11)is formed by the labyrinth. It presents a bulge called promontory which is due to the basal coil of cochlea; oval window into which is fixed the footplate of stapes; round window or the fenestra cochleae which is covered by the secondary tympanic membrane. Above the oval window is the canal for facial nerve. Its bony covering may sometimes be congenitally dehiscent and the nerve may lie exposed making it very vulnerable to injuries or infection. Above the canal for facial nerve is the prominence of lateral semicircular canal. Just anterior to the oval window, the medial wall presents a hook-like projection called processus cochleariformis. The tendon of tensor tympani takes a turn here to get attachment to the neck of malleus. The cochleariform process also marks the level of the genu of the facial nerve which is an important landmark for surgery of the facial nerve. Medial to the pyramid is a deep recess called sinus tympani, which is bounded by the subiculwm below and the ponticulus above (Figure 1.10).
The lateral wall is formed largely by the tympanic membrane and to a lesser extent by the bony outer attic wall called scutum. The tympanic membrane is semitransparent and forms a "window" into the middle ear. It is possible to see some structures of the middle ear through the normal tympanic membrane, e.g. the long process of incus, incudostapedial joint and the round window.

It is a large, air-containing space in the upper part of mastoid and communicates with the attic through the aditus. Its roof is formed by tegmen antri, which is a continuation of the tegmen tympani and separates it from the middle cranial fossa. The lateral wall of antrum is formed by a plate of bone which is on an average 1.5 cm thick in the adult. It is marked externally on the surface of mastoid by suprameatal (MacEwen's) triangle (Figure 1.12).
Figure
1.11
Medial
wall
of
middle
ear.
1.
Promontory
7.
Ponticulus
2.
Processus
cochleariformis
8.
Sinus
tympani
3.
CN
VII
9.
Subiculum
4. Oval window
10.
Round
window
5. Horizontal canal
11.
Tympanic
plexus
6. Pyramid

Aditus is an opening through which the attic communicates with the antrum. The bony prominence of the horizontal canal lies on its medial side while the fossa incudis, to which is attached the short process of incus, lies laterally. Facial nerve courses just below the aditus.

(FIGURE
1.13)
The mastoid consists of bone cortex with a "honeycomb" of air cells underneath. Depending on development of air cell, three types of mastoid have been described.
- 1. Well-pneumatized or cellular. Mastoid cells are well-
developed and intervening septa are thin.
Figure 1.12 MacEwen's (suprameatal) triangle. It is bounded by temporal line (a), posterosuperior segment of bony external auditory canal (b) and the line drawn as a tangent to the external canal (c). It is an important landmark to locate the mastoid antrum in mastoid surgery.
- 2. Diploetic. Mastoid consists of marrow spaces and a few air cells.
3.
Sclerotic or
acellular.
There are
no cells
or marrow
spaces.
With any type of mastoid pneumatization, antrum is always present. In sclerotic mastoids, antrum is usually small and
- the sigmoid sinus is anteposed.
Depending on the location, mastoid air cells are divided into:
oom
Zygomatic cells (in the root of zygoma).
Tegmen cells (extending into the tegmen tympani). NO
Perisinus cells (overlying the sinus plate). oo
Retrofacial cells (round the facial nerve).
Perilabyrinthine cells (located above, below and behind the labyrinth, some of them pass through the arch of superior semicircular canal. These cells may communicate with the petrous apex).
6. Peritubal (around the eustachian tube. Along with hypotym-
panic cells
they also
communicate with
the petrous
apex).
7.
Tip cells
(which are
quite large
and lie
medial and
lateral to
the digastric
ridge in
the tip
of mastoid).
- 8. Marginal cells (lying behind the sinus plate and may extend into the occipital bone).
9.
Squamosal cells
(lying in
the squamous
part of
temporal bones).

Figure 1.14 Korner's septum (A) as seen on mastoid exploration, (B) the antrum which lies deep to it. in coronal section of mastoid; in its presence there is difficulty in locating
Abscesses may form in relation to these air cells and may sometimes be located far from the mastoid region.

tympanic membrane. The lateral process forms a knob-like projection on the outer surface of the tympanic membrane and gives attachment to the anterior and posterior malleal (malleolar) folds.
Mastoid develops from the squamous and petrous bones. The petrosquamosal suture may persist as a bony plate—the Korner's septum, separating superficial squamosal cells from the deep petrosal cells. Korner's septum is surgically important as it may cause difficulty in locating the antrum and the deeper cells; and thus may lead to incomplete removal of disease at mastoidectomy (Figure 1.14). Mastoid antrum cannot be reached unless the Korner's septum has been removed.

There are three ossicles in the middle ear—the malleus, incus and stapes.
The malleus has head, neck, handle (manubrium), a lateral and an anterior process. Head and neck of malleus lie in the attic. Manubrium is embedded in the fibrous layer of the
The incus has a body and a short process, both of which lie in the attic, and a long process which hangs vertically and attaches to the head of stapes.
The stapes has a head, neck, anterior and posterior crura, and a footplate. The footplate is held in the oval window by annular ligament.
The ossicles conduct sound energy from the tympanic membrane
to the
oval window
and then
to the
inner ear
fluid.

There are two muscles—lensor tympani and the stapedius; the former attaches to the neck of malleus and tenses the tympanic membrane while the latter attaches to the neck of
stapes and helps to dampen very loud sounds thus preventing noise trauma to the inner ear. Stapedius is a second arch muscle and is supplied by a branch of CN VII while tensor tympani develops from the first arch and is supplied by a branch of mandibular nerve (V3).

It lies on the promontory and is formed by (i) tympanic branch of glossopharyngeal and (ii) sympathetic fibres from the plexus round the internal carotid artery. Tympanic plexus supplies innervation to the medial surface of the tympanic membrane, tympanic cavity, mastoid air cells and the bony eustachian tube. It also carries secretomotor fibres for the parotid gland. Section of tympanic branch of glossopharyngeal nerve can be carried out in the middle ear in cases of Frey's syndrome.
Course of secretomotor fibres to the parotid:
Inferior salivary nucleus ~ CN IX ~ Tympanic branch ~ Tympanic plexus ~ Lesser petrosal nerve > Otic ganglion ~ Auriculotemporal nerve > Parotid gland.

It is a branch of the facial nerve which enters the middle ear through posterior canaliculus, and runs on the medial surface of the tympanic membrane between the handle of malleus and long process of incus, above the attachment of tendon of tensor tympani. It carries taste from anterior two-thirds of tongue and supplies secretomotor fibres to the submaxillary and sublingual salivary glands.

Mucous membrane of the nasopharynx is continuous with that of the middle ear, aditus, antrum and the mastoid air cells. It wraps the middle ear structures—the ossicles, muscles, ligaments and nerves—like peritoneum wraps various viscera in the abdomen—raising several folds and dividing the middle ear into various compartments. Middle ear contains nothing but the air; all the structures lie outside the mucous membrane. Histologically, the eustachian tube is lined by ciliated epithelium, which is pseudostratified columnar in the cartilaginous part, columnar in the bony part with several mucous glands in the submucosa. Tympanic cavity is lined by ciliated columnar epithelium in its anterior and inferior part which changes to cuboidal type in the posterior part. Epitympanum and mastoid air cells are lined by flat, nonciliated epithelium.

Middle ear is supplied by six arteries, out of which two are the main, i.e.
1. Anterior tympanic branch of maxillary artery which supplies tympanic membrane.
2. Stylomastoid branch of posterior auricular artery which supplies middle ear and mastoid air cells.
Four minor vessels are:
1. Petrosal branch of middle meningeal artery (runs along greater petrosal nerve).
2. Superior tympanic branch of middle meningeal artery traversing along the canal for tensor tympani muscle.
3. Branch of artery of pterygoid canal (runs along eustachian tube).
4. Tympanic branch of internal carotid.
Veins drain into pterygoid venous plexus and superior petrosal sinus.

Lymphatics from the middle ear drain into retropharyngeal and parotid nodes while those of the eustachian tube drain into retropharyngeal group (see Table 1.1).

The internal ear or the labyrinth is an important organ of hearing and balance. It consists of a bony and a membranous labyrinth. The membranous labyrinth is filled with a clear fluid called endolymph while the space between membranous and bony labyrinths is filled with perilymph.

It consists of three parts: the vestibule, the semicircular canals and the cochlea.
1. Vestibule. It is the central chamber of the labyrinth. In its lateral wall lies the oval window. The inside of its medial wall presents two recesses, a spherical recess, which lodges the saccule, and an elliptical recess, which lodges the utricle. Below the elliptical recess is the opening of aqueduct of vestibule
through which passes the endolymphatic duct. In the posterosuperior part of vestibule are the five openings of semicircular canals (Figure 1.16C).
2. Semicircular canals. They are three in number, the lateral, posterior and superior, and lie in planes at right angles to one another. Each canal has an ampullated end which opens independently into the vestibule and a nonampullated end. The nonampullated ends of posterior and superior canals unite to form a common channel called crus commune. Thus, the three canals open into the vestibule by five openings.
3. Cochlea. The bony cochlea is a coiled tube making 2.5 to 2.75 turns round a central pyramid of bone called modiolus. The base of modiolus is directed towards internal acoustic meatus and transmits vessels and nerves to the cochlea. Around the modiolus and winding spirally like the thread of a screw, is a thin plate of bone called osseous spiral lamina. It divides the bony cochlea incompletely and gives attachment to the basilar membrane. The bony bulge in the medial wall of middle ear, the promontory, is due to the basal coil of the cochlea. The bony cochlea contains three compartments:

(a) Scala vestibuli, (b) Scala tympani, (c) Scala media or the membranous cochlea (Figure 1.17). The scala vestibuli and scala tympani are filled with perilymph and communicate with each other at the apex of cochlea through an opening called helicotrema. Scala vestibuli is closed by the footplate of stapes which separates it from the air-filled middle ear. The scala tympani is closed by secondary tympanic membrane; it is also connected with the subarachnoid space through the aqueduct of cochlea (Figure 1.18).
Superior
SCC
Figure
1.17
Section through
cochlea to
show scala
media
(cochlear duct),
scala vestibuli
and scala
tympani.
Figure 1.18 Diagrammatic representation of perilymphatic system. CSF passes into scala tympani through aqueduct of cochlea.

It consists of the cochlear duct, the utricle and saccule, the three semicircular ducts, and the endolymphatic duct and sac.
1. Cochlear duct (Figure 1.17). Also called membranous cochlea or the scala media. It is a blind coiled tube. It appears triangular on cross-section and its three walls are formed by:
(a)
- the basilar membrane, which supports the organ of Corti;
(b)
the
Reissner’s membrane,
which separates
it from
the scala
vestibule;
and
- (c) the stria vascularis, which contains vascular epithelium and is concerned with secretion of endolymph.
Cochlear duct is connected to the saccule by ductus reuniens (Figure 1.16B). The length of basilar membrane increases as we proceed from the basal coil to the apical coil. It is for this reason that higher frequencies of sound are heard at the basal coil while lower ones are heard at the apical coil.
2. Utricle and saccule. The utricle lies in the posterior part of bony vestibule. It receives the five openings of the three semicircular ducts. It is also connected to the saccule through utriculosaccular duct. The sensory epithelium of the utricle is called macula and is concerned with linear acceleration and deceleration. The saccule also lies in the bony vestibule, anterior to the utricle and opposite the stapes footplate. Its sensory epithelium is also called macula. Its exact function is not known. It probably also responds to linear acceleration and deceleration. In Méniére's disease, the distended saccule lies against the stapes footplate and can be surgically decompressed by perforating the footplate.
3. Semicircular ducts. They are three in number and cor respond exactly to the three bony canals. They open in the utricle. The ampullated end of each duct contains a thickened ridge of neuroepithelium called crista ampullaris.

4. Endolymphatic duct and sac. Endolymphatic duct is formed by the union of two ducts, one each from the saccule and the utricle. It passes through the vestibular aqueduct. Its terminal part is dilated to form endolymphatic sac, which lies between the two layers of dura on the posterior surface of the petrous bone.
for
Endolymphatic sac is surgically important. It is exposed drainage or shunt operation in Méniére's disease.

There are two main fluids in the inner ear: perilymph and endolymph.
1. Perilymph resembles extracellular fluid and is rich in Na ions. It fills the space between the bony and the membranous labyrinth. It communicates with CSF through the aqueduct of cochlea which opens into the scala tympani near the round window. In fact this duct is not a direct communication but contains connective tissue resembling arachnoid through which perilymph percolates. There are two views regarding the formation of perilymph: (i) It is a filtrate of blood serum and is formed by capillaries of the spiral ligament and (ii) it is a direct continuation of CSF and reaches the labyrinth via aqueduct of cochlea.
2. Endolymph fills the entire membranous labyrinth and resembles intracellular fluid, being rich in K ions. It is secreted by the secretory cells of the stria vascularis of the cochlea and by the dark cells (present in the utricle and also near the ampullated ends of semicircular ducts). There are two views regarding its flow: (i) longitudinal, i.e. endolymph from the cochlea reaches saccule, utricle and endolymphatic duct and gets absorbed through endolymphatic sac, which lies in the subdural space and (ii) radial, i.e. endolymph is secreted by stria vascularis and also gets absorbed by the stria vascularis. This view presumes that endolymphatic sac is a vestigial structure in man and plays no part in endolymph absorption. Composition of endolymph, perilymph and CSF is given in Table 1.2.

The entire labyrinth receives its arterial supply through labyrinthine artery, which is a branch of anterior-inferior cerebellar artery but sometimes from the basilar. In the internal auditory canal it divides in the manner shown in 1.19 and 1.20.
Figures Venous drainage is through three veins, namely internal auditory vein, vein of cochlear aqueduct and vein of vestibular aqueduct, which ultimately drain into inferior petrosal sinus and lateral venous sinus.

Blood supply
to the
supply to
middle ear
inner ear
and bony
is independent
otic of
capsule, and
blood there
is no
cross circulation
between the
two.
- 2. Blood supply to cochlea and vestibular labyrinth is segmental, therefore, independent ischaemic damage can occur to these organs causing either cochlear or vestibular symptoms.

Auricle. First branchial cleft is the precursor of external auditory canal. Around the 6th week of embryonic life, a series of six tubercles appear around the first branchial cleft. They progressively coalesce to form the auricle (Figure 1.21). Tragus develops from the tubercle of the first arch while the rest of the pinna develops from the remaining five tubercles of the second arch. Faulty fusion between the first and the second arch tubercles causes preauricular sinus or cyst, which is commonly seen between the tragus and crus of helix. By the 20th week, pinna achieves adult shape. Initially, the pinna is located low on the side of the neck and then moves on to a more lateral and cranial position.
External auditory meatus. It develops from the first branchial cleft. By about the 16th embryonic week, cells proliferate from the bottom of ectodermal cleft and form a meatal plug. Recanalization of this plug forms the epithelial lining of the bony meatus. Recanalization begins from the deeper part near the tympanic membrane and progresses outwards, and that explains why deeper meatus is sometimes developed
1.
Figure
1.21
branchial
Development of
pinna.
Six hillocks
of
His around
cleft and
parts of
pinna which
first develop
from them.
while there is atresia of canal in the outer part. External ear canal is fully formed by the 28th week of gestation.
Tympanic membrane. It develops from all the three germinal layers. Outer epithelial layer is formed by the ectoderm, inner mucosal layer by the endoderm and the middle fibrous layer by the mesoderm.
Middle ear cleft. The eustachian tube, tympanic cavity, attic, antrum and mastoid air cells develop from the endoderm of tubotympanic recess which arises from the first and partly from the second pharyngeal pouches (Figure 1.22).
Malleus and incus are derived from mesoderm of the first arch while the stapes develop from the second arch except its footplate and annular ligament which are derived from the otic capsule.

Membranous inner ear. Development of the inner ear starts in the 3rd week of fetal life and is complete by the 16th week. Ectoderm in the region of hindbrain thickens to form an auditory placode, which is invaginated to form auditory vesicle or the otocyst. The latter then differentiates into the endolymphatic duct and sac; the utricle, the semicircular ducts; and saccule and the cochlea. Development of phylogenetically older part of labyrinth—pars superior (semicircular canals and utricle) takes place earlier than pars inferior (saccule and cochlea).
The embryologic source and the time of development of external and middle ears are quite independent of the development of the inner ear. It is therefore not unusual to see malformed and nonfunctional inner ear in the presence of normal external and middle ears, and vice versa.
The cochlea is developed sufficiently by 20 weeks of gestation (Table 1.3) and the fetus can hear in the womb of the mother. This probably explains how Abhimanyu, while still unborn, could have heard the conversation between his mother and father (Arjuna) in the legend given in the Great Indian epic of Mahabharata written thousands of years ago.

Organ of Corti is the sense organ of hearing and is situated on the basilar membrane. Important components of the organ of Corti are:
1. Tunnel of Corti, which is formed by the inner and outer rods. It contains a fluid called cortilymph. The exact function of the rods and cortilymph is not known.
2. Hair cells. They are important receptor cells of hearing and transduce sound energy into electrical energy. Inner hair cells form a single row while outer hair cells are arranged in three or four rows. Inner hair cells are richly supplied by afferent cochlear fibres and are probably more important in the transmission of auditory impulses. Outer hair cells mainly receive efferent innervation from the olivary complex and are concerned with modulating the function of inner hair cells. Differences between inner and outer hair cells are in Table 2.1.
given
3. Supporting cell. Deiters' cells are situated between the outer hair cells and provide support to the latter. Cells of Hensen lie outside the Deiters' cells.
4. Tectorial membrane. It consists of gelatinous matrix with delicate fibres. It overlies the organ of Corti. The shearing force between the hair cells and tectorial membrane produces the stimulus to hair cells.

Ninety-five per cent of afferent fibres of spiral ganglion supply the inner hair cells while only five per cent supply the outer hair cells. Efferent fibres to the hair cells come from the olivocochlear bundle. Their cell bodies are situated in superior olivary complex. Each cochlea sends innervation to both sides of the brain.

Hair cells are innervated by dendrites of bipolar cells of spiral ganglion which is situated in Rosenthal's canal (canal running along the osseous spiral lamina). Axons of these bipolar cells form the cochlear division of CN VII and end in the cochlear nuclei, the dorsal and ventral, on each side of the medulla. Further course of auditory pathways is complex. From cochlear nuclei, the main nuclei in the ascending auditory pathways, sequentially, from below
upwards are:
Superior olivary complex
Nucleus of lateral lemniscus NO
Inferior colliculus OO
Medial geniculate body
Auditory cortex oO
The auditory fibres travel via the ipsilateral and contralateral routes and have multiple decussation points. Thus each ear is represented in both cerebral hemispheres. The area of cortex, concerned with hearing is situated in the superior temporal gyrus (Brodmann's area 41). For auditory pathways, remember the mnemonic E.COLI-MA: Eighth nerve, Cochlear nuclei, Olivary complex, Lateral lemniscus, Inferior colliculus, Medial geniculate body and Auditory cortex.

Any vibrating object causes waves of compression and rarefaction and is capable of producing sound. In the air, at 20°C and at sea level, sound travels at a speed of 344 m (1120 ft) per second. It travels faster in liquids and solids than in the air. Also, when sound energy has to pass from air to liquid medium, most of it is reflected because of the impedance offered by the liquid.

A sound signal in the environment is collected by the pinna, passes through external auditory canal and strikes the tympanic membrane. Vibrations of the tympanic mem- brane are transmitted to stapes footplate through a chain of ossicles coupled to the tympanic membrane. Movements of stapes footplate cause pressure changes in the labyrin- thine fluids, which move the basilar membrane. This stim- ulates the hair cells of the organ of Corti. It is these hair cells which act as transducers and convert the mechani- cal energy into electrical impulses, which travel along the auditory nerve. Thus, the mechanism of hearing can be broadly divided into:
1. Mechanical conduction of sound (conductive appa- ratus).
2. Transduction of mechanical energy to electrical impulses (sensory system of cochlea).
3. Conduction of electrical impulses to the brain (neural pathways).
Figure
2.1
organ of
Corti.
1.

A person under water cannot hear any sound made in the air because 99.9% of the sound energy is reflected away from the surface of water because of the impedance offered by it. A similar situation exists in the ear when air-conducted sound has to travel to cochlear fluids. Nature has compensated for this loss of sound energy by interposing the middle ear which converts sound of greater amplitude but lesser force, to that of lesser amplitude but greater force. This function of the middle ear is called impedance matching mechanism or the transformer action.
It is accomplished by:
- (a) Lever action of the ossicles. Handle of malleus is 1.3 times longer than long process of the incus, providing a mechanical advantage of 1.3.
(b)
Hydraulic action
of tympanic
membrane.
The area
of tym-
panic membrane is much larger than the area of stapes
Trapezoid body
Figure
2.2
Auditory pathways
from the
right cochlea.
Note bilateral
route through brainstem and bilateral cortical representation.
footplate, the average ratio between the two being 21:1. As the effective vibratory area of tympanic membrane is only two-thirds, the effective areal ratio is reduced to 14:1, and this is the mechanical advantage provided by the tympanic membrane (Figure 2.3).
The product of areal ratio and lever action of ossicles is 18:1. According to some workers (Wever and Lawrence) out of a total of 90 mm? area of human tympanic membrane, only 55 mm? is functional and given the area of stapes footplate (3.2 mm?), the areal ratio is 17:1 and total transformer ratio (17x 1.3) is 22.1.
membrane
Figure 2.3 Transformer action of the middle ear. Hydraulic effect of tympanic membrane and lever action of ossicles combine to compensate the sound energy lost during its transmission from air to liquid medium.
3000 Hz
800-1600 Hz
800 Hz
500-2000 Hz
External
auditory
canal
Tympanic
membrane
Middle
ear
Ossicular chain
(c)
Curved membrane
effect.
Movements of
tympanic mem-
brane are
more where

malleus at
the handle
periphery is
attached.
than at
the
This too
centre provides
some leverage.
Phase differential between oval and round windows. Sound waves striking the tympanic membrane do not reach the oval and round windows simultaneously. There is a preferential pathway to the oval window because of the ossicular chain. Thus, when oval window is receiving wave of compression, the round window is at the phase of rarefaction. If the sound waves were to strike both the windows simultaneously, they would cancel each other's effect with no movement of the perilymph and no hearing. This acoustic separation of windows is achieved by the presence of intact tympanic membrane and a cushion of air in the middle ear around the round window. Phase differential between the windows contributes 4 dB when tympanic membrane is intact.
Natural resonance of external and middle ear. Inherent anatomic and physiologic properties of the external and middle ear allow certain frequencies of sound to pass more easily to the inner ear due to their natural resonances. Natural resonance of external ear canal is 3000 Hz and that of middle ear 800 Hz. Frequencies most efficiently transmitted by ossicular chain are between 500 and 2000 Hz while that by tympanic membrane is 800-1600 Hz. Thus greatest sensitivity of the sound transmission is between 500 and 3000 Hz and these are the frequencies most important to man in dayto-day conversation (Table 2.2).

Movements of the stapes footplate, transmitted to the cochlear fluids, move the basilar membrane and set up
Figure 2.4 Frequency localization in the cochlea. Higher frequencies are localized in the basal turn and then progressively decrease towards the apex.
shearing force between the tectorial membrane and the hair cells. The distortion of hair cells gives rise to cochlear microphonics, which trigger the nerve impulse.
A sound wave, depending on its frequency, reaches maximum amplitude on a particular place on the basilar membrane and stimulates that segment (travelling wave theory of von Bekesy). Higher frequencies are represented in the basal turn of the cochlea and the progressively lower ones towards the apex (Figure 2.4).

Hair cells get innervation from the bipolar cells of spiral ganglion. Central axons of these cells collect to form the cochlear nerve which goes to the ventral and dorsal cochlear nuclei. From there, both crossed and uncrossed fibres travel to the superior olivary nucleus, lateral lemniscus, inferior colliculus, medial geniculate body and finally reach the auditory cortex of the temporal lobe.

Four types of potentials have been recorded; three from the cochlea and one from CN VIII fibres. They are:
1.
Endocochlear potential
2.
Cochlear microphonic
3. Summating potential
[—
4
4.
Compound action
potential
“}—
from nerve fibres
1. Endocochlear potential. It is a direct current (DC) potential recorded from scala media. It is +80 mV and is generated from the stria vascularis by Na*/K*-ATPase pump and provides source of energy for cochlear transduction (Figure 2.5). It is present at rest and does not require sound stimulus. This potential provides a sort of "battery" to drive the current through hair cells when they move in response to a sound stimulus.
2. Cochlear microphonic (CM). When basilar membrane moves in response to sound stimulus, electrical resistance at the tips of hair cells changes allowing flow of K* through hair cells and produces voltage fluctuations called cochlear microphonic. It is an alternating current (AC) potential.
3. Summating potential (SP). It is a DC potential and follows "envelope" of stimulating sound. It is produced by hair cells.
from cochlea
Figure 2.5 Davis' battery model of cochlear transduction. Scala media has a DC potential of 180 mV. Stimulation of hair cells produces intracellular potential of 240 mV. This provides flow of current of 120 mV through the top of hair cells.
It may be negative or positive. SP has been used in diagno- sis of Méniére’s disease. It is superimposed on VIII nerve action potential. Both CM and SP are receptor potentials as seen in other sensory end-organs. They differ from action potentials in that: (i) they are graded rather than all or none phenom- enon, (ii) have no latency, (iii) are not propagated and (iv) have no postresponse refractory period. 4. Compound action potential. It is an all or none response of auditory nerve fibres.

They are located in the ampullated ends of the three semicircular ducts. These receptors respond to angular acceleration.

They are located in otolith organs (i.e. utricle and saccule). Macula of the utricle lies in its floor in a horizontal plane. Macula of the saccule lies in its medial wall in a vertical plane. They sense position of head in response to gravity and linear acceleration.
(a) Structure of a crista (Figure 2.6). It is a crest-like mound of connective tissues on which lie the sensory epithelial cells. The cilia of the sensory hair cells project into the cupula, which is a gelatinous mass extending from the surface of crista to the ceiling of the ampulla and forms a water tight partition, only to be displaced to one or the other side like a swing door, with movements of endolymph. The gelatinous mass of cupula consists of polysaccharide and contains canals into which project the cilia of sensory cells.
Hair cells are of two types (Figure 2.7). Type Icells are flaskshaped with a single large cup-like nerve terminal surrounding the base. Type I cells are cylindrical with multiple nerve
Figure 2.6 Structure of ampullary end of semicircular duct. Over the crista lie sensory hair cells interspersed with supporting cells. Hair from sensory cells project into the gelatinous substance of cupula.
Figure 2.7 Sensory hair cells of the vestibular organs. Type | (left) and Type Il (right).
terminals at the base. From the upper surface of each cell, project a single hair, the kinocilium and a number of other cilia, the stereocilia. The kinocilium is thicker and is located on the edge of the cell. Sensory cells are surrounded by supporting cells which show microvilli on their upper ends.

(b) Structure of a macula. A macula consists mainly of two parts: (i) a sensory neuroepithelium, made up of type I and type II cells, similar to those in the crista; (ii) an otolithic membrane, which is made up of a gelatinous mass and on the top, the crystals of calcium carbonate called otoliths or otoconia (Figure 2.8). The cilia of hair cells project into the gelatinous layer. The linear, gravitational and head tilt movements cause displacement of otolithic membrane and thus stimulate the hair cells which lie in different planes.

Vestibular or Scarpa's ganglion is situated in the lateral part of the internal acoustic meatus. It contains bipolar cells. The distal processes of bipolar cells innervate the sensory
epithelium of the labyrinth while its central processes aggre-
gate to form the vestibular nerve.

The fibres of vestibular nerve end in vestibular nuclei and some go to the cerebellum directly.
Vestibular nuclei are four in number, the superior, medial,
lateral and descending. Afferents to these nuclei come from:
- 1. Peripheral vestibular receptors (semicircular canals, utri-
cle and saccule)
Cerebellum go bo
Reticular formation
Spinal cord RB
Contralateral vestibular nuclei Ot
Thus, information received from the labyrinthine receptors is
integrated with
information from
other somatosensory
systems.
Efferents from vestibular nuclei go to:
1. Nuclei of CN HI, IV, VI via medial longitudinal bundle. It is the pathway for vestibulo-ocular reflexes and _ this explains the genesis of nystagmus.
2. Motor part of spinal cord (vestibulospinal fibres). This coordinates the movements of head, neck and body in the maintenance of balance.
3. Cerebellum (vestibulocerebellar fibres). It helps to coordinate input information to maintain the body balance. 4. Autonomic nervous system. This explains nausea, vomiting, palpitation, sweating and pallor seen in vestibular disorders (e.g. Méniére's disease).
Vestibular nuclei of the opposite side. a
6. Cerebral cortex (temporal lobe). This is responsible for subjective awareness of motion.

Vestibular system is conveniently divided into:
- 1. Peripheral, which is made up of membranous labyrinth (semicircular ducts, utricle and saccule) and vestibular nerve.
Figure 2.9 Rotation test. At the end of rotation to the right, semicircular canals stop but endolymph continues to move to the right, i.e. towards the left ampulla but away from the right, causing nystagmus to the left.
- 2. Central, which is made up of nuclei and fibre tracts in the central nervous system to integrate vestibular impulses with other systems to maintain body balance.

They respond to angular acceleration and deceleration. The three canals lie at right angles to each other but the one which lies at right angles to the axis of rotation is stimulated the most. Thus horizontal canal will respond maximum to rotation on the vertical axis and so on. Due to this arrangement of the three canals in three different planes, any change in position of head can be detected. Stimulation of semicircular canals produces nystagmus and the direction of nystagmus is determined by the plane of the canal being stimulated. Thus, nystagmus is horizontal from horizontal canal, rotatory from the superior canal and vertical from the posterior canal.
The stimulus to semicircular canal is flow of endolymph which displaces the cupula. The flow may be towards the cupula (ampullopetal) or away from it (ampullofugal), better called utriculopetal and utriculofugal. Ampullopetal flow is more effective than ampullofugal for the horizontal canal. The quick component of nystagmus is always opposite to the direction of flow of endolymph. Thus, if a person is rotated to the right for sometime and then abruptly stopped, the endolymph continues to move to the right due to inertia (i.e. ampullopetal for left canal), the nystagmus will be horizontal and directed to the left (Figure 2.9). Remember nystagmus is in the direction opposite to the direction of flow of endolymph.

reflexes and motor outputs to the eyes (vestibulo-ocular reflex), neck (vestibulocervical reflex), and trunk and limbs (vestibulospinal reflex) to maintain new position of head and body, but if any component of push and pull system of one side is disturbed for a longer time due to disease, vertigo and ataxia will develop.

Disorientation in space causes vertigo or dizziness and can arise from disorders of any of the three systems: vestibular, visual or somatosensory. Normally, the impulses reaching the brain from the three systems are equal and opposite. If any component on one side is inhibited or stimulated, the information reaching the cortex is mismatched, resulting in disorientation and vertigo. The vestibular inhibition on one side (e.g. acute vestibular failure, labyrinthectomy, Méniére's disease, VIIIth nerve section) causes vertigo. Similarly, stimulation of labyrinth by thermal or rotational stimulus causes vertigo. Dizziness can similarly result from the ocular causes, e.g. high errors of refraction or acute extraocular muscle paralysis with diplopia.
Vertigo and its causes are discussed in detail in Chapter 7.

It is characterized by nausea, vomiting, pallor and sweating during sea, air, bus or car travel in certain susceptible individuals. It can be induced by both real and apparent motion and is thought to arise from the mismatch of information reaching the vestibular nuclei and cerebellum from the visual, labyrinthine and somatosensory systems. It can be controlled by the usual labyrinthine sedatives.

This section aims to introduce certain terms which are frequently used in audiology and acoustics.
Sound. It is a form of energy produced by a vibrating object. A sound wave consists of compression and rarefaction of molecules of the medium (air, liquid or solid) in which it travels. Velocity of sound is different in different media. In the air, at 20°C, at sea level, sound travels 344 m (1120 ft) per second, and is faster in liquid and still faster in a solid medium.
Frequency. It is the number of cycles per second. The unit of frequency is Hertz (Hz) named after the German scientist Heinrich Rudolf Hertz. A sound of 1000 Hz means 1000 cycles per second.
Pure tone. A single frequency sound is called a pure tone, e.g. a sound of 250, 500 or 1000 Hz. In pure tone audiometry, we measure the threshold of hearing in decibels for various pure tones from 125 to 8000 Hz.
Complex sound. Sound with more than one frequency is called a complex sound. Human voice is a complex sound.
Pitch. It is a subjective sensation produced by frequency of sound. Higher the frequency, greater is the pitch.
Overtones. A complex sound has a fundamental frequency, i.e. the lowest frequency at which a source vibrates. All frequencies above that tone are called the overtones. The latter determine the quality or the timbre of sound.
Intensity. It is the strength of sound which determines its loudness. It is usually measured in decibels. At a distance of 1 of m, intensity
Whisper
=
30dB
Normal conversation
=
60dB
Shout
=
90dB
Discomfort of the ear
=
120dB
Pain in the ear
130
dB
Loudness. It is the subjective sensation produced by intensity. More the intensity of sound, greater the loudness.

Decibel (dB). It is 1/10th of a bel and is named after Alexander Graham Bell, the inventor of telephone. It is not an absolute figure but represents a logarithmic ratio between two sounds, namely the sound being described and the reference sound. Sound can be measured as power, i.e. watts/cm? or as pressure, i.e. dynes/cm?. In audiology, sound is measured as sound pressure level (SPL). It is compared with the reference sound which has an SPL of 0.0002 dynes/cm? or 20 pPa (micropascals), which roughly corresponds to the threshold of hearing in normal subjects at 1000 Hz. Decibel notation was introduced in audiology to avoid dealing with large figures of sound pressure level (0.0002 dynes/cm? at normal threshold of hearing to 200 dynes/cm? which causes pain in the ear. The latter is 1,000,000 times the former).
Formula for decibel is
S)= sound being described
So= reference sound
(because power of sound is proportional to square of SPL)
or
20
log
Ifa sound has an SPL of 1000, i.e. (10%) times the reference sound, it is expressed as 20 x 3 = 60 dB. Similarly, a sound of 1,000,000, ie. (10°) times the reference sound SPL is expressed simply as 120 dB and so on.
Noise. It is defined as an aperiodic complex sound. There are three types of noise:
(a)
White noise.
It contains
all frequencies
in audible
spec- trum
and is
comparable to
the white
light which
tains all
the colours
of the
visible spectrum.
broad-band noise and is used for masking.
Narrow band
noise.
It is
white noise
with certain
cies, above
Thus, band
it has
white in
pure and
below
a
the frequency
noise.
tone
Speech given
range
It is
used noise,
smaller to
audiometry.
noise.
speech range
It is
a
noise
(300-3000
con-
It is
a
frequen- filtered
than mask
out.

the broad-
the having
Hz).
test frequency
frequencies
All other
in the
frequencies are
filtered out.
Masking. It is a phenomenon to produce inaudibility of one sound by the presentation of another. In clinical audiometry, one ear is kept busy by a sound while the other is being tested. Masking of nontest ear is essential in all bone conduction tests, but for air conduction tests, it is required only when difference of hearing between two ears exceeds 40 dB.
(b)
(c)
i.e. 40 dB HL, but for one with a hearing loss of say 30 dB, it would be 30 + 40, i.e. 70 dB HL. In other words, sensation level refers to the sound which will produce the same sensation, as in normally hearing person. In speech audiometry, discrimination scores are tested at 30-40 dB SL. Stapedial reflex is elicited with a sound of 70-100 dB SL.
Most comfortable level (MCL). It is the intensity level of sound that is most comfortable for the person.
Loudness discomfort level. It is the level of sound which produces discomfort in the ear. Usually, it is 90-105 dB SL. It is important to find the loudness discomfort level of a person when prescribing a hearing aid.
Dynamic range. It is the difference between the most comfortable level and the loudness discomfort level. The dynamic range is reduced in patients with positive recruitment phenomenon, as is the case in cochlear type of hearing loss.
Sound level meter. It is an instrument to measure level of noise and other sounds. Sound level meters have different weighting networks (e.g. A, B or C) for different sensitivities at different frequencies. When describing a sound measured by a sound level meter, the weighting network must be indicated.
Noise levels are often expressed as dB(A) which refers to sound pressure level measured with 'A' network where the low and extremely high frequencies are given much less weightage compared to those in the middle range which are more important and are responsible for noise-induced hearing loss.
(d)

1. Conductive hearing loss. It is caused by any disease process interfering with the conduction of sound from the external ear to the stapediovestibular joint. Thus the cause may lie in the external ear (obstructions), tympanic membrane (perforation), middle ear (fluid), ossicles (fixation or disruption) or the eustachian tube (obstruction).
2. Sensorineural (SN) hearing loss. It results from lesions of the cochlea (sensory type) or VIIIth nerve and its central connections (neural type). The term retrocochlear is used when hearing loss is due to lesions of VIIIth nerve, and central deafness, when it is due to lesions of central auditory connections.

tive and
sensorineural deafness
in the
same ear.
There is
air-bone gap
impairment of
bone loss.
Mixed hearing
indicating conductive
conduction loss
is seen
indicating in
element, and
sensorineural some
cases of
otosclerosis and
chronic suppurative
otitis media.
While assessing the auditory function it is important to

(a)
Type of
hearing loss
(conductive, sensorineural
or mixed).
- (b) Degree of hearing loss (mild, moderate, moderately severe, severe, profound or total).
(c)
Site of
lesion.
If conductive,
the lesion
may be
at exter-
nal ear,
tympanic eustachian
membrane, tube.
Clinical etry
can be
middle examination
helpful sensorineural,
to find
find retrocochlear
the out
site ear,
ossicles or
and tympanom-
of whether
or central.
Special differentiate
Cause of
hearing loss.
matic, the
such lesion
tests these
types.
The infective,
ototoxic, cause
neoplastic, vascular
may be
congenital, degenerative,
or autoimmune
lesions.
If is
cochlear, of
hearing will
be trau-
metabolic, process.
history and
laboratory investigations
Detailed are
required.

Hearing of an individual can be tested by clinical and audiometric tests.

1. Finger friction test
2. Watch test
air conduction is louder and heard twice as long as through the bone conduction route. To test bone conduction (BC), the footplate of vibrating tuning fork is placed firmly on the mastoid bone. Cochlea is stimulated directly by vibrations conducted through the skull bones. Thus, BC is a measure of the cochlear function only. The clinically useful tuning fork tests include:
(a) Rinne test. In this test air conduction of the ear is compared with its bone conduction. A vibrating tuning fork is placed on the patient's mastoid and when he stops hearing, it is brought beside the meatus. If he still hears, AC is more than BC. Alternatively, the patient is asked to compare the loudness of sound heard through air and bone conduction. Rinne test is called positive when AC is longer or louder than BC. It is seen in normal persons or those having sensorineural deafness. A negative Rinne (BC > AC) is seen in conductive deafness. A negative Rinne indicates a minimum air-bone gap of 15-20 dB.
A prediction of air-bone gap can be made if tuning forks of 256, 512 and 1024 Hz are used.
e
A
Rinne test
equal or
negative for
256
Hz but
positive for
512
Hz indicates
A
Rinne
1024
air-bone test
negative
Hz
A
Rinne gap
for indicates
256
air-bone negative
for and
1024
of
20-30
and dB.
512
gap all
Hz of
the indicates
Remember three
air-bone that
a
negative
Hz but
30-45
dB.
tuning gap
Rinne forks
of
45-60
for
256, of
256, dB.
512
and
1024
a
minimum
AB
gap of
15,
30,
45
dB, respectively.

False negative Rinne. It is seen in severe unilateral sensorineural hearing loss. Patient does not perceive any sound of tuning fork by air conduction but responds to bone conduction testing. This response to bone conduction is, in reality, from the opposite ear because of transcranial transmission of sound. In such cases, correct diagnosis can be made by masking the nontest ear with Barany's noise box while testing for bone conduction. Weber test will further help as it gets lateralized to the better ear.
(b) Weber test. In this test, a vibrating tuning fork is placed in the middle of the forehead or the vertex and the patient is asked in which ear the sound is heard. Normally, it is heard equally in both ears. It is lateralized to the worse ear in conductive deafness and to the better ear in sensorineural deafness. In weber test, sound travels directly to the cochlea via bone. Lateralization of sound in weber test with a tuning fork of 512 Hz implies a conductive loss of 15-25 dB in ipsilateral ear or a sensorineural loss in the contralateral ear.
(c) Absolute bone conduction (ABC) test. Bone conduction is a measure of cochlear function. In ABC test, patient's bone conduction is compared with that of the examiner (presuming that the examiner has normal hearing). External auditory meatus of both the patient and examiner should be occluded (by pressing the tragus inwards) to prevent ambient noise entering through AC route. In conductive deafness, the patient and the examiner hear the fork for the same duration of time. In sensorineural deafness, the patient hears the fork for a shorter duration.
(d)
Schwabach’s test.
Here again
BC
of patient
is compared
with that
of the
meatus is
lute bone
rineural normal
not occluded.
hearing
It has
conduction deafness
Table person
the same
test.
(examiner)
but significance
Schwabach and
lengthened
4.1
summarizes as
abso- is
reduced in
conductive the
in senso-
deafness.
interpretation of
tuning fork

(e) Bing test. It is a test of bone conduction and examines the effect of occlusion of ear canal on the hearing. A vibrating tuning fork is placed on the mastoid while the examiner alternately closes and opens the ear canal by pressing on the tragus inwards. A normal person or one with sensorineural hearing loss hears louder when ear canal is occluded and softer when the canal is open (Bing positive). A patient with conductive hearing loss will appreciate no change (Bing negative).
Figure 4.1 Tuning fork tests. (A) Testing for air conduction. (B) Testing for bone conduction. (C) Weber test.
(f) Gelle's test. It is also a test of bone conduction and examines the effect of increased air pressure in ear canal on the hearing. Normally, when air pressure is increased in the ear canal by Siegel's speculum, it pushes the tympanic membrane and ossicles inwards, raises the intralabyrinthine
Rinne Weber ABC, Normal = AC > BC (Rinne positive) Not lateralized Same as examiner's Equal. Rinne Weber ABC, Conductive deafness = BC > AC (Rinne negative) Lateralized to poorer ear Same as examiner's Lengthened. Rinne Weber ABC, SN deafness = AC > BC Lateralized to better Reduced Shortened. , Normal = . , Conductive deafness = . , SN deafness = ear. Schwabach, Normal = . Schwabach, Conductive deafness = . Schwabach, SN deafness = 
512
¢
e
pressure and causes immobility of basilar membrane and decreased hearing, but no change in hearing is observed when ossicular chain is fixed or disconnected. Gelle's test is performed by placing a vibrating fork on the mastoid while changes in air pressure in the ear canal are brought about by Siegel's speculum. Gelle's test is positive in nor mal persons and in those with sensorineural hearing loss. It is negative when ossicular chain is fixed or disconnected. It was a popular test to find out stapes fixation in otosclerosis but has now been superceded by tympanometry.

An audiometer is an electronic device which produces pure tones, the intensity of which can be increased or decreased in 5 dB steps (Figure 4.2). Usually air conduction thresholds are measured for tones of 125, 250, 500, 1000, 2000, 4000 and 8000 Hz and bone conduction thresholds for 250, 500, 1000, 2000 and 4000 Hz. The amount of intensity that has to be raised above the normal level is a measure of the degree of hearing impairment at that frequency. It is charted in the form of a graph called audiogram. The threshold of bone conduction is a measure of cochlear function. The difference in the thresholds of air and bone conduction (A-B gap) is a measure of the degree of conductive deafness. It may be noted that audiometer is so calibrated that the hearing of a normal person, both for air and bone conduction, is at 0 dB and there is no A-B gap, while tuning fork tests normally show AC > BC. When difference between the two ears is 40 dB or above in air conduction thresholds, the better ear is masked to avoid getting a shadow curve from the nontest better ear. Similarly, masking is essential in all bone conduction studies. Masking is done by employing narrow-band noise to the nontest ear.

(a)
It is
a
measure of
threshold of
hearing by
air and
bone conduction and thus the degree and type of hearing loss.
(b) A record can be kept for future reference.
- (c) Audiogram is essential for prescription of hearing aid.
(d)
Helps to
find degree
of handicap
for medicolegal
purposes.
(e) Helps to predict speech reception threshold.
Figure
4.2
Two-room audiometry setup. Audiometrician watches responses of the patient sitting across a glass partition.

In this test, the patient's ability to hear and understand speech is measured. Two parameters are studied: (i) speech reception threshold and (ii) discrimination score.
(a) Speech reception threshold (SRT). It is the minimum intensity at which 50% of the words are repeated correctly by the patient. A set of spondee words (two syllable words with equal stress on each syllable, e.g. baseball, sunlight, daydream, etc.) is delivered to each ear through the headphone of an audiometer. The word lists are delivered in the form of recorded tapes or monitored voice and their intensity varied in 5 dB steps till half of them are correctly heard. Normally, SRT is within 10 dB of the average of pure tone threshold of three speech frequencies (500, 1000 and 2000 Hz). An SRT better than pure tone average by more than 10 dB suggests a functional hearing loss.
(b) Speech discrimination score. Also called speech recognition or word recognition score. It is a measure of patient's ability to understand speech. Here, a list of phonetically balanced (PB) words (single syllable words, e.g. pin, sin, day, bus, etc.) is delivered to the patient's each ear separately at 30-40 dB above his SRT and the percentage of words correctly heard by the patient is recorded. In normal persons and those with conductive hearing loss a high score of 90-100% can be obtained (Figure 4.3A, B and Table 4.2).
Figure
4.3
Speech audiogram.
A—PB
B—PB
score in
a
normal score
in runs
parallel to
C—Cochlear person
conductive that
100%
hearing of
a
normal
SNHL.
D—Roll over
curve:
increases further.
PB
PB
at loss
person.
max is
max at
at
70
dB
80
dB.
and
30
dB.
100%
at then
PB
scores
70
dB.
attains
This curve
a
decline plateau.
as intensity

Table 4.2, 1 = Ability to understand speech and its relation to speech discrimination (SD) score. A list of 50 PB words is presented and the number correctly heard is multiplied by 2., 1 = A list of 50 PB words is presented and the number correctly heard is multiplied by 2.. SD score, 1 = Ability to understand speech. 90-100%, 1 = Normal. 76-88%, 1 = Slight difficulty. 60-74%, 1 = Moderate difficulty. 40-58%, 1 = Poor. <40%, 1 = Very poor

PB max. Instead of using a single suprathreshold intensity of 30-40 dB above SRT as described above, it is better to chart PB scores against several levels of speech intensity and find the maximum score (PB max) a person can attain. Also note the intensity of sound at which PB max is attained. It is a useful test clinically to set the volume of hearing aid (Figure 4.3C). Maximum volume of hearing aid should not be set above PB max.
Roll over phenomenon. It is seen in retrocochlear hearing loss. With increase in speech intensity above a particular level, the PB word score falls rather than maintain a plateau as in cochlear type of sensorineural hearing loss (Figure 4.3D). Thus speech audiometry is useful in several ways:
- (i) To find speech reception threshold which correlates well with average of three speech frequencies of pure tone audiogram.
(ii) To differentiate organic from nonorganic (functional)
- (iii) To find the intensity at which discrimination score is best. This is helpful for fitting hearing aid and setting
a its volume for maximum discrimination.
- (iv) To differentiate a cochlear from a retrocochlear sensorineural hearing loss.

It is a self-recording audiometry where various pure tone frequencies automatically move from low to high while the patient controls the intensity through a button. Two tracings, one with continuous and the other with pulsed tone, are obtained. The tracings help to differentiate a cochlear from a retrocochlear and an organic from a functional hearing loss.
Various types of tracings obtained are:
Type I
Continuous and pulsed tracings overlap. Seen
in
normal
hearing
or
conductive
hearing
loss.
Continuous and pulsed tracings overlap up to
1000 Hz and then continuous tracing falls. Seen in cochlear loss.
Continuous tracing falls below pulsed tracing
at 100-500 Hz even up to 40-50 dB. Seen in retrocochlear/neural lesion.
Continuous tracing falls below pulsed lesion at
up
to
1000
Hz
by
more
than
25
dB.
Seen
in
retrocochlear/neural
lesion.
Continuous
tracing
is
above
pulsed
one.
Seen
in nonorganic hearing loss.
Type II
Type II
Type IV
Type V
Bekesy audiometry is seldom performed these days.

It is an objective test, widely used in clinical practice and is particularly useful in children. It consists of:
(a) Tympanometry
(b) Acoustic reflex measurements
(a) Tympanometry. It is based on a simple principle, i.e. when a sound strikes tympanic membrane, some of the sound energy is absorbed while the rest is reflected. A stiffer tympanic membrane would reflect more of sound energy than a compliant one. By changing the pressures in a sealed external auditory canal and then measuring the reflected sound energy, it is possible to find the compliance or stiff ness of the tympano-ossicular system and thus find the healthy or diseased status of the middle ear.
Essentially, the equipment consists of a probe which snugly fits into the external auditory canal and has three channels: (i) to deliver a tone of 220 Hz, (ii) to pick up the reflected sound through a microphone and (iii) to bring about changes in air pressure in the ear canal from positive to normal and then negative (Figure 4.5). By charting the compliance of tympano-ossicular system against various pressure changes, different types of graphs called tympanograms are obtained which are diagnostic of certain middle ear pathologies.

Type A
Normal tympanogram.
Type As
Compliance
is
lower
at
or
near
ambient
air
pressure. Seen in fixation of ossicles, e.g. oto- sclerosis or malleus fixation.
Type AD
High compliance at or near ambient pressure.
Seen in ossicular discontinuity or thin and lax tympanic membrane.
Type B
A flat or dome-shaped graph. No change in
with
pressure
changes.
Seen
in
middle
ear
fluid
or
thick
tympanic
membrane.
Type C
Maximum compliance occurs with negative pressure in excess of 100 mm H,O. Seen in retracted tympanic membrane and may show some fluid in middle ear.
Testing function of eustachian tube. Tympanometry has also been used to find function of eustachian tube in cases of intact or perforated tympanic membrane. A negative or a positive pressure (—200 or +200 mm H,O) is created in the middle ear and the person is asked to swallow five times in 20 s. The ability to equilibrate the pressure indicates normal tubal function. The test can also be used to find the patency of the grommet placed in the tympanic membrane in cases of serous otitis media.
(b) Acoustic reflex. It is based on the fact that a loud sound, 70-100 dB above the threshold of hearing of a particular ear, causes bilateral contraction of the stapedial muscles which can be detected by tympanometry. Tone can be delivered to one ear and the reflex picked from the same or the contralateral ear. The reflex arc involved is:
Ipsilateral. CN VIII — ventral cochlear nucleus + CN VII nucleus ipsilateral stapedius muscle.
Contralateral. CN VIIL > ventral cochlear nucleus > contralateral medial superior olivary nucleus > contralateral CN VII nucleus — contralateral stapedius muscle (Figure 4.7).
Figure 4.4 (A) Impedance audiometry in progress. (B) Impedance audiometer.

Figure 4.5 Principle of impedance audiometry. (A) Oscillator to produce a tone of 220 Hz. (B) Air pump to increase or decrease air pressure in the air canal. (C) Microphone to pick up and measure sound pressure level reflected from the tympanic membrane.

(i)
To test
the hearing
in infants
and young
children.
It is
an objective method.
- (ii) To find malingerers. A person who feigns total deafness and does not give any response on pure tone audiometry but shows a positive stapedial reflex is a malingerer.
(iii)
To detect
cochlear pathology.
Presence of
stapedial reflex
at lower
intensities, e.g.
40-60
dB
than the
usual
70
dB
indi- cates
recruitment and
thus
a
cochlear type
of hearing
loss.
(iv) To detect VITth nerve lesion. If a sustained tone of 500 or 1000 Hz, delivered 10 dB above acoustic reflex threshold, for a period of 10 s, brings the reflex amplitude to 50%, it shows abnormal adaptation and is indicative of VIlIth nerve lesion (stapedial reflex decay).
(v) Lesions of facial nerve. Absence of stapedial reflex when hearing is normal indicates lesion of the facial nerve, proximal to the nerve to stapedius. The reflex can also be used to find prognosis of facial paralysis as the appearance of reflex, after it was absent, indicates return of function and a favourable prognosis.
(vi) Leston of brainstem. If ipsilateral reflex is present but the contralateral reflex is absent, lesion is in the area of crossed pathways in the brainstem.
Figure
4.6
Types of
tympanograms.
A—Normal.
As—Reduced compliance
at ambient
pressure
(otosclerosis).
stands for shallow tympanogram but remember for stiffness.
AbD—Increased compliance
at ambient
pressure
(ossicular tinuity).
‘d’
stands for
deep tympanogram.
Remember ossicular
chain.
B—Flat or dome-shaped (fluid in middle ear).
C—Maximum compliance at pressures more than -200 mm H2O (negative pressure in middle ear), e.g. eustachian tube obstruction or early stage of otitis media with effusion.
Figure
4.7
Acoustic reflex.
discon- disruption
of
‘s’

Physical volume of ear canal. Acoustic immittance can also measure the physical volume of air between the probe tip and tympanic membrane. Normally it is up to 1.0 mL in children and 2 mL in adults. Any increase in volume, >2 mL in children and >2.5 mL in adults, indicates perforation of the tympanic membrane (because middle ear volume is added up to the volume of external ear canal). This has also been used to find patency of the ventilation tube.

It is a phenomenon of abnormal growth of loudness. The ear which does not hear low intensity sound begins to hear greater intensity sounds as loud or even louder than normal hearing ear. Thus, a loud sound which is tolerable in normal ear may grow to abnormal levels of loudness in the recruiting ear and thus becomes intolerable. The patients with recruitment are poor candidates for hearing aids. Recruitment is typically seen in lesions of the cochlea (e.g. Méniére's disease and presbycusis) and thus helps to differentiate a cochlear from a retrocochlear sensorineu-

Alternate binaural loudness balance test is used to detect recruitment in unilateral cases. A tone, say of 1000 Hz, is played alternately to the normal and the affected ear and the intensity in the affected ear is adjusted to match the loudness in normal ear. The test is started at 20 dB above the threshold of deaf ear and then repeated at every 20 dB rise until the loudness is matched or the limits of audiometer reached. In conductive and neural deafness, the initial difference is maintained throughout while in cochlear lesions, partial, complete or over-recruitment may be seen (Figure 4.8).

Patients with cochlear lesions distinguish smaller changes in intensity of pure tone better than normal persons and those with conductive or retrocochlear pathology. SISI test is thus used to differentiate a cochlear from a retrocochlear lesion.
In this test, a continuous tone is presented 20 dB above the threshold and sustained for about 2 min. Every 5 s, the tone is increased by | dB and 20 such blips are presented. Patient indicates the blips heard. In conductive deafness, SISI score is seldom more than 15%; it is 70-100% in cochlear deafness and 0-20% in nerve deafness.

It is a measure of nerve fatigue and is used to detect retrocochlear lesions. Normally, a person can hear a tone continuously for 60 s. In nerve fatigue, he stops hearing earlier. The threshold tone decay test is simple and is performed in the following manner:
A tone of 4000 Hz is presented at 5 dB above the patient's threshold of hearing, continuously for a period of 60 s. If patient stops hearing earlier, intensity is increased by another 5 dB. The procedure is continued till patient can hear the tone continuously for 60 s, or no level exists above the threshold where tone is audible for full 60 s. The result is expressed as number of dB of decay. A decay more than 25 dB is diagnostic of a retrocochlear lesion.

It is an objective test which measures electrical activity in the auditory pathways in response to auditory stimuli. It requires special equipment with an averaging computer. There are several components of evoked electric response but only two have gained clinical acceptance. They are:
(a)
Electrocochleography (EcoG). It measures electrical potentials arising in the cochlea and CN VIII in response to auditory stimuli within first 5 ms. The response is in the form of three phenomena: cochlear microphonics, summating potentials and the action potential of VUlIth nerve. The recording electrode is usually a thin needle passed through the tympanic membrane onto the promontory. In adults, it can be done under local anaesthesia but in children or anxious persons sedation or general anaesthesia is required. Sedation does not interfere in these responses. EcoG is useful (i) to find threshold of hearing in young infants and children within 5-10 dB and (ii) to differentiate lesions of cochlea from those of the VIIth nerve.
Normally the ratio between the amplitude of summating potential to the action potential is less than 30%. An increase in this ratio is indicative of Méniére's disease (Figure 4.9).
Auditory brainstem response (ABR). Also called BAER or BAEP (brainstem auditory evoked response or potential) or BERA (brainstem evoked response audiometry) is to elicit brainstem responses to auditory stimulation by clicks or tone bursts. It is a noninvasive technique to find the integrity of central auditory pathways through the VHIth nerve, pons and midbrain. In this method, electrical potentials are generated in response to several click stimuli or tone bursts and picked up from the vertex by surface electrodes. It measures hearing sensitivity in the range of 1000-4000 Hz. In a normal person, seven waves are produced in the first 10 ms. The first, third and fifth waves are most stable and are used in measurements. The waves are studied for absolute latency, interwave latency (usually between wave I and V) and the amplitude (Figure 4.10).
(b)
The exact anatomic site of neural generators for various waves is disputed but the latest studies indicate the following sites:
Wave I
Wave
II

hearing sensitivity in severe to profoundly deaf infants. ASSR is useful in such situations. It is, like ABR, an electrophysiological test which uses steady state pure tone signals instead of transient signals of tone bursts or clicks used in ABR. The steady state signals are also modulated rapidly in amplitude and frequency and thus gives a frequency-specific audiogram. Hearing losses exceeding 80 dB can be detected. It can help in selection of children for cochlear implantation at an early age.
Wave III
Wave IV
Wave V
Distal part of CN VIII
Proximal part brainstem of CN VIII near the
Cochlear nucleus
Lateral lemniscus
Waves VI and VII
Inferior colliculus
As aide memorie remember the mnemonic EE COLI
an (eight, eight, cochlear nucleus, olivary complex, lateral lemniscus, inferior colliculus) compare E COLI-MA in pathways of hearing.
ABR is used:
- (i) As a screening procedure for infants.
(ii)
To determine
the threshold
of hearing
in infants;
also in children and adults who do not cooperate and in malingerers.
(iii) To diagnose retrocochlear pathology particularly acoustic neuroma.
(iv) To diagnose brainstem pathology, e.g. multiple sclerosis or pontine tumours.
(vi)
To monitor
CN
VIII
intraoperatively in
surgery of
acoustic neuromas
to preserve
the function
nerve.

Though ABR, conducted with tone bursts of various frequencies, can produce frequency-specific thresholds of hearing in infants to fit hearing aid at a young age, it has limitations. It cannot test hearing losses above 80 dB. It cannot detect of
cochlear

They are low-intensity sounds produced by outer hair cells of a normal cochlea and can be elicited by a very sensitive microphone placed in the external ear canal and analyzed by a computer. Sound produced by outer hair cells travels in a reverse direction: outer hair cells > basilar membrane —> perilymph — oval window — ossicles > tympanic membrane — ear canal. OAEs are present when outer hair cells are healthy and are absent when they are damaged and thus help to test the function of cochlea. They do not disappear in VIIIth nerve pathology as cochlear hair cells are normal.
Types of OAEs. Broadly OAEs are of two types: spontaneous and evoked. The latter are elicited by a sound stimulus.
- (a) Spontaneous OAEs. They are present in healthy normal hearing persons where hearing loss does not exceed 30 dB. They may be absent in 50% of normal persons.
(b)
Evoked
OAEs.
They are
further divided
into two
types depending
on
(i)
Transient the
sound evoked
A
series of
stimulus
OAEs click
used
(TEOAEs).
stimuli to
elicit them.
Evoked are
presented by
clicks.
at
80-85
dB
SPL
(sound pressure
level)
and response
recorded.
- (ii) Distortion product OAEs (DPOAEs). Two tones are simultaneously presented to the cochlea to produce distortion products. They have been used to test hearing in the range of 1000-8000 Hz.

(a) OAEs are used as a screening test of hearing in neonates and to test hearing in uncooperative or mentally challenged individuals after sedation. Sedation does not interfere with OAEs.
(b) They help to distinguish cochlear from retrocochlear hearing loss. OAEs are absent in cochlear lesions, e.g. ototoxic sensorineural hearing loss. They detect ototoxic effects earlier than pure tone audiometry.
(c) OAEs are also useful to diagnose retrocochlear pathology, especially auditory neuropathy. Auditory neuropathy is a neurologic disorder of CN VIII. Audiometric tests, e.g. SNHL for pure tones, impaired speech discrimination score, absent or abnormal auditory brainstem response, show a retrocochlear type of lesion but OAEs are normal.
OAEs are absent in 50% of normal individuals, lesions of cochlea, middle ear disorders (as sound travelling in reverse direction cannot be picked up) and when hearing loss exceeds 30 dB.

Patients with central auditory disorders have difficulty in hearing in noisy surroundings or when the speech is
distorted and not clearly spoken. Three different types of speech discrimination tests are used.
(a) Monotic test. It is presented with speech message which is distorted. Patients with lesions of brain and cortex have difficulty to understand the message.
(b) Dichotic test. Two different speech messages are pre- sented simultaneously, one to each ear and patient is asked to identify both. Staggered spondaic word test is the one more often used. Pairs of spondaic words along with digits or nonsense words are simultane- ously presented to the ears. Patients with temporal lobe lesions will have difficulty identifying these words when presented to the ear opposite to that of the side of lesion.
(c) Binaural tests. They are used to identify integration of information from both ears. Such tests are normal in cortical lesions but affected in lesions of brainstem and thus help to localize the site of lesion. Most common test used is binaural masking level difference test.
Central auditory tests are not used routinely.
8. HEARING ASSESSMENT IN INFANTS AND CHILDREN (SEE P. 118)

Any disease process which interferes with the conduction of sound to reach cochlea causes conductive hearing loss. The lesion may lie in the external ear and tympanic membrane, middle ear or ossicles up to stapediovestibular joint.
The characteristics of conductive hearing loss are:
Negative Rinne test, i.e. BC > AC.
Weber lateralized to poorer ear. Normal absolute bone conduction. ON
Low frequencies affected more. oR
Audiometry shows bone conduction better than air conduction with airbone gap. Greater the air-bone gap, more is the conductive loss (Figure 5.1).
. Loss is not more than 60 dB. nm
Speech discrimination is good.

The cause may be congenital (Table 5.1) or acquired (Table 5.2).

- 1. Complete obstruction of ear canal:
30
- 2. Perforation of tympanic membrane (It
varies and is directly proportional to the size of perforation):
- 3. Ossicular interruption with intact drum:
dB
4. Ossicular interruption with perforation:
5. Malleus fixation:
38 dB
10-25 dB
6. Closure of oval window:
60 dB
Note here that ossicular interruption with intact drum causes more loss than ossicular interruption with perforated drum.

Most cases of conductive hearing loss can be managed by medical or surgical means. Treatment of these conditions is discussed in respective sections. Briefly, it consists of:
1. Removal of canal obstructions, e.g. impacted wax, for-
eign body, osteoma or exostosis, keratotic mass, benign or malignant tumours, or meatal atresia.
2. Removal of fluid. Myringotomy with or without grommet insertion.
3. Removal of mass from middle ear. Tympanotomy and removal of small middle ear tumours or cholesteatoma behind intact tympanic membrane.
4. Stapedectomy, as in otosclerotic fixation of stapes footplate.
5. Tympanoplasty. Repair of perforation, ossicular chain or both.
6. Hearing aid. In cases, where surgery is not possible, refused or has failed.

It is an operation to (i) eradicate disease in the middle ear and (ii) to reconstruct hearing mechanism. It may be combined with mastoidectomy if disease process so demands. Type of middle ear reconstruction depends on the damage present in the ear. The procedure may be limited only to repair of tympanic membrane (myringoplasty) , or to reconstruction of ossicular chain (ossiculoplasty), or both (tympanoplasty). Reconstructive surgery of the ear has been greatly facilitated by development of operating microscope, microsurgical instruments and biocompatible implant materials.
From the physiology of hearing mechanism, the following principles can be deduced to restore hearing surgically:
- (a) An intact tympanic membrane, to provide large hydrau-
lic ratio between the tympanic membrane and stapes footplate.
- (b) Osstcular chain, to conduct sound from tympanic membrane to the oval window.
(c)
Two functioning windows, one on the scala vestibuli (to receive sound vibrations) and the other on the scala
ear
Figure
5.1
loss
(A)
Audiogram with
A-B
gap.
of right
(B)
Symbols used
Meatal atresia
Fixation of stapes footplate
Fixation of malleus head
Ossicular discontinuity
© Congenital cholesteatoma

Table 5.2, 1 = Acquired causes of conductive hearing loss. External ear Middle ear, 1 = Any obstruction in the ear canal, e.g. wax, foreign body, furuncle, acute inflammatory swelling, benign or malignant tumour or atresia of canal. (a) Perforation of tympanic membrane, traumatic or infective (b) Fluid in the middle ear, e.g. acute otitis media, serous otitis media or haemotympanum (c) Mass in middle ear, e.g. benign or malignant tumour (d) Disruption of ossicles, e.g. trauma to ossicu- lar chain, chronic suppurative otitis media, cholesteatoma (e) Fixation of ossicles, e.g. otosclerosis, tympa- nosclerosis, adhesive otitis media (f) Eustachian tube blockage, e.g. retracted tym- panic membrane, serous otitis media
tympani (to act as a relief window). If it is only one win- dow,
as in
stapes fixation
or closure
of round
window, there
will be
no in
movement conductive
Acoustic hearing
of loss.
separation not
reach of
two both
windows, the
achieved by
windows providing
preferential cochlear
fluids resulting
so that
sound simultaneously.
an intact
pathway to
one does
It can
be tympanic
membrane, window
(usually the
oval)
in showing
conductive audiogram
charting.
(d)
hearing
(e)
(f)
by providing ossicular chain and by the presence of air in the middle ear.
Functioning eustachian tube, to provide aeration to the middle ear.
A
functioning sensorineural
and VIth nerve.
Types of Tympanoplasty
Wullstein classified tympanoplasty into five types (Figure 5.2).
TypeI
Defect is perforation of tympanic membrane which is repaired with a graft. It is also called myringoplasty.
Defect
is
perforation
of
tympanic
membrane
with
erosion

of
malleus.
Graft
is
placed
on
the
incus
or
remnant
of
malleus.
Malleus and incus are absent. Graft is placed directly on the stapes head. It is also called myrin- gostapediopexy or columella tympanoplasty.
Only
the
footplate
of
stapes
is
present.
It
is
exposed
to
the
external
ear,
and
graft
is
placed
between
the
oval
and
round
windows.
A
narrow
middle
ear
(cavum
minor)
is
thus
created
to
have
an
air
pocket
around
the
round
window.
Amucosa-lined
space
extends
from
the
eustachian
tube
to
the
round
window.
Sound
waves
in
this
case
act
directly
on
the
footplate
while
the
round
window
has
been
shielded.
Stapes footplate is fixed but round window is functioning. In such cases, another window is created on horizontal semicircular canal and covered with a graft. Also called fenestration operation.
Type II
Type III
Type IV
Type V
Several modifications have appeared in the above classification and they mainly pertain to the types of ossicular reconstruction.

It is repair of tympanic membrane. Graft materials of choice are temporalis fascia or the perichondrium taken from the patient. Sometimes, homografts such as dura, vein, fascia or cadaver tympanic membrane are also used. Repair can be done by two techniques—the underlay or the overlay. In the underlay technique, margins of perforation are freshened and the graft placed medial to perforation or tympanic annulus, if large, and is supported by gelfoam in the middle ear (Figure 5.3A). In the overlay technique, the graft is placed lateral to fibrous layer of the tympanic membrane after carefully removing all squamous epithelium from the lateral surface of tympanic membrane remnant (Figure 5.3B and Chapter 82).

Ossicles are essential for transmission of sound from tympanic membrane to labyrinth. Several types of prosthesis are available to replace ossicles depending on the ossicular defects (Table 5.3). Autograft ossicles can be sculptured to bridge the gap. Homograft preserved ossicles with or without tympanic membrane have been used but are difficult to procure and have danger of transmission of disease (Figure 5.4).
apparatus, i.e.
the cochlea

Type III (Myringostapediopexy)
Type
IV
Figure 5.3 Myringoplasty. (A) Underlay technique—fascia graft is under the anterior annulus. It is supported by gelfoam in the middle ear to prevent medial displacement. (B) Overlay technique—fascia graft lies lateral to anterior annulus onto the anterior bony canal wall. It is placed medial to malleus handle to prevent lateralization.
Autograft, Material = • Incus, head of malleus • Cortical bone from mastoid. Autograft, Remarks = • Risk of harbouring disease • Low cost • Easily available. Allograft, Material = • Plastipore (polyeth- ylene sponge) • Hydroxyapatite (HA) implants • Titanium implants • Glass isomer • Teflon prosthesis • HA (50%) + Titanium (50%) • HA + Silicon (flex-HA). Allograft, Remarks = • Readymade • Easy to store and use • Costly • Likely to be extruded. Homograft, Material = (HAPEX) • Preserved ossicles only • Ossicles with tym- panic membrane. Homograft, Remarks = • Difficult to procure • Risk of disease transmission
Type
Il
Type
V
(Fenestration)
Cc
Figure
5.4
Ossicular reconstruction.
Sculptured autograft
or homograft
ossicles
Modified have
been used.
incus graft
(A)
(B)
Malleus—Footplate leus
Malleus—Stapes malleus
assembly.
handle with
stapes tympanic
membrane assembly.
stapes
Modified footplate.
(TM)
malleus
(C)
Modified to
stapes head.
head.
connecting incus
mal- connecting
Malleus is
missing.
(D)
Modified incus
TM
to stapes
footplate.
At the time of ossicular reconstruction in chronic otitis media, one should ensure:
¢
Middle ear
is healthy
and free
of mucosal
disease and

cholesteatoma.
Eustachian tube
function is
good.
Atelectatic shows poor eustachian tube function.
In cases
of canal
wall-up mastoidectomy
toma or
active mucosal
disease, done
the procedure
middle ear
for cholestea-
is delayed
for about
6
months to
ensure ear
is free
of disease.
Primary ossicular reconstruction can be performed in:
¢ Traumatic ossicular disruption
¢ Fixation of ossicles
© Canal wall down procedures when there is no mucosal dis-
ease or cholesteatoma
¢
Types of prosthesis (Figure 5.5)
1. Incus prosthesis. Used when incus is missing but handle of malleus and stapes with superstructure are present and functional.
2. Incus-stapes prosthesis. Used when incus and stapes superstructure are missing. Malleus and stapes footplate are functional.
3. Partial ossicular replacement prosthesis (PORP). Used
when malleus
and incus
are absent.
Stapes is
present and
mobile.
PORP
is placed
between tympanic
4. Total ossicular replacement prosthesis (TORP). Used
when malleus, incus and stapes superstructure are absent. Only the stapes footplate is present and is mobile.

Sensorineural hearing loss (SNHL) results from lesions of the cochlea, VIIth nerve or central auditory pathways. It may be present at birth (congenital) or start later in life (acquired).
The characteristics of sensorineural hearing loss are:
A positive Rinne test, i.e. AC > BC. Weber lateralized to better ear. wen
Bone conduction reduced on Schwabach and absolute
bone conduction tests.
More often involving high frequencies. ou
No gap between air and bone conduction curve on audiometry (Figure 5.6).
Loss may exceed 60 dB. Speech discrimination is poor. One
There is difficulty in hearing in the presence of noise.

It is present at birth and is the result of anomalies of the inner ear or damage to the hearing apparatus by prenatal or perinatal factors (see p. 115).

It appears later in life. The cause may be genetic or nongenetic. The genetic hearing loss may manifest late (delayed onset) and may affect only the hearing, or be a part of a larger syndrome affecting other systems of the body as well (syndromal). Common causes of acquired SNHL include:
1. Infections of labyrinth—viral, bacterial or spirochaetal
2. Trauma to labyrinth or VIIIth nerve, e.g. fractures of
temporal bone or concussion of the labyrinth or the ear surgery
. Noise-induced hearing loss
. Ototoxic drugs BA
Presbycusis
. Méniére's disease Acoustic neuroma WATIDHD
Sudden hearing loss
. Familial progressive SNHL SO
. Systemic disorders, e.g. diabetes, hypothyroidism, kidney disease, autoimmune disorders, multiple sclerosis, blood dyscrasias. —

1. History. It is important to know whether disease is congenital or acquired, stationary or progressive, associated with other syndromes or not, involvement of other members of the family and possible aetiologic factors.
2. Severity of deafness (mild, moderate, moderately severe, severe, profound or total). This can be found out on audiometry.
- 3. Type of audiogram. Whether loss is high frequency, low frequency, mid-frequency or flat type.
4. Site of lesion, i.e. cochlear, retrocochlear or central.
5. Laboratory tests. They depend on the aetiology suspected, e.g. X-rays or CT scan of temporal bone for evidence of bone destruction (congenital cholesteatoma, glomus tumour, middle ear malignancy or acoustic neuroma), blood counts (leukaemia), blood sugar (diabetes), serology for syphilis, thyroid functions (hypothyroidism), kidney function tests, etc.

Early detection of SNHL is important as measures can be taken to stop its progress, reverse it or to start an early rehabilitation programme, so essential for communication. Syphilis of the inner ear is treatable with high doses of penicillin and steroids with improvement in hearing. Hearing loss of hypothyroidism can be reversed with replacement therapy. Serous labyrinthitis can be reversed by attention to middle ear infection. Early management of Méniére's disease can prevent further episodes of vertigo and hearing loss. SNHL due to perilymph fistula can be corrected surgically by sealing the fistula in the oval or round window with fat. Ototoxic drugs should be used with care and discontinued if causing hearing loss. In many such cases, it may be possible to regain hearing, total or partial, if the drug is stopped. Noiseinduced hearing loss can be prevented from further deterioration if the person is removed from the noisy surroundings. Rehabilitation of hearing impaired with hearing aids and other devices is discussed in Chapter 20.

It may be viral, bacterial or syphilitic.
1. Viral labyrinthitis. Viruses usually reach the inner ear by blood stream affecting stria vascularis and then the endolymph and organ of Corti. Measles, mumps and cytomegaloviruses are well-documented to cause labyrinthitis. Several other viruses, e.g. rubella, herpes zoster, herpes simplex, influenza and Epstein-Barr are clinically known to cause deafness but direct proof of their invasion of labyrinth is lacking.
2. Bacterial. Bacterial infections reach labyrinth through the middle ear (tympanogenic) or through CSF (meningogenic). Labyrinthitis as a complication of middle ear infection is discussed on page 45. Sensorineural hearing loss following meningitis is a well-known clinical entity. Bacteria can invade the labyrinth along nerves, vessels, cochlear aqueduct or the endolymphatic sac. Membranous labyrinth is totally destroyed.
3. Syphilitic. Sensorineural hearing loss is caused both by congenital and acquired syphilis. Congenital syphilis is of two types: the early form, manifesting at the age of 2 or the late form, manifesting at the age of 8-20 years. Syphilitic involvement of the inner ear can cause:
(a) Sudden sensorineural hearing loss, which may be unilateral or bilateral. The latter is usually symmetrical in high frequencies or is a flat type.
(b) Méniére's syndrome with episodic vertigo, fluctuating hearing loss, tinnitus and aural fullness—a picture simulating Méniére's disease.
(c)
Hennebert’s sign.
A
positive fistula
sign in
of
a
fistula.
This is
due to
fibrous adhesions
stapes footplate
and the
membranous labyrinth.
- (d) Tullio phenomenon in which loud sounds produce vertigo.

Diagnosis of otosyphilis can be made by other clinical evidence of late acquired or congenital syphilis (interstitial keratitis, Hutchinson's teeth, saddle nose, nasal septal perforation and frontal bossing) and the laboratory tests. Fluorescent treponema-absorption test (FTA-ABS) and venereal disease research laboratory (VDRL) or rapid plasma reagin (RPR) tests from CSF are useful to establish the diagnosis. Treatment of otosyphilis includes i.v. penicillin and steroids.

It is a genetic disorder in which there is progressive degeneration of the cochlea starting in late childhood or early adult life. Hearing loss is bilateral with flat or basin-shaped audiogram but an excellent speech discrimination.

Various drugs and chemicals can damage the inner ear and cause sensorineural hearing loss, tinnitus and sometimes vertigo (Table 5.4).
1. Aminoglycoside antibiotics. Streptomycin, gentamicin and tobramycin are primarily vestibulotoxic. They selectively destroy type I hair cells of the crista ampullaris but, administered in large doses, can also damage the cochlea. Neomycin, kanamycin, amikacin, sisomycin and dihydrostreptomycin are cochleotoxic. They cause selective destruction of outer hair cells, starting at the basal coil and progressing onto the apex of cochlea.
Patients particularly at risk are those:
(a) having impaired renal function,
(b) elderly people above the age of 65,
(c) concomitantly receiving other ototoxic drugs,
(d) who have already received aminoglycoside antibiotics,
(e)
who are
receiving high
doses of
ototoxic drugs
with high
of drug,
and
- (f) who have genetic susceptibility to aminoglycosides. Here the antibiotic binds to the ribosome and interferes with protein synthesis, thus causing death of the cochlear cells.
Symptoms of ototoxicity, hearing loss, tinnitus and/or giddiness may manifest during treatment or after completion of the treatment (delayed toxicity).
2. Diuretics. Furosemide, bumetanide and ethacrynic acid are called loop diuretics as they block transport of sodium and chloride ions in the ascending loop of Henle. They are known to cause oedema and cystic changes in the stria vascularis of the cochlear duct. In most cases, the effect is reversible but permanent damage may occur. Hearing loss
may be bilateral and symmetrical or sometime sudden in onset.

3. Salicylates. Symptoms of salicylate ototoxicity are tinnitus and bilateral sensorineural hearing loss particularly affecting higher frequencies. Site of lesion testing indicates cochlear involvement, but light and electron microscopy have failed to show any morphologic changes in the hair cells. Possibly they interfere at enzymatic level. Hearing loss due to salicylates is reversible after the drug is discontinued. SNHL has also been noted with other NSAIDs, e.g. naproxen, piroxicam and ketorolac but is reversible.
4. Quinine. Ototoxic symptoms due to quinine are tinnitus and sensorineural hearing loss, both of which are reversible. Higher doses may cause permanent loss. The symptoms generally appear with prolonged medication but may occur with smaller doses in those who are susceptible. Congenital deafness and hypoplasia of cochlea have been reported in children whose mothers received this drug during the first trimester of pregnancy. Ototoxic effects of quinine are due to vasoconstriction in the small vessels of the cochlea and stria vascularis.
5. Chloroquine and hydroxychloroquine. Effect is similar to that of quinine and cause reversible SNHL. Sometimes permanent deafness can result.
6.
Cytotoxic drugs.
Nitrogen mustard,
cisplatin and
carbo- platin
can cause
cells cochlear
of the
cochlea.
7.
Deferoxamine substance
(Desferrioxamine).
used in
who receive
have
They affect
the outer
hair
It the
treatment is
an iron-chelating
high iron
thalassaemic blood
load.
transfusions
Like cisplatin
in turn
and aminoglycosides,
damage.
deferoxamine also causes high-frequency sensorineural hearing loss. Onset of hearing loss is sudden or delayed. It is permanent but in some cases it can be reversible when the drug is discontinued. It causes toxicity to nerves; children are affected more.
8.
Miscellaneous.
Isolated
cases
of
deafness
have
been
reported
with
erythromycin,
ampicillin
and

chlorampheni-
col,
indomethacin,
phenylbutazone,
ibuprofen,
tetanus
antitoxin,
propranolol
and
propylthiouracil.
Alcohol,
tobacco
and
marijuana
also
cause
damage
to
the
inner ear.
9. Topical ear drops. Topical use of drugs in the middle ear can also cause damage to the cochlea by absorption through oval and round windows. Deafness has occurred with the use of chlorhexidine which was used in the preparation of ear canal before surgery or use of ear drops containing amino- glycoside antibiotics, e.g. neomycin, framycetin and gentami- cin. Ototoxic potential is also present in ear drops containing polymyxin B, propylene glycol and antifungal agents. Use only approved ototopical drops for middle ear infection.

Hearing loss associated with exposure to noise has been wellknown in boiler makers, ironand coppersmiths, and artillery men. Lately, noise trauma has assumed greater significance because of its being an occupational hazard; the compensations asked for and the responsibilities thrust upon the employer and the employee to conserve hearing. Hearing loss caused by excessive noise can be divided into two groups:
1. Acoustic trauma. Permanent damage to hearing can be caused by a single brief exposure to very intense sound without this being preceded by a temporary threshold shift. Also called impulse noise, such noise can arise from an explosion, gun fire or a powerful cracker and may reach or cross 140 dB. Noise level ofa gun or rifle may reach 140-170 dB SPL (sound pressure level). Such brief and loud noises mechanically damage organ of Corti, tear Reissner's membrane, rupture hair cells and allowing mixing of perilymph and endolymph. A severe blast, in addition, may concomitantly damage the tympanic membrane and disrupt ossicles further adding conductive loss. Impulse noise may be as brief as 0.2 ms. No impulse noise more than 140 dB (A) is permitted.
2. Noise-induced hearing loss (NIHL). Hearing loss, in this case, follows chronic exposure to less intense sounds than seen in acoustic trauma and is mainly a hazard of noisy occupations.
(a)
Temporary threshold
shift
(TTS).
The hearing
is impaired
immediately after
an interval
exposure of
to
2
weeks.
a
few to
minutes
Amount intensity,
frequency
Permanent
(b)
noise but
recovers to
a
few of
TTS
depends and
threshold shift
duration.
(PTS).
even on
up the
noise—its impairment
is permanent
and does
not recover
at all.
The damage caused by noise trauma depends on several factors:
(i)
Frequency of noise. A frequency of 2000-3000 Hz causes more damage than lower or higher frequencies.

90, Permitted daily exposure (h) = 8.0. 92, Permitted daily exposure (h) = 6.0. 95, Permitted daily exposure (h) = 4.0. 97, Permitted daily exposure (h) = 3.0. 100, Permitted daily exposure (h) = 2.0. 102, Permitted daily exposure (h) = 11/2. 105, Permitted daily exposure (h) = 1.0. 110, Permitted daily exposure (h) = 1/2. 115, Permitted daily exposure (h) = 1/4
85 AB rule of time intensity states that "any rise of 5 dB noise level will reduce the permitted noise exposure time to half."
(ii)
Intensity and
duration of
noise.
As the
intensity increases,
permissible time
for gives
the exposure
permissible sity
levels limits
for the
of safety
Continuous vs
more of
is reduced.
Table
5.5
time ear.
interrupted harmful.
Susceptibility of
the varies
in noise.
Continuous individual.
different
Degree individuals.
(v)
Pre-existing ear
disease.
A noise of 90 dB (A) SPL, 8 h a day for 5 days per week is the maximum safe limit as recommended by Ministry of Labour, Govt. of India, Model Rules under Factories Act (Table 5.5). No exposure in excess of 115 dB (A) is to be permitted. No impulse noise of intensity greater than 140 dB (A) is permitted.
The Noise Pollution (Regulation and Control) Rules 2000, Ministry of Environment and Forest, Govt. of India has defined permissible limits of noise for various zones or areas (Table 5.6). According to which silence zone is 100 m around the premises of hospitals, nursing homes, educational institutions and courts. Also manufacture, sale and use of fire crackers generating sound level above 125 dB (AI) or 145 dB (C) pk from 4 m distance from the point of bursting are not permitted (Environment Protection Rules 2006) [dB (AI) = A-weighted impulse sound pressure level in decibels; dB (C) pk = C-weighted peak sound pressure in decibels].

The audiogram in NIHL shows a typical notch, at 4 kHz, both for air and bone conduction (Figure 5.7). It is usually symmetrical on both sides. At this stage, patient complains of high-pitched tinnitus and difficulty in hearing in noisy surroundings but no difficulty in day-to-day hearing. As the duration of noise exposure increases, the notch deepens and also widens to involve lower and higher frequencies. Hearing impairment becomes clinically apparent to the patient when the frequencies of 500, 1000 and 2000 Hz (the speech frequencies) are also affected.
NIHL causes damage to hair cells, starting in the basal turn of cochlea. Outer hair cells are affected before the inner hair cells.
for various
inten- noise
of
TTS
is and
PTS
(iii)
(iv)
Table 5.6, 1 = Permissible limits of noise as per the Noise Pollution (Regulation and Control) Rules 2000, Ministry of Environment and Forest, Govt. of India Day (6 am to 10 pm) Night (10 pm to 6. Table 5.6, 2 = Permissible limits of noise as per the Noise Pollution (Regulation and Control) Rules 2000, Ministry of Environment and Forest, Govt. of India Day (6 am to 10 pm) Night (10 pm to 6. Zone/Area, 1 = Limits in dB(A) Leq a. Zone/Area, 2 = am) Limits in dB(A) Leq. Industrial, 1 = 75. Industrial, 2 = 70. Commercial, 1 = 65. Commercial, 2 = 55. Residential, 1 = 55. Residential, 2 = 45. Silence, 1 = 50. Silence, 2 = 40

dB
in loss
Hearing
Figure 5.7 Early case of noise-induced hearing loss. Note dip at 4000 Hz.
Noise-induced hearing loss is preventable. Persons who have to work at places where noise is above 85 dB (A) should have pre-employment and then annual audiograms for early detection. Ear protectors (ear plugs or ear muffs) should be used where noise levels exceed 85 dB (A). They provide protection up to 35 dB (see Table 5.7). If hearing impairment has already occurred, rehabilitation is similar to that employed for other sensorineural hearing losses.
3. Nonauditory effects of noise. Apart from hearing loss, noise can affect other systems of the body. It interferes with
rest and sleep causing chronic fatigue and stress. Through activation of the autonomic nervous system and pituitary— adrenal axis, it causes annoyance and irritability. Hypertension and peptic ulcer have also been attributed to it. It also adversely affects task performance where communication through speech is required. Laryngeal problems have been noticed in workers who have to speak loudly in persistently noisy surroundings.

Immune-mediated inner ear disease (Syn. autoimmune SNHL) causes progressive bilateral sensorineural hearing loss. It occurs between 40 and 50 years with equal incidence in both sexes. Nearly 50% of patients also experience vestibular symptoms like disequilibrium, motion intolerance, positional or episodic vertigo. About 15% of patients have evidence of other autoimmune disorder such as ulcerative colitis, systemic lupus, rheumatoid arthritis or multiple sclerosis. Moscicki et al. defined the condition as:
'Bilateral SNHL > 30 dB at any frequency and evidence of progression in at least one ear on two serial audiograms that are done at equal to or less than 3 months apart. Progression is defined as threshold shift of = 15 dB at one frequency or 10 dB at two or more consecutive frequencies or significant change in speech discrimination'.

1. Audiogram. To establish above criteria, repeated audiograms can be taken at one month intervals. Audiogram may show loss at high and low frequencies.
2. Speech audiogram. Speech discrimination is affected though threshold of pure tones remains the same.
3. Evoked response audiometry. To exclude acoustic neuroma or multiple sclerosis.
Contrast-enhanced MRI. *
5. Blood tests to exclude systemic autoimmune disorders. Total and differential counts, ESR, rheumatoid factor, antinuclear antibodies, C3 and C4 compliment levels, Raji cell assay for circulating immune complexes.
6. Western blot essay for anti-Hsp 70 (anti-heat shock protein 70) antibodies. Antigen used in this test is crude protein extract from bovine renal cells. It is not a specific test for diagnosis but correlates to both active disease and steroid responsiveness.

Prednisolone
1
mg/kg/day
up
to
a
total
of
60
mg/day
(for
adults)
for
4
weeks.
Sometimes
response
is
late.
If
no
response
is
seen
in
4
weeks,
steroid
is
tapered
off
in
12
days.
Responders
continue
till
a
plateau
is
reached
and
then
con-
tinue
on
maintenance
dose
of
10-20
mg
every
other
day
for
about
6
months.
Side
effects
and
risks
of
long-term
steroid
therapy
should
be
kept
in
mind.
Those who cannot take steroids can be given methotrex- ate 15 mg/week for 6-8 weeks and if the patient responds, continue it for 6 months. If no response is obtained for 6-8 weeks trial, drug is discontinued.
Alternative to methotrexate is cyclophosphamide but it is toxic.
more
Other treatments include intratympanic steroid injection, IgG injection and plasmapheresis.
systemic

Sudden SNHL is defined as 30 dB or more of SNHL over at least three contiguous frequencies occurring within a period of 3 days or less. Mostly it is unilateral. It may be accompanied by tinnitus or temporary spell of vertigo.

Most often the cause of sudden deafness remains obscure, in which case it is called the idiopathic variety. In such cases, three aetiological factors are considered—viral, vascular or the rupture of cochlear membranes. Spontaneous perilymph fistulae may form in the oval or round window. Other aetiological factors which cause sudden deafness and must be excluded are listed below. Remember the mnemonic "In The Very Ear Too No Major Pathology."
1. Infections. Mumps, herpes zoster, meningitis, encephali- tis, syphilis, otitis media.
2.
Trauma.
Head
injury,
ear
operations,
noise
trauma,
baro-
trauma,
spontaneous
rupture
of
cochlear
3. Vascular. Haemorrhage (leukaemia), embolism or throm- bosis of labyrinthine or cochlear artery or their vasospasm. They may be associated with diabetes, hypertension, poly- cythaemia, macroglobinaemia or sickle cell trait.
4. Ear (otologic). Méniére’s disease, Cogan’s syndrome, large vestibular aqueduct.
5. Toxic. Ototoxic drugs, insecticides.
6. Neoplastic. Acoustic neuroma. Metastases in cerebello- pontine angle, carcinomatous neuropathy.
7. Miscellaneous. Multiple sclerosis, hypothyroidism, sarcoidosis.
- 8. Psychogenic.

As far as possible, the aetiology of sudden hearing loss should be discovered by detailed history, physical examination and laboratory investigations. The investigations may include audiometry, vestibular tests, imaging studies of temporal bones, sedimentation rate, tests for syphilis, diabetes, hypothyroidism, blood disorders and lipid profiles. Some cases may require exploratory tympanotomy where perilymph fistula is strongly suspected. Where the cause still remains obscure, treatment is empirical and consists of:
- 1. Bed rest.
2. Steroid therapy. Prednisolone 40-60 mg in a single morning dose for 1 week and then tailed off in a period of 3 weeks. Steroids are anti-inflammatory and relieve oedema. They have been found useful in idiopathic sud- den hearing loss of moderate degree.
3.
Inhalation
of
carbogen
(5%
CO»
+
95%
Og).
It
increases
cochlear
blood
flow
and
improves
4. Vasodilator drugs.
5. Low molecular weight dextran. It decreases blood viscos-
ity.
It
is
contraindicated
in
cardiac
failure
and
bleeding
disorders.
6. Hyperbaric oxygen therapy. Available only in selected cen- tres, hyperbaric oxygen raises concentration of oxygen in
labyrinthine fluids and improves cochlear function (see p358).
7. Low-salt diet and a diuretic. It is empirical and has same benefit as in cases of Méniére's disease.
8. Intratympanic steroids therapy. It raises the local concentration of steroids in cochlear fluids, thus avoiding side effects of systemic therapy.

Many treatment protocols have been suggested for idiopathic sensorineural sudden hearing loss but none has shown significant benefit over the benefit of spontaneous recovery which occurs in 50-60% cases within first 2 weeks. of the drugs, dextran 40, vasodilators, carbogen inhalation (5% COx, with 95% Og), diatrizoate meglumine, have shown significant benefit.
Generally prescribed medicines include:
1. Steroids.
2. Inhalation of carbogen.
3. Low-salt diet and a diuretic.
4. Hyperbaric oxygen.

Fortunately, about half the patients of idiopathic sensorineural hearing loss recover spontaneously within 15 days. Chances of recovery are poor after 1 month. Severe hearing loss and that associated with vertigo have poor prognosis. Younger patients below 40 and those with moderate losses have better prognosis (see Table 5.8).

Sensorineural hearing loss associated with physiological aging process in the ear is called presbycusis. It usually manifests at the age of 65 years but may do so early if there is hereditary predisposition, chronic noise exposure or generalized vascular disease.
Four pathological types of presbycusis have been identified.
1. Sensory. This is characterized by degeneration of the organ of Corti, starting at the basal coil and progressing gradually to the apex. Higher frequencies are affected but speech discrimination remains good.
2. Neural. This is characterized by degeneration of the cells of spiral ganglion, starting at the basal coil and progressing to the apex. Neurons of higher auditory pathways may also be affected. This manifests with high tone loss but speech discrimination is poor and out of proportion to the pure tone loss.
3. Strial or metabolic. This is characterized by atrophy of stria vascularis in all turns of cochlea. In this, the physical and chemical processes of energy production are affected. It runs in families. Audiogram is flat but speech discrimination is good.
4. Cochlear conductive. This is due to stiffening of the basilar membrane thus affecting its movements. Audiogram is sloping type.
Patients of presbycusis have great difficulty in hearing in the presence of background noise though they may hear well in quiet surroundings. They may complain of speech
being heard but not understood. Recruitment phenomenon is positive and all the sounds suddenly become intolerable when volume is raised. Tinnitus is another bothersome problem and in some it is the only complaint. Patients of presbycusis can be helped by a hearing aid. They should also have lessons in speech reading through visual cues. Curtailment of smoking and stimulants like tea and coffee may help to decrease tinnitus.

In this type of hearing loss, there is no organic lesion. It is either due to malingering or is psychogenic. In the former, usually there is a motive to claim some compensation for being exposed to industrial noises, head injury or ototoxic medication. Patient may present with any of the three clinical situations:
(i) Total hearing loss in both ears, (ii) total loss in only one ear or (iii) exaggerated loss in one or both ears. The responsibility of the physician is to find out: Is the patient malingering? If so, what is his actual threshold of hearing? This is accomplished by:
1. High index of suspicion. Suspicion further rises when the patient makes exaggerated efforts to hear, frequently making requests to repeat the question or placing a cupped hand to the ear.
2. Inconsistent results on repeat pure tone and speech audiometry tests. Normally, the results of repeat tests are within +5 dB. A variation greater than 15 dB is diagnostic of NOHL.
3. Absence of shadow curve. Normally, a shadow curve can be obtained while testing bone conduction, if the healthy ear is not masked. This is due to transcranial transmission of sound to the healthy ear. Absence of this curve in a patient complaining of unilateral deafness is diagnostic of NOHL.
4. Inconsistency in PTA and SRT. Normally, pure tone average (PTA) of three speech frequencies (500, 1000 and 2000 Hz) is within 10 dB of speech reception threshold (SRT). An SRT better than PTA by more than 10 dB points to NOHL.
5. Stenger test. It can be done with a pair of identical tuning forks or a double-channel audiometer. Principle involved is that, if a tone of two intensities, one greater than the other, is delivered to two ears simultaneously, only the ear which receives tone of greater intensity will hear it. To do this test, take two tuning forks of equal frequency, strike and keep

them say 25 cm from each ear. Patient will claim to hear it in the normal ear. Now bring the tuning fork on the side of feigned deafness to within 8 cm, keeping the tuning fork on the normal side at the same distance. The patient will deny hearing anything even though tuning fork on normal side is where it could be heard earlier. A person with true deafness should continue to hear on the normal side. Patient should be blindfolded this test.
during
This same test can be performed with a two-channel audi- ometer using pure tone or speech signals.
6.
Acoustic reflex
threshold.
Normally, stapedial
elicited at
70-100
dB
SL.
If patient
claims reflex
total deafness
is but
the reflex can be elicited, it indicates NOHL.
7. Electric response audiometry (ERA). It is very useful in NOHL and can establish hearing acuity of the person to within 5-10 dB of actual thresholds.

Hearing loss is impairment of hearing and its severity may vary from mild to severe or profound, while the term deafness is used, when there is little or no hearing at all. In some countries, this rigid differentiation is not made. They use the term deafness to denote any degree of hearing loss irrespective of its severity. In 1980, WHO recommended that the term "deaf" should be applied only to those individuals whose hearing impairment is so severe that they are unable to benefit from any type of amplification. A similar definition is used in India while extending benefits to the hearing handicapped.

(Ministry of Social Welfare, Government of India—Scheme of Assistance to Hearing Handicap).
"The deaf are those in whom the sense of hearing is non- functional for ordinary purposes of life." They do not hear/
understand sounds
at all
even with
amplified speech.
The cases
included in
the category
will be
those having
loss more
than
90
dB
in the
better ear
hearing
(profound impair
ment) or total loss of hearing in both ears.
The partially hearing are defined as those falling under any one of the following categories:
Hearing acuity
More than 30 but not more than 45
dB in better ear
More than 45 but not more than 60
More than 60 but not more than 90

WHO (1980) recommended the following classification on the basis of pure tone audiogram taking the average of the
of loss. cent of population has 210 110
Figure 5.8 Classification hearing Ninety-five per thresholds between and dB HL.
thresholds of hearing for frequencies of 500, 1000 and 2000 Hz with reference to ISO: R. 389-1970 (International Calibration of Audiometers).
DEGREE OF HEARING LOSS (FIGURE 5.8)
1. Mild
26-40 dB
2. Moderate
41-55 dB
3. Moderately severe
56-70 dB
4. Severe
71-91 dB
5. Profound
More than 91 dB
6. Total
From this it is implied that there is no apparent impairment of hearing from 0 to 25 dB.
The disability to understand speech with different degrees of hearing loss is given in Table 5.9.

When a disease process strikes an organ or a system it causes an impairment either in structure or function, but this impairment may or may not become clinically manifested. When impairment affects the ability to perform certain functions in the range considered normal for that individual it is called disability. The disability further restricts the duties and roles expected from an individual by society and is called a

To exemplify, injury (disease) to the ear may result in hear- ing impairment which, depending on its severity, will affect the individual's ability to hear and perform certain activities (disability) and will be termed handicap by the society:
Disease > Impairment — Disability > Handicap.
0-25, Degree of impairment (WHO classification) Ability to = Not significant. 0-25, understand speech = No significant difficulty with faint speech. 26-40, Degree of impairment (WHO classification) Ability to = Mild. 26-40, understand speech = Difficulty with faint speech. 41-55, Degree of impairment (WHO classification) Ability to = Moderate. 41-55, understand speech = Frequent difficulty with normal speech. 56-70, Degree of impairment (WHO classification) Ability to = Moderately severe. 56-70, understand speech = Frequent difficulty even with loud speech. 71-91, Degree of impairment (WHO classification) Ability to = Severe. 71-91, understand speech = Can understand only shouted or amplified speech. Above 91, Degree of impairment (WHO classification) Ability to = Profound. Above 91, understand speech = Usually cannot understand even amplified speech

Sometimes it is desired to express the impairment and handicap in terms of percentage for the purposes of compensation. Different countries and professional bodies have adopted their own system to calculate this percentage.
One of the methods to find hearing handicap is given below:
In the above calculation only three speech frequencies (500, 1000 and 2000 Hz) are taken into account but it is felt that frequency of 3000 Hz is important for hearing in the presence of noise and should also be taken into account. American Academy of Ophthalmology and Otolaryngology recommends and takes into account the average of four frequencies 500, 1000, 2000 and 3000 Hz when calculating the handicap.
(i) Take an audiogram and calculate the average of thresholds of hearing for frequencies of 500, 1000 and 2000 Hz say = A.
(ii)
Deduct from
it
25
dB
(as there
is no
impairment up
to
25 dB), ie. A-25.
(iii) Multiply it by 1.5, ie. (A-25) x 1.5.
This is the percentage of hearing impairment for that ear. Similarly calculate the percentage of hearing impairment for the other ear.
Total percentage handicap of an individual
Government of India reserved certain percentage of vacancies in Group C and D in favour of the physically handicapped and has extended certain other benefits. It has also recommended the classification based on percentage of impairment and the test required to be performed (see Table 5.10). (Brochure on Reservations and Concessions for Physically Handicapped in Central Govt. Services published by Ministry of Personnel, Public Grievances and Pensions, Dept. of Personnel and Training.)

Unilateral loss of hearing, even though total, does not produce a serious handicap or affect speech but it impairs localization of the sound source, difficulty in discrimination of speech in the presence of background noise and some difficulty at a meeting or in classroom when the speaker is on the side of affected ear. It should also alert the individual that he does not have a "spare or reserve ear" and has to take all precautions for the safety of the only hearing ear; also the surgeon should be careful when he is called upon to operate on this only hearing ear. Bone-anchored hearing aids are the treatment of choice for management of single-sided deafness (see p. 123).

Speech discrimination
1.
I.
Mild hearing impairment
26-40
dB
in
better
ear
80-100% in better ear
Less
than
40%
2.
Il.
Moderate hearing impairment
41-55
dB
in
better
ear
50-80% in better ear
40-50%
3
Ul.
Severe hearing impairment
56-70 dB hearing impairment
40-50%
50-75%
in better ear
4,
IV.
(a) Total deafness
No
hearing
No discrimination
100%
(b) Near total deafness
91 dB and above in better ear
-do-
100%
(c) Profound hearing impairment
71 to 90 dB
Less
than
40%
in
betterear
75-100%
(Pure
tone
average
of
hearing
in
500,
1000
and
2000
Hz
by
air
should
be
taken
as
basis
for
consideration
as
per
the
test
recommendations.)
Further it should be noted that:
(a)
When
there
is
only
an
island
of
hearing
present
in
one
or
two
in
better
ear,
it
should
be
considered
as
total
loss
of
hearing.
(b)
Wherever
there
is
no
response
(NR)
at
any
of
the
3
(500,
100,
2000
Hz),
it
should
be
as
to
130
dB
loss
for
the
purposes
of
of
disability
and
in
arriving
at
the
average.
This
is
based
on
the
fact
that
maximum
intensity
limits
in
most
of
the
audiometers
is
110
dB
and
some
have
facilities
for
20
dB
for
testing.
Il.
Recommendations
about
the
categories
of

(Hearing
impairment-Physical
aspect
only-Test
recommended).
(a)
Pure
tone
audiometry
(ISO
R
389-1970
at
present,
is
being
used
as
Audiometric
Standard
in
most
of
the
audiometers.
Hence
the
used
in
testing
should
be
accordingly
calibrated).
Three
frequency
average
at
500,
1000
and
2000
Hz
by
Air
Conduc-
tion
(AC),
will
be
used
for
categorization.
(b)
Wherever
possible
the
pure
tone
audiometric
results
should
be
supplemented
by
the
speech
discrimination
score—tested
at
sen-
sation
level
(SL),
i.e.
the
speech
discriminations
test
is
conducted
at
30-40
dB?
the
patient’s
hearing
The
stimuli
used
be
either
phonetically
balance
words
(PB)
of
the
particular
language
or
its
material.
At
present
only
a
few
Indian
languages
have
standard
speech
material
for
testing.
Hence
wherever
the
standardized
test
material
is
not
available,
either
Indian
English
Test
could
be
made
use
of
with
English
knowing
population
or
material
to
PB
be
used.
(c)
Wherever
children
are
tested
and
pure
tone
is
not
possible,
free
field
testing
should
be
employed.

Category |
No special benefits.
Category
Il
Considered for Hearing Aids at free or concessional costs only.
Category Ill
Hearing aids, free of cost or at concessional rates. Job reservation—benefit of special Employment Exchange.
Scholarships at School. Single language formula.
Category IV
Hearing Aids—facilities of reservation-special employment exchange. Special facilities in schools like scholarships.
Hearing aids—exemption from 3 language formula (to study in recommended single language).
It
is felt that for consideration of admission under special category for courses conducted by institutions like Indian Institute of Technol- ogy (IIT), Industrial Training Institute (ITI) and others, categories | and II only should be considered for reservation of seats, provided they fulfill the other educational stipulations for the course.
We have considered the different types of hearing affection, i.e. conductive versus sensorineural, and agree that the disability will be judged by the conditions prevalent in the patient at the time of referral and examination. In case of failure of surgery or other thera- peutic interventions, the patient will be considered and categorized on the basis of the recommended tests.
Left blank is the original recommendations; has been added by the author.

Assessment of vestibular functions can be divided into two groups:

Nystagmus is an important sign in the evaluation of vestibular system. It is defined as involuntary, rhythmical, oscillatory movement of eyes. It may be horizontal, vertical or rotatory. Vestibular nystagmus has a slow and a fast component, and by convention, the direction of nystagmus is indicated by the direction of the fast component. Intensity of nystagmus is indicated by its degree (Table 6.1).
To elicit nystagmus, patient is seated in front of the examiner or lies supine on the bed. The examiner keeps his finger about 30 cm from the patient's eye in the central position and moves it to the right or left, up or down, but not moving at any time more than 30° from the central position to avoid gaze nystagmus. Presence of spontaneous nystagmus always indicates an organic lesion.
Vestibular nystagmus is called peripheral, when it is due to lesion of labyrinth or VIIIth nerve and central, when lesion is in the central neural pathways (vestibular nuclei, brainstem, cerebellum).
Irritative lesions of the labyrinth (serous labyrinthitis) cause nystagmus to the side of lesion. Paretic lesions (purulent labyrinthitis, trauma to labyrinth, section of VIIIth nerve) cause nystagmus to the healthy side. Nystagmus of peripheral origin can be suppressed by optic fixation by looking ata fixed point, and enhanced in darkness or by the use of Frenzel glasses (+20 dioptre glasses) both of which abolish optic fixation.

Nystagmus of central origin cannot be suppressed by optic fixation. Purely torsional nystagmus indicates lesion of the brainstem/vestibular nuclei and is seen in syringomyelia. Vertical downbeat nystagmus indicates lesion at craniocervical region such as Arnold—Chiari malformation or degenerative lesion of the cerebellum. Vertical upbeat nystagmus is seen in lesions at the junction of pons and medulla or pons and midbrain. Pendular nystagmus is either congenital or acquired. The latter is seen in multiple sclerosis. Pendular nystagmus may also be disconjugate, i.e. vertical in one eye and horizontal in the other. Table 6.2 shows differences in the nystagmus of peripheral and central lesions.

The patient is asked to stand with feet together and arms by the side with eyes first open and then closed. With the eyes open, patient can still compensate the imbalance but with eyes closed, vestibular system is at more disadvantage. In peripheral vestibular lesions, the patient sways to the side of lesion. In central vestibular disorder, patient shows instability. If patient can perform this test without sway, "sharpened Romberg test" is performed. In this the patient stands with one heel in front of toes and arms folded across the chest. Inability to perform the sharpened Romberg test indicates vestibular impairment.

The patient is asked to walk along a straight line to a fixed point, first with eyes open and then closed. In case of uncompensated lesion of peripheral vestibular system, with eyes closed, the patient deviates to the affected side.

The past-pointing, falling and the slow component of nystagmus are all in the same direction. If there is acute vestibular failure, say on the right side, nystagmus is to the left but the past-pointing and falling will be towards the right, i.e. towards side of the slow component.

This test is particularly useful when patient complains of vertigo in certain head positions. It also helps to differentiate a peripheral from a central lesion.

Patient sits on a couch. Examiner holds the patient's head, turns it 45° to the right and then places the patient in a supine position so that his head hangs 30° below the horizontal (Figure 6.1). Patient's eyes are observed for nystagmus. The test is repeated with head turned to left and then again in straight head-hanging position. Four parameters of nystagmus are observed: latency, duration, direction and fatiguability (see Table 6.2). In benign paroxysmal positional vertigo, nystagmus appears after a latent period of 2-20 s, lasts for less than a minute and is always in one direction, i.e. towards the ear that is undermost. On repetition of the test, nystagmus may still be elicited but lasts for a shorter period. On subsequent repetitions it disappears altogether, i.e, nystagmus is fatiguable. Patient also complains of vertigo when the head is in critical position.
Figure 6.1 Hallpike manoeuvre.
In central lesions (tumours of IVth ventricle, cerebellum, temporal lobe, multiple sclerosis, vertebrobasilar insufficiency or raised intracranial tension) nystagmus is produced immediately, as soon as the head is in critical position without any latency and lasts as long as head is in that critical position. Direction of nystagmus also varies in different test positions (direction changing) and is nonfatiguable on repetition of test
(Table 6.2).

All cases of giddiness should be tested for cerebellar disorders. Disease of the cerebellar hemisphere causes:
Asynergia (abnormal finger-nose test) . Dysmetria (inability to control range of motion) wnNr
Adiadochokinesia (inability to perform rapid alternating movements)
4. Rebound phenomenon (inability to control movement
of extremity
when opposing
forceful restraint
is suddenly
released)
Midline disease of cerebellum causes:
1. Wide base gait
2. Falling in any direction
3. Inability to make sudden turns while walking
4. Truncal ataxia
Nystagmus observed in midline or hemispheral disorders of cerebellum includes gaze evoked nystagmus, rebound nystagmus and abnormal optokinetic nystagmus.

The basis of this test is to induce nystagmus by thermal stimulation of the vestibular system. Advantage of the test is that each labyrinth can be tested separately. Patient is also asked whether vertigo induced by the caloric test is qualitatively similar to the type experienced by him during the episode of vertigo. If yes, it proves labyrinthine origin of vertigo.
1. Modified Kobrak test. It is a quick office procedure. Patient is seated with head tilted 60° backwards to place horizontal canal in vertical position. Ear is irrigated with ice water for 60 s, first with 5 mL and if there is no response, 10, 20 and 40 mL. Normally, nystagmus beating towards the opposite ear will be seen with 5 mL of ice water. If response is seen with increased quantities of water between 5 and 40 mL, labyrinth is considered hypoactive. No response to 40mL of water indicates dead labyrinth.
2. Fitzgerald—Hallpike test (bithermal caloric test). In this test, patient lies supine with head tilted 30° forward so that horizontal canal is vertical (Figure 6.2). Ears are irrigated for 40 s alternately with water at 30°C and at 44°C (i.e. 7° below and above normal body temperature) and eyes observed for appearance of nystagmus till its end point. Time taken from the start of irrigation to the end point of nystagmus is recorded and charted on a calorigram (Figure 6.3). If no nystagmus is elicited from any ear, test is repeated with water at 20°C for 4 min before labelling the labyrinth dead. A gap of 5 min should be allowed between two ears. Cold water induces nystagmus to opposite side and warm water to the same side (remember mnemonic COWS: cold-opposite, warm-same). Depending on response to the caloric test, we can find canal paresis or dead labyrinth, directional preponderance, i.e. nystagmus is more in one particular direction than in the other, or both canal and directional response as duration of nystagmus) elicited from a particular canal (labright or left, after stimulation with cold and warm is less than that from the opposite side. It can also be expressed as percentage of the total response from both ears.

paresis preponderance. (a) Canal paresis. It indicates that (measured yrinth), water
Response from the left ear
—
Lg0
+
Lag
+
Rg9
+
Raa
Response from the right ear
Rego + R44 X 100
=
— Lo + L44 + Rgo + Rag where Lgo is the response from left side with water at 30°C and L4, is response from left ear after stimulation with warm water at 44°C. Less or no response from a particular side is indicative of depressed function of the ipsilateral labyrinth, vestibular nerve or vestibular nuclei and is seen in Méniére's disease, acoustic neuroma, postlabyrinthectomy or vestibular nerve section.
(b) Directional preponderance. It takes into consideration the duration of nystagmus to the right or leftirrespective of whether it is elicited from the right or left labyrinth. We know that
Lg + L44 X 100
=
Figure 6.2 Fitzgerald—Hallpike test. (A) Patient is in supine position and head raised by 30° to make horizontal canal vertical. (B) Position of canal and the direction of flow of endolymph.
right beating nystagmus is caused by Lyy and Ry, and left beating nystagmus is caused by Rap and Ly44. Therefore,
If the nystagmus is 25-30% or more on one side than the other, it is called directional preponderance to that side. It is believed that directional preponderance occurs towards the side of a central lesion, away from the side in a peripheral lesion; however, it does not help to localize the lesion in central vestibular pathways.
Canal paresis and directional preponderance can also be

Canal paresis on one side with directional preponderance to the opposite side is seen in unilateral Méniére's disease while canal paresis with directional preponderance to ipsilateral side is seen in acoustic neuroma.
3. Cold-air caloric test. This test is done when there is perforation of tympanic membrane because irrigation with water in such a case with perforation is contraindicated. The test employs Dundas Grant tube, which is a coiled copper tube
44
Figure
6.3
Calorigram.
A. Normal. Responses from both labyrinths are almost same with both cold and warm water. Cold water is usually a stronger stimulus than the warm one.
B. Left canal paresis. Responses from left labyrinth both with cold and warm water are much less compared to those from right.
C. Left directional preponderance. Nystagmus to left is 230 s compared to the one directed to right at 140.
wrapped in cloth. The air in the tube is cooled by pouring ethyl chloride and then blown into the ear. It is only a rough qualitative test.

It is a method of detecting and recording of nystagmus, which is spontaneous or induced by caloric, positional, rotational or optokinetic stimulus. The test depends on the presence of corneoretinal potentials which are recorded by placing electrodes at suitable places round the eyes. The test is also useful to detect nystagmus, which is not seen with the naked eye. It also permits to keep a permanent record of nystagmus.

Patient is asked to follow a series of vertical stripes on a drum moving first from right to left and then from left to right. Normally it produces nystagmus with slow component in the direction of moving stripes and fast component in the opposite direction. Optokinetic abnormalities are seen in brainstem and cerebral hemisphere lesions. Thus this test is useful to diagnose a central lesion.

Patient is seated in Barany's revolving chair with his head tilted 30° forward and then rotated 10 turns in 20 s. The chair is stopped abruptly and nystagmus observed. Normally there is nystagmus for 25-40 s. The test is useful as it can be performed in cases of congenital abnormalities where ear canal has failed to develop and it is not possible to perform the caloric test. Disadvantage of the test is that both the labyrinths are simultaneously stimulated during the rotation process and cannot be tested individually. The test has now been made more sophisticated by the use of torsion swings, electronystagmography and computer analysis of the results.

It is the only vestibular test which helps in differentiating an end organ lesion from that of vestibular nerve. Patient stands with his feet together, eyes closed and arms outstretched and then a current of 1 mA is passed to one ear. Normally, person sways towards the side of anodal current. Body sway can be studied by a special platform.

It is a method to evaluate vestibular function by measuring postural stability and is based on the fact that maintenance of posture depends on three sensory inputs—visual, vestibular and somatosensory. It uses either a fixed or a moving platform. Visual cues can also be varied. The clinical application of posturography is still under investigation.

of vestibular cause and are divided
Disorders system vertigo into:
The manoeuvre consists of five positions:
- 1. Peripheral, which involve vestibular end organs and their Ist order neurons (i.e. the vestibular nerve). The cause lies in the internal ear or the VIIIth nerve. They are responsible for 85% of all cases of vertigo.
2. Central, which involve central nervous system after the entrance of vestibular nerve in the brainstem and involve vestibulo-ocular, vestibulospinal and other central nervous system pathways.
Table 7.1 lists the common causes of vertigo of peripheral and central origin.

1. Ménieére's disease (endolymphatic hydrops). It is characterized by vertigo, fluctuating hearing loss, tinnitus and sense of pressure in the involved ear. Vertigo is of sudden onset, lasts for a few minutes to 24 h or so. (The disease has been discussed on p. 100).
2. Benign paroxysmal positional vertigo (BPPV). It is characterized by vertigo when the head is placed in a certain critical position. There is no hearing loss or other neurologic symptoms. Positional testing establishes the diagnosis and helps to differentiate it from positional vertigo of central origin (Table 7.1). Disease is caused by a disorder of posterior semicircular canal though many patients have history of head trauma and ear infection.
It has been demonstrated that otoconial debris, consisting of crystals of calcium carbonate, is released from the degenerating macula of the utricle and floats freely in the endolymph. When it settles on the cupula of posterior semicircular canal in a critical head position, it causes displacement of the cupula and vertigo. The vertigo is fatiguable on assuming the same position repeatedly due to dispersal of the otoconia but can be induced again after a period of rest. Thus, typical history and Hallpike manoeuvre establishes the diagnosis.
The condition can be treated by performing Lpley's manoeuvre. The principle of this manoeuvre is to reposition the otoconial debris from the posterior semicircular canal back into the utricle. The doctor stands behind the patient and the assistant on the side. The patient is made to sit on the table so that when he is made to lie down, his head is beyond the edge of the table as is done in Dix-Hallpike manoeuvre. His face is turned 45° to the affected side.
¢ Position 1. With the head turned 45°, the patient is made to lie down in head-hanging position (Dix-Hallpike manoeuvre). It will cause vertigo and nystagmus. Wait till vertigo and nystagmus subside.
¢ Position 2. Head is now turned so that affected ear is up.
¢
Position
3.
The
whole
body
and
head
are
now
rotated
away
from
the
affected

ear
to
a
lateral
recumbent
position
in
a
face-down
position.
¢ Position 4. Patient is now brought to a sitting position with head still turned to the unaffected side by 45°.
¢ Position 5. The head is now turned forward and chin brought down 20°.
There should be a pause at each position till there is no nystagmus or there is slowing of nystagmus, before changing to the next position. After manoeuvre is complete, patient should maintain an upright posture for 48 h. Eighty per cent of the patients will be cured by a single manoeuvre. If the patient remains symptomatic, the manoeuvre can be repeated. A bone vibrator placed on the mastoid bone helps to loosen the debris.
3. Vestibular neuronitis. It is characterized by severe vertigo of sudden onset with no cochlear symptoms. Attacks may last from a few days to 2 or 3 weeks. It is thought to occur due to a virus that attacks vestibular ganglion. Management of acute attack is similar to that in Méniére’s disease. The disease is usually self-limiting.
4. Labyrinthitis. It has been discussed in detail on p. 80.
© Circumscribed labyrinthitis is seen in cases of unsafe type of chronic suppurative otitis media (CSOM) and fistula test is positive.
¢ Serous labyrinthitis is caused by trauma or infection (viral or bacterial) adjacent to inner ear but without actual inva- sion. There is severe vertigo and sensorineural hearing loss. A partial or full recovery of inner ear functions is possible if treated early.
© Purulent labyrinthitis is a complication of CSOM. There is actual bacterial invasion of inner ear with total loss of cochlear and vestibular functions. Vertigo in this condi- tion is due to acute vestibular failure. There is severe nau- sea and vomiting. Nystagmus is seen to the opposite side due to destruction of the affected labyrinth.
5. Vestibulotoxic drugs. Several drugs cause ototoxicity by damaging the hair cells of the inner ear. Some primar- ily affect the cochlear while others affect the vestibular

labyrinth. Aminoglycoside antibiotics particularly streptomycin, gentamicin and kanamycin have been shown to affect hair cells of the crista ampullaris and to some extent those of the maculae. Certain other drugs which cause dizziness or unsteadiness are antihypertensives, labyrinthine sedatives, oestrogen preparations, diuretics, antimicrobials (nalidixic acid, metronidazole) and antimalarials. However, their mode of action may be different.
6. Head trauma. Head injury may cause concussion of labyrinth, completely disrupt the bony labyrinth or VIIth nerve, or cause a perilymph fistula. Severe acoustic trauma, such as that caused by an explosion, can also disturb the vestibular end organ (otoliths) and result in vertigo.
7. Perilymph fistula. In this condition, perilymph leaks into the middle ear through the oval or round window. It can follow as a complication of stapedectomy, or ear surgery when stapes is accidentally dislocated. It can also result from sudden pressure changes in the middle ear (e.g. barotrauma, diving, forceful Valsalva) or raised intracranial pressure (weightlifting or vigorous coughing). A perilymph fistula causes intermittent vertigo and fluctuating sensorineural hearing loss, sometimes with tinnitus and sense of fullness in the ear (compare Méniére's disease).
8. Syphilis. Syphilis of inner ear, both acquired and congenital, causes dizziness in addition to sensorineural hearing loss. Late congenital syphilis usually manifesting between 8 and 20 years, mimics Méniére's disease with episodes of acute vertigo, sensorineural hearing loss and tinnitus. Hennebert's sign, i.e. a positive fistula test in the presence of an intact tympanic membrane, is present in congenital syphilis. Neurosyphilis (tertiary acquired) can cause central type of vestibular dysfunction.

9. Acoustic neuroma. It has been classified in peripheral vestibular disorders as it arises from CN VIII within internal acoustic meatus. It causes only unsteadiness or vague sensation of motion. Severe episodic vertigo, as seen in the end organ disease, is usually missing (for details refer Chapter 18).
Other tumours of temporal bone (e.g. glomus tumour, carcinoma of external or middle ear and secondaries), destroy the labyrinth directly and cause vertigo.

1. Vertebrobasilar insufficiency. It is a common cause of central vertigo in patients over the age of 50 years. There is transient decrease in cerebral blood flow. Common cause is atherosclerosis. Ischaemia in these patients may also be precipitated by hypotension or neck movements when cervical osteophytes press on the vertebral arteries during rotation and extension of head.
Vertigo is abrupt in onset, lasts several minutes and is associated with nausea and vomiting. Other neurological symptoms like visual disturbances, drop attacks, diplopia, hemianopia, dysphagia and hemiparesis resulting from ischaemia to other areas of brain may also accompany vertigo.
Some patients only complain of intermittent attacks of dizziness or vertigo on lateral rotation and extension of head.
2. Posteroinferior cerebellar artery syndrome (Wallenberg syndrome). Thrombosis of the posterior inferior cerebellar artery cuts off blood supply to lateral medullary area. There is violent vertigo along with diplopia, dysphagia, hoarseness of voice, Horner syndrome, sensory loss on ipsilateral side of face and contralateral side of the body, and ataxia. There may be horizontal or rotatory nystagmus to the side of the lesion.
3. Basilar migraine. Migraine is a vascular syndrome producing recurrent headaches with symptom-free intervals. Headache is usually unilateral and of the throbbing type. Basilar artery migraine produces occipital headache, visual disturbances, diplopia and severe vertigo which is abrupt and may last for 5-60 min. Basilar migraine is common in adolescent girls with strong menstrual relationship and positive family history.
4. Cerebellar disease. Cerebellum may be affected by haemorrhage (hypertension), infarction (occlusion of arterial supply), infection (otogenic cerebellar abscess) or tumours (glioma, teratoma or haemangioma). Acute cerebellar disease may Cause severe vertigo, vomiting and ataxia simulating an acute peripheral labyrinthine disorder. Tumours are slow growing and produce classical features of cerebellar disease, i.e. incoordination, past-pointing, adiadochokinesia, rebound phenomenon and wide-based gait.

5. Multiple sclerosis. It is a demyelinating disease affecting young adults. Vertigo and dizziness are common complaints. There are other multiple neurological signs and symptoms, e.g. blurring or loss of vision, diplopia, dysarthria, paraesthesia and ataxia. Spontaneous nystagmus may be seen. Acquired pendular nystagmus, dissociated nystagmus and vertical upbeat nystagmus are important features in diagnosis.
6. Tumours of brainstem and floor of IVth ventricle. Gliomas and astrocytomas may arise from pons and midbrain; medulloblastoma, ependymomas, epidermoid cysts or teratomas may arise from floor of IVth ventricle. These tumours cause other neurological signs and symptoms in addition to vertigo and dizziness. Positional vertigo and nystagmus may
also be the presenting features. CT scan and magnetic resonance imaging are useful in their diagnosis.
7. Epilepsy. Vertigo may occur as an aura in temporal lobe epilepsy. The history of seizure and/or unconsciousness following the aura may help in the diagnosis. Sometimes, vertigo is the only symptom of epilepsy and that may pose a difficult diagnostic problem. Electroencephalography may show abnormalities during the attack.
8. Cervical vertigo. Vertigo may follow injuries of neck 7-10 days after the accident. It is usually provoked with movements of neck to the side of injury. Examination shows tenderness of neck, spasms of cervical muscles and limitation of neck movements. X-rays show loss of cervical lordosis. Exact mechanism of cervical vertigo is not known. It may be due to disturbed vertebrobasilar circulation, involvement of sympathetic vertebral plexus or alteration of tonic neck reflexes.

1. Ocular vertigo. Normally, balance is maintained by integrated information received from the eyes, labyrinths and somatosensory system. A mismatch of information from any of these organs causes vertigo and in this case from the eyes. Ocular vertigo may occur in case of acute extraocular muscle paresis or high errors of refraction.
2. Psychogenic vertigo. This diagnosis is suspected in patients suffering from emotional tension and anxiety. Often other symptoms of neurosis, e.g. palpitation, breathlessness, fatigue, insomnia, profuse sweating and tremors are also present. Symptom of vertigo is often vague in the form of floating or swimming sensation or light headedness. There is no nystagmus or hearing loss. Caloric test shows an exaggerated response.

be corrected surgically any time after the age of 6 years, if cosmetic appearance so demands.
The pinna may be afflicted by congenital, traumatic, inflammatory or neoplastic disorders.

The developmental abnormalities of the pinna may be just minor variations from the normal or major abnormalities.
1. Anotia. It is complete absence of pinna and lobule, and usually forms part of the first arch syndrome (Figure 8.1).
2. Microtia (Figure 8.2). It is a major developmental anom- aly.
Degree of
microtia may
vary.
It is
frequently associated
with anomalies
nal ear.
of external
The condition
auditory may
canal, be
middle unilateral
ing loss
is frequent.
Peanut ear
is
a
form and
or inter-
bilateral.
of microtia.
3. Macrotia. It is excessively large pinna.
4. Bat ear (Syn. prominent ear or protruding ear). This is an abnormally protruding ear. The concha is large with poorly developed antihelix and scapha. The deformity can
Figure
8.1
Anotia. Note total absence of pinna and external auditory
Hear-
5. Cup ear or lop ear. It is hypoplasia of upper third of the auricle. Upper portion of helix or pinna is cupped. Cockleshell ear or snail-shell ear are greater deformities of cup ear.
6. Cryptotia (Syn. pocket ear). Upper third of the auricle is embedded under the scalp skin. It can be corrected by mobilizing the pinna to normal position and covering the raw area by a skin graft.
7. Coloboma. There is a transverse cleft in the pinna in the middle.
8. Minor deformities. Absence of tragus, Darwin's tubercle, additional folds (Stahl's ear), and Satyr ear.
¢ Darwin's tubercle is a pointed tubercle on the upper part of helix and represents apex of pinna of lower animals.
- ¢ In Stahl's ear, helix which should normally be folded is flat and the upper crus of antihelix is duplicated and reaches rim of helix. It can be corrected by a mould in the first 6 weeks of life.

9. Deformities of ear lobule. They are absence of lobule, large lobule, bifid lobule or a pixed (attached) lobule.
10. Preauricular tags or appendages. They are skin-covered tags that appear on a line drawn from the tragus to the angle of mouth. They may contain small pieces of cartilage (Figure 8.3).
11. Preauricular pit or sinus. Preauricular pit is a depression in front of the crus of helix or above the tragus. Preauricular sinus is an epithelial track and is due to incomplete fusion of tubercles. It may get repeatedly infected causing purulent discharge. Abscess may also form. Treatment is surgical excision of the track if the sinus gets repeatedly infected (Figure 8.4).

1. Haematoma of the auricle. It is collection of blood between the auricular cartilage and its perichondrium. Often it is the result of blunt trauma seen in boxers, wrestlers and rugby players. Extravasated blood may clot and then organize, resulting in a typical deformity called Cauliflower ear (pugilistic or boxer's ear) (Figure 8.5). If haematoma gets infected, severe perichondritis may set in.
Treatment is aspiration of the haematoma under strict aseptic precautions and a pressure dressing, carefully packing all concavities of the auricle to prevent reaccumulation. Aspiration may need to be repeated. When aspiration fails, incision and drainage should be done
Figure 8.3 Preauricular appendages.
Infected preauricular sinus with pus exuding from the
and pressure applied by dental rolls tied with through and through sutures. All cases should receive prophylactic antibiotics.
2. Lacerations (Figure 8.6). They are repaired as early as possible. The perichondrium is stitched with absorbable sutures. Special care is taken to prevent stripping of perichondrium from cartilage for fear of avascular necrosis. Skin is closed with fine nonabsorbable sutures. Broad-spectrum antibiotics are given for 1 week.
3. Avulsion of pinna. When pinna is still attached to the head by a small pedicle of skin, primary reattachment should be considered and it is usually successful. Completely avulsed pinna can be reimplanted in selected cases by the microvascular techniques; in others, the skin of the avulsed segment of pinna is removed and the cartilage implanted under the postauricular skin for later reconstruction.
4. Frostbite. Injury due to frostbite varies between erythema and oedema, bullae formation, necrosis of skin and subcutaneous tissue, and complete necrosis with loss of the affected part.
Cauliflower ear (pugilistic or boxer's ear).
Figure 8.7 Keloid following piercing of pinna for ornaments.
Treatment of a frostbitten ear consists of:
(a) rewarming with moist cotton pledgets at a temperature of 38-42°C,

(b) application of 0.5% silver nitrate soaks for superficial infection,
(c) analgesics for pain; rapid rewarming of frostbitten ear causes considerable pain,
(d)
(e)
(f)
protection of bullae from rupture, systemic antibiotics for deep infection, and
surgical debridement
should wait
several months
true demarcation
dead and
living as
appears quite
late.
5. Keloid of auricle. It may follow trauma or piercing of the ear for ornaments. Usual sites are the lobule or helix (Figure 8.7). Surgical excision of the keloid usually results in recurrence. Recurrence of keloid can be avoided by preand postoperative radiation with a total dose of 600-800 rad delivered in four divided doses. Some prefer local injection of steroid after excision.

1. Perichondritis (Figure 8.8). It results from infection secondary to lacerations, haematoma or surgical incisions. It can also result from extension of infection from diffuse otitis externa or a furuncle of the meatus. Pseudomonas and mixed flora are the common pathogens.
Initial symptoms are red, hot and painful pinna which feels stiff. Later abscess may form between the cartilage and perichondrium with necrosis of cartilage as the cartilage survives only on the blood supply from its perichondrium. Treatment in early stages consists of systemic antibiotics and local application of 4% aluminium acetate compresses. When abscess has formed, it must be drained promptly and culture and sensitivity of the pus obtained. Incision is made in the natural fold and devitalized cartilage removed. Some prefer to place a catheter in the abscess and administer a continuous drip of antibiotics, selected by culture and sensitivity for 7-10 days.
2. Relapsing polychondritis. It is a rare autoimmune disorder involving cartilage of the ear. Other cartilages, septal, laryngeal, tracheal, costal may also be involved. The entire
Figure 8.8 Perichondritis.
auricle except its lobule becomes inflamed and _ tender. External ear canal becomes stenotic. Treatment consists of high doses of systemic steroids.
3. Chondrodermatitis nodularis chronica helicis. Small painful nodules appear near the free border of helix in men about the age of 50 years. Nodules are tender and the patient is unable to sleep on the affected side. Treatment is excision of the nodule with its skin and cartilage.

The diseases of external auditory canal are grouped as:
Trauma
Tumours
Miscellaneous conditions

1. Atresia of external canal. Congenital atresia of the meatus may occur alone or in association with microtia. When it occurs alone, it is due to failure of canalization of the ectodermal core that fills the dorsal part of the first branchial cleft. The outer meatus, in these cases, is obliterated with fibrous tissue or bone while the deep meatus and the tympanic membrane are normal. Atresia with microtia is more common. It may be associated with abnormalities of the middle ear, internal ear and other structures.
2. Collaural fistula. This is an abnormality of the first branchial cleft. The fistula has two openings: one situated in the neck just below and behind the angle of mandible and the other in the external canal or the middle ear. The track of
the fistula traverses through the parotid in close relation to the facial nerve.

Minor lacerations of canal skin result from Q-tip injury (scratching the ear with hair pins, needles or matchstick) or unskilled instrumentation by the physician. They usually heal without sequelae.
Major lacerations result from gunshot wounds, automobile accidents or fights. The condyle of mandible may force through the anterior canal wall. These cases require careful treatment. Aim is to attain a skin-lined meatus of adequate diameter. Stenosis of the ear canal is a common complication.

e¢ Eczematous otitis externa
¢ Seborrhoeic otitis externa
¢ Neurodermatitis
(a) Furuncle (localized acute otitis externa). A furuncle is a staphylococcal infection of the hair follicle. As the hair are confined only to the cartilaginous part of the meatus, furuncle is seen only in this part of meatus. Usually single, the furuncles may be multiple.
Patient usually presents with severe pain and tenderness which are out of proportion to the size of the furuncle. Movements of the pinna are painful. Jaw movements, as in chewing, also cause pain in the ear. A furuncle of posterior meatal wall causes oedema over the mastoid with obliteration of the retroauricular groove. Periauricular lymph nodes (anterior, posterior and inferior) may also be enlarged and tender.
Treatment in early cases, without abscess formation, consists of systemic antibiotics, analgesics and local heat. An ear pack of 10% ichthammol glycerine provides splintage and reduces pain. Hygroscopic action of glycerine reduces oedema, while ichthammol is mildly antiseptic. If abscess has formed, incision and drainage should be done.
In case of recurrent furunculosis, diabetes should be excluded, and attention paid to the patient's nasal vestibules which may harbour staphylococci and the infection transferred by patient's fingers. Staphylococcal infections of the skin as a possible source should also be excluded and suitably treated.
(b) Diffuse otitis externa. It is diffuse inflammation of meatal skin which may spread to involve the pinna and epidermal layer of tympanic membrane.
Aetiology. Disease is commonly seen in hot and humid climate and in swimmers. Excessive sweating changes the pH of meatal skin from that of acid to alkaline which favours growth of pathogens. Two factors commonly responsible for this condition are:
(i) trauma to the meatal skin and
(ii) invasion by pathogenic organisms.

Trauma can result from scratching the ear canal with hair pins or matchsticks, unskilled instrumentation to remove foreign bodies or vigorous cleaning of ear canal after a swim when meatal skin is already macerated. Break in continuity of meatal lining sets the ground for organisms to invade.
Common organisms responsible for otitis externa are Staphylococcus aureus, Pseudomonas pyocyaneus, Bacillus proteus and Escherichia coli but more often the infection is mixed.
Some cases of otitis externa are secondary to infection of the middle ear, or allergic sensitization to the topical ear drops used for chronic suppurative otitis media.
Clinical features.
Diffuse otitis
externa may
be acute
or chronic
with varying
degrees of
severity.
Acute phase is characterized by hot burning sensation in the ear, followed by pain which is aggravated by movements of jaw. Ear starts oozing thin serous discharge which later becomes thick and purulent. Meatal lining becomes inflamed and swollen. Collection of debris and discharge accompanied with meatal swelling gives rise to conductive hearing loss. In severe cases, regional lymph nodes become enlarged and tender with cellulitis of the surrounding tissues.
Chronic phase is characterized by irritation and strong desire to itch. This is responsible for acute exacerbations and reinfection. Discharge is scanty and may dry up to form crusts. Meatal skin which is thick and swollen may also show scaling and fissuring. Rarely, the skin becomes hypertrophic leading to meatal stenosis (chronic stenotic otitis externa) .
Treatment. Acute phase is treated as follows:
(i) Ear toilet. It is the most important single factor in the treatment of diffuse otitis externa. All exudate and debris should be meticulously and gently removed. Special attention should be paid to anteroinferior meatal recess, which forms a blind pocket where discharge is accumulated. Ear toilet can be done by dry mopping, suction clearance or irrigating the canal with warm, sterile normal saline.

- (ii) Medicated wicks. After thorough toilet, a gauze wick soaked in antibiotic steroid preparation is inserted in the ear canal and patient advised to keep it moist by instilling the same drops twice or thrice a day. Wick is changed daily for 2-3 days when it can be substituted by ear drops. Local steroid drops help to relieve oedema, erythema and prevent itching. Aluminium acetate (8%) or silver nitrate (3%) are mild astringents and can be used in the form of a wick to form a protective coagulum to dry-up an oozing meatus.
(iii) Antibiotics. Broad-spectrum systemic antibiotics are used when there is cellulitis and acute tender lymphadenitis. (iv) Analgesics. For relief of pain.
Chronic phase. Treatment aims at (i) reduction of meatal swelling so that ear toilet can be effectively done and
(ii)
alleviation of
itching so
that scratching
is stopped
and further
recurrences controlled.
A gauze wick soaked in 10% ichthammol glycerine and inserted into the canal helps to reduce swelling. This is followed by ear toilet with particular attention to anteroinferior meatal recess. Itching can be controlled by topical application of antibiotic steroid cream.
When the meatal skin is thickened to the point of obstruction and resists all forms of medical treatment, i.e. chronic stenotic otitis externa, it is surgically excised, bony meatus is widened with a drill and lined by split-skin graft.
(c) Otomycosis. Otomycosis is a fungal infection of the ear canal that often occurs due to Aspergillus niger, A. fumigatus or Candida albicans. It is seen in hot and humid climate of tropical and subtropical countries. Secondary fungal growth is also seen in patients using topical antibiotics for treatment of otitis externa or middle ear suppuration.
The clinical features of otomycosis include intense itching, discomfort or pain in the ear, watery discharge with a musty odour and ear blockage. The fungal mass may appear white, brown or black and has been likened to a wet piece of filter paper.

Examined with an otoscope, A. niger appears as blackheaded filamentous growth, A. fumigatus as pale blue or green and Candida as white or creamy deposit. Meatal skin appears sodden, red and oedematous.
Treatment consists of thorough ear toilet to remove all discharge and epithelial debris which are conducive to the growth of fungus. It can be done by syringing, suction or mopping. Specific antifungal agents can be applied. Nystatin (100,000 units/mL of propylene glycol) is effective against Candida. Other broad-spectrum antifungal agents include clotrimazole and povidone iodine. Two per cent salicylic acid in alcohol is also effective. It is a keratolytic agent which removes superficial layers of epidermis, and along with that, the fungal mycelia growing into them. Antifungal treatment should be continued for a week even after apparent cure to avoid recurrences. Ear must be kept dry. Bacterial infections are often associated with otomycosis and treatment with an antibiotic/steroid preparation helps to reduce inflammation and oedema and thus permitting better penetration of antifungal agents.
(d) Otitis externa haemorrhagica. It is characterized by formation of haemorrhagic bullae on the tympanic membrane and deep meatus. It is probably viral in origin and may be seen in influenza epidemics. The condition causes severe pain in the ear and blood-stained discharge when the bullae rupture, Treatment with analgesics is directed to give relief from pain. Antibiotics are given for secondary infection of the ear canal, or middle ear if the bulla has ruptured into the middle ear.
- (e) Herpes zoster oticus. It is characterized by formation of vesicles on the tympanic membrane, meatal skin, concha and postauricular groove. The VIIth and VHIth cranial nerves may be involved.

(f) Malignant (necrotizing) otitis externa. It is an inflammatory condition caused by pseudomonas infection usually in the elderly diabetics, or in those on immunosuppressive drugs. Its early manifestations resemble diffuse otitis externa but there is excruciating pain and appearance of granulations in the ear canal. Facial paralysis is common. Infection may spread to the skull base and jugular foramen causing multiple cranial nerve palsies. Anteriorly, infection spreads to temporomandibular fossa, posteriorly to the mastoid and medially into the middle ear and petrous bone.
Diagnosis. Severe otalgia in an elderly diabetic patient with granulation tissue in the external ear canal at its cartilaginous-bony junction should alert the physician of necrotizing otitis externa. CT scan may show bony destruction but is often not helpful. Gallium-67 is more useful in diagnosis and follow-up of the patient. It is taken up by monocytes and reticuloendothelial cells, and is indicative of soft tissue infection. It can be repeated every 3 weeks to monitor the disease and response to treatment. Technetium 99 bone scan reveals bone infection but test remains positive for a year or so and cannot be used to monitor the disease.
Treatment. It consists of:
(i)
Control of
diabetes.
(ii)
Toilet of
ear canal.
Remove discharge,
debris and
gran- ulations
or any
dead tissue
or bone.

(iii) Antibiotic treatment against causative organism, which in most ears is P. aeruginosa, but sometimes other organisms which can be found by culture and sensitivity. Antibiotic treatment is continued for 6-8 weeks, sometimes more. Antibiotics found effective are:
e Gentamicin combined with ticarcillin. They are given intravenously. Gentamicin is both ototoxic and nephrotoxic, and ticarcillin may produce penicillin-like reactions.
e Third-generation cephalosporins, e.g. ceftriaxone 1-2 g/day iv. or ceftazidime 1-2 g/day i.v. are usually combined with an aminoglycoside.
© Quinolones (ciprofloxacin, ofloxacin and levofloxacin) are also effective and can be given orally. They can be combined with rifampin. Ciprofloxacin 750 mg OD orally can be used. Oral therapy with quinolones obviates the need for admission for i.v. injections.
If patient is not responsive, culture and sensitivity of ear discharge should guide the surgeon.
Prolonged antibiotic treatment has replaced radical surgery and resections done earlier for this condition.
(g) Eczematous otitis externa. It is the result of hypersensitivity to infective organisms or topical ear drops such as chloromycetin or neomycin, etc. It is marked by intense irritation, vesicle formation, oozing and crusting in the canal. Treatment is withdrawal of topical antibiotic causing sensitivity and application of steroid cream.
(h) Seborrhoeic otitis externa. It is associated with seborrhoeic dermatitis of the scalp. Itching is the main complaint. Greasy yellow scales are seen in the external canal, over the lobule and postauricular sulcus. Treatment consists of ear toilet, application of a cream containing salicylic acid and sulfur, and attention to the scalp for seborrhoea.

(i) Neurodermatitis. It is caused by compulsive scratching due to psychological factors. Patient's main complaint is intense itching. Otitis externa of bacterial type may follow infection of raw area left by scratching. Treatment is sympathetic psychotherapy and that meant for any secondary infection. Ear pack and bandage to the ear are helpful to prevent compulsive scratching.
(j) Primary cholesteatoma of external auditory canal. In contrast to middle ear cholesteatoma, squamous epithelium of the external canal invades its bone. Usually there is some abnormality of bone of external canal which is conducive for epithelium to invade it. It may be post-traumatic or postsurgical. Clinical features include purulent otorrhoea and pain; tympanic membrane being normal. Granulations associated with sequestrated bone need histological examination to differentiate it from carcinoma, necrotizing otitis externa and a benign sequestrum.
Treatment consists of removal of necrotic bone and cholesteatoma, and lining the defect with fascia.

1. Impacted wax or cerumen. Wax is composed of secretion of sebaceous glands, ceruminous glands, hair, desquamated epithelial debris, keratin and dirt. Sebaceous and ceruminous (modified sweat glands) glands open into the space of the hair follicle (Figure 8.9). Sebaceous glands provide fluid rich in fatty acids while secretion of ceruminous gland is rich in lipids and pigment granules. Secretion of both these glands mixes with the desquamated epithelial cells and keratin shed from the tympanic membrane and deep bony meatus to form wax. Wax has a protective function as it lubricates the ear canal and entraps any foreign material that happens to enter the canal. It has acidic pH and is bacteriostatic and fungistatic. Normally, only a small amount of wax is secreted, which dries up and is later expelled from the meatus by movements of the jaw. As some people sweat more than others, the activity of ceruminous glands also varies; excessive wax may be secreted and deposited as a plug in the meatus. Certain other factors like narrow and tortuous ear canal, stiff hair or obstructive lesion of the canal, e.g. exostosis, may favour retention of wax. It may dry up and form a hard impacted mass. Patient usually presents with impairment of hearing or sense of blocked ear. Tinnitus and giddiness may result from impaction of wax against the tympanic membrane. Reflex cough due to stimulation of auricular branch of vagus may sometimes occur. The onset of these symptoms may be sudden when water enters the ear canal during bathing or swimming and the wax swells up. Long standing impacted wax may ulcerate the meatal skin and result in granuloma formation (wax granuloma).
Treatment of wax consists in its removal by syringing or instrumental manipulation. Hard impacted mass may sometimes require prior softening with wax solvents.
Technique of syringing the ear. Patient is seated with ear to be syringed towards the examiner. A towel is placed round his neck. A kidney tray is placed over the shoulder and held snugly by the patient. Patient's head is slightly tilted over the tray to collect the return fluid.

Pinna is pulled upwards and backwards and a stream of water from the ear syringe is directed along the posterosuperior wall of the meatus. Pressure of water, built up deeper to the wax, expels the wax out (Figure 8.10). If wax is tightly impacted, it is necessary to create a space between it and the meatal wall for the jet of water to pass, otherwise syringing will be ineffective or may even push the wax deeper. Ear canal should be inspected from time to time to see if all wax has been removed. Unnecessary syringing should be avoided.
At the end of the procedure, ear canal and tympanic membrane must be inspected and dried with a pledget of cotton. Any ulceration seen in meatal wall as a result of impacted wax is protected by application of suitable antibiotic ointment. Normally, boiled tap water cooled to body temperature is used. If it is too cold or too hot it would stimulate the labyrinth, as in caloric testing, and cause vertigo. Too much force used in syringing may rupture the tympanic membrane especially when it has already been weakened by previous disease. Patient complains of intense pain and may become giddy and even faint. It is necessary before syringing to ask the patient for any past history of ear discharge or an existing perforation. A quiescent otitis media may be reactivated by syringing. Instrumental manipulation. It should always be done by skilled hands and under direct vision. Cerumen hook, scoop or JobsonHorne probe are often used. First, a space is created between the wax and meatal wall, the instrument is passed beyond the wax, and whole plug then dragged out in a single piece. If it breaks, syringing may be used to remove the fragments. Occasionally, if the wax is too hard and impacted, to be removed by syringing or instruments, it should be softened by drops of 5% sodium bicarbonate in equal parts of glycerine and water instilled two or three times a day for a few
Figure 8.10 (A) Syringing of ear. (B) Illustration to show how jet of water expels the wax or a foreign body.

days. Hydrogen peroxide, liquid paraffin or olive oil may also achieve the same result. Commercial drops containing ceruminolytic agents like paradichlorobenzene 2% can also be used and above methods tried again.
2. Foreign bodies of ear. (a) Nonliving. Children may insert a variety of foreign bodies in the ear; the common ones often seen are: a piece of paper or sponge, grain seeds (rice, wheat, maize), slate pencil, piece of chalk or metallic ball bearings. An adult may present with a broken end of matchstick used for scratching the ear or an overlooked cotton swab. Vegetable foreign bodies tend to swell up with time and get tightly impacted in the ear canal or may even suppurate.
Maggots in the ear. Flies may be attracted to the foul-smelling ear discharge and lay eggs which hatch out into larvae called maggots. They are commonly seen in the month of August, September and October. There is severe pain with swelling round the ear and blood-stained watery discharge. Maggots may be seen filling the ear canal. Treatment consists of instilling chloroform water to kill the maggots, which can later be removed by forceps. Usually, such patients have discharging ears with perforation of the tympanic membrane and syringing may not be advisable.
Methods of removing a foreign body include:

(ii)
Syringing
(iii)
Suction
(iv) Microscopic removal with special instruments
(v)
Postaural approach
Soft and irregular foreign bodies like a piece of paper, swab or a piece of sponge can be removed with fine crocodile forceps.
Most of the seed grains and smooth objects can be removed with syringing. Smooth and hard objects like steel ball bearing should not be grasped with forceps as they tend to move inwards and may injure the tympanic membrane. In all impacted foreign bodies or in those where earlier attempts at extraction have been made, it is preferable to use general anaesthetic and an operating microscope. Occasionally, postaural approach is used to remove foreign bodies impacted in deep meatus, medial to the isthmus or those which have been pushed into the middle ear.
Unskilled attempts at removal of foreign bodies may lacerate the meatal lining, damage the tympanic membrane or the ear ossicles.
(b) Living. Flying or crawling insects like mosquitoes, beetles, cockroach or an ant may enter the ear canal and cause intense irritation and pain. No attempt should be made to catch them alive. First, the insect should be killed by instilling oil (a household remedy), spirit or chloroform water. Once killed, the insect can be removed by any of the methods described above.
3. Keratosis obturans. Collection of a pearly white mass of desquamated epithelial cells in the deep meatus is called keratosis obturans. This, by its pressure effect, causes absorption of bone leading to widening of the meatus so much so that facial nerve may be exposed and paralyzed.
(a) Aetiology. It is commonly seen between 5 and 20 years and may affect one or both ears. It may sometimes be associated with bronchiectasis and chronic sinusitis. Normally, epithelium from surface of tympanic membrane migrates onto the posterior meatal wall. Failure of this migration or obstruction to migration caused by wax may lead to accumulation of the epithelial plug in the deep meatus. (b) Clinical features. Presenting symptoms may be pain in
the ear,
hearing loss,
tinnitus and
sometimes ear
discharge.

On examination, ear canal may be full of pearly white mass of keratin material disposed in several layers. Removal of this mass may show widening of bony meatus with ulceration and even granuloma formation.
(c) Treatment. Keratotic mass is removed either by syringing or instrumentation, similar to the techniques employed for impacted wax. Secondary otitis externa may be present and should be treated. Patient should be periodically checked and any reaccumulations removed. Recurrence can be checked to some extent by the use of keratolytic agent such as 2% salicylic acid in alcohol.
4. Acquired atresia and stenosis of meatus. It can result from:
(a) Infections, e.g. chronic otitis externa—an important cause (Figure 8.11).
Figure 8.11 Meatal stenosis following chronic otitis externa.
(b) Trauma, e.g. lacerations, fracture of tympanic plate, surgery on ear canal or mastoid.
(c) Burns—thermal or chemical.
Treatment is meatoplasty. Using a postaural incision, scar tissue and thickened meatal skin are excised, bony meatus is enlarged and the raw meatal bone is covered with pedicled flaps from meatus or split-skin grafts.

Diseases of
tympanic membrane
may be
primary or
second- ary to conditions affecting external ear, middle ear or eusta-
chian tube.
Normal tympanic
colour with
at the
tip seen
membrane.
a
concavity of
in the
lies
It is
shiny on
its lateral
malleus, the
umbo.
anteroinferior above
the and
surface,
A
bright quadrant
lateral ish.
process
Transparency seen
pearly more
cone grey
in marked
of
(Figure of
malleus varies.
Some through
a
transparent membrane
is mobile
light
8.12).
and middle
ear membrane.
when can
be
Attic is
area slightly
pink- structures
can
A
be normal
tested with
tympanic pneumatic
otoscope or Siegle's speculum.
1. Retracted tympanic membrane. It appears dull and lustreless. Cone of light is absent or interrupted. Handle of malleus appears foreshortened. Lateral process of malleus becomes more prominent. Anterior and posterior malleal folds become sickle shaped (Figure 8.13). A retracted tympanic membrane is the result of negative intratympanic pressure when the eustachian tube is blocked.
2. Myringitis bullosa. It is a painful condition characterized by formation of haemorrhagic blebs on the tympanic membrane and deep meatus. It is probably caused by a virus or Mycoplasma pneumoniae.
3. Herpes zoster oticus. It is a viral infection involving geniculate ganglion of facial nerve. It is characterized by appearance of vesicles on the tympanic membrane, deep meatus, concha and retroauricular sulcus. It may involve VIIth (more often) and the VIIIth cranial nerves.
4. Myringitis granulosa. Nonspecific granulations form on the outer surface of tympanic membrane. It may be associated with impacted wax, long-standing foreign body or external ear infection.

Figure 8.12 Normal tympanic membrane of the right side. Note a bright cone of light at 5'o clock.
Figure 8.13 Retracted tympanic membrane.

(a)
Trauma due
to
a
hair pin,
matchstick or
unskilled attempts
to remove
a
foreign body.
Sudden change
in air
pressure, e.g.
a
slap or
a
kiss on
the ear or a sudden blast. Forceful Valsalva may rupture
a
thin atrophic
membrane.
Pressure by
a
fluid column,
forceful syringing.
(d)
Fracture of
temporal bone.
Treatment. In a majority of cases, edges of perforation get inverted towards the middle ear. In such cases, the ear should be examined under operating microscope and the edges of perforation repositioned and splinted (see p. 407). Injuries of tympanic membrane may be associated with facial paralysis or subluxation of stapes (vertigo and nystagmus) and sensorineural hearing loss. In such cases, urgent exploration may be required.
6. Atrophic tympanic membrane. A normal tympanic membrane consists of outer epithelial, middle fibrous and inner mucosal layer. In serous otitis media, the middle fibrous e.g.
diving, water
sports or
(b)
(c)
layer gets absorbed leaving a thin drumhead which easily gets collapsed with eustachian tube insufficiency. A perforation of tympanic membrane also heals only by epithelial and mucosal layers without the intervening fibrous layer.
7. Retraction pockets and atelectasis. When the tympanic membrane is thin and atrophic, a segment of it or the entire membrane may collapse inwards due to eustachian tube insufficiency. It may form a retraction pocket or get plastered onto promontory and also wrap round the ossicles. A deep retraction pocket may accumulate keratin debris and form a cholesteatoma.

8. Tympanosclerosis. It is hyalinization and later calcification in the fibrous layer of tympanic membrane. It appears as chalky white plaque. Mostly, it remains asymptomatic. It is frequently seen in cases of serous otitis media as a complication of ventilation tube. Tympanosclerosis mostly affects tympanic membrane but may be seen involving ligaments, joints of ossicles, muscle tendons and submucosal layer of middle ear cleft, and interferes in the conduction of sound.
9. Perforations. They may be central, attic or marginal and are associated with chronic otitis media (see p. 80).

helps to keep the tube closed and thus protect it from the reflux of nasopharyngeal secretions.
Eustachian tube, also called auditory or pharyngotympanic tube, connects nasopharynx with the tympanic cavity. In an adult, it is about 36 mm long and runs downwards, forwards and medially from its tympanic end, forming an angle of 45° with the horizontal. It is divided into two parts: bony, which is posterolateral, forms one-third (12 mm) of the total length and fibrocartilaginous, which is anteromedial, forms two-thirds (24mm). The two parts meet at isthmus which is the narrowest part of the tube (Figure 9.1). The fibrocartilaginous part of the tube is made of a single piece of cartilage folded upon itself in such a way that it forms the whole of medial lamina, roof and a part of the lateral lamina; the rest of its lateral lamina is made of fibrous membrane.
The tympanic end of the tube is bony, measures 5 x 2 mm and is situated in the anterior wall of middle ear, a little above the level of floor. The pharyngeal end of the tube is slit-like, vertically. The cartilage at this end raises an elevation called torus tubarius, which is situated in the lateral wall of the nasopharynx, 1—1.25 cm behind the posterior end of inferior turbinate.

Three muscles are related to the tube: tensor veli palatini, levator veli palatini and salpingopharyngeus. The medial fibres of the tensor veli palatini are attached to the lateral lamina of the tube and when they contract help to open the tubal lumen. These fibres have also been called dilator tubae muscle. The exact role of the levator veli palatini and the salpingopharyngeus muscles to open the tube is uncertain. It is believed that the levator veli palatini muscle, which runs inferior and parallel to the cartilaginous part of the tube forms a bulk under the medial lamina and during contraction pushes it upward and medially thus assisting in opening the tube.
The elastin hinge. The cartilage, at the junction of medial, and lateral lamina at the roof, is rich in elastin fibres which form a hinge. By its recoil it helps to keep the tube closed when no longer acted upon by dilator tubae muscle.
Ostmann's pad of fat. It is a mass of fatty tissues related laterally to the membranous part of the cartilaginous tube. It also

Histologically, the mucosa shows pseudostratified ciliated columnar epithelium interspersed with mucous secreting goblet cells. Submucosa, particularly in the cartilaginous part of the tube, is rich in seromucinous glands. The cilia beat in the direction of nasopharynx and thus help to drain secretions and fluid from the middle ear into the nasopharynx.

Tympanic branch of cranial nerve (CN) IX supplies sensory as well as parasympathetic secretomotor fibres to the tubal mucosa. Tensor veli palatini muscle is supplied by mandibular branch of trigeminal (V3) nerve. Levator veli palatini and salpingopharyngeus muscles receive motor nerve supply through pharyngeal plexus (cranial part of CN XI
through vagus).

The eustachian tube of infants is wider, shorter and more horizontal; thus infections from the nasopharynx can easily reach the middle ear. Even the milk may regurgitate into the middle ear if the infants are not fed in head-up position (see Table 9.1).

Physiologically, eustachian tube performs three main functions:
1. Ventilation and thus regulation of middle ear pressure.
2. Protection against (i) nasopharyngeal sound pressure and (ii) reflux of nasopharyngeal secretions.
3. Clearance of middle ear secretions.
1. Ventilation and regulation of middle ear pressure. For normal hearing, it is essential that pressure on two sides of the tympanic membrane should be equal. Negative or positive pressure in the middle ear affects hearing. Thus, eustachian tube should open periodically to equilibrate the air pressure in the middle ear with the ambient pressure. Normally, the eustachian tube remains closed and opens
intermittently during swallowing, yawning and sneezing. Posture also affects the function; tubal opening is less efficient in recumbent position and during sleep due to venous engorgement. Tubal function is also poor in infants and young children and thus responsible for more ear problems in that age group. It usually normalizes by the age of 7-10 years.
Figure 9.1 Horizontal section through the eustachian tube showing bony and cartilaginous parts, isthmus, tympanic and pharyngeal ends.
2. Protective functions. Abnormally, high sound pressures from the nasopharynx can be transmitted to the middle ear if the tube is open thus interfering with normal hearing. Normally, the eustachian tube remains closed and protects the middle ear against these sounds. A normal eustachian tube also protects the middle ear from reflux of nasopharyngeal secretions into the middle ear. This reflux occurs more readily if the tube is wide in diameter (patulous tube), short in length (as in babies) or the tympanic membrane is perforated (cause for persistence of middle ear infections in cases of tympanic membrane perforations). High pressures in the nasopharynx can also force nasopharyngeal secretions into the middle ear, e.g. forceful nose blowing, closed-nose swallowing as in the presence of adenoids or bilateral nasal obstruction.

Figure 9.2 Vertical section through eustachian tube. Note: Cartilage of the tube forms medial wall, roof and part of lateral wall. Elastin is situated in the roof at the junction of medial and lateral laminae and helps the medial laminae to regain its original position of closure. (A) Eustachian tube is closed in resting position. (B) Tube is open when tensor veli palatini (dilator tubae) muscle contracts.

Length, Differences between infant and adult eustachian tube.Infant = 13-18 mmat birth (about half as long as in adult). Length, Differences between infant and adult eustachian tube.Adult = 36 mm(31-38 mm). Direction, Differences between infant and adult eustachian tube.Infant = More horizontal. At birth, it forms an angle of 10° with the horizontal. At age 7 and later it is 45°. Direction, Differences between infant and adult eustachian tube.Adult = Forms an angle of 45° with the horizontal. Angulation at isthmus, Differences between infant and adult eustachian tube.Infant = No angulation. Angulation at isthmus, Differences between infant and adult eustachian tube.Adult = Angulation present. Bony versus cartilaginous part, Differences between infant and adult eustachian tube.Infant = Bony part is slightly longer than one-third of the total length of the tube and is relatively wider. Bony versus cartilaginous part, Differences between infant and adult eustachian tube.Adult = Bony part one-third; cartilaginous part two-thirds. Tubal cartilage, Differences between infant and adult eustachian tube.Infant = Flaccid. Retrograde reflux of nasopharyngeal secretions can occur. Tubal cartilage, Differences between infant and adult eustachian tube.Adult = Comparatively rigid. Remains closed and protects the middle ear from the reflux. Density of elastin at the hinge, Differences between infant and adult eustachian tube.Infant = Less dense; tube does not efficiently close by recoil. Density of elastin at the hinge, Differences between infant and adult eustachian tube.Adult = Density of elastin more and helps to keep the tube closed by recoil of cartilage. Ostmann's pad of fat, Differences between infant and adult eustachian tube.Infant = Less in volume. Ostmann's pad of fat, Differences between infant and adult eustachian tube.Adult = Large and helps to keep the tube closed

3. Clearance of middle ear secretions. Mucous membrane of the eustachian tube and anterior part of the middle ear is lined by ciliated columnar cells. The cilia beat in the direction of nasopharynx. This helps to clear the secretions and debris in the middle ear towards the nasopharynx. The clearance function is further augmented by active opening and closing of the tube.

1. Valsalva test. The principle of this test, as also of politzerization, is to build positive pressure in the nasopharynx so that air enters the eustachian tube. To do this test, patient pinches his nose between the thumb and index finger, takes a deep breath, closes his mouth and tries to blow air into the ears. If air enters the middle ear, the tympanic membrane will move outwards, which can be verified by otoscope or the microscope. In the presence of a tympanic membrane perforation, a hissing sound is produced or if discharge is also present in the middle ear, cracking sound will be heard. Failure of this test does not prove blockage of the tube because only about 65% of persons can successfully perform this test. This test should be avoided (i) in the presence of atrophic scar of tympanic membrane which can rupture and (ii) in the presence of infection of nose and nasopharynx where infected secretions are likely to be pushed into the middle ear causing otitis media.
2. Politzer test. This test is done in children who are unable to perform Valsalva test. In this test, olive-shaped tip of the Politzer's bag is introduced into the patient's nostril on the side of which the tubal function is desired to be tested. Other nostril is closed, and the bag compressed while at the same time the patient swallows (he can be given sips of water) or says "ik, ik, ik." By means of an auscultation tube, connecting the patient's ear under test to that of the examiner, a hissing sound is heard if tube is patent. Compressed air can also be used instead of Politzer's bag. The test is also used therapeutically to ventilate the middle ear.

the tip of which is bent, is passed along the floor of nose till it reaches the nasopharynx. Here it is rotated 90° medially and gradually pulled back till it engages on the posterior border of nasal septum (Figure 9.3A). It is then rotated 180° laterally so that the tip lies against the tubal opening (Figure 9.3B). A Politzer's bag is now connected to the catheter and air insufflated. Entry of air into the middle ear is verified by an auscultation tube. The procedure of catheterization should be gentle as it is known to cause complications such as:
(a) Injury to eustachian tube opening which causes scarlater.
ring
(b) Bleeding from the
(c) Transmission of nasal and nasopharyngeal infection into the middle ear otitis media.
nose. causing
- (d) Rupture of atrophic area of tympanic membrane if too much pressure is used.
4. Toynbee's test. While the above three tests use a positive pressure, Toynbee's manoeuvre causes negative pressure. It is a more physiological test. It is performed by asking the patient to swallow while nose has been pinched. This draws air from the middle ear into the nasopharynx and causes inward movement of tympanic membrane, which is verified by the examiner otoscopically or with a microscope.
5. Tympanometry (also called inflation—deflation test). In this test, positive and negative pressures are created in the external ear canal and the patient swallows repeatedly. The ability of the tube to equilibrate positive and negative pressures to the ambient pressure indicates normal tubal function. The test can be done both in patients with perforated or intact tympanic membranes (see p. 24).
6. Radiological test. A radio-opaque dye, e.g. hypaque or lipoidal instilled into the middle ear through a pre-existing perforation and X-rays taken should delineate the tube and any obstruction. The time taken by the dye to reach the nasopharynx also indicates its clearance function. This test is no longer popular now.

3. Catheterization. In this test, nose is first anaesthetized by topical spray of lignocaine and then a eustachian tube catheter,
7. Saccharine or methylene blue test. Saccharine solution is placed into the middle ear through a pre-existing
perforation. The time taken by it to reach the pharynx and impart a sweet taste is also a measure of clearance function. Similarly, methylene blue dye can be instilled into the middle ear and the time taken by it to stain the pharyngeal secretions can be noted.
Indirect evidence of drainage/clearance function is established when ear drops instilled into the ear with tympanic membrane perforation cause bad taste in throat.
8. Sonotubometry. A tone is presented to the nose and its recording taken from the external canal. The tone is heard louder when the tube is patent (compare patulous eustachian tube). It also tells the duration for which the tube remains open. It is a noninvasive technique and provides information on active tubal opening. Accessory sounds produced in the nasopharynx, during swallowing, may interfere with the test results. The test is under development.

1. Tubal blockage. Normally, eustachian tube is closed. It opens intermittently during swallowing, yawning and sneezing through the active contraction of tensor veli palatini muscle. Air, composed of oxygen, carbon dioxide, nitrogen and water vapour, normally fills the middle ear and mastoid. When tube is blocked, first oxygen is absorbed, but later other gases, CO, and nitrogen also diffuse out into the blood. This results in negative pressure in the middle ear and retraction of tympanic membrane. If negative pressure is still further increased, it causes "locking" of the tube with collection of transudate and later exudate and even haemorrhage. Effects of acute and long-term tubal blockage are shown in Table 9.2.
Eustachian tube obstruction can be mechanical, functional or both. Mechanical obstruction can result from (i) intrinsic causes such as inflammation or allergy or (ii) extrinsic causes such as tumour in the nasopharynx or adenoids. Functional obstruction is caused by collapse of the tube due to increased cartilage compliance, which resists opening of the tube or failure of active tubal-opening mechanism due to poor function of tensor veli palatini. The common clinical conditions which can cause tubal obstruction are listed in Table 9.3.
Symptoms of tubal occlusion include otalgia, which may be mild to severe, hearing loss, popping sensation, tinnitus and disturbances of equilibrium or even vertigo.
Signs of tubal occlusion will vary and depend upon the acuteness of the condition and severity. They include retracted tympanic membrane, congestion along the handle of malleus and the pars tensa, transudate behind the tympanic membrane, imparting it an amber colour and sometimes a fluid level with conductive hearing loss. In severe cases, as in barotrauma, tympanic membrane is markedly retracted with haemorrhages in subepithelial layer, haemotympanum or sometimes a perforation.
2. Adenoids and eustachian tube function. Adenoids cause tubal dysfunction by:
(a) Mechanical obstruction of the tubal opening.
(b) Acting as reservoir for pathogenic organisms.
(c)

Incases ofallergy, mast cells of the adenoid tissue release inflammatory mediators which cause tubal blockage.
Thus, adenoids can cause otitis media with effusion or recurrent acute otitis media. Adenoidectomy can help both these conditions.
3. Cleft palate and tubal function. Tubal function is dis- turbed in cleft palate patients due to:
(a) Abnormalities of torus tubarius, which shows high elas- tin density making tube difficult to open.
(b)
Tensor
veli
palatini
muscle
does
not
insert
into
the
torus
tubarius
in
40%
cases
of
cleft
palate
and
where
it
does insert, its function is poor.
Otitis media with effusion is common in these patients. Even after repair of the cleft palate deformity, many of them require insertion of grommets to ventilate the middle ear.
4. Down syndrome and tubal function. Function of eusta- chian tube is defective possibly due to poor tone of tensor veli palatini muscle and abnormal shape of nasopharynx. Children with this syndrome are prone to frequent otitis media or otitis media with effusion.

Acute tubal blockage
Absorption of ME gases
Negative pressure in ME
a of TM
ee in ME/haemorrhage (acute OME)
Prolonged tubal blockage/dysfunction
OME an watery or mucoid discharge)
ae
ear/perforation
a pocket/cholesteatoma
pee incudostapedial joint
ME, middle ear; TM, tympanic membrane; OME, otitis media with
effusion.
e Upper respiratory infection (viral or bacterial)
e Allergy
¢ Sinusitis
Nasal polyps
Deviated nasal septum
Hypertrophic adenoids
Nasopharyngeal tumour/mass
Cleft palate e Submucous cleft palate
¢ Down syndrome e Functional

In ventilation of the middle ear cleft, air passes from eustachian tube to mesotympanum, from there to attic, aditus, antrum and mastoid air cell system. Mesotympanum communicates with the attic via anterior and posterior isthmi, situated in membranous diaphragm between the mesotympanum and the attic. Anterior isthmus is situated between tendon of tensor tympani and the stapes. Posterior isthmus is situated between tendon of stapedius muscle and pyramid, and the short process of incus. In some cases, middle ear can also communicate directly with the mastoid air cells through the retrofacial cells. Any obstruction in the pathways of ventilation can cause retraction pockets or atelectasis of tympanic membrane, e.g.
1. Obstruction of eustachian tube > Total atelectasis of tympanic membrane.
2. Obstruction in middle ear > Retraction pocket in posterior part of middle ear while anterior part is ventilated.
3. Obstruction of isthmi — Attic retraction pocket.
4. Obstruction at aditus + Cholesterol granuloma and col-
lection of mucoid discharge in mastoid air cells, while middle ear and attic appear normal.
Depending on the location of pathologic process, other changes such as thin atrophic tympanic membrane, partial or total (due to absorption of middle fibrous layer), cholesteatoma, ossicular necrosis and tympanosclerotic changes may also be found.
Principles of management of retraction pockets and atelectasis of middle ear would entail correction/repair of the irreversible pathologic processes and establishment of the ventilation.

In this condition, the eustachian tube is abnormally patent. Most of the time it is idiopathic but rapid weight loss, pregnancy especially third trimester, or multiple sclerosis can also cause it.
Patient’s chief
complaints are
hearing his
own voice
(autophony), even
disturbing.
Due the
his own
to nasopharynx
much so
that abnormal
breath sounds,
potency, are
the inspiration
and exaggerated
easily which
pressure transmitted
movements is
very changes
to of
the middle
tympanic expiration;
if these
patient nostril.
breathes can
in ear
be seen
movements after
closing are
so with
further the
opposite
Acute condition of patulous tube is self-limiting and does not require treatment. In others, weight gain, oral administration of potassium iodide is helpful but some long-standing cases may require cauterization of the tubes or insertion of a grommet.

Pharyngeal end of the eustachian tube can be examined by posterior rhinoscopy, rigid nasal endoscope or flexible nasopharyngoscope. The extrinsic causes which obstruct this end can be excluded
(Figure 9.4). Tympanic end of the tube can be examined by microscope or endoscope, if there is a pre-existing perforation. Eustachian tube endoscopy or middle ear endoscopy can be done with very fine flexible endoscopes. Simple examination of tympanic membrane with otoscope or microscope may reveal retraction pockets or fluid in the middle ear. Similarly, movements of tympanic membrane with respiration point to patulous eustachian tube. Further assessment of function of the tube can be made by Valsalva, politzerization, Toynbee and other tests already described. Aetiologic causes of eustachian tube dysfunction can be assessed by thorough nasal examination including endoscopy, tests of allergy, CT scan of temporal bones and of paranasal sinuses. MRI may be required to exclude multiple sclerosis in patulous eustachian tube.

Torus tubarius
Figure 9.4 Endoscopic view of nasopharynx showing torus tubarius in the right lateral wall of nasopharynx. Note also the fossa of Rosenmiller which lies behind it. Fossa of Rosenmiller is the commonest site for the origin of carcinoma nasopharynx.
Nasal septum

It is an acute inflammation of middle ear by pyogenic organisms. Here, middle ear implies middle ear cleft, i.e. eustachian tube, middle ear, attic, aditus, antrum and mastoid air cells.

It is more common especially in infants and children of lower socioeconomic group. Typically, the disease follows viral infection of upper respiratory tract but soon the pyogenic organisms invade the middle ear.

1. Via eustachian tube. It is the most common route. Infection travels via the lumen of the tube or along subepithelial peritubal lymphatics. Eustachian tube in infants and young children is shorter, wider and more horizontal and thus may account for higher incidence of infections in this age group. Breast or bottle feeding in a young infant in horizontal position may force fluids through the tube into the middle ear and hence the need to keep the infant propped up with head a little higher. Swimming and diving can also force water through the tube into the middle ear.
2. Via external ear. Traumatic perforations of tympanic membrane due to any cause open a route to middle ear infection.
3. Blood-borne. This is an uncommon route.

Anything that interferes with normal functioning of eustachian tube predisposes to middle ear infection. It could be:
1. Recurrent attacks of common cold, upper respiratory tract infections and exanthematous fevers like measles, diphtheria or whooping cough.
Infections of tonsils and adenoids. Chronic rhinitis and sinusitis. Nasal allergy. oO go
Tumours of nasopharynx, packing of nose or nasopharynx for epistaxis.
Cleft palate. >
Bacteriology. Most common organisms in infants and young children are Streptococcus pneumoniae (30%), Haemophilus influenzae (20%) and Moraxella catarrhalis (12%). Other organisms include Streptococcus pyogenes, Staphylococcus aureus and sometimes Pseudomonas aeruginosa. In about 18-20%, no growth is seen. Many strains of H. influenzae and M. catarrhalis are B-lactamase producing.

The disease runs through the following stages:
Amoxicillin Ampicillin Co-amoxiclav Erythromycin Cefaclor (II generation) Cefixime (III generation) Cefpodoxime proxetil Ceftibuten (III generation) Co-trimoxazole (Trimethoprim + Sulfamethoxazole), Trade names = Novamox, Biomox Biocillin Augmentin, Enhancin Emycin, Althrocin Keflor, Distaclor Taxim-0, Biotax-0 Cepodem, Cefoprox Procadax Ciplin, Septran. Amoxicillin Ampicillin Co-amoxiclav Erythromycin Cefaclor (II generation) Cefixime (III generation) Cefpodoxime proxetil Ceftibuten (III generation) Co-trimoxazole (Trimethoprim + Sulfamethoxazole), Total daily dose = 40 mg/kg 50-100 mg/kg 40 mg/kg 30-50 mg/kg 20 mg/kg 8 mg/kg 10 mg/kg (max. 400 mg/day) 9 mg/kg 8 mg (TMP) + 40 mg (SMZ)/kg. Amoxicillin Ampicillin Co-amoxiclav Erythromycin Cefaclor (II generation) Cefixime (III generation) Cefpodoxime proxetil Ceftibuten (III generation) Co-trimoxazole (Trimethoprim + Sulfamethoxazole), Divided dose = 3 4 2-3 4 2-3 1 or 2 2 1 2
membrane with a yellow spot on its summit. Tenderness may be elicited over the mastoid antrum.
X-rays of mastoid will show clouding of air cells because of exudate.
4.
Stage of
resolution.
The tympanic
membrane
2. Decongestant nasal drops. Ephedrine nose drops (1% in adults and 0.5% in children) or oxymetazoline (Nasivion) or xylometazoline (Otrivin) should be used to relieve eustachian tube oedema and promote ventilation of middle ear.
ruptures with

release tory
process early
of pus
and begins
or subsidence
to of
resolve.
if the
infection without
rupture
Symptoms.
symptoms.
If
Inflamma- proper
was treatment
mild, of
resolution tympanic
With is
started may
start membrane.
evacuation of
pus, earache
is even
relieved, fever
comes down
and child
feels better.
Signs. External auditory canal may contain blood-tinged discharge which later becomes mucopurulent. Usually, a small perforation is seen in anteroinferior quadrant of pars tensa. Hyperaemia of tympanic membrane begins to subside with return to normal colour and landmarks.
5. Stage of complication. If virulence of organism is high or resistance of patient poor, resolution may not take place and disease spreads beyond the confines of middle ear. It may lead to acute mastoiditis, subperiosteal abscess, facial paralysis, labyrinthitis, petrositis, extradural abscess, meningitis, brain abscess or lateral sinus thrombophlebitis.

1. Antibacterial therapy (Table 10.1). It is indicated in all cases with fever and severe earache. As the most common organisms are S. pneumoniae and H. influenzae, the drugs which are effective in acute otitis media are ampicillin (50 mg/kg/day in four divided doses) and amoxicillin (40 mg/ kg/day in three divided doses). Those allergic to these penicillins can be given cefaclor, co-trimoxazole or erythromycin. In cases where f-lactamase-producing H. influenzae or M. catarrhalis are isolated, antibiotics like amoxicillin clavulanate, augmentin, cefuroxime axetil or cefixime may be used. Antibacterial therapy must be continued for a minimum of 10 days, till tympanic membrane regains normal appearance and hearing returns to normal. Early discontinuance of therapy with relief of earache and fever, or therapy given in inadequate doses may lead to secretory otitis media and residual hearing loss.
3. Oral nasal decongestants. Pseudoephedrine (Sudafed) 30 mg twice daily or a combination of decongestant and antihistaminic (Triominic) may achieve the same result without resort to nasal drops which are difficult to administer in children.
4. Analgesics and antipyretics. Paracetamol helps to relieve pain and bring down temperature.
5. Ear toilet. If there is discharge in the ear, it is dry-mopped with sterile cotton buds and a wick moistened with antibiotic may be inserted.
6.
Dry local
heat helps
to relieve
pain.
7. Myringotomy. It is incising the drum to evacuate pus and is indicated when (i) drum is bulging and there is acute pain, (ii) there is an incomplete resolution despite antibiotics when drum remains full with persistent conductive deafness and (iii) there is persistent effusion beyond 12 weeks. All cases of acute suppurative otitis media should be carefully followed till drum membrane returns to its normal appearance and conductive deafness disappears (Figure 10.1).

It is a variety of acute suppurative otitis media, often seen in children suffering from measles, scarlet fever or influenza. Causative organism is p-haemolytic streptococcus. There is rapid destruction of whole of tympanic membrane with its annulus, mucosa of promontory, ossicular chain and even mastoid air cells. There is profuse otorrhoea. In these cases, healing is followed by fibrosis or ingrowth of squamous epithelium from the meatus (secondary acquired cholesteatoma). Treatment is early institution of antibacterial therapy. It is continued for at least 7-10 days, even if response is seen early. Cortical mastoidectomy may be indicated if medical treatment fails to control or the condition gets complicated by acute mastoiditis.

This is an insidious condition characterized by accumulation of nonpurulent effusion in the middle ear cleft. Often the effusion is thick and viscid but sometimes it may be thin and serous. The fluid is nearly sterile. The condition is commonly seen in school-going children.

Two main mechanisms are thought to be responsible.
1. Malfunctioning of eustachian tube. Eustachian tube fails to aerate the middle ear and is also unable to drain the fluid.
2. Increased secretory activity of middle ear mucosa. Biop- sies of middle ear mucosa in these cases have confirmed
increase in number of mucus or serous-secreting cells.

1. Malfunctioning of eustachian tube. The causes are:
(a) Adenoid hyperplasia.
(b) Chronic rhinitis and sinusitis.
(c) Chronic tonsillitis. Enlarged tonsils mechanically
obstruct the movements of soft palate and interfere with the physiological opening of eustachian tube.
(d)
Benign and
malignant tumours
of nasopharynx.
This cause
should always
be excluded
in unilateral
serous otitis media in an adult.
- (e) Palatal defects, e.g. cleft palate, palatal paralysis.
2. Allergy. Seasonal or perennial allergy to inhalants or foodstuff is common in children. This not only obstructs eustachian tube by oedema but may also lead to increased secretory activity as middle ear mucosa acts as a shock organ in such cases.
3. Unresolved otitis media. Inadequate antibiotic therapy in acute suppurative otitis media may inactivate infection but fail to resolve it completely. Low-grade infection lingers on. This acts as stimulus for mucosa to secrete more fluid. The number of goblet cells and mucous glands also increase. Recent increase in the incidence of this disease seems to be due to this factor.
4. Viral infections. Various adenoand rhinoviruses of upper respiratory tract may invade middle ear mucosa and stimulate it to increased secretory activity.

1. Symptoms. The disease affects children of 5-8 years of age. The symptoms include:
(a)
Hearing loss.
This is
the presenting
and sometimes
the only
symptom.
It is
insidious in
onset and
rarely exceeds 40 dB. Deafness may pass unnoticed by the par-
ents and
may be
accidentally discovered
during audio-
metric screening tests.
Delayed and
defective speech.
Because of
hearing development of speech is delayed or defective.
Mild
(b)
(c)
earaches.
There may
be history
of upper
loss, respira-
tory tract infections with mild earaches.
2. Otoscopic findings. Tympanic membrane is often dull and opaque with loss of light reflex. It may appear yellow, or bluish in colour.
grey Thin leash of blood vessels may be seen along the handle of malleus or at the periphery of tympanic membrane and differs from marked congestion of acute suppurative otitis media. Tympanic membrane may show varying degree of retraction. Sometimes, it may appear full or slightly bulging in its posterior part due to effusion. Fluid level and air bubbles may be seen when fluid is thin and tympanic membrane transparent (Figure 10.2). Mobility of the tympanic membrane is restricted.

1. Tuning fork tests show conductive hearing loss.
2. Audiometry. There is conductive hearing loss of 20-40
dB.
there
is
sensorineural
hearing
loss due to fluid pressing on the round window mem-
brane. This disappears with evacuation of fluid.
Figure
10.2
on Valsalva.
3.
Impedance
Otitis media with effusion. Note appearance of bubbles audiometry.
It is
an objective
test useful
in infants
children.
Presence compliance
of and
flat fluid
is curve
with
a
indicated by
shift to
nega- tive
side.
- 4. X-ray mastoids. There is clouding of air cells due to fluid.

The aim of treatment is removal of fluid and prevention of its recurrence.

(a) Decongestants. Topical decongestants in the form of nasal drops, sprays or systemic decongestants help to relieve oedema of eustachian tube.
(b) Antiallergic measures. Antihistaminics or sometimes steroids may be used in cases of allergy. If possible, allergen should be found and desensitization done.
(c) Antibiotics. They are useful in cases of upper respiratory tract infections or unresolved acute suppurative otitis media.
(d) Middle ear aeration. Patient should repeatedly perform Valsalva manoeuvre. Sometimes, politzerization or eustachian tube catheterization has to be done. This helps to ventilate middle ear and promote drainage of fluid. Children can be given chewing gum to encourage repeated swallowing which opens the tube.

When fluid is thick and medical treatment alone does not help, fluid must be surgically removed.
(a) Myringotomy and aspiration of fluid. An incision is made in tympanic membrane and fluid aspirated with suction. Thick mucus may require installation of saline or a mucolytic agent like chymotrypsin solution to liquefy mucus before it can be aspirated. Sometimes, two incisions are made in the tympanic membrane, one in the anteroinferior and the other in the anterosuperior quadrant to aspirate thick, glue-like secretions (Figure 10.3) on "beer-can" principle.
(b) Grommet insertion. If myringotomy and aspiration combined with medical measures have not helped and fluid recurs, a grommet is inserted to provide continued aeration
Figure 10.3 To aspirate thick mucus, two incisions may be required in the tympanic membrane.
Cc
Figure
10.4
Grommet in
B
the tympanic
membrane
(A
and
B), the
grommet
(C).
of middle ear (Figure 10.4). It is left in place for weeks or months or till it is spontaneously extruded.
(c) Tympanotomy or cortical mastoidectomy. It is sometimes required for removal of loculated thick fluid or other associated pathology such as cholesterol granuloma.
(d) Surgical treatment of causative factor. Adenoidectomy, tonsillectomy and/or wash-out of maxillary antra may be required. This is usually done at the time of myringotomy.

1. Atrophic tympanic membrane and atelectasis of the middle ear. In prolonged effusions, there is dissolution of fibrous layer of tympanic membrane. It becomes thin and atrophic and retracts into the middle ear.
2. Ossicular necrosis. Most commonly, long process of incus gets necrosed. Sometimes, stapes superstructure also gets necrosed. This increases the conductive hearing loss to more than 50 dB.
3. Tympanosclerosis. Hyalinized collagen with chalky deposits may be seen in tympanic membrane, around the ossicles or their joints, leading to their fixation.
4. Retraction pockets and cholesteatoma. Thin atrophic part of pars tensa may get invaginated to form retraction
pockets pockets may the attic region.
or cholesteatoma. Similar be seen in
5. Cholesterol granuloma. This is due to stasis of secretions
in middle ear and mastoid.

Infants and children between the age of 6 months and 6 years may get recurrent episodes of acute otitis media. Such episodes may occur four to five times in a year. Usually, they occur after acute upper respiratory infection, the child being free of symptoms between the episodes. Recurrent middle infections may sometimes be superimposed upon an existing middle ear effusion. Sometimes, the underlying cause is recurrent sinusitis, velopharyngeal insufficiency, hyper trophy of adenoids, infected tonsils, allergy and immune deficiency. Feeding the babies in supine position without propping up the head may also cause the milk to enter the middle ear directly that can lead to middle ear infection.
Management of such children involves:
Finding the cause and eliminating it, if possible. noe
. Antimicrobial prophylaxis. Amoxicillin (20 mg/kg for 3-6 months) or sulfisoxazole have been used but they pre- vent only 1-2 bouts of otitis media in a year and have the disadvantage of creating antimicrobial resistance or hypersensitivity reaction and thus not preferred by many
in
favour
of
early
insertion
of
tympanostomy
tubes.
3. Myringotomy and insertion of tympanostomy tube. If the child has 4 bouts of acute otitis media in 6 months or 6 bouts in 1 year, insertion of a tympanostomy tube is recommended.
4. Adenoidectomy with or without tonsillectomy.
5. Management of inhalant or food allergy.

It is a nonsuppurative condition resulting from failure of eustachian tube to maintain middle ear pressure at ambient atmospheric level. The usual cause is rapid descent during air flight, underwater diving or compression in pressure chamber.

Eustachian tube allows easy and passive egress of air from middle ear to the pharynx if middle ear pressure is high. In
the reverse situation, where nasopharyngeal air pressure is high, air cannot enter the middle ear unless tube is actively opened by the contraction of muscles as in swallowing, yawning or Valsalva manoeuvre. When atmospheric pressure is higher than that of middle ear by critical level of 90 mm Hg, eustachian tube gets "locked," i.e. soft tissues of pharyngeal end of the tube are forced into its lumen. In the presence of eustachian tube oedema, even smaller pressure differentials cause "locking" of the tube. Sudden negative pressure in the middle ear causes retraction of tympanic membrane, hyperaemia and engorgement of vessels, transudation and haemorrhages.
Sometimes, though rarely, there is rupture of labyrinthine membranes with vertigo and sensorineural hearing loss.

Severe earache, hearing loss and tinnitus are common complaints. Vertigo is uncommon. Tympanic membrane appears retracted and congested. It may get ruptured. Middle ear may show air bubbles or haemorrhagic effusion. Hearing loss is usually conductive but sensorineural type of loss may also be seen.

The aim is to restore middle ear aeration. This is done by catheterization or politzerization. In mild cases, decongestant nasal drops or oral nasal decongestant with antihistaminics are helpful. In the presence of fluid or failure of the above methods, myringotomy may be performed to "unlock" the tube and the fluid.
aspirate

Aero-otitis can be prevented by the following measures:
1.
Avoid
air
travel
in
the
presence
of
upper
infection or allergy.
2. Swallow repeatedly during descent. Sucking sweets or chewing gum is useful.
3. Do not permit sleep during descent as number of swal- lows normally decrease during sleep.
4. Autoinflation of the tube by Valsalva should be per- formed descent.
intermittently during
5. Use vasoconstrictor nasal spray and a tablet of antihis- taminic and systemic decongestant, half an hour before descent in persons with previous history of this episode.
6. In recurrent barotrauma, attention should be paid to nasal polyps, septal deviation, nasal allergy and chronic sinus infections.

Normally, middle ear cleft is lined by different types of epithelium in different regions: ciliated columnar in the anterior and inferior part, cuboidal in the middle part and pavement-like in the attic. The middle ear is nowhere lined by keratinizing squamous epithelium. It is the presence of latter type of epithelium in the middle ear or mastoid that constitutes a cholesteatoma. In other words, cholesteatoma is a "skin in the wrong place." The term cholesteatoma is a misnomer because it neither contains cholesterol crystals nor it is a tumour to merit the suffix "oma." However, the term has been retained because of its wider usage. Essentially, cholesteatoma consists of two parts: (i) the matrix, which is made up of keratinizing squamous epithelium resting on a thin stroma of fibrous tissues and (ii) a central white mass, consisting of keratin debris produced by the matrix (Figure 11.1). For this reason, it has also been named epidermosis or keratoma.

Genesis of cholesteatoma is a matter of debate. Any theory of its genesis must explain how squamous epithelium appeared in the middle ear cleft. The various views expressed are:
1. Presence of congenital cell rests.
2. Invagination of tympanic membrane from the attic or posterosuperior part of pars tensa in the form of retrac- tion pockets (Figure 11.2) (Wittmaack’s theory). The outer surface of tympanic membrane is lined by stratified squa- mous epithelium which after invagination forms the matrix of cholesteatoma and lays down keratin in the pocket.
3. Basal cell hyperplasia (Ruedi’s theory). The basal cells of germinal layer of skin proliferate under the influ- ence of infection and lay down keratinizing squamous epithelium.
4. Epithelial invasion (Habermann’s theory). The epithelium from the meatus or outer drum surface grows into the middle ear through a pre-existing perforation especially of the marginal type where part of annulus tympanicus has already been destroyed.
5. Metaplasia (Sade’s theory). Middle ear mucosa, like respi- ratory mucosa elsewhere, undergoes metaplasia due to repeated infections and transforms into squamous epithelium.

(FIGURE
11.3)
The cholesteatoma is classified into:
- 1. Congenital
2. Acquired, primary
3. Acquired, secondary
1. Congenital cholesteatoma. It arises from the embryonic epidermal cell rests in the middle ear cleft or temporal bone. Congenital cholesteatoma occurs at three important sites: middle ear, petrous apex and the cerebellopontine angle, and produces symptomatology depending on its location. A middle ear congenital cholesteatoma presents as a white mass behind an intact tympanic membrane and causes conductive hearing loss. It may sometimes be discovered on routine examination of children or at the time of myringotomy. It may also spontaneously rupture through the tympanic membrane and present with a discharging ear indistinguishable from a case of chronic suppurative otitis media.
2. Primary acquired cholesteatoma (Figure 11.2). It is called primary as there is no history of previous otitis media or a pre-existing perforation. Theories on its genesis are:
(a) Invagination of pars flaccida. Persistent negative pressure in the attic causes a retraction pocket which accumulates keratin debris. When infected, the keratin mass expands towards the middle ear. Thus, attic perforation is in fact the proximal end of an expanding invaginated sac.
(b)
Basal cell
hyperplasia.
There is
proliferation of
the basal
layer of
induced
Expanding by
subclinical cholesteatoma
childhood then
breaks through
forming an
attic perforation.
- (c) Squamous metaplasia. Normal pavement epithelium of attic undergoes metaplasia, keratinizing squamous epithelium due to subclinical infections. Such a change has also been demonstrated in cases of otitis media with effusion.
3. Secondary acquired cholesteatoma. In these cases, there is already a pre-existing perforation in pars tensa. This is often associated with posterosuperior marginal perforation or sometimes large central perforation. Theories on its genesis include:

(a) Migration of squamous epithelium. Keratinizing squamous epithelium of external auditory canal or outer surface of tympanic membrane migrates through the
(b)
{
TO
NN
Epithelial invasion through posterosuperior perforation
Figure 11.2 Genesis of cholesteatoma.
perforation into the middle ear. Perforations, involying tympanic annulus as in acute necrotizing otitis media, are more likely to allow in-growth of squamous epithelium. Metaplasia. Middle ear mucosa undergoes metaplasia due to repeated infections of middle ear through the pre-existing perforation.

Once cholesteatoma enters the middle ear cleft, it invades the surrounding structures, first by following the path of least resistance, and then by enzymatic bone destruction.
Figure
11.1
Schematic structure
of cholesteatoma.
_
Sn fy
or
Retraction pocket
fy ff
a
Basal cell
hyperplasia
f

Chronic suppurative otitis media (CSOM) is a long-standing infection of a part or whole of the middle ear cleft characterized by ear discharge and a permanent perforation. A perforation becomes permanent when its edges are covered by squamous epithelium and it does not heal spontaneously. A permanent perforation can be likened to an epithelium-lined fistulous track.

Incidence of CSOM is higher in developing countries because of poor socioeconomic standards, poor nutrition and lack of health education. It affects both sexes and all age groups. In India, the overall prevalence rate is 46 and 16 persons per thousand in rural and urban population, respectively. It is also the single most important cause of hearing impairment in rural population.

Clinically, it is divided into two types:
1. Tubotympanic. Also called the safe or benign type; it involves anteroinferior part of middle ear cleft, ie. eustachian tube and mesotympanum and is associated with a central perforation. There is no risk of serious complications.
2.
Atticoantral.
Also called
unsafe or
dangerous type;
it involves
posterosuperior and
mastoid)
part of
and is
the associated
perforation.
The eroding
process osteitis.
Table disease
with cleft
(i.e.
attic, an
attic is
often associated
such as
cholesteatoma,
Risk of
11.1
shows antrum
or
a
marginal with
a
bone- granulations
is high
in this
differences
CSOM.

The disease starts in childhood and is therefore common in that age group.
1. It is the sequela of acute otitis media usually following exanthematous fever and leaving behind a large central perforation.
The perforation becomes permanent and_ permits repeated infection from the external ear. Also the middle ear mucosa is exposed to the environment and gets sen- sitized to dust, pollen and other aeroallergens causing persistent otorrhoea.
2. Ascending infections via the eustachian tube. Infec- tion from tonsils, adenoids and infected sinuses may be responsible for persistent or recurring otorrhoea. Ascending infection to middle ear occurs more easily in the presence of infection.
3. Persistent mucoid otorrhoea is sometimes the result of allergy to ingestants such as milk, eggs, fish, etc.

The tubotympanic disease remains localized to the mucosa and, that too, mostly to anteroinferior part of the middle ear cleft. Like any other chronic infection, the processes of healing and destruction go hand in hand and either of them may take advantage over the other, depending on the virulence of organism and resistance of the patient. Thus, acute exacerbations are not uncommon. The pathological changes seen in this type of CSOM are:
1. Perforation of pars tensa. It is a central perforation and its size and position varies (Figure 11.4).
2.
Middle ear
mucosa.
It may
be normal
when disease
is qui-
escent or
is active.
3.
Polyp.
inflamed inactive.
It is
A
polyp mucosa
and presents
to oedematous
is and
velvety
a
smooth mass
which in
has the
pink, fleshy
4.
Ossicular protruded
external polyp
chain.
show some
degree seen
of oedematous
through canal.
It is
and
a
perforation usually
in
It pale
atticoantral is
of of incus.
Perforation of tympanic membrane
Figure 11.4 Perforation of tympanic membrane. Note: Attic and posterosuperior marginal perforation are seen in dangerous type of CSOM and are often associated with cholesteatoma. Stratified squamous epithelium from the external auditory canal can grow into the middle ear in any type of marginal perforation by immigration and form a cholesteatoma. Therefore, all marginal perforations are considered dangerous. Central are considered safe as cholesteatoma is not associated with them.
perforations usually usually
disease intact
necrosis, in
contrast
(Figure and
mobile particularly
of
11.5).
but the
long may
process between
or variety.
the two
types of
Figure 11.5 presenting in the ear canal. (A) Polyp in the ear canal. (B) Schematic illustration of a polyp arising from the promontory passing through the perforation and
Figure 11.6 Classification of chronic otitis media.

5. Tympanosclerosis. It is hyalinization and subsequent calcification of subepithelial connective tissue. It is seen in remnants of tympanic membrane or under the mucosa of middle ear. It is seen as white chalky deposit on the promontory, ossicles, joints, tendons and oval and round windows. Tympanosclerotic masses may interfere with the mobility of these structures and cause conductive deafness.
media may also have patches of tympanosclerosis in tympanic membrane, or in middle ear involving promontory, ossicles, tendons of stapedius and tensor tympani. Fibrotic tissue may appear in middle ear. It is always associated with some degree of conductive hearing loss.
6. Fibrosis and adhesions. They are the result of healing process and may further impair mobility of ossicular chain or block the eustachian tube.

Pus culture in both types of aerobic and anaerobic CSOM may show multiple organisms. Common aerobic organisms are Pseudomonas aeruginosa, Proteus, Escherichia coliand Staphylococcus aureus, while anaerobes include Bacteroides fragilis and anaerobic Streptococci.

Tubotympanic disease of middle ear is a mucosal disease with no evidence of invasion of squamous epithelium. It is called "active? when there is a perforation of pars tensa with inflammation of mucosa and mucopurulent discharge. It is called "inactive? when there is a permanent perforation of pars tensa but middle ear mucosa is not inflamed and there is no discharge. Permanent perforation implies that squamous epithelium on the external surface of pars tensa and mucosa lining its inner surface have fused across its edge. Healed chronic otitis media is the condition when tympanic membrane has healed (usually by two layers), is atrophic and easily retracted if there is negative pressure in the middle ear. Healed otitis
Atticoantral disease has been called squamosal disease of middle ear. It may be "inactive? when there are retraction pockets in pars tensa (usually the posterosuperior region) or pars flaccida. There is no discharge but there is a possibility of squamous debris in retraction pockets to become infected and start discharging. Some retraction pockets are shallow and self-cleansing. "Active" squamosal disease of middle ear implies presence of cholesteatoma of posterosuperior region of pars tensa or in the pars flaccida. It erodes bone, forms granulation tissue and has purulent offensive discharge (Figure 11.6).

1. Ear discharge. It is nonoffensive, mucoid or mucopurulent, constant or intermittent. The discharge appears mostly at time of upper respiratory tract infection or on accidental entry of water into the ear.
2. Hearing loss. It is conductive type; severity varies but rarely exceeds 50 dB. Sometimes, the patient reports of a paradoxical effect, ie. hears better in the presence of discharge than when the ear is dry. This is due to "round window shielding effect' produced by discharge which helps to maintain phase differential. In the dry ear with perforation, sound waves strike both the oval and round windows simultaneously, thus cancelling each other's effect (see physiology of hearing).
In long standing cases, cochlea may suffer damage due to absorption of toxins from the oval and round windows and hearing loss becomes mixed type.
Central perforation
(anterior)
Total perforation
with destruction
of even
the fibrous
annulus
Figure 11.7
Types of perforations in the tympanic membrane seen in CSOM.
3. Perforation. Always central, it may lie anterior, posterior or inferior to the handle of malleus. It may be small, medium or large or extending up to the annulus, i.e. subtotal (Figure 11.7).
4, Middle ear mucosa. It is seen when the perforation is large. Normally, it is pale pink and moist; when inflamed it looks red, oedematous and swollen. Occasionally, a polyp may be seen.

1. Examination under microscope (Figure 11.8). It is essential in every case and provides useful information regarding presence of granulations, in-growth of squamous epithelium from the edges of perforation, status of ossicular chain, tympanosclerosis and adhesions. An ear which appears dry may show hidden discharge under the microscope. Rarely, cholesteatoma may coexist with a central perforation and can be seen under a microscope.
2. Audiogram. It gives an assessment of degree of hearing loss and its type. Usually, the loss is conductive but a sensorineural element be may present.
3. Culture and sensitivity of ear discharge. It helps to select proper antibiotic ear drops.
4. Mastoid X-rays/CT scan temporal bone. Mastoid is usually sclerotic but may be pneumatized with clouding of air cells. There is no evidence of bone destruction. Presence of bone destruction is a feature of atticoantral disease.

The aim is to control infection and eliminate ear discharge and at a later stage to correct the hearing loss by surgical means.
1. Aural toilet. Remove all discharge and debris from the ear. It can be done by dry mopping with absorbent cotton buds, suction clearance under microscope or irrigation (not
Figure 11.8 Examination of the ear under microscope.
forceful syringing) with sterile normal saline. Ear must be dried after irrigation.
2. Ear drops. Antibiotic ear drops containing neomycin, polymyxin, chloromycetin or gentamicin are used. They are combined with steroids which have local antiinflammatory effect. To use ear drops, patient lies down with the diseased ear up, antibiotic drops are instilled and then intermittent pressure applied on the tragus for antibiotic solution to reach the middle ear. This should be done three or four times a day. Acid pH helps to eliminate pseudomonas infection, and irrigations with 1.5% acetic acid are useful.
Care should be taken as ear drops are likely to cause maceration of canal skin, local allergy, growth of fungus or resistance of organisms. Some ear drops are potentially ototoxic.
o@®
Subtotal perforation
(medium sized)
Attic perforation
Posterosuperior marginal
3. Systemic antibiotics. They are useful in acute exacerbation of chronically infected ear, otherwise role of systemic antibiotics in the treatment of CSOM is limited.
4. Precautions. Patients are instructed to keep water out of the ear during bathing, swimming and hair wash. Rubber inserts can be used. Hard nose blowing can also push the infection from nasopharynx to middle ear and should be avoided.
5. Treatment of contributory causes. Attention should be paid to treat concomitantly infected tonsils, adenoids, maxillary antra and nasal allergy.
6. Surgical treatment. Aural polyp or granulations, if present, should be removed before local treatment with antibiotics. It will facilitate ear toilet and permit ear drops to be used effectively. An aural polyp should never be avulsed as it may be arising from the stapes, facial nerve or horizontal canal and thus lead to facial paralysis or labyrinthitis.

7. Reconstructive surgery. Once ear is dry, myringoplasty with or without ossicular reconstruction can be done to restore hearing. Closure of perforation will also check repeated infection from the external canal.

It involves posterosuperior part of middle ear cleft (attic, antrum, posterior tympanum and mastoid) and is associated with cholesteatoma, which, because of its bone eroding properties, causes risk of serious complications. For this reason, the disease is also called unsafe or dangerous type.

Aetiology of atticoantral disease is same as of cholesteatoma and has been discussed earlier. It is seen in sclerotic mastoid, and whether the latter is the cause or effect of disease is not yet clear.

Atticoantral diseases are associated with the following pathological processes:

2. Osteitis and granulation tissue. Osteitis involves outer attic wall and posterosuperior margin of the tympanic ring. A mass of granulation tissue surrounds the area of osteitis and may even fill the attic, antrum, posterior tympanum and mastoid. A fleshy red polypus may be seen filling the meatus.
3.
Ossicular necrosis.
It is
common in
atticoantral disease.
Destruction may
may also
be limited
involve the
entire greater
stapes ossicular
to the
long superstructure,
chain.
than in
disease the
cholesteatoma process
of handle
Therefore, of
bridges incus
of hearing
tubotympanic type.
the gap
caused or
malleus or
loss is
always
Occasionally, by
the destroyed
ossicles and
is not
apparent
(cholesteatoma hearer).
4. Cholesterol granuloma. It is a mass of granulation tissue with foreign body giant cells surrounding the cholesterol crystals. It is a reaction to long-standing retention of secretions or haemorrhage, and may or may not coexist with cholesteatoma. When present in the mesotympanum, behind an intact drum, the latter appears blue.

1. Ear discharge. Usually scanty, but always foul-smelling due to bone destruction. Discharge may be so scanty that the patient may not even be aware of it. Total cessation of discharge from an ear which has been active till recently should be viewed seriously, as perforation in these cases might be sealed by crusted discharge, inflammatory mucosa or a polyp, obstructing the free flow of discharge. Pus, in these cases, may find its way internally and cause complications.
2. Hearing loss. Hearing is normal when ossicular chain is intact or when cholesteatoma, having destroyed the ossicles, bridges the gap caused by destroyed ossicles (cholesteatoma hearer). Hearing loss is mostly conductive but sensorineural element may be added.
- 3. Bleeding. It may occur from granulations or the polyp when cleaning the ear.

1. Perforation. It is either attic or posterosuperior marginal type(Figure 11.7). A small attic perforation may be missed due to presence of a small amount of crusted discharge. Sometimes, the area of perforation is masked by a small granuloma.
- 2. Retraction pocket. An invagination of tympanic membrane is seen in the attic or posterosuperior area of pars tensa. Degree of retraction and invagination varies. In early stages, pocket is shallow and self-cleansing but later when pocket is deep, it accumulates keratin mass and gets infected.
Stages of retraction pockets. There are four stages of tympanic membrane retraction.
- (a) Stage I. Tympanic membrane is retracted but does not contact the incus. It is a mild form of retraction.
(b)
Stage
I.
Tympanic membrane
is retracted
deep and
contacts the
incus;
middle
Stage
III.
Also membrane
ear mucosa
called comes
Middle ear
middle ear
be lifted
middle to
lie space
is mucosa
from balloons
on the
totally or
is not
ear atelectasis.
promontory affected.
Tympanic and
ossicles.
partially obliterated
is intact.
Tympanic the
promontory up
when panic
membrane layer
but membrane
with suction
N2O
is used
during is
thin because
has been
absorbed
In these
structure cases
tip.
It anaesthesia.
its collagenous
due to
long process
are absorbed.
can also
Tym- middle
prolonged of
retraction.
incus and
stapes
Placement super-
of
a
ventilation tube
helps to
restore the
position of
tympanic membrane.

- (d) Stage IV. Also called adhesive otitis media. Tympanic membrane is very thin and wraps the promontory and ossicles. There is no middle ear space, mucosal lining of the middle ear is absent and tympanic membrane gets adherent to the promontory. Retraction pockets are formed which may collect keratin plugs and form cholesteatoma. Erosion of the long process of incus and stapes superstructure is common in such cases.
3. Cholesteatoma. Pearly-white flakes of cholesteatoma can be
sucked from
the retraction
pockets.
Suction clearance
(c)
and examination under operating microscope forms an important part of the clinical examination and assessment of any type of CSOM.

1. Examination under microscope. All patients of chronic middle early disease should be examined under microscope (Figure 11.8). It may reveal presence of cholesteatoma, its site and extent, evidence of bone destruction, granuloma, condition of ossicles and pockets of discharge.
- 2. Tuning fork tests and audiogram. They are essential for preoperative assessment and to confirm the degree and type of hearing loss.
3. X-ray mastoids/CT scan temporal bone. They indicate extent of bone destruction and degree of mastoid pneumatization. They are useful to indicate a low-lying dura or an anteposed sigmoid sinus when operation is being contemplated on a sclerotic mastoid. Cholesteatoma causes destruction in the area of attic and antrum (key area), better seen in lateral view. CT scan of temporal bone gives more information and is preferred to X-ray mastoids.
- 4. Culture and sensitivity of ear discharge. It helps to select proper antibiotic for local or systemic use.

1. Pain. Pain is uncommon in uncomplicated CSOM. Its presence is considered serious as it may indicate extradural, perisinus or brain abscess. Sometimes, it is due to otitis externa associated with a discharging ear.
2. Vertigo. It indicates erosion of lateral semicircular canal which may progress to labyrinthitis or meningitis. Fistula test should be performed in all cases.
3. Persistent headache. It is suggestive of an intracranial complication.
4. Facial weakness indicates erosion of facial canal.
5. A listless child refusing to take feeds and easily going to sleep (extradural abscess).
- 6. Fever, nausea and vomiting (intracranial infection).
7. Irritability and neck rigidity (meningitis).
8. Diplopia (Gradenigo syndrome) petrositis.

Meatus
Dependence
Recurrence or residual disease
Second look surgery
Patients limitations
Auditory rehabilitation
Normal appearance
Does not require routine cleaning
High rate of recurrent or residual cholesteatoma
Requires second look surgery after
6 months or so to rule out cholesteatoma
No limitation. Patient allowed swimming
Easy to wear a hearing aid if needed

10. Abscess round the ear (mastoiditis).
It is not uncommon for a patient of CSOM, residing in a far-flung village, where medical facilities are poor, to go to a doctor for the first time, presenting with complications. It then demands urgent attention and emergency medical or surgical treatment.

1. Surgical. It is the mainstay of treatment. Primary aim in surgical treatment is to remove the disease and render the ear safe, and second in priority is to preserve or reconstruct hearing but never at the cost of the primary aim. Two types of surgical procedures are done to deal with cholesteatoma:
- (a) Canal wall down procedures. They leave the mastoid cavity open into the external auditory canal so that the diseased area is fully exteriorized. The commonly performed operations for atticoantral disease are atticotomy, modified radical mastoidectomy and rarely, the radical mastoidectomy (see operative surgery).
(b)
Canal wall up procedures. Here disease is removed by combined approach through the meatus and mastoid but retaining the posterior bony meatal wall intact, thereby avoiding an open mastoid cavity. It gives dry ear and permits easy reconstruction of hearing mechanism. However, there is danger of leaving some cholesteatoma behind. Incidence of residual or recurrent cholesteatoma in these cases is very high and therefore long-term follow-up is essential. Some surgeon's even advise routine re-exploration in all cases after 6 months or so. Canal wall up procedures are advised only in selected cases. In combined approach or intact canal wall mastoidectomy, disease is removed both permeatally, and through cortical mastoidectomy and posterior tympanotomy approach, in which a window is created between the mastoid and middle ear, through the facial recess, to reach sinus tympani (see p. 5).
See Table 11.2 for the comparison of canal wall up and canal wall down procedures.
2.
Reconstructive surgery.
Hearing can
be restored
by myringoplasty
or tympanoplasty.
of
It can
primary surgery
or as
a
second be
stage done
at the
time procedure.

Widely open meatus communicating with mastoid
Dependence on doctor for cleaning mastoid cavity once or twice a year
Low rate of recurrence or residual disease and thus a safe procedure
Not required
Swimming can lead to infection of mastoid cavity and it is thus curtailed
Problems in
fitting
a
hearing and
mastoid cavity
which aid
due to
sometimes large
gets meatus
infected
3. Conservative treatment. It has a limited role in the management of cholesteatoma but can be tried in selected cases, when cholesteatoma is small and easily accessible to suction clearance under operating microscope. Repeated suction clearance and periodic checkups are essential. It can also be tried out in elderly patients above 65 and those who are unfit for general anaesthesia or those refusing surgery. Polyps and granulations can also be surgically removed by cup forceps or cauterized by chemical agents like silver nitrate or trichloroacetic acid. Other measures like aural toilet and dry ear precautions are also essential.

In most of the cases, infection is secondary to pulmonary tuberculosis; infection reaches the middle ear through eustachian tube. Sometimes, it is blood-borne from tubercular focus in the lungs, tonsils, cervical or mesenteric lymph nodes. Disease is mostly seen in children and young adults.

The process is slow and insidious. Tubercles appear in the submucosal layers of middle ear cleft and caseate. There is painless necrosis of tympanic membrane. Multiple perforations may form which coalesce to form a single large perforation. Middle ear and mastoid get filled with pale granulations. Caries of bone and ossicles may occur leading to complications. Mastoiditis, facial paralysis, postauricular fistula, osteomyelitis with formation of bony sequestra and profound hearing loss are often seen in these cases.

1.
Painless ear
absent in
cases foul-smelling
of tubercular
because
2.
Perforation.
ber, of
Earache is
the
Multiple are
seen in
ease.
These underlying
characteristically
Discharge is
often bone
perforations, pars
tensa may
destruction.
two and
form or
three
a
coalesce into
in num-
classical
a
single sign
of dis-
large perforation
then it
becomes indistinguishable
from nonspecific
CSOM.
3. Hearing loss. There is severe hearing loss, out of proportion to symptoms. Mostly conductive, it may have sensorineural component due to involvement of labyrinth.
4. Facial paralysis. It isa common complication and may come unexpectedly. This may be the presenting feature in a child.

In the presence of secondary pyogenic infection, tubercular otitis media may be indistinguishable from chronic suppurative otitis media. Culture of ear discharge for tubercle bacilli, histopathological examination of granulations and X-ray chest, and other evidence of tuberculosis in the body help to confirm the diagnosis. Presently DNA probe and PCR (polymerase chain reaction) from the ear discharge can give early diagnosis in 3-7 days.

1. Systemic antitubercular therapy as being carried for primary disease.
2. Local treatment in the form of aural toilet and control of secondary pyogenic infection.
3. Mastoid surgery indicated for complications. Healing is delayed in tuberculous cases. Wound breakdown and fistula formation are common. Reconstructive surgery of middle ear is delayed till antitubercular therapy has been completed.

Itis a rare condition. Spirochaetes reach middle ear through eustachian tube when syphilitic lesions are present in the nose or nasopharynx. Infection may also be blood-borne. Sensory end organs of the inner ear and their nerves are soon invaded by spirochaetes leading to profound sensorineural hearing loss, tinnitus and vertigo. Bone necrosis and sequestrum formation are common and they lead to foetid ear discharge. Secondary pyogenic infection may occur, giving a clinical picture very much like chronic suppurative otitis media.
Definite diagnosis of syphilitic otitis media can only be made by specific treponemal antigen tests such as treponemal pallidum immobilization (TPI) test and fluorescent treponemal antibody absorption test (FTA-ABS). VDRL and RPR (reactive plasma reagin) tests are nonspecific but useful to monitor disease, however false positive tests may occur.
Treatment consists of antisyphilitic therapy with attention to aural toilet and control of secondary infection. Surgery may be required for removal of sequestra.

Though there is a general decline in the incidence of complications, they are still frequently seen in India. The causes are poor socioeconomic conditions, lack of education and awareness about healthcare (middle ear discharge is still being considered merely a nuisance rather than a potentially dangerous condition), and lack of availability of trained specialists in the far-flung rural areas where transportation facilities are still inadequate.

1. Age. Most of the complications occur in the first decade of life or in the elderly when the patient's resistance is low.
2. Poor socioeconomic group. Several factors such as overcrowding, poor health education and personal hygiene, and limited access to healthcare play an important part.
3. Virulence of organisms. Many organisms are developing resistance to antibiotics and acute infections are either not controlled or progress to subacute or chronic otitis media. Insufficient dose, less effective drug or insufficient period of administration of antibiotic can cause complications. Streptococcus pneumoniae type III (earlier called pneumococcus type III) is very virulent due to production of autolysin and pneumolysin. Haemophilus influenzae is developing resistance to B-lactam antibiotics and chloramphenicol. Other resistant strains are Pseudomonas aeruginosa and methicillin resistant aureus.
Staphylococcus
4. Immune-compromised host. Patients suffering from AIDS, uncontrolled diabetes, transplant patients receiving immunosuppressive drugs and cancer patients receiving chemotherapy are more prone to develop complications.
5. Preformed pathways. /nfection can easily travel beyond the middle ear cleft if preformed pathways exist, e.g. dehiscence
of bony facial canal, previous ear surgery, fracture of tem- poral
bone, stapedectomy,
perilymph fistula
or congenitally
enlarged aqueduct of vestibule (as in Mondini abnormality of inner ear) or dehiscence in the floor of middle ear.
6. Cholesteatoma. Osteitis or granulation tissue in chronic otitis media destroys the bone and helps infection to penetrate deeper.
In acute and chronic middle ear infection, disease pro- cess
is limited
only to
the mucoperiosteal
lining of
the cleft
but if
it spreads
into the
bony walls
of the
various complications can arise.
cleft or
beyond it,

1. Direct bone erosion. In acute infections, it is the process of hyperaemic decalcification. In chronic infection, it may be osteitis, erosion by cholesteatoma or granulation tissue.
2. Venous thrombophlebitis. Veins of Haversian canals are connected with dural veins which in turn connect with dural venous sinuses and superficial veins of brain. Thus, infection from the mastoid bone can cause thrombophlebitis of venous sinuses and even cortical vein thrombosis. This mode of spread is common in acute infections.

(a)
Congenital
dehiscences,
e.g.
in
bony
facial
canal,
floor of middle ear over the jugular bulb.
(b) Patent sutures, e.g. petrosquamous suture.
(c) Previous skull fractures. The fracture sites heal only fibrous scar which infection.
by permits
(d) Surgical defects, e.g. stapedectomy, fenestration and mastoidectomy with exposure of dura.
(e) Oval and round windows.
(f) Infection from labyrinth travel along internal
can acoustic meatus, aqueducts of the vestibule and that of the cochlea to the meninges.

Complications of otitis media are classified into two main groups (Figure 12.1):

1. Mastoiditis
2. Petrositis
3. Facial paralysis
4. Labyrinthitis

1. Extradural abscess
2. Subdural abscess
3. Meningitis
4. Brain abscess
5. Lateral sinus thrombophlebitis
6. Otitic hydrocephalus.
Figure 12.1 Complications of otitis media.

They are the direct result of middle ear infection and should
be differentiated from complications. They include:
Ossicular erosion NO
Atelectasis and adhesive otitis media OO
Tympanosclerosis
Cholesteatoma formation Or
Conductive hearing loss due to ossicular erosion or fixation ND
Sensorineural hearing loss
Speech impairment SOW
Learning disabilities
The last two are secondary to loss of hearing in the develop- mental phase of the infant or child.

Inflammation of mucosal lining of antrum and mastoid air cell system is an invariable accompaniment of acute otitis media and forms a part of it. The term "mastoiditis" is used when infection spreads from the mucosa, lining the mastoid air cells, to involve bony walls of the mastoid air cell system.

Acute mastoiditis usually accompanies or follows acute suppurative otitis media, the determining factors being high virulence of organisms or lowered resistance of the patient due to measles, exanthematous fevers, poor nutrition or associated systemic disease such as diabetes.
Acute mastoiditis is often seen in mastoids with wellair cell Children are affected more. Beta- developed system. haemolytic streptococcus is the most common causative
organism though other organisms responsible for acute otitis media may also be seen. Very often, anaerobic organisms are also associated with mastoiditis and need antibacterial therapy against them.

Two main pathological processes are responsible:
1. Production of pus under tension.
2. Hyperaemic decalcification and osteoclastic resorption of bony walls.
Extension of inflammatory process to mucoperiosteal lining of air cell system increases the amount of pus produced due to large surface area involved. Drainage of this pus, through a small perforation of tympanic membrane and/or eustachian tube, cannot keep pace with the amount being produced. Swollen mucosa of the antrum and attic also impede the drainage system resulting in accumulation of pus under tension. Hyperaemia and engorgement of mucosa causes dissolution of calcium from the bony walls of the mastoid air cells
(hyperaemic decalcification).
Both these combine destruction and processes to cause coalescence of mastoid air cells, converting them into a single irregular cavity filled with pus (empyema of mastoid). Pus may break through mastoid cortex leading to subperiosteal abscess which may even burst on surface leading to a discharging fistula (Figure 12.2A, B).

Symptoms. They are similar to that of acute suppurative otitis media. In a case of acute middle ear infection, it is the change in the character of these symptoms which is significant and a pointer to the development of acute mastoiditis.
- 1. Pain behind the ear. Pain is seen in acute otitis media but it subsides with establishment of perforation or treatment with antibiotics. It is the persistence of pain, increase in its intensity or recurrence of pain, once it had subsided. These are of
significant pointers pain.
2.
3.
Fever.
It of
acute ment
is the
otitis persistence
Figure 12.2 (A) Mastoid fistula. (B) Burst mastoid abscess exuding pus.
or media,
that in
recurrence of
fever in
a
case spite
points
Ear discharge.
and of
the
In increases
cease development
mastoiditis, in
antibiotic of
treat- mastoiditis.
discharge purulence.
due to
In obstruction
would worsen.
in
a
case becomes
some profuse
cases, to
its drainage
Any persistence
of acute
otitis discharge
but may
other of
discharge media,
points symptoms
beyond to
3
weeks, mastoiditis.

1. Mastoid tenderness. This is an important sign. Tenderness is elicited by pressure over the middle of mastoid process, at its tip, posterior border or the root of zygoma. Tenderness elicited over the suprameatal triangle may not be diagnostic of acute mastoiditis as it is seen even in cases of the acute otitis media due to inflammation of mastoid antrum (antritis). Tenderness should always be compared with that of the healthy side.
2. Ear discharge. Mucopurulent or purulent discharge, often pulsatile (light-house effect), may be seen coming through a central perforation of pars tensa.
3. Sagging of posterosuperior meatal wall. It is due to periostitis of bony party wall between the antrum and deeper posterosuperior part of bony canal.
4. Perforation of tympanic membrane. Usually, a small perforation is seen in pars tensa with congestion of the rest of tympanic membrane. Perforation may sometimes appear as a nipple-like protrusion. Sometimes, tympanic membrane is intact but dull and opaque especially in those who have received inadequate antibiotics.
5. Swelling over the mastoid. Initially, there is oedema of periosteum, imparting a smooth "ironed out" feel over the mastoid. Later retroauricular sulcus becomes obliterated and pinna is pushed forwards and downwards. When pus bursts through bony cortex, a subperiosteal fluctuant abscess is formed (Figure 12.3).
6. Hearing loss. Conductive type of hearing loss is always present.
7. General findings. Patient appears ill and toxic with low-grade fever. In children, fever is high with a rise in pulse rate.

1. Blood counts show polymorphonuclear leucocytosis.
2. Erythrocyte sedimentation rate is usually raised.
Figure 12.3 Mastoid abscess. Note that pinna is pushed downwards and forwards.
3. X-ray mastoid. There is clouding of air cells due to collection of exudate in them. Bony partitions between air cells become indistinct, but the sinus plate is seen as a distinct outline. In later stages, a cavity may be seen in the mastoid.
4, Ear swab for culture and sensitivity.

1. Suppuration of mastoid lymph nodes. Scalp infection may cause mastoid lymph node enlargement and then suppuration leading to abscess formation, but in such cases there is no history of preceding otitis media, ear discharge or deafness. Abscess is usually superficial.
2.
Furunculosis of
meatus.
It is
differentiated from
acute mastoiditis by:
(a) Absence of preceding acute otitis media.
(b) Painful movements of pinna; pressure over the tragus or below the cartilaginous part of meatus causes excruciating pain.
(c) Swelling of meatus is confined to the cartilaginous part only.
abscesses seen from behind.
(d) Discharge is never mucoid or mucopurulent. Mucoid element in discharge can only come from the middle ear and not from the external ear which is devoid of mucus-secreting glands.
(e) Enlargement of preor postauricular lymph nodes.
(f) Conductive hearing loss is usually mild and is due to the occlusion of meatus.
(g) An absolutely normal looking tympanic membrane excludes possibility of acute mastoiditis.
(h) X-ray mastoid with clear air-cell system excludes acute mastoiditis. Sometimes, difficulty arises when air-cell system appears hazy due to superimposed soft tissue swelling in cases of furunculosis.

1. Hospitalization of the patient. Patient is hospitalized if not already done.
2. Antibiotics. In the absence of culture and sensitivity, start with amoxicillin or ampicillin. Specific antimicrobial is started on the receipt of sensitivity report. Since anaerobic organisms are often present, chloramphenicol or metronidazole is added.
3. Myringotomy. When pus is under tension it is relieved by wide myringotomy (see operative surgery). Early cases of acute mastoiditis respond to conservative treatment with antibiotics alone or combined with myringotomy.

(a) Subperiosteal abscess.
(b) Sagging of posterosuperior meatal wall.
(c) Positive reservoir sign, i.e. meatus immediately fills with pus after it has been mopped out.
(d) No change in condition of patient or it worsens in spite
of adequate medical treatment for 48 h.
(e)
Mastoiditis, leading
to complications,
e.g.
facial paralysis,
labyrinthitis, intracranial
complications, etc.
Aim of cortical mastoidectomy is to exenterate all the mas- toid
air cells
and remove
any pockets
of pus.
Adequate antibiotic
treatment must
following mastoidectomy.
be continued
at least
for
5
days

Subperiosteal abscess
Labyrinthitis NO
Facial paralysis
Petrositis OR
Extradural abscess
Subdural abscess
Meningitis Brain abscess OID
9. Lateral sinus thrombophlebitis
10. Otitic hydrocephalous.

1. Postauricular abscess (Figure 12.3). This is the commonest abscess that forms over the mastoid. Pinna is displaced forwards, outwards and downwards. In infants and children, abscess forms over the MacEwen's triangle; pus in these cases travels along the vascular channels of lamina cribrosa.
2. Zygomatic abscess. It occurs due to infection of zygomatic air cells situated at the posterior root of zygoma. Swelling appears in front of and above the pinna (Figure 12.4A,B). There is associated oedema of the upper eyelid. In these cases, pus collects either superficial or deep to the temporalis muscle.
3. Bezold abscess. It can occur following acute coalescent mastoiditis when pus breaks through the thin medial side of the tip of the mastoid and presents as a swelling in the upper part of neck. The abscess may (i) lie deep to sternocleidomastoid, pushing the muscle outwards, (ii) follow the posterior belly of digastric and present as a swelling between the tip of mastoid and angle of jaw, (iii) be present in upper part of posterior triangle, (iv) reach the parapharyngeal space or (v) track down along the carotid vessels (Figure 12.5). Clinical features. Onset is sudden. There is pain, fever, a tender swelling in the neck and torticollis. Patient gives history of purulent otorrhoea.
A Bezold abscess should be differentiated from:
(a) acute upper jugular lymphadenitis.
(b) abscess or a mass in the lower part of the parotid gland.
(c)
an infected branchial cyst.
(d) parapharyngeal abscess.
(e) jugular vein thrombosis.
Figure 12.5 Bezold abscess. Pus bursting through the medial side of the tip of mastoid and collecting under the sternomastoid or digastric triangle.
A computed tomography (CT) scan of the mastoid and swelling of the neck may establish the diagnosis. Treatment

(a) Cortical mastoidectomy for coalescent mastoiditis with careful exploration of the tip for a fistulous opening into the soft tissues of the neck. (b) Drainage of the neck abscess through a separate incision and putting a drain in the dependent part. (c) Administration of intravenous antibiotics guided by the culture and sensitivity report of the pus taken at the time of surgery.
4, Meatal abscess (Luc abscess). In this case, pus breaks through the bony wall between the antrum and exter nal osseous meatus. Swelling is seen in deep part of bony meatus. Abscess may burst into the meatus.
5. Behind the mastoid (Citelli's abscess). Abscess is formed behind the mastoid more towards the occipital bone (compare postauricular mastoid abscess which forms over the mastoid). Some authors consider abscess of the digastric triangle, which is formed by tracking of pus from the mastoid tip, as the Citelli's abscess.
6. Parapharyngeal or retropharyngeal abscess. This results from infection of the peritubal cells due to acute coalescent mastoiditis.

It is a condition of slow destruction of mastoid air cells but without the acute signs and symptoms often seen in acute mastoiditis. There is no pain, no discharge, no fever and no mastoid swelling but mastoidectomy may show extensive destruction of the air cells with granulation tissue and dark gelatinous material filling the mastoid. It is not surprising to find erosion of the tegmen tympani and sinus plate with an extradural or perisinus abscess.

The condition often results from inadequate antibiotic therapy in terms of dose, frequency and duration of administration. Most often it results from use of oral penicillin given in cases of acute otitis media when acute symptoms subside but smouldering infection continues in the mastoid.

Patient is often a child, not entirely feeling well, with mild pain behind the ear but with persistent hearing loss. Tympanic membrane appears thick with loss of translucency. Slight tenderness may be elicited over the mastoid. Audiometry shows conductive hearing loss of variable degree. X-ray of mastoid will reveal clouding of air cells with loss of cell outline.

Cortical mastoidectomy with full doses of antibiotics is the treatment of choice. This may cause tympanic membrane to return to normal with improvement in hearing.

Spread of infection from middle ear and mastoid to the petrous part of temporal bone is called petrositis. It may be associated with acute coalescent mastoiditis, latent mastoiditis or chronic middle ear infections.

Like mastoid, petrous bone may be of three types: pnewmalized with air cells extending to the petrous apex, diploic containing only marrow spaces and sclerotic. Pneumatization of petrous apex occurs in only 30% of cases with cells extending from the middle ear or mastoid to the petrous apex. Usually two cell tracts are recognized:
- 1. Posterosuperior tract which starts in the mastoid and runs behind or above the bony labyrinth to the petrous apex; some cells even pass through the arch of superior semicircular canal to reach the
apex.
- 2. Anteroinferior tract which starts at the hypotympanum near the eustachian tube runs around the cochlea to reach the petrous apex.
Infective process runs along these cell tracts and reaches the petrous apex. Pathological process is similar to that of coalescent mastoiditis forming epidural abscess at the petrous apex involving cranial nerve VI and trigeminal ganglion.

Gradenigo syndrome is the classical presentation, and consists of a triad of (i) external rectus palsy (VIth nerve palsy), (ii) deep-seated ear or retro-orbital pain (Vth nerve involvement) and (iii) persistent ear discharge. It is uncommon to see the full triad these days.
Persistent ear discharge with or without deep-seated pain in spite of an adequate cortical or modified radical mastoidectomy also points to petrositis.
Fever, headache, vomiting and sometimes neck rigidity may also be associated. Some patients may get facial paralysis and recurrent vertigo due to involvement of facial and statoacoustic nerves.
Diagnosis of petrous apicitis requires both CT scan and MRI. CT scan of temporal bone will show bony details of the petrous apex and the air cells while MRI helps to differentiate diploic marrow-containing apex from the fluid or pus.

Cortical, modified radical or radical mastoidectomy is often required if not already done. The fistulous tract should be found out, which is then curetted and enlarged to provide free drainage. Tract of posterosuperior cells starts in the
Trautmann’s triangle or the attic. Tract of anterior cells is situated near the tympanic opening of eustachian tube and passes above the carotid artery, anterior to the cochlea. In the latter case, radical mastoidectomy is required. Suitable intravenous antibacterial therapy should
pre- cede and follow surgical intervention. Most cases of acute petrositis can now be cured with antibacterial therapy alone. It should be given in initial high doses and continued for 4-5 days, even after complete disappearance of symptoms.

It can occur as a complication of both acute and chronic otitis media.

Facial nerve is normally well-protected in its bony canal. Sometimes, the bony canal is dehiscent and the nerve lies just under the middle ear mucosa. It is in these cases that inflammation of middle ear spreads to epi- and perineurium causing facial paralysis. Facial nerve function fully recovers if acute otitis media is controlled with systemic antibiotics. Myringotomy or cortical mastoidectomy may sometimes be required.

Facial paralysis in chronic otitis media either results from cholesteatoma or from penetrating granulation tissue. Cho- lesteatoma destroys bony canal and then causes pressure on the nerve, further aided by oedema of associated inflam- matory process. Facial paralysis is insidious but slowly pro- gressive. Treatment is urgent exploration of the middle ear and mastoid. Facial canal is inspected from the geniculate ganglion to the stylomastoid foramen. If granulation tissue or cholesteatoma has entered the bony canal, the latter is uncapped in the area of involvement. Granulation tissue surrounding the nerve is removed but if it actually invades the nerve sheath, it is left in place. Ifa segment of the nerve has been destroyed by the granulation tissue, resection of nerve and grafting are better left to a second stage when infection has been controlled and fibrosis has matured.

There are three types of labyrinthitis:
1. Circumscribed labyrinthitis
2. Diffuse serous labyrinthitis
3. Diffuse suppurative labyrinthitis
CIRCUMSCRIBED LABYRINTHITIS (FISTULA OF LABYRINTH)
There is thinning or erosion of bony capsule of labyrinth, usually of the horizontal semicircular canal.
Aetiology. The causes are:
1. Chronic suppurative otitis media with cholesteatoma is
the most common cause.
2. Neoplasms of middle ear, e.g. carcinoma or glomus
tumour.
3. Surgical or accidental trauma to labyrinth.
Clinical features. A part of membranous labyrinth is exposed and becomes sensitive to pressure changes. Patient complains
of transient vertigo often induced by pressure on tragus, cleaning the ear or while performing Valsalva manoeuvre. It is diagnosed by “fistula test” which can be performed in two ways.
1. Pressure on tragus. Sudden inward pressure is applied on the tragus. This increases air pressure in the ear canal and stimulates the labyrinth. Patient will complain of ver- tigo. Nystagmus may also be induced with quick compo- nent towards the ear under test.
2. Siegel’s speculum. When positive pressure is applied to ear canal, patient complains of vertigo usually with nystagmus. The quick component of nystagmus would be towards the affected ear (ampullopetal displacement of cupula).
Ampullopetal flow of endolymph (as also ampullopetal displacement of cupula) whether in rotation, caloric or fis- tula test causes nystagmus to same side.
If negative pressure is applied, again it would induce ver-
tigo and nystagmus but this time the quick component of nystagmus would be directed to the (opposite) healthy side due to ampullofugal displacement of cupula.
Treatment. In chronic suppurative otitis media or choles- teatoma, mastoid exploration is often required to eliminate the cause. Systemic antibiotic therapy should be instituted before and after operation to prevent spread of infection into the labyrinth.

It is diffuse intralabyrinthine inflammation without pus for- mation and is a reversible condition if treated early.

1. Most often it arises from pre-existing circumscribed laby- rinthitis associated with chronic middle ear suppuration or cholesteatoma.
2. In acute infections of middle ear cleft, inflammation spreads through annular ligament or the round window.
3. It can follow stapedectomy or fenestration operation.
Clinical features. Mild cases complain of vertigo and nau- sea but in severe cases, vertigo is worse with marked nausea, vomiting and even spontaneous nystagmus. Quick compo- nent of nystagmus is towards the affected ear. As the inflammation is diffuse, cochlea is also affected with some degree of sensorineural hearing loss. Serous labyrinthitis, if not checked, may pass onto suppu- rative labyrinthitis with total loss of vestibular and cochlear function.

e Medical
1. Patientis put to bed, his head immobilized with affected ear above.
2. Antibacterial therapy is given in full doses to control infection.
3. Labyrinthine sedatives, e.g. prochlorperazine (Stemetil)
or
dimenhydrinate
(Dramamine),
are
given
for
symp-
tomatic
relief
of
vertigo.
4. Myringotomy is done if labyrinthitis has followed acute otitis media and the drum is bulging. Pus is cultured for specific antibacterial therapy.
¢ Surgical. Cortical mastoidectomy (in acute mastoiditis) or modified radical mastoidectomy (in chronic middle ear infection or cholesteatoma) will often be required to treat the source of infection. Medical treatment should always precede surgical intervention.

This is diffuse pyogenic infection of the labyrinth with permanent loss of vestibular and cochlear functions.
Aetiology. It usually follows serous labyrinthitis, pyogenic organisms entering through a pathological or surgical fistula.
Clinical features. There is severe vertigo with nausea and vomiting due to acute vestibular failure. Spontaneous nystagmus will be observed with its quick component towards the healthy side. Patient is markedly toxic. There is total loss of hearing. Relief from vertigo is seen after 3-6 weeks due to adaptation.
Treatment. It is same as for serous labyrinthitis. Rarely, drainage of the labyrinth is required, if intralabyrinthine suppuration is acting as a source of intracranial complications, e.g. meningitis or brain abscess.

It is collection of pus between the bone and dura. It may occur both in acute and chronic infections of middle ear.

In acute otitis media, bone over the dura is destroyed by hyperaemic decalcification, while in chronic otitis media it is destroyed by cholesteatoma and in such a case the pus comes to lie directly in contact with dura. Spread of infection can also occur by venous thrombophlebitis; in this case, bone over the dura remains intact. An extradural abscess may lie in relation to dura of middle or posterior cranial fossa or outside the dura of lateral venous sinus (perisinus abscess). The affected dura may be covered with granulations or appear unhealthy and discoloured.

Most of the time extradural or perisinus abscesses are asymptomatic and silent, and are discovered accidently during cortical or modified radical mastoidectomy.
However, their presence is suspected when there is:
Persistent headache on the side of otitis media.
Severe pain in the ear. ON
General malaise with low-grade fever.
Pulsatile purulent ear discharge. oR
Disappearance of headache with free flow of pus from
the ear (spontaneous abscess drainage).
Diagnosis is made on contrast-enhanced CT or MRI.

1. Cortical or modified radical or radical mastoidectomy. It is often required to deal with the causative disease process.
Extradural abscess is evacuated by removing overlying bone till the limits of healthy dura are reached. Cases where bony plate of tegmen tympani or sinus plate is intact but there is suspicion of an abscess, the intact bony plate is deliberately removed to evacuate any collection of pus.
2. An antibiotic cover should be provided for a minimum of 5 days and patient closely observed for any further complications, such as sinus thrombosis, meningitis or brain abscess.

Infection spreads from the ear by erosion of bone and dura or by thrombophlebitic process in which case intervening bone remains intact. Pus rapidly spreads in subdural space and comes to lie against the convex surface of cerebral hemisphere causing pressure symptoms. With time, the pus may get loculated at various places in subdural space.

Signs and symptoms of subdural abscess are due to (i) meningeal irritation, (ii) thrombophlebitis of cortical veins of cerebrum and (iii) raised intracranial tension.
1. Meningeal irritation. There is headache, fever (102°F or more), malaise, increasing drowsiness, neck rigidity and positive Kernig's sign.
2. Cortical venous thrombophlebitis. Veins over the cerebral hemisphere undergo thrombophlebitis leading to aphasia, hemiplegia and hemianopia. There may be Jacksonian type of epileptic fits which may increase to give a picture of status epilepticus.
3. Raised intracranial tension. There is papilloedema, ptosis and dilated pupil (IIIrd nerve involvement), and involvement of other cranial nerves. CT scan or MRI is required for diagnosis.

Lumbar puncture should not be done as it can cause herniation of the cerebellar tonsils. It is a neurological emergency. Aseries of burr holes or a craniotomy is done to drain subdural empyema. Intravenous antibiotics are administered to control infection. Once infection is under control, attention is paid to causative ear disease which may require mastoidectomy.

It is inflammation of leptomeninges (pia and arachnoid) usually with bacterial invasion of CSF in subarachnoid space. It is the most common intracranial complication of otitis media. It can occur in both acute and chronic otitis media. In infants and children, otogenic meningitis usually follows acute otitis media while in adults it is due to chronic middle ear infection.

Blood-borne infection is common in infants and children; in adults, it follows chronic ear disease, which spreads by bone erosion or retrograde thrombophlebitis. In the latter
case, it may be associated with an extradural abscess or granulation tissue.
In one-third of the patients with meningitis, another intracranial complication may coexist.

Symptoms and signs of meningitis are due to (i) presence of infection, (ii) raised intracranial tension, and (iii) meningeal and cerebral irritation. Their severity will vary with the extent of disease.
1. There is rise in temperature (102-104°F) often with chills and rigors.
Headache. bo
Neck rigidity. yo
Photophobia and mental irritability. OR
Nausea and vomiting (sometimes projectile).
Drowsiness which may progress to delirium or coma. ND
Cranial nerve palsies and hemiplegia.
Examination will show (i) neck rigidity, (ii) positive Kernig's sign (extension of leg with thigh flexed on abdomen causing pain), (iii) positive Brudzinski's sign (flexion of neck causes flexion of hip and knee), (iv) tendon reflexes are exaggerated initially but later become sluggish or absent and (v) papilloedema (usually seen in late stages).

CT or MRI with contrast will help to make the diagnosis. It may also reveal another associated intracranial lesion. Lumbar puncture and CSF examination establish the diagnosis. CSF is turbid, cell count is raised and may even reach 1000/mL with predominance of polymorphs; protein level is raised, sugar is reduced and chlorides are diminished. CSF is always cultured to find the causative organisms and their antibiotic sensitivity.

Medical.
Medical treatment
takes precedence
over surgery.
Antimicrobial therapy directed against aerobic and anaer- obic
organisms should
be instituted.
Culture and
sensitivity of
CSF
will further
aid in
the choice
of antibiotics.
Corticosteroids combined with antibiotic therapy further helps to reduce neurological or audiological complications.
Surgical. Meningitis following acute otitis media may require myringotomy or cortical mastoidectomy. Meningitis following chronic otitis media with cholesteatoma will require radical or modified radical mastoidectomy.
Surgery is undertaken as soon as general condition of patient permits. It may be done urgently, if there has been no satisfactory response to medical treatment.

Fifty per cent of brain abscesses in adults and twenty-five per cent in children are otogenic in origin. In adults, abscess usually follows chronic suppurative otitis media with cholesteatoma, while in children, it is usually the result of acute otitis media. Cerebral abscess is seen twice as as cerebellar abscess.
frequently

Cerebral abscess develops as a result of direct extension of middle ear infection through the tegmen or by retrograde thrombophlebitis, in which case the tegmen will be intact. Often it is associated with extradural abscess.
Cerebellar abscess also develops as a direct extension through the Trautmann's triangle or by retrograde thrombophlebitis. This is often associated with extradural abscess, perisinus abscess, sigmoid sinus thrombophlebitis or labyrinthitis.

Both aerobic and anaerobic organisms are seen. Aerobic ones include pyogenic staphylococci, Streptococcus pneumoniae, Streptococcus haemolyticus, Proteus mirabilis, Escherichia coli and Pseudomonas aeruginosa. Common among the anaerobic ones are the Peptostreptococcus and Bacteroides fragilis. Haemophilus influenzae is rarely seen.

Brain abscess develops through four stages.
- 1. Stage of invasion (initial encephalitis). It often passes unnoticed as symptoms are slight. Patient may have headache, low-grade fever, malaise and drowsiness.
2. Stage of localization (latent abscess). There are no symptoms during this stage. Nature tries to localize the pus by formation of a capsule. The stage may last for several weeks.
3. Stage of enlargement (manifest abscess). Abscess begins to enlarge. A zone of oedema appears round the abscess and is responsible for aggravation of symptoms. Clinical features at this stage are due to:
(a) Raised intracranial tension.
(b) Disturbance of function in the cerebrum or cerebellum, causing focal symptoms and signs.
4. Stage of termination (rupture of abscess). An expanding abscess in the white matter of brain ruptures into the ventricle or subarachnoid space resulting in fatal meningitis.

Brain abscess is often associated with other complications, such as extradural abscess, perisinus abscess, meningitis, sinus thrombosis and labyrinthitis, and thus the clinical picture may be overlapping.
Clinical features can be divided into:
1. those due to raised intracranial tension.
2. those due to area of brain affected. They are the localizing features.
1. Symptoms and signs of raised intracranial tension
(a) Headache. Often severe and generalized, worse in the morning.
(b)
Nausea
and
vomiting.
The
latter
is
usually
projectile.
Seen
more
often
in
cerebellar
lesions.
(c)
Level
of
consciousness.
Lethargy,
which
progresses
to
stupor
and
finally
coma.
(d) Papilloedema is absent in early cases. Appears late when raised intracranial tension has persisted for 2-3 weeks. Appears early in cerebellar abscess.
(e) Slow pulse and subnormal temperature.
Figure 12.6 CT scan showing left-sided cerebellar abscess.

Figure 12.7 CT scan of right-sided otogenic cerebral abscess.

(a), 1 = Temporal lobe abscess. , 1 = (i) Nominal aphasia. If abscess involves dominant hemi- sphere, i.e. left hemisphere in right-handed persons, patient fails to tell the names of common objects such as key, pen, etc. but can demonstrate their use.. , 1 = (ii) Homonymous hemianopia. This is due to pressure on the optic radiations. Visual field, opposite to the side of lesion, is lost. It can be elicited by confron- tation test, by standing in front of the patient and comparing his visual field with that of the exam- iner, or by perimetry. The defect is usually in the upper, but sometimes in the lower quadrants.. , 1 = (iii) Contralateral motor paralysis. In the usual upward spread of abscess, face is involved first followed by the armandleg. Inward spread, towards internal capsule, involves the leg first followed by the armandtheface. (iv) Epileptic fits. Involvement of uncinate gyrus causes. (b), 1 = transtentorial herniation. Cerebellar abscess (Figure 12.6) (i) Headache involves suboccipital region and may be. , 1 = (ii) Spontaneous nystagmus is common and irregular and generally to the side of lesion. (iii) Ipsilateral hypotonia and weakness.. , 1 = (iv) Ipsilateral ataxia . Patient staggers to the side of lesion. (v) Past-pointing and intention tremor can be elicited by finger nose test. (vi) Dysdiadochokinesia. Rapid pronation and supina- tion of the forearm shows slow and irregular move-. , 1 = ments on the affected side.

1. Skull X-rays are useful to see midline shift, if pineal gland is calcified, and also reveals gas in the abscess cavity. They have been replaced by CT scan.
2.
CT
scan is
the single
most important
means of
investiga- tion
and
12.7).
helps to
find
It dural
also the
reveals abscess,
ther site
and size
of associated
sigmoid improved
3.
X-ray an
abscess complications
sinus thrombosis,
the diagnosis.
mastoids or
CT
scan of
(Figure such
etc.
the temporal
MRI
bone as
extra- has
for fur-
evalu- ation
ear disease.
4, Lumbar puncture. Great care should be exercised while doing lumbar puncture because of the risk of coning. CSF will show some rise in pressure, increase in protein content but normal glucose level. White cell count of CSF is raised but is much less than seen in cases of meningitis. CSF contains polymorphs or lymphocytes depending on the acuteness of lesion.

Medical. High doses of antibiotics are given parenterally. As the infection is often mixed, antibiotics may be combined. Chloramphenicol and third generation cephalosporins are usually effective. Bacteroides fragilis, an obligate anaerobe, often seen in brain abscess, responds to metronidazole. Aminoglycoside antibiotics, e.g. gentamicin, may be required if infection suspected is pseudomonas or proteus. Culture of discharge from the ear may be helpful in the choice of antibiotic.
Raised intracranial
tension can
be lowered
by dexametha-
sone, 4 mg i.v. 6 hourly or mannitol 20% in doses of 0.5 g/kg body weight.
Discharge from the ear should be treated by suction clearance and use of topical ear drops.
Neurosurgical. Abscess is approached through a sterile field. Options include: (i) repeated aspiration through a burr hole, (ii) excision of abscess and (iii) open incision of
the abscess and evacuation of pus. The choice of surgical procedure is left to the judgement of the neurosurgeon. If abscess is treated by aspiration, it should be followed by repeat CT or MRI scans to see if it diminishes in size. An expanding abscess, or one that does not decrease in size, may require excision. Pus recovered from the abscess should be cultured and its sensitivity discovered. Penicillin can be instilled into the abscess after aspiration.
Otologic. Associated ear disease which caused the brain abscess needs attention. Acute otitis media might have resolved with the antibiotics given for the abscess. Chronic otitis media would require radical mastoidectomy to remove the irreversible disease and to exteriorize the infected area. Surgery of the ear is undertaken only after the abscess has been controlled by antibiotics and neurosurgical treatment.

It is an inflammation of inner wall of lateral venous sinus with formation of an intrasinus thrombus.

It occurs as a complication of acute coalescent mastoiditis, masked mastoiditis or chronic suppuration of middle ear and cholesteatoma.

The pathological process can be divided into the following stages:
1.
perisinus abscess.
Abscess forms
in rela-
tion to
outer plate
dural wall
of may
have been
or cholesteatoma.
the sinus.
destroyed
Overlying by
bony coalescent
Sometimes, it
bone remains
intact dural
erosion when
route of
infection was
by thrombophlebitic
process.
2. Endophlebitis and mural thrombus formation. Inflammation spreads to inner wall of the venous sinus with deposition of fibrin, platelets and blood cells leading to thrombus formation within the lumen of sinus.
3.
Obliteration of
sinus lumen
and intrasinus
abscess.
Mural thrombus
enlarges
Organisms to
occlude may
invade abscess
which the
sinus the
lumen thrombus
may release
infected completely.
causing emboli
intrasinus into
the blood
stream causing
septicaemia.
4. Extension of thrombus. Though central part of thrombus breaks down due to intrasinus abscess, thrombotic process continues both proximally and distally. Proximally, it may spread to confluence of sinuses and to superior sagittal sinus or cavernous sinus, and distally, into mastoid emissary vein, to jugular bulb or jugular vein.

In acute infections, haemolytic streptococcus, pneumococcus or staphylococcus are common. These days, majority of cases of thrombophlebitis are seen in chronic infection with cholesteatomas, and the organisms found are Bacillus proteus, Pseudomonas pyocyaneus, Escherichia coli and Staphylococci.
6. Culture and sensitivity of ear swab.

1. Septicaemia and pyaemic abscesses in lung, bone, joints
or
subcutaneous
tissue.
2. Meningitis and subdural abscess.
3. Cerebellar abscess.
4.
of
jugular
bulb
and
jugular
vein
with
involve-
ment
of
[Xth,
Xth
and
XIth
cranial
nerves.
5. Cavernous sinus thrombosis. There would be chemosis, proptosis, fixation of eyeball and papilloedema.
6. Otitic hydrocephalus, when thrombus extends to sagittal sinus via confluens of sinuses.

1.
Intravenous
antibacterial
therapy.
Choice
of
antibiotic
will
depend
on
sensitivity
of
organism
and
tolerance
of
the
patient.
can
be
changed
after
culture
and
sensitiv-
ity
report
is
should
be
continued
at
least
for
a
week
after
the
operation,
which
is
invariably
required.

or
modified
radical
mastoidectomy
is
performed,
depend-
ing
on
whether
sinus
thrombosis
has
complicated
acute
or
chronic
middle
ear
disease.
Sinus
bony
plate
is
removed
to
expose
the
dura
and
drain
the
perisinus
abscess.
An infected clot or intrasinus abscess may be present
and
must
be
drained.
In
such
cases,
sinus
dura
is
already
destroyed
or
may
appear
unhealthy
and
discoloured
with
granulations
on
its
surface.
Dura
is
incised
and
the
infected
clot
and
abscess
drained.
Before
incision
in
the
dura,
sinus
is packed, above and below, by inserting a pack between the bone and dura of sinus to control bleeding.
Healthy
red
clot
beyond
the
abscess
at
either
end
of
sinus
should
not
be
disturbed.
Pack
is
removed
5-6
days
postop-
eratively and wound secondarily closed.
3. Ligation of internal jugular vein. It is rarely required these days. It is indicated when antibiotic and surgical treatment have failed to control embolic phenomenon and rigors, or tenderness and swelling along jugular vein is spreading.
4,
therapy.
It
is
rarely
required
and
used
when
thrombosis is extending to cavernous sinus.
5. Supportive treatment. Repeated blood transfusions may be required to combat anaemia and improve patient's resistance.

It is characterized by raised intracranial pressure with normal CSF findings. It is seen in children and adolescents with acute or chronic middle ear infections.

Lateral sinus thrombosis accompanying middle ear infection causes obstruction to venous return. If thrombosis extends to superior sagittal sinus, it will also impede the function of arachnoid villi to absorb CSF. Both these factors result in raised intracranial tension.

1. Severe headache, sometimes intermittent, is the pre- senting feature. It may be accompanied by nausea and vomiting.
2. Diplopia due to paralysis of VIth cranial nerve.
3. Blurring of vision due to papilloedema or optic atrophy.

1. Papilloedema may be 5-6 diopters, sometimes with patches of exudates and haemorrhages.
2. Nystagmus due to raised intracranial tension.
3. Lumbar puncture. CSF pressure exceeds 300 mm H,O (normal 70-120 mm H,O). It is otherwise normal in cell, protein and sugar content and is bacteriologically sterile.

The aim is to reduce CSF pressure to prevent optic atrophy and blindness. This is achieved medically by acetazolamide and corticosteroids and repeated lumbar puncture or placement of a lumbar drain. Sometimes, draining CSF into the peritoneal cavity (lumboperitoneal shunt) is necessary. Middle ear infection may require antibiotic therapy and mastoid exploration to deal with sinus thrombosis.

It may be pertinent to review the anatomy of the labyrinth and introduce the terminology often used to describe it:, 1 = It may be pertinent to review the anatomy of the labyrinth and introduce the terminology often used to describe it:. 1., 1 = Otic labyrinth. Also called membranous labyrinth or endolymphatic labyrinth. It consists of utricle, saccule, cochlea, semicircular ducts, endolymphatic duct and sac. It is filled with endolymph.. 2., 1 = Periotic labyrinth or perilymphatic labyrinth (or space). It surrounds the otic labyrinth and is filled with perilymph. It includes vestibule, scala tympani, scala vestibuli, peri- lymphatic space of semicircular canals and the periotic duct, which surrounds the endolymphatic duct of otic labyrinth.. 3., 1 = Otic capsule. It is the bony labyrinth. It has three layers.. (b), 1 = Enchondral. Develops from the cartilage and later ossifies into bone. It is in this layer that some islands of cartilage are left unossified that later give rise to otosclerosis.. (c), 1 = Periosteal. Covers the bony labyrinth.. Otic capsule or the bony labyrinth ossifies from 14 centres, the first one appears in the region of cochlea at 16 weeks and, 1 = Otic capsule or the bony labyrinth ossifies from 14 centres, the first one appears in the region of cochlea at 16 weeks and

Otosclerosis, more aptly called otospongiosis, is a primary disease of the bony labyrinth. In this, one or more foci of irregularly laid spongy bone replace part of normally dense enchondral layer of bony otic capsule. Most often, otosclerotic focus involves the stapes region leading to stapes fixation and conductive deafness. However, it may involve certain other areas of the bony labyrinth where it may cause neurosensory loss or no symptoms at all.

The exact cause of otosclerosis is not known; however, the following facts have been documented.
Anatomical basis. Bony labyrinth is made of enchondral- bone
which is
subject to
little change
in life.
But sometimes,
in this
due hard
bone, to
new there
certain spongy
are nonspecific
bone.
areas of
factors
One such
area is
cartilage are
rests which
activated the
to form
/fissula ante
a
fenestram

1. Stapedial otosclerosis. Stapedial otosclerosis causing stapes fixation and conductive deafness is the most common variety. Here lesion starts just in front of the oval window in an area called "fissula ante fenestram." This is the site of predilection (anterior focus). Lesion may start behind the oval window (posterior focus), around the margin of the stapes footplate (circumferential), in the footplate but annular ligament being free (biscuit type). Sometimes, it may completely obliterate the oval window niche (obliterative type) (Figure 13.1).
2. Cochlear otosclerosis. Cochlear otosclerosis involves region of round window or other areas in the otic capsule, and may cause sensorineural hearing loss probably due to liberation of toxic materials into the inner ear fluid.
- 3. Histologic otosclerosis. This type of otosclerosis remains asymptomatic and causes neither conductive nor sensorineural hearing loss.
Figure
13.1
Anterior focus.
Types of
stapedial
(B)
Posterior
(D)
Biscuit type
(thick

Grossly, otosclerotic
yellow.
otosclerosis.
focus.
(A)
(C)
plate).
Circumferential.
(E)
Obliterative.
lesion
Sometimes, cularity,
appears it
is in
which chalky
red case,
in colour
the due
otosclerotic
white, greyish
or to
increased focus
vas- is
active and
rapidly progressive.
Microscopically, spongy bone appears in the normally dense enchondral layer of otic capsule. In immature active lesions, there are numerous marrow and vascular spaces with plenty of osteoblasts and osteoclasts and a lot of cement substance which stains blue (blue mantles) with haematoxylin-eosin stain. Mature foci show less vascularity and laying of more bone and more of fibrillar substance than cementum, and is stained red.

1. Hearing loss. This is the presenting symptom and usually starts in twenties. It is painless and progressive with insidious onset. Often it is bilateral conductive type.
2. Paracusis Willisii. An otosclerotic patient hears better in noisy than in quiet surroundings. This is because a normal person will raise his voice in noisy surroundings.
3. Tinnitus. It is more commonly seen in cochlear otosclerosis and in active lesions.
4. Vertigo. It is an uncommon symptom.
5. Speech. Patient has a monotonous, well-modulated soft speech.

1. Tympanic membrane is quite normal and mobile. Sometimes, a reddish hue may be seen on the promontory through the tympanic membrane (Schwartze sign). This is indicative of active focus with increased vascularity.
2. Eustachian tube function is normal.
3. Tuning fork tests show negative Rinne (i.e. BC > AC) first for 256 Hz and then 512 Hz and still later, when stapes
dB
in loss
Figure 13.2 Otosclerosis left ear. Note dip at 2000 Hz in bone conduction (Carhart's notch).
fixation is complete, for 1026 Hz. Weber test will be lateralized to the ear with greater conductive loss. Absolute bone conduction may be normal. It is decreased in cochlear otosclerosis with sensorineural loss.
Pure tone audiometry shows loss of air conduction, more for

Bone conduction
in bone
conduction quencies
is normal.
curve.
but notch
(5
In
It maximum
dB
and
5
at dB
at at
500
Hz, is
2000
10
4000
Hz)
dB
at some
cases, different
there is
a
at
Hz and
is
1000
(Figure
13.2).
different called
Hz,
15
dip fre-
Carhart’s dB
at
Carhart’s
2000
Hz notch
disap- pears
after successful
stapedectomy.
Mixed hearing loss is not uncommon in otosclerosis.
- There is loss in bone conduction with air-bone gap.
Speech audiometry
reveals normal
discrimination except
in those
with cochlear
Tympanometry may
involvement.
be shows
a
curve of
normal ossicular
absent when
stapes in
early stiffness.
is fixed
Stapedial
(see p.
24).
score but
later becomes
cases reflex

Otosclerosis should be differentiated from other causes of conductive deafness particularly serous otitis media, adhesive otitis media, tympanosclerosis, attic fixation of head of malleus, ossicular discontinuity or congenital stapes fixation.

Medical. There is no medical treatment that cures otosclerosis. Sodium fluoride has been tried to hasten the maturity of active focus and arrest further cochlear loss, but controversies exist and this treatment is not recommended generally.
Surgical. Siapedectomy/stapedotomy with a placement of prosthesis is the treatment of choice. Here the fixed otosclerotic stapes is removed and a prosthesis inserted between the incus and oval window (Figure 13.3). Prosthesis employed may be a teflon piston, stainless steel piston, platinum—teflon or titanium-teflon piston (Figure 13.4). In 90% of patients, there is good improvement in hearing after stapedectomy.
Selection of patients for stapes surgery. Hearing threshold for
air conduction
should be
30
dB
or worse.
(It is
this level
when patient
starts feeling
socially handicapped.)
Average air-bone gap should be at least 15 dB with Rinne negative for 256 and 512 Hz.
Speech discrimination score should be 60% or more.

1. The only hearing ear.
2. Associated Méniére's disease. When there is history of vertigo with clinical evidence of Méniére's disease in an otosclerotic patient, there are more chances of sensorineural loss after
hearing stapedectomy.
3. Young children. Recurrent eustachian tube dysfunction is common in children. It can displace the prosthesis or cause acute otitis media. Also the growth of otosclerotic focus is faster in children leading to reclosure of oval window.
4. Professional athletes, high construction workers, divers and frequent air travellers. Stapes surgery has the risk to cause postoperative vertigo and/or dizziness and thus interfere with their profession; or frequent air pressure changes may damage the hearing or cause severe vertigo.
5.
Those who
work in
noisy surroundings.
After stapedectomy,
they would
be more
get sensorineural
hear- ing
loss due
to noise
trauma.
- 6. Otitis externa, tympanic membrane perforation and exostosis are relative contraindications. Stapedectomy can be done after they have been treated first for above conditions. Similarly, stapedectomy is avoided during pregnancy.
The operation is preferably done under local anaesthesia.

- 1. Meatal incision and elevation of the tympanomeatal flap.
2.
Exposure of
stapes area.
This may
require removal
of pos-
terosuperior bony overhang of the canal.
3. Removal of stapes superstructure.
4. Creation of a hole in the stapes footplate (stapedotomy)
or removal of a part of footplate (stapedectomy).
5. Placement of prosthesis.
6. Repositioning the tympanomeatal flap.
Figure
13.3
(A)
Before removal
of stapes.
(B)
Stapes removed
and replaced
by
a
teflon piston.
Figure 13.4 Stapes prostheses. (A) Teflon piston. (B) Platinum-teflon piston. (C) Titanium-teflon piston.

1. Tear of tympanomeatal flap and later perforation of tympanic membrane
2. Injury to chorda tympani with taste disturbance particularly if opposite chorda was earlier injured
. Incus dislocation . Injury to facial nerve ov B® OO
. Vertigo
(a) Early in postoperative period (intraoperative trauma, serous labyrinthitis, long prosthesis)
(b) Late due to perilymph fistula and benign paroxysmal
positional vertigo
. Perilymph fistula/granuloma
. Conductive loss ND
(a) Short prosthesis
(b) Loose prosthesis
(c) Displacement of prosthesis
(d)
Incus erosion
(late)
8. Sensorineural hearing loss
(a) Intraoperative trauma
(b) Labyrinthitis
(c) Perilymph fistula/granuloma
9.
Dead ear
Two per cent of patients undergoing this operation may suffer sensorineural loss. Slowly progressive high frequency
loss is seen in long-term follow-up. One in 200 patients may
get a totally “dead” ear.
Stapes mobilization is no longer done these days as it gives
temporary results; refixation being quite common.
Lempert’s fenestration operation is almost outdated now. Here
an alternative window is created in the lateral semicircular
canal to function for the obliterated oval window. It has the disadvantage of a postoperative mastoid cavity and an inherent hearing loss of 25 dB which cannot be corrected.
Hearing aid. Patients who refuse surgery or are unfit for surgery can use hearing aid. It is an effective alternative.

Facial nerve runs from pons to parotid. It is a mixed nerve having motor and a sensory root. The latter is also called the nerve of Wrisberg and carries secretomotor fibres to the lacrimal gland and salivary glands, and brings fibres of taste and general sensation. Thus there are two efferent and two afferent pathways. Components of the facial nerve include:
1. Special visceral efferent forms the motor root and supplies all the muscles derived from the second branchial arch, i.e. all the muscles of facial expression, auricular muscles (now vestigial), stylohyoid, posterior belly of digastric and the stapedius.
2. General visceral efferent supplies secretomotor fibres to lacrimal, submandibular and sublingual glands and the smaller secretory glands in the nasal mucosa and the palate.
3. Special visceral afferent brings taste from the anterior two-thirds of tongue via chorda tympani and soft and hard palate via greater superficial petrosal nerve. Taste is carried to the nucleus of tractus solitarius.
4. General somatic afferent brings general sensation from the concha, posterosuperior part of external canal and the tympanic membrane. These fibres account for vesicular eruption in herpes zoster infection of the geniculate ganglion. It also brings proprioceptive sensation from the facial muscles.

brainstem at pontomedullary junction, travels through pos- terior cranial fossa and enters the internal acoustic meatus. At the fundus of the meatus (lateral most part of meatus), the nerve enters the bony facial canal, traverses the tem- poral bone and comes out of the stylomastoid foramen. Here it crosses the styloid process and divides into terminal branches. The course of the nerve (Figure 14.2) can thus be divided into three parts.
1. Intracranial part. From pons to internal acoustic meatus (15-17 mm).
2. Intratemporal part. From internal acoustic meatus to sty- lomastoid foramen. It is further divided into:
(a) Meatal segment (8-10 mm). Within internal acoustic meatus.
(b) Labyrinthine segment (4.0 mm). From fundus of meatus to the geniculate ganglion where nerve takes a turn poste- riorly forming a “genu.” The nerve in the labyrinthine segment has the narrowest diameter (0.61—0.68 mm) and the bony canal in this segment is also the narrow- est. Thus oedema or inflammation can easily compress the nerve and cause paralysis. This is also the shortest segment of the nerve.
(c) Tympanic or horizontal segment (11.0 mm). From geniculate ganglion to just above the pyramidal eminence. It lies above the oval window and below the lateral semicircular canal.
(d) Mastoid or vertical segment (13.0 mm). From the pyramid to stylomastoid foramen. Between the tympanic and mastoid segments is the second genu of the nerve.

Motor nucleus of the nerve is situated in the pons. It receives fibres from the precentral gyrus. Upper part of the nucleus which innervates forehead muscles receives fibres from both the cerebral hemispheres, while the lower part of nucleus which supplies lower face gets only crossed fibres from one hemisphere. The function of forehead is preserved in supranuclear lesions because of bilateral innervation. Facial nucleus also receives fibres from the thalamus by alternate routes and provides involuntary control to facial muscles. The emotional movements such as smiling and crying are thus preserved in supranuclear palsies because of these fibres from the thalamus (Figure 14.1).

Motor fibres take origin from the nucleus of VIIth nerve, hook round the nucleus of VIth nerve and are joined by the sensory root (nerve of Wrisberg). Facial nerve leaves the
- 3. Extracranial part. From stylomastoid foramen to the ter mination of its peripheral branches.

1. Greater superficial petrosal nerve. It arises from geniculate ganglion and carries secretomotor fibres to lacrimal gland and the glands of nasal mucosa and palate.
2. Nerve to stapedius. It arises at the level of second genu and supplies the stapedius muscle.
3. Chorda tympani. It arises from the middle of vertical segment, passes between the incus and neck of malleus, and leaves the tympanic cavity through petrotympanic fissure. It carries secretomotor fibres to submandibular and sublingual glands and brings taste from anterior two-thirds of tongue.
Figure 14.1 Forehead receives bilateral innervation and is thus saved in supranuclear paralysis. Emotional movements controlled by thalamo-nuclear fibres are also preserved.
B
Figure 14.2 (A) Course of facial nerve. Intratemporal part consists of four segments: meatal (1), labyrinthine (2), tympanic (3) and mastoid (4). (B) Branches of facial nerve on face.
4, Communicating branch. It joins auricular branch of vagus and supplies the concha, retroauricular groove, posterior meatus and the outer surface of tympanic membrane.
- 5. Posterior auricular nerve. It supplies muscles of pinna, occipital belly of occipitofrontalis and communicates with auricular branch of vagus.
6. Muscular branches to stylohyoid and posterior belly of digastric.
7. Peripheral branches. The nerve trunk, after crossing the styloid process, forms two divisions, an upper temporofacial and a lower cervicofacial, which further divide into smaller branches. These are the temporal, zygomatic, buccal, mandibular and cervical and together form pes anserinus (goosefoot). They supply all the muscles of facial expression.

It is derived from four blood vessels: (i) Anterior-inferior cerebellar artery supplies the nerve in cerebellopontine angle; (ii) labyrinthine artery, branch of anterior-inferior cerebellar artery, which supplies the nerve in internal auditory canal; (iii) superficial petrosal artery, a branch of middle meningeal artery, which supplies geniculate ganglion and the adjacent region; and (iv) stylomastoid artery, branch of posterior auricular artery, which supplies the mastoid and tympanic segment. All the arteries form an external plexus which lies in the epineurium and feeds a deeper intraneural internal plexus (Figure 14.3).

For middle ear and mastoid surgery
1. Processus cochleariformis. It demarcates the geniculate ganglion which lies just anterior to it. Tympanic segment of the nerve starts at this level.
2. Oval window and horizontal canal. The facial nerve runs
above the
(stapes)
and below
the horizontal
canal.
3. Short process of incus. Facial nerve lies medial to the short process of incus at the level of aditus.
4. Pyramid. Nerve runs behind the pyramid and the posterior tympanic sulcus.
5. Tympanomastoid suture. In vertical or mastoid segment,
nerve runs
behind this
suture.
- 6. Digastric ridge. The nerve leaves the mastoid at the anterior end of digastric ridge.
For parotid surgery (Figure 14.4)
1. Cartilaginous pointer. The nerve lies 1 cm deep and slightly anterior and inferior to the pointer. Cartilaginous pointer is a sharp triangular piece of cartilage of the pinna and "points" to the nerve.
2. Tympanomastoid suture. Nerve lies 6-8 mm deep to this suture.
3. Styloid process. The nerve crosses lateral to styloid process.
4. Posterior belly of digastric. If posterior belly of digastric
muscle is traced backwards along its upper border to its attachment to the digastric groove, nerve is found to lie between it and the
styloid process.

1. Bony dehiscence. This is the most common anomaly. Dehiscence (absence of bony cover) occurs most commonly in tympanic segment over the oval window. It also occurs near the region of geniculate ganglion or in the
Figure 14.3 Blood supply of facial nerve. (i) Cerebellopontine angle: Anterior-inferior cerebellar artery. (ii) Internal auditory canal: Labyrinthine artery. (iii) Geniculate ganglion and adjacent facial nerve: Superficial petrosal. (iv) Mastoid segment: Stylomastoid artery. Thus both carotid and vertebrobasilar systems supply the nerve and meet at labyrinthine segment.
region of
retrofacial mastoid
cells.
A
dehiscent nerve
is prone
to injury
at the
time of
surgery or
gets easily
mastoid and
middle ear
2.
Prolapse of nerve. The dehiscent nerve may prolapse over the stapes and make stapes surgery or ossicular reconstruction difficult.
3. Hump. The nerve may make a hump posteriorly near the horizontal canal making it vulnerable to injury while exposing the antrum during mastoid surgery.
4. Bifurcation and trifurcation. The vertical part of facial
nerve divides
into two
or three
branches, each
occu-
pying a separate canal and exiting through individual foramen.
5. Bifurcation and enclosing the stapes. The nerve divides
proximal to oval window—one part passing above and the other below it and then reuniting.
- 6. Between oval and round windows. Just before oval window the nerve crosses the middle ear passing between oval and round windows.
Anomalies of the nerve are more common in congenital ears; utmost care should be taken while operating cases of
microtia or other congenital conditions of the ear.
Figure 14.4 Surgical landmarks of the facial nerve in parotid surgery.

From inside out, a nerve fibre consists of axon, myelin sheath, neurilemma and endoneurium. A group of nerve fibres is enclosed in a sheath called perineurium to form a fascicle and the fascicles are bound together by epineurium (Figure 14.6).

Degree of nerve injury will determine the regeneration of nerve and its function. Earlier nerve injuries were divided into:
- 1. Neurapraxia, a conduction block, where flow of axoplasm
through the axons was partially obstructed.
2. Axonotmesis—injury to axons.
3. Neurotmesis—injury to nerve.
Sunderland classified nerve injuries into five degrees of severity based on anatomical structure of the nerve and this classification is now widely accepted.
- 1°= Partial block to flow of axoplasm; no morphological changes are seen. Recovery of function is complete (neurapraxia).
2°=
Loss of axons, but endoneurial tubes remain intact.
During recovery,
axons will
grow into
their respective
tubes, and the result is good (axonotmesis).
Injury to
endoneurium.
one tube
can grow
into another.
(neurotmesis).
Injury to
perineurium will
impair
Injury
3°=
4°=
5°=
in regeneration
to epineurium
Synkinesis addition
axons of
can to
above.
of in
fibres
(partial addition
occur
Scarring transection).
to above
(complete nerve
transection).
The first three degrees are seen in viral and inflammatory disorders while fourth and fifth are seen in surgical or accidental trauma to the nerve or in neoplasms.

These tests are useful to differentiate between neurapraxia and degeneration of the nerve. They also help to predict
prognosis and indicate time for surgical decompression of the nerve.
1. Minimal nerve excitability test. The nerve is stimulated at steadily increasing intensity till facial twitch is just noticeable. This is compared with the normal side. There is no difference between the normal and paralyzed side in conduction block. In other injuries, where degeneration sets in, nerve excitability is gradually lost. When the difference between two sides exceed 3.5 m amp, the test is positive for degeneration. Degeneration of fibres cannot be detected earlier than 48-72 h of its commencement.
2. Maximal stimulation test (MST). This test is similar to the minimal nerve excitability test but instead of measuring the threshold of stimulation, the current level which gives maximum facial movement is determined and compared with the normal side. Response is visually graded as equal, decreased or absent. Reduced or absent response with maximal stimulation indicates degeneration and is followed by incomplete recovery.
3. Electroneuronography (ENoG). It is a sort of evoked electromyography. The facial nerve is stimulated at the stylomastoid foramen and the compound muscle action
Figure 14.6 Structure of a nerve. (A) Cross section of nerve. (B) Structure of nerve fibre, longitudinal and cross-sectional views.
potentials are picked up by the surface electrodes. Supramaximal stimulation is used to obtain maximal action potentials. The responses of action potentials of the paralyzed side are compared with that of the normal side on similar stimulation and thus percentage of degenerating fibres is calculated. Studies reveal that degeneration of 90% occurring in the first 14 days indicates poor recovery of function. Faster rate of degeneration occurring in less than 14 days has a still poorer prognosis. ENoG is most useful between 4 and 21 days of the onset of complete paralysis.
4, Electromyography (EMG). This tests the motor activity of facial muscles by direct insertion of needle electrodes usu-: " . . . ally in orbicular oculi and orbicularis oris muscles and the recordings are made during rest and voluntary contraction of muscle.
In a normal resting muscle, biphasic or triphasic poten- tials are seen every 30-50 ms.

In a denervated muscle spontaneous involuntary action potentials
called fibrillation
potentials are
seen.
They appear
14-21
days nerve
after after
denervation.
oe injury,
With
_
regeneration
.
:
reinnervation polyphasic
rer fibrillation
potentials.
cal evidence
They
‘
appear of
facial potentials
°
6-12
weeks function
and thus
:
prior provide
the of
the replace
.
to clini-
earliest evidence
of recovery.
Voluntary contraction causes motor discharge. Dimin- ished or no response to voluntary contraction is seen after

Electromyography is useful in planning reanimation proce- dures.
normal or
polyphasic potentials
after
1
year of
injury indicates
isno are
that reinnervation
need for
reanimation and
is taking
intact there
potentials seen,
it indicates
place
If motor
end fibrillation
plates but
no evidence
of reinnervation
and need
for nerve
substitution.
Electrical silence indicates atrophy of motor end plates and need for
muscle transfer procedures rather than nerve substitution.
Thus ENoG and EMG are complimentary and help to prognosticate
in cases
of facial
paralysis and
in deciding
for reanimation,
i.e.
nerve substitution
muscle transposition or sling operation.

The cause may be central or peripheral. The peripheral lesion may involve the nerve in its intracranial, intratemporal or extratemporal parts. Peripheral lesions are more common and about two-thirds of them are of the idiopathic variety (Table 14.1).
versus

Sixty to seventy-five per cent of facial paralysis is due to Bell palsy. It is defined as idiopathic, peripheral facial paralysis or paresis of acute onset. Both sexes are affected with equal frequency. Any age group may be affected though incidence rises with increasing age. A positive family history is present in 6-8% of patients. Risk of Bell palsy is more in diabetics (angiopathy) and pregnant women (retention of fluid).

1. Viral infection. Most of the evidence supports the viral aetiology due to herpes simplex, herpes zoster or the Epstein— Barr virus. Other cranial nerves may also be involved in Bell palsy which is thus considered a part of the total picture of polyneuropathy.
2. Vascular ischaemia. It may be primary or secondary. Primary ischaemia is induced by cold or emotional stress. Secondary ischaemia is the result of primary ischaemia which causes increased capillary permeability leading to exudation of fluid, oedema and compression of microcirculation of the nerve.
3. Hereditary. The fallopian canal is narrow because of hereditary predisposition and this makes the nerve susceptible to early compression with the slightest oedema. Ten per cent of the cases of Bell palsy have a positive family history.
4. Autoimmune disorder. T-lymphocyte changes have been observed.

Onset is sudden. Patient is unable to close his eye. On attempting to close the eye, eyeball turns up and out (Bell phenomenon). Saliva dribbles from the angle of mouth. Face becomes asymmetrical. Tears flow down from the eye (epiphora). Pain in the ear may precede or accompany the nerve paralysis. Some complain of noise intolerance (stapedial paralysis) or loss of taste (involvement of chorda tympani). Paralysis may be complete or incomplete. Bell palsy is recurrent in 3-10% of patients.

Diagnosis is always by exclusion. All other known causes of peripheral facial paralysis should be excluded. This requires careful history, complete otological and head and neck examination, X-ray studies, blood tests such as total count, peripheral smear, sedimentation rate, blood sugar and serology.
Nerve excitability tests are done daily or on alternate days and compared with the normal side to monitor nerve degeneration.
Localizing the site of lesion (topodiagnosis) helps in establishing the aetiology and also the site of surgical decompression of nerve, if that becomes necessary.

1.
Reassurance.
- 2. Relief of ear pain by analgesics.
Figure 14.7 Facial paralysis left side. Compare with normal side.
3. Care of the eye as outlined on p. 98. Eye must be protected against exposure keratitis. 4. Physiotherapy or massage of the facial muscles gives psychological support to the patient. It has not been shown to influence recovery. Active facial movements are encouraged when there is return of some movement to the facial muscles.

¢ Steroids. Their utility has not been proved beyond doubt in carefully controlled studies. Prednisolone is the drug of choice. If patient reports within 1 week, the adult dose of prednisolone is 1 mg/kg/day divided into morning and evening doses for 5 days. Patient is seen on the fifth day. If paralysis is incomplete or is recovering, dose is tapered during the next 5 days. If paralysis remains complete, the same dose is continued for another 10 days and thereafter tapered in next 5 days (total of 20 days). Contraindications to use of steroids include pregnancy, diabetes, hypertension, peptic ulcer, pulmonary tuberculosis and glaucoma. Steroids have been found useful to prevent incidence of synkinesis, crocodile tears and to shorten the recovery time of facial paralysis. Steroids can be combined with acyclovir for Herpes zoster oticus or Bell palsy.
- © Other drugs. Vasodilators, vitamins, mast cell inhibitors and antihistaminics have not been found useful.
Surgical treatment. Nerve decompression relieves pressure on the nerve fibres and thus improves the microcirculation of the nerve. Vertical and tympanic segments of nerve are decompressed. Some workers have suggested total decompression including labyrinthine segment by postaural and middle fossa approach.

Eighty-five to ninety per cent of the patients recover fully. Ten to fifteen per cent recover incompletely and may be left with some stigmata of degeneration. Recurrent facial palsy may not recover fully. Prognosis is good in incomplete Bell palsy (95% complete recovery) and in those where clinical recovery starts within 3 weeks of onset (75% complete recovery).
Figure 14.8 Ramsay—Hunt syndrome. Note facial palsy and small vesicles in the concha of the right side.

It is also an idiopathic disorder consisting of a triad of facial paralysis, swelling of lips and fissured tongue. Paralysis may
be recurrent. Treatment is the same as for Bell palsy.
¢ Recurrent facial palsy. Recurrent facial palsy is seen in
Bell palsy
(3-10%
cases),
Melkersson syndrome,
diabetes, sarcoidosis
and tumours.
Recurrent palsy
on the
same side
may be caused by a tumour in 30% of cases.
¢ Bilateral facial paralysis. Simultaneous bilateral facial paralysis may be seen in Guillain-Barré syndrome, sarcoidosis, sickle cell disease, acute leukaemia, bulbar palsy, leprosy and some other systemic disorders.

HERPES ZOSTER OTICUS (RAMSAY-HUNT SYNDROME)
There is facial paralysis along with vesicular rash in the external auditory canal and pinna (Figure 14.8). There may also be anaesthesia of face, giddiness and hearing impairment due to involvement of Vth and VIIth nerves. Treatment is
the same as for Bell palsy.
Infections of Middle Ear (see p. 80)
Malignant Otitis Externa (see p. 52)

Fractures of temporal bone may be longitudinal, transverse or mixed (Figure 14.9). Facial palsy is seen more often in transverse fractures (50%). Paralysis is due to intraneural haematoma, compression by a bony spicule or transection of nerve. In these cases, it is important to know whether paralysis was of immediate or delayed onset. Delayed onset paralysis is treated conservatively like Bell palsy while immediate onset paralysis may require surgery in the form of decompression, re-anastomosis of cut ends or cable nerve graft (Table 14.2).

Facial nerve is injured during stapedectomy, tympanoplasty or mastoid surgery. Paralysis may be immediate or delayed
and treatment is the same as in temporal bone trauma.
Figure 14.9 (A) Longitudinal fracture runs along the axis of petrous pyramid. Typically, it starts at squamous part of temporal bone, runs through roof of external ear canal and middle ear towards the petrous apex, and to foramen lacerum. (B) Transverse fracture. It runs across the axis of petrous. Typically, it begins at foramen magnum, passes through occipital bone, jugular fossa, petrous pyramid ending in middle cranial fossa. It may pass medial, lateral or through the labyrinth.
Sometimes, nerve is paralyzed due to pressure of packing on the exposed nerve and this should be relieved first.
Operative injuries to facial nerve can be avoided if atten- tion is paid to the following:
(a)
Anatomical knowledge
of the
course of
facial nerve,
possible variations and anomalies and its surgical land- marks.
Cadaver dissections
should be
part of the training in ear surgery.
Always working
along the
course across
it.
Constant irrigation
injury.
Use when
diamond of
drilling burr
when nerve
to and
never avoid
working thermal
near the
Gentle handling
of the
nerve when
it is
nerve.
exposed, ing any pressure of instruments on the nerve.
avoid-
(b)
(c)
(d)
Frequency
Type of injury
Fracture line
Bleeding from ear
Cerebrospinal fluid otorrhoea
Structures injured
Hearing loss
Vertigo

More common (80%)
Parietal blow
Runs parallel to long axis of petrous pyramid. Starts at squamous part of temporal bone to end at
foramen lacerum
Common, due to injury to tegmen and tympanic membrane
Present, often mixed with blood
Tegmen, ossicles and tympanic membrane
Conductive
Less often; due to concussion
Less (20%), delayed onset. Nerve is injured in tympanic segment, distal to geniculate ganglion

Less common (20%)
Occipital blow
Runs across the petrous. Starts at foramen magnum or jugular foramen towards the foramen spinosum
Absent because tympanic membrane is intact.
Haemotympanum may be seen
Absent or unmanifested

Sensorineural
Severe, due to injury to labyrinth or CN VIII
Most common (50%). Immediate onset. Injury to nerve in meatal or labyrinthine segment proximal to
geniculate ganglion.
(e) Not to remove any granulations that penetrate the nerve.
(f)
Using magnification;
never to
work on
facial nerve
without an operating microscope.

Facial nerve may be injured in surgery of parotid tumours or deliberately excised in malignant tumours. Accidental injuries in the parotid region can also cause facial paralysis. Application of obstetrical forceps may also result in facial paralysis in the neonate due to pressure on the extratemporal of nerve.
part

Carcinoma of external or middle ear, glomus tumour, rhabdomyosarcoma and metastatic tumours of temporal bone, all result in facial paralysis. Facial nerve neuroma occurs anywhere along the course of nerve and produces paralysis of gradual or sudden onset. It is treated by excision and nerve grafting. High-resolution CT scan and gadoliniumenhanced MRI is very useful for facial nerve tumour.

Facial paralysis with tumour of the parotid almost always implies malignancy (see Tumours of salivary glands).

Peripheral facial paralysis is mostly of idiopathic variety but always
needs exclusion
of diabetes,
hypothyroidism, leukae-
mia, sarcoidosis,
periarteritis nodosa,
Wegener’s granuloma-
tosis, leprosy,
syphilis and
demyelinating disease.

It is
caused by
cerebrovascular accidents
thrombosis or
embolism), tumour
(haemorrhage, or
an abscess.
It causes
paralysis of
only the
lower half
of face
on the
contralateral side. Forehead movements are retained due to bilateral
innervation
of
frontalis muscle. Involuntary emotional
movements and the tone of facial muscles are also retained.

All the
muscles of
the face
on the
involved side
are para-
lyzed. Patient is unable to frown, close the eye, purse the lips or whistle.
A lesion at the level of nucleus is identified by associated paralysis of VIth nerve.
A lesion at cerebellopontine angle is identified by the pres- ence of vestibular and auditory defects and involvement of
other cranial nerves such as Vth, [Xth, Xth and XIth.
A lesion in the bony canal, from internal acoustic meatus to stylomastoid foramen, can be localized by topodiagnostic tests.
A lesion outside the temporal bone, in the parotid area, affects only
the motor
nerve.
It may
sometimes be
incomplete as
some branches
of the
nerve may
not involved in tumour or trauma.

The following tests are useful in finding the site of lesion in paralysis of lower motor neuron.
1. Schirmer test. It compares lacrimation of the two sides. A strip of filter paper is hooked in the lower fornix of each eye and the amount of wetting of strip measured. Decreased lacrimation indicates lesion proximal to the geniculate ganglion as the secretomotor fibres to lacrimal gland leave at the geniculate ganglion via greater superficial petrosal nerve.
2. reflex. reflex is lost in lesions above
Stapedial Stapedial the nerve to stapedius. It is tested by tympanometry.
3. Taste test. It can be measured by a drop of salt or sugar solution placed on one side of the protruded tongue, or by electrogustometry. Impairment of taste indicates lesion above the chorda tympani.
4, Submandibular salivary flow test. It also measures func- tion
of chorda
tympani.
Polythene tubes
are passed
into both Wharton ducts and drops of saliva counted during one
be
Figure 14.10 Topographical localization of VlIth nerve lesions. (A) Suprageniculate or transgeniculate lesion. Secretomotor fibres to the lacrimal gland leave at the geniculate ganglion and are interrupted in lesions situated at/or proximal to geniculate ganglion. (B) Suprastapedial lesions cause loss of stapedial reflex and taste but preserve lacrimation. (C) Infrastapedial lesions cause loss of taste but preserve stapedial reflex and lacrimation. (D) Infrachordal lesions cause loss of facial motor function only.
minute period. Decreased salivation shows injury above the chorda.

Peripheral facial paralysis due to any cause may result in any of the following complications:
1. Incomplete recovery. Facial asymmetry persists. Eye cannot be closed resulting in epiphora. A weak oral sphincter causes drooling and difficulty in taking food.
2. Exposure keratitis. Eye cannot be closed, tear film from the cornea evaporates causing dryness, exposure keratitis and corneal ulcer. This is worse when tear production is also affected. It can be prevented by use of artificial tears (methylcellulose drops) every 1-2 h, eye ointment and proper cover for the eye at night.
Temporary tarsorrhaphy may also be indicated. Eye closure can also be improved by using gold-weight implant sutured to the tarsal plate deep to levator palpebrae muscle.
3. Synkinesis (mass movement). When the patient wishes to close the eye, corner of mouth also twitches or vice versa. It is due to cross innervation of fibres; there is no treatment.
4. Tics and spasms. They are the result of faulty regeneration of fibres. Involuntary movements are seen on the affected side of the face.
Figure 14.11 Hemifacial spasm. Note all the facial muscles and platysma in spasm. Picture taken during paroxysm of clonic contractions.
5. Contractures. They result from fibrosis of atrophied muscles or fixed contraction of a group of muscles. They affect movements of face but facial symmetry at rest is good.
6. Crocodile tears (gustatory lacrimation). There is unilateral lacrimation with mastication. This is due to faulty regeneration of parasympathetic fibres which now supply lacrimal gland instead of the salivary glands. It can be treated by section of greater superficial petrosal nerve or tympanic neurectomy.
7. Frey's syndrome (gustatory sweating). There is sweating and flushing of skin over the parotid area during mastication. It results from parotid surgery.
8. Psychological and social problems. Drooling during eat- ing and drinking and impairment of speech cause social problems.

They are characterized by involuntary twitching of facial muscles on one or both sides.
1. Hemifacial spasm. It is characterized by repeated, uncontrollable twitchings of facial muscles on one side (Figure 14.11). It is of two types (i) essential or idiopathic, where cause is not known and (ii) secondary, where cause is acoustic neuroma, congenital cholesteatoma or glomus tumour. Many cases of hemifacial spasm are due to irritation of the nerve because of a vascular loop at the cerebellopontine angle. Microvascular decompression through posterior fossa craniotomy has met with high success rate in these cases. Idiopathic type has been treated by selective section of the branches of facial nerve in the parotid or by puncturing the facial nerve with a needle in its tympanic segment.
Botulinum toxin has been used in the affected muscle. It blocks the neuromuscular junction by preventing release of acetylcholine.
2. Blepharospasm. Twitchings and spasms are limited to orbiculars oculi muscles on both sides. The eyes are closed due to muscle spasms causing functional blindness. The cause is uncertain, but probably lies in the basal ganglia. It is treated by selective section of nerves supplying muscles around the eye on both sides.
Botulinum-A toxin injected into the periorbital muscles gives relief for 3-6 months. Injection can be repeated, if necessary.

1. Decompression. The nerve may be compressed by oedema, haematoma or a fractured bone in its intratemporal part. The bony canal is exposed and uncapped. The sheath of nerve is also slit to relieve pressure due to oedema or intraneural haematoma.
2.
End-to-end anastomosis.
This is
done when
the gap
between severed
tres.
It is
ends of
the
a
suitable procedure
nerves is
only
a
few for
extratemporal millime-
part of
the

Méniére's disease, also called endolymphatic hydrops, is a disorder of the inner ear where the endolymphatic system is distended with endolymph. It is characterized by (i) vertigo, (ii) sensorineural hearing loss, (iii) tinnitus and (iv) aural fullness.

The main pathology is distension of endolymphatic system, mainly affecting the cochlear duct (scala media) and the saccule, and to a lesser extent the utricle and semicircular canals. The dilatation of cochlear duct is such that it may completely fill the scala vestibuli; there is marked bulging of Reissner's membrane, which may even herniate through the helicotrema into the apical part of scala tympani (Figure 15.1). The distended saccule may come to lie against the stapes footplate. The utricle and saccule may show outpouchings into the semicircular canals.

The main pathology in Méniére's disease is distension of endolymphatic system due to increased volume of endolymph. This can result either from increased production of endolymph or its faulty absorption or both. Normally, endolymph is secreted by stria vascularis, fills the membranous labyrinth and is absorbed through the endolymphatic sac (see p. 10 for inner ear fluids).
The exact cause of Méniére's disease is not yet known. Various theories have been postulated (Figure 15.2).
1. Defective absorption by endolymphatic sac. Normally, endolymph is carried by the endolymphatic duct to the sac where it is absorbed. Defective absorption by the sac may be responsible for raised endolymph pressure. Experimental obstruction of endolymphatic sac and its duct also produces hydrops. Ischaemia of sac has been observed in cases of Méniére's disease undergoing sac surgery, indicating poor vascularity and thus poor absorption by the sac. Distension of membranous labyrinth leads to rupture of Reissner's membrane and thus mixing of perilymph with endolymph, which is thought to bring about an attack of vertigo.
2. Vasomotor disturbance. There is sympathetic overactivity resulting in spasm of internal auditory artery and/or its branches, thus interfering with the function of cochlear or vestibular sensory neuroepithelium. This is responsible for deafness and vertigo. Anoxia of capillaries of stria vascularis

Age and sex. Disease is commonly seen in the age group of 35-60 years. Males are affected more than females. Usually, disease is unilateral but the other ear may be affected after a few years.
Cardinal symptoms of Méniére's disease are (i) episodic vertigo, (ii) fluctuating hearing loss, (iii) tinnitus and (iv) sense of fullness or pressure in the involved ear.
1. Vertigo. It comes in attacks. The onset is sudden. Patient gets a feeling of rotation of himself or his environment. Sometimes, there is feeling of "to and fro" or "up and down" movement. Attacks come in clusters, with periods of spontaneous remission lasting for weeks, months or years. Usually, an attack is accompanied by nausea and vomiting with ataxia and nystagmus. Severe attacks may be accompanied by other symptoms of vagal disturbances such as abdominal cramps, diarrhoea, cold sweats, pallor and bradycardia. Usually, there is no warning symptom of an oncoming attack of vertigo but sometimes the patient may feel a sense of fullness in the ear, change in character of tinnitus or discomfort in the ear which herald an attack.
Some cases of Méniére's disease show Tullio phenomenon. It is a condition where loud sounds or noise produce vertigo and is due to the distended saccule lying against the stapes footplate. This phenomenon is also seen when there are three functioning windows in the ear, e.g. a
Figure
Figure 15.1 (A) Normal cochlear duct. (B) Cochlear duct is distended with endolymph pushing the Reissner's membrane into scala vestibuli.
15.2
Aetiologic factors
and symptomatology
of
Méniére’s disease (endolymphatic hydrops).
fenestration of horizontal canal in the presence of a mobile stapes.
2. Hearing loss. It usually accompanies vertigo or may precede it. Hearing improves after the attack and may be normal during the periods of remission. This fluctuating nature of hearing loss is quite characteristic of the disease. With recurrent attacks, improvement in hearing during remission may not be complete; some hearing loss being added in every attack leading to slow and progressive deterioration of hearing which is permanent.

- © Distortion of sound. Some patients complain of distorted hearing. A tone of a particular frequency may appear normal in one ear and of higher pitch in the other leading to diplacusis. Music appears discordant.
¢
Intolerance to
loud sounds.
Patients of
Méniére’s disease
can- not
tolerate amplification
of sound
due to
recruitment phenomenon.
They are
poor candidates
for hearing
aids.
3. Tinnitus. It is low-pitched roaring type and is aggravated during acute attacks. Sometimes, it has a hissing character. It may persist during periods of remission. Change in intensity and pitch of tinnitus may be the warning symptom of attack.
4. Sense of fullness or pressure. Like other symptoms, it also fluctuates. It may accompany or precede an attack of vertigo.
5. Other features. Patients of Méniére's disease often show signs of emotional upset due to apprehension of the repetition of attacks. Earlier, the emotional stress was considered to be the cause of Méniére's disease.

1. Otoscopy. No abnormality is seen in the tympanic membrane.
2.
Nystagmus.
It is
seen only
during acute
attack.
The quick
component of nystagmus is towards the unaffected ear.
3. Tuning fork tests. They indicate sensorineural hearing loss. Rinne test is positive, absolute bone conduction is reduced in the affected ear and Weber is lateralized to the better ear.

1. Pure tone audiometry. There is sensorineural hearing loss. In early stages, lower frequencies are affected and the curve is of rising type. When higher frequencies are involved curve becomes flat or a falling type (Figure 15.3).
2.
Speech audiometry.
Discrimination score
is usually
55-85%
between much
the attacks
impaired
3.
during
Special but
discrimination and
immediately audiometry
nature of
disease cochlear
tests.
and following
thus lesions,
help e.g.
acoustic the
to differentiate
neuroma ability
is an
attack.
cochlear from
(Table retro-
15.1).
(a)
Recruitment test
is positive.
- (b) SISI (short increment sensitivity index) test. SISI score is better than 70% in two-thirds of the patients (normal 15%).
(c)
Tone decay
test.
Normally, there
is decay
of less
than
20
dB.
4, Electrocochleography. It shows changes diagnostic of Méniére's disease. Normally, ratio of summating potential (SP) to action potential (AP) is 30%. In Méniére's disease, SP/AP ratio is greater than 30% (Figure 15.4).
Pure tone audiogram Speech discrimination score Roll over phenomenon Recruitment SISI score Threshold tone decay test Stapedial reflex Stapedial reflex decay (page 97) BERA, Normal = Normal 90-100% Absent Absent 0-15% 0-15 dB Present Normal Normal interval between wave I & V. Pure tone audiogram Speech discrimination score Roll over phenomenon Recruitment SISI score Threshold tone decay test Stapedial reflex Stapedial reflex decay (page 97) BERA, Cochlear lesion = Sensorineural hearing loss Below 90% Absent Present Over 70% Less than 25 dB Present Normal Normal interval between wave I & V. Pure tone audiogram Speech discrimination score Roll over phenomenon Recruitment SISI score Threshold tone decay test Stapedial reflex Stapedial reflex decay (page 97) BERA, Retrocochlear lesion = Sensorineural hearing loss Very poor Present Absent 0-20% Above 25 dB Absent Abnormal Wave V delayed or absent

5. Caloric test. It shows reduced response on the affected side in 75% of cases. Often, it reveals a canal paresis on the affected side (most common) but sometimes there is directional preponderance to healthy side or a combination of both canal paresis on the affected side and directional preponderance on the opposite side.
of fullness in the ear. The test has a diagnostic and prognostic value. These days, glycerol test is combined with electrocochleography.
6. Glycerol test. Glycerol is a dehydrating agent. When given orally, it reduces endolymph pressure and thus causes an improvement in hearing.
Patient is given glycerol (1.5 mL/kg) with an equal amount of water and a little flavouring agent or lemon juice. Audiogram and speech discrimination scores are recorded before and 1-2 h after ingestion of glycerol. An improvement of 10 dB in two or more adjacent octaves or gain of 10% in discrimination score makes the test positive. There is also improvement in tinnitus and in the sense

1. Cochlear hydrops. Here, only the cochlear symptoms and signs of Méniére's disease are present. Vertigo is absent. It is only after several years that vertigo will make its appearance. It is believed that in these cases, there is block at the level of ductus reuniens, thereby confining the increased endolymph pressure to the cochlea only (Figure 15.5).
2. Vestibular hydrops. Patient gets typical attacks of episodic vertigo while cochlear functions remain normal. It is only
with time that a typical picture of Méniére's disease will develop. Many of the cases of vestibular Méniére's disease are labelled "recurrent vestibulopathy" as endolymphatic hydrops could not be demonstrated in the study of temporal bones in such cases.
3. Drop attacks (Tumarkin's otolithic crisis). In this, there is a sudden drop attack without loss of consciousness. There is no vertigo or fluctuations in hearing loss. Patient gets a feeling of having been pushed to the ground or poleaxed. It is an uncommon manifestation of Méniére's disease and occurs either in the early or late course of disease. Possible mechanism is deformation of the otolithic membrane of the utricle or saccule due to changes in the endolymphatic pressure.
4, Lermoyez syndrome. Here symptoms of Méniére's disease are seen in reverse order. First there is progressive deterioration of hearing, followed by an attack of vertigo, at which time the hearing recovers.

Méni€re's disease is an idiopathic condition while Méniére's syndrome, though resembling Méniére's disease clinically (episodic vertigo, fluctuating hearing loss, tinnitus and ear fullness), results from a variety of conditions such as trauma (head injury or ear surgery), viral infections (following measles or mumps), syphilis (congenital or late acquired), Cogan's syndrome, otosclerosis or autoimmune disorders. It is also called secondary Méniére's disease.

Committee on Hearing and Equilibrium of the American Acad- emy of Otolaryngology—Head and Neck Surgery (AAOHNS) classified the diagnosis of Méniére’s disease as follows:
1. Certain. Definite Méniére’s histopathology. disease confirmed by
2.
Definite.
Two
or
more
definitive
episodes
of
vertigo lasting 20 min or longer.
(a) Audiometrically documented hearing loss on at least
one occasion.
(b) Tinnitus or aural fullness in the affected ear.
(c) All other causes excluded.
3. Probable
(a) One definitive episode of vertigo.
(b) Audiometrically documented hearing loss on at least
(c) Tinnitus or aural fullness in the treated ear.
(d) Other causes excluded.
4. Possible
(a)
Episodic
vertigo
of
Méniére’s
type
without
docu-
mented hearing loss (vestibular variant) or
(b) Sensorineural hearing loss, fluctuating or fixed, with disequilibrium but without definitive episodes (cochlear variant).
(c) Other causes excluded.

This can be done in certain and definite cases of Méniére's dis- ease. It is based on average of the pure tone thresholds at 0.5,
1, 2, 3 kHz (rounded to nearest whole) of the worst audiogram during interval of 6 months before treatment (see Table 15.2).

1. Reassurance. Patient anxiety can be relieved by reassurance and by explaining the true nature of disease. This is particularly important in acute attack.
2. Cessation of smoking. Nicotine causes vasospasm. Smoking should be completely stopped. For some patients, this may be the only treatment necessary.
3. Low salt diet. Patient should take salt-free diet as far as possible. No extra salt should be permitted. Salt intake should not exceed 1.5-2.0 g/day.
4. Avoid excessive intake of water.
5. Avoid over-indulgence in coffee, tea and alcohol.
6. Avoid stress and bring a change in lifestyle. Mental relax- ation exercises and yoga are helpful to decrease stress.
7. Avoid activities requiring good body balance. As the attack of Méniére's disease is abrupt, sometimes with no warning symptom, professions such as flying, underwater diving or working at great heights should be avoided.

During the acute attack, there is severe vertigo with nausea and vomiting. Patient is apprehensive. Head movements provoke giddiness. Therefore, treatment would consist of:
1.
Reassurance and
psychological support
to allay
worry and
anxiety.
104
2. Bed rest with head supported on pillows to prevent excessive movements.
3. Vestibular sedatives to relieve vertigo. They should be administered intramuscularly or intravenously, if vomiting precludes oral administration. Drugs useful in acute attack are dimenhydrinate (Dramamine), promethazine theoclate (Avomine) or prochlorperazine (Stemetil).
Diazepam (Valium or Calmpose) 5-10 mg may be given intravenously. It has a tranquillizing effect and also suppresses the activity of medial vestibular nucleus.
In some patients, acute attack can be stopped by atropine, 0.4 mg, given subcutaneously.

- (a) Inhalation of carbogen (5% COxg with 95% Og). It is a good cerebral vasodilator and improves labyrinthine circulation.
(b) Histamine drip. Histamine diphosphate, 2.75 mg dissolved in 500 mL of glucose, given as i.v. drip at a slow rate is also a good vasodilator and helps to control acute attacks. Adverse effects of histamine include tachycardia, disturbances of cardiac rhythm, hypotension, hyperthermia and bronchospasm. It should not be used on empty stomach and is contraindicated in asthmatics.

When patient presents after the acute attack, the treatment consists of:
1. Vestibular sedatives. Prochlorperazine (Stemetil) 10 mg, thrice a day, orally for two months and then reduced to 5 mg thrice a day for another month.
2. Vasodilators. Nicotinic acid, 50 mg, is taken about an hour before meals thrice a day. The dose can be increased slowly to achieve flushing of skin.
Betahistine (Vertin) 8-16 mg, thrice a day, given orally, also increases labyrinthine blood flow by releasing histamine in the body.
3. Diuretics. Sometimes, diuretic furosemide, 40 mg tablet, taken on alternate days with potassium supplement helps to control recurrent attacks, if not controlled by vasodilators or vestibular sedatives.
4. Propantheline bromide (Probanthine), 15 mg, thrice a day, can be given alone or in combination with vasodilator and is quite effective.
5. Elimination of allergen. Sometimes, a food or inhalant allergen is responsible for such attacks. It should be found and eliminated or desensitization done.
6. Hormones. Investigations should be directed to find any endocrinal disorder such as hypothyroidism, and appropriate replacement therapy given. Control of stress by change in lifestyle is important to prevent recurrent attacks. About 80% of the patients can be effectively managed by medical therapy alone.
INTRATYMPANIC GENTAMICIN THERAPY (CHEMICAL LABYRINTHECTOMY)
Gentamicin is mainly vestibulotoxic. It has been used in daily or biweekly injections into the middle ear. Drug is
absorbed through the round window and causes destruction of the vestibular labyrinth. Total control of vertigo spells has been reported in 60-80% of patients with some relief from symptoms in others. Hearing loss, sometimes severe and profound, has been reported in 4—30% of patients treated with this mode of therapy.

It is a small wick made of polyvinyl acetate and measures 1 mm x 9 mm. It is meant to deliver drugs from external canal to the inner ear and thus avoid repeated intratympanic injections. It requires a tympanostomy tube (grommet) to be inserted into the tympanic membrane and the wick is passed through it. When soaked with drug, wick delivers it to the round window to be absorbed into the inner ear. It has been used to deliver steroids in sudden deafness and gentamicin to destroy vestibular labyrinth in Méniére's disease.

It is used only when medical treatment fails.
1. Conservative procedures. They are used in cases where vertigo is disabling but hearing is still useful and needs to be preserved. They are:
- (a) Decompression of endolymphatic sac.
(b) Endolymphatic shunt operation. A tube is put, connecting endolymphatic sac with subarachnoid space, to drain excess endolymph.
(c)
Sacculotomy
(Fick’s operation).
It is
puncturing the
sac- cule
with
a
needle saccule
lies through
close penetrated.
stapes to
stapes
Cody’s
a
stainless steel
tack would
cule tack
cause when
it claimed
A
distended footplate
tack and
can procedure
consists through
be easily
of placing
the stapes
periodic gets
decompression distended.
to have
be reproduced
The of
the
Both shown
these operations
good by
sacculotomy others
is results
instead of
saccule, drained
into procedure
sac- were
but they
could thus
abandoned.
similar cochlear
the not
Cochleo- procedure
in which,
duct perilymph
is is
punctured
(otic-periotic performed
with and
shunt).
a
curved needle
The passed
round window
to puncture
cochlear duct.
(d) Section of vestibular nerve. The nerve is exposed by retrosigmoid or middle cranial fossa approach and selectively sectioned. It controls vertigo but preserves hearing.
(e) Ultrasonic destruction of vestibular labyrinth. Cochlear function is preserved.
2. Destructive procedures. They totally destroy cochlear and vestibular function and are thus used only when cochlear function is not serviceable.
- ¢ Labyrinthectomy. Membranous labyrinth is completely destroyed either by opening through the lateral semicircular canal by transmastoid route or through the oval window by a transcanal approach. This gives relief from the attacks of vertigo.

3. Intermittent low-pressure pulse therapy [Meniett device therapy (Figure 15.6)]. It is observed that intermittent positive pressure delivered to inner ear fluids brings relief
from the symptoms of Méniére's disease. Not only there is improvement in vertigo, tinnitus and ear fullness, but hearing may also improve. Intermittent positive pressure waves can be delivered through an instrument called Meniett device which has been approved by FDA. A prerequisite for such a therapy is to perform a myringotomy and insert a ventilation tube so that the device when coupled to the external ear canal can deliver pressure waves to the round window membrane via the ventilation tube. Pressure waves pass through the perilymph and cause reduction in endolymph pressure by redistributing it through various communication channels such as the endolymphatic sac or the blood vessels (Figure 15.6). Some believe they regulate secretion of endolymph by the stria vascularis.
Patient can self-administer the treatment at home. It may require a few months before complete remission of disease is obtained. Meniett device therapy has been recommended for patients who have failed medical treatment and the surgical options are being considered.

Of all the cases of ear carcinoma, 85% occur on the pinna,
10% in the external canal and 5% in the middle ear.
Tumours of the external ear may arise from the pinna or
external auditory canal (Table 16.1).

1. Preauricular sinus or cyst. This results from faulty union of hillocks of the first and second branchial arches during the development of pinna. Preauricular sinus presents as a small opening in front of the crus of helix. It has a branching tract lined by squamous epithelium which when blocked results in a retention cyst. Patient usually presents with a cyst which is infected. Surgery is indicated if there is unsightly swelling or infection. Cyst or sinus tract must be excised completely to avoid recurrence.
2. Sebaceous cyst. Common site is postauricular sulcus or below and behind the ear lobule. Treatment is total surgical excision.
3. Dermoid cyst. Usually presents as a rounded mass over the upper part of mastoid behind the pinna.
4. Keloid. It often follows trauma such as piercing the ear lobule for ornaments or a surgical incision (Figure 16.1). There is a genetic susceptibility. Black races are more often affected. Keloid presents as a pedunculated tumour. Treatment is
surgical excision with injection of triamcinolone into the sur gical site or immediate postoperative radiation of 300 rads.
5. Haemangiomas. They are congenital tumours often seen in childhood. Other parts of face and neck may also be involved. They are of two types:
(a)
Capillary haemangioma.
It is
a
mass of
capillary-sized blood vessels and may present as a "port-wine stain." It
does not regress spontaneously.
Cavernous haemangioma (also called strawberry tumour).
of endothelial-lined
spaces filled
with blood.
It increases
rapidly during
the first
year but
regresses there-
after and may completely disappear by the fifth year.
6. Papilloma (wart). It may present as a tufted growth or flat grey plaque and is rough to feel. It is viral in origin. Treatment is surgical excision or curettage with cauterization of its base.

7. Cutaneous horn. It is a form of papilloma with heaping up of keratin and presents as horn-shaped tumour. It is often seen at the rim of helix in elderly people. Treatment is surgical excision.
8. Keratoacanthoma. It is a benign tumour clinically resembling a malignant one. It presents as a raised nodule with a
Figure 16.1
Keloid following piercing of ear lobule for an ear ring.
(b)
central crater. Initially, it grows rapidly but slowly regresses leaving a scar. Treatment is excision biopsy.
9. Neurofibroma. It presents as a nontender, firm swelling and may be associated with von Recklinghausen disease. Treatment is surgical excision, if tumour occludes ear canal or presents a cosmetic problem.

1. Squamous cell carcinoma. The site of predilection is the helix (Figure 16.2). It may present as a painless nodule or an ulcer with raised everted edges and indurated base. Metastases to regional lymph nodes occur very late. Disease is more common in males in their fifties who had prolonged exposure to direct sunlight. Faircomplexioned people are more prone. Treatment. Small lesions with no nodal metastases are excised locally with 1 cm of healthy area around it. Larger lesions of the pinna or those coming within 1 cm of external auditory canal and lesions with nodal metastases may require total amputation of the pinna, often with en bloc removal of parotid gland and cervical lymph nodes.
2. Basal cell carcinoma. The common sites are the helix and the tragus. It is more common in men beyond 50 years of age. It presents as a nodule with central crust, removal of which results in bleeding. Ulcer has a raised or beaded edge. Lesion often extends circumferentially into the skin but may penetrate deeper, involving cartilage or bone. Lymph node metastases usually do not occur.
Treatment. Superficial lesions, not involving cartilage, can be irradiated and cosmetic deformity avoided. Lesions involving cartilage may require surgical excision as in cases of squamous cell carcinoma.
3. Melanoma. It may occur anywhere over the auricle. It is more common in men of light complexion who are exposed to sun. Metastases are seen in 16-50% of the cases. Treatment. Superficial melanoma, less than 1 cm in diameter, situated over the helix, is managed by wedge resection and primary closure.
Figure 16.2 Squamous cell carcinoma of pinna.
Superficial melanoma, larger than | cm, infiltrative melanomas, melanoma of posterior auricular surface or concha and all recurrent melanomas are treated by resection of pinna, parotidectomy and radical neck dissection.

1. Osteoma. It arises from cancellous bone and presents as a single, smooth, bony, hard, pedunculated tumour, often arising from the posterior wall of the osseous meatus, near its outer end (Figure 16.3). Treatment is surgical removal by fracturing through its pedicle or removal with a drill.
2. Exostoses. They are multiple and bilateral, often presenting as smooth, sessile, bony swellings in the deeper part of the meatus near the tympanic membrane. They arise from compact bone. Exostosis is often seen in persons exposed to entry of cold water in the meatus as in divers and swimmers. Males are affected three times more than females.
Treatment. When small and asymptomatic, no treatment is necessary. Larger ones, which impair hearing or cause retention of wax and debris, may be removed with high speed drill to restore normal sized meatus. Exostoses may extend deeply and lie in close relation to the facial nerve. Therefore, use of gouge and hammer should be avoided.
3. Ceruminoma. It is a tumour of modified sweat glands which secrete cerumen. It presents as a smooth, firm, skincovered polypoid swelling in outer part of the meatus, generally attached to the posterior or inferior wall. It obstructs the meatus leading to retention of wax and debris. Malignant type outnumbers the benign by 2:1 ratio.
Treatment. Tumour has a tendency to recur, therefore wide surgical excision should be done and patient regularly followed up. Some of the ceruminomas are malignant and if there is any suspicion of malignancy on histology, postoperative radiotherapy should be given.
Figure 16.3
108
=
SECTION!
—
DISEASES
OF
EAR
4. Sebaceous adenoma. It arises from sebaceous glands of the meatus and presents as a smooth, skin-covered swelling in the outer meatus. Treatment is surgical excision. 5. Papilloma. Similar to the one seen on the pinna.

1.
Squamous cell
carcinoma.
of long-standing
the meatus
or be
Most ear
a
secondary often,
It may
it is
arise extension
seen in
primarily from
cases from
the middle
ear carcinoma.
Presenting symptoms are blood staining of hitherto mucopurulent or purulent discharge and severe earache.
Examination may show an ulcerated area in the meatus or a bleeding polypoid mass or granulations. Facial nerve may
be paralyzed because of local extension of disease through posterior meatal wall or its spread into the middle ear. Regional lymph nodes (preauricular, postauricular, infraauricular and upper deep cervical) may be involved. Treatment is en bloc wide surgical excision with postoperative radiation.
2. Basal cell and adenocarcinomas. They can rarely arise from the meatus. Clinical picture is similar to that of squamous cell variety. Diagnosis is made only on biopsy. Treatment is wide surgical excision and postoperative radiation.
3. Malignant ceruminoma. Malignant type is twice as common as benign.
4, Malignant melanoma. Rare tumour.

Tumours of middle ear and mastoid can be divided into:
1. PRIMARY TUMOURS
(a) Benign: Glomus tumour
(b) Malignant: Carcinoma, sarcoma
2. SECONDARY TUMOURS
- (a) From adjacentareas, e.g. nasopharynx, external meatus and the parotid.
(b)
Metastatic, e.g.
from carcinoma
of bronchus,
breast, kidney, thyroid, prostate and gastrointestinal tract.

It is the most common benign neoplasm of middle ear and is so-named because of its origin from the glomus bodies. The latter resemble carotid body in structure and are found in the dome of jugular bulb or on the promontory along the course of tympanic branch of IXth cranial nerve (Jacobson's nerve). The tumour consists of paraganglionic cells derived from the neural crest.

The tumour is often seen in the middle age (40-50 years).
Females are affected five times more.
It is
a
benign, nonencapsulated
but neoplasm.
Its rate
of growth
is very
may pass
before there
is any
extremely slow
change vascular
from the
years initial
symp- toms. Tumour is locally invasive.
Microscopically, it shows masses or sheets of epithelial cells which have large nuclei and a granular cytoplasm. There is abundance of thin-walled blood sinusoids with no contractile muscle coat, accounting for profuse bleeding from the tumours.
For purposes of diagnosis and treatment, two types are differentiated.
1. Glomus jugulare. They arise from the dome of jugular bulb, invade the hypotympanum and jugular foramen, causing neurological signs of IXth to XIIth cranial nerve involvement. They may compress jugular vein or invade its lumen.
2. Glomus tympanicum. They arise from the promontory of the middle ear and cause aural symptoms, sometimes with facial paralysis.
1. Tumour may initially fill the middle ear and later per- forate through the tympanic membrane to present as a vascular polyp.
It may invade labyrinth, petrous pyramid and the mastoid. yn
It may invade jugular foramen and the base of skull, caus- ing [Xth to XIIth cranial nerve palsies.
4. By spread through eustachian tube, it may present in the nasopharynx.
5. It may spread intracranially to the posterior and middle cranial fossae.
6. Metastatic spread to lungs and bones is rare, but seen in 4% of cases. Metastatic lymph node enlargement can also
occur,

In 90% of cases, symptoms pertain to the ear.
1. When tumour is intratympanic. Earliest symptoms are hearing loss and tinnitus. Hearing loss is conductive and slowly progressive. Tinnitus is pulsatile and of swishing character, synchronous with pulse and can be temporarily stopped by carotid pressure.
Otoscopy shows a red reflex through intact tympanic membrane. "Rising sun" appearance is seen when tumour arises from the floor of middle ear. Sometimes, tympanic membrane appears bluish and may be bulging.
"Pulsation sign" (Brown sign) is positive, i.e. when ear canal pressure is raised with Siegel's speculum, tumour pulsates vigorously and then blanches; reverse happens with the release of pressure.
2.
When tumour
presents as
a
polyp.
to hearing
loss and
tinnitus, there
is history
of profuse
bleeding from
the ear
either spontaneously
or on
attempts to
clean it.
Dizziness or vertigo and facial paralysis may appear. Ear- ache
is less
common than
in carcinoma
of the
external and
middle ear, and helps to differentiate them from it.
Otorrhoea may occur due to secondary infection and the condition
may simulate
otitis media
with polyp.
Examination reveals a red, vascular polyp filling the meatus. It bleeds readily and profusely on manipulation or at biopsy.
3. Cranial nerve palsies. This is a late feature appearing several years after aural symptoms. IXth to XIIth cranial nerves may be paralyzed. There is dysphagia and hoarseness with unilateral paralysis of the soft palate, pharynx
110
(EX,
X)
and vocal
cord
(X)
with weakness
of the
trapezius and sternomastoid muscles (XI) and atrophy of half of tongue (XII).
Tumour may present as a mass over the mastoid or in the nasopharynx.
Signs of intracranial involvement may also occur.
4,
Audible bruit.
At all
stages, auscultation
with stethoscope

over the
mastoid may
reveal systolic
bruit.
Some glomus tumours secrete catecholamines and produce symptoms like headache, sweating, palpitation, hypertension and anxiety, and require further investigations.
5. Rule of 10s. Remember that 10% of the tumours are familial, 10% multicentric and up to 10% functional, i.e. they secrete catecholamines.

In addition to thorough history and physical examination, the patient is checked-up to find out the extent of tumour, other associated glomus tumours and serum levels of catecholamines or their breakdown products in urine (vanillylmandelic acid, metanephrine, etc.). Investigations include:
1. Computed tomography (CT) scan head. Using bone window, 1 mm thin sections are cut. It helps to distinguish glomus tympanicum from the glomus jugulare tumour by identification of caroticojugular spine which is eroded in the latter. CT scan also helps to differentiate it from the aberrant carotid artery, high or dehiscent jugular bulb.
2. MRI. It gives soft tissue extent of tumour. Magnetic resonance angiography and venography further help to delineate invasion of jugular bulb and vein or compression of the carotid artery.
3. CT head and MRI combined together provide an excellent preoperative guidance in the differential diagnosis of petrous apex lesions.
4. Four-vessel angiography. It is necessary when CT head shows involvement of jugular bulb, carotid artery or intradural extension. It also helps to delineate any other glomus tumour (as they may be multiple), find the feeding vessels or embolization of tumour if required.
5. Brain perfusion and flow studies. They are necessary when tumour is pressing on internal carotid artery. If the case needs surgery, brain perfusion and adequacy of contralateral internal carotid artery and circle of Willis can be assessed. If needed, xenon blood flow and isotope studies are done for precise blood flow, and the risk of stroke and need for surgical replacement of internal carotid artery.
6.
Embolization.
In large
tumours, embolization
of feeding
vessels
1-2
days before
operation helps
to reduce
blood loss.
7. Biopsy. Preoperative biopsy of the tumour for diagnosis is never done. Clinical and radiologic features are very characteristic to make diagnosis. Tumour is very vascular and bleeds profusely. There is also likelihood of injuring the high jugular bulb or aberrant internal carotid artery if diagnosis is mistaken.

It consists of:
1. Surgical removal.
2. Radiation.
3. Embolization.
- 4. Combination of the above techniques.
SURGICAL APPROACHES TO GLOMUS TUMOURS
1. Transcanal approach. Suited for limited glomus tympanicum tumour where entire circumference of the tumour is visible, only tympanotomy will suffice.
2. Hypotympanic approach. Suited for tumours limited to promontory with extension to hypotympanum but not into the mastoid. A superiorly based tympanomeatal flap is raised by postauricular approach. Bony inferior tympanic ring is drilled away to see the lower limit of tumour.
3.
Extended facial
recess approach.
Used for
glomus tym-
panicum extending
into mastoid
but not
into the
jugular bulb.
If extensive,
modified radical
operation is
done.
4. Mastoid-neck approach. Used for glomus jugulare tumours not extending to internal carotid artery, posterior cranial fossa or neck.
- 5. Infratemporal fossa approach of Fisch. Used for large glomus jugulare tumours.
6.
Transcondylar approach.
Used for
tumours extending
towards foramen
magnum.
jugulare tumours.
tion with
Usually
It they
gives exposure
Radiation approach
treatment reduce
its vascularity
for are
to condyle
does not
and inoperable
surgery recurrent
glomus craniocervical
and cure
arrest tumours,
or for
its residual
older junc-
jugular tubercle.
the growth.
tumours, individuals
tumour but
may
Radiation is
recurrences where
extensive skull
used after
base surgery
is not
Embolization is used to reduce the vascularity of tumour before surgery or is the sole treatment in the inoperable patients who have received radiation.

It is a rare condition, there being one case in 20,000 new patients examined, but it is the commonest primary middle ear malignancy.

It affects age group of 40-60 years and is slightly more common in females. Most cases (75%) have associated long-standing ear discharge. Chronic irritation may be the causative factor in such cases. Some cases are seen in radical mastoid cavities. Primary carcinoma of mastoid air cells is also seen in radium dial painters.

Tumour may arise primarily from middle ear or be an extension of carcinoma of the deep meatus. Squamous cell variety
Figure 17.1 Squamous cell carcinoma of middle ear and mastoid in a 3-year-old child. He also had facial paralysis.
is by far the most common (Figure 17.1). Adenocarcinoma may occasionally be seen; it arises from the glandular elements of middle ear.
Spread of tumour. To begin with, carcinoma destroys ossicles, facial canal, internal ear, jugular bulb, carotid canal or deep bony meatus and mastoid. It may spread in petrous pyramid towards its apex. Dura is usually resistant. It may spread to the parotid gland, temporomandibular joint, infratemporal fossa and down the eustachian tube to nasonode occurs late.
pharynx. Lymph enlargement

Patient often presents with clinical picture simulating chronic suppurative otitis media. However, the following features in age group of 40-60 years may arouse suspicion of malignancy:
- 1. Chronic foul-smelling discharge especially when blood stained.
Pain which is usually severe and comes at night. bo
Facial palsy. go
Friable, haemorrhagic granulations or polyp. yA
Appearance of or increase in hearing loss or vertigo. Ot

Definitive diagnosis is made only on biopsy. Extent of disease is judged by clinical and radiological examination. CT scan and angiography are useful in the assessment of disease.
Figure 17.2 A 4-year-old child with rhabdomyosarcoma of the right middle ear and mastoid. He also had facial palsy on the same side.

A combination of surgery and radiotherapy gives better results. Surgery consists of radical mastoidectomy, subtotal or total petrosectomy depending on the extent of tumour. Radiotherapy alone is given as a palliative measure when tumour involves cranial nerves ([Xth to XIIth) or spreads into the cranial cavity or the nasopharynx.

¢ Rhabdomyosarcoma. It is a rare tumour, mostly affecting children. It arises from the embryonic muscles tissue or the pluripotential mesenchyme. In early stages, it mimics chronic suppurative otitis media with ear discharge, polyp or granulations. Facial palsy occurs early (Figure 17.2). Diagnosis is made only on biopsy. Prognosis is poor. A combination of radiation and chemotherapy is the treatment of choice. Surgery is done in selected localized lesions.
¢ Other sarcomas. Osteosarcoma, lymphoma, fibrosarcoma and chondrosarcoma are rare. Distant metastases are seen in the lungs or bone. Prognosis is poor.

Tumours of external auditory meatus, parotid gland or nasopharynx may invade middle ear cleft either through the or bone erosion.
preformed pathways Sometimes, temporal bone is the site of distant metastases in advanced cases of carcinoma of the breast, bronchus, prostate, kidney or gastrointestinal tract.

Acoustic
neuroma
is
also
known
as
vestibular schwannoma,
neurilemmoma or eighth nerve tumour.

Acoustic neuroma constitutes 80% of all cerebellopontine angle tumours and 10% of all the brain tumours.

2. Cochleovestibular symptoms. They are the earliest symptoms when tumour is still intracanalicular and are caused by pressure on cochlear or vestibular nerve fibres or on the internal auditory artery. Progressive unilateral sensorineural hearing loss, often accompanied by tinnitus, is the presenting symptom in majority of cases. There is marked difficulty in understanding speech, out of proportion to the pure tone hearing loss. This feature is characteristic of acoustic neuroma. Some patients may get sudden hearing loss. Vestibular symptoms are imbalance or unsteadiness. True vertigo is seldom seen.
It is a benign, encapsulated, extremely slow-growing tumour of the VIIIth nerve. Microscopically, it consists of elongated spindle cells with rod-shaped nuclei lying in rows or palisades. Bilateral tumours are seen in patients with neurofibromatosis.

The tumour almost always arises from the Schwann cells of the vestibular, but rarely from the cochlear division of VIIth nerve within the internal auditory canal (Figure 18.1). As it expands, it causes widening and erosion of the canal and then appears in the cerebellopontine angle. Here, it may grow anterosuperiorly to involve Vth nerve or inferiorly to involve the [Xth, Xth and XIth cranial nerves. In later stages, it causes displacement of brainstem, pressure on cerebellum and raised intracranial tension (Figure 18.2). The growth of the tumour is extremely slow and the history may extend over several years.

Depending on the size, the tumour is classified as:
1. Intracanalicular (when it is confined to internal auditory canal)
Small size (up to 1.5 cm)
Medium size (1.5-4 cm) ge
Large size (over 4 cm)
bo

1. Age and sex. Tumour is mostly seen in age group of 40-60 years. Both sexes are equally affected.
112

¢ Vth nerve. This is the earliest nerve to be involved. There is reduced corneal sensitivity, numbness or paraesthesia of face. Involvement of this nerve indicates that the tumour is roughly 2.5 cm in diameter and occupies the cerebellopontine angle.
e Vilth nerve. Sensory fibres are affected early. There is hypoaesthesia of posterior meatal wall (Hitzelberger's sign), loss of taste (as tested by electrogustometry) and reduced lacrimation on Schirmer test. Motor fibres are more resistant and are affected late. Delayed blink reflex may be an early manifestation.
e [Xth and Xth nerves. There is dysphagia and hoarseness due to palatal, pharyngeal and laryngeal paralysis.
© Other cranial nerves. XIth and XUth, UIrd, [Vth and VIth are affected when tumour is very large.
4.
Brainstem involvement.
There is
ataxia, weakness
and numb-
ness of
the arms
and legs
with exaggerated
tendon reflexes.
They are
seen when
long motor
tracts are
involved.
- 5. Cerebellar involvement. Pressure symptoms on cerebellum are seen in large tumours. This is revealed by fingernose test, knee-heel test, dysdiadochokinesia, ataxic gait and inability to walk along a straight line with tendency to fall to the affected side.
6.
Raised intracranial tension. This is also a late feature.
There is
headache, nausea,
vomiting, diplopia
due to
VIth- nerve
involvement and
papilloedema with
blurring of
vision.

Attempts should be made to diagnose the tumour in its otological phase when it is still intracanalicular. This is possible when all cases of unilateral sensorineural hearing loss with tinnitus or imbalance are carefully evaluated.
1. Audiological tests. See Table 15.1 for difference between cochlear and retrocochlear lesions.
(a) Pure tone audiometry will show sensorineural hearing loss, more marked in high frequencies.
(b) Speech audiometry shows poor speech discrimination and this is disproportionate to pure tone hearing loss. Roll-over phenomenon, i.e. reduction of discrimination score when loudness is increased beyond a particular limit is most commonly observed.
(c) Recruitment phenomenon is absent.
(d) Short Increment Sensitivity Index (SISI) test will show a score of 0-20% in 70-90% of cases.
(e) Threshold tone decay test shows retrocochlear type of lesion.

3.
Vestibular tests.
Caloric test
will show
absent response
small, in
caloric test
4.
96%
of may
be tests.
patients.
normal.
Complete examination
diminished
When or
tumour is
very of
cranial nerves,
cerebellar functions,
brainstem signs
of pyramidal
Figure 18.1 Inner aspect of lateral end of internal auditory canal with
and sensory tracts should be done. Fundus is examined for blurring of disc margins or papilloedema.

(a)
Plain
X-rays
(transorbital, tovertical
views)
However,
Computed even
0.5
by
Stenver’s,
Towne’s give
positive findings
small intracanalicular
tomography and
in
80%
tumours
(CT)
scan.
cm into
of are
A
the
a
CT
scan.
the submen-
patients.
not tumour
posterior
If intrameatal
replaced combined
fossa with
tumour earlier
and myodil
MRI
can methods
of meatography.
with gadolinium
and is
contrast.
the roma.
gold standard
detected.
that can
projects be
intrathecal detected
air, even
be detected.
CT
scan has
pneumoencephalography
It is
superior to
CT
scan for
diagnosis
Intracanalicular tumour,
of acoustic
of even
a
neu- few
millime- tres,
can be
easily diagnosed
by this
method.
- (d) Vertebral angiography. This is helpful to differentiate acoustic neuroma from other tumours of cerebellopontine angle when doubt exists.
6. Evoked response audiometry (BERA). It is very useful in the diagnosis of retrocochlear lesions. In the presence of VIIth nerve tumour, a delay of > 0.2 ms in wave V between two ears is significant (see p. 26).
7. CSF examination. Protein level is raised. Lumbar puncture is usually avoided. Important tests for work-up of acoustic neuroma are given below:
Pure tone audiometry.
Speech discrimination score.
Roll-over curve.
- Stapedial reflex decay.
structures passing through different areas.
Evoked response audiometry.
MRI with contrast.
Figure 18.2 Acoustic neuroma and its expansion. (A) Intracanalicular. (B) Tumour extending into cerebellopontine angle. (C) Tumour pressing
(b)
(c)

Acoustic neuroma should be differentiated from the cochlear pathology (i.e. Méniére's disease) and other cerebellopontine angle tumours, e.g. meningioma, primary cholesteatoma and arachnoidal cyst (Table 18.1).

Surgical removal of the tumour is the treatment of choice. Surgical approach will depend upon the size of tumour.
The various approaches are:
- 1. Middle cranial fossa approach.
2. Translabyrinthine approach.
- 3. Suboccipital (retrosigmoid) approach.
4. Combined translabyrinthine-suboccipital approach.

Conventional radiotherapy by external beam has no role in the treatment of acoustic neuromas due to low tolerance of the central nervous system to radiation.
e Acoustic neuroma
- ¢ Meningioma
© Epidermoid (cholesteatoma)
- e Arachnoid cyst
e Schwannoma of other cranial nerves (e.g. CN V >VII>IX,
X, Xl)
e Aneurysm
- © Glomus tumour
e Metastasis
X-knife or Gamma knife surgery. It is a form of stereotactic radiotherapy where radiation energy is converged on the tumour, thus minimizing its effect on the surrounding normal tissue. This causes arrest of the growth of the tumour and also reduction in its size. It can be used in patients who refuse surgery or have contraindications to surgery or in those with a residual tumour.
X-knife surgery is done through linear accelerator and knife through a Cobalt-60 source.
gamma
Cyber knife. Itis an improvement over the above. It is totally frameless and more accurate. It uses real-time image guidance technology through computer-controlled robotics.

Hearing loss in a child may develop from causes before birth (prenatal), during birth (perinatal) or thereafter (postnatal).

They may pertain to the infant or the mother.
1.
Infant factors.
An infant
may be
born with
inner ear
anomalies due
to genetic
or nongenetic
causes.
Anomalies may affect inner ear alone (nonsyndromic) or may form
part of a syndrome (syndromic).
Anomalies affecting the inner ear may involve only the membranous labyrinth or both the membranous and bony
labyrinths. They include:
- (a) Scheibe dysplasia. It is the most common inner ear anomaly. Bony labyrinth is normal. Superior part of membranous labyrinth (utricle and semicircular ducts) is also normal. Dysplasia is seen in the cochlea and saccule; hence also called cochleosaccular dysplasia. It is inherited as an autosomal recessive nonsyndromic trait.
(b)
Alexander dysplasia.
It affects
only the
basal turn
of membranous
cochlea.
affected.
Residual
Thus only
hearing high
frequencies is
present in
are low
frequencies and
can be
exploited by
amplification with
hearing aids.
(c) Bing-Siebenmann dysplasia. There is complete absence of membranous labyrinth.
(d) Michel aplasia. There is complete absence of bony and membranous labyrinth. Even the petrous apex is absent but external and middle ears may be completely

Children with profound (>90 dB loss) or total deafness fail to develop speech and have often been termed deaf-mute or deaf and dumb. However, these children have no defect in their speech producing apparatus. The main defect is deafness. They have never heard speech and therefore do not develop it. In lesser degrees of hearing loss, speech does develop but is defective. The period from birth to 5 years of life is critical for the development of speech and language, therefore, there is need for early identification and assessment of hearing loss and early rehabilitation in infants and children. It was observed that children whose hearing loss was observed and managed before 6 months of age had higher scores of vocabulary, better expressive and comprehensive language skills than those diagnosed and managed after 6 months of age emphasizing the importance of early identification and treatment.
unaffected. No hearing aids or cochlear implantation
can be used.
(e)
Mondini
dysplasia.
Only
basal
coil
is
present
or
cochlea
is
1.5
turns.
There
is
incomplete
partition
between
the
scalae
due
to
absence
of
osseous
spiral
lamina.
Condi-
tion
is
unilateral
or
This
deformity
may
be
seen
in
Pendred,
Waardenburg,
branchio-oto-renal,
Treacher-Collins
and
Wildervanck
syndromes.
(f)
Enlarged
vestibular
aqueduct.
Vestibular
aqueduct
is
enlarged
(>2
mm),
endolymphatic
sac
is
also
enlarged
and
can
be
seen
on
Ty
MRI.
It
causes
early
onset
senso-
rineural
hearing
loss
which
is
progressive.
Vertigo
may
be
present.
Perilymphatic
fistula
may
occur.
(g)
Semicircular
canal
malformations.
Both
superior
and
lateral
or
only
lateral
semicircular
canal
malformations
may
be

(a) Infections during pregnancy.
(b) Drugs during pregnancy.
(c) Radiation to mother in the first trimester.
(d) Other factors.
Syndromes commonly associated with hearing loss are given in Table 19.1.
(a)
Infections during
developing fetus
loviruses, herpes
mnemonic, pregnancy.
Infections are
toxoplasmosis, type
which rubella,
1
and
2
and affect
the cytomega-
syphilis.
TORCHES.
Drugs during
mycin, pregnancy.
Streptomycin, amikacin,
the pregnant
age quinine
mother, the
Remember gentamicin,
or chloroquine,
cross the
cochlea.
tobra- when
given placental
barrier
Thalidomide not
only to
and affects
causes abnormalities
of limbs,
heart, ear
face,
- (c) Radiation to mother in the first trimester.
(d)
Other factors.
Nutritional deficiency,
diabetes, lip
dam- but
and also
palate.
toxaemia and
thyroid deficiency.
Maternal alcoholism
atogenic to the developing auditory system.

They relate to causes during birth or in early neonatal period. They are as follows.
1. Anoxia. It damages the cochlear nuclei and causes haemor- rhage
into the
ear.
Placenta praevia,
prolonged labour,
cord round
the neck
and prolapsed
cord can
all cause
fetal anoxia.
2. Prematurity and low birth weight. Born before term or with birth weight less than 1500 g (3.3 Ib).
115
is also
ter-
(b)

15. Goldenhar syndrome (Facio-auriculo- vertebral dysplasia) or (oculo-auriculo- vertebral [OAV] syndrome), Features = • Facial asymmetry • Low set ears, atresia of ear canal • Cardiac abnormalities • Preauricular tags/pits • Hemivertebrae in cervical region • Epibulbar dermoid • Coloboma of upper lid. 15. Goldenhar syndrome (Facio-auriculo- vertebral dysplasia) or (oculo-auriculo- vertebral [OAV] syndrome), Type of hearing loss = Mixed or conductive. 15. Goldenhar syndrome (Facio-auriculo- vertebral dysplasia) or (oculo-auriculo- vertebral [OAV] syndrome), Onset (Congenital/ delayed) = Congenital. 15. Goldenhar syndrome (Facio-auriculo- vertebral dysplasia) or (oculo-auriculo- vertebral [OAV] syndrome), Type of inheritance = AD or sporadic Extra chromosome Multifactorial (genetic and environmental). 16. Down syndrome (Trisomy 21), Features = • Microcephaly • Mental retardation/delayed development • Short stature • Epicanthal folds • Stenosis of ear canal • High incidence of serous otitis media • Atlanto-axial instability. 16. Down syndrome (Trisomy 21), Type of hearing loss = Conductive. 16. Down syndrome (Trisomy 21), Onset (Congenital/ delayed) = Congenital. 16. Down syndrome (Trisomy 21), Type of inheritance = Extra chromosome
Figure 19.1 Waardenburg syndrome. Note white forelock, hetero- chromia iridis and depigmentation of skin.
3. Birth injuries, e.g. forceps delivery. They may cause intra- cranial haemorrhage with extravasation of blood into the inner ear.
4. Neonatal jaundice. Bilirubin level greater than 20 mg% damages the cochlear nuclei.
5. Neonatal meningitis.
6. Sepsis.
7. Time spent in neonatal ICU.
8. Ototoxic drugs used for neonatal meningitis or septicaemia.

- 1. Genetic. Though deafness is genetic, it manifests later
in childhood
or adult
life.
Deafness may
occur alone
as in
familial progressive sensorineural deafness or in association with certain syndromes, e.g. Alport, Klippel-Feil, Hurler, etc.
2. Nongenetic. They are essentially same as in adults and include:
- (a) Viral infections (measles, mumps, varicella, influenza), meningitis and encephalitis.
(b) Secretory otitis media.
(c)
Ototoxic drugs.
(d)
Trauma, e.g. fractures of temporal bone, middle ear surgery or perilymph leak.
- (e) Noise-induced deafness.

This may require a detailed history of prenatal, perinatal or postnatal causes, family history, physical examination and certain on the cause investigations depending suspected.
1.
Suspicion of
is suspected
if
(i)
the child
sleeps through
loud noises
unperturbed or
fails to
startle to
loud sounds,
(ii)
fails to
develop speech
at
1-2 years. A partially hearing child may have a defective
118
speech and perform poorly in school and be labelled mentally retarded. It is essential that all children at risk for hearing loss should be screened and followed.
. Risk factors for hearing loss in children (Recommendations of Joint Committee on Infant Hearing—updated to 1994).
(a) Family history of hearing loss.
(b) Prenatal infections (TORCHES).
(c) Craniofacial anomalies including those of pinna and ear canal.
(d) Birth weight less than 1500 g (3.3 Ibs).
(e) Hyperbilirubinaemia requiring exchange transfusion.
(f) Ototoxic medications included but not limited to aminoglycosides used in multiple courses or in combination with loop diuretics.
(g) Bacterial meningitis.
(h) 'Apgar score of 0-4 at 1 min or 0-6 at 5 min.
(i) Mechanical ventilation for 5 days or longer.
(j) Stigmata or other findings associated with a syndrome known to include sensorineural and/or conductive hearing loss.

Assessment of auditory function in neonates, infants and children demands special techniques. They are grouped under the following heads (Table 19.2):
1.
Screening procedures.
They are
employed to
test hearing
in
“high-risk”
response infants
and to
the children
sound with
one
On the
are based
signal.
or more
contrary, ing
loss had
50%
no universal
on infant’s
It is
now risk
observed factors
of children
risk factor.
neonatal behavioural
that have
95%
of normal
with
This hearing.
sensorineural leads
screening for
hear- to
a
programme early
Two important
tests are
to study
otoacoustic of
emissions
(OAEs) and auditory brainstem responses (ABR).
- (a) OAEs are generated at outer hair cells and can be picked up from the external ear as the energy produced by them travels in reverse direction from outer hair cells — ossicles — tympanic membrane — ear canal where it is picked up. OAEs are absent if outer hair cells in the cochlea are nonfunctional or there is middle ear effusion or canal debris due to meconium which may persist for 3-4 days. They are normal even when VIIIth
nerve is
nonfunctional.
Thus can
be used
in the
diag- nosis
of neuropathy
of
VIIIth nerve.
- (b) ABRs are generated in response to sound stimulus presented to the ear and picked up from the scalp. With a response of 30-35 dB nHL, the infant who passes the test and the hearing is considered normal. Infants who fail these tests are followed up with repeat tests.
Arousal test. A high-frequency narrow band noise is presented for 2 s to the infant when he is in light sleep. A nor mal hearing infant can be aroused twice when three such stimuli are presented to him.

'Developed by Virginia Apgar, an anaesthesiologist. It takes note of five items: heart rate, respiratory effort, muscle tone, reflex irritability and colour; noted at 1 and 5 min after birth. Low score is 0 and highest 10, i.e. 2 marks for each item.

° ABR/OAEs
° Arousal test
« Auditory response cradle
Behaviour observation audiometry
° Moro's reflex
© Cochleopalpebral reflex
° Cessation reflex
¢ Distraction techniques (6-18 months)
Conditioning techniques (7 months — 2 years)
« Visual reinforcement audiometry
Se eeeetiomenn(cestcan)
© Objective tests
° ABR
° Otoacoustic emissions
© Impedance audiometry
Auditory response cradle is a screening device for newborns, where baby is placed in a cradle and his behaviour (trunk and limb movement, head jerk and respiration) in response to auditory stimulation are monitored by transducers. It can screen babies with moderate, severe or profound hearing loss. 2. Behaviour observation audiometry. Auditory signal presented to an infant produces a change in behaviour, e.g. alerting, cessation of an activity, widening of eyes or facial grimacing. Moro's reflex is one of them and consists of sudden movement of limbs and extension of head in response to sound of 80-90 dB. In cochleopalpebral reflex, the child responds by a blink to a loud sound. In cessation reflex, an infant stops activity or starts crying in response to a sound of 90 dB.
3. Distraction techniques are used in children 6-7 months old. The child at this age turns his head to locate the source of sound. In this test, the child is seated in his mother's lap, an assistant distracts the child's attention while the examiner produces a sound from behind or from one side to see if the child tries to locate it. Sounds used are high frequency rattle (8 kHz), low-frequency hum, whispered sound as "S, S, S", xylophone, warbled tones or narrow band noise (500-4000 Hz).

(a) Visual reinforcement audiometry (VRA). It is a conditioning technique in which child is trained to look for an auditory stimulus by turning his head. This behaviour is reinforced by a flashing light or an animated toy. This test helps to determine the hearing threshold using standard audiometric techniques. The auditory stimulus is delivered by headphones or better still by insert earphones which are accepted better and are also light weight. Test is well-suited between the developmental age of 6 months to 2 years.
(b)
Play audiometry.
The child
is conditioned
to per
form an
act such
ting
a
ring bucket
on
a
as placing
post each
time rect
a
marble or
in
a
box, putting
he performance
of hears
a
plastic
a
sound the
act is
block signal.
reinforced
Each with
put- in
a
cor praise,
encouragement or reward. Ear specific thresholds can be determined by standard audiometric techniques. This test can be used in children with developmental age of 2-4 or 5 years.
(c) Speech audiometry. The child is asked to repeat the names of certain objects or to point them out on the pictures. The voice can be gradually lowered. In this way, hearing level and speech discrimination can be tested. The test can also be used to examine the child's expressive ability when he is asked to name the toys like horse, duck or objects like cup, plate, etc.

(i)
Electrocochleography.
It can
ity to
within
20
dB.
requiring
But measure
it placement
of auditory
is an
invasive sensitiv-
procedure electrodes

Auditory brainstem response. It is not a direct test of hearing but correlates highly with the pure-tone thresholds. Identifiable waveforms in ABR are generally present 10-20 dB above behavioural threshold. ABR provides an ear-specific information as sound stimulus can be presented to each ear separately by headphones or ear inserts. It is an objective test and can be done under sedation as the latter has no effect on ABR. ABR is used both as a screening test and as a definitive hearing assessment test in children. In a screening test, a response to a click stimulus of less than 40 dB nHL or less is the criterion of passing the test. To find hearing threshold in an infant, ABR tracing is obtained first at higher sound stimulus and then gradually lowered till wave V is just identifiable but repeatable.
(b) Otoacoustic emissions (see p. 27). Transient evoked emissions (TEOAEs) are absent in ears where hearing loss exceeds 30 dB. Distortion product emissions (DPOAEs) are absent when hearing loss exceeds 50 dB.
(c) Impedance audiometry. Normally, stapedius muscle contracts reflexly in response to a sound of 70-100 dB HL and this reflex can be recorded. Absence of acoustic reflex indicates middle ear disorder, retrocochlear hearing loss or severe to profound SNHL. Used with behaviour audiometry, acoustic reflexes are useful component to cross-check. Absence of acoustic reflex, but a normal tympanometry with parental concern for hearing loss suggests possibility of SNHL of severe to profound degree. Absence of acoustic reflex but an abnormal tympanogram generally indicates conductive loss. Since ABR and OAEs provide more information, use of acoustic reflexes in assessment of paediatric testing has declined.
OAEs and ABR have been used both in screening programmes and in hearing evaluation in infants and children.

It is essential to know the degree and type of hearing loss, and other associated handicaps such as blindness or mental retardation and whether hearing loss is prelingual (before development of speech) or postlingual. Aetiology of hearing loss remains obscure in about half the cases. Aims of habilitation of any hearing-impaired child are development of speech and language, adjustment in society and useful employment in a vocation.
1. Parental guidance. It is a great emotional shock for parents to learn that their child is deaf. They should be dealt with sympathetically, so as to accept the child. They should be told of child's disability and how to care for it. Habilitation of the deaf demands a lot from parents: care and periodic replacement of hearing aid, change of ear-moulds as child grows, follow-up visits for re-evaluation, education at home and the selection of vocation.
2. Hearing aids. Most deaf children have a small but useful portion of residual hearing which can be exploited by amplification of sound. Hearing aids should be prescribed as early as possible. If necessary, binaural aids, one for each ear, can be used. Hearing aids help to develop lipreading also.

4. Development of speech and language. Communication is a two way process, depending on the receptive and expressive skills. Reception of information is through visual, auditory or tactile faculties while expression is through oral or written speech or the manual sign language. In the hearing impaired, auditory faculty is poor or totally absent (Figure 19.2). Thus, for proper communication, there is need either to improve hearing through amplification of the residual hearing or cochlear implants; and in the absence of the feasibility of developing the auditory faculty, one has to develop visual or tactile means of communication.
(a) Auditory-oral communication. This is the method used by a normal person and is the best way of communication. In the deaf, it can be used in those with moderate to severe hearing loss or those who are postlingually deaf. Hearing aids are provided to augment auditory reception. At the same time, training is also imparted in speech reading, i.e. to read movements of lips, face, and natural gestures of hand and body. Expressive skill is encouraged through oral speech.
(b) Manual communication. It makes use of the sign language or finger-spelling method but has the disadvantage that abstract ideas are difficult to express and general public does not understand it.
(c)
Total communication. It uses all modalities of sensory input, i.e. auditory, visual, tactile and kinaesthetic. Such children are taught to develop oral speech, lipreading and sign language. All children with prelingual severe to profound deafness, should undergo training in this form of communication. Vibrotactile aids are useful for those who are totally deaf and also blind. These aids are attached to the child's hand or sternum and the vibrations of speech are perceived through tactile sensation.
5. Education of the deaf. There are residential and day schools for the hearing impaired. Some children with
Figure 19.2 The faculties of a hearing-impaired person which can be utilized for receptive and expressive skills in communication.
moderate hearing loss can be integrated into schools for
the normal hearing children with preferential seating in the class.
Radio hearing aids have revolutionized education of
the deaf. In this device, the microphone and transmitter are worn by the teacher and the receiver and amplifier by

the child. With this system, the child can hear the teacher's voice better, without being disturbed by environmental noises. 6. Vocational guidance. The deaf are sincere and good workers. Given the opportunity, commensurate with their ability, they can be usefully employed in several vocations.

All hearing-impaired individuals need some sort of aural rehabilitation avail- for communication. The various means able to them are:
1. Instrumental devices
(a) Hearing aids
(i) Conventional hearing aids
(ii) Bone-anchored hearing aids
(iii) Implantable hearing aids (vibrant soundbridge)
(b) Implants
(i) Cochlear implants
(ii) Auditory brainstem implants
(c) Assistive devices for the deaf
2.
Training
(a) Speech (lip) reading
(b) Auditory training
(c) Speech conservation

A hearing aid is a device to amplify sounds reaching the ear. Essentially, it consists of three parts: (i) a microphone, which picks up sounds and converts them into electrical impulses, (ii) an amplifier, which magnifies electrical impulses and (iii) a receiver, which converts electrical impulses back to sound. This amplified sound is then carried through the earmould to the membrane.
tympanic

Air conduction hearing aid. In this, the amplified sound is transmitted via the ear canal to the tympanic membrane.
Bone conduction hearing aid. Instead of a receiver, it has a bone vibrator which snugly fits on the mastoid and directly stimulates the cochlea. This type of aid is especially useful in persons with actively draining ears, otitis externa or atresia of the ear canal when ear inserts cannot be worn.
Most of the aids are air conduction type. They can be:
1.
Body-worn types.
Most common
type
(Figure
20.1A);
microphone and
one case
worn the
ear level.
fication amplifier
at the
along chest
level
This with
type with
the while
of minimal
aid battery
receiver allows
high feedback.
are in
is situated
degree
It is
useful at
of ampli-
deaf persons
or children
with congenital
deafness.
- 2. Behind-the-ear (BTE) types. Here microphone, ampli-
fier, receiver and battery are all in one unit which is worn
behind
the
ear.
It
is
coupled
to
the
ear
canal
with
a
tubing
and
an
earmould.
It
is
useful
for
slight
to
moderate
cases
of
hearing
loss
the
high
frequency
ones.
Spectacle
types.
It
is
a
of
the
“behind-the-
ear”
type
and
the
unit
is
housed
in
the
auricular
part
of
the spectacle frame. It is useful to persons who need both
eye
glasses
for
vision
and
a
hearing
aid.
It
is
not
very
pop-
ular
now.
In-the-ear
(ITE)
types.
The
entire
hearing
aid
is
housed
in
an
earmould
which
can
be
worn
in
the
ear.
It
is
use-
ful
for
mild
to
moderate
hearing

losses
with
flat
configu-
ration.
They
are
very
popular
because
of
their
cosmetic
appeal.
Canal types (ITC and CIC). The hearing aid is so small that the entire aid can be worn in the ear canal with- out projecting into the concha. For using this aid, it is required that the ear canal should be large and wide, and patient should have the dexterity to manipulate the min- ute controls in the aid. It is useful for mild to moderate cases of hearing loss of high frequency (1-4 kHz).
Two types are available: in the canal (ITC) and another still
smaller and invisible type, completely in the canal (CIC).

Any individual who has a hearing problem that cannot be helped by medical or surgical means is a candidate for hear- aid.
ing
1. Sensorineural hearing loss, which interferes with day-to- day activities of a person. Hearing aid may not suit all such persons because of the intolerable distortion of sound in some, particularly in those with recruitment.
Deaf
children
should
be
fitted
with
hearing
aid
as
early
as
possible
for
of
speech
and
learning.
In
severely deaf children, binaural aids (one for each ear and individually fitted) are more useful. Training in lip reading is given simultaneously.
Conductive deafness. Most of such persons can be helped by surgery but hearing aid is prescribed when surgery is refused or not feasible or has failed.

While fitting a hearing aid, consideration is given to:
1. Degree of hearing loss.
2.
3. Type of hearing loss (conductive or sensorineural).
Configuration
of
hearing
loss
(type
of
affected).
Presence of recruitment.
Uncomfortable loudness level.
Figure 20.1 Various types of hearing aids. (A) Body-worn. (B) Behind-the-ear type. (C) Spectacle type. (| D) In-the-ear type.
Condition of the outer and middle ear.
Cosmetic acceptance of the aid.
Age and dexterity of patient. One
Type of earmould. Sel
The type of fitting, whether it is monoaural (one aid only), binaural (one aid for each ear), binaural with y-connection (one aid but two receivers, one for each ear) or the contralateral routing of signals type.
CROS (contralateral routing of signals). In this type, microphone is fitted on the side of the deaf ear and the sound thus picked up is passed to the receiver placed in the better ear. This is useful for persons with one ear severely impaired and helps in sound localization coming from the side of the deaf ear. Now bone-anchored hearing aids (see infra) are being preferred for single-sided deafness and have replaced the use of CROS aids.

Bone-anchored hearing aid is a type of hearing aid which is based on the principle of bone conduction. It is primarily suited to people who have conductive hearing loss, unilateral hearing loss and those with mixed hearing loss who cannot otherwise wear "in the ear" or "behind the ear" hearing aids.
Figure 20.2 Appearances when sound processor is attached to abutment.
Bone-anchored hearing aids use a surgically implanted abutment to transmit sound by direct conduction through bone to the cochlea, bypassing the external auditory canal and middle ear (Figure 20.2). BAHA has three components: (i) titanium fixture (ii) titanium abutment and (iii) sound processor (Figure 20.3).
The titanium fixture is surgically embedded in the skull bone with abutment exposed outside the skin. The titanium fixture bonds with the surrounding tissue in a process called osseointegration. The sound processor is attached to the abutment once osseointegration is complete which usually takes 2-6 months after implantation. The BAHA device transmits vibrations to the external abutment which further vibrates the skull and cochleae.
Candidacy profile. Bone-anchored hearing aids can be used in:
1.
People who
have chronic
inflammation or
infection of
the ear
canal and
cannot wear
standard
“in the
ear”
air- conduction
- 2. Children with malformed or absent outer ear and ear
canals as
in microtia
or canal
atresia.
- 3. Single-sided deafness (see Table 20.1).

In the past, the contralateral routing of signal (CROS) hearing aid was the only option available for rehabilitation of patients with single-sided deafness. Poor performance and aesthetic considerations limited the use of CROS aids. The BAHA device can now be implanted on the side of the deaf ear, and it transmits the sound by means of bone conduction to the contralateral cochlea. The BAHA is fixed on the deaf side and collects sound waves to transmit to healthy cochlea of the other side. This process eliminates the headshadow effect and allows for hearing from both sides of the head. The BAHA substantially improves speech recognition in quiet and in noise compared with the CROS aids.
Surgery. The surgery is typically performed in a single stage in adults. About 3 months are allowed for osseointegration before the sound processor can be attached. A two-stage procedure is recommended in children in whom the fixture is placed into the bone in the first stage. After about 6 months to allow for osseointegration, a second-stage operation is done to connect the abutment through the skin to the fixture.
Complications of BAHA are few and may include occasional failure to osseointegrate the implant and local infections and inflammation at the implant site.

Implantable middle ear hearing aids represent a new category of hearing devices that work on a direct drive principle. Rather than delivering acoustic energy into the external auditory canal (as with traditional hearing aid systems),
1.
2.
When air-conduction
°
Canal
(AC)
atresia, congenital
hearing aid
cannot or
acquired, be
used:
not amenable
to treatment.
- ° Chronic ear discharge, not amenable to treatment.
°
Excessive feedback
and discomfort
from air-conduction
hearing aid.
Conductive or
mixed hearing
loss, e.g.
tympanosclerosis where surgery is contraindicated.
3.
Single-sided hearing loss.
direct drive middle ear implant systems use mechanical vibrations delivered directly to the ossicular chain, while leaving the ear canal completely open.
Implantable middle ear devices are generally available in two types:
- 1. Piezoelectric devices. Piezoelectric devices operate by passing an electric current into a piezoceramic crystal, which changes its volume and thereby produce a vibratory signal. This piezoelectric transducer in turn is coupled to the ossicles and drives the ossicular chain by vibration.
Examples of such devices are Envoy, middle-ear transducer (MET or also called otologic device), Rion and totally integrated cochlear amplifier (TICA).
- 2. Electromagnetic hearing devices. Electromagnetic hearing devices function by passing an electric current into a coil, which creates a magnetic flux that drives an adjacent magnet. The small magnet is attached to one of the ossicles of the middle ear to convey vibrations to the cochlea. An example of such a device is the vibrant soundbridge device (previously known as the Symphonix device; now being manufactured by MED-EL).

Vibrant soundbridge device. The vibrant soundbridge is a semi-implantable device made of two components: an internal and an external. The internal component is called vibrating ossicular prosthesis (VORP) and is made up of three parts: the receiver, floating mass transducer (FMT) and a conductor link between the two. FMT is connected to the incus (Figures 20.4 and 20.5).
The external component is called the audio processor which is worn behind the ear. The audio processor contains a microphone that picks up sound from the environment and transmits it across the skin by radiofrequency waves to the internal receiver.
Candidacy profile. Appropriate candidates for direct drive middle ear hearing devices include adults aged 18 years and older with moderate-to-severe sensorineural hearing loss. Candidates should have experience of using traditional hearing aids and should have a desire for an alternative hearing system.
Often, patients who are interested in seeking direct drive middle ear hearing devices have experienced dissatisfaction regarding the sound quality of their current hearing aids. Other problems these patients feel with these aids are discomfort due to the occlusion effect of the canal, wax occluding hearing aid mould and wax impaction of the external auditory canal, inability to wear traditional hearing aids
due to sensitive ear canal skin and the inability to overcome acoustic feedback issues (see Table 20.2 for disadvantages of conventional hearing aids).
Procedure. The internal device is surgically implanted. The procedure is conducted under general anaesthesia. The receiver of the implant is positioned under the skin over the mastoid bone via a standard cortical mastoidectomy and posterior tympanotomy approach; the ossicular chain is visualized and the FMT is attached to the long process of the incus. The middle ear structures are not modified. Therefore, there is no significant impact on the residual hearing of the patient.
Six to eight weeks after the procedure, the patient is fitted with the external audio processor that attaches magnetically to the back of the ear. The processor is then programmed.
Advantages. A direct drive system provides mechanical energy directly to the ossicles, bypassing the ear canal and the tympanic membrane. This eliminates many of the inherent issues of conventional hearing aids such as occlusion, feedback, discomfort and wax related problems. One major advantage of direct drive devices is the ability to provide

improved sound quality to the hearing-impaired subjects particularly in noisy environments.

A cochlear implant is an electronic device that can provide useful hearing and improved communication abilities for persons who have severe to profound sensorineural hearing loss and who cannot benefit from hearing aids. A cochlear implant works by producing meaningful electrical stimulation of the auditory nerve where degeneration of the hair cells in the cochlea has progressed to a point such that amplification provided by hearing aids is no longer effective. Various cochlear implants are shown in Figures 20.6-20.8.

Acochlear implant has an external and internal component.
1. External component. It consists of an external speech processor and a transmitter. The speech processor may be body worn or behind the ear type; the latter being preferred.
2. Internal component. It is surgically implanted and comprises the receiver/stimulator package with an electrode array.
Sound is picked up by the microphone in the speech processor. The speech processor analyses and codes sounds into electrical pulses. The processor uses a variety of coding strategies to deliver meaningful speech parameters from the acoustic stimulus to the nerve. Examples of such strategies are simultaneous analogue strategy (SAS), continuous interleaved sampling (CIS), spectral peak (SPEAK) and advanced combination encoder (ACE).
° Cosmetically unacceptable due to visibility.
e Acoustic feedback.
© Spectral distortion.
© Occlusion of external auditory canal.
© Collection of wax in the canal and blockage of insert.
¢ Sensitivity of canal skin to earmoulds.
e Problem to use in discharging ears.
Figure 20.4 Vibrant soundbridge middle ear implant.
The electrical impulses are sent from the processor to the transmitting coil which in turn sends the signal to the surgically implanted receiver/stimulator via radiofrequency. The receiver/stimulator decodes the signal and transmits it to the electrode array. Current day implants are multichannel processors with the electrode having a linear array of electrode contacts used to deliver multiple channels of current to different places along the basilar membrane. The electrode array which has been placed in the scala tympani of the cochlea stimulates the spiral ganglion cells. The auditory
Figure
20.6
MED-EL
cochlear implants.
(A)
MED-EL
C-401.
(B)
Sonata model
with ear
level speech
processor.
Figure 20.7 Nucleus cochlear implant (Cochlear Corporation) with ear level speech processor.
Figure
20.8
Advanced bionics cochlear implant system.
nerve is thus stimulated and sends these electrical pulses to the brain which are finally interpreted as sound.
Candidacy profile. Cochlear implants may be used both in children and adults. The following criteria help define candidacy for cochlear implantation:
rR

- Bilateral severe to profound sensorineural hearing loss.
DO
Little or no benefit from hearing aids.
No medical contraindication for surgery. Go
Realistic expectation.
Good family and social support toward habilitation.
Adequate cognitive function to be able to use the device. D
Candidates with such hearing impairment may be defined as prelingual or postlingual depending on whether they were deafened before or after the acquisition of speech and language.
In children who have hearing impairment at birth or early in childhood, early intervention with hearing aids or a cochlear implant is vital for auditory stimulus. Auditory deprivation, i.e. lack of auditory stimulus in the early developmental period causes degeneration in the central auditory pathways. This will limit the benefit in terms of speech and language acquisition following cochlear implantation.

Factors that predict a successful clinical outcome are:
1. Previous auditory experience (postlingual patients or prior use of hearing aids).
2. Younger age at implantation (especially for prelingual children).
3. Shorter duration of deafness.
4. Neural plasticity within the auditory system.
Multichannel implants are the standard today and perform much better than single-channel devices. Postlingual children or adults achieve very good benefit. They develop the ability to recognize speech with no or minimal lip reading or visual cues. They eventually can also use the telephone. Prelingually deafened children also develop good speech understanding and language acquisition over a period of time. This can take a couple of years and requires constant auditoryverbal training. Early age at implantation ensures better results and children can be implanted at 12 months of age. Prelingually deafened adults with no or little prior auditory experience obtain very limited benefit from cochlear implantation. They will however obtain sound awareness.

Thorough evaluation of the patient is very critical in the selection of candidates for a cochlear implant. The main purpose is to determine if the patient is medically and audiologically suitable for an implant. It also helps the clinicians to predict and counsel the family regarding the expected outcomes following the procedure. Medical evaluation through detailed history and physical examination is necessary to confirm fitness for a general anaesthetic. The necessary preanaesthetic tests will be required to be carried out. All candidates must be fully vaccinated against meningitis (particularly Haemophilus influenzae type B, Pneumococcus and in some areas Meningococcus).
Figure 20.9 Principle of cochlear implant.
Imaging of the temporal bone, cochlea, auditory nerve and brain is carried out using CT and MRI. This is required to provide an image of the structure of the cochlea and help identify any anomalies or pathology that may complicate the implantation process.
as not to disrupt the blood supply. Usually, a two-layered approach is chosen utilizing a flap of skin and subcutaneous tissue, followed by a second layer of musculoperiosteal flap. A pocket is created under the second flap and a well or recess is drilled in the bone to house the receiver/stimulator.
Audiological evaluation may include some or all of the following depending on the age of the patient:
Pure tone audiogram
Speech discrimination tests
Tympanometry
Otoacoustic emissions (OAE)
Auditory brainstem responses (ABR)
Auditory steady state responses (ASSR)
A hearing aid trial and evaluation is mandatory in determining the candidacy for cochlear implantation. This may include aided free-field sound detection thresholds, as well as aided speech perception and discrimination scores. Speech and language evaluation is required to assess the child's communicative status and to determine any developmental language or articulation disorders. This will also form a baseline for further evaluations postimplantation to help assess progress and identify areas of deficit in speech perception. This in turn would aid in the programming of the patient's device.
Psychological evaluation is performed where there may be concerns regarding the cognitive status or mental function of the patient. This is also important to identify children who may have disabilities other than hearing loss. This may provide information that is important when counselling parents about expectations following cochlear implantation.

The principle of cochlear implant surgery is to place the electrode array within the scala tympani of the cochlea. This allows the electrodes to be in close proximity to the spiral ganglion cells and their dendrites (that lie in the modiolus and osseous spiral lamina of the cochlea, respectively).
Surgery is carried out under general anaesthesia and is similar to mastoid surgery. Once the patient is positioned, prepped and draped, the position of the device is marked and the incision planned. Flaps are elevated carefully so
There are broadly two surgical techniques to approach the cochlea for implantation: (i) The facial recess approach where a simple cortical mastoidectomy is done first and the short process of the incus and the lateral semicircular canal are identified. The facial recess is opened by performing a posterior tympanotomy. The stapes, promontory and round window niche are identified. Cochleostomy is then performed anteroinferior to the round window membrane to a diameter of 1.0-1.6 mm depending on the electrode to be used. (ii) The pericanal techniques where a tympanomeatal flap is elevated to perform a cochleostomy either by endaural or postaural approach. In the pericanal techniques a bony tunnel is drilled along the external canal towards the middle ear. The examples of pericanal techniques include the Veria and suprameatal recess approach.
The device is placed in the "well" created and is secured with ties. The electrode array is gently and gradually inserted through the cochleostomy till complete insertion has been achieved. Electrophysiological testing is carried out to check that the electrode impedances and telemetry responses are satisfactory.
The wound is closed in layers and a mastoid bandage

Postoperative Mapping (Programming) of Device and Habilitation
Activation of the implant is done 3-4 weeks after implantation. Following this the implant is "programmed" or "mapped." Mapping is done on a regular basis during postoperative rehabilitation to fine-tune the processor and get the best performance as the patient gets used to hearing with the implant.
(Re) Habilitation is an essential part for those who have undergone cochlear implantation. All patients need auditoryverbal therapy. In auditory-verbal therapy, the emphasis is laid on making the child listen and speak like a normal per- son
use lip
reading and
visual cues.
Learning
to listen takes time and requires concerted efforts from the patient, the family and the person providing habilitation services.
Table 20.3 summarizes the complications of cochlear implant surgery.

This implant is designed to stimulate the cochlear nuclear complex in the brainstem directly by placing the implant in the lateral recess of the fourth ventricle. Such an implant is needed when CN VIII has been severed in surgery of vestibular schwannoma. In these cases, cochlear implants are obviously of no use. In unilateral acoustic neuroma, ABI is not necessary as hearing is possible from the contralateral side but in bilateral acoustic neuromas as in NF9, rehabilitation is required by ABI.
Brainstem implant is similar to "Nucleus" multichannel cochlear implant except that the multielectrode array is attached to a Dacron mesh, which is placed on the brainstem. Receiver/stimulator has a removable magnet so that MRI can be safely performed in such cases if need arises. ABIs help in communication, awareness and recognition of environmental sounds; however, they are not as efficient as multichannel cochlear implants. Only limited numbers of such implants have been performed in the world and are under constant technological developments.

Hearing-impaired persons should enjoy life as best as normally hearing persons do. For this, devices are needed to help him to listen in special difficult situations, warn him of danger signals and help him to telecommunicate with his family and friends who are far away from him. These devices can thus be divided into three groups:

They are not hearing aids but devices which help the hearing impaired to listen efficiently in the presence of background noise, over the telephone, in auditoriums or theatres. They may be used by the person individually or are meant for a group.
the technology
used, they
are grouped
as hard-wired
system, induction
loops,
AM
(amplitude modu-
lation), FM (frequency modulation) or infrared signals.

A hearing-impaired person may not hear a telephone or a doorbell, a baby crying in another room, an alarm clock or the noise of a smoke detector. Alerting devices are useful in such situations. They produce an extra loud sound signal or relay the signal to an area closer to the individual. A "hearing dog" is one such simple device. The dog is trained to bark loudly at the sound of a doorbell or cry of a baby to alert his master. It is a helpful companion for the hearing impaired. For people with severe to profound or total deafness, even these devices which produce extra loud sound may not be useful. They need assistive signalling devices where the sound (as of doorbell, telephone, alarm clock, baby crying) is changed into a light signal or vibrations. Alarm clock with flashing lights or those devices which produce strong vibrations to awaken the individual or even shake his bed are also available.

A telephone amplifier can be attached to the hand set of a telephone, residential or public, to amplify the sound. A telephone coupler is a device that can be connected to the telephone and the signal produced is picked up by the hearing aid. For the profoundly or totally deaf individuals, telecommunication devices for the deaf (TDDs) can be used. They convert typed message into sounds that can be transmitted over the standard telephone lines, and at the other end another TDD converts these sound signals back into typewritten messages. Email and short message services (SMS) on mobile phones have made life easier for the hearing impaired. Closed-caption television decoder can be attached to television sets to provide them cues to enjoy news, movies and other programmes.

Earlier called lip-reading, it is an integrated process to understand speech by studying movements of lips, facial expression, gestures and the probable context of conversation. The skill of speech reading is not only useful for the totally deaf but also useful for those hearing-impaired individuals who have high-frequency loss and difficulty in hearing in noisy surroundings.

It enhances listening skill and is used with speech reading. The patient is exposed to various listening situations with different degrees of difficulty and taught selectively to concentrate on speech sounds.
and
Auditory training is useful for those using hearing aids cochlear implants.

In sudden, severe or profound hearing loss, the person loses the ability to monitor his own speech production. As a result, defects arise in articulation, resonance, pitch and the volume of voice. Speech conservation aims to educate such a person to use his tactile and proprioceptive feedback to monitor his systems speech production.

Pain in the ear can be due to causes occurring locally in the ear or referred to it from remote areas.
cranial nerves; and from Cy (lesser occipital) and Cy and Cs (greater auricular), pain may be referred from these remote areas (Figure 21.1).

1. External ear. Furuncle, impacted wax, otitis externa, otomycosis, myringitis bullosa, herpes zoster and malignant neoplasms.
2. Middle ear. Acute otitis media, eustachian tube obstruction, mastoiditis, extradural abscess, aero-otitis media and carcinoma middle ear.

As ear receives nerve supply from Vth (auriculotemporal branch), [Xth (tympanic branch) and Xth (auricular branch)

(a)
Dental.
Caries
tooth,
apical
abscess,
impacted
molar,
malocclusion and Costen syndrome.!
(b) Oral cavity. Benign or malignant ulcerative lesions of
oral cavity or tongue.
(c)
Temporomandibular joint disorders. Bruxism, osteoar-
thritis, recurrent dislocation and ill-fitting denture.
!
Costen syndrome. It is due to abnormality of temporomandibular joint (TMJ) with defective bite. It manifests with tinnitus, vertigo and earache, and also causes blocked feeling in the ear. There is associated pain in ipsilateral, frontal, parietal and occipital region. Treatment includes analgesics, hot fermentation and slow TMJ exercises. Defective bite can be corrected an orthodontist.
by
(d) Sphenopalatine neuralgia.
2. Via [Xth cranial nerve
(a)
Oropharynx.
Acute
tonsillitis,
peritonsillar
abscess,
tonsillectomy. Benign or malignant ulcers of soft pal-
ate, tonsil and its pillars.
(b) Base of tongue. Tuberculosis or malignancy.
(c) Elongated styloid process.
3. Via Xth cranial nerve. Malignancy or ulcerative lesion
of vallecula, epiglottis, larynx or laryngopharynx and oesophagus.
4. Via C, and C; spinal nerves. Cervical spondylosis, injuries of cervical spine and caries spine.

When no cause has been discovered, pain may be functional in origin but the patient should be kept under observation
with periodic re-evaluation.
Otalgia is a symptom. It is essential to find its cause before specific treatment can be instituted.
Tinnitus is ringing sound or noise in the ear. The characteristic feature is that the origin of this sound is within the patient. Usually, it is unilateral but may also affect both ears. It may vary in pitch and loudness and has been variously described by the patient as roaring, hissing, swishing, rustling or clicking type of noise. Tinnitus is more annoying in quiet surroundings, particularly at night, when the masking effect of ambient noise from the environment is lost.

Two types of tinnitus are described:
1. Subjective, which can only be heard by the patient.
2. Objective, which can even be heard by the examiner with the use of a stethoscope.

Subjective tinnitus
may have
its origin
in the
middle ear,
inner system.
Systemic hypertension
ear,
VIIth disorders
and nerve
like certain
or the
anaemia, drugs
external ear,
arteriosclerosis, may
act through
the inner
ear or
central auditory
pathways.
In the
conduc- tive hearing loss, the patient may hear abnormal noises in
the head during eating, speaking or even respiration.
Objective tinnitus is seen less frequently. Vascular lesions, e.g.
glomus tumour
or carotid
artery aneurysm
cause swish-
ing tinnitus
ily synchronous
abolished
Venous hum
with by
pressure can
pulse.
on the
It can
be common
stopped by
temporar- carotid
artery.
pressure on
the neck
veins.
Tinnitus synchronous with respiration may occur due to abnormally
patent eustachian
tube.
Palatal myoclonus
pro- duces
clicking of
soft sound
palate due
to clonic
and can
be contraction
easily diagnosed.
of the
muscles
Clonic contraction
of muscles
of middle
ear
(stapedius and
tensor tympani)
may cause tinnitus which is often difficult to diagnose.
Sometimes, tinnitus is psychogenic and no cause can be found in the ear or central nervous system.
Tinnitus should be differentiated from auditory halluci- nations
in which
a
person hears
voices or
other organized
sounds like
that of
music.
It is
seen in
psychiatric disorders.

Tinnitus is a symptom and not a disease. Where possible, its cause should be discovered and treated. Sometimes, even
the treatment of cause may not alleviate tinnitus. When no
cause is found, management of tinnitus includes:
1.
Reassurance
and
psychotherapy.
Many
times
the
patient
has
to learn to live with tinnitus.
2. Techniques of relaxation and biofeedback.
3.
Sedation
and
tranquillizers.
They
may
be
needed
in
initial
stages till patient has adjusted to the symptom.
4.
Masking
of
tinnitus.
Tinnitus
is
more
annoying
at
bedtime
when the surroundings are quite. Use of a fan, loudly

clicking clock or a similar device may mask the tinnitus and help the patient to go to sleep. Use of a hearing aid, in persons with hearing loss, not only improves hearing but also provides a masking effect.
Tinnitus maskers can be used in patients who have no hearing loss. They are worn like a hearing aid. Use of tinnitus masker for a short time may provide, in some individuals, a symptom-free period for several hours due to the phenomenon of residual inhibition.

It is a combination of a hearing aid and a masker in one device. Looks like a hearing aid. Both hearing aid and masking device have independent volume controls.

Jastreboff from University of Maryland described a neurophysiologic model for generation of tinnitus and the basis for habituation therapy. It presumes that tinnitus does not cause as much annoyance as the emotional reactions generated from the limbic and autonomic systems. His therapeutic model aims to attenuate connections between auditory, limbic and autonomic nervous systems and thus create tinnitus habituation. It occurs at two levels.
1. Habituation of reaction. It is uncoupling of brain and body from reactions to tinnitus.
2. Habituation of tinnitus. It is blocking the tinnitus-related neuronal activity to reach level of consciousness. With this therapy patients suffering from tinnitus lose awareness of tinnitus and also do not get annoyed even when do have tinnitus.
negative they
Therapy consists of two major components: (i) counselling and (ii) sound therapy.
Counselling. It is important to educate the patient about tinnitus, its mechanism of generations, perception of tinnitus at subcortical and cortical levels and the plasticity of brain which can habituate any sensory stimuli. Limbic system (emotions) and autonomic system (body reactions) are the primary sources of negative reactions to tinnitus, i.e. sleep disturbance, inability to concentrate, annoyance, anxiety and depression and not the tinnitus per se.
Sound therapy. Patient is exposed to environmental sounds, music radio, television, or use of hearing aids (in case he suffers from hearing loss). In general, he should avoid silent environment. To produce external sound for habituation, sound generators are used which produce continuous low-level, broad-band noise for at least 8 h a day. Sound, here is used not for masking the tinnitus but is adjusted to remain at a low level, for habituation. TRT needs a long period of 18-24 months but gives a significant improvement in more than 80% of patients.

23.
24.
25.
26.
27. Acute and Chronic Rhinitis
28. Granulomatous of
29.
30.
31.
Anatomy of Nose
Physiology of Nose
Diseases of External Nose and
Nasal Vestibule
Nasal Septum and Its Diseases
Diseases Nose
Miscellaneous Disorders of Nasal
Cavity
Allergic Rhinitis
Vasomotor and Other Forms of Nonallergic Rhinitis
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
Nasal Polypi
Epistaxis
Trauma to the Face
Anatomy and Physiology of
Paranasal Sinuses
Acute Sinusitis
Chronic Sinusitis
Complications of Sinusitis
Benign and Malignant Neoplasms of Nasal Cavity
Neoplasms of Paranasal Sinuses
Proptosis

Itis pyramidal in shape with its root up and the base directed downwards. Various terms used in its description are shown in Figure 23.1. Nasal pyramid consists of osteocartilaginous framework covered by muscles and skin.
- 3. Lesser alar (or sesamoid) cartilages. Two or more in number. They lie above and lateral to alar cartilages. The various cartilages are connected with one another and with the adjoining bones by perichondrium and periosteum. Most of the free margin of nostril is formed of fibrofatty tissue and not the alar cartilage.

Upper one-third of the external nose is bony while lower twothirds are cartilaginous. The bony part consists of two nasal bones which meet in the midline and rest on the upper part of the nasal process of the frontal bones and are themselves held between the frontal processes of the maxillae (Figure 23.2).

1. Upper lateral cartilages. They extend from the undersurface of the nasal bones above, to the alar cartilages below. They fuse with each other and with the upper border of the septal cartilage in the midline anteriorly. The lower free edge of upper lateral cartilage is seen intranasally as limen vestibuli or nasal valve on each side.
4. Septal cartilage. Its anterosuperior border runs from under the nasal bones to the nasal tip. It supports the dorsum of the cartilaginous part of the nose. In septal abscess or after excessive removal of septal cartilage as in submucosal resection (SMR) operation, support of nasal dorsum is lost and a supratip depression results.

Osteocartilaginous framework of nose is covered by muscles which bring about movements of the nasal tip, ala and the overlying skin. They are the procerus, nasalis (transverse and alar parts), levator labii superioris alaeque nasi, anterior and posterior dilator nares and depressor septi.

- . Lower lateral cartilages (alar cartilages). Each alar cartilage is U-shaped. It has a lateral crus which forms the ala and a medial crus which runs in the columella. Lateral crus overlaps lower edge of upper lateral cartilage on each side. no
The skin over the nasal bones and upper lateral cartilages is thin and freely mobile while that covering the alar cartilages is thick and adherent, and contains many sebaceous glands. It is the hypertrophy of these sebaceous glands which gives rise to a lobulated tumour called rhinophyma (see p. 144).
Figure
23.1
Various parts of nose and related facial structures.
Figure 23.3 Structures on lateral wall of nose.

It is divided into right and left nasal cavities by nasal septum. Each nasal cavity communicates with the exterior through naris or nostril and with the nasopharynx through posterior nasal aperture or the choana. Each nasal cavity consists of a skin-lined portion—the vestibule and a mucosa-lined portion, the nasal cavity proper.

Anterior and inferior part of nasal cavity is called vestibule. It is lined by skin and contains sebaceous glands, hair follicles and the hair called vibrissae. Its upper limit on the lateral wall is marked by limen nasi (also called nasal valve).
1. Nasal valve. It is bounded laterally by the lower border of upper lateral cartilage and fibrofatty tissue and anterior end of inferior turbinate, medially by the cartilaginous nasal septum, and caudally by the floor of pyriform aperture. The angle between the nasal
2. inspiration.
septum and lower border of upper lateral cartilage is nearly 30°. Nasal valve area. It is the cross-sectional area bounded by the structures forming the valve. It is the least crosssectional area of nose and regulates airflow and resistance on

Each nasal cavity has a lateral wall, a medial wall, a roof and a floor.

Three and occasionally four turbinates or conchae mark the lateral wall of nose. Conchae or turbinates are scrolllike bony projections covered by mucous membrane. The spaces below the turbinates are called meatuses (Figures 23.3 and 23.4).
Inferior turbinate. It is a separate bone and below it, into the inferior meatus, opens the nasolacrimal duct guarded at its terminal end by a mucosal valve called Hasner's valve.
Hiatus semilunaris
Figure 23.5 Lateral wall of nose. Middle turbinate is reflected upwards to show structures of the middle meatus.
Middle turbinate. It is an ethmoturbinal—a part of ethmoid bone. It is attached to the lateral wall by a bony lamella called ground or basal lamella. Its attachment is not straight but in an S-shaped manner. In the anterior third, it lies in sagittal plane and is attached to lateral edge of cribriform plate. In the middle third, it lies in frontal plane and is attached to lamina papyracea while in its posterior third, it runs horizontally and forms roof of the middle meatus and is attached to lamina papyracea and medial wall of maxillary sinus.
Middle meatus. It shows several important structures which are important in endoscopic surgery of the sinuses (Figure 23.5). Uncinate process is a hook-like structure running in from anterosuperior to posteroinferior direction. Its posterosuperior border is sharp and runs parallel to anterior border of bulla ethmoidalis; the gap between the two is called hiatus semilunaris (inferior). It is a two-dimensional space of 1-2 mm width.
The ostia of various sinuses draining anterior to basal lamella form anterior group of paranasal sinuses while those which open posterior and superior to it form the posterior group.
The anteroinferior border of uncinate process is attached to the lateral wall. Posteroinferior end of uncinate process is attached to inferior turbinate dividing the membranous part of lower middle meatus into
Figure 23.6 Upper attachment of uncinate process: (A) into lamina papyracea, (B) into skull base and (C) into middle turbinate thus affecting drainage of frontal sinus.
Figure

23.7
(A)
through middle
meatus.
Uncinate process
forms the
medial wall
and floor
of the
infundibulum.
(B)
Coronal sec-
tion showing
relationships of
uncinate process,
bulla ethmoidalis,
middle turbinate,
maxillary sinus,
orbit and
cribriform plate.
anterior and posterior fontanelle. The fontanel area is devoid of bone and consists of membrane only and leads into maxillary sinus when perforated. Upper attachment of uncinate process shows great variation and may be inserted into the lateral nasal wall, upwards into the base of skull or medially into the middle turbinate (Figure 23.6). This also accounts for variations in drainage of frontal sinus.
The space limited medially by the uncinate process and frontal process of maxilla and sometimes lacrimal bone, and laterally by the lamina papyracea is called infundibulum. Natural ostium of the maxillary sinus is situated in the lower part of infundibulum. Accessory ostium or ostia of maxillary sinus are sometimes seen in the anterior or posterior fontanel (Figure 23.7).
Bulla ethmoidalis. It is an ethmoidal cell situated behind the uncinate process. Anterior surface of the bulla forms the posterior boundary of hiatus semilunaris. Depending on pneumatization, bulla may be a pneumatized cell or a solid bony prominence. It may extend superiorly to the skull base and posteriorly to fuse with ground lamella. When there is a space above or behind the bulla, it is called suprabullar or retrobullar recesses, respectively (Figure 23.8). The suprabullar and retrobullar recesses together form the lateral sinus (sinus lateralis of Grunwald). The lateral sinus is thus bounded superiorly by the skull base, laterally
Figure 23.8 Axial view showing middle meatus and its structure. Note also the retrobullar recess.

by lamina papyracea, medially by middle turbinate and inferiorly by the bulla ethmoidalis. Posteriorly the sinus lateralis may extend up to basal lamella of middle turbinate. The cleft-like communication between the bulla and skull base and opening into middle meatus is also called hiatus semilunaris superior in contrast to hiatus semilunaris inferior referred to before.
Atrium of the middle meatus. It is a shallow depression lying in front of middle turbinate and above the nasal vestibule.
Agger nasi. It is an elevation just anterior to the attachment of middle turbinate. When pneumatized it contains air cells, the agger nasi cells, which communicate with the frontal recess. An enlarged agger nasi cell may encroach on frontal recess area, constricting it and causing mechanical obstruction to frontal sinus drainage.
Pneumatization of middle turbinate leads to an enlarged ballooned out middle turbinate called concha bullosa. It drains into frontal recess directly or through agger nasi cells. Haller cells are air cells situated in the roof of maxillary sinus. They are pneumatized from anterior or posterior ethmoid cells. Enlargement of Haller cells encroaches on ethmoid infundibulum, impeding draining of maxillary sinus.
Superior turbinate. It is also an ethmoturbinal and is situated posterior and superior to middle turbinate. It may also get pneumatized by one or more cells. It forms an important landmark to identify ostium of sphenoid sinus which lies medial to it.
Superior meatus. It is a space below the superior turbinate. Posterior ethmoid cells open into it. Number of posterior ethmoid cells varies from 1 to 5. Onodi cell is a posterior ethmoidal cell which may grow posteriorly by the side of sphenoid sinus or superior to it for as much distance as 1.5 cm from the anterior surface of sphenoid. Onodi cell is surgically important as the optic nerve may be related to its lateral wall.
Sphenoethmoidal recess.
It is
situated above
the superior
turbinate. Sphenoid sinus opens into it.
Supreme turbinate. It is sometimes present above the superior turbinate and has a narrow meatus beneath it.

The ostium of sphenoid sinus is situated in the sphenoethmoidal recess medial to the superior or supreme turbinate. It can be located endoscopically about 1 cm above the upper margin of posterior choana close to the posterior border of the septum.

Anterior sloping part of the roof is formed by nasal bones, posterior sloping part is formed by the body of sphenoid bone and the middle horizontal part is formed by the cribriform plate of ethmoid through which the olfactory nerves enter the nasal cavity.

It is formed by palatine process of the maxilla in its anterior three-fourths and horizontal part of the palatine bone in its posterior one-fourth.

1. Vestibule. It is lined by skin containing hair, hair follicles and sebaceous glands.
2. Olfactory region. Upper one-third of lateral wall (up to superior concha), corresponding part of the nasal septum and the roof of nasal cavity form the olfactory region. Here, mucous membrane is paler in colour.
3. Respiratory region. Lower two-thirds of the nasal cavity form the respiratory region. Here mucous membrane shows variable thickness being thickest over nasal conchae especially at their ends, quite thick over the nasal septum but very thin in the meatuses and floor of the nose. It is highly vascular and also contains erectile tissue. Its surface is lined by pseudostratified ciliated columnar epithelium which contains plenty of goblet cells. In the submucous layer of mucous membrane are situated serous, mucous, both serous and mucous secreting glands, the ducts of which open on the surface of mucosa.

1. Olfactory nerves. They carry sense of smell and supply olfactory region of nose. They are the central filaments of the olfactory cells and are arranged into 12-20 nerves which pass through the cribriform plate and end in the olfactory bulb. These nerves can carry sheaths of dura, arachnoid and pia with them into the nose. Injury to these nerves can open CSF space leading to CSF rhinorrhoea or meningitis (Figure 23.9).
2. Nerves of common sensation. They are:
(a) Anterior ethmoidal nerve.
(b) Branches of sphenopalatine ganglion.
(c) Branches of infraorbital nerve. They supply vestibule of nose both on its medial and lateral side.
Most of the posterior two-thirds of nasal cavity (both septum and lateral wall) are supplied by branches of sphenopalatine ganglion which can be blocked by placing a pledget of cotton soaked in anaesthetic solution near the sphenopalatine foramen situated at the posterior extremity of middle turbinate. Anterior ethmoidal nerve which supplies anterior and superior part of the nasal cavity (lateral wall and septum) can be blocked by placing the pledget high up on the inside of nasal bones where the nerve enters.
3. Autonomic nerves. Parasympathetic nerve fibres supply the nasal glands and control nasal secretion. They come from greater superficial petrosal nerve, travel in the nerve of pterygoid canal (vidian nerve) and reach the sphenopalatine ganglion where they relay before reaching the nasal cavity. They also supply the blood vessels of nose and cause vasodilation.
Sympathetic nerve fibres come from upper two thoracic segments of spinal cord, pass through superior cervical ganglion, travel in deep petrosal nerve and join the parasympathetic fibres of greater petrosal nerve to form the nerve of pterygoid canal (vidian nerve). They reach the nasal cavity without relay in the sphenopalatine ganglion. Their stimulation causes vasoconstriction. Excessive rhinorrhoea in cases

Figure 23.9 Nerve supply of nose. (A) Lateral wall. Sohenopalatine ganglion situated at the posterior end of middle turbinate supplies most of posterior two-thirds of nose. (B) Nerves on the medial wall.
of vasomotor and allergic rhinitis can be controlled by section of the vidian nerve.

Both the internal and external carotid systems supply the nose. Details of blood supply are given on p. 176.
Lymphatics from the external nose and anterior part of nasal cavity drain into submandibular lymph nodes while those from the rest of nasal cavity drain into upper jugular nodes either directly or through the retropharyngeal nodes. Lymphatics of the upper part of nasal cavity communicate with subarachnoid space along the olfactory nerves.

Functions of the nose are classified as:
. Respiration. Air-conditioning of inspired air. De
. Protection of lower airway.
Vocal resonance.
. Nasal reflex functions. Olfaction. Qo

Nose is the natural pathway for breathing. Mouth breathing is an acquired act through learning. So natural is the instinct to breath through the nose that a newborn infant with choanal atresia may asphyxiate to death if urgent measures are not taken to relieve it. The nose also permits breathing and eating to go on simultaneously.
During quiet respiration, inspiratory air current passes through middle part of nose between the turbinates and nasal septum. Very little air passes through inferior meatus or olfactory region of nose (Figure 24.1). Therefore, weak odorous substances have to be sniffed before they can reach the olfactory area.
During expiration, air current follows the same course as during inspiration, but the entire air current is not expelled directly through the nares. Friction offered at limen nasi converts it into eddies under cover of inferior and middle turbinates and this ventilates the sinuses through the ostia.
Anterior end
of inferior
turbinate undergoes
swelling and
shrinkage thus
regulating inflow
of air.
Nasal cycle. Nasal mucosa undergoes rhythmic cyclical congestion and decongestion, thus controlling the airflow through nasal chambers. When one nasal chamber is working, total nasal respiration, equal to that of both nasal chambers, is carried out by it. Nasal cycle varies every 2-4 h and may be characteristic of an individual.

Nose is aptly called the "air-conditioner" for lungs. It filters and purifies the inspired air and adjusts its temperature and humidity before the air passes to the lungs.
1.
Filtration and
purification.
Nasal vibrissae
at the
entrance of
nose act
cotton.
adhere as
Finer to
the filters
to sift
particles larger
like mucus
which particles
dust, is
pollen spread
like
a
like fluffs
of and
sheet bacteria
all over
the surface of the mucous membrane. The front of the nose can filter particles up to 3 um, while nasal mucus traps particles as fine as 0.5-3.0 pm. Particles smaller than 0.5 ym seem to pass through the nose into lower airways without difficulty.
2. Temperature control of the inspired air. It is regulated by large surface of nasal mucosa which is structurally adapted to perform this function. This mucous membrane, particularly in the region of middle and inferior turbinates and adjacent parts of the septum, is highly vascular with cavernous venous spaces or sinusoids which control the blood flow, and this increases or decreases the size of turbinates. This also makes an efficient "radiator" mechanism to warm up the cold air. Inspired air which may be at 20°C or 0°C or even at subzero temperature is heated to near body temperature (37°C) in one-fourth of second, the time that the air takes to pass from the nostril to the nasopharynx. Similarly, hot air is cooled to the level of body temperature.
3. Humidification. This function goes on simultaneously with the temperature control of inspired air. Relative humidity of atmospheric air varies depending on climatic conditions. Air is dry in winter and saturated with moisture in summer months. Nasal mucous membrane adjusts the relative humidity of the inspired air to 75% or more. Water, to saturate the inspired air, is provided by the nasal mucous membrane which is rich in mucous and serous secreting glands. About 1000 mL of water is evaporated from the surface of nasal mucosa in 24h.

Moisture is essential for integrity and function of the ciliary epithelium. At 50% relative humidity, ciliary function stops in 8-10 min. Thus, dry air predisposes to infections of the respiratory tract. Humidification also has a significant effect on gas exchange in the lower airways. In nasal obstruction, gaseous exchange is affected in the lungs, leading to rise in pCOg, causing apnoeic spells during sleep; it also decreases pOo.

1. Mucociliary mechanism. Nasal mucosa is rich in goblet cells, secretory glands both mucous and serous. Their secretion forms a continuous sheet called mucous blanket spread over the normal mucosa. Mucous blanket consists of a superficial mucus layer and a deeper serous layer, floating on the top of cilia which are constantly beating to carry it like a “conveyer belt” towards the nasopharynx
and carry
organisms and
dust particles.
(Figure 24.2). It moves at a speed of 5-10 mm/min and the complete sheet of mucus is cleared into the pharynx every 10-20 min. The inspired bacteria, viruses and dust particles are entrapped on the viscous mucous blanket and then carried to the nasopharynx to be swallowed. Presence of turbinates almost doubles the surface area to perform this function. About 600-700 mL of nasal secretions are produced in 24 h.
In mammals, cilia beat 10-20 times per second at room temperature. They have a rapid "effective stroke" and a slow "recovery stroke." In the former, the extended cilia reach mucus layer while in the recovery stroke, they bend and travel slowly in the reverse direction in the thin serous layer, thus moving the mucous blanket in only one direction. In immotile cilia syndrome, cilia are defective and cannot beat effectively, leading to stagnation of mucus in the nose and sinuses and bronchi causing chronic rhinosinusitis and bronchiectasis. Movements of cilia are affected by drying, drugs (adrenaline), excessive heat or cold, smoking, infections and noxious fumes like sulfur dioxide and carbon dioxide.
2. Enzymes and immunoglobulins. Nasal secretions also contain an enzyme called muramidase (lysozyme) which kills bacteria and viruses. Immunoglobulins IgA and IgE, and interferon are also present in nasal secretions and provide immunity against upper respiratory tract infections.
3. Sneezing. It is a protective reflex. Foreign particles which irritate nasal mucosa are expelled by sneezing. Copious
flow of nasal secretions that follows irritation by noxious substance helps to wash them out.

The pH of nasal secretion is nearly constant at 7. The cilia and the lysozyme act best at this pH. Alteration in nasal pH, due to infections or nasal drops, seriously impairs the functions of cilia and lysozyme.
So efficient are the functions of nose that 500 cubic feet of air, that we breathe every 24 h, is filtered, humidified, adjusted to proper temperature and cleared of all the dust, bacteria and viruses before reaching the lungs.

Nose forms a resonating chamber for certain consonants in speech. In phonating nasal consonants (M/N/NG), sound passes through the nasopharyngeal isthmus and is emitted through the nose. When nose (or nasopharynx) is blocked, speech becomes denasal, i.e. M/N/NGare uttered as B/D/G, respectively. It is to be remembered that in Hindi alphabets, last letter of a "varga" (ort : GG 7, US aah: wg, @ 8, 7 Uat: U, &, 4, 4, 4) is substituted by its third letter. Thus, an affected person utters @@T for AMT and aa for AT. Reverse is true in velopharyngeal insufficiency where 4M is substituted for @Tet.

Several reflexes are initiated in the nasal mucosa. Smell of a palatable food cause reflex secretion of saliva and gastric juice. Irritation of nasal mucosa causes sneezing. Nasal function is closely related to pulmonary functions through nasobronchial and nasopulmonary reflexes. It has been observed that nasal obstruction leads to increased pulmonary resistance and is reversed when nasal obstruction is surgically treated. Nasal packing in cases of epistaxis or after nasal surgery leads to lowering of pOy which returns to normal after removal of the pack. Pulmonary hypertension or cor pulmonale can develop in children with long-standing nasal obstruction due to tonsil and adenoid hypertrophy and can be reversed after removal of the tonsils and adenoids.

Sense of smell is well-developed in lower animals to give warning of the environmental dangers but it is comparatively less important in man. Still it is important for pleasure and for enjoying the taste of food. When nose is blocked, food tastes bland and unpalatable. Vapours of ammonia are never used to test the sense of smell as they stimulate fibres of the trigeminal nerve and cause irritation in the nose rather than stimulate the olfactory receptors.
1. Olfactory pathways. Smell is perceived in the olfactory region of nose which is situated high up in the nasal cayity. This area contains millions of olfactory receptor cells. Peripheral process of each olfactory cell reaches the mucosal surface and is expanded into a ventricle with several cilia on it. This acts as a sensory receptor to receive odor ous substances. Central processes of the olfactory cells are grouped into olfactory nerves which pass through the cribriform plate of ethmoid and end in the mitral cells of the olfactory bulb. Axons of mitral cells form olfactory tract and carry smell to the prepyriform cortex and the amygdaloid nucleus where it reaches consciousness. Olfactory system is also associated with autonomic system at the hypothalamic level.
2. Disorders of smell. It is essential for the perception of smell that the odorous substance be volatile and that it should reach the olfactory area unimpeded. Also necessary are the healthy state of olfactory mucosa and the integrity of neural pathways, i.e. olfactory nerves, olfactory bulb and tract and the cortical centre of olfaction.
Anosmia is total loss of sense of smell while hyposmia is partial loss. They can result from nasal obstruction due to nasal polypi, enlarged turbinates or oedema of mucous membrane as in common cold, allergic or vasomotor rhinitis. Anosmia is also seen in atrophic rhinitis, a degenerative disorder of nasal mucosa; peripheral neuritis (toxic or influenzal); injury to olfactory nerves or olfactory bulb in fractures of anterior cranial fossa; and intracranial lesions like abscess, tumour or meningitis which cause pressure on olfactory tracts.

Parosmia is perversion of smell; the person interprets the odours incorrectly. Often these persons complain of disgusting odours. It is seen in the recovery phase of postinfluenzal anosmia and the probable explanation is misdirected regeneration of nerve fibres. Intracranial tumour should be excluded in all cases of parosmia.
Sense of smell can be tested by asking the patient to smell common odours such as lemon, peppermint, rose, garlic or cloves from each side of the nose separately, with eyes closed. Quantitative estimation (quantitative olfactometry) requires special equipment.

Aim of these operations is only the appearance of nose but also its function.
to correct not outer

The nasal skin may be invaded by streptococci or staphylococci leading to a red, swollen and tender nose. Sometimes, it is an extension of infection from the nasal vestibule. Treatment is systemic antibacterials, hot fomentation and analgesics.

Depressed nasal dorsum may involve bony, cartilaginous or both bony and cartilaginous components of nasal dorsum (Figure 25.1). Nasal trauma causing depressed fractures is the most common aetiology. It can also result from excessive removal of septum in submucous resection, destruction of septal cartilage by haematoma or abscess, sometimes by leprosy, tuberculosis or syphilis. The deformity can be cor rected by augmentation rhinoplasty by filling the dorsum with cartilage, bone or a synthetic implant. If depression is only cartilaginous, cartilage is taken from the nasal septum or auricle and laid in a single or multiple layers. If deformity involves both cartilage and bone, cancellous bone from the iliac crest is the best. Autografts (taken from the same individual) are preferred to allografts (taken from other individuals or cadavers). Saddle deformity can also be corrected by synthetic implants of silicone or teflon but they are likely to be extruded.

This may also involve the bone or cartilage or both bone and cartilage. It can be corrected by reduction rhinoplasty which consists of exposure of nasal framework by careful raising of the nasal skin by a vestibular incision, removal of hump and narrowing of the lateral walls by osteotomies to reduce the left removal.
widening by hump

In crooked nose, the midline of dorsum from frontonasal angle to the tip is curved in a Cor S-shaped manner. In a deviated nose, the midline is straight but deviated to one side

Usually, these deformities are traumatic in origin. Injuries sustained during birth, neonatal period or childhood, but not immediately recognized, will also develop into these deformities with the growth of nose. The deviated or crooked nose can be corrected by rhinoplasty or septorhinoplasty.

They may be congenital, benign or malignant (Table 25.1).
1. CONGENITAL TUMOURS
(a) Dermoid cyst (Figure 25.3). It is of two types:
© Simple dermoid. It occurs as a midline swelling under the skin but in front of the nasal bones. It does not have any external opening.
Figure
25.2
but
Nasal bridge
is
S-shaped in
crooked nose.
It is
deviated to
one side
in deviated
nose.
straight
¢ Dermoid with a sinus. It is seen in infants and children and is represented by a pit or a sinus in the midline of the dorsum of nose. Hair may be seen protruding through the sinus opening. In these cases, the sinus track may lead to a dermoid cyst lying under the nasal bone in front of upper part of nasal septum or may have an intracranial dural connection. In those with intracranial extension, sinus tract passes through the cribriform plate or foramen caecum and is attached to dura or has other intracranial connection. Meningitis occurs if infection travels along this path. Treatment of such cysts may necessitate splitting of the nasal bones to remove any extension in the upper part of the nasal septum. A combined neurosurgical—otolaryngologic approach is required in those extending intracranially so as to close simultaneously any bony defect through which the fistulous tract passed (Figure 25.4).
(b) Encephalocele or meningoencephalocele. It is herniation of brain tissue along with its meninges through a congenital bony defect. An extranasal meningoencephalocele presents as a subcutaneous pulsatile swelling in the midline at the root of nose (nasofrontal variety), side of nose (nasoethmoid variety) or on the anteromedial aspect of the orbit (naso-orbital variety) .
Swellings show cough impulse and may be reducible. Treatment is neurosurgical; severing the tumour stalk from

the brain and repairing the bony defect through which herniation has taken place.
(c) Glioma. It is a nipped off portion of encephalocele during embryonic development. Most of them (60%) are extranasal and present as firm subcutaneous swellings on the bridge, side of nose or near the inner canthus. Some of them are purely intranasal (30%), while 10% are both intraand extranasal. Extranasal gliomas are encapsulated and can be easily removed by external nasal approach.

They arise from the nasal skin and include papilloma (skin wart), haemangioma, pigmented naevus, seborrhoeic keratosis, neurofibroma or tumour of sweat glands.
Rhinophyma or potato tumour is a slow-growing benign tumour due to hypertrophy of the sebaceous glands of the tip of nose often seen in cases of long-standing acne rosacea. It presents as a pink, lobulated mass over the nose with superficial vascular dilation; mostly affects men past middle age (Figure 25.4). Patient seeks advice because of the unsightly appearance of the tumour, or obstruction to breathing and vision due to large size of the tumour. Treatment consists of paring down the bulk of tumour with sharp knife or carbon dioxide laser and the area allowed to reepithelialize. Sometimes, tumour is completely excised and the raw area skin grafted.
Figure
25.4
Rhinophyma.
Figure 25.3 Types of dermoids. (A) Simple dermoid beneath the skin. (B) Dermoid with an external pit or sinus. It lies in front of septum and

(a) Basal cell carcinoma (rodent ulcer) (Figure 25.5). This is the most common malignant tumour involving skin of nose (87%), equally affecting males and females in the age group of 40-60 years. Common sites on the nose are the tip and the ala. It may present as a cyst or papulo-pearly nodule or an ulcer with rolled edges. It is very slow growing and remains confined to the skin for a long time. Underlying cartilage or bone may get invaded. Nodal metastases are extremely rare. Treatment depends on the size, location and depth of the tumour. Early lesion can be cured by cryosurgery, irradiation or surgical excision with 3-5 mm of healthy skin around the palpable borders of the tumour.
Lesions which are recurrent, extensive or with involvement of cartilage or bone are excised and the surgical defect closed by local or distant flaps or a prosthesis.
(b) Squamous cell carcinoma (epithelioma). This is the second most common malignant tumour (11%), equally affecting both sexes in 40-60 age group. It occurs as an infiltrating nodule or an ulcer with rolled out edges affecting side of nose or columella (Figure 25.6). Nodal metastases are seen in 20% of cases.
Figure 25.5 Basal cell carcinoma of the nose.
Early lesions respond to radiotherapy; more advanced lesions or those with exposure of bone or cartilage require wide surgical excision and plastic repair of the defect. Enlarged regional lymph nodes will require block dissection.
(c) Melanoma. This is the least common variety. Clinically, it is superficially spreading type (slow growing) or nodular invasive type. Treatment is surgical excision.

It is an acute infection of the hair follicle by Staphylococcus aureus. Trauma from picking of the nose or plucking the nasal vibrissae is the usual predisposing factor.
The lesion is small but exquisitely painful and tender. Inflammation may spread to the skin of nasal tip and dor sum which become red and swollen. The furuncle may rupture spontaneously in the nasal vestibule.
Treatment of furuncle consists of anal- warm compresses, gesics to relieve pain, and topical and systemic antibiotics directed against staphylococcus. If a fluctuant area appears, incision and drainage can be done. In no case should the furuncle be squeezed or prematurely incised because of the danger of spread of infection to cavernous sinus through venous thrombophlebitis.
A furuncle of nose may complicate into cellulitis of the lip or septal abscess.
upper

It is diffuse dermatitis of nasal vestibule. Nasal discharge, due to any cause such as rhinitis, sinusitis or nasal allergy, coupled with trauma of handkerchief, is the usual predisposing factor. The causative organism is S. aureus. Vestibulitis may be acute or chronic.
In acute form, vestibular skin is red, swollen and tender; crusts and scales cover an area of skin erosion or excoriation. The upper lip may also be involved (Figure 25.8).
In chronic form, there is induration of vestibular skin with painful fissures and crusting.
Figure 25.8 Acute vestibulitis (left side).
Treatment consists of cleaning the nasal vestibule of all crusts and scales with cotton applicator soaked in hydrogen peroxide and application of antibiotic-steroid ointment. The latter should always be continued for a few more days, even after the apparent cure, as the condition is likely to relapse. A chronic fissure can be cauterized with silver nitrate. Attention should be paid to the cause of nasal discharge.

Accidental or surgical trauma to the nasal tip or vestibule can lead to web formation and stenosis of anterior nares. In Young's operation, vestibular skin flaps are raised to create deliberate closure of nares in the treatment of atrophic rhinitis (seep. 153). Destructive inflammatory lesions of nose also cause stenosis. Earlier, several cases of vestibular stenosis resulted from smallpox (Figure 25.9).
Congenital atresia of anterior nares due to noncanalization of epithelial plug is a rare condition.
Stenosis of nares can be corrected by reconstructive plas- tic procedures.

1. Nasoalveolar cyst presents a smooth bulge in the lateral wall and floor of nasal vestibule. The cyst can be ye
Figure 25.10 Nasoalveolar cyst as seen during operation.
excised by sublabial approach preserving the integrity of vestibular skin (Figure 25.10).
Papilloma or wart may be single or multiple, pedunculated or sessile. Treatment is surgical excision under local anaesthesia.
Squamous cell carcinoma arises from the lateral wall of the vestibule and may extend into nasal floor, columella and
upper lip. It can metastasize to the parotid and submandibular nodes. Treatment is surgical excision or irradiation.

depression of lower part of nose and drooping of the nasal tip.
Nasal septum consists of three parts:
1. Columellar septum. It is formed of columella containing the medial crura of alar cartilages united together by fibrous tissue and covered on either side by skin.
2. Membranous septum. It consists of double layer of skin with no bony or cartilaginous support. It lies between the columella and the caudal border of septal cartilage. Both columellar and membranous parts are freely movable from side to side.
3. Septum proper. It consists of osteocartilaginous framework, covered with nasal mucous membrane. Its principal constituents are (Figure 26.1):
(a) the perpendicular plate of ethmoid,
(b) the vomer and
(c) alarge septal (quadrilateral) cartilage wedged between the above two bones anteriorly. Other bones which make minor contributions at the periphery are crest of nasal bones, nasal spine of frontal bone, rostrum of sphenoid, crest of palatine bones and the crest maxilla, and the anterior nasal spine of maxilla.
Septal cartilage not only forms a partition between the right and left nasal cavities but also provides support to the tip and dorsum of cartilaginous part of nose. Its destruction, e.g. in septal abscess, injuries, tuberculosis or excessive removal during septal surgery, leads to
Crest of palatine bone
Figure
26.1.
Anatomy of nasal septum.
Septal cartilage lies in a groove in the anterior edge of vomer and rests anteriorly on anterior nasal spine. During trauma, it may get dislocated from anterior nasal spine or vomerine groove causing caudal septal deviation or septal spur, respectively. This compromises the nasal airway. Septal cartilage is also intimately related to the upper lateral cartilages of nose and is in fact fused with them in the upper third. For this reason septal deviation may be associated with deviation of cartilaginous part of external nose.
Blood Vessels of Nasal Septum (see Chapter 33). Nerve Supply of Nasal Septum (see Chapter 23).

Little's area or Kiesselbach's plexus. This is the vascular area in the anteroinferior part of nasal septum just above the vestibule. Anterior ethmoidal, sphenopalatine, greater palatine and septal branch of superior labial arteries and their corresponding veins form an anastomosis here. This is the commonest site for epistaxis. This is also the site for origin of the "bleeding polypus" (haemangioma) of nasal septum.

Trauma inflicted on the nose from the front, side or below can result in injuries to the nasal septum. The septum may buckle on itself, fracture vertically, horizontally or be crushed to pieces as in a smashed nose. The fractured pieces of septum may overlap each other or project into the nasal cavity through mucosal tears. Fracture of the septal cartilage or its dislocation from the vomerine groove, can result from trauma to the lower nose without associated fractures of nasal bones. Septal injuries with mucosal tears cause profuse epistaxis while those with intact mucosa result in septal haematoma which, if not drained early, will cause absorption of the septal cartilage and saddle nose deformity. "Jarjaway" fracture of nasal septum results from blows from the front; it starts just above the anterior nasal spine and runs horizontally backwards just above the junction of septal cartilage with the vomer (Figure 26.2A). "Chevallet" fracture of septal cartilage results from blows from below; it runs vertically from the anterior nasal spine upwards to the junction of bony and cartilaginous dorsum of nose (Figure 26.2B).
Figure 26.2 Septal fracture showing: (A) Jarjaway type. (B) Chevallet type.

Early recognition and treatment of septal injuries is essential. Haematomas should be drained. Dislocated or fractured septal fragments should be repositioned and supported between mucoperichondrial flaps with mattress sutures and nasal packing. Fractures of nasal pyramid are often complicated with fractures of the septum and both should be treated concomitantly.

Septum is important in supporting the lower part of the external nose. If its injuries are ignored, they would result in deviation of the cartilaginous nose, or asymmetry of nasal tip, columella or the nostril.

Trauma and errors of development form the two important factors in the causation of deviated septum.
1. Trauma. A lateral blow on the nose may cause displacement of septal cartilage from the vomerine groove and maxillary crest, while a crushing blow from the front may cause buckling,
Figure
26.3
DNS
high-arched palate.
twisting, fractures and duplication of nasal septum with telescoping of its fragments. Injuries to the nose commonly occur in childhood but are often overlooked. Even the history may not be forthcoming. Trauma may also be inflicted at birth during difficult labour when nose is pressed during its passage through the birth canal. Birth injuries should be immediately attended to as they result in septal deviation later in life.
2. Developmental error. Nasal septum is formed by the tectoseptal process which descends to meet the two halves of the developing palate in the midline. During the primary and secondary dentition, further development takes place in the palate, which descends and widens to accommodate the teeth. Unequal growth between the palate and the base of skull may cause buckling of the nasal septum. In mouth breathers, as in adenoid hypertrophy, the palate is often highly arched and the septum is deviated (Figure 26.3). Similarly, DNS may be seen in cases of cleft lip and palate and in those with dental abnormalities.
3. Racial factors. Caucasians are affected more than black Americans.
4. Hereditary factors. Several members of the same family have deviated nasal may septum.

Deviation may involve only the cartilage, bone or both the cartilage and bone.
Figure 26.5 Anterior dislocation. Caudal border of septal cartilage projects into right naris.
1. Anterior dislocation. Septal cartilage may be dislocated into one of the nasal chambers. This is better appreciated by looking at the base of nose when patient's head is tilted backwards (Figure 26.5).
2. C-shaped deformity. Septum is deviated in a simple curve to one side. Nasal chamber on the concave side of the nasal septum will be wider and may show compensatory hypertrophy of turbinates.
3. S-shaped deformity. Either in vertical or anteroposterior plane. Such a deformity may cause bilateral nasal obstruction. 4. Spurs. A spur is a shelf-like projection often found at the junction of bone and cartilage. A spur may press on the lateral wall and gives rise to headache. It may also predispose to repeated epistaxis from the vessels stretched on its convex surface.
5. Thickening. It may be due to organized haematoma or overriding of dislocated septal fragments.

DNS can involve any age and sex. Males are affected more than females.
1. Nasal obstruction. Depending on the type of septal deformity, obstruction may be unilateral or bilateral. Respiratory currents pass through upper part of nasal cavity, therefore, high septal deviation cause nasal obstruction more than lower ones.
When examining a case of nasal obstruction, one should ascertain the site of obstruction in the nose. It could be (i) vestibular (caudal septal dislocation, synechiae or stenosis), (ii) at the nasal valve (synechiae, usually postrhinoplasty), (iii) attic (along the upper part of nasal septum due to high septal deviation; (iv) turbinal (hypertrophic turbinates or concha bullosa) and (v) choanal (choanal atresia or a choanal polyp). Unilateral choanal atresia may be missed in infancy and childhood. Choanal polyp may be missed on the anterior rhinoscopy unless posterior rhinoscopy or nasal endoscopy is done.
Cottle test.
It is
used in
nasal obstruction
due to
abnormality of
the nasal
valve.
In this
test, cheek
is drawn
laterally while
the
Figure 26.6 Cottle test: On pulling the cheek away from the midline, the nasal valve opens, increasing the airflow from that side of the nasal cavity.
patient breathes quietly. If the nasal airway improves on the test side, the test is positive and indicates abnormality of the vestibular component of nasal valve (Figure 26.6).
2. Headache. Deviated septum, especially a spur, may press on the lateral wall of nose giving rise to pressure headache. 3. Sinusitis. Deviated septum may obstruct sinus ostia resulting in poor ventilation of the sinuses. Therefore, it forms an important cause to predispose or perpetuate sinus infections. 4, Epistaxis. Mucosa over the deviated part of septum is exposed to the drying effects of air currents leading to formation of crusts, which when removed causes bleeding. Bleeding may also occur from vessels over a septal spur.

5. Anosmia. Failure of the inspired air to reach the olfactory region may result in total or partial loss of sense of smell.
6. External deformity. Septal deformities may be associated with deviation of the cartilaginous or both the bony and cartilaginous dorsum of nose, deformities of the nasal tip or columella.
7. Middle ear infection. DNS also predisposes to middle ear infection.

Minor degrees of septal deviation with no symptoms are commonly seen in patients and require no treatment. It is only when deviated septum produces mechanical nasal obstruction or the symptoms given above that an operation is indicated.

It is generally done in adults under local anaesthesia. It consists of elevating the mucoperichondrial and mucoperiosteal flaps on either side of the septal framework by a single incision made on one side of the septum, removing the
150
deflected parts of the bony and cartilaginous septum, and then repositioning the flaps (see section on Operative Surgery for details).

It is a conservative approach to septal surgery. In this operation, much of the septal framework is retained. Only the most deviated parts are removed. Rest of the septal framework is corrected and repositioned by plastic means. Mucoperichondrial/periosteal flap is generally raised only on one side of the septum, retaining the attachment and blood supply on the other. Septoplasty has now almost replaced SMR operation (see Chapter 87).
Septal surgery is usually done after the age of 17 so as not to interfere with the growth of nasal skeleton. However, if a child has severe septal deviation causing marked nasal obstruction, conservative septal surgery (septoplasty) can be performed to provide a good airway.

It is collection of blood under the perichondrium or periosteum of the nasal septum (Figure 26.7). It often results from nasal trauma or septal surgery. In bleeding disorders, it may occur spontaneously.

Bilateral nasal obstruction is the commonest presenting symptom. This may be associated with frontal headache and a sense of pressure over the nasal bridge.
Examination reveals smooth rounded swelling of the septum in both the nasal fossae. Palpation may show the mass to be soft and fluctuant.

Small haematomas can be aspirated with a wide bore ster ile needle. Larger haematomas are incised and drained by a small anteroposterior incision parallel to the nasal floor. Excision of a small piece of mucosa from the edge of incision gives better drainage. Following drainage, nose is packed on both sides to prevent reaccumulation. Systemic antibiotics should be to abscess.
given prevent septal

Septal haematoma, if not drained, may organize into fibrous tissue leading to a permanently thickened septum. If secondary infection supervenes, it results in septal abscess with necrosis of cartilage and depression of nasal dorsum.

Mostly, it results from secondary infection of septal haema- toma. Occasionally, it follows furuncle of the nose or upper lip.
It may also follow acute infection such as typhoid or measles.
Figure 26.7 Septal haematoma.
Figure 26.8 Septal abscess.

There is severe bilateral nasal obstruction with pain and tenderness over the bridge of nose. Patient may also complain of fever with chills and frontal headache. Skin over the nose may be red and swollen. Internal examination of nose reveals smooth bilateral swelling of the nasal septum (Figure 26.8). Fluctuation can be elicited in this swelling. Septal mucosa is often congested. Submandibular lymph nodes may also be enlarged and tender.

Abscess should be drained as early as possible. Incision is made in the most dependent part of the abscess and a piece of septal mucosa excised. Pus and necrosed pieces of cartilage are removed by suction. Incision may require to be reopened daily for 2-3 days to drain any pus or to remove any necrosed pieces of cartilage. Systemic antibiotics are started as soon as diagnosis has been made and continued at least for a of 10
period days.

Necrosis of septal cartilage often results in depression of the cartilaginous dorsum in the supratip area and may require augmentation rhinoplasty 2-3 months later. Necrosis of
Figure 26.9 Septal perforation.
septal flaps may lead to septal perforation. Meningitis and cavernous sinus thrombosis following septal abscess, though rare these days, can be a serious complication.

1. Traumatic perforations. Trauma is the most common cause. Injury to mucosal flaps during SMR, cauterization of septum with chemicals or galvanocautery for epistaxis and habitual nose picking are the common forms of trauma. Occasionally, septum is deliberately perforated to put ornaments.

- (a) Septal abscess.
(b) Nasal myiasis.
(c) Rhinolith or neglected foreign body causing pressure necrosis.
(d) Chronic granulomatous conditions like lupus, tuberculosis and leprosy cause perforation in the cartilaginous part while syphilis involves the bony part. In these cases, evidence of the causative disease may also be seen in other systems of the body.
(e) Wegener's granuloma is a midline destructive lesion which may cause total septal destruction.
Figure 26.10 Septal button for closure of perforation.

(a)
Prolonged use of steroid sprays in nasal allergy.
(b)
Cocaine addicts.
(c)
Workers in certain occupations, e.g. chromium plating,
dichromate or soda ash (sodium carbonate) manufac-
ture or those exposed to arsenic or its compounds.
4, Idiopathic. In many cases, there is no history of trauma or previous disease and the patient may even be unaware of the existence of a perforation.

Small anterior perforations cause whistling sound during inspi- ration or expiration. Larger perforations develop crusts which
obstruct the nose or cause severe epistaxis when removed.

An attempt should always be made to find out the cause before treatment of perforation. This may require biopsy from the granulations or biopsy of the edge of the perforation. Inactive small perforations can be surgically closed by plastic flaps. Larger perforations are difficult to close. Their treatment is aimed to keep the nose crust-free by alkaline nasal douches and application of a bland ointment. Sometimes, a thin silastic button can be worn to get relief from the symptoms (Figure 26.10).

form in the nose, which with attempted removal causes bleeding.
Acute rhinitis can be viral, bacterial or irritative type.

1. Common cold (coryza)
¢ Aetiology. It is caused by a virus. The infection is usually contracted through airborne droplets. Several viruses (adenovirus, picornavirus and its subgroups such as rhinovirus, coxsackie virus and enteric cytopathic human orphan virus) are responsible. Incubation period is 1-4 days and illness lasts for 2-3 weeks.
© Clinical features. To begin with, there is burning sensation at the back of nose soon followed by nasal stuffiness, rhinorrhoea and sneezing. Patient feels chilly and there is low-grade fever. Initially, nasal discharge is watery and profuse but may become mucopurulent due to secondary bacterial invasion. Secondary invaders include Streptococcus haemolyticus, pneumococcus, Staphylococcus, Haemophilus influenzae, Klebsiella pneumoniae and Moraxella catarrhalis.
e Treatment. Bed rest is essential to cut down the course of illness. Plenty of fluids are encouraged. Symptoms can be easily controlled with antihistaminics and nasal decongestants. Analgesics are useful to relieve headache, fever and myalgia. Nonaspirin containing analgesics are preferable as aspirin causes increased shedding of virus. Antibiotics
when secondary
infection supervenes.
- © Complications. The disease is usually self-limiting and resolves spontaneously after 2-3 weeks, but occasionally, complications such as sinusitis, pharyngitis, tonsillitis, bronchitis, pneumonia and otitis media may result.
2. Influenzal rhinitis. Influenza viruses A, B or C are respon- sible.
Symptoms and
signs are
similar to
those of
common cold. Complications due to bacterial invasion are common.
3. Rhinitis associated with exanthemas. Measles, rubella and chickenpox are often associated with rhinitis which precedes exanthemas by 2-3 days. Secondary infection and are more and severe.
complications frequent

Nonspecific infections. It may be primary or secondary. Primary bacterial rhinitis is seen in children and is usually the result of infection with pneumococcus, streptococcus or staphylococcus. A greyish white tenacious membrane may bacterial rhinitis is the result of bacterial infec-
tion supervening acute
Secondary viral rhinitis.
Diphtheritic rhinitis. Diphtheria of nose is rare these days. It may be primary or secondary to faucial diphtheria and may occur in acute or chronic form. A greyish membrane is seen covering the inferior turbinate and the floor of nose; membrane is tenacious and its removal causes bleeding. Excoriation of anterior nares and upper lip may be seen. Treatment is isolation of the patient, systemic penicillin and diphtheria antitoxin.

This form of acute rhinitis is caused by exposure to dust, smoke or irritating gases such as ammonia, formaline, acid fumes, etc. or it may result from trauma inflicted on the nasal mucosa during intranasal manipulation, e.g. removal of a foreign body. There is an immediate catarrhal reaction with sneezing, rhinorrhoea and nasal congestion. The symptoms may pass off rapidly with removal of the offending agent or may persist for some days if nasal epithelium has been damaged. Recovery will depend on the amount of and the infection that epithelial damage supervenes.

Chronic nonspecific inflammations of nose include:
. Chronic simple rhinitis. Hypertrophic rhinitis. Ne
. Atrophic rhinitis. oo
Rhinitis sicca.
. Rhinitis caseosa.
CUR

Recurrent attacks of acute rhinitis in the presence of predis- posing factors leads to chronicity. The predisposing factors are:
1. Persistence of nasal infection due to sinusitis, tonsillitis and adenoids.
2. Chronic irritation from dust, smoke, cigarette smoking, snuff, etc.
3. Nasal obstruction due to deviated nasal septum, synechia leading to persistence of discharge in the nose.
4. Vasomotor rhinitis.
5. Endocrinal or metabolic factors, e.g. hypothyroidism, excessive intake of carbohydrates and lack of exercise.

Simple chronic rhinitis is an early stage of hypertrophic rhinitis. There is hyperaemia and oedema of mucous membrane with hypertrophy of seromucinous glands and increase in goblet cells. Blood sinusoids particularly those over the turbinates are distended.

1. Nasal obstruction. Usually worse on lying and affects the dependent side of nose.
2. Nasal discharge. It may be mucoid or mucopurulent, thick and viscid and often trickles into the throat as postnasal drip. Patient has a constant desire to blow the nose or clear the throat.
3. Headache. It is due to swollen turbinates impinging on the nasal septum.
4. Swollen turbinates. Nasal mucosa is dull red in colour. Turbinates are swollen; they pit on pressure and shrink with application of vasoconstrictor drops (this differentiates the condition from hypertrophic rhinitis). Middle turbinate may also be swollen and impinge on the septum.
5. Postnasal discharge. Mucoid or mucopurulent discharge is seen on the posterior pharyngeal wall.

1. Treat the cause with particular attention to sinuses, ton- sils, adenoids, allergy, personal habits (smoking or alco- hol indulgence), environment or work situation (smoky or dusty surroundings).
2. Nasal irrigations with alkaline solution help to keep the nose free from viscid secretions and also remove superfi- cial infection.
3. Nasal decongestants help to relieve nasal obstruction and improve sinus ventilation. Excessive use of nasal drops and sprays should be avoided because it may lead to rhi- nitis medicamentosa. A short course of systemic steroids helps to wean the patients already addicted to excessive use of decongestant drops or sprays.
4. Antibiotics help to clear nasal infection and concomitant sinusitis.

It is characterized by thickening of mucosa, submucosa, seromucinous glands, periosteum and bone. Changes are more marked on the turbinates.

Common causes are recurrent nasal infections, chronic sinusitis, chronic irritation of nasal mucosa due to smoking, industrial irritants, prolonged use of nasal drops and vasomotor and rhinitis.
allergic

Nasal obstruction is the predominant symptom. Nasal discharge is thick and sticky. Some complain of headache, heaviness of head or transient anosmia.

Examination shows hypertrophy of turbinates. Turbinal mucosa is thick and does not pit on pressure. It shows little shrinkage with vasoconstrictor drugs due to presence of underlying fibrosis. Maximum changes are seen in the inferior turbinate. It may be hypertrophied in its entirety or only at the anterior end, posterior end or along the inferior border giving it a mulberry appearance.

Attempt should be made to discover the cause and remove it. Nasal obstruction can be relieved by reduction in size of turbinates. The various methods are:
1. Linear cauterization.
2. Submucosal diathermy.
3. Cryosurgery of turbinates.
4.
Partial or total turbinectomy. Hypertrophied inferior turbinate can be partially removed at its anterior end, inferior border or posterior end. Middle turbinate, if hypertrophied, can also be removed partially or totally. Excessive removal of turbinates should be avoided as it leads to persistent crusting.
5. Submucous resection of turbinate bone. This removes bony obstruction but preserves turbinal mucosa for its function.
6. Lasers have also been used to reduce the size of turbinates.

This is seen in cases of marked deviation of septum to one side. The roomier side of the nose shows hypertrophy of inferior and middle turbinates. This is an attempt on the part of nature to reduce the wide space to overcome the ill effects of drying and crusting that always attend wider nasal space. Hypertrophic changes in these cases are not reversible with the correction of nasal septum and often require reduction of turbinates at the time of septal surgery.

It is a chronic inflammation of nose characterized by atrophy of nasal mucosa and turbinate bones. The nasal cavities are roomy and full of foul-smelling crusts. Atrophic rhinitis is of two types: primary and secondary.

Aetiology (Remember Mnemonic HERNIA)
The exact cause is not known. Various theories advanced regarding its causation are:
2. Endocrinal disturbance. Disease usually starts at puberty,
3 . Racial factors. White and yellow races are more suscep- tible than natives of equatorial Africa.
1. Hereditary factors. Disease is known to involve more than one member in the same family.
involves
females
more
than
males,
the
crusting
and
foe-
tor
with
disease
tends
to
cease
after
meno-
pause;
these
factors
have
raised
the
possibility
of
disease
being
an
endocrinal
disorder.
Nutritional deficiency. Disease may be due to deficiency of vitamin A, D or iron or some other dietary factors.
5.
The fact
that ern
countries raises
the is
rarely possibility
Infective.
is decreasing
seen in
well-to-do of
some nutritional
Various cases
of organisms
atrophic rhinitis
bacillus), in
west- families
have been
such diphtheroids,
staphylococci as
cultured
Klebsiella
Proteus and
streptococci ered
to be
secondary rather
than vulgaris,
from ozaenae,
(Perez
Escherichia but
they coli,
are invaders
all consid-
responsible the
primary causative
for foul
organisms smell
of the
disease.
- 6. Autoimmune process. The body reacts by a destructive process to the antigens released from the nasal mucosa. Viral infection or some other unspecified agents may trigger antigenicity of nasal mucosa.

Ciliated columnar epithelium is lost and is replaced by stratified squamous type. There is atrophy of seromucinous glands, venous blood sinusoids and nerve elements. Arteries in the mucosa, periosteum and bone show obliterative endarteritis. The bone of turbinates undergoes resorption causing widening of nasal chambers. Paranasal sinuses are small due to their arrested development.

Disease is commonly seen in females and starts around puberty. There is foul smell from the nose making the patient a social outcast though patient himself is unaware of the smell due to marked anosmia (merciful anosmia) which accompanies these degenerative changes. Patient may complain of nasal obstruction in spite of unduly wide nasal chambers. This is due to large crusts filling the nose. Epistaxis may occur when the crusts are removed.
Examination shows nasal cavity to be full of greenish or greyish black dry crusts covering the turbinates and septum. Attempts to remove them may cause bleeding. When the crusts have been removed, nasal cavities appear roomy with atrophy of turbinates so much so that the posterior wall of nasopharynx can be easily seen. Nasal turbinates may be reduced to mere ridges. Nasal mucosa appears pale. Septal perforation and dermatitis of nasal vestibule may be present. Nose may show a saddle deformity.
Atrophic changes may also be seen in the pharyngeal mucosa which may appear dry and glazed with crusts (atrophic pharyngitis, p. 256).
Similar changes may occur in the larynx with cough and hoarseness of voice (atrophic laryngitis).
Hearing impairment may be noticed because of obstruction to eustachian tube and middle ear effusion.
Paranasal sinuses are usually small and underdeveloped with thick walls. They appear opaque on X-ray. Antral wash is difficult to perform due to thick walls of the sinuses.

The disease persists for years but there is a tendency to recover spontaneously in middle age.

It may be medical or surgical.
1. Medical. Complete cure of the disease is not yet possible.
Treatment aims at maintaining nasal hygiene by removal of crusts and the associated putrefying smell, and to further check crust formation.
(a) Nasal irrigation and removal of crusts. Warm normal saline or an alkaline solution made by dissolving a teaspoonful of powder containing soda bicarbonate 1| part, sodium biborate 1 part, sodium chloride 2 parts in 280 mL of water is used to irrigate the nasal cavities. The solution is run through one nostril and comes out from the other. It loosens the crusts and removes thick tenacious discharge. Care should be taken to avoid pushing the fluid into the sinuses and eustachian tube. Initially, irrigations are done two or three times a day but later once in every 2 or 3 days is sufficient. Hard crusts may be difficult to remove by irrigation. They are first loosened and then mechanically removed with forceps or suction.
(b) 25% glucose in glycerine. After crusts are removed, nose is painted with 25% glucose in glycerine. This inhibits the growth of proteolytic organisms which are responsible for foul smell.
(c) Local antibiotics. Spraying or painting the nose with appropriate antibiotics help to eliminate secondary infection. Kemicetine™ antiozaena solution contains chloromycetin, oestradiol and vitamin Dy and may be found useful.
(d) Oe6estradiol spray. Helps to increase vascularity of nasal mucosa and regeneration of seromucinous glands.
(e) Placental extract injected submucosally in the nose may provide some relief.
(f) Systemic use of streptomycin. 1 g/day for 10 days has given good results in reducing crusting and odour. It is effective against Klebsiella organisms.
(g) Potassium iodide given by the mouth promotes and liquefies nasal secretion.

(a)
Young’s operation.
pletely just
They are
within opened
mucosa
Both the
the nasal
after may
revert
Modified nostrils
vestibule
6
months to
Young’s bilateral
are by
or closed
raising later.
normal and
nasal
In com-
flaps.
these crusting
To obstruction,
aims to
partially avoid
cases, reduced.
the discomfort
modified close
the
Young’s nostrils.
It of
also claimed
to give
the same
benefit as
Young’s.
Narrowing the nasal cavities. Nasal chambers are very wide in atrophic rhinitis and air currents dry up secretions leading to crusting. Narrowing the size of the nasal airway helps to relieve the symptoms. Among the techniques followed, some are:
(i) Submucosal injection of teflon paste.
(ii) Insertion of fat, cartilage, bone or teflon strips under the mucoperiosteum of the floor and lateral wall of nose and the mucoperichondrium of the septum.
(iii)
Section and
medial displacement
of lateral
wall of
nose.

Specific infections like syphilis, lupus, leprosy and rhinoscleroma may cause destruction of the nasal structures leading to atrophic changes. Atrophic rhinitis can also result from
(b)
long-standing purulent sinusitis, radiotherapy to nose or excessive surgical removal of turbinates.
Unilateral atrophic rhinitis. Extreme deviation of nasal septum may be accompanied by atrophic rhinitis on the wider side.

Itis also a crust-forming disease seen in patients who work in hot, dry and dusty surroundings, e.g. bakers, ironand goldsmiths. Condition is confined to the anterior third of nose particularly of the nasal septum. Here, the ciliated columnar epithelium undergoes squamous metaplasia with atrophy of seromucinous glands. Crusts form on the anterior part of septum and their removal causes ulceration and epistaxis, and may lead to septal perforation.
Treatment consists of correction of the occupational surroundings and application of bland ointment or one with an antibiotic and steroid to the affected part. Nose pricking and forcible removal of crusts should be avoided. Nasal douche, like the one used in cases of atrophic rhinitis, is useful.

It is an uncommon condition, usually unilateral and mostly affecting males.
Nose is filled with offensive purulent discharge and cheesy material. The disease possibly arises from chronic sinusitis with collection of inspissated cheesy material. Sinus mucosa becomes granulomatous. Bony walls of sinus may be destroyed, requiring differentiation from malignancy. Treatment is removal of debris and granulation tissue and free drainage of the affected sinus. Prognosis is good.

Various granulomatous lesions involving the nose are listed in Table 28.1. They are the result of bacterial or fungal infections or due to causes not yet clear. Many of these lesions may be manifestations of systemic diseases, which should always be looked for while making the diagnosis. Biopsy of the lesion is also essential, not only to establish the correct diagnosis of granulomatous disease but also to exclude a neoplasm, in which many of these diseases may clinically simulate.

It is a chronic granulomatous disease caused by Gramnegative bacillus called Klebsiella rhinoscleromatis or Frisch bacillus. The disease is endemic in several parts of the world. In India, it is seen more often in the northern than in the southern parts.

The disease starts in the nose and extends to nasopharynx, oropharynx, larynx (mostly subglottic region), trachea and bronchi. Mode of infection is unknown. Both sexes of any be affected.
age may

- 1. Atrophic stage. It resembles atrophic rhinitis and is characterized by foul-smelling purulent nasal discharge and crusting.
2.
Granulomatous stage.
Granulomatous nodules
form in
nasal mucosa.
There is
also subdermal
lower part
nose and
upper lip
giving
a
"woody" feel (Figure 28.1). Nodules are painless and nonulcerative.
- 3. Cicatricial stage. This causes stenosis of nares, distortion of upper lip, adhesions in the nose, nasopharynx and oropharynx. There may be subglottic stenosis with respiratory distress.

Biopsy shows infiltration of submucosa with plasma cells, lymphocytes, eosinophils, Mikulicz cells and Russell bodies. The latter two are diagnostic features of the disease (Figure 28.2). Mikulicz cells are large foam cells with a central nucleus and vacuolated cytoplasm containing causative bacilli. Russell bodies are homogenous eosinophilic inclusion bodies found in the plasma cells. They occur due to accumulation of immunoglobulins secreted by the plasma cells. The causative organisms can be cultured from the biopsy material.

Both streptomycin (1 g/day) and tetracycline (2 g/day) are given together for a minimum period of 4—6 weeks and repeated, if necessary, after | month. Treatment is stopped
Figure 28.2 Rhinoscleroma showing foamy Mikulicz cells (arrow) and lymphocytic infiltration (arrowheads) (H&E, x400). Mikulicz cells contain Gram-negative bacteria which can be better appreciated in sections stained with Giemsa stain and examined under oil immer- sion lens.
only when two consecutive cultures from the biopsy material are negative. Steroids can be combined to reduce fibrosis. Surgical treatment may be required to establish the airway and correct nasal deformity.

Nasal syphilis is of two types: acquired and congenital.
1. Acquired. It occurs as:
(a)
Primary.
It manifests
as primary
chancre of
the vestibule
of nose.
It is
rare.
(b) Secondary. Rarely recognized. It manifests as simple rhinitis with crusting and fissuring in the nasal vestibule. Diagnosis is suggested by the presence of mucous patches in the pharynx, skin rash, fever and generalized lymphadenitis.
(c) Tertiary. This is the stage in which nose is commonly involved. Typical manifestation is the formation of a gumma on the nasal septum. Later, the septum is destroyed both in its bony and cartilaginous parts. Perforation may also appear in the hard palate. There is offensive nasal discharge with crusts. Bony or cartilaginous sequestra may be seen. Bridge of the nose collapses causing a saddle nose deformity.

(a) Early form. It is seen in the first 3 months of life and manifests as "snuffles." Soon the nasal discharge becomes purulent. This is associated with fissuring and excoriation of the nasal vestibule and of the upper lip. (b) Late form. Usually manifests around puberty. Clinical picture is similar to that seen in tertiary stage of acquired syphilis. Gummatous lesions destroy the nasal structures. Other stigmata of syphilis such as corneal opacities, deafness and Hutchinson's teeth are also present.

It is made on serological tests (VDRL) and biopsy of the tissue with special stains to demonstrate Treponema pallidum.

Penicillin is the drug of choice: benzathine penicillin 2.4 million units i.m. every week for 3 weeks with a total dose of 7.2 million units. Nasal crusts are removed by irrigation with alkaline solution. Bony and cartilaginous sequestra should also be removed. Cosmetic deformity is corrected after disease becomes inactive.

Syphilis can lead to vestibular stenosis, perforations of nasal septum and hard palate, secondary atrophic rhinitis and saddle nose deformity.

Primary tuberculosis of nose is rare. More often it is second- ary to lung tuberculosis. Anterior part of nasal septum and anterior end of inferior turbinate are the sites commonly involved. First, there is nodular infiltration followed later by ulceration and perforation of nasal septum in its cartilagi- nous part.
Diagnosis can be made on biopsy and special staining of for acid fast bacilli and culture of organisms.
sections
Treatment is antitubercular drugs.

It is a low-grade tuberculous infection commonly affecting nasal vestibule or the skin of nose and face. The skin lesions manifest characteristically as brown, gelatinous nodules called "apple-jelly" nodules. In the vestibule, it presents as chronic vestibulitis. Perforation may occur in the cartilaginous part of nasal septum.
It is difficult to isolate tubercle bacilli by culture, however, biopsy of the lesion is useful to make the diagnosis.
Treatment is the same as for tuberculosis of nose.

Leprosy is very common in the tropics and is widely prevalent in India. It is caused by Mycobacterium leprae. The nose is involved as a part of systemic disease, more often in the lepromatous than tuberculoid or dimorphous forms of disease.
Infection starts in the anterior part of nasal septum and anterior end of inferior turbinate. Initially, there is excessive nasal discharge with red and swollen mucosa. Later, crusting and bleeding supervene. Nodular lesions on the septum may ulcerate and cause perforation. Late sequelae of disease are atrophic rhinitis, depression of bridge of nose and destruction of anterior nasal spine with retrusion of the columella (Figure 28.3).
Figure 28.3
158
Diagnosis can be made from the scrapings of nasal mucosa and biopsy. Acid-fast lepra bacilli can be seen in the foamy appearing histiocytes called lepra cells.
Treatment is with dapsone, rifampin and isoniazid. Reconstruction procedures are required when disease is inactive.

It is a chronic granulomatous disease caused by Rhinosporidium seeberi and affects both man and animals.

Most of the cases come from India, Sri Lanka and Pakistan though cases have been reported from Africa (Kenya, Tanzania, Rwanda, Burkinafaso, Chad and Egypt), South America (Argentina, Brazil), North America, Europe and Canada. No case is reported from Australia.
In India, disease is more common in the southern states. It is prevalent in the states of Tamil Nadu, Kerala, Madhya Pradesh, Chhattisgarh, Puducherry and Andhra Pradesh. A few cases are also reported from Punjab and Haryana.
Disease is also seen to involve animals such as cows, bulls, horses, mules and dogs where men and animals share the same infected ponds.

It has long been considered to be a fungus but it has been difficult to classify this organism. It has not been cultured so far. Some consider it to be a protozoa or a fish parasite belonging to DRIP clade (Dermocystidium, Rosette agent, Ichthyophonus and Psorospermum).

Three stages have been recognized in the life cycle of the organism: trophic stage, development of sporangia and production of endospores (Figure 28.6).
Figure 28.4
Rhinosporidiosis presenting as (A) a polypoidal mass protruding through the naris and (B) multiple sites of involvement, viz. nose, conjunctiva and tongue.
Figure
28.5
(A) Histologic section showing rhinosporidiosis (blue arrow) evoking mixed inflammatory response (H&E, x40). (B) Histologic section
showing sporangium
(blue arrow)
which is
fully packed
with immature
sporoblasts at
the periphery
and mature
ones at
the centre
1. Trophic stage. The endospore is oval or rounded, 6-8 pm in size, clear cytoplasm, vesicular nucleus with a nucleolus and a covering of chitin. It gradually increases in size, begins to divide cytoplasm and nucleus forming small endospores by several divisions. Trophocyte becomes large filled with young endospores.
2. Development of sporangium. The mature trophocyte then develops into sporangium. A sporangium is 200250 um in diameter, contains 12,000—16,000 endospores. It has a thick wall consisting of two layers: outer chitinous and inner cellulose
layer. Endospores mature with the formation of mucoid and chitinous wall. Sporangium filled with thousands of endospores develops a germinal pore ready to burst and liberate the endospores.
3. Production of endospores. Sporangium filled with endospores develops high internal pressure and ruptures liberating endospores into the surrounding tissue. If internal pressure is not high, spores are liberated one by one without breaking the wall. After liberation endospores start their life as trophic stage. Some endospores are carried by lymphatic channels to the blood stream to cause disseminated form of disease.

The disease mostly affects nose and nasopharynx; other sites such as lip, palate, conjunctiva, epiglottis, larynx, trachea, bronchi, skin, vulva and vagina may also be affected.
The disease is acquired through contaminated water of ponds also frequented by animals. In the nose, the disease presents as a leafy, polypoidal mass, pink to purple in colour and attached to nasal septum or lateral wall. Sometimes, it extends into the nasopharynx and may hang behind the soft palate. The mass is very vascular and bleeds easily on touch. Its surface is studded with white dots representing the sporangia of fungus.
In early stages, the patient may complain of nasal discharge which is often blood tinged and nasal stuffiness. Sometimes, frank epistaxis is the only presenting complaint.

This is made on biopsy. It shows several sporangia, oval or round in shape and filled with spores which may be seen bursting through its chitinous wall. It has not been possible to culture the organism or transfer the disease to experimental animals.

Complete excision of the mass with diathermy knife and cauterization of its base. Recurrence may occur after surgical excision. Not many drugs are effective against the disease. Dapsone has been tried with some success.

The usual causative organisms are Aspergillus niger, A. fumigatus or A. flavus. They invade nasal tissues when host's defence mechanisms are compromised due to immunosuppressive drugs.

Clinical features are those of acute or subacute rhinitis or sinusitis. A black or greyish membrane is seen in the nasal mucosa. Exploration of maxillary sinus reveals a fungus ball containing semisolid cheesy-white or blackish material. The organisms can be seen on special staining for fungus.

Surgical debridement of the involved tissues and antifungal drugs, e.g. Amphotericin B. Repeated irrigation of the involved area with application of 1% solution of gentian violet is also useful.

It is fungal infection of nose and paranasal sinuses which may prove rapidly fatal. It is seen in uncontrolled diabetics or in those taking immunosuppressive drugs. From the nose and sinuses, infection can spread to orbit, cribriform plate, meninges and brain. The rapid destruction associated with the disease is due to affinity of the fungus to invade the arteries and cause endothelial damage and thrombosis. Typical finding is the presence of a black necrotic mass filling the nasal cavity and eroding the septum and hard palate. Special stains help to identify the fungus in tissue sections.
Treatment is by amphotericin B and surgical debride- ment
of the
affected tissues
and control
of underlying
predisposing cause.

Other fungal infections of nose such as candidiasis, histoplasmosis, blastomycosis, etc. are rare.

It is a systemic disorder of unknown aetiology involving mainly the upper airways, lungs, kidneys and the skin.
160
—_
It should be differentiated from nonhealing midline granuloma because the treatment of the two is different.
quite

Early symptoms of Wegener's granulomatosis include clear or blood-stained nasal discharge which later becomes purulent. The patient often complains of "persistent cold" or "sinus." Nasal findings include crusting, granulations, septal perforation and a saddle nose. Destruction may also involve eyes, orbit, palate, oral cavity or oropharynx. Middle ear can also be involved.
General systemic symptoms include anaemia, fatigue, night sweats and migratory arthralgias.
Involvement of lung is manifested by cough and sometimes haemoptysis. X-ray chest may show a single or multiple cavity lesions.
Sooner or later, kidneys are also involved. Urine examination will show red cells, casts and albumin. Serum creatinine level is raised. Renal failure is the usual cause of death in these patients.

Biopsy from the nose is diagnostic. It shows necrosis and ulceration of mucosa, epithelioid granuloma and necrotizing vasculitis involving small arteries or veins. Erythrocyte sedimentation rate is raised.

It consists of systemic steroids and cytotoxic drugs. Cyclophosphamide and azathioprine, both are found effective.

They are mostly seen in children and may be organic or inorganic. Pieces of paper, chalk, button, pebbles and seeds are the common objects. Pledgets of cotton or swabs may be accidentally left in the nose.

Patient may present immediately if the history of foreign body is known. If overlooked, the child presents with unilateral nasal discharge which is often foul smelling and occasionally bloodstained. It is a dictum that "If a child presents with unilateral, foul-smelling nasal discharge, foreign body must be excluded." Occasionally, a radiograph of the nose is useful to confirm and localize a foreign body if it is radio-opaque. In addition to overlooked foreign body in the nose, other important causes for unilateral blood-stained discharge in a child are rhinolith, nasal diphtheria, nasal myiasis and acute or chronic unilateral sinusitis.

Pieces of paper or cotton swabs can be easily removed with a pair of forceps. Rounded foreign bodies can be removed by passing a blunt hook (a eustachian catheter is a good instrument) past the foreign body and gently dragging it forward along the floor. In babies and uncooperative children, general anaesthesia with cuffed endotracheal tube is used. Patient is placed in Rose's position, a pack is inserted into the nasopharynx and the foreign body retrieved with a forceps or a hook. Foreign bodies lodged far behind in the nose may need to be pushed into the nasopharynx before removal. A nasal endoscope is very useful to locate the foreign body and carefully remove it.

A foreign body left in the nose may result in:
1. nasal infection and sinusitis.
2. rhinolith formation.
3. inhalation into the tracheobronchial tree.

It is stone formation in the nasal cavity. A rhinolith usually forms around the nucleus of a small exogenous foreign body, blood clot or inspissated secretions by slow deposition of calcium and magnesium salts. Over a period of time, it grows into a large, irregular mass which fills the nasal cavity and then may cause pressure necrosis of the septum and/or lateral wall of nose.

Rhinoliths are more common in adults. Its common presentation is unilateral nasal obstruction and foul-smelling discharge which is very often bloodstained. Frank epistaxis and neuralgic pain may result from ulceration of the surrounding mucosa.
On examination, a grey brown or greenish-black mass with irregular surface and stony hard feel is seen in the nasal cavity between the septum and turbinates. It is often brittle and a portion of it may break off while manipulating. Sometimes it is surrounded by granulations.

They are removed under general anaesthesia. Most of them can be removed through anterior nares. Large ones need to be broken into pieces before removal. Some particularly hard and irregular ones require lateral rhinotomy.

Maggots are larval forms of flies. They are seen to infest nose, nasopharynx and paranasal sinuses causing extensive destruction (Figures 29.1 A, B, C and 29.2). Flies, particularly of the genus Chrysomyia, are attracted by the foul-smelling discharge emanating from cases of atrophic rhinitis, syphilis, leprosy or infected wounds and lay eggs, about 200 at a time, which within 24 h hatch into larvae. In India, they are mostly seen from the month of August to October.
Figure 29.1 Maggots nose. (A) Swelling of nose and puffy eyelids with serosanguinous nasal discharge. (B) Maggots have practically destroyed the cheek and eye in this old and neglected lady. (C) Perforation of palate (arrow).
Figure 29.2 (A) The maggot. (B) The fly responsible for maggots.

In the first 3 or 4 days maggots produce intense irritation, sneezing, lacrimation and headache. Thin blood-stained discharge oozes from the nostrils. The eyelids and lips become puffy. Till this time patient is not aware of maggots. He may present simply as a case of epistaxis. It is only on the third or fourth day that the maggots may crawl out of the nose. Patient has foul smell surrounding him. Maggots cause extensive destruction to nose, sinuses, soft tissue of face, palate and the eyeball. Fistulae may form in the palate or around the nose. Death may occur from meningitis.

All visible maggots should be picked up with forceps. Many of them try to retreat into darker cavities when light falls on them. Instillation of chloroform water and oil kills them. Nasal douche with warm saline is used to remove slough, crusts and dead maggots. A patient with maggots should be isolated with a mosquito net to avoid contact with flies which can perpetuate this cycle. All patients should receive instruction for nasal hygiene before leaving the hospital.

Adhesion formation between the nasal septum and turbinates by scar tissue is often the result of injury to opposing surfaces of nasal mucosa. It can result from intranasal operations such as septal surgery, polypectomy, removal of foreign bodies, reduction of nasal fractures, endoscopic sinus surgery or even intranasal packing. Severe infections which cause ulcerative lesions in the nose can also lead to synechia formation.
Nasal synechia (Figure 29.3) often cause nasal obstruction or may impede drainage from the sinuses resulting in sinusitis, headache and nasal discharge.
Figure 29.3 Nasal synechia left.
Treatment is removal of synechia and prevention of the opposing raw surfaces to come into contact with each other by placing a thin silastic or a cellophane sheet between them. This is changed every two or three days till healing is complete.

It is due to persistence of bucconasal membrane and may be unilateral or bilateral, complete or incomplete, bony (90%) or membranous (10%). Unilateral atresia is more common and may remain undiagnosed until adult life. Bilateral atresia presents with respiratory obstruction as the newborn, being a natural nose breather, does not breathe from mouth. Diagnosis of choanal atresia can be made by (i) presence of mucoid discharge in the nose, (ii) absence of air bubbles in the nasal discharge, (iii) failure to pass a catheter from nose to pharynx, (iv) putting a few drops of a dye (methylene blue) into the nose and seeing its passage into the pharynx, or (v) flexible nasal endoscopy, (vi) installing radio-opaque dye into the nose and taking a lateral film, and (vii) computed tomography (CT) scan in axial plane is more useful.
Emergency management may be required in bilateral choanal atresia to provide an airway. A feeding nipple with a large hole provides a good oral airway (McGovern's technique) and obviates the need for tracheostomy. Definitive treatment consists of correction of atresia by transnasal or transpalatal approach. The latter is usually done at one and a half years. Choanal atresia can be corrected by using nasal endoscopes and drill. Removal of a part of posterior nasal septum transnasally is another option to treat such cases.

Leakage of CSF into the nose is called CSF rhinorrhoea. It may be clear fluid or mixed with blood as in acute head injuries.
(c)
sphenoid sinus,
(d)
(temporal fracture).

CSF forms a jacket of fluid round the brain and spinal cord acting as a buffer against sudden jerks. It is secreted by choroid plexuses in the lateral, third and fourth ventricles and is absorbed into the dural venous sinuses by arachnoid villi. Villi have one-way valve mechanism allowing CSF of the subarachnoid space to be absorbed into the blood but not vice versa. Total volume of CSF varies from 90 to 150 mL. It is secreted at the rate of about 20 mL/h (350-500 mL/ day). Thus total CSF is replaced three to five times every day. Normal CSF pressure at lumbar puncture is 50-150 mm H,O. CSF pressure rises on coughing, sneezing, nose blowing, straining on stools or lifting heavy weight—activities which should be avoided in cases of CSF leak or after its repair.

CSF from anterior cranial fossa reaches the nose via (i) cribriform plate, (ii) roof of ethmoid air cells or (iii) frontal sinus. CSF from middle cranial fossa follows injuries to sphenoid sinus. In fractures of temporal bone, CSF reaches the middle ear and then escapes through the eustachian tube into the nose (CSF otorhinorrhoea) (Figure 29.4).

History, CSF fluid = Nasal or sinus surgery, head injury or intracranial tumour. History, Nasal secretion = Sneezing, nasal stuffiness, itching in the nose or lacrimation. Flow of discharge, CSF fluid = A few drops or a stream of fluid gushes down when bending forward or straining; cannot be sniffed back. Flow of discharge, Nasal secretion = Continuous, no effect of bending forward or straining. Can be sniffed back. Character of discharge Taste, CSF fluid = Thin, watery and clear Sweet. Character of discharge Taste, Nasal secretion = Slimy (mucus) or clear (tears) Salty. Sugar content, CSF fluid = More than 30 mg/dL (Compare with sugar in CSF after lumbar puncture as sugar is less in CSF in meningitis.). Sugar content, Nasal secretion = Less than 10 mg/dL. Presence of β 2 transferrin, CSF fluid = Always present. It is specific for CSF. Presence of β 2 transferrin, Nasal secretion = Always absent

There is history of clear watery discharge from the nose on bending the head or straining. It may be seen on rising in the morning when patient bends his head (reservoir sign—fluid which had collected in the sinuses, particularly sphenoid, empties into the nose). CSF rhinorrhoea should be differentiated from nasal discharge of allergic or vasomotor rhinitis as the former is sudden, gushes in drops when bending and cannot be sniffed back. Nasal discharge, because of its mucus content, also stiffens the handkerchief (Table 29.1).
CSF rhinorrhoea after head trauma is mixed with blood and shows double target sign when collected on a piece of filter paper. It shows central red spot (blood) and peripheral lighter halo.
Nasal endoscopy can help to localize CSF leak in some cases. Otoscopic/microscopic examination of the ear may reveal fluid in the middle ear in cases of otorhinorrhoea.

Beta-2 transferrin is a protein seen in CSF and not in the nasal discharge. Its presence is a specific and sensitive test and requires only a few drops of CSF. The specimen of nasal discharge is tested for this protein. Perilymph and aqueous humour are the only other fluids which contain this protein. Another protein called beta trace protein is also specific for CSF and is widely used in Europe. It is secreted by meninges and choroid plexus. Facilities to test these proteins are not easily available everywhere. Glucose testing by oxidase or biochemical estimation are no used.
peroxidase longer

1. High-resolution CT scan. Cuts are taken at 1-2 mm. Both coronal and axial cuts are important to see the bony defects. Axial cuts show any defects of frontal or sphe- noid sinus.
2. MRI. T>-weighted image MRI is useful in depicting the site of leak. It requires that CSF leak is active at the time of scan. It is a noninvasive test. It is indicated also if encephalocele or intracranial pathology is suspected.
Intrathecal fluorescein study. It can be done preopera- tively to diagnose the site or intraoperatively at the time of repair. It is an invasive procedure. Only 0.25-0.5 mL of 5% fluorescein diluted with 10 mL of CSF is injected. ge
Patient lies in 10° head down position for sometime. Dye can be detected intranasally with the help of endoscope. Dye appears bright yellow but when seen with a blue filter it appears fluorescent green. One should examine olfactory cleft (cribriform plate), middle meatus (frontal and ethmoidal sinuses), sphenoethmoidal recess (sphenoid sinus) and area of torus tubarius (temporal bone fracture) to localize the lesion.
Use of intrathecal radioactive substances has been abandoned.
4. CT cisternogram. It requires intrathaecal injection of iohexol and a CT scan to localize the site in cases when beta-2 transferrin cannot be done. Now it is not favoured by many.

Early cases of post-traumatic CSF rhinorrhoea can be managed by conservative measures such as bed rest, elevating the head of the bed, stool softeners, and avoidance of nose blowing, sneezing and straining. Prophylactic antibiotics can be used to prevent meningitis. Acetazolamide decreases CSF formation. These measures can be combined with lumbar drain if indicated.
Surgical repair can be done by the following:
1. Neurosurgical intracranial approach.
2. Extradural approaches such as external ethmoidec- tomy for cribriform plate and ethmoid area, trans-septal sphenoidal approach for sphenoid and osteoplastic flap approach for frontal sinus leak.
3. Transnasal endoscopic approach. With the advent of endoscopic surgery for nose and sinuses, most of the leaks from the anterior cranial fossa and sphenoid sinus can be managed endoscopically with a success rate of 90% with first attempt. Principles of repair include:
(a)
Defining
the
sites
of
bony
defect
(Figure
29.5).
Itcan
be
(i) Cribriform plate
(ii) Lateral lamina close to anterior ethmoid artery
(iii)
Roof
of
ethmoid
(iv) Frontal sinus leak
(v) Sphenoid sinus
(b) Preparation of graft site.
(c) Underlay grafting of the fascia extradurally followed by placement of mucosa (as a free graft or pedicled flap) (Figure 29.6).
(d) If bony defect is larger than 2 cm, it is repaired with cartilage (from nasal septum or auricular concha) followed by placement of mucosa.
(e) Placement of surgicel and gelfoam further strengthens the area. This is followed by a high antibiotic smeared nasal pack.
Sometimes fat
from the
thigh or
abdomen is
used plug
the defect
in place
of fascia
graft.
(f) Lumbar drain if CSF pressure is high.
(g) Antibiotics

CSF leak from frontal sinus often requires osteoplastic flap, operation and obliteration of the sinus with fat.
to

It is an IgE-mediated immunologic response of nasal mucosa to airborne allergens and is characterized by watery nasal discharge, nasal obstruction, sneezing and itching in the nose. This may also be associated with symptoms of itching in the eyes, palate and pharynx. Two clinical types have been recognized:

1. Seasonal. Symptoms appear in or around a particular season when the pollens of a particular plant, to which the patient is sensitive, are present in the air.
2. Perennial. Symptoms are present throughout the year.

Inhalant allergens. They may be seasonal or perennial. Seasonal allergens include pollens from trees, grasses and weeds. They vary geographically. The knowledge of pollen appearing in a particular area and the season in which they occur is important. Their knowledge also helps in skin tests. Perennial allergens are present throughout the year regardless of the season. They include molds, dust mites, cockroaches and dander from animals. Dust includes dust mite, insect parts, fibres and animal danders. Dust mites live on skin scales and other debris and are found in the beddings, mattresses, pillows, carpets and upholstery.
Inhaled allergens produce specific IgE antibody in the genetically predisposed individuals. This antibody becomes fixed to the blood basophils or tissue mast cells by its Fc end (Figure 30.1). On subsequent exposure, antigen combines with IgE antibody at its Fab end. This reaction produces degranulation of the mast cells with release of several chemical mediators, some of which already exist in the preformed state while others are synthesized afresh. These mediators (Figure 30.2) are responsible for symptomatology of allergic disease. Depending on the tissues involved, there may be vasodilation, mucosal oedema, infiltration with eosinophils, excessive secretion from nasal glands or smooth muscle contraction. A "priming affect" has also been described, i.e. mucosa earlier sensitized to an allergen will react to smaller doses of subsequent specific allergen. It also gets "primed" to other nonspecific antigens to which patient was not exposed (Figure 30.3). Nonspecific nasal hyper-reactivity is seen in patients of allergic rhinitis. There is increased nasal response to normal stimuli resulting in sneezing, rhinorrhoea and nasal congestion. Clinically, allergic response occurs in two phases:
Genetic predisposition plays an important part. Chances of children developing allergy are 20 and 47%, respectively, if one or both parents suffer from allergic diathesis.

1. Acute or early phase. It occurs immediately within 5-30 min, after exposure to the specific allergen and consists of sneezing, rhinorrhoea nasal blockage and/or bronchospasm. It is due to release of vasoactive amines like histamine.
2. Late or delayed phase. It occurs 2-8 h after exposure to allergen without additional exposure. It is due to
Figure 30.1 (A) Structure of IgE antibody. Fc end is attached to the mast cell or blood basophil while Fab end is the antigen binding site. (B) Release of mediator substances from mast cell producing symptoms of nasal allergy. One antigen bridges two adjacent molecules of IgE antibody.
infiltration of inflammatory cells—eosinophils, neutrophils, basophil, monocytes and CD, + T cells at the site of antigen deposition causing swelling, congestion and thick secretion. In the event of repeated or continuous exposure to allergen, acute phase symptomatology overlaps the late phase.

There is no age or sex predilection. It may start in infants as young as 6 months or older people. Usually the onset is at 12-16 years of age.
The cardinal symptoms of seasonal nasal allergy include paroxysmal sneezing, 10-20 sneezes at a time, nasal obstruction, watery nasal discharge and itching in the nose. Itching may also involve eyes, palate or pharynx. Some may get bronchospasm. The duration and severity of symptoms may vary with the season.
Symptoms of perennial allergy are not so severe as that of the seasonal type. They include frequent colds, persistently stuffy nose, loss of sense of smell due to mucosal oedema, postnasal drip, chronic cough and hearing impairment due to eustachian tube blockage or fluid in the middle ear. Signs of allergy may be seen in the nose, eyes, ears, pharynx or larynx.
¢ Nasal signs include transverse nasal crease—a black line across the middle of dorsum of nose due to constant upward rubbing of nose simulating a salute (allergic salute), pale and oedematous nasal mucosa which may appear bluish. Turbinates are swollen. Thin, watery or mucoid is
discharge usually present.
- © Ocular signs include oedema of lids, congestion and cobblestone appearance of the conjunctiva, and dark circles under the eyes (allergic shiners).
©
Otologic signs
include retracted
or serous
as
a
result of
eustachian tube
blockage.
e Pharyngeal signs include granular pharyngitis due to hyperplasia of submucosal lymphoid tissue. A child with perennial allergic rhinitis may show all the features of prolonged mouth breathing as seen in adenoid hyperplasia.
¢ Laryngeal signs include hoarseness and oedema of the vocal cords.

New Allergic Rhinitis and Its Impact on Asthma (ARIA) classification (Table 30.1). It is based on duration and symptoms of disease. Duration of symptoms is subdivided into intermittent or persistent and severity of disease into mild, moderate or severe.
This new system of classification helps in treatment guidelines.
A detailed history and physical examination is helpful, and also gives clues to the possible allergen. Other causes of nasal stuffiness should be excluded.

Figure by allergic or nonspecific stimuli.
symptomatology allergy.

Intermittent: Symptoms are present
— Less than 4 days a week or
— For less than 4 weeks
° Persistent: Symptoms are present
— More than 4 days a week or
— For more than 4 weeks
- ¢ Severity of disease
*
Mild:
of the
following
— Sleep disturbance
—
Impairment of
daily activities,
symptoms are
leisure
- — Impairment of school or work
—
Troublesome symptoms
- ° Moderate to severe: One or more of the above symptoms
Nasal smear. It shows large number of eosinophils in allergic rhinitis. Nasal smear should be taken at the time of clinically active disease or after nasal challenge test. Nasal eosinophilia is also seen in certain nonallergic rhi- nitis, e.g. NARES (nonallergic rhinitis with eosinophilia syndrome).
Skin tests. These tests help to identify specific allergen. They are prick, scratch and intradermal tests.
(a) Skin prick test. This is an excellent method to demon- strate the allergen. A drop of concentrated allergen solution is placed on the volar surface of the forearm or back and a sharp needle pricked into the dermis through the drop. It introduces the allergen into the dermis. A positive reaction is manifested by the for- mation of a central wheal and a surrounding zone of erythema (flare) within 10-15 min.
Simultaneously
a
control
test
is
performed
with
histamine
and
the
diluent
used
in
allergen
solution.
Specific IgE measurements. It is an in vitro test to find the specific allergen. There is a good correlation between the skin tests and specific IgE measure- ments. However both false positive and false nega- tive results can occur. It is therefore recommended to correlate the two tests with clinical symptoms. (b)
Radioallergosorbent test (RAST). It is an in vitro test and measures specific IgE antibody concentration in the patient’s serum.

Nasal provocation test. A crude method is to challenge the nasal mucosa with a small amount of allergen placed at the end of a toothpick and asking the patient to sniff into each nostril and to observe if allergic symptoms are repro- duced. More sophisticated techniques are available now.

Nasal allergy may cause:
1. Recurrent sinusitis because of obstruction to the sinus ostia.
2. Formation of nasal polypi in about 2%.
3 Serous otitis media.
Orthodontic problems and other ill-effects of prolonged
mouth breathing especially in children.
sport
5. Bronchial asthma. Patients of nasal allergy have four times more risk of developing bronchial asthma. Twenty to thirty per cent of patients with rhinitis have asthma.

Treatment can be divided into:
1. Avoidance of allergen.
2. Treatment with drugs.
3. Immunotherapy.
1. Avoidance of allergen. This is most successful if the anti- gen involved is single. Removal of a pet from the house, encasing the pillow or mattress with plastic sheet, change of place of work or sometimes change of job may be required. A particular food article to which the patient is found aller- gic can be eliminated from the diet.

(a) Antihistaminics. They control rhinorrhoea, sneezing and nasal itch. All antihistaminics have the side effect of drowsiness; some more than the other. The dose and type of the antihistaminic has to be individualized. If one antihistaminic is not effective, another may be tried from a different class.
(b)
(c) Corticosteroids. Oral corticosteroids are very effective in controlling the symptoms of allergic rhinitis but their use should be limited to acute episodes which have not been controlled by other measures. They have several systemic side effects.
(d)
Sympathomimetic
drugs
(oral
or
topical).
Alpha-adrenergic
drugs
constrict
blood
vessels
and
reduce
nasal
conges-
tion
and
oedema.
They
also
cause
central
nervous
sys-
tem
stimulation
and
are
often
given
in
with
antihistaminics
to
counteract
drowsiness.
Pseu-
doephedrine
and
phenylephrine
are
often
combined
with
for
oral
Topical
use
of
drugs
causes
nasal
decongestion.
Phenylephrine,
oxymetazoline
and
xylo-
metazoline
are
often
used
to
relieve
nasal
obstruction,
but
are
notorious
to
cause
severe
rebound
congestion.
Patient
resorts
to
using
more
and
more
of
them
to
relieve
nasal
obstruction.
This
vicious
cycle
leads
to
rhi-
nitis
medicamentosa.

Topical steroids such as beclomethasone dipropio- nate, budesonide, flunisolide acetate, fluticasone and mometasone inhibit recruitment of inflammatory cells into the nasal mucosa and suppress late-phase allergic reaction, are used as aerosols and are very effective in the control of symptoms. They have also been used in rhinitis medicamentosa while withdrawing topical use of decongestant nasal drops. Topical steroids have fewer systemic side effects but their continuous use may cause mucosal atrophy and even septal perforation. It is wise to break their use for 1-2 weeks every 2-3 months. They may also promote growth of fungus.
Sodium
cromoglycate.
It
stabilizes
the
mast
cells
and
pre-
vents
them
from
degranulation
despite
the
formation
of
IgE-antigen
complex.
It
is
used
as
2%
solution
for
nasal
drops
or
spray
or
as
an
aerosol
powder.
It
is
useful
both
in
seasonal
and
perennial
allergic
rhinitis.
(e) Anticholinergics. They block rhinorrhoea both of the allergic and nonallergic rhinitis. Ipratropium bromide has been used as nasal spray to control rhinorrhoea. There are no systemic side effects.
(f) Leukotriene receptor antagonists. They include montelu- kast, pranlukast and zafirlukast. They block cysteinyl leukotriene type receptors. They are well-tolerated and have few side effects.
(g) Anti-lgk. It reduces the IgE level and has an anti- inflammatory effect. Omalizumab is such a drug. It is indicated in children above 12 years who have moder- ate to severe asthma. It is not yet approved for allergic rhinitis.

3. Immunotherapy. Immunotherapy or hyposensitization is used when drug treatment fails to control symptoms or produces intolerable side effects. Allergen is given in gradu- ally increasing doses till the maintenance dose is reached. Immunotherapy suppresses the formation of IgE. It also raises the titre of specific IgG antibody. Immunotherapy has to be given for a year or so before significant improvement of symptoms can be noticed. It is discontinued if uninter rupted treatment for 3 years shows no clinical improvement.
Subcutaneous immunotherapy is often used but now sub- lingual and nasal routes are also being employed. The latter can be used with doses 20-100 times greater than used by the subcutaneous route.
A
step
care
approach
is
recommended
by
ARIA
for
aller-
gic
rhinitis
¢ Oral antihistamines or intranasal cromolyn sodium is rec- ommended for mild intermittent disease.
¢ For allergic symptoms of moderate severity or for per- sistent disease intranasal corticosteroids can be used as monotherapy.
¢
For
severe
symptoms,
therapy
with
oral
non-
sedating
antihistamines
and
intranasal
steroids
is
used.
¢
For
severe
and
persistent
symptoms
in
spite
of
the
above
treatment
a
short
course
of
oral
steroids
and
immuno-
therapy
is
recommended.
¢ If nasal obstruction persists a short course of intranasal decongestant can be used. Oral decongestant can be com- bined with antihistamines.
e Avoid allergen and irritants in all forms of disease. Nonal- lergic rhinitis can coexist with allergic rhinitis. Nonspe- cific stimuli produce allergic rhinitis-like symptoms due to hyper-reactivity of nasal mucosa.

Itis nonallergic rhinitis but clinically simulating nasal allergy with symptoms of nasal obstruction, rhinorrhoea and sneezing. One or the other of these symptoms may predominate. The condition usually persists throughout the year and all the tests of nasal allergy are negative.

Nasal mucosa has rich blood supply. Its vasculature is similar to the erectile tissue in having venous sinusoids or "lakes" which are surrounded by fibres of smooth muscle which act as sphincters and control the filling or emptying of these sinusoids. Sympathetic stimulation causes vasoconstriction and shrinkage of mucosa, while parasympathetic stimulation causes vasodilation and engorgement. Overactivity of parasympathetic system also causes excessive secretion from the nasal glands.
Autonomic nervous system is under the control of hypothalamus and therefore emotions play a great role in vasomotor rhinitis. Autonomic system is unstable in cases of vasomotor rhinitis. Nasal mucosa is also hyper-reactive and responds to several nonspecific stimuli, e.g. change in temperature, humidity, blasts of air, small amounts of dust or smoke.

1. Paroxysmal sneezing. Bouts of sneezing start just after getting out of the bed in the morning.
2.
Excessive
rhinorrhoea.
This
accompanies
sneezing
or
this
may
be
the
only
symptom.
It
is
profuse
and
watery
and
may
even
wet
several
handkerchiefs.
The
nose
may
drip
when
the
patient
leans
forward
and
this
may
need
to
be
differentiated
from
CSF
rhinorrhoea
(see
p.
163).
3. Nasal obstruction. This alternates from side to side. Usu- ally more marked at night. It is the dependent side of nose which is often blocked when lying on one side. 4. Postnasal
drip.

Nasal mucosa over the turbinates is generally congested and hypertrophic. In some, it may be normal.

Long-standing cases or VMR develop nasal polypi, hypertrophic rhinitis and sinusitis.
MEDICAL
1.
Avoidance
of
physical
factors
which
provoke
symptoms,
e.g.
sudden
change
in
humidity,
blasts
of
air
or
dust.
2. Antihistaminics and oral nasal decongestants are helpful in relieving nasal obstruction, sneezing and rhinorrhoea.
3.
Topical
steroids
(e.g.
beclomethasone
dipropionate,
budesonide
or
fluticasone),
used
as
spray
or
aerosol,
are
useful
to
control
symptoms.
4. Systemic steroids can be given for a short time in very severe Cases.
5. Psychological factors should be removed. Tranquillizers may be needed in some patients.

1.
Nasal
can
be
relieved
by
measures
which
reduce the size of nasal turbinates (see hypertrophic rhini- tis). Other associated causes of nasal obstruction, e.g. polyp, deviated nasal septum, should also be corrected.
2. Excessive rhinorrhoea, not corrected by medical therapy and bothersome to the patient, can be relieved by sec- tioning the parasympathetic secretomotor fibres to nose (vidian neurectomy).

Nasal mucosa responds to several different stimuli produc- ing symptoms of rhinitis. Some of these conditions have acquired specific eponyms. Some authorities categorize them under the catch-all term of vasomotor rhinitis.
1. Drug-induced rhinitis. Several antihypertensive drugs such as reserpine, guanethidine, methyl dopa and propran- olol are sympathetic blocking agents and cause nasal stuffi- ness. Some anticholinesterase drugs, e.g. neostigmine, used in the treatment of myasthenia gravis, have acetylcholine like action and cause nasal obstruction. Contraceptive pills also cause nasal obstruction because of oestrogens.
2. Rhinitis medicamentosa. Topical decongestant nasal drops are notorious to cause rebound phenomenon. Their excessive use causes rhinitis. It is treated by withdrawal of nasal drops, short course of systemic steroid therapy and in some cases, surgical reduction of turbinates, if they have become hypertrophied.
3. Rhinitis of pregnancy. Pregnant women may develop persistent rhinitis due to hormonal changes. Nasal mucosa
becomes oedematous and blocks the airway. Some may develop secondary infection and even sinusitis. In such cases, care should be taken while prescribing drugs. Generally, local measures such as limited use of nasal drops, topical steroids and limited surgery (cryosurgery) to turbinates are sufficient to relieve the symptoms. Safety of the developing fetus is not established for newer antihistaminics and they should be avoided.
4. Honeymoon rhinitis. This usually follows sexual excitement leading to nasal stuffiness.
5. Emotional rhinitis. Nose may react to several emotional stimuli. Psychological states like anxiety, tension, hostility, humiliation, resentment and grief are all known to cause rhinitis. Treatment is proper counselling for psychological adjustment. Imipramine, which has both antidepressant and anticholinergic effects, has been found useful.
6. Rhinitis due to hypothyroidism. Hypothyroidism leads to hypoactivity of the sympathetic system with predominance of
parasympathetic activity causing nasal stuffiness and "colds." Replacement of thyroid hormone relieves the condition.

7. Gustatory rhinitis. Spicy and pungent food may in some people produce rhinorrhoea, nasal stuffiness, lacrimation, sweating and even flushing of face. This is a cholinergic response to stimulation of sensory receptors on the palate. Spicy food, particularly the red pepper, contains capsaicin which is known to stimulate sensory nerves. It can be relieved by ipratropium bromide nasal spray (an anticholinergic), a few minutes before meals.
8. Nonairflow rhinitis. It is seen in patients of laryngectomy and tracheostomy. Nose is not used for airflow and the turbinates become swollen due to loss of vasomotor control. Similar changes are also seen in nasopharyngeal obstruction due to choanal atresia or adenoidal hyperplasia, the latter having the additional factor of infection due to stagnation of discharge in the nasal cavity which should otherwise drain freely into the nasopharynx.

Nasal polypi are non-neoplastic masses of oedematous nasal
or sinus mucosa.They are divided into two main varieties:
1. Bilateral ethmoidal polypi.
2. Antrochoanal polyp.

Aetiology of nasal polypi is very complex and not wellunderstood. They may arise in inflammatory conditions of nasal mucosa (rhinosinusitis) , disorders of ciliary motility or abnormal composition of nasal mucus (cystic fibrosis). Various diseases associated with the formation of nasal polypi are:
1. Chronic rhinosinusitis. Polypi are seen in chronic rhino- sinusitis of both allergic and nonallergic origin. Nonal- lergic rhinitis with eosinophilia syndrome (NARES) is a form of chronic rhinitis associated with polypi.
2. Asthma. Seven per cent of the patients with asthma of atopic or nonatopic origin show nasal polypi.
3.
Aspirin
intolerance.
Thirty-six
per
cent
of
the
patients
with
aspirin
intolerance
may
show
polypi.
Samter’s
triad
consists
of
nasal
polypi,
asthma
and
aspirin
4.
Cystic
fibrosis.
Twenty
per
cent
of
patients
with
cystic
fibrosis
form
polypi.
It
is
due
to
abnormal
mucus.
5. Allergic fungal sinusitis. Almost all cases of fungal sinus- itis form nasal polypi.
6. Kartagener syndrome. This consists of bronchiectasis sinusitis, situs inversus and ciliary dyskinesis.
7. Young syndrome. It consists of sinopulmonary disease and azoospermia.
8. Churg-Strauss syndrome. Consists of asthma, fever, eosinophilia, vasculitis and granuloma.
9. Nasal mastocytosis. It is a form of chronic rhinitis in which nasal mucosa is infiltrated with mast cells but few eosinophils. Skin tests for allergy and IgE levels are normal.

Nasal mucosa, particularly in the region of middle meatus and turbinate, becomes oedematous due to collection of extracellular fluid causing polypoidal change. Polypi which are sessile in the beginning become pedunculated due to gravity and excessive sneezing.
In early stages, surface of nasal polypi is covered by ciliated columnar epithelium like that of normal nasal mucosa but later it undergoes a metaplastic change to transitional and squamous type on exposure to atmospheric irritation. Submucosa shows large intercellular spaces filled with serous fluid. There is also infiltration with eosinophils and round cells.

Multiple nasal polypi always arise from the lateral wall of nose, usually from the middle meatus. Common sites are uncinate process, bulla ethmoidalis, ostia of sinuses, medial surface and edge of middle turbinate. Allergic nasal polypi almost never arise from the septum or the floor of nose.

1. Multiple polypi can occur at any age but are mostly seen in adults.
2. Nasal stuffiness leading to total nasal obstruction may be the presenting symptom.
3. Partial or total loss of sense of smell.
4. Headache due to associated sinusitis.
5. Sneezing and watery nasal discharge due to associated allergy.
6. Mass protruding from the nostril.

On anterior rhinoscopy, polypi appear as smooth, glistening, grape-like masses often pale in colour. They may be sessile or pedunculated, insensitive to probing and do not bleed on touch. Often they are multiple and bilateral. Long-standing cases present with broadening of nose and increased intercanthal distance. A polyp may protrude from the nostril and appear pink and vascular simulating neoplasm (Figure 32.1). Nasal cavity may show purulent discharge due to associated sinusitis.
Probing of a solitary ethmoidal polyp may be necessary to differentiate it from hypertrophy of the turbinate or cystic middle turbinate.

Diagnosis can be easily made on clinical examination. Computed tomography (CT) scan of paranasal sinuses is essential to exclude the bony erosion and expansion suggestive of neoplasia. Simple nasal polypi may sometimes be associated
with malignancy underneath, especially in people above 40 years and this must be excluded by histological examination of the suspected tissue. CT scan also helps to plan surgery.

1.
Early polypoidal
changes with
oedematous mucosa
may
revert to normal with antihistaminics and control of allergy.
2. Ashort course of steroids may prove useful in case of people who cannot tolerate antihistaminics and/or in those with asthma and polypoidal nasal mucosa. They may also be used to prevent recurrence after surgery. Contraindications to use of steroids, e.g. hypertension, peptic ulcer, diabetes, pregnancy and tuberculosis should be excluded.

1. Polypectomy. One or two polyps which are pedunculated can be removed with snare. Multiple and sessile polypi require special forceps.
2. Intranasal ethmoidectomy. When polypi are multiple and sessile, they require uncapping of the ethmoidal air cells by intranasal route, a procedure called intranasal ethmoidectomy.
3. Extranasal ethmoidectomy. This is indicated when polypi recur after intranasal procedures and surgical landmarks are ill-defined due to previous surgery. Approach is through the medial wall of the orbit by an external incision, medial to medial canthus.
4. Transantral ethmoidectomy. This is indicated when infection and polypoidal changes are also seen in the maxillary antrum. In this case, antrum is opened by Caldwell—Luc approach and the ethmoid air cell approached through the medial wall of the antrum. This procedure is also superceded by endoscopic sinus surgery.
5. Endoscopic sinus surgery. These days, ethmoidal polypi are removed by endoscopic sinus surgery more popularly called functional endoscopic sinus surgery (FESS). It is done with various endoscopes of 0°, 30° and 70° angulation. Polypi can be removed more accurately when ethmoid cells are removed, and drainage and ventilation provided to the other involved sinuses such as maxillary, sphenoidal or frontal.
Figure 32.1 A polyp protruding from the left nostril in a patient with

This polyp arises from the mucosa of maxillary antrum near its accessory ostium, comes out of it and grows in the choana and nasal cavity. Thus it has three parts.
1. Antral, which is a thin stalk.
2. Choanal, which is round and globular.
3. Nasal, which is flat from side to side.

Exact cause is unknown. Nasal allergy coupled with sinus infection is incriminated. Antrochoanal polypi are seen in children and young adults. Usually they are single and unilateral.

Unilateral nasal obstruction is the presenting symptom. Obstruction may become bilateral when polyp grows into the nasopharynx and starts obstructing the opposite choana (Tables 32.1 and 32.2). Voice may become thick and dull due to hyponasality. Nasal discharge, mostly mucoid, may be seen on one or both sides.

As the antrochoanal polyp grows posteriorly, it may be missed on anterior rhinoscopy. When large, a smooth greyish mass covered with nasal discharge may be seen. It is soft and can be moved up and down with a probe. A large polyp may protrude from the nostril and show a pink congested look on its exposed part (Figure 32.2).

Bilateral vestibulitis
Collapsing nasal alae
Stenosis of nares
Congenital atresia of nares

Acute rhinitis (viral and bacterial)
Chronic rhinitis and sinusitis
Rhinitis medicamentosa
Allergic rhinitis
Hypertrophic turbinates
DNS
Nasal
polypi
Atrophic rhinitis
Rhinitis
sicca
Septal haematoma
Septal abscess
Bilateral
choanal
atresia

Adenoid hyperplasia
Large choanal polyp
Thornwaldt’s cyst
Adhesions between soft palate and posterior
pharyngeal wall
Large benign and malignant tumours
32.2 Antrochoanal the left nostril in
Figure polyp projecting through a 14-year-old patient.
Posterior rhinoscopy may reveal a globular mass filling
the choana or the nasopharynx. A large polyp may hang
down behind the soft palate and present in the oropharynx
(Figure 32.3 A,B).
Examination of the nose with an endoscope may reveal
a
choanal or antrochoanal polyp hidden posteriorly in the
nasal cavity (Figure 32.4).
See Table 32.3 for differences between antrochoanal and ethmoidal polypi.

1. A blob of mucus often looks like a polypus but it would
disappear on blowing the nose.
2. Hypertrophied middle turbinate is differentiated by
its
pink
appearance
and
hard
feel
of
bone
on
probe
testing.
3. Angiofibroma has history of profuse recurrent epistaxis.
It is firm in consistency and easily bleeds on probing.
4. Other neoplasms may be differentiated by their fleshy
pink
appearance,
friable
nature
and
their
tendency
to
bleed.
Figure 32.4 Endoscopic view of a choanal polyp right side.

Age
Common in children
Common in adults
Aetiology
Infection
Allergy or multifactorial
Number
Solitary
Multiple
Laterality
Unilateral
Bilateral
Origin
Maxillary sinus near the ostium
Ethmoidal sinuses, uncinate process, middle turbinate
and middle meatus
Growth
Grows backwards to the choana; may hang down
Mostly grow anteriorly and may present at the nares
behind the soft palate
Size
and
Trilobed with antral, nasal and choanal parts.
Usually small and grape-like masses
shape
Choanal part may protrude through the choana and fill
the nasopharynx obstructing both sides
Recurrence
Uncommon, if removed completely
Common
Treatment
Polypectomy; endoscopic removal or Caldwell-Luc
Polypectomy. Endoscopic surgery or ethmoidectomy
operation if recurrent
(which may be intranasal, extranasal or transantral)
X-rays of paranasal sinuses may show opacity of the involved antrum. X-ray (lateral view), soft tissue nasopharynx, reveals
a globular swelling in the postnasal space. It is differenti-

ated from
angiofibroma by
of
a
column of
air behind the polyp.

An antrochoanal polyp is easily removed by avulsion either
through the nasal or oral route. Recurrence is uncommon
after complete removal. In cases which do recur, Caldwell-
Luc operation may be required to remove the polyp com-
pletely from the site of its origin and to deal with coexistent
maxillary sinusitis. These days, endoscopic sinus surgery has
superceded other modes of polyp removal. Caldwell—Luc is avoided.
operation
1. If a polypus is red and fleshy, friable and has granular surface, especially in older patients, think of malignancy.
2. Simple nasal polyp may masquerade a malignancy under-
neath. Hence all polypi should be subjected to histology.
3. A simple polyp in a child may be a glioma, an encepha-
locele or a meningoencephalocele. It should always be
aspirated and fluid examined for CSF. Careless removal
of such polyp would result in CSF rhinorrhoea and
meningitis.
4. Multiple nasal polypi in children may be associated with mucoviscidosis.
5. Epistaxis and orbital symptoms associated with a polyp should always arouse the suspicion of malignancy.

Bleeding from inside the nose is called epistaxis. It is fairly common and is seen in all age groups—children, adults and older people. It often presents as an emergency. Epistaxis is a sign and nota disease per se and an attempt should always be made to find any local or constitutional cause.

(FIGURES 33.1 AND 33.2)
Nose is richly supplied by both the external and internal carotid systems, both on the septum and the lateral walls.

1. Anterior ethmoidal
artery | Branches of ophthalmic
2. Posterior ethmoidal artery J artery

1. Sphenopalatine artery (branch of maxillary artery) gives
nasopalatine and posterior medial nasal branches.
2. Septal branch of greater palatine artery (branch of max- illary artery).
3. Septal branch of superior labial artery (branch of facial artery).

INTERNAL CAROTID SYSTEM
Branches of
ophthalmic artery
2. Posterior ethmoidal

1. Posterior lateral nasal
—
From sphenopalatine
branches
artery
2. Greater palatine artery —
From maxillary artery
3. Nasal branch of anterior —
From infraorbital
superior dental
branch of maxillary
artery
4. Branches of facial artery to nasal vestibule

It
is
situated
in
the
anterior
inferior
part
of
nasal
sep-
tum,
just
above
the
vestibule.
Four
arteries—anterior
eth-
moidal, septal branch of superior labial, septal branch of
sphenopalatine and the greater palatine, anastomose here to form a vascular plexus called “Kiesselbach’s plexus.” This area is exposed to the drying effect of inspiratory current and to finger nail trauma, and is the usual site for epistaxis in children and adults.
young
Retrocolumellar vein. This vein runs vertically downwards just behind the columella, crosses the floor of nose and joins venous plexus on the lateral nasal wall. This is a common
site of venous bleeding in young people.

It is a plexus of veins situated inferior to posterior end of inferior turbinate. It is a site of posterior epistaxis in adults.

They may be divided into:
1. Local, in the nose or nasopharynx.
2. General.
3. Idiopathic.

1. Trauma. Finger nail trauma, injuries of nose, intranasal surgery, fractures of middle third of face and base of of violent sneeze.
skull, hard-blowing nose,
2. Infections
(a) Acute: Viral rhinitis, nasal diphtheria, acute sinusitis.
(b) Chronic: All crust-forming diseases, e.g. atrophic rhini- tis, rhinitis sicca, tuberculosis, syphilis septal perforation,
granulomatous lesion of the nose, e.g. rhinosporidiosis.
3. Foreign bodies
(a) Nonliving: Any neglected foreign body, rhinolith.
(b) Living: Maggots, leeches.
4. Neoplasms of nose and paranasal sinuses.
(a) Benign: Haemangioma, papilloma.
(b) Malignant: Carcinoma or sarcoma.
5. Atmospheric changes. High altitudes, sudden decom-
pression (Caisson disease) .
6. Deviated nasal septum.

1. Adenoiditis.
2. Juvenile angiofibroma.
3. Malignant tumours.
septum.

1. Cardiovascular system. Hypertension, arteriosclerosis, mitral stenosis, pregnancy (hypertension and hormonal).
2. Disorders of blood and blood vessels. Aplastic anaemia, leukaemia, thrombocytopenic and vascular purpura, haemophilia, Christmas disease, scurvy, vitamin K deficiency and hereditary haemorrhagic telangectasia.
3. Liver disease. Hepatic cirrhosis (deficiency of factor II, VU, IX and X).
Kidney disease. Chronic nephritis. al
5. Drugs. Excessive use of salicylates and other analgesics (as for joint pains or headaches), anticoagulant therapy (for heart disease).
6. Mediastinal compression. Tumours of mediastinum (raised venous pressure in the nose).
7. Acute general infection. Influenza, measles, chickenpox, whooping cough, rheumatic fever, infectious mononucleosis, typhoid, pneumonia, malaria and dengue fever.
8. Vicarious menstruation (epistaxis occurring at the time
of menstruation).
Many times the cause of epistaxis is not clear.

1. Little's area. In 90% cases of epistaxis, bleeding occurs from this site.
2. Above the level of middle turbinate. Bleeding from above
the middle
turbinate and
corresponding area
on the
sep-
tum is often from the anterior and posterior ethmoidal vessels (internal carotid system).
3. Below the level of middle turbinate. Here bleeding is
from the
sphenopalatine artery.
It may
be hid-
den, lying
require lateral
to middle
or inferior
infrastructure of
turbinate these
turbinates and
may for
localization of
the bleeding
site and
placement of
packing to
control it.
4. Posterior part of nasal cavity. Here blood flows directly into the pharynx.
5. Diffuse. Both from septum and lateral nasal wall. This is often seen in general systemic disorders and blood dyscrasias.
6. Nasopharynx.

When blood flows out from the front of nose with the patient
in sitting position.

Mainly the blood flows back into the throat. Patient may
swallow it and later have a “coffee-coloured” vomitus. This may erroneously be diagnosed as haematemesis.
. . . The differences between the two types of epistaxis are
tabulated herewith (Table 33.1).

In any case of epistaxis, it is important to know:
Mode of onset. Spontaneous or finger nail trauma.
Duration and frequency of bleeding. Amount of blood loss. ON
Side of nose from where bleeding is occurring.
Whether bleeding is of anterior or posterior type. Oo
Any known bleeding tendency in the patient or family.
7.
History
Figure 33.3 Methods of anterior nasal packing. (A) Packing in vertical layers. (B) Packing in horizontal layers.
of known
medical ailment
(hypertension, leukae-
mia, mitral
valve disease,
cirrhosis and
nephritis).
- 8. History of drug intake (analgesics, anticoagulants, etc.).

Most of the time, bleeding occurs from the Little's area and can be easily controlled by pinching the nose with thumb and index finger for about 5 min. This compresses the vessels of the Little's area. In Trotter's method patient is made to sit, leaning a little forward over a basin to spit any blood and breathe quietly from the mouth. Cold compresses should be applied to the nose to cause reflex vasoconstriction.

This is useful in anterior epistaxis when bleeding point has been located. The area is first topically anaesthetized and the bleeding point cauterized with a bead of silver nitrate or coagulated with electrocautery.

In cases of active anterior epistaxis, nose is cleared of blood clots by suction and attempt is made to localize the bleeding site. In minor bleeds, from the accessible sites, cauterization of the bleeding area can be done. If bleeding is profuse and/or the site of bleeding is difficult to localize, anterior packing should be done. For this, use a ribbon gauze soaked with liquid paraffin. About 1 m gauze (2.5 cm wide in adults and 12 mm in children) is required for each nasal cavity. First, few centimetres of gauze are folded upon itself and inserted along the floor and then the whole nasal cavity is packed tightly by layering the gauze from floor to the roof and from before backwards. Packing can also be done in vertical layers from back to the front (Figure 33.3). One or both cavities may need to be packed. Pack can be removed after 24 h, if bleeding has stopped. Sometimes, it has to be kept for 2-3 days; in that case, systemic antibiotics should be given to prevent sinus infection and toxic shock syndrome.

It is required for patients bleeding posteriorly into the throat. A postnasal pack is first prepared by tying three silk ties to a piece of gauze rolled into the shape of a cone. A rubber catheter is passed through the nose and its end brought out from the mouth (Figure 33.4). Ends of the silk threads are tied to it and catheter withdrawn from nose. Pack, which follows the silk thread, is now guided into the nasopharynx with the index finger. Anterior nasal cavity is now packed and silk threads tied over a dental roll. The third silk thread is cut short and allowed to hang in the oropharynx. It helps in easy removal of the pack later. Patients requiring postnasal pack should always be hospitalized. Instead of postnasal pack, a Foley's catheter size 12-14 F can also be used. After insertion balloon is inflated with 5-10 mL of saline. The bulb is inflated with saline and pulled forward so that choana is blocked and then an anterior nasal pack is kept in the usual manner. These days nasal balloons are also available (Figure 33.5). A nasal balloon has two bulbs, one for the postnasal space and the other for nasal cavity.

Using topical or general anaesthesia, bleeding point is localized with a rigid endoscope. It is then cauterized with a malleable unipolar suction cautery or a bipolar cautery. The procedure is effective with less morbidity and decreased hospital stay. The procedure has a limitation when profuse bleeding does not permit localization of the bleeding point.

In case of persistent or recurrent bleeds from the septum, just elevation of mucoperichondrial flap and then repositioning it back helps to cause fibrosis and constrict blood vessels. SMR operation can be done to achieve the same result or remove any septal spur which is sometimes the cause of epistaxis.

1. External carotid. When bleeding is from the external carotid system and the conservative measures have failed, ligation of external carotid artery above the origin of superior thyroid artery should be done. It is avoided these days in favour of embolization or ligation of more peripheral branches of sphenopalatine artery.
Figure 33.4 Technique of postnasal pack.
2. Maxillary artery. Ligation of this artery is done in uncontrollable posterior epistaxis. Approach is via Caldwell—Luc operation. Posterior wall of maxillary sinus is removed and the maxillary artery or its branches are blocked by applying clips. This procedure is now superceded by transnasal endoscopic sphenopalatine artery ligation.
3. Ethmoidal arteries. In anterosuperior bleeding above the middle turbinate, not controlled by packing, anterior and posterior ethmoidal arteries, which supply this area, can be ligated. The vessels are exposed in the medial wall of the orbit by an external ethmoid (Lynch) incision.

The procedure can be done with rigid endoscopes under topical anaesthesia with sedation or under a general anaesthesia. A mucosal flap is lifted in posterior part of lateral nasal wall, sphenopalatine artery (SPA) is localized as it exits the foramen and closed with a vascular clip. Distal branches of the artery can be additionally cauterized and the flap then reposited. Anterior ethmoidal artery can also be ligated by Lynch incision as an adjunctive procedure. SPA ligation gives high success in control of refractory posterior bleed.

It is done by an interventional radiologist through femoral artery catheterization. Internal maxillary artery is localized and the embolization is performed with absorbable gelfoam and/ or polyvinyl alcohol or coils. Both ipsilateral or bilateral embolizations may be required for unilateral epistaxis because of cross circulation. Embolization is generally a safe procedure but may have potential risks like cerebral thromboembolism, haematoma at local site. Ethmoidal arteries cannot be embolized.

- 1. Make the patient sit up with a back rest and record any
blood loss taking place through spitting or vomiting.
Figure 33.5 Epistaxis balloon for posterior epistaxis. Posterior balloon (A) is inflated with 10 mL and anterior balloon (B) with 30 mL. Catheter provides nasal airway.
2. Reassure the patient. Mild sedation should be given.
3. Keep check on pulse, BP and respiration.
4. Maintain haemodynamics. Blood transfusion may be required.
5. Antibiotics may be given to prevent sinusitis, if pack is to be kept beyond 24 h.
6. Intermittent oxygen may be required in patients with
bilateral packs because of increased pulmonary resistance from reflex.
nasopulmonary
- 7. Investigate and treat the patient for any underlying local
or general cause.
Hereditary haemorrhagic telangiectasia. It occurs on the anterior part of nasal septum and is the cause of recurrent bleeding. It can be treated by using Argon, KTP or Nd: YAG laser. The procedure may require to be repeated several times in a year as telangectasia recurs in the surrounding mucosa. Some cases require septodermoplasty where anterior part of septal mucosa is excised and replaced by a split-skin graft.

Injuries of face may involve soft tissues, bones or both. The majority of facial injuries are caused by automobile accidents. Others result from sports, personal accidents, assaults and fights. The management of facial trauma can be divided into:
1. General management.
2. Soft tissue injuries and their management.
3. Bone injuries and their management.

1. Airway. Maintenance of airway should receive the highest priority. Airway is obstructed by loss of skeletal support, aspiration of foreign bodies, blood or gastric contents or swelling of tissues. Airway is secured by intubation or the tracheostomy.
2. Haemorrhage. Injuries of face may bleed profusely.
Bleeding
should
be
stopped
by
pressure
or
ligation
of
vessels.
3. Associated injuries. Facial injuries may be associated with injuries of head, chest, abdomen, neck, larynx, cervical spine or limbs and should be attended too.

Wound is thoroughly cleaned of any dirt, grease or foreign matter. The lacerations are closed by accurate approximation of each layer.

Parotid tissue, if exposed, is repaired by suturing. Injuries of parotid duct are more serious. Both ends of the duct are identified and sutured over a polyethylene tube with fine suture. The tube is left for 3 days to 2 weeks.

If severed, the facial nerve is exposed by superficial parotidectomy and cut ends are approximated with 8-0 or 10-0 silk under magnification.

The face can be divided into three regions:
1. Upper third. Above the level of supraorbital ridge.
2. Middle third. Between the supraorbital ridge and the upper teeth.
3. Lower third. Mandible and the lower teeth.
The various fractures encountered in these regions listed in Table 34.1.

Frontal sinus fractures may involve anterior wall, posterior wall or the nasofrontal duct.
1. Anterior wall fractures may be depressed or comminuted. Defect is mainly cosmetic. Sinus is approached through a wound in the skin if that is present, or through a brow incision. The bone fragments are elevated, taking care not to strip them from the periosteum. The interior of the sinus is always inspected to rule out fracture of the posterior wall.
2. Posterior wall fractures may be accompanied by dural tears, brain injury and CSF rhinorrhoea. They may require neurosurgical consultation. Dural tears can be covered by temporalis fascia. Small sinuses can be obliterated with fat.
3. Injury to nasofrontal duct causes obstruction to sinus drain- age and may later be complicated by a mucocele. In such cases, make a large communication between the sinus and the nose. Small sinuses can be obliterated with fat after removing the sinus mucosa completely.

Ridge fractures often cause periorbital ecchymosis, flattening of the eyebrow, proptosis or downward displacement of eye. Fragment of bone may also be pushed into the orbit and get impacted. Ridge fractures require open reduction through an incision in the brow or transverse skin line of the forehead.
are

They may be depressed or linear, with or without separa- tion. They often extend into the orbit. Brain injury and cerebral oedema are commonly associated with each other and require neurosurgical consultation.

Fractures of nasal bones are the most common because of the projection of nose on the face. Traumatic forces may act from the front or side. Magnitude of force will determine the depth of injury.

1. Depressed. They are due to frontal blow. Lower part of nasal bones which is thinner, easily gives way. A severe frontal blow will cause "open-book fracture" in which nasal septum is collapsed and nasal bones splayed out. Still, greater forces will cause comminution of nasal bones and even the frontal processes of maxillae with flattening and widening of nasal dorsum.
2. Angulated. A lateral blow may cause unilateral depression of nasal bone on the same side or may fracture both the nasal bones and the septum with deviation of nasal bridge. Nasal fractures are often accompanied by injuries of nasal septum which may be simply buckled, dislocated or fractured into several pieces. Septal haematoma may form.

1. Swelling of nose. Appears within few hours and may obscure details of examination.
2. Periorbital ecchymosis.
3. Tenderness.
4. Nasal deformity. Nose may be depressed from the front or side, or the whole of the nasal pyramid deviated to one side.
Crepitus and mobility of fractured fragments.
Nasal obstruction due to septal injury or haematoma.
Lacerations of the nasal skin with of nasal bones
exposure and cartilage may be seen in compound fractures.
Epistaxis. SI
Figure 34.1 Types of fractures. (A) Normal. (B) Frontal blow causing depressed fracture or open-book fracture. (C) Lateral blow causing deviation of nasal bridge or depression of one nasal bone.

Diagnosis is best made on physical examination. X-rays may or may not show fracture (Figure 34.2). Patient should not be dismissed as having no fracture because X-rays did not reveal it. X-rays should include Waters' view, right and left lateral views and occlusal view.

Simple fractures without displacement need no treatment; others may require closed or open reduction. Presence of oedema interferes with accurate reduction by closed methods. Therefore, the best time to reduce a fracture is before the appearance of oedema, or after it has subsided, which is usually in 5-7 days. It is difficult to reduce a nasal fracture after 2 weeks because it heals by that time. Healing is faster in children and therefore earlier reduction is imperative.

tained by either frontal or lateral blow can be reduced by a straight blunt elevator guided by digital manipulation from outside.
Laterally, displaced nasal bridge can be reduced by firm digital pressure in the opposite direction. Impacted fragments sometimes require disimpaction with Walsham or Asch's forceps before realignment. Septal fractures are also reduced by Asch's forceps. Septal haematoma, if present, must be drained.
Simple fractures may not require intranasal packing. Unstable fractures require intranasal packing and external splintage.
Figure 34.2 Fractured nasal bone (arrow) as seen in radiograph.
2. Open reduction. Early open reduction in nasal fractures is rarely required. This is indicated when closed methods fail. Certain septal injuries can be better reduced by open methods. Healed nasal deformities resulting from nasal trauma can be corrected by rhinoplasty or septorhinoplasty.

Direct force over the nasion fractures nasal bones and displaces them posteriorly. Perpendicular plate of ethmoid, ethmoidal air cells and medial orbital wall are fractured and driven posteriorly. Injury may involve cribriform plate, frontal sinus, frontonasal duct, extraocular muscles, eyeball and the lacrimal apparatus. Medial canthal ligament may be avulsed.

1. Telecanthus, due to lateral displacement of medial orbital wall.
2. Pug nose. Bridge of nose is depressed and tip turned up.
3. Periorbital ecchymosis.
4. Orbital haematoma due to bleeding from anterior and posterior ethmoidal arteries.
5.
CSF
leakage due
to fracture
of cribriform
plate and
dura.
- 6. Displacement of eyeball.

Various facial films will be required to assess the extent of fracture and injury to other facial bones. Computed tomography (CT) scans are more useful.

1. Closed reduction. In uncomplicated cases, fracture is reduced with Asch's forceps and stabilized by a wire passed through fractured bony fragments and septum and then tied over the lead plates. Intranasal packing is given. Splint- ing is kept for 10 days or so.
Figure 34.3 Fracture zygoma left.
2. Open reduction. This is required in cases with extensive comminution of nasal and orbital bones, and those complicated by other injuries to lacrimal apparatus, medial canthal ligaments, frontal sinus, etc.
An H-type incision gives adequate exposure of the fractured area. This can be extended to the eyebrows if access to frontal sinuses is also required.
Nasal bones are reduced under vision and bridge height is achieved. Medial orbital walls can be reduced. Medial canthal ligaments, if avulsed, are restored with a through and through wire. Intranasal packing may be required to restore the contour. When bone comminution is severe, restoration of medial canthal ligaments and lacrimal apparatus should receive preference over reconstruction of nasal contour.

After nasal bones, zygoma is the second most frequently fractured bone. Usually, the cause is direct trauma. Lower segment of zygoma is pushed medially and posteriorly resulting in flattening of the malar prominence and a step deformity at the infraorbital margin. Zygoma is separated at its three processes (Figure 34.3). Fracture line passes through zygomaticofrontal suture, orbital floor, infraorbital margin and foramen, anterior wall of maxillary sinus and the zygomaticotemporal suture. Orbital contents may herniate into the maxillary sinus.

1. Flattening of malar prominence.
Step deformity of infraorbital margin. bo
Anaesthesia in the distribution of infraorbital nerve. Go
Trismus, due to depression of zygoma on the underlying coronoid process. yp
5. Oblique palpebral fissure, due to the displacement of lat- eral palpebral ligament.
6. Restricted ocular movements, due to entrapment of infe- rior rectus muscle. It may cause diplopia.
7. Periorbital emphysema, due to escape of air from the
maxillary sinus on nose blowing.

Waters' or exaggerated Waters' view shows the fracture and displacement the best. Maxillary sinus may show clouding due to the presence of blood. Comminution with depression of orbital floor and herniation of orbital contents cannot be seen on plain X-rays. CT scan of the orbital will be more useful.

Only displaced fractures require treatment. Open reduction and internal wire fixation gives best results. Fracture is exposed at the frontozygomatic suture through lateral brow incision and reduced by passing an elevator behind the zygoma. Wire fixation is done at frontozygomatic suture and infraorbital margin. The latter is exposed by a separate incision in the lower lid. Fracture of orbital floor can also be repaired through this incision.
Transantral approach is less favourable. Antrum_ is exposed as in Caldwell—Luc operation, blood is aspirated, fracture reduced and then stabilized by a pack in the antrum. Fractures of orbital floor can also be reduced. Antral pack is removed in about 10 days through the buccal incision, which is left open at the end of operation, or through the intranasal antrostomy route.

Zygomatic arch generally breaks into two fragments which get depressed. There are three fracture lines, one at each end and third in the centre of the arch.

Characteristic features are depression in the area of zygomatic arch, local pain aggravated by talking and chewing, trismus or limitation of the movements of mandible due to impingement of fragments on the condyle or coronoid process.

Arch fractures are best seen on submentovertical view of the skull. Waters' view is also taken.

A vertical incision is made in the hair-bearing area above or in front of the ear, cutting through temporal fascia. An elevator is passed deep to temporal fascia and carried under the depressed bony fragments which are then reduced. Fixation is usually not required as the fragments remain stable.

Zygomatic and Le Fort II maxillary fractures are always accompanied by fractures of orbital floor. Isolated fractures of orbital floor, when a large blunt object strikes the globes, are called "blow out fractures." Orbital contents may herniate into the antrum
(Figure 34.4).

1. Ecchymosis of lid, conjunctiva and sclera.
2. Enophthalmos with inferior displacement of the eyeball.
This becomes apparent when oedema subsides.
3. Diplopia, which may be due to displacement of the eye-
ball or entrapment of inferior rectus and inferior oblique muscles.
4. Hypoaesthesia or anaesthesia of cheek and upper lip, if infraorbital nerve is involved.

Waters' view shows a convex opacity bulging into the antrum from above (tear-drop opacity). CT scans may confirm the diagnosis (Figure 34.5). Entrapment of inferior rectus and inferior oblique muscles is diagnosed by asking the patient to look up and down, or by the traction test. The latter is performed by grasping the globe and passively rotating it to check for restriction of its movements.

Indications for surgery include enophthalmos and persistent diplopia due to entrapment of muscle. Orbital floor fractures can be satisfactorily reduced by a finger passed into the antrum through a transantral approach. A pack can be kept in the antrum to support the fragments. Infraor bital approach, through a skin crease of the lower lid, can also be used either alone or in combination with transantral approach. Badly comminuted fractures of orbital floor can be repaired by a bone graft from the iliac crest, nasal septum or the anterior wall of the antrum. Silicon or teflon sheets have also been used to reconstruct the orbital floor but autogenous grafts are preferable.
Figure
34.4
Blow out fracture with herniation of orbital contents into the maxillary sinus.
Figure 34.5
CT scan showing blow out fracture of right orbital floor.

1. Le Fort I (transverse) fracture runs above and parallel to the palate. It crosses lower part of nasal septum, maxillary antra and the pterygoid plates.
2. Le Fort II (pyramidal) fracture passes through the root of
nose, lacrimal
bone, floor
of orbit,
upper part
of maxil-
lary sinus and pterygoid plates. This fracture has some features common with the zygomatic fractures.
- 3. Le Fort III (craniofacial dysjunction). There is complete
separation of
facial bones
from the
cranial bones.
The fracture
line passes
junction, superior
through root
orbital of
nose, ethmofrontal
fissure, lateral
frontozygomatic and
temporozygomatic upper part of pterygoid plates.

1. Malocclusion of teeth with anterior open bite.
2. Elongation of midface.
3. Mobility in the maxilla.
4. CSF rhinorrhoea. Cribriform plate is injured in Le Fort II
and Le Fort II fractures.

X-rays, helpful in diagnosis of maxillary fractures are Waters' view, posteroanterior view, lateral view and the CT scans.
Figure
34.6
They help to delineate fracture lines and the displacement of fragments.

Treatment of maxillary fractures is complex. Immediate attention is paid to restore the airway and stop severe haem- orrhage from maxillary artery or its branches. For good cosmetic and functional results, fractures should be treated as early as the patient’s condition permits. Associated intracranial and cervical spine injuries may delay specific treatment.
Fixation of maxillary fractures can be achieved by:
1. Interdental wiring.
2. Intermaxillary wiring using arch bars.
3. Open reduction and interosseous wiring as in zygomatic fractures.
4. Wire slings from frontal bone, zygoma or infraorbital rim to the teeth or arch bars.

Fractures of mandible have been classified according to their location (Figure 34.7). Condylar fractures are the most common. They are followed, in frequency, by fractures of the angle, body and symphysis (mnemonic CABS). Fractures of the ramus, coronoid and alveolar processes are uncommon. Multiple fractures are seen as frequently as single ones. Most of the mandibular fractures are the result of direct trauma; however, condylar fractures are caused by indirect trauma to the chin or opposite side of the body of mandible. Displacement of mandibular fractures is determined by (i) the pull of muscles attached to the fragments, (ii) direction of fracture line and (iii) bevel of the fracture.

Fractures of maxilla: (A) Le Fort |, (B) Le Fort Il and
(C) Le Fort Ill.
In fractures of condyle, if fragments are not displaced, pain and trismus are the main features and tenderness is elicited at the site of fracture. If fragments are displaced, there is in addition, malocclusion of teeth and deviation of jaw to the opposite side on opening the mouth.
Most of the fractures of angle, body and symphysis can be diag-
nosed by intraoral and extraoral palpation. Step deformity,
Figure 34.7 Fractures of mandible (Dingman's classification). Condylar fractures are the most common, followed by those of the angle, body wall
of orbit,
sutures and
the
186
malocclusion of teeth, ecchymosis of oral mucosa, tender ness at the site of fracture and crepitus may be seen.

X-rays useful in mandibular fractures are PA view of the skull (for condyle), right and left oblique views of mandible and the view.
panorex

Both closed and open methods are used for reduction and fixation of the mandibular fractures.
In closed methods, interdental wiring and intermaxillary fixation are useful. External pin fixation can also be used. In open methods, fracture site is exposed and fragments fixed by direct interosseous wiring. This is further strengthened by a wire tied in a figure of eight manner. These days, compression plates are available to fix the fragments. With their use, prolonged immobilization and intermaxillary fixation can be avoided.
Condylar fractures are also treated by intermaxillary fixation with arch bars and rubber bands. Sometimes, open reduction and interosseous wiring may be required in adult edentulous patients with bilateral condylar fractures or in fractures of children.
Immobilization of mandible beyond 3 weeks, in condylar fractures, can cause ankylosis of temporomandibular joints. Therefore, intermaxillary wires are removed and jaw exercises started. If occlusion is still disturbed, intermaxillary wires are reapplied for another week and the process repeated till the bite and jaw movements are normal.

It is a communication between the antrum and oral cavity. The fistulous opening may be situated on the alveolus or gingivolabial sulcus.

1.
Dental extraction
of second
times is
premolar second
antral and
cavity and
the most
and upper
third)
their important
cause.
molars
(first are
closely extraction
and related
may lead
to
Roots some-
to the
fistula
formation. Presence of apical tooth abscess predis-
poses to it.
2. Failure of sublabial incision to heal after Caldwell—Luc operation.
3. Erosion of antrum by carcinoma.
4. Fractures or penetrating injuries of maxilla.
5. Osteitis of maxilla, syphilis or malignant granuloma.

1. Regurgitation of food. Food or fluids pass from oral cav- ity into the antrum and thence into the nose.
2. Discharge. Antrum is always infected. Foul-smelling dis- charge is seen, filling the nose or exuding from the fistu- lous opening into the mouth.
3. Inability to build positive or negative pressure in the mouth. Patient will have difficulty to blow the wind instru- ments or drink through a straw. To drink through a straw, negative pressure has to be created in the oral cavity. This cannot be done in the presence of an oroantral fistula as air gets drawn from nose to antrum to oral cavity. Reverse is true when blowing wind instruments; instead of build- ing a positive pressure in the oral cavity, air is blown out from the oral cavity to antrum and out through the nose.

A probe can be passed from the fistulous opening in the oral cavity into the antrum.

Recent fistula. When fistula is discovered immediately after tooth extraction and there is no infection or a retained tooth in the antrum, conservative treatment with suturing of gum margins and a course of antibiotics is effective.
Chronic fistula or a large fistula. It requires surgical repair by a palatal or a buccal flap. Maxillary sinusitis is first treated by repeated irrigations and antibiotics. Squamous-lined fis- tulous track is excised, bony edges of the fistula are smooth- ened and prepared for the flaps to sit properly. Caldwell—Luc operation may be required to remove a retained tooth root or a foreign body, clear the antrum of diseased mucosa and to provide a nasoantral window for free drainage. Some fis- tulas are better closed by a dental obturator. The latter also permits observation of antral cavity particularly in those treated for cancer.

Paranasal sinuses are air-containing cavities in certain bones of skull. They are four on each side. Clinically, paranasal sinuses have been divided into two groups:
1. Anterior group. This includes maxillary, frontal and anterior ethmoidal. They all open in the middle meatus and their ostia lie anterior to basal lamella of middle turbinate.
2. Posterior group. This includes posterior ethmoidal sinuses which open in the superior meatus and the sphenoid sinus which opens in sphenoethmoidal recess.

It is the largest of paranasal sinuses and occupies the body of maxilla. It is pyramidal in shape with base towards lateral wall of nose and apex directed laterally into the zygomatic process of maxilla and sometimes in the zygomatic bone itself (Figure 35.1). On an average, maxillary sinus has a capacity of 15 mL in an adult. It is 33 mm high, 35 mm deep and 25 mm wide.

¢ Anterior wall is formed by facial surface of maxilla and is related to the soft tissues of cheek.
¢ Posterior wall is related to infratemporal and pterygopalatine fossae.
¢ Medial wallis related to the middle and inferior meatuses. At places, this wall is thin and membranous. It is related to uncinate process, anterior and posterior fontanelle, and inferior turbinate and meatus.
¢ Floor is formed by alveolar and palatine processes of the maxilla and is situated about 1 cm below the level of floor of nose (Figure 35.1). Usually it is related to the roots of second premolar and first molar teeth. Depending on the age of the person and pneumatization of the sinus, the roots of all the molars, sometimes the premolars and canine, are in close relation to the floor of maxillary sinus separated from it by a thin lamina of bone or even no bone at all. Oroantral fistulae can result from extraction of any of these teeth. Dental infection is also an important cause of maxillary sinusitis.
Ostium of the maxillary sinus is situated high up in medial wall and opens in the posteroinferior part of ethmoidal infundibulum into the middle meatus. It is unfavourably situated for natural drainage. An accessory ostium is also present behind the main ostium in 30% of cases.
¢ Roof of the maxillary sinus is formed by the floor of the orbit. It is traversed by infraorbital nerve and vessels.

Each frontal sinus is situated between the inner and outer tables of frontal bone, above and deep to the supraorbital margin. It varies in shape and size and is often loculated by incomplete septa. The two frontal sinuses are often asymmetric and the intervening bony septum is thin and often obliquely placed or may even be deficient. Frontal sinus may be absent on one or both sides or it may be very large extending into orbital plate in the roof of the orbit. Its average dimensions are: height 32 mm, breadth 24 mm and depth 16 mm (remember code 8, i.e. 8 x 4, 8 x 3. and 8 x 2). Anterior wall of the sinus is related to the skin over the forehead; inferior wall, to the orbit and its contents; and posterior wall to the meninges and frontal lobe of the brain.
Drainage of the frontal sinus is through its ostium into the frontal recess. In fact frontal sinus, its ostium and the frontal recess form an hour glass structure. Frontal recess is situated in the anterior part of middle meatus and is bounded by the middle turbinate (medially), lamina papyracea (laterally), agger nasi cells (anteriorly) and bulla ethmoidalis (posteriorly). It may be encroached by several anterior ethmoidal cells, which may obstruct its ventilation and drainage and lead to sinusitis. Frontal recess drains into the infundibulum or medial to it, depending on the superior attachment of the uncinate process (refer to Figure 23.6).
Figure
35.1
showing relationship
of maxillary
and ethmoidal
sinuses sinus
is about
to
1
cm orbit
and below
the the
nasal floor
of cavity.
Floor nose.
—
Due to encroachment of small air cells in the frontal recess, the drainage pathway may be reduced to a straight or more often tortuous pathway which was earlier called nasofrontal duct. It is an erroneous term as no true duct exists.

Ethmoidal sinuses are thin-walled air cavities in the lateral masses of ethmoid bone. Their number varies from 3 to 18. They occupy the space between upper third of lateral nasal wall and the medial wall of orbit. Clinically, ethmoidal cells are divided by the basal lamina into an anterior ethmoid group which opens into the middle meatus and posterior ethmoid group which opens into the superior meatus and into if that be supreme meatus, present. Roof of the ethmoid is formed by medial extension of the orbital plate of the frontal bone, which shows depressions on its undersurface, called fovea ethmoidalis. The lateral wall is formed by a thin plate of bone called lamina papyracea.

Important ethmoid cells in the anterior group include:
1. Agger nasi cells — present in the agger nasi ridge.
2. Ethmoid bulla — forms the posterior boundary of the hia-
tus semilunaris.
3. Supraorbital cells.
4. Frontoethmoid cells — situated in the area of the frontal recess and may encroach the frontal sinus.
5. Haller cells — situated in the floor of the orbit.

The posterior group of ethmoid sinuses lies posterior to the basal lamina of middle concha. They are 1-7 in number and open into superior meatus or in the supreme meatus, when present. One important cell of this group is sphenoethmoid cell, also called the Onodi cell. It is the most posterior cell of this group and extends along the lamina papyracea, lateral or superior to the sphenoid and may extend 1.5 cm behind the anterior face of sphenoid. Optic nerve and sometimes the carotid artery are related to it laterally and are in danger during endoscopic surgery.
At birth anterior ethmoids are 5 x 2 x 2: mm and posterior ethmoids are 5 x 4 x 2 mm. They attain their adult size by the 12th
year.

It occupies the body of sphenoid. The two, right and left sinuses, are rarely symmetrical and are separated by a thin bony septum which is often obliquely placed and may even be deficient (compare frontal sinus) (Figures 35.2 and 35.3). Ostium of the sphenoid sinus is situated high up in the anterior wall and opens into the sphenoethmoidal recess, medial to the superior or supreme turbinate. It may be slit like, oval or round and can be seen endoscopically. In adults, it is situated about 1.5 cm from the upper border of choana. The average distance from the anterior nasal spine to the ostium is about 7 cm.
An adult sphenoid sinus is about 2 cm high, 2 cm deep and 2 cm wide, but its pneumatization varies. In some cases pneumatization may extend into greater or lesser wing of sphenoid, pterygoid or clivus, i.e. basilar part of occipital bone.
Figure 35.2 Coronal section of sphenoid sinuses. Note the reliefs made by various structures in the cavity of sphenoid sinus. Optic nerve forms the superolateral ridge.
Figure 35.3 Relations of sphenoid sinus.

Lateral wall of the sphenoid is related to the optic nerve and carotid artery. The opticocarotid recess can be seen in between the two. It may extend laterally when the anterior clinoid processes are also pneumatized. Maxillary nerve may be related to lower part of the lateral wall of sphenoid. The optic nerve and internal carotid artery are usually covered by a thin bone, but sometimes this bony covering may be dehiscent, and then these structures lie exposed, covered only by mucosa.
Floor of the sinus is related to the Vidian nerve. Relation of the roof can be divided into two parts. Anterior part of the roof is related to the olfactory tract, optic chiasma and frontal lobe, while posterior part is related to the pituitary gland in the sella turcica and laterally to the cavernous sinus. Posterior wall of the sphenoid forms the clivus.
Relations of the sphenoid sinus are important in endoscopic skull base surgery.

Paranasal sinuses are lined by mucous membrane which is continuous with that of the nasal cavity through the ostia of sinuses. It is thinner and less vascular compared to that of the nasal cavity. Histologically, it is ciliated columnar

The lymphatics of maxillary, ethmoid, frontal and sphenoid sinuses form a capillary network in their lining mucosa and collect with lymphatics of nasal cavity. Then
Maxillary, Development and growth of paranasal sinuses.Status at birth = Present at birth Volume 6-8 mL. Maxillary, Development and growth of paranasal sinuses.Growth = Rapid growth from birth to 3 years and from 7-12 years. Adult size - 15 years. Maxillary, Development and growth of paranasal sinuses.First radiologic evidence = 4-5 months after birth. Ethmoid, Development and growth of paranasal sinuses.Status at birth = • Present at birth • Anterior group: 5 × 2 × 2 mm • Posterior group: 5 × 4 × 2 mm. Ethmoid, Development and growth of paranasal sinuses.Growth = Reach adult size by 12 years. Ethmoid, Development and growth of paranasal sinuses.First radiologic evidence = 1 year. Frontal, Development and growth of paranasal sinuses.Status at birth = Not present. Frontal, Development and growth of paranasal sinuses.Growth = Invades frontal bone at the age of 4 years. Size increases until teens and complete development by 20 years. Frontal, Development and growth of paranasal sinuses.First radiologic evidence = 6 years. Sphenoid, Development and growth of paranasal sinuses.Status at birth = Not present. Sphenoid, Development and growth of paranasal sinuses.Growth = Reaches sella turcica by the age of 7 years, dorsum sellae by late teens and basisphenoid by adult age Reaches full size between 15 years to adult age. Sphenoid, Development and growth of paranasal sinuses.First radiologic evidence = 4 years
Figure 35.4 Mucociliary clearance of paranasal sinuses. (A) Maxillary sinus. (B) Frontal sinus. (C) Anterior and posterior group of sinuses. See text for details.
epithelium with goblet cells which secrete mucus. Cilia are more marked near the ostia of sinuses and help in drainage of mucus into the nasal cavity.

Growth of sinuses continues during childhood and early adult life. Radiologically, maxillary sinuses can be identified at 4-5 months, ethmoids at 1 year, frontals at 6 years and sphenoids at 4 years (Table 35.1).

Paranasal sinuses develop as outpouchings from the mucous membrane of lateral wall of nose. At birth, only the maxillary and ethmoidal sinuses are present and are large enough to be clinically significant.
190
they drain into lateral retropharyngeal and/or jugulodigastric nodes.

Ventilation of paranasal sinuses takes place through their ostia. During inspiration, air current causes negative pressure in the nose. This varies from -6 mm to -200 mm H,O, depending on the force of inspiration. During expiration, positive pressure is created in the nose and this sets up eddies which ventilate the sinuses. Thus, ventilation of sinuses is paradoxical; they are emptied of air during inspiration and filled with air during expiration. This is just the reverse of what takes place in lungs which fill during inspiration and empty during expiration.

Maxillary sinus. Mucus from all the walls of the maxillary sinus—anterior, medial, posterior, lateral and roof—is trans- ported by the cilia to the natural ostium and then through it into the middle meatus (Figure 35.4A). Mucus always drains from the natural ostium, even though accessory ostia be pres- ent in the fontanelle. It is also observed that inferior meatal antrostomy made in Caldwell—Luc operation provides ven- tilation to the sinuses, but it does not help in mucociliary clearance which still takes place through the natural ostium.
Frontal sinus. Mucociliary clearance of the frontal sinus is unique (see Figure 35.4B). Mucus travels up along the interfrontal septum, along the roof of the lateral wall, along the floor and then exits through the natural ostium. At two points, one just above the ostium and other in the frontal recess, part of the mucus recycles through the sinus and this
may carry infection of the frontal recess and sinuses drain- ing into it, towards the frontal sinus. Circulation is anticlock- wise in the right and clockwise in the left frontal sinus.
Sphenoid sinus. Mucociliary clearance is towards its ostium into the sphenoethmoidal recess.
Ethmoid sinus. Mucus from anterior group of ethmoid sinuses joins that from the frontal and maxillary sinuses and travels towards eustachian tube, passing in front of torus tubarius into the nasopharynx. Mucus from posterior eth- moids drains into superior or supreme meatus and then joins the mucus from the sphenoid sinus in the sphenoeth- moidal recess, passes above and behind the torus tubarius into the nasopharynx (Figure 35.4C).
It is noted that infected discharge from the anterior group of sinuses, passes behind the posterior pillars and causes hyper- trophy of lateral pharyngeal bands. Discharge from posterior group of sinuses spreads over the posterior pharyngeal wall.

It is not clear why nature provided paranasal sinuses. Prob- able functions are:
1. Airconditioning of the inspired air by providing large surface area over which the air is humidified and warmed.
2. To provide resonance to voice.
3. To act as thermal insulators to the delicate
protect struc- tures in the orbit and the cranium from variations of intranasal temperature.
To lighten the skull bones. »
5. To provide extended surface for olfaction; olfactory mucosa is situated in the upper part of nasal cavity and extends over ethmoid as well.
To provide local immunologic defence against microbes. To buffers against and thus brain No
act as trauma protect against injury, e.g. frontal, ethmoid and sphenoid sinuses.
Acute inflammation of sinus mucosa is called acute sinusitis. The sinus most commonly involved is the maxillary followed in turn by ethmoid, frontal and sphenoid. Very often, more than one sinus is infected (multisinusitis). Sometimes, all the sinuses of one or both sides are involved simultaneously (pansinusitis unilateral or bilateral).
Sinusitis may be "open" or "closed" type depending on whether the inflammatory products of sinus cavity can drain freely into the nasal cavity through the natural ostia or not. A "closed" sinusitis causes more severe symptoms and is also likely to cause complications.

1. Nasal infections. Sinus mucosa is a continuation of nasal mucosa and infections from nose can travel directly by continuity or by way of submucosal lymphatics. Most common cause of acute sinusitis is viral rhinitis followed by bacterial invasion.
2.
Swimming
and
diving.
Infected
water
can
enter
the
sinuses
through
their
ostia.
High
content
of
chlorine
gas
in
swim-
ming
pools
can
also
set
up
chemical
3.
Trauma.
Compound
fractures
or
penetrating
injuries
of
sinuses—frontal,
maxillary
and
ethmoid—may
permit
direct
infection
of
sinus
mucosa.
barotrauma
may
be
followed
by
4. Dental infections. This applies to maxillary sinus. Infection from the molar or premolar teeth or their extraction may be followed by acute sinusitis.

1. Obstruction to sinus ventilation and drainage. Normally, sinuses are well-ventilated. They also secrete small amount of mucus, which by ciliary movement, is directed towards the sinus ostia from where it drains into the nasal cavity. Any factor(s) which interfere with this function can cause sinusitis due to stasis of secretions in the sinus. They are:
(a) Nasal packing
(b) Deviated septum
(c) Hypertrophic turbinates
(d) Oedema of sinus ostia due to allergy or vasomotor rhinitis
(e) Nasal polypi
(f) Structural abnormality of ethmoidal air cells
(g) Benign or malignant neoplasm.
2. Stasis of secretions in the nasal cavity. Normal secretions of nose may not drain into the nasopharynx because of their viscosity (cystic fibrosis) or obstruction (enlarged adenoids, choanal atresia) and get infected.
3. Previous attacks of sinusitis. Local defences of sinus mucosa are already damaged.

¢ Environment. Sinusitis is common in cold and wet climate. Atmospheric pollution, smoke, dust and overcrowding also predispose to sinus infection.
¢ Poor general health. Recent attack of exanthematous fever (measles, chickenpox, whooping cough), nutritional deficiencies and systemic disorders (diabetes, immune deficiency syndromes).

Most cases of acute sinusitis start as viral infections followed soon by bacterial invasion. The bacteria most often responsible for acute suppurative sinusitis are Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pyogenes, Staphylococcus aureus and Klebsiella pneumoniae. Anaerobic organisms and mixed infections are seen in sinusitis of dental origin.

Acute inflammation
of sinus
mucosa dation
of fluid,
outpouring increased
activity the
virulence ity
of be
causes hyperaemia,
of of
serous polymorphonuclear
and of
organisms, the
exu- cells
and mucous
glands.
Depending defences
sinus ostium
to drain
mild the
(nonsuppurative)
exudate or
of the
host the
exudates, or
is serous;
severe later
purulent.
lining.
Severe
Failure sinus
it infections
of ostium
and tions.
on and
capabil- the
disease
(suppurative).
may cause
to drain
destruction of
Dental infections
in suppurative
its are
sinusitis.
Infections chapter.
of individual
may
Initially, become
mucopurulent destruction
results bony
of mucosal
in empyema
walls leading
very of
the to
complica- fulminating
sinuses and
are dealt
soon result
within this

1. Most commonly, it is viral rhinitis which spreads to involve the sinus mucosa. This is followed by bacterial invasion.
2. Diving and swimming in contaminated water.
3. Dental infections are important source of maxillary sinusitis. Roots of premolar and molar teeth are related to the floor of sinus and may be separated only by a thin layer of mucosal covering. Periapical dental abscess may burst into the sinus; or the root of a tooth, during extraction, may be pushed into the sinus. In case of oroantral fistula, following tooth extraction, bacteria from oral cavity enter the maxillary sinus.
4. Trauma to the sinus such as compound fractures, penetrating injuries or gunshot wounds may be followed by sinusitis.

One or more of the predisposing factors enumerated for sinusitis in general may be responsible for acute or recurrent infection.

Clinical features depend on (i) severity of inflammatory process and (ii) efficiency of ostium to drain the exudates. Closed ostium sinusitis is of greater severity and leads more often to complications.
1. Constitutional symptoms. It consist of fever, general malaise and body ache. They are the result of toxaemia.
2. Headache. Usually, this is confined to forehead and may thus be confused with frontal sinusitis.
3. Pain. Typically, it is situated over the upper jaw, but may be referred to the gums or teeth. For this reason patient may primarily consult a dentist. Pain is aggravated by stooping, coughing or chewing. Occasionally, pain is referred to the ipsilateral supraorbital region and thus may simulate frontal sinus infection.
4, Tenderness. Pressure or tapping over the anterior wall of antrum produces pain.
5.
Redness and
oedema of
cheek.
Commonly seen
in chil-
dren.
The lower
eyelid may
become puffy.
6. Nasal discharge. Anterior rhinoscopy/nasal endoscopy shows pus or mucopus in the middle meatus. Mucosa of the middle meatus and turbinate may appear red and swollen.
Postural test.
If no
pus seen
in the
middle meatus,
it is
decongested with
constrictor and
sinus turned
a
pledget the
up.
of cotton
patient is
made
Examination to
after soaked
sit with
10-15
with
a
vaso- the
affected min
may show
discharge in
the middle
meatus.
7. Postnasal discharge. Pus may be seen on the upper soft palate on posterior rhinoscopy or nasal endoscopy.

© Transillumination test. Affected sinus will be found opaque.
¢ X-rays. Waters' view will show either an opacity or a fluid level in the involved sinus. Computed tomography (CT) scan is the preferred imaging modality to investigate the sinuses.

1.
Antimicrobial
drugs.
and
amoxicillin
are
quite
effective
and
cover
a
wide
range
of
Eryth-
romycin
or
doxycycline
or
cotrimoxazole
are
equally
effective
and
can
be
given
to
those
who
are
sensitive
to
penicillin.
B-lactamase-producing
strains
of
H.
influenzae
and
M.
catarrhalis
may
necessitate
the
use
of
amoxicillin/
clavulanic
acid
or
cefuroxime
axetil.
Sparfloxacin
is
also
effective,
and
has
the
advantage
of
single
daily
dose.
2.
Nasal
decongestant
drops.
One
per
cent
ephedrine
or
0.1%
xylo-
or
are
used
as
nasal
drops
or
sprays
to
decongest
sinus
ostium
and
encourage
drainage.
3. Steam inhalation. Steam alone or medicated with men-
thol
or
Tr.
Benzoin
Co.
provides
symptomatic
relief
and
encourages
sinus
drainage.
should
be
given
15-20
min
after
nasal
decongestion
for
better
penetration.
4. Analgesics. Paracetamol or any other suitable analgesic should be given for relief of pain and headache.
5. Hot fomentation. Local heat to the affected sinus is often soothing and helps in the resolution of inflammation.

Antral lavage. Most cases of acute maxillary sinusitis respond to medical treatment. Lavage is rarely necessary. It is done only when medical treatment has failed and that too only under cover of antibiotics.

1. Acute maxillary sinusitis may change to subacute or chronic sinusitis.
2. Frontal sinusitis. Due to obstruction of frontal sinus drainage pathway because of oedema.
3. Osteitis or osteomyelitis of the maxilla.
4. Orbital cellulitis or abscess. Infection spreads to the orbit because of oedema either directly from the roof of maxillary sinus or indirectly, after involvement of ethmoid sinuses.

1. Usually follows viral infections of upper respiratory tract followed later by bacterial invasion.
2. Entry of water into the sinus during diving or swimming.
3. External trauma to the sinus, e.g. fractures or penetrat- ing injuries.
4.
Oedema
of
middle
meatus,
secondary
to
ipsi-
lateral
maxillary
or
ethmoid
sinus
Predisposing factors, pathology and bacteriology are the as in acute sinusitis in
same general.

1. Frontal headache. Usually severe and localized over the affected sinus. It shows characteristic periodicity, i.e. comes up on waking, gradually increases and reaches its
peak by about mid day and then starts subsiding. It is also called “office headache” because of its presence only dur- ing the office hours.
2. Tenderness. Pressure upwards on the floor of frontal sinus, just above the medial canthus, causes exquisite pain. It can also be elicited by tapping over the anterior wall of frontal sinus in the medial part of supraorbital region.
3. Oedema of upper eyelid with suffused conjunctiva and photophobia.
4. Nasal discharge. A vertical streak of mucopus is seen high up in the anterior part of the middle meatus. This may be absent if the ostium is closed with no drainage. Nasal mucosa is inflamed in the middle meatus.
X-rays. Opacity of the affected sinus or fluid level can be seen. Both Waters' and lateral views should be taken. CT scan is the preferred modality.

This is same as for acute maxillary sinusitis, i.e. antimicrobials, decongestion of the sinus ostium for drainage and analgesics. A combination of antihistaminic with an oral nasal decongestant (pseudoephedrine or phenylephrine hydrochloride) is useful. Placing a pledget of cotton soaked in a vasoconstrictor in the middle meatus, once or twice daily, helps to relieve ostial oedema and promotes sinus drainage and ventilation. If patient shows response to medical treatment and pain is relieved, treatment is continued for full 10 days to 2 weeks.

Trephination of frontal sinus. If there is persistence or exacerbation of pain or pyrexia in spite of medical treatment for 48 h, or if the lid swelling is increasing and threatening orbital cellulitis, frontal sinus is drained externally. A 2 cm long horizontal incision is made in the superomedial aspect of the orbit below the eyebrow (Figure 36.1). Floor of frontal sinus is exposed and a hole drilled with a burr. Pus is taken for culture and sensitivity, and a plastic tube inserted and fixed. Sinus can now be irrigated with normal saline two or three times daily until frontonasal duct
becomes patent. This can be determined by adding a few drops of methylene blue to the irrigating fluid and its exit seen through the nose. Drainage tube is removed when frontonasal duct becomes patent.

1. Orbital cellulitis.
2. Osteomyelitis of frontal bone and fistula formation.
3. Meningitis, extradural abscess or frontal lobe abscess, if infection breaks through the posterior wall of the sinus.
4. Chronic frontal sinusitis, if the acute infection is neglected or improperly treated.

Acute ethmoiditis is often associated with infection of other sinuses. Ethmoid sinuses are more often involved in infants and young children.

1. Pain. It is localized over the bridge of the nose, medial and deep to the eye. It is aggravated by movements of the eye ball.
2. Oedema of lids. Both eyelids become puffy and swollen.
There
is
increased
lacrimation.
Orbital
cellulitis
is
an
early
complication
in
such
cases.
3. Nasal discharge. On anterior rhinoscopy, pus may be
seen
in
middle
or
superior
meatus
depending
on
the
of
anterior
or
posterior
group
of
ethmoid
sinuses.
- 4. Swelling of the middle turbinate.

Medical treatmentis the same as for acute maxillary sinusitis. Visual deterioration and exophthalmos indicate abscess in the posterior orbit and may require drainage of the ethmoid sinuses into the nose through an external ethmoidectomy incision.

1. Orbital cellulitis and abscess.
2. Visual deterioration and blindness due to involvement of
optic nerve.
Cavernous sinus thrombosis. ge
Extradural abscess, meningitis or brain abscess.

Isolated involvement of sphenoid sinus is rare. It is often a part of pansinusitis or is associated with infection of poste- rior ethmoid sinuses.

1.
Headache.
Usually localized
to the
occiput or
vertex.
Pain may also be referred to the mastoid region.
- 2. Postnasal discharge. It can only be seen on posterior rhiA streak of be the roof and
noscopy. pus may seen on posterior wall of nasopharynx or above the posterior end of middle turbinate.
X-rays. Opacity or fluid level may be seen in the sphenoid sinus. Lateral view of the sphenoid sinus is taken in supine or prone position and is helpful to demonstrate the fluid level. CT scan is more useful.

Mucocele of the sphenoid sinus or its neoplasms may clinically simulate features of acute infection of sphenoid sinus and should always be excluded in any case of isolated sphenoid sinus involvement.

Sinus infection lasting for months or years is called chronic sinusitis. Most important cause of chronic sinusitis is failure of acute infection to resolve.

Acute infection destroys normal ciliated epithelium impairing drainage from the sinus. Pooling and stagnation of secretions in the sinus invites infection. Persistence of infection causes mucosal changes, such as loss of cilia, oedema and polyp formation, thus continuing the vicious cycle (Figure 37.1).

In chronic infections, process of destruction and attempts at healing proceed simultaneously. Sinus mucosa becomes thick and polypoidal (hypertrophic sinusitis) or undergoes atrophy (atrophic sinusitis). Surface epithelium may show desquamation, regeneration or metaplasia. Submucosa is infiltrated with lymphocytes and plasma cells and may show microabscesses, granulations, fibrosis or polyp formation.

Mixed aerobic and anaerobic organisms are often present.
Clinical features are often vague and similar to those of acute sinusitis but of lesser severity. Purulent nasal discharge is the commonest complaint. Foul-smelling discharge suggests anaerobic infection. Local pain and headache are often not marked except in acute exacerbations. Some patients complain of nasal stuffiness and anosmia.

1. X-ray of the involved sinus may show mucosal thickening or opacity.
2. X-rays after injection of contrast material may show soft tissue changes in the sinus mucosa.
3. Computed tomography (CT) scan is particularly useful in
ethmoid and
sphenoid sinus
infections and
has replaced
studies with
contrast materials.
- 4. Aspiration and irrigation: Finding of pus in the sinus is confirmatory.

It is essential to search for underlying aetiological factors
which obstruct sinus drainage and ventilation. A work-up for
nasal
allergy
may
be
required.
Culture
and
of
sinus
discharge helps in the proper selection of an antibiotic.
Initial treatment of chronic sinusitis is conservative, includ-
ing antibiotics, decongestants, antihistaminics and sinus
Figure
37.1
Causative factors
and pathophysiology
of chronic
irrigations. More often, some form of surgery is required either to provide free drainage and ventilation or radical surgery to remove all irreversible diseases so as to provide wide or to obliterate the sinus.
Recently, endoscopic sinus surgery is replacing radical on the sinuses and provides good drainage and It also avoids external incisions.
drainage operations ventilation.

1. Antral puncture and irrigation. Sinus cavity is irrigated with a cannula passed through the inferior meatus. Removal of pus and exudates helps the sinus mucosa to revert to normal.
2. Intranasal antrostomy. It is indicated if sinus irrigations fail to resolve infection. A window is created in the inferior meatus to provide aeration to the sinus and its free drainage.
3. Caldwell—Luc operation. In this operation, antrum is entered through its anterior wall by a sublabial incision. All irreversible diseases are removed and a window is created between the antrum and inferior meatus.
Details of the above operations are described in the section on Operative Surgery.

1. Intranasal drainage operations. Correction of deviated septum, removal of a polyp or anterior portion of middle turbinate, or intranasal ethmoidectomy provide drainage through the frontonasal duct. Treatment of associated maxillary sinusitis also helps to resolve chronic frontal sinusitis.
Trephination of frontal sinus (see p. 193). gon
External frontoethmoidectomy (Howarth's or Lynch operation). The frontal sinus is entered through its floor by a curvilinear incision round the inner margin of the orbit. Diseased mucosa is removed, ethmoid cells exenterated and a new frontonasal duct created.
4. Osteoplastic flap operation. It may be unilateral or bilateral. A coronal or a brow incision is used. The anterior wall of frontal sinus is reflected as an osteoplastic flap, based inferiorly. The diseased tissues are removed and the sinus drained through a new frontonasal duct. If it is desired to obliterate the sinus, all diseased as well as healthy mucosa are stripped off and the sinus obliterated with fat.

1. Intranasal ethmoidectomy. This operation is done for chronic ethmoiditis with polyp formation. The ethmoid air cells and the diseased tissue are removed between the middle turbinate and the medial wall of orbit by the intranasal route. The frontal and sphenoid sinuses can also be drained by this operation.
2. External ethmoidectomy. In this operation, ethmoid sinuses are approached through medial orbital incision. Access can also be obtained to sphenoid and frontal sinuses and the operation is called _fronto-spheno-ethmoidectomy.

Sphenoidotomy. Access to the sphenoid sinus can be obtained by removal of its anterior wall. This is accomplished by external ethmoidectomy or trans-septal approach, usually the former, because of the coexistence of ethmoid disease with chronic sphenoiditis.

Many different species of fungi are found to involve the paranasal sinuses; the more common being the Aspergillus, Alternaria, Mucor or Rhizopus. They may involve single or multiple sinuses. Four different varieties of fungal infection of sinuses are seen:

1. Fungal ball. It is due to implantation of fungus into an otherwise healthy sinus which on CT shows a hyperdense area with no evidence of bone erosion or expansion. Maxillary sinus is the most commonly involved followed by sphenoid, ethmoid and the frontal in that order. Treatment is surgical removal of the fungal ball and adequate drainage of the sinus. No antifungal therapy is required. 2. Allergic fungal sinusitis. It is an allergic reaction to the causative fungus and presents with sinunasal polyposis and mucin. The latter contains eosinophils, CharcotLeyden crystals and fungal hyphae. There is no invasion of the sinus mucosa with fungus. Usually more than one sinus are involved on one or both sides. CT scan shows mucosal thickening with hyperdense areas. There may be expansion of the sinus or bone erosion due to pressure, but no fungal invasion. Treatment is endoscopic surgical clearance of the sinuses with provision of drainage and ventilation. This is combined with pre and postoperative systemic steroids.
3. Chronic invasive sinusitis. Here the fungus invades into the sinus mucosa. There is bone erosion by fungus. Patient presents with chronic rhinosinusitis. CT scan shows thickened mucosa with opacification of sinus and bone erosion. Patient may have intracranial or intraorbital invasion. Histopathology shows fungal invasion of submucosa and granulomatous reaction with multinucleated giant cells. Treatment consists of surgical removal of the involved mucosa, bone and soft tissues followed by antifungal therapy with iv. amphotericin B. Up to a total of 2-3 g of the drug is given. This is followed by itraconazole therapy for 12 months or more monitored by serial CT or MRI scans. 4. Fulminant fungal sinusitis. It is an acute presentation and is mostly seen in immunocompromised or diabetic individuals. Common fungal species are Mucor or Aspergillus (Figure 37.2).

e Mucor causes rhinocerebral disease. Due to invasion of the blood vessels, mucor fungus causes ischaemic necrosis presenting as a black eschar, involving inferior turbinate, palate or the sinus. It spreads to the face, eye, skull base and the brain. Treatment is surgical debridement of necrotic tissue and i.v. amphotericin B.
e Aspergillus infection can also cause acute fulminant sinusitis with tissue invasion. Such patients present with acute sinusitis and develop sepsis and other sinus complications. Unlike Mucor infection, there is no black eschar. Treatment is antifungal therapy and surgery.
Figure 37.2 Aspergillus fungus. Note septate hyphae with acute angle branching (arrow). (A) H&E stain, x200. (B) Gomori methenamine silver stain, x200.
Figure 37.3 Functional endoscopic surgery of paranasal sinuses
without monitor and camera.

Better understanding of the pathophysiology of recurrent and chronic sinusitis and the fact that most of the changes are reversible, if proper drainage and ventilation is provided to the sinuses has, in more recent years, led to the development of endoscopic surgery of sinuses. This has further been made possible by advances in technology, such as development of:
- 1. Rigid endoscopes, which provide better illumination and magnification and permit visualization of structures situated at different
angles.
Figure 37.4 Endoscopic surgery in progress. Endoscope and other surgical instruments are passed through the nose and surgery performed by looking at the monitor.
2.
Microsurgical instruments,
which permit
precise and
lim- ited surgery, directed at specific sites, to remove obstruction to the sinus ostia.
Endoscopes can also be passed through a cannula into the maxillary sinus to visualize its interior and take accurate biopsies or deal with certain pathological conditions such as small cysts and polyps.
With endoscopic surgery, it is now possible to cure selected cases of chronic and recurrent infections of the frontal, maxillary, ethmoid and sphenoid sinuses without resort to external operations (see section on Operative Surgery).
2.

As long as infection is confined only to the sinus mucosa, it is called sinusitis. Complications are said to arise when infection spreads into or beyond the bony wall of the sinus (see Table 38.1 and Figure 38.1).

blocked. The sinus fills with mucus and its bony walls get expanded due to expansile process. CT scan and MRI can help in the diagnosis. A polyp, tumour or trauma in the middle meatus may also obstruct the sinus ostium to cause a mucocele. Mucocele of sphenoid sinus or sphenoethmoidal mucocele arises from slow expansion and destruction of sphenoid and posterior ethmoid sinuses. Clinical features are those of superior

The sinuses commonly affected by mucocele in the order of frequency
are the
frontal, ethmoidal,
maxillary and
sphe- noidal. There are two views in the genesis of a mucocele:
1.
Chronic obstruction
to sinus
ostium resulting
in accumu-
lation of secretions which slowly expand the sinus and destroy its bony walls.
Cystic dilatation
of mucous
gland of
the sinus
mucosa due
to obstruction
of is
surrounded its
duct.
by normal
In this
sinus case,
wall mucosa.
of
The mucocele
contents of
mucocele are
sterile.
Mucocele of the frontal sinus (Figure 38.2) usually presents in the superomedial quadrant of the orbit (90%) and displaces the eyeball forward, downward and laterally. The swelling is cystic and nontender; egg-shell crackling may be elicited. Sometimes, it presents as a cystic swelling in the forehead (10%). Patient's complaints are usually mild and may include headache, diplopia and proptosis. Imaging of the frontal sinus usually reveals clouding of the sinus with loss of scalloped outline which is so typical of the normal frontal sinus (see Figure 38.3). Treatment is frontoethmoidectomy with free drainage of frontal sinus into the middle meatus.
Mucocele of ethmoid sinuses causes expansion of the medial wall of the orbit, displacing the eyeball forward and laterally. In addition, it may cause a bulge in the middle meatus of nose. A mucocele of the ethmoid can be drained by an intranasal operation, uncapping the ethmoidal bulge and establishing free drainage. Sometimes, it may require external ethmoid operation.
Mucous retention cyst of the maxillary sinus presents as a retention cyst due to obstruction of the duct of seromucinous gland and usually does not cause bone erosion. It is asymptomatic and is observed as an incidental finding on radiographs. No treatment is generally required for asymptomatic retention cysts as most of them regress spontaneously over a period of time.

Mucocele of the maxillary sinus can occur as a complica- tion
of chronic
sinus inflammation
when its
ostium is
Figure
38.1
Complications of sinusitis.
orbital fissure syndrome (involvement of CN III, IV, VI and ophthalmic division of V) or orbital apex syndrome which is superior orbital fissure syndrome with additional involvement of optic and maxillary division of trigeminal nerve. Exophthalmos is always present and the pain is localized to the orbit or forehead. Some may complain of headache in the occiput or vertex. Treatment is external ethmoidectomy with sphenoidotomy. Anterior wall of the sphenoid sinus is removed, cyst wall uncapped and its fluid contents evacuated.
Pyocele or mucopyocele is similar to mucocele but its contents are purulent. It can result from infection of a mucocele of of the sinuses.
any
Figure
38.2
Mucocele of
frontal sinus.
Note swelling
above the
medial canthus of left eye (arrow).
Figure
38.3
CT scan of mucocele of the left frontoethmoid region.
Note the
left eyeball
has been
displaced downwards
and laterally
Endoscopic surgery has replaced external operation of the sinuses for treatment of all mucocele or mucopyoceles of various sinuses.

Osteomyelitis is infection of bone marrow and should be differentiated from osteitis which is infection of the compact bone. Osteomyelitis, following sinus infection, involves either the maxilla or the frontal bone.
1. Osteomyelitis of the maxilla. It is more often seen in infants and children than adults because of the presence of spongy bone in the anterior wall of the maxilla. Infection may start in the dental sac and then spread to the maxilla, but less often, it is primary infection of the maxillary sinus. Clinical features are erythema, swelling of cheek, oedema of lower lid, purulent nasal discharge and fever. Subperiosteal abscess followed by fistulae may form in infraorbital region (Figure 38.4), alveolus or palate, or in zygoma. Sequestration of bone may occur. Treatment consists of large doses of antibiotics, drainage of any abscess and removal of the sequestra.
Osteomyelitis of
maxilla may
cause damage
to tempo-
rary or
permanent tooth-buds,
maldevelopment of
maxilla, oroantral
fistula, persistently
draining sinus
or epiphora.
- 2. Osteomyelitis of frontal bone (Figure 38.5). It is more often seen in adults as frontal sinus is not developed in infants and children. Osteomyelitis of frontal bone results from acute infection of frontal sinus either directly or through the venous spread. It can also follow trauma or surgery of frontal sinus in the presence of acute infection. Pus may form externally under the periosteum as soft doughy swelling (Pott's puffy tumour), or internally as an extradural abscess. Treatment consists of large doses of antibiotics, drainage of abscess and trephining of frontal sinus through its floor. Sometimes, it requires removal
Figure 38.4
Osteomyelitis of maxilla with fistula formation in infra- orbital region (arrow).
- of sequestra and necrotic bone by raising a scalp flap through a coronal incision (Figure 38.5).

Orbit and its contents are closely related to the ethmoid, frontal and maxillary sinuses, but most of the complications, however, follow infection of ethmoids as they are separated from the orbit only by a thin lamina of bone— lamina papyracea. Infection travels from these sinuses either by osteitis or as thrombophlebitic process of ethmoidal veins.
Orbital complications include:
1. Inflammatory oedema of lids. This is only reactionary. There is no erythema or tenderness of the lids which characterises lid abscess. It involves only preseptal space, ie. lies in front of orbital septum. Eyeball movements and vision are normal. Generally, upper lid is swollen in frontal, lower lid in maxillary, and both upper and lower lids in ethmoid sinusitis.
2. Subperiosteal abscess. Pus collects outside the bone under the periosteum. A subperiosteal abscess from
Figure 38.5
Case of chronic frontal sinusitis presenting with a fistula in the floor of the sinus.
ethmoids forms on the medial wall of orbit and displaces the eyeball forward, downward and laterally; from the frontal sinus, abscess is situated just above and behind the medial canthus and displaces the eyeball downwards and laterally; from the maxillary sinus, abscess forms in the floor of the orbit and displaces the eyeball upwards and forwards.

3. Orbital cellulitis. When pus breaks through the periosteum and finds its way into the orbit, it spreads between the orbital fat, extraocular muscles, vessels and nerves. Clinical features will include oedema of lids, exophthalmos, chemosis of conjunctiva and restricted movements of the eye ball. Vision is affected causing partial or total loss which is sometimes permanent. Patient may run high fever. Orbital cellulitis is potentially dangerous because of the risk of meningitis and cavernous sinus thrombosis.
4. Orbital abscess. Intraorbital abscess usually forms along lamina papyracea or the floor of frontal sinus. Clinical picture is similar to that of orbital cellulitis. Diagnosis can be easily made by CT scan or ultrasound of the orbit. Treatment is i.v. antibiotics and drainage of the abscess and that of the sinus (ethmoidectomy or trephination of frontal sinus).
5. Superior orbital fissure syndrome. Infection of sphenoid sinus can rarely affect structures of superior orbital fissure. Symptoms consist of deep orbital pain, frontal headache and progressive paralysis of CN VI, II and IV, in that order.
6. Orbital apex syndrome. It is superior orbital fissure syndrome with additional involvement of the optic nerve and maxillary division of the trigeminal (Vs) (Figure 38.6).

Frontal, ethmoid and sphenoid sinuses are closely related to anterior cranial fossa and infection from these can cause:
Meningitis and encephalitis
Extradural abscess Subdural abscess ON
Brain abscess oR
Cavernous sinus thrombosis.

Aetiology. Infection of paranasal sinuses, particularly those of ethmoid and sphenoid and less commonly the frontal,
and orbital complications from these sinus infections can cause thrombophlebitis of the cavernous sinus(es). Other sources of infection are listed in Table 38.2. The valveless nature of the veins connecting the cavernous sinus causes easy spread of infection.
Clinical features. Onset of cavernous sinus thrombophlebitis is abrupt with chills and rigors. Patient is acutely ill. Eyelids get swollen with chemosis and proptosis of eyeball. Cranial nerves IJ, IV and VI which are related to the sinus get involved individually and sequentially causing total ophthalmoplegia. Pupil becomes dilated and fixed, optic disc shows congestion and oedema with diminution of vision. Sensation in the distribution of V, (ophthalmic division of CN V) is diminished. CSF is usually normal. Condition needs to be differentiated from orbital cellulitis (Table 38.3). CT scan is useful for this.
Treatment. It consists of i.v. antibiotics and attention to the focus of infection, drainage of infected ethmoid or sphenoid sinus. Blood culture should be taken before starting antibiotic therapy. Role of anticoagulants is not clear.

In suppurative sinusitis, discharge constantly flows into the and can cause or pharynx aggravate:
- 1. Otitis media (acute or chronic).
2.
Pharyngitis and
tonsillitis.
Hypertrophy phoid
bands yngitis)
behind is
the posterior
indicative unilateral
and sinusitis
of affect
the may
also of
lateral pillars
chronic side
cause of
the recurrent
(lateral sinusitis.
lym- phar-
It may
involved sinus.
tonsillitis or
be
Chronic granular
pharyngitis.
3.
Persistent laryngitis and tracheobronchitis. Sinusitis may be associated with recurrent laryngitis, bronchiectasis and asthma but the latter are not necessarily caused by sinusitis.

The role of sinus infection to act as focus of infection is doubtful. A few conditions such as polyarthritis, tenosynovitis, fibrositis and certain skin diseases may respond to elimination of infection in the sinuses. However, sinus infection, if present in these cases, is treated on its own merit.

Both benign and malignant tumours of the nasal cavity (Table 39.1) per se are uncommon. Very often their separa- tion from tumours of paranasal sinuses is difficult except in early stages. In addition to primary tumours, nasal cavity can be invaded by growths from paranasal sinuses, nasopharynx, cranial or buccal cavity.
Benign lesions are usually smooth, localized and covered with mucous membrane. Malignant ones are usually friable, have a granular surface and tend to bleed easily.

1. Squamous papilloma. Verrucous lesions similar to skin warts can arise from the nasal vestibule or lower part of nasal septum. They may be single or multiple, pedunculated or sessile (Figure 39.1). Treatment is local excision with cauterization of the base to prevent recurrence. They can also be treated by cryosurgery or laser.
2. Inverted papilloma (Transitional cell papilloma or Ringertz tumour or Schneiderian papilloma). It is a tumour of the nonolfactory mucosa of nose (Schneiderian membrane) and paranasal sinuses. Most common site of origin is lateral wall of nose in the middle meatus; less commonly it arises from the maxillary, frontal or sphenoid sinus (Figure 39.2). It is so named because hyperplastic papillomatous tissue grows into the stroma rather than in exophytic manner (Figure 39.3). Human papilloma virus is thought to be responsible for its aetiology. Clinically, men are affected more than women in the age group of 40-70. It is almost always unilateral and presents with nasal obstruction, nasal discharge and epistaxis. It can invade sinuses or orbit. Orbital involvement causes proptosis, diplopia and lacrimation.
On examination
of nose
or endoscopy,
it presents
as
a
pale polypoidal
mass resembling
Computed tomography
imaging
(MRI)
MRI
also from
the
Care
a
simple nasal
(CT)
show and
helps actual
should ated
with to
differentiate tumour
polypus or
magnetic and
extent polypi.
resonance of
associated mass.
Biopsy be
taken it
or even
for their
removal.
Treatment.
It
Medial can
ing approach.
the lesion.
secretions is
essential as
in sinus
for diagnosis.
polypi may
might maxillectomy
have been
is the
treatment by
lateral rhinotomy
These days
endoscopic be
associ- operated
of or
choice.
sublabial approach
deglov- is

preferred.
In
10-15%
of cases,
it is
malignancy.
Wider external surgical approaches may be required for tumour extending to the frontal sinus or orbit. Recurrence can occur. Radiotherapy is not advised as it may induce malignancy.
3. Pleomorphic adenoma. Rare tumour, usually arises from the nasal septum. Treatment is wide surgical excision.
4. Schwannoma. Schwannoma is an uncommon benign tumour arising from the nose or paranasal sinuses. The latter include ethmoid, maxillary and sphenoid sinuses. It arises from the Schwann cells of nerve sheath.
Clinically it presents a rounded mass, firm in consistency, yellowish in colour and may show blood vessels running on its surface. It can cause pressure necrosis of the surrounding bones. Imaging techniques, CT and MRI, are useful to show the extent. Diagnosis is made on biopsy. Treatment is surgical excision. They can be removed by endoscopic surgery or by external approaches.

e
Squamous
papilloma
©
Carcinoma
Inverted
papilloma
»
Squamous
cell
carcinoma
e
Pleomorphic
adenoma
°
Adenocarcinoma
e
Schwannoma
e
Malignant
melanoma
°¢
Meningioma
e
Esthesioneuroblastoma
e
Haemangioma
e
Haemangiopericytoma
e
Chondroma
e
Lymphoma
e
Angiofibroma
©
Solitary
plasmacytoma
e
Encephalocele
e
Various
types
of
sarcoma
°
Glioma
e
Dermoid
Figure 39.1
Squamous papilloma nose, left side.
5. Meningioma. It is an uncommon tumour found intrana-
sally. Treatment is surgical excision by lateral rhinotomy.
6. Haemangioma. It may be:
(a) Capillary haemangioma (bleeding polypus of the septum). It is a soft, dark red, pedunculated or sessile tumour arising from the anterior part of nasal septum (Figure 39.4). Usually it is smooth but may become ulcerated and present with recurrent epistaxis and nasal obstruction. Treatment is local excision with a cuff of surrounding mucoperichondrium.
(b) Cavernous haemangioma. It arises from the turbinates on the lateral wall of nose. It is treated by surgical excision with preliminary cryotherapy. Extensive lesions may require radiotherapy and surgical excision.
7. Chondroma. It can arise from the ethmoid, nasal cavity or nasal septum. Pure chondromas are smooth, firm and lobulated. Others may be mixed type fibro-, osteoor angiochondromas. Treatment is surgical excision. For recurrent or large tumours, wide excision should be done because of their tendency to malignant transformation after repeated interference.
8. Angiofibroma. It is included in nasal tumours because its primary site of origin is supposed to be posterior part of nasal cavity near the sphenopalatine foramen (see p. 246).

9. Intranasal meningoencephalocele. It is herniation of brain tissues and meninges through foramen caecum or cribriform plate. It presents as a smooth polyp in the upper part of nose between the septum and middle turbinate, usually in infants and young children. The mass increases in size on crying or straining. Unless care is taken, it may be misdiagnosed as a simple polyp and mistakenly avulsed, resulting in cerebrospinal fluid rhinorrhoea or meningitis. For the same reason biopsy should not be taken. CT scan is essential to demonstrate a defect in the base of skull. Treatment is frontal craniotomy, severing the stalk from the brain, and repair of dural and bony defect. Intranasal mass is removed as secondary procedure after cranial defect has sealed.
10. Gliomas. Of all the gliomas, 60% are extranasal, 30% are intranasal and 10% both intra and extranasal. They are seen in infants and children. An intranasal glioma presents as a firm polyp sometimes protruding at the anterior nares.
Figure 39.2 (A) Inverted papilloma in a 79-year-old male (right side). (B) CT scan of the same.
Figure 39.3 Histological section of a Schneiderian papilloma showing ribbons of thickened epithelial proliferations growing downwards into the stroma (H&E, x40).
Figure
39.4
Bleeding polypus arising from right side of nasal
septum.
11. Nasal dermoid. It presents as widening of upper part of nasal septum with splaying of nasal bones and hypertelorism. A pit or a sinus may be seen in the midline of nasal dorsum with hair protruding from the opening.

1. Carcinoma of nasal cavity. Primary carcinoma per se is rare. It may be an extension of maxillary or ethmoid carcinoma. Squamous cell variety is the most common, seen in about 80% of cases. Rest may be adenoid cystic carcinoma or an adenocarcinoma.
(a) Squamous cell carcinoma. It may arise from the vestibule, anterior part of nasal septum or the lateral wall of nasal cavity. Most of them are seen in men past 50 years of age.
(i) Vestibular. It arises from the lateral wall of nasal vestibule and may extend into the columella, nasal floor and upper lip with metastases to parotid nodes.
(ii) Septal. Mostly arises from mucocutaneous junction and causes burning and soreness in the nose. It has often been termed "nose-picker's cancer." Usually, it is of low-grade malignancy.
(iii) Lateral wall. This is the site most commonly involved. Easily extends into ethmoid or maxillary sinuses. Grossly, it presents as a polypoid mass in the lateral wall of nose. Metastases are rare. Treatment is combination of radiotherapy and surgery.
(b) Adenocarcinoma and adenoid cystic carcinoma. They arise from the glands of mucous membrane or minor salivary glands and mostly involve upper part of the lateral wall of nasal cavity.
2. Malignant melanoma. Usually seen in persons about 50 years of age. Both sexes are equally affected. Grossly, it presents as a slaty-grey or bluish-black polypoid mass. Within the nasal cavity, most frequent site is anterior part of nasal septum followed by middle and inferior turbinate. Amelanotic varieties are nonpigmented. Tumour spreads by lymphatics and blood stream. Cervical nodal metastases may be present at the time of initial examination. Treatment is wide surgical excision. Immunological defences of the patient play a great role in the control of this disease. Radiotherapy and chemotherapy suppress the immune processes and are avoided. A 5-year survival rate of 30% can be expected after wide surgical excision.

3. Esthesioneuroblastoma (Syn. olfactory neuroblastoma). Also called olfactory placode tumour as it arises from the olfactory epithelium in the upper third of nose. Bimodal peaks of incidence at 10-20 and 50-60 years are seen. Most common symptoms are unilateral nasal obstruction and epistaxis. When tumour invades orbit and the surrounding structures, other symptoms like proptosis, headache, epiphora, diplopia and blurred vision can also arise. Lymph node metastases in the neck can occur in 10-15%. Intranasal or endoscopic examination of nose reveals a friable cherry-red, polypoidal mass in the upper third of nasal cavity. It is a vascular tumour and biopsy should not be immediately attempted unless imaging studies have been done. High-resolution CT scan shows the extent of lesion. It may show destruction or erosion of the cribriform plate or
Figure
39.5
Rhabdomyosarcoma of the nose in a 2%-years-old
male child.
orbital wall. MRI with enhancement will reveal extension into the orbit or intracranially.
Biopsy of tumour reveals true nature of the tumour. It may be low grade with formation of pseudorosettes or high grade with nuclear pleomorphism but no rosette formation. It may require special staining to differentiate from other tumours.
It should
be differentiated
from lymphoma,
melanoma, plasmacytoma, rhabdomyosarcoma, undifferentiated carcinoma and neuroendocrine carcinoma.
Treatment. Treatment protocols differ between institutions. They are:
(a) Craniofacial resection with adjuvant radiation. (b) Preoperative radiation followed by craniofacial resection. (c) Preoperative chemotherapy and radiation followed by craniofacial resection for advanced lesions extending to orbit, cribriform plate and intracranially. Neck nodes if present are also radiated.
Craniofacial resection is done by osteoplastic flap exposing the anterior cranial fossa while facial approach is through lateral rhinotomy or midfacial degloving. En-block removal of tumour can thus be accomplished. Defect in the base of skull is repaired by pericranial flap.

4, Haemangiopericytoma. It is a rare tumour of vascular origin. It arises from the pericyte—a cell surrounding the capillaries. It is usually seen in the age group of 60-70 and presents with epistaxis. Brisk bleeding may occur on biopsy. The tumour may be benign or malignant but it cannot be distinguished histologically. Treatment is wide surgical excision. Radiotherapy is used for inoperable or recurrent lesions.
5. Lymphoma. Rarely a non-Hodgkin lymphoma presents on the septum.
6. Plasmacytoma. Solitary plasmacytoma without generalized osseous disease may be seen in the nasal cavity. It predominantly affects males over 40 years. Treatment is by radiotherapy followed 3 months later by surgery if total regression does not occur. Long-term follow-up is essential to exclude development of multiple myeloma.
7. Sarcomas. Osteogenic sarcoma, chondrosarcoma, rhabdomyosarcoma (Figure 39.5), angiosarcoma, malignant histiocytoma are other rare tumours affecting the nose.

Paranasal sinuses may be affected by both benign and malig- nant neoplasms but the latter are much more common.
3. Histology. More than 80% of the malignant tumours are of squamous cell variety. Rest are adenocarcinoma, adenoid cystic carcinoma, melanoma and various types of sarcomas (Figures 40.3 and 40.4).

1. Osteomas. They are most commonly seen in the frontal sinus followed in turn by those of ethmoid and maxillary. They may remain asymptomatic, being discovered incidentally on X-rays (Figure 40.1). Treatment is indicated when they become symptomatic, causing obstruction to the sinus ostium, formation of mucocele, pressure symptoms due to their growth in the orbit, nose or cranium.
2. Fibrous dysplasia. In this condition, bone is replaced by fibrous tissue; mostly involves maxillary but sometimes the ethmoid and frontal sinuses. Patient seeks advice for disfigurement of the face, nasal obstruction and displacement of the eye. Treatment is surgical resculpturing of the involved bone to achieve a good cosmetic and functional result (Figure 40.2).
3. Ossifying fibroma. Seen in young adults. The tumour can be shelled out easily.
4. Ameloblastoma (adamantinoma). It is a locally aggressive tumour that arises from the odontogenic tissue and invades the maxillary sinus. Treatment is surgical excision. Other rare tumours include inverted papilloma, meningioma and haemangioma (see Chapter 39).

1. Incidence. Cancer of nose and paranasal sinuses constitutes 0.44% of all body cancers in India (0.57% in males and 0.44% in females). Most frequently involved are the maxillary sinuses followed in turn by ethmoids, frontal and sphenoid.
2. Aetiology. Cause of sinus malignancy is largely unknown. People working in hardwood furniture industry, nickel refining, leather work and manufacture of mustard gas have shown higher incidence of sinunasal cancer. Cancer of the maxillary sinus is common in Bantus of South Africa where locally made snuff is used, which is found rich in nickel and chromium.
Workers of furniture industry develop adenocarcinoma of the ethmoids and upper nasal cavity, while those engaged in nickel refining get squamous cell and anaplastic carcinoma.

It arises from the sinus lining and may remain silent for a long time giving only vague symptoms of "sinusitis." It then spreads to destroy the bony confines of the maxillary sinus and invades the surrounding structures.
CLINICAL FEATURES (FIGURE 40.5)
Disease is common in 40-60 age group with preponderance in males.
1. Early features of maxillary sinus malignancy are nasal stuffiness, blood-stained nasal discharge, facial paraesthesias or pain and epiphora. These symptoms may be missed or simply treated as sinusitis.
- 2. Late features will depend on the direction of spread and extent of growth.
Figure 40.1
Osteoma right frontal sinus (arrow).
—
Figure 40.3 Photomicrograph showing adenocarcinoma having glandular pattern with neoplastic epithelial cells lining them. The cells contain intracytoplasmic mucin (black arrow) (H&E, x200).
Figure 40.4 Photomicrograph showing well-differentiated squamous cell carcinoma with pearl formation (H&E, x200).
Antroethmoidal carcinoma left side. Note (A) swelling of left cheek and (B) expansion of alveolus and palate.

3. Medial spread to nasal cavity gives rise to nasal obstruction, discharge and epistaxis. It may also spread into anterior and posterior ethmoid sinuses and that is why most antral malignancies are antroethmoidal in nature.
4. Anterior spread causes swelling of the cheek and later invasion of the facial skin.
5. Inferior spread causes expansion of alveolus with dental pain, loosening of teeth, poor fitting of dentures, ulceration of gingiva and swelling in the hard palate.
6. Superior spread invades the orbit causing proptosis, diplopia, ocular pain and epiphora.
7. Posterior spread is into pterygomaxillary fossa, pterygoid plates and the muscles causing trismus. Growth may also spread to the nasopharynx, sphenoid sinus and base of skull.
8. Intracranial spread can occur through ethmoids, cribriform plate or foramen lacerum.
9. Lymphatic spread. Nodal metastases are uncommon and occur only in the late stages of disease. Submandibular and upper jugular nodes are enlarged. Maxillary and ethmoid sinuses drain primarily into retropharyngeal nodes, but these nodes are inaccessible to palpation.
10.
Systemic metastases
are rare.
May be
seen in
the lungs
(most commonly)
and occasionally
in bone.

- 1. Radiograph of sinuses. Opacity of the involved sinus with expansion and destruction of the bony walls.
2.
(CT)
scan.
If available,
this is
the best
noninvasive scan
method should
be done
to find
both in
the axial
extent of
disease.
and coronal
CT
planes.
It also
helps in
the staging
- 3. Biopsy. If growth presents in the nose or mouth, biopsy can be easily taken. In early cases, with suspicion of malignancy, sinus should be explored by Caldwell-Luc operation. Direct visualization of the site of tumour in the sinus also helps in staging of the tumour.
Endoscopy of the nose and maxillary sinus will provide detailed examination. An accurate biopsy can also be taken. This route is preferred to Caldwell-Luc approach.

There is no universally accepted classification for maxillary carcinoma.
1. Ohngren's classification. An imaginary plane is drawn, extending between medial canthus of eye and the angle of mandible (Figure 40.6). Growths situated above this plane (suprastructural) have a poorer prognosis than those below it (intrastructural).
2. AJCC (American Joint Committee on Cancer) classification (Tables 40.1—40.3). AJCC classification is only for squamous cell carcinoma and does not include nonepithelial tumours of lymphoid tissue, soft tissue, cartilage and bone. Histopathologically, squamous cell carcinoma is further graded into:
(a) Well-differentiated,
(b) Moderately differentiated and
(c) Poorly differentiated.
In histopathology, note should also be made of vascular
or perineural invasion.
3. Lederman's classification (Figure 40.7). It uses two horizontal lines of Sebileau; one passing through the floors of orbits and the other through floors of antra, thus dividing the area into:
(a) Suprastructure. Ethmoid, sphenoid and frontal sinuses and the olfactory area of nose.
(b)
Mesostructure.
Maxillary sinus
and the
respiratory part
of nose.
- (c) Infrastructure. Containing alveolar process. This classification further uses vertical lines, extending down the medial walls of orbit to separate ethmoid sinuses and nasal fossa from the maxillary sinuses.
The student may note here that suprastructure and infrastructure of Lederman's classification is not the same as in Ohngren's classification.

Histologically, nature of malignancy is important in deciding the line of treatment as is the location and extent of disease.
Figure 40.6 Ohngren's line extends from medial canthus of eye to the angle of mandible. Growths anteroinferior to this plane (infrastructural) have a better prognosis than those posterosuperior to it (suprastructural).
Lederman's classification.

Ty
Tumour limited to maxillary sinus mucosa with no erosion or destruction of bone.
To
Tumour
causing
bone
erosion
or
including
extension
into
the
hard
palate
and/or
middle
nasal
meatus,
except
exten-
sion
to
posterior
wall
of
maxillary
sinus
and
pterygoid
plates.
T3
Tumour
invades
any
of
the
following:
bone
of
the
posterior
wall
of
maxillary
sinus,
tissues,
floor
or
medial
wall
of
orbit, pterygoid fossa and ethmoid sinuses.
T4a
|
Tumour
invades
anterior
orbital
contents,
skin
of
cheek,
pterygoid
plates,
infratemporal
fossa,
cribriform
plate,
sphenoid
or
frontal
sinuses.
T4p
|
Tumour
invades
any
of
the
orbital
apex,
dura,
brain,
middle
cranial
fossa,
cranial
nerves
other
than
maxillary
division
of
trigeminal
nerve
(V2),
nasopharynx
or
clivus.

Nx
Regional lymph nodes cannot be assessed.
No
No regional lymph node metastasis.
N,
Metastasis in a single ipsilateral lymph node, 3 cm or less in greatest dimension.
No
Metastasis in a single ipsilateral lymph node, more than 3 cm but not more than 6 cm in greatest dimension; or in multiple ipsi-
lateral lymph nodes, none more than 6 cm in greatest dimension; or in bilateral or contralateral lymph nodes, none more than 6 cm in greatest dimension.
No,
in
a
single
ipsilateral
lymph
node,
more
than
3
cm
but
not
more
than
6
cm
in
greatest
dimension.
Nz,
in
multiple
lymph
nodes,
none
more
than
6
cm
in
greatest
Nz,
Metastasis in bilateral or contralateral lymph nodes, none more than 6 cm in greatest dimension.
Ng
Metastasis in a lymph node, more than 6 cm in greatest dimension.

M,
Distant metastasis cannot be assessed.
Mo,
No distant metastasis.
M,
Distant metastasis.
Source: AJCC, Cancer Staging Manual, fifth ed. Chicago, 2002.
Early
T 1, 40.3 Classification of cancer of nasal cavity and ethmoid sinuses (AJCC, 2002) = Tumour restricted to any one subsite, with or without bony invasion. T 2, 40.3 Classification of cancer of nasal cavity and ethmoid sinuses (AJCC, 2002) = Tumour invading two subsites in a single region or extending to involve an adjacent region within the nasoethmoidal complex, with or without bony invasion. T 3, 40.3 Classification of cancer of nasal cavity and ethmoid sinuses (AJCC, 2002) = Tumour extends to invade the medial wall or floor of the orbit, maxillary sinus, palate or cribriform plate. T 4a, 40.3 Classification of cancer of nasal cavity and ethmoid sinuses (AJCC, 2002) = Tumour invades any of the following: anterior orbital contents, skin of nose or cheek, minimal extension to anterior cranial fossa, pterygoid plates, sphenoid or frontal sinuses. T 4b, 40.3 Classification of cancer of nasal cavity and ethmoid sinuses (AJCC, 2002) = Tumour invades any of the following: orbital apex, dura, brain, middle cranial nerves other than (V 2 ), nasophar- ynx or clivus
cases with
Stage
I
and
II
squamous cell
carcinomas are
with surgery
equal results.
modalities
(Figures
T3
and of
may be
40.8
T4
and
40.9)
lesions radiation
and given
dose are
surgery.
preoperatively of
radiation of
radiation treated
Radiation or
is postoperatively.
5500
cGy.
used conformal
is
5000-5500
radiotherapy of
radiotherapy reduce
or radiation
by combined
in such
cases
Preoperative
Similarly postoperative
dose cGy.
Now three-dimensional
and intensity-modulated

cover larger
side effects
techniques tumour
of volumes
and radiation
to optic
nerves help
to and
lens by
providing accurate
and homogenous
radiation dose.
Chemoradiation, i.e. chemotherapy and radiation con- comitantly has also been used for large and inoperable
tumours by
different workers
with
5
year survival
of more
than
60%.
Intra-arterial infusion
of
5-Fu or
5-Fu with concomitant radiation has also been used with good
results in
preference to
deformities created
by exten-
sive surgery associated with advanced malignancy.

Survival diminishes with the stage of tumour. Overall 5 year survival is about 40-50%. However, advances are being made in the multimodal therapy with improved techniques
of radiation delivery with the hope to improve results and protect injury to lens and optic nerve.

Ethmoid sinuses are often involved from extension of the primary growth of the maxillary sinus. Primary growth of ethmoid sinuses per se is not common.

1. Early features include nasal obstruction, blood-stained nasal discharge and retro-orbital pain.
2. Late features are broadening of the nasal root, lateral displacement of eyeball and diplopia (Figure 40.10). Extension through cribriform plate may cause meningitis.
3. Nodal involvement is not common. Upper nodes may be involved.
Figure 40.8 Weber—-Fergusson's incision used in maxillectomy.

CT scan is essential to know the extent of disease and intracranial spread. In early cases, treatment is preoperative radiation, followed by lateral rhinotomy and total ethmoidectomy. If cribriform plate is involved, anterior cranial fossa is exposed by a neurosurgeon and total exenteration of the growth in one piece is accomplished by what is called craniofacial resection.

Frontal sinus malignancies are uncommon and are seen in the age group of 40-50 years with male predominance (5:1).
Figure 40.10 Carcinoma ethmoid.
Figure 40.9
(A) Maxillectomy with orbital exenteration
on the
right side.
(B)
Same patient
after rehabilitation
with prosthesis and an artificial eye.
a
maxillary

Pain and swelling of the frontal region are the presenting features. Growths may erode through the floor of frontal
sinus and
present as
a
swelling above
the medial
canthus.
Growths of frontal sinus may extend through the ethmoids into the orbit. Dura of anterior cranial fossa be involved may
if growth penetrates the posterior wall of the sinus.

Frontal sinus malignancy is treated by preoperative radiation followed by surgery. Surgery includes frontal sinusotomy
with ethmoid and orbital exenteration. Neurosurgical approach may be required to resect the dura of anterior cranial fossa, if involved.

Primary malignancy of the sinus is rare. It has to be differentiated from the inflammatory lesions in this area. Plain X-rays, CT scan and biopsy through sphenoidotomy are essential to know the nature and extent of disease. Radiotherapy is the mainstay of treatment.
Orbit has rigid walls; any space occupying lesion of the orbit causes eyeball to protrude forward or also displace in some other direction, i.e. medial, lateral, up or down depending on location in the orbit. Proptosis should be differentiated from pseudoproptosis, i.e. apparently proptosed eyeball though it is normal in position. This happens with enophthalmos of the contralateral eye due to previous forgotten trauma such as orbital blowout fractures. Lid retraction and high myopia can also make the eyeball look proptosed. Proptosis can be measured by exophthalmometer.

Table 41.1 shows the various conditions causing proptosis and can be remembered by the acronym of VEIN. Various conditions of concern to the ENT surgeon include orbital cellulitis or orbital abscess, subperiosteal abscess, fungal infections of sinuses, Graves ophthalmopathy, benign and malignant neoplasms of nose and paranasal sinuses, such as angiofibroma, inverted papilloma, nasal polyposis, mucoceles, esthesioneuroblastoma, paranasal sinus malignancies and trauma following endoscopic surgery. Some important diseases are described below.
1.
Idiopathic orbital
As the
name indicates,
cause is
uncertain.
specific lacrimal
It may
be diffused
structures in
gland, ritis).
the orbit,
sclera
(scleritis)
Patient complains
eye movements.
of dull
Proptosis or
localized e.g.
muscles or
optic to
(myositis), nerve
orbital is
(perineu- pain
seen especially
in on
70-80%
of patients.
CT
scan with
enhancement shows
enlargement of
the
affected structures. An important feature is involvement of muscle and its tendon attached to the globe and differentiates it from thyroid-related disease where only muscle belly is involved but not its tendon. Biopsy shows nonspecific inflammation without evidence of vasculitis. Treatment is oral steroids. In some cases, immunosuppression with cyclophosphamide, cyclosporine or radiotherapy may be required.
- . Graves ophthalmopathy. This is the most common cause of bilateral and sometimes unilateral proptosis. Patient is hyperthyroid but sometimes he is euthyroid or even hypothyroid. Lid retraction and lid lag may be present with chemosis and lid oedema. CT scan is useful to differentiate it from idiopathic orbital inflammation (vide supra). Visual loss can occur. Extreme proptosis causes corneal ulceration and may require orbital decompression which nowadays can be done endoscopically through the nose.
.
Haemangioma of
orbit.
It can
be cavernous
or capillary.
Cavernous haemangioma

in adult.
It of
18-67
is the
is more
common years.
It lateral
manifests proptosis.
without
CT/MRI
associated it.
It is
an in
benign females
as tumour
in the
painless, reveals
age group
progressive,
a
round inflammation
encapsulated enhances
uni- or
oval or
infiltration mass.
Intraconal on
i.v.
contrast.
Treatment mass
around in
location is
complete and
excision with
its capsule
by lateral
orbitotomy.
- . Capillary haemangioma. Most common tumour of orbit in infants and children. May be isolated or associated with a lesion on the upper lid or elsewhere on the skin. Most of them involute by age 7. CT/MRI with contrast

Vascular. Venous varix, cavernous haemangioma, carotid-cavernous fistula
Endocrinal. Graves' disease which may cause bilateral or sometimes even unilateral proptosis
Inflammations and infections. Idiopathic orbital inflammation (pseudotumour of orbit), orbital cellulitis or abscess, mucormycosis or aspergillosis of sinuses, Wegener's granulomatosis, inflammations of lacrimal gland.
Neoplastic. Tumours (both benign and malignant) or tumour-like conditions arising from the orbital contents or its adjoining structures.
Orbit contains
eyeball, optic
nerve, muscles,
nerves, blood
vessels and
lacrimal gland
and tumour
and tumour-like
conditions can
arise from
them.
They also
Primary arise
tumours schwannoma,
of from
orbit, paranasal
sinuses its
glioma, rimal
gland.
walls or
adnexa retinoblastoma,
Malignant and
cranial
(lid, lacrimal
fibrous tumours
include cavity
gland and
and dysplasia,
osteoma, rhabdomyosarcoma,
invade the
orbit.
conjunctiva).
Dermoid histiocytosis
X, lymphoma,
orbital leukaemic
cyst, cavernous
or capillary
meningioma, deposits,
haemangioma, pleomorphic
malignant adenoma
tumours of
of lac-
melanoma of
choroid.
- (b) Tumours of paranasal sinuses. Mucocele of frontal or ethmoidal sinuses, inverted papilloma, angiofibroma, malignant tumours of sinuses.
Tumours from cranial cavity. Meningioma of the sphenoid ridge.
- (c) Metastatic tumours. Carcinoma breast (most common), lung, prostate, kidney, thyroid, gastrointestinal tract.
and
(a)

is diagnostic. Tumour is nonencapsulated and infiltrates the surrounding structures. Local or systemic steroids to involute the mass. Total excision is not
help possible. 5. Venous varix of the orbit. It presents with positional proptosis and congestion. Proptosis can also be induced by Valsalva manoeuvre. A carotid-cavernous fistula is either spontaneous or traumatic; it presents with pulsatile proptosis, bruit, visual loss, dilated and arterialized blood vessels in the conjunctiva or limbus.
6. Lymphoma. It is the most common malignant tumour of adults. It may be isolated or associated with systemic disease. Most of the patients are between 50 and 70 years with female preponderance. It presents as painless progressive exophthalmos. Usually lesions are located anteriorly and can be palpated or seen under the conjunctiva. Most of them are extraconal. CT shows a homogenous tumour without bone involvement. Biopsy is necessary to differentiate it from the benign lymphoid or other tumours. Isolated lymphoma can be treated by radiation alone while systemic ones require chemotherapy in addition to orbital radiation.
7.
Rhabdomyosarcoma.
It is
the most
common primary
malig- nant
tumour of
orbit
6-7
years ents
of age.
as to
It in
can painless
but the
or adjoining
extraconal.
be children
occur even
progressive paranasal
and is
usually in
the seen
newborn.
proptosis sinuses.
CT
is taken.
helpful
Treatment and
at
It pres-
can
It may
in spread
be diagnosis.
is radiation
intraconal
Biopsy should
and chemotherapy.
Five- year
survival of
90%
can be
achieved in
localized disease.

- 8. Dermoid cyst. It is the most common benign tumour of orbit in children. It is due to the trapped ectoderm and occurs at suture lines. Deep dermoids of orbit arise from the sphenoethmoid or sphenozygomatic sutures. They may remain asymptomatic till adult age. They present with painless, progressive proptosis with globe displacement. CT orbit
may show a cyst with pressure effects (Figure 41.1). Large cysts may communicate with temporal fossa, paranasal sinuses or the cranial cavity. Treatment is surgical excision. 9. Tumours of optic nerve. Glioma of optic nerve is usually seen in children and may be associated with neurofibromatosis. It causes progressive proptosis and visual loss. Meningioma of optic nerve (from arachnoid sheath) occurs mostly in the middle-aged women leading to proptosis and visual loss.

A case of proptosis requires a detailed history including onset, duration and progression. Associated illnesses (thyroid disease, tumours of nose or paranasal sinuses, systemic disorders such as leukaemia, lymphoma, Wegener's granulomatosis) should be looked for. Pain is a feature of inflammation or infection. Visual loss may be present and should be documented.
Physical examination should include type of proptosis (straight forward or with globe displacement), condition of the conjunctiva (swelling, chemosis), scleral appearance, ocular movements and vision. Note should also be made if the proptosis is pulsatile or associated with change in position of the head or appears on performing Valsalva (venous varix).
CT and MRI are important and give clue to the type of tumour (intraconal/extraconal), smooth or infiltrative, location in the orbit and its extent, any changes in the adjoining bone or extension to sinuses or cranial cavity. They can help to differentiate thyroid orbitopathy from the idiopathic orbital inflammation.
Ultrasonography may be required to find abscess or cystic lesions.
FNAC or biopsy of the lesion may be required for histologic diagnosis.
Dermoid cyst of the right orbit: (A) axial view and (B) coronal view (arrows). Patient presented with a mild proptosis.
Systemic diseases causing orbital lesions should be investigated as mandated by history and clinical examination and relevant investigation.

Imaging techniques help to make a diagnosis. Biopsy of the lesion can be taken depending on its location in the orbit. Anteriorly located lesions can be approached by lid or conjunctival incision. Excisional biopsy is useful in encapsulated and well-circumscribed lesions such as dermoid, cavernous haemangioma and pleomorphic adenoma of the lacrimal gland. All cases causing proptosis do not require surgery. Medical treatment includes antibiotics in orbital cellulitis, steroids in pseudotumour, chemotherapy for lymphoma,
42.
Anatomy of Oral Cavity
45.
Non-neoplastic Disorders
43.
Common Disorders of Oral Cavity
of Salivary Glands
44.
Tumours of Oral Cavity
46.
Neoplasms of Salivary Glands

The oral cavity extends from the lips to the oropharyngeal isthmus, i.e. up to the level of anterior pillar of tonsils. It is divided into the following sites (Figure 42.1):
1. Lips. They form anterior boundary of the oral vestibule.
2. Buccal or cheek mucosa. It lines the inner surface of
cheeks and
lips, and
extends up
to pterygomandibular
raphe.
Anteriorly, it
extends to
the meeting
line of
lips.
3. Gums (gingivae). They surround the teeth and cover the upper and lower alveolar ridges.
4. Retromolar trigone. It is a triangular area of mucosa covering anterior surface of the ascending ramus of mandible. Its base is posterior to the last molar while its apex is adjacent to the tuberosity of maxilla.
. Hard palate. It forms roof of the oral cavity. Do
. Oral tongue. Only anterior two-thirds of tongue are included in the oral cavity. Posterior one-third or base of tongue is situated behind the circumvallate papillae and forms part of the oropharynx. Oral tongue is divided into tip, lateral borders, dorsum and the undersurface.
best seen by displacing the lateral surface of tongue in medial direction with the help of a tongue depressor.

1. Lips. Lower. Medial portion of lower lip drains into submental and lateral portion to submandibular nodes. Upper. Drain into preauricular, infraparotid and submandibular nodes.
2. Buccal mucosa. Submental and submandibular nodes.
3. Upper and lower alveolar ridges. Buccal aspect of mucosa drains into submental and submandibular nodes.
Lingual aspect of upper alveolus drains into upper deep cervical and lateral retropharyngeal nodes.
Lingual aspect of lower alveolus drains into submandibular nodes.
4. Hard palate. Upper deep cervical and lateral retropharyngeal nodes. Anterior part of palate drains into submandibular nodes.
7. Floor of mouth. It is a crescent-shaped area between the gingivae and undersurface of tongue. Anterior portion of the floor is best seen when patient raises the tip of tongue to touch the hard palate. Frenulum and sublingual papillae with openings of submandibular ducts can be easily seen. Lateral portion of floor of mouth is
5. Floor of mouth. Anterior portion of floor of mouth drains into submandibular nodes. Lymphatics from this area also cross the midline.
Posterior portion drains into deep cervical nodes.
Tongue. Tip of tongue drains into submental and juguloomohyoid nodes, lateral portion drains into ipsilateral, submandibular and deep cervical nodes. Central portion and base drain into cervical nodes of both sides.
upper 6. deep

Some of the common ulcers are described in this chapter. The causes of the ulcers of oral cavity are listed in Table 43.1.

1. Herpangina. It is a coxsackie viral infection mostly affecting children. To begin with, multiple small vesicles appear on the faucial pillars, tonsils, soft palate and uvula. They rupture to form ulcers which are usually 2-4 mm in size, have a yellow base and red areola around them. They seldom persist beyond 1 week.
(b) Secondary or recurrent herpes chiefly affects adults. It is milder in form as adults have some immunity to this virus. Most commonly, it involves the vermilion border of the lip (herpes labialis) but less often lesions appear intraorally on the hard palate and gingiva. In recurrent herpes, it is presumed that virus lies dormant in the trigeminal ganglion and, when reactivated, travels along peripheral sensory nerves to involve oropharyngeal mucosa. Precipitating factors include emotional stress, fatigue, fever, pregnancy or immune deficiency states. Treatment is mostly symptomatic. Acyclovir, 200 mg, five times a day for 5 days helps to cut down the course of recurrent herpes labialis.
2. Herpetic gingivostomatitis. Also known as orolabial herpes. It is caused by herpes simplex virus and is of two types: primary and secondary.
(a) The primary infection affects children and is character ized by clusters of multiple vesicles which soon rupture to form ulcers. Any part of the oral cavity may be affected. Constitutional symptoms like fever, malaise and headache may accompany sore throat and lymphadenopathy.

1., 1 = Infections. 1., 2 = Infections. , 1 = (a). , 2 = Viral. Herpangina, herpes simplex (primary and secondary), hand, foot and mouth disease. 2., 1 = Immune disorders. Aphthous ulcer, Behçet syndrome. 2., 2 = Immune disorders. Aphthous ulcer, Behçet syndrome. 3., 1 = Trauma. 3., 2 = Trauma. , 1 = (a) (b) (c). , 2 = Physical. Cheek bite, jagged tooth, ill-fitting denture Chemical. Silver nitrate, phenol, aspirin burns Thermal. Hot food or fluid, reverse smoking. 4., 1 = Neoplasms. 4., 2 = Neoplasms. 5., 1 = Skin disorders. Erythema multiforme, lichen planus, benign mucous membrane pemphigoid, bullous pemphigoid, lupus erythematosus. 5., 2 = Skin disorders. Erythema multiforme, lichen planus, benign mucous membrane pemphigoid, bullous pemphigoid, lupus erythematosus. 6., 1 = Blood disorders. Leukaemia, agranulocytosis, pancytope- nia, cyclic neutropenia, sickle cell anaemia. 6., 2 = Blood disorders. Leukaemia, agranulocytosis, pancytope- nia, cyclic neutropenia, sickle cell anaemia. 7., 1 = Drug allergy. Mouth washes, toothpaste, etc. Reactions to systemic drugs. 7., 2 = Drug allergy. Mouth washes, toothpaste, etc. Reactions to systemic drugs. 8., 1 = Vitamin deficiencies. 8., 2 = Vitamin deficiencies. 9., 1 = Miscellaneous. Radiation mucositis, cancer chemotherapy, diabetes mellitus, uraemia. 9., 2 = Miscellaneous. Radiation mucositis, cancer chemotherapy, diabetes mellitus, uraemia
3. Hand, foot and mouth disease. It is also a viral infection affecting children. Oral lesions are seen on the palate, tongue and buccal mucosa. Vesicles also develop on the skin of hands, feet and sometimes buttocks.

1. Vincent infection (Acute necrotizing ulcerative gingivitis) . It is similar to Vincent's angina. Causative organisms are the same (a fusiform bacillus and a spirochaete—Borrelia vincentii). More often the disease affects young adults and middle-aged persons. It starts at the interdental papillae and then spreads to free margins of the gingivae which get covered with necrotic slough. Gingivae also become red and oedematous. Similar ulcer and necrotic membrane may also form over the tonsil (Vincent's angina). Diagnosis is made by smear from the affected area. Treatment is systemic antibiotics (penicillin or erythromycin and metronidazole), frequent mouth washes (with sodium bicarbonate solution) and attention to dental hygiene.
2. Specific bacterial infections. Tuberculosis, syphilis and actinomycosis may present as chronic ulcers.

Moniliasis (candidiasis). It is caused by Candida albicans and occurs in two forms:
- 1. Thrush. It appears as white grey patches on the oral mucosa and tongue. When wiped off, they leave an erythematous mucosa. The condition is seen in infants and children. Adults are also affected when they are suffering from systemic malignancy and diabetes or taking broad-spectrum antibiotics, cytotoxic drugs, steroids or radiation.
Figure 43.1 Multiple aphthous ulcers on the uvula and faucial pillars (arrowheads).
- 2. Chronic hypertrophic candidiasis. Also called candidal leukoplakia. The lesion appears as white patch which cannot be wiped off. Mostly affects anterior buccal mucosa just behind the angle of mouth.
Thrush can be treated by topical application of nystatin or clotrimazole. Hypertrophic form usually requires excisional surgery.

1. Aphthous ulcers. They are recurrent and superficial, usually involving movable mucosa, i.e. inner surfaces of lips, buccal mucosa, tongue, floor of mouth and soft palate, while sparing mucosa of the hard palate and gingivae. In the minor form, which is more common, ulcers are 2-10 mm in size and multiple with a central necrotic area and a red halo (Figure 43.1). They heal in about 2 weeks without leaving a scar. In the major form, ulcer is very big, 2-4 cm in size and heals with a scar but is soon followed by another ulcer.
Aetiology of aphthous ulcers is unknown. It may be an autoimmune process, nutritional deficiency (vitamin By, folic acid and iron), viral or bacterial infection, food allergies or due to hormonal changes or stress.
Aphthous ulcers can be differentiated from viral ulcers by their frequent recurrence, involvement of movable mucosa as on the soft palate or cheek, and the absence of constitutional symptoms like fever, malaise and enlargement of cervical nodes.
Treatment consists of topical application of steroids and cauterization with 10% silver nitrate. In severe cases, 250 mg of tetracycline dissolved in 50 mL of water is given as mouth rinse and then to be swallowed, four times a day. Local pain can be relieved with lignocaine viscous.
2. Behcet syndrome (Oculo-oro-genital syndrome). It is characterized by a triad of (i) aphthous-like ulcers in the oral cavity, (ii) genital ulcerations and (iii) uveitis. The edge of the ulcer is characteristically punched out. There may also be lesions of the skin, joints and central nervous system.
Figure 43.2 A large exophytic mass with ulceration at the top on the left side of tongue in a 58-year-old male, habituated to chewing tobacco and "pan." It was due to sharp gagged teeth; healed on extraction of teeth.

Traumatic ulcer. A traumatic ulcer on the lateral border of tongue may be due to jagged tooth or ill-fitting denture, on the buccal mucosa due to cheek bite and on the palate due to injury with a foreign object such as pencil or toothbrush (Figures 43.2 and 43.3).
Similarly, acute ulcerative lesions of oral and oropharyngeal mucosa can result from accidental ingestion of acids or alkalies or hot fluids.
Aspirin burn is seen in the buccal sulcus when a tablet of aspirin is kept against a painful tooth to get relief from toothache.

Malignancies of the oral cavity or oropharynx may present as chronic ulcers. Though most commonly it is squamous cell carcinoma, it could be carcinoma of minor salivary glands or non-Hodgkin lymphoma.

1. Erythema multiforme. It is a disease of rapid onset involving the skin and mucous membranes, either of which may be involved alone. The aetiology is unknown but may be associated with drug allergy (sulfonamides) or recent herpes simplex infection. Oral mucosal lesions consist of vesicles or bullae which soon rupture to form ulcers covered with pseudomembrane. Any area of oral mucosa is involved but the common sites are lips, buccal mucosa and tongue. The lesions bleed easily. The distinctive feature is to form haemorrhagic crusts on the lips. Skin lesions consist of erythematous patches on the palms, soles and extensor surfaces of the extremities. Oral lesions may occur without skin involvement in 25% of patients. The disease is self-limiting and management is mainly supportive. Steroids are used to treat the severe form.
2. Pemphigus vulgaris. It is an autoimmune disorder affecting older age group (50-70 years). Oral lesions are seen in 50% of the cases and may precede skin lesions.
Figure 43.3 Ulcer on lateral border of tongue simulating carcinoma (arrowheads). It was caused by a sharp jagged tooth (A) and healed completely following tooth extraction (B).
Oral ulcerations are superficial and involve palate, buccal mucosa and tongue. Treatment consists of systemic steroids and cytotoxic drugs.
3. Benign mucous membrane pemphigoid (BMMP). It is also an autoimmune disorder. Mucosal lesions involve cheek, gingivae and palate. Conjunctiva is the next important site. Lesion starts as a bulla filled with clear or haemorrhagic fluid which ruptures to form superficial ulceration covered with shaggy collapsed mucosa. Skin lesions may be absent. Treatment consists of steroids.
4. Lichen planus. Oral lesions are seen with or without skin lesions. Skin lesions are pruritic, purple, polygonal papules. They are seen on the forearms and medial side of thigh. Oral lesions occurs in two forms:
(a) Reticular. White striae forming lace-like pattern are seen on the buccal mucosa on both sides. They are and no treatment.

asymptomatic require (b) Erosive. It is characterized by painful ulceration on the buccal mucosa, gingiva or lateral tongue. Each ulcer is surrounded by a keratotic periphery. Treatment consists of topical steroids.
5. Chronic discoid lupus erythematosus. Oral lesions are almost always associated with skin lesions. Oral lesions are similar to those of erosive form of lichen planus.

Blood dyscrasias cause ulcerations in the oral cavity and pharynx. Due to lack of defence mechanism, e.g. granulocytes, infections quickly supervene causing ulcers. Acute leukaemia is mainly of two types—acute lymphoblastic type, which occurs in young children and acute myeloid type, occurring in the middle aged or the elderly. Both cause hypertrophy of gums with ulceration and bleeding. Agranulocytosis is characterized by ulcerations in throat with severe neutropenia (Figure 43.4). Cyclical neutropenia is a
Figure 43.4 A 58-year-old man developed ulcerations on the palate when white cell count fell to 2500 per cubic mm on cancer chemotherapy with cisplatin and 5-fluorouracil.
condition with periodic falls in neutrophil count when the person becomes prone to infections and oral ulceration. In pancytopenia, there is a drop in RBC count, white cell count and platelets.
When suspected, blood dyscrasias are investigated by peripheral blood film, blood counts and bone marrow aspiration.

Systemic administration of drugs like penicillin, tetracycline, sulfa drugs, barbiturates, phenytoin, etc. may cause erosive, vesicular or bullous lesions in the oral cavity. Contact stomatitis may occur due to local reaction to mouth washes, lozenges, chewing gum, toothpastes or to prosthetic dental materials. Oral lesions may vary from erythema to vesicles and bullae formation.

Radiation mucositis. It follows radiation of oral cavity or oro- pharynx for cancer. At first, the mucosa becomes red and then forms spotty areas of mucositis which coalesce to form large ulcerated areas covered by slough. Mucositis of cancer chemotherapy can be caused by drugs like methotrexate, 5-FU and bleomycin. It manifests as ery- thema, oedema and ulceration.

1. Median rhomboid glossitis. It is red rhomboid area, devoid of papillae, seen on the dorsum of tongue in front of foramen caecum. It is a developmental anomaly that occurs due to persistence of tuberculum impar, which fails to invaginate. Recent studies reveal this condition to be due to chronic candida infection. The condition is asymptomatic and no treatment is necessary.
2. Geographical tongue. It is characterized by erythematous areas, devoid of papillae, surrounded by an irregular keratotic white outline (Figure 43.5). The lesions keep changing their shape and hence the condition is also called "migratory glossitis." The condition is asymptomatic and may not require any treatment.
3. Hairy tongue. Due to excessive formation of keratin, the filiform papillae on the dorsum of the tongue become elongated. They get coloured, brown or black, due to chromogenic bacteria and look like hair. Smoking seems to be one of the factors. Treatment consists of scraping the lesions with a tongue cleaner, application of half-strength hydrogen peroxide and improving the general nutritional status of the patient by vitamins. Causative factors, if known, should be removed.
4. Fissured tongue. It may be congenital or seen in cases of syphilis, deficiency of vitamin B complex or anaemia. Congenital fissuring associated facial palsy is seen in Melkersson— Rosenthal syndrome.
5. Ankyloglossia (tongue tie) (Figure 43.6). True tongue tie which produces symptoms is uncommon. If tongue can be protruded beyond the lower incisors, it is unlikely to cause speech defects. A mobile tongue is important to maintain orodental hygiene—to clean the debris and prevent formation of dental plaques. Treatment of any significant tongue tie is transverse release and vertical closure. Thin mucosal folds can be simply incised.
6. Fordyce spots. They are aberrant sebaceous glands present under the buccal or labial mucosa and shine through it as yellowish or yellow-brown spots. They are seen with equal frequency in both males and females and are considered normal.

7. Nicotine stomatitis. This disorder is seen in smokers particularly those in the habit of reverse smoking. Palatal mucosa shows pin-point red spots in the centre of umbili- cated
papular lesions.
They are
due to
the
2.
Figure 43.5 Geographical tongue.
Figure 43.6 Tongue tie.
minor salivary glands and their duct openings as a reaction to the heat of the smoke. The nicotine stomatitis is a misnomer as nicotine is not the cause. Management is elimination of smoking.

Oral submucous fibrosis (OSF) is a chronic insidious process characterized by juxtaepithelial deposition of fibrous tissue in the oral cavity and pharynx. The condition was first described in India by Joshi in 1953. The disease is widely seen in India, Pakistan, Taiwan, Sri Lanka, Nepal and Thailand due to habit of betel-nut chewing.

1.
Socioeconomic status.
In tus
has been
India, associated
lesions fibrosis.
with like
leukoplakia,
This access
to poor
socioeconomic higher
risk sta-
of precancerous
erythroplakia is
related to
medical care.
Tobacco chewing.
It is
a
major risk
factor in
submucous fibrosis as it is in lesions of leukoplakia and erythroplakia.
and education,
submucous diet,
lifestyle and
3. Areca nuts. Areca nuts are chewed alone, with tobacco or in the form of pan (containing lime, catechu and other ingredients on a betel leaf). Betel quid without tobacco also increases the risk of oral precancerous lesions, but causes higher risk for oral submucous fibrosis relative to leukoplakia, erythroplakia or multiple precancerous lesions. International agency for research on cancer has classified betel quid without tobacco also as a carcinogen for humans.
4. Alcohol. It is observed that drinking increases the risk of leukoplakia by 1.5-fold, OSF by 2-fold and that of erythroplakia by 3-fold.
5. Nutritional. Deficiency of vitamins and micronutrients has been suggested. Therapy of OSF with vitamin A, zinc and antioxidants has shown some beneficial effect. Lesser intake of fruits and vegetables has been associated with oral premalignant lesions.
6. Immune process. OSF is considered a cell-mediated immune reaction to arecoline in areca nuts. It may also reflect a localized collagen disorder or an autoimmune process in the oral cavity.
7. Multifactorial. Several factors may operate together in the causation of OSF. Habit of betel-nut chewing, drinking or smoking tobacco coupled with dietary deficiencies may have synergistic effect.

Histopathology in early cases of OSF shows presence of polymorphonuclear leukocytes, eosinophils and a few lymphocytes while advanced cases show lymphocytes and plasma cells. Immunochemistry of inflammatory cells showed higher population of activated T-lymphocytes especially the T-helper/inducer lymphocytes but minor population of B-cells and macrophages. Later studies also showed significant increase in number of T-lymphocytes, macrophages and high CD4+ to CD8+ lymphocyte ratio in the subepithelial connective tissue suggesting that OSF is a cellular immune response. Small number of B-lymphocytes suggests minor role of humoral immunity in OSF. In advanced stages, there was severe fibrosis and loss of vascularity in the lamina propria and submucosa. The process may extend deeper into muscle layers also. Activated macrophages and T-lymphocytes produce fibrogenic cytokines which act on mesenchymal cells to produce fibrosis. Also certain cytokines liberated by T-lymphocytes upregulate synthesis of collagen but downregulate collagenase production further promoting fibrosis. It is thus believed that OSF is due to increased production of collagen and its decreased degradation in subepithelial layers of the oral mucosa (Figure 43.7).

The basic change is fibroelastotic transformation of connective tissues in lamina propria associated with epithelial atrophy, sometimes preceded by vesicle formation. In later stages, when fibrosis is marked, there is progressive trismus and difficulty to protrude the tongue.
Leukoplakia and squamous cell carcinoma may be associated with submucous fibrosis possibly because of common aetiological factors involved.
It is a premalignant condition and malignant transforma- tion has been seen in 3-7.6% of cases.

1. Age and sex. No age or sex is immune but the disease mostly affects age group of 20-40 years.
2. Symptoms. Patient often presents with:
(a) Intolerance to chillies and spicy food.
(b) Soreness of mouth with constant burning sensation; wors-
ened during meals particularly of pungent spicy type.
(c) Repeated vesicular eruption on the palate and pillars.
(d) Difficulty to open the mouth fully.
(e) Difficulty to protrude the tongue.
3. Findings. Changes of submucous fibrosis are most marked over (i) soft palate, (ii) faucial pillars and (iii) buccal mucosa (Figure 43.8). In initial stages, there is patchy redness of mucous membrane with formation of vesicles which rupture to form superficial ulcers.
In later stages, when fibrosis develops in the submucosal layers, there is blanching of mucosa with loss of suppleness. Fibrotic bands can be seen and felt in the affected areas. Fibrosis and scarring has also been demonstrated in the underlying muscle leading to further restrictive mobility of soft palate, tongue and jaw. Trismus is progressive, so much so that patient may not be able to put his finger in the mouth or brush his teeth. Orodental hygiene is affected badly and teeth become carious. Examination of oral cavity is difficult particularly to rule out other associated premalignant lesions or malignancy.

1. Steroids. Topical injection of steroids into the affected
area is more effective than their systemic use as it also has the advantage of fewer side effects. It may be combined
Figure
43.8
Submucous fibrosis. (A) Note the blanched appearance of the soft palate and faucial pillars. (B) Marked trismus due to submucous fibrosis.
with hylase. Dexamethasone 4 mg (1 mL) combined with hylase, 1500 IU in 1 mL is injected into the affected area
biweekly for 8-10 weeks. This brings marked improve- ment in symptoms and relieves trismus.
2. Avoid irritant factors, e.g. areca nuts, pan, tobacco, pungent foods, etc.
3. Treat existent anaemia or vitamin deficiencies.
4. Encourage jaw opening exercises.

It is indicated in advanced cases to relieve trismus. Various surgical techniques used are:
1. Simple release of fibrosis and skin grafting. There is high recurrence rate due to graft contracture.
ene Surgical excision and buccal fat pad graft. Superficial temporal fascia flap and split-skin graft. Coronoidectomy and temporal muscle myotomy.
Bilateral tongue flaps. Requires flap division at a second stage.
Nasolabial flaps. They are small to cover the defect com- pletely, cause facial scar and require division of flaps at sec- ond stage.
Island palatal mucoperiosteal flap. It is based on greater
palatine
artery.
Possible
only
in
selected
cases.
Requires
extraction
of
second
molar
for
the
flap
to
sit
without
ten-
sion. Not suitable for bilateral cases.
Bilateral radial forearm free flap. It is bulky and hair bearing. May require debulking procedure, third molar may require extraction.

The tumours of oral cavity can be classified as follows:
1. Benign tumours
(a) Solid
(b) Cystic
2. Premalignant lesions
3. Malignant lesions
(a) Carcinoma
(b) Nonsquamous malignant lesions

1. Papilloma. Papillomas are common in the oral cavity. Peak incidence is in the third to fifth decades. Most of them appear on the soft and hard palate, uvula, tongue and lips. Mostly they are less than 1 cm in size, pedunculated and white in colour. Their surface is irregular but sometimes smooth. Treatment is excisional biopsy. Recurrence is rare.
2. Fibroma (fibroepithelial polyp). It is a smooth, mucosacovered pedunculated tumour, usually about 1 cm in size and soft to firm in consistency. It can occur anywhere in the oral or oropharyngeal mucosa (Figure 44.1). The usual cause is chronic irritation. It is easily treated by conservative surgical excision.
3. Haemangioma. Mucosal haemangiomas can occur in the oral cavity or oropharynx (Figure 44.2). They are mostly seen in children. Three types of haemangiomas are known: capillary, cavernous and mixed. When haemangiomas are present at birth or in young children, they should be observed for some period as spontaneous regression can occur.
of
40-50
years, haemangioma-like
dilated veins
(phlebostasis)
may occur
on the
oral or
lingual mucosa.
An infected haemangioma may be difficult to differentiate from a pyogenic granuloma. Haemangiomas that are large and persistent or those which continue to grow are problematic. Use of cryosurgery or laser is not possible in large diffuse lesions. Sclerotherapy has also not been found effective. However, microembolization alone or as a preoperative adjunct to surgery has been found very useful.
4. Lymphangioma. Lymphangiomas mostly involve anterior two-thirds of tongue. They may involve the tongue diffusely and cause macroglossia or may present as localized soft swelling which is compressible. They do not involute spontaneously. Small lesions can be excised surgically. Symptomatic large lesions can be partially excised to reduce the bulk. Total excision of these lesions is not possible.
5. Torus. It is a submucosal bony outgrowth. It may involve the
hard palate

or mandible.
Palatine torus
is more
com- mon
as
a
narrow ridge,
solitary nodule
lobulated mass in the midline of the hard palate.
Mandibular tori
project from
the lingual
aspect gingiva,
near innocuous
the bicuspid
and resection
area and
is indicated
are bilateral.
only or
a
of
Tori when
they the
are inter
fere with
speech, mastication
or fitting
of dentures.
6. Pyogenic granuloma (Figure 44.3). It is a reactive granuloma usually occurs in response to trauma or chronic irritation. It mostly involves anterior gingivae but sometimes the other sites such as tongue, buccal mucosa or lips. Usually it is soft, smooth, reddish to purple mass which bleeds on touch. Treatment is surgical excision. Recurrence is unlikely after complete excision.
7.
Pregnancy granuloma.
It is
clinically and
histologically similar
to pyogenic
trimester ended.
granuloma.
It is
It usually
and regresses
excised only
if starts
once it
persists in
the first
pregnancy after
has
It is
likely to
recur if
operated during
8. Granular cell myoblastoma or granular cell tumour. Most of these tumours occur in the oral cavity and the site of predilection is tongue. Earlier they were thought to arise from the muscle (hence called myoblastoma) but are now considered to be derived from Schwann cells. The tumour presents as a firm submucosal nodule. Treatment is conservative surgical excision. Recurrence is uncommon.
Congenital epulis is also a granular cell tumour involving of future incisors in female infants.
the gums
9. Minor salivary gland neoplasms. Pleomorphic adenoma is the most common. Site of predilection is soft or hard palate but can occur anywhere in the oral cavity. It presents as a painless submucosal nodule. Treatment is wide surgical excision because of the high incidence of recurrence.

10. Solitary fibrous tumour. Though most common in the pleura, this tumour has been seen in the oral tongue and buccal mucosa, and rarely also in the nasopharynx, sinonasal tract, soft palate, retromolar trigone, salivary gland and thyroid. It is a benign tumour.
Clinically it presents as a painless, slow-growing, welldemarcated, mobile, submucosal tumour. Mean age at presentation is 49 years with female preponderance.
It arises from the mesenchyme and is histologically composed of spindle cells arranged in haphazard manner with thick collagen bundles between the cells. It may show capillary proliferation and pericystic pattern and thus need to be differentiated from haemangiopericytoma. Immunohistochemistry helps to differentiate them from neurofibroma, leiomyoma and other spindle cell tumours.
Treatment is complete surgical excision.

1. Mucocele. Most common site is the lower lip (Figure 44.4). Itis a retention cyst of minor salivary glands of the lip. The lesion appears as a soft and cystic mass of bluish colour. Treatment is surgical excision.
2. Ranula (Figure 44.5). It is a cystic translucent lesion seen in the floor of mouth on one side of the frenulum and pushing the tongue up. It arises from the sublingual salivary gland due to obstruction of its ducts. Some ranulae extend into the neck (plunging type).
Treatment is complete surgical excision if small, or marsupialization, if large. Often it is not possible to excise the ranula completely because of its thin wall or ramifications in various tissue planes.
Dermoid. A sublingual dermoid is median or lateral, situated above the mylohyoid. It shines through the mucosa as a white mass in contrast to the translucent nature of the ranula. A submental dermoid develops below the mylohyoid and presents as a submental swelling behind the chin.

1. Leukoplakia. WHO defined leukoplakia as a clinical white patch that cannot be characterized clinically or pathologically as any other disease. It is a clinical definition and does not take pathology into consideration. Other white lesions of oral mucosa, i.e. lichen planus, discoid lupus erythematosus, white spongy nevus and candidiasis are excluded.
(a) Aetiological factors include smoking, tobacco chewing, alcohol abuse particularly, if combined with smoking. Chronic trauma can also occur due to ill-fitting dentures or cheek bites. It may also be associated with submucous fibrosis, hyperplastic candidiasis or Plummer-Vinson syndrome.
(b) Sites involved. Buccal mucosa and oral commissures are the most common sites. It may however involve floor of mouth, tongue, gingivobuccal sulcus and the mucosal surface of lip. Buccal mucosa is the most common site in India (Figure 44.6).
(c) Age and sex. Mostly, it is seen in the fourth decade, males are affected two to three times more often.
(d) Clinical types. (i) Homogenous variety presents with a smooth or wrinkled white patch. It is less often
Figure 44.2 (A) Haemangioma on the lateral border of tongue. (B) Multiple haemangioma involving both lips, buccal mucosa and tongue in a
Figure 44.4 Mucocele of the lower lip.
Figure 44.6
Leukoplakia on lateral border of tongue.
variety presents
thematous malignancy.
(ii)
as white
patches base.
(iii)
where leukoplakia
and has
Nodular
(speckled)
or nodules
Erosive is
erosions on
ery-
(erythroleukoplakia)
interspersed and
variety with
erythroplakia fissures.
The latter
two varieties
have higher
malignant transformation.
- (e) Histology. About 25% of leukoplakias may show some
form of
epithelial dysplasia
from mild
to severe.
Higher the
grade of
dysplasia more

are the
chances of
its going
into malignant
change.
- (f) Malignant potential. The chances of leukoplakia becoming malignant are cited from 1 to 17.5%. On an average about 5% become malignant. Malignant potential varies according to the site and type of leukoplakia, and the duration of follow-up.
(g)
Management
(i)
Many of
the if
causative
In lesions
agent lesions
change, will
disappear is
removed.
with higher
a
biopsy
In
(ii)
(iii)
suspicious potential
is taken
small spontaneously
for to
malignant rule
out lesions,
surgical excision
or ablation
with laser
or cryotherapy
can be
done.
2. Erythroplakia. Similar to leukoplakia, which is a white patch, erythroplakia is a red patch or plaque on the mucosal surface. Red colour is due to decreased keratinization and as a result the red vascular connective tissue of the submucosa shines through. There is no sex predilection. Most common sites are lower alveolar mucosa, gingivobuccal sulcus and the floor of the mouth. Most of lesions of erythroplakia show severe dysplasia, carcinoma in situ or a frank invasive carcinoma when first seen. Malignant potential is 17 times higher than in leukoplakia. Grossly, the lesion may be of three varieties— homogenous, speckled or granular, and erythroplakia, interspersed with areas of leukoplakia (often indistinguishable from erythroleukoplakia, type of leukoplakia). Treatment is excision biopsy and follow-up.
- 3. Melanosis and mucosal hyperpigmentation. Benign pigmented lesions of oral mucosa may transform into malignant melanomas; however, the incidence of this change is not known. About one-fourth of mucosal melanomas may resemble benign lesions and hence biopsy may become mandatory.

Compared to western countries, India has high incidence of oral cancers. Age adjusted incidence rate in India is 44.8 and 23.7 in males and females, respectively, compared to 11.2/100,000 in USA. Several aetiological factors are responsible. (6-S aetiology, i.e. smoking, spirits, sharp jagged tooth, sepsis, syndrome of Plummer-Vinson and syphilitic glossitis.)
- 1. Smoking. Incidence of oral cancer is six times more in smokers than in nonsmokers. In certain parts of India, there is an unusual habit of reverse smoking where burning end of the "churat" (rolled tobacco leaf) is put in the mouth. This gives high incidence of cancer of the hard palate.
2. Tobacco chewing. Powdered tobacco, mixed with lime, is placed in some part of the vestibule of the mouth. Carcinoma develops at the site of the quid. Chewing "pan" and keeping the quid in the vestibule is largely responsible for oral cancer in India.
3. Alcohol. Cancer of upper aerodigestive tract occurs six times more in heavy drinkers as compared to nondrinkers.
4. Dietary deficiencies. Their role in genesis of cancer has not been definitely established. Riboflavin deficiency may be responsible for cancer in alcoholics. Paterson—Brown— Kelly syndrome also called Plummer—Vinson syndrome (iron deficiency anaemia) is responsible for cancer of the oral cavity and hypopharynx.
5. Dental sepsis, jagged sharp teeth and ill-fitting dentures. All these cause chronic irritation and may lead to development of cancer.

1. Mucosal lip (from junction of skin—vermilion border to line of contact of upper and lower lip).
2. Buccal mucosa (includes mucosa of cheek and inner surface of lips up to line of contact of opposing lip).
Anterior two-thirds of tongue (oral tongue). v0
Hard palate. Bf
Lower alveolar ridge. ot
Upper alveolar ridge.
Floor of mouth.
Retromolar trigone.
GID
Clinical presentation and treatment of cancer of the oral cavity at different sites are described below:

Mostly, it is squamous cell carcinoma, often seen in males in the age group of 40-70 years. Lower lip is more often involved. Site of predilection is between the midline and commissure of the lip. Lesion is of exophytic or ulcerative type. Lymph node metastases develop late. Submental and submandibular nodes are the first to be involved; other deep cervical nodes may also get involved later.
Treatment is surgical excision with adequate safety margin of healthy tissue and plastic repair of the defect. Lymph node metastases require block dissection.
Radiotherapy also gives good results in early cases.

Buccal mucosa covers a large area. It extends from the meeting point of lips in front to the pterygomandibular raphe behind and from upper gingivobuccal sulcus to the lower one.
Carcinoma of buccal mucosa is very common. Its inci- dence is next only to tongue cancer. Equally seen in both sexes.
Site of origin. Most common site is the angle of mouth or the line of occlusion of upper and lower teeth. It may also arise from the buccal sulcus where "pan" or tobacco quid is kept. As the whole of buccal mucosa is "condemned," carcinoma may be multicentric.
Gross appearance. Lesion may be exophytic or ulceroinfil- trative; the latter may infiltrate deeply. Exophytic type may be associated with erythroleukoplakia. Buccal mucosa is also
the most common site for verrucous carcinoma which is a white papillary growth with considerable keratinization. Local spread. From its site of origin, the lesion may spread deeply involving submucosa + muscle + subcutaneous fat — skin. Involvement of buccinator muscle or anterior masseter causes trismus. Tumour may spread radially from its site of origin and involve angle of the mouth and lip anteriorly, retromolar trigone and medial pterygoid posteriorly, upper gingivobuccal sulcus and maxilla superiorly, lower gingivobuccal sulcus and alveolar ridge and gums inferiorly. Lymphatic spread. Nodal involvement occurs in about 50% of cases. Submandibular and later the upper jugular nodes may get involved. Upper jugular nodes may also be involved, directly skipping the submandibular group. Clinical features. Early lesions are asymptomatic. Pain and bleeding are seen when lesions are ulcerative and invade deeply. Involvement of the buccinator, masseter or the pterygoid muscles causes trismus. Fungating mass over the cheek, or a foul-smelling bleeding mass in the oral cavity are late features.
Figure 44.7 Carcinoma upper lip and oral commissure. Note associated leukoplakia.

Figure 44.8 Carcinoma buccal mucosa.
Histological type. Squamous cell carcinoma is the most Tumours can also arise from minor salivary glands as in tumours.
Investigations. Biopsy of the lesion for histological type of growth. Computed tomography scan for involvement of (mandible or maxilla) and extension into infratemporal common. with histology salivary gland the bone fossa.

(a) Stage I(T; No). Surgical excision.
(b) Stage IT (T, No). (i) Radiotherapy to primary lesion and also nodes if bone is not involved. (ii) If bone (maxilla/ mandible) is involved or growth infiltrates the muscle, surgery is the treatment of choice. It involves excision of the growth, marginal or segmental mandibulectomy (or partial maxillectomy) and reconstruction of the area with skin or mucosal flaps.
(c) Stage III and IV. Surgical resection, reconstruction with skin and/or myocutaneous flaps and postoperative radiotherapy to the site of lesion and nodes. Surgical resection is combined with neck dissection if nodes are clinically palpable.

Carcinoma involving anterior two-thirds of tongue is commonly seen in men in the age group of 50-70 years. It may also occur in younger age group and in females. It may also develop on a pre-existing leukoplakia, long-standing dental ulcer or syphilitic glossitis (Figure 44.9). Vast majority are squamous cell type.
Site. Most common site is middle of the lateral border or the
ventral aspect
of the
tongue.
Uncommonly, the
tip or

Spread. Locally, it may infiltrate deeply into the lingual musculature causing ankyloglossia or may spread to the floor of mouth, alveolus and mandible. Lymph node metastases go to the submandibular and upper jugular nodes (from the lateral border of tongue) and to the submental and jugulo-omohyoid group (from the tip). Bilateral or contralateral nodal involvement can also occur.
Clinically, cancer of the oral tongue presents as:
- (a) An exophytic lesion like a papilloma (Figure 44.10).
(b) A nonhealing ulcer with rolled edges, greyish white shaggy base and induration (Figure 44.11).
(c)
A
submucous nodule
with induration
of the
surround- ing
tissue.
Figure 44.9 Carcinoma lateral border of the tongue (arrow). Note associated leukoplakia of floor of mouth (double arrows).
Figure 44.10 Exophytic growth on the right lateral border of tongue in a 60-year-old male. It was squamous cell carcinoma.
Figure 44.11 Ulcerative type of squamous cell carcinoma of tongue in a 40-year-old female.

(a)
Early lesions
are painless
and remain
asymptomatic
a
long time.
(b) Pain in the tongue locally at the site of ulcer.
(c)
Pain in
the ipsilateral
ear;
it is
due to
common supply
of the
temporal)
tongue
(lingual from
nerve)
the mandibular
nal nerve.
(d) A lump in the mouth.
- (e) Enlarged lymph node mass in the neck.
(f)
Dysphagia, difficulty
to protrude
the tongue,
for nerve
ear and
(auriculo division
of the
trigemi- slurred
speech and
the mouth
are late
features.
For staging, see TNM classification (Tables 44.2 and 44.3).
Treatment. Aim of treatment is to treat primary tumour in
control neck
disease
(nodal metastasis)
and preserve function of the tongue as much as possible.
Small tumours (T|No) give equal results if treated with or radiotherapy surgery.
T,No tumours can also be treated by radiotherapy includ- ing
the neck
nodes to
eliminate micrometastases.
They can
also be
treated by
with prophylactic
neck dissection.
Stage III or IV tumours require combined treatment with surgery and postoperative radiotherapy. It gives better results than either modality alone. Block dissection neck is always done.
Depending on the size and extent of the primary lesion of the
tongue, surgery
may consist
of hemiglossectomy
includ- ing
a
portion of
the floor
of mouth,
mandibulectomy and
block segmental
or dissection
so-called "commando operation."

Itis either squamous cell or glandular variety; the latter being more common. Glandular variety arises from minor salivary glands of the palate and may be adenoid cystic, mucoepidermoid or adenocarcinoma. It is common in India especially in people who have the habit of reverse smoking, i.e. keeping the burning end of didi or cigar in the mouth. Both men and women are affected.
Cancer starts as a superficial ulcer with rolled out edges and gives no symptoms except painless irregularity on the palate felt by the tongue. It may spread to the gingiva, lip, soft palate or invade the bone of hard palate, floor of the nasal cavity or the antrum. Lymphatic metastases may spread to the submandibular and upper jugular nodes. Cancer palate should be differentiated from cancer of maxillary antrum or nose which has spread to the palate.
Treatment. Small tumours are resected along with the underlying bone, larger ones require partial maxillectomy. If nodes are enlarged, block dissection is also combined. Surgical defect in the palate, left after excision of the growth, is closed by a suitable prosthesis.

It is also called gingival carcinoma; it is mostly seen in men. Usual site of involvement is lower jaw behind the first molar. Tumour may spread to the cheek, floor of mouth, retromolar trigone or the hard palate. Gingival cancer may invade the underlying bone and then spread rapidly along hemi-
of neck
nodes—the
Stage
|
Stage
II
Stage
III
Stage
IVA
Stage
IVB
Stage
IVC
the neurovascular bundle. Nodal metastases go to submandibular and upper jugular nodes.
Treatment. Radiotherapy is avoided because of the risk of radio-osteonecrosis. Surgery is the treatment of choice. Early mucosal lesion on the lower alveolus is treated by local excision with marginal resection of the mandible. Extensive lesions require wide excision which may necessitate segmental or hemimandibulectomy. Block dissection may be
Figure 44.12 Ulcerative squamous cell carcinoma in the floor of oral cavity (arrow) in a 55-year-old male.
combined if nodes are also palpable. Upper alveolar lesions may require partial maxillectomy.

Squamous cell carcinoma is the most common. It affects males more than females in ratio of 4:1. Typically, lesions start anteriorly near the opening of submandibular duct which may get obstructed, leading to enlargement of submandibular gland (Figure 44.12).
Usually, the lesion is ulcerative or infiltrative type and spreads locally into the adjoining areas such as ventral aspect of the tongue, lingual gingiva, mandibular periosteum or deeply into the floor of mouth and submental space. Lymphatic metastases go to submandibular nodes. Lesions of the floor of mouth remain asymptomatic for a long time or cause soreness or irregularity in the floor of the mouth. A swelling in the submandibular region may be either due to obstructive enlargement of submandibular salivary gland or lymph node metastases and this may require differentiation. Treatment. Small lesions without involvement of tongue, lingual gingiva or nodes can be treated by surgical excision or radiotherapy with equal results. Larger lesions with extension to the tongue, gingiva or mandible require wide excision including marginal or segmental mandibular resection. Block dissection is indicated when cervical nodes show clinical evidence of metastases. Prophylactic neck dissection or irradiation is advised for No neck in stage II cancer because of high incidence of micrometastases (40%), stage IIT and IV cancers and

Involvement of retromolar trigone may be primary or secondary to extension of growths from the gingiva, floor of mouth, buccal mucosa or the arch.
excision palatine Treatment depends on the extent of lesion. Wide surgical often combined with block dissection is required.

About 15% of patients with carcinoma of the oral cavity have multiple primary cancers affecting the upper aerodigestive tract. This is because of the common risk factors such
Figure 44.13 Mixed salivary tumour palate.
as smoking and alcohol simultaneously operating at various sites.

In addition to carcinoma, other malignant lesions that involve the oral cavity are:
1. Minor salivary gland tumours. In one series, 80-90% of all minor salivary gland tumours were malignant. Palate is the most common site but can involve tongue, cheek, lip, gums and floor of mouth (Figure 44.13).
Adenoid cystic variety is the most common (40%). Next in frequency are the adenocarcinoma (30%) and mucoepidermoid carcinoma (20%). Treatment is wide surgical excision along with block dissection, if the neck nodes are positive.
2. Melanoma. Mucosal melanomas of oral cavity and oropharynx are rare. Peak age incidence is the sixth decade; males are affected more (2:1). Palate and gingiva are the most common sites. They appear as areas of higher pigmentation and later may ulcerate and bleed. Amelanotic variety is also seen. Both cervical nodal and distant metastases are seen. Treatment of choice is wide surgical excision including underlying bone. Local recurrence is common. Prognosis is poor with 5-year cure rate of only 15%.
3. Lymphoma. Lymphomas can involve oral cavity or oropharynx, majority of them occurring in the palatine tonsils. Males are affected more. Usual presentation is that of a smooth, submucosal bulky mass which is occasionally ulcerated. They are mostly of non-Hodgkin variety. Cervical nodes may be involved in 40-70% of the patients. Treatment is radiation, alone or in combination with chemotherapy.
4. Kaposi sarcoma. It is a vascular tumour, multifocal in origin, primarily affecting skin but may occur in the oral cavity. Its incidence is high in AIDS (acquired immune deficiency syndrome) patients. The lesion appears as a reddish purple nodule or a plaque mostly on the palate. Microscopically, it
230
consists of spindle cells with haemorrhagic cleft-like spaces. Treatment is not satisfactory. Kaposi sarcoma in non-AIDS patients may respond to chemotherapy but its response in patients suffering from AIDS is poor (see also p. 371).

It is the use of certain pharmacological agents to halt, delay or reverse the process of carcinogenesis. It has been used to prevent oral premalignant lesions to develop into cancer or to prevent the development of second primary cancers after the main primary cancer has been treated. Agents used have been vitamin A, beta carotene, alpha tocopherol (vitamin E), selenium and natural or synthetic retinoids such as 13-cis retinoic acid. Beta carotene and vitamin A induced remission of oral leukoplakia is seen in 25-50% of patients. Similarly, in a controlled trial, 13-cis retinoic acid reduced the incidence of second primary lesions in the aerodigestive tract. The beneficial effect of these agents may be limited to the duration of treatment only.
In addition to their use in head and neck, retinoids have shown significant chemopreventive activity in cancers of lung, skin, cervix, bladder and ovary. Trials are also being conducted in Cox-2 inhibitors (e.g. celecoxib) in the prevention of oral premalignant lesions.

of IgM also indicates recent infection. IgM is present in 100% patients by day 5.
Itis a viral infection caused by paramyxovirus. Disease is contracted by droplet infection and fomites. Children are most often affected but adults can also contract the disease. Incubation period is 2-3 weeks (7-23 days). Patient is infective even before the appearance of clinical manifestations and remains so 7-10 days after parotid swelling subsides. Virus is excreted through salivary, nasal and urinary excretions.

Initial period of viraemia causes fever (up to 103°F), malaise, anorexia and muscular pain. Parotid swelling may appear only on one side. Other parotid gland may be enlarged simultaneously or after some time. Submandibular and sublingual salivary glands may also be enlarged but isolated involvement of submandibular gland is rare. Swelling subsides in about a week.

© Orchitis with painful and tender testis, on one but uncommonly both sides, may occur. Sterility following mumps is rare.
© Ophritis causes lower abdominal pain. Female sterility is almost never seen.
¢ Pancreatitis causes pain in abdomen.
© Aseptic meningitis or meningoencephalitis may occur with or without the salivary gland involvement. Headaches, neck stiffness and drowsiness may occur.
© Unilateral sensorineural hearing loss can occur due to involvement of the labyrinth. Sudden deafness has been noticed.
¢ Other complications include thyroiditis, myocarditis, nephritis and arthritis.

Usually clinical; difficulties arise when parotids are not enlarged.
1. Serum and urinary amylase are raised during the first week of parotitis.
2. Serology. Serum IgG and IgM are measured as early as possible and after 10-14 days of illness. Presence of IgG indicates past exposure and possible immunity. However rise in IgG titre more than four times from acute to convalescent serum indicates recent infection. Similarly presence

Parotitis is treated by proper hydration, rest, analgesics and cold or hot compresses over the parotid to relieve pain. Food which encourages salivary flow should be avoided as they cause pain. Parotid swelling persists for about 1 week. Orchitis is treated by cold compresses and support to the scrotum, and administration of analgesics. Steroids have not been found useful.

An infant has maternal immunity for 1 year. After that immunization can be given by MMR (Mumps, Measles, Rubella) vaccine at the age of 15 months. Older children, adolescents and adults who were not protected by MMR and have not had mumps, and are in contact with children should receive monoclonal mumps or MMR vaccine.
Mumps immunoglobulin is of no value as a prophylaxis or in established disease.

It is most commonly seen in the elderly, debilitated and dehydrated patients. Dry mouth due to any cause is a predisposing factor. Staphylococcus aureus is the usual causative organism though other Gram-positive and anaerobic organisms have also been observed. Usual route of infection is from the mouth the Stensen's duct.
through

The onset is sudden with severe pain and enlargement of gland (Figure 45.1). Movements of jaw aggravate the pain. Opening of the Stensen's duct is swollen and red and may be discharging pus or the latter can also be expressed by gentle pressure over the gland. Patient is usually febrile and toxaemic.

White cell count shows leukocytosis with increase in polymorphs. Causative organisms should be identified and their sensitivity established by culture of blood and the pus collected from the opening of the parotid duct.

It consists of appropriate antibiotics, preferably administered through i.v. route, adequate hydration, measures to promote salivary flow and attention to oral hygiene. If temperature does not subside and there is progressive induration of the gland, in spite of adequate medical management, surgical drainage should be done.

This usually involves parotid gland which shows recurrent bacterial infection. During acute exacerbation, parotid is enlarged and tender, and pus can be expressed from its
Figure 45.1 Acute suppurative parotitis on left side in a 58-year-old female.
|
duct. Between the acute episodes, gland is firm and slightly enlarged. Culture of pus from the duct reveals staphylococci or streptococci. Sialography shows normal duct system. Treatment of acute episode is similar to that of acute bacterial sialadenitis. Between the attacks, patient is instructed to keep good oral hygiene, avoid drugs which dry oral mucosa and use sialogogues to promote salivation.

As the name implies, there is dilatation of the ductal system, leading to stasis of secretions, which predisposes to infection. Clinically, sialectasis resembles chronic recurrent sialadenitis, but can be differentiated from it by sialography. Different degrees of dilatation of the ductal system—punctuate, globular or cavitary types—may be seen. Sialectasis may be congenital, associated with granulomatous disease or autoimmune disease such as Sjogren's syndrome.

Tuberculosis, sarcoidosis and actinomycosis may involve the salivary glands. Tubercular infection may involve parenchyma or lymph nodes of the parotid and present as a nontender mass. Sometimes, overlying skin undergoes necrosis leading to a fistula formation. Surgical excision of the involved tissue and antitubercular treatment usually control the disease. Uveoparotid fever is due to sarcoidosis of the parotid. It is characterized by fever, enlargement of the parotid and lacrimal glands, chorioretinitis, and cranial nerve palsies.
Actinomycosis of parotid is uncommon. It may present as an acute abscess with sinus formation discharging sulfur-like granules, or as an indolent swelling in the parotid. Treatment is surgical drainage and large doses of penicillin or tetracycline.

Calculi may form in the ducts of submandibular or parotid glands. They are formed by the deposition of calcium phosphate on the organic matrix of mucin or cellular debris. About 90% of the stones are seen in submandibular and 10% in the parotid. Stones may form in the duct or parenchyma of the gland. The presenting feature is intermittent swelling of the involved gland, and pain due to obstruction to outflow of saliva. Sometimes, stone is visible at the duct opening or can be palpated. About 80% of the stones are radio-opaque and can be seen on appropriate X-rays (Figures 45.2 and 45.3). Sialography may be required for radiolucent stones. Stones in peripheral part of submandibular or parotid ducts can be removed intraorally, while those at the hilum or in the parenchyma require excision of the gland.

It is an autoimmune disorder involving exocrine glands of the body. It may be primary or secondary.
1. Primary Sjogren's syndrome consists of xerostomia and xerophthalmia and is due to involvement of salivary and lacrimal glands. Parotid is the most often involved gland. It has also been known as benign lymphoepithelial lesion of parotid or Mikulicz's disease. Both sexes are equally involved.
2. Secondary Sjogren's syndrome consists of three major components: (i) keratoconjunctivitis sicca (due to involvement of lacrimal gland); (ii) xerostomia (due to involvement of
Figure 45.3 duct (arrow).
A radio-opaque stone seen in the right submandibular salivary glands and minor mucous glands of the oral cayity); (iii) autoimmune connective tissue disorder, usually the rheumatoid arthritis, sometimes the systemic lupus erythematosus (SLE). Often there is bilateral swelling of the salivary glands. It is also more common in females (90%).
Diagnosis depends on history and physical examination of keratoconjunctivitis and xerostomia. Schirmer's test may be done to prove decreased tear formation. Biopsy of lower lip for evidence of involvement of minor salivary glands. SS-A an SS-B antibodies are necessary for diagnosis. Raised erythrocyte sedimentation rate, positive rheumatoid factor and positive antinuclear antibodies tests help to exclude associated rheumatoid arthritis or SLE.

The tumours of major or minor salivary glands are either from epithelial or mesenchymal tissues. Larger the size of salivary gland, more are the chances of a tumour being benign. Eighty per cent of parotid, 50-60% of submandibular and only about 25% of other minor salivary gland tumours are benign. In other words, chances of malignant tumours in minor salivary glands are higher.
myxochondroid or chondroid and its proportion to the epithelial element may vary.
Rapid growth, restricted mobility, fixity of overlying skin, pain and facial nerve involvement indicate the possibility of tumour being malignant.
Table 46.1 shows benign and malignant tumours of saliglands.
vary

It is the most common benign tumour of salivary glands. It can arise from the parotid, submandibular or other minor salivary glands. In the parotid it usually arises from its tail. It can also arise from the deep lobe of the parotid and present as a parapharyngeal tumour in the oropharynx.
Pleomorphic adenomas
are slow-growing
tumours and
may be
ally seen
females.
in the
They thelial
and at
initial third
or presentation.
fourth are
called decade,
with
“mixed mesenchymal
tumours”
elements
They are
usu- propensity
because are
for both
seen epi-
in histology.
The stroma
of the
tumour may
be mucoid,
fibroid, vascular,
Though tumour is encapsulated, it sends pseudopods into the surrounding gland which are left behind if the tumour is simply shelled out. It is therefore essential that surgical excision of the tumour should include normal gland tissue around it. In the parotid, it amounts to superficial parotidectomy.

They are commonly seen between fifth and seventh decade with preponderance in males (5:1). They mostly involve the tail of the parotid and are bilateral in 10% of the patients. They may be multiple. Adenolymphoma is a rounded, encapsulated tumour, at times cystic, with mucoid or brownish fluid. Histologically, epithelial and lymphoid elements are seen. Treatment is superficial parotidectomy though they can be enucleated without danger of recurrence.

They arise from acidophilic cells called oncocytes and comprise less than 1% of all salivary gland tumours. Mostly seen in the elderly, they usually do not grow larger than 5 cm and involve the superficial lobe of parotid. Benign oncocytomas are cystic rather than solid. Malignant oncocytomas are also seen. Oncocytomas show increased uptake of technetium-99. Treatment for parotid oncocytomas is also superficial parotidectomy.

Haemangiomas are the most common benign tumours of the parotid in children, predominantly affecting females. Most of them are discovered at birth, grow rapidly in the neonatal period and then involute spontaneously. Cutaneous haemangioma may coexist in 50% of the patients. They are soft and painless and increase in size with crying or straining. Overlying skin may show bluish discoloration. Surgical excision is indicated if they do not regress spontaneously.

They are less common and may involve parotid and submandibular glands. On palpation, they feel soft and cystic. They do not regress spontaneously and are surgically excised.
Lipoma and neurofibroma are rare.

Some pathologists do not consider it to be malignant and call it mucoepidermoid tumour and not cancer, but it is known to metastasize and kill. Generally, it is slow-growing but can invade the facial nerve. Histologically, there are areas of mucin-producing cells and the squamous cells, and hence the name. Greater the epidermoid element, more malignant is the behaviour of the tumour. The tumours have been further classified as low grade and high grade. Low-grade tumours have good prognosis (90%, 5 years survival rate), high-grade tumours are more aggressive and have poor prognosis (30%, 5 years survival rate). Low-grade tumours are more common in children.
Behaviour of mucoepidermoid tumours of minor salivary glands is more aggressive and akin to adenoid cystic carcinoma, but in the major salivary glands they behave like pleomorphic adenoma.
Low-grade tumours of the parotid are treated by superficial or total parotidectomy, depending on the location of the tumour. Facial nerve is preserved.
High-grade tumours being more aggressive are treated by total parotidectomy. Facial nerve may be sacrificed if invaded by the tumour. Some surgeons also combine radical neck dissection because of high incidence of microscopic spread of the tumour.

It is a slow-growing tumour but infiltrates widely into the tissue planes and muscles. It also invades perineural spaces and lymphatics and thus causes pain and VIIth nerve paralysis. It can metastasize to lymph nodes. Local recurrences after surgical excision are common and can occur as late as 10-20 years after surgery. Distant metastases go to the lung, brain and bone.
Treatment is radical parotidectomy with largest cuff of normal tissue around the boundaries of the tumour.
grossly
Radical neck is not done unless nodal metastases are present. Postoperative radiation is given if margins of the resected specimen are not free of tumour.

It is a low-grade tumour which appears similar to a benign mixed tumour. It presents as a small, firm, movable and encapsulated tumour, sometimes bilateral. Metastases are rare. A conservative approach of superficial or total parotidectomy is adopted.

More often it arises in minor salivary glands. It is highly aggressive locally and sends distant metastasis.

There are two varieties of this tumour:
1. Carcinoma developing in pre-existing benign mixed tumour and
2. A"de novo" tumour.
The latter has much shorter history. Rapid growth and pain developing in a benign tumour should always arouse a suspicion of malignant change. Treatment of malignant tumour is radical parotidectomy. Facial nerve sacrificed during operation is grafted immediately.

It is a rapidly growing tumour that infiltrates, causes pain and ulcerates through the skin. It can metastasize to neck nodes. Treatment is radical parotidectomy which may include cuff of muscle or even a portion of mandible, temporal bone and the involved skin. Radical neck is combined if nodal metastases are present. Surgery is followed by postoperative radiation to primary site and the neck.

It is a rare, but aggressive tumour. It has a tendency to spread rapidly, causes pain, becomes fixed to skin and ulcerates. It causes facial paralysis and cervical nodal metastasis. Treatment is wide excision, radical neck and postoperative radiation.

It is a rare tumour usually associated with systemic disease, but may occasionally be a primary tumour. Treatment is same as for other lymphomas.

Rarely other sarcomas, e.g. rhabdomyosarcoma may arise from the parotid.

Frey's syndrome arises as a complication of parotid surgery usually manifesting several months after the operation. It is characterized by sweating and flushing of the preauricular skin during mastication causing nuisance to the person or social embarrassment. It is the result of aberrant innervation of sweat glands by parasympathetic secretomotor fibres which were destined for the parotid. Now instead of causing salivary secretion from the parotid, they cause secretion from the sweat glands. The condition can be treated by tympanic neurectomy which intercepts these parasympathetic fibres at the level of middle ear. Some people like to place a sheet of fascia lata between the skin and the underlying fat to prevent secretomotor fibres reaching the sweat glands. Subcutaneous infiltration of botulinum toxin has also been used to alleviate the symptoms. Generally, no treatment other than reassurance is required in most of these patients.

47.
48.
49.
50.
Anatomy and Physiology of Pharynx
Adenoids and Other
Inflammations of Nasopharynx
Tumours of Nasopharynx
Acute and Chronic Pharyngitis
51.
52.
53.
54.
55.
Acute and Chronic Tonsillitis
Head and Neck Space Infections
Tumours of Oropharynx
Tumours of the Hypopharynx and Pharyngeal Pouch
Snoring and Sleep Apnoea

Pharynx is a conical fibromuscular tube forming upper part of the air and food passages. It is 12-14 cm long, extending from base of the skull (basiocciput and basisphenoid) to the lower border of cricoid cartilage where it becomes continuous with the oesophagus. The width of pharynx is 3.5 cm at its base and this narrows to 1.5 cm at pharyngo-oesophageal junction, which is the narrowest part of digestive tract apart from the appendix.

From within outwards it consists of four layers:
2. Pharyngeal aponeurosis (pharyngobasilar fascia)
3. Muscular coat
4. Buccopharyngeal fascia
1. Mucous membrane. It lines the pharyngeal cavity and is continuous with mucous membrane of eustachian tubes, nasal cavities, mouth, larynx and oesophagus. The epithelium is ciliated columnar in the nasopharynx and stratified squamous elsewhere. There are numerous mucous glands scattered in it.
2. Pharyngeal aponeurosis (pharyngobasilar fascia). It is a fibrous layer which lines the muscular coat and is particu-
larly thick near the base of skull but is thin and indistinct inferiorly. It fills up the gap left in the muscular coat near the base of skull.
3. Muscular coat. It consists of two layers of muscles with three muscles in each layer.
- (a) External layer. It contains superior, middle and inferior
constrictor muscles.
(b) Internal layer. It contains stylopharyngeus, salpingopharyngeus and palatopharyngeus muscles.
4. Buccopharyngeal fascia. It covers outer surface of the constrictor muscles and in the upper part, it is also prolonged forwards to cover the buccinator muscles. Above the upper border of superior constrictor, it blends with pharyn- geal aponeurosis.

Inferior constrictor muscle has two parts: thyropharyngeus with oblique fibres and cricopharyngeus with transverse fibres. Between these two parts exists a potential gap called Killian's dehiscence. It is also called "gateway of tears" as perforation can occur at this site during oesophagoscopy. This is also the site for herniation of pharyngeal mucosa in cases of pharyngeal pouch.

Scattered throughout the pharynx in its subepithelial layer is the lymphoid tissue which is aggregated at places to form masses, collectively called Waldeyer’s ring. The masses are:
. Nasopharyngeal tonsil or the adenoids Ne
. Palatine tonsils or simply the tonsils
. Lingual tonsil OF
. Tubal tonsils (in fossa of Rosenmiiller)
. Lateral pharyngeal bands ov
. Nodules (in posterior pharyngeal wall). a>

There are two potential spaces in relation to the pharynx where abscesses can form.
1. Retropharyngeal space, situated behind the pharynx and
extending from the base of skull to the bifurcation of tra-
chea (see p. 265).
2. Parapharyngeal space, situated on the side of pharynx. It contains carotid vessels, jugular vein, last four cranial nerves and cervical sympathetic chain (see p. 267).

Anatomically, divided into (Figure 47.3): pharynx is three _ parts
1. Nasopharynx
2. Oropharynx
3. Hypopharynx or laryngopharynx.

Nasopharynx is the uppermost part of the pharynx and therefore, also called epipharynx. It lies behind the nasal cavi- ties and extends from the base of skull to the soft palate or the level of the horizontal plane passing through the hard palate (Figure 47.4).
1. Roof of the nasopharynx is formed by basisphenoid and basiocciput.
Figure
47.1
it
pharyngeal wall.
From within
outwards consists
of
(a)
mucous membrane,
(b)
pharyngobasilar fascia,
(c)
muscular coat
and
(d)
buccopharyngeal fascia.
Figure
47.2
Waldeyer’s ring.
Figure 47.3
Divisions of pharynx and the vertebrae related to their
Figure
47.4
Rathke’s pouch
is represented
by
a
dimple, high
in nasopharynx.
Inferior to
this, within
the adenoid
mass, is
the naso-
pharyngeal bursa.

2. Posterior wall is formed by arch of the atlas vertebra covered by prevertebral muscles and fascia. Both the roof and the posterior wall imperceptibly merge with each other.
3. Floor is formed by the soft palate anteriorly but is deficient posteriorly. It is through this space, the nasopharyngeal isthmus, that the nasopharynx communicates with the oropharynx.
4. Anterior wallis formed by posterior nasal apertures or choanae, separated from each other by the posterior border of the nasal septum. Posterior ends of nasal turbinates and meatuses are seen in this wall.
5. Lateral wall. Each lateral wall presents the pharyngeal opening of eustachian tube situated 1.25 cm behind the posterior end of inferior turbinate. It is bounded above and behind by an elevation called torus tubarius raised by the cartilage of the tube. Above and behind the tubal elevation is a recess called fossa of Rosenmiiller, which is the commonest site for origin of carcinoma (Figure 47.5). A ridge extends from the lower end of torus tubarius to the lateral pharyngeal wall and is called salpingopharyngeal fold (Figure 47.9). Itis raised by the corresponding muscle.

It is a subepithelial collection of lymphoid tissue at the junction of roof and posterior wall of nasopharynx and causes the overlying mucous membrane to be thrown into radiating folds. It increases in size up to the age of 6 years and then gradually atrophies.

It is an epithelial-lined median recess found within the adenoid mass and extends from pharyngeal mucosa to the periosteum of the basiocciput. It represents the attachment of notochord to the pharyngeal endoderm during embryonic life. When infected, it may be the cause of persistent postnasal discharge or crusting. Sometimes an abscess can form in the bursa (Thornwaldt's disease) .

It is represented clinically by a dimple above the adenoids and is reminiscent of the buccal mucosal invagination,
Figure 47.5 Endoscopic view of nasopharynx showing torus tubarius in the lateral wall of nasopharynx. Note also the fossa of Rosenmiller which lies behind it. Fossa of Rosenmiller is the commonest site for the origin of carcinoma nasopharynx.
to form the anterior lobe of pituitary. A craniopharyngioma may arise from it.

It is collection of subepithelial lymphoid tissue situated at the tubal elevation. It is continuous with adenoid tissue and forms a part of the Waldeyer's ring. When enlarged due to infection, it causes eustachian tube occlusion.

It is a space between the base of the skull and upper free border of superior constrictor muscle. Through it enters (i) the eustachian tube, (ii) the levator veli palatini, (iii) tensor veli palatini and (iv) ascending palatine artery—branch of the facial artery (Figure 47.1).

It is a mucosal ridge raised by fibres of palatopharyngeus. It encircles the posterior and lateral walls of nasopharyngeal isthmus. Soft palate, during its contraction, makes firm contact with this ridge to cut off nasopharynx from the oropharynx during the deglutition or speech.

Functionally, nasopharynx is the posterior extension of nasal cavity. It is lined by pseudostratified ciliated columnar epithelium.

Lymphatics of the nasopharynx, including those of the adenoids and pharyngeal end of eustachian tube, drain into upper deep cervical nodes either directly or indirectly through retropharyngeal and parapharyngeal lymph nodes. They also drain into spinal accessory chain of nodes in the posterior triangle of the neck. Lymphatics of the nasopharynx may also cross midline to drain into contralateral lymph nodes.

- 1. Acts as a conduit for air, which has been warmed and humidified in the nose, in its passage to the larynx and trachea.
2. Through the eustachian tube, it ventilates the middle ear and equalizes air pressure on both sides of tympanic membrane. This function is for
important hearing. 3. Elevation of the soft palate against posterior pharyngeal wall and the Passavant’s ridge helps to cut off nasophar- ynx from oropharynx. This function is important during swallowing, vomiting, gagging and speech. 4. Acts as a resonating chamber during voice production. Voice disorders are seen in nasopharyngeal obstruction and velopharyngeal incompetence (see Chapter 63). 5. Acts as a drainage channel for the mucus secreted by nasal and nasopharyngeal glands.

Oropharynx extends from the plane of hard palate above to the plane of hyoid bone below. It lies opposite the oral cavity with which it communicates through oropharyngeal isthmus. The latter is bounded above, by the soft palate; below, by the upper surface of tongue; and on either side, by palatoglossal arch (anterior pillar).

1. Posterior wall. It is related to retropharyngeal space and lies opposite the second and upper part of the third cervical vertebrae.
2. Anterior wall. It is deficient above, where oropharynx communicates with the oral cavity, but below it presents:
- (a) Base of tongue, posterior to circumvallate papillae.
(b)
Lingual tonsils,
one on
either side,
situated in
the of
tongue.
They following
may show
compensatory tonsillectomy
Valleculae.
or
They between
are the
glottis.
base
Each epiglottic
base enlargement
may be
the seat
cup-shaped of
tongue is
of infection.
depressions and
anterior bounded
fold medially
(Figure
47.6).
They lying
surface of
epi- by
the median
glosso- by
pharyngoepiglottic are
the seat
fold of
retention cysts.

(a) Palatine (faucial) tonsil (for details, see p. 257).
(b) Anterior pillar (palatoglossal arch) formed by the palatoglossus muscle.
(c) Posterior pillar (palatopharyngeal arch) formed by the palatopharyngeus muscle.
Both anterior and posterior pillars diverge from the soft palate and enclose a triangular depression called tonsillar fossa in which is situated the palatine tonsil (Figure 47.7). Boundary between oropharynx above and the hypopharynx below is formed by upper border of epiglottis and the pharyngoepiglottic folds.

Lymphatics from the oropharynx drain into upper jugular chain particularly the jugulodigastric (tonsillar) node. The soft palate, lateral and posterior pharyngeal walls and the base of tongue also drain into retropharyngeal and parapharyngeal nodes and from there to the jugulodigastric and posterior cervical group. The base of tongue may drain bilaterally.
(c)
Median and
lateral
Figure
47.6
Base of
tongue and
valleculae.
Figure
47.7
Various structures
seen in
the oropharynx.

1. As a conduit for passage of air and food.
2. Helps in the pharyngeal phase of deglutition.
3. Forms part of vocal tract for certain speech sounds.
4. Helps in appreciation of the taste. Taste buds are pres-
ent in
the base
of tongue,
soft palate,
anterior pillars
and

- 5. Provides local defence and immunity against harmful intruders into the air and food passages. This function is subserved by subepithelial masses of lymphoid tissues scattered as Waldeyer's ring. They are strategically placed at the portals of air and food entry and act as protective sentinels. B-lymphocytes in the germinal centres of the follicles produce secretory antibodies of IgA class whereas T-lymphocytes in parafollicular region produce cell-mediated immunity against various viruses, bacteria and fungi. Pathogens which happen to enter into these lymphoid masses are dealt by IgM and IgG antibodies
secreted by plasma cells.
Figure
47.8
Normal pyriform
fossae right
and left
(arrows).
They show better when patient phonates.

Hypopharynx is the lowest part of the pharynx and lies behind and partly on the sides of the larynx. Its superior limit is the plane passing from the body of hyoid bone to the posterior pharyngeal wall, while the inferior limit is lower border of cricoid cartilage where hypopharynx becomes continuous with oesophagus. Hypopharynx lies opposite the third, fourth, fifth, sixth cervical vertebrae. Clinically, it is subdivided into three regions—the pyriform sinus, postcricoid region and the posterior pharyngeal wall.
1.
Pyriform sinus
(fossa).
It lies
on either
side of
the larynx
and extends from pharyngoepiglottic fold to the upper end of oesophagus.
It is bounded laterally by the thyrohyoid membrane and the thyroid cartilage and medially by the aryepiglottic fold, posterolateral surfaces of arytenoid and cricoid cartilages (Figure 47.8). It forms the lateral channel for food. Foreign bodies may lodge in the pyriform fossa. Internal laryngeal nerve runs submucosally in the lateral wall of the sinus and thus is easily accessible for local anaesthesia. It is also through this nerve that pain is referred to the ear in carcinoma of the pyriform sinus.
2. Postcricoid region. It is the part of the anterior wall of laryngopharynx between the upper and lower borders of cricoid lamina. It is a common site for carcinoma in females suffering from Plummer-Vinson syndrome (Figure 47.9).
3. Posterior pharyngeal wall. It extends from the level of hyoid bone to the level of cricoarytenoid joint.

Pyriform sinus is richly supplied by lymphatics which exit through
the thyrohyoid
drain into
the upper jugular chain.
Lymphatics of the posterior wall terminate in the lateral pharyngeal or parapharyngeal nodes and thence to the cervical nodes.
deep lymph
Lymphatics of postcricoid region also drain into the parapharyngeal nodes but may also drain into nodes of supraclavicular and paratracheal chain.
Figure 47.9 Pharynx opened from behind showing structures related to nasopharynx, oropharynx and laryngopharynx.
Rich lymphatic network of pyriform fossae explains the high frequency with which nodal metastases are seen in carcinoma of this region.

Laryngopharynx, like oropharynx, is a common pathway for air and food, provides a vocal tract for resonance of
certain speech sounds and helps in deglutition. There is coordination between contraction of pharyngeal muscles and relaxation of cricopharyngeal sphincter at the upper end of oesophagus. Lack of this coordination, i.e. failure of cricopharyngeal sphincter to relax when pharyngeal muscles are contracting causes hypopharyngeal diverticulum.

The nasopharyngeal tonsil, commonly called "adenoids", is situated at the junction of the roof and posterior wall of the nasopharynx. It is composed of vertical ridges of lymphoid tissue separated by deep clefts (Figure 48.1). Covering epithelium is of three types: ciliated pseudostratified columnar, stratified squamous and transitional. Unlike palatine tonsils, adenoids have no crypts and no capsule. Adenoid tissue is present at birth, shows physiological enlargement up to the age of 6 years, and then tends to atrophy at puberty and almost completely disappears by the age of 20.
Blood supply. Adenoids receive their blood supply from:
1. Ascending palatine branch of facial.
2. Ascending pharyngeal branch of external carotid.
3. Pharyngeal branch of the third part of maxillary artery.
4. Ascending cervical branch of inferior thyroid artery of thyrocervical trunk.
Lymphatics from the adenoid drain into upper jugular nodes directly or indirectly via retropharyngeal and parapharyngeal nodes.
Nerve supply is through CN IX and X. They carry sensa- tion.
Referred pain
to ear
due to
adenoiditis is
also medi-
ated through them.

Adenoids, 1 = are subject to physiological enlargement. in, 1 = childhood. Certain children have a tendency to
Figure 48.1
Adenoid mass after removal with curette. Note ridges of lymphoid tissue separated by deep clefts.
generalized lymphoid hyperplasia in which adenoids also take part.
Recurrent attacks of rhinitis, sinusitis or chronic tonsillitis may cause chronic adenoid infection and hyperplasia.
to
Allergy of the upper respiratory tract may also contribute the enlargement of adenoids.

Symptoms and signs depend not merely on the absolute size of
mass but
are relative
to the
available space
in the nasopharynx.
Enlarged and infected adenoids may cause nasal, aural or general symptoms.

(a)
Nasal obstruction
is to
mouth the
with feeding
commonest symptom.
Nasal or
obstruction suckling
in
This leads
also interferes
a
child.
As respiration
feeding cannot
take place
simultaneously, and
a
child with
adenoid enlargement fails to thrive.
- (b) Nasal discharge. It is partly due to choanal obstruction, as the normal nasal secretions cannot drain into nasopharynx and partly due to associated chronic rhinitis. The child often has a wet bubbly nose.
(c)
Sinusitis. Chronic maxillary sinusitis is commonly associated with adenoids. It is due to persistence of nasal discharge and infection. Reverse is also true that a primary maxillary sinusitis may lead to infected and enlarged adenoids.
(d) Epistaxis. When adenoids are acutely inflamed, epistaxis can occur with nose blowing.
(e) Voice change. Voice is toneless and loses nasal quality due
to nasal obstruction.

(a) Tubal obstruction. Adenoid mass blocks the eustachian tube leading to retracted tympanic membrane and conductive hearing loss.
(b)
Recurrent attacks
of acute
may occur
due to
spread of
infection
Chronic
(c)
suppurative via
the otitis
eustachian media
tube.
may fail
to resolve
in the
infected adenoids.
(d) Serous otitis media. Adenoids form an important cause of serous otitis media in children. The waxing and waning size of adenoids causes intermittent eustachian tube obstruction with fluctuating hearing loss. Impedance audiometry helps to identify the condition.
(b)
Figure 48.2 Adenoid facies. Patient is a mouth breather.

(a)
Adenoid facies.
Chronic breathing
lead adenoid
facies.
nasal to
The expression,
child open
teeth obstruction
and facial
appearance has
an mouth,
and hitched
in appearance
48.2).
elongated prominent
up upper
due
Hard palate
to mouth
called face
with and
crowded lip.
Nose disuse
in atrophy
these cases
dull upper
gives
a
pinched- of
alaenasi is
highly
(Figure arched
as the
moulding action
of the
tongue on
palate is
lost.
Pulmonary hypertension. Long-standing nasal obstruc- tion due to adenoid hypertrophy can cause pulmonary hypertension and cor pulmonale.
(c)
Aprosexia, i.e. lack of concentration.

Examination of postnasal space is possible in some young children and an adenoid mass can be seen with a mirror. A rigid or a flexible nasopharyngoscope is also useful to see details of the nasopharynx in a cooperative child. Soft tissue lateral radiograph of nasopharynx will reveal the size of adenoids and also the extent to which nasopharyngeal air space has been compromised (Figure 48.3). Detailed nasal examination should always be conducted to exclude other causes of nasal obstruction.

When symptoms are not marked, breathing exercises, decongestant nasal drops and antihistaminics for any co-existent nasal allergy can cure the condition without resort to surgery. When symptoms are marked, adenoidectomy is done. Indications and details of the operation are discussed in the section on operative surgery.
Figure 48.3 Enlarged adenoids (arrows) in a 7-year-old girl. There is very little breathing space in the nasopharynx.

Acute infection of the nasopharynx may be an isolated infection confined to this part only or be a part of the generalized upper airway infection. It may be caused by viruses (common cold, influenza, parainfluenza, rhino or adenovirus) or bacteria (especially streptococcus, pneumococcus or Haemophilus influenzae).

Dryness and burning of the throat above the soft palate is usually the first symptom as is commonly noted in common cold. This is followed by pain and discomfort localized to the back of nose with some difficulty on swallowing. In severe infections, there is pyrexia and enlarged cervical lymph nodes. Examination of nasopharynx reveals congested and swollen mucosa often covered with whitish exudate.

Mild cases clear up spontaneously. Some analgesic may be required for relief of pain and discomfort. In severe cases with general symptoms, systemic antibiotic or chemotherapy may be necessary. In children, there is associated adenoiditis which causes nasal obstruction and requires nasal decongestant drops.

It is often associated with chronic infections of nose, paranasal sinuses and pharynx. It is commonly seen in heavy smokers,
drinkers and those exposed to dust and fumes.

Postnasal discharge and crusting with irritation at the back of nose is the most common complaint. Patient has a constant desire to clear the throat by hawking or inspiratory snorting (forcibly drawing nasal secretions back into the throat). Examination of nasopharynx reveals congested mucosa and mucopus or dry crusts. In children, adenoids are often enlarged and infected (chronic adenoiditis).

Chronic infections of the nose, paranasal sinuses and oro- pharynx should be attended to. Excessive smoking and drinking should be corrected. Preventive measures should be
taken to
avoid dust
and fumes.
Alkaline nasal
douche helps
to remove crusts and mucopus. Steam inhalations are soothing.

It is infection of the pharyngeal bursa which is a median recess representing attachment of notochord to endoderm
of the primitive pharynx. Pharyngeal bursa is located in the midline of posterior wall of the nasopharynx in the adenoid mass.

1. Persistent postnasal discharge with crusting in the nasopharynx.
2. Nasal obstruction due to swelling in the nasopharynx.
3. Obstruction to eustachian tube and _ serous otitis media.
4. Dull type of occipital headache.
Recurrent sore throat. ox
6. Low-grade fever.
Examination would reveal a cystic and fluctuant swelling
in the posterior wall of nasopharynx. It may also show crusts
in the nasopharynx due to dried up discharge.

Antibiotics are given to treat infection and marsupializa- tion of the cystic swelling and adequate removal of its lining membrane.

Benign and malignant tumours of the nasopharynx are listed in Table 49.1.

It is a rare tumour, though it is the commonest of all benign tumours of nasopharynx.

The exact cause is unknown. As the tumour is predominantly seen in adolescent males in the second decade of life, it is thought to be testosterone dependent. Such patients have a hamartomatous nidus of vascular tissue in the nasopharynx and this is activated to form angiofibroma when male sex hormone appears.

The site of origin of the tumour is still a matter of dispute. Earlier it was thought to arise from the roof of nasopharynx or the anterior wall of sphenoid bone but now it is believed to arise from the posterior part of nasal cavity close to the superior margin of sphenopalatine foramen. From here the tumour grows into the nasal cavity, nasopharynx and into the pterygopalatine fossa, running behind the posterior wall of maxillary sinus which is pushed forward as the tumour grows. Laterally, it extends into pterygomaxillary fossa and thence to fossa and cheek.

Angiofibroma, as the name implies, is made up of vascular and fibrous tissues: the ratio of the two components may vary. Mostly, the vessels are just endothelium-lined spaces with no elastic or muscle coat. This accounts for the severe bleeding as the vessels lose the ability to contract; also the bleeding cannot be controlled by application of adrenaline
(Figure 49.1).

Nasopharyngeal fibroma is a benign tumour but locally invasive and destroys the adjoining structures. It may extend into:
1. Nasal cavity causing nasal obstruction, epistaxis and nasal discharge.
2. Paranasal sinuses. Maxillary, sphenoid and ethmoid sinuses can all be invaded.
4. Orbits giving rise to proptosis and “frog-face deformity.” It enters through the inferior orbital fissure and also destroys apex of the orbit. It can also enter the orbit
through superior orbital fissure.
5. Cranial cavity. It can extend into:
(a) Anterior cranial fossa through roof of ethmoids or
plate.
(b)
Middle
cranial
fossa
through
erosion
of
floor
of
middle
cranial
fossa
or
indirectly
by
invading
the
sphenoid
sinus
and
sella
turcica.
In
the
former
case,
tumour
lies
lateral
to
internal
carotid
artery
and
in
the latter case medial to the artery.

1. Age and sex. Tumour is seen almost exclusively in males in the age group of 10-20 years. Rarely, it may be seen in older people and females.
2. Profuse, recurrent and spontaneous epistaxis. This is the most common presentation. Patient may be markedly anaemic due to repeated blood loss.
3. Progressive nasal obstruction and denasal speech. It is due to mass in the postnasal space.
4. Conductive hearing loss and otitis media with effusion. It occur due to obstruction of eustachian tube.
5.
Mass
in
the
nasopharynx.
Tumour
is
sessile,
lobulated
or
smooth
and
obstructs
one
or
both
choanae.
It
is
pink
or
purplish
in
colour.
Consistency
is
firm
but
digital
palpation
should
never
be
done
until
at
the
time
of
6. Other clinical features like broadening of nasal bridge, proptosis, swelling of cheek, infratemporal fossa or involvement of IInd, [rd, [Vth and VIth cranial nerves will depend on the extent of tumour (Figure 49.2).

e Angiofibroma
e Carcinoma nasopharynx
¢ Choanal polyp
e Lymphoma
e¢ Squamous papilloma
e Rhabdomyosarcoma
e Thornwaldt’s cyst
e Chordoma
e Pleomorphic adenoma
e Plasmacytoma
¢ Craniopharyngioma
e Haemangiopericytoma
e Paraganglioma
e Malignant salivary gland
e
Hamartoma
tumours
© Congenital tumours
e Melanoma
° Hairy © Teratoma
polyp
© Epignathi
3. Pterygomaxillary fossa, infratemporal fossa and cheek.
246

It is mostly based on clinical picture. Biopsy of the tumour is attended with profuse bleeding and is, therefore, avoided. If it is essential to differentiate it from other tumours, biopsy can be done under general anaesthesia with all arrangements to control bleeding and transfuse blood.

- 1.
Computed tomography (CT) scan of the head with contrast enhancement is now the investigation of choice (Figure 49.3). It has replaced conventional radiographs. It shows the extent of tumour, bony destruction or displacements. Anterior bowing of the posterior wall of
.
often called
antral sign
sign, is pathognomic of angiofibroma.
Magnetic resonance
imaging
(MRI)
to
CT
scans and
ent shows
intracranially any
is soft
in the
tissue infratemporal
or
Holman-Miller complementary
extensions pres-
fossa or
in the
orbit.
. Carotid angiography shows the extent of tumours, its vascularity and feeding vessels which mostly come from the external carotid system. In very large tumours or those with intracranial extension vessels may also come from internal carotid system. Embolization of vessels can be done at this time to decrease bleeding at operation. Feeders from only the external carotid system can be embolized. Resection of tumour should not be delayed beyond 2448 h of embolization to avoid vascularization from the contralateral side.
. Arrangement for blood transfusion. Though blood may not be required during surgery if successful embolization is done, 2-3 units of blood should be available and kept in reserve after grouping and cross matching.

Surgery. Surgical excision is the treatment of choice though radiotherapy and chemotherapy singly or in combination have also been used. Spontaneous regression of the tumour with advancement of age, as thought previously, does not occur and no wait and watch policy should be adopted. Surgical approaches used to remove angiofibroma, depending on its origin and extensions, are listed below.
1. Transpalatine (Figure 49.4)
2. Transpalatine + Sublabial (Sardana's approach)
3. Lateral rhinotomy with medial maxillectomy
(a) Via facial incision
(b) Via degloving approach
4. Endoscopic removal
5. Transmaxillary (Le Fort I) approach
6. Maxillary swing approach or facial translocation approach, or Wei's operation
7. Infratemporal fossa approach
8. Intracranial—extracranial approach
Transpalatal approach is used for tumours confined to the nasopharynx. It can be extended into Sardana's approach if the tumour extends laterally. These days such tumours can be removed endoscopically. Wide access to pterygomaxillary fossa can also be obtained by removing the anterior wall of maxillary sinus along with parts of pyriform aperture of nose through osteotomies and later reconstruction at the end of operation with plates. This avoids depression and of the face (Table 49.2).
to a wider access to remove tumours which extend into maxillary and ethmoid sinuses and pterygopalatine fossa. For tumours of infratemporal fossa maxillary swing approach also called facial translocation has been used. Here an osteoplastic flap with entire cheek and maxilla is raised as a single unit, which is later reconstructed. Most of the intracranial extensions are extradural and can be removed easily with the deformity Transmaxillary Le Fort I approach has also been used give

A. Nose and nasopharynx
B. Nose, nasopharynx maxillary antrum and pterygopalatine fossa
C. As in B + Infratemporal fossa
D. As in C + Cheek extension
E. As in B + C + Intracranial
F. Residual or recurrent disease (extracranial)
G. Intracranial residual or recurrent extracranial approaches but tumours extending intradurally or to the cavernous sinus require help of the neurosurgeon. Preoperative embolization of the tumour reduces its blood supply and causes less bleeding, if tumour removal is performed within 24-48 h of embolization before collaterals have time to develop. Preoperative angiography also helps to find any feeders from internal carotid system. Before the era of tumour embolization, oestrogens were used systemically to reduce blood supply. Similarly cryotherapy was also used. Recurrence of juvenile angiofibroma is not uncommon and has been reported in up to 35%.
Management of recurrent tumour. Various options used are:
- (a) Observation. The tumour may spontaneously regress.
(b)
Revision surgery
and removal.
If it
recurs even
after revision
surgery, radiotherapy
Radiation.
may be
Reduces the
subsides over
time.
rence is
surgically
Radiotherapy.
blood supply
and
It is
also used
inaccessible.
Radiotherapy mode
of treatment.
fractions has
been
A
dose is
delivered diate.
Tumour even
up of
in
3-3.5
regresses to
3
years.
extension
3000
when the
tumour tumour
used recur-
as
a
primary to
weeks.
slowly
3500
cGy
Response in
about
Radiotherapy of
disease is
in is
15-18
not
a
year, also
when tumour
used derives
for its
imme- sometimes
intracranial blood
supply from
the internal
carotid system.
Recurrent angiofibromas have also been treated by radio- therapy.

Intensity modulated
radiotherapy—a newer
mode of
treatment—may be
employed.
Treatment with
radiotherapy that
all large
tumours is
with intracranial
radiation while
Some believe
extension others
reserve should
it for
be recurrent
inoperable tumours.
Radiation to
nasopharynx in
the young
has the risk of development of malignancy at a later age.
Hormonal therapy. Since the tumour occurs in young males at puberty, probably activated by testosterone, hormonal

Transpalatal or endoscopic
Lateral rhinotomy with medial maxillectomy
OR
Endoscopic
OR
Le Fort |
Extended lateral rhinotomy
OR
Infratemporal fossa approach
OR
Maxillary swing approach
Combined intracranial and extracranial approach (craniotomy +
one of the extracranial approaches)
OR
Radiation if intracranial part is inaccessible
Observation OR repeat surgery or radiation if inaccessible
Stereotactic radiation (X or gamma knife)
(c)
250
therapy as the primary or adjunctive treatment has been used. Diethylstilbestrol and flutamide (an androgen blocker) have been used in the past to arrest the growth but no significant regression has been observed in practice.
Chemotherapy. Very aggressive recurrent tumours and resid- ual lesions have been treated by chemotherapy. Doxorubicin, vincristine and dacarbazine have been used in combination. Chemotherapy and radiotherapy can arrest the growth and cause some tumour regression but not total tumour eradication.

They are very rare and arise from the roof or lateral wall of nasopharynx. They include:
1. Congenital tumours. They are seen at birth and are six times more common in females than males. Various types include:
(a) Hairy polyp. A dermoid with skin appendages.
(b) True teratoma. Having elements derived from all the three germ layers.
(c) Epignathi. Having well-developed fetal parts.
Pleomorphic adenoma. Chordoma. Derived from the notochord. Hamartoma. Malformed normal tissue, e.g. haemangioma. Choristoma. Mass of normal tissues at an abnormal site. Paraganglioma. Oo oo

EPIDEMIOLOGY AND GEOGRAPHIC DISTRIBUTION
Nasopharyngeal cancer is a multifactorial disease. Its inci-
dence and geographic distribution depends on several fac-
tors such as genetic susceptibility, environment, diet and habits.
personal
Nasopharyngeal cancer is most common in China par ticularly in southern states and Taiwan.

Its incidence in North American whites is 0.25% of all cancers, while it is 18% in American Chinese. Chinese born in America have lesser incidence than those born in China. Burning of incense or wood (polycyclic hydrocarbon), use of preserved salted fish (nitrosamines) along with vitamin C deficient diet (vitamin C blocks nitrosification of amines and is thus protective) may be other factors operative in China.
Nasopharyngeal cancer is uncommon in India and con- stitutes only 0.41% (0.66% in males and 0.17% in females) of all cancers except in the North East region where people are predominantly of Mongoloid origin. People in Southern China, Taiwan and Indonesia are more prone to this cancer.

The exact aetiology is not known. The factors responsible
are:
1. Genetic. Chinese have a higher genetic susceptibility to nasopharyngeal cancer. Even after migration to other countries they continue to have higher incidence.
2. Viral. Epstein-Barr (EB) virus is closely associated with nasopharyngeal cancer. Specific viral markers are being developed to screen people in high-incidence areas. EB virus has two important antigens: viral capsid antigen (VCA) and early antigen (EA). IgA antibodies of EA are highly specific for nasopharyngeal cancer but have sen- sitivity of only 70-80% while IgA antibodies of VCA are more sensitive but less specific. AgA antibodies against both EA and VCA should be done for screening of for cancer.
patients nasopharyngeal
3. Environmental. Air pollution, smoking of tobacco and opium, nitrosamines from dry salted fish, smoke from burning of incense and wood have all been incriminated.

Squamous cell carcinoma in various grades of its differentia- tion or its variants as transitional cell carcinoma and lym- phoepithelioma is the most common (85%). Lymphomas constitute 10% and the rest 5% are rhabdomyosarcoma, malignant mixed salivary tumour or malignant chordoma. On the basis of histology, as seen on light microscopy, WHO has lately reclassified epithelial growths into three types (see Table 49.3).

Type | (25%)
Type Il (12%)
Keratinizing carcinoma
Nonkeratinizing differentiated carcinoma
Squamous cell carcinoma
¢ Transitional cell carcinoma
¢ Intermediate cell carcinoma
e Lymphoepithelial carcinoma (Regaud type is tumour with malignant
tissues in nests)
Type Ill (63%)
Nonkeratinizing
undifferentiated carcinoma
Anaplastic carcinoma
Clear cell carcinoma
e Lymphoepithelial carcinoma (Schminke type is tumour with diffusely
distributed malignant tissue)
¢ Spindle cell carcinoma
Note: All the types are squamous cell carcinoma when seen under electron microscope. Special stains for epithelial and lymphoid markers are differentiate them from
required to lymphomas.
Grossly, the tumour presents in three forms:
1. Proliferative. When a polypoid tumour fills the nasopharynx, it causes obstructive nasal symptoms.
2. Ulcerative. Epistaxis is the common symptom.
3. Infiltrative. Growths infiltrate submucosally.

Local spread. Commonest site of origin in the nasopharynx is the fossa of Rosenmiller. Anterior spread causes blockage of choana and nasal cavity and inferior spread is towards oropharynx and hypopharynx, lateral spread involves parapharyngeal space and infratemporal fossa through the sinus of Morgagni, upward spread is towards intracranial structures. Foramen lacerum and foramen ovale provide direct route of spread to middle cranial fossa causing diplopia or ophthalmoplegia. VIth cranial nerve is the first to be involved. Spread along the posterior skull base involves jugular foramen (CN IX, X, XI), hypoglossal canal (CN XI) or sympathetic nerve (Horner syndrome). These structures can also be involved secondary to involvement of parapharyngeal space. Involvement of pterygoid muscles causes trismus.
Lymphatic spread. Nasopharynx is rich in lymphatics and an early lymphatic spread is seen in cervical nodes. Ipsilateral nodes are involved more often but contralateral or bilateral nodes can also get involved. Lymphatic spread may be direct to these nodes or indirectly through involvement of retropharyngeal or parapharyngeal nodes. Involvement of retropharyngeal nodes also causes neck stiffness and torticollis.
Distant metastases. Lung, bone and liver are the most common sites involved.

e Age. It is mostly seen in fifth to seventh decades but may involve younger age groups. It is not uncommon to see cancer of nasopharynx in twenties and thirties.
e Sex. Males are three times more prone than females.
Symptomatology is divided into four main groups:
1. Nasal. Nasal obstruction, nasal discharge, denasal speech (rhinolalia clausa) and epistaxis.
2. Otologic. Due to obstruction of eustachian tube, there is conductive hearing loss, serous or suppurative otitis media. Tinnitus and dizziness may occur. Presence of unilateral serous otitis media in an adult should raise suspicion of nasopharyngeal growth. Rarely, tumour grows up the tube into the middle ear.
3. Ophthalmoneurologic. This occurs due to extension of tumour to the surrounding regions. Nearly all the cranial nerves may be involved.
Squint and diplopia due to involvement of CN VI, ophthalmoplegia (CN III, IV and VI), facial pain and reduced corneal reflex may (invasion of CN V through foramen lacerum) occur. Tumours may directly invade the orbit leading to exophthalmos and blindness (CN II at the apex of the orbit). Involvement of [Xth, Xth and XIth cranial nerves may occur, constituting jugular foramen syndrome. Usually, this is due to pressure of enlarged lateral retropharyngeal lymph nodes on these nerves in the neck. CN XII may be involved due to extension of growth to hypoglossal canal. Horner syndrome may occur due to involvement of cervical sympathetic chain. Nasopharyngeal cancer can cause conductive deafness (eustachian tube blockage), ipsilateral temporoparietal neuralgia (involvement of CN V) and palatal paralysis (CN X)— collectively called Trotter's triad.
Figure
49.5
Routes of spread (green area) and clinical features (blue area) of nasopharyngeal cancer.

4. Cervical nodal metastases. This may be the only manifestation of nasopharyngeal cancer. A lump of nodes is found between the angle of jaw and the mastoid and some nodes along the spinal accessory in the posterior triangle of neck. Nodal metastases are seen in 75% of the patients, when first seen, about half of them with bilateral nodes.
5. Distant metastases involve bone, lung, liver and other sites. Distant metastases may be present at the time of diagnosis. Presenting symptoms and signs of nasopharyngeal cancer in order of frequency are:
Cervical lymphadenopathy (most common) (60-90%)
Epistaxis
Cranial nerve
of these
Headache
Earache
Neck pain

1. Endoscopic evaluation. This can be done under local anaesthesia using rigid or flexible endoscopes. Growth may be proliferative, ulcerative or infiltrative submucosal. Biopsy can be taken.
2. Imaging studies
(a) CT scan/MRI nasopharynx and neck. High-resolution, contrast-enhanced CT of neck and nasopharynx is the study of choice. It reveals primary growth, erosion of skull base and clivus, extensions to parapharyngeal, retropharyngeal and intracranial regions. Neck nodes can also be seen. MRI is better for softtissue extension.
(b) X-ray/CT chest for secondaries lung.
(c) CT abdomen or ultrasound abdomen for secondaries liver.
(d) Positron emission tomography scan. It is getting popular to show metastases anywhere in the body.
3. Biopsy. It can be done under local or general anaesthesia using endoscopes. In case growth is not visible, but highly suspected because of metastatic nodes, blind biopsies from multiple sites in nasopharynx can be taken. A strip of mucosa from fossa of Rosenmuller or posterior wall of nasopharynx can be taken. It may require transpalatal exposure of nasopharynx. General anaesthesia is preferred if occult primary is suspected.
4. Audiogram. A baseline audiogram is important. It not only establishes diagnosis of serous otitis media but is also important for side effects of radiation and chemotherapy which can cause sensorineural hearing loss.

WHO classified nasopharyngeal carcinoma on histopathological basis into three types (Table 49.3). Type III is the most common in North America. However, the frequency of different histopathological types may differ from country to country. These types have also been correlated to titres of EB virus and also in their response to radiotherapy. It is palsies.
CN
VI
paralysis is
the most
common observed that type II and type III are associated with higher titres of EB virus and have higher local control rates with radiotherapy.

1. Radiotherapy. It is the treatment of choice for nasopha- ryngeal cancer. Stage I and II are treated by radiotherapy alone while stage III and IV require concomitant radia- tion and chemotherapy or radiation followed by chemo- therapy. External beam radiation of 6000-7000 cGy can be delivered by linear accelerator to the primary and both sides of neck. More advanced techniques of radiotherapy such as three-dimensional conformal radiotherapy and intensity modulated radiotherapy (IMRT) are now being used more and more. They allow higher dose delivery to the tumour with reduced damage to the adjacent nor- mal structures such as spinal cord, brainstem and parotid glands. IMRT has also been used for recurrent disease where conventional radiotherapy produces more serious side effects such as transverse myelitis.
2. Chemotherapy. Some stage III and IV cancers of naso- pharynx can be cured by radiotherapy alone but cure rate is doubled when chemotherapy is combined with radiotherapy. Chemotherapy can be given concomitantly or postradiotherapy. Cisplatin or cisplatin with 5-FU have been used. Chemotherapy has also been found useful to control metastases from lymphoepithelioma and undif- ferentiated carcinoma of nasopharynx. Goal of chemo- radiotherapy in nasopharyngeal carcinoma is to improve local control of tumour and to treat distant metastases.
3.
Treatment
of
recurrent
and
residual
(persistent)
disease.
This
can
occur
in
neck
nodes
or
in
the
(a) Positive nodes in the neck. They require radical neck dis- section with removal of sternocleidomastoid muscle, CN XI and internal jugular vein. Modified neck dis- section is not preferred as extensive disease has been seen on histopathology even when only a single node was present. Bilateral neck disease may require bilat- eral neck dissection but with preservation of internal jugular vein to avoid cerebral and facial oedema.
Before treating positive nodes in the neck one should make sure that no disease exists in the nasopharynx and there are no distant metastases.

(b) Recurrent or residual (persistent) disease in the nasophar- ynx. First it should be evaluated by CT and MRI to see the size, location and regional extent or infiltration. It can be treated by
(i) Second course of external radiation. IMRT has been used. Second course of radiation is more hazardous and causes injury to brainstem, eye, ear, pituitary gland and temporal lobe.
(ii) Brachytherapy. It can deliver high dose to the tumour with less radiation to the surrounding structures. Gold grains (Gold 198) have been used.
(iii) Nasopharyngectomy. It can be done by various ways such as by (i) endoscopic approach, (ii) lateral rhinotomy and medial maxillectomy, (iii) maxillary swing or (iv) Le Fort I approach.
Before undertaking nasopharyngectomy exclude extension of growth intracranially, to parapharyngeal space, or around the internal carotid artery.

Figure 49.6 Supraclavicular fossa (or Ho's triangle) is bounded by medial (A) and lateral (B) ends of clavicle and the point (C) where neck
meets the shoulder. It includes caudal portions of levels IV and V.

1. Lymphomas. Non-Hodgkin’s type is more common than Hodgkin’s Almost all B-cell T-cell lympho-
type. are type. mas are seen in Asian population.
2. Rhabdomyosarcoma. Commonly seen in children. Embryonal rhabdomyosarcoma presents as a polypoid mass in the nasopharynx.
3. Plasmacytoma. It may be solitary or part of generalized
multiple myelomatosis.
4. Chordoma (from remnant of notochord).
Adenoid cystic carcinoma (from minor salivary glands). an
6. Melanoma (rare).

Acute pharyngitis is very common and occurs due to varied aetiological factors like viral, bacterial, fungal or others (Table 50.1). Viral causes are more common. Acute streptococcal pharyngitis (due to Group A beta-haemolytic streptococci) has received more importance because of its aetiology in rheumatic fever and_poststreptococcal glomerulonephritis.

Pharyngitis may occur in different grades of severity. Milder infections present with discomfort in the throat, some malaise and low-grade fever. Pharynx in these cases is congested but there is no lymphadenopathy. Moderate and severe infections present with pain in throat, dysphagia, headache, malaise and high fever. Pharynx in these cases shows erythema, exudate and enlargement of tonsils and lymphoid follicles on the posterior pharyngeal wall. Very severe cases show oedema of soft palate and uvula with enlargement of cervical nodes. It is not possible, on clinical examination, to differentiate viral from bacterial infections but, viral infections are generally mild and are accompanied by rhinorrhoea and hoarseness while the bacterial ones are severe. Gonococcal pharyngitis is mild and may even be asymptomatic.

Culture of throat swab is helpful in the diagnosis of bacterial pharyngitis. It can detect 90% of Group A streptococci. Diphtheria is cultured on special media. Swab from a suspected case of gonococcal pharyngitis should be cultured immediately without delay. Failure to get any bacterial growth suggests a viral aetiology.

1. General measures. Bed rest, plenty of fluids, warm saline gargles or pharyngeal irrigations and analgesics form the mainstay of treatment.
Local discomfort in the throat in severe cases can be relieved by lignocaine viscous before meals to facilitate swallowing.
2. Specific treatment. Streptococcal pharyngitis (Group A, beta-haemolyticus) is treated with penicillin G, 200,000 to 250,000 units orally four times a day for 10 days or benzathine penicillin G, 600,000 units once i.m. for patient, 60 lb in weight and 1.2 million units once i.m. for patient >60 Ib. In penicillin-sensitive individuals, erythromycin, 20-40 mg/kg body weight daily, in divided oral doses for 10 days is equally effective. Diphtheria is treated by diphtheria antitoxin and administration of penicillin or erythromycin (see p. 260). Gonococcal pharyngitis responds to conventional doses of penicillin or tetracycline.

1. Herpangina. It is caused by Group A coxsackie virus and mostly affects children. Characteristic features include fever, sore throat and vesicular eruption on the soft palate and pillars. Vesicles are small and surrounded by a zone of erythema.
2. Infectious mononucleosis. It is caused by Epstein-Barr virus. It affects older children and young adults, and is characterized by fever, sore throat, exudative pharyngitis, lymphadenopathy, splenomegaly and hepatitis.
3. Cytomegalovirus. It mostly affects immunosuppressed transplant patients. Clinically, it mimics infectious mononucleosis but heterophil antibody test is negative.
4. Pharyngoconjunctival fever. It is caused by an adenovirus and is characterized by sore throat, fever and conjunctivitis. There may be pain in abdomen, mimicking appendicitis.
5. Acute lymphonodular pharyngitis. It is usually caused by a coxsackie virus and characterized by fever, malaise and sore throat. White-yellow solid nodules appear on the posterior pharyngeal wall in this type of pharyngitis.
6. Measles and chickenpox also cause pharyngitis. Measles is characterized by the appearance of Koplik's spots (white spots surrounded by red areola) on the buccal mucosa opposite the molar teeth. The spots appear 3-4 days before the appearance of rash.

Candida infection of the oropharynx can occur as an extension of oral thrush. It is seen in patients who are immunosuppressed, debilitated or taking high doses of antimicrobials. Often patient complains of pain in the throat with dysphagia. Nystatin is the drug of choice.

Chlamydia trachomatis infection causes acute pharyngitis and can be treated by erythromycin or sulfonamides.

e
Rhinoviruses
°
Streptococcus
(Group
A,
© Candida albicans
Toxoplasmosis (parasitic, rare)
e Influenza
beta-haemolyticus)
° Chlamydia trachomatis
e Parainfluenza
e Diphtheria
e Measles and chickenpox
° Gonococcus
°
Coxsackie
virus
e
Herpes
simplex
¢
Infectious
mononucleosis
° Cytomegalovirus
Toxoplasmosis is caused by Toxoplasma gondii, an obligate intracellular parasite. This infection is very rare.

It is a chronic inflammatory condition of the pharynx. Pathologically, it is characterized by hypertrophy of mucosa, seromucinous glands, subepithelial lymphoid follicles and even the muscular coat of the pharynx.
Chronic pharyngitis is of two types:
1. Chronic catarrhal pharyngitis.
2. Chronic hypertrophic (granular) pharyngitis.

A large number of factors are responsible:
1. Persistent infection in the neighbourhood. In chronic rhinitis and sinusitis, purulent discharge constantly trick- les down the pharynx and provides a constant source of infection. This causes hypertrophy of the lateral pharyn- geal bands.
chronic
tonsillitis
and
dental
sepsis
are
also
for
chronic
pharyngitis
and
recurrent
sore
throats.
2. Mouth breathing. Breathing through the mouth exposes the pharynx to air which has not been filtered, humidi- fied and adjusted to body temperature thus making it more susceptible to infections. Mouth breathing is due to:
(a) Obstruction in the nose, e.g. nasal polypi, allergic or vasomotor rhinitis, turbinal hypertrophy, deviated septum or tumours.
(b) Obstruction in the nasopharynx, e.g. adenoids and tumours.
(c) Protruding teeth which prevent apposition of lips.
(d) Habitual, without any organic cause.
3. Chronic irritants. Excessive smoking, chewing of tobacco
and
pan,
heavy
drinking
or
highly
spiced
food
can
all
lead
to
chronic
4. Environmental pollution. Smoky or dusty environment or irritant industrial fumes may also be responsible for chronic pharyngitis.
5. Faulty voice production. Less often realized but an impor- tant cause of chronic pharyngitis is the faulty voice produc- tion. Excessive use of voice or faulty voice production seen in certain professionals or in “pharyngeal neurosis” where
person resorts to constant throat clearing, hawking or snorting, and that may cause chronic pharyngitis, especially of hypertrophic variety.

Severity of symptoms in chronic pharyngitis varies from per- son to person.
1. Discomfort or pain in the throat. This is especially noticed in the mornings.
2. Foreign body sensation in throat. Patient has a constant desire to swallow or clear his throat to get rid of this “foreign body.”
3. Tiredness of voice. Patient cannot speak for long and has to make undue effort to speak as throat starts aching. The voice may also lose its quality and may even crack.
4. Cough. Throat is irritable and there is tendency to cough. Mere opening of the mouth may induce retching or gagging.

1. Chronic catarrhal pharyngitis. In this, there is a conges- tion of posterior pharyngeal wall with engorgement of ves- sels; faucial pillars may be thickened. There is increased mucus secretion which cover mucosa.
may pharyngeal
2. Chronic hypertrophic (granular) pharyngitis
(a) Pharyngeal wall appears thick and oedematous with congested mucosa and dilated vessels.
(b) Posterior pharyngeal wall may be studded with red-
dish
nodules
(hence
the
term
granular
pharyngitis).
These
nodules
are
due
to
hypertrophy
of
subepi-
thelial
lymphoid
follicles
normally
seen
in
pharynx
(Figure 50.1).
(c) Lateral pharyngeal bands become hypertrophied.
(d) Uvula may be elongated and appear oedematous.

1. In every case of chronic pharyngitis, aetiological factor should be sought and eradicated.
2. Voice rest and speech therapy is essential for those with faulty voice production. Hawking, clearing the throat fre- quently or any other such habit should be stopped.
3. Warm saline gargles, especially in the morning, are sooth- ing and relieve discomfort.
Granular Note: Reddish nodules on the
Figure 50.1 pharyngitis. posterior pharyngeal wall.
» Mandl's paint may be applied to pharyngeal mucosa.
5. Cautery of lymphoid granules is suggested. Throat is
sprayed with local anaesthetic and granules are touched with
10-25%
silver nitrate.
Electrocautery or
diathermy of nodules may require general anaesthesia.

It is a form of chronic pharyngitis often seen in patients of atrophic rhinitis. Pharyngeal mucosa along with its mucous glands shows atrophy. Scanty mucus production by glands leads to formation of crusts, which later get infected giving rise to foul smell.

Dryness and discomfort in throat are the main complaints. Hawking and dry cough may be present due to crust formation. Examination shows dry and glazed pharyngeal mucosa often covered with crusts.

This is same as for coexistent atrophic rhinitis. Aim is to remove the crusts and promote secretion. The crusts can be removed by spraying the throat with alkaline solution, or pharyngeal irrigation. MandlI's paint applied locally has a soothing effect. Potassium iodide, 325 mg, administered orally for a few days helps to promote secretion and prevent crusting.

It is a benign condition characterized by horny excrescences on the surface of tonsils, pharyngeal wall or lingual tonsils. They appear as white or yellowish dots. These excrescences are the result of hypertrophy and keratinization of epithelium. They are firmly adherent and cannot be wiped off. There is no accompanying inflammation nor any constitutional symptoms and thus can be easily differentiated from acute follicular tonsillitis. The disease may show spontaneous regression and may not require any specific treatment except for reassurance to the patient.

The tonsil is supplied by five arteries (Figure 51.3).
. Tonsillar branch of facial artery. This is the main artery. . Ascending pharyngeal artery from external carotid. Oo DO
. Ascending palatine, a branch of facial artery.
. Dorsal linguae branches of lingual artery. B®
. Descending palatine branch of maxillary artery. COU

Veins from the tonsils drain into paratonsillar vein which joins the common facial vein and pharyngeal venous plexus.

Lymphatics from the tonsil pierce the superior constrictor and drain into upper deep cervical nodes particularly the jugulodigastric (tonsillar) node situated below the angle of mandible.

Lesser palatine branches of sphenopalatine ganglion (CN V) and glossopharyngeal nerve provide sensory nerve supply.

They act as sentinels to guard against foreign intruders like viruses, bacteria and other antigens coming into contact through inhalation and ingestion. There are two mechanisms:
1. Providing local immunity.
2. Providing a surveillance mechanism so that entire body is prepared for defence.
Both these mechanisms are operated through humoral and cellular immunity.
1. Local immunity. Tonsils and adenoids are lined by squamous epithelium, surface area of which is further increased by several crypts of tonsils and folds of adenoid. This epithelium is specialized and contains M-cells, antigen processing cells and micropores. Through them antigenic material is brought into contact with subepithelially situated lymphoid follicles. Follicles have a germinal centre rich in B-cells and a mantel zone rich in large lymphocytes. B-cells when stimulated change to plasma cells and produce antibodies. Bacteria and viruses are also phagocytosed by the macrophages
and destroyed. Low-dose antigens and chronic infections are dealt with in this manner.
2. Surveillance mechanism. It identifies the intruder and alerts the body for wider response. If the dose of antigen is high, B-cells of the germinal centre proliferate and undergo hyperplasia and also enter the blood stream. Complex
Figure 51.1 Primary and secondary crypts of tonsils.
Figure 51.2 Relations of tonsil. Tonsil is related laterally to its capsule (1), loose areolar tissue containing paratonsillar vein (2), superior constrictor muscle (3), styloglossus (4), glossopharyngeal nerve (5), facial artery (6), medial pterygoid muscle (7), angle of mandible (8) and submandibular salivary gland (9), pharyngobasilar fascia (10), bucco- pharyngeal fascia (11).
Figure
51.3
Arterial supply of tonsil.

immune system comes into play with antigen processing cells, memory cells, dendritic cells, macrophages, T-helper and T-suppressor cells. Antibodies produced by the plasma cells prepare the antigen to be phagocytosed by neutrophils and other phagocytes. The antibodies also get attached to macrophages and give them enhanced ability to catch the antigens.
Tonsils are most active from 4 to 10 years of age. Involution begins after puberty resulting in decrease of B-cell production and relative increase in ratio of T to B cells. There has been a common notion that removal of tonsils and adenoids will impair the integrity of immune system and make the patient susceptible to poliovirus or increase the incidence of Hodgkin disease in them. This has not been substantiated by clinical and epidemiological observations. Removal of tonsil and adenoid has also not affected general immune surveillance function. Tonsil and adenoid, however, should only be removed on specific indications.

Primarily, the tonsil consists of (i) surface epithelium which is continuous with the oropharyngeal lining, (ii) crypts which are tube-like invaginations from the surface epithelium and (iii) the lymphoid tissue. Acute infections of tonsil may involve these components and are thus classified as:
1. Acute catarrhal or superficial tonsillitis. Here tonsillitis is a part of generalized pharyngitis and is mostly seen in viral infections.
2. Acute follicular tonsillitis. Infection spreads into the crypts which become filled with purulent material, presenting at the openings of crypts as yellowish spots (Figure 51.4). 3. Acute parenchymatous tonsillitis. Here tonsil substance is affected. Tonsil is uniformly enlarged and red.
4. Acute membranous tonsillitis. It is a stage ahead of acute follicular tonsillitis when exudation from the crypts coalesces to form a membrane on the surface of tonsil.
Figure 51.4 Acute follicular tonsillitis.

Acute tonsillitis often affects school-going children, but also affects adults. It is rare in infants and in persons who are above 50 years of age.
Haemolytic streptococcus is the most commonly infecting organism. Other causes of infection may be staphylococci, pneumococci or H. influenzae. These bacteria may primarily infect the tonsil or may be secondary to a viral infection.

The symptoms vary with severity of infection. The predominant symptoms are:
1. Sore throat.
2. Difficulty in swallowing. The child may refuse to eat anydue to local thing pain.
3. Fever. It may vary from 38 to 40°C and may be associated with chills and rigors. Sometimes, a child presents with an unexplained fever and it is only on examination that an acute tonsillitis is discovered.
4. Earache. It is either referred pain from the tonsil or the result of acute otitis media which may occur as a
complication.
5. Constitutional symptoms. They are usually more marked than seen in simple pharyngitis and may include headache, general body aches, malaise and constipation. There may be abdominal pain due to mesenteric lymphadenitis simulating a clinical picture of acute appendicitis.

1. Often the breath is foetid and tongue is coasted.
2. There is hyperaemia of pillars, soft palate and uvula.
3. Tonsils are red and swollen with yellowish spots of purulent material presenting at the opening of crypts (acute follicular tonsillitis) or there may be a whitish membrane on the medial surface of tonsil which can be easily wiped away with a swab (acute membranous tonsillitis, Figure 51.5). The tonsils may be enlarged and congested so much so that they almost meet in the midline along with some
Figure 51.5 Acute follicular tonsillitis. Note pus beads on the surface of left tonsil. On the right pus beads have coalesced together to form a membrane.
oedema of the uvula and soft palate (acule parenchymatous tonsillitis).
- 4. The jugulodigastric lymph nodes are enlarged and tender.

1. Patient is put to bed and encouraged to take plenty of fluids.
2. Analgesics (aspirin or paracetamol) are given according to the age of the patient to relieve local pain and bring down the fever.
3. Antimicrobial therapy. Most of the infections are due to Streptococcus and penicillin is the drug of choice. Patients allergic to penicillin can be treated with erythro-
mycin. Antibiotics should be continued for 7-10 days.

1. Chronic tonsillitis with recurrent acute attacks. This is due to incomplete resolution of acute infection. Chronic infection may persist in lymphoid follicles of the tonsil in the form of microabscesses.
Peritonsillar abscess. po
. Parapharyngeal abscess. go
Cervical abscess due to suppuration of jugulodigastric lymph nodes. A
5. Acute otitis media. Recurrent attacks of acute otitis media may coincide with recurrent tonsillitis.
6.
Rheumatic
fever.
Often
seen
in
with
due
to
Group
A
beta-haemolytic
Streptococci.
Acute glomerulonephritis. Rare these days. on
. Subacute bacterial endocarditis. Acute tonsillitis in a patient
with
valvular
heart
disease
may
be
by
endo-
carditis.
It
is
usually
due
to
viridans

1. Membranous tonsillitis. It occurs due to pyogenic organisms. An exudative membrane forms over the medial surface of the tonsils, along with the features of acute tonsillitis.
2. Diphtheria. Unlike acute tonsillitis which is abrupt in onset, diphtheria is slower in onset with less local discomfort, the membrane in diphtheria extends beyond the tonsils, on to the soft palate and is dirty grey in colour. It is adherent and its removal leaves a bleeding surface. Urine may show albumin. Smear and culture of throat swab will reveal
Corynebacterium diphtheriae.
3. Vincent angina. It is insidious in onset with less fever and less discomfort in throat. Membrane, which usually forms over one tonsil, can be easily removed revealing an irregular ulcer on the tonsil. Throat swab will show both the organisms typical of disease, namely fusiform bacilli and spirochaetes.
4. Infectious mononucleosis. This often affects young adults. Both tonsils are very much enlarged, congested and covered with membrane. Local discomfort is marked. Lymph nodes are enlarged in the posterior triangle of neck along with splenomegaly. Attention to disease is attracted because of failure of the antibiotic treatment. Blood smear may show
—
more than 50% lymphocytes, of which about 10% are atypical. White cell count may be normal in the first week but rises in the second week. Paul-Bunnell test (mono test) will show high titre of heterophil antibody.
5. Agranulocytosis. It presents with ulcerative necrotic lesions not only on the tonsils but elsewhere in the oropharynx. Patient is severely ill. In acute fulminant form, total leucocytic count is decreased to <2000/cu mm or even as low as 50/cu mm and polymorph neutrophils may be reduced to 5% or less. In chronic or recurrent form, total count is reduced to 2000/cu mm with less marked granulocytopenia.

6. Leukaemia. In children, 75% of leukaemias are acute lymphoblastic and 25% acute myelogenous or chronic, while in adults 20% of acute leukaemias are lymphocytic and 80% nonlymphocytic.
Peripheral blood shows TLC >100,000/cu mm. It may be normal or less than normal. Anaemia is always present and may be progressive. Blasts cells are seen on examination of the bone marrow.
7. Aphthous ulcers. They may involve any part of oral cavity or oropharynx. Sometimes, it is solitary and may involve the tonsil and pillars. It may be small or quite large and alarming. It is very painful.
8. Malignancy tonsil (see p. 269).
9. Traumatic ulcer. Any injury to oropharynx heals by formation of a membrane. Trauma to the tonsil area may occur accidently when hit with a toothbrush, a pencil held in mouth or fingering in the throat. Membrane appears within 24 h.

Diagnosis of ulceromembranous lesion of throat thus requires:
1. History.
2. Physical examination.
3. Total and differential counts (for agranulocytosis, leu-
kaemia, neutropenia, infectious mononucleosis).
4. Blood smear (for atypical cells).
5. Throat swab and culture (for pyogenic bacteria, Vincent angina, diphtheria candidal infection).
Bone marrow aspiration or needle biopsy. No
Other tests. Paul-Bunnell or mono spot test and biopsy
of the lesion.

It is an acute specific infection caused by the Gram-positive bacillus, C. diphtheriae. It spreads by droplet infection. Incubation period is 2-6 days. Some persons are "carriers" of this disease, i.e. they harbour organisms in their throat but have no symptoms.

Children are affected more often though no age group is immune. Oropharynx is commonly involved and the larynx and nasal cavity may also be affected.
In the oropharynx, a greyish white membrane forms over the tonsils and spreads to the soft palate and posterior pharyngeal wall. It is quite tenacious and causes bleeding when removed. Cervical lymph nodes, particularly the jugulodigastric, become enlarged and tender, sometimes presenting a "bull-neck" appearance. Patient is ill and toxaemic but fever seldom rises above 38°C.

Exotoxin produced by C. diphtheriae is toxic to the heart and nerves. It causes myocarditis, cardiac arrhythmias and acute cir- culatory failure.
Neurological complications usually appear a few weeks after infection and include paralysis of soft palate, diaphragm and ocular muscles.
In the larynx, diphtheritic membrane may cause airway obstruction.

Treatment of diphtheria is started on clinical suspicion without waiting for the culture report. Aim is to neutralize the free exotoxin still circulating in the blood and to kill the organisms producing this exotoxin. Dose of antitoxin is based on the site involved and the duration and severity of disease. It is 20,000-40,000 units for diphtheria in less than 48 h, or when the membrane is confined to the tonsils only; and 80,000-120,000 units, if disease has lasted longer than 48 h, or the membrane is more extensive. Antitoxin is given by iv. infusion in saline in about 60 min. Sensitivity to horse serum should be tested by conjunctival or intracutaneous test with diluted antitoxin and adrenaline should be at hand for any immediate hypersensitivity. In the presence of hypersensitivity reaction, desensitization should be done. Antibiotics used are benzyl penicillin 600 mg 6 hourly for 7 days. Erythromycin is used in penicillin-sensitive individuals (500 mg 6 hourly orally).

1. It may be a complication of acute tonsillitis. Pathologi-
cally,
microabscesses
walled
off
by
fibrous
tissue
have
been
seen
in
the
lymphoid
follicles
of
the
tonsils.
2. Subclinical infections of tonsils without an acute attack.
3. Mostly affects children and young adults. Rarely occurs after 50 years.
4. Chronic infection in sinuses or teeth may be a predispos- factor.
ing

1. Chronic follicular tonsillitis. Here tonsillar crypts are full of infected cheesy material which shows on the surface as yellowish spots.
2. Chronic parenchymatous tonsillitis. There is hyperpla- sia of lymphoid tissue. Tonsils are very much enlarged
Figure 51.6 Parenchymatous tonsillitis. The two tonsils are almost touching each other causing problems of deglutition, speech and respiration.
and may interfere with speech, deglutition and respira- tion (Figure 51.6). Attacks of sleep apnoea may occur.
Long-standing cases develop features of cor pulmonale.
3. Chronic fibroid tonsillitis. Tonsils are small but infected, with history of repeated sore throats.

1. Recurrent attacks of sore throat or acute tonsillitis.
2. Chronic irritation in throat with cough.
3. Bad taste in mouth and foul breath (halitosis) due to pus in crypts.
4. Thick speech, difficulty in swallowing and choking spells at night (when tonsils are large and obstructive).

1. Tonsils may show varying degree of enlargement. Some- times they meet in the midline (chronic parenchymatous type).
2. There may be yellowish beads of pus on the medial sur- face of tonsil (chronic follicular type).
3.
Tonsils
are
small
but
pressure
on
the
anterior
pillar
expresses
frank
pus
or
cheesy
material
(chronic
fibroid
type).
4.
Flushing
of
anterior
pillars
compared
to
the
rest
of
the
pharyngeal
mucosa
is
an
important
sign
of
chronic
ton-
sillar
5. Enlargement of jugulodigastric lymph nodes is a reliable sign of chronic tonsillitis. During acute attacks, the nodes enlarge further and become tender.

1. Conservative treatment consists of attention to general health, diet, treatment of coexistent infection of teeth, nose and sinuses.
2. Tonsillectomy is indicated when tonsils interfere with
speech,
deglutition
and
or
cause
recurrent
attacks
(see
Chapter
93).
Figure 51.7 A tonsillar cyst right side in a 55-year-old male (arrow).

- . Peritonsillar abscess. Ne
. Parapharyngeal abscess.
- . Intratonsillar abscess. OO
. Tonsilloliths.
- . Tonsillar cyst.
ok
- . Focus of infection in rheumatic fever, acute glomerulonephritis, eye and skin disorders. a
Tonsilloliths (calculus of the tonsil). It is seen in chronic tonsil- litis when its crypt is blocked with retention of debris. Inor-
ganic salts
of calcium
and magnesium
are then
deposited leading to formation of a stone. It may gradually enlarge and then ulcerate through the tonsil.
Tonsilloliths are seen more often in adults and give rise to local
discomfort or
foreign body
sensation.
They are
by palpation
ment is
simple be
indicated or
removal for
which cannot
be
Intratonsillar removed.
abscess.
substance of
It is
tonsil.
opening accumulation
It usually
in pain
and ment
acute follicular
gritty feeling
the on
stone probing.
or tonsillectomy,
sepsis or
for the
deeply
Treat- if
that set
of stone
pus follows
within blocking
dysphagia.
Tonsil is
administration abscess
of the
the
There appears
is crypt
marked swollen
of if
required;
later
Tonsillar cyst.
It is
due appears
as
a
yellowish antibiotics
and local
red.
and tonsillectomy
Treat- drainage
should to
blockage of
swelling over
of the
be performed.
a
tonsillar the
tonsil.
crypt and
Very symptomless.
It can
be easily
drained
(Figure often
51.7).

1.
Acute
lingual
Acute
infection
of
a
lingual
ton-
sil
gives
rise
to
dysphagia
and
feeling
of
lump
in
the
throat.
On
with
a
laryngeal
mirror,
lingual
tonsil
may
appear
enlarged
and
congested,
some-
times
studded
with
follicles
like
the
ones
seen
in
acute
fol-
licular
Cervical
lymph
nodes
may
be
enlarged.
Treatment
is
by
antibiotics.
2.
Hypertrophy
of
lingual
tonsils.
Mostly,
it
is
a
compen-
satory
of
lymphoid
tissue
in
response
to
repeated
in
tonsillectomized
patients.
Usual
it is
unilateral dysphagia, pain in the tongue, excessive salivation and some degree of trismus. Protrusion of the tongue is painful. Jugulodigastric nodes will be enlarged and tender. It is a potentially dangerous condition as laryngeal oedema can easily follow.
Diagnosis is made by mirror examination and palpation of the base of tongue. Treatment is by antibiotics, analgesics, proper hydration and incision and drainage of the abscess.

It is suppuration of the parotid space. Deep cervical fascia splits into two layers, superficial and deep, to enclose the parotid gland and its associated structures. Parotid space lies deep to its superficial layer.
Contents of parotid space include parotid gland and its associated parotid lymph nodes, facial nerve, external carotid artery and retromandibular vein. Fascial layer is very thick superficially but very thin on the deep side of the parotid gland where parotid abscess can burst to form a parapharyngeal abscess and thence spread to the mediastinum.

Dehydration, particularly in postsurgical cases and debilitated patients, with stasis of salivary flow is the predisposing cause. Infection from the oral cavity travels via the Stenson's duct to invade the parotid gland. Multiple small abscesses may form in the parenchyma. They may then coalesce to form a single abscess.

Most common organism is Staphylococcus aureus but Streptococci, anaerobic organisms and rarely the Gram-negative have been cultured.
organisms

Usually follows 5-7 days after operation. There is swelling, redness, indurations and tenderness in the parotid area and at the angle of mandible. Parotid abscess is usually unilateral, but bilateral abscesses may occur. Fluctuation is difficult to elicit due to thick capsule. Opening of the Stenson's duct becomes congested and may exude pus on pressure over the parotid. Patient is toxic, running high fever and dehydrated.

Diagnosis of the abscess can be made by ultrasound or computed tomography scan. More than one loculi of pus may be seen. Aspiration of abscess can be done for culture and sensitivity of the causative organisms.

Correct the dehydration, improve oral hygiene and promote salivary flow. Intravenous antibiotics are instituted.
Surgical drainage under local or general anaesthesia is carried out by a preauricular incision as employed for parotidectomy. Skin flap is raised to expose surface of the gland, and the abscess or abscesses are bluntly opened working parallel to the branches of the VIIth nerve. Skin incision is loosely approximated over a drain and allowed to heal by secondary intention.

Submandibular space lies between mucous membrane of the floor of mouth and tongue on one side and superficial layer of deep cervical fascia extending between the hyoid bone and mandible on the other (Figure 52.1). It is divided into two compartments by the mylohyoid muscle:
1. Sublingual compartment (above the mylohyoid).
2. Submaxillary and submental compartment (below the mylohyoid).
The two compartments are continuous around the posterior border of mylohyoid muscle.
Ludwig's angina is infection of submandibular space.

1. Dental infections. They account for 80% of the cases. Roots of premolars often lie above the attachment of mylohyoid and cause sublingual space infection while roots of the molar teeth extend up to or below the mylohyoid line and primarily cause submaxillary space infection (Figure 52.2).
2. Submandibular sialadenitis, injuries of oral mucosa and fractures of the mandible account for other cases.

Mixed infections involving both aerobes and anaerobes are common. Alpha-haemolytic Streptococci, Staphylococci and bacteroides groups are common. Rarely Haemophilus influenzae, Escherichia coliand Pseudomonas are seen.

There is marked difficulty in swallowing (odynophagia) with varying degrees of trismus. When infection is localized to the sublingual space, structures in the floor of mouth are swollen and tongue seems to be pushed up and back.
Figure 52.2 Roots of molar teeth project below and those of premolars above the attachment of mylohyoid muscle.
When infection spreads to submaxillary space, submental and submandibular regions become swollen and tender, and impart woody-hard feel. Usually, there is cellulitis of the tissues rather than frank abscess. Tongue is progressively pushed upwards and backwards threatening the airway.
Laryngeal oedema may appear (Figure 52.3).

1. Systemic antibiotics.
2. Incision and drainage of abscess.
(a) Intraoral—if infection is still localized to sublingual
space.
- (b) External—if infection involves submaxillary space. A transverse incision extending from one angle of mandible to the other is made with vertical opening of midline musculature of tongue with a blunt haemostat. Very often it is serous fluid rather than frank that is encountered.
pus
3. Tracheostomy, if airway is endangered.

1. Spread of infection to parapharyngeal and retropharyngeal spaces and thence to the mediastinum.
Figure 52.3 Ludwig's angina in a 7-year-old child.
- 2. Airway obstruction due to laryngeal oedema, or swelling
and pushing back of the tongue.
3. Septicaemia.
4. Aspiration pneumonia.

It is a collection of pus in the peritonsillar space which lies between the capsule of tonsil and the superior constrictor muscle.

Peritonsillar abscess usually follows acute tonsillitis though it may arise de novo without previous history of sore throats. First, one of the tonsillar crypts, usually the crypta magna, gets infected and sealed off. It forms an intratonsillar abscess which then bursts through the tonsillar capsule to set up peritonsillitis and then an abscess.
Culture of pus from the abscess may reveal pure growth of Streptococcus pyogenes, S. aureus or anaerobic organisms. More often the growth is mixed, with both aerobic and anaerobic organisms.

Peritonsillar abscess mostly affects adults and rarely the children though acute tonsillitis is more common in children. Usually, it is unilateral though occasionally bilateral abscesses are recorded. Clinical features are divided into:
- 1. General. They are due to septicaemia and resemble any acute infection. They include fever (up to 104°F), chills and rigors, general malaise, body aches, headache, nausea and constipation.

(a) Severe pain in throat. Usually unilateral.
(b) Odynophagia. It is so marked that the patient cannot even swallow his own saliva which dribbles from the angle of his mouth. Patient is usually dehydrated.
(c) Muffled and thick speech, often called "hot potato voice."
(d) Foul breath due to sepsis in the oral cavity and poor hygiene.
(e) Ipsilateral earache. This is referred pain via CN IX
which supplies both the tonsil and the ear.
(f) Trismus due to spasm of pterygoid muscles which are
in close proximity to the superior constrictor.

1.
The
tonsil,
pillars
and
soft
palate
on
the
involved
side
are
congested
and
swollen.
Tonsil
itself
may
not
appear
enlarged
as
it
gets
buried
in
the
oedematous
pillars
(Figure
52.4).
2. Uvula is swollen and oedematous and pushed to the opposite side.
3. Bulging of the soft palate and anterior pillar above the tonsil.
4. Mucopus may be seen covering the tonsillar region.
5. Cervical lymphadenopathy is commonly seen. This involves jugulodigastric lymph nodes.
6. Torticollis. Patient keeps the neck tilted to the side of abscess.

1. Hospitalization.
2. Intravenous fluids to combat dehydration.
3.
Antibiotics.
Suitable antibiotics
in large
i.v.
doses to
cover both aerobic and anaerobic organisms.
- 4. Analgesics like paracetamol are given for relief of pain
and to
lower the
temperature.
Sometimes, stronger
anal- gesics
like pethidine
may be
required.
Aspirin is
avoided because
of the
danger of
bleeding.
- 5. Oral hygiene should be maintained by hydrogen peroxide or saline mouth washes.
The above conservative measures may cure peritonsillitis. Ifa frank abscess has formed, incision and drainage will be required.
¢ Incision and drainage of abscess. A peritonsillar abscess is opened at the point of maximum bulge above the upper pole of tonsil or just lateral to the point of junction of anterior pillar with a line drawn through the base of uvula (Figure 52.5). With the help of a guarded knife, a small stab incision is made and then a sinus forceps inserted to open the abscess. Putting the sinus forceps the following day may also be necessary to drain any reaccumulation.
¢ Interval tonsillectomy. Tonsils are removed 4-6 weeks folan attack of lowing quinsy.
e Abscess or hot tonsillectomy. Some people prefer to do "hot"tonsillectomy instead of incision and drainage. Abscess tonsillectomy has the risk of rupture of the abscess during anaesthesia and excessive bleeding at the time of operation.

Rare with modern therapy.
1. Parapharyngeal abscess (a peritonsillar abscess is a potential parapharyngeal abscess) .
2. Oedema of larynx. Tracheostomy may be required.
3. Septicaemia. Other complications like endocarditis, nephritis, brain abscess may occur.
Figure 52.4 Peritonsillar abscess left side.
Figure 52.5 Peritonsillar abscess. Site of drainage is just lateral to the junction of vertical line through anterior pillar and horizontal line through base of uvula.
4, Pneumonitis or lung abscess. Due to aspiration of pus,
if
spontaneous rupture of abscess has taken place.
5. Jugular vein thrombosis.
6. Spontaneous haemorrhage from carotid artery or jugular vein.

¢ Retropharyngeal space. It lies behind the pharynx between the buccopharyngeal fascia covering pharyngeal constrictor muscles and the prevertebral fascia. It extends from the base of skull to the bifurcation of trachea. The space is divided into two lateral compartments (spaces of Gillette) by a fibrous raphe (Figure 52.6). Each lateral space contains retropharyngeal nodes which usually disappear at 3-4 years of age. Parapharyngeal space communicates with the retropharyngeal space. Infection of retropharyn- geal space can pass down behind the oesophagus into the mediastinum.
¢ Prevertebral space. It lies between the vertebral bodies posteriorly and the prevertebral fascia anteriorly. It extends from the base to skull of coccyx. Infection of this space
Figure 52.6 Spaces in relation to pharynx where abscesses can form.
usually comes from the caries of spine. Abscess of this space produces a midline bulge in contrast to abscess of retropharyngeal space which causes unilateral bulge.

It is commonly seen in children below 3 years. It is the result of suppuration of retropharyngeal lymph nodes secondary to infection in the adenoids, nasopharynx, posterior nasal sinuses or nasal cavity. In adults, it may result from penetrating injury of posterior pharyngeal wall or cervical oesophagus. Rarely, pus from acute mastoiditis tracks along the undersurface of petrous bone to present as retropharyngeal abscess.

1.
Dysphagia and
breathing are
prominent symp-
toms as the abscess obstructs the air and food passages.
2. Stridor and croupy cough may be present.
3. Torticollis. The neck becomes stiff and the head is kept extended.
4. Bulge in posterior pharyngeal wall. Usually seen on one side of the midline.
Radiograph of soft tissue, lateral view of the neck shows widening of prevertebral shadow and possibly even the presence of gas (Figure 52.7).

1. Incision and drainage of abscess. This is usually done without anaesthesia as there is risk of rupture of abscess during intubation. Child is kept supine with head low. Mouth is opened with a gag. A vertical incision is given in the most fluctuant area of the abscess. Suction should always be available to prevent aspiration of pus.
2. Systemic antibiotics. Suitable antibiotics are given.
Figure 52.7 Retropharyngeal abscess. Radiograph of soft tissue, lateral view neck showing widening of prevertebral space with gas formation (arrow).
3. Tracheostomy. A large abscess may cause mechanical obstruction to the airway or lead to laryngeal oedema. Tracheostomy becomes mandatory in these cases.

It is tubercular in nature and is the result of (i) caries of cer- vical spine or (ii) tuberculous infection of retropharyngeal
Figure 52.8 Spaces of head and neck seen in coronal section. Mucosa (1), pharyngobasilar fascia (2), buccopharyngeal fascia (3), superior constrictor muscle (4), superficial layer of deep cervical fascia enclosing submandibular gland (5), parotid gland (6), masseter muscle (7), temporalis muscle (8) and medial pterygoid muscle (9).
lymph nodes secondary to tuberculosis of deep cervical nodes. The former presents centrally behind the prevertebral fascia while the latter is limited to one side of midline as in true retropharyngeal abscess behind the buccopharyngeal fascia.

Patient may complain of discomfort in throat. Dysphagia, though present, is not marked. Posterior pharyngeal wall shows a fluctuant swelling centrally or on one side of midline. Neck may show tuberculous lymph nodes. In cases with caries of cervical spine, X-rays are diagnostic.

- 1. Incision and drainage of abscess. It can be done through a
vertical incision along the anterior border of sternomas- toid (for low abscess) or along its posterior border (for
high abscess)
.
2. Full course of antitubercular therapy should be given.

(Syn. Abscess of pharyngomaxillary or lateral pharyngeal space.)

Parapharyngeal space is pyramidal in shape with its base at the base of skull and its at the bone.
apex hyoid

- ¢ Medial. Buccopharyngeal fascia covering the constrictor
muscles.
- ¢ Posterior. Prevertebral fascia covering prevertebral mus-
cles and transverse processes of cervical vertebrae.
- ¢ Lateral. Medial pterygoid muscle, mandible and deep surface of
parotid gland.
Styloid process and the muscles attached to it divide the parapharyngeal space into anterior and posterior compartments. Anterior compartment is related to tonsillar fossa medially and medial pterygoid muscle laterally. Posterior compartment is related to posterior part of lateral pharyngeal wall medially and parotid gland laterally. Through the posterior compartment pass the carotid artery, jugular vein, IXth,
Xth, XIth, XIIth cranial nerves and sympathetic trunk.
It also contains upper deep cervical nodes.
Parapharyngeal space communicates with other spaces, viz. retropharyngeal, submandibular, parotid, carotid and visceral (Table 52.1).

Infection of parapharyngeal space can occur from:
1. Pharynx. Acute and chronic infections of tonsil and ade- noid, bursting of peritonsillar abscess.
2. Teeth. Dental infection usually comes from the lower last molar tooth.
3. Ear. Bezold abscess and petrositis.
4. Other spaces. Infections of parotid, retropharyngeal and
submaxillary spaces.
5. External trauma. Penetrating injuries of neck, injection of local anaesthetic for tonsillectomy or mandibular
nerve block.

Clinical features depend on the compartment involved.
Anterior compartment infections produce a triad of symp- toms: (i) prolapse of tonsil and tonsillar fossa, (ii) trismus

pharyngeal space or pharyngo- maxillary space ), Extent = Base of skull to hyoid bone and submandibular gland. Parapharyngeal space ( Lateral pharyngeal space or pharyngo- maxillary space ), Location = Buccopharyngeal fascia covering lateral aspect of pharynx medially, and fascia covering pterygoid muscles, mandible and parotid gland laterally. Parapharyngeal space ( Lateral pharyngeal space or pharyngo- maxillary space ), Source of infection = • Peritonsillar abscess • Parotid abscess • Submandibular gland infection • Masticator space abscess. Masticator space, Extent = Base of skull to lower border of mandible. Masticator space, Location = Between superficial layer of deep cervical fascia and the muscles of mastication- masseter, medial and lateral pterygoids insertion of temporalis muscle and the mandible. Masticator space, Source of infection = Infection of third molar
(due to spasm of medial pterygoid muscle) and (iii) external swelling behind the angle of jaw. There is marked odynophagia associated with it.
Posterior compartment
involvement produces
(i)
bulge of
pharynx behind
IX,
X, the
posterior
XI, and
ing of
XII
parotid pillar,
and region.
sympathetic
There
(ii)
paralysis chain,
is minimal
of
CN
and
(iii)
trismus swell-
or tonsillar
prolapse.
Fever, odynophagia, sore throat, torticollis (due to spasm of prevertebral muscles) and signs of toxaemia are common to both compartments.

1. Acute oedema of larynx with respiratory obstruction.
2. Thrombophlebitis of jugular vein with septicaemia.
3. Spread of infection to retropharyngeal space.
4. Spread of infection to mediastinum along the carotid space.
5. Mycotic aneurysm of carotid artery from weakening of its
wall by
purulent material.
It may
involve common
carotid or
internal carotid
artery.
6. Carotid blow out with massive haemorrhage.

1. Systemic antibiotics. Intravenous antibiotics may become necessary to combat infection.
2. Drainage of abscess. This is usually done under gen-
eral
If
the
trismus
is
marked,
preoperative
tracheostomy
becomes
Abscess
is
drained
by
a
horizontal
incision,
made
2-3
cm
below
the
angle
of
mandible.
Blunt
dissection
along
the
inner
surface
of
medial
pterygoid
muscle
towards
styloid
process
is
carried
out
and
abscess
evacuated.
A
drain
is
inserted.
Transoral
drainage
should
never
be
done
due
to
dan-
ger
of
injury
to
great
vessels
which
pass
through
this
space.

They are far less common compared to malignant tumours. The common ones are described here.

It is usually pedunculated, arises from the tonsil, soft palate or faucial pillars. Often asymptomatic, it may be discovered accidentally by the patient or the physician. When large, it causes local irritation in the throat. Treatment is surgical excision.

It can occur on the palate, tonsil, posterior and lateral pharyngeal wall. It may be of capillary or cavernous type. Capillary haemangioma or asymptomatic cavernous haemangioma may be left alone. It is treated only if it is increasing in size or giving symptoms of bleeding and dysphagia. Treatment is diathermy coagulation or injection of sclerosing agents. Cryotherapy or laser coagulation is very effective.

It is mostly seen submucosally on the hard or soft palate. It is potentially malignant and should be excised totally.

It is usually seen in the vallecula. It is yellow in appearance and may be pedunculated or sessile. When large, it causes foreign body sensation in the throat. Treatment is surgical excision, if pedunculated; or incision and drainage with removal of its cyst wall.
Lipoma, fibroma and neuroma are other rare benign tumours.
Gross appearances of the tumour can be divided into four
types:
1. Superficially spreading
2. Exophytic
3. Ulcerative
4. Infiltrative
The first two types are seen in the palatine arch; they are rarely associated with metastasis. Ulcerative and infiltrative types often involve the base of tongue and tonsil. They have poor prognosis and deeply invade the adjoining structures and have marked tendency for regional metastasis.
Histologically, the tumours may be:
1. Squamous cell carcinoma. Shows various grades of differen- tiation (well, moderately or poorly differentiated) and is the most common variety.
2. Lymphoepithelioma. A poorly differentiated variant of the
above,
with
admixture
of
which
do
not
show
any
features
of
This
is
often
seen
in
tonsil,
base
of
tongue
and
vallecula.
3. Adenocarcinoma. It arises from minor salivary glands. It is mostly seen on the palate and fauces.
4. Lymphomas. Both Hodgkin and non-Hodgkin lympho- mas arise from the tonsil and base of tongue. They are seen in the young adults and sometimes in the children. Enlarged cervical nodes may coexist.
TNM classification. It is similar to the one used in cancer of the oral cavity. (see Table 53.2). Treatment. Treatment of oropharyngeal cancer depends upon the site and extent of disease, patient’s general con- dition, philosophy and experience of the treating surgeon and facilities available at a particular centre. The various options are:
Surgery alone
Radiation alone NO
Combination of surgery and radiotherapy GO
Chemotherapy alone or as an adjunct to surgery or radiotherapy ~A
Palliative therapy. on

The common sites of malignancy in the oropharynx are: (Table 53.1)
1. Posterior one-third (or base) of tongue.
2. Tonsil and tonsillar fossa.
3. Faucial palatine arch, i.e. soft palate and anterior pillar.
4. Posterior and lateral pharyngeal wall.

This is commonly seen in India (Figure 53.1). The lesion remains asymptomatic for a long time and patient pres- ents when metastases in cervical nodes make their appear- ance. Earlier symptoms of sore throat, feeling of lump in the throat and slight discomfort on swallowing are often

¢ Base of tongue
¢ Tonsil and tonsillar fossa e Faucial arch
e Pharyngeal wall

Tumour 2 cm or less in greatest dimension
To
Tyb
Tumour more than 2 cm but not more than 4 cm in greatest dimension
Tumour more than 4 cm in greatest dimension
Tumour invades the larynx, deep/extrinsic muscle of tongue,
medial pterygoid,
or mandible
Tumour invades
lateral pterygoid
plates, lateral
nasopharynx, or
encases carotid artery

Regional lymph nodes cannot be assessed
No regional lymph node metastasis
Metastasis in a single ipsilateral lymph node, 3 cm or less in greatest dimension
Metastasis in a single ipsilateral lymph node, more than 3 cm but not more than 6 cm in greatest dimension, or in multiple ipsilateral lymph nodes, none more than 6 cm in greatest dimension, or in bilateral or contralateral lymph nodes, none more than 6 cm in greatest dimension
Metastasis in a single ipsilateral lymph node more than
3 cm but not more than 6 cm in greatest dimension
Metastasis in multiple ipsilateral lymph nodes, none more than 6 cm in greatest dimension
Metastasis in bilateral or contralateral lymph nodes, none more than 6 cm in greatest dimension
No
Noa
Nop
Noo
N3
in
a
lymph node
more than
6
cm in
est dimension

Mx
Distant metastasis cannot be assessed
Mo
No distant metastasis
M,
Distant metastasis

0
Tis
|
Il
Ul
IVA
IVC
Ty
To
T3
Ty
To
Ts
Tya
Tya
T,
To
Ts
Tya
Tab
Any
Any
No
No
No
No
N,
Ny
N,
No
Ny
No
No
No
No
Any
N3
Any great-
muscle, pterygoid
base of
skull or
T
T
N
N
Mo
Mo
Mo
Mo
Mo
Mo
Mo
Mo
Mo
Mo
Mo
Mo
Mo
Mo
Mo
M,
Figure 53.1 An exophytic growth at the base of tongue.
ignored or attributed to lingual tonsils. Late features are referred pain in the ear, dysphagia, bleeding from the mouth and change in the quality of speech (hot potato voice).

1. Local. Lesions are deeply infiltrative and spread to the rest
of tongue
musculature, epiglottis
and pre-epiglottic
space, tonsil and its pillars, and hypopharynx.
2. Lymphatic. Seventy per cent of the cases show cervical metastases either unilateral or bilateral at the time of ini-
tial consultation.
Jugulodigastric nodes
are the
first to
be involved.
3. Distant metastases. Bones, liver and lungs may be involved.

Lesions can be seen on indirect laryngoscopy but palpation of the tumour should never be omitted. Palpation under anaesthesia when tissues are relaxed gives better idea of the degree of infiltration of tissues. Lesion is usually far more extensive than it appears on mirror examination. Computed tomography scan is recommended for tumour and nodal staging. Biopsy is essential to know its histology.

Treatment may vary from centre to centre, some favouring radiotherapy, others surgery and still others radiotherapy followed by salvage surgery.
Tumours which are radiosensitive such as anaplastic car- cinoma,
lymphoepithelioma or
lymphoma are
treated by
radiotherapy to the primary and neck nodes.
For
T;
and
Ty squamous
cell carcinoma
with
No or
N;
neck, surgical
block dissection
is preferred
and if neck dissection specimen reveals a stage more than Nj, postoperative radiation is added.
Ts and T, lesions require surgical excision with mandibu- lar resection, neck dissection and postoperative radiation.
T, lesions, which also extend into anterior two-thirds of tongue
or vallecula,
require extensive
surgery with
total glossectomy
and laryngectomy
in addition
to the
block dissection.
Chemotherapy may be combined with radiotherapy and surgery in such cases.
For advanced cancers, in patients with poor health, only palliation with radioor chemotherapy may be required.
They often end up into tracheostomy and gastrostomy in the terminal phase to restore their air and food passages and for relief of strong analgesics pain.

Squamous cell carcinoma is the most common and presents as an ulcerated lesion with necrotic base (Figure 53.2). Lymphomas may present as unilateral tonsillar enlargement with or without ulceration and may simulate indolent peritonsillar abscess (Figure 53.3).

1. Local. Tumour may spread locally to soft palate and pillars, base of tongue, pharyngeal wall and hypopharynx. It may invade pterygoid muscles and mandible resulting in pain and trismus. Parapharyngeal space may also get invaded.
2. Lymphatic. Fifty per cent of the patients have initial cervical node involvement at the time of presentation. Jugulodigastric nodes are the first to be involved.
3. Distant metastases. They are seen in late cases.

Persistent sore throat, difficulty in swallowing, pain in the ear or lump in the neck are the presenting symptoms. Later on, bleeding from the mouth, fetor oris and trismus occur.
may

Palpation of tonsillar area should never be omitted to find the extent of tumour. Biopsy is essential for histological typing.

1. Radiotherapy. Early by radiotherapy along and radiosensitive tumours are treated
with irradiation of cervical nodes.
2. Surgery. Excision of the tonsil can be done for early superficial lesions. Larger lesions and those which invade bone require wide surgical excision with hemimandibulectomy and neck dissection (commando operation).
3. Combination therapy. Surgery may be combined with preor postoperative radiation. Chemotherapy may be given as an adjunct to surgery or radiation.

Soft palate, uvula and anterior tonsillar pillar comprise the faucial arch. Carcinoma in these sites is often of squamous cell variety. Lesions are superficially spreading and well-differentiated with late tendency for nodal metastases (Figure 53.4). Thus they behave more like carcinomas of the oral cavity.
Spread may occur locally to the contiguous structures or lymph nodes. Upper deep cervical and submandibular nodes may be involved.
Patients with palatine arch cancer usually present with persistent sore throat, local pain or earache. Growth may have been noticed by the patient while using the mirror, or by his physician while examining his throat or by the dentist.
Treatment is irradiation or surgical excision.
Figure 53.2
soft
Squamous cell carcinoma involving tonsil, pillar and palate.
Figure 53.3
Lymphoma tonsil presenting as unilateral tonsillar
enlargement.
Figure 53.4 An ulcerative lesion of palatine arch. It was welldifferentiated squamous cell carcinoma.

Lesions remain asymptomatic for a long time. They may spread submucosally to the adjoining areas such as tonsil, soft palate, tongue, nasopharynx or hypopharynx. They may also invade parapharyngeal space or anterior spinal ligaments. Sixty per cent of patients may have lymph node metastases. Bilateral nodal involvement is common.
Treatment is irradiation or surgical excision of growth with skin grafting. This is often combined with block dissection when nodes are palpable. Access to posterior pharyngeal wall is through lateral pharyngotomy with or without mandibular osteotomy.

Parapharyngeal space is described on p. 267 (refer Figure 52.6). It lies lateral to the pharynx. Both benign and malignant tumours are seen. They cause a bulge in lateral pharyngeal wall of oropharynx and distort the pillars and soft palate, and thus mimic neoplasms of the oropharynx. Commonly seen tumours are those from the deep lobe of parotid, neurogenic (e.g. neurilemmoma), chemodectoma
(from carotid body), lipoma or aneurysm of internal carotid artery.

It is due to elongated styloid process or calcification of stylohyoid ligament. Patient complains of pain in tonsillar fossa and upper neck which radiates to the ipsilateral ear. It gets aggravated on swallowing. Diagnosis can be made by transoral palpation of the styloid process in the tonsillar fossa and by a radiograph such as anteroposterior view with open mouth or lateral view of skull. Many persons may have elongated styloid process but remain asymptomatic and do not need treatment. Symptomatic styloid process can be excised by transoral or cervical approach.

Benign tumours. They are exceptionally uncommon and include papilloma, adenoma, lipoma, fibroma and leiomyoma. They present as smooth well-defined masses which are sometimes pedunculated and mobile.
Malignant tumours. Carcinoma of the hypopharynx is very common in India. Practically, most of the tumours are squamous cell type with various grades of differentiation. The various subsites involved are: (i) pyriform sinus, (ii) postcricoid region and (iii) posterior pharyngeal wall, in that order of frequency.

It constitutes 60% of all hypopharyngeal cancers, mostly affecting males above 40 years of age. Growth is either exophytic or ulcerative and deeply infiltrative. Because of the large size of the pyriform sinus, growths of this region remain asymptomatic for a long time. Metastatic neck nodes may be the first to attract attention.

1. Locally, the growth may spread upwards to the vallecula and base of tongue; downwards to postcricoid region; medially to aryepiglottic folds and ventricles. It may infiltrate into the thyroid cartilage, thyroid gland or may present as a soft tissue mass in the neck.
2. Lymphatic spread occurs early. Pyriform fossa has a rich lymphatic network. Seventy-five per cent of the patients have cervical nodal metastases when first seen, with half of them having bilateral involvement. Upper and middle group of jugular cervical nodes are often involved. Sometimes, nodes make their appearance long after the primary has been eradicated.
3. Distant metastases often occur late and may be seen in lung, liver and bones.

Early symptoms are few. Something sticking in the throat and "pricking sensation" on swallowing may be the earliest symptoms. Referred otalgia, pain on swallowing and increasing dysphagia may follow. A mass of lymph nodes high up in the neck may be the first sign. Hoarseness and laryngeal obstruction indicate laryngeal oedema or spread of disease to the larynx.
Growth and its extent can often be seen on mirror examination. Sometimes, pooling of secretions obstructs the view. Barium swallow and CT scan are helpful to evaluate the extent of growth and status of lymph nodes. Endoscopic examination is necessary for biopsy and accurate assessment of the extent of growth and also to find out any synchronous primary at any other site.

Early growth without nodes can be cured by radiotherapy with the advantage of preserving the laryngeal function. If growth is limited to pyriform fossa and does not extend to postcricoid region, total laryngectomy and partial pharyngectomy is done. Remaining pharynx can be primarily closed. This is often combined with elective or prophylactic block dissection of lymph nodes. If growth extends to postcricoid region, total laryngectomy and pharyngectomy is done along with block dissection. Pharyngo-oesophageal segment is reconstructed with myocutaneous flaps or stomach pull-up. Planned postoperative radiotherapy can be given routinely to all cases. Patients with no palpable nodes (No neck) can also be given radiotherapy avoiding block dissection.

This constitutes 30% of laryngopharyngeal malignancies. Paterson—Brown-Kelly (Plummer-Vinson) syndrome characterized by hypochromic microcytic anaemia is an important aetiological factor as one-third of patients of postcricoid carcinoma be from it.
may suffering

Usually an ulcerative type of lesion arises from postcricoid region. Local spread often occurs in an annular fashion causing marked dysphagia. Growths may invade cervical oesophagus, arytenoids or recurrent laryngeal nerve at cricoarytenoid joint.
Lymphatic spread involves paratracheal lymph nodes and may be bilateral due to the midline nature of lesions. They may not be clinically palpable.

Females are usually affected, sometimes in the early age group of twenties and thirties. Progressive dysphagia is the predominant presenting symptom. This may cause
progressive malnutrition and weight loss. Sometimes, voice change and aphonia may be produced due to infiltration of recurrent laryngeal nerve or posterior cricoarytenoid muscles affecting vocal cord mobility.

Postcricoid growths may not be visible on indirect laryngoscopy. Oedema and erythema of the postcricoid region and pooling of secretions in the hypopharynx are suggestive of growth. Laryngeal crepitus, felt normally while moving larynx over the cervical spine, may be lost.
Lateral soft tissue radiograph of the neck may show an increased prevertebral shadow. Barium swallow is essential to find the lower extent of the disease. Endoscopy is always done to take biopsy and assess the extent of lesion.

Prognosis is poor both with irradiation and surgical treatment. Some prefer to give radiotherapy initially. It has the advantage of preserving laryngeal function. Failed cases are subjected to laryngo-pharyngo-oesophagectomy with stomach pull-up or colon transposition to reconstruct pharyngo-oesophageal segment. Many feel that initial surgery, if feasible, gives better results.

This is the least common of laryngopharyngeal malignancy constituting only 10% of them. They are mostly seen in males above 50 years of age.

Growth is usually exophytic but may be ulcerative. It remains localized until late and then spreads to the prevertebral fascia, muscles and vertebrae.
Lymphatic spread is usually bilateral due to midline nature of the lesion. Fifty per cent of the patients with cancer of posterior pharyngeal wall have nodal metastasis on their initial examination. Retropharyngeal nodes, though not clinically palpable, may also be involved.

Dysphagia or spitting of blood may be the presenting symptom. Some may present with a palpable mass of nodes in the neck without any symptoms pointing to the primary tumour.

Indirect mirror examination often reveals the tumour. Lateral soft tissue radiography may show vertical extent and thickness of the tumour and any involvement of cervical vertebrae. Endoscopy is essential for biopsy and accurate assessment of the tumour and to find any synchronous primary at any other site.

Early lesions, particularly exophytic, can be treated by radiotherapy with preservation of laryngeal function. Early small lesions can also be excised surgically via lateral pharyngotomy and primary repair with equally good results. Advanced lesions may require laryngopharyngectomy and block dissection of neck with repair of the food channel. Gross 5-year cure rate is only 19%.

Also called hypopharyngeal diverticulum or Zenker's diverticulum, it is a pulsion diverticulum where pharyngeal mucosa herniates through the Killian's dehiscence—a weak area between two parts of the inferior constrictor (Figures 54.1 and 54.2).

Exact cause is not known. It is probably due to spasm of cricopharyngeal sphincter or its incoordinated contractions during the act of deglutition. It is usually seen after 60 years of age.

Herniation of pouch starts in the midline. It is at first behind the oesophagus and then comes to lie on its left. Mouth of the sac is wider than the opening of oesophagus and food preferentially enters the sac.

Dysphagia is the prominent feature. It appears after a few swallows when the pouch gets filled with food, and presses
Figure 54.1 Hypopharyngeal (Zenker's) diverticulum. Hypopharyngeal mucosa herniates through the Killian's dehiscence—a weak area between two parts of inferior constrictor muscle.
Figure
Potential sites for hypopharyngeal diverticulum.
on the oesophagus. Gurgling sound is produced on swallowing. Undigested food may regurgitate at night, when patient is recumbent, causing cough and aspiration pneumonia. Patient is often malnourished due to dysphagia. Patients with pharyngeal pouch may have associated hiatus hernia. Rarely carcinoma can develop in long-standing cases of pha- ryngeal pouch.

1. Excision of pouch and cricopharyngeal myotomy. This is done through cervical approach.
2.
Dohlman’s
The
partition
wall
between
the
oesophagus
and
the
pouch
is
divided
by
diathermy
through
an
endoscope.
This
is
done
in
poor
risk
debili-
tated
patients.
3. Endoscopic laser treatment. It is similar to Dohlman’s procedure. Partition between the pouch and oesophagus is divided by COg laser using operating microscope.

It is an undesirable disturbing sound that occurs during sleep. It is estimated that 25% of adult males and 15% of adult females snore. Its prevalence increases with age.

¢ Sleep apnoea. It is cessation of breathing that lasts for 10 s or more during sleep. Less than five such episodes is normal.
¢ Apnoea index. It is number of episodes of apnoea in 1 h.
¢ Hypopnoea. It is reduction of airflow. Some define it as drop of 50% of airflow from the base line associated with an EEG defined arousal or 4% drop in oxygen saturation.
¢ Respiratory disturbance index (RDI). Also called apnoea— hypopnoea index. It is the number of apnoea and hypopnoea events per hour. Normally RDI is less than five. Based on RDI, severity of apnoea has been classified as mild, 5-14; moderate, 15-29; and severe = 30.
¢ Arousal. Transient awakening from sleep as a result of apnoea or respiratory efforts.
¢ Arousal index. It is number of arousal events in 1 h. Less than four is normal.
¢ Sleep efficiency. Minutes of sleep divided by minutes in bed after lights are turned off.
¢ Multiple sleep latency test or nap study. Patient is given four or five scheduled naps usually in the daytime. Latency period from wakefulness to the onset of sleep and rolling eye movement (REM) sleep are measured. It is performed when narcolepsy is suspected or daytime sleepiness is evaluated objectively.

Muscles of pharynx are relaxed during sleep and cause partial
Breathing against
obstruction causes
vibrations of soft palate, tonsillar pillars and base of tongue producing
sound.
Sound as
loud as
90
dB
has been
recorded during
snoring.
Snoring may be primary, i.e. without association with obstructive sleep apnoea (OSA) or complicated, i.e. associated with OSA. Primary snoring is not associated with excessive daytime sleepiness and has apnoea—hypnoea index of less than five.
In children most common cause is adenotonsillar hypertrophy. In adults cause of snoring could be in the nose or nasopharynx such as septal deviation, turbinate hypertrophy, nasal valve collapse, nasal polypi or tumours; in oral cavity and oropharynx such as elongated soft palate and uvula, tonsillar enlargement, macroglossia, retrognathia, large base of tongue; or its tumour; in the larynx and laryngopharynx such as laryngeal stenosis or omega-shaped epiglottis. Other causes include obesity and thick neck with collar size exceeding 42 cm. Use of alcohol, sedatives and hypnotics aggravates snoring due to muscle relaxation.

Sites of snoring may be soft palate, tonsillar pillars or hypopharynx. It may vary from patient to patient and even in the same patient thus making surgical correction a difficult decision. Sometimes sites of snoring are multiple even in the same patient.

Excessive loud snoring is socially disruptive and forms snoring-spouse syndrome and is the cause of marital discord sometimes leading to divorce. In addition, a snorer with obstructive sleep apnoea may manifest with:
Excessive daytime sleepiness
Morning headaches
General fatigue
Memory loss
Irritability and depression
Decreased libido
Increased risk of road accidents
Table 55.1 shows an Epworth sleepiness scale.

1. Avoidance of alcohol, sedatives and hypnotics.
2. Reduction of weight.
3. Sleeping on the side rather than on the back.
4. Removal of obstructing lesion in nose, nasopharynx, oral cavity, hypopharynx and larynx. Radiofrequency has been used for volumetric reduction of tissues of turbi- nates, soft palate and base of tongue.
5. Performing uvulopalatoplasty (UPP) surgically with cold knife or assisted with radiofrequency (RAUP) or laser (LAUP).

© Sitting and reading
e Watching TV
Sitting inactive in a public place (e.g. theatre
or in a meeting)
e Being a passenger in a car for 1 h without
break
e Lying down to rest in the afternoon when
circumstances permit
Sitting and talking to someone
Sitting quietly after a lunch without alcohol
Sitting
in
a
car
while
stopped
in
traffic
for
a
few minutes
O =never dozing off; 1 = slight chance of dozing off; 2 = moderate chance of dozing off; 3 = high chance of dozing off.

Apnoea means no breathing at all. There is no movement of air at the level of nose and mouth. It is of three types.
1. Obstructive. There is collapse of the upper airway result- ing in cessation of airflow. Other factors may be obstruc- tive conditions of nose, nasopharynx, oral cavity and oropharynx, base of tongue or larynx.
2. Central. Airways are patent but brain fails to signal the muscles to breathe.
3. Mixed. It is combination of both types.

Apnoea during sleep causes hypoxia and retention of carbon dioxide which leads to pulmonary constriction leading to congestive heart failure, bradycardia and cardiac hypoxia leading to left heart failure, and cardiac arrhythmias sometimes leading to sudden death. During sleep apnoea, there are frequent arousals which cause sleep fragmentation, daytime sleepiness and other manifestations. Table 55.2 lists the consequences of obstructive sleep apnoea.

A normal healthy adult sleeps for 7-8 h. Sleep occurs in two phases: non-REM and REM. The two phases occur in semiregular cycles, each cycle lasting for 90-120 min. There are thus three or four cycles of sleep.

It forms 75-80% of sleep and occurs in four stages:
1. Stage I. Transition from wakefulness to sleep. It constitutes 2-5% of sleep. EEG shows decrease of alpha and increase of theta waves. Muscle tone is less. Person can be easily aroused from this stage.
2. Stage II. Characterized by sleep spindles or 'K' complexes and decrease in muscle tone. It constitutes 45-55% of sleep.
- 3. Stage III. Forms 3-8% of sleep, characterized by delta waves. It is deep sleep.

Congestive heart failure/cor pulmonale
Polycythaemia and hypertension
Atrial and ventricular arrhythmias and left heart failure
Attacks of angina
Snoring spouse syndrome
Loss of memory
Traffic accidents
4. Stage IV. Forms 10-15% of sleep, characterized by delta waves. It is deep, most restful sleep.

Forms 20-25% of total sleep, characterized by rapid eye movements, increased autonomic activity with erratic cardiac and respiratory movements. Dreaming occurs in this stage but muscular activity is decreased so that dreams are not enacted.
See Table 55.3 for differences between non-REM and
REM sleep.

Patient's bed partner gives more reliable information than the patient himself because latter does not know what happened during sleep. History should include snoring during sleep, restless disturbed sleep, gasping, choking or apnoeic events and sweating. In the daytime, there is history of excessive daytime sleepiness (Epworth sleepiness scale is more often used, see Table 55.1) and fatigue, irritability, morning headaches, memory loss and impotence. Also one should elicit history of body position during sleep, use of alcohol, sedatives and caffeine intake, mouth breathing and history of menopause or having hormonal replacement therapy.

Risk factors include male gender, obesity and age above 40 years.
1. Body mass index. It is calculated by dividing body weight in kilograms by height in metres squared. Normal BMI, 18.5-24.9; overweight, 25-29%; and obesity, 30-34.9. Obese patients need to reduce weight.
2. Collar size. Neck circumference at the level of crico- thyroid membrane is measured. Collar size should not exceed 42 cm in males and 37.5 cm in females.
3. Complete head and neck examination. Look for tonsillar hypertrophy, retrognathia, macroglossia, elon- gated soft palate and uvula, base of tongue tumours, septal deviation, nasal polyps, turbinate hypertrophy and nasal valve collapse. Also examine nasopharynx and larynx.
4. Muller’s manoeuvre. A flexible endoscope is passed through the nose and the patient asked to inspire vigor- ously with nose and mouth completely closed. Look for

Duration, Differences between non-REM and REM sleep.Non-REM = 75-80% of sleep. Duration, Differences between non-REM and REM sleep.REM = 20-25% of sleep. Eye movements, Differences between non-REM and REM sleep.Non-REM = No eye rolling. Eye movements, Differences between non-REM and REM sleep.REM = Rapid conjugate eye movements. Autonomic activity, Differences between non-REM and REM sleep.Non-REM = Less autonomic activity gives slow heart rate, low BP, slow and steady respiration. Autonomic activity, Differences between non-REM and REM sleep.REM = Increased autonomic activity with fluctuations in BP, heart rate and respiration. Brain activity, Differences between non-REM and REM sleep.Non-REM = Minimal. Brain activity, Differences between non-REM and REM sleep.REM = Brain is active (REM sleep is also called activated brain in a paralyzed person). Muscular activity, Differences between non-REM and REM sleep.Non-REM = Functional but less. Muscular activity, Differences between non-REM and REM sleep.REM = Decreased. Since muscles are relaxed, snoring and OSA occurs in this stage. EEG, Differences between non-REM and REM sleep.Non-REM = Passes from alpha to delta waves from stage I to IV. EEG, Differences between non-REM and REM sleep.REM = Mixed frequency, low-voltage waves with occasional bursts of saw-tooth waves. Dreaming, Differences between non-REM and REM sleep.Non-REM = No. Dreaming, Differences between non-REM and REM sleep.REM = Yes
collapse of the soft tissues at the level of base of tongue and just above the soft palate. Level of pharyngeal obstruction can be found.
changes but those with cor pulmonale as a result of severe OSA may require permanent tracheostomy.
Systemic examination
is done
to look
for hypertension,
con- gestive
heart failure,
pedal oedema,
truncal obesity
and any
sign of
hypothyroidism.
Cephalometric radiographs are taken for craniofacial anomalies and tongue base obstruction.
Polysomnography. It is the "gold standard" for diagnosis of sleep apnoea and records various parameters which include:

EEG (electroencephalography)—to look for non-REM or REM sleep and stages of non-REM sleep.
ECG (electrocardiography)—for heart rate and rhythm.
e¢ EOM (electroculogram)—for rolling eye movements.
e EMG (electromyography)—recorded from submental and tibialis anterior muscle.
Pulse oximetry—to assess oxygen saturation of blood to know lowest SaOyg during sleep.
Nasal and oral airflow—for episodes of apnoea and hypopnoea.
Sleep position—helps
to know
whether apnoea/hypop-
noea episodes
occur position.
¢
Blood pressure.
- © Oesophageal pressure. Not done in all laboratories. Negative oesophageal pressure helps to know degree of breathing efforts made by the patient.
Split-night polysomnography.
In this
study, the
first part
of night
is used
part of
in usual
polysomnography night
positive is
used in
while titration
airway because
of pressures
pressure episodes
the second
of
(CPAP).
of sleep
half of
pressures for
night
CPAP
the second
for
It is
not apnoea
and occur
are should
continuous recommended
more thus
ideally often
in missed.
be done
on
Titration
a
second night.
Polysomnography can differentiate between primary snoring, pure OSA and central sleep apnoea.

1.
Change in
lifestyle.
Those mal
symptoms can
be with
treated mild
with disease
weight and
loss and
mini- dietary
in supine
or lateral
recumbent
- (a) Use of alcohol in the evening aggravates OSA. Sedatives/hypnotics taken at night also have the same effect.
(b)
Smoking should
(c) Reduction of weight is helpful.
2. Positional therapy. Patient should sleep on the side as supine position may cause obstructive apnoea. A rubber ball can be fixed to the back of shirt to prevent adopting supine position.
3. Intraoral devices. They alter the position of mandible or tongue to open the airway and relieve snoring and sleep apnoea. Mandible advancement device (MAD) keeps the mandible forward while tongue retaining device (TRD) keeps tongue in anterior position during sleep. They help improve or abolish snoring. MAD is also useful in retrognathic patients.
4.
CPAP
(continuous positive
airway pressure).
It provides
pneumatic splint
to mum
airway airway
cm
H,O.
for sleep
About device
40%
increases device
its calibre.
to open
study of
cumbersome and
Opti- the
is airway
usually patients
and find
difficult kept
the use
to carry
is at
5-20
of
CPAP
with them
when travelling
and thus
stop using
it.
When CPAP is not tolerated, a BiPAP (bilevel positive airway pressure) device is used. It delivers positive pressure at two fixed levels—a higher inspiratory and a lower expiratory pressure. Now an autotitrating PAP (APAP) is also available which continuously adjusts the pressure. Their disadvantages are same as those of CPAP.

It is
indicated
Permanent ment
but it
is of
its own.
for failed
or noncompliant
tracheostomy is
the
“gold not
accepted
It is
usually not
socially and
a
preferred medical
therapy.
standard”
of treat-
has complications
option
Surgical procedures
used in
OSA
include:
- 1. Tonsillectomy and/or adenoidectomy.
2.
Nasal surgery.
may or
the aggravating
deviated factor
nasal septum,
tion of
turbinate be
the for
OSA.
Septoplasty removal
size of
nasal help
to by
patients.
primary to
polyps relieve
reduc- nasal
• Weight reduction • Avoidance of alcohol, sedatives and smoking • Positional therapy • Intraoral devices • Mandibular advancement device • Tongue retention device • CPAP or BiPAP or APAP, Surgical = • Tonsil and adenoid surgery (children) • Nasal surgery: septoplasty, turbinate reduction, polypectomy • Palate surgery • Uvulopalatoplasty (UPP) • Uvulopalatopharyngoplasty (UPPP) • Advancement pharyngoplasty • Tongue base surgery: lingual tonsillectomy, laser midline glossectomy • Tongue base radiofrequency reduction • Mandibular osteotomy with genioglossus advancement • Hyoid myotomy and suspension • Hyoid bone suspended to lower border of mandible • Hyoid bone suspended to upper border of thyroid cartilage • Maxillomandibular osteotomy and advancement • Tracheostomy-the gold standard
It helps to pull the base of tongue anteriorly. Along with this procedure, the hyoid bone is freed from its inferior musculature and suspended from lower border of mandible by wires. This also helps to pull the base of tongue anteriorly.
5. Tongue base radiofrequency. Radiofrequency (RF) is used in five to six sittings to reduce the size of tongue. RF needle is inserted submucosally. It coagulates tissue and causes scarring thus reducing the size of tissue.
6.

Maxillomandibular advancement
osteotomy.
Osteoto- mies
are performed
on
Osteotomy mandibular
of the
maxilla
These osteotomies
plates and
screws.
selected cases
ramus is
like
a
and
Le are
then
This but
has fixed
surgical the
Fort in
anterior procedure
disadvantage maxilla.
I
position with
is effective
of causing
in aesthetic
facial changes.
See Table 55.4 for summary of management of OSA.
56.
57.
58.
59.
60.
Anatomy and Physiology of Larynx
Laryngotracheal Trauma
Acute and Chronic Inflammations
of Larynx
Congenital Lesions of Larynx and
Stridor
Laryngeal Paralysis
61.
62.
63.
64.
65.
Benign Tumours of Larynx
Cancer Larynx
Voice and Speech Disorders
Tracheostomy and Other
Procedures for Airway
Foreign Bodies of Air Passages

The larynx lies in front of the hypopharynx opposite the third to sixth cervical vertebrae. It moves vertically and in anteroposterior direction during swallowing and phonation. It can also be passively moved from side to side producing a characteristic grating sensation called laryngeal crepitus. In an adult, the larynx ends at the lower border of Cg vertebra.

Larynx has three unpaired and three paired cartilages.
Unpaired: Thyroid, cricoid and epiglottis.
Paired: Arytenoid, corniculate and cuneiform.
. Thyroid. It is the largest of all (Figure 56.1). Its two alae meet anteriorly forming an angle of 90° in males and 120° in females. Vocal cords are attached to the middle of thyroid angle. Most of laryngeal foreign bodies are arrested above the vocal cords, i.e. above the middle of thyroid cartilage and an effective airway can be provided by piercing the cricothyroid membrane—a procedure called cricothyrotomy.
2. Cricoid. It is the only cartilage forming a complete ring. Its posterior part is expanded to form a lamina while it is narrow an arch.
— anteriorly forming
. Epiglottis. It is a leaf-like, yellow, elastic cartilage forming anterior wall of laryngeal inlet. It is attached to the body of hyoid bone by hyoepiglottic ligament, which divides it into suprahyoid and infrahyoid epiglottis. A stalk-like process of epiglottis (petiole) attaches the epiglottis to the thyroid angle just above the attachment of vocal cords. Anterior surface of epiglottis is separated from thyrohyoid membrane and upper part of thyroid cartilage by a potential space filled with fat—the pre-epiglottic space. The space may be invaded in carcinoma of supraglottic larynx or the base of tongue. vo
Posterior surface
of epiglottis
is concavoconvex—con-

cave above but convex below forming a bulge called tubercle of epiglottis, which obstructs view of anterior commissure when examining larynx by indirect laryngoscopy. Epiglottic cartilage shows several pits which lodge the mucous glands. It may also show perforations providing direct communication between the laryngeal surface of epiglottis and pre-epiglottic space. Supraglottic cancers can spread through them to pre-epiglottic space. Epiglottis is not essential for swallowing and can be amputated in carcinoma with little aspiration.
- 4. Arytenoid cartilages. They are paired. Each arytenoid cartilage is pyramidal in shape. It has a base which articulates
with cricoid cartilage; a muscular process, directed laterally to give attachment to intrinsic laryngeal muscles; a vocal process directed anteriorly, giving attachment to vocal cord; and an apex which supports the corniculate cartilage.
5. Corniculate cartilages (of Santorini) (Corn = horn). They are paired. Each articulates with the apex of arytenoid cartilage as if forming its horn.
6. Cuneiform cartilages (of Wrisberg). They are rod shaped. Each is situated in aryepiglottic fold in front of corniculate cartilage and provides passive supports to the fold.
Thyroid, cricoid and most of the arytenoid cartilages are hyaline cartilages whereas epiglottis, corniculate, cuneiform and tip of arytenoid near the corniculate cartilage are elastic fibrocartilage. Hyaline cartilages can undergo ossification; it begins at the age of 25 years in thyroid, a little later in cricoid and arytenoids, and is complete by 65 years of age. Calcification seen in these cartilages can be confused with foreign bodies of oesophagus or larynx on X-rays.

Cricoarytenoid joint. It is a synovial joint surrounded by capsular ligament. It is formed between the base of arytenoid and a facet on the upper border of cricoid lamina. Two types of movements occur in this joint: (i) rotatory, in which arytenoid cartilage moves around a vertical axis, thus abducting or adducting the vocal cord; (ii) gliding movement, in which one arytenoid glides towards the other cartilage or away from it, thus closing or opening the posterior part of glottis.
Figure
56.1__Laryngeal framework.
Cricothyroid joint. It is also a synovial joint. Each is formed by the inferior cornua of thyroid cartilage with a facet on the cricoid cartilage. Cricoid cartilage rotates at these joints on a transverse axis which passes transversely through these joints.

Membrane and ligaments of larynx. The term extrinsic is used when membrane or ligament attaches to the structures outside the larynx, i.e. to the hyoid bone or trachea. The term intrinsic is used for membranes joining within the larynx but not extending to hyoid bone or trachea.

(FIGURE
56.1)
(a) Thyrohyoid membrane. It connects thyroid cartilage to
hyoid bone. It is pierced by superior laryngeal vessels and internal laryngeal nerve.
(b) Cricotracheal membrane. It connects cricoid cartilage to
the first tracheal ring.
(c) Hyoepiglottic ligament. It attaches epiglottis to hyoid
bone (Figure 56.2).

- (a) Cricovocal membrane. It is a triangular fibroelastic mem-
brane.
Its upper
border is
free and
stretches between
middle of
thyroid angle
to the
vocal process
of ary-
tenoid and
forms the
vocal ligament
(Figure
56.2).
Its lower border attaches to the arch of cricoid car- tilage.
From its
lower attachment
proceeds upwards
and medially
and thus,
with its
fellow on
the opposite
side, forms
conus elasticus
(Figure 56.3) where subglottic foreign bodies sometimes get impacted.
Quadrangular membrane.
It lies
deep to
mucosa of
ary- epiglottic
folds between
and is
the epiglottic
lower border
forms the
not well-defined.
and
It stretches
arytenoid cartilages.
vestibular ligament
Its which
lies in the false cord.
Figure
56.2
Sagittal section
of larynx
showing cricovocal
and quadrangular
space.
boundaries of
the pre-epiglottic
(b)
- (c) Cricothyroid ligament. The anterior part of cricothyroid
membrane is thickened to form the ligament and its lateral part forms the cricovocal membrane.
- (d) Thyroepiglottic ligament. It attaches epiglottis to thyroid cartilage.

They are of two types: intrinsic, which attach laryngeal cartilages to each other and extrinsic, which attach larynx to the surrounding structures.
1. Intrinsic muscles. They may act on vocal cords or laryngeal inlet.
(a) Acting on vocal cords (Figures 56.4 and 56.5)
e
Abductors:
Posterior cricoarytenoid e Adductors:
©
Tensors:
Lateral cricoarytenoid
Interarytenoid (transverse arytenoid)
Thyroarytenoid (external part)
Cricothyroid
Vocalis (internal part of thyroarytenoid)
Figure
56.3
of larynx.
Lower free
edge of
the quadrangular
edge of
of membrane
lies in
the cricovocal
membrane conus
elasticus false
forms by
the cord
while the
vocal cricovocal
upper ligament.
membranes of
free
Note two
sides.
Figure
56.4
Laryngeal muscles and their action.
(b) Acting on laryngeal inlet (Figure 56.5)
© Openers of laryngeal inlet.
Thyroepiglottic (part of thyroarytenoid)
Interarytenoid (oblique part). Aryepiglottic
(posterior oblique part of interarytenoids)
- 2. Extrinsic muscles. They connect the larynx to the neighbouring structures and are divided into elevators or of
depressors larynx.
- (a) Elevators. Primary elevators act directly as they are attached to the thyroid cartilage and include stylopharyngeus, salpingopharyngeus, palatopharyngeus and
thyrohyoid.
Secondary elevators act indirectly as they are attached to
the hyoid
bone and
include mylohyoid
(main), digastric,
stylohyoid and
geniohyoid.
- (b) Depressors. They include sternohyoid, sternothyroid
and omohyoid.

Laryngeal cavity starts at the laryngeal inlet where it communicates with the pharynx and ends at the lower border of cricoid cartilage where it is continuous with the lumen of trachea. Two pairs of folds, vestibular and vocal, divide the cavity into three parts, namely the vestibule, the ventricle and the subglottic space.
Inlet of larynx. It is an oblique opening bounded anteriorly by free margin of epiglottis; on the sides, by aryepiglottic folds and posteriorly by interarytenoid fold (Figure 56.6).
Vestibule. It extends from laryngeal inlet to vestibular folds. Its anterior wall is formed by posterior surface of epiglottis; sides by the aryepiglottic folds and posterior wall by mucous membrane over the anterior surface of arytenoids.
Ventricle (sinus of larynx). It is a deep elliptical space between vestibular and vocal folds, also extending a short distance above and lateral to vestibular fold. The saccule is a diverticulum of mucous membrane which starts from the anterior part of ventricular cavity and extends upwards between vestibular folds and lamina of thyroid cartilage.
- © Closers of laryngeal inlet:
Figure
56.6
Laryngeal inlet
and intrinsic
muscles of
larynx as
seen from
behind.
Figure 56.7 Rima glottidis. Note anterior two-thirds of vocal cord is membranous and posterior one-third cartilaginous, and the space between them is called phonatory glottis and respiratory glottis, respectively.
When abnormally enlarged and distended, it may form a laryngocele—an air containing sac which may present in the neck. There are many mucous glands in the saccule, which help to lubricate the vocal cords.
Subglottic space (infraglottic larynx). It extends from vocal cords to lower border of cricoid cartilage.

Vestibular folds (false vocal cords). Two in number; each is a fold of mucous membrane extending anteroposteriorly across the laryngeal cavity. It contains vestibular ligament, a few fibres of thyroarytenoideus muscle and mucous glands.
Vocal folds (true vocal cords). They are two pearly white sharp bands extending from the middle of thyroid angle to the vocal processes of arytenoids. Each vocal cord consists of a vocal ligament which is the true upper edge of cricovocal membrane covered by closely bound mucous membrane with scanty subepithelial connective tissue.
Glottis (rima glottidis). It is the elongated space between vocal cords anteriorly, and vocal processes and base of arytenoids posteriorly (Figure 56.7).
Anteroposteriorly, glottis is about 24 mm in men and
16 mm in women. It is the narrowest part of laryngeal cavity. Anterior two-thirds of glottis are formed by membranous cords while posterior one-third by vocal processes of arytenoids. Size and shape of glottis varies with the movements of
vocal cords. Anterior two-thirds of glottis is also called phonatory glottis as it is concerned with phonation but posterior one-third called respiratory glottis.

It lines the larynx and is loosely attached except over the posterior surface of epiglottis, true vocal cords and corniculate and cuneiform cartilages. Epithelium of the mucous membrane is ciliated columnar type except over the vocal cords and upper part of the vestibule where it is stratified squamous type. Mucous glands are distributed all over the mucous lining and are particularly numerous on the posterior surface of epiglottis, posterior part of the aryepiglottic folds and in the saccules. There are no mucous glands in the vocal folds.

Supraglottic larynx above the vocal cords is drained by lymphatics, which pierce the thyrohyoid membrane and go to upper deep cervical nodes. Infraglottic larynx below the vocal cords is drained by lymphatics which pierce cricothyroid membrane and go to prelaryngeal and pretracheal nodes and thence to lower deep cervical and mediastinal nodes. Some vessels pierce through cricotracheal membrane and drain directly into lower deep cervical nodes.
carcinoma
There are practically no lymphatics in vocal cords, hence of this site rarely shows lymphatic metastases.

1. Pre-epiglottic space of Boyer (Figures 56.2 and 56.8). It is bounded by upper part of thyroid cartilage and thyrohyoid membrane in front, hyoepiglottic ligament above and infrahyoid epiglottis and quadrangular membrane behind. Laterally, it is continuous with paraglottic space. It is filled with fat, areolar tissue and some lymphatics.
2. Paraglottic space. It is bounded by the thyroid cartilage laterally, conus elasticus inferomedially, the ventricle and quadrangular membrane medially, and mucosa of pyriform fossa posteriorly (Figures 56.3 and 56.8). It is continuous with pre-epiglottic space. Growths which invade this space can present in the neck through cricothyroid space.
- 3. Reinke's space. Under the epithelium of vocal cords is a potential space with scanty subepithelial connective tissues. It is bounded above and below by the arcuate lines, in front
Figure 56.8
Paraglottic and pre-epiglottic spaces communicate with each other.
by anterior commissure, and behind by vocal process of arytenoid. Oedema of this space causes fusiform swelling of the membranous cords (Reinke's oedema).

Laryngeal mucosa develops from the endoderm of the cephalic part of foregut. Laryngeal cartilages and muscles develop from the mesenchyme. Development of other structures is as follows:
Superior laryngeal nerve, a branch of vagus, is 4th arch nerve and supplies cricothyroid and constrictors of pharynx. Recurrent laryngeal nerve is 6th arch nerve and supplies all the intrinsic muscles of larynx.

The larynx of an infant differs considerably from that of an adult and has a great clinical significance.
1. Infant's larynx is positioned high in the neck level of glottis being opposite to C3 or C4 at rest and reaches Cl or C2 during swallowing. This high position allows the epiglottis to meet soft palate and make a nasopharyngeal channel for nasal breathing during suckling. The milk feed passes separately over the dorsum of tongue and the sides of epiglottis, thus allowing breathing and feeding to go on simultaneously.
2. Laryngeal cartilages are soft and collapse easily. Epiglottis is omega shaped and arytenoids relatively large covering significant portion of the posterior glottis.
3. Thyroid cartilage in an infant is flat. It also overlaps the cricoid cartilage and is in turn overlapped by the hyoid bone. Thus cricothyroid and thyrohyoid spaces are narrow and not easily discernible as landmarks when performing tracheostomy.
4. Infant's larynx is small and conical. The diameter of cricoid cartilage is smaller than the size of glottis, making subglottis the narrowest part. It has a bearing in the selection of paediatric endotracheal tube.
In adults,
subglottic-glottic dimensions
are approxi-
mately same
and larynx
is cylindrical.
- 5. Submucosal tissues of infant's larynx are comparatively loose and easily undergo oedematous change with trauma or inflammation leading to obstruction.
Infant's larynx shows two spurts in growth. In the first 3 years of life, larynx grows in width and length, and thus obviates the need for any airway surgery in certain congenital anomalies. The second spurt in growth occurs during adolescence when the thyroid angle develops. The length of vocal cords then increases leading to voice changes associated with puberty (see puberphonia). With growth of the neck, larynx gradually descends to adult level; the vocal cords lying opposite C;.
In childhood, vocal cord is 6 mm in females and 8 mm in males. It increases to 15-19 mm in adult female and 17-23 in adult male.

The larynx performs the following important functions:
1. Protection of lower airways
2. Phonation
3. Respiration
4. Fixation of the chest.

Phylogenetically, this is the earliest function to develop; voice production is secondary. The larynx protects the lower passages in three different ways:
1. Sphincteric closure of laryngeal opening.
2. Cessation of respiration.
3. Cough reflex.
When food is swallowed, its entry into air passage is prevented by closure of three successive sphincters consisting of (i) laryngeal inlet (aryepiglottic folds, tubercle of epiglottis and arytenoids, approximately closing the laryngeal inlet completely), (ii) false cords and (iii) true cords, which close the glottis. Thus, no foreign matter meant to be swallowed or accidentally vomited can enter the larynx. Respiration temporarily ceases through a reflex gener- ated by afferent fibres of ninth nerve, when food comes
in contact with posterior pharyngeal wall or the base of tongue.
Cough is an important and powerful mechanism to dislodge and expel a foreign particle when it comes into contact with respiratory mucosa. Larynx is aptly called watch-dog of lungs as it immediately "barks" at the entry of any foreign intruder.

Larynx is like a wind instrument. Voice is produced by the following mechanism (aerodynamic myoelastic theory of voice
production):
1. Vocal cords are kept adducted.
2. Infraglottic air pressure is generated by the exhaled air from the lungs due to contraction of thoracic and abdominal muscles.
3. The air force open the cords and is released as small
puffs which vibrate the vocal cords and produce sound
which
is
amplified
by
mouth,
pharynx,
nose
and
chest.
This sound is converted into speech by the modulatory
action of lips, tongue, palate, pharynx and teeth.
Intensity of sound depends on the air pressure produced by the lungs while pitch depends on the frequency with which the vocal cords vibrate.

Larynx regulates flow of air into the lungs. Vocal cords abduct during inspiration and adduct during expiration.

When larynx is closed, chest wall gets fixed and various thoracic and abdominal muscles can then act best. This function is important in digging, pulling and climbing. Coughing, vomiting, defaecation, micturition and childbirth also require a fixed thoracic cage against a closed glottis.

1. Most common cause is automobile accidents when neck
strikes
against
the
steering
wheel
or
the
instrument
panel.
Blow or kick on the neck. . Neck striking against a stretched wire or cable. Strangulation. OU 9 DO
. Penetrating injuries with sharp instruments or gunshot wounds.

The degree and severity of damage will vary from slight bruises externally or the tear and laceration of mucosa inter nally to a comminuted fracture of the laryngeal framework. The wound may be compounded externally due to break in the skin or internally by mucosal tears. Laryngeal fractures are common after 40 years of age because of calcification of the laryngeal framework. In children, cartilages are more resilient and escape injury.
Pathological changes that may be seen in laryngeal
trauma are:
1. Haematoma and oedema of supraglottic or subglottic region.
2. Tears in laryngeal or pharyngeal mucosa leading to sub- cutaneous emphysema.
3. Dislocation of cricoarytenoid joints. The arytenoid cartilage may be displaced anteriorly, dislocated or avulsed.
4.
of
cricothyroid
joint.
This
may
cause
recur-
rent
laryngeal
nerve
paralysis,
which
traverses
just
behind
this
joint.
5. Fractures of the hyoid bone.
6. Fractures of thyroid cartilage. They may be vertical or transverse. Fracture of upper part of thyroid cartilage may result in avulsion of epiglottis and one or both false
cords.
Fractures
of
lower
part
of
thyroid
cartilage
may
dis-
place
or
disrupt
the
true
vocal
cords.
Fractures of cricoid cartilage. on
Fractures of upper tracheal rings.
9. Trachea may separate from the cricoid cartilage and retract into upper mediastinum. Injury to recurrent laryngeal nerve is often associated with laryngotracheal separation.

Symptoms of laryngotracheal injury would vary, greatly depending on the structures damaged and the severity of include:
damage. They
1. Respiratory distress.
2. Hoarseness of voice or aphonia.
3.
Painful
and
difficult
This
is
by
aspiration of food.
4. Local pain in the larynx. More marked on speaking or swallowing.
5. Haemoptysis, usually the result of tears in laryngeal or tracheal mucosa.
External signs include:
1. Bruises or abrasions over the skin.
2. Palpation of the laryngeal area is painful.
3. Subcutaneous emphysema due to mucosal tears. It may increase on coughing.
4. Flattening of thyroid prominence and contour of ante-
rior
cervical
region.
Thyroid
notch
may
not
be
palpable.
5. Fracture displacements of thyroid or cricoid cartilage
or
hyoid
bone.
Gap
may
be
felt
between
the
fractured
6. Bony crepitus between fragments of hyoid bone, thyroid or cricoid cartilages may sometimes be elicited.
7. Separation of cricoid cartilage from larynx or trachea.

1. Indirect laryngoscopy. If patient’s condition permits, this is the most valuable examination. It may reveal location and degree of oedema, haematoma, mucosal lacerations, posterior displacement of epiglottis, exposed fragments of cartilage, asymmetry of glottis or laryngeal inlet.
2.
Direct
laryngoscopy.
It
is
rarely
informative
in
early
period
following
injury.
If
it
may
precipitate
distress
and
immediate
tracheos-
tomy.
Fibreoptic
laryngoscopy
gives
improved
visualiza-
tion
and
has
replaced
direct
in
recent
years.
3. X-rays. Soft tissue lateral film of the neck is very useful and may reveal subcutaneous emphysema, swelling of laryngeal mucosa, displacement of epiglottis, fracture displacements of hyoid bone, thyroid and cricoid carti- lages or change in the configuration of air column.
288
SECTION V — DISEASES OF LARYNX AND TRACHEA
4. CT scan. It is very valuable in assessing moderately severe or severe injuries of larynx. Presently three-dimensional CT is found more useful in trauma.
laryngeal 5. Associated injuries. It is essential to examine for other injuries like injury to head, cervical spine, chest, abdo- men and extremities. X-ray chest for pneumothorax and gastrograffin swallow for oesophageal tears may be required.

1. Patient should be hospitalized and observed for respira- tory distress.
2. Voice rest is essential.
3. Humidification of inspired air is essential.
4. Steroid therapy should be started immediately and in full dose. It helps to resolve oedema and haematoma and prevent scarring and stenosis.
5. Antibiotics are given to prevent perichondritis and carti- lage necrosis.

1. Tracheostomy. Endotracheal intubation in cases of laryn- geal trauma may be difficult and hazardous. Tracheos- tomy is preferred in these cases.
2. Open reduction. Ideally, it is done 3-5 days after injury and if possible should not be delayed beyond 10 days.
(a) Fractures of hyoid bone, thyroid or cricoid cartilage can be wired and replaced in their anatomic posi- tions. Miniplates made of titanium can be used for immobilization of cartilaginous fragments.
(b) Mucosal lacerations are repaired with catgut and any loose fragments of cartilage removed.
(c) Epiglottis is anchored in its normal position and if already avulsed, may be excised.
(d) Arytenoid cartilages can be repositioned in their normal position or may be removed if completely avulsed.
(e) In laryngotracheal separation, end-to-end anastomo- sis can be done.
(f) Internal splintage of laryngeal structures may be required. It is done with a laryngeal stent, or silicone tube which may have to be left for 2-6 weeks on an average.
(g) Webbing of anterior commissure can be prevented by a silastic keel.

1. Laryngeal stenosis, which may be supraglottic, glottic or subglottic.
2. Perichondritis and laryngeal abscess.
3. Vocal cord paralysis.

The infectious typeis more common and usually follows upper respiratory infection. To begin with, it is viral in origin but soon bacterial invasion takes place with Streptococcus pneumoniae, Haemophilus influenzae and haemolytic Streptococci or Staphylococcus aureus. Exanthematous fevers like measles, chickenpox and whooping cough are also associated with laryngitis. The noninfectious typeis due to vocal abuse, allergy, thermal or chemical burns to larynx due to inhalation or ingestion of various substances, or laryngeal trauma such as endotracheal intubation.

Symptoms are usually abrupt in onset and consist of:
1. Hoarseness which may lead to complete loss of voice.
2. Discomfort or pain in throat, particularly after talking.
3. Dry, irritating cough which is usually worse at night.
4. General symptoms of head cold, rawness or dryness of throat, malaise and fever if laryngitis has followed viral infection of upper respiratory tract.
5.
When
there
is
secondary
infection
with
fever
and
toxaemia
or
purulent
expectoration.
Analgesics. To relieve local pain and discomfort. no
Steroids. Useful in laryngitis following thermal or chemi- cal burns.
Acute membranous laryngitis. This condition is similar to acute membranous tonsillitis and is caused by pyogenic nonspecific organisms. It may begin in the larynx or may be an extension from the pharynx. It should be differentiated from
laryngeal diphtheria.

It is an acute inflammatory condition confined to supraglottic structures, i.e. epiglottis, aryepiglottic folds and arytenoids. There is marked oedema of these structures which obstruct the may airway.

It is a serious condition and affects children of 2-7 years of age but can also affect adults. H. influenzae B is the most common organism responsible for this condition in children.

Laryngeal appearances vary with severity of disease. In early stages, there is erythema and oedema of epiglottis, aryepiglottic folds, arytenoids and ventricular bands, but the vocal cords appear white and near normal and stand out in contrast to surrounding mucosa, betraying the degree of hoarseness patient has. Later, hyperaemia and swelling increase. Vocal cords also become red and swollen. Subglottic region also gets involved. Sticky secretions are seen between the cords and interarytenoid region. In case of vocal abuse, submucosal haemorrhages may be seen in the vocal cords.
1. Onset of symptoms is abrupt with rapid progression.
2. Sore throat and dysphagia are the common presenting symptoms in adults.
3. Dyspnoea and stridor are the common presenting symp-
toms in children. They are rapidly progressive and may prove fatal unless relieved.
4. Fever may go up to 40°C. It is due to septicaemia. Patient’s condition may rapidly deteriorate.

1. Vocal rest. This is the most important single factor. Use of voice during acute laryngitis may lead to incomplete or delayed recovery.
2. Avoidance of smoking and alcohol.
3. Steam inhalations. It is done with Tr. Benzoin Co, oil of eucalyptus or pine are soothing and loosen viscid secretions.
4. Cough sedative. To suppress troublesome irritating cough.
1. Depressing the tongue with a tongue depressor may
show
red
and
swollen
epiglottis.
Indirect
laryngos-
copy
may
show
oedema
and
congestion
of
supraglot-
tic
This
is
avoided
for
fear
of
precipitating
complete
It
is
better
done
in operation theatre where facilities for intubation are available.
2. Lateral soft tissue X-ray of neck may show swollen epiglot- tis (thumb sign).
1. Hospitalization. Essential because of the danger of respi- ratory obstruction.
2. Antibiotics. Ampicillin or third generation cephalospo- rin are effective against H. influenzae and are given by parenteral route (i.m. or i.v.) without waiting for results of throat swab and blood culture.
3. Steroids. Hydrocortisone or dexamethasone is given in
appropriate doses i.m. or i.v. They relieve oedema and
may obviate need for tracheostomy.
4, Adequate hydration. Patient may require parenteral fluids.
5. Humidification and oxygen. Patient may require mist tent or a croupette.
6. Intubation or tracheostomy. It may be required for respi- ratory obstruction.

It is an inflammatory condition of the larynx, trachea and
bronchi; more common than acute epiglottitis.

Mostly, it is viral infection (parainfluenza type I and II)
affecting children between 6 months and 3 years of age.
Male children are more often affected. Secondary bacte-
rial infection by Gram-positive cocci soon supervenes.

The loose areolar tissue in the subglottic region swells up
and causes respiratory obstruction and stridor. This, cou-
pled
with
thick
tenacious
secretions
and
crusts,
may
com-
pletely occlude the airway.

Disease starts as upper respiratory infection with hoarseness
and croupy cough. There is fever of 39-40°C. This may be

followed by difficulty in breathing and inspiratory type of stridor. Respiratory difficulty may gradually increase with signs of upper airway obstruction, i.e. suprasternal and inter- costal recession. Differences between acute epiglottitis and acute laryngo-tracheo-bronchitis are given in Table 58.1.

1. Hospitalization is often essential because of the increas- ing difficulty in breathing.
2. Antibiotics like ampicillin 50 mg/kg/day in divided doses are effective against secondary infections due to Gram-positive cocci and H. influenzae.
3. Humidification helps to soften crusts and tenacious secretions which block tracheobronchial tree.
4. Parenteral fluids are essential to combat dehydration.
5. Steroids, e.g. hydrocortisone 100 mg i.v. may be useful to relieve oedema.
6. Adrenaline, racemic adrenaline administered via a respi- rator is a bronchodilator and may relieve dyspnoea and avert tracheostomy.
7. Intubation/ tracheostomy is done, should respiratory obstruction increase in spite of the above measures. Tra- cheostomy is done if intubation is required beyond 72 h. Assisted
ventilation may be required.

Mostly, it is secondary to faucial diphtheria affecting chil- dren below 10 years of age. Incidence of diphtheria in gen- eral is declining due to widespread use of immunization.

Effects of laryngeal diphtheria are due to:
1. Formation of a tough pseudomembrane over the larynx and trachea which may completely obstruct the airway.
Acute laryngo-tracheo-bronchitis (or group)
Causative organism
Haemophilus influenzae type B
Age
2-7 years
Pathology
Supraglottic larynx
Prodromal symptoms
Absent
Onset
Sudden
Fever
High
Patient’s look
Toxic
Cough
Usually absent
Stridor
Present and may be marked
Odynophagia
Present, with drooling of secretions
Radiology
«Thumb sign on lateral view
Parainfluenza virus type | and II
3 months to 3 years
Subglottic area
Present
Slow
Low grade or no fever
Nontoxic
Present (barking seal-like)
Present
Steeple sign on anteroposterior view of neck
Treatment
Humidified oxygen, third generation cephalosporin
Humidified O, tent, steroids
(ceftriaxone) or amoxicillin
@Examination of larynx and radiographs are avoided lest complete obstruction is precipitated. Examination is done in the operation theatre where immediate intubation can be done.
2. Exotoxin liberated by bacteria leading to myocarditis and various
neurological complications.

¢ General symptoms. Onset is insidious with low-grade fever (100-101°F), sore throat and malaise but patient is very toxaemic with tachycardia and thready pulse.
e
Laryngeal
symptoms.
Hoarse
voice,
croupy
cough,
inspi-
ratory
stridor,
dyspnoea
with
marked
upper
airway
¢
Membrane.
Greyish
white
membrane
is
seen
on
the
tonsil,
pharynx
and
soft
palate.
It
is
adherent
and
its
removal
leaves
a
bleeding
surface.
Similar
membrane
is
seen
over
the
larynx
and
trachea.
¢ Cervical lymphadenopathy. Characteristic “bull-neck” may be seen.

Laryngeal diphtheria is mostly secondary to faucial diphthe- ria. Diagnosis is always clinical but confirmed by smear and culture of Corynebacterium diphtheriae. Treatment is started on clinical suspicion.

1. Diphtheria antitoxin. Dose depends on clinical severity and duration of illness, and varies from 20,000 to 100,000 units i.v. route as saline infusion after a test dose. It neu-
tralizes free toxin circulating in the blood.
2. Antibacterials. Benzylpenicillin, 500,000 units im. every 6h for 6 days, is effective against diphtheria bacilli. Eryth- romycin can be given to those who are allergic to penicillin.
3.
Maintenance
of
airway.
Tracheostomy
may
become
essen-
tial.
Direct
laryngoscopy,
removal
of
diphtheritic
mem-
brane
and
can
be
done.
Intubation
relieves
and
can
make
trache-
ostomy easy.
4. Complete bed rest. Complete bed rest for 2-4 weeks is essential to guard against effects of myocarditis.

1. Asphyxia and death due to airway obstruction.
2. Toxic myocarditis and circulatory failure.
3. Palatal paralysis with nasal regurgitation.
4. Laryngeal and pharyngeal paralysis.

Often termed “oedema glottidis” in the past, it involves the supraglottic and subglottic region where laryngeal mucosa is loose. Oedema of the vocal cords occurs rarely because of the sparse subepithelial connective tissue.

1. Infections
(a) Acute epiglottitis, laryngo-tracheo-bronchitis, tuber- culosis or syphilis of larynx.
(b) Infection in neighbourhood, e.g. peritonsillar abscess, retropharyngeal abscess and Ludwig’s angina.
2. Trauma. Surgery of tongue, floor of mouth, laryngeal trauma, foreign body, endoscopy especially in children, intubation, thermal or caustic burns or inhalation or irri- tant or fumes.
gases
3. Neoplasms. Cancer of larynx or laryngopharynx often associated with deep ulceration.
4. Allergy. Angioneurotic oedema or anaphylaxis.
Radiation. For cancer of larynx or pharynx.
6. Systemic diseases. Nephritis, heart failure or myxoedema.

1. Airway obstruction. Degree of respiratory distress varies. Tracheostomy may become essential.
2. Inspiratory stridor.
3. Indirect laryngoscopy. It shows oedema of supraglot-
tic or subglottic region. Children may require direct laryngoscopy.

If there is airway obstruction, intubation of larynx or tra- cheostomy will be immediately required. Less severe cases are treated conservatively and treatment will depend on the cause. An injection of adrenaline (1:1000) 0.3-0.5ml i.m., repeated in 15 min if necessary, is useful in allergic or angioneurotic oedema. Steroids are useful in epiglottitis, laryngo-tracheo-bronchitis or oedema due to traumatic allergic or postradiation causes.

It is a diffuse inflammatory condition symmetrically involv- ing the whole larynx, i.e. true cords, ventricular bands,
interarytenoid region and root of the epiglottis.

1. It may follow incompletely resolved acute simple laryngi- tis or its recurrent attacks.
2. Presence of chronic infection in paranasal sinuses, teeth
and
tonsils
and
the
chest
are
important
contributory
causes.
3. Occupational factors, e.g. exposure to dust and fumes
such as in miners, strokers, gold or iron smiths and work-
ers in chemical industries.
4. Smoking and alcohol.
5. Persistent trauma of cough as in chronic lung diseases.
6. Vocal abuse.

1.
Hoarseness.
This
is
the
commonest
complaint.
Voice
becomes
easily
tired
and
patient
becomes
aphonic
by
the
end of the day.
2. Constant hawking. There is dryness and intermittent tick- ling in the throat and patient is compelled to clear the throat repeatedly.
3. Discomfort in the throat.
4. Cough. It is dry and irritating.
Laryngeal examination. There is hyperaemia of laryngeal structures. Vocal cords appear dull red and rounded. Flecks of viscid mucus are seen on the vocal cords and interarytenoid region.

1. Eliminate infection of upper or lower respiratory tract. Infection in the sinuses, tonsils, teeth or chronic chest infection (bronchitis, bronchiectasis, tuberculosis, etc.) should be treated.
2. Avoidance of irritating factors. E.g. smoking, alcohol or polluted environment, dust and fumes.
3. Voice rest and speech therapy. Voice rest has to be pro- longed for weeks or months. Patient should receive train- ing in proper use of voice.
4. Steam inhalations. They help to loosen secretions and give relief.
5. Expectorants. They help to loosen viscid secretions and give relief from hawking.

It may be either a diffuse and symmetrical process or a localized one, the latter appearing like a tumour of the larynx. Localized variety presents as dysphonia plica ventricularis, vocal nodules, vocal polyp, Reinke's oedema and contact ulcer. (They have been described in the relevant sections.)

Pathological changes start in the glottic region and later may extend to ventricular bands, base of epiglottis and even subglottis. Mucosa, submucosa, mucous glands and in later stages intrinsic laryngeal muscles and joints may be affected. Initially, there is hyperaemia, oedema and cellular infiltration in the submucosa. The pseudostratified ciliated epithelium of respiratory mucosa changes to squamous type, and squamous epithelium of the vocal cords to hyperplasia and keratinization. The mucous glands suffer hypertrophy at first but later undergo atrophy with diminished secretion and dryness of larynx.

This disease mostly affects males (8:1) in the age group of 30-50 years.
Hoarseness, constant desire to clear the throat, dry cough,
tiredness
of
voice
and
in
throat
when
the
voice
has
been
used
for
an
extended
period
of
time
are
the
com-
mon presenting symptoms.
Examination. On examination, changes are often diffuse and symmetrical.
1. Laryngeal mucosa, in general, is dusky red and thickened.
2.
Vocal
cords
appear
red
and
swollen.
Their
edges
lose
sharp
demarcation
and
appear
rounded.
In
late
stages,
cords become bulky and irregular giving nodular appearance.
3. Ventricular bands appear red and swollen and may be mistaken for prolapse or eversion of the ventricle.
4. Mobility of cords gets impaired due to oedema and infil-
tration,
and
later
due
to
muscular
atrophy
or
arthritis
of
the
cricoarytenoid
joint.

1. Conservative. Same as for chronic laryngitis without hyperplasia.
2. Surgical. Stripping of vocal cords, removing the hyper- plastic and oedematous mucosa, may be done in selected cases. Damage to underlying vocal ligament should be care- fully avoided. One cord is operated at a time.

It is bilateral symmetrical swelling of the whole of membranous part of the vocal cords, most often seen in middle-aged men and women. This is due to oedema of the subepithelial space (Reinke's space) of the vocal cords. Chronic irritation of vocal cords due to misuse of voice, heavy smoking, chronic sinusitis and laryngopharyngeal reflex are the probable aetiological factors. It can also occur in myxoedema.

Hoarseness is the common symptom. Patient uses false cords for voice production and this gives him a low-pitched and rough voice. On indirect laryngoscopy, vocal cords appear as fusiform swellings with pale translucent look. Ventricular bands may appear hyperaemic and hypertrophic and may hide the view of the true cords.

1.
Decortication
of
the
vocal
cords,
i.e.
removal
of
strip
of
epithelium,
is
done
first
on
one
side
and
3-4
weeks
later
on the other.
Voice rest.
Speech therapy for proper voice production.

It is a form of chronic hypertrophic laryngitis affecting pos- terior part of larynx in the region of interarytenoid and pos- terior part of the vocal cords.
Clinically, patient presents with hoarseness or husky voice and irritation in the throat. Indirect laryngoscopy reveals heaping up of red or grey granulation tissue in the interarytenoid region and posterior thirds of vocal cords; the latter sometimes showing ulceration due to constant hammering of vocal processes as in talking, forming what is called “contact ulcer.” The condition is bilateral and sym- metrical. It does not undergo malignant change. However, biopsy of the lesion is essential to differentiate the lesion
from carcinoma and tuberculosis. Aetiology is uncertain. It is mostly seen in men who indulge in excessive alcohol and smoking. Other factors are excessive forceful talking and gastro-oesophageal reflux disease where posterior part of larynx is being constantly bathed with acid juices from the stomach. Treatment is removal of granulation tissue under oper- ating microscope which may require repetition, control of acid reflux and speech therapy.

It is characterized by atrophy of laryngeal mucosa and crust formation. Condition is often seen in women and is associated with atrophic rhinitis and pharyngitis.
Common symptoms include hoarseness of voice which temporarily improves on coughing and removal of crusts. Dry irritating cough and sometimes dyspnoea is due to obstructing crusts.
Examination shows atrophic mucosa covered with foul- smelling
crusts.
When crusts
have been
expelled, mucosa
may show
excoriation and
bleeding.
Crusting may
also be
seen in
the trachea.
Treatment is elimination of the causative factor and humidification. Laryngeal sprays with glucose in glycerine or oil of pine are comforting and help to loosen the crusts. Associated nasal and pharyngeal conditions will require attention. Expectorants containing ammonium chloride or iodides also to loosen the crusts.
help

It is almost always secondary to pulmonary tuberculosis, mostly affecting males in middle age group. Tubercle bacilli reach the larynx by bronchogenic or haematogenous routes.

Disease affects posterior part of larynx more than anterior. Parts affected are: (i) interarytenoid fold, (ii) ventricular bands, (iii) vocal cords and (iv) epiglottis, in that order. Tubercle bacilli, carried by sputum from the bronchi, settle and penetrate the intact laryngeal mucosa particularly in the interarytenoid region (bronchogenic spread). This leads to formation of submucosal tubercles which may later caseate and ulcerate. Laryngeal mucosa appears red and swollen due to cellular infiltration (pseudoedema). Stages of perichondritis and cartilage necrosis are not commonly seen these days.

They would greatly depend on the stage of tuberculosis. Weakness of voice is the earliest symptom followed by hoarseness. Ulceration in the larynx gives rise to severe pain which may radiate to the ears. Swallowing is painful with marked in later dysphagia stages.

1. Hyperaemia of the vocal cord in its whole extent or con- fined to posterior part with impairment of adduction is the first sign.
2. Swelling in the interarytenoid region giving a mamillated appearance.
3. Ulceration appearance. of vocal cord giving mouse-nibbled
4. Superficial ragged ulceration on the arytenoids and interarytenoid region.
5. Granulation tissue in interarytenoid region or vocal pro- cess of arytenoid.
Pseudoedema of the epiglottis “turban epiglottis.” No
Swelling of ventricular bands and aryepiglottic folds.
8. Marked pallor of surrounding mucosa.

In addition to X-ray chest and sputum examination, biopsy of laryngeal lesion is essential to exclude carcinoma and differentiate it from other condition.

Treatment is the same as for pulmonary tuberculosis. Voice rest is important.

It is an indolent tubercular infection associated with lupus of nose and pharynx. Unlike tuberculosis of larynx which mostly affects posterior parts, lupus involves the anterior part of larynx. Epiglottis is involved first and may be completely destroyed by the disease. The lesion spreads to aryepiglottic folds and sometimes to ventricular bands. Lupus of larynx is a painless and often an asymptomatic condition and may be discovered on routine laryngeal examination in cases of lupus of nose. There is no pulmonary tuberculosis. Treatment is antitubercular drugs. Prognosis is good.

It is a rare condition now. Only gumma of tertiary stage is sometimes seen. It may occur in any part of the larynx and present as a smooth swelling which may later ulcerate. Diagnosis is only on biopsy and serological tests. Laryngeal stenosis is a frequent complication.

It is a rare condition and is often associated with leprosy of the skin and nose. It presents as diffuse nodular infiltration of epiglottis, aryepiglottic folds and arytenoids. Lesions may ulcerate. It is associated with nasal leprosy. Diagnosis is made on biopsy from the lesion. Deformity of the laryngeal inlet and stenosis are the end results of this disease after healing.

combined with steroids to prevent fibrosis. Subglottic stenosis is a frequent complication requiring subsequent reconstructive surgery.

Fungal infections such as candidiasis, histoplasmosis and blastomycosis may rarely affect the larynx. Diagnosis is usu-
ally made on biopsy and on finding a similar lesion in other parts of the body.

Laryngomalacia (congenital laryngeal stridor)
Congenital vocal cord paralysis
Congenital subglottic stenosis
Laryngeal web
Subglottic haemangioma
Laryngo-oesophageal cleft
Laryngocele
Laryngeal cyst
ecoeooeeee
3. Congenital subglottic stenosis. It is due to abnormal thickening of cricoid cartilage or fibrous tissue seen below the vocal cords. Child may remain asymptomatic till upper respiratory infection causes dyspnoea and stridor. Cry is normal as in laryngomalacia. Diagnosis is made when subglottic diameter is less than 4 mm in full-term neonate (normal 4.5-5.5 mm) or 3 mm in premature neonate (normal 3.5 mm). Many cases of congenital stenosis improve as the larynx grows but some may require surgery.
1. Laryngomalacia (congenital laryngeal stridor). It is the most common congenital abnormality of the larynx. It is characterized by excessive flaccidity of supraglottic larynx which is sucked in during inspiration producing stridor and sometimes cyanosis. Stridor is increased on crying but subsides on placing the child in prone position; cry is normal. The condition manifests at birth or soon after, and usually disappears by 2 years of age. Direct laryngoscopy shows elongated epiglottis, curled upon itself (omega-shaped @), floppy aryepiglottic folds and prominent arytenoids. Flexible laryngoscope is very useful to make the diagnosis. Laryngomalacia cannot be diagnosed in a paralyzed patient. Mostly, treatment is conservative. Tracheostomy may be required for some cases of severe respiratory obstruction (Figure 59.1). Supraglottoplasty is required in cases of severe laryngomalacia.
2. Congenital vocal cord paralysis. It results from birth trauma when recurrent laryngeal nerve is stretched during breech or forceps delivery or can result from anomalies of the central nervous system.
Figure 59.1
Laryngomalacia. Note: epiglottis is folded longitudinally

4. Laryngeal web (Figure 59.2). It is due to incomplete recanalization of larynx. Mostly, the web is seen between the vocal cords and has a concave posterior margin. Presenting features are airway obstruction, weak cry or aphonia dating from birth. Treatment depends on the thickness of the web. Thin webs can be cut with a knife or COg laser. Thick ones may require excision via laryngofissure and placement of a silicon keel and subsequent dilatations.
5. Subglottic haemangioma. Though congenital, patient is asymptomatic till 3-6 months of age when haemangioma begins to increase in size. About 50% of the children have associated cutaneous haemangiomas. Patient may present with stridor but has a normal cry. Agitation of the patient or crying may increase airway obstruction due to venous filling. Direct laryngoscopy shows reddish-blue mass below the vocal cords. Biopsy is sometimes, not always, associated with haemorrhage. Some patients have associated mediastinal haemangioma.
Depending on individual case, the treatment is:
(a) Tracheostomy and observation, as many haemangiomas involute spontaneously. (b) Steroid therapy. Dexamethasone 1 mg/kg/day for 1 week and then prednisolone 3 mg/kg in divided doses for | year.
- (c) CQOg laser excision, if lesion is small.
6. Laryngo-oesophageal cleft. It is due to failure of the fusion of cricoid lamina. Patient presents with repeated aspiration and pneumonitis. Coughing, choking and cyanosis are present at the time of feeding.
7. Laryngocele. It is dilatation of laryngeal saccule and extends between thyroid cartilage and the ventricle. It may be internal, external or combined. Treatment is endoscopic or external excision.

8. Laryngeal cyst. It arises in the aryepiglottic fold and appears as bluish, fluid-filled smooth swelling in the supraglottic larynx. Respiratory obstruction may necessitate tracheostomy. Needle aspiration or incision and drainage of cyst provide an emergency airway. Treatment is deroofing the cyst or excision with COg laser.

Stridor is noisy respiration produced by turbulent airflow through the narrowed air passages. It may be heard during inspiration, expiration or both (Figure 59.3).
¢ Inspiratory stridor is often produced in obstructive lesions
of supraglottis or pharynx, e.g. laryngomalacia or retropharyngeal abscess.
¢ Expiratory stridor is produced in lesions of thoracic trachea,
primary and
secondary bronchi,
e.g.
bronchial foreign
body, and
tracheal stenosis.
- ¢ Biphasic stridor is seen in lesions of glottis, subglottis and cervical trachea, e.g. laryngeal papillomas, vocal cord paralysis and subglottis stenosis.

Stridor may arise from lesions of nose, tongue, mandible, pharynx, larynx or trachea and bronchi. Common causes of stridor in infants and children are given below:

e
Infectious mononucleosis
a . Nose. Choanal atresia in newborn.
. Tongue. Macroglossia due to cretinism, haemangioma or lymphangioma, dermoid at base of tongue, lingual thyroid. no
3.
Mandible.
Micrognathia,
Pierre-Robin syndrome.
In these
cases, stridor
is due
to falling
back of
tongue.
- 4. Pharynx. Congenital dermoid, adenotonsillar hypertro-
phy, retropharyngeal abscess, tumours.
- 5. Larynx.
(a) Congenital. Laryngeal web, laryngomalacia, cysts, vocal cord paralysis, subglottic stenosis.
e
(b)
Figure
59.3
Types of
stridor and
their site
of origin.
(b) Inflammatory. Epiglottitis, laryngotracheitis, diphtheria, tuberculosis.
(c) Neoplastic. Haemangioma and juvenile multiple papillomas, carcinoma in adults.
(d)
Traumatic.
Injuries of
larynx, foreign
bodies, oedema
following endoscopy,
(e) Neurogenic. Laryngeal paralysis due to acquired lesions.
(f) Miscellaneous. Tetanus, tetany, laryngismus stridulus.
6. Trachea and bronchi
(a) Congenital. Atresia, stenosis, tracheomalacia.
(b) Inflammatory. Tracheobronchitis.
- (c) Neoplastic. Tumours of trachea.
(d)
Foreign body,
stenosis trachea
(e.g.
ing prolonged intubation or tracheostomy).
- 7. Lesions outside respiratory tract
(a)
Congenital.
Vascular rings
(cause gia),
oesophageal tula,
dyspha- tracheo-oesophageal

goitre,
Inflammatory.
cystic fis-
hygroma.
Retropharyngeal and
_retro-oesopha- geal
abscess.
(c) Traumatic. FB oesophagus compression). (secondary tracheal
(d) Tumours. Masses in neck.

Stridor is a physical sign and not a disease. Attempt should always be made to discover the cause. It is important to elicit:
1. Time of onset. To find whether cause is congenital or acquired.
2. Mode of onset. Sudden onset (foreign body, oedema), gradual and progressive (laryngomalacia, subglottic haemangioma, juvenile papillomas).
3. Duration. Short (foreign body, oedema, infections), long
(laryngomalacia, laryngeal
stenosis, subglottic
haeman- gioma, anomalies of tongue and jaw).
- 4. Relation to feeding. Aspiration in laryngeal paralysis,
oesophageal atresia, laryngeal cleft, vascular ring, foreign body oesophagus.
Cyanotic spells. Indicate need for airway maintenance.
Laryngeal trauma. Blunt injuries to larynx, intubation, endoscopy.
Aspiration or ingestion of a foreign body. ND
follow-

1. Stridor is always associated with respiratory distress. There may be recession in suprasternal notch, sternum, intercostal spaces and epigastrium during inspiratory efforts.
2. Note whether stridor is inspiratory, expiratory or bipha- sic which indicates the probable site of obstruction.
3. Note associated characteristics of stridor.
(a) Snoring or snorting sound—nasal or nasopharyn- geal cause.
(b) Gurgling sound and muffled voice—pharyngeal cause.
(c) Hoarse cry or voice—laryngeal cause at vocal cords. Cry is normal in laryngomalacia and subglottic stenosis.
(d) Expiratory wheeze—bronchial obstruction.
4. Associated fever indicates infective condition, e.g. acute laryngitis, epiglottitis, laryngo-tracheo-bronchitis or diphtheria.
5. Stridor of laryngomalacia, micrognathia, macroglossia and innominate artery compression disappears when baby lies in prone position.
6. Sequential auscultation with unaided ear and with stetho-
scope
over
the
nose,
open
mouth,
neck
and
the
chest
helps
to
localize
the
probable
site
of
origin
of
stridor.
7. Examination of nose, tongue, jaw and pharynx and lar- ynx can exclude local pathology in these areas. In adults, indirect laryngoscopy can be done easily while infants and children require flexible fibreoptic laryngoscopy.
Flexible fibreoptic laryngoscopy. It can be done under topi- cal anaesthesia as an outdoor procedure and allows exami- nation of nose, nasopharynx and larynx. It helps in the diagnosis of laryngomalacia, vocal cord paralysis, laryngeal papillomas, laryngeal cysts and congenital anomalies of lar- ynx, e.g. laryngeal web or clefts.

History and clinical examination will dictate the type of tests required.
1. Soft tissue lateral and PA view of neck and X-ray chest in PA and lateral view help in diagnosing the foreign bodies of the airway.
2.
X-ray
chest
in
and
expiratory
phases
or
a
flu-
oroscopy
of
chest
help
to
diagnose
radiolucent
foreign
bodies.
3. CT scan with contrast is helpful for mediastinal mass and
other
congenital
vascular
anomalies
compressing
the
tra-
chea
or
bronchi,
e.g.
anomalous
innominate
artery,
dou-
ble aortic arch or an anomalous left pulmonary artery forming a sling around the trachea.
4. Angiography may be needed for above vascular anoma- lies before operation.
5. Oesophagogram with contrast may be needed for tra- cheobronchial fistula or aberrant vessels or oesophageal atresia.

Microlaryngoscopy and bronchoscopy under general anaesthe- sia. This procedure is done in operation theatre with full preparation for resuscitative measures to deal with respira- tory distress. Patient is monitored for oxygen saturation, pulse, blood pressure and electrocardiography. Services of an expert anaesthetist are essential. Anaesthesia is induced with insufflation and i.v. route established. Patient is kept on spontaneous respiration.
After a quick and short direct laryngoscopy, broncho- scope is inserted to examine the air passage from the sub- glottis to bronchi for any obstruction. Secretions can be collected for culture and sensitivity, crusts and foreign body if any removed. After bronchoscopy, child is intubated and examination of larynx or oesophagus can be done.
Microlaryngoscopy
can
be
done
without
with
patient
on
breathing
and
oxygen
and
gases
being
delivered
through
a
catheter
via
the
laryngoscope.
Magnification
can
be
provided
with
telescope
or
microscope.

Once the diagnosis has been made, treatment of exact cause can be planned.

Motor. All the muscles which move the vocal cord (abduc- tors, adductors or tensors) are supplied by the recurrent laryngeal nerve except the cricothyroid muscle. The latter receives its innervation from the external laryngeal nerve— a branch of superior laryngeal nerve.
Sensory. Above the vocal cords, larynx is supplied by inter- nal laryngeal nerve—a branch of superior laryngeal, and below the vocal cords by recurrent laryngeal nerve.
Recurrent laryngeal nerve. Right recurrent laryngeal nerve arises from the vagus at the level of subclavian artery, hooks around it and then ascends between the trachea and oesophagus. The left recurrent laryngeal nerve arises from the vagus in the mediastinum at the level of arch of aorta, loops around it and then ascends into the neck in the tracheo-oesophageal groove. Thus, left recurrent laryngeal nerve has a much longer course which makes it more prone to paralysis compared to the right one (Figure 60.1).
Superior laryngeal nerve. It arises from inferior ganglion of the vagus, descends behind internal carotid artery and, at the level of greater cornua of hyoid bone, divides into external and internal branches. The external branch supplies cricothyroid muscle while the internal branch pierces the thyrohyoid membrane and supplies sensory innervation to the larynx and hypopharynx.

Laryngeal paralysis may be unilateral or bilateral, and may involve:
1.
Recurrent laryngeal nerve.
2. Superior laryngeal nerve.
3. Both recurrent and superior laryngeal nerves (combined
or complete paralysis).

In topographical manner, the causes are:
1. Supranuclear. Rare.
2. Nuclear. There is involvement of nucleus ambiguus in
the medulla. The causes are vascular, neoplastic, motor neurone disease, polio and syringobulbia. In nuclear
lesions, there would be associated paralysis of other cranial nerves and neural pathways.
- 3. High vagal lesions. Vagus nerve may be involved in the
skull, at the exit from jugular foramen or in parapharyn- geal space (Table 60.1).
- Low vagal or recurrent laryngeal nerve (Table 60.2). Systemic causes. Diabetes, syphilis, diphtheria, typhoid, Oe
streptococcal or viral infections, lead poisoning.
- 6. Idiopathic. In about 30% of cases, cause remains obscure.

Unilateral injury to recurrent laryngeal nerve results in ipsilateral paralysis of all the intrinsic muscles except the cricothyroid. The vocal cord thus assumes a median or
artery
paramedian position and does not move laterally on deep inspiration (Table 60.2). There are many theories to explain the median or paramedian position of the cord. One is Semon's law which states that, in all progressive organic lesions, abductor fibres of the nerve, which are phylogenetically newer, are more susceptible and thus the first to be paralyzed compared to adductor fibres. The other explanation is Wagner and Grossman hypothesis which states that cricothyroid muscle which receives innervation from superior laryngeal nerve keeps the cord in paramedian position due to its adductor function.
The aetiology of recurrent laryngeal nerve paralysis is given in Table 60.3. Bronchogenic carcinoma is an important cause of left recurrent paralysis and should always be excluded by X-ray chest, bronchoscopy and biopsy unless the other cause is obvious.

Unilateral recurrent laryngeal paralysis may pass undetected as about one-third of the patients are asymptomatic. Others have some change in voice but no problems of aspiration or airways obstruction. The voice in unilateral paralysis gradually improves due to compensation by the healthy cord which crosses the midline to meet the paralyzed one.
2.
3.
4.

1.
Generally occurs
due recovers
no treatment
to is
required opposite
in healthy
6-12
ever injection
months of
the voice
in the
Laryngoplasty as
compensation cord.
it gelfoam
waiting is
or paralysis
advisable fat
period.
type not
take place.
Laryngoplasty type
posterior glottis
Teflon
I
can
I
be used
with is
also injection
if arytenoid
incompetent.
has been
used in
the past
to wait.
How- be
used to
improve compensation
does adduction
is done
to if
medialize the
cord permanently
but is
not favoured
these days.
Median, Location of the cord from midline = Midline. Median, Situation in.Health = Phonation. Median, Situation in.Disease = RLN paralysis. Paramedian, Location of the cord from midline = 1.5 mm. Paramedian, Situation in.Health = Strong whisper. Paramedian, Situation in.Disease = RLN paralysis. Intermediate (cadaveric), Location of the cord from midline = 3.5 mm. This is neutral position of cricoarytenoid joint. Abduction and adduction take place from this position. Intermediate (cadaveric), Situation in.Health = -. Intermediate (cadaveric), Situation in.Disease = Paralysis of both recurrent and superior laryngeal nerves. Gentle abduction, Location of the cord from midline = 7 mm. Gentle abduction, Situation in.Health = Quiet respiration. Gentle abduction, Situation in.Disease = Paralysis of adductors. Full abduction, Location of the cord from midline = 9.5 mm. Full abduction, Situation in.Health = Deep inspiration. Full abduction, Situation in.Disease = -
can

Neuritis or surgical trauma (thyroidectomy) are the most important causes. The condition is often acute.

As all the intrinsic muscles of larynx are paralyzed, the vocal cords lie in median or paramedian position due to unopposed action of cricothyroid muscles (Figure 60.2).

As both the cords lie in median or paramedian position, the airway is inadequate causing dyspnoea and stridor but the voice is good. Dyspnoea and stridor become worse on exertion or during an attack of acute laryngitis.

Tracheostomy. Many cases of bilateral abductor paralysis require tracheostomy as an emergency procedure or when they develop upper respiratory tract infection. In long-standing cases, the choice is between a permanent tracheostomy with a speaking valve or a surgical procedure to lateralize the cord. The former relieves stridor, preserves good voice but has the disadvantage of a tracheostomy hole in the neck. The latter relieves airway obstruction but at the expense of a good voice; however, there is no tracheostomy hole in the neck.
Widening the respiratory airway without a permanent tracheostomy (endoscopic or through external cervical approach). Aim is to widen the respiratory airway through larynx. This can be achieved by (i) arytenoidectomy with suture, (ii) arytenoidopexy (fixing the arytenoid in lateral position), (iii) lateralization of vocal cord and (iv) laser cor dectomy (removal of one cord). These operations have now been replaced by less invasive techniques such as:
- 1. Transverse cordotomy (Kashima operation). Soft tissue at the junction of membranous cord and vocal process of
arytenoid is excised laterally with laser. This provides good airway. In case airway is still insufficient more tissue can be removed at subsequent operation.
2. Partial arytenoidectomy. Medial part of arytenoid is excised with laser. Sometimes only the vocal process of arytenoid is ablated.
3. Reinnervation procedures. These have been used to innervate paralyzed posterior cricoarytenoid muscle by implant- ing a nerve—muscle pedicle of sternohyoid or omohyoid muscle with its nerve supply from ansa hypoglossi. These procedures have not been very successful.
4. Thyroplasty type II. It creates lateral expansion of larynx and is similar to vocal cord lateralization. Quality of voice may not be good.

Isolated lesions of this nerve are rare; usually, it is a part of combined paralysis. Paralysis of superior laryngeal nerve causes paralysis of cricothyroid muscle and ipsilateral anaesthesia of the larynx above the vocal cord. Paralysis of cricothyroid can also occur when external laryngeal nerve is involved in thyroid surgery, tumours, neuritis or diphtheria.

Voice is weak and pitch cannot be raised with decreased ability to sing. Anaesthesia of the larynx on one side may pass unnoticed or cause occasional aspiration. Laryngeal findings include:
1. Askew position of glottis as anterior commissure is rotated to the healthy side.
2. Shortening of cord with loss of tension. The paralyzed cord appears wavy due to lack of tension.
3. Flapping of the paralyzed cord. As tension of the cord is lost, it sags down during inspiration and bulges up during expiration.
4. Electromyography of the cricothyroid muscle helps to diagnose the condition.

This is an uncommon condition. Both the cricothyroid muscles are with anaesthesia of paralyzed along upper larynx.

Important causes include surgical or accidental trauma, neuritis (mostly diphtheritic), pressure by cervical nodes or involvement in a neoplastic process.

Presence of both paralysis and bilateral anaesthesia causes inhalation of food and pharyngeal secretions giving rise to and fits. Voice is weak and cough choking husky.

It depends on the cause. Cases due to neuritis may recover spontaneously. Patients with repeated aspiration may
require tracheostomy with a cuffed tube and an oesophageal feeding tube. Epiglottopexy is an operation to close the laryngeal inlet to protect the lungs from repeated aspiration. It is a reversible procedure.

This causes paralysis of all the muscles of larynx on one side except the interarytenoid which also receives innervation from the opposite side.

Thyroid surgery is the most common cause when both recurrent and external laryngeal nerves of one side may be involved.
It may also occur in lesions of nucleus ambiguus or that of the vagus nerve proximal to the origin of superior laryngeal nerve. Thus, lesion may lie in the medulla, posterior cranial fossa, jugular foramen or parapharyngeal space.

As all the muscles of larynx on one side are paralyzed, vocal cord will lie in the cadaveric position, i.e. 3.5 mm from the midline (Table 60.2). The healthy cord is unable to approximate the paralyzed cord, thus causing glottic incompetence. This results in hoarseness of voice and aspiration of liquids through the glottis. Cough is ineffective due to air waste.

1. Speech therapy. With proper speech therapy, the healthy cord may compensate the loss of function of paralyzed vocal cord across the midline.
by moving 2. Procedures to medialize the cord. In uncompensated cases, aim is to bring the paralyzed cord towards the midline so that healthy cord can meet it. This is achieved by: (a) Injection of teflon paste lateral to the paralyzed cord. This is done by direct laryngoscopy under local anaesthesia. Now thyroplasty is the preferred procedure.
(b) Thyroplasty type I. Vocal cord is medialized towards midline for opposite cord to meet. This can be combined with arytenoids adduction procedure. Thyroplasty is done by creating a window in the thyroid cartilage and placing a silicon or other prosthesis to medialize the cord. Operation can be done under local anaesthesia.

Both recurrent and superior laryngeal nerves on both sides are paralyzed. This is a rare condition. As all the laryngeal muscles are paralyzed, both cords lie in cadaveric position. There is also total anaesthesia of the larynx.

1. Aphonia. As cords do not meet at all.
2. Aspiration. This is due to incompetent glottis and laryngeal anaesthesia.
3. Inability to cough. This is due to inability of the cords to meet. This results in retention of secretions in the chest.
4. Bronchopneumonia. This is due to repeated aspirations and retention of secretions.

1. Tracheostomy. Essential to remove pulmonary secretions and inhaled material.
2. Gastrostomy. It will prevent aspiration and maintain nutrition.
3.
Epiglottopexy.
It
is
an
operation
in
which
is
folded
backwards
and
fixed
to
the
arytenoids
so
as
to prevent aspiration into the lungs. It is a reversible procedure.
4.
Vocal
cord
plication.
Larynx
is
opened
by
laryngofis-
sure.
Mucosa
of
the
true
and
false
cords
is
removed
and
then
they
are
approximated
with
sutures.
This
procedure
helps
to
prevent
and
can
be
reversed
when
required.
5. Total laryngectomy. May be needed in those where cause
is
and
irreversible
and
speech
is
unservice-
able.
Laryngectomy
will
prevent
repeated
and
lung
6. Diversion procedures. Trachea is separated at third or fourth rings and its upper segment (laryngotracheal) is anastomosed to oesophagus while the lower end is brought out as tracheostome for breathing. Aspirated material now finds its way to oesophagus. This operation is done in intractable
aspiration.

It may be unilateral or bilateral. Unilateral paralysis is more common. The cause may be birth trauma or congenital anomaly of a great vessel or heart. Bilateral paralysis may be due to hydrocephalus or Arnold—Chiari malformation, intracerebral haemorrhage during birth, meningocoele, or cerebral or nucleus ambiguus agenesis. The patient of bilateral paralysis presents with features of bilateral abductor paralysis and respiratory obstruction necessitating tracheostomy.

Several surgical procedures have been designed to improve the quality of voice. They include:
1. Excision of benign or malignant lesions by microlaryn- geal surgery or laser.
2. Injection of vocal cord with teflon paste or gelfoam to augment and medialize the paralyzed cord so that the opposite healthy cord can easily approximate.
3. Thyroplasty. Isshiki divided thyroplasty procedures into four categories to produce functional alteration of vocal cords.
(a) Type I. It is medial displacement of vocal cord as is achieved in teflon paste injection.
(b) Type II. It is lateral displacement of vocal cord and is used to improve the airway.

(c) Type III. Itis used to shorten (relax) the vocal cord. Relaxation of vocal cord lowers the pitch. This procedure is done in mutational falsetto or in those who have undergone gender transformation from female to male.
(d) Type IV. This procedure is used to lengthen (tighten) the vocal cord and elevate the pitch. It converts male character of voice to female and has been used in gender transformation. It is also used when
vocal cord is lax and bowing due to aging process or trauma.
- 4. Laryngeal reinnervation procedures. In this, a segment of anterior belly of omohyoid muscle, carrying its nerve (ansa hypoglossi) and vessels, is implanted into the thyroarytenoid muscle after making a window in thyroid cartilage. It is supposed to innervate the paralyzed thyroarytenoid muscle.

Benign tumours of the larynx are not as common as the malignant ones. They are divided into: (i) non-neoplastic and (ii) neoplastic (Table 61.1).
hyperplasia forming a nodule. In the early stages, the nodules appear soft, reddish and oedematous swellings but later they become greyish or white in colour.
Patients with vocal nodules complain of hoarseness. Vocal fatigue and pain in the neck on prolonged phonation are other common symptoms.
Early cases of vocal nodules can be treated conservatively by educating the patient in proper use of voice. With this treatment, many nodules in children disappear completely. Surgery is required for large nodules or nodules of long standing in adults. They are excised with precision under operating microscope either with cold instruments or laser avoiding any trauma to the underlying vocal ligament (Figure 61.3).
Figure 61.1 Vocal nodules. Typically, they form at the junction of anterior one-third with posterior two-thirds of vocal cord.
Figure 61.2
Vocal nodules.

They are not true neoplasms but are tumour-like masses
which form as a result of infection, trauma or degeneration.
They are seen more frequently than true benign neoplasms.
They are further divided into solid and cystic.

They appear symmetrically on the free edge of vocal cord, at the junction of anterior one-third, with the posterior twothirds, as this is the area of maximum vibration of the cord and thus subject to maximum trauma (Figures 61.1 and 61.2). Their size varies from that of pin-head to half a pea. They are the result of vocal trauma when person speaks in unnatural low tones for prolonged periods or at high intensities. They mostly affect teachers, actors, vendors or pop singers. They are also seen in school going children who are too assertive and talkative.
Pathologically, trauma to the vocal cord in the form of vocal abuse or misuse causes oedema and haemorrhage in the submucosal space. This undergoes hyalinization and fibrosis. The overlying epithelium also undergoes
Figure 61.3 Note the set up for microlaryngeal surgery.
Speech therapy and re-education in voice production are essential to prevent their recurrence.

It is also the result of vocal abuse or misuse. Other contributing factors are allergy and smoking. Mostly, it affects men in the age group of 30-50 years. Typically, a vocal polyp is unilateral arising from the same position as vocal nodule. It is soft, smooth and often pedunculated. It may flop up and down the glottis during respiration or phonation. Hoarseness is a common symptom. Large polyp may cause dyspnoea, stridor or intermittent choking. Some patients complain of diplophonia (double voice) due to different vibratory frequencies of the two vocal cords.
Vocal polyp is caused by sudden shouting resulting in haemorrhage in the vocal cord and subsequent submucosal oedema. Treatment is surgical excision under operating microscope followed by speech therapy.

This is due to collection of oedema fluid in the subepithelial space of Reinke. Usual cause is vocal abuse and smoking. Both vocal cords show diffuse symmetrical swellings. Treatment is longitudinal incision in the cord and removal of gelatinous fluid. Re-education in voice production and cessation of smoking are essential to prevent recurrence.

This is again due to faulty voice production in which vocal processes of arytenoids hammer against each other resulting in ulceration and granuloma formation. Some cases are due to gastric reflux. Chief complaints are hoarse voice, a constant desire to clear the throat and pain in the throat which is worse on phonation. Examination reveals unilateral or bilateral ulcers on the vocal processes of arytenoids with mucosal congestion over the arytenoid cartilages. There may be granuloma formation.
Management consists of

(b) Speech therapy to stop throat clearing and correct the pitch of voice.
(c) Inhaled steroids or intralesional injection of steroid to correct inflammation and size of granuloma. Microlaryngeal surgery may be needed to remove granuloma.

It results from injury to vocal processes of arytenoids due to rough intubation, use of large tube or prolonged presence of tube between the cords. Mucosal ulceration is followed by granuloma formation over the exposed cartilage. Usually, they are bilateral involving posterior thirds of true cords. They present with hoarseness and if large, dyspnoea as well. Treatment is voice rest and endoscopic removal of the granuloma.

This is also a localized form of epithelial hyperplasia involving upper surface of one or both vocal cords. It appears as a white plaque or warty growth on the cord without affecting its mobility. It is regarded as a precancerous condition because "carcinoma in situ" frequently supervenes. Hoarseness is the common presenting symptom. Treatment is stripping of vocal cords and subjecting the tissues to histology for any malignant change. Chronic laryngeal irritants as the aetiological factors should be sought and eliminated.

It mostly affects men in the age group of 50-70 years. Amyloid deposits involve vocal cord, ventricular band, subglottic area or trachea. It presents as a submucosal mass. Presenting symptoms are hoarseness or breathing difficulty. Systemic disease like multiple myeloma should be excluded. Diagnosis is made on biopsy and special staining. Treatment of localized deposits is by surgical removal. Prognosis is good.

1. Ductal cysts. Most often they are retention cysts due to blockage of ducts of seromucinous glands of laryngeal mucosa. They are seen in the vallecula, aryepiglottic fold, false cords, ventricles and pyriform fossa. They may remain asymptomatic if small, or cause hoarseness, cough, throat pain and dyspnoea, if large (Figure 61.4).
Sometimes, an intracordal cyst may occur on the true cord. It is similar to an epidermoid inclusion cyst.
2. Saccular cysts. Obstruction to the orifice of saccule causes retention of secretion and distension of saccule which presents as a cyst in laryngeal ventricle. Anterior saccular cysts present in the anterior part of ventricle and obscure part of vocal cord. Lateral saccular cysts, which are larger, extend into the false cord, aryepiglottic fold and may even appear in the neck through thyrohyoid membrane just as laryngoceles do.
3. Laryngocele. It is an air-filled cystic swelling due to dilatation of the saccule (Figure 61.5). A laryngocele may be:
(a)
Internal which
is confined
within the
larynx and
pres- ents
as distension
of false
cord and
aryepiglottic fold.
(b) External in which distended saccule herniates through the thyroid membrane and presents in neck.
(c) Combined or mixed in which both internal and external components are seen.
A laryngocele is supposed to arise from raised transglottic air as in trumpet players, glass-blowers or weight lifters.
pressure
Figure 61.4 (A) Aryepiglottic cyst. It caused intermittent laryngeal obstruction. (B) Cyst after removal.
Figure
61.5
Laryngocele mixed type with internal and external components.

A laryngocele presents with hoarseness, cough and if large, obstruction to the airway. An external laryngocele presents as a reducible swelling in the neck which increases in size on coughing or performing Valsalva (Figure 61.6). Diagnosis can be made by indirect laryngoscopy, and soft tissue AP and lateral views of neck with Valsalva. CT scan helps to find the extent of lesion. Treatment is surgical excision through an external neck incision. Marsupialization of an internal laryngocele can be done by laryngoscopy but there are chances of recurrence. A laryngocele in an adult may be associated with carcinoma which causes obstruction of saccule.

Except for laryngeal papillomas which constitute about 80% of the total occurrence of neoplasms of the larynx, others are uncommon.
Figure 61.6 Laryngocele left side as seen on Valsalva (arrow).
Figure 61.7 Supraglottic papillomatosis.

They can be divided into (i) juvenile and (ii) adult-onset types.

Juvenile papillomatosis is the most common benign neoplasm of the larynx in children. It is viral in origin and is caused by human papilloma DNA virus type 6 and 11. It is presumed that affected children got the disease at birth from their mothers who had vaginal human papilloma virus disease.
Papillomas mostly affect supraglottic and glottic regions of larynx but can also involve subglottis, trachea and bronchi (Figure 61.7). Children who had tracheostomy for respiratory distress due to laryngeal papillomas have higher incidence of tracheal and stomal involvement due to seeding. DNA virus particles have been found in the cells of basement membrane of respiratory mucosa and may account for widespread involvement and recurrence.
Patient, often a child, between the age of 3 and 5 years presents with hoarseness or aphonia with respiratory difficulty or even stridor. Diagnosis is made by flexible fibreoptic laryngoscopy and later confirmed by direct laryngoscopy and biopsy. Papillomas are known for recurrence but rarely undergo malignant change.
Treatment consists of microlaryngoscopy and COg laser excision avoiding injury to vocal ligament. Recurrence is common and procedure needs to be repeated several times. In the absence of facilities of CO, laser, tumour can be removed under microscope with cup forceps or a debrider similar to the one used in endoscopic nasal surgery. Aim of therapy is to maintain a good airway, preserve voice and avoid recurrence.
Besides surgery, various medical therapies are being used an adjuvants. Interferon alpha-2a has shown promising results but has several side effects including fever, chills, myalgia, arthralgia, headache, loss of weight and suppression of bone marrow. Similarly 13-cis-retinoic acid has been used. This too has several side effects.

Usually, it is single, smaller in size, less aggressive and does not recur after surgical removal. It is common in males (2:1) in the age group of 30-50 years and usually arises from the anterior half of vocal cord or anterior commissure. Treatment is the same as for juvenile type.

Most of them arise from cricoid cartilage though they also occur on thyroid or arytenoid cartilages. They may present in the subglottic area causing dyspnoea or may grow outward from the posterior plate of cricoid and cause sense of lump in throat and dysphagia. They affect men four times more than women in the age group of 40-60 years. CT scan is helpful and delineates its extent. Biopsy is required for diagnosis. Use of CO, laser is more helpful in taking biopsy of this hard tumour. Treatment consists of excision by laryngofissure or lateral pharyngotomy approach depending on the location of the tumour. Large and recurrent tumours require laryngectomy.

Infantile haemangioma involves the subglottic area and presents with stridor in the first 6 months of life. About 50% of such children have haemangiomas elsewhere in the body particularly in the head and neck area. They tend to involute spontaneously but a tracheostomy may be needed to relieve respiratory obstruction if airway is compromised. Most of them are of capillary type and can be vaporized with COg laser. Adult haemangiomas involve vocal cord or supraglottic larynx. They are cavernous type and cannot be treated with laser. They are left alone if asymptomatic. For larger ones causing symptoms, steroid or radiation therapy may be employed.

It arises from Schwann cells and is often submucosal. Overlying epithelium shows pseudoepitheliomatous hyperplasia, which may on histology, resemble well-differentiated carcinoma. Treatment is removal under microscope. Recurrence can occur if not excised completely.

Other rare benign laryngeal tumours include rhabdomyoma, neurofibroma, neurilemmoma, lipoma or fibroma.

Cancer larynx constitutes 2.63% of all body cancers in India. It is ten times more common in males than in females (4.79% versus 0.47%). Its incidence is 3.29 new cases in males and 0.42 new cases in females per 100,000 popula- tion (National Cancer Registry, ICMR, April 2005 report). Recently, its incidence in females has increased in western countries due to more women taking to smoking. Disease is mostly seen in the age group of 40-70 years but younger people in thirties may occasionally be affected.

Both tobacco and alcohol are well-established risk factors in laryngeal cancer. Cigarette smoke contains benzopyrene and other hydrocarbons which are carcinogenic in man. Combination of alcohol and smoking increases the risk 15-folds compared to each factor alone (2-3 folds). Previous radiation to neck for benign lesions or laryngeal papilloma may induce laryngeal carcinoma. Japanese and Russian workers have reported cases of familial laryngeal malignancy incriminating genetic factors. Occupational exposure to asbestos, mustard gas and other chemical or petroleum products has also been related to the genesis of laryngeal cancer but without conclusive evidence.

About
90-95%
of
laryngeal
malignancies
are
squamous
cell
carcinoma
with
various
grades
of
differentiation.
Cordal
lesions are often well-differentiated while supraglottic ones are anaplastic.
The rest 5-10% of lesions include verrucous carcinoma,
spindle
cell
carcinoma,
malignant
salivary
gland
tumours
and sarcomas.
Site

Supraglottis
e
Suprahyoid
(both
lingual
and
laryngeal
surfaces)
Infrahyoid
e
Aryepiglottic folds (laryngeal aspect only)
e
Arytenoids
e
Ventricular
bands
(or
false
cords)
True
vocal
cords
including
anterior
and
pos-
terior
commissure
Subglottis
up
to
lower
border
of
cricoid
cartilage
Glottis
Figure 62.1 According to AJCC, glottis extends from the horizontal plane passing through lateral margin of ventricle at its junction with superior surface of the vocal cord to 1 cm below it. Subglottis extends from lower limit of glottis to lower border of cricoid cartilage.

Ty
Tumour limited to one subsite of supraglottis with normal vocal cord mobility.
To
Tumour invades mucosa of more than one adjacent subsites of supraglottis or glottis or region outside the supraglottis
(e.g., mucosa of base of tongue, vallecula, medial wall of pyriform sinus) without fixation of the larynx.
T3
Tumour
limited
to
larynx
with
vocal
cord
fixation
and/or
invades
any
of
the
postcricoid
area,
pre-epiglottic
tissues,
paraglottic space and/or minor thyroid cartilage invasion.
Ta
Tumour
invades
through
the
thyroid
cartilage
and/or
invades
tissues
beyond
the
larynx
(e.g.,
trachea,
soft
tissues
of
neck
including deep extrinsic muscle of tongue, strap muscles, thyroid or oesophagus).
T,b
Tumour invades prevertebral space, encases carotid artery or invades mediastinal structures.

Ty
Tumour limited to vocal cord(s) (may involve anterior or posterior commissures) with normal mobility.
Ta
Tumour limited to one vocal cord.
Tyb
Tumour
involves
both
vocal
cords.
To
Tumour extends to supraglottis and/or subglottis, and/or with impaired vocal cord mobility.
T3
Tumour
limited
to
the
larynx
with
vocal
cord
fixation
and/or
invades
paraglottic
space
and/or
minor
thyroid
cartilage
erosion.
Tya
Tumour
invades
through
thyroid
cartilage
and/or
invades
tissues
beyond
the
larynx
(e.g.,
trachea,
soft
tissues
of
neck
includ-
ing deep extrinsic muscles of the tongue, strap muscles, thyroid, or oesophagus).
T,b

Ty
Tumour limited to the subglottis.
To
Tumour extends to vocal cord(s) with normal or impaired mobility.
T3
Tumour limited to larynx with vocal cord fixation.
Tya
Tumour invades cricoid or thyroid cartilage and/or invades tissues beyond the larynx (e.g., trachea, soft tissues
of neck including deep extrinsic muscle of tongue, strap muscles, thyroid or oesophagus).
T,b
Source: Greene FL, Page DL, Fleming ID, et al. (editors). American Joint Committee on Cancer Staging Manual, 6th edition, New York: Springer-Verlag, 2002.

Nx
0
Ti
No
Mo
No
|
Ty
No
Mo
Ny
Metastasis in a single ipsilateral lymph node, 3 cm
ll
To
No
Mo
or less in greatest dimension.
Hl
T3
No
Mo
No
Metastasis in a single ipsilateral lymph node, more
Ty
Ny
Mo
than 3 cm but not more than 6 cm in greatest
To
Ny
Mo
dimension,
or
multiple
lymph
nodes,
T3
Ny
Mo
none more than 6 cm in greatest dimension, or
IVA
Ta
No
Mo
bilateral
or
contralateral
lymph
nodes,
none
more
Ta
N,
Mo
than
6
cm
in
greatest
Ty
No
Mo
Noa
Metastasis in a single ipsilateral lymph node more
To
No
Mo
than
3
cm
but
not
more
than
6
cm
in
greatest
T3
No
Mo
Tsa
No
Mo
Nob
Metastasis in multiple ipsilateral lymph nodes, none
IVB
T,b
Any
N
Mo
more
than
6
cm
in
greatest
Any
T
Ng
Mo
Noc
Metastasis in bilateral or contralateral lymph nodes,
IVC
Any
T
Any
N
M,
none
more
than
6
cm
in
greatest

N3
Metastasis in a lymph node more than 6 cm in
greatest dimension.
Grade
1:
Well-differentiated
Grade
2:
Moderately differentiated

Grade
3:
Poorly differentiated
Myx
Mo
M,

Supraglottic cancer is less frequent than glottic cancer. Majority of lesions are seen on epiglottis, false cords followed by aryepiglottic folds, in that order.
Spread. Cancer of supraglottic region may spread locally and invade the adjoining areas, i.e. vallecula, base of tongue and pyriform fossa. Cancer of infrahyoid epiglottis and anterior ventricular band may extend into pre-epiglottic space and penetrate the thyroid cartilage.
Nodal metastases occur early. Upper and middle jugular nodes are often involved. Bilateral metastases may be seen in cases of epiglottic cancer.
Symptoms. Supraglottic growths are often silent. Hoarseness is a late symptom. Throat pain, dysphagia and referred pain in the ear or mass of lymph nodes in the neck may be the presenting features. Weight loss, respiratory obstruction and halitosis are late features.

In vast majority of cases, laryngeal cancer originates in the glottic region. Free edge and upper surface of vocal cord in its anterior and middle third is the most frequent site (Figures 62.2 and 62.3).
Spread. Locally, the lesion may spread anteriorly to anterior commissure and then to the opposite cord; posteriorly to vocal process and arytenoid region; upward to ventricle and false cord; and downwards to subglottic region. Vocal cord mobility is unaffected in early stages.
Fixation of vocal cord indicates spread of disease to thyroarytenoid muscle and is a bad prognostic sign.
There are few lymphatics in vocal cords and nodal metastases are practically never seen in cordal lesions unless the disease spreads beyond the region of membranous cord.
Symptoms.
Hoarseness of
voice is
an early
sign because
lesions of
cord affect
its vibratory
capacity.
It is
because of
this that
glottic cancer
is detected
early.
Increase in size of growths with accompanying oedema or cord fixation may cause stridor and laryngeal obstruction.

Subglottic region extends from glottic area to lower border of cricoid cartilage. Lesions of this region are rare.
Spread.
Growth starts
on one
side of
subglottis and
may spread
around downwards
the to
the is
late and
Subglottic anterior
trachea.
that is
wall to
Upward why
hoarseness growths
roid gland
can and
spread side
to the
vocal is
not an
invade cricothyroid
ribbon
Lymphatic muscles
metastases of
go or
cords early
symptom.
membrane, neck.
to prelaryngeal,
pretracheal, paratracheal
and lower
jugular nodes.
Symptoms. The earliest presentation of subglottic cancer may be stridor or laryngeal obstruction but this is often late and by this time disease has already spread sufficiently to encroach the airway.
Hoarseness in
subglottic cancer
indicates spread
of dis-
ease to
the undersurface
arytenoid muscle
nerve at
of vocal
cords, or
the infiltration
involvement the
ture of
cricoarytenoid subglottic
of of
thyro- recurrent
joint.
laryngeal
Hoarseness growth.
is
a
late fea-
thy-
(b)
(c)
vallecula,
Figure 62.2 Cancer larynx. (A) Supraglottic. (B) Glottic. (C) Subglottic.
Figure 62.3 Laryngeal cancer involving supraglottic, glottic and subglottic area as seen on rigid endoscopy.

1. History. Symptomatology of glottic, subglottic and supraglottic lesions would vary and is described under appropriate heads. It is a dictum that any patient in cancer age group having persistent or gradually increasing hoarseness for 3 weeks must have laryngeal examination to exclude cancer.

(a)
Appearance of
lesion.
the site
of origin.
(i)
(ii)
(iii)
(iv)
Lesions ofsuprahyoid
while those
Lesions
epiglottis of
infrahyoid of
vocal ulcer
or lesion
will are
usually epiglottis
cord may
thickening.
Lesions of
anterior granulation
tissue.
Lesions of
subglottic mucosal
nodule,
Vocal cord
region appear
commissure vary
with exophytic
are ulcerative.
as raised
nodule, may
appear mostly
mobility.
indicates involving
Impairment deeper
appear as
as
a
the or
raised sub-
anterior fixation
infiltration cle,
of vocal
into cricoarytenoid
geal thyroarytenoid
joint or
invasion nerve
and is
an
Extent of
disease.
tongue and
important
Spread of
recurrent sign.
of disease
pyriform fossa
to should
be half.
cord mus-
laryn- base
noticed.
of
310
- 3. Flexible fibreoptic or rigid laryngoscopy or video laryngoscopy. It isan outdoor procedure and allows detailed documentation of laryngeal pathology.
4, Examination of neck. It is done to find (i) extralaryngeal spread of disease and (ii) nodal metastasis. Growths of anterior commissure and subglottic region spread through cricothyroid membrane and may produce a midline swelling. They may also invade the thyroid cartilage and cause perichondritis when cartilage will be tender on palpation. Thyroid gland and strap muscles may also be invaded.
Search should be made for metastatic lymph nodes, their size and number; and also if they are mobile or fixed, unilateral, bilateral or contralateral.

(a) Xray chest. It is essential for coexistent lung disease (e.g. tuberculosis), pulmonary metastasis or mediastinal nodes.
(b) Soft tissue lateral view neck. Extent of lesions of epiglottis, aryepiglottic folds, arytenoids and involvement of preepiglottic space may be seen. Destruction of thyroid cartilage may be seen. This is now superceded by computed tomography (CT) scan and magnetic resonance imaging (MRI).
(c) CT scan. It is a very useful investigation to find the extent of tumour, invasion of pre-epiglottic or paraglottic space, destruction of cartilage and cervical lymph node involvement.
(d) MRI. More useful in recurrent cancers after radiotherapy.
6. Direct laryngoscopy. It is done to see (i) the hidden areas of larynx and (ii) extent of disease.
Hidden areas of the larynx include infrahyoid epiglottis, anterior commissure, subglottis and ventricle, which may not be clearly seen by mirror examination making direct laryngoscopy essential.
7. Microlaryngoscopy. For small lesions of vocal cords, laryngoscopy is done under microscope to better visualize the lesion and take more accurate biopsy specimens without damaging the cord.
8. Supravital staining and biopsy. It is useful in selection of the site of biopsy in leukoplakic lesion. Toluidine blue is applied to the laryngeal lesion and then washed with saline and examined under the operating microscope. Carcinoma in situ and superficial carcinomas take up the dye while leukoplakia does not. Thus, it helps to select the area for biopsy in a leukoplakic patch.

It depends upon the site of lesion, extent of lesion, pres- ence or absence of nodal and distant metastases. Treatment consists of:
1. Radiotherapy
2.
Surgery
(a) Conservation laryngeal surgery
(b) Total laryngectomy
3. Combined therapy. Surgery with pre- or postoperative radiotherapy
4. Endoscopic COg laser excision
5. Organ preservation
1. Radiotherapy. Curative radiotherapy is reserved for early lesions which neither impair cord mobility nor invade cartilage or cervical nodes. Cancer of the vocal cord without impairment of its mobility gives a 90% cure rate after irradiation and has the advantage of preservation of voice. Superficial exophytic lesions, especially of the tip of epiglottis, and aryepiglottic folds give 70-90% cure rate. Radiotherapy does not give good results in lesions with fixed cords, subglottic extension, cartilage invasion and nodal metastases. These lesions require surgery.

(a)
Conservation surgery. Earlier total laryngectomy was done for most of the laryngeal cancers and the patient was left with no voice and a permanent tracheostome. Lately, there has been a trend for conservation laryngeal surgery which can preserve voice and also avoids a permanent tracheal opening. However, few cases would be suitable for this type of surgery and they should be carefully selected. Conservation surgery includes:
(i) Excision of vocal cord after splitting the larynx (cordectomy via laryngofissure).
(ii) Excision of vocal cord and anterior commissure region (partial frontolateral laryngectomy).
(iii) Excision of supraglottis, i.e. epiglottis, aryepiglottic folds, false cords and ventricle—a sort of transverse section of larynx above the vocal cords (partial horizontal laryngectomy).
(b) Total laryngectomy. The entire larynx including the hyoid bone, pre-epiglottic space, strap muscles and one or more rings of trachea are removed. Pharyngeal wall is repaired and lower tracheal stump sutured to the skin for breathing.
Laryngectomy may be combined with block dissection for nodal metastasis.
Total laryngectomy is conditions: indicated in the following
(i) Ts lesions (i.e. with cord fixed)
(ii)
All
Ty lesions
(iii) Invasion of thyroid or cricoid cartilage
(iv) Bilateral arytenoid cartilage involvement
(v) Lesions of posterior commissure
(vi) Failure after radiotherapy or conservation surgery
(vii) Transglottic cancers, i.e. tumours involving supraglottis and glottis across the ventricle, causing fixation of the vocal cord.
It is contraindicated in patients with distant metastasis.
3. Combined therapy. Surgical ablation may be combined with preor postoperative radiation to decrease the incidence of recurrence. Preoperative radiation may also render fixed nodes resectable.

4, Endoscopic resection with COz laser. Carcinoma of the mobile membranous vocal cord is traditionally treated with radiotherapy. Now such lesions can be precisely excised with COg laser under microscope with the same good results. Laser excision has the advantages of lower cost, lower duration of treatment and morbidity.
Similar T, lesions of the supraor infrahyoid epiglottis with or without neck nodes have been treated with CO,
laser. Cervical nodes in such cases are managed surgically with appropriate neck dissection. Endoscopic COg laser is getting popular at some centres where facilities of CO, laser and expertise are available. 5. Organ preservation. To avoid total laryngectomy in stage III and IV, trials were conducted for laryngeal preservation. They showed that induction chemotherapy followed by radiotherapy or concurrent chemoradiation showed better locoregional control of disease than laryngectomy with postoperative radiotherapy. It also had the advantage of preservation of laryngeal function. However concurrent chemoradiation causes more toxicity and proper selection of cases should be made. Such studies of organ preservation may also improve quality of life.

Carcinoma in situ. It is best treated by transoral endoscopic COz laser. If laser is not available, stripping of vocal cord is done under microscope and tissue subjected to biopsy. If biopsy shows invasive carcinoma, radiotherapy is given. If biopsy confirms only carcinoma in situ, treatment is regular follow-up.

- T;—carcinoma. Radiotherapy is the treatment of choice. If radiotherapy is refused or not available, excision of cord by endoscopic COg laser or laryngofissure is performed.
T;—carcinoma with
extension to
anterior commissure.
Radio- therapy
is the
best tolateral
choice.
partial follow-up.
In the
absence laryngectomy
If it
fails,
as above
but
T
of this,
is done
total laryngectomy
surgery
No—It implies
cord(s), with
fron- regular
is performed.
arytenoid.
is tumour
anterior cess
of
Treatment preferred.
of the
glottic commissure
the arytenoid
tic or
subglottic is
same region,
i.e.
and/or with
vocal vocal
extension regions
pro- to
but
Treatment
(see
Figure
62.4).
1.
Is mobility
of vocal
cord normal
or impaired?
- 2. Is there involvement of anterior commissure and/or arytenoid?
If cord is mobile and anterior commissure and arytenoid are not involved, radiotherapy gives good results. Such patients are kept under regular follow-up. If disease recurs, total laryngectomy is performed. Some surgeons will still consider partial vertical laryngectomy to preserve voice in such radiation-failed cases.
If anterior
commissure and/or
arytenoid are
involved or
cord mobility
because is
impaired, of
which the
would form
possibility entail
radiotherapy is
not of
developing total
of laryngectomy.
conservation gectomy
surgery or
the frontolateral
voice.
up
Such preferred
perichondritis
In such
such as
laryngectomy patients
and are
converted
In
No to
cases, some
vertical is
done also
kept under
total neck,
in metastasis
are less
laryngectomy

Ty carcinoma,
than hemilaryn-
regular if
disease chances
25%, therefore
follow- recurs.
of occult
nodal prophylactic
neck with
no depends
supraglot- lymph
on node
two factors
supraglottic
dissection is not done. However, if radiation is considered the mode of treatment, for the primary, upper neck nodes are included in the radiation field.
Cord mobility is important in determining the outcome of Ty lesions. Normal cord mobility suggests growth is only limited to the surface. Impaired mobility indicates deeper invasion into intrinsic laryngeal muscles or paraglottic space and thus poor response to radiation. Invasion of paraglottic or subglottic space is also associated with undetected invasion of laryngeal cartilages and hence poor survival results. With radiation, cure rate of Ts lesions, with normal cord mobility, is 86% and it drops to 63% if cord mobility is impaired.
Ts and Ty, glottic carcinomas are best treated by total laryngectomy. It is combined with neck dissection if nodes are palpable. More advanced Ty lesions are treated by combined therapy, i.e. surgery with postoperative radiotherapy or only palliative treatment.
Subglottic cancer. Early lesions T; and Ty» are treated by radiotherapy. Ts; and Ty lesions require total laryngectomy and postoperative radiation. Radiation portal should also include superior mediastinum.
Supraglottic cancer. Following factors are considered in the treatment option:
Status of cervical lymph nodes
Mobility of cord WN
Age of the patient
Status of lung functions Cartilage invasion Subsite of supraglottis involved NOOR
Status of pre-epiglottic space involvement
T, lesions
respond well
to radiation.
They excised
with
Ty lesions
COg laser.
are treated
by can
also laryngectomy
be with
or without
neck dissection
if lung
good.
If lung
function is poor, radiotherapy can be given to the primary and the nodes.
Ts; and Ty lesions often require total laryngectomy with neck dissection and postoperative radiotherapy to neck.

Figure 62.5 (A) Electrolarynx. (B) A laryngectomized patient using the electrolarynx to produce sound.

speak six to ten words before reswallowing air. Voice is rough but loud and understandable.
2. Artificial larynx. It is used in those who fail to learn oesophageal speech.
(a)
Electrolarynx.
It is
ble device.
Its
a
transistorized, vibrating
of the
neck and
battery disc
is held
a
low-pitched operated
soft sound
porta- tissues
in the
hypopharynx which
is further
modulated into
speech by
the tongue, lips, teeth and palate (Figure 62.5A,B).
Transoral pneumatic device. Another type of artificial larynx is a transoral device. Here vibrations produced in a rubber diaphragm are carried by a plastic tube into the back of the oral cavity where sound is converted into speech by modulators. This is a pneumatic type of device and uses expired air from the tracheostome to vibrate the diaphragm.
(b)
3.
Tracheo-oesophageal speech.
Here attempt
is made
to carry
air from
trachea to
oesophagus or
hypopharynx by
the creation
of skin-lined
fistula or
by placement
of an
artificial prosthesis. The vibrating column of air entering the pharynx
is then
speech.
This technique
has the
disad- vantage
of food
entering
(Blom-Singer the
trachea.
or
Panje)
are being
These days
used to
shunt prosthesis
air from
tra- chea
to the
oesophagus.
They have
inbuilt valves
which work
only in one direction thus preventing problems of aspiration.
After laryngectomy, patient loses his speech completely. Various methods by which communication can be established are listed in Table 62.3.
1. Oesophageal speech. In this, patient is taught to swallow air and hold it in the upper oesophagus and then slowly eject it from the oesophagus into the pharynx. Patient can

laryngectomized patients
¢ Written language (pen and paper)
Aphonic lip speech (by trapping air in buccal cavity; often combined with sign language)
Oesophageal speech
Electrolarynx
Transoral pneumatic device
Tracheo-oesophageal speech
e Blom-Singer prosthesis
e Panje prosthesis

Hoarseness is defined as roughness of voice resulting from variations of periodicity and/or intensity of consecutive
sound waves.
For production of normal voice, vocal cords should:
1. Be able to approximate properly with each other.
2. Have a proper size and stiffness.
3. Have an ability to vibrate regularly in response to air column.
Any condition that interferes with the above functions causes hoarseness.
(a)
Loss of
approximation may
be seen
in vocal
cord paralysis
or fixation
or
a
tumour coming
in between
the vocal
cords.
(b) Size of the cord may increase in oedema of the cord or a tumour; there is a decrease in partial surgical excision or fibrosis.
(c) Stiffness may decrease in paralysis, increase in spastic dysphonia or fibrosis.
Cords may not be able to vibrate properly in the presence of congestion, submucosal haemorrhages, nodule or a polyp.

Hoarseness is a symptom and not a disease per se. The causes of hoarseness are summarized in Table 63.1.

1.
History.
Mode
of
onset
and
duration
of
illness,
patient’s
occupation,
habits
and
complaints
are
impor-
tant
and
would
often
help
to
elucidate
the
cause.
Any
hoarseness
persisting
for
more
than
3
weeks
deserves
examina-
tion
of
larynx.
should
be
excluded
in
patients
above
40
years.
2. Indirect laryngoscopy. Many of the local laryngeal causes can be diagnosed.
3.
of
neck,
chest,
and
neu-
rological
system
would
help
to
find
cause
for
laryngeal
4. Laboratory investigations and radiological examination should be done as per dictates of the cause suspected on clinical examination.
5.
Direct
and
microlaryngoscopy
help
in
detailed
examination,
biopsy
of
the
lesions
and
assess-
ment of the mobility of cricoarytenoid joints.
6.
Bronchoscopy and
oesophagoscopy may
be required
in cases of paralytic lesions of the cord to exclude malignancy.

Here voice
by ventricular
folds which
have rough,
taken over
the low-pitched
be secondary
(false of
unpleasant.
to impaired
paralysis, fixation,
bands true
cords.
cords)
Voice
Ventricular function
in these
situations excision
try is
voice the
true cord
or tumours.
to compensate
may such
as
Ventricular or
assume pho-
natory function
of true
cords.
Functional type of ventricular dysphonia occurs in normal larynx. Here cause is psychogenic. In this type, voice begins normally but soon becomes rough when false cords usurp the function of true cords. Diagnosis is made on indirect laryngoscopy; the false cords are seen to approximate partially or completely and obscure the view of true cords
—
on phonation. Ventricular dysphonia secondary to laryngeal disorders is difficult to treat but the functional type can be helped through voice therapy and psychological counselling.

It is a functional disorder mostly seen in emotionally labile females in the age group of 15-30 years. Aphonia is usually sudden and unaccompanied by other laryngeal symptoms. Patient communicates with whisper. On examination, vocal cords are seen in abducted position and fail to adduct on phonation; however, adduction of vocal cords can be seen on coughing, indicating normal adductor function. Even though patient is aphonic, sound of cough is good. Treatment given is to reassure the patient of normal laryngeal function and psychotherapy.

Normally, childhood voice has a higher pitch. When the larynx matures at puberty, vocal cords lengthen and the voice changes to one of lower pitch. This is a feature exclusive to males. Failure of this change leads to persistence of childhood high-pitched voice and is called puberphonia. It is seen in boys who are emotionally immature, feel insecure and show excessive fixation to their mother. Psychologically, they shun to assume male responsibilities though their physical and sexual development is normal. Treatment is training the body to produce low-pitched voice. Pressing the thyroid prominence in a backward and downward direction relaxes the overstretched cords and low tone voice can be produced (Gutzmann's pressure test). The patient pressing on his larynx learns to produce low tone voice and then trains himself to produce syllables, words and numbers. Prognosis is good.

It is weakness of voice due to fatigue of phonatory muscles. Thyroarytenoid and interarytenoids or both may be affected. It is seen in abuse or misuse of voice or following laryngitis. Patient complains of easy fatiguability of voice. Indirect shows three characteristic laryngoscopy findings:
1. Elliptical space between the cords in weakness of thyroarytenoid.
2. Triangular gap near the posterior commissure in weakness of interarytenoid.
3. Key-hole appearance of glottis when both thyroarytenoid and interarytenoids are involved (Figure 63.1).
Treatment is voice rest and vocal hygiene, emphasizing on of voice rest after excessive use of voice.
periods

Dysphonia can be divided into three types: adductor, abductor and mixed.

The adductor muscles of larynx go into spasm causing vocal cords to go into adduction. Voice becomes strained or strangled, and phonation is interrupted in between leading to voice breaks. Larynx is however morphologically normal. Severity of the condition differs from mild and intermittent symptoms to those with moderate or severe dysphonia. Flexible fibreoptic laryngoscopy is useful during which patient's speech, sustained phonation and respiratory activities are studied. Patient may have tremors of larynx, palate and pharynx.
Aetiology of the condition is uncertain but one should exclude neurological conditions such as Parkinsonism, myoclonus, pseudobulbar palsy, multiple sclerosis, cerebellar disorders, tardive dyskinesia and amyotrophic lateral sclerosis. CT scan and MRI are not useful but help to rule out neurological conditions.
Treatment consists of botulinum toxin injections in the thyroarytenoid muscle on one or both sides to relieve spasm. Percutaneous electromyography (EMG) guided route through cricothyroid space is preferred. Dose of botulinum toxin depends on severity of the condition. Toxin injections relieve voice breaks due to spasms and improve airflow but the benefit lasts only up to 16 weeks or so when repeat injection may be needed. Sometimes, if dose of toxin is not regulated it may cause breathiness of voice and discomfort to swallow.
Voice therapy is useful to improve voice and the duration of benefit. Voice therapy alone without injection does not help much. Earlier disease was considered to be psychological in origin but psychotherapy was not found useful. Section of recurrent laryngeal to paralyze the cord/cords has been used in the past but it interferes with glottic closure leading to breathy and weak voice and swallowing discomfort. This treatment is still used when injection treatment fails and the spasms are severe.

It is due to spasms of posterior cricoarytenoid muscle (the only abductor) and thus keeping the glottis open. Patient gets a breathy voice or breathy breaks in voice. The condition is gradually progressive and the symptoms get aggravated during periods of stress or when patient uses telephone.

It is more complex, both the adductor and abductor function may be affected.

It is lack of nasal resonance for words which are resonated in
the nasal cavity, e.g. m, n, ng.
It is due to blockage of the nose or nasopharynx. Impor-
tant causes are listed in Table 63.2.

It is
seen when
certain words
which have
little nasal
nance are
of the
resonated through
nasopharynx to
nose.
cut off
The from
defect oropharynx
reso- is
in failure
or abnormal
Table 63.2, 1 = Causes of hyponasality and hypernasality. Common cold Nasal allergy Nasal polypi Nasal growth Adenoids Nasopharyngeal mass Familial speech pattern Habitual, 1 = Velopharyngeal insufficiency Congenitally short soft palate Submucous palate Large nasopharynx Cleft of soft palate Paralysis of soft palate Postadenoidectomy Oronasal fistula Familial speech pattern Habitual speech pattern
communication between the oral and nasal cavities. The causes are listed in Table 63.2.

It is a disorder of fluency of speech and consists of hesitation to start, repetitions, prolongations or blocks in the flow of speech. When well-established, a stutterer may develop secondary mannerisms such as facial grimacing, eye blink and abnormal head movements. Normally, most of the children have dysfluency of speech between 2 and 4 years. If too much attention is given or child reprimanded by parents and peers, this behaviour pattern may become fixed and child may develop into an adult stutterer. Stuttering can be prevented by proper education of the parents, not to overreact to child's dysfluency in early stages of speech development. Treatment of an established stutterer is speech therapy and psychotherapy to improve his image as a speaker and reduce his fear of dysfluency.

Tracheostomy is making an opening in the anterior wall of trachea and converting it into a stoma on the skin surface.
term tracheotomy
has been
interchangeably used
but the
latter actually
means opening
the trachea,
which is a step in the tracheostomy operation.

1.
Alternative pathway
for breathing.
This circumvents
any obstruction in the upper airway from lips to the tracheostome.
- 2. Improves alveolar ventilation. In cases of respiratory
insufficiency, alveolar ventilation is improved by:
(a) Decreasing the dead space by 30-50% (normal dead is 150
space mL).
(b) Reducing the resistance to airflow.
3. Protects the airways. By using cuffed tube, tracheobron-
chial tree is protected against aspiration of:
(a) Pharyngeal secretions, as in case of bulbar paralysis or coma.
(b) Blood, as in haemorrhage from pharynx, larynx or maxillofacial injuries. With tracheostomy, pharynx and larynx can also be packed to control bleeding.
4. Permits removal of tracheobronchial secretions. When patient is unable to cough as in coma, head injuries, respiratory paralysis; or when cough is painful, as in chest injuries or upper abdominal operations, the tracheobronchial airway can be kept clean of secretions by repeated suction through the tracheostomy, thus avoiding need for repeated bronchoscopy or intubation which is not only traumatic but also requires expertise.
5. Intermittent positive pressure respiration (IPPR). If IPPR is required beyond 72 h, tracheostomy is superior to intubation.
6. To administer anaesthesia. In cases where endotracheal intubation is difficult or impossible as in laryngopharyngeal growths or trismus.

There are three main indications (Table 64.1)
1. Respiratory obstruction.
2. Retained secretions.
3. Respiratory insufficiency.

Emergency tracheostomy
Elective or tranquil tracheostomy
Permanent tracheostomy
Percutaneous dilatational tracheostomy
Mini tracheostomy (cricothyroidotomy)
1. Emergency tracheostomy. It is employed when airway obstruction is complete or almost complete and there is an urgent need to establish the airway. Intubation or laryngot- omy are either not possible or feasible in such cases.
2. Elective tracheostomy (syn. tranquil, orderly or routine tracheostomy). This is a planned, unhurried procedure. Almost all operative surgical facilities are available, endotra- cheal tube can be put and local or general anaesthesia can be given. It is of two types:
(a)
Therapeutic,
to
relieve
remove
tracheobronchial
or
give
assisted
(b)
Prophylactic,
to
guard
against
anticipated
or
of
blood
or
secre-
tions
such
as
in
extensive
surgery
of
tongue,
floor
of
mouth,
mandibular
resection
or
laryngofissure.
Elective tracheostomy is often temporary and is closed when indication is over.
3. Permanent This be for cases
tracheostomy. may required
of bilateral abductor paralysis or laryngeal stenosis. In laryn- gectomy or laryngopharyngectomy, lower tracheal stump is brought to surface and stitched to the skin.
Tracheostomy has also been divided into high, mid

or low. A high tracheostomy is done above the level of thyroid isthmus (isthmus lies against II, III and IV tracheal rings). It violates the first ring of trachea. Tracheostomy at this site can cause perichondritis of the cricoid cartilage and sub- glottic stenosis and is always avoided. Only indication for high tracheostomy is carcinoma of larynx because in such cases, total larynx anyway would ultimately be removed and a fresh tracheostome made in a clean area lower down. A mid tracheostomy is the preferred one and is done through the II or III ring and would entail division of the thyroid isthmus or its retraction upwards or downwards to expose this part of trachea. A low tracheostomy is done below the level of isthmus. Trachea is deep at this level and close to several large vessels; also there are difficulties with tracheostomy tube which impinges on suprasternal notch.

Whenever possible, endotracheal intubation should be done before tracheostomy. This is specially important in infants and children.
Position. Patient lies supine with a pillow under the shoulders so that neck is extended. This brings the trachea forward.
Anaesthesia. No anaesthesia is required in unconscious patients or when it is an emergency procedure. In conscious patients, 1-2% lignocaine with epinephrine is infiltrated in the line of incision and the area of dissection. Sometimes, general anaesthesia with intubation is used.

1.
A
vertical incision
is extending
nal notch.
be used
made from
cricoid
This in
cartilage is
the in
rapid most
emergency access
tion.
the midline
to just
favoured and
with elective
minimum
A
transverse incision,
above of
neck, the
incision ster-
and procedures.
of bleeding
5
cm can
It gives
and tissue
long, made
dissec- two
fingers’
Figure
64.1
incision.
(B)
Skin incisions
in tracheostomy.
(A)
Vertical
Transverse incision.
Figure
64.2
Mid tracheostomy.
Thyroid isthmus
is divided
ligated.
breadth above
the sternal
It has
notch the
can advantage
midline and
be used
of
a
in elective
cosmetically better
scar
(Figure
64.1).
2. After incision, tissues are dissected in the midline. Dilated veins are either displaced or ligated.
3. Strap muscles are separated in the midline and retracted laterally.
4. Thyroid isthmus is displaced upwards or divided between the clamps, and suture ligated.
5. A few drops of 4% lignocaine are injected into the trachea to suppress cough when trachea is incised.
6.
Trachea is
fixed with
a
hook and
opened with
a
verti- cal
incision and
second in
the region
rings.
opening.
The chondritis
of third
This is
then first
of
(Figure
64.2).
tracheal cricoid
and fourth
converted ring
cartilage or
into
a
is never
divided with
stenosis third
circular as
peri- can
result
¢ Infants below 1 year (mostly congenital lesions)

© Subglottic haemangioma
° Subglottic stenosis
e Laryngeal cyst
° Glottic web
° Bilateral vocal cord paralysis
© Children (mostly inflammatory or traumatic lesions)
° Acute laryngo-tracheo-bronchitis
©
e Laryngeal oedema (chemical/thermal injury)
e External laryngeal trauma
¢ Prolonged intubation
e Juvenile laryngeal papillomatosis
7. Tracheostomy tube of appropriate size is inserted and secured by tapes (see p. 461 for different types and size of tracheostomy tubes).
8. Skin incision should not be sutured or packed tightly as it may lead to development of subcutaneous emphysema.
9. Gauze dressing is placed between the skin and flange of the tube around the stoma.

Important conditions requiring tracheostomy in this age group are listed in Table 64.2.
Great care and caution is required when doing tracheostomy in infants and children lest it is attended with complications that are avoidable.
1. Trachea of infants and children is soft and compressible and its identification may become difficult and the sur- geon may easily displace it and go deep or lateral to it injuring recurrent laryngeal nerve or even the carotid. It is always useful to have an endotracheal tube or a bronchoscope inserted into trachea before operation. Tracheostomy in infants and children is preferably done under general anaesthesia.
2. During positioning, do not extend the neck too much as this pulls structures from chest into the neck and thus injury may occur to pleura, innominate vessels and thymus or the tracheostomy opening may be made too low near suprasternal notch.
3. Before incising trachea, silk sutures are placed in the trachea, on either side of midline.
4. Tracheal lumen is small, do not insert knife too deep; it will injure posterior tracheal wall or even oesophagus causing tracheo-oesophageal fistula.
5. Trachea is simply incised, without excising a circular piece of tracheal wall.
6. Avoid infolding of anterior tracheal wall when inserting the tracheostomy tube.
7. Selection of tube is important. It should be of proper diameter, length and curvature. A long tube impinges on the carina or right bronchus. With high curvature, lower end of tube impinges on anterior tracheal wall while upper part compresses the tracheal rings or cricoid (see Appendix II, p. 451).
Figure
64.3
Tracheotomized patient of laryngeal cancer with suction- aid tracheostomy tube and receiving oxygen through a side port.
8. Use soft silastic or portex tube. Metallic tubes cause more trauma.
9.
Take
a
X-ray
of
the
neck
and
chest
to
ascertain the position of the tracheostomy tube.

1. Constant supervision. After tracheostomy, constant supervision of the patient for bleeding, displacement or blocking of tube and removal of secretions is essential. A nurse or patient’s relative should be in attendance. Patient is given a bell or a paper pad and a pencil to communicate.
2. Suction. Depending on the amount of secretion, suction may be required every half an hour or so; use sterile cath- eters with a Y-connector to break suction force. Suction injuries to tracheal mucosa should be avoided. This is done by applying suction to the catheter only when with- drawing it (Figure 64.3).
3. Prevention of crusting and tracheitis. This is achieved by
(a) Proper humidification, by use of humidifier, steam tent, ultrasonic nebulizer or keeping a boiling kettle in the room.
(b) If crusting occurs, a few drops of normal or hypo- tonic saline or Ringer’s lactate are instilled into the trachea every 2-3 h to loosen crusts. A mucolytic agent such as acetylcysteine solution can be instilled to liquify tenacious secretions or to loosen the crusts.
4.
Care
of
tube.
Inner
cannula
should
be
removed
and
cleaned
as
and
when
indicated
for
the
first
3
days.
Outer
tube,
unless
blocked
or
displaced,
should
not
be
removed
for
3-4
days
to
allow
a
track
to
be
formed
when
tube
placement
will
become
easy.
After
3-4
days,
outer
tube
can
be
removed
and
cleaned
every
day.
If cuffed tube is used, it should be periodically deflated to prevent pressure necrosis or dilatation of trachea.
Decannulation. Tracheostomy tube should not be kept longer than necessary. Prolonged use of tube leads to tra- cheobronchial infections, tracheal ulceration, granulations, stenosis and unsightly scars.

To decannulate a patient, tracheostomy tube is plugged
and the patient closely observed. If the patient can toler ate it for 24 h, tube can be safely removed. In children, the above procedure is done using a smaller tube. After tube removal, wound is taped and patient again closely observed.
Healing of the wound takes place within a few days or a week.
Rarely a secondary closure of wound may be required.
Observe the following principles when decannulating an infant or a young child:
- 1. Decannulate in the operation theatre where services of a trained nurse and an anaesthetist are available.
2.
Equipment for
reintubation should
be available
imme- diately.
of
a
good headlight,
laryngoscope, proper-sized
and
a
tracheostomy tray.
- 3. After decannulation, watch the child for several hours for respiratory distress, tachycardia and colour oxymetry is very useful to monitor oxygen saturation. It may require blood gas determinations. When attempts at decannulation are not look for the cause. It be:
successful, may
(a) Persistence of the condition for which tracheostomy was done.
(b) Obstructing granulations around the stoma or below it where tip of the tracheostomy tube had been impinging.
(c) Tracheal oedema or subglottic stenosis.
(d) Incurving of tracheal wall at the site of tracheostome.
(e) Tracheomalacia.
(f) Psychological dependence on tracheostomy and inability to tolerate the resistance of the upper airways.
A case of difficult decannulation may require endoscopic examination of the larynx, trachea and bronchi preferably under magnification using telescopes or a flexible

1. Immediate (at the time of operation):
(a) Haemorrhage.
(b) Apnoea. This follows opening of trachea in a patient who had prolonged respiratory obstruction. This is due to sudden washing out of CO, which was acting as a respiratory stimulus. Treatment is to administer 5% COg in oxygen or assisted ventilation.
(c) Pneumothorax due to injury to apical pleura.
(d) Injury to recurrent laryngeal nerves.
(e) Aspiration of blood.
(f) Injury to oesophagus. This can occur with tip of knife while incising the trachea and may result in tracheooesophageal fistula.
2. Intermediate (during first few hours or days):
(a) Bleeding, reactionary or secondary.
(b) Displacement of tube.
(c) Blocking of tube.
(d) Subcutaneous emphysema.
(e) Tracheitis and tracheobronchitis with crusting in trachea.
(f) Atelectasis and lung abscess.
(g) Local wound infection and granulations.
3. Late (with prolonged use of tube for weeks and months):
(a) Haemorrhage, due to erosion of major vessel.
(b) Laryngeal stenosis, due to perichondritis of cricoid cartilage.
(c) Tracheal stenosis, due to tracheal ulceration and infection.
(d) Tracheo-oesophageal fistula, due to prolonged use of cuffed tube or erosion of trachea by the tip of tracheos- tomy tube.
(e) Problems of decannulation. Seen commonly in infants and children.
(f) Persistent tracheocutaneous fistula.
(g)
Problems
of
scar.
Keloid
or
unsightly
scar.
(h) Corrosion of tracheostomy tube and aspiration of its fragments into the tracheobronchial tree.

When airway obstruction is so marked as to allow no time to do an orderly tracheostomy, following measures are taken:
- 1. Jaw thrust. Lifting the jaw forward and extending the neck improves the airway by displacing the soft tissues. Neck extension should be avoided in spinal injuries.
2. Oropharyngeal airway. It displaces the tongue anteriorly and relieves soft tissue obstruction. Ventilation can be carried out by face mask placed snugly over the face and covering both nose and mouth. Ambu bag can be used for inflation of air or oxygen.
- 3. Nasopharyngeal airway (trumpet). It is inserted transnasally into the posterior hypopharynx and relieves soft tissue obstruction caused by the tongue and pharynx. It is better tolerated than oropharyngeal airway in awake patients.
4. Laryngeal mask airway. It is a device with a tube and a triangular distal end which fits over the laryngeal inlet (Figure 64.4). Oxygen can be delivered directly into the trachea. Though most commonly used for nonemergent airway control, it can be used as an alternative if standard mask ventilation is inadequate and intubation unsuccessful (see Appendix II on Instruments).
5. Transtracheal jet ventilation. It is an invasive procedure. An intravenous catheter of 12 or 14 gauge with a syringe attached is inserted into the cricothyroid membrane and directed caudally. Once intraluminal placement is confirmed by aspiration, needle is withdrawn leaving the catheter in position and jet ventilation started. In thin individuals where trachea can be palpated, catheter can be inserted easily. Expiration of air should be insured otherwise pulmonary barotrauma with pneumothorax, pneumomediastinum and surgical emphysema can result.
6. Endotracheal intubation. This is the most rapid method.
Larynx is visualized with a laryngoscope and endotracheal
tube or a bronchoscope inserted. No anaesthesia is required. This helps to avoid a hurried tracheostomy in which complication rate is higher. After intubation, an orderly tracheostomy can be performed.

7. Cricothyrotomy or laryngotomy or mini tracheostomy. This is a procedure for opening the airway through the cricothyroid membrane. Patient's head and neck is extended, lower border of thyroid cartilage and cricoid ring are identified. Skin in this area is incised vertically and then cricothyroid membrane cut with a transverse incision. This space can be kept open with a small tracheostomy tube or by inserting the handle of knife and turning it at right angles if tube is not available. It is essential to perform an orderly tracheostomy as soon as possible because perichondritis, subglottic oedema and laryngeal stenosis can follow prolonged laryngotomy.
"Mini tracheostomy is an emergency procedure to buy time to allow patient to be carried to operation theatre. Commercial emergency kits are also available for this. As an elective procedure it has been done to clear the bronchial secretions following thoracic surgery."
Cricothyroid needle puncture is a procedure where a large-bore intravenous catheter is introduced through the cricothyroid membrane. It is only an emergency procedure till patient can be intubated or tracheostomized. The procedure does not provide adequate ventilation.
8. Emergency tracheostomy. Technique of emergency tracheostomy is as follows: Patient's neck is extended, trachea identified and fixed between surgeon's left thumb and index finger. A vertical incision is made from lower border of thyroid to suprasternal notch cutting through skin and subcutaneous tissues. Lower border of cricoid cartilage is identified and a transverse incision made in pretracheal fascia. The thyroid isthmus dissected down to expose upper three tracheal rings. Vertical tracheal incision is made in second and third rings, opened with a haemostat and the tube inserted. Bleeding can be controlled by packing with gauze.
Emergency tracheostomy on a struggling patient with inadequate lighting, suction and instruments is fraught with many complications. If possible, an endotracheal tube should be put for a more orderly procedure to be carried out.

This type of tracheostomy is done in ICU where patient is already intubated and being monitored. It is done under sedation. Neck is extended with a pad under the shoulders. Neck is prepared and draped and 1.5-2 cm incision is made 2 cm below the lower border of cricoid. Trachea is exposed by dissection and palpation. Thyroid isthmus is pushed down. Now a small caliber flexible bronchoscope, to which a camera has been attached, is passed through the endotracheal tube to monitor the passage of the needle, guide wire and dilator/s. It is important to enter the trachea in the midline and avoid any lateral entry. Entry into the trachea is made between second and third rings. After dilatation tracheostomy tube is inserted.
Advantages of the procedure include: (i) No need to transport the patient to operation theatre, (ii) avoiding operation theatre (OT) expenses, and (iii) avoiding ICU nosocomial infections to be carried to OT and earlier discharge of patient.
The procedure is avoided in patients who are obese, have a neck mass, difficult to intubate, difficult to extend the neck, larynx and trachea are not easily palpable or have uncorrectable coagulopathies.
Complications of the procedure include paratracheal entry of dilator or tracheostomy tube into the lumen, haemorrhage, damage to posterior tracheal wall and surgical emphysema.

Children are more often affected; more than half of them are below 4 years. Accidents occur when they suddenly inspire during play or fight while having something in the mouth. In children, peanut is the most common vegetable foreign body; others include almond seed, peas, beans, gram or wheat seed, watermelon seed, pieces of carrot or apple. Nonvegetable matters include plastic whistle, plastic toys, safety pins, nails, all-pin, twisted wires or ball bearings. In adults, foreign bodies are aspirated during coma, deep sleep or alcoholic intoxication. Loose teeth or denture may be aspirated during anaesthesia.

1. Nonirritating type. Plastic, glass or metallic foreign bod- ies are relatively nonirritating and may remain symptom- less for a long time.
2.
Irritating
type.
Vegetable
or
foreign
bodies
like
peanuts,
beans,
seeds,
etc.
set
up
a
diffuse
violent
reaction
lead-
ing
to
and
oedema
of
the
mucosa—a
condition
called
“vegetal
bronchitis.”
They
also
swell
up
with
time
causing
airway
and
later
suppuration
in
the
lung.
Areca
nut
is
a
common
foreign
body
in
Rajasthan
(India)
due
to
habit
of
chewing
these
nuts
frequently,
peanuts
are
common
in
USA,
water-
melon
seeds
in
Egypt
and
pumpkin
seeds
in
Greece.

Symptomatology of foreign body is divided into three stages:
1.
Initial period
of choking,
gagging and
wheezing.
This lasts
for ashort
time.
in
the larynx
may or
further
2.
Symptomless the
be coughed
down interval.
foreign in
The body
out or
it may
the tracheobronchial
lodge tree.
mucosa adapts
initial symptoms
to disappear.

A foreign body aspirated into air passage can lodge in the larynx, trachea or bronchi. Site of lodgement would depend on the size, shape and nature of the foreign body. A large foreign body, unable to pass through the glottis, will lodge in the supraglottic area while the smaller one will pass down through the larynx into the trachea or bronchi. Foreign bodies with sharp points, e.g. pins, needles, fish bones, etc. can stick anywhere in the larynx or tracheobronchial tree.
Symptomless interval will vary with the size and nature of the foreign body. It may last a few hours or a few weeks.
3. Later symptoms. They are caused by airway obstruction, inflammation or trauma induced by the foreign body and would depend on the site of its lodgement (Table 65.1).
(a)
Laryngeal foreign
body.
A
large foreign
body may
totally obstruct
the airway
resuscitative leading
measures obstructive
to are
sudden taken
foreign the
throat, dyspnoea,
body hoarseness
will of
wheezing
Tracheal and
foreign duce
body.
cough seed
and may
death urgently.
cause unless
A
partially discomfort
voice, croupy
or cough,
haemoptysis.
A
sharp
(Figure foreign
body haemoptysis.
move carina
and ble
slap”
A
loose up
and down
and
“palpatory also
be will
trachea of
vocal thud.”
present.
Bronchial
It is
best foreign
right bronchus
the pain
in aphonia,
65.1).
only pro-
body between
cords causing
Asthmatoid heard
body.
Most because
tracheal
a
lumen.
lobar like
the
“audi- wheeze
at patient’s
open foreign
it is
may mouth.
bodies wider
A
and foreign
or segmental
it may
produce only
body bronchus
a
check ingress

enter more
the in
may line
totally with
obstruct causing
valve of
air obstructive
but, not
emphysema.
the
For atelectasis
obstruction, or
allowing egress,
thus leading
pathogenesis to
and clinical
picture of
body, see
Figure
65.2.
Emphysematous bulla may rupture causing spontaneous pneumothorax. A foreign body may also shift from one side to the other causing change in the physical signs. A retained foreign body in the lung may later give rise to pneumonitis, bronchiectasis or lung abscess.
(b)
(c)
Figure 65.1 Laryngeal foreign bodies. (1) Safety pin. (2) An all pin.
(3, 4) A twisted wire. (5) A fruit seed.
Figure 65.2 Types of bronchial obstruction by a foreign body. (A) Partial obstruction. Air can pass in and out, causing only wheeze. (B) One way obstruction. Air can go in (during inspiration) but not out, causing emphysema of lungs. (C) Total obstruction. Air can neither go in nor out, causing obstructive atelectasis. (D) One way obstruction (reverse of B). Air can only go out, causing atelectasis. Dark pink shows normal size of lung while lighter pink indicates effect of obstruction.

. . . It can be made by detailed history of the foreign body "ingestion", physical examination of the neck and chest. A history of sudden onset of coughing, wheezing and dimin-. soe . ished entry of air into the lungs on auscultation forms a : : ae en classical triad. There should be a high index of suspicion in : . : : children with wheezing, stridor, cough or asthma and those : : . : : : with recurrent chest infections being treated with steroids d antibiotics. Radiology i helpful and AN ADIOUCs. Tolosy 1s very helpiu.
1. Soft tissue posteroanterior and lateral view of the neck in its extended position. This can show radio-opaque and sometimes even the radiolucent foreign bodies in the larynx and trachea (Figures 65.3 to 65.5). A coin or a flat
2.
Figure
65.3
A
radiolucent fruit
seed
(chiku)
seen in
subglottic of larynx (arrow). Patient has a tracheostomy (dashed arrows).
Figure 65.4 PA view chest showing a nail in the right bronchus.
foreign body in trachea lies edge on in PA view and flat on lateral view.
Plain
X-ray chest
in posteroanterior
and lateral
views
(Figure
65.6)
(a)
gu
It may
a
show shape
(b)
(c)
.
the radio-opaque
.
location.
Lobar or
segmental
. : tion by foreign body).
:
.
Unilateral hyperinflation
entire lung
shift to
:
body—its
:
atelectasis
(complete wos
size, obstruc-
of lobe
(if ball
opposite roscopy
valve side
or
X-rays
:
or segment
obstruction).
is seen
taken or
Mediastinal in
hyperinflation.
during
Fluo- inspiration
and ration
are helpful.
(d) Pneumomediastinum or pneumothorax.
expi- region

Figure 65.5 PA view chest showing a broken piece of Fuller's tracheostomy tube in the left bronchus.
Figure
65.6
X-ray chest showing a spring from a ball pen in the right bronchus.
(e)
A
normal
X-ray
chest.
In
early
cases
within
24
h
or
a
foreign
body
causing
partial
with
full
ingress
and
egress
of
air
does
not
produce
any
sign.
(f)
Pneumonitis/bronchiectasis.
Prolonged
stay
of
for-
eign
body
may
cause
atelectasis,
pneumonitis
or
bronchiectasis.
3. X-ray chest at the end of inspiration and expiration. Atel- ectasis and obstructive emphysema can be seen. They are indirect evidence of radiolucent foreign bodies.
Posteroanterior chest view in expiration is most useful in bronchial foreign bodies. It shows obstructive emphy- sema on the side of foreign body with mediastinal shift to opposite side. If this is not possible as in children, a lateral decubitus film is taken—a hyperinflated lung is indicative of foreign body. Obstructive emphysema is an early sign while atelectasis comes late.
4.
Fluoroscopy/videofluoroscopy.
Evaluation
during
inspi-
ration
and
can
be
made.
5. CT chest.

Laryngeal foreign body. A large bolus of food obstructed above the cords may make the patient totally aphonic,
Figure 65.7 Heimlich manoeuvre. Sudden thrust directed upwards and backwards, below the epigastrium, squeezes the air from the lungs, sufficient to dislodge a foreign body.
unable to cry for help. He may die of asphyxia unless immediate first aid measures are taken. The measures consist of pounding on the back, turning the patient upside down and following Heimlich manoeuvre. These measures should not be done if patient is only partially obstructed, for fear of causing total obstruction.
Heimlich manoeuvre. Stand behind the person and place your
arms around
his lower
chest and
give four
abdominal thrusts.
The residual
air in
the lungs
may dislodge
the for-
eign body
providing some
airway
(Figure
65.7).
Cricothyrotomy or emergency tracheostomy should be done if Heimlich manoeuvre fails. Once acute respiratory emergency is over, foreign body can be removed by direct laryngoscopy or by laryngofissure, if impacted.
Tracheal and bronchial foreign bodies can be removed by bronchoscopy with full preparation and under general anaesthesia. Emergency removal of these foreign bodies is not indicated unless there is airway obstruction or they are of the vegetable nature (e.g. seeds) and likely to swell up.
Methods to remove tracheobronchial foreign body:
1.
Conventional rigid bronchoscopy.
2.
Rigid bronchoscopy with telescopic aid.
3. Bronchoscopy with C-arm fluoroscopy.
4. Use of Dormia basket or Fogarty’s balloon for rounded objects.
5.
first
and
then
bronchoscopy
through
the
tracheostome.
6. Thoracotomy and bronchotomy for peripheral foreign bodies.
7. Flexible fibreoptic bronchoscopy in selected adult patients.
Equipment for foreign body removal include:
1.
Bronchoscope,
for
the
age
of
patient
and
a
size smaller and the other a size larger (see p. 461).

2. Telescope or optical forceps.
3. Two laryngoscopes.
4.
Foreign
body
forceps,
Dormia
basket,
Fogarty’s
catheter
and a syringe to inflate it.
66.
Thyroid Gland and Its Disorders

The thyroid gland consists of two lobes and an isthmus, and weighs between 15 and 25 g in adults. Each lobe is conical in shape with apex directed upwards and measures 5 cm in length, 3 cm in breadth and 2 cm anteroposteriorly. Isthmus lies against the IInd and IIIrd tracheal rings. A small pyramidal lobe extends from the isthmus, usually close to the left lobe, towards the hyoid bone (Figure 66.1).

True capsule surrounds the gland and sends septa into the thyroid tissue. False capsule is the loose areolar tissue derived from the middle layer of deep cervical fascia which ensheaths larynx, trachea and thyroid.

It is a condensation of fascia connecting the cricoid and the Ist and sometimes the IInd tracheal ring to the posteromedial aspect of each thyroid lobe. Recurrent laryngeal nerve (RLN) (or its two branches, if the nerve divides extralaryngeally) passes deep to the ligament or through it. Small branches of the inferior thyroid artery lie close to the ligament and bleed during surgery. Extreme care is required to control bleeding to avoid injury to recurrent laryngeal nerve or its branches. The ligament may also contain a small amount of thyroid tissue which is left behind at the site and is responsible for the radionuclide uptake in the thyroid bed and raised thyroglobulin level after thyroidectomy has been done for cancer.

It is also a condensation of pretracheal fascia and connects superior-anterior-medial portion of thyroid lobe and the isthmus of gland to the laryngotracheal complex. It also contains moderate-sized vessels.

Its external branch supplies cricothyroid muscle and, if injured, causes bowing and inferior placement of vocal cord with consequent loss of pitch. Superior thyroid artery and vein(s) are closely related to it. When ligating these vessels, be as close to the upper pole of thyroid as possible. Downward traction on the thyroid gland further helps to avoid injury to this nerve.
It is a branch of vagus. On the right side, it hooks round the subclavian artery and passes behind the carotid sheath. On the left, it hooks round the aortic arch, lateral to the ligament of ductus arteriosus and enters the neck behind the carotid sheath. Its relations to inferior thyroid artery are important. The nerve runs superficial, deep or through the branches of the artery to reach the posterior suspensory ligament. RLN is closer to trachea on the left than on the right side.

It is an anomalous RLN. It does not have a recurrent course. After origin from the vagus, it runs directly medially to supply the larynx running along the inferior thyroid artery. It does not hook around the subclavian on the right or aortic arch on the left. The anomaly is more common on the right side and is often associated with anomalous right subclavian artery which in such cases arises from the left side of descending aorta and runs behind the oesophagus.

Two main arteries supply the gland. Inferior thyroid artery is a branch of thyrocervical trunk. It passes behind the carotid sheath and supplies the thyroid and parathyroid glands. Superior thyroid artery is a branch directly from the external carotid and runs close to the external branch of superior laryngeal nerve. Sometimes a third artery called thyroidea ima also supplies the gland.

Three veins drain the thyroid gland on each side. Superior thyroid vein from the upper pole drains directly into the internal jugular vein. Middle thyroid vein emerges from lateral surface of gland and drains into the internal jugular vein. Inferior thyroid veins are multiple, form a plexus which drains into the right and left brachiocephalic veins.

Thyroid gland drains into the central compartment— prelaryngeal, pretracheal and paratracheal nodes (level VI), into superior mediastinum (level VII) and also into level II, Ill and IV nodes. Nodes are important when treating thyroid malignancies (Figure 66.2).
Figure 66.1 Anatomy of thyroid gland. (Modified with permission from Becker and Stucchi, Essentials of Surgery, 1st ed, Elsevier, 2005).

anywhere from the hyoid bone above to the superior mediastinum below. It descends along the thymus gland.
During thyroid surgery, these glands should be identified and preserved in benign disease. Superior parathyroids are more constant in location than inferior. Superior parathyroid is located above the inferior thyroid artery, posterior to RLN and close to cricoid cartilage along the posterior border of thyroid gland. Inferior parathyroid is located below the level of inferior thyroid artery and usually lies anterior to RLN. However inferior parathyroid may be located

Sternohyoid, sternothyroid and omohyoid muscles receive their motor nerve supply from the ansa hypoglossi, which supplies them in their lower half. If strap muscles need division for exposure of large goitres, they are transected in their upper part to preserve their innervation.
Figure 66.3 A 15-year-old female with lingual thyroid (arrow) (A); CT scan sagittal view of the same (B).
a

It is bounded medially by trachea and oesophagus, laterally by retracted strap muscles, and superiorly by the lower pole of thyroid. Its apex is directed interiorly at thoracic inlet. RLN runs through this triangle from lateral to medial side on the right and straight up along tracheoesophageal groove on the left.

It is seen in 1:3000 to 1:4000 patients of thyroid disease. It may be the only thyroid tissue or be present in addition to normal thyroid or an ectopic thyroid. When large it causes airway obstruction or difficulty to swallow. It is seen as a mass at the base of tongue on indirect laryngoscopy. It should be differentiated from other masses occurring at the base of tongue, i.e. lymphoma, squamous cell carcinoma, minor salivary gland tumour, lingual tonsil or rarely thyroglossal cyst. Surgical removal should be done, if lingual thyroid causes symptoms of airway obstruction or dysphagia by suprahyoid transpharyngeal approach. It requires lifelong replacement of thyroid hormones, if it was the only thyroid tissue.

Thyroid gland contains two types of cells: (i) follicular cells which synthesize and liberate T, (thyroxine) and Ts (triiodothyronine) and (ii) parafollicular 'C' cells which liberate calcitonin which has a calcium lowering effect (Figure 66.4).
Synthesis and release of thyroid hormones take place through five steps:
1. Active uptake of iodide.
2. Oxidation of iodide to iodine and binding of iodine to tyrosine molecule. The enzyme responsible for oxidation and binding is thyroid peroxidase.
3. Coupling of iodotyrosines to form T, and Ts. Two molecules of di-iodotyrosines (DIT) form T, and coupling one molecule of monoiodotyrosine (MIT) with one molecule of di-iodotyrosine form T 3. Again thyroid peroxidase is responsible for coupling and iodination.
DIT, MIT, Ty, Ts and thyroglobulin are bound together and form the colloid which is stored in the follicles of the thyroid gland. 4. At the time of secretion, colloid is taken up by thyroid cells and the peptide bonds between thyroglobulin and iodinated residues are broken by proteases to release Ty, Ts, DIT and MIT.
- 5. Uncoupled iodinated tyrosines (MIT and DIT) are deiodinated by enzyme iodotyrosine deiodinase and the iodine thus liberated is recycled. However, the enzyme does not act on iodinated tyrosine thus Ty and Ts are not affected.
In the congenital absence of this iodotyrosine deiodinase enzyme, MIT and DIT are not deiodinated and appear in urine with loss of iodine causing iodine deficiency. Ts is the active thyroid hormone but thyroid gland provides only 20% if it. Rest of the 80% of Ts is formed in the peripheral tissues by deiodination of T,. Ts is three times more potent than Ty. Synthesis and release of thyroid hormone is under the control of hypothalamus-pituitary—thyroid axis (Figure 66.5).

Hypothalamus secretes thyrotropin releasing hormone (TRH), which stimulates pituitary to release thyroid stimulating hormone (TSH). TSH acts on thyroid gland for synthesis and releases T, and Ts. In the feedback mechanism, T, and Ts hormones inhibit formation and release of TRH and TSH.
Figure 66.5 Hypothalamus-pituitary—thyroid axis. TRH, thyrotropin releasing hormone; TSH, thyroid stimulating hormone (aka thyrotropin); T,, thyroxine; T3, tri-iodothyronine.
Important terms used in thyroid disease are discussed in
Box 66.1.

Hashimoto disease is an autoimmune disorder. Antibodies develop against thyroglobulin and thyroid peroxidase and lead to hypothyroidism and raised TSH. The thyroid parenchyma is diffusely infiltrated with lymphocytes and fibrotic septae extend into the parenchyma. Thyroid size may be normal, enlarged or small. Multiple or a single regenerative nodules may form. Disease is more common in females and diagnosis can be made by measuring the level of antibodies against thyroglobulin and thyroid peroxidase. Nodules in Hashimoto disease may develop into a lymphoma or sometimes papillary cell carcinoma. Treatment of Hashimoto disease is thyroxine therapy to combat hypothyroidism.
Autoantibodies (TPO and Tg antibodies)
J
Thyroid inflammation with destruction of thyroid tissue and

Multiple micronodules or a large nodule (can change to lymphoma or papillary cancer)
Responds to exogenous thyroid

It is
due to
low levels
is the
most disease,
common total
of thyroid
cause.
or for
hormones.
Other subtotal
lymphoma for
or
Iodine causes
deficiency include
thyroidectomy, head
Graves’
and neck
disease.
cancers
Certain ism,
e.g.
amiodarone, drugs
lithium,
Hashimoto radiation
to neck
as or
can induce
hypothyroid- para-aminosalicylic
acid or
antithyroid drugs
or goitrogenic
substances in
diet.
Symptoms and signs of hypothyroidism are listed in
Table 66.1.
It can be treated by giving exogenous thyroid hormone. Hypothyroidism can also occur in neonates (1:5000) and thus there is need to test them after birth. Cretinism manifests after several months of extrauterine life. It causes lethargy, stunted growth, mental retardation and hearing loss. Neonatal hypothyroidism or cretinism can arise from inadequate iodine in mother's diet, administration antithyroid drugs or radioactive iodine to mother to treat her thyrotoxicosis or agenesis of thyroid in the infant. It is therefore essential for all pregnant mothers to maintain a euthyroid state.

It is an autoimmune disorder presenting clinically with feature of hyperthyroidism, goitre, ophthalmopathy and uncommonly dermopathy. Women are affected much more than men (5:1 to 10:1). Both genetic and environmental factors play their role in the causation of disease. It is caused by antibodies against TSH receptors. When antibodies react with their receptors, thyroid cells are stimulated to form excess thyroid hormone. Diagnosis is made on clinical features of hyperthyroidism (Table 66.2) and laboratory tests. TSH is suppressed and T, (free and bound) is raised. Causes of hyperthyroidism are listed in Table 66.3.

Thyroid cancer constitutes 2-3 new cases per 100,000 every year. It is two to four times more common in females than males. Genetic factors also play a part in their development. Various malignant neoplasms of the thyroid are listed in Table 66.4.

It is the most common cancer constituting 65-70% of all thyroid cancers. Majority of them are seen in third and fourth decade with two to three times preponderance in females. It is also seen in children even with cervical and distant metastases but has a favourable prognosis. It arises
Graves' disease (autoimmune disorder)
Toxic multinodular goitre
Autonomous nodule
TSH-secretory pituitary
Functioning thyroid
Exogenous tumour
cancer/
intake of
metastases thyroid
hormone
(thyrotoxicosis factitia)
¢
Thyroiditis
from follicular cells of thyroid and consists of fibrovascular stalk with cancerous follicular cells forming a papilla hence the name. Cells have abundant pale cytoplasm with typical nuclei. The latter are folded or grooved with intranuclear cytoplasm. Nucleoli are prominent and give typical Orphan Annie eye appearance. These features help to diagnose the tumour at fine needle aspiration cytology (FNAC). Papillary
carcinoma may undergo cystic change and also present laminated calcium bodies called psammoma bodies.

- 1. Ionizing radiation. Low-dose radiation for head and neck several
As a fallout from nuclear exposure as happened in Chernobyl accident, and Hiroshima and Nagasaki. Ionradiation causes mutation.
especially years previously. izing gene
2. Familial. Five to ten per cent of patients with papillary thyroid carcinoma (PTC) have family history of thyroid cancer. Cowden syndrome consists of multiple hamartomas, breast tumours, skin tags and follicular or papillary cancer. Gardner's syndrome consists of familial colonic polyposis with thyroid cancer. Papillary carcinoma is seen in areas with adequate iodine intake unlike follicular carcinoma which is more common in low-iodine intake areas with endemic goitre. Tumour may be multicentric in origin either because of intrathyroidal lymphatic spread or de novo multicentric origin.
Papillary cancer may spread locally and involve surrounding structures, e.g. strap muscles, trachea, oesophagus and recurrent laryngeal nerves.
It has a tendency to spread by lymphatics unlike the follicular carcinoma which spreads by blood stream. About one-third of the patients may have lymph node involvement at the time of presentation. Sometimes metastatic lymph nodes are palpable but the primary in the thyroid is occult and not clinically palpable.
Distant metastases are less common than in other forms of thyroid cancer but may be seen at the time of diagnosis. Pulmonary metastases are common.
Cells of papillary carcinoma may be functional and may show ability to concentrate iodine, secrete thyroglobulin and even produce thyroid hormones. They also express TSH receptors on their surface. All these features have clinical bearing in the management of these tumours.

- PTC may present with the following symptoms:
1. An asymptomatic mass in the thyroid.
2. Metastatic nodes in the neck. About one-third of patients have palpable neck nodes.
3. Depending on local invasion, mass in the thyroid may produce symptoms of local invasion of strap muscles, tra- chea, oesophagus or laryngeal nerves.
4. Pulmonary or bone metastases with or without a mass in the neck; occult primary of the thyroid may present with metastases.

History, clinical examination and FNAC are important. Ultrasound of the thyroid and the neck is important to look for other lesions in thyroid (as the disease is multifocal) and also involvement of neck nodes. Thyroid function tests may reveal hyperthyroidism though most of the patients are euthyroid. X-ray chest may show pulmonary metastases. CT / MRI may be required for extent of disease or retrosternal extension.

1. Microcarcinoma, which is less than 1.5 cm, hardly palpa- ble clinically with no capsular invasion or cervical nodes and mostly discovered incidentally at the operation, is treated with lobectomy with isthmusectomy.
2. Intrathyroidal tumour more than 1.5 cm with no nodule on contralateral side and no palpable neck nodes, also requires a lobectomy with isthmusectomy.
3. Gross disease in both lobes seen on preoperative ultra- sound or on palpation at the time of operation with no cervical nodes requires a total or near total thyroidectomy.
4. High-risk patients require total thyroidectomy.
5. Tracheal invasion requires tracheal segmental excision and repair in addition to the excision of growth.
6. Cervical lymph node dissection is done if nodes are pal- pable. There is no role of elective neck dissection.

After total thyroidectomy, disease may sometimes be left in the ligament of Berry, pyramidal lobe or superior poles of thyroid and requires postoperative radioiodine ablation.

It arises
from the
follicular cells
about
10-15%
of of
the thyroid.
thyroid malignancies.
It
Usually constitutes
presents at
age
50
and is
in females
(3:1).
Clinically, it presents either as a solitary thyroid nodule or a rapid increase in a pre-existent nodule.
More often it spreads by blood stream and thus may have
distant metastases
at presentation
in
10-15%
Unlike papillary
cancer, common
and if
it
FNAC
may after
lymph node
occurs, indicates
be surgical
be diagnosed
invasion.

1. If diagnosed as follicular neoplasm on FNAC (not can- cer), lobectomy with isthmusectomy should be done. Include also the pyramidal lobe.
If lobectomy specimen is reported as carcinoma, com-
pletion
of
thyroidectomy
is
done
i.e.
removal
of
other
lobe as well. It can be followed by radioiodine scan and ablation of metastatic disease.
2. If diagnosed follicular carcinoma on FNAG, total thyroid- ectomy should be performed.
3. If nodule size is greater than 4 cm in an elderly person and is reported as follicular neoplasm, perform total thyroidectomy due to the risk of its being carcinoma.
Neck
is
done
only
if
nodes
are
palpable.
Pro-
phylactic neck dissection has no role.
Prognosis of follicular carcinoma is poor if age at pre-
sentation
is
more
than
50
years,
size
is
more
than
4
cm
or
distant metastases are present.

It is also called oncocytic carcinoma. Oncocytes are large cells which are rich in mitochondria and stain pink. It may be reported
removal involvement
extensive as
follicular of
the as
the latter
is less
spread locally.
neoplasm.
specimen
It is
that only
carcinoma requires
capsular can
or vascular
multifocal, bilateral and spreads to regional nodes or sends distant metastases. Its behavior is more aggressive than follicular carcinoma. These tumours do not take up radioactive iodine as well as is seen in follicular carcinomas.
Aggressive of thyroid cancer: Hurthle cell > Follicular >
Papillary cell cancer.
Clinically, it presents as a thyroid nodule. Mean age at presentation is slightly more than in follicular carcinoma. FNAC may show Hurthle cells but cannot differentiate benign Hurthle cell adenoma from carcinoma. It is only on histologic findings of capsular or vascular invasion that diagnosis of carcinoma can be made.

Hurthle cell adenoma is a benign tumour and requires lobectomy and isthmusectomy only. If capsular or vascular invasion is seen on histopathology a completion thyroidectomy should be done.
If it
is reported
as
Hurthle cell
carcinoma on
FNAC
a
total thyroidectomy
is done.
with
Lateral clearance
nodes, if
palpable, of
paratracheal
a
neck nodes
dissection is
also performed.
Hurthle
In the follow-up, technetium scan should be done as cells do not take up radioiodine.
Prognosis is worse than in follicular or papillary carcinoma.

It represents less than 5% of all thyroid cancers. Mostly affects patients in the age group of 60-80 years, is very rare before 50 years. Affects women more than men in the ratio of 3:2. It has an aggressive behaviour, grows rapidly to involve the surrounding structures causing hoarseness, stridor dyspnoea, dysphagia and thoracic inlet obstruction. Cervical lymph node involvement is seen in 80% of patients at presentation. Due to its aggressive nature, cervical nodal mass and thyroid mass may fuse together and it may be difficult to distinguish the two. Unlike thyroid lymphoma, which is painless, anaplastic carcinoma causes a painful mass. Distant metastases can lodge in long bones and brain and are present in 50% of patients at presentation. Treatment is unsatisfactory. Median survival in only a few months. Surgery, radiation and chemotherapy have a limited role. Palliation in the form of tracheostomy and nutritional is the treatment.
support only
Figure
66.6
Types of
medullary cancer
of thyroid.

It arises from the parafollicular C cells of the thyroid which are neuroectodermal in origin. Hence it may be associated with other tumours of neuroectodermal origin as in MEN Ila and MEN IIb. Most medullary carcinomas are located in the middle and upper thirds of thyroid lobes which are derived from the ultimobranchial bodies having C cells. They comprise about 5% of thyroid malignancies.
Clinically, medullary carcinoma presents with a neck mass with cervical nodes in the age group of 50-60 years. Both sexes may be equally involved. As tumour is aggressive and invades locally, it causes pain, dyspnoea, dysphagia and hoarseness. Distant metastases may be seen in the mediastinum, lung and bone at the time of presentation in about half the cases.
Medullary carcinoma can be sporadic (more common) or familial. The latter may be associated with MEN type IIA and IIB (see Table 66.5) or without any endocrinopathy (Figure 66.6).
Parafollicular cells secrete calcitonin and a carcinoembryonic antigen. Levels of calcitonin have been used in the diagnosis of medullary carcinoma and in postsurgical followup for recurrent or residual tumours.
Sporadic carcinoma presents as a unifocal lesion and is not associated with other endocrinal pathologies. Familial type presents at a younger age and is multicentric and bilateral. Familial type is of two types: one, without association with other endocrinopathy and the other associated with multiple endocrinopathies (MEN type IIA and IIB) (Table 66.5).
A variant of medullary thyroid cancer is mixed medullary cancer in which both follicular and C cells are seen. Its behavior and treatment are same as medullary cancer.
Diagnosis is made on FNAC and elevated levels of calcitonin. Patients should be tested for mutation of RET protooncogene which is present in peripheral lymphocyte DNA. Investigate also for other endocrinopathies by measuring levels of serum calcium (for parathyroid) and 24-h urine for catecholamines/metanephrine for pheochromocytoma. Relevant imaging studies are also done.

Screening of all first degree relatives and children is required for RET-proto-oncogene mutations in cases of medullary cancer associated with MEN type IIA and IIB. All children with medullary cancer and those with MEN type ITA should undergo thyroidectomy by age of 6 years. Those with MEN type IIB should have surgery within first year of life.

Because of aggressive nature and multicentricity of the lesion, total thyroidectomy is the treatment of choice. It is combined with removal of nodes in level VI even if not involved but if level VI nodes are involved, comprehensive neck dissection (level II to V) is also done. If primary lesion is more than 2 cm, ipsilateral elective neck dissection should be done. Level VII nodes should also be removed.
Pheochromocytoma, if associated, should be removed before thyroidectomy to avoid hypertensive crisis during operation.
Postoperative follow-up is done by measuring calcitonin levels
for any
residual, recurrent
or metastatic
disease.
Radioactive iodine and chemotherapy are not effective.
Role of external beam radiation is also controversial.

Most of the thyroid lymphomas are B-cell non-Hodgkin type. They are seen in the age group of 60-80 years and are more common in females (ratio of 3:1). Pre-existing Hashimoto disease predisposes to lymphoma formation.
Clinically, a lymphoma presents like an anaplastic thyroid cancer, with rapidly growing painless thyroid mass which invades the surrounding structures leading to hoarseness, stridor, dyspnoea, dysphagia and thoracic inlet obstruction. Cervical lymph nodes are enlarged. Lymph node enlargement can occur in other regions of body as well depending on the stage of disease. Patient is often hypothyroid.
Histologically, lymphoma needs to be differentiated from anaplastic carcinoma and Hashimoto disease by immunohistochemistry. Therefore it may require FNAB or isthmusectomy to have enough tissue; the latter also decompresses the trachea to relieve airway obstruction.
Treatment depends on the stage of disease. Surgery, external beam radiation and chemotherapy have been used depending on the stage. Role of surgery may be limited to
open biopsy only or a tracheostomy or removal of early disease when it is localized to thyroid (stage I) or thyroid and the neck nodes (stage II). Radiotherapy can also be used in such cases of stage I and II but for a disease of stage III (both sides of diaphragm) or stage IV (disseminated disease) radiotherapy and chemotherapy are combined.

Malignant disease of breast, kidney, lung, and squamous cell cancers of head and neck and melanomas can send metastases to the thyroid and present as a nodule. FNAC is helpful in diagnosis. Metastases from undifferentiated cancers may be difficult to distinguish from anaplastic carcinoma of the thyroid.

Clinically, palpable nodules are seen in about 4-7% of people. Their incidence increases as the age advances. About 5-10% of the nodules may be malignant. Risk of a nodule being malignant increases if the patient received radiation in childhood. Incidence of a thyroid nodule being malignant also increases in patients over the age of 50-60 years.
A solitary thyroid nodule can be:
- — Colloid nodule (also called adenomatous nodule)
2.
Adenoma
(a) Follicular adenoma
(b) Hurthle cell adenoma
3. Thyroid cyst (cystic change in colloid nodule or follicular adenoma)
Regenerative nodule OOS
Dominant nodule (in multinodular goitre)
Autonomous (or toxic) nodule Carcinoma ND
The main consideration in a thyroid nodule is to pick up cases of carcinoma. Of all the palpable nodules, only about will be
5-10% malignant.

It is a benign condition, clinically resembling an adenoma, hence called adenomatous. It is due to hyperplasia of follicular cells with the follicles filled with colloid. TSH level is normal but hyperplasia of follicular cells is attributed to their being more sensitive to the action of TSH. Lowering the TSH by administering exogenous thyroid hormone (suppressive therapy) is used to treat such nodules but it is difficult to predict which nodules will respond to this form of therapy.

It is a well-demarcated, encapsulated and true benign neoplasm of follicular cells. It may undergo cystic degeneration, haemorrhage, calcification and fibrosis. It is difficult to differentiate it from follicular carcinoma on FNAC because diagnosis of carcinoma requires vascular and/or capsular invasion which is possible only on histology of the mass removed.

Hurthle cells are oncocytes, rich in mitochondria. Like follicular adenoma, it is also well-demarcated and encapsulated. An adenoma of Hurthle cell cannot be differentiated from Hurthle cell carcinoma on FNAC as the diagnosis of latter requires capsular and/or vascular invasion.

About one-fourth of the thyroid nodules present as a cyst or a cystic component in a nodule. It may be:
1. Simple thyroid cyst.
2. Haemorrhage in colloid nodule.
3. Papillary carcinoma with cystic change.
4. Parathyroid cyst.
5. Thyroglossal duct cyst.
Cystic nature of the thyroid mass can be identified on ultrasound. Aspiration of clear fluid is indicative of parathyroid cyst; fluid in such cases is tested for parathormone. Aspiration of brown fluid indicates haemorrhage in the colloid nodule; red or bloody aspirate indicates papillary cancer. FNAC should be done from the solid component of the cyst under ultrasound guidance in masses with both solid and cystic components. Thyroid cysts larger than 4 cm are surgically excised; those smaller than 4 cm are aspirated and suppressed with thyroid hormones (suppression therapy) and if they recur surgical excision should be done (Figure 66.7).

Clinically, only one nodule is palpable though gland has multiple nodules. Other nodules are diagnosed at ultrasound or direct palpation of gland at the time of the surgery. A dominant nodule seen in a multinodular goitre may be malignant.

It is seen in Hashimoto disease. Patient is hypothyroid and TSH level is either raised or at the upper limit of normal. TSH stimulates the follicular cells to form a nodule.

It is a single hot nodule in the thyroid and is so-called because it functions independently of TSH. It is due to mutation in TSH receptors on follicular cells, which function independently and cause proliferation of follicular cells and their enhanced function. Thyroid scan is diagnostic which reveals high uptake by the nodule but the rest of the gland shows low uptake. It causes thyrotoxicosis which is usually mild. TSH level is low. Treatment is total lobectomy with
Age < 20 and > 45 years
Male gender
History of previous radiation to neck
Family history of thyroid cancer
Rapid growth
Pain
Compressive or invasive features such as stridor, dyspnoea and dysphagia
Fixed and hard lesion
Size of nodule > 4 cm
Recurrent or rapidly filling cyst after aspiration
Nodules that occur in Graves' or Hashimoto thyroiditis
isthmusectomy or thyroid ablation with radioactive iodine; the former being preferred. In Europe, it has been treated by injection of ethanol into the nodule under ultrasound guidance especially in patients unfit for surgery or unwilling for radioiodine therapy. Ethanol injection may need to be repeated in some patients. It has the risk of diffusion to recurrent laryngeal nerve and cause its paralysis.

Thyroid malignancies may present as a solitary nodule (see Table 66.6).

1. History is important. Patient may present with large thy- roid nodule or be referred because an incidental nodule is discovered on imaging. A solitary thyroid nodule or a dominant nodule in a multinodular goitre has the risk of being malignant. Risk factors for a nodule being malig- nant are shown in Table 66.6.
2.
Physical
Concern
for
malignancy
increases
when
(a) Nodule is larger than 4 cm.
(b) Fixed to skin or underlying structures.
(c) Firm to feel.
(d)
There
are
lymph
nodes
in
the
neck
at
lev-
els
VI,
I,
III,
IV
and
V.
(e) examination reveals fixed vocal cord.
Laryngoscopic 3. family history medullary
Thyroid function. TSH, T4 and T3 are measured. They are normal in a colloid nodule. Fall in TSH or rise of T4 (total or free) and T3 indicate hyperfunctioning nodule. Estimation of calcitonin level is indicated in patients with of carcinoma.
4. Ultrasound of thyroid. It is a useful test and identifies:
(a) Small nodules which cannot be palpated or nodules
factors for malignancy exist, surgery may be required (Figure 66.8).
in obese
neck.

(b) Multiple nodules.
(c) Accurate size of nodule(s) and their location.
(d) Cystic or solid nature of nodules.
(e) Associated cervical lymph nodes.
(f) Vascularity of thyroid gland or nodules.
5. Thyroid scan. Technetium (°° Tc), !°9I and !8!I have been used. It can differentiate between cold and hot nodules. Most of the nodules are cold (80%) with only 5% being hot. Chances of malignancy in cold nodules is about 10% while it is only 1% in hot nodules.
6. Fine needle aspiration cytology or biopsy (FNAC/FNAB). Sensitivity and specificity of FNAC for diagnosis of thyroid masses is high—up to 90%. Ultrasound-guided FNAC/ FNAB further increases the accuracy of cytologic diagnosis. The aspiration may be reported as benign, malignant or indeterminate. Follicular or Hurthle cell tumours are reported as neoplasms because FNAC cannot differentiate between benign and malignant lesions. Insufficient material collected on FNAC may require repeat procedure or observation and periodic evaluation. If risk
7. CT/MRI. They are not used routinely but are very help-
ful in
evaluating the
size and
extent of
retrosternal goi-
tres.
It may
also show
degree of
tracheal compression.

Enlargement of thyroid and formation of nodule(s) are due to trophic effect of TSH on thyroid cells. Suppressing TSH with exogenous T, is assumed to reduce nodule formation. A nonrandomized study showed reduction of nodule in 30% of patients. But other studies which were randomized did not corroborate it. There is also no consensus how long to give therapy, how much to suppress TSH, whether nodule will decrease or only stabilize in size. Suppressive therapy has also the risk of cardiac arrhythmias and decrease in bone density in such patients who are usually euthyroid. In general suppressive therapy is not preferred.

Broadly there are four indications for thyroid surgery which can be remembered by four C’s.
1. Cancer thyroid.
2.
Suspicion
of
cancer,
e.g.
thyroid
neoplasm
on
FNAC
and
thyroid nodule with risk factors.
3.
Compressive
symptoms,
e.g.
pressure
on
trachea
or
oesophagus or veins causing dyspnoea or dysphagia. Sub-
sternal
extension
causing
thoracic-inlet
syndrome
with
positive Pemberton’s sign, i.e. raising the arms above head
causes respiratory distress, suffusion of face and neck vein engorgement.
4. Cosmetic. A large nodule or a multinodular goitre.
To this may be added the fifth indication of hyperthyroid-
ism, such as autonomous nodule or Graves’ disease.

1. Detailed history.
3. Thyroid profile. TSH, T, and Ts.
4. Indirect laryngoscopy. For vocal cord paralysis or a com-
pensated vocal cord function.
5. FNAC. To know the histology.
6. TPO antibodies. When indicated for Graves’ disease.
7.
Level
of
calcitonin.
When
medullary
carcinoma
is
8 9. . Serum calcium level. As a baseline.
10
lL
19.
.
Ultrasound
thyroid/neck.
For
size
and
number
of
thy-
roid nodules and status of lymph nodes in the neck.
Thyroid scan. If indicated, e.g. for autonomous nodule.
CT chest. For retrosternal goitre.
Investigations for surgical fitness:
¢ Haemogram.
Urine; routine and microscopic.
Blood
sugar
(F).
Blood
urea/creatinine.
X-ray
chest.
ECG.
Cardiac
echo
(if
required).

Hemithyroidectomy or thyroid lobectomy, Description = Complete removal of one thyroid lobe with isthmus. Hemithyroidectomy or thyroid lobectomy, Indication = • Benign nodule • Intrathyroidal cancer. Subtotal thyroidectomy, Description = Removal of more than one half of the thyroid lobe on each side and also isthmus leaving 3 g on each sides. Subtotal thyroidectomy, Indication = • Thyrotoxicosis • Multinodular goitre. Near total thyroidectomy, Description = Removal of complete thyroid lobe on one side along with isthmus and almost complete removal of lobe on other side leaving only a small part (1 g) of it to protect parathyroid and RLN. Near total thyroidectomy, Indication = For some malignancies. Total thyroidectomy, Description = Complete removal of both lobes and isthmus. Total thyroidectomy, Indication = • Medullary carcinoma • Hurthle cell carcinoma • Follicular carcinoma • High-risk patient with papillary carcinoma. Isthmusectomy, Description = Complete removal of isthmus. Isthmusectomy, Indication = • Nodule of isthmus • For diagnostic biopsy. Completion thyroidectomy, Description = Removal of remaining thyroid gland to convert it into total or subtotal thyroidectomy after ominous histology is received. Completion thyroidectomy, Indication = If total lobectomy proves malignant with capsular/vascular invasion, other lobe is excised.

A horizontal incision in the skin crease below the collar line. In females with heavy breasts incision is a bit higher so that it does not drag to manubrium to cause hypertrophic scar. Incision is made from one sternomastoid muscle to the opposite one. It cuts through skin and subcutaneous tissues.

Platysma is then cut in the same incision with diathermy and the upper and lower flaps are developed. The upper flap should reach the thyroid notch and the lower one up to the clavicle.

Midline is defined as a vertical incision made in fascia separating the two sternohyoid muscles. Sternothyroid muscles are separated from the thyroid gland.

The normal lobe is inspected and palpated for any undetected nodularity.

Identify and ligate the middle thyroid vein which drains into the internal jugular vein. Inferior thyroid veins which form a venous plexus in front of the trachea are ligated and cut. Thyroidea ima artery may lie in front of trachea; it is identified and

Branches of inferior thyroid artery are found close to the thyroid. They are ligated medially preserving blood supply to parathyroids. Recurrent laryngeal nerve may pass anterior, posterior or through the branches of the artery and care is taken to preserve it.
RLN is injured near the ligament of Berry when cautery or artery forceps is used indiscriminately while cutting the ligament of Berry to free the thyroid lobe.

Division of sternothyroid muscle near its upper part helps to ligate superior thyroid artery and vein. They should be ligated individually. External branch of superior laryngeal nerve lies posteromedial to thyroid vessels and should be identified and preserved. Traction inferiorly on the thyroid helps to save the nerve.

THEIR BLOOD SUPPLY
Branches of inferior thyroid artery are ligated distally as close to thyroid as possible to preserve parathyroid blood supply. Also examine the thyroid specimen and if parathyroids have been removed, they are identified, separated, sliced and implanted in the sternocleidomastoid muscle. Before implantation, histological confirmation of parathyroid tissue is made by frozen section.

Isthmus is separated from the trachea, doubly clamped and divided. Cut surface of isthmus on contralateral side is ligated with 3/0 chronic catgut by continuous interlocking suture for haemostasis.

Wound is irrigated with saline or betadine solution. Suction drainage is used to avoid haematoma formation.

Anaesthetist performs Valsalva manoeuvre by intermittent positive pressure for any venous ooze and if none is observed, wound is repaired. Strap muscles are approximated with 3/0 vicryl. Platysma is also approximated and subcutaneous sutures applied with 3/0 vicryl and then subcuticular sutures used for good cosmetic result. Steristrips are then applied to further strengthen the incision lines.

1. Haematoma. It can be avoided by ligation of vessels, at the time of surgery and performing Valsalva at the end of operation to check for venous ooze. A haematoma can compress the airway and should be identified early and evacuated.
2. Airway obstruction. Tracheotomy may be required. Compression by haematoma, tracheomalacia and laryngeal oedema or myxoedematous cords also cause obstruction to airway.
Injury to recurrent laryngeal nerve. Injury to superior laryngeal nerve. Wound infection. or Ye
Hypocalcaemia. Removal or devascularization of parathyroid glands causes numbness and tingling of lips, hands and feet. In such cases calcium level may be less than 8.0 mg/dL. Critical period is 24-96 h after operation. Always check for serum calcium levels postoperatively. It may require calcium and vitamin D supplementation by oral or i.v. route depending on severity of hypocalcaemia. D>
Pneumothorax. Due to injury to pleura in lower neck. N
8. Hypothyroidism. It is usually seen 4—6 weeks after operation. This would require long-term thyroid replacement.

It is a fibromuscular tube, about 25 cm long in an adult. It extends from the lower end of pharynx (Cg) to the cardiac end of stomach (T;) (Figure 67.1). It runs vertically but inclines to the left from its origin to thoracic inlet and again from T; to oesophageal opening in the diaphragm. It shows three normal constrictions and it is important to know their location at oesophagoscopy. They are:
The upper oesophageal sphincter starts at the upper border of oesophagus and is about 3-5 cm in length and functions during the act of swallowing. The lower oesophageal sphincter is situated at lower portion of oesophagus. It is also 3-5 cm in length and functions to prevent oesophageal reflux. Middle portion of oesophagus shows active peristalsis. The waves are weaker in the upper part, becoming gradually stronger towards the lower portion.
1. At pharyngo-oesophageal junction (Cg)—15 cm from the upper incisors.
2. At crossing of arch of aorta and left main bronchus (T4)—25 cm from upper incisors.
3. Where it pierces the diaphragm (T}))—40 cm from upper incisors.
Foreign bodies in the oesophagus can be held up at these constrictions.
The wall of oesophagus consists of four layers. From are:
within outwards, they
(a) Mucosa, which is stratified lined by squamous
(b) Submucosa, which connects mucosa to muscular layer.
(c)
Muscular layer,
which has
inner circular
and outer
lon- gitudinal
fibres.
thickened
Circular to
form fibres
a
at the
cardiac sphincter.
lower
The end
upper are
third of
oesophagus has
striated, the
lower third
smooth, and the middle third both striated and smooth muscle fibres (Figure 67.2).
(d) Fibrous layer, which forms loose covering of oesophagus.

Parasympathetic fibres come from vagus nerves (X) and sympathetic fibres from the sympathetic trunk.

The cervical, thoracic and abdominal parts drain respectively into deep cervical, posterior mediastinal and gastric nodes.

Manometric studies have shown two high pressure zones in oesophagus and they form the physiological sphincters.
Figure 67.1 Anatomy of oesophagus and levels of normal constrictions from the upper incisors.

The act of swallowing is divided into three phases:
1. Oral or buccal phase. The food which is placed in the mouth is chewed, lubricated with saliva, converted into a bolus and then propelled into the pharynx by elevation of the tongue against the palate.
2. Pharyngeal phase. It is initiated when the bolus of food comes into contact with pharyngeal mucosa. A series of reflex actions take place carrying the food past oroand laryngopharynx into the oesophagus. The communications into nasopharynx, oral cavity and larynx are cut off.
(a)
(b)
(c)
(d)
_—
Closure of nasopharynx. Soft palate contracts against the Passavant's ridge on the posterior pharyngeal wall and completely cuts off the nasopharynx from the oropharynx.
Closure of
oropharyngeal isthmus.
The entry
of food
back into
oral cavity
against is
prevented the
muscles.
Closure of
larynx.
by palate
by contraction
and sphincteric
Aspiration temporary
geal of
tongue action
of palatoglossal
into the
cessation larynx
of inlet
by respiration,
contraction of
false and
base of
of true
cords, is
prevented closure
aryepiglottic and
tongue.
tection
The to
larynx backwards
Contraction role
is of
laryn- folds,
rising of
larynx of
not when
epiglottis clear
food but
it passes
of pharyngeal
ryngeus.
Relaxation timed
and into
the into
the closure
under in
the providing
pro- is
seen to
deflect pyriform
muscles of
synchronous oesophagus
fossae.
of cricopharyngeus
cricopha- muscles
that food
passes during
muscles.
3.
Oesophageal phase.
the
After cricopharyngeal
movements contraction
food sphincter
of oesophagus
ach.

is so
from pharynx
of enters
pharyngeal the
closes and
take
Gastro-oesophageal oesophagus
the bolus
sphincter relaxes
well before
permits fluids
of to
pass.
oesophagus, the
peristaltic down
at the
peristaltic
Bolus of
food the
stom- lower
wave is
passed end
of reaches
and by
contraction peristaltic
waves and
then the
sphincter closes.
Regurgitation of food back from stomach into oesopha- gus is prevented by:
(i)
tone of gastro-oesophageal sphincter,
(ii)
negative intrathoracic pressure,
(iii)
pinch-cock effect of diaphragm,
(iv)
mucosal folds,
(v)
oesophagogastric angle and
(vi)
slightly positive intra-abdominal pressure.

All oral feeds are stopped immediately. Nutrition is main- tained through i.v. route. Massive doses of antibiotics are
given i.v. to combat infection.
Early perforations of cervical oesophagus can be man- aged
by conservative
measures;
drainage is
required only
if suppuration
develops.
Retrovisceral space
and/or upper
mediastinum can
be drained
neck.
Rupture of thoracic oesophagus is more serious and con- servative
treatment rarely
succeeds.
If diagnosis
is made
early
(within pleural
6
h), cavity
not possible;
perforation drained.
is surgically
surgery is
then repaired
is delayed,
restricted and
repair to
drainage infected area.

Acids, alkalies or other chemicals may be swallowed accidentally in children or taken with the purpose of suicide in adults.

Severity of oesophageal burns depends on the nature of corrosive substance, its quantity and concentration and the duration of its contact with the oesophageal wall. Alkalies are more destructive and penetrate deep into the layers of the oesophagus. With lye burns, entire oesophagus and stomach may slough off causing fatal mediastinitis and peritonitis.
Oesophageal burns run through three stages:
_ Stages of acute necrosis.
2. Stage of granulations: Slough separates leaving granulating ulcer.
3. Stage of stricture: Stricture formation begins at 2 weeks and continues for 2 months or longer.

Evaluate the patient and determine the type of caustic ingested, signs and symptoms of shock, upper airway obstruction, mediastinitis, peritonitis, acid-base imbalance, and associated burns of face, lips and oral cavity. Take X-ray of the chest and soft tissue lateral view of neck.
is of
the
It is acute inflammation of the oesophagus and can be due to (i) ingestion of hot liquids, (ii) ingestion of caustic or corrosive agents, (iii) laceration due to swallowed foreign body, or trauma of oesophagoscopy, (iv) monilial infection of oesophagus from thrush in the oral cavity and (v) systemic disorder like pemphigus. Patient complains of dysphagia, retrosternal burning or haematemesis. Diagnosis can be made from history, X-ray studies and oesophagoscopy.

Perforation or the rupture of oesophagus results from:
1. Instrumental trauma, i.e. oesophagoscopy or dilatation of strictures with bougies. The common site or rupture in these cases is just above the upper sphincter; sometimes it is the lower oesophagus near the hiatus.
2. Spontaneous rupture. This usually follows vomiting and involves mostly the lower third of oesophagus. Postemetic rupture of all the layers of oesophagus is called Boerhaave syndrome.

Early diagnosis is imperative, as mediastinitis, resulting from
rupture, can
rapidly prove
fatal.
com- plaining
of pain
in the
neck or
interscapular region,
following an oesophagoscopy, should be suspected of a perforation.
The features of cervical oesophageal rupture are pain, fever, difficulty to swallow and local tenderness, along with signs of surgical emphysema in the neck.
The features of thoracic oesophageal rupture are pain, referred to the interscapular region, fever 102-104°F (39-40°C), signs of shock, surgical emphysema in the neck, crunching sound over the heart (Hamman's sign, because of air in the mediastinum) and pneumothorax.
X-rays of the chest and neck are essential. They may reveal widening of the mediastinum and retrovisceral space, surgical emphysema, pneumothorax, pleural effusion or gas under the diaphragm.

1. Hospitalize the patient.
2. Treat shock and acid-base imbalance by i.v. fluids and electrolytes. Monitor urine output for renal failure.
. Relieve pain. OO
. Relieve airway obstruction. Tracheostomy may be required. 5. Neutralization of the corrosive by appropriate weak acid or alkali, given by mouth, can be done but is effective only if done within first 6 h. He
6. Parenteral antibiotics should be started immediately and continued for 3-6 weeks depending on the degree of burns.
7. Pass a nasogastric tube. It is useful to feed the patient and to maintain oesophageal lumen.
8. Oesophagoscopy. Some advocate an early oesophagos- copy within 2 days to know if burns in the oesophagus have occurred and if so, their degree and extent so as to plan further treatment. Oesophagoscope is not passed beyond the first severe circumferential burn.
9. Steroids should be started within 48-96 h and contin- ued for 4-6 weeks to prevent stricture.
10. Follow the patient with oesophagogram and oesopha- goscopy every 2 weeks, till healing is complete, for the development of any stricture.
11. Ifstricture develops it can be treated by:
(a) Oesophagoscopy and prograde dilatations, if permeable.
(b) Gastrostomy and retrograde dilatation, if imperme- able.
(c) Oesophageal reconstruction or by-pass, if dilatations are impossible.
12. Patients of corrosive injuries of oesophagus may require life-long follow-up.

The strictures usually occur when muscular coat of the is The common causes are:
oesophagus damaged.
1. Burns due to corrosive substances or hot fluids.
2. Trauma to oesophageal wall due to impacted foreign bodies or instrumentation or external injuries.
Ulcerations due to reflux oesophagitis. Ulcerations due to diphtheria or typhoid. Sites of surgical anastomosis. or Ye
Congenital, usually in the lower third. D>

Dysphagia, first to solids and then to liquids, is the common complaint. When obstruction is complete, regurgitation and cough may occur. Patient is malnourished. Barium swallow establishes the diagnosis. Oesophagos- copy is required to exclude malignancy.

1. Prograde dilatation with bougies. It should be done under direct vision through oesophagoscope. Dilatations may be required frequently.
2. Gastrostomy. It helps to feed the patients and give rest to the inflamed area above the strictures. After a few days, when inflammation subsides, lumen may become visible and prograde dilatation can be restored. Patient can be given a thread to swallow, which is recovered from the stomach, and prograde or retrograde bouginage can be done.
3. Surgery. Excision of strictured segment and reconstruction of food passage using stomach, colon or jejunum.

It is displacement of stomach into the chest through oesophageal opening of the diaphragm. Most patients are elderly, past 40 years. This disorder is of two types:
1. Sliding. Stomach is pushed into the thorax, in line with the oesophagus. Reflux oesophagitis is common and may give rise to ulceration and stenosis. Haemateme- sis may occur. It is caused by raised intra-abdominal pressure.
2. Paraoesophageal. A part of the stomach along with its peritoneal covering passes up into the thorax by the side of oesophagus. The gastro-oesophageal junction still remains below the diaphragm and the angle between oesophagus and stomach is maintained. There is no reflux oesophagitis in this type of hernia. The main symp- tom is dyspnoea on exertion due to position of stomach in the thorax and sometimes bleeding.
Diagnosis of both types of hiatus hernia can be made by swallow.
barium

Mainly it is surgical; the hernia is reduced and diaphragmatic opening repaired. Early cases and those unfit for surgery may be treated conservatively to reduce reflux oesophagitis by measures such as (i) sleeping with head and chest raised, (ii) avoidance of smoking, (iii) use of drugs that reduce acidity (antacids and proton pump inhibitors), (iv) reduction of obesity and (v) attention to the causes which raises intra-abdominal pressure.

Classical features of this syndrome include dysphagia, iron-deficiency anaemia, glossitis, angular stomatitis, koilonychia (spooning of nails) and achlorhydria. There is atrophy of the mucous membrane of the alimentary tract.
Predominantly, it affects females past 40 years. Barium swallow shows a web in the postcricoid region and the same can be seen on oesophagoscopy. It is due to subepithelial fibrosis in this region.
About 10% of the cases with this syndrome will develop postcricoid carcinoma. It also predisposes to the develop- ment of carcinoma in the tongue, buccal mucosa, pharynx, oesophagus and the stomach.

1.
To
correct
anaemia
by
oral/parenteral
iron.
Serum
levels
of
iron
are
more
important
than
level.
Asso-
ciated
Byy
and
Bg
should
also
be
corrected.
2. Dilatation of the webbed area by oesophageal bougies.

It is a functional disorder where the patient complains of “lump” in the throat. There is no true dysphagia. In fact, feeling of lump is more marked between the meals rather than during a meal. Such a patient may have fear of cancer in the throat. Clinical examination of the pharynx, larynx and base of tongue is normal.
Treatment is reassurance to the patient when no cause has found.
been

They can be divided into:
1. Hypermotility disorder, e.g. cricopharyngeal spasm, dif- fuse oesophageal spasm, nut cracker oesophagus.
2. Hypomotility disorders, e.g. cardiac achalasia, gastro- oesophageal reflux, scleroderma, amyotrophic lateral sclerosis.
They may involve the upper sphincter, lower sphincter or the body of oesophagus.

It is caused by failure of the upper oesophageal sphincter to relax properly. There is incoordination between relaxation of the upper oesophageal sphincter and simultaneous contraction of the pharynx. The common causes are cerebrovascular accidents, Parkinson's disease, bulbar polio, multiple sclerosis and muscular dystrophies.

It is characterized by strong nonperistaltic contractions of the body of oesophagus while sphincteric relaxation is normal. The symptoms consist of dysphagia or odynophagia with substernal chest pain, simulating angina pectoris. Barium swallow may show segmented oesophageal spasms giving a rosary bead or a cork-screw type of oesophagus, though it may be normal in some. Manometry shows normal relaxation of the sphincter on swallowing. The treatment is dilatation of lower oesophagus. Severe cases may require myotomy of oesophagus from the arch of aorta to lower sphincter.

These are strong, high amplitude oesophageal contractions but the contractions remain peristaltic (compare diffuse oesophageal spasm where contractions are nonperistaltic). It causes and substernal dysphagia pain.

It is characterized by the absence of peristalsis in the body of oesophagus and high resting pressure in lower oesophageal sphincter; the latter also does not relax dur- ing swallowing.
The symptoms of cardiac achalasia include dysphagia, which is more to liquids than solids (reverse of that seen in malignancy or strictures) and regurgitation of swallowed food particularly at night.
The diagnosis is made by (i) radiography (barium swallow shows dilated oesophagus with narrowed rat tail lower end), sometimes also called bird-beak appearance; (ii) manomet- ric studies (low pressure in the body of oesophagus and high pressure at lower sphincter and failure of the sphincter to relax); (iii) endoscopy (to exclude benign stricture or any development of carcinoma which is a common complica- tion of this disorder.
The treatment of choice is the modified Heller’s operation (myotomy of the narrowed lower portion of the oesopha- gus). Forceful pneumatic dilatation of the lower oesophagus can be done in those unfit for
surgery.

It is due to decreased function of lower oesophageal sphincter thus permitting regurgitation of gastric contents into oesophagus. Other causes of gastro-oesophageal reflux are pregnancy, hiatus hernia, scleroderma, excessive use of tobacco and alcohol, and drugs that relax the smooth muscle (anticholinergic, beta-adrenergic drugs and calcium-channel blockers) .
The symptoms of oesophageal reflux include substernal pain, heartburn and regurgitation.
The treatment consists of:
Elevation of the head of bed at night.
Avoiding food at least 3 h before bedtime. NO
Antacids. OO
Drugs that increase tone of lower oesophageal sphincter, e.g. metoclopramide. A
Hg receptor antagonists, e.g. cimetidine and ranitidine. ao
Avoiding smoking, alcohol, caffeine, chocolates, mints
and carbonated drinks.
7. Antireflux surgery, e.g. Nissen’s fundoplication.
COMPLICATIONS OF GASTRO-OESOPHAGEAL REFLUX
1. Oesophagus
© Oesophagitis, haemorrhage. oesophageal mucosal erosion and
¢ Benign oesophageal stricture.
¢ Barrett’s oesophagus (normal squamous epithelium of oesophagus is replaced by columnar epithelium as a result of continuous inflammation). It is a precancerous condition.
2. Lung
e Aspiration pneumonia.
e Asthma.
¢ Bronchiectasis.
3. Larynx
¢ Posterior laryngitis causing vague pain in throat, hoarse- ness and repeated throat clearing.
e Pachydermia laryngis.
¢ Contact ulcers and granulomas.
° Posterior glottic stenosis.
¢ Paroxysmal laryngospasm.
¢ Carcinoma larynx.
4. Ear
¢ Otitis media with effusion.
5. Miscellaneous
¢ Globus hystericus.

It is a systemic collagen disorder primarily neural, but secondarily weakening the smooth muscles of the lower two-thirds of oesophagus and the lower oesophageal sphincter. Dysphagia may precede cutaneous lesions. Barium swallow shows absence of peristalsis in distal twothirds of the oesophagus. Many of these patients have hiatus hernia, or reflux oesophagitis and may develop stricture in distal part of the oesophagus due to recurrent inflammation.

It occurs at the junction of squamous and columnar epithelium at the lower end of oesophagus and has also been called lower oesophageal ring. Usually seen in patients above 50 years of age. Cause is unknown. Symptomatic patients complain of intermittent dysphagia and some may even present with bolus obstruction. It may be associated with hiatus hernia. Treatment is oesophageal dilatation.

Benign neoplasms are rare compared to malignant ones.
Leiomyoma is the most common and accounts for twothirds of all the benign neoplasms. It arises from the smooth muscle and grows in the wall of oesophagus. Dysphagia is produced when tumour exceeds the diameter of 5 cm. Barium swallow shows an ovoid filling defect. Endoscopy reveals a submucosal swelling. Biopsy should not be taken. Treatment is enucleation of the tumour by thoracotomy.
Mucosal polyps, lipomas, fibromas and haemangiomas are other benign tumours. They are often pedunculated and present in the oesophageal lumen. Endoscopic removal is avoided because of the danger of oesophageal perforation. Treatment is excision surgical by oesophagotomy.

Incidence of oesophageal carcinoma is high in China, Japan, USSR and South Africa. In India, it constitutes 3.6% of all body cancers in the rich and 9.13% of those in the poor.

Smoking and alcohol consumption are high-risk fac- tors and so are some particular dietary habits. In India,
high incidence is associated with tobacco chewing and smoking. About 5% of oesophageal cancers arise in the pre-existing pathological lesions, such as benign strictures, hiatus hernia, cardiac achalasia and diverticula. Plummer-Vinson syndrome is another factor.
predisposing

Squamous cell carcinoma is the most common (93%). Adenocarcinoma (3%) is also seen, but in the lower oesophagus, and may be an upward extension of the gastric carcinoma. Other types are rare.

1. Direct. The lesion may fill the lumen and infiltrate the wall of oesophagus. It may also spread to the adjoining structures such as the trachea, left bronchus, aorta or pericardium. Involvement of the recurrent laryngeal nerves causes aspiration problems.
2. Lymphatic. Depending on the site involved, cervical, mediastinal or coeliac nodes may be involved. Cervical and thoracic lesions also spread to supraclavicular nodes. "Skip lesions" may also occur due to spread through the submucosal lymphatics.
3. Blood borne. Metastases may develop in the liver, lungs, bone and brain.

- 1. Early symptoms. They include substernal discomfort and preference for soft or liquid food.
2. Progressive dysphagia and emaciation. Dysphagia first
to solids
and then
to liquids.
Patient loses
weight and
becomes emaciated.
3. Pain. Usually signifies extension of tumour beyond the walls of oesophagus. It is referred to the back.
- 4. Aspiration problem. Spread of cancer may cause laryngeal paralysis or fistulae formation leading to cough, hoarseness of voice, aspiration pneumonia and mediastinitis.

1. Barium swallow. It shows narrow and irregular oesoph- ageal lumen, without proximal dilatation of the oesophagus.
2.
Oesophagoscopy.
Useful
to
see
the
site
of
involvement,
extent
of
the
lesion
and
to
take
biopsy.
Flexible
fibre-
optic
oesophagoscopy
obviates
the
need
for
general
and
gives
a
magnified
view.
3. Bronchoscopy. It helps to exclude extension of growth into the trachea and bronchi.
4. CT scan. It is useful to assess the extent of disease and nodal metastases.

Surgery of upper two-thirds of oesophagus is difficult due to great vessels and involvement of mediastinal nodes. Radio- therapy is the treatment of choice. Surgery is the preferred method of treatment for cancer of lower one-third. The affected segment, with a wide mar- gin of oesophagus proximally and the fundus of stomach distally, can be excised with primary reconstruction of the food channel.
346
SECTION VII — DISEASES OF OESOPHAGUS
In advanced lesions, only palliation is possible. An alterna-
4. Laser surgery: Oesophageal growth is burnt with Nd: YAG laser to provide a food channel. Chemotherapy is used only as a palliative measure in the locally advanced or disseminated disease.
tive food channel can be provided by:
1. A by-pass operation.
2. Oesophageal intubation with Celestin or Mousseau-
Barbin or a similar tube.
PROGNOSIS
3. Permanent gastrostomy or a feeding jejunostomy.
Five-year survival is not more than 5-10%.
Dysphagia is difficulty in swallowing. The term odynophagia is used when swallowing causes pain. The latter is more marked in ulcerative and inflammatory lesions of food passages-oral cavity, oropharynx and oesophagus.

The cause of dysphagia may be preoesophageal (i.e. due to disturbance in the oral or pharyngeal phase of deglutition), or oesophageal (when disturbance is in oesophageal phase). This classification is clinically useful as most of the preoesophageal causes can be easily excluded by physical examination while oesophageal ones require investigation.

1. Oral phase. Normally, food must be masticated, lubricated with saliva, converted into a bolus by movements of tongue and then pushed into the pharynx by elevation of the tongue against the hard palate. Any disturbance in these events will cause dysphagia. Thus cause may be:
(a) Disturbance in mastication. Trismus, fractures of mandible, tumours of the upper or lower jaw and disorders of temporomandibular joints.
(b) Disturbance in lubrication. Xerostomia following radio-
therapy, Mikulicz's disease.
(c) Disturbance in mobility of tongue. Paralysis of tongue, painful ulcers, tumours of tongue, lingual abscess, total glossectomy.
(d) Defects of palate. Cleft palate, oronasal fistula.
(e)
Lesions of
buccal cavity
and floor
of mouth.
Stomatitis, ulcerative
lesions,
Ludwig’s angina.
2. Pharyngeal phase. For a normal swallow, food should enter the pharynx and then be directed towards oesophageal opening. All unwanted communications into the nasopharynx, larynx, oral cavity should be closed. Disturbances in this phase can arise from:
- (a) Obstructive lesions of pharynx, e.g. tumours of tonsil, soft palate, pharynx, base of tongue, supraglottic larynx, or even obstructive hypertrophic tonsils.
(b)
Inflammatory conditions,
e.g.
acute tonsillitis,
peritonsil- lar abscess, retro or parapharyngeal abscess, acute epiglottitis, oedema larynx.
(c) Spasmodic conditions, e.g. tetanus, rabies.
(d) Paralytic conditions. Paralysis of soft palate due to diphtheria, bulbar palsy, cerebrovascular accidents. They cause regurgitation into the nose.
Paralysis of larynx, lesions of vagus and bilateral superior laryngeal nerves cause aspiration of food into the larynx.

The lesions may lie in the lumen, in the wall or outside the wall of oesophagus.
1.
Lumen.
to
lumen
can
occur
in
atresia,
for-
eign
body,
strictures,
benign
or
malignant
tumours.
2. Wall. It can be acute or chronic oesophagitis, or motility disorders. The latter are:
(a) Hypomotility disorders, e.g. achalasia, scleroderma, amyotrophic lateral sclerosis.
(b) Hypermotility disorders, e.g. cricopharyngeal spasm, diffuse oesophageal spasm.
3. Outside the wall. The lesions cause obstruction by press- ing on the oesophagus from outside:
(a) Hypopharyngeal diverticulum (see p. 274).
(b)
Hiatus
hernia.
(c) Cervical osteophytes (Figure 69.1).
(d) Thyroid lesions, e.g. enlargement, tumours, Hashi- moto thyroiditis.
(e) Mediastinal lesions, e.g. tumours of mediastinum, lymph node enlargement, aortic aneurysm, cardiac enlargement.
(f) Vascular rings (dysphagia lusoria).

1. History. A detailed history is of paramount importance. Ascertain, if dysphagia is of:
(a) Sudden onset: Foreign body or impaction of food on a pre-
existing stricture or malignancy, neurological disorders.
(b) Progressive: Malignancy.
(c)
Intermittent:
Spasms
or
spasmodic
episodes
over
an
organic lesion.
(d)
More to liquids: Paralytic lesions.
(e) More to solids and progressing even to liquids: Malig- nancy or stricture.
(f) Intolerance to acid food or fruit juices: Ulcerative lesions.
Note any associated symptoms, e.g. regurgitation and heart burn (hiatus hernia); regurgitation of undigested food while lying down, with cough at night (hypopharyngeal diverticulum); aspiration into lungs (laryngeal paralysis); aspiration into the nose (palatal paralysis).
2. Clinical examination. Examination of oral cavity, orophar- ynx, and larynx and hypopharynx can exclude most of the
(c)
Figure 69.1 (A & B) Cervical osteophytes causing dysphagia. Barium swallow shows pressure on oesophagus by large osteophytes.
pre-oesophageal causes of dysphagia. Examination of the neck, chest and nervous system, including cranial nerves should also be undertaken.
3. Blood examination. Haemogram is important in the diagnosis and treatment of Plummer-—Vinson syndrome and to know the nutritional status of the patient.

- (a) X-ray chest. To exclude cardiovascular, pulmonary and mediastinal diseases.
5. Manometric and pH studies. A pressure transducer along with a pH electrode and an open-tipped catheter is introduced into the oesophagus to measure the pressures in the oesophagus and at its sphincters. Acid reflux into the oesophagus is measured by pH electrode. It also measures the effectiveness of oesophagus to clear the acid load after acid solution is put in the oesophagus. These studies help in motility disorders, gastro-oesophageal reflux and to find whether oesophageal spasms are spontaneous or acid induced.
(b)
Lateral view
neck.
To exclude
cervical osteophytes
and any
soft tissue
lesions
Barium of
swallow.
It cardiac
postcricoid is
achalasia, oesophageal
or retropharyngeal
useful in
the strictures,
diagnosis space.
of malignancy,
diverticula, spasms.
hiatus
Combined trol
or cineradiography,
motility hernia
with or
fluoroscopic it
can help
disorders of
oesophageal in
wall con-
or of
sphincters.
6. Oesophagoscopy. It gives direct examination of oesophageal mucosa and permits biopsy specimens. Flexible fibreoptic or rigid scopes can be used.
7. Other investigations. Bronchoscopy (for bronchial carcinoma), cardiac catheterization (for vascular anomalies), thyroid scan (for malignant thyroid) may be required, depending on the case.
An ingested foreign body (FB) may lodge in:
1. The tonsil.
2. The base of tongue/vallecula.
3. Posterior pharyngeal wall
4. The pyriform fossa.
5. The oesophagus.
1 -Tonsil. Usually, it is a sharp fish bone or a needle in one of the tonsillar crypts. It can be easily observed by oropharyngeal examination and removed.

2. Base of tongue or vallecula. Here again it is usually the fish bone or a needle. It can be observed by mirror examination. It can be removed as an office procedure by asking the patient to hold his own tongue while examiner holds a large laryngeal mirror or an endoscope in one hand and a curved forceps in the other. Sometimes a sharp needle in the base of tongue may get totally embedded into its substance due to repeated attempts to feel. It can be diagnosed by radiology and may require pharyngotomy to extract it. Once in muscular layers, it has a chance to migrate and an early removal is indicated.
3. Posterior pharyngeal wall. A wire, a needle or a staple can get transfixed to posterior pharyngeal wall. It happens when these objects are accidently taken with food. Most of them can be seen with oropharyngeal examination under good illumination and removed with a forceps.
4. Pyriform fossa. Fish bone, chicken or a mutton bone, needle or a denture may lodge in the pyriform fossa. Small foreign bodies can be removed under local anaesthetic with a curved forceps as described above. Large impacted foreign bodies or those in children should be removed by endoscopy under general anaesthesia.
5. Oesophagus. Usual foreign bodies that get lodged in the oesophagus are a coin, piece of meat, chicken bone, denture, safety pin or a marble. Sometimes other object like nails, screws, plastic objects or pieces of glass may also be seen. Disc batteries are also becoming common these days due to their wide spread usage. Most of oesophageal foreign bodies lodge just below the cricopharyngeal sphincter. If they lodge lower down, an underlying condition such as congenital or acquired stricture or a malignancy (in adults) should be suspected and/or a follow-up barium swallow should be done when oedema due to foreign body removal has subsided.

- 1. Age. Children are most often affected. Nearly 80% are below 5 years. They have a tendency to put anything in

vA
0
the
mouth.
Playing
while
eating
is
another
factor.
Educa-
tion
of
parents
is
important
to
prevent
such
accidents
in
toddlers
and
young
children.
2. Loss of protective mechanism. Use of upper denture pre-
vents
tactile
sensation
and
a
foreign
body
is
swallowed
undetected.
Loss
of
consciousness,
epileptic
seizures,
deep
sleep
or
alcoholic
intoxication
are
other
factors.
3. Carelessness. Poorly prepared food, improper mastica- tion, hasty eating and drinking.
4.
Narrowed oesophageal lumen. Pieces of food may be held up in cases of oesophageal stricture or carcinoma. The first symptom of carcinoma oesophagus may be sud- den obstruction from a foreign body such as a piece of meat, fruit or vegetable.
5. Psychotics. Foreign body may be swallowed with an attempt to commit suicide.

By far the commonest site is at or just below the cricopharyngeal sphincter. Flat objects like coins are held up at the sphincter while others are held in the upper oesophagus just below the sphincter due to poor peristalsis. Foreign bodies which pass the sphincter can be held up at the next narrowing at bronchoaortic constriction or at the cardiac end. Sharp or pointed objects lodge anywhere in the oesophagus.
Once object passes the oesophagus it is likely to pass per rectum but sometimes it gets obstructed at pylorus, duodenum, terminal ileum, ileocaecal junction, caecum, sigmoid colon or even at the rectum. Size and shape of the object and its nature, sharp or pointed plays an important part in its lodgement in oesophagus or lower down.

1. History of initial choking or gagging.
2. Discomfort or pain located just above the clavicle on the
right
or
left
of
trachea.
Discomfort
increases
on
attempts
to
swallow.
Local
may
point
to
the
site
of
FB
in
cervical
but
not
so
in
lower
3. Dysphagia. Obstruction to swallowing may be partial or total. Partial obstruction becomes total with time due to oedema.
4. Drooling of saliva. It is seen in cases of total obstruction. Saliva may be aspirated causing pneumonitis.
5. Respiratory distress. Impacted foreign body in the upper oesophagus compresses posterior wall of trachea causing respiratory obstruction especially in children. Laryngeal oedema can develop.
6. Substernal or epigastric pain. It may occur due to oesophor
ageal spasm incipient perforation. 7. In partial obstruction, patient may still be taking normal food with little or no discomfort for a few days. Even X-rays may be normal. No complacency should be observed and an endoscopic examination performed when history and physical examination strongly suggest a foreign body.

1. Tenderness in the lower part of neck on the right or left of trachea.
2. Pooling of secretions in the pyriform fossa on indirect
They do
not disappear
on swallowing.
- 3. Sometimes a foreign body may be seen protruding from the oesophageal opening in the postcricoid region.

Posteroanterior and lateral views of neck and similar views of the chest including abdomen are taken. They reveal most of the radio-opaque foreign bodies and their location (Figures 70.1-70.3). Foreign bodies of the oesophagus lie in the coronal plane in PA view and edge on in the lateral view. It is just the reverse in tracheal foreign bodies because of orientation of vocal cords. Radiolucent foreign bodies may show as an air bubble in cervical oesophagus in X-ray soft tissue lateral view of neck. Failure to see a foreign body on X-ray does not rule it out as small fish bones, pieces of wood or plastics are radiolucent. Barium swallow is avoided as it may spill over into the larynx and thus delay the subsequent endoscopic procedure and also make it more difficult. Also look for multiple foreign bodies (as coins). A disc battery may elude as it may cast a double shadow or stacked coin appearance.

1. Endoscopic removal. Most of the foreign bodies in oesophagus can be removed by oesophagoscopy under general anaesthesia. Both rigid and flexible scopes have been used to remove foreign bodies from the oesophagus. Rigid oesophagoscope, appropriate for the size of patient with proper type of forceps is preferred. Soft (meat pieces without bone, vegetable matter) and blunt objects can be removed with flexible scopes (see Table 70.1 for comparison of the two procedures). A hypopharyngeal speculum resembling a laryngoscope with long blade is less traumatic and more convenient to use for foreign bodies lodged near the upper sphincter.
2. Cervical oesophagotomy. Impacted foreign bodies or those with sharp hooks such as partial dentures located above thoracic inlet may require removal through an incision in the neck and opening of cervical oesophagus.
3. Transthoracic oesophagotomy. For impacted foreign bodies of thoracic oesophagus, chest is opened at the appropriate level.
Figure 70.2 A fish bone in the oesophagus. Also note the presence of a bubble of gas (arrow).
Foreign body food passage. (A) PA view showing 50 paisa coin. (B) Lateral view of the same.
A foreign body which has passed the pylorus of stomach may pass through rest of gastrointestinal tract without difficulty; stool should be examined daily for 3-4 days for spontaneous expulsion. Patient should take a normal diet and no purgative should be administrated to hasten the passage of foreign body. Operative interference is required when:
- (a) Patient complains of pain and tenderness in abdomen.
(b)
is not
showing any
progress on
periodic
X-rays taken at a few days interval.
Figure 70.3
Two metallic foreign bodies. A large ring along with a
~—-
(c) Objects are sharp and likely to penetrate or get obstructed, e.g. nails, pins, needles, sharp bones, den- ture fragments, razors and long thin wires.
(d)
Foreign
body
is
5
cm
or
longer
(e.g.
hair
pin)
in
a
child
of
2
years;
it

is
unlikely
to
pass
through
turns
of
duo-
denum.
A
disc
battery
larger
than
1.5
cm
in
a
child
of
6
years
and
remaining
in
stomach
for
48
h.
(e) There is pyloric stenosis.

1. Respiratory obstruction. This is due to tracheal compres- sion by the FB in the oesophagus, or laryngeal oedema especially in infants and children.
2. Perioesophageal cellulitis and abscess. It occurs in the neck.
3. Perforation. Sharp objects may perforate the oesopha- geal wall, setting up mediastinitis, pericarditis or empy- ema. They may perforate the aorta and prove fatal.
4. Tracheo-oesophageal fistula. Rare.
5. Ulceration and stricture. Overlooked foreign bodies may cause slow ulceration and stricture formation.

Ingestion of disc batteries is becoming common because of their
widespread use
in hearing
aids, toys,
calculators and
other electronic
potassium devices.
hydroxide them
to cause
They contain
and mercury
oesophageal sodium
hydroxide, which
injury.
one place
causes complications
leaks
Prolonged like
through sojourn
at stricture,
perforation, tracheo-oesophageal
fistula, mediastinitis
and death.
It is observed that a disc battery causes damage to mucosa in
1
h, muscle
coat in
2-4
h
and perforation
of the
oesopha- gus
in
8-12
h, therefore
it should
be removed
promptly from
small piece (arrows).
the oesophagus. If lodged in stomach, a radiographic follow
352
up is conducted every 4-7 days and parents instructed to observe stool daily for spontaneous passage. If patient is a child under 6 years and battery size is 1.5 cm or more, followup X-ray examination is done after 48 h of ingestion and if the battery is still in stomach, it is removed endoscopically.

- e It is not recommended to remove oesophageal foreign bodies by Foley's or balloon catheter as they can
be aspirated. Removal under direct vision is always preferable.
Do not try to push foreign bodies down into the stomach for spontaneous expulsion later.
Use
of
papain,
a
meat
tenderizer,
is
not
if
a
bolus
of
meat
is
stuck
up.
It
can
digest
the
oesophageal
wall.
Also
sometimes
meat
contains
bone
which
is
not
digested.
Do not use glucagon to relax lower oesophageal sphincter for foreign body to pass. It does not relax a stricture or oesophageal ring if foreign body is held due to that.

(COg or Helium—Neon) or liquid (pumped inorganic dye in a glass tube). Various types of lasers are given in Table 71.1.

LASER is an acronym for Light Amplification by Stimulated Emission of Radiation. Principle. Normally, an atom is in a stable form, i.e. the electrons, equal to the number of protons, are revolving around the nucleus in a fixed orbit. When given energy, electrons change their orbits away from the nucleus and the atoms are then called "excited" but this excited state of atoms does not last long. The atoms soon release their absorbed energy automatically (spontaneous emission) and return to their original state. If photons are made to strike these excited atoms, the decay of the atoms is accelerated and both the incident and the absorbed photons are released (stimulated emission). This stimulated radiation is amplified with the help of mirrors. Thus, lasers are electromagnetic radiations. They have specific wavelength, which depends upon the type of lasing medium such as argon, carbon dioxide, Nd:YAG, helium, etc.

Depending upon the lasing medium, various types of lasers with differing wavelength can be created. Lasing medium can be solid (ruby, Nd: YAG or potassium titanyl phosphate); gas

When a laser hits the tissue it can meet the following fates (Figure 71.1):
- . Reflection. Part or whole of laser light is reflected back. _
2.
Absorption.
Laser energy
by the
tissue.
It is
the absorbed
tissues.
Scatter.
etration deep
energy which
scatters into
the produces
in tissues
becomes its
effect and
on its
pen- limited.
Shorter the
wavelength, more
of the
energy is
scattered.
- 4. Transmission. The light is transmitted through the tissue without causing any effect on tissues through which it passed. Argon laser has been used to coagulate retinal vessels without any damage to cornea, lens or the vitreous.
Lasers which are reflected or transmitted through the tissue do not cause any effect on tissues.
Effect of laser on the tissues depends on the absorbed energy. At a temperature of 60°C, there is protein denaturation but tissues can recover. At 80°C there is degradation of collagen tissue and at 100°C, cells and their pericellular water convert into heat that causes tissue ablation. Thus lasers can be used to cut (make incision), coagulate blood vessels or vaporize the tissue. When a burn is created by laser beam,
Figure
71.1
ye
it always causes some degree of collateral damage. Zones of tissue damage can be divided into (Figure 71.2):
1.
Zone
of
vaporization.
A
crater
is
created
due
to
tissue
ablation and vaporization leaving behind only a few flakes of carbon.
2. Zone of thermal necrosis. This is just adjacent to the above zone. There is tissue necrosis. Small blood vessels, nerves and lymphatics are sealed.
3. Zone of thermal conductivity and repair. This zone recovers with time.
Wavelength
488-514 nm
532
nm

e
Argon
© KTP (Potassium titanyl phosphate)
e
Nd:
YAG
(Neodymium:
yttrium
aluminium
garnet)
*
COs
(Carbon
dioxide)
©
Ho:
YAG
(Holmium:
YAG)
°
Er:
YAG
(Erbium:
YAG)
©
Diode laser
e
Tunable
dye
lasers
1060 nm
10,600
2100 nm
2960
nm
600-1000 nm
577 nm

Depending on the wavelength, laser energy produces the following effects:
1. Photothermal. It produces heat energy which is used to cut, coagulate or vaporize tissues.
2. Photoacoustic. It can be used to break stones and has been used in lithotripsy.
3. Photochemical. Ultraviolet lasers with wavelength of 248 and 312 nm can ionize DNA and RNA, respectively and are carcinogenic. This effect of specific lasers (e.g. argon tunable dye laser) has been used in photodynamic ther-
apy to selectively destroy cancerous tissue.
4. Photodissociation (LASIK lasers). Photodissociation breaks C-C bonds, divides collagen without heating it, e.g. excimer laser used in LASIK procedures to reshape cornea for refractive errors.

Visible lasers. Visible light has a wavelength of 400-700 nm (more precisely 380-760). Lasers falling in this range of wavelength are visible lasers. They have different colours from violet to red (VIBGYOR). As the laser light is visible, they do not require a separate aiming beam to focus them. Argon laser (488-514 nm) has a blue colour. KTP laser (512 nm) has a blue-green colour (Table 71.2).
Invisible lasers. Lasers in ultraviolet zone (1-380 nm) and infrared zone (>760 nm) are not visible. Infrared lasers are further divided into near-infrared lasers (760-2500 nm) and mid-infrared lasers (2500-50,000 nm). There are no far-infrared lasers (50,000-1,000,000 nm).
Lasers which can be transmitted through optical fibres.
Argon
KTP
Nd: YAG
Er:
YAG
Ho: YAG
Figure 71.2 Zones of tissue destruction caused by lasers.
Invisible spectrum
—

Lasers approved by FDA for otological work include:
Argon -514 nm
KTP -532 nm
COzg — 10,600
- Er: YAG — 2960
Otologic lasers have been used to vaporize small glomus tumours, acoustic neuromas, small A-V malformation, granulation tissue or adhesions in the middle ear. Lasers have also been used to do a myringotomy, drilling a hole in incus or malleus for ossicular reconstruction, welding of grafts in tympanoplasty or coagulating membranous posterior semicircular canal in benign paroxysmal positional vertigo and in stapes surgery to make a hole in stapes footplate.

They are important when operating with lasers.
1. Wavelength of laser. Properties of lasers depend on their wavelength.
2.
Power.
It
is
output
from
the
machine
and
is
measured
in
Watts.
Higher
the
power,
more
is
the
energy
delivered
to
tissues.
3. Exposure time. It is measured in seconds.
4. Spot size. It is the area exposed to beam. Spot size is the min-
imum
at
focal
length.
Focused
beam
is
used
for
cutting
and
decofocussed
beam
for
or
ablation
of
tissues.
5. Power density. It is power delivered per unit area of spot size and is measured in Watts/cm2. It indicates intensity of beam.
Power density (measured in watts per cm?)
= = Area of spot size in cm
Power in watts
6. Fluence or radiant exposure. It is power density multi- plied by duration of exposure in seconds and measured in joules/cm’. It indicates total energy delivered to tissues unit area.
per
Fluence (measured in joules) =
Power density (measured in watts) x Time (seconds)

1. Continuous mode. It provides constant stable energy; as the active medium is continuously kept in a stimulated mode.
2. Pulsed mode. Gives interrupted beam as the active medium is intermittently activated for a short time.
3. Q-switched mode. Provides very short pulses in a controlled manner. Pulses range between 10 ns and 10 ps.

Articulated arm is used for COg laser as it does not pass through optical fibres.
© Optical fibres require micromanipulator or a hand piece at the end of an articulated arm containing reflective mirrors to direct the light when working through a microscope.
¢ Optical fibres. Near-infrared (Nd: YAG) and visible lasers (argon and KTP 512) can pass through optical fibres. Invisible lasers require an aiming beam (e.g. He-Ne) of visible light to locate the spot.

Advantages include precise incision, easy and rapid ablation of tissues, excellent haemostasis, and minimal postoperative and oedema of tissues.
pain Some lasers can be passed through optical fibres and can thus be used through flexible endoscopes, straight or curved tubes to ablate tumours situated in difficult locations in the tracheobronchial tube or nasal crevices or clefts. Disadvantages include high cost in the purchase of equipment and its maintenance, special training in operating with lasers, hazards in the use of laser requiring special precautions, and safety measures and special anaesthesia requirements to avoid fires.

Clinical use of lasers is determined by:
1. Wavelength of laser
2. Selective absorptive property of tissues
3. Ability of laser to pass through flexible optical fibre
4. Mode of delivery (continuous wave mode or pulsed mode)
Argon laser. It lies in the visible spectrum, wavelength 488— 514 nm, blue-green in colour, easily transmitted through clear fluids, e.g. cornea, lens and vitreous humour. It is absorbed by haemoglobin and pigmented tissues, and thus it is used to treat port—wine strain, haemangioma and telangiectasias. When focused on a small point, it can vaporize the target tissue. It has been used to create a hole in stapes footplate but requires a drop of blood for its absorption at that site so that it is not reflected by white bone of stapes footplate. It can be delivered by optical fibres.
KTP laser. It lies in visible spectrum, wavelength 532 nm, properties are similar to Argon laser, absorbed by haemoglobin, can be delivered through optical fibres. Clinically it has been used in stapes surgery, endoscopic sinus surgery to remove polyps or inverted papillomas and vascular lesions,
microlaryngeal surgery for excision of polyps, cysts, papillomas, contact ulcers, laryngoceles and early malignant lesions. It has also been used to remove tracheobronchial lesions through bronchoscope.
Nd: YAG. It has a wavelength of 1064 nm (double that of KTP), lies in near-infrared zone of electromagnetic spectrum and is invisible; thus it requires a separate aiming beam of visible light and can pass through flexible optical fibres. It can pass through clear fluids but is absorbed by pigmented tissue and thus has been used in the eye and urinary bladder. It creates nearly 4 mm zone of necrosis and thermal coagulation both in depth and laterally, therefore useful for coagulation of blood vessels or control bleeding.
Clinically it has been used to debulk tracheobronchial and oesophageal lesions for palliation, hereditary hemorrhagic telangiectasia and turbinectomy.

COyz laser. Wavelength 10,400 nm, invisible, requires an aiming beam of helium-neon laser, cannot pass through flexible optical fibres, and requires articulated arm with a series of reflective mirrors to direct the beam to the target area. Requires a micromanipulator if working through an operating microscope, can be absorbed by water or clear glass. Ordinary glass or lenses of the microscope can also absorb the rays as well as the glasses worn by the operator. It is absorbed by tissues high in water and is not colour dependent. Reflection and scatter through tissues is minimum. It causes minimal effect on adjacent tissues in depth and laterally. COg laser is the workhorse laser and has been widely used in ENT. It can cut precisely, coagulate bleeders and vaporize tissues.
Clinically it has been used in laryngeal surgery to excise vocal nodules, polyps, cysts, granulomas or juvenile laryngeal papillomas. It cuts precisely and a spot size of 0.3 mm can be achieved; lesion is first delineated and then dissected with microlaryngeal instruments. Microflaps can be raised to treat Reinke's oedema. It is also used for leukoplakia, T, lesion of vocal cord or localized lesions of supraepiglottis and infraepiglottis. Transverse cordotomy and endoscopic partial or complete arytenoidectomy can also be done in bilateral abductor paralysis.
In the
oropharynx, it
has been
used to
excise benign
or malignant
lesions.
Laser tonsillectomy
can be
done in
coagulopathies.
Plastic surgeons have used it to remove benign and malignant lesions of skin and to vaporize naevi and tattoos.
Diode laser. It has a wavelength of 600-1000 nm. It can be delivered by optical fibres. It is moderately absorbed by melanin and haemoglobin. Diode lasers have been used in turbinate reduction, laser-assisted stapedectomy and mucosa-intact tonsillar ablation.

Itis important to observe safety precautions in the use of laser as they can cause damage to eyes (retina, cornea or lens), skin and airways or cause endotracheal tube fires which can be catastrophic. The measures to be taken include:
1. Education of the staff. All personnel working with lasers, doctors, nurses and technical staff of the operation theatre should be educated about the safe use of lasers and their hazards.
2. Protection of eyes and skin. All the staff working in the operation theatre should use wavelength-specific glasses. Glasses should also have side protectors to avoid damage by any reflected rays. Plain glasses for CO, laser, blue-green for Nd: YAG and amber coloured for Argon laser are used. Wavelength-specific glasses are available with all the lasers. Lasers with visible or nearinfrared range of electromagnetic spectrum (400-1400 nm) damage the retina and those with ranges less than 400 nm and more than 1400 nm damage the cornea and sometimes even the lens causing corneal opacities or cataracts. Patient's eye should be protected by double layer of saline-soaked cotton eye pads. All other exposed parts of face should be covered by salinesoaked wet towels.
3. Endotracheal tubes. Wavelength-specific endotracheal tubes are available. Rubber tubes are better than PVC ones; the latter are less resistant to laser beam and also produce more damage if accidental fires take place; also their breakdown products are more toxic. Tubes can be wrapped with reflective aluminium foils to avoid burns. Colourless or silicon tubes can be used with Nd: YAG laser but they should not have any black or dark lettering or a lead-lined marking along the side.
Cuff of the tube should be inflated with methyleneblue-mixed saline and then covered with wet cottonoids. In case cuff is accidently damaged by laser, blue colour will alert the surgeon and the tube can be replaced.
4. Anaesthetic gases. Halothane or enflurane are used as they are noninflammable. Nitrous oxide gas, being oxidizing, is not used.
Concentration of oxygen to ventilate the patient should be commensurate with oxygenation of patient and should not exceed 40%. Oxygen can be mixed with nitrogen, helium or air.

5. Evacuation of smoke. In addition to suction tube being used by the surgeon to aspirate blood and secretions, another suction tube should be available to remove smoke and steam created by vaporization of tissues. It should not spread into the OT and be inhaled by OT staff. Smoke may be mutagenic and has also been shown to contain virus particles from tissue vaporization of viral papillomas.
6. Tube fires. This is a dreaded complication and several cases are on record. In case of tube fire, stop ventilation immediately, pour saline with a syringe and withdraw the tube simultaneously. Re-establish the airway with a new endotracheal tube. Perform a bronchoscopy to assess the degree of damage to the tracheobronchial tube and give intravenous steroids. Carefully follow the patient postoperatively with repeat bronchoscopies.
It is necessary therefore that a bowl filled with saline, along with a syringe similarly filled, be kept on the table when using lasers.

1. Display a sign outside OT “Lasers In Use.”
2. Close the OT door. No entry or exit of staff permitted.
3. Protective glasses, specific for the wavelength of laser, being used should be worn by the surgeon, nurses and other OT personnel. Glasses should have side protectors.
—
(a) COg laser. Plain clear glasses. Optics of microscope are also protective.
(b) Argon or KTP lasers. Amber colour or orange-yellow glasses.
(c) Nd: YAG. Blue-green glasses.
Wet saline pads are placed on the eyes. oO
Wet saline-soaked towels for face or exposed parts of skin.
6. Use wavelength specific endotracheal tube or wrap the tube with aluminium foil.
7. Use methylene blue-coloured saline for cuff of endotra- cheal tube.
8. Use noninflammable gases such as enflurane. Oxygen concentration in inhaled gases should not exceed 40%. Do not use NoO.
9. Keep a bowl and a syringe filled with saline in readiness in case of tube fires.

It is an upcoming newer modality of treating cancer of skin, larynx, nasopharynx, tumours of aerodigestive tract and endobronchial tumours. It is based on the principle of injecting a photosensitizing agent which is taken up preferentially by the tumour cells and then exposing the site to a specific wavelength of the laser. Laser activates the photosensitizing agent which brings about destruction of cancer cells but spares the normal tissues. Photodynamic therapy has also been used for recurrences after surgery, radiation or chemotherapy. Photosensitizing agents used intravenously include haematoporphyrin derivative (for head and neck cancers) and photosan-3 (for endobronchial tumours). Topical sensitizer, delta aminolevulinic acid, has been used for skin cancers (basal cell carcinoma and Bowen disease). Laser often used in photodynamic therapy is argon tunable dye laser with a wavelength of 630 nm. It also has the advantage of delivery through flexible fibres. Also, by changing the dye, lasers with different wavelengths can be produced. Patients receiving photodynamic therapy should avoid exposure to sunlight and use sun-protective clothing to avoid photosensitive skin reactions which may continue for several weeks.

Radiowaves have been used surgically to reduce the volume of tissues. It has been used on inferior turbinates to relieve nasal obstruction; on soft palate to relieve primary snoring, upper airway resistance and sleep apnoea; and on the base of tongue to relieve sleep apnoea. It has also been used for the treatment of

The radiofrequency (RF) device generates electromagnetic waves of very high frequency between 350 kHz and 4 MHz. Usually 460 kHz is used. RF is delivered through various probes according to the site of ablation. The probe, inserted into the tissues, causes ionic agitation, heats up the tissues which result in protein coagulation and tissue necrosis but no charring. Later scar formation occurs in 3 weeks with reduction in size of tissue. Usually the temperature is controlled between 80 and 85°C. The essential parameters of radiofrequency are the power (in Watts), temperature (degrees of Celsius), resistance (in Ohms), treatment time (in seconds) and total energy delivered in joules (i.e. Watt x seconds); they can all be controlled in the device.
Using different types of electrodes, radiofrequency has also been used to perform tonsillotomy, microlaryngeal surgery (to remove granulomas, papillomas, cysts), myringotomy, uvulopalatoplasty, correction of rhinophyma and cosmetic removal of skin lesions. Radiofrequency is used to cut and coagulate tissues with minimal lateral tissue damage and charring. It is a minimally invasive technique and surgery can be performed as an outdoor procedure. Complications are few. The procedures are cost effective.

Hyperbaric oxygen therapy (HBOT) is a treatment modality involving the intermittent inhalation of 100% oxygen in chambers pressurized above 1 atmosphere absolute (ATA). ATA is the unit of pressure and 1 ATA is equal to 760 mm of mercury or pressure at sea level. When all pressures to which a person is exposed are summed up, the result is called atmospheres absolute. (Committee on Hyperbaric Medicine, Undersea & Hyperbaric Medicine Society 1976).
HBOT has been used as an adjunctive therapy for sudden sensorineural hearing loss (SSNHL) as it raises the amount of oxygen in the inner ear by diffusion, which activates cell metabolism leading to restoration of ionic balance and electrophysiological functions of cochlea.
Due of lack of definite cause of SSNHL, its treatment is largely empirical and includes use of a wide variety of therapies like systemic and intratympanic steroids, vasodilators, antiviral and anticoagulants to counteract possible inflammatory mechanism modifying hydrostatic pressure and improving cochlear blood flow. The possible final goal of any treatment modality of SSNHL is the restoration of oxygen tension in the cochlea to encourage healing and return of hearing to normal levels.
The high spontaneous recovery rate of SSNHL and its low incidence make validation of empirical treatment modalities difficult. HBOT in recent year has gained relevance for treating SSNHL in combination with other agents. The Undersea & Hyperbaric Medicine Society (UHMS) after a review of data available across the world has approved the use of HBOT in SSNHL in October 2011.

Over the last two decades, animal studies and clinical trials have produced reasonable scientific evidence or wellvalidated clinical experience. This has led to a renaissance of HBO and produced a set of indications for which HBO is beneficial (Figure 71.4).

1. Healing in problem wounds, diabetic or venous
2. Necrotizing soft tissue damage including malignant oti-
tis externa
3. Radiation tissue damage
4, Carbon monoxide poisoning
5. Crush injury and other acute traumatic ischaemia
- 6. Decompression sickness
supports the use of HBOT within 2 weeks of symptom
7. Air/gas embolism
8 Compromised skin grafts and flaps
9. Osteomyelitis
10 Thermal burns
11. Clostridial myonecrosis
12. Intracranial abscess
13. Exceptional blood loss (anaemia)
14. Sensorineural hearing loss

. Bell palsy
- Burns DO
. Anoxic encephalopathy 99
- . Traumatic brain injury Stroke OUR
. Spinal cord injury ND
- . Cerebral palsy/Autism

Patients with moderate to profound SSNHL (2 41 dB) who present within 14 days of symptom onset should be considered for HBOT. While patients presenting after this time may experience improvement when treated with HBOT, the medical literature suggests that early intervention is associated with improved outcomes. The best evidence onset.

The recommended treatment profile consists of 100% Ox at 2.0-2.5 atmospheres absolute for 90 min daily for 10-20 treatments. However, the optimal number of HBO treat- ments will vary, depending on the severity and duration of symptomatology and the response to treatment.

There is no formal detailed cost analysis for SSNHL in the literature. However, the World Health Organization (WHO) has described the cost impact of hearing loss. Hearing impairment makes it difficult to obtain, perform and keep jobs, and the hearing impaired are often stigmatized and socially isolated. Adult onset hearing loss is the 15th leading cause of burden of disease, and is projected to move up to 7th by the year 2030 (WHO, 2008). Although additional studies are recommended to further define the pathology and optimize the treatment of SSNHL, based on the current medical evidence, the use of HBOT outweighs the risk. Furthermore, significantly improving a patient's hearing and minimizing the social and economic burden of this disease outweighs treatment costs.

Rapid freezing of tissues to temperatures of —30°C and below and their slow thawing causes destruction. This fact has been used to treat various lesions of the head and neck including benign, premalignant and malignant neoplasms. Agents used in freezing the tissue are used either by an open method (liquid nitrogen spray or carbon dioxide snow) or through a closed system such as a cryoprobe. A cryoprobe is based on Joule-Thomson effect, ie. rapid expansion of compressed gas through a small hole produces cooling. Probes in current use produce a tip temperature of —70°C or below and are available in different sizes and designs to suit the area of cryoapplication. Some probes also have thermocouples which can be inserted into the tissue to monitor the temperature. The clinically available closed systems employ liquid nitrogen, nitrous oxide or carbon dioxide.

Cryotherapy can be applied under local or light general anaesthesia. Sometimes, no anaesthesia is used as freezing itself causes numbness. The area to be frozen should be insulated. A suitable cryoprobe is applied into or upon the tissues and the latter frozen quickly for 3-8 min and then allowed to thaw slowly. The procedure is repeated once or twice. Area frozen should include a margin of normal tissue. A thermocouple can be implanted to ensure freezing at an adequate depth. After cryotherapy, the area is allowed to heal by secondary intention. The necrotic slough falls off in 3-6 weeks. Repeat cycles of cryotherapy may be required to achieve the desired result.

1. Useful in poor risk patients and can be applied without anaesthesia or under local anaesthesia.
2. Useful in patients with bleeding disorders or coagulopathies.
3. Can be used in multiple cancers, palliation of
recur- rent cancers where second course of radiation is not advisable.
Causes minimal post-treatment discomfort or pain. Ot
Causes minimal scarring. Can be used at sites, notorious for keloid formation, e.g. presternal region.
6. It is an outpatient procedure.
1. No tissue is available for biopsy in case of small lesions.
2. Not possible to assess margins of tumour to know whether free of malignant cells.
3. No control on depth of freezing.
4. When used for skin lesions, cryotherapy causes depigmen- tation and loss of hair due to destruction of hair follicles.
5. Anaesthesia of the part is required when lesion is near the nerve, e.g. ulnar or digital.
With the advent of laser therapy, many of the indications for cryotherapy will be reduced; however, its lower cost will be an important factor in developing countries.

Radiotherapy forms an important modality to treat head and neck cancer. It causes irreparable damage to DNA and thus stops multiplication of cells. It also induces apoptosis and the cell gradually dies in a programmed manner. The cell may still maintain its shape but has been so damaged that it cannot multiply. It may be used:
1. Alone, to cure early cancers and thus preserve the func- tion of the organ, e.g. glottic cancer.
2. As an adjuvant to surgery, given pre- or postoperatively, to increase the survival rate in more advanced cancers.
3. In combination with chemotherapy to treat cancer and preserve the function of the organ. Concurrent radiation and chemotherapy have been used in advanced cancers of the oral cavity, larynx, oropharynx and hypopharynx.
4. As a palliative measure, in advanced lesions, to control local symptoms of pain, bleeding or obstruction to air and food channels, when total control of disease cannot be anticipated.
Radiotherapy for benign lesions. Earlier it was used for ton- sil-adenoid and papilloma larynx and acne. This led to cancers in the long term and has been abandoned. It has also been used in the treatment of certain benign vascular lesions where risks of surgery are great, e.g. angiofibroma or glomus tumour and also to control excessive scar formation in keloids. It is sometimes used in idiopathic orbital inflammation. Gamma knife or cyberknife have been used in acoustic neuroma.

1. Photon beams. These are the most common forms of radiation, both X-rays and gamma rays fall in this category (Figure 73.1). A photon is considered a "packet" of electromagnetic radiations. X-rays are produced by X-ray machines when high energy electrons bombard a metallic target. Gamma rays are emitted by radioactive sources, e.g. cobalt 60.
2. Electron beams. These are second most common forms of radiation. Their main characteristic is rapid dose build up and sharp dose fall off with very little scatter. They are used to boost up the radiation dose to the target area avoiding radiation to adjoining vital structures, e.g. spinal cord. They are produced by linear accelerator, betatron and microtron (Figure 73.2).
3. Particle radiation, e.g. neutrons, protons or pions. They are still under investigation. Neutron radiation is being used in malignant salivary gland tumours.

1. External beam therapy or teletherapy. It uses photon or electron beams which are projected to the target area through the skin.
2. Brachytherapy. It uses radioactive materials close to the target area. It has the advantages of (i) delivering high dose to the target area and (ii) continuous radiation rather than intermittent. Continuous low-dose radiation is found to be more effective than intermediate or high-dose treatment to slowly proliferating or hypoxic cells. Brachytherapy is delivered in three ways.
(a) Interstitial. Radioactive material is implanted into the tumour.
(b) Intracavitary. Placing the radiation source into the cavity, e.g. nasopharynx or maxillary antrum.
(c) Surface moulds. Applying the radioactive source directly
to the
surface of
tumour with
the help
of
a
mould.
Two types of radioactive materials are used for brachytherapy; permanent ones, which have a short half-life and temporary ones, which have long half-life and need removal. Various materials with short-life include gold seeds 198, half-life 2.7 days; iodine seeds 125, half-life 60 days; pallidium103, half-life 17 days. Long-life implants include radium 226 (needles), half-life 1620 years; cesium 137 (tubes and needles), half-life 30 years; iridium 192 (wires and seeds), half-life 74 days.
Brachytherapy can be used to deliver full dose of radiation or only to boost radiation after external beam radiation. There is presently the revival of interest in brachytherapy due to development of safer radionuclides, better delineation of the tumour area by CT/MRI, computerized dosimetry and after-loading techniques which reduce radiation hazards to the radiation oncologist.

3. Conformal radiotherapy. It is a three-dimensional radiotherapy that conforms to the size and shape of the tumour to deliver maximum dose to the target area, least dose to the surrounding normal tissue and sharp cut off to critical areas. This has been possible by development of CT/MRI to better define the tumour size and the computer technology. It has been used in the nasopharynx to deliver high dose and avoid the harmful effects on the spinal cord, optic nerves and salivary glands.
4. Intensity modulated radiotherapy (IMRT). It is a form of conformal radiotherapy. The primary tumour, its lymphatic drainage and critical structures are defined and each
ao
Figure 73.2 Novalis—Tx linear accelerator. Dual energy with photon and electron beam and 6D robotic couch. It is used for both cancerous and noncancerous lesions. It generates photons (6 and 15 MV) and electrons (6, 9, 12, 15 and 18 MeV). It can be used for:
- 3D CRT -3D conformal radiotherapy
IMRT — Intensity-modulated radiotherapy
IGRT -Image-guided radiotherapy
SRT -Stereotactic radiotherapy
SRS -Stereotactic radiosurgery (single session), e.g. acoustic neuroma and trigeminal neuralgia
- SBRT -Stereotactic body radiotherapy, e.g. to spine, liver, lung, prostate, etc.
(Courtesy Drs GK Jadhav and Sapna Manocha Verma, Indraprastha Apollo Hospital, Delhi).
—
is exposed by computer-controlled beams of radiation in depth, intensity and duration in a programmed manner that is fed in the computer. It has the greatest advantage of delivering a high tumour dose with least side effects. It is a recent advance in the treatment of cancer of the nasopharynx, larynx and paranasal sinuses.

5. Cyberknife stereotactic radiation. It is an emerging radiotherapy technique used in patients with recurrent head and neck cancers, who have already received a full course of radiation up to 7000-7600 centigrays (cGy) and no further radiation is possible, due to acute and late complications. Cyberknife stereotactic radiation is delivered by 6-million volts (MV) linear accelerator mounted on a robotic arm under image guidance. It delivers precise radiation to target area, avoiding surrounding normal tissues.

Higher the energy of radiation, deeper does it penetrate. Earlier, X-ray machines produced energy in kilovolts (kV) and could be used for superficial tumours of the skin or lip. Now, machines can produce radiations of high energy in MV. They have greater penetrating power and can be used for deep-seated tumours sparing untoward effects on the skin and bone. The various machines used for radiation are:
1. Kilovoltage machines. They produce X-rays of 50-400 kV. They were the earliest machines used and can be divided into superficial 5-150 kV or orthovoltage 200-400 kV X-ray machines.
2. Cobalt 60 machine. It is the most commonly used source for head and neck cancer. It uses radioactive cobalt source, which produces gamma rays of 1.17 and 1.33 MeV (fixed). The source has its natural decay time and needs replacement after every 5 years.
3. Linear accelerator, betatron or microtron. They are megavoltage machines which work on electricity and produce radiation of 4-25 MV. They can produce both photon or electron beams depending on whether an intervening metallic target is used in machine or not. Today most departments use dual
Superficial therapy Orthovoltage therapy Megavoltage therapy Low energy Medium energy High energy Cobalt 60 therapy, Radiation = X-rays X-rays X-rays γ -rays or electrons X-rays electron beam Same Same γ -rays. Superficial therapy Orthovoltage therapy Megavoltage therapy Low energy Medium energy High energy Cobalt 60 therapy, Voltage = 50-150 kV 200-400 kV More than1 MV Usually 4-6 MV 6-15 MV 15-25 MV 1.17 or 1.33 MeV (fixed)
ky, kilovolts; MeV, million electron voltage; MV, million volts.
energy linear accelerators. They can produce low-energy megavoltage X-rays (4-6 MV), high-energy megavoltage (1525 MV) and also electron beams. Megavoltage therapy has the advantages of

(a) Sparing the skin and thus reducing the skin reactions. (b) Deep penetration of photon beam. Deeper the location of tumour, higher is the megavoltage beam used. (c) Isodose distribution, i.e. providing uniformity of the dose delivered.
Electron beams are used for superficial tumours as in cancers of skin or lip and thus sparing deeper tissues. Different ranges of electron beams depending on the depth of tumour can also be used. Because of their sharp cut off property, they are used to spare important structures such as spinal cord and brain.
4. Radioactive material. Earlier, radium 226 was used in the form of needles but now its use has been replaced by safer radionuclides, e.g. cesium 137 (in the form of pellets), iridium 192 (in the form of wire), gold 198 and iodine 125 (in the form of seeds or grain).
Unit of radiation. Earlier, rad (radiation absorbed dose) was the unit of radiation. Now it has been replaced by an international unit—Gray. One Gray (Gy) is equivalent to one joule of energy deposited per kilogram of material.
One Gy equals 100 rads or 100 cGy.
Table 73.1 gives the energy range and type of radiation in different types of radiotherapy.

It is delivered by teletherapy, brachytherapy or by combination of both. Dose will depend on extent of the tumour and its lymphatic field and tolerance of normal tissue in the adjoining area. Usual dose delivered is:
¢ 6000—6500 cGy for small tumours.
© 6500—7000 cGy for large tumours.
¢ 7000—7500 cGy for massive tumours.
Usually shrinking fields are used, i.e. initial dose covers cancer area and its lymphatic field, subsequently the portals are reduced to cover the tumour area only.
. Radiation alone Effective in early cases; preserves function
2.
Combined with
surgery
°
Preoperative radiation
e
Postoperative radiation
Used in
advanced cases
Postradiotherapy, concurrent chemoradiation
Palliative radiation
Massive inoperable tumours, poor condition of the patient and distant metastases
.
.

It has several advantages:
(a) Itreduces the tumour bulk making questionably resectable tumour to the one definitely resectable.
(b) Vascularity and oxygenation of tumour has not been interfered and response is better than would be the case in the scarred area after surgery.
(c)
Lymphatics are
blocked, therefore
tumour dissemina-
tion would
be less
during surgery.
Also it
will reduce
the risk
of distant
metastases.
(d) Helps to eliminate microscopic disease beyond tumour mass and occult metastases in lymph nodes.
(e) Treatment portals are smaller than would be required in postoperative radiation for residual tumour or one with positive margins.
Preoperative radiation has disadvantages also. It reduces the vitality of tissues and interferes with healing process and thus increases the chances of flap necrosis, fistula formation and carotid blow-out. Other disadvantage is limitation to give postoperative radiation in case the surgical margins are reported positive after surgery as the patient has already received radiation.
Preoperative dose is usually 4500 cGy delivered in 4-5
weeks. It is sufficient to eradicate nearly 90% of micrometastases. Higher doses interfere with wound healing.

1. It is more effective as the bulk of disease has been removed at surgery.
2. Extent of tumour has been defined at surgery and radiation is given to the suspected areas of residual disease or areas of positive margins.
3. Surgical resection is technically easier and postoperative healing better.
4. A greater dose of radiation can be delivered to target area adjusted on the basis of residual disease and positive margins.
5. There are few complications of flap necrosis, wound dehiscence and infection as surgery is done on nonradiated tissues.

- 1. Blood supply to the tissues is interfered with due to fibrosis after surgery and the cancer cells are hypoxic and do not respond well.
2.
Ifsurgical complications
occur, postoperative
radiation is
delayed which
may allow
tumour cells
to regrow.
Results of
radiation are
poor, if
it gets
delayed beyond
6
weeks.
- 3. Tumour cells are squeezed into blood vessels and lymphatics at the time of surgery, increasing the chances of distant metastases.
Postoperative radiation is indicated in all cases when
(a) Margins are positive or too close.
(b) There is involvement of bone or cartilage.
(c) Lymph nodes show extracapsular invasion.
(d) There is involvement of multiple nodes and their size exceeds 3 cm.

Chemotherapy has been combined with radiation with the aim of (i) creating a synergistic effect to kill cells and (ii) to eliminate micrometastasis which may cause distant metastasis. Combination of modalities has resulted in better tumour response and disease-free interval and organ preservation but no improvement in overall survival of patients. Induction chemotherapy (given before radiation) and concurrent chemotherapy (given simultaneously) have shown good results in organ preservation when given to cases of laryngeal cancer. Chemoradiation has shown both improved survival time and organ preservation in cancer of larynx. Trials are also being conducted to preserve function of the hypopharynx, oral cavity, base of tongue and tonsils by chemoradiation to avoid mutilating surgery in this area.

When cancer is too advanced, has distant metastases, or the condition of the patient is too poor to undergo surgery due to poor nutrition or other factors such as heart, lung, liver or kidney disease, only palliative treatment is possible.

Radiotherapy can be given alone or in combination with surgery. This can be decided by
1. Type of radiation whether it is curative or palliative. Palliative treatment is chosen in advanced lesions, patient unfit for surgery due to poor nutrition or associated advanced disease of heart, lung, liver or kidney. Palliative treatment can control local symptoms of pain or pressure on air or food passages.
2. Size and site of tumour and status of regional lymph nodes. It is necessary to know the lymphatic drainage of the area, which may need to be included in the field of radiation even when lymph nodes are not involved. The extent of tumour can be found by CT and MRI. Smaller tumours can be treated with radiotherapy or surgery with equal results. However radiation helps to preserve the function. Larger tumours may require both radiation and surgery.
3. Histology and nature of tumour. Histology of the tumour is important to know its grade of differentiation and whether the tumour is rapidly proliferating or having a slow growth. Ulcerative and infiltrative tumours respond poorly. Lymphoid tissue tumours respond quickly. Anaplastic tumours and those of embryonal origin respond well but recur early. 4. General condition of the patient. Radiation causes nausea, vomiting, oral ulcerations or dysphagia and the nutrition of the patient may further worsen with radiation.

Complications of radiotherapy depend on the site of radiation, total dose delivered and the daily fraction of radiation used. High total dose and larger daily fractions cause more complications.
—

1
- . Radiation sickness (loss of appetite and nausea)
2
3.
.
Mucositis
.
4.
.
5.
Xerostomia
Skin reactions (erythema, dry or wet desquamation)
Laryngeal oedema
6.
.
Candida infections
- . Haematopoietic suppression 7.
8.
.
Acute transverse
myelitis
(rare)

OMNOAKRWN
. Permanent xerostomia
. Skin changes (atrophy of skin, subcutaneous fibrosis)
.
Decaying of teeth (secondary to xerostomia)
. Osteoradionecrosis (mandible more than maxilla)
. Cartilage necrosis
. Trismus (fibrosis of TM joint and muscles)
.
Transverse myelitis
(Lhermitte syndrome)
.
.
.
Radiation retinopathy
Endocrinal and
cataract deficit
(thyroid, pituitary)
Serous otitis
media and
sensorineural hearing
loss and
vestibular symptoms
.
Radiation-induced malignancy (thyroid cancer, osteosarcoma of orbit)
. Brain injury (Somnolence syndrome and brain necrosis)
Complications following early or late (Table 73.3).

1. Nutrition. This is particularly important in cancers of oral cavity and pharynx. Diet, rich in protein, vitamins and iron should be given. Nasogastric tube may be used where necessary. Blood transfusions may be required, if haemoglobin level is low.
2. Care of teeth. Any patient who is a candidate for irradiation particularly in the area of mandible should have dental evaluation and get all questionable teeth extracted before radiation. Wound must heal before radiation is started. Xerostomia adds to caries of teeth. Osteoradionecrosis of the mandible may follow extraction of teeth in an irradiated mandible.
3. Care of skin. Skin reactions are common with orthovoltage or electron beam therapy to skin but the modern megavoltage therapy has a skin-sparing effect. During radiotherapy, patient is advised in the care of irradiated skin. He should:
(a) keep the area dry and avoid washing with soap and water.
(b) avoid exposure to sunlight.
(c)
avoid wet
shaving.
radiation can be divided into
(d)
not use
adhesive plaster
for dressings;
skin at
the time
of removal.
cover
(e)
the area
with soft
cloth, like
it silk,
provide free aeration of the skin.
- (f) not use abrasive dressing or clothing.
For moist desquamation, an antibiotic ointment is useful. Topical steroid creams can be used to relieve itching and pain.

4. Care of oral cavity. Mucositis and xerostomia are common complications. They also interfere with feeding. Patient should avoid alcohol, tobacco or highly spiced food to lessen further trauma. Irritating mouth washes which contain alcohol should not be used. Milk of magnesia can be applied to the area of mucositis to give protective coating. It also neutralizes the acid pH and prevents caries of teeth. Pain and discomfort of mucositis can be relieved by use of lignocaine 10% to enable the patient to eat. Acute radiation mucositis usually persists 8-12 weeks after radiation. Amifostine has been found to decrease the incidence of acute and chronic xerostomia but is not widely used because of its severe side effects and high cost.
5. Care against infection. Patients receiving radiotherapy are generally debilitated and easily develop Candida infection of the oral cavity and pharynx. It can be treated by topical application of nystatin or clotrimazole. Mycelia may lie protected beneath organic debris and may require antifungal systemic therapy.
peels which
off the
should

Chemotherapy may be used alone or in combination with other modalities of treatment. Most of the head and neck malignan- cies are squamous cell cancers and the drugs found effective are methotrexate, cisplatin, bleomycin and 5-fluorouracil. Adriamycin has been used for certain nonsquamous carcino- mas (e.g. adenoid cystic carcinoma) and dacarbazine for mela- nomas. Lymphomas of the head and neck, both Hodgkin and non-Hodgkin types, are also treated by chemotherapy because of their multifocal origin and widespread involvement.

Palliative chemotherapy. Cytotoxic drugs, singly or in com- bination, are used to treat advanced, recurrent or metastatic disease with an aim to relieve the symptoms and to prolong life in some of them.
¢ When used before surgery or radiation, it is called induc-
tion or anterior chemotherapy. It helps to reduce tumour bur-
den
and micrometastases that can occur at the time of
surgery or in the period before radiation.
¢ When used simultaneously with radiotherapy, it acts as a
radiosensitizer
to
cells
which
are
otherwise
radioresistant.
e¢ When used after surgery or radiation, it is called
posterior
chemotherapy and is aimed to cure micrometastases.

Methotrexate, cisplatin, bleomycin and 5-fluorouracil have been used as single agents in various dosage forms. They have also been used in combination with other drugs with the object to improve overall response rate and duration of response. A trend is emerging that combination of two or more drugs improves the response rate and definitely improves the quality of patient's life but it has failed to improve the duration of response.

Commonly used anticancer drugs and their side effects are listed in Table 74.1.
Patient who is a candidate for cancer chemotherapy should be worked up in the following manner:
1. History and clinical examination (exclude kidney, heart
2. Haematological tests
ene
and lung disease)
e Haemoglobin
e Total and differential
count
e Platelet count
As a baseline, as many
of the drugs are
myelosuppressive
Urine exam
Biochemistry
¢ Blood urea nitrogen 8
. . Methotrexate and cisplatin
° Creatinine
. are nephrotoxic
e Liver function tests
Radiology
e X-ray chest (Bleomycin causes interstitial pulmonary fibrosis)
e CT scan or MRI where indicated
]
For
extent
of
° Ultrasound of liver/spleen
}
disease
Pulmonary function tests (for Bleomycin)
ECG (for Adriamycin)
Audiogram
(Cisplatin
causes
high
frequency
hearing
loss)
Nutritional status

Most of the drugs act on rapidly dividing cells and therefore include also normal cells as those of hair follicles, gastrointestinal mucosa and bone marrow causing alopecia, stomatitis, nausea, vomiting, diarrhoea, anaemia, leukopenia and thrombocytopenia. Some drugs have selective action on kidney (methotrexate, cisplatin), nerves (vincristine and cisplatin), heart (adriamycin) and bladder (cyclophosphamide).

mL/min. 10. Paclitaxel, used anticancer Type of neoplasm = Squamous cell cancer of head and neck. 10. Paclitaxel, drugs and their side Conventional dose a = 135-350 mg/m 2 as 3 h infusion every 3 weeks. 10. Paclitaxel, effects Side effects = Neutropenia and infection. Peripheral neuritis. 10. Paclitaxel, Remarks = Colony stimulating agents to counteract neutropenia
to weight
and height
of
a
person.

Acquired immunodeficiency syndrome is caused by retroviruses. Those infecting the human beings are of two types: (i) HIV type I—which is the most common and very pathogenic and (ii) HIV type I—which is less common and less pathogenic. Once virus enters the body, it attacks T-lymphocytes and other cells which have CD4 surface marker. CD4 T-lymphocytes are normally associated with helper-inducer function of the immune system. With the fall in CD4 lymphocytes below 500 cells/mm, (normal 600-1500 cells/mm*), the immune system starts breaking down with the appearance of opportunistic infections and unusual malignancies, when it is called AIDS. When CD4cell count falls below 200 cells/mm*, death occurs within 2-3 years.
In India, HIV disease was first documented in 1986 in sex workers in Chennai (erstwhile Madras) and Ministry of Health and Family welfare launched AIDS Control Pro- gramme in 1987. In 1992, NACO (National AIDS control organization) established state level societies (SACS-State AIDS control societies) in various states, union territories and three cities. With the concerted effort, NACO has brought 57% reduction in new HIV cases in the last decade with 2.74 lakh cases in 2000 to 1.6 lakh cases in 2011. HIV prevalence has steadily declined from 0.41% in 2001 to 0.35% in 2006 and 0.25% in 2011.
Prevalence of HIV/AIDS, according to NACO, in the year
2007 and 2011 is shown in Table 75.1.
As on 2011, 86% of patients were in the age group of 15- 49 years and 7% were children, below 15 years. Of all infec- tions, 61% are in males and 39% in females. About 1.48 lakh people have died due to AIDS related causes in 2011. Four states where disease is highly prevalent are Andhra Pradesh, Maharashtra, Karnataka and Tamil Nadu.

HIV infection is transmitted through:
1. Sexual contact—homosexual or heterosexual.
2. Use of nonsterile needles, syringes or other skin-piercing instruments.
3. Blood and blood products.
4. Infected mother to infant—During pregnancy, during
birth, and via breast milk.
High-risk groups include (i) heterosexually promiscu-
ous
(ii)
homosexuals
(iii)
prostitutes
and
truck
drivers
(iv)
IV
drug
users
(v)
recipients
of
blood
and
blood
products
(haemophilics,
thalassaemia
patients
and
those
undergoing dialysis and (vi) children born to HIV infected
mothers.
Major hazard to healthcare workers is from blood and
body fluids like amniotic, pleural, peritoneal or pericardial fluid.
Risk of acquiring infection from the specimens of urine, stool, saliva, sputum, tears, sweat and vomitus is negligible unless are
they visibly bloody.

AIDS virus was isolated in 1983 and was called HTLV II/ LAV (human T-cell lymphotropic virus type III or lymphoadenopathy associated virus). Later its name was changed to HIV
(Human Immunodeficiency Virus).

1. Lipid membrane has two layers.
2. Envelope glycoproteins
(a) Glycoprotein 120 (helps virus to bind to host cell).
(b) Glycoprotein 41 (helps in fusion of viral and cellular
membranes).
3. Viral core proteins
(a) Matrix protein p17
(b) Capsid protein p24
(c) Nucleocapsid protein p6, p7
(d) Single-stranded RNA: Two copies
(e) Viral enzymes
(i) Reverse transcriptase
(ii) _Integrase
(iii)
Protease

1. Virus enters the body of an individual through various
modes
of
transmission.
It
binds
to
CD
4+
receptors
situ-
ated on the surface of helper T-cells and macrophages.
Such receptors are also present on monocytes, macro- and CNS dendritic cells.
phages
2.
Fusion
of
virus
to
cell
membrane
allows
the
viral
core
to
be injected into the host cell.
3. Reverse transcriptase, an enzyme present in viral core, changes viral RNA to DNA and the latter migrates to host genome.
Figure
4. Viral integrase helps viral DNA to integrate into host's genome and the latter is then called a provirus.
5. Provirus directs synthesis of new HIV particles.
6. During RNA transcription, there is also formation of protein precursors or polyproteins which are cleaved by proteases to form functional viral proteins. The latter increase the infectivity of the new virus particles.
The new
viruses originating
from the
host cells
bind to
new cells and the cycle goes on.
Immunodeficiency arises from loss of helper T-lymphocytes, which play an important role in cell-mediated immunity. Monocytes harbour the virus and disseminate disease but their number does not change.

After exposure, the disease runs through the following stages:
1. Initial viraemia. Primary infection with HIV, first causes viraemia which produces mild clinical disease like fever, headache, body aches and pains, macular skin rash and lymph nodes enlargement. This picture resembles infections like mononucleosis and subsides in 1-2 weeks. The virus is then taken up by lymphoid organs like lymph nodes, tonsils, adenoids and spleen. Initial plasma viraemia lasts for a few weeks and then no virus can be detected in plasma.
2. Latent period. This is the asymptomatic period and may last for a variable period, on an average 10 years. In up to 5-10% of cases latent period may be 15 years or more.
They are called long-term survivors or long-term nonprogressors. In some cases, about 10%, latent period is short, nearly 3 years. They are called rapid progressors. During this period no virus is detectable in plasma though it is replicating in the lymphoid tissue and the CD4 T-helper cell number and function is deteriorating. Antibody test becomes positive in 2-4 months of infection.
- 3. Advanced disease. It starts after several years. The CD4 T-cell count falls below 200 cells/mm? and patient becomes susceptible to opportunistic infections. There are clinical signs and symptoms of AIDS and death may occur within 2 years.

HIV infection causes loss of helper T-cell population, which is important in cell-mediated immunity. As the T-cell counts gradually diminishes, morbidity and mortality due to HIV increases.
Three types of lesions are seen:
- 1. Opportunistic infections. All types of infection can occur—viral, bacterial, protozoal or mycobacterial. They can involve any area of ear, nose and throat, head and neck, and central nervous system.
2.
Unusual malignancies.
Kaposi sarcoma
(KS)
and lym-
phomas are
branes common.
KS
or viscera.
ear or
KS
can involve
may external
occur be
ear in
canal), oral,
nasal, laryngeal
mucosa.
skin, seen
in mucous
the skin
mem- of
face neck
or extremities.
nasopharyngeal,
KS
Non-Hodgkin
(nose,
It can
also oropharyngeal
causes obstructive
lymphoma can
or symptoms.
involve nodal
and extra-
nodal sites
(see infra).
Hodgkin lymphoma
is less
common.
- 3. Neurological disorders. They can be due to primary HIV infection or opportunistic organisms. Primary HIV infection of CNS can cause encephalopathy (AIDS dementia complex), myelopathy, peripheral neuropathy and cranial nerve involvement, most often VIth but occasionally Vth and VIIth.
HIV
manifestations in
different areas
in
ENT
and
Head
&
Neck are given below.
1. Ear. Viral, bacterial or fungal infections which can involve external, middle or internal ear are:
e Kaposi sarcoma
e Seborrhoeic dermatitis of external canal
e Malignant otitis externa
e Serous otitis media
e Acute otitis media
e Pseudomonas and candida infection of the external and middle ear
e Mycobacterial infections
e
Sensorineural hearing
loss—due to
viral infection
auditory nerve
or cochlea
and demyelination

e Herpes zoster (Ramsay—Hunt syndrome)
2. Nose and paranasal sinuses
e Herpetic lesions of nose
e Recurrent sinusitis
e Chronic sinus infection
¢ Fungal sinusitis
of of
CNS
¢ Kaposi sarcoma
e Lymphomas-B cell type
¢ Burkitt lymphoma
3. Oral cavity and oropharynx
e Candidal infection of oral cavity can be thrush-like, atrophic or hypertrophic forms of candidiasis. Can- dida infection also involves oropharynx, hypophar- ynx or oesophagus. They cause difficulty and painful swallowing.
e Herpetic lesions of palate, buccal mucosa, lips or gums. Such lesion may form large ulcers
Giant aphthous ulcers
Adenotonsillar hypertrophy.
Generalized lymphadenopathy
Kaposi sarcoma of palate
Non-Hodgkin lymphoma of tonsil or tongue
Hairy leukoplakia (see infra)
° Gingivitis
4. Larynx
e Laryngitis—fungal, viral (herpes simplex, cytomegalo- virus) or tubercular
° Kaposi sarcoma
¢ Non-Hodgkin lymphoma
5. Salivary Glands
© Parotitis
e Xerostomia
e Diffuse parotid enlargement
e Lymphoepithelial cysts of parotid. They arise from parotid nodes, often on both sides.
6. Neck
Lymphadenopathy. It could be only a follicular hyperplasia or due to a disease such as tuberculosis, histoplasmosis, toxoplasmosis or non-Hodgkin or Hodgkin lymphoma.

It is a multicentric neoplasm which may involve any part of the skin, mucosa or the viscera. There is excessive proliferation of spindle cells of vascular origin. It is noninvasive and respects the fascial planes. In the oral cavity, Kaposi sarcoma is mostly seen in the palate, but may occur on the tongue or gingiva or on the posterior wall of the pharynx. It appears purplish in colour and may need to be differentiated from angioma or pyogenic granuloma. It can occur at any stage of HIV infection, even in those with normal CD4 counts. Size of the tumour may vary from a few mm to several centimetres. Diagnosis is based on biopsy which may show proliferation of spindle cells, endothelial cells, extravasation of red blood cells and haemosiderin-laden macrophages. Treatment includes localized radiation, intralesional vinblastine or cryotherapy. Systemic chemotherapy may be given in those with multiple lesions.

HIV patients have high incidence of lymphomas. B-cell lymphomas are more common (90%) and many are due to Epstein-Barr virus. Risk of lymphomas increases as disease progresses generally in patient with CD4+ count less than
200/mm3. CNS lymphomas occur in late stages of the disease while systemic ones can occur early. Both nodal and extranodal sites can be involved; the latter include nose and paranasal sinuses, tonsils, nasopharynx, tongue, orbit and larynx. They also involve gastrointestinal tract, lung and bone marrow. Cervical lymphadenopathy can occur.
Hodgkin lymphoma is less common.

It is a white, vertically corrugated lesions on the anterior part of the lateral border of tongue. It is probably caused by Epstein-Barr virus. It develops early and gives clue to HIV infection. AIDS develops in 50% of such patients in 16 months or 80% in 30 months. Differential diagnosis includes leukoplakia, carcinoma in situ, hypertrophic candidiasis or lichen planus. Biopsy should be done to confirm.

Diagnostic tests are based on identification of antibodies or viral antigens. Antibodies are formed within 3 months (2 weeks—12 weeks) of infection.
1. ELISA test (enzyme-linked immunosorbent assay). It is a very sensitive test (sensitivity more than 95.5%).
2. Western blot. It is a confirmatory test and specific for HIV antibodies.
3. CD4 count. Normal count is 600-1500/mm°. Decreasing counts indicate immune compromise and the correspond- ing risk for development of opportunistic infections and malignancies. Disease has been classified according to CD4 counts such as (i) less than 500 cells/mm; (ii) 200-499 cells/mm or (iii) less than 200 cells/mm?; AIDS-defining illnesses appear when CD4 count fall below 200/mm°.
4. p24 antigen assay. It detects p24 core protein of HIV. The test is positive even prior to seroconversion. High levels of p24 antigen are present before development of antibodies and are useful in those suspected of acute HIV syndrome.
5. PCR tests. They determine HIV-RNA. Two important tests are reverse transcriptase PCR and branched DNA assay. They determine number of copies of RNA per mil- lilitre of plasma and indicate viral load.

Doctors, particularly the surgeons, nurses and laboratory staff handling the blood, blood-stained body fluids and other secretions may contract the disease as occupational hazard. They should follow the universal precautions (vide infra) considering that every sample they handle is potentially infected. The risk is due to:
1. Needle-stick injury. Hollow needle (e.g. injection nee- dle) is more dangerous than solid needle (e.g. suture needle). The risk is. 0.3%, i.e. (1:300).
2. Cuts with contaminated knife or other sharp instruments.
3. Exposure of open wound to infected blood or body fluid. Entry of virus can also occur through an area of dermatitis.
4.
Large
mucous
membrane
exposure,
e.g.
by
splatter
of
blood,
amniotic
fluid,
etc.
Risk
is
0.09%.
5. Exposure of skin to infected blood and body fluids. Use of gloves and gown/coat is protective.

Several factors need to be taken into account before a prophylactic treatment is started.
1. Hollow needle vs solid needle. Hollow needles (injection needle) have more chance to introduce infected blood.
Superficial vs deep cut. Deeper cuts are more dangerous. no
go
Source patient
having or
not having
HIV
and if
yes, how
serious is
his condition
the antiretroviral
due drugs
to advanced
being
HIV
used disease
by him
(Ist or
and
IInd line)
for the
possibility of
exposure to
drug resistant
virus.
- 4. Condition of healthcare worker, e.g. pregnancy and other diseases he/she suffer from and medication he/she is using for such ailments.
First step after exposure is to wash the area thoroughly with water and apply an antiseptic. ELISA test should be done as soon as possible to establish negative base line and to repeat the test at 6 weeks, 3 months and 6 months for any seroconversion.
If decision is made to start drug prophylaxis, it is recommended to start two drugs of nucleoside analogue reverse transcriptase inhibitor group for 4 weeks for routine exposures (zidovudine and lamivudine or lamivudine and stavudine) and three drug regimen for high-risk exposure. Treatment should be started immediately certainly not later than 24 h. Seroconversion has been noted in spite of this prophylaxis, therefore avoidance of exposure by universal precaution is the best.

¢ Wash hands before and after patient or specimen contact.
¢ Handle the blood of all patients as potentially infectious.
Wear gloves for potential contact with blood and body
fluids.
All
sharps
like
blades,
needles,
etc.
to
be
put
in
impermeable container and destroyed.
¢ During operation, knife to be passed to the surgeon in a tray.
¢ Place used syringes immediately in a nearby impermeable
container; DO NOT recap, bend or manipulate needle in anyway!
¢ Use double gloves where they are likely to be pierced as
in
fracture
surgery.
Quality
of
gloves
is
important
as
many
of them may have holes. Impermeable gloves are also available.
¢ Wear protective eyewear and mask if splatter with blood or body fluids is anticipated (e.g. bronchoscopy, oral surgery).
¢ Wear gowns when splash with blood or body fluids is anticipated.
¢ Handle all linen soiled with blood and/or body secretions as potentially infectious.
¢ Refrain from patient care if you suffer from exudative or weeping skin lesion or dermatitis.
¢ Process all laboratory specimens as potentially infectious.
e Wear mask to protect against TB and other respiratory organisms (HIV is not airborne).

There are four major classes of antiretroviral drugs:
1. Nucleoside reverse transcriptase inhibitors (prevent con- version of RNA to DNA.).
e Zidovudine (AZT)
e Didanosine (ddI)
e Zalcitabine (ddC)
e Stavudine (d4T)
e Lamivudine (3TC)
2. Non-nucleoside reverse transcriptase inhibitors (pre- vent conversion of RNA to DNA). They bind to reverse transcriptase.
e Delavirdine
e Nevirapine
e
Efavirenz
e Tenofovir (nucleotide analogue)
3. Protease inhibitors (prevent cleavage of viral proteins into their functional forms by binding to viral protease enzyme.
e Saquinavir
e Ritonavir
e Indinavir
4. Fusion inhibitors (interfere with entry of virus into target cells). They bind to HIV-gp 41
e Enfuvirtide
Antiretroviral drugs can prevent progression of HIV to AIDS. Drug resistance can occur. Second line drugs are more expensive. Toxicity of drugs should be kept in mind. Drug interactions are common and should be avoided. Combination drug therapy is more effective and prevents drug resistance; it is the standard of treatment today to com- bine two or more drugs.
pti te) me a |S
76.
Clinical Methods in ENT
77.
Neck Masses

Evaluation of the patient with disease of ear, nose and throat requires skill in eliciting a meaningful history and masterly dexterity in the examination of darker cavities of the ear, nose, pharynx and larynx. A student is expected to learn this by regular practice in ENT clinics.

(FIGURE
76.1)
The requirements of equipment in a clinic may vary but the essential instruments for routine examination are listed here.
1. Bull’s eye lamp. It provides a powerful source of light. The lamp can be tilted, rotated, raised or lowered according to the needs.
. Head mirror. It is a concave mirror used to reflect light from the Bull’s eye lamp onto the part being examined. It has a focal length of approximately 25 cm. The exam- iner sees through the hole in the centre of the mirror. Diameter of the mirror is 89 mm (3'4") and that of the central hole is 19 mm (3/4").
Tongue depressors. Different sizes for children and adults should be available. It is used in the examination of oral cavity and oropharynx.
Nasal specula. Two types are commonly used, namely Thudicum and Vienna types. The size of the nasal spec- ulum is selected according to the age of the patient and size of the nostril.
. Laryngeal mirrors. They are used to examine the lar- ynx and laryngopharynx. Various sizes, from 6 to 30 mm diameter, are available. To prevent fogging, a mirror is always warmed over a spirit lamp or by dipping it in hot water and then tested on the back of hand before inser- tion into the mouth.
Postnasal mirror. It is used to examine the nasopharynx and posterior part of nasal cavity. Like laryngeal mirror, it is also warmed and tested on the back of hand before use.
Ear specula. Various sizes are available to suit different sizes of the ear canal. The largest speculum which can be conveniently inserted in the ear canal should be used.
. Siegle’s speculum. Essential in examination of tympanic membrane; it gives magnified view of tympanic mem- brane and helps to test its mobility. It is also used to elicit the fistula sign.
10. Jobson-Horne’s probe. One end of the probe is used to
11. Blunt probe. It is used for palpation in the nasal cavity or ear canal.
12. Tilley’s or Hartman’s forceps. It is used in packing of ear canal or nasal cavity.

14. Otoscope. It is an electric or battery operated device with a magnifying glass. Sometimes it has an arrange- ment to attach a bulb to function as Siegel’s speculum. It is useful for detailed examination of the ear. It is an essential instrument to examine the ear of an infant, a child or a bedridden patient.
15. Spirit lamp. It is used to warm the laryngeal or postnasal mirror (Figure 76.2).
16.
17.
18.
Tuning forks. Commonly used tuning fork has a fre- quency of 512 Hz. Forks of other frequencies, e.g. 256 and 1024 Hz should also be available.
form
a
cotton
bud
to
clean
the
ear
of
discharge
and
the
other
end
(with
ring
curette)
is
used
to
remove
the
wax.
. Eustachian catheter. It is used to test patency of the eustachian tube and can also be used to remove for- eign bodies from the nose. To test the patency of eustachian tube, the nose is first anaesthetized, the catheter is then passed along the floor of nose into the nasopharynx, turned medially and then slightly with- drawn till it engages on the posterior free border of the nasal septum. At this point, it is rotated 180° laterally to lie against the opening of eustachian tube. A bulb is attached and air insufflated. If the tube is patent, air enters the middle ear and can be detected by an auscul- tation tube which connects patient’s ear to that of the examiner (see p. 59).
Gloves.
They
are
essential
for
intraoral
palpation.
Spray.
It
is
used
to
apply
local
to
abolish
the
gag
reflex.
Suction apparatus. To clear the ear or nose of discharge or blood for detailed examination.

1.
History
of
present
illness.
A
patient
presents
with
cer
tain
presenting
complaints.
They
are
asked
in
detail,
with
reference
to
the
duration
of
symptoms,
their
onset,
progression,
severity
and
other
accompanying
Inquiry
should
also
be
made
of
any
systemic
disease
the
patient
may
be
suffering
from,
e.g.
diabetes,
hypertension,
coronary
artery
disease,
liver
or
kidney
dis-
ease,
or
a
bleeding
disorder.
Also
find
out
about
the
treat-
ment
patient
has
taken
or
is
still
taking
for
the
present
ailment.
Figure 76.1A Left to right. (1) Tuning fork. (2) Siegel's speculum. (3) Otoscope. (4) Head mirror. (5) Barany's noise box.
Figure 76.1B (1) Jobson-Horne probe. (2) Vienna nasal speculum. (3) Hartmann's packing forceps. (4) Wilde's packing forceps. (5) Eustachian tube catheter. (6) Thudicum nasal speculum. (7) Laryngeal mirror. (8) Postnasal mirror. (9) Blunt probe. (10) Ear specula. (11) Lacks tongue depressor.
2. History of past illness. It includes history of similar complaints in the past, treatment taken, history of any operation which the patient has undergone and allergy to any drug.
3. Personal history. Inquire about the patient's profession
and nature
of job,
personal habits
(smoking, chewing
pan or
tobacco, sive
use use
of tea
or of
alcohol)
coffee).
and
It is
food habits

also slump in the seat. Bull's eye lamp is kept on the left at the level of his shoulder. The examiner uses a head mirror to reflect light from the Bull's eye lamp on to the area of examination. A head mirror gives good illumination and permits freedom to use both hands for other activities. Some prefer to use a head light in place of Bull's eye lamp and a head mirror.
important
(exces- to
know about
his activities,
exercise, or
sedentary habits.
- 4. Family history. Some diseases have a genetic basis, e.g. otospongiosis, certain types of sensorineural hearing loss and autoimmune disorders while others are the result of close contact between different members of the family, e.g. tuberculosis, syphilis, pediculosis, scabies, etc.

The patient is examined in a semi-dark room. He is seated on a stool or chair opposite the examiner and is made to sit erect
leaning slightly forward towards the examiner. He should not

A patient with ear disease presents with one or more of the following complaints:
1. Hearing loss.
2. Tinnitus.
3. Dizziness or vertigo.
4. Ear discharge.
5. Earache.
6. Itching in the ear.
7. Deformity of the pinna.
8. Swelling around the ear.
The details of history of these symptoms particularly in reference to the onset, duration, progression and severity should be noted.

It includes examination of:
1. Pinna and the surrounding area.
2. External auditory canal
(a) Without speculum
(b) With speculum
Figure 76.2
Warming a laryngeal mirror over a spirit lamp. Warm only the glass side of the mirror.
3. Tympanic membrane.
4, Middle ear.
5. Mastoid.
6. Eustachian tube.
7. Facial nerve and other cranial nerves.
1. Pinna and the surrounding area. The pinna is examined
by inspection
and palpation.
Both of
its surfaces,
and the medial, should be examined.
Look for
size
(microtia, macrotia);
of contour,
redness cauliflower
(furuncle vesicles
ear);
or shape
position
(abnormalities
(bat abscess);
in concha
retroauricular ear).
Also look
for
(haematoma, groove
(herpes abscess)
;
zoster);
scars
(trauma or
operation);
ulceration or
neoplasm.
Also examine the area above, in front, below and behind the
pinna and
look for
a
swelling
(mastoid or
zygomatic abscess,
neoplasm sinus);
fistula or
lymph
(mastoid nodes);
fistula)
sinus scar
(endaural
(preauricular or
postaural scar
due to
previous operation).
Palpation of pinna is essential to look for raised temperature (perichondritis or abscess); thickness of tissues (perichondritis); fluctuation (seroma or abscess) and tenderness. Movement of pinna is painful in furunculosis of the external canal.

(a)
Examination without a speculum. This is an important part of the examination and precedes introduction of speculum. The pinna is pulled upwards and backwards while the tragus is pulled forwards to spread open the meatus. Look for the size of meatus (narrow or wide), contents of lumen (wax, debris, discharge or polyp) or swelling of its wall (furuncle, neoplasm).
(b)
Examination with a speculum. Once the size of the meatus is known, proper speculum is selected and introduced (Figure 76.3). Use the largest speculum that can easily enter the canal. Look for wax, debris, discharge, polyp, granulations, exostosis, benign or malignant neoplasm, sagging of posterosuperior area (coalescent mastoiditis).
3. Examination of tympanic membrane. Normal tympanic membrane is pearly white in colour and semi-transparent and obliquely set at the medial end of the meatus. It has two parts—pars tensa and pars flaccida, both of which should
be carefully examined. Its various landmarks are shown in Figure 76.4. A tympanic membrane is examined for:
(a) Colour. Red and congested in acute otitis media, bluish in secretory otitis media or haemotympanum. A chalky plaque is seen in tympanosclerosis.
(b) Position. Tympanic membrane may be retracting or bulging. General retraction is seen in tubal occlusion, retraction pockets are seen in attic or posterosuperior region and may collect epithelial flakes. Sometimes, tympanic membrane is very thin, deeply retracted and is fixed to promontory as in adhesive otitis media.
Bulging tympanic
seen in
acute otitis
media, haemotympanum
or neoplasm
of middle
ear which
has not
yet perforated
the drum.

(c) Surface of tympanic membrane. It may show vesicles or bullae (herpes zoster or myringitis bullosa), a perforation (acute or chronic otitis media). A perforation may be central (in pars tensa) or attic (in pars flaccida) or marginal (at the periphery involving the annulus). A central perforation may be small, medium, subtotal or total.
(d) Mobility. It is tested with a Siegle's speculum (Figure 76.5). A normal tympanic membrane is mobile.
Figure 76.4 Landmarks of a normal tympanic membrane (right side).
Figure
76.5
Use of Siegel's speculum to see mobility of tympanic membrane.
Restricted mobility is seen in the presence of fluid or adhesions in the middle ear. An atrophic segment of membrane be tympanic may hypermobile.
4, Examination of middle ear. Normally, middle ear cannot be examined directly. When tympanic membrane is semitransparent, some structures can be seen through it. In the presence of a perforation, it is possible to know the condition of middle ear mucosa and any in-growth of squamous epithelium from the edges of the perforation.
5.
Examination of
mastoid.
Look for
a
swelling
(abscess or
enlarged nodes),
obliteration of
retroauricular groove
(furun- cle),
fistula
(burst abscess),
scar
(previous operation)
.
Normally, mastoid surface feels irregular on palpation.
These irregularities
are
“ironed out”
feels smooth
in periosteal
inflammation as
in
Tenderness of
mastoid is
subperiosteal seen
abscess.
in mastoiditis.
It is
elicited

(a) Over the antrum (just above and behind the meatus). (b) Over the tip.
- (c) Over the part between the mastoid tip and mastoid antrum.
6.
eustachian tube.
Tympanic orifice
of eustachian
tube ear
if there
can be
seen is
geal in
perforation opening
Function of
of of
the tube
can be
tube the
anterior tympanic
seen can
be part
of middle
membrane.
by posterior
tested of
a
perforation, ear
7.
Examination coexist
air tries
to blow
of facial
purative of
otitis externa,
media, tumours
It is
essential by
Pharyn- rhinoscopy.
Valsalva manoeuvre.
can be
felt with
nerve.
the ear,
mouth
Paralysis
In to
escape from
and of
e.g.
herpes of
to test
acute zoster
external oticus,
or middle
for facial
nose closed.
may or
chronic malignant
ear nerve
disease.

(a) Voice test
(b) Tuning fork tests
e Rinne test
© Weber test
e Schwabach test
e Absolute bone conduction test.

(a) Spontaneous nystagmus
(b) Fistula test (p. 41)
(c) Positional tests (p. 42).

A patient with disease of the nose and paranasal sinuses
presents with one or more of the following complaints:
1.
Nasal
2.
Nasal
in sup-
otitis and
trauma.
every case
of ear
Figure 76.6 (A) Anterior rhinoscopy. (B) Technique of holding a Thudicum nasal speculum.
Postnasal drip. vo
3.

Sneezing. OP
Epistaxis.
Headache or facial pain. Swelling or deformity. Disturbances of smell. OND
Snoring. SO
Change in voice (hyperor hyponasality). _
A detailed history of these symptoms with special regard to their onset, duration, progression, severity should be asked. They are discussed in the relevant sections of the book.

Nasal examination includes:
Examination of external nose.
. Examination of vestibule.
Anterior rhinoscopy. go
. Posterior rhinoscopy. UR
Functional examination of nose.

Examine the skin and osteocartilaginous framework of nose both by inspection and palpation.
Skin is examined for signs of inflammation (furuncle, septal abscess), scars (operation or trauma), sinus (congenital dermoid), swelling (dermoid or glioma) or a neoplasm (basal cell or squamous cell carcinoma).
Osteocartilaginous framework is examined for deformity, e.g. deviated or twisted nose, hump, depressed bridge, bifid or pointed tip, destruction of nose (trauma, syphilis, cancer). Palpation of nose is done to find raised temperature, fixity of skin, thickening of soft tissues, tenderness, fluctuation or crepitation.

It is the anterior skin-lined part of nasal cavity having vibrissae and can be easily examined by tilting the tip of nose upwards. It is examined for a furuncle, a fissure (chronic rhinitis), crusting, dislocated caudal end of the septum, and tumours (cyst, papilloma or carcinoma).
Technique. Patient is seated facing the examiner. A Thudicum or Vienna type of speculum is used to open the vestibule. The speculum is held in the left hand (by a right-handed person) (Figure 76.6). It should be fully closed while introducing and partially open when removing from the nose to avoid catching the hair. Light is focussed at different sites in the nose to examine the nasal septum, roof, floor and the lateral wall. For this, patient's head may need to the tilted in different directions. Look for the following points:
(a)
Nasal passage.
Narrow
(septal deviation
or hypertrophy
of turbinates,
growth)
Septum.
Deviation and
wide or
(haematoma spur,
or abscess)
(atrophic ulcer,
and rhinitis).
perforation, growth
swelling
(rhinosporidiosis, haemangioma).
(c) Floor of nose. Defect (cleft palate or fistula), swelling (dental cyst), neoplasm (haemangioma) or granulations (foreign body or osteitis).
(d)
Roof.
Usually not
seen except
in cases
of atrophic
rhinitis.
(e)

Lateral wall. Look at the turbinates and meatuses. Only the inferior and middle turbinates and their corresponding meatuses can be visualized. Examine the colour of mucosa (congested in inflammation and pale in allergy), size of turbinates (enlarged and swollen in hypertrophic rhinitis, small and rudimentary in atrophic rhinitis), discharge (discharge in the middle meatus indicates infection of maxillary, frontal or anterior ethmoidal sinuses), mass (polyp, rhinosporidiosis, carcinoma). A probe test should be done. It ascertains the site of attachment, consistency, mobility and sensitiveness of the mass. Attachment of the mass is found by passing the probe on all its surfaces. Bleeding during probing indicates vascular nature of the mass.

Technique. Patient sits facing the examiner, opens his mouth and breathes quietly from the mouth. The examiner depresses the tongue with a tongue depressor and introduces posterior rhinoscopic mirror, which has been warmed and tested on the back of hand (Figure 76.7). The mirror
(b)
Figure 76.7 Technique of posterior rhinoscopy.
Figure 76.8 Structures seen on posterior rhinoscopy.
is held like a pen and carried behind the soft palate. With- out
touching it
on the
posterior third
of tongue
to avoid
gag reflex, light from the head mirror is focussed on the rhinoscopic
mirror which
further illuminates
the part
to be
examined.
Patient’s relaxation
is important
so that
soft pal-
ate does not contract.
Structures normally seen on postrhinoscopy are shown in Figure 76.8. Look for the following:
(a) Choanal polyp or atresia.
(b) Hypertrophy of posterior ends of inferior turbinates.
(c)
Discharge in
the middle
meatus.
It is
seen in
infections of maxillary, frontal or ethmoidal sinuses. Discharge
above the middle turbinate indicates infection of the posterior ethmoid or the sphenoid sinuses.

Test for patency of the nose and sense of smell.
(a) Patency of nose. (i) Spatula test. A clean cold tongue depressor is held below the nostrils to look for the area of
mist formation,
exhales
(Figure
76.9), the
two sides are compared.
(ii) Cotton-wool test. A fluff of cotton is held against each nostril and its movements are noticed when patient inhales or exhales.
(b) Sense of smell. A simple test is to ask the patient to identify the smell of a solution or substance held before
the nostril
while keeping
the eyes
closed.
Each nostril
is tested separately. Common substances used are the clove oil,
Figure
76.9
Testing the
patency of
nose.
Note the
area of
mist for-
mation on the tongue depressor when patient exhales from nose.
Figure 76.10 Testing for tenderness of maxillary sinus by pressure
on the canine fossa.
peppermint, coffee and essence of rose. Ammonia stimulates the fibres of CN V and is not used to test the sense of smell.

1. Maxillary sinus
2. Frontal sinus
3. Ethmoid sinuses
4. Sphenoid sinus

It is examined by inspection, palpation and_ trans-
illumination.
Maxillary sinus has five walls and except for the posterior,
all other walls can be examined directly.
Examine:
(a) _ lip, eye
the soft tissues of cheek, lower lid and the molar
region,
(b) the orbit and its contents, and the vision,
(c) the vestibule of mouth by everting the lip,
(d)
upper alveolus, teeth and palate,
(e)
the nose by anterior and posterior rhinoscopy,
(f)
tenderness
by
pressure
over
the
canine
fossa
(Figure
76.10).
Transillumination of maxillary sinus is done by placing a specially made light source centrally in the mouth and closing the lips. Normally, a crescent of light in the inferior fornix and glow in the pupil, equally bright on both sides, can be seen. In the presence of pus, thickened mucosa or a neoplasm, the affected side does not transmit light. This test has limited value and has practically been abandoned in favour of X-rays.

It is also examined by inspection, palpation and transillumination.
Frontal sinus has three walls: anterior, posterior and floor. the anterior wall and floor lend themselves to external
Only examination.
(a) External examination. For this, examine the forehead, root of nose, orbital margins, the orbit and its contents. Look for redness, swelling, fistula, proptosis and displacement of the eye balls.
Tenderness of the frontal sinus can be elicited by pressure or percussion with a finger on its anterior wall above the medial part of eyebrow, or by pressing upwards on its floor above the medial canthus
(Figure 76.11).
(b) Examination of nose. Nose should be examined by ante- rior as well as posterior rhinoscopy for evidence of discharge in the middle meatus and for any neoplasm.
Transillumination is done by placing a small light source in the superomedial angle of the orbit and observing the transmission of light from the anterior wall of the sinus. It is compared on both sides. Transillumination of frontal sinus is of limited value and has practically been abandoned in favour of X-rays.

They are divided into two groups: the anterior and posterior. The former drains below the middle turbinate and the latter above it. They are examined by inspection and palpation.
(a) External examination. It includes examination of orbit, upper and lower eye lids, root of nose, eye ball and vision. Tenderness can be elicited only in the anterior ethmoid sinuses. This is done by gentle pressure applied on the medial wall of orbit just behind the root of nose. The area is tender in acute ethmoiditis.
(b) Nasal examination. Anterior rhinoscopy reveal ability mass.
may pus, polypi, or growth in the middle meatus (anterior group of ethmoid sinuses) or between the middle turbinate and the septum (posterior group of ethmoid sinuses). Probe test should be done to find the consistency, attachment and friof the
Posterior rhinoscopy may reveal pus or growth, below or the middle turbinate.
above

Sphenoid sinus lies deep and is not easy to examine directly.
Sometimes, its anterior wall can be seen in atrophic rhinitis or in marked deviation of the septum to the opposite side.
(a) Anterior rhinoscopy. Sphenoid sinus opens in the sphe- noethmoidal recess. Attention should therefore be paid to
Figure 76.11 Testing for tenderness of frontal sinus.
the findings in the olfactory fissure near the roof of nose. It may show discharge, crusts, polyp or growth. A probe can be used to palpate the mass.
(b) Posterior rhinoscopy. It may reveal pus in the nasopharynx or the choana, above the middle or superior turbinate. A growth or a polyp may also be seen.

A patient with disease of nasopharynx presents with:
1. Nasal obstruction.
Postnasal discharge. po
Epistaxis. 99
Deafness (tubal block). B®
Cranial nerve palsies. Ov
Enlargement of lymph nodes in the neck. D
A detailed history of these symptoms regarding their onset, duration, progression and severity should be asked.
(These have been discussed in the relevant sections.)

Clinical examination of nasopharynx includes:
1. Anterior rhinoscopy.
2. Posterior rhinoscopy.
3. Other methods.
(a) Digital examination
(b) Endoscopy
(c) Retraction of soft palate with catheters and mirror
4. Cranial nerves.
5. Cervical lymph nodes.

It is possible to see only a small part of the nasopharynx on anterior rhinoscopy. The view can be facilitated by decon-
gestion of nasal and turbinal mucosa with vasoconstrictors.

The technique is described on p. 378. Structures to be examined are:
(a) Anterior wall. Posterior border of nasal septum, choa- nae, posterior ends of turbinates and their meatuses.
(b) Lateral walls. Torus tubarius, opening of eustachian tube, pharyngeal recess.
(c) Floor. Upper surface of soft palate.
(d) Roof of posterior wall.
Only a small part of nasopharynx can be seen in the mir- ror at one time. The examiner tilts the mirror in different directions to see all the walls of the nasopharynx and then mentally reconstitutes the entire picture.
Abnormal findings in the nasopharynx include:
(a) Discharge. It may be seen below the middle turbinate (anterior group of sinuses) or above the middle turbi- nate (posterior group of sinuses).
(b) Crusting. Atrophic rhinitis or nasopharyngitis.
(c)
Mass
(i) Smooth pale mass—antrochoanal polyp.
(ii) Pink lobulated mass—angiofibroma.
(iii) Irregular bleeding mass—carcinoma.
(iv)
Smooth
swelling
in
the
roof—Thornwaldt’s
cyst
or
abscess.
(v) Irregular mass with radiating folds-adenoids.
(vi) Irregular mass filling the lower part of choana— mulberry hypertrophy of inferior turbinate.
(d) Bleeding. Due to posterior nasal or nasopharyngeal pathology.

(a) Digital examination. It is a quick method to examine the nasopharynx by palpation but is uncomfortable for the patient. The examiner stands behind and to the right of the patient, invaginates patient’s cheek with his left fin- ger and inserts right index finger behind the soft palate into the nasopharynx. He first examines the posterior border of the nasal septum, then the choana, lateral wall and finally the posterior wall of nasopharynx. Adenoids, antrochoanal polyp and other masses in the nasopharynx can be examined. Avoid this examination if angiofibroma is suspected.
(b) Endoscopy. A rigid nasal endoscope is passed through the nose after local anaesthesia and decongestion of nasal mucosa. It gives a bright and magnified view of the naso- pharyngeal structures. Using endoscopes with different angles of view, it is possible to examine structures situated at an angle. Flexible nasopharyngoscope can also be used. It is also passed through the nose and gives a magnified view.
(c) Retraction of soft palate with catheters and mirror exam- ination. This method is reserved for difficult cases where view of nasopharynx is not obtained by other methods. It requires good local or general anaesthesia.
A soft rubber catheter is passed through each nostril
and
recovered
from
the
oropharynx.
Both
ends
of
catheter
are
held
together
and
clamped.
In
this
way,
soft
palate
is
retracted
forwards.
Now
a
mirror
can
be
introduced
and
the
nasopharynx examined.

Malignancy of nasopharynx can involve any of the CN II to XII, more often CN IX, X and XI.
5. EXAMINATION OF CERVICAL LYMPH NODES
It is not unusual for nasopharyngeal malignancy to present primarily as a lymph node mass in the neck. Lymph nodes commonly involved are upper internal jugular and those along the accessory nerve in the posterior triangle of the neck.

Oral cavity extends from the lips to the level of anterior tonsillar pillars. Structures included in it are:
Lips.
Buccal mucosa. Gums and teeth. GON
Hard palate. OUR
Anterior two-thirds of tongue.
Floor of mouth. NS

A patient with disease of the oral cavity may present with one or more of the following complaints:
1. Pain. It may be localized to a particular site in the oral cavity, e.g. tooth, tongue, buccal mucosa, floor of mouth, etc. Sometimes, pain is referred to the ear from pathology in the oral cavity.
2. Disturbance of salivation. Xerostomia (dryness of mouth) can result from mouth breathing, irradiation or generalized disease of the salivary glands. Excessive salivation can result from ulcers of mouth and pharynx, poor orodental hygiene, ill-fitting denture and iodide therapy.
3.
Disturbance of
taste.
Sweet, sour
and salt
tastes are
appre- ciated
by taste
Patient buds
on may
the complain
taste, diminished
cases anterior
two-thirds of
unilateral or
perverted may
be local
tongue, or
injury
Trismus.
of or
tongue.
bilateral taste.
on to
There important
ones ative
lesions, the
tongue, chorda
Lesions e.g.
tympani are
several related
dental illa,
and causes
to the
abscess, malignant
oral trauma
lesions of
loss of
in heavily
or the
these coated
facial of
trismus cavity
nerve.
but the
include to
mandible tongue,
ulcer- or
buccal max-
mucosa and
retromolar trigone
that have
infiltrated deeply.
5. Lesion or oral cavity. Patient can easily see several parts of his oral cavity in the mirror and present with an abnormal growth, coating of tongue, a cleft (lip or palate) or a fistula (oroantral). It is not unusual for some patients of cancerophobia to fix their attention on the circumvallate papillae as cancer.

Examine both the lips—the upper and lower, by inspection and palpation. Each lip has an outer (cutaneous), an inner
4.
(mucosal) surface and a vermilion border. Look for any swellings, vesicles, ulcers, crusts, scars, unilateral or bilateral clefts.

It can be examined by asking the patient to open the mouth and by retracting the cheek with a tongue depressor. Exam- ine the mucosa of cheek and vestibule of mouth. Look for:
(a)
Change in
colour.
(b)
Change in surface appearance, e.g. ulceration, vesicles or bullae (pemphigus), white stria (lichen planus), blanched appearance with submucosal scars (submucous fibrosis), leukoplakia, erythroplakia, pigmentation, atrophic change in mucosa, swelling or growth. Opening of parotid duct is seen opposite the upper second molar tooth. It may be red and swollen with secretions flowing through it on massage of parotid gland (viral or suppurative parotitis).

Examine the gums and teeth in both the upper and lower jaws. Outer surface of gums is examined by retracting the cheeks and lips and the inner surface by pushing the tongue with a away tongue depressor.
(a) Red and swollen gums. Gingivitis.
(b)
Ulcerated gums
covered with
a
membrane.
or Vincent infections.
(c) Hyperplasia. Pregnancy or dilantin therapy.
(d) Growths. Benign or malignant neoplasms.
(e) Loose teeth. Maxillary or mandibular periodontitis. growth,
(f)
Carious infected
tooth or
teeth.
Cause of
maxillary sinusitis if upper, and Ludwig's angina, if lower.
Malocclusion.
(g)
Fractures of
mandible or
of teeth
illa, abnormalities of temporomandibular joint.

Look for:
(a) Cleft palate
(b) Oronasal fistula
(c) High-arched palate
(d) Bulge
(e) Bony growth in midline
(f) Mass or ulcer
Trauma or syphilis
Mouth breathers
Tumours of palate, nose or antrum
Torus palatinus
Cancer

Only oral tongue (anterior two-thirds) is included in the oral cavity. First, examine the tongue in its natural position and then ask the patient to protrude it, move it to the right and left and then up. Examine the tip, dorsum, lateral bor- ders and undersurface.
(a)
Large
size.
Macroglossia,
haemangioma,
lymphangi-
oma, cretinism, oedema or abscess.
(b) Inability to protrude. Congenital ankyloglossia, cancer
tongue or floor of mouth, painful ulcer, abscess.
(c)
Deviation
on
protrusion.
Paralysis
CN
XII
on
the
side
of deviation.
(d)
Bald
tongue.
Iron-deficiency
anaemia,
median
rhom-
boid
glossitis
(single
patch
in
midline
on
the
dorsum),
geographical tongue.
Viral ulcers
max-
Figure 76.12 Palpation of oral cavity.
(e)
Fissures.
(Melkersson
syndrome),
syphilitic.
A
single
nonhealing
fissure
may
be
malignant.
(f)
Ulcers.
Aphthous
traumatic
(jagged
tooth
or
denture),
malignant, syphilitic or tubercular.
(g) White thick patch or plaque. Leukoplakia.
(h) Proliferative growth. Malignancy.

Examine anterior part which lies under the tongue and two lateral gutters. Lateral gutters are better examined by two tongue depressors; one retracting the tongue and the other, the cheek.
Opening of the submandibular duct is seen as a raised papilla on either side of the frenulum.
(a)
Short
frenulum.
tongue-tie).
(b) Scar. Trauma or corrosive burn.
(c)
Ulcer.
Trauma,
erosion
of
submandibular
duct
stone,
aphthous ulcer, malignancy.
(d) Swelling. Ranula, sublingual dermoid, calculus of sub-
duct,
benign
or
malignant
tumours,
Lud-
wig’s angina.

Look for the inflammation due to impaction of last molar tooth or a malignant lesion of this area.
ankyloglossia
(i.e.

All lesions of the oral cavity, particularly of the tongue, floor of mouth, cheek, lip and palate, must be palpated. A swelling in the floor of mouth should be examined by bimanual palpation, to differentiate a swelling of submandibular (Figure 76.12) salivary gland from that of submandibular lymph nodes.

Oropharynx lies opposite the oral cavity. It starts at the level of anterior pillars and is bounded above by the junction of hard and soft palate and below by the V-shaped row of circumvallate papillae.
Structures included in it are:
1. Tonsils and pillars
2. Soft palate
3. Base of tongue
4. Posterior pharyngeal wall

A disease of the oropharynx can disturb swallowing, phonation, respiration and hearing. A patient with disease of oropharynx presents with one or more of the following complaints:
1. Sore throat. Acute or chronic tonsillitis, pharyngitis, ulcerative lesions of pharynx, etc.
2. Odynophagia (painful swallowing). Ulcers, peritonsillar or retropharyngeal abscess, lingual tonsillitis, etc.
3. Dysphagia (difficulty in swallowing). Tonsillar enlargements; parapharyngeal tumour; benign or malignant disease of tonsils, base of tongue or posterior pharyngeal wall; paralysis of soft palate; globus hystericus.
4. Change in voice. Paralysis of palate causes hypernasality. Space-occupying lesions of the oropharynx cause muffled or hot-potato voice.
5. Earache. Benign ulcers or malignant lesions of the base of tongue, tonsil, pillars and palate cause referred pain in the ipsilateral ear.
6. Snoring. Large tonsils and other oropharyngeal lesions may obstruct respiration and cause snoring or sleep apnoea syndrome.
7. Halitosis (bad smell from the mouth). In the oropharynx, cause may be infected tonsils, postnasal discharge or malignancy of oral cavity or oropharynx.
8. Hearing loss. A conductive hearing loss due to disturbance of eustachian tube function can result from enlarged tonsils (which interfere with movements of soft palate), cleft palate, submucous palate, palatal paralysis, recurrent pharyngitis or tonsillitis.
9. Abnormal appearance. A patient may notice an abnor mal finding while looking at his throat in the mirror and then consult the doctor. It is not unusual for patient to be concerned about hypertrophic circumvallate papillae and have fear of cancer.

First, examine the oropharynx by asking the patient to open the mouth widely. Tongue depressor is used when this preliminary examination is unsatisfactory, or when it is required to displace the tongue to one side to examine tonsillolingual sulcus, or to press on the tonsils to look for the contents of tonsillar crypts. The base of tongue is examined by laryngeal mirror. Following structures of oropharynx are carefully examined.

(i) Presence. Look for presence or absence of tonsils.
(ii) Size: Large and obstructive, small or embedded.
(iii) Symmetry. Unilateral or bilateral enlargement.
(iv) Crypts. White or yellow spots at the openings of crypts (follicular tonsillitis), white excrescences not easily wiped off (keratosis).
(v) Membrane. Diphtheria, Vincent’s angina, membra- nous tonsillitis, etc.
(vi) Ulcer. Cancer, Vincent’s angina, tuberculosis, ulcerat- ing tonsillolith.
(vii)
Mass.
Cystic
(retention
cyst),
pedunculated
or
ses-
sile
solid
mass
(papilloma,
fibroma),
proliferative
growth
(cancer).
(viii) Bulge. Peritonsillitis, parapharyngeal abscess, para- pharyngeal tumour.
Pressure
on
the
anterior
pillar
with
the
edge
of
tongue
depressor
may
express
cheesy
material
from
the
crypts
(normal)
of
frank
fluid
pus
(septic
tonsil).
Palpation of the tonsil with a gloved finger is essential to know the consistency of the mass (hard in malignancy or ton- sillolith), pulsation in tonsillar area (internal carotid artery aneurysm), palpation for an elongated styloid process.
(b) Pillars. Uniform congestion of the pillars, tonsils and pharyngeal mucosa is seen in acute tonsillitis. Conges- tion of only the pillars may be a sign of chronic tonsillitis. Ulceration or proliferative growth may be an extension of malignancy from the tonsil base of tongue or the retromolar trigone.

Look for redness (peritonsillitis), bulge or swelling. Normally, uvula is the midline. It becomes oedematous and displaced to the opposite side in peritonsillar abscess. Note movements of soft palate when the patient says “Aa.” Deviation of the uvula and soft palate to the healthy side is a sign of vagal paralysis. This may be associated with paralysis of posterior pharyngeal wall which shows a “curtain effect” (the paralyzed side moves like a sliding curtain to the healthy side).
A bifid uvula may be a sign of submucous cleft palate. In such cases, a notch can be palpated in the hard palate at its junction with soft palate in the midline.

It can be seen directly. Look for lymphoid nodules (granular pharyngitis), purulent discharge trickling down the posterior pharyngeal wall (sinusitis), hypertrophy of lateral pharyn- geal bands just behind the posterior pillars (chronic sinus- itis), thin glazed mucosa and crusting (atrophic pharyngitis).

Posterior one-third of tongue forms the base of tongue and lies between the V-shaped row of circumvallate papillae and the valleculae. Valleculae are two shallow depressions which lie between the base of tongue and the epiglottis.
Base
of
tongue
and
valleculae
are
best
examined
by
indi-
rect laryngoscopy and finger palpation.
(a) Indirect laryngoscopy. Look for the colour of mucosa (normal or congested); prominent veins, varicosities at the base of tongue or lingual thyroid, ulceration (malignancy, tuberculosis or syphilis), solid swelling (lingual tonsil, lin- gual thyroid, lymphoma, carcinoma base of tongue), cystic swelling (vallecular cyst, dermoid or thyroglossal cyst).
(b) Palpation of base of tongue. It should never be omitted. Extent of tumour which infiltrates deeper into the tongue is better appreciated by palpation than by inspection. If the patient fails to relax sufficiently, palpation should be done
under general anaesthesia. When palpating any structure in the oropharynx in a child, the examiner should invaginate the patient's cheek between his teeth with finger of the opposite hand to prevent biting on the examiner's finger.

A patient with disease of the larynx presents with one or more of the following complaints:
1. Disorders of voice. e.g. hoarseness aphonia, puberpho- nia or easy fatiguability of voice.
2. Respiratory obstruction.
3. Cough and expectoration.
4. Repeated clearing of throat (chronic laryngitis, benign or malignant tumours of larynx).
5.
Pain
in
throat
(ulcerative
lesions
of
larynx,
of
laryngeal
cartilages,
arthritis
of
laryngeal
joints).
6. Dysphagia (epiglottitis, aspiration of secretions due to laryngeal paralysis).
7. Mass in the neck (cervical nodes, direct extension of growth, laryngocele).

Clinical examination of larynx includes:
External examination of larynx. >
Indirect laryngoscopy. ON
Flexible or rigid fibreoptic endoscopy.
Assessment of voice. oR
Assessment of cervical lymph nodes.

Both inspection and palpation are employed.
Look for:
(a) Redness of skin (abscess, perichondritis).
(b) Bulge or swelling (extension of growth or enlarged lymph nodes).
(c) Widening of larynx (growth of pyriform fossa).
(d) Surgical emphysema (accidental or surgical trauma).
(e) Change in contour or displacement of laryngeal structures (trauma or neoplasm). Palpate the hyoid bone, thyroid cartilage, thyroid notch, cricoid cartilage, and the tracheal rings.
(f) Movements of larynx. Normally, larynx moves with deglutition. It can also be moved from side to side producing a characteristic grating sound (laryngeal crepitus). Fixity of larynx indicates inflammation of infiltration of growth into the surrounding structures. Loss of larynis due to carcinoma.
geal crepitus postcricoid

Technique. Patient is seated opposite the examiner. He should sit erect with the head and chest leaning slightly towards the examiner. He is asked to protrude his tongue which is wrapped in gauze and held by the examiner between the thumb and middle finger. Index finger is used to keep the upper lip or moustache out of the way (Figure 76.13).
Gauze piece is used to get a firm grip of the tongue and to protect it against injury by the lower incisors. Laryngeal mirror (size 4—6) which has been warmed and tested on the back of hand is introduced into the mouth and held firmly against the uvula and soft palate. Light is focussed on the laryngeal mirror and patient is asked to breathe quietly. To see movements of the cords, patient is asked to take deep inspiration (abduction of cords), say "Aa" (adduction of cords) and "Eee" (for adduction and tension). Movements of both the cords are compared.
Structures seen on indirect laryngoscopy (Figure 76.14). Indirect laryngoscopy permits examination of structures of the and
oropharynx, larynx laryngopharynx.
¢ Larynx. Epiglottis, aryepiglottic folds, arytenoids, cuneiform and corniculate cartilages, ventricular bands, ventricles, true cords, anterior commissure, posterior commissure, subglottis and rings of trachea.
¢ Laryngopharynx. Both pyriform fossae, postcricoid region, posterior wall of laryngopharynx.
¢ Oropharynx. Base of tongue, lingual tonsils, valleculae, medial and lateral glossoepiglottic folds.

(a) Flexible endoscopy. In difficult cases, where laryngeal examination cannot be performed with a mirror due to anatomical abnormalities or intolerance of mirror by the patient, a flexible rhinolaryngoscope can be used. It is passed through the nose under local anaesthesia and gives
Figure
76.13
Indirect laryngoscopy.
a good view of the larynx, laryngopharynx, subglottis and even upper trachea. It is an outdoor procedure.
(b)
Rigid endoscopy.
For this
purpose,
a
rigid fibreoptic
telescope is used. It gives a clear, wide-angle view of the lar- ynx and laryngopharynx. It is also an outdoor procedure.
Stroboscopy. A stroboscope is a device which emits light in pulses,
of which
can be
set by
the examiner.
If frequency of pulses is same at which vocal cords are moving,
the latter appear stationary giving more time to study the cord.
If frequency
of pulses
is more
or less
than that
of vocal
cord movements,
the cords
are seen
in slow
motion.
Stroboscopes are synchronized with rigid or fibreoptic endoscopes and
the vocal
cord movements
can be
recorded on
video
(video stroboscopy).
Stroboscopy has
been found
very useful
in diag-
nosis of laryngeal paralysis, completeness of glottic closure during phonation, very small early laryngeal cancer, vocal cord scarring, laryngeal cyst versus polyp and sulcus vocalis.

The examiner should make note of the quality of voice of the
patient such
as hoarse,
rough, breathy,
bitonal, dys-
phonic, whispered or feeble.

No examination of the larynx and hypopharynx is complete without thorough search for cervical lymph nodes.

(f) Facial
2. Lateral cervical nodes. They include nodes, superficial and deep to sternocleidomastoid muscle and in the poste- rior triangle.
(a) Superficial external jugular group
(b) Deep group
(i) Internal jugular chain (upper, middle and lower
groups)
(ii) Spinal accessory chain
(iii) Transverse cervical chain

(a) Anterior jugular chain
(b) Juxtavisceral chain
(i) Prelaryngeal
(ii) Pretracheal
(iii) Paratracheal

(a) Submental nodes. They lie on the mylohyoid muscle in
the submental triangle, 2-8 in number.
Afferents come from the chin, middle part of lower lip,
anterior gums, anterior floor of mouth and tip of tongue.
Efferents go to submandibular nodes and internal jugular
chain.
(b) Submandibular nodes. They lie in submandibular tri- angle in relation to submandibular gland and facial artery. Afferents come from lateral part of the lower lip, upper lip, cheek, nasal vestibule and anterior part of nasal cavity, gums, teeth, medial canthus, soft palate, anterior pillar, anterior part of tongue, submandibular and sublingual salivary glands and floor of mouth. Efferents go to internal jugular chain.
(c) Parotid nodes. They lie in relation to the parotid salivary gland and are extraglandular and intraglandular. Preauricu- lar and infraauricular nodes are part of the extraglandular
group.
Figure
76.15
of head
and neck.
Figure 76.16 Medial and lateral groups of retropharyngeal lymph nodes seen from behind. Node of Rouviere is the uppermost node of lateral group.
(b) Juxtavisceral chain. It consists of prelaryngeal, pretracheal, and paratracheal nodes.
Prelaryngeal node (Delphian node) lies on cricothyroid membrane and drains subglottic region of larynx and pyriform sinuses.
Pretracheal nodes lie in front of the trachea, deep to pre- tracheal
fascia, and
drain thyroid
gland and
Efferents from
these nodes
go to
paratracheal, lower
inter nal
jugular and
anterior mediastinal
nodes.
Paratracheal nodes (recurrent nerve chain) lie along recurrent laryngeal nerve and drain the thyroid lobes, subglottic larynx, trachea and cervical oesophagus.

(a) Retropharyngeal nodes. They lie behind the pharynx and are divided into lateral and medial groups. Lateral group lies at the level of atlas, close to the base of skull. Most superior node of the lateral group is called node of Rouviere (Figure 76.16).
Medial group lies near the midline but at a little lower level. Retropharyngeal nodes drain the nasal cavity, paranasal sinuses, hard and soft palate, nasopharynx, posterior wall of the pharynx and send efferents to the upper internal jugular group.
(b) Sublingual nodes. They lie deep along the lingual vessels and drain anterior part of the floor of mouth and ventral surface of tongue. Lymphatics from these nodes end in the submandibular or upper jugular nodes.

Examination of neck nodes is important, particularly in head and neck malignancies and a systematic approach should be followed.
Neck nodes are better palpated while standing at the back of the patient. Neck is slightly flexed to achieve relaxation of muscles (Figure 76.17). The nodes are examined in the following manner so that none is missed.
1. Upper horizontal chain. Examine submental, submandibular, parotid, facial, postauricular and occipital nodes.
2. External jugular chain. It lies superficial to sternomastoid.
Figure 76.17 Examination of neck nodes.
- 3. Internal jugular chain. Examine the upper, middle and
lower groups. Many of them lie deep to sternomastoid muscle which may need to be displaced posteriorly.
. Spinal accessory chain.
. Transverse cervical chain. OF
. Anterior jugular chain. STD
. Juxtavisceral chain. Prelaryngeal, pretracheal and para-
tracheal nodes.
When a node or nodes are palpable, look for the following points:
1. Location of nodes.
2. Number of nodes.
3. Size.
4. Consistency. Metastatic nodes are hard; lymphoma nodes are firm and rubbery; hyperplastic nodes are soft. Nodes of metastatic melanoma are also soft.
. Discrete or matted nodes. Ot
. Tenderness. Inflammatory nodes are tender. SID
. Fixity to overlying skin or deeper structures. Mobility should be checked both in the vertical and horizontal planes.

- IA Submental nodes, which lie in the submental triangle, i.e. between right and left anterior bellies of digastric muscles and the bone.
hyoid
- IB Submandibular nodes, lying between anterior and posterior bellies of digastric muscle and the lower border of the body of mandible.
Level
|
¢ Submental (IA)
¢ Submandibular (IB)
Upper jugular
Mid jugular
Lower jugular
Posterior triangle group (spinal accessory
and transverse cervical chains)
¢
Prelaryngeal
e
Pretracheal
e
Paratracheal
Nodes of upper mediastinum
Level
Il
Level
Ill
Level
IV
Level
V
Level
VI
Level VII
Figure 76.18
Classification of neck nodes according to levels.
Levels are shown in Table 76.1.

They are located along the upper third of jugular vein, i.e. between the skull base above and the level of lower border of hyoid bone (or bifurcation of carotid artery) below.

They are located along the middle third jugular vein, from the level of hyoid bone above, to the level of lower border of cricoid cartilage (or where omohyoid muscle crosses the below.
jugular vein)

They are located along the lower third of jugular vein; from lower border of cricoid cartilage to the clavicle. Virchow's node is included into this level.

They are located in the posterior triangle, i.e. between posterior border of sternocleidomastoid (anteriorly), anterior border of trapezius (posteriorly) and the clavicle below. They include lymph nodes of spinal accessory chain, transverse cervical nodes and supraclavicular nodes. Level V nodes are further subdivided into upper, middle and lower, corresponding to planes that define levels II, II and IV.
Figure 76.19 Supraclavicular fossa (or Ho's triangle) is bounded by medial and lateral ends of clavicle and the point where neck meets the shoulder. Nodes in this zone include nodes of lower parts of levels IV and V.

They are located between the medial borders of sternocleidomastoid muscles (or carotid sheaths) on each side, hyoid bone above and suprasternal notch below. They include preand nodes.
laryngeal, pretracheal paratracheal

They are located below the suprasternal notch and include nodes of the upper mediastinum.
Lymph nodes of supraclavicular zone or fossa (Ho's triangle) (Figure 76.19). Supraclavicular zone is situated between (i) upper border of medial end of clavicle, (ii) upper border of lateral end of clavicle and (iii) point where neck meets the shoulder. Nodes in this triangle are important in carcinoma of the nasopharynx. Metastases in these nodes, irrespective of their size, would place them in Ns category (AJCC, 1977). Nodes in this zone include lower part of levels IV and V.

Retropharyngeal
Facial
Preauricular
Postauricular (mastoid)
Intraparotid
Suboccipital

It is a procedure to remove lymph nodes and the surrounding fibrofatty tissues from the neck, to eradicate metastases to cervical lymph nodes from cancer of the aerodigestive tract.

1. Radical neck dissection.
2. Modified radical neck dissection.
(a) Type I—Preserves CN XI
(b) Type II—Preserves CN XI and internal jugular vein
(c) Type II—Preserves CN XI, internal jugular vein and
sternocleidomastoid muscle.
3. Selective neck dissection
(a) Supraomohyoid (or anterolateral) (removes level I
to III)
(b) Lateral (removes nodes in level II, III, IV)
(c)
Posterolateral
(removes
level
II
to
V
suboccipital
and
postauricular nodes)
(d) Anterior compartment (removes level VI nodes)
4. Extended neck dissection (vide infra).

In this procedure, all lymph nodes, extending from the man- dible above to the clavicle below and from lateral border of sternomastoid, hyoid bone and contralateral anterior belly of digastric, medially, to the anterior border of trapezius poste- are removed. The dissection would include:
riorly, specimen
1. Lymph nodes of submental, submandibular, upper, middle and lower jugular, and lateral (posterior) trian- gle regions, i.e. level I to V along with its fibrofatty tissue.
Sternomastoid muscle. bo
Internal jugular vein. yo
Spinal accessory nerve. OR
Submandibular salivary gland.
Tail of the parotid. ND
Omohyoid muscle.
It saves following structures:
¢ Carotid artery.
¢ Brachial plexus, phrenic nerve, vagus nerve, cervical sym- pathetic chain, marginal mandibular branch of facial, lin- gual and hypoglossal nerves.
Radical neck dissection does not remove nodes of post- auricular, suboccipital, parotid (except those in the tail), facial, retropharyngeal and paratracheal regions.
Incision used in radical neck dissection (Figure 76.20) will depend on the incision being used to remove the primary growth and whether patient received any preoperative
radiation.
Commonly, the incisions used are:
1.
Schobinger
2. Mc Fee
Hockey-stick ge
4. Extensions from Gluck-Sorenson’s incision, used for lar-
yngectomy with neck dissection (Figure 76.21).
Contraindications to radical neck dissection include:
1. Untreatable primary cancer.
2. Distant metastases.
3. Inoperable neck nodes when they are fixed to important
4. Medical illness which makes the patient unfit for major
surgery.

It is similar to radical neck dissection but with preservation of one or more of the following structures:
1. Spinal accessory nerve
2. Internal jugular vein
3. Sternocleidomastoid muscle.
Figure 76.20 Incisions commonly used in radical neck dissection. (A) Schobinger. (B) Mc Fee. (C) Hockey-stick.
¢ or anterolateral. Removes levels I to III,
Supraomohyoid usually done in cancer of oral cavity.
e Lateral. Removes levels II to IV.
Used in cancer of pharynx, hypopharynx and larynx.
¢ Posterolateral. Removes levels II to V with postauricular
and occipital nodes.
Used in cancer or melanoma of skin, located in poste-
rior scalp or posterior upper neck behind the line passing
through the tragus.
¢ Anterior compartment. Removes nodes at level VI, i.e. pre-
tracheal, paratracheal and prelaryngeal.
Used in differentiated thyroid cancer, subglottic, cervical
tracheal or hypopharyngeal cancers.
Figure
76.21
Gluck-Sorenson’s incision
used for
laryngec- tomy. Dotted lines showing extensions of incisions for radical neck dissection.

It consists of preservation of one or more lymph node groups and all the three nonlymphatic structures, i.e. spinal acces-
sory, sternocleidomastoid muscle and internal jugular vein.

It consists of any of the neck dissections as described and further extended to include additional lymph node groups or nonlymphatic structures or both. Additional lymph node groups include retropharyngeal, parotid or level VI nodes and nonlymphatic structures may include external carotid artery, hypoglossal nerve, parotid gland and levator scapulae, etc. that are not routinely included in that dissection.

Clinically, neck masses can be divided into: (i) those in the midline (Figure 77.1) and (ii) those in the lateral aspect of neck (Figure 77.2). The latter can be grouped according to triangles of neck. Only the clinically important ones are described in this

It presents as a cystic midline swelling, usually affecting young children but can occur at any age (Figure 77.3). It is usually rounded with a diameter of 2-4 cm. It increases in size with upper respiratory tract infection. Sometimes it presents as a draining sinus if it has burst due to infection or has been surgically drained. Because of the attachment of thyroglossal duct to foramen caecum at the base of tongue, it moves with tongue protrusion.
During development, thyroid anlage starts at foramen caecum, passes through base of tongue and then descends in front, behind or through the hyoid bone to form the thyroid gland. Thyroglossal cyst can occur anywhere in the course of thyroid duct (Figure 77.4). It may contain the only functioning thyroid tissue in the body. Rarely carcinoma develops in the cyst.
It presents as a midline submental swelling but does not move on protrusion of the tongue as it is not attached to foramen caecum. Sometimes it arises from the floor of mouth and needs differentiation from ranula. Treatment is surgical excision. A midline dermoid is also seen just above the suprasternal notch.

There are two to eight nodes situated in the submental triangle between the platysma and mylohyoid muscle. They drain chin, middle part of lower lip, incisor region of gingiva, anterior floor of mouth and the tip of tongue. When enlarged, the draining areas should be looked for infections or malignancy.

Treatment is complete surgical excision, including with it the body of hyoid bone and core of tongue tissue around the tract in the suprahyoid tongue base to the foramen caecum (Sistrunk's operation). Simple excision of cyst without removal of its tract leads to recurrence.
They belong to juxtavisceral chain of nodes and lie in front of the larynx and trachea. They drain the larynx and trachea, thyroid isthmus and anteromedial aspect of thyroid lobes. In case of enlargement of the above nodes, draining areas should be examined.
Figure
77.1
Midline swellings of neck.

Figure 77.2 Lateral swellings of neck as seen in different triangles of neck.
Figure
77.4
Sites of
thyroglossal duct
cysts.
1.
Base of
tongue,
2.
Suprahyoid,
3.
Subhyoid
(most common),
and 5. In front of cricoid.
4.
On the
thyroid cartilage
Figure 77.5 Left-sided branchial sinus discharging pus.
fistula. A second arch branchial sinus has an external opening at the junction of lower and middle of the anterior border of sternomastoid and may exude mucoid discharge. It may have an internal opening in the tonsillar fossa. When both internal and external openings are present, it is called a branchial fistula.
Treatment of branchial cyst is surgical excision along with its tract, if present.

(FIGURE 77.5)
A second arch fistula has a typical course, the knowledge of which can help in the total surgical extirpation of the tract. It has:
1. An external opening along the anterior border of sternocleidomastoid muscle.
2. A tract which ascends just deep to deep cervical fascia along the carotid artery.
3. The tract passes deep to second arch structures, i.e. external carotid artery, stylohyoid and posterior belly of digastric but superficial to third arch structure, i.e. internal carotid artery (the tract passes between internal and external carotid arteries). It also runs superficial to hypoglossal nerve.
4. Pierces the pharyngeal wall and ends in the tonsillar fossa.
Complete excision of the tract can be accomplished by
step-ladder incisions.
Third branchial cleft sinus is uncommon. Its external opening is at the same place as second cleft sinus but internal opening is situated in pyriform sinus. Tract passes behind both internal and external carotid vessels but is superficial to vagus and hypoglossal nerves.

It is a pseudocyst caused by extravasation of mucus from obstruction to sublingual salivary gland. It presents as an isolated swelling in the submandibular area and is transilluminant. Sometimes plunging ranula coexists with a ranula in the floor of mouth. Treatment is total excision along with removal of sublingual gland.

It arises from the chemoreceptor cells in the carotid body, hence also called chemodectoma. Mostly presents after 40 years. It is a very slow-growing tumour and the history of mass in the neck may extend into several years. It presents as a painless swelling which is pulsatile. Bruit can be heard with a stethoscope. It moves from side to side but not vertically. It may extend into the parapharyngeal space and present in the oropharynx (Figure 77.6).
Contrast-enhanced CT and MRI with gadolinium are diagnostic and also show the extent of the tumour. MRI angiography shows splaying of internal and external carotid arteries (Lyre's sign). Some tumours are functional and secrete catecholamines. Hence serum catecholamines and urinary metanephrines and vanillylmandelic acid (VMA) should be estimated. Fine-needle aspiration cytology (FNAC) or biopsy should not be done because of the vascularity of tumour.
Treatment is surgical when the patient is younger than 50 years and surgically fit, or when the tumour extends into the oropharynx causing difficulty in speech, swallowing or breathing. Radiotherapy is also effective and is used in older patients and those unfit for surgery or those who refuse surgery or have a metastatic disease.

These tumours present in the upper neck near the angle of mandible or retromandibular area. They may also be seen intraorally displacing the tonsil, lateral pharyngeal wall and soft palate medially. Though majority of these tumours are of salivary gland origin (pleomorphic adenomas being the most common) others like schwannoma, neurofibroma, lipoma, haemangioma, paraganglioma or lymph node metastasis in parapharyngeal nodes are also seen. Diagnosis can be established by imaging techniques and FNAC.

Balls of
cells
(Zellballen of
chief cells)
Figure 77.6 (A) A 23-year-old female patient with carotid body tumour where surgery was earlier attempted. (B) MRI neck showing the tumour. (C) MRI angiography. Note splaying of the external carotid artery (ECA) and internal carotid artery (ICA). This is also called Lyre's sign. (D) Tumour after removal. (E) Histopathology of tumour showing a Zellballen pattern having chief cells and sustentacular cells (H&E, x200).
When inflamed due to infection, it becomes painful and
Injection of sclerosing agents is not favoured as it makes increases in size. Spontaneous regression is unpredictable.
Treatment is surgical excision with preservation of neural and
vascular structures.
Complete excision
may not
be pos-
sible in a single operation. Bipolar diathermy is useful. Rup- ture
of cyst
makes dissection
Recurrence rate
after surgical excision is only 5% if whole tumour is removed mac-
roscopically but it is 50% if some part is left. Cystic hygroma causing respiratory distress may be aspirated or may require tracheostomy to relieve respiratory obstruction.
later dissection more difficult.

Mass due to tubercular lymph nodes in the neck is very common in India. Any lymph node group can be involved. It can occur in any age or sex. Involved lymph node may be single, multiple or matted due to periadenitis. Tubercular abscess
—-
Figure 77.7 (A) Cystic hygroma neck in a 27-year-old male. (B) CT scan neck of the same point.
Figure 77.8 Multiple tubercular nodes in the neck.
may form when node(s) caseate. It may become adherent to the skin and underlying structures or a draining tubercular sinus may develop (Figures 77.8 and 77.9).
Diagnosis is usually made by FNAC or lymph node biopsy which reveals a granulomatous lesion. Sometimes acid fast bacilli (AFB) can be demonstrated. AFB from the aspirated or biopsy material can be cultured and sensitivity established, to be prepared for multidrug resistant lesions. X-ray chest, skin test and work-up for other nodal group involvement should be done. Tuberculosis is also becoming more common due to AIDS.
Treatment consists of initial 2 months course of four drugs (rifampicin, isoniazid, pyrazinamide and ethambutol) followed by 4 months course of rifampicin and isoniazid. Nodes may initially increase in size during treatment before they finally subside. Surgical excision of lymph node mass or abscess is occasionally required when drug treatment fails.

Any lymph node group can be involved depending on the site of primary malignancy. Upper cervical lymph nodes are commonly involved in malignancies of upper aerodigestive tract. Nasopharyngeal malignancies spread to accessory chain of nodes in the posterior triangle. In many cases primary malignant lesion is not discernible (occult primary), and in such cases the most common sites are tonsil, base of tongue, nasopharynx and pyriform sinus. Node(s) in supraclavicular area should alert the surgeon to the possibility of an infraclavicular primary in the lung, breast, stomach, colon, kidney, ovary and testis.

Both Hodgkin and non-Hodgkin lymphomas may present with cervical lymphadenopathy. Other lymphatic structures of the Waldeyer ring may also be involved and cause symptoms of dysphagia, serous otitis media or respiratory obstruction. In such cases, other lymph nodes in the axilla, groin and abdomen should be examined in addition to spleen and liver enlargement.

Occasionally an extra rib may arise from the seventh cervical vertebra and end anteriorly by attaching to the first rib. This rib may produce a bony hard lump in the supraclavicular region. Most often it is seen on the right but may be present on the left or is bilateral.
Subclavian artery and brachial plexus which normally pass between anterior and middle scalene muscles over the first rib have now to pass over the cervical rib (a vertebral space higher) and are thus compressed. It produces neurological or vascular symptoms. Patient may complain of tingling sensation or numbness along the upper side of forearm and hand due to compression of the lower part of brachial plexus. When subclavian artery is compressed, hand becomes cold and numb with intermittent claudication of upper limb. Due to arterial compression an aneurysm may develop with mural thrombus which may shoot emboli to the distal arterial system of the upper limb. Cervical rib, if asymptomatic, does not require treatment
=.
Figure 77.9 (A) Caseating tubercular suprasternal node forming abscess. (B) Tubercular nodes in supraclavicular area (same patient).
but symptomatic ones are excised by supraclavicular or transaxillary approach.

head is tilted on the ipsilateral shoulder. A mass can be palpated in the sternocleidomastoid muscle on physical examination. In long-standing cases, asymmetry of face and head can develop as a sequel.
Mostly seen in the newborns due to birth trauma. Fibrosis and later shortening of the sternocleidomastoid muscle causes torticollis. Face is turned to opposite side but the
Treatment is passive exercises of the neck in early stages. Surgery is done when the condition is persistent and likely to cause facial hemihypoplasia. It consists of division of sternomastoid muscle.

It is incision of the tympanic membrane with the purpose to drain suppurative or nonsuppurative effusion of the
middle ear or to provide aeration in case of malfunctioning eustachian tube. Ventilation tube (grommet) may also be
required in the latter case.

1. Acute suppurative otitis media
(a) Severe earache with bulging tympanic membrane.
(b) Incomplete resolution with opaque drum and persis-
tent conductive deafness.
(c) Complications of acute otitis media, e.g. facial paraly-
sis, labyrinthitis or meningitis with bulging tympanic membrane.
Serous otitis media. gon
Aero-otitis media (to drain fluid and “unlock” the eusta-
chian tube).
4. Atelectatic ear (grommet is often inserted for long-term aeration).

Suspected intratympanic glomus tumour. Myringotomy in
these cases can cause profuse bleeding. Tympanotomy is

In infants and children, always use general anaesthesia.
For adults, general anaesthesia is used only when tympanic
membrane is acutely inflamed. If there is no inflammation,
myringotomy
can be done under local anaesthesia or no
anaesthesia at all.

Ear canal is cleaned of wax and debris.
2. Operation is ideally performed under operating micro-
scope using a sharp myringotome and a good suction apparatus.
3. In acute suppurative otitis media, a circumferential inci-
sion is made in the posteroinferior quadrant of tympanic
membrane,
midway
between
handle
of
malleus
and
tym-
panic
annulus,
avoiding
injury
to
incudostapedial
joint
(Figure 78.1A).
Figure 78.1 (A) Circumferential incision used in acute suppurative otitis media. (B) Radial incision used in serous otitis media.
Figure 78.2 Grommet in right tympanic membrane.
- 4. In serous otitis media, a small radial incision is given in
the posteroinferior
or anteroinferior
quadrant and
all the effusion sucked out (Figure 78.1B).
When ventilation tube is to be inserted, incision should be just enough to admit the tube (Figure 78.2).

- 1. When tympanic membrane is thick, incision may remain
only in the superficial layers of drum head without cutting through its entire thickness.
- 2. Incision in the posterior meatal wall. This may happen when distinction between drum head and _ posterior meatal wall is lost, when both are inflamed.

Daily mopping of ear discharge will be required in cases of
acute suppurative otitis media. In serous otitis media, just
leave a wad of cotton wool for 24—48 h.
Drum incisions usually heal rapidly. No water should be
permitted to enter the ear canal for at least 1 week, and
if
a grommet has been inserted, entry of water is prevented so
long as grommet is in position.

1. Injury to incudostapedial joint or stapes.
2. Injury to jugular bulb with profuse bleeding, if jugular bulb is high and floor of the middle ear dehiscent.
3. Middle ear infection.

It is an operation in which reconstructive procedure is limited to repair of tympanic membrane perforation.

It is an operation to eradicate disease in the middle ear and to reconstruct the hearing mechanism without mastoid surgery, with or without tympanic membrane grafting. This means ossicular reconstruction only or ossicular reconstruction with myringoplasty.

It is an operation to eradicate disease in both the mastoid and middle ear cavity, and to reconstruct the hearing mechanism with or without tympanic membrane grafting.

MASTOIDECTOMY
OR
SCHWARTZ
OPERATION)
It is an exenteration of all accessible mastoid air cells preserving the posterior meatal wall.

It is an operation to eradicate disease of the attic and mastoid, both of which are exteriorized into the external auditory canal by removal of the posterior meatal and lateral attic walls. Tympanic membrane remnant, functioning ossicles and the reversible mucosa and function of the eustachian tube are preserved. These structures are necessary to reconstruct hearing mechanism at the time of surgery or in a second-stage operation.

It is an operation to eradicate disease of the middle ear and mastoid in which mastoid, middle ear, attic and the antrum are exteriorized into the external ear by removal of posterior meatal wall. All remnants of tympanic membrane, malleus, incus (not the stapes), chorda tympani and the mucoperiosteal lining are removed, and the opening of eustachian tube closed by packing a piece of muscle or cartilage into the eustachian tube.
Meatoplasty is an operation in which a crescent of conchal cartilage is excised to widen the meatus. It is invariably combined with all canal wall down procedures, i.e. modified radical and radical mastoidectomies for easy access to mastoid cavity for periodic inspection and cleaning. It is also done as an isolated procedure in a sagging auricle seen in older people. Sagging auricle obstructs the ear canal and causes hearing loss and retention of wax.

It is an operation to eradicate mastoid disease, when present, and to obliterate the mastoid cavity. Obliteration of mastoid cavity is done with pedicled temporalis muscle or musculofascial tissue raised as flaps.

1. Endomeatal or transcanal approach. It is used to raise a tympanomeatal flap in order to expose the middle ear. Rosen's incision is the most commonly used for stapedectomy. It requires the meatus and canal to be wide enough to work. It consists of two parts: (i) a small vertical incision at 12 o'clock position near the annulus and (ii) a curvilinear incision starting at 6 o'clock position to meet the first incision in the posterosuperior region of the canals, 5-7 mm away from the annulus (Figure 79.1) Posterior meatal canal skin is raised in continuity with tympanic membrane, after dislocating the annulus from the sulcus. It gives a good view of the middle ear and ossicles. Stapes, if still covered by posterosuperior overhang of bony meatus, can be exposed by removing this part of the overhang. This incision is also used commonly for exploratory tympanotomy to find cause for conductive hearing loss, inlay myringoplasty or ossicular reconstruction.

(a)
Excision of osteomas or exostosis of ear canal.
(b)
(c)
(d)
Large tympanic membrane perforations.
Attic cholesteatomas
with limited
extension into
the antrum.
Modified radical mastoidectomy where disease is lim- ited to attic, antrum and part of mastoid.
is made incision
Endaural approach through Lempert's (Figure 79.2). It consists of two parts:
Lempert I. It is semicircular incision, made from 12 o'clock to 6 o'clock position in the posterior meatal wall at the bony-cartilaginous junction.
Figure
79.2
Endaural
(Lempert’s)
incision.
(A)
Incision in
the canal
and incisura
terminalis.
(B)
Magnified view
of
A.
Note position
of
Lempert
|
and
Lempert
II
Lempert II. Starts from the first incision at 12 o'clock and then passes upwards in a curvilinear fashion between tragus and the crus of helix. It passes through the incisura terminalis and thus does not cut the cartilage. Both mastoid and external canal can be done.
surgery
- 3. Postaural (or Wilde's) incision (Figure 79.3). It starts at
the highest
attachment of
the pinna,
follows the
curve of
retroauricular groove,
lying mastoid
tip.
In the
mastoid infants
process and
is not
1
cm behind
children it,
and up
developed to
and ends
2
at the
years of
the facial
age, nerve
lies exposed
near its
exit, and
the incision
therefore is
slant- ing posteriorly, avoiding lower part of the mastoid. Some surgeons prefer to make the postaural incision in the sulcus

(retroauricular groove). Postaural incision is used for:
(a) Cortical mastoidectomy.
(b) Modified radical and radical mastoidectomy.
when extends anterior to
(c) Tympanoplasty: perforation handle of malleus.
Figure
79.4
Cortical mastoidectomy.
Posterior meatal
wall is
left intact.
(d) Exposure of CN VII in vertical segment.
(e) Surgery of endolymphatic sac.

Cortical mastoidectomy, known as simple or complete mastoidectomy or Schwartz operation, is complete exenteration of all accessible mastoid air cells and converting them into a single cavity. Posterior meatal wall is left intact (Figure 79.4). Middle ear structures are not disturbed.

1. Acute coalescent mastoiditis.
2. Incompletely resolved acute otitis media with reservoir
sign.
3. Masked mastoiditis.
4. As an initial step to perform:
(a) endolymphatic sac surgery
(b) decompression of facial nerve
(c) translabyrinthine or retrolabyrinthine procedures for acoustic neuroma.
Figure 79.5 shows the various structures and landmarks after cortical seen mastoidectomy.

Patient lies supine with face turned to one side and the ear to be operated uppermost.

1.
Incision.
A
curved postaural
incision about
1
cm behind but parallel to the retroauricular sulcus, start-
ing at
pinna to
the mastoid
tip
(Figure 79.3B).
In infants and children up to 2 years, the incision is short and more horizontal. This is to avoid cutting facial nerve which
is superficial in the lower part of mastoid (Figure 79.3C).
Incision cuts through soft tissues up to the periosteum.
Temporalis muscle is not cut in the incision.
2. Exposure of lateral surface of mastoid and MacEwen's triangle. Periosteum is incised in the line of first incision. A horizontal incision may be made along the lower border of temporalis muscle for more exposure. Periosteum is scraped from the surface of mastoid and posterosuperior margin of osseous meatus. Tendinous fibres of sternomastoid are sharply cut and scraped down. A self-retaining mastoid retractor is applied.
3. Removal of mastoid cortex and exposure of antrum. Mastoid cortex is removed with burr, or gouge and hammer. Mastoid antrum is exposed in the area of suprameatal triangle (MacEwen's triangle). In an adult, antrum lies 12-15 mm from the surface. Horizontal semicircular canal is identified. Keep in mind the presence of Korner's septum which would need removal to the antrum.
explore
4. Removal of mastoid air cells. All accessible mastoid air cells are removed leaving behind the bony plate of tegmen tympani above, sinus plate behind and posterior meatal wall in front.
5. Removal of mastoid tip and finishing the cavity. Lateral wall of the mastoid tip is removed, exposing muscle fibres of posterior belly of digastric. Zygomatic cells situated in the root of zygoma, retrosinus cells lying between sinus plate and cortex, behind the sinus, are removed. A finished cavity should have bevelled edges so that soft tissue can easily sit in and obliterate the cavity.

6. Closure of wound. Mastoid cavity is thoroughly irrigated with saline to remove bone dust and the wound is closed in two layers. A rubber drain may be left at the lower end of incision for 24-48 h in case of infection or excessive bleeding. A meatal pack should be kept to avoid stenosis of ear canal. Mastoid is dressing applied.

1.
Antibiotics started
preoperatively are
continued postop-
eratively for at least 1 week. Culture swab taken from the mastoid,
during operation,
may dictate
a
change in
the antibiotic.
Drain,
2.
if put,
is removed
in
24-48
h
and done.
- 3. Stitches are removed on the sixth day.

1. Injury to facial nerve.
2. Dislocation of incus.
3. Injury to horizontal semicircular canal. Patient will have
postoperative giddiness and nystagmus.
4. Injury to sigmoid sinus with profuse bleeding.
5. Injury to dura of middle cranial fossa.
6. Postoperative wound infection and wound breakdown.
sterile dressing

Radical mastoidectomy is a procedure to eradicate disease from the middle ear and mastoid without any attempt to reconstruct hearing. Posterior meatal wall is removed and the entire area of middle ear, attic, antrum and mastoid is converted into a single cavity. All remnants of tympanic membrane, ossicles (except stapes footplate) and mucoperiosteal lining are removed (Figure 80.1). Eustachian tube is obliterated by a piece of muscle or cartilage. Aim of the operation is to permanently exteriorize the diseased area for inspection and cleaning. The radical mastoidectomy is infrequently required these days.

1. When all cholesteatoma cannot be safely removed, e.g. that invading eustachian tube, round window niche, perilabyrinthine or hypotympanic cells.
2. If previous attempts to eradicate chronic inflammatory disease or cholesteatoma have failed.
As an approach to petrous apex. go
Removal of glomus tumour.
Carcinoma middle ear. Radical mastoidectomy followed by radiotherapy is an alternative to en bloc removal of temporal bone in carcinoma middle ear. ou

Mostly, general anaesthesia is given. Local anaesthesia can be used in selected cases.
Figure
80.1
Radical mastoidectomy.
The entire
area of
mastoid, middle
erated ear,
and attic
and antrum
no attempt
is exteriorized.
is made
to reconstruct
Eustachian the
hearing tube
is oblit-

Same as for cortical mastoidectomy.
8. Meatoplasty. A flap, based laterally at the concha is raised from posterior and superior meatal wall and turned into the mastoid cavity to cover the area of the facial ridge. This helps in the epithelialization of the mastoid cavity. A piece of conchal cartilage can be removed to enlarge the meatus and to facilitate inspection and access to cavity.
9. Obliteration of the cavity. If mastoid cavity is very large, it may be obliterated with temporalis muscle or other soft tissues, taking care that no vestige of disease (cholesteatoma) is buried underneath.
10. Closure of wound. The cavity is packed with ribbon gauze, impregnated with an antibiotic/antiseptic and the wound is closed with interrupted sutures. Mastoid dressing is applied.

1. Dressing. First dressing is done on third or fourth day. Replace the outer gauze and cotton and look for any signs of perichondritis or infection of meatal pack. Second dressing is done on sixth or seventh day when stitches are removed and meatal pack is changed.
Figure
80.3.
Endaural incision.
Thereafter, change the pack at weekly intervals or leave the cavity unpacked with regular suction and cleaning till epithelialization is complete.
2. Antibiotic. A suitable antibiotic is given for about a week.
3. Cavity care. Usually, cavity is fully epithelialized in 2-3 months. It should be periodically checked (every 4-6 months) in the first year and then annually for removal of any debris or infection. Any granulation tissue which delays epithelialization is removed or cauterized.

Facial paralysis.
Perichondritis of pinna. ON
Injury to dura or sigmoid sinus.
Labyrinthitis, if stapes gets dislocated. TOP
Severe conductive deafness of 50 dB or more. This is due
to removal of all ossicles and tympanic membrane.
- Cavity problems. Twenty-five per cent of the cavities do not heal and continue to discharge, requiring regular aftercare. >
It is a modification of radical mastoidectomy where as much of the hearing mechanism as possible is preserved. The dis-

ease process which is often localized to the attic and antrum is removed and the whole area fully exteriorized into the
meatus by
removal of
meatal and
lateral attic
wall (Figure 81.1).

1. Cholesteatoma confined to the attic and antrum.
2. Localized chronic otitis media.
Irreversibly damaged tissues are removed, preserving the rest to conserve or reconstruct hearing mechanism.

Mostly general, local anaesthesia can be used in selected cases.
Figure 81.1 Modified radical mastoidectomy. Posterior meatal wall is removed to exteriorize the diseased area into the meatus and the hearing mechanism reconstructed.

- Incision, postaural or endaural. . Retraction of soft tissues and exposure of mastoid area. Removal of cortical bone and exposure of antrum and attic. wn
Steps 2 and 3 are the same as in radical mastoidectomy.
Removal of diseased tissue. Cholesteatoma, granulations or unhealthy mucosa is removed. Incus and head of malleus often require removal, if cholesteatoma engulfs them or extends medial to them. They are preserved if possible. Lateral attic wall is removed to fully exteriorize the attic. >
Facial ridge is lowered. Mastoid cavity is smoothened with polishing burr, removing any overhangs and then irrigated with normal saline. os
. Reconstruction of hearing mechanism. Pars tensa of ~
and middle
ear, if
healthy, are
left undisturbed.
If disease
the irreversible
tympanic extends
membrane can
also into
removed.
or ossicular
be done
only
Reconstruction chain,
(mastoidectomy of
if damaged,
with tympanoplasty
operation).
- Meatoplasty and closure of wound are same as in radical mastoidectomy. ge

Closure of perforation of pars of the tympanic brane is called myringoplasty. It has the advantage of:
tensa mem-
1. restoring the hearing loss and in some cases the tinnitus.
2. checking repeated infection from external auditory canal and eustachian tube (nasopharyngeal infection ascends more easily via eustachian tube in the presence of perforation than otherwise).
checking aeroallergens reaching the exposed middle ear mucosa, leading to persistent ear discharge. ye
Myringoplasty should be differentiated from type I tympa-
noplasty. Though both refer to repair of tympanic membrane,
tympanoplasty
entails
exposure
of
the
middle
ear
to
inspect
the middle ear and also ensure ossicular integrity.
Myringoplasty can be combined with ossicular reconstruc-
tion when it is called tympanoplasty.
Physiologic principles for middle ear reconstruction are 29.
discussed on p.

Active discharge from the middle ear. wo
. Nasal allergy. It should be brought under control before surgery.
. Otitis externa. Ag
Ingrowth of squamous epithelium into the middle ear.
In
such
cases,
excision
of
squamous
epithelium
from
the
middle
ear
or
a
tympanomastoidectomy
may
be
required.
. When the other ear is dead or not suitable for hearing aid rehabilitation. or
. Children below 3 years. a

Supine with face turned to one side; the ear to be operated is
up.
Graft materials used are:
1. Temporalis fascia (most common)
2. Areolar fascia overlying the temporal fascia
3. Perichondrium from the tragus
4. Cartilage
5. Vein
6. Periosteum
Incision for exposure of tympanic membrane depends on the size of the ear canal; it may be endomeatal, endaural or postaural.

1. Harvesting the graft, e.g. of temporalis fascia; or perichondrium from the tragus.
2. Preparing the tympanic membrane for grafting. An incision is made along the edge of perforation and the ring of epithelium removed. Remove also a strip of mucosal layer from the inner side of perforation.
3. Inspecting the middle ear. A stapes-type incision is made and the tympanomeatal flap raised to see the integrity and mobility of the ossicular chain and to ensure that no squamous epithelium has grown into the middle ear.
4. Placing the graft. Middle ear is packed with gelfoam soaked with an antibiotic. A proper-sized graft is placed so that its edges extend under the margins of perforation all round and a small part also extends over the posterior canal wall. Tympanomeatal flap is replaced. An underlay technique has the advantage that the squamous epithelium is not buried in the middle ear (Figure 82.1).

1. Temporal fascia or perichondrial graft is harvested as above.
2. Incision is made in the meatus as shown in Figure 82.2
and meatal skin raised along with all epithelium from the
Figure
82.1
Myringoplasty.
Underlay technique—fascia
under the
anterior annulus.
It is
supported ear to prevent medial displacement.
graft by
gelfoam in
the is
middle
Figure 82.2 Overlay technique. (A) Incision to raise medial meatal skin with tympanic membrane epithelium. (B) Placement of graft. (C) Replacement of skin.
Figure 82.3 Myringoplasty. Overlay technique—fascia graft lies lateral to anterior annulus onto the anterior bony canal wall. It is placed medial to malleus handle to prevent lateralization.
outer surface of tympanic membrane remnant and preserved to be used later.
3. Graft placed on the outer surface of tympanic membrane. A slit is made in the graft to tuck it under the handle of malleus (Figure 82.3).
4. Meatal skin removed earlier is now replaced, covering the periphery of the graft.
5. Ear canal packed with gelfoam and then with a small antibiotic pack.
A modification of the overlay technique is to place the anterior edge of fascia graft under the annulus after removing the epithelium from its undersurface. This prevents blunting of anterior canal which is seen as a complication of overlay technique.
6. Closure of endaural or postaural incision.
7. Mastoid dressing.

1. Stitches are removed after 5-6 days.
2. Ear pack is removed after 5-6 days without disturbing the
gelfoam.
3. Patient is seen at 3 and 6 weeks after operation.
4. Complete epithelialization of graft takes 6-8 weeks.

1. Middle ear becomes narrow.
2. Graft may get adherent to the promontory.
3. Anteriorly, graft may lose contact from the remnant of
tympanic membrane leading to anterior perforation.

1. Blunting of the anterior sulcus.
2. Epithelial pearls. They are epidermal cysts, when squamous epithelium is buried under the graft.
3. Lateralization of graft. Graft loses contact from the mal-
leus handle
resulting in
conductive loss.
It is
prevented by tucking the graft under the handle.

1. Splintage. It is used in fresh traumatic perforations. The torn edges of the perforation are carefully everted under the microscope and splinted with absorbable gelfoam placed in the middle ear through the tear. Smaller tears can be splinted on the outer surface of the tympanic membrane with a piece of cigarette paper, gelfilm or silicon sheet.
2. Cautery patching. This is useful in small, long-standing central perforations where the margins have become epithelialized and chronic. In this procedure, margins of the perforation are cauterized with 50% trichloracetic acid to remove the epithelialized edge (or freshened with a fine pick used for myringoplasty) and then supported with a cigarette paper moistened with 1% phenol in glycerine. This procedure can be repeated at 2 weeks interval. Instead of cigarette paper, other material such as steristrip, gelfilm or silicone sheets have also been used.
3. Fat-graft myringoplasty. It is also used to close small perforations. After local anaesthesia, edges of perforation are freshened with 1 mm stapes hook. The inside of perforation is also scrapped. A small piece of fat harvested from the ear lobule is plugged into the perforation like an hourglass. Over a time, the fat graft adheres and closes the perforation.

This procedure involves puncturing the medial wall of maxillary sinus in the region of inferior meatus and irrigating the sinus.

1. Chronic and subacute maxillary sinusitis with dual pur-
pose of: (i) confirming the diagnosis and (ii) washing out the
pus.
. To collect the specimen of the antral contents for culno
ture and or examination to exclude
sensitivity, cytological early malignancy.

In adults, local anaesthesia is preferred. A pack of 4% lignocaine with adrenaline is kept in inferior meatus for 10-15 min. In children, general anaesthesia is required. Area of middle meatus should be decongested to open the maxillary ostium for easy return of fluid.

Sitting position is preferred in all adults, when using local anaesthesia. When using general anaesthesia, patient is
placed in tonsillectomy position.

The lateral wall of inferior meatus is punctured with Lichtwitz trocar and cannula at a point 1.5-2.0 cm from anterior end of inferior turbinate and near the attachment of concha with lateral wall. Here, the bone is very thin and can be easily pierced. Trocar and cannula are directed towards the homolateral ear. The nasoantral wall pierces with a "crack." Now remove the trocar and advance the cannula till it reaches the opposite antral wall and then withdraw a little. The antrum can now be irrigated with normal saline at 37°C with a 20 mL or Higginson's syringe (Figure 83.1). Syringing is continued till return is clear. After the puncture is over, cannula is removed and a pack kept in the inferior meatus to control bleeding.

1. Thin amber-coloured fluid, flowing from cannula imme-
diately on puncture and containing cholesterol crystals, indicates presence of antral cyst.
2. Blobs of mucopus in washings indicate hyperplastic sinusitis.
3. Presence of foul-smelling pus which easily mixes with irri-
gating fluid indicates suppuration. In such cases, antral
wash may be repeated once or twice a week.

a . Pack is removed after about an hour.
2.
should
be
given
for
5-6
days
in
cases
of
suppuration.
3. Nasal decongestant drops should be used to improve patency of the ostium.
4. Analgesics may be required for headache or postopera-
tive pain.

1. Swelling of cheek. This is due to faulty technique. In this case, cannula lies in the soft tissues over the anterolateral wall of the maxilla and has failed to pierce the nasoantral wall.
2. Orbital injury and cellulites. If trocar and cannula pierces the roof of antrum.
3. Puncture of the posterior antral wall. This would cause swelling in posterior part of cheek.
Note: This operation is now being performed less often. Most surgeons prefer to get a CT to find the middle meatal pathology causing obstruction to sinus ostium and deal with sinus disease and meatal pathology together by functional endoscopic sinus surgery (FESS) at the same time.
4. Bleeding. It occurs due to injury to nasal mucosa.
5. Air embolism. It is rare but may prove fatal. This compli- cation can be prevented by avoiding insufflation of air into the antrum after lavage.
Intranasal inferior meatal antrostomy is a process of making an opening in the nasoantral wall of the inferior meatus by intranasal route. This operation is now rarely required and has been superseded by functional endoscopic sinus surgery.

1.
Irreversible change
in sinus
hypertrophy.
2. Presence of osteitis.
3. Suspicion of malignancy.

Same as in submucous resection (SMR) operation (see Chapter 86).

Inferior turbinate is fractured medially and upwards with a large periosteal elevator. Nasoantral wall of inferior meatus is perforated with a curved haemostat and then this open- ing is enlarged, forwards with Kerrison’s bone forceps or
mucosa, e.g.
polypoidal reverse backbiting forceps and backwards with Luc's or straight through cut forceps. Opening should be 1.5-2 cm in diameter and as close to the floor of nose as possible (Figure 84.1). Intrasinus pus/debris is removed by suction. Packing into the sinus and nose may be required if there is severe bleeding.

Few complications.
- 1. Postoperative bleeding.
2. Injury to nasolacrimal duct.
Note: These days, intranasal antrostomy is performed in the middle meatus. Middle meatal antrostomy is more physiological and is performed with nasal endoscopes and other surgical instruments used in functional endoscopic sinus surgery.
Caldwell-Luc operation is a process of opening the maxillary antrum through canine fossa by sublabial approach and dealing with the pathology inside the antrum. Operation is also called anterior antrostomy as access to maxillary sinus is made through anterior wall of the sinus.

1. Chronic maxillary sinusitis with irreversible changes in the sinus mucosa.
. Removal of foreign bodies or root of a tooth.
. Dental cyst. Ww
Oroantral fistula. WA
. Suspected neoplasm in the antrum and its biopsy.
. Recurrent antrochoanal polyp. . Fracture of maxilla or blow-out fractures of the orbit. DOrID
. As an approach to ethmoids (Horgan's transantral ethmoidectomy).
9. Approach to pterygopalatine fossa for ligation of maxillary artery.
10. Vidian neurectomy.
3. Opening the antrum. Using cutting burr or gouge and hammer, a hole is made in the antrum. Opening is enlarged using Kerrison’s punch.
4. Dealing with pathology. Once maxillary antrum has been
opened,
pathology
is
removed.
Diseased
antral
mucosa
can
be
removed
with
elevators,
curettes
and
forceps.
Cyst,
benign tumour, foreign body or a polyp is removed.
5. Making nasoantral window. A curved haemostat is pushed
into the antrum from the inferior meatus and then this
opening is enlarged with Kerrison’s and side-biting forceps to make a window <1.5 cm in diameter.
6. Packing the antrum. Ribbon gauze, impregnated with liquid
paraffin or Furacin™ (Furacin™ is 0.2% w/w nitrofurazone)
ointment
can
be
packed
in
the
antrum
and
its
end
brought
out
from
the
nasoantral
window
into
the
nose.
Intrasinus
packing
is done if there is severe bleeding. Pack is also kept in the nose.
7. Closure of wound. Sublabial incision is closed with one or two catgut sutures.

General anaesthesia with cuffed endotracheal tube and a pharyngeal pack. Can be done under local anaesthesia.

Reclining with head-end of the table raised. Patient lies supine with face turned slightly to the opposite side.

1. Incision. A horizontal incision with its ends upward is made below the gingivolabial sulcus, from lateral incisor to the second molar (Figure 85.1). It cuts through mucous membrane and
periosteum.
2. Elevation of flap. The mucoperiosteal flap is raised from the canine fossa to the infraorbital nerve avoiding injury to the nerve.

1. Ice packs over the cheek in the first 24 h prevent oedema, haematoma and discomfort to the patient.
2. Packing in the sinus and nose can be removed in 24-48 h.
3. Antibiotics are given for 5-7 days.
4.
Patient
should
avoid
blowing
his
nose
for
2
weeks
to
avoid surgical emphysema.
5. Maxillary sinus is irrigated through the antrostomy with saline. Patient can do it himself with Higginson’s syringe.

1. Postoperative bleeding. This can be controlled by nasal pack.
.
Anaesthesia
of
the
cheek
due
to
stretching
of
infraorbital
nerve. It may last for a few weeks or months.
. Anaesthesia of teeth. . Injury to nasolacrimal duct. . Sublabial fistula. ov oo
. Osteomyelitis of maxilla (rare). D>
no

Management of chronic maxillary sinusitis, removal of
antrochoanal polyp, biopsy of maxillary sinus tumours,
A
Figure operation.
(A)
(B)
position antrostomy.
ligation of maxillary artery for posterior epistaxis and drainage of maxillary sinus in closure of oroantral fistula can all be done through endoscopic surgery. CaldwellLuc operation these days may be indicated in limited situations as (i) initial step in medial maxillectomy, (ii) in management of complex midfacial fractures and to repair orbital floor fractures, (iii) removal of foreign bodies which cannot be removed by endoscopic approach and (iv) in management and staging of carcinoma of the palate.
Note: Ever since the advent of endoscopic sinus surgery, indications for Caldwell—Luc operation have decreased. Functional endoscopic sinus surgery can achieve all that can be done through Caldwell—Luc operation.

1. Deviated nasal septum (DNS) causing symptoms of nasal obstruction and recurrent headaches.
DNS
causing
to
of
paranasal
sinuses
and
middle
ear,
resulting
in
recurrent
sinusitis
and
otitis
media.
Recurrent epistaxis from septal spur. 90
As a part of septorhinoplasty for cosmetic correction of external nasal deformities.
As a preliminary step in hypophysectomy (trans-septal trans-sphenoidal approach) or vidian neurectomy (trans- septal approach). on
nn

1.
Patients
below
17
years
of
age.
In
such
cases,
a
conserva-
tive surgery (septoplasty) should be done.
Acute episode of respiratory infection. no
Bleeding diathesis. ge
Untreated diabetes or hypertension.

Local preferred. in children and apprehensive adults.
anaesthesia is General anaesthesia is used

1. Infiltration of nasal septum. It is done in its subperichon- 2%
drial planes with xylocaine and 1:50,000 adrenaline.
2. Incision. A curvilinear incision with forward convex- ity is made at the mucocutaneous junction on the left side of the septum. It cuts only through the mucosa and perichondrium.
3. Elevation of mucoperichondrial and periosteal flap. Plane of dissection is important. It should be beneath the perichondrium and periosteum (Figure 86.1A).
4. Incision of the cartilage. Cartilage is incised just posterior to the first incision. Avoid cutting the opposite mucoperi- chondrium, otherwise, it will result in perforation.
5. Elevation of opposite mucoperichondrium and perios- teum. With the elevator passed through the cartilage inci- sion, mucoperichondrial and periosteal flap is raised from
the opposite side of the septum (Figure 86.1B).
6. Removal of cartilage and bone. Now working between the two flaps, cartilage and bone are removed. Cartilage can be removed with Ballenger swivel knife and bone with Luc’s forceps. Bony spur or ridge can be removed with gouge and hammer. Preserve a strip of cartilage about 1 cm wide along the dorsal and caudal border of the septum to prevent col- lapse of the bridge of nose or retraction columella (Figure 86.2).
7. Stitching. One or two catgut or silk stitches are applied in the initial mucoperichondrial incision.
8. Packing. Ribbon gauze, smeared with an antibiotic oint-
ment
or
liquid
paraffin,
is
packed
in
each
nasal
cavity
to
pre-
vent
collection
of
blood
between
the
flaps.
Nasal
dressing
is
applied.

1. Patient is placed in semi-sitting position to prevent 00z- ing of blood. Outer nasal dressing is changed if soaked in blood.
2. A soft diet should be taken in the first two postopera-
tive
days
to
minimize
active
mastication
which
causes
bleeding.
Pain, if any, should be controlled with analgesics. G9
Antibiotic cover is given for 5-6 days.
Nasal packs are gently removed after 24 h and thereaf- ter, decongestant nasal drops and steam inhalations are given daily for 5-6 days. ot
6. Silk stitch, if any, is removed on 5th or 6th day.
7. Patient should avoid trauma to the nose for several days.

- 1. Bleeding. It may require repacking, if severe.
2. Septal haematoma. Evacuate the haematoma and give intranasal packing on both sides of septum for equal pressure.
A13
Figure
86.1
Submucous resection
of nasal
septum.
(A)
Incision elevation
of flap
on the
left.
(B)
Elevation of
flap on
the right
and after
incis- ing the septal cartilage. (C) Closing the incision.
3. Septal abscess. This can follow infection of septal haematoma.
4. Perforation. When tears occur on opposing side of mucous membrane.
5. Depression of bridge. Usually occurs in supratip area due to too much removal of cartilage along the dorsal border.
6. Retraction of columella. Often seen when caudal strip of cartilage is not preserved.
7. Persistence of deviation. It usually occurs due to inadequate surgery and may require revision operation.
. Flapping of nasal septum. Rarely seen, when too much of septal framework has been removed. Septum, which now consists of two mucoperichondrial flaps, moves to the right or left with respiration. co
. Toxic shock syndrome. It is rare after septal surgery. It can follow staphylococcal (sometimes streptococcal) infection and is characterized by nausea, vomiting, purulent secretions, hypotension and rash. It should be diagnosed early. It is treated by removal of packing, hydrating the patient, maintaining blood pressure and administering proper antibiotics. Oo

These days SMR operation has been replaced by septoplasty. As much of the cartilage or bone as possible should be retained. Sometimes straight pieces of bone or cartilage can be put back between the mucosal flaps. Only indication for SMR is when cartilage or bone from the septum is required for a graft.
s
Septoplasty is a conservative approach to septal surgery; as much of the septal framework as possible is retained. Mucoperichondrial/ periosteal flap is generally raised only on one side. This operation has almost replaced the SMR operation.

1. Deviated septum causing nasal obstruction on one or both sides.
As a part of septorhinoplasty for cosmetic reasons. ODD
. Recurrent epistaxis usually from the spur.
Sinusitis due to septal deviation. wR
. Septal deviation making contact with lateral nasal wall and causing headaches.
. For approach to middle meatus or frontal recess in endo- a
scopic sinus surgery when deviated septum obstructs the view and access to these areas.
Access to endoscopic dacryocystorhinostomy operation in some cases. N
As an approach to pituitary fossa (trans-septal trans- sphenoidal approach). es
Septal deviation causing sleep apnoea or hypopnoea syndrome. a

1. Acute nasal or sinus infection.
2. Untreated diabetes.
3. Hypertension.
4. Bleeding diathesis.

1. Infiltrate the septum with 1% lignocaine with adrenaline, 1:100,000.
D . Correct the bony septum by removing the deformed
Or . Remove maxillary crest to realign the septal cartilage.
. In cases of deviated septum, make a slightly curvilinear incision, 2-3 mm above the caudal end of septal cartilage on the concave side (Killian’s incision). In case of caudal dislocation, a transfixion or hemitransfixion (Freer’s) incision is made. The latter is septocolumellar incision between caudal end of septal cartilage and columella.
. Raise mucoperichondrial/mucoperiosteal flap on one side only.
. Separate septal cartilage from the vomer and ethmoid
plate
and
raise
mucoperiosteal
flap
on
the
opposite
side
of
septum.
parts. Deformed septal cartilage is corrected by various methods, such as:
(a) Scoring on the concave side (Figure 87.1).
(b) Cross-hatching or morselizing.
(c) Shaving.
(d) Wedge excision.
Further
manipulations
like
realignment
of
nasal
spine,
separation
of
septal
cartilage
from
upper
lateral
carti-
lages, implantation of cartilage strip in the columella or the dorsum of nose may be required.
. Trans-septal sutures are placed to coapt mucoperichon- drial flaps.
. Nasal pack.

1. Septal surgery is a daycare surgery and the patient can go home after he fully recovers from effects of seda- tion with no postoperative nausea or bleeding. Patients with obstructive sleep apnoea should better be observed overnight.
Avoid strenuous exercise as it may cause bleeding.
. Pack, if kept is removed the next day and patient be
instructed
not
to
blow
the
nose
or
sneeze
hard.
Secre-
tions
can
be
drawn
backwards
into
the
throat
by
snorting
rather
than
blowing
the
nose.
. Saline spray or steam inhalation are encouraged after
pack removal.
. Xylo- or oxymetazoline drops are used if nose becomes
stuffy.
. Nasal splints, if used, are removed on fourth to eighth day and gentle suction of nose is done.
. Patient should avoid trauma to nose, wipe the nose gently and in no case push the nose from one side to another.
Figure 87.1 Septal cartilage is straightened by scoring the cartilage on the concave side to remove interlocked cartilage stresses (A), or by shaving the convex side of cartilage (B). Dislocated septal cartilage can be replaced in the maxillary groove or on the anterior nasal spine by excision of the cartilage along the floor of nose and fixing it with a suture (C).

Same as for SMR operation.
1. Bleeding.
2. Septal haematoma and abscess.
3. Septal perforation.
5. Saddle nose deformity.
6. Columellar retraction.
7.
Persistence
of
septal
deviation,
or
external
nasal
deformity.
8. Cerebrospinal fluid rhinorrhoea (rare) occurs if perpen- dicular plate of ethmoid is avulsed.
9. Toxic shock syndrome.
4. Supratip depression.

Like anterior and posterior rhinoscopy, endoscopy of nose and nasopharynx helps in the diagnosis of diseases of nose, paranasal sinuses (PNS) and the nasopharynx. Because of the brighter illumination, magnification and angled view provided by the endoscopes, it is possible to examine all clefts and crevices of the nose and nasopharynx. It is an important part of examination of nose and nasopharynx.

_ . To diagnose any disease of the nose and PNS.
. To diagnose source of bleeding in epistaxis. bo
. To assess response to medical or surgical treatment of the nose and PNS disease. oo
. To take a precise biopsy from nose and nasopharynx. ~

Topical anaesthesia with 4% xylocaine and a vasoconstrictor (oxymetazoline), first as a nasal spray and then nasal packs.

1. 4mm 30° endoscope
2.7 mm 30° endoscope
|
Required when nasal
2.7mm 70° endoscope J passages are narrow
. Freer’s elevator or elevator with a suction channel DO
. Suction tips OOo
. Biopsy forceps B®
. Antifog solution or Savlon™ to prevent fogging of the endoscopic lens.
ou

After nasal packs are removed, endoscopy is performed by three passes:
1. First obtain a general view of the nasal cavity. Look for any septal deviation or spurs and their size, mucous or purulent discharge in the nasal cavity and colour of the nasal mucous membrane.
2. Pass the endoscope along the floor of nose into the naso-
pharynx
and
examine:
(i)
opening
of
tube,
(ii)
walls
of
nasopharynx,
(iii)
upper
surface
of
soft
palate
and
uvula
and
(iv)
opening
of
tube
of
opposite
side.
To
see
these
endoscope
is
rotated.
3. Withdraw the endoscope slightly and examine the mar- gins of choana and posterior ends of turbinates.
4. Withdraw endoscope slowly and at the same time exam- ine inferior meatus for opening of nasolacrimal duct and Hasner’s valve. Slight pressure over the lacrimal sac may express a drop or two of lacrimal fluid through the naso- lacrimal opening.
SECOND PASS (EXAMINATION OF THE SPHENOETHMOIDAL RECESS, SUPERIOR MEATUS AND OPENINGS OF SPHENOID SINUS AND POSTERIOR ETHMOIDAL CELLS) (FIGURE 88.1)
Endoscope is passed medial to middle turbinate to examine posterior part of middle turbinate, sphenoethmoidal
418
recess, superior turbinate and meatus, openings of posterior ethmoid cells (in the superior meatus) and opening of sphenoid sinus in the posterior wall of sphenoethmoidal recess between the nasal septum and superior turbinate.

Endoscope is passed from the front into the middle meatus. Sometimes middle turbinate needs to be displaced medially or 2.7 mm 30° endoscope have to be used. Examine uncinate process, bulla ethmoidalis, hiatus semilunaris, sinus of the turbinate (cavity on lateral side of middle turbinate) and the frontal recess.
Sometimes middle meatus is better entered from behind where the space is wider than from the front and structures are seen from behind forward, e.g. basal lamina, bulla ethmoidalis, hiatus semilunaris, sinus of the turbinate, and uncinate process and the frontal recess.

Occasionally bleeding can occur due to suction or manipulation of instruments. It is usually mild and easily controlled by vasoconstrictor nasal drops.

Endoscopic surgery has made a great contribution towards management of sinus disease. Indications for conventional
operations like those of Caldwell-Luc, frontal sinus opera- tions and external ethmoidectomy have greatly reduced.
Endoscopic surgery is minimally invasive surgery and does not
require skin
incisions or
removal of
intervening bone
to access the disease. In the sinuses, ventilation and drainage
Repair of CSF leak.
. Pituitary surgery.
Optic nerve decompression.
. Orbital decompression for Graves’ disease.
Control of posterior epistaxis (endoscopic clipping of
sphenopalatine artery).
Choanal atresia. so
SNOW
of the
sinuses is
established preserving
the nasal
and sinus
mucosa and
its function
of mucociliary
Advances in endoscopic surgery have been possible due to:
1. Development of better optics.
2. Improved brighter illumination.
3.
Development of microsurgical instruments to work with the endoscopes and precise removal of tissue with sharp cuts without stripping the mucosa.
4. Concomitant developments in imaging techniques like
CT and MRI to precisely define the area of pathology.
5. Introduction of powered instrumentation in the form of soft-tissue shavers also called microdebriders (to remove nasal polyps, soft-tissue masses or mucosa) help reduce bleeding to a great extent while bone-cutting drills help endoscopic surgery of frontal sinus, lacrimal sac, etc. to remove bony obstruction.
6. The latest advancement has been the computer-assisted image-guided navigational surgery in difficult cases or revisional surgery when landmarks are not easy to identify.

OWIHD
1. Chronic bacterial sinusitis unresponsive to adequate medical treatment.
. Recurrent acute bacterial sinusitis.
Polypoid rhinosinusitis (diffuse nasal polyposis) . . Fungal sinusitis with fungal ball or nasal polypi. oo
Antrochoanal polyp.
. Mucocele of frontoethmoid or sphenoid sinus.
Control of epistaxis by endoscopic cautery.
. Removal of foreign body from the nose or sinus.
Endoscopic septoplasty.

1. Removal of benign tumours, e.g. inverted papillomas or angiofibromas.
2. Orbital abscess or cellulitis management.
3. Dacryocystorhinostomy.

. Inexperience and lack of proper instrumentation. ne
Disease inaccessible by endoscopic procedures, e.g. lat-
eral frontal sinus disease and stenosis of internal opening
of frontal sinus.
. Osteomyelitis. O°
Threatened intracranial or intraorbital complication. &

General anaesthesia is preferred by most of the surgeons. Local anaesthesia with iv. sedation can be used in adults when limited work is to be done.

Patient lies flat in supine position with head resting on a ring or head rest. Some also prefer to raise it by 15°.

Two surgical techniques are followed:
- 1. Anterior to posterior (Stammberger's technique). In this
technique surgery proceeds from uncinate process back- ward to sphenoid sinus. Advantage of this technique is to
tailor the extent of surgery to the extent of disease.
. Posterior to anterior (Wigand's technique). Surgery starts no
at the sphenoid sinus and proceeds anteriorly along the base of skull and medial orbital wall. This is mostly done in extensive polyposis or in revisional sinus surgery.

1. Remove the pledgets of cotton kept for nasal decongestion and topical anaesthesia.
Figure 89.1 Endoscopic surgery in progress. Endoscope and other surgical instruments are passed through the nose and surgery performed by looking at the monitor.
Figure 89.2 Sites of injection (indicated by stars) just anterior to uncinate process on the lateral wall of right side of nose.
2. Inspect the nose with 4mm 0° endoscope or do complete nasal endoscopy if not already done.
3. Inject submucosally 1% lignocaine with 1:100,000 adrenaline under endoscopic control (Figure 89.2):
(a) On the lateral wall, near the upper end of middle turbinate.
(b) On the lateral wall, just below the first injection.
(c) On the lateral wall, just above the inferior turbinate.
(d) In the middle turbinate, posterior aspect.
(e) Posterior aspect of nasal septum.
4. Replace cotton pledgets and repeat injections on the opposite side if bilateral functional endoscopic sinus surgery (FESS) is to be done.
Medialize the middle turbinate and identify the uncinate process and bulla ethmoidalis. If middle turbinate is large, partial or total turbinectomy is performed. In case of concha bullosa, lateral lamella is removed. Definitive surgical steps include:
- 1. Uncinectomy. Uncinate process is incised with sickle knife and removed with Blakesley forceps.
2.
Identification and
maxillary ostium.
Maxillary ostium
posterior lies
to lower
ized, it
is enlarged
posteriorly with
a
above the
inferior third
of uncinate
anteriorly turbinate
process.
and
Once with
a
local- backbiting
through cut-straight
forceps forceps.
or

3. Bullectomy. Bulla ethmoidalis is penetrated with curette or Blakesley forceps and removed. Avoid injury to medial orbital wall, skull base or anterior ethmoidal artery.
4. Penetration of basal lamella and removal of posterior ethmoid cells. Basal lamella is the dividing thin bony septum between anterior and posterior ethmoid cells. It is penetrated in the lower and medial part with a small curette and then removed with Blakesley forceps. Posterior ethmoid cells are exenterated. Optic nerve is at risk if Onodi cell is present. Onodi cell is a posterior ethmoid cell which extends into the sphenoid bone lateral and superior to the sphenoid sinus.
5. Clearance of frontal recess and frontal sinusotomy. If frontal sinus is clear on CT scan and patient also does not suffer from frontal headaches, nothing need to be done. In the event of frontal sinus disease, frontal recess is cleared and frontal sinus drainage established. Opening of frontal sinus is situated lateral to attachment of middle turbinate, medial to medial orbital wall, anterior to anterior ethmoidal artery and posterior to agger nasi cell(s). Surgery in the area of frontal recess is challenging as any disrespect to the mucosa in this area would lead to stenosis of frontal sinus opening with mucocele formation or recurrent frontal sinusitis.
6. Sphenoidotomy. This step is done after clearance of posterior ethmoid cells or after frontal sinusotomy. It is omitted if sinus is healthy. In this procedure anterior wall of sphenoid sinus is removed, and pus and inspissated material from within the sinus removed. There are two ways to remove the anterior sinus wall:
(a) By entering the sphenoid sinus anterior and inferior to the ethmoid cavity created by the above steps.
(b) By enlarging the opening of sphenoid sinus with Blakesley forceps or J-curette. Sinus opening is identified after removal of the posterior-inferior portion of superior turbinate near the nasal septum and about 1.0 cm above the upper border of posterior choana.

7. Nasal packs. Finally the nasal packs are applied, if septal surgery has also been done with FESS or to stop any bleeding from the nasal cavity.

It is individualized according to the extent of surgery done.
1. Removal of nasal packs. Nasal packs, if kept, are removed at the time of discharge 24 h after the operation.
2. Antibiotics. An intraoperative intravenous antibiotic (amoxiclav, cephalosporin or quinolone) is administered and then continued for 7-10 days by oral route.
Antihistaminics. For allergic patients. go
Analgesics. For relief of postoperative pain.
Nasal irrigations. Saline irrigations are started after 1 week postoperatively to remove blood clots, crusts and secretions and continued once or twice a day for 1 week. ot
6. Steroid nasal sprays. Required in cases of nasal allergy or those operated for nasal polyps.
7.
Endoscopic removed
toilet.
by lateral
suction to
nose middle
Blood clots,
and forceps
turbinate.
is divided
be crusts
and from
the
Any with
adhesion suction.
Suction lary
sinus with
clearance
Healthy can
debris ethmoid
formation are
area in
mucosa be
a
curved is
a
painful done
the should
from cannula.
process, within
Since topical
not the
maxil- the
endoscopic nasal
anaesthetic with
a
decongestant is
sprayed before
the procedure.
Patient pays weekly visits for inspection of the cavity for 4 weeks and thereafter as required till mucosalization of the cavity is complete.

They are similar to conventional surgery of ethmoid com- plex and can be divided into major and minor. Mostly they
involve orbit
or skull
base, or
are of
general nature
(see
Table 89.1). Many of the complications are preventable by careful surgical technique.

It is direct visualization of larynx and hypopharynx.
the operation table. Neck is flexed on thorax and the head extended on atlanto-occipital joint (barking-dog position) .

1. When indirect laryngoscopy is not possible as in infants and young children, and the symptomatology points to larynx and/or hypopharynx, e.g. hoarseness, dyspnoea, stridor and dysphagia.
2. When indirect laryngoscopy has not been successful,
e.g. due to excessive gag reflex or overhanging epiglottis obscuring a part or complete view of the larynx.
3. To examine hidden areas of:
(a) Hypopharynx. Base of tongue, valleculae and lower part of pyriform fossa.
(b)
Larynx.
Infrahyoid epiglottis,
anterior commissure,
ventricles and subglottic region.
- 4. To find the extent of growth and take a biopsy.

1. Removal of benign lesions of larynx, e.g. papilloma, fibroma, vocal nodule, polyp or cyst.
. Removal of foreign bodies from larynx and hypopharynx. NO
. Dilatation of laryngeal strictures. oo

1.
Diseases or injuries of cervical spine.
2. Moderate or marked respiratory obstruction unless the
airway has been provided by tracheostomy.
3. Recent coronary occlusion or cardiac decompensation.

General anaesthesia is preferred though this procedure can be performed under local anaesthesia. In infants and young children, no anaesthesia may be required if procedure is for diagnostic purpose.

Patient lies supine. Head is elevated by 10-15 cm by plac- ing a pillow under the occiput or by raising head flap of

- 1. A piece of gauze is placed on the upper teeth to protect
them against
trauma.
2. Laryngoscope is lubricated with a little autoclaved liquid paraffin or jelly.
3. Laryngoscope is held by the handle in the left hand. Right hand is used to retract the lips and guide the laryngoscope and to handle suction and instruments.
4. Laryngoscope is introduced by one side of the tongue
which is
pushed to
side till
of tongue
is reached.
It is
then moved
to the
lifted forward to bring the epiglottis in view.
- 5. Laryngoscope is now advanced behind the epiglottis and
lifted forward
without levering
it on
the upper
teeth or
jaw
(Figure
90.1).
This gives
good view
of the
interior of
the larynx.
- 6. If anterior commissure laryngoscope is being used, its tip
can be
advanced further
ventricular bands
to examine
the ventricles
and anterior
commissure.
It
- can be passed between the vocal cords to examine the subglottic region.
7. Following structures are examined serially: Base of tongue, right and left valleculae, epiglottis, (its tip, lingual and laryngeal surfaces), right and left pyriform sinuses,
Figure 90.1
aryepiglottic folds, arytenoids, postcricoid region, both false cords, anterior and posterior commissure, right and left ventricles, right and left vocal cords and subglottic area. Mobility of vocal cords should also be observed.
A right-angled telescope can be used to see the undersur- face of vocal cords and the walls of the subglottis. After the procedure is completed, laryngoscope is withdrawn and lips and teeth examined for
any injury.

1. Patient is kept in coma position to prevent aspiration of blood or secretions.
2. Patient’s respiration should be watched for any laryngeal spasm and cyanosis.
3. Trauma to larynx, especially if repeated attempts at laryngoscopy have been made. It may lead to laryngeal oedema and distress.
respiratory 4. Bleeding may occur from the operative site. Patient may spit blood. Care should be taken to prevent aspiration.

1. Injury to lips and tongue if they are nipped between the teeth and the laryngoscope.
2. Injury to teeth. They may get dislodged and fall into pharynx.
3. Bleeding.
4. Laryngeal oedema.

1. To find out the cause for wheezing, haemoptysis or
unexplained
cough
for
more
than
4
weeks.
2. When X-ray chest shows:
(a) Atelectasis of a segment, lobe or entire lung.
(b) Opacity localized to a segment or lobe of lung.
(c) Obstructive emphysema—to exclude foreign body.
(d) Hilar or mediastinal shadows.
. Vocal cord palsy. go
4. Collection of bronchial secretions for culture and sensi-
tivity tests, acid fast bacilli, fungus and malignant cells.

1. Removal of foreign bodies.
2. Removal of retained secretions or mucus plug in cases of head injuries, chest trauma, thoracic or abdominal sur- comatosed
gery, or patients.

General anaesthesia with no endotracheal tube or with only a small bore catheter is often preferred. It can also be done under surface anaesthesia.
topical

There are two methods to introduce bronchoscope:
1. Direct method. Here bronchoscope is introduced directly the
through glottis. 2. Through laryngoscope. Here glottis is first exposed with the help of a spatular type laryngoscope and then the bronchoscope is introduced through the laryngoscope into the trachea. Laryngoscope is then withdrawn. This method is useful in infants and young children, and in
adults who have short neck and thick tongue.

1. A piece of gauze or a dental guard is placed on the upper teeth for their protection against injury.
2. Proper-sized bronchoscope is lubricated with a swab of autoclaved liquid paraffin or jelly. It is held by the shaft in surgeon’s right hand in a pen-like fashion. Fingers of the left hand are used to retract the upper lip and guide
the bronchoscope.
3. Now looking through the scope, tip of epiglottis is identi-
fied
first
and
the
scope
passed
behind
it
and
the
lifted
forward
to
expose
the
glottis.
Now
bronchoscope
is
rotated
90°
clockwise
so
that
its
bevelled
tip
is
in
the
axis
of
glottis
to
ease
its
entry
into
the
trachea.
Once
trachea
is entered, scope is rotated back to the original position.
4. Bronchoscope is gradually advanced and the entire
tra- cheobronchial tree examined. Axis of bronchoscope should be made to correspond with axes of the trachea and bronchi. To achieve this, head and neck are flexed to the left when examining the right bronchial tree and vice versa.
Openings of all the segmental bronchi in both the
lungs are examined in seriatim.
5. Direct vision, right angled and retrograde telescopes can
be used for magnification and detailed examination.
. Biopsy of the lesion of suspicious area can be taken. ND
. Secretions can be collected for exfoliative cytology, or bacteriologic examination.

1. Keep the patient in humid atmosphere. 2. Watch for respiratory distress. This could be due to laryn- geal spasm or subglottic oedema if the procedure had been unduly prolonged or the bronchoscope introduced repeatedly. Inspiratory stridor and suprasternal retrac- tion will indicate need for tracheostomy.

1. Injury to teeth and lips.
2. Haemorrhage from the biopsy site.
3. Hypoxia and cardiac arrest.

1. Select proper size of bronchoscope according to patient’s age (see Table Al in Appendix II).
2. Do not force bronchoscope through closed glottis.
2. Route
3. Structures seen
4. Bedside exam
5. Ventilation
6. Foreign body removal
7.
Biopsy
8. Jaw and neck abnormalities or
injuries
9. Control of haemorrhage
10. Cost of procedure

Topical
Nasal or through endotracheal tube
e Nasal cavity
e Undistorted view of supraglottic and glottic
areas
e Segmental and even subsegmental bronchi
Possible
Poor control of ventilation especially in children.
Airway is compromised by scope
¢
Difficult
©
Contraindicated in children
Only small piece can be taken
Useful
Difficult

General
Oral only
e
Nose
not
seen
¢
Supraglottic area is difficult to evaluate
e Only segmental bronchi can be reached
Not possible
Better control of ventilation especially in children
°
Easy
¢
Ventilation can be done through scope
¢
Ideal
for
foreign
body
removal
in
children
and
adults
Larger piece can be taken
Technically difficult procedure
¢ Better control
e Useful in massive haemoptysis
¢
Tip of bronchoscope can be used to press the
bleeding area
More, require operation theatre facilities with
costs of operation theatre and anaesthesia
3. Repeated removal and introduction of bronchoscope should be avoided.
4. Procedure should not be prolonged beyond 20 min in
infants and children, otherwise it may cause subglottic
oedema in postoperative period.

These days flexible fibreoptic bronchoscopy has replaced
rigid bronchoscopy for diagnostic procedures particularly
in adults. It provides magnification and better illumina-
tion, and because of the smaller size of scope permits
examination of subsegmental bronchi. It is also easy to use in patients with neck or jaw abnormalities and injuries where rigid bronchoscopy may almost be impossible technically. This procedure can be performed under topical anaesthesia and is very useful for bedside examination of the critically ill patients. The suction/biopsy channel provided in the fibrescope helps to remove secretions, inspissated plugs of mucus or even small foreign bodies. Flexible bronchoscope can also be easily passed through endotracheal tube or the tracheostomy opening. However, it has limited utility in children because of the problems of ventilation. Table 91.1 gives the comparative advantages and disad- vantages of flexible and rigid bronchoscopy.

Oesophagoscopy is of three types:
1. Rigid oesophagoscopy.
2. Flexible fibreoptic oesophagoscopy.
3. Transnasal oesophagoscopy.

Same as for direct laryngoscopy. Patient lies supine, head is elevated by 10-15 cm, neck flexed on chest and head extended at atlanto-occipital joint. The purpose of this posi- tion is to attain the axes of mouth, pharynx and oesophagus in a straight line to pass the rigid tube easily. This position can be achieved with the help of an assistant or a special head rest.

1. To investigate cause for dysphagia, e.g. cancer oesopha- gus, cardiac achalasia, strictures, oesophagitis, divertic- ulae, etc.
. To find cause for retrosternal burning, e.g. reflux oesophagitis or hiatus hernia.
To find cause for haematemesis, e.g. oesophageal varices.
Secondaries neck with unknown primary (as a part of panendoscopy).

1. Removal of a foreign body.
2. Dilatation in case of oesophageal strictures or cardiac achalasia.
3. Endoscopic removal of benign lesions, e.g. fibroma, pap- illoma, cysts, etc.
4. Insertion of Souttar’s or Mousseau-Barbin tube in pallia- tive treatment of oesophageal carcinoma.
5. Injection of oesophageal varices.

1. Trismus—makes the procedure technically difficult.
2. Disease of cervical spine, e.g. cervical trauma, spon- dylosis, tuberculous spine, osteophytes and kyphosis. They make rigid oesophagoscopy technically difficult. Flexible fibreoptic oesophagoscopy is performed in these cases.
. Receding mandible.
Aneurysm of aorta for fear of rupture and fatal haemorrhage.
Advanced heart, liver or kidney disease may be a relative contraindication.

General anaesthesia with orotracheal intubation with tube in the left corner of the mouth. It can be performed under local anaesthesia in selected individuals.

1. A piece of gauze is placed over the upper teeth to protect them or a dental guard.
2. Oesophagoscope is lubricated with a swab of autoclaved liquid paraffin or jelly.
3. The oesophagoscope is held by its proximal end in a pen-
like
fashion
and
into
the
mouth
by
the
right
side
of
the
tongue
and
then
towards
the
middle
of
its
dorsum.
Now there are four basic steps:
1. Identification of arytenoids. Once oesophagoscope has been introduced to the back of tongue, it is advanced gently by the left thumb and index finger. Epiglottis is first seen, then the endotracheal tube and a little further down arytenoids can be identified.
2. Passing the cricopharyngeal sphincter. Keeping the tip of oesophagoscope strictly in the midline, behind the larynx, it is lifted with movements of left thumb to open the hypopharynx. With slow but sustained pressure, the sphincter will open and then the tip of oesophagoscope can be guided easily into the oesophagus. Never apply force to open the sphincter. Sometimes, a fine bougie can be used to find the lumen. An additional dose of muscle relaxant may be required if sphincter does not open. Once oesophagus has been entered, it is easier to advance the scope, provided oesophageal lumen is kept constantly in view.
3. Crossing the aortic arch and left bronchus. In an adult, this natural narrowing lies about 25 cm from the incisors. Aortic pulsation can be seen. When crossing this area, head of the patient is slightly lowered so that oesopha- geal lumen is in line with that of the scope.
4. Passing the cardia. Head and shoulders remain below the level of the table, head being slightly higher than the shoulders and moved slightly to the right. At this stage, the oesophagoscope points to the left anterior-superior iliac spine. Cardia is identified by its redder and more velvety or rugose mucosa.

Never forget to inspect the oesophageal wall again when the is withdrawn.
oesophagoscope

1. Sips of plain water followed by usual diet may be given in an uneventful oesophagoscopy.
2. Patient is watched for pain in the interscapular region,
surgical emphysema of neck and abrupt rise of tempera-
ture. They indicate oesophageal perforation.

1. Injury to lips and teeth.
2. Injury to arytenoids.
3. Injury to pharyngeal mucosa. They are all the result of careless technique and can be avoided.
4. Perforation of oesophagus. Most often it occurs at the
site
of
Killian’s
dehiscence
(near
cricopharyngeal
sphinc-
ter)
when
undue
force
has
been
used
to
pass
the
oesoph-
agoscope.
Surgical
emphysema
develops
within
an
hour
or
so
and
the
patient
complains
of
pain
in
the
interscapu-
lar
region.
This
may
be
by
abscess
in
retro-
space
or
mediastinum.
5. Compression of trachea. Oesophagoscope may press
on
posterior
tracheal
wall,
especially
in
children,
caus-
ing
to
and
cyanosis.
Treatment
is
immediate
withdrawal
of
oesophagoscope.

Its main
advantage over
the rigid
oesophagoscopy is
that is
an outdoor
thesia procedure,
and can
spine does
be used
not require
it general
in or
jaw where
The oesophagus,
ined patients
rigid endoscopy
stomach in
one sitting.
provided anaes-
with abnormalities
is technically
and duodenum
Good of
can all
illumination by
sis of
the taking
the fibrescope
mucosal and
helps in
of ies
or affecting
precision biopsies,
benign tumours,
even injection
(Figure be
exam- magnification
accurate these
removal dilatation
92.1).
In diagno-
sites of
and small
of webs
varices with
cases of
malignant permits
foreign bod-
or strictures
sclerosing and
agents disease,
oesophageal stent
can be
placed as
a
palliative measure.
The procedure is performed under local anaesthesia with or without intravenous sedation. The patient lies in left lateral position and fibrescope is passed through a plastic mouth prop into the pharynx, postcricoid
Figure 92.1 Flexible fibreoptic oesophagoscopy.
area and oesophagus, insufflating air as the endoscope is advanced, to open the lumen of oesophagus. These days flexible fibreoptic oesophagoscopy has practically replaced rigid oesophagoscopy except in some cases of foreign bodies.

In contrast to flexible fibreoptic oesophagoscopy, which is performed through the oral route by gastroenterologists, transnasal oesophagoscopy is performed through nose. This flexible fibrescope has a working channel of 2mm and the air can also be inflated through it to distend the walls of oesophagus to look for any lesion in its mucosal folds. Oesophagus can be examined up to gastric fundus. It is being used:
1. to look for pathology in oesophagus in cases of dysphagia.
2. as a part of panendoscopy in the work-up of a cancer patient to look for a second primary and take a biopsy.
3. to remove foreign bodies from the oesophagus.
4. to perform tracheoesophageal puncture for oesophageal speech in laryngectomized patient.
5. to take a laryngeal biopsy.

1. Recurrent infections of throat. This is the most common
indication.
Recurrent infections are further defined as:
(a) Seven or more episodes in 1 year, or
(b) Five episodes per year for 2 years, or
(c) Three episodes per year for 3 years, or
(d) Two weeks or more of lost school or work in 1 year.
2. Peritonsillar abscess. In children, tonsillectomy is done 4-6 weeks after abscess has been treated. In adults, second attack of peritonsillar abscess forms the absolute indication.
3. Tonsillitis. It causes febrile seizures.
4. Hypertrophy of tonsils. It causes:
(a) airway obstruction (sleep apnoea),
(b) difficulty in deglutition and
(c) interference with speech.
5. Suspicion of malignancy. A unilaterally enlarged tonsil may be a lymphoma in children and an epidermoid car- cinoma in adults. An excisional biopsy is done.

1. Diphtheria carriers, who do not respond to antibiotics.
2. Streptococcal carriers, who may be the source of infec- tion to others.
3. Chronic tonsillitis with bad taste or halitosis which is unresponsive to medical treatment.
4. Recurrent streptococcal tonsillitis in a patient with valvu- lar heart disease.

1. Palatopharyngoplasty which is done for sleep apnoea syndrome.
2.
neurectomy.
Tonsil
is
removed
first
and then IX nerve is severed in the bed of tonsil.
3. Removal of styloid process.

1. Haemoglobin level less than 10 g%.
2. Presence of acute infection in upper respiratory tract, even acute tonsillitis. Bleeding is more in the presence of acute infection.
Children under 3 years of age. They are poor surgical risks. ge
. Overt or submucous cleft palate. UR
Bleeding disorders, e.g. leukaemia, purpura, aplastic
anaemia or haemophilia.
At the time of epidemic of polio. SD
. Uncontrolled systemic disease, e.g. diabetes, cardiac dis-
ease, hypertension or asthma.
. Tonsillectomy is avoided during the period of menses. co

Usually done under general anaesthesia with endotracheal intubation. In adults, it may be done under local anaesthesia.

Rose’s position, i.e. patient lies supine with head extended by placing a pillow under the shoulders. A rubber ring is placed under the head to stabilize it (Figure 93.1). Hyperex- tension should be avoided.
always

1. Boyle—Davis mouth gag is introduced and opened. It is held in place by Draffin’s bipods or a string over a pulley 93.2 and
(Figures 93.3).
. Tonsil is grasped with tonsil-holding forceps and pulled medially. no
. Incision is made in the mucous membrane where it oo
reflects from the tonsil to anterior pillar. It may be
extended
along
the
upper
pole
to
mucous
membrane
between
the
tonsil
and
posterior
pillar.
. A blunt curved scissor may be used to dissect the tonsil »~
from the peritonsillar tissue and separate its upper pole.
. Now the tonsil is held at its upper pole and traction downwards and Dissection is contin- OL
applied medially.
ued
with
tonsillar
dissector
or
scissors
until
lower
pole
is
reached (Figure 93.4).
. Now wire loop of tonsillar snare is threaded over the ton- a
sil
on
to
its
pedicle,
tightened,
and
the
pedicle
cut
and
the
tonsil
removed.
A gauze sponge is placed in the fossa and pressure applied for a few minutes. n
Bleeding points are tied with silk. Procedure is repeated on the other side. ge

(a)
Keep the
patient in
coma position
until fully
recovered from anaesthesia.
(b) Keep a watch on bleeding from the nose and mouth.
(c)
Keep check
on vital
signs, e.g.
pulse, respiration
and blood pressure.
2. Diet. When patient is fully recovered he is permitted to take liquids, e.g. cold milk or ice cream. Sucking of ice cubes gives relief from pain. Diet is gradually built from soft to solid food. They may take custard, jelly, soft boiled eggs or slice of bread soaked in milk on the second day. Plenty of fluids should be encouraged.
3. Oral hygiene. Patient is given Condy's or salt water gargles three to four times a day. A mouth wash with plain water after every feed helps to keep the mouth clean.
4. Analgesics. Pain, locally in the throat and referred to the ear, can be relieved by analgesics like paracetamol. An analgesic can be given half an hour before meals. Avoid aspirin and ibuprofen as it can cause bleeding due to decrease in platelet adhesiveness.
5. Antibiotics. A suitable antibiotic can be given orally or by injection for a week.
Patient is usually sent home 24 h after operation unless there
is some
resume his
normal duties within 2 weeks.
Other methods for tonsillectomy (Table 93.1).
1.
2.
Guillotine method.
Largely abandoned.
It can
only when
tonsils are
scarred by
repeated
Electrocautery.
mobile and
tonsil bed
Both has
been used.
unipolar
It and
reduces blood
bipolar loss
be done
has not
been electrocautery
but causes
thermal injury
to tissues.
Figure 93.1 Rose's position for tonsillectomy. Neck is extended by a sand bag under the shoulders and the head is supported on a ring.
Figure 93.3 Tonsillectomy. Boyle-Davis mouth gag is supported on Draffin's bipods.

Figure 93.2 Set of instruments for tonsillectomy. (1) Knife in kidney tray, (2) and (3) toothed and nontoothed Waugh's forceps, (4) tonsil holding forceps, (5) tonsil dissector and anterior pillar retractor, (6) Luc's forceps, (7) scissor, (8) curved artery forceps, (9) Negus artery forceps, (10) tonsillars nare, (11) Boyle-Davis mouth gag with three sizes of tongue blades, (12) Doyen's mouth gag, (13) adenoid curette, (14) tonsil swabs, (15) nasopharyngeal pack, (16) towel clips.

Misceeronenceneetncetconnca)
© Guillotine method
© Intracapsular (capsule preserving) tonsillectomy with debrider
° Harmonic scalpel (ultrasound)
e Plasma-mediated ablation technique
* Cryosurgical technique

° Electrocautery
° Laser tonsillectomy or tonsillotomy (CO, or KTP)
* Coblation tonsillectomy
e Radiofrequency
3. Laser tonsillectomy. It is indicated in coagulation disor ders. Both KTP-512 and COg lasers have been used but the former is preferred. Technique is similar to the one used in dissection method.
4. Laser tonsillotomy. Another method is laser tonsillotomy, which aims to reduce the size of tonsils. It is indicated in patients who are unable to tolerate general anaesthesia. Tonsils are reduced by laser ablation up to anterior pil- lars in stages repeated by applications.
5.
Intracapsular
tonsillectomy.
With
the
use
of
powered
instruments
(debrider)
tonsil
is
removed
but
its
capsule
is
preserved
in
the
hope
to
reduce
pain.
6.
Harmonic
scalpel.
It
uses
to
cut
and
coagulate
tissues.
It
is
a
cold
method
and
causes
less
tissue
damage
and
less
pain
compared
to
electrocautery
technique.
7. Plasma-mediated ablation technique. In this ablation method, protons are energized to break molecular bonds between tissues. It is a cold method and does not cause thermal injury.
8. Coblation tonsillectomy.
9. Cryosurgical technique. Tonsil is frozen by application of cryoprobe and then allowed to thaw. Two applications, each . : . : . of 3-4 min, are applied. Tonsillar tissue will undergo necro- 5 . . : sis and later fall off leaving a granulating surface. Bleeding . . : is less due to thrombosis of vessels caused by freezing.

- 1. Primary haemorrhage. Occurs at the time of operation.
It can be controlled by pressure, ligation or electrocoagu- lation of the bleeding vessels.
Snare
Figure 93.4 (A) Tonsil being dissected from its bed. (B) The pedicle at the lower pole of tonsil being cut with a snare.
2. Reactionary haemorrhage. Occurs within a period of 24 h and can be controlled by simple measures such as removal of the clot, application of pressure or vasocon- tet . . er strictor. Presence of a clot prevents the clipping action of the superior constrictor muscle on the vessels which pass through it (compare postpartum uterine bleed- ing). If above measures fail, ligation or electrocoagula- tion of the bleeding vessels can be done under general anaesthesia.
3. Injury to tonsillar pillars, uvula, soft palate, tongue or superior constrictor muscle due to bad _ surgical technique.
4. Injury to teeth.
5. Aspiration of blood.
6.
Facial
oedema.
Some
patients
get
oedema
of
the
face
of
the
eyelids.
7. Surgical emphysema. Rarely occurs due to injury to supe-
rior constrictor muscle.

1.
Secondary
haemorrhage.
Usually
seen
between
the
fifth
to
tenth
day.
It
is
the
result
of
sep-
sis
and
premature
of
the
membrane.
Usu-
ally, it is heralded by bloodstained sputum but may be profuse.
Simple measures like removal of clot, topical appli- cation of dilute adrenaline or hydrogen peroxide with pressure usually suffice. For profuse bleeding, general anaesthesia is given and bleeding vessel is electroco- agulated. Ligation of bleeding vessel is difficult due to friable tissue. Sometimes, approximation of pil- lars with mattress sutures may be required. External carotid ligation may also be required if bleeding is uncontrollable.
of
blood
or
plasma,
depending
on
blood
loss, is given. Systemic antibiotics are given for control of infection
2.
Infection.
Infection
of
tonsillar
fossa
may
lead
to
para-
we
.
abscess
or
otitis
media.
se . . 3. Lung complications. Aspiration of blood, mucus or tissue fi . ragments may cause atelectasis or lung abscess.
se . 4. Scarring in soft palate and pillars.
5.
Tonsillar
remnants.
Tonsil
tags
or
tissue,
left
due
to
inad-
equate
surgery,
may
get
repeatedly
infected.
6.
of
lingual
tonsil.
This
is
a
late
and
is
compensatory
to
loss
of
palatine
tonsils.
Sometimes, lymphoid tissue is left in the plica triangu- laris near the lower pole of tonsil, which later gets hyper- trophied. Plica triangularis should therefore be removed
during tonsillectomy.

Adenoidectomy may be indicated alone or in combination with tonsillectomy. In the latter event, adenoids are removed first and the nasopharynx packed before starting tonsillectomy.

1. Adenoid hypertrophy causing snoring, mouth breath- ing, sleep apnoea syndrome or speech abnormalities, i.e. (rhinolalia clausa).
Recurrent rhinosinusitis. yon
. Chronic otitis media with effusion associated with ade- noid hyperplasia.
. Recurrent ear discharge in benign CSOM associated with adenoiditis/adenoid hyperplasia. »
. Dental malocclusion. Adenoidectomy does not correct dental abnormalities but will prevent its recurrence after orthodontic treatment. or

- 1. Cleft palate or submucous palate. Removal of adenoids
causes velopharyngeal insufficiency in such cases.
2. Haemorrhagic diathesis.
3. Acute infection of upper respiratory tract.

as for of neck should
Same tonsillectomy. Hyperextension always be avoided.

1. Boyle-Davis mouth gag is inserted. Before actual removal of adenoids, nasopharynx should always be examined by retracting the soft palate with curved end of the tongue depressor and by digital palpation, to confirm the diag- nosis, to assess the size of adenoids mass and to push the lateral adenoid masses towards the midline. A laryngeal mirror helps to assess the size and extent of adenoid mass.
- 2. Proper size of "adenoid curette with guard" is introduced into the nasopharynx till its free edge touches the posterior border of nasal septum and is then pressed backwards to engage the adenoids. At this level, head should be slightly flexed to avoid injury to the odontoid process.
oo
.
With gentle
sweeping movement,
adenoids are
shaved off
(Figure
94.1), with
smaller behind
Lateral curettes;
are masses
small removed
with tags
punch are
similarly of
removed lymphoid
forceps.
tissue
Take left
care not
to injure
pharyngeal ends
tubes.
- Haemostasis is achieved by packing the area for someal
time.
Persistent bleeders
are electrocoagulated
under
- vision. If bleeding is still not controlled, a postnasal pack is left for 24 h.

These days adenoids can be removed more precisely by using a debrider under endoscopic control.

Same as in tonsillectomy. There is no dysphagia and patient is up and about early.

- 1. Haemorrhage. It usually seen in immediate postopera-
tive period. Nose and mouth may be full of blood or the

gon Injury to eustachian tube opening.
only indication may be vomitus of dark-coloured blood which the patient had been swallowing gradually in the postoperative period. Rising pulse rate is another indi- cator. Treatment is same as for preoperative haemor- rhage. Postnasal pack under general anaesthesia is often required.
Injury to pharyngeal musculature and vertebrae. This is due to hyperextension of neck and undue pres- sure of curette. Care should be taken when operating patients of Down syndrome as 10-20% of them have atlantoaxial instability. An X-ray neck in extension
No Nasopharyngeal stenosis. It occurs due to scarring.
and flexion should be taken to rule out atlantoaxial instability.
Grisel syndrome. Patient complains of neck pain and develops torticollis. Mostly it is due to spasm of paraspi- nal muscles, but can be due to atlantoaxial dislocation requiring cervical collar and even traction.
Velopharyngeal insufficiency. It is necessary to check for submucous cleft palate by inspection and palpation before removal of adenoids.
Recurrence. This is due to regrowth of adenoid tissue left behind.

1.
Law’s view
(Figure
95.1).
Itis lateral
oblique view
of mastoid.
Patient lies in such a way that sagittal plane of the skull is parallel to the film and X-ray beam is projected 15°cephalo-
caudal. Structures seen are:
(a) External auditory canal superimposed on internal auditory canal.
(b) Mastoid air cells.
(c) Tegmen.
(d) Lateral sinus plate.
(e) Temporomandibular joint.
"Key area" of the mastoid, i.e. attic, aditus and antrum are not well seen.
2. Schuller's view. Similar to Law's view but cephalocaudal beam makes an angle of 30° to the sagittal.
Structures seen are:
(a) External canal superimposed on internal canal.
(e) Condyle of mandible.
(f) Sinodural angle.
- (g) Antrum and upper part of attic (i.e. key area).
This view is taken to see the extent of pneumatization, destruction of intercellular septa (as in mastoiditis), loca-
tion of sinus plate and tegmen, cholesteatoma and longitu- dinal fracture of petrous pyramid.
3. Stenver's view. This view is taken in such a way that long axis of the petrous bone lies parallel to the film.
(a) Entire petrous pyramid.
(b) Arcuate eminence.
(c) Internal auditory meatus.
(d) Labyrinth with its vestibule.
(e) Cochlea.
- (f) Mastoid antrum.
4. Towne's view. It is anteroposterior view with 30° tilt from above and in front. It shows both petrous pyramids which
can be
compared.
(a) Arcuate eminence and superior semicircular canal.
(b) Mastoid antrum.
(c) Internal auditory canal.

(d) Tympanic cavity.
(f) External auditory canal.
This view is usually taken for acoustic neuroma and apical petrositis.
5. Transorbital view (Figure 95.2). This view is taken with occiput on the film with orbitomeatal line at right angles to
the film. X-ray beam passes through the orbit.
(a) Internal auditory canal.
(b) Cochlea.
(c) Labyrinth.
(d) Entire petrous pyramid projected through the orbit.
Since both petrous pyramids are seen, structures on one side can be compared with those of the opposite side.
This view is usually done for acoustic neuroma and petrous pyramid.
6. Submentovertical view. This view is taken with vertex near the film and X-ray beam projected at right angles to the film from the submental area.
(a) External auditory canal.
(b)
Middle ear
cleft, ice.
mastoid eustachian tube.
(d) Sphenoid sinus.
(e) Foramen ovale and spinosum.
(f) Carotid canal.

1. Waters' view (Figures 95.3-95.5) (occipitomental view or nose-chin position). It is taken in such a way that nose and
chin of
touch the
film while
X-ray beam
is pro-
jected from behind. Waters' view with open mouth is pre- ferred as it also shows sphenoid sinus. In this view, petrous
bones are projected below the maxillary antra.
(a) Maxillary sinuses (seen best).
(b) Frontal sinuses.
(c) Sphenoid sinus (if the film is taken with open mouth).
(d) Zygoma.
(e) Zygomatic arch.
(f)
Nasal bone.
(g) Frontal process of maxilla.
cells, middle
ear and
(h) Superior orbital fissure.
(i) Intratemporal fossa.
2. Caldwell view (Occipitofrontal view or nose-forehead position). The view is taken with nose and forehead touch- ing the film and X-ray beam is projected 15—20° caudally.
(a) Frontal sinuses (seen best).
(b) Ethmoid sinuses.
(c) Maxillary sinuses.
(d)
Frontal
process
of
zygoma
and
zygomatic
process
of
frontal bone.
(e) Superior margin of orbit and lamina papyracea.
(f) Superior orbital fissure.
(g) Foramen rotundum (inferolateral to superior orbital fissure).
3. Lateral view. Lateral side of the skull lies against the film
and beam is from the other
X-ray projected perpendicular side. Structures seen are:
(a) Anterior and posterior extent of sphenoid, frontal and maxillary sinuses.
(b)
Sella
turcica.
(c) Ethmoid sinuses.
(d) Alveolar process.
(e) Condyle and neck of mandible.
4. Submentovertical (basal) view. This view is taken as
described earlier.

(a)
Sphenoid,
posterior
ethmoid
and
maxillary
sinuses
(seen best in that order).
(b)
Zygoma.
(c) Zygomatic arch.
(d) Mandible along with coronoid and condyloid processes.
5. Right and left oblique views. They are taken to see the
posterior
ethmoid
sinuses
and
the
optic
foramen
of
the
cor
responding side.

1. Lateral view of nasal bones (Figure 95.8). Both right and left lateral views should be taken. Fracture line and depres- sion or elevation of the fractured segment is seen. Lower part of nasal bones is thin and fractured more frequently. Groove for ethmoidal nerve and vessels can be seen running down- wards and forwards and may look like fracture line.
2. Occlusal view of nasal bone. In this the X-ray film is held between the teeth, and central beam is projected perpen- dicular to the film. Both sides of the nasal pyramid are seen. Fracture line and lateral displacement of the nasal pyramid is seen clearly.
3. Waters’ view. It gives end-on view of nasal arch. Fractures of right and left nasal bones and their lateral displacement can be seen.

1. Lateral view of neck. It is an extremely useful view in ENT.
In a normal person, it shows:
(a) Outline of base of tongue.
(b) Vallecula.
(c) Hyoid bone.
(d) Epiglottis and aryepiglottic folds.
(e) Arytenoids.
(f) False and true cords with ventricle in between them.
(g) Thyroid and cricoid cartilages.
(h) Subglottic space and trachea.
(i) Prevertebral soft tissues.
(j) Cervical spine.
(k) Pretracheal soft tissues and thyroid.
This view helps in the diagnosis of:
(a) Foreign bodies of larynx, pharynx and upper oesopha- gus and to differentiate a foreign body of the airway from
that of the food passage.
(b) Acute epiglottitis (Thumb sign).
(c) Retropharyngeal abscess.
(d)
Position
of
tube,
T-tube
or
laryngeal
stent.
(e) Laryngeal stenosis.
(f) Fractures of larynx and hyoid bone and _ their displacement.
(g) Compression of trachea by thyroid or retropharyngeal masses.
(h)
Caries
of
cervical
spine,
with
retropharyn-
geal abscess or osteophytes in cervical vertebrae or injuries of spine.
2. Anteroposterior view of neck (Figure 95.10). This view is useful to differentiate a foreign body of larynx from that of oesophagus. It is also done to see any compression or displacement of trachea by lateral neck masses, e.g. thyroid tumours or enlargement.
3. Soft tissue lateral view of nasopharynx (Figure 95.26). This is usually taken to assess soft tissue masses in the nasopharynx, e.g. adenoids, antrochoanal polyp or angiofibroma.
The structures seen are:

(a) Nasal cavity. A foreign body in the nose can be seen. Interruption of air column from nose to nasopharynx may indicate a tumour or choanal atresia.
(b) Soft palate.
(c)
Roof
and
posterior
wall
of
Adenoid
mass
may
be
seen
arising
from
posterosuperior
wall
of
naso-
pharynx
and
compromising
the
airway.
Antrochoanal polyp may also show a soft tissue density, but usually a column of air is seen between the mass and posterior wall of nasopharynx. This column of air differ- entiates antrochoanal polyp from other nasopharyngeal masses arising from posterosuperior wall of nasopharynx, e.g. angiofibroma.
Figure
95.1
B
Law’s view
(A)
Note normal
structures and
pneumatization on
the right.
(B)
On the
left side
mastoid is
destruction (arrows) of the "key area" due to cholesteatoma.
Figure 95.2 Transorbital view. Note both petrous pyramids are projected through the orbits. Internal auditory meatus and labyrinths are seen on both sides. Internal auditory canals of both sides are normal in shape and dimensions (arrows).
sclerotic with
Figure
95.3
Waters' view with open mouth. Note haziness of left maxillary
sinus due
to mucosal
(arrow).
Other sinuses
appear normal.
Figure 95.5 Waters' view showing a globular cyst in the right (arrow) and mucosal hypertrophy in the left maxillary sinus.
Figure 95.4 Waters' view. Note opacity in right maxillary sinus due to sinusitis (arrow).
Figure 95.6 Lateral view of nasal bones showing fracture.
Figure 95.7 Waters' view showing carcinoma right maxillary sinus. Note: (i) Soft tissue expansion. (ii) Bony destruction of walls of maxillary sinus.
Figure

95.8 Lateral view—nasal bones. Note fracture of nasal bone
(arrow).
Figure 95.9
Occlusal view showing a stone in right submandibular duct.
Figure 95.10 PA view. A radio-opaque foreign body (coin) in the oesophagus at the level of thoracic inlet.
Figure 95.11 Lateral view neck showing a foreign body (coin) in oesophagus. Note the end-on position of coin in this position.
Figure 95.12
PA view—neck and chest showing a ring at the level of thoracic inlet.
Figure 95.13 (A) PA view—neck and chest with denture at the level of thoracic inlet. Denture is radiolucent but two metallic hooks are visible (arrow). (B) Lateral view—neck of the same patient showing metallic hooks at thoracic inlet (arrow).
Figure 95.14
Open safety pin in the oesophagus. Note its point is directed downwards.
Figure 95.15 X-ray lateral view neck showing a chicken bone in the oesophagus opposite C7 and Cg (arrows).
Figure 95.16 PA view chest. A radio-opaque foreign body (nail) in the right bronchus.
Figure
95.18
PA
chest in
a
child with
a
radiolucent foreign
body
(a peanut)
in right
bronchus.
Note:
(i)
Collapse of
right lung
and shift
of mediastinum to the same side. (ii) Raised dome of diaphragm on the
right. (iii) Emphysema left lung.
Figure
95.17
Soft tissue
lateral view
neck showing
a
fruit
(Chiku)
in the
subglottic region
of larynx
(arrow).
Note:
The seed
patient needed tracheostomy (black arrow) to relieve obstruction.
Figure 95.19 PA view chest showing a hair pin in the right bronchus.

Figure 95.20 Soft tissue lateral view neck showing retropharyngeal and retro-oesophageal abscess due to a fish bone in a 25-year-old female. Note: (i) Increased prevertebral soft tissue shadow (maximum against C7). (ii) Compression of trachea compromising the airway. (iii) Straightening of cervical spine. (iv) Vertebral bodies and intervertebral spaces are normal.
Figure 95.22 Radiograph (lateral view neck) showing retropharyngeal abscess. Note increase in width of prevertebral soft tissues opposite Cg and also gas shadow due to gas-producing organisms. To diagnose retropharyngeal abscess, the prevertebral soft tissue shadow should exceed width of body of Cy» (or should be more than 7 mm) in both children and adults opposite C2, Opposite Cg, the soft tissue shadow should exceed 14mm in children less than 15 years and
22
mm in
adults.
Figure 95.21 Hypodermic injection needle in the trachea in an adult. Sudden gasp due to a pat on the back from an unnoticed friend when he was busy picking his teeth.
Figure 95.23 Xeroradiograph (lateral view neck). Note laryngeal stenosis involving supraglottic, glottic and subglottic regions.
Figure
95.24
Soft tissue radiograph (lateral neck) showing tracheal compression due to thyroid mass.
Figure
95.26
Soft tissue lateral view—nasopharynx. Note a large adenoid mass almost completely obstructing the nasopharyngeal air-
way (arrowheads).
Figure
95.25
Lateral view neck showing calcification in the thyroid gland.
Figure
95.27
Barium swallow
in
a
patient with
Note irregular narrowing of lower oesophagus due to carcinoma.
Figure 95.28 Cardiac achalasia (megaoesophagus). There is a failure of lower oesophageal sphincter to relax with dilatation of oesophagus due to stasis of food. Achalasia is due to degeneration of ganglion cells of Auerbach's plexus.

Appendix |
Some Memorable
Nuggets for Rapid
Review
Appendix II
Instruments

. Acute epiglottitis in children is caused by Haemophilus influenzae type B. It produces a typical “Thumb sign” on lateral X-ray film. Ampicillin was considered the drug of choice but now many organisms have become resis- tant to it and ceftriaxone is preferred.
. Acute fulminant fungal sinusitis is an invasive sinusitis and is commonly seen in diabetics, HIV infected patients and transplant patients receiving chemotherapy for immu- nosuppression. Therapy in such cases should be urgent, aggressive surgical debridement and amphotericin-B.
. Acute laryngotracheobronchitis or croup is a viral infec- tion caused by parainfluenzae type 1, 2, and sometimes 3. Critical area involved is subglottic larynx producing oedema with stridor and respiratory distress. X-ray (PA view) larynx shows typical “steeple sign” but X-rays are avoided as any manipulation may precipitate acute obstruction.
. Adenocarcinoma of ethmoid is mostly seen in those exposed to wood dust.
. Adenoid facies, seen in adenoid hyperplasia, consists of crowded teeth, high-arched palate and underdevel- oped pinched nostrils.
. Ammonia is not used to test sense of smell as it stimu- lates fibres of trigeminal nerve supplying the nose and not the olfactory ones.
. Angio-oedema. Deficiency of C, esterase inhibitor causes angio-oedema. Deficiency of this inhibitor causes increased levels of C; esterase. This leads to anaphylatoxins which cause capillary permeability and oedema. Deficiency of C, esterase inhibitor is an inher- ited condition.
. Area of adult tympanic membrane is 90 mm?, of which only 55 mm? is functional. Area of stapes footplate is 3.2 mm?. Area ratio (or hydraulic ratio) is 17:1. Accord- ing to other workers, functional area is 45 mm? and area ratio 14:1.

. Arnold nerve. It is auricular branch of jugular gan- glion of vagus. It enters the mastoid canaliculus and exits the tympanomastoid suture and supplies skin of posterior part of pinna on medial surface of concha, and posterior wall of external auditory canal and tym- panic membrane.
"Note: For details and better understanding, the reader is advised to refer the relevant sections of the book.
10. Arnold—Chiari malformation. Cerebellar tonsils proj- ect through the foramen magnum.
ll.
12. Barrett’s oesophagus. In the distal oesophagus, squa- mous epithelium is replaced by columnar epithelium (similar to the one that lines the stomach). It is due to gastroesophageal reflux disease. Carcinoma can develop in 2-5% of patients with Barrett's oesophagus.
13. Battle's sign is ecchymosis over the mastoid seen in fractures of temporal bone.
14. Bell's phenomenon is seen in lower motor neuron paralysis of CN VII. The eyeball turns up and out when trying to close the eye.
15. Bezold abscess. It is an abscess under the mastoid and/ or in the digastric triangle when empyema of mastoid bursts through its medial side of the tip.
16. Bill's island. It is a thin plate of bone left on sigmoid sinus when it is to be retracted during surgery. It should be differentiated from Bill's bar.
17. Blue drum is seen in haemotympanum (due to tem- poral bone fracture), glue ear, glomus tumour or hae- mangioma of middle ear.
18. Boerhaave syndrome. It is spontaneous rupture of oesophagus due to severe vomiting.
19. Broyles' ligament. Small ligament which connects both the vocal cords at the anterior commissure to the thy- roid cartilage.
20. Bryce's sign. Seen in laryngocele. When the swelling is pressed, a gurgling sound is produced.

21. Carcinoma of nasopharynx is caused, among other factors, by Epstein-Barr virus. Most common site of origin is fossa of Rosenmiller (pharyngeal recess). Most common histological variety is squamous cell car- cinoma and radiotherapy is the treatment of choice.
22. Carhart's notch is seen in otosclerosis. Bone conduc- tion curve shows maximum loss at 2000 Hz.
23. Caroticotympanic artery is a branch of internal carotid artery. It anastomoses with branches of external carotid system in the middle ear.
24. CHARGE syndrome consists of Coloboma, Heart defects, choanal Atresia, Retarded growth, Genital hypoplasia and Ear anomalies.
Axis
of
ossicular
rotation
passes
between
anterior
pro-
cess
of
malleus
to
short
process
of
incus.

petrositis-a complication of otitis media.. 49., 1 = Grisel syndrome. It is atlanto-axial dislocation. Can follow adenoid surgery or nasopharyngeal infections. Manifests with painful neck and torticollis.. 50., 1 = Griesinger's sign is oedema over the mastoid and is seen in lateral sinus thrombosis. It is due to thrombo- sis of mastoid emissary vein impeding venous drainage and thus causing oedema over the mastoid.. 51., 1 = Gutmann's pressure test. It is done in puberphonia. Pressing on the thyroid prominence in a backward and downward direction relaxes the overstretched vocal cords and thus low-pitched voice can be produced.. 52., 1 = Habenula perforata. It is the area where branches of cochlear nerve enter the cochlea. Openings may be wide leading to a perilymph gusher in stapes surgery. This condition is associated with enlarged internal acoustic meatus and stapes fixation of congenital ori- gin. It is an X-linked disease and can be diagnosed on CT.
53. Hennebert's phenomenon. Dysequilibrium following nose blowing or lifting a heavy object. Seen in peri- lymph fistula (do not confuse with Hennebert's sign).
54. Hennebert's sign is a positive fistula sign in the absence of fistula. Seen in congenital syphilis due to formation of adhesions between stapes footplate and saccule. Also seen in some cases of Méniére’s disease.
55. Hidden areas of the larynx include infrahyoid epiglot- tis, anterior commissure, subglottis, ventricle and apex of pyriform fossa.
56. Horner syndrome consists of ptosis, miosis (constric- tion of pupil), anhidrosis and enophthalmos due to paralysis of cervical sympathetics.

57. Hyrtl's fissure, also called tympanomeningeal hiatus, it is an embryonic remnant that connects CSF space to middle ear just anterior and inferior to the round win- dow. It runs parallel to cochlear aqueduct. It can be a source of congenital CSF otorrhoea or meningitis from middle ear infections. Normally it gets obliterated.
58. In any case of unilateral otitis media with effusion in an adult, rule out nasopharyngeal pathology especially the carcinoma.
60. Inverted papilloma or Ringertz tumour arises from the lateral wall of nose. It is characterized by squamous or transitional cell epithelium with fibrovascular stroma. Inward growth of epithelium towards stroma lends the name of inverted papilloma to it. It is associated with squamous cell carcinoma in 10-15% of patients.
61. Jacobson's nerve (Syn. tympanic branch of glosso- pharyngeal nerve). Forms tympanic plexus, supplies mucosa of the middle ear cleft. Its preganglionic para- sympathetic fibres leave the middle ear as lesser petro- sal nerve and supply the parotid gland. Section of this nerve is sometimes done to treat Frey’s syndrome.
62. Jugular foramen syndrome is paralysis of CN IX, X and XI. It is seen in carcinoma nasopharynx, glomus jugu- lare, large acoustic neuroma or thrombophlebitis of jugular bulb.
63. Kallmann syndrome is hypogonadism. anosmia and congenital
64. Kartagener syndrome consists of recurrent sinusitis, bronchiectasis and situs inversus. Ciliary motility is dis- turbed. Electron microscope shows absence of dye in side arms in A-tubules of nasal respiratory mucosa.
65. Korner's septum, sometimes seen during mastoid sur- gery, is a bony plate separating superficial squamous cells from the deeper petrosal air cells. Antrum lies deep to it.
66. Krause's nodes are lymph nodes situated in the jugular foramen. Enlargement of these nodes compresses on CN IX, X and XI causing jugular foramen syndrome.

. In Fitzgerald—Hallpike (bithermal caloric) test, ther- mal stimulation occurs in the horizontal semicircu- lar canal. Cold water (30°C) causes nystagmus to the opposite side while warm water (44°C) to the same side. Remember COWS (Cold-Opposite-Warm-Same) .
67 . Larynx has three important spaces: pre-epiglottic, para- glottic and Reinke's. The first two are important because they are invaded by carcinoma arising in the laryngeal mucosa. Reinke's space is often affected by oedema and causes polypoid degeneration of vocal cords.
68. Lermoyez syndrome is a variant of Méniére’s disease. Patient first gets hearing loss and tinnitus. An attack of vertigo follows and relieves tinnitus and improves hearing.
69. Lever ratio between the handle of malleus and the long process of incus is 1.3:1.
70. Lhermitte's sign. A rare sign seen after radiation of cervical spine. Electrical current-like sensation is felt in both arms, dorsal spine and both legs on flexing the neck.
71. Lyre's sign. It is splaying apart of internal and external carotid arteries on angiogram in cases of carotid body tumour of the neck.
72. Malleus and incus are derived from the first arch. Sta- pes develops from second arch except its footplate and annular ligament which are derived from the otic capsule.
73. Marcus Gunn pupil. This is due to interruption of afferent papillary pathway due to retrobulbar neuri- tis or any other optic nerve disease. When light is put on the diseased side, the pupils of both sides remain dilated but when the light is put on the healthy side it constricts both the pupils. In the latter case pupil on diseased side constricts due to consensual light reflex because the efferent pathway on the diseased side is normal. It is an important sign during endoscopic sinus surgery for any injury to the optic nerve.
74. Marcus Gunn syndrome. Opening of mouth causes opening of eyes and closing of mouth causes ptosis. This is due to associated movements following facial paralysis and faulty regeneration of facial nerve fibres.

75. Mastoid antrum lies 12-15 mm deep from the surface of suprameatal triangle in an adult. The thickness of the bone overlying the antrum is only 2 mm at birth and then increases at the rate of 1 mm every year.
76. Mastoid tip does not develop till 2 years; hence postau- ral incision to open the mastoid before this age needs to be modified to avoid injury to the facial nerve.
77. Michel aplasia. There is total lack of development of inner ear. It can be confused radiologically with laby- rinthine ossification and can be distinguished by MRI which may show membranous labyrinth in the latter.
78. Modiolus is the central bony axis of cochlea and mea- sures 5 mm in length.
79. Most common organism responsible for acute bacte- rial sinusitis is Streptococcus pneumoniae followed by Hae- mophilus influenzae. Anaerobic and mixed infections are seen in sinusitis of dental origin.
80. Most common organisms in acute otitis media are S. pneumoniae, H. influenzae and Moraxella catarrhalis in decreasing order.

111. Russell bodies. Seen in rhinoscleroma. Plasma cells are seen to contain rounded eosinophilic structures on histopathology.
112. Samter's triad consists of nasal polypi, bronchial asthma and aspirin sensitivity.
113. Schaumann's bodies. Seen in sarcoid granuloma.
114. Schneiderian membrane (mucosa). It is another name for respiratory mucosa of nose and consists of pseudostratified ciliated columnar cells.
115. Schwartz sign is a pink reflex, seen through intact tympanic membrane, in the area of oval window. It indicates active otosclerosis
usually during pregnancy.
116. Sinodural angle, also called Citelli's angle, is situated between the sigmoid sinus and middle fossa dura plate.
117. Sipple's syndrome. Consists of medullary carcinoma of thyroid, pheochromocytoma and parathyroid hyperplasia or adenoma.
118. Sluder's neuralgia. It is characterized by neuralgic pain in lower half of face with nasal congestion, rhinorrhoea and increased lacrimation. It is due to neuralgia of sphenopalatine ganglion.
119. Solid angle is the area where three bony semicircular canals meet.
120. Structures of ear fully formed by birth are middle ear, malleus, incus, stapes, labyrinth and the cochlea.
121. Tone decay, also called auditory fatigue, is change in auditory threshold when a continuous tone is presented to the ear. It is seen in acoustic neuroma and other retrocochlear lesions.
122. Trautmann's triangle is bounded by the bony labyrinth anteriorly, sigmoid sinus posteriorly and the dura or superior petrosal sinus superiorly (Figure 76.4).
123. Treatment of choice for antrochoanal polyp in a child is functional endoscopic sinus surgery (FESS) or intranasal polypectomy. Caldwell—Luc operation is avoided.
124. Trotter (or Sinus of Morgagni) syndrome or triad is seen in nasopharyngeal carcinoma which spreads laterally to involve the sinus of Morgagni involving mandibular nerve. It is characterized by:

(a) Conductive hearing loss (due to eustachian tube obstruction).
(b) Ipsilateral immobility of soft palate.
(c) Neuralgic pain in the distribution of V3.
Trismus and preauricular fullness may be associated with the above.

125 . Tullio phenomenon. Loud sound produces vertigo. It is seen in congenital syphilis or when three functioning windows are present in the ear, e.g. fistula of semicircular canal or fenestration operation in the presence of mobile footplate of stapes.
126. Turban epiglottis is due to oedema and infiltration of the epiglottis and is caused by laryngeal tuberculosis. Lupus—a form of tuberculosis, on the other hand, eats away and destroys the epiglottis.
127. Tympanic membrane develops from all the three germinal layers: ectoderm (outer epithelial layer) mesoderm (middle fibrous layer) and endoderm (inner mucosal layer).
128. Ventricle of Morgagni (Syn. laryngeal ventricle between true and false cords).
129. Vertical and anteroposterior dimensions of middle ear are 15 mm each while transverse dimension is 2 mm at mesotympanum, 6 mm above at the epitympanum and 4 mm below in the hypotympanum. Thus, middle ear is the narrowest between the umbo and promontory.
130. Vocal nodules occur at the junction of anterior with middle third of vocal cords as this is the maximum vibratory area during speech.
131. Volume of middle ear varies between 0.5 and 1.0 mL. It is important when giving intratympanic injections, e.g. gentamicin in Méniére's disease and steroids in sudden deafness.
132. Vomeronasal organ. It is a vestigial structure earlier related to smell. It can sometimes be visualized as a pit on the anteroinferior part of nasal septum.
133. Wallenberg syndrome (posterior inferior cerebellar artery syndrome) or lateral medullary syndrome is due to thrombosis of posterior inferior cerebellar artery causing ischaemia of lateral part of medulla. It is characterized by:
(a) Vertigo, nausea and vomiting
(b)
(c)
Dysphagia
(d)
Dysphonia
- (e) Ataxia with tendency to fall to the involved side
(f)
Loss of
pain and

temperature sensation
on same
side of
face and
contralateral side
of limbs.
134. Woodruff's plexus. It is a plexus of veins situated inferior to posterior end of inferior turbinate. It is a site of posterior epistaxis in adults.
135. Wrisberg's cartilage. Another name for cuneiform cartilage situated in aryepiglottic fold. It is fibroelastic cartilage and does not undergo calcification.
This section intends to introduce only the commonly used instruments in operative surgery and is by no means an exhaustive list. Other instruments used for routine diagnostic ENT examination are dealt with on p. 374.

Myringotome. Used for myringotomy.
Mollison's mastoid retractor. Used in mastoidectomy to retract soft tissues after incision and elevation of flaps. It is selfretaining and haemostatic.
Jansen's self-retaining mastoid retractor. Used in mastoidectomy similar to Mollison's retractor.
Lempert's endaural retractor. Used for endaural approach to ear surgery. It has two lateral blades which retract the flaps and a third central blade with holes. The central blade retracts the temporalis muscle. The central blade can be fixed to the body of the retractor by its hole.
Lempert's endaural speculum. It is like Vienna model nasal speculum but curved. It is used to spread open the meatus when giving local injection or making an endaural incision.
Lempert's curette (scoop). Used for removal of bony septa and granulations in mastoid surgery.
MacEwen's curette and cell seeker. Used in mastoid surgery to explore the air cells with one end and to curette the intervening septa and granulations with the other.
Farabeuf periosteal elevator. Used for elevation of periosteum from the mastoid cortex in mastoidectomy.

Lichtwitz trocar and cannula. Used for proof puncture (antral lavage). Puncture is done in the inferior meatus as this site is easily accessible and safe.
Tilley's harpoon. Used for intranasal antrostomy in the inferior meatus. Its advantage lies in the removal of the bony chips when the instrument is withdrawn so that they do not fall in the sinus cavity. It is rarely used these days.
Tilley's antral burr. Used to enlarge and smoothen the hole made by harpoon in intranasal inferior meatal antrostomy. No longer used now.
Rose's sinus douching cannula. Used in irrigation of maxillary sinus, which already has a nasoantral window due to intranasal antrostomy or Caldwell—Luc operation. Direction of the tip is indicated by the hook outside.
Luc's forceps. Used in Caldwell—Luc operation (to remove mucosa), submucosal resection (SMR) operation (to remove bone or cartilage), polypectomy (to grasp and avulse polyp) and to take biopsy from the nose or throat.
Nasal snare (Krause's). Used for removal of nasal polypi. Polyp is engaged in the wire loop and avulsed. Its use has greatly declined now. Wire used in this snare is 30 SWG.
St. Clair Thomson's nasal speculum. It has long blades which are concave from inside. Used in nasal surgery, e.g. SMR operation or septoplasty.
Killian's long-bladed nasal speculum. Used in SMR or septoplasty operation to keep mucoperiosteal flaps away.
Tilley's dressing forceps. Used for nasal packing, ear dressing, removal of foreign bodies from the nose. It has a box joint.
Hartmann's dressing forceps. Similar to above forceps. It has a screw joint. The jaw is serrated and grooved.
Wilde's dressing forceps. Used for packing the nasal cavity or ear canal. It acts on spring action.

Ballenger swivel knife. Used in removal of septal cartilage in SMR operation. The blade of knife revolves automatically and changes direction when cutting the cartilage anteroposteriorly downwards, and posteroanteriorly. Different sizes of blades are also shown. Can be used to harvest septal cartilage as a graft for reconstruction.
Killian's nasal gouge (bayonet-shaped). Used for removal of septal spurs or bony crests and ridges in SMR operation.
A
Freer's double-ended elevator. Used for elevation of mucoperichondrium or periosteum in SMR or septoplasty operation.
C
Walsham's forceps. Used for disimpacting and reducing fractures of nasal bone.
Asch's septum forceps. Used for reducing fractures of nasal septum.

Boyle-Davis mouth gag. Used for opening the mouth and retracting the tongue anteriorly. Tongue blades of various sizes can be interchanged according to the age of the patient. It is used for various operations on the oral cavity (palate surgery), oropharynx (tonsillectomy, surgery of soft palate, pharyngoplasty), and nasopharynx (adenoidectomy, excision of angiofibroma).
Doyen's mouth gag. Used to keep the mouth open for intraoral surgery when retraction of the tongue is not required or desirable. Mostly used for tongue surgery. It is applied on one side of the mouth on molar teeth.
Jenning's mouth gag. Use is similar to the one above. It is applied in the centre of the mouth.
Draffin's bipod. Each pod has four rings. They can be assembled to vary the height at which the tongue blade of the BoyleDavis mouth gag can be suspended. The lower end of each pod can be placed in one of the several depressions in Magauran's plate (vide infra).
Tonsil holding forceps (Denis Browne's). Used for holding the tonsil during tonsillectomy by dissection method.
Tonsil dissection forceps with teeth (Waugh's). For incision in mucous membrane and dissection of tonsil.
Tonsil dissector and anterior pillar retractor. One end is used to dissect the tonsil and the other to retract the anterior pillar to the fossa for inspect any bleeding point.
Tonsil artery forceps (straight and curved). Straight forceps is used to catch the bleeding point and curved one is used as replacement forceps before tying with a ligature.
Negus artery forceps. Its tip is sharply curved. The forceps is used as replacement forceps to ligate the bleeding point.
Eves' tonsil snare. Used for tonsillectomy. After the tonsil has been dissected till its lower pole, snare is passed round the tonsil to engage the pedicle and then firmly closed. It crushes and cuts the pedicle thereby minimizing bleeding. Wire used in snare is number 25 SWG.

Peritonsillar abscess forceps. Used for drainage of peritonsillar abscess. Not used these days; instead a number 11 blade covered with micropore plaster except its tip and distal part is used.
St. Clair Thompson's adenoid curette with guard. Used in adenoidectomy. Curette shaves off the adenoid mass while the guard holds the tissue and prevents its slipping.
Leighton tonsillotome (Guillotine)

Tracheal hook (blunt and sharp). Blunt tracheal hook (A) is used to retract thyroid isthmus upwards or downwards to expose the trachea. Sharp tracheal hook (B) is applied to lower border of cricoid cartilage to stabilize the trachea and prevents its movements during respiration when making incision in the tracheal wall.
Tracheal dilator. Used to keep open the tracheal edges after incision in the trachea so that tracheostomy tube can be easily inserted. A curved artery forceps can be easily used in place of a tracheal dilator.
Laryngoscope. Used for direct laryngoscopy (diagnostic or therapeutic). It has a single or a twin light carrier which can be connected to a cold-light source through a flexible cable. Older models have a small electric bulb which works on batteries or a transformer. There are several models of laryngoscope. The size of laryngoscope used will vary with the age of the patient.

. Laryngoscope
. Chest support OON
. Suction tip and other instruments
. Fibreoptic cord
. Various types of microlaryngeal instruments ao
. Specimen bottle
Oesophagoscope. Used for diagnostic or therapeutic oesophagoscopy. Length of oesophagoscope and its lumen vary with the age of the patient. Mechanism of illumination is similar to that of laryngoscope. Handle, at the proximal end of oesophagoscope, indicates the direction of the bevel at the distal end. Illustrated below is Negus oesophagoscope with twin proximal lighting.
Bronchoscope. Similar to oesophagoscope but has openings at the distal part of the tube which help in ventilation of contralateral lung or side bronchi. Its distal tip is also bevelled to facilitate its passage between the cords. Size of bronchoscope will vary with the age of the patient. For indications and technique of bronchoscopy see p. 424. Size of bronchoscope for different age groups is given in Table Al.

Preterm-1 month, Inner diameter of tracheostomy tube (mm) = 2.5-3.0. Preterm-1 month, Size of bronchoscope tube (mm) = 2.5. 1-6 months, Inner diameter of tracheostomy tube (mm) = 3.5. 1-6 months, Size of bronchoscope tube (mm) = 3.0. 6-18 months, Inner diameter of tracheostomy tube (mm) = 4. 6-18 months, Size of bronchoscope tube (mm) = 3.5. 18 months to 3 years, Inner diameter of tracheostomy tube (mm) = 4.5. 18 months to 3 years, Size of bronchoscope tube (mm) = 4.0. 3-6 years, Inner diameter of tracheostomy tube (mm) = 5. 3-6 years, Size of bronchoscope tube (mm) = 4.5. 6-9 years, Inner diameter of tracheostomy tube (mm) = 5.5. 6-9 years, Size of bronchoscope tube (mm) = 5.0. 9-12 years, Inner diameter of tracheostomy tube (mm) = 6.0. 9-12 years, Size of bronchoscope tube (mm) = 6.0. 12-14 years, Inner diameter of tracheostomy tube (mm) = 7.0. 12-14 years, Size of bronchoscope tube (mm) = 6.0
Smallest flexible fibreoptic bronchoscope is 2 mm diameter. It can be easily passed through endotracheal tube of 3-3.5 mm inner diameter.
Ventilation bronchoscope. Various sizes are available for infants, children and adults.
Proximal end of bronchoscope consists of the following:
. Prism light deflector where fibreoptic cord is connected. . Main wP
portal of
the tube
instruments like
section tube
or forceps
for biopsy
or forceps
removal or a telescope can be passed.

. For introducing flexible instruments like fine suction tube or flexible forceps or for jet ventilation. SO
. For connecting to any type of respirator for ventilation or assisted respiration, any of the openings B, C or D can be closed with a suitable plug.

Paediatric ventilation bronchoscope sizes
© 20cm x 2.5mm
° 20 cm x 3.0 mm
20 cm x 3.5 mm
30 cm x 3.5 mm
30 cm x 4.0 mm
30 cm x 5.0 mm
¢
30cm
x
6.0
mm
Adult ventilation bronchoscope sizes
¢ 43cm x 6.5 mm
° 43cm x 7.5mm
° 43cm x 8.5 mm
Tracheostomy tubes for adults. Various types available are:
1. Uncuffed and cuffed tubes.
2. Double cuff tube. Each cuff can be inflated alternately to prevent pressure necrosis at one site.
for foreign
body
Fenestrated tube. Single or multiple holes are situated at the upper curvature. The hole(s) help in speech production or in weaning from tracheostomy. Fenestrated tube is used in children for decannulation.
Adjustable flange long tube. Extra length tracheostomy tubes are used when pretracheal tissues are thick or swollen or to by-pass a growth or stenosis in trachea. Flange in these cases can be adjusted.
Single lumen tube. There is no inner cannula
Double lumen tube. They have an inner cannula inside an outer cannula. It is easier to remove, clean and replace the inner cannula, keeping outer cannula in place for breathing.
Suction-aid tracheostomy tubes. They have a small tube ending above the cuff to suck out pharyngeal secretion and prevent their aspiration.
Tracheostomy with speaking valve. A valve is fitted at the outer end of tracheostomy tube. It allows ingress of air when breathing in but closes when breathing out. In the latter situation air finds its way to vocal cords to produce sound. It is used in long-term treatment of bilateral abductor paralysis or laryngeal stenosis. Digital closure of tracheostomy tube to speak is thus avoided.
Classification of tubes according to the material they are made of. A tracheostomy tube may be made of:

GON Silver. An alloy of silver, copper and phosphorus, e.g. Fuller, Negus or Jackson's tube. PVC (polyvinyl chloride). They are disposable, single use tubes and thermolabile, and thus adjust to tracheal lumen. Silicone. Bacteria and secretions do not adhere to the tube and there is minimum of crusting.
~A Siliconized PVC. It has the properties of both PVC and silicon, i.e. it is thermolabile and adjusts to tracheal wall while silicon prevents crusting.
5. Silastic. It is soft and nonirritating, and minimizes crusting.
6. Armoured tubes. They are plastic tubes reinforced by a spiral or rings of stainless steel. They are not easily kinked.
Fuller's tracheostomy tube. It consists of an outer tube and an inner tube, the latter being slightly longer. Outer tube is made of two blades, which when pressed together, can be easily introduced into the tracheostomy opening. Inner tube has a hole in the centre so that patient can still have a chance to breathe from the larynx even when tube is blocked at its outer end.
Jackson's tracheostomy tube. It has three parts: outer tube, inner tube and an obturator. Outer tube is not split, inner tube can be fixed to the shield of the outer tube by a lock. The obturator helps in the introduction of tube into the trachea.
Cuffed tracheostomy tube. When cuff is inflated, it prevents aspiration of pharyngeal secretions into the trachea. It can also prevent air-leak. It is used when there is danger of aspiration of pharyngeal secretions as in unconscious patient or when
patient is put on a respirator. Cuff should be deflated every 2 h for 5 min to prevent damage to trachea and cartilage necrosis. Nowadays, tubes with two cuffs are available and inflation of the cuff can be alternated to avoid cuff pressure at one site in trachea.

Cuffed suction — aid tracheostomy tube. It is like an ordinary cuffed tube but also has a suction tube which reaches above the cuff. It helps to suck out pharyngeal secretion collected above the cuff. Suction should always be done before deflating the cuff so that accumulated pharyngeal secretions do not get aspirated into the trachea.

Size of tube = "se +4= (Internal diameter in mm)
Length of the tube = Size of tube x 3 (in cm)
For example, in a 4-year-old child size of the tube = ' +4=5mm
Length of the tube = 5 x 3=15 cm
Roughly size of the tube is size of the child's little finger.
How to select size of tracheostomy tube in adults
Tracheostomy tube for adults is selected by size (or number) of the tube. Larger the size (number) greater is the inner diameter (see Table Al). In adults, tubes of inner diameter varying between 6 and 9 or 10 mm are used. Sometimes size of tube is expressed in French gauge (FG), which is 3.14 times the outer diameter of the tube.
FG = outer diameter x x (x = 3.14 or approx 3)
For example, a tube of 36 FG will have an outer diameter of nearly 12.0 mm. Size of Jackson's or Negus tube is usually indicated by FG.
Laryngeal mask airway. Laryngeal mask airway (LMA) is a device with a tube and a laryngeal mask which fits over the supraglottic region. Size of mask is selected according to the weight of the patient. The cuff of mask is first deflated and
positioned over the larynx and later inflated. It is used where face mask is ineffective and intubation of the larynx difficult. Other advantages of LMA include:
¢ To intubate the patient with endotracheal tube (less than 6 mm inner diameter) directly or to first pass a stylet and then rail-road endotracheal tube.
¢ To pass flexible bronchoscope for fibreoptic assessment of airway and then pass the stylet.
LMA is not as effective as endotracheal tube to prevent aspiration of gastric secretions. Its use is contraindicated in obstruction in the area of glottis and subglottis, and cannot be used in patients with trismus.
A
ABC test. See Absolute bone conduction test
ABI.
See
Auditory brainstem
implant

ABR.
See
response
Abscess acute mastoiditis and, 78-79, 78f-79f
Bezold,
78,
79f,
446
extradural, 81
intratonsillar, 261
behind mastoid (Citelli), 79
meatal (Luc), 79
nasal septum and septal, 150-151, 150f orbital,
200
otogenic brain, 82-84, 83f parapharyngeal,
266f-267f,
267-268
parapharyngeal or retropharyngeal, 79
parotid,
263
peritonsillar, 264-265, 265f postauricular,
78
retropharyngeal,
265-266, acute,
266,
266f chronic,
266-267
subdural,
81
subperiosteal,
200
zygomatic,
78,
78f£
Absolute bone conduction test (ABC test),

Acellular mastoid cells, 6
Acinic cell
235
Acoustic neuroma,
46
112
clinical features
of,
112
differential diagnosis of, 114, 114t incidence
of,
112
investigations and diagnosis of, 112-114
origin and
growth of,
112,
113f pathology
of,
112
114
Acoustic reflex test, 24, 25f
Acoustic trauma,
34
Acoustics, 19-20
(AIDS),
369-370
Actinomycin-D, 368t
Acute suppurative parotitis, 231-232, 232f
Adenocarcinomas,
108,
235
of ethmoid,
446
Adenoid facies, 446
Adenoidectomy, 431-432, 431f
Adenoids aetiology of, 243
anatomy and physiology of, 243, 243f clinical features of, 243-244, 244f
cystic carcinoma
of,
235
diagnosis of, 244, 244f eustachian tube disorders and, 60
treatment of, 244
Adenolymphoma,
234
Aditus,
5
ad antrum, 6
266f
26f
B
BAHA. See Bone-anchored hearing aid
Basal cell
107-108,
145,
145f
Basal lamella,
420
Bat ear, 48
Battle's sign, 446
Behaviour observation audiometry, 118
Behcet syndrome,
218
Bekesy audiometry,
Bell palsy, 95, 95f£
Bell's phenomenon,
446
Benign mucous membrane pemphigoid

Benign paroxysmal positional vertigo
(BPPV),
45
Berry's ligament,
326
Betatron,
364
Bilateral ethmoidal polypi, 172-173, 175t
Bill's island,
446
Bing test, 22
Bing-Siebenmann dysplasia, 115
24
Bithermal caloric test, 43, 43f-44f, 448
Bleomycin, 368t
Blepharospasm,
99
Blow out fractures, 184, 184f
Blue drum,
446
BMMP. See Benign mucous membrane pemphigoid
Body equilibrium,
Boerhaave syndrome, 446
Boil,
145,
145f
Bone-anchored hearing aid (BAHA),
122-123,
122f-123f,
123t
Bony cochlea, 9, 10f
Bony labyrinth,
9-11,
9f
BPPV. See Benign paroxysmal positional vertigo
Brachytherapy,
362
Brainstem, tumours
of,
Branchial cyst, 391-392
Branchial sinus
or fistula,
Branchio-oto-renal
392, syndrome,
Bronchial foreign
body,
Bronchoscopy flexible
fibreoptic, rigid,
424-425,
392f
116t-117t
321,
322f,
425,
425t
Broyles'ligament,
446
Bryce's sign,
446
Buccal mucosa, 216, 216f carcinoma of, 226-227, 226f
examination of, 382
Bulla ethmoidalis, 137-138, 137f-139f
Bullectomy,
420
c
Caldwell—Luc operation, 411-412, 412f
Caloric test,
43-44,
43f—44f,
102
Calorigram, 43, 44f
Canal for
Candidiasis.
5
See
Moniliasis
Capillary haemangioma,
Cardiac
211-212
achalasia,
344,
443f
Carhart's notch,
87,

Caroticotympanic
87f,
446
artery,
446
Carotid body
tumour,
392,
393f
Catheterization, of eustachian tube, 59, 59f
Cauliflower ear, 49, 49f
Cavernous sinus thrombosis, 201, 201t
CD4 count, 371
Cellulitis, orbital,
200,
201t
Central auditory tests, 28
Cerebellar disease,
46
Cerebellar dysfunction, test of, 42
Cerebrospinal fluid.
See
CSF
Ceruminoma,
107
Cervical lymph
nodes anterior, 386
examination of, 381, 385
lateral, 386
not clinically palpable, 386, 386f
Cervical rib,
394-395
Cervical vertigo, 47
CHARGE
syndrome,
Cheek mucosa,
216,
Chemodectoma,
446
216f
392,
Chemotherapy,
230,
393f
250,
252
drugs used
in,
367,
368t pretreatment work-up of patient for, 367
radiotherapy with,
365
single agent
compared to
multidrug combination therapy, 367
367
types of,
367
Chest, fixation
of,
286
Chevallet fracture, 147, 148f
Chloroquine, 34
425t
323
46-47
of,
18
Contralateral routing of signals (CROS), 122
Corniculate cartilages,
282
168
Costen syndrome,
447
Cottle test, 149, 149f
Cowden syndrome,
331
CPAP. See Continuous positive airway pressure
381
Cricoarytenoid joint,
282,
449
Cricoid,
282
Cricopharyngeal spasm,
Cricothyroid joint,
344
283
Cricothyrotomy, 320
Crista,
16,
16f
Crocodile tears, 98
Crooked or deviated nose, 143, 143f

CROS. See Contralateral routing of signals
Crouzon syndrome,
116t-117t
Crus commune,
9
Cryoimmunization,
360
Cryosurgery advantages
of, disadvantages
technique of,
uses
361
of,
361
360
of,
360
CSF
rhinorrhoea, aetiology
163-165
of,
163
diagnosis of,
164
laboratory tests
for,
164
site for,
164
nasal secretions compared to, 164t physiology
of,
163
sites of
leakage,
163,
163f treatment
of,
164-165,
165f
CSOM. See Chronic suppurative otitis media
Cuneiform cartilages,
282
Cup ear,
48
Cutaneous horn,
106
Cyclophosphamide, 368t
Cylindroma,
235
Cystic fibrosis,
172
Cystic hygroma, 392-393, 394f
Cystic lesions,
304-305,
305f
Cytotoxic drugs,
D
Dacarbazine,
368t
Dalrymple's sign,
Dandy
447
syndrome, dB.
See
Deaf,
447
Decibel definition
Deaf of,
38
child aetiology
of,
115-117
postnatal causes,
117
prenatal causes, 115, 116t-117t education
of,
119-120
evaluation of
assessment methods for, 118-119, 118t causation for, 117-118
management of, 119-120, 120f
Decannulation,
318-319
Decibel
(dB),
19
Deferoxamine,
34
Dehydration,
360
Denaturation,
360
Dermoid cyst, 106, 143, 144f, 212, 212f, 224
sublingual,
390
Deviated nasal septum (DNS), 148-150
aetiology of,
148
treatment for,
149-150
types of, 148-149, 148f-149f
Diagnostic nasal endoscopy, 417-418, 417f

Diphtheria, 259
Diploetic mastoid cells, 6
34,
34t
Disability, 38
Disc batteries, ingestion of, 351-352
Diuretics, 33-34, 34t
Dizziness, 18. See also Vertigo
DNS. See Deviated nasal septum
Dominant nodule,
334
Donaldson's line,
447
Down syndrome
eustachian tube disorders and, 60
hearing loss and, 116t-117t
Doxorubicin, 368t
Drop attacks, 103
Drumhead. See Tympanic membrane
Ductal cysts,
304,
305f
Dynamic range, 20
Dysphagia aetiology of, 347
barium swallow in patient with, 442f investigations for, 347-348
lusoria,
447
oesophageal causes of, 347, 348f preoesophageal
causes of,
347
Dysphonia, 314-315
abductor, 314-315
adductor,
314
mixed,
314
ventricular, 313-314
Dysphonia plica ventricularis (ventricular dysphonia), 313-314
E
Eagle syndrome. See Styalgia
Ear.
See also
External ear;
Internal
Labyrinth; Middle ear.
development of,
11-12
examination of
functional,
377
physical, 376-377, 376f-377£
symptomatology,
375-376

facial paralysis from surgery of, 96-97
foreign bodies of, 54
instruments for, 451-452
internal,
9-11
middle,
4-8
operations of
surgical approaches to, 400-401, 401f terminology
for,
400
syringing of, 53, 54f wax,
53,
53f-54f
Ear, nose and throat (ENT). See also specific anatomy

equipment for, 374, 375f general setup and patient position for, 375
history taking
for,
374-375
HIV
manifestations in,
370-371
lymph nodes of head and neck, classifica- tion of, 385-386, 385f
radiofrequency surgery in, 358
Ear lobule deformities, 48
Ear surgery, lasers in, 356
Earache.
See
Otalgia
EcoG., See Electrocochleography
Elastic fibrocartilage,
447
Electrocochleography (EcoG), 26, 27f, 101,

Electromagnetic spectrum, lasers and,
355-356,
355f
Electromyography
(EMG),
94
Electron beams, 362, 363f
Electroneuronography (ENoG), 93-94
Electronystagmography, 44
ELISA test, 371
ear;
Embolization, 180
EMG. See Electromyography
Encephalocele, 144
Endaural incision, 400, 401f
Endocochlear potential, 15, 16f
Endolymph, 10, 11t, 447
Endolymphatic duct,
10
Endolymphatic hydrops. See Méniére's disease
Endolymphatic sac,
10,
447
Endomeatal incision,
400,
401f
Endoscopic sinus surgery, 173, 197, 197f anaesthesia
for,
419
complications with, 421, 421t contraindications
of,
419
indications of, 419
position for, 419
postoperative care for, 420-421
steps of, 419-420, 420f techniques
in,
419,
420F
advanced,
419
Endoscopy,
381
diagnostic nasal, 417-418, 417f flexible,
384-385
rigid, 385
End-to-end anastomosis, 99
ENOoG.
See
Electroneuronography
ENT. See Ear, nose and throat
Epiglottis,
282,
450
acute, 289-290, 290t, 446
Epilepsy,
47
Epistaxis anterior nasal packing for, 179, 179f
causes of
general,
177
local, 176-177
cauterization for, 179
178,
178t first
aid for,
179
general measures in, 180
ligation of vessels and, 179-180
posterior nasal packing for, 179, 180f sites of, 177-178, 449
SMR
operation for,
179
Epithelioma. See Squamous cell carcinoma
Epley's manoeuvre,
45
Epworth sleepiness
scale,
277t
Equilibrium, maintenance of body, 18

Erythema multiforme,
218
Erythroplakia,
225
Esthesioneuroblastoma, 204
Ethmoidal sinuses,
188
examination of,
380
malignant neoplasms
of, prognosis
of,
209
treatment of, 209
mucociliary clearance of, 189f, 190
Eustachian tube,
447
anatomy of,
57,
58f catheterization of, 59, 59f
60
adenoids causing, 60
cleft palate
and,
60,
447
and,
60
tubal blockage, 60, 60t examination of, 61, 61f, 377

middle ear clearance secretions, 59
protective, 58
tests for,
59-60
ventilation and middle ear pressure regulation,
57-58
infant compared to adult, 57, 58t lining of, 57
of of,
209
209,
209f of,
15,
Eustachian tube (Continued)
muscles related to, 57, 58f nerve supply of, 57
patulous, 61
retraction pockets and, 61
Evoked response audiometry, 26-27, 27f, 119
Exostoses,
107,
447
Exposure keratitis, 98
External beam therapy, 362
External ear, 2-4
diseases of, 48-56
natural resonance
nerve supply of, 4
tumours of,
106-108
External ear canal. See Meatus, external auditory
atresia of, 50
congenital disorders in, 50-51
exostosis of, 447
inflammations of, 51-53
primary cholesteatoma of, 53
trauma to, 51
Extranasal ethmoidectomy,
Extrinsic muscles, 284
F
Face injuries
bone injuries and, 181
fractures of
lower third, 182t, 185-186
middle third,
182f,
182t,
185-186
upper third,
181-182, general
182t of,
soft tissue,
181
Facial lacerations, 181

anatomy and functions of, 90-94
blood supply to, 91, 92f branches of, 90-91
course of, 90, 91f electrodiagnostic tests for, 92-94
examination of, 377
hyperkinetic disorders of, 98-99
intracranial part
of,
90
intratemporal part of, 90
nerve structure in, 92, 94f nucleus of, 90, 91f
severed, 181
severity of injury to, 92
surgery of,
99
landmarks of, 91, 92f variation and anomalies of, 91-92, 93f

causes of, 94-97, 94t
Bell palsy, 95, 95f ear or mastoid surgery, 96-97
herpes zoster
oticus,
96,
96f
96
neoplasms, 97
temporal bone fractures, 96, 96f, 97t

complications following, 98
end-to-end anastomosis for, 99
facial lesion localization in, 97-98
hypoglossal-facial anastomosis for, 99
nerve graft in, 99
otitis media complications and, 80
peripheral,
97
topodiagnostic tests for intratemporal part lesions in, 97-98, 98f
tubercular otitis media and, 74
Facial recess,
5
Falling, 42
False vocal cords, 284
181
173
15t
Faucial diphtheria, 260
Fibroma, 223, 224f
Fibrous dysplasia, 205, 206f
Fick's operation,
104
Fistula branchial,
collaural,
392,
392f
50-51
oroantral,
186
perilymph,
46
test,
41
Fitzgerald—Hallpike test,
368t
Follicular adenoma,
Follicular
333
Fordyce spots,
Foreign of
331
220
bodies air
passages, aetiology
441f—442f of,
321
321
of,
322-323, management
nature of,
322f-323f of,
323,
321
bronchus, ear,
54
food passages
349
complications of,
323f
349-350
of, disc
batteries
351
and, investigations
351-352
of,
350, of,
site lodgement
larynx,
350f-351f
350-351, of,
440f nasal
cavity,
161
oesophagus,
437f-439f submandibular
in of
of in
in in
in in
duct,
438f thoracic
inlet,
438f-439f
Fractures.
See blow
out, also
Temporal
184,
Chevallet, bone
184f
147, of
face lower
third, middle
148f
182t, third,

frontal bone,
frontal
185-186
182-185,
182f,
181-182,
182t
182
sinus,
Jarjaway,
147, of
mandible, maxilla,
181
148f
185-186,
184,
185f nasal,
182-183,
X-rays, of
182f-183f
434
147-148
aetiopathogenesis of,
147, complications
of, treatment
of, naso-orbital,
148
183
of orbital
floor, supraorbital
184,
184f ridge,
181
of zygoma,
183-184, zygomatic
183f arch,
184
Frenzel manoeuvre,
Frequency,
447
19
cochlea localization
normal speech,
of, range
of,
20
20
Frey's syndrome,
98,
236,
447
Frontal bone fractures, 182
Frontal sinus,
187-188
380, malignant
neoplasms clinical
features of,
treatment
380f of,
209-210
210
of,
210
mucocele of,
198-199,
199f mucociliary clearance of, 189f, 190
osteoma of,
205,
205f
148
182t
351t
349
440f
43,
43f-44f,
185f
15,
148f
15f
448
fractures.
Frontal sinus fractures, 181
Frost bite, 49-50
Functional aphonia, 314
Fungal ball, 196
Furuncle, 51, 145, 145f
Fusion inhibitors, 372
G
Gait,
41
Galen's anastomosis,
Galvanic test,
44
Gardner's syndrome,
Gastro-oesophageal
331
reflux,
Gelle's test,
22-23,
447
Gingivae.
See
Glandular
Gums tumours,
Glioma,
306
144,
203
Globus pharyngeus,
Glomus
344
tumour,

109-110
aetiology and
pathology clinical
features diagnosis
of, of,
344-345
109
109-110
of,
110
spread of,
treatment
Glottis,
284,
Glue ear,
109
of,
110
284f
64
Glycerol test,
Goldenhar
102
syndrome,
Gradenigo syndrome,
Granular cell
tumour,
Granulomatous
116t-117t
447
223, diseases
bacterial,
156-158, fungal,
of
157t
157t,
158-159
of unspecified
cause,
157t,
Graves ophthalmopathy,
Graves's
306
nose
159-160
211
disease,
330,
330t
Griesinger's sign,
Grisel syndrome,
Grommet
447
447
insertion,
Gums
(gingivae),
65,
65f
216, examination
216f of,
382
Gustatory lacrimation,
Gustatory sweating,
98
98,
236
Gutmann's pressure
test,
447
447

BAHA,
122-123, conventional,
121-122
disadvantages of
conventional, fitting
of,
121-122
implantable,
123-124, indications
for, types
of,
121
121,
122f
Hearing assessment,
21-28
tests for,
clinical tests
finger
23-26
for,
21-23
friction speech
tuning
(voice)
test,
21
test, fork
watch tests,
test, special
21
tests for,
Hearing impaired,
Cochlear
21
21-23,
22f,
26-28
rehabilitation implants;
assistive devices
implants
Hearing for,
127
for,
124-127
ABI,
127
instrumental devices
training for,
for,
127
Hearing level
(HL),
autoimmune inner
20
ear of,
conductive,
29,
21, aetiology
of, in
450
29,
30t different
lesions apparatus,
of
29
management of,
fluctuating,
29-32
and,
447
handicap degree
labyrinth
Méniére's mixed,
in,
39,
116t-117t
40t inflammations
causing, disease
and,
101
21
noise trauma
and, noise-induced,
34-36,
449
34
permissible exposure
permissible preventing,
in, limits
in,
35,
35t non-organic,
37-38
in otitis
media, ototoxic
72
drugs and,
presbycusis and,
sensorineural,
37
21
32
diagnosis
33-34,
32-33
of,
32
familial progressive,
33
of,
33
social and
legal specific
aspects forms
SSNHL, of,
of,
33-37
358-359
sudden,
36-37
36

prognosis of,
treatment
36-37
37,
37t of,
37
syndromes associated
with,
116t-117t tubercular otitis media and, 74
unilateral,
39
Heimlich manoeuvre,
Helicot,
9
Hemifacial spasm,
98,
Hennebert's
323,
98f phenomenon,
448
sign,
448
Hereditary haemorrhagic
Herpangina, telangiectasia,
217
Herpes zoster
oticus, facial
paralysis
52,
55
from,
96,
Herpetic
96f gingivostomatitis,
Hiatus hernia, 343
217
323f
35,
35,
35t
34t
38-39
33
35t
121-124
disease
38, and,
38f conductive
123t
124t
22t,
64
of.
See aids.
also
36
124f
180
Hitzelberger sign, 449
HIV. See Human immunodeficiency virus
HL. See Hearing level
Hoarseness, 313, 313t
Hormonal therapy, 249-250
Horner syndrome,
448
Human immunodeficiency virus (HIV)
AIDS
patients and,
369-370
antiretroviral drugs
for,
372
course of,
370
diagnostic tests for, 371
ENT
manifestations of,
370-371
healthcare workers and infection of,

management of, 372
universal precautions for, 372
HIV-I virion, structure of, 369, 370f
India history and epidemiology with, 369,
370t life cycle of, 369-370
transmission modes of, 369
Hump nose,
143
adenoma,
334
331-332
Hydraulic ratio,
446
Hydrochloroquine,
Hyperbaric oxygen

Hypernasality,
315,
315t
Hyperthyroidism, 330, 330t
Hypoglossal-facial anastomosis,
Hyponasality,
315,
Hypopharynx anatomy
of,
241,
(HBOT),
99
315t
241f-242f functions of, 242
lymphatic drainage of, 241-242
273-274
postcricoid region
273-274
posterior pharyngeal
wall carcinoma,
pyriform sinus carcinoma, 273
Hypopnoea,
276
Hypothyroidism,
100
rhinitis due
to,
171
symptoms and signs of, 329-330, 330t
Hyrtl's fissure,
448
I
Idiopathic orbital inflammation, 211
Immunotherapy, 169
Impairment,
38
Impedance audiometry,
Incisura terminalis,
2,
3f
Incus,
7
Infectious mononucleosis, 259-260
Inferior turbinate,
135
Inflammatory oedema of lids, 200
Instruments ear,
451-452
for hearing impaired rehabilitation,
121-124
larynx and
trachea, nose,
453-455
throat,
456-459
for tinnitus,
131
Intermittent low-pressure
105f£
Internal ear,
9-11.
pulse
See also
autoimmune disease
of, therapy,
Labyrinth.
36
bony labyrinth
of,
9,
9f fluids and circulation in, 10
membranous labyrinth of, 9f, 10
Intranasal ethmoidectomy, 173
459-464
34
therapy
24-26,
25f,
65,
119
104,

Intranasal meningoencephalocele,
203
Intratonsillar abscess, 261
Intratympanic gentamicin therapy, 104
Intratympanic muscles,
7-8
Intrinsic muscles, 283-284, 283f-284f
Intubation granuloma, 304
J
Jacobson's nerve, 448
Jarjaway fracture, 147, 148f
Jaw thrust,
319
Jervell and Lange-Nielson syndrome,
116t-117t
Jugular chain, anterior, 386
Jugular foramen syndrome, 448
Juvenile nasopharyngeal
angiofibroma,
246-250,
247f-248f,
Juxtavisceral chain, 386
K
Kallmann syndrome,
Kaposi sarcoma,
448
229-230,
Kartagener syndrome,
371
172,
448
Keloid,
106,
106f
Keratoacanthoma, 106-107
Keratosis obturans, 54, 304
Kiesselbach's plexus, 147
Killian's dehiscence,
238
Killian's polyp.
See
Antrochoanal
173-175
Kilovoltage machines, 364
Klippel-Feil syndrome,
116t-117t
Korner's septum, 7, 448
Krause's nodes,
448
249t polyp,

direct,
297,
310
anaesthesia for, 422
complications with,
423
contraindications of, 422
indications of,
422
position for,
422
procedure for,
423
for,
422-423, flexible
fibreoptic,
422f
297
indirect,
309
structures seen in, 384, 384f technique for, 384, 384f
micro,
310
Laryngotracheal trauma
aetiology of, 287
clinical features of, 287
complications of, 288
diagnostic evaluation of, 287-288
pathology of, 287
288
Laryngotracheobronchitis,
Larynx anatomy
artificial, of,
282-285
312,
312f benign
tumours of
neoplastic,
303t,
305-306
non-neoplastic, 303-305, 303t rare,
306
cancer of,
449
307
classification and staging of, 307, 307f,
307t-308t diagnosis
of,
309-310
epidemiology of,
307
glottic,
309,
309f,
311,
311f histopathology of, 307-309
subglottic,
309,
311
supraglottic,
309, treatment
311
of, cartilages
310-311
of,
282, cavity
of,
282f
284-285, congenital
284f legions
of,
295, cyst
of,
295
diphtheria of, 290-291
embryological development of, 285
384-385
foreign bodies in, 440f haemangiomas
of,
306
hidden areas
of, infraglottic,
284
inlet of,
284, instruments
284f for,
459-464
joints of,
282-283
leprosy of,
293
lupus of,
293
lymphatic drainage
membranes of,
285
of,
282f-283f, mucous
membrane muscles
mycosis of,
283
285

of,
283-284, of,
283f-284f
294
nerve supply
oedema of,
of,
285,
291
paediatric, paralysis
285-286
of causes
of,
298,
299t classification
of,
298
combined
(complete), phonosurgery
301
for,
301-302
recurrent, bilateral,
recurrent,
300,
300f unilateral,
of superior
298-300, nerve,
300-301
physiology of,
286
scleroma of, 294
sinus of, 284
448
290,
298,
290t,
298f
446
295f-296f
299t

spaces of, 283f, 285, 285f syphilis of, 293
total laryngectomy for, 310
vocal rehabilitation after, 312, 312f, 312t tuberculosis
of,
293
vestibule of, 284
Lasers advantages
Argon, and
disadvantages
356
clinical applications
COs,
357
diode,
357
in ear
surgery,
356
electromagnetic spectrum

invisible,
355
KTP,
356-357
operational parameters
photodynamic and,
of,
356
therapy with,
principle of,
354-358
properties and
effects safety
precautions tissues
358
of,
355
in use
effects and
354f-355f types
of,
354,
355t visible,
355f,
355
YAG,
357
Lateral sinus thrombophlebitis, 84-85
Lateral wall
of nose,
in,
176,

Lederman's classification,
207,
207f
Leiomyoma,
345
Lempert's incision, 400, 401f
Leprosy, of
nose,
157-158,
157f
Lermoyez syndrome,
Leukaemia,
103,
260
Leukoplakia,
224-225, hairy,
371
Leukotriene receptor
Lever ratio,
448
225f,
304
antagonists,
448
Lhermitte's sign,
Lichen
448
planus,
219
Lids, inflammatory
Linear accelerator,
Lingual oedema
169
of,
200
364
thyroid,
328,
Lips,
216,
216f carcinoma
of classification
of,
228t clinical
presentation
226, sites
226f of,
226-229
staging of,
228t examination
of,
Little's area,
381-382
147
in,
176
Loudness,
19
Loudness discomfort level, 20
Ludwig's angina,
263-264,
264f
Lupus erythematosus,
Lupus chronic
vulgaris of
nose,
Lymphangioma,
223,
Lymphoma,
204,
Non-Hodgkin, thyroid, 333
Lyre's sign, 448
M
MacEwen's triangle, 5, 6f
Macrotia,
48
Macula,
10
16,
16f-17f
Maggots, nose,
161-162,
162f
Malignant ceruminoma, 108
discoid,
157
234
212,
371
229, and
328f of,
357-358
damage from,
354-355, of,
356-357
236, of,
394
356
355-356, treatment
of,
219
78f-79f
Meatus, nasal
middle,
137f, atrium
of, superior,
136-137,
136f-137f
138
138
Median rhomboid
glossitis,
Medullary carcinoma,

Melanoma,
220
332-333,
107,
145,
229
malignant,
204
Melanosis,
225
96
Membranous labyrinth,
9f, of,
12,
12t
Méniére's disease,
aetiology
45
of,
100, clinical
101f of,
of,
103
101
101
of,
101-102,
Méniére's syndrome
pathology staging
102f,
332t
102t compared
of,
100,
101f
103,
103t of
acute attack
chronic management,
103-104
phase general
surgery management,
measures,
104
103
and, variants
104-105, of,
vertigo
102-103,
105f
103f and,
100-101
Méniére's syndrome,
compared
Méniére's to,
103
Meningioma,
203
Meningitis,
81-82
Meningoencephalocele,
Metastatic lymph
nodes,
368t
Michel aplasia,
Microtia, test,
115,
48,
48f
Microtron,
364
Microwick,
Middle ear,
anterior
104
4-8, wall
blood
450
of, supply
5, of,
cleft development
of, lining
of,
8
divisions of,
62-66
4,
5f tube
in,
59
eustachian clearance
tube
57-58
floor of,
5,
5f lateral
wall of,
5
lymphatic drainage of, 8, 9t medial
wall of,
5,
6f natural
resonance ossicles
of, of,
7,
8f,
14
posterior wall
roof of,
5, of,
5,
5f transformer
action tumours

of carcinoma,
110-111
109
glomus,
109-110
sarcomas,
111,
111f secondary,
111
volume of,
450
Middle turbinate,
Migraine, basilar,
Mikulicz cells,
Minimal
Modified
136
46
156,
156f nerve
excitability
Kobrak test,
test,
43
15,
15t
14
regulating
377
144
394
59-60
448
5£
8
to,
103
100-101
12,
6f of,
10
12f disease
of secretions
pressure in,
93
Modiolus, 9, 448
Mondini dysplasia, 115
(candidiasis)
,
217
Most comfortable level (MCL), 20
Motion sickness, 18
Mouth, floor
of,
216, carcinoma
of, examination
229, of,
216f
229f
382
MST.
See
Maximal stimulation
test
Mucocele, 224, 225f
Mucoepidermoid carcinoma, 235-236
Mucormycosis, 159, 449
Mucosal hyperpigmentation, 225
Mucous retention
cysts,
198-199,
Miller's manoeuvre, 449
Multiple sclerosis, 46
Multiple sleep latency test, 276
Mumps
(viral parotitis),
231
Myringitis bullosa,
55
Mpyringitis granulosa,
Myringoplasty,
29-30,
55
31f,
400
407
contraindications for, 406
fat-graft,
407
406
postoperative care for, 406
technique for,
406-407,
407f
Myringotomy,
65, anaesthesia
65f for,
398
399
contraindications of, 398
398
operation steps of, 398, 398f pitfalls
of,
398-399
postoperative care for, 399

fractures of, 147-148
aetiopathogenesis of, 147, 148f complications of, 148
treatment of, 148
perforation of,
151, septal
abscess and,
151f
150-151, septal
haematoma and,
150,
Nasal synechia, 162-163, 163f
Naso-orbital fractures,
183
Nasopharyngeal airway, 319
Nasopharyngeal angiofibroma,
249t
Nasopharyngeal bursa,
Nasopharyngeal cancer
aetiology of, 250
classification of, 252, 253t clinical features of, 251-252, 251f
epidemiology and geographic distribution of,
250-252
pathology of, 250-251, 250t spread
of,
251,
251f treatment of, 252
Nasopharyngeal tonsil,
Nasopharyngitis acute,
244
chronic, 244-245
Nasopharynx.
See also
Adenoids acute
nasopharyngitis of,
244
238-240,
239f bilateral obstruction of, 174t
carcinoma of,
446
chronic nasopharyngitis of, 244-245
epithelial lining
of,
240
380-381
functions of, 240
lymphatic drainage of, 240
pharyngeal bursitis
and,
245
tumours of
benign,
246-250,
246t malignant, 246t, 250-253
X-ray, soft
tissue lateral
view
Neck.
See also
Head infections.
dissection,
388-389
classification of, 388-389
extended, 389
modified, 388-389
radical,
388,
389f selective,
389
masses of,
390-396,
390f-391f nodes
classification by level of, 387-388,
387f-388f, 387t examination
of,
386-387,
X-ray, anteroposterior
X-ray,

387f view
of, lateral
view of,
434
Neoplasms,
97
Neurodermatitis, 52
Neurofibroma, 107
Nicotine stomatitis, 220
Noise,
19
Noise trauma, 34-36
Noise-induced hearing loss, 34
permissible exposure in, 35, 35t permissible limits in, 35, 35t
preventing,
35,
35t
Non-nucleotide reverse transcriptase inhibi- tors,
372
Non-organic hearing loss, 37-38
Non-REM sleep, 277, 278t
Nose.
See
Ear, nose
and throat
air-conditioning of inspired air by, 140
anatomy of, 133-139
bilateral obstruction of, 174t
434
and neck
of,
434
space
239,
239
150f
150f
246-250,
239f

blood supply in, 139, 176, 177f-178f cellulitis of, 143
deformities of,
143
examination of, 378-379
functional, 379, 379f symptomatology for, 377-378
external,
134
diseases of,
143-145
examination of, 378
tumours of, 143-145, 144t fractures of, 182-183, 182f-183f
granulomatous diseases of bacterial,
156-158,
157t fungal, 157t, 158-159
of unspecified cause, 157t, 159-160
instruments for,
453-456
internal,
135-138
lining membrane of, 138-139
lateral wall of nose, blood supply in, 176,
178f leprosy
of,
157-158,
157f lower
airway protection
by, lupus vulgaris of, 157
of,
139
maggots in, 161-162, 162f musculature of, 134
nerve supply of, 139f, 138-139
olfaction and, 142
osteocartilaginous framework of, 134,

respiration in, 140, 141f skin
of,
134
syphilis of, 157
tuberculosis of, 157
unilateral obstruction of, 173t vestibule
of,
135
bilateral obstruction of, 174t diseases of, 145-146
378
lining membrane of, 138
tumours of, 146
vocal resonance and, 141
Nucleoside reverse transcriptase inhibitors,
372
Nutcracker oesophagus, 344

caloric test and, 43-44, 43f44f cerebellar dysfunction test and, 42
degree of,
42t electronystagmography and, 44
Fistula test and, 41
Hallpike manoeuvre for, 42, 42f, 42t optokinetic test and, 44
past-pointing and falling with, 42
positional,
42t spontaneous,
41
°
OAEs.
See
Otoacoustic
emissions
sleep
apnoea
(OSA)
consequences of, 277t
of,
279t
pathophysiology of, 277
Ocular vertigo, 47
Oesophageal spasm, diffuse, 344
Oesophageal speech, 312
Oesophagoscopy
flexible fibre optic, 427, 427f
rigid,
426-427
transnasal,
427
Oesophagotomy
cervical, 350
transthoracic, 350
140-141,
141f
Oesophagus.
See also
acute oesophagitis
anatomy
Dysphagia and,
342
of,
340,
340f-341f
Barrett's,
446
benign structures of, 343
carcinoma of, 345-346
corrosive burns
of,
342-343, dysphagia
caused by,
347,
348f foreign body in, 437f-439f
globus pharyngeus
and,
344
hiatus hernia and, 343
of, motility
disorders
340
of,
344-345
neoplasms of,
345-346
nerve supply of, 340
nut-cracker,
344
342
physiology of, 340
Plummer-Vinson
449
swallowing phases
Ohngren's line,
207, syndrome
and,
341
207f
Olfaction, 142
Olfactory neuroblastoma, 204
Olfactory pathways,
142
Oncocytoma,
234
Oncovin,
368t
Optokinetic test,

44
216f
225-226
226
sites of,
226-229
of,
381-382
miscellaneous of,
216
lesions nonsquamous
of,
220
malignant
229-230, palpation
of, radiation
and tumours
of benign,
223-224
chemoprevention for, 230
223
cystic lesions,
224
malignant lesions, 225-230
premalignant lesions,
224-225
solid,
223-224
ulcers of,
217-220
blood disorders,
causes of,
217t drug
allergy,
219
immune disorders, 218, 218f infection,
217-218
miscellaneous,
220
neoplasms,
218
skin disorders,
trauma,
218-219
218,
219f submucous
Oral fibrosis

220-221
clinical features of, 221, 222f pathogenesis of, 221, 221f
pathology of,
221
treatment
Orbital of,
221
apex syndrome,
200,
200f
Orbital cellulitis,
200,
201t
Orbital floor fractures, 184, 184f
Organ of Corti, 13
Oroantral fistula, 186
Oropharyngeal airway, 319
382,
382f care
of,
366
lesions
219, and
219f and,
343-344, treatment
of, of,
(OSF),
Otosclerosis,
86-89
aetiology of, 86
differential diagnosis of, 88
pathology of, 87
signs of, 87, 87f sites of stapedial, 449
87
88-89
types of, 86, 87f
Ototoxic drugs, hearing loss and, 33-34, 34t
Oval window,
5,
9
round window phase differential between,
15
Overtones,
P
P24
antigen assay,
19
371
Pachydermia laryngis,
292-293
Palatine arch, carcinoma of, 271, 271f
Papillary cystadenoma
lymphomatosum,
Papillary thyroid
Papilloma,
106,
108, inverted,
202,
Schneiderian,
330-331
223,
269
203f,
448
202,
Squamous,
202,
203f,
202f adult-onset,
306
juvenile, 305-306, 305f, 449
transitional cell,
202,
203f
Paraglottic space,
Paranasal
283f, sinuses.
Frontal sinus;
Sphenoid
285,
285f
See also
Ethmoidal
Maxillary sinus.
187-190
development and
growth examination
of,
379-380
functional endoscopic

190
fungal infections of, 196-197
of,
189-190
198-199, mucociliary
clearance mucous
membrane neoplasms
of, of
benign,
205
malignant, 205-210, 206f physiology
of,
190
risk factors associated with, 449
X-ray of
Caldwell view
of, of,
434
434,
437f right
and left
oblique views
of, submentovertical view of, 434
Waters'
view of,
433,
436f
Parapharyngeal abscess,
Parapharyngeal
266f-267f, tumours,
Parathyroid glands,
Parotid abscess,
272,
392
327
263
Parotid gland
and duct,
181
Pars flaccida, of tympanic membrane, 2, 4f
Pars tensa,
membrane,
2,
3f—4f
Passavant's ridge,
240
Past-pointing,
42
Paterson—Brown-Kelly syndrome,

PB
max.
See
Phonetically
PCR
tests,
371
Pemberton's manoeuvre,
Pemphigus vulgaris,
218
Perichondritis,
50,
50f
Perilymph,
10,
11t fistula,
46
Peripheral receptors, 16, 16f-17f
Peritonsillar abscess, 264-265, 265f
199f of,
190
188-189
surgery sinuses;
sinus;
Sinusitis;
of,
189,
189t of,
197,
434
267-268
450
234
343-344, balanced
449
max
Petrositis, 79-80
Pharyngeal bursitis, 245
Pharyngeal pouch
aetiology of, 274
clinical features of, 274-275
pathology of, 274
sites of, 274-275, 274f treatment
of,
275
Pharyngeal wall,
238, lateral,
carcinoma posterior
271-272,
239f of,
271-272
274
examination of, 383
Pharyngitis,
201
acute,
254-255,
255t atrophic,
256
chronic, 255-256, 256f fungal,
254
keratosis,
256
viral infections causing, 254
Pharyngotympanic tube.
See
Pharynx.
See also
Hypopharynx;
ynx;
Oropharynx anatomy
of,
238
divisions of, 238-242, 239f spaces
of,
238
Phonasthenia,
314,
314f
Phonation,
286
Phonetically balanced max (PB max), 24
Photodynamic therapy,
358
Photon beams,
Pierre-Robin
362,
363f sequence,
Pillars, examination
Pinna,
116t-117t, of,
383
2.
See also
Auricle.
avulsion of,
49
11, diseases
of,
12f
48-50
auricle trauma,
49-50
congenital disorders,

48-49
disorders, examination
of, nerve
supply of,
50
376
4
Pitch,
19
Plasmacytoma, 204
Pleomorphic adenoma, 202, 234, 269
Plummer-Vinson syndrome, 343-344, 449
Politzer test,
59
Polychondritis, relapsing,
Polypectomy,
173
Polysomnography, split-night,
278
Ponticulus,
5
Positional test. See Hallpike manoeuvre
Postaural incision,
401,
401f
Postcricoid region,
Posterior inferior
273-274
cerebellar artery
syndrome
(Wallenberg syndrome),
46,
450
Posterior suspensory ligament, 326
Posturography,
44
Pouch of
Luschka,
Preauricular sinus,
449
49, tags
or
Pre-epiglottic space

granuloma, 223
rhinitis,
170-171
Prelaryngeal nodes,
390
Preoperative open biopsy, 449
Presbycusis,
37
Pretracheal nodes, 390
Processus cochleariformis, 5
Promontory, 5
Proof puncture. See Antral lavage
49f,
106
appendages,
48, of
Boyer,
283f,
49f
285,
50
278
Nasophar-
449
Q
Quinine,
34
Quinsy. See Peritonsillar abscess
R
See also
cancer treatment
cyberknife modes
of,
364-365, stereotactic,
dose,
364
infection prevention
364
for, nutrition and, 366
care with,
particle,
362
postoperative, preoperative,
365
365
skin care
with, sources
of, teeth
366
364, care
364t with,
366
types of
beams,
362, unit of, 364, 364t
Radioactive material,
364
Radioallergosorbent test (RAST), 168
Radiofrequency surgery
in
ENT,
358
Radiological test,
59
Radiotherapy,
249, chemotherapy
252.
See also
combined complications
with, of,
conformal, 362
curative,
310
intensity modulated, 362-364
modes of,
362-364
for palliation,
patient
365
care during,
planning,
365
surgery and,
365
Ramsay—Hunt syndrome.

Ranula,
224,
225f plunging,
392
RAST. See Radioallergosorbent test
Rathke's pouch,
239-240,
449
RDI. See Respiratory disturbance index
Recruitment,
26,
26f,
449
Recurrent laryngeal nerve, 326
Regenerative nodule,
334
Reinke's space,
285,
448
oedema of,
304
REM sleep, 277, 278t
Respiration,
286
Respiratory disturbance index (RDI),

Retromolar trigone, 216, 216f examination
of,
382
Retropharyngeal abscess, 265-266, 266f acute, 266, 266f
chronic,
266-267
Retropharyngeal nodes, 386, 386f
Rhabdomyosarcoma,
111,
111f,
212
See
365-366,
365
366t
363f
366
366
366
364t
Salivary glands (Continued)
Sjogren's syndrome and, 233
tumours of benign, 234, 234t
234t,
235-236
minor,
223,
Samter's triad,
229,
450
Sarcoidosis,
160
Scarpa's ganglion, 16-17
Schatzki's ring, 345
Schaumann's bodies, 450
Scheibe dysplasia,
115
Schirmer test,
97
Schneiderian papilloma,
450
Schwabach's test,
22,
22t
Schwannoma, 202
Schwartz sign,
450
Scleroderma,
345
Sclerotic mastoid cells, 6
Screamer's nodes,
303-304,
Scutum,
5
Sebaceous adenoma,
Sebaceous
202,
203f,
303f-304f
108
cyst,
106
Semicircular canals,
9
malformations of,
115
stimulation of flow in, 17, 17f
Semicircular ducts,
10
Sensation level
Septal
(SL),
20
abscess,
150-151,
Septal haematoma,
Septoplasty,
150,
150
indications for,
415
postoperative care for, 415-416
postoperative complications
with, technique
for,
Septum
415,
416f of
nose.
See
Short increment

Sialadenitis, chronic
Sialectasis,
232
Sicca syndrome,
233
Sigmoid sinus thrombophlebitis, 84-85
Singer's nodes,
303-304,
303f-304f
Sinus of Morgagni, 239f, 240
Sinus tympani,
5
Sinusitis acute
aetiology bacterial,
ethmoid, of,
191
448
193
frontal,
192-193, fulminant
fungal, maxillary,
pathology
192
of,
191
sphenoid,
193-194
trephination in,
193, allergic
fungal, chronic
diagnosis
196
of, of,
193f
195
195
invasive,
196
195
pathophysiology of,
surgery for,
196
195-196
of, descending
198f,
198t infections,
focal infections,
intracranial,
201
201
200-201
local,
198-200
orbital, 200, 200f fulminant
fungal,
196
fungal,
447
noninvasive forms of, 449
index test
232
229f
150f
150f
193f
446
195,
195f
416
Subiculum, 5
Sublingual nodes, 386
Submandibular salivary flow test,
97-98
Submental nodes,
390
Submucous resection operation (SMR.
operation),
149-150
complications with, 413-414
for epistaxis,
179
indications for, 413
postoperative care for, 413
present status of, 414
steps of, 413, 414f
Subperiosteal abscess,
200
Sudden sensorineural hearing loss (SSNHL),

Summating potential (SP), 15-16
Superior laryngeal nerve, 326
Superior orbital fissure syndrome, 200
Superior turbinate,
138
Supporting cell,
13
Suppurative parotitis,
acute,
231-232,
Suprameatal triangle, 5
Supraorbital ridge fractures, 181
Swallowing phases,
oesophagus and,
also
Dysphagia
Sympathomimetic drugs,
Synkinesis,
98
Syphilis,
46
nasal,
157
Syringing, of ear, 53, 54f
T
Taste test, 97
T-cell lymphoma,
TDDs.
160
See
Telecommunication deaf
Tectorial membrane,
13
Teeth examination of, 382
radiation and care of, 366
Tegmen antri,
5
Telecommunication devices for deaf (TDDs),
127
Teletherapy,
362
Temporal bone
fractures facial paralysis from, 96, 96f, 97t
X-ray of
Law's view
Schuller's of,
433, view
of,
Stenver's view
of,
435f
433
433
submentovertical view
Towne's view of, 433
transorbital view
of, of,
433,
433
435f
Tensor tympani,
7-8
TESPAL. See Transnasal endoscopic sphenopalatine artery ligation
Thermal shock,
360
Thornwaldt's disease, 245
Threshold tone decay test, 26
Throat.
See
Ear, nose
and throat
Thymic cyst,
391
Thyroglossal duct cyst, 390, 391f
Thyroid cyst,
334,
334f
Thyroid gland,
282,
282f anatomy of, 326b-329b, 327f-328f
arteries of, 326
benign disorders of, 329-330, 330t capsules of, 326
lymphatic drainage of, 326, 327f£
lymphoma of,
333
malignant disorders of, 330-333, 330t metastases of, 333

physiology of, 328-329, 328f-329f
341.
168
devices
232f
See for
Thyroid gland (Continued)
surgery indications for, 336
preoperative work-up for, 336-337
steps for,
337
types of,
veins
336-337, of,
326
Thyroid nodule
evaluation of,
334-335,
334t, management of, 335, 335f
types of,
333-335
Tinnitus,
101
causes of,
130, instrument
for, objective,
130t
131
130
retraining subjective,
treatment therapy,
130
of, types
130-131
of,
130
Tissue destruction,
Tone decay,
mechanism
450
Tongue,
216,
of,
227-228, at
base of
335f of,
360
227f, tongue,
269-271, of,
base,
382
383-384
fissured,
220
geographical, hairy,
220
miscellaneous tie,
220,
220f
Tonsillar cyst,
261
Tonsillar fossa,
Tonsillectomy anaesthesia
for,
271,
220,
220f lesions
of,
271f
428
contraindications
430
for, indications
for,
428
428, postoperative
429f care
steps for,
for,
428-429, techniques
429-430,
429f-430f for,
429,

acute aetiology
of,
259
complications of, 259
signs of,
259
treatment
259
of,
259
types of,
258-260, chronic,
260-261,
258f
261f membranous,
259
Tonsilloliths,
261
Tonsils candidal
infection of,
260
carcinoma of, 271, 271f examination
of,
383
lingual, diseases
of,
261-262
palatine anatomy of, 257-258, 258f
257-258
Topical ear
drops,
Torus,
223
Toxic shock
syndrome,
34
414
Toynbee's test, 59
Tracheal foreign body, 321, 323, 441f
Tracheal instruments,
459-464
Tracheobronchitis,
201
Tracheo-oesophageal speech, 312
Tracheostomy,
316-320
complications in,
319
elective,
316
emergency, 316, 320
functions of, 316
of,
430t
428
227t-228t
270f
336t
131
220
430t
259-260
(Continued)
in infants
316,
317t and
children,
318,
318t mini,
320
operation steps in, 317-318, 317£
percutaneous dilatational,
320
permanent,
316
318f technique
for, types
of,
316
Tracheostomy tube
for adults,
461
care of,
318
cuffed,
462-463
Fuller's,
462
Jackson's,
462
size of,
461t,
Transantral
463
ethmoidectomy,
Transitional
173
cell papilloma,
Transnasal endoscopic
202,
203f sphenopalatine
ligation
(TESPAL),
Transtracheal
180
jet ventilation,
Traumatic ulcer,
319
218,
Trautmann's
219f,

260
triangle,
450
syndrome,
Trotter syndrome,
True vocal
116t-117t
450
cords,
284
Tubal tonsil,
240
Tubercular lymph
394f-395f
Tuberculosis, of
nose, nodes,
157
Tullio phenomenon,
450
Tumarkin's otolithic crisis, 103
Tuning fork
tests,
21-23,
22f,
22t,
Tunnel of
Corti,
13
Tympanic membrane,
atrophic,
55-56
cautery-patching development
of, of,
12
55-56
376-377, layers
of,
3,
4f nerve supply of, 4
normal,
55,
55f pars flaccida of, 2, 4f
pars tensa
of,
2,
3f-4f perforations
of, procedures
for retracted,
56
closure
55, retraction
55f pockets

splintage of,
407
traumatic rupture
tympanosclerosis of,
and
64
450.
2-3,
407
See
377£
of,
407
55
of,
56
Tympanic plexus,
8
Tympanometry, 24, 25f, 59
Tympanoplasty with
mastoidectomy,
400
without mastoidectomy, 400
principles of, 29-32
types of,
30
Tympanosclerosis,
56
U
Uncinectomy, 420
Undifferentiated carcinoma, 236
UPP.
See
Uvulopalatoplasty
Upper horizontal chain nodes, 385-386
Usher syndrome, 116t-117t
Utricle,
10,
18
Uvulopalatoplasty (UPP), 279
also
317
after,
462
318-319,
393-394, in,
artery

Valleculae,
383-384
Valsalva test,
Van der
59
Hoeve
Vascular stasis,
Vasomotor syndrome,
360
rhinitis
Venous varix,
(VMR), of
orbit,
Ventricle,
284,
450
Vertebrobasilar insufficiency,
Vertigo,
18
BPPV,
45
central vestibular

cervical,
47
Méniére's disease
ocular,
47
116t-117t
170
46
disorders causing,
and,
100-101
45-46, disorders
46t psychogenic,
47
Vestibular aqueduct,
Vestibular folds,
284
hydrops,
102-103
nerve,
16-17
neuronitis,
45
system,
16-17
assessment of
laboratory for,
41-42
tests for,
central
43-44
connections disorders
of, of
central,
45-47, peripheral,
46t
45-46, peripheral
receptors physiology
of,
Vestibule,
9
of larynx,
of
284
nose,
135
bilateral obstruction
diseases
46t of,
16,
16f-17f of,
of,
145-146
378
lining membrane
of,
146
Vestibulitis,
145-146,
146f
Vestibulotoxic drugs,
45-46
Vibrating ossicular prosthesis (VORP), 123, 124f
Vincent angina,
259
Vincent infection,
Vincristine,
217
368t
Viral parotitis.
VMR.
See
Mumps
See
Vasomotor
congenital paralysis
rhinitis of,
295,
301
mouse-nibbled appearance
of,
449
polypoid degeneration of, 292
vocal nodules
of,
303-304,
303f-304f
Vocal folds,
Vocal
Vocal
Voice
284
nodules,
450
polyp,
304
assessment,
Voice
385
313-315
Vomeronasal organ,
VORP.
See
450
Vibrating ossicular
prosthesis

Waardenburg syndrome, 116t-117t, 117f
Waldeyer's
ring,
238,
239f
Wallenberg syndrome. See Posterior inferior
cerebellar artery syndrome
Warthin
tumour,
234
Wax,
ear,
53,
53f-54f
Wax
granuloma,
53
Weber
test,
22,
22t
Wegener's granulomatosis, 159-160
Well-pneumatized mastoid cells, 6
174t
138
17-18
17
causing,
115
45-47,
212
enlarged,
Western blot test, 371
Wildervanck syndrome, 116t-117t
Wilde's incision, 401, 401f
Woodruff's plexus, 450
blood supply in, 176
Wrisberg's cartilage,
450
Wrisberg's nerve, 449
Xx
X-ray mastoids,
65
nasal fractures,
434
nasopharynx, soft tissue lateral view, 434
neck, anteroposterior view, 434
Y
Young syndrome, 172
Zygoma fractures, 183-184, 183f
Zygomatic arch fractures, 184

Scanning Electron Micrographs by Kathy Tosney
Ultrasound Images by Nancy Chescheir and Hytham Imseis
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Langman’s medical embryology. — 12th ed. / T.W. Sadler.
p- ;.cm.
Medical embryology
Includes index.
ISBN 978-1-4511-1342-6
1. Embryology, Human—Textbooks. 2. Abnormalities, Human—Textbooks. I. Langman, Jan. Medical
embryology. II. Title. III. Title: Medical embryology.
[DNLM: 1. Embryology. 2. Congenital Abnormalities. QS 604]
QM601.L35
2012
612.6'4—dc23
2011025451

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has
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of
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and
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accepted
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However,
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Dr. Tom Kwasigroch for his wonderful friendship, excellence in teaching, and dedication to his students.
Special thanks: To Drs. David Weaver and Roger Stevenson for all of their help with the clinical material, including providing many of the clinical figures.
To Dr. Sonja Rasmussen for her help in reviewing all of the clinical correlations and for her editorial assistance.
expert
very student will be affected by pregnancy, either their mother's, since what happens in the womb does not, necessarily, stay in the womb, or by someone else's. As health care professionals you will often encounter women of childbearing age who may be pregnant, or you may have children of your own, or maybe it is a friend who is pregnant. In any case, pregnancy and childbirth are relevant to all of us, and unfortunately, these processes often culminate in negative outcomes. For example, 50% of all embryos are spontaneously aborted. Further more, prematurity and birth defects are the leading causes of infant mortality and major contributors to disabilities. Fortunately, new strategies can improve pregnancy outcomes, and health care professionals have a major role to play in implementing these initiatives. However, a basic knowledge of embryology is essential to the success of these strategies, and with this knowledge, every health care professional can play a role in providing healthier babies. To accomplish its goal of providing a basic understanding of embryology and its clinical relevance, Langman's Medical Embryology retains its unique approach of combining an economy of text with excellent diagrams and clinical images. It stresses the clinical importance of the subject by providing numerous clinical examples that result from abnormal embryological events. The following pedagogic features and updates in the 12th edition help facilitate student learning.

Embryology is organized into two parts. The first provides an overview of early development from gametogenesis through the embryonic period. Also included in this section are chapters on placental and fetal development as well as prenatal diagnosis and birth defects. The second part of the text provides a description of the fundamental processes of embryogenesis for each organ system.
Clinical
Correlates:
to describ-
ing normal events, each chapter contains clinical correlates that appear in highlighted boxes. This material is designed to demonstrate the clinical relevance of embryology and the importance of understanding key developmental events as a first step to improving birth outcomes and having healthier babies. Clinical pictures and case descriptions are used to provide this information and this material has been increased and updated in this edition.
Genetics: Because of the increasingly important roll of genetics and molecular biology in embryology and the study of birth defects, basic genetic and molecular principles are discussed. The first chapter provides an introduction to molecular pathways and defines key terms in genetics and molecular biology. Then, throughout the text, major signaling pathways and genes that regulate embryological development are identified and discussed.
Extensive
Art
Program:
Nearly
400
illustra- tions are used to enhance understanding of the text, including four-color line drawings, scanning electron micrographs, and clinical pictures. Additional color pictures of clinical cases have been added to enhance the clinical correlate sections.
Summary:
At the
end of
each chapter
is
a
sum- mary
that serves
described as
in detail
are highlighted
a
concise review
throughout and
Problems key
elements assist
of the
key the
chapter.
defined points
Key terms
in to
of the
of student
the an
material.
these summaries.
Solve:
Problems each
chapter in
related to
are assessing
their
Detailed appendix
at
Glossary:
in the
provided to
understanding answers
the
A
the back
of back
glossary the
book are
of the
book.
of key
and terms
has is
located been
expanded extensively.

thePoint Web site: This site for students and instructors provides the full text of the book and its figures online; an interactive question bank of USMLE board-type questions; and Simbryo animations that demonstrate normal embryological events and the origins of some birth defects. Simbryo offers six vector art animation modules to illustrate the complex, three-dimensional aspects of embryology. Modules include an overview of the normal stages of early embryogenesis, plus development of the head and neck and the genitourinary, cardiovascular, and pulmonary systems. Teaching aids for instructors will also be provided in the form of an image bank and a series of lectures on the major topics in embryology presented in PowerPoint with accompanying notes. I hope you find this edition of Langman' Medical Embryology to be an excellent resource for learning embryology and its clinical significance. Together the textbook and online site, thePoint, are designed to provide a user-friendly and innovative approach to understanding the subject.
TW. Sadler
Cc
O
N
T
Preface viii
Introduction / Embryology: Clinical Relevance and
Historical Perspective xii

Chapter | / Introduction to Molecular
Regulation and Signaling 3
Gene Transcription 3
Other Regulators of Gene Expression
5
Induction and Organ Formation 5
Cell Signaling 6
Chapter 2 / Gametogenesis: Conversion
of Germ Cells into Male and Female
Gametes 10
Primordial Germ Cells 10
The Chromosome Theory of Inheritance
||
Morphological Changes During Maturation
of the Gametes 21
Chapter 3 / First Week of Development:
Ovulation to Implantation 29
Ovarian Cycle 29
Fertilization 32
Cleavage 37
Blastocyst Formation 37
Uterus at Time of Implantation 39
Chapter 4 / Second Week of
Development: Bilaminar Germ Disc 43
Day 8 43
Day 9 43
Days || and 12 44
Day 13 46
Chapter 5 / Third Week of
Development: Trilaminar Germ Disc 51
Gastrulation: Formation of Embryonic Mesoderm
and Endoderm 51
Formation of the Notochord 51
Establishment of the Body Axes 52
Fate Map Established During Gastrulation 57
Growth of the Embryonic Disc 57
Further Development of the Trophoblast 59
E
N
T
S
Chapter 6 / Third to Eighth Weeks: The
Embryonic Period 63
Derivatives of the Ectodermal Germ Layer 63
Derivatives of the Mesodermal Germ Layer 70
Derivatives of the Endodermal Germ Layer 78
Patterning of the Anteroposterior Axis: Regulation by Homeobox Genes 81
External Appearance During the Second Month 81
Chapter
7
/
The
Gut
Tube and
the
Body
Cavities 86
A Tube on Top of a Tube 86
Formation of the Body Cavity 87
Serous Membranes 88
Diaphragm and Thoracic Cavity 90
Formation of the Diaphragm 92
Chapter 8 / Third Month to Birth: The
Fetus and Placenta 96
Development of the Fetus 96
Fetal Membranes and Placenta 100
Chorion Frondosum and Decidua Basalis 102
Structure of the Placenta 103
Amnion and Umbilical Cord 107

Placental Changes at the End of Pregnancy 108
Amniotic Fluid 109
Fetal Membranes in Twins 110
Parturition (Birth) 115
Chapter 9 / Birth Defects and Prenatal
Diagnosis 117
Birth Defects 117
Prenatal Diagnosis 125
Fetal Therapy 128
Part 2 Systems-Based
Embryology 13!
Chapter 10 / The Axial Skeleton 133
Skull 133
Vertebrae and the Vertebral Column 142
Ribs and Sternum 144
Chapter II / Muscular System 145
Striated Skeletal Musculature 145
Innervation of Axial Skeletal Muscles 146
Skeletal Muscle and Tendons 148
Molecular Regulation of Muscle Development 148
Patterning of Muscles 148
Head Musculature 148
Limb Musculature 148
Cardiac Muscle 149
Smooth Muscle 149
Chapter 12/ Limbs 151
Limb Growth And Development 151
Limb Musculature 152
Chapter 13 / Cardiovascular
System 162
Establishment and Patterning of the Primary Heart
Field 162
Formation and Position of the Heart Tube 164
Formation of the Cardiac Loop 166
Molecular Regulation of Cardiac Development 169
Development of the Sinus Venosus 170
Formation of the Cardiac Septa 171
Formation of the Conducting System of the

Vascular Development 185
Circulation Before and After Birth 195
Chapter 14 / Respiratory System 201
Formation of the Lung Buds 201
Larynx 203
Trachea, Bronchi, And Lungs 203
Maturation of the Lungs 205
Chapter I5 / Digestive System 208
Divisions of the Gut Tube 208
Molecular Regulation of Gut Tube
Development 209
Mesenteries 210
Foregut 211
Molecular Regulation of Liver Induction 219
Pancreas 221
Midgut 222
Hindgut 229
Chapter 16 / Urogenital System 232
Urinary System 232
Genital System 243
Chapter |7 / Head and Neck 260
Pharyngeal Arches 262
Pharyngeal Pouches 266
Pharyngeal Clefts 268
Molecular Regulation of Facial Development 268
Tongue 273
Thyroid Gland 274
Face 275
Intermaxillary Segment 278
Secondary Palate 278
Nasal Cavities 282
Teeth 283
Molecular Regulation of Tooth Development 285
Chapter 18 / Central Nervous
System 287
Spinal Cord 288
Brain 297
Molecular Regulation of Brain Development 308
Cranial Nerves 313
Autonomic Nervous System 315
Chapter 19/ Ear 321
Internal Ear 321
Middle Ear 324
External Ear 325
Chapter 20 / Eye 329
Optic Cup and Lens Vesicle 329
Retina, Iris, and Ciliary Body 331
Lens 333
Choroid, Sclera, and Cornea 333
Vitreous Body 333
Optic Nerve 334
Molecular Regulation of Eye Development 334
Chapter 21 / Integumentary System 339
Skin 339
Hair 341
Sweat Glands 342
Mammary Glands 342

Answers to Problems 347
Figure Credits 357
Glossary of Key Terms 361
Index 371
Placode: A local thickening in the embryonic ectoderm layer that develops into a sensory organ or ganglion.

There once was a flat sheet of cells
That were stumpy and ugly as hell;
But one day they arose, stood tall on their toes, and declared they were the best cells of all.
Presumptuously they cried that their lineage was high and right proudly they bragged of their codes;
But soon it was clear, they weren't like the ear and they were nixed in their dreams as placodes.
Semantics, they screamed, please maintain our dreams, but their pleas were unheeded and late;
And now to this day in repast they must lay as a misconstrued, flat neural plate!
TW.
Sadler
Twin Bridges, MT

From a single cell to a baby in 9 months (Fig. 1.1A,B); a developmental process that represents an amazing integration of increasingly complex phenomena. The study of these phenomena is called embryology, and the field includes investigations of the molecular, cellular, and structural factors contributing to the formation of an organism. These studies are important because they provide knowledge essential for creating health care strategies for better reproductive outcomes. Thus, our increasingly better understanding of embryology has resulted in new techniques for prenatal diagnoses and treatments, therapeutic procedures to circumvent problems with infertility, and mechanisms to prevent birth defects, the leading cause of infant mortality. These improvements in prenatal and reproductive health care are significant not only for their contributions to improved birth outcomes but also for their long-term effects postnatally. In fact, both our cognitive capacity and our behavioral characteristics are affected by our prenatal experiences, and factors such as maternal smoking, nutrition, stress, diabetes, etc., play a role in our postnatal health. Furthermore, these experiences, in combination with molecular and cellular factors, determine our potential to develop certain adult diseases, such as cancer and cardiovascular disease. Thus, our prenatal development produces many ramifications affecting our health for both the short and long term, making the study of embryology and fetal development an important topic for all health care professionals. Also, with the exception of a few specialties, most physicians and health care workers will have an opportunity to interact with women of childbearing age, creating the potential for these providers to have a major impact on the outcome of these developmental processes and their sequelae.

The process of progressing from a single cell through the period of establishing organ primordia
(the first 8 weeks of human development) is called the period of embryogenesis (sometimes called the period of organogenesis); the period from that point on until birth is called the fetal period, a time when differentiation continues while the fetus grows and gains weight. Scientific approaches to study embryology have progressed over hundreds of years. Not surprisingly, anatomical approaches dominated early investigations. Observations were made, and these became more sophisticated with advances in optical equipment and dissection techniques. Comparative and evolutionary studies were part of this equation as scientists made comparisons among species and so began to understand the progression of developmental phenomena. Also investigated were offspring with birth defects, and these were compared to organisms with normal developmental patterns. The study of the embryological origins and causes for these birth

In the 20th century, the field of experimental embryology blossomed. Numerous experiments were devised to trace cells during development to determine their cell lineages. These approaches included observations of transparent embryos from tunicates that contained pigmented cells that could be visualized through a microscope. Later, vital dyes were used to stain living cells to follow their fates. Still later in the 1960s, radioactive labels and autoradiographic techniques were employed. One of the first genetic markers also arose about this time with the creation of chickquail chimeras. In this approach, quail cells, which have a unique pattern to their heterochromatin distribution around the nucleolus, were grafted into chick embryos at early stages of development. Later, host embryos were examined histologically, and the fates of the quail cells were determined. Permutations of this approach included development of antibodies specific to quail cell antigens that greatly assisted in the identification of these cells. Monitoring cell fates with these and other techniques provides valuable information about the origins of different organs and tissues.
recognized independently by two clinicians, W. Lenz and W. McBride and showed that the conceptus was vulnerable to maternal factors that crossed the placenta. Soon, numerous animal models demonstrating an association between environmental factors, drugs, and genes provided further insights between developmental events and the origin of birth defects.
Today, molecular approaches have been added to the list of experimental paradigms used to study normal and abnormal development. Numerous means of identifying cells using reporter genes, fluorescent probes, and other marking techniques have improved our ability to map cell fates. Using other techniques to alter gene expression, such as knockout, knock-in, and antisense technologies has created new ways to produce abnormal development and allowed the study of a single gene's function in specific tissues. Thus, the advent of molecular biology has advanced the field of embryology to the next level, and as we decipher the roles of individual genes and their interplay with environmental factors, our understanding of normal and abnormal developmental processes progresses.

olecular biology has opened the doors to new ways to study embryology and to enhance our understanding of normal and abnormal development. Sequencing the human genome, together with creating techniques to investigate gene regulation at many levels of complexity, has taken embryology to the next level. Thus, from the anatomical to the biochemical to the molecular level, the story of embryology has progressed, and each chapter has enhanced our knowledge.
string of DNA and is referred to as heterochromatin. For transcription to occur, this DNA must be uncoiled from the beads. In this uncoiled state, chromatin is referred to as euchromatin.
There are approximately 23,000 genes in the human genome, which represents only one fifth of the number predicted prior to completion of the Human Genome Project. Because of various levels of regulation, however, the number of proteins derived from these genes is closer to the original predicted number of genes. What has been disproved is the one-gene—oneprotein hypothesis. Thus, through a variety of mechanisms, a single gene may give rise to many proteins.
Gene expression can be regulated at several levels: (1) different genes may be transcribed, (2) nuclear deoxyribonucleic acid (DNA) transcribed from a gene may be selectively processed to regulate which RNAs reach the cytoplasm to become messenger RNAs (mRNAs), (3) mRNAs may be selectively translated, and (4) proteins made from the mRNAs may be differentially modified.

Genes are contained in a complex of DNA and proteins (mostly histones) called chromatin, and its basic unit of structure is the nucleosome (Fig. 1.1). Each nucleosome is composed of an octamer of histone proteins and approximately 140 base pairs of DNA. Nucleosomes themselves are joined into clusters by binding of DNA existing between nucleosomes (linker DNA) with other histone proteins (H1 histones; Fig. 1.1). Nucleosomes keep the DNA tightly coiled, such that it cannot be transcribed. In this inactive state, chromatin appears as beads of nucleosomes on a
Genes reside within the DNA strand and contain regions called exons, which can be translated into proteins, and introns, which are interspersed between exons and which are not transcribed into proteins (Fig. 1.2). In addition to exons and introns, a typical gene includes the following: a promoter region that binds RNA polymerase for the initiation of transcription; a transcription initiation site; a translation initiation site to designate the first amino acid in the protein; a translation termination codon; and a 3' untranslated region that includes a sequence (the poly A addition site) that assists with stabilizing the mRNA, allows it to exit the nucleus, and permits it to be translated into protein (Fig. 1.2). By convention, the 5' and the 3' regions of a gene are specified in relation to the RNA transcribed from the gene. Thus, DNA is transcribed from the 5' to the 3' end, and the promoter region is upstream from the transcription initiation site (Fig. 1.2). The promoter region, where the RNA polymerase binds, usually contains the sequence TATA, and this site
Figure 1.1 Drawing showing nucleosomes that form the basic unit of chromatin. Each nucleosome consists of an octamer of histone proteins and approximately 140 base pairs of DNA. Nucleosomes are joined into clusters by linker DNA and other histone proteins.

Figure |.2 Drawing of a "typical" gene showing the promoter region containing the TATA box; exons that contain DNA sequences that are translated into proteins; introns; the transcription initiation site; the translation initiation site that designates the code for the first amino acid in a protein; and the 3' untranslated region that includes the poly A addition site that participates in stabilizing the mRNA, allows it to exit the nucleus, and permits its translation into a protein.
is called the TATA box (Fig. 1.2). In order to bind to this site, however, the polymerase requires additional proteins called transcription factors (Fig. 1.3). Transcription factors also have a specific DNA-binding domain plus a transactivating domain that activates or inhibits transcription of the gene whose promoter or enhancer it has bound. In combination with other proteins, transcription factors activate gene expression by causing the DNA nucleosome complex to unwind, by releasing the polymerase so that it can transcribe the DNA template, and by preventing new nucleosomes from forming.
appropriate tissue. Enhancers act by altering chromatin to expose the promoter or by facilitating binding of the RNA polymerase. Sometimes, enhancers can inhibit transcription and are called silencers. This phenomenon allows a transcription factor to activate one gene while silencing another by binding to different enhancers. Thus, transcription factors themselves have a DNAbinding domain specific to a region of DNA plus a transactivating domain that binds to a promoter or an enhancer and activates or inhibits the gene regulated by these elements.
Enhancers are regulatory elements of DNA that activate utilization of promoters to control their efficiency and the rate of transcription from the promoter. Enhancers can reside anywhere along the DNA strand and do not have to reside close to a promoter. Like promoters, enhancers bind transcription factors (through the transcription factor's transactivating domain) and are used to regulate the timing of a gene's expression and its cell-specific location. For example, separate enhancers in a gene can be used to direct the same gene to be expressed in different tissues. The PAX6 transcription factor, which participates in pancreas, eye, and neural tube development, contains three separate enhancers, each of which regulates the gene's expression in the

Methylation of cytosine bases in the promoter regions of genes represses transcription of those genes. Thus, some genes are silenced by this mechanism. For example, one of the X chromosomes in each cell of a female is inactivated (X chromosome inactivation) by this methylation mechanism. Similarly, genes in different types of cells are repressed by methylation, such that muscle cells make muscle proteins (their promoter DNA is mostly unmethylated), but not blood proteins (their DNA is highly methylated). In this manner, each cell can maintain its characteristic differentiated state. DNA methylation is also responsible for genomic imprinting in
Figure 1.3 Drawing showing binding of RNA polymerase II to the TATA box site of the promoter region of a gene. This binding requires a complex of proteins plus an additional protein called a transcription factor. Transcription factors have their own specific DNA-binding domain and function to regulate gene expression.
which only a gene inherited from the father or the mother is expressed, while the other gene is silenced. Approximately 40 to 60 human genes are imprinted and their methylation patterns are established during spermatogenesis and oogenesis. Methylation silences DNA by inhibiting binding of transcription factors or by altering histone binding resulting in stabilization of nucleosomes and tightly coiled DNA that cannot be transcribed.

The initial transcript of a gene is called nuclear RNA (nRNA) or sometimes premessenger RNA. nRNA is longer than mRNA because it contains introns that are removed (spliced out) as the nRNA moves from the nucleus to the cytoplasm. In fact, this splicing process provides a means for cells to produce different proteins from a single gene. For example, by removing different introns, exons are "spliced" in different patterns, a process called alternative splicing (Fig. 1.4). The process is carried out by spliceosomes, which are complexes of small nuclear RNAs (snRNAs) and proteins that recognize specific splice sites at the 5' or the 3' ends of the nRNA. Proteins derived from the same gene are called splicing isoforms (also called splice variants or alternative splice forms), and these afford the opportunity for different cells to use the same gene to make proteins specific for that cell type. For example, isoforms of the WT1 gene have different functions in gonadal versus kidney development.
Even after a protein is made (translated), there may be post-translational modifications that affect its function. For example, some proteins have to be cleaved to become active, or they might have to be phosphorylated. Others need to combine with other proteins or be released from sequestered sites or be targeted to specific cell regions. Thus, there are many regulatory levels for synthesizing and activating proteins, such that although only 23,000 genes exist, the potential number of proteins that can be synthesized is probably closer to five times the number of genes.

Organs are formed by interactions between cells and tissues. Most often, one group of cells or tissues causes another set of cells or tissues to change their fate,a process called induction. In each such interaction, one cell type or tissue is the inducer that produces a signal, and one is the responder to that signal. The capacity to respond to such a signal is called competence, and competence requires activation of the responding tissue by a competence factor. Many inductive interactions occur between epithelial and mesenchymal cells and are called epithelial-mesenchymal interactions (Fig. 1.5). Epithelial cells are joined together in tubes or sheets, whereas mesenchymal cells are fibroblastic in appearance and dispersed in extracellular matrices (Fig. 1.5). Examples of epithelial-mesenchymal interactions include the following: gut endoderm and surrounding mesenchyme to produce gut-derived organs, including the liver and pancreas; limb mesenchyme with overlying ectoderm (epithelium) to produce limb outgrowth and differentiation; and endoderm of the ureteric bud and mesenchyme from the metanephric blastema to produce nephrons in the kidney. Inductive interactions can also occur between two epithelial tissues,
Figure 1.4 Drawing of a hypothetical gene illustrating the process of alternative splicing to form different proteins from the same gene. Spliceosomes recognize specific sites on the initial transcript of nRNA from a gene. Based on these sites, different introns are "spliced out" to create more than one protein from a single gene. Proteins derived from the same gene are called splicing isoforms.
Figure |.5 Drawing illustrating an epithelial-mesenchymal interaction. Following an initial signal from one tissue, a second tissue is induced to differentiate into a specific structure. The first tissue constitutes the inducer, and the second is the responder. Once the induction process is initiated, signals (arrows) are transmitted in both directions to complete the differentiation process.
such as induction of the lens by epithelium of the optic cup. Although an initial signal by the inducer to the responder initiates the inductive event, crosstalk between the two tissues or cell types is essential for differentiation to continue (Fig. 1.5, arrows).

Cell-to-cell signaling is essential for induction, for conference of competency to respond, and for crosstalk between inducing and responding cells. These lines of communication are established by paracrine interactions, whereby proteins synthesized by one cell diffuse over short distances to interact with other cells, or by juxtacrine interactions, which do not involve diffusable proteins. The diffusable proteins responsible for paracrine signaling are called paracrine factors or growth and differentiation factors (GDFs).

Paracrine Signaling
Paracrine factors act by signal transduction pathways either by activating a pathway directly or by blocking the activity of an inhibitor of a pathway (inhibiting an inhibitor, as is the case with hedgehog signaling). Signal transduction pathways include a signaling molecule (the ligand) and a receptor (Fig. 1.6). The receptor spans the cell membrane and has an extracellular domain (the ligand-binding region), a transmembrane domain, and a cytoplasmic domain. When a ligand binds its receptor, it induces a conformational change in the receptor that activates its cytoplasmic domain. Usually, the result of this activation is to confer enzymatic activity to the receptor, and most often this activity is a kinase that can phosphorylate other proteins using ATP as a substrate. In turn, phosphorylation activates these proteins to phosphorylate additional proteins, and thus a cascade of protein interactions is established that ultimately activates a transcription factor. This transcription factor then activates or inhibits gene expression. The pathways are numerous and
Figure 1.6 Drawing of a typical signal transduction pathway involving a ligand and its receptor. Activation of the receptor is conferred by binding to the ligand. Typically, the activation is enzymatic involving a tyrosine kinase, although other enzymes may be employed. Ultimately, kinase activity results in a phosphorylation cascade of several proteins that activates a transcription factor for regulating gene expression.
complex and in some cases are characterized by one protein inhibiting another that in turn activates another protein (much like the situation with hedgehog signaling).

Juxtacrine signaling is mediated through signal transduction pathways as well but does not involve diffusable factors. Instead, there are three ways juxtacrine signaling occurs: (1) A protein on one cell surface interacts with a receptor on an adjacent cell in a process analogous to paracrine signaling (Fig. 1.6). The Notch pathway represents an example of this type of signaling. The Notch receptor protein extends across the cell membrane and binds to cells that have Delta, Serrate, or Jagged proteins in their cell membranes. Binding of one of these proteins to Notch causes a conformational change in the Notch protein such that part of it on the cytoplasmic side of the membrane is cleaved. The cleaved portion then binds to a transcription factor to activate gene expression. Notch signaling is especially important in neuronal differentiation, blood vessel specification, and somite segmentation. (2) Ligands in the extracellular matrix secreted by one cell interact with their receptors on neighboring cells. The extracellular matrix is the milieu in which cells reside. This milieu consists of large molecules secreted by cells including collagen, proteoglycans (chondroitin sulfates, hyaluronic acid, etc.), and glycoproteins, such as fibronectin and laminin. These molecules provide a substrate for cells on which they can anchor or migrate. For example, laminin and type IV collagen are components of the basal lamina for epithelial cell attachment, and fibronectin molecules form scaffolds for cell migration. Receptors that link extracellular molecules such as fibronectin and laminin to cells are called integrins. These receptors "integrate" matrix molecules with a cell's cytoskeletal machinery (e.g., actin microfilaments) thereby creating the ability to migrate along matrix scaffolding by using contractile proteins, such as actin. Also, integrins can induce gene expression and regulate differentiation as in the case of chondrocytes that must be linked to the matrix to form cartilage. (3) There is direct transmission of signals from one cell to another by gap junctions. These junctions occur as channels between cells through which small molecules and ions can pass. Such communication is important in tightly connected cells like epithelia of the gut and neural tube because they allow these cells to act in concert. The junctions themselves are

made of connexin proteins that form a channel, and these channels are "connected" between adjacent cells.
It is important to note that there is a great amount of redundancy built into the process of signal transduction. For example, paracrine signaling molecules often have many family members such that other genes in the family may compensate for the loss of one of their counterparts. Thus, the loss of function of a signaling protein through a gene mutation does not necessarily result in abnormal development or death. In addition, there is crosstalk between pathways, such that they are intimately interconnected. These connections provide numerous additional sites to regulate signaling.

There are a large number of paracrine signaling factors acting as ligands, which are also called GDFs. Most are grouped into four families, and members of these same families are used repeatedly to regulate development and differentiation of organ systems. Furthermore, the same GDFs regulate organ development throughout the animal kingdom from Drosophila to humans. The four groups of GDFs include the fibroblast growth factor (FGF), WNT, hedgehog, and transforming growth factor-B (TGF-B) families. Each family of GDFs interacts with its own family of receptors, and these receptors are as important as the signal molecules themselves in determining the outcome of a signal.

Originally named because they stimulate the growth of fibroblasts in culture, there are now approximately two dozen FGF genes that have been identified, and they can produce hundreds of protein isoforms by altering their RNA splicing or their initiation codons. FGF proteins produced by these genes activate a collection of tyrosine receptor kinases called fibroblast growth factor receptors (FGFRs). In turn, these receptors activate various signaling pathways. FGFs are particularly important for angiogenesis, axon growth, and mesoderm differentiation. Although there is redundancy in the family, such that FGFs can sometimes substitute for one another, individual FGFs may be responsible for specific developmental events. For example, FGF8 is important for development of the limbs and parts of the brain.

The hedgehog gene was named because it coded for a pattern of bristles on the leg of Drosophila that resembled the shape of a hedgehog. In
mammals, there are three hedgehog genes, Desert, Indian, and sonic hedgehog. Sonic hedgehog is involved in a number of developmental events including limb patterning, neural tube induction and patterning, somite differentiation, gut regionalization, and others. The receptor for the hedgehog family is Patched, which binds to a protein called Smoothened. The Smoothened protein transduces the hedgehog signal, but it is inhibited by Patched until the hedgehog protein binds to this receptor. Thus, the role of the paracrine factor hedgehog in this example is to bind to its receptor to remove the inhibition of a transducer that would normally be active, not to activate the transducer directly.

There are at least 15 different WNT genes that are related to the segment polarity gene, wingless in Drosophilia. Their receptors are members of the frizzled family of proteins. WNT proteins are involved in regulating limb patterning, midbrain development, and some aspects of somite and urogenital differentiation among other actions.

The TGF-B superfamily has more than 30 members and includes the TGF-Bs, the bone morphogenetic proteins, the activin family, the Miillerian inhibiting factor (MIF, anti-Millerian hormone), and others. The first member of the family, TGF-B1, was isolated from virally transformed cells. TGF-B members are important for extracellular matrix formation and epithelial branching that occurs in lung, kidney, and salivary gland development. The BMP family induces bone formation and is involved in regulating cell division, cell death (apoptosis), and cell migration among other functions.

Another group of paracrine signaling molecules important during development are neurotransmitters, including serotonin and norepinephrine, that act as ligands and bind to receptors just as proteins do. These molecules are not just transmitters for neurons, but also provide important signals for embryological development. For example, serotonin (5HT) acts as a ligand for a large number of receptors, most of which are G protein—coupled receptors. Acting through these receptors, 5HT regulates a variety of cellular functions, including cell proliferation and migration, and is important for establishing laterality, gastrulation, heart development, and other processes during early stages of differentiation. Norepinephrine also acts through receptors and appears to play a role in apoptosis (programmed cell death) in the interdigital spaces and in other cell types.
_
During the past century, embryology has progressed from an observational science to one involving sophisticated technological and molecular advances. Together, observations and modern techniques provide a clearer understanding of the origins of normal and abnormal development and, in turn, suggest ways to prevent and treat birth defects. In this regard, knowledge of gene function has created entire new approaches to the subject.

There are approximately 23,000 genes in the human genome, but these genes code for approximately 100,000 proteins. Genes are contained in a complex of DNA and proteins called chromatin, and its basic unit of structure is the nucleosome. Chromatin appears tightly coiled as beads of nucleosomes on a string and is called heterochromatin. For transcription to occur, DNA must be uncoiled from the beads as euchromatin. Genes reside within strands of DNA and contain regions that can be translated into proteins, called exons, and untranslatable regions, called introns. A typical gene also contains a promoter region that binds RNA polymerase for the initiation of transcription; a transcription initiation site, to designate the first amino acid in the protein; a translation termination codon; and a 3' untranslated region that includes a sequence (the poly A addition site) that assists with stabilization of the mRNA. The RNA polymerase binds to the promoter region that usually contains the sequence TATA, the TATA box. Binding requires additional proteins called transcription factors. Methylation of cytosine bases in the promoter region silences genes and prevents transcription. This process is responsible for X chromosome inactivation whereby the expression of genes on one of the X chromosomes in females is silenced and also for genomic imprinting in which either a paternal or a maternal gene's expression is repressed.
Different proteins can be produced from a single gene by the process of alternative splicing that removes different introns using spliceosomes. Proteins derived in this manner are called splicing isoforms or splice variants. Also, proteins may be altered by post-translational modifications, such as phosphorylation or cleavage.
Induction is the process whereby one group of
cells or
tissues
(the inducer)
causes another
neurotransmitters, such as serotonin (5HT) and norepinephrine, also act through paracrine signaling, serving as ligands and binding to receptors to produce specific cellular responses. Juxtacrine factors may include products of the extracellular matrix, ligands bound to a cell's surface, and direct cell-to-cell communications.

- 1. What is meant by "competence to respond" as part of the process of induction? What tissues are most often involved in induction? Give two examples.
2.
Under normal
conditions,
FGFs and
their receptors
(FGFRs)
of the
skull sutures.
and development
How be
might disrupted?
paracrine for
of the
these growth
cranial signaling
Do these
or pathways
juxtacrine think
of
a
FGF
way might
pathways involve
signaling?
Can that
loss be
of you
expression circumvented?
of one

Development begins with fertilization, the process by which the male gamete, the sperm, and the female gamete, the oocyte, unite to give rise to a zygote. Gametes are derived from primordial germ cells (PGCs) that are formed in the epiblast during the second week and that move to the wall of the yolk sac (Fig. 2.1). During the fourth week, these cells begin to migrate from the yolk sac toward the developing gonads, where they arrive by the end of the fifth week. Mitotic divisions increase their number during their migration and also when they arrive in the gonad. In preparation for fertilization, germ cells undergo gametogenesis, which includes meiosis, to reduce the number of chromosomes and cytodifferentiation to complete their maturation.
Figure 2.1 An embryo at the end of the third week, showing the position of PGCs in the wall of the yolk sac, close to the attachment of the future umbilical cord. From this location, these cells migrate to the developing gonad.

Teratomas are tumors of disputed origin that often contain a variety of tissues, such as bone, hair, muscle, gut epithelia, and others. It is thought that these tumors arise from pluripotent stem cells that can differentiate into any of the three germ layers or their derivatives. Some evidence suggests that PGCs that have strayed from their normal migratory paths could be responsible for some of these tumors (Fig. 2.2). Another source may be epiblast cells that give rise to all three germ layers during gastrulation (See Fig. 5.9, p. 58 and p. 59).
Figure 2.2 Oropharnyngeal teratoma. These tumors may arise from PGCs or from epiblast cells (see Chapter 5), both of which are pluripotent. Tissues within the tumors include derivatives of all three germ layers and may include gut, bone, skin, teeth, and so forth.

Traits of a new individual are determined by specific genes on chromosomes inherited from the father and the mother. Humans have approximately 23,000 genes on 46 chromosomes. Genes on the same chromosome tend to be inherited together and so are known as linked genes. In somatic cells, chromosomes appear as 23 homologous pairs to form the diploid number of 46. There are 22 pairs of matching chromosomes, the autosomes, and one pair of sex chromosomes. If the sex pair is XX, the individual is genetically female; if the pair is XY, the individual is genetically male. One chromosome of each pair is derived from the maternal gamete, the oocyte, and one from the paternal gamete, the sperm. Thus, each gamete contains a haploid number of 23 chromosomes, and the union of the gametes at fertilization restores the diploid number of 46.

Mitosis is the process whereby one cell divides, giving rise to two daughter cells that are genetically identical to the parent cell (Fig. 2.3). Each daughter cell receives the complete complement of 46 chromosomes. Before a cell enters mitosis, each chromosome replicates its deoxyribonucleic acid (DNA). During this replication phase,
II
chromosomes are extremely long, they are spread diffusely through the nucleus, and they cannot be recognized with the light microscope. With the onset of mitosis, the chromosomes begin to coil, contract, and condense; these events mark the beginning of prophase. Each chromosome now consists of two parallel subunits, chromatids, that are joined at a narrow region common to both called the centromere. Throughout prophase, the chromosomes continue to condense, shorten, and thicken (Fig. 2.3.4), but only at prometaphase do the chromatids become distinguishable (Fig. 2.3B). During metaphase, the chromosomes line up in the equatorial plane, and their doubled structure is clearly visible (Fig. 2.3C). Each is attached by microtubules extending from the centromere to the centriole, forming the mitotic spindle. Soon, the centromere of each chromosome divides, marking the beginning of anaphase, followed by migration of chromatids to opposite poles of the spindle. Finally, during telophase, chromosomes uncoil and lengthen, the nuclear envelope reforms, and the cytoplasm divides (Fig. 2.3D-—F). Each daughter cell receives half of all doubled chromosome material and thus maintains the same number of chromosomes as the mother cell.

Meiosis is the cell division that takes place in the germ cells to generate male and female gametes,
Figure 2.3 Various stages of mitosis. In prophase, chromosomes are visible as slender threads. Doubled chromatids become
clearly visible
as individual
units during
metaphase.At no
time during
division do
members of
a
chromosome pair
sperm and egg cells, respectively. Meiosis requires two cell divisions, meiosis I and meiosis II, to reduce the number of chromosomes to the haploid number of 23 (Fig. 2.4). As in mitosis, male and female germ cells (spermatocytes and primary oocytes) at the beginning of meiosis I replicate their DNA so that each of the 46 chromosomes is duplicated into sister chromatids. In contrast to mitosis, however, homologous chromosomes then align themselves in pairs, a process called synapsis. The pairing is exact and point for point except for the XY combination. Homologous pairs then separate into two daughter cells, thereby reducing the chromosome number from diploid to haploid. Shortly thereafter, meiosis II separates sister chromatids. Each gamete then contains 23 chromosomes.

paired homologous chromosomes (Fig. 2.4C). Segments of chromatids break and are exchanged as homologous chromosomes separate. As separation occurs, points of interchange are temporarily united and form an X-like structure, a chiasma (Fig. 2.4C). The approximately 30 to 40 crossovers (one or two per chromosome) with each meiotic I division are most frequent between genes that are far apart on a chromosome.
As a result of meiotic divisions:

° crossover, which redistributes genetic material
e
random distribution
of homologous
mosomes to the daughter cells
Each germ
cell contains
a
haploid number
of chromosomes,
so that
at fertilization
diploid number of 46 is restored.

Crossovers, critical events in meiosis I, are the interchange of chromatid segments between
Also during meiosis, one primary oocyte gives rise to four daughter cells, each with 22 plus
Figure 2.4 First and second meiotic divisions. A. Homologous chromosomes approach each other. B. Homologous chromosomes pair, and each member of the pair consists of two chromatids. C. Intimately paired homologous chromosomes interchange chromatid fragments (crossover). Note the chiasma. D. Double-structured chromosomes pull apart. E. Anaphase of the first meiotic division. F,G. During the second meiotic division, the double-structured chromosomes split at the centromere.At completion of division, chromosomes in each of the four daughter cells are different from each other.
the chro-
e
Figure 2.5 Events occurring during the first and second maturation divisions. A. The primitive female germ cell (primary oocyte) produces only one mature gamete, the mature oocyte. B. The primitive male germ cell (primary spermatocyte) produces four spermatids, all of which develop into spermatozoa.
1 X chromosomes (Fig. 2.54). Only one of these develops into a mature gamete, however, the oocyte; the other three, the polar bodies, receive little cytoplasm and degenerate during subsequent development. Similarly, one primary spermatocyte gives rise to four daughter cells, two with 22 plus 1 X chromosomes and two with 22 plus 1 Y chromosomes (Fig. 2.5B). In contrast to oocyte formation, however, all four develop into mature gametes.

Chromosomal abnormalities, which may be numerical or structural, are important causes of birth defects and spontaneous abortions. It is estimated that 50% of conceptions end in spontaneous abortion and that 50% of these abortuses have major chromosomal abnormalities. Thus, approximately 25% of conceptuses have a major chromosomal defect. The most common chromosomal abnormalities in abortuses are 45, X (Turner syndrome), triploidy, and trisomy 16. Chromosomal abnormalities account for 10% of major birth defects, and gene mutations account for an additional 8%.

The normal human somatic cell contains 46 chromosomes; the normal gamete contains 23. Normal somatic cells are diploid, or 2n; normal gametes are haploid, or n. Euploid refers to any exact
multiple of n (e.g., diploid or triploid). Aneuploid refers to any chromosome number that is not euploid; it is usually applied when an extra chromosome is present (trisomy) or when one is missing (monosomy). Abnormalities in chromosome number may originate during meiotic or mitotic divisions. In meiosis, two members of a pair of homologous chromosomes normally separate during the first meiotic division, so that each daughter cell receives one member of each pair (Fig. 2.6A). Sometimes, however, separation does not occur (nondisjunction), and both members of a pair move into one cell (Fig. 2.6B,C).As a result of nondisjunction of the chromosomes, one cell receives 24 chromosomes, and the other receives 22 instead of the normal 23.When, at fertilization, a gamete having 23 chromosomes fuses with a gamete having 24 or 22 chromosomes, the result is an individual with either 47 chromosomes (trisomy) or 45 chromosomes (monosomy). Nondisjunction, which occurs during either the first or the second meiotic division of the germ cells, may involve the
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Figure 2.6 A. Normal maturation divisions. B. Nondisjunction in the first meiotic division. C. Nondisjunction in the second meiotic division.
autosomes or sex chromosomes. In women, the incidence of chromosomal abnormalities, including nondisjunction, increases with age, especially at 35 years and older.
13, 14, 15, 21, and 22 because they cluster during meiosis.
Occasionally, nondisjunction occurs during mitosis (mitotic nondisjunction) in an embryonic cell during the earliest cell divisions. Such conditions produce mosaicism, with some cells having an abnormal chromosome number and others being normal. Affected individuals may exhibit few or many of the characteristics of a particular syndrome, depending on the number of cells involved and their distribution.

Sometimes chromosomes break, and pieces of one chromosome attach to another. Such translocations may be balanced, in which case breakage and reunion occur between two chromosomes, but no critical genetic material is lost and individuals are normal; or they may be unbalanced, in which case part of one chromosome is lost, and an altered phenotype is produced. For example, unbalanced translocations between the long arms of chromosomes 14 and 21 during meiosis | or Il produce gametes with an extra copy of chromosome 21, one of the causes of Down syndrome (Fig. 2.7). Translocations are particularly common between chromosomes
TRISOMY 21 (DOWN SYNDROME) Down syndrome is caused by an extra copy of chromosome 21 (trisomy 21) (Fig. 2.8). Features of children with Down syndrome include growth retardation; varying degrees of intellectual disability; craniofacial abnormalities, including upward slanting eyes, epicanthal folds (extra skin folds at the medial corners of the eyes), flat facies, and small ears; cardiac defects; and hypotonia (Fig. 2.9). These individuals also have an increased chance of developing leukemia, infections, thyroid dysfunction, and premature aging. Furthermore, an increased frequency and earlier onset of Alzheimer's disease is observed among persons with Down syndrome. In 95% of cases, the syndrome is caused by trisomy 21 resulting from meiotic nondisjunction, and in 75% of these instances, nondisjunction occurs during oocyte formation. The incidence of Down syndrome is approximately one in 2,000 conceptuses for women under age 25. This risk increases with maternal age to | in 300 at age 35 and | in 100 at age 40.
In approximately 4% of cases of Down syndrome, there is an unbalanced translocation
Figure 2.7 A. Translocation of the long arms of chromosomes 14 and 21 at the centromere. Loss of the short arms is not clinically significant, and these individuals are clinically normal, although they are at risk for producing offspring with unbalanced translocations. B. Karyotype of translocation of chromosome 21 onto 14, resulting in Down syndrome.

between chromosome 21 and chromosome 13, 14, 15, or 21 (Fig. 2.7).The final 1% is caused by mosaicism resulting from mitotic nondisjunction. These individuals have some cells with a normal chromosome number and some that are aneuploid. They may exhibit few or many of the characteristics of Down syndrome.
and malformations of the skeletal system. The incidence of this condition is approximately | in 5,000 newborns. Eighty-five percent are lost between 10 weeks of gestation and term, whereas those born alive usually die by 2 months of age. Approximately 5% live beyond | year.
TRISOMY 18 Patients with trisomy 18 show the following features: intellectual disability, congenital heart defects, low-set ears, and flexion of fingers and hands (Fig. 2.10). In addition, patients frequently show micrognathia, renal anomalies, syndactyly,
TRISOMY 13 The main abnormalities of trisomy 13 are intellectual disability, holoprosencephaly, congenital heart defects, deafness, cleft lip and palate, and eye defects, such as microphthalmia, anophthalmia, and coloboma (Fig. 2.11). The incidence of this abnormality is approximately | in 20,000 live
Figure 2.8 Karyotype of trisomy 21, Down syndrome.
Figure 2.9 A. Child with Down syndrome. Note the flat broad face, oblique palpebral fissures, and protruding tongue. Children with Down syndrome usually have some degree of intellectual disability and many have cardiac defects. B. Another characteristic of these children is a broad hand with a single transverse (simian) crease.
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Figure 2.10 Child with trisomy 18. Note the low-set ears, small mouth, deficient mandible (micrognathia), flexion of the hands, and absent and/or hypoplasia of the radius and ulna.
births, and more than 90% of the infants die in the first month after birth. Approximately 5% live beyond | year.

KLINEFELTER SYNDROME The clinical features of Klinefelter syndrome, found only in males and usually detected by amniocentesis, are sterility, testicular atrophy, hyalinization of the seminiferous tubules, and usually gynecomastia. The cells have 47 chromosomes with a sex chromosomal complement of the XXY type, and a sex chromatin (Barr) body is found in 80% of cases. (Barr body: formed by condensation of an inactivated X chromosome; a Barr body is also present in normal females because one of the X chromosomes is normally inactivated). The incidence is approximately | in 500 males. Nondisjunction of the XX homologues is the most common causative event. Occasionally, patients with Klinefelter syndrome have 48 chromosomes: 44 autosomes and 4 sex chromosomes (48, XXXY). Although intellectual
Figure 2.11 Child with trisomy 13. Note the bilateral cleft lip, the sloping forehead, and anophthalmia.
disability is not generally part of the syndrome, the more X chromosomes there are, the more likely there will be some degree of cognitive impairment.
TURNER SYNDROME Turner syndrome, with a 45, X karyotype, is the only monosomy compatible with life. Even then, 98% of all fetuses with the syndrome are spontaneously aborted. The few that survive are unmistakably female in appearance (Fig. 2.12) and are characterized by the absence of ovaries (gonadal dysgenesis) and short stature. Other common associated abnormalities are webbed neck, lymphedema of the extremities, skeletal deformities,and a broad chest with widely spaced nipples. Approximately 55% of affected females are monosomic for the X and chromatin body negative because of nondisjunction. In 80% of these females, nondisjunction in the male gamete is the cause. In the remainder of females, structural abnormalities of the X chromosome or mitotic nondisjunction resulting in mosaicism are the causes.
TRIPLE X SYNDROME Patients with triple X syndrome (47, XXX) often go undiagnosed because of their mild physical features. However, these girls frequently have problems with speech

Figure 2.12 Patient with Turner syndrome. A. At birth. Note the loose skin at the posterior of the neck caused by the remains of a cystic hygroma (fluid-filled cyst), the short neck, malformed ears, and swelling in the hand B and the foot C caused by lymphedema. D. At 6 years of age, the webbed neck is prominent and the nipples are widely spaced with a broad chest.
and self-esteem. They have two sex chromatin bodies in their cells.

Structural chromosome abnormalities, which involve one or more chromosomes, usually result from chromosome breakage. It has been suggested that breaks are caused by environmental factors, such as viruses, radiation, and drugs, but the evidence is inconclusive. The result of breakage depends on what happens to the broken pieces. In some cases, the broken piece of a chromosome is lost, and the infant with partial deletion of a chromosome is abnormal. A well-known syndrome, caused by partial deletion of the short arm of chromosome 5, is
the cri-du-chat syndrome. Affected infants have a cat-like cry, microcephaly (small head), intellectual disability, and congenital heart disease. Many other relatively rare syndromes are known to result from a partial chromosome deletion.
Microdeletions, spanning only a _ few contiguous genes, may result in microdeletion syndrome or contiguous gene syndrome. Sites where these deletions occur, called contiguous gene complexes, are usually identified by fluorescence in situ hybridization (FISH; see p. 20). An example of a microdeletion occurs on the long arm of chromosome I5 (I5ql1—I5qI3 [Note: chromosomes have a long arm, designated "gq," and a short arm, designated "p,'" based on the position of the centromere.]). When the microdeletion occurs on the maternal chromosome, it results in Angelman's syndrome, and the children have intellectual disability, cannot speak, exhibit poor motor development, and are prone to unprovoked and prolonged periods of
Figure 2.13 Patient with Angelman's syndrome resulting from a microdeletion on maternal chromosome 15. If the defect is inherited on the paternal chromosome, Prader—Willi's syndrome occurs (Fig. 2.14).

laughter (Fig. 2.13). If the microdeletion occurs on the paternal chromosome, Prader-Willi syndrome results. Affected individuals are characterized by hypotonia, obesity, intellectual disability, hypogonadism, and undescended testes (Fig. 2.14). Characteristics that are differentially expressed depending upon whether the genetic material is inherited from the mother or the father are examples of genomic imprinting. Other contiguous gene syndromes may be inherited from either parent, including Miller-Dieker syndrome (lissencephaly, developmental delay, seizures, and cardiac and facial abnormalities resulting from a deletion at 17p13) and most cases of 22q1 1 syndrome (palatal defects, conotruncal heart defects, speech delay, learning disorders, and schizophrenia-like disorder resulting from a deletion in 22q1 1).
Fragile sites are regions of chromosomes that demonstrate a propensity to separate or break under certain cell manipulations. For example,
Figure 2.14 Patient with Prader-Willi syndrome resulting from a microdeletion on paternal chromosome 15. If the defect is inherited on the maternal chromosome, Angelman's syndrome occurs (Fig. 2.13).
fragile sites can be revealed by culturing lymphocytes from a patient in folate-deficient medium. Although numerous fragile sites have been defined and consist of CGG repeats, only those in the FMRI gene on the long arm of the X chromosome (Xq27) have been correlated with an altered phenotype that is called the fragile X syndrome. Greater than 200 repeats occur in the promoter region of the gene in affected individuals compared to 6 to 54 repeats in normal subjects. Fragile X syndrome is characterized by intellectual disability, large ears, prominent jaw, and large testes. The syndrome occurs in | per 5,000 individuals and, since it is an X-linked condition, males are affected almost exclusively, which may account for the preponderance of males among the cognitively impaired. Fragile X syndrome is second only to Down syndrome as a cause of intellectual disability due to genetic abnormalities.

Many congenital malformations in humans are inherited, and some show a clear Mendelian pattern of inheritance. Many birth defects are directly attributable to a change in the structure or function of a single gene, hence the name single gene mutation. This type of defect is estimated to account for approximately 8% of all human malformations.
With the exception of the X and Y chromosomes in the male, genes exist as pairs, or alleles, so that there are two doses for each genetic determinant: one from the mother and one from the father. If a mutant gene produces an abnormality in a single dose, despite the presence of a normal allele, it is a dominant mutation. If both alleles must be abnormal (double dose) or if the mutation is X-linked (occurs on the X chromosome) in the male, it is a recessive mutation. Variations in the effects of mutant genes may be a result of modifying factors.
The application of molecular biological techniques has increased our knowledge of genes responsible for normal development. In turn, genetic analysis of human syndromes has shown that mutations in many of these same genes are responsible for some congenital abnormalities and childhood diseases. Thus, the link between key genes in development and their role in clinical syndromes is becoming clearer.
In addition to causing congenital malformations, mutations can result in inborn errors of metabolism. These diseases, among which phenylketonuria, homocystinuria, and galactosemia are the best known, may be accompanied by or cause various degrees of intellectual disability if proper diets and medical care are not instituted.

Cytogenetic analysis is used to assess chromosome number and integrity. The technique requires dividing cells, which usually means establishing cell cultures that are arrested in metaphase by chemical treatment. Chromosomes are Giemsa-stained to reveal light and dark banding patterns (G-bands; Fig. 2.7) unique for each chromosome. Each band represents 5 to 10 x 10° base pairs of DNA, which may include a few to several hundred genes. Recently, high-resolution metaphase banding techniques have been developed that demonstrate greater numbers of bands representing even smaller pieces of DNA, thereby facilitating diagnosis of small deletions.
Molecular techniques, such as FISH, use specific DNA probes to identify ploidy for a few selected chromosomes and for detecting microdeletions. Fluorescent probes are hybridized to chromosomes or genetic loci using cells on a slide, and the results are visualized with a fluorescence microscope (Fig. 2.15).
Microarrays use spots of specific DNA sequences (probes) attached to a solid surface, usually glass or silicon (Affimetrix chips). These probes may be a short sequence from a gene or other DNA element that are used to hybridize a cDNA or cRNA sample (the target sample). Hybridization of probe-target sequences are detected and quantified using fluorescence or other reporter techniques. Results can detect single nucleotide polymorphisms, mutations, and changes in expression levels. Some companies now offer such techniques commercially for anyone who wants their genome tested or sequenced.
Figure 2.15 A. FISH, using a probe for chromosome 21 (red dots). Note that there are three red dots in each cell, indicating trisomy 21 (Down syndrome).The green dots represent a control probe for chromosome 1|3.Two cells are superimposed on the lower right, giving the impression of the presence of multiple probes. B. FISH analysis of 22q1 | deletion syndrome. The green signals identify chromosome 22; the red signal represents FISH probe N25, which is in the ql region. It is present on only one of the pairs of chromosome 22 indicating the other has the 22qI1 deletion.

epithelial cells, known as follicular cells, originate from surface epithelium covering the ovary.

Oogenesis is the process whereby oogonia differentiate into mature oocytes.

Once PGCs have arrived in the gonad of a genetic female, they differentiate into oogonia (Fig. 2.16A,B). These cells undergo a number of mitotic divisions, and by the end of the third month, they are arranged in clusters surrounded by a layer of flat epithelial cells (Figs. 2.17 and 2.18). Whereas all of the oogonia in one cluster are probably derived from a single cell, the flat
The majority of oogonia continue to divide by mitosis, but some of them arrest their cell division in prophase of meiosis I and form primary oocytes (Figs. 2.16C and 2.17A). During the next few months, oogonia increase rapidly in number, and by the fifth month of prenatal development, the total number of germ cells in the ovary reaches its maximum, estimated at 7 million. At this time, cell death begins, and many oogonia as well as primary oocytes degenerate and become atretic. By the seventh month, the majority of oogonia have degenerated except for a few near the surface. All surviving primary oocytes have entered prophase of meiosis I, and most of them are individually surrounded by a layer of flat
Figure 2.16 Differentiation of PGCs into oogonia begins shortly after their arrival in the ovary. By the third month of development, some oogonia give rise to primary oocytes that enter prophase of the first meiotic division. This prophase may last 40 or more years and finishes only when the cell begins its final maturation. During this period, it carries 46 double-structured chromosomes.
Figure 2.17 Segment of the ovary at different stages of development. A. Oogonia are grouped in clusters in the cortical part of the ovary. Some show mitosis; others have differentiated into primary oocytes and entered prophase of the first meiotic division. B. Almost all oogonia are transformed into primary oocytes in prophase of the first meiotic division. C. There are no oogonia. Each primary oocyte is surrounded by a single layer of follicular cells, forming the primordial follicle. Oocytes have entered the diplotene stage of prophase, in which they remain until just before ovulation. Only then do they enter metaphase of the first meiotic division.
follicular epithelial

cells
(Fig.
2.17B).
A
primary oocyte,
together with
its surrounding
flat epithelial
cells, is
known as
a
primordial follicle
(Fig.
2.18).
Maturation of Oocytes Continues at Puberty
Near the
time of
birth, all
primary oocytes
started prophase
ing of
into metaphase,
a
resting ized
meiosis
I, but
they stage
instead enter
during by
a
Primary lacy
oocytes finish
their first
This arrested
ration network
the prophase
have of
proceed- diplotene
stage, that
of is
character- chromatin
remain arrested
meiotic state
(Fig.
in prophase
division before
2.17C).
and puberty
by do
not is
oocyte reached.
matu- inhibitor
(OMI),
a
small peptide
secreted by follicular cells. The total number of primary oocytes at birth is estimated to vary from 600,000 to 800,000. During childhood, most oocytes become atretic; only approximately 40,000 are present by the beginning of puberty, and fewer than 500 will be ovulated. Some oocytes that reach maturity late in life have been dormant in the diplotene stage of the first meiotic division for 40 years or more before ovulation. Whether the diplotene stage is the most suitable phase to protect the oocyte against environmental influences is unknown.The fact that the risk of having children with chromosomal abnormalities increases with maternal age indicates that primary oocytes are vulnerable to damage as they age.
Figure 2.18 A. Primordial follicle consisting of a primary oocyte surrounded by a layer of flattened epithelial cells. B. Early primary or preantral stage follicle recruited from the pool of primordial follicles.As the follicle grows, follicular cells become cuboidal and begin to secrete the zona pellucida, which is visible in irregular patches on the surface of the oocyte. C. Mature primary (preantral) follicle with follicular cells a stratified of cells around the and the of a well-defined zona forming layer granulosa oocyte presence pellucida.

At puberty, a pool of growing follicles is established and continuously maintained from the supply of primordial follicles. Each month, 15 to 20 follicles selected from this pool begin to mature. Some of these die, while others begin to accumulate fluid in a space called the antrum, thereby entering the antral or vesicular stage (Fig. 2.19). Fluid continues to accumulate such that, immediately prior to ovulation, follicles are quite swollen and are called mature vesicular follicles or Graffian follicles (Fig. 2.19B). The antral stage is the longest, whereas the mature vesicular stage encompasses approximately 37 hours prior to ovulation.
As development continues, fluid-filled spaces appear between granulosa cells. Coalescence of these spaces forms the antrum, and the follicle is termed a vesicular or an antral follicle. Initially, the antrum is crescent-shaped, but with time, it enlarges (Fig. 2.19). Granulosa cells surrounding the oocyte remain intact and form the cumulus oophorus. At maturity, the mature vesicular (Graafian) follicle may be 25 mm or more in diameter. It is surrounded by the theca interna, which is composed of cells having characteristics of steroid secretion, rich in blood vessels, and the theca externa, which gradually merges with the ovarian connective tissue (Fig. 2.19).

As primordial follicles begin to grow, surrounding follicular cells change from flat to cuboidal and proliferate to produce a stratified epithelium of granulosa cells, and the unit is called a primary follicle (Fig. 2.18B,C). Granulosa cells rest on a basement membrane separating them from surrounding ovarian connective tissue (stromal cells) that form the theca folliculi. Also, granulosa cells and the oocyte secrete a layer of glycoproteins on the surface of the oocyte, forming the zona pellucida (Fig. 2.18C). As follicles continue to grow, cells of the theca folliculi organize into an inner layer of secretory cells, the theca interna, and an outer fibrous capsule, the theca externa. Also, small, finger-like processes of the follicular cells extend across the zona pellucida and interdigitate with microvilli of the plasma membrane of the oocyte. These processes are important for transport of materials from follicular cells to the oocyte.
With each ovarian cycle, a number of follicles begin to develop, but usually only one reaches full maturity. The others degenerate and become atretic. When the secondary follicle is mature, a surge in luteinizing hormone (LH) induces the preovulatory growth phase. Meiosis I is completed, resulting in formation of two daughter cells of unequal size, each with 23 double-structured chromosomes (Fig. 2.20A,B). One cell, the secondary oocyte, receives most of the cytoplasm; the other, the first polar body, receives practically none. The first polar body lies between the zona pellucida and the cell membrane of the secondary oocyte in the perivitelline space (Fig. 2.20B). The cell then enters meiosis II but arrests in metaphase approximately 3 hours before ovulation. Meiosis II is completed only if the oocyte is fertilized; otherwise, the cell degenerates approximately 24 hours after ovulation. The first polar body may undergo a second division (Fig. 2.20C).

Figure 2.19 A. Vesicular (antral) stage follicle. The oocyte, surrounded by the zona pellucida, is off center; the antrum has developed by fluid accumulation between intercellular spaces. Note the arrangement of cells of the theca interna and the theca externa. B. Mature vesicular (Graafian) follicle. The antrum has enlarged considerably, is filled with follicular fluid, and is surrounded by a stratified layer of granulosa cells. The oocyte is embedded in a mound of granulosa cells, the cumulus oophorus.
Figure 2.20 Maturation of the oocyte. A. Primary oocyte showing the spindle of the first meiotic division. B. Secondary oocyte and first polar body. The nuclear membrane is absent. C. Secondary oocyte showing the spindle of the second meiotic division. The first polar body is also dividing.

Maturation of Sperm Begins at Puberty Spermatogenesis, which begins at puberty, includes all of the events by which spermatogonia are transformed into spermatozoa. At birth, germ cells in the male infant can be recognized in the sex cords of the testis as large, pale cells surrounded by supporting cells (Fig. 2.21). Supporting cells, which are derived from the surface epithelium of the testis in the same manner as follicular cells, become sustentacular cells, or Sertoli cells (Fig. 2.21B).
Shortly before puberty, the sex cords acquire a lumen and become the seminiferous tubules. At about the same time, PGCs give rise to spermatogonial stem cells. At regular intervals, cells emerge from this stem cell population to form type A spermatogonia, and their production marks the initiation of spermatogenesis. Type A cells undergo a limited number of mitotic divisions to form clones of cells. The last cell division produces type B spermatogonia, which then divide to form primary spermatocytes (Figs. 2.21B and 2.22). Primary
Figure 2.21 A. Cross section through primitive sex cords of a newborn boy showing PGCs and supporting cells.
B.
Cross section
through
a
seminiferous tubule
at puberty.
Note the
different stages
of spermatogenesis
and that
considerably greater than depicted in this figure.
divi-

spermatocytes then enter a prolonged prophase (22 days) followed by rapid completion of meiosis I and formation of secondary spermatocytes. During the second meiotic division, these cells immediately begin to form haploid spermatids (Figs. 2.21B to 2.23). Throughout this series of events, from the time type A cells leave the stem cell population to formation of spermatids, cytokinesis is incomplete, so that successive cell generations are joined by cytoplasmic bridges. Thus, the progeny of a single type A spermatogonium form a clone of germ cells that maintain contact throughout differentiation (Fig. 2.22). Furthermore, spermatogonia and spermatids remain embedded in deep recesses of Sertoli cells throughout their development (Fig. 2.21B). In this manner, Sertoli cells support and protect the germ cells, participate in their nutrition, and assist in the release of mature spermatozoa.
binding to Sertoli cells stimulates testicular fluid production and synthesis of intracellular androgen receptor proteins.

The series of changes resulting in the transformation of spermatids into spermatozoa is spermiogenesis. These changes include (1) formation of the acrosome, which covers half of the nuclear surface and contains enzymes to assist in penetration of the egg and its surrounding layers during fertilization (Fig. 2.24); (2) condensation of the nucleus; (3) formation of neck, middle piece, and tail; and (4) shedding of most of the cytoplasm as residual bodies that are phagocytized by Sertoli cells. In humans, the time required for a spermatogonium to develop into a mature spermatozoon is approximately 74 days, and approximately 300 million sperm cells are produced daily.
Spermatogenesis is regulated by LH production by the pituitary gland. LH binds to receptors on Leydig cells and stimulates testosterone production, which in turn binds to Sertoli cells to promote spermatogenesis. Follicle-stimulating hormone (FSH) is also essential because its
When fully formed, spermatozoa enter the lumen of seminiferous tubules. From there, they are pushed toward the epididymis by contractile elements in the wall of the seminiferous tubules. Although initially only slightly motile, spermatozoa obtain full motility in the epididymis.

In humans and in most mammals, one ovarian follicle occasionally contains two or three clearly distinguishable primary oocytes (Fig. 2.25A). Although these oocytes may give rise to twins or triplets, they usually degenerate before reaching maturity. In rare cases, one primary oocyte contains two or even three nuclei (Fig. 2.25B). Such binucleated or trinucleated oocytes die before reaching maturity.
ao at
In contrast to atypical oocytes, abnormal spermatozoa are seen frequently, and up to 10% of all spermatozoa have observable defects. The head or the tail may be abnormal, spermatozoa may be giants or dwarfs, and sometimes they are joined (Fig. 2.25C). Sperm with morphologic abnormalities lack normal motility and probably do not fertilize oocytes
Figure 2.25 Abnormal germ cells. A. Primordial follicle with two oocytes. B. Trinucleated oocyte. C. Various types of abnormal
spermatozoa.
PGCs appear in the wall of the yolk sac in the fourth week and migrate to the indifferent gonad (Fig. 2.1), where they arrive at the end of the fifth week. In preparation for fertilization, both male and female germ cells undergo gametogenesis, which includes meiosis and cytodifferentiation. During meiosis I, homologous chromosomes pair and exchange genetic material; during meiosis II, cells fail to replicate DNA, and each cell is thus provided with a haploid number of chromosomes and half the amount of DNA of a normal somatic cell (Fig. 2.4). Hence, mature male and female gametes have 22 plus X or 22 plus Y chromosomes, respectively.
Birth defects may arise through abnormalities in chromosome number or structure and from single gene mutations. Approximately 10% of major birth defects are a result of chromosome abnormalities, and 8% are a result of gene mutations. Trisomies (an extra chromosome) and monosomies (loss of a chromosome) arise during mitosis or meiosis. During meiosis, homologous chromosomes normally pair and then separate. If separation fails (nondisjunction), however, one cell receives too many chromosomes, and one receives too few (Fig. 2.6).

The incidence of abnormalities of chromosome number increases with age of the mother, particularly with mothers aged 35 years and older. Structural abnormalities of chromosomes include large deletions (cri-du-chat syndrome) and microdeletions. Microdeletions involve contiguous genes that may result in defects such as Angelman's syndrome (maternal deletion, chromosome 15q11—-15q13) or Prader-Willi syndrome (paternal deletion, 15q11—15q13). Because these syndromes depend on whether the affected genetic material is inherited from the mother or the father, they are also an example of imprinting. Gene mutations may be dominant (only one gene of an allelic pair has to be affected to produce an alteration) or recessive (both allelic gene pairs must be mutated). Mutations responsible for many birth defects affect genes involved in normal embryological development.
In the female, maturation from primitive germ cell to mature gamete, which is called oogenesis, begins before birth; in the male, it is called spermatogenesis, and it begins at puberty. In the female, PGCs form oogonia. After repeated mitotic divisions, some of these arrest in prophase of meiosis I to form primary oocytes. By the seventh month, many oogonia have become atretic, and only primary oocytes
remain surrounded by a layer of follicular cells derived from the surface epithelium of the ovary (Fig. 2.17). Together, they form the primordial follicle. At puberty, a pool of growing follicles is recruited and maintained from the finite supply of primordial follicles. Thus, every month, 15 to 20 follicles begin to grow, and as they mature, they pass through three stages: (1) primary or preantral, (2) vesicular or antral, and (3) mature vesicular or Graafian follicle. The primary oocyte remains in prophase of the first meiotic division until the secondary follicle is mature. At this point, a surge in LH stimulates preovulatory growth: Meiosis I is completed, and a secondary oocyte and polar body are formed. Then, the secondary oocyte is arrested in metaphase of meiosis II approximately 3 hours before ovulation and will not complete this cell division until fertilization.

In the male, primordial cells remain dormant until puberty, and only then do they differentiate into spermatogonia. These stem cells give rise to primary spermatocytes, which through two successive meiotic divisions produce four spermatids (Fig. 2.5). Spermatids go through a series of changes (spermiogenesis) (Fig. 2.24), including (1) formation of the acrosome; (2) condensation of the nucleus; (3) formation of neck, middle piece, and tail; and (4) shedding of most of the cytoplasm. The time required for a spermatogonium to become a mature spermatozoon is approximately 74 days.

1.
What
is
the
most
common
cause
of
abnor-
mal
chromosome
number?
Give
an
example
of a clinical syndrome involving abnormal
numbers of chromosomes.
2.
In
addition
to
numerical
abnormalities,
what
types of chromosomal alterations occur?
3. What is mosaicism, and how does it occur?

At puberty, the female begins to undergo regular monthly cycles. These sexual cycles are controlled by the hypothalamus. Gonadotropinreleasing hormone (GnRH), produced by the hypothalamus, acts on cells of the anterior lobe (adenohypophysis) of the pituitary gland, which in turn secrete gonadotropins. These hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), stimulate and control cyclic changes in the ovary.
FSH also stimulates maturation of follicular (granulosa) cells surrounding the oocyte. In turn, proliferation of these cells is mediated by growth differentiation factor 9,a member of the transforming growth factor-B (TGFB) family. In cooperation, theca interna and granulosa cells produce estrogens: theca interna cells produce androstenedione and testosterone, and granular cells convert these hormones to estrone and 17 B-estradiol. As a result of this estrogen production,
At the beginning of each ovarian cycle, 15 to 20 primary-stage (preantral) follicles are stimulated to grow under the influence of FSH. (The hormone is not necessary to promote development of primordial follicles to the primary follicle stage, but without it, these primary follicles die and become atretic.) Thus, FSH rescues 15 to 20 of these cells from a pool of continuously forming primary follicles (Figs. 3.1 and 3.2). Under normal conditions, only one of these follicles reaches full maturity, and only one oocyte is discharged; the others degenerate and become atretic. In the next cycle, another group of primary follicles is recruited, and again, only one follicle reaches maturity. Consequently, most follicles degenerate without ever reaching full maturity. When a follicle becomes atretic, the oocyte and surrounding follicular cells degenerate and are replaced by connective tissue, forming a corpus atreticum.

During ovulation, some women feel a slight pain, called mittelschmerz (German for "middle pain") because it normally occurs near the middle of the menstrual cycle. Ovulation is also generally accompanied by a rise in basal temperature, which can be monitored to aid couples in becoming pregnant or preventing pregnancy. Some women fail to
e The uterine endometrium enters the follicular or proliferative phase;
e Thinning of the cervical mucus occurs to allow passage of sperm; and
e The anterior lobe of the pituitary gland is stimulated to secrete LH.
At midcycle, there is an LH surge that:
e Elevates concentrations of maturation-promoting factor, causing oocytes to complete meiosis I and initiate meiosis II;
¢ Stimulates production of progesterone by follicular stromal cells (luteinization); and
© Causes follicular rupture and ovulation.
In the days immediately preceding ovulation, under the influence of FSH and LH, the vesicular follicle grows rapidly to a diameter of 25 mm to become a mature vesicular (graafian)
ovulate because of a low concentration of gonadotropins. In these cases, administration of an agent to stimulate gonadotropin release, and hence ovulation, can be employed. Although such drugs are effective, they often produce multiple ovulations, so that the likelihood of multiple pregnancies is 10 times higher in these women than in the general population.
“7
Figure 3.2 A. Primordial follicle. B. Growing follicle. C. Vesicular follicle. Every day from the pool of primordial follicles A, some begin to develop into growing follicles B, and this growth is independent of FSH. Then, as the cycle progresses, FSH secretion recruits growing follicles to begin development into vesicular (antral) follicles. C. During the last few days of maturation of vesicular follicles, estrogens, produced by follicular and thecal cells, stimulate increased production of LH by the pituitary gland (Fig. 3.1), and this hormone causes the follicle to enter the mature vesicular (graafian) stage, to complete meiosis |, and to enter meiosis II, where it is arrested in metaphase approximately 3 hours before ovulation.

follicle. Coincident with final development of the vesicular follicle, there is an abrupt increase in LH that causes the primary oocyte to complete meiosis I and the follicle to enter the preovulatory mature vesicular stage. Meiosis II is also initiated, but the oocyte is arrested in metaphase approximately 3 hours before ovulation. In the meantime, the surface of the ovary begins to bulge locally, and at the apex, an avascular spot, the stigma, appears. The high concentration of LH increases collagenase activity, resulting in digestion of collagen fibers surrounding the follicle. Prostaglandin levels also increase in response to the LH surge and cause local muscular contractions in the ovarian wall. Those contractions extrude the oocyte, which together with its surrounding granulosa cells from the region of the cumulus oophorus breaks free (ovulation) and floats out of the ovary (Fig. 3.3). Some of the cumulus oophorus cells then rearrange themselves around the zona pellucida to form the corona radiata (Figs. 3.2B to 3.6).

After ovulation, granulosa cells remaining in the wall of the ruptured follicle, together with cells from the theca interna, are vascularized by surrounding vessels. Under the influence of LH, these cells develop a yellowish pigment and change into lutein cells, which form the corpus luteum and secrete estrogens and progesterone (Fig. 3.3C). Progesterone, together with some estrogen, causes the uterine mucosa to enter the progestational or secretory stage in preparation for implantation of the embryo.
A
Figure 3.3 A. Mature vesicular follicle bulging at the ovarian surface. B. Ovulation. The oocyte, in metaphase of meiosis Il, is discharged from the ovary together with a large number of cumulus oophorus cells. Follicular cells remaining inside the collapsed follicle differentiate into lutean cells. C. Corpus luteum. Note the large size of the corpus luteum, caused by hypertrophy and accumulation of lipid in granulosa and theca interna cells. The remaining cavity of the follicle is filled with fibrin.
Figure 3.4 Relation of fimbriae and ovary. Fimbriae collect the oocyte and sweep it into the uterine tube.

Shortly before ovulation, fimbriae of the uterine tube sweep over the surface of the ovary, and the tube itself begins to contract rhythmically. It is thought that the oocyte, surrounded by some granulosa cells (Figs. 3.3B and 3.4), is carried into the tube by these sweeping movements of the fimbriae and by motion of cilia on the epithelial lining. Once in the tube, cumulus cells withdraw their cytoplasmic processes from the zona pellucida and lose contact with the oocyte.
Once the oocyte is in the uterine tube, it is propelled by peristaltic muscular contractions of the tube and by cilia in the tubal mucosa with the rate of transport regulated by the endocrine status during and after ovulation. In humans, the fertilized oocyte reaches the uterine lumen in 3 to 4
approximately days.

If fertilization does not occur, the corpus luteum reaches maximum development approximately 9 days after ovulation. It can easily be recognized as a yellowish projection on the surface of the ovary. Subsequently, the corpus luteum shrinks because of degeneration of lutean cells (luteolysis) and forms a mass of fibrotic scar tissue, the corpus albicans. Simultaneously, progesterone production decreases, precipitating menstrual bleeding. If the oocyte is fertilized, degeneration of the corpus luteum is prevented by human chorionic gonadotropin, a hormone secreted by the syncytiotrophoblast of the developing embryo. The corpus luteum continues to grow and forms the corpus luteum of pregnancy (corpus luteum graviditatis). By the end of the third month, this structure may be one third to one half of the total size of the ovary. Yellowish luteal cells continue to secrete progesterone until the end of the fourth month; thereafter, they regress slowly as secretion of progesterone by the trophoblastic component of the placenta becomes adequate for maintenance of pregnancy. Removal of the corpus luteum of pregnancy before the fourth month usually leads to abortion.

Fertilization, the process by which male and female gametes fuse, occurs in the ampullary region of the uterine tube. This is the widest part of the tube and is close to the ovary (Fig. 3.4). Spermatozoa may remain viable in the female reproductive tract for several days.
Only 1% of sperm deposited in the vagina enter the cervix, where they may survive for many hours. Movement of sperm from the cervix to the uterine tube occurs by muscular contractions of the uterus and uterine tube and very little by their own propulsion. The trip from cervix to oviduct can occur as rapidly as 30 minutes or as slow as 6 days. After reaching the isthmus, sperm become less motile and cease their migration. At ovulation, sperm again
become tractants
the egg,
ization motile,
perhaps produced
because by
cumulus and
swim usually
to fertilize
in to
occurs.
the oocyte
the female
genital
(1)
capacitation and
tion to
acquire this
Capacitation the
cells of
chemoat- surrounding
the ampulla,
where
Spermatozoa fertil-
are not
immediately upon
tract but
able arrival
must
(2)
the undergo
acrosome capability.
is
a
period female
of reproductive
lasts approximately
tract
7
conditioning that
reac- in
in the
human hours.
Thus, speeding
to the
ampulla is not an advantage, since capacitation has not yet occurred and such sperm are not capable of fertilizing the egg. Much of this conditioning during capacitation occurs in the uterine tube and involves epithelial interactions between the sperm and the mucosal surface of the tube. During this time, a glycoprotein coat and seminal plasma proteins are removed from the plasma membrane that overlies the acrosomal region of the spermatozoa. Only capacitated sperm can pass through the corona cells and undergo the acrosome reaction.

Figure 3.5 A. Scanning electron micrograph of sperm binding to the zona pellucida. B. The three phases of oocyte penetration. In phase |, spermatozoa pass through the corona radiata barrier; in phase 2, one or more spermatozoa penetrate the zona pellucida; in phase 3, one spermatozoon penetrates the oocyte membrane while losing its own plasma membrane. Inset shows normal spermatocyte with acrosomal head cap.
Figure 3.6 A. Oocyte immediately after ovulation, showing the spindle of the second meiotic division. B. A spermatozoon has penetrated the oocyte, which has finished its second meiotic division. Chromosomes of the oocyte are arranged in a vesicular nucleus, the female pronucleus. Heads of several sperm are stuck in the zona pellucida. C. Male and female pronuclei. D,E. Chromosomes become arranged on the spindle, split longitudinally, and move to opposite poles. F. Two-cell stage.
The acrosome reaction, which occurs after binding to the zona pellucida, is induced by zona proteins. This reaction culminates in the release of enzymes needed to penetrate the zona pellucida, including acrosinand trypsin-like substances (Fig. 3.5).
The phases of fertilization include
Phase 1, penetration of the corona radiata
Phase 2, penetration of the zona pellucida
Phase 3, fusion of the oocyte and sperm cell membranes

enzymes (acrosin) allows sperm to penetrate the zona, thereby coming in contact with the plasma membrane of the oocyte (Fig. 3.5). Permeability of the zona pellucida changes when the head of the sperm comes in contact with the oocyte surface. This contact results in release of lysosomal enzymes from cortical granules lining the plasma membrane of the oocyte. In turn, these enzymes alter properties of the zona pellucida (zona reaction) to prevent sperm penetration and inactivate species-specific receptor sites for spermatozoa on the zona surface. Other spermatozoa have been found embedded in the zona pellucida, but only one seems to be able to penetrate the oocyte (Fig. 3.6).
Of the 200 to 300 million spermatozoa normally deposited in the female genital tract, only 300 to 500 reach the site of fertilization. Only one of these fertilizes the egg. It is thought that the others aid the fertilizing sperm in penetrating the barriers protecting the female gamete. Capacitated sperm pass freely through corona cells (Fig. 3.5).

The zona is a glycoprotein shell surrounding the egg that facilitates and maintains sperm binding and induces the acrosome reaction. Both binding and the acrosome reaction are mediated by the ligand ZP3, a zona protein. Release of acrosomal

The initial adhesion of sperm to the oocyte is mediated in part by the interaction of integrins on the oocyte and their ligands, disintegrins, on sperm. After adhesion, the plasma membranes of the sperm and egg fuse (Fig. 3.5). Because the plasma membrane covering the acrosomal head cap disappears during the acrosome reaction, actual fusion is accomplished between the oocyte membrane and the membrane that covers the posterior region of the sperm head (Fig. 3.5). In the human, both the head and the tail of the spermatozoon enter the cytoplasm of the oocyte, but
Figure 3.7 A. Phase contrast view of the pronuclear stage of a fertilized human oocyte with male and female pronuclei. B. Two-cell stage of human zygote.
the plasma membrane is left behind on the oocyte surface. As soon as the spermatozoon has entered the oocyte, the egg responds in three ways:
cellular and molecular events associated with early embryogenesis.
1 Cortical and zona reactions. As a result of the release of cortical oocyte granules, which contain lysosomal enzymes, (1) the oocyte membrane becomes impenetrable to other spermatozoa, and (2) the zona pellucida alters its structure and composition to prevent sperm binding and penetration. These reactions prevent polyspermy (penetration of more than one spermatozoon into the oocyte).
2 Resumption of the second meiotic division. The oocyte finishes its second meiotic division immediately after entry of the spermatozoon. One of the daughter cells, which receives hardly any cytoplasm, is known as the second polar body; the other daughter cell is the definitive oocyte. Its chromosomes (22 plus X) arrange themselves in a vesicular nucleus known as the female pronucleus (Figs. 3.6 and 3.7).
3 Metabolic activation of the egg. The activating factor is probably carried by the spermatozoon. Activation encompasses the initial

Barrier methods of contraception include the male condom, made of latex and often containing chemical spermicides, which fits over the penis, and the female condom, made of polyurethane, which lines the vagina. Other barriers placed in the vagina include the diaphragm, the cervical cap, and the contraceptive sponge.
The spermatozoon, meanwhile, moves forward until it lies close to the female pronucleus. Its nucleus becomes swollen and forms the male pronucleus (Fig. 3.6); the tail detaches and degenerates. Morphologically, the male and female pronuclei are indistinguishable, and eventually, they come into close contact and lose their nuclear envelopes (Fig. 3.7A). During growth of male and female pronuclei (both haploid), each pronucleus must replicate its DNA. If it does not, each cell of the two-cell zygote has only half of the normal amount of DNA. Immediately after DNA synthesis, chromosomes organize on the spindle in preparation for a normal mitotic division. The 23 maternal and 23 paternal (double) chromosomes split longitudinally at the centromere, and sister chromatids move to opposite poles, providing each cell of the zygote with the normal diploid number of chromosomes and DNA (Fig. 3.6D,E).As sister chromatids move to opposite poles, a deep furrow appears on the surface of the cell, gradually dividing the cytoplasm into two parts (Figs. 3.6F and 3.7B).
Hormonal methods are another commonly used form of contraception. These approaches provide the female hormones estrogen and/or progestin. These hormones produce their effects by inhibiting ovulation (by preventing the release of FSH and LH from the pituitary gland), changing the lining of the uterus, and thickening cervical mucus, making it difficult for sperm to enter the uterus.
Hormonal contraception can be provided through birth control pills, a skin patch, vaginal ring, injection, or implant. There are two types of contraceptive pills: The first is a combination of estrogen and the progesterone analogue progestin; the second is comprised of progestin alone. Both pills are effective, but one may suit some women better than others for various health-related issues.

A male "pill" has been developed and tested in clinical trials. It contains a synthetic androgen that prevents both LH and FSH secretion and either stops sperm production (70% to 90% of men) or reduces it to a level of infertility.
The intrauterine device (IUD) is a small T-shaped unit and there are two types: hormonal and copper. The hormonal device releases progestin that causes thickening of cervical mucus to prevent sperm from entering the uterus. Also, it may make sperm less active and both eggs and sperm less viable. The copper type releases copper into the uterus that prevents fertilization or inhibits attachment of the fertilized egg to the uterine wall. It also helps prevent sperm from entering the uterine tubes.
Emergency contraceptive pills (ECPs) are used as birth control measures that may prevent pregnancy if taken 120 hours after sexual intercourse.These pills may be administered as high doses of progestin alone or in combination with estrogen (Plan B). Other types of ECPs (mifepristone [RU486] and ulipristal acetate [ella]) act as antihormonal agents. Also, mifepristone is effective as an abortifacient if taken after the time of implantation.
Sterilization is another form of birth control. The method for men is a vasectomy, which prevents the release of sperm by blocking the ductus deferens, the tube that transports sperm from the testes to the penis. The sterilization method for women is tubal sterilization in which the uterine tubes are blocked or ligated. These procedures for both men and women can be reversed in some cases.

Infertility is a problem for 15% to 30% of couples. Male infertility may be a result of insufficient numbers of sperm and/or poor motility. Normally, the ejaculate has a volume of 2 to 6 mL, with as many as 100 million sperm per milliliter. Men with 20 million sperm per milliliter or 50 million sperm per total ejaculate are usually fertile. Infertility in a woman may be due to a number of causes, including occluded uterine tubes (most commonly caused by pelvic inflammatory disease), hostile cervical mucus, immunity to spermatozoa, absence of ovulation, and others.
One percent of all pregnancies in the United States occur using assisted reproductive technology (ART). Offspring from these conceptions show increases in prematurity (<37 weeks' gestation), low birth weight (<2,500 g), very low birth weight (<1,500g), and some types of birth defects. Most of these adverse outcomes are caused by increased rates of multiple births (twins, triplets, etc.), which occur more commonly among ART pregnancies. Recent studies indicate, however, that even among singleton births after ART, there are increases in preterm birth and malformed infants. Some of the approaches used for ART include the following:
In vitro fertilization (IVF) of human ova and embryo transfer is the standard procedure used by laboratories throughout the world. Follicle growth in the ovary is stimulated by administration of gonadotropins. Oocytes are recovered by laparoscopy from ovarian follicles with an aspirator just before ovulation when the oocyte is in the late stages of the first meiotic division. The egg is placed in a simple culture medium, and sperm are added immediately. Fertilized eggs are monitored to the eight-cell stage and then placed in the uterus to develop to term.
The success rate of IVF depends upon maternal age. Approximately 30% of couples will conceive after one attempt if the woman is younger than 35 years of age. The rate drops to 25% for women aged 35 to 37, 17% for those aged 38 to 40, and to <5% for those over 40, In addition to these relatively low success rates, the technique is also associated with a higher rate of congenital malformations. To increase chances of a successful pregnancy, four or five ova are collected, fertilized, and placed in the uterus. This approach sometimes leads to multiple births.

The frequency of multiple births depends on maternal age (with a higher incidence in younger women) and the number of embryos transferred. For a 20to 29-year-old woman and three embryos transferred, the risk is 46%. Multiple births are a disadvantage because they have high rates of morbidity and mortality.
Severe male infertility, in which the ejaculate contains very few live sperm (oligozoospermia) or even no live sperm (azoospermia), can be overcome using intracytoplasmic sperm injection. With this technique, a single sperm, which may be obtained from any point in the male reproductive tract, is injected into the cytoplasm of the egg to cause fertilization. This approach offers couples an alternative to using donor sperm for IVF. The technique carries an increased risk for fetuses to have Y chromosome deletions but no other chromosomal abnormalities.
e
Two-cell stage
Four-cell stage
Morula
Figure 3.8 Development of the zygote from the two-cell stage to the late morula stage. The two-cell stage is reached approximately 30 hours after fertilization; the four-cell stage is reached at approximately 40 hours; the |2to |6-cell stage is reached at approximately 3 days; and the late morula stage is reached at approximately 4 days. During this period, blastomeres are surrounded by the zona pellucida, which disappears at the end of the fourth day.
The main results of fertilization are as follows:

chromosomes, half
from the
father and
half from
the mother.
a
new
Hence, combination
the zygote
of chromosomes
contains different
from both
parents.

An
X-carrying sperm
produces female
(XX)
embryo, produces
a
male and
a
(XY)
chromosomal sex
Y-carrying embryo.
of the
a
sperm
Therefore, embryo
the is
determined at
fertilization.
e
Initiation of cleavage. Without fertilization, the oocyte usually degenerates 24 hours after ovulation.
blastomeres (Fig. 3.8). Until the eight-cell stage, they form a loosely arranged clump (Fig. 3.9). After the third cleavage, however, blastomeres maximize their contact with each other, forming a compact ball of cells held together by tight junctions (Fig.3.9B).This process, compaction, segregates inner cells, which communicate extensively by gap junctions, from outer cells. Approximately 3 days after fertilization, cells of the compacted embryo divide again to form a 16-cell morula (mulberry). Inner cells of the morula constitute the inner cell mass, and surrounding cells compose the outer cell mass. The inner cell mass gives rise to tissues of the embryo proper, and the outer cell mass forms the trophoblast, which later contributes to the placenta.

Once the zygote has reached the two-cell stage, it undergoes a series of mitotic divisions, increasing the numbers of cells. These cells, which become smaller with each cleavage division, are known as

About the time the morula enters the uterine cavity, fluid begins to penetrate through the zona pellucida into the intercellular spaces of the inner cell mass. Gradually, the intercellular spaces
Figure 3.9 Scanning electron micrographs of A uncompacted and B compacted eight-cell mouse embryos. In the uncompacted state, outlines of each blastomere are distinct, whereas after compaction, cell-cell contacts are maximized,
Figure 3.10 A. Section of a 107-cell human blastocyst showing inner cell mass and trophoblast cells. B. Schematic representation of a human blastocyst recovered from the uterine cavity at approximately 4.5 days. Blue, inner cell mass or embryoblast; green, trophoblast. C. Schematic representation of a blastocyst at the sixth day of development showing trophoblast cells at the embryonic pole of the blastocyst penetrating the uterine mucosa. The human blastocyst begins to penetrate the uterine mucosa by the sixth day of development.

become confluent, and finally, a single cavity, the blastocele, forms (Fig. 3.10A,B). At this time, the embryo is a blastocyst. Cells of the inner cell mass, now called the embryoblast, are at one pole, and those of the outer cell mass, or trophoblast, flatten and form the epithelial wall of the blastocyst (Fig. 3.10A,B). The zona pellucida has disappeared, allowing implantation to begin. In the human, trophoblastic cells over the embryoblast pole begin to penetrate between the epithelial cells of the uterine mucosa on about the sixth day (Fig. 3.10C). New studies suggest that L selectin on trophoblast cells and its carbohydrate receptors on the uterine epithelium mediate initial attachment of the blastocyst to the uterus. Selectins are carbohydrate-binding _ proteins involved in interactions between leukocytes and endothelial cells that allow leukocyte "capture" from flowing blood. A similar mechanism is now proposed for "capture" of the blastocyst from the uterine cavity by the uterine epithelium. Following capture by selectins, further attachment and invasion by the trophoblast involve integrins, expressed by the trophoblast and the extracellular matrix molecules laminin and fibronectin. Integrin receptors for laminin promote attachment, while those for fibronectin stimulate migration. These molecules also interact along signal transduction pathways to regulate trophoblast differentiation, so that implantation is the result of mutual trophoblastic and endometrial action. Hence, by the end of the first week

Embryonic stem cells (ES cells) are derived from the inner cell mass of the embryo. Because these cells are pluripotent and can form virtually any cell or tissue type, they have the potential for curing a variety of diseases, including diabetes, Alzheimer's and Parkinson's diseases, anemias, spinal cord injuries, and many others. Using animal model research with stem cells has been encouraging. For example, mouse ES cells in culture have been induced to form insulin-secreting cells, muscle and nerve stem cells, and glial cells. In whole animals, ES cells have been used to alleviate the symptoms of Parkinson's disease and to improve motor ability in rats with spinal cord injuries.
ES cells may be obtained from embryos after IVF,a process called reproductive cloning. This approach has the disadvantage that the cells may cause immune rejection, because they would not be genetically identical to their hosts. The cells could be modified to cir cumvent this problem, however. Another issue with this approach is based on ethical considerations, as the cells are derived from viable embryos.
As the field of stem cell research progresses, scientific advances will provide more genetically compatible cells, and the approaches will be less controversial. Most recently, techniques have been devised to take nuclei from adult cells (e.g., skin) and introduce them into enucleated oocytes. This approach is called therapeutic cloning or somatic nuclear transfer. Oocytes are stimulated to differentiate into blastocysts, and ES cells are harvested. Because the cells are derived from the host, they are compatible genetically, and because fertilization is not involved, the technique is less controversial.
of development, the human zygote has passed through the morula and blastocyst stages and has begun implantation in the uterine mucosa.

The wall of the uterus consists of three layers:
1 Endometrium or mucosa lining the inside wall
2 Myometrium, a thick layer of smooth muscle
4

Adult tissues contain stem cells that may also prove valuable in treating diseases. These cells are restricted in their ability to form different cell types and, therefore, are multipotent, not pluripotent, although scientists are finding methods to circumvent this disadvantage. Adult stem cells isolated from rat brains have been used to cure Parkinson's disease in rats, suggesting that the approach has promise. Disadvantages of the approach include the slow rates of cell division characteristic of these cells and their scarcity, which makes them difficult to isolate in sufficient numbers for experiments.

The exact number of abnormal zygotes formed is unknown because they are usually lost within 2 to 3 weeks of fertilization, before the woman realizes she is pregnant, and therefore are not detected. Estimates are that as many as 50% of pregnancies end in spontaneous abortion and that half of these losses are a result of chromosomal abnormalities. These abortions are a natural means of screening embryos for defects, reducing the incidence of congenital malformations. Without this phenomenon, approximately 12% instead of 2% to 3% of infants would have birth defects.
With the use of a combination of IVF and polymerase chain reaction, molecular screening of embryos for genetic defects is being conducted. Single blastomeres from early-stage embryos can be removed, and their DNA can be amplified for analysis. As the Human Genome Project provides more sequencing information, and as specific genes are linked to various syndromes, such procedures will become more commonplace.
- 3 Perimetrium, the peritoneal covering lining the outside wall (Fig. 3.11)
From puberty (11 to 13 years) until menopause (45 to 50 years), the endometrium undergoes changes in a cycle of approximately 28 days under hormonal control by the ovaries. During this menstrual cycle, the uterine endometrium passes through three stages, the
1 Follicular or proliferative phase
2 Secretory or progestational phase
3 Menstrual phase (Figs. 3.12 and 3.13)
Endometrium (progestational stage)

Figure 3.11 Events during the first week of human development. |, oocyte immediately after ovulation; 2, fertilization, approximately 12 to 24 hours after ovulation; 3, stage of the male and female pronuclei; 4, spindle of the first mitotic division; 5, two-cell stage (approximately 30 hours of age); 6, morula containing 12 to 16 blastomeres (approximately 3 days of age); 7, advanced morula stage reaching the uterine lumen (approximately 4 days of age); 8, early blastocyst stage (approximately 4.5 days of age; the zona pellucida has disappeared); and 9, early phase of implantation (blastocyst approximately 6 days of age). The ovary shows stages of transformation between a primary follicle and a preovulatory follicle as well as a corpus luteum. The uterine endometrium is shown in the progestational stage.
The proliferative phase begins at the end of the menstrual phase, is under the influence of estrogen, and parallels growth of the ovarian follicles. The secretory phase begins approximately 2 to 3 days after ovulation in response to progesterone produced by the corpus luteum. If fertilization does not occur, shedding of the endometrium (compact and spongy layers) marks the beginning of the menstrual phase. If fertilization does occur, the endometrium assists in implantation and contributes to formation of the placenta. Later in gestation, the placenta assumes the role of hormone production, and the corpus luteum degenerates.
At the time of implantation, the mucosa of the uterus
is in
the secretory
phase
(Fig.
3.12), during
which time
uterine glands
and arteries
become coiled
and the
tissue becomes
succulent.
As
a
Figure 3.12 Changes in the uterine mucosa correlated with those in the ovary. Implantation of the blastocyst has caused development of a large corpus luteum of pregnancy. Secretory activity of the endometrium increases gradually as a result of
Figure
3.13
Changes in
mucosa
(endometrium)
changes in
the ovary
during
a
regular menstrual
cycle without

result, three distinct layers can be recognized in the endometrium: a superficial compact layer, an intermediate spongy layer, and a thin basal layer (Fig. 3.12). Normally, the human blastocyst implants in the endometrium along the anterior or posterior wall of the body of the uterus, where it becomes embedded between the openings of the glands (Fig. 3.12).
If the oocyte is not fertilized, venules and sinusoidal spaces gradually become packed with blood cells, and an extensive diapedesis of blood into the tissue is seen. When the menstrual phase begins, blood escapes from superficial arteries, and small pieces of stroma and glands break away. During the following 3 or 4 days, the compact and spongy layers are expelled from the uterus, and the basal layer is the only part of the endometrium that is retained (Fig. 3.13). This layer, which is supplied by its own arteries, the basal arteries, functions as the regenerative layer in the rebuilding of glands and arteries in the proliferative phase (Fig. 3.13).
_-~
With each ovarian cycle, a number of primary follicles begin to grow, but usually only one reaches full maturity, and only one oocyte is discharged at ovulation. At ovulation, the oocyte is in metaphase of the second meiotic division and is surrounded by the zona pellucida and some granulosa cells (Fig. 3.4). Sweeping action of tubal fimbriae carries the oocyte into the uterine tube. Before spermatozoa can fertilize the oocyte,
UU
they must
undergo
1 Capacitation, during which time a glycoprotein coat and seminal plasma proteins are removed from the spermatozoon head
2 The acrosome reaction, during which acrosinand trypsin-like substances are released to penetrate the zona pellucida
During fertilization, the spermatozoon must penetrate
42 Part | General Embryology
1 The corona radiata
2 The zona pellucida
3 The oocyte cell membrane (Fig. 3.5)
As soon as the spermatocyte has entered the oocyte,
1 The oocyte finishes its second meiotic divi- sion and forms the female pronucleus

2 The zona pellucida becomes impenetrable to other spermatozoa
3 The head of the sperm separates from the tail, swells, and forms the male pronucleus (Figs. 3.6 and 3.7)
After both pronuclei have replicated their DNA, paternal and maternal chromosomes intermingle, split longitudinally, and go through a mitotic division, giving rise to the two-cell stage. The results of fertilization are
1
Restoration
of
the
diploid
number
of
2 Determination of chromosomal sex

Cleavage is a series of mitotic divisions that results in an increase in cells, blastomeres, which become smaller with each division. After three divisions, blastomeres undergo compaction to become a tightly grouped ball of cells with inner and outer layers. Compacted blastomeres divide to form a 16-cell morula. As the morula
enters the uterus on the third or fourth day after fertilization, a cavity begins to appear, and the blastocyst forms. The inner cell mass, which is formed at the time of compaction and will develop into the embryo proper, is at one pole of the blastocyst. The outer cell mass, which surrounds the inner cells and the blastocyst cavity, will form the trophoblast.
The the time of implantation is in
uterus at the secretory phase, and the blastocyst implants in the endometrium along the anterior or pos- terior wall (Fig. 3.12). If fertilization does not occur, then the menstrual phase begins, and the spongy and compact endometrial layers are shed. The basal layer remains to regenerate the other layers during the next cycle (Fig. 3.13).

1. What is the role of the corpus luteum, and what is its origin?
2. What are the three phases of fertilization, and what reactions occur once fusion of the sperm and oocyte membranes takes place?
3. What are the primary causes of infertility in men and women?
4. A woman has had several bouts of pelvic inflammatory disease and now wants to have children; however, she has been having diffi- culty becoming pregnant. What is likely to be the problem, and what would you suggest?

his chapter gives a day-by-day account of the major events of the second week of development; however, embryos of the same fertilization age do not necessarily develop at the same rate. Indeed, considerable differences in rate of growth have been found even at these early stages of development.

At the eighth day of development, the blastocyst is partially embedded in the endometrial stroma. In the area over the embryoblast, the trophoblast has differentiated into two layers: (1) an inner layer of mononucleated cells, the cytotrophoblast, and (2) an outer multinucleated zone without distinct cell boundaries, the syncytiotrophoblast (Figs. 4.1 and 4.2). Mitotic figures are found in the cytotrophoblast but not in the syncytiotrophoblast. Thus, cells in the cytotrophoblast divide and migrate into the syncytiotrophoblast, where they fuse and lose their individual cell membranes. Cells of the inner cell mass or embryoblast also differentiate into two layers: (1) a layer of small cuboidal cells adjacent to the blastocyst cavity, known as the hypoblast layer, and (2) a layer of high columnar cells adjacent to the amniotic cavity, the epiblast layer (Figs. 4.1 and 4.2). Together, the layers form a flat disc.At the same time, a small cavity appears within the epiblast. This cavity enlarges to become the amniotic cavity. Epiblast cells adjacent to the cytotrophoblast are called amnioblasts; together with the rest of the epiblast, they line the amniotic cavity (Figs. 4.1 and 4.3). The endometrial stroma adjacent to the implantation site is edematous and highly vascular. The large, tortuous glands secrete abundant glycogen and mucus.

The blastocyst is more deeply embedded in the endometrium, and the penetration defect in the surface epithelium is closed by a fibrin coagulum (Fig. 4.3). The trophoblast shows considerable progress in development, particularly at the embryonic pole, where vacuoles appear in the syncytium. When these vacuoles fuse, they form
Figure 4.1 A 7.5-day human blastocyst, partially embedded in the endometrial stroma. The trophoblast consists of an inner layer with mononuclear cells, the cytotrophoblast, and an outer layer without distinct cell boundaries, the syncytiotrophoblast. The embryoblast is formed by the epiblast and hypoblast layers. The amniotic cavity appears as a small cleft.
Figure 4.2 Section of a 7.5-day human blastocyst (x 100). Note the multinucleated appearance of the syncytiotrophoblast, large cells of the cytotrophoblast, and slit-like amniotic cavity.
large lacunae,
and this
phase of
trophoblast devel-

opment is
thus known
as the
lacunar stage
(Fig.
4.3).
At the abembryonic pole, meanwhile, flat- tened
cells probably
originating from
the hypo-
blast form
(Heuser’s)
a
thin membrane,
of the
that lines
cytotrophoblast together
exocoelomic the
(Fig.
4.3).
with the
hypoblast, the
inner surface
This membrane,
forms the
lining of
exocoelomic cavity,
or primitive
yolk sac.
By the 11th to the 12th day of development, the blastocyst is completely embedded in the endometrial stroma, and the surface epithelium almost entirely covers the original defect in the uterine wall (Figs. 4.4 and 4.5). The blastocyst now produces a slight protrusion into the lumen of the uterus. The trophoblast is characterized by lacunar spaces in the syncytium that form an
Figure 4.3 9-day human blastocyst. The syncytiotrophoblast shows a large number of lacunae. Flat cells form the exocoelomic
The bilaminar
disc consists
a
layer of
columnar epiblast
cells and
a
layer of
cuboidal hypoblast
membrane.
of
Maternal
sinusoids
Figure 4.4 Human blastocyst of approximately 12 days. The trophoblastic lacunae at the embryonic pole are in open connection with maternal sinusoids in the endometrial stroma. Extraembryonic mesoderm proliferates and fills the space
between the exocoelomic membrane and the inner aspect of the trophoblast.
intercommunicating network.
This network
is endothelial
particularly evident
at the
embryonic pole;
at the
abembryonic pole, the trophoblast still consists mainly of cytotrophoblastic cells (Figs. 4.4 and 4.5).
Concurrently, cells of the syncytiotrophoblast lining
These capillaries,
dilated, lacunae
are known
become of
the which
maternal capillaries.

are congested
as sinusoids.
continuous and
The syncytial
with the
sinusoids, and
maternal blood
enters the
lacunar system
penetrate deeper into the stroma and erode the
(Fig. 4.4). As the trophoblast continues to erode
Primitive yolk sac
Extraembryonic mesoderm
Figure 4.5 Fully implanted 12-day human blastocyst (x 100). Note maternal blood cells in the lacunae, the exocoelomic
more and more sinusoids, maternal blood begins to flow through the trophoblastic system, establishing the uteroplacental circulation. In the meantime, a new population of cells appears between the inner surface of the cytotrophoblast and the outer surface of the exocoelomic cavity. These cells, derived from yolk sac cells, form a fine, loose connective tissue, the extraembryonic mesoderm, which eventually fills all of the space between the trophoblast externally and the amnion and exocoelomic membrane internally (Figs. 4.4 and 4.5). Soon, large cavities develop in the extraembryonic mesoderm, and when these become confluent, they form a new space known as the extraembryonic cavity, or chorionic cavity (Fig. 4.4). This space surrounds the primitive yolk sac and amniotic cavity, except where the germ disc is connected to the trophoblast by the connecting stalk (Fig. 4.6). The extraembryonic mesoderm lining the cytotrophoblast and amnion is called the extraembryonic somatic mesoderm; the lining covering the yolk sac is known as the extraembryonic splanchnic mesoderm (Fig. 4.4).

Growth of the bilaminar disc is relatively slow compared with that of the trophoblast; consequently, the disc remains very small (0.1 to 0.2 mm). Cells of the endometrium, meanwhile, become polyhedral and loaded with glycogen and lipids; intercellular spaces are filled with extravasate, and the tissue is edematous. These changes, known as the decidua reaction, at first are confined to the area immediately surrounding the implantation site but soon occur throughout the endometrium.

By the 13th day of development, the surface defect in the endometrium has usually healed. Occasionally, however, bleeding occurs at the implantation site as a result of increased blood flow into the lacunar spaces. Because this bleeding occurs near the 28th day of the menstrual
Figure 4.6 A |3-day human blastocyst. Trophoblastic lacunae are present at the embryonic as well as the abembryonic pole, and the uteroplacental circulation has begun. Note the primary villi and the extraembryonic coelom or chorionic
Figure 4.7 Section through the implantation site of a 13-day embryo. Note the amniotic cavity, yolk sac, and chorionic cavity. Most of the lacunae are filled with blood.
cycle, it may be confused with normal menstrual bleeding and, therefore, may cause inaccuracy in determining the expected delivery date.
The trophoblast is characterized by villous structures. Cells of the cytotrophoblast proliferate locally and penetrate into the syncytiotrophoblast, forming cellular columns surrounded by syncytium. Cellular columns with the syncytial covering are known as primary villi (Figs. 4.6 and 4.7) (see Chapter 5, p. 59).
(Figs. 4.6 and 4.7). This yolk sac is much smaller than the original exocoelomic cavity, or primitive yolk sac. During its formation, large portions of the exocoelomic cavity are pinched off. These portions are represented by exocoelomic cysts, which are often found in the extraembryonic coelom or chorionic cavity (Figs. 4.6).
In the meantime, the hypoblast produces additional cells that migrate along the inside of the exocoelomic membrane (Fig. 4.4). These cells proliferate and gradually form a new cavity within the exocoelomic cavity. This new cavity is known as the secondary yolk sac or definitive yolk sac

The syncytiotrophoblast is responsible for hormone production (see Chapter 8,p.105), including human chorionic gonadotropin (hCG). By the end of the second week, quantities of this hormone are sufficient to be detected by radioimmunoassays, which serve as the basis for pregnancy testing.
Because 50% of the implanting embryo's genome is derived from the father, it is a foreign body that potentially should be rejected by the maternal system, similar to rejection of a transplanted organ. A pregnant woman's immune system needs to change in order for her to tolerate the pregnancy. How this occurs is not well understood, but it appears that a shift from cell-mediated immunity to humoral (antibody mediated) immunity occurs and that this shift
Meanwhile, the extraembryonic coelom expands and forms a large cavity, the chorionic cavity. The extraembryonic mesoderm lining the inside of the cytotrophoblast is then known as the chorionic plate. The only place where extraembryonic mesoderm traverses the chorionic cavity is in the connecting stalk (Fig. 4.6). With development of blood vessels, the stalk becomes the umbilical cord.
protects the embryo from rejection. However, the immune system alterations place pregnant women at increased risk for certain infections, such as influenza, which explains the increased risk of death from these infections in pregnant women. In addition, manifestations of autoimmune disease may change during pregnancy. For example, multiple sclerosis and rheumatoid arthritis, primarily cell-mediated conditions, show improvement during pregnancy, whereas women with systemic lupus erythrematosis (a predominantly antibody-mediated immune disorder) are more severely affected when pregnant.
Abnormal implantation sites sometimes occur even within the uterus. Normally, the human blastocyst implants along the anterior or posterior wall of the body of the uterus. Occasionally, the blastocyst

implants close to the internal os (opening) (Fig. 4.8) of the cervix, so that later in development, the placenta bridges the opening (placenta previa) and causes severe, even life-threatening bleeding in the second part of pregnancy and during delivery.
Occasionally, implantation takes place outside the uterus, resulting in an extrauterine pregnancy, or ectopic pregnancy. Ectopic pregnancies may occur at any place in the abdominal cavity, ovary, or uterine tube (Fig. 4.8). Ninety-five percent of ectopic pregnancies occur in the uterine tube, however, and most of these are in the ampulla (80%; Fig. 4.9).In the abdominal cavity, the blastocyst most frequently attaches itself to the peritoneal lining of the rectouterine cavity, or pouch of Douglas (Fig. 4.10). The blastocyst may also attach itself to the peritoneal covering of the intestinal tract or to the omentum. Sometimes, the blastocyst develops
Figure 4.8 Abnormal implantation sites of the blastocyst. |, implantation in the abdominal cavity (1.4%; the ovum most frequently implants in the rectouterine cavity [pouch of Douglas; Fig. 4.10] but may implant at any place covered by peritoneum); 2, implantation in the ampullary region of the tube (80%); 3, tubal implantation (12%); 4, interstitial implantation (0.2%; e.g., in the narrow portion of the uterine tube); 5, implantation in the region of the internal os, frequently resulting in placenta previa (0.2%); and 6, ovarian implantation (0.2%).
in the ovary proper, causing a primary ovarian pregnancy. Ectopic pregnancies occur in 2% of all pregnancies and account for 9% of all pregnancy related deaths for the mother. In most ectopic pregnancies, the embryo dies about the second month of gestation and may result in severe hemorrhaging in the mother.

Abnormal blastocysts are common. For example, in a series of 26 implanted blastocysts varying in age from 7.5 to 17 days recovered from patients of normal fertility, 9 (34.6%) were abnormal. Some consisted of syncytium only; others showed varying degrees of trophoblastic hypoplasia. In two, the embryoblast was absent, and in some, the germ disc showed an abnormal orientation.
their trophoblast was so inferior that the corpus luteum could not have persisted. These embryos probably would have been aborted with the next menstrual flow and, therefore, pregnancy would not have been detected. In some cases, however, the trophoblast develops and forms placental membranes, although little or no embryonic tissue is present. Such a condition is known as a hydatidiform mole. Moles secrete high levels of hCG and may produce benign or malignant (invasive mole, choriocarcinoma) tumors.
It is likely that most abnormal blastocysts would not have produced any sign of pregnancy because
Genetic analysis of hydatidiform moles indicates that although male and female pronuclei may be genetically equivalent, they may be different functionally. This evidence is derived from the fact that although cells of moles are diploid, their entire genome is paternal. Thus, most moles arise from
Figure 4.9 Tubal pregnancy. Embryo is approximately 2 months old and is about to escape through a rupture in the tubal wall.
fertilization of an oocyte lacking a nucleus followed by duplication of the male chromosomes to restore the diploid number. These results also suggest that paternal genes regulate most of the development of the trophoblast, because in moles this tissue differentiates even in the absence of a female pronucleus.
Other examples of functional differences in maternal and paternal genes are provided by the observation that certain genetic diseases depend on whether the defective or missing gene is inherited from the father or the mother. For example, a microdeletion on chromosome 15 inherited from a father causes
Figure 4.10 Midline section of bladder, uterus, and rectum shows an abdominal pregnancy in the rectouterine (Douglas) pouch.

Prader-Willi syndrome (a condition characterized by hypotonia, intellectual disability, hypogonadism, and obesity), whereas inheriting the same deletion from the mother causes Angelman's syndrome (a condition characterized by seizures, little to no speech, paroxysms of laughter, and severe intellectual disability). This phenomenon, in which there is differential modification and/or expression of homologous alleles or chromosome regions depending on the parent from whom the genetic material is derived, is known as genomic imprinting. Less than 1% of human genes are believed to be imprinted (see Chapter 2, p. 19).
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At the beginning of the second week, the blastocyst is partially embedded in the endometrial stroma. The trophoblast differentiates into (1) an inner, actively proliferating layer, the cytotrophoblast, and (2) an outer layer, the syncytiotrophoblast, which erodes maternal tissues (Fig. 4.1). By day 9, lacunae develop in the syncytiotrophoblast. Subsequently, maternal sinusoids are eroded by the syncytiotrophoblast, maternal blood enters the lacunar network, and by the end of the second week, a primitive uteroplacental circulation begins (Fig. 4.6). The cytotrophoblast, meanwhile, forms cellular columns penetrating into and surrounded by the syncytium. These columns are primary villi. By the end of the second week, the blastocyst is completely embedded, and the surface defect in the mucosa has healed (Fig. 4.6).

The inner cell mass or embryoblast, meanwhile, differentiates into (1) the epiblast and (2) the hypoblast, together forming a bilaminar disc (Fig. 4.6). Epiblast cells give rise to amnioblasts that line the amniotic cavity superior to the epiblast layer. Hypoblast cells are continuous with the exocoelomic membrane, and together they surround the primitive yolk sac (Fig. 4.4). By the end of the second week, extraembryonic mesoderm fills the space between the trophoblast and the amnion and exocoelomic membrane internally. When vacuoles develop in this tissue, the extraembryonic coelom or chorionic cavity forms (Fig. 4.6). Extraembryonic mesoderm lining the cytotrophoblast and amnion is extraembryonic somatic mesoderm; the lining surrounding the yolk sac is extraembryonic splanchnic mesoderm (Fig. 4.6).
The second week of development is known as the week of 2's:
- 1 The trophoblast differentiates into 2 layers; the cytotrophoblast and syncytiotrophoblast
Preimplantation and postimplantation reproductive failure occurs often. Even in some fertile women under optimal conditions for pregnancy, 15% of oocytes are not fertilized, and 10% to 15% start cleavage but fail to implant. Of the 70% to 75% that implant, only 58% survive until the second week, and 16% of those are abnormal. Hence, when the first expected menstruation is missed, only 42% of the eggs exposed to sperm are surviving. Of this percentage, a number will be aborted during subsequent weeks, and a number will be abnormal at the time of birth.
2 The embryoblast forms 2 layers; the epiblast and hypoblast
3 The extraembryonic mesoderm splits into 2 layers; the somatic and splanchnic layers
4 2 cavities form; the amniotic and yolk sac cavities.

Implantation occurs at the end of the first week. Trophoblast cells invade the epithelium and underlying endometrial stroma with the help of proteolytic enzymes. Implantation may also occur outside the uterus, such as in the rectouterine pouch, on the mesentery, in the uterine tube, or in the ovary (ectopic pregnancies).

1. The second week of development is known as the week of 2's. Formation of what structures supports this statement?
2. During implantation, the trophoblast is invading maternal tissues, and because it contains approximately 50% paternal genes, it is a foreign body. Why is the conceptus not rejected by an immunologic response from the mother's system?
3. A woman who believes she is pregnant complains of edema and vaginal bleeding. Examination reveals high plasma hCG concentrations and placental tissue, but no evidence of an embryo. How would you explain this condition?
4. A young woman who has missed two menstrual periods complains of intense abdominal pain. What might an initial diagnosis be, and how would you confirm it?

The most characteristic event occurring during the third week of gestation is gastrulation, the process that establishes all three germ layers (ectoderm, mesoderm, and endoderm) in the embryo. Gastrulation begins with formation of the primitive streak on the surface of the epiblast (Figs. 5.1 and 5.2A). Initially, the streak is vaguely defined (Fig. 5.1), but in a 15to 16-day embryo, it is clearly visible as a narrow groove with slightly bulging regions on either side. The cephalic end of the streak, the primitive node, consists of a slightly elevated area surrounding the small primitive pit (Fig. 5.2). Cells of the epiblast migrate toward the primitive streak (Fig. 5.2). Upon arrival in the region of the streak, they become flask-shaped, detach from the epiblast, and slip beneath it (Fig. 5.2B,C). This inward movement is known as invagination. Cell migration and specification are controlled by fibroblast growth factor 8 (FGF8), which is synthesized by streak cells themselves. This growth factor controls cell movement by downregulating E-cadherin, a protein that normally binds epiblast cells together. FGF8 then controls cell specification into the mesoderm by regulating Brachyury (T) expression. Once the cells have invaginated, some displace the hypoblast, creating the embryonic endoderm, and others come to lie between the epiblast and newly created endoderm to form mesoderm. Cells remaining in the epiblast then form ectoderm. Thus, the epiblast, through the process of gastrulation, is the source of all of the germ layers (Fig. 5.2B), and cells in these layers will give rise to all of the tissues and organs in the embryo.

As more and more cells move between the epiblast and hypoblast layers, they begin to spread laterally and cranially (Fig. 5.2). Gradually, they migrate beyond the margin of the disc and establish contact with the extraembryonic mesoderm covering the yolk sac and amnion. In the cephalic direction, they pass on each side of the prechordal plate. The prechordal plate itself forms between the tip of the notochord and the oropharyngeal membrane and is derived from some of the first cells that migrate through the node in the midline and move in a cephalic direction. Later, the prechordal plate will be important for induction of the forebrain (Figs. 5.2 and 5.3). The oropharyngeal membrane at the cranial end of the disc consists of a small region of tightly adherent ectoderm and endoderm cells that represents the future opening of the oral cavity.

Prenotochordal cells invaginating in the primitive node move forward cranially in the midline until they reach the prechordal plate (Fig. 5.3). These prenotochordal cells become intercalated in the hypoblast so that for a short time, the midline of the embryo consists of two cell layers that form the notochordal plate (Fig. 5.3B). As the hypoblast is replaced by endoderm cells moving in at the streak, cells of the notochordal plate proliferate and detach from the endoderm. They then form a solid cord of cells, the definitive notochord (Fig. 5.3C), which underlies the neural tube and serves as the basis for the axial skeleton. Because elongation of the notochord is a dynamic process, the cranial end forms first, and caudal regions are added as the primitive streak assumes a more caudal position. The notochord and prenotochordal cells extend cranially to the prechordal plate (an area just caudal to the oropharyngeal membrane) and caudally to the primitive pit. At the point where the pit forms an indentation in the epiblast, the neurenteric canal temporarily connects the amniotic and yolk sac cavities (Fig. 5.3A).
The cloacal membrane is formed at the caudal end of the embryonic disc (Fig. 5.2A). This membrane, which is similar in structure to the oropharyngeal membrane, consists of tightly adherent ectoderm and endoderm cells with no intervening mesoderm. When the cloacal membrane appears, the posterior wall of the yolk sac forms a small diverticulum that extends
Figure 5.1 A. Implantation site at the end of the second week. B. Representative view of the germ disc at the end of the second week of development. The amniotic cavity has been opened to permit a view of the dorsal side of the epiblast. The hypoblast and epiblast are in contact with each other, and the primitive streak forms a shallow groove in the caudal region of the embryo.

into the connecting stalk. This diverticulum, the allantoenteric diverticulum, or allantois, appears around the 16th day of development (Fig. 5.3.4). Although in some lower vertebrates the allantois serves as a reservoir for excretion products of the renal system, in humans, it remains rudimentary but may be involved in abnormalities of bladder development (see Chapter 16, p. 240).

anterior visceral endoderm (AVE), expresses genes essential for head formation, including the transcription factors OTX2, LIM1, and HESX1, and the secreted factors cerberus and lefty, which inhibit nodal activity in the cranial end of the embryo. These genes establish the cranial end of the embryo before gastrulation. The primitive streak itself is initiated and maintained by expression of Nodal, a member of the transforming growth factor-B (TGF-B) family (Fig. 5.4). Once the streak is formed, Nodal upregulates a number of genes responsible for formation of dorsal and ventral mesoderm and head and tail structures. Another member of the TGF-f family, bone morphogenetic protein 4 (BMP4), is secreted throughout the embryonic disc (Fig. 5.4). In the presence of this protein and FGF, mesoderm
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Figure 5.3 Schematic views illustrating formation of the notochord, whereby prenotochordal cells migrate through the primitive streak, become intercalated in the endoderm to form the notochordal plate, and finally detach from the endoderm to form the definitive notochord. Because these events occur in a cranial-to-caudal sequence, portions of the definitive notochord are established in the head region first. A. Drawing of a sagittal section through a 17-day embryo. The most cranial portion of the definitive notochord has formed, while prenotochordal cells caudal to this region are intercalated into the endoderm as the notochordal plate. Note that some cells migrate ahead of the notochord. These mesoderm cells form the prechordal plate that will assist in forebrain induction. B. Schematic cross section through the region of the notochordal plate. Soon, the notochordal plate will detach from the endoderm to form the definitive notochord. C. Schematic view showing the definitive notochord.
Figure 5.4 Sagittal section through the node and primitive streak showing the expression pattern of genes regulating the craniocaudal and dorsoventral axes. Cells at the prospective cranial end of the embryo in the AVE express the transcription factors OTX2, LIMI, and HESX! and the secreted factor cerberus that contribute to head development and establish the cephalic region. Once the streak is formed and gastrulation is progressing, BMP4 is secreted throughout the bilaminar disc and acts with FGF to ventralize mesoderm into intermediate and lateral plate mesoderm. Goosecoid, expressed in the node, regulates chordin expression, and this gene product, together with noggin and follistatin, antagonizes the activity of BMP4, dorsalizing mesoderm into notochord and paraxial mesoderm for the head region. Later, expression of the Brachyury (T) gene antagonizes BMP4 to dorsalize mesoderm into notochord and paraxial mesoderm in caudal regions of the embryo.

will be ventralized to contribute to kidneys (intermediate mesoderm), blood, and body wall mesoderm (lateral plate mesoderm). In fact, all mesoderm would be ventralized if the activity of BMP4 were not blocked by other genes expressed in the node. For this reason, the node is the organizer. It was given that designation by Hans Spemann, who first described this activity in the dorsal lip of the blastopore, a structure analogous to the node, in Xenopus embryos. Thus, chordin (activated by the transcription factor Goosecoid), noggin, and follistatin antagonize the activity of BMP4.As a result, cranial mesoderm is dorsalized into notochord, somites, and somitomeres (Fig. 5.4). Later, these three genes are expressed in the notochord and are important in neural induction in the cranial region.
As mentioned, Nodal is involved in initiating and maintaining the primitive streak. Similarly, HNF-3B maintains the node and later induces regional specificity in the forebrain and midbrain areas. Without HNF-3B, embryos fail to gastrulate properly and lack forebrain and midbrain structures. As mentioned previously, Goosecoid activates inhibitors of BMP4 and contributes to regulation of head development. Overor underexpression of this gene in laboratory animals results in severe malformations of the head region, including duplications, similar to some types of conjoined twins (Fig. 5.5).
Figure 5.5 Conjoined twins. If the gene Goosecoid is overexpressed in frog embryos, the result is a two-headed tadpole. Perhaps overexpression of this gene explains the origin of this type of conjoined twins.

Regulation of dorsal mesoderm formation in middle and caudal regions of the embryo is controlled by the Brachyury (T) gene expressed in the node, notochord precursor cells, and notochord. This gene is essential for cell migration through the primitive streak. Brachyury encodes a sequence-specific DNA_ binding protein that functions as a transcription factor. The DNA-binding domain is called the T-box, and there are more than 20 genes in the T-box family. Thus, mesoderm formation in these regions depends on this gene product, and its absence results in shortening of the embryonic axis (caudal dysgenesis). The degree of shortening depends on the time at which the protein becomes deficient.

Laterality (left-right-sidedness) also is established early in development and is orchestrated by a cascade of signal molecules and genes. When the primitive streak appears, FGF8 is secreted by cells in the node and primitive streak and induces expression of Nodal, but only on the left side of the embryo (Fig. 5.6A). Later, as the neural plate is established, FGF8 maintains Nodal expression in the lateral plate mesoderm, as well as LEFTY2, and both of these genes upregulate PITX2. PITX2 is a homeobox-containing transcription factor responsible for establishing left-sidedness (Fig. 5.6B) and its expression is repeated on the left side of the heart, stomach, and gut primordia as these organs are assuming their normal asymmetrical body positions. If the gene is expressed ectopically (e.g., on the right side), this abnormal expression results in laterality defects, including situs inversus and dextrocardia (placement of the heart to the right side; see p. 57). Simultaneously, LEFTY is expressed on the left side of the floor plate of the neural tube and may act as a barrier to prevent left-sided signals from crossing over. Sonic hedgehog (SHH) may also function in this role as well as serving as a repressor for left-sided gene expression on the right. The Brachyury (T) gene, encoding a transcription factor secreted by the notochord, is also essential for expression of Nodal, LEFTY-1, and LEFTY-2 (Fig. 5.6B). Importantly, the neurotransmitter serotonin (5HT) also plays a critical role in this signaling cascade that establishes laterality. 5HT is concentrated on the left side, probably because it is broken down by its metabolizing enzyme monoamine oxidase (MAO) on the right, and is upstream from FGF8 signaling (Fig. 5.6B). Alterations in 5HT signaling result in situs inversus, dextrocardia, and a variety of heart defects (see Clinical Correlations, p. 57). Genes regulating right-sided development are not as well defined, although expression of the

Figure 5.7 Dorsal view of the germ disc showing the primitive streak and a fate map for epiblast cells. Specific regions of the epiblast migrate through different parts of the node and streak to form mesoderm. Thus, cells migrating at the cranialmost part of the node will form the notochord (n); those migrating more posteriorly through the node and cranialmost aspect of the streak will form paraxial mesoderm (pm; somitomeres and somites); those migrating through the next portion of the streak will form intermediate mesoderm (im; urogenital system); those migrating through the more caudal part of the streak will form lateral plate mesoderm (Ipm; body wall); and those migrating through the most caudal part will contribute to extraembryonic mesoderm (eem; chorion).
transcription factor Snail is restricted to the right lateral plate mesoderm and probably regulates effector genes responsible for establishing the right side. Why the cascade is initiated on the left remains a mystery, but the mechanism may involve cilia on cells in the node that beat to create a gradient of Nodal toward the left or by a signaling gradient established by gap junctions and small ion transport.

Regions of the epiblast that migrate and ingress through the primitive streak have been mapped, and their ultimate fates have been determined (Fig. 5.7). For example, cells that ingress through the cranial region of the node become prechordal plate and notochord; those migrating at the lateral edges of the node and from the cranial end of the streak become paraxial mesoderm; cells migrating through the midstreak region become intermediate mesoderm; those migrating through the more caudal part of the streak form lateral plate mesoderm; and cells migrating through the caudalmost part of the streak contribute to extraembryonic mesoderm (the other source of this tissue is the primitive yolk sac [hypoblast]).

The beginning of the third week of development, when gastrulation is initiated, is a highly sensitive stage for teratogenic insult. At this time, fate maps can be made for various organ systems, such as the eyes and brain anlage, and these cell populations may be damaged by teratogens. For example, animal studies indicate that high doses of alcohol at this stage kill cells in the anterior midline of the germ disc, producing a deficiency of the midline in craniofacial structures and resulting in holoprosencephaly (see Chapter 17, p. 279). In such a child, the forebrain is small, the two lateral ventricles often merge into a single ventricle,and the eyes are close together (hypotelorism). Because this stage is reached 2 weeks after fertilization, it is approximately 4 weeks from the last menses. Therefore, the woman may not recognize that she is pregnant, having assumed that menstruation is late and will begin shortly. Consequently, she may not take precautions she would normally consider if she knew she was pregnant.

The embryonic disc, initially flat and almost round, gradually becomes elongated, with a broad cephalic and a narrow caudal end (Fig. 5.24). Expansion of the embryonic disc occurs mainly in the cephalic region; the region of the primitive streak remains more or less the same size. Growth and elongation of the cephalic part of the disc are caused by a continuous migration of cells from the primitive streak region in a cephalic direction. Invagination of surface cells in the primitive streak and their subsequent migration forward and laterally continues until the end of the fourth week. At that stage, the primitive streak shows regressive changes, rapidly shrinks, and soon disappears.
That the primitive streak at the caudal end of the disc continues to supply new cells until the end of the fourth week has an important bearing on development of the embryo. In the cephalic part, germ layers begin their specific differentiation by the middle of the third week, whereas in the caudal part, differentiation begins by the end of the fourth week. Thus gastrulation, or formation of the germ layers, continues in caudal segments while cranial structures are differentiating, causing the embryo to develop cephalocaudally.
Gastrulation itself may be disrupted by genetic abnormalities and toxic insults. In caudal dysgenesis (sirenomelia), insufficient mesoderm is formed in the caudalmost region of the embryo. Because this mesoderm contributes to formation of the lower limbs, urogenital system (intermediate mesoderm), and lumbosacral vertebrae, abnormalities in these structures ensue.
Affected individuals exhibit a variable range of defects, including hypoplasia and fusion of the lower limbs, vertebral abnormalities, renal agenesis, imperforate anus, and anomalies of the genital organs (Fig. 5.8A,B). In humans, the condition is associated with maternal diabetes and other causes. In mice, abnormalities of Brachyury (T), WNT, and engrailed genes produce a similar phenotype.
Situs inversus is a condition in which transPosition of the viscera in the thorax and abdomen occurs. Despite this organ reversal, other structural abnormalities occur only slightly more frequently in these individuals. Approximately 20% of patients with complete situs inversus also

Figure 5.8 A,B. Two examples of sirenomelia (caudal dysgenesis). Loss of mesoderm in the lumbosacral region has resulted in fusion of the limb buds and other defects.
Figure 5.9 Sacrococcygeal teratoma probably resulting from remnants of the primitive streak. These tumors may become malignant and are most common in female fetuses.
have bronchiectasis and chronic sinusitis because of abnormal cilia (Kartagener's syndrome). Interestingly, cilia are normally present on the ventral surface of the primitive node and may be involved in left-right patterning during gastrulation
(see p. 55). Other conditions of abnormal sidedness are known as laterality sequences. Patients with these conditions do not have complete situs inversus, but appear to be predominantly bilaterally left-sided or right-sided. The spleen reflects the
differences; those with left-sided bilaterality have polysplenia, and those with right-sided bilaterality have asplenia or hypoplastic spleen. Patients with laterality sequences are also likely to have other malformations, especially heart defects.
The neurotransmitter serotonin (5HT) is an important signal molecule for establishing laterality, and animal studies show that disrupting SHT signaling results in cases of situs inversus, dextrocardia,and heart defects (see Chapter | 3). Recent results also show that children born to mothers who take antidepressants from the class of drugs called selective serotonin reuptake inhibitors (SSRIs) have an increased risk of having a variety of heart

By the beginning of the third week, the trophoblast is characterized by primary villi that consist of a cytotrophoblastic core covered malformations, thus providing additional evidence for 5HT's importance in establishing laterality.

Sometimes, remnants of the primitive streak persist in the sacrococcygeal region. These clusters of pluripotent cells proliferate and form tumors, known as sacrococcygeal teratomas, which commonly contain tissues derived from all three germ layers (Fig. 5.9). This is the most common tumor in newborns, occurring with a frequency of one in 37,000. Teratomas may also arise from primordial germ cells that fail to migrate to the gonadal ridge (see p. 10).
by a syncytial layer (Figs. 5.10 and 5.11). During further development, mesodermal cells penetrate the core of primary villi and grow toward the decidua. The newly formed structure is known as a secondary villus (Fig. 5.11).
Figure 5.10 A 13-day-old implantation site showing primary villi of the trophoblastic shell just beginning to be invaded by mesoderm from the chorionic plate.
By the end of the third week, mesodermal cells in the core of the villus begin to differentiate into blood cells and small blood vessels, forming the villous capillary system (Fig. 5.11).The villus is now known as a tertiary villus or a definitive placental villus. Capillaries in tertiary villi make contact with capillaries developing in the mesoderm of the chorionic plate and in the connecting stalk (Figs. 5.12 and 5.13). These vessels, in turn, establish contact with the intraembryonic circulatory system, connecting the placenta and the embryo. Hence, when the heart begins to beat in the fourth week of development, the villous system is ready to supply the embryo proper with essential nutrients and oxygen. Meanwhile, cytotrophoblastic cells in the villi penetrate progressively into the overlying syncytium until they reach the maternal endometrium.

Figure 5.12 Presomite embryo and the trophoblast at the end of the third week. Tertiary and secondary stem villi give the trophoblast a characteristic radial appearance. Intervillous spaces, which are found throughout the trophoblast, are lined with syncytium. Cytotrophoblastic cells surround the trophoblast entirely and are in direct contact with the endometrium. The the chorionic of the stalk.
embryo is suspended in cavity by means connecting
Chapter
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Figure 5.13 Longitudinal section through a villus at the end of the fourth week of development. Maternal vessels penetrate the cytotrophoblastic shell to enter intervillous spaces, which surround the villi. Capillaries in the villi are in contact with vessels in the chorionic plate and in the connecting stalk, which in turn are connected to intraembryonic vessels.
Here they establish contact with similar extensions of neighboring villous stems, forming a thin outer cytotrophoblast shell (Figs. 5.12 and 5.13). This shell gradually surrounds the trophoblast entirely and attaches the chorionic sac firmly to the maternal endometrial tissue (Fig. 5.12). Villi that extend from the chorionic plate to the decidua basalis (decidual plate: the part of the endometrium where the placenta will form; see Chapter 8) are called stem or anchoring villi. Those that branch from the sides of stem villi are free (terminal) villi, through which exchange of nutrients and other factors will occur. The chorionic cavity, meanwhile, becomes larger, and by the 19th or the 20th day, the embryo is attached to its trophoblastic shell by a narrow connecting stalk (Fig. 5.12). The connecting stalk later develops into the umbilical cord, which forms the connection between the placenta and embryo.
remain in the epiblast form ectoderm. Hence, epiblast gives rise to all three germ layers in the embryo, ectoderm, mesoderm, and endoderm, and these layers form all of the tissues and organs (Figs. 5.2 and 5.3).

The most characteristic event occurring during the third week is gastrulation, which begins with the appearance of the primitive streak, which has at its cephalic end the primitive node. In the region of the node and streak, epiblast cells move inward (invaginate) to form new cell layers, endoderm and mesoderm. Cells that do not migrate through the streak but
Prenotochordal cells invaginating in the primitive pit move forward until they reach the prechordal plate. They intercalate in the endoderm as the notochordal plate (Fig. 5.3). With further development, the plate detaches from the endoderm, and a solid cord, the notochord, is formed. It forms a midline axis, which will serve as the basis of the axial skeleton (Fig. 5.3). Cephalic and caudal ends of the embryo are established before the primitive streak is formed. Thus, cells in the hypoblast (endoderm) at the cephalic margin of the disc form the AVE, which expresses head-forming genes, including OTX2, LIM1, and HESX1 and the secreted factor cerberus. Nodal, a member of the TGF-f family of genes, is then activated and initiates and maintains the integrity of the node and streak. In the presence of FGF, BMP4 ventralizes mesoderm during gastrulation so that it forms intermediate and lateral plate mesoderm. Chordin, noggin, and follistatin antagonize BMP4 activity and dorsalize mesoderm to form the notochord and somitomeres in the head region. Formation of these structures in more caudal regions is regulated by the Brachyury (T) gene (Fig. 5.4A). Laterality (left-right asymmetry) is regulated by a cascade of signaling molecules and genes. FGF8, secreted

1. A 22-year-old woman consumes large quantities of alcohol at a party and loses consciousness; 3 weeks later, she misses her second consecutive period. A pregnancy test is positive. Should she be concerned about the effects of her binge-drinking episode on her baby?
2. An ultrasound scan detects a large mass near the sacrum of a 28-week female fetus. What might the origin of such a mass be, and what type of tissue might it contain?
3. On ultrasound examination, it was determined that a fetus had well-developed facial and thoracic regions, but caudal structures were abnormal. Kidneys were absent, lumbar and sacral vertebrae were missing, and the hindlimbs were fused. What process may have been disturbed to cause such defects?
4. A child has polysplenia and abnormal positioning of the heart. How might these two abnormalities be linked developmentally, and when would they have originated? Should you be concerned that other defects might be present? What genes might have caused this event, and when during embryogenesis would it have been initiated?
he embryonic period, or period of organogenesis, occurs from the third to the eighth weeks of development and is the time when each of the three germ layers, ectoderm, mesoderm, and endoderm, gives rise to a number of specific tissues and organs. By the end of the embryonic period, the main organ systems have been established, rendering the major features of the external body form recognizable by the end of the second month.

At the beginning of the third week of development, the ectodermal germ layer has the shape of a disc that is broader in the cephalic than in the caudal region (Fig. 6.1). Appearance of the notochord and prechordal mesoderm induces the overlying ectoderm to thicken and form the neural plate (Fig. 6.2A,B). Cells of the plate make up the neuroectoderm, and their induction represents the initial event in the process of neurulation.

Upregulation of fibroblast growth factor (FGF) signaling together with inhibition of the activity of bone morphogenetic protein 4 (BMP4), a transforming growth factor-B (TGF-B) family member responsible for ventralizing ectoderm and mesoderm, causes induction of the neural plate. FGF signaling probably promotes a neural pathway by an unknown mechanism while it represses BMP transcription and upregulates expression of chordin and noggin, which inhibit BMP activity. In the presence of BMP4, which permeates the mesoderm and ectoderm of the gastrulating embryo, ectoderm is induced to form epidermis, and mesoderm forms intermediate and lateral plate mesoderm. If ectoderm is protected from exposure to BMPs, its "default state" is to become neural tissue. Secretion of three other molecules, noggin, chordin, and follistatin, inactivates BMP. These three proteins are present in the organizer (primitive node), notochord, and prechordal mesoderm. They neuralize ectoderm by inhibiting BMP and cause mesoderm to become notochord and paraxial mesoderm (dorsalizes mesoderm); however, these neural inducers induce only forebrain and midbrain types of tissues. Induction of caudal neural plate structures (hindbrain and spinal cord) depends on two secreted proteins, WNT3a and FGF. In addition, retinoic acid (RA) appears to play a role in organizing the cranial-to-caudal axis because it can cause respecification of cranial segments into more caudal ones by regulating expression of homeobox genes (see p. 78).

Neurulation is the process whereby the neural plate forms the neural tube. By the end of the third week, the lateral edges of the neural plate become elevated to form neural folds, and the depressed midregion forms the neural groove (Fig. 6.2). Gradually, the neural folds approach each other in the midline, where they fuse (Fig. 6.3A4,B). Fusion begins in the cervical region (fifth somite) and proceeds cranially and caudally (Fig. 6.3C,D).As a result, the neural tube is formed. Until fusion is complete, the cephalic and caudal ends of the neural tube communicate with the amniotic cavity by way of the anterior (cranial) and posterior (caudal) neuropores, respectively (Figs. 6.3C,D and 6.4A). Closure of the cranial neuropore occurs at approximately day 25 (18to 20-somite stage), whereas the posterior neuropore closes at day 28 (25-somite stage) (Fig. 6.4B). Neurulation is then complete, and the central nervous system is represented by a closed tubular structure with a narrow caudal portion, the spinal cord, and a much broader cephalic portion characterized by a number of dilations, the brain vesicles (see Chapter 18).
22 days
23 days
c
23
days
Figure 6.3 A. Dorsal view of an embryo at approximately day 22. Seven distinct somites are visible on each side of the neural tube. B. Dorsal view of a human embryo at 2! days. C. Dorsal view of an embryo at approximately day 23. Note the pericardial bulge on each side of the midline in the cephalic part of the embryo. D. Dorsal view of a human embryo at 23 days.

by active
migration and
displacement to
enter
As the neural folds elevate and fuse, cells at the lateral
border or
crest of
the neuroectoderm
begin to dissociate from their neighbors. This cell
population, the
neural crest
(Figs.
6.5
and
6.6), will
undergo an
epithelial-to-mesenchy- mal transition as it leaves the neuroectoderm
the underlying
mesoderm.
(Mesoderm refers
to cells
the epiblast
and extraem-
bryonic tissues. Mesenchyme refers to loosely organized embryonic connective tissue regardless
of origin.)
Crest cells
from the
trunk region
leave the neuroectoderm after closure of the neural
Figure 6.4 A. Lateral view of a 14-somite embryo (approximately 25 days). Note the bulging pericardial area and the first and second pharyngeal arches. B. The left side of a 25-somite embryo approximately 28 days old. The first three pharyngeal arches and lens and otic placodes are visible.
tube and migrate along one of two pathways: (1) a dorsal pathway through the dermis, where they will enter the ectoderm through holes in the basal lamina to form melanocytes in the skin and hair follicles, and (2) a ventral pathway through the anterior half of each somite to become sensory ganglia, sympathetic and enteric neurons, Schwann's cells, and cells of the adrenal medulla (Fig. 6.5). Neural crest cells also form and migrate from cranial neural folds, leaving the neural tube before closure in this region (Fig. 6.6). These cells contribute to the craniofacial skeleton, as well as neurons for cranial ganglia, glial cells, melanocytes, and other cell types (Table 6.1, p.69). Neural crest cells are so fundamentally important and contribute to so many organs and tissues that they are sometimes referred to as the fourth germ layer. Evolutionarily, these cells appeared at the dawn of vertebrate development and expanded this group extensively by perfecting a predatory lifestyle.

concentrations of BMPs, together with FGF and WNT proteins, induce PAX3 and other transcription factors that "specify" the neural plate border (Fig. 6.5A). In turn, these transcription factors induce a second wave of transcription factors, including SNAIL and FOXD3, which specify cells as neural crest, and SLUG, which promotes crest cell migration from the neuroectoderm. Thus, the fate of the entire ectodermal germ layer depends on BMP concentrations: High levels induce epidermis formation; intermediate levels, at the border of the neural plate and surface ectoderm, induce the neural crest; and very low concentrations cause formation of neural ectoderm. BMPs, other members of the TGF-B family, and FGFs regulate neural crest cell migration, proliferation, and differentiation, and abnormal concentrations of these proteins have been associated with neural crest defects in the craniofacial region of laboratory animals (see Chapter 17).
Molecular Regulation of Neural Crest Induction Induction of neural crest cells requires an interaction at the junctional border of the neural plate and surface ectoderm (epidermis) (Fig. 6.5). Intermediate concentrations of BMPs are established at this boundary compared to neural plate cells that are exposed to very low levels of BMPs and surface ectoderm cells that are exposed to very high levels. The proteins noggin and chordin regulate these concentrations by acting as BMP inhibitors. The intermediate
By the time the neural tube is closed, two bilateral ectodermal thickenings, the otic placodes and the lens placodes, become visible in the cephalic region of the embryo (Fig. 6.4B). During further development, the otic placodes invaginate and form the otic vesicles, which will develop into structures needed for hearing and maintenance of equilibrium (see Chapter 19). At approximately the same time, the lens placodes appear. These placodes also invaginate and, during the fifth week, form the lenses of the eyes (see Chapter 20).
Neural crest
Sympathetic ganglion
Developing suprarenal
gland eR.
Dorsal root
ganglion
Urogenital ridge
cells

Figure 6.5 Formation and migration of neural crest cells in the spinal cord. A,B. Crest cells form at the tips of neural folds and do not migrate away from this region until neural tube closure is complete. C. After migration, crest cells contribute to a heterogeneous array of structures, including dorsal root ganglia, sympathetic chain ganglia, adrenal medulla, and other tissues (Table 6.1, p. 69). D. In a scanning electron micrograph, crest cells at the top of the closed neural tube can be seen migrating away from this area.
Figure 6.6 Drawing shows the migratory paths of neural crest
cells in
the head
region.These cells
leave the
crests of
the neural folds prior to neural tube closure and migrate to form structures in the face and neck (blue area). | to 6,
pharyngeal arches; V, VIl, IX, and X, epibranchial placodes.
In general terms, the ectodermal germ layer gives rise to organs and structures that maintain
contact with the outside world:
e The central nervous system;
e The peripheral nervous system;
e
The sensory epithelium of the ear, nose, and eye; and
e The epidermis, including the hair and nails.
In addition, it gives rise to:
e Subcutaneous glands,
e The mammary glands,
¢ The pituitary gland,
- e And enamel of the teeth.
Preaortic ganglion
Enteric ganglia

Connective tissue and bones of the face and skull
Cranial nerve ganglia (see Table 17.2)
C cells of the thyroid gland
Conotruncal septum in the heart
Odontoblasts
Dermis in face and neck
Spinal (dorsal root) ganglia
Sympathetic chain and preaortic ganglia
Parasympathetic ganglia of the gastrointestinal tract
Schwann cells
Glial cells
Meninges (forebrain)
Melanocytes
Smooth muscle cells to blood vessels of the face and forebrain

Neural tube defects (NTDs) result when neural tube closure fails to occur. If the neural tube fails to close in the cranial region, then most of the brain fails to form, and the defect is called anencephaly (Fig. 6.7A). If closure fails anywhere from the cervical region caudally, then the defect is called spina bifida (Fig. 6.7B,C). The most common site for spina bifida to occur is in the lumbosacral region (Fig. 6.7C), suggesting that the closure process in this area may be more susceptible to genetic and/ or environmental factors. Anencephaly is a lethal defect, and most of these cases are diagnosed prenatally and the pregnancies terminated. Children with spina bifida lose a degree of neurological function based on the spinal cord level of the lesion and its severity.
Occurrence of these types of defects is common and varies by different regions. For example, prior to fortification of enriched flour with folic acid in the United States, the overall rate was | in 1,000 births, but in North and South Carolina, the rate was | in 500 births. In parts of China, rates were as high as | in 200 births.Various genetic and environmental factors account for the variability, but regardless of the region rates have been reduced significantly
following folic acid administration. For example, rates throughout the United States are now approximately | in 1,500 births. It is estimated that 50% to 70% of NTDs can be prevented if women take 400 Ug of folic acid daily (the dose present in most multivitamins) beginning 3 months prior to conception and continuing throughout pregnancy. Because 50% of pregnancies are unplanned, it is
Figure 6.7 Examples of NTDs, which occur when closure of the neural tube fails. A. Anencephaly.

recommended that all women of childbearing age take a multivitamin containing 400 jg of folic acid daily. If a woman has had a child with an NTD or if there is a history of such defects in her family, it is recommended that she take 400 tg of folic acid daily and then 4,000 jg per day starting | month before she tries to become pregnant and continuing through the first 3 months of pregnancy.
Figure 6.7 (Continued) B,C. Infants with spina bifida. Most cases occur in the lumbosacral region. Fifty to seventy percent of all NTDs can be prevented by the vitamin folic acid.

or visceral mesoderm layer (Figs. 6.8C,D and 6.9).
Initially, cells of the mesodermal germ layer form a thin sheet of loosely woven tissue on each side of the midline (Fig. 6.8). By approximately the 17th day, however, cells close to the midline proliferate and form a thickened plate of tissue known as paraxial mesoderm (Fig. 6.8). More laterally, the mesoderm layer remains thin and is known as the lateral plate. With the appearance and coalescence of intercellular cavities in the lateral plate, this tissue is divided into two layers (Fig. 6.8B,C):
e A layer continuous with mesoderm covering the amnion, known as the somatic or parietal mesoderm layer, and
e A layer continuous with mesoderm covering the yolk sac, known as the splanchnic
Together, these layers line a newly formed cavity, the intraembryonic cavity, which is continuous with the extraembryonic cavity on each side of the embryo. Intermediate mesoderm connects paraxial and lateral plate mesoderm (Figs. 6.8B,D and 6.9).

By the beginning of the third week, paraxial mesoderm begins to be organized into segments. These segments, known as somitomeres, first appear in the cephalic region of the embryo, and their formation proceeds cephalocaudally. Each somitomere consists of mesodermal cells arranged in concentric whorls around the center of the unit. In the head region, somitomeres form
Figure 6.8 Transverse sections showing development of the mesodermal germ layer. A. Day 17.B. Day 19.C. Day 20. D. Day 21.The thin mesodermal sheet gives rise to paraxial mesoderm (future somites), intermediate mesoderm (future excretory units), and the lateral plate, which is split into parietal and visceral mesoderm layers lining the intraembryonic cavity.
in association with segmentation of the neural arises
in the
occipital region
of the
embryo at
plate into neuromeres and contribute to mes- enchyme in the head (see Chapter 17). From the
occipital region caudally, somitomeres further organize
into somites.
The first
pair of
somites approximately
the
20th day
(Fig. 6.2C,D). From here, new somites appear in
craniocaudal sequence
(Fig.
6.10)
at
a
rate of
approximately three
pairs per
day until,
Somite
Intermediate mesoderm
(parietal layer)
(visceral layer)
at the
end of the fifth week, 42 to 44 pairs are present (Figs. 6.4B and 6.10). There are 4 occipital, 8 cervical, 12 thoracic, 5 lumbar, 5 sacral, and 8 to 10 coccygeal pairs. The first occipital and the last five to seven coccygeal somites later disappear, while the remaining somites form the axial skeleton (see Chapter 10). Because somites appear with a specified periodicity, the age of an embryo can be accurately determined during this early time period by counting somites (Table 6.2, p. 72).

at high concentrations cranially and decreases in concentration caudally, whereas the combination of FGF8 and WNT3a proteins is expressed at higher concentrations caudally and lower ones cranially. These overlapping expression gradients control the segmentation clock and activity of the NOTCH pathway.

Molecular Regulation of Somite Formation Formation of segmented somites from unsegmented presomitic (paraxial) mesoderm (Fig. 6.10) is dependent upon a segmentation clock established by cyclic expression of a number of genes. The cyclic genes include members of the Notch and WNT signaling pathways that are expressed in an oscillating pattern in presomitic mesoderm. Thus, Notch protein accumulates in presomitic mesoderm destined to form the next somite and then decreases as that somite is established. The increase in Notch protein activates other segment-patterning genes that establish the somite. Boundaries for each somite are regulated by retinoic acid (RA) and a combination of FGF8 and WNT3a. RA is expressed
When somites first form from presomitic mesoderm, they exist as a ball of mesoderm (fibroblast-like) cells. These cells then undergo a process of epithelization and arrange themselves in a donut shape around a small lumen (Fig. 6.11). By the beginning of the fourth week, cells in the ventral and medial walls of the somite lose their epithelial characteristics, become mesenchymal (fibroblast-like) again, and shift their position to surround the neural tube and notochord. Collectively, these cells form the sclerotome that will differentiate into the vertebrae and ribs (see Chapter 10). Cells at the dorsomedial and ventrolateral edges of the upper region of the somite form precursors for muscle cells, while cells between these two groups form the dermatome (Fig. 6.11B). Cells from both muscle precursor groups become mesenchymal again and migrate beneath the dermatome to create

20, Number of Somites = 1-4. 21, Number of Somites = 4-7. 22, Number of Somites = 7-10. 23, Number of Somites = 10-13. 24, Number of Somites = 13-17. 25, Number of Somites = 17-20. 26, Number of Somites = 20-23. 27, Number of Somites = 23-26. 28, Number of Somites = 26-29. 30, Number of Somites = 34-35
the dermomyotome (Fig. 6.11C,D). In addition, cells from the ventrolateral edge migrate into the parietal layer of lateral plate mesoderm to form most of the musculature for the body wall (external and internal oblique and transversus abdominis muscles) and most of the limb muscles (Fig. 6.11B; see Chapter 11). Cells in the dermomyotome ultimately form dermis for the skin of the back and muscles for the back, body wall (intercostal muscles), and some limb muscles (see Chapter 11).
Each myotome and dermatome retains its innervation from its segment of origin, no matter where the cells migrate. Hence, each somite forms its own sclerotome (the tendon cartilage and bone component), its own myotome (providing the segmental muscle component), and its own dermatome, which forms the dermis of the back. Each myotome and dermatome also has its own segmental nerve component.

Molecular Regulation of Somite Differentiation Signals for somite differentiation arise from surrounding structures, including the notochord, neural tube, epidermis, and lateral plate mesoderm (Fig. 6.12). The secreted protein products of the noggin genes and sonic hedgehog (SHH), produced by the notochord and floor plate of the neural tube, induce the ventromedial portion of the somite to become sclerotome. Once induced, sclerotome cells express the transcription factor PAX1, which initiates the cascade of cartilageand bone-forming genes for vertebral formation. Expression of PAX3, regulated by WNT proteins from the dorsal neural tube, marks the dermomyotome region of the somite. WNT proteins from the dorsal neural tube also target the dorsomedial portion of the somite, causing it to initiate expression of the muscle-specific gene MYF5 and to form primaxial muscle precursors. Interplay between the inhibiting protein BMP4 (and probably FGFs) from the lateral plate mesoderm and activating WNT products from the epidermis direct the dorsolateral portion of the somite to express another muscle-specific gene, MYOD, and to form primaxial and abaxial muscle precursors. The midportion of the dorsal epithelium of the somite is directed by neurotrophin 3 (NT-3), secreted by the dorsal region of the neural tube, to form dermis.

Intermediate mesoderm, which temporarily connects paraxial mesoderm with the lateral plate (Figs. 6.8D and 6.9), differentiates into urogenital structures. In cervical and upper thoracic regions, it forms segmental cell clusters (future nephrotomes), whereas more caudally, it forms an unsegmented mass of tissue, the nephrogenic cord. Excretory units of the urinary system and the gonads develop from this partly segmented, partly unsegmented intermediate mesoderm (see Chapter 16).

Lateral plate mesoderm splits into parietal (somatic) and visceral (splanchnic) layers, which line the intraembryonic cavity and surround the organs, respectively (Figs. 6.8C,D, 6.9, and 6.13A). Mesoderm from the parietal layer, together with overlying ectoderm, forms the lateral body wall folds (Fig. 6.13). These folds, together with the head (cephalic) and tail (caudal)
73
Figure 6.11 Stages in the development of a somite. A. Mesoderm cells that have undergone epithelization are arranged around a small cavity. B. Cells from the ventral and medial walls of the somite lose their epithelial arrangement and migrate around the neural tube and notochord. Collectively, these cells constitute the sclerotome that will form the vertebrae and ribs. Meanwhile, cells at the dorsomedial and ventrolateral regions differentiate into muscle precursor cells, while cells that remain between these locations form the dermatome. B. Both groups of muscle precursor cells become mesenchymal and migrate beneath the dermatome to form the dermomyotome B,C while some cells from the ventrolateral group also migrate into the parietal layer of lateral plate mesoderm. B. Eventually, dermatome cells also become mesenchymal and migrate beneath the ectoderm to form the dermis of the back D.

folds, close the ventral body wall. The parietal layer of lateral plate mesoderm then forms the dermis of the skin in the body wall and limbs, the bones and connective tissue of the limbs, and the sternum. In addition, sclerotome and muscle precursor cells that migrate into the parietal layer of lateral plate mesoderm form the costal cartilages, limb muscles, and most of the body wall muscles (see Chapter 11). The visceral layer of lateral plate mesoderm, together with embryonic endoderm, forms the wall of the gut tube (Fig. 6.13B). Mesoderm cells of the parietal layer surrounding the intraembryonic cavity form thin membranes, the mesothelial membranes, or serous membranes, which will line the peritoneal, pleural, and pericardial cavities and secrete serous fluid (Fig. 6.13B). Mesoderm cells of the visceral layer form a thin serous membrane around each organ (see Chapter 7).
Figure 6.12 Expression patterns of genes that regulate somite differentiation. Sonic hedgehog (SHH) and noggin, secreted by the notochord and floor plate of the neural tube, cause the ventral part of the somite to form sclerotome and to express PAX, which in turn controls chondrogenesis and vertebrae formation. WNT proteins from the dorsal neural tube activate PAX3, which demarcates the dermomyotome.WNT proteins also direct the dorsomedial portion of the somite to differentiate into muscle precursor cells and to express the muscle-specific gene MYF5.The mid-dorsal portion of the somite is directed to become dermis by NT-3 expressed by the dorsal neural tube.Additional muscle precursor cells are formed from the dorsolateral portion of the somite under the combined influence of activating WNT proteins and inhibitory BMP4 protein, which together activate
MyoD expression.

Blood cells and blood vessels also arise from mesoderm. Blood vessels form in two ways: vasculogenesis, whereby vessels arise from blood islands (Fig. 6.14) and angiogenesis, which entails sprouting from existing vessels. The first blood islands appear in mesoderm surrounding the wall of the yolk sac at 3 weeks of development and slightly later in lateral plate mesoderm and other regions (Fig. 6.15). These islands arise from mesoderm cells that are induced to form and blood cell formation.
Although the first blood cells arise in blood islands in the wall of the yolk sac, this population is transitory. The definitive hematopoietic stem cells are derived from mesoderm surrounding the aorta in a site near the developing mesonephric kidney called the aortagonad-mesonephros region (AGM). These cells colonize the liver, which becomes the major hematopoietic organ of the embryo and
Figure 6.13 A. Cross section through a 21-day embryo in the region of the mesonephros showing parietal and visceral mesoderm layers. The intraembryonic cavities communicate with the extraembryonic cavity (chorionic cavity). B. Section at the end of the fourth week. Parietal mesoderm and overlying ectoderm form the ventral and lateral body wall. Note the peritoneal (serous) membrane.
Figure 6.14 Blood vessels form in two ways: vasculogenesis (top), in which vessels arise from blood islands and angiogenesis (bottom), in which new vessels sprout from existing ones. During vasculogenesis, FGF2 binds to its receptor on subpopulations of mesoderm cells and induces them to form hemangioblasts. Then, under the influence of VEGF acting through two different receptors, these cells become endothelial and coalesce to form vessels. Angiogenesis is also regulated by VEGE, which stimulates proliferation of endothelial cells at points where new vessels will sprout from existing ones. Final modeling and stabilization of the vasculature are accomplished by PDGF and TGF-B.
Figure 6.15 Extraembryonic blood vessel formation in the villi, chorion, connecting stalk, and wall of the yolk sac in a

fetus from approximately the second to seventh months of development. Stem cells from the liver colonize the bone marrow, the definitive bloodforming tissue, in the seventh month of gestation, and thereafter, the liver loses its blood-forming function.

FGF2 induces blood island development from competent mesoderm cells that form hemangioblasts. Hemangioblasts are directed to form blood cells and vessels by vascular endothelial growth factor (VEGF), which is secreted by surrounding mesoderm cells. The signal to express VEGF may involve HOXB5, which upregulates the VEGF receptor FLK1 (Fig. 6.14). Hemangioblasts in the center of blood islands form hematopoietic stem cells, the precursors of all blood cells, whereas peripheral hemangioblasts differentiate into angioblasts, the precursors to blood vessels. These angioblasts proliferate and are eventually induced to form endothelial cells by VEGF secreted by surrounding mesoderm cells (Fig. 6.14). This same factor then regulates coalescence of these endothelial cells into the first primitive blood vessels.
Once the process of vasculogenesis establishes a primary vascular bed, which includes the dorsal aorta and cardinal veins,

Capillary hemangiomas are abnormally dense collections of capillary blood vessels that form the most common tumors of infancy, occurring in approximately 10% of all births. They may occur anywhere but are often associated with craniofacial structures (Fig. 6.16A). Facial lesions may be focal or diffuse, with diffuse lesions causing more secondary complications, including ulcerations, scarring, and airway obstruction (mandibular hemangiomas;
additional vasculature is added by angiogenesis, the sprouting of new vessels (Fig. 6.14). This process is also mediated by VEGF, which stimulates proliferation of endothelial cells at points where new vessels are to be formed. Maturation and modeling of the vasculature are regulated by other growth factors, including platelet-derived growth factor (PDGF) and TGF-f, until the adult pattern is established. Specification of arteries, veins, and the lymphatic system occurs soon after angioblast induction. SHH, secreted by the notochord, induces surrounding mesenchyme to express VEGF. In turn, VEGF expression induces the Notch pathway (a transmembrane receptor pathway), which specifies arterial development through expression of ephrinB2 (ephrins are ligands that bind to Eph receptors in a pathway involving tyrosine kinase signaling). In addition to specifying arteries, expression of ephrinB2 suppresses venous cell fate. Notch signaling also upregulates expression of EPHB4, a vein-specific gene, but how this gene and others specify venous development is not clear. On the other hand, PROX1, a homeodomain-containing transcription factor, appears to be the master gene for lymphatic vessel differentiation. Vessel outgrowth is patterned, not random, and appears to involve guidance factors similar to those employed by the nervous system.
Fig. 6.16B). Insulin-like growth factor 2 is highly expressed in the lesions and may be one factor promoting abnormal vessel growth. Whether or not VEGF plays a role has not been determined
Figure 6.16 A. Focal capillary hemangioma. B. Diffuse capillary hemangioma involving the oral cavity.

The gastrointestinal tract is the main organ system derived from the endodermal germ layer. This germ layer covers the ventral surface of the embryo and forms the roof of the yolk sac (Fig. 6.16A).With development and growth of the brain vesicles, however, the embryonic disc begins to bulge into the amniotic cavity. Lengthening of the neural tube now causes the embryo to curve into the fetal position as the head and tail regions (folds) move ventrally (Fig. 6.17). Simultaneously, two lateral body wall folds form and also move ventrally to close the ventral body wall (Fig. 6.18). As the head and tail and two lateral folds move ventrally, they pull the amnion down with them, such that the embryo lies within the amniotic cavity (Figs. 6.17 and 6.18). The ventral body wall closes completely except for the umbilical region where the connecting stalk and yolk sac duct remain attached (Figs. 6.17 and 6.19). Failure of the lateral body folds to close the body wall results in ventral body wall defects (see Chapter 7).
larger portion of the endodermal germ layer is incorporated into the body of the embryo to form the gut tube. The tube is divided into three regions: the foregut, midgut, and hindgut (Fig. 6.17C). The midgut communicates with the yolk sac by way of a broad stalk, the vitelline (yolk sac) duct (Fig. 6.17D).This duct is wide initially, but with further growth of the embryo, it becomes narrow and much longer (Figs. 6.17D and 6.18B).
As a result of cephalocaudal growth and closure of the lateral body wall folds a continuously

At its cephalic end, the foregut is temporarily bounded by an ectodermal-endodermal membrane called the oropharyngeal membrane (Fig. 6.17A,C). This membrane separates the stomadeum, the primitive oral cavity derived from ectoderm, from the pharynx, a part of the foregut derived from endoderm. In the fourth week, the oropharngeal membrane ruptures, establishing an open connection between the oral cavity and the primitive gut (Fig. 6.17D). The hindgut also terminates temporarily at an ectodermal—endodermal membrane, the cloacal membrane (Fig. 6.17C). This membrane separates the upper part of the anal canal, derived from endoderm, from the lower part, called the proctodeum, which is formed by an invaginating pit lined by ectoderm. The membrane breaks
Figure 6.18 Cross sections through embryos at various stages of development to show the effect of lateral folding on the endoderm-lined cavity. A. Folding is initiated. B. Transverse section through the midgut to show the connection between the gut and yolk sac. C. Section just below the midgut to show the closed ventral abdominal wall and gut suspended from the dorsal abdominal wall by its mesentery. Arrows, lateral folds.
down in
the seventh
week to
create the
opening for
the anus.
Another important result of cephalocaudal growth and lateral folding is partial incorporation of the allantois into the body of the embryo, where it forms the cloaca (Fig. 6.19A).The distal portion of the allantois remains in the connecting stalk. By the fifth week, the yolk sac duct, allantois, and umbilical vessels are restricted to the umbilical region (Fig. 6.19).
in its posterior wall and later migrate to the gonads to form eggs and sperm (see Chapter 16). Hence, the endodermal germ layer initially forms the epithelial lining of the primitive gut and the intraembryonic portions of the allantois and vitelline duct (Fig. 6.19A). During further development, endoderm gives rise to:

The role of the yolk sac is not clear. It may function as a nutritive organ during the earliest stages of development prior to the establishment of blood vessels. It also contributes some of the first blood cells, although this role is very transitory. One of its main functions is to provide germ cells that reside
¢ The epithelial lining of the respiratory tract; e The parenchyma of the thyroid, parathyroids, liver, and pancreas (see Chapters 15 and 17); e The reticular stroma of the tonsils and the thymus; e The epithelial lining of the urinary bladder and the urethra (see Chapter 16); and e The epithelial lining of the tympanic cavity and auditory tube (see Chapter 19).
Figure 6.19 Sagittal sections through embryos showing derivatives of the endodermal germ layer. A. Pharyngeal pouches, epithelial lining of the lung buds and trachea, liver, gallbladder, and pancreas. B. The urinary bladder is derived from the cloaca and, at this stage of development, is in open connection with the allantois.
Anterior
3'
EN
5'
Posterior
Late
High RA response
Low RA response
Figure 6.20 Drawing showing the arrangement of homeobox genes of the Antennapedia (ANT-C) and Bithorax (BX-C) classes of Drosophila and conserved homologous genes of the same classes in humans. During evolution, these genes have been duplicated, such that humans have four copies arranged on four different chromosomes. Homology between Drosophila genes and those in each cluster of human genes is indicated by color. Genes with the same number, but positioned on different chromosomes, form a paralogous group. Expression of the genes is in a cranial-to-caudal direction from the 3' (expressed early) to the 5' (expressed later) end as indicated in the fly and mouse embryo diagrams. RA modulates expression of these genes, with those at the 3' end more to the being responsive compound.
Figure 6.21 A. Lateral view of a 28-somite human embryo.The main external features are the pharyngeal arches and somites.

Homeobox genes are known for their homeodomain, a DNA-binding motif, the homeobox. They code for transcription factors that activate cascades of genes regulating phenomena such as segmentation and axis formation. Many homeobox genes are collected into homeotic clusters, although other genes also contain the homeodomain. An important cluster of genes specifying the craniocaudal axis is the homeotic gene complex Hom-C in Drosophila. These genes, which contain the Antennapedia and Bithorax classes of homeotic genes, are organized on a single chromosome as a functional unit. Thus, genes specifying more cranial structures lie at the 3' end of the DNA and are expressed first, with genes controlling posterior development expressed sequentially and lying increasingly toward the 5' end (Fig. 6.20). These genes are conserved in humans, existing as four copies, HOXA, HOXB, HOXC, and HOXD, which are arranged and expressed like those in Drosophila. Thus, each cluster lies on a separate chromosome, and the genes in each group are numbered 1 to 13 (Fig. 6.20). Genes with the same number, but belonging to different clusters, form a paralogous group, such as HOXA4, HOXB4, HOXC4, and HOXD4. The pattern of expression of these genes, along with evidence from knockout experiments in which mice are created that lack one or more of these genes, supports the hypothesis that they play a role in cranial-to-caudal patterning of the derivatives of all three germ layers. For example, an overlapping expression pattern of the HOX code exists in the somites and vertebrae, with genes located more toward the 3' end in each cluster being expressed in and regulating development of more cranial segments (Fig. 6.20).

5-8, ApproximateAge (wk) = 5. 10-14, ApproximateAge (wk) = 6. 17-22, ApproximateAge (wk) = 7. 28-30, ApproximateAge (wk) = 8
81

At the end of the fourth week, when the embryo has approximately 28 somites, the main external features are the somites and pharyngeal arches (Fig. 6.21). The age of the embryo is therefore usually expressed in somites (Table 6.2, p. 72). Because counting somites becomes difficult during the second month of development, the age of the embryo is then indicated as the crownrump length (CRL) and expressed in millimeters (Table 6.3, p. 81). CRL is the measurement from the vertex of the skull to the midpoint between the apices of the buttocks.
During the second month, the external appearance of the embryo is changed by an increase in head size and formation of the limbs, face, ears, nose, and eyes. By the beginning of the fifth week, forelimbs and hindlimbs appear as paddle-shaped buds (Fig. 6.22). The former are located dorsal to the pericardial swelling at the level of the fourth cervical to the first thoracic somites, which explains their innervation by the brachial plexus. Hindlimb buds appear slightly later just caudal to attachment of the umbilical stalk at the level of the lumbar and upper sacral somites. With further growth, the terminal portions of the buds flatten, and a circular constriction separates them from the proximal, more cylindrical segment (Fig. 6.23). Soon, four radial grooves separating five slightly thicker areas appear on the distal portion of the buds, foreshadowing formation of the digits (Fig. 6.23).
Figure 6.22 Human embryo (CRL 9.8 mm, fifth week)
(*29.9).The forelimbs are paddle-shaped.
Figure 6.24 Human embryo (CRL 21 mm, seventh week) (x4).The chorionic sac is open to show the embryo in its amniotic
sac.
The yolk
sac, umbilical
cord, and
vessels in
the chorionic
plate of
the placenta
are clearly
visible.
Note the
size of

Most major organs and organ systems are formed during the third to eighth weeks. This period, which is critical for normal development, is therefore called the period of organogenesis or embryogenesis. Stem cell populations are establishing each of the organ primordia, and these interactions are sensitive to insult from genetic and environmental influences. Thus, from the third to eighth weeks is the time when most gross structural birth
“<e defects are induced. Unfortunately, the mother may not realize she is pregnant during this critical time, especially during the third and fourth weeks, which are particularly vulnerable. Consequently, she may not avoid harmful influences, such as cigarette smoking and alcohol. Understanding the main events of organogenesis is important for identifying the time that a particular defect was induced and, in turn, determining possible causes for the malformation (see Chapter 9).
These grooves, known as rays, appear in the hand region first and shortly afterward in the foot, as the upper limb is slightly more advanced in development than the lower limb. While fingers and toes are being formed (Fig. 6.24), a second constriction divides the proximal portion of the buds into two segments, and the three parts characteristic of the adult extremities can be recognized (Fig. 6.25).
Figure 6.25 Human embryo (CRL 25 mm, seventh to eighth weeks). The chorion and the amnion have been opened. Note the size of the head, the eye, the auricle of the ear, the well-formed toes, the swelling in the umbilical cord caused by intestinal loops, and the yolk sac in the chorionic cavity.
84
Part
|
General
Embryology
Z_U
The embryonic period, which extends from the third to the eighth weeks of development, is the period during which each of the three germ layers, ectoderm, mesoderm, and endoderm, gives rise to its own tissues and organ systems. As a result of organ formation, major features of body form are established (Table 6.4, p. 84).
The ectodermal germ layer gives rise to the organs and structures that maintain contact with the outside world:
¢ Central nervous system;
¢ Peripheral nervous system;
¢ Sensory epithelium of ear, nose, and eye;
¢ Skin, including hair and nails; and
e
Pituitary, mammary,
and sweat
enamel of

the teeth.
Induction of
inactivation of
cranial the
neural the
region, chordin,
growth inactivation
and follistatin
plate glands
and is
factor regulated
BMP4.
is by
In caused
secreted the
by noggin,
by the
node, noto-
BMP4 in the hindbrain and spinal cord regions is effected by WNT3a and FGE In the absence of inactivation, BMP4 causes ectoderm to become epidermis and mesoderm to ventralize to form intermediate and lateral plate mesoderm.
Important components of the mesodermal germ layer are paraxial, intermediate, and lateral plate mesoderm. Paraxial mesoderm forms somitomeres, which give rise to mesenchyme of the head and organize into somites in occipital and caudal segments. Somites give rise to the myotome (muscle tissue), sclerotome (cartilage and bone), and dermatome (dermis of the skin), which are all supporting tissues of the body. Signals for somite differentiation are derived from surrounding structures, including the notochord, neural tube, and epidermis. The notochord and floor plate of the neural tube secrete Sonic hedgehog (SHH), which induces the sclerotome.
Two muscle-forming
regions differentiate:
One is
induced somite
in the
dorsomedial by
proteins region
of the
by chord,
and prechordal
mesoderm.
Inactivation of
WNT
portion of
the neural
dorsal tube.
The other
is induced

(mm) = 4.0-6.0. 28-30, Figure = 6.8 B. 28-30, Characteristic Features = Fourth visceral arch formed; hindlimb buds appear; otic vesicle and lens placode. 31-35, Somites = . 31-35, Length (mm) = 7.0-10.0. 31-35, Figure = 6.22. 31-35, Characteristic Features = Forelimbs paddle-shaped; nasal pits formed; embryo tightly C-shaped. 36-42, Somites = . 36-42, Length (mm) = 9.0-14.0. 36-42, Figure = 6.23. 36-42, Characteristic Features = Digital rays in hand and foot plates; brain vesicles prominent; external auricle forming from auricular hillocks; umbilical herniation initiated. 43-49, Somites = . 43-49, Length (mm) = 13.0-22.0. 43-49, Figure = 6.24. 43-49, Characteristic Features = Pigmentation of retina visible; digital rays separating; nipples and eyelids formed; maxillary swellings fuse with medial nasal swellings as upper lip forms; prominent umbilical herniation. 50-56, Somites = . 50-56, Length (mm) = 21.0-31.0. 50-56, Figure = 6.25. 50-56, Characteristic Features = Limbs long, bent at elbows, knees; fingers, toes free; face more human-like; tail disappears; umbilical herniation persists to end of third month
in the ventrolateral region of the somite by a combination of BMP4 and FGF, secreted by lateral plate mesoderm, and by WNT proteins, secreted by the overlying ectoderm.

The dorsal midportion of the somite becomes dermis under the influence of neurotrophin 3, secreted by the dorsal neural tube (Fig. 6.12). Mesoderm also gives rise to the vascular system (i.e., the heart, arteries, veins, lymph vessels, and all blood and lymph cells). Furthermore, it gives rise to the urogenital system: kidneys, gonads, and their ducts (but not the bladder). Finally, the spleen and cortex of the suprarenal glands are mesodermal derivatives.
The endodermal germ layer provides the epithelial lining of the gastrointestinal tract, respiratory tract, and urinary bladder. It also forms the parenchyma of the thyroid, parathyroids, liver, and pancreas. Finally, the epithelial lining of the tympanic cavity and auditory tube originates in the endodermal germ layer.
Craniocaudal patterning of the embryonic axis is controlled by homeobox genes. These genes, conserved from Drosophila, are arranged in four clusters, HOXA, HOXB, HOXC, and HOXD, on four different chromosomes. Genes toward the 3' end of the chromosome control development of more cranial structures; those more toward the 5' end regulate differentiation of more posterior structures. Together, they regulate patterning of the hindbrain and axis of the embryo (Fig. 6.20).
As a result of formation of organ systems and rapid growth of the central nervous system, the initial flat embryonic disc begins to lengthen and to form head and tail regions (folds) that cause the embryo to curve into the fetal position. The embryo also forms two lateral body wall folds that grow ventrally and close the ventral body wall. As a result of this growth and folding, the amnion is pulled ventrally and the embryo lies within the amniotic cavity (Fig. 6.17). Connection with the yolk sac and placenta is maintained through the vitelline duct and umbilical cord, respectively.

1. Describe the process of neurulation and include definitions for the terms neural folds, neural tube, and neural tube closure. Where is neural tube closure initiated and how does it proceed? What week in gestation is the process completed? What happens if neural tube closure fails cranially? Caudally? What is an NTD and how can most be prevented?
2. What is the embryological origin of neural crest cells? Are they ectodermal, mesodermal, or endodermal in origin? To what structures do they contribute? What protein is primarily responsible for their induction?
3. From what germ layer are somites formed? How are they organized, and what tissues do they form?
4. What are the two ways that blood vessels arise? What growth factor plays a key role in early blood cell and vessel formation? What type of tumor is caused by abnormal prolif~ erations of capillary blood vessels?
5. What are the major subdivisions of the gut tube, and what germ layer gives rise to these parts? What structure forms a connection from the midgut to the yolk sac? What membranes close the gut tube cranially and caudally?
6. Why are the third to eighth weeks of embryogenesis so important for normal development and the most sensitive for induction of structural defects?

During the third and fourth weeks the top layer (ectoderm) of the trilaminar embryonic disc forms the neural plate that rolls up into a tube to form the brain and spinal cord by the process called neurulation (see Chapter 6, p. 67). Almost simultaneously, the ventral layer (endoderm) rolls down to form the gut tube, such that the embryo consists of a tube on top ofa tube: the neural tube dorsally and the gut tube ventrally (Fig. 7.1). The middle layer (mesoderm) holds the two tubes together and the lateral plate component of this mesoderm layer also splits into visceral (splanchnic) and parietal (somatic) layers. The visceral layer rolls ventrally and is intimately connected to the gut tube; the parietal layer, together with the overlying ectoderm, forms the lateral body wall folds (one on each side of the embryo), which move ventrally and meet in the midline to close the ventral body wall (Fig. 7.1).The space between visceral and parietal layers of lateral plate mesoderm is the primitive body cavity, which at this early stage is a continuous cavity, since it
Figure 7.1 Transverse sections through embryos at various stages of closure of the gut tube and ventral body wall. A. At approximately 19 days, intercellular clefts are visible in the lateral plate mesoderm. B. At 20 days, the lateral plate is divided into somatic and visceral mesoderm layers that line the primitive body cavity (intraembryonic cavity). C. By 21 days, the primitive body cavity (intraembryonic cavity) is still in open communication with the extraembryonic cavity. D. By 24 days, the lateral body wall folds, consisting of the parietal layer of lateral plate mesoderm and overlying ectoderm are approaching each other in the midline. E. At the end of the fourth week, visceral mesoderm layers are continuous with parietal layers as a double-layered membrane, the dorsal mesentery. Dorsal mesentery extends from the caudal limit of the foregut to the end of the hindgut.
has not yet been subdivided into the pericardial, pleural, and abdominopelvic regions.

At the end of the third week, intraembryonic mesoderm differentiates into paraxial mesoderm, which forms somitomeres and somites that play a major role in forming the skull and vertebrae; intermediate mesoderm, which contributes to the urogenital system; and lateral plate mesoderm, which is involved in forming the body cavity (Fig. 7.1). Soon after it forms as a solid mesodermal layer, clefts appear in the lateral plate mesoderm that coalesce to split the solid layer into two (Fig. 7.1B): (1) the parietal (somatic) layer adjacent to the surface ectoderm and continuous with the extraembryonic parietal mesoderm layer over the amnion. Together, the parietal (somatic) layer of lateral plate mesoderm and overlying ectoderm are called the somatopleure; (2) the visceral
(splanchnic) layer adjacent to endoderm forming the gut tube and continuous with the visceral layer of extraembryonic mesoderm covering the yolk sac (Figs. 7.1B). Together, the visceral (splanchnic) layer of lateral plate mesoderm and underlying endoderm are called the splanchnopleure. The space created between the two layers of lateral plate mesoderm constitutes the primitive body cavity. During the fourth week, the sides of the embryo begin to grow ventrally forming two lateral body wall folds (Fig. 7.1B and C). These 'olds consist of the parietal layer of lateral plate mesoderm, overlying ectoderm, and cells from adjacent somites that migrate into the mesoderm ayer across the lateral somitic frontier (see Chapter 11, p. 143). As these folds progress, the endoderm ayer also folds ventrally and closes to form the gut tube (Fig. 7.1D and E). By the end of the fourth week, the lateral body wall folds meet in the midline and fuse to close the ventral body wall (Fig. 7.1C-E). This closure is aided by growth of the head and tail regions (folds) that cause the embryo to curve into the fetal position (Fig. 7.2). Closure

Figure 7.2 Midsagittal sections of embryos at various stages of development showing cephalocaudal folding and its effects upon position of the heart, septum transversum, yolk sac,and amnion. Note that, as folding progresses, the opening of the gut tube into the yolk sac narrows until it forms a thin connection, the vitelline (yolk sac) duct, between the midgut and the yolk sac D. Simultaneously, the amnion is pulled ventrally until the amniotic cavity nearly surrounds the embryo. A. 17 days. B. 22 days. C. 24 days. D. 28 days. Arrows: head and tail folds.
of the ventral body wall is complete except in the region of the connecting stalk (future umbilical cord). Similarly, closure of the gut tube is complete except for a connection from the midgut region to the yolk sac called the vitelline (yolk sac)duct (Fig. 7.2D). This duct is incorporated into the umbilical cord, becomes very narrow (Fig. 8.16, p. 106), and degenerates with the yolk sac between the second and third months of gestation. (Note that throughout the process of body cavity and gut tube development, the parietal and visceral layers of lateral plate mesoderm are continuous with each other at the junction of the gut tube with the posterior body wall [Fig. 7.1D,E}).

Some cells of the parietal layer of lateral plate mesoderm lining the body wall of the primitive embryonic cavity become mesothelial and form the parietal layer of the serous membranes

Ventral body wall defects occur in the thorax, abdomen, and pelvis and involve the heart (ectopia cordis), abdominal viscera (gastroschisis), and/or urogenital organs (bladder or cloacal exstrophy), depending upon the location and size of the abnormality. The malformations are due to a failure of the ventral body wall to close and probably involve the lateral body wall folds to a greater extent than the head and tail folds. Thus, one or both of the lateral body wall folds fail to progress ventrally or there are abnormalities in the fusion process once they meet in the midline. An omphalocele also represents a ventral body wall defect; however, its primary cause is not due to inhibition of body wall closure. Instead, this abnormality occurs when a portion of the gut tube fails to return to the abdominal cavity following its normal herniation into the umbilical cord (see p. 220).
Ectopia cordis occurs when lateral body wall folds fail to close the midline in the thoracic region causing the heart to lie outside the body cavity (Fig. 7.3A). Sometimes, the closure defect begins at the caudal end of the sternum and extends into the upper abdomen resulting in a spectrum of abnormalities called Cantrell pentalogy. This spectrum includes ectopia cordis, defects in the anterior region of the diaphragm, absence of the pericardium, defects in the sternum, and abdominal wall defects including omphalocele and gastroschisis.

lining the outside of the peritoneal, pleural, and pericardial cavities. In a similar manner, some cells of the visceral layer of lateral plate mesoderm form the visceral layer of the serous membranes covering the abdominal organs, lungs, and heart (Fig. 7.1E). Visceral and parietal layers are continuous with each other as the dorsal mesentery (Fig. 7.1E), which suspends the gut tube from the posterior body wall into the peritoneal cavity. Dorsal mesentery extends continuously from the caudal limit of the foregut to the end of the hindgut. Ventral mesentery exists only from the caudal foregut to the upper portion of the duodenum and results from thinning of mesoderm of the septum transversum, a block of mesoderm that forms connective tissue in the liver and the central tendon of the diaphragm (see Figs. 7.2D and 7.5). These mesenteries are double layers of peritoneum that provide a pathway for blood vessels, nerves, and lymphatics to the organs.
(Note, omphaloceles that may occur in Cantrell pentalogy are secondary to the body wall closure defect, not primary. The closure defect reduces the size of the abdominal cavity and prevents the return of the intestinal loops from the umbilical cord; see p. 220).
Gastroschisis occurs when body wall closure fails in the abdominal region (Fig. 7.3B).As a result, intestinal loops herniate into the amniotic cavity through the defect, which usually lies to the right of the umbilicus. The incidence of gastroschisis is increasing (3.5/10,000), and it is most common in infants from thin women younger than 20 years of age. The defect can be detected by fetal ultrasound and by elevated a-fetoprotein (AFP) concentrations in maternal serum and the amniotic fluid. The malformation is not associated with chromosome abnormalities, but other defects occur in 15% of cases. Affected loops of bowel may be damaged by exposure to amniotic fluid, which has a corrosive effect, or by twisting around each other (volvulus) and compromising their blood supply.

Bladder and cloacal exstrophy results from abnormal body wall closure in the pelvic region. Bladder exstrophy represents a less severe closure defect in this region and only the bladder is exposed (Fig. 7.3C; in males, the penis may be involved and epispadius [a split in the dorsum of the penis; see Chapter 16, p. 252] is common). Cloacal exstrophy results from a more severe

Cc
D
Examples of ventral body wall defects due to failure of the ventral body wall to close.
A. Ectopia cordis. The heart lies outside the thorax, and there is a cleft in the thoracic wall. B. Gastroschisis. Intestines have herniated through the abdominal wall to the right of the umbilicus, the most common location for this defect. C. Bladder exstrophy. Closure in the pelvic region has failed. In males, the defect usually includes a split in the dorsum of the penis, a defect called epispadius. D. Cloacal exstrophy.A larger closure defect in which most of the pelvic region has failed to close, leaving the bladder, part of the rectum, and the anal canal exposed.
failure of body wall closure in the pelvis such that the bladder and rectum, which are derived from the cloaca (see Chapter 16, p. 238), are exposed (Fig. 7.3D).
Omphalocele represents another ventral body wall defect (Fig. 7.4), but it does not arise from a failure in body wall closure. Instead, it originates when portions of the gut tube (the midgut) that normally herniates into the umbilical cord during the sixth to the tenth weeks (physiological umbilical herniation) fails to return to the abdominal cavity (see Chapter 15, p. 220). Subsequently, loops of bowel, and other viscera, including the liver, may herniate into the defect. Since the umbilical cord is covered by a reflection of the amnion, the defect is covered by this epithelial layer. (In contrast, loops of bowel in gastroschisis are not covered by amnion because they herniate through the abdominal wall directly into the amniotic cavity.) Omphalocele, which occurs in 2.5/10,000 births, is associated with high mortality rates and severe malformations, including cardiac abnormalities and neural tube defects. Furthermore, chromosome abnormalities are present in 15% of cases. Like gastroschisis, omphaloceles are associated with elevated AFP concentrations.
B

Figure 7.4 Examples of omphaloceles, a defect that occurs when loops of bowel, that normally herniate into the umbilical cord during the sixth to the tenth weeks of gestation (physiological umbilical herniation), fail to return to the body cavity. A. Drawing showing loops of herniated bowel within the umbilical cord that have failed to return to the abdominal cavity. The bowel is covered by amnion because this membrane normally reflects onto the umbilical cord. B. Infant with an omphalocele. The defect is associated with other major malformations and chromosome abnormalities.

pericardioperitoneal canals, on each side of the foregut (Fig. 7.5B).
The septum transversum is a thick plate of mesodermal tissue occupying the space between the thoracic cavity and the stalk of the yolk sac (Fig. 7.5A,B). The septum is derived from visceral (splanchnic) mesoderm surrounding the heart and assumes its position between the primitive thoracic and abdominal cavities when the cranial end of the embryo grows and curves into the fetal position (Fig. 7.2B-D).This septum does not separate the thoracic and abdominal cavities completely, but leaves large openings, the
When lung buds begin to grow, they expand caudolaterally within the pericardioperitoneal canals (Fig. 7.5C). As a result of the rapid growth of the lungs, the pericardioperitoneal canals become too small, and the lungs begin to expand into the mesenchyme of the body wall dorsally, laterally, and ventrally (Fig. 7.5C). Ventral and lateral expansion is posterior to the pleuropericardial folds. At first, these folds appear as small ridges projecting into the primitive undivided thoracic cavity (Fig. 7.5C). With expansion of the lungs, mesoderm
Figure 7.5 A. Drawing showing the ventral view of an embryo at 24 days of gestation. The gut tube is closing, the anterior and posterior intestinal portals are visible, and the heart lies in the primitive pleuropericardial cavity, which is partially separated from the abdominal cavity by the septum transversum (arrow). B. Portion of an embryo at approximately 5 weeks with parts of the body wall and septum transversum removed to show the pericardioperitoneal canals. Note the size and thickness of the septum transversum and liver cords penetrating the septum. C. Growth of the lung buds into the pericardioperitoneal canals. Note the pleuropericardial folds.

of the body wall splits into two components (Fig. 7.6): (1) the definitive wall of the thorax and (2) the pleuropericardial membranes, which are extensions of the pleuropericardial folds that contain the common cardinal veins and phrenic nerves. Subsequently, descent of the heart and positional changes of the sinus venosus shift the common cardinal veins toward the midline, and the pleuropericardial membranes are drawn out in mesentery-like fashion (Fig. 7.6A). Finally, they fuse with each other and with the root of the lungs, and the thoracic cavity is divided into the definitive pericardial cavity and two pleural cavities (Fig. 7.6B).
A
Figure
7.6
A.
Transformation of
the pericardioperitoneal
canals into
the pleural
cavities and
pleuropericardial membranes.
Note
Mesenchyme of
the body
wall the
splits pleuropericardial
folds into
the containing
pleuropericardial the
membranes common
and cardinal
definitive vein
body and
wall.
B.
the phrenic
The nerve.
thorax fusion
of the
pleuropericardial folds
with each
other and
with the
root of
the lungs.
Note the
the after
phrenic now in the fibrous pericardium. The right common cardinal vein has developed into the superior vena cava.
In the adult, the pleuropericardial membranes form the fibrous pericardium.

Although the pleural cavities are separate from the pericardial cavity, they remain in open communication with the abdominal (peritoneal) cavity by way of the pericardioperitoneal canals (Fig. 7.5B). During further development, the opening between the prospective pleural and peritoneal cavities is closed by crescent-shaped folds, the pleuroperitoneal folds, which project into the caudal end of the pericardioperitoneal canals (Fig. 7.7A). Gradually, the folds extend medially and ventrally, so that by the seventh week, they fuse with the mesentery of the esophagus and with the septum transversum (Fig. 7.7B). Hence, the connection between the pleural and peritoneal portions of the body cavity is closed by the pleuroperitoneal membranes. Further expansion of the pleural cavities relative to mesenchyme of the body wall adds a peripheral rim to the pleuroperitoneal membranes (Fig. 7.7C). Once this rim is established, myoblasts originating from somites at cervical segments three to five (C,_,) penetrate the membranes to form the muscular part of the diaphragm.
Figure 7.7 Development of the diaphragm. A. Pleuroperitoneal folds appear at the beginning of the fifth week. B. Pleuroperitoneal folds fuse with the septum transversum and mesentery of the esophagus in the seventh week, separating the thoracic cavity from the abdominal cavity. C. Transverse section at the fourth month of development.An additional rim derived from the wall forms the most of the body peripheral part diaphragm.
nerve,

A congenital diaphragmatic hernia, one of the more common malformations in the newborn (I per 2,000), is most frequently caused by failure of one or both of the pleuroperitoneal membranes to close the pericardioperitoneal canals (Fig. 7.8). In that case, the peritoneal and pleural cavities are continuous with one another along the posterior body wall. This hernia allows abdominal viscera to enter the pleural cavity. In 85% to 90% of cases, the hernia is on the left side, and intestinal loops, stomach, spleen, and part of the liver may enter the thoracic cavity (Fig. 7.8). The abdominal viscera in the chest push the heart anteriorly and compress the lungs, which are commonly hypoplastic. A large defect is associated with a high rate of mortality (75%) from pulmonary hypoplasia and dysfunction. Occasionally, a small part of the muscular fibers of the diaphragm fails to develop, and a hernia may remain undiscovered until the child is several years old. Such a defect, frequently seen in the anterior portion of the diaphragm, is a parasternal hernia. A small peritoneal sac containing intestinal loops may enter the chest between the sternal and costal portions of the diaphragm (Fig. 7.8A).
Another type of diaphragmatic hernia, esophageal hernia, is thought to be due to congenital shortness of the esophagus. Upper portions of the stomach are retained in the thorax, and the stomach is constricted at the level of the diaphragm.
Figure 7.8 Congenital diaphragmatic hernia. A. Abdominal surface of the diaphragm showing a large defect of the pleuroperitoneal membrane. B. Hernia of the intestinal loops and part of the stomach into the left pleural cavity. The heart and mediastinum are frequently pushed to the right, and the left lung is compressed. C. Radiograph of a newborn with a large defect in the left side of the diaphragm.Abdominal viscera have entered the thorax through the defect.
Thus, the diaphragm is derived from the following structures:
- e the septum transversum, which forms the

central tendon of the diaphragm;
e the two pleuroperitoneal membranes;
© muscular components from somites at cervical
segments three to five; and
- e the mesentery of the esophagus, in which the crura of the diaphragm develop (Fig. 7.7C).
During the fourth week, the septum transversum lies opposite cervical somites, and nerve components of the third, fourth, and fifth cervical segments of the spinal cord grow into the septum. At first, the nerves, known as phrenic nerves, pass into the septum through the pleuropericardial folds (Fig. 7.5B). This explains why further expansion of the lungs and descent of the septum shift the phrenic nerves that innervate the diaphragm into the fibrous pericardium (Fig. 7.6). Although the septum transversum lies opposite cervical segments during the fourth week, by the sixth week, the developing diaphragm is at the level of thoracic somites. The repositioning of the diaphragm is caused by rapid growth of the dorsal part of the embryo (vertebral column), compared with that of the ventral part. By the beginning of the third month, some of the dorsal bands of the diaphragm originate at the level of the first lumbar vertebra.
The phrenic nerves supply the diaphragm with its motor and sensory innervation. Since the most peripheral part of the diaphragm is derived from mesenchyme of the thoracic wall, it is generally accepted that some of the lower intercostal (thoracic) nerves contribute sensory fibers to the peripheral part of the diaphragm.
__

At the end of the third week, the neural tube is elevating and closing dorsally, while the gut tube is rolling and closing ventrally to create a "tube on top of a tube." Mesoderm holds the tubes together and the lateral plate mesoderm splits to form a visceral (splanchnic) layer associated with the gut and a parietal (somatic) layer that, together with overlying ectoderm, forms the lateral body wall folds. The space between the visceral and parietal layers of lateral plate mesoderm is the primitive body cavity (Fig. 7.1). When the lateral body wall folds move ventrally and fuse in the midline, the body cavity is closed, except in the region of the connecting stalk (Figs. 7.1 and 7.2). Here the gut tube maintains an attachment to the yolk sac as the yolk sac
1.

A
newborn infant
cannot dies.
An autopsy
defect reveals
on the
the breathe
a
and soon
large left
intestines side,
diaphragmatic with
occupying the
stomach the
and left
side of
the thorax.
Both lungs
are severely
hypoplastic.
What is the embryological basis for this defect?
- -A child is born with a large defect lateral to the umbilicus. Most of the large and the small bowel protrude through the defect
and are
not covered
embryological by
amnion.
basis should
you be
for
What is
this abnormality,
concerned that
the and
other malfor-
mations may
be present?
- . Explain why the phrenic nerve, which sup-
plies motor
fibers to
the dia-
phragm, originates
when most
from of
cervical the
diaphragm
From which
cervical nerve originate?
segments is
in segments
does the

The period from the beginning of the ninth week to birth is known as the fetal period. It is characterized by maturation of tissues and organs and rapid growth of the body. The length of the fetus is usually indicated as the crownrump length (CRL) (sitting height) or as the crown-heel length (CHL), the measurement from the vertex of the skull to the heel (standing height). These measurements, expressed in centimeters, are correlated with the age of the fetus in weeks or months (Table 8.1). Growth in length is particularly striking during the third, fourth, and fifth months, while an increase in weight is most striking during the last 2 months of gestation. In general, the length of pregnancy is considered to be 280 days, or 40 weeks after the onset of the last normal menstrual period (LNMP) or, more accurately, 266 days or 38 weeks after fertilization. For the purposes of the following discussion, age is calculated from the time of fertilization and is expressed in weeks or calendar months.

One
of
the
most
striking
changes
taking
place
during
fetal
life
is
the
relative
slowdown
in
growth
of
the
head
compared
with
the
rest
of
the
body.
At the beginning of the third month, the head constitutes approximately half of the CRL (Figs. 8.1 and 8.2). By the beginning of the fifth month, the size of the head is about one third of the CHL, and at birth, it is approximately one quarter of the CHL (Fig. 8.2). Hence, over time, growth of the body accelerates but that of the head slows down. During the third month, the face becomes more human looking (Figs. 8.3 and 8.4). The eyes, initially directed laterally, move to the ventral aspect of the face, and the ears come to lie close to their definitive position at the side of the head (Fig. 8.3). The limbs reach their relative length in comparison with the rest of the body, although the lower limbs are still a little shorter and less well developed than the upper extremities. Primary ossification centers are present in the long bones and skull by the 12th week. Also by the 12th week, external genitalia develop to such a degree that the sex of the fetus can be determined by external examination (ultrasound). During the sixth week, intestinal loops cause a large swelling (herniation) in the umbilical cord, but by the 12th week, the loops have withdrawn into the abdominal cavity. At the end of the third month, reflex activity can be evoked in aborted fetuses, indicating muscular activity. During the fourth and fifth months, the fetus lengthens rapidly (Fig. 8.5 and Table 8.1),

9-12, Growth in Length andWeight During the Fetal Period.CRL (cm) = 5-8. 9-12, Growth in Length andWeight During the Fetal Period.Weight (g) = 10-45. 13-16, Growth in Length andWeight During the Fetal Period.CRL (cm) = 9-14. 13-16, Growth in Length andWeight During the Fetal Period.Weight (g) = 60-200. 17-20, Growth in Length andWeight During the Fetal Period.CRL (cm) = 15-19. 17-20, Growth in Length andWeight During the Fetal Period.Weight (g) = 250-450. 21-24, Growth in Length andWeight During the Fetal Period.CRL (cm) = 20-23. 21-24, Growth in Length andWeight During the Fetal Period.Weight (g) = 500-820. 25-28, Growth in Length andWeight During the Fetal Period.CRL (cm) = 24-27. 25-28, Growth in Length andWeight During the Fetal Period.Weight (g) = 900-1,300. 29-32, Growth in Length andWeight During the Fetal Period.CRL (cm) = 28-30. 29-32, Growth in Length andWeight During the Fetal Period.Weight (g) = 1,400-2,100. 33-36, Growth in Length andWeight During the Fetal Period.CRL (cm) = 31-34. 33-36, Growth in Length andWeight During the Fetal Period.Weight (g) = 2,200-2,900. 37-38, Growth in Length andWeight During the Fetal Period.CRL (cm) = 35-36. 37-38, Growth in Length andWeight During the Fetal Period.Weight (g) = 3,000-3,400
Figure 8.1 A 9-week fetus. Note the large head size compared with that of the rest of the body. The yolk sac and long vitelline duct are visible in the chorionic cavity. Note the umbilical cord and herniation of intestinal loops. One side of the chorion has many villi (chorion frondosum), while the other side is almost smooth (chorion laeve).

and at the end of the first half of intrauterine life, its CRL is approximately 15 cm, about half the total length of the newborn. The weight of the fetus increases little during this period and by the end of the fifth month is still <500 g.The fetus is covered with fine hair, called lanugo hair; eyebrows and head hair are also visible. During the fifth month, movements of the fetus can be felt by the mother.
During the second half of intrauterine life, weight increases considerably, particularly during the last 2.5 months, when 50% of the fullterm weight (approximately 3,200 g) is added. During the sixth month, the skin of the fetus
Figure 8.2 Size of the head in relation to the rest of the body at various stages of development.
97
Figure 8.3 An | |-week fetus. The umbilical cord still shows a swelling at its base, caused by herniated intestinal
loops. The skull of this fetus lacks the normal smooth contours. Fingers and toes are well developed.
Figure 8.5 An |8-week fetus connected to the placenta by its umbilical cord. The skin of the fetus is thin because of lack of
Figure 8.4 A |2-week fetus in utero. Note the extremely thin skin and underlying blood vessels. The face has all of the human characteristics, but the ears are still primitive. Movements begin at this time but are usually not felt by the mother.

Taste buds appear, Age (Wk) = 7. Swallowing, Age (Wk) = 10. Respiratory movements, Age (Wk) = 14-16. Sucking movements, Age (Wk) = 24. Some sounds can be heard, Age (Wk) = 24-26. Eyes sensitive to light a, Age (Wk) = 28. a Recognition of form and color occurs postnatally., Age (Wk) = a Recognition of form and color occurs postnatally.
is reddish and has a wrinkled appearance because of the lack of underlying connective tissue. A fetus born early in the sixth month has great difficulty surviving. Although several organ systems are able to function, the respiratory system and the central nervous system have not differentiated sufficiently, and coordination between the two systems is not yet well established. By 6.5 to 7 months, the fetus has a CRL of about 25 cm and weighs approximately 1,100 g. If born at this time, the infant has a 90% chance of surviving. Some developmental events occurring during the first 7 months are indicated in Table 8.2.
During the
last
2
months, the
fetus obtains
well-rounded contours
tion of
subcutaneous intrauterine
as the
fat life,
the fatty
substance secretory
skin
(vernix products
At the
has the
body, from
end
(Fig.
result
8.6).
of
By is
covered caseosa)
sebaceous of
largest the
ninth deposi-
the end
of by
a
whitish, composed
of glands.
month, circumference
sage through
the fact
with the
birth weight
g, of
its
50
CRL
cm.
canal.
a
normal is
about
Sexual the
of all
parts regard
At fetus
skull of
to the
the its
time is
36
cm, and
characteristics and
the testes
should be
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pas- birth,
of
3,000
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3,400
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are pronounced,
scrotum.

The date of birth is most accurately indicated as 266 days, or 38 weeks, after fertilization. The oocyte is usually fertilized within 12 hours of ovulation; however, sperm deposited in the reproductive tract up to 6 days prior to ovulation can survive to fertilize oocytes. Thus, most pregnancies occur when sexual intercourse occurs within a 6-day period that ends on the day of ovulation. A pregnant woman usually will see her obstetrician when she has missed two successive menstrual bleeds. By that time, her recollection about coitus is usually vague, and it is readily understandable that the day of fertilization is difficult to determine.
99
Figure 8.6 A 7-month fetus. This fetus would be able to survive. It has well-rounded contours as a result of deposition of subcutaneous fat. Note the twisting of the umbilical cord.
The obstetrician calculates the date of birth as 280 days or 40 weeks from the first day of the LNMP. In women with regular 28-day menstrual periods, the method is fairly accurate, but when cycles are irregular, substantial miscalculations may be made. An additional complication occurs when the woman has some bleeding about 14 days after fertilization as a result of erosive activity by the implanting blastocyst (see Chapter 4, Day 13, p. 44). Hence, the day of delivery is not always easy to determine. Most fetuses are born within 10 to 14 days of the calculated delivery date. If they are born much earlier, they are categorized as premature; if born later, they are considered postmature.
Occasionally, the age of an embryo or small fetus must be determined. By combining data on the onset of the last menstrual period with fetal length, weight, and other morphological characteristics typical for a given month of development, a reasonable estimate of the age of the fetus can be formulated. A valuable tool for assisting in this determination is ultrasound, which can provide an accurate (1 to 2 days) measurement of CRL during the 7th to 14th weeks. Measurements commonly used in the 16th to 30th weeks are biparietal diameter (BPD), head and abdominal circumference, and femur length. An accurate determination of fetal size and age is important for managing pregnancy, especially if the mother has a small pelvis or if the baby has a birth defect.

There is considerable variation in fetal length and weight, and sometimes these values do not correspond with the calculated age of the fetus in months or weeks. Most factors influencing length and weight are genetically determined, but environmental factors also play an important role.
The average size of a newborn is 2,500 to 4,000 g (6 to 9 Ib) with a length of 51 cm (20 in). The term low birth weight (LBW) refers to a weight <2,500 g, regardless of gestational age. Many infants weigh <2,500 g because they are preterm (born before 37 weeks of gestation). In contrast, the terms intrauterine growth restriction (IUGR) and small for gestational age (SGA) take into account gestational age.
IUGR is a term applied to infants who do not attain their optimal intrauterine growth. These infants are pathologically small and at risk for poor outcomes. Infants who are SGA have a birth weight that is below the 10th percentile for their gestational age. These babies may be pathologically small (they may have IUGR) or they may be constitutionally small (healthy, but smaller in size). The challenge is to differentiate the two conditions so that healthy, but small babies are not subjected to high-risk protocols used for babies with IUGR.
Approximately | in 10 babies have IUGR and therefore have an increased risk of neurological problems, congenital malformations, meconium aspiration, hypoglycemia, hypocalcemia, and respiratory distress syndrome (RDS). There are also long-term effects on these infants. For

The placenta is the organ that facilitates nutrient and gas exchange between the maternal and fetal compartments. As the fetus begins the ninth week of development, its demands for nutritional and other factors increase, causing major changes in the placenta. Foremost among these is an increase in surface area between maternal and fetal components to facilitate exchange. The disposition of fetal membranes is also altered as production of amniotic fluid increases.
example, babies with IUGR have been shown to have a greater chance as adults to develop a metabolic disorder later in life, such as obesity, hypertension, hypercholesterolemia, cardiovascular disease, and type 2 diabetes (called Barker's hypothesis).
The incidence of IUGR is higher in blacks than in whites. Causative factors include chromosomal abnormalities; teratogens; congenital infections (rubella, cytomegalovirus, toxoplasmosis, and syphilis); poor maternal health (hypertension and renal and cardiac disease); the mother's nutritional status and socioeconomic level; her use of cigarettes, alcohol, and other drugs; placental insufficiency; and multiple births (e.g., twins, triplets).
The major growth-promoting factor during development before and after birth is insulin-like growth factor-I (IGF-I), which has mitogenic and anabolic effects. Fetal tissues express IGF-I, and serum levels are correlated with fetal growth. Mutations in the IGF-I gene result in IUGR, and this growth retardation is continued after birth. In contrast to the prenatal period, postnatal growth depends on growth hormone (GH). This hormone binds to its receptor (GHR), activating a signal transduction pathway and resulting in synthesis and secretion of IGF-I. Mutations in the GHR result in Laron's dwarfism, which is characterized by marked short stature, and sometimes blue sclera. These individuals show little or no IUGR, because IGF-I production does not depend on GH during fetal development.

The fetal component of the placenta is derived from the trophoblast and extraembryonic mesoderm (the chorionic plate); the maternal component is derived from the uterine endometrium. By the beginning of the second month, the trophoblast is characterized by a great number of secondary and tertiary villi, which give it a radial appearance (Fig. 8.7). Stem (anchoring) villi extend from the mesoderm of the chorionic plate to the cytotrophoblast shell. The surface of the villi is formed by the syncytium, resting on a layer
Figure 8.7 Human embryo at the beginning of the second month of development.At the embryonic pole, villi are numerous and well formed; at the abembryonic pole, they are few in number and poorly developed.
of cytotrophoblastic cells that in turn cover a core of vascular mesoderm (Fig. 8.8A,C). The capillary system developing in the core of the villous stems soon comes in contact with capillaries of the chorionic plate and connecting stalk, thus giving rise to the extraembryonic vascular system.
quantities of maternal blood to intervillous spaces (Figs. 8.7 and 8.8).
Maternal blood is delivered to the placenta by spiral arteries in the uterus. Erosion of these maternal vessels to release blood into intervillous spaces (Figs. 8.7 and 8.8) is accomplished by endovascular invasion by cytotrophoblast cells. These cells, released from the ends of anchoring villi (Figs. 8.7 and 8.8), invade the terminal ends of spiral arteries, where they replace maternal endothelial cells in the vessels' walls, creating hybrid vessels containing both fetal and maternal cells. To accomplish this process, cytotrophoblast cells undergo an epithelial-to-endothelial transition. Invasion of the spiral arteries by cytotrophoblast cells transforms these vessels from small-diameter, high-resistance vessels to larger-diameter, lowresistance vessels that can provide increased

During the following months, numerous small extensions grow out from existing stem villi and extend as free villi into the surrounding lacunar or intervillous spaces. Initially, these newly formed free villi are primitive (Fig. 8.8C), but by the beginning of the fourth month, cytotrophoblastic cells and some connective tissue cells disappear. The syncytium and endothelial wall of the blood vessels are then the only layers that separate the maternal and fetal circulations (Fig. 8.8B,D). Frequently, the syncytium becomes very thin, and large pieces containing several nuclei may break off and drop into the intervillous blood lakes. These pieces, known as syncytial knots, enter the maternal circulation and usually degenerate without causing any symptoms. Disappearance of cytotrophoblastic cells progresses from the smaller to larger villi, and although some always persist in large villi, they do not participate in the exchange between the two circulations.
Figure 8.8 Structure of villi at various stages of development. A. During the fourth week. The extraembryonic mesoderm penetrates the stem villi in the direction of the decidual plate. B. During the fourth month. In many small villi, the wall of the capillaries is in direct contact with the syncytium. C,D. Enlargement of the villus as shown in Figures 8.8A,B.

Preeclampsia is a condition characterized by maternal hypertension and proteinuria due to reduced organ perfusion and occurs in approximately 5% of pregnancies. The condition may Progress to eclampsia, which is characterized by seizures. Preeclampsia begins suddenly anytime from approximately 20 weeks' gestation to term and may result in fetal growth retardation, fetal death, or death of the mother. In fact, preeclampsia is a leading cause of maternal mortality in the United States and is completely reversible by delivery of the baby. However, delivery too early puts the infant at risk for complications related to preterm birth. Despite many years of research, the cause of preeclampsia is unknown. The condition

In the early weeks of development, villi cover the entire surface of the chorion (Fig. 8.7). As pregnancy advances, villi on the embryonic appears to be a trophoblastic disorder related to failed or incomplete differentiation of cytotrophoblast cells, many of which do not undergo their normal epithelial-to-endothelial transformation. As a result, invasion of maternal blood vessels by these cells is rudimentary. How these cellular abnormalities lead to hypertension and other problems is not clear. Risk factors for preeclampsia include, preeclampsia in a previous pregnancy, nulliparity (first pregnancy), obesity, family history of preeclampsia, multiple gestation (twins or more), and medical conditions, such as hypertension and diabetes. Preeclampsia also commonly occurs in women with hydatidiform moles (see Chapter 4, p. 48) in which case it occurs early in pregnancy.
pole continue to grow and expand, giving rise to the chorion frondosum (bushy chorion). Villi on the abembryonic pole degenerate, and by the third month, this side of the chorion, now known as the chorion laeve, is smooth (Figs. 8.9 and 8.10A).
Figure 8.9 A 6-week embryo.The amniotic sac and chorionic cavity have been opened to expose the embryo, showing the bushy appearance of the trophoblast at the embryonic pole in contrast to small villi at the abembryonic pole. Note the connecting stalk and yolk sac with its extremely long vitelline duct.

The difference between the embryonic and abembryonic poles of the chorion is also reflected in the structure of the decidua, the functional layer of the endometrium, which is shed during parturition. The decidua over the chorion frondosum, the decidua basalis, consists of a compact layer of large cells, decidual cells, with abundant amounts of lipids and glycogen. This layer, the decidual plate, is tightly connected to the chorion. The decidual layer over the abembryonic pole is the decidua capsularis (Fig. 8.10A). With growth of the chorionic vesicle, this layer becomes stretched and degenerates. Subsequently, the chorion laeve comes into contact with the uterine wall (decidua parietalis) on the opposite side of the uterus, and the two fuse (Figs. 8.10 to 8.12), obliterating the uterine lumen. Hence, the only portion of the chorion participating in the exchange process is the chorion frondosum, which, together with the decidua basalis, makes up the placenta. Similarly, fusion of the amnion and chorion to form the amniochorionic membrane obliterates the chorionic cavity (Fig. 8.10A,B). It is this membrane that ruptures during labor (breaking of the water).

By the beginning of the fourth month, the placenta has two components: (1) a fetal portion, formed by the chorion frondosum and (2) a maternal portion, formed by the decidua basalis (Fig. 8.10B). On the fetal side, the placenta is bordered by the chorionic plate (Fig. 8.13); on its maternal side, it is bordered by the decidua basalis, of which the decidual plate is most intimately incorporated into the placenta. In the junctional zone, trophoblast and decidual cells intermingle. This zone, characterized by decidual and syncytial giant cells, is rich in amorphous extracellular material. By this time, most cytotrophoblast cells have
Fused decidua
parietalis,
Figure 8.10 Relation of fetal membranes to wall of the uterus. A. End of the second month. Note the yolk sac in the chorionic cavity between the amnion and chorion.At the abembryonic pole, villi have disappeared (chorion laeve). B. End of the third month. The amnion and chorion have fused, and the uterine cavity is obliterated by fusion of the chorion laeve and the decidua parietalis.

degenerated. Between the chorionic and decidual plates are the intervillous spaces, which are filled with maternal blood. They are derived from lacunae in the syncytiotrophoblast and are lined with syncytium of fetal origin. The villous trees grow into the intervillous blood lakes (Figs.8.8 and 8.13). During the fourth and fifth months, the decidua forms a number of decidual septa, which project into intervillous spaces but do not reach the chorionic plate (Fig. 8.13).These septa have a core of maternal tissue, but their surface is covered by a layer of syncytial cells, so that at all times, a syncytial layer separates maternal blood in intervillous lakes from fetal tissue of the villi. As a result of this septum formation, the placenta is divided into a number of compartments, or cotyledons (Fig. 8.14). Because the decidual septa do not reach the chorionic plate, contact between intervillous spaces in the various cotyledons is maintained.
Figure 8.11 A 19-week fetus in its natural position in the uterus, showing the umbilical cord and placenta. The lumen of the uterus is obliterated. In the wall of the uterus is a large growth, a myofibroma.
As a result of the continuous growth of the fetus and expansion of the uterus, the placenta also enlarges. Its increase in surface area
Figure 8.12 A 23-week fetus in the uterus. Portions of the wall of the uterus and the amnion have been removed to show the fetus. In the background are placental vessels converging toward the umbilical cord. The umbilical cord is tightly wound around the abdomen, possibly causing abnormal fetal position in the uterus (breech position).
Figure
8.13
The placenta
in the
second half
The cotyledons
separated by
the decidual
(maternal) septa. Most of the intervillous blood returns to the maternal circulation by way of the endometrial veins.A small portion enters neighboring cotyledons. The intervillous spaces are lined by syncytium.

roughly parallels that of the expanding uterus, and throughout pregnancy, it covers approximately 15% to 30% of the internal surface of the uterus. The increase in thickness of the placenta results from arborization of existing villi and is not caused by further penetration into maternal tissues.
vessels, converge toward the umbilical cord (Fig. 8.144). The chorion, in turn, is covered by the amnion. Attachment of the umbilical cord is usually eccentric and occasionally even marginal. Rarely, however, does it insert into the chorionic membranes outside the placenta (velamentous insertion).

Cotyledons receive their blood through 80 to 100 spiral arteries that pierce the decidual plate and enter the intervillous spaces at more or less regular intervals (Fig. 8.13). Pressure in these arteries forces the blood deep into the intervillous spaces and bathes the numerous small villi of the villous tree in oxygenated blood. As the pressure decreases, blood flows back from the chorionic plate toward the decidua, where it enters the endometrial veins (Fig. 8.13). Hence, blood from the intervillous lakes drains back into the maternal circulation through the endometrial veins.

At full term, the placenta is discoid with a diameter of 15 to 25 cm, is approximately 3 cm thick, and weighs about 500 to 600 g. At birth, it is torn from the uterine wall and, approximately 30 minutes after birth of the child, is expelled from the uterine cavity as the afterbirth. When the placenta is viewed from the maternal side, 15 to 20 slightly bulging areas, the cotyledons, covered by a thin layer of decidua basalis, are clearly recognizable (Fig. 8.14B). Grooves between the cotyledons are formed by decidual septa.
The fetal surface of the placenta is covered entirely by the chorionic plate. A number of large arteries and veins, the chorionic
Collectively, the intervillous spaces of a mature placenta contain approximately 150 mL
Figure 8.14 A full-term placenta. A. Fetal side. The chorionic plate and umbilical cord are covered by amnion. B. Maternal side showing the cotyledons. In one area, the decidua has been removed. The maternal side of the placenta is always carefully inspected at birth, and frequently one or more cotyledons with a whitish appearance are present because of excessive fibrinoid formation and infarction of a group of intervillous lakes.
of blood, which is replenished about three or four times per minute. This blood moves along the chorionic villi, which have a surface area of 4 to 14 m*. Placental exchange does not take place in all villi, however, only in those that have fetal vessels in intimate contact with the covering syncytial membrane. In these villi, the syncytium often has a brush border consisting of numerous microvilli, which greatly increases the surface area and consequently the exchange rate between maternal and fetal circulations

Because some fetal blood cells escape across the placental barrier, there is a potential for these cells to elicit an antibody response by the mother's immune system. The basis for this response is the fact that more than 400 red blood cell antigens have been identified, and although most do not cause problems during pregnancy, some can stimulate a maternal antibody response against fetal blood cells. This process is an example of isoimmunization, and if the maternal response is sufficient, the antibodies will attack and hemolyze fetal red blood cells, resulting in hemolytic disease of the fetus and newborn. Previously, the disease was called erythroblastosis fetalis because in some cases severe hemolysis stimulated an increase in production of fetal blood cells called erythroblasts. However, this severity of anemia occurs only in a few cases such that hemolytic disease of the fetus and newborn is a more appropriate terminology. In rare cases, the anemia becomes so severe that fetal hydrops (edema and effusions into the body cavities) occurs, leading to fetal death (Fig. 8.15). Most severe cases are caused by antigens from the CDE (Rhesus) blood group system. The D or Rh antigen is the most dangerous, because immunization can result from a single exposure and occurs earlier and with greater severity with each succeeding pregnancy. The maternal antibody response occurs in cases when the fetus is D(Rh) positive and the mother is D(Rh) negative and is elicited when fetal red blood cells enter the maternal system because of small areas of bleeding at the surface of placental villi or at birth. This condition can be prevented by screening women at their first prenatal visit for Rh blood type and for the presence of anti-D antibodies to determine if she has been sensitized previously. In Rh-negative women without anti-D antibodies, recommendations include treatment with Rh

(Fig. 8.8D).The placental membrane, which separates maternal and fetal blood, is initially composed of four layers: (1) the endothelial lining of fetal vessels, (2) the connective tissue in the villus core, (3) the cytotrophoblastic layer, and (4) the syncytium (Fig. 8.8C). From the fourth month on, the placental membrane thins because the endothelial lining of the vessels comes in intimate contact with the syncytial membrane, greatly increasing the rate of exchange (Fig. 8.8D). Sometimes called the
immunoglobulin at 28 weeks' gestation; following times when fetal-maternal mixing of blood may have occurred (e.g., after amniocentesis or pregnancy loss); and after delivery if the newborn is found to be Rh positive. Since the introduction of Rh immunoglobulin in 1968, hemolytic disease in the fetus and newborn in the United States has almost been eliminated.
Antigens from the ABO blood group can also elicit an antibody response, but the effects are much milder than those produced by the CDE group. About 20% of all infants have an ABO maternal incompatibility, but only 5% will be clinically affected. These can be effectively treated postnatally.
Figure 8.15 Fetal hydrops caused by the accumulation of fluid in fetal tissues.
placental barrier, the placental membrane is not a true barrier, as many substances pass through it freely. Because the maternal blood in the intervillous spaces is separated from the fetal blood by a chorionic derivative, the human placenta is considered to be of the hemochorial type. Normally, there is no mixing of maternal and fetal blood. However, small numbers of fetal blood cells occasionally escape across microscopic defects in the placental membrane.

Main functions of the placenta are (1) exchange of metabolic and gaseous products between maternal and fetal bloodstreams and (2) production of hormones.

Exchange of gases—such as oxygen, carbon dioxide, and carbon monoxide—is accomplished by simple diffusion. At term, the fetus extracts 20 to 30 mL of oxygen per minute from the maternal circulation, and even a short-term interruption of the oxygen supply is fatal to the fetus. Placental blood flow is critical to oxygen supply, as the amount of oxygen reaching the fetus primarily depends on delivery, not diffusion.

Exchange of nutrients and electrolytes, such as amino acids, free fatty acids, carbohydrates, and vitamins, is rapid and increases as pregnancy advances.

Immunological competence begins to develop late in the first trimester, by which time the fetus makes all of the components of complement. Immunoglobulins consist almost entirely of maternal immunoglobulin G (IgG), which begins to be transported from mother to fetus at approximately 14 weeks. In this manner, the fetus gains passive immunity against various infectious diseases. Newborns begin to produce their own IgG, but adult levels are not attained until the age of 3 years.

By the end of the fourth month, the placenta produces progesterone in sufficient amounts to maintain pregnancy if the corpus luteum is removed or fails to function properly. In all probability, all hormones are synthesized in the syncytial trophoblast. In addition to progesterone, the placenta produces increasing amounts of estrogenic hormones, predominantly estriol, until just before the end of pregnancy, when a maximum level is reached. These high levels of

Maternal steroidal hormones readily cross the placenta. Other hormones, such as thyroxine, do so only at a slow rate. Some synthetic progestins rapidly cross the placenta and may masculinize female fetuses. Even more dangerous was the use of the synthetic estrogen diethylstilbestrol (DES), which easily crosses the placenta. This compound produced clear-cell carcinoma of the vagina and abnormalities of the cervix and uterus in females and in the testes of males in individuals who were exposed to the compound during their intrauterine life (see Chapter 9).
Although the placental barrier is frequently considered to act as a protective mechanism against damaging factors, many viruses—such as rubella, cytomegalovirus, Coxsackie, variola, varicella, measles, and poliomyelitis virus—traverse the placenta without difficulty. Once in the fetus, some viruses cause infections, which may result in cell death and birth defects (see Chapter 9).
Unfortunately, most drugs and drug metabolites traverse the placenta without difficulty, and many cause serious damage to the embryo (see Chapter 9). In addition, maternal use of heroin and cocaine can cause habituation in the fetus.
estrogens stimulate uterine growth and development of the mammary glands.
During the first 2 months of pregnancy, the syncytiotrophoblast also produces human chorionic gonadotropin (hCG), which maintains the corpus luteum. This hormone is excreted by the mother in the urine, and in the early stages of gestation, its presence is used as an indicator of pregnancy. Another hormone produced by the placenta is somatomammotropin (formerly placental lactogen). It is a growth-hormone-like substance that gives the fetus priority on maternal blood glucose and makes the mother somewhat diabetogenic. It also promotes breast development for milk production.

The oval line of reflection between the amnion and embryonic ectoderm (amnio—ectodermal junction) is the primitive umbilical ring. At the fifth week of development, the following structures pass through the ring (Fig. 8.16A,C): (1) the connecting stalk, containing the
Figure 8.16 A.A 5-week embryo showing structures passing through the primitive umbilical ring. B. The primitive umbilical cord of a 10-week embryo. C. Transverse section through the structures at the level of the umbilical ring. D. Transverse section through the primitive umbilical cord showing intestinal loops protruding in the cord.
allantois and the umbilical vessels, consisting of two arteries and one vein; (2) the yolk stalk (vitelline duct), accompanied by the vitelline vessels; and (3) the canal connecting the intraembryonic and extraembryonic cavities (Fig. 8.16C). The yolk sac proper occupies a space in the chorionic cavity, that is, the space between the amnion and chorionic plate (Fig. 8.168).
During further development, the amniotic cavity enlarges rapidly at the expense of the chorionic cavity, and the amnion begins to envelop the connecting and yolk sac stalks, crowding them together and giving rise to the primitive umbilical cord (Fig. 8.16B). Distally, the cord contains the yolk sac stalk and umbilical vessels. More proximally, it contains some intestinal loops and the remnant of the allantois (Fig. 8.16B,D).The yolk sac, found in the chorionic cavity, is connected to the umbilical cord by its stalk. At the end of the third month, the amnion has expanded so that it comes in contact with the chorion, obliterating the chorionic cavity (Fig. 8.10B). The yolk sac then usually shrinks and is gradually obliterated.

The abdominal cavity is temporarily too small for the rapidly developing intestinal loops, and some of them are pushed into the extraembryonic space in the umbilical cord. These extruding intestinal loops form a physiological umbilical hernia (see Chapter 15). At approximately the end of the third month, the loops are withdrawn into the body of the embryo, and the cavity in the cord is obliterated. When the allantois and the vitelline duct and its vessels are also obliterated, all that remains in the cord are the umbilical vessels surrounded by Wharton's jelly. This tissue, which is rich in proteoglycans, functions as a protective layer for the blood vessels. The walls of the arteries are muscular and contain many elastic fibers, which contribute to a rapid constriction and contraction of the umbilical vessels after the cord is tied off.

At the end of pregnancy, a number of changes that occur in the placenta may indicate reduced exchange between the two circulations. These
changes include (1) an increase in fibrous tissue in the core of the villus, (2) thickening of basement membranes in fetal capillaries, (3) obliterative changes in small capillaries of the villi, and (4) deposition of fibrinoid on the surface of the villi in the junctional zone and in the chorionic plate. Excessive fibrinoid formation frequently causes infarction of an intervillous lake or sometimes of an entire cotyledon. The cotyledon then assumes a whitish appearance.

The amniotic cavity is filled with a clear, watery fluid that is produced in part by amniotic cells but is derived primarily from maternal blood. The amount of fluid increases from approximately

At birth, the umbilical cord is approximately | to 2 cm in diameter and 50 to 60 cm long. It is tortuous, causing false knots. Length of a cord reflects the amount of intrauterine movement of the fetus, and shortened cords have been observed in fetal movement disorders and with intrauterine constraint. An extremely long cord may encircle the neck of the fetus, usually without increased risk, whereas a short one may cause difficulties during delivery by pulling the placenta from its attachment in the uterus.
Normally, there are two arteries and one vein in the umbilical cord. In one in 200 newborns, however, only a single umbilical artery is present, and these babies have approximately a 20% chance of having cardiac and other vascular defects. The missing artery either fails to form (agenesis) or degenerates early in development.

Occasionally, tears in the amnion result in amniotic bands that may encircle part of the fetus, particularly the limbs and digits. Amputations, ring constrictions, and other abnormalities, including craniofacial deformations, may result (Fig. 8.17). Origin of the bands is unknown.
30 mL at 10 weeks of gestation to 450 mL at 20 weeks to 800 to 1,000 mL at 37 weeks. During the early months of pregnancy, the embryo is suspended by its umbilical cord in this fluid, which serves as a protective cushion. The fluid (1) absorbs jolts, (2) prevents adherence of the embryo to the amnion, and (3) allows for fetal movements. The volume of amniotic fluid is replaced every 3 hours. From the beginning of the fifth month, the fetus swallows its own amniotic fluid, and it is estimated that it drinks about 400 mL a day, about half of the total amount. Fetal urine is added daily to the amniotic fluid in the fifth month, but this urine is mostly water, because the placenta is functioning as an exchange for metabolic wastes. During childbirth, the amniochorionic membrane forms a hydrostatic wedge that helps to dilate the cervical canal.

Hydramnios or polyhydramnios is the term used to describe an excess of amniotic fluid (1,500 to 2,000 mL), whereas oligohydramnios refers to a decreased amount (<400 mL). Both conditions are associated with an increase in the incidence of birth defects. Primary causes of hydramnios include idiopathic causes (35%), maternal diabetes (25%), and congenital malformations, including central nervous system disorders (e.g., anencephaly) and gastrointestinal defects (atresias, e.g., esophageal) that prevent the infant from swallowing the fluid. Oligohydramnios is a rare occurrence that may result from renal agenesis. The lack of fluid in the amniotic cavity may constrict the fetus and cause club foot or there may be too little fluid for the fetus to "breathe" into its lungs resulting in lung hypoplasia.
Premature rupture of the membranes (PROM) refers to rupture of the membranes before uterine contractions begin and occurs in 10% of pregnancies. Preterm PROM occurs before 37 completed weeks of pregnancy, occurs in 3% of pregnancies, and is a common cause of preterm labor. Causes of preterm PROM are unknown, but risk factors include a previous pregnancy affected by prematurity or PROM, black race, smoking, infections, and severe polyhydramnios.
Figure 8.17 Limb abnormalities caused by amniotic bands. A. Limb constriction ring. B. Digit amputation (big toe) and ring constriction (second toe).

The frequency of multiple gestation (e.g., twins, triplets, etc.) has increased substantially in recent years and now accounts for over 3% of all live births in the United States. The rate of twin births has increased to a high of 32.6 per 1,000 births in 2008. The reasons for this increase are twofold: The increasing age of mothers at the time of their infants' birth and the increasing use of fertility treatments, including assisted reproductive technologies (ART).

Approximately 90% of twins are dizygotic, or fraternal, and their incidence increases with maternal age (doubling at age 35) and with fertility procedures, including ART. They result from simultaneous shedding of two oocytes and fertilization by different spermatozoa. Because the two zygotes have totally different genetic constitutions, the twins have no more resemblance than any other brothers or sisters. They may or may not be of different sex. The zygotes implant individually in the uterus, and usually each develops its own placenta, amnion, and chorionic sac (Fig. 8.18A). Sometimes, however, the two placentas are so close together that they fuse.
Similarly, the walls of the chorionic sacs may also come into close apposition and fuse (Fig. 8.18B). Occasionally, each dizygotic twin possesses red blood cells of two different types (erythrocyte mosaicism), indicating that fusion of the two placentas was so intimate that red cells were exchanged.

The second type of twins, which develops from a single fertilized ovum, is monozygotic, or identical, twins. The rate for monozygotic twins is 3 to 4 per 1,000. They result from splitting of the zygote at various stages of development. The earliest separation is believed to occur at the two-cell stage, in which case two separate zygotes develop. The blastocysts implant separately, and each embryo has its own placenta and chorionic sac (Fig. 8.19A). Although the arrangement of the membranes of these twins resembles that of dizygotic twins, the two can be recognized as partners of a monozygotic pair by their strong resemblance in blood groups, fingerprints, sex, and external appearance, such as eye and hair color.
Splitting of the zygote usually occurs at the early blastocyst stage. The inner cell mass splits
sion, large anastomoses shunt more blood to one of the partners than to the other.
Figure 8.19 Possible relations of fetal membranes in monozygotic twins. A. Splitting occurs at the two-cell stage, and each embryo has its own placenta, amniotic cavity, and chorionic cavity. B. Splitting of the inner cell mass into two completely separated groups. The two embryos have a common placenta and a common chorionic sac but separate amniotic cavities. C. Splitting of the inner cell mass at a late stage of development. The embryos have a common placenta, a common amniotic cavity, and a common chorionic cavity.

into two separate groups of cells within the same blastocyst cavity (Fig. 8.19B). The two embryos have a common placenta and a common chorionic cavity but separate amniotic cavities (Fig. 8.19B). In rare cases, the separation occurs at the bilaminar germ disc stage, just before the appearance of the primitive streak (Fig. 8.19C). This method of splitting results in formation of two partners with a single placenta and a common chorionic and amniotic sac. Although the twins have a common placenta, blood supply is usually well balanced. Although triplets are rare (about one per 7,600 pregnancies), birth of quadruplets, quintuplets, and so forth is rarer. In recent years, multiple births have occurred more frequently in mothers given gonadotropins (fertility drugs) for ovulatory failure.

Twin pregnancies have a high incidence of perinatal mortality and morbidity and an increased risk for preterm delivery. Approximately 60% of twins are born preterm and also have a high incidence of being low birth weight. Both of these factors put twin pregnancies at great risk and twin pregnancies have an infant mortality rate three times higher than that for singletons.
The incidence of twinning may be much higher than the number observed at birth because twins are conceived more often than they are born. Many twins die before birth, and some studies indicate that only 29% of women pregnant with twins actually give birth to two infants. The term vanishing twin refers to the death of one fetus. This disappearance, which occurs in the first trimester or the early second trimester, may result from resorption or formation of a fetus papyraceus (Fig. 8.20).
Another problem leading to increased mortality among twins is twin—-twin transfusion syndrome, which occurs in 15% of monochorionic monozygotic
Figure 8.20 Fetus papyraceus. One twin is larger, and the other has been compressed and mummified, hence the term papyraceus.
pregnancies. In this condition, placental vascular anastomoses, which occur in a balanced arrangement in most monochorionic placentas, are formed, so that one twin receives most of the blood flow, and flow to the other is compromised.As a result, one twin is larger than the other (Fig. 8.21). The outcome is poor, with the death of both twins occurring in 50% to 70% of cases.

At later stages of development, partial splitting of the primitive node and streak may result in formation of conjoined twins. These twins are classified according to the nature and degree of their union (Figs. 8.22 and 8.23). Occasionally, monozygotic twins are connected only by a common skin bridge or by a common liver bridge. The type of twins formed depends on when and to what extent abnormalities of the node and streak occurred. Misexpression of genes, such as Goosecoid, may also result in conjoined twins. Many conjoined twins have survived, including the most famous pair, Chang and Eng, who were joined at the abdomen and who traveled to England and the United States on exhibitions in the mid1800s. Finally settling in North Carolina, they farmed and fathered 21 children with their two wives.
Figure 8.21 Monozygotic twins with twin transfusion syndrome. Placental vascular anastomoses produced unbalanced blood flow to the two fetuses.
(continued from
previous page)
Craniopagus
Figure 8.23 Examples of conjoined twins. A. Dicephalus (two heads). B. Craniopagus (joined at the head and
Figure 8.22 Thoracopagus, pygopagus, and craniopagus twins (pagus; fastened). Conjoined twins can be separated only if they have no vital parts in common.
thorax).
In brother sister pairs of dizygotic twins, testosterone from the male twin can affect development of the female. Thus, females in such pairs tend to have square jaws, larger teeth, perform better on spacial-ability tests and have better ball skills than most girls. They are 15% less likely to get married and they have fertility problems, producing 25% fewer children.

For the first 34 to 38 weeks of gestation, the uterine myometrium does not respond to signals for parturition (birth). During the last 2 to 4 weeks of pregnancy, however, this tissue undergoes a transitional phase in preparation for the onset of labor. Ultimately, this phase ends with a thickening of the myometrium in the upper region of the uterus and a softening and thinning of the lower region and cervix.
Labor itself is divided into three stages: (1) effacement (thinning and shortening) and dilatation of the cervix (this stage ends when the cervix is fully dilated), (2) delivery of the fetus, and (3) delivery of the placenta and fetal membranes. Stage 1 is produced by uterine contractions that force the amniotic sac against the cervical canal like a wedge, or if the membranes have ruptured, then pressure will be exerted by the presenting part of the fetus, usually the head. Stage 2 is also assisted by uterine contractions, but the most important force is provided by increased intra-abdominal pressure from contraction of

Factors initiating labor are not known and may involve "retreat from maintenance of pregnancy," in which pregnancy-supporting factors (e.g., hormones) are withdrawn, or active induction caused by stimulatory factors targeting the uterus. Probably, components of both phenomena are involved. Unfortunately, a lack of knowledge about these factors has restricted progress in preventing preterm birth. Preterm birth (delivery before 37 completed weeks) of premature infants occurs in approximately 12% of births in the United States and is a leading cause of infant mortality and also contributes significantly to morbidity. It is caused by preterm PROM, premature onset of labor, or pregnancy complications requiring premature delivery. Risk factors include previous preterm birth, black race; multiple gestations; infections, such as periodontal disease and bacterial vaginosis; and low maternal body-mass index.
abdominal muscles. Stage 3 requires uterine contractions and is aided by increasing intraabdominal pressure.
As the uterus contracts, the upper part retracts, creating a smaller and smaller lumen, while the lower part expands, thereby producing direction to the force. Contractions usually begin about 10 minutes apart; then, during the second stage of labor, they may occur <1 minute apart and last from 30 to 90 seconds. Their occurrence in pulses is essential to fetal survival, as they are of sufficient force to compromise uteroplacental blood flow to the fetus.
ea
The fetal period extends from the ninth week of gestation until birth and is characterized by rapid growth of the body and maturation of organ systems. Growth in length is particularly striking during the third, fourth, and fifth months (approximately 5 cm_ per month), whereas increase in weight is most striking during the last 2 months of gestation (approximately 700 g per month) (Table 8.1, p. 94). Most babies weigh between 2,700 and 4,000 g (6 to 9 Ib) at birth. Those babies weighing <2,500 g (5 Ib 8 oz) are considered low birth weight; those below 1,500 g (3 Ib 5 oz) are considered very low birth weight. [UGR is a term applied to babies who do not achieve their genetically determined potential size and are pathologically small. This group is distinct from babies that are healthy, but are below the 10th percentile in weight for their gestational age and are classified as SGA.

A striking change is the relative slowdown in the growth of the head. In the third month, it is about half the size of the CRL. By the fifth month, the size of the head is about one third of the CHL, and at birth, it is one quarter of the CHL (Fig. 8.2).
During the fifth month, fetal movements are clearly recognized by the mother, and the fetus is covered with fine, small hair.
A fetus born during the sixth or the beginning of the seventh month has difficulty surviving, mainly because the respiratory and central nervous systems have not differentiated sufficiently.
In general, the length of pregnancy for a full-term fetus is considered to be 280 days, or 40 weeks after onset of the last
menstruation, or,more accurately, 266 days or 38 weeks after fertilization.
The placenta consists of two components: (1) a fetal portion, derived from the chorion frondosum or villous chorion, and (2) a maternal portion, derived from the decidua basalis. The space between the chorionic and decidual plates is filled with intervillous lakes of maternal blood. Villous trees (fetal tissue) grow into the maternal blood lakes and are bathed in them. The fetal circulation is at all times separated from the maternal circulation by (1) a syncytial membrane (a chorion derivative) and (2) endothelial cells from fetal capillaries. Hence, the human placenta is of the hemochorial type.
Intervillous lakes of the fully grown placenta contain approximately 150 mL of maternal blood, which is renewed three or four times per minute. The villous area varies from 4 to 14 m', facilitating exchange between mother and child.
Main functions of the placenta are (1) exchange of gases; (2) exchange of nutrients and electrolytes; (3) transmission of maternal antibodies, providing the fetus with passive immunity; (4) production of hormones, such as progesterone, estradiol, and estrogen (in addition, it produces hCG and somatomammotropin); and (5) detoxification of some drugs.

The amnion is a large sac containing amniotic fluid in which the fetus is suspended by its umbilical cord. The fluid (1) absorbs jolts, (2) allows for fetal movements, and (3) prevents adherence of the embryo to surrounding tissues. The fetus swallows amniotic fluid, which is absorbed through its gut and cleared by the placenta. The fetus adds urine to the amniotic fluid, but this is mostly water. An excessive amount of amniotic fluid (hydramnios) is associated with anencephaly and esophageal atresia, whereas an insufficient amount (oligohydramnios) is related to renal agenesis.
The umbilical cord, surrounded by the amnion, contains (1) two umbilical arteries, (2) one umbilical vein, and (3) Wharton's jelly, which serves as a protective cushion for the vessels.
Fetal membranes in twins vary according to their origin and time of formation. Two thirds of twins are dizygotic, or fraternal; they have two amnions, two chorions, and two placentas, which sometimes are fused. Monozygotic twins usually have two amnions, one chorion, and one placenta. In cases of conjoined twins, in which the fetuses are not entirely split from each other, there is one amnion, one chorion, and one placenta.
Signals initiating parturition (birth) are not clear, but preparation for labor usually begins between 34 and 38 weeks. Labor itself consists of three stages: (1) effacement and dilatation of the cervix, (2) delivery of the fetus, and (3) delivery of the placenta and fetal membranes.

1. An ultrasound at 7 months’ gestation shows too much space (fluid accumulation) in the amniotic cavity. What is this condition called, and what are its causes?
2.
Later
in
her
a
woman
realizes
that
she
was
probably
exposed
to
toluene
in
the
workplace
during
the
third
week
of
gestation
but
tells
a
fellow
worker
that
she
is
not
concerned
about
her
baby
because
the
placenta
protects
her
infant
from
toxic
factors
by
acting
as
a
barrier.
Is
she
correct?

Birth defect, congenital malformation, and congenital anomaly are synonymous terms used to describe structural, behavioral, functional, and metabolic disorders present at birth. Terms used to describe the study of these disorders are teratology (Gr. teratos; monster) and dysmorphology. Dysmorphologists are usually within a department of clinical genetics. Major structural anomalies occur in approximately 3% of liveborn infants and birth defects are a leading cause of infant mortality, accounting for approximately 25% of infant deaths. They are the fifth leading cause of years of potential life lost prior to age 65 and a major contributor to disabilities. They are also nondiscriminatory; the frequencies of birth defects are the same for Asians, African Americans, Latin Americans, Whites, and Native Americans.
In 40% to 45% of persons with birth defects, the cause is unknown. Genetic factors, such as chromosome abnormalities and mutant genes, account for approximately 28%; environmental factors produce approximately 3% to 4%; a combination of genetic and environmental influences (multifactorial inheritance) produces 20% to 25%; and twinning causes 0.5% to 1%.
Minor anomalies occur in approximately 15% of newborns. These structural abnormalities, such as microtia (small ears), pigmented spots, and short palpebral fissures, are not themselves detrimental to health but, in some cases, are associated with major defects. For example, infants with one minor anomaly have a 3% chance of having a major malformation; those with two minor anomalies have a 10% chance; and those with three or more minor anomalies have a 20% chance. Therefore, minor anomalies serve as clues for diagnosing more serious underlying defects. In particular, ear anomalies are easily recognizable indicators of other defects and are observed in virtually all children with syndromic malformations.
Malformations occur during formation of structures, for example, during organogenesis. They may result in complete or partial absence of a structure or in alterations of its normal configuration. Malformations are caused by environmental and/or genetic factors acting independently or in concert. Most malformations have their origin during the third to eighth weeks of gestation (Fig. 9.1).
Disruptions result in morphological alterations of already formed structures and are caused by destructive processes. Vascular accidents leading to transverse limb defects and defects produced by amniotic bands are examples of destructive factors that produce disruptions (Fig. 9.2).

Deformations result from mechanical forces that mold a part of the fetus over a prolonged period. Clubfeet, for example, are caused by compression in the amniotic cavity (Fig. 9.3). Deformations often involve the musculoskeletal system and may be reversible postnatally.
A syndrome is a group of anomalies occurring together that have a specific common cause. This term indicates that a diagnosis has been made and that the risk of recurrence is known. In contrast, association is the nonrandom appearance of two or more anomalies that occur together more frequently than by chance alone, but the cause has not been determined. An example is the VACTERL association (vertebral, anal, cardiac, tracheoesophageal, renal, and limb anomalies). Although they do not constitute a diagnosis, associations are important because recognition of one or more of the components promotes the search for others in the group.

Until the early 1940s, it was assumed that congenital defects were caused primarily by hereditary factors. With the discovery by N. Gregg that rubella (German measles) affecting a mother

Figure 9.1 Graph showing the times in gestation versus the risks of birth defects being induced. The most sensitive time is the embryonic period during the third to eighth weeks. The fetal period begins at the end of the eighth week and extends to term. During this time, the risk for gross structural defects being induced decreases, but organ systems may still be affected. For example, the brain continues to differentiate during the fetal period, such that toxic exposures may cause learning disabilities or intellectual disability. The fact that most birth defects occur prior to the eighth week makes it imperative to initiate birth defects prevention strategies prior to conception. Unfortunately, most women do not appear for their first prenatal visit until the eighth week, which is after the critical time for prevention of most birth defects.
during early pregnancy caused abnormalities in the embryo, it suddenly became evident that congenital malformations in humans could also be caused by environmental factors. In 1961, observations by W. Lenz linked limb defects to the sedative thalidomide and made it clear that drugs could also cross the placenta and produce birth defects (Fig. 9.4). Since that time, many agents have been identified as teratogens (factors that cause birth defects) (Table 9.1, p. 118).
Defects produced by amniotic bands as examples of disruptions. A. Cleft lip. B. Toe amputations. C. Finger amputations. Strands of ammion may be swallowed or become wrapped around structures causing various disruption-type
Figure 9.2 defects. The origin of the bands of amniotic tissue is unknown.
Figure 9.3 Abnormal positioning of the lower limbs and clubfeet as examples of deformations. These defects are probably caused by oligohydramnios (too little amniotic fluid).

Factors determining the capacity of an agent to produce birth defects have been defined and set forth as the principles of teratology. They include the following:
1 Susceptibility to teratogenesis depends on the genotype of the conceptus and the manner in which this genetic composition interacts with the environment. The maternal genome is also important with respect to drug metabolism, resistance to infection, and other biochemical and molecular processes that affect the conceptus.
2 Susceptibility to teratogens varies with the developmental stage at the time of exposure. The most sensitive period for inducing birth defects is the third to eighth weeks of gestation, the period of embryogenesis. Each organ system may have one or more stages of susceptibility. For example, cleft palate can be induced at the blastocyst stage (day 6), during gastrulation (day 14), at the early limb bud stage (fifth week), or when the palatal shelves are forming (seventh week). Furthermore, whereas most abnormalities are produced during embryogenesis, defects may also be induced before or after this period; no stage of development is completely safe (Fig. 9.1).
3 Manifestations of abnormal development depend on dose and duration of exposure to a teratogen.
4 Teratogens act in specific ways (mechanisms) on developing cells and tissues to initiate abnormal embryogenesis (pathogenesis). Mechanisms may involve inhibition of a specific biochemical or molecular process; pathogenesis may involve cell death, decreased cell proliferation, or other cellular phenomena.
5 Manifestations of abnormal development are death, malformation, growth retardation, and functional disorders.
Figure 9.4 A,B. Examples of phocomelia. Limb defects characterized by loss of the long bones of the limb. These defects

ears, mandibular hypoplasia, cleft palate, heart defects. Industrial solvents, 1 = Low birth weight, craniofacial and neural tube defects. Organic mercury, 1 = Neurological symptoms similar to those of cerebral palsy. Lead, 1 = Growth retardation, neurological disorders. Hormones, 1 = Hormones. Androgenic agents, 1 = Masculinization of female genitalia: fused labia, clitoral hypertrophy (ethisterone, norethisterone). DES, 1 = Malformation of the uterus, uterine tubes, and upper vagina; vaginal cancer; malformed testes. Maternal diabetes, 1 = Various malformations; heart and neural tube defects most common. Maternal obesity, 1 = Neural tube defects, heart defects, omphalocele. SSRIs, Selective serotonin reuptake inhibitors;ACE, angiotensin-converting enzyme; DES, diethylstilbestrol., 1 = SSRIs, Selective serotonin reuptake inhibitors;ACE, angiotensin-converting enzyme; DES, diethylstilbestrol.
Infectious Agents use of a vaccine have nearly eliminated congenital
Infectious agents that cause birth defects (Table
9.1) include a number of viruses. Birth defects due to rubella (German measles) during preg-
nancy (congenital rubella syndrome) used to be a major problem, but development and widespread
malformations from this cause.
Cytomegalovirus is a serious threat. Often, the mother has no symptoms, but the effects on
the fetus can be devastating. The infection can cause serious illness at birth and is sometimes
fatal. On the other hand, some infants are asymptomatic at birth, but develop abnormalities later, including hearing loss, visual impairment, and intellectual disability.

Herpes simplex virus and varicella virus can cause birth defects. Herpes-induced abnormalities are rare and usually infection is transmitted to the child during delivery, causing severe illness and sometimes death. Intrauterine infection with varicella causes scarring of the skin, limb hypoplasia, and defects of the eyes and central nervous system. The occurrence of birth defects after prenatal infection with varicella is infrequent and depends on the timing of the infection. Among infants born to women infected before 13 weeks' gestation, 0.4% are malformed; whereas the risk increases to 2% among infants whose mothers are infected during 13 to 20 weeks of

Malformations apparently do not occur following maternal infection with measles, mumps, hepatitis, poliomyelitis, echovirus, coxsackie virus, and influenza, but some of these infections may cause spontaneous abortion or fetal death or may be transmitted to the fetus. For example, coxsackie B virus may cause an increase in spontaneous abortion, while measles and mumps may cause an increase in early and late fetal death and neonatal measles and mumps. Hepatitis B has a high rate of transmission to the fetus, causing fetal and neonatal hepatitis; whereas hepatitis A, C, and E are rarely transmitted transplacentally. Echoviruses seem to have no adverse effects on the fetus. Also, there is no evidence that immunizations against any of these diseases harm the fetus.
A
complicating factor
introduced by
these and
other infectious
agents
(cause fevers),
is that
and most
are elevated
(hyperthermia)
caused external
sources, body
by fevers
such atogenic.
as hot
Characteristically, by
elevated pyrogenic
temperature or
possibly by
tubs and
saunas, is
ter- neurulation
temperatures and
such as
anencephaly and
is affected
neural tube
spina bifida,
defects, are
produced.
Toxoplasmosis can
cause birth
defects.
Poorly cooked meat; feces of domestic animals, especially cats; and soil contaminated with feces can carry the protozoan parasite Toxoplasmosis gondii. A characteristic feature of fetal toxoplasmosis infection is cerebral calcifications. Other features that may be present at birth include microcephaly (small head), macrocephaly (large head), or hydrocephalus (an increase in cerebrospinal fluid in the brain). In a manner similar to cytomegalovirus, infants who appear normal at birth may later develop visual impairment, hearing loss, seizures, and intellectual disability.

Ionizing radiation kills rapidly proliferating cells, so it is a potent teratogen, producing virtually any type of birth defect depending upon the dose and stage of development of the conceptus at the time of exposure. Radiation from nuclear explosions is also teratogenic. Among women survivors pregnant at the time of the atomic bomb explosions over Hiroshima and Nagasaki, 28% spontaneously aborted, 25% gave birth to children who died in their first year of life, and 25% gave birth to children who had severe birth defects involving the central nervous system. Similarly, the explosion of the nuclear reactor at Chernobyl, which released up to 400 times the amount of radiation as the nuclear bombs, has also resulted in an increase in birth defects throughout the region. Radiation is also a mutagenic agent and can lead to genetic alterations of germ cells and subsequent malformations.

The role of chemical agents and pharmaceutical drugs (medications) in the production of abnormalities in humans is difficult to assess for two reasons: (1) most studies are retrospective, relying on the mother's memory for a history of exposure, and (2) pregnant women take a large number of medications. A National Institutes of Health study discovered that pregnant women, on average, took four medications during pregnancy. Only 20% of pregnant women used no drugs during their pregnancy. Even with this widespread use of medications during pregnancy, insufficient information is available to judge the safety of approximately 90% of these drugs if taken during pregnancy.
On the other hand, relatively few of the many drugs used during pregnancy have been positively identified as being teratogenic. One example is thalidomide, an antinauseant and sleeping pill. In 1961, it was noted in West Germany that the frequency of amelia and meromelia (total or partial absence of the extremities), a rare abnormality that was usually inherited, had suddenly increased (Fig. 9.2). This observation led to examination of the prenatal histories of affected children and to the discovery that many mothers had taken thalidomide early in pregnancy. The causal relation between thalidomide and meromelia was discovered only because the drug produced such an unusual abnormality. If the defect had been a more common type, such as cleft lip or heart malformation, the association with the drug might easily have been overlooked.
The discovery that a drug like thalidomide could cross the placenta and cause birth defects was revolutionary and led directly to the science of teratology and founding of the Teratology Society. Today, thalidomide is still in use as an immunomodulatory agent in the treatment of people with AIDS and other immunopathological diseases such as leprosy, lupus erythematosis, and graft versus host disease. Limb defects still occur in babies exposed to the drug, but it is now clear that other malformations are produced as well. These abnormalities include heart malformations, orofacial clefts, intellectual disability, autism, and defects of the urogenital and gastrointestinal systems.

Other drugs with teratogenic potential include the anticonvulsants diphenylhydantoin (phenytoin), valproic acid, and trimethadione, which are used by women who have seizure disorders. Specifically, trimethadione and diphenylhydantoin produce a broad spectrum of abnormalities that constitute distinct patterns of dysmorphogenesis known as the trimethadione and fetal hydantoin syndromes. Facial clefts are particularly common in these syndromes. The anticonvulsant valproic acid increases the risk for several defects, including atrial septal defects, cleft palate, hypospadius, polydactyly, and craniosynostosis, but the highest risk is for the neural tube defect, spina bifida. The anticonvulsant Carbamazepine also has been associated with an increased risk for neural tube defects and possibly other types of malformations.
Antipsychotic and antianxiety agents (major and minor tranquilizers, respectively) are suspected producers of congenital malformations. The antipsychotics phenothiazine and lithium have been implicated as teratogens. Although evidence for the teratogenicity of phenothiazines is conflicting, an association between lithium and congenital heart defects, especially Ebstein anomaly, is better documented, although the risk is small. Antidepressant drugs that work as selective serotonin reuptake inhibitors (SSRIs) appear to cause birth defects, especially of the heart, and also may produce an increase in spontaneous abortions. These compounds, which include fluoxetine (Prozac), paroxetine (Paxil), and others, may act by inhibiting serotonin (5HT) signaling important for establishing laterality (left-right sidedness) and for heart develop- ment (see Chapter 13).
Mycophenolate mofetil (MMEF) is an immunosuppressant drug used to prevent rejection in organ transplant patients. Use of the drug in pregnancy has resulted in spontaneous abortions and birth defects, including cleft lip and palate, microtia (small ears), microcephaly, and heart defects.

The anticoagulant warfarin is teratogenic. Infants born to mothers with first trimester exposures typically have skeletal abnormalities, including nasal hypoplasia, abnormal epiphyses in their long bones, and limb hypoplasia. In contrast, the anticoagulant heparin does not appear to be teratogenic.
Antihypertensive agents that inhibit angiotensin-converting enzyme (ACE inhibitors) produce growth retardation, renal dysfunction, fetal death, and oligohydramnios if exposures occur during the second or third trimester. Effects of exposure to these compounds in the first trimester are less clear.
Caution has also been expressed regarding a number of other compounds that may damage the embryo or fetus. The most prominent among these are propylthiouracil and potassium iodide (goiter and intellectual disability), streptomycin (hearing loss), sulfonamides (kernicterus), the antidepressant imipramine (limb deformities), tetracyclines (bone and tooth anomalies), amphetamines (oral clefts and cardiovascular abnormalities), and quinine (hearing loss).

One of the increasing problems in today's society is the effect of social drugs, such as LSD (lysergic acid diethylamide), PCP (phencyclidine, or "angel dust''), marijuana, alcohol, and cocaine. In the case of LSD, limb abnormalities and malformations of the central nervous system have been reported. A comprehensive review of more than 100 publications, however, led to the conclusion that pure LSD used in moderate doses is not teratogenic and does not cause genetic damage. A similar lack of conclusive evidence for teratogenicity has been described for marijuana, PCP, and cocaine. There is a well-documented association between maternal alcohol ingestion and congenital abnormalities. Because alcohol may induce a broad spectrum of defects, ranging from intellectual disability to structural abnormalities of the brain (microcephaly, holoprosencephaly), face, and heart, the term fetal alcohol spectrum disorder (FASD) is used to refer to any alcohol-related defects. Fetal alcohol syndrome (FAS) represents the severe end of the spectrum and includes structural defects, growth deficiency, and intellectual disability (Fig. 9.5). Alcohol-related neurodevelopmental disorder (ARND) refers to cases with evidence of involvement of the central nervous system that do not meet the diagnostic criteria for FAS. The incidence of FAS and ARND together has been estimated to be 1 in 100 live births. Furthermore, alcohol is the leading cause of intellectual disability. It is not clear how much alcohol is necessary to cause a developmental problem. The
Figure 9.5 Characteristic features of a child with Fetal alcohol syndrome (FAS), including an indistinct philtrum, thin upper lip, depressed nasal bridge, short nose, and flat midface.
dose and timing during gestation are both critical, but there is probably no "safe" level. Even binge drinking (>5 drinks per sitting) at a critical stage of development appears to increase the risk for birth defects, including orofacial clefts.
Cigarette smoking has been linked to an increased risk for orofacial clefts (cleft lip and cleft palate). It also contributes to intrauterine growth retardation and premature delivery.

Isotretinoin (Accutane), an analogue of vitamin A, has been shown to cause a characteristic pattern of malformations known as the isotretinoin embryopathy. The drug is prescribed for the treatment of cystic acne and other chronic dermatoses, but it is highly teratogenic and can produce virtually any type of malformation. Even topical retinoids, such as etretinate, may have the potential to cause abnormalities. Vitamin A itself may be teratogenic at high doses, based on animal studies and the fact that isotretinoin is a closely related compound. Precisely how much is potentially harmful (>10,000 or >25,000 IU) is controversial, but the amount of vitamin A typically contained in multivitamins (2,000 to 8,000 IU) is below these doses, unless an individual takes more than one multivitamin a day.

Androgenic Agents In the past, synthetic progestins were frequently used during pregnancy to prevent abortion. The progestins ethisterone and norethisterone have considerable androgenic activity, and many cases of masculinization of the genitalia in female embryos have been reported. The abnormalities consist of an enlarged clitoris associated with varying degrees of fusion of the labioscrotal folds.
Endocrine disrupters are exogenous agents that interfere with the normal regulatory actions of hormones controlling developmental processes. Most commonly, these agents interfere with the action of estrogen through its receptor to cause developmental abnormalities of the central nervous system and reproductive tract. For some time, it has been known that the synthetic estrogen diethylstilbestrol (DES), which was used to prevent abortion, raised the incidence of carcinomas of the vagina and cervix in women exposed to the drug in utero. Furthermore, a high percentage of these women had reproductive dysfunction caused in part by congenital malformations of the uterus, uterine tubes, and upper vagina. Male embryos exposed in utero can also be affected, as evidenced by an increase in malformations of the testes and abnormal sperm analysis among these individuals. In contrast to women, however, men do not demonstrate an increased risk of developing carcinomas of the genital system.
Today, environmental estrogens are a concern, and numerous studies to determine their effects on the unborn are under way. Decreasing sperm counts and increasing incidences of testicular cancer, hypospadias, and other abnormalities of the reproductive tract in humans, together with documented central nervous system abnormalities (masculinization of female brains and feminization of male brains) in other species with high environmental exposures, have raised awareness of the possible harmful effects of these agents. Many are formed from chemicals used for industrial purposes and from pesticides.
Oral Contraceptives Birth control pills, containing estrogens and progestogens, appear to have a low teratogenic potential. Because other hormones such as DES produce abnormalities, however, use of oral contraceptives should be discontinued if pregnancy is suspected.
Cortisone Experimental work has repeatedly shown that cortisone injected into mice and rabbits at certain stages of pregnancy causes a high percentage of cleft palates in the offspring. Some recent epidemiologic studies also suggest that
women who take corticosteroids during pregnancy are at a modestly increased risk for having a child with an orofacial cleft.

Diabetes Disturbances in carbohydrate metabolism during pregnancy in diabetic mothers cause a high incidence of stillbirths, neonatal deaths, abnormally large infants, and congenital malformations. The risk of congenital anomalies in children born to mothers with pregestational diabetes (diabetes diagnosed before pregnancy; both type 1 [insulin dependent] and type 2 [non-insulin dependent}) is three to four times that for offspring of nondiabetic mothers and has been reported to be as high as 80% in the offspring of diabetics with long-standing disease. The increased risk is for a wide variety of malformations, including neural tube defects and congenital heart defects. There is also a higher risk for caudal dysgenesis (sirenomelia: see Figure 5.8, p. 58).
Factors responsible for these abnormalities have not been delineated, although evidence suggests that altered glucose levels play a role and that insulin is not teratogenic. In this respect, a significant correlation exists between the severity and duration of the mother's disease and the incidence of malformations. Also, strict control of maternal glucose levels beginning before conception and continuing throughout gestation reduces the occurrence of malformations to incidences approaching those in the general population.
The risk for birth defects associated with gestational diabetes (diabetes that is first diagnosed during pregnancy) is less clear, with some, but not all studies showing a slightly increased risk. Given that the onset of gestational diabetes is believed to be after the critical period for inducing structural birth defects (3 to 8 weeks gestation), some investigators have suggested that any observed increased risk may be due to the fact that some women diagnosed with gestational diabetes probably had diabetes before pregnancy, but it was not diagnosed.
Phenylketonuria Mothers with phenylketonuria (PKU), in which the enzyme phenylalanine hydroxylase is deficient or reduced, resulting in increased serum concentrations of phenylalanine, are at risk for having infants with intellectual disability, microcephaly, and cardiac defects. Women with PKU who maintain their low-phenylalanine diet prior to conception and throughout pregnancy reduce the risk to their infants to that observed in the general population.

Although many nutritional deficiencies, particularly vitamin deficiencies, have been proven to be teratogenic in laboratory animals, the evidence for specific cause and effects in humans is more difficult to document. One example is endemic cretinism, caused by iodine deficiency and characterized by stunted mental and physical growth. Recent evidence also indicates that methyl-deficient diets alter expression of imprinted genes and may result in birth defects and diseases, such as cancer postnatally. Finally, recent studies show that poor maternal nutrition prior to and during pregnancy contributes to low birth weight and birth defects and that severe starvation during pregnancy is associated with a two to threefold increase in schizophrenia in the offspring.

Obesity has reached epidemic proportions in the United States and has nearly doubled in the past 15 years. In 2007 to 2008, over one-third of women of reproductive age were obese (body mass index >30).
Prepregnancy obesity, is associated with a twofold increased risk for having a child with a neural tube defect. Causation has not been determined but may relate to maternal metabolic disturbances affecting glucose, insulin, or other factors. Prepregnancy obesity also increases the risk for having a baby with a heart defect, omphalocele, and multiple congenital anomalies.

Hypoxia induces congenital malformations in a great variety of experimental animals. Whether the same is valid for humans remains to be seen. Although children born at relatively high altitudes are usually lighter in weight and smaller than those born near or at sea level, no increase in the incidence of congenital malformations has been noted. In addition, women with cyanotic cardiovascular disease often give birth to small infants but usually without gross congenital malformations.

Several years ago, researchers in Japan noted that a number of mothers with diets consisting mainly of fish had given birth to children with multiple neurological symptoms resembling cerebral palsy. Further examination revealed that the fish contained an abnormally high level of organic mercury, which was spewed into Minamata Bay and other coastal waters of Japan by large industries. Many of the mothers did
not show any symptoms themselves, indicating that the fetus was more sensitive to mercury than the mother. In the United States, similar observations were made when seed corn sprayed with a mercury-containing fungicide was fed to hogs and the meat was subsequently eaten by pregnant women. Likewise, in Iraq, several thousand babies were affected after mothers ate grain treated with mercurycontaining fungicides.
Lead has been associated with increased abortions, growth retardation, and neurological disorders.

A number of studies have indicated that exposures to chemicals and other agents, such as ethylnitrosourea and radiation, can cause mutations in male germ cells. Epidemiological investigations have linked paternal occupational and environmental exposures to mercury, lead, solvents, alcohol, cigarette smoking, and other compounds to spontaneous abortion, low birth weight, and birth defects. Advanced paternal age is a factor for an increased risk for some types of structural birth defects, Down syndrome, and new autosomal dominant mutations. Interestingly,

Many birth defects can be prevented. For example, supplementation of salt with iodine eliminates intellectual disability and bone deformities resulting from cretinism. Placing women with diabetes or PKU under strict metabolic control prior to conception reduces the incidence of birth defects in their offspring. Folate supplementation lowers the incidence of neural tube defects, such as spina bifida and anencephaly, and also reduces the risk for hyperthermia-induced abnormalities. Avoidance of alcohol during all stages of pregnancy reduces the incidence of birth defects. An essential component of all prevention strategies is to initiate interventions prior to conception.
It is important for physicians prescribing medications to women of childbearing age to consider the possibility of pregnancy and the potential teratogenicity of the compounds. Hundreds of children have been born with severe birth defects produced by retinoids (isotretinoin embryopathy), compounds used for the treatment of cystic acne (isotretinoin, Accutane). Because patients with acne are usually young and may be sexually active, these agents must be used cautiously some studies have shown that men younger than 20 also have an increased risk of fathering a child with a birth defect, but results from other studies have been inconclusive. Even transmission of paternally mediated toxicity is possible through seminal fluid and from household contamination from chemicals brought home on work clothes by the father.

The perinatologist has several approaches for assessing growth and development of the fetus in utero, including ultrasound, maternal serum screening, amniocentesis, and chorionic villus sampling (CVS). In combination, these techniques are designed to detect malformations, genetic abnormalities, overall fetal growth, and complications of pregnancy, such as placental or uterine abnormalities. The use and development of in utero therapies have heralded a new concept in which the fetus is now a patient.

Ultrasonography is a relatively noninvasive technique that uses high-frequency sound waves reflected from tissues to create images. The approach may be transabdominal or transvaginal, with the latter producing images with higher resolution (Fig. 9.6). In fact, the technique, which was first developed in the 1950s, has advanced to a degree whereby detection of blood flow in major vessels, movement of heart valves, and flow of fluid in the trachea and bronchi are possible. The technique is safe and commonly used, with approximately 80% of pregnant women in the United States receiving at least one scan.
Important parameters revealed by ultrasound include characteristics of fetal age and growth; presence or absence of congenital anomalies; status of the uterine environment, including the amount of amniotic fluid (Fig. 9.7.4); placental position and umbilical blood flow; and whether multiple gestations are present (Fig. 9.7B). All of these factors are then used to determine proper approaches for management of the pregnancy.
Determination of fetal age and growth is crucial in planning pregnancy management, especially for low-birth-weight infants. In fact, studies show that ultrasound-screened and -managed pregnancies with low-birth-weight _ babies reduced the mortality rate by 60% compared with an unscreened group. Fetal age and growth are assessed by crown-rump length during the 5th to the 10th weeks of gestation. After that, a combination of measurements—including the biparietal diameter (BPD) of the skull, femur
Figure 9.6 Examples of the effectiveness of ultrasound in imaging the embryo and fetus. A. A 6-week embryo. B. Lateral view of the fetal face. C. Hand. D. Feet.
length, and abdominal circumference—are used (Fig. 9.8). Multiple measurements of these parameters over time improve the ability to determine the extent of fetal growth.
Congenital malformations that can be determined by ultrasound include the neural tube defects anencephaly and spina bifida (see Chapter 18); abdominal wall defects, such as omphalocele and gastroschisis (see Chapter 15); and heart (see Chapter 13) and facial defects, including cleft lip and palate (see Chapter 17).

Ultrasound can also be used to screen for Down syndrome and some other chromosome-related abnormalities through a test called nuchal translucency. This test involves measurement of the translucent space at the posterior of the baby's neck, where fluid accumulates when Down syndrome and some other abnormalities are present. The test is performed at 11 to 14 weeks of pregnancy. Information from this test, combined with maternal serum screening test results and the mother's age, can be combined to provide a risk estimate. Then, based on this risk assessment, a woman can decide whether she wants invasive testing, such as amniocentesis, which would provide a definitive diagnosis.
Figure 9.7 A. Ultrasound image showing position of the fetal skull and placement of the needle into the amniotic cavity
Figure 9.8 Ultrasounds showing measures used to assess embryonic and fetal growth. A. Crown-rump (CR) length in a 7-week embryo. B. Biparietal (B-P) diameter of the skull. C. Abdominal circumference. D. Femur length (F-L).

A search for biochemical markers of fetal status led to development of maternal serum screening tests. One of the first of these tests assessed serum a-fetoprotein (AFP) concentrations. AFP is produced normally by the fetal liver, peaks at approximately 14 weeks, and "leaks" into the maternal circulation via the placenta. Thus, AFP concentrations increase in maternal serum during the second trimester and then begin a steady decline after 30 weeks of gestation. In cases of neural tube defects and several other abnormalities, including omphalocele, gastroschisis, bladder exstrophy, amniotic band syndrome, sacrococcygeal teratoma, and intestinal atresia, AFP levels increase in amniotic fluid and maternal serum. In other instances, AFP concentrations decrease, as, for example, in Down syndrome, trisomy 18, sex chromosome abnormalities, and triploidy. AFP screening, combined with testing other second trimester markers (e.g., human chorionic gonadotropin (HCG), unconjugated estriol, and inhibin A) can increase the detection rate for birth defects using these serum screening studies.

During amniocentesis, a needle is inserted transabdominally into the amniotic cavity (identified by
ultrasound;
Fig.
9.7.4), and
20
to
30 mL of fluid is withdrawn. Because of the amount of fluid required, the procedure is not usually performed before 14 weeks' gestation, when sufficient quantities are available without endangering the fetus. Recent studies suggest that the risk of fetal loss related to the procedure is as low as 1 in 300 to 500, but may be even less for individuals and centers highly skilled in the technique. The fluid itself'is analyzed for biochemical factors, such as AFP and acetylcholinesterase. In addition, fetal cells, sloughed into the amniotic fluid, can be recovered and used for metaphase karyotyping and other genetic analyses (see Chapter 2). Unfortunately, the harvested cells are not rapidly dividing, and therefore, cell cultures containing mitogens must be established to provide sufficient metaphase cells for analysis. Thus, results are available 1 to 2 weeks after the procedure. Once chromosomes are obtained, major chromosomal alterations, such as translocations, breaks, trisomies, and monosomies, can be identified. With special stains (Giemsa) and high-resolution techniques, chromosome-banding patterns can be determined. Furthermore, with recent advances in molecular biology, more sophisticated molecular analyses using polymerase chain reaction (PCR) and genotyping assays have increased the level of detection for genetic abnormalities.

In cases of fetal anemia produced by maternal antibodies or other causes, blood transfusions for the fetus can be performed. Ultrasound is used to guide insertion of a needle into the umbilical cord vein, and blood is transfused directly into the fetus.

Treatment for infections, fetal cardiac arrhythmias, compromised thyroid function, and other medical problems is usually provided to the mother and reaches the fetal compartment after crossing the placenta. In some cases, however, agents may be administered to the fetus directly by intramuscular injection into the gluteal region or via the umbilical vein.

Because of advances in ultrasound and surgical procedures, operating on fetuses has become possible. Because of risks to the mother, infant, and subsequent pregnancies, however, procedures are only performed in centers with well-trained teams and only when there are no reasonable alternatives. Several types of surgeries may be performed, including placing shunts to remove fluid from organs and cavities. For example, in obstructive urinary disease of the urethra, a shunt may be inserted into the fetal bladder. One problem is diagnosing the condition early enough to prevent renal damage. Ex utero surgery, in which the uterus is opened and the fetus is operated on directly, has been used for repairing congenital diaphragmatic hernias, removing cystic (adenomatoid) lesions in the lung, and repairing spina bifida defects. Also, in recent years, fetal intervention has become available for certain congenital heart defects. At this time, however, most fetal surgical interventions are considered experimental and are undergoing randomized clinical trials to determine their effectiveness.

Because the fetus does not develop any immunocompetence before 18 weeks' gestation, it may be possible to transplant tissues or cells before this time without rejection. Research in this field is focusing on hematopoietic stem cells for treatment of immunodeficiency and hematologic disorders. Gene therapy for inherited metabolic diseases, such as Tay-Sachs and cystic fibrosis, is also being investigated.
__~
Various agents (Table 9.1, p. 118) and genetic factors are known to cause congenital malformations and approximately 3% of all live-born infants will have a birth defect. Agents that cause birth defects include viruses, such as rubella and cytomegalovirus; radiation; drugs, such as thalidomide, aminopterin, anticonvulsants, antipsychotics, and antianxiety compounds; social drugs, such as cigarettes, and alcohol; hormones, such as DES; and maternal diabetes. Effects of teratogens depend on the maternal and fetal genotype, the stage of development when exposure occurs, and the dose and duration of exposure of the agent. Most major malformations are produced during the period of embryogenesis (teratogenic period; third to eighth weeks),
but in stages before and after this time, the fetus is also susceptible, so that no period of gestation is completely free of risk. Prevention of many birth defects is possible, but it depends on beginning preventative measures before conception and increasing physicians' and women's awareness of the risks.

Many techniques are available to assess the growth and developmental status of the fetus. Ultrasound can accurately determine fetal age, growth parameters, and can detect many malformations. Maternal serum screening for AFP and other markers can indicate the presence of a neural tube defect or other abnormalities. Combinations of maternal serum screening and ultrasound to detect nuchal translucency can be used for detecting Down syndrome and some other chromosome related abnormalities. Amniocentesis is a procedure in which a needle is placed into the amniotic cavity and a fluid sample is withdrawn. This fluid can be analyzed biochemically and also provides cells for culture and genetic analysis. Chorionic villus sampling (CVS) involves aspirating a tissue sample directly from the placenta to obtain cells for genetic analysis. Previously, invasive procedures, such as amniocentesis and CVS, were offered only to women at higher risk, such as women of advanced maternal age (35 years and older), a history of neural tube defects in the family, previous gestation with a chromosome abnormality, chromosome abnormalities in either parent, and a mother who is a carrier for an X-linked disorder. In recent years, risks associated with these procedures have decreased and, consequently, these procedures have been made more widely available.
Modern medicine has also made the fetus a patient who can receive treatment, such as transfusions, medications for disease, fetal surgery, and gene therapy.

1. Amniocentesis reveals an elevated AFP level. What should be included in a differential diagnosis, and how would a definitive one be made?
2. A 40-year-old woman is approximately 8 weeks pregnant. What tests are available to determine whether her unborn child has Down syndrome? What are the risks and advantages of each technique?
3. Why is it important to determine the sta- tus of an infant prenatally? What maternal or family factors might raise your concern about the well-being of an unborn infant?
4.
What
factors
influence
the
action
of
a
teratogen?
5. A young woman in only the third week of her pregnancy develops a fever of 104°F but refuses to take any medication because she is afraid that drugs will harm her baby. Is she correct?
6. A young woman who is planning a fam- ily seeks advice about folic acid and other vitamins. Should she take such a supplement, and if so, when and how much?
7. A young insulin-dependent diabetic woman who is planning a family is concerned about the possible harmful effects of her disease on her unborn child. Are her concerns valid, and what would you recommend?
e axial skeleton includes the skull, vertebral column, ribs, and sternum. In general, the skeletal system develops from paraxial and lateral plate (parietal layer) mesoderm and from neural crest. Paraxial mesoderm forms a segmented series of tissue blocks on each side of the neural tube, known as somitomeres in the head region and somites from the occipital region caudally. Somites differentiate into a ventromedial part, the sclerotome, and a dorsolateral part, the dermomyotome. At the end of the fourth week, sclerotome cells become polymorphous and form loosely organized tissue, called mesenchyme, or embryonic connective tissue (Fig. 10.1). It is characteristic for mesenchymal cells to migrate and to differentiate in many ways. They may become fibroblasts, chondroblasts, or osteoblasts (boneforming cells).

(see Chapter 12). Neural crest cells in the head region also differentiate into mesenchyme and participate in formation of bones of the face and skull. The remainder of the skull is derived from occipital somites and somitomeres. In some bones, such as the flat bones of the skull, mesenchyme in the dermis differentiates directly into bone, a process known as intramembranous ossification (Fig. 10.2). In most bones, however, including the base of the skull and the limbs, mesenchymal cells first give rise to hyaline cartilage models, which in turn become ossified by endochondral ossification (Fig. 10.3). The following paragraphs discuss development of the most important bony structures and some of their abnormalities.
The bone-forming capacity of mesenchyme is not restricted to cells of the sclerotome, but occurs also in the parietal layer of the lateral plate mesoderm of the body wall. This layer of mesoderm forms bones of the pelvic and shoulder girdles, limbs, and sternum

The skull can be divided into two parts: the neurocranium, which forms a protective case around the brain, and the viscerocranium, which forms the skeleton of the face.
Figure 10.1 Development of the somite. A. Paraxial mesoderm cells are arranged around a small cavity. B. As a result of further differentiation, cells in the ventromedial wall lose their epithelial arrangement and become mesenchymal. Collectively, they are called the sclerotome. Cells in the ventrolateral and dorsomedial regions form muscle cells and also migrate beneath the remaining dorsal epithelium (the dermato me) to form the myotome.
Figure 10.2 Skull bones of a 3-month-old fetus show the spread of bone spicules from primary ossification centers in the flat bones of the skull.

The neurocranium is most conveniently divided into two portions: (1) the membranous part, consisting of flat bones, which surround the brain as a vault, and (2) the cartilaginous part, or chondrocranium, which forms bones of the base of the skull.

The membranous portion of the skull is derived from neural crest cells and paraxial mesoderm as indicated in Figure 10.4. Mesenchyme from these two sources invests the brain and undergoes intramembranous ossification. The result is formation of a number of flat, membranous bones that are characterized by the presence of needle-like bone spicules. These spicules progressively radiate from primary ossification centers toward the periphery (Fig. 10.2). With further growth during fetal and postnatal life, membranous bones enlarge by apposition of new layers on the outer surface and by simultaneous osteoclastic resorption from the inside.

At birth, the flat bones of the skull are separated from each other by narrow seams of connective tissue, the sutures, which are also derived from two sources: neural crest cells (sagittal suture) and paraxial mesoderm (coronal suture). At points where more than two bones meet, sutures are wide and are called fontanelles (Fig. 10.5). The most prominent of these is the anterior fontanelle, which is found where the two parietal and two frontal bones meet. Sutures and fontanelles allow the bones of the skull to overlap (molding) during birth. Soon after birth, membranous bones move back to their original positions, and the skull appears large and round. In fact, the size of the vault is large compared with the small facial

Several sutures and fontanelles remain membranous for a considerable time after birth. The bones of the vault continue to grow after birth, mainly because the brain grows. Although a 5to 7-year-old child has nearly all of his or her cranial capacity, some sutures remain open until adulthood. In the first few years after birth, palpation of the anterior fontanelle may give valuable information as to whether ossification of the skull is proceeding normally and whether
Figure 10.5 Skull of a newborn, seen from above A and the right side B. Note the anterior and posterior fontanelles and posterior fontanelle closes about 3 months after birth; the anterior fontanelle closes around the middle of the sutures. The second year. Many disappear during
Figure 10.6 Dorsal view of the chondrocranium, or base of the skull, in the adult showing bones formed by endochondral ossification. Bones that form rostral to the rostral half of the sella turcica arise from neural crest and constitute the prechordal (in front of the notochord) chondrocranium (blue). Those forming posterior to this landmark arise from paraxial mesoderm (chordal chondrocranium) (red).
intracranial pressure is normal. In most cases, the anterior fontanelle closes by 18 months of age, and the posterior fontanelle closes by 1 to 2 months of age.
Cartilaginous Neurocranium or Chondrocranium The cartilaginous neurocranium or chondrocranium of the skull initially consists of a number of separate cartilages. Those that lie in front of the rostral limit of the notochord, which ends at the level of the pituitary gland in the center of the sella turcica, are derived from neural crest cells. They form the prechordal chondrocranium. Those that lie posterior to this limit arise from occipital sclerotomes formed by paraxial mesoderm and form the chordal chondrocranium. The base of the skull is formed when these cartilages fuse and ossify by endochondral ossification (Figs. 10.3 and 10.6).

The viscerocranium, which consists of the bones of the face, is formed mainly from the first two pharyngeal arches (see Chapter 17). The first arch gives rise to a dorsal portion, the maxillary process, which extends forward beneath the region of the eye and gives rise to the maxilla, the zygomatic bone, and part of the temporal bone (Fig. 10.7). The ventral portion, the mandibular process, contains the Meckel cartilage. Mesenchyme around the Meckel cartilage condenses and ossifies by intramembranous ossification to give rise to the mandible. The Meckel cartilage disappears except in the sphenomandibular ligament. The dorsal tip of the mandibular process, along with that of the second pharyngeal arch, later gives rise to the
incus, the malleus, and the stapes (Fig. 10.7). Ossification of the three ossicles begins in the fourth month, making these the first bones to become fully ossified. Mesenchyme for formation of the bones of the face is derived from neural crest cells, including the nasal and lacrimal bones (Fig. 10.4).

Neural crest cells originating in the neuroectoderm form the facial skeleton and part of the skull. These cells also constitute a vulnerable population as they leave the neuroectoderm; they are often a target for teratogens. Therefore, it is not surprising that craniofacial abnormalities are common birth defects (see Chapter 17).

In some cases, the cranial vault fails to form (cranioschisis), and brain tissue exposed to amniotic fluid degenerates, resulting in anencephaly. Cranioschisis is caused by failure of the cranial neuropore to close (Fig. 10.8A). Children with such severe skull and brain defects cannot survive. Children with relatively small defects in the skull through which meninges and/or brain tissue
At first, the face is small in comparison with the neurocranium. This appearance is caused by (1) virtual absence of the paranasal air sinuses and (2) the small size of the bones, particularly the jaws. With the appearance of teeth and development of the air sinuses, the face loses its babyish characteristics.
herniate (cranial meningocele and meningoencephalocele, respectively) (Fig. 10.8B) may be treated successfully. In such cases, the extent of neurological deficits depends on the amount of damage to brain tissue.

Another important category of cranial abnormalities is caused by premature closure of one or more sutures. These abnormalities are collectively known as craniosynostosis, which occurs in one in 2,500 births and is a feature of more than 100 genetic syndromes. The shape of the skull depends on which of the sutures closed prematurely. Early closure of the sagittal suture (57% of cases) results in frontal and occipital expansion, and the skull becomes long and narrow (scaphocephaly) (Fig. 10.9). Premature closure of the coronal sutures results in a short skull called brachycephaly (Fig. 10.1 0A). If the coronal sutures close prematurely on one side only, then the result is an asymmetric flattening
Figure 10.8 A. Child with anencephaly. Cranial neural folds fail to elevate and fuse, leaving the cranial neuropore open. The skull never forms, and brain tissue degenerates. B. Patient with meningocele. This rather common abnormality may be successfully repaired.
Figure 10.9 Craniosynostosis involving the sagittal suture. A. Child with scaphocephaly caused by early closure of the sagittal suture. Note the long narrow shape of the head with prominent frontal and occipital regions. B,C computed tomography (CT) scans of the skull showing the long narrow shape of the head with bossing of the frontal and occipital regions. B. caused by premature closure of the sagittal suture C.
of the skull called plagiocephaly (Fig. 10.10B,C). Regulation of suture closure involves secretion of various isoforms of transforming growth factor-f.
Fibroblast growth factors (FGFs) and fibroblast growth factor receptors (FGFRs) also play important roles in skeletal development. There are many members of the FGF family and their receptors. Together, they regulate cellular events, including proliferation, differentiation, and migration. Signaling is mediated by the receptors, which are transmembrane tyrosine kinase receptors, each of which has three extracellular immunoglobulin domains, a transmembrane segment, and a cytoplasmic tyrosine kinase domain. FGFRI and FGFR2

are coexpressed in prebone and precartilage regions, including craniofacial structures; FGFR3 is expressed in the cartilage growth plates of long bones and in the occipital region. In general, FGFR2 increases proliferation, and FGFR! promotes osteogenic differentiation, whereas the role of FGFR3 is unclear. Mutations in these receptors, which often involve only a single amino acid substitution, have been linked to specific types of craniosynostosis (FGFrl, FGFR2, and FGFR3) and several forms of Skeletal dysplasia (FGFR3) (Table 10.1). In addition to these genes, mutations in the transcription factor MSX2, a regulator of parietal bone growth, causes Boston-type craniosynostosis, which can

HOXAI3, Phenotype = Small, short digits, divided uterus, hypospadias. HOXD13, Chromosome = 2q31. HOXD13, Abnormality = Synpolydactyly. HOXD13, Phenotype = Fused, multiple digits. TBX5, Chromosome = 12q24.1. TBX5, Abnormality = Upper limb and heart defects. TBX5, Phenotype = Digit defects, absent radius, limb bone hypoplasia, atrial and ventricular septal defects, conduction abnormalities. COLIAI and COLIA2, Chromosome = . COLIAI and COLIA2, Abnormality = Limb defects, blue sclera. COLIAI and COLIA2, Phenotype = Shortening, bowing, and hypomineralization of the long bones, blue sclera. Fibrillin (FBNI), Chromosome = I15q15-21. Fibrillin (FBNI), Abnormality = Marfan’s syndrome. Fibrillin (FBNI), Phenotype = Long limbs and face, sternal defects (pectus excavatum and carinatum), dilation and dissection of the ascending aorta, lens dislocation
affect a number of bones and sutures. The TWIST] gene codes for a DNA-binding protein and plays a role in regulating proliferation. Mutations in this gene cause Saethre—Chotzen's syndrome characterized by proliferation and premature differentiation in the coronal suture, causing craniosynostosis, and syndactyly (Fig. 10.10).
primarily affects the long bones (Fig. 10.1 1A). Other skeletal defects include a large skull (megalocephaly) with a small midface (Fig. 10.1 1B), short fingers, and accentuated spinal curvature. ACH is inherited as an autosomal dominant, and 90% of cases appear sporadically due to new mutations.

Achondroplasia (ACH), the most common form of skeletal dysplasia (1 per 20,000 live births),
Thanatophoric dysplasia is the most common neonatal lethal form of skeletal dysplasia (1 per 40,000 live births). There are two types; both are autosomal dominant. Type | is characterized by short,
Figure 10.10 Craniosynostosis involving the coronal sutures. A. Child with brachycephaly caused by early closure of both coronal sutures. Note the tall shape of the skull with flattened frontal and occipital regions. B. Child with plagiocephaly resulting from premature closure of the coronal suture on one side of the skull. C. CT scan of the skull showing plagiocephaly resulting from premature closure of the coronal suture on one side.
curved femurs with or without cloverleaf skull; type II individuals have straight, relatively long femurs and severe cloverleaf skull caused by craniosynostosis (Fig. 10.12). Another term for cloverleaf skull is kleeblattschadel. It occurs when all of the sutures close prematurely, resulting in the brain growing through the anterior and sphenoid fontanelles.
type of ACH. Common to all of these forms of skeletal dysplasias are mutations in FGFR3 causing abnormal endochondral bone formation, so that growth of the long bones and the base of the skull are adversely affected.

Hypochondroplasia, another autosomal dominant form of skeletal dysplasia, appears to be a milder
Cleidocranial dysostosis is an example of a generalized dysplasia of osseus and dental tissues that is characterized by late closure of the fontenalles
Figure 10.11 A. Nine-year-old child with Achondroplasia (ACH) showing a large head, short extremities, short fingers, and protruding abdomen. B. Lateral view of the patient's head showing a prominent forehead and midfacial hypoplasia.
Figure 10.12 Patient with cloverleaf skull characteristic of thanatophoric dwarfism type II. The shape of the skull results from abnormal growth of the cranial base, caused by a mutation in FGFR3, followed by craniosynostosis. The sagittal, coronal, and lambdoid sutures are commonly involved.
Figure 10.13 Child with cleidocranial dysostosis with generalized skeletal dysplasias. One characteristic of the condition is delayed closure of the fontanelles and decreased mineralization of the cranial sutures, such that the head appears larger due to bossing of the frontal, parietal, and occipital bones. Other parts of the skeleton are affected as well and often the clavicles are underdeveloped or missing, as in this case.
and decreased mineralization of the cranial sutures resulting in bossing (enlargement) of the frontal, parietal, and occipital bones (Fig. 10.13). Other parts of the skeleton are affected as well and often times the clavicles are underdeveloped or missing.

Acromegaly is caused by congenital hyperpituitarism and excessive production of growth hormone. It is characterized by disproportional enlargement of the face, hands, and feet. Sometimes, it causes more symmetrical excessive growth and gigantism.

Microcephaly is usually an abnormality in which the brain fails to grow and, as a result, the skull fails to expand (Fig. 10.14). Many children with microcephaly are severely intellectually disabled.
Figure 10.14 Child with microcephaly showing a small head due to the fact that the brain failed to grow to its normal size. One cause for this abnormality is exposure to alcohol in utero. In most cases, microcephaly is associated with significant intellectual disabilities.
142
Part
Il
Figure
10.15
A.
showing the
developing regions
of
a
somite.
Sclerotome cells
are dispersing
to migrate
around the
and notochord
to contribute
to vertebral
various components.

Vertebrae form from the sclerotome portions of the somites, which are derived from paraxial mesoderm (Fig. 10.15A). A typical vertebra consists of a vertebral arch and foramen (through which the spinal cord passes), a body, transverse processes, and usually a spinous process (Fig. 10.15B). During the fourth week, sclerotome cells migrate around the spinal cord and notochord to merge with cells from the opposing somite on the other side of the neural tube (Fig. 10.15.4). As development continues, the sclerotome portion of each somite also undergoes a process called resegmentation. Resegmentation occurs when the caudal half of each sclerotome grows into and fuses with the cephalic half of each subjacent sclerotome (arrows in Fig. 10.16.A,B).Thus, each vertebra is formed from the combination of the caudal half of one somite and the cranial half of its neighbor. Patterning of the shapes of the different vertebrae is regulated by HOX genes.
Mesenchymal cells between cephalic and caudal parts of the original sclerotome segment do not proliferate but fill the space between two precartilaginous vertebral bodies. In this way, they contribute to formation of the intervertebral disc (Fig. 10.16B). Although the notochord regresses
Figure 10.16 Formation of the vertebral column at various stages of development. A. At the fourth week of development, sclerotomic segments are separated by less dense intersegmental tissue. Note the position of the myotomes, intersegmental arteries, and segmental nerves. B. Proliferation of the caudal half of one sclerotome proceeds into the intersegmental mesenchyme and cranial half of the subjacent sclerotome (arrows). Note the appearance of the intervertebral discs. C. Vertebrae are formed by the upper and lower halves of two successive sclerotomes and the intersegmental tissue. the intervertebral and can move the vertebral column.
Myotomes bridge discs, therefore, formation.
B.
Example of
a

typical vertebra
showing its
entirely in the region of the vertebral bodies, it persists and enlarges in the region of the intervertebral disc. Here it contributes to the nucleus pulposus, which is later surrounded by circular fibers of the annulus fibrosus. Combined, these two structures form the intervertebral disc (Fig. 10.16C).
Resegmentation of sclerotomes into definitive vertebrae causes the myotomes to bridge the intervertebral discs, and this alteration gives them the capacity to move the spine (Fig. 10.16C). For the

The process of formation and rearrangement of segmental sclerotomes into definitive vertebrae is complicated, and it is fairly common to have two successive vertebrae fuse asymmetrically or have half a vertebra missing, a cause of scoliosis (lateral curving of the spine). Also, the number of vertebrae is frequently more or less than the norm. In Klippel-Feil sequence, the cervical vertebrae are fused causing reduced mobility and a short neck.
One of the most serious vertebral defects is the result of imperfect fusion or nonunion of the vertebral arches. Such an abnormality, known as cleft vertebra (spina bifida), may involve only the bony vertebral arches, leaving the spinal cord same reason, intersegmental arteries, at first lying between the sclerotomes, now pass midway over the vertebral bodies. Spinal nerves, however, come to lie near the intervertebral discs and leave the vertebral column through the intervertebral foramina.
As the vertebrae form, two primary curves of the spine are established: the thoracic and sacral curvatures. Later, two secondary curves are established: the cervical curvature, as the child learns to hold up his or her head and the lumbar curvature, which forms when the child learns to walk.
intact. In these cases, the bony defect is covered by skin, and no neurological deficits occur (spina bifida occulta). A more severe abnormality is spina bifida cystica, in which the neural tube fails to close, vertebral arches fail to form, and neural tissue is exposed.Any neurological deficits depend on the level and extent of the lesion (Fig. 10.17). This defect, which occurs in one per 2,500 births, may be prevented, in many cases, by providing mothers with folic acid prior to conception (see Chapter 6, p. 69). Spina bifida can be detected prenatally by ultrasound, and if neural tissue is exposed, amniocentesis can detect elevated levels of «.-fetoprotein in the amniotic fluid. (For the various types of spina bifida, see Fig. 6.7, p. 69-70.)

Figure 10.17 Ultrasound scans of the vertebral columns in a normal infant A and one with spina bifida B aged 4 months. The cleft vertebrae are readily apparent (arrows).

The bony portion of each rib is derived from sclerotome cells that remain in the paraxial mesoderm and that grow out from the costal processes of thoracic vertebrae. Costal cartilages are formed by sclerotome cells that migrate across the lateral somitic frontier into the adjacent lateral plate mesoderm (see Chapter 11 for a description of the lateral somitic frontier). The sternum develops independently in the parietal layer of lateral plate mesoderm in the ventral body wall. Two sternal bands are formed in the parietal (somatic) layer of lateral

Occasionally extra ribs are formed, usually in the lumbar or cervical regions. Cervical ribs occur in approximately 1% of the population and are usually attached to the seventh cervical vertebra. Because of its location, this type of rib may impinge on the brachial plexus or the subclavian artery, resulting in varying degrees of anesthesia in the limb.

Cleft sternum is a very rare defect and may be complete or located at either end of the sternum. Thoracic organs are covered only by skin and soft tissue. The defect arises when the sternal bands fail to grow together in the midline. Hypoplastic ossification centers and premature fusion of sternal segments also occur particularly in infants with congenital heart defects (20% to 50%). Multiple manubrial ossification centers occur in 6% to 20% of all children but are especially common in those with Down syndrome.
Pectus excavatum is the term for a depressed sternum that is sunken posteriorly. Pectus carinatum refers to a flattening of the chest bilaterally with an anteriorly projecting sternum. The projection of the sternum resembles the keel of a boat. Both defects may result from abnormalities of ventral body wall closure or formation of the costal cartilages and sternum.
plate mesoderm on either side of the midline, and these later fuse to form cartilaginous models of the manubrium, sternebrae, and xiphoid process.
ZL
The skeletal system develops from mesenchyme, which is derived from the mesodermal germ layer and from the neural crest. Some bones, such as the flat bones of the skull, undergo intramembranous ossification; that is, mesenchyme cells are directly transformed into osteoblasts (Fig. 10.2). In most bones, such as the long bones of the limbs, mesenchyme condenses and forms hyaline cartilage models of bones (Fig. 10.3). Ossification centers appear in these cartilage models, and the bone gradually ossifies by endochondral ossification. The skull consists of the neurocranium and the viscerocranium (face). The neurocranium includes a membranous portion, which forms the cranial vault, and a cartilaginous portion, the chondrocranium, which forms the base of the skull. Neural crest cells form the face, part of the cranial vault, and the prechordal part of the chondrocranium (the part that lies rostral to the pituitary gland). Paraxial mesoderm forms the remainder of the skull.

The vertebral column and ribs develop from the sclerotome compartments of the somites, and the sternum is derived from mesoderm in the ventral body wall. A definitive vertebra is formed by condensation of the caudal half of one sclerotome and fusion with the cranial half of the subjacent sclerotome (Fig. 10.16).
The many abnormalities of the skeletal system include vertebral (spina bifida), cranial (cranioschisis and craniosynostosis), and facial (cleft palate) defects. Major malformations of the limbs are rare, but defects of the radius and digits are often associated with other abnormalities (syndromes).

1. Why are cranial sutures important? Are they involved in any abnormalities?
2. Explain the origin of scoliosis as a vertebral anomaly. What genes might be involved in this abnormality?
ith the exception of some smooth muscle tissue (see later), the muscular system develops from the mesodermal germ layer and consists of skeletal, smooth, and cardiac muscle. Skeletal muscle is derived from paraxial mesoderm, which forms somites from the occipital to the sacral regions and somitomeres in the head. Smooth muscle differentiates from visceral splanchnic mesoderm surrounding the gut and its derivatives and from ectoderm (pupillary, mammary gland, and sweat gland muscles). Cardiac muscle is derived from visceral splanchnic mesoderm surrounding the heart tube.

Head musculature (see Chapter 17) is derived from seven somitomeres, which are partially segmented whorls of mesenchymal cells derived from paraxial mesoderm (see Chapter 6, p. 72). Musculature of the axial skeleton, body wall, and limbs is derived from somites, which initially form as somitomeres and extend from the occipital region to the tail bud. Immediately after segmentation, these somitomeres undergo a process of epithelization and form a "ball" of epithelial cells with a small cavity in the center (Fig. 11.1A). The ventral region of each somite then becomes mesenchymal again and forms the sclerotome (Fig. 11.1B—D), the bone-forming cells for the vertebrae and ribs. Cells in the upper region of the somite form the dermatome and two muscle-forming areas at the ventrolateral (VLL) and dorsomedial (DML) lips (or edges), respectively (Fig. 11.1B). Cells from these two areas migrate and proliferate to form progenitor muscle cells ventral to the dermatome, thereby forming the dermomyotome (Figs. 11.1B,C and 11.2). Some cells from the VLL region also migrate into the adjacent parietal layer of the lateral plate mesoderm (Fig. 11.1B).
Here they form infrahyoid, abdominal wall (rectus abdominus, internal and external oblique, and transversus abdominus), and limb muscles. The remaining cells in the myotome form muscles of the back, shoulder girdle, and intercostal muscles (Table 11.1, p. 145). Initially, there is a well-defined border between each somite and the parietal layer of lateral plate mesoderm called the lateral somitic frontier (Fig. 11.1B).This frontier separates two mesodermal domains in the embryo:
1. The primaxial domain that comprises the region around the neural tube and contains only somite-derived (paraxial mesoderm) cells.
2 The abaxial domain that consists of the parietal layer of lateral plate mesoderm together with somite cells that have migrated across the lateral somitic frontier.

Muscle cells that cross this frontier (those from the VLL edge of the myotome) and enter the lateral plate mesoderm comprise the abaxial muscle cell precursors and receive many of their signals for differentiation from lateral plate mesoderm (Fig. 11.3); those that remain in the paraxial mesoderm and do not cross the frontier (the remaining VLL cells and all of the DML cells) comprise the primaxial muscle cell precursors and receive many of their developmental signals from the neural tube and notochord (Fig. 11.3). Regardless of their domain, each myotome receives its innervation from spinal nerves derived from the same segment as the muscle cells.
The lateral somitic frontier also defines the border between dermis derived from dermatomes in the back and dermis derived from lateral plate mesoderm in the body wall. It also defines a border for rib development, such that the bony components of each rib are derived from primaxial sclerotome cells and the cartilaginous parts of those ribs that attach to the sternum are derived from sclerotome cells that migrate across the lateral somitic frontier (abaxial cells).

Figure | 1.1 Cross-sectional drawings showing the stages of development in a somite. A. Mesoderm cells become epithelial and are arranged around a small lumen. B. Cells in the ventral and medial walls of the somite lose their epithelial characteristics and migrate around the neural tube and notochord, and some move into the parietal layer of lateral plate mesoderm. Collectively, these cells constitute the sclerotome. Cells at the DML and VLL regions of the somite form muscle cell precursors. Cells from both regions migrate ventral to the dermatome to form the dermomyotome.VLL cells also migrate into the parietal layer of lateral plate mesoderm across the lateral somitic frontier (green line). In combination, somitic cells and lateral plate mesoderm cells constitute the abaxial mesodermal domain, while the primaxial mesodermal domain only contains somitic cells (paraxial mesoderm). C. Together, dermatome cells and the muscle cells that associate with them form the dermomyotome. D. The dermomyotome begins to differentiate: Myotome cells contribute to primaxial muscles, and dermatome cells form the dermis of the back.

Thoracoabdominal region, Primaxial = Prevertebral Intercostals. Thoracoabdominal region, Abaxial = Pectoralis major and minor External oblique. Upper limb, Primaxial = Rhomboids. Upper limb, Abaxial = Distal limb muscles. Upper limb, Primaxial = Levator scapulae. Upper limb, Abaxial = Distal limb muscles. Upper limb, Primaxial = Latissimus dorsi. Upper limb, Abaxial = Distal limb muscles. Lower limb a, Primaxial = Prevertebral Intercostals. Lower limb a, Abaxial = All lower limb muscles
innervation. The description does not preclude the fact that epaxial (above the axis) muscles (back muscles) are innervated by dorsal primary rami, whereas hypaxial (below the axis) muscles (body wall and limb muscles) are innervated by ventral primary rami (Fig. 11.4).
Figure
|
1.3
Expression patterns
of genes
that regulate
somite differentiation.
gin, secreted
Sonichedgehog by
the notochord
neural tube,
cause sclerotome
(SHH)
and floor
the and
ventral to
chondrogenesis concentrations
part express
and
PAX, vertebral
of
SHH
tube activate
PAX3,
WNT
form and
plate of
the which
of somite
in formation.
proteins which
from nog-
the to
form turn
controls
WNT
the demarcates
also muscle
specific direct
precursor gene
is
MYF5.
directed to
secreted and
low dorsal
neural the
dermatome.
the
DML
portion cells
and of
the somite
to
The dermatome
become by
the dermis
dorsal ence
of activating
tein activates
region
WNT
MyoD
to create
express portion
of by
neurotrophin neural
tube.
proteins the
the muscle-
somite
3
(NT-3)
The combined
and expression
a

in second
group influ-
inhibitory the
of
BMP4
Ventrolateral muscle
pro-
(VLL)
cell precursors.
Figure 11.4 Cross section through half the embryo showing innervation to developing musculature. Epaxial (true back muscles) are innervated by dorsal (posterior) primary rami. Hypaxial muscles (limb and body wall) are innervated by ventral (anterior) primary rami.

During differentiation, precursor cells, the myoblasts, fuse and form long, multinucleated muscle fibers. Myofibrils soon appear in the cytoplasm, and by the end of the third month, cross-striations, typical of skeletal muscle, appear. A similar process occurs in the seven somitomeres in the head region rostral to the occipital somites. However, somitomeres never segregate into recognizable regions of sclerotome and dermomyotome segments prior to differentiation. Tendons for the attachment of muscles to bones are derived from sclerotome cells lying adjacent to myotomes at the anterior and posterior borders of somites. The transcription factor SCLERAXIS regulates development of tendons.

Genes regulating muscle development have recently been identified. Bone morphogenetic protein 4 (BMP4) and probably fibroblast growth factors from lateral plate mesoderm, together with WNT proteins from adjacent ectoderm, signal VLL cells of the dermomyotome to express the muscle-specific gene MyoD (Fig. 11.3). BMP4 secreted by ectoderm cells induces production of WNT proteins by the dorsal neural tube at the same time that low concentrations of sonic hedgehog (SHH) proteins, secreted by the notochord and floor plate of the neural tube, reach the DML cells of the dermomyotome. Together these proteins induce expression of MYF5 and MyoD in these cells (note that SHH does not play a role in specifying VLL cells). Both MyoD and MYF5 are members of a family of transcription factors called myogenic regulatory factors (MRFs),
and this group of genes activates pathways for muscle development.

Patterns of muscle formation are controlled by connective tissue into which myoblasts migrate. In the head region, these connective tissues are derived from neural crest cells; in cervical and occipital regions, they differentiate from somitic mesoderm; and in the body wall and limbs, they originate from the parietal layer of lateral plate mesoderm.

All voluntary muscles of the head region are derived from paraxial mesoderm (somitomeres and somites), including musculature of the tongue, eye (except that of the iris, which is derived from optic cup ectoderm), and that associated with the pharyngeal (visceral) arches (Table 11.2, p. 146, and Fig. 11.2). Patterns of muscle formation in the head are directed by connective tissue elements derived from neural crest cells.

The first indication of limb musculature is observed in the seventh week of development as a condensation of mesenchyme near the base of the limb buds (Fig. 11.2). The mesenchyme is derived from dorsolateral cells of the somites that migrate into the limb bud to form the muscles. As in other regions, connective tissue dictates the pattern of muscle formation, and this tissue is derived from the parietal layer of lateral plate mesoderm, which also gives rise to the bones of the limb (see chapter 12).
Somitomeres 1 and 2, Muscles = Superior, medial, ventral recti. Somitomeres 1 and 2, Innervation = Oculomotor (III). Somitomere 3, Muscles = Superior oblique. Somitomere 3, Innervation = Trochlear (IV). Somitomere 4, Muscles = Jaw closing. Somitomere 4, Innervation = Trigeminal (V). Somitomere 5, Muscles = Lateral rectus. Somitomere 5, Innervation = Abducens (VI). Somitomere 6, Muscles = Jaw opening, other second arch. Somitomere 6, Innervation = Facial (VII). Somitomere 7, Muscles = Stylopharyngeus. Somitomere 7, Innervation = Glossopharyngeal (IX). Somites 1 and 2, Muscles = Intrinsic laryngeals. Somites 1 and 2, Innervation = Vagus (X). Somites 2-5 a, Muscles = Tongue. Somites 2-5 a, Innervation = Hypoglossal (XII)

Cardiac muscle develops from splanchnic mesoderm surrounding the endothelial heart tube. Myoblasts adhere to one another by special attachments that later develop into intercalated discs. Myofibrils develop as in skeletal muscle, but myoblasts do not fuse. During later development, a few special bundles of muscle cells with irregularly distributed myofibrils become visible. These bundles, Purkinje's fibers, form the conducting system of the heart.

Smooth muscle for the dorsal aorta and large arteries is derived from lateral plate mesoderm and neural crest cells. In the coronary arteries, smooth muscle originates from proepicardial

Partial or complete absence of a muscle is common and usually not debilitating. Examples include partial or complete absence of the palmaris longus, serratus anterior, or quadratus femoris muscles.A more serious defect is called Poland sequence that occurs in | per 20,000 individuals and is
Figure 11.5 Poland sequence.The pectoralis minor and part of the pectoralis major muscles are missing on the patient's left side. Note displacement of the nipple and areola.
cells (see Chapter 13) and neural crest cells (proximal segments). Smooth muscle in the wall of the gut and gut derivatives is derived from the splanchnic layer of lateral plate mesoderm that surrounds these structures. Only the sphincter and dilator muscles of the pupil and muscle tissue in the mammary and sweat glands are derived from ectoderm.
Serum response factor (SRF) is a transcription factor responsible for smooth muscle cell differentiation. This factor is upregulated by growth factors through kinase phosphorylation pathways. Myocardin and myocardin-related transcription factors (MRTFs) then act as coactivators to enhance the activity of SRE thereby initiating the genetic cascade responsible for smooth muscle development.
characterized by absence of the pectoralis minor and partial loss of the pectoralis major (usually the sternal head) muscles (Fig. 11.5). The nipple and areola are absent or displaced, and there are often digital defects (syndactyly [fused digits] and brachydactyly [short digits]) on the affected side. The disfiguring nature of the defects can be problematic, especially in females because of breast development.
Partial or completeabsence ofabdominal musculature is called prune belly syndrome (Fig. | 1.6). Usually, the abdominal wall is so thin that organs are visible and easily palpated. This defect is sometimes associated with malformations of the urinary tract and bladder, including urethral obstruction. These defects cause an accumulation of fluid that distends the abdomen, resulting in atrophy of the abdominal muscles.

Muscular dystrophy is the term for a group of inherited muscle diseases that cause progressive muscular wasting and weakness. There are a large number of these types of diseases of which Duchenne's muscular dystrophy (DMD) is the most common (I per 4,000 male births). The disease is inherited as X-linked recessive such that males are much more often affected than females. Both DMD and Becker's muscular dystrophy (BMD) are caused by mutations

Figure 11.6 Prune belly syndrome: a distended abdomen from atrophy of abdominal wall musculature.
Z_
Most muscles arise from the mesoderm. Skeletal muscles are derived from paraxial mesoderm, including (1) somites, which give rise to muscles of the axial skeleton, body wall, and limbs, and (2) somitomeres, which give rise to muscles of the head. Progenitor cells for muscle tissues are derived from the ventrolateral (VLL) and dorsomedial (DML) edges (lips) of the prospective dermomyotome. Cells from both regions contribute to formation of the myotome. Some cells from the VLL also migrate across the lateral somitic frontier into the parietal layer of the lateral plate mesoderm. This frontier or border separates two mesodermal domains in the embryo: (1) the primaxial domain that surrounds the neural tube and contains only somite-derived cells (paraxial mesoderm) and (2) the abaxial domain that consists of the parietal layer of lateral plate mesoderm in combination with somite-derived cells that migrate across the frontier into this region (Fig. 11.1). Abaxial muscle precursor cells differentiate into infrahyoid, abdominal wall (rectus abdominus, external and internal obliques, transversus abdominus), and limb muscles. Primaxial muscle precursor cells form muscles of the back, some muscles of the shoulder girdle, and intercostal muscles (Table 11.1, p. 145). Muscles of the back (epaxial muscles) are innervated by dorsal primary rami; muscles of the limbs and body wall (hypaxial muscles) are innervated by ventral primary rami. Molecular in the gene for dystrophin on the X chromosome. However, in DMD, no functional dystrophin is made and the disease is much more severe with an earlier onset (<5 years of age versus 8 to 25 years of age in BMD). Dystrophin is a cytoplasmic protein that forms a dystrophin-associated protein complex linking the cytoskeleton to the extracellular matrix.

signals for muscle cell induction arise from tissues adjacent to prospective muscle cells. Thus, signals from lateral plate mesoderm (BMPs) and overlying ectoderm (WNTs) induce VLL cells; while signals from the neural tube and notochord (SHH and WNTs) induce DML cells. Connective tissue derived from somites, parietal mesoderm, and neural crest (head region) provides a template for establishment of muscle patterns. Most smooth muscles and cardiac muscle fibers are derived from splanchnic mesoderm. Smooth muscles of the pupil, mammary gland, and sweat glands differentiate from ectoderm.

1. Muscle cells are derived from what two regions of the somite? Which region contributes to the abaxial mesodermal domain? What muscles form from the abaxial and primaxial domains?
2. In examining a newborn female infant, you note that her right nipple is displaced toward the axilla and that the right anterior axillary fold is nearly absent. What is your diagnosis?
3. Patterning of muscles is dependent on what type of tissue?
4. How do you explain the fact that the phrenic nerve, which originates from cervical segments 3, 4, and 5, innervates the diaphragm in the thoracic region?

The limbs, including the shoulder and pelvic girdles, comprise the appendicular skeleton. At the end of the fourth week of development, limb buds become visible as outpocketings from the ventrolateral body wall (Fig. 12.1A). The forelimb appears first followed by the hindlimb 1 to 2 days later. Initially, the limb buds consist of a mesenchymal core derived from the parietal (somatic) layer of lateral plate mesoderm that will form the bones and connective tissues of the limb, covered by a layer of cuboidal ectoderm. Ectoderm at the distal border of the limb thickens and forms the apical ectodermal ridge (AER) (Fig. 12.2 see also Fig. 12.9A). This ridge exerts an inductive influence on adjacent mesenchyme, causing it to remain as a population of undifferentiated, rapidly proliferating cells, the progress zone. As the limb grows, cells farther from the influence of the AER begin to differentiate into cartilage and muscle. In this manner, development of the limb proceeds proximodistally.

In 6-week-old embryos, the terminal portion of the limb buds becomes flattened to form the handand footplates and is separated from the proximal segment by a circular constriction (Fig. 12.1B). Later, a second constriction divides the proximal portion into two segments, and the main parts of the extremities can be recognized (Fig. 12.1C). Fingers and toes are formed when cell death in the AER separates this ridge into five parts (Fig. 12.3A). Further formation of the digits depends on their continued outgrowth under the influence of the five segments of ridge ectoderm, condensation of the mesenchyme to form cartilaginous digital rays, and the death of intervening tissue between the rays (Fig. 12.3B,C). Development of the upper and lower limbs is similar except that morphogenesis of the lower limb is approximately 1 to 2 days behind that of the upper limb. Also, during the seventh week of gestation, the limbs rotate in opposite directions. The upper limb rotates 90° laterally, so that the extensor muscles lie on the lateral and posterior surface, and the thumbs lie laterally, whereas the lower limb rotates approximately 90 degrees medially, placing the extensor muscles on the anterior surface and the big toe medially.
While the external shape is being established, mesenchyme in the buds begins to condense, and these cells differentiate into chondrocytes (Fig. 12.4). By the sixth week of development, the first hyaline cartilage models, foreshadowing the bones of the extremities, are formed by these chondrocytes (Figs. 12.4 and 12.5). Joints are formed in the cartilaginous condensations when chondrogenesis is arrested, and a joint interzone is induced. Cells in this region increase in number and density, and then a joint cavity is formed by cell death. Surrounding cells differentiate into a joint capsule. Factors regulating the positioning of joints are not clear, but the secreted molecule WNT14 appears to be the inductive signal.

Ossification of the bones of the extremities, endochondral ossification, begins by the end of the embryonic period. Primary ossification centers are present in all long bones of the limbs by the 12th week of development. From the primary center in the shaft or diaphysis of the bone, endochondral ossification gradually progresses toward the ends of the cartilaginous model (Fig. 12.5).
At birth, the diaphysis of the bone is usually completely ossified, but the two ends, the epiphyses, are still cartilaginous. Shortly thereafter, however, ossification centers arise in the epiphyses. Temporarily, a cartilage plate remains between the diaphyseal and epiphyseal ossificaion centers. This plate, the epiphyseal plate, plays an important role in growth in the length of the bones. Endochondral ossification proceeds on both sides of the plate (Fig. 12.5). When the b P ¢. one has acquired its full length, the epiphyseal ates disappear, and the epiphyses unite with the shaft of the bone.
In long bones, an epiphyseal plate is found on each extremity; in smaller bones, such as the phalanges, it is found only at one extremity; and
Figure
12.1
the limb
buds in
human embryos.
A.
At
5
weeks.
B.
At
6
weeks.
C.
At
8
weeks.
Hindlimb development lags behind forelimb development by | to 2 days.
in irregular bones, such as the vertebrae, one or more primary centers of ossification and usually several secondary centers are present.
skull) also form from interzone regions, but in this case the interzone remains as a dense fibrous structure.

Synovial joints between bones begin to form at the same time that mesenchymal condensations initiate the process of forming cartilage. Thus, in the region between two chondrifying bone primordia, called the interzone (for example between the tibia and femur at the knee joint), the condensed mesenchyme differentiates into dense fibrous tissue. This fibrous tissue then forms articular cartilage, covering the ends of the two adjacent bones; the synovial membranes; and the menisci and ligaments within the joint capsule (e.g., the anterior and posterior cruciate ligaments in the knee). The joint capsule itself is derived from mesenchyme cells surrounding the interzone region. Fibrous joints (e.g., the sutures in the

Limb musculature is derived from dorsolateral cells of the somites that migrate into the limb to form muscles and, initially, these muscle components are segmented according to the somites from which they are derived (Fig. 12.6). However, with elongation of the limb buds, the muscle tissue first splits into flexor and extensor components (Fig. 12.7) and then additional splittings and fusions occur, such that a single muscle may be formed from more than one original segment. The resulting complex pattern of muscles is determined by connective tissue derived from lateral plate mesoderm.
A. Longitudinal section through the limb bud of a chick embryo, showing a core of mesenchyme covered a layer of ectoderm that thickens at the distal border of the limb to form the AER. In humans, this occurs during the week of development. B. External view of a chick limb at high magnification showing the ectoderm and the specialized
Figure 12.2 by fifth region at the tip of the limb called the AER.
segments. As soon as the buds form, ventral primary rami from the appropriate spinal nerves penetrate into the mesenchyme.At first, each ventral ramus enters with dorsal and ventral branches derived from its specific spinal segment, but soon branches in their respective divisions begin to unite to form large dorsal and ventral nerves (Fig. 12.7). Thus, the radial nerve, which supplies the extensor musculature, is formed by a combination of the dorsal segmental branches, whereas the ulnar and median nerves, which supply the flexor musculature, are formed by a combination of the ventral branches. Immediately after the nerves have entered the limb buds, they establish an intimate contact with the differentiating mesodermal condensations, and the early contact between the nerve and muscle cells is a prerequisite for their complete functional differentiation.
Spinal nerves not only play an important role in differentiation and motor innervation of the limb musculature, but also provide sensory innervation for the dermatomes. Although the original dermatomal pattern changes with growth and rotation of the extremities, an orderly sequence can still be recognized in the adult (Fig. 12.8).
Figure 12.4 A. Lower extremity of an early 6-week embryo, illustrating the first hyaline cartilage models. B,C. Complete

Figure 12.5 Endochondral bone formation. A. Mesenchyme cells begin to condense and differentiate into chondrocytes. B. Chondrocytes form a cartilaginous model of the prospective bone. C,D. Blood vessels invade the center of the cartilaginous model, bringing osteoblasts (black cells) and restricting proliferating chondrocytic cells to the ends (epiphyses) of the bones. Chondrocytes toward the shaft side (diaphysis) undergo hypertrophy and apoptosis as they mineralize the surrounding matrix. Osteoblasts bind to the mineralized matrix and deposit bone matrices. Later, as blood vessels invade the epiphyses, secondary ossification centers form. Growth of the bones is maintained by proliferation of chondrocytes in the growth plates.
Figure
12.7
As muscle
cells move
into the
limb, they
split into
ments.
dorsal
Muscles
(extensor)
and are
initially divide
to compartments.
innervated form
ventral
(flexor)
by dorsal
ventral and
Ultimately, dorsal
and ventral
ventral nerves.
compart- primary
ventral branches
divisions from
unite into
rami that
branches to
these their
respective large
dorsal and
Figure 12.8 Forelimbs with their sensory innervation to the dermatomes represented. Note that sensory innervation to the limb maintains a segmental pattern reflecting the embryological origin of each dermatome and its innervation.

Positioning of the limbs along the craniocaudal axis in the flank regions of the embryo is regulated by the HOX genes expressed along this axis. These homeobox genes are expressed in overlapping patterns from head to tail (see Chapter 6, p. 81), with some having more cranial limits than others. For example, the cranial limit of expression of HOXB8 is at the cranial border of the forelimb, and misexpression of this gene alters the position of these limbs.
Once positioning along the craniocaudal axis is determined, growth must be regulated along the proximodistal, anteroposterior, and dorsoventral axes (Fig. 12.9). Limb outgrowth, which occurs first, is initiated by TBX5 and FGF10 in the forelimb and TBX4 and FGF10 in the hindlimb secreted by lateral plate mesoderm cells (Fig. 12.9A). Once outgrowth is initiated, bone morphogenetic proteins (BMPs), expressed in ventral ectoderm, induce formation of the AER by signaling through the homeobox gene MSX2. Expression of Radical fringe (a homologue of Drosophila fringe), in the dorsal half of the limb ectoderm, restricts the location of the AER to the distal tip of the limbs. This gene induces expression of SER2, a homologue of Drosophila serrate, at the border between cells that are expressing Radical fringe and those that are not. It is at this border that the AER is established. Formation of the border itself is assisted by expression of Engrailed-1 in ventral ectoderm cells, because this gene represses expression of Radical fringe. After the ridge is established, it expresses FGF4 and FGF8, which maintain the progress zone, the rapidly proliferating population of mesenchyme cells adjacent to the ridge (Fig. 12.9). Distal growth of the limb is then affected by these rapidly proliferating cells under the influence of the FGFs. As growth occurs, mesenchymal cells at the proximal end of the progress zone become farther away from the ridge and its influence and begin to slow their division rates and to differentiate.
Patterning of
the anteroposterior
axis of
the limb

is regulated by the zone of polarizing activity (ZPA), a cluster of cells at the posterior border of the limb near the body wall (Fig. 12.9B). These cells produce retinoic acid (vitamin A), which initiates expression of sonic hedgehog (SHH), a secreted factor that regulates the anteroposterior axis.
Thus, for example, digits appear in the proper order, with the thumb on the radial (anterior) side. As the limb grows, the ZPA moves distalward to remain in proximity to the posterior border of the AER. Misexpression of retinoic acid or SHH in the anterior margin of a limb containing a normally expressing ZPA in the posterior border results in a mirror image duplication of limb structures (Fig. 12.10).
The dorsoventral axis is also regulated by BMPs in the ventral ectoderm, which induce expression of the transcription factor EN1. In turn, EN7 represses WNT7a expression, restricting it to the dorsal limb ectoderm. WNT7a is a

Figure 12.10 Experimental procedure for grafting a new ZPA from one limb bud into another using chick embryos. The result is the production of a limb with mirror image duplication of the digits much like that shown in the adjacent figure, indicating the role of the ZPA in regulating anteroposterior patterning of the limb. SHH protein is the molecule secreted by the ZPA responsible for this regulation.
secreted factor that induces expression of LMX1, a transcription factor containing a homeodomain, in the dorsal mesenchyme (Fig. 12.9C). LMX1 specifies cells to be dorsal, establishing the dorsoventral components. In addition, WNT7a maintains SHH expression in the ZPA and therefore

Radiologists use the appearance of various ossification centers to determine whether a child has reached his or her proper maturation age. Useful information about bone age is obtained from ossification studies in the hands and wrists of children. Prenatal analysis of fetal bones by ultrasonography provides information about fetal growth and gestational age.

Limb malformations occur in approximately 6 per 10,000 live births, with 3.4 per 10,000 affecting the upper limb and 1.1 per 10,000 affecting the lower. These defects are often associated with other birth defects involving the craniofacial, cardiac, and genitourinary systems. Abnormalities of the limbs vary greatly, and they may be represented by partial (meromelia) or complete absence (amelia) of one or more of the extremities indirectly affects anteroposterior patterning as well. These two genes are also intimately linked in signaling pathways in Drosophila, and this interaction is conserved in vertebrates. In fact, all of the patterning genes in the limb have feedback loops. Thus, FGFs in the AER activate SHH in the ZPA, while WNT7a maintains the SHH signal.
Although patterning genes for the limb axes have been determined, it is the HOX genes that regulate the types and shapes of the bones of the limb (Fig. 12.9D). This HOX gene expression is dependent upon the combinatorial expression of FGF, SHH, and WNT7a genes that cause HOX expression in three phases in the limb that correspond to the proximal (stylopod: humerus and femur), middle (zeugopod: radius and ulna; tibia and fibula), and distal (autopod: hand and foot) regions. Genes of the HOXA and D clusters are the primary determinants in the limb accounting for patterning the bones. Thus, misexpression of either of these two genes may result in limb truncations and anterior—posterior duplications. Just as in the craniocaudal axis of the embryo, HOX genes are nested in overlapping patterns of expression that somehow regulate patterning (Fig. 12.9D). Factors determining forelimb versus hindlimb are the transcription factors TBX5 (forelimbs) and TBX4 together with PITX1 (hindlimbs).

(Fig. 12.11.A). Sometimes, the long bones are absent, and rudimentary hands and feet are attached to the trunk by small, irregularly shaped bones (phocomelia, a form of meromelia) (Fig. 12.11B). Sometimes, all segments of the extremities are present but abnormally short (micromelia).
Although these abnormalities are rare and mainly hereditary, cases of teratogen-induced limb defects have been documented. For example, many children with limb malformations were born between 1957 and 1962. Many mothers of these infants had taken thalidomide, a drug widely used as a sleeping pill and antinauseant. It was subsequently established that thalidomide causes a characteristic syndrome of malformations consisting of absence or gross deformities of the long bones, intestinal atresia, and cardiac anomalies. Studies indicate that the fourth and the fifth weeks of gestation are the most sensitive period for induction of the limb defects. Because the drug
Figure 12.11 A. Child with unilateral amelia and multiple defects of the left upper limb. B. Patient with a form of meromelia called phocomelia. The hands are attached to the trunk by irregularly shaped bones.
is now being used to treat AIDS and cancer patients, distribution of thalidomide is carefully regulated to avoid its use by pregnant women.
A different category of limb defects involves the digits. Sometimes, the digits are shortened (brachydactyly; Fig. 12.12A). If two or more fingers or toes are fused, it is called syndactyly (Fig. 12.12B). Normally, mesenchyme between prospective digits in handand footplates is removed by cell death (apoptosis). In 1 per 2,000 births, this process fails, and the result is fusion between two or more digits. The presence of extra fingers or toes is called polydactyly (Fig. 12.12C). The extra digits frequently lack proper muscle connections. Abnormalities involving polydactyly are usually bilateral, whereas absence of a digit (ectrodactyly), such as a thumb, usually occurs unilaterally.

role of the HOX genes in limb development is illustrated by two abnormal phenotypes produced by mutations in this family of genes: mutations in HOXA23 result in hand-foot— genital syndrome, characterized by fusion of the carpal bones and small short digits. Affected females often have a partially (bicornuate) or a completely (didelphic) divided uterus and abnormal positioning of the urethral orifice. Affected males may have hypospadias. The genital defects are due to the fact that HOXA13 plays an important role in development of the cloaca into the urogenital sinus and anal canal (see Chapter 16). Mutations in HOXD13 result in a combination of syndactyly and polydactyly (synpolydactyly).
Cleft hand and foot consists of an abnormal cleft between the second and fourth metacarpal bones and soft tissues. The third metacarpal and phalangeal bones are almost always absent, and the thumb and index finger and the fourth and fifth fingers may be fused (Fig. 12.12D). The two parts of the hand are somewhat opposed to each other.
A number of gene mutations have been identified that affect the limbs and sometimes other structures (see Table 10.1, p. 137). The
TBX5 mutations (chromosome 12q24.1) result in Holt-Oram syndrome, characterized by upper limb abnormalities and heart defects consistent with a role for this gene in upper limb and heart development. Virtually all types of limb defects affecting the upper limb have been observed, including absent digits, polydactyly, syndactyly, absent radius, and hypoplasia of any of the limb bones. Heart defects include atrial and ventricular septal defects and conduction abnormalities.
Osteogenesis imperfecta is characterized by shortening, bowing, and hypomineralization of the long bones of the limbs that can result in fractures and blue sclera
2 Digital defects. A. Brachydactyly, short digits. B. Syndactyly, fused digits. C. Polydactyly, extra digits. D. Cleft ead ce of these defects may involve either the hands or feet or both.

(Fig. 12.13). Several types of osteogenesis imperfecta occur, ranging from persons with a mildly increased frequency of fractures to a severe form that is lethal in the neonatal period. In most cases, dominant mutations in the COL1A1 or COL1A2 genes that are involved in production of type I collagen cause the abnormalities.
Marfan syndrome is caused by mutations in the fibrillin (FBN1) gene located on chromosome 15q21.1. Affected individuals are usually tall and slender with long thin limbs and a long thin face. Other characteristics include sternal defects (pectus excavatum or carinatum), joint hyperflexibility, dilatation and/or dissection of the ascending aorta, and dislocation of the lens of the eye.
Arthrogryposis or congenital joint contractures (Fig. 12.14) usually involves more than one joint and may be caused by neurological defects (motor horn cell deficiency, meningomyelocele), muscular abnormalities (myopathies, muscle agenesis), joint and contiguous tissue
T 3 Newborn with osteogenesis imperfecta. Note the shortness and bowing of the limbs.
Figure 12.14 Infant with arthrogryposis (congenital joint contractures).
problems (synostosis, abnormal development), and fetal crowding and constraint (multiple births, oligohydramnios). For example, clubfoot is often caused by constraint in utero from too little amniotic fluid (oligohydramnios).
Congenital absence or deficiency of the radius is usually a genetic abnormality observed with malformations in other structures, such as craniosynostosis—radial aplasia syndrome (Baller-Gerold syndrome). These individuals have synostosis in one or more cranial sutures, absence of the radius, and other defects.
Amniotic bands may cause ring constrictions and amputations of the limbs or digits (Fig. 12.15; see also Fig. 8.17, p. 107). The origin of the bands is not clear, but they may represent adhesions between the amnion and affected structures in the fetus. Other investigators believe that bands originate from tears in the amnion that detach and surround part of the fetus.

Transverse limb deficiencies are limb defects in which proximal structures are intact, but structures distal to a transverse plane are partially or completely absent (Fig. 12.16). The defects may be due to disruption of the AER or its signaling or to vascular abnormalities, such as thromboses or vasoconstriction.
Figure 12.15 Digit amputations resulting from amniotic bands.
Figure 12.16 Transverse limb deficiency, which may be caused by disruptions to the AER or to vascular abnormalities.
Z_
Limbs form at the end of the fourth week as buds along the body wall adjacent to specific spinal segments determined by HOX genes (upper limb, C5—T2; lower limb, L2-S2).The apical ectodermal ridge (AER) at the distal border of the limb regulates limb outgrowth by secreting FGFs that maintain a region of rapidly dividing cells immediately adjacent to the ridge called the progress zone. The zone of polarizing activity (ZPA) located at the posterior border of the limb secretes sonic hedgehog (SHH) and controls anterior—posterior patterning (thumb to little finger).
Bones of the limb form by endochondral ossification and are derived from the parietal layer of lateral plate mesoderm. Muscle cells migrate from somites in a segmental fashion and segregate into dorsal and ventral muscle groups. Later fusion and splitting of these groups into different muscles distorts the original segmental pattern.
Congenital hip dislocation consists of underdevelopment of the acetabulum and head of the femur. It is rather common and occurs mostly in female newborns. Although dislocation usually occurs after birth, the abnormality of the bones develops prenatally. Because many babies with congenital hip dislocation are breech deliveries, it has been thought that breech posture may interfere with development of the hip joint. It is frequently associated with laxity of the joint capsule.
Muscles are innervated by ventral primary rami that split into dorsal and ventral branches. The dorsal and ventral branches eventually unite into dorsal and ventral nerves to innervate the dorsal (extensor) and ventral (flexor) compartments, respectively.

Digits form when apoptosis (programmed cell death) occurs in the AER to separate this structure into five separate ridges. Final separation of the digits is achieved by additional apoptosis in the interdigital spaces. Many digital defects occur that are related to these patterns of cell death, including polydactyly, syndactyly, and clefts (Fig 12.12).

- 1. If you observe congenital absence of the radius or digital defects, such as absent thumb or polydactyly, would you consider examining the infant for other malformations? Why?

The vascular system appears in the middle of the third week, when the embryo is no longer able to satisfy its nutritional requirements by diffusion alone. Progenitor heart cells lie in the epiblast, immediately adjacent to the cranial end of the primitive streak. From there, they migrate through the streak and into the splanchnic layer of lateral plate mesoderm where they form a horseshoe-shaped cluster of cells called the primary heart field (PHF) cranial to the neural folds (Fig. 13.1). As the progenitor heart cells migrate and form the PHF during days 16 to 18, they are specified on both sides from lateral to medial to become the atria, left ventricle, and most of the right ventricle (Fig.13.1.A) Patterning of these cells occurs at the same time that laterality (left-right sidedness) is being established for the entire embryo and this process and the signaling pathway it is dependent upon (Fig. 13.2) is essential for normal heart development.
The remainder of the heart, including part of the right ventricle and outflow tract (conus cordis and truncus arteriosus), is derived from the secondary heart field (SHF).This field of cells appears slightly later (days 20 to 21) than
Cloacal membrane
Figure 13.2 Dorsal view of a drawing of a 16-day embryo showing the laterality pathway.The pathway is expressed in lateral plate mesoderm on the left side and involves a number of signaling molecules, including serotonin (SHT), which result in expression of the transcription factor PITX2, the master gene for left sidedness.This pathway specifies the left side of the body and also programs heart cells in the primary and SHFs.The right side is specified as well, but genes responsible for this patterning have not been completely determined. Disruption of the pathway on the left results in laterality abnormalities, including many heart defects.
blasts. cavity (Fig. 13.1B,C). addition the cardiogenic other
Pharyngeal arches

those in the PHE resides in splanchnic mesoderm ventral to the posterior pharynx, and is responsible for lengthening the outflow tract (see Fig. 13.3). Cells in the SHF also exhibit laterality, such that those on the right side contribute to the left of the outflow tract region and those on the left contribute to the right. This laterality is determined by the same signaling pathway that establishes laterality for the entire embryo (Fig. 13.2) and explains the spiraling nature of the pulmonary artery and aorta and ensures that the aorta exits from the left ventricle and the pulmonary artery from the right ventricle.
Once cells establish the PHE they are induced by the underlying pharyngeal endoderm to form cardiac myoblasts and blood islands that will form blood cells and vessels by the process of vasculogenesis (Chapter 6, p. 75). With time, the islands unite and form a horseshoe-shaped endothelial-lined tube surrounded by myoThis region is known as the cardiogenic region; the intraembryonic (primitive body) cavity over it later develops into the pericardial
In to region, blood islands appear bilaterally, parallel, and close to the midline of the embryonic shield. These islands form a pair of longitudinal vessels, the dorsal aortae.
Figure 13.3 Drawing showing the SHF that lies in splanchnic mesoderm at the posterior of the pharynx. The SHF provides cells that lengthen the outflow region of the heart, which includes part of the right ventricle and the outflow tract (conus cordis and truncus arteriosus). Neural crest cells, migrating from cranial neural folds to the heart through pharyngeal arches in this region, regulate the SHF by controlling FGF concentrations. Disruption of the SHF causes shortening of the outflow tract region, resulting in outflow tract defects.

Establishing laterality during gastrulation (see Chapter 5, p. 55-56) is essential for normal heart development because it specifies cells contributing to and patterning the right and left sides of the heart. The process requires a signaling cascade that includes serotonin (SHT) as a key molecule in initiating the pathway (Fig. 13.2). SHT is concentrated on the left side of the embryo, in part because of a high concentration of its degrading enzyme, monoamine oxidase (MAO), on the right side near the primitive node. Nodal and FGF also accumulate on the left side and together with SHT propagate signals that culminate in expression of PITX2, the master gene for left sidedness. The right side is also specified, but signals responsible for this event have not been as well established.
Cardiac progenitor cells are also specified at this time both for the parts of the heart they will form and their left-right sidedness by the laterality pathway. Thus, this period (days 16 to 18) is critical for heart development and experimental studies show

Initially, the central portion of the cardiogenic area is anterior to the oropharyngeal membrane and the neural plate (Fig. 13.4A). With closure of the neural tube and formation of the brain vesicles, however, the central nervous system grows cranially so rapidly that it extends over the central cardiogenic region and the future pericardial cavity (Fig. 13.4). As a result of growth of the brain and cephalic folding of the embryo, the oropharyngeal membrane is pulled forward, while the heart and pericardial cavity move first to the cervical region and finally to the thorax (Fig. 13.4).
As the embryo grows and bends cephalocaudally, it also folds laterally (Fig. 13.5). As a result, the caudal regions of the paired cardiac tube merge except at their caudalmost ends (Fig. 13.6). Simultaneously, the central part of the horseshoe-shaped tube expands to form the future outflow tract and ventricular regions. Thus, the heart becomes a continuous expanded tube consisting of an inner endothelial lining and an outer myocardial layer (Fig. 13.5C). It receives venous drainage at its caudal pole and begins to pump blood out of the first aortic arch into the dorsal aorta at its cranial pole (Figs. 13.6 and 13.7).
that disruption of the laterality pathway causes many different types of heart defects, including dextrocardia (right-sided heart), ventricular septal defects (VSDs), atrial septal defects (ASDs), double outlet right ventricle (DORV; both the aorta and pulmonary artery exit the right ventricle), and outflow tract defects, such as transposition of the great vessels, pulmonary stenosis, and others. Laterality defects of the heart, such as atrial and ventricular isomerisms (both atria or both ventricles have similar characteristics instead of the normal left-right differences) and inversions (the characteristics of the atria or ventricles are reversed), also occur because of disruptions in specifying left and right sidedness in the progenitor heart cells.

The importance of laterality in normal heart development may explain the apparent role of antidepressants of the selective serotonin reuptake inhibitor (SSRIs) class that have been linked to an increase in heart defects. These compounds alter SHT concentrations, which may disrupt 5HT signaling in the laterality pathway causing the malformations.
The developing heart tube bulges more and more into the pericardial cavity. Initially, however, the tube remains attached to the dorsal side of the pericardial cavity by a fold of mesodermal tissue, the dorsal mesocardium (Figs. 13.5C and 13.7). No ventral mesocardium is ever formed. With further development, the dorsal mesocardium disappears, creating the transverse pericardial sinus, which connects both sides of the pericardial cavity. The heart is now suspended in the cavity by blood vessels at its cranial and caudal poles (Fig. 13.7).
During these events, the myocardium thickens and secretes a thick layer of extracellular matrix, rich in hyaluronic acid, which separates it from the endothelium (Figs. 13.5C). In addition, mesothelial cells on the surface of the septum transversum form the proepicardium near the sinus venous at its caudal end and migrate over the heart to form most of the epicardium. The remainder of the epicardium is derived from mesothelial cells originating in the outflow tract region. Thus, the heart tube consists of three layers: (1) the endocardium, forming the internal endothelial lining of the heart; (2) the myocardium, forming the muscular wall; and (3) the epicardium or visceral pericardium, covering the outside of the tube. This outer layer is responsible for formation of the coronary arteries, including their endothelial lining and smooth muscle.
Chapter
13
System
165

Figure 13.6 Frontal view of an embryo showing the heart in the pericardial cavity and the developing gut tube with the anterior and posterior intestinal portals. The original paired tubes of the heart primordial have fused into a single tube except at their caudal ends, which remain separate. These caudal ends of the heart tube are embedded in the septum transversum, while the outflow tract leads to the aortic sac and aortic arches.
The heart tube continues to elongate as cells are added from the SHF to its cranial end (Fig. 13.3). This lengthening process is essential for normal formation of part of the right ventricle and the outflow tract region (conus cordis and truncus arteriosus that form part of the aorta and pulmonary artery), and for the looping process. If this lengthening is inhibited, then a variety of outflow tract defects occur, including DORV (both the aorta and pulmonary artery arise from the right ventricle), VSDs, tetralogy of Fallot (see Fig. 13.31), pulmonary atresia (see Fig. 13.33B), and pulmonary stenosis. The SHF is regulated by neural crest cells that control concentrations of FGFs in the area and pass nearby the SHF in the pharyngeal arches as they migrate from the hindbrain to septate the outflow tract (compare Fig. 13.3 with Fig. 13.27). As the outflow tract lengthens, the cardiac tube begins to bend on day 23.The cephalic portion of the tube bends ventrally, caudally, and to the right (Fig. 13.8); and the atrial (caudal) portion shifts dorsocranially and to the left (Figs. 13.8 and 13.9A). This bending, which may be due to cell shape changes, creates the cardiac loop. It is complete by day 28. While the cardiac loop is forming, local expansions become visible throughout the length of the tube. The
Primitive pericardial
cavity
Septum transversum
}
Anterior intestinal
portal

Figure 13.8 Formation of the cardiac loop. A. 22 days. B. 23 days. C. 24 days. D. Frontal view of the heart tube undergoing looping in the pericardial cavity. The primitive ventricle is moving ventrally and to the right, while the atrial region is moving dorsally and to the left (arrows).
atrial portion, initially a paired structure outside the pericardial cavity, forms a common atrium and is incorporated into the pericardial cavity (Fig. 13.8). The atrioventricular junction remains narrow and forms the atrioventricular canal, which connects the common atrium and the early embryonic ventricle (Fig. 13.10). The bulbus cordis is narrow except for its proximal third. This portion will form the trabeculated part of the right ventricle (Figs. 13.8 and 13.10). The midportion, the conus cordis, will form the outflow tracts of both ventricles. The distal part of the bulbus, the truncus arteriosus, will form the roots and proximal portion of the aorta and pulmonary artery (Fig. 13.10). The junction between the ventricle and the bulbus cordis, externally indicated by the bulboventricular sulcus (Fig. 13.8C), remains narrow. It is called the primary interventricular foramen (Fig. 13.10). Thus, the cardiac tube is organized by regions along its craniocaudal axis from the conotruncus to the right ventricle to the left ventricle to the atrial region, respectively (Fig. 13.84—C). Evidence suggests that organization of these segments is regulated by homeobox genes in a manner similar to that for the craniocaudal axis of the embryo (see Chapter 6, p. 81). At the end of loop formation, the smoothwalled heart tube begins to form primitive trabeculae in two sharply defined areas just proximal and distal to the primary interventricular foramen (Fig. 13.10). The bulbus temporarily remains smooth walled. The primitive ventricle, which is now trabeculated, is called the primitive left

Figure 13.9 Heart of a 5-mm embryo (28 days). A. Viewed from the left. B. Frontal view. The bulbus cordis is divided into the truncus arteriosus, conus cordis, and trabeculated part of the right ventricle. Broken line, pericardium.
ventricle. Likewise, the trabeculated proximal third of the bulbus cordis is called the primitive right ventricle (Fig. 13.10). The conotruncal portion of the heart tube, initially on the right side of the pericardial cavity, shifts gradually to a more medial position. This change in position is the result of formation of two transverse dilations of the atrium, bulging on each side of the bulbus cordis (Figs. 13.9B, and 13.10).

Dextrocardia is a condition where the heart lies on the right side of the thorax instead of the left and it occurs when the heart loops to the left instead of the right. The defect may be induced during gastrulation, when laterality is established,

Signals from anterior (cranial) endoderm induce a heart-forming region in overlying splanchnic mesoderm by inducing the transcription factor NKX2.5.The signals require secretion of BMPs 2 and 4 secreted by the endoderm and lateral plate mesoderm. Concomitantly, the activity of WNT proteins (3a and 8), secreted by the neural tube, must be blocked because they normally inhibit heart development. Inhibitors (crescent and cerberus) of the WNT proteins are produced by endoderm cells immediately adjacent to heartforming mesoderm in the anterior half of the embryo. The combination of bone morphogenetic protein (BMP) activity and WNT inhibition by crescent and cerberus causes expression of NKX2.5, the master gene for heart development (Figs. 13.1 and 13.11). BMP expression also upregulates expression of FGF8 that is important for the expression of cardiac-specific proteins.
<a or slightly later when cardiac looping occurs. Dextrocardia occurs with situs inversus, a complete reversal of asymmetry in all organs, or may be associated with laterality sequences in which only some organ positions are reversed (see Chapter 5).
sinus venosus and atria. Following this initial exposure to RA, these structures express the gene for retinaldehyde dehydrogenase, which allows them to make their own RA and commits them to becoming caudal cardiac structures. Lower concentrations of RA in more anterior cardiac regions (ventricles and outflow tract) contribute to specification of these structures. The importance of RA in cardiac signaling explains why the compound can produce a variety of cardiac defects.
NKX2.5 contains a homeodomain and is a homologue of the gene tinman that regulates heart development in Drosophila. TBX5 is another transcription factor that contains a DNA-binding motif known as the T-box. Expressed later than NKX2. 5, it plays an important role in septation.
Once the cardiac tube is formed, the venous portion is specified by retinoic acid (RA) produced by mesoderm adjacent to the presumptive

Cardiac looping is dependent upon several factors, including the laterality pathway and expression of the transcription factor PITX2 in lateral plate mesoderm on the left side. PITX2 may play a role in the deposition and function of extracellular matrix molecules that control looping. In addition, NKX2.5 upregulates expression
Figure 13.11 Heart induction. BMPs secreted by the endoderm and lateral plate mesoderm in combination with inhibition of WNT expression by crescent in the anterior half of the embryo induce expression of NKX2.5 in the heart-forming region of the lateral plate mesoderm (splanchnic layer). NKX2.5 is then responsible for heart induction.
of HAND1 and HAND2, transcription factors that are expressed in the primitive heart tube and that later become restricted to the future left and right ventricles, respectively. Downstream effectors of these genes participate in the looping phenomenon. HAND 1 and HAND2, under the regulation of NKX2.5, also contribute to expansion and differentiation of the ventricles.

In the middle of the fourth week, the sinus venosus receives venous blood from the right and left sinus horns (Fig. 13.12A). Each horn receives blood from three important veins: (1) the vitelline or the omphalomesenteric vein, (2) the umbilical vein, and (3) the common cardinal vein. At first, communication between the sinus and the atrium is wide. Soon, however, the entrance of the sinus shifts to the right (Fig. 13.12B). This shift is caused primarily by left-to-right shunts of blood, which occur in the venous system during the fourth and fifth weeks of development. With obliteration of the right umbilical vein and the left vitelline vein during the fifth week, the left sinus horn rapidly loses its importance

(Fig. 13.12B). When the left common cardinal vein is obliterated at 10 weeks, all that remains of the left sinus horn is the oblique vein of the left atrium and the coronary sinus (Fig. 13.13). As a result of left-to-right shunts of blood, the right sinus horn and veins enlarge greatly. The right horn, which now forms the only communication between the original sinus venosus and the atrium, is incorporated into the right atrium to form the smooth-walled part of the right atrium (Fig. 13.14). Its entrance, the sinuatrial orifice, is flanked on each side by a valvular fold, the right and left venous valves (Fig. 13.14). Dorsocranially, the valves fuse, forming a ridge known as the septum spurium (Fig. 13.14). Initially the valves are large, but when the right sinus horn is incorporated into the wall of the atrium, the left venous valve and the septum spurium fuse with the developing atrial septum (Fig. 13.14B).The superior portion of the right venous valve disappears entirely. The inferior portion develops into two parts: (1) the valve of the inferior vena cava and (2) the valve of the coronary sinus (Fig. 13.14B). The crista terminalis forms the dividing line between the original trabeculated part of the right atrium and the smooth-walled part (sinus venarum), which originates from the right sinus horn (Fig. 13.14).
Figure 13.12 Dorsal view of two stages in the development of the sinus venosus at approximately 24 days. A and 35 days. B. Broken line, the entrance of the sinus venosus into the atrial cavity. Each drawing is accompanied by a scheme to show in transverse section the great veins and their relation to the atrial cavity. ACV, anterior cardinal vein; PCV, posterior cardinal vein; UV, umbilical vein; VIT V, vitelline vein; CCV, common cardinal vein. (See also Fig. | 3.43.)
Figure 13.13 Final stage in development of the sinus venosus and great veins.

Figure 13.14 Ventral view of coronal sections through the heart at the level of the atrioventricular canal to show development of the venous valves. A. 5 weeks. B. Fetal stage. The sinus venarum (blue) is smooth walled; it derives from the right sinus horn. Arrows, blood flow.

The major septa of the heart are formed between the 27th and 37th days of development, when the embryo grows in length from 5 mm to approximately 16 to 17 mm. One method by which a septum may be formed involves two actively growing masses of tissue that approach each other until they fuse, dividing the lumen into two separate canals (Fig. 13.15.4,B). Such a septum may also be formed by active growth of a single tissue mass that continues to expand until it reaches the opposite side of the lumen (Fig. 13.15C). Formation of such tissue masses depends on synthesis and deposition of extracellular matrices and cell proliferation. The masses, known as endocardial cushions, develop in the atrioventricular and conotruncal regions. In these locations, they assist in formation of the atrial and ventricular

(membranous portion) septa, the atrioventricular canals and valves, (Fig. 13.16) and the aortic and pulmonary channels (See Fig. 13.19). Because of their key location, abnormalities in endocardial cushion formation may cause cardiac malformations, including atrial and ventricular septal defects (VSDs) and defects involving the great vessels (i.e., transposition of the great vessels, common truncus arteriosus, and of
The other manner in which a septum is formed does not involve endocardial cushions. If, for example, a narrow strip of tissue in the wall of the atrium or ventricle should fail to grow while areas on each side of it expand rapidly, a narrow ridge forms between the two expanding portions (Fig. 13.15D,E). When growth of the expanding portions continues on either side of the narrow portion, the two walls approach each other and eventually merge, forming a septum (Fig. 13.15F).
Figure 13.15 A,B. Septum formation by two actively growing ridges that approach each other until they fuse. C. Septum formed by a single actively growing cell mass. D-F. Septum formation by merging two expanding portions of the wall of the heart. Such a septum never completely separates two cavities.
Such a septum never completely divides the original lumen but leaves a narrow communicating canal between the two expanded sections. It is usually closed secondarily by tissue contributed by neighboring proliferating tissues. Such a septum partially divides the atria and ventricles.

At the end of the fourth week, a sickle-shaped crest grows from the roof of the common atrium into the lumen. This crest is the first portion of the septum primum (Figs. 13.144 and 13.16A,B). The two limbs of this septum extend toward the endocardial cushions in the atrioventricular canal. The opening between the lower rim of the septum primum and the endocardial cushions is the ostium primum (Fig. 13.16A,B). With further development, extensions of the superior and inferior endocardial cushions grow along the edge of the septum primum, closing the ostium primum (Fig. 13.16C,D). Before closure is complete, however, cell death produces perforations in the upper portion of the septum primum. Coalescence of these perforations forms the ostium secundum, ensuring free blood flow from the right to the left primitive atrium (Fig. 13.16B,D).
new crescent-shaped fold appears. This new fold, the septum secundum (Fig. 13.16C,D), never forms a complete partition in the atrial cavity (Fig. 13.165 G). Its anterior limb extends downward to the septum in the atrioventricular canal. When the left venous valve and the septum spurium fuse with the right side of the septum secundum, the free concave edge of the septum secundum begins to overlap the ostium secundum (Fig. 13.16E,F). The opening left by the septum secundum is called the oval foramen (foramen ovale). When the upper part of the septum primum gradually disappears, the remaining part becomes the valve of the oval foramen. The passage between the two atrial cavities consists of an obliquely elongated cleft (Fig. 13.16E-G) through which blood from the right atrium flows to the left side (arrows in Figs. 13.14B and 13.16E).
When the lumen of the right atrium expands as a result of incorporation of the sinus horn, a

After birth, when lung circulation begins and pressure in the left atrium increases, the valve of the oval foramen is pressed against the septum secundum, obliterating the oval foramen and separating the right and left atria. In about 20% of cases, fusion of the septum primum and septum secundum is incomplete, and a narrow oblique cleft remains between the two atria. This condition is called probe patency of the oval foramen; it does not allow intracardiac shunting of blood.

While the primitive right atrium enlarges by incorporation of the right sinus horn, the primitive left atrium is likewise expanding. Initially, a single embryonic pulmonary vein develops as an outgrowth of the posterior left atrial wall, just to the left of the septum primum (Fig. 13.17A). This vein gains connection with veins of the developing lung buds. During further development, the pulmonary vein and its branches are incorporated into the left atrium, forming the large smooth-walled part of the adult atrium. Although initially one vein enters the left atrium, ultimately, four pulmonary veins enter (Fig. 13.17B) as the branches are incorporated into the expanding atrial wall.

In the fully developed heart, the original embryonic left atrium is represented by little more than the trabeculated atrial appendage, while the smooth-walled part originates from the pulmonary veins (Fig. 13.17). On the right side, the original embryonic right atrium becomes the trabeculated right atrial appendage containing the pectinate muscles, and the smooth-walled sinus venarum originates from the right horn of the sinus venosus.
At the end of the fourth week, two mesenchymal cushions, the atrioventricular endocardial cushions, appear at the anterior and posterior borders of the atrioventricular canal (Figs. 13.18 and 13.19). Initially, the atrioventricular canal gives access only to the primitive left ventricle and is separated from the bulbus cordis by the bulbo (cono) ventricular flange (Fig. 13.10). Near the end of the fifth week, however, the posterior extremity of the flange terminates almost midway along the base of the superior endocardial cushion and is much less prominent than before (Fig. 13.19). Since the atrioventricular canal enlarges to the right, blood passing through the atrioventricular orifice now has direct access to the primitive left as well as the primitive right ventricle. In addition to the anterior and posterior endocardial cushions, the two lateral atrioventricular cushions appear on the right and left borders of the canal (Figs. 13.18 and 13.19). The anterior and posterior cushions, in the meantime, project further into the lumen and fuse, resulting in a complete division of the canal into right and left atrioventricular orifices
Figure 13.17 Coronal sections through the heart to show development of the smooth-walled portions of the right and left atria. Both the wall of the right sinus horn (blue) and the pulmonary veins (red) are incorporated into the heart to form the
smooth-walled parts
of the
atria.
septum
|
Interventricular

Figure 13.19 Frontal section through the heart of a day-35 embryo.At this stage of development, blood from the atrial cavity enters the primitive left ventricle as well as the primitive right ventricle. Note development of the cushions in the atrioventricular canal. Cushions in the truncus and conus are also visible. Ring, primitive interventricular foramen. Arrows, blood flow.

cords (Fig. 13.20B). Finally, muscular tissue in the cords degenerates and is replaced by dense connective tissue. The valves then consist of connective tissue covered by endocardium.They are connected to thick trabeculae in the wall of the ventricle, the papillary muscles, by means of chordae tendineae (Fig. 13.20C). In this manner, two valve leaflets, constituting the bicuspid (or mitral) valve, form in the left atrioventricular canal, and three, constituting the tricuspid valve, form on the right side.
Dense

Heart and vascular abnormalities make up the largest category of human birth defects and are present in 1% of live born infants. The incidence among stillborns is 10 times as high. It is estimated that 12% of babies with heart defects have a chromosomal abnormality and, conversely, that 33% of babies with a chromosomal abnormality have a heart defect. In some conditions, such as trisomy 18, the incidence of heart defects is 100%. Approximately 2% of heart defects are due to environmental agents, but most are caused by a complex interplay between genetic and environmental influences (multifactorial causes). Classic examples of cardiovascular teratogens include rubella virus and thalidomide. Others include RA (Accutane), alcohol, and many other compounds. Maternal diseases, such as insulin-dependent diabetes, have also been linked to cardiac defects.
Targets for genetic or teratogen-induced heart defects include heart progenitor cells from the PHF and SHF, neural crest cells, endocardial cushions, and other cell types important for heart development. The fact that the same malformation can result from disrupting different targets (e.g., transposition of the great arteries can result from disruption of the SHF or neural crest cells) means that heart defects are heterogeneous in origin and difficult to classify epidemiologically.
Genes regulating cardiac development are being identified and mapped, and mutations that result in heart defects are being discovered. For example, mutations in the heart-specifying gene NKX2.5, on chromosome 5q35, can produce ASDs (secundum type), tetralogy of Fallot, and atrioventricular conduction delays in an autosomal dominant fashion. Mutations in the TBX5 gene result in Holt-Oram syndrome, characterized by preaxial (radial) limb abnormalities and ASDs. Defects in the muscular portion of the interventricular septum may also occur. Holt-Oram syndrome is one of a group of heart-hand syndromes illustrating that the same genes may participate in multiple developmental processes. For example, TBX5 regulates forelimb development and plays a role in septation of the heart. Holt-Oram syndrome is inherited as ok
an autosomal dominant trait with a frequency of 1/100,000 live births.

Mutations in a number of genes regulating production of sarcomere proteins cause hypertrophic cardiomyopathy that may result in sudden death in athletes and the general population. The disease is inherited as an autosomal dominant, and most mutations (45%) target the B-myosin heavy-chain gene (14qI1.2). The result is cardiac hypertrophy due to disruption in the organization of cardiac muscle cells (myocardial disarray), which may adversely affect cardiac output and/or conduction.
Ventricular inversion is a defect in which the morphologic left ventricle is on the right and connects to the right atrium through a mitral valve. The morphologic right ventricle is on the left side and connects to the left atrium through the tricuspid valve. The defect is sometimes called L-transposition of the great arteries because the pulmonary artery exits the morphologic left ventricle and the aorta exits the morphologic right ventricle. However, the arteries are in their normal positions, but the ventricles are reversed. The abnormality arises during the establishment of laterality and specification of the left and right sides of the heart by the laterality pathway.
Atrial septal defect (ASD) is a congenital heart abnormality with an incidence of 6.4/10,000 births and with a 2:1 prevalence in female to male infants. One of the most significant defects is the ostium secundum defect, characterized by a large opening between the left and right atria. It may be caused by excessive cell death and resorption of the septum primum (Fig. 13.21B,C) or by inadequate development of the septum secundum (Fig. 13.21D,E). Depending on the size of the opening, considerable intracardiac shunting may occur from left to right.
The most serious abnormality in this group is complete absence of the atrial septum (Fig. 13.21F). This condition, known as common atrium or cor triloculare biventriculare, is always associated with serious defects elsewhere in the heart.
Occasionally, the oval foramen closes during prenatal life. This abnormality, premature closure

Figure 13.21 A. Normal atrial septum formation. B,C. Ostium secundum defect caused by excessive resorption of the septum primum. D,E. Similar defect caused by failure of development of the septum secundum. F. Common atrium, or cor triloculare biventriculare, resulting from complete failure of the septum primum and septum secundum to form.
of the oval foramen, leads to massive hypertrophy of the right atrium and ventricle and underdevelopment of the left side of the heart. Death usually occurs shortly after birth.
Endocardial cushions of the atrioventricular canal not only divide this canal into a right and left orifice, but also participate in formation of the membranous portion of the interventricular septum and in closure of the ostium primum (Fig 13.16).This region has the appearance of a cross, with the atrial and ventricular septa forming the post, and the atrioventricular cushions the crossbar (Fig. 13.16E). The integrity of this cross is an important sign in ultrasound scans of the heart. Whenever the atrioventricular cushions fail to fuse, the result is a persistent atrioventricular canal, combined with a defect in the cardiac septum (Fig. |3.22A).This septal defect has an atrial and a ventricular component, separated by abnormal valve leaflets in the single atrioventricular orifice (Fig. 13.22B,C).
Occasionally, endocardial cushions in the atrioventricular canal partially fuse. The result is a defect in the atrial septum, but the interventricular septum is closed (Fig. 13.22D,E). This defect, the ostium primum defect, is usually combined with a cleft in the anterior leaflet of the tricuspid valve (Fig. 13.220).

Tricuspid atresia, which involves obliteration of the right atrioventricular orifice (Fig. 13.23), is characterized by the absence or fusion of the tricuspid valves. Tricuspid atresia is always associated with (1) patency of the oval foramen, (2) VSD, (3) underdevelopment of the right ventricle, and (4) hypertrophy of the left ventricle.
Ebstein anomaly is a condition where the tricuspid valve is displaced toward the apex of the right ventricle. The valve leaflets are abnormally positioned, and the anterior one is usually enlarged. As a result, there is hypertrophy of the right atrium with a small right ventricle.
Figure 13.22 A. Persistent common atrioventricular canal. This abnormality is always accompanied by a septum defect in the atrial as well as in the ventricular portion of the cardiac partitions. B. Valves in the atrioventricular orifices under normal conditions. C. Split valves in a persistent atrioventricular canal. D,E. Ostium primum defect caused by incomplete fusion of the atrioventricular endocardial cushions.
Figure 13.23 A. Normal heart. B. Tricuspid atresia. Note the small right ventricle and the large left ventricle.
Septum Formation in the Truncus Arteriosus and Conus Cordis
During the fifth week, pairs of opposing ridges appear in the truncus. These ridges, the truncus swellings, or cushions, lie on the right superior wall (right superior truncus swelling) and on the left inferior wall (left inferior truncus swelling) (Fig. 13.19). The right superior truncus swelling grows distally and to the left, and the left inferior truncus swelling grows distally and to the right. Hence, while growing toward the aortic sac, the swellings twist around each other, foreshadowing the spiral course of the future septum (Fig. 13.24). After complete fusion, the ridges form the aorticopulmonary septum, dividing the truncus into an aortic and a pulmonary channel.

dorsal and left ventral walls of the conus cordis (Figs. 13.19 and 13.24).The conus swellings grow toward each other and distally to unite with the truncus septum. When the two conus swellings have fused, the septum divides the conus into an anterolateral portion (the outflow tract of the right ventricle) (Fig. 13.25) and a posteromedial portion (the outflow tract of the left ventricle) (Fig. 13.26).
When the truncus swellings appear, similar swellings (cushions) develop along the right
Neural crest cells, originating in the edges of the neural folds in the hindbrain region, migrate through pharyngeal arches 3, 4, and 6 to the outflow region of the heart, which they invade (Fig. 13.27). In this location, they contribute to endocardial cushion formation in both the conus cordis and truncus arteriosus. These neural crest cells also control cell production and lengthening of the outflow tract region by the SHE Therefore, outflow tract defects may
c
Figure 13.24 Development of the conotruncal ridges (cushions) and closure of the interventricular foramen. Proliferations of the right and left conus cushions, combined with proliferation of the anterior endocardial cushion, close the interventricular foramen and form the membranous portion of the interventricular septum. A. 6 weeks (12 mm). B. Beginning of the seventh week (14.5 mm). C. End of the seventh week (20 mm).
Figure 13.25 Frontal section through the heart of a 7-week embryo. Note the conus septum and position of the pulmonary valves.
Figure 13.27 Drawing showing the origin of neural crest cells in the hindbrain and their migration through pharyngeal arches 3,4, and 6 to the outflow tract of the heart. In this location, they contribute to septation of the conus cordis and truncus arteriosus.

occur by several mechanisms: direct insults to the SHF; insults to neural crest cells that disrupt their formation of the conotruncal septum; insults to neural crest cells that disrupt their signals to the SHE which they regulate. Heart defects caused by these mechanisms include tetralogy of Fallot (Fig. 13.31), pulmonary stenoses, persistent (common) truncus arteriosus (Fig. 13.32), and transposition of the great vessels (Fig. 13.33). Since neural crest cells also contribute to craniofacial development, it is not uncommon to see facial and cardiac abnormalities in the same individual (see Chapter17, p. 269-270).

By the end of the fourth week, the two primitive ventricles begin to expand. This is accomplished by continuous growth of the myocardium on the outside and continuous diverticulation and trabecula formation on the inside (Figs. 13.19 and 13.26).
The medial walls of the expanding ventricles become apposed and gradually merge, forming the muscular interventricular septum (Fig. 13.26). Sometimes, the two walls do not merge completely, and a more or less deep apical cleft between the two ventricles appears. The space between the free rim of the muscular ventricular septum and the fused endocardial cushions permits communication between the two ventricles.
The interventricular foramen, above the muscular portion of the interventricular septum, shrinks on completion of the conus septum (Fig. 13.24). During further development, outgrowth of tissue from the anterior (inferior) endocardial cushion along the top of the muscular interventricular septum closes the foramen (Fig. 13.16E,F). This tissue fuses with the abutting parts of the conus septum. Complete closure of the interventricular foramen forms the membranous part of the interventricular septum (Fig. 13.16F).

When partitioning of the truncus is almost complete, primordia of the semilunar valves become visible as small tubercles found on the main truncus swellings. One of each pair is assigned to the pulmonary and aortic channels, respectively (Fig. 13.28). A third tubercle appears in both channels opposite the fused truncus swellings. Gradually, the tubercles hollow out at their upper surface, forming the semilunar valves (Fig. 13.29). Recent evidence shows that neural crest cells contribute to formation of these valves.
Transverse sections the truncus arteriosus at the level of the semilunar valves at weeks 5.A.
hMoW
Figure 13.29 Longitudinal sections through the semilunar valves at weeks 6. A. 7.B. and 9. C. of development. The upper surface is hollowed (arrows) to form the valves.
OW

Ventricular septal defects (VSDs) involving the membranous or muscular portion of the septum (Fig. 13.30) are the most common congenital cardiac malformation, occurring as an isolated condition in 12/10,000 births. Most (80%) occur in the muscular region of the septum and resolve as the child grows. Membranous VSDs usually represent a more serious defect and are often associated with abnormalities in partitioning of the conotruncal region. Depending on the size of the opening, blood carried by the pulmonary artery may be 1.2 to |.7 times as abundant as that carried by the aorta.
Tetralogy of Fallot, the most frequently occurring abnormality of the conotruncal region (Fig. 13.31), is due to an unequal division of the conus resulting from anterior displacement of the conotruncal septum. Displacement of the septum produces four cardiovascular alterations: (1) a narrow right ventricular outflow region, pulmonary infundibular stenosis; (2) a large defect of the interventricular septum; (3) an overriding aorta that arises directly above the septal defect; and (4) hypertrophy of the right ventricular wall because of higher pressure on the right side. Tetralogy of Fallot, which is not fatal, occurs in 9.6/10,000 births.
Persistent (common) truncus arteriosus results when the conotruncal ridges fail to form such that no division of the outflow tract occurs. (Fig. 13.32). In such a case, which occurs in 0.8/10,000 births, the pulmonary artery arises some distance above the origin of the undivided truncus. Since the ridges also participate in formation of the interventricular septum, the persistent truncus is always accompanied by a defective interventricular septum. The undivided truncus thus overrides both ventricles and receives blood from both sides.

Transposition of the great vessels occurs when the conotruncal septum fails to follow its normal spiral course and runs straight down (Fig. 13.33A). As a consequence, the aorta originates from the right ventricle, and the pulmonary artery originates from the left ventricle. This condition, which occurs in 4.8/10,000 births, sometimes is
Figure 13.30 A. Normal heart. B. Isolated defect in the membranous portion of the interventricular septum. Blood from the left ventricle flows to the right through the interventricular foramen (arrows).
Figure 13.31 Tetralogy of Fallot. A. Surface view. B. The four components of the defect: pulmonary stenosis, overriding aorta, interventricular septal defect, and hypertrophy of the right ventricle.
associated with a defect in the membranous part of the interventricular septum. It is usually accompanied by an open ductus arteriosus. Since the SHF and neural crest cells contribute to the formation and septation of the outflow tract, respectively, insults to these cells contribute to cardiac defects involving the outflow tract.
hypoplasia, parathyroid dysfunction, and cardiac abnormalities involving the outflow tract, such as persistent truncus arteriosus and tetralogy of Fallot. Craniofacial malformations are often associated with heart defects because neural crest cells play important roles in the development of both the face and heart.
DiGeorge sequence is an example of the 22q!1 deletion syndrome (see Chapter 1I7, p. 269) characterized by a pattern of malformations that have their origin in abnormal neural crest development. These children have facial defects, thymic
Valvular stenosis of the pulmonary artery or aorta occurs when the semilunar valves are fused for a variable distance. The incidence of the abnormality is similar for both regions, being approximately 3 to 4/10,000 births. In the case of a
Figure 13.32 Persistent truncus arteriosus. The pulmonary artery originates from a common truncus. A. The septum in the truncus and conus has failed to form. B. This abnormality is always accompanied by an interventricular septal defect.

Figure 13.33 A. Transposition of the great vessels. B. Pulmonary valvular atresia with a normal aortic root. The only access route to the lungs is by way of a patent ductus arteriosus.
valvular stenosis of the pulmonary artery, the trunk of the pulmonary artery is narrow or even atretic (Fig. 12.33B). The patent oval foramen then forms the only outlet for blood from the right side of the heart. The ductus arteriosus, always patent, is the only access route to the pulmonary circulation.
When fusion of the semilunar aortic valves is complete—aortic valvular atresia (Fig. |3.34B)— the aorta, left ventricle, and left atrium are markedly underdeveloped. The abnormality is usually accompanied by an open ductus arteriosus, which delivers blood into the aorta.
In aortic valvular stenosis (Fig. |3.34A), fusion of the thickened valves may be so complete that only a pinhole opening remains. The size of the aorta itself is usually normal.
Ectopia cordis is a rare anomaly in which the heart lies on the surface of the chest. It is caused by failure of the embryo to close the ventral body wall (see Chapter 7, p. 87).
Figure 13.34 A. Aortic valvular stenosis. B. Aortic valvular atresia. Arrow in the arch of the aorta indicates direction of blood flow. The coronary arteries are supplied by this retroflux. Note the small left ventricle and the large right ventricle.

Initially, the pacemaker for the heart lies in the caudal part of the left cardiac tube. Later, the sinus venosus assumes this function, and as the sinus is incorporated into the right atrium, pacemaker tissue lies near the opening of the superior vena cava. Thus, the sinuatrial node is formed.
The atrioventricular node and bundle (bundle of His) are derived from two sources: (1) cells in the left wall of the sinus venosus and (2) cells from the atrioventricular canal. Once the sinus venosus is incorporated into the right atrium, these cells lie in their final position at the base of the interatrial septum.

Blood vessel development occurs by two mechanisms: (1) vasculogenesis in which vessels arise by coalescence of angioblasts and (2) angiogenesis whereby vessels sprout from existing vessels. The major vessels, including the dorsal aorta and cardinal veins, are formed by vasculogenesis. The remainder of the vascular system then forms by angiogenesis. The entire system is patterned by guidance cues involving vascular endothelial growth factor (VEGF) and other growth factors (see Chapter 6, p. 75).

When pharyngeal arches form during the fourth and fifth weeks of development, each arch receives its own cranial nerve and its own artery (see Chapter 17). These arteries, the aortic arches, arise from the aortic sac, the most distal part of the truncus arteriosus (Figs. 13.10 and 13.35). The aortic arches are embedded in mesenchyme of the pharyngeal arches and terminate in the right and left dorsal aortae. (In the region of the arches, the dorsal aortae remain paired, but caudal to this region, they fuse to form a single vessel.) The pharyngeal arches and their vessels appear in a cranial-to-caudal sequence, so that they are not all present simultaneously. The aortic sac contributes a branch to each new arch as it forms, giving rise to a total of five pairs of arteries. (The fifth arch either never forms or forms incompletely and then regresses. Consequently, the five arches are numbered J, II, III, IV, and VI [Figs. 13.36 and 13.37A].) During further development, this arterial pattern becomes modified, and some vessels regress completely.
Division of the truncus arteriosus by the aorticopulmonary septum divides the outflow channel of the heart into the ventral aorta and the pulmonary trunk. The aortic sac then forms right and left horns, which subsequently give rise to the brachiocephalic artery and the proximal segment of the aortic arch, respectively (Fig. 13.37B,C). By day 27, of the first aortic arch has most disappeared (Fig. 13.36), although a small portion persists to form the maxillary artery. Similarly, the second aortic arch soon disappears. The remaining portions of this arch are the hyoid and stapedial arteries. The third arch is large; the fourth and sixth arches are in the process of formation. Even though the sixth arch is not completed, the primitive pulmonary artery is already present as a major branch (Fig. 13.36).

Figure 13.36 A. Aortic arches at the end of the fourth week. The first arch is obliterated before the sixth is formed. B. Aortic arch system at the beginning of the sixth week. Note the aorticopulmonary septum and the large pulmonary arteries.
In the 29-day embryo, the first and second aortic arches have disappeared (Fig. 13.36B).The third, fourth, and sixth arches are large. The conotruncal region has divided so that the sixth arches are now continuous with the pulmonary trunk.
loses its connection with the dorsal aorta and disappears. On the left, the distal part persists during intrauterine life as the ductus arteriosus. Table 13.1 summarizes the changes and derivatives of the aortic arch system.
With further development, the aortic arch system loses its original symmetrical form, as shown in Figure 13.37A and establishes the definitive pattern illustrated in Figure 13.37B,C. This representation may clarify the transformation from the embryonic to the adult arterial system. The following changes occur:
The third aortic arch forms the common carotid artery and the first part of the internal carotid artery. The remainder of the internal carotid is formed by the cranial portion of the dorsal aorta. The external carotid artery is a sprout of the third aortic arch.
The fourth aortic arch persists on both sides, but its ultimate fate is different on the right and left sides. On the left, it forms part of the arch of the aorta, between the left common carotid and the left subclavian arteries. On the right, it forms the most proximal segment of the right subclavian artery, the distal part of which is formed by a portion of the right dorsal aorta and the seventh intersegmental artery (Fig. 13.37B).
The fifth
aortic arch
either never
forms or
forms incompletely
and then
regresses.
The sixth aortic arch, also known as the pulmonary arch, gives off an important branch that grows toward the developing lung bud (Fig. 13.37B). On the right side, the proximal part becomes the proximal segment of the right pulmonary artery. The distal portion of this arch

A number of other changes occur along with alterations in the aortic arch system: (1) the dorsal aorta between the entrance of the third and fourth arches, known as the carotid duct, is obliterated (Fig. 13.38); (2) the right dorsal aorta disappears between the origin of the seventh intersegmental artery and the junction with the left dorsal aorta (Fig. 13.38); (3) cephalic folding, growth of the forebrain, and elongation of the neck push the heart into the thoracic cavity. Hence, the carotid and brachiocephalic arteries elongate considerably (Fig. 13.37C).As a further result of this caudal shift, the left subclavian artery, distally fixed in the arm bud, shifts its point of origin from the aorta at the level of the seventh intersegmental artery (Fig. 13.37B) to an increasingly higher point until it comes close to the origin of the left common carotid artery (Fig. 13.37C); (4) as a result of the caudal shift of the heart and the disappearance of various portions of the aortic arches, the course of the recurrent laryngeal nerves becomes different on the right and left sides. Initially, these nerves, branches of the vagus, supply the sixth pharyngeal arches. When the heart descends, they hook around the sixth aortic arches and ascend again to the larynx, which accounts for their recurrent course. On the right, when the distal part of the sixth aortic arch and the fifth aortic arch disappear, the recurrent laryngeal nerve moves up and hooks around the right subclavian

Figure 13.37 A. Aortic arches and dorsal aortae before transformation into the definitive vascular pattern. B. Aortic arches and dorsal aortae after the transformation. Broken lines, obliterated components. Note the patent ductus arteriosus and position of the seventh intersegmental artery on the left. C. The great arteries in the adult. Compare the distance between the place of origin of the left common carotid artery and the left subclavian in B and C.After disappearance of the distal part of the sixth aortic arch (the fifth arches never form completely), the right recurrent laryngeal nerve hooks around the right subclavian artery. On the left, the nerve remains in place and hooks around the ligamentum arteriosum.
TABLE 13.1, 1 = Derivatives of the Aortic Arches. Arch, 1 = Arterial Derivative. 1, 1 = Maxillary arteries. 2, 1 = Hyoid and stapedial arteries. 3, 1 = Common carotid and first part of the internal carotid arteries a. 4 Left side, 1 = Arch of the aorta from the left common carotid to the left subclavian arteries b. Right side, 1 = Right subclavian artery (proximal portion) c. 6 Left side, 1 = Left pulmonary artery and ductus arteriosus. Right side, 1 = Right pulmonary artery. a Remainder of the internal carotid arteries are derived from the dorsal aorta; the external carotid arteries sprout from the third aortic arch., 1 = a Remainder of the internal carotid arteries are derived from the dorsal aorta; the external carotid arteries sprout from the third aortic arch.. b The proximal portion of the aortic arch is derived from the left horn of the aortic sac; the right horn of this sac forms the brachiocephalic artery., 1 = b The proximal portion of the aortic arch is derived from the left horn of the aortic sac; the right horn of this sac forms the brachiocephalic artery.
Figure 13.38 Changes from the original aortic arch system.

artery. On the left, the nerve does not move up, since the distal part of the sixth aortic arch persists as the ductus arteriosus, which later forms the ligamentum arteriosum (Fig. 13.37).
Vitelline and Umbilical Arteries
After birth, the proximal portions of the umbilical arteries persist as the internal iliac and superior vesical arteries, and the distal parts are obliterated to form the medial umbilical ligaments.
The vitelline arteries, initially a number of paired vessels supplying the yolk sac (Fig. 13.35), gradually fuse and form the arteries in the dorsal mesentery of the gut. In the adult, they are represented by the celiac and superior mesenteric, arteries. The inferior mesenteric arteries are derived from the umbilical arteries. These 3 vessels supply derivatives of the foregut, midgut, and hindgut, respectively.
The umbilical arteries, initially paired ventral branches of the dorsal aorta, course to the placenta in close association with the allantois (Fig. 13.35). During the fourth week, however, each artery acquires a secondary connection with the dorsal branch of the aorta, the common iliac artery, and loses its earliest origin.

Coronary arteries are derived from two sources: (1) angioblasts formed from sprouts off the sinus venosus that are distributed over the heart surface by cell migration and (2) the epicardium itself. Some epicardial cells undergo an epithelial-to-mesenchymal transition induced by the underlying myocardium. The newly formed mesenchymal cells then contribute to endothelial and smooth muscle cells of the coronary arteries. Neural crest cells also contribute smooth muscle cells along the proximal segments of these arteries. Connection of the coronary arteries to the aorta occurs by ingrowth of arterial endothelial cells from the arteries into the aorta. By this mechanism, the coronary arteries "invade" the aorta.

Under normal conditions, the ductus arteriosus is functionally closed through contraction of its muscular wall shortly after birth to form the ligamentum arteriosum. Anatomical closure by means of intima proliferation takes | to 3 months. A patent ductus arteriosus, one of the most frequently occurring abnormalities of the great vessels (8/10,000 births), especially in premature infants, either may be an isolated abnormality or may accompany other heart defects (Figs. 13.31A and 13.33). In particular, defects that cause large differences between aortic and pulmonary pressures may cause increased blood flow through the ductus, preventing its normal closure.
circulation between the proximal and distal parts of the aorta is established by way of large intercostal and internal thoracic arteries. In this manner, the lower part of the body is supplied with blood. Classic clinical signs associated with this condition include hypertension in the right arm concomitant with lowered blood pressure in the legs.
In coarctation of the aorta (Fig. 13.39A,B), which occurs in 3.2/10,000 births, the aortic lumen below the origin of the left subclavian artery is significantly narrowed. Since the constriction may be above or below the entrance of the ductus arteriosus, two types (preductal and postductal) may be distinguished. The cause of aortic narrowing is primarily an abnormality in the media of the aorta, followed by intima proliferations. In the preductal type, the ductus arteriosus persists; whereas in the postductal type, which is more common, this channel is usually obliterated. In the latter case, collateral

Abnormal origin of the right subclavian artery (Fig. 13.40A,B) occurs when the artery is formed by the distal portion of the right dorsal aorta and the seventh intersegmental artery. The right fourth aortic arch and the proximal part of the right dorsal aorta are obliterated. With shortening of the aorta between the left common carotid and left subclavian arteries, the origin of the abnormal right subclavian artery finally settles just below that of the left subclavian artery. Since its stem is derived from the right dorsal aorta, it must cross the midline behind the esophagus to reach the right arm. This location does not usually cause problems with swallowing or breathing, since neither the esophagus nor the trachea is severely compressed.
With a double aortic arch, the right dorsal aorta persists between the origin of the seventh intersegmental artery and its junction with the left dorsal aorta (Fig. 13.41).A vascular ring surrounds the trachea and esophagus and commonly
Figure 13.39 Coarctation of the aorta. A. Preductal type. B. Postductal type. The caudal part of the body is supplied by large hypertrophied intercostal and internal thoracic arteries.
Figure 13.40 Abnormal origin of the right subclavian artery. A. Obliteration of the right fourth aortic arch and the proximal portion of the right dorsal aorta with persistence of the distal portion of the right dorsal aorta. B. The abnormal right subclavian artery crosses the midline behind the esophagus and may compress it.
compresses these structures, causing difficulties in breathing and swallowing.
In a right aortic arch, the left fourth arch and left dorsal aorta are obliterated and replaced by the corresponding vessels on the right side. Occasionally, when the ligamentum arteriosum lies on the left side and passes behind the esophagus, it causes complaints with swallowing.

An interrupted aortic arch is caused by obliteration of the fourth aortic arch on the left side (Fig. 13.42A,B). It is frequently combined with an abnormal origin of the right subclavian artery. The ductus arteriosus remains open, and the descending aorta and subclavian arteries are supplied with blood of low oxygen content. The aortic trunk supplies the two common carotid arteries.
Figure 13.41 Double aortic arch. A. Persistence of the distal portion of the right dorsal aorta. B. The double aortic arch forms a vascular ring around the trachea and esophagus.
Figure 13.42 A. Obliteration of the fourth aortic arch on the right and left and persistence of the distal portion of the right dorsal aorta. B. Case of interrupted aortic arch. The aorta supplies the head; the pulmonary artery, by way of the ductus arteriosus, supplies the rest of the body.

In the fifth week, three pairs of major veins can be distinguished: (1) the vitelline veins, or omphalomesenteric veins, carrying blood from the yolk sac to the sinus venosus; (2) the umbilical veins, originating in the chorionic villi and carrying oxygenated blood to the embryo; and (3) the cardinal veins, draining the body of the embryo proper (Fig. 13.43).
cords growing into the septum interrupt the course of the veins, and an extensive vascular network, the hepatic sinusoids, forms (Fig. 13.44).

Before entering the sinus venosus, the vitelline veins form a plexus around the duodenum and pass through the septum transversum. The liver
With reduction of the left sinus horn, blood from the left side of the liver is rechanneled toward the right, resulting in an enlargement of the right vitelline vein (right hepatocardiac channel). Ultimately, the right hepatocardiac channel forms the hepatocardiac portion of the inferior vena cava. The proximal part of the left vitelline vein disappears (Fig. 13.45, B). The anastomotic network around the duodenum develops into a single vessel, the portal vein (Fig. 13.45). The superior mesenteric vein, which
Figure 13.43 Main components of the venous and arterial systems in a 4-mm embryo (end of the fourth week).
Figure
13.44
the vitelline
and umbilical
veins during
the
A.
fourth and
B.
fifth weeks.
Note the
around the
duodenum, formation
of the
hepatic sinusoids,
and drains the primary intestinal loop, derives from the right vitelline vein. The distal portion of the left vitelline vein also disappears (Fig. 13.45.4,B).

plexus initiation
of left-to-right
shunts between
veins.
sinusoidal plexus of the liver. After birth, the left umbilical vein and ductus venosus are obliterated and form the ligamentum teres hepatis and ligamentum venosum, respectively.
Initially, the umbilical veins pass on each side of the liver, but some connect to the hepatic sinusoids (Fig. 13.444,B). The proximal part of both umbilical veins and the remainder of the right umbilical vein then disappear, so that the left vein is the only one to carry blood from the placenta to the liver (Fig. 13.45). With the increase of the placental circulation, a direct communication forms between the left umbilical vein and the right hepatocardiac channel, the ductus venosus (Fig. 13.45.4,B).This vessel bypasses the

Initially, the cardinal veins form the main venous drainage system of the embryo. This system consists of the anterior cardinal veins, which drain the cephalic part of the embryo, and the posterior cardinal veins, which drain the rest of the embryo. The anterior and posterior veins join before entering the sinus horn and form the short common cardinal veins. During the fourth week, the cardinal veins form a symmetrical system (Fig. 13.43).
Figure 13.45 Development of vitelline and umbilical veins in the A second and B third months. Note formation of the ductus venosus, portal vein, and hepatic portion of the inferior vena cava. The splenic and superior mesenteric veins enter
Figure 13.46 Development of the inferior vena cava, azygos vein, and superior vena cava. A. Seventh week. The anastomosis lies between the subcardinals, supracardinals, sacrocardinals, and anterior cardinals. B. The venous system at birth showing the three components of the inferior vena cava.
During the fifth to the seventh weeks, a number of additional veins are formed: (1) the subcardinal veins, which mainly drain the kidneys; (2) the sacrocardinal veins, which drain the lower extremities; and (3) the supracardinal veins, which drain the body wall by way of the intercostal veins, taking over the functions of the posterior cardinal veins (Fig. 13.46).
the fourth week of development and ultimately form the internal jugular veins (Fig 13.46). External jugular veins are derived from a plexus of venous vessels in the face and drain the face and side of the head to the subclavian veins.

Formation of the vena cava system is characterized by the appearance of anastomoses between left and right in such a manner that the blood from the left is channeled to the right side. The anastomosis between the anterior cardinal veins develops into the left brachiocephalic vein (Fig. 13.46A,B). Most of the blood from the left side of the head and the left upper extremity is then channeled to the right. The terminal portion of the left posterior cardinal vein entering into the left brachiocephalic vein is retained as a small vessel, the left superior intercostal vein (Fig. 13.46B). This vessel receives blood from the second and third intercostal spaces. The superior vena cava is formed by the right common cardinal vein and the proximal portion of the right anterior cardinal vein. The anterior cardinal veins provide the primary venous drainage of the head during
The anastomosis between the subcardinal veins forms the left renal vein. When this communication has been established, the left subcardinal vein disappears, and only its distal portion remains as the left gonadal vein. Hence, the right subcardinal vein becomes the main drainage channel and develops into the renal segment of the inferior vena cava (Fig. 13.46B).
The anastomosis between the sacrocardinal veins forms the left common iliac vein (Fig. 13.46B).The right sacrocardinal vein becomes the sacrocardinal segment of the inferior vena cava. When the renal segment of the inferior vena cava connects with the hepatic segment, which is derived from the right vitelline vein, the inferior vena cava, consisting of hepatic, renal, and sacrocardinal segments, is complete.
With obliteration of the major portion of the posterior cardinal veins, the supracardinal
veins assume a greater role in draining the body wall. The 4th to 11th right intercostal veins empty into the right supracardinal vein, which together with a portion of the posterior cardinal vein forms the azygos vein (Fig. 13.46).

The complicated development of the vena cava may account for the fact that deviations from the normal pattern are common. Also, the fact that the original pattern of venous return is established bilaterally and then shifts to the right probably accounts for the fact that vena cava abnormalities are often observed in individuals with laterality (sidedness) defects.
A double inferior vena cava occurs when the left sacrocardinal vein fails to lose its connection with the left subcardinal vein (Fig. 13.47A).The left common iliac vein may or may not be present, but the left gonadal vein remains as in normal conditions.
On the left, the 4th to 7th intercostal veins enter into the left supracardinal vein, and the left supracardinal vein, then known as the hemiazygos vein, empties into the azygos vein (Fig. 13.46B).
caudal part of the body reaches the heart by way of the azygos vein and superior vena cava. The hepatic vein enters into the right atrium at the site of the inferior vena cava. Usually, this abnormality is associated with other heart malformations.
Left superior vena cava is caused by persistence of the left anterior cardinal vein and obliteration of the common cardinal and proximal part of the anterior cardinal veins on the right (Fig. |3.48A). In such a case, blood from the right is channeled toward the left by way of the brachiocephalic vein. The left superior vena cava drains into the right atrium by way of the left sinus horn, that is, the coronary sinus.
Absence of the inferior vena cava arises when the right subcardinal vein fails to make its connection with the liver and shunts its blood directly into the right supracardinal vein (Figs. 13.46 and 13.47B). Hence, the bloodstream from the
A double superior vena cava is characterized by the persistence of the left anterior cardinal vein and failure of the left brachiocephalic vein to form (Fig. 13.48B). The persistent left anterior cardinal vein, the left superior vena cava, drains into the right atrium by way of the coronary sinus.

Figure 13.47 A. Double inferior vena cava at the lumbar level arising from the persistence of the left sacrocardinal vein. B. Absent inferior vena cava. The lower half of the body is drained by the azygos vein, which enters the superior vena cava. The hepatic vein enters the heart at the site of the inferior vena cava.

Before birth, blood from the placenta, about 80% saturated with oxygen, returns to the fetus by way of the umbilical vein. On approaching the liver, most of this blood flows through the ductus venosus directly into the inferior vena cava, short-circuiting the liver. A smaller amount enters the liver sinusoids and mixes with blood from the portal circulation (Fig. 13.49). A sphincter mechanism in the ductus venosus, close to the entrance of the umbilical vein, regulates flow of umbilical blood through the liver sinusoids. This sphincter closes when a uterine contraction renders the venous return too high, preventing a sudden overloading of the heart.
From the left atrium, where it mixes with a small amount of desaturated blood returning from the lungs, blood enters the left ventricle and ascending aorta. Since the coronary and carotid arteries are the first branches of the ascending aorta, the heart musculature and the brain are supplied with well-oxygenated blood. Desaturated blood from the superior vena cava flows by way of the right ventricle into the pulmonary trunk. During fetal life, resistance in the pulmonary vessels is high, such that most of this blood passes directly through the ductus arteriosus into the descending aorta, where it mixes with blood from the proximal aorta. After coursing through the descending aorta, blood flows toward the placenta by way of the two umbilical arteries. The oxygen saturation in the umbilical arteries is approximately 58%.
After a short course in the inferior vena cava, where placental blood mixes with deoxygenated blood returning from the lower limbs, it enters the right atrium. Here, it is guided toward the oval foramen by the valve of the inferior vena cava, and most of the blood passes directly into the left atrium. A small amount is prevented from doing so by the lower edge of the septum secundum, the crista dividens, and remains in the right atrium. Here, it mixes with desaturated blood returning from the head and arms by way of the superior vena cava.

During its course from the placenta to the organs of the fetus, blood in the umbilical vein gradually loses its high oxygen content as it mixes with desaturated blood. Theoretically, mixing may occur in the following places (Fig. 13.49I-V): in the liver (J), by mixture with a small amount of blood returning from the portal system; in the inferior vena cava (IJ), which carries deoxygenated blood returning from the lower extremities, pelvis, and kidneys; in the right atrium (I/]), by mixture with blood returning from the head and limbs; in the left atrium (IV), by mixture with blood
Figure 13.49 Fetal circulation before birth. Arrows, direction of blood flow. Note where oxygenated blood mixes with deoxygenated blood in: the liver (I), the inferior vena cava (II), the right atrium (Ill), the left atrium (IV), and at the entrance of the ductus arteriosus into the descending aorta (V).
returning from the lungs; and at the entrance of the ductus arteriosus into the descending

Changes in the vascular system at birth are caused by cessation of placental blood flow and the beginning of respiration. Since the ductus arteriosus closes by muscular contraction of its wall, the amount of blood flowing through the lung vessels increases rapidly. This, in turn, raises pressure in the left atrium. Simultaneously, pressure in the right atrium decreases as a result of interruption of placental blood flow.The septum primum is then apposed to the septum secundum, and functionally, the oval foramen closes.
To summarize, the following changes occur in the vascular system after birth (Fig. 13.50):
Closure of the umbilical arteries, accomplished by contraction of the smooth musculature in their walls, is probably caused by thermal and mechanical stimuli and a change in oxygen tension. Functionally, the arteries close a few minutes after birth, although the actual obliteration of the lumen by fibrous proliferation may take 2 to 3 months. Distal parts of the umbilical arteries form the medial umbilical ligaments, and the proximal portions remain open as the superior vesical arteries (Fig. 13.50).

venosus occurs shortly after that of the umbili- cal arteries. Hence, blood from the placenta may
Figure 13.50 Human circulation after birth. Note the changes occurring as a result of the beginning of respiration and interruption of placental blood flow. Arrows, direction of blood flow.
enter the newborn for some time after birth. After obliteration, the umbilical vein forms the ligamentum teres hepatis in the lower margin of the falciform ligament. The ductus venosus, which courses from the ligamentum teres to the inferior vena cava, is also obliterated and forms the venosum.

Closure of the ductus arteriosus by contraction of its muscular wall occurs almost immediately after birth; it is mediated by bradykinin, a substance released from the lungs during initial inflation. Complete anatomical obliteration by proliferation of the intima is thought to take 1 to 3 months. In the adult, the obliterated ductus arteriosus forms the ligamentum arteriosum. Closure of the oval foramen is caused by an increased pressure in the left atrium, combined with a decrease in pressure on the right side. The first breath presses the septum primum against the septum secundum. During the first days of life, however, this closure is reversible. Crying by the baby creates a shunt from right to left, which accounts for cyanotic periods in the newborn. Constant apposition gradually leads to fusion of the two septa in about 1 year. In 20% of individuals, however, perfect anatomical closure may never be obtained (probe patent foramen ovale).

The lymphatic system begins its development later than the cardiovascular system, not appearing until the fifth week of gestation. Lymphatic vessels arise as sac-like outgrowths from the endothelium of veins. Six primary lymph sacs
are formed: two jugular, at the junction of the subclavian and anterior cardinal veins; two iliac, at the junction of the iliac and posterior cardinal veins; one retroperitoneal, near the root of the mesentery; and one cisterna chyli, dorsal to the retroperitoneal sac. Numerous channels connect the sacs with each other and drain lymph from the limbs, body wall, head, and neck. Two main channels, the right and left thoracic ducts, join the jugular sacs with the cisterna chyli, and soon an anastomosis forms between these ducts. The thoracic duct then develops from the distal portion of the right thoracic duct, the anastomosis, and the cranial portion of the left thoracic duct. The right lymphatic duct is derived from the cranial portion of the right thoracic duct. Both ducts maintain their original connections with the venous system and empty into the junction of the internal jugular and subclavian veins. Numerous anastomoses produce many variations in the final form of the thoracic duct.
Specification of the lymphatic lineage is regulated by the transcription factor PROX1 that upregulates lymphatic vessel genes and downregulates blood vessel genes. A critical gene that is upregulated is VEGFR3 that is the receptor for the paracrine factor VEGFC. This protein causes PROX1 expressing endothelial cells to sprout from existing veins to initiate growth of lymphatic vessels.
s_-

On approximately day 16, heart progenitor cells migrate through the primitive streak to a position cranial to the neural folds where they establish a horseshoe-shaped region in the splanchnic layer of lateral plate mesoderm called the primary heart field (PHF) (Fig. 13.1). As they migrate, these cells are specified by the laterality pathway (Fig. 13.2) to contribute to right and left sides of the heart and to form specific heart regions, including the atria, left ventricle, and part of the right ventricle (Fig. 13.1A). The remainder of the heart, including part of the right ventricle, conus cordis, and truncus arteriosus (the outflow tract), is derived from cells in the secondary heart field (SHF) (Fig. 13.3). The SHF lies in splanchnic mesoderm near the floor of the posterior part of the pharynx and is regulated by neural crest cells that migrate through pharyngeal arches in this region (Figs. 13.3 and 13.27). Disruption of the laterality pathway results in many different types of heart defects, while disruption of the SHF
results in defects of the outflow tract, including transposition of the great arteries, pulmonary stenosis, DORYV, and others.

Induction of the cardiogenic region is initiated by anterior endoderm underlying progenitor heart cells, and causes the cells to become myoblasts and vessels. BMPs secreted by this endoderm in combination with inhibition of WNT expression induces expression of NKX2.5 the master gene for heart development. Some cells in the PHF become endothelial cells and form a horseshoe-shaped tube, while others form myoblasts surrounding the tube. By the 22nd day of development, lateral body wall folds bring the two sides of the horseshoe (Fig. 13.5) toward the midline where they fuse (except for their caudal [atrial] ends) to form a single, slightly bent heart tube (Fig. 13.8) consisting of an inner endocardial tube and a surrounding myocardial mantle (Fig. 13.5C). During the fourth week, the heart undergoes cardiac looping. This process causes the heart to fold on itself and assume its normal position in the left part of the thorax with the atria posteriorly and the ventricles in a more anterior position. Failure of the heart to loop properly results in dextrocardia and the heart lies on the right side. Dextrocardia can also be induced at an earlier time when laterality is established.
Septum formation in the heart in part arises from development of endocardial cushion tissue in the atrioventricular canal (atrioventricular cushions) and in the conotruncal region (conotruncal swellings). Because of the key location of cushion tissue, many cardiac malformations are related to abnormal cushion morphogenesis.

Septum Formation in the Atrium. The septum primum, a sickle-shaped crest descending from the roof of the atrium, begins to divide the atrium in two but leaves a lumen, the ostium primum, for communication between the two sides (Fig. 13.16). Later, when the ostium primum is obliterated by fusion of the septum primum with the endocardial cushions, the ostium secundum is formed by cell death that creates an opening in the septum primum. Finally, a septum secundum forms, but an interatrial opening, the oval foramen, persists. Only at birth, when pressure in the left atrium increases, do the two septa press against each other and close the communication between the two. Abnormalities in the atrial septum may vary from total absence (Fig. 13.21) to a small opening known as probe patency of the oval foramen.
Septum Formationin theAtrioventricular
Canal. Four endocardial cushions surround
the atrioventricular canal. Fusion of the opposing superior and inferior cushions divides the orifice into right and left atrioventricular canals (Fig. 13.16B-D). Cushion tissue then becomes fibrous and forms the mitral (bicuspid) valve on the left and the tricuspid valve on the right (Fig. 13.16F). Persistence of the common atrioventricular canal (Fig. 13.22) and abnormal formation of the valves are defects that occur due to abnormalities in this endocardial cushion tissue.
Septum Formation in the Ventricles. The interventricular septum consists of a thick muscular part and a thin membranous portion (Figs. 13.16F and 13.26) formed by (1) the inferior endocardial atrioventricular cushion, (2) the right conus swelling, and (3) the left conus swelling (Fig. 13.24). In many cases, these three components fail to fuse, resulting in an open interventricular foramen. Although this abnormality may be isolated, it is commonly combined with other compensatory defects (Figs. 13.30 and 13.31).

Septum Formation in the Bulbus. The bulbus is divided into (1) the truncus (aorta and pulmonary trunk), (2) the conus (outflow tract of the aorta and pulmonary trunk), and (3) the smooth-walled portion of the right ventricle. The truncus region is divided by the spiral aorticopulmonary septum into the two main arteries (Fig. 13.24). The conus swellings divide the outflow tracts of the aortic and pulmonary channels and with tissue from the inferior endocardial cushion, close the interventricular foramen (Fig. 13.24). Many vascular abnormalities, such as transposition of the great vessels and pulmonary valvular atresia, result from abnormal division of the conotruncal region; their origin may involve neural crest cells that contribute to septum formation in the conotruncal region (Fig. 13.27).
The aortic arches lie in each of the five pharyngeal arches (Figs. 13.35 and 13.37). Four important derivatives of the original aortic arch system are (1) the carotid arteries (third arches); (2) the arch of the aorta (left fourth aortic arch); (3) the pulmonary artery (sixth aortic arch), which during fetal life is connected to the aorta through the ductus arteriosus; and (4) the right subclavian artery formed by the right fourth aortic arch, distal portion of the right dorsal aorta, and the seventh intersegmental artery (Fig. 13.37B).The most common vascular aortic arch abnormalities include (1) open ductus arteriosus and coarctation of the aorta (Fig. 13.39) and (2) persistent right aortic arch and abnormal right subclavian artery (Figs. 13.40 and 13.41), which may cause respiratory and swallowing complaints.
The vitelline arteries initially supply the yolk sac but later form the celiac and superior mesenteric arteries. The inferior mesenteric arteries are derived from the umbilical arteries. These 3 arteries supply the foregut, midgut, and hindgut regions, respectively.

The paired umbilical arteries arise from the common iliac arteries. After birth, the distal portions of these arteries are obliterated to form the medial umbilical ligaments, whereas the proximal portions persist as the internal iliac and vesicular arteries.
Venous System. Three systems can be recognized: (1) the vitelline system, which develops into the portal system; (2) the cardinal system, which forms the caval system; and (3) the umbilical system, which disappears after birth. The complicated caval system is characterized by many abnormalities, such as double inferior and superior vena cava and left superior vena cava (Fig. 13.48), which are also associated with laterality defects.
Changes at Birth. During prenatal life, the placental circulation provides the fetus with its oxygen, but after birth, the lungs take on gas exchange. In the circulatory system, the following changes take place at birth and in the first postnatal months: (1) the ductus arteriosus closes; (2) the oval foramen closes; (3) the umbilical vein and ductus venosus close and remain as the ligamentum teres hepatis and ligamentum venosum; and (4) the umbilical arteries form the medial umbilical ligaments.
Lymphatic System. The lymphatic system develops later than the cardiovascular system, originating from the endothelium of veins as five sacs: two jugular, two iliac, one retroperitoneal, and one cisterna chyli. Numerous channels form to connect the sacs and provide drainage from other structures. Ultimately, the thoracic duct forms from anastomosis of the right and left thoracic ducts, the distal part of the right thoracic duct, and the cranial part of the left thoracic duct. The right lymphatic duct develops from the cranial part of the right thoracic duct.
200
Part
II
Systems-Based

- 1. A prenatal ultrasound of a 35-year-old
woman in
her
12th week
of gestation
an abnormal
image of
of
a
the four-chambered
typical view
cross,
a
portion piece
is missing.
fetal heart.
provided reveals
Instead by
just
What the
cross, and
what below
the the
structures defect
cross- constitute
does this
infant probably have?
- 2. A child is born with severe craniofacial
defects and transposition of the great vessels.
3. What type of tissue is critical for dividing the heart into four chambers and the outflow tract into pulmonary and aortic channels?
What cell population may play a role in both
abnormalities, and what type of insult might
have produced this effect?
. A patient complains about having difficulty swallowing. What vascular abnormality or abnormalities might produce this complaint? What is its embryological origin?

When the embryo is approximately 4 weeks old, the respiratory diverticulum (lung bud) appears as an outgrowth from the ventral wall of the foregut (Fig. 14.1A).The appearance and location of the lung bud are dependent upon an increase in retinoic acid (RA) produced by adjacent mesoderm. This increase in RA causes upregulation of the transcription factor TBX4 expressed in the endoderm of the gut tube at the site of the respiratory diverticulum. TBX4 induces formation of the bud and the continued growth and differentiation of the lungs. Hence, epithelium of the internal lining of the larynx, trachea, and bronchi, as well as that of the lungs, is entirely of endodermal origin. The cartilaginous, muscular, and connective tissue components of the trachea and lungs are derived from splanchnic mesoderm surrounding the foregut.
Initially, the lung bud is in open communication with the foregut (Fig. 14.1B). When the diverticulum expands caudally, however, two longitudinal ridges, the tracheoesophageal ridges, separate it from the foregut (Fig. 14.2A). Subsequently, when these ridges fuse to form the tracheoesophageal septum, the foregut is divided into a dorsal portion, the esophagus, and a ventral portion, the trachea and lung buds (Fig. 14.2B,C). The respiratory primordium maintains its communication with the pharynx through the laryngeal orifice (Fig. 14.2D).
Openings of pharyngeal pouches
Figure 14.1 A. Embryo of approximately 25 days' gestation showing the relation of the respiratory diverticulum to the heart, stomach, and liver. B. Sagittal section through the cephalic end of a 5-week embryo showing the openings of the pharyngeal pouches and the laryngotracheal orifice.

Figure 14.2 A-C. Successive stages in development of the respiratory diverticulum showing the tracheoesophageal ridges and formation of the septum, splitting the foregut into esophagus and trachea with lung buds. D. The ventral portion of the pharynx seen from above showing the laryngeal orifice and surrounding swelling.

Abnormalities in partitioning of the esophagus and trachea by the tracheoesophageal septum result in esophageal atresia with or without tracheoesophageal fistulas (TEFs). These defects occur in approximately |/3,000 births, and 90% result in the upper portion of the esophagus ending in a blind pouch and the lower segment forming a fistula with the trachea (Fig. |4.3A). lsolated esophageal atresia (Fig. 14.3B) and H-type TEF without esophageal atresia (Fig. 14.3C) each account for 4% of these defects. Other variations (Fig. 14.3D,E) each account for approximately 1% of these defects. These abnormalities are associated with other birth defects, including cardiac abnormalities, which occur in 33% of these cases. In this regard, TEFs are a component of the VACTERL association (Vertebral anomalies, Anal atresia, Cardiac defects, Tracheoesophageal fistula, Esophageal atresia, Renal anomalies, and Limb defects), a collection of defects of unknown
Figure 14.3 Various types of esophageal atresia and/or TEFs. A. The most frequent abnormality (90% of cases) occurs with the upper esophagus ending in a blind pouch and the lower segment forming a fistula with the trachea. B. Isolated esophageal atresia (4% of cases). C. H-type TEF (4% of cases). D,E. Other variations (each |% of cases).
causation, but occurring more frequently than predicted by chance alone.
A complication of some TEFs is polyhydramnios, since in some types of TEF, amniotic fluid,

The internal lining of the larynx originates from endoderm, but the cartilages and muscles originate from mesenchyme of the fourth and sixth pharyngeal arches. As a result of rapid proliferation of this mesenchyme, the laryngeal orifice changes in appearance from a sagittal slit to a T-shaped opening (Fig. 14.4A). Subsequently, when mesenchyme of the two arches transforms into the thyroid, cricoid, and arytenoid cartilages, the characteristic adult shape of the laryngeal orifice can be recognized (Fig. 14.4B).
At about the time that the cartilages are formed, the laryngeal epithelium also proliferates rapidly, resulting in a temporary occlusion of the lumen. Subsequently, vacuolization and recanalization produce a pair of lateral recesses, the laryngeal ventricles. These recesses are bounded by folds of tissue that differentiate into the false and true vocal cords.
Since musculature of the larynx is derived from mesenchyme of the fourth and sixth pharyngeal arches, all laryngeal muscles are innervated by branches of the tenth cranial nerve, the vagus nerve: The superior laryngeal nerve innervates derivatives of the fourth pharyngeal arch, and the recurrent laryngeal nerve innervates derivatives of the sixth pharyngeal arch.
when swallowed does not pass to the stomach and intestines.Also, gastric contents and/or amniotic fluid at birth may enter the trachea through a fistula, causing pneumonitis and pneumonia.
(For further details on the laryngeal cartilages, see Chapter 17, p. 263.)

During its separation from the foregut, the lung bud forms the trachea and two lateral outpocketings, the bronchial buds (Fig. 14.2B,C). At the beginning of the fifth week, each of these buds enlarges to form right and left main bronchi. The right then forms three secondary bronchi, and the left, two (Fig. 14.5.4), thus foreshadowing the three lobes of the lung on the right side and two on the left (Fig. 14.5B,C).
With subsequent growth in caudal and lateral directions, the lung buds expand into the body cavity (Fig. 14.6). The spaces for the lungs, the pericardioperitoneal canals, are narrow. They lie on each side of the foregut and are gradually filled by the expanding lung buds. Ultimately, the pleuroperitoneal and pleuropericardial folds separate the pericardioperitoneal canals from the peritoneal and pericardial cavities, respectively, and the remaining spaces form the primitive pleural cavities (see Chapter 7). The mesoderm, which covers the outside of the lung, develops into the visceral pleura. The somatic mesoderm layer, covering the body wall from the inside, becomes the parietal pleura (Fig. 14.6A). The space
Figure 14.4 Laryngeal orifice and surrounding swellings at successive stages of development. A. 6 weeks. B. 12 weeks.
Figure 14.5 Stages in development of the trachea and lungs. A. 5 weeks. B. 6 weeks. C. 8 weeks.
Figure 14.6 Expansion of the lung buds into the pericardioperitoneal canals.At this stage, the canals are in communication with the peritoneal and pericardial cavities. A. Ventral view of lung buds. B. Transverse section through the lung buds showing the pleuropericardial folds that will divide the thoracic portion of the body cavity into the pleural and pericardial cavities.
between the parietal and visceral pleura is the pleural cavity (Fig. 14.7).

Figure 14.7 Once the pericardioperitoneal canals separate from the pericardial and peritoneal cavities, respectively, the lungs expand in the pleural cavities. Note the visceral and parietal pleura and definitive pleural cavity. The visceral pleura extends between the lobes of the lungs.
During further development, secondary bronchi divide repeatedly in a dichotomous fashion, forming 10 tertiary (segmental) bronchi in the right lung and 8 in the left, creating the bronchopulmonary segments of the adult lung. By the end of the sixth month, approximately 17 generations of subdivisions have formed. Before the bronchial tree reaches its final shape, however, an additional six divisions form during postnatal life. Branching is regulated by epithelial-mesenchymal interactions between the endoderm of the lung buds and splanchnic mesoderm that surrounds them. Signals for branching, which emit from the mesoderm, involve members of the fibroblast growth factor family. While all of these new subdivisions are occurring and the bronchial tree is developing, the lungs assume a more caudal position, so that by the time of birth, the
TABLE 14.1 Maturation of the Lungs, 1 = TABLE 14.1 Maturation of the Lungs. TABLE 14.1 Maturation of the Lungs, 2 = TABLE 14.1 Maturation of the Lungs. Pseudoglandular period, 1 = 5-16 wk. Pseudoglandular period, 2 = Branching has continued to form terminal bronchioles. No respiratory bronchioles or alveoli are present.. Canalicular period, 1 = 16-26 wk. Canalicular period, 2 = Each terminal bronchiole divides into two or more respiratory bronchioles, which in turn divide into three to six alveolar ducts.. Terminal sac period, 1 = 26 wk to birth. Terminal sac period, 2 = Terminal sacs (primitive alveoli) form, and capillaries establish close contact.. Alveolar period, 1 = 8 mo to childhood. Alveolar period, 2 = Mature alveoli have well-developed epithelial endothelial (capillary) contacts.
bifurcation of the trachea is opposite the fourth thoracic vertebra.

Up to the seventh prenatal month, the bronchioles divide continuously into more and smaller canals (canalicular phase) and the vascular supply increases steadily (Fig. 14.8A). Terminal bronchioles divide to form respiratory bronchioles and each of these divides into three to six alveolar ducts (Fig. 14.8B). The ducts end in terminal sacs (primitive alveoli) that are surrounded by flat alveolar cells in close contact with neighboring capillaries (Fig. 14.8B). By the end of the seventh month, sufficient numbers of mature alveolar sacs and capillaries are present to guarantee adequate gas exchange, and the premature infant is able to survive (Fig. 14.9) (Table 14.1).
During the last 2 months of prenatal life and for several years thereafter, the number of terminal sacs increases steadily. In addition, cells lining the sacs, known as type I alveolar epithelial cells, become thinner, so that surrounding capillaries protrude into the alveolar sacs (Fig. 14.9). This intimate contact between epithelial and endothelial cells makes up the blood-air barrier. Mature alveoli are not present before birth. In addition to endothelial cells and flat alveolar epithelial cells, another cell type develops at the end of the sixth month. These cells, type II alveolar epithelial cells, produce surfactant, a phospholipid-rich fluid capable of lowering surface tension at the air— alveolar interface.
Before birth, the lungs are full of fluid that contains a high chloride concentration, little protein, some mucus from the bronchial glands,
Figure 14.8 Histological and functional development of the lung. A. The canalicular period lasts from the | 6th to the 26th week. Note the cuboidal cells lining the respiratory bronchioli. B. The terminal sac period begins at the end of the sixth and beginning of the seventh prenatal month. Cuboidal cells become very thin and intimately associated with the endothelium of blood and lymph capillaries or form terminal sacs (primitive alveoli).

Figure 14.9 Lung tissue in a newborn. Note the thin squamous epithelial cells (also known as alveolar epithelial cells, type I) and surrounding capillaries protruding into mature alveoli.
and surfactant from the alveolar epithelial cells (type II). The amount of surfactant in the fluid increases, particularly during the last 2 weeks before birth.

Surfactant is particularly important for survival of the premature infant.When surfactant is insufficient, the air—water (blood) surface membrane tension becomes high, bringing great risk that alveoli will collapse during expiration. As a result, respiratory distress syndrome (RDS) develops. This is a common cause of death in the premature infant. In these cases, the partially collapsed alveoli contain a fluid with a high protein content, many hyaline membranes, and lamellar bodies, probably derived from the surfactant layer. RDS, which was previously known as hyaline membrane disease, accounts for approximately 20% of deaths among newborns. Treatment of preterm babies with artificial surfactant as well as treatment of mothers with premature labor with glucocorticoids to stimulate surfactant production have reduced the mortality associated with RDS.
Although many abnormalities of the lung and bronchial tree have been described (e.g.,blind-ending
As concentrations of surfactant increase during the 34th week of gestation, some of this phospholipid enters the amniotic fluid and acts on macrophages in the amniotic cavity. Once "activated," evidence suggests that these macrophages migrate across the chorion into the uterus where they begin to produce immune system proteins, including interleukin-1B (IL-1B). Upregulation of these proteins results in increased production of prostaglandins that cause uterine contractions. Thus, there may be signals from the fetus that participate in initiating labor and birth.
Fetal breathing movements begin before birth and cause aspiration of amniotic fluid. These movements are important for stimulating lung development and conditioning respiratory muscles. When respiration begins at birth, most of the lung fluid is rapidly resorbed by the blood and lymph capillaries, and a small amount is probably expelled via the trachea and bronchi during delivery. When the fluid is resorbed from alveolar sacs, surfactant remains deposited as a thin phospholipid coat on alveolar cell membranes. With air entering alveoli during the first breath, the surfactant coat prevents development of an air—water (blood) interface with high surface tension. Without the fatty surfactant layer, the alveoli would collapse during expiration (atelectasis).

trachea with absence of lungs and agenesis of one lung), most of these gross abnormalities are rare. Abnormal divisions of the bronchial tree are more common; some result in supernumerary lobules. These variations of the bronchial tree have little functional significance, but they may cause unexpected difficulties during bronchoscopies.
More interesting are ectopic lung lobes arising from the trachea or esophagus. It is believed that these lobes are formed from additional respiratory buds of the foregut that develop independent of the main respiratory system.
Most important clinically are congenital cysts of the lung, which are formed by dilation of terminal or larger bronchi. These cysts may be small and multiple, giving the lung a honeycomb appearance on radiograph, or they may be restricted to one or more larger ones. Cystic structures of the lung usually drain poorly and frequently cause chronic infections.
Respiratory movements after birth bring air into the lungs, which expand and fill the pleural cavity. Although the alveoli increase somewhat in size, growth of the lungs after birth is due primarily to an increase in the number of respiratory bronchioles and alveoli. It is estimated that only one-sixth of the adult number of alveoli are present at birth. The remaining alveoli are formed during the first 10 years of postnatal life through the continuous formation of new primitive alveoli.
_
into thin, flat cells, type I alveolar epithelial cells, intimately associated with blood and lymph capillaries. In the seventh month, gas exchange between the blood and air in the primitive alveoli is possible. Before birth, the lungs are filled with fluid with little protein, some mucus, and surfactant, which is produced by type II alveolar epithelial cells and which forms a phospholipid coat on the alveolar membranes. At the beginning of respiration, the lung fluid is resorbed except for the surfactant coat, which prevents the collapse of the alveoli during expiration by reducing the surface tension at the air— blood capillary interface. Absent or insufficient surfactant in the premature baby causes respiratory distress syndrome (RDS) because of collapse of the primitive alveoli (hyaline membrane disease).

Growth of the lungs after birth is primarily due to an increase in the number of respiratory bronchioles and alveoli and not to an increase in the size of the alveoli. New alveoli are formed during the first 10 years of postnatal life.

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the yolk by of the vitelline duct,
As a result of cephalocaudal and lateral folding of the embryo, a portion of the endoderm-lined yolk sac cavity is incorporated into the embryo to form the primitive gut. Two other portions of the endoderm-lined cavity, the yolk sac and the allantois, remain outside the embryo (Fig. 15.1A-D).
In the cephalic and caudal parts of the embryo, the primitive gut forms a blind-ending tube, the foregut and hindgut, respectively. The middle part, the midgut, remains temporally connected to sac means or yolk stalk (Fig. 15.1D). Development of the primitive gut and its derivatives is usually discussed in four sections: (a) The pharyngeal gut, or pharynx, extends from the oropharyngeal membrane to the respiratory diverticulum and is part of the foregut; this section is particularly important for development of the head and neck and is discussed in Chapter 17. (b) The remainder of the foregut lies caudal to the pharyngeal tube and extends as far caudally as the liver outgrowth. (c) The midgut begins caudal to the liver bud and extends to the junction
Figure 15.1 Sagittal sections through embryos at various stages of development demonstrating the effect of cephalocaudal and lateral folding on the position of the endoderm-lined cavity. Note formation of the foregut, midgut, and hindgut. A. Presomite embryo. B. Embryo with seven somites. C. Embryo with 14 somites. D. At the end of the first month.

of the right two-thirds and left third of the transverse colon in the adult. (d) The hindgut extends from the left third of the transverse colon to the cloacal membrane (Fig. 15.1). Endoderm forms the epithelial lining of the digestive tract and gives rise to the specific cells (the parenchyma) of glands, such as hepatocytes and the exocrine and endocrine cells of the pancreas. The stroma (connective tissue) for the glands is derived from visceral mesoderm. Muscle, connective tissue, and peritoneal components of the wall of the gut also are derived from visceral mesoderm.

Regional specification of the gut tube into different components occurs during the time that the lateral body folds are bringing the two sides of the tube together (Figs. 15.2 and 15.3). Specification is initiated by a concentration gradient of retinoic acid (RA) from the pharynx, that is exposed to little or no RA, to the colon, that sees the highest concentration of RA. This RA gradient causes transcription factors to be expressed in different regions of the gut tube. Thus, SOX2 "specifies" the esophagus and stomach; PDX1, the duodenum; CDXC, the small intestine; and CDXA, the large intestine and rectum (Fig. 15.2A). This initial patterning is stabilized by reciprocal interactions between the endoderm and visceral mesoderm adjacent to the gut tube (Fig. 15.2BD).This epithelial-mesenchymal interaction is initiated by sonic hedgehog (SHH) expression throughout the gut tube. SHH expression upregulates factors in the mesoderm that then determine the type of structure that forms from the gut tube, such as the stomach, duodenum,
Figure 15.2 Diagrams showing molecular regulation of gut development. A. Color-coded diagram that indicates genes responsible for initiating regional specification of the gut into esophagus, stomach, duodenum, etc. B-D. Drawings showing an example from the midgut and hindgut regions indicating how early gut specification is stabilized. Stabilization is effected by epithelialI-mesenchymal interactions between gut endoderm and surrounding visceral (splanchnic) mesoderm. Endoderm cells initiate the stabilization process by secreting SHH, which establishes a nested expression of HOX genes in the mesoderm.This interaction results in a genetic cascade that regulates specification of each gut region as is shown for the small and large intestine regions in these diagrams.

Figure 15.3 Transverse sections through embryos at various stages of development. A. The intraembryonic cavity, bordered by visceral and somatic layers of lateral plate mesoderm, is in open communication with the extraembryonic cavity. B. The intraembryonic cavity is losing its wide connection with the extraembryonic cavity. C. At the end of the fourth week, visceral mesoderm layers are fused in the midline and form a double-layered membrane (dorsal mesentery) between right and left halves of the body cavity.Ventral mesentery exists only in the region of the septum transversum (not shown).
small intestine, etc. For example, in the region of the caudal limit of the midgut and all of the hindgut, SHH expression establishes a nested expression of the HOX genes in the mesoderm (Fig. 15.2D). Once the mesoderm is specified by this code, then it instructs the endoderm to form the various components of the midand hindgut regions, including part of the small intestine, cecum, colon, and cloaca (Fig. 15.2).

Portions of the gut tube and its derivatives are suspended from the dorsal and ventral body wall by mesenteries, double layers of peritoneum that enclose an organ and connect it to the body wall. Such organs are called intraperitoneal, whereas organs that lie against the posterior body wall and are covered by peritoneum on their anterior surface only (e.g., the kidneys) are considered retroperitoneal. Peritoneal ligaments are double layers of peritoneum (mesenteries) that pass from one organ to another or from an organ to the body wall. Mesenteries and ligaments provide pathways for vessels, nerves, and lymphatics to and from abdominal viscera (Figs. 15.3 and 15.4). Initially the foregut, midgut, and hindgut are in broad contact with the mesenchyme of the posterior abdominal wall (Fig. 15.3). By the fifth week, however, the connecting tissue bridge has
artery
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Figure 15.4 Primitive dorsal and ventral mesenteries. The liver is connected to the ventral abdominal wall and to the stomach
by the
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omentum, respectively.
superior mesenteric
artery runs
mes-
The
Figure 15.5 Embryos during the fourth A and fifth B weeks of development showing formation of the gastrointestinal tract and the various derivatives originating from the endodermal germ layer.

narrowed, and the caudal part of the foregut, the midgut, and a major part of the hindgut are suspended from the abdominal wall by the dorsal mesentery (Figs. 15.3C and 15.4), which extends from the lower end of the esophagus to the cloacal region of the hindgut. In the region of the stomach, it forms the dorsal mesogastrium or greater omentum; in the region of the duodenum, it forms the dorsal mesoduodenum; and in the region of the colon, it forms the dorsal mesocolon. Dorsal mesentery of the jejunal and ileal loops forms the mesentery proper. Ventral mesentery, which exists only in the region of the terminal part of the esophagus, the stomach, and the upper part of the duodenum (Fig. 15.4), is derived from the septum transversum. Growth of the liver into the mesenchyme of the septum transversum divides the ventral mesentery into (a) the lesser omentum, extending from the lower portion of the esophagus, the stomach, and the upper portion of the duodenum to the liver and (b) the falciform ligament, extending from the liver to the ventral body wall (Fig. 15.4; see Chapter 7).

When the embryo is approximately 4 weeks old, the respiratory diverticulum (lung bud) appears at the ventral wall of the foregut at the border with the pharyngeal gut (Fig. 15.5). The tracheoesophageal septum gradually partitions this diverticulum from the dorsal part of the foregut (Fig. 15.6). In this manner, the foregut
Successive stages in development of the respiratory diverticulum and esophagus through partitioning of the
divides into a ventral portion, the respiratory primordium (see Chapter 14), and a dorsal portion, the esophagus.
At first, the esophagus is short (Fig. 15.5.4), but with descent of the heart and lungs, it lengthens

Esophageal atresia and/or tracheoesophageal fistula results either from spontaneous posterior deviation of the tracheoesophageal septum or from some mechanical factor pushing the dorsal wall of the foregut anteriorly. In its most common form, the proximal part of the esophagus ends as a blind sac, and the distal part is connected to the trachea by a narrow canal just above the bifurcation (Fig. 15.7A). Other types of defects in this region occur much less frequently (Fig. 15.7B-E) (see Chapter 14).
rapidly (Fig. 15.5B). The muscular coat, which is formed by surrounding splanchnic mesenchyme, is striated in its upper two-thirds and innervated by the vagus; the muscle coat is smooth in the lower third and is innervated by the splanchnic plexus.
Atresia of the esophagus prevents normal passage of amniotic fluid into the intestinal tract, resulting in accumulation of excess fluid in the amniotic sac (polyhydramnios). In addition to atresias, the lumen of the esophagus may narrow, producing esophageal stenosis, usually in the lower third. Stenosis may be caused by incomplete recanalization, vascular abnormalities, or accidents that compromise blood flow. Occasionally, the esophagus fails to lengthen sufficiently, and the stomach is pulled up into the esophageal hiatus through the diaphragm. The result is a congenital hiatal hernia.

The stomach appears as a fusiform dilation of the foregut in the fourth week of development (Fig. 15.8). During the following weeks, its appearance and position change greatly as a result of the different rates of growth in various regions of its wall and the changes in position of surrounding organs. Positional changes of the stomach are most easily explained by assuming that it rotates around a longitudinal and an anteroposterior axis (Fig. 15.8).
Figure
15.8
A-C.
the stomach
along its
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as seen
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D,E.
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The stomach rotates 90° clockwise around its longitudinal axis, causing its left side to face anteriorly and its right side to face posteriorly (Fig. 15.8A—C). Hence, the left vagus nerve, initially innervating the left side of the stomach, now innervates the anterior wall; similarly, the right nerve innervates the posterior wall. During this rotation, the original posterior wall of the stomach grows faster than the anterior portion, forming the greater and lesser curvatures (Fig. 15.8C). The cephalic and caudal ends of the stomach originally lie in the midline, but during further growth, the stomach rotates around an anteroposterior axis, such that the caudal or pyloric part moves to the right and upward, and the cephalic or cardiac portion moves to the left and slightly downward (Fig. 15.8D,E).The stomach thus assumes its final position, its axis running from above left to below right. Since the stomach is attached to the dorsal body wall by the dorsal mesogastrium and to the ventral body wall by the ventral mesogastrium (Figs. 15.4 and 15.9A), its rotation and disproportionate growth alter the position of these mesenteries. Rotation about the longitudinal axis pulls the dorsal mesogastrium to the left, creating a space behind the stomach called the omental bursa (lesser peritoneal sac) (Figs. 15.9 and 15.10). This rotation also pulls the ventral mesogastrium to the right. As this process continues in the fifth week of development, the spleen primordium appears as a mesodermal proliferation between the two leaves of the dorsal mesogastrium (Figs. 15.10 and 15.11). With continued rotation of the stomach, the dorsal mesogastrium lengthens, and the portion between the spleen and dorsal midline swings to the left and fuses with the peritoneum of the posterior abdominal wall (Figs. 15.10 and 15.11). The posterior leaf of the dorsal mesogastrium and the peritoneum along this line of fusion degenerate. The spleen, which remains intraperitoneal, is then connected to the body wall in the region of the left kidney by the lienorenal ligament and to the stomach by the gastrolienal ligament

(Figs. 15.10 and 15.11). Lengthening and fusion of the dorsal mesogastrium to the posterior body wall also determine the final position of the pancreas. Initially, the organ grows into the dorsal mesoduodenum, but eventually its tail extends into the dorsal mesogastrium (Fig. 15.10A). Since this portion of the dorsal mesogastrium fuses with the dorsal body wall, the
stomach
Figure 15.9 A. Transverse section through a 4-week embryo showing intercellular clefts appearing in the dorsal mesogastrium. B,C. The clefts have fused, and the omental bursa is formed as an extension of the right side of the intraembryonic cavity behind the stomach.
vein
Figure 15.10 A. The positions of the spleen, stomach, and pancreas at the end of the fifth week. Note the position of the spleen and pancreas in the dorsal mesogastrium. B. Position of spleen and stomach at the | 1th week. Note formation of the omental bursa (lesser peritoneal sac).
Figure 15.11 Transverse sections through the region of the stomach, liver, and spleen, showing formation of the omental bursa (lesser peritoneal sac), rotation of the stomach, and position of the spleen and tail of the pancreas between the two leaves of the dorsal mesogastrium.With further development, the pancreas assumes a retroperitoneal position.
Figure 15.12 A. Derivatives of the dorsal mesentery at the end of the third month. The dorsal mesogastrium bulges out on the left side of the stomach, where it forms part of the border of the omental bursa. B. The greater omentum hangs down from the greater curvature of the stomach in front of the transverse colon.

tail of the pancreas lies against this region (Fig. 15.11). Once the posterior leaf of the dorsal mesogastrium and the peritoneum of the posterior body wall degenerate along the line of fusion, the tail of the pancreas is covered by peritoneum on its anterior surface only and therefore lies in a retroperitoneal position. (Organs, such as the pancreas, that are originally covered by peritoneum, but later fuse with the posterior body wall to become retroperitoneal, are said to be secondarily retroperitoneal.) As a result of rotation of the stomach about its anteroposterior axis, the dorsal mesogastrium bulges down (Fig. 15.12). It continues to grow down and forms a double-layered sac extending over the transverse colon and small intestinal loops like an apron (Fig. 15.134). This doubleleafed apron is the greater omentum; later, its layers fuse to form a single sheet hanging from the greater curvature of the stomach (Fig. 15.138). The posterior layer of the greater omentum also fuses with the mesentery of the transverse colon (Fig. 15.13B).
The lesser omentum and falciform ligament form from the ventral mesogastrium, which itself is derived from mesoderm of the septum transversum.When liver cords grow into the septum, it thins to form (a) the peritoneum
Figure 15.13 A. Sagittal section showing the relation of the greater omentum, stomach, transverse colon, and small intestinal loops at 4 months. The pancreas and duodenum have already acquired a retroperitoneal position. B. Similar section as in A in the newborn. The leaves of the greater omentum have fused with each other and with the transverse mesocolon. The transverse mesocolon covers the duodenum, which fuses with the posterior body wall to assume a retroperitoneal position.
Figure 15.14 A.A 3-mm embryo (approximately 25 days) showing the primitive gastrointestinal tract and formation of the liver bud. The bud is formed by endoderm lining the foregut. B. A 5-mm embryo (approximately 32 days). Epithelial liver cords penetrate the mesenchyme of the septum transversum.

of the liver; (b) the falciform ligament, extending from the liver to the ventral body wall; and (c) the lesser omentum, extending from the stomach and upper duodenum to the liver (Figs. 15.14 and 15.15). The free margin of the falciform ligament contains the umbilical vein (Fig. 15.10A), which is obliterated after birth to form the round ligament of the liver (ligamentum teres hepatis). The free margin of the lesser omentum connecting the duodenum and liver (hepatoduodenal ligament) contains the bile duct, portal vein, and hepatic artery (portal triad). This free margin also forms the roof of the epiploic foramen of Winslow, which is the opening connecting the omental bursa (lesser sac) with the rest of the peritoneal cavity (greater sac) (Fig. 15.16).
A
Figure 15.15 A.A 9-mm embryo (approximately 36 days). The liver expands caudally into the abdominal cavity. Note condensation of mesenchyme in the area between the liver and the pericardial cavity, foreshadowing formation of the diaphragm from part of the septum transversum. B.A slightly older embryo. Note the falciform ligament extending between the liver and the anterior abdominal wall and the lesser omentum extending between the liver and the foregut (stomach and duodenum). The liver is entirely surrounded by peritoneum except in its contact area with the diaphragm. This is the bare area of the liver.
Figure 15.16 Lesser omentum extending from the liver to the lesser curvature of the stomach (hepatogastric ligament) and to the duodenum (hepatoduodenal ligament). In its free margin anterior to the omental foramen (epiploic foramen of Winslow) are the hepatic artery, portal vein, and bile duct (portal triad).

The terminal part of the foregut and the cephalic part of the midgut form the duodenum. The junction of the two parts is directly distal to the origin of the liver bud (Figs. 15.14 and 15.15). As

Pyloric stenosis occurs when the circular and, to a lesser degree, the longitudinal musculature of the stomach in the region of the pylorus hypertrophies. One of the most common abnormalities of the stomach in infants, pyloric stenosis was previously believed to develop only during fetal life, despite the fact that most cases present 3 to 5 days after birth. However, recent data indicate that a postnatal exposure (e.g., treatment with erythromycin in the newborn period) substantially increases the risk for pyloric stenosis, suggesting that in some cases the defect may develop later. Pyloric stenosis is characterized by an extreme narrowing of the pyloric lumen, and the passage of food is obstructed, resulting in severe projectile vomiting. In a few cases, the pylorus is atretic. Other malformations of the stomach, such as duplications and a prepyloric septum, are rare.
the stomach rotates, the duodenum takes on the form of a C-shaped loop and rotates to the right. This rotation, together with rapid growth of the head of the pancreas, swings the duodenum from its initial midline position to the right side of the abdominal cavity (Figs. 15.10A and 15.17). The duodenum and head of the pancreas press against the dorsal body wall, and the right surface of the dorsal mesoduodenum fuses with the adjacent peritoneum. Both layers subsequently disappear, and the duodenum and head of the pancreas become fixed in a retroperitoneal position. The entire pancreas thus obtains a retroperitoneal position. The dorsal mesoduodenum disappears entirely except in the region of the pylorus of the stomach, where a small portion of the duodenum (duodenal cap) retains its mesentery and remains intraperitoneal.
During the second month, the lumen of the duodenum is obliterated by proliferation of cells in its walls. However, the lumen is recanalized shortly thereafter (Fig. 15.18A,B). Since the foregut is supplied by the celiac artery and the midgut is supplied by the superior mesenteric artery, the duodenum is supplied by branches of both arteries (Fig. 15.14).

The liver primordium appears in the middle of the third week as an outgrowth of the endodermal epithelium at the distal end of the foregut
Figure 15.17 Transverse sections through the region of the duodenum at various stages of development.At first, the duodenum and head of the pancreas are located in the median plane. A, but later, they swing to the right and acquire a retroperitoneal position. B.
(Figs. 15.14 and 15.15). This outgrowth, the hepatic diverticulum, or liver bud, consists of rapidly proliferating cells that penetrate the septum transversum, that is, the mesodermal plate between the pericardial cavity and the stalk of the yolk sac (Figs. 15.14 and 15.15). While hepatic cells continue to penetrate the septum, the connection between the hepatic diverticulum and the foregut (duodenum) narrows, forming the bile duct. A small ventral outgrowth is formed by the bile duct, and this outgrowth gives rise to the gallbladder and the cystic duct (Figs. 15.15). During further development, epithelial liver cords intermingle with the vitelline and umbilical veins, which form hepatic sinusoids. Liver cords differentiate into the parenchyma (liver cells) and form the lining of the biliary ducts. Hematopoietic cells, Kupffer cells, and connective tissue cells are derived from mesoderm of the septum transversum.
Figure 15.18 Upper portion of the duodenum showing the solid stage. A and cavity formation. B produced by recanalization.
When liver cells have invaded the entire septum transversum, so that the organ bulges caudally into the abdominal cavity, mesoderm of the septum transversum lying between the liver and the foregut and the liver and the ventral abdominal wall becomes membranous, forming the lesser omentum and falciform ligament, respectively. Together, having formed the peritoneal connection between the foregut and the ventral abdominal wall, they are known as the

Mesoderm on ferentiates into visceral peritoneum except on its cranial surface (Fig. 15.15B). In this region, the liver remains in contact with the rest of the original septum transversum. This portion of the septum, which consists of densely packed mesoderm, will form the central tendon of the diaphragm. The surface of the liver that is in contact with the future diaphragm is never covered by peritoneum; it is the bare area of the liver (Fig. 15.15).
of the
liver dif-
In the 10th week of development, the weight of the liver is approximately 10% of the total body weight. Although this may be attributed partly to the large numbers of sinusoids, another important factor is its hematopoietic function. Large nests of proliferating cells, which produce red and white blood cells, lie between hepatic cells and walls of the vessels. This activity gradually subsides during the last
2 months of intrauterine life, and only small hematopoietic islands remain at birth. The weight of the liver is then only 5% of the total body weight.
Another important function of the liver begins at approximately the 12th week, when bile is formed by hepatic cells. Meanwhile, since the gallbladder and cystic duct have developed and the cystic duct has joined the hepatic duct to form the bile duct (Fig. 15.15), bile can enter the gastrointestinal tract. As a result, its contents take on a dark green color. Because of positional changes of the duodenum, the entrance of the bile duct gradually shifts from its initial anterior position to a posterior one, and consequently, the bile duct passes behind the duodenum (Figs. 15.19 and 15.20).

All of the foregut endoderm has the potential to express liver-specific genes and to differentiate into liver tissue. However, this expression is blocked by factors produced by surrounding tissues, including ectoderm,noncardiac mesoderm, and particularly the notochord (Fig. 15.21). The action of these inhibitors is blocked in the prospective hepatic region by fibroblast growth factors (FGEF2) secreted by cardiac mesoderm and by blood vessel-forming endothelial cells adjacent to the gut tube at the site of liver bud outgrowth. Thus, the cardiac mesoderm together with neighboring vascular endothelial cells "instructs" gut endoderm to express liver-specific genes by inhibiting an inhibitory factor of these same genes. Other factors participating in this "instruction" are bone morphogenetic proteins (BMPs) secreted by the septum transversum. BMPs appear to enhance the competence of prospective liver endoderm to respond to FGF2. Once this "instruction" is received, cells in the liver field differentiate into both hepatocytes and biliary cell lineages, a process that is at least partially regulated by hepatocyte nuclear transcription factors (HNF3 and 4).
Figure 15.20 A. Pancreas during the sixth week of development. The ventral pancreatic bud is in close contact with the dorsal pancreatic bud. B. Fusion of the pancreatic ducts. The main pancreatic duct enters the duodenum in combination with the bile duct at the major papilla. The accessory pancreatic duct (when present) enters the duodenum at the minor papilla.

Figure 15.21 Diagrams of the cardiacand hepatic-forming regions illustrating induction of liver development.All of the gut endoderm has the potential to form liver tissue, but this capacity is repressed by inhibitors secreted by neighboring mesoderm, ectoderm, and the notochord. Stimulation of hepatic development is achieved by secretion of BMPs by the septum transversum and FGF2 by cardiac mesoderm. BMPs enhance the competence of prospective liver endoderm to respond to FGF2. Then, FGF2 inhibits activity of the inhibitors, thereby specifying the hepatic field and initiating liver development. This interaction demonstrates that not all inductive processes are a result of direct signaling by an inducing molecule, but instead may occur by removal of a repressor signal.

Variations in liver lobulation are common but not clinically significant. Accessory hepatic ducts and duplication of the gallbladder (Fig. 15.22) are also common and usually asymptomatic. However, they become clinically important under pathological conditions. In some cases the ducts, which pass through a solid phase in their development, fail to recanalize (Fig. 15.22). This defect, extrahepatic biliary atresia, occurs in
1/15,000 live births. Among patients with extrahepatic biliary atresia, 15% to 20% have patent proximal ducts and a correctable defect, but the remainder usually die unless they receive a liver transplant. Another problem with duct formation lies within the liver itself; it is intrahepatic biliary duct atresia and hypoplasia. This rare abnormality (1/100,000 live births) may be caused by fetal infections. It may be lethal but usually runs an extended benign course.
Figure 15.22 A. Obliteration of the bile duct resulting in distention of the gallbladder and the hepatic ducts distal to the obliteration. B. Duplication of the gallbladder.

The pancreas is formed by two buds, dorsal and ventral, originating from the endodermal lining of the duodenum (Fig. 15.19). Whereas the dorsal pancreatic bud is in the dorsal mesentery, the ventral pancreatic bud is close to the bile duct (Fig. 15.19). When the duodenum rotates to the right and becomes C-shaped, the ventral pancreatic bud moves dorsally in a manner similar to the shifting of the entrance of the bile duct (Fig. 15.19). Finally, the ventral bud comes to lie immediately below and behind the dorsal bud (Fig. 15.20). Later, the parenchyma and the duct systems of the dorsal and ventral pancreatic buds fuse (Fig. 15.20B). The ventral bud forms the uncinate process and inferior part of the head of the pancreas. The remaining part of the gland is derived from the dorsal bud. The main pancreatic duct (of Wirsung) is formed by the distal part of the dorsal pancreatic duct and the entire ventral pancreatic duct (Fig. 15.20B). The proximal part of the dorsal pancreatic duct either is obliterated or persists as a small channel, the accessory pancreatic duct (of Santorini). The main pancreatic duct, together with the bile duct, enters the duodenum at the site of the major papilla; the entrance of the accessory duct (when present) is at the site of the minor papilla. In

The ventral pancreatic bud consists of two components that normally fuse and rotate around the duodenum so that they come to lie below the dorsal pancreatic bud. Occasionally, however, the right portion of the ventral bud migrates along its normal route, but the left migrates in the opposite direction. In this manner, the duodenum is surrounded by pancreatic tissue, and an annular pancreas is about 10% of cases, the duct system fails to fuse, and the original double system persists.
In the third month of fetal life, pancreatic islets (of Langerhans) develop from the parenchymatous pancreatic tissue and scatter throughout the pancreas. Insulin secretion begins at approximately the fifth month. Glucagonand somatostatin-secreting cells also develop from parenchymal cells. Visceral mesoderm surrounding the pancreatic buds forms the pancreatic connective tissue.

Fibroblast growth factor 2 (FGF2) andactivin (a TGF-B family member) produced by the notochord and endothelium of the dorsal aorta repress SHH expression in gut endoderm destined to form the dorsal pancreatic bud. The ventral bud is induced by visceral mesoderm. As a result, expression of the pancreatic and duodenal homeobox 1 (PDX) gene is upregulated. Although all of the downstream effectors of pancreas development have not been determined, it appears that expression of the paired homeobox genes PAX4 and 6 specifies the endocrine cell lineage, such that cells expressing both genes become (insulin), 5 (somatostatin), and y (pancreatic polypeptide) cells; whereas those expressing only PAX6 become & (glucagon) cells.
formed (Fig. 15.23). The malformation sometimes constricts the duodenum and causes complete obstruction.
Accessory pancreatic tissue may be anywhere from the distal end of the esophagus to the tip of the primary intestinal loop. Most frequently, it lies in the mucosa of the stomach and in Meckel's diverticulum, where it may show all of the histological characteristics of the pancreas itself.
Figure 15.23 Annular pancreas. The ventral pancreas splits and forms a ring around the duodenum, occasionally resulting in duodenal stenosis.
Figure 15.24 Embryo during the sixth week of development, showing blood supply to the segments of the gut and formation and rotation of the primary intestinal loop. The superior mesenteric artery forms the axis of this rotation and supplies the midgut. The celiac and inferior mesenteric arteries supply the foregut and hindgut, respectively.

In the 5-week embryo, the midgut is suspended from the dorsal abdominal wall by a short mesentery and communicates with the yolk sac by way of the vitelline duct or yolk stalk (Figs. 15.1 and 15.24). In the adult, the midgut begins immediately distal to the entrance of the bile duct into the duodenum (Fig. 15.15) and terminates at the junction of the proximal twothirds of the transverse colon with the distal third. Over its entire length, the midgut is supplied by the superior mesenteric artery (Fig. 15.24). Development of the midgut is characterized by rapid elongation of the gut and its mesentery, resulting in formation of the primary intestinal loop (Figs. 15.24 and 15.25).At its apex, the loop remains in open connection with the yolk sac by way of the narrow vitelline duct (Fig. 15.24). The cephalic limb of the loop develops into the distal part of the duodenum, the jejunum, and part of the ileum. The caudal limb becomes the lower portion of the ileum, the cecum, the appendix, the ascending colon, and the proximal two-thirds of the transverse colon.

Development of the primary intestinal loop is characterized by rapid elongation, particularly of the cephalic limb. As a result of the rapid growth and expansion of the liver, the abdominal cavity temporarily becomes too small to contain all the
Figure 15.25 A. Primary intestinal loop before rotation (lateral view). The superior mesenteric artery forms the axis of the loop. Arrow, counterclockwise rotation. B. Similar view as in A showing the primary intestinal loop after 180° counterclockwise rotation. The transverse colon passes in front of the duodenum.
Figure 15.26 Umbilical herniation of the intestinal loops in an embryo of approximately 8 weeks (crown-rump length, 35 mm). Coiling of the small intestinal loops and formation of the cecum occur during the herniation. The first 90° of rotation occurs during herniation; the remaining 180° occurs during the return of the gut to the abdominal cavity in the third month.
intestinal loops, and they enter the extraembryonic cavity in the umbilical cord during the sixth week of development (physiological umbilical herniation) (Fig. 15.26).

Coincident with growth in length, the primary intestinal loop rotates around an axis formed by the superior mesenteric artery (Fig. 15.25). When viewed from the front, this rotation is counterclockwise, and it amounts to approximately 270° when it is complete (Figs. 15.24 and 15.25). Even during rotation, elongation of the small intestinal loop continues, and the jejunum and ileum form a number of coiled loops (Fig. 15.26). The large intestine likewise lengthens considerably but does not participate in the coiling phenomenon. Rotation occurs during herniation (about 90°), as well as during return of the intestinal loops into the abdominal cavity (remaining 180°) (Fig. 15.27).
Figure 15.27 A. Anterior view of the intestinal loops after 270° counterclockwise rotation. Note the coiling of the small intestinal loops and the position of the cecal bud in the right upper quadrant of the abdomen. B. Similar view as in A with the intestinal loops in their final position. Displacement of the cecum and appendix caudally places them in the right lower quadrant of the abdomen.
Figure 15.28 Successive stages in development of the cecum and appendix. A. 7 weeks. B. 8 weeks. C. Newborn.

During the 10th week, herniated intestinal loops begin to return to the abdominal cavity. Although the factors responsible for this return are not precisely known, it is thought that regression of the mesonephric kidney, reduced growth of the liver, and expansion of the abdominal cavity play important roles.
peritoneum of the posterior abdominal wall (Fig. 15.30). After fusion of these layers, the ascending and descending colons are permanently anchored in a retroperitoneal position. The appendix, lower end of the cecum, and sigmoid colon, however, retain their free mesenteries
The proximal portion of the jejunum, the first part to reenter the abdominal cavity, comes to lie on the left side (Fig. 15.27A). The later returning loops gradually settle more and more to the right. The cecal bud, which appears at about the sixth week as a small conical dilation of the caudal limb of the primary intestinal loop, is the last part of the gut to reenter the abdominal cavity. Temporarily, it lies in the right upper quadrant directly below the right lobe of the liver (Fig. 15.27A). From here, it descends into the right iliac fossa, placing the ascending colon and hepatic flexure on the right side of the abdominal cavity (Fig. 15.27B). During this process, the distal end of the cecal bud forms a narrow diverticulum, the appendix (Fig. 15.28).
Since the appendix develops during descent of the colon, its final position frequently is posterior to the cecum or colon. These positions of the appendix are called retrocecal or retrocolic, respectively (Fig. 15.29).

The mesentery of the primary intestinal loop, the mesentery proper, undergoes profound changes with rotation and coiling of the bowel. When the caudal limb of the loop moves to the right side of the abdominal cavity, the dorsal mesentery twists around the origin of the superior mesenteric artery (Fig. 15.24). Later, when the ascending and descending portions of the colon obtain their definitive positions, their mesenteries press against the

The fate of the transverse mesocolon is different. It fuses with the posterior wall of the greater omentum (Fig. 15.30) but maintains its mobility. Its line of attachment finally extends from the hepatic flexure of the ascending colon to the splenic flexure of the descending colon (Fig. 15.30B).
The mesentery of the jejunoileal loops is at first continuous with that of the ascending colon (Fig. 15.30A). When the mesentery of the ascending mesocolon fuses with the posterior abdominal wall, the mesentery of the jejunoileal loops obtains a new line of attachment that extends from the area where the duodenum becomes intraperitoneal to the ileocecal junction (Fig. 15.30B).
Figure 15.29 Various positions of the appendix. In about 50% of cases, the appendix is retrocecal or retrocolic.
Figure 15.30 Frontal view of the intestinal loops with A and after removal of B the greater omentum. Gray areas, parts of the dorsal mesentery that fuse with the posterior abdominal wall. Note the line of attachment of the mesentery proper.

Normally, the ascending colon, except for its most caudal part (approximately 1 in), fuses to the posterior abdominal wall and is covered by peritoneum on its anterior surface and sides. Persistence of a portion of the mesocolon gives rise to a mobile cecum. In the most extreme form, the mesentery of the ascending colon fails to fuse with the posterior body wall. Such a long mesentery allows abnormal movements of the gut or even volvulus of the cecum and colon. Similarly, incomplete fusion of the mesentery with the posterior body wall may give rise to retrocolic pockets behind the ascending mesocolon.A retrocolic hernia is entrapment of portions of the small intestine behind the mesocolon.

Omphalocele (Fig. 15.31.4,B) involves herniation of abdominal viscera through an enlarged umbilical ring. The viscera, which may include liver, small and large intestines, stomach, spleen, or gallbladder, are covered by amnion. The origin of the defect is a failure of the bowel to return to the body cavity from its physiological herniation during the 6th to 10th weeks. Omphalocele occurs in 2.5/10,000 births and is associated with a high rate of mortality
(25%) and severe malformations, such as cardiac anomalies (50%) and neural tube defects (40%). Approximately 15% of live-born infants with omphalocele have chromosomal abnormalities.
Gastroschisis (Fig. 15.31C) is the term applied to a protrusion of abdominal contents through the body wall directly into the amniotic cavity. It occurs lateral to the umbilicus usually on the right, and the defect is most likely due to abnormal closure of the body wall around the connecting stalk (see Chapter 7). Viscera are not covered by peritoneum or amnion, and the bowel may be damaged by exposure to amniotic fluid. Gastroschisis occurs in 1/10,000 births but is increasing in frequency, especially among young women (<20 years old). The reason for this increase and why the defect is more prevalent in babies born to younger women is not known. Unlike omphalocele, gastroschisis is not associated with chromosome abnormalities or other severe defects, so the survival rate is excellent. Volvulus (rotation of the bowel) resulting in a compromised blood supply may, however, kill large regions of the intestine and lead to fetal death.

In 2% to 4% of people, a small portion of the vitelline duct persists, forming an outpocketing of the ileum, Meckel's diverticulum
Figure 15.31 A. Omphalocele showing failure of the intestinal loops to return to the body cavity after physiological herniation. The herniated loops are covered by amnion. B. Omphalocele in a newborn. C. Newborn with gastroschisis. Loops of bowel extend through a closure defect in the ventral body wall and are not covered by amnion (see Chapter 7).
or ileal diverticulum (Fig. 15.32A). In the adult, this diverticulum, approximately 40 to 60 cm from the ileocecal valve on the antimesenteric border of the ileum, does not usually cause any symptoms. However, when it contains heterotopic pancreatic tissue or gastric mucosa, it may cause ulceration, bleeding, or even perforation. Sometimes, both ends of the vitelline duct transform into fibrous cords, and the middle portion forms a large
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Figure 15.32 Remnants of the vitelline duct. A. Meckel's, or ileal, diverticulum combined with fibrous cord (vitelline ligament). B. Vitelline cyst attached to the umbilicus and wall of the ileum by vitelline ligaments. C. Vitelline fistula connecting the lumen of the ileum with the umbilicus.
Figure 15.33 A. Abnormal rotation of the primary intestinal loop. The colon is on the left side of the abdomen, and the small intestinal loops are on the right. The ileum enters the cecum from the right. B. The primary intestinal loop is rotated 90° clockwise (reversed rotation). The transverse colon passes behind the duodenum.

cyst, an enterocystoma, or a vitelline cyst (Fig. 15.32B). Since the fibrous cords traverse the peritoneal cavity, intestinal loops may twist around the fibrous strands and become obstructed, causing strangulation or volvulus. In another variation, the vitelline duct remains patent over its entire length, forming a direct communication between the umbilicus and the intestinal tract. This abnormality is known as an umbilical fistula, or a vitelline fistula (Fig. 15.32C). A fecal discharge may then be found at the umbilicus.

Malrotation of the intestinal loop may result in twisting of the intestine (volvulus) and a compromise of the blood supply. Normally, the primary intestinal loop rotates 270° counterclockwise. Occasionally, however, rotation amounts to 90° only. When this occurs, the colon and cecum are the first portions of the gut to return from the umbilical cord, and they settle on the left side of the abdominal cavity (Fig. 15.33A). The later returning loops then move more and more to the right, resulting in a

Reversed rotation of the intestinal loop occurs when the primary loop rotates 90° clockwise. In this abnormality, the transverse colon passes behind the duodenum (Fig. 15.33B) and lies behind the superior mesenteric artery.
Duplications of intestinal loops and cysts may occur anywhere along the length of the gut tube. They are most frequently found in the region of the ileum, where they may vary from a long segment to a small diverticulum. Symptoms usually occur early in life, and 33% are associated with other defects, such as intestinal atresias, imperforate anus, gastroschisis, and omphalocele. Their origin is unknown, although they may result from abnormal proliferations of gut parenchyma.

Atresias and stenoses may occur anywhere along the intestine. Most occur in the duodenum, fewest occur in the colon, and equal numbers occur in the jejunum and ileum (1/1,500 births). Atresias in the upper duodenum are probably due to a lack of recanalization (Fig. 15.18). From the distal portion of the duodenum caudally, however, stenoses and atresias were thought to be caused by vascular "accidents" that resulted in compromised blood flow and tissue necrosis in a section of the gut resulting in the defects. It was suggested that these accidents could be caused by malrotation, volvulus, gastroschisis, omphalocele, and other factors. However, new evidence suggests that problems with gut differentiation can also cause these defects. Thus, misexpression of some HOX genes and of genes and receptors in the FGF family result in gut atresias. In 50% of cases, a region of the bowel is lost, and in 20%, a fibrous cord remains (Fig. 15.34A,B). In another 20%, there is narrowing, with a thin diaphragm separating the larger and smaller
Figure 15.34 The most commonly occurring bowel atresias and stenoses. A. the most common, occurs in 50% of cases; B and C occur in 20% each of cases, and D occurs in 5% of cases. They may be caused by problems with expression of HOX and FGFs and certain FGF receptors during gut differentiation or by vascular accidents; those in the upper duodenum may be caused by a lack of recanalization.Atresias A-C occur in 95% of cases, and stenoses D in only 5%.

pieces of bowel (Fig. 15.34C). Stenoses and multiple atresias account for the remaining 10% of these defects, with a frequency of 5% each (Fig. 15.34D). Apple peel atresia accounts for 10% of atresias. The atresia is in the proximal jejunum, and the intestine is short, with the portion distal to the lesion coiled around a mesenteric remnant (Fig. 15.35). Effects of atresias on newborns depend on the amount of bowel that has been damaged and its location. Some babies with extensive gut involvement have low birth weight and other abnormalities.
Figure 15.35 Apple peel atresia, which occurs in the jejunum and accounts for 10% of bowel atresias. The affected portion of the bowel is coiled around a remnant of mesentery.

The hindgut gives rise to the distal third of the transverse colon, the descending colon, the sigmoid, the rectum, and the upper part of the anal canal. The endoderm of the hindgut also forms the internal lining of the bladder and urethra (see Chapter 16).

The terminal portion of the hindgut enters into the posterior region of the cloaca, the primitive anorectal canal; the allantois enters into the anterior portion, the primitive urogenital sinus (Fig 15.36A). The cloaca itself is an endoderm-lined cavity covered at its ventral boundary by surface ectoderm. This boundary between the endoderm and the ectoderm forms the cloacal membrane (Fig. 15.36). A layer of mesoderm, the urorectal septum, separates the region between the allantois and hindgut. This septum is derived from the merging of mesoderm covering the yolk sac and surrounding the allantois (Figs. 15.1 and 15.36). As the embryo grows and caudal folding continues, the tip of the urorectal septum comes to lie close to the cloacal membrane (Fig. 15.36B,C). At the end of the seventh week, the cloacal membrane ruptures, creating the anal opening for the hindgut and a ventral opening for the urogenital sinus. Between the two, the tip of the urorectal septum forms the perineal body (Fig. 15.36C). The upper part (two-thirds) of the anal canal is derived from endoderm of the hindgut; the lower part (one-third) is derived from ectoderm around the proctodeum (Fig. 15.36B,C). Ectoderm in the region of the proctodeum on the surface of part of the cloaca proliferates and invaginates to create the anal pit (Fig. 15.37D). Subsequently, degeneration of the cloacal membrane (now called the anal membrane) establishes continuity between the upper and lower parts of the anal canal. Since the caudal part of the anal canal originates from ectoderm, it is supplied by the inferior rectal arteries, branches of the internal pudendal arteries. However, the cranial part of the anal canal originates from endoderm and is therefore supplied by the superior rectal artery, a continuation of the inferior mesenteric artery, the artery of the hindgut.The junction between the endodermal and ectodermal regions of the anal canal is delineated by the pectinate line,

just below the anal columns. At this line, the epithelium changes from columnar to stratified squamous epithelium.
Figure 15.36 Cloacal region in embryos at successive stages of development. A. The hindgut enters the posterior portion of the cloaca, the future anorectal canal; the allantois enters the anterior portion, the future urogenital sinus. The urorectal septum is formed by merging of the mesoderm covering the allantois and the yolk sac (Fig. 14.1D). The cloacal membrane, which forms the ventral boundary of the cloaca, is composed of ectoderm and endoderm. B. As caudal folding of the embryo continues, the urorectal septum moves closer to the cloacal membrane. C. Lengthening of the genital tubercle pulls the urogenital portion of the cloaca anteriorly; breakdown of the cloacal membrane creates an opening for the hindgut and one for the urogenital sinus. The tip of the urorectal septum forms the perineal body.

Rectourethral and rectovaginal fistulas, which occur in 1/5,000 live births, may be caused by abnormalities in formation of the cloaca and/or the urorectal septum. For example, if the cloaca is too small, or if the urorectal septum does not extend far enough caudally, then opening of the hindgut shifts anteriorly leading to an opening of the hindgut into the urethra or vagina (Fig. 15.37A,B). Rectoanal fistulas and atresias vary in severity and may leave a narrow tube or fibrous remnant connected to the perineal surface (Fig. 15.37C). These defects are probably due to misexpression of genes during epithelialmesenchymal signaling.Imperforate anus occurs
when the anal membrane fails to breakdown (Fig. 15.37D).
Congenital megacolon is due to an absence of parasympathetic ganglia in the bowel wall (aganglionic megacolon or Hirschsprung disease). These ganglia are derived from neural crest cells that migrate from the neural folds to the wall of the bowel. Mutations in the RET gene, a tyrosine kinase receptor involved in crest cell migration (see Chapter 17), can result in congenital megacolon. In most cases, the rectum is involved, and in 80%, the defect extends to the midpoint of the sigmoid. In only 10% to 20% are the transverse and right-side colonic segments involved, and in 3%, the entire colon is affected.
Figure 15.37 Urorectal. A and rectovaginal. B fistulas that result from incomplete separation of the hindgut from the urogenital sinus by the urorectal septum. These defects may also arise if the cloaca is too small, which causes the opening of the hindgut to shift anteriorly. C. Rectoperineal (fistula). These defects probably result from misexpression of genes during epithelial-mesenchymal signaling in this region. D. Imperforate anus resulting from failure of the anal membrane to break down.
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The epithelium of the digestive system and the parenchyma of its derivatives originate in the endoderm; connective tissue, muscular components, and peritoneal components originate in the mesoderm. Different regions of the gut tube such as the esophagus, stomach, duodenum, etc. are specified by a RA gradient that causes transcription factors unique to each region to be expressed (Fig. 15.2A). Then, differentiation of the gut and its derivatives depends upon reciprocal interactions between the gut endoderm (epithelium) and its surrounding mesoderm (an epithelial-mesenchymal interaction). HOX genes in the mesoderm are induced by SHH secreted by gut endoderm and regulate the craniocaudal organization of the gut and its derivatives. The gut system extends from the oropharyngeal membrane to the cloacal membrane (Fig. 15.5) and is divided into the pharyngeal gut, foregut, midgut, and hindgut. The pharyngeal gut gives rise to the pharynx and related glands (see Chapter 17).

The foregut gives rise to the esophagus, the trachea and lung buds, the stomach, and the duodenum. proximal to the entrance of the bile duct. In addition, the liver, pancreas, and biliary apparatus develop as outgrowths of the endodermal epithelium of the upper part of the duodenum (Fig. 15.15). Since the upper part of the foregut is divided by a septum (the tracheoesophageal septum) into the esophagus posteriorly and the trachea and lung buds anteriorly, deviation of the septum may result in abnormal openings between the trachea and esophagus. The epithelial liver cords and biliary system growing out into the septum transversum (Fig. 15.15) differentiate into parenchyma. Hematopoietic cells (present in the liver in greater numbers before birth than afterward), the Kupffer cells, and connective tissue cells originate in the mesoderm.The pancreas develops from a ventral bud and a dorsal bud that later fuse to form the definitive pancreas (Figs. 15.19 and 15.20). Sometimes, the two parts surround the duodenum (annular pancreas), causing constriction of the gut (Fig. 15.23).
The midgut forms the primary intestinal loop (Fig. 15.24), gives rise to the duodenum distal to the entrance of the bile duct, and continues to the junction of the proximal two-thirds of the transverse colon with the distal third. At its apex, the primary loop remains temporarily in open connection with the yolk sac through the vitelline duct. During the sixth week, the loop grows so rapidly that it protrudes into the umbilical cord (physiological herniation) (Fig. 15.26). During the 10th week, it returns into the abdominal cavity. While these processes are occurring, the midgut loop rotates 270° counterclockwise (Fig. 15.27). Remnants of the vitelline duct, failure of the midgut to return to the abdominal cavity, malrotation, stenosis, and duplication of parts of the gut are common abnormalities.

The hindgut gives rise to the region from the distal third of the transverse colon to the upper part of the anal canal; the distal part of the anal canal originates from ectoderm. The hindgut enters the posterior region of the cloaca (future anorectal canal), and the allantois enters the anterior region (future urogenital sinus). The urorectal septum will divide the two regions (Fig. 15.36) and breakdown of the cloacal membrane covering this area will provide communication to the exterior for the anus and urogenital sinus. Abnormalities in the size of the posterior region of the cloaca shift the entrance of the anus anteriorly, causing rectovaginal and rectourethral fistulas and atresias (Fig. 15.37).
The anal canal itself is derived from endoderm (cranial part) and ectoderm (caudal part). The caudal part is formed by invaginating ectoderm around the proctodeum. Vascular supply to the anal canal reflects its dual origin. Thus, the cranial part is supplied by the superior rectal artery from the inferior mesenteric artery, the artery of the hindgut, whereas the caudal part is supplied by the inferior rectal artery, a branch of the internal pudendal artery.

1. Prenatal ultrasound showed polyhydramnios at 36 weeks, and at birth, the infant had excessive fluids in its mouth and difficulty breathing. What birth defect might cause these conditions?
2. Prenatal ultrasound at 20 weeks revealed a midline mass that appeared to contain intestines and was membrane bound. What diagnosis would you make, and what would be the prognosis for this infant?
3. At birth, a baby girl has meconium in her vagina and no anal opening. What type of birth defect does she have, and what was its embryological origin?
divided into two entirely different components: the urinary system and the genital system. Embryologically and anatomically, however, they are intimately interwoven. Both develop from a common mesodermal ridge (intermediate mesoderm) along the posterior wall of the abdominal cavity, and initially, the excretory ducts of both systems enter a common cavity, the cloaca. unctionally, the urogenital system can be

Three slightly overlapping kidney systems are formed in a cranial-to-caudal sequence during intrauterine life in humans: the pronephros, mesonephros, and metanephros. The first of these systems is rudimentary and nonfunctional; the second may function for a short time during the early fetal period; the third forms the permanent kidney.

At the beginning of the fourth week, the pronephros is represented by 7 to 10 solid cell groups in the cervical region (Figs. 16.1 and 16.2). These groups form vestigial excretory units, nephrotomes, that regress before more caudal ones are formed. By the end of the fourth week, all indications of the pronephric system have disappeared.

The mesonephros and mesonephric ducts are derived from intermediate mesoderm from upper thoracic to upper lumbar (L3) segments (Fig. 16.2). Early in the fourth week of development, during regression of the pronephric system, the first excretory tubules of the mesonephros appear. They lengthen rapidly, form an S-shaped loop, and acquire a tuft of capillaries that will form a glomerulus at their medial extremity (Fig. 16.3A). Around the glomerulus, the tubules form Bowman's capsule, and together these
Figure 16.1 Transverse sections through embryos at various stages of development showing formation of nephric tubules. A. 21 days. B. 25 days. Note formation of external and internal glomeruli and the open connection between the intraembryonic cavity and the nephric tubule.
Segmented intermediate
mesoderm
Figure 16.2 A. Relationship of the intermediate mesoderm of the pronephric, mesonephric, and metanephric systems. In cervical and upper thoracic regions, intermediate mesoderm is segmented; in lower thoracic, lumbar, and sacral regions, it forms a solid, unsegmented mass of tissue, the nephrogenic cord. Note the longitudinal collecting duct, formed initially by the pronephros but later by the mesonephros (Mesonephric duct). B. Excretory tubules of the pronephric and mesonephric systems in a 5-week embryo.
structures constitute a renal corpuscle. Laterally, the tubule enters the longitudinal collecting duct known as the mesonephric or wolffian duct 16.2 and

In the middle of the second month, the mesonephros forms a large ovoid organ on each side of the midline (Fig. 16.3). Since the developing gonad is on its medial side, the ridge formed by both organs is known as the urogenital ridge (Fig. 16.3). While caudal tubules are still differentiating, cranial tubules and glomeruli show degenerative changes, and by the end of the second month, the majority have disappeared. In the male, a few of the caudal tubules and the mesonephric duct persist and participate in formation of the genital system, but they disappear in the female.

The third urinary organ, the metanephros or permanent kidney, appears in the fifth week. Its excretory units develop from metanephric mesoderm (Fig. 16.4) in the same manner as in the mesonephric system. The development of the duct system differs from that of the other kidney systems.
Collecting ducts of the permanent kidney develop from the ureteric bud, an outgrowth of the mesonephric duct close to its entrance to the cloaca (Fig. 16.4). The bud penetrates the metanephric tissue, which is molded over its distal end as a cap (Fig. 16.4). Subsequently, the bud dilates, forming the primitive renal pelvis, and splits into cranial and caudal portions, the future major calyces (Fig. 16.54,B).
Each calyx forms two new buds while penetrating the metanephric tissue. These buds continue to subdivide until 12 or more generations of tubules have formed (Fig. 16.5). Meanwhile, at the periphery, more tubules form until the end of the fifth month. The tubules of the second order enlarge and absorb those of the third and fourth generations, forming the minor calyces of the renal pelvis. During further development, collecting tubules of the fifth and successive generations elongate considerably and converge on the minor calyx, forming the renal pyramid (Fig. 16.5D). The ureteric bud gives rise to the ureter, the renal pelvis, the major and minor calyces, and approximately 1 to 3 million collecting tubules.
233
Excretory tubule
Figure 16.3 A. Transverse section through the urogenital ridge in the lower thoracic region of a 5-week embryo showing formation of an excretory tubule of the mesonephric system. Note the appearance of Bowman's capsule and the gonadal ridge. The mesonephros and gonad are attached to the posterior abdominal wall by a broad urogenital mesentery. B. Relation of the gonad and the mesonephros. Note the size of the mesonephros. The mesonephric duct (wolffian duct) runs along the lateral side of the mesonephros.

Figure 16.5 Development of the renal pelvis, calyces, and collecting tubules of the metanephros. A. 6 weeks. B. At the end of the sixth week. C. 7 weeks. D. Newborn. Note the pyramid form of the collecting tubules entering the minor calyx.

Each newly formed collecting tubule is covered at its distal end by a metanephric tissue cap (Fig. 16.6A). Under the inductive influence of the tubule, cells of the tissue cap form small vesicles, the renal vesicles, which in turn give rise to small S-shaped tubules (Fig. 16.6B,C). Capillaries grow into the pocket at one end of the S and differentiate into glomeruli. These tubules, together with their glomeruli, form nephrons, or excretory units. The proximal end of each nephron forms Bowman's capsule, which is deeply indented by a glomerulus (Fig. 16.6C,D).The distal end forms an open connection with one of the collecting tubules, establishing a passageway from Bowman's capsule to the collecting unit. Continuous lengthening of the excretory tubule results in formation of the
Figure 16.6 Development of a metanephric excretory unit. Arrows, the place where the excretory unit (blue) establishes an open communication with the collecting system (yellow), allowing flow of urine from the glomerulus into the collecting
proximal convoluted tubule, loop of Henle, and distal convoluted tubule (Fig. 16.6E,F). Hence, the kidney develops from two sources: (1) metanephric mesoderm, which provides excretory units and (2) the ureteric bud, which gives rise to the collecting system.
Nephrons are formed until birth, at which time there are approximately 1 million in each kidney. Urine production begins early in gestation, soon after differentiation of the glomerular capillaries, which start to form by the 10th week. At birth, the kidneys have a lobulated appearance, but the lobulation disappears during infancy as a result of further growth of the nephrons, although there is no increase in their number.

As with most organs, differentiation of the kidney involves epithelial mesenchymal interactions. In this example, epithelium of the ureteric bud from the mesonephros interacts with mesenchyme of the metanephric blastema (Fig. 16.7). The mesenchyme expresses WT1, a transcription factor that makes this tissue competent to respond to induction by the ureteric bud. WT7 also regulates production of glialderived neurotrophic factor (GDNF) and hepatocyte growth factor (HGF, or scatter factor) by the mesenchyme, and these proteins stimulate branching and growth of the ureteric buds (Fig. 16.7A).The tyrosine kinase receptors RET, for GDNE and MET, for HGE are synthesized by the epithelium of the ureteric buds, establishing signaling pathways between the two tissues. In turn, the buds induce the mesenchyme via fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 7 (BMP7) (Fig. 16.7A). Both of these growth factors block apoptosis and stimulate proliferation in the metanephric mesenchyme while maintaining production of WT1. Conversion of the mesenchyme to an epithelium for nephron formation is also mediated by the ureteric buds through expression of WNT9B and WNT6, which upregulate PAX2 and WNT4 in the metanephric mesenchyme. PAX2 promotes condensation of the mesenchyme preparatory to tubule formation, while WNT¢4 causes the condensed mesenchyme to epithelialize and form tubules (Fig. 16.7B). Because of these interactions, modifications in the extracellular matrix also occur. Thus, fibronectin, collagen I, and collagen III are replaced with laminin and type IV collagen, characteristic of an epithelial basal lamina (Fig. 16.7B). In addition, the cell adhesion molecules syndecan and E-cadherin, which are essential for condensation of the mesenchyme into an epithelium, are synthesized.
tubule

Figure 16.7 Genes involved in differentiation of the kidney. A. WT!, expressed by the mesenchyme, enables this tissue to respond to induction by the ureteric bud. GDNF and HGF, also produced by the mesenchyme, interact through their receptors, RET and MET, respectively, in the ureteric bud epithelium, to stimulate growth of the bud and maintain the interactions. The growth factors FGF2 and BMP7 stimulate proliferation of the mesenchyme and maintain WT! expression. B. WNT9B and WNT6 secreted by branches of the ureteric bud epithelium cause upregulation of PAX2 and WNT4 in the surrounding mesenchyme. In turn, these genes cause the mesenchyme to epithelialize (PAX2) and to then form tubules (WNT4). Changes in the extracellular matrix also occur, such that laminin and type IV collagen form a basement membrane (orange) for the epithelial cells.

Wilms' tumor is a cancer of the kidneys that usually affects children by 5 years of age but may also occur in the fetus.Wilms' tumor is due to mutations in the WTI gene on I||pI3, and it may be associated with other abnormalities and syndromes. For example, WAGR syndrome is characterized by Wilms' tumor, aniridia, gonadoblastomas (tumors of the gonads), and mental retardation (intellectual disability). The constellation of defects is due to a microdeletion in chromosome || that includes both the PAX6 (aniridia) and WT! genes that are only 700 kb apart. Similarly, Denys-Drash syndrome consists of renal failure, ambiguous genitalia, and Wilms' tumor.

Renal dysplasias and agenesis are a spectrum of severe malformations that represent the primary diseases requiring dialysis and transplantation in the first years of life. Multicystic dysplastic kidney is one example of this group of abnormalities in which numerous ducts are surrounded by undifferentiated cells. Nephrons fail to develop, and the ureteric bud fails to branch, so that the collecting ducts never form. In some cases, these defects cause involution of the kidneys and renal agenesis. Renal agenesis may also arise if the interaction between the metanephric mesoderm and the ureteric bud fails to occur. Normally, during the interaction, GDNF produced by the metanephric mesoderm produces branching and growth of the ureteric bud. Thus, mutations in genes that regulate GDNF expression of signaling may result in renal agenesis. Examples include the gene SALLI, responsible for TownesBrock syndrome; PAX2 that causes renal coloboma syndrome; and EYA/ that results in branchio-otorenal syndrome. Bilateral renal agenesis, which occurs in 1/10,000 births, results in renal failure. The baby presents with Potter sequence, characterized by anuria, oligohydramnios (decreased volume of amniotic fluid), and hypoplastic lungs secondary to the oligohydramnios. In 85% of cases, other severe defects, including absence or abnormalities of the vagina and uterus, vas deferens, and seminal vesicles, accompany this condition. Common associated defects in other systems include cardiac anomalies, tracheal and duodenal atresias, cleft lip and palate, and brain abnormalities. Because of the oligohydramnios, the uterine cavity is compressed resulting in a characteristic appearance of the fetus, including a flattened face (Potter facies) and club feet.

In congenital polycystic kidney disease (Fig. 16.8), numerous cysts form. It may be inherited as an autosomal recessive or autosomal dominant disorder or may be caused by other factors. Autosomal recessive polycystic kidney disease (ARPKD), which occurs in 1/5,000 births, is a progressive disorder in which cysts form from collecting ducts. The kidneys become very large, and renal failure occurs in infancy or childhood. In autosomal dominant polycystic kidney disease (ADPKD), cysts form from all segments of the nephron and usually do not cause renal failure until adulthood. The autosomal dominant disease is more common (1/500 to 1/1,000 births), but less progressive than the autosomal recessive disease. Both types of disease are linked to mutations in genes that encode proteins localized in cilia and that are important for ciliary function. These abnormalities belong to a growing group of diseases called the ciliopathies that are due to mutations in cilia related proteins. These disorders include Bardet-Biedal syndrome, characterized by renal cysts, obesity, intellectual disability, and limb defects, and Meckel Gruber syndrome, characterized by renal cysts, hydrocephalus, miropthalmia, cleft palate, absence of the olfactory tract, and polydactyly. Since cilia are present on most cell types and in most tissues, many organ systems can be affected by abnormalities in ciliary structure and function.

Duplication of the ureter results from early splitting of the ureteric bud (Fig. 16.9). Splitting may be partial or complete, and metanephric tissue may be divided into two parts, each with its own renal pelvis and ureter. More frequently, however, the two parts have a number of lobes in common as a result of intermingling of collecting tubules. In rare cases, one ureter opens into the bladder, and the other is ectopic, entering the vagina, urethra, or vestibule (Fig. 16.9C). This abnormality results from development of two ureteric buds. One of the buds usually has a normal position, whereas the abnormal bud moves down together with the mesonephric duct. Thus, it has a low, abnormal entrance in the bladder, urethra, vagina, or epididymal region.
Figure 16.8 Surface view of a fetal kidney with multiple cysts characteristic of polycystic kidney disease.
Figure 16.9 A,B.A complete and a partial double ureter. C. Possible sites of ectopic ureteral openings in the vagina, urethra, and vestibule. D,E. Photomicrographs of complete and partial duplications of the ureters (U). Arrows, duplicated hilum; B, bladder; K, kidneys; ML, median umbilical ligament.

The kidney, initially in the pelvic region, later shifts to a more cranial position in the abdomen. This ascent of the kidney is caused by diminution of body curvature and by growth of the body in the lumbar and sacral regions
(Fig. 16.10). In the pelvis, the metanephros receives its arterial supply from a pelvic branch of the aorta. During its ascent to the abdominal level, it is vascularized by arteries that originate from the aorta at continuously higher levels. The lower vessels usually degenerate, but some may remain.
Figure 16.10 A-C. Ascent of the kidneys. Note the change in position between the mesonephric and metanephric systems. The mesonephric system degenerates almost entirely, and only a few remnants persist in close contact with the gonad. In both male and female embryos, the gonads descend from their original level to a much lower position.

During their ascent, the kidneys pass through the arterial fork formed by the umbilical arteries, but occasionally, one of them fails to do so. Remaining in the pelvis close to the common iliac artery, it is known as a pelvic kidney (Fig. 16.1 1A). Sometimes, the kidneys are pushed so close together during their passage through the arterial fork, that the lower poles fuse, forming a horseshoe kidney (Fig. 16.11B,C). The horseshoe kidney is usually at the level of the lower lumbar vertebrae, since its ascent is prevented by the root of the inferior mesenteric artery (Fig. 16.11B). The ureters arise from the anterior surface of the kidney and pass ventral to the isthmus in a caudal direction. Horseshoe kidney is found in 1/600 people.
Accessory renal arteries are common; they derive from the persistence of embryonic vessels that formed during ascent of the kidneys. These arteries usually arise from the aorta and enter the superior or inferior poles of the kidneys.
Figure 16.11 A. Unilateral pelvic kidney showing the position of the adrenal gland on the affected side. B,C. Drawing and photomicrograph, respectively, of horseshoe kidneys showing the position of the inferior mesenteric artery.

The definitive kidney formed from the metanephros becomes functional near the 12th week. Urine is passed into the amniotic cavity and mixes with the amniotic fluid. The fluid is swallowed by the fetus and recycles through the kidneys. During fetal life, the kidneys are not responsible for excretion of waste products, since the placenta serves this function.

During the fourth to seventh weeks of development, the cloaca divides into the urogenital sinus anteriorly and the anal canal posteriorly (Fig. 16.12) (see Chapter 15, p. 227). The urorectal septum is a layer of mesoderm between the primitive anal canal and the urogenital sinus. The tip of the septum will form the perineal body (Fig. 16.12C). Three portions of the urogenital sinus can be distinguished: The upper and largest part is the urinary bladder (Fig. 16.13A). Initially, the bladder is continuous with the allantois, but when the lumen of the allantois is obliterated, a thick fibrous cord, the urachus, remains and connects the apex of the bladder with the umbilicus (Fig. 16.13B). In the adult, it forms the median umbilical ligament. The next part is a rather narrow canal, the pelvic part of the urogenital sinus, which in the male gives rise to the prostatic and membranous parts of the urethra. The last part is the phallic part of the urogenital sinus. It is flattened from side to side, and as the genital tubercle grows, this part of the sinus will be pulled ventrally (Fig. 16.13A). (Development of the phallic part of the urogenital sinus differs greatly between the two sexes.)
During differentiation of the cloaca, the caudal portions of the mesonephric ducts are absorbed into the wall of the urinary bladder (Fig. 16.14). Consequently, the ureters, initially outgrowths from the mesonephric ducts, enter the bladder separately (Fig. 16.14B). As a result of ascent of the kidneys, the orifices of the ureters move
Figure absorbed
16.12
Divisions of
the cloaca
into the
urogenital sinus
and anorectal
canal.
The mesonephric
duct is
gradually into
the wall
of the
urogenital sinus,
and the
ureters enter
C.
8
weeks.

farther cranially; those of the mesonephric ducts move close together to enter the prostatic urethra and in the male become the ejaculatory ducts (Fig. 16.14C,D). Since both the mesonephric ducts and ureters originate in the mesoderm, the mucosa of the bladder formed by incorporation of the ducts (the trigone of the bladder) is also mesodermal. With time, the mesodermal lining of the trigone is replaced by endodermal epithelium, so that finally, the inside of the bladder is completely lined with endodermal epithelium.
The epithelium of the urethra in both sexes originates in the endoderm; the surrounding connective and smooth muscle tissue is derived from visceral mesoderm. At the end of the third month, epithelium of the prostatic urethra begins to proliferate and forms a number of outgrowths that penetrate the surrounding mesenchyme. In the male, these buds form the prostate gland (Fig. 16.13B). In the female, the cranial part of the urethra gives rise to the urethral and paraurethral glands.
Figure 16.13 A. Development of the urogenital sinus into the urinary bladder and definitive urogenital sinus. B. In the male, the definitive urogenital sinus develops into the penile urethra. The prostate gland is formed by buds from the urethra, and seminal vesicles are formed by budding from the ductus deferens.
separately.
A.
At the
end of
the fifth
week.
B.
7
weeks.
241
Figure 16.14 Dorsal views of the bladder showing the relation of the ureters and mesonephric ducts during development. Initially, the ureters are formed by an outgrowth of the mesonephric duct A, but with time, they assume a separate entrance into the urinary bladder B—D. Note the trigone of the bladder formed by incorporation of the mesonephric ducts C,D.

When the lumen of the intraembryonic portion of the allantois persists, a urachal fistula may cause urine to drain from the umbilicus (Fig. 16.15A). If only a local area of the allantois persists, secretory activity of its lining results in a cystic dilation, a urachal cyst (Fig. 16.15B). When the lumen in the upper part persists, it forms a urachal sinus. This sinus is usually continuous with the urinary bladder (Fig. 16.15C).
lateral body wall folds to close in the midline in the pelvic region (see Chapter 7, p. 87). This anomaly is rare, occurring in 2/100,000 live births.
Exstrophy of the bladder (Fig. 16.16A) is a ventral body wall defect in which the bladder mucosa is exposed. Epispadias is a constant feature (Fig. 16.35), and the open urinary tract extends along the dorsal aspect of the penis through the bladder to the umbilicus. Exstrophy of the bladder is probably due to failure of the
Exstrophy of the cloaca (Fig. 16.16B) is a more severe ventral body wall defect in which progression and closure of the lateral body wall folds are disrupted to a greater degree than is observed in bladder exstrophy (see Chapter 7, p. 87). In addition to the closure defect, normal development of the urorectal septum is altered, such that anal canal malformations and imperforate anus occur (see Chapter 15, p. 228). Furthermore, since the body folds do not fuse, the genital swellings are widely spaced resulting in defects in the external genitalia (Fig. 16.16B). Occurrence of the defect is rare (1/30,000).
Figure 16.15 A. Urachal fistula. B. Urachal cyst. C. Urachal sinus. The sinus may or may not be in open communication with the urinary bladder.
Figure 16.16 A. Exstrophy of the bladder. B. Cloacal exstrophy in a newborn.

do not acquire male or female morphological characteristics until the seventh week of development.
Sex differentiation is a complex process that involves many genes, including some that are autosomal. The key to sexual dimorphism is the Y chromosome, which contains the testis-determining gene called the SRY (sex-determining region on Y) gene on its short arm (Yp11). The protein product of this gene is a transcription factor initiating a cascade of downstream genes that determine the fate of rudimentary sexual organs. The SRY protein is the testis-determining factor; under its influence, male development occurs; in its absence, female development is established.

Although the sex of the embryo is determined genetically at the time of fertilization, the gonads
Gonads appear initially as a pair of longitudinal ridges, the genital or gonadal ridges (Fig. 16.17). They are formed by proliferation of the epithelium and a condensation of underlying mesenchyme. Germ cells do not appear in the genital ridges until the sixth week of development.
Primordial germ cells originate in the epiblast, migrate through the primitive streak, and by the third week reside among endoderm cells in the wall of the yolk sac close to the allantois (Fig. 16.18A). During the fourth week, they migrate by ameboid movement along the dorsal mesentery of the hindgut (Fig. 16.18A,B), arriving at the primitive gonads at the beginning of the fifth week and invading the genital ridges in the sixth week. If they fail to reach the ridges, the gonads
Figure 16.17 A. Relation of the genital ridge and the mesonephros showing location of the mesonephric duct. B. Transverse section the and at the level indicated in A.
through mesonephros genital ridge
Figure 16.18 A.A 3-week embryo showing the primordial germ cells in the wall of the yolk sac close to the attachment of the allantois. B. Migrational path of the primordial germ cells along the wall of the hindgut and the dorsal mesentery into the genital ridge.
do not develop. Hence, the primordial germ cells have an inductive influence on development of the gonad into ovary or testis. Shortly before and during arrival of primordial germ cells, the epithelium of the genital ridge proliferates, and epithelial cells penetrate the underlying mesenchyme. Here they form a number of irregularly shaped cords, the primitive sex cords (Fig. 16.19). In both male and female embryos, these cords are connected to surface epithelium, and it is impossible to differentiate between the male and female gonad. Hence, the gonad is known as the indifferent gonad.

the gland, the cords break up into a network of tiny cell strands that later give rise to tubules of the rete testis (Fig. 16.20A,B). During further development, a dense layer of fibrous connective tissue, the tunica albuginea, separates the testis cords from the surface epithelium (Fig. 16.20). In the fourth month, the testis cords become horseshoe-shaped, and their extremities are continuous with those of the rete testis (Fig. 16.20B). Testis cords are now composed of primitive germ cells and sustentacular cells of Sertoli derived from the surface epithelium of the gland. Interstitial cells of Leydig, derived from

If the embryo is genetically male, the primordial germ cells carry an XY sex chromosome complex. Under influence of the SRY gene on the Y chromosome, which encodes the testisdetermining factor, the primitive sex cords continue to proliferate and penetrate deep into the medulla to form the testis or medullary cords (Figs. 16.20A and 16.21). Toward the hilum of the original mesenchyme of the gonadal ridge, lie between the testis cords. They begin development shortly after onset of differentiation of these cords. By the eighth week of gestation, Leydig cells begin production of testosterone and the testis is able to influence sexual differentiation of the genital ducts and external genitalia. Testis cords remain solid until puberty, when they acquire a lumen, thus forming the seminiferous tubules. Once the seminiferous tubules
Figure 16.20 A. Transverse section through the testis in the eighth week, showing the tunica albuginea, testis cords, rete testis, and primordial germ cells. The glomerulus and Bowman's capsule of the mesonephric excretory tubule are degenerating. B. Testis and genital duct in the fourth month. The horseshoe-shaped testis cords are continuous with the rete testis cords. Note the ductuli efferentes (excretory mesonephric tubules), which enter the mesonephric duct.
are canalized, they join the rete testis tubules, which in turn enter the ductuli efferentes. These efferent ductules are the remaining parts of the excretory tubules of the mesonephric system. They link the rete testis and the mesonephric or wolffian duct, which becomes the ductus deferens (Fig. 16.20B).

In female embryos with an XX sex chromosome complement and no Y chromosome, primitive sex cords dissociate into irregular cell clusters (Figs. 16.21 and 16.22A).These clusters, containing groups of primitive germ cells, occupy the medullary part of the ovary. Later, they disappear and are replaced by a vascular stroma that forms the ovarian medulla (Fig. 16.22). The surface epithelium of the female gonad, unlike that of the male, continues to proliferate. In the seventh week, it gives rise to a second generation of cords, cortical cords, which penetrate the underlying mesenchyme but remain close to the surface (Fig. 16.22A). In the third month, these cords split into isolated cell clusters. Cells in these clusters continue to proliferate and begin to surround each oogonium with a layer of epithelial cells called follicular cells. Together, the oogonia and follicular cells constitute a primordial follicle (Fig. 16.22B; see Chapter 2, p. 22). It may thus be stated that the genetic sex of an embryo is determined at the time of fertilization, depending on whether the spermatocyte carries an X or aY chromosome. In embryos with an XX sex chromosome configuration, medullary cords of the gonad regress, and a secondary generation of cortical cords develops (Figs. 16.21 and 16.22).In embryos with an XY sex chromosome complex, medullary cords develop into testis cords, and secondary cortical cords fail to develop (Figs. 16.20 and 16.21).
245
Figure 16.22 A. Transverse section of the ovary at the seventh week, showing degeneration of the primitive (medullary) sex cords and formation of the cortical cords. B. Ovary and genital ducts in the fifth month. Note degeneration of the medullary cords. The excretory mesonephric tubules (effere! nt ductules) do not communicate with the rete. The cortical zone of the ovary contains groups of oogonia surrounded by follicular cells.

Indifferent Stage
Initially, both male and female embryos have two pairs of genital ducts: mesonephric (wolffian) ducts and paramesonephric (miillerian) ducts. The paramesonephric duct arises as a longitudinal invagination of the epithelium on the anterolateral surface of the urogenital ridge (Fig. 16.23). Cranially, the duct opens into the abdominal cavity with a funnel-like structure. Caudally, it first runs lateral to the mesonephric duct, then crosses it ventrally to grow caudomedially (Fig. 16.23). In the midline, it comes in close contact with the paramesonephric duct from the opposite side. The two ducts are initially separated by a septum but later fuse to form the uterine canal (Fig. 16.24A). The caudal tip of the combined ducts projects into the posterior wall of the urogenital sinus, where it causes a small swelling, the paramesonephric or miillerian tubercle (Fig. 16.24A). The mesonephric ducts open into the urogenital sinus on either side of the miillerian tubercle.

SRY is a transcription factor and the master gene for testes development. It appears to act in conjunction with the autosomal gene SOX9, a transcriptional regulator, that can also induce testes differentiation (Fig. 16.25 for a potential pathway for these genes). SOX9 is known to bind to the promoter region of the gene for antimiillerian hormone (AMH; also called miillerian inhibiting
Figure 16.23 Genital ducts in the sixth week in the male A and female B. The mesonephric and paramesonephric ducts are present in both. Note the excretory tubules of the mesonephros and their relation to the developing gonad in both sexes.

Figure 16.24 A. Genital ducts in the female at the end of the second month. Note the paramesonephric (miillerian) tubercle and formation of the uterine canal. B. Genital ducts after descent of the ovary. The only parts remaining from the mesonephric system are the epoophoron, paroophoron, and Gartner's cyst. Note the suspensory ligament of the ovary, ligament of the ovary proper, and round ligament of the uterus substance [MIS]) and probably regulates this gene's expression. Initially, SRY and/or SOX9 induce the testes to secrete FGF9 that acts as a chemotactic factor that causes tubules from the mesonephric duct to penetrate the gonadal ridge. Without penetration by these tubules, differentiation of the testes does not continue. Next, SRY either directly or indirectly (through SOX9) upregulates production of steroidogenesis factor 1 (SF1) that stimulates differentiation of Sertoli and Leydig cells. SF1 working with SOX9 elevates the concentration of AMH leading to regression of the paramesonephric (miillerian) ducts. In Leydig cells, SF1 upregulates the genes for enzymes that synthesize testosterone. Testosterone enters cells of target tissues where it may remain intact or be converted to dihydrotestosterone by a 5-@ reductase enzyme. Testosterone and dihydrotestosterone bind to a specific high-affinity intracellular receptor, and this hormone receptor complex is transported to the nucleus where it binds to DNA to regulate transcription of tissue-specific genes and their protein products. Testosterone receptor complexes mediate virilization of the mesonephric ducts to form the vas deferens, seminal vesicles, efferent ductules, and epididymis. Dihydrotestosterone receptor complexes modulate differentiation of the male external genitalia (Fig. 16.26). WNT is the ovary-determining gene.This gene upregulates DAX1, a member of the nuclear hormone receptor family, that inhibits the

function of SOX9. In addition, WNT4 regulates expression of other genes responsible for ovarian differentiation, but these target genes have not been identified. One target may be the TAFII105 gene, whose protein product is a subunit for the TATA-binding protein for RNA polymerase in ovarian follicular cells. Female mice that do not synthesize this subunit do not form ovaries.
Estrogens are also involved in sexual differentiation and under their influence, the paramesonephric (miillerian) ducts are stimulated to form the uterine tubes, uterus, cervix, and upper vagina. In addition, estrogens act on the external genitalia at the indifferent stage to form the labia majora, labia minora, clitoris, and lower vagina (Fig. 16.26).

As the mesonephros regresses, a few excretory tubules, the epigenital tubules, establish contact with cords of the rete testis and finally form the efferent ductules of the testis (Fig. 16.27). Excretory tubules along the caudal pole of the testis, the paragenital tubules, do not join the cords of the rete testis (Fig. 16.27). Their vestiges are collectively known as the paradidymis.
Except for the most cranial portion, the appendix epididymis, the mesonephric ducts persist and form the main genital ducts (Fig. 16.27). Immediately below the entrance of the efferent
MALES
(XY)

Figure 16.25 Schematic showing genes responsible for differentiation of the testes and ovaries. In both males and females, SOX9 and WNT4 are expressed in the gonadal ridges. In males, the expression of SRY upregulates SOX9, which in turn activates expression of SF/ and other genes responsible for testes differentiation, while inhibiting expression of WNT4. In females, the uninhibited expression of WNT4 upregulates DAX/ that in turn inhibits SOX9 expression. Then, under the continued influence of WNT4, other downstream target genes (perhaps TAFII/05) induce ovarian differentiation.
ductules, the mesonephric ducts elongate and become highly convoluted, forming the (ductus) epididymis. From the tail of the epididymis to the outbudding of the seminal vesicle, the mesonephric ducts obtain a thick muscular coat and form the ductus deferens. The region of the ducts beyond the seminal vesicles is the ejaculatory duct. Under the influence of antimullerian hormone (AMH) produced by sertoli cells, paramesonephric ducts in the male degenerate except for a small portion at their cranial ends, the appendix testis (Fig. 16.27B).
249
Figure 16.27 A. Genital ducts in the male in the fourth month. Cranial and caudal (paragenital tubule) segments of the mesonephric system regress. B. Genital ducts after descent of the testis. Note the horseshoe-shaped testis cords, rete testis, and efferent ductules entering the ductus deferens. The paradidymis is formed by remnants of the paragenital mesonephric tubules. The paramesonephric duct has degenerated except for the appendix testis. The prostatic utricle is an outpocketing from the urethra.

The paramesonephric ducts develop into the main genital ducts of the female. Initially, three parts can be recognized in each duct: (1) a cranial vertical portion that opens into the abdominal cavity, (2) a horizontal part that crosses the mesonephric duct, and (3) a caudal vertical part that fuses with its partner from the opposite side (Fig. 16.24). With descent of the ovary, the first two parts develop into the uterine tube (Fig. 16.24B), and the caudal parts fuse to form the uterine canal. When the second part of the paramesonephric ducts moves mediocaudally, the urogenital ridges gradually come to lie in a transverse plane (Fig. 16.28A4,B). After the ducts fuse in the midline, a broad transverse pelvic fold is established (Fig. 16.28C).This fold, which extends from the lateral sides of the fused paramesonephric ducts toward the wall of the pelvis, is the broad ligament of the uterus. The uterine tube lies in its upper border, and the ovary lies on its posterior surface (Fig. 16.28C).The uterus and broad ligaments divide the pelvic cavity into the uterorectal pouch and the uterovesical pouch. The fused paramesonephric ducts give rise to the corpus and cervix of the uterus.
paramesonephric
Figure 16.28 Transverse sections through the urogenital ridge at progressively lower levels. A,B. The ducts approach each other in the midline and fuse. C. As a result of fusion, a transverse fold, the broad ligament of the uterus, forms in the pelvis. The gonads come to lie at the posterior aspect of the transverse fold.
Figure 16.29 Formation of the uterus and vagina. A. 9 weeks. Note the disappearance of the uterine septum. B. At the end of the third month. Note the tissue of the sinovaginal bulbs. C. Newborn. The fornices and the upper portion of the vagina are formed by vacuolization of the paramesonephric tissue, and the lower portion of the vagina is formed by vacuolization of the sinovaginal bulbs.

They are surrounded by a layer of mesenchyme that forms the muscular coat of the uterus, the myometrium, and its peritoneal covering, the perimetrium.
Thus, the vagina has dual origin, with the

Shortly after the solid tip of the paramesonephric ducts reaches the urogenital sinus (Figs. 16.29A and 16.30A), two solid evaginations grow out from the pelvic part of the sinus (Figs. 16.29B and 16.30B). These evaginations, the sinovaginal bulbs, proliferate and form a solid vaginal plate. Proliferation continues at the cranial end of the plate, increasing the distance between the uterus and the urogenital sinus. By the fifth month, the vaginal outgrowth is entirely canalized. The wing-like expansions of the vagina around the end of the uterus, the vaginal fornices, are of paramesonephric origin (Fig. 16.30C).
a upper portion derived from the uterine canal and the lower portion derived from the urogenital sinus. The lumen of the vagina remains separated from that of the urogenital sinus by a thin tissue plate, the hymen (Figs. 16.29C and 16.30C), which consists of the epithelial lining of the sinus and a thin layer of vaginal cells. It usually develops a small opening during perinatal life. The female retain of the may some remnants cranial and caudal excretory tubules in the mesovarium, where they form the epoophoron and paroophoron, respectively (Fig. 16.24B). The mesonephric duct disappears except for a small cranial portion found in the epoophoron and occasionally a small caudal portion that may be found in the wall of the uterus or vagina. Later in life, it may form Gartner's cyst (Fig. 16.24B).
Figure 16.30 Sagittal sections showing formation of the uterus and vagina at various stages of development. A. 9 weeks.

Duplications of the uterus result from lack of fusion of the paramesonephric ducts in a local area or throughout their normal line of fusion. In its extreme form, the uterus is entirely double (uterus didelphys) (Fig. 16.31A); in the least severe form, it is only slightly indented in the middle (uterus arcuatus) (Fig. 16.31B). One of the relatively common anomalies is the uterus bicornis, in which the uterus has two horns entering a common vagina (Fig. 16.31C). This condition is normal in many mammals below the primates.
In patients with complete or partial atresia of one of the paramesonephric ducts, the rudimentary part lies as an appendage to the well-developed side. Since its lumen usually does not communicate with the vagina, complications are common (uterus bicornis unicollis with one rudimentary horn) (Fig. 16.31D). If the atresia involves both sides, an atresia of the cervix may result (Fig. 16.316). If the sinovaginal bulbs fail to fuse or do not develop at all, a double vagina or atresia of the vagina, respectively, results (Fig. 16.31A,F).In the latter case,a small vaginal pouch originating from the paramesonephric ducts usually surrounds the opening of the cervix.
Indentation
Figure 16.31 Main abnormalities of the uterus and vagina, caused by persistence of the uterine septum or obliteration of the lumen of the uterine canal.

folds anteriorly and anal folds posteriorly (Fig. 16.32B,C).
In the third week of development, mesenchyme cells originating in the region of the primitive streak migrate around the cloacal membrane to form a pair of slightly elevated cloacal folds (Fig. 16.32A). Cranial to the cloacal membrane, the folds unite to form the genital tubercle. Caudally, the folds are subdivided into urethral
In the meantime, another pair of elevations, the genital swellings, becomes visible on each side of the urethral folds. These swellings later form the scrotal swellings in the male (Fig. 16.33) and the labia majora in the female (Fig. 16.36B). At the end of the sixth week, however, it is impossible to distinguish between the two sexes.

Figure 16.32 Indifferent stages of the external genitalia. A. Approximately 4 weeks. B. Approximately 6 weeks. C. In utero photograph of a 56-day embryo showing continued growth of the genital tubercle and elongation of the urethral folds that have not yet initiated fusion. The genital swellings remain indistinct.

Development of the external genitalia in the male is under the influence of androgens secreted
by the fetal testes and is characterized by rapid elongation of the genital tubercle, which is now called the phallus (Figs. 16.33A and 16.34A). During this elongation, the phallus pulls the urethral folds forward so that they form the
Figure 16.33 A. Development of external genitalia in the male at 10 weeks. Note the deep urethral groove flanked by the urethral folds. B. Transverse sections through the phallus during formation of the penile urethra. The urogenital groove is bridged by the urethral folds. C. Development of the glandular portion of the penile urethra. D. Newborn.
Figure 16.34 A. In utero photograph of the genitalia of a male fetus at 12 weeks. Note that the urethral folds are fusing and that the scrotal swellings are enlarging to merge in the midline. B. Genitalia of a female fetus at | 1 weeks. Note that the urethral folds, which will become the labia minora, have not fused and that the genital swellings that are forming the labia majora are widely separated.
lateral walls of the urethral groove. This groove extends along the caudal aspect of the elongated phallus but does not reach the most distal part, the glans. The epithelial lining of the groove, which originates in the endoderm, forms the urethral plate (Fig. 16.33B).
when ectodermal cells from the tip of the glans penetrate inward and form a short epithelial cord. This cord later obtains a lumen, thus forming the external urethral meatus (Fig. 16.33C).
At the end of the third month, the two urethral folds close over the urethral plate, forming the penile urethra (Figs. 16.33B and 16.34.A).This canal does not extend to the tip of the phallus. This most distal portion of the urethra is formed during the fourth month,

In hypospadias, fusion of the urethral folds is incomplete, and abnormal openings of the urethra occur along the inferior aspect of the penis, usually near the glans, along the shaft, or near the base of the penis (Fig. 16.35). In rare cases, the urethral meatus extends along the scrotal raphe. When fusion of the urethral folds fails entirely, a wide sagittal slit is found along the entire length of the penis and the scrotum. The two scrotal swellings then closely resemble the labia majora. The incidence of hypospadias is
The genital swellings, known in the male as the scrotal swellings, arise in the inguinal region. With further development, they move caudally, and each swelling then makes up half of the scrotum. The two are separated by the scrotal septum (Figs. 16.33D and 16.34A).
3 to 5/1,000 births, and this rate represents a doubling over the past 15 to 20 years. Reasons for the increase are not known, but one hypothesis suggests it could be a result of a rise in environmental estrogens (endocrine disruptors; see Chapter 8).
Epispadias is a rare abnormality (1/30,000 births) in which the urethral meatus is found on the dorsum of the penis (Fig. 16.35D). Although epispadias may occur as an isolated defect, it is most often associated with exstrophy of the bladder and abnormal closure of the ventral body wall (Fig. 16.16).
Figure 16.35 A. Hypospadias showing the various locations of abnormal urethral orifices. B. Patient with glandular hypospadias. The urethra is open on the ventral surface of the glans penis. C. Patient with hypospadius involving the glans and shaft of the penis. D. Epispadias combined with exstrophy of the bladder. Bladder mucosa is exposed.

Micropenis occurs when there is insufficient androgen stimulation for growth of the external genitalia. Micropenis is usually caused by primary hypogonadism or hypothalamic or pituitary dysfunction. By definition, the penis is 2.5 standard deviations below the mean in length as measured along the dorsal surface from the pubis to the tip with the penis stretched to resistance. Bifid penis or double penis may occur if the genital tubercle splits.

Estrogens stimulate development of the external genitalia of the female. The genital tubercle elongates only slightly and forms the clitoris (Figs. 16.34B and 16.36A); urethral folds do not fuse, as in the male, but develop into the labia minora. Genital swellings enlarge and form the labia majora. The urogenital groove is open and forms the vestibule (Figs. 16.34B and 16.36B). Although the genital tubercle does not elongate extensively in the female, it is larger than in the male during the early stages of development. In fact, using tubercle length as a criterion (as monitored by ultrasound) has resulted in mistakes in identification of the sexes during the third and fourth months of gestation.
Figure 16.36 Development of the external genitalia in the female at 5 months A and in the newborn B.

Since sexual development of males and females begins in an identical fashion, it is not surprising that abnormalities in differentiation and sex determination occur. Ambiguous genitalia (Fig. 16.37) may appear as a large clitoris or a small penis. Thus, a child may be born with a typically female appearance, but with a large clitoris (clitoral hypertrophy) or typically male with a small penis that is open on its ventral surface (hypospadius). In some cases, these abnormalities result in individuals with characteristics of both sexes and they may be called hermaphrodites. However true hermaphrodites have both male and female gonadal tissues and such individuals have not been described in humans.
Figure 16.37 Male (46,XY) infant with ambiguous genitalia. Note partial fusion of the scrotal swellings and a small penis with hypospadius.
Instead, these individuals have ovotestes that have both testicular and ovarian tissue. They may be typically female or typically male or in between in terms of genital development. In 70% of cases, the karyotype is 46,XX, and there is usually a uterus. External genitalia are ambiguous or predominantly female, and most of these individuals are raised as females.
Sometimes, the genotypic (chromosomal) sex does not match the phenotype (physical appearance). For example, the most common cause of sexual ambiguity is congenital adrenal hyperplasia (CAH). Biochemical abnormalities in the adrenal glands result in decreased steroid hormone production and an increase in adrenocorticotropic hormone (ACTH). In most cases, 2-hydroxylation is inhibited. Females with this condition can have a range of sexual characteristics varying from partial masculinization with a large clitoris to virilization and a male appearance. In a rarer form of CAH, there is a 17a-hydroxylase deficiency resulting in females having female internal and external anatomy at birth, but failure of secondary sex characteristics to appear at puberty due to an inability of the adrenals or ovaries to produce sex hormones. Consequently, there is no breast development or growth of pubic hair. In males with |7a-hydroxylase deficiency, virilization is inhibited.

Another cause of sexual ambiguity is the androgen insensitivity syndrome (AIS). Individuals withAlS are males (have aY chromosome and testes), but there is a lack of androgen receptors or failure of tissues to respond to receptor-dihydrotestosterone
complexes. Consequently, androgens produced by the testes are ineffective in inducing differentiation of male genitalia. Since these patients have testes and MIS is present, the paramesonephric system is suppressed, and uterine tubes and uterus are absent. In patients with complete androgen insensitivity syndrome (CAIS) a vagina is present, but usually short or poorly developed. The testes are frequently found in the inguinal or labial regions, but spermatogenesis does not occur. Furthermore, there is an increased risk of testicular tumors, and 33% of these individuals develop malignancies prior to age 50. Other patients have mild androgen insensitivity syndrome (MAIS) or partial androgen insensitivity syndrome (PAIS) forms of the disorder. With the mild form, there is virilization to varying degrees, but with the partial form ambiguous genitalia may be present, including clitoromegaly or a small penis with hypospadius. Testes are usually undescended in these cases.
5-o-reductase deficiency (5-ARD) is another condition that causes ambiguous genitalia in males and is due to an inability to convert testosterone to dihydrotestosterone because of a lack of the reductase enzyme. Without dihydrotestosterone external genitalia do not develop normally and may appear male, but be underdeveloped with

Toward the end of the second month, the urogenital mesentery attaches the testis and mesonephros to the posterior abdominal wall (Fig. 16.3A). With degeneration of the mesonephros, the attachment serves as a mesentery for the gonad (Fig. 16.28B). Caudally, it becomes ligamentous and is known as the caudal genital ligament. Also extending from the caudal pole of the testis is a mesenchymal condensation rich in extracellular matrices, the gubernaculum (Fig. 16.38). Prior to descent of the testis, this band of mesenchyme terminates in the inguinal region between the differentiating internal and external abdominal oblique muscles. Later, as the testis begins to descend toward the internal inguinal ring, an extra-abdominal portion of the gubernaculum forms and grows from the inguinal region toward the scrotal swellings. When the testis passes through the inguinal canal, this extraabdominal portion contacts the scrotal floor (the gubernaculum forms in females also, but in normal cases, it remains rudimentary).
hypospadius, or they may appear to be female with clitoromegaly.
Other conditions may be associated with abnormal sexual differentiation. For example, Klinefelter syndrome, with a karyotype of 47,XXY (or other variants, e.g., XXXY), is the most common sex chromosome disorder, occurring with a frequency of 1/1,000 males. Patients may have decreased fertility, small testes, and decreased testosterone levels. Gynecomastia (enlarged breasts) is present in approximately 33% of affected individuals. Nondisjunction of the XX homologues is the most common causative factor.

In gonadal dysgenesis, oocytes are absent, and the ovaries appear as streak gonads. Individuals are phenotypically female but may have a variety of chromosomal complements, including XY. XY female gonadal dysgenesis (Swyer syndrome) results from point mutations or deletions of the SRY gene. Individuals appear to be normal females, but do not menstruate and do not develop secondary sexual characteristics at puberty. Patients with Turner syndrome also have gonadal dysgenesis. They have a 45,X karyotype and short stature, high-arched palate, webbed neck, shield-like chest, cardiac and renal anomalies, and inverted nipples (see Chapter 2, p. 17).
Factors controlling descent of the testis are not entirely clear. It appears, however, that outgrowth of the extra-abdominal portion of the gubernaculum produces intra-abdominal migration, that an increase in intra-abdominal pressure due to organ growth produces passage through the inguinal canal, and that regression of the extra-abdominal portion of the gubernaculum completes movement of the testis into the scrotum. Normally, the testes reach the inguinal region by approximately 12 weeks' gestation, migrate through the inguinal canal by 28 weeks, and reach the scrotum by 33 weeks (Fig. 16.38). The process is influenced by hormones, including androgens and MIS. During descent, blood supply to the testis from the aorta is retained, and testicular vessels extend from their original lumbar position to the testis in the scrotum.
Independently from descent of the testis, the peritoneum of the abdominal cavity forms an evagination on each side of the midline into the ventral abdominal wall. This evagination, the
Figure 16.38 Descent of the testis. A. During the second month. B. In the middle of the third month. Peritoneum lining the body cavity evaginates into the scrotal swelling, where it forms the vaginal process (tunica vaginalis). C. In the seventh month. D. Shortly after birth.

processus vaginalis, follows the course of the gubernaculum testis into the scrotal swellings (Fig. 16.38B). Hence, the processus vaginalis, accompanied by the muscular and fascial layers of the body wall, evaginates into the scrotal swelling, forming the inguinal canal (Fig. 16.39).
The testis descends through the inguinal ring and over the rim of the pubic bone and is present in the scrotum at birth. The testis is then covered by a reflected fold of the processus vaginalis (Fig. 16.38D). The peritoneal layer covering the testis is the visceral layer of the tunica
|
Figure 16.39 Drawing showing the coverings of the testes that are derived from constituents of the abdominal wall. These coverings are formed as the testes migrate through the wall in route from their retroperitoneal location in the
vaginalis; the remainder of the peritoneal sac forms the parietal layer of the tunica vaginalis (Fig. 16.38D). The narrow canal connecting the lumen of the vaginal process with the peritoneal cavity is obliterated at birth or shortly thereafter.
In addition to being covered by peritoneal layers derived from the processus vaginalis, the testis becomes ensheathed in layers derived from the anterior abdominal wall through which it

The connection between the abdominal cavity and the processus vaginalis in the scrotal sac normally closes in the first year after birth (Fig. 16.38D). If this passageway remains open, intestinal loops may descend into the scrotum, causing a congenital indirect inguinal hernia (Fig. 16.40A). Sometimes, obliteration of this passageway is irregular, leaving small cysts along its course. Later, these cysts may secrete fluid, forming a hydrocele of the testis and/or spermatic cord (Fig. |6.40B).
In 97% of male newborns, testes are present in the scrotum before birth. In most of the remainder, descent will be completed during the first 3 months postnatally. However, in <1% of infants, one or both testes fail to descend. The condition is called cryptorchidism and may be caused by decreased androgen (testosterone) production. The undescended testes fail to produce mature spermatozoa, and the condition is associated with a 3% to 5% incidence of renal anomalies.

Descent of the gonads is considerably less in the female than in the male, and the ovaries finally settle just below the rim of the true pelvis.The cranial genital ligament forms the suspensory ligament of the ovary, whereas the caudal genital ligament forms the ligament of the ovary proper and the round ligament of the uterus (Fig. 15.24). The latter extends into the labia majora.
s_
The urinary and genital systems both develop from mesodermal tissue. Three urinary systems develop in a temporal sequence from cranial to caudal segments:
The pronephros, which forms in the cervical region, is vestigial.
passes. Thus, the transversalis fascia forms the internal spermatic fascia, the internal abdominal oblique muscle gives rise to the cremasteric fascia and muscle, and the external abdominal oblique muscle forms the external spermatic fascia (Fig. 16.39). The transversus abdominis muscle does not contribute a layer, since it arches over this region and does not cover the path of migration.
Figure 16.40 A. Inguinal hernia.The vaginal process remains in open communication with the peritoneal cavity. In such a case, portions of the intestinal loops often descend toward and occasionally into the scrotum, causing an inguinal hernia. B. Hydrocele.
The mesonephros, which forms in the thoracic and lumbar regions, is large and is characterized by excretory units (nephrons) and its own collecting duct, the mesonephric or wolffian duct. In the human, it may function briefly, but most of the system disappears. Ducts and tubules from the mesonephros form the conduit for sperm from the testes to the urethra. In the female, these ducts regress.

The metanephros, or permanent kidney, develops from two sources. It forms its own excretory tubules or nephrons like the other systems, but its collecting system originates from the ureteric bud, an outgrowth of the mesonephric duct. This bud gives rise to the ureter, renal pelvis, calyces, and the entire collecting system (Fig. 16.5). Connection between the collecting and excretory tubule systems is essential for normal development (Fig. 16.6). WT1, expressed by
the mesenchyme, makes this tissue competent to respond to induction by the ureteric bud. Interactions between the bud and mesenchyme occur through production of GDNF and HGF by the mesenchyme with their tyrosine kinase receptors RET and MET, respectively, produced by the ureteric epithelium. PAX2 and WNT4, produced by metanephric mesenchyme, cause epithelialization and excretory tubule differentiation (Fig. 16.7). Early division of the ureteric bud may lead to bifid or supernumerary kidneys with ectopic ureters (Fig. 16.9). Abnormal positions of the kidney, such as pelvic and horseshoe kidney, are also well-known defects (Fig. 16.11).

The genital system consists of (1) gonads or primitive sex glands, (2) genital ducts, and (3) external genitalia. All three components go through an indifferent stage in which they may develop into either a male or a female. The SRY gene on the Y chromosome produces testes-determining factor and regulates male sexual development. Genes downstream from SRY include SOX9 and steroidogenesis factor (SF1) that stimulate differentiation of Sertoli and Leydig cells in the testes. Expression of the SRY gene causes (1) development of the medullary (testis) cords, (2) formation of the tunica albuginea, and (3) failure of the cortical (ovarian) cords to develop. WNT is the master gene for ovarian development. It upregulates DAX1 that inhibits the expression of SOX9. Then, WNT4 together with other downstream genes causes formation of ovaries with (1) typical cortical cords, (2) disappearance of the medullary (testis) cords, and (3) failure of the tunica albuginea to develop (Fig. 16.21). When primordial germ cells fail to reach the indifferent gonad, the gonad remains indifferent or is absent.
The indifferent duct system and external genitalia develop under the influence of hormones. Testosterone produced by Leydig cells in the testes stimulates development of the mesonephric ducts (vas deferens, epididymis), whereas mullerian inhibiting substance (MIS) produced by Sertoli cells in the testes causes regression of the paramesonephric ducts (female duct system). Dihydrotestosterone stimulates development of the external genitalia, penis, scrotum, and prostate (Fig. 16.26). Estrogens influence development of the paramesonephric female system, including the uterine tube, uterus, cervix, and upper portion of the vagina. They also stimulate differentiation of the external genitalia, including the clitoris, labia, and lower portion of the vagina (Fig. 16.26). Errors in production of or sensitivity to hormones of the testes lead to a predominance of female characteristics under influence of the maternal and placental estrogens.

1., 1 = During development of the urinary system, three systems form.What are they, and what parts of each, if any, remain in the newborn?. 2., 1 = At birth, an apparently male baby has no tes- ticles in the scrotum. Later, it is determined that both are in the abdominal cavity.What is the term given to this condition? Explain the embryological origin of this defect.. 3., 1 = It is said that male and female external geni- talia have homologies.What are they, and what are their embryological origins?. 4., 1 = After several years of trying to become preg- nant, a young woman seeks consultation. Examination reveals a bicornate uterus. How could such an abnormality occur?
esenchyme for formation of the head M region is derived from paraxial and lateral plate mesoderm, neural crest, and thickened regions of ectoderm known as ectodermal placodes. Paraxial mesoderm (somites and somitomeres) forms a large portion of the membranous and cartilaginous components of the neurocranium (skull) (Fig. 17.1; see also Chapter 10 and Fig. 10.6), all voluntary muscles of the craniofacial region (see Table 17.1, p. 262), the dermis and connective tissues in the dorsal region of the head, and the meninges caudal to the prosencephalon. Lateral plate mesoderm forms the laryngeal cartilages (arytenoid and cricoid) and connective tissue in this region. Neural crest cells originate in the neuroectoderm of forebrain, midbrain, and hindbrain regions and migrate ventrally into the pharyngeal arches and rostrally around the forebrain and optic cup into the facial region (Fig. 17.2). In these locations, they form the entire viscerocranium (face) and parts of the membranous and cartilaginous regions of the neurocranium (skull) (Fig. 17.1; see also Chapter 10 and Fig. 10.6) They also form all other tissues in these regions, including cartilage, bone, dentin, tendon, dermis, pia and arachnoid, sensory neurons, and glandular connective tissue.

Cells from ectodermal placodes, together with neural crest, form neurons of the fifth, seventh, ninth, and tenth cranial sensory ganglia.
The most distinctive feature in development of the head and neck is the presence of pharyngeal arches (the old term for these structures is branchial arches because they somewhat resemble the gills [branchial] of a fish). These arches appear in the fourth and fifth weeks of development and contribute to the characteristic external appearance of the embryo (Table 17.1, p. 262 and Fig. 17.3). Initially, they consist of bars of mesenchymal tissue separated by deep clefts known as pharyngeal clefts (Fig. 17.3C; see also Fig. 17.6). Simultaneously, with development of the arches and clefts, a number of outpocketings, the pharyngeal pouches, appear along the lateral walls of the pharynx, the most cranial part of the foregut (Fig. 17.4; see also Fig. 17.6). The pouches penetrate the surrounding mesenchyme, but do not establish an open communication with the external clefts (Fig. 17.6). Hence, although development of pharyngeal arches, clefts, and pouches resembles formation of gills in fishes and amphibians, in the human embryo, real gills are never formed. Therefore, the term pharyngeal (arches, clefts, and pouches) has been adopted for the human embryo.
17.1 Skeletal structures of the head and face. Mesenchyme for these structures is derived from neural crest
Figure (blue), lateral plate mesoderm (yellow), and paraxial mesoderm (somites and somitomeres) (red).
Figure 17.2 Migration pathways of neural crest cells from forebrain, midbrain, and hindbrain regions into their final locations (shaded areas) in the pharyngeal arches and face. Regions of ectodermal thickenings (placodes), which will assist crest cells in formation of the fifth (V), seventh (VII), ninth (IX), and tenth (X) cranial sensory ganglia, are also illustrated.

Figure 17.3 Development of the pharyngeal arches. A. 25 days. B. 28 days. C. 5 weeks.
Figure 17.4 Pharyngeal pouches as outpocketings of the foregut and the primordium of the thyroid gland and aortic
Pharyngeal arches not only contribute to formation of the neck, but also play an important role in formation of the face. At the end of the fourth week, the center of the face is formed by the stomodeum, surrounded by the first pair of pharyngeal arches (Fig. 17.5). When the embryo is 42 days old, five mesenchymal prominences can be recognized: the mandibular prominences (first pharyngeal arch), caudal to the stomodeum; the maxillary prominences (dorsal portion of the first pharyngeal arch), lateral to the stomodeum; and the frontonasal prominence, a slightly rounded elevation cranial to the stomodeum. Development of the face is later complemented by formation of the nasal prominences (Fig. 17.5). In all cases, differentiation of structures derived from arches, pouches, clefts, and prominences is dependent on epithelial—mesenchymal interactions.

Each pharyngeal arch consists of a core of mesenchymal tissue covered on the outside by surface ectoderm and on the inside by epithelium of endodermal origin (Fig. 17.6). In addition to mesenchyme derived from paraxial and lateral plate mesoderm, the core of each arch receives substantial numbers of neural crest cells, which migrate into the arches to contribute to skeletal components of the face. The original mesoderm of the arches gives rise to the musculature of the face and neck. Thus, each pharyngeal arch is characterized by its own muscular components. The muscular components of each arch have their own cranial nerve, and wherever the muscle cells migrate, they carry their nerve component with them (Figs. 17.6 and 17.7). In addition, each arch has its own arterial component (Figs. 17.4 and 17.6). (Derivatives of the pharyngeal arches and their nerve supply are summarized in Table 17.1.
Figure 17.5 A. Frontal view of an embryo of approximately 24 days. The stomodeum, temporarily closed by the oropharyngeal membrane, is surrounded by five mesenchymal prominences. B. Frontal view of a slightly older embryo showing rupture of the oropharyngeal membrane and formation of the nasal placodes on the frontonasal prominence. C. Scanning electron micrograph of a human embryo similar to that shown in B.
Figure 17.6 Drawing shows the pharyngeal arches cut in cross section. Each arch consists of a mesenchymal core derived from mesoderm and neural crest cells and each is lined internally by endoderm and externally by ectoderm. Each arch also contains an artery (one of the aortic arches) and a cranial nerve and each will contribute specific skeletal and muscular components to the head and neck. Between the arches are pouches on the inner surface and clefts externally.

Figure 17.7 Each pharyngeal arch is supplied by its own cranial nerve. The trigeminal nerve supplying the first pharyngeal arch has three branches: the ophthalmic, maxillary, and mandibular. The nerve of the second arch is the facial nerve; that of the third is the glossopharyngeal nerve. The musculature of the fourth arch is supplied by the superior laryngeal branch of the vagus nerve, and that of the sixth arch, by the recurrent branch of the vagus nerve.

The first pharyngeal arch consists of a dorsal portion, the maxillary process, which extends forward beneath the region of the eye, and a ventral portion, the mandibular process, which contains Meckel's cartilage (Figs. 17.5 and 17.8A). During further development, Meckel's cartilage disappears except for two small portions at its dorsal end that persist and form the incus and malleus (Figs. 17.8B and 17.9). Mesenchyme of the maxillary process gives rise to the premaxilla, maxilla, zygomatic bone, and part of the temporal bone through membranous ossification (Fig. 17.8B). The mandible is also formed by membranous ossification of mesenchymal tissue surrounding Meckel's cartilage. In addition, the first arch contributes to formation of the bones of the middle ear (see Chapter 19).

Muscles of the arches do not always attach to the bony or cartilaginous components of their own arch but sometimes migrate into surrounding regions. Nevertheless, the origin of these muscles can always be traced, since their nerve supply is derived from the arch of origin.

Musculature of the first pharyngeal arch includes the muscles of mastication (temporalis, masseter, and pterygoids), anterior belly of the digastric, mylohyoid, tensor tympani, and tensor palatini. The nerve supply to the muscles of the first arch is provided by the mandibular branch of the trigeminal nerve (Fig. 17.7). Since mesenchyme from the first arch also contributes to the dermis of the face, sensory supply to the skin of the face is provided by ophthalmic,
The cartilage of the second or hyoid arch (Reichert's cartilage) (Fig. 17.8B) gives rise to the stapes, styloid process of the temporal bone, stylohyoid ligament, and ventrally, the lesser horn and upper part of the body of the hyoid bone (Fig. 17.9). Muscles of the hyoid arch are the stapedius, stylohyoid, posterior belly of the digastric, auricular, and muscles of facial expression. The facial nerve, the nerve of the second arch, supplies all of these muscles.

The cartilage of the third pharyngeal arch produces the lower part of the body and greater horn of the hyoid bone (Fig. 17.9). The musculature is limited to the stylopharyngeus muscles. These muscles are innervated by the glossopharyngeal nerve, the nerve of the third arch (Fig. 17.7).

arch). 4-6, Derivatives of the Pharyngeal Arches andTheir Innervation.Muscles = Cricothyroid, levator palatine, constrictors of pharynx Intrinsic muscles of larynx. 4-6, Derivatives of the Pharyngeal Arches andTheir Innervation.Skeleton = Laryngeal cartilages (thyroid, cricoid, arytenoid, corniculate, cuneiform)
Figure 17.8 A. Lateral view of the head and neck region demonstrating the cartilages of the pharyngeal arches participating in formation of the bones of the face and neck. B. Various components of the pharyngeal arches later in development. Some of the components ossify; others disappear or become ligamentous. The maxillary process and Meckel's cartilage are replaced by the maxilla and mandible, respectively, which develop by membranous ossification.

Arches
Cartilaginous components of the fourth and sixth pharyngeal arches fuse to form the thyroid,
cricoid, arytenoid, corniculate, and cuneiform car- tilages of the larynx (Fig. 17.9). Muscles of the
fourth arch (cricothyroid, levator palatini, and constrictors of the pharynx) are innervated by
the superior laryngeal branch of the vagus, the nerve of the fourth arch. Intrinsic muscles of the
larynx are supplied by the recurrent laryngeal branch of the vagus, the nerve of the sixth arch.
Figure 17.9 Definitive structures formed by the cartilaginous components of the various pharyngeal arches.

The human embryo has four pairs of pharyngeal pouches; the fifth is rudimentary (Figs. 17.6 and 17.10). Since the epithelial endodermal lining of the pouches gives rise to a number of important organs, the fate of each pouch is discussed separately. Derivatives of the pharyngeal pouches are summarized in Table 17.2.
The first pharyngeal pouch forms a stalk-like diverticulum, the tubotympanic recess, which comes in contact with the epithelial lining of the first pharyngeal cleft, the future external auditory meatus (Fig. 17.10). The distal portion of the diverticulum widens into a sac-like structure, the primitive tympanic or middle ear cavity, and the proximal part remains narrow, forming the
Figure 17.10 A. Development of the pharyngeal clefts and pouches. The second arch grows over the third and fourth arches, burying the second, third, and fourth pharyngeal clefts. B. Remnants of the second, third, and fourth pharyngeal clefts form the cervical sinus, which is normally obliterated. Note the structures formed by the various pharyngeal pouches.
auditory (eustachian) tube. The lining of the tympanic cavity later aids in formation of the tympanic membrane or eardrum (see Chapter 19).

The epithelial lining of the second pharyngeal pouch proliferates and forms buds that penetrate into the surrounding mesenchyme. The buds are secondarily invaded by mesodermal tissue, forming the primordium of the palatine tonsils (Fig. 17.10). During the third and fifth months, the tonsil is infiltrated by lymphatic tissue. Part of the pouch remains and is found in the adult as the tonsillar fossa.

The third and fourth pouches are characterized at their distal extremity by a dorsal and a ventral wing (Fig. 17.10). In the fifth week, epithelium of the dorsal region of the third pouch differentiates into the inferior parathyroid gland, while the ventral region forms the thymus (Fig. 17.10). Both gland primordia lose their connection with the pharyngeal wall, and the thymus then migrates in a caudal and a medial direction, pulling the inferior parathyroid with it (Fig. 17.11). Although the main portion of the thymus moves rapidly to its final position in the anterior part of the thorax, where it fuses with its counterpart from the opposite

Epithelium of the dorsal region of the fourth pharyngeal pouch forms the superior parathyroid gland. When the parathyroid gland loses contact with the wall of the pharynx, it attaches itself to the dorsal surface of the caudally migrating thyroid as the superior parathyroid gland (Fig. 17.11). The ventral region of the fourth pouch gives rise to the ultimobranchial body, which is later incorporated into the thyroid gland. Cells of the ultimobranchial body give rise to the parafollicular, or C, cells of the thyroid gland. These cells secrete calcitonin, a hormone involved in regulation of the calcium level in the blood (Table 17.2).

1, Derivatives = Tympanic (middle ear) cavity. , Derivatives = Auditory (eustachian) tube. 2, Derivatives = Palatine tonsils. , Derivatives = Tonsillar fossa. 3, Derivatives = Inferior parathyroid gland. , Derivatives = Thymus. 4, Derivatives = Superior parathyroid gland ultimobranchial body (parafollicular [C] cells of the thyroid gland)

The 5-week embryo is characterized by the presence of four pharyngeal clefts (Fig. 17.6), of which only one contributes to the definitive structure of the embryo. The dorsal part of the first cleft penetrates the underlying mesenchyme and gives rise to the external auditory meatus (Figs. 17.10 and 17.11). The epithelial lining at the bottom of the meatus participates in formation of the eardrum (see Chapter 19).
Active proliferation of mesenchymal tissue in the second arch causes it to overlap the third and fourth arches. Finally, it merges with the epicardial ridge in the lower part of the neck (Fig. 17.10), and the second, third, and fourth clefts lose contact with the outside (Fig. 17.10B). The clefts form a cavity lined with ectodermal epithelium, the cervical sinus, but with further development, this sinus disappears.

Neural crest cells arise from neuroepithelial cells adjacent to the surface ectoderm all along the edges of the neural folds. Bone morphogenetic protein (BMP) signaling is important in establishing this edge region and then regulates WNT1 expression to cause prospective crest cells to undergo an epithelial-to-mesenchymal transition and begin their migration into the surrounding mesenchyme. In the hindbrain, crest cells originate in a specific pattern from segments called rhombomeres (Fig. 17.12). There are eight of these segments in the hindbrain (R1—R8), and neural crest cells from specific segments migrate to populate specific pharyngeal arches. These crest cells migrate in three streams: Those from R1 and R2 migrate to the first arch along with crest cells from the caudal
Figure 17.12 Drawing showing the pathways of neural crest cell migration from the cranial neural folds into the face and pharyngeal arches. From the hindbrain region, crest cells migrate from segments called rhombomeres. Rhombomeres express a specific pattern of HOX genes (the midbrain and rhombomeres | and 2 express the homeodomain-containing transcription factor OTX2; see also Fig. 18.31), and neural crest cells carry these expression patterns into the pharyngeal arches. Also, notice that there are three streams of crest cells and that rhombomeres 3 and 5 do not contribute many (if any) cells to these streams. The three streams are important because they provide guidance cues for cranial nerves growing back from their to establish connections in the hindbrain also ganglia (see Fig. 18.40).

Figure 17.13 A,B. Drawings showing the gene expression patterns in pharyngeal arch endoderm and mesenchyme. Endoderm is responsible for patterning the skeletal derivatives of the arches, but the response of the mesenchyme to these signals is dictated by the genes that the mesenchyme expresses. Gene expression in the endoderm of the pouches shows a specific pattern: FGF8 is expressed in the anterior region of each pouch with BMP7 expressed in the posterior region; SHH is expressed in the posterior region of pouches 2 and 3, while PAX! is expressed in the dorsal-most area of each pouch. A,B. Mesenchymal expression patterns are established by neural crest cells that migrate into the arches and carry the genetic code from their rhombomeres of origin (or also from the midbrain in the case of the first arch) to the arches B (see also Figs. 17.12 and 18.31).
midbrain region; crest from R4 migrate to the second arch; and cells from R6 and R7 migrate to arches 4 to 6 (Fig. 17.12). Segregation of the three streams is assisted by the fact that very few crest cells form from R3 and R5 segments and those that do enter adjoining streams of cells to migrate. Three distinct streams are important because they provide axonal guidance cues for axons from ganglia forming in the head and neck region, including the trigeminal, geniculate, vestibuloacoustic, petrosal, and nodose ganglia. These ganglia are formed from a combination of crest cells and cells from placodes in this region (see Chapter 18). Axons from the trigeminal ganglion enter the hindbrain at R2; those from the geniculate and vestibuloacoustic at R4; and those from the petrosal and nodose at R6 and R7, thus accounting for three streams of crest cells. No axons project to R3 or R5.

Neural crest cells that populate the pharyngeal arches form the skeletal components characteristic of each arch. Previously, it was thought that neural crest cells regulated patterning of these skeletal elements, but now it is clear that this process is controlled by pharyngeal pouch endoderm. Formation of the pharyngeal pouches occurs prior to neural crest migration and takes place even in the absence of crest cells. Pouches are formed by migration of endoderm cells laterally, and this migration is stimulated by fibroblast growth factors (FGFs). As pouches form, they express a very characteristic pattern of genes (Fig. 17.13). BMP7 is expressed in the posterior endoderm of each pouch; FGF8 lies in the anterior endoderm; and PAX17 expression is restricted to the dorsal-most endoderm of each pouch. In addition, SHH is expressed in the posterior endoderm of the second and third pouches. These expression patterns then regulate differentiation and patterning of pharyngeal arch mesenchyme into specific skeletal structures. This process, however, is also dependent on the mesenchyme and represents another example of an epithelial-mesenchymal interaction. In this case, the response of the mesenchyme to endodermal signals is dependent on transcription factors expressed in that mesenchyme. These
transcription factors include HOX genes and others carried by neural crest cells into the arches. Crest cells acquire their specific gene expression patterns from the rhombomeres of their origin (Fig. 17.12). The pattern of rhombomeres itself is established by a nested code of HOX gene expression in the hindbrain (see Chapter 18) that crest cells carry with them when they migrate. The first arch is HOX-negative but does express OTX2, a homeodomain-containing transcription factor that is expressed in the midbrain; the second arch expresses HOXA2; and arches 3 to 6 express members of the third paralogous group of HOX genes, HOXA3, HOXB3,and HOXD3 (Fig. 17.13B).The different expression patterns of these

Since glandular tissue derived from the pouches undergoes migration, it is not unusual for accessory glands or remnants of tissue to persist along the transcription factors allow each arch to respond differently to signals emanating from pouch endoderm, such that the first arch forms the maxilla and mandible, the second arch, the hyoid bone, etc.
The remainder of the skeleton of the face, the midand upper facial regions, also is derived from neural crest cells that migrate into the frontonasal prominence (Fig. 17.12). In this region, signals emanating from the surface ectoderm and the underlying areas of the neuroepithelium dictate the fate of the mesenchyme. Again, it appears that SHH and FGF8 play major roles in patterning this area, but the specific genetic interactions are not known.
pathway. This is true particularly for thymic tissue, which may remain in the neck, and for the parathyroid glands. The inferior parathyroids are more variable in position than the superior ones and are sometimes found at the bifurcation of the common carotid artery.
Figure 17.14 A. Lateral cervical cyst opening at the side of the neck by way of a fistula. B. Lateral cervical cysts and fistulas in front of the sternocleidomastoid muscle. Note also the region of preauricular fistulas. C. A lateral cervical cyst opening into the pharynx at the level of the palatine tonsil.

Branchial fistulas occur when the second pharyngeal arch fails to grow caudally over the third and fourth arches, leaving remnants of the second, third, and fourth clefts in contact with the surface by a narrow canal (Fig. 17.14A). Such a fistula, found on the lateral aspect of the neck directly anterior to the sternocleidomastoid muscle, usually provides
drainage for a lateral cervical cyst (Fig. 17.14B). These cysts, remnants of the cervical sinus, are most often just below the angle of the jaw (Fig. 17.15), although they may be found anywhere along the anterior border of the sternocleidomastoid muscle. Frequently, a lateral cervical cyst is not visible at birth but becomes evident as it enlarges during childhood.
Internal branchial fistulas are rare; they occur when the cervical sinus is connected to the lumen of the pharynx by a small canal, which usually opens in the tonsillar region (Fig. 17.14C). Such a fistula results from a rupture of the membrane between the second pharyngeal cleft and pouch at some time during development.
Figure 17.15 Patient with a lateral cervical cyst. These cysts are always on the lateral side of the neck in front of the sternocleidomastoid muscle. They commonly lie under the angle of the mandible and do not enlarge until later in life.

Neural crest cells (Fig. 17.2) are essential for formation of much of the craniofacial region. Consequently, disruption of crest cell development results in craniofacial malformations. Since neural crest cells also contribute to the conotruncal endocardial cushions, which septate the outflow tract of the heart into pulmonary and aortic channels, and to regulation of the secondary heart field and its contribution of cells to development of the cardiac outflow tract, many infants with craniofacial defects also have cardiac abnormalities, including
Persistent truncus arteriosus, tetralogy of Fallot, and transposition of the great vessels. Unfortunately, crest cells appear to be a particularly vulnerable cell population and are easily killed by compounds such as alcohol and retinoic acid. Examples of craniofacial defects involving crest cells include the following:
Treacher Collins syndrome (mandibulofacial dysostosis) is characterized by malar hypoplasia due to underdevelopment of the zygomatic bones, mandibular hypoplasia, down-slanting palpebral fissures, lower eyelid colobomas, and malformed external ears. Mutations in the TCOF/ gene are responsible for most cases. The product of this gene is a nucleolar protein called treacle that appears to be involved in neural crest cell differentiation. Treacher Collins syndrome is inherited as an autosomal dominant trait, with 60% of cases arising as new mutations. However, phenocopies can be produced in laboratory animals following exposure to teratogenic doses of retinoic acid, suggesting that some cases in humans may be caused by teratogens.

Robin sequence may occur independently or in association with other syndromes and malformations. Like Treacher Collins syndrome, Robin sequence alters first-arch structures, with development of the mandible most severely affected. Infants usually have a triad of micrognathia, cleft palate, and glossoptosis (posteriorly placed tongue) (Fig. 17.16A). Robin sequence may be due to genetic or environmental factors. It may also occur as a deformation, as for example, when the chin is compressed against the chest in cases of oligohydramnios. The primary defect includes poor growth of the mandible and, as a result, a posteriorly placed tongue that fails to drop from between the palatal shelves, preventing their fusion. Robin sequence occurs in approximately 1/8,500 births.
22q11.2 deletion syndrome has several presentations, including DiGeorge syndrome (Fig. 17.16B), DiGeorge anomaly, velo-cardio-facial syndrome (Fig. 17.16C), Shprintzen syndrome, conotruncal anomaly face syndrome, and congenital thymic aplasia and hypoplasia. The defects are the result of a deletion on the long arm of chromosome 22 and occur in approximately 1/4000 births. The syndrome is characterized by a variety of malformations and degrees of severity, but infants most often present with congenital heart defects, mild facial dysmorphology, learning disabilities, and frequent infections due to thymic hypoplasia or aplasia that disrupts the immune system's T-cell-mediated response. Many of the infants also have seizures due to hypocalcemia caused by abnormal

development of the parathyroid glands. Origin of the defects is due to disruption of neural crest cells that contribute to all of the affected structures. Even the thymus and parathyroid defects are related to neural crest cells, since these cells contribute the mesenchyme into which endoderm from the pharyngeal pouches
migrates. Endoderm cells from the pouches make the thymus and parathyroid cells (see Fig. 17.10) and neural crest derived mesenchyme makes the connective tissue. Without this mesenchyme, the normal epithelial (endodermal)-mesenchymal interaction essential for differentiation of the glands does not occur.
Figure 17.16 Patients with craniofacial defects thought to arise from insults to neural crest cells. A. Robin sequence. Note the very small mandible (micrognathia) that keeps the tongue from "dropping" out of the way of the palatal shelves resulting in cleft palate. B,C. Examples of 22q| 1.2 deletion syndrome: DiGeorge syndrome. B. Note the small mouth, nearly smooth philtrum, micrognathia, prominent nasal bridge, and posteriorly rotated ears;Velo-cardio-facial syndrome. C. This patient shows mild facial dysmorphology, including mild malar hypoplasia, micrognathia, prominent upper lip, and large ears. D. Hemifacial microsomia (oculoauriculovertebral spectrum, or Goldenhar syndrome). Note the abnormal ear with skin tags and the small chin.

Oculoauriculovertebral spectrum (Goldenhar syndrome) includes a number of craniofacial abnormalities that usually involve the maxillary, temporal, and zygomatic bones, which are small and flat. Ear (anotia, microtia), eye (tumors and dermoids in the eyeball), and vertebral (fused and hemivertebrae, spina bifida) defects are commonly observed in these patients (Fig. 17.16D).Asymmetry is present in 65% of the cases, which occur in 1/5,600 births. Other malformations, which occur in 50% of cases, include cardiac abnormalities, such as tetralogy of Fallot and ventricular septal defects. Causes of hemifacial microsomia are unknown.
Figure 17.17 Ventral portion of the pharyngeal arches seen from above showing development of the tongue. | to IV, the cut pharyngeal arches. A. 5 weeks (~6 mm). B. 5 months. Note the foramen cecum, site of origin of the thyroid primordium.

The tongue appears in embryos of approximately 4 weeks in the form of two lateral lingual swellings and one medial swelling, the tuberculum impar (Fig. 17.17A). These three swellings originate from the first pharyngeal arch. A second median swelling, the copula, or hypobranchial eminence, is formed by mesoderm of the second, third, and part of the fourth arch. Finally, a third median swelling, formed by the posterior part of the fourth arch, marks development of the epiglottis. Immediately behind this swelling is the laryngeal orifice, which is flanked by the arytenoid swellings (Fig. 17.17).
As the lateral lingual swellings increase in size, they overgrow the tuberculum impar and merge, forming the anterior two-thirds, or body, of the tongue (Fig. 17.17). Since the mucosa covering the body of the tongue originates from the first pharyngeal arch, sensory innervation to this area is by the mandibular branch of the trigeminal nerve. The body of the tongue is separated from the posterior third by a V-shaped groove, the terminal sulcus (Fig. 17.17B).
The posterior part, or root, of the tongue originates from the second, third, and parts of the fourth pharyngeal arch. The fact that sensory innervation to this part of the tongue is supplied by the glossopharyngeal nerve indicates that tissue of the third arch overgrows that of the second.
The epiglottis and the extreme posterior part of the tongue are innervated by the superior laryngeal nerve, reflecting their development from the fourth arch. Some of the tongue muscles probably differentiate in situ, but most are derived from myoblasts originating in occipital somites. Thus, tongue musculature is innervated by the hypoglossal nerve.
The general sensory innervation of the tongue is easy to understand. The body is supplied by the trigeminal nerve, the nerve of the first arch;

In ankyloglossia (tongue-tie), the tongue is not freed from the floor of the mouth. Normally, extensive cell degeneration occurs, and the frenulum is the only tissue that anchors the tongue to the floor of the mouth. In the most common form of ankyloglossia, the frenulum extends to the tip of the tongue.
that of the root is supplied by the glossopharyngeal and vagus nerves, the nerves of the third and fourth arches, respectively. Special sensory innervation (taste) to the anterior two thirds of the tongue is provided by the chorda tympani branch of the facial nerve, while the posterior third is supplied by the glossopharyngeal nerve.

The thyroid gland appears as an epithelial proliferation in the floor of the pharynx between the tuberculum impar and the copula at a point later indicated by the foramen cecum (Figs. 17.17 and 17.18A). Subsequently, the thyroid descends in front of the pharyngeal gut as a bilobed diverticulum
(Fig. 17.18). During this migration, the thyroid remains connected to the tongue by a narrow canal, the thyroglossal duct. This duct later disappears.
With further development, the thyroid gland descends in front of the hyoid bone and the laryngeal cartilages. It reaches its final position in front of the trachea in the seventh week (Fig. 17.18B). By then, it has acquired a small median isthmus and two lateral lobes. The thyroid begins to function at approximately the end of the third month, at which time the first follicles containing colloid become visible. Follicular cells produce the colloid that serves as a source of thyroxine and triiodothyronine. Parafollicular, or C, cells derived from the ultimobranchial body (Fig. 17.10) serve as a source of calcitonin.
Figure 17.18 A. The thyroid primordium arises as an epithelial diverticulum in the midline of the pharynx immediately caudal to the tuberculum impar. B. Position of the thyroid gland in the adult. Broken line, the path of migration.

A thyroglossal cyst may lie at any point along the migratory pathway of the thyroid gland but is always near or in the midline of the neck. As indicated by its name, it is a cystic remnant of the thyroglossal duct. Although approximately 50% of these cysts are close to or just inferior to the body of the hyoid bone (Figs. 17.19 and 17.20), they may also be found at the base of the tongue or close to the thyroid cartilage.
Sometimes, a thyroglossal cyst is connected to the outside by a fistulous canal, a thyroglossal fistula. Such a fistula usually arises secondarily after rupture of a cyst but may be present at birth.
Aberrant thyroid tissue may be found anywhere along the path of descent of the thyroid gland. It is commonly found in the base of the tongue, just behind the foramen cecum, and is subject to the same diseases as the thyroid gland itself.

Figure 17.19 Thyroglossal cysts. These cysts, most frequently found in the hyoid region, are always close to the midline.
Figure 17.20 Thyroglossal cyst. These cysts, which are remnants of the thyroglossal duct, may be anywhere along the migration pathway of the thyroid gland. They are commonly found behind the arch of the hyoid bone.An important diagnostic characteristic is their midline location.

Atthe end ofthe fourth week, facial prominences consisting primarily of neural crest-derived mesenchyme and formed mainly by the first pair of pharyngeal arches appear. Maxillary prominences can be distinguished lateral to the stomodeum, and mandibular prominences can be distinguished caudal to this structure (Fig. 17.21). The frontonasal prominence, formed by proliferation of mesenchyme ventral to the brain vesicles, constitutes the upper border of the stomodeum. On both sides of the frontonasal prominence, local thickenings of the surface ectoderm, the nasal (olfactory) placodes, originate under inductive influence of the ventral portion of the forebrain (Fig. 17.21).
During the fifth week, the nasal placodes invaginate to form nasal pits. In so doing, they create a ridge of tissue that surrounds each pit and forms the nasal prominences.The prominences on the outer edge of the pits are the lateral nasal prominences; those on the inner edge are the medial nasal prominences (Fig. 17.22).
Otic placode
prominences.
B.
|0-week embryo.
C.
Photograph of
a
human embryo
at
a
stage similar
to that
in
A.
Initially, the maxillary and lateral nasal prominences are separated by a deep furrow, the nasolacrimal groove (Figs. 17.22 and 17.23). Ectoderm in the floor of this groove forms a solid epithelial cord that detaches from the overlying ectoderm. After canalization, the cord forms the nasolacrimal duct; its upper end widens to form the lacrimal sac. Following detachment of the cord, the maxillary and lateral nasal prominences merge with each other. The nasolacrimal duct then runs from the medial corner of the eye to the inferior meatus of the nasal cavity, and the maxillary prominences enlarge to form the cheeks and maxillae.
Figure 17.24 A. Intermaxillary segment and maxillary processes. B. The intermaxillary segment giving rise to the philtrum of the upper lip, the median part of the maxillary bone with its four incisor teeth, and the triangular primary palate.

The nose is formed from five facial prominences (Fig. 17.23): the frontal prominence gives rise to the bridge; the merged medial nasal prominences provide the crest and tip;and the lateral nasal prominences form the sides (alae) (Table 17.3).

As a result of medial growth of the maxillary prominences, the two medial nasal prominences merge not only at the surface but also at a deeper level. The structure formed by the two merged prominences is the intermaxillary segment. It is composed of (1) a labial component, which forms the philtrum of the upper lip; (2) an upper jaw component, which carries the four incisor teeth; and (3) a palatal component, which forms the triangular primary palate (Fig. 17.24). The intermaxillary segment is continuous with the rostral portion of the nasal septum, which is formed by the frontal prominence.
Although the primary palate is derived from the intermaxillary segment (Fig. 17.24), the main part of the definitive palate is formed by two shelf-like outgrowths from the maxillary prominences. These outgrowths, the palatine shelves, appear in the sixth week of development and are directed obliquely downward on each side of the tongue (Fig. 17.25). In the seventh week, however, the palatine shelves ascend to attain a horizontal position above the tongue and fuse, forming the secondary palate (Figs. 17.26 and 17.27).
Anteriorly, the shelves fuse with the triangular primary palate, and the incisive foramen is the midline landmark between the primary and secondary palates (Fig. 17.27B). At the same time as the palatine shelves fuse, the nasal septum grows down and joins with the cephalic aspect of the newly formed palate (Fig. 17.27).
Figure 17.25 A. Frontal section through the head of a 6.5-week embryo. The palatine shelves are in the vertical position on each side of the tongue. B. Ventral view of the palatine shelves after removal of the lower jaw and the tongue. Note the clefts between the primary triangular palate and the palatine shelves, which are still vertical.
Figure 17.26 A. Frontal section through the head of a 7.5-week embryo. The tongue has moved downward, and the palatine shelves have reached a horizontal position. B. Ventral view of the palatine shelves after removal of the lower jaw and tongue. The shelves are horizontal. Note the nasal septum.

Figure 17.27 A. Frontal section through the head of a 10-week embryo. The two palatine shelves have fused with each other and with the nasal septum. B. Ventral view of the palate. The incisive foramen forms the midline between the primary and secondary palate.

Cleft lip and cleft palate are common defects that result in abnormal facial appearance and difficulties with speech. The incisive foramen is considered the dividing landmark between anterior and posterior cleft deformities. Those anterior to the incisive foramen include lateral cleft lip, cleft upper jaw, and cleft between the primary and secondary palates (Figs. |7.28,B-D and 17.29A,B). Such defects are due to a partial or complete lack of fusion of the maxillary prominence with the medial nasal prominence on one or both sides. Those that lie posterior to the incisive foramen include cleft (secondary) palate and cleft uvula (Figs. 17.28E and 17.29C). Cleft palate results from a lack of fusion of the palatine shelves, which may be due to smallness of the shelves, failure of the shelves to elevate, inhibition of the fusion process itself, or failure of the tongue to drop from between the shelves because of micrognathia. The third category is formed by a combination of clefts lying anterior as well as posterior to the incisive foramen (Fig. 17.28F). Anterior clefts vary in severity from a barely visible defect in the vermilion of the lip to extension into the nose (Fig. |7.29A). In severe cases, the cleft extends to a deeper level, forming a cleft of the upper jaw, and the maxilla is split between the lateral incisor and the canine tooth. Frequently, such a cleft extends to the incisive foramen (Fig. 17.28CF). Likewise, posterior clefts vary in severity from cleavage of the entire secondary palate (Fig. 17.28E and 17.29C) to cleavage of the uvula only.
Oblique facial clefts are produced by failure of the maxillary prominence to merge with its corresponding lateral nasal prominence. When this occurs, the nasolacrimal duct is usually exposed to the surface.
D
E
F

Figure 17.28 Ventral view of the palate, gum, lip, and nose. A. Normal. B. Unilateral cleft lip extending into the nose. C. Unilateral cleft involving the lip and jaw and extending to the incisive foramen. D. Bilateral cleft involving the lip and jaw. E. Isolated cleft palate. F. Cleft palate combined with unilateral anterior cleft lip.
Figure 17.29 A. Lateral Cleft lip (compare to Fig. |7.28C). B. Bilateral cleft lip (compare to Fig. 17.28D). C. Cleft palate (compare to Fig. 17.28E). D. Midline cleft lip

Photographs demonstrating normal and abnormal development involving the midline of the face and brain. A. Normal face of an embryo at the beginning of the sixth week. Note the distance between the nasal openings (vertical lines) and between the medial nasal prominences (Xs). B. Face of a newborn with a midline cleft lip. In this case, there is insufficient tissue in the midline to fill in the space between the medial nasal prominences. C. Face of a 6-week embryo showing an extensive deficiency of midline tissue.As a consequence, the medial nasal prominences have not formed, and there is a single nasal opening created by fusion of the two lateral nasal prominences.The deficiency of midline tissue is reflected in the brain as well, and as a result, the head is narrow, and the eyes are positioned more anteriorly and slightly caudal to the nasal opening. D. Face of a newborn with holoprosencephaly characterized by such an extensive deficiency of midline tissue that the eyes have fused (synophthalmia), and a proboscis with a single nasal opening has formed from fusion of the lateral nasal processes.An upper lip is formed by fusion of the maxillary prominences. The head is narrow, and the brain would have a single ventricle (holoprosencephaly) due to such a loss of midline tissue that the two lateral ventricles fused. The condition may be caused by mutations in sonic hedgehog (SHH; the gene that establishes the midline), by altered cholesterol biosynthesis, and by maternal exposure to teratogens, such as alcohol, in the third week of development (see also Chapter 18).
Median (midline) cleft lip, a rare abnormality, is caused by incomplete merging of the two medial nasal prominences in the midline. (Fig. 17.29D and
Fig. 17.30B). Infants with midline clefts are often cognitively impaired and may have brain abnormalities that include varying degrees of loss of

midline structures. Loss of midline tissue may be so extensive that the lateral ventricles fuse (holoprosencephaly) (Fig. 17.30CD). These defects are induced very early in development, at the beginning of neurulation (days 19-21) when the midline of the forebrain is being established (see Chapter 18).
Most cases of cleft lip with or without cleft palate are multifactorial. These conditions are usually classified as: (1) cleft lip with or without cleft palate; and (2) cleft palate and are thought to be etiologically and pathogenetically distinct.. Cleft lip with or without cleft palate (approximately 1/700 births) occurs more frequently in males (65%) than in females and its incidence varies among populations. Asians and Native Americans have some of the highest rates (3.5/1,000) while African Americans have the lowest (1/1,000).

The frequency of isolated cleft palate is lower than that of cleft lip (1/1,500 births), occurs more often in females (55%) than in males. In females, the palatal shelves fuse approximately | week later than in males, which may be related to why isolated cleft palate occurs more frequently in females than in males.
Causes of cleft lip with or without cleft palate are not well defined. Some cases are syndromic and associated with certain syndromes and genes. Others are nonsyndromic, but associated with some of the same genes that cause syndromes, such as IRF6 (Van der Woude syndrome) and MSXI. Still other cases are caused by exposure to teratogenic compounds, such as anticonvulsant medications, particularly valproic acid. Cigarette smoking during pregnancy also increases the risk for having a baby with orofacial clefts.
oral cavity by way of the newly formed foramina, the primitive choanae (Fig. 17.31C).
During the sixth week, the nasal pits deepen considerably, partly because of growth of the surrounding nasal prominences and partly because of their penetration into the underlying mesenchyme (Fig. 16.31A). At first, the oronasal membrane separates the pits from the primitive
These choanae lie on each side of the midline and immediately behind the primary palate. Later, with formation of the secondary palate and further development of the primitive nasal chambers (Fig. 17.31D), the definitive choanae lie at the junction of the nasal cavity and the pharynx.
Figure 17.31 A. Sagittal section through the nasal pit and lower rim of the medial nasal prominence of a 6-week embryo. The primitive nasal cavity is separated from the oral cavity by the oronasal membrane. B. Similar section as in A showing the oronasal membrane breaking down. C. A 7-week embryo with a primitive nasal cavity in open connection with the oral cavity. D. Sagittal section through the face of a 9-week embryo showing separation of the definitive nasal and oral cavities by the primary and secondary palate. Definitive choanae are at the junction of the oral cavity and the pharynx.
Figure
17.32
the tooth
at successive
stages of
A.
Bud stage;
8

weeks.
B.
Cap stage;
10
weeks.
C.
Bell stage;
3
months.
D.
6
months.
Paranasal air sinuses develop as diverticula of the lateral nasal wall and extend into the maxilla, ethmoid, frontal, and sphenoid bones. They reach their maximum size during puberty and contribute to the definitive shape of the face.

The shape of the face is determined not only by expansion of the paranasal sinuses but also by growth of the mandible and maxilla to accommodate the teeth. Teeth themselves arise from an epithelial-mesenchymal interaction between overlying oral epithelium and underlying mesenchyme derived from neural crest cells. By the sixth week of development, the basal layer of the epithelial lining of the oral cavity forms a C-shaped structure, the dental lamina, along the length of the upper and lower jaws. This lamina subsequently gives rise to a number of dental buds (Fig. 17.32), 10 in each jaw, which form the primordia of the ectodermal components of the teeth. Soon, the deep surface of the buds invaginates, resulting in the cap stage of tooth development (Fig. 17.32B). Such a cap consists of an outer layer, the outer dental epithelium, an inner layer, the inner dental epithelium, and a central core of loosely woven tissue, the stellate reticulum. The mesenchyme, which originates in the neural crest in the indentation, forms the dental papilla (Fig. 17.32B).
As the dental cap grows and the indentation deepens, the tooth takes on the appearance of a bell (bell stage) (Fig. 17.32C). Mesenchyme cells of the papilla adjacent to the inner dental layer differentiate into odontoblasts, which later produce dentin. With thickening of the dentin layer, odontoblasts retreat into the dental papilla, leaving a thin cytoplasmic process (dental process) behind in the dentin (Fig. 17.32D). The odontoblast layer persists throughout the life of the tooth and continuously provides predentin. The remaining cells of the dental papilla form the pulp of the tooth (Fig. 17.32D).
In the meantime, epithelial cells of the inner dental epithelium differentiate into ameloblasts (enamel formers). These cells produce long enamel prisms that are deposited over the dentin (Fig. 17.32D). Furthermore, a cluster of these cells in the inner dental epithelium forms the enamel knot that regulates early tooth development (Fig. 17.32B).

Enamel is first laid down at the apex of the tooth and from here spreads toward the neck. When the enamel thickens, the ameloblasts retreat into the stellate reticulum. Here they regress, temporarily leaving a thin membrane (dental cuticle) on the surface of the enamel. After eruption of the tooth, this membrane gradually sloughs off.
Figure 17.33 The tooth just before birth A and after eruption B.
Formation of the root of the tooth begins when the dental epithelial layers penetrate into the underlying mesenchyme and form the epithelial root sheath (Fig. 17.32D). Cells of the dental papilla lay down a layer of dentin continuous with that of the crown (Fig. 17.33). As more and more dentin is deposited, the pulp chamber narrows and finally forms a canal containing blood vessels and nerves of the tooth.
position With further lengthening of the the mesenchyme gives rise to the periodontal ligament (Fig. 17.33), which holds the tooth firmly in and functions as a shock absorber.
root, crown is gradually pushed through the overlying tissue layers into the oral cavity (Fig. 17.33B).The eruption of deciduous or milk teeth occurs 6 to 24 months after birth.
Mesenchymal cells on the outside of the tooth and in contact with dentin of the root differentiate into cementoblasts (Fig. 17.33A). These cells produce a thin layer of specialized bone, the cementum. Outside of the cement layer,
Buds for the permanent teeth, which lie on the lingual aspect of the milk teeth, are formed during the third month of development. These buds remain dormant until approximately the sixth year of postnatal life (Fig. 17.34). Then they begin to grow, pushing against the underside
Left anterolateral view
Figure 17.34 Replacement of deciduous teeth with permanent teeth in a child. |, incisor; C, canine; PM, premolar; MI, M2, M3; Ist, 2nd, and 3rd molars. (From Moore, KL and Dalley,AF Clinically Oriented Anatomy, 5th ed. Figure 7.47B, p. 993. Lippincott Williams & Wilkins, Baltimore: 2006.)

of the milk teeth and aiding in the shedding of them. As a permanent tooth grows, the root of the overlying deciduous tooth is resorbed by osteoclasts.

Teeth are present only in vertebrates and parallel the evolutionary appearance of the neural crest. Tooth development represents a classic example of an epithelial-mesenchymal interaction, in this case between the overlying epithelium and underlying neural crest-derived mesenchyme. Regulation of tooth patterning from incisors to molars is generated by a combinatorial expression of HOX genes expressed in the mesenchyme. With respect to each tooth's individual development, the epithelium governs differentiation to the bud stage, at which time this regulatory function is transferred to the mesenchyme. Signals for development involve growth factors including WNTs, bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs); the secreted factor sonic hedgehog (SHH); and transcription factors, such as MSX1 and 2, that interact in a complex pathway to produce cell differentiation and patterning for each tooth. Teeth also appear to have a signaling center that represents the "organizer" for tooth development much like the activity of the node during gastrulation (see Chapter 5). This organizer region is called the enamel knot, and it appears in a circumscribed region of the dental epithelium at the tips of the tooth buds. It then enlarges at the cap stage into a tightly packed group of cells but undergoes apoptosis (cell death) and disappears by the end of this stage (Fig. 17.32B). While it is present, it expresses FGF4, SHH, and BMP2

Natal teeth have erupted by the time of birth. Usually, they involve the mandibular incisors, which may be abnormally formed and have little enamel.
Teeth may be abnormal in number, shape, and size. They may be discolored by foreign substances, such as tetracyclines, or be deficient in enamel, a condition often caused by vitamin D deficiency (rickets). Many factors affect tooth development, including genetic and environmental influences.
and 4. FGF4 may regulate outgrowth of cusps much as it participates in limb outgrowth produced by the apical ectodermal ridge; while BMP4 may regulate the timing of apoptosis in knot cells.
s_-
Pharyngeal (branchial) arches, consisting of bars of mesenchymal tissue separated by pharyngeal pouches and clefts, give the head and neck their typical appearance in the fourth week (Fig. 17.3). Each arch contains its own artery (Fig. 17.4), cranial nerve (Fig. 17.7), muscle element, and cartilage bar or skeletal element (Figs. 17.8 and 17.9; Table 17.1, p. 269). Endoderm of the pharyngeal pouches gives rise to a number of endocrine glands and part of the middle ear. In subsequent order, the pouches give rise to (1) the middle ear cavity and auditory tube (pouch 1), (2) the stroma of the palatine tonsil (pouch 2), (3) the inferior parathyroid glands and thymus (pouch 3), and (4) the superior parathyroid glands and ultimobranchial body (pouches 4 and 5) (Fig. 17.10).
Pharyngeal clefts give rise to only one structure, the external auditory meatus.

Patterning of the skeletal elements of the pharyngeal arches is regulated by gene expression in pharyngeal pouch endoderm. The process involves epithelial-mesenchymal signaling with endoderm of the pouches sending signals to the responding tissue, the mesenchyme. Mesenchymal gene expression is initially determined by homeodomain-containing transcription factors (OTX2 and HOX genes) carried by pharyngeal arches by migrating neural crest cells. Crest cells originate from the caudal midbrain and from segments in the hindbrain called rhombomeres. These genes respond to endodermal signals and dictate the type of skeletal elements that form.
The thyroid gland originates from an epithelial proliferation in the floor of the tongue and descends to its level in front of the tracheal rings in the course of development.
The paired maxillary and mandibular prominences and the frontonasal prominence are the first prominences of the facial region. Later, medial and lateral nasal prominences form around the nasal placodes on the frontonasal prominence. All of these structures are important, since they determine, through fusion and specialized growth, the size and integrity of the mandible, upper lip, palate, and nose (Table 17.3). Formation of the upper lip occurs by fusion of

the two maxillary prominences with the two medial nasal prominences (Figs. 17.22 and 16.23). The intermaxillary segment is formed by merging of the two medial nasal prominences in the midline. This segment is composed of (1) the philtrum; (2) the upper jaw component, which carries the four incisor teeth; and (3) the palatal component, which forms the triangular primary palate. The nose is derived from (1) the frontonasal prominence, which forms the bridge; (2) the medial nasal prominences, which provide the crest and tip; and (3) the lateral nasal prominences, which form the alae (Fig. 17.23). Fusion of the palatal shelves, which form from the maxillary prominences, creates the hard (secondary) and soft palate. A series of cleft deformities may result from partial or incomplete fusion of these mesenchymal tissues, which may be caused by hereditary factors and drugs (diphenylhydantoin).
The adult form of the face is influenced by development of paranasal sinuses, nasal conchae, and teeth. Teeth develop from epithelial-mesenchymal interactions —_ between oral epithelium and neural crest—derived mesenchyme. Enamel is made by ameloblasts (Figs. 17.32 and 17.33). It lies on a thick layer of dentin produced by odontoblasts, a neural crest derivative. Cementum is formed by cementoblasts, another mesenchymal derivative found in the root of the tooth. The first teeth (deciduous teeth or milk teeth) appear 6 to 24 months after birth, and the definitive or permanent teeth, which supplant the milk teeth, are formed during the third month of development (Fig. 17.34).

1. Why are neural crest cells considered such an important cell population for craniofacial development?
2. You are called as a consultant for a child with a very small mandible and ears that are repre- sented by small protuberances bilaterally. The baby has had numerous episodes of pneumo- nia and is small for its age. What might your diagnosis be, and what might have caused these abnormalities?
3. A child is born with a median cleft lip. Should you be concerned about any other abnormalities?
4. A child presents with a midline swelling beneath the arch of the hyoid bone. What might this swelling be, and what is its basis embryologically?
e central nervous system (CNS) appears at the beginning of the third week as a slippershaped plate of thickened ectoderm, the neural plate, in the mid-dorsal region in front of the primitive node. Its lateral edges soon elevate to form the neural folds (Fig. 18.1).
(Fig. 18.3B). Final closure of the cranial neuropore occurs at the 18 to 20-somite stage (25th day); closure of the caudal neuropore occurs approximately 3 days later.
With further development, the neural folds continue to elevate, approach each other in the midline, and finally fuse, forming the neural tube (Figs. 18.2 and 18.3). Fusion begins in the cervical region and proceeds in cephalic and caudal directions (Fig. 18.3). Once fusion is initiated, the open ends of the neural tube form the cranial and caudal neuropores that communicate with the overlying amniotic cavity (Fig. 18.3B). Closure of the cranial neuropore proceeds cranially from the initial closure site in the cervical region (Fig. 18.3A) and from a site in the forebrain that forms later. This latter site proceeds cranially, to close the rostralmost region of the neural tube, and caudally to meet advancing closure from the cervical site

The cephalic end of the neural tube shows three dilations, the primary brain vesicles: (1) the prosencephalon, or forebrain; (2) the mesencephalon, or midbrain; and (3) the rhombencephalon, or hindbrain (Fig. 18.4). Simultaneously, it forms two flexures: (1) the cervical flexure at the junction of the hindbrain and the spinal cord and (2) the cephalic flexure in the midbrain region (Fig. 18.4).
'When the embryo is 5 weeks old, the prosencephalon consists of two parts: (1) the telencephalon, formed by a midportion and two lateral outpocketings, the primitive cerebral hemispheres; and (2) the diencephalon, characterized by outgrowth of the optic vesicles (Fig. 18.5). A deep furrow, the rhombencephalic isthmus, separates the mesencephalon from the rhombencephalon.
Figure 18.1 A. Dorsal view of a late presomite embryo at approximately 18 days. The amnion has been removed, and the neural plate is clearly visible. B. Dorsal view at approximately 20 days. Note the somites and the neural groove and neural folds.
Figure 18.2 A-C. Transverse sections through successively older embryos showing formation of the neural groove, neural tube, and neural crest. Cells of the neural crest migrate from the edges of the neural folds and develop into spinal and cranial sensory ganglia. D. Scanning electron micrograph of a chick embryo showing the neural tube and neural crest cells migrating from the dorsal region of the tube (compare with B and C).
The rhombencephalon also consists of two parts: (1) the metencephalon, which later forms the pons and cerebellum and (2) the myelencephalon. The boundary between these two portions is marked by the pontine flexure (Fig. 18.5).
lumen becomes very narrow and is then known as the aqueduct of Sylvius. Each lateral ventricle communicates with the third ventricle through the interventricular foramina of Monro (Fig. 18.5).

The lumen of the spinal cord, the central canal, is continuous with that of the brain vesicles. The cavity of the rhombencephalon is the fourth ventricle, that of the diencephalon is the third ventricle, and those of the cerebral hemispheres are the lateral ventricles (Fig. 18.5). The lumen of the mesencephalon connects the third and fourth ventricles. This

Neuroepithelial, Mantle, and Marginal Layers
The wall of a recently closed neural tube consists of neuroepithelial cells. These cells extend over
the entire thickness of the wall and form a thick
Figure 18.3 A. Dorsal view of a human embryo at approximately day 22. Seven distinct somites are visible on each side of the neural tube. B. Dorsal view of a human embryo at approximately day 23. The nervous system is in connection with the amniotic cavity through the cranial and caudal neuropores.
Figure 18.4 Drawing of a sagittal section through the brain at approximately 28 days of human development. Three brain vesicles represent the forebrain (F), midbrain (M), and hindbrain (H).
Figure 18.5 Drawing of a sagittal section through the brain at approximately 32 days of human development. The three original brain vesicles have segregated into the telencephalon, diencephalon, mesencephalon, metencephalon, and myelencephalon.
Figure 18.6 A. Section of the wall of the recently closed neural tube showing neuroepithelial cells, which form a pseudostratified epithelium extending over the full width of the wall. Note the dividing cells at the lumen of the tube. B. Scanning electron micrograph of a section of the neural tube of a chick embryo similar to that in A.
pseudostratified epithelium (Fig. 18.6). Junctional complexes at the lumen connect them. During the neural groove stage and immediately after closure of the tube, they divide rapidly, producing more and more neuroepithelial cells. Collectively, they constitute the neuroepithelial layer or neuroepithelium.
A longitudinal groove, the sulcus limitans, marks the boundary between the two.The dorsal and ventral midline portions of the neural tube, known as the roof and floor plates, respectively, do not contain neuroblasts; they serve primarily as pathways for nerve fibers crossing from one side to the other.

Once the neural tube closes, neuroepithelial cells begin to give rise to another cell type characterized by a large round nucleus with pale nucleoplasm and a dark-staining nucleolus. These are the primitive nerve cells, or neuroblasts (Fig. 18.7). They form the mantle layer, a zone around the neuroepithelial layer (Fig. 18.8). The mantle layer later forms the gray matter of the cord.

The outermost layer of the spinal cord, the marginal layer, contains nerve fibers emerging from neuroblasts in the mantle layer. As a result of myelination of nerve fibers, this layer takes on a white appearance and therefore is called the white matter of the spinal cord (Fig. 18.8).

As a result of continuous addition of neuroblasts to the mantle layer, each side of the neural tube shows a ventral and a dorsal thickening. The ventral thickenings, the basal plates, which contain ventral motor horn cells, form the motor areas of the spinal cord; the dorsal thickenings, the alar plates, form the sensory areas (Fig. 18.8A).
In addition to the ventral motor horn and the dorsal sensory horn, a group of neurons accumulates between the two areas and forms a small intermediate horn (Fig. 18.8B). This horn, containing neurons of the sympathetic portion of the autonomic nervous system (ANS), is present only at thoracic (T1-T12) and upper lumbar levels (L2 or L3) of the spinal cord.

Neuroblasts, or primitive nerve cells, arise exclusively by division of the neuroepithelial cells. Initially, they have a central process extending to the lumen (transient dendrite), but when they migrate into the mantle layer, this process disappears, and neuroblasts are temporarily round and apolar (Fig. 18.9A). With further differentiation, two new cytoplasmic processes appear on opposite sides of the cell body, forming a bipolar neuroblast (Fig. 18.9B).The process at one end of the cell elongates rapidly to form the primitive axon, and the process at the other end shows a number of cytoplasmic arborizations, the primitive dendrites (Fig. 18.9C).
Figure
Chapter
18
Central
Nervous
System
18.7
Section of
the neural
tube at
a
slightly more
advanced stage
than in
Figure
18.6.
The major
portion
291
of the
wall consists
of neuroepithelial
cells.
On the
periphery, immediately
adjacent to
limiting membrane,
neuroblasts form. These cells, which are produced by the neuroepithelial cells in ever-increasing numbers, will form the mantle layer.
The cell is then known as a multipolar neuroblast and with further development becomes the adult nerve cell or neuron. Once neuroblasts form, they lose their ability to divide. Axons of neurons in the basal plate break through the marginal zone and become visible on the ventral aspect of the cord. Known collectively as the ventral motor root of the spinal nerve, they
Figure 18.8 A,B. Two successive stages in the development of the spinal cord. Note formation of ventral motor and
Figure 18.10 A. Motor axons growing out from neurons in the basal plate and centrally and peripherally growing fibers of nerve cells in the dorsal root ganglion. B. Nerve fibers of the ventral motor and dorsal sensory roots join to form the trunk of the spinal nerve. C. Scanning electron micrograph of a cross section through the spinal cord of a chick embryo. The ventral horn and ventral motor root are differentiating.
conduct motor impulses from the spinal cord to the muscles (Fig. 18.10). of

Axons neurons in the dorsal sensory horn (alar plate) behave differently from those in the ventral horn. They penetrate into the marginal layer of the cord, where they ascend to either higher or lower levels to form association neurons.

The majority of primitive supporting cells, the gliablasts, are formed by neuroepithelial cells after production of neuroblasts ceases. Gliablasts migrate from the neuroepithelial layer to the mantle and marginal layers. In the mantle layer, they differentiate into protoplasmic astrocytes and fibrillar astrocytes (Fig. 18.11). These cells are situated between blood vessels and neurons where they provide support and serve metabolic functions.
Another type of supporting cell possibly derived from gliablasts is the oligodendroglial cell. This cell, which is found primarily in the marginal layer, forms myelin sheaths around the ascending and descending axons in the marginal layer.

During elevation of the neural plate, a group of cells appears along each edge (the crest) of the neural folds (Fig. 18.2). These neural crest cells are ectodermal in origin and extend throughout the length of the neural tube. Crest cells migrate laterally and give rise to sensory ganglia (dorsal root ganglia) of the spinal nerves and other cell types (Fig. 18.2).
During further development, neuroblasts of the sensory ganglia form two processes (Fig. 18.10A). The centrally growing processes penetrate the dorsal portion of the neural tube. In the spinal cord, they either end in the dorsal horn or ascend through the marginal layer to one of the higher brain centers. These processes are known collectively as the dorsal sensory root of the spinal nerve (Fig. 18.10B). The peripherally growing processes join fibers of the ventral motor roots and thus participate in formation of the trunk of the spinal nerve. Eventually, these processes terminate in the sensory receptor organs. Hence, neuroblasts of the sensory ganglia derived from neural crest cells give rise to the dorsal root neurons.
In the second half of development, a third type of supporting cell, the microglial cell, appears in the CNS. This highly phagocytic cell type is derived from vascular mesenchyme when blood vessels grow into the nervous system (Fig. 18.11). When neuroepithelial cells cease to produce neuroblasts and glia blasts, they differentiate into ependymal cells lining the central canal of the spinal cord.
In addition to forming sensory ganglia, cells of the neural crest differentiate into sympathetic neuroblasts, Schwann cells, pigment cells, odontoblasts, meninges, and mesenchyme of the pharyngeal arches (see Table 6.1, p. 69).

Motor nerve fibers begin to appear in the fourth week, arising from nerve cells in the basal plates (ventral horns) of the spinal cord. These fibers
Figure 18.11 Origin of the nerve cell and the various types of glial cells. Neuroblasts, fibrillar and protoplasmic astrocytes, and ependymal cells originate from neuroepithelial cells. Microglia develop from mesenchyme cells of blood vessels as
Figure 18.12 A. Motor horn cell with naked rootlet. B. In the spinal cord, oligodendroglia cells surround the ventral rootlet; outside the spinal cord, Schwann cells begin to surround the rootlet. C. In the spinal cord, the myelin sheath is formed by oligodendroglia cells; outside the spinal cord, the sheath is formed by Schwann cells.
collect into bundles known as ventral nerve roots (Fig. 18.10). Dorsal nerve roots form as collections of fibers originating from cells in dorsal root ganglia (spinal ganglia). Central processes from these ganglia form bundles that grow into the spinal cord opposite the dorsal horns. Distal processes join the ventral nerve roots to form a spinal nerve (Fig. 18.10). Almost immediately, spinal nerves divide into dorsal and ventral primary rami. Dorsal primary rami innervate dorsal axial musculature, vertebral joints, and the skin of the back. Ventral primary rami innervate the limbs and ventral body wall and form the major nerve plexuses (brachial and lumbosacral).

Schwann cells myelinate the peripheral nerves with each cell myelinating only a single axon. These cells originate from neural crest, migrate peripherally, and wrap themselves around axons, forming the neurilemma sheath (Fig. 18.12). Beginning at the fourth month of fetal life, many nerve fibers take on a whitish appearance as a result of deposition of myelin, which is formed by repeated coiling of the Schwann cell membrane around the axon (Fig. 18.12C). The myelin sheath surrounding nerve fibers in the spinal cord has a completely different origin, the oligodendroglial cells (Fig. 18.12B,C). Unlike Schwann cells, a single oligodendrocyte can myelinate up to 50 axons. Although myelination of nerve fibers in the spinal cord begins in approximately the fourth month of intrauterine life, some of the motor fibers descending from higher brain centers to the spinal cord do not become myelinated until the first year of postnatal life. Tracts in the nervous system become myelinated at about the time they start to function.

In the third month of development, the spinal cord extends the entire length of the embryo, and spinal nerves pass through the intervertebral foramina at their level of origin (Fig. 18.13). With increasing age, however, the vertebral column and dura lengthen more rapidly than the neural tube, and the terminal end of the spinal cord gradually shifts to a higher level. At birth, this end is at the level of the third lumbar vertebra (Fig. 18.13C). As a result of this disproportionate growth, spinal nerves run obliquely from their segment of origin in the spinal cord to the corresponding level of the vertebral column. The dura remains attached to the vertebral column at the coccygeal level.
In the adult, the spinal cord terminates at the level of L2-L3, whereas the dural sac and subarachnoid space extend to S2. At the end of the cord, a threadlike extension of pia mater passes caudally, goes through the dura, which provides a covering layer at S2 and extends to the first coccygeal vertebra. This structure is called the filum terminale, and it marks the tract of regression of the spinal cord as well as providing support for the cord (the part covered by dura and extending from S2, to the coccyx is also called the coccygeal ligament). Nerve fibers below the terminal end of the cord collectively constitute the cauda equina. When cerebrospinal fluid is tapped during a lumbar puncture, the needle is inserted at the lower lumbar level (L4—L5), avoiding the lower end of the cord.

Dorsal (sensory) and ventral (motor) regions of the developing spinal cord are dependent upon concentration gradients between members of the transforming growth factor beta (TGF-) family of growth factors secreted in the dorsal neural tube and sonic hedgehog (SHH) secreted by the notochord and floor plate (Fig. 18.144). Initially, bone morphogenetic proteins (BMP) 4 and 7 are secreted by ectoderm overlying the neural tube, and the presence of these proteins establishes a second signaling center in the roof plate. Then, BMP4 in the roof plate induces a cascade of TGF-B proteins, including BMP5, BMP7, activin, and dorsalin in the roof plate and surrounding area. This cascade is organized in time and space such that a concentration gradient of these factors is established. As a result, cells near the roof plate are exposed to the highest concentrations with more ventrally positioned cells seeing less and less of these factors.
Figure 18.14 A,B. Drawings illustrating the molecular regulation of neuron differentiation in the spinal cord. A. Initially, BMP4 and 7 secreted in the ectoderm overlying the neural tube establish a signaling center in the roof plate. Then, BMP4 in the roof plate upregulates a cascade of TGF-B proteins, including BMPS and 7, activin, and dorsalin in this region. Similarly, SHH secreted by the notochord establishes additional SHH signaling in the floor plate. In this manner, an overlapping gradient involving the dorsal and ventral factors is established in the neural tube. B. The gradient established by TGF-B proteins and SHH activates transcription factors that regulate neuronal differentiation. For example, high concentrations of TGF-f in the dorsal neural tube activate PAX3 and 7 that control sensory neuron differentiation. High concentrations of SHH and very low concentrations of TGF-f near the floor plate activate NKX2.2 and NKXé6./ and ventral neuron formation. Slightly higher concentrations of TGF-B and slightly lower concentrations of SHH activate NKX6./ and PAX6 and differentiation of ventral motor neurons and so on.

Similar events occur in the ventral region of the neural tube, only the signaling molecule is SHH. This factor is first expressed in the notochord followed by the establishment of a second signaling center in the floor plate (Fig. 18.14). As a result, there is a diminishing concentration of SHH from the ventral to the dorsal region of the neural tube.
Thus, two overlapping concentrations are established between TGF-B family members and SHH. These gradients then activate transcription factors that regulate differentiation of sensory and motor neurons. For example, high concentrations of TGF-f factors and very low concentrations of SHH in the dorsal neural
tube activate PAX3 and 7 that controls sensory neuron differentiation (Fig. 18.14B). Likewise, high concentrations of SHH and very low concentrations of TGF-B molecules in the ventralmost region result in activation of NKX2.2 and NKX6.1 and ventral neuron formation. Immediately dorsal to this region, where slightly

Most defects of the spinal cord result from abnormal closure of the neural folds in the third and fourth weeks of development. The resulting abnormalities, neural tube defects (NTDs), may involve the meninges, vertebrae, muscles, and skin. The birth prevalence of NTDs, including spina bifida and anencephaly, varies among different populations and may be as high as 1/200 births in some areas, such as Northern China. The birth prevalence of NTDs in the United States has decreased by approximately 25% to 1/1,500 births since fortification of flour with folic acid was instituted in 1998.
lower concentrations of SHH and higher concentrations of TGF-B molecules occur, expression of NKX6.1 and PAX6 is initiated, and these transcription factors induce differentiation of ventral motor horn cells. These interactions continue to produce all the different types of neurons in the spinal cord.
the vertebral arches and may or may not involve underlying neural tissue. Spina bifida occulta is a defect in the vertebral arches that is covered by skin and normally does not involve underlying neural tissue (Fig. 18.154). Most often, the defect occurs in the sacral region (S|—S,) and is sometimes marked by a patch of hair overlying the affected region. The defect, which is due to a lack of fusion of the vertebral arches, affects about 10% of otherwise normal people. The malformation is not usually detected at birth and does not cause disability. Often, the defect is first noticed as an incidental finding diagnosed when an x-ray of the back is performed.
Spina bifida is a general term for NTDs affecting the spinal region. It consists of a splitting of
Other types of spina bifida include meningocele and myelomeningocele. These are severe

Figure 18.15 A-E. Drawings illustrating a variety of NTDs involving the spinal cord. The term spina bifida applies to all of the defects because the bony arch of one or more vertebrae has failed to fuse dorsal to the spinal cord. In some cases, the bony defect is covered by skin (spina bifida occulta; A), but the spinal cord is intact. Often, this defect is noticeable because of a patch of dark hair that grows over the area. In cases of meningoceles B, only a fluid-filled sac of meninges protrudes from the defect, whereas myelominigoceles include neural tissue in the sac C. Rachischisis refers to NTDs in which the neural tube fails to close, resulting in spina bifida and exposure of neural tissue that often becomes necrotic D,E. Rachischisis may occur in the spinal cord or brain regions of the neural tube and represents the most severe type of abnormality. Most spinal cord defects occur in the lumbosacral area, and 50% to 70% of all NTDs can be prevented by maternal use of folic acid (400 1g daily) prior to and during pregnancy.

NTDs in which neural tissue and/or meninges protrude through a defect in the vertebral arches and skin to forma cyst-like sac (Fig. 18.15). Most lie in the lumbosacral region and result in neurological deficits, but they are usually not associated with intellectual disability. In some cases, only fluid-filled meninges protrude through the defect (meningocele) (Fig. 18.15B); in others, neural tissue is included in the sac (myelomeningocele) (Fig. 18.15C). Occasionally, the neural folds do not elevate but remain as a flattened mass of neural tissue (spina bifida with myeloschisis or rachischisis) (Figs. 18.15D,E and 18.16). Hydrocephaly requiring intervention develops in 80% to 90% of children born with severe NTDs and is often related to the presence of an Arnold-Chiari malformation (herniation of part of the cerebellum into the foramen magnum), which obstructs the flow of cerebrospinal fluid and causes the hydrocephalus. Herniation of the cerebellum occurs because the spinal cord is tethered to the vertebral column due to its abnormal development. As the vertebral column lengthens, tethering of the cord pulls the cerebellum into the foramen magnum, cutting off the flow of cerebrospinal fluid. Hydrocephalus can be treated by inserting a ventriculoperitoneal shunt, which allows drainage of the cerebrospinal fluid from one of the cerebral ventricles to the peritoneal cavity.
Spina bifida can be diagnosed prenatally by ultrasound and by determination of alphafetoprotein levels in maternal serum and amniotic fluid. The vertebra can be visualized by 12 weeks of gestation, and defects in closure of the vertebral arches can be detected. Experimental treatment for the defect is to perform surgery in utero as early as 22 weeks of gestation. The fetus is exposed by an incision into the uterus, the defect is repaired, and the infant is placed back in the uterus.

Hyperthermia, valproic acid, and hypervitaminosis A produce NTDs, as do a large number of other teratogens. The origin of most NTDs is multifactorial, and the likelihood of having a

The brain is sometimes divided into the brain stem (consisting of the myelencephalon, pons from the metencephalon, and the mesencephalon) and the higher centers (cerebellum and
Figure 18.16 Patient with a severe spina bifida involving several vertebrae in the lumbosacral region.
child with such a defect increases significantly once one affected offspring is born. Recent evidence proves that folic acid (folate) reduces the occurrence of NTDs by as much as 50% to 70% if 400 jig is taken daily beginning at least 1 month prior to conception and continuing through early pregnancy.
Since approximately 50% of all pregnancies are unplanned, it is recommended that all women of child-bearing age take a multivitamin containing 400 pg of folic acid daily. Furthermore, women who have had a child with an NTD or who have a family history of NTDs should take 400 ug of folic acid daily and then 4,000 pg (4 mg) per day starting at least 1 month before conception through the first 3 months of pregnancy.
cerebral hemispheres). The brain stem is a direct continuation of the spinal cord and has a similar organization. Thus, distinct basal and alar plates, representing motor and sensory areas, respectively, are found on each side of the midline. However, the higher centers reflect almost
Figure 18.17 Lateral view of the brain vesicles in an 8-week embryo (crown-rump length ~27 mm).The roof plate of the rhombencephalon has been removed to show the intraventricular portion of the rhombic lip. Note the origin of the cranial nerves.
none of this basic pattern and, instead, show accentuation of the alar plates and regression of the basal plates.

the pontine flexure to the rhombencephalic isth- mus (Figs. 18.5 and 18.17).

The myelencephalon is a brain vesicle that gives
The rhombencephalon consists of the myelencephalon, the most caudal of the brain vesicles, and the metencephalon, which extends from rise to the medulla oblongata. It differs from
the spinal cord in that its lateral walls are everted
(Fig. 18.18). Alar and basal plates separated by the sulcus limitans can be clearly distinguished.
Figure 18.18 A. Dorsal view of the floor of the fourth ventricle in a 6-week embryo after removal of the roof plate. Note the alar and basal plates in the myelencephalon. The rhombic lip is visible in the metencephalon. B,C. Position and differentiation of the basal and alar plates of the myelencephalon at different stages of development. Note formation of the nuclear groups in the basal and alar plates. Arrows, path followed by cells of the alar plate to the olivary nuclear complex. The
The basal plate, similar to that of the spinal cord, contains motor nuclei. These nuclei are divided into three groups: (1) a medial somatic efferent group, (2) an intermediate special visceral efferent group, and (3) a lateral general visceral efferent group (Fig. 18.18C;Table 18.1). The somatic efferent group contains motor neurons, which form the cephalic continuation of the anterior horn cells. Since this group continues rostrally into the mesencephalon, it is called the somatic efferent motor column. In the myelencephalon, it includes neurons of the hypoglossal (XII) nerve that supply the tongue musculature. In the metencephalon and the mesencephalon, the column contains neurons of the abducens (VI) (Fig. 18.19), trochlear (IV), and oculomotor(III) nerves (Fig. 18.23), respectively. These nerves supply the eye musculature.

The special visceral efferent group extends into the metencephalon, forming the special visceral efferent motor column. Its motor neurons supply striated muscles of the pharyngeal arches. In the myelencephalon, the column is represented by neurons of the accessory (XI), vagus (X), and glossopharyngeal (IX) nerves.
Somatic efferent
(VI)
Figure 18.19 Transverse section through the caudal part of the metencephalon. Note the differentiation of the various motor and sensory nuclear areas in the basal and alar plates, respectively, and the position of the rhombic lips, which project partly into the lumen of the fourth ventricle and partly above the attachment of the roof plate. Arrows, direction of migration of the nuclei.
The general visceral efferent group contains motor neurons that supply involuntary musculature of the respiratory tract, intestinal tract, and heart.
The alar plate contains three groups of sensory relay nuclei (Fig. 18.18C; Table 18.1). The most lateral of these, the somatic afferent (general sensory) group, receives sensations of pain, temperature, and touch from the pharynx by way of the glossopharyngeal nerve (IX). The intermediate, or special afferent, group receives impulses from taste buds of the tongue, palate, oropharynx, and epiglottis and from the vestibulocochlear nerve (VIII) for hearing and balance. The medial, or general visceral afferent, group receives interoceptive information from the gastrointestinal tract and heart.
The roof plate of the myelencephalon consists of a single layer of ependymal cells covered by vascular mesenchyme, the pia mater (Fig. 18.18C). The two combined are known as the tela choroidea. Because of active proliferation of the vascular mesenchyme, a number of sac-like invaginations project into the underlying ventricular cavity (Figs. 18.18C). These tuft-like invaginations form the choroid plexus, which produces cerebrospinal fluid.

The metencephalon, similar to the myelencephalon, is characterized by basal and alar plates (Fig. 18.19). Two new components form (1) the cerebellum, a coordination center for posture and movement (Fig. 18.20), and (2) the pons, the pathway for nerve fibers between the spinal cord and the cerebral and cerebellar cortices.
Each basal plate of the metencephalon (Fig. 18.19; Table 18.1) contains three groups of motor neurons: (1) the medial somatic efferent group, which gives rise to the nucleus of the abducens nerve; (2) the special visceral efferent group, containing nuclei of the trigeminal and facial nerves, which innervate the musculature of the first and second pharyngeal arches; and (3) the general visceral efferent group, with axons that supply the submandibular and sublingual glands.
The marginal layer of the basal plates of the metencephalon expands as it makes a bridge for nerve fibers connecting the cerebral cortex and cerebellar cortex with the spinal cord. Hence, this portion of the metencephalon is known as the pons (bridge). In addition to nerve fibers, the pons contains the pontine nuclei, which originate in the alar plates of the metencephalon and myelencephalon (Fig. 18.19, arrows).
The alar plates of the metencephalon contain three groups of sensory nuclei: (1) a lateral somatic afferent group, which contains neurons of the trigeminal nerve; (2) the special afferent group; and (3) the general visceral afferent group (Fig. 18.19;Table 18.1).

The dorsolateral parts of the alar plates bend medially and form the rhombic lips (Fig. 18.18). In the caudal portion of the metencephalon, the rhombic lips are widely separated, but immediately below the mesencephalon, they approach each other in the midline (Fig. 18.20). As a result of a further deepening of the pontine flexure, the rhombic lips compress cephalocaudally and form the cerebellar plate (Fig. 18.20). In a 12-week embryo, this plate shows a small midline portion, the vermis, and two lateral portions, the hemispheres. A transverse fissure soon separates the nodule from the vermis and the lateral
Figure 18.20 A. Dorsal view of the mesencephalon and rhombencephalon in an 8-week embryo. The roof of the fourth ventricle has been removed, allowing a view of its floor. B. Similar view in a 4-month embryo. Note the choroidal fissure and the lateral and medial apertures in the roof of the fourth ventricle.
Figure 18.21 Sagittal sections through the roof of the metencephalon showing development of the cerebellum. A. 8 weeks (~30 mm). B. 12 weeks (70 mm). C. 13 weeks. D. 15 weeks. Note formation of the external granular layer on the surface of the cerebellar plate B,C. During later stages, cells of the external granular layer migrate inward to mingle with Purkinje cells and form the definitive cortex of the cerebellum. The dentate nucleus is one of the deep cerebellar nuclei. Note the anterior and posterior velum.
flocculus from the hemispheres (Fig. 18.20B). This flocculonodular lobe is phylogenetically the most primitive part of the cerebellum. Initially, the cerebellar plate consists of neuroepithelial, mantle, and marginal layers (Fig. 18.214). During further development, a number of cells formed by the neuroepithelium migrate to the surface of the cerebellum to form the external granular layer. Cells of this layer retain their ability to divide and form a proliferative zone on the surface of the cerebellum (Fig. 18.21B,C).

In the sixth month of development, the external granular layer gives rise to various cell types. These cells migrate toward the differentiating Purkinje cells (Fig. 18.22) and give rise to granule cells. Basket and stellate cells are produced by proliferating cells in the cerebellar white matter. The cortex of the cerebellum, consisting of Purkinje cells, Golgi II neurons, and neurons produced by the external granular layer, reaches its definitive size after birth (Fig. 18.22B).The deep cerebellar nuclei, such as the dentate nucleus, reach their final position before birth (Fig. 18.21D).
Figure 18.22 Stages in development of the cerebellar cortex. A. The external granular layer on the surface of the cerebellum forms a proliferative layer from which granule cells arise. They migrate inward from the surface (arrows). Basket and stellate cells derive from proliferating cells in the cerebellar white matter. B. Postnatal cerebellar cortex showing differenti- ated Purkinje cells, the molecular layer on the surface, and the internal granular layer beneath the Purkinje cells.
Figure 18.23 A,B. Position and differentiation of the basal and alar plates in the mesencephalon at various stages of development. Arrows in A indicate the path followed by cells of the alar plate to form the nucleus ruber and substantia nigra. Note the various motor nuclei in the basal plate.

In the mesencephalon (Fig. 18.23), each basal plate contains two groups of motor nuclei: (1) a medial somatic efferent group, represented by the oculomotor and trochlear nerves, which innervate the eye musculature and (2) a small general visceral efferent group, represented by the nucleus of Edinger-Westphal, which innervates the sphincter pupillary muscle (Fig. 18.23B). The marginal layer of each basal plate enlarges and forms the crus cerebri. These crura serve as pathways for nerve fibers descending from the cerebral cortex to lower centers in the pons and spinal cord. Initially, the alar plates of the mesencephalon appear as two longitudinal elevations separated by a shallow midline depression (Fig. 18.23). With further development, a transverse groove divides each elevation into an anterior (superior) and a posterior (inferior) colliculus (Fig. 18.23B).The posterior colliculi serve as synaptic relay stations for auditory reflexes; the anterior colliculi function as correlation and reflex centers for visual impulses. The colliculi are formed by waves of neuroblasts migrating into the overlying marginal zone. Here they are arranged in layers (Fig. 18.23B).

The prosencephalon consists of the telencephalon, which forms the cerebral hemispheres and the diencephalon, which forms the optic cup and stalk, pituitary, thalamus, hypothalamus, and epiphysis.

The diencephalon, which develops from the median portion of the prosencephalon (Figs. 18.5 and 18.17), is thought to consist of a roof plate and two alar plates but to lack floor and basal plates (interestingly, SHH, a ventral midline marker, is expressed in the floor of the diencephalon, suggesting that a floor plate does exist). The roof plate of the diencephalon consists of a single layer of ependymal cells covered by vascular mesenchyme. Together, these layers give rise to the choroid plexus of the third ventricle (Fig. 18.30). The most caudal part of the roof plate develops into the pineal body, or epiphysis. This body initially appears as an epithelial thickening in the midline, but by the seventh week, it begins to evaginate (Figs. 18.24 and 18.25). Eventually, it becomes a solid organ on the roof of the mesencephalon (Fig. 18.30) that serves as a channel through which light and darkness affect endocrine and behavioral rhythms. In the adult, calcium is frequently deposited in the epiphysis and then serves as a landmark on radiographs of the skull.

The alar plates form the lateral walls of the diencephalon. A groove, the hypothalamic sulcus, divides the plate into a dorsal and a ventral region, the thalamus and hypothalamus, respectively (Figs. 18.24 and 18.25).
As a result of proliferative activity, the thalamus gradually projects into the lumen of the diencephalon. Frequently, this expansion is so great that thalamic regions from the right and left sides fuse in the midline, forming the massa intermedia, or interthalamic connexus.
The hypothalamus, forming the lower portion of the alar plate, differentiates into a number of nuclear areas that regulate the visceral functions, including sleep, digestion, body temperature, and emotional behavior. One of these groups, the
Figure 18.24. A. Medial surface of the right half of the prosencephalon in a 7-week embryo. B. Transverse section through the prosencephalon at the level of the broken line in A.The corpus striatum bulges out in the floor of the lateral ventricle and the foramen of Monro.
mamillary body, forms a distinct protuberance on the ventral surface of the hypothalamus on each side of the midline (Figs. 18.24A and 18.25A).

—
The hypophysis,
or pituitary
gland, two
completely different
parts:
(1)
outpocketing of
the stomodeum
develops from
an ectodermal
(primitive
Figure 18.25. A. Medial surface of the right half of the telencephalon and diencephalon in an 8-week embryo. B,C.
oral
Figure 18.26 A. Sagittal section through the cephalic part of a 6-week embryo showing Rathke's pouch as a dorsal outpocketing of the oral cavity and the infundibulum as a thickening in the floor of the diencephalon. B,C. Sagittal sections through the developing hypophysis in the | Ith and 16th weeks of development, respectively. Note formation of the pars tuberalis encircling the stalk of the pars nervosa.
cavity) immediately in front of the oropharyngeal membrane, known as Rathke's pouch and (2) a downward extension of the diencephalon, the infundibulum (Fig. 18.26).
When the embryo is approximately 3 weeks old, Rathke's pouch appears as an evagination of the oral cavity and subsequently grows dorsally toward the infundibulum. By the end of the second month, it loses its connection with the oral cavity and is then in close contact with the infundibulum.
During further development, cells in the anterior wall of Rathke's pouch increase rapidly in number and form the anterior lobe of the hypophysis, or adenohypophysis (Fig. 18.26B). A small extension of this lobe, the pars tuberalis, grows along the stalk of the infundibulum and eventually surrounds it (Fig. 18.26C). The posterior wall of Rathke's pouch develops into the pars intermedia, which in humans seems to have little significance.
The infundibulum gives rise to the stalk and the pars nervosa, or posterior lobe of the hypophysis (neurohypophysis) (Fig. 18.26C). It is composed of neuroglial cells. In addition, it

Occasionally,a small portion of Rathke's pouch persists in the roof of the pharynx as a pharyngeal hypophysis. Craniopharyngiomas arise from remnants of Rathke's pouch. They may form within the sella turcica or along the stalk of the pituitary but usually lie above the sella. They may cause hydrocephalus and pituitary dysfunction (e.g., diabetes insipidus, growth failure).
contains a number of nerve fibers from the hypothalamic area.

The telencephalon, the most rostral of the brain vesicles, consists of two lateral outpocketings, the cerebral hemispheres, and a median portion, the lamina terminales (Figs. 18.5, 18.24, and 18.25). The cavities of the hemispheres, the lateral ventricles, communicate with the lumen of the diencephalon through the interventricular foramina of Monro (Fig. 18.24).

The cerebral hemispheres arise at the beginning of the fifth week of development as bilateral evaginations of the lateral wall of the prosencephalon (Fig. 18.24). By the middle of the second month, the basal part of the hemispheres (ie., the part that initially formed the forward extension of the thalamus) (Fig. 18.24A) begins to grow and bulges into the lumen of the lateral ventricle and into the floor of the foramen of Monro (Figs. 18.24B and 18.25A,B). In transverse sections, the rapidly growing region has a striated appearance and is therefore known as the corpus striatum (Fig. 18.25B).
In the region where the wall of the hemisphere is attached to the roof of the diencephalon, the wall fails to develop neuroblasts and remains very thin (Fig. 18.24B). Here the hemisphere wall consists of a single layer of ependymal cells covered by vascular mesenchyme, and together, they form the choroid plexus. The choroid plexus should have formed the roof of the hemisphere, but as a result of the disproportionate growth of the various parts of the hemisphere, it protrudes into the lateral ventricle along the choroidal fissure (Figs. 18.25 and 18.27). Immediately above the choroidal fissure, the wall of the
Figure 18.27 A. Medial surface of the right half of the telencephalon and diencephalon in a 10-week embryo.
B. Transverse section through the hemisphere and diencephalon at the level of the broken line in A.
hemisphere thickens, forming the hippocampus (Figs. 18.24B and 18.25B). This structure's primary function is olfaction, and it bulges into the lateral ventricle.

With further expansion, the hemispheres cover the lateral aspect of the diencephalon, mesencephalon, and cephalic portion of the metencephalon (Figs. 18.27 and 18.28). The corpus striatum (Fig. 18.24B), being a part of the wall of the hemisphere, likewise expands posteriorly and is divided into two parts: (1) a dorsomedial portion, the caudate nucleus, and (2) a ventrolateral portion, the lentiform nucleus (Fig. 18.27B). This division is accomplished by axons passing to and from the cortex of the hemisphere and breaking through the nuclear mass of the corpus striatum. The fiber bundle thus formed is known as the internal capsule (Fig. 18.27B). At the same time, the medial wall of the hemisphere and the lateral wall of the diencephalon fuse, and the caudate nucleus and thalamus come into close contact (Fig. 18.27B).
Continuous growth of the cerebral hemispheres in anterior, dorsal, and inferior directions results in the formation of frontal, temporal, and occipital lobes, respectively. As growth in the region overlying the corpus
Figure 18.28 Development of gyri and sulci on the lateral surface of the cerebral hemisphere. A. 7 months. B. 9 months.
striatum slows, however, the area between the frontal and temporal lobes becomes depressed and is known as the insula (Fig. 18.284). This region is later overgrown by the adjacent lobes and at the time of birth is almost completely covered. During the final part of fetal life, the surface of the cerebral hemispheres grows so rapidly that a great many convolutions (gyri) separated by fissures and sulci appear on its surface (Fig. 18.28B).
nasal placodes differentiate into primary sensory neurons of the nasal epithelium, which has axons that grow and make contact with secondary neurons in the developing olfactory bulbs (Fig. 18.29). By the seventh week, these contacts are well established. As growth of the brain continues, the olfactory bulbs and the olfactory tracts of the secondary neurons lengthen, and together they constitute the olfactory nerve (Fig. 18.30).

The cerebral cortex develops from the pallium (Fig. 18.24B), which has two regions: (1) the paleopallium, or archipallium, immediately lateral to the corpus striatum (Fig. 18.25B), and (2) the neopallium, between the hippocampus and the paleopallium (Figs. 18.25B and 18.27B). In the neopallium, waves of neuroblasts migrate to a subpial position and then differentiate into fully mature neurons. When the next wave of neuroblasts arrives, they migrate through the earlier-formed layers of cells until they reach the subpial position. Hence, the early-formed neuroblasts obtain a deep position in the cortex, while those formed later obtain a more superficial position.
At birth, the cortex has a stratified appearance due to differentiation of the cells in layers. The motor cortex contains a large number of pyramidal cells, and the sensory areas are characterized by granular cells.
Differentiation of the olfactory system is dependent on epithelial-mesenchymal interactions. These occur between neural crest cells and ectoderm of the frontonasal prominence to form the olfactory placodes (see Chapter 17, p. 273) and between these same crest cells and the floor of the telencephalon to form the olfactory bulbs (Fig. 18.29). Cells in the

In the adult, a number of fiber bundles, the commissures, which cross the midline, connect the right and left halves of the hemispheres. The most important fiber bundles make use of the lamina terminalis (Figs. 18.244 and 18.25A). The first of the crossing bundles to appear is the anterior commissure. It consists of fibers connecting the olfactory bulb and related brain areas of one hemisphere to those of the opposite side (Figs. 18.27A and 18.30).
The second commissure to appear is the hippocampal commissure, or fornix commissure. Its fibers arise in the hippocampus and converge on the lamina terminalis close to the roof plate of the diencephalon. From here, the fibers continue, forming an arching system immediately outside the choroid fissure, to the mamillary body and the hypothalamus.
The most important commissure is the corpus callosum. It appears by the 10th week of development and connects the nonolfactory areas of the right and the left cerebral cortices. Initially, it forms a small bundle in the lamina terminalis (Fig. 18.27A). As a result of continuous expansion of the neopallium, however, it extends first anteriorly and then posteriorly, arching over the thin roof of the diencephalon (Fig. 18.30).
Figure 18.29 A. Sagittal section through the nasal pit and lower rim of the medial nasal prominence of a 6-week embryo. The primitive nasal cavity is separated from the oral cavity by the oronasal membrane. B. Similar section as in A toward the end of the sixth week, showing breakdown of the oronasal membrane. C. At 7 weeks, neurons in the nasal epithelium have extended processes that contact the floor of the telencephalon in the region of the developing olfactory bulbs. D. By 9 weeks, definitive oronasal structures have formed, neurons in the nasal epithelium are well differentiated, and secondary neurons from the olfactory bulbs to the brain begin to lengthen. Together, the olfactory bulbs and tracts of the secondary neurons constitute the olfactory nerve (Fig. 18.30).

In addition to the three commissures developing in the lamina terminalis, three more appear. Two of these, the posterior and habenular commissures, are just below and rostral to the stalk of the pineal gland. The third, the optic chiasma, which appears in the rostral wall of the diencephalon, contains fibers from the medial halves of the retinae (Fig. 18.30).

Cerebrospinal fluid (CSF) is secreted by the choroid plexuses in the brain ventricles. These plexuses are modifications of the ependymal layer and produce approximately 400 to 500 mL of CSF per day. The fluid circulates through the brain ventricles leaving the lateral ventricles, for example, through the interventricular foramina, entering the third ventricle, then passing through the cerebral aqueduct into the fourth ventricle (Fig. 18.30). Some CSF enters the spinal canal and some exits the fourth ventricle through its median and lateral apertures to enter the subarachnoid space that surrounds the CNS. CSF is absorbed into the venous system from the subarachnoid space through arachnoid granulations, especially those that project into the superior sagittal sinus. CSF "floats" the brain and, thus, provides a cushion for the brain and gives it buoyancy so that its weight does not compress cranial nerves against the inside of the skull.

Anteroposterior (craniocaudal) patterning of the CNS begins early in development, during gastrulation and neural induction (see Chapters 5 and 6). Once the neural plate is established, signals for segregation of the brain into forebrain, midbrain, and hindbrain regions are derived from homeobox genes expressed in the notochord, prechordal plate, and neural plate. The hindbrain has eight segments, the rhombomeres, that have variable expression patterns of the Antennapedia class of homeobox genes, the HOX genes (see Chapter 6, p. 81). These genes are expressed in overlapping (nested) patterns, with genes at the most 3' end of a cluster having more anterior boundaries and paralogous genes having identical expression domains (Fig. 18.81). Genes at the 3' end are also expressed earlier than those at the 5' end, so that a temporal relation to the expression pattern is established. These genes, then, confer positional value along the anteroposterior axis of the hindbrain, determine the identity of the rhombomeres, and specify their derivatives. How this regulation occurs is not clear, although retinoids (retinoic acid) play a critical role in regulating HOX expression. For example, excess retinoic acid shifts HOX gene expression anteriorly and causes more cranial rhombomeres to differentiate into more caudal types. Retinoic acid deficiency results in a small hindbrain. There is also a differential response to retinoic acid by the HOX genes; those at the 3' end of the cluster are more sensitive than those at the 5' end.

Specification of the forebrain and midbrain areas is also regulated by genes containing a homeodomain. However, these genes are not of the Antennapedia class, whose most anterior boundary of expression stops at rhombomere 3. Thus, new genes have assumed the patterning role for these regions of the brain, which evolutionarily constitute the "new head." At the neural plate stage, LIM1, expressed in the prechordal plate, and OTX2, expressed in the neural plate, are important for designating the forebrain and midbrain areas, with LIM1 supporting OTX2 expression. (These genes are also expressed at the earliest stages of gastrulation, and they assist in specifying the entire cranial region of the epiblast.) Once the neural folds and pharyngeal arches appear, additional homeobox genes, including OTX1, EMX1, and EMX2, are expressed in specific and in overlapping (nested) patterns in the midand forebrain regions and specify the identity of these areas. Once these boundaries are established, two additional organizing centers appear: the anterior neural ridge (ANR) at the junction of the cranial border of the neural plate and nonneural ectoderm (Fig. 18.32) and the isthmus (Fig. 18.33) between the hindbrain and midbrain. In both locations, fibroblast growth factor 8 (FGF8) is the key signaling molecule, inducing subsequent gene expression that regulates differentiation. In the ANR at the four-somite stage, FGF8 induces expression of FOXG1, a transcription factor
(Fig. 18.32). FOXG1 then regulates development of the telencephalon (cerebral hemispheres) and regional specification within the forebrain, including the basal telencephalon and the retina. In the isthmus at the junction between the midbrain and hindbrain territories, FGF8 is expressed in a ring around the circumference of this location (Fig. 18.33). FGF8

induces expression of engrailed 1 and 2 (EN1 and EN2), two homeobox-containing genes, expressed in gradients radiating anteriorly and posteriorly from the isthmus. EN1/ regulates development throughout its expression domain, including the dorsal midbrain (tectum) and anterior hindbrain (cerebellum), whereas EN2 is involved only in cerebellar development.
Figure 18.33 Organizing center in the rhombencephalic isthmus at the boundaries between the midbrain (M) and hindbrain (H).This region secretes FGF8 in a circumferential ring that induces expression of engrailed | and 2 (EN/ and EN2) in gradients anteriorly and posteriorly from this area. EN/ regulates development of the dorsal midbrain, and both genes participate in formation of the cerebellum. WNT/, another gene induced by FGF8, also assists in development of the
FGF8 also induces WNT1 expression in a circumferential band anterior to the region of FGF8 expression (Fig. 18.33). WNT1 interacts with EN1 and EN2 to regulate development of this region, including the cerebellum. In fact, WNT1 may assist in early specification of the midbrain area since it is expressed in this region at the neural plate stage. FGF8 is also expressed at this early time in mesoderm underlying the midbrain—hindbrain junction and may therefore regulate WNT expression and initial patterning of this region. The constriction for the isthmus is slightly posterior to the actual midbrain—hindbrain junction, which lies at the caudal limit of OTX2 expression.

Dorsoventral (mediolateral) patterning also occurs in the forebrain and midbrain areas. Ventral patterning is controlled by SHH just as it is throughout the remainder of the CNS. SHH, secreted by the prechordal plate, induces expression of NKX2.1, a homeodomain-containing gene that regulates development of the hypothalamus. Importantly, SHH signaling requires cleavage of the protein, and the carboxy-terminal portion executes this process. Following cleavage of the SHH protein, cholesterol is covalently linked to the carboxy-terminus of the amino terminal product. The amino terminal portion

Holoprosencephaly (HPE) refers to a spectrum of abnormalities in which a loss of midline structures results in malformations of the brain and face. In severe cases, the lateral ventricles merge into a single telencephalic vesicle (alobar HPE), the eyes are fused, and there is a single nasal chamber along with other midline facial defects (Fig. 18.34). In less severe cases, some division of the prosencephalon into two cerebral hemispheres occurs, but there is incomplete development of midline structures. Usually, the olfactory bulbs and tracts and the corpus callosum are hypoplastic or absent. In very mild cases, sometimes the only indication that some degree of HPE has occurred is the presence of a single central incisor. HPE occurs in 1/15,000 live births but is present in 1/250 pregnancies that end in early miscarriage. Mutations in SHH, the gene that regulates establishment of the ventral midline in the CNS, result in some forms of HPE. Another cause is defective retains all of the signaling properties of SHH, and its association with cholesterol assists in its distribution.
Dorsal (lateral) patterning of the neural tube is controlled by bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) expressed in the nonneural ectoderm adjacent to the neural plate. These proteins induce expression of MSX1 in the midline and repress expression of FOXG1 (Fig. 18.32). Once the neural tube is closed, BMP2 and 4 are expressed in the roof plate, and these proteins regulate expression of the transcription factor LHX2 in the cortex.This expression then initiates a cascade of genes to pattern this region.
Expression patterns of genes regulating anterior—posterior (craniocaudal) and dorsoventral (mediolateral) patterning of the brain overlap and interact at the borders of these regions. Furthermore, various brain regions are competent to respond to specific signals and not to others. For example, only the cranial part of the neural plate expresses NKX2.1 in response to SHH. Likewise, only the anterior neural plate produces FOXG1 in response to FGF8; midbrain levels express EN2 in response to the same FGF8 signal. Thus, a competence to respond also assists in specifying regional differences.

cholesterol biosynthesis leading to SmithLemli-Opitz syndrome. These children have craniofacial and limb defects, and 5% have HPE. Smith-Lemli-Opitz's syndrome is an autosomal recessive condition due to abnormalities in 7-dehydrocholesterol reductase, which metabolizes 7-dehydrocholesterol to cholesterol. Many of the defects, including those of the limbs and brain, may be due to abnormal SHH signaling, as cholesterol is necessary for this gene to exert its effects. Other genetic causes include mutations in the transcription factors sine occulis homeobox 3 (SIX3), TG-interacting factor (TGIF), and the zinc finger protein (ZIC2).
Schizencephaly is a rare disorder in which large clefts occur in the cerebral hemispheres, sometimes causing a loss of brain tissue. Mutations in the homeobox gene EMX2 appear to account for some of these cases.
Ossification defects in bones of the skull can result in meningoceles, meningoencephaloceles, and meningohydroencephaloceles.
Figure 18.34 Child with holoprosencephaly (HPE). Note that a loss of midline tissue has resulted in a midline cleft lip, lack of nasal tissue, and eyes that are too close together (hypotelorism). In the brain, the loss of midline tissue causes the lateral ventricles to merge into a single chamber. Mutations in the gene SHH, which specifies the midline of the CNS at neural plate stages, is one cause for this spectrum of abnormalities.
The most frequently affected bone is the squamous part of the occipital bone, which may be partially or totally lacking. If the opening of the occipital bone is small, only meninges bulge through it (meningocele), but if the defect is large, part of the brain and even part of the ventricle may penetrate through the opening into the meningeal sac (Figs. 18.35 and 18.36). The latter two malformations are known as meningoencephalocele and meningohydroencephalocele, respectively. These defects occur in 1/12,000 births.

Exencephaly is characterized by failure of the cephalic part of the neural tube to close. As a result, the vault of the skull does not form, leaving the malformed brain exposed. Later, this tissue degenerates, leaving a mass of necrotic tissue.This defect is called anencephaly, although the brain stem remains intact (Fig. 18.37A). In some cases, the closure defect of the neural tube extends caudally into the spinal cord, and the abnormality is called craniorachischisis (Fig. 18.37B). Again, there is anencephaly, but with a large defect involving the spine. Because anencephalic fetuses lack a swallowing reflex, the last 2 months of pregnancy are characterized by polyhydramnios. The abnormality can be recognized on ultrasound, as the vault of the skull is absent. Anencephaly occurs in 1/5,000 births and is more common in females than in males. Like spina bifida, many of these cases can be prevented by having women take 400 ug of folic acid per day before and during early pregnancy.
Hydrocephalus is characterized by an abnormal accumulation of cerebrospinal
Figure 18.35 A. Profile of a child with a skull defect in the occipital region with protruding meninges and/or neural tissue. B—D. Drawings illustrating various types of skull defects in which meninges (meningocele; B) or meninges with neural tissue (meningoencephalocele, C; and meningohydroencephalocele, D) protrude through a bony defect. The defects usually occur in the occipital region, but may involve other areas of the skull, such as the frontonasal region. In most cases, the origin of these defects is due to abnormal neural tube closure, and many can be prevented by maternal use of folic acid (400 ig daily) prior to and during pregnancy.

fluid (CSF) within the ventricular system. In most cases, hydrocephalus in the newborn is due to an obstruction of the aqueduct of Sylvius (aqueductal stenosis). This prevents the cerebrospinal fluid of the lateral and third ventricles from passing into the fourth ventricle and from there into the subarachnoid space, where it would be resorbed. As a result, fluid accumulates in the lateral ventricles and presses on the brain and bones of the skull. Because the cranial sutures have not yet fused, spaces between them widen as the head expands. In extreme cases, brain tissue and bones become thin and the head may be very large (Fig. 18.38).
Microcephaly describes a cranial vault that is smaller than normal (Fig. 18.39). Because the size of the cranium depends on growth of the brain, the underlying defect is in brain development. Causation of the abnormality is varied; it may be genetic (autosomal recessive) or caused by prenatal insults, such as infection or exposure to drugs or other teratogens. Intellectual disability occurs in more than half the cases.
Fetus with a large occipital meningoencephalocele. Some infants with smaller defects can survive with surgery, and their degree of neurological deficits depends on the amount of neural tissue that is abnormal or lost.
Fetal infection by toxoplasmosis may result in cerebral calcification, intellectual disability,
A. Fetus with anencephaly (absent brain) due to a lack of closure of the cranial neural folds. Once the folds fail to close, neural tissue is disorganized and is exposed to amniotic fluid, which causes necrosis and loss of tissue. This defect is always fatal,and most pregnancies with such cases are terminated. B. Fetus with anencephaly and craniorachischisis. The neural tube has failed to close in cranial and upper spinal cord regions resulting in massive necrosis of neural tissue. The defects illustrated in A and B can be prevented by maternal use of folic acid (400 1g daily) prior to and during pregnancy.

Figure 18.38 Child with severe hydrocephalus. Because the cranial sutures had not closed, pressure from the accumulated cerebrospinal fluid enlarged the head, thinning the bones of the skull and cerebral cortex.
hydrocephalus, or microcephaly. Likewise, exposure to radiation during the early stages of development may produce microcephaly. Hyperthermia (fever) produced by maternal infection or by sauna baths and hot tubs may cause spina bifida and anencephaly.
The aforementioned abnormalities are the most serious ones, and they may be incompatible with life. A great many other defects of the CNS may occur without much external manifestation. For example, the corpus callosum may be partially or completely absent without much functional disturbance. Likewise, partial or complete absence of the cerebellum may result in only a slight disturbance of coordination. On

By the fourth week of development, nuclei for all 12 cranial nerves are present. All except the olfactory (I) and optic (II) nerves arise from the brain stem, and of these, only the oculomotor (III) arises outside the region of the hindbrain. In the hindbrain, proliferation centers in the neuroepithelium establish eight distinct segments, the rhombomeres. These rhombomeres give rise to motor nuclei of cranial nerves IV, V, VI, VII, EX, X, XI, and XII (Figs. 18.17 and 18.40). Establishment of this segmental pattern appears to be directed by mesoderm collected into somitomeres beneath the overlying neuroepithelium.
Motor neurons for cranial nuclei are within the brainstem, while sensory ganglia are outside
Figure 18.39 Child with microcephaly. This abnormality, due to poor growth of the brain, is frequently associated with intellectual disability.
the other hand, cases of severe intellectual disability may not be associated with morphologically detectable brain abnormalities. Intellectual disability may result from genetic abnormalities (e.g., Down syndrome) or from exposures to teratogens, including infectious agents (rubella, cytomegalovirus, toxoplasmosis). The leading cause of intellectual disability is, however, maternal alcohol abuse.
of the brain. Thus, the organization of cranial nerves is homologous to that of spinal nerves, although not all cranial nerves contain both motor and sensory fibers (Table 18.2).
Cranial nerve sensory ganglia originate from ectodermal placodes and neural crest cells. Ectodermal placodes include the nasal, otic, and four epibranchial placodes represented by ectodermal thickenings dorsal to the pharyngeal (branchial) arches (Table 18.3; see also Fig. 17.2). Epibranchial placodes contribute to ganglia for nerves of the pharyngeal arches (V, VII, IX, and X). Parasympathetic (visceral efferent) ganglia are derived from neural crest cells, and their fibers are carried by cranial nerves III, VI, IX, and X (Table 18.2).

Figure 18.40 Segmentation patterns in the brain and mesoderm that appear by the 25th day of development. The hindbrain (coarse stipple) is divided into eight rhombomeres (r/—r8), and these structures give rise to the cranial motor nerves (m). P|—P4, pharyngeal (branchial) arches; t, telencephalon; d, diencephalon; m, mesencephalon.

TABLE 18.2 Origins of Cranial Nerves andTheir Composition.Innervation = Stylopharyngeus
Cranial Nerve
Vagus (X)
Spinal Accessory (XI)

Brain Region
Innervation
Type
GVE (otic ganglion)
SA (inferior ganglion)
GVA (superior ganglion)
GSA (superior ganglion)
SVE (branchiomotor)
GVE (ganglia at or near viscera)
SE
Palate and epiglottis (taste)
Pharynx, larynx, trachea, heart, esophagus, stomach, intestines
Base of tongue, external auditory meatus
Constrictor m. pharynx, intrinsic m. larynx, sup. two-thirds esophagus
Trachea, bronchi, digestive tract, heart
Sternocleidomastoid, trapezius m.
Soft palate, pharynx (with X)
Hypoglossal (XII)
Myelencephalon
SE
M. of tongue (except palatoglossus)
SE, general somatic efferent; SVE, special visceral efferent (supplying striated muscles derived from the pharyngeal [branchial] arches).GVE, general visceral efferent; GVA, general visceral afferent; SA, special afferent; GSA, general somatic afferent; ant., anterior; ext., external;; inf., inferior; med., medial; m., muscle; post., posterior; Sup., superior;

In the
fifth week,
cells originating
in ral
crest of
each side
the of
thoracic the
immediately
Functionally, the Autonomic nervous system (ANS)
can be
two parts:
a
sympathetic por-
tion in
the thoracolumbar
region and
a
parasym- spinal
region cord
behind the
neu- migrate
toward the
dorsal aorta
pathetic portion
in the
cranial and
sacral regions.
on the
region
(Fig.
18.41).
Here they
form
a
bilateral chain
of segmentally

epibranchial placodes. Vagus (X), Ganglion = Vagal parasympathetic (visceral efferent). Vagus (X), Origin = Hindbrain neural crest
—
Figure 18.41 Formation of the sympathetic ganglia.A portion of the sympathetic neuroblasts migrates toward the proliferating mesothelium to form the medulla of the suprarenal gland.
arranged sympathetic ganglia interconnected by longitudinal nerve fibers. Together, they form the sympathetic trunks on each side of the vertebral column (Fig. 18.42). From their position in the thorax, neuroblasts migrate toward the cervical and lumbosacral regions, extending the sympathetic trunks to their full length. Although initially the ganglia are arranged segmentally, this arrangement is later obscured, particularly in the cervical region, by fusion of the ganglia.
Some sympathetic neuroblasts migrate in front of the aorta to form preaortic ganglia, such as the celiac and mesenteric ganglia (Fig. 18.42B). Other sympathetic cells migrate to the heart, lungs, and gastrointestinal tract, where they give rise to sympathetic organ plexuses (Fig. 18.42).
Once the sympathetic trunks have been established, nerve fibers originating in the visceroefferent column (lateral horn) of the thoracolumbar segments (T1-L2-L3) of the spinal cord penetrate the ganglia of the trunks (Fig. 18.42). Some of these nerve fibers synapse at the same levels in the sympathetic trunks or pass through the trunks to preaortic or collateral ganglia (Fig. 18.42). They are known as preganglionic fibers, have a myelin sheath, and stimulate the sympathetic ganglion cells. Passing from spinal nerves to the sympathetic ganglia, they form the white communicating rami. Because the visceroefferent column extends only from the first thoracic to the second or third lumbar segment of the spinal cord, white rami are found only at these levels.

Axons of the sympathetic ganglion cells, the postganglionic fibers, have no myelin sheath. They either pass to other levels of the sympathetic trunk or extend to the heart, lungs, and intestinal tract (Fig. 18.42A, broken lines). Other fibers, the gray communicating rami, pass from the sympathetic trunk to spinal nerves and from there to peripheral blood vessels, hair, and sweat glands. Gray communicating rami are found at all levels of the spinal cord.

The suprarenal gland develops from two components: (1) a mesodermal portion, which forms the cortex, and (2) an ectodermal portion, which forms the medulla. During the fifth week of development, mesothelial cells between the root of the mesentery and the developing gonad begin to proliferate and penetrate the underlying mesenchyme (Fig. 18.41). Here, they differentiate into large acidophilic organs, which form the fetal cortex, or primitive cortex, of the suprarenal gland (Fig. 18.43). Shortly afterward, a second wave of cells from the mesothelium penetrates the mesenchyme and surrounds the original acidophilic cell mass. These cells, smaller than those of the first wave, later form the definitive cortex of the gland (Fig. 18.43A,B). After birth, the fetal cortex regresses rapidly except for its outermost layer, which differentiates into the reticular zone. The adult structure of the cortex is not achieved until puberty.
While the fetal cortex is being formed, cells originating in the sympathetic system (neural crest cells) invade its medial aspect, where they are arranged in cords and clusters. These cells give rise to the medulla of the suprarenal gland. They stain yellow-brown with chrome salts and hence are called chromaffin cells (Fig. 18.43). During embryonic life, chromaffin cells are scattered widely throughout the embryo, but in the adult, the only persisting group is in the medulla of the adrenal glands.

Neurons in the brainstem and the sacral region (S2-S4) of the spinal cord give rise to preganglionic parasympathetic fibers. Fibers from nuclei in the brainstem travel via the oculomotor (III), facial (VII), glossopharyngeal (IX), and vagus (X) nerves. Postganglionic fibers arise from neurons (ganglia) derived from neural crest cells and pass to the structures they innervate (e.g., pupil of the eye, salivary glands, viscera).
Figure 18.43 A. Chromaffin (sympathetic) cells penetrating the fetal cortex of the suprarenal gland. B. Later in development, the definitive cortex surrounds the medulla almost completely.

Congenital megacolon (Hirschsprung disease) results from a failure of parasympathetic ganglia to form in the wall of part or all of the colon and rectum because the neural crest cells fail to migrate. Most familial cases of Hirschsprung disease are caused by mutations in the RET gene, which codes for a cell membrane tyrosine kinase receptor. This gene on chromosome 10q11 is essential for neural crest cell migration. The ligand for the receptor is glial cell-derived neurotrophic growth factor secreted by mesenchyme cells through s_-
The CNS originates in the ectoderm and appears as the neural plate at the middle of the third week (Fig. 18.1). After the edges of the plate fold, the neural folds approach each other in the midline to fuse into the neural tube (Figs. 18.2 and 18.3).The cranial end closes at approximately day 25, and the caudal end closes at day 28. The CNS then forms a tubular structure with a broad cephalic portion, the brain, and a long caudal portion, the spinal cord. Failure of the neural tube to close results in defects such as spina bifida (Figs. 18.15 and 18.16) and anencephaly (Fig. 18.37), defects that can be prevented by folic acid.
which crest cells migrate. Receptor ligand interactions then regulate crest cell migration. Consequently, if there are abnormalities in the receptor, migration is inhibited, and no parasympathetic ganglia form in affected areas. The rectum is involved in nearly all cases, and the rectum and sigmoid are involved in 80% of affected infants. The transverse and ascending portions of the colon are involved in only 10% to 20%. The colon is dilated above the affected region, which has a small diameter because of tonic contraction of noninnervated musculature.

The spinal cord, which forms the caudal end of the CNS, is characterized by the basal plate containing the motor neurons, the alar plate for the sensory neurons, and a floor plate and a roof plate as connecting pathways between the two sides (Fig. 18.8). Spinal nerves form from each segment of the spinal cord. These nerves have their motor nuclei in the basal plate (inside the cord) and their sensory cell bodies in spinal ganglia derived from neural crest cells (outside the cord). SHH ventralizes the neural tube in the spinal cord region and induces the floor and basal plates. Bone morphogenetic proteins 4 and 7, expressed in nonneural ectoderm, maintain and upregulate expression of PAX3 and PAX7 in the alar and roof plates.

The brain can be divided into the brain stem, which is a continuation of the spinal cord and resembles that structure in its organization of basal and alar plates, and the higher centers, the cerebellum and cerebral hemispheres, which accentuate the alar plates. After closure of the neural tube, the brain consists of three vesicles: the rthombencephalon (hindbrain), mesencephalon (midbrain), and prosencephalon (forebrain). Later, these primary vesicles subdivide into five different regions. The rhombencephalon is divided into (1) the myelencephalon, which forms the medulla oblongata (this region has a basal plate for somatic and visceral efferent neurons and an alar plate for somatic and visceral afferent neurons) (Fig. 18.18) and (2) the metencephalon, with its typical basal (efferent) and alar (afferent) plates (Fig. 18.19). This brain vesicle is also characterized by formation of the cerebellum (Fig. 18.20), a coordination center for posture and movement, and the pons, the pathway for nerve fibers between the spinal cord and the cerebral and the cerebellar cortices (Fig. 18.19). The mesencephalon, or midbrain, does not subdivide and resembles the spinal cord with its basal efferent and alar afferent plates. The mesencephalon's alar plates form the anterior and posterior colliculi as relay stations for visual and auditory reflex centers, respectively (Fig. 18.23). The prosencephalon also subdivides into the diencephalon posteriorly and the telencephalon anteriorly. The diencephalon consists of a thin roof plate and a thick alar plate in which the thalamus and hypothalamus develop (Figs. 18.24 and 18.25). It participates in formation of the pituitary gland, which also develops from Rathke's pouch (Fig. 18.26). Rathke's pouch forms the adenohypophysis, the intermediate lobe, and pars tuberalis, and the diencephalon forms the posterior lobe; the neurohypophysis, which contains neuroglia and receives nerve fibers from the hypothalamus.

The telencephalon consists of two lateral outpocketings, the cerebral hemispheres, and a median portion, the lamina terminalis (Fig. 18.27). The lamina terminalis is used by the commissures as a connection pathway for fiber bundles between the right and left hemispheres (Fig. 18.30). The cerebral hemispheres, originally two small outpocketings (Figs. 18.24 and 18.25), expand and cover the lateral aspect of the diencephalon, mesencephalon, and metencephalon (Figs. 18.26-18.28). Eventually, nuclear regions of the telencephalon come in close contact with those of the diencephalon (Fig. 18.27).
The ventricular system, containing cerebrospinal fluid, extends from the lumen in the spinal cord to the fourth ventricle in the rhombencephalon, through the narrow aqueduct in the mesencephalon and to the third ventricle in the diencephalon. By way of the foramina of Monro, the ventricular system extends from the third ventricle into the lateral ventricles of the cerebral hemispheres. Cerebrospinal fluid is produced in the choroid plexuses of the third, fourth, and lateral ventricles. Blockage of cerebrospinal fluid in the ventricular system or subarachnoid space results in hydrocephalus.

The brain is patterned along the anteroposterior (craniocaudal) and dorsoventral (mediolateral) axes. HOX genes pattern the anteroposterior axis in the hindbrain and specify rhombomere identity. Other transcription factors containing a homeodomain pattern the anteroposterior axis in the forebrain and midbrain regions, including LIM1 and OTX2. Two other organizing centers, the anterior neural ridge and the rhombencephalic isthmus, secrete FGF8, which serves as the inducing signal for these areas. In response to this growth factor, the cranial end of the forebrain expresses FOXG1, which regulates development of the telencephalon, and the isthmus expresses engrailed genes that regulate differentiation of the cerebellum and the roof of the midbrain. As it does throughout the CNS, SHH, secreted by the prechordal plate and notochord, ventralizes the forebrain and midbrain areas. Bone morphogenetic proteins 4 and 7, secreted by nonneural ectoderm, induce and maintain expression of dorsalizing genes.
There are 12 cranial nerves, and most of these originate from the hindbrain. Motor neurons for each of the nerves are located within the brain, whereas sensory neurons originate outside of the brain from ectodermal placodes and neural crest cells (Tables 18.2 and 18.3,). In this regard, organization of the sensory and motor cell bodies for these nerves is similar to that for spinal nerves.

The autonomic nervous system (ANS) consists of sympathetic and parasympathetic components. This is a 2-neuron system with preganglionic and postganglionic fibers. Preganglionic neurons for the sympathetic system lie in the intermediate (lateral) horns of the spinal cord from T1 to L2-3; its postganglionic neurons lie in the sympathetic trunks and collateral (preaortic) ganglia along the aorta. Parasympathetic preganglionic neurons have their nuclei in the brainstem (associated with cranial nerves III, VII, [X, and X) and in the sacral region of the spinal cord (S2—4); postganglionic nuclei reside in ganglia that usually lie close to the organs they innervate. Neural crest cells form all of the for the ANS.
ganglia
320
Part
Il

1. How are cranial nerves and spinal nerves similar? How are they different?
2.
3. At what level is a spinal tap performed? From an embryological standpoint, why is this possible?
What
come
together
to
form
a
spinal
nerve?
What
is
the
between
a
dorsal
root,
ventral
root,
dorsal
primary
ramus,
and
ventral
primary
ramus?
What
types
of
fibers
(sensory
or
motor)
are
found
in
each
of
these
structures?
4. What is the embryological basis for most
NTDs? Can they be diagnosed prenatally?
Are there any means of prevention?
Prenatal ultrasound reveals an infant with an enlarged head and expansion of both lateral ventricles. What is this condition called, and what might have caused it?
‘What
are
the
two
parts
of
the
autonomic
nervous
system
(ANS)?
Where
are
their
preganglionic
neurons
located?
What
cells
give
rise
embryologically
to
their
postganglionic neurons?
n the adult, the ear forms one anatomic unit serving both hearing and equilibrium. In the embryo, however, it develops from three distinctly different parts: (1) the external ear, the sound-collecting organ; (2) the middle ear, a sound conductor from the external to the internal ear; and (3) the internal ear, which converts sound waves into nerve impulses and registers changes in equilibrium.
each vesicle divides into (1) a ventral component that gives rise to the saccule and cochlear duct and (2) a dorsal component that forms the utricle, semicircular canals, and endolymphatic duct (Figs. 19.3 to 19.6). Together, these epithelial structures form the membranous labyrinth.

The first indication of the developing ear can be found in embryos of approximately 22 days as a thickening of the surface ectoderm on each side of the rhombencephalon (Fig. 19.1). These thickenings, the otic placodes, invaginate rapidly and form the otic or auditory vesicles (otocysts) (Fig. 19.2). During later development,
Saccule, Cochlea, and Organ of Corti In the sixth week of development, the saccule forms a tubular outpocketing at its lower pole (see Fig. 19.3C-E). This outgrowth, the cochlear duct, penetrates the surrounding mesenchyme in a spiral fashion until the end of the eighth week, when it has completed 2.5 turns (Fig. 19.3D,E). Its connection with the remaining portion of the saccule is then confined to a narrow pathway, the ductus reuniens (Fig. 19.3E; see also Fig. 19.8).
19.1 A.An embryo at the end of the fourth week of development showing the otic and optic vesicles. B. Region
Figure of the rhombencephalon showing the otic placodes in a 22-day embryo.
Figure 19.2 A-C. Transverse sections through the region of the rhombencephalon showing formation of the otic vesicles. A. 24 days. B. 27 days. C. 4.5 weeks. Note the statoacoustic ganglia.

Mesenchyme surrounding the cochlear duct soon differentiates into cartilage (Fig. 19.4). In the 10th week, this cartilaginous shell undergoes vacuolization, and two perilymphatic spaces, the scala vestibuli and scala tympani, are formed (Fig. 19.4B, C).The cochlear duct is then separated from the scala vestibuli by the vestibular membrane and from the scala tympani by the basilar membrane (Fig. 19.4C). The lateral wall of the cochlear duct remains attached to the surrounding cartilage by the spiral ligament, whereas its median angle is connected to and partly supported by a long cartilaginous process, the modiolus, the future axis of the bony cochlea (Fig. 19.4B). Initially, epithelial cells of the cochlear duct are alike (Fig. 19.4A). With further development, however, they form two ridges: the inner ridge, the future spiral limbus, and the outer ridge (Fig. 19.4B). The outer ridge forms one row of inner and three or four rows of outer hair cells, the sensory cells of the auditory system (Fig. 19.5). They are covered by the tectorial membrane, a fibrillar gelatinous substance attached to the spiral limbus that rests with its tip on the hair cells (Fig. 19.5). The sensory cells and tectorial membrane together constitute the organ of Corti. Impulses received by this organ are transmitted to the spiral ganglion and then to the nervous system by the auditory fibers of cranial nerve VIII (Figs. 19.4 and 19.5).

During the sixth week of development, semicircular canals appear as flattened outpocketings of the utricular part of the otic vesicle (Fig. 19.64, B). Central portions of the walls of these outpocketings eventually appose each other (Fig. 19.6B,C) and disappear, giving rise to three semicircular canals (Fig. 19.6; see also Fig. 19.8). Whereas one end of each canal dilates to form the crus ampullare, the other, the crus nonampullare, does not widen (Fig. 19.6). Because two of the latter type fuse, however, only five crura enter the utricle, three with an ampulla and two without. Cells in the ampullae form a crest, the crista ampullaris, containing sensory cells for
Figure 19.3 A,B. Development of the otocyst showing a dorsal utricular portion with the endolymphatic duct and a ventral
saccular portion.
C-E.
Cochlear duct
at
6,
7,and
8
weeks, respectively.
Note formation
of the
ductus reuniens
and
Figure
Figure 19.4 Development of the scala tympani and scala vestibuli. A. The cochlear duct is surrounded by a cartilaginous shell. B. During the | 0th week, large vacuoles appear in the cartilaginous shell. C. The cochlear duct (scala media) is separated from the scala tympani and the scala vestibuli by the basilar and vestibular membranes, respectively. Note the auditory nerve fibers and the spiral (cochlear) ganglion.
maintenance of equilibrium. Similar sensory areas, the
maculae acusticae,
develop in
the walls
of the
utricle and
saccule.
Impulses generated
in sen-
sory cells of the cristae and maculae as a result of a change in position of the body are carried to
19.5
the organ
of
Corti.
A.
10
weeks.
B.
Approximately
5
months.
C.
Full-term infant.
Note the

Figure 19.6 Development of the semicircular canals. A. 5 weeks. B. 6 weeks. C. 8 weeks. D-F. Apposition, fusion, and disappearance, respectively, of the central portions of the walls of the semicircular outpocketings. Note the ampullae in the semicircular canals.
cells of this ganglion are derived from the neural crest. The ganglion subsequently splits into cochlear and vestibular portions, which supply sensory cells of the organ of Corti and those of the saccule, utricle, and semicircular canals, respectively.
cavity, and the proximal part remains narrow and forms the auditory tube (eustachian tube) (Figs. 19.7B and 19.8), through which the tympanic cavity communicates with the nasopharynx.

The malleus and incus are derived from cartilage of the first pharyngeal arch, and the stapes is derived from that of the second arch (Fig. 19.9.4). Although the ossicles appear during the first half of fetal life, they remain embedded in mesenchyme until the eighth month (Fig. 19.9B), when the surrounding tissue dissolves (Figs. 19.7, 19.8, and 19.9B).The endodermal epithelial lining of the primitive tympanic cavity then extends along the wall of the newly developing space. The

The tympanic cavity, which originates in the endoderm, is derived from the first pharyngeal pouch (Figs. 19.2 and 19.7).This pouch expands in a lateral direction and comes in contact with the floor of the first pharyngeal cleft. The distal part of the pouch, the tubotympanic recess, widens and gives rise to the primitive tympanic
Figure 19.7 A. Transverse section of a 7-week embryo in the region of the rhombencephalon, showing the tubotympanic recess, the first pharyngeal cleft, and mesenchymal condensation, foreshadowing development of the ossicles. B. Middle ear showing the cartilaginous precursors of the auditory ossicles. Thin yellow line in mesenchyme indicates future expansion of the Note the meatal from the meatus to the primitive tympanic cavity. plug extending primitive auditory tympanic cavity.
Figure 19.8 Ear showing the external auditory meatus, the middle ear with its ossicles, and the inner ear.
tympanic cavity is now at least twice as large as before. When the ossicles are entirely free of surrounding mesenchyme, the endodermal epithelium connects them in a mesentery-like fashion to the wall of the cavity (Fig. 19.9B). The supporting ligaments of the ossicles develop later within these mesenteries.
Because the malleus is derived from the first pharyngeal arch, its muscle, the tensor tympani, is innervated by the mandibular branch of the trigeminal nerve. The stapedius muscle, which is attached to the stapes, is innervated by the facial nerve, the nerve to the second pharyngeal arch.
the epithelium of the tympanic cavity invades the bone of the developing mastoid process, and epithelium-lined air sacs are formed (pneumatization). Later, most of the mastoid air sacs come in contact with the antrum and tympanic cavity. Expansion of inflammations of the middle ear into the antrum and mastoid air cells is a common complication of middle ear infections.

During late fetal life, the tympanic cavity expands dorsally by vacuolization of surrounding tissue to form the tympanic antrum. After birth,
The external auditory meatus develops from the dorsal portion of the first pharyngeal cleft (Fig. 19.7A). At the beginning of the
Figure 19.9 A. Derivatives of the first three pharyngeal arches. Note the malleus and incus at the dorsal tip of the first arch and the stapes at that of the second arch. B. Middle ear showing the handle of the malleus in contact with the eardrum.The stapes will establish contact with the membrane in the oval window. The wall of the tympanic cavity is lined with
third month, epithelial cells at the bottom of the meatus proliferate, forming a solid epithelial plate, the meatal plug (Fig. 19.7B). In the seventh month, this plug dissolves, and the epithelial lining of the floor of the meatus participates in formation of the definitive eardrum. Occasionally, the meatal plug persists until birth, resulting in congenital deafness.

The eardrum is made up of (1) an ectodermal epithelial lining at the bottom of the auditory meatus, (2) an endodermal epithelial lining of the tympanic cavity, and (3) an intermediate layer of connective tissue (Fig. 19.9B) that forms the fibrous stratum. The major part of the eardrum is firmly attached to the handle of the malleus
(Figs. 19.8 and 19.9B), and the remaining portion forms the separation between the external auditory meatus and the tympanic cavity.

The auricle develops from six mesenchymal proliferations at the dorsal ends of the first and second pharyngeal arches, surrounding the first pharyngeal cleft (Fig. 19.10). These swellings (auricular hillocks), three on each side of the external meatus, later fuse and form the definitive auricle (Fig. 19.10). As fusion of the auricular hillocks is complicated, developmental abnormalities of the auricle are common. Initially, the external ears are in the lower neck region (Fig. 19.10A,B), but with development of the mandible, they ascend to the side of the head at the level of the eyes.
Figure 19.10 A. Drawing of a 6-week-old embryo showing a lateral view of the head and six auricular hillocks surrounding the dorsal end of the first pharyngeal cleft. B. Six-week-old human embryo showing a stage of external ear development similar to that depicted in A. Note that hillocks |, 2,and 3 are part of the mandibular portion of the first pharyngeal arch and that the ear lies horizontally at the side of the neck. At this stage, the mandible is small.As the mandible grows anteriorly and posteriorly, the ears, which are located immediately posterior to the mandible, will be repositioned into their characteristic location at the side of the head. C-E. Fusion and progressive development of the hillocks into the adult auricle.

Congenital hearing loss may be caused by abnormal development of the membranous and bony labyrinths or by malformations of the auditory ossicles and eardrum. In the most extreme cases, the tympanic cavity and external meatus are absent.
Many types of congenital hearing loss are caused by genetic factors, but environmental factors may
also interfere with normal development of the internal and middle ear. Rubella and cytomegalovirus infections during pregnancy can cause hearing loss. Another teratogen that can cause hearing loss is isotretinoin (Accutane), which produces a variety of ear defects.
External ear defects are common; they include minor and severe abnormalities (Fig. 19.11). They are significant from the standpoint of the psychological and emotional trauma they may cause
Figure 19.11 External ear defects A. Almost complete absence (anotia) of the external ear. B. A small ear (microtia) with abnormal features. C. Abnormal ear with preauricular appendages (skin tags). Note also the slight depression and small hillock along the line of the mandible. These are remnants of ear development and indicate the path of the ear as it moved to its normal position due to growth of the mandible. D. Preauricular pit.
and for the fact that they are often associated with other malformations. Thus, they serve as clues to examine infants carefully for other abnormalities. All of the frequently occurring chromosomal syndromes and most of the less common ones have ear anomalies as one of their characteristics.

The ear consists of three parts that have different origins but that function as one unit. The internal ear originates from the otic vesicle, which in the fourth week of development detaches from surface ectoderm. This vesicle divides into a ventral component, which gives rise to the saccule and cochlear duct and a dorsal component, which gives rise to the utricle, semicircular canals, and endolymphatic duct (Figs. 19.3, 19.6, and 19.8). The epithelial structures thus formed are known collectively as the membranous labyrinth. Except for the cochlear duct, which forms the organ of Corti, all structures derived from the membranous labyrinth are involved with equilibrium.
The middle ear, consisting of the tympanic cavity and auditory tube, is lined with epithelium of endodermal origin and is derived from the first pharyngeal pouch. The auditory tube extends between the tympanic cavity and nasopharynx. The ossicles, which transfer sound from the tympanic membrane to the oval window, are derived from the first (malleus and incus) and second (stapes) pharyngeal arches (Fig. 19.9).
Preauricular appendages and pits (Fig. 19.11CD) are skin tags and shallow depressions, respectively, anterior to the ear. Pits may indicate abnormal development of the auricular hillocks, whereas appendages may be caused by accessory hillocks. Like other external ear defects, both are sometimes associated with other malformations.
The external auditory meatus develops from the first pharyngeal cleft and is separated from the tympanic cavity by the tympanic membrane (eardrum). The eardrum consists of (1) an ectodermal epithelial lining, (2) an intermediate layer of mesenchyme, and (3) an endodermal lining from the first pharyngeal pouch.
The auricle develops from six mesenchymal hillocks (Fig. 19.10) along the first and second pharyngeal arches. Defects in the auricle are often associated with other congenital malformations.

1. The otic placode plays a major role in formation of the internal (inner ear). What is a placode, and where does the otic placode form? What structures does it contribute to the inner ear?
2. What is the embryologic origin of the tympanic (middle ear) cavity, auditory tube, and tympanic membrane (eardrum)?
3. A newborn has bilateral microtia. Should you be concerned about other malformations? What cell population might be involved in the embryologic origin of the defect?

The developing eye appears in the 22-day embryo as a pair of shallow grooves on the sides of the fore-
brain (Fig. 20.1). With closure of the neural tube, these grooves form outpocketings of the forebrain,
the optic vesicles. These vesicles subsequently
come in contact with the surface ectoderm and
induce changes in the ectoderm necessary for
lens formation (Fig. 20.1). Shortly thereafter, the
optic vesicle begins to invaginate and forms the
double-walled optic cup (Figs. 20.1 and 20.24).
Otic
Figure 20.1 A. Embryo at the end of 4 weeks of development showing the otic and optic vesicles. B. Transverse section through the forebrain of a 22-day embryo (~14 somites) showing the optic grooves. C. Transverse section through the forebrain of a 4-week embryo showing the optic vesicles in contact with the surface ectoderm. Note the slight thickening of the ectoderm (lens placode). D. Transverse section through the forebrain of a 5-mm embryo showing invagination of the optic vesicle and the lens placode.
Lens pit
Lens
vesicle
Figure 20.2 A. Ventrolateral view of the optic cup and optic stalk of a 6-week embryo. The choroid fissure on the undersurface of the optic stalk gradually tapers off. B. Transverse section through the optic stalk as indicated in A, showing the hyaloid artery in the choroid fissure. C. Section through the lens vesicle, the optic cup, and optic stalk at the plane of the choroid fissure. D. Scanning electron micrograph through the eye at 6 weeks of development. The lens vesicle has not quite finished detaching from the surface ectoderm, and the two layers of the optic cup have formed. E. Scanning electron micrograph through the eye at 6.5 weeks of development. The lens is completely detached from the surface ectoderm and will soon start to form lens fibers.

Figure 20.3 Section through the eye of a 7-week embryo. The eye primordium is completely embedded in mesenchyme. Fibers of the neural retina converge toward the optic nerve.
The inner and outer layers of this cup are initially separated by a lumen, the intraretinal space (Fig. 20.2B), but soon this lumen disappears, and the two layers appose each other (Fig. 20.2D,E). Invagination is not restricted to the central portion of the cup but also involves a part of the inferior surface (Fig. 20.2A) that forms the choroid fissure. Formation of this fissure allows the hyaloid artery to reach the inner chamber of the eye (Fig. 20.3; see also Fig. 20.7). During the seventh week, the lips of the choroid fissure fuse, and the mouth of the optic cup becomes a round opening, the future pupil.
cells, including the outer nuclear layer, inner nuclear layer, and ganglion cell layer (Fig. 20.4). On the surface is a fibrous layer that contains axons of nerve cells of the deeper layers. Nerve fibers in this zone converge toward the optic stalk, which develops into the optic nerve (Fig. 20.3). Hence, light impulses pass through most layers of the retina before they reach the rods and cones.
During these events, cells of the surface ectoderm, initially in contact with the optic vesicle, begin to elongate and form the lens placode (Fig. 20.1). This placode subsequently invaginates and develops into the lens vesicle. During the fifth week, the lens vesicle loses contact with the surface ectoderm and lies in the mouth of the optic cup (Figs. 20.2C-E and 20.3).

The outer layer of the optic cup, which is characterized by small pigment granules, is known as the pigmented layer of the retina (Fig. 20.2D,E; see also Fig. 20.6). Development of the inner (neural) layer of the optic cup is more complicated. The posterior four-fifths, the pars optica retinae, contains cells bordering the intraretinal space (Fig. 20.3) that differentiate into light-receptive elements, rods and cones (Fig. 20.4). Adjacent to this photoreceptive layer is the mantle layer, which, as in the brain, gives rise to neurons and supporting
The anterior fifth of the inner layer, the pars ceca retinae, remains one cell layer thick. It later divides into the pars iridica retinae, which forms the inner layer of the iris, and the pars ciliaris retinae, which participates in formation of the ciliary body (Figs. 20.5 and 20.6).
Meanwhile, the region between the optic cup and the overlying surface epithelium is filled with loose mesenchyme (Figs. 20.2C and 20.6). The sphincter and dilator pupillae muscles form in this tissue (Fig. 20.5). These muscles develop from the underlying ectoderm of the optic cup. In the adult, the iris is formed by the pigment-containing external layer, the unpigmented internal layer of the optic cup, and a layer of richly vascularized connective tissue that contains the pupillary muscles (Fig. 20.5).
The pars ciliaris retinae is easily recognized by its marked folding (Figs. 20.5B and 20.6). Externally, it is covered by a layer of mesenchyme that forms the ciliary muscle; on the inside, it is connected to the lens by a network of elastic fibers, the suspensory ligament or zonula (Fig. 20.6). Contraction of the ciliary muscle changes tension in the ligament and controls curvature of the lens.
Figure 20.4 Various layers of the pars optica retinae in a fetus of approximately 25 weeks.
Figure
20.5
the iris
and ciliary
body.
The rim
of the
optic cup
is covered

by mesenchyme,
in which
the
Figure 20.6 Section through the eye of a 15-week fetus showing the anterior chamber, iridopupillary membrane, inner and outer vascular layers, choroid, and sclera.

Shortly after formation of the lens vesicle (Fig. 20.2C), cells of the posterior wall begin to elongate anteriorly and form long fibers that gradually fill the lumen of the vesicle (Fig. 20.3). By the end of the seventh week, these primary lens fibers reach the anterior wall of the lens vesicle. Growth of the lens is not finished at this stage, however, since new (secondary) lens fibers are continuously added to the central core.

At the end of the fifth week, the eye primordium is completely surrounded by loose mesenchyme (Fig. 20.3). This tissue soon differentiates into an inner layer comparable with the pia mater of the brain and an outer layer comparable with the dura mater. The inner layer later forms a highly vascularized pigmented layer known as the choroid; the outer layer develops into the sclera and is continuous with the dura mater around the nerve

the sclera, the substantia propria of the cornea (Fig. 20.6). The anterior chamber itself is lined by flattened mesenchymal cells. Hence, the cornea is formed by (1) an epithelial layer derived from the surface ectoderm, (2) the substantia propria or stroma, which is continuous with the sclera, and (3) an epithelial layer, which borders the anterior chamber. The iridopupillary membrane in front of the lens disappears completely. The posterior chamber is the space between the iris anteriorly and the lens and ciliary body posteriorly. The anterior and posterior chambers communicate with each other through the pupil and are filled with fluid called the aqueous humor produced by the ciliary process of the ciliary body. The clear aqueous humor circulates from the posterior chamber into the anterior chamber providing nutrients for the avascular cornea and lens. From the anterior chamber, the fluid passes through the scleral venous sinus (canal of Schlemm) at the iridocorneal angle where it is resorbed into the bloodstream. Blockage of the flow of fluid at the canal of Schlemm is one cause of glaucoma.
Differentiation of mesenchymal layers overlying the anterior aspect of the eye is different. The anterior chamber forms through vacuolization and splits the mesenchyme into an inner layer in front of the lens and iris, the iridopupillary membrane, and an outer layer continuous with

Mesenchyme not only surrounds the eye primordium from the outside but also invades the inside of the optic cup by way of the choroid
fissure. Here, it forms the hyaloid vessels, which during intrauterine life supply the lens and form the vascular layer on the inner surface of the retina (Fig. 20.6). In addition, it forms a delicate network of fibers between the lens and retina. The interstitial spaces of this network later fill with a transparent gelatinous substance, forming the vitreous body (Fig. 20.6). The hyaloid vessels in this region are obliterated and disappear during fetal life, leaving behind the hyaloid canal.

The optic cup is connected to the brain by the optic stalk, which has a groove, the choroid fissure, on its ventral surface (Figs. 20.2 and 20.3). In this groove are the hyaloid vessels. The nerve fibers of the retina returning to the brain lie among cells of the inner wall of the stalk (Fig. 20.7). During the seventh week, the choroid fissure closes, and a narrow tunnel forms inside the optic stalk (Fig. 20.7B). As a result of the continuously increasing number of nerve fibers, the inner wall of the stalk grows, and the inside and outside walls of the stalk fuse (Fig. 20.7C). Cells of the inner layer provide a network of neuroglia that support the optic nerve fibers.
The optic stalk is thus transformed into the optic nerve. Its center contains a portion of the hyaloid artery, later called the central artery of the retina. On the outside, a continuation of the choroid and sclera, the pia arachnoid and dura layer of the nerve, respectively, surround the optic nerve.

PAX6 is the key regulatory gene for eye development. It is a member of the PAX (paired box) family of transcription factors and contains two DNA-binding motifs that include a paired domain and a paired-type homeodomain. Initially, this transcription factor is expressed in a band in the anterior neural ridge of the neural plate before neurulation begins (Fig. 20.8A,B; see also Fig. 18.32).At this stage, there is a single eye field that later separates into two optic primordia (Fig. 20.8B). The signal for separation of this field is sonic hedgehog (SHH) expressed in the prechordal plate. SHH expression upregulates PAX2 in the center of the eye field and downregulates PAX6 (Fig. 20.8C). Later, this pattern is maintained so that PAX2 is expressed in the optic stalks and PAX6 is expressed in the optic cup and overlying surface ectoderm that forms the lens. As development proceeds, it appears that PAX6 is not essential for optic cup formation. Instead, this process is regulated by interactive signals between the optic vesicle and surrounding mesenchyme and the overlying surface ectoderm in the lens-forming region (Fig. 20.9). Thus, fibroblast growth factors (FGF) from the surface ectoderm promote differentiation of the neural (inner layer) retina, while transforming growth factor B(TGF-B), secreted by surrounding mesenchyme, directs formation of the pigmented (outer) retinal layer. Downstream from these gene products, the transcription factors MITF and CHX10 are expressed and direct differentiation of the pigmented and neural layer, respectively (Fig. 20.9). Thus, the lens ectoderm is essential for proper formation of the optic cup, such that without a lens placode, no cup invagination occurs.
Differentiation of the lens depends on PAX6, although the gene is not responsible for inductive activity by the optic vesicle. Instead, PAX6 acts in the surface ectoderm to regulate lens development (Fig. 20.9C). This expression upregulates the transcription factor SOX2 and also maintains PAX6 expression in the prospective lens ectoderm. In turn, the optic vesicle secretes

BMP-4, which also upregulates and maintains SOX2 expression as well as expression of LMAF, another transcription factor (Fig. 20.9C). Next, the expression of two homeobox genes, SIX3 and PROX1, is regulated by PAX6. The combined expression of PAX6, SOX2, and LMAF
initiates expression of genes responsible for lens crystallin formation, including PROX1. SIX3 also acts as a regulator of crystallin production by inhibiting the crystallin gene. Finally, PAX6, acting through FOX3, regulates cell proliferation in the lens.
O
MITF
Figure 20.9 Drawing showing molecular regulation of epithelial-mesenchymal interactions responsible for patterning eye development. A. Once PAX6 establishes the eye field, FGFs, secreted by surface ectoderm in the prospective lens-forming region overlying the optic vesicle, promote differentiation of the neural retinal layer; while members of the TGF-B family, secreted by surrounding mesenchyme, promote differentiation of the pigmented retinal layer. These external signals cause regionalization of the inner and outer layers of the optic cup and upregulate downstream genes, including CHX10 and MITF, that regulate continued differentiation of these structures B,C. In addition to its role in determining the eye fields, PAX6 regulates lens development. Thus, PAX6 upregulates SOX2 expression in the prospective lens, while BMP4 secreted by the outer vesicle upregulates the transcription factor LMAF. Once this gene is activated, PAX6 induces expression of the homeodomain-containing genes SIX3 and PROX!.The combined expression of PAX6, SOX2, LMAF,and PROX! leads to crystallin formation. SIX3 assists in regulating this process by inhibiting the crystallin gene.

Coloboma may occur if the choroid fissure fails to close. Normally, this fissure closes during the seventh week of development (Fig. 20.7). When it does not, a cleft persists. Although such a cleft is usually in the iris only—coloboma iridis (Fig. 20.10A)—it may extend into the ciliary body, the retina, the choroid, and the optic nerve. Coloboma is a common eye abnormality frequently associated with other eye defects. Colobomas (clefts) of the eyelids may also occur. Mutations in the PAX2 gene have
Figure 20.10 A. Coloboma iris. B. Persistence of the iridopupillary membrane.
been linked with optic nerve colobomas and may play a role in the other types as well. Renal defects also occur with mutations in PAX2 as part of the renal coloboma syndrome (see Chapter 16).
The iridopupillary membrane (Fig. 20.10B) may persist instead of being resorbed during formation of the anterior chamber.
Congenital cataracts cause the lens to become opaque during intrauterine life. Although this anomaly is usually genetically determined, many children born to mothers who had rubella (German measles) between the fourth and seventh weeks of pregnancy had cataracts. If the mother is infected after the seventh week of pregnancy, the lens escapes damage, but the child may have hearing loss as a result of cochlear abnormalities. Because of the MMR (measles, mumps, and rubella) vaccine, congenital rubella syndrome has been nearly eradicated in the United States.
The hyaloid artery may persist to form a cord or cyst. Normally, the distal portion of this vessel degenerates, leaving the proximal part to form the central artery of the retina.
In microphthalmia, the eye is too small; the eyeball may be only two thirds of its normal volume. Usually associated with other ocular abnormalities, microphthalmia can result from intrauterine infections, such as cytomegalovirus and toxoplasmosis.
Anophthalmia is absence of the eye. In some cases, histological analysis reveals some ocular tissue. The defect is usually accompanied by severe cranial abnormalities.

Congenital aphakia (absence of the lens) and aniridia (absence of the iris; Fig. 20.11) are rare anomalies that result from disturbances in induction and development of tissues responsible for formation of these structures. Mutations
Figure 20.11 Patient with aniridia (absence of the iris), which can be due to mutations in PAX6.
Figure 20.12 Synophthalmia.The eyes are fused because loss of midline structures prevented the eye fields from separating. Such babies also have severe cranial defects, including holoprosencephaly (see Chapter 18).
in PAX6 result in aniridia and may also contribute to anophthalmia and microphthalmia.
Cyclopia (single eye) and synophthalmia (fusion of the eyes) comprise a spectrum of defects in which the eyes are partially or completely fused (Fig. 20.12).The defects are caused by a loss of midline tissue that may occur as early as days 19 to 21 of gestation or at later stages when facial development is initiated. This loss results in underdevelopment of the forebrain and frontonasal prominence. These defects are invariably associated with a brain defect called holoprosencephaly, in which the cerebral hemispheres are partially or completely merged into a single telencephalic vesicle. Factors associated with holoprosencephaly include alcohol, maternal diabetes, mutations in SHH, and abnormalities in cholesterol metabolism that may disrupt SHH signaling (see Chapter 18).
__
downregulating PAX6, restricting this gene's expression to the optic cup and lens. Epithelial— mesenchymal interactions between prospective lens ectoderm, optic vesicle, and surrounding mesenchyme then regulate lens and optic cup differentiation (Figs. 20.8 and 20.9).

- 1. A newborn has unilateral aphakia (absent lens). What is the embryological origin of this defect?
2.
In taking
a
history of
a
young woman
in her
10th week
concerned of
gestation, that
she rubella
you may
have sometime
eighth during
weeks of
become contracted
the fourth
her types
of defects
to pregnancy.
might
What be
her offspring?
- 3. Physical examination of a newborn reveals clefts in the lower portion of the iris bilaterally. What is the embryological basis for this defect? What other structures might be involved?

The skin is the largest organ in the body and has a dual origin: (1) A superficial layer, the epidermis, develops from the surface ectoderm. (2) A deep layer, the dermis, develops from the underlying mesenchyme.

Initially, the embryo is covered by a single layer of ectodermal cells (Fig. 21.1A). In the beginning of the second month, this epithelium divides, and a layer of flattened cells, the periderm, or epitrichium, is laid down on the surface (Fig.21.1B). With further proliferation of cells in the basal layer, a third, intermediate zone is formed (Fig. 21.1C). Finally, at the end of the fourth month, the epidermis acquires its definitive arrangement, and four layers can be distinguished (Fig. 21.1D):
A
which are reflected on the surface of the skin in the fingerprint.
thick spinous
layer consists
of large
poly- hedral cells containing fine tonofibrils.
The granular layer contains small keratohya- lin granules in its cells.
The horny
layer, forming
like surface
of of
closely the
the epidermis,
packed dead
tough scale-
is made
cells up
containing keratin.
The basal layer, or germinative layer, is responsible for production of new cells. This layer later forms ridges and hollows,
Cells of the periderm are usually cast off during the second part of intrauterine life and can be found in the amniotic fluid. During the first 3 months of development, the epidermis is invaded by cells arising from the neural crest. These cells synthesize melanin pigment in melanosomes. As melanosomes accumulate, they are transported down dendritic processes of melanocytes and are transferred intercellularly to keratinocytes of the skin and hair bulb. In this manner, pigmentation of the skin and hair is acquired.
Figure
21.1
the skin
at various
stages of
D.
Birth.
A.
5
weeks.
B.
7
weeks.
C.
4
months.

A large number of pigmentary disorders occur, and these can be classified as diseases of melanocyte development, function, and survival. Examples of abnormalities of melanocyte function include piebaldism (patchy absence of hair pigment) and Waardenburg syndrome (WS), which feature patches of white skin and hair. There are several types of WS, but they share some common characteristics, including patches of white hair (usually a forelock), heterochromia irides (eyes of different colors), white patches of skin, and deafness. The defects arise because of faulty migration or proliferation of neural crest cells (absence of melanocytes derived from these cells in the stria vascularis in the cochlea accounts for deafness in these diseases). Some types of WS result from mutations in PAX3, including WSI and WS3.
Diseases of melanocyte function include the various forms of albinism characterized by globally reduced or absent pigmentation in the skin, hair, and eyes. These cases are classified as different types of oculocutaneous albinism (OCA). In most cases, abnormalities of melanin synthesis or processing produce the abnormalities.
Vitiligo results from a loss of melanocytes due to an autoimmune disorder. There is patchy loss of pigment from affected areas, including the skin and overlying hair and the oral mucosa. Vitiligo is also associated with other autoimmune diseases, particularly of the thyroid.

The epidermal ridges that produce typical patterns on the surface of the fingertips, palms of the hand, and soles of the feet are genetically determined. They form the basis for many studies in medical genetics and criminal investigations (dermatoglyphics). In children with chromosomal abnormalities, the epidermal pattern on the hand and fingers is sometimes used as a diagnostic tool.

Dermis is derived from mesenchyme that has three sources: (1) lateral plate mesoderm supplying cells for dermis in the limbs and body wall, (2) paraxial mesoderm supplying cells for dermis in the back, and (3) neural crest cells supplying cells for dermis in the face and neck. During the third and fourth months, this tissue, the corium (Fig. 21.1D), forms many irregular papillary structures, the dermal papillae, which project upward into the epidermis. Most of these papillae contain a small capillary or a sensory nerve end organ. The deeper layer of the dermis, the subcorium, contains large amounts of fatty tissue.
At birth, the skin is covered by a whitish paste, the vernix caseosa, formed by secretions from sebaceous glands and degenerated epidermal cells and hairs. It protects the skin against the macerating action of amniotic fluid.

Ichthyosis, excessive keratinization of the skin, is characteristic of a group of hereditary disorders that are usually inherited as an autosomal recessive trait but may also be X-linked. In severe cases, ichthyosis may result in a grotesque appearance, as in the case of a harlequin fetus (Fig. 21.2).
Figure 21.2 Ichthyosis in a harlequin fetus with massive thickening of the keratin layer, which cracks to form fissures between thickened plaques.

Hairs begin development as solid epidermal proliferations from the germinative layer that penetrates the underlying dermis (Fig. 21.3). At their terminal ends, hair buds invaginate. The invaginations, the hair papillae, are rapidly filled with mesoderm in which vessels and nerve endings develop (Fig. 21.3B,C). Soon, cells in the center of the hair buds become spindle-shaped and keratinized, forming the hair shaft, while peripheral cells become cuboidal, giving rise to the epithelial hair sheath (Fig. 21.3B,C).
attached to the dermal root sheath. The muscle is the arrector pili muscle. Continuous proliferation of epithelial cells at the base of the shaft pushes the hair upward, and by the end of the third month, the first hairs appear on the surface in the region of the eyebrow and upper lip. The first hair that appears, lanugo hair, is shed at about the time of birth and is later replaced by coarser hairs arising from new hair follicles.
The dermal root sheath is formed by the surrounding mesenchyme.A small smooth muscle, also derived from mesenchyme, is usually
The epithelial wall of the hair follicle usually shows a small bud penetrating the surrounding mesoderm (Fig. 21.3C). Cells from these buds form the sebaceous glands. Cells from the central region of the gland degenerate, forming a fat-like substance (sebum) secreted into the hair follicle, and from there, it reaches the skin.
Figure 21.3 Development of a hair and a sebaceous gland. A. 4 months. B. 6 months. C. Newborn.

Hypertrichosis (excessive hairiness) is caused by an unusual abundance of hair follicles. It may be localized to certain areas of the body, especially the lower lumbar region covering a spina bifida occulta defect or may cover the entire body (Fig. 21.4).
Atrichia, the congenital absence of hair, is usually associated with abnormalities of other ectodermal derivatives, such as teeth and nails.

There are two types of sweat glands: eccrine and apocrine. Eccrine sweat glands form in the skin over most parts of the body beginning as buds from the germinative layer of the epidermis. These buds grow into the dermis, and their end coils to form the secretory parts of the glands. Smooth muscle cells associated with the glands also develop from the epidermal buds. These glands function by merocrine mechanisms (exocytosis) and are involved in temperature control.
Apocrine sweat glands develop anywhere there is body hair, including the face, axillae, and pubic region. They begin to develop during puberty and arise from the same epidermal buds that produce hair follicles. Hence, these sweat glands open onto hair follicles instead of skin. The sweat produced by these glands contains lipids, proteins, and pheromones, and odor originating from this sweat is due to bacteria that break down these products. It should be noted that these glands classified as apocrine because a portion of the secretory cells is shed and incorporated into the secretion.
Figure 21.4 Child with hypertrichosis.

Mammary glands are modified sweat glands and first appear as bilateral bands of thickened epidermis called the mammary lines or mammary ridges. In a 7-week embryo, these lines extend on each side of the body from the base of the forelimb to the region of the hindlimb (Fig. 21.5C). Although the major part of each mammary line disappears shortly after it forms, a small portion in the thoracic region persists and penetrates the underlying mesenchyme (see Fig. 21.5.4). Here it forms 16 to 24 sprouts, which in turn give rise to small, solid buds. By the end of prenatal life, the epithelial sprouts are canalized and form the lactiferous ducts. Initially, the lactiferous ducts open into a small epithelial pit (Fig. 21.5B). Shortly after birth, this pit is transformed into the nipple by proliferation of the underlying mesenchyme. At birth, lactiferous ducts have no alveoli and therefore no secretory apparatus. At puberty, however, increased concentrations of estrogen and progesterone stimulate branching from the ducts to form alveoli and secretory cells.
Figure 21.5 A,B. Sections through the developing mammary gland at the third and eighth months, respectively. C. Positions of accessory nipples (blue line, mammary line).

Polythelia is a condition in which accessory nipples have formed resulting from the persistence of fragments of the mammary line (Fig. 21.5C). Accessory nipples may develop anywhere along the original mammary line (Fig. 21.6) but usually appear in the axillary region.
Polymastia occurs when a remnant of the mammary line develops into a complete breast.
Inverted nipple is a condition in which the lactiferous ducts open into the original epithelial pit that has failed to evert.
ZU
The skin and its associated structures, hair, nails, and glands, are derived from surface ectoderm. Melanocytes, which give the skin its color, are derived from neural crest cells, which migrate into the epidermis. The production of new cells
Figure 21.6 Child with bilateral polythelia (supernumary nipples).
occurs in the germinative layer. After moving to the surface, cells are sloughed off in the horny layer (Fig. 21.1). The dermis, the deep layer of the skin, is derived from lateral plate mesoderm and from dermatomes of the somites.
Hairs develop from downgrowth of epidermal cells into the underlying dermis. By about

20 weeks, the fetus is covered by downy hair, lanugo hair, which is shed at the time of birth. Sebaceous glands, sweat glands, and mammary glands all develop from epidermal Proliferations. Supernumerary nipples (polythelia) and breasts (polymastia) are relatively common (Figs. 21.5 and 21.6).
1.

A
woman appears
to have
accessory nipples
in her axilla and on her abdomen bilaterally.
What is the embryological basis for these additional nipples, and why do they occur in
these locations?
1. During the process of induction, one group of cells or tissues (the inducer) causes another group (the responder) to change its fate. The responding cells must have the competence to respond, which is conferred by a competency factor. Most inductive processes during embryo development involve epithelial—mesenchymal interactions, and these include the kidneys, gut derivatives, limbs, and many others.
. Signaling by fibroblast growth factors (FGFs), which are part of the family of growth and differentiation factors, is by paracrine mechanisms and it can be disrupted at many levels. For example, even subtle alterations of the ligand and/or its receptor can alter signaling because of the high degree of specificity between these proteins. Such alterations might be caused by gene mutations, as in fact has happened with the FGF receptors, resulting in skull defects (see Chapter 10).Also,ifany of the proteins in the signaling cascade downstream from receptor activation have been altered, then normal signaling may be disrupted. Similarly, modifications of the transcription factors or their DNA-binding sites can alter the quality or quantity of protein products. Fortunately, there is redundancy built into the system that can circumvent alterations in the pathways. The simplest example is the fact that in some cases, one FGF protein can substitute for another.
(OR
1.
The most
mosome common
cause number
either for
abnormal is
nondisjunction meiosis
or mitosis.
sons, chromosomes
division.
chro- during
For unknown
fail to
Nondisjunction
II
results and
separate rea-
during during
in half
of the
meiosis gametes
half having
If fertilization
ing two
having copies
occurs
a
chromosome somy
with of
results;
if it
two copies
cell
I
or no
copy
a
chromosome.
a
gamete
a
lack- normal
occurs one,
mono- between
and
a
normal
a
one, gamete

trisomy results. Trisomy 21 (Down syndrome), the most common numerical abnormality resulting in birth defects (intellectual disability, abnormal facies, heart malformations), is usually caused by nondisjunction in the mother and occurs most frequently in children born to women older than 35 years of age, reflecting the fact that the risk of meiotic nondisjunction increases with increasing maternal age. Other trisomies that result in syndromes of abnormal development involve chromosomes 8, 9, 13, and 18. Monosomies involving autosomal chromosomes are fatal, but monosomy of the X chromosome (Turner syndrome) is compatible with life. This condition is usually (80%) a result of nondisjunction during meiosis of paternal chromosomes and is characterized by infertility, short stature, webbing of the neck, and other defects. Karyotyping of embryonic cells obtained by amniocentesis or chorionic villus biopsy (see Clinical Correlates in Chapter 9) can detect chromosome abnormalities prenatally.
Chromosomes sometimes break, and the pieces may create partial monosomies or trisomies or become attached (translocated) to other chromosomes. Translocation of part of chromosome 21 onto chromosome 14, for example, accounts for approximately 4% of cases of Down syndrome. Chromosomes may also be altered by mutations in single genes. The risk of chromosomal abnormalities is increased by maternal and paternal age over 35 years.
. Mosaicism occurs when an individual has two or more cell lines that are derived from a single zygote but that have different genetic characteristics. The different cell lines may arise by mutation or by mitotic nondisjunction during cleavage, as in some cases of Down syndrome.
Ca
1.
The role
of the
corpus luteum
is to
produce hormones
pregnancy pregnancy
for and
until then
the preparing
the uterus
for maintaining
placenta that
becomes fully
348

functional (approximately the beginning of the fourth month). Initially, progesterone is the primary hormone produced, and it causes the uterus to enter the progestational (secretory) phase. Later, both estrogen and progesterone are produced to maintain pregnancy. The corpus luteum originates from the theca interna (derived from ovarian stromal cells) and from granulosa cells that remain in the ovary after ovulation.
2. The three phases of fertilization are: (1) penetration of the corona radiata, (2) penetration of the zona pellucida, and (3) fusion of the oocyte and sperm cell membranes. Once fusion occurs, the egg undergoes the cortical and zona reactions to prevent polyspermy. Cortical granules next to the oocyte plasma membrane release lysosomal enzymes that alter the cell membrane and the zona pellucida, such that additional sperms cannot enter the egg.
3. Infertility occurs in approximately 20% of married couples. A major cause of infertility in women is blockage of the uterine (fallopian) tubes caused by scarring from repeated pelvic inflammatory disease; in men, the primary cause is low sperm count. In vitro fertilization (IVF) techniques can circumvent these problems, although the success rate (approximately 20%) is low.
4. Pelvic inflammatory diseases, such as gonorrhea, are a major cause of occluded oviducts (uterine tubes). Although the patient may be cured, scarring closes the lumen of the tubes and prevents the passage of sperm to the oocyte and of the oocyte to the uterine cavity. IVF can overcome the difficulty by fertilizing the woman's oocytes in culture and transferring them to her uterus for implantation.

1. The second week is known as the week of twos because the trophoblast differentiates into two layers, the syncytiotrophoblast and cytotrophoblast; the embryoblast differentiates into two layers, the epiblast and hypoblast; the extraembryonic mesoderm splits into two layers, the splanchnic (visceral) and somatic (parietal) layers; and two cavities, the amniotic and yolk sac cavities, form.
2. Itis not clear why the conceptus is not rejected by the maternal system. Recent evidence suggests that secretion of immunosuppressive molecules, such as cytokines and proteins,
and expression of unrecognizable antigens of the major histocompatibility complex protect the conceptus from rejection. In some cases, maternal immunological responses do adversely affect pregnancy, as in some cases of autoimmune disease. Thus, patients with systemic lupus erythematosus have poor reproductive outcomes and histories of multiple spontaneous abortions. It has not been conclusively shown that maternal antibodies can cause birth defects.
3. In some cases, trophoblastic tissue is the only tissue in the uterus, and embryo-derived cells are either absent or present in small numbers. This condition is termed a hydatidiform mole, which, because of its trophoblastic origin, secretes human chorionic gonadotropin and mimics the initial stages of pregnancy. Most moles are aborted early in pregnancy, but those containing remnants of an embryo may remain into the second trimester. If pieces of trophoblast are left behind following spontaneous abortion or surgical removal of a mole, cells may continue to proliferate and form tumors known as invasive moles, or choriocarcinoma. Because early trophoblast development is controlled by paternal genes, it is thought that the origin of moles may be from fertilization of an ovum without a nucleus.
4. The most likely diagnosis is an ectopic pregnancy in the uterine tube, which can be confirmed by ultrasound. Implantation in a uterine tube results from poor transport of the zygote and may be a result of scarring. As with Down syndrome, the frequency of ectopic pregnancy increases with maternal age over 35.
(CRB

- 1. Unfortunately, consuming large quantities of alcohol at any stage during pregnancy may adversely affect embryonic development. In this case, the woman has exposed the embryo during the third week of gestation (assuming that fertilization occurred at the midpoint of the menstrual cycle), at the time of gastrulation. This stage is particularly vulnerable to insult by alcohol and may result in fetal alcohol syndrome (intellectual disablitiy, abnormal facies) (see Chapter 9). Although fetal alcohol syndrome is most common in offspring of alcoholic mothers, no safe levels of blood alcohol concentration have been established for embryogenesis. Therefore, because alcohol causes birth defects and is the leading cause

of intellectual disability, it is recommended that women who are planning a pregnancy or who are already pregnant refrain from use of any alcohol.
2. Such a mass is probably a sacrococcygeal teratoma. These tumors arise as remnants of the primitive streak, usually in the sacral region. The term teratoma refers to the fact that the tumor contains different types of tissues. Because it is derived from the streak, which contains cells for all three germ layers, it may contain tissues of ectoderm, mesoderm, or endoderm origin. Such tumors are three times as common in female fetuses as in male fetuses.
3. The baby has a severe form of caudal dysgenesis called sirenomelia (mermaid-like). Sirenomelia, which occurs in varying degrees, is probably caused by abnormalities in gastrulation in caudal segments. It was initially termed caudal regression, but it is clear that structures do not regress; they simply do not form. Also known as caudal agenesis and sacral agenesis, sirenomelia is characterized by varying degrees of flexion, inversion, lateral rotation, and occasional fusion of the lower limbs; defects in lumbar and sacral vertebrae; renal agenesis; imperforate anus; and agenesis of internal genital structures except the testes and ovaries. Its cause is unknown. It occurs sporadically but is most frequently observed among infants of diabetic mothers.
4. This patient has left-sided laterality sequence and should be evaluated for additional defects. Sidedness is established at the time of primitive streak formation (gastrulation) and is regulated by genes, such as Nodal, and PITX2 which become restricted in their expression. Partial reversal of left-right asymmetry is more often associated with other defects than complete asymmetry (situs inversus).

1. Cells that remain in the epiblast form the ectodermal germ layer. The central region of this layer along the embryonic axis differentiates into the neural plate, and in the third and fourth weeks of gestation, the edges of this plate begin to elevate and form the neural folds. The folds roll up into a tube by fusing in the midline dorsally. Closure of the folds begins in the cervical region and zippers from this point cranially and caudally. Completion
of the closure process occurs at the end of the fourth week of gestation (28 days). The entire process of fold formation, elevation, and closure is called neurulation. Neural tube defects occur when the closure process fails in one or more regions. If it fails cranially, the result is anencephaly; if caudally, the defect is called spina bifida. Seventy percent of these defects can be prevented if women take 400 ug of folic acid daily beginning at least 3 months prior to conception and continuing throughout pregnancy. Because 50% of pregnancies are unplanned, it is recommended that all women of childbearing age take a multivitamin containing 400 jg of folic acid daily.
Neural crest cells are ectodermal in origin, arising from the edges (crests) of the neural folds. In cranial regions, they migrate from the folds prior to neural tube closure; whereas in caudal regions (spinal cord), they migrate after closure. BMPs are the key proteins that establish the neural fold border by upregulating PAX3. PAX3 and other transcription factors then initiate a genetic cascade that specifies crest cells. Two important components of the cascade are the transcription factors FOXD3, that specifies crest cells, and SLUG that promotes crest cell migration. Crest cells form many structures, including the bones, connective tissues, and dermis of the face, cranial nerve ganglia, sympathetic and parasympathetic ganglia, melanocytes, and conotruncal septa in the heart (see Table 6.1, p. 69).

. Somites form from the paraxial portion of the mesodermal germ layer. They first appear as segmental blocks of loosely organized mesoderm (somitomeres) along the axis of the embryo. The cells then undergo an epithelialization process to form somites that consist of a ventral portion, the sclerotome, and a dorsal portion that has two types of cells: the dermatome (central part) and myotome (medial and lateral parts). Cells in the myotome regions proliferate and migrate beneath the dermatome to form the dermomyotome. Eventually, all the cells in the somite lose their epithelial characteristics and become mesenchymal again. Sclerotome cells migrate to form the vertebrae and ribs, myotomes form skeletal muscle, and the dermatomes form the dermis of the back.
. Blood vessels form by vasculogenesis, whereby cells in blood islands coalesce to form endothelial tubes; and by angiogenesis, whereby vessels form by sprouting from existing vessels. Vascular endothelial growth factor (VEGF)
350
stimulates both types of vessel development. In some cases, overproliferation of capillaries causes tumors called hemangiomas, but it is not clear whether or not overexpression of VEGF is involved in their origin.
- 5. The gut tube has three divisions: the foregut, midgut, and hindgut. The midgut maintains a connection to the yolk sac called the vitelline (yolk sac) duct, and this structure does not close completely until later in development. The opening into the pharyngeal gut is closed by the oropharyngeal membrane that degenerates in the fourth week; the opening into the hindgut is closed by the cloacal membrane that degenerates in the seventh week.
6.

- Development during the third to eighth weeks is critical because this is when cell populations responsible for organ formation are established and when organ primordia are being formed. Early in the third week, gastrulation begins to provide cells that constitute the three germ layers responsible for organogenesis. Late in the third week, differentiation of the central nervous system is initiated, and over the next 5 weeks, all of the primordia for the major organ systems will be established. At these times, cells are rapidly proliferating, and critical cell-cell signals are occurring. These phenomena are particularly sensitive to disruption by outside factors, such as environmental hazards, pharmaceutical agents, and drugs of abuse. Thus, exposure to such factors may result in abnormalities known as birth defects or congenital malformations.
(ERB
1. Failure of the left pleuroperitoneal membrane to close the pericardioperitoneal canal on that side is responsible for the defect. This canal is larger on the left than on the right, closes later, and therefore, may be more susceptible to abnormalities. The degree of hypoplasia of the lungs resulting from compression by abdominal viscera determines the fate of the infant. Treatment requires surgical repair of the defect, and attempts to correct the malformation in utero have been made.
2. The defect is gastroschisis. It occurs because of a weakness in the body wall caused by abnormal closure of the ventral body wall. Because the bowel is not covered by the amnion, it may become necrotic because of exposure to the amniotic fluid. It is also possible for the bowel loops to twist around themselves (volvulus),
cutting off their blood supply and producing an infarction. Gastroschisis is not associated with genetic abnormalities and only 15% have other malformations. Therefore, if damage to the bowel is not too extensive, survival rates are good.

- 3. During the fourth week, the septum transversum, which forms the central tendon of the diaphragm, lies opposite cervical segments three to five (C3 to C5).As the embryo grows and the headfold curves ventrally, the position of the septum transversum (diaphragm) shifts caudally into the thoracic cavity. Musculature for the diaphragm is derived from the original cervical segments located at its site of origin, however. Therefore, because muscle cells always carry the nerve from their site of origin to wherever they migrate, it is the phrenic nerve from C3, C4, and C5 that innervates the diaphragm (C3, C4, and C5 keep the diaphragm alive).

1. An excess of amniotic fluid is called hydramnios or polyhydramnios, and many times (35%) the cause is unknown (idiopathic). A high incidence (25%) is also associated with maternal diabetes and with birth defects that interfere with fetal swallowing, such as esophageal atresia and anencephaly.
2. No. She is not correct. The placenta does not act as a complete barrier, and many compounds cross freely, especially lipophilic substances, such as toluene and_ alcohol. Furthermore, early in pregnancy, the placenta is not completely developed, and the embryo is particularly vulnerable. These early weeks are also very sensitive to insult by compounds such as toluene, which causes toluene embryopathy.

1. Neural tube defects, such as spina bifida and anencephaly, produce elevated o-fetoprotein (AFP) levels, as do abdominal defects, such as gastroschisis and omphalocele. Maternal serum AFP levels are also elevated, so that they may be used as a screen to be confirmed by amniocentesis. Ultrasonography is used to confirm the diagnosis.
2. Because Down syndrome is a chromosomal abnormality resulting most commonly from

trisomy 21 (see Chapter 2), cells for chromosomal analysis can be collected by amniocentesis or chorionic villus biopsy (CVS). CVS has the advantage that sufficient cells can be obtained immediately to do the analysis; whereas cells collected by amniocentesis, which is not usually done prior to 14 weeks' gestation, must be cultured for approximately 2 weeks to obtain sufficient numbers. The risk of fetal loss following CVS is 1%, which is about twice as high as that of amniocentesis.
. Status of the fetus is critical for managing pregnancy, delivery, and postnatal care. Size, age, and position are important for determining the time and mode of delivery. Knowing whether birth defects are present is important for planning postnatal care. Tests for determining fetal status are dictated by maternal history and factors that increase risk, such as exposure to teratogens, chromosome abnormalities in either parent, advanced maternal age, or the birth of a previous infant with a birth defect.
Factors that influence the action of a teratogen are: (1) genotype of the mother and conceptus, (2) dose and duration of exposure to the agent, and (3) stage of embryogenesis when exposure occurs. Most major malformations are produced during the embryonic period (teratogenic period), the third to eighth weeks of gestation. Stages prior to this time, however, including the preimplantation period, and after the eighth week (fetal period) remain susceptible. The brain, for example, remains sensitive to insult throughout the fetal period. No stage of pregnancy is free of risk from teratogenic insult.
. The woman is correct that drugs may be teratogenic. Severe hyperthermia such as this, however, is known to cause neural tube defects (spina bifida and anencephaly) at this stage of gestation. Therefore, one must weigh the risk of teratogenicity of an antipyretic agent with a low teratogenic potential, such as lowdose aspirin, against the risk of hyperthermia. Interestingly, malformations have been associated with sauna-induced hyperthermia. No information about exercise-induced hyperthermia and birth defects is available, but strenuous physical activity (running marathons) raises body temperature significantly and probably should be avoided during pregnancy.
6. Because more than 50% of pregnancies are unplanned, all women of childbearing age

should consume 400 pg of folic acid daily as a supplement to prevent neural tube defects. Ifa woman has not been taking folate and is planning a pregnancy, she should begin the supplement 3 months prior to conception and continue throughout gestation. Folic acid is nontoxic even at high doses, can prevent up to 70% of neural tube defects, and may prevent conotruncal heart defects and facial clefts.
- . The woman's concerns are valid, as infants of insulin-dependent diabetic mothers have an increased incidence of birth defects, including a broad spectrum of minor and major anomalies. Placing the mother under strict metabolic control using multiple insulin injections prior to conception and throughout pregnancy, significantly reduces the incidence of abnormalities and affords the greatest opportunity for a normal pregnancy. A similar scenario occurs with women who have phenylketonuria. Strict management of these patients' disease prior to conception and during pregnancy virtually eliminates the risk of congenital defects in the offspring. Both situations stress the need for planning pregnancies and for avoiding potential teratogenic exposures, especially during the first 8 weeks of gestation, when most defects are produced.

- 1. Cranial sutures are fibrous regions between flat bones of the skull. Membranous regions between the flat bones are known as _fon-tanelles, the largest of which is the anterior fontanelle (soft spot). These sutures and fontanelles permit: (1) molding of the head as it passes through the birth canal and (2) growth of the brain. Growth of the skull, which continues after birth as the brain enlarges, is greatest during the first 2 years of life. Premature closure of one or more sutures (craniosynostosis) results in deformities in the shape of the head, depending on which sutures are involved. Craniosynostosis is often associated with other skeletal defects, and evidence suggests that genetic factors are important in the causation (see Table 10.1, p. 137). Defects of the long bones and digits are often associated with other malformations and should prompt a thorough examination of all systems. Clusters of defects that occur simultaneously with a common cause are called syndromes, and limb anomalies, especially of the radius and digits, are common components of such clusters.
352
Diagnosis of syndromes is important in determining recurrence risks and thus in counseling parents about subsequent pregnancies.
2. Formation of the vertebrae is a complex process involving growth and fusion of the caudal portion of one sclerotome with the cranial portion of an adjacent one. Not surprisingly, mistakes occur, and they result in fusions and increases and decreases in the number of vertebrae (Klippel-Feil sequence). In some cases, only half a vertebra forms (hemivertebra), resulting in asymmetry and lateral curvature of the spine (scoliosis). HOX (homeobox) genes that pattern the vertebra may have mutations that cause part of one not to form properly. Scoliosis may also be caused by weakness of back muscles.
GEGEN

1. Muscle cells are derived from the ventrolateral (VLL) and dorsomedial (DML) lips (edges) of the somites. Cells from both regions contribute to formation of the dermomyotome, and in addition, some cells from the VLL lips migrate across the lateral somitic frontier into the parietal layer of lateral plate mesoderm. Together, these cells and lateral plate mesoderm constitute the abaxial mesodermal domain, while paraxial mesoderm around the neural tube forms the primaxial mesodermal domain. Muscles derived from the primaxial domain include the back muscles, some neck muscles, some muscles of the shoulder girdle, and the intercostal muscles. The abaxial domain forms the remainder of the axial and limb muscles (see Table 11.1, p. 145).
2. There is absence of the pectoralis minor and partial or complete absence of the pectoralis major muscle. The defect known as Poland anomaly is the most likely diagnosis. Poland anomaly is often associated with shortness of the middle digits (brachydactyly) and digital fusion (syndactyly). Loss of the pectoralis major muscle produces little or no loss of function, because other muscles compensate. The defect's disfiguring characteristics can be quite concerning, however, especially in females.
3. Patterning for muscles depends on connective tissue that forms from fibroblasts. In the head, with its complicated pattern of muscles of facial expression, neural crest cells direct patterning; in cervical and occipital regions, connective tissue from somites directs it; and in
the body wall and limbs, somatic mesoderm directs it.
- 4. Innervation for muscles is derived from the vertebral level from which the muscle cells originate, and this relation is maintained regardless of where the muscle cells migrate. Thus, myoblasts forming the diaphragm originate from cervical segments 3, 4, and 5, migrate to the thoracic region, and carry their nerves with them.
(GEESE

- 1. Defects of the long bones and digits are often associated with other malformations and should prompt a thorough examination of all systems. Clusters of defects that occur simultaneously with a common cause are called syndromes, and limb anomalies, especially of the radius and digits, are common components of such clusters. Diagnosis of syndromes is important in determining recurrence risks and thus in counseling parents about subsequent pregnancies.
GEGEN
1. A four-chambered view is sought in ultrasound scans of the heart. The chambers are divided by the atrial septum superiorly, the ventricular septum inferiorly, and the endocardial cushions surrounding the atrioventricular canals laterally. Together, these structures form a cross with integrity readily visualized by ultrasound. In this case, however, the fetus probably has a ventricular septal defect, the most commonly occurring heart malformation, in the membranous portion of the septum. The integrity of the great vessels should also be checked carefully, because the conotruncal septum dividing the aortic and pulmonary channels must come into contact with the membranous portion of the interventricular septum for this structure to develop normally.
2. Because neural crest cells contribute to much of the development of the face and to the conotruncal septum, these cells have probably been disrupted. Crest cells may have failed to migrate to these regions, failed to proliferate, or may have been killed. Retinoic acid (vitamin A) is a potent teratogen that targets neural crest cells among other cell populations. Because retinoids are effective in treating acne, which is common in young women

of childbearing age, great care should be employed before prescribing the drug to this cohort.
3. Endocardial cushion tissue is essential for proper development of these structures. In the common atrioventricular canal, the superior, the inferior, and two lateral endocardial cushions divide the opening and contribute to the mitral and tricuspid valves in the left and right atrioventricular canals. In addition, the superior and inferior cushions are essential for complete septation of the atria by fusion with the septum primum and of the ventricles by forming the membranous part of the interventricular septum. Cushion tissue in the conus and truncus forms the conotruncal septum, which spirals down to separate the aorta and pulmonary channels and to fuse with the inferior endocardial cushion to complete the interventricular septum. Therefore, any abnormality of cushion tissue may result in a number of cardiac defects, including atrial and ventricular septal defects, transposition of the great vessels, and other abnormalities of the outflow tract.
4. In the development of the vascular system for the head and neck, a series of arterial arches forms around the pharynx. Most of these arches undergo alterations, including regression, as the original patterns are modified. Two such alterations that produce difficulty swallowing are: (1) double aortic arch, in which a portion of the right dorsal aorta (that normally regresses) persists between the seventh intersegmental artery and its junction with the left dorsal aorta, creating a vascular ring around the esophagus; and (2) right aortic arch, in which the ascending aorta and the arch form on the right. If in such cases the ligamentum arteriosum remains on the left, it passes behind the esophagus and may constrict it.

1. This infant most likely has some type of tracheoesophageal atresia with or without a tracheoesophageal fistula. The baby cannot swallow, and this condition results in polyhydramnios. The defect is caused by abnormal partitioning of the trachea and esophagus by the tracheoesophageal septum. These defects are often associated with other malformations, including a constellation of vertebral
anomalies, anal atresia, cardiac defects, renal anomalies, and limb defects known as the VACTERL association.
2. Babies born before 7 months of gestation do not produce sufficient amounts of surfactant to reduce surface tension in the alveoli to permit normal lung function. Consequently, alveoli collapse, resulting in respiratory distress syndrome. Administration of steroids during pregnancy and use of artificial surfactants have improved the prognosis for these infants.
CEE

1. The baby most likely has some type of esophageal atresia and/or tracheoesophageal fistula. In 90% of these cases, the proximal part of the esophagus ends in a blind pouch, and a fistula connects the distal part with the trachea. Polyhydramnios results because the baby cannot swallow amniotic fluid. Aspiration of fluids at birth may cause pneumonia. The defect is caused by an abnormal partitioning of the respiratory diverticulum from the foregut by the tracheoesophageal septum.
2. The most likely diagnosis is an omphalocele resulting from a failure of herniated bowel to return to the abdominal cavity at 10 to 12 weeks of gestation. Because the bowel normally herniates into the umbilical cord, it is covered by amnion. This situation is in contrast to gastroschisis, in which loops of bowel herniate through an abdominal wall defect and are not covered by amnion. The prognosis is not good, because 25% of infants with omphalocele die before birth, 40% to 88% have associated anomalies, and approximately 15% show chromosomal abnormalities. If no other complicating defects are present, surgical repair is possible, and in experienced hands, survival is 100%.
3. This infant has an imperforate anus with a rectovaginal fistula, part of an anorectal atresia complex. She appears to have a high anorectal atresia, because the fistula connects the rectum to the vagina, accounting for meconium (intestinal contents) in this structure. The defect was probably caused by a cloaca that was too small, so that the cloacal membrane was shortened posteriorly. This condition causes the opening of the hindgut to shift anteriorly. The smaller the cloaca is posteriorly, the farther anteriorly the hindgut opening shifts, resulting in a higher defect.
354

1.
The three systems to form are the pronephros, mesonephros, and metanephros—all derivatives of the intermediate mesoderm. They form in succession in a cranial-to-caudal sequence. Thus, the pronephros forms in cervical segments at the end of the third week but is rudimentary and rapidly regresses. The mesonephros, which begins early in the fourth week, extends from thoracic to upper lumbar regions. It is segmented in only its upper portion and contains excretory tubules that connect to the mesonephric (wolffian) duct. This kidney also regresses but may function for a short time. It is more important because the tubules and collecting ducts contribute to the genital ducts in the male. Collecting ducts near the testes form the efferent ductules, whereas the mesonephric duct forms the epididymis, ductus deferens, and ejaculatory duct. In the female, these tubules and ducts degenerate, because maintaining them depends on testosterone production. The metanephros lies in the pelvic region as a mass of unsegmented mesoderm (metanephric blastema) that forms the definitive kidneys. Ureteric buds grow from the mesonephric ducts and, on contact with the metanephric blastema, induce it to differentiate. The ureteric buds form collecting ducts and ureters, while the metanephric blastema forms nephrons (excretory units), each of which consists of a glomerulus (capillaries) and renal tubules.

2. Both the ovaries and testes develop in the abdominal cavity from intermediate mesoderm along the urogenital ridge. Both also descend by similar mechanisms from their original position, but the uterus prevents migration of the ovary out of the abdominal cavity. In the male, however, a mesenchymal condensation, the gubernaculum (which also forms in females but attaches to the uterus), attaches the caudal pole of the testis, first to the inguinal region and then to the scrotal swellings. Growth and retraction of the gubernaculum, together with increasing intra-abdominal pressure, cause the testis to descend. Failure of these processes causes undescended testes, known as cryptorchidism. Approximately 2% to 3% of term male infants have an undescended testicle, and in 25% of these, the condition is bilateral. In many cases, the undescended testis descends by age 1. If it does not, testosterone administration (because this hormone is thought to play a role in
descent) or surgery may be necessary. Fertility may be affected if the condition is bilateral.
3. Male and female external genitalia pass through an indifferent stage during which it is impossible to differentiate between the two sexes. Under the influence of testosterone, these structures assume a masculine appearance, but the derivatives are homologous between males and females. These homologies include: (1) the clitoris and penis, derived from the genital tubercle; (2) the labia majora and scrotum, derived from the genital swellings that fuse in the male; and (3) the labia minora and penile urethra, derived from the urethral folds that fuse in the male. During early stages, the genital tubercle is larger in the female than in the male, and this has led to misidentification of sex by ultrasound.
4. The uterus is formed by fusion of the lower portions of the paramesonephric (miillerian) ducts. Numerous abnormalities have been described; the most common consists of two uterine horns (bicornuate uterus). Complications of this defect include difficulties in becoming pregnant, high incidence of spontaneous abortion, and abnormal fetal presentations. In some cases, a part of the uterus has a blind end (rudimentary horn), causing problems with menstruation and abdominal pain.
GEGEN

- 1. Neural crest cells are important for craniofacial development because they contribute to so many structures in this region. They form all of the bones of the face and the anterior part of the cranial vault and the connective tissue that provides patterning of the facial muscles. They also contribute to cranial nerve ganglia, meninges, dermis, odontoblasts, and stroma for glands derived from pharyngeal pouches. In addition, crest cells from the hindbrain region of the neural folds migrate ventrally to participate in septation of the conotruncal region of the heart into aortic and pulmonary vessels. Unfortunately, crest cells appear to be vulnerable to a number of compounds, including alcohol and retinoids, perhaps because they lack catalase and superoxide dismutase enzymes that scavenge toxic free radicals. Many craniofacial defects result from insults on neural crest cells and may be associated with cardiac abnormalities because of the contribution of these cells to heart morphogenesis.

The child may have DiGeorge anomaly, which is characterized by these types of craniofacial defects and partial or complete absence of thymic tissue. Loss of thymic tissue compromises the immune system, resulting in numerous infections. Damage to neural crest cells is the most likely cause of the sequence, because these cells contribute to development of all of these structures, including the stroma of the thymus. Teratogens, such as alcohol, have been shown to cause these defects experimentally.
Children with midline clefts of the lip often have intellectual disabilities. Median clefts are associated with loss of other midline structures, including those in the brain. In its extreme form, the entire cranial midline is lost, and the lateral ventricles of the cerebral hemispheres are fused into a single ventricle, a condition called holoprosencephaly. Midline clefts, induced as the cranial neural folds begin to form (approximately days 19 to 21), result from the loss of midline tissue in the prechordal plate region.
The child most likely has a thyroglossal cyst that results from incomplete regression of the thyroglossal duct. These cysts may form anywhere along the line of descent of the thyroid gland as it migrates from the region of the foramen cecum of the tongue to its position in the neck. A cyst must be differentiated from ectopic glandular tissue, which may also remain along this pathway.

1.
Cranial and
spinal they
differ in
consistent nerves
that in
rons for
cranial their
both nervous
from are
homologues, nerves
are much
composition.
lie in
system;
the nervous
synapse neural
system.
Motor basal
plates but
less neu-
of and
sensory crest,
the central
ganglia, lie
outside
Fibers on
spinal neurons
cord
II, and
XI,
VII])
and in
and brain.
are
XII)
IX, and
pathetic from
the sensory
the
Three entirely
are
X)
have derived
central neurons
alar plates
cranial of
the nerves
sensory;
entirely motor,
four motor;
sensory, fibers;
and and
(I,
(IV,
VI, three
and one
(III)
has
(VII, parasym-
only components.
motor
In contrast,
each spinal
nerve has
motor and
sensory fibers.

The components that come together to form a spinal nerve are the dorsal and ventral roots, which contain sensory (afferent) and motor
(efferent) fibers, respectively. Cell bodies for motor neurons are located in the ventral horns of the spinal cord; whereas those of sensory neurons reside outside of the spinal cord in dorsal root ganglia and are derivatives of neural crest cells. Therefore, spinal nerves contain both motor and sensory fibers. Each spinal nerve is very short and divides almost immediately at each intervertebral foramen into a dorsal primary ramus (to back muscles) and a ventral primary ramus (to limb and body wall muscles). Each of these rami is a mixed nerve containing both motor and sensory fibers.
. A spinal tap is performed between vertebra L4 and vertebra L5, because the spinal cord ends at the L2 to L3 level. Thus, it is possible to obtain cerebrospinal fluid at this level without damaging the cord. The space is created because after the third month, the cord, which initially extended the entire length of the vertebral column, does not lengthen as rapidly as the dura and vertebral column do, so that in the adult, the spinal cord ends at the L2 to L3 level.
. The embryological basis for most neural tube defects is inhibition of closure of the neural folds at the cranial and caudal neuropores. In turn, defects occur in surrounding structures, resulting in anencephaly, some types of encephaloceles, and spina bifida cystica. Severe neurological deficits accompany abnormalities in these regions. Neural tube defects, which occur in approximately 1 in 1,500 births, may be diagnosed prenatally by ultrasound and findings of elevated levels of a-fetoprotein in maternal serum and amniotic fluid. Recent evidence has shown that daily supplements of 400 wg of folic acid started 3 months prior to conception prevent up to 70% of these defects.

. This condition, hydrocephalus, results from a blockage in the flow of cerebrospinal fluid from the lateral ventricles through the foramina of Monro and the cerebral aqueduct into the fourth ventricle and out into the subarachnoid space, where it would be resorbed. In most cases, blockage occurs in the cerebral aqueduct in the midbrain. It may result from genetic causes (X-linked recessive) or viral infection (toxoplasmosis, cytomegalovirus).
. The autonomic nervous system is composed of the sympathetic and parasympathetic systems. The sympathetic portion has its preganglionic neurons located in the intermediate horn of the spinal cord from T1 to L2. The parasympathetic portion has a craniosacral
356
origin with its preganglionic neurons in the brain and spinal cord (S2 to $4). The cranial outflow is carried by cranial nerves III, VII, IX, and X. Postganglionic cell bodies for both systems are derived from neural crest cells.
/

1. A placode is a region of cuboidal ectoderm that thickens by assuming a columnar shape. The otic placodes form on both sides of the hindbrain and then invaginate to form otic vesicles. Placodes give rise to sensory organs, and the otic placodes are no exception. Thus, from the otic vesicle, tubular outpocketings form and differentiate into the saccule, utricle, semicircular canals, and the endolymphatic and cochlear ducts. Together, these structures constitute the membranous labyrinth of the internal ear.
2. The tympanic (middle ear) cavity and auditory tube are derivatives of the first pharyngeal pouch and are lined by endoderm.The pouch expands laterally to incorporate the ear ossicles and create the middle ear cavity, while the medial portion lengthens to form the auditory tube that maintains an open connection to the pharynx. The tympanic membrane (eardrum) forms from tissue separating the first pharyngeal pouch from the first pharyngeal cleft. It is lined by endoderm internally and ectoderm externally with a thin layer of mesenchyme in the middle.
3. Microtia involves defects of the external ear that range from small but well-formed ears to absence of the ear (anotia). Other defects occur in 20% to 40% of children with microtia or anotia, including the oculoauriculovertebral spectrum (hemifacial microsomia), in which case the craniofacial defects may be asymmetrical. Because the external ear is derived from hillocks on the first two pharyngeal arches, which are largely formed by neural crest cells, this cell population plays a role in most external ear malformations.

- 1. The lens forms from a thickening of ectoderm (lens placode) adjacent to the optic
cup. Lens induction may begin very early, but contact with the optic cup plays a role in this process as well as in maintenance and differentiation of the lens. Therefore, if the optic cup fails to contact the ectoderm or if the molecular and cellular signals essential for lens development are disrupted, a lens will not form.
2. Rubella is known to cause cataracts, microphthalmia, congenital deafness, and cardiac malformations. Exposure during the fourth to eighth week places the offspring at risk for one or more of these birth defects.
3. As the optic cup reaches the surface ectoderm, it invaginates, and along its ventral surface, it forms a fissure that extends along the optic stalk. It is through this fissure that the hyaloid artery reaches the inner chamber of the eye. Normally, the distal portion of the hyaloid artery degenerates, and the choroid fissure closes by fusion of its ridges. If this fusion does not occur, colobomas occur. These defects (clefts) may occur anywhere along the length of the fissure. If they occur distally, they form colobomas of the iris; if they occur more proximally, they form colobomas of the retina, choroid, and optic nerve, depending on their extent. Mutations in PAX2 can cause optic nerve colobomas and may be responsible for other types as well. Also, mutations in this gene have been linked to renal defects and renal coloboma syndrome.
(aeesnsy
- 1. Mammary gland formation begins as budding of epidermis into the underlying mesenchyme. These buds normally form in the pectoral region along a thickened ridge of ectoderm, the mammary or milk line. This line or ridge extends from the axilla into the thigh on both sides of the body. Occasionally, accessory sites of epidermal growth occur, so that extra nipples (polythelia) and extra breasts (polymastia) appear. These accessory structures always occur along the milk line and usually in the axillary region. Similar conditions also occur in males.
gene expression. J Anat 1994;184:15.
Figure
6.21A,B
Courtesy
of
the
Carnegie

Collection,
National
Museum
of
Health
and
Medicine,
Washington,
DC.
Figure
6.22
Courtesy
of
Dr.
E.
Blechschmidt,
of
Anatomy,
of
Gottingen.
Figure
6.23
Courtesy
of
Dr.
E.
of
Anatomy,
of
Figure
6.24
Reprinted
with
from
Hamilton
WJ,
Mossman
HW.
Human
Embryology.
Baltimore,
MD:
Williams & Wilkins; 1972.
Figure
7.3A—C
Courtesy
of
Dr.
Roger
Stevenson,
Greenwood
Genetic
Center,
Greenwood,
SC.
Figure
7.3D
Courtesy
of
Dr.
David
D.
Weaver,
of
Medical
and
Molecular
Genetics,
Indiana
School
of
Medicine
Figure
7.4B
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
7.8C
Courtesy
of
Dr.
Don
Nakayama,
of
Surgery,
of
North
Carolina.
Figure
8.4
Courtesy
of
Dr.
E.
of
Anatomy,
of
Figure
8.15
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
8.17A,B
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
8.20
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
8.21
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure

8.23A,B
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
9.2
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
9.3
Courtesy
of
Dr.
David
D.
Weaver,
of
Medical
and
Molecular
Genetics,
Indiana
School
of
Medicine.
Figure 9.4A,B Courtesy of Dr. Roger Stevenson, Greenwood Genetic Center, Greenwood, SC.
Figure 9.5 Courtesy of Dr. Roger Stevenson, Greenwood Genetic Center, Greenwood, SC.
Figure
9.6A—D
Courtesy
of
Dr.
Hytham
Imseis,
of
Obstetrics
and
Gynecology,
Mountain
Area
Health Education Center, Asheville, NC.
Figure
9.7A,B
Courtesy
of
Dr.
Hytham
Imseis,
of
and
Mountain
Area
Figure
9.8A—D
Courtesy
of
Dr.
Hytham
Imseis,
of
and
Mountain
Area
Health
Education
Center,
Asheville,
NC.
Figure
10.3
Modified
from
Gilbert
SE
Developmental
Biology.
Sunderland,
MA:
Sinauer
Associates,
Inc.;
2010.
Figure 10.8A,B Courtesy of Dr. Roger Stevenson, Greenwood Genetic Center, Greenwood, SC.
Figure
10.9A
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
10.9B,C
Courtesy
of
Dr.
Michael
L.
Cunningham,
Division
of
Craniofacial
Medicine,
Children’s
Center,
of
Washington.
Figure
10.10A

Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
10.10B
Courtesy
of
Dr.
J.
Jane,
of
Neurosurgery,
of
Virginia.
Figure
10.10C
Courtesy
of
Dr.
Michael
L.
Division
of
Medicine,
Center,
of
Figure
10.11A,B
Courtesy
of
Dr.
David
D.
Weaver,
of
Medical
and
Molecular
Genetics,
Indiana
School
of
Medicine.
Figure 10.12 Courtesy of Dr. Roger Stevenson, Greenwood Genetic Center, Greenwood, SC.
Figure
10.13
Courtesy
of
Dr.
David
D.
Weaver,
of
Medical
and
Molecular
Genetics,
Indiana
School
of
Medicine.
Figure
10.14
Courtesy
of
Dr.
David
D.
Weaver,
of
Medical
and
Molecular
Genetics,
Indiana
University School of Medicine.
Figure
10.15B
Reprinted
with
from
Moore
KL,
Dalley
AF.
Clinically
Oriented
Anatomy.
5th
ed.
Philadelphia:
Williams
and
Wilkins;
2006.
Figure
10.17A,B
Courtesy
of
Dr.
Nancy
Chescheir,
of
and
of
North
Carolina.
Figure 11.5 Courtesy of Dr. Roger Stevenson, Greenwood Genetic Center, Greenwood, SC.
Figure
11.6
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
12.2A,B
Courtesy
of
Dr.
K.
W.
Tosney,
Molecular,
Cellular,
and

Biology
Department,
University of Michigan.
Figure 12.5 Modified from Gilbert SE Developmental Biology. Sunderland, MA: Sinauer Associates, Inc.; 2010.
Figure
12.8
Reprinted
with
from
Moore
KL,
Dalley
AE
Oriented
Anatomy.
5th
ed.
Philadelphia, PA: Lippincott Williams & Wilkins.
Figure
12.9D
Shubin
N,
Tabin
C,
Carroll
S.
Fossils,
genes
and
the
evolution
of
animal
limbs.
Nature
1997;388:639-648.
Figure 12.10 Courtesy of Dr. Roger Stevenson, Greenwood Genetic Center, Greenwood, SC.
Figure
12.11A
Courtesy
of
Dr.
David
D.
Weaver,
of
Medical
and
Molecular
Genetics,
Indiana
Figure 12.11B Courtesy of Dr. Roger Stevenson, Greenwood Genetic Center, Greenwood, SC.
Figure 12.12A-D Courtesy of Dr. Roger Stevenson, Greenwood Genetic Center, Greenwood, SC.
Figure 12.13 Courtesy of Dr. David D. Weaver, Department of Medical and Molecular Genetics, Indiana
School
of
Medicine.
Figure
12.14
Courtesy
of
Dr.
David
D.
Weaver,
of
Medical
and
Molecular
Genetics,
Indiana
School
of
Medicine.
Figure
12.15
Courtesy
of
Dr.
David
D.
Weaver,
of
Medical
and
Molecular
Genetics,
Indiana
Figure 12.16 Courtesy of Dr. Roger Stevenson, Greenwood Genetic Center, Greenwood, SC.
Figure
15.16
Reprinted
with
from
Agur
AMR.
Grant’
Atlas
of
Anatomy.
10th
ed.
MD:
Williams
&
Wilkins;
1999:107.

Figure 15.22 Modified from Gilbert SE Developmental Biology. Sunderland, MA: Sinauer; 2006.
Figure
15.31B,C
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure 15.35 Courtesy of Dr. D. Nakayama, Department of Surgery, University of North Carolina.
Figure 16.8 Courtesy of Dr. Roger Stevenson, Greenwood Genetic Center, Greenwood, SC.
Figure
16.9D,E
Reprinted
with
from
Stevenson
RE,
Hall
JG,
Goodman
RM,
eds.
Human
Malformations
and
Related
Anomalies.
New
York,
NY:
Oxford
Press;
1993.
Figure
16.11
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
16.16A
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
16.16B
Courtesy
of
Dr.
David
D.
Weaver,
of
Medical
and
Molecular
Genetics,
Indiana
School
of
Medicine.
Figure 16.32C Reprinted with permission from Jirasek J. An Atlas of the Human Embryo and Fetus. London:
Taylor and Francis Books Ltd.; 2001.
Figure 16.34A,B Reprinted with permission from Jirasek J. An Atlas of the Human Embryo and Fetus. London:
Figure 16.35B,C Courtesy of Dr. Roger Stevenson, Greenwood Genetic Center, Greenwood, SC.
Figure 16.37 Courtesy of Dr. David D. Weaver, Department of Medical and Molecular Genetics, Indiana
Figure
17.5C
Reprinted
with
from
Jirasek
J.
An
Atlas
of
the
Human
Embryo
and
Fetus.
London:
Taylor
and
Francis
Books
Ltd.;
2001.

Figure 17.15 Courtesy of Dr. A. Shaw, Department of Surgery, University of Virginia.
Figure 17.16A-D Courtesy of Dr. David D. Weaver, Department of Medical and Molecular Genetics, Indiana
School
of
Medicine.
Figure 17.20 Courtesy of Dr. A. Shaw, Department of Surgery, University of Virginia.
Figure 17.21C Reprinted with permission from Jirasek J. An Atlas of the Human Embryo and Fetus. London:
Figure 17.23C Reprinted with permission from Jirasek J. An Atlas of the Human Embryo and Fetus. London:
Taylor
and
Francis
Books
Ltd.;
2001.
Figure 17.29A,D Courtesy of Dr. David D. Weaver, Department of Medical and Molecular Genetics, Indiana
Figure
17.30A,C
Reprinted
with
from
Jirasek
J.
An
Atlas
of
the
Human
Embryo
and
Fetus.
London:
Taylor
and
Francis
Books
Ltd.;
2001.
Figure 17.30B,D Courtesy of Dr. Roger Stevenson, Greenwood Genetic Center, Greenwood, SC.
Figure 17.34 Reprinted with permission from Moore KL, Dalley AE Clinically Oriented Anatomy. 5th ed.
Figure
18.2D
Courtesy
of
Dr.
K.W.Tosney,
Cellular,
and
Biology
Figure
18.6B
Courtesy
of
Dr.
K.W.
Tosney,
Cellular,
and
Biology
of
Michigan.
Figure
18.10C
Courtesy
of
Dr.
K.W.
Tosney,
Cellular,
and
Biology
of
Michigan.
Figure 18.16 Courtesy of Dr. Roger Stevenson, Greenwood Genetic Center, Greenwood, SC.
Figure
18.32
Redrawn

from
Rubenstein
JLR,
Beachy
PA.
Patterning
of
the
embryonic
forebrain.
Curr
Opin
Neurobiol 1998;8:18-26.
Figure
18.34
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
18.36
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
18.37A,B
Courtesy
of
Dr.
Roger
Greenwood
Genetic
Center,
SC.
Figure
18.38
Courtesy
of
Dr.
J.
Warkany.
Reprinted
with
from
Warkany
J.
Malformations.
Chicago, IL: Year Book Medical Publishers; 1971.
Figure 18.39 Courtesy of Dr. David D. Weaver, Department of Medical and Molecular Genetics, Indiana
Figure 18.42B Reprinted with permission from Moore KL, Dalley AF. Clinically Oriented Anatomy. 5th ed.
Philadelphia, PA: Lippincott Williams and Wilkins; 2006.
359
L
K
moO
<u
A
Abaxial
domain
Mesodermal
domain
comprised
of
the
parietal
layer
of
lateral
plate
mesoderm
and
somite
cells
from
the
myotome
and
sclerotome
regions
that
migrate across the lateral somitic frontier.
Acrosome
reaction
Release
of
enzymes
from
the
acro-
some
on
the
head
of
sperm
that
assists
in
sperm
pen-
etration
of
the
zona
pellucida.
Zona
proteins
induce
the
reaction
following
sperm
binding.
Adenohypophysis
Anterior
portion
of
the
pituitary
derived
from
Rathke’s
pouch.
Alar
plates

Sensory
area
in
the
dorsal
region
of
the
spinal
cord
and
brain.
Allantois
Vestigial
structure
that
serves
as
a
and
waste
storage
organ
for
avian
embryos.
It
extends
from
the
ventral
region
of
the
urogenital
sinus
to
the
umbilicus.
Later,
its
distal
portion,
called
the
urachus,
becomes
a
fibrous
cord
and
forms
the
median
umbili-
cal
ligament.
If
it
remains
patent,
then
it
may
form
a
urachal
fistula
or
cyst
in
this
region.
Alternative
splicing
Process
of
removing
(‘splicing
out”)
introns
to
create
different
proteins
from
the
same
gene.
Alveolar
cells
Cells
lining
the
alveoli.
Type
I
cells
are
involved
in
gas
exchange.
Type
II
cells
produce
surfactant.
Amelia
Complete
absence
of
a
limb.
Amniocentesis
Procedure
used
to
withdraw
amniotic
fluid
for
analysis
of
factors,
such
as
o-fetoprotein
(AFP)
and
cells
(chromosomes),
which
provides
infor-
mation
about
the
status
of
the
fetus.
Amniochorionic
membrane
Membrane
formed
when
expansion
of
the
amniotic
cavity
obliterates
the
chorionic
cavity
causing
the
amnion
to
contact
the
chorion
and
the
two
to
fuse.
The
amniochorionic
membrane
serves
as
a
hydrostatic
wedge
during
the
of
labor.

Amnion
Membrane
derived
from
the
epiblast
that
sur-
rounds
the
fluid-filled
amniotic
cavity
around
the
embryo
and
fetus.
The
fluid
cushions
the
fetus
and
forms
a
wedge
to
assist
with
dilation
of
the
cervix
during
labor.
The
fluid
itself
can
be
used
for
analysis
of
fetal
well-being.
Amniotic
bands
Pieces
of
amnion
that
tear
loose
and
can
wrap
themselves
around
digits
and
limbs
causing
constrictions
and
amputations
or
can
be
swallowed
by
the
fetus
causing
disruptions
in
facial
The
origin
of
bands
is
unknown.
Anencephaly
Neural
tube
defect
in
which
the
cranial
neural
folds
fail
to
close,
leading
to
tissue
degenera-
tion
and
little
or
no
formation
of
higher
brain
centers,
cerebral
cortex,
etc.
The
abnormality
is
lethal,
but
70%
S
A
T
of these
of defects
can
400
pg of
folic to
conception
Angiogenesis be
prevented by
acid beginning
Formation from
existing daily
maternal
2
to
3
use months
prior throughout
of blood
vessels.
Annulus fibrosis
vertebral disc.
Anterior visceral
endoderm
Outer ring
of fibrous
endoderm cells
disc responsible
secretion pregnancy.
vessels by
tissue
(AVE)
at the
cranial for
inducing of
sprouting in
an inter-
Collection end
of the
transcription
LIM1, and
HESX1.
Antimiillerian hormone
inhibiting substance
causes head

factors of
the bilaminar
region through
including,
Another term
produced regression
ducts
Aortic by
of in
males.
arch sal
Branch aorta
the
OTX2, for
miillerian-
Sertoli miillerian
from traveling
arch.
Initially, in
cells that
(paramesonephric)
the aortic
the center
there considerable
patterns for
Apical ectodermal
ectoderm are
five remodeling
sac to
the dor-
of pairs,
to the
head and
ridge at
the outgrowth
each pharyngeal
but form
these undergo
definitive neck,
aorta,
(AER)
distal of
the tip
limb erating
population the
progress
Apoptosis of
zone.
Programmed the
digits.
Atresia
Congenital example,
cell vascular
Layer of
the of
limb by
maintaining adjacent
thickened that
controls
a
rapidly mesoderm
death, absence
gut atresia.
Autonomic thetic
and parasympathetic
trol smooth
Basal prolif-
cells, for
example, of
an opening
called between
or lumen,
Composed of
for the
nervous muscle
and sympa-
systems glands.
plates
Motor spinal
area cord
Bladder and
brain.
exstrophy lack
of
Ventral closure
of pelvic
in the
body the
lateral resulting
the defect.
Blastocyst
Stage tation
where sphere
of embryogenesis
outer with
inner that
ventral portion
wall defect
body con-
of the
caused wall
in folds
protrusion of
at the
trophoblast by
in the
time the
bladder of
implan- cells
a
small group
of cell
mass, at
one
Bone morphogenetic
the pole.
proteins transforming
growth as
signal molecules
events, including
tem,
(BMPs)
form
a

fluid-filled embryoblast
cells, the
Members factor
B
for
a
family number
dorsalizing participating
of that
serve of
morphogenetic the
central in
nervous sys-
etc.
362
Glossary
Bowman’s of
Key capsule
each proximal
Terms
Cup-shaped convoluted
rounds
a
structure tubule
glomerulus.
Brachycephaly
Type of
coronal sutures
close short
head shape.
Brachydactyly
Short
Brain vesicles
spaces in
the
Once that
craniosynostosis at
the end
partially of
sur- in
which prematurely
resulting digits.
the neural
brain fill
mary brain
vesicles:
mesencephalon tube
with the
in
a
closes, fluid
to form
the prosencephalon
(midbrain);
(hindbrain).
These definitive
tall, expanded
three pri-
(forebrain);
and rhombencephalon
three primary
vesicles:
the telencephalon
lon vesicles
The form
five prosencephalon
and does
not divides
diencephalon;
divide;
the and
metencephalon
Brainstem into
the mesencepha-
the rhombencephalon
and forms
myelencephalon.
“Lower”
centers myelencephalon,
mesencephalon.
Cc
Capacitation
A
period
of
sperm
conditioning
in
the
female
reproductive
tract
lasting
about
7
hours
that
is
required
for
sperm
to
be
able
to
fertilize
an
egg.
Cardiac
looping
Bending
of
the
heart
tube
positions
the
heart
in
the
left
thoracic
region
and
creates
the
“typical”
heart
shape
with
the
atria
posterior
to
the
ventricles.
Cardinal
veins
System
of
anterior,
posterior,
and

com-
mon
cardinal
veins
that
drain
the
head
and
body
of
the
embryo
in
the
late
third
and
early
fourth
weeks.
Caudal
dysgenesis
Also
called
sirenomelia
or
mermaid
syndrome,
it
is
caused
by
insufficient
of
mesoderm
by
the
primitive
streak.
there
are
not
enough
cells
to
form
the
lower
part
of
the
body
so
that
the
legs
are
fused.
Renal
agenesis
is
usually
the
cause
of
death.
The
defects
are
most
often
observed
in
infants
from
insulin-dependent
diabetics.
Cerebral
aqueduct
(of
Sylvius)
Lumen
of
the
mesen-
cephalon
that
connects
the
third
and
fourth
It
is
often
the
site
for
that
impede
the
flow
of
cerebrospinal
fluid
and
cause
hydrocephalus.
Chondrocranium
Part
of
the
neurocranium
that
forms
the
base
of
the
skull
and
arises
by
first
establish-
ing
cartilage
models
for
the
bones
(endochondral
ossification).
Chorion
Multilayered
structure
of
the
somatic
layer
of
extraembryonic
mesoderm,
cytotrophoblast,
and
syncytiotrophoblast.
It
the
fetal
por-
tion
of
the
placenta,
including
the
villi
and
villus
lakes.
Chorionic
cavity
Space
formed
between
the
extraem-
bryonic
mesoderm
lining
the
cytotrophoblast
(somatic

mesoderm)
and
that
surrounding
the
yolk
sac
and
embryo
(splanchnic
mesoderm).
The
chorionic
cavity
will
eventually
be
obliterated
by
expansion
of
the
amniotic
cavity
and
fusion
of
the
amnion
with
the
chorion.
Chorion
frondosum
(leafy
chorion)
Embryonic
side
of the chorion, where villi form.
of the
brain, including
pons of
the metencephalon,
the and
the
Chorion
laeve
(smooth
chorion)
Abembryonic
side
of
the
chorion,
where
villi
regress,
leaving
a
smooth
surface.
Choroid
plexuses
Vascularized
formed
in
the
lateral,
third,
and
fourth
ventricles
that
produce
cere-
brospinal
fluid.
Cloaca
Common
chamber
for
the
hindgut
and
urinary
systems.
Its
anterior
portion
forms
the
sinus,
and
its
posterior
portion
forms
the
anus.
Cloacal
membrane
(plate)
Membrane
formed
at
the
caudal
end
of
the
embryo
from
adhesion
between
epiblast
and
hypoblast
cells.
Later,
it
covers
the
cloaca
and
breaks
down
to
form
openings
into
the
sinus
and
anus.
Coloboma
Defect
in
the
eye
due
to
closure
of
the
optic
fissure.
Usually,
these
defects
are
restricted
to
the
iris.
Compaction
Process
whereby
cells
of
the
morula
stage

form
tight
junctions
to
seal
in
for
forming
and
pumping
fluid
into
the
blastocyst
cavity.
Connecting
stalk
connection
that
con-
nects
the
embryo
to
the
placenta.
It
contains
the
allan-
tois
and
umbilical
vessels
and
will
be
into
the
umbilical
cord
with
the
yolk
sac
(vitelline)
stalk
(duct).
malformation
Synonymous
with
the
term
birth
defect,
it
refers
to
any
structural,
behavioral,
func-
tional, or metabolic disorder present at birth.
Cotyledons
Compartments
(15
to
20)
in
the
placenta
formed
when
decidual
septa
grow
into
the
intervillous
spaces.
These
septa
never
reach
the
chorionic
plate
so
that
there
is
between
cotyledons.
Cranial
nerves
(CNs)
Twelve
pairs
of
nerves
with
the
brain,
with
all
but
two
(the
olfactory
and
optic)
originating
from
the
brainstem.
Craniosynostosis
Premature
closure
of
one
or
more
cra-
nial
sutures,
leading
to
an
abnormally
shaped
skull.
A
major
cause
is
mutations
in
fibroblast
growth
factor
receptors
(FGFRs).
Crista
terminalis
Ridge
of
tissue
in
the
right
atrium
between
the
original
trabeculated
part
of
the
right
atrium
and
the
smooth-walled
part
derived
from
the
sinus
venosus.
Cryptorchidism
Failure
of

one
or
both
testes
to
descend
to
the
scrotum.
Cytotrophoblast
trophoblast.
Proliferative
inner
layer
of
the

Deformations
Altered
of
caused
by
mechanical
forces,
for
example,
clubfeet
resulting
from
too
little
room
in
the
amniotic
cavity.
Dermatome
Dorsal
portion
of
each
somite
that
forms
the
dermis
of
the
skin
of
the
back.
Dermatomes
are
segmented
and
supplied
by
spinal
nerves
from
the
seg-
ments
at
which
they
originated.
This
segmental
pat-
tern
is
maintained
as
they
migrate
over
the
body.
Thus,
each
region
that
they
occupy
on
the
skin
is
also
called
a
dermatome nerve
and is
that originally
innervated supplied
the of
the somite.
- Diaphysis Shaft of the long bones.
Derived from
(forebrain)
hypothalamus, posterior
caudal and
by the
same dermatome
portion forms
lobe stalks
(nerves), and
of spinal
region of
the the
thalamus, the
other
Dihydrotestosterone responsible
for the
duct and
external
Diploid
The in
pituitary, structures.
Converted from
differentiation optic
testosterone and
of genitalia.
normal somatic
as chromosome
cells.
In
23
homologous of
46.
Disruptions
Term used
from destructive
had formed
that these
cells, pairs
the mesonephric
complement present
chromosomes to
form to
describe processes
normally, cause
bowel limb
or appear
the diploid
number birth
that alter
for defects
resulting
a
example, atresias
and digit
amputations.
Dizygotic twins
common
Twins formed
form of
Dorsal twinning
mesentery pending
structure vascular
amniotic after
it accidents
bands from
that cause
two
(66%).
Double the

gut tube
the lower
end as
the gut
mesentery posterior
of grows
layer of
peritoneum from
the and
are lost
body and
colon.
Dorsal primary
innervates ramus
intrinsic maxial
muscle
as the
dorsal esophagus
body wall
to the
rotates, some
portions wall,
rectum.
parts of
the that
is, parts
Branch back
cells,
Sensory ganglion
to of
muscles, and
skin fibers
the spinal
Dysmorphology of
the gut
of most
sus- from
Later, dorsal
fuse the
a
spinal derived
over the
passing cord.
Study of
ment, and
the prevention
dysmorphologist back.
from
a
dorsal causes,
of birth
is
a
clinical department.
Ectoderm
One of
skin, the
central structures.
Ectopia cordis
lack
Ventral of
closure thoracic
three prognoses,
defects.
to the
duodenum nerve
that from
pri- root
treat-
Usually, geneticist
basic nervous
in germ
layers system,
body hair,
wall of
the region
lateral causing
cavity.
Ectopic
Something that
is not
in its
normal example, an embryo's implantation site.
Ectrodactyly
Missing digits.
Efferent ductules
to the
Tubules mesonephric
from the
tubules that
duct seminiferous
are for
tubules derived
connect the
the
a
a
genetics that
and forms
many defect
body the
resulting wall
heart other
from folds
in to
lie the
outside position,
the for
rete testis
passage to
from nephric
kidney.
Embryogenesis
Another of
sperm the
nephric epididymis.
tubules
The of
the term
for meso-
organogenesis, mean-
ing the
period of
organ formation
from approximately

the third to eighth weeks after fertilization.
eggs, the
Endocardial connective
responsible cushions
Structures tissue
covered consisting
by endothelium
for the
heart.
Endochondral ossification
bone by
most septation
363
of loose
that processes
are occurring
Mechanism first
establishing lowed
by ossification.
characteristic in
for
a
This of
the forming
cartilaginous type
bones the
skull.
Endoderm
One the
gut and
of the
model fol-
of bone
the three
its basic
Enhancers
Regulatory utilization
elements of
promoters, and
regulate
Epiblast the
Dorsal inar
germ ment.
of limbs
germ is
and base
layers that
DNA
that control
promoter rate
of transcription.
(top)
layer disc
during
The hypoblast
of the
embryo
Epibranchial of
cells comprising
the forms
are derived
placodes derm
lying
Four dorsal
sensory second
of form
activate efficiency,
the week
bilam- of
the ventral
develop- layer.
from the
All epiblast.
thickened to
ganglia for
Highly mesonephric
the regions
pharyngeal cranial
tissues of
ecto- arches
nerves that
V,VII, convoluted
duct
Epiphyseal plate
diaphysis region
and used
for
IX, form
and derived
X.
from sperm
Cartilaginous and
epiphysis ues
to produce
cation storage.
region of
the long
bone growth
until
Then the
these
Epiphysis bones
plates between
the the
bones that
contin- by
endochondral have
acquired disappear
End of
the
Epiploic foramen
lesser ossifi-
their full
(close).
long bones.
(of and
greater at
the free
Winslow)
sacs margin
duodenum
Opening in
the length.
between abdominal
of and
the the

lesser the
common bile
liver.
duct, cavity
omentum
In the
located between
its ventral
hepatic
(the portal
triad).
Epithelial-mesenchymal interactions
virtually every
organ is
formed.
ectoderm and
derm artery,
the border
and lie
portal vein
Process whereby
Examples underlying
and surrounding
and include
mesenchyme, mesenchyme,
metanephric limb
gut endo-
ureter mesenchyme,
and forth
between differentiation.
Exon
Region of
a
protein.
Falciform attaches
Fibroblast in
a
ligament gene
etc.
these cell
types that
can
F
Part the
liver to
growth large
involved family
of the
factors epithelium
Signals pass
back to
regulate be
transcribed ventral
organ into
a
mesentery body
wall.
(FGFs)
having in
a
Signal over
number ing
formation
Mutations
15
members.
of of
the in
embryological sutures
that proteins
They are
events, and
bones their
receptors of
craniofacial abnormalities,
craniosynostosis.
Fistula
An abnormal
(FGFRs)
includ- of
the cause
including many
passageway.
Folic acid
A
“B”
vitamin that
can prevent
approxi- mately 70% of neural tube defects if taken as a 400-p1g
skull.
a
variety forms
of
364
Glossary supplement
of by
to conception
Fontanelle
Wide than
two tanelle,
Key
Terms mothers
beginning and
continuing sutures
bones
2
to
3
months throughout
in the
skull meet.
sometimes the
two
Foramen
The called
parietal and
cecum thirds
and ing
the prior
created where
two
Pit at
posterior site
Foramen permits
the one
of
“soft is
the more

anterior spot,”
frontal bones
junction third
origin of
ovale
Opening shunting
fetal fon-
located where
meet.
of the
of the
the thyroid
in of
blood development.
Foregut
Part of
the pharynx
just ing
to
a
point esophagus,
gut anterior
tongue two
represent- gland.
the interatrial
from tube
proximal right
septum that
to beginning
to just
the distal
stomach, addition
to which
all
Fossa to
and the
lungs, left
during caudal
lung bud
the part
liver to
the and
extend- bud.
It forms
of the
the duodenum,
liver, gallbladder,
form ovalis
from diverticula
Depression atrial
septum septum
in and
pancreas,
(buds)
off on
the right
formed secundum
the foramen
are ovale
Gastroschisis
Ventral lack
of closure
abdominal of
region and
sometimes
Gastrulation when
side the
pressed is
closed the
gut tube.
of septum
against at

body wall
resulting other
Process layers
from through
in the
inter- primum
each and
other birth.
defect resulting
body and
from wall
folds protrusion
organs through
of forming
the mesoderm.
Germ layers
Three derm,
and trulation.
basic endoderm
These embryo.
Glomerulus
Tuft capsule
the the
a
in the
of intestines
defect.
three involving
primitive streak
primary movement
to form
cell layers
derived layers
germ of
cells endoderm
of ectoderm,
from form
all of
capillaries the
of the
and meso-
process of
structures formed
at the
end of
Gray rami
communicantes ganglionic
gas- in
the in
the
Bowman convoluted
Connections sympathetic
fibers pathetic
trunks levels
to spinal
of
Greater the
spinal omentum
from tubule.
carrying from
ganglia nerves.
cord.
Double dorsal
mesentery intestines
from serves
layer
Gray post-
in the
rami exist
of and
the as
greater
a
storage of
infection
Greater peritoneum
extending sym-
at all
formed down
over curvature
site for
(the police
sac
Most tion
of the
tum.
The foramen
of fat
and can
officer of
lesser the
sac two
of the
It wall
the abdominal
lying sacs
off pockets
abdomen).
cavity dorsal
with excep-
to are
(of the
connected
Winslow).
Growth factors
are
Proteins usually
secreted by
receptors that
and on
via act
as signal
have target
Gubernaculum cells.
Condensation ing
from the
assists in
of mesenchyme
testis to
the floor
descent abdominal
wall to
of the
the lesser
omen- the
epiploic molecules
that signals
transduced extend-
of the
scrotum testis

scrotum.
from that
their
Haploid
Term somes
used in
H
to denote
the present
gametes in
somatic
Hindgut
Part one-third
of
(23),
(diploid)
of the
of the
the gut
tube transverse
anal the
canal.
descending the
number which
is of
half cells.
extending colon
It forms
from to
the part
colon, and
the upper
part the
of the
Holoprosencephaly
Defect sue
for the
face and
lateral ventricles
Homeobox genes
homeobox, of
the sigmoid
the chromo-
number the
distal upper
portion transverse
colon, anal
canal.
where brain
so much
has been
fuse together
colon, the
rectum, midline
lost that
and
a
specific within
a
region are
appear factors
tis- the
as two
one.
that
DNA-binding called
important establishing
motif the
homeodomain.
for patterning
different the
origin and
type limbs,
and other
Hydatidaform contain
a
(sequence)
These the
genes embryonic
regions of
the brain,
of gut
axis, determining
derivatives, similar
patterning phenomena.
mole
Trophoblast but
with no
embryo.
and probably
egg forms
Moles placental
express arise
only from
followed fertilization
by duplication
somes to
restore concentrations
the tissue,
paternal genes
of an
enucleated of
the paternal
a
diploid number.
of human
may become
Moles chorionic
invasive
Hydrocephalus
(malignant).
Increased in
the brain
leading
Usually due
the fluid,
amounts of
to to
a
block which
aqueduct most
of nial
Sylvius sutures
sometimes relieved.
Hyperplasia
An
Hypertrophy in
have not
chromo- secrete
high gonadotropin

and cerebrospinal
fluid intracranial
in the
pressure.
circulatory often
pattern occurs
in the
the mesencephalon.
fused, of
cerebral
If the
great child’s
the cra-
head proportions
increase if
the in
An cell
number.
increase
Hypoblast
Ventral
Contributes in
layer size
of to
of
a
part the
formation bryonic
mesoderm
Hypospadias of
but not
An opening
tral aspect
the of
penis or
Process tissue
causes their
or enlarges,
not organ.
bilaminar the
germ yolk
to sac
of the
urethra scrotum.
whereby another
fate.
is the
Thus, set
one responder.
Inguinal canal
Oblique abdomen
to female
fetuses
Inner cell
cell one
population of
cells disc.
extraem- the
embryo.
along the
ven- of
or cells
tissues type
is the
passageway the
scrotum as
well.
mass
Cluster of
the blastocyst
develops.
Intermaxillary segment
processes;
it includes
lip, the
upper teeth,
and for
to inducer,
or
a
change and
from one
the the
of testes.
cells and
from segregated
which lower
Forms to
in one
the pole
entire
Formed from
the philtrum
jaw component
the embryo
medial region
housing palate.
the nasal
of the
upper four
incisor

Juxtacrine does
signaling not
(1)
Type use
diffusable
A
protein of
cell-to-cell proteins.
(ligand)
its receptor
on extracellular
signaling
There are
on another
matrix another,
and junctions.
Karyotype
Lateral horn
for the
Chromosomal one
cell that
three types:
surface cell
surface, secreted
(3)
Direct reacts
(2)
Ligands by
one transmission
cell with
in interact
of

makeup of
an individual.
L
Origin of
sympathetic region
of neurons
nervous the
Lateral
(intermediate system
spinal plate
cord columns)
lying in
from
T1
to
L1-L2.
mesoderm splits
Mesoderm-derived into
splanchnopleure
(parietal)
cavity.
Lateral somitic
and frontier
the cells
parietal from
tissue that
(visceral)
and somatopleure
layers surrounding
the
Border layer
of lateral
the each
somite lateral
organs and
each somite
plate myotome
and migrate
plate domain.
Laterality sequences
during across
mesoderm mesoderm.
sclerotome the
and
Right gastrulation
Patients
Some regions
frontier to
with and
in with
the defects
primarily bilaterally
Lesser omentum
part it
form left
sides third
of enter
the the
are week
in sidedness,
right of
abaxial established
such or
left that
they are
sided,
Double of
the layer
ventral liver
to the
curvature mesentery
proximal of
have of
peritoneum and
end of
the the
Lesser sac
stomach.
Space municates
behind with
(greater sac)
Ligand
A
the the
extending duodenum
lesser rest
omentum of
via signal
the laterality
forming from
the and
lesser that
the epiploic
molecule.
abdominal foramen
com- cavity
Winslow).
(of the
lateral the
with signals
via gap
365
M
to two daughter cells each with 46 chromosomes.
366
Monozygotic twins
Splitting may
mation of
Twins occur
the time
of inner
Morphogen formed
at the
germ disc,
cell mass
Molecule induce
cells from
two-cell
a
single stage
but usually
formation.
secreted at
to induce
differentiate.
more centration
than
The

a
distance same
one cell
type gradient.
Miillerian inhibiting
miillerian substance
hormone.
causes regression
oocyte.
or after
takes for-
place at
that morphogen
by establishing
the can
can
a
con-
Another
Produced of
term for
by anti-
Sertoli the
ducts in
male fetuses.
Derived the
rhombencephalon medulla
oblongata.
Myelomeningocele
Protrusion cord
tissue through
spina bifida.
Myotome
Muscle myocytes
forming derived
medial regions
These miillerian
cells and
from the
caudal portion
of
(hindbrain)
and of
meninges
a
defect in
the region
from that
cells remain
the intrinsic
muscles back
and
Nephron
Functional proximal
vertebral of
a
somite the
ventrolateral coalesce
forms the
and spinal
arch called
formed by
and beneath
in the
dorso- the
primaxial muscles,
some dermatome.
domain intercostal
muscles of

unit and
distal
form at
of the
kidney convoluted
and form
and the
upper cervical
limb girdle.
consisting tubules,
and cells
a
Cells the
migrate to
tips
(“crest”)
other spinal
of the
the neuroepithelium
of regions
ganglia, face,
septa some
cranial
(enteric the
of the
loop of
Henle, that
neural to
folds and
form bones
for then
many and
the nerve
ganglia), nerable
for why
outflow structures,
tract ganglia,
ganglia melanocytes,
to teratogenic
many of
the for
etc.
the gut
These insult
and children
diac defects.
Neurocranium case
with
Part of
the around
the the
brain viscerocranium
the facial
skull that
(the other
cells rovide
heart, tube
are
a
clefts also
forms vul-
rationale have
car-
a
part of
or face).
membranous neurocranium,
skull, and
the cartilaginous
cranium, forming
Neuromeres protective
the skull
is of
two or
flat bones
neurocranium the
base of
Brain meres.
They where
they
Neuropores parts,
of the
or chondro-
the skull.
especially are
associated prominent
called in
rhombomeres.
Cranial tube
that and
exist from
folds is
initiated portions
caudal openings
the until
time that
it of
the closing
Neurulation
Process into
the neural
week and
with somito-
the hindbrain
in closure
is complete,
of that
neural of
tube.
ends close
the tube
anencephaly tube.
transforming
Neurulation at
28
days.
results
Failure the
begins of
in neural
and
Notochord
An immediately

spina the
neural the
neural is,
unclosed neural
plate in
the the
tube bifida.
extended ventral
to column
of the
neural defects,
third folds
to including
midline floor
plate of
cells lying
end ing
the brain
system of
the neural
and extending
spinal cord.
from
It plate,
and the
spinal the
somites cord,
to ecule
the hypophysis
the form
for these
Nucleosome vertebrae.
phenomena
Basic to
for induc-
(motor)
region sclerotome
of the
portion
The major
is sonic
unit taining
a
of structure
complex mately
of
140
base pairs
Nucleus pulposus
intervertebral of
signal mol-
hedgehog
(SHH).
of histone
chromatin, con-
DNA.
Central disc
chord cells.
Omentum
Fold to
the gelatinous
derived and
approxi- portion
from proliferation

of peritoneum
liver
(lesser the
transverse
Omental passing
omentum)
colon bursa
posterior of
an of
from noto-
the or
from and
the beyond
(lesser to
the connects
to peritoneal
Winslow).
Omphalocele
Ventral of
(greater peritoneal
stomach stomach
to omentum).
sac)
Space by
gut rest
of the
sac)
through created
rotation.
peritoneal the
body physiologically
This space
cavity epiploic
wall herniated
to the
body cavity
Organogenesis
(greater foramen
defect caused
loops in
the of
tenth
Period primordia
week.
are from
eighth organs
when the
usually considered
of the
third week
of week
are most
sensitive most
birth defects
Oropharyngeal
This to
the is
the to
insult, occurs.
membrane cranial
end of
epiblast and
ing
Membrane the
germ disc
hypoblast of
the oral
ynx cells.
cavity develops.
membrane).
Outer cell
mass and
cover the
Paracrine signaling
another
(The and
(of by
failure bowel
to return
organ to
be end
of time
when induction
formed by
of at
adhesion
Later, and
breaks old
Cells that
term surround
inner cell
Type where
proteins short
distances the
between it
covers the
down open-
as the
phar- was
buccopharyngeal the
mass and
that

of signaling
synthesized to
interact
Paramesonephric blastocyst
cavity will
form from
by with
other ducts
(Miillerian parallel
abdominal one
one cells.
ducts)
duct and
cavity tal
sinus.
to the
posterior
These the
uterus, the
ducts uterine
vagina
Paraxial regress
the cell
to cell
diffuse
Ducts extend
wall in
of the
(Fallopian)
in female
that from
the male
tubes, fetus
and the
urogeni- but
form upper
fetuses.
mesoderm the
Mesoderm-derived axis
of the
embryo and
somitomeres.
Parenchyma
The specific
together by
tissue responsible
for forming
cells of
a
connective
Parietal
Pertaining
Parturition to
the
Birth.
tissue gland
called wall
of part
of along
somites or
organ the
held stroma.
any cavity.
368

endoderm., 1 = . layer of endoderm., 2 = . layer of endoderm., 3 = . A narrowing of a canal or orifice., 1 = . A narrowing of a canal or orifice., 2 = . A narrowing of a canal or orifice., 3 = . The ectodermally lined primitive oral cav-, 1 = . The ectodermally lined primitive oral cav-, 2 = . The ectodermally lined primitive oral cav-, 3 = . ity , separated from the pharynx by the oropharyngeal, 1 = . ity , separated from the pharynx by the oropharyngeal, 2 = . ity , separated from the pharynx by the oropharyngeal, 3 = . membrane, that later breaks down., 1 = . membrane, that later breaks down., 2 = . membrane, that later breaks down., 3 = . Connective tissue of glands., 1 = . Connective tissue of glands., 2 = . Connective tissue of glands., 3 = . Phospholipid made by alveolar type II cells, 1 = . Phospholipid made by alveolar type II cells, 2 = . Phospholipid made by alveolar type II cells, 3 = . that reduce surface tension in alveoli, which is essen-, 1 = . that reduce surface tension in alveoli, which is essen-, 2 = . that reduce surface tension in alveoli, which is essen-, 3 = . , 1 = Stroma Surfactant. , 2 = Stenosis Stomodeum. , 3 = Splanchnopleure
the end of the sixth month, making it difficult for pre-
mature infants born before this time to survive.
Suture Narrow seams of connective tissue that separate
the flat bones of the skull allowing molding of the
skull through the birth canal and growth and expan-
sion as the brain grows.
Sympathetic
trunks
Paired
collections
of
ganglia lying on the posterior body wall lateral to the
vertebral
bodies.
Sometimes
called
chain
ganglia.
Syncytiotrophoblast
Outer
multinucleated
layer
of
the
trophoblast
that
serves
to
invade
the

endometrium
of
the uterus.
Syndactyly Fusion of one or more digits.
Syndrome A group of abnormalities occurring together
that
have
a
known
cause,
for
example,
Down
syn-
drome and fetal alcohol syndrome (FAS).
T
Telencephalon
prosencephalon hemispheres.
Teratogen
A
factor that
causes
a
birth most
forms portion
the of
cerebral defect,
such as
a
drug or environmental toxicant.
Teratology Science that studies the origin, causes, and prevention of birth defects.
Teratoma
Tumor-containing derivatives
from all
three germ layers. They may arise from remnants of the
primitive streak or from germ cells that do not migrate successfully
to the
gonadal ridges.
The most
common ones
are caudal
region.
Thyroglossal duct
teratomas arising
Duct formed
in along
the buttocks
the path
roid migration
extending in
of thy-
from the
foramen cecum in the tongue to the neck.
Tracheoesophageal septum Septum that separates the trachea from the gut tube.
Transcription factors
Proteins that
have
DNA-binding sites that regulate the expression of downstream genes.
Throphoblast
Outer cell
layer surrounding
the blasto-
cyst from which placental tissues are derived.
U
Urachus
Vestigial
remnant
of
the
allantois
from
the
ven-
tral
surface
of
the
sinus
to
the
umbilicus
that normally regresses to a fibrous cord, forming the
median umbilical ligament. Sometimes it may remain
patent to form a urachal fistula or cyst.
Urogenital
ridge
Bilateral
epithelial-covered
elevation
of
mesoderm
that
lies
in
the
lower
thoracic and lumbar regions and that forms the meso-
nephric kidneys and the gonads.
from the
369

Urorectal
septum
Wedge
of
mesoderm
that
grows
down
between
the
hindgut
and
primitive
sinus, partially separating these two structures. The
caudal end of the septum forms the perineal body.
Uterovesical pouch Depression between the vagina and
the bladder.
Vv
Vasculogenesis Formation of blood vessels from blood
islands in situ.
Ventral mesentery Double layer of peritoneum derived
from the septum transversum and extending from the
liver to the ventral body wall (the falciform ligament)
and from the liver to the stomach and duodenum
(lesser omentum).
Ventral primary ramus Ventral branch of a spinal nerve
that innervates limb and trunk muscles except the
intrinsic (“true”) back muscles, which are innervated
by dorsal primary rami.
Ventral root Motor fibers passing from ventral horn cells
in the spinal cord to a spinal nerve.
Visceral Relating to the organs of the body.
Viscerocranium Part of the skull that comprises the
bones
of
the
face
(the
other
part
of
the
skull
is
the
neurocranium).
Vitelline duct Connection between the yolk sac and
the
primary
loop
of
the
midgut
through
the
stalk.
Failure
of
this
duct
to
degenerate
results
in
fistulas
and
diverticula
(Meckel’s
diverticu-
lum) from the small intestine to the umbilicus.

White
rami
communicantes
Connections
carrying
preganglionic sympathetic fibers from spinal nerves to
the sympathetic trunks. White rami exist only at levels
T1-12.
Y
Yolk sac Structure located ventral to the bilaminar germ
disc derived from the hypoblast. It is the site of origin
of
the
first
blood
cells
and
remains
attached
to
the
midgut
via
the
vitelline
(yolk
sac)
duct
until
late
in
Z
Zone of polarizing activity (ZPA) Population of
mesoderm
cells
at
the
posterior
border
of
the
limb
next to the apical ectodermal ridge that regulates
anterior—posterior patterning of the limb.

neurocranium, 135, 135-136, 136
viscerocranium,
136,
136-137
Axons,
269,
293,
316
Azoospermia,
36b
Azygos vein,
193,

Baller—Gerold syndrome,
Bardet-Biedal
194
160b syndrome,
Barker's hypothesis,
Barr body,
Basal
100b
17b lamina,
7
Basal plates,
290,
299t
Basilar membrane, 322, 323
Basket cells,
301
Becker's muscular dystrophy (BMD), 149b
Bicuspid valve,
175
Bifid penis,
254)
Bilaminar germ
Bile duct,
Biliary disc,
218,
219
atresia,
Birth.
See also
breathing
220b
Parturition movements,
control
(see
206
Contraceptive low
weight, postmature,
methods)
100b
99
premature,
99
preterm,
109b time
of,
99
Birth defects,
83b chemical
agents, environmental
120t, factors,
heart,
176b-178b heavy
metals,
124-125
hormones,
120r, hyperthermia,
123-124
121
hypoxia,
124
infectious agents,
nutritional
120-121, deficiencies,
obesity,
124
pharmaceutical drugs,
pharyngeal region,
physical agents,
prevention
120¢
124
121-123
270b—-273b
120¢
of,
125b radiation,
121
and spontaneous
teratology, abortions,
principles types
of,
117
viral infections,
Bladder,
240-241, defects
13)-21b of,
119
121
242
of,
242b-243b exstrophy
Blastocyst abnormal,
of,
88,
89,
242b,
48)
embedded in,
43
formation,
37-39, implantation
38
of,
39,
Blastomeres,
37,
Blood cells
75
molecular regulation, 77
Blood types,
106b
Blood
BMP.
Body
Body
Body
Bone vessels,
75,
See
Bone
75-77,
76
morphogenetic axes,
formation cavities,

protein of,
52,
54-56,
87-88, wall
defects, age,
87
225b
157b
(BMP)
55,57
37
40
237b
43-50
121-123
117-125
243
Connective tissue cells, 218
256b
Connexin proteins,
Conotruncal
7
endocardial
Contiguous gene
cushions, syndrome,
Contraceptive methods
barrier methods,
emergency
35)
contraceptive hormonal
methods, intrauterine
pills
35b-36b device,
male pill,
36b sterilization,
36b
Conus cordis,
167,
Conus septum,
181
Copula,
273
Cor triloculare biventriculare, 176b, 177
Cornea,
333,
333
Coronary arteries, 188
Coronary sinus,
170
Corpus atreticum,
Corpus
Corpus
29
callosum,
306, striatum,
303,
Cortex development,
Cortical cords,
245
Cortisone,
123-124
Cotyledons,
105
Cranial defects,
Cranial
310b-313b meningocele,
Cranial
137, nerves,
313-315
origins,
314t-315t
Cranial neuropores,
287
Craniofacial defects,
137b-141b skeleton,
37
Craniopagus twins,
114
Craniopharyngiomas,
304b
Craniorachischisis,
311b
Cranioschisis,
137b-138b
Craniosynostosis,
138,
137b-138b radial
aplasia syndrome,
Cremasteric fascia,
Cricothyroid,
160)
258
265
Cri-du-chat syndrome,
Crista ampullaris,
Crista
19)
322,
323
dividens,
195
Crista terminalis,
Crossovers,
170
12
Crosstalk,
6

Crown-heel length (CHL), 96
Crown-rump length
(CRL),
81, ultrasound
measurement,
Crus ampullare,
Crus
125
322,
324
cerebri,
302
Crus nonampullare,
Cryptorchidism,
322,
258b
Cumulus oophorus,
23
CVS. See Chorionic villus sampling (CVS)
Cyclopia,
337b
Cystic acne, 125b
Cystic duct,
218
Cytodifferentiation,
10
Cytomegalovirus,
120
Cytoskeletal machinery,
7
Cytotrophoblast,
43,
43
D
Deafness,
327b
Decidua basalis, 102-103, 103
Decidua
reaction,
46
Decidual
plate,
61,
103
Deciduous teeth, 284, 284
Definitive
choanae,
282
313b
304
306,
306
137b
36b
179,
180,
181
271
19b
324
(ECPs),
96
36b
Definitive cortex, 316
Definitive placental villus, 60, 60
Deletion, chromosome, 18)
Dendrite,
290
Dental buds, 283
Dental cuticle, 283
Dental epithelium,
Dental lamina,
283
Dental papilla,
Dentate
283, nucleus,
283
301
Dentin,
283
Denys-Drash syndrome, 237b
Deoxyribonucleic acid
(DNA)
genes,
3
mitosis,
11
Dermomyotome,
Desert hedgehog,
fetus,
96-99,
971,
97-99,
99t growth, length and weight, 95, 95t
of hands,
151
histological and
horizons, functional,
99, of
99t limbs,
151-152,
152
molecular regulation, 154-157, 156
metanephric excretory
unit,
235
monthly changes
time of
birth, week
1, week

during,
99
29-42
2,
43-50
week
3,
51-62
Dextrocardia,
169b
Diabetes,
124,
176b
Diaphragm,
90-92, development
96-99,
91,
92,218
of, formation
of, hernias,
93b
Diaphragmatic
Diaphysis,
151
Dicephalus twins,
92
92-94.
hernia,
93,
93
114
Diencephalon,
302-304,
Diethylstilbestrol,
303,
107b,
123
DiGeorge sequence,
183b
Diphenylhydantoin,
Diplotene stage,
22
Distal convoluted
Dizygotic tubule,
twins,
110,
111
domain,
Dominant mutation,
Dorsal
4
20b mesentery,
Dorsal
Dorsal
211
mesocardium, mesogastrium,
Dorsal
164
211,213
root ganglia,
293
Double penis,
254
Down syndrome,
125
21, karyotype,
146
16
oocyte formation,
physical
14b features,
16
Duchenne’s muscular
dystrophy
(DMD),
Ductuli efferentes,
245
Ductus arteriosus, 186, 188, 189, 195
closure of,
197
Ductus deferens, 245, 248
Ductus reuniens,
321,
322
Ductus venosus,
192,
195
closure of, 196-197
Duodenum formation of, 217, 218
Dwarfism,
137b-141b
1201,
97t,
97-99,
1496
lungs,
205
283
133
8
122
236
304
99t
E
Ear external,
internal,
325-326
321-324
middle,
324-325
Eardrum, 267, 325, 326
Ebstein anomaly,
177b
E-cadherin,
236
Ectoderm embryonic

period, thickenings,
63
67
Ectodermal germ
neural layer
crest neural
cells,
66-68
induction, neural
63
plate,
63
neuroectoderm, neurulation,
63
63,
65
Ectodermal placodes,
Ectopia cordis,
Ectopic
88,
313
89, lung
lobes,
Ectopic pregnancy,
Ectopic
184b
2066
48b, thymic
49
tissue,
Ectrodactyly,
158b
Edinger-Westphal
Efferent ductules,
Ejaculatory ducts,
Embryo fifth
week,
108
presomite, somite,
60,
67
third week,
10
Embryoblast,
38
Embryogenesis teratology,
119
Embryonic disc
Embryonic growth,
period,
270b nucleus,
247
241,
248
57
63-85
Embryonic stem
cells
(ES
cells),
39b
Emergency contraceptive pills (ECPs), 36)
Enamel knot,
283
Endocardial cushions,
Endochondral
171,
177b bone
Endocrine disrupters,
123
Endoderm,
5
embryonic period, 63
germ layer,
78-79,
78-80
germ layers, gastrulation, 51
Endometrium,
39
Engrailed 1 and 2 (EN1 and EN2), 309
Enhancers,
4
Enterocystoma.
See
Eph receptors,
77
ephrinB2,
77
Epiblast layer, 43, 43
Epibranchial placodes,
Epicardial ridge,
Epigenital
Epispadias,
268
tubules,
247
151,
302
253b,
254
Epithelial endodermal
lining,
266
Epithelial-mesenchymal interactions, 5, 6, 209
Epithelization,
72
skeletal muscles,
145
Epoophoron,
250
Erythroblastosis fetalis, 106b

Vitelline cyst
313
64
302
133,
134,
154
Erythrocyte
Esophageal mosaicism,
atresia,
202b, types
of,
202b
Esophageal hernia, 93
Esophagus,
211,
211-212
abnormalities, atresia,
212b
Estriol,
107
unconjugated,
247
3,
212b
127
8
Euploid,
13b
Eustachian tube, 263, 324
Exemcephaly,
3116
Exocoelomic
Exocoelomic cavity,
44, cysts,
47,
44
47
Exons,
3
External auditory meatus, 266, 268, 325-326
External ear defects, 327, 327b
External genitalia
indifferent stage, 251, 252
male, 252, 252-253, 253
Extraembryonic mesoderm,
43
Extrahepatic biliary atresia, 220b
Extrauterine pregnancy,
48)
Eye abnormality, 336b-337b
choroid, sclera,
and cornea,
333,
333
development, molecular regulation, 334, 335
lens,
333
optic cup and lens vesicle, 329, 330, 331
optic nerve,
334
retina, iris, and ciliary body, 331
vitreous body, 333, 333-334

Face, 275-278
Facial clefts,
279b
Facial development, 268-273
Facial nerves,
300,
325
Facial
False prominences,
275
knots,
109b
Fate map,
56,
57
Fertilization,
11
acrosome reaction,
34
ampullary region, uterine tube, 32
capacitation,
33
corona radiata, penetration, 34
cortical and
zona reactions,
35
egg, metabolic
activation,
35
oocyte and sperm cell membranes, penetration, 34-35
second meiotic
division,
35
zona pellucida, penetration, 34
Fetal alcohol
spectrum disorder,
122
Fetal alcohol syndrome (FAS), 122
Fetal circulation,
195-196,
196
Fetal
Fetal
Fetal hydantoin
syndromes, hydrops,
106,
106b membranes,
100-101
Fetal period,
96
development, 96-99, 961, 97-99, 99t surgery,
129
Fetal therapy,
129
Fetal transfusion,
129
FGE See Fibroblast growth factor (FGF)
Fibrillar astrocytes,
293,
293
Fibroblast growth factor (FGF), 51, 138b, 219, 221, 236, 285,
308
neural induction, 63
proteins,
72
122
110
212b
Fibroblast growth factor receptors (FGFRs), 138b
Fibroblasts,
133
Fibrous joints, 152
Filum
terminale,
295
Flocculonodular lobe, 301
Fluorescence in situ hybridization (FISH), 19b-21b, 21
Folate
supplementation,
125)
Folic
acid
(folate)
69,
69b,
70,
297b
Follicle-stimulating hormone (FSH), 26
ovarian
cycle,
29
cells,
21,
245,
274
Follicular/proliferative phase, 39, 40
Follistatin,

55
Fontanelles, 135, 135
Footplate
limbs,
151
Foramen
cecum,
274
Foramen
ovale,
172
Forebrain.
See
Prosencephalon
Foregut
duodenum,
217,
218
esophagus,
271,
211-212
liver
and
gallbladder,
217-219
stomach, 212-216, 213-215
Fornix
commissure,
306
Fragile
X
syndrome,
20b
Fraternal
twins.
See
Dizygotic
twins
Free
(terminal)
villi,
61
Frontonasal prominence, 275
G
20b
Gallbladder
abnormalities of, 220b
duplication of, 220, 220b
formation
of,
217-219
Gametes
abnormal, 27b
maturation, 21-26
Gametogenesis
chromosome theory of inheritance, 11-21
morphological
changes,
maturation,
21-27
primordial germ cells, 10, 10, 10b
Gap
junctions,
7
Gartner's
cyst,
250
Gastroschisis,
88,
89,
225b,
226
Gastrulation
body axes, establishment, 52, 54-56, 55,57
embryonic
disc
growth,
57
embryonic mesoderm and endoderm, formation, 51, 52,
53
fate
map
establishment,
56,
57
notochord
51-52,
54
teratogenesis,
57b-59b
trophoblast development, 57—61
GDNE See Glial-derived neurotrophic factor (GDNF)
Gene expression, regulation of, 4
Gene
20b
Gene
therapy,
129
Gene
chromatin,
3,
3
DNA methylation represses transcription, 4-5
enhancers,
4
euchromatin,
3
exons,
3
heterochromatin,
3
histone
proteins,
3
introns,
3

linker
DNA,
3
nucleosome,
3,
3
Gene
promoter
region,
3,
4
RNA polymerase, 5
silencers, 4
TATA box, 4, 4
factor,
4,
4
site,
3
site,
3
Genetic
diagnostic techniques, identification, 20b-21b
Genetic variability,
12
Genital ducts
female, 247, 249-250
indifferent stage, 246, 246
male, 247-248, 249
molecular regulation, 246-247
Genital ridge,
243
Genital swellings, 251
Genital tubercle, 251
Genomic imprinting, 19)
Germ cells
abnormal,
27
formation of, 243, 244
primordial,
245
German measles, 117, 120
Giemsa-stained chromosomes, 20b
Glaucoma,
333
Gliablasts, 293, 293
Glial cells, 293, 293
Glial-derived neurotrophic factor (GDNF), 236
Glossopharyngeal nerves, 264, 273, 300
Glucagon,
221
Glycoproteins,
7
Goiter, 122
Goldenhar syndrome. See Oculoauriculovertebral spectrum
Gonadal dysgenesis,
17,
256b
Gonadotropin releasing hormone (GnRH), 29
Gonadotropins, 29
Gonads ovary, 245, 246
testis, 244-245, 245
Goosecoid,
55
Graffian follicles, 23, 23
Granule cells, 301
Granulosa cells, 23, 23
Gray communicating
rami,
316,
Growth hormone (GH), 1006
Gubernaculum, 256
Gut,
86,
86
atresias, 227b—228b, 228
divisions of, 208-209
molecular regulation, 209, 209-210
rotation, defects of, 227b
Gyri,
306,
306
H
Habenular commissures, 307
Hair,
341,
341

Hand-foot—genital syndrome, 158b
Handplate,
151
Hands,
153
Head face, 275-278
facial development, 268-273
intermaxillary segment,
278
musculature,
146, nasal
cavities, pharyngeal
148,
282-283, arches,
148¢
282,
262-266
clefts,
268
317
283
pharyngeal pouches,
266-268
secondary palate,
278,
278,
279
skeletal structures of, 260
teeth,
283-285
thyroid gland, 274-275
tongue, 273-274
Hearing loss,
327b

atria of, 172-174, 172-174
conducting system,
185
defects,
164b,
176b-178b,
182b-184b development
of,
164,
165,
169-170
establishment and patterning, 162-163
induction, 169
molecular regulation of, 169-170
muscles of,
149
septum formation, 181
Heart tube, 164, 165, 166
Heart-hand syndromes,
176b
Hedgehog
Indian,
8
sonic, 55, 73, 155, 209, 285, 334
types, 8
Hemangioblasts,
75
Hematopoietic stem cells, 75, 218
Hemiazygos vein,
193,
194
Hemolytic disease, fetus and newborn, 106b
Heparin, 122
Hepatic diverticulum, 218
Hepatic sinusoids, 191
Hepatocyte growth factor (HGF), 236
Hepatocyte nuclear transcription factors, 219
Hermaphrodites,
255b
Herniation physiological, 222-223
retraction of, 224
Herpes simplex virus, 121
Heterochromatin,
3,
8
Hiatal hernia.,
212b
Hindbrain. See Rhombencephalon
Hindgut,
229,
229
230b
Hip dislocation, 161b
Hippocampus,
305,
306
Hirschsprung disease, 230b. See also Megacolon
(HPE),
57,
282b,
310b,
337b
Holt—Oram syndrome,
158),
176)
Homeobox genes (HOX), 155, 285, 308
neural induction,
63
80,
81
Homocystinuria, 20b
Homologous chromosomes,
11,
12
Hormone receptor complex, 247
Horseshoe kidney, 239b, 240
Human chorionic gonadotropin (hCG), 47)
model,
133,
151,
153
Hyaline membrane disease, 206)
7

Hydatidiform mole, 48b
Hydraminios,
109b
Hydrocephalus, 297b, 311b
Hymen,
250
Hyoid artery, 185
Hyperthermia,
120¢
Hypertrophic cardiomyopathy, 176b
Hypoblast layer,
43,
43
Hypobranchial eminence,
273
Hypochondroplasia, 140b
Hypoglossal nerve, 273, 299
Hypophysis, 303-304, 304, 304b
Hypoplasia,
220b
Hypoplastic ossification centers, 144
Hypospadias, 253b, 254
Hypothalamus,
302,
303
sulcus, 302, 303
Hypoxia, 124
I
Ichythyosis, 340, 340b
Ileal diverticulum. See Meckel's diverticulum
Imperforate anus, 230, 230b
Implantation abnormal, 47b
uterus and,
39-40
In vitro fertilization (IVF), 36)
Inborn errors,
20)
Incisive foramen, 279b
Incus,
137,
324
Indian hedgehog, 8
Induction, 8-9
Inferior parathyroid gland, 267, 267
Inferior vena cava, 191, 193
defects of, 194, 194b
Infertility,
36b
Infundibulum, 304, 304
Inguinal hernia,
258,
258b
Inheritance, chromosome theory of autosomes,
11
diploid,
11
fertilization,
11
haploid,
11
homologous pairs, 11
linked genes,
11
meiosis,
11-13
mitosis, oocyte,
11
11
sex chromosomes,
sperm,
11
Insula,
306,
306
Insulin,
221
Insulin-like growth factor-I (IGF-I), 100b
Integrins juxtacrine signaling,
7
Intellectual disability,
313)
Interleukin-1f,
206
Intermaxillary segment, 278, 278
Intermediate mesoderm,
70
Internal branchial fistulas, 271)

Internal capsule, 305, 305
Interstitial cells of Leydig, 244
Interthalamic connexus,
302,
303
Interventricular foramen,
167,
168,
181
Interventricular foramina of Monro, 288
Intestinal loop
duplications mesenteries
of, of,
227b
224,
225
primary,
222
reversed rotation of, 227, 227b
Intracytoplasmic sperm injection, 36b
Intrahepatic biliary
duct atresia,
220b
Intrauterine growth restriction (IUGR), 1006
Introns,
8
Invagination germ layers, gastrulation, 51, 53
Todine,
124
Iridopupillary membrane, 333, 337b
Iris, 331, 332
Islets of Langerhans, 221
11
Isoimmunization,
106b
1201,
123
embryopathy, 123, 125b
Isthmus,
308
Jooued proteins,
7
Joint contractures. See Arthrogryposis
Jugular veins, 193
Junctional zone, 103
Juxtacrine interactions,
Juxtacrine signaling, 7

Kartagener's syndrome, 58b
Karyotyping, 15), 16b
Keratinization, 341
Kidney abnormal
location of,
239b—240b ascent of, 239
collecting system of, 233, 235
defects of, 237b-238b excretory system of, 235-236
function of, 240
horseshoe, 239b, 240
molecular regulation of, 236, 236
pelvic,
239,239b position of, 238
systems of, 232-242
Kinase,
6,
6
Kleeblattschadel, 140b
Klinefelter syndrome,
17b,
256b
Klippel-Feil sequence, 143b
Kupffer cells, 219

L
selectin,
38
Labia majora, 251
Labial component, 278
Labor, 115
Lacrimal sac, 277
Lacunar stage, 44, 44
Lamina terminalis, 306
Laminin,
7
Lanugo hair,
97
Laron's dwarfism, 100b
Laryngeal orifice, 201, 273
Larynx, 203, 203
Lateral cervical cyst, 271b
Lateral cleft
lip,
279b,
280
Lateral lingual swellings, 273
Lateral plate
skeletal system, 133
Laterality,
164b sequences, 58b, 169b
Lead,
120¢
Left inferior truncus swelling, 179
LEFTY-2,
55
Lens,
333
placodes, 67
vesicle, 329, 330, 331
Lentiform nucleus, 305, 305
Levator palatini,
265
Ligamentum arteriosum, 188, 189)
Ligamentum teres
hepatis,
192,
197
Ligamentum venosum, 192
Limbs defects, 157b-161b
development of, 151-152, 152
6
Limbs
growth and
development, musculature
Linked genes,
Lithium,
120r, of,
146,
148,
11
122
Liver abnormalities,
220b formation of, 217-219
molecular regulation,
219,
Lobster claw
Loop deformity,
of
Henle,
236
Low birth
weight,
L-transposition
100b of
great
Lumbar puncture,
Lungs,
203-205,
295
204
buds,
201,
204
congenital cysts,
206b ectopic
lobes, expansion
cysts,
206b
206b of,
histological
204
201, and
maturation
201,
202
functional of,
development,
205-207, premature
birth,
Luteinization,
205r
206b
29
Luteinizing hormone
ovarian cycle,
(LH),
29
Luteolysis,

Maculae acusticae,
323
Malleus,
137,
324
Mamillary body, 303, 303
Mammary glands
abnormalities of,
343b
342,
343
Mandible,
136
Mandibular branch, 273
Mandibular process,
136
Mandibular prominences,
Mandibulofacial
262, dysostosis.
Mantle layer,
290,
Marfan
See
297
syndrome,
Massa intermedia,
Mastoid
159b
302, process,
303
325
Maternal alcohol
Maternal abuse,
313b serum
Mature screening,
vesicular
Maxilla,
136,
127
follicles,
23,
264
Maxillary artery,
185
process,
136
prominences, 271
Meckel cartilage,
135,
Meckel
264
Gruber syndrome,
Meckel’s diverticulum,
Medial
237b
225b—226b, nasal
Median prominences,
nerve,
275
153
Medulla oblongata,
Medullary
298
cords.
See
Testis
Megacolon congenital, 230b, 318b
Meiosis crossovers,
12
germ cells,
11
homologous chromosomes,
polar bodies,
12-13
23
220
158b-159b,
159
arteries,
176b
151-152,
152-157
23
122
275
Treacher
12
226
152
205
Collins syndrome
primary oocytes,
spermatocytes,
12
12
synapsis,
12
Melanocytes,
67
Meningocele,
137,
137b,
296b
137b
Meningohydroencephalocele,
Menstrual phase,
39,
40,
41
Mercury,
120¢
Meromelia,
157b
Mesencephalon,
302
Mesenchyme,
66,
Mesenteric
133
artery,
222
Mesenteric ganglia,

Mesenteries,
316
210,
210-211
225)
loops,

embryonic period,
224,
225
63
germ layers,
gastrulation, intermediate,
57,
73
lateral,
57
lateral plate, 73-74
paraxial,
57
Mesodermal germ
blood and
blood intermediate
layer vessels,
mesoderm, lateral
plate mesoderm,
paraxial
51
75,
311,
310b
76,
77
73
73-74
mesoderm,
Mesonephric
70-73, duct,
246,
Mesonephros,
72,
246
232-233, genital
ridge,
243
Metabolic activation,
Metanephros,
35
233,
234
Metencephalon,
298
Methylation,
DNA,
4-5
20b
Microcephaly,
141,
Microdeletion
141b syndrome,
312b
Microglial cell,
Micromelia,
293
157b
Micropenis,
254)
Microphthalmia,
Microtia,
327
Midbrain.
See
Middle
11
Mesencephalon ear,
Midgut,
324-325,
324,
325
rotation
Mild of,
223, androgen
224
insensitivity
Milk teeth.
See
Miller—Dieker syndrome
Deciduous teeth
syndrome,
19b
Mitosis,
11,
17
nondisjunction,
14b spindle,
11
Mitral valve,
175
Mobile cecum,
Molecular
225)
regulation cell
signaling,
6-8
gene transcription
and signaling
(see also
(see induction
and organ
Cell-to-cell signaling)
Gene transcription)
Organogenesis)
regulators, gene expression,
5
Moles,
48b
Monoamine oxidase (MAO), 55
Monosomy,
136
Monozygotic twins, 108, 112, 112
Morula,
37
Mosaicism, 14b
5-6
(see also
337b
233,

19b
234
73t,
74,
75
(MAIS),
256b
Motor cortex, 306
Motor innervation, 64
Miillerian duct. See Paramesonephric duct
Mulkticystic dysplastic kidney, 237b
Muscles absence of, 149b-150b
cardiac, 149
dilator pupillae, 331
head,
148
innervation of, 146-147, 147
limb,
148
molecular regulation, 148
patterning of,
148
skeletal orgins
of,
147¢
striated,
145
and tendons, 148
smooth,
149
Muscular dystrophy, 1496
Muscular interventricular septum, 181
Mutations dominant,
206
gene,
20b recessive,
20b
Mycophenolate mofetil,
122
Mycophenylate,
120¢
Myelencephalon, 288, 298-300, 298, 299,
314t-315t,
319
Myelin,
294
Myelination,
294
Myelomeningocele, 297b
Myoblasts,
148
Myocardial disarray, 176b
Myocardin,
149
Myocardin-related transcription factors
(MRTF5),
149
Myocardium,
164
Myogenic regulatory factors (MRFs), 148
Myometrium,
39,
250
Myotome,
73,
145
N
Nasal cavities, 282-283, 282
Nasal (olfactory) placodes, 275
Nasal prominences,
275
Nasal septum,
278,
Nasolacrimal duct,
Natal teeth,
285b
Neck,
260-286.
See
Neopallium,
279
277
also
306,
Nephrogenic
306
cord,
cells, craniofacial
73
137b, defects,
271b,
271b derivatives,
69t ectodermal germ layer, 66-70
heart development,
179
muscle formation,
67
148
parasympathetic nervous
system, spinal

cord,
293
sympathetic nervous system, 315
Neural folds,
neurulation,
63
Neural groove,
63
Neural induction, molecular regulation of, 63
Neural plate,
287
66
63
Neural tube defects (NTDs)
Head
293,
313,
317
315t anencephaly,
69, spina
bifida,
69b
69b, spinal
cord,
Neurilemma
70
296b-297b sheath,
294
Neuroblasts,
290,
297
Neurocranium cartilaginous,
136
membranous,
135-136
Neuroectoderm,
63
Neuroepithelial cells, 288, 290
Neuromeres,
71
Neurons,
291
Neuropores, 287
Neurotransmitter serotonin (5HT), 60b
Neurulation,
63,
86
Nipple,
342
Noggin,
55
genes, somite
differentiation, neural
induction,
Nondisjunction,
63
13b-14b
Norethisterone,
123
Nose,
278
Notch pathway, 7,77
Notochord formation
allantoenteric diverticulum/allantois,
cloacal membrane,
51
definitive notochord,
neurenteric canal,
51
notochordal plate,
plate,
51
prechordal plate,
51
prenotochordal cells, 51
Nuchal translucency, 126
Nuclear hormone receptor family, 247
Nuclear RNA (nRNA), 5, 5
Nucleosome,
3,
3
Numerical abnormalities,
13b-18b
Nutritional deficiency, 124
Oo
Obesity,
124
Oblique facial
clefts,
279b
Occipital somites,
273
Oculoauriculovertebral spectrum, 273b
Oculomotor nerves, 299, 302
Odontoblasts,
283,
283

Olfactory bulbs, 306, 307
Olfactory nerve,
306
Olfactory placodes, 306, 306
Oligodendroglial cells, 293, 293, 294
Oligohydramnios,
99b
Oligozoospermia,
36b
Omental bursa,
213
Omentum, 215-216, 216
Omphalocele,
90b,
90,
225b,
Omphalomesenteric
226
veins.
Oocyte,
10
formation, maturation
145
inhibition,
Oogenesis,
21-23
Oogonia,
21,
22
Optic chiasma, 307, 307
Optic cup, 329, 330, 331
Optic nerve,
334
Optic stalk,
334,
334
Optic vesicles, 329, 329
Oral contraceptives,
123
Organ formation.
See
Organ of
Corti,
322
22
See
Vitelline
51
51
73
52,
54
veins
Organic mercury, 120f, 124
Organogenesis.
See also
Embryonic competence
factor,
5
crosstalk,
6
epithelial-mesenchymal interactions,
5
Oronasal membrane,
282,
282
Oropharyngeal teratoma, 10, 10b
Oropharyngeal membrane,
51,
78
heart tube,
164
Ossicles,
324-325
Ossification endochondral,
133, intramembranous,
134,
133, membranous,
135
133
Osteogenesis imperfecta,
Ostium primum
Ostium
151
134
158b-159b, defect,
177, secundum
Otic placodes,
Otic
178
defect,
67, vesicles,
67
Oval foramen,
closure
172
of,
197
Ovarian cycle,
corpus
29
albicans, corpus
32
atreticum,
29
corpus luteum,
31,
37

follicle-stimulating hormone
(FSH),
29
gonadotropin releasing hormone (GnRH), 29
luteinization,
29
luteinizing hormone
oocyte transport,
37, ovulation,
29,
29h, proliferative
(LH),
29
32,
32
30-34, phase,
sexual cycles,
Ovarian
29
medulla,
Ovary descent
of,
258
Ovulation,
29
P
Pacemaker,
Palate cleft,
117
component,
278, primary,
278
278
Palatine shelves,
Palatine
278, tonsils,
Paleopallium,
278
278
267
306,
Pallium,
306
Pancreas abnormalities
of, development
306
221b of,
219
221
islets,
221
molecular regulation of, 221
Pancreatic and duodenal homeobox 1 (PDX) gene, 221
Papillary muscles,
175
Paracrine interactions,
Paracrine
6
6-7
Paradidymis,
247
Parafollicular cells,
267
Paragenital tubules,
Paramesonephric
247
duct,
Paranasal air
sinuses,
246,
283
Parasternal hernia,
93b
Parasympathetic nervous system, 317, 318b
Parathyroid gland,
267
ectopic,
270b
245
31
29
176,
321
177
period
5,
185
246,
247
159
6
Paraxial mesoderm, 70-73
Polycystic kidney
disease,
238,
238b
Polydactyly,
158b,
159
Polyhydramnios, 212b, 311b. See also Hydramnios
Polymastia,
343b
Polymerase chain reaction (PCR), 39b
Polyspermy,
35
Polythelia,
343b
Pons,
300
Pontine nuclei, 300
Posterior commissures, 307

Postganglionic fibers, 316, 317, 317
Postmature birth,
99
Potter sequence, 237b
Pouch of
Douglas,
48b,
49
Prader—Willi's syndrome, 19, 19b
Preaortic ganglia,
316,
317
Preauricular appendages,
327,
Prechordal chondrocranium,
Prechordal
328b
136
plate,
51
Preeclampsia,
102b
Preganglionic parasympathetic fibers, 317
Pregnancy abnormalities
in,
47
placenta,
108-109
Premature birth
lungs,
206b
Premature rupture of membranes (PROM), 109
Premaxilla,
264
Prenatal diagnosis
amniocentesis,
127
CVS,
128
maternal serum screening, 127
ultrasonography, 125-126, 126, 127
Preterm birth,
115)
Primary heart field (PHF), 162
Primary oocytes,
12
Primary villi, 46, 47,59, 59
Primitive node,
gastrulation,
51
Primitive pit, gastrulation, 51, 53
Primordial follicle,
22
Primordial germ cells (PGCs), 10, 10, 10b, 21, 21, 27,

Probe patency,
172
Probe patent foramen ovale, 197
Processus vaginalis,
257
Proctodeum,
78,
229,
Progenitor heart
Progesterone,
229
cells,
107
Progress zone,
151
phase,
Pronephros,
Pronucleus,
29,
232,
162
41,
232
35
Prophase,
11
Prosencephalon diencephalon,
telencephalon,
302-304
304-306
Prostate gland,
241
Proteoglycans,
7
Protoplasmic astrocytes, 293, 293
Proximal convoluted
tubule,
236
Prune belly
syndrome,
149},
150
Pulmonary channel,
179
Pulmonary infundibular stenosis, 182b, 183
Pulmonary vein,
174
Purkinje’s fibers,
Pygopagus twins,
Pyloric stenosis,
Q
Quinine,
122
149
114
217b
41

Rachischisis, 297b
Radial nerve,
153
Radical fringe,
155
Rathke's pouch, 304, 304
Rays,
83
Recessive mutations, 20b
Rectoanal atresias,
230,
230b
Rectourethral fistula, 230, 230b
Rectouterine cavity,
49b,
49
Rectovaginal fistula,
Recurrent laryngeal
Reichert’s cartilage,
Renal
230,
230b nerves,
186
264
agenesis,
Renal
Renal
Renal
237b coloboma
syndrome, corpuscle,
dysplasias,
233
237b
Renal tumors,
237b-238b
Resegmentation,
142
Respiratory bronchioles,
205
distress syndrome
diverticulum,
RET
gene,
318b
Rete testis,
244
Retina,
331
Retinoic acid (RA), 63, 155, 169, 308
125)
Retrocecal appendix, 224, 224
Retrocolic hernia,
225b
Rh antigen,
106)
Rhombencephalon cerebellum,
300-301
metencephalon,
300
myelencephalon, 298-300
Rhombic lips,
300
Rhombomeres,
268,
Ribs,
144
144b
Rickets. See Vitamin D deficiency
Right atrial
appendage,
174
Right superior truncus swelling, 179
RNA
3
Robin sequence,
271b
Roof plate,
302
Rubella,
119

Saccule,
321-322
Sacrococcygeal teratoma, 58, 59b
Scala tympani,
322,
323
Scala vestibuli,
322,
323
Scaphocephaly,
1376,
138
Scatter factor. See Hepatocyte growth factor (HGF)
Schwann cells,
67,
294
Sclera, 333, 333
Sclerotome, 72, 133, 145, 146
Scoliosis,
145)
Scrotal swellings, 253
Sebaceous glands,
341
Sebum,
341
Secondary heart field (SHF), 162
Secondary palate,
278,
279
Secondary villus,
59,
59
Secondary yolk sac/definitive yolk sac, 47, 47
Secretory/progestational phase, 39, 40
Segmentation clock, 72
322,
324
Semilunar valves, 181, 181, 182
Seminiferous tubules,
24,
244
308
201,
337)
(RDS),
206b
201,
202
Sensory ganglia, 67,293
Sensory innervation,
94,273
Sensory relay
nuclei,
300
Septum secundum,
172
Septum spurium, 170
55
Serous membranes, 86, 88
Sertoli cells, 24
Serum response factor (SRF), 149
11
Sex glands, sexual differentiation, 248
Sexual cycles, 29
Shprintzen syndrome, 271b
Signal transduction pathways juxtacrine
7
paracrine signaling, 6-7
Signaling cell,
6-7
juxtacrine,
7
paracrine,
6-7
4
Sine occulis homeobox 3 (SIX3), 310b
Sinuatrial node,
185
Sinuatrial orifice,
170
Sinus venarum,
170,
174
Sinus venosus, 170, 170, 171
Sinusoids,
45,
45
Sirenomelia,
57b,

Situs inversus,
Skeletal
58
57b, dysplasia,
Skeletal muscles
head,
148
innervation of, 146-147
limb,
148
origins of,
148t patterning
of, striated,
148
145
and tendons,
148
Skeletal structures of, 260
Skin keratinization of, 341
layers of,
339
Skull development
of neurocranium,
135,
135
newborn,
135,
135-136
viscerocranium, 136, 136-137
Small for
gestational age
(SGA),
Smith-Lemli-Optiz
Smoking,
123
Smooth muscle, 149
Solvents,
120t
Somatic efferent, 299
Somatic nuclear
transfer,
Somatomammotropin,
1006
310b
39b
107
Somatopleure,
87
Somites development,
146
development of, 133, 133
differentiation,
72-73
72
145,
73
146
Somitomeres,
70,
133,
145
Sonic hedgehog (SHH), 55, 155, 209
eye development,
334
somite differentiation,
73
tooth development, 285
syndrome,
169b
139b—140b,
139¢
Special sensory innervation, 274
Sperm,
10
Spermatic cord, 258, 258b
Spermatids,
26
Spermatocytes, 12
Spermatogenesis, 24, 24-26, 26
Spermatozoa,
24
Spermiogenesis, 26, 26
Sphincter mechanism,
195
Sphincter pupillary muscle, 302
296b cystica,
143b occulta,
143b
alar plates,
290
basal plates, 290
glial cells,
293
gray matter, 290
mantle layer, 290, 291
molecular regulation, 295-296
myelination, 294
nerve cells, 290-291, 293

neural crest cells, 293
neural tube defects (NTDs), 296b-297b neuroblasts,
290,
291
neuroepithelial layer, 290
neuroepithelium,
290
63
positional changes, 294, 295
spinal nerves,
293-294
sulcus limitans,
290
287
Spinal nerves,
293-294
Spinous processes,
Spiral
142
ligament,
322
Spiral limbus,
322
Splanchnic mesoderm, 145
Splanchnic/ visceral mesoderm layer, 70
Splanchnopleure,
87
Spleen,
214
Spliceosomes,
5,
5
Splicing isoforms,
5,
5
Spontaneous abortions, chromosomal and genetic factors,

Stapedial artery, 185
Stapedius muscle,
324
Stapes,
137,
324
Statoacoustic ganglion, 323
Stellate cells,
301
Stellate reticulum, 283
Stem cell
adult,
39b embryonic, 39b
hematopoietic,
75
transplantation, 128
Stem villi,
61
Stenoses,
227b—228b,
228
Sternocleidomastoid muscle, 270b
Sternum,
144
defects of,
1446
Steroidogenesis factor 1 (SF1), 247
Stomach abnormalities of, 217b
formation of, 212-216, 213-215
Stomodeum,
303,
304
Striated muscles,
Striated
299
skeletal musculature,
145,
146,
Structural chromosome
Stylopharyngeus muscles, 264
147,
147¢
18b—20b
Subclavian artery
abnormal origin of, 189), 190
Sulci,
306
Sulcus limitans,
290
Superior laryngeal nerve, 273
Superior parathyroid
gland,
267
Superior vena
cava,
193, defects
of,
194b,
Suprarenal gland,
193
195
316-317
205,
206b
Sustentacular cells of Sertoli, 244
Sutures,
135
Sweat glands,
342
Swyer syndrome,
256b nervous
Synapsis, system,
organ plexuses,
12
Syncytial knots,
101
Syncytiotrophoblast, 43, 43, 44
Syncytium,
101
Syndactyly,
158,
Syndecan,
236
Synophthalmia,
Synovial
159
337b joints,
Synovial
Syphilis,
152
membranes,
120¢
T
TATA
box,
T-box,
4,
4
55
Teeth abnormalities
of,
285

formation of, 283-285, 283, 284
Tela choroidea,
300
Telecephalic vesicle,
Telencephalon,
287,
Temporal bone,
Tendons,
148
Tensor palatini,
264
Teratogenesis gastrulation,
57b-59b
Teratogens,
120t mechanisms,
119
Teratology principles
of,
Teratomas,
119
10,
10b
Terminal sulcus,
Tertiary villus,
273
60
Testis descent
of,
256-258, formation
hydrocele
257
of,
244-245, of,
245
258b
244
285)
Tetralogy of
Fallot,
171,
182b,
183
TG-interacting factor (TGIF), 310)
Thalamus,
302,
303
Thalidomide,
118,
120t,
157b heart
defects,
176b
Thanatophoric dysplasia, 139b—-140b, 141
Theca folliculi,
23
Therapeutic cloning, 39b
Thoracic cavity, 90-92, 91, 92
Thoracic duct,
198
Thoracopagus twins, 114
Thyroglossal duct, 274, 274b, 275
Thyroglossal fistula,
274b,
275
324
264
310b
302
152
322
315-316
316
Thyroid
of,
274b
formation
of,
270,
270
Thyroxine,
274
Time
of
birth,
99
Tongue,
273-274
Tongue-tie,
273b
Tonsillar
fossa,
267
Tooth
285)
Toxoplasmosis,
1201,
121
Trabeculated
atrial
174
Trachea,
203-205,
204
Tracheoesophageal
fistulas
(TEFs),
202b-203b,

septum,
212,
212b
factors,
4,
4
Transforming
growth
factor-B
(TGF-B),
52,
54
follistatin,
63
Transient
dendrite,
290
Translocations,
14b
Transmembrane tyrosine kinase receptors, 138)
Transplantation
stem
cell,
128
Transposition
of
great
vessels,
182b,
184
Transversalis
fascia,
258
Transverse
limb
deficiencies,
160b,
161
pericardial
sinus,
164
processes,
142
Treacher Collins syndrome, 271b
Treacle,
271b
Tricuspid
atresia,
177b,
178
Tricuspid
valve,
175
nerves,
300
Triiodothyronine,
274
Trilaminar
germ
disc,
51—62.
See
also
Trimethadione,
120¢,
122
Triple
X
syndrome,
17b-18b
Trisomy,
13b
13,
15b,
17,
17b
18,
15b,
17
21
(see
Down
syndrome)
Trochlear
nerves,
302
Trophoblast,
37,
38
Truncus
arteriosus,
179,
181
persistent,
182b,
183
Truncus
swellings,
179
Tubal
49
Tubotympanic recess, 266, 324
‘Tumors
renal,
237b—238b
Wilms’,
237b
Tunica
albuginea,
244
Tunica
vaginalis,
257-258
Turner
syndrome,
17),
18,
256b
22q11.2
deletion
syndrome,
271b
22q11
syndrome,
19b

Twins
conjoined,
113b,
114
dizygotic,
110,
111
fetal membranes in, 110, 111, 112
monozygotic,
110,
112,
112
Twin-twin
transfusion
syndrome,
113b
‘Tympanic
antrum,
325
Tympanic
cavity,
324,
324
Tympanic
membrane,
325,
326
Tyrosine
kinase
receptor,
318b
Tyrosine
kinase
77
U
Ulnar nerve, 153
Ultimobranchial body, 267, 274
Ultrasound,
99
prenatal diagnosis, 125, 125-126, 126
Umbilical arteries,
185,
188
196
Umbilical cord, 107-108, 108
connecting stalk,
61
47,
Umbilical
47
fistula,
Umbilical
Umbilical closure
Uncinate
226, herniation,
veins,
192, of,
196-197
process,
221
Upper jaw
278,
Urachal cyst, 242, 242b
Urachus,
240
fistula,
242, sinus,
242b
242,
242b
Ureter, duplication
Urethra,
240-241
Urethral groove,
253
Urinary bladder,
Urogenital ridge,
Urogenital sinus,
240
230,
229, of,
Urorectal
237b,
278
238
234
240
241
septum,
Uterine canal,
240
246
Uterine tube,
249
Uteroplacental circulation,
Uterorectal pouch,
Uterovesical
249
pouch,
Uterus arcuatus,
bicornis,
251,
249
251b
251,
251b defects
of,
251b duplications
of,
251b formation of, 249-250
implantation basal
arteries, basal
41
layer,
40,
41

compact layer,
40,
41
endometrium,
39
follicular/proliferative phase, 39, 40
menstrual phase,
39,
40,
41,
41
myometrium,
39
perimetrium,
39,
40
proliferative phase,
41,
41
secretory/progestational phase, 39, 40
spongy layer,
40,
41
Uterus didelphys,
Vv

atresia, 251, 251b defects of, 251b
formation of, 250, 250
fornices,
250
Valproic acid, 1201, 122
Valvular stenosis
pulmonary artery, 183b-184b
Vanishing twin, 113b
251,
251b
227b
223,
223
192
of,
46
Varicella
virus,
121
Vascular accidents, 227b
Vascular
growth
factor
(VEGF),
77,
185
Vasculogenesis,
75,
185
Venous
system
cardinal veins, 192-194
defects
of,
194b-195b
umbilical
veins,
192,
192
vitelline veins, 191-192, 192
Ventral
aorta,
185
Ventral body wall defects, 78, 88b—90b
Ventral
mesentery,
211,
218
inversion,
176b
Ventricular septal defects (VSDs), 182, 182b
Vermis,
300
Vertebrae
cleft,
143b
defects,
143b
development, 142, 142-143
Vertebral
column,
142,
142-143
fibers
of
cranial
nerve
VIII,
323
membrane,
322,
323
Villi
chorionic sampling of, 128
free,
61
primary,
47
Virilization,
247
Viscerocranium,
136,
136-137
Visceroefferent
column,
316
Vitamin
A,
123,
155
embryopathy,
123
Vitamin
D
285)
Vitelline
arteries,
185,
188
Vitelline
cyst,
226,
227b
Vitelline
duct,
78,
88,
222,
222
of,
225b-227b
Vitelline
fistula,
226,
227b
Vitelline
veins,

191-192,
192
Vitreous
body,
333,
333-334
Vocal
cords,
203
Volvulus,
227b

Waardenburg
syndrome
(WS),
340,
340b
WAGR
syndrome,
237b
Warfarin,
1201,
122
Wharton’s
jelly,
108
Wilms’ tumor, 237b
WNT signaling pathways, 72
Wolffian duct, 233, 233, 246, 246
x
X chromosome inactivation,
4
X-linked
recessive
disease,
149b
Z
Zinc finger protein (ZIC2), 310b
Zona pellucida, 22, 23
Zone of polarizing activity (ZPA), 155
Zygomatic
bone,
136,
264
Zygotes,
10
abnormal,
39b